key: cord-341472-29opvzrj authors: curley, gerard f.; laffey, john g. title: future therapies for ards date: 2014-12-04 journal: intensive care med doi: 10.1007/s00134-014-3578-z sha: doc_id: 341472 cord_uid: 29opvzrj nan despite more than 150 randomized clinical trials (rcts) of multiple potential therapies, the only interventions for acute respiratory distress syndrome (ards) that reduce mortality are those that minimize ventilator-induced lung injury [1] . this 'translational failure' may have a number of explanations. firstly, ards is a syndrome, and interventional trials in ards generally include a heterogenous patient group with a wide spectrum of disease etiology and disease severity. second, deficits exist in our understanding of key aspects of the pathogenesis of ards. notwithstanding these challenges, a number of promising therapies are currently under investigation for ards, and offer hope for the future. aspirin platelets are important in ards pathogenesis. in preclinical studies, aspirin reduces thromboxane a 2 , p-selectin, and platelet-derived chemokine (e.g., ccl5 and cxcl4) production, reduces platelet-neutrophil aggregates and neutrophil extracellular traps, and enhances anti-inflammatory lipid mediators such as 15-epilipoxin a4. aspirin reduces the risk of developing ards in critically ill patients [2] . a clinical study of aspirin in human volunteers undergoing endotoxin inhalation (arena nct01659307) and a rct of aspirin for ards prevention [3] are ongoing (table 1) . hmg coa-reductase inhibitors (statins) exert diverse 'pleiotropic' effects beyond their 'pharmacologic' effect in cholesterol reduction, including anti-inflammatory and endothelial protective effects. results from both preclinical and observational studies support a potential role for statins in ards. simvastatin improved pulmonary and systemic organ function in a phase 1/2 rct in ards [4] , but two larger phase 2/3 trials of statin therapy, carried out in ireland/uk [5] and the usa [6], respectively, did not demonstrate benefit. rosuvastatin, a hydrophilic statin, did not improve clinical outcomes in sepsis-associated ards and may have increased hepatic and renal dysfunction [6] . the lipophilic statin simvastatin did not worsen hepatic or renal function, it non-significantly reduced mortality, but it did not increase the number of ventilator-free days (vfd, the primary outcome) [5] . a definitive large trial of simvastatin, powered for mortality as a primary outcome, may be warranted. activation of coagulation plays a key role in the pathogenesis of ards, resulting in alveolar fibrin deposition which impairs gas exchange. in pre-clinical studies, heparin has been found to reduce alveolar fibrin deposition and exert anti-inflammatory effects. in one small rct, heparin decreased the number of vfd in patients at risk for ards [7] . further studies investigating the efficacy of nebulized heparin in patients at risk of ards (actrn12612000418875) ( table 1 ) are underway. interferon beta (ifn-b) increases endothelial expression of cd73, the rate-limiting enzyme in the conversion of adenosine monophosphate to adenosine, which in turn binds to pulmonary a2b receptors and exerts multiple protective effects in pre-clinical models. in a recent openlabel dose-escalation study, only two (8 %) of 26 ards patients treated with 10 lg per day of ifn-b-1a died by day 28, compared to a 32 % mortality in a parallel control group [8] . although the study was not randomized or blinded, and there were some baseline differences between the treated and control cohorts, further investigation of ifn-b for ards is warranted. tumor necrosis factor receptor 1 blockade tumor necrosis factor (tnf) exerts its effects by binding to one of two tnf receptors, designated tnfr1 and tnfr2. tnf-activated pro-inflammatory pathways and the programmed cell death pathways that result in tissue injury are largely mediated through tnfr1, while tnfr2 signaling plays a role in tissue repair and angiogenesis. promising pre-clinical data support the efficacy of anti-tnfr1 monoclonal antibodies [9] . in one study, inhaled anti-tnfr1 antibodies decreased the pulmonary inflammation induced by endotoxin in healthy volunteers [10] . early phase studies in ards patients are awaited. angiotensin converting enzyme 2 angiotensin-converting enzyme (ace) cleaves angiotensin-i to generate angiotensin-ii, which causes vasoconstriction, inflammation, and increased vascular permeability via type 1 (at1r) and type 2 receptors. ace-2, a homolog of ace, cleaves a single residue from ang-ii to generate ang1-7 [11] , which blocks many at1r-mediated actions. imai et al. [11] found that ace, ang-ii, and at1r function as lung injury-promoting factors, whereas ace-2 protects the lung from injury. ace2 is a receptor for severe acute respiratory syndrome-coronavirus (sars-cov), while sars-cov induces downregulation of ace2, which is an important step in the development of severe lung failure [12] . in addition, mortality is increased in patients with ards who have the ace dd phenotype, which results in greater ace activity [13] . a human phase i/ii clinical trial of recombinant human ace2 therapy in patients with early ards is in progress (nct01597635) ( table 1) . adrenomedullin adrenomedullin (am), an endogenous 52 amino acid peptide belonging to the calcitonin gene-related peptide family, is expressed in multiple tissues, including endothelial cells, and plays a crucial role in endothelial barrier integrity. am acts via binding of the calcitonin receptorlike receptor, thereby raising intracellular camp levels in endothelial cells and reducing myosin light chain (mlc) phosphorylation. thus, am may prevent endothelial contraction and intercellular gap formation [14] . am expression is upregulated in inflammatory diseases including ards and sepsis, and endogenous am may contribute to the protection of vascular function in inflammation [14] . am therapy reduces pulmonary permeability injury and decreases inflammation in experimental ards and sepsis. the committee for orphan medicinal products of the european medicines agency (ema) recently recommended am as an orphan drug for the treatment of ards (ema/comp/104704/ 2010). clinical trials with am are in the planning stage. keratinocyte growth factor (kgf) is a fibroblast growth factor expressed predominantly by mesenchymal cells, and its receptor (kgfr) is expressed on epithelial cells and macrophages. results from pre-clinical studies suggest that intra-tracheal kgf reduces lung injury induced by hyperoxia, ventilator-induced lung injury, and bacterial pneumonia. in a recent study, kgf treatment (palifermin ò ) increased markers of type ii alveolar epithelial cell proliferation and increased alveolar concentrations of reparative proteases and the anti-inflammatory cytokine il-1ra following endotoxin inhalation by volunteers [15] . a phase ii clinical trial of palifermin ò in ards has recently been concluded (isrctn95690673), and the results are awaited (table 1) . mesenchymal stem/stromal cells mesenchymal stem/stromal cells (mscs) can regulate both the innate and adaptive immune systems and can modulate macrophage phenotype, inhibit the production of inflammatory cytokines by activated cd4 and cd8 t cells, and stimulate the generation of foxp3? regulatory t cells, potentially reducing pro-inflammatory cytokines in ards [16] . mscs directly attenuate bacterial sepsis, the commonest and most severe cause of ards, by enhancing macrophage phagocytosis and increasing antimicrobial peptide secretion, thereby increasing bacterial clearance [16] . mscs also repair the injured lung following ventilation-induced lung injury, via paracrine mechanisms [17, 18] . a recent pilot study of msc therapy for ards demonstrated no adverse effects [19] . a phase 1/2, open-label, dose-escalation, multi-center clinical trial of allogeneic bm-mscs in patients with moderate to severe ards is underway in the usa (nct01775774) ( table 1) . although there have been many failed therapies to date, new therapies based on improved understanding of the mechanisms implicated in the development of ards are emerging, and may provide a treatment option in the near future. effects of interventions on survival in acute respiratory distress syndrome: an umbrella review of 159 published randomized trials and 29 meta-analyses prehospitalization antiplatelet therapy is associated with a reduced incidence of acute lung injury: a population-based cohort study us critical illness and injury trials group: lung injury prevention with aspirin study group (usciitg: lips-a) (2012) lung injury prevention with aspirin (lips-a): a protocol for a multicentre randomised clinical trial in medical patients at high risk of acute lung injury a randomized clinical trial of hydroxymethylglutarylcoenzyme a reductase inhibition for acute lung injury (the harp study) simvastatin in the acute respiratory distress syndrome rosuvastatin for sepsisassociated acute respiratory distress syndrome nebulized heparin is associated with fewer days of mechanical ventilation in critically ill patients: a randomized controlled trial the effect of intravenous interferon-beta-1a (fp-1201) on lung cd73 expression and on acute respiratory distress syndrome mortality: an open-label study selective inhibition of intra-alveolar p55 tnf receptor attenuates ventilatorinduced lung injury a novel tnfr1-targeting domain antibody attenuates pulmonary inflammation in a human model of lung injury, via actions on the lung micro-vascular endothelium angiotensin-converting enzyme 2 protects from severe acute lung failure a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury ace i/d but not agt (-6)a/g polymorphism is a risk factor for mortality in ards adrenomedullin and endothelial barrier function keratinocyte growth factor promotes epithelial survival and resolution in a human model of lung injury therapeutic potential and mechanisms of action of mesenchymal stromal cells for acute respiratory distress syndrome effects of intratracheal mesenchymal stromal cell therapy during recovery and resolution after ventilator-induced lung injury mesenchymal stem cells enhance recovery and repair following ventilator-induced lung injury in the rat treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study acknowledgments j. laffey and g. curley are funded by the key: cord-016869-pzwlxtd6 authors: pal, subrata title: the lung and its transplantation and artificial replacement date: 2013-01-08 journal: design of artificial human joints & organs doi: 10.1007/978-1-4614-6255-2_15 sha: doc_id: 16869 cord_uid: pzwlxtd6 the human thoracic cavity houses a pair of lungs, the left lung and the right lung. the left lung is slightly smaller (since the heart is placed a bit to the left in the body) and has two lobes, and the right lung is bigger, with three lobes. they are spongy and elastic organs that are broad at the bottom and taper at the top. they consist of air sacs, the alveoli. many alveoli group together and open into a common space. from this space arise the alveolar ducts, which join together to form bronchioles. the bronchioles connect them to the respiratory tract. the lungs also have blood vessels, the branches of the pulmonary artery and veins (fig. 15.1). apart from the lungs, several associated organs and structures together form the respiratory system. the respiratory system is closely linked with the circulatory system, as the transport of the gases takes place through blood. the lung and its transplantation and artificial replacement the respiratory system starts with the nose, which covers the nasal cavity. the nasal cavity opens to the atmosphere through the openings called the nostrils. the nasal cavity is divided into two portions by a cartilagenous septum and is lined by fine hairs that filter the dust particles from the air. the nasal cavity is separated from the mouth by hard and soft palates that form its floor. it opens into the region called the pharynx (fig. 15.2) . the pharynx is a common passage to both food and air. this allows more air whenever required and also allows passage of air in case the nose is blocked. the pharynx continues into the glottis. the glottis is the narrow opening into the larynx. it is guarded by a flap of tissue called the epiglottis. several folds of elastic connective tissue are embedded into the posterior end of the glottis. they are called the vocal cords. these extend into the larynx. the larynx is also called the voice box. the vocal cords stretch across the larynx and vibrate when the air passes through them. this vibration produces various sounds. the coordinated movement of the lips, cheeks, tongue, and jaws produce specific sounds that result in speech. speech is an ability that only humans are gifted with, the larynx is held open with the help of cartilages. the "adam's apple" is a prominent cartilage of the larynx. the larynx continues as the trachea after the cords. the trachea is also called the windpipe. the trachea is held open with the help of c-shaped cartilagenous rings. the open ends of the rings are located toward the esophagus, also known as the foodpipe. the trachea is situated in front of the esophagus. the cartilages keep the larynx and trachea from collapsing even when there is no air in them. the trachea then branch into two main branches called bronchi. each bronchus is also supported by the cartilagenous rings. the bronchus then branches into several bronchioles. the bronchioles progressively lose the cartilages as they become narrower. the bronchioles end as fine tubules called the alveolar ducts ( fig. 15.3) . each alveolar duct opens into an alveolar sac. an alveolar sac is the extended region into which a group of alveoli or air sacs open. each alveolus is a saclike structure lined by a single layer of epithelial cells. it is bound on the outside by a network of capillaries. all the alveoli on one side are enclosed by the membrane called the pleural membrane and constitute a lung. the pulmonary artery from the heart containing impure blood enters the lungs and branches into minute capillaries that surround the alveoli. these capillaries then join together to form the pulmonary vein, which carries the purified blood back to the heart. the common composition of atmospheric air that we breathe in is nitrogen-78%, oxygen-21%, carbon dioxide-0.03-0.04%, hydrogentraces and noble gases in traces. thus, the air naturally contains nearly 500 times more oxygen than carbon dioxide. this oxygen-rich air is taken in by the nostrils. in the nasal cavity, it is filtered by the fine hairs in the nose. the cavity also has a rich supply of blood vessels that keep the air warm. this air then enters the pharynx, then the larynx, and then into the trachea. the trachea and the bronchi are lined with ciliated epithelial cells and secretory cells (goblet cells). the secretory cells secrete mucus, which moistens the air as it passes through the respiratory tract, and also trap any fine particles of dust or bacteria that have escaped the hairs of the nasal cavity. the cilia beat with an upward motion such that the foreign particles along with the mucus is sent to the base of the buccal cavity, from where it may be either swallowed or coughed out ( fig. 15 .4). the air from the bronchus then enters the bronchioles and then the alveoli. the alveoli form the respiratory surface in humans. the capillaries lining the alveoli have blood that has a low concentration of oxygen. so the oxygen from the air easily diffuses into the blood through the thin barrier of the alveolus wall. similarly, since the concentration of carbon dioxide is quite high in the blood, the gas easily diffuses out into the alveolar space. from here, the air-which has a comparatively higher concentration of carbon dioxide than the air that entered it-leaves the lungs (fig. 15 .5). the human lung is a very important and strong organ that has to withstand a strong and continuous assault stemming from a wide range of environmental conditions and the lifestyle in our modern world, which cause a multitude of diseases and conditions, as outlined here: • lung transplantation is a surgical procedure to totally or partially replace a patient's diseased lung with a donor's lung. while lung transplants carry certain associated risks, they can also extend life expectancy and enhance the quality of life for end-stage pulmonary patients. lung transplantation is the therapeutic measure of last resort for patients with end-stage lung disease who have exhausted all other available treatments without improvement. a variety of conditions may make such surgery necessary. as of 2005, the most common reasons for lung transplantation in the united states were the following [1] : 27% from chronic obstructive pulmonary disease (copd), including emphysema 16% from idiopathic pulmonary fibrosis 14% from cystic fibrosis 12% from idiopathic (formerly known as "primary") pulmonary hypertension 5% from alpha 1-antitrypsin deficiency 2% due to replacing previously transplanted lungs that have failed after a period 12% from other causes. a lobe transplant is a surgery in which part of a living donor's lung is removed and used to replace part of a recipient's diseased lung. this procedure usually involves the donation of lobes from two different people, thus replacing a single lung in the recipient. donors who have been properly screened should be able to maintain a normal quality of life despite the reduction in lung volume. many patients can be helped by the transplantation of a single healthy lung. the donated lung typically comes from a donor who has been pronounced brain-dead. certain patients may require both lungs to be replaced. this is especially the case for people with cystic fibrosis, due to the bacterial colonization commonly found within such patients' lungs; if only one lung were transplanted, bacteria in the native lung could potentially infect the newly transplanted organ. some respiratory patients may also have severe cardiac disease that itself would necessitate a heart transplant. these patients can be treated by a surgery in which both lungs and the heart are replaced by organs from a donor or donors. first performed in 1987, this type of transplant typically involves the transplantation of a heart and lungs into a recipient. prior to operating on the recipient, the transplant surgeon inspects the donor lung(s) for signs of damage or disease. if the lung or lungs are approved, then the recipient is connected to an intervenous line and various monitoring equipment, including pulse oximetry. the patient will be given general anesthesia, and a machine ventilator will breathe for the patient. the donors are usually road accident victims whose organs can be transplanted within 5-6 h of death. there are a large number of patients who are waiting for such transplantation from donors. potkey of vamc cleveland, ohio (2009), recently developed one model of artificial lungs. he indicated more than 35 million americans are living with chronic lung disease; it is responsible for nearly 350,000 deaths every year in the united states alone [1] . acute respiratory distress syndrome has a mortality rate of 50% and affects 1.50 lakh americans each year [2] . many patients waiting for lung transplants die while on the waiting list. to help combat these problems, artificial lungs have been developed with the goal of replacing or supplementing the respiratory function of the lung. artificial lungs mimic the function of real lungs, adding oxygen to, and removing carbon dioxide from, the blood. in all cases, however, the performance of artificial lungs is still significantly lower than that of natural lungs. the human lung is a remarkable organ, providing a maximum gas exchange rate for both o 2 and co 2 of 2-6 l/min [3] . on the other hand, current artificial lungs are only capable of a maximum gas exchange rate of 0.25-0.40 l/min, limiting their use to the short-term respiratory support for patients at rest. this insufficiency is due to the smaller surface area, smaller surface-area-to-volume ratio, and greater membrane thickness of artificial lungs compared to the human lung [3] . recent advances in the micromachining of silicone elastomer (pdms) have made possible the creation of a new highly efficient artificial lung (fig. 15.6 ) with feature sizes similar to or better than those of the human lung. such a micromachined artificial lung would have an improved gas exchange performance compared to its conventional counterparts, potentially resulting in increased clinical use. silicone has been used as the membrane material in some commercially available artificial lungs due to its biocompatibility, durability, stability, and high permeability to oxygen and carbon dioxide. however, these devices have limited gas-exchange capability mainly due to the membrane's thickness (>50 μm). a significant advantage of silicone membranes is that blood plasma leakage does not occur as it does in microporous hollow fiber oxygenators [3] . in fact, hollow fiber oxygenators are sometimes coated with a thin layer of silicone in order to reduce plasma leakage and increase the device lifetime. due to these advantages, silicone was utilized as the membrane material in this study (fig. 15.7) . lung, artificial: basic principles and current applications new design for a pumping artificial lung development of a total artificial lung 1. describe the structure and function of natural lungs. what conditions in a patient create the requirement for an artificial lung? 2. describe how to develop an artificial lung. indicate the volume of blood and oxygen and other gases to be handled. develop a schematic diagram for an artificial lung. key: cord-344206-53g7yjf9 authors: ray, archita; jaiswal, ashish; dutta, joytri; singh, sabita; mabalirajan, ulaganathan title: a looming role of mitochondrial calcium in dictating the lung epithelial integrity and pathophysiology of lung diseases date: 2020-09-21 journal: mitochondrion doi: 10.1016/j.mito.2020.09.004 sha: doc_id: 344206 cord_uid: 53g7yjf9 with the increasing appreciation of mitochondria in modulating cellular homeostasis, various disease biology researchers have started exploring the detailed role of mitochondria in multiple diseases beyond neuronal and muscular diseases. in this context, emerging shreds of evidence in lung biology indicated the meticulous role of lung epithelia in provoking a plethora of lung diseases in contrast to earlier beliefs. as lung epithelia are ceaselessly exposed to the environment, they need to have multiple protective mechanisms to maintain the integrity of lung structure and function. as ciliated airway epithelium and type 2 alveolar epithelia require intense energy for executing their key functions like ciliary beating and surfactant production, it is no surprise that defects in mitochondrial function in these cells could perturb lung homeostasis and engage in the pathophysiology of lung diseases. on one hand, intracellular calcium plays a central role in executing key functions of lung epithelia, and on the other hand maintenance of intracellular calcium needs the buffering role of mitochondria. thus, the regulation of mitochondrial calcium in lung epithelia seems to be critical in lung homeostasis and could be decisive in the pathogenesis of various lung diseases. since time immemorial, lung health has been underestimated even though the lung is a 48 vulnerable organ as it has to face numerous environmental irritants. though air pollution 49 levels touched well beyond the safety limit and have silently killed so many lives hitherto, 50 we didn't make sufficient efforts to reduce air pollution. this is because there is no acute 51 mortality owing to air pollution. as an outcome of this, people across the world have 4 (carafoli 2010) . however, in a living cell, mitochondrial respiration could be the main 89 inducer for the calcium uptake process. thus, mitochondrial respiration and its calcium 90 uptake are exclusively interdependent. earlier functional studies indicated that mitochondria 91 can uptake a large amount of calcium in the presence of phosphate whereas its uptake could 92 be limited in the absence of phosphate (carafoli 2010 ). later, a number of proteins had been 93 discovered that regulate the influx and efflux of calcium across mitochondria in both 94 directions (finkel et al. 2015) . the mitochondrial calcium uniporter (mcu) complex, 95 na + /ca 2+ exchanger, and h + /ca 2+ exchanger are the major proteins that help mitochondrial 96 calcium migrate back and forth (finkel et al. 2015) . to enter into the mitochondria, 97 cytosolic ca 2+ has to cross the outer mitochondrial membrane (omm) and inner swelling and ultimately release of cytochrome c from mitochondria to the cytosol to initiate 122 cellular apoptosis (sebag et al. 2018) . 123 the cytosolic calcium concentration is tightly regulated in the eukaryotic cell. calcium 124 ions which play the crucial role of second messenger in the signalling pathways are 125 controlled by various pumps, exchangers, channels and numerous binding proteins. 126 endoplasmic reticulum (er) and golgi bodies are the reservoirs of calcium in the cells. the lung homeostasis depends on the elimination of exogenous irritants. in this defence 168 mechanism of safeguarding, the airway epithelium is robust enough to protect the entire lung. the airway epithelium has one effective strategy, mucociliary apparatus, for efficacious 170 protection, and this is based on the trapping of foreign particles inside the "gel" or "viscous" 171 layer followed by continuous upward ciliary beating to propel the foreign particles out of the 172 airway (ridley and thornton 2018). it seems that mitochondria have a major role in the 173 effectiveness of mucociliary apparatus in two ways: a) by providing energy for the ciliary 174 beating and b) by less uptake of intracellular calcium into the mitochondria. among various 175 regulatory mechanisms for ciliary beat frequency, the increase of intracellular calcium is 176 crucial. more importantly, calcium is also important to coordinate the synchronization of 177 ciliary beats of subsequent airway epithelial cells of the larger airway (schmid and salathe 178 2011). as increased intracellular calcium is a requisite for the ciliary beat, the reduction in 179 intracellular calcium by mitochondrial uptake theoretically will reduce ciliary beating. this 180 was demonstrated by acetylcholine that has the capacity to inhibit ciliary beating through 181 increased mitochondrial uptake of intracellular calcium. all these indicate the feasible role of 182 mitochondria and mitochondrial calcium towards lung homeostasis. the densely located mitochondria in close proximity to the axoneme basal body of 184 cilia seem to provide energy continuously for the movement of cilia. so cristae was found in the ciliated epithelia of these severe asthmatic patients (thomas et al. 194 2010). it is well acclaimed that mitochondrial swelling is the end product of mitochondrial 195 calcium overload. interleukin-13 (il-13), a pleiotropic th2 cytokine, is known to promote mitochondrial 239 ros production, matrix ca 2+ uptake, and apoptosis in the respiratory epithelium (sebag et al. the copd is a debilitating lung condition characterized by airway inflammation (chronic 315 bronchitis), destruction of lung tissue (emphysema) and small airway remodelling (cloonan 316 and choi 2016). although among the copd patients, 20% are non-smokers, the majority of 317 them have a previous history of cigarette smoking. the status of mitochondria and 318 mitochondrial calcium in the pathogenesis of copd is extensively investigated. in lung disease biology, most of the currently available therapeutics is based on the earlier 678 conceptual changes (chu and drazen 2005) . for example, asthma was considered as a 679 psychosomatic disorder or "asthma nervosa" in hippocrates period and as a result "mind 680 control" was one of major therapeutic option for asthma in those times. this is partially true 681 now also because neurogenic axis that causes sudden bronchospasm when the asthma patient 682 feels either greatest joy or sad. later, in 20th century "asthma nervosa" concept was updated 683 with "airway smooth muscle involvement" followed with "airway hyperresponsiveness" and 684 then with "airway inflammation". these conceptual changes led to introduction of anti short-term cigarette smoke exposure leads to 785 metabolic alterations in lung alveolar cells rejuvenating cellular respiration for optimizing 788 respiratory function: targeting mitochondria mitochondria: at the crossroads of regulating lung epithelial cell 792 function in chronic obstructive pulmonary disease mitochondrial dysfunction increases allergic airway inflammation dysfunction of mitochondria ca 2+ uptake in cystic fibrosis airway epithelial 799 cells systematic identification of mcu modulators by orthogonal interspecies 804 chemical screening comparison of eight 15-807 lipoxygenase (lo) inhibitors on the biosynthesis of 15-lo metabolites by human 808 neutrophils and eosinophils pgc1α and mitochondrial metabolism-emerging concepts and 810 relevance in ageing and neurodegenerative disorders letm1: essential for mitochondrial biology and cation 812 homeostasis? histone 3.3 participates in a self-sustaining cascade of apoptosis that contributes to 815 the progression of chronic obstructive pulmonary disease the role of mitochondria in aging calcium, atp, 820 and ros: a mitochondrial love-hate triangle airway remodeling in asthma: update on mechanisms and therapeutic 823 approaches pgc1α repression in ipf 826 fibroblasts drives a pathologic metabolic, secretory and fibrogenic state the fateful encounter of mitochondria with calcium: how did it 829 happen? obesity, 831 mitochondrial dysfunction, and obstructive lung disease. chapter in a book titled 832 health-care preparedness 835 and health-care worker protection in covid-19 pandemic asthma: one hundred years of treatment and onward mitochondria in lung disease inflammation alters regional mitochondrial ca2+ in human airway smooth 842 muscle cells mitochondria and cystic fibrosis transmembrane conductance 844 regulator dialogue: some news an intriguing involvement of 846 mitochondria in cystic fibrosis airway remodeling in asthma: what really 848 matters the ins and outs of mitochondrial calcium blockade of nox2 and stim1 signaling 855 limits lipopolysaccharide-induced vascular inflammation redox homeostasis, 858 oxidative stress and mitophagy the machineries, regulation and cellular functions of 860 mitochondrial calcium calcium 863 channel blocker reduces airway remodeling in severe asthma. a proof-of-concept 864 study calcium and ros: a mutual 866 interplay mitochondrial calcium uniporter regulates pgc-1α expression to mediate 869 metabolic reprogramming in pulmonary fibrosis abnormalities in airway epithelial junction formation in chronic obstructive 872 pulmonary disease should we abandon the notion that calcium channel 874 blockers are potentially useful for asthma endothelial mitochondria determine rapid barrier failure in chemical lung injury complex i and ii 880 are required for normal mitochondrial ca 2+ homeostasis ip3 receptor-mediated 882 calcium signaling and its role in autophagy in cancer 884 ds16570511 is a small-molecule inhibitor of the mitochondrial calcium 885 uniporter mitochondrial dysfunction in human primary alveolar type ii cells in 888 emphysema the interface between er and mitochondria: molecular 890 compositions and functions mitochondrial dna damage 893 initiates acute lung injury and multi-organ system failure evoked in rats by intra-894 tracheal pseudomonas aeruginosa mitochondrial redox system, dynamics, and 896 dysfunction in lung inflammaging and copd the role of mitochondrial-related nuclear genes in age-899 related common disease mitochondrial fission 901 and fusion: a dynamic role in aging and potential target for age-related 902 disease mcu-induced mitochondrial calcium uptake promotes mitochondrial 905 biogenesis and colorectal cancer growth mitochondrial structural changes and dysfunction are associated with 908 experimental allergic asthma esculetin restores 910 mitochondrial dysfunction and reduces allergic asthma features in experimental 911 murine model linoleic acid metabolite drives severe asthma by causing airway epithelial injury causes airway epithelial injury in asthma mitochondrial dysfunction in metabolic syndrome and 922 ageing and the border between health 924 and disease accelerated ageing of the lung in copd: new 926 concepts role of calcium and calcium antagonists in airway function role of mitochondria-associated er membranes in 932 calcium regulation in cancer-specific settings calcium homeostasis in aging neurons myriad functions of stanniocalcin-1 stc1) cover multiple therapeutic targets in the complicated pathogenesis of 938 idiopathic pulmonary fibrosis (ipf) mitochondrial regulation of airway smooth 941 muscle functions in health and pulmonary diseases mitochondria and aging: a role for the mitochondrial 944 transition pore mitochondrial damage pathways in ventilator induced lung injury (vili): 946 an update mitochondrial dysfunction in airway 948 disease redox regulation of motile cilia in airway disease induction of apoptosis with tobacco smoke 952 and related products in a549 lung epithelial cells in vitro mitochondrial dysfunction and oxidative stress in asthma: implications 954 for mitochondria-targeted antioxidant therapeutics the mitochondrial barriers 957 segregate agonist-induced calcium-dependent functions in human airway 958 mucins: the frontline defence of the lung mitochondrial ca2+-dependent nlrp3 activation exacerbates the pseudomonas 963 aeruginosa-driven inflammatory response in cystic fibrosis pharmacological modulation of mitochondrial 967 calcium uniporter controls lung inflammation in cystic fibrosis ca2+ transfer from 970 the er to mitochondria: when, how and why mitochondrial calcium: transport and 973 modulation of cellular processes in homeostasis and cancer the risk for lung cancer incidence with calcium 977 channel blockers: a systematic review and meta-analysis of observational 978 studies. drug safety contrasting effects of adipokines on the cytokine production 981 by primary human bronchial epithelial cells: inhibitory effects of ciliary beat co-ordination by calcium inhibition of the mitochondrial calcium uniporter prevents il-13 and allergen-987 mediated airway epithelial apoptosis and loss of barrier function calcium flux and endothelial dysfunction 990 during acute lung injury: a stimulating target for therapy new 993 insights into the role of mitochondria in aging: mitochondrial dynamics and more emerging role of 12/15-lipoxygenase (alox15) in human 996 pathologies regulation and functions of 15-lipoxygenases in human 998 calcium channel blockers and asthma shared epithelial pathways to lung repair 1001 and disease the mitochondrial basis of aging and age-related disorders mitochondrial dysfunction in the aged lung and copd: a role 1006 for mitochondrial calcium ciliary dysfunction and ultrastructural abnormalities are 1009 features of severe asthma bronchial 1012 smooth muscle remodeling involves calcium-dependent enhanced mitochondrial 1013 biogenesis in asthma cftr activity and mitochondrial 1015 function mam (mitochondria-associated membranes) in mammalian cells: lipids 1017 and beyond tobacco smoke 1019 induces both apoptosis and necrosis in mammalian cells: differential effects of the basic machineries for 1022 mitochondrial protein quality control novel targets for mitochondrial medicine calcium promotes membrane association of reticulocyte 1026 15-lipoxygenase a selective and cell-permeable mitochondrial 1030 calcium uniporter (mcu) inhibitor preserves mitochondrial bioenergetics after 1031 hypoxia/reoxygenation injury mitochondrial dysfunction in neurodegenerative 1033 diseases and drug targets via apoptotic signaling transport of calcium ions into 1035 mitochondria mito-porter for mitochondrial delivery and 1037 mitochondrial functional analysis epithelial barrier integrity and overdrive activation of alveolar epithelial cells in 1040 the pathogenesis of acute respiratory distress syndrome and lung fibrosis pathobiology of cigarette smoke-induced chronic 1043 obstructive pulmonary disease mitochondrial calcium uniporter as a target of microrna-1046 340 and promoter of metastasis via enhancing the warburg effect lanthanum chloride impairs spatial learning and memory by inducing 1050 [ca2+]m overload, mitochondrial fission-fusion disorder and excessive mitophagy in 1051 hippocampal nerve cells of rats. metallomics. 12, 592-606. key: cord-291145-rdg31p17 authors: rice, shawn j.; hyland, victoria; behera, madhusmita; ramalingam, suresh s.; bunn, paul; belani, chandra p. title: guidance on the clinical management of e-cigarette or vaping associated lung injury (evali)? date: 2020-08-29 journal: j thorac oncol doi: 10.1016/j.jtho.2020.08.012 sha: doc_id: 291145 cord_uid: rdg31p17 in the summer of 2019 there was a rise in clusters of adolescents and young adults in the united states reporting to emergency departments with acute respiratory distress related to e-cigarette or vaping usage. the number of patients with e-cigarette or vaping associated lung injury (evali) continued to rise through the summer before peaking in september 2019. through the efforts of state and federal public health agencies, officials were able to define the condition, identify the relationship of the respiratory injury to tetrahydrocannabinol (thc)-containing products, and stem the rise in new cases. in this report, we present a comprehensive review of the clinical characteristics and features of evali patients and present guidelines for patient care and management to inform and navigate clinicians who may encounter these patients in their clinical practice. beginning in early 2012 a disturbing trend was building in the background of the public health arena, and it would develop into one of the largest public health mysteries of the decade. sporadic cases of lung injury associated with vaping began in 2012 1 , and in july of 2019 clusters of young people (ages 10-19 years) began entering hospital emergency departments (ed) with complaints of shortness of breath and chest pains associated with vaping 2 . while the early cases are not necessarily reflective of the etiology of the 2019 evali series, they suggest that unregulated development of inhaled devices have the potential for significant health consequences in some proportion to individual risk coupled with the breadth of distribution. the cases in the summer of 2019 were particularly worrisome because the increase in ed visits was primarily among young males from up to 10 states with no known cause for respiratory distress or illness 3 . e-cigarette and vaping associated lung injury (evali) usually presents with symptoms of dyspnea, fever, nausea, and cough 4 . a common theme in these patients is that they reported the use of electronic-cigarette (e-cigarette) or vaping products within 90 days of their ed visit. through february 18 2020, 2807 cases and 68 deaths from a mysterious lung injury have been reported in the united states of america and involved all 50 states 5 . the outbreak of evali was primarily limited to the usa. these cases developed before the current covid-19 pandemic although many of the symptoms and radiographic findings are j o u r n a l p r e -p r o o f overlapping. while it is possible to diagnose viral infections with pcr testing, the current state of testing in the us is prone to false negatives and positives for both pcr-based and antibodybased methods. therefore, careful consideration must be applied when evali and covid-19 are the possible diagnoses. identifying the evali causative agent and characterizing the lung injury from vaping became a top public health priority. vaping or e-cigarettes involves high-temperature heating of a liquid to generate an aerosol vapor that can deliver substances like nicotine or thc to the lungs. generally, these liquids contain flavoring agents, diluents, nicotine, thc, nicotine and thc, or less commonly, neither nicotine nor thc; the ingredients can vary greatly in the chemical composition, especially between manufacturers or homemade mixtures. it became apparent early in the outbreak that most patients (86-92%) admitted to using thc vaping liquids within three months of developing the disease 3,4 . this proved to be an important observation, as laboratory analyses of these vaping liquids sought to identify candidate agents resulting in evali. vitamin e acetate, medium-chain triglyceride oil, and other lipids rose to the top of the list of causative agents 4 . by mid-december 2019 a causative agent for evali was finally identified. using bronchoalveolar-lavage (bal) fluid from evali patients and healthy donors who have not used thc-containing products, researchers used isotope dilution mass spectrometry and identified vitamin e acetate in over 90% of evali cases versus none in the healthy controls. according to the center for disease control and prevention (cdc), new cases of evali in the usa have continued to decline through february 2020, since its' peak in mid-september 2019. guidelines on the clinical management of evali is important for these acutely ill patients. a timely diagnosis and treatment course can improve patient experiences and outcomes. here we present a comprehensive review of evali to aid physicians in the management of these patients. a pubmed search was performed on march 21 using the keywords "vaping", "lung injury" to collect references that included evali and those that may report respiratory injuries prior to the naming of the condition evali, leading to retrieval of 116 j o u r n a l p r e -p r o o f articles. review articles and commentaries were excluded from the dataset of articles. we reviewed the remaining literature with a focus on the patient characteristics, clinical features, and guidance for patient care and management. in 2019, clusters of patients with severe acute respiratory symptoms began to appear in the us among midwest states (minnesota, illinois, indiana, and wisconsin) and western states (utah and california). the respective states and national health agencies created the lung injury response clinical working group, to track and monitor these patients resulting in the emergence of numerous case studies and reports. these clusters indicated a larger issue existed. we identified 27 case reports or case series related to the description of evali patients in our dataset of evali literature from 2017-2020 as shown in table s1 . while these studies provide insight into the characteristics of evali patients, the limited number of patients in these studies does not allow for definitive conclusive recommendations about the disease and its' management. therefore, we focused our attention on the larger observational studies. the cdc established case definitions for the surveillance of evali to identify and track patients (table 1) . cases are classified as confirmed or probable based on the presence or absence of signs of other etiologies for the respiratory symptoms, i.e. other infectious agents. probable cases are those where infectious agents may present in a patients airway, but the clinical team deems that the agent is likely not the cause of the respiratory symptoms. evali cases began to rise in the usa throughout the summer of 2019 before peaking in september, and they continued to decline through february of 2020 ( figure 1a ). as of february 18, 2020, 2807 evali patients have been reported to the cdc and 68 confirmed deaths have j o u r n a l p r e -p r o o f been attributed to the disease ( figure 1b ). evali cases have been identified in all 50 states, and deaths have occurred in 29 states and the district of columbia ( figure 1b ). the cdc published monthly reports on the characteristics of evali cases for the us and summaries for limited state clusters have also been reported, which are summarized in table 2 . patients across all the datasets tended to be male (62-79%), less than 30 years old (median age 23-27 years) and have used thc-containing products within the last 90 days (76.9-91%). affected cases were primarily non-hispanic whites (46-79%) and hispanic (12-47%), with california having a lower number of non-hispanic whites (46%) and a higher proportion of hispanic (47%) cases, which may be due to the small sample size (n=160) and demographics of the state 27 . @. these were early and partial studies that apparently were published quickly to get the word out to the general population. the sharp and sustained decline in new evali cases is a testament to the effectiveness of this approach in putting a halt to the outbreak. 1 vaping or e-cigarette use among evali patients 2 as e-cigarette and vaping became associated with lung injury in youths, it is important to 3 understand how this group of patients were using these devices. evali patients report using 4 various types of vaping liquids containing products of thc, nicotine, and cannabidiol (cbd) 34 . for evali patients in the us, most use thc or nicotine-containing products daily, 74% and 6 85%, respectively 34 . the use of thc containing products more than 5 times a day is associated 7 with a higher risk for developing evali (aor 3.1, 95% ci 1.6-6.0) 31 . the source of products 8 differed between thc and nicotine-containing liquids. most patients report getting thc 9 products from only informal sources (78%), i.e. family or friends, while most nicotine products 10 were acquired from commercial sources (69%) 34 . youths, <18 years, primarily acquire either 11 thc or nicotine products through informal channels. this is problematic because informal 12 sources can be dangerous and also difficult to track. a comparative analysis of symptomatic it is worth noting that while most cases of evali report use of thc containing products, a 18 minority of cases (8-24%) report use of only nicotine products or neither nicotine nor thc 19 products (2-7%)( table 2) . some of this discrepancy may be attributable to patient recall error, j o u r n a l p r e -p r o o f secondhand inhalation, or some other unknown explanation, but it could suggest that there may 1 be two or more agents able to effect evali symptoms. the cdc recommends that people 2 abstain from e-cigarette and vaping products, but if one must use such a product, they should 3 be acquired through formal commercial channels. table 2 . while the cdc reports provide an overview of characteristics across 10 the entire us, three state-level studies provide more in-depth clinical and outcome data. therefore, we will focus our discussion on the clinical presentation of evali patients of illinois 12 and wisconsin 32 , utah 33 , and california 27 datasets ( the above histological and imaging features represent observations from adult patients, 20 but since evali also affects many adolescent patients it is important to consider this group of 21 patients separately. adolescents tend to display similar features as adult patients with evali, but additional common imaging findings are noted in these patients, including pleural effusions 23 (50%), mild thickening of the bronchial wall(50%), and enlarged lymph nodes (75%) 44 . in 14 these findings support the assertion that vitamin e acetate is a likely cause of evali, but they 15 do not rule out other possible agents, as some evali patients did not have detectable vitamin e 16 acetate in bal samples and 8-24% evali patients report smoking nicotine only (table 2 ). vitamin e acetate is used as a diluent for vaping products that contain thc because it has the 18 advantageous characteristics of being inexpensive and tasteless 54 . while vitamin e acetate seems to be a strong candidate for causing evali, our 20 understanding of the mechanism behind the lung damage is limited. vitamin e acetate can form 21 a non-covalent complex with thc in aerosols produced during vaping, which would allow the 22 complex to be delivered to the lungs 55 . however, the effect of this complex on the lung is not 23 known and warrants further investigation to establish a possible mechanism of lung toxicity. vaping involves aerosolizing a liquid containing many constituents (nicotine, thc, flavorings, 3 mice were exposed to77.3 to 167.5 µg vitamin e acetate, propylene glycol and vegetable glycol 4 (pg-vg), or air. following a two week exposure period, bal fluid and lung tissue were 5 harvested from the mice. mice exposed to vitamin e acetate display similar phenotype to 6 human evali patients including a rise in albumin levels, and elevated leukocytes and lipid-7 laden macrophages in bal fluid, relative to the pg-vg and air groups. mice exposed to vitamin 8 e acetate displayed profuse oil red o stained alveolar macrophages within the pneumocytes of 9 the alveoli. this model could be used to prove that vitamin e acetate does in fact cause evali, 10 establish a mechanism of action, and identify other agents that cause lung injury. symptoms should be evaluated for evali using definitions in table 1 . the cdc offers 20 suggestions on questions to ask during the initial clinical assessment to obtain information 21 essential for an evali diagnosis (https://www.cdc.gov/tobacco/basic_information/e-22 cigarettes/severe-lung-disease/healthcare-providers/pdfs/dont-forget-to-ask-assessing-the-riskof-lung-injury-508.pdf). once evali is suspected, patients should be assigned for inpatient or 4 during hospitalization, patients should abstain from e-cigarette and vaping products, and 5 initiation of antimicrobial agents and corticosteroids as the condition warrants. the previous steps are important for managing the acute effects of evali, but 7 generating a thorough plan for discharging the patient may be important for reducing 8 readmission and death 40,58 . the cdc has established an "evali discharge readiness 9 checklist" to aid clinicians in the discharge planning process 10 (https://www.cdc.gov/tobacco/basic_information/e-cigarettes/pdfs/evali-discharge-readiness-11 checklist-508.pdf). the treatment team should screen patients for substance abuse disorders 12 and provide the patient with contact information for the appropriate support that the patients will vaping-associated lung injury severe e-cigarette, or vaping requiring venovenous extracorporeal membrane oxygenation anaerobic 6 necrotizing pneumonia: another potential life-threatening complication of vaping? a case of vaping-associated 9 respiratory bronchiolitis-associated interstitial lung disease secondary 11 to electronic nicotine delivery system use confirmed with open lung biopsy secondhand smoke from electronic cigarette resulting in 14 hypersensitivity pneumonitis a 19-year-old man with vaping-associated lung injury tree-in-bloom acute lung injury induced by vaping cannabis oil life-threatening bronchiolitis related to electronic cigarette use in a 21 canadian youth a patient from mexico with vaping-associated 23 lung injury, seizures and renal failure 1e10 anti-idiotype vaccine in non-small cell lung cancer: experience in 26 stage iiib/iv patients discordant bilateral 28 bronchoalveolar lavage findings in a patient with acute eosinophilic pneumonia 29 associated with counterfeit tetrahydrocannabinol oil vaping cytologic features of vaping-induced lung injury: a 32 case report severe lung injury associated with use of e-cigarette, or vaping characteristics of hospitalized and nonhospitalized patients 37 in a nationwide outbreak of e-cigarette, or vaping, product use-associated lung injury -38 united states characteristics of e-cigarette, or vaping, products used by patients with 41 associated lung injury and products seized by law enforcement -minnesota patient characteristics and product use behaviors among persons with 1 e-cigarette, or vaping, product use-associated lung injury -indiana characteristics of persons who report using only nicotine-containing 5 products among interviewed patients with e-cigarette, or vaping pulmonary illness related to e-cigarette use in illinois and wisconsin -9 final report clinical presentation, treatment, and short-term outcomes of lung 11 injury associated with e-cigarettes or vaping: a prospective observational cohort study update: product, substance-use, and demographic characteristics of 14 hospitalized patients in a nationwide outbreak of e-cigarette, or vaping, product use-15 risk factors for e-cigarette, or vaping, product use-associated lung 18 injury (evali) among adults who use e-cigarette, or vaping, products -illinois covid-19: potential implications for individuals with substance use 22 disorders reduce your risk of serious lung disease caused by corona 24 virus by quitting smoking and vaping covid-19 and smoking: is nicotine the hidden link? smoking upregulates angiotensin-converting enzyme-2 receptor: a 29 potential adhesion site for novel coronavirus sars-cov-2 (covid-19) characteristics of patients experiencing rehospitalization or death 32 after hospital discharge in a nationwide outbreak of e-cigarette, or vaping, product 33 use-associated lung injury -united states pathology of vaping-associated lung injury imaging findings of vaping-associated lung injury lung biopsy findings in severe pulmonary illness associated 40 use (vaping) e-cigarette, or 1 vaping, product use-associated lung injury in adolescents: a review of imaging features pediatric chest radiographic and ct findings of electronic 4 cigarette or vaping product use-associated lung injury (evali) pulmonary lipid-laden macrophages and vaping more on the pathology of vaping-9 associated lung injury testing for lipid-laden macrophages in bronchoalveolar lavage fluid to 11 diagnose vaping-associated pulmonary injury. are we there yet? the lipid-laden alveolar macrophage as a marker of 14 aspiration in parenchymal lung disease cut-off values and significance of oil red o-positive cells in 17 bronchoalveolar lavage fluid e-cigarette or vaping product use-associated lung injury: what is the role 20 of cytologic assessment? lipid-laden macrophages as biomarkers of 22 vaping-associated lung injury vitamin e acetate in bronchoalveolar-lavage fluid associated with 25 evali analysis of cannabinoid-containing fluids in illicit vaping cartridges 27 recovered from pulmonary injury patients: identification of vitamin e acetate as a 28 major diluent hydrogen bonding between 30 tetrahydrocannabinol and vitamin e acetate in unvaped, aerosolized, and condensed 31 aerosol e-liquids potential for release of pulmonary toxic ketene from vaping 34 pyrolysis of vitamin e acetate an animal model of inhaled vitamin e acetate and evali-like lung 37 injury update: interim guidance for health care professionals evaluating 39 and caring for patients with suspected e-cigarette, or vaping lung injury and for reducing the risk for rehospitalization and death following hospital 41 discharge -united states key: cord-267979-k70gnrdw authors: yıldız-peköz, ayca; ehrhardt, carsten title: advances in pulmonary drug delivery date: 2020-09-23 journal: pharmaceutics doi: 10.3390/pharmaceutics12100911 sha: doc_id: 267979 cord_uid: k70gnrdw pulmonary drug delivery represents an attractive, non-invasive administration option. in addition to locally acting drugs, molecules that are intended to produce systemic effects can be delivered via the pulmonary route. several factors need to be considered in the context of delivering drugs to or via the lungs—in addition to the drug itself, its formulation into an appropriate inhalable dosage form of sufficient stability is critical. it is also essential that this formulation is paired with a suitable inhaler device, which generates an aerosol of a particle/droplet size that ensures deposition in the desired region of the respiratory tract. lastly, the patient’s (patho-) physiology and inhalation manoeuvre are of importance. this special issue brings together recent advances in the areas of inhalation device testing, aerosol formulation development, use of in vitro and in silico models in pulmonary drug deposition and drug disposition studies, and pulmonary delivery of complex drugs, such as vaccines, antibiotics and peptides, to or via the lungs. drugs have been inhaled for therapeutic and recreational purposes since ancient times. the development of modern-day inhalers, e.g., pressurised metered-dose inhalers (pmdis) and more recently, dry powder inhalers (dpis), jet and vibrating mesh nebulisers (vmns), and soft mist inhalers (smis), has given pulmonary drug delivery a momentum boost that transformed a therapeutic niche into a market predicted to hit us$41.5 billion by 2026 [1] . when delivering medicines to or via the lungs, not only the drug but also the formulation, the device and the patient need to be considered. we attempted to cover all these topics, while at the same time putting a spotlight on the up and coming areas of pulmonary drug delivery research. for this special issue, we selected 24 publications written by 131 authors hailing from 21 countries-many of whom are regular contributors to our pulmonary drug delivery workshop series (www.pulmonarydrugdelivery. org). due to the variety of subjects covered, this has become the most comprehensive special issue published by pharmaceutics to date. adorni and colleagues studied 13 different nebulisers taking into account their aerosol output, aerosol output rate, mass median aerodynamic diameter (mmad) and fine particle fraction according to the european standard en 13544-1, using sodium fluoride as a reference formulation [2] . a correlation between the aerosol quality and the nebulization rate was identified. the respirable delivered dose and respirable dose delivery rate were also determined. this study might be helpful when choosing a nebuliser depending on the drug, therapy regime and patient. pmdis account for two-thirds of sold inhalers, however, due to technological advancements and environmental concerns, dpis emerged as the preferred medical device for the treatment of a diverse range of respiratory disorders. many dpis contain powder mixtures of coarse carrier particles and micronised drug particles with aerodynamic particle diameters of 1-5 µm. it is estimated that only 10-15% of the drug reaches the deep lung while 20% of the drug is lost in the oropharyngeal sphere and 65% is not released from the carrier due to interparticulate adhesive forces. lechanteur and evrard have reviewed carrier-free particles, which are characterized by a sugar-based core encompassed by a corrugated shell layer produced by spray drying [3] . special attention is given to the relation between the morphology (characterized by a corrugated surface) and lung deposition performance. a different approach to overcoming the limitations of conventional carrier-based dry powders was followed by benke et al. [4] . they report the development of an interactive physical blend of a surface-modified carrier and spray-dried meloxicam potassium with suitable shape and size for pulmonary delivery. the nonsteroidal anti-inflammatory drug was used with the intention to provide local anti-inflammatory effects to decrease the progression of cystic fibrosis (cf) and chronic obstructive pulmonary disease (copd). in vitro and in silico studies resulted in high lung deposition, confirming that the interparticle interactions were indeed reduced in the novel formulation. rashid and co-workers, on the other hand, followed the traditional approach and formulated a lactose carrier-based dry powder formulation of glucagon for pulmonary delivery [5] . they investigated l-leucine and magnesium stearate as dispersibility enhancers and found the highest fine particle fraction (fpf) for the formulation to contain mg stearate (36%) and large carrier lactose, whereas leucine was not a suitable excipient for the pulmonary delivery of glucagon. liquid formulation of fluticasone instead of dry powders were studied by dogbe et al., in order to improve the biopharmaceutical performance of the drug [6] . the study compares liposomes and cyclodextrin (cyd) complexes in vitro and in vivo in mice. the in vitro tests showed no cytotoxic effects of either formulation. fluticasone liposomes resulted in up to 30-times higher lung concentration in comparison with free drug after intranasal administration. fluticasone hydroxypropyl-cyclodextrin complexes also showed higher lung accumulation than the free form after inhalation, however, this effect was not as pronounced as those observed with the liposomes. seven publications in this special issue cover various aspects of the inhalation of anti-infectives to treat lung infections. inhaling antibiotics allows for high target site concentrations, whilst minimising systemic exposure and toxicity. nonetheless, only a handful of antibiotics are currently marketed as nebulisable solutions or dry powders, and almost exclusively for the use in cf. future inhaled antibiotic trials should therefore focus on disease areas outside of cf, e.g., non-cf bronchiectasis, drug-resistant non-tuberculous mycobacterial infections, ventilator-associated pneumonia, post-transplant airway infections and tuberculosis (tb). therefore, an increased number of drugs as well as novel drugs must be studied as well as other formulations. banaschewski and hofmann [7] have reviewed research into completed inhaled development programs, as well as ongoing research into inhaled therapies for both non-tb mycobacterial lung disease and tb. they conclude that preclinical and clinical studies have shown that inhalation therapy, complementary to current guidance-based therapy strategies, are clinically beneficial for all types of mycobacterial infections. however, an open-minded approach should be followed to continue investigating potential additions to the antimycobacterial therapeutic arsenal. in two papers, sibum and colleagues report the formulation, characterisation and stability testing of high-dose dry powders of isoniazid with little or no excipient for the treatment of tb [8, 9] . initially, isoniazid was jet milled and spray dried with and without the excipient l-leucine. however, milling isoniazid did not yield a suitable formulation and spray drying the pure drug resulted in particles too large for pulmonary administration. when 5% l-leucine was added, respirable particles could be produced by spray drying but their storage stability was poor at higher relative humidity [8] . the stability was later improved by using trileucine instead of l-leucine. the optimal formulation contained 3% trileucine w/w and had a maximum fine particle dose of 58 mg when a nominal dose of 80 mg was dispersed from the cyclops ® dry powder inhaler [9] . in a case also using isoniazid, wyszogrodzka-gaweł et al. developed a theranostic approach to tb treatment and diagnosis that allows for imaging of the lungs by mri [10] . metal-organic framework (mof) fe-mil-101-nh 2 nanoparticles were loaded with isoniazid using factorial design of spray-drying with poly(lactide-co-glycolide) and leucine. the formulation thus obtained had mri contrast capabilities, aerodynamic properties suitable for lung delivery, modified drug release and was taken up by macrophages. rossi and co-workers, in an attempt to treat mycobacterial lung infections, studied inhalable antibiotic powders targeting alveolar macrophages [11] . their sodium hyaluronate-based formulation contained two antibiotics (i.e., rifampicin and isoniazid) and the efflux pump inhibitor, verapamil and was produced by spray drying. the sub-micron-sized particles had a high fine particle fraction, showed a sustained release profile, were not toxic towards macrophages and achieved more than 80% reduction in bacterial viability in susceptible and resistant m. tuberculosis strains in vitro. isoniazid was introduced in 1952. bedaquiline, on the other hand is a relatively novel oral anti-tb drug that was approved in the us in 2012 by fast-track accelerated approval and is on the world health organization's list of essential medicines. bedaquiline, however, has a black-box warning of increased risk of death and arrhythmias. hence, momin et al. developed inhalable bedaquiline dry powder particles with the intention of reducing the systemic side-effects [12] . bedaquiline was processed by spray drying and the resulting microparticles were stable during one-month of storage. spray-dried bedaquiline was non-toxic in respiratory epithelial cell cultures and effectively inhibited the growth of m. tuberculosis in vitro. antimicrobial peptides (amps) are being considered as alternatives to conventional antibiotics. amps do not only have direct antimicrobial activity, but also modulate the immune system and wound repair, making them of interest in cf therapy. forde and colleagues studied whether prodrugs of amps (pro-amps) can be delivered by vmn and whether modifications of pro-amp had an effect on the delivery [13] . nebulisation did not alter amps' physical characteristics and antimicrobial activity. approximately 25% of the nominal dose was delivered in a spontaneous breathing setting, with higher delivery rates observed in a mechanically-ventilated model. these results demonstrated the feasibility of amp delivery using a vmn and also that the prodrug modification is not detrimental. vaccines against bacterial diseases may directly reduce antibiotic use through reduction of disease incidence. thus, immunisation has the potential to reduce antibiotic use. vaccine delivery via mucosal surfaces is an interesting alternative to parenteral vaccination and in many cases resembles the route taken by the microorganism when entering the body. hellfritsch and scherließ in their review provide an introduction to respiratory vaccination, formulation approaches and application strategies [14] . another disease that could benefit from the advantages that inhalation therapy offers in terms of reduced systemic drug burden is lung cancer. parvathaneni et al. in their study investigated the anti-tumour effects of liposomally-encapsulated pirfenidone in vitro [15] . pirfenidone, a repurposed anti-fibrotic drug, was encapsulated in cationic liposomes. the formulation was successfully aerosolised by a jet nebuliser and showed promising anti-tumour effects in various human lung cell lines compared to free pirfenidone. particle deposition in the lungs is associated with the breathing patterns of the patient and also pathophysiological changes due to lung diseases. in their study, farkas and colleagues measured realistic inhalation profiles of mild, moderate, and severe copd patients and simulated the deposition patterns of the symbicort ® turbuhaler ® in comparison to data generated from healthy control subjects [16] . they found an association between the amount of drug deposited within the lungs and disease severity. the results from this study suggest that to receive a similar lung concentration, severe copd patients would require much higher doses than healthy individuals. tailoring the shape and size of fibre-like aerosols to achieve targeted pulmonary drug delivery with increased deposition efficiency is an interesting concept. shachar-berman et al. calculated the transport and deposition characteristics of fibres under physiological inhalation conditions in silico using computational fluid dynamics (cfd) simulations [17] . aerosol deposition was quantified as a function of the equivalent diameter (dp) and geometrical aspect ratio (ar). they found that high ar fibres in the narrow range of dp = 6-7 µm mainly deposited in the upper airways, whereas fibres in the range of dp = 4-6 µm penetrated all the way to distal lung regions. to prolong the duration of the effect in the lungs, increasing the drug's affinity to lung tissue is an important strategy for drug development. however, differences in lung structure and blood flow affect local pulmonary drug disposition. himstedt and co-workers studied regional lung distribution of four drugs (i.e., salmeterol, fluticasone propionate, linezolid and indomethacin) after intravenous administration in rats [18] . in addition, a semi-mechanistic model was employed to describe the observed tissue drug concentrations. the in silico model was able to explain the pulmonary pharmacokinetics of the two neutral and one basic model drug based on their tissue specific affinities (kp) and organ blood flow. the pulmonary pk of indomethacin, however, could not be modelled, suggesting that acidic drugs have different pulmonary pk characteristics. in their paper, salomon et al. studied the activity of carnitine transporter octn2, which is associated with asthma and other inflammatory lung diseases. they studied freshly isolated human alveolar type i (ati)-like epithelial cells in primary culture and several respiratory epithelial cell models [19] . [ 3 h]-acetyl-l-carnitine uptake and pharmacological inhibition was determined in ati-like, ncl-h441, a549 and calu-3 cells. it was concluded that octn2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium, however none of the tested cell lines are optimal surrogates for primary cells in carnitine transport studies. pulmonary drug delivery research is usually mainly concerned with administering aerosols to the lungs. the non-deposited, exhaled dose, however, can be a significant health hazard in both clinical and homecare settings. in two publications, mcgrath and colleagues used nebulised albuterol sulphate solution when they investigated fugitive aerosol emissions from two commercially available nebulisers in combination with an open or valved facemask or using a mouthpiece with and without a filter [20] and during high flow nasal cannula (hfnc, see below for more on hfnc) therapy [21] , respectively. it was shown that the mmad of the fugitively-emitted aerosols was less than 1 µm, while the initially generated aerosols were between 2 and 5 µm. a facemask combination resulted in the highest time-averaged fugitively-emitted aerosol concentrations, whereas a filter on the exhalation port of the mouthpiece yielded the lowest concentrations. in the hfnc study, fugitive aerosol emissions were influenced by the interface type, patient and supplemental gas-flow rate, with fugitive aerosol mmad decreasing with an increasing flow rate. these findings are important in developing policy and best practice for risk mitigation from fugitive emissions. 'foamy' alveolar macrophages (fam) may be indicators of drug-induced phospholipidosis. currently, orally administered amiodarone is used to induce pulmonary phospholipidosis. patel et al. in their study investigated if pulmonary delivery of amiodarone in rats could be established as a novel phospholipidosis-induced fam model in comparative inhalation toxicology [22] . a high dose of aerosolised amiodarone caused transient pulmonary inflammation, however, only oral delivery resulted in fam. high flow nasal cannula (hfnc) is widely utilized to support critically ill adults, paediatrics and neonates. through the continuous delivery of oxygen at high flow rates that meet or exceed patients' inspiratory flow, hfnc improves oxygenation, respiratory rates, patient comfort, and tolerance during therapy. as hfnc becomes more widely employed, the technique is also being considered for aerosol drug delivery. ji et al. have identified the ratio of nasal cannula gas flow to patient inspiratory flow as a primary independent predictor of inhaled dose. when the ratio was <1, the inhaled dose was higher than those with ratio > 1. the inhaled dose was also more consistent with quiet and distressed breathing with ratio < 1 [23] . in a separate study, alcoforado and co-workers observed that both flow and active heated humidity inversely impacted aerosol delivery through hfnc. nonetheless, aerosol administration across the range of commonly used flows can provide measurable levels of lung drug deposition in healthy adult subjects [24] . ji and colleagues retrospectively analysed study data on hfnc-delivery epoprostenol (iepo), utilised to improve oxygenation in mechanically ventilated patients with severe hypoxemia comorbid with pulmonary hypertension or right heart dysfunction [25] . their data suggest that iepo via hfnc can improve oxygenation in adult patients and supports the need for a larger prospective randomised control trial to further evaluate the efficacy of iepo via hfnc. in this special issue, a cross-section of current research in the field of pulmonary drug delivery is published. there is still a lot of work to be done in the areas of inhaler devices and formulation development, particularly, with regards to dry powder and colloidal systems. a severe limitation in this field of research is the small number of excipients fda/ema-approved for inhalation. topical delivery of antibiotics appears to be an area that has attracted a lot of interest in recent years and is likely to make an even bigger impact in the treatment of pulmonary infections in the future. moreover, viral lung diseases such as covid-19 are a challenge and delivering antivirals by inhalation might be an approach worth considering. in addition to infectious diseases, conditions such as lung cancer are being actively researched in the context of inhalation drug delivery. the respiratory route can also be utilised to achieve mucosal vaccination against bacterial or viral infections. furthermore, there is a tangible shift from lab-based experiments towards in silico studies, e.g., in the areas of deposition modelling and physiology-based pharmacokinetic modelling. in the foreseeable future, however, the computer-based approached will need to be based on real-life data generated in actual experiments in the lab or the clinical setting. in the context of data generation, scientists should focus on novel techniques to study the fate of inhaled drugs, in order to allow in vivo/in vitro correlations and predictions. the authors declare no conflict of interest. inhalation delivery of complex drugs-the next steps aerosolization performance of jet nebulizers and biopharmaceutical aspects influence of composition and spray-drying process parameters on carrier-free dpi properties and behaviors in the lung: a review development of an innovative, carrier-based dry powder inhalation formulation containing spray-dried meloxicam potassium to improve the in vitro and in silico aerodynamic properties excipient interactions in glucagon dry powder inhaler formulation for pulmonary delivery pharmaceutical benefits of fluticasone propionate association to delivery systems: in vitro and in vivo evaluation inhaled antibiotics for mycobacterial lung disease characterization and formulation of isoniazid for high-dose dry powder inhalation dispersibility and storage stability optimization of high dose isoniazid dry powder inhalation formulations with l-leucine or trileucine an inhalable theranostic system for local tuberculosis treatment containing an isoniazid loaded metal organic framework fe-mil-101-nh2-from raw mof to drug delivery system sodium hyaluronate nanocomposite respirable microparticles to tackle antibiotic resistance with potential application in treatment of mycobacterial pulmonary infections inhalable dry powder of bedaquiline for pulmonary tuberculosis: in vitro physicochemical characterization, antimicrobial activity and safety studies vibrating mesh nebulisation of pro-antimicrobial peptides for use in cystic fibrosis mucosal vaccination via the respiratory tract systematic development and optimization of inhalable pirfenidone liposomes for non-small cell lung cancer treatment simulation of airway deposition of an aerosol drug in copd patients in silico optimization of fiber-shaped aerosols in inhalation therapy for augmented targeting and deposition across the respiratory tract towards a quantitative mechanistic understanding of localized pulmonary tissue retention-a combined in vivo/in silico approach based on four model drugs octn2-mediated acetyl-l-carnitine transport in human pulmonary epithelial cells in vitro investigation of the quantity of exhaled aerosols released into the environment during nebulisation investigation of fugitive aerosols released into the environment during high-flow therapy comparison of oral, intranasal and aerosol administration of amiodarone in rats as a model of pulmonary phospholipidosis the ratio of nasal cannula gas flow to patient inspiratory flow on trans-nasal pulmonary aerosol delivery for adults: an in vitro study impact of gas flow and humidity on trans-nasal aerosol deposition via nasal cannula in adults: a randomized cross-over study epoprostenol delivered via high flow nasal cannula for icu subjects with severe hypoxemia comorbid with pulmonary hypertension or right heart dysfunction key: cord-332650-05oz5zwz authors: fiorelli, silvia; massullo, domenico; ibrahim, mohsen; piccioni, federico; andreetti, claudio; vanni, camilla; rocco, monica; rendina, erino angelo; menna, cecilia title: perspectives in surgical and anaesthetic management of lung cancer in the era of coronavirus disease 2019 (covid-19) date: 2020-08-26 journal: eur j cardiothorac surg doi: 10.1093/ejcts/ezaa295 sha: doc_id: 332650 cord_uid: 05oz5zwz early in 2020, coronavirus disease 2019 (covid-19) quickly spread globally, giving rise to a pandemic. in this critical scenario, patients with lung cancer need to continue to receive optimal care and at the same be shielded from infection with the potentially severe acute respiratory syndrome coronavirus 2. upgrades to the prevention and control of infection have become paramount in order to lower the risk of hospital contagion. aerosol-generating procedures such as endotracheal intubation or endoscopic procedures may expose health care workers to a high risk of infection. moreover, thoracic anaesthesia usually requires highly complex airway management procedures because of the need for one-lung isolation and one-lung ventilation. therefore, in the current pandemic, providing a fast-track algorithm for scientifically standardized diagnostic criteria and treatment recommendations for patients with lung cancer is urgent. suggestions for improving existing contagion control guidelines are needed, even in the case of non-symptomatic patients who possibly are responsible for virus spread. a covid-19-specific intraoperative management strategy designed to reduce risk of infection in both health care workers and patients is also required. in december 2019, coronavirus disease 2019 (covid19) emerged and disseminated rapidly throughout china and many other countries worldwide [1] . lockdown measures including limitations on people's movements and cancellation of non-essential activities designed to reduce the spread of covid-19 have been introduced globally. elective surgery should be limited in order to reduce patient traffic and to avoid virus spread [2] . in italy, hospitals limited operations to those for class a diseases (diseases that require hospitalization within 30 days for clinical conditions that can potentially worsen rapidly to the point of becoming an emergency or, in any case, seriously affect the prognosis of the patient). lung cancer remains the most common cause of cancer deaths worldwide, representing a relevant health care burden [3] . if it is essential to continue to guarantee adequate care to patients with lung cancer, it is also important to protect this frail population from infection. therefore, in the current pandemic situation, several aspects of the surgical treatment of lung cancer, patient selection and perioperative management should be highlighted and reconsidered. delaying surgical treatment even for early-stage lung cancer could be inappropriate because no one knows how long the pandemic will last. even though no prospective or well-designed studies can provide answers to these questions, the following observations, which are based on the experiences of extremely skilled, highvolume thoracic surgical departments whose management teams are continuously working on and updating their procedures, can serve as useful guidelines. during the covid-19 pandemic, it is relevant to consider carefully the lung cancer surgical centre to which patients are referred, taking into account the different levels of complexity of lung cancer management in terms of diagnostic processes and surgical indications. it is important to know whether the surgical centre deals with stage iiia patients and whether its surgeons perform extended lung resections, including bronchovascular reconstructions; carinal sleeve resections; heart, great vessel, chest wall and spine reconstructions; and post-chemotherapy resections. it has been reported that high-volume hospitals have higher lung resection rates and operate on patients who are older and have more comorbidities. nevertheless, these patients have better surgical outcomes than younger patients with fewer comorbidities, most notably in the early postoperative period. higher-volume hospitals have more specialized infrastructures, are more likely to have dedicated thoracic surgeons on site and can be expected to have staff with advanced skills in the management of all patients with lung cancer, including anaesthetists. higher hospital volumes may increase the relevant experience and maintain the skills of surgeons who perform complex lung cancer resections [4] . during the pandemic, it is paramount to avoid sending patients to surgical centres where locally advanced lung tumours are not regularly treated or that lack management by multidisciplinary teams (oncologists, radiotherapists and surgeons). moreover, the medical institution should adopt a covid-19 dedicated therapeutic pathway for patients whose test results are positive for severe acute respiratory syndrome coronavirus (sars-cov-2). they should be separated from non-infected patients or from patients who are suspected of being infected [covid-dedicated wards, intensive care units (icus), radiological areas and operating rooms separated from covid-free areas]. alternatively, some hospitals should be committed to treating covid-19 patients exclusively (hub hospitals), whereas other medical centres can continue to treat ordinary non-infected cases (covid-19-free spoke hospitals) [5] . the following suggestions and perspectives are provided by a european high-volume referral centre where locally advanced lung cancer patients are treated surgically in a covid-19 hospital that guarantees a covid-free therapeutic pathway. during the pandemic, the process of diagnosing and treating patients affected by lung space-occupying lesions has to be performed carefully in order to appropriately select patients suitable for surgery. if fluorodeoxyglucose positron emission tomography (pet) plays an essential role in normal clinical practice in selecting the actual neoplastic cases, it is even more important during this worldwide crisis that it be the main diagnostic tool for excluding benign lesions and identifying malignant neoplasms. moreover, bronchoscopy should be avoided when possible [6] as should any redundant staging procedure that could potentially lead to infection of other patients or medical staff. however, bronchoscopy is necessary to determine the surgical strategy when a bronchial or carinal reconstruction is planned. hence, we need a fast-track algorithm for diagnostic and therapeutic strategies ( fig. 1a -c) to be used in patients with stage ia-iiia lung cancer, to guide the use of computed tomography (ct) and fluorodeoxyglucose pet scans and the pretest risk of malignancy and cytological/histological diagnostic procedures ( fig. 2a) . a practical diagnostic algorithm for approaching the solitary pulmonary nodule, stratifying clinical risk factors in a standardized manner and combining this information with radiological signs would point the physician towards a benign or malignant cause and recommend invasive tests for the nodules likely to be malignant. several sources or references are routinely used to calculate the probability of malignancy, taking into consideration the following factors: age, smoking habit, haemoptysis, history of malignancy, nodule diameter, location, edge characteristics, growth rate, cavity wall thickness, calcification, contrast enhancement on ct scan >15 hu and pet scan results. a convenient and reliable way of performing this assessment is by using a calculator available online at www.chestx-ray.com, taking into account the likelihood ratios. when the probability that the lesion is benign is >70% [3, 7] , elective surgery should be postponed for 3 months. if the probability that the lesion is malignant is high, a cytological/histological diagnostic procedure should be considered. if the lesion is diagnosed as a lung cancer, the patient should undergo surgery. conversely, if a diagnosis of lung cancer is not reached, followup studies should be performed within 1 month. sars-cov-2 interstitial pneumonia is characterized by multifocal bilateral glass-like opacities [8] . these radiological features need special attention because, when there is an early-stage or a resolution-phase infection, a differential diagnosis with lepidic lung adenocarcinoma should be considered ( fig. 2b and c) . obviously, clinical history and radiological features should be considered simultaneously to reach a diagnostic conclusion. a pure ground-glass opacity (ggo) can be radiologically clear or have a solid component, easily detected on a ct scan. generally, ggos produce negative findings on pet scans, so a fluorodeoxyglucose pet scan has a limited role in the ggo characterization process. a needle biopsy is a poor diagnostic tool for ggos; there is no decrease in life expectancy when it is performed [9, 10] . according to published reports, a follow-up period could be necessary to rule out changes in both pure and solid-component ggos [11] . for a mixed ggo, there is no unanimous consensus for surgical indication or follow-up. thus, in light of the unknown evolution of a pure ggo or a predominantly solid-component ggo lesion (solid component <50%), we recommend a 3-month follow-up period during the covid-19 outbreak. conversely, a predominantly solidcomponent ggo (>50%) should be considered for surgical resection without further characterization (fig. 1a) , always considering the risk-benefit ratio for each patient. in the wake of the covid-19 pandemic, navigating cancer is challenging. however, some fixed surgical indications should be clear to avoid delayed treatment potentially causing tumour progression and even endangering the patient's life. high suspicion of lung cancer and a histological diagnosis of lung cancer. all patients with a high probability of lung cancer or a recently received lung cancer diagnosis should undergo surgical resection. to minimize inter-regional movement, patients should be encouraged to go to the nearest thoracic surgery centre; only those patients affected by locally advanced lung cancer should be referred to high-volume, highly skilled thoracic surgery centres for fast-track diagnostic and therapeutic procedures. all patients receiving multimodal treatment, including surgery, should be considered for lung resection. patients with lung cancer who are being treated with induction chemo-or radiotherapy while waiting for surgical treatment should be considered for a fast-track surgical procedure. conversely, surgery for patients who need adjuvant chemotherapy could be delayed. studies have shown that starting chemotherapy at 57-127 days after surgery did not increase mortality (hazard ratio 1.037) [12] . the thoracic surgery outcomes research network (an american multi-institutional cooperative of experts) [13] recently published a covid-19 guide for triage of operations for thoracic malignancies, suggesting that patients with solid or predominantly solid (>50%) ggos or lung cancer/presumed lung cancer > _2 cm should not defer surgery. however, they recommend that patients with small lesions (ggos with solid component <50% or lung cancer <2 cm) should postpone surgery for 3 months. according to our experience as a european surgical centre unexpectedly struck by the covid-19 surge, because we do not know the impact, timeline and duration of surge, it is not appropriate to delay surgery 'a priori' as long as hospital resources are still intact during the pandemic. for this reason, we recommend using pet to identify lesions that should be operated on right away. in pet-negative lesions with a high probability to be cancer, all the diagnostic procedures to reach a certain preoperative diagnosis of lung cancer (bronchoscopy, endobronchial ultrasound or ct guided needle biopsy) should be considered to carefully select patients who should have the surgical procedures. palliative surgical treatment for lung cancer. patients with stage iv lung cancer who require palliative treatment should be identified and referred without delay. breathlessness due to pleural effusion (m1 stage) may be relieved by fluid removal via needle aspiration or a narrow-bore indwelling catheter [3, 14] . patients affected by recurrent pleural effusions should be offered talc pleurodesis for longer-term benefit. although talc slurry is a viable choice for patients who are not suitable for thoracoscopy, video-assisted thoracic surgery procedures with talc poudrage are strongly indicated for patients with stage iv lung cancer even during the covid-19 outbreak because of the significant qualityof-life improvement and the favourable benefit/risk ratio. life-threatening lung cancer. centrally or endobronchially located lung cancer could represent a life-threatening condition causing haemoptysis or respiratory failure ( fig. 2a) . urgent assessment for and treatment with operative procedures should be considered during the covid-19 pandemic. rigid bronchoscopy, mechanical dilatation or neodymiumyttrium-aluminium-garnet laser to remove obstructions and airway stenting are suggested to maintain/re-establish airway patency, thereby providing immediate relief for the patient and allowing him or her to undergo chemo-or radiotherapy. preoperative clinical assessment should be performed by evaluating performance status and lung function [15] . during the covid-19 pandemic, spirometry could increase the risk of contagion for patients and medical staff. initial guidance from several respiratory societies [16] suggests avoiding airway challenge testing, which actively induces cough. therefore, analysis of blood gases and spot-check oximetry could be sufficient for preoperative assessment, thereby limiting spirometry to patients with previous respiratory failure, to unfit patients or to patients for whom extended surgical resection is planned. these societies also recommend wiping down equipment and surrounding areas after each patient, cleaning the department at the end of each day and providing patients with tissues to cough into and disposing of the tissues in an appropriate clinical waste bin. to screen and carefully select patients undergoing spirometry and then elective lung surgery, it can be helpful to administer a telephone covid-19 anamnestic questionnaire, following a patient's screening algorithm before hospitalization ( fig. 1b and c). aerosol-generating procedures (tracheal intubation, bronchoscopy, tracheotomy, non-invasive ventilation, cardiopulmonary resuscitation, manual ventilation) [17] put health care workers at high risk of contagion and need reinforced airborne precautions. using adequate personal protective equipment (ppe) and taking appropriate precautions help lower the risk of infection from aerosol-generating procedures [18] . despite the fact that several recommendations for intraoperative management have been determined for patients known to be or suspected of being infected with covid-19, no specific standardized protective measures are recommended for patients with no suspicion of infection. specific suggestions should be implemented in order to prevent hospital acquired infections because of possible viral transmission from asymptomatic or minimally symptomatic patients. an experienced anaesthesiologist should use level 3 protection airborne precautions [19, 20] including mask respirators (e.g. n95, ffp2 or equivalent standard) [21] when performing endotracheal intubation. particulate respirators, designed to block 95-99% of aerosol particles and recommended during management of covid-19 patients, should be routinely used during thoracic anaesthesia. in fact, because high-complexity airway management is often required with thoracic anaesthesia and because lung isolation could be necessary, additional precautions are mandatory. despite the fact that the use of respirator masks is critical when treating covid-19 patients and that ppe could become scarce during the pandemic, advanced airway management during thoracic anaesthesia needs mandatory reinforcement of airborne precautions. to limit ppe consumption and to reduce the number of health care workers exposed, unnecessary operating room staff should be eliminated. in this setting, the double lumen tube should be preferred, because it is easier and faster to position than the bronchial blocker [22] . the position of the double lumen tube can be checked by auscultation and by observation of the movements of the chest wall, which differs from a bronchial blocker, which requires the use of a bronchoscope for proper positioning. during the covid-19 pandemic, the use of a bronchoscope should be avoided and reserved for predicting difficult airway management situations (awake fibre-optic intubation with single lumen endotracheal tube and bronchial blocker) or for tube misplacement that cannot be managed with minimal double lumen tube advancement or withdrawal. they also recommended avoiding procedures that could cause aerosolization and potential virus spreading [19] . a visual summary of the suggested precautions during thoracic anaesthesia is shown in fig. 3 . it is beneficial to avoid unnecessary aerosol therapy during the postoperative hospital stay in order to limit the spread of the virus. the patient's discharge from the hospital can be managed by providing an indwelling chest tube connected to a heimlich valve in order to contain contaminated air or pleural effusion from the lung. during the pandemic, postoperative follow-up can be managed through a telemedicine service or by the general practitioner remaining in contact with the surgeon. due to the disruption caused by the covid-19 pandemic, a huge number of elective surgical procedures will be cancelled or postponed, risking inappropriately treated lung cancer and an increase in the time-related mortality rate [23] . governments should develop recovery plans and implement strategies to rapidly restore surgical activity in situations in which elective procedures were severely curtailed. in their communication of 14 may 2020, the world health organization warns that the new coronavirus may be here to stay and that it is impossible to predict when the pandemic might be controlled [24] . thus, during the covid-19 pandemic, patient selection guidelines for lung surgery need to be revisited. differential diagnosis from benign diseases (including a possible sars-cov-2 infection) is mandatory. the present perioperative suggestions may help to accurately select patients undergoing lung cancer surgery, guaranteeing a rapid diagnostic and therapeutic process. intraoperative precautions and adequate ppe should also be applied to the management of all patients to prevent cross infections. when we embraced the foregoing recommendations, our high-volume thoracic surgery centre reported a 30% decrease in surgical procedures in a month (6 march-7 april), from 120 patients to 85, mostly due to the allocation of resources from the institutional administration. during the observation period, 88 patients were evaluated, 85 patients (96.6%) were operated on, 1 patient (1.1%) was deferred because of the presence of fever and contact with confirmed cases of covid-19 at the time of questionnaire and 2 patients (2.2%) voluntarily waived surgery. the icu admission rate was 3.5% (3 patients). thus, it is important to highlight the sustainability of early-stage lung cancer surgery when the icu admission rate is low. to date, no cases of sars-cov-2 infection were recorded among the medical staff or the patients who were operated on during their hospital stay. this favourable outcome may have been determined by the appropriate screening of patients and the improved precautions adopted. clinical features of patients infected with 2019 novel coronavirus in wuhan, china managing covid-19 in surgical systems lung cancer: diagnosis and management: summary of updated nice guidance high procedure volume is strongly associated with improved survival after lung cancer surgery a call to action becomes practice: cardiac and vascular surgery during covid-19 pandemic based on the lombardy emergency guidelines expert consensus for bronchoscopy during the epidemic of 2019 novel coronavirus infection (trial version) reasons for healthcare workers becoming infected with novel coronavirus disease 2019 (covid-19) in china coronavirus disease 2019 (covid-19): a perspective from china outcomes of unresected ground-glass nodules with cytology suspicious for adenocarcinoma comparisons of clinical outcomes in patients with and without a preoperative tissue diagnosis in the persistent malignant-looking, ground-glass-opacity nodules natural history of pure ground-glass opacity after longterm follow-up of more than 2 years association of delayed adjuvant chemotherapy with survival after lung cancer surgery thoracic surgery outcomes research network, inc. covid-19 guidance for triage of operations for thoracic malignancies: a consensus statement from thoracic surgery outcomes research network national collaborating centre for cancer. the diagnosis and treatment of lung cancer (update). nice clinical guideline 24 2nd esmo consensus conference on lung cancer: early-stage nonsmall-cell lung cancer consensus on diagnosis, treatment and follow-up artp guidelines for covid-19 outbreak of a new coronavirus: what anaesthetists should know anesthetic management of patients with suspected or confirmed 2019 novel coronavirus infection during emergency procedures the societã  italiana di anestesia analgesia rianimazione e terapia intensiva (siaarti) airway research group, and the european airway management society. the italian coronavirus disease 2019 outbreak: recommendations from clinical practice clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance joint statement on the use of ppe by anesthesia professionals during the covid-19 pandemic airway management in anesthesia for thoracic surgery: a "real life" observational study elective surgery cancellations due to the covid-19 pandemic: global predictive modelling to inform surgical recovery plans impossible to predict when covid-19 pandemic will be controlled conflict of interest: none declared. european journal of cardio-thoracic surgery thanks henning a. gaissert, jeffrey a. hagen and the other, anonymous reviewer(s) for their contribution to the peer review process of this article. key: cord-352532-xqphom6x authors: papanikolaou, ilias c; sharma, om p title: 1 tropical lung diseases date: 2013-12-31 journal: hunter's tropical medicine and emerging infectious disease doi: 10.1016/b978-1-4160-4390-4.00001-1 sha: doc_id: 352532 cord_uid: xqphom6x nan the term "tropics" refers to the region of the earth lying between the tropic of cancer and the tropic of capricorn. in the tropics, warm climate, poverty, lack of education, and poor sanitation provide an ideal environment for pathogens, vectors and intermediate hosts to flourish [1] . in this vast landmass, respiratory infections are a major cause of morbidity and mortality in children and adults [2] . in a typical tropical clinic, 20-40% of outpatients have respiratory complaints, and 20-30% of inpatients have lung disease (table 1-1) [2] . many tropical patients suffer from lung diseases that are found worldwide, e.g. asthma, bronchiectasis, chronic obstructive lung disease, hiv infection-related lung disease, and lung cancer. numerous dust diseases, e.g. silicosis, asbestosis, byssinosis, hypersensitivity pneumonitis, and diseases due to microbial contamination of agricultural products, remain under-recognized. diseases associated with pulmonary symptoms and infection that are concentrated in the tropics include malaria, pulmonary schistosomiasis, melioidosis, paragonimiasis, echinococcal cysts, tropical eosinophilia, and diseases related to nutritional deficiencies [3] . in addition, individuals who come in contact with birds or animals may develop zoonoses such as tularemia, psittacosis, q fever and leptospirosis [4] . in the tropics, indoor air pollution caused by biomass fuels used for cooking and heating of the homes and huts is an important cause of obstructive lung disease and chronic lung infections [5] . the following are the common tropical pulmonary conditions: l pneumonia: typical and atypical l eosinophilic pneumonias and tropical pulmonary eosinophilia l bronchiectasis, asthma and chronic obstructive pulmonary disease (copd) l pleural effusion l nontuberculous granulomatous lung disease l occupational lung diseases. a reasonable approach to the patient with lung disease in the tropic starts with age, occupational exposure, physical examination, hiv status, chest x-ray and blood tests. in children, bacterial pneumonia is the most common and life-threatening disorder. known immunodeficiency suggests tuberculosis, fungi and opportunistic pathogens. peripheral blood eosinophilia with either a pleural effusion or diffuse parenchymal consolidation may suggest a parasitic infection, or, when combined with wheezing, tropical pulmonary eosinophilia. worldwide diseases like copd may affect nonsmoking individuals due to indoor pollutants. streptococcus pneumoniae is the most common bacterial cause of pneumonia. upper respiratory involvement often precedes the onset of pneumococcal pneumonia, which is characterized by fever, chills, malaise and sweating. the patient is flushed and febrile with a rapid pulse and respiratory rate. dyspnea is associated with a nonproductive cough, and sputum, if present, may be thick, tenacious or "rusty". severe pleuritic chest pain causing tachypnea and grunting respiration is often present. such symptoms are abrupt in young, immunocompetent patients ( fig.1.1 ) [6] . in elderly patients, symptoms may be few and can be dominated by confusion, delirium and prostration [7] . physical examination of the affected lung, usually the lower lobe, reveals diminished lung expansion, impaired percussion note, decreased breath sounds, crepitations (crackles/rales) and bronchial breath sounds. cyanosis is common and a herpes simplex eruption may be seen on the lips. with proper treatment, most patients with pneumococcal pneumonia improve clinically and radiographically within 1-2 weeks. when resolution occurs, fever subsides within a week as the temperature decreases following a crisis pattern ( fig. 1.2a) . delayed resolution is seen in smokers, the elderly, and in those with poor nutrition, diabetes or other comorbid illnesses. staphylococcal pneumonia (staphylococcus aureus), accounts for 2-10% of acute community-acquired pneumonias. it is an important cause of pneumonia in children, the elderly, patients recovering from influenza, people with diabetes mellitus, and those who are immunocompromised. methicillin-resistant staphylococcus aureus (mrsa) causes illness in 1% of cases of upper or lower respiratory tract infection in the community and in 10% of patients who are hospitalized. patients with staphylococcal pneumonia are usually ill with high fever, shaking chills, chest pain, cough and purulent sputum. chest x-ray films show patchy consolidation and cavities. sputum examination is an important aid in the diagnosis of pneumonia. color, amount, consistency and odor are helpful: mucopurulent sputum is commonly found in bacterial pneumonia or bronchitis; scanty watery sputum is often noted in atypical pneumonia; "rusty" sputum is seen in pneumococcal pneumonia; and currant-jelly or dark-red sputum suggests klebsiella pneumoniae. foul-smelling expectoration is associated with anaerobic infections due to aspiration, ilias c papanikolaou, om p sharma give an inhaled bronchodilator for 5 days* • soothe the throat and relieve the cough with a safe remedy • if coughing for more than 3 weeks or if having recurrent wheezing, refer for assessment for tb or asthma • advise the mother when to return immediately • follow-up in 2 days • if wheezing (even if it disappeared after rapidly acting bronchodilator) give an inhaled bronchodilator for 5 days* • soothe the throat and relieve the cough with a safe remedy • if coughing for more than 3 weeks or if having recurrent wheezing, refer for assessment for tb or asthma • advise the mother when to return immediately • follow-up in 5 days if not improving a blood count usually reveals leukocytosis in bacterial pneumonia, leukopenia in viral infection, and eosinophilia in parasitic infestation. when available, chest x-ray is extremely helpful ( table 1 -2). tuberculosis is omnipresent in the tropics; upper lobe lesions with or without cavities strongly suggest tuberculosis. in children, the integrated management of childhood illness (imci) guidelines for treating pneumonia are recommended (see fig. 1 .1) [8] . nevertheless, a patient's illness has to be assessed based on geography, prevalence of potential etiologies, virulence of the organism, and the drug sensitivity pattern (box 1.1). in some areas, particularly papua new guinea, south africa and spain, resistance of the pneumococcus to penicillin is common. for children with non-severe pneumonia, the world health organization (who) recommends oral trimethoprim-sulfamethoxazole (tmp-smx) or oral amoxicillin for 5 days [9] . in severe pneumonia in hospitalized children, the policy in low-income countries is to first give benzylpenicillin injections, changing the therapy to oral amoxicillin when the child responds. in very severe pneumonia, in children in low-income settings, chloramphenicol may be given first with benzylpenicillin and gentamicin in combination as an alternative [10, 11] . atypical pneumonia is caused by mycoplasma pneumoniae, chlamydia pneumoniae, legionella spp., viruses, tuberculosis, fungi and parasites. this syndrome is not extensively studied in the tropics because of the expense involved in culturing and isolating various organisms and obtaining serologic and immunologic tests. mycoplasma pneumoniae infections occur worldwide, affecting mostly school-aged children and young adults. a typical patient with mycoplasma pneumonia is an older child or young adult with an insidious onset of fever, malaise, tightness of the chest, and dry brassy cough. constitutional symptoms are out of proportion to the respiratory symptoms. hemoptysis, pleural pain and gastrointestinal symptoms are uncommon. the tropical physician should be aware of the non-respiratory manifestations of mycoplasma infection, including anemia, myringitis, stevens-johnson syndrome, hepatitis and neuritis [12] (see table 1 -2). leptospirosis is common in tropical areas where sanitation is poor and water supply primitive. epidemics of leptospirosis occur after high rainfall in monsoon seasons when the water supply is contaminated by sewage or animal urine. about half of the patients with leptospirosis have fever, cough, hemoptysis and pneumonitis [13] . other features are jaundice, conjunctivitis and impaired renal function. melioidosis, caused by burkholderia pseudomallei, is endemic in southeast asia (vietnam, cambodia, myanmar), northern australia and west africa. melioidosis is hyperendemic in northern australia, and in parts of northeastern thailand it is an important cause of fatal community-acquired pneumonia [14] . patients become infected while wading through fields, paddies, and flooded roads. clinical presentation is protean and nonspecific. the radiologic picture of upper lobe infiltration and cavity formation can be indistinguishable from tuberculosis [15] . diagnosis requires isolation of the organism. the mortality rate ranges from 20% to 50% but is higher in hivinfected and immunocompromised hosts. respiratory symptoms of cough and chest pain in typhoid are present in up to 50% of cases at the onset of the disease. pulmonary infiltrates may be associated with positive sputum cultures for salmonella typhi. a fever chart showing continuous fever is highly suggestive of enteric fever. diagnosis may be difficult without blood and stool culture facilities. in brucellosis, the lungs are involved in about 5% to 10% of cases, usually following inhalation of organisms. abnormalities include bronchopneumonia, solitary or multiple lung nodes, miliary interstitial lung disease, lung abscess and pleural effusion. organisms can be identified on stains or sputum cultures. tularemia is a generalized infection caused by francisella tularensis and occurs after skin or mucous membrane contact with infected mammals or through the bite of an arthropod, usually a tick or biting fly. diagnosis should be considered in the presence of a skin ulcer associated with fever, generalized lymphadenopathy, cough and signs of pneumonia. pneumonia, either primary from inhalation of an infected aerosol or secondary to systemic infection, occurs in about 20% of cases. children with malnutrition and edema should be admitted to hospital pneumonic plague is less common than either bubonic or septicemic disease. nevertheless, fatal bronchopneumonia can occur without lymphadenopathy and is characterized by watery, bloody sputum. a sputum gram stain can show bipolar stunted rods. pneumonic plague and tularemic pneumonia should be considered when a severe, rapidly progressive bronchopneumonia is reported in an endemic area, and "typical" bacterial pneumonias have been ruled out. in slaughterhouses, meat-processing plants, and areas with sheep and goat husbandry, q fever (coxiella burnetii) can cause epidemics of atypical pneumonia. inhalation of dried infected material is the chief source, and fever, headache and dry cough are the main symptoms. occasionally, the sputum is blood-streaked. bornholm disease (caused by coxsackieviruses and occasionally other enteroviruses), also known as epidemic pleurodynia or devil's grip, causes chest discomfort and cough. widespread epidemics of bornholm disease occur in the pacific islands and south africa. in 2002-2003, an unusual coronavirus was responsible for more than 8000 cases of a severe acute respiratory syndrome (sars) that spread via international travel across continents from its origin in guandong province, china. the sars coronavirus was previously unknown in humans; a possible reservoir was identified in civet cats and raccoons. after droplet inhalation of the virus, there was an incubation period of 2-7 days, then fever, cough, malaise and headache occurred. pulmonary inflammation was characterized by desquamation of pneumocytes, hyaline membrane formation and acute respiratory distress syndrome (ards). the chest x-ray showed diffuse opacities or consolidation, especially in the lower lung fields. recovery could be slow and some patients developed fibrosis. mortality was 10-20%, with the elderly and those with cardiovascular problems being especially at risk. kawasaki disease occurs in children under 5 years of age. this acute multisystem disease of unknown cause is characterized by fever of 5 days duration and four of five clinical features: non-purulent conjunctivitis; injected (or fissured) lips or pharynx or strawberry tongue; cervical adenopathy; a maculopapular rash; and changes in the extremities (erythema and edema of the palms and soles, associated with desquamation). pneumonitis occurs in 10% of the children and coronary artery dilatation and aneurysms in 20-25% of untreated cases. in brazil there has been a seasonal rise of the condition at the beginning and end of the monsoon season [16] . cryptococcus neoformans and c. gatti are saprophytic fungi distributed worldwide and are particularly abundant in soil contaminated by pigeon droppings in the tropics as well as in temperate countries. pulmonary infection results from inhalation of the organisms from environmental sources [17] . systemic helminth infection usually elicits eosinophilia and increased ige. although eosinophilia can be a clue to a pulmonary helminth infestation, the definitive diagnosis requires demonstration of ova or larvae in sputum, bronchial alveolar lavage fluid, pleural fluid or lung biopsy [18] . loeffler's syndrome refers to "simple" pulmonary eosinophilia with no or minimal systemic and pulmonary symptoms. in many helminth infestations (ascaris, strongyloidiasis, hookworm), the larvae migrate through the lung and can cause fever, cough, dyspnea, wheezing, hemoptysis and lung infiltrate. schistosomes cause two clinical syndromes. in acute disease, immature schistosomula pass through the lung, and can lead to fever, eosinophilia and pulmonary infiltrate. in chronic schistosomiasis, especially when portal hypertension has led to venous shunts, eggs can bypass the liver and plug pulmonary capillaries and arterioles, producing granuloma and pulmonary hypertension. radiographs may show dilated pulmonary arteries ( fig. 1.3 ). in paragonimiasis, the lung is the predominantly involved organ. the diagnosis must be considered in a patient from southeast asia with cough, hemoptysis (which is recurrent in >80% of cases), a pulmonary cavity and pleural effusion. tropical pulmonary eosinophilia, typically in india and other south asian countries, causes immunologic hyperresponsiveness to wuchereria bancrofti, brugia malayi or other microfilariae. clinical presentation consists of nocturnal cough, wheezing, fever and weight loss. chest radiographs show diffuse interstitial miliary infiltrates ( fig. 1.4) ; there is a high eosinophil count. in developed countries, serum ige and antifilarial antibodies can be used to confirm the diagnosis (table 1-3) . bronchiectasis is a chronic, debilitating condition. dilatation and distortion of the airways leads to impaired mucociliary clearance, which encourages bacterial colonization and bronchial inflammation. patients have fever, chronic cough, mucopurulent sputum, hemoptysis (table 14) , wheezing, dyspnea and malaise (box 1.2) . the primary cause of copd is smoking l copd affects men and women equally l copd is not curable but can be prevented the diagnosis of bronchiectasis in developed countries is confirmed by computed tomography of the chest (fig. 1.5) ; whereas, in the tropics, the diagnosis is mainly clinical and depends upon a compatible history, presence of finger clubbing, sputum that settles into three layers (mucoid or frothy, mucopurulent, and purulent) and a chest x-ray, if available. treatment includes regular chest percussion, broadspectrum antibiotics for exacerbations, and influenza and pneumococcal vaccinations. the incidence of asthma in the tropics is low for unclear reasons; however, the disease remains underdiagnosed and untreated. "all that wheezes is not asthma" is a dictum that is true in the tropics, as there are many entities that cause wheezing and difficulty in breathing, including tropical eosinophilia and mitral stenosis. asthma monitoring in the tropics can be achieved by using an inexpensive peak flow meter. treatment should fit the frequency and severity of attacks. betaagonists and cromolyn sodium (sodium cromoglycate) are usually available. oral corticosteroids in short courses can be used to control severe episodes; however, long-term use of systemic corticosteroids, without adequate monitoring, is not safe. aerosol inhalers are of great value but they are expensive, difficult to use, and require painstaking teaching. chronic obstructive lung disease is a progressive disease which is characterized by airway obstruction that is only partially reversible by bronchodilator therapy. the term copd encompasses chronic bronchitis and emphysema. once a common disease of men, copd is now as frequent in women because of increased tobacco use and the widespread use of dung and biomass for indoor cooking and heating in low-income countries (box 1.3) . the most common symptoms are dyspnea and chronic cough. it is mild and occurs only on heavy exertion. with progression of airway obstruction, patients become more short of breath and eventually cannot breathe at rest. physical examination in the early stage is normal, but in advanced disease, prolonged expiration and expiratory wheezes are audible. in severe cases, the thoracic cage becomes barrelshaped with increased anterior-posterior diameter; percussion note is hyperresonant. when chest x-ray and pulmonary function testing are not available, a peak-flow meter is an inexpensive device to assess severity of airway obstruction and monitor the response to treatment. cessation of smoking is essential. oral theophylline and beta-agonist drugs control symptoms. antibiotics (ampicillin, tetracycline and sulfa drugs) are available to treat copd exacerbations in the tropics. pleural effusion is a frequent condition with variable clinical signs and symptoms. small effusions can remain silent and are often detected only on chest radiography. large effusions are associated with dyspnea and diminished chest movements on the affected side. vocal fremitus is reduced; percussion note is stony dull; and auscultation reveals diminished breath sounds and decreased vocal resonance. sometimes, bronchial breathing is heard at the upper level of dullness. in addition there may be a pleural friction sound. exudative effusions typically have cell counts, protein and biochemical markers opposite to those of transudates. exudates can be further classified into neutrophilic, lymphocytic and eosinophilic. neutrophilic exudates may be due to bacterial infection, gastrointestinal diseases, pulmonary embolism, collagen-vascular diseases (cvd) and asbestos-related benign effusion. pleural effusion occurs in about 50% cases of pneumonia, and can progress to a complicated effusion (pleural fluid ph<7.2, positive gram stain) or to an empyema, both necessitating pleural fluid drainage with a chest tube thoracostomy in addition to antibiotic treatment. empyema can occur in pneumococcal, staphylococcal (most often) and klebsiella infections. a right-sided pleural effusion may be associated with amebic liver abscess. the disease presenting with the highest pleural fluid lymphocytosis is tuberculous pleuritis; however, early in the course, there can be a neutrophilic exudate. a large volume of pleural fluid should be obtained for examination for acid-fast bacilli. in about one-third of cases, the tuberculin skin test is negative initially and converts to positive after 2-4 weeks. knowledge of the hiv status of a patient with pleural effusion, if positive, significantly inclines to a tuberculosis. an eosinophilic exudate is more common in the tropics. endemic parasitic and fungal infections are major causes of such an effusion. ascariasis, echinococcosis and paragonimiasis are some of the causative parasitic infections. paragonimiasis is associated with low pleural fluid glucose and low ph. fungal diseases responsible for such an effusion are histoplasmosis, cryptococcosis and coccidioidomycosis. in the absence of chest x-ray or biopsy evidence, it is not possible to diagnose pulmonary involvement due to sarcoidosis and other granulomatous diseases. consequently, in the tropics, these disorders remain undiagnosed. the possibility of sarcoidosis should be considered in a patient with dyspnea, uveitis, hepatosplenomegaly, peripheral lymphadenopathy, chronic skin lesions, and a chest x-ray film showing bilateral hilar adenopathy [18] . the occupational disorders result from human social activity, and as such are preventable. the dusts that provoke occupational disorders can be classified into: those that induce granulomatous reaction (e.g. beryllium, talc and organic antigens); those that cause fibrosis (e.g. silica, asbestos and coal); and those that cause neither inflammation nor fibrosis, thus remaining inert (e.g. iron, barium and tin) ( podoconiosis is an endemic nonfilarial elephantiasis occurring in individuals exposed to red clay soil derived from alkaline rock. a chronic and debilitating disease, it exerts a large economic burden. the silica particles are found in the skin, lymph nodes and lymphatics of affected and unaffected individuals. these individuals have reduced lung function as compared with adults living in areas of low silica concentration [19] . immunological aspects of tropical lung disease pneumonia: the forgotten killer of children. geneva: unicef/ who parasitic lung infections tropical infections and the lung pulmonary disease world health organization, family and community health cluster, department of child and adolescent health and development. consultative meeting to review evidence and research priorities in the management of acute respiratory infections (ari) influence of age on symptoms and presentation in patients with community acquired pneumonia integrated management of childhood illness (imci) for high hiv settings clinical efficacy of co-trimoxazole versus amoxicillin twice daily for treatment of pneumonia: a randomised controlled clinical trial in pakistan chloramphenicol alone versus chloramphenicol plus penicillin for severe pneumonia in children chloramphenicol versus benzylpenicillin and gentamicin for the treatment of severe pneumonia in children in papua new guinea: a randomised trial approach to the patient with tropical pulmonary disease pulmonary complications of leptospirosis the epidemiology of melioidosis in australia and papua new guinea melioidosis after brief exposure: a serological survey in us marines kawasaki disease: a clinical and epidemiological study of 70 children in brazil lung biology in health and disease: tropical lung disease lung biology in health and disease: tropical lung disease assessment of respiratory function in patients with podoconiosis key: cord-261640-ehc123p7 authors: smith, maxwell l.; gotway, michael b.; crotty alexander, laura e.; hariri, lida p. title: vaping-related lung injury date: 2020-10-27 journal: virchows arch doi: 10.1007/s00428-020-02943-0 sha: doc_id: 261640 cord_uid: ehc123p7 the use of electronic nicotine delivery systems has increased in popularity dramatically over the past decade. although lung diseases caused by vaping have been reported since the modern invention of the electronic cigarette, in the summer of 2019, patients began to present to health care centers at epidemic levels with an acute respiratory illness relating to vaping, which the center for disease control termed e-cigarette or vaping product use-associated lung injury (evali). this review discusses electronic nicotine delivery systems as well as the etiology, clinical presentation, imaging findings, pathologic features, treatment, and long-term consequences of evali. we conclude with the practical impact evali has had on the practice of pathology. in the summer of 2019, an acute, mysterious, and deadly respiratory illness related to vaping emerged, primarily in young patients, in the usa. cases increased dramatically and peaked in late september 2019. the center for disease control and prevention (cdc) termed the disease causing this epidemic ecigarette or vaping product use-associated lung injury (evali). prior to evali, vaping had been associated with a variety of different pulmonary presentations ranging from lipoid pneumonia to diffuse alveolar hemorrhage, but at low numbers. in this review, we discuss electronic nicotine delivery systems (ends) as well as the etiology, clinical presentation, imaging findings, pathologic features, treatment, and long-term consequences of evali. we conclude with a discussion on the practical impact evali has had on the practice of pathology. electronic nicotine delivery systems ends, also known as e-cigarettes and vaping devices, were originally developed as a replacement device for conventional tobacco cigarette smokers [1] . however, their success in the arena of smoking cessation has been very limited, and they remain unapproved as cessation tools due to a lack of data demonstrating efficacy relative to currently approved nicotine replacement therapies [2] . the aerosols produced by e-cigarettes are known to cause a variety of deleterious health effects, although more research and long-term studies are still needed [2] . e-devices have rapidly evolved since entering the international market in 2013, with vape pens, box mods, and pod-based devices being the most commonly used vaping devices in 2020 [3] . although e-cigarettes are used in conjunction with conventional tobacco by many cigarette smokers (dual users), their sole use in young adults and adolescents has skyrocketed [4] . this is concerning as use of tobacco products had been declining worldwide for over 50 years, and now, a new generation of nicotine addicts is being created through these novel vaping devices through the use of appealing flavors and packaging [5] . even more concerning is that children and teenagers who use ecigarettes are more likely to smoke conventional tobacco [6] . in mid to late 2019, vaping caused a novel disease in the usa which rapidly reached epidemic levels, termed evali. thousands of e-cigarette users, predominantly males aged 13-34, developed respiratory, gastrointestinal, and systemic symptoms after vaping [7] . testing of both e-liquids vaped and samples from the airways of those affected found vitamin e acetate (vea) [8] . vea is a clear viscous solution that was used as a cutting agent to increase tetrahydrocannabinol (thc) dealer profits. mixtures of vea and thc oils were used in the production of black market and gray market vaping devices and cartridges in the spring of 2019 [9] . when vea is heated to the typical temperatures of an ends, it decomposes into the highly toxic ketene gas [10] . when tested in animals, vea caused acute lung injury when inhaled through e-cigarette aerosols, confirming it as the probable chemical responsible for evali [11, 12] . while there is substantial evidence associating vea with many cases of evali, there are other adulterants that likely are responsible in a subset of cases. in patients presenting with evali, the main symptoms include shortness of breath, cough, chest pain, diarrhea, abdominal pain, fever, and fatigue [13, 14] . symptoms occur anywhere from hours to weeks prior to presentation. laboratory tests commonly reveal an elevated erythrocyte sedimentation rate and c-reactive protein level, transaminitis, and leukocytosis [13] . to meet the cdc criteria for a "confirmed" evali case, patients must have vaped within 90 days before symptom onset, have bilateral infiltrates on chest imaging, have a negative evaluation for infection, and have no other plausible alternative diagnoses. cases of "probable" evali share similar criteria, except that infection may be present, but the clinical team caring for the patient has high confidence that infection is not the primary cause for the patient's respiratory condition. a wide arrange of imaging abnormalities have been described in the setting of evali, most commonly acute lung injury (ali) and organizing pneumonia, as well as imaging patterns resembling non-fibrotic hypersensitivity pneumonitis (hp) and acute eosinophilic pneumonia (aep) [15] [16] [17] [18] . these features include multifocal or diffuse ground-glass opacities, often with areas of organizing consolidation ( fig. 1a and b) . the ground-glass opacities often show predilection to the central regions and show a variable cephalocaudad distribution. imaging manifestations resembling lipoid pneumonia, diffuse alveolar hemorrhage (dah), and respiratory bronchiolitisinterstitial lung disease (rb-ild) have also been suggested as manifestations of evali, but these patterns overlap with the more commonly reported evali patterns, particularly ali and hp [15] [16] [17] [18] . correlating the pathologic features with the patterns of imaging abnormalities suggests that the majority of imaging findings are related to varying degrees of ali, sometimes airway centered and upper lobe predominant due to the inhaled nature of the injury, with or without features of organization. subpleural sparring is frequently encountered but is a non-specific finding. the imaging findings of evali can closely resemble hp, leading to an incorrect presumptive diagnosis based on imaging alone (fig. 1c) . in these patients, high-resolution computed tomography (hrct) shows upper lobe predominant of diffuse, poorly defined centrilobular ground-glass opacity nodules and/or opacities which have been shown to correspond to acute and organizing lung injury pathologically. despite the published reports of lipoid pneumonia as a mechanism of injury in evali, no well-characterized radiologic and pathologic cases have been published. most of the clinicopathologic diagnoses of lipoid pneumonia have been based on the finding of lipid-laden macrophages in bal cytology specimens (discussed in detail below). to date, there cases of evali present to pathology in a variety of ways, including cytologic preparations of bronchoalveolar lavage (bal) specimens, transbronchial biopsies, cryobiopsies, and surgical lung biopsies. the primary consideration in the clinical differential is often infection, and thus, many cases have associated microbiology specimens. bal specimens from evali patients are mostly inflammatory with the majority of inflammatory cells being macrophages. rare neutrophils, lymphocytes, and eosinophils may be encountered. the macrophages are unique in that their cytoplasm is often distended with mostly fine cytoplasmic vacuoles of similar size (fig. 2a ) [19] . enlarged cytoplasmic vacuoles and variably sized vacuoles, features seen in exogenous lipoid pneumonia, are encountered much less frequently. oil red-o staining highlights the vacuoles to be composed of lipid material (fig. 2b) . the lipid-laden macrophage that was first feature associated with e-cigarette use in 2012 based only on clinical presentation, imaging findings, presence of lipidladen macrophages, and a history of e-cigarette use [20] . no confirmatory biopsy was performed in this initial report, and the patient had many risk factors for pulmonary injury other than vaping. perhaps due to the precedent set in the literature, the finding of lipid-laden macrophages in bal specimens in the early stage of the evali epidemic in 2019 led investigators to conclude that the lung injury was related to a form of exogenous lipoid pneumonia related to vaping [17, 21, 22] . the fine cytoplasmic vacuoles encountered in the macrophages actually represent the accumulation of endogenous cellular lipid material from epithelial injury, a process referred to as endogenous lipoid pneumonia. endogenous lipoid pneumonia is seen in a variety of settings including acute lung injury, obstructed airways, and infections [23] [24] [25] . transbronchial, cryo-, and surgical wedge biopsies all show similar features in the setting of evali. the predominant pathologic pattern falls along the spectrum of acute lung injury [26, 27] , the natural progression of which has been well described [28] . in very early stages of acute lung injury, the only visible histologic finding may be edema and slightly enlarged pneumocytes. this is followed by the formation of hyaline membranes and fibrin deposition in the exudative phase. the proliferative, or organizing, phase shows the infiltration of inflammatory cells into the injurious process with subsequent polyps of organizing immature fibroblastic tissue. from a pathologic perspective, evali cases may show features of any of the patterns of acute lung injury, including diffuse alveolar damage, acute fibrinous and organizing pneumonia (afop), and organizing pneumonia (fig. 3) . some cases show distinctly airway-centered pathology, while others appear as more of a diffuse process (fig. 4) . the relative presence of exudative and organizing features, along with the distribution of injury likely depends on (a) the temporal relationship between the toxic exposure (or repetitive toxic exposure) and the biopsy and (b) the severity/dose of the exposure. cases of diffuse alveolar damage in the organizing phase may mimic cellular non-specific interstitial pneumonia (fig. 4c) . intra-alveolar, or airspace, macrophages are described as a relatively consistent finding, although this feature is not always prominent [26, 27] . when prominent, the histologic features mirror what has been described in the bal findings of evali. the macrophages show finely vacuolated cytoplasm, occasionally with pigment or pigmented debris (fig. 5) . rare descriptions of additional patterns of lung injury have been described, including giant cell interstitial pneumonia that is a form of pneumoconiosis related to hard metal exposure in one case report, the presence of giant cell interstitial pneumonia on lung biopsy was hypothesized to be from the combustion and aerosolization of metal elements of the vaping apparatus, which was supported by investigation with inductively coupled plasma mass spectrometry [29] . interestingly, energydispersive x-ray spectroscopy on the actual tissue samples failed to reveal evidence of tungsten or cobalt. to date, no cases of histologic confirmed exogenous lipoid pneumonia have been described in association with vaping [26, 27] . the histology of exogenous lipoid pneumonia includes the deposition of variably sized lipid droplets into the interstitium with an associated foreign body giant cell response and often a component of fibrosis (fig. 6) . in comparison to evali, the lipid droplets of exogenous lipoid pneumonia are typically much larger and have more significant variability in size. similarly, despite the radiologic features in some cases that seem to suggest subacute hypersensitivity pneumonitis, no cases of histologically confirmed hypersensitivity pneumonitis have been described in the literature in association with vaping. subacute hp is classically described as a centrilobular alveolitis with airway centered, poorly formed interstitial granulomas. hp results from exposure to inhaled organic antigens with an associated hypersensitivity response. in considering the inhalational mechanism of injury between subacute hp and evali, it is not surprising that the radiologic features overlap. however, the histologic distinction between evali and hp is typically straightforward. all cases of ali, regardless of the pathologic pattern, should include testing for infectious organisms with acid-fast bacilli and grocott's methenamine silver (gms) stains at a minimum. if there is significant necrosis and neutrophilic inflammation, viral immunohistochemistry may also be considered (cytomegalovirus, herpes virus, adenovirus). oil red-o does not have a clear role in the histologic assessment and work-up of lung specimens, especially considering that fresh, unprocessed tissue must be used for effective oil red-o. despite the low sensitivity and specificity of oil red-o, it remains on many clinical fig. 6 a, b the histologic distinction between exogenous lipoid pneumonia and electronic cigarette or vaping-associated lung injury (evali) is dramatic and distinctive. exogenous lipoid pneumonia shows numerous lipid vacuoles a, most of which are much larger than individual cells. there is associated fibrosis in which many of the droplets are embedded. occasional macrophages contain lipid droplets within their cytoplasm. however, the droplets are much larger and more variable (arrow) compared to evali, h&e, × 100. larger lipid vacuoles are surrounded by several multinucleated giant cells and a foreign body giant cell reaction (arrowhead) b, a feature not seen in evali, h&e, × 200 fig. 5 a, b airspace macrophages seen in biopsy specimens from patients with electronic cigarette or vapingassociated lung injury. marked accumulation of macrophage with distended and foamy cytoplasm is frequently encountered (arrow) a, h&e, × 400. some of the macrophages may have pigment or pigmented material in the cytoplasm b, h&e, × 200 algorithms [30] for the pathologic work-up suspected evali. however, given its lack of utility, oil red-o is not necessary for diagnostic purposes in suspected evali cases, cytology or surgical pathology specimens. the spectrum of histologic acute lung injury produces a long differential diagnosis, including infection, connective tissue disease-associated ild, adverse drug/toxin reaction, acute eosinophilic pneumonia, diffuse alveolar hemorrhage, foreign material, acute exacerbation of underlying ild, subacute hypersensitivity pneumonitis, and idiopathic disease. this requires detailed histologic inspection in an attempt to identify histologic clues to an etiology and close clinical correlation. acute lung injury pattern with distinct airway centricity, prominent foamy macrophages, pigment, and an absence of other histology to suggest alternatives should be enough to at least consider the possibility of evali. however, the diagnosis can only be made confidently following correlation with a history of exposure to ends. given the novelty of evali, treatment has not been studied outside of observational studies and case series, and therefore, the optimal treatment is not known. the vast majority of the documented patients with evali required hospitalization (9 5%), although this likely represents significant bias towards patients with more severe disease [7] . in a large series of 98 evali patients, 76% required supplemental oxygen, 22% required non-invasive ventilation, and 26% required intubation and mechanical ventilation, with extracorporeal membrane oxygenation (ecmo) rarely being needed [17] . systemic glucocorticoid use was reported in the majority of patients with evali [17, 21] . however, the studies investigating the efficacy of systemic glucocorticoids were observational, and studies formally investigating their efficacy in a controlled prospective manner have not been conducted. although over sixty deaths have been documented, many patients with evali may have resolution of symptoms upon cessation of vaping [31] . according to the cdc as of february 18, 2020, 2807 patients have been hospitalized with evali in the usa with 68 confirmed evali-associated deaths [32] . evali is a serious respiratory illness that, in its most severe form, manifests as acute respiratory distress syndrome [7, 17] . studies investigating imaging abnormalities in ards survivors have shown that the disease can resolve completely. the degree of resolution reported among different studies varies widely, with between 25 and 85% of ards survivors reported to have residual fibrotic changes on chest imaging [33, 34] . therefore, it is hypothesized that a fraction of evali survivors may develop similar chronic fibrotic changes, but longterm studies will be needed to investigate this hypothesis. clinical and imaging follow-up will be critical to ensure resolution of the pathologic process. case reports have suggested that there may be residual lung dysfunction, mainly diffusion abnormalities, present for at least up to 2 months after discharge [35, 36] . however, follow-up studies must be conducted to determine whether these abnormalities persist long term. it is also well-known that many of the chronic pathologic sequelae linked to tobacco cigarette smoking, such as emphysema, smoking-related interstitial fibrosis, and lung cancer, were not characterized until many years, and even decades, after the habit became popularized, [37] . it may take a similar amount of time before the long-term pathologic sequelae of ecigarette use become apparent. the introduction of evali as a new diagnostic consideration when patients present with acute or subacute respiratory symptoms has had multiple practical impacts for practicing pathologists. first, while the general histologic findings lack specificity for evali, it is critical that pathologists consider and report evali as a possible etiology when confronted with a lung biopsy showing acute lung injury, especially in the younger demographic. many patients are reluctant to admit to vaping, and many do not equate vaping with smoking, so the standard social history questions regarding smoking may miss potential vaping and dabbing exposures. a note in the pathology report specifically suggesting vape-related lung injury as a possible etiology may be the impetus for the clinician to question further regarding inhalational exposures. like many cases of interstitial lung disease, clinicopathologic correlation is essential. second, pathologists must be aware that the clinical and imaging presentation of some patients with evali can strongly mimic subacute hypersensitivity pneumonitis [38] , where both disorders can present with poorly defined, ground-glass attenuation centrilobular nodules or multifocal ground-glass opacity. in this setting, lung biopsy can be very helpful in arriving at the correct diagnosis, as the pathologic pattern seen in hp and evali are quite distinct. rather than the cellular interstitial infiltrates with lymphocytes and/or vague poorly formed granulomas typical of subacute hp, the pathologic pattern of evali shows features of acute lung injury without the presence of granulomatous inflammation. finally, the recent coronavirus disease of 2019 (covid-19) pandemic has resulted in further diagnostic challenges related to evali. there is a tremendous amount of overlap between the imaging findings of covid-19 and evali [14] . however, the movement towards widespread pre-procedural testing for covid has made it less likely for pulmonary specimens to be obtained from unrecognized covid-19-positive patients. additionally, the societal changes associated with the pandemic, including stay-at-home orders, isolation, and untoward economic impacts, have resulted in increased anxiety. the impact of covid-19 on smoking and vaping habits remains unknown. the university of utah group has identified the first rise in evali cases since the fall of 2019 [39] . these data support the ongoing nature of the evali epidemic with new cases still being reported. however, the cdc stopped tracking new cases in february of 2020 making it difficult to know the true current prevalence. evali is a new consideration for patients presenting with acute and subacute respiratory illnesses. the imaging appearance is diverse, ranging from bilateral opacities similar to dad to centrilobular nodules resembling hp. the pathologic features primarily include the spectrum of acute lung injury, including dad, afop, and organizing pneumonia. some cases may be accentuated in the centrilobular regions, similar to the imaging findings. prior to evali, vaping had been associated with a wide variety of pulmonary presentations including lipoid pneumonia, acute respiratory distress syndrome, and diffuse alveolar hemorrhage. however, the majority of the cases of evali are likely related to vaping vea. it remains possible that other forms of pulmonary illness related to vaping of other chemical compounds will contribute to be identified, and they may not fall into the spectrum of what has currently been described for evali. while the overall prevalence has decreased dramatically since the peak in september, 2019, the health care system must remain vigilant in screening for potentially harmful pathologic sequelae associated with the practice of vaping. electronic cigarettes: the new face of nicotine delivery and addiction the evolving landscape of electronic cigarettes: a systematic review of recent evidence vaping versus juuling: how the extraordinary growth and marketing of juul transformed the us retail ecigarette market tobacco use among middle and high school students -united states flavored e-cigarette use and progression of vaping in adolescents electronic cigarette use and progression from experimentation to established smoking characteristics of hospitalized and nonhospitalized patients in a nationwide outbreak of e-cigarette, or vaping, product use-associated lung injury -united states vitamin e acetate in bronchoalveolar-lavage fluid associated with evali vape cart additive makers pull products as others go dark potential for release of pulmonary toxic ketene from vaping pyrolysis of vitamin e acetate an animal model of inhaled vitamin e acetate and evali-like lung injury what are the mechanisms underlying vaping-induced lung injury? nih workshop report: ecigarette or vaping product use associated lung injury (evali): developing a research agenda radiologic, pathologic, clinical, and physiologic findings of electronic cigarette or vaping product useassociated lung injury (evali): evolving knowledge and remaining questions imaging of vapingassociated lung disease imaging findings of vaping-associated lung injury pulmonary illness related to e-cigarette use in illinois and wisconsin -final report respiratory failure caused by lipoid pneumonia from vaping e-cigarettes 2020) e-cigarette or vaping product use-associated lung injury: what is the role of cytologic assessment? an unexpected consequence of electronic cigarette use outbreak of electronic-cigarette-associated acute lipoid pneumonia -north carolina pulmonary lipid-laden macrophages and vaping cut-off values and significance of oil red o-positive cells in bronchoalveolar lavage fluid the lipid-laden alveolar macrophage as a marker of aspiration in parenchymal lung disease testing for lipid-laden macrophages in bronchoalveolar lavage fluid to diagnose vaping-associated pulmonary injury. are we there yet? pathology of vaping-associated lung injury lung biopsy findings in severe pulmonary illness associated with e-cigarette use (vaping) the pathologist's approach to acute lung injury giant cell interstitial pneumonia secondary to cobalt exposure from e-cigarette use e-cigarette, or vaping, product use associated lung injury (evali): case series and diagnostic approach identifying, tracking, and treating lung injury associated with e-cigarettes or vaping acute respiratory distress syndrome: ct abnormalities at long-term follow-up imaging of acute respiratory distress syndrome impaired lung function following e-cigarette or vaping product use associated lung injury in the first cohort of hospitalized adolescents the importance of antivaping vigilance-evali in seven adolescent pediatric patients in northeast ohio health effects of cigarette smoking 2020) e-cigarette or vaping product useassociated lung injury: a review for pathologists diagnosing evali in the time of covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions drs. hariri, gotway, crotty alexander, and smith: drafting and critical review of the manuscript and reading and approving the final version.data availability data derived from currently existing medical literature and authors' experience. key: cord-313785-8tipkksu authors: d'ettorre, gabriella; ceccarelli, giancarlo; marazzato, massimiliano; campagna, giuseppe; pinacchio, claudia; alessandri, francesco; ruberto, franco; rossi, giacomo; celani, luigi; scagnolari, carolina; mastropietro, cristina; trinchieri, vito; recchia, gregorio egidio; mauro, vera; antonelli, guido; pugliese, francesco; mastroianni, claudio maria title: challenges in the management of sars-cov2 infection: the role of oral bacteriotherapy as complementary therapeutic strategy to avoid the progression of covid-19 date: 2020-07-07 journal: front med (lausanne) doi: 10.3389/fmed.2020.00389 sha: doc_id: 313785 cord_uid: 8tipkksu background: gastrointestinal disorders are frequent in covid-19 and sars-cov-2 has been hypothesized to impact on host microbial flora and gut inflammation, infecting intestinal epithelial cells. since there are currently no coded therapies or guidelines for treatment of covid-19, this study aimed to evaluate the possible role of a specific oral bacteriotherapy as complementary therapeutic strategy to avoid the progression of covid-19. methods: we provide a report of 70 patients positive for covid-19, hospitalized between march 9th and april 4th, 2020. all the patients had fever, required non-invasive oxygen therapy and presented a ct lung involvement on imaging more than 50%. forty-two patients received hydroxychloroquine, antibiotics, and tocilizumab, alone or in combination. a second group of 28 subjects received the same therapy added with oral bacteriotherapy, using a multistrain formulation. results: the two cohorts of patients were comparable for age, sex, laboratory values, concomitant pathologies, and the modality of oxygen support. within 72 h, nearly all patients treated with bacteriotherapy showed remission of diarrhea and other symptoms as compared to less than half of the not supplemented group. the estimated risk of developing respiratory failure was eight-fold lower in patients receiving oral bacteriotherapy. both the prevalence of patients transferred to icu and mortality were higher among the patients not treated with oral bacteriotherapy. conclusions: a specific bacterial formulation showed a significant ameliorating impact on the clinical conditions of patients positive for sars-cov-2 infection. these results also stress the importance of the gut-lung axis in controlling the covid-19 disease. understanding the invasive process of sars-cov-2 is essential. we know that the entry points for the virus into the body, such as ace2 receptors, are enzymes that are linked to intestinal cells. coronaviruses constantly change their binding patterns as they evolve, and the potential target in the lungs also varies, but not in the small intestine, where it remains constant. the cells of the intestinal mucosa (enterocytes) could, therefore, be a reservoir for coronaviruses (1) . in the acute phase, only 10% of coronavirus disease patients present virus cdna in the blood, but almost 50% of them excrete it in the stools. the infectious form of the virus was even identified several times, suggesting that the orofecal route is a mode of contamination (1) . the gut involvement might explain the wide variation in viral load from one test to another in the same person as if the virus were hiding there (2) . chinese researchers have investigated changes in the microbiota in the patients who have died for covid-19 infection. the sequencing of their microbiota revealed a significant decrease in bifidobacteria and lactobacilli, the main families of symbiotic bacteria, as well as an increase in opportunistic bacteria such as corynebacterium or ruthenibacterium (1) . intestinal dysbiosis has a long-reaching immune impact on the pulmonary immune system (3) , and hence might be an additional risk for respiratory distress induced by in this context, the use of oral bacteriotherapy might be an option. some strains of lactobacilli and bifidobacteria have a protective role against influenza virus, rhinovirus, respiratory syncytial virus, adenovirus, and pneumovirus (4, 5) . we report here our observation on patients supplemented with oral bacteriotherapy in addition to the current anti-covid-19 treatment (hydroxychloroquine, azithromycin, tocilizumab). the comparison group was covid-19 positive subjects not treated with oral bacteriotherapy, hospitalized in the same clinic at the same time. our results stress the importance of the gut-lung axis in the control of the covid-19 illness (6, 7). the patients evaluated in this study were hospitalized at the department of infectious diseases, policlinico umberto abbreviations: abx, antibiotics; alt, alanine aminotransferase; alt, aspartate aminotransferase; ci, confidence interval; covid-19, coronavirus disease 19; cpap, continuous positive airway pressure; ecmo, extracorporeal membrane oxygenation; fdr, false discovery rate; gla, gut lung axis; hb, hemoglobin; hcq, hydroxychloroquine; hiv, human immunodeficiency virus; ho-1, heme oxygenase-1; icu, intensive care unit; irq, interquartile range; nrf2, nuclear factor erythroid 2p45-related factor 2; ob-, oral bacteriotherapy not administered group; ob+, oral bacteriotherapy administered group; ros, reactive oxygen species; scfa, short chain fatty acids; tcz, tocilizumab. i, "sapienza" university of rome, italy, between march 9th, 2020 and april 4th, 2020 (sub-intensive care unit for covid -19) . ethical approval was obtained from ethics committee of policlinico umberto i (approval number/id prot. 109/20209). all the patients were staying at home before their referral to the emergency department, and from there to our department. oropharyngeal and nasopharyngeal swabs for diagnosis of covid-19 were performed in duplicate for sars-cov-2 e and s gene by a reverse transcriptase polymerase chain reaction (rt-pcr). all the patients were positive for covid-19 and met the following clinical criteria: fever: > 37.5 • c, need of non-invasive oxygen therapy, and ct lung involvement on imaging more than 50%. they were diagnosed with symptomatic covid-19 disease state, which, however, at the time of evaluation did not require endotracheal intubation and invasive mechanical ventilation. oxygen therapy was delivered via venturi mask in spontaneous breathing patients; if hypoxemia persisted continuous positive airway pressure (cpap) was applied. dyspnea was defined as "a subjective experience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity" (8) . acute diarrhea was defined as a stool with increased water content, volume, or frequency that lasts <14 days (9) . highresolution ct scan was used to identify lung involvement according to the official diagnosis and treatment protocol (6th edition) declared by the national health commission of china. typical ct findings of covid-19 are (1) ground-glass opacities, (2) consolidation, (3) reticular pattern, (4) crazy paving pattern (10) . patients with severe acute hypoxemia due to covid-19 pneumonia and in need for invasive mechanical ventilation were referred to the intensive care unit (icu) of policlinico umberto i. since there are, currently, no coded therapies or guidelines for the medical treatment of covid-19, the patients were treated with hydroxychloroquine (hcq) 200 mg bid, antibiotics (abx) (azithromycin 500 mg) and tocilizumab (tcz) dosage is 8 mg/kg (up to a maximum of 800 mg per dose) with an interval of 12 h for two times, eventually plus oxygen. in addition to the above treatments, randomly chosen patients initiated oral bacteriotherapy on march 13th. for each patient, the charlson comorbidity index (11), the oxygensupport requirement, as well as, laboratory values comprising alanine aminotransferase (alt), aspartate aminotransferase (alt), hemoglobin (hb), ph, hydrogen carbonate (hco − 3 ), lactic acid and arterial carbon anhydride pressure (paco 2 ) were determined at baseline. the observed partial pressure of arterial oxygen (pao 2 ), the fraction of inspired oxygen fio 2 , the disappearance of symptoms associated to covid-19, adverse events, and the number of patients transferred to icu were collected at 24 h, 48 h, 72 h, and 7 days from the start of oral bacteriotherapy and hospitalization for all the patients independently from the treatments. patients were considered positive for respiratory failure when the determined pao 2 /fio 2 ratio was <300. since this is a retrospective real-life emergency data collection, some laboratory data were unavailable. in particular, in the case of significant clinical and respiratory gas exchange improvement, sometimes the clinician has not repeated the follow up blood gas analysis, considering it an unnecessary painful invasive procedure. the formulation administered in this study contained: streptococcus thermophilus dsm 32345, l.acidophilus dsm 32241, l. helveticus dsm 32242, l. paracasei dsm 32243, l. plantarum dsm 32244, l. brevis dsm 27961, b. lactis dsm 32246, b. lactis dsm 32247. ormendes sa, lausanne, switzerland which gifted the product sivomixx r (sivobiome r in usa) is responsible for the standardization of the product in terms of enzymatic content, biochemical and immunological profile. the oral bacteriotherapy involved the use of 2,400 billion bacteria per day. the formulation was administered in three equal doses per day. no sample-size calculations were performed. the categorical variables were compared using the χ 2 test and showed as absolute frequencies and percentage. the shapiro-wilk test was used to test the normality of distribution of continuous variables. when they were not normally distributed, logarithmic transformation was performed in accordance to boxcox transformation with −0.25≤λ≤0.25. for normally distributed continuous variables, mean values between two groups were compared by student's ttest and showed as mean ± sd (standard deviation); for data not normally distributed, the mann-whitney test was used and indicated as median (25th−75th). the longitudinal analysis of data relative to respiratory failure in relation to the "not treated vs. treated" group was performed by a general linear mixed model with the glimmix procedure considering the binary as distribution and logit as link function. the benjamini-hochberg false discovery rate (fdr) correction was used to account for multiple hypothesis testing when necessary. a p < 0.05 was considered statistically significant. all statistical analyses were performed by using sas v.9.4 and jmp v. 14 (sas institute inc., cary, nc, usa). data relative to 70 subjects positive to the sars-cov-2 test (median age, 59 years, interquartile range, 50-70) were collected during period march 9th-april 4th , 2020. none of the patients had recently traveled to china or south korea or iran. patients were unable to confirm if they had contact with persons infected with covid-19 and were also not accurate in recalling the exact duration of the symptoms before hospital admission. the proportion of females (29, 41 .4%) was lower respect to the percentage of males (41, 58.6%). symptomatology of patients at admission was: fever (66, 94.3%), cough (54, 77.1%), dyspnea (44, 62.9%), headache (11, 15.7%), asthenia (15, 21 .4%), myalgia (4, 5.7%), diarrhea (33, 47.1%), while 56 (80.0%), presented comorbidities in a range of 1 to 6. a group of 28 subjects received oral bacteriotherapy (ob+), while another group of 42 individuals not supplemented with oral bacteriotherapy (ob-) was the comparison group. table 1 shows the characteristics of patients at admittance. no statistically significant differences were observed between the ob+ group and the ob-one respect to sex, age, ast, alt, hb, body mass index (bmi), and charlson comorbidity index at baseline. no significant differences between groups were also found for respiratory parameters as well as for the proportion of subjects presenting diarrhea, fever, cough, dyspnea, headache, asthenia, and myalgia. furthermore, all patients had clinical and radiological signs compatible with covid-19 pneumonia and needed respiratory assistance in the hospital setting but not resuscitation support. the two groups of patients were homogeneous respect to the proportion of subjects needing non-invasive oxygen support delivered via venturi mask in spontaneous breathing or by continuous positive airway pressure (cpap). notably, at admittance, a significantly higher proportion of patients with respiratory failure was present in the group treated with oral bacteriotherapy respect to the ob-one (ob-11/42, 26.2%; ob+ 14/28, 50%; p = 0.042). therefore, all enrolled patients were classifiable in stage iii (severe pneumonia-severe covid-19) of the syndromic classification proposed by the italian society of anesthesia and resuscitation (siaarti) (12) . for what concerns drug therapies, both groups did not differ for number, type, and combinations of administered drugs during the period of hospitalization ( table 2 ). the median time from diagnosis to the start of oral bacteriotherapy administration was 1 day (min 0-max 2) and duration of treatment was 14 days for all patients. the oral bacterial administration was associated with the disappearance of diarrhea in all the patients within 7 days. interestingly, a large proportion of ob+ subjects (6/14, 42.9%) solved diarrhea within 24 h and almost the totality (13/14, 92.9%) within 3 days (figure 1a) . also, the other signs and symptoms-fever, asthenia, headache, myalgia, and dyspneaconsidered cumulatively, presented a similar trend, more evident from the second day of bacteriotherapy ( figure 1b) . notably, less than half of patients not treated with bacteriotherapy experienced the disappearance of diarrhea or other symptoms within 7 days. for what concerns the respiratory outcome, by applying the general linear mixed model with the glimmix procedure, we observed a significant difference in the evolution of respiratory outcome between the ob+ group and the ob-one (p < 0.001). after 7 days of treatment, the calculated model showed an 8-fold significantly decreased risk to evolve a respiratory failure, with the need of resuscitation support i.e., in need for prone ventilation or extracorporeal membrane oxygenation (ecmo) for patients administered with bacteriotherapy respect to the ob-individuals (figure 2) . qt interval prolongation, hepatic and renal abnormalities, and immunosuppression were monitored carefully, also for the propensity of the qt interval to increase in patients treated with azithromycin. no adverse events were recorded. patients treated with tocilizumab reported a sense of asthenia after the administration of the drug and a reduction of the blood pressure, which did not require any medical treatment. as of april 4th, 2020, although not statistically significant, the ob-group showed a higher prevalence of patients transferred to the icu for mechanical ventilation (ob-vs. ob+; 2/42, 4.8% vs. 0/28, 0.0%) or coming to a lethal outcome (obvs. ob+; 4/42, 9.5% vs. 0/28, 0.0%). the observed prevalence of patients with lethal outcome within the control group was in line with that (mean ± sd 9.4 ± 1.7%;) recorded in italy in the period march 9th-april 4th, 2020 (13) . all the patients also treated with bacteriotherapy survived the covid-19 illness, and none required invasive mechanical ventilation and icu admission. this report comes from doctors in the "trenches" during the italian war against the covid-19 infection. the urgency of the covid-19 pandemic shifted the balance between waiting for evidence before deciding whether to administer therapy or creating evidence during routine patient care, in favor of the second choice. the united states food and drug administration (fda) has permitted an emergency-use authorization to prescribe the hydroxychloroquine (14) . the who, cdc, and fda have not taken a position on the use of tocilizumab in covid-19, even though china's national health commission recommends it for use in covid-19 patients but only if elevated il-6 levels are present (15) . also, italian clinicians are utilizing a variety of empirical approaches to managing covid-19, in a "learn while doing" method (16) . there is an urgent need to determine which interventions against covid-19 are the best, but in the absence of clinical trials to guide the management, not collecting the data from the use of off-label therapies it is a missed opportunity. here we report a "snapshot" on 70 patients hospitalized at the department of infectious diseases between march 9th and april 4th, 2020. a group of patients has been treated with hydroxychloroquine, tocilizumab, and antibiotics alone or in combination, while, a second group of subjects were administered with oral bacteriotherapy in addition to the standard drug therapy. results evidenced a worse survival, as well as, a higher risk of transfer to an intensive resuscitation for the patient not supplemented with bacteriotherapy respect to the supplemented one. also, the estimated risk to develop respiratory failure during covid-19 course was more than eight times lower in the group treated with oral bacteriotherapy respect to the not treated one. as for the other signs and symptoms associated with covid-19, i.e., diarrhea, fever, cough, dyspnea, asthenia, myalgia a the benjamini hochberg fdr correction was used to account for multiple hypothesis testing. statistical significance between the group at alpha level 0.05 was also reported. figure 2 | analysis of the longitudinal data for the respiration variable in relation to the "ob-vs. ob+" group performed by glimmix. for each time point, the odds ratio, the confidence interval 95% and the statistical significance were reported. significant improvement is already evident as early as after 24-48 h after the start of the bacteriotherapy. there are potential anatomical communications and complex pathways involving the gut-lung axis (gla) (5) . the mesenteric lymphatic system is the pathway between the lungs and the intestine, through which intact bacteria, their fragments or metabolites can cross the intestinal barrier to reach systemic circulation and influence the pulmonary immune response (17) (18) (19) . intestinal metabolites significantly affect not only local intestinal immunity but also other organs through the lymphatic and circulatory system. for example, short chain fatty acids (scfa) produced primarily by bacterial fermentation of dietary fiber, act in the lungs as signaling to attenuate inflammatory and allergic responses (20, 21) . mice with scfa receptor deficiency show increased inflammatory responses in experimental models of asthma (19) . human cells possess antioxidative defense systems for their protection against reactive oxygen species (ros) generated by viruses; however, viral infections often inhibit such a response (22) . there is no reason to believe that that this is not true also for covid-19 (23) . we hypothesized that in patients infected by covid-19, a bacterial formulation with the "appropriate" biochemical and immunological profile might trigger several protective biological functions. the bacterial strains present in the product we administered enhance the production of both the nuclear factor erythroid 2p45-related factor 2 (nrf2) and its target heme oxygenase-1 (ho-1) (24). these molecules exert antiviral activity through a reduction of oxidative stress. nrf2 and ho-1 have significant antiviral activity against a wide variety of viruses, including human immunodeficiency virus (hiv), influenza virus, respiratory syncytial virus, dengue virus, and ebola virus among others (25) (26) (27) (28) (29) . notably, beneficial properties of ho-1 expression have been reported for viruses that produce lung disease. mice that overexpress ho-1 in the lungs display less inflammatory cell infiltration into the lungs and decreased apoptosis of respiratory epithelial cells, as compared to control mice. therefore, ho-1 expression prevents an exacerbated immune response in this tissue and subsequent damage (26) . the collection of clinical data, examination, and nursing of covid-19 patients is challenging for the risk of virus transmission. the covid-19 is present in the stools, even in discharged patients, with potential recurrence and transmission of the virus (30, 31) . our initiative aimed to modulate the gut-lung axis, facilitate patient management and possibly determine the outcome of lung infection. oral bacteriotherapy has shown a statistically significant impact on the clinical conditions of covid-19 patients. having considered the different outcomes and unethical to deprive a percentage of covid-19 patients of the chance to get oral bacteriotherapy, we did not include more patients or extended the time of observation. pending the results of confirmatory clinical trials, this report is aimed at providing an interim suggestion for improving the management of the covid-19 illness, keeping in mind that different bacterial preparations may have quite different outcomes (32). all datasets presented in this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by ethics committee of policlinico umberto i. the patients/participants provided their written informed consent to participate in this study. gd'e, gce, and mm contributed substantially to the conception and design of the study and interpretation and wrote the manuscript. gca, cp, fa, fr, gr, lc, cs, cm, vt, ger, and vm contributed substantially to the the acquisition of data and the analysis. ga, fp, and cmm drafted or provided critical revision of the article and provided final approval of the version to publish. gr contributed substantially to data interpretation. authors wish to thank prof. claudio de simone for suggesting the rationale of the study and the dosage of administered oral bacteriotherapy. we thank all those in the intensive care covid-19 study group and in the infectious diseases covid-19 study group of la sapienza university of rome who assisted in the care of the patients in this program. we express our solidarity with those who are or have been ill with covid-19 the health care workers on the front lines of this pandemic. the small intestine, an underestimated site of sars-cov-2 infection: from red queen effect to probiotics immunodepletion with hypoxemia: a potential high-risk subtype of coronavirus disease 2019. medrxiv protective microbiota: from localized to long-reaching co-immunity. front immunol probiotic effects on cold and influenza-like symptom incidence and duration in children the commensal microbiota and viral infection: a comprehensive review the gut-lung axis in health and respiratory diseases: a place for interorgan and inter-kingdom crosstalks the role of the lung microbiota and the gut-lung axis in respiratory infectious diseases dyspnoea: a multidimensional and multidisciplinary approach acute diarrhea chest ct manifestations of new coronavirus disease 2019 (covid-19): a pictorial review a new method of classifying prognostic comorbidity in longitudinal studies: development and validation percorso assistenziale per il paziente affetto da covid-19 sezione 1 -procedure area critica -versione 02 source: italian civil protection department covid-19: interim guidance on management pending empirical evidence surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) optimizing the trade-off between learning and doing in a pandemic desired turbulence? gut-lung axis, immunity, and lung cancer contributions of the intestinal microbiome in lung immunity gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis microbiome-driven allergic lung inflammation is ameliorated by short-chain fatty acids diet, microbiota and gut-lung connection viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis covid-19: melatonin as a potential adjuvant treatment effects of the probiotic formulation slab51 in in vitro and in vivo parkinson's disease models hemin activation ameliorates hiv-1 infection via heme oxygenase-1 induction adenovirus-mediated transfer of heme oxygenase-1 cdna attenuates severe lung injury induced by the influenza virus in mice heme oxygenase-1 modulates human respiratory syncytial virus replication and lung pathogenesis during infection human heme oxygenase 1 is a potential host cell factor against dengue virus replication the cytoprotective enzyme heme oxygenase-1 suppresses ebola virus replication enteric involvement of severe acute respiratory syndromeassociated coronavirus infection diet supplementation, probiotics, and nutraceuticals in sars-cov-2 infection: a scoping review the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 d'ettorre, ceccarelli, marazzato, campagna, pinacchio, alessandri, ruberto, rossi, celani, scagnolari, mastropietro, trinchieri, recchia, mauro, antonelli, pugliese and mastroianni. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-349226-xzlc1pni authors: khatiwada, saroj; subedi, astha title: lung microbiome and coronavirus disease 2019 (covid-19): possible link and implications date: 2020-08-05 journal: hum microb j doi: 10.1016/j.humic.2020.100073 sha: doc_id: 349226 cord_uid: xzlc1pni coronavirus disease 2019 (covid-19) is a rapidly emerging disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the disease begins as an infection of lungs, which is self-limiting in the majority of infections; however, some develop severe respiratory distress and organ failures. lung microbiome, though neglected previously have received interest recently because of its association with several respiratory diseases and immunity. lung microbiome can modify the risk and consequences of covid-19 disease by activating an innate and adaptive immune response. in this review, we examine the current evidence on covid-19 disease and lung microbiome, and how lung microbiome can affect sars-cov-2 infection and the outcomes of this disease. to date there is no direct evidence from human or animal studies on the role of lung microbiome in modifying covid-19 disease; however, related studies support that microbiome can play an essential role in developing immunity against viral infections. future studies need to be undertaken to find the relationship between lung microbiome and covid-19 disease. the rapid outbreak of the coronavirus disease has crippled global health and economy [1] . as of 21 st july 2020, it has affected 188 countries and infected 14,727,753 people causing death of 610,560 patients [2] . the rapid increase in the number of severely ill patients after covid-19 infection across the globe has resulted in heavy investment in health care infrastructures and medical supplies. to stop the spread of the virus, many countries have imposed a strict international travel ban and put the population into home restriction [3] . the covid-19 disease is caused by a virus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which emerged in wuhan, china at the end of 2019 [4] . the disease is highly transmissible among persons through aerosols, and evidence shows the virus can remain viable for up to 3 days on plastic and stainless steel, all of which facilitates the easy propagation of this virus [5] . the usual symptoms of covid-19 include fever, dry cough, and tiredness. some patients may have aches and pains, nasal congestion, anosmia, sore throat or diarrhea [6] . a large majority of patients may not develop any symptoms after the viral infection, thereby acting as a hidden carrier for this devastating disease [7] . the common complications of covid-19 that lead to death include acute respiratory failure (arf), pneumonia, acute respiratory distress syndrome (ards), acute liver injury, acute cardiac injury, acute kidney injury, septic shock, disseminated intravascular coagulation, blood clots and rhabdomyolysis [8] [9] [10] [11] . the development of pneumonia or acute respiratory illness is consistently observed after covid19 infection with the majority of the patient showing lesions involving bilateral lungs, which is often the common cause of covid-19 death [10, 12, 13] . microbiome refers to the collective genomes of all the microorganisms. it includes every organism (not only bacteria, but also archaea, fungi and viruses), dna and rna species, and 4 proteins that can affect health and development of diseases [14] . microbiota refers to the community of microorganisms, including bacteria, viruses, fungi, and protozoans, that live in a host body [15] . the covid-19 disease begins with the invasion of lungs by sars-cov-2 virus, and the major complications that develop subsequently are related to lung infection and immune response generation, therefore, lung microbiome might play an important role from initiation to the progression of this disease [16] . this review focuses on the potential relationship between covid-19 disease and lung microbiome. we summarized the most important findings on lung microbiome and its role in immunity and lung disease including covid-19. we searched for the keywords (sars-covfor the articles until 20 july 2020. all the original research articles reporting microbiome in covid-19 were included. we highlight the role of the lung microbiome, and how the microbiome can modulate sars-cov-2 attack along with the consequences of viral attack. finally, we seek the potential role of lung microbiome and immunity in severity of covid-19 disease. we also discuss the relevant studies supporting the role of microbiome against viral infection and disease complications. until very recently, the lung was considered to be a sterile site free from bacteria [17] . being an environment with favourable temperature, moisture and mucus, and a large surface area that is in regular contact with the external environment, the lung is indeed a microbiome rich site [18] . lung microbiome is more dynamic and transient than that of the gastrointestinal tract because of bidirectional movement of air and mucus [19, 20] . in a healthy human, the lung microbiota has a low density but harbours a prominent diversity of interacting microbiota. at the phylum level, firmicutes, bacteroidetes and proteobacteria are the most common phyla. at the genus level, prevotella, veillonella and streptococcus are the predominant microorganisms [17, 21] . the composition of lung microbiome is mainly determined by microbial immigration, elimination, and relative growth rates of its members. in lung diseases, these factors can change, and therefore overgrowth of one species with reduction of microbial diversity occurs (shown in figure 1 ) [22] . during lung pathology, drastic changes in the local environment occur, creating a favourable environment for a specific microbial growth. in one of the common lung disease, asthma, bronchoscopy samples have shown an increased abundance of haemophilus, neisseria, fusobacterium, and porphyromonas [23, 24] . recent evidence shows microbiota administration through the oral or intranasal route as probiotics can lower allergic inflammation seen in asthma, highlighting the intricate relationship between microbiota and asthma [25] . in another common lung disease, chronic obstructive pulmonary disease (copd), lactobacillus, fusobacteria, leptotrichia and fusobacterium were observed in abundant numbers [26, 27] . the most abundant bacteria seen in cystic fibrosis were pseudomonas, staphylococcus, stenotrophomonas, achromobacter, and streptococcus [28, 29] . recent evidence shows that lung microbiome is altered in lung cancer, and the microbiome can influence cancer development and progression [30, 31] . emerging evidence shows the relationship between lung microbiota and susceptibility to pneumonia [32] . pneumonia is a widespread lung disease characterised by infection of lung alveoli leading to an infiltration of inflammatory cells and fluids into the alveoli. this disease can be caused by bacteria, virus, fungi or protozoans [33] . streptococcus pneumoniae and haemophilus influenzae type b are the most common bacterial causative agents, whereas, 6 respiratory syncytial virus (rsv) is the most common viral agent for pneumonia [34, 35] . the endotracheal aspirates of patients with ventilator associated pneumonia had a higher bacterial load but a lower abundance of bacterial class, bacilli, than matched controls [36] . similarly, the microbiota profile of endotracheal aspirates was different between the mechanically ventilated pneumonia group and the non-pneumonia group. in the intubated patients with pneumonia, pseudomonas, corynebacterium, and rothia were more abundant, while streptococcus and prevotella were less abundant as compared to those without pneumonia [37] . the abundance of firmicutes phyla was reduced in the patients with interstitial pneumonia including the overall phyla richness as compared to healthy group. the abundance of streptococcus was significantly reduced in interstitial pneumonia patients, while prevotella and veillonella were enriched in comparison to healthy volunteers [38] . in addition, the microbial diversity and composition tended to differ according to the causative agent of pneumonia, which suggests that lung microbiome can vary according to the type of pneumonia [39] . overall, pneumonia is characterized by a shift in lung microbiome, with domination by pneumonia etiologic agent, high microbial biomass, and lowering of microbial diversity [33] . the composition and diversity of upper respiratory tract and lung microbiome have been found to shift in response to respiratory viral infections in humans and animals [40] [41] [42] . some of the common viruses invading respiratory tract and lungs include rhinovirus, influenza virus, adenovirus, parainfluenza virus and rsv. these respiratory viral infections are quite common during childhood, and they often increase susceptibility to secondary bacterial infection of lungs [41] . the upper respiratory tract microbiome of rhinovirus and rsv infected infants was found to be largely dominated by moraxella, streptococcus, corynebacterium, haemophilus, and dolosigranulum [43] . in another study, rhinovirus infection was found to increase the relative abundance of haemophilus and neisseria, both of which are associated with secondary lung infections [44] . in mice, influenza virus infection of lungs caused a shift in the 7 composition and diversity of microbial community. the viral infection changed the dominant bacterial class from alphaproteobacteria to gammaproteobacteria and actinobacteria, and this was followed by an increase in the relative abundance of streptococcus and staphylococcus [45] . in summary, the lung microbiome is highly dynamic, and infections (bacterial, viral) can lead to rapid alterations in the lung microbiome, and the microbial communities can play a critical role in those infections (shown in figure 1 ). the alveolar surface is the largest surface area in direct contact to the external environment, so it is constantly exposed to invading microorganisms. therefore, it harbours several lines of defence against potential infections. a continuous layer of pulmonary epithelial cells constitutes a physical and biological barrier for inhaled substances and pathogens. the mucus coating of the pulmonary epithelium, proteolytic enzymes, defence proteins (immunoglobulins, 8 lactoferrin, defensins) and lysozymes present on the surface and in the fluids around alveoli protect against infection [18] . pulmonary epithelial cells are in close contact with cells of the immune system, and they also secrete a wide range of cytokines and chemokines. they express cell surface receptors called pattern-recognition receptors (prrs), such as the toll-like receptors (tlrs), which enable them to recognize pathogen-associated molecular patterns (pamps) from viruses, bacteria, fungi, protozoa, and multicellular parasites [46] . apart from the above mentioned immune mechanisms, lung contains immune cells such as dendritic cells, macrophages (alveolar and interstitial), lymphocytes (t-cells, γδ t cells, nk cells, b-cells), innate lymphoid cells and neutrophils that support innate and adaptive immune mechanism [47]. all these immune cells are involved in eliminating antigens and pathogens from the respiratory tract through phagocytosis or activation of effectors molecules that remove antigens. regulatory t cells resident within the lungs are vital for the maintenance of immune tolerance to airborne particles, and they are directly influenced by local microbiota [46] . another type of t cells, called lung resident memory γδ t cells provide a rapid immune response at barrier surfaces to recall antigens previously encountered through lung mucosa, which is apart from its role in cytokine and interferon production to stop viral and bacterial infection [48] . despite the existing immunity mechanisms, lung infection is unavoidable. in the next section, we will discuss how sars-cov-2 initiates lung infection and escape the immune response. in the respiratory tract, sars-cov-2 bind to the specific host receptors called angiotensinconverting enzyme 2 (ace2). this virus has an envelope-anchored spike protein that facilitates virus entry into host cells by first binding to ace2. the virus then enters the host cell through endocytosis [49] . inside the cells, the viral genome replicates and makes necessary proteins, 9 which assemble to form new virions. the newly released virions can invade other lung cells [4, 50] . the stress of viral production will lead to cell death, which in turn will trigger a series of immune responses and initiate inflammation reflected as lymphopenia, elevated c reactive protein (crp) and erythrocyte sedimentation rate [13] . the viral rnas and pamps are detected by the prrs, such as tlrs inside the cells [51] . the recognition will lead to the activation of the transcription factor nuclear factor-κb (nf-κb) and interferon regulatory factor 3 (irf3) to induce proinflammatory cytokines such as interferonα and tumour necrosis factor-beta (tnf-β). the production of type i interferons (ifn) will cause suppression of viral multiplication and prevent the severity of disease [52] . increase in interleukin-6 (il-6), crp, and chemokines such as interferon-γ-inducible protein 10 (ip-10), monocyte chemoattractant protein 1 (mcp-1), macrophage inflammatory protein 1-alpha (mip-1a), and tumour necrosis factor-alpha (tnf-α) were observed in covid-19 patients [10, 53] . if the body immune response cannot stop the sars-cov-2 multiplication, then a massive degree of lung injury occurs. this degree of damage can generate a substantial immune response from immune effector cells which is characterized by a large amount of cytokines (ifn-α, ifn-γ, il-1β, il-6, il-12, il-18, il-33, and tnf-α) and chemokines (ccl2, ccl3, ccl5, cxcl8, cxcl9, and cxcl10) in the body [54] . this condition of hyperinflammation manifested as 'cytokine storm' is called as covid-19 ards. this is one of the most dangerous and potentially life-threatening events related to covid-19 disease [55] . besides, sars-cov-2 infection damages the pulmonary lining altering the composition of normal microbiota, and can promote the growth of specific bacteria to initiate secondary pneumonia [41] . the formation of double-membrane vesicles that lack prrs, where the virus replicates, leading to failure of recognition by immune cells and therefore avoid host detection [54] . another feature of this virus, eight surface proteins, can block the action of interferon i, causing the cells to fail to stop viral multiplication [56] . the virus has also developed a strategy to stop host cell recognition by mimicking host cell capping machinery [52] . the sars-cov-2 viral infection occurs amid the local environment of diverse microbiota; therefore, it is apparent that lung microbiota can have an impact on the initiation, development, and progression of the covid-19 disease. lungs are at the frontline of immunity as they are constantly exposed to a wide variety of external environment. the microbiome has a principal role in shaping pulmonary immunity, and healthy lung has a vast array of microbiota. microbes boost innate and adaptative immunity (site-specific in lungs and systemic), release factors which assist respiratory functions, and prevent an invasion of lungs from pathogens [57] . the normal microbiota restricts the growth of harmful pathogens that may make their way into the lungs. this growth restriction may occur by several mechanisms, such as, limiting nutrients access and secreting growth inhibitors [58] . activation of lung immune cells that initiates innate and adaptive immunity requires microbe exposure [59] . the function of one of the vital effector and regulator cells of the lung, γδ t cells, depends on pathogen invasion [48, 60] . evidence shows that exposure to certain bacterial strains in neonate protects against excessive airway inflammation through the alteration of immune cells [61] . furthermore, microbial exposure is required for developing innate immunity, as children exposed to the microbial rich environment had a lower rate of asthma and allergic sensitization, and stronger immunity [62] . one of the products of lung's normal flora showed an allergo-protective effect in the animal model of airway inflammation [63] . in summary, previous studies highlight that lung immunity development requires exposure to diverse microbes (shown in figure 1 ). [67] . moreover, this study showed the severity of covid-19 disease is directly correlated with the predominance of opportunistic pathogens (clostridium ramosum and clostridium hathewayi) and inversely with the predominance of beneficial commensals (alistipes onderdonkii and bacteroides ovatus) [67] . similarly, covid-19 patients had increased proportions of opportunistic fungal pathogens, candida albicans, candida auris, and aspergillus flavus as compared to controls [66] . in summary, the role of gut microbiome against sars-cov-2 infection and disease severity is rapidly emerging, and therefore gut microbiota may be developed as therapeutics for the management of covid-19 disease in future. till date, only two studies have analysed the lung microbiome of covid-19 patients [64, 65] . in one of the study, bronchoalveolar lavage fluid from covid-19 patients (n=8), communityacquired pneumonia patients (n=25) and healthy controls (n=20) was investigated, and a significant difference in microbiota composition was observed [65] . both covid-19 and community-acquired pneumonia patients had enrichment of pathogenic and commensal bacteria, indicating a degree of microbial dysbiosis in both diseased states [65] . in the other study, the lung post-mortem biopsies from 20 deceased covid-19 patients were used to study lung microbiome. the most common bacterial genera were acinetobacter, chryseobacterium, burkholderia, brevundimonas, sphingobium, and enterobacteriaceae. the most common fungal genera were cutaneotrichosporon, followed by issatchenkia, wallemia, cladosporium, alternaria, dipodascus, mortierella, aspergillus, naganishia, diutina, and candida [64] . therefore, a mix of bacterial and fungal infection was seen in the covid-19 patients of this study. overall, there is very little information about the composition of lung microbiome in covid-19 patient. further studies with bigger sample size are required to know more precisely the composition and the role of lung microbiome in the severity of sars-cov-2 infections. although there is no direct evidence if lung microbiome can affect potential sars-cov-2 infections and the outcomes of this disease, yet several lines of evidence support that bacteria in lungs have important roles in this disease. firstly, microbiota dwelling on the respiratory surface can acts as a barrier, thereby preventing viral attachment to host cells. secondly, microbiota prime the lung immunity, which will fight against viral infection, and exposure to 13 a diverse range of microbiota may build a wider immunity. such phenomenon is particularly common in the gut, where gut microbiota can protect against potential flu viral infections [68] . in mice, the nasal application of a respiratory normal flora, corynebacterium pseudodiphtheriticum, increased the tlr3 antiviral response against rsv and enhanced the production of tnfα, il-6, ifnγ, and ifnβ [69] . another respiratory tract flora, staphylococcus aureus, protected against influenza induced lung injury [70] . recent evidence of the association between covid-19 disease severity with the gut microbiota seems highly promising [67] . thus, it appears that lung microbiome may modify propensity to sars-cov viral infection. till date, it is unknown if lung microbiota can modify the risk of developing severe respiratory complications such as ards after sars-cov-2 infection. the dysbiosis of lung microbiome may contribute to covid-19 ards because microbial dysbiosis is found to provoke a dysregulated immune response leading to inflammation [22, 55, 71] . in the ards unrelated to covid-19 disease, lung microbiome is found to be enriched with gut-associated bacteria, which demonstrates the relationship between lung microbiome and ards [72] . recently, the altered microbiome (increase in bacterial burden and a decrease in alpha diversity) was shown to be associated with inflammation and mortality in ards patients. the change in microbiota population (a reduction in betaproteobacteria and an increase in staphylococcus, streptococcus and enterobacteriaceae) was significantly associated with serum cytokine il-6 [73] . lung microbiome seems to have a critical role in severely ill patients because a recent study found that lung microbiota predicts the clinical outcome and death in those patients [74] . this study showed that increased lung bacterial burden and lung enrichment with gut-associated bacteria were predictive of adverse consequences of ards. thus, in covid-19 ards, the microbiome may have a significant role in determining the severity of the disease, and the outcomes of ards. in the future, studies will find if the presence of a specific bacterial population is associated with low risk for sars-cov-2 infection and ards development. based on the current evidence, it appears that immunity is at the forefront for protection against covid-19 disease. immunity development is a continuous process and requires exposure to a range of microbiota and environments [75] . exposure of human population to diverse air microbes that may occur in unhygienic environment is believed to develop more robust immunity, and this may partly account for the variation in the covid-19 death rate among countries [76] . several other factors such as the method of delivery at birth, air microbiota of the immediate environment, prenatal and childhood exposures to microbes, respiratory viral illness, indoor and outdoor pollution can have a critical influence in the microbiome composition and immunity development in children [46] . exposure to air with high air microbial load is found to reduce asthma risk in children [77] . in many developed countries, over the years, there has been a change in diet, sanitary conditions, antibiotics use, exposure to environmental chemicals and the change in the living environment, all of which may have ultimately reduced the exposure to diverse microbes. this factor is believed to be one of the principal factors for the current rise in the number of autoimmune lung diseases, and weak immunity [32, 75] . till date little is known about the role of lung microbiome in covid-19 disease. future studies are highly warranted to solve the complex relationship between lung immunity, lung microbiome and sars-cov-2 infection along with this disease complications. because of the therapeutic benefits of microbiota as probiotics, future studies should also focus on the potential benefits and applicability of microbiota transplantation for the covid-19 disease. we also emphasize collecting bronchoalveolar samples from the covid-19 infected patients and perform 16s rrna gene sequencing in a large sample size. we conclude that lung microbiome can have a profound impact on the susceptibility to sars who. coronavirus disease (covid-2019) situation reports 2020 wuhan coronavirus (2019-ncov) global cases the socio-economic implications of the coronavirus and covid-19 pandemic: a review covid-19 infection: origin, transmission, and characteristics of human coronaviruses aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 who. q&a on coronaviruses (covid-19) 2020 covid-19: four fifths of cases are asymptomatic, china figures indicate coronavirus disease 2019 coronavirus disease 2019: retrospective study clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiology and clinical features of covid-19: a review of current literature clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study. the lancet infectious diseases clinical characteristics of 3,062 covid-19 patients: a meta-analysis the lung microbiome in health and disease the role of the lung microbiota and the gut-lung axis in respiratory infectious diseases covid-19 pathophysiology: a review the microbiome and the respiratory tract respiratory microbiome and epithelial interactions shape immunity in the lungs the respiratory tract microbiome and lung inflammation: a two-way street the lung microbiome: new principles for respiratory bacteriology in health and disease spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography the role of the microbiome in exacerbations of chronic lung diseases features of the bronchial bacterial microbiome associated with atopy, asthma, and responsiveness to inhaled corticosteroid treatment disordered microbial communities in asthmatic airways probiotics against airway allergy: host factors to consider the lung microbiome in moderate and severe chronic obstructive pulmonary disease the lung tissue microbiome in chronic obstructive pulmonary disease sputum dna sequencing in cystic fibrosis: non-invasive access to the lung microbiome and to pathogen details the lower airway microbiota in early cystic fibrosis lung disease: a longitudinal analysis interaction between the microbiome and tp53 in human lung cancer characterization of microbiome in bronchoalveolar lavage fluid of patients with lung cancer comparing with benign mass like lesions the lung microbiome and its role in pneumonia integrative physiology of pneumonia comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia identification of respiratory microbiota markers in ventilator-associated pneumonia the dynamics of respiratory microbiota during mechanical ventilation in patients with pneumonia alveolar microbiota profile in patients with human pulmonary tuberculosis and interstitial pneumonia streptococcus pneumoniae caused different microbial structure and correlation network in lung microbiota differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia patients with coronavirus disease 2019 or h1n1 influenza. clinical infectious diseases differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy changes in microbiota during experimental human rhinovirus infection dynamic changes in the microbiome and mucosal immune microenvironment of the lower respiratory tract by influenza virus infection lung homeostasis: influence of age, microbes, and the immune system lung defences: an overview lung-resident gammadelta t cells and their roles in lung diseases receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment coronavirus infections and immune responses severe acute respiratory syndrome coronavirus an overview of viral structure and host response epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study molecular immune pathogenesis and diagnosis of covid-19 the cytokine storm in covid-19: an overview of the involvement of the chemokine/chemokine-receptor system sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. current opinion in virology the influence of lung microbiota on lung carcinogenesis, immunity, and immunotherapy innate immunity in the lungs the lung microbiota: role in maintaining pulmonary immune homeostasis and its implications in cancer development and therapy lung microbiota promotes tolerance to allergens in neonates via pd-l1 innate immunity and asthma risk in amish and hutterite farm children farm-derived grampositive bacterium staphylococcus sciuri w620 prevents asthma phenotype in hdm-and ova-exposed mice the lung tissue microbiota features of 20 deceased patients with covid-19 genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease alterations in fecal fungal microbiome of patients with covid-19 during time of hospitalization until discharge alterations in gut microbiota of patients with covid-19 during time of hospitalization tonic interferon signals in lung stromal cells protect from influenza virus infection respiratory commensal bacteria corynebacterium pseudodiphtheriticum improves resistance of infant mice to respiratory syncytial virus and streptococcus pneumoniae superinfection bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages the impact of lung microbiota dysbiosis on inflammation enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome unique patterns of lower respiratory tract microbiota are associated with inflammation and hospital mortality in acute respiratory distress syndrome lung microbiota predict clinical outcomes in critically ill patients role of the microbiota in immunity and inflammation hay fever, hygiene, and household size exposure to environmental microorganisms and childhood asthma we, the authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work not applicable key: cord-329066-9xo5zztv authors: yuan, kai; feng, yanyan; wang, hesong; zhao, lu; wang, wei; wang, ting; feng, yuyin; huang, guangrui; xu, anlong title: fgl2 is positively correlated with enhanced antitumor responses mediated by t cells in lung adenocarcinoma date: 2020-03-13 journal: peerj doi: 10.7717/peerj.8654 sha: doc_id: 329066 cord_uid: 9xo5zztv lung cancer is the most common malignant tumor, accounting for 25% of cancer-related deaths and 14% of new cancers worldwide. lung adenocarcinoma is the most common type of pulmonary cancer. although there have been some improvements in the traditional therapy of lung cancer, the outcome and prognosis of patients remain poor. lung cancer is the leading cause of cancer-related deaths worldwide, with 1.8 million new cases being diagnosed each year. precision medicine based on genetic alterations is considered a new strategy of lung cancer treatment that requires highly specific biomarkers for precision diagnosis and treatment. fibrinogen-like protein 2 (fgl2) plays important roles in both innate and adaptive immunity. however, the diagnostic value of fgl2 in lung cancer is largely unknown. in this study, we systematically investigated the expression profile and potential functions of fgl2 in lung adenocarcinoma. we used the tcga and oncomine datasets to compare the fgl2 expression levels between lung adenocarcinoma and adjacent normal tissues. we utilized the gepia, prognoscan and kaplan-meier plotter databases to analyze the relationship between fgl2 expression and the survival of lung adenocarcinoma patients. then, we investigated the potential roles of fgl2 in lung adenocarcinoma with the timer database and functional enrichment analyses. we found that fgl2 expression was significantly lower in lung adenocarcinoma tissue compared with adjacent normal tissue. a high expression level of fgl2 was correlated with better prognostic outcomes of lung adenocarcinoma patients, including overall survival and progression-free survival. fgl2 was positively correlated with the infiltration of immune cells, including dendritic cells, cd8(+) t cells, macrophages, b cells, and cd4(+) t cells, in lung adenocarcinoma. functional enrichment analyses also showed that a high expression level of fgl2 was positively correlated with enhanced t cell activities, especially cd8(+) t cell activation. thus, we propose that high fgl2 expression, which is positively associated with enhanced antitumor activities mediated by t cells, is a beneficial marker for lung adenocarcinoma treatment outcomes. lung cancer is a malignant lung tumor with unrestrained cell proliferation in the lung (schwartz & cote, 2016) . as a highly prevalent and invasive disorder in women and men, more than 1.8 million people are diagnosed with lung cancer every year (torre, siegel & jemal, 2016) . lung cancer accounts for almost 25% of cancer-related deaths and 14% of new cancers worldwide. although there have been some improvements in the therapy and diagnosis of lung cancer, the outcome and prognosis of patients remain poor. the 5-year survival rate varies from 4-17% depending on regional and stage differences. almost 1.6 million people die from lung cancer yearly. the lack of effective first-line drugs, the nonoptimal administration route and the formation of resistant tumors might be correlated with the poor prognostic survival of lung cancer patients (pilkington et al., 2015) . lung cancer is generally classified into two kinds of histopathology groups: non-small cell lung cancer and small cell lung cancer (latimer, 2018) . as the most common type of lung cancer, lung adenocarcinoma belongs to the non-small cell lung cancer group. fifty percent of non-small cell lung cancer patients have a lung adenocarcinoma. it is also the most common form of lung cancer in asian countries. the symptoms and signs of lung adenocarcinoma are similar to those of other types of lung cancer (zappa & mousa, 2016) . shortness of breath and persistent cough are the main symptoms of lung adenocarcinoma patients. the category of lung adenocarcinoma includes a variety of subtypes, such as acinar predominant adenocarcinoma, lepidic predominant adenocarcinoma, and micropapillary predominant adenocarcinoma. radiotherapy, chemotherapy, surgical resection, and immunotherapy are common therapies employed to treat lung adenocarcinoma. however, because of the difficulties in diagnosing early lung adenocarcinoma, the average five-year survival rate is only approximately 18% (hirsch et al., 2017) . fibrinogen-like protein 2 (fgl2) plays important roles in both innate and adaptive immunity (liu, liu & chen, 2017) . the fgl2 protein is located on the surface of endothelial cells and macrophages (yang & hooper, 2013) . cd8 + and cd4 + t cells constitutively secrete fgl2 protein to induce an inflammatory response. several disorders, including severe acute respiratory syndrome (sars), abortion and allograft rejection, are correlated with fgl2 (hsieh et al., 2010; zhao et al., 2013) . in the area of cancer research, previous studies have found that altered fgl2 gene expression contributes to immune surveillance evasion in murine renal carcinoma (birkhäuser et al., 2013) . moreover, fgl2 contributes to glioblastoma multiforme (gbm) progression by stimulating immunosuppression mechanisms (yan et al., 2015) . however, the diagnostic value of fgl2 in lung cancer is largely unknown. in this study, we systematically explored the potential roles of fgl2 in lung adenocarcinoma. data downloaded from the tcga dataset and pnas were used to compare the fgl2 expression levels between lung adenocarcinoma and adjacent normal tissues. three bioinformatics databases, including gepia, prognoscan and kaplan-meier plotter, were adopted to analyze the relationship of fgl2 expression and the survival of lung adenocarcinoma patients. the timer database was used to discover the association between the immune status and fgl2 expression in lung adenocarcinoma. functional enrichment analyses, including gene ontology (go), kyoto encyclopedia of genes and genomes (kegg) pathway and gsea, were used to explore the potential functions of fgl2 in lung adenocarcinoma development. the normalized fpkm (fragments per kilobase per million mapped reads) values were downloaded from the cancer genome atlas (tcga) data portal (https://portal.gdc. cancer.gov). normalized rna-seq datasets were used as input. microarray mrna data of lung adenocarcinoma were downloaded from proc. natl. acad. sci. usa (pnas) (https://www.pnas.org/) (bhattacharjee et al., 2001) and the geo database (gse32863). the microarray data were log2 transformed. fgl2 expression was compared between lung cancer and normal adjacent tissues. statistical significance was calculated with spss 20.0. detailed information of included patients are listed in table s1 . the gepia, prognoscan and kaplan-meier plotter databases were used to evaluate the prognostic potential of fgl2 in lung adenocarcinoma. the gepia (gene expression profiling interactive analysis) database is a new web server (http://gepia.cancer-pku.cn/) for cancer and normal gene expression profiling and interactive analyses. gsea was first introduced at 2003. some concerns appeared immediately after gsea was proposed (tamayo et al., 2016) . the concerns or limitations were list as follows: the null distribution of gsea is superfluous and very hard to be worth calculating. the kolmogorov-smirnov-like statistic is not as sensitive as original. the results of gsea are dependent on the algorithm clusters the genes, and the number of clusters being analyzed. the prognoscan database is a new database (http://dna00.bio.kyutech.ac.jp/prognoscan/) used to explore the relation between patient prognosis and gene expression with large collections of tumor microarray datasets. it is a useful platform to evaluate potential tumor markers in cancer research. the kaplan-meier plotter database (http://kmplot.com/analysis/) is a useful online tool used to assess the effects of specific genes on cancer prognosis and can estimate survival from lifetime data. detailed information of included patients are listed in table s1 . the timer database is a useful web tool that can be used to conduct a comprehensive analysis of tumor-infiltrating immune cells. this tool can evaluate the relationship between the immune status and fgl2 mrna expression in the lung adenocarcinoma microenvironment. the immune status includes inflammatory cells and the immune gene marker sets of immune cells. the timer database was used to measure the correlation between fgl2 mrna expression and the infiltration of immune cells, including b cells, cdt cells, cd4 + cells, macrophages and dendritic cells. furthermore, the timer database was used to measure the correlation between fgl2 mrna expression and the immune gene marker sets of immune cells. gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) analyses were used to analyze the potential function of fgl2 with the database for annotation, visualization and integrated discovery (david). go analysis is a powerful bioinformatics tool used to determine biological processes (describing the physiological or cellular role carried out by the fgl2), cellular components (cc) and molecular functions (mf). related data were downloaded from the bioinformatics database tcga data portal and used to compare the mrna expression level of fgl2 in lung adenocarcinoma and normal adjacent tissues. the results showed that the mean fpkm value of fgl2 in lung adenocarcinoma was 3.266, which was significantly lower than that in normal adjacent tissue (3.958, p < 0.001) (fig. 1a) . microarray data from pnas and the geo also showed that the fgl2 mrna expression level was lower in lung adenocarcinoma tissue than in normal adjacent tissue (figs. 1b-1c). we used three databases (prognoscan, gepia, kaplan-meier plotter) to analyze the prognostic value of fgl2 in lung adenocarcinoma. in the gepia database, high fgl2 expression was correlated with better overall survival (os) in lung cancer (os hr = 0.61, log-rank p = 0.0016, cutoff-high = 50%) ( fig. 2a ). in the prognoscan database, an analysis of the cohort gse13213 showed that a high fgl2 mrna level was related to better overall survival in lung adenocarcinoma (os hr = 0.68, 95% ci = 0.48 to 0.96, cox p = 0.029471) (fig. 2b ). in the kaplan-meier plotter database, high fgl2 mrna expression was correlated with better overall survival and progression-free survival in lung we used the timer database to explore the correlation between immune cell infiltration and fgl2 expression in lung adenocarcinoma. as shown in fig. 3a , the fgl2 expression level was positively correlated with b cell infiltration (r = 0.409, p = 5.79e−21), cd8 + t cell infiltration (r = 0.539, p = 1.37e−47), cd4 + t cell infiltration (r = 0.379, p = 1.97e−17), macrophage infiltration (r = 0.540, p = 3.87e−38) and dendritic cell infiltration (r = 0.718, p = 1.44e−78) in lung adenocarcinoma. then, we further investigated the correlation between the fgl2 mrna level and the subtypes of immune cell infiltration with gsea ( fig. 3b ). fgl2 expression was positively correlated with the infiltration of effector memory cd8 + t cells, activated cd8 + t cells, activated cd4 + t cells, type 1 t helper cells, effector memory cd4 + t cells, central memory cd8 + t cells, immature dendritic cells and natural killer t cells. to validate the relationship between fgl2 expression and immune cell infiltration, we also investigated the correlation between the immune marker sets of immune cells and fgl2 expression in lung adenocarcinoma with the timer and gepia databases. as shown in table 1 , data from the timer database indicated that fgl2 expression was positively correlated with most of the immune marker sets. for example, fgl2 was positively corelated with the t cell gene markers cd3d (cor = 0.532, p = 1.97e−37), cd3e (cor = 0.583, p = 3.25e−46), and cd2 (cor = 0.646, p = 1.59e−59); cd8 + t cell data from the gepia database also showed similar results to that of the timer database. detailed information is listed in table 2 . we performed go and kegg pathway analyses with data obtained from the tcga dataset. go analysis indicated that fgl2-correlated genes were enriched in the immune response, the adaptive immune response, the positive regulation of t cell proliferation, the positive regulation of interferon-gamma production, the positive regulation of tumor necrosis factor production, t cell activation, the interferon-gamma-mediated signaling pathway, t cell costimulation, t cell differentiation, the t cell receptor signaling pathway, antigen processing and presentation of exogenous peptide antigen via mhc class i, tap-dependent, etc (fig. 4a) . kegg pathway analysis showed that fgl2 was correlated with genes involved in cell adhesion, natural killer cell-mediated cytotoxicity, the tnf signaling pathway, the t cell receptor signaling pathway, antigen processing and presentation, etc. (fig. 4b) . we also conducted gsea to explore the biological functions of fgl2 in lung adenocarcinoma. fgl2 expression was positively related to the inflammatory response, the interferon alpha response, the interferon gamma response, tnf alpha signaling via nf-κb, complement, and apoptosis (fig. 4c ). lung adenocarcinoma is a type of non-small cell lung cancer and the most common histologic type of lung cancer. in lung adenocarcinoma, there is a variety of divergent molecular, pathologic, and clinical spectra (testa, castelli & pelosi, 2018) . the common signs of lung adenocarcinoma include weight loss, dyspnea, chest pain, and cough. the extrapulmonary manifestations include hypercalcemia of the malignancy and hypertrophic pulmonary osteoarthropathy. tobacco smoking is a risk factor for lung adenocarcinoma (song et al., 2017) . in addition to smoking, gene mutations are also important mutagenic factors of lung adenocarcinoma (ding et al., 2008) . somatic mutations might influence tumor suppressor genes and oncogenes in lung adenocarcinoma. recent opinions on the treatment of lung adenocarcinoma have changed from traditional chemotherapy to precision medicine based on the genetic alterations of cancer (herbst, previous studies have found that many gene mutations contribute to lung adenocarcinoma. the mutated genes include egfr, kras, tp53, stk11, nf1, and keap1. the egfr-activating mutation frequency varies depending on ethnicity and region. egfr is correlated with the cell proliferation, invasion, survival, and angiogenesis of tumors (sharma et al., 2007) . oral egfr tyrosine kinase inhibitors such as gefitinib and erlotinib prolonged progression-free survival and the objective response rate compared with traditional chemotherapy (mok et al., 2009; shepherd et al., 2005) . the inactivation of keap1 in kras mutations is related to the inhibition of glutaminase in lung cancer (romero et al., 2017) . tp53 mutations are commonly found in advanced-grade lung adenocarcinoma (ahrendt et al., 2003) . precision medicine also provides genes that are beneficial for the progression and survival of cancers. fgl2 is an important factor in regulating the immune system. fgl2 is upregulated in gbm, promoting gbm development by suppressing dendritic cell activities (yan et al., 2015) . however, the diagnostic value of fgl2 in lung cancer is largely unknown. in this study, we systematically investigated the expression profile and potential functions of fgl2 in lung adenocarcinoma. first, we evaluated the expression of fgl2 in lung adenocarcinoma tissue and adjacent normal tissue. data obtained from the tcga dataset and the oncomine database indicated that fgl2 expression was significantly lower in lung adenocarcinoma tissue than in adjacent normal tissue. this implied that fgl2 might be a beneficial biomarker of lung adenocarcinoma. second, we analyzed the relationship of the fgl2 level and the prognostic survival of lung adenocarcinoma patients with three bioinformatics datasets. a high fgl2 mrna level was correlated with better prognostic outcomes of lung adenocarcinoma, including overall survival and progression-free survival. these results indicate that fgl2 might negatively regulate the progression of lung adenocarcinoma. then, we explored the correlation between fgl2 expression and the immune status in the tumor microenvironment of lung adenocarcinoma. fgl2 expression was positively correlated with immune cell infiltration and immune marker sets in lung adenocarcinoma. the immune cells included cd8 + t cells, cd4 + t cells, macrophages, b cells and dendritic cells. cd8 + t cells (often called cytotoxic t lymphocytes or ctls) are very important for tumor surveillance. cd8 + t cells use three major mechanisms to kill tumor cells. the first is the secretion of cytokines (primarily tnf-α and ifn-γ). our results showed that high fgl2 expression was positively correlated with ifn-γ production and signaling and tnf-α production and signaling. the second major function is the production and release of cytotoxic granules, which mainly contain perforin 1 (prf1) and granzymes. our results showed that the expression level of fgl2 was positively correlated with the expression levels of prf1, granzyme k (gzmk ), gzma, gzmh, gzmb, and gzmm. the third antitumor function of cd8 + t cells is to induce the apoptosis of tumor cells via fas/fas ligand (faslg) interactions. our results showed that the expression level of fgl2 was positively correlated with the expression levels of fas and faslg. these results show that high fgl2 expression is closely related to enhanced cd8 + t cell-mediated antitumor activities. dendritic cells (dcs) are considered important factors that provide protective immunity against lung adenocarcinoma (wang, huang & li, 2019) . inactive dcs are correlated with the poor prognosis of lung cancer patients. dcs present antigens to activate antitumor t cells. mature dcs in lung cancer express high levels of cytokines and costimulatory molecules (cd40/80/86) to activate t cells (macri et al., 2018) . mhc type ii molecules on dcs promote the activation of cd4 + t cells. mhc type 1 molecules on dcs promote the activation of cd8 + t cells (liu & cao, 2015) . except for activating t cells, dcs can also recruit and activate nk cells by secreting c-c chemokine receptor type 5 (ccr5) at the tumor site (liu et al., 2008) . in our study, we found that fgl2 expression was positively corelated to the infiltration of cd4 + t cells, cd8 + t cells, macrophages, b cells and dcs in lung adenocarcinoma. in addition, the fgl2 level was positively correlated with several subtypes of immune cells, including effector memory cd8 + t cells, activated cd8 + t cells, activated cd4 + t cells, type 1 t helper cells, effector memory cd4 + t cells, central memory cd8 + t cells, immature dendritic cells and natural killer t cells. these results indicate that fgl2 plays an important role in antitumor immunity by enhancing antitumor activities in lung adenocarcinoma. there were some low correlation values for certain gene markers assayed to ascertain correlation between immune markers and fgl2 expression. as for b cells, table 1 indicated the correlation of b cells gene markers varied from 0.21-0.316. in table 2 , the correlation of b cells gene markers varied from 0.24-0.29. in addition, fig. 3a indicated the correlation between fgl2 and b cells was 0.409. the results showed that fgl2 has not possessed strong correlation with b cells in the tumor microenvironment of lung adenocarcinoma. tumor infiltrating b cells appear in every stage of lung cancer and play critical roles in shaping tumor progression. however, the b cells functions in antitumor immunity of lung cancer are controversial (wang et al., 2019) . some studies demonstrated that tumor infiltrating b cells have protective effects on anti-tumor immunity in lung cancer, while other studies revealed that tumor infiltrating b cells have inhibitory effects on antitumor immunity in lung cancer. owning to the b cells functions in antitumor immunity is dualistic, fgl2 might possessed poor or moderate correlation with b cells in the tumor microenvironment of lung adenocarcinoma. apart from b cells, fig. 3a showed correlation between fgl2 and cd4 + t cells was 0.373. this result indicated that the correlation between fgl2 and cd4 + t cells was moderate at best. previous studies found that the role of cd4 + t cells in antitumor activity of lung cancer is dualistic (zheng, hu & yao, 2017) . some types of cd4 + t cells impair the functions of cytotoxic t lymphocytes to promote the tumor development, while some types of cd4 + t cells induce the activation of cytotoxic t lymphocytes to exert antitumor immune response. this might explain the reason that correlation between fgl2 and cd4 + t cells was poor and moderate in lung adenocarcinoma. finally, we performed functional enrichment analysis to explore the biological functions of fgl2 in lung adenocarcinoma. go and kegg analyses indicated that fgl2-correlated genes are mainly enriched in pathways involved in t cell proliferation/differentiation/activation and antigen processing and presentation. gsea showed that fgl2 expression was positively correlated with enhanced tumor killing. these results further indicate that fgl2 enhances antitumor activities in lung adenocarcinoma. in the research conducted by zhu et al. titled ''stroma-derived fibrinogen-like protein 2 activates cancer-associated fibroblasts to promote tumor growth in lung cancer'', they found that fgl2 could promote tumor growth of lung cancer by activating cancer-associated fibroblasts using a mouse model of lewis lung carcinoma (zhu et al., 2017) . in the research conducted by yan et al. (2019) titled ''fgl2 promotes tumor progression in the cns by suppressing cd103 + dendritic cell differentiation'', they found fgl2 accelerated tumor progression of gbm by suppressing cd103 + dendritic cell differentiation. in our study, we extracted data of lung adenocarcinoma patients from different clinical databases such as tcga, prognoscan, and timer. we found that fgl2 expression was significantly lower in lung adenocarcinoma tissue compared with adjacent normal tissue. a high expression level of fgl2 was correlated with better prognostic outcomes of lung adenocarcinoma patients. we speculated that fgl2 might play differential roles in distinct models of cancer. in the mouse model of lewis lung carcinoma, fgl2 induced an activated and protumorigenic phenotype of cancer-associated fibroblasts in the tumor microenvironment (tme). cancer-associated fibroblasts, also known as tumor-associated fibroblast, promote tumorigenic features by producing cytokines, or initiating extracellular matrix remodeling in the tumor microenvironment (erdogan & webb, 2017) . the cytokines could disrupt normal cell functions, such as normal cell cycle regulation to active their pro-tumor actions (öhlund, elyada & tuveson, 2014) . in addition, cancer-associated fibroblasts produce and secret angiogenic factors such as fibroblast growth factor (fgf), and vascular endothelial growth factor (vegf) to stimulate angiogenesis supporting the formation of tumors and the proliferation of cancer cells and metastasis (shiga et al., 2015) . in the mouse model of brain tumor, yan et al. demonstrated that fgl2 promotes gbm tumor progression by suppressing cd103 + dendritic cell differentiation. the function of dendritic cells and their subtypes in gbm has not been elucidated clearly (srivastava et al., 2019) . dendritic cells might interplay with other types of immune cells including macrophages, t cells, and microglia in the tumor microenvironment (tme). some researchers considered that certain subsets of dendritic cells recognize and present tumor antigens to induce the t cells immune responses (d'agostino et al., 2012) . if the cd103 + dendritic cell differentiation was suppressed, the antigen processing functions of dendritic cells might be impaired in gbm. so, fgl2 might promote gbm tumor progression by suppressing cd103 + dendritic cell differentiation. in our study, we demonstrated fgl2 was positively correlated with t cells, especially cd8 + t cells activation in the tumor microenvironment of lung adenocarcinoma patients. cd8 + t cells, also known as cytotoxic t lymphocytes (ctls), play important role of antitumor immune response in the tumor microenvironment (aerts & hegmans, 2013) . ctls could recognize and kill tumor cells by the complex formation of t-cell receptor (tcr) and human leukocyte antigen class (hla). tcr and related signaling molecules could activate the transduction cascade to induce immune synapse and stimulate antitumor responses of ctls (farhood, najafi & mortezaee, 2019) . then, the activated ctls produce and secret cytotoxic granules such as granzymes and perforin into the targeted tumor cells. adhesive and co-stimulatory molecules including cd80/cd86, cd11a/cd18, and lymphocyte function-associated antigen 1 (lfa-1) are important in the process of tcr-mediated ctls antitumor effects (durgeau et al., 2018) . ctl-associated antigen 4 (ctla-4) and programmed death-1 receptor (pd-1)-ligand (pd-l1) are checkpoint receptors could be targeted to relieve ctls exhaustion. antigen presenting cells (apcs) such as dendritic cells (dcs) and macrophages could activate naïve cd8 + t cells by binding the tcrs with cd3 and other costimulatory molecules. fgl2 is mainly expressed in apcs such as dcs and macrophages. so, we infer that fgl2 might activate ctls functions to exert anti-tumor effects by stimulating apcs to bind naïve cd8 + t cells. in brief, fgl2 might play different roles in different types of cancer models. in lung cancer animal model conducted by zhu et al., fgl2 might promote tumor progress by activating cancer-associated fibroblasts in tumor microenvironment. in gbm animal model, fgl2 promotes gbm progress by suppressing cd103 + dendritic cell differentiation. in clinical databases of lung cancer patients, fgl2 exhibited antitumor activities by activating ctls in the tumor microenvironment of lung cancer. there are no significant differences in the expression levels of fgl2 between patients with or without egfr or kras mutations in the tcga database. there is no significant correlation between fgl2 and other genes implicated in lung adenocarcinoma such as egfr, kras, tp53, stk11, nf1, and keap1. we speculated that fgl2 might indirectly affect those genes in lung adenocarcinoma by changing the immune status in the tumor environment. egfr regulates several signaling transduction cascades such as mapk, jnk, and akt signaling pathways, leading to tumor cell proliferation, cell cycle progression, angiogenesis, and metastasis (bethune et al., 2019) . egfr-targeted therapy such as egfr tyrosine kinase inhibitors (egfr-tkis) alters the tumor microenvironment in lung cancer (matsumoto et al., 2019) . egfr-tkis could increase cytotoxic cd8 + t cells and dendritic cells in the tumor environment of lung cancer (jia et al., 2019) . fgl2 also increase cytotoxic cd8 + t cells and dendritic cells in the tumor environment of lung cancer. so, fgl2 might affect egfr by influencing immune status in tumor environment of lung cancer. kras is important in promoting cell survival and growth in tumor cells. almost 30% patients with lung adenocarcinoma are positive for kras gene mutation (tomasini et al., 2016) . kras is a strong initiator of tumorigenesis in lung adenocarcinoma. it is also a predictive response to targeted therapy of lung adenocarcinoma (dias carvalho et al., 2018) . kras is related with immune-suppressed state by regulating components of adaptive and innate immune response (cullis, das & bar-sagi, 2018) . mutant kras could up-regulate immunosuppressive cells in tumor such as myeloid-derived suppressor cells (mdscs), cd4 + foxp3 + t regulatory cells, and cd19 + il10 + b regulatory cells. these immunosuppressive cells could suppress the activities of tumoricidal cells such as cd8 + t cytotoxic cells, natural killer (nk) cells in the tumor microenvironment. on the contrary, fgl2 could increase the levels of tumoricidal cells such as cd8 + t cytotoxic cells, natural killer (nk) cells in the tumor microenvironment. so, fgl2 affect kras by influencing immune status in tumor environment of lung cancer. tp53 is regarded as tumor suppressor gene that it could prevent genome mutation. tp53 could activate dna repair process and arrest cell proliferation by holding cell cycle (uehara & tanaka, 2018) . mutation of tp53 leads to tumor escape from senescence and apoptosis. activation of tp53 could increase the levels of tumor-infiltrating leukocytes such as cd8 + t cells in tumor microenvironment (guo et al., 2017) . fgl2 also increases the levels of tumor-infiltrating leukocytes such as cd8 + t cells in tumor microenvironment. so, fgl2 possesses synergic effects with tp53 to enhance antitumor immunity in tumor microenvironment. stk11, known as liver kinase b1 (lkb1), regulates cell polarity and regarded as tumor suppressor. loss of stk11 would lead to cell polarity disorganization and induce tumor growth. koyama et al. found that loss of stk11/lkb1 induced neutrophil recruitment and inflammatory mediator production to suppress the t cells in tumor environment of lung cancer (koyama et al., 2016) . in addition, stk11/lkb1 inactivated mutations were related with reduced expression of pd-1 ligand pd-l1 in tumor cells. so, we infer that fgl2 exert synergic effects with stk11 to enhance the cytotoxic t lymphocytes activities in tumor environment of lung adenocarcinoma. nf1 is regarded as a tumor suppressor in lung cancer negatively regulates ras signaling pathway. nf1 mutations present in similar patients populations with kras mutation (redig et al., 2016) . we infer that fgl2 affect kras by influencing immune status in tumor environment of lung adenocarcinoma. so, fgl2 might affect nf1 function by influencing immune status in tumor environment of lung adenocarcinoma. keap1 has been shown to interact with nuclear factor erythroid 2-related factor 2 (nrf2). keap1-nrf2 pathway play important role in oxidative response by inducing anti-inflammatory and antioxidant effects. keap1 mutation is correlated with poor prognosis of lung cancer (frank et al., 2018) . aberrant keap1-nrf2 pathway activity alters the immune microenvironment of lung adenocarcinoma. keap1 mutation is associated reduced leukocyte infiltration of tumor microenvironment in lung adenocarcinoma (thorsson et al., 2018) . we infer that fgl2 affects the keap1 effect by influencing immune status in tumor environment of lung adenocarcinoma. so, we infer some indirect correlations might explain the mechanism of fgl2 affecting other genes. fgl2 might affect other genes functions by influencing immune status in tumor environment of lung adenocarcinoma. in this study, we explored the expression profile and potential effects of fgl2 in lung adenocarcinoma. we found that fgl2 expression was significantly lower in lung adenocarcinoma tissue than in adjacent normal tissue. high fgl2 mrna expression was correlated with better prognostic outcomes of lung adenocarcinoma patients, including overall survival and progression-free survival. these results indicate that fgl2 might function as a negative regulator of lung adenocarcinoma. then, we investigated the potential mechanism of fgl2 in lung adenocarcinoma. fgl2 was positively correlated with the infiltration of immune cells, including cd8 + t cells, cd4 + t cells, macrophages, b cells and dendritic cells, in lung adenocarcinoma. these results imply that fgl2 exerts its antitumor effects by enhancing immune cell infiltration in lung adenocarcinoma. go and kegg functional enrichment analyses and gsea also showed that fgl2 expression was positively correlated with enhanced tumor killing. thus, we propose that fgl2, which is positively associated with enhanced antitumor activities mediated by t cells, is a beneficial marker for lung adenocarcinoma treatment outcomes. in this study, we used bioinformatic analysis to discover the potential roles of fgl2 in lung adenocarcinoma. in the future studies, in vivo and in vitro experiments will carry out to demonstrate the role of fgl2 in modulating the t cell-mediated immune response in lung adenocarcinoma. soluble fibrinogen like protein 2 (sfgl2), the novel effector molecule for immunoregulation intratumoral dendritic cells in the anti-tumor immune response dendritic cell subsets impact of tumor microenvironment on the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors in patients with egfr-mutant non-small cell lung cancer gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma fibroblast heterogeneity in the cancer wound a systematic review of the clinical effectiveness of first-line chemotherapy for adult patients with locally advanced or metastatic non-small cell lung cancer clinical and molecular characteristics of nf1-mutant lung cancer keap1 loss promotes kras-driven lung cancer and results in dependence on glutaminolysis epidemiology of lung cancer epidermal growth factor receptor mutations in lung cancer erlotinib in previously treated non-small-cell lung cancer the authors declare there are no competing interests. • kai yuan and yanyan feng performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.• hesong wang, lu zhao, wei wang, ting wang and yuyin feng analyzed the data, prepared figures and/or tables, and approved the final draft.• guangrui huang and anlong xu conceived and designed the experiments, prepared figures and/or tables, and approved the final draft. the following information was supplied regarding data availability:the code is available in the supplemental files. supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.8654#supplemental-information. key: cord-336560-m5u6ryy9 authors: boudewijns, robbert; thibaut, hendrik jan; kaptein, suzanne j. f.; li, rong; vergote, valentijn; seldeslachts, laura; de keyzer, carolien; bervoets, lindsey; sharma, sapna; van weyenbergh, johan; liesenborghs, laurens; ma, ji; jansen, sander; van looveren, dominique; vercruysse, thomas; jochmans, dirk; wang, xinyu; martens, erik; roose, kenny; de vlieger, dorien; schepens, bert; van buyten, tina; jacobs, sofie; liu, yanan; martí-carreras, joan; vanmechelen, bert; wawina-bokalanga, tony; delang, leen; rocha-pereira, joana; coelmont, lotte; chiu, winston; leyssen, pieter; heylen, elisabeth; schols, dominique; wang, lanjiao; close, lila; matthijnssens, jelle; van ranst, marc; compernolle, veerle; schramm, georg; van laere, koen; saelens, xavier; callewaert, nico; opdenakker, ghislain; maes, piet; weynand, birgit; cawthorne, christopher; velde, greetje vande; wang, zhongde; neyts, johan; dallmeier, kai title: stat2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in sars-cov-2 infected hamsters date: 2020-07-02 journal: biorxiv doi: 10.1101/2020.04.23.056838 sha: doc_id: 336560 cord_uid: m5u6ryy9 since the emergence of sars-cov-2 causing covid-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. in search for key targets of effective therapeutics, robust animal models mimicking covid-19 in humans are urgently needed. here, we show that productive sars-cov-2 infection in the lungs of mice is limited and restricted by early type i interferon responses. in contrast, we show that syrian hamsters are highly permissive to sarscov-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which stat2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. finally, we assess sars-cov2-induced lung pathology in hamsters by micro-ct alike used in clinical practice. our results reveal the importance of stat2-dependent interferon responses in the pathogenesis and virus control during sars-cov-2 infection and may help rationalizing new strategies for the treatment of covid-19 patients. sars-cov-2 belongs to the family of coronaviridae, which contains a large group of viruses that are constantly circulating in animals and humans. illness in humans caused by coronaviruses is mostly mild and manifested by respiratory or digestive problems as leading symptoms 1 . however, some coronaviruses, such as sars-cov-1, mers-cov and the recent sars-cov-2, have been responsible for serious outbreaks of severe and lethal respiratory disease 2, 3 . unlike the previous outbreaks with sars-cov-1 and mers-cov, the current sars-cov-2 outbreak has evolved to the largest global health threat to humanity in this century. the unprecedented scale and rapidity of the current pandemic urges the development of efficient vaccines, antiviral and anti-inflammatory drugs. a key step in expediting this process is to have animal models that recapitulate and allow to understand viral pathogenesis, and that can in particular be used to identify new drug targets and preclinically assess preventive and therapeutic countermeasures. acute respiratory disease caused by sars-cov-1 and mers infections is characterized by a dysregulated inflammatory response in which a delayed type i interferon (ifn) response promotes the accumulation of inflammatory monocyte-macrophages [4] [5] [6] . the severe lung disease in covid-19 patients seems to result from a similar overshooting inflammatory response 7 . however, because even non-human primates do not fully replicate covid-19, little information and no appropriate animal models are currently available to address this hypothesis 8 . to address this knowledge gap, we compared the effect of sars-cov-2 infection in wild-type (wt) mice of different lineages (balb/c and c57bl/6) and syrian hamsters, as well as a panel of matched transgenic mouse and hamster strains with a knockout (ko) of key components of adaptive and innate immunity. we used an original patient isolate of sars-cov-2 (betacov/belgium/ghb-03021/2020) that was passaged on huh7 and vero e6 cells for these studies ( fig. s1 and fig. s2a ). for full characterization and to exclude possible contaminants, we performed deep sequencing on the inoculum that was used to infect the animals (fig. s2a) . no adventitious agents could be detected (data not shown). however, two in-frame deletions in the n-terminal domain and the furin-cleavage site of spike (s) glycoprotein (9aa and 5aa, respectively) had occurred between cell culture passage p4 (mixed population of 85% wt genomes and 15% (9+5aa del) mutant genomes) and p6 (100% (9+5aa del) mutant genomes) [9] [10] [11] , likely as adaptation to growth in vero e6 cells in vitro (fig. s2b ). to first examine whether adaptive immunity contributed to the susceptibility to sars-cov-2 infection, we inoculated wt (immune-competent) and scid mice (lacking functional t and b cells) from the same balb/c background intranasally with a high 2 × 10 5 tcid50 viral dose (p4 virus) (fig. 1a ). on day 3 p.i., a viral rna peak in the lungs was observed ( fig. 1b and fig. s3 ) with no obvious differences in viral loads (fig. 1b) nor lung pathology ( fig. 1d and fig. s4a and s4b ) between wt and scid mice. these data indicate that mice that lack the human ace2 receptor 12 , can in principle be infected with sars-cov-2, although inefficiently and likely transiently, as also observed for sars-cov-1 4,13 . however, adaptive immunity did not markedly contribute to this low susceptibility. interferons are the prototypic first-line innate immune defense against viral infections. to evaluate interferons, we compared viral rna levels and lung pathology in wt c57bl/6 mice, and c57bl/6 mice with a genetic ablation of their type i (ifnar1 -/-) and iii interferon (ifn) receptors (il28r -/-) (fig. 1a ). ifnar1 -/mice showed an enhanced replication of sars-cov-2 in the lung on day 3 p.i. compared to both wt and il28r -/mice (fig. 1c ). similar to balb/c mice, overall viral loads were low. ifnar1 -/mice that were treated prior to infection with human convalescent sars-cov-2 patient serum or plasma that contains spike-specific antibodies (fig. 1e, fig. s4 ) had a 3-10-fold reduction in viral loads depending on the patient donor. this provides further evidence for active, although inefficient virus replication in ifnar1 -/mice. wt and knockout (ifnar1 -/-, il28r -/-) mouse strains, all on c57bl/6 background, presented consistently with only a mild lung pathology. however, ifnar1 -/mice showed increased levels of intra-alveolar hemorrhage, sometimes accompanied by some peribronchiolar inflammation ( fig. 1d and fig. s5a and s5b ). passive transfer of hcs did not result in an obvious improvement in histopathological scores ( [18] [19] [20] . likewise, hcs treatment modulated, at least to some extent, the observed gene expression patterns ( fig. 1f and cgas (mb21d1, p=0.094) mrna levels. in summary, our data are in line with restriction of sars-cov-2 infection by the interferon system in mice, and also suggest limited inflammatory responses in the lungs of mice, in contrast to covid-19 in humans 21 . taken together, mice were considered as a poor model to study covid-19 pathogenesis, or to assess the efficacy of vaccines and treatments. in contrast, syrian hamsters have been reported to be highly susceptible to sars-cov-1 22 and sars-cov-2 23 and might thus provide a small animal model to study sars-cov-induced pathogenicity and the involvement of the immune response in aggravating lung disease. in contrast to mice, intranasal inoculation of sars-cov-2 in wt hamsters resulted in high viral rna loads (fig. 2b, fig. s7 ) a proxy used for the quantification of viral loads (see fig. s9c ), and in actual infectious titers (fig. 2c) in the lungs, i.e. roughly 4 log10 higher than in ifnar -/mice (fig. 2c) . also, a marked lung pathology [median cumulative score (mcs) 9 out of maximal score of 18; iqr=8.5-10.5 (p4 virus)] characterized by a multifocal necrotizing bronchiolitis, massive leukocyte infiltration and edema was observed in infected hamsters but not in mice ( fig. 2d and fig. s8a -c). this resembles histopathological findings in humans suffering from severe bronchopneumonia 24 . in order to investigate the roles of type i and iii ifn in the pathogenesis of sars-cov-2 infection, we compared virus replication levels and lung pathology in wt hamsters and hamsters with ablated signal transducer and activator of transcription 2 (stat2 -/lacking type i and iii ifn signaling) 25, 26 and il28r expression (il28r-a -/lacking ifn type iii signaling) ( fig. 2a) . of note, these receptor knockouts did not affect ace2 expression in hamster lungs (fig. s9a ), while interferon-stimulated genes (isg) 27 such as mx-2 (strongly induced by ifnα/stat2 signaling) and ip-10 (induced by both type i and type ii ifns) showed a differential expression pattern when comparing the different genotypes, triggered by sars-cov-2 infection (fig. s9b) . importantly, lower baseline expression of mx-2 and ip-10 and failure to respond to sars-cov-2 infection by mx-2 upregulation in stat2 -/hamsters confirmed the functional knockout. as expected, il28r-a -/hamsters showed an intermediate phenotype between that of wt and stat2 -/concerning their antiviral response. for many respiratory viruses, including sars-cov-1, type i and iii interferon signaling has been described to play an important role in restricting infection 28 . no marked differences were observed in viral rna levels in the lung of wt, stat2 -/or il28r-a -/hamsters (fig. 2b) . however, stat2 -/hamsters had higher titers of infectious virus in the lung (fig. 2c ), high titer viremia (measured by rt-qpcr and virus titration) 29 and fig. s8c ). matrix metalloprotease (mmp)-9 levels, which may serve as a sensitive marker for the infiltration and activation of neutrophils in inflamed tissues 31, 32 , were markedly elevated in the lungs of all infected hamsters (fig. 2g) . however, higher mmp-9 levels were found in stat2 -/animals, thereby inversely correlating with the histological findings (fig. 2d ). in addition, biomarkers elevated in critically ill covid-19 patients 2,7,33 such as the cytokines il-6, il-10 and ifn- were not found to be elevated in the serum of infected hamsters (fig. s10b) , although mrna levels of ip-10 (cxl10) were upregulated in the lungs of sars-cov-2 infected hamsters as reported for other cytokine/chemokines downstream of ifn- 23 (fig. s9b) . nonetheless, infected stat2 -/and il28r-a -/had clearly increased levels of il-6 and il-10 in their lungs (fig. s10a ). such an inverse correlation between biomarkers and pathology in wt versus stat2 -/hamsters is in line with findings in mouse models of sars-cov-1 infection in which pathology correlated with the induction and dysregulation of alternatively activated "wound-healing" monocytes/macrophages 4,6 . to assess the utility of the hamsters for testing the effect of therapeutic interventions on sars-cov-2 replication, wt hamsters were treated with human convalescent plasma or a neutralizing sars-cov-1 and sars-cov-2specific single-domain antibody fc fusion construct (vhh-72-fc) 34 prior to infection (fig. 2h) . unlike a single dose of convalescent plasma, which did not significantly reduce viral load in the lungs, pre-treatment with vhh-72-fc reduced viral loads in the lung ~10 5 -fold compared to untreated control animals, validating hamsters as preclinical model for testing anti-sars-cov-2 therapies. the lack of readily accessible serum markers or the absence of overt disease symptoms in hamsters prompted us to establish a non-invasive means to score for lung infection and sars-cov-2 induced lung disease by computed tomography (ct) as used in standard patient care to aid covid-19 diagnosis with high sensitivity and monitor progression/recovery 7, 33, 35, 36 . similar as in humans 37 , semiquantitative lung pathology scores were obtained from high-resolution chest micro-ct scans of freebreathing animals 38 the increase in replication of sars-cov-2 seen in il28r-a -/hamsters, on one hand, combined with a tempered inflammatory response and lung injury as compared to wt hamsters, on the other hand, is in line with the role of type iii ifn plays during respiratory virus infections, including sars-cov-1 53 . this observation also suggests that in humans pegylated ifn-lambda 54,55 (or similar modulators of innate immunity) may possibly be considered to protect medical staff and other frontline workers from sars-cov-2 infection or to dampen symptoms in critically ill patients 56 . in conclusion, hamsters may be preferred above mice as infection model for the preclinical assessment of antiviral therapies, of convalescent serum transfer and of approaches that aim at tempering the covid-19 immune pathogenesis in critically ill patients 21, 57 . the latter may be achieved by repurposing anti-inflammatory drugs 58 such as il-6 receptor antagonists (e.g. tocilizumab) 59 , or small molecule jak/stat inhibitors (e.g. ruxolitinib or tofacitinib). educated by our finding that stat2 signaling plays a dual role in also limiting viral dissemination, targeting the virus-induced cytokine response and overshooting of macrophage activation may need to be complemented by (directly acting) antivirals 60 . wild-type syrian hamsters (mesocricetus auratus) were purchased from janvier laboratories. all other mouse (c57bl/6, ifnar1 -/-, il28r -/-, balb/c and scid) and hamster (stat2 -/and il28r-a -/-) strains were bred in-house. six-to eight-weeks-old female mice and wild-type hamsters were used throughout the study. knock-out hamsters were used upon availability; seven-to twelve-week old female stat2 -/hamsters; five-to seven-week-old il28r-a -/hamsters. vero e6 (african green monkey kidney, kind gift from peter bredenbeek, lumc, nl) and huh7 (human hepatoma, jcrb0403) cells were maintained in minimal essential medium (gibco) supplemented with 10% fetal bovine serum (integro), 1% bicarbonate (gibco), and 1% l-glutamine (gibco). for maintenance of calu-3 cells (human airway epithelium, kind gift from lieve naesens, ku leuven, be), the above medium was supplemented with 10mm hepes (gibco). all assays involving virus growth were performed using 2% (vero e6 and huh7) or 0.2% (calu-3) fetal bovine serum instead of 10%. sars-cov-2 strain betacov/belgium/ghb-03021/2020 (epi isl 407976|2020-02-03) recovered from a nasopharyngeal swab taken from a rt-qpcr-confirmed asymptomatic patient returning from wuhan, china beginning of february 2020 62 was directly sequenced on a minion platform (oxford nanopore) as described previously 63 antibody vhh-72-fc was administered i.p. at a dose of 20mg/kg 1 day prior to infection. vhh-72-fc was expressed in expicho cells (thermofisher scientific) and purified from the culture medium as described 34 . briefly, after transfection with pcdna3.3-vhh-72-fc plasmid dna, followed by incubation at 32c and 5% co2 for 6-7 days, the vhh-72-fc protein in the cleared cell culture medium was captured on a 5 ml mabselect sure column (ge healthcare), eluted with a mcilvaine buffer ph 3, neutralized using a saturated na3po4 buffer, and buffer exchanged to storage buffer (25 mm l-histidine, 125 mm nacl). the antibody's identity was verified by protein-and peptide-level mass spectrometry. animals were euthanized at different time-points post-infection, organs were removed and lungs were homogenized manually using a pestle and a 12-fold excess of cell culture medium (dmem/2%fcs). rna extraction was performed from homogenate of 4 mg of lung tissue with rneasy mini kit (qiagen), or 50µl of serum using the nucleospin kit (macherey-nagel), according to the manufacturer's instructions. other organs were collected in rnalater (qiagen) and homogenized in a bead mill (precellys) prior to extraction. of 100µl eluate, 4µl was used as template in rt-qpcr reactions. rt-qpcr was performed on a lightcycler96 platform (roche) using the itaq universal probes one-step rt-qpcr kit (biorad) with primers and probes (table s1) infectious virus were used to express the amount of rna as normalized viral genome equivalent (vge) copies per mg tissue, or as tcid50 equivalents per ml serum, respectively. the mean of housekeeping gene β-actin was used for normalization. the relative fold change was calculated using the 2 -δδct method 66 . after extensive transcardial perfusion with pbs, lungs were collected, extensively homogenized using manual disruption (precellys24) in minimal essential medium (5% w/v) and centrifuged (12,000 rpm, 10min, 4°c) to pellet the cell debris. infectious sars-cov-2 particles were quantified by means of endpoint titrations on confluent vero e6 cell cultures. viral titers were calculated by the spearman-kärber method and expressed as the 50% tissue culture infectious dose (tcid50) per 100mg tissue. to study differential gene expression, rna was extracted from lung tissues using trizol, subjected to cdna synthesis (high capacity cdna reverse transcription kit, thermo fisher scientific), and qpcr using a custom taqman qrt-pcr array (thermo fisher scientific) of 30 genes known to be activated in response to virus infection 16 , as well as two housekeeping genes (table s2) for histological examination, the lungs were fixed overnight in 4% formaldehyde and embedded in paraffin. tissue sections (4 µm) were stained with hematoxylin and eosin to visualize and score for lung damage. calu-3 (human airway epithelial) cells were plated at 5×10 4 cytokine levels in lung homogenates and serum of hamsters were determined by elisa for ifn- (eha0005), il-6 (eha0008) and il-10 (eha0006) following the manufacturer's instructions (wuhan fine biotech co., ltd). the levels of gelatinase b/metalloproteinase (mmp)-9 present in lung homogenates were analyzed using gelatin zymography 68 , essentially as described previously 69 . for quantification of zymolytic bands internal control samples were spiked into each sample. equivalent hamster enzyme concentrations were calculated with the use of known amounts of recombinant human pro-mmp-9 and recombinant human pro-mmp-9δoghem as standards 70 . hamsters were anaesthetized using isoflurane (iso-vet) (2-3% in oxygen) and installed in prone position into the x-cube micro-ct scanner (molecubes) using a dedicated imaging bed. respiration was monitored throughout. a scout view was acquired and the lung was selected for a non-gated, helical ct acquisition using the high-resolution ct protocol, with the following parameters: 50 kvp, 960 exposures, 32 ms/projection, 350 µa tube current, rotation time 120 s. data were reconstructed using a regularized statistical (iterative) image reconstruction algorithm using nonnegative least squares 71 , using an isotropic 100 µm voxel size and scaled to hounsfield units (hus) after calibration against a standard air/water phantom. the spatial resolution of the reconstruction was estimated at 200 µm by minimizing the mean squared error between the 3d reconstruction of the densest rod in a micro-ct multiple density rod phantom (smart scientific) summed in the axial direction and a digital phantom consisting of a 2d disk of 17.5 mm radius that was post-smoothed with gaussian kernels using different full width half maxima (fwhm), after aligning the symmetry axis of the rod to the z-axis. visualization and quantification of reconstructed micro-ct data was performed with dataviewer and ctan software (bruker micro-ct). as primary outcome parameter, a semi-quantitative scoring of micro-ct data was performed as previously described 38, 39, 72 with minor modifications towards optimization for covid-19 lung disease in hamsters. in brief, visual observations were scored (from 0 -2 depending on severity, both for parenchymal and airway disease) on 5 different, predefined transversal tomographic sections throughout the entire lung image for both lung and airway disease by two independent observers (l.s. and g.v.v.) and averaged. scores for the 5 sections were summed up to obtain a score from 0 to 10 reflecting severity of lung and airway abnormalities compared to scans of healthy, wt control hamsters. as secondary measures, image-derived biomarkers (nonaerated lung volume, aerated lung volume, total lung volume, the respective densities within these volumes and large airways volume) were quantified as in 38, 72 for a manually delineated voi in the lung, avoiding the heart and main blood vessels. the threshold used to separate the airways and aerated (grey value 0-55) from non-aerated lung volume (grey value 56-255) was set manually on an 8-bit greyscale histogram and kept constant for all data sets. il28r -/-(n=5) mice. at the indicated time intervals p.i., viral rna levels were determined by rt-qpcr, normalized against β-actin mrna levels and transformed to estimate viral genome equivalents (vge) content per weight of the lungs ( figure s2 ). for heat-inactivation, sars-cov-2 was incubated for 30min at 56°c. dotted line indicates lower limit of quantification (lloq). the data shown are means ± sem. (d) histopathological scoring of lungs for all different mouse strains. mice were sacrificed on day 3 p.i. and lungs were stained with h&e and scored for signs of lung damage (inflammation and hemorrhage). scores are calculated as percentage of the total maximal score. "no score" means not contributing to theoretical full cumulative score of 100%. numbers (n) of animals analyzed per condition are given in the inner circle. (e) viral rna levels in ifnar1 -/mice after treatment with anti-sars-cov-2 serum or plasma. mice were either left untreated (ic, infection control), or treated intraperitoneally one day before infection with convalescent serum (patient #1), convalescent plasma (patient #2) or with negative control plasma (patient #3 nc, negative control) and sacrificed on day 3 p.i. viral rna levels were determined in the lungs, normalized against βactin and fold-changes were calculated using the 2 (-δδcq) method compared to mean of ic. the data shown are means ± sem. (f) heatmap showing gene expression profiles of 30 selected marker genes in the lungs of uninfected and infected ifnar1 -/mice that were either left untreated or treated with convalescent serum from patient #1 (n=3 per group). analysis performed on day 3 p.i. the scale represents fold change compared to non-infected animals. statistical significance between groups was calculated by the nonparametric two-tailed mann-whitney u-test (ns = not significant, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001). wt, stat2 -/and il28r-a -/hamster strains were intranasally inoculated with 2 × 10 5 tcid50 of passage 4 or 2 × 10 6 of passage 6 sars-cov-2. outcomes derived from inoculation with passage 4 or passage 6 sars-cov-2 is designated by circles (p4) or squares (p6). on the indicated days post inoculation (d.p.i.), organs and blood were collected to determine viral rna levels, infectious viral load and score for lung damage. viral loads in the indicated organs were quantified by rt-qpcr (b, e and f) or virus titration (c). (b,f) viral rna levels in the indicated organs were normalized against β-actin mrna levels and transformed to estimate viral genome equivalents (vge) content per weight of the lungs ( figure s5 ). (c) infectious viral loads in the lung are expressed as the number of infectious virus particles per 100 mg of lung tissue. (e) viral rna levels in the blood were calculated from a standard of infectious virus and expressed as tcid50 equivalents per ml blood. dotted lines indicate lower limit of quantification (lloq) or lower limit of detection (llod) (d) histopathological scoring of lungs. hamsters were sacrificed on day 4 p.i. with passage 4 sars-cov-2 and lungs were stained with h&e and scored for signs of lung damage (apoptotic bodies, necrotizing bronchiolitis, edema, pneumonia and inflammation). scores are calculated as percentage of the total maximal score. (g) levels of matrix metalloproteinase (mmp)-9 levels in lung homogenates of sars-cov-2 infected hamsters, relative to non-infected controls of the same strain. statistical significance was calculated between infected and non-infected animals within each group. values for infected animals (n=7 each) compiled from two independent experiments using either p4 (n=3, circles) and p6 (n=4, squares) sars-cov-2. (h) viral rna levels in hamsters after treatment with convalescent sars-cov-2 plasma or with a previously described antibody. hamsters were either left untreated (ic, infection control, n=5) or treated with a single-domain antibody (vhh-72-fc, n=4), convalescent plasma (patient #2, n=4) or negative control plasma (patient #3 nc, negative control, n=4) and sacrificed on day 4 p.i. viral rna levels were determined in the lungs, normalized against β-actin and fold-changes were calculated using the 2 (-δδcq) method compared to the mean of ic. the data shown are means ± sem. statistical significance between groups was calculated by the nonparametric two-tailed mann-whitney u-test (ns p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001). . 2c) . lines indicate matched samples. the data shown are means ± sem. statistical significance between groups was calculated by the nonparametric two-tailed mann whitney u-test (ns p > 0.05, * p < 0.05). d wrote the original draft with input from co-authors developed the stat2 -/-and il28r-a -/-hamster strains are named as inventors on us patent application no. 62/988,610, entitled ''coronavirus binders are named as inventors on us patent application no. 62/991,408, entitled ''sars-cov-2 virus binders origin and evolution of pathogenic coronaviruses a novel coronavirus from patients with pneumonia in china clinical progression of patients with covid-19 in shanghai dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection clinical features of patients infected with 2019 novel coronavirus in wuhan comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model the proximal origin of sars-cov-2 probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak cryo-em structure of the 2019-ncov spike in the prefusion conformation sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor neutralizing antibody against severe acute respiratory syndrome (sars)-coronavirus spike is highly effective for the protection of mice in the murine sars model treatment of 5 critically ill patients with covid-19 with convalescent plasma complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis small molecule inhibitors of tbk1 serve as adjuvant for a plasmidlaunched live-attenuated yellow fever vaccine interferon-stimulated genes: what do they all do? a genetic ifn/stat1/fas axis determines cd4 t stem cell memory levels and apoptosis in healthy controls and adult t-cell leukemia patients treatment of multiple sclerosis patients with interferon-beta primes monocyte-derived macrophages for apoptotic cell death an evolutionary recent ifn/il-6/cebp axis is linked to monocyte expansion and tuberculosis severity in humans reducing mortality from 2019-ncov: host-directed therapies should be an option severe acute respiratory syndrome coronavirus infection of golden syrian hamsters simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility pathological findings of covid-19 associated with acute respiratory distress syndrome efficient gene targeting in golden syrian hamsters by the crispr/cas9 system stat2 knockout syrian hamsters support enhanced replication and pathogenicity of human adenovirus, revealing an important role of type i interferon response in viral control validation of assays to monitor immune responses in the syrian golden hamster (mesocricetus auratus) combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread detectable 2019-ncov viral rna in blood is a strong indicator for the further clinical severity gastrointestinal symptoms of 95 cases with sars-cov-2 infection neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential gelatinase b: a tuner and amplifier of immune functions clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging longitudinal micro-ct provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection the trinity of covid-19: immunity, inflammation and intervention longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients attenuated sars-cov-2 variants with deletions at the s1/s2 junction sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism immune response in stat2 knockout mice modeling severe fever with thrombocytopenia syndrome virus infection in golden syrian hamsters: importance of stat2 in preventing disease and effective treatment with favipiravir zika virus infection of adult and fetal stat2 knock-out hamsters constitutive type i interferon modulates homeostatic balance through tonic signaling anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection a transcriptional regulatory atlas of coronavirus infection of human cells lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections new treatment options for delta virus: is a cure in sight? lambda interferons come to light: dual function cytokines mediating antiviral immunity and damage control covid-19 and emerging viral infections: the case for interferon lambda the cytokine storm of severe influenza and development of immunomodulatory therapy immune status could determine efficacy of covid-19 therapies targeting interleukin-6 signaling in clinic coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease xgenome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes first cases of coronavirus disease 2019 (covid-19) in the who european region accounting for population structure reveals ambiguity in the zaire ebolavirus reservoir dynamics modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis modular approach to customize sample preparation procedures for viral metagenomics analysis of relative gene expression data using real-time quantitative pcr and the 2-δδct method inhibitors of the interferon response enhance virus replication in vitro zymography methods for visualizing hydrolytic enzymes analysis of gelatinases in complex biological fluids and tissue extracts differential diagnosis of autoimmune pancreatitis from pancreatic cancer by analysis of serum gelatinase levels iterative ct reconstruction using shearlet-based regularization radiosafe micro-computed tomography for longitudinal evaluation of murine disease models we thank kathleen van key: cord-295156-trzkb9ne authors: cheong, dorothy h.j.; daniel tan, w.s.; fred wong, w.s.; tran, thai title: anti-malarial drug, artemisinin and its derivatives for the treatment of respiratory diseases date: 2020-05-13 journal: pharmacol res doi: 10.1016/j.phrs.2020.104901 sha: doc_id: 295156 cord_uid: trzkb9ne artemisinins are sesquiterpene lactones with a peroxide moiety that are isolated from the herb artemisia annua. it has been used for centuries for the treatment of fever and chills, and has been recently approved for the treatment of malaria due to its endoperoxidase properties. progressively, research has found that artemisinins displayed multiple pharmacological actions against inflammation, viral infections, and cell and tumour proliferation, making them effective against diseases. moreover, it has displayed a relatively safe toxicity profile. the use of artemisinins against different respiratory diseases has been investigated in lung cancer models and inflammatory-driven respiratory disorders. these studies revealed the ability of artemisinins in attenuating proliferation, inflammation, invasion, and metastasis, and in inducing apoptosis. artemisinins can regulate the expression of pro-inflammatory cytokines, nuclear factor-kappa b (nf-κb), matrix metalloproteinases (mmps), vascular endothelial growth factor (vegf), promote cell cycle arrest, drive reactive oxygen species (ros) production and induce bak or bax-dependent or independent apoptosis. in this review, we aim to provide a comprehensive update of the current knowledge of the effects of artemisinins in relation to respiratory diseases to identify gaps that need to be filled in the course of repurposing artemisinins for the treatment of respiratory diseases. in addition, we postulate whether artemisinins can also be repurposed for the treatment of covid-19 given its anti-viral and anti-inflammatory properties. artemisinins are sesquiterpene lactones with a peroxide moiety that are isolated from the herb artemisia annua. it has been used for centuries for the treatment of fever and chills, and has been recently approved for the treatment of malaria due to its endoperoxidase properties. progressively, research has found that artemisinins displayed multiple pharmacological actions against inflammation, viral infections, and cell and tumour proliferation, making them effective against diseases. moreover, it has displayed a relatively safe toxicity profile. the use of artemisinins against different respiratory diseases has been investigated in lung cancer models and inflammatory-driven respiratory disorders. these studies revealed the ability of artemisinins in attenuating proliferation, inflammation, invasion, and metastasis, and in inducing apoptosis. artemisinins can regulate the expression of pro-inflammatory cytokines, nuclear factor-kappa b (nf-κb), matrix metalloproteinases (mmps), vascular endothelial growth factor (vegf), promote cell cycle arrest, drive reactive oxygen species (ros) production and induce bak or bax-dependent or independent apoptosis. in this review, we aim to provide a comprehensive update of the current knowledge of the effects of artemisinins in relation to respiratory diseases to identify gaps that need to be filled in the course of repurposing artemisinins for the treatment of respiratory diseases. in addition, we postulate whether artemisinins can also be repurposed for the treatment of covid-19 given its anti-viral and anti-inflammatory properties. atp-binding cassette subfamily member 2 ace2 angiotensin-converting enzyme 2 aec alveolar epithelial cells ahr airway hyperresponsiveness aif apoptosis-inducing factor ali acute lung injury ap respiratory diseases refer to any disease or disorder of the airways and the lungs that interfere with respiration [1] . the respiratory system, comprising of the nose, nasal cavities and the lung, is the sole internal system that is exposed to the external environment. hence, it is easily susceptible to environmental agents (such as bacterial or viral infections, smoking, air pollution or cold weather [2] ), that can cause respiratory diseases. importantly, chronic respiratory diseases are a great cause of j o u r n a l p r e -p r o o f concern as approximately one billion people suffer from them while four million succumb to these illnesses prematurely every year [2] . the forum of international respiratory societies identified chronic obstructive pulmonary disorder (copd), asthma, acute respiratory infections, and lung cancer as the top few respiratory diseases that heavily burden society [2] . in brief, copd is an obstructive lung disease characterized by long term breathing issues and poor airflow. it affects 200 million people and is the fourth leading cause of death worldwide [3] . it is currently being treated using inhaled bronchodilators and glucocorticoids [4] . asthma is a condition in which the airways narrow and swell, and is accompanied by increased mucus production. it affects 300 million people worldwide [5] . the cause of asthma is still unknown, and it is furthermore uncurable. treatment is limited to symptom relief using inhaled corticosteroids and bronchodilators [5] . acute respiratory infections include pneumonia and viral respiratory infections. annually, respiratory tract infections like influenza kill 250,000 to 500,000 people and cost billions of dollars. in addition, it occasionally causes epidemics that threaten the health of the global population [6] . lung cancer is a malignant lung tumour that is characterized by uncontrolled cell growth in the lung tissues, and this growth can spread to other parts of the body, causing death. it has the highest fatality rate amongst the major cancers, killing more than 1.4 million people a year [7] . patients are diagnosed and their disease is classified into different stages where earlier-stage patients are treated with surgery to remove the lung tumour, while late-stage patients are treated with chemotherapy or radiotherapy but often succumb to the disease [2] . currently, the global approach to managing respiratory diseases is to provide better healthcare, reduce environmental pollution, and to create public awareness of the prevalence and risks of such diseases. research in this field explores the causes of these respiratory diseases, prognostic markers to better diagnose patients and new therapies that can maintain and contain the disease [2] . nonetheless, a lot more work will need to be done to find safer and more effective treatment methods. natural products have been used to treat respiratory diseases as far back as 2600bc with the first records indicating that oils of cedrus (cedar), commiphora (myrrh), cupressus sempervirens (cypress) and glycyrrhiza glabra (licorice) were being used to treat inflammation, coughs, and colds [8] . male newborns of the indian tribes of southern california were bathed in hot salvia ashes as it was believed to provide lifetime immunity from all respiratory diseases [9] . in 1952, erythromycin, derived from saccharopolyspora erythraea, was launched commercially for bacterial infections affecting the upper j o u r n a l p r e -p r o o f respiratory tract [10] . umckaloabo contains root extract of pelargonium sidoides and was marketed in 1897 against tuberculosis but was later superseded by antibiotics. in the 2000s, it regained popularity for the treatment of acute bronchitis and is now one of the most commonly prescribed childhood medications [11] . today, many natural-based products are still being investigated for its beneficial properties against respiratory diseases. in this review, we will provide a comprehensive update of the current knowledge of artemisinin, and its derivatives, for the treatment of various respiratory diseases. artemisinin is a sesquiterpene lactone with a peroxide constituent [12] . it is isolated from the leafy parts of artemisia annua, a herb and medicinal plant that has been used for the treatment of chills and fever for centuries [13] . in the 1960s, the search for new anti-malarial drugs began in lieu of the increasing resistance of plasmodium falciparum to chloroquine. artemisinin, also known as qinghaosu, was first isolated. dihydroartemisinin (dha) was subsequently the first generation of derivatives, made by modifying the carbonyl groups into hydroxyl groups [12] . others like the more water-soluble artesunate and more oil-soluble artemether and arteether followed [14] . these derivatives were ten times more potent than artemisinin [12] , with artesunate having a more favourable pharmacokineticpharmacodynamic profile [15] . they are also more easily produced [12] (table 1) . artemisinins and its derivates are selectively taken up by parasites-infected erythrocytes and later localized in the parasite membranes, including that of the mitochondria, digestive vacuole and the parasite limiting membrane [12, 14] . all forms of the drug contain an endoperoxide bridge (c-o-o-c) that is crucial for its antimalarial activity, where the compound itself is catalyzed by heme or iron to form free radicals. these free radicals then alkylate malaria membrane-associated proteins, killing the parasite [14] . artemisinin and its derivatives are found to be effective against different severities of malaria, especially those resistant to traditional gold standard drugs. they are highly efficacious, requiring only nanomolar concentrations in vitro [14] . they are also fast-acting, showing therapeutic potential as early as 20 hours after administration. moreover, artemisinins display a relatively safe toxicity profile, with the ld50 being 4223 mg/kg. in addition, whilst there was some evidence for neurotoxicity in neuronal cells and animals at high dosages, this was never reported in humans despite the wide usage of the drug [12, 14] . apart from its anti-malarial effects, artemisinin and its derivatives also exhibited additional properties in other diseases. for example, artesunate had anti-cancer effects as shown by its cytotoxic activity against 55 cancer cell lines through its regulation of various processes, including dna damage and repair, apoptosis, and proliferation [16, 17] . artesunate displayed anti-inflammatory properties, as seen by its attenuation of the production of interleukin (il)-1β, il-6 and il-8 in tumour necrosis factor (tnf)α-stimulated rheumatoid arthritis fibroblast-like synoviocytes (ra fls) via the regulation of nf-κb and phosphoinositide 3 kinase (pi3k) pathways [18] . it also displayed anti-viral properties where artemisinin inhibited the replication of human cytomegalovirus (hcmv) through a reduction in the dna binding activity of nf-κb and sp1, and subsequently downstream activities of akt1 and p70s6k. [19] . many of these pathophysiological processes are also present in respiratory diseases. thus, artemisinin and its derivatives could potentially be repurposed for the treatment of respiratory diseases as well. the effects of artemisinin and its derivatives have been examined in various in vitro models (tables 2-5 ) and these include: inhibition of cell proliferation; inductions of cell cycle arrest and apoptosis; inhibition of inflammation and oxidative stress; inhibition of angiogenesis, invasion and metastasis, and chemosensitization of cancer cells to chemotherapeutic agents. the anti-proliferative effect of artemisinin and its derivatives are observed in a variety of lung cancer cell lines, including the non-small cell lung cancer (nsclc) cell lineslung adenocarcinoma a549 [20] [21] [22] [23] [24] , pc-9 [21] , pc-14 [25] , h1299 [22] , astc-a-1 [26] [27] [28] and spc-a-1 [29] cells, squamous carcinoma sk-mes-1 cells and large cell lung cancer nci-h661 cells [29] . interestingly, one study noted that low concentrations (1.25-5 μg/l) of artesunate were unable to prevent the proliferation of a549 cells [30] , suggesting that there may be a therapeutic window by which the drug would have anti-proliferative effects. j o u r n a l p r e -p r o o f in addition, artesunate was found to have anti-proliferative effects in non-cancer cell types. pretreatment with artesunate reduced mitogen-stimulated increases in cyclin d1 protein expression and cell number in both asthmatic and non-asthmatic human cultured airway smooth muscle (asm) cells. this effect was mediated by reductions in p-akt and p-p70s6k protein expressions, which were not observed with dexamethasone treatment [31] . in hcmv, artesunate, but not ganciclovir, reduced the proliferation rates of infected human embryonic lung fibroblasts (helf) [32] . it is worthy to note that they did not have cyotoxic effects on healthy, non-diseased cells, such as in normal human lung fibroblast wi-38 cells [21, 33] , non-cancerous human dermal fibroblasts ccd-1108sk cells [34] and normal hepatic l-02 cells [35] . this finding lends support for the desired clinical property of arteminismins in regimes where inhibiton of cell growth is needed (such as in the cancer setting) without affecting the healthy, non-disease state condition. various studies have shown that artemisinins induce cell cycle arrest at different phases in lung and nasopharyngeal cancer. artemisinin, artesunate, and dha inhibited cell proliferation in a549 and h1299 cells via cell cycle arrest in the g1 phase [22] , with corresponding downregulation of p-akt, p-glycogen and h1299 cells [34] . collectively, these studies show that whilst artemisinins bring about cell cycle arrest, caution should be taken into the study of artemisinins on cell cycle arrest as they affect different stages of the cell cycle and the effects may be cell-type dependent. the apoptotic effects of artemisinins and its derivatives are largely observed in lung cancer cell lines and have been found to induce both the intrinsic and extrinsic pathways of apoptosis (table 3) . dha induces apoptosis in a549 and pc-9 cells. the glycolytic metabolism was attenuated, together with the inhibition of glucose uptake, and lactate and atp production. dha treatment also reduced the levels of p-s6 ribosomal protein, p-mammalian target of rapamycin (mtor), and glucose transporter (glut)1. these effects were enhanced together with the glycolysis inhibitor 2-deoxy-d-glucose (2dg), inducing apoptosis through the activation of caspases 3, 8, and 9, cytochrome c and apoptosis-inducing factor (aif), without raising ros levels [21] . other studies found that artemisinins and its derivatives interestingly, xiao and colleagues observed that blockade of caspases 8 or 9, but not caspase 3, largely inhibited the pro-apoptotic effects of artemisinin [26, 27] . in contrast, gao and colleagues found that silencing either caspase led to almost no activation of all three caspases, suggesting the role of an amplification loop among these caspases [45] . in the latter, there was no loss of mitochondrial membrane potential and cytochrome c release, but an activation of smac and aif release [45] . artesunate similarly induced ros-mediated apoptosis through the release of smac and aif, but this was accompanied by the loss of mitochondrial membrane potential. here, inhibiting caspases 8 or 9 did not have any effect whilst silencing aif did prevent artesunate-induced apoptosis [44]. certain differences regarding the apoptotic effects involving bak and bax were uncovered. xiao and colleagues found that silencing bax and bak by rnai did not have any effect on artemisinin-induced apoptosis, suggesting a bax/bak-independent apoptotic process [26, 27] . however, others observed that silencing bak, but not bax inhibited artesunate-induced apoptosis and aif release. in fact, artesunate was found to only activate bak, not bax [44, 45] . on the other hand, silencing pro-apoptotic bax, but not bak hampered dha-induced apoptosis [39] . interestingly, whilst artesunate treatment in astc-a-1 and a549 cells did not induce a significant levels of ferrous ion and endogenous oxidation stress in a549 cells [35] . together, these studies show that artemisinins induce apoptosis but utilize very different pathways to induce apoptosis even within the same cell lines itself. several studies have indicated the anti-inflammatory effects of artemisinins in vitro with most studies mainly in vivo (table 4 ). beas-2b cells were found to be insensitive to dexamethasone after being exposed to cigarette smoke extract (cse) and tnf-α stimulation. treatment with artesunate was able to reverse this effect and restore hdac2 deactivation that was induced by cse [50] . beas-2b cells artemisinin-daumone hybrid 15 (artd) was able to inhibit the invasion and metastasis of a549 cells, coupled with downregulation of e2f transcription factor 1 (e2f1) and hepatocyte nuclear factor 4 alpha (hnf4a), and upregulation of tumour-suppressive activating transcription factor 3 (atf3) [58] . these show that artemisinins have the potential to impair angiogenesis and metastasis, but its effects were largely explored in the lung cancer setting. thus, it would be interesting to see if similar effects are observed in other respiratory diseases that are implicated with angiogenic and metastatic events. multiple studies have shown that artemisinin and its derivatives could chemosensitize other drugs. 10 µg/ml of dha and 10 µg/ml of doxorubicin was found to be the most optimal concentrations that could reduce a549 cell viability [59] . dha promoted the cytotoxic and apoptotic levels of carboplatin in llc cells via the phosphorylation of p38 [36] . dha together with abt-263 could activate bax-dependent apoptosis in nsclc cells. this was because dha induced downregulation of survivin and an upregulation of bim, contributing to cotreatment-induced cytotoxicity. also, dha downregulated mcl-1 expression which is responsible for drug resistance to abt-263. this anti-tumour effect was also observed in vivo on h1975 xenograft growth in nude mice [48] . similarly, dha also upregulated bax expression in the presence of gefitinib in h1975 cells, alongside an attenuation of p-akt, p-mtor, psignal transducers and activators of transcription (stat)3 and bcl-2 to prevent migration and invasion [38] . surprisingly, the jnk inhibitor sp600125 synergistically promoted dha-induced cell apoptosis in a549 and astc-a-1 cells by activating bax translocation, mitochondrial membrane depolarisation, cytochrome c release and caspase 3 and 9, unlike its usual anti-apoptotic function that suppresses c-jun n-terminal kinase (jnk) and bax [60] . dha also interestingly promoted dictamine-induced apoptosis via a caspase 3-mediated pathway in a549 cells, even though dictamine alone induces s phase cell cycle arrest at low concentrations and cell apoptosis at higher concentrations without the j o u r n a l p r e -p r o o f involvement of caspases or mitochondria [61] . dha and cisplatin ablated cell proliferation and induced apoptosis in both a549 and cisplatin-insensitive a549/ddp cells [56] . dha could also reverse the high resistance of a549 cells to arsenic trioxide to reduce cell viability and promote cell death via higher levels of ros and dna damage, with no adverse effects on normal human bronchial epithelial cells [62] . on the contrary, apoptosis triggered by a combination of dha and gemcitabine in a549 cells was not associated with additional generation of ros as compared to either treatments alone. instead, the combination strongly activated both the bak-mediated intrinsic apoptosis pathway as well as the fascaspase 8-mediated extrinsic apoptosis pathway [63] . moreover, dha can enhance radiosensitization in glc-82 lung cancer cells, inducing apoptosis with heightened expressions of p53 and p21, and lowered expression of bcl-2 [37] . dha coupled with a low dose of ionizing radiation led to irreparable g2/m phase cell cycle arrest as well as apoptosis due to ros generation and the activation of caspases 3 and 8 [39] . in sclc, pre-treatment with transferrin sensitized the multi-resistant h69vp phenotype to artemisinin as they had double the number of transferrin receptors. this combination induced dna fragmentation and apoptosis [64] . treating a549 cells with chloroquine prior to artesunate treatment synergistically promoted cell death, where an increase in the sub-g1 population of cells was observed, and the build-up of acidic vacuoles and ros resulted in cytochrome c release followed by caspase 3mediated apoptosis [33] . conversely, artesunate did not induce a549 cell apoptosis when administered alone or in the presence of local radiotherapy. instead, it induced g2/m phase cell cycle arrest, with heightened no protein, and lessened cyclin b1 and cdc2 mrna expression [40] . all these studies show that various therapies can be used in conjunction with artemisinins to promote its therapeutic effectiveness. the effects of artemisinins in in vivo models are summarized in tables 2-5 and these include disease models of pulmonary fibrosis, acute lung injury (ali), asthma, copd, lung cancer, and npc. in general, the underlying mechanisms implicated in these models include inhibition of oxidative stress, inflammation, airway remodelling features, and tumour formation. artemisitene reduced bleomycin-induced acute inflammatory responses through the activation of the nrf2 pathway, as seen by a reduction in the total number of inflammatory cells, neutrophils, macrophages and lymphocytes, together with lower il-4, il-6, tumomur growth factor (tgf)β and monocyte chemoattractant protein-1 (mcp-1) mrna expressions [66] . artesunate treatment attenuated lung injury in paraquat-intoxicated rats via reductions in tgfβ1, il-10 and tnf-α [70] . 30 mg/kg of artesunate suppressed total, eosinophil and neutrophil inflammatory cell counts as well in an ovainduced model of allergic asthma [68] . it also reduced il-8 levels and total inflammatory and neutrophil cell counts that were increased in a 40 days cigarette smoke-induced lung oxidative damage mouse model. il-8 levels were similarly lowered by artesunate in 16hbe cells exposed to cigarette smoke extract [69] . interestingly, the activation of nlrp3 inflammasome was dependent on pulmonary ros generation accompanied by higher asc and caspase 1 levels [53]. in another study, liu and colleagues also found that artesunate suppressed many rir-stimulated factors involved in lung inflammation, including the production of serum and pulmonary no, mda, macrophage inflammatory protein 2 (mip-2) and prostaglandin e2 (pge2), and attenuated nf-κb translocation [72] . artesunate also protected against sepsis-induced lung injury by reducing il-6 and tnf-α levels in both the serum and balf. in the lung tissues, artesunate suppressed cyclooxygenase-2 (cox-2), inos and nf-κb levels and activated nrf2 through and increase in ho-1 expression and enzymatic activity [73] . the effect of artesunate on copd was similar to that of ali where artesunate dose-dependently suppressed total and differential whether or not changes in inflammation brought about by artesunate was associated with changes in lung function parameters have been reported by one group. here, artesunate treatment in mice exposed to cigarette smoke and ova saw a reduction in methacholine-induced ahr, with efficacies similar to the extent produced by dexamethasone [50] . both artesunate and dha treatment in an ova-induced model of allergic asthma brought about a reduction in ahr [74, 75] . another study found that 120 µg of artesunate relieved ova-induced airway resistance with comparable efficacy to 3 µg of salbutamol through an increase in [ca 2+ ]i and reduced traction force in airway smooth muscle cells, mediated by bitter taste receptor signaling [78] . however, the concentration and dose used in this study are high; suggesting the need to explore whether the same effects could be observed at the lower therapeutic range. artemisinin was also able to improve the behavior scores (sneezing, nasal rubbing) in a mouse model of allergic rhinitis, where mice were given nasal drip of 500 µg of ova [77] . j o u r n a l p r e -p r o o f artemisitene inhibited bleomycin-induced collagen and hydroxyproline expression in mice. the expression of key players of fibrosis, smooth muscle (sm)-α actin and tgfβ were also reduced in bleomycin-treated mice [66] . similar observations were made with dha, which reduced the szapiel fibrotic score and hydroxyproline content with comparable efficacy to dexamethasone in bleomycininduced pulmonary fibrosis in rats [79] . another study also showed that dha treatment reduced interstitial fibrosis, leukocyte infiltration, collagen deposition and sm-α actin expression in lung tissues with heightened e-cadherin expression. the reduction in sm-α actin, normally heightened in the event of oxidative stress, was also seen in dha-treated rat alveolar epithelial cells (aecs) cultured in hypoxia, which shows that dha could inhibit the hypoxia-induced increase in myofibroblastic-like process [65] . artesunate attenuated bleomycin-induced pulmonary fibrosis in sprague dawley rats through a reduction in pro-fibrotic proteins such as tgfβ1, smad3, heat shock protein 47 (hsp47), sm-α actin, and collagen i [80] . the same group also observed that artesunate upregulated mmp2 and mmp9 expressions while reducing tissue inhibitor of metalloproteinases (timp) and timp2 levels, which then contributed to a decrease in collagen iv protein expression, which is otherwise heightened in bleomycininduced pulmonary fibrosis [81] . artemisinin, artesunate, and dha inhibited processes that contribute to tumour malignancy, including migration, invasion, cancer stem cells and epithelial-mesenchymal transition (emt) transition. this was through attenuation of the wnt/β-catenin pathway that contributes to tumour cell proliferation and malignancy, as seen by a reduction in wnt5-a/b protein level and a simultaneous increase in naked cuticle homolog 2 (nkd2) and axis inhibition protein 2 (axin2) that eventually led to a drop in β-catenin levels [22] . artesunate post-treatment also reportedly prevented tgfβ1-induced emt in rle-6tn alveolar epithelial cells by reducing p-smad3 and smad3 and upregulating smad7 protein expressions [82] . artesunate impaired tumour growth and metastasis in a chicken embryo metastasis model, secreted levels of mmp9 and cathepsin k that contribute to the bone-resorbing activity [58] . in addition, oral administration of artemisinin inhibited lymph node and lung metastasis, with no effect on tumour growth in a llc mouse model, promoting longer survival. tumour lymphangiogenesis was also inhibited, with corresponding reduction in vegf-c levels [57] . interestingly, studies done by two different groups found that a combination of dha with either cisplatin or onconase could more effectively ablate the density of the microvasculature and microvessels in an a549 mouse xenograft model [29, 56] . studies by wong and colleagues found that artesunate mitigated mucus hypersecretion via a reduction in muc5ac mrna expression in the lung tissues of ova-challenged asthmatic mice [74, 75] . whether or not similar effects on mucus production and muc5ac expression can be observed using a more clinically relevant allergen such as house dust mite remains to be observed. unfortunately, not much research has looked at the effect of artemisinins on mucus production and alleviating it would be beneficial since excessive mucus production occurs in many lung diseases and impede on patients' comfort levels. tong and colleagues observed that artemisinin, dha and artesunate were all able to reduce tumour growth in an a549-induced mouse xenograft model via inhibition of the wnt-5a/b/β-catenin signaling pathway [22] . artesunate dose-dependently attenuated a549 xenograft growth in mice with a reduction in egfr, akt and atp-binding cassette subfamily member 2 (abcg2) mrna and protein expressions [83] . in addition, artesunate radiosensitized tumour cells to the effects of local radiotherapy [40] . conversely, another group found that 10 mg/kg of artesunate was not sufficient to inhibit a549-induced xenograft growth in mice, although it could potentially block invasion as observed by a reduction in j o u r n a l p r e -p r o o f icam-1 and mmp9 protein abundance [30] . unexpectedly, oral administration of either dlae or artesunate was able to inhibit a549 xenograft growth but only dlae was able to inhibit pc-9 induced tumour growth [34] . the tumour-inhibiting rate of dha as studied in nude mice bearing a549 cells was 54.3% [24] . a combination of dha and abt-263 reduced xenograft growth in nude mice [48] . using an acute model of allergic asthma in mice, artesunate pre-treatment was found to reduce the area of smα actin positive cells in the airways and cyclin d1 protein expression [31] . currently, only one study has investigated the use of artesunate for lung cancer in humans. adding on 120 mg/day of artesunate treatment to vinorelbine and cisplatin chemotherapy was found to promote better disease control and slow the time to disease progression as compared to advanced stage nsclc patients treated with vinorelbine and cisplatin chemotherapy alone. however, no significant differences to short term survival rate, mean survival time and one-year survival rates were observed. importantly, this treatment combination did not produce significant toxic effects [84] . the recent coronavirus disease 2019 (covid-19) pandemic has affected and taken many lives [85] . since vaccines against the novel sars-cov-2 virus, which causes covid-19, may take a long time to be developed, many are repurposing drugs for its treatment. chloroquine (cq) and hydroxychloroquine (hcq) are anti-malarial drugs being tested for covid-19 [86, 87] that have also been used against autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (sle). whilst hcq has displayed a safer toxicity profile than cq [86] , there are still side effects that are of concern. one example is cardiac toxicity, which would be especially dangerous for patients with pre-existing health conditions, like that of cardiovascular diseases, as they would have poorer prognosis for covid-19 [88] . whilst the anti-malarial mode of action of artemisinins are different from cq or hcq, their immunomodulant effects against inflammatory disorders and viral replications are overlapping. traditionally, artemisinins have been used for the treatment of fevers, and could be useful given that 83.3% of patients with covid-19 have fever [89] . given its ability to reduce tnf-α and il-6, key j o u r n a l p r e -p r o o f mediators of acute respiratory distress syndrome (ards) that leads to the worsening of covid-19 patient conditions [90] , artemisinins may be a promising therapy. other molecular targets of artemisinin and its derivatives, as shown in fig. 1 , may also be involved in the pathogenesis of covid-19 and thus may have other benefits that may not yet be known. moreover, artemisinins are known to display a safe toxicity profile so higher doses can be prescribed with less worry about potential side effects. with the understanding that cq and hcq are affective against viruses due to the ph altering activities that affect viral replication, artemisinins could alternatively be used as adjunct therapy to lower the dose required of cq or hcq, and reduce side effects, while also suppressing the cytokine storm. unfortunately, no study to date has investigated the effects or interactions of artemisinins on the angiotensin-converting enzyme 2 (ace2) receptor, that is known to be the critical binding cellular receptor of sars-cov-2 [91] . this can greatly influence the favourability of trying out the effectiveness of artemisinins for covid-19. artemisinins have been used for a long time with high efficacies and relatively safe toxicity profiles. some groups have looked into modifications to artemisinins in order to improve its efficacy and lower the risk of toxic side effects. dha was observed to display poor water solubility and short plasma halflife. dai and colleagues connected dha with a multiarm polyethylene glycol (peg) to produce peg-dha and found that it was 82 to 163 times more water-soluble and its blood circulation half-time was 5.75 to 16.75 times that of dha, all while retaining or improving its anti-cancer efficacy [92] . sun and colleagues encapsulated dha with gelatin or hyaluronan nanoparticles using an electrostatic field system and observed that it inhibited proliferation and promoted apoptosis of a549 cells better than dha [93] . dha loaded with nanostructured lipid carriers (dha-nlc) resided more greatly in organs such as the lung, liver, spleen, brain, and muscle, and less in the heart and kidneys, promoting sustained-release and better drug-targeted effects, therefore allowing for lower dosages and systemic toxic side effects [94] . a c-10 acetal artemisinin synthesized using the sonogashira cross-coupling reaction displayed higher growth inhibition of a549 cells compared to artemisinin. however, it only had moderate effects on other cancer cell lines such as breast, prostate, and neuroblastoma [95] . lastly, yang and colleagues noted that transferrin receptors were overexpressed in cancer cells. they, therefore, developed adducts of transferrin with artemisinin, dha or artesunate and found that their j o u r n a l p r e -p r o o f anti-cancer effects were stronger in a549 cells with improved cellular uptake, whilst having minimal effects on normal human liver hl-7702 cells [96] . we present here an up-to-date overview of the current knowledge of artemisinins and its derivatives as potential therapeutic targets for the treatment of respiratory diseases. fig. 1 ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. respiratory disease forum of international respiratory societies, respiratory diseases in the world world health organization, global surveillance, prevention and control of chronic respiratory diseases pocket guide for asthma management and prevention estimates of worldwide burden of cancer in 2008: globocan biodiversity: a continuing source of novel drug leads desert plants and people a historical overview of natural products in drug discovery pelargonium sidoides extract for treating acute respiratory tract infections the development of the antimalarial drugs with new type of chemical structure--qinghaosu and dihydroqinghaosu anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/akt pathway artemisinin and the antimalarial endoperoxides: from herbal remedy to targeted chemotherapy. microbiological reviews artemisinins: artemisinin, dihydroartemisinin, artemether and artesunate, in milestones in drug therapy molecular modes of action of artesunate in tumor cell lines the anti-malarial artesunate is also active against cancer anti-malarial agent artesunate inhibits tnf-alpha-induced production of proinflammatory cytokines via inhibition of nf-kappab and pi3 kinase/akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes the antiviral activities of artemisinin and artesunate the selectivity of artemisinin-based drugs on human lung normal and cancer cells dihydroartemisinin inhibits glucose uptake and cooperates with glycolysis inhibitor to induce apoptosis in non-small cell lung carcinoma cells artemisinin and its derivatives can significantly inhibit lung tumorigenesis and tumor metastasis through wnt/beta-catenin signaling dihydroartemisinin inhibits cell proliferation via akt/gsk3beta/cyclind1 pathway and induces apoptosis in a549 lung cancer cells the role of calcium, p38 mapk in dihydroartemisinin-induced apoptosis of lung cancer pc-14 cells amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of astc-a-1 cells artemisinin induces caspase-8/9-mediated and bax/bakindependent apoptosis in human lung adenocarcinoma (astc-a-1) cells dihydroartemisinin (dha) induces caspase-3-dependent apoptosis in human lung adenocarcinoma astc-a-1 cells combination of onconase and dihydroartemisinin synergistically suppresses growth and angiogenesis of non-small-cell lung carcinoma and malignant mesothelioma effects of artesunate on the invasion of lung adenocarcinoma a549 cells and expression of icam-1 and mmp-9 the antimalarial drug artesunate inhibits primary human cultured airway smooth muscle cell proliferation human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ros dependent apoptosis dried leaf artemisia annua efficacy against nonsmall cell lung cancer synthesis and in vitro antitumor evaluation of dihydroartemisinincinnamic acid ester derivatives dihydroartemisinin sensitizes lewis lung carcinoma cells to carboplatin therapy via p38 mitogen-activated protein kinase activation dihydroartemisinin and gefitinib synergistically inhibit nsclc cell growth and promote apoptosis via the akt/mtor/stat3 pathway ionizing radiation potentiates dihydroartemisinininduced apoptosis of a549 cells via a caspase-8-dependent pathway artesunate enhances radiosensitivity of human non-small cell lung cancer a549 cells via increasing no production to induce cell cycle arrest at g2/m phase artemisinin-daumone hybrid inhibits cancer cell-mediated osteolysis by targeting cancer cells and osteoclasts in vitro and in vivo inhibition of tumor cell viability by combined dihydroartemisinin and doxorubicin treatment, and the underlying mechanism the jnk inhibitor sp600125 enhances dihydroartemisinin-induced apoptosis by accelerating bax translocation into mitochondria in human lung adenocarcinoma cells dihydroartemisinine enhances dictamnine-induced apoptosis via a caspase dependent pathway in human lung adenocarcinoma a549 cells dihydroartemisinin sensitizes human lung adenocarcinoma a549 cells to arsenic trioxide via apoptosis synergistic induction of apoptosis in a549 cells by dihydroartemisinin and gemcitabine transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells dihydroartemisinin alleviates oxidative stress in bleomycininduced pulmonary fibrosis artemisitene activates the nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury untargeted proteomics and systems-based mechanistic investigation of artesunate in human bronchial epithelial cells anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma effects of artesunate on cigarette smoke-induced lung oxidative damage in mice and the expression of nrf2 and the possible mechanism artesunate attenuates lung injury in paraquat-intoxicated rats via downregulation of inflammatory cytokines artesunate inhibits renal ischemia reperfusion-stimulated lung inflammation in rats by activating ho-1 pathway. inflammation artesunate protects against sepsis-induced lung injury via heme oxygenase-1 modulation. inflammation anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/akt pathway dihydroartemisinin suppresses ovalbumin-induced airway inflammation in a mouse allergic asthma model dihydroartemisinin ameliorated ovalbumin-induced asthma in mice via regulation of mir-183c. medical science monitor : international medical journal of experimental and clinical research effect of artemisinin and neurectomy of pterygoid canal in ovalbumininduced allergic rhinitis mouse model artesunate attenuates airway resistance in vivo and relaxes airway smooth muscle cells in vitro via bitter taste receptor-dependent calcium signalling dihydroartemisinin supresses inflammation and fibrosis in bleomycine-induced pulmonary fibrosis in rats anti-profibrotic effects of artesunate on bleomycin-induced pulmonary fibrosis in sprague dawley rats artesunate modulates expression of matrix metalloproteinases and their inhibitors as well as collagen-iv to attenuate pulmonary fibrosis in rats relationship between artesunate influence on the process of tgf-beta1 induced alveolar epithelial cells transform into mesenchymal cells and on idiopathic pulmonary fibrosis the effects of artesunate on the expression of egfr and abcg2 in a549 human lung cancer cells and a xenograft model artesunate combined with vinorelbine plus cisplatin in treatment of advanced non-small cell lung cancer: a randomized controlled trial world health organisation. coronavirus disease (covid-19) pandemic hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial candidate drugs against sars-cov-2 and covid-19 chloroquine and hydroxychloroquine to treat covid-19: between hope and caution clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis could chloroquine /hydroxychloroquine be harmful in coronavirus disease 2019 (covid-19) treatment? a pneumonia outbreak associated with a new coronavirus of probable bat origin novel multiarm polyethylene glycol-dihydroartemisinin conjugates enhancing therapeutic efficacy in non-small-cell lung cancer enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells dihydroartemisinin loaded nanostructured lipid carriers (dha-nlc): evaluation of pharmacokinetics and tissue distribution after intravenous administration to rats synthesis of a novel series of artemisinin dimers with potent anticancer activity involving sonogashira cross-coupling reaction enhanced delivery of artemisinin and its analogues to cancer cells by their adducts with human serum transferrin orally formulated artemisinin in healthy fasting vietnamese male subjects: a randomized, four-sequence, open-label, pharmacokinetic crossover study review of the clinical pharmacokinetics of artesunate and its active metabolite dihydroartemisinin following intravenous, intramuscular, oral or rectal administration determination of artemisitene in rat plasma by ultra-performance liquid chromatography/tandem mass spectrometry and its application in pharmacokinetics key: cord-286771-77hs34jm authors: cruces, pablo; retamal, jaime; hurtado, daniel e.; erranz, benjamín; iturrieta, pablo; gonzález, carlos; díaz, franco title: a physiological approach to understand the role of respiratory effort in the progression of lung injury in sars-cov-2 infection date: 2020-08-10 journal: crit care doi: 10.1186/s13054-020-03197-7 sha: doc_id: 286771 cord_uid: 77hs34jm deterioration of lung function during the first week of covid-19 has been observed when patients remain with insufficient respiratory support. patient self-inflicted lung injury (p-sili) is theorized as the responsible, but there is not robust experimental and clinical data to support it. given the limited understanding of p-sili, we describe the physiological basis of p-sili and we show experimental data to comprehend the role of regional strain and heterogeneity in lung injury due to increased work of breathing. in addition, we discuss the current approach to respiratory support for covid-19 under this point of view. severe acute respiratory syndrome coronavirus 2 (sars-cov2) pandemic has pushed health systems' response to its maximum capacity. in many countries, the surge of cases has exceeded the facilities, technological, and human resources availability at all levels of care. intensive care units have been overcrowded due to swarming of severe cases in a few weeks, where acute respiratory failure (arf) and acute respiratory distress syndrome (ards) are the main cause of admission. protective lowtidal volume (vt) mechanical ventilation (mv), including delivering a physiologic low vt adjusted by ideal body weight, is currently the standard of care for patients requiring invasive respiratory support, like moderate and severe ards. the surge of patients presenting with sars-cov2 has led to an unprecedented demand of mechanical ventilators, surprising the whole world with a shortage of equipment unthinkable just 6 months ago. due to high demand of invasive mv in many hospitals, mechanical ventilators have become a scarce or nonexistent resource, and other respiratory support strategies have been used, including high flow nasal cannula (hfnc), non-invasive ventilation (niv), and other alternative devices. specific indications for their use are not well defined, consensus guidelines are controversial and frequently they are not followed in clinical practice. the risk of healthcare professional's infection due to aerosolization was suggested as a strong contraindication for hfnc and niv at the beginning of pandemic, contributing in some degree to the shortage of invasive mechanical ventilators. as pandemic reached peak of cases, use of non-invasive devices became widespread. cohort studies form china, italy and north america [1] [2] [3] , showed niv use between 10 and 30% of patients, but when considering single center and small case series it ranges from 0 to 69%. although non-invasive respiratory support may prevent invasive mv, failure of this approach may lead to morbidity and mortality [4] [5] [6] [7] [8] [9] [10] [11] [12] . some patients will remain dyspneic, breathing spontaneously, with or without respiratory support. currently, indirect information suggests that vigorous and dysregulated respiratory effort may be a promoter of lung injury, a phenomenon known as "patient self-induced lung injury" (p-sili) [13] [14] [15] . biomechanical framework for amplification of lung damage: stress and strain the lung can be described as a pre-stressed network of viscoelastic tissue elements deformed by surface tension and the action of respiratory musculature. this characteristic allows deformation in a time-dependent manner upon applied pressure and return to its initial configuration once the pressure is relieved [16] . breathing produces a phenomenon of continuous cyclic strain deformation throughout life, where the applied pressure is inspiratory pressure. in biomechanical terms, deformation in the lung is measured in terms of strain, defined as the relative change in volume normalized by a reference volume. this biomechanical property can be defined for the whole lung (global strain) as the ratio between the vt and a reference volume, usually the volume of air at the end of passive expiration, and the functional residual capacity (frc). correspondingly, the force acting on a surface unit, producing its deformation, is the stress. the transpulmonary pressure corresponds to the stress in the lung. strain and stress in the lung tissue are closely related to each other through a constitutive relation (stress = tissue elastance*strain). both are considered to play an important role in the onset and development of ventilator induced lung injury (vili). high values (non-physiological) of strain, measured as pulmonary tissue deformation relative to volume change, are known to be harmful to the lung and to increase mortality in ards patients under mv [17] . indeed, improved clinical outcomes observed in ards patients due to lower vt corresponds to a reduction of the lung deformation because of mv [18] . these compelling and well-established findings have directed the attention of several groups to understand the regional mechanisms of deformation in mechanically ventilated patients. understanding the global strain in the lung has allowed the identification of thresholds of safer vt to prevent vili, currently present in guidelines and consensuses [17] . in injured lungs, there is a wide spectrum of tissue aeration, producing inhomogeneity of ventilation. lung inhomogeneity has been recently proposed as a vili promoter in ards patients, given the fact that lung injury can occur despite the use of recommended vt and pressures, parameters that are considered to be safe in the ventilation of healthy lungs. the concept of stress raisers may explain these findings. the term stress raisers refer to those additional regional factors capable of intensifying the damage. stress raisers produce amplification of the stress applied in certain localized regions of the lung, like the areas of high inhomogeneity of ventilation [19] [20] [21] [22] . the deleterious effects of high regional strain in the lung was confirmed recently in a swine model of injurious mv, where lung zones of increased regional strain had a spatial correlation with areas of tissue inflammation [23] . this study highlights the relevance of a better understanding of the spatio-temporal progression of regional strain, supporting that strain is a relevant and prominent determinant of vili [23] [24] [25] . the heterogeneous distribution of opening pressures throughout the lung results in an overstretch of the aerated lung zones ("baby lung") and also in collapsed (poorly aerated) regions due to repetitive cycles of recruitment-derecruitment. the generation of injurious mechanical forces is inevitable when invasive mv is applied, due to the heterogeneous nature of ards and the inflation/deflation dynamics of the lungs. there is a coupling between the applied mechanical stimuli and the biochemical response of lung cells, a biological process called mechanotransduction [26, 27] . mechanotransduction can be a pathway of lung injury when the mechanical stimuli are excessive, triggering an inflammatory response in the lung. amplification of lung damage, i.e., vili, depends on the level of energy dissipated by the lung parenchyma and its deformation. the lung does not discriminate the origin of these forces that can be generated by mv or by the respiratory muscles. in this way, biomechanical mechanisms that cause p-sili can occur with or without mv. there is strong evidence that spontaneous ventilation during mv has a role in progression of lung injury [28] . although spontaneous breathing has proved beneficial in the treatment of mild ards patients, opposite effects occurred when lung injury was severe. spontaneous breathing amplified the damage in severe lung injury, increasing transpulmonary pressures, atelectasis, cyclic collapse, and histological signs of damage [28] [29] [30] [31] [32] . the paradox of spontaneous breathing and lung damage can be explained by the solid-like biomechanical behavior of injured lungs. some of the mechanisms described for lung injury from spontaneous effort are increased lung stress/strain, increased lung perfusion, and patient ventilator asynchrony. the generation of vigorous diaphragm contractions induces high negative pleural pressures that will be dissipated along the visceral pleura surface in a homogeneous shape (fluid behavior) in case of healthy lungs, but this dissipation is uneven in case of ards lungs and stress is concentrated in the interphase of collapsed and ventilated lung (solid behavior). this increment in local lung stress has been associated with higher lung inflammation in the dependent lung regions in experimental models. in addition, increment of venous return and oscillations in pulmonary blood flow could favor lung edema production, and finally patient-ventilator dyssyncronies as reverse triggering are associated with increments of vt that may induce vili [30, 33, 34] . there has been a particular emphasis on interventions to prevent mv in recent years, such as hfnc and niv, maintaining spontaneous ventilation and avoiding vili [35] [36] [37] [38] [39] . experimental studies and indirect clinical information have given a counter point to this approach, suggesting that spontaneous unregulated ventilatory effort for extended periods of time can also induce progression of the lung damage [13, 14] . in spite of these facts, it may be counterintuitive the current recommendation to avoid or deliberately delay the start of the mv. currently, the knowledge of p-sili in extubated patients is limited. p-sili occurs in healthy lungs without mv, in some conditions, like an intense increase in minute ventilation ( e). stress failure of blood-gas barrier after forced training in racehorses was described by west et al. in 1993 [40] . similar findings have been described in elite athletes after prolonged high intensity exercise (i.e., triathletes, marathon runners, and swimmers), which in fact can led to pulmonary edema, in absence of cardiac alterations. after intense exercise, bronchoscopic samples have found higher concentration of red blood cells, total proteins, albumin, and inflammatory cells (neutrophils), mimicking the findings in other mammals [41] [42] [43] [44] . these alterations can be correlated to the ones described by mascheroni et al. in an experimental ovine study [45] . the authors observed a serious deterioration in pulmonary function after 3.5-13 h of pharmacologically induced hyperventilation in spontaneously breathing animals without lung disease. these alterations were prevented by mv and sedoparalysis. this study confirms that vigorous spontaneous ventilation can affect the lung and controlled mv can prevent or attenuate the damage of the lung in this setting [45] . the alterations in lung function in this experiment were inversely proportional to the exposure time to hyperventilation. as authors point out, they could not discriminate if only the "mechanical stress" was responsible for these observations. off note is that during the observation period, animals were intubated (infraglotic artificial airway) and without positive pressure ventilation. this experimental design may have contributed to the deterioration of lung function by promoting lung atelectasis. for example, hedenstierna et al. described that perioperative atelectasis collapse can easily reach 50% of the total lung tissue after a few minutes even in uneventful anesthesia [46] . atelectasis could contribute to p-sili by two main mechanisms: reduction of frc and subsequent increment in dynamic strain during tidal ventilation and generation of heterogeneous lung tissue [17, 19, 47, 48] . recently, we developed a 4d tomographic study that employs image-based biomechanical analysis [49] to unveil the volumetric distribution of regional deformation of the whole lung in subjects without mv. in healthy sedated rats under (unassisted) spontaneously breathing, we observed volumetric regional strain and strain heterogeneity, quantifying the magnitude of these deformation indices and its progression in time [50] . given the fact that regional strain and heterogeneity are present during a normal respiratory cycle without harming the lung leads to the question: why p-sili does not develop in normal lungs deformed by physiologic vt? the answer probably is related to many factors, as the amount transpulmonary pressure generated, alveolar-capillary barrier indemnity and the magnitude and topographic distribution of dissipated energy on the lungs. a possible explanation might be that the susceptibility to p-sili depends on the size of the frc, prior to injury induced by high global strain. loss of normally aerated lung volume has two main effects: less lung available for tidal deformation and increased force of diaphragmatic contraction. for a same vt, a lung with lower frc is inherently more susceptible to global regional strain. reduced lung volume has important effects on diaphragm position and function. cephalad displacement results in a greater curvature of the diaphragm and an increase in the size of the zone of apposition. further, diaphragmatic fibers are lengthened, augmenting its capability of generate force during the contraction. if respiratory neuromuscular function is intact, then increased drive translates into stronger diaphragm contraction and larger "swings" of negative pressure. this has been demonstrated in laboratory studies, in which spontaneous effort was greater in more severe lung injury. stronger spontaneous effort is linearly related to larger degrees of pendelluft, as well as greater tidal recruitment and regional strain (fig. 1) . in a follow-up experimental study, we compared animals with acute lung injury under controlled mv and spontaneously breathing without mv. lung injury was induced by lung lavage in rats, followed by 3 h of spontaneous breathing or low vt-mv. micro-ct images were acquired at the beginning and at the end of the observation period, and 4d regional strain maps were constructed. we found a marked tomographic progression of the nonaerated-tissue compartment, and a reduction of the normal-tissue compartment, in accordance to de-recruitment phenomenon. additionally, we found a significant progression of regional fig. 2 regional volumetric strain maps in a 3-h murine model of patient self-inflicted lung injury randomized to two groups: group i: subjects with induced lung injury on low tidal volume mechanical ventilation at the beginning of the experiment (t1) and at the end of the experiment (t3) (upper left and right panels). group ii: subjects with induced lung injury on spontaneous breathing (no mechanical ventilation) at the beginning of the experiment (t1) and at the end of the experiment (t3) (lower left and right panels). progression of regional strain and heterogeneity in time is observed in spontaneous breathing, which reaches volumetric strain levels of up to 80%. regional strain distribution remains more uniform and homogeneous in low tidal volume mechanical ventilation volumetric strain and heterogeneity after spontaneous breathing. in contrast, low vt-mv had limited progression of the regional strain and heterogeneity at the end of the study (fig. 2) [51] . lung heterogeneity has been associated with ards severity and mortality [19] . peri atelectatic alveoli, as mead et al. described in a theoretical model of alveolar interdependence, can concentrate tension until 4times in comparison with the global tension applied to the system [52] . some years ago, our group showed that the peri-atelectatic region in a rat-model of injurious mv presented more inflammation and alveolar disruption than the rest of lung [53] . if we project the alveolar interdependence to heterogeneous lung with multiple collapsed regions, we can explain this as a trigger of inflammation during spontaneous ventilation. our group is currently working on topographic correlation of areas of strain and inflammation in the p-sili model. we measured gene expression pathways on lung tissue homogenate and lung histology. preliminary results are supportive of our hypothesis. regions-ofinterest (roi) with high regional strain had increased expression of genes involved in apoptosis, il-2 signaling, g-protein signaling, activation of ligand-activated ion channels, coagulation, and inflammation, among others, compared to rois with low regional strain (taqman™ array rat inflammation 96-well plates, cat. no. 4414081, thermo fisher scientific, usa) (fig. 3) . a similar gene expression was identified in areas of high stretch in mechanically ventilated rats in a high global lung strain model [24] . off note is that in our p-sili model, animals under spontaneous breathing had higher degree of histopathological damage compared to low vt-mv, specifically alveolar wall disruption and hemorrhage, hyperemia, and leucocyte infiltration (fig. 4) . interestingly, although fig. 3 variation of gene expression in high strain and low strain regions of the lung in a murine model of patient self-inflicted lung injury. a representative images of in vivo/ex vivo fit between tomographic maps of regional strain and 3d digitized frozen lungs. red areas represent high strain regions, while the green/blue areas represent low strain regions in spontaneous breathing. low and high strain regions from the same frozen lung were cut, homogenized, and the rna purified. b gene expression variation of inflammation/pathological mechanotransduction between regions of high and low regional strain. the genes that increased their expression in regions of high deformation were tnf superfamily member 13b (tnfsf13b, > 8 times), interleukin-2 receptor subunit beta (il2rb, > 6 times), phosphodiesterase 4a (pde4a,~3 times), 5hydroxytryptamine receptor 3a (htr3a), plasma kallikrein (klkb1), and leukotriene c4 synthase (ltc4s). these genes are involved in apoptosis, il-2 signaling, g-protein signaling, activation of ligand-activated ion channels, coagulation, and inflammation, respectively biomechanical phenomena and gene expression are regional, lung damage was diffuse. a possible explanation for this is that many of the biomarkers mentioned are water soluble and easily diffusible in plasma and respiratory secretions, so they can be secreted locally, but their consequences are more diffuse, and even at distance. rationale of non-invasive support in arf due to sars-cov2: hypothetical fear vs common practice gattinoni et al. recently described two phenotypes in patients with sars-cov2, "non-ards" type 1 (or type l), and ards, type 2 (or type h) [12, 54] . type 1 refers to initial covid-19 pneumonia, characterized by low elastance, low v/q ratio, low lung weight, and low recruitability. on the contrary, type 2 fulfills classic criteria of ards. in a small case series of 16 patients, the authors described that patients switched from type 1 to type 2 after 1 week of non-invasive support. authors proposed that facing high respiratory drive, p-sili is responsible to progression from type 1 to type 2 covid-19 phenotypes. our initial experimental data suggest that one mechanism of the clinical observation of gattinoni et al. may be due to regional lung volumetric deformation and pathological mechanotransduction induced by high strain-spontaneous breathing. as the lung does not discriminate the origin of the force that produce volumetric deformation, whether that can be generated by mechanical ventilation (vili) or the respiratory muscles. under this point of view, this last mechanism can be more precisely describe as "effort-induced lung injury", instead of p-sili. as some authors have pointed out, the type 1 and type 2 phenotypes are an oversimplification of arf due to sars-cov2, as it is not possible to attribute to a single mechanism the complexity of covid-19. thus, respiratory support, non-invasive and invasive, cannot be decided on a single parameter to prevent potential complications and decrease morbidity and mortality. pathophysiology of covid-19 respiratory failure [55] explains why patients with covid-19 usually present with moderate to severe hypoxemia, so it seems appropriate to use standard oxygen therapy, hfnc and niv as initial respiratory support. due to the discordance of hypoxemia and respiratory distress, it is important to have in mind that previous studies that showed that stratified by severity hypoxemia high vt (greater than 9.5 ml/kg [56] or 9 ml/kg [57] ) predicts failure of niv support. niv failure has been associated to mortality [5] , where high global strain may have a role on progression in lung injury. interestingly, a study showed that the use of the helmet as an interface for niv was associated with a better outcome than the traditional interface. whether the possibility to deliver higher peep could be part of the explanation is not known [7] . high peep could reduce the respiratory drive, the negative pressure swings and global/regional strain due to caudal displacement and shortening of the diaphragm muscle. in this way, sartini et al. recently described the effects of niv and prone position cycles in patients with covid-19 respiratory failure [58] . they found a significant decrease fig. 4 representative images of lung histology of a 3-h murine experimental study where subjects were randomized to three groups: group i: subjects with normal (uninjured lungs) on spontaneous breathing (no mechanical ventilation) (a, b). group ii: subjects with induced lung injury on low vt mechanical ventilation (c, d). group iii: subjects with induced lung injury on spontaneous breathing (no mechanical ventilation) (f-j). in the first image set, no edema or perivascular infiltration is appreciated at ×100 (a) and ×200 (b). in the second image set, minimal amount of perivascular fluid is occasionally observed at ×100 (c-e). in the third image set, we observed alveolar wall disruption and hemorrhage at ×400 (f), perivascular edema and hemorrhage at ×200 (g), intense hyperemia in lung parenchyma vascular bed with signs of initial perivascular edema and leucocyte infiltration at ×200 (h), intense hyperemia and perivascular accumulation of leucocytes at ×100 (i), and perivascular accumulation of polymorphonuclear cell leucocytes and lymphoid cells at ×400 (j) in respiratory rate and an improvement of oxygenation parameters. it is impossible to asses isolated respiratory function in covid-19 respiratory failure as well as other causes ards patients. a clear example in the study of carteaux et al. where immunosuppression and severity also where associated to niv failure [56] . as explained before, hypoxemia is infrequently the primary cause of respiratory distress, so it is important to consider other factors as well as non-respiratory organ disfunctions, like acute kidney injury, myocardial, and severe endothelial dysfunction; all of which are common in sars-cov2 [59] [60] [61] [62] . the correct assessment of these factors gives a unique opportunity to non-respiratory treatments for covid-19. hfnc has shown remarkable results as primary respiratory support in de novo arf [63] , improving oxygenation and decreasing escalation of care and intubation rate when compared to standard oxygen therapy [63] [64] [65] . the benefit may result from the decrease of the anatomic dead space, reducing the ventilatory demand and work of breathing (wob) [66] . in covid-19, hfnc has been shown to be safe, well tolerated and it has a synergistic effect when combined with other treatments like prone position [67] [68] [69] [70] [71] . prone position has been extensively studied in patients with ards and invasive mv, showing an improvement of oxygenation due to many mechanisms, like improving frc, ventilation/perfusion heterogeneity, diaphragm motion in dorsal regions, increasing regional ventilation in dependent lung regions, among others [72, 73] . some principles of prone position can be applied to awake extubated patients, but physiology is still not known in depth. it has been demonstrated as a safe intervention, and currently, it is widely used in emergency room, general wards as well as icu settings [58, [68] [69] [70] [71] . the physiological concepts of hfnc, niv, and prone position can also be applied to patients with hypoxemic arf secondary to sars-cov2 infection. comorbidities are highly relevant in the selection of the selected noninvasive support strategy (i.e., morbid obesity, copd, chronic heart failure). awake prone position could attenuate p-sili by reducing distending pressures, and negative swings of intrathoracic pressure, and more importantly, an increase in frc. theoretically, these mechanisms can improve alveolar interdependence phenomena by decreasing global strain and heterogeneity. a pragmatic approach to arf due to sars-cov2 some authors have highlighted the importance of a physiologic approach to sars-cov2 arf [74] [75] [76] [77] [78] . we strongly recommend a conservative approach to respiratory failure due to sars-cov2. hypoxemia alone (as well as all derived parameters, like p/f ratio) should not precipitate intubation, and pao 2 as low as 50 to 60 mmhg can be tolerated when there is no evidence of low end-organ perfusion or signs of dysoxia. all obvious indications of invasive mv, like hemodynamic instability, alteration of consciousness, should be carefully assessed over time. in our experience, in most of these patients, intubation can be prevented using timely non-invasive support, and treating identified or suspected complications early. a special consideration is to prevent fluid overload in these patients, although most of the time initial presentation is some degree of dehydration when acute kidney injury is not present. off note is that ctscans do not change our usual management. when patients develop an increase of wob (respiratory rate greater than 35 breaths per minute, increase of respiratory muscles work, severe dyspnea and shortness of breath), a promptly evaluation of secondary causes is assessed and treated [78] [79] [80] [81] , including end-organ failure, early suspicion of bacterial superinfection, and thromboembolic events. this is in line with the concept of multidimensional dyspnea assessment coined by banzett et al. [79, 81] . we acknowledge that there is no clear threshold to decide invasive mv, even considering hypoxemia and increased wob. a special limitation is that there is no standardized measurement of respiratory distress in covid-19 respiratory failure. given these facts, there are many aspects of p-sili still in debate and our current understanding is very limited on the role of p-sili in progression of lung disease [78] . the tipping point might be the tolerance of the patient to noninvasive measures and the response to treatment, although it is also a subjective decision. mv is a lifesaving intervention in many situations, but it carries a high risk of complications. p-sili during mv can occur in situations where high respiratory drive cannot be controlled. a short course of deep sedation and neuromuscular blocker (nmb), with daily assessment of discontinuation (nmb-holiday) is recommended [82, 83] . weaning in arf-covid-19 also needs a special consideration. we have observed many situations where increase wob due to high e demand, unrelated to the course of covid-19 pneumonia, can prompt weaning failure, prolonging mv duration, or ultimately extubation failure. usual situations include severe fever due to systemic unresolved inflammation, delirium, superinfection, drug withdrawal, and acidosis. clinicians need to prevent them before weaning and extubation (i.e., early initiation of antipsychotics, early discontinuation of benzodiazepines infusions, temperature control, etc.). all of these aspects are used to grasp our gestalt of arf due to sars-cov2. it includes a multisystemic evaluation (not only respiratory system) to decide appropriate respiratory support, invasive or non-invasive, and correction of other factors that increased e demand and wob [79] . after intubation, usual care to prevent complications are instituted [75, 77] , and conditions for success weaning and extubation are assessed daily, to prevent an excessive duration of mv and morbidity and mortality associated [77, 82, 84] . to our understanding, p-sili might be one of the many factors that can explain progression of lung disease in covid-19. we showed preliminary experimental data that regional lung strain and heterogeneity can be identified in acutely injured lungs under unassisted spontaneous breathing. they are capable to induce progression of lung collapse, inflammation, and progressive alterations of lung mechanics. early use of controlled low vt mv may prevent this progression. given the current pandemic due to sars-cov2 and shortage of medical resources, like mechanical ventilators, the physicians must balance the interventions on respiratory support based on the pathophysiology of arf, but also they must take into account that progression of disease severity might be a consequence of inadequate respiratory support in subjects with high work of breathing, even when it is not primary driven by respiratory failure. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region icu and ventilator mortality among critically ill adults with coronavirus disease 2019 clinical characteristics of coronavirus disease 2019 in china non-invasive ventilation in community-acquired pneumonia and severe acute respiratory failure association between noninvasive ventilation and mortality among older patients with pneumonia effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial noninvasive ventilation of patients with acute respiratory distress syndrome. insights from the lung safe study failure of high-flow nasal cannula therapy may delay intubation and increase mortality cardiac arrest and mortality related to intubation procedure in critically ill adult patients: a multicenter cohort study clinical practice and risk factors for immediate complications of endotracheal intubation in the intensive care unit: a prospective, multiple-center study covid-19 pneumonia: different respiratory treatments for different phenotypes? mechanical ventilation to minimize progression of lung injury in acute respiratory failure ventilation-induced lung injury exists in spontaneously breathing patients with acute respiratory failure: yes spontaneous breathing in early acute respiratory distress syndrome: insights from the large observational study to understand the global impact of severe acute respiratory failure study balancing forces: architectural control of mechanotransduction lung stress and strain during mechanical ventilation: any safe threshold? the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome lung inhomogeneity in patients with acute respiratory distress syndrome the future of mechanical ventilation: lessons from the present and the past regional physiology of ards the role of three-dimensionality and alveolar pressure in the distribution and amplification of alveolar stresses does regional lung strain correlate with regional inflammation in acute respiratory distress syndrome during nonprotective ventilation? an experimental porcine study the link between regional tidal stretch and lung injury during mechanical ventilation deterioration of regional lung strain and inflammation during early lung injury mechanotransduction in the lungs towards prevention of ventilatorinduced lung injury: is mechanotransduction the answer? the comparison of spontaneous breathing and muscle paralysis in two different severities of experimental lung injury spontaneous breathing during lung-protective ventilation in an experimental acute lung injury model: high transpulmonary pressure associated with strong spontaneous breathing effort may worsen lung injury fifty years of research in ards. spontaneous breathing during mechanical ventilation. risks, mechanisms, and management when to promote spontaneous respiratory activity in acute respiratory distress patients? the role of spontaneous effort during mechanical ventilation: normal lung versus injured lung spatial patterns and frequency distributions of regional deformation in the healthy human lung spontaneous effort causes occult pendelluft during mechanical ventilation high-flow nasal oxygen therapy and noninvasive ventilation in the management of acute hypoxemic respiratory failure high-flow nasal cannula oxygen therapy versus conventional oxygen therapy in patients with acute respiratory failure: a systematic review and meta-analysis of randomized controlled trials high flow nasal cannula oxygen versus noninvasive ventilation in adult acute respiratory failure: a systematic review of randomized-controlled trials the clinical practice of high-flow nasal cannula oxygen therapy in adults: a japanese cross-sectional multicenter survey can high-flow nasal cannula reduce the rate of endotracheal intubation in adult patients with acute respiratory failure compared with conventional oxygen therapy and noninvasive positive pressure ventilation?: a systematic review and meta-analysis stress failure of pulmonary capillaries in racehorses with exercise-induced pulmonary hemorrhage highly athletic terrestrial mammals: horses and dogs intense exercise impairs the integrity of the pulmonary blood-gas barrier in elite athletes airway inflammation in nonasthmatic amateur runners bronchial epithelial damage after a half-marathon in nonasthmatic amateur runners acute respiratory failure following pharmacologically induced hyperventilation: an experimental animal study mechanisms of atelectasis in the peri-operative period reabsorption atelectasis in a porcine model of ards: regional and temporal effects of airway closure, oxygen, and distending pressure lung stress and strain during mechanical ventilation: any difference between statics and dynamics? improving the accuracy of registration-based biomechanical analysis: a finite element approach to lung regional strain quantification mapping regional strain in anesthetized healthy subjects during spontaneous ventilation progresión de la deformación regional pulmonar y heterogeneidad es mayor en respiración espontánea en comparación con ventilación mecánica controlada en lesión pulmonar aguda experimental stress distribution in lungs: a model of pulmonary elasticity non-lobar atelectasis generates inflammation and structural alveolar injury in the surrounding healthy tissue during mechanical ventilation covid-19 pneumonia: ards or not? severe covid-19 disease: rather avds than ards? failure of noninvasive ventilation for de novo acute hypoxemic respiratory failure: role of tidal volume predictors of intubation in patients with acute hypoxemic respiratory failure treated with a noninvasive oxygenation strategy respiratory parameters in patients with covid-19 after using noninvasive ventilation in the prone position outside the intensive care unit kidney disease is associated with in-hospital death of patients with covid-19 management of acute kidney injury in patients with covid-19 covid 19 and heart failure: from infection to inflammation and angiotensin ii stimulation. searching for evidence from a new disease endothelial cell infection and endotheliitis in covid-19 high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure high-flow nasal cannula in critically ill subjects with or at risk for respiratory failure: a systematic review and meta-analysis early high-flow nasal cannula oxygen therapy in adults with acute hypoxemic respiratory failure in the ed: a before-after study physiologic effects of high-flow nasal cannula in acute hypoxemic respiratory failure prone positioning in high-flow nasal cannula for covid-19 patients with severe hypoxemia: a pilot study efficacy and safety of early prone positioning combined with hfnc or niv in moderate to severe ards: a multi-center prospective cohort study early self-proning in awake, non-intubated patients in the emergency department: a single ed's experience during the covid-19 pandemic prone positioning in conscious patients on medical wards: a review of the evidence and its relevance to patients with covid-19 infection use of prone positioning in nonintubated patients with covid-19 and hypoxemic acute respiratory failure prone positioning in acute respiratory distress syndrome prone position for acute respiratory distress syndrome. a systematic review and meta-analysis basing respiratory management of covid-19 on physiological principles acute respiratory failure in covid-19: is it "typical a proposed lung ultrasound and phenotypic algorithm for the care of covid-19 patients with acute respiratory failure respiratory pathophysiology of mechanically ventilated patients with covid-19: a cohort study caution about early intubation and mechanical ventilation in covid-19 multidimensional dyspnea profile: an instrument for clinical and laboratory research the affective dimension of laboratory dyspnea: air hunger is more unpleasant than work/ effort validation of the swedish multidimensional dyspnea profile (mdp) in outpatients with cardiorespiratory disease neuromuscular blockers in the acute respiratory distress syndrome: a meta-analysis guidelines on the management of acute respiratory distress syndrome a systematic review of the impact of sedation practice in the icu on resource use, costs and patient safety publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the plataforma experimental bio-ct, faculty of dentistry, from universidad de chile (fondequip eqm150010), for performing the μ-ct analysis. authors' contributions pc and fd conceived the idea of the manuscript. pc took the lead in writing the manuscript, in consultation with fd. jr, deh, and pi were the main reviewers of p-sili during mechanical ventilation section, in consultation with pc and fd. deh and pi were responsible of preliminary results of volumetric strain deformation section. pc and be were the primary reviewers of p-sili without positive pressure ventilation, in consultation with fd. cg analyzed and wrote the preliminary results of histology, in consultation with pc and fd. fd, pc, be, and deh contributed to the discussion in accordance with the preliminary results. all authors were involved in manuscript preparation, and they provided critical feedback to the analysis and discussion. pc and fd are the guarantors of and take responsibility for the content of the manuscript. all authors made substantial contributions to the research, provided final approval of the version to be published, and have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. all authors have read and approved the manuscript. fondo nacional de desarrollo científico y tecnológico grant (fondecyt) # 1160631 to pc, be and deh. ethics approval and consent to participate not applicable consent for publication not applicable the authors have no conflict of interest to disclose. key: cord-288371-uyj4iske authors: arrieta, oscar; cardona, andrés f.; lara, luis; heredia, david; barrón, feliciano; zatarain-barrón, zyanya lucia; lozano, francisco; de lima, vladmir cordeiro; maldonado, federico; corona-cruz, francisco; ramos, maritza; cabrera, luis; martin, claudio; corrales, luis; cuello, mauricio; arroyo-hernández, marisol; aman, enrique; bacon, ludwing; baez, renata; benitez, sergio; botero, antonio; burotto, mauricio; caglevic, christian; ferraris, gustavo; freitas, helano; kaen, diego lucas; lamot, sebastián; lyons, gustavo; mas, luis; mata, andrea; mathias, clarissa; muñoz, alvaro; patane, ana karina; oblitas, george; pino, luis; raez, luis e.; remon, jordi; rojas, leonardo; rolfo, christian; ruiz-patiño, alejandro; samtani, suraj; viola, lucia; viteri, santiago; rosell, rafael title: recommendations for detection, prioritization, and treatment of thoracic oncology patients during the covid‐19 pandemic: the thocoop cooperative group date: 2020-06-20 journal: crit rev oncol hematol doi: 10.1016/j.critrevonc.2020.103033 sha: doc_id: 288371 cord_uid: uyj4iske the world currently faces a pandemic due to sars-cov-2. relevant information has emerged regarding the higher risk of poor outcomes in lung cancer patients. as such, lung cancer patients must be prioritized in terms of prevention, detection and treatment. on may 7th, 45 experts in thoracic cancers from 11 different countries were invited to participate. a core panel of experts regarding thoracic oncology care amidst the pandemic gathered virtually, and a total of 60 initial recommendations were drafted based on available evidence. by may 16th, 44 experts had agreed to participate, and voted on each recommendation using a delphi panel on a live voting event. consensus was reached regarding the recommendations (>66% strongly agree/agree) for 57 questions. strong consensus (>80% strongly agree/agree) was reached for 45 questions. patients with lung cancer represent a particularly vulnerable population during this time. special care must be taken to maintain treatment while avoiding exposure. at the end of 2019, an outbreak of cases of atypical pneumonia was documented in china. the etiological agent was identified as sars-cov-2 (severe acute respiratory syndrome coronavirus 2), and the disease was later known as covid19 . from that initial site, covid-19 has been transmitted in an accelerated j o u r n a l p r e -p r o o f in another study of 105 cancer patients infected with covid-19, reported in the 2020 aacr (dai) the authors documented that lung cancer (22 cases corresponding to 21%) was the most common type of cancer in these series, followed by gastrointestinal, breast cancer, thyroid and hematological malignancies. compared with a control group without malignancies of 536 patients with covid-19, cancer patients had a higher risk of death, (or = 2.34 p = 0.03), admission to the icu (or = 2.84, p = <0.01), development of serious symptoms (or = 2.79, p = <0.1) and a higher probability of requiring mechanical ventilation [5] . patients with lung cancer (n=22) included in this analysis represented the group with the second highest risk of complications, preceded only by hematological tumors, 4 died (18.8%), 6 (27.7%) were admitted to the icu, 11 (50%) developed serious symptoms and 4 (18.8%) required mechanical ventilation. it has become seemingly clear that the covid-19 pandemic is a global health problem, with increasing trends in most world regions, including america. as such, public policies have been implemented to counteract the effect on health systems. given the exponential increase in cases, hospitals have undergone considerable adaptations to offer care for patients with covid-19, many of which require intensive care management. however, this refocusing has affected the care of patients with other serious diseases, including cancer care. in this regard, the urgent need for an adequate allocation and rational use of health systems is evident. the who states that "governments and health systems have an obligation to ensure, to the best of their ability, adequate provision of health care for al". when this guarantee is flailing due to the current pandemic, the prioritization and rational use of resources should, to the best of our ability, be based upon evidence-based recommendations, particularly in time-sensitive conditions. delivery of standard-of-care for every patient at any moment should be the goal of all health providers. in the extreme case where saturated or collapsed health care systems challenge the status quo, a guideline of evidence-based recommendations which can be implemented provisionally without impacting longterm outcomes can aid decision making in the clinical setting. furthermore, the access to potentially curative oncological surgeries has been reduced significantly. in italy during the year of 2019, 371,000 cancer cases were diagnosed of which 80% were surgical candidates, and due to the covid-19 pandemic, the number of surgeries has decreased in the last 30 days [6] . this change and prioritization of health systems in the care of patients with covid-19 has led professionals in the field of oncology to make decisions about which patients should receive oncological treatment. there are already some guidelines and consensus, for the attention of patients with cancer in multiple neoplasia. these guidelines attempt to stratify patients according to risk categories to determine which patients may benefit the most from immediate therapy, and identify those whose clinical scenario will not change because of delayed treatment [7] . this consensus is an effort by a multidisciplinary team of key opinion leaders who are currently faced with the challenge of providing standard-of-care for thoracic malignancies patients in the setting of overwhelmed or collapsed health systems. these recommendations seek to propose management algorithms applicable to a population of patients with thoracic malignancies who might face delays or shortages due to the pandemic. in this way we seek to improve risk stratification, prioritize treatments and reduce complications in the current scenario. it is of utmost important to stress that these should only be considered when the scenario merits it, and not as routine choices. furthermore, some of these treatment recommendations might be influenced by differences in socio-economic conditions and regulatory approvals of cancer drugs between countries as well as specific j o u r n a l p r e -p r o o f government restrictions during the covid19 outbreak. additionally, it is imperative that each clinical decision be made considering the baseline characteristics of the patient, including age and comorbidities. on may 7th, 45 experts (medical oncology, surgeons, pulmonologists and radiotherapists) in thoracic cancers from 11 different countries were invited to participate in this project. by may 16th, 44 had agreed to participate, with a total of 44 experts for the final consensus. a core of 6 experts gathered on may 9th in order to compose a set of clinically relevant questions in the covid-19 pandemic setting. each question was voted on in terms of relevance, application, frequency and available evidence. in the end, a total of 60 questions were considered for inclusion in the voting panel. all questions were thoroughly reviewed in the literature in order to compose an evidence-based recommendation for each. questions in which evidence is scarce or controversial were only considered if they were highly clinically meaningful or frequent scenarios in the current setting. one question was deleted post-voting due to controversial evidence pertaining to the recommendation, leaving a total of 59 questions and recommendations. a modified delphi process was used to establish consensus about whether and how to adapt clinical care during this pandemic for patients with thoracic j o u r n a l p r e -p r o o f malignancies. on may 16th at 10 a.m., all invited experts who agreed to participate logged on to a live voting platform. during this meeting, participants had access to a voting tool in which they would state whether they agreed or disagreed with each recommendation. the delphi scale for each recommendation included the following options "totally disagree; strongly disagree; slightly disagree; neither agree nor disagree; slightly agree; agree; strongly agree; totally agree". during this session, a moderator read each question and displayed in a screen the recommendation, once this was done, panelists had 6 minutes to vote on an 8question section, once the voting was completed the moderator moved to the next section until all recommendations had been voted on. questions for the delphi process were stratified according to 16 main categories which are all clinical scenarios frequently assessed in a thoracic oncology unit, or by any practice which routinely treats patients with thoracic cancers, all questions are summarized in table 1 . once the voting was complete, all answers were categorized into three main categories for each recommendation, and the percentages of each are presented in table 1 . for this purpose, "totally agree" and "strongly agree" were categorized into category 1, "agree" and "slightly agree" into category 2, and "totally disagree; strongly disagree; slightly disagree; neither agree nor disagree" into category 3. a threshold of 66% for agreement (categories 1 and 2) or disagreement (category 3) was required for each question to reach consensus and a threshold of 80% for strong consensus. the criteria mentioned above are recommended for hospital discharge and termination of contact isolation after covid-19 infection [20] . however, positive sars cov 2 rt-pcr results were reported even 13 days after clinical symptoms mentioned resolved [21] . this may be related to the immune status of each patient, use of glucocorticoids, and time of virus clearance [22] . esmo recommends restarting treatment after 2 negative rt-pcr tests with a one week interval following clinical [14] . vats and thoracotomy approaches [25] . also, there is information from nonrandomized phase i and ii studies comparing sbrt with surgery, a conjunct j o u r n a l p r e -p r o o f analysis of these trials showed a better os at 3 years in patients treated with sbrt [25] . so, sbrt could be considered an option in these patients [26] . if the patient is also candidate of adjuvant rt (r1 or pathological n2), the treatment could be administered after the completion of chemotherapy, or delayed till 3 months after surgery. in a review evaluation of 3500 of the national cancer database, the patients that received sequential chemoradiation had a better survival compared with the group of concomitant chemoradiation [27] [28] [29] . recommendation: hypofractionated radiotherapy is not recommended in this clinical scenario. although hypofractioning is gaining acceptance, high technology is necessary in order to implement this modality. the dose should be 50-60gy in 25-30 fractions [30, 31] . recommendation: every patient should be considered for chemoradiation the standard treatment is chemoradiation followed by durvalumab in patients who did not progress during the next 42 days. the two-year survival of stage iii nsclc patients ranges from 24-55% [32] , but increases to 66% with concomitant j o u r n a l p r e -p r o o f chemoradiation followed by durvalumab [33] . hypofractionation is not recommended concurrently with chemotherapy. recommendation: a concurrent approach is preferred, although sequential treatment can be kept in mind for specific cases in which toxicity is considerable. concurrent chemoradiation over sequential treatment is superior in terms of os, but the benefit is modest and the toxicities could be higher, so the risk for immunosuppression in the pandemic could be less for the sequential therapy [34, 35] . the choice of treatment should be made based on clinical features like patients´ symptoms, rate of disease progression, disease burden, (i.e. patients with hilar tumors or vascular compression) could be treated with rt [36] . no hypofractionation or only mild hypofractionation (i.e. 50gy in 20fx) is recommended. while this modality could prolong the treatment duration it can delay the time to chemoradiation with the expected immunosuppressive effects and daily visits. recommendation: g-csf should not be routinely used, only if neutropenia develops and represents an issue. in this scenario the use of g-csf is associated with a higher probability of toxicity when administered during chemoradiation, however, a review readdressed this question for the safety of this combination [40] . recommendation: hypofractionated schedules in this clinical setting are not currently fully supported by available evidence. nonetheless, the approach could be an option for treatment in some patients that have access to the technology. shorter courses of rt are associated with less immunosuppression in other cancers, though currently evidence for lung cancer in this clinical setting is scarce and therefore, an evidence-based recommendation cannot be made [40] . this approach could, however, diminish the risk of infection by minimizing the number of visits to the hospital to receive treatment [38, 39] . nonetheless, hypofractionated schedules increase risk of radiation pneumonitis and should be decided in case by case scenario [41] . in many retrospective cohorts, delays on treatment have an impact on prognosis, especially in earlier stages [42] . similarly, in advanced disease, shorter delays were correlated with poorer outcomes. the association reflects the biology of the disease, as symptomatic patients often receive expedited treatment to control symptoms [43, 44] . considering the highly effective therapies available in the firstline setting with a clear impact in os, pfs, and orr its necessary to make a counterbalance considering risk and benefits [45] . patients that have oligometastatic disease with low disease burden may be suitable for an aggressive approach (surgery or radiotherapy, ablative techniques) to all metastatic lesions looking for a curative intention strategy [46] . docetaxel has a modest orr benefit compared with best supportive care (less than 10%) and a median pfs of 2-3 months. meanwhile, pemetrexed has demonstrated similar efficacy but a more favorable toxicity profile [45] . considering though this has not been evaluated in this particular scenario, many studies have shown evidence regarding the use of the serum level of cea as a prognostic and predictive factor for recurrence and death [47] [48] [49] . guidelines do not recommend determination of serum cea, however, considering potential delays in response evaluation during this pandemic, it could provide valid information. [49] 10.0 non oncogene-driver mutations and suitable for immunotherapy immunotherapy be considered a safe treatment during the pandemic? recommendation: immunotherapy should be administered to all candidate patients. until now, no evidence of an increased mortality has been documented, and a recent study shows pd-1 blockade in lung cancer is not associated with increased severity of covid-19. theoretically patients under immunotherapy could be more immunocompetent than non-users, thus potentially a greater inflammatory response could be established. cytokine release syndrome (crs) is a rare complication seen with car-t cells therapy or pd-1 inhibitors characterized by an increased level of il-6 and ifnγ [50] . the acute respiratory distress syndrome (ards) is one of the most lethal complications in almost one third of patients in this pandemic, due to a secondary cytokine storm that produce a hyperactivation of t-cells that contribute to the j o u r n a l p r e -p r o o f severe immune injury. this proinflammatory state in the covid-19 patients could progress to an acute inflammatory distress syndrome ards or even to multiorgan failure [51, 52] . nonetheless, current evidence suggests that pd-1 blockade does not impact the severity of covid-19 in patients with lung cancer [53] . recently in a model-based approach pembrolizumab 400mg 6-weekly 6w was compared with 3-weekly 3w approved regimens in terms of pharmacokinetic and security. the 6w regimen had similar predicted exposure, likewise, fewer than 1% of patients had transiently lower concentrations compared to 3w regimens, nonpeak concentrations over the security dose of 10mg/kg [54] . nivolumab 480mg 4w regimen is recommended too based in a success pharmacokinetic (pk) analyses comparing with 3mg/kg and flat dose of 240mg 2w [55] . in addition, durvalumab 1500mg 4-weekly regimen has been explored in the caspian trial in extensive sclc [37] and it is being tested in the ongoing pacific (2,4,5 and 6) trials [56] with an acceptable safety. additionally, atezolizumab can be administered 1680 q4w, a dosing regimen that has been shown to be interchangeable with 1200 q3w, but offers patients longer visit intervals [57] . all the regimens are fda approved. recommendation: patients on tkis should be monitored for pulmonary symptoms in every visit or telephone call, but patients suitable to receive tkis must do so. one concern is the increased risk of pneumonitis in patients with nsclc during tkis treatment. based on a recent metanalysis the overall incidence of egfr-tki pneumonitis was 1.12% in patients without prior exposure to egfr-tki, and 1.13% in egfr-tki retreatment group. grade ≥3 pneumonitis was presented in 0.81% of patients in the total cohort [58] . likewise, all grade and grade ≥3 pneumonitis were reported in 2.14% and 1.55% respectively, of patients with an alk inhibitors [59] . further, data from the alk in lung cancer trial of brigatinib in 1st line (alta-1l) showed patients treated with brigatinib and crizotinib presented with g3/4 interstitial lung disease or pneumonitis in 3% and 0.7% of cases, respectively [60] . chemotherapy has shown to prolong os in patients with ps2. also, a metanalysis confirmed the benefit of platinum-based regimens compared with monotherapy in this population, at the cost of an increase in hematologic toxicity, more grade 3-4 anemia and neutropenia [65, 66] . regarding platinum therapy, superiority of carboplatin-based combination over monotherapy has been reported in two large j o u r n a l p r e -p r o o f phase iii trials with an acceptable toxicity profile [67, 68] . therefore, platinumpreferably carboplatin doublets could be considered in eligible ps 2 patients, but during this pandemic the risk of contracting the infection should be considered. tkis demonstrated in many phases 3 trials a more favorable toxicity profile compared with chemotherapy. additionally, they help achieve longer responses. on the other hand, treatment beyond progression in slow progressive disease could be employed as a valid strategy. considering risks during pandemic, longer monitorization of these patients could be considered an option, preferable with intermediate evaluation with telemedicine or phone calls as back-up resources. regimens with immunotherapy treatment could be followed with 4-weekly outpatient visits rather than 3-weekly? recommendations: patients on chemotherapy or combination treatments with immunotherapy could be monitored with 4-weekly outpatient visits. it is not recommended to delay the studies in these patients. however, once a patient has been diagnosed to have extensive stage disease, further standing is not required, except for brain imaging [74] . recommendation: if surgery times are prolonged, chemotherapy or sbrt can be used instead. only 5% of patients present in early stages are candidates for surgical treatment, and the decision to carry out surgery should be discussed by a multidisciplinary team, since a complete evaluation of the mediastinum is required, this in addition recommendation: the omission/delay of chemotherapy treatment is not recommended due to the high rate of growth. due to the high rate of tumor growth that occurs in the sclc and that it is considered a systemic disease from the start, the omission/delay of standard chemotherapy treatment is not recommended in these patients [77] . recommendation: delaying the starting time of rt treatment is not recommended due to the high rate of growth; moreover, early initiation has a benefit in survival. the use of concurrent chemotherapy with radiotherapy is the standard of treatment for patients with limited disease due to the impact on survival. in patients with limited disease, the use of etoposide and cisplatin with radiotherapy has response rates of 70% to 90% with a 5-year survival of 25% to 30%. use of concurrent vs. sequential therapy has been questioned [76] , but several studies, including a cochrane study, have shown benefit for the early start of rt [78] . therefore, if j o u r n a l p r e -p r o o f possible, early radiation initiation is recommended, but if toxicity is an important issue a sequential approach could be an option. recommendation: considering the current situation, the best option is to delay the administration of radiotherapy until 6 months after the start of the adjuvant treatment without having a significant impact on oncological outcomes. sclc patients are a high-risk population for developing brain metastases, which are associated with poor survival. as such, it is not recommended to suspend the administration of pci in these patients. one could, however, consider delaying the administration of the therapy until 6 months after the start of the adjuvant without significantly impacting on the outcomes. pci has been shown to be effective in a meta-analysis of seven randomized studies that included 978 patients. it showed a reduction in the incidence of metastases (relative risk [rr] 0.46; 95% ci 0.38-0.57) and a decrease in mortality (rr 0.84; 95% ci 0.73-0.97). in the same study, a subgroup analysis showed there was no difference in mortality when starting radiotherapy less than 6 or more than 6 months after starting chemotherapy treatment, there was only a higher risk of developing brain metastases in patients that started pci later than 6 months [79] . regular contrastenhanced cranial mri follow up should be available if pci is not performed. recommendation: due to lower probability of developing hematological toxicity, cisplatin should be considered. the substitution of cisplatin for carboplatin could be considered due to its different toxicity profile, however taking into account that the neutropenia rate is increased, and that the current evidence shows that the use of granulocyte colony stimulating factor in conjunction with chemotherapy and radiotherapy increases the toxicity. in recommendation: pci can be omitted because of the controversial benefit in survival. in extensive disease the benefit of prophylactic radiotherapy to the brain is controversial due to discordant results. in addition to the current systemic treatment, the recommendation is that it could be omitted to decrease the risks of the patients by offering follow-up only. in malignant pleural mesothelioma (mpm) is a relatively low-frequency tumor, nonetheless worldwide incidence has been rising during the last decade, and it is likely that this trend will continue. the exposure to asbestos is accountable for approximately 80% of mpm cases [87] . following this pattern, several large-scale epidemiological analyses predicts that incidence in europe and latin america will reach its highest historic level in 2020 [88] . screening for mesothelioma in high risk population has been studied and is not recommended based on the absence of benefit in mortality. patients with mesothelioma often present with high symptoms burden that require attention promptly, even in early stages. considering the limited access to surgery and hospitalization risks, neoadjuvant treatment could be preferred over adjuvant approaches during the pandemic. extrapleural pneumonectomy is associated with increased mortality and morbidity, despite this, it is a valid procedure considering pandemic situation, total pleurectomy is considered safer [90, 91] . recommendation: we currently do recommend trimodal treatment, including radiotherapy. this is a fast-progressing tumor which is radiosensitive and therefore os benefit could be achieved with trimodal therapy. the efficacy of trimodal treatment using chemotherapy surgery and hemithoracic radiotherapy has been explored in some retrospective analyses and phase 2 trials in patients who complete the treatment has been reported [92] [93] [94] [95] [96] . however, an important increased risk of developing pneumonitis up to 30% has to be assumed [97] . recommendation: patients could be considered for deferring cytotoxic therapy if they are asymptomatic and have a low burden of disease. some guidelines recommend deferring treatment in asymptomatic patients with good functional status with unresectable disease, considering starting treatment after clinical or radiographical progression. this could be a good option for selected patients that could be tracked to identify symptoms of progression during pandemic [89] . recommendation: consider starting first-line treatment in every symptomatic patient preferably with a carboplatin-based regimen in combination with pemetrexed. comparison data has emerged between cisplatin versus carboplatin regimens combined with pemetrexed in medically inoperable population. oncological outcomes in a cohort of more than 1,700 patients were similar [96] . guidelines recommend carboplatin-based regimen even in patients with good functional status j o u r n a l p r e -p r o o f ps 0-1. options for patients who are not candidates for platinum could be monotherapy regimens with pemetrexed or vinorelbine with poorer outcomes [98, 99] . recommendation: due to the lack of efficacy in the available data, pemetrexed maintenance therapy is not be recommended. regarding bevacizumab, we do not consider the benefit could outweigh the risks of covid-19 infection. two trials, and one phase 3 trial have demonstrated an os benefit with bevacizumab both during the induction phase and as maintenance. remarkably, the median os benefit does not exceed the 3 months, at the cost of increased grade 3-4 adverse events [100] . additionally, considering the extra visits, omitting maintenance during the pandemic is advised [101] . the efficacy of pemetrexed maintenance therapy is not well stablished and should not be recommended as well. recommendation: second-line treatment in asymptomatic patients could be delayed until clinical or radiological progression, considering risks during the pandemic. limited data are available to guide second-line treatment and beyond. prognosis in patients who progress is ominous, and a standard of care is not available. limited evidence from phase ii trials have identified subgroups of patients who will benefit j o u r n a l p r e -p r o o f from receiving subsequent therapy. in the other hand checkpoint inhibitors therapy is emerging in this scenario and could be used for symptomatic patients, independently of pdl1-expression. a monotherapy strategy with pembrolizumab [102] [103] [104] or nivolumab [105] could represent safer options when compared with the combined treatment with nivolumab and ipilimumab [106, 107] . in the midst of the covid-19 pandemic, oncologists will need to weigh the risks of death and morbidity from covid-19 against the magnitude of benefit of intended cancer therapies. early estimates from china suggest an overall case fatality rate of 2%, increasing to 8% for 70-79 year-olds, and 15% for those ≥80 years of age [108] . case fatality rates (cfr) are also markedly higher among patients with comorbidities, 11% for cardiovascular disease, 7% for diabetes and 6% for chronic respiratory disease. patients with cancer are among those most vulnerable to severe illness from respiratory viral infections [109] . have not yet been strongly affected by this pandemic. there is scarce information about the potential consequences of modifications to the standard of care. additionally, until now, the impact, timeline, and duration of pandemic remain unknown; consequently, the uncertainty for cancer patients regarding their treatment will last longer. however, with this pandemic having reached all areas across the globe, there is an increasing need for guidance for all oncologists to optimize resources, until this current crisis is over. with some luck, in the short future, we will recover the certainty and security to treat cancer patients using the established standard of care. dr. oscar arrieta reports personal fees from pfizer, grants and personal fees from astra zeneca, grants and personal fees from boehringer ingelheim, personal fees from lilly, personal fees from merck, personal fees from bristol myers squibb, grants and personal fees from roche, outside the submitted work. all the remaining authors declare no conflict of interest. j o u r n a l p r e -p r o o f centro de investigación y manejo del cáncer (cimca) hospital vivián pellas coordinador de la sección oncología centro oncológico riojano integral hospital de rehabilitacion respiratoria maría ferrer unidad oncológica molecular peruana thoracic oncology program memorial cancer institute, memorial healthcare system centro integral oncología clara campal bacelona clínica colsanitas speaker: msd, roche, astra zeneca, bms, novartis. advisory and consulting: msd viteri reports personal fees and non-financial support from roche, personal fees from bms, non-financial support from osepharma, personal fees from msd, non-financial support from merck serono outside the submitted work reports personal fees from astrazeneca principal investigator: bms consulting or advisory role, company: astrazeneca; travel, accommodations, expenses company: mundipharma, boehringer ingelheim, astra zeneca dr. luis mas: speaker bristol-myers squibb and the foundation for clinical and applied cancer research -ficmac., other from pfizer radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study covid-19 in latin america: the implications of the first confirmed case in brazil case-fatality rate and characteristics of patients dying in relation to covid-19 in italy clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan patients with cancer appear more vulnerable to sars-cov-2: a multi-center study during the covid-19 outbreak the need of covid19 free hospitals to maintain cancer care cancer guidelines during the covid-19 pandemic clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china rational use of face masks in the covid-19 pandemic correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study how we treat patients with lung cancer during the sars-cov-2 pandemic: primum non nocere. esmo open lymphopenia in cancer patients and its effects on response to immunotherapy: an opportunity for combination with cytokines? the role of a cachexia grading system in patients with non-small cell lung cancer treated with immunotherapy: implications for survival surgical operations during the covid-19 outbreak: should elective surgeries be suspended? cancer patients in sars-cov-2 infection: a nationwide analysis in china sensitivity of chest ct for covid-19: comparison to rt-pcr positive rt-pcr test results in patients recovered from covid-19 early corticosteroid treatment for severe pneumonia caused by 2009 h1n1 influenza virus adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage ib&#x2013;iiia non-small-cell lung cancer (adjuvant navelbine international trialist association [anita]): a randomised controlled trial. the lancet oncology thoracoscopic lobectomy: report on safety, discharge independence, pain, and chemotherapy tolerance. the journal of thoracic and cardiovascular surgery long-term survival after lobectomy for non-small cell lung cancer by videoassisted thoracic surgery versus thoracotomy long-term follow-up on nrg oncology rtog 0915 (ncctg n0927): a randomized phase 2 study comparing 2 stereotactic body radiation therapy schedules for medically inoperable patients with stage i peripheral non-small cell lung cancer 30 gy or 34 gy? comparing 2 single-fraction sbrt dose schedules for stage i medically inoperable non-small cell lung cancer lung adjuvant cisplatin evaluation: a pooled analysis by the lace collaborative group association of delayed adjuvant chemotherapy with survival after lung cancer surgery practice recommendations for lung cancer radiotherapy during the covid-19 pandemic: an estro-astro consensus statement alternative multidisciplinary management options for locally advanced non-small cell lung cancer during the covid-19 global pandemic the iaslc lung cancer staging project: proposals for revision of the tnm stage groupings in the forthcoming (eighth) edition of the tnm classification for lung cancer durvalumab after chemoradiotherapy in stage iii non-small-cell lung cancer. reply sequential vs. concurrent chemoradiation for stage iii non-small cell lung cancer: randomized phase iii trial rtog 9410 meta-analysis of concomitant versus sequential radiochemotherapy in locally advanced non-small-cell lung cancer predicting the need for palliative thoracic radiation after first-line chemotherapy for advanced nonsmall cell lung carcinoma durvalumab plus platinum&#x2013;etoposide versus platinum&#x2013;etoposide in first-line treatment of extensive-stage small-cell lung cancer (caspian): a randomised, controlled, open-label, phase 3 trial. the lancet phase iii study of the eastern cooperative oncology group (ecog 2597): induction chemotherapy followed by either standard thoracic radiotherapy or hyperfractionated accelerated radiotherapy for patients with unresectable stage iiia and b non-small-cell lung cancer induction chemotherapy followed by chemoradiotherapy compared with chemoradiotherapy alone for regionally advanced unresectable stage iii non-small-cell lung cancer: cancer and leukemia group b chemoradiation and granulocyte-colony or granulocyte macrophagecolony stimulating factors (g-csf or gm-csf): time to think out of the box? abnormal pulmonary function tests predict the development of radiation-induced pneumonitis in advanced non-small cell lung cancer effect of time to treatment on survival in non-small cell lung cancer treatment delays in non-small cell lung cancer and their prognostic implications effect of delays on prognosis in patients with non-small cell lung cancer metastatic non-small cell lung cancer: esmo clinical practice guidelines for diagnosis, treatment and follow-up comparison of multi-fraction versus single-fraction stereotactic body radiation therapy (sbrt) for symptomatic bone metastasis: results of the stat rt and stat rad phase i/ii prospective trials brain metastasis development and poor survival associated with carcinoembryonic antigen (cea) level in advanced non-small cell lung cancer: a prospective analysis usefulness of serum carcinoembryonic antigen (cea) in evaluating response to chemotherapy in patients with advanced non small-cell lung cancer: a prospective cohort study carcinoembryonic antigen and cytokeratin-19 fragments for assessment of therapy response in non-small cell lung cancer: a systematic review and meta-analysis cytokine release syndrome during sequential treatment with immune checkpoint inhibitors and kinase inhibitors for metastatic melanoma advances in the research of cytokine storm mechanism induced by corona virus disease severe cytokine release syndrome in a patient receiving pd-1-directed therapy. pediatr blood cancer impact of pd-1 blockade on severity of covid-19 in patients with lung cancers a six-weekly dosing schedule for pembrolizumab in patients with cancer based on evaluation using modelling and simulation thoracoscopic surgery versus thoracotomy for lung cancer: short-term outcomes of a randomized trial pacific-2: phase 3 study of concurrent durvalumab and platinumbased chemoradiotherapy in patients with unresectable, stage iii nsclc alternative dosing regimens for atezolizumab: an example of model-informed drug development in the postmarketing setting pneumonitis in advanced non-small-cell lung cancer patients treated with egfr tyrosine kinase inhibitor: meta-analysis of 153 cohorts with 15,713 patients: metaanalysis of incidence and risk factors of egfr-tki pneumonitis in nsclc the incidence of alk inhibitor-related pneumonitis in advanced non-smallcell lung cancer patients: a systematic review and meta-analysis. lung cancer brigatinib versus crizotinib in alk-positive non-small-cell lung cancer first-line erlotinib therapy until and beyond response evaluation criteria in solid tumors progression in asian patients with epidermal growth factor receptor mutation-positive non-small-cell lung cancer: the aspiration study epidermal growth factor receptor tyrosine kinase inhibitors beyond progressive disease: a retrospective analysis for japanese patients with activating egfr mutations continued treatment with gefitinib beyond progressive disease benefits patients with activating egfr mutations. lung cancer systemic treatment options for brain metastases from non-small-cell lung cancer what can platinum offer yet in the treatment of ps2 nsclc patients? a systematic review and meta-analysis treatment of advanced non-small-cell lung cancer patients with ecog performance status 2: results of an european experts panel carboplatin and weekly paclitaxel doublet chemotherapy compared with monotherapy in elderly patients with advanced non-small-cell lung cancer: ifct-0501 randomised randomized phase iii trial of single-agent pemetrexed versus carboplatin and pemetrexed in patients with advanced non-small-cell lung cancer and eastern cooperative oncology group performance status of 2 phase ii trial of single-agent oral vinorelbine in elderly (> or =70 years) patients with advanced non-small-cell lung cancer and poor performance status metronomic oral vinorelbine as first-line treatment in elderly patients with advanced non-small cell lung cancer: results of a phase ii trial (move trial) telemedicine for cancer patients during covid-19 pandemic: between threats and opportunities hyperprogressive disease in patients with advanced non-small cell lung cancer treated with pd-1/pd-l1 inhibitors or with single-agent chemotherapy immune-related adverse events with immune checkpoint inhibitors in thoracic malignancies: focusing on non-small cell lung cancer patients characteristics and outcomes of small cell lung cancer patients diagnosed during two lung cancer computed tomographic screening programs in heavy smokers american college of chest physicians evidence-based clinical practice guidelines a meta-analysis of thoracic radiotherapy for small-cell lung cancer systematic review evaluating the timing of thoracic radiation therapy in combined modality therapy for limited-stage small-cell lung cancer time between the first day of chemotherapy and the last day of chest radiation is the most important predictor of survival in limited-disease small-cell lung cancer prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission carboplatin-or cisplatin-based chemotherapy in first-line treatment of small-cell lung cancer: the cocis meta-analysis of individual patient data first-line atezolizumab plus chemotherapy in extensive-stage small-cell lung cancer role of thoracic consolidation radiation in extensive stage small cell lung cancer: a systematic review and meta-analysis of randomised controlled trials use of thoracic radiotherapy for extensive stage small-cell lung cancer: a phase 3 randomised controlled trial prophylactic cranial irradiation in extensive small-cell lung cancer prophylactic cranial irradiation versus observation in patients with extensive-disease small-cell lung cancer: a multicentre, randomised, open-label, phase 3 trial a systematic analysis of efficacy of second-line chemotherapy in sensitive and refractory small-cell lung cancer malignant pleural mesothelioma: esmo clinical practice guidelines for diagnosis, treatment and follow-up malignant pleural mesothelioma: history, controversy and future of a manmade epidemic treatment of malignant pleural mesothelioma: american society of clinical oncology clinical practice guideline a systematic review and meta-analysis of surgical treatments for malignant pleural mesothelioma extra-pleural pneumonectomy versus no extra-pleural pneumonectomy for patients with malignant pleural mesothelioma: clinical outcomes of the mesothelioma and radical surgery (mars) randomised feasibility study multicenter phase ii trial of neoadjuvant pemetrexed plus cisplatin followed by extrapleural pneumonectomy and radiation for malignant pleural mesothelioma trimodality treatment of malignant pleural mesothelioma: an institutional review long-term results in malignant pleural mesothelioma treated with neoadjuvant chemotherapy, extrapleural pneumonectomy and intensity-modulated radiotherapy trimodality therapy with induction chemotherapy followed by extrapleural pneumonectomy and adjuvant high-dose hemithoracic radiation for malignant pleural mesothelioma pemetrexed plus cisplatin or pemetrexed plus carboplatin for chemonaïve patients with malignant pleural mesothelioma: results of the international expanded access program phase ii study of hemithoracic intensity-modulated pleural radiation therapy (imprint) as part of lung-sparing multimodality therapy in patients with malignant pleural mesothelioma active symptom control with or without chemotherapy in the treatment of patients with malignant pleural mesothelioma (ms01): a multicentre randomised trial phase ii study of pemetrexed with and without folic acid and vitamin b12 as front-line therapy in malignant pleural mesothelioma bevacizumab for newly diagnosed pleural mesothelioma in the mesothelioma avastin cisplatin pemetrexed study (maps): a randomised, controlled, open-label, phase 3 trial phase ii study of pemetrexed and carboplatin plus bevacizumab as first-line therapy in malignant pleural mesothelioma clinical safety and activity of pembrolizumab in patients with malignant pleural mesothelioma (keynote-028): preliminary results from a non-randomised, openlabel, phase 1b trial 03 long-term overall survival for patients with malignant pleural mesothelioma on&#xa0;pembrolizumab enrolled in keynote-028 pembrolizumab as palliative immunotherapy in malignant pleural mesothelioma programmed death 1 blockade with nivolumab in patients with recurrent malignant pleural mesothelioma nivolumab or nivolumab plus ipilimumab in patients with relapsed malignant pleural mesothelioma (ifct-1501 maps2): a multicentre, open-label, randomised, non-comparative, phase 2 trial ipilimumab and nivolumab in the treatment of recurrent malignant pleural mesothelioma (initiate): results of a prospective, single-arm, phase 2 trial characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention respiratory viral infections in patients with cancer or undergoing hematopoietic cell transplant cancer is associated with severity and mortality of patients with covid-19 a systematic review and meta-analysis hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states treatment guidance for lung cancer patients during the covid-19 pandemic localized (i-iiia) mesothelioma? patients with localized diseased must be discussed in a multidisciplinary session aimed for curative intent, considering neoadjuvant chemotherapy followed by surgery (preferably total pleurectomy/decortication). do you have any consideration for trimodality (cht, surgery & rt) treatment? we currently do recommend trimodal treatment, including radiotherapy. this is a fast-progressing tumor which is radiosensitive and therefore os benefit could be achieved with trimodal therapy. key: cord-322756-ouvn71r9 authors: chow, michael y.t.; qiu, yingshan; lam, jenny k.w. title: inhaled rna therapy: from promise to reality date: 2020-09-04 journal: trends pharmacol sci doi: 10.1016/j.tips.2020.08.002 sha: doc_id: 322756 cord_uid: ouvn71r9 rna-based medicine is receiving growing attention for its diverse roles and potential therapeutic capacity. the largest obstacle in its clinical translation remains identifying a safe and effective delivery system. studies investigating rna therapeutics in pulmonary diseases have rapidly expanded and drug administration by inhalation allows the direct delivery of rna therapeutics to the target site of action while minimizing systemic exposure. in this review, we highlight recent developments in pulmonary rna delivery systems with the use of nonviral vectors. we also discuss the major knowledge gaps that require thorough investigation and provide insights that will help advance this exciting field towards the bedside. michael y.t. chow, 1,2,3 yingshan qiu, 1,3 and jenny k.w. lam 1, * rna-based medicine is receiving growing attention for its diverse roles and potential therapeutic capacity. the largest obstacle in its clinical translation remains identifying a safe and effective delivery system. studies investigating rna therapeutics in pulmonary diseases have rapidly expanded and drug administration by inhalation allows the direct delivery of rna therapeutics to the target site of action while minimizing systemic exposure. in this review, we highlight recent developments in pulmonary rna delivery systems with the use of nonviral vectors. we also discuss the major knowledge gaps that require thorough investigation and provide insights that will help advance this exciting field towards the bedside. the diverse roles of rna in the body have led to the emergence of different approaches to harnessing rna for therapeutic use. rna therapeutics can be broadly divided into three functional classes: (i) inhibition of gene expression [e.g., small interfering rna (sirna; see glossary), microrna (mirna), and antisense oligonucleotide (aso)]; (ii) protein encoding (e.g., mrna); and (iii) protein targeting (e.g., rna aptamers) [1] . despite their diverse mechanisms of action, it is no secret that the biggest barrier to all types of rna therapeutic is delivery; that is, to bring therapeutic rna molecules into the target cells effectively in a safe and reproducible manner. with the us food and drug administration (fda) approval of the first two sirna therapeutics, patisiran and givosiran, both of which target hepatic disorders, the field of rna therapy is ready to look for applications beyond the liver [2, 3] . there is an increasing number of studies that report the potential of rna in treating a range of lung diseases including asthma [4] , cystic fibrosis (cf) [5] , lung cancer [6] , and respiratory infections [7] . inhalation of aerosol is an efficient way to deliver rna to the lung by maximizing local concentration while minimizing systemic exposure. aln-rsv01, designed to treat respiratory syncytial virus (rsv) infection, was the first sirna candidate to be delivered through the pulmonary route in clinical trials in 2008 [8, 9] . since then, several clinical trials on inhaled rna therapy have been initiated (box 1). however, no inhaled rna therapeutic has yet been approved for use in clinics. naked rna for inhalation rna is a negatively charged, hydrophilic macromolecule that is incapable of permeating the cell membrane. it is vulnerable to degradation before reaching the target sites due to the abundance of rnase in the body. therefore, it has to rely on delivery vectors to protect it from premature degradation and facilitate its cell entry. interestingly, it has been known for over a decade that naked rna, including both sirna and mrna, can be transfected in the lung following pulmonary delivery, as shown in many in vivo studies [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . given that the lung comprises various cell types with distinct functions, it is crucial to understand which cell types are susceptible to naked rna transfection for effective clinical translation. to address this issue, ng et al. carried out a comprehensive investigation on the distribution and activity of naked sirna in the lung of mice following intratracheal administration [21] . the silencing activity of naked sirna was most prominent in lung epithelial cells, dendritic cells, and alveolar macrophages. similar observations were reported by other groups in which the activity of naked sirna was primarily found in lung epithelial cells but not the endothelial cells [23] , making it attractive for use in lung conditions affecting these cell types without systemic exposure, for example in rsv infection and cf. ng et al. stressed that chemical modification is crucial for naked sirna to induce effective gene silencing by improving metabolic stability and reducing immunostimulation [21] . using modified naked mrna, tiwari et al. successfully developed a mrna-based approach to express neutralizing antibodies in the lung via intratracheal delivery to prevent rsv infection in mice [19] . the authors also compared naked mrna with the use of polyethyleneimine (pei, a synthetic cationic polymer discussed later) derivatives as delivery vectors and noticed that the transfection efficiency of naked mrna was either better than, or comparable to, these polymers. despite the promising effect of pulmonary naked rna delivery, the exact mechanism of how naked rna crosses the cell membrane barrier in the lung remains unclear, although it has been suggested that the pulmonary surfactant has a significant role in facilitating rna uptake [25, 26] . some studies also showed that the use of delivery vectors could significantly improve rna transfection compared with naked rna in the airways [27] [28] [29] [30] [31] . the development of safe and effective inhaled delivery systems in parallel is paramount currently. with the recent success of sirna in the clinic and the intensive investigation of mrna in clinical trials, including mrna vaccines against coronavirus disease 2019 (covid-19) [32, 33] , we believe that these two rna candidates are likely to be the first to enter the clinic for treating lung diseases. therefore, in this review, we focus on the pulmonary delivery of sirna and mrna. we glossary aerosol: suspension of solid particles or liquid droplets in gas. in humans, drug delivery via the pulmonary route has to be administered in the form of an aerosol. antisense oligonucleotide (aso): short single-stranded dna or rna (~20 nucleotides in length) that binds to a target mrna through complementary base pairing, activating rnase h that leads to degradation of mrna, thereby preventing the translation of mrna into protein. cell-penetrating peptide (cpp): short cationic or amphipathic, natural or synthetic peptide (usually <30 amino acids) that is developed to deliver large cargoes, such as proteins, peptides, and nucleic acids, into cells by promoting cellular uptake. chemical modification: strategy to improve the stability and/or reduce immunogenicity of rna by modifying the structure of rna while maintaining the biological activity of the molecules (e.g., methylation of the ribose 2'-oh group; alteration of the bases; modification of phosphodiester backbone, etc.). intratracheal administration: introduction of substances directly into the trachea, either through the oral cavity via intubation or through a surgical procedure that creates an incision in the trachea (tracheotomy). microrna (mirna): a short rna (~21-25 nucleotides in length) that is partially complementary to multiple messenger rna (mrna), preventing the translation of mrna into protein through the rna interference mechanism. mrna vaccine: mrna that encodes the target antigen to elicit immune responses in the body. naked rna: rna that is not associated with any delivery vectors or transfection agents, such as polymers and lipids. nebulization: conversion of liquid medications into a spray of fine droplets that can be breathed in by the patient. nonviral vector: agent or vehicle that transports nucleic acids into the cells without involving the use of viruses. parenteral: nonoral route of drug administration; usually refers to injection or infusion of drug directly into the body, bypassing the skin and mucous membranes. rna aptamer: singled-stranded rna oligonucleotide that serves as ligand and binds to specific targets (e.g., proteins aln-rsv01 is a naked sirna targeting the rsv nucleocapsid protein for the treatment of the associated viral respiratory infection. it was the first sirna investigated for pulmonary delivery in clinical trials. rsv causes significant illness in immunocompromised patients following lung transplantation, and bronchiolitis obliterans syndrome (bos) is the major cause of morbidity and mortality in these patients [101] . the phase i clinical trial (nct00496821) started in 2007 and demonstrated that aln-rsv01 was well tolerated following intranasal administration. the phase iib clinical trial (nct01065935) showed that aerosolized aln-rsv01 was effective in reducing the incidence of new or progressive bos in lung transplant patients with rsv infection following inhalation. although aln-rsv01 failed to progress to a phase iii trial, it marked an important milestone of inhaled rna therapy [8, [101] [102] [103] . excellair is an sirna targeting spleen tyrosine kinase (syk), which is involved in the inflammatory response in the lung epithelium [104] . it was investigated for the treatment of asthma by inhalation. the phase i trial began in 2009. there was little information published about the outcome of the study, although it was reported that the drug was well tolerated in patients with asthma. the phase ii trial was discontinued in 2015 [105] . eluforsen is a single-stranded rna aso targeting cf transmembrane conductance regulator (cftr) for inhalation to patients with f508del cf. the phase i clinical trial (nct02564354) initiated in 2015 showed that cftr activity was restored after intranasal administration of eluforsen. the phase ib clinical trial (nct02532764) was completed in 2017 and demonstrated that inhaled eluforsen was safe, well tolerated, and improved respiratory symptoms in patients with f508del cf [106, 107] . however, no further clinical development is planned for this candidate. mrt5005 is the first inhaled mrna candidate for cf and delivers mrna encoding fully functional cftr protein. the phase i/ii clinical trial was initiated in may 2018 to test the safety and tolerability of mrt5005. patients with cf received mrna encoding fully function cftr protein through nebulization. interim results were encouraging, showing that mrt5005 was well tolerated at low and mid-dose levels (8-16 mg) with no serious adverse events reported at any dose level (up to 24 mg). there was a marked improvement of lung function in patients after single dose of mrt5005 at the middose level ii . in early 2020, the fda granted fast track and rare pediatric disease designations for mrt5005 for the treatment of cf iii,iv . discuss and highlight the recent advances of nonviral vector-based pulmonary rna delivery systems development, gather what we have learnt from these studies, and identify the major gaps of knowledge. with these, we provide insights and direction to move the field forward. many rna delivery vectors have been developed for pulmonary delivery and their major functions are to facilitate the uptake of rna by target cells and to protect rna from premature degradation. selected recent studies with different rna delivery vectors that have demonstrated in vivo transfection in animal models are summarized in table 1 . we highlight some important studies in each category and discuss them in more detail herein. lipid-based delivery systems due to their high transfection efficiency, ease of synthesis, and low batch variability, lipids were popular as transfection agents during the early years of gene therapy studies for various routes of administration [34, 35] . the transfection efficiency and toxicity of lipid-based systems are affected by the composition of lipids and the ratio of lipids to rna. typically, cationic lipids, such as n-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (dotma) and 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (dotap), are used to form lipoplexes with, or encapsulate, rna [36] . the addition of neutral helper lipids, such as dioleoyl phosphatidylethanolamine (dope) and cholesterol, in the lipoplexes provide the ability to facilitate rna complexation, increase stability of the lipoplexes, and reduce toxicity [37, 38] . one major problem associated with lipid-based systems for pulmonary delivery is their poor structural stability because they readily fuse with pulmonary surfactants in the airways [39] , leading to premature release of rna before cellular uptake. lipid nanoparticles (lnps), which comprise cationic lipids, cholesterol, and polyethylene glycol (peg), have been developed to improve the structural stability of lipid-based systems [35, 40] (table 1) . with proper design and engineering, they can encapsulate rna efficiently. lnps are already in use in the clinic for parenteral injection of sirna [41] and also are in an ongoing clinical trial to deliver mrna (mrt5005) to the lung through nebulization for the treatment of cf, with encouraging early results (box 1). there are two main categories of polymers: natural and synthetic polymers. natural polymers have the advantages of excellent biocompatibility, biodegradability, and safety profiles [42] . derived from the shells of crustaceans, the natural polymer chitosan is commonly investigated for pulmonary delivery due to its mucoadhesive and mucopermeable properties, enabling it to cross the mucus layer in the airways efficiently [43] . it can be used to form polyplexes with rna or as a coating layer on the surface of nanoparticles [44, 45] (table 1) . however, chitosan is limited by its poor solubility at physiological ph and relatively low transfection efficiency [42] . to overcome these problems, water-soluble chitosan derivatives, such as piperazine-substituted chitosan, were developed and found to be efficient for pulmonary sirna delivery in healthy mice [46] (table 1) . furthermore, inhalable chitosan/sirna dry powder formulations were successfully prepared by supercritical drying [47] and spray-freeze drying [48] (table 1) . both studies demonstrated a gene-silencing effect of the powder formulations in lung tissues following intratracheal administration in mouse models of lung cancer, taking these delivery systems one step closer to clinical application. among the synthetic polymers, pei is extensively studied due to its high cationic charge density, good aqueous solubility, and wide ph-buffering capacity [49] . its high versatility allows it to be functionalized to achieve specific targeting. for example, transferrin-pei was used to target activated t cells in the lung, particularly t helper 2 cells, as potential therapy for asthma by reducing and nucleotides) with high affinity and specificity. small interfering rna (sirna): short double-stranded rna (~21-23 nucleotides in length with two 3'overhang nucleotides) in which the antisense strand binds to the target mrna through complementary base pairing, preventing the translation of mrna into protein via rnai. transfection: a process of introducing nucleic acids into cells artificially. (table 1) . however, the relatively high toxicity of this nonbiodegradable polymer remains a considerable concern, even with a low-molecular-weight pei, rendering it difficult to be translated for clinical application. thus, biodegradable synthetic polymers were developed to address this issue. for example, in a recent study, hyperbranched poly(beta amino esters) (hpbaes) [52] were used to deliver mrna through nebulization, with promising gene expression observed in the lung epithelium of mice without local or systemic toxicity after repeated dosing (table 1) . to enhance rna delivery efficiency for lung cancer therapy, yan et al. used a combinatorial library of functional polyester and high-throughput screening to identify matching cancer cells for specific targeting [27, 53] (table 1) . after screening a library of >500 polyester candidates, synthetic amine a13 modified polyester pe4k-a13 was found to be potent for sirna delivery to mice with lung tumors [27] (table 1) . cell-penetrating peptides (cpps) have attracted increasing attention for rna delivery due to their versatility and cell entry ability [54] . the design of peptide sequence can be inspired by natural peptides, proteins with known functions, or by computational simulation [55, 56] . the sequence of amino acids determines the properties of peptides, such as structure, charges, solubility, and polarity, which further affect the interaction with rna, cellular uptake, toxicity, and transfection efficiency. one of the drawbacks of cpps is the lack of cell specificity, which can be addressed by introducing cell-targeting sequences [57] . furthermore, natural l-amino acids are susceptible to protease degradation; although strategies such as replacing l-amino acids with d-analogs has been proposed, this approach may lower the efficiency of the peptides [58] . although many cpps appeared to be promising candidates for delivering rna to the lung, only a few have shown in vivo transfection. welch et al. developed disulfide-constrained cyclic amphipathic peptides that form complexes with sirna with good transfection efficiency because the disulfide bond reduction in the cytosol facilitated the release of the cargo as well as proteolytic clearance. efficient gene knockdown was also observed in lung tissues of healthy mice following pulmonary delivery [28] ( [25, 31] . the pegylated kl4 peptide was also used to formulate mrna as inhalable dry powder by spray-drying and spray-freeze drying techniques, with efficient gene expression observed in the lung of healthy mice [31] (table 1) . to overcome the limitations of a single class of delivery vector, hybrid delivery systems, which are defined as the combination of two or more delivery vectors into a single entity, have been investigated and developed. this formulation strategy aims to increase the strengths of these delivery vectors while decreasing their disadvantages, with lower toxicity compared with their precursors [59] . the combination of lipids with polymers intends to address the problems of poor structural stability associated with lipids and the low biocompatibility of polymers. thanki et al. developed lipid-polymer nanoparticles (lpns) comprising a poly(lactic-co-glycolic acid) (plga) matrix core coated with lipidoid, with sirna localized in both the core and the shell [60] . this design compensated the low sirna-loading capacity of plga by introducing cationic lipidoids while controlling the rate of sirna release through degradation of the polymer. enhanced lung retention upon pulmonary administration of the sirna-loaded lpns was observed in animal studies, suggesting their potential for controlled sirna delivery in the lung (table 1) (table 1 ). this hybrid system demonstrated effective gene silencing in animal models of pulmonary fibrosis with good stability and low toxicity. another common hybrid strategy is to combine lipids with peptides, with the latter serve as a hydrophilic group and facilitate cellular uptake and transportation. for example, cationic liposomes comprising dotma/dope were blended with epithelial-targeting peptides to deliver sirna to the lung of healthy mice, leading to successful silencing of the epithelial sodium channel at the airway epithelium [62, 63] (table 1 ). the results demonstrated the potential application of this hybrid system for cf therapy. similar to lipid-peptide hybrids, the major aim of combining polymers with peptides is to enhance the cellular transport efficiency. polymers are usually conjugated covalently with the peptides. feldmann et al. developed a polymer system called viper (virus-mediated polymer for endosomal escape), which comprised a cationic methacrylated-based copolymer for sirna binding and a membrane lytic peptide melittin, to facilitate endosomal escape [64] ( table 1 ). the hybrid system demonstrated more effective gene-silencing effects in the lung of healthy mice compared with the unmodified polymer system. guan et al. developed a multimodular synthetic peptide with anchor, cationic, and targeting moieties that can form complexes with biocompatible poloxamine-based copolymers and mrna via self-assembly [65] (table 1) . these ternary complexes showed excellent mrna expression in the lungs of mice with cf with negligible toxicity, making them an attractive gene delivery system for cf and other lung diseases. functionalized nanoparticles in addition to the aforementioned conventional delivery vectors, inorganic metal-based nanoparticles have also been evaluated for the pulmonary delivery of rna. however, these nanoparticles cannot act as transfection vectors by themselves due to their lack of rna-binding ability. therefore, they are usually functionalized on the surface to enhance the transfection efficiency. for example, gold nanoparticles are attractive delivery vectors because of their ease of synthesis and conjugation as well as their superior stability [66] . gold nanoparticles were conjugated with sirna through gold-thiol bonds and functionalized with m2pep, a peptide that selectively targets tumor associated macrophages [67] (table 1) . specific gene-silencing effects were observed in targeted macrophages following intratracheal administration in a mouse lung tumor model. it has been suggested that pulmonary surfactant facilitates the delivery of polymer-based delivery systems in the lung [68] . to take advantage of this phenomenon, pulmonary surfactant and surfactant protein b-coated dextran-based nanoparticles were developed for sirna delivery, with successful gene-silencing effects observed in healthy mice and in a model of acute lung injury (ali), respectively, following pulmonary administration [29, 69] (table 1) . exosomes are extracellular vesicles secreted in many cell types. they are involved in communication through the transfer of substances, such as lipids, proteins, and nucleic acids, between cells [70, 71] . they have many desirable features for rna delivery, including high biocompatibility with low inherent toxicity and immunogenicity [72] . zhang et al. presented a new approach to delivering small rna to the lung with the use of exosomes. sirna was loaded into serum-derived exosomes via calcium-mediated transfection. following intratracheal administration, the sirnaloaded exosomes were successfully taken up by lung macrophages to achieve specific gene silencing in a mouse model of ali [73] (table 1) . we carried out a survey of studies published between 2015 and early 2020 that reported sirna or mrna transfection following pulmonary delivery in animals ( figure 1) . a pubmed search was performed using the search terms 'sirna' or 'mrna', 'pulmonary delivery', 'intratracheal', 'inhalation', 'nebulization', and with the filters 'last 5 years' (publication date) and 'other animals' (species) . in total, 53 articles were included in the survey [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [27] [28] [29] [30] [31] 36, 38, 40, [45] [46] [47] [48] [50] [51] [52] [53] [60] [61] [62] [63] [64] [65] 67, 69, [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] . each article was categorized manually according to the type of rna delivery vector used or naked rna; the animal model (disease or healthy); the method of administration to animal; and the type of rna (sirna or mrna). while polymer was the most commonly used rna delivery vector due to its high versatility and ease of preparation, the hybrid delivery system is gaining popularity, many of which include a targeting peptide to improve specificity. with the successful transfection of naked rna in the lung and the technological advances in chemical modifications that greatly enhance the stability and reduce the immune activation of rna [88, 89] , many researchers opt for the use of naked rna to transfect lung tissues due to its simplicity ( figure 1a ). this approach can also eliminate the risk of toxicity and immunogenicity associated with the delivery vectors. it is particularly popular when researchers are interested in examining the biological function of a particular protein rather than its therapeutic potential, such as the role of yes-associated protein (yap), ribosomal protein s3 (rps3), and pi3k/sgk1 pathway in ali [13, 14, 18] . nevertheless, the successful use of naked rna should not lead us to discard the use of delivery vectors for clinical applications. the transfection efficiency of naked rna may not be robust enough for therapeutic use. also, as mentioned earlier, the cellular uptake mechanism of naked rna in the airways is still unclear. it has been speculated that lung lining fluid and pulmonary surfactants have a critical role in facilitating the transportation of rna into cells [68] . the composition of lining fluids could vary substantially among patients and pathological conditions. indeed, it has already been observed that pulmonary surfactant protein and lipid composition change significantly as a result of aging [90] . lung diseases, such as cf, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (copd), are also associated with surfactant deficiency and altered lipid compositions [91] [92] [93] . consequently, the transfection efficiency of naked rna could be easily influenced by the age and disease status of the patients, leading to problems of reproducibility. moreover, naked rna lacks active targeting ability. given that the lung tissues contain a variety of cell types, it would be desirable to use a delivery vector with a targeting moiety to enhance specificity while reducing transfection in unintended cell types. the potential of rna therapeutics to treat a range of lung diseases prompts us to examine the possibility of developing a universal delivery platform that can be adopted by different rna therapeutics for different conditions. while such a system would accelerate the clinical translation of inhaled rna therapy, delivery barriers could be unique to a specific physiological condition. in that case, it would be more effective to have a carefully designed system to overcome these specific sets of barriers. for example, patients with cf have thick and excessive mucus in the airway; hence, a delivery vector with excellent mucus penetration ability would be favorable. moreover, in preclinical studies, healthy animals are sometimes used to evaluate the efficiency of a delivery vector, with the use of reporter or housekeeping genes as the rna target ( figure 1b) . this approach may run into a similar problem in that a delivery vector may perform differently in a healthy animal versus a disease model due to the different delivery barriers in the airways. furthermore, a non-disease rna target (which is generally used to evaluate the efficiency of a delivery vector) does not provide any information regarding the pharmacodynamics properties of the proposed therapy. even if a disease model is used, there is still the challenge of the clinical translation because many lung disease models have limitations that prevent their direct translation to human disease [94] [95] [96] [97] . for instance, asthma is a complex condition that is observed exclusively in humans. the most common model used in preclinical studies is murine allergic airway inflammation. however, the distribution of lung inflammation in mice is different from human asthma, and the animals develop tolerance after repeated allergen exposure [97] . therefore, a collaborative approach between formulation scientists, pharmacologists and clinicians will facilitate the development of a clinically relevant delivery system for inhaled rna therapy. many studies have focused on the development of rna delivery vectors, but the translation of these vectors into suitable dosage forms for clinical application is currently lacking. here, we summarize the critical steps for the development of successful pulmonary rna delivery system ( figure 2 , key figure) and discuss the areas/factors that should be focused on. in general, it is desirable to use delivery vectors that exhibit cell-targeting properties with the ability to overcome the specific barriers associated with the disease concerned. delivery vectors that are nonbiodegradable or with a low rna-loading capability would be less attractive. a thorough understanding of the excretion pathway is required to ensure that the delivery vectors would not accumulate in the body, especially when synthetic or nonbiodegradable materials are used. vectors with high loading capacity could avoid the use of excessive excipients, thereby minimizing the risk of toxicity. moreover, to evaluate the in vivo efficacy of the delivery system, rna is often administered intratracheally to the lung of animals either as large droplets instilled by pipette or as a fine spray aerosolized by a microsprayer or similar device ( figure 1c ). clearly, these administration methods are impractical for human use. for clinical practice, rna drugs can be delivered either as liquid aerosols through nebulization or as dry powder for inhalation ( figure 2 ). while the former can deliver high doses of liquid formulations over a period of time (typically~10-20 min, depending on the dose), the latter is more portable and convenient to use. however, there are stringent requirements for particles to be suitable for inhalation in clinical settings and animal studies cannot reflect the 'inhalability' of a formulation. surprisingly, only a few studies have evaluated the aerosol performance of rna formulations designed for inhalation [31, 48, 51] (box 2). a good understanding of the aerosol properties of the formulation could boost the chance of successful clinical translation ( figure 2 ). moreover, nebulizers or dry powder inhalers are needed to generate aerosol for inhalation. given that both devices could damage the fragile rna molecules (especially single-stranded mrna) due to the high shear stress during aerosolization, it is essential to examine the rna integrity, in terms of physical structure and biological activity, in the aerosolized particles ( figure 2 ). it is unclear whether the rna dose is optimized in in vivo preclinical studies because the doseresponse relationship of rna following pulmonary delivery is often not reported. dose trends optimization is critical for clinical translation not only for maximizing the therapeutic efficacy, but also for the practicality of administration. the amount of excipient (which may be included in the formulation to improve stability or to enhance aerosol performance) needs to be considered carefully because this can also affect the final liquid volume or powder mass to be administered. when rna is delivered through nebulization, a high dose would increase the volume and, hence, the administration time, leading to the increased risk of rna degradation due to prolonged exertion of shear stress on the rna molecules. for dry powder formulations, there is a constraint on the amount of powder that could be inhaled by a patient each time. currently, tobiâ® podhalerâ® (tobramycin inhalation powder) approved for treating pseudomonas aeruginosa infection in patients with cf, has the highest inhaled dose of~30 mg per actuation [98] . the rna dose has to be taken into consideration during the development of an effective delivery system that is fit for purpose. another area that requires attention during development of a pulmonary rna delivery system is understanding the biodistribution and pharmacokinetic profile of the inhaled rna formulation ( figure 2 ). many studies focus on the evaluation of gene expression in the lung tissues as a whole. while naked rna is shown to manipulate gene expression primarily in the lung epithelial cells and macrophages [21] , the sites where nanoparticulate rna delivery vectors exert their biological activity are less clear. for delivery vectors that rely on nonspecific cellular uptake mechanisms, rna could be effectively taken up by various cell types and absorbed into the systemic circulation. therefore, it is critical that biodistribution as well as pharmacokinetic profiles are thoroughly investigated. furthermore, the long-term toxicity of the delivery system also needs to be carefully evaluated. rna therapeutics have great potential in lung diseases. here, we have discussed nonviral delivery systems developed for sirna and mrna therapeutics in the lung and briefly summarized the what is the exact cellular uptake mechanism of naked rna in the airways? what are the factors governing its uptake? what are the delivery barriers to different rna delivery systems in different respiratory diseases? is it possible to have a universal pulmonary delivery platform that can be used to deliver different types of rna therapeutics? how can we protect the integrity of rna molecules against shear and thermal stresses effectively during the aerosolization and drying process? how do we improve the translation of inhaled rna therapy from animal studies to clinical applications? the most important factor that affects the aerosol performance of an inhaled formulation is particle size, which is most suitably expressed in aerodynamic diameter. aerodynamic diameter is defined as the diameter of a sphere of density 1 g/cm 3 that has the same settling velocity in still air as the particle of interest. it is generally accepted that particles with aerodynamic diameter between 1 and 5 î¼m are optimal for lung deposition [108] . larger particles tend to deposit at the back of the throat and get swallowed subsequently, whereas smaller particles are likely to be exhaled. this criterion applies to both liquid and powder aerosol [109] . the aerodynamic particle size distribution of an aerosol is often described in terms of mass median aerodynamic diameter (mmad) and geometric standard deviation (gsd) [109] . the method of choice for measuring particle size distribution of inhaled products is the cascade impactor (ci), which operates on the principle of inertia impaction [110] . ci comprises multiple stages and separates particles according to their aerodynamic diameters. large particles with high inertia are unable to follow the airstream and impact on earlier stages, whereas small particles remain in the airstream and flow to the next stage, where the process is repeated. the two parameters commonly reported from ci are emitted dose and fine particle dose. the former refers to the total dose that has exited the dispersion device, while the latter represents the amount of aerosol with an aerodynamic diameter below a certain threshold (typically 5 î¼m) [111] . these can also be expressed in fractions relative to the loaded dose or recovered dose. an important feature with the use of ci for measuring particle size is that a dispersion device (a nebulizer for liquid dosage forms or a dry powder inhaler for solid dosage forms) needs to be connected to generate the aerosol [112] . given that the choice of device can have a dramatic impact on the aerosol properties of formulations, it is crucial to identify a suitable device to maximize the aerosol performance of a given formulation. there are different designs of ci, but only three are currently listed in both european pharmacopoeia and united states pharmacopoeia: the andersen cascade impactor (aci), the next generation impactor (ngi), and the multi-stage liquid impinger (msli) [112] . current preclinical and clinical state of the field. currently, it appears that the development of mrna therapeutics is lagging behind that of sirna therapeutics ( figure 1d ), possibly because of the relatively poor stability of the long single-stranded rna molecule. with technological advances in rna modification that improve the stability, specificity, and safety of rna therapeutics [88, 89] and the recent success of sirna therapeutics, we believe that both sirna and mrna will enter the clinic for the treatment of respiratory diseases in the near future. however, some crucial questions remain to be addressed before a successful rna inhalation delivery system can be realized (see outstanding questions). in light of the current covid-19 pandemic, development of an inhaled version of mrna vaccine is an area that deserves more attention. currently, there are several clinical studies (clinical trial no i .-nct03164772; nct03908671; nct02662634; nct03076385; and nct03345043) that demonstrate the safety and efficacy of mrna vaccines for the treatment of and protection against lung cancer and influenza, respectively [99, 100] . mrna vaccines for covid-19 are being explored in several different clinical trials (clinical trial no.: nct04283461; nct04470427; nct04405076; nct04449276; nct04480957; nct04380701; and nct04368728). these mrna vaccine candidates are designed to be administered through parenteral injection. if any of these are successful, we believe that an inhaled version of the successful mrna vaccine will be an area to explore because it will provide a non-invasive route of administration with the possibility of self-administration, especially dry powder formulations, which show superior stability. the challenges of manufacture and scale-up, including the production of rna, delivery vectors, and the loading of rna into the vectors without losing their physicochemical properties and biological activities, also need to be overcome. overall, a safe and efficient rna delivery system to the lung remains the key to successful clinical translation. rna therapies explained gene-silencing technology gets first drug approval after 20-year wait givosiran: first approval t-cell targeted pulmonary sirna delivery for the treatment of asthma new approaches to genetic therapies for cystic fibrosis overcoming multiple drug resistance in lung cancer using sirna targeted therapy advancements in nucleic acid based therapeutics against respiratory viral infections aln-rsv01 for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients rna interference therapy in lung transplant patients infected with respiratory syncytial virus intratracheal administration of small interfering rna targeting fas reduces lung ischemiareperfusion injury mrna-mediated gene supplementation of toll-like receptors as treatment strategy for asthma in vivo ribosomal protein s3 gene silencing protects against experimental allergic asthma insulin ameliorates pulmonary edema through the upregulation of epithelial sodium channel via the pi3k/sgk1 pathway in mice with lipopolysaccharide induced lung injury yes-associated protein (yap) signaling regulates lipopolysaccharide-induced tissue factor expression in human endothelial cells duox1 mediates persistent epithelial egfr activation, mucous cell metaplasia, and airway remodeling during allergic asthma small interfering rna targeting nf-kappab attenuates lipopolysaccharide-induced acute lung injury in rats fluorescence-and computed tomography for assessing the biodistribution of sirna after intratracheal application in mice ribosomal protein s3 gene silencing protects against cigarette smoke-induced acute lung injury engineered mrna-expressed antibodies prevent respiratory syncytial virus infection herpes virus entry mediator (hvem) expression promotes inflammation/organ injury in response to experimental indirect-acute lung injury intratracheal administration of sirna triggers mrna silencing in the lung to modulate t cell immune response and lung inflammation establishment of an evaluation method for gene silencing by serial pulmonary administration of sirna and pdna powders: naked sirna inhalation powder suppresses luciferase gene expression in the lung blockade of endothelial, but not epithelial, cell expression of pd-l1 following severe shock attenuates the development of indirect acute lung injury in mice expression of therapeutic proteins after delivery of chemically modified mrna in mice from pulmonary surfactant, synthetic kl4 peptide as effective sirna delivery vector for pulmonary delivery nanocarrier lipid composition modulates the impact of pulmonary surfactant protein b (sp-b) on cellular delivery of sirna aerosol delivery of stabilized polyester-sirna nanoparticles to silence gene expression in orthotopic lung tumors functional delivery of sirna by disulfide-constrained cyclic amphipathic peptides hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of sirna to murine alveolar macrophages targeted delivery of chil3/chil4 sirna to alveolar macrophages using ternary complexes composed of hmg and oligoarginine micelles effective mrna pulmonary delivery by dry powder formulation of pegylated synthetic kl4 peptide tailoring mrna vaccine to balance innate/adaptive immune response an mrna vaccine against sars-cov-2 -preliminary report non-viral gene therapy: gains and challenges of non-invasive administration methods lipid nanoparticle systems for enabling gene therapies combinatorial treatment of idiopathic pulmonary fibrosis using nanoparticles with prostaglandin e and sirna(s) small interfering rna from the lab discovery to patients' recovery intratumoral delivery and therapeutic efficacy of nanoparticle-encapsulated anti-tumor sirna following intrapulmonary administration for potential treatment of lung cancer the lord of the lungs: the essential role of pulmonary surfactant upon inhalation of nanoparticles expression kinetics of nucleosidemodified mrna delivered in lipid nanoparticles to mice by various routes lipid nanoparticles enabling gene therapies: from concepts to clinical utility natural polysaccharides for sirna delivery: nanocarriers based on chitosan, hyaluronic acid, and their derivatives chitosan-based nanomaterials for drug delivery chitosan-based sirna delivery systems chemically modified hcftr mrnas recuperate lung function in a mouse model of cystic fibrosis water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of sirna histological quantification of gene silencing by intratracheal administration of dry powdered small-interfering rna/chitosan complexes in the murine lung intratracheal administration of sirna dry powder targeting vascular endothelial growth factor inhibits lung tumor growth in mice polyethylenimine: a versatile, multifunctional non-viral vector for nucleic acid delivery targeted delivery of sirna to activated t cells via transferrin-polyethylenimine (tf-pei) as a potential therapy of asthma development of spray-freeze-dried sirna/pei powder for inhalation with high aerosol performance and strong pulmonary gene silencing activity inhaled nanoformulated mrna polyplexes for protein production in lung epithelium functional polyesters enable selective sirna delivery to lung cancer over matched normal cells versatility of cell-penetrating peptides for intracellular delivery of sirna cell penetrating peptides: the potent multicargo intracellular carriers recent advances in cell penetrating peptide-based anticancer therapies cancer targeting peptides cell-penetrating peptides: design strategies beyond primary structure and amphipathicity non-viral transfection vectors: are hybrid materials the way forward? mechanistic profiling of the release kinetics of sirna from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration self-assembled micelle interfering rna for effective and safe targeting of dysregulated genes in pulmonary fibrosis delivery of enac sirna to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis effective silencing of enac by sirna delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung in vitro and in vivo delivery of sirna via viper polymer system to lung cells self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis five years of sirna delivery: spotlight on gold nanoparticles dual targeted immunotherapy via in vivo delivery of biohybrid rnai-peptide nanoparticles to tumourassociated macrophages and cancer cells barrier or carrier? pulmonary surfactant and drug delivery surfactant protein b (sp-b) enhances the cellular sirna delivery of proteolipid coated nanogels for inhalation therapy exosomal mirnas in lung diseases: from biologic function to therapeutic targets biological function of exosomes as diagnostic markers and therapeutic delivery vehicles in carcinogenesis and infectious diseases exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges exosome-mediated small rna delivery: a novel therapeutic approach for inflammatory lung responses rnai-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancercell killing pulmonary codelivery of doxorubicin and sirna by ph-sensitive nanoparticles for therapy of metastatic lung cancer the spacer arm length in cellpenetrating peptides influences chitosan/sirna nanoparticle delivery for pulmonary inflammation treatment biokinetic studies of non-complexed sirna versus nano-sized pei f25-lmw/sirna polyplexes following intratracheal instillation into mice multi-functionalized carbon dots as theranostic nanoagent for gene delivery in lung cancer therapy a single methylene group in oligoalkylamine-based cationic polymers and lipids promotes enhanced mrna delivery the suppression of metastatic lung cancer by pulmonary administration of polymer nanoparticles for co-delivery of doxorubicin and survivin sirna pulmonary delivery of polyplexes for combined pai-1 gene silencing and cxcr4 inhibition to treat lung fibrosis biodistribution/biostability assessment of sirna after intravenous and intratracheal administration to mice, based on comprehensive analysis of in vivo/ex vivo/ polyacrylamide gel electrophoresis fluorescence imaging increased survival by pulmonary treatment of established lung metastases with dual stat3/cxcr4 inhibition by sirna nanoemulsions treatment of acute lung injury and earlyand late-stage pulmonary fibrosis with combination emulsion sirna polyplexes strategy to enhance lung cancer treatment by five essential elements: inhalation delivery, nanotechnology, tumor-receptor targeting, chemo-and gene therapy the impact of microfluidic mixing of triblock micelleplexes on in vitro / in vivo gene silencing and intracellular trafficking a new combination therapy for asthma using dual-function dexamethasone-conjugated polyethylenimine and vitamin d binding protein sirna chemical and structural modifications of rnai therapeutics modified mrna as an alternative to plasmid dna (pdna) for transcript replacement and vaccination therapy molecular composition of the alveolar lining fluid in the aging lung pulmonary surfactant dysfunction in pediatric cystic fibrosis: mechanisms and reversal with a lipid-sequestering drug surfactant abnormalities in idiopathic pulmonary fibrosis, hypersensitivity pneumonitis and sarcoidosis decreased surfactant lipids correlate with lung function in chronic obstructive pulmonary disease (copd) animal models of asthma: value, limitations and opportunities for alternative approaches mouse models in squamous cell lung cancer: impact for drug discovery exploring animal models that resemble idiopathic pulmonary fibrosis animal models of asthma: utility and limitations tobramycin inhalation powder (tobi podhaler) for the treatment of lung infection in patients with cystic fibrosis a phase i/iia study of the mrna-based cancer immunotherapy cv9201 in patients with stage iiib/iv non-small cell lung cancer preclinical and clinical demonstration of immunogenicity by mrna vaccines against h10n8 and h7n9 influenza viruses new therapies for acute rsv infections: where are we? evaluation of the safety, tolerability and pharmacokinetics of aln-rsv01, a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus clinical status of duplex rna oligonucleotide therapy for obstructive and restrictive respiratory diseases antisense oligonucleotide eluforsen improves cftr function in f508del cystic fibrosis antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in f508del cystic fibrosis pulmonary delivery of therapeutic sirna pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications pulmonary drug delivery measurement of aerodynamic particle size distribution of orally inhaled products by cascade impactor: how to let the product specification drive the quality requirements of the cascade impactor key: cord-356174-40k6m7l0 authors: ducloyer, mathilde; gaborit, benjamin; toquet, claire; castain, louise; bal, antonin; arrigoni, pierre paul; lecomte, raphaël; clement, renaud; sagan, christine title: complete post-mortem data in a fatal case of covid-19: clinical, radiological and pathological correlations date: 2020-08-06 journal: int j legal med doi: 10.1007/s00414-020-02390-1 sha: doc_id: 356174 cord_uid: 40k6m7l0 a 75-year-old man presented to a french hospital with a 4-day fever after returning from a coronavirus disease-19 (covid-19) cluster region. a reverse-transcription polymerase chain reaction test was positive for severe acute respiratory syndrome coronavirus-2 (sars cov-2) using a nasopharyngeal swab sample. after he returned home and a telephone follow-up, he was found deceased 9 days after first showing symptoms. whole-body, non-enhanced, post-mortem computed tomography (pmct) and a forensic autopsy were performed approximately 48 h after death, with sanitary precautions. the pmct showed bilateral and diffuse crazy-paving lung opacities, with bilateral pleural effusions. post-mortem virology studies detected the presence of sars-cov-2 (b.1 lineage) in the nasopharynx, plasma, lung biopsies, pleural effusion and faeces confirming the persistence of viral ribonucleic acid 48 h after death. microscopic examination showed that severe lung damage was responsible for his death. the main abnormality was diffuse alveolar damage, associated with different stages of inflammation and fibrosis. this case is one of the first to describe complete post-mortem data for a covid-19 death and highlights the ability of pmct to detect severe involvement of the lungs before autopsy in an apparently natural death. the present pathology results are concordant with previously reported findings and reinforce the disease pathogenesis hypothesis of combined viral replication with an inappropriate immune response. most patients infected with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) experience a mild form of the illness, but 5% experience a severe form resulting in 1.4% mortality [1] . the pathophysiological mechanisms of these severe infections are poorly understood [2] . sars-cov-2 infection has been reported to induce both direct organ damage [3, 4] and an inappropriate immune response that results in viral sepsis [5] . infectious sars-cov-2 virus particles have been isolated from respiratory samples [6] , but to date, there are few postmortem reports about the possibility of broad dissemination of viral particles and possible impact on different organs [7, 8] . such findings are essential for a better understanding of the disease and to provide new perspectives for future treatment (e.g. anti-viral and immunomodulator) trials. here we report the full autopsy results for a 75-year-old man deceased from coronavirus disease-19 . a 75-year-old man was admitted to a covid-19 testing unit in a french regional hospital early in the pandemic. he presented for virus screening after experiencing 4 days of fever after returning from a disease cluster area. a family member was also hospitalized for sars-cov-2 infection. he had no relevant medical history, and his only medication was a laxative. upon arrival, blood pressure, heart rate and cardio-pulmonary auscultation were normal. the patient did not complain of dyspnoea, and oximetry with room air was 95%. he presented with hyperthermia (38°c), moderate asthenia, diarrhoea and drowsiness. his body-mass index was normal (25 kg/m 2 ). sars-cov-2 screening was performed by nasopharyngeal swab and covid-19 was confirmed 24 h later by real-time reverse-transcription polymerase-chain reaction (rt-pcr). the threshold for hospitalization was not met, so the patient returned home with clinical monitoring instructions and follow-up by telephone. the blood sample taken 3 days before death showed lymphopenia (770/mm 3 ) and an increase in c-reactive protein (163 mg/l). polymorphonuclear neutrophils were slightly increased (7.38 10 9 /l), and electrolytes were normal. the telephone follow-up reported symptom stability and no fever on the seventh day following the first symptoms. on the ninth day after illness onset, the patient was found deceased in his bed at home by a close relative. a forensic investigation was required by a french prosecutor. the present report was written in accordance with the helsinki declaration on medical research. the first investigation was whole-body, non-enhanced, postmortem computed tomography (pmct) imaging approximately 24 h after death, with specific disinfection of the ct scanner. pmct was performed using an aquilion prime scanner (canon medical systems corporation, otawara, tochigi, japan), with soft tissue, bone and pulmonary parenchyma reconstructions. scan parameters were 135 k, modulated mas and a 1-mm slice thickness. a radiologist trained for pulmonary diseases and a forensic radiologist examined the images using a carestream pacs station (carestream health, rochester, ny, usa) with multiplanar reconstructions. after the pmct, a complete forensic autopsy was performed by a forensic pathologist and a forensic assistant, in a negative room pressure. access to the autopsy room was strongly limited during the examination according to published recommendations [9] . personal protective equipment included a filtering facepiece type 2 (ffp2) medical mask, a scrub hat, two pairs of surgical gloves separated by a pair of cut-resistant gloves and a tyvek® dupont coverall covered by a surgical gown, protective eyewear and rubber boots. normal autopsy technique was used, except that the trachea was clamped as a precaution before extracting the heart and lungs as a block. organs were studied both in situ and individually on a dissection table. the following samples were taken. (1) for virology purposes: a nasopharyngeal swab, percutaneous lumbar puncture sample, transthoracic blood puncture sample, rectal swab, pleural effusion sample, lung biopsy sample. (2) for bacteriology purposes: blood samples for culture and a lumbar puncture sample. (3) for histology: samples of the liver, kidneys, small intestine, colon, brain, heart and lungs were fixed in formalin. specialized disinfection of the autopsy room and the material it contained was performed using a disinfecting detergent (anios oxy'floor, anios laboratories, lille-hellemmes, france) on the floors, the ceiling and surfaces. every piece of equipment, used or unused, was discarded after the autopsy. the samples were conditioned in triple packaging, which was disinfected before being taken out of the autopsy room. pathological examination was performed on each organ. microscopic examinations of the lungs were performed on the central and peripheral areas of each lobe, using sections stained with haematoxylin and eosin and a cd3 (t cell marker) immunohistochemical staining. real-time rt-pcr was performed using the nasopharyngeal swab, plasma, pleural effusion, lung biopsy, cerebrospinal fluid and faecal swab samples. nasopharyngeal swab, pleural effusion and plasma sample were also sent to the national reference center for respiratory viruses (lyon, france) for sequencing. an amplicon-based approach developed by the artic network (https://artic.network/ncov-2019) and combined with oxford nanopore technologies sequencing (oxford, uk) was used. brain, cervical and abdominal areas showed no abnormalities other than normal post-mortem changes. no signs of putrefaction or traumatic injuries to bone or soft tissues were observed. examination of the thorax revealed bilateral pleural effusions but no pericardial effusion. a bilateral, multilobar crazypaving pattern was observed in the lungs, defined by groundglass opacity and intralobular and perilobular reticulations (fig. 1) . approximately 85% of lung parenchyma was affected. the subpleural parenchyma was slightly consolidated, but the anterior portion of each lung field was spared. the trachea and bronchi were filled with liquid. the oesophagus presented with global and marked distension with undetermined spongiform contents. a few centimetre-sized nodes were present in the mediastinum, notably in the pre-tracheal area. the post-mortem autopsy delay was estimated to be 48 h. external examination of the body showed no traumatic injuries to the skin. internal exploration of the chest confirmed the bilateral pleural effusions totaling 200 ml of serohaematic fluid. the heart weighted 470 g and showed a moderate dilatation of the right ventricle, without increase of the myocardial wall thickness. gross examination showed non-obstructive atherosclerotic plaques in the coronary arteries and the aortic bifurcation. both lungs were slightly dense, but the liver, spleen, intestines, kidneys, pancreas and brain were macroscopically normal. sars-cov-2 was detected in all the samples except in cerebrospinal fluid. to quantify sars-cov-2 viral load, we used an external standard curve that revealed a higher viral load in respiratory samples (7.05 log 10 copies/10 6 cells in nasopharyngeal swab, 5.52 log 10 copies/10 6 cells in lung biopsy and 5.15 log 10 copies/10 6 cells in pleural effusion sample) than in plasma (4.5 log 10 copies/10 6 cells). the percentage of sars-cov-2 genome covered was 99.6%, 98.9% and 92.2% for the nasopharyngeal swab, pleural effusion and plasma sample, respectively. multiple sequence alignment revealed that consensus sequences generated from the three samples were identical. using the pangolin web application (https://pangolin.cog-uk.io), the sequences were assigned to the b.1 lineage currently circulating in europe. bacteriological blood analysis identified gram-positive cocci (staphylococcus schelfeiferi and s. epidermidis) considered to be consequent to post-mortem proliferation. the right and left lungs weighed 1044 g and 834 g, respectively. the main lung injury was bilateral diffuse alveolar damage. it appeared heterogeneous with identifiable stages: an acute stage (fig. 2) defined by scattered or diffuse hyaline membranes, associated in some areas with alveolar oedema, an alveolar eosinophil exudate and a few vacuolated macrophages; and a more organized stage (fig. 3 ) defined by parenchymal collapse and enlargement of alveolar septa, filled with incorporated alveolar fibrin deposits, relatively pronounced hyperplasia of type-2 pneumocytes, very sparse multinucleated giant cells and minor fibroblast proliferation. squamous cell metaplasia was not observed. immunohistology identified slight-to-moderate interstitial or perivascular infiltration by inflammatory tcd3+ lymphocytes. alveolar infiltration by neutrophils was not observed and both capillaries and arterioles were thrombosis-free. other pathological findings included mild hepatic steatosis and confirmed a mild coronary artery atherosclerosis. heart sections showed no evidence of myocarditis but displayed some scattered wavy fibres. no other microscopic anomalies were found in other organs, especially brain and kidney vasculitis or disseminated intravascular coagulation. currently, post-mortem covid-19 pathology and virology findings are rare. clinical and forensic covid-19 autopsies are usually not required for such deaths, especially when such diagnoses are made before death and given the considerable logistical constraints deployed to limit the risk of virus transmission. here we report complete post-mortem data following the natural evolution of untreated covid-19. considering the subject's clinical history and absence of other acute or chronic diseases detected by histological fig. 1 post-mortem computed tomography and pulmonary parenchyma reconstruction. axial (a) and sagittal (b) views showing diffuse, bilateral and panlobar ground-glass opacities associated with interlobular and intralobular septal thickening, subpleural consolidations and bilateral pleural effusions (asterisk). the anterior portions of both lungs were more likely to be spared examination, the main hypothesis was that death resulted from the consequences of sars-cov-2 infection. diffuse alveolar damage led to hypoxemia, resulting in polypnea. cardiac arrest may have occurred when compensatory mechanisms were exhausted. cardiac abnormalities, asymptomatic during the individual's lifetime, may also have been responsible for a poorer hemodynamic response to hypoxia. electrolyte disturbances may have been associated. pulmonary embolism, frequently observed during the evolution of the disease [10] , was excluded by both gross and microscopic examinations. the histological findings were consistent with those of previous studies [7, 8, 11] , and very similar to those viral pneumonia findings associated with middle east respiratory syndrome and sars-cov-1 [12] . in addition to diffuse alveolar damage, different microscopic patterning was observed that may illustrate the progression and pathogenesis of the disease. in some areas, an acute exudate pattern (fibrin deposition and/ or alveolar exudates) appeared to be the consequence of earlystage damage to the capillary endothelium and alveolar epithelium. in other areas, there was a more organized pattern, with enlargement of alveolar septa and pronounced desquamated type-2 pneumocyte hyperplasia, fibrin deposition/ incorporation into alveolar septa and some interstitial fibrosis with minimal fibroblast proliferation. these changes suggest the beginning of a healing process 1 week after disease progression. moreover, the presence of a t lymphocyte infiltrate may reflect an early immune response that increases extracellular matrix remodelling and contributed to the fibrosis. these results reinforce the hypothesis of direct pathogenesis due to viral replication combined with an excessive immune response [13, 14] . the autopsy allowed an analysis of almost all the organs, but there was no evidence for multi-organ damage. concerning the post-mortem virology data, this case demonstrated that rna from sars-cov-2 was still detectable in blood, faeces, the lungs and the upper airways more than 48 h after death. given the current status of viral persistence on inanimate surfaces [15] , this finding should be interpreted cautiously: viral presence is not the same as viral infectivity. only viral cultures from post-mortem samples would reliably demonstrate viral survival in the deceased. no support was found for neurological involvement during the infection [16, 17] as virus was not detected in cerebrospinal fluid. sequencing brought no arguments for a variation of the viral sequence between airway and plasma. the european lineage was the one expected in this part of the world [18] . in addition, we have demonstrated the first post-mortem imaging of a covid-19 deceased person. normally, there are many post-mortem changes, and some can mimic pulmonary diseases observed in the living [19] . the most frequent post-mortem changes in the lungs are diffuse ground-glass opacities sometimes associated with posterior consolidation. these features are very close to those described in covid-19 chest ct scans and could imitate or mask them [20] . for this reason, pmct would probably be inefficient for detecting moderate or asymptomatic covid-19 in a deceased person. in the present case, the predominant pattern described as crazy paving was indicative of covid-19, especially as there was no other pre-existing pulmonary pathology [21] [22] [23] . the very extensive distribution in both lungs was reminiscent of previously described white lungs in severe cases [24] . pleural effusion is an uncommon finding in moderate covid-19 but is associated with severe and refractory covid-19 pneumonia diffuse alveolar damage in a more organized stage. note the enlargement of the alveolar septa, fibrin deposition and incorporation, intraalveolar exudates and a type-2 pneumocyte hyperplasia (arrow) representing patchy, interstitial chronic inflammation [25] . in contrast, tracheal and bronchial findings should not be used for diagnosis as they are subject to many post-mortem changes due to passive mobilization of gastric fluid [26] . the present data highlight the potential for pmct to orient a pathologist investigating an apparently natural death, measures for preventing contamination of personnel and facilities and guidance for forensic pathologists to harvest appropriate virology and histology samples. collecting post-mortem information adds to the knowledge base for this new virus. this report provides additional descriptions of microscopic findings in covid-19 and illustrates different stages of disease progression. it also highlights the possible role of pmct for detecting severe and potentially fatal covid-19, provided that images are read by a trained forensic radiologist. it also confirms virology data on postmortem viral persistence in the respiratory tract and supports recommendations for infection prevention when performing an autopsy. further cases are needed for a better understanding of, treatment of, and hopefully prevention of covid-19. clinical characteristics of coronavirus disease 2019 in china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus epidemiology and immune pathogenesis of viral sepsis china novel coronavirus investigating and research team (2020) a novel coronavirus from patients with pneumonia in china pathological findings of covid-19 associated with acute respiratory distress syndrome pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer autopsy in suspected covid-19 cases acute pulmonary embolism associated with covid-19 pneumonia detected by pulmonary ct angiography pulmonary postmortem findings in a series of covid-19 cases from northern italy: a two-centre descriptive study lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore how the sars coronavirus causes disease: host or organism? sars-cov-2 and viral sepsis: observations and hypotheses. the lancet s014067362030920x persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents nervous system involvement after infection with the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients balloux f (2020) emergence of genomic diversity and recurrent mutations in sars-cov-2 post-mortem ct imaging of the lungs: pathological versus non-pathological findings chest ct manifestations of new coronavirus disease 2019 (covid-19): a pictorial review relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 ct imaging features of 2019 novel coronavirus (2019-ncov) radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study clinical characteristics of refractory covid-19 pneumonia in wuhan, china. clin infect dis: ciaa270 the significance of the postmortem discovery of gastric contents in the air passages publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors gratefully acknowledge jean-louis van grimberghe and dr. david lebosse for their support. conflict of interest the authors declare that they have no conflict of interest. key: cord-355122-x3v80bdp authors: desterke, christophe; turhan, ali g.; bennaceur-griscelli, annelise; griscelli, frank title: pparγ cistrome repression during activation of lung monocyte-macrophages in severe covid-19 date: 2020-09-25 journal: iscience doi: 10.1016/j.isci.2020.101611 sha: doc_id: 355122 cord_uid: x3v80bdp the molecular mechanisms of cytokine storm in patients with severe covid-19 infections are poorly understood. to uncover these events, we performed transcriptome analyses of lung biopsies from covid-19 patients, revealing a gene enrichment pattern similar to that of pparγ-knockout macrophages. single-cell gene expression analysis of bronchoalveolar lavage fluids revealed a characteristic trajectory of pparγ-related disturbance in the cd14+/cd16+ cells. we identified a correlation with the disease severity and the reduced expression of several members of the pparγ complex such as ep300, rxra, rara, sumo1, nr3c1, ccdc88a. chip-seq analyses confirmed repression of the pparγ-rxra-nr3c1 cistrome in covid-19 lung samples. further analysis of protein-protein networks highlighted an interaction between the pparγ-associated protein sumo1 and a nucleoprotein of the sars virus. overall, these results demonstrate for the first time, the involvement of the pparγ complex in severe covid-19 lung disease and suggest strongly its role in the major monocyte / macrophage-mediated inflammatory storm. the pandemic caused by the novel sars-cov-2 coronavirus has rapidly become the chief public health challenge for many countries around the world. respiratory complications have been well documented in patients with this disease . this coronavirus harbors a viral s-protein which, during infection, binds with the human protein receptor ace2 (p. . ace2 is abundant in lung, heart, kidney, and adipose tissue and, thus, the binding of the s-protein to ace2 enables membrane fusion and the introduction of sars-cov-2 rna into these cells (turner et al., 2004) . the mean incubation period of the disease is about 3 to 9 days and about 18% of cases remain asymptomatic (nishiura et al., 2020) . about 41.8% of patients develop acute respiratory distress syndrome (ards), for which diabetes mellitus can be a contributing factor. other comorbidities associated with ards include hypertension, cardiovascular disease, and chronic kidney disease (c. . the severity of the disease is also age-associated, with the risk of the mortality increasing in patients over 60 years old (wu and mcgoogan, 2020) . one of the potential consequences of sars-cov-2 infection is an uncontrolled immune response in the lungs, which can require treatment in an intensive care unit. this immune response, associated with a cytokine storm, is heterogenetic and variable among individuals, and is still not well understood (mcgonagle et al., 2020) . it also remains unclear why the heterogenetic immune response to covid-19 worsens in certain patients; as yet, we lack adequate knowledge to predict which cases will evolve from mild to severe infection. in this work, we investigated the immune infiltration in biopsied lung tissues from covid-19 patients using different bioinformatics approaches, including whole transcriptome experiments, single-cell transcriptome characterization, and investigations of the epigenetic landscape. mechanistically, we observed major deregulation of the innate immune response in these lung samples. in particular, we identified notable dysregulation of the pparγ-dependent genetic program in macrophages, which is associated with an increase in the inflammation-promoting program in monocyte-macrophage cells. pparγ repression was found in different independent cohorts and was strongly correlated in multiple patients with disease severity at the single-cell level in the cd14-cd16 myeloid population. pparγ is a member of peroxisome proliferator-activated receptor family with pparα and pparβ.they have common structure comprising an amino terminal activation function-1 domain, a dna binding domain, a hinge domain and a conserved activation function-2 domain which allow ligand binding (nolte et al., 1998) . pparγ was firstly described as important regulator of adipocyte differentiation and could have some unsaturated fatty acids, eicosapentaenoic acids and oxidized lipids as natural ligands (bell-parikh et al., 2003) . in immune system and lung, pparγ is expressed in various cell types such as monocytes and macrophages (standiford et al., 2005) , dendritic cells (gosset et al., 2001) , epithelial airway cells (hetzel et al., 2003) . during lung inflammation, pparγ could repressed transcription factors that regulated expression of pro-inflammatory molecules like nfkb1, stat1 and ap1 (honda et al., 2004; straus and glass, 2007) . in our work, pparγ repression in lung was associated with the induction of several inhibitory immune checkpoints, the lipopolysaccharide-sensitive tlr2 receptor, and hla class i molecules, and these relationships were all associated with disease severity. in an epigenetic analysis of patients with severe covid-19, we detected the repression of components of pparγ-associated cistrome, including nr3c1 and rxra, which had rgs12 as target in its second promoter. transcriptome data of human lung biopsy samples from the dataset gse147507 (blanco-melo et al., 2020) were subjected to gene-set enrichment analysis using the 'hallmarks' gene set from the msigdb database. as compared to healthy donor samples, the majority of samples from covid-19 lungs were significantly enriched in genes associated with innate immunity, such as the interferon alpha response (with a normalized enrichment score (nes) of +7.72, p-value<0.001; supplemental figure 1a ), tnfa signaling via nfkb (nes of +4.88, p-value<0.001; figure 1 ), complement cascade (nes of +3.51, p-value<0.001; supplemental figure 1a ), and reactive oxygen species pathway (nes of + 2.78, p-value<0.001; supplemental figure 1a ). likewise, these tissues were also enriched in genes linked with il-6/jak signaling, which plays a role in both innate and adaptive immunity (nes= +2.49, ; supplemental figure 1b ). furthermore, we observed dramatic enrichment in the interferon gamma response, which is involved in the adaptive immune response (nes=+8.11, p-value<0.001; supplemental figure 1c ). taken together, changes in the expression patterns of these immune-related genes enabled us to clearly differentiate, via unsupervised classification (euclidean distances), between lung samples from covid-19 patients and those from healthy donors (supplemental figure 1d) . these results suggested that in covid-19 lung samples there was a dramatic upregulation of the interferon gamma response, which was accompagnied by a large innate immune response. indeed, a network analysis of this immune-activated program revealed centralized connections around the interferon gamma response ( figure 1a) , with some targets shared with the interferon alpha response. a large part of the network was organized around aspects of the innate response such as interferon alpha, tnfa, the complement cascade, and reactive oxygen species (ros). the majority of the immune network ( figure 1a ) was composed of connections between enzymes, followed by signaling molecules, receptors, and transcription factors. we detected expression changes for a few ligands including chemokines and interleukins ( figure 1a) , which were mainly induced by the ifnα and ifnγ response. to validate these results, we obtained single-cell transcriptome data from the bronchoalveolar lavage fluid of healthy donors (n=6, green) and patients with mild covid-19 (n=3, blue) or severe covid-19 (n=3, red) from the dataset gse145926. these data were merged together for the purpose of umap dimensionality reduction ( figure 1b ) ; after canonical correlation and filtration, the merged transcriptome analysis comprised 90696 cells, with a representative proportion of each subgroup ( figure 1c) . in this analysis, some of the main subpopulations identified were: t lymphocytes that expressed cd3e and/or cd8a (supplemental figure 2 , at the top of the umap in figure 1b) , with a reduction in the cd8a cluster size in severe covid-19 samples (supplemental figure 2) ; natural killer cells that expressed nkg7, for which the cluster size increased in severe disease ( figure 1d, supplemental figure 2 , at the top right of the umap in figure 1b) , some epithelial cells that expressed krt8 (supplemental figure 3 , on the right of the umap in figure 1b) , and some b lymphocytes expressing ms4a1 alias cd20 (supplemental figure 3 , on the top of the umap figure 1b ). this analysis also confirmed a major increase in the monocyte-macrophage markers cd14 and cd16 (alias fcgr3a; italiani and bora schi, 2014) with increased disease severity (figure 1d, 1e, 1f) . these results suggest a major role for the j o u r n a l p r e -p r o o f innate immune response in the lungs of covid-19 patients, which intensifies with disease severity. a differentially expressed gene (deg) analysis was performed on lung biopsies from covid-19 patients and healthy donors; this revealed widespread repression of many gene pathways in covid-19 lungs (supplemental figures 4a-4b) , which could affect major functionalities of the cells in this organ. these results were consistent with those of a gene-set enrichment analysis (supplemental figure 5) . specifically, the gene-set enrichment analysis (performed using the 'hallmarks' gene set of the msigdb database) highlighted repression of the mitosis spindle and p53 pathway (cell cycle gatekeeper) in samples of covid-19 lungs compared to those of healthy donors (nes = -3.45 and -2.77, respectively, with p-value<0.001, supplemental figure 5a ). we also detected the repression of signaling pathways that are implicated in stem cell functionality, such as the notch pathway (nes = -1.70, p-value <0.001, supplemental figure 5b ) and the beta-catenin signaling pathway (nes = -2.17, p-value<0.001, supplemental figure 5b ). furthermore, heme metabolism was also repressed in these samples .001, supplemental figure 5c ). these results suggest that in covid-19 lung pathology there are major defects in important functionalities linked with tissue homeostasis, such as the regulation of cell proliferation, stem cell signaling, and metabolism. of the 108 genes that were found to be upregulated in covid-19 lung samples (supplemental table 1 ), the majority were implicated in immune functionalities. in particular, the deg analysis highlighted ifi6 as the most upregulated molecule in covid-19 samples, and it was expressed in individual cells at progressively higher levels in more severe cases (supplemental figure 4c) . a landscape immune score analysis, performed on lung biopsies with the xcell algorithm and downstream limma analysis (supplemental table 2 ), revealed a significant increase in immune score in covid-19 lung samples, representing increases in neutrophil, monocyte, m1 macrophage, and adaptive dendritic cell infiltrations (supplemental figure 4d) . at single cell level, cd68 macrophage m1 marker was confirmed to be increase in bronchoalveolar lavage fluid of mild covid-19 as compared to healthy donor samples but it was not case in severe covid-19. in contrast expression cd163 macrophage m2 marker seem to be constant between different groups of samples (supplemental figure 6) . in order to characterize the immune infiltration of covid-19 lungs, transcriptome data from lung biopsies were cross-integrated with transcriptome data from human hematopoietic and non-hematopoietic tissues (gse76340) (pont et al., 2016) , which were taken to be representative of the micro-environment of the lung samples. the resulting cross-matrix comprised 170 whole transcriptome analyses and was submitted to the cross-batch normalization algorithm combat to correct batch error (supplemental figure 7 , supplemental table 3). after mathematical correction, the normalized matrix was reduced to the 108 genes that were found to be upregulated in covid-19 lung samples (supplemental table 1 ). an unsupervised prinicipal component analysis revealed major differentiation between healthy donor lungs and covid-19 lungs along the first principal axis (p-value = 3.215239e-29, supplemental figure 4e ). mononuclear cells, monocytes, and macrophages were found in positions similar to the covid-19 lung samples, suggesting major infiltrations in this tissue (supplemental figure 4e ) and confirming the results of the 'xcell' immune score analysis (supplemental figure 4c ). in this principal component analysis, lung samples from healthy donors clustered together with normal primary bronchial epithelial cells, as well as endothelial cells and fibroblasts, suggesting that these cell populations are not implicated in the immune infiltration of covid-19 lungs. hematopoietic stem cells, b-cells, t-cells, and dendritic cells were found in intermediary positions between healthy donor and covid-19 lung samples, which suggested that these cells played only a minor role in the immune infiltration (supplemental figure 4e) . we also confirmed that the microarray batches coming from different datasets used in this analysis did not influence the results, as they were found to be well-distributed across the first principal axis (supplemental figure 8) . immune checkpoints have been widely discussed as new therapeutic targets for the regulation of the immune system in cancer therapy (pardoll, 2012) . in the case of covid-19, a patient's prognosis has been found to depend, at least in part, on the immune response . for this reason, we next investigated the expression of immune checkpoints in covid-19 lung biopsies compared to healthy donor samples, and constructed an expression heatmap with unsupervised classification that enabled us to differentiate covid19 samples (cova and covb, covid-19 lung biopsy replicate a and b) from those of healthy donors (hd1 and hd2, lung biopsies from healthy donors 1 and 2) (supplemental figure 9a) . the majority of inhibitory immune checkpoints, including ceacam1, ido1, lgals9, cybb, pdl1 (alias cd274), and cd47, were found to be overexpressed in covid-19 lung samples (supplemental figure 9a) . some of these, such as cd47, lgals9, havcr2, ido1, and cd274, were confirmed to be induced during severe disease progression (supplemental figure 9b ) at a single-cell level in the cd14-cd16 subpopulation (supplemental figures 9c, 9d and supplemental figure 10) . these results suggest a potential modification of communication among immune cells in covid-19 lungs. instead, our investigation of stimulatory immune checkpoints did not differentiate among covid-19 and healthy donor lung samples (supplemental figure 9e ). in bronchoalveolar lavage fluid at the single-cell level, cd48 and cd40 were found to be expressed in covid-19 lung tissue; cd48 was associated with increased disease severity (supplemental figure 9f ) but cd40 was not (supplemental figure 11 ). in cases of severe covid-19, cd48 was found to be expressed in the cd14-cd16 subpopulation (supplemental figure 9g) . these results mainly suggest that immune cell communication, which is regulated by immune checkpoints, may be modified during infection by covid-19, particularly by the upregulation of inhibitory checkpoints. when we examined the transcriptomes of the covid-19 lung biopsies, we observed that, compared to data from healthy donors, there was a notable increase in antigen-presenting molecules from the hla class i subcategory, including hla-a, hla-b, and hla-c. we also detected an increase, though lower in magnitude, in antigen-presenting molecules from the class ii subcategory: hla-dpa1, hla-dqa1, and hla-drb5 (figure 2a) . the strong expression of hla class i molecules was confirmed with the single-cell transcriptome data obtained from bronchalveolar lavage fluid, especially in the case of severe covid-19 ( figure 2b ). specifically, hla-b was highly expressed in all lymphoid and myeloid cell subpopulations in the lungs of the patient with severe covid-19 ( figure 2c ). when we examined the expression of toll-like receptors (tlrs), we found no evidence for the induction of the tlr7 sensor in covid-19 lung samples, but did observe upregulation of tlr4, which is sensitive to lipopolysaccharides ( figure 2d ). in bronchoalveolar lavage fluid at the single-cell level, expression of the lipopolysaccharide (lps)-sensors tlr2 and tlr4 was found to be induced in covid-19 lungs ( figure 2e ), but expression of tlr7 was unchanged. tlr2 expression appeared to increase with disease severity, especially in the cd14-cd16 cell populations ( figure 2f ), while tlr4 induction was less dramatic ( figure 2g ). these results provide evidence for cd14-cd16 myeloid infiltration in covid-19 lung samples, with a substantial induction of hla class i-presenting molecules and the tlr2 lps sensor. to further characterize the transcriptome of covid-19 lung biopsies, specifically with respect to the lympho-myeloid lineages, we performed transcriptome immune cell deconvolution on this subset of the bulk transcriptome data ( figure 3a ). we observed that covid-19 samples were segregated from those of healthy donors by a notable differentiation in lymphoid and myeloid components along the first principal axis (p-value=2.003047e-17, figure 3a ). covid-19 samples were distinguished by positive correlations with myeloid markers (blue barplot, figure 3b ) and a single negative correlation with one lymphoid ifitm1 (pink barplot, figure 3b ). these results confirmed the strong influence of the myeloid signature, particularly when compared to that of the lymphoid signature, in covid-19 lung samples. we observed a similar pattern at the single-cell level in the bronchoalveolar lavage liquid of patients with severe covid-19 ( figure 3c) , especially with the markers ccl7, fcerg1, and s100a11. in patients with severe covid-19, s100a11 was found to be mainly induced in the cd14-cd16 subpopulations ( figure 3d ). fcerg1 (fc fragment of ige receptor ig) is known to be expressed in monoyctes and macrophages (bournazos et al., 2016) , and here its induction was also found to be more restricted to the cd14-cd16 cell subpopulations ( figure 3e ). these results suggested that, along with hla and tlrs, some myeloid markers are also induced in the cd14-cd16 cell subpopulation during severe covid-19. a gene-set enrichment analysis performed using the msigdb immune gene subset revealed that, compared to samples from healthy donors, covid-19 samples were enriched in genes that had previously been found to be upregulated in macrophages following knockout of pparγ (nes = +3.20, p-value<0.001, figure 4a ). consistent with this, we determined that pparγ expression was in fact lower in covid-19 lung samples compared to those from healthy donors ( figure 4b ). by unsupervised classification, an expression heatmap confirmed that the genes involved in this pparγ-dependent program in macrophages were also overexpressed in covid-19 lung samples ( figure 4c ). we specifically identified interferon-related genes such as ifi6, ifitm2, and ifi44, tnf-related molecules such as tnfaip3, interleukins such as il1b, chemokines such as ccl20, molecules associated with reactive oxygen species such as sod2 (superoxide dismutase 2), and immune checkpoints like cd48 ( figure 4c ). single-cell transcriptome data from bronchoalveolar lavage fluid confirmed the drastic repression of pparγ in patients with severe covid-19 ( figure 4d ) that was exacerbated in the cd14-cd16 cell subpopulations ( figure 4e ). to investigate this further, we examined patterns of pparγ-dependent gene expression in single-cell transcriptomes of the cd14+/cd16+ cell subset of bronchoalveolar lavage fluid from healthy donors (n=6) and patients with severe covid-19 (n=3, as compared to six healthy donor lungs) ( figure 5a ); this subset comprised 559 cells from healthy donors and 1134 cells from patients with severe covid-19. in this cell compartment, pparγ-negative cells were representative of severe covid-19 patients and pparγ-positive cells were representative of samples from healthy donors ( figure 5b ). likewise, unsupervised principal component analysis differentiated, along the first principal axis, between cells from patients with severe covid-19 and those from healthy donors (figure 5c ), and identified a specific gene expression signature that characterized these myeloid cells ( figure 5d ). for example, cd16+/cd14+/pparγ-negative cells from patients with severe covid-19 especially repressed some membrane molecules like the tetraspanin cd9, the transferrin receptor tfrc, the cell junction molecule jaml, and the free fatty acid receptor ffar4 ( figure 5e ). in this cell compartment, inflammatory transcription factors such as nfkbia and mafb, as well as superoxide dismutase (sod2), were overexpressed in severe covid-19 samples ( figure 5f ). upon closer examination of the cd14+/cd16+ cell compartment ( figure 6a) , it was also possible to subdivide the cell population based on pparγ expression: high (over 8 copies of pparγ per cell), medium (between 3 and 8 copies of pparγ per cell), and low (fewer than 3 copies of pparγ per cell) ( figure 6b ). we constructed a pseudotime expression trajectory based on pparγ expression, and this replicated the distribution of cells from severe covid-19 samples and from healthy donor samples (figure 6c) , as well as the distribution of cells in the pparγ-expression categories ( figure 6d ). an expression heatmap of the pseudotime pparγ trajectory enabled the identification of 6 clusters of molecules; clusters 1 and 5, in particular, closely followed the same pattern of pseudotime expression as pparγ ( figure 6e ). within these clusters, we were intrigued to find retn alias resistin, which is known to be associated with diseases such as noninsulin-dependent diabetes mellitus and acquired generalized lipodystrophy ( figure 6f ), fabp4, a fatty-acid binding molecule ( figure 6f ), and mrc1, which is implicated in the recognition of complex carbohydrate structures on glycoproteins ( figure 6f ). in the same population of cells, we also confirmed a pparγ-dependent induction trajectory of the inhibitory immune checkpoint ido1 (figure 6g) . these results suggest that deregulation of this pparγdependent program plays a central role in the macrophage response during severe covid-19 lung infection. in particular, the cd14+/cd16+ cell compartment of bronchoalveolar lavage fluids demonstrated a characteristic immune disturbance in samples from patients with severe covid-19. to further investigate the dysregulation of the pparγ-dependent macrophage program in the lungs of covid-19 patients, we constructed an interactome of the pparγ network and examined the changes in expression among groups. to do this, pparγ interactions were collected from the innatedb database of immune network interactions (breuer et al., 2013) ( figure 7a ). of the gene partners associated with pparγ, we found evidence in the singlecell transcriptomes (bronchoalveolar lavage fluid) for changes in expression associated with covid-19 infection (figure 7b) , specifically in sumo1, nr3c1, klf6, rxra, cebpd, and plagl1. at the single-cell level, a strong correlation was found between pparγ and sumo1 (r=0.47, figure 7c ), pparγ and nr3c1 (r=0.44, figure 7c) , and pparγ and rxra (r=0.35, figure 7c) . a weaker correlation was detected between pparγ and klf6 (r=0.13, figure 7d ) in covid-19 patients, and indeed, we did observe a strong activation of klf6 associated with the severe cases of the disease (figure 7e ) in all lympho-myeloid cell compartments. we constructed another pparγ interactome using the intact protein database ( figure 7f ) and, again, found evidence of relationships between pparγ and some of its partners at the single-cell level in the bronchoalveolar lavage fluid of covid-19 patients ( figure 7g ). for example, pparγ expression was strongly correlated with that of ep300 (r=0.29, figure 7h ), ccdc88a (r=0.60, figure 7h ) and rxra (r=0.45, figure 7h ) in covid-19 cases. the expression of optn did not demonstrate a strong correlation to pparγ (r=0.15, figure 7i) ; however, its expression did progressively increase in the cd14-cd16 myeloid compartment with increased disease severity ( figure 7j) . these results suggest that, along with pparγ, some of its associated proteins are also affected by covid-19, particularly in the cd14-cd16 cell population in severe cases of the disease. the corepression of pparγ-associated dna binding partners such as nr3c1, rara, rxra, and ep300 could be evidence for coregulation of the entire pparγ cistrome in the cd14-cd16 cell population during severe covid-19. we retrieved data from a pparγ chip-sequencing experiment performed on the thp-1 monocyte cell line (gene expression omnibus (geo) sample gsm624141) (pott et al., 2012) from the cistrome project database and processed the dataset using version hg38 of the human genome. promoter mapping was performed with the beta cistrome application +/-100 kb around transcription starting sites (wang et al., 2013) . this analysis confirmed the presence of a well-conserved signal in the mammalian promoter database ( figure 8a ). when we integrated the pparγ chip-seq data with the pparγ-repressed signature found in the lungs of covid-19 patients (supplemental table 2 ), we were able to identify a genomic program with a landscape of both distal and proximal promoters ( figure 8b and supplemental table 4 ). to confirm the relationships between the pparγ-nr3c1-rxra cistrome and the pparγ repression signature of the covid-19 transcriptomes, chip-seq data obtained from the thp-1 monocyte cell line were retrieved for both nr3c1 (geo sample gsm2661793) (rollins et al., 2017) and rxra (geo sample gsm624142) (pott et al., 2012) from the cistrome project database. we also collected thp-1 chip-seq data for polr2a (geo sample gsm1905827) (yu et al., 2015) to highlight transcriptional activity in this program. using the 'deeptools' pipeline (ramírez et al., 2014) , all of these chip-seq experiments were integrated with the pparγ-repressed signature from covid-*19 lungs in order to identify common promoter signals +/-5 kb around transcription starting sites. as shown in figure 8c , the proximal pparγ-repressed epigenetic program in monocytes shared its normal transcriptionally active promoters with rxra and nr3c1. to characterize the active promoters in monocytes, we retrieved thp-1 chip-seq experiments for histone h3 lysine 27 acetylation (geo sample gsm2544237) (phanstiel et al., 2017) from the cistrome project database. from the pparγ-repressed program (supplemental table 4 and figure 8b ), rgs12 was confirmed to present a pparγ signal in its 2 nd promoter; its activity was demonstrated with the h3k27ac histone mark and binding of rna polymerase ii ( figure 8d) , suggesting that this target belongs to the pparγ-repressed program during covid-19 in the lungs. at the single-cell level, rgs12 was confirmed to be repressed in the cd14-cd16 cell compartment in covid-19 lungs, especially in the severe form of the disease ( figure 8e ). in order to investigate the interactions of the pparγ complex with the human sars virus, we explored the virushostnet 2.0 database (guirimand et al., 2015) using the deregulated partners of pparγ that we identified (supplemental table 5 ). by integrating these interactions with the pparγ network, we were able to to create a virus-host network ( figure 8f ). using this, we determined that within the pparγ-repressed complex, only sumo1 is capable of interacting with a sars virus, through the nucleoprotein ncap_cvhsa of the human coronavirus. this result suggests that, during lung infection, repression of the pparγ complex may be mediated via the interaction of sumo1 with the virus, and that sumoylation function may also be affected. the covid-19 pandemic has already affected hundreds of thousands of people and has become the greatest health challenge worldwide. the range of clinical presentations varies from asymptomatic and mild clinical symptoms to acute respiratory-distress syndrome (ards) and death. respiratory complications have been well described in this disease, which is caused by infection with the virus sars-cov-2 (severe acute respiratory syndrome coronavirus 2). diabetes mellitus has been identified as a factor that contributes to the development of ards, while other associated comorbidities include hypertension, cardiovascular disease, and chronic kidney disease (c. . in addition, a link has been demonstrated between obesity and covid-19 cases that require invasive mechanical ventilation (simonnet et al., 2020) . in this work, by comparing the transcriptomes of covid-19 lung biopsies with those of healthy donors, we were able to characterize the immune infiltration in this tissue using different bioinformatics approaches. the major finding of our work is the discovery of a link between the severity of covid-19 and repression of the pparγ complex. in general, we observed that the adaptive immune response to the disease appeared to be driven by the induction of interferon gamma, while the innate response was largely the result of the induction of interferon alpha, tnf alpha, the complement cascade, and reactive oxygen species. immune scoring of covid-19 lung biopsies revealed major myeloid infiltration, specifically by monocytes, m1 macrophages, and neutrophils. instead, the lymphoid scores for this sample were not significant, which suggests that the role of the adaptive immune response is less important than that of the innate response. this finding was confirmed by immune deconvolution analysis of the transcriptome data from covid-19 lung samples. the sars-cov-2 coronavirus is a single-, positive-stranded rna virus enveloped in a lipid bilayer (f. . the lipid bilayer fuses with the host cell membrane, releasing rna into the cytoplasm and resulting in the translation of various viral proteins. the replicated rna genome and synthesized viral proteins reassemble into new viruses, which burst out of the cell (qinfen et al., 2004) . when we analyzed the induction of toll-like receptors (tlrs) in covid-19 lung samples, only the lipopolysaccharide sensor tlr2 was found to be upregulated with respect to controls, which is consistent with detection of the coronavirus envelope. the tlr7 sensor did not seem to be affected by covid-19 infection in our samples, which suggests a lack of presentation of genetic material from the virus in the lungs; this is consistent with our other data demonstrating inefficiency in the adaptive immune response to this disease. compared to the virus that caused the sars outbreak in 2003, the virus that causes covid-19 uses the same mechanism to enter host cells, but at a slower speed. however, sars-cov-2 accumulates to higher concentrations in the body compared to sars-cov. this explains why covid-19 has a longer incubation period and is more contagious, while sars instead presents with more symptoms and increased disease severity . immune scoring of covid-19 lung biopsies revealed the infiltration of monocytes/macrophages in this tissue, and gene-set enrichment analysis confirmed a major role for this type of cells in the immune program described in covid-19 lungs. several types of cytokines and chemokines belong to this dysregulated macrophage program. as in macrophage activation syndrome (mas), macrophages in the lungs of covid-19 patients could play a central role in the non-adapted lung immune response through their contributions to cytokine storm. in covid-19 lung samples, we detected an increase in interleukin 1 (il1a and il1b) and its receptor il1r2. il-1 is a pro-inflammatory cytokine produced primarily by monocytes and macrophages. it is present in the inactive form, pro-il-1β, but, upon monocyte/macrophage activation it is cleaved by caspase-1 and becomes biologically active. via signaling through its receptor, il-1β causes lymphocyte and endothelial activation as well as the production of other inflammatory cytokines including il-6 (v. . il-6 signaling, through the jak cascade, was found to be enriched in covid-19 lung samples compared to healthy donor tissues and expression of il-6st was likewise upregulated. il-6 is a pleotropic cytokine produced in the early stages of inflammation and is central in driving the acute-phase response. one study of patients with mas demonstrated that il-6 produced activated macrophages in tissue obtained from liver biopsies (billiau et al., 2005) . for this reason, treatment with tocilizumab monoclonal antibody, which blocks il-6 receptors, has been proposed to neutralize the cytokine storm that can occur during severe cases of covid-19 . within the il-6 pathway, we found that the scoc3 (suppressor of cytokine signaling 3) protein was upregulated in covid-19 lung samples and, at the same time, we detected an induction of inhibitory immune checkpoints such as pdl1. il-6 was reported to signal, via scos3, for the induction of its receptor pd1 during a cytokine storm, which was then found to neutralize the cellmediated antiviral response (velazquez-salinas et al., 2019). among the inhibitory immune checkpoints that were upregulated in covid-19 lungs, there is an fda-approved targeted therapy (consisting of an antagonistic monoclonal antibody) for only one, cd274. however, a potential therapeutic strategy of targeting pdl1/pd1 in order to manipulate the adaptive immune response could be dangerous in covid-19, because a variety of fatal adverse events related to the respiratory system have been recorded, including but not limited to pneumonia and respiratory failure . in the lungs of covid-19 patients, we detected a strong induction of the tnf-alpha response using gene-set enrichment analysis. tnf-alpha is a pleomorphic cytokine that has been implicated in the pathogenesis of several inflammatory diseases. it is produced largely by monocytes and macrophages that are activated by toll-like receptor ligands such as endotoxins as well as cytokines such as il-18, and stimulates local endothelial cells as well as lymphocytes (shenoi and wallace, 2010) . il-18 was also found to be upregulated in covid-19 lung biopsies compared to controls; this is a unique cytokine in the il-1 family because it is constitutively present in keratinocytes, epithelial cells, and blood monocytes (puren et al., 1999) . in addition to promoting secretion of tnfα and chemokines by macrophages, il-18 also induces production of the pro-inflammatory cytokine ifnγ by nk cells and t cells (dinarello, 2007) . indeed, using gene-set enrichment analysis, we observed substantial activation of the ifn-gamma response in covid-19 lung samples, the primary function of which is to strongly activate monocytes and macrophages (schroder et al., 2004) . activated macrophages are divided into several general classes based upon their respective stimuli and their resulting polarization, with m1 macrophages driven by ifn-gamma into a classical proinflammatory phenotype that is characterized by increased microbicidal ability, heightened responses to tlr ligands, and upregulated antigen processing and presentation. consistent with this, we found that the immune scores of covid-19 lung biopsies highlighted significant infiltration of macrophages of the m1 type. these cells are potent producers of pro-inflammatory cytokines, including il-6, il-12 and il-23, as well as the chemokines ip-10, mig, and itac, which recruit polarized th1 cells in addition to nk cells (mosser, 2003) (mantovani et al., 2004) . there is also evidence that ifnγ may be a critical driver of hemophagocytosis by these activated macrophages (zoller et al., 2011) . this could be connected to the reduction in heme metabolism-related activity we observed in the transcriptome of covid-19 lung samples. this dysregulation in secreted effectors in the lungs of covid-19 patients places monocytes/macrophages at the center of the cytokine storm that has been observed in severe cases of this disease. this was further corroborated by our finding that a gene-set enrichment analysis performed on covid-19 lung samples highlighted upregulation in a transcriptional program that was characteristic of macrophage cells in which pparγ has been knocked out. specifically, this program involved the activation of interferon-related genes, including several cytokines and chemokines. we determined that the expression of pparγ was indeed downregulated in covid-19 lung samples, which confirmed the origin of this macrophage dysregulation. previous research has shown that pparγ in macrophages limits pulmonary inflammation and promotes host recovery following respiratory viral infection (huang et al., 2019b) . in a murine influenza infection model with genetic-induced obesity (db/db), it was shown that the transcription factor pparγ was downregulated in the lung macrophages of db/db mice after influenza infection. the treatment of 15-deoxy-δ12, 14-prostaglandin j2 (15d-pgj2), a ppar-γ agonist, largely rescued the survival of db/db mice after influenza infection (huang et al., 2019a) . as was demonstrated in h1n1 infection, there could be a link between pparγ and macrophages that drives inflammation in covid-19 patients (josset et al., 2012) . it is possible that restoring higher levels of pparγ expression in macrophages could attenuate the severe immune response that has been linked with these innate cells. several pparγ agonists exist on the market (in phases ii/iii) for this purpose and they have already been proposed as an alternative therapy to target the cytokine storm (ciavarella et al., 2020) . in an animal model, it has been shown that loss of pparγ can delay monocyte differentiation into macrophages and increase a pro-inflammatory phenotype with long-term lps stimulation, characterized by an elevated production of the pro-inflammatory cytokines tnf-α, il1-β, il-6, and il-12, and reduced production of the anti-inflammatory cytokine il-10 (heming et al., 2018) . pparγ has also been implicated in the homeostasis of lipid metabolism in macrophages (li et al., 2000) and ppar receptors have been characterized as a crossroad between lipid metabolism and inflammation (chinetti et al., 2000) . in mice, systemic pparγ deletion provokes severe type-2 diabetes (gilardi et al., 2019) and pparγ has been associated with type-2 diabetes in humans via a genome-wide association study (gwas) of finns (scott et al., 2007, p. 2) . within the hospitalized covid-19 population, individuals with diabetes are over-represented (f. and this disorder has been defined as a major comorbidity of covid-19 . it is possible that the link between covid-19 and type-2 diabetes could be pparγ. furthermore, genetic polymorphism in pparγ has also been linked to body mass index (bmi) (fornage et al., 2005) via gwas analysis by the framingham heart study 100k project (fox et al., 2007) . pparγ could also represent a link with the obesity comorbidity found with covid-19 that could reach 44 percent (richardson et al., 2020) . the epigenetically repressed cistrome program identified in covid-19 lungs extended to proteins of the classical retinoic acid pathway, such as rxra, but also to the glucocorticoid receptor nr3c1; this result could suggest changes in the sensitivity of macrophages to glucocorticoids via their anti-inflammatory action on this cell type (mylka et al., 2018) . inside the pparγ interactome, optineurin was found to be induced during severe covid-19; this molecule has been characterized as an interleukin-1 receptor-associated kinase 1-binding protein and has been implicated in inflammatory signaling (tanishima et al., 2017) . finally, within the pparγ-associated repressed protein network, we identified sumo1 as capable of interacting with a nucleoprotein of the human sars virus. pparγ sumoylation is important for the lps response and transregulation of proinflammatory cytokines, via ncor occupancy of kappab binding sites (jennewein et al., 2008) . sumoylation could act as a brake on pparγ-associated repression of the transcriptional activation of inflammatory response genes in macrophages (g. . in this work, the immune response to covid-19 infection in the lungs was investigated; using different approaches, we identified dysfunction in m1 monocytes/macrophages in the innate response process. this dysfunction was characterized by repression of pparγ, which may play a key role in the cytokine storm of inflammatory monocytes/macrophages in the sars-cov-2-infected lung. limitations of the study: integrative analysis through different bioinformatics methods on lung transcriptomes of covid19 diseased patients, enabled the characterization of the lung innate response as major in this pathological tissue. a fundamental deregulation of pparγ disruption was found in the innate immune response through monocyte-macrophage cells. repression of pparγ was confirmed at single cell level in the severity of the disease. network analysis on the pparγ interacting complex revealed also the disruption of some of its partners. pparγ is an essential molecule implicated in macrophage activation and glucose, fatty acid, cholesterol metabolism: these mechanisms could link to the severity of the disease highly correlated to diabetes and obesity. interestingly, pparγ is a druggable target. this bioinformatics study on disturbed immune markers and immune cell deconvolution was validated at single cell level in an independent cohort of samples. epigenetic integration in the analysis allowed to confirm the genome landscape implication of pparγ-nr3c1-rxra cistrome repression in severe lung covid19 diseases. sumo1 that represses the pparγ partner in covid19 diseased lungs was also found to interact with the sars virus. the discovery of this original mechanism could help to better understand the huge innate inflammatory processes in covid19 diseased lungs as well as the associated risk factors found in this disease. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, frank griscelli (franck.grisceli@gustaveroussy.fr). this study did not generate specific reagents. we obtained transcriptomes from covid-19 lung biopsies from dataset gse147507 in the gene expression omnibus database (https://www.biorxiv.org/content/10.1101/2020.03.24.004655v1) and now publish in cell on 28th may 2020 (https://www.sciencedirect.com/science/article/pii/s009286742030489x?via%3dihub) (blanco-melo et al., 2020) . 'volcanoplot' software was developed to analyze the significance of immune scores; the corresponding r functions are available at the web address: https://github.com/cdesterke/volacanoimmune. chip2heat.sh software was developed in shell bash (bourne-again shell) and implemented with the deeptools pipeline, specifically computematrix and plotheatmap, with the promoter plot saved in different formats: png, pdf, svg. this software can be downloaded at: https://github.com/cdesterke/chip2heat. the r code for the single-cell analyses for marker validation is detailed in the transparent methods section of the supplemental information and also provided at the following address: https://github.com/cdesterke/covid19sc. additional resources with interactive web interface of the single cell analysis: to facilitate data exploration of disturb cd14+/cd16+ double population in bronchoalveolar lavage fluid of severe covid19 patients, we developed an interactive web interface available at https://cdesterke.shinyapps.io/covid19lung/. this website was built with flexdasboard and shiny application inclusion and with graphical interactivity display by r plotly. this data dashboard allows exploring expression of biomarkers found on pparγ trajectory in lung monocyte-macrophage disrupted cell population in severe covid19. user need to select gene id on the left sidebar and application will display expression of this selected marker with interactivity on tsne graph, number of positive cells for this marker will be display in value box at top right of the dashboard, also expression by group will be display on violinplot, finally statistical summary (mean and standard deviation: sd) will display by group of samples. mendeley dataset associated to this manuscript: doi: doi:10.17632/3xnypzvcf7.1 -supplemental data s1: r bioinformatics code for single cell transcriptome analysis, related to figures from 1 to 8: doi: http://dx.doi.org/10.17632/3xnypzvcf7.1#file-14fb92f7-2a84-478c-a1fc-99d41668f1e4 -supplemental table 3 : list of gene expression omnibus (geo) transcriptome samples included to performed integrative immune deconvolution of covid-19 lung biopsy, related to figure 3: doi: http://dx.doi.org/10.17632/3xnypzvcf7.1#file-39267130-458c-41d8-8bf7-26ae18b717d1 -supplemental table 4 : pparγ integrative repressed program in covid-19 lung transcriptome, related to the figure 8: doi: http://dx.doi.org/10.17632/3xnypzvcf7.1#file-03b71cec-f317-439c-9176-170edd6ac7e0 -supplemental table 5 signalpromoter plot of pparγ chip-seq data from thp-1 integrated with the repression signature found in covid-19 lung transcriptomes (size of the circle is proportional to the negative logratio found in the covid-19 lung transcriptome, mapping +/-100kb around transcription starting sites (tss)); c. promoter heatmap for pparγ-rxra-nr3c1 cistrome and polr2a from chip-seq data from thp-1 adapted to the repression signature found in the covid-19 lung transcriptome (mapping +/-5kb around transcription starting sites (tss)); d. pparγ chip-seq signal found in the promoter of rgs12, mapped with polr2a and histone h3 k27 acetylation in thp-1 cells; e. umap representation of single-cell transcriptome (bronchoalveolar lavage fluid) expression of rgs12 in hds or patients with mild or severe covid-19 ; f. human-sars protein-protein interactome network, with integration of the pparγ complex affected in covid-19 lung infection (edges represent connections found in virushostnet database). sod2 ifitm2 ifi6 s100a8 il1b lcp1 marcks aqp9 rsad2 tnfaip3 hspa1a pnrc1 zfp36l1 ccl20 bcl6 fosl2 adm nfkbiz gramd1a atp6v1c1 smg7 sms fndc3b ptx3 gng2 cd48 pim2 ncf4 cnn2 irak3 apobec3a lcn2 oasl slamf7 herc5 chi3l1 eif2ak2 fpr1 mxd1 cfb ptk2b samsn1 ifi44 slc39a8 fcgr2a lyn adamdec1 scn1b upb1 abca7 retn sgpp2 maml2 qpct vnn3 prok2 vnn2 lilrb2 tnip3 casp5 tnfsf14 slc6a6 abca1 nsmaf usf2 slamf1 tcf7l2 plagl2 slc35a2 -pparγ expression is repressed in inflammatory lungs of severe covid19 patients -pparγ trajectory is disrupted in bronchoalveolar cd14+/cd16+ cells of covid19 patients -we report here the epigenetics repression of pparγ-nr3r1-rxra cistrome in this setting. -sumo1, as repressed pparγ partner interacts with nucleoprotein of the human sars virus biosynthesis of 15-deoxy-delta12,14-pgj2 and the ligation of ppargamma macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, ifn-gamma-producing lymphocytes and il-6-and tnfalpha-producing macrophages imbalanced host response to sars-cov-2 drives development of covid-19 the role and function of fcγ receptors on myeloid cells innatedb: systems biology of innate immunity and beyond--recent updates and continuing curation peroxisome proliferator-activated receptors (ppars): nuclear receptors at the crossroads between lipid metabolism and inflammation pharmacological (or synthetic) and nutritional agonists of ppar-γ as candidates for cytokine storm modulation in covid-19 disease interleukin-18 and the pathogenesis of inflammatory diseases inverse effects of the ppar(gamma)2 pro12ala polymorphism on measures of adiposity over 15 years in african americans and whites. the cardia study genome-wide association to body mass index and waist circumference: the framingham heart study 100k project why tocilizumab could be an effective treatment for severe covid-19? systemic pparγ deletion in mice provokes lipoatrophy, organomegaly, severe type 2 diabetes and metabolic inflexibility peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells virhostnet 2.0: surfing on the web of virus/host molecular interactions data the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status peroxisome proliferator-activated receptor-γ modulates the response of macrophages to lipopolysaccharide and glucocorticoids inhibition of mmp-9 expression by ppargamma activators in human bronchial epithelial cells peroxisome proliferatoractivated receptor gamma is expressed in airways and inhibits features of airway remodeling in a mouse asthma model targeting peroxisome proliferator-activated receptor-gamma decreases host mortality after influenza infection in obese mice ppar-γ in macrophages limits pulmonary inflammation and promotes host recovery following respiratory viral infection sumoylation of peroxisome proliferator-activated receptor gamma by apoptotic cells prevents lipopolysaccharide-induced ncor removal from kappab binding sites mediating transrepression of proinflammatory cytokines implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic 2009 h1n1 influenza a virus after host adaptation peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in ldl receptor-deficient mice early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia single-cell landscape of bronchoalveolar with covid-19 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the chemokine system in diverse forms of macrophage activation and polarization the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease the many faces of macrophage activation the autophagy receptor sqstm1/p62 mediates anti-inflammatory actions of the selective nr3c1/glucocorticoid receptor modulator compound a (cpda) in macrophages estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) ligand binding and coactivator assembly of the peroxisome proliferator-activated receptor-gamma the blockade of immune checkpoints in cancer immunotherapy a sumoylation-dependent pathway mediates transrepression of inflammatory response genes by ppar-gamma role of interleukin-1 (il-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to il-1 blockade static and dynamic dna loops form ap-1-bound activation hubs during macrophage development microarray gene expression analysis to evaluate cell type specific for immunotherapy of hematological malignancies pparg binding landscapes in macrophages suggest a genome-wide contribution of pu.1 to divergent pparg binding in human and mouse gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells the life cycle of sars coronavirus in vero e6 cells deeptools: a flexible platform for exploring deep-sequencing data presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the glucocorticoid-induced phosphorylation by cdk9 modulates the coactivator functions of transcriptional cofactor grip1 in macrophages interferon-gamma: an overview of signals, mechanisms and functions a genomewide association study of type 2 diabetes in finns detects multiple susceptibility variants tumor necrosis factor inhibitors in the management of juvenile idiopathic arthritis: an evidence-based review high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation peroxisome proliferator-activated receptor-{gamma} as a regulator of lung inflammation and repair anti-inflammatory actions of ppar ligands: new insights on cellular and molecular mechanisms identification of optineurin as an interleukin-1 receptor-associated kinase 1-binding protein and its role in regulation of myd88-dependent signaling ace2: from vasopeptidase to sars virus receptor the role of interleukin 6 during viral infections target analysis by integration of transcriptome and chip-seq data with beta fatal adverse events associated with programmed cell death ligand 1 inhibitors: a systematic review and meta-analysis risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease a new coronavirus associated with human respiratory disease in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention effective treatment of severe covid-19 patients with tocilizumab rna polymerase ii-associated factor 1 regulates the release and phosphorylation of paused rna polymerase ii clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin association of blood glucose control and outcomes in patients with covid-19 and pre-existing type 2 diabetes hemophagocytosis causes a consumptive anemia of inflammation we thank the association "vaincre le cancer nrb" (https://www.vaincrelecancer-nrb.org/) which made it possible to finance the computer equipment used to carry out these analyses.this work was performed with grants from the anr "programme d'investissements d'avenir" of the ingestem national infrastructure key: cord-308461-4lhh3du0 authors: ueki, hiroshi; wang, i-hsuan; zhao, dongming; gunzer, matthias; kawaoka, yoshihiro title: multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon impress date: 2020-01-29 journal: nat protoc doi: 10.1038/s41596-019-0275-y sha: doc_id: 308461 cord_uid: 4lhh3du0 in vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. in this protocol, we describe a methodology for analyzing influenza virus–infected lung in vivo by two-photon imaging microscopy. we describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) for multicolor analysis. setup of this model typically takes ~30 min and enables the observation of influenza virus–infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. this imaging system, which we termed two-photon impress (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo. in vivo two-photon imaging is an analytical approach that can be used to visualize cell dynamics and hemodynamics in organs or tissues of live animals. information in real time obtained by using this approach, such as changes in cell behavior and morphology, tissue localization, and blood flow, has revealed highly sophisticated and dynamic systems of living organisms. during in vivo imaging, the blood circulation in the tissue being observed is maintained; therefore, this technique is also effective for analyzing the migration and invasion of immune cells in the inflammatory environment. observations in physiological environments deepen our understanding of host response mechanisms under both steady-state and disease conditions. computed tomography, x-ray, and ivis spectrum (an in vivo imaging system) imaging methods have been used as non-invasive approaches; however, these techniques have low spatiotemporal resolution and have been able to estimate only the site of inflammation in an organ 1, 2 . therefore, it is impossible to observe cellular responses of the immune system using these approaches. by contrast, a two-photon excitation laser microscope, the light source of which is a near-infrared laser that produces low damage to cells but has long-reaching depth in tissue, enables us to capture the movement of cells in living animals at high resolution. two-photon imaging has been in use in biological science since the 1990s; it has progressed at a remarkable rate, and observation methods for various organs, including brain, liver, and lymph nodes, have been reported 3, 4 . in this protocol, we describe how to use it to image virus-infected lungs. we have previously demonstrated that this protocol works by using mice infected with mouse-adapted seasonal influenza virus (h1n1) or highly pathogenic avian influenza virus (h5n1) 5 . the lung, which is a respiratory organ, has contact with the outside environment and is an important organ for research on immunity to infectious diseases. in the seventeenth century, marcello malpighi discovered pulmonary capillaries and alveoli in the frog lung by using optical microscopy 6 ; now fluorescent reporter mice facilitate the study of disease models in conjunction with two-photon excitation microscopy (table 1) . however, a challenge encountered when imaging the lung is that it is constantly moving during respiration. the lung has been stabilized in several ways during in vivo observation by microscopy, including bronchus clamping, prolonged apnea, gluing, and suction 7, 8 ; however, it is difficult to reduce motion artifacts due to lung respiratory movement under physiological conditions and hence to obtain high-quality images. bronchus clamping can suppress respiratory motion artifacts of the lung lobe 9, 10 ; however, it is not suitable for long-term observation because it causes severe hypoxia. although prolonging apnea is less invasive [11] [12] [13] , it does not allow researchers sufficient time to observe the lung for image acquisition by two-photon excitation microscopy, and the quality of the images tends to deteriorate over time. gluing addresses the above limitations 14, 15 ; however, it can induce shear force injury and inflammation, which affect the interpretation of results. a suction window, which is currently the most commonly used stabilizing system during lung imaging, achieves moderate immobilization of the lung and high-quality images [16] [17] [18] [19] ; however, the observation period is limited to ≤12 h. ex vivo imaging of lungs and in vivo imaging of trachea have also been performed as complementary methods 8 . each of these methods has its advantages and disadvantages, and it is important to select and optimize the method best suited to the goal of the experiments and disease model. in vivo observation of lungs has been performed using various lung disease and experimental models, including bacterial infection, allergen inoculation, tumor metastasis, and lipopolysaccharide (lps)-induced sepsis (table 1) . however, for viral respiratory diseases, such as influenza, other than an observation in a methodology report 20 , only analyses of the trachea in vivo [21] [22] [23] and isolated lungs had been performed 24 , with no analysis of the lung in vivo, until our recent publication 5 (table 1) . unlike ex vivo methods, which involve isolated or sliced lungs, in vivo imaging using two-photon excitation microscopy of live animals enables researchers to observe hemodynamics, migration and extravasation of immune cells, as well as interactions among immune cells during influenza virus infection. however, it is technically demanding to perform two-photon excitation microscopy of live influenza virus-infected lung, which exhibits severe inflammation, requiring the development of highly sophisticated, less invasive instruments and surgical techniques. in addition, when observing animals infected with pathogenic viruses, specialized facilities and instruments are frequently required to avoid the spread of the virus. furthermore, because many types of immune cells infiltrate the infected lung in an inflammatory environment, it is necessary to distinguish the target immune cells from the infected cells by using fluorescent labels in the infected microenvironment. to detect multiple fluorescent signals excited simultaneously by a two-photon excitation laser, fluorochromes with different spectra and equal brightness must be selected; however, there is currently no comprehensive database of fluorescent reagents, fluorescent reporter viruses, and reporter mouse lines available for lung in vivo imaging. we therefore also provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) [25] [26] [27] for multicolor analysis under pathological conditions in this protocol. our system uses suction-based lung stabilization 16, 28 to improve an existing in vivo two-photon imaging system for influenza virus-infected lung as a model of an acute inflammatory respiratory disease 5 . we have successfully used c57bl/6 mice and transgenic mice of the c57bl/6 background (6-to 10-week-old males and females). by using our method, described in detail here, it is possible to visualize and analyze the behavior of immune cells and their interactions with infected cells during an influenza virus infection, which creates an acute inflammatory environment. a limitation of two-photon excitation microscopy is that the observation depth that can be achieved is a maximum of~70 μm. therefore, we cannot observe the bronchial region. this limitation is linked to the wavelength of the infrared laser and detector capability of the microscope. however, as laser technology develops, the observation depth achievable using this method will improve. in this protocol, we describe the application of this methodology to influenza virus infection of the lungs because this is what we have used it for previously. this protocol could be applied not only to studies of the early stages of inflammation due to infection or other causes, but also to analyses of tissue regeneration mechanisms in lungs that are in the process of recovering from infection or other protocol nature protocols injuries. the information provided will also be useful to those using two-photon imaging analysis for the evaluation of the effects of drugs and vaccines, as well as biological events in the lungs and other organs (e.g., liver, spleen) 5 . moreover, with minor modifications, the approach could be applied to analyses of other respiratory diseases, including other infectious models (e.g., severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers)), pulmonary fibrosis, and tumor metastasis. . dna fingerprinting showed that this cell line has the same origin as one obtained from atcc (cat. no. ccl-34, rrid:cvcl_0422) ! caution all viruses and infected animals should be handled in accordance with your institution's biosafety regulations. all work on highly pathogenic avian influenza viruses must be performed under biosafety level 3 (bsl3) regulations. accordingly, all our in vivo imaging studies were performed in the bsl3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan c critical the cells should be regularly checked to ensure that they are not contaminated with mycoplasma. • isoflurane (msd animal health) ! caution isoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature (18-25°c). • sevoflurane (maruishi pharmaceutical) ! caution sevoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature. c critical all reagents should be prepared under sterile conditions. fluorescent reagents should be protecting from light during the setup procedure because they are light sensitive. to prepare 10 mg/ml of tamoxifen solution in sunflower seed oil, dissolve 100 mg of tamoxifen in 1 ml of ethanol (99.5%) and add 9 ml of sunflower seed oil. after adding the ethanol and sunflower seed oil, mix well by vortexing and sonication. this solution can be stored in a refrigerator (2-8°c) for a week. ! caution tamoxifen powder should be handled in a hood. to avoid inhalation and contact with skin, wear rubber gloves and a surgical mask. prepare a solution at a concentration of 2 mg/ml in sterile 1× pbs or saline, make aliquots in 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl (100 μg) of fluorescent dextran i.v. per mouse. qtracker 655 vascular labels immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and inject 50 μl i.v. at a concentration of 0.1 μm. prepare a solution at a concentration of 1 × 10 8 beads/ml in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for long periods (~3 months). immediately before use, mix well and inject 50 μl i.v. per mouse. qdot 655 wga immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and i.v. inject 50 μl. prepare a solution at a concentration of 100 μm in sterile 1× pbs or saline, dispense the solution into dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of fluorescent dextran i.v. per mouse. divide the 5 mm dmso stock solution into dark 1.5-ml microtubes and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 50 μm in sterile 1× pbs or saline and i.v. inject 50 μl per mouse. prepare a solution at a concentration of 100 mm in sterile 1× pbs or saline, dispense the solution in dark 1.5-ml microtubes, and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare a solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of the solution i.v. per mouse. prepare a working solution according to the vendor's manual, dissolve pan caspase in vivo probe in 5 μl of dmso, and add 55 μl of 1× injection buffer (from the kit). inject 60 μl of the solution i.v. per mouse within 1 h of preparation. prepare a working solution according to the vendor's manual, dissolve 100 μl of pkh26pcl in 900 μl of ethanol and store at room temperature for up to 3 months. immediately before use, prepare a solution at a concentration of 10 μm in sterile dilution buffer (from the kit) and inject 50 μl intranasally per mouse. cellrox green, orange, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 250 μm. lysotracker blue, green, red, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 100 μm. mitotracker orange cmtmros, red cm-h2xros, and red fm immediately before use, dilute 50 μg of mitotracker in 1 ml of dmso and inject 50 μl i.v. at a concentration of 100 μm. c critical the mitotracker solution should be prepared fresh each time immediately before use. prepare the solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a solution at a concentration of 10 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare the solution at a concentration of 10 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a working solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare each solution at a concentration of 1 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 1 week. immediately before use, prepare solutions at a concentration of 100 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. dilute fluorescent antibodies to a concentration of 1 µg per 10 μl with sterile 1× pbs or saline and inject 50 μl i.v. per mouse. ! caution it should be noted that antibody staining may affect the target cell behavior; for example, at a high dose (~200 μg), antibodies may neutralize cell activities and/or cause antibody-dependent cytotoxic activity [35] [36] [37] . in our studies, we use 5 µg of antibody for brightness screening because inoculation of fluorochrome-conjugated anti-ly-6g antibody at low doses (1-40 µg) into mice does not affect neutrophil recruitment 38 . the contribution of ly-6g, which is expressed predominantly on murine neutrophils, to recruitment during inflammation remains a matter of debate. it has been reported that low-dose antibody treatment inhibited ly-6g ligation and the recruitment of neutrophils to the site of inflammation 39 ; however, a more recent study indicated that ly-6g knockout did not affect either neutrophil differentiation or recruitment to the site of inflammation in catchup mice 32 . therefore, a low dose of anti-ly-6g antibody is used in our protocol. laser path adjustment system an overview of the laser path adjustment system is shown in fig. 1 . our two-photon excitation laser (chameleon vision ii) unit is placed on an anti-vibration table outside the bsl3 facility. the laser beam enters the bsl3 room, where the two-photon excitation scanning microscope is located, through a window (composed of a small glass window (wg12012-b) and a planar window (rs seal)) connecting the inside and the outside of the bsl3 facility (fig. 1c,d) . the laser path connecting the laser source unit and the two-photon excitation microscope is adjusted by automated laser beam alignment and the aligna 4d stabilization system is adjusted with two active mirrors. ! caution this system adjusts the laser path passing from the outside to the inside of the bsl3 facility for maintenance purposes, so there is no need for this setup unless you are using pathogens that require bsl3 containment. heat is generated when the laser source unit is running, so keep the temperature and humidity constant by using air conditioning equipment. ! caution the system should be operated only by users trained to deal with unenclosed high-power invisible beams and should be placed in an appropriate enclosure with interlocking doors. two-photon excitation laser scanning microscopy system for in vivo imaging of virus-infected mouse lungs in a bsl3 facility a schematic of the arrangement of the in vivo lung imaging system for virus-infected mouse is shown in fig. 2a, and layout examples are shown in fig. 2b -g. this in vivo lung imaging system is based on the upright microscope lsm 780 nlo system, which is equipped with four different lasers (excitation at 405, 488, 543, and 633 nm) for confocal imaging and a two-photon excitation laser (excitation at 630-1,050 nm). to be able to perform the surgical procedure on the mouse, we replaced the sample stage with a large, flat one (microscope stage for in vivo experiment) as shown in fig. 2b ,c. to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser should be set at 910 nm. all fluorescent spectra between the 410-and 695-nm wavelengths can be detected using a 20× water immersion lens, and we record signals in lambda image stacks (0.13 frames per s, 1,024 × 1,024 pixels) and acquire z-stack images with z-depths of 5 μm (total of 10-μm z-depth). we perform spectral separation of the acquired lambda stacks by using the linear unmixing function of the zen software. although the lsm 780 microscope system is controlled by a primary personal computer, we recommended adding >64 gb of ram for appropriate imaging analysis. we keep the mice on a heated stage on the sample stage and record their vital signs using a labox-1 pulse oximeter. to observe the lungs of the mice with a thoracotomy, we place the ventilator with an airway pressure monitor and anesthesia machine for rodents in appropriate positions on the stage. we installed high-efficiency particulate air (hepa) filters in the exhalation duct of the ventilation system (fig. 2b,d) , and the operator wore a positive pressure mask (versaflo faceshields) and a tyvek suit (fig. 2e-g) to avoid exposure to the viruses. ! caution the wavelength and power of the excitation laser should be adjusted appropriately according to the experimental conditions. increasing the laser power enhances target signals and enables detection of second-harmonic generation (shg), in which structures with repeating patterns lead to the formation of a signal. shg is a useful phenomenon for visualizing collagen fibers in the lung without staining; however, it should be noted that the autofluorescence of lung tissue is also enhanced under excessive excitation conditions ( supplementary fig. 1 ). when using this protocol, we did not perform experiments under which shg occurs, in order to minimize autofluorescence; it is better to adjust the laser power according to the experimental purpose. when the wavelength of the excitation laser is too short, the autofluorescence signal becomes very strong and it is difficult to observe properly. by contrast, when the laser wavelength is too long, it becomes difficult to obtain a signal because of the short excitation energy (supplementary fig. 2 ). ! caution although color separation of emission using a conventional optical band-pass filter is also available for this protocol, multispectral imaging is a useful approach for simultaneously analyzing multiple targets by eliminating tissue autofluorescence and identifying fluorescent labels with overlapping spectra 40, 41 . in vivo two-photon imaging is performed under conditions of single stimulation with a two-photon excitation laser; limitations exist regarding available fluorescent reagents/proteins for multiple labeling of target cells and lung architecture. therefore, we recommend using a multispectral approach to produce crosstalk-free images of fluorescence with overlapping spectra that cannot be separated by using band-pass filters. before starting experiments, it is necessary to collect spectral signatures of the emission signal of each fluorescent reagent and protein as reference spectra under the same excitation condition as will be used in the experiment. to observe the mouse lung using an upright microscope, it is necessary to prepare a thoracic suction window to immobilize the lung. in the bsl3 facility, animal experiments must be performed while wearing two or three layers of latex gloves; therefore, the thoracic suction window was designed for easy handling, even in the bsl3 facility, and to be minimally invasive for the infected animals (fig. 3a-c and supplementary fig. 3 ). to position a cover glass for each observation, flatten the upper surface of the thoracic suction window so that a commercially available cover glass will fit. this device is also designed to reduce concavity and convexity as much as possible so that blood containing virus cannot accumulate. connect the thoracic suction window to an aspirator through a waste tank and a suction regulator. to prevent the spread of virus-containing aerosols, install hepa filters between the waste tank and the suction regulator as shown in fig. 3d . starting up the imaging system equipment • timing 20-30 min 2 on the day of analysis, turn on the two-photon excitation laser and the aligna 4d control unit placed outside the bsl3 facility, and verify that they are working. c critical the aligna 4d control unit needs to be kept on. 3 wearing a tyvek suit, positive pressure mask, and gloves according to the guidelines for the bsl3 facility, enter the bsl3 facility where the imaging system is housed. ? troubleshooting 4 turn on the microscope controllers, confocal lasers, and the computer for the two-photon excitation microscope and the aligna 4d system. 5 launch the microscope control software zen and turn on the lasers, including the two-photon excitation laser. 6 launch the aligna 4d control software kangoo and adjust the laser path connecting the laser source unit and the microscope (supplementary fig. 4) . ? troubleshooting 7 wrap the hot plate with aluminum foil, turn it on, and keep it at 35°c. sterilize the surgical area and tools with 70% ethanol and place all instruments within easy reach. animal anesthesia • timing 2-3 min 8 turn on the gas anesthesia vaporizer and supply 5% isoflurane to a mouse anesthesia induction chamber. 9 anesthetize the influenza virus-infected mouse with 5% isoflurane in a mouse anesthesia induction chamber. subsequently, transfer the mouse to the hot plate while supplying 2% isoflurane via an anesthetic mask. ? troubleshooting 10 inject the chosen fluorescent dyes and antibodies via the retro-orbital plexus (as shown in supplementary video 1) using an insulin syringe. tables 4 and 5 show the brightness levels of antibodies and fluorescence of dyes, respectively, in vivo. ! caution when working with viruses in a bsl3 containment, it is not safe to use needles, so we avoid them as much as possible, which is a standard precaution in high-containment laboratories. in addition, in the bsl3 facility, animal experiments must be performed wearing two or three layers of latex gloves. tail-vein administration is a common method; however, it is not easy to perform these procedures with so many layers of gloves. use tweezers to hold down the mouse to make the administration route. when an infected animal is not used, an administration route can be created via the tail vein or the jugular vein. ? troubleshooting surgical procedure • timing 10-15 min c critical before experimenting with infected animals, practice the surgical procedures with euthanized animals. 11 place the mouse on its back and tape the anterior limbs with adhesive tape (fig. 4a) . 12 using straight scissors, cut the skin beneath the chin in the middle and expose the trachea (fig. 4b) . insert a tracheal cannula and intubate the mouse to facilitate mechanical ventilation with a ventilator (fig. 4c) . turn on the ventilator, ventilate the mouse at a respiratory rate of 120 breaths per min, and apply positive-end expiratory pressure (peep;~6 cm h 2 o) and a tidal volume of 0.5 ml. deliver isoflurane continuously at 2% to maintain anesthesia. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. 13 place the mouse in the right lateral decubitus position and re-fix its anterior limbs with the tape (fig. 4d) . make an incision in the skin at the left axilla using straight scissors, straight iris scissors, and hooked forceps (fig. 4e) . ! caution carefully change the mouse's position in order to avoid cannula drop off. 14 expose the left lung lobe by surgical intercostal incision between ribs 3 and 4, and keep it exposed by using retractors (fig. 4f) . the brightness of each fluorescent protein during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. the brightness of each fluorochrome during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. af, alexa fluor; nd, not detected. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. c critical because lungs infected with viruses often shrink, secure a large field of surgical view so that the suction window can reach it. 15 place the mouse beneath the objective lens and connect a device to monitor the heart rate of the mouse (we use a labox-1 pulse oximeter). starting up the thoracic vacuum window system • timing 2-3 min 16 turn on the aspirator connected to the thoracic suction window. 17 fix the thoracic suction window to the holding block at a 90°angle and put a round cover glass on the tip of the suction device. ? troubleshooting 18 turn on the suction pressure regulator and adjust the suction pressure to 25-30 mmhg. 19 lower the thoracic suction window gently to immobilize the mouse lungs (fig. 4g,h) . the thoracic suction window should cause the lung to stick to the cover glass because of negative pressure. ! caution carefully move the suction window so as not to scratch the objective. 20 position the objective lens above the thoracic window. 21 put water drops on the cover glass by using a pasteur pipette and lower the objective lens to the thoracic suction window (fig. 4i) . unmixing of spectrum data and analyzing the images • timing 1-2 h per sample 24 to unmix the spectrum data, prepare a reference image of each spectrum in advance. to make a reference image, acquire each fluorescent dye or protein separately without any co-staining and analyze the single fluorescent spectrum. we use the linear unmixing module of the zen software for separating spectrum data; however, other commercial or open-source software is available (table 6 ). 25 subject unmixed time-series stacks to image registration to correct for tissue drifts and respiratory artifacts. this step is critical to certain analyses, such as long-term tracking of individual cells or subcellular structures. in some cases, a reference channel is required for determining the shift and distortion of the objects. in our studies, we use time-series stacks of blood vessels or collagens for such use, because their localizations are constant over time without substantial changes in shape or structure during the observation. ! caution some image registration algorithms may cause spatial distortion. choose algorithms that generate corrected data suitable for your subsequent analyses, especially when examination of the shape and structure of cells and tissues is required. 26 analyze the movies as required for your experiment. troubleshooting advice can be found in table 7 . step 1, infection: 10-20 min anticipated results the imaging system described in this protocol enables the observation of the behavior of virusinfected cells and immune cells in infected lungs in real time. typical images of influenza virus-infected lung are shown in fig. 5a and supplementary video 2. when observing while using a multicolor fluorescent label, it is easier to analyze the detected images if the brightness level of each fluorophore is adjusted to make them similar. it is better to choose fluorescent dyes or proteins that possess high fluorescence stability for long-term observations (tables 2, 4 and 5). we have found that use of ma-cerulean-viruses or ma-venus-viruses for infection produces influenza virus-infected cells with sufficient brightness (table 3) . for labeling immune cells and alveolar cells, we have achieved good results by using the fluorochrome phycoerythrin (pe) for antibody staining and rosa-tdtomato 42 or -mtfp1 33 mice that were crossed with cell-specific cre-expressing mice. if using reporter mice expressing a fluorescent protein such as gfp, which is regulated by an endogenous promoter, the expression level of the fluorescent protein should be confirmed. to visualize the lung structure, we use texas-red dextran or qtracker 655 vascular labels for the red to far-infrared channel. mice die during anesthesia the level of anesthesia is too high decrease the concentration of anesthesia as soon as the mouse shows loss of righting reflex 10 mice regain consciousness during anesthesia the level of anesthesia is too low confirm the concentration of anesthesia; administer the reagents again after a brief pause 15 no heart rate is measured the monitoring probe is mispositioned make sure that the monitoring probe is in the appropriate place 17 the cover glass falls off the cover glass does not hold on the suction device put water droplets on the tip of the suction device and then place the cover glass on it influenza virus-infected lungs are infiltrated by numerous immune cells, including neutrophils and monocytes [43] [44] [45] . an immune cell-specific reporter mouse line can be used to visualize cells infiltrating the alveoli and cells in blood vessels, whereas it is preferable to label intravascular cells by intravenous administration of fluorochrome-conjugated antibodies 5, 46, 47 . consistent with previous reports, intravenously injected antibodies will label only the cells in contact with the blood flow and not those in extravascular regions under our experimental conditions 5 . by administering a fluorescently labeled antibody against neutrophils into neutrophil reporter mice, we can observe the behavior of both the neutrophils infiltrating the influenza-infected lungs and the neutrophils in blood vessels separately (fig. 5b) . to observe the interaction between different kinds of infiltrating immune cells, such as neutrophils and monocytes, double-reporter mice expressing fluorescent proteins with different spectra but similar brightness have a major advantage (fig. 5c and supplementary video 3) . co-infection of the host with different strains of influenza virus can lead to the emergence of reassortant viruses. by infecting mice with color-flu viruses that produce different fluorescence spectra, we detected alveolar epithelial cells that simultaneously expressed two fluorescent proteins in vivo (fig. 6) . visualization of co-infected cells might enable us to better understand the reassortment process of influenza viruses in vivo. in summary, the use of this in vivo imaging system for infected animal and multicolor imaging enables us to analyze pathology and immune cell dynamics at the cellular level, which would not be possible by using conventional histopathology methods. this imaging system thus provides a novel and useful approach for investigating viral pathogenicity. further information on research design is available in the nature research reporting summary linked to this article. the data that support this study are available from the corresponding author upon reasonable request. the matlab scripts are available at https://github.com/kawaokalab/ueki_pnas_2018. for all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or methods section. the exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement a statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly the statistical test(s) used and whether they are one-or two-sided only common tests should be described solely by name; describe more complex techniques in the methods section. a description of all covariates tested a description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons a full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) and variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) for null hypothesis testing, the test statistic (e.g. f, t, r) with confidence intervals, effect sizes, degrees of freedom and p value noted give p values as exact values whenever suitable. for hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes estimates of effect sizes (e.g. cohen's d, pearson's r), indicating how they were calculated our web collection on statistics for biologists contains articles on many of the points above. policy information about availability of computer code to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser was set at 910 nm. all fluorescent spectra between 410 and 695 nm wavelengths were detected using a 20x water immersion lens (carl zeiss ag, germany) and the signals were recorded in lambda image stacks. we use the linear unmixing module of zen software for separating spectrum data. unmixed time-series stacks are subjected to image registration to correct for tissue drifts and respiratory artefacts. a reference channel is required for determining the shift and distortion of the objects. in our studies, we employ time-series stacks of blood vessels or collagens for such use, as their localizations are constant over time without significant changes in shapes or structures during the observation. for manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. we strongly encourage code deposition in a community repository (e.g. github). see the nature research guidelines for submitting code & software for further information. policy information about availability of data all manuscripts must include a data availability statement. this statement should provide the following information, where applicable: -accession codes, unique identifiers, or web links for publicly available datasets -a list of figures that have associated raw data -a description of any restrictions on data availability the data that support this study are available from the corresponding author upon reasonable request. october 2018 field-specific reporting please select the one below that is the best fit for your research. if you are not sure, read the appropriate sections before making your selection. for a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf all studies must disclose on these points even when the disclosure is negative. only one sample was shown as a representative example that can be obtained by using the imaging protocol. data exclusions no data was excluded since one representative image was shown. no repeated measurements were performed in this paper since one image has been shown as a representative image by using the imaging protocol. • microsurgery straight scissors (13.5 cm • microsurgery bulldog forceps (brc, cat. no. 70052-30cii/r) • insulin syringes (0.5 ml, 100 u, 30 gauge × 10 mm; nipro, cat. no. 08277) • pasteur pipettes (bd falcon, cat. no. 357575) • customized surgical retractor • thoracic suction window (sakura seiki, custom made) stage for mounting a thoracic suction window (sakura seiki, custom made) • suction regulator (iwaki, cat. no. 1450050) • cover glass (matsunami glass, cat. no. c013001) • hot plate (hipet, cat • confocal microscope system (zeiss, model no. lsm 780 nlo) • infrared laser (coherent, model no. chameleon vision ii) • 20× water immersion lens (zeiss, plan-apochromat model) • beam-pointing stabilizer (tem messtechnik, model no. aligna 4d system high-efficiency particulate air (hepa) filters (vacushield; pall, cat. no. 4402) • artificial ventilator (shinano, cat • airway pressure monitor (shinano) • gas anesthesia vaporizer • mouse anesthesia induction chamber • mouse anesthesia mask • positive pressure mask (versaflo faceshields softwear iii) • surgical gloves rs seal (roxtec, cat labox-1) highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread consecutive ct in vivo lung imaging as quantitative parameter of influenza vaccine efficacy in the ferret model calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links in vivo fluorescence microscopy: lessons from observing cell behavior in their native environment in vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs marcello malpighi and the discovery of the pulmonary capillaries and alveoli live imaging of the lung live imaging of the pulmonary immune environment examination of the pulmonary circulation with the microscope color-coded real-time cellular imaging of lung t-lymphocyte accumulation and focus formation in a mouse asthma model pathophysiological role of endothelins in pulmonary microcirculatory disorders due to intestinal ischemia and reperfusion intravital microscopy of the murine pulmonary microcirculation surfactant protein a mediates pulmonary clearance of staphylococcus aureus in vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation dap12 expression in lung macrophages mediates ischemia/reperfusion injury by promoting neutrophil extravasation stabilized imaging of immune surveillance in the mouse lung two-photon imaging within the murine thorax without respiratory and cardiac motion artifact visualization of immediate immune responses to pioneer metastatic cells in the lung the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors t cell response in the lung following influenza virus infection neutrophil trails guide influenza-specific cd8 + t cells in the airways live imaging of influenza infection of the trachea reveals dynamic regulation of cd8 + t cell motility by antigen imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy three phases of cd8 t cell response in the lung following h1n1 influenza infection and sphingosine 1 phosphate agonist therapy multi-spectral fluorescent reporter influenza viruses (color-flu) as powerful tools for in vivo studies molecular determinants of virulence and stability of a reporter-expressing h5n1 influenza a virus amino acid changes in pb2 and ha affect the growth of a recombinant influenza virus expressing a fluorescent reporter protein live imaging of the lung a transgenic mouse line that retains cre recombinase activity in mature oocytes irrespective of the cre ransgene transmission insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes a multifunctional teal-fluorescent rosa26 reporter mouse line for cre-and flp-mediated recombination trackmate: an open and extensible platform for single-particle tracking the role of neutrophils during mild and severe influenza virus infections of mice excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis the effects of acute neutrophil depletion on resolution of acute influenza infection, establishment of tissue resident memory (trm), and heterosubtypic immunity antibodies against neutrophil ly6g do not inhibit leukocyte recruitment in mice in vivo ly6g ligation blocks recruitment of neutrophils via a β2-integrin-dependent mechanism spectral imaging: principles and applications multispectral imaging in biology and medicine: slices of life a robust and high-throughput cre reporting and characterization system for the whole mouse brain regulating the adaptive immune response to respiratory virus infection a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection innate immunity to influenza virus infection intravascular staining for discrimination of vascular and tissue leukocytes in vivo compartmental analysis of leukocytes in mouse lungs a study of the pulmonary circulation by the transillumination method pulmonary capillary recruitment during airway hypoxia in the dog the behavior of the arterioles and capillaries of the lung capillary recruitment during airway hypoxia: role of pulmonary artery pressure capillaroscopic appearance of the pulmonary alveoli in the living dog diffusing capacity of the lung during hypoxia: role of capillary recruitment intrapulmonary blood flow redistribution during hypoxia increases gas exchange surface area the normal behavior of the pulmonary blood vessels with observations on the intermittence of the flow of blood in the arterioles and capillaries an experimental model for simultaneous quantitative analysis of pulmonary micro-and macrocirculation during unilateral hypoxia in vivo a thoracic window for observation of the lung in a living animal microscopic observation of the lung in vivo precapillary oxygenation contributes relevantly to gas exchange in the intact lung pulmonary microcirculatory observations in vivo under physiological conditions direct measurement of pulmonary capillary transit times intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries measurement of lung microvascular pressure in the intact anesthetized rabbit by the micropuncture technique emergency granulopoiesis promotes neutrophil-dendritic cell encounters that prevent mouse lung allograft acceptance capillary perfusion patterns in single alveolar walls measurement of microhemodynamics in the ventilated rabbit lung by intravital fluorescence microscopy donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction leukocyte kinetics in pulmonary microcirculation: intravital fluorescence microscopic study the pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis temporal capillary perfusion patterns in single alveolar walls of intact dogs aspirin-triggered 15-epi-lipoxin a4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice sites of leukocyte sequestration in the pulmonary microcirculation cxcr4 identifies transitional bone marrow premonocytes that replenish the mature monocyte pool for peripheral responses contribution of selectins to leucocyte sequestration in pulmonary microvessels by intravital microscopy in rabbits lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli leukocyte margination in alveolar capillaries: interrelationship with functional capillary geometry and microhemodynamics neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy pulmonary microvascular changes during sepsis: evaluation using intravital videomicroscopy intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model in vivo measurement of the mouse pulmonary endothelial surface layer real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation direct visualisation of microparticles in the living lung spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to t cells in the lung two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts intravital imaging allows real-time characterization of tissue resident eosinophils quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged sickle cell disease mice initial host response to bacteria in the murine lung differs between pseudomonas aeruginosa, staphylococcus aureus and streptococcus pneumoniae inkt cell emigration out of the lung vasculature requires neutrophils and monocyte-derived dendritic cells in inflammation a new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats the lung is a host defense niche for immediate neutrophil-mediated vascular protection an experimental rat model for studying pulmonary microcirculation by in vivo videomicroscopy bispecific antibody targets multiple pseudomonas aeruginosa evasion mechanisms in the lung vasculature alveolar dynamics in acute lung injury: heterogeneous distension rather than cyclic opening and collapse maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury leukotriene b4-mediated neutrophil recruitment causes pulmonary capillaritis during lethal fungal sepsis α-toxin induces platelet aggregation and liver injury during staphylococcus aureus sepsis neutrophil mobilization via plerixafor-mediated cxcr4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow transfusion of human platelets treated with mirasol pathogen reduction technology does not induce acute lung injury in mice a new model of lung metastasis for intravital studies visualizing the function and fate of neutrophils in sterile injury and repair in vivo subcellular resolution optical imaging in the lung reveals early metastatic proliferation and motility two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches patrolling monocytes control tumor metastasis to the lung pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window cancer cells induce metastasis-supporting neutrophil extracellular dna traps a permanent window for the murine lung enables high-resolution imaging of cancer metastasis embryonic stem cells and mice expressing different gfp variants for multiple non-invasive reporter usage within a single animal cre reporter strains produced by targeted insertion of eyfp and ecfp into the rosa26 locus a global double-fluorescent cre reporter mouse in vivo depletion of cd11c + dendritic cells abrogates priming of cd8 + t cells by exogenous cell-associated antigens notch-rbp-j signaling controls the homeostasis of cd8 -dendritic cells in the spleen zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages absence of mhc class ii on cdcs results in microbial-dependent intestinal inflammation identification of a dendritic cell receptor that couples sensing of necrosis to immunity a macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse analysis of fractalkine receptor cx(3)cr1 function by targeted deletion and green fluorescent protein reporter gene insertion fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis generation of cd4creer(t(2)) transgenic mice to study development of peripheral cd4-t-cells notch2 integrates signaling by the transcription factors rbp-j and creb1 to promote t cell cytotoxicity b lymphocyte-specific, cre-mediated mutagenesis in mice genetic analysis of basophil function in vivo lethal influenza infection in the absence of the natural killer cell receptor gene ncr1 high-efficiency type ii cell-enhanced green fluorescent protein expression facilitates cellular identification, tracking, and isolation hyperspectral phasor analysis enables multiplexed 5d in vivo imaging the orfeo toolbox remote sensing image processing software optimizing imaging parameters for the separation of multiple labels in a fluorescence image spectral imaging and linear un-mixing enables improved fret efficiency with a novel gfp2-yfp fret pair blind source separation techniques for the decomposition of multiply labeled fluorescence images image matching as a diffusion process: an analogy with maxwell's demons diffeomorphic demons: efficient non-parametric image registration automated motion artifact removal for intravital microscopy, without a priori information nonrigid registration using free-form deformations: application to breast mr images imart software for correction of motion artifacts in images collected in intravital microscopy automated filtering of intrinsic movement artifacts during twophoton intravital microscopy removing physiological motion from intravital and clinical functional imaging data software tools for single-cell tracking and quantification of cellular and molecular properties cellprofiler: image analysis software for identifying and quantifying cell phenotypes icy: an open bioimage informatics platform for extended reproducible research additional information supplementary information is available for this peer review information nature protocols thanks megan macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank s. watson for editing the manuscript. we thank k. iwatsuki-horimoto, l. wu, s. fukuyama, y. matsuzawa, and k. miyake randomization no randomization is included in this paper since one image has been shown as a representative image by using the imaging protocol. blinding was not relevant to this study which is describing a imaging protocol and anticipated results. we require information from authors about some types of materials, experimental systems and methods used in many studies. here, indicate whether each material, system or method listed is relevant to your study. if you are not sure if a list item applies to your research, read the appropriate section before selecting a response. all antibodies used are commercialized and the fluorescence has been tested in this study. the information is included in table 4 . animals and other organisms policy information about studies involving animals; arrive guidelines recommended for reporting animal research laboratory animals six-ten-week-old c57bl/6 mice (japan slc, inc.) and transgenic mouse lines were used in this study. all animal care and experiments conformed to the guidelines for animal experiments of the university of tokyo, and were approved by the animal research committee of the university of tokyo (pa17-31 and pa17-17). all in vivo imaging studies were performed in the biosafety level 3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan. field-collected samples not applicable. all experiments with mice were performed in accordance with the university of tokyo's regulations for animal care and use and were approved by the animal experiment committee of the institute of medical science, the university of tokyo.note that full information on the approval of the study protocol must also be provided in the manuscript. key: cord-353013-7cx0gnum authors: deng, pengbo; hu, chengping; li, yuanyuan; cao, liming; yang, huaping; li, min; an, jian; jiang, juan; gu, qihua title: bronchial fistula: rare complication of treatment with anlotinib date: 2020-10-20 journal: zhongguo fei ai za zhi doi: 10.3779/j.issn.1009-3419.2020.102.40 sha: doc_id: 353013 cord_uid: 7cx0gnum background and objective: anlotinib is a newly developed small molecule multiple receptor tyrosine kinase (rtk) inhibitor that was approved for the treatment of patients with lung cancer in china. we aim to report 3 cases of rare complication of anlotinib-bronchial fistula (bf) during the treatment of lung cancer patients and summarize the possible causes. methods: we collected three patients who developed bf due to anlotinib treatment, and conducted a search of medline and pubmed for medical literature published between 2018 and 2020 using the following search terms: "anlotinib, " "lung cancer, " and "fistula." results: our literature search produced two case reports (three patients) which, in addition to our three patients. we collated the patients' clinical characteristics including demographic information, cancer type, imaging features, treatment received, risk factors for anlotinib related bf, and treatment-related outcomes. the six patients shared some common characteristics: advanced age, male, concurrent infection symptoms, diabetes mellitus (dm), advanced squamous cell and small cell lung cancers, centrally located tumors, tumor measuring ≥5 cm in longest diameter, and newly formed tumor cavitation after multi-line treatment especially after receiving radiotherapy. fistula types included broncho-pericardial fistula, broncho-pleural fistula, and esophago-tracheobronchial fistula. six patients all died within 6 months. conclusion: although anlotinib is relatively safe, it is still necessary to pay attention to the occurrence of bf, a rare treatment side effect that threatens the quality of life and overall survival of patients. anlotinib, therefore, requires selective use and close observation of high-risk patients. the application of anti-tumor angiogenesis and growth drugs in the treatment of lung cancer is widely recognized. anlotinib is a newly developed small molecule multiple receptor t y rosi ne k i na se (rt k s) i n h ibitor for ora l medication that targets vascular endothelial growth factor receptor (vegfr) 2/3, platelet-derived growth factor receptors (pdgfr) α/β, fibroblast growth factor receptors (fgfr) 1-4, c-kit and ret [1] , and has inhibitory effects on tumor angiogenesis. it was approved by china food and drug administration (cfda) for the treatment of patients with both non-small cell lung cancer (nsclc) and small cell lung cancer (sclc) and progression after >2 lines of chemotherapy. in december 2015 and june 2016, anlotinib was certified by the us food and drug administration (fda) for the treatment of ovarian cancer and soft tissue sarcoma. although anlotinib has fewer side effects than other small molecule drugs that target vascular function and inhibit tumor angiogenesis, it still has a 20% incidence of grade 3 or 4 treatment-related adverse events, and a dose reduction or suspension of treatment is usually required [2, 3] . the complications of bronchial fistula (bf) are not stated in clinical trials and cfda labeling, however, we have identified in our clinic several cases of patients with postanlotinib treatment-related bf, from which three cases were selected and combined with three cases reported by others, and their patient characteristics collated and analyzed to guide future clinical applications of anlotinib. case 1: a 69-year-old chinese male smoker presented to the local hospital with a persistent dry cough for nine months, hoarseness for two months, and shortness of breath for four days. a computed tomography (ct) scan of the chest showed that the left main bronchus was thickened and occluded, and the left hilar was occupied by a tumor (28 mm×31 mm) associated with the left pulmonary artery and invasion of the left atrium ( fig 1a) . electron microscopy revealed a left bronchial lesion, and its biopsy revealed a squamous cell carcinoma. electron microscopy guided biopsy of the left bronchial lesion provided a histopathological diagnosis of squamous cell lung cancer in july 2018. the patient refused chemotherapy and was instead treated with anlotinib (orally, 12 mg once daily on day 1 to 14 of a 21-day cycle). after the use of anlotinib for 1.5 mon, the patient' s symptoms improved, and a ct scan identified possible cavitation in the left upper lung tumor ( fig 1b) . however, another 1.5 months later, the patient worsened and displayed the following: shortness of breath, ct indicating the upper left lung tumor was larger than the before (55 mm×35 mm), with cavitation, as well as communication with the left main bronchus and pericardial cavity with large amounts of gas buildup, suggesting the formation of a possible bronchopericardial fistula (bpcf) (fig 1c) . a second bronchoscopy showed a huge fistula at the left main bronchus, a huge cavity was seen with a visible heartbeat on the inner wall ( fig 1d-fig1f ). the patient was treated with anti-infectives, given nutritional supplementation, and gas drained through a catheter inserted into his pericardium for 5 d, after which his ct showed a significant reduction of gas and his pericardium was partially conglutinated (fig 1d) . the patient, who was in poor condition, was required to return to the local hospital for supportive treatment, and eventually died due to tumor progression 2 months later. case 2: a 63-year-old chinese male smoker diagnosed with squamous cell cancer of the right lung [epidermal growth factor receptor (egfr) 19del, stage ivb] in 2016, with a history of diabetes mellitus (dm) (poorly controlled), who successively received the following: four series of chemotherapy cycles with gemcitabine (gem)+carboplatin (cbp); four months of targeted therapy (icotinib); 36 gy (3 gy×12 fractions) sequential radiation therapy on the lung tumor and mediastinal lymph node metastasis; and two chemotherapy cycles with paclitaxel (ptx); was started on anlotinib (orally, 12 mg once daily on day 1 to 14 of a 21-day cycle) in 2018 for four months. before initiating anlotinib, the patient's ct scan revealed a tumor in the central right upper lung (55 mm×65 mm) with an invasion of the right main bronchus and obstructive pneumonia in the right upper lung (fig 2a) . the patient's symptoms were alleviated transiently, and his ct revealed huge cavitation in the upper right lung tumor (fig 2b) . after four months of anlotinib treatment (in october 2018), the patient worsened and began coughing pyohemosputum (100 ml/d), blood sputum and fever, and his ct displayed a right broncho-pleural fistula (bpf) with liquid pneumothorax formation ( fig 2c) . staphylococcus aureus and stenotrophomonas maltophilia were cultured in the patient's sputum, and actinomyces cariestus was cultured in the pleural drainage f luid. the patient received closed thoracic drainage and multiple anti-bacterial therapies, after which the fever and drainage decreased. however, the patient died two months later due to tumor progression, failure of the fistula to close, and worsened infection. case 3: a 49-year-old chinese male smoker complained of dry cough for three months. a ct scan showed an upper left lung tumor (92 mm×89 mm) with left upper lung atelectasis, along with mediastinal lymphadenopathy (short axis=46 mm), and multiple thin-walled cavitation lesions in the left lower lung considered to be lung cancer metastases ( fig 3a, fig 3b) . bronchoscopy showed an endobronchial lesion in the left upper lobe bronchus, with an invasion of the left lower lobe bronchus and the left main stem. the patient was diagnosed in december 2019 with small cell lung cancer by endobronchial biopsy and confirmed to have multiple metastases to the pleura, pericardium, liver, and spleen. meanwhile, the patient was also diagnosed with active viral hepatitis b and poorly controlled dm. the patient received anti-tumor treatment with etoposide (vp-16), cbp, plus anlotinib (orally, 12 mg once daily on day 1 to 14 of a 21 d cycle) while receiving blood glucose regulation and anti-hbv treatment. ten days after completing his first course of treatment, the patient suddenly developed left chest pain, increased shortness of breath, cough, and sputum, without fever. due to the emergence of the coronavirus disease 2019 (covid-19) epidemic in china, he did not go to the hospital for examination promptly and continued to insist on oral treatment with anlotinib at home. six weeks later, his ct scan depicted smaller left upper lung tumor and mediastinal lymph nodes but also showed left lower lung atelectasis and a worm-like cavity, with an incomplete cavity wall and communication with the pleural cavity that led to the formation of a liquid pneumothorax ( fig 3c) . closed thoracic drainage was completed and a large amount of purulent fluid and gas were drained, which was an indication www.lungca.org klebsiella pneumoniae was also cultured in the pleural drainage fluid. the patient died 2.5 months later due to tumor progression a n d w o r s e n e d infection. we conduc ted a detailed search of the literature (written in engl ish) published be t we en 2 018 a nd 2 0 2 0 i n m e d l i n e a nd pubmed usi ng the following search criteria: "anlotinib," " lu ng ca ncer," a nd "fistula". the search ret r ieved t wo ca se reports of three lung cancer patients with f i s t u l a s re l at e d to the use of anlotinib. w e r e a d t h e f u l l te x t s, su m ma r i zed a n d a n a l y z e d t h e general information, t u mor-related data a n d c o m b i n e d t reat ment of t hese t h r e e p a t i e n t s , i n addition to data from ou r t h ree pat ients, and tr ied to der ive s o m e m e a n i n g f u l conjectures. t h e p a t i e n t s ' d e m o g r a p h i c information, cancer t y p e , i m a g i n g features, treatment, r i s k f a c t o r s o f anlotinib related bf www.lungca.org and outcome, etc. are shown in tab 1 [4, 5] . among the six cases in this review (including our case report), the mean age was 59.7-year-old (ranging from 49 yr to 69 yr), and 83.8% (5/6) of them are over 50 yr. the ratio of men to women was 5:1. smoking and non-smoking patients were equally divided (ratio 3:3). in our cases, two of the three patients had poorly controlled dm, and three had concurrent symptoms of infection when the bronchial fistula developed, including fever (2/3) and purulent sputum (3/3). five patients (83.3%) were diagnosed with squamous cell nsclc and one (16.7%) was diagnosed with sclc, all (100.0%) of them were central type. all patients (100.0%) had reached stage iiib and above at the start of anlotinib treatment, five of whom (83.3%) were in a metastatically advanced stage. the tumors on the ct showed just one case had a tumor with a <5 cm longest diameter, while the others were ≥5 cm (83.3%). before anlotinib treatment, only one patient had tumor cavitation, however, after anlotinib treatment, three more patients formed new cavities, with cavity formation rate reached 66.7%. the tumor of the majority of the patients (66.7%, 4/6) measured less than 1 cm from the pleura, except for two patients with esophago-tracheobronchial fistula (etbf). the number of patients treated with anlotinib in 1 st , 2 nd and 3 rd line treatment (or greater) was two, one, and three respectively, indicating 83.3% (5/6) patients received ≥2 line treatment. 33.3% (2/6) patients had received thoracic radiation therapy (trt). the types of bronchial fistulas the six patients developed were bpf (three patients), etbf (two patients), and one extremely rare bpcf. after the emergence of bf, all patients (100.0%) died within six months, with an average survival time of 2.67 months (ranging from 0.5 mon-6 mon). anlotinib is a novel orally administered multi-rtk inhibitor that inhibits tumor angiogenesis and growth [6] , which was demonstrated to have manageable toxicity, longer circulation times and broad-spectrum anti-tumor potential [7] . anlotinib was approved by cfda as a candidate drug for third-line treatment of advanced nsclc on may 9, 2018, and for third-line treatment of sclc on september 4, 2019. the above is based on a randomized double-blind controlled phase iii clinical trial called alter0303, whose results confirmed that anlotinib as a third-line treatment for nsclc patients can significantly improve their overall sur vival rate with controllable side effects [2] . a nother randomized phase ii study called alter1202 indicated that anlotinib appeared to provide significant progressionfree survival (pfs) and disease control rate (dcr) benefits for patients with sclc who progressed after two lines of chemotherapy [8, 9] . the above clinical studies found that hypertension, elevated thyroid-stimulating hormone, hand-and-foot syndrome, elevated thyroglobulin, elevated total cholesterol, and diarrhea [2, 3] were frequently observed during treatment with anlotinib. w hile hypertension, hyponatremia, and hemoptysis are the most common adverse events reported by patients with squamous cell lung cancer [10] , bf has not been www.lungca.org bf is a rare, life-threatening condition that seriously affects the quality of life of patients with lung cancer. it includes et bf, bpf, broncho-med iast i na l f ist u la (bmf), and the extremely rare bpcf. it can be caused by a tumor invasion but is more common in tumor patients after chemotherapy, radiotherapy, and inter ventional treatments [11, 12] . the incidence of malignant etbf has been reported to be 0.16% to 0.3% for lung cancer [13, 14] , with a median survival time (mst) from diagnosis of only 6 weeks in the absence of appropriate treatment [13] . bpf is more common in lung cancer patients after surgery, directly caused by lung cancer invasion is rarely reported which usually combined with infection [15] . in recent years, the use of anti-tumor vascular targeting drugs has also been found to cause bf. in the earliest clinical studies of bevacizumab, the first anti-angiogenic drug used in clinics, it was found that bevacizumab used in the treatment of lung cancer often caused bf, of which etbf was the most common [16] , bpf and bmf [17] were also have been reported. in phase ii clinical trials of bevacizumab in combination with chemotherapy and radiation for the treatment of sclc and nsclc, both groups had a very high incidence of bf (2/29, 2/5 respectively), and the death of one patient prompted early trial closures [18] . they consider that the unique role of bevacizumab in inhibiting angiogenesis and consequently delaying wound healing were likely accounts for this rare and serious effect. therefore, as anlotinib has a similar antiangiogenic effect with bevacizumab, when a patient develops a bf during the treatment of anlotinib, we should carefully consider the correlation between anlotinib and the bf formation. generally, we considered that anti-angiogenic drugs can lead to necrotic cavity formation due to ischemia in the center of the tumor. in our review, five patients (83.3%) had a tumor longer than 5 cm in diameter. and four of the five patients with large tumors formed a necrotic cavity after receiving treatment with anlotinib, leading to rupture of the cavity wall after continuous use. suggesting that we need to be highly vigilant when using anlotinib to treat large tumors, especially when new cavities appear. however, there were two cases of etbf that did not appear to display new necrotic cavities, possibly because the trachea is adjacent to the esophagus, similar to the central tumor, so only slight necrosis of the tube wall will form a fistula. therefore, patients with etbf may evade typical necrotic cavities after the use of anlotinib. a case of etbf caused by bevacizumab reported and summarized 9 similar cases, which concluded that trt had been suggested to be the most significant risk factor for bevacizumab-related etbf, also including damage to the esophagus and bulky subcarinal lymph node [16] . also, in our two cases, two had bulky subcarinal lymph node, and one had a history of trt, indicating trt and bulky subcarinal lymph node, suggesting that these two factors is also related to etbf, caused by anlotinib. acquired etbf should be suspected whenever patients with mediastinal metastasis develop suspicious clinical symptoms, for example, drinking waterwww.lungca.org induced choking cough, especially coughing up food just eaten. the diagnosis relies on an esophagram, ct, and videof luoroscopy. fistulas that occur in the bronchi and its distal end may be difficult to plug with a stent implant and an occlude, and the chance of surgical management for advanced lung cancer patients is almost impossible. however, for etbf, a fully covered self-expandable metallic stent has been demonstrated as a superior alternative treatment owing to its high efficiency, which immediately relieved the patient's symptoms after palliative treatment [4] . two of t he t h ree bpf pat ient s we ev a lu ated had pathogenic bacteria cultured from sputum or drainage fluid, including staphylococcus aureus, actinomycetes and klebsiella pneumoniae, which can all cause necrotizing pneumonia, lung abscess, and empyema, especially in tumor patients with cavities; this may also be one of the important causes of bf. it has been reported that patients with lung cancer have decreased immunit y after chemotherapy and an actinomycetes infection may finally lead to the occurrence of bpf [15] . therefore, for lung cancer patients who have a lot of purulent sputum after anlotinib treatment, we need to actively identify the pathogens and utilize anti-infective treatments to prevent infections from aggravating tumor necrosis. meanwhile, two of our three patients have poorly controlled dm, which causes decreased immunity, making them prone to co-infection. as we know, central lung cancer easily causes obstructive pneumonia, and anlotinib causes tumor necrosis. in this situation, once the patients with dm develop pulmonary infections, it will be more difficult to control. this may be one of the possible risk factors for the formation of bf and also needs to be paid attention to. 83.8% (5/6) of our bf patients are over 50 yr, and half of them are over 60 yr, which can be correlated to the higher incidence of lung cancer in people over 50. also, the incidence of bf is significantly higher in males than in females, and all lung cancer types are squamous cell carcinoma and small cell lung cancer, which was more common in men. from the perspective of cancer type and location, squamous cell carcinoma and small cell lung cancer are more likely to be located at the hilum and easily advance to the mediastinum and trachea, causing an invasion of the airway mucosa, necrosis, and bleeding, leading to the defect of the tracheal wall, and fistulas are also easily formed. five of them were diagnosed by bronchoscopy which further confirmed that the lesions affected the trachea. in our review, four of the six patients (66.7%) were undergoing treatment with ≥2 lines of therapy, and three were treated with more than four lines (50%). multi-line treatment means that the patient has a difficult to control tumor, has an impaired immune system, is in poor general condition, and may have cachexia. these characteristics of patients in advanced tumor stages may be the reason that the tumor is more easily necrotic, less repairable, and more likely to cause secondary infection when treated with anlotinib. two patients also received trt, which is a recognized risk factor that can cause bf, especially etbf and bmf [19] [20] [21] , suggesting that we need to pay attention to the patient's radiotherapy history before initiating anlotinib therapy. bpcf in lung cancer patient is extremely rare, only a few cases have been reported [22] . in one case, a patient with squamous carcinoma, stage iiia, acquired bpcf after chemotherapy and trt, after recovered from bpcf, he continued received trt and chemotherapy, subsequently treated with bevacizumab and cetuximab. three months later, the patient reappeared bpcf and died of fatal hemoptysis quickly. in this case, the role of bevacizumab in the recurrence of bpcf needs to be paid attention to. in our patient cases, there was a patient with bpcf, who had the following characteristics which may be the risk factors for bpcf development: the tumor was located in the left hilar, close to the mediastinal pleura and pericardium; the patient had poorly controlled dm; although he was using anlotinib as the 1 st line treatment, he was in poor general condition (ps score 3) with cachexia; and a necrotic cavity formed in the tumor after treatment with anlotinib. based on the patient's previously discussed characteristics, when lung cancer patients have exacerbated shortness of breath and chest tightness during the use of anlotinib, in addition to considering other common causes, it is necessary to pay attention to the possibility of bpcf. prompt puncture drainage and active anti-infective treatments can promote pericardial adhesion closure, which is impor tant for improving the prognosis of patients. in general, once a patient develops a bf during the use of anlotinib, the survival time will decrease significantly, and there will no longer be an opportunity for further chemotherapy, radiotherapy, and vascular targeted therapy. most patients die in a short time due to the hard-to-heal fistulas, recurrent infections that are difficult to control, and tumor progression. it has been repor ted that an adenocarcinoma lung cancer patient developed a bmf after 1 st line chemotherapy, radiotherapy, 2 nd line chemotherapy plus bevacizumab, then used nivolumab (an immunecheckpoint inhibitor) as follow-up treatment. to heal the bf, the treatment requires sufficient anti-tumor efficacy, low risk of bacterial infection, and low risk of weakening the airway wall integrity. immune checkpoint inhibition is effective for nsclc but rarely causes necrosis, is less toxic, as well as less likely to lead to infections compared to cytotoxic drugs [23] [24] [25] [26] . nivolumab successfully controlled the bmf and the tumor of this patient and prolonged the survival time of the patient for 28 months [27] . a lso, if patients have sensitive gene www.lungca.org mutations, targeted therapy should be considered as one of the alternatives. a lthough the data provided by anlotinib in current clinical trials indicate it is relatively safe, it is still necessary to pay attention to the occurrence of adverse reactions in clinical applications, such as bf. the incidence of bronchial fistula caused by anlotinib in lung cancer is extremely rare, but it seriously affects the quality of life and overall survival of patients. therefore, we need to use it selectively and closely observe high-risk patients. it's possible risk factors include: (1) ≥50 years old male, (2) dm and infection, (3) central lung cancer, squamous cell carcinoma or sclc, (4) advanced stage, (5) long diameter of tumor≥5 cm and cavity formation, (6) multi-line treatment, and (7) thoracic radiation therapy. a nlotinib inhibits angiogenesis v ia suppressing the activation of vegfr2, pdgfrβ and fgfr1 effect of anlotinib as a third-line or further treatment on overall survival of patients with advanced non-small cell lung cancer: the alter 0303 phase 3 randomized clinical trial anlotinib as a third-line therapy in patients with refractory advanced non-small-cell lung cancer: a multicentre, randomised phase ii trial (alter0302) esophago-tracheobronchia l f istula fol low ing treatment of anlotinib in advanced squamous cell lung cancer: two case reports bronchopleural fistula in squamous cell lung cancer following anlotinib treatment: a case report preclinical characterization of anlotinib, a highly potent and selective vascular endothelial growth factor receptor-2 inhibitor safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors anlotinib as third-line or further-line treatment inrelapsed sclc: a multicentre, randomized, double-blind phase 2 trial effect of anlotinib in advanced small cell lung cancer patients previously received chemoradiotherapy: a subgroup analysis in alter 1202 trial subgroup analysis of histolog y in a lter0303: a nlotinib hydrochloride as 3 rd line and further line treatment in refractory advanced nsclc patients (pts) malignant esophagorespiratory fistula: management options and survival tracheal-oesophageal fistula in a patient with lung cancer tracheoesophageal fistula tracheoesophageal fistula due to cancer bronchoepidural fistula in a man with actinomycosis complicated non-small cell lung cancer tracheomediastinal fistula: rare complication of treatment with bevacizumab tracheoesophageal fistula formation in patients with lung cancer treated with chemoradiation and bevacizumab spontaneous pneumothorax due to bronchopleural fistula following reirradiation for locoregionally recurrent squamous cell lung cancer tracheo-parenchymal fistula following concurrent chemo-radiation for stage iii nsclc tracheo-esophageal fistula with bevacizumab after mediastinal radiation nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer pembrolizumab versus docetaxel for previously treated, pd-l1-positive, advanced non-small-cell lung cancer (keynote-010): a randomised controlled trial atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (oa k): a phase 3, open-label, multicentre randomised controlled t r ia l . l a ncet bronchial fistula: rare complication of treatment with anlotinib t h i s m a n u s c r i p t w a s s u p p o r t e d b y n a t i o n a l nat u r a l s c ienc e fou nd at ion of c h i n a (to pen g bo deng pb, cao lm and yang hp conceived and designed the study. deng pb, li yy and jiang j collecting the data. deng pb, li m, an j and gu qh analyzed the data. deng pb, li yy, cao lm and hu cp provided critical inputs on design, analysis, and interpretation of the study. all the authors had access to the data. all authors read and approved the final manuscript as submitted. our study has already approved by examination and approval documents for scientific research projects medical ethics committee of xiangya hospital, central south university. key: cord-021744-x320625f authors: thompson, mark s. title: systemic approach to differential diagnosis date: 2017-11-17 journal: small animal medical differential diagnosis doi: 10.1016/b978-0-323-49830-2.00002-0 sha: doc_id: 21744 cord_uid: x320625f nan protein concentration is greater than 2.5 g/dl nucleated cell count ranges from 400-10,000/ml predominant cell type on cytology is the small lymphocyte (also see neutrophils, macrophages, plasma cells, and mesothelial cells) triglyceride concentration of pleural fluid is greater than that of serum (definitive test) signs of these two types of allergies are similar. atopy tends to occur primarily in young adults, whereas food hypersensitivity can begin at any age. atopy is usually seasonal at first but may become less seasonal. causes mechanistic • secretory • osmotic • permeability (exudative) • dysmotility • mixed temporal • acute • chronic anatomic • extraintestinal • small intestinal • large intestinal • diffuse pathophysiologic • biochemical • allergic • inflammatory • neoplastic etiologic • bacteria • dietary • fungal • idiopathic • parasitic • viral causal • exocrine • thrombocytopenia-increased buccal mucosal bleeding time (bmbt), decreased platelet count (plt), normal activated partial thromboplastin time (aptt), normal prothrombin time (pt), normal fibrin degradation products (fdp) • platelet dysfunction (e.g., aspirin treatment)-increased bmbt, normal plt, increased aptt, normal, pt, normal fdp • intrinsic pathway defect (e.g., hemophilia a or b)-normal bmbt, normal plt, increased aptt, normal pt, normal fdp • factor vii deficiency-normal bmbt, normal plt, normal aptt, increased pt, normal, fdp • multiple factor defects (e.g., vitamin k antagonism)-normal bmbt, normal plt, increased aptt, increased pt, normal fdp • common pathway defect (e.g., factor x deficiency)-normal bmbt, normal plt, increased aptt, increased pt, normal fdp • dic-increased bmbt, decreased plt, increased aptt, increased pt, increased fdp • von willebrand disease-increased bmbt, normal plt, normal aptt, normal pt, normal fdp differential diagnosis acute onset of coughing, sneezing, nasal discharge, ocular discharge low-grade fever secondary commensal bacterial infections leading to mucopurulent discharge and productive cough may lead to pneumonia with high fever, inappetence, productive cough, and increased respiratory effort variable incubation period, prodromal phase: nervousness, anxiety, paresthesia progress to forebrain signs ("furious" form of rabies): irritability, restlessness, pica, photophobia, increased saliva production with decreasing ability to swallow, hyperesthesia progressing to incoordination, seizures, and death may also progress to "dumb" form: paralysis, lower motor disease, leading to coma, respiratory paralysis, and death pseudorabies suspected to be result from ingestion of infected raw pork neurologic dysfunction: ataxia, abnormal papillary light response, restlessness, trismus, cervical rigidity, ptyalism, tachypnea, excoriation from pruritus of head and neck; vomiting, diarrhea; most dogs die within 48 hours hacking cough with gagging, easily elicited with tracheal palpation; cough may be paroxysmal, usually subsides within 7-10 days, and may lead to secondary bacterial or mycoplasmal infection • bun/creatinine-increased in 50%-65% of dogs and in 33% (cr) and 57% (bun) in cats. usually prerenal due to dehydration and hypotension. may be secondary to intrinsic renal failure (sepsis and immune-complex). • potassium-decreased in 20% of cases in dogs and 56% in cats. increased loss in vomiting and due to renal loss with fluid therapy plus reduced intake and aldosterone release caused by hypovolemia. • sodium-can be increased, decreased, or normal. increase usually caused by dehydration, decrease caused by losses secondary to vomiting. • calcium-commonly decreased in cats, rarely in dogs, rarely increased in both dogs and cats. reduction is a poor prognostic indicator in cats, but has no prognostic significance in dogs. may be caused by saponification in peripancreatic fat and glucagon release stimulating calcitonin. • chloride-very commonly decreased in dogs. loss in gi secretions in vomiting. • phosphate-often increased in dogs; uncommonly increased or decreased in cats. increase usually due to reduced renal excretion secondary to renal compromise. decrease (in cats) due to treatment for diabetes mellitus. • glucose-increased in 40%-88% of dogs and decreased in up to 40%. increased in 64% of cats, rarely decreased. increase due to decreased insulin and increased glucagon, cortisol, and catecholamines. decrease caused by sepsis or anorexia. • albumin-increased in 39%-50% and decreased in 17% of dogs. increased in 8%-30% and decreased in 40% of cats. increase due to dehydration. decrease due to gut loss, malnutrition, concurrent hepatic disease, or renal loss. • hepatocellular enzymes (alt, ast)-increased in 61% of dogs and 68% of cats. hepatic necrosis and vacuolation due to sepsis, local effects of pancreatitis +/-concurrent hepatic disease in cats. • cholestatic enzymes (alp and ggt)-increased in 79% of dogs and 50% of cats. biliary obstruction due to acute or chronic pancreatitis +/-concurrent cholangitis +/-lipidosis in cats; steroid-induced alp in dogs. • bilirubin-increased in 53% of dogs and 64% of cats (same causes as ggt and alp). • cholesterol-increased in 48%-80% of dogs and 64% of cats. can be due to cholestasis; unclear if cause or effect. • triglycerides-commonly increased in dogs. unclear if cause or effect. • neutrophils-increased in 55%-60% of dogs, increased in 30% and decreased in 15% of cats. increased due to inflammatory response. decreased in some cats due to consumption, may be a poor prognostic indicator. • hematocrit-increased in about 20% and decreased in 20% of both dogs and cats. increased due to dehydration and decreased due to anemia of chronic disease or gastric ulceration. • platelets-commonly decreased in severe cases in dogs. decreased due to circulating proteases +/à dic. failure of normal closure of neural tube: vary in severity from clinically inapparent (agenesis of corpus callosum) to severe (anencephaly) lissencephaly: failure of normal migration of neurons in development of cerebral cortex; leads to abnormal appearance of sulci and gyri (most often seen in lhasa apso) cerebellar hypoplasia: seen most often in cats after in utero panleukopenia infection; rarely seen with parvovirus infection of developing cerebellum in dogs; may be isolated malformation without infection chiari-like malformations: protrusion of cerebellar vermis through foramen magnum (cavalier king charles spaniel, other dog breeds) hydrocephalus: congenital hydrocephalus seen most often in toy and brachycephalic breeds; suggests hereditary basis; often congenital stenosis or aplasia of mesencephalic aqueducts • confirm and remove small uroliths from bladder or urethra. • obtain uroliths for quantitative analysis and culture. • retrieve uroliths from bladder or urethra using stone forceps or stone basket. • fragment uroliths with laser lithotripsy. • fill bladder before and after voiding urohydropropulsion to remove small uroliths. should be bilaterally symmetric, have a smooth and regular surface, have soft parenchyma, and not be painful to touch. • radiography of limited value for providing an actual diagnosis but may provide information about size, shape, contour, and location of the prostate. prostatomegaly may cause dorsal displacement of the colon and cranial displacement of the urinary bladder. mineralization with neoplasia, bacterial prostatitis, and abscessation may be apparent. • prostatic ultrasound is the most useful and practical imaging method. normal prostate should have smooth borders and homogenous parenchymal pattern of moderate echogenicity. ultrasound also offers the opportunity for guided aspirates and core biopsy sampling for culture, cytology, and histopathology. • ct and mri can evaluate size, shape, and homogeneity of prostate and allow evaluation of intrapelvic lesions, metastatic spread, and ureteral obstruction. • definitive diagnosis requires cytologic, histologic, or bacteriologic evaluation of a prostate sample. samples can be obtained using procedures such as semen collection, prostatic massage and wash, brush technique, fine needle aspiration, and biopsy. • stop use of nephrotoxic drugs. • if proteinuria is insignificant (trace to 1 + dipstick reading and urine specific gravity >1.035), there is no need for further workup. • perform urinalysis to exclude hemorrhage, infection, or inflammation as cause of proteinuria. if these conditions are present, do urine culture. if these conditions are not present, do urine protein/creatinine ratio. • perform serum chemistry and cbc. evaluate serum albumin and globulin. • marked proteinuria ratio (up/uc >3) with quiet sediment and normal globulins or a polyclonal gammopathy is consistent with renal glomerular disease (glomerulonephritis, amyloidosis). rule out causes of glomerulonephropathy such as heartworm disease, hepatozoonosis, immune-mediated diseases such as sle, chronic infectious diseases such as borreliosis, felv, fiv, ehrlichiosis, other chronic inflammatory diseases, neoplasia, and hyperadrenocorticism. • if no underlying disease found, may need renal biopsy to assess for glomerulonephritis or amyloidosis • proteinuria detected by precipitation testing but not dipstick or proteinuria associated with a monoclonal gammopathy may be caused by bence jones proteins. this requires a search for osteolytic or lymphoproliferative lesions. ehrlichiosis may mimic myeloma. if ehrlichia negative, protein electrophoresis in indicated. a monoclonal gammopathy suggests myeloma. clinical findings, dogs afast as an extension of the physical exam dh view • dh and cc views are most common positive sites in lowscoring dogs and cats. • useful for detecting pericardial effusion (racetrack sign) and pleural effusion • advantage: less air interference than transthoracic tfast views • assessment of the weak or collapsed patient's volume status by observing dynamics of caudal vena cava (cvc) as it passes through diaphragm • may be indicative of right-sided heart failure, pericardial effusion/tamponade in acute collapse/weakness • may be indicative of volume overload, third spacing, primary gallbladder disease, and pancreatitis in the less acute patient • liver masses, cysts, and diffuse or irregular changes in echogenicity may be appreciated • least gravity-dependent view where air would rise to (pneumoabdomen) and fluid only at this site may be retroperitoneal rather than intraabdominal • acoustic window into the abdominal and retroperitoneal space for free abdominal fluid and retroperitoneal fluid • splenic masses and diffuse or irregular changes in echogenicity may be appreciated. • left kidney may appreciate variety of pathology, including hydronephrosis, pyelectasia, cortical cysts, perinephric cysts, masses, mineralization, calculi, and mineralization. • may be able to also see right kidney in small dogs and cats through the sr view • cc and dh views are most common positive site in low-scoring dogs and cats. • urinary bladder may appreciate variety of lesions such as calculi, masses, wall thickening/abnormalities, and emphysema. • spleen and small intestine most often visible here • splenic masses and diffuse or irregular changes in echogenicity may be appreciated. • small intestine pathology may be appreciated, including dilated loops (ileus, obstruction), wall thickening, masses, and related lymph nodes. • the name of this view is a misnomer because the liver and right kidney are not typically imaged. • liver and right kidney are normally not present at the level of the umbilicus unless they are enlarged. • stomach is not visible at the level of the umbilicus unless it's distended. • the hr umbilical view completes afast and is likely the region to perform abdominocentesis in higher-scoring dogs and cats. • patients may be positioned in right or left lateral recumbency, especially if tfast exam pcs views immediately follow the afast exam in stable patients, and in respiratorycompromised patients, and for the chest tube site (cts) view; sternal recumbency or standing positioning is safer and preferred for the entire tfast examination. • ultrasound probe is placed in five positions: 1. diaphragmatico-hepatic (dh) view-immediately caudal to the xiphoid (same as afast dh view). useful for detecting pericardial effusion (racetrack sign) and pleural effusion. left and right cts views-at the level of the seventh to eighth intercostal spaces at the highest point, upper third of the thorax, where lung may be visualized on the dorsolateral thoracic wall in the absence of pneumothorax, and where the cap of air would rise in the presence of pneumothorax • if evidence of lung against the thoracic wall is observed sonographically, then pneumothorax is effectively ruled out. • if there is no evidence that lung is against the thoracic wall, then the lung point is searched for where the transition zone is between pneumothorax and lung recontacting the thoracic wall. 3. left and right pcs (pericardial site) view-over the heart at the level of the fifth and sixth intercostal spaces in gravity-dependent regions of the thorax • pcs views are used for quick assessment of lungs, heart, pleural, and pericardial spaces. tfast as an extension of the physical exam • part of both afast and tfast examinations-see afast dh view • useful for sonographic confirmation of pleural effusion and pericardial effusion (racetrack sign) • less air interference than tfast transthoracic pcs views, liver and gallbladder provide acoustic window into thorax • allows for assessment of volume status by observing dynamics of the caudal vena cava (see dh view in afast as an extension of exam). • the most recent, most accurate described methodology by the originator of the vet blue is to begin by finding the transition zone in a standing (or sternal) patient at the cts/cd view where abdominal contents and lung are viewed over an intercostal space, then sliding toward the head two intercostal spaces to begin the vet blue at the cd view (point 1). • from the cd view (point 1) draw an imaginary line to the elbow. halfway from the cd to the elbow is the ph view (point 2), and at the elbow is the md view (point 3) and then in the axillary area as the final cr view. if the heart is in view at the md, slide the probe directly dorsally until over lung for the md view, and define the cr view by finding the transition of lung and thoracic inlet, then sliding caudally over the first two intercostal spaces. if a gator sign orientation is not observed, then you cannot be assured lung is being imaged. vet blue lung ultrasound findings in progression from most to least aerated/most consolidated • dry lung-glide sign with a-lines (reverberation artifact) at lung line indicates dry lung at the lung periphery. the confounder: a-lines with no glide sign consistent with pneumothorax • wet lung ulrs (also called b-lines)-hyperechoic streaks that oscillate with respiration and extend to the far field, obliterating a-lines • shred sign-deviation of the lung line (pulmonary-pleural line) and within the deviation hyperechoic foci of air movement seen in bronchi. comparable to a radiographic air bronchogram. indicates lung consolidation/infiltration. • tissue sign-more severe consolidation/infiltration where no air movement is present. referred to as hepatization of lung. • nodule sign-anechoic round (nodule) often with a hyperechoic far border and acoustic enhancement through the far field as a ulr. vet blue differential diagnosis for patients with respiratory signs with dry lung in all fields • upper airway disease (laryngeal paralysis, collapsing trachea) • airway obstruction (mass) • feline asthma • chronic obstructive pulmonary disease • pulmonary thromboembolism • centrally located lung lesion away from lung line, therefore missed by vet blue uveitis differential diagnosis in the dog and cat systemic infection bacterial • bacteremia or septicemia (d, c) • bartonellosis (d, c) • leptospirosis (d) • borreliosis (d) • brucellosis (d) rickettsial • ehrlichiosis (d, c) • rocky mountain spotted fever (d) viral • canine adenovirus-1 (d) • felv (c) • fiv (c) • fip (c) histoplasmosis (d, c) • coccidiomycosis (d, c) • cryptomycosis (d, c) • aspergillosis (d) algal • protothecosis parasitic • aberrant nematode larval migration • toxocara (ocular larval migrans) (d, c) • dirofilaria larvae (d) leishmaniasis (d, c) immune-mediated uveitis • idiopathic anterior uveitis (d, c) • lens-induced uveitis (d, c) • canine adenovirus vaccine reaction (d) • uveodermatologic syndrome (d) (primarily akita and arctic breeds) primarily golden retrievers) mammary tumors • mixed tumors (fibroadenomas) • adenomas • mesenchymal tumors • malignant mammary tumors • solid carcinomas • tubular adenocarcinomas • papillary adenocarcinomas • anaplastic carcinomas • sarcomas (rare) • most feline mammary tumors are adenocarcinomas bb pellets or shot may be confused with small mammary masses) prostatic disease differential diagnosis benign prostatic hyperplasia (bph) acute prostatitis chronic prostatitis abscess cyst prostatic neoplasia • adenocarcinoma most common • transitional cell carcinoma second most common • urothelial carcinoma • primary and metastatic hemangiosarcoma • history of lower urinary tract signs, penile discharge, hematuria, dysuria, tenesmus, obstipation, ribbon stools, stiff gait. severe systemic signs suggest sepsis or systemic inflammation raises suspicion of acute prostatitis • digital rectal examination along with caudal abdominal palpation is a noninvasive initial screening test. the prostate acquired • toxic (escherichia coli endotoxin) • drugs (glucocorticoids, chemotherapeutics) • metabolic disease (hypokalemia, hypercalcemia) • tubular injury or loss active urine sediment • good body condition • hyperkalemia (if oliguric) • normal to increased hematocrit • enlarged kidneys • potentially severe metabolic acidosis • severe clinical signs for level of dysfunction • normal-sized parathyroid glands (ultrasound appearance) chronic renal failure • history of previous renal disease • history of polyuria/polydipsia • small irregular kidneys • nonregenerative anemia • normal to hypokalemia • normal to mild metabolic acidosis • inactive urine sediment • weight loss/cachexia • mild clinical signs for level of dysfunction • enlarged parathyroid glands calculi (nephroliths or nephrolith fragments that have migrated into the ureter) acute pain preemptive scoring system (examples in each category) chronic pain assessment acute pain assessment subjective evaluation of pain in animals relies on observation and interpretation of animal behavior. pain may be indicated by loss of normal behaviors or appearance of abnormal behaviors thrashing • lethargic, withdrawn, dull, obtunded • may ignore environmental stimuli • abnormal sleep-wake cycle, inability to sleep • may bite, lick, or chew painful area • adopt abnormal body positions to cope with pain, hunched posture, "prayer position" • abnormal tail position • lameness, abnormal gait • anorexia, reluctant to eliminate • ears held back, eyes wide open with dilated pupils or closed with a dull appearance • disuse or guarding of painful area • vocalization • may become more aggressive and resist handling or palpation or may become more timid and seek increased contact with sit very quietly, and pain may be missed by those looking for more active signs of pain acute pain preemptive scoring system (examples in each category) minor procedures: no pain • physical examination, restraint • radiography • suture removal, cast application, bandage change moderate surgeries: moderate pain • ovariohysterectomy, castration, caesarean section major surgeries: severe pain • fracture repair, cruciate ligament repair • thoracotomy, laminectomy, exploratory laparotomy • limb amputation • ear canal ablation chronic pain assessment clinical signs of chronic pain depend on underlying cause and pathologic state. in treatment (e.g.,osteoarthritic dog that experiences acute pain after excessive strenuous activity) examinations (focused assessment with sonography for trauma rule-outs include: • right-sided volume overload • pulmonary hypertension • right-sided heart failure • dilated cardiomyopathy 2. an attenuated cvc with little variability in its diameter supports a low cvp and hypovolemia; cvc is flat • rule-outs include causes of profound hypovolemia, including hypovolemia and distributive shock the cvc normally has a change in its diameter of between 30% and 50%; in the ballpark of normal with a "bouncing" appearance • gallbladder is often adjacent the diaphragm on dh view • may be displaced by an enlarged liver • may be difficult to image with diaphragmatic hernia or gallbladder rupture, calculi/mineralization, or emphysema • feline gallbladders are more difficult to image on dh view. • gallbladder wall edema used to visualize the heart, pericardial space, and pleural space • assess for pericardial or pleural effusion combining with the dh view increase depth so that the heart is seen in its entirety to avoid false positives from mistaking right ventricle/other heart chambers for effusion views: left ventricular short axis view to assess volume status and contractility; long axis four-chamber view to assess for right-sided conditions thoracic surgery, lung lobe aspirate, thoracoscopy, tracheal wash, thoracentesis probe is positioned as described at the tfast cts view but then moved through three more views bilaterally • vet blue has eight total acoustic views (four views bilaterally) cd (caudodorsal lung region)-same as tfast cts view, upper third of the thorax at the level of the eighth to ninth intercostal spaces directly above the xiphoid near the highest point where lung may be visualized on the dorsolateral thoracic wall ph (perihilar lung region)-sixth to seventh intercostal space, middle third of the thorax md (middle lung region)-fourth to fifth intercostal space, lower third of the thorax cr (cranial lung region)-second to third intercostal space gfast triad for volume status and patient monitoring gfast, the name for the use of afast and its fluid scoring system, tfast and vet blue combined, may be used for rapid patient volume status assessment during, before, and after fluid resuscitation • characterization of cvc and hepatic veins for estimation of cvp (see earlier); forms of shock (e.g., hypovolemic/distributive shock/cardiogenic/obstructive shock) • tfast-assessment of cardiac views for volume and contractility, right-and left-sided conditions (see tfast) • vet blue-presence of wet lung screens for left-sided cardiac overload, and the pattern-based approach and vet blue lung ultrasound signs help determine chf, pneumonia, neoplasia, granulomatous conditions, pte (pulmonary thromboembolism), and others fast ultrasound edited by gregory r. lisciandro, dvm, dipl. abvp, dipl. acvecc of hill country veterinary specialists, fastvet.com and editor of textbook focused ultrasound techniques for the small animal practitioner amphotericin b ethylene • patients are placed in either lateral recumbency; right is preferred because the basic echo views, gallbladder, caudal vena cava, left kidney are more easily imaged. dorsal recumbency should not be used because of increased patient respiratory and hemodynamic stress. ultrasound probe is placed in four regions of abdomen:1. diaphragmatic-hepatic (dh) view-at the level of the xiphoid, images the diaphragm, liver, gallbladder, caudal vena cava, pleural space, pericardial space, and lung 2. spleno-renal (sr) view-images spleen, left kidney, abdominal and retroperitoneal space 3. cysto-colic (cc) view-images bladder; however, an airfilled colon can confound imaging 4. hepato-renal (hr) umbilical view-images small intestine and spleen • probe is fanned in longitudinal (sagittal); transverse is not necessary • purpose is quick assessment of afast target organs and detection of free abdominal and retroperitoneal fluid. blood rapidly defibrinates in blunt trauma and nontrauma so it is seen as anechoic black triangulations. penetrating trauma is different initially because blood often clots and blends in as soft tissue. abdominal fluid score (afs) blunt trauma (think medical first)• afs 1 and 2 are major injury, small volume bleeder-no blood transfusion needed, not expected to be anemic (pcv >35%) if intraabdominal bleeding only. reassess by afast and afs (abdominal fluid score) to monitor for changes minimally 4 hours postadmission and sooner if unstable. if afs stays 1-2 but pcv drops, look for bleeding at another site (retroperitoneal, pleural space, fracture site, external). • afs 3 and 4 are major injury, large volume bleeder (afs [3] [4] or becomes . expect anemia (pcv <35%), use graduated fluid therapy (one third shock dose) and repeat titrated fluid challenges needed. with severe anemia (pcv <25%) blood transfusion is often necessary, and surgery uncommon. • blood from ripping, tearing, crushing tends to clot, making it blend with adjacent tissue and difficult to detect by afast. with time clotted blood will defibrinate and become visible as black anechoic triangulations. serial exams are key and should be performed as often as needed until certain the patient is medical and not surgical. • afs 3 and 4 initially or on serial exams need surgical ligation of bleeding. • afs 1 and 2 that stays 1 or 2 with serial exams is not surgical. • afs 3 and 4 that are not anemic still need surgical exploration; waiting may lead to need for transfusion and additional risk and expense. nontraumatic hemoabdomen (variably medical and surgical) • bleeding intraabdominal tumor, spleen most common, pcv generally low normal or low. surgical problem. • useful for ruling out pneumothorax and for surveying for lung lesions. • probe is placed perpendicular to the long axis of the adjacent ribs in order to image the intercostal space • the orientation obtained is referred to as the gator (alligator) sign from the image created by rounded rib heads as the gator's eyes and the intercostal space, a white line, as the gator bridge of its nose, likened to a partially submerged alligator peering at the sonographer. • glide sign-normal to-and-fro motion of lung along the intercostal space or more specifically the movement of parietal and visceral pleural ruling out pneumothorax. absence of the glide sign suggests pneumothorax. • ultrasound lung rockets (ulr, also called b-lines)-hyperechoic streaks that extend from pleural line through the far field that oscillate like a pendulum in synchrony with respiration 1. trauma-associated ulrs immediately rule out pneumothorax at that level of the thorax and support lung contusions until proven otherwise. 2. in nontrauma ulrs (also called b-lines) represent various forms of alveolar-interstitial edema, including left-sided chf, hemorrhage, variety of pneumonias, inflammation, as more common causes (see vet blue) • step sign-deviation from the expected linear pulmonarypleural interface 1. chest wall trauma or disease (intercostal tears, fractured ribs, subpleural hematoma) 2. pleural space disease (effusion, diaphragmatic hernia, masses) • lung point-location or transition zone at which collapsed lung secondary to pneumothorax recontacts thoracic wall 1. move probe ventrally to middle, then ventral or lower third of the thorax with patient standing or sternal until evidence of lung against the thoracic wall is found, then move incrementally dorsally until lung is lost to determine the exact lung point 2. use the lung point to assess and monitor pneumothorax; upper one third trivial to mild; middle one third is moderate; lower one third is severe. key: cord-348672-e34103b1 authors: zhang, jiaqi; bai, wenliang; guo, chao; liu, lei; wang, guige; huang, cheng; chen, yeye; zhang, ye; li, shanqing title: postoperative short-term outcomes between sublobar resection and lobectomy in patients with lung adenocarcinoma date: 2020-10-01 journal: cancer manag res doi: 10.2147/cmar.s266376 sha: doc_id: 348672 cord_uid: e34103b1 background: to investigate postoperative temporary consequences of the enrolled patients with lung adenocarcinoma. patients and methods: we analyzed the clinical data of patients with lung adenocarcinoma admitted by the same surgical team of peking union medical college hospital (pumch) from july 2019 to december 2019. statistical methods including propensity score matching (psm) analysis was used to analyze the differences among them. results: a total of 108 patients were enrolled, including 50 patients with sublobar resection and 58 patients with lobectomy. before psm, there were statistically significant differences in age (p=0.015), hospitalization costs (p=0.042), lymphadenectomy (p=0.000), pathological staging (p=0.000), number of lymph nodes removed (p=0.000), number of positive lymph nodes (p=0.034), chest drainage duration (p=0.000), total chest drainage (p=0.000), length of postoperative hospital stays (p=0.000), postoperative d-dimer level (p=0.030) and perioperative lymphocyte margin (lm) (p=0.003) between sublobar resection and lobectomy. after psm, there were statistical differences in number of lymph nodes removed (p=0.000), chest drainage duration (p=0.031) and total chest drainage (p=0.002) between sublobar resection and lobectomy. whether with psm analysis or not, there were no significant differences in other blood test results, such as inflammation indicators, postoperative neutrophil-lymphocyte ratio (nlr), albumin level, perioperative activity of daily living (adl) scale scoring margin, complications, postoperative admission to intensive care unit (icu) and readmission within 30 days. nlr was associated with total chest drainage (p=0.000), length of postoperative hospital stays (p=0.000), postoperative d-dimer level (p=0.050) and adl scale scoring margin (p=0.003) between sublobar resection and lobectomy. conclusion: sublobar resection, including wedge resection and segmentectomy, was as safe and feasible as lobectomy in our study, and they shared similar short-term outcomes. postoperative nlr could be used to detect the clinical outcomes of patients. secondary resectability of pulmonary function (srpf) should be the main purpose of sublobar resection. according to global cancer statistics in 2018, lung cancer, accounting for 11.6% of all cancers, has been the most common cancer and the leading cause of cancerrelated death. 1 in recent years, with the progress of cancer screening and decline in smoking, the population of lung cancer-related deaths has declined. early diagnosis and early treatment significantly benefit the overall survival of lung cancer patients. the latest us cancer statistics reported that the five-year survival rate of lung cancer was 19%. 2, 3 the continuous optimization of surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy has gradually improved the survival of lung cancer patients. surgical treatment progresses rapidly. based on considerations such as minor damage and preservation of normal lung function, and as a result of the early detection of lung cancer, sublobar resection gradually emerged. studies have shown that sublobar resection had a good protective effect on patients' residual lung function. 4 however, the surgical damage evaluation of patients with different surgical methods is inconclusive, and the impact of different lung resections on the short-term outcomes of patients is not yet clear. considering that adenocarcinoma dominated a great part in lung cancer, the purpose of this study was to retrospectively analyze the short-term outcomes of lung adenocarcinoma patients who underwent different lung resections. we retrospectively evaluated a consecutive series of patients who underwent lung resection for lung adenocarcinoma in the department of thoracic surgery, pumch from july 2019 to december 2019. the institutional review board of pumch approved the study. written informed consent was obtained from all eligible patients. all patients underwent systemic evaluation before surgery, including clinical staging evaluation and cardiopulmonary function evaluation. preoperative cancer staging was assessed by positron emission tomography/computed tomography (ct), or chest and abdomen ct combined with enhanced brain magnetic resonance imaging/ct and whole-body bone scintigraphy. the eighth edition ajcc/ uicc lung cancer stage classification was used for staging. patients included met the following criteria: (1) surgeons were senior physicians in the same surgical team with similar oncological and surgical principles (one surgical team, consisting of a professor who is the director of thoracic surgery (>10 years) and three attending physicians); (2) video-assisted thoracic surgery (vats) was performed in all patients; (3) postoperative paraffin pathology confirmed lung adenocarcinoma or carcinoma in situ. exclusion criteria included the following: (1) preoperative neoadjuvant therapy; (2) preoperative systemic evaluation considered malignant pleural effusion, n3 lymph node metastasis or extrathoracic metastases; (3) mediastinal mass resection, esophageal repair and other nonpulmonary operations were performed simultaneously; (4) metastatic lung cancer; (5) simultaneous bilateral surgery. vats was a priority in all operations. if an unexpected situation occurred during the operation, such as major bleeding, the operation would be converted to thoracotomy. lobectomy and sublobar resection were conducted according to the nccn guidelines. 5 sublobar resection was conducted if the nodule was ≤2 cm and it met one of the following criteria: adenocarcinoma in situ, groundglass opacity (ggo) >50% or doubling time ≥400 days. segmentectomy was preferred over wedge resection. in sublobar resection, including wedge resection and segmentectomy, stapler was used for dividing intersegmental plane. systematic lymph node dissection was usually performed in lobectomy, and systematic lymph node sampling was often conducted in sublobar resection. preoperative preparations included smoking and alcohol cessation, respiratory function training and patients' education. patients fasted for eight hours before surgery, and resumed oral fluid intake six hours after surgery. blood was monitored on the first morning after operation. a bedside chest radiograph was conducted on the first day after surgery to assess lung retention and chest drainage status. early mobilization with underground activities at least 2~3 times a day was encouraged, following by normal diet. lower fat intake and appropriate protein and electrolyte supplementation during perioperative period were recommended. drugs for pain relief, phlegm reduction, atomization and preventing infection were routinely given to patients after surgery. according to the patient's serum albumin level, we administrated them with human albumin to ensure a normal level. if chest drainage was less than 200 ml/d after surgery, and there were no obvious air leak and chylothorax, the chest tube would be removed. a chest radiograph would be taken on the first day after extubation. patients without obvious pneumothorax and pleural effusion could be considered for discharge. all patients were scored with the activity of daily living (adl) scale by specialized nurses when they were admitted to and discharged from hospital. outpatient follow-up for all patients was conducted about one month later. data were collected from all enrolled patients comprising basic information, surgical information, pathology, perioperative blood test results, chest drainage status, hospitalization schedule, complications, adl scale score, postoperative admission to icu, readmission within 30 days and hospitalization costs (before settlement of medical insurance). we calculated body mass index (bmi). blood test results include white blood cell (wbc) counting, neutrophil counting, lymphocyte counting, hemoglobin concentrations, platelet counting, albumin level and d-dimer level. we calculated the nlr, neutrophil-albumin ratio, platelet-d-dimer ratio, platelet-albumin ratio, d-dimer margin, albumin margin, neutrophil margin, lymphocyte margin (lm), platelet margin, wbc margin. with respect to complications, we listed several common or serious ones, including acute coronary syndrome (acs), pulmonary embolism (pe), atrial fibrillation (af), pneumonia, air leak and chylothorax. to minimize selection bias between two groups, a propensity score matching (psm) analysis was performed. the ratio of patients in each group was 1:1, the match tolerance was therefore set at 0.02, and the following variables were used for the psm analysis: sex, age, bmi, and pathological staging. statistical analysis was performed using spss 23.0 software (ibm corp., armonk, ny, usa). the measurement data were expressed as x ±s. one-way anova test or rank sum test was used to compare the differences between measurement data. count data were expressed as percentages, and comparisons between groups were performed using chi-squared test. a two-tailed p<0.05 was considered statistically significant. the correlation of measurement data was analyzed by rank correlation test. a total of 108 patients with lung adenocarcinoma were enrolled, including 31 males and 77 females, with average age of (57.09±12.00) years. among them, 50 patients had sublobar resection and 58 patients underwent lobectomy. patients were classified into two groups according to different lung resections, and the demographics of patients in each group are shown in table 1 . all patients had no perioperative blood transfusion. female patients accounted for 71.30% of enrolled patients, but there was no statistically significant difference in sex between sublobar resection and lobectomy (p=0.316). there was statistical difference in terms of age (p=0.015). there were statistically significant differences in lymphadenectomy (p=0.000) and pathological staging (p=0.001) between sublobar resection and lobectomy. but among pathological results, spread through air spaces (stas) (p=0.916), vascular invasion (p=0.103), visceral pleural involvement (p=0.331), and bronchus invasion (p=0.073) showed no statistically significant differences between sublobar resection and lobectomy. there were a larger number of lymph nodes removed and number of positive lymph nodes in patients with lobectomy than that in patients with sublobar resection (p=0.000, p=0.034, respectively). between sublobar resection and lobectomy, there were statistically significant differences in chest drainage duration (p=0.000), total chest drainage (p=0.000), length of postoperative hospital stays (p=0.000), and hospitalization costs (p=0.042). but there were no statistical differences in duration of operation (p=0.063) and bleeding volume during surgery (p=0.181). postoperative d-dimer level (p=0.030) and perioperative lm (p=0.003) were statistically different between sublobar resection and lobectomy. there were no statistically significant differences in nlr (p=0.170) and other test results between them. among 108 patients, three had af, four had air leak, one had acs and one suffered chylothorax. two patients were admitted to icu after operation because of acs and one patient was admitted to icu because of anaphylactic shock. three patients returned to hospital within 30 days owing to recurrent pleural effusion, influenza a virus infection and lower extremity deep vein thrombosis, respectively. after symptomatic treatment, they recovered well without serious consequences. there were no significant differences among sublobar resection and lobectomy in terms of postoperative complications, postoperative admission to icu (p=0.473), readmission within 30 days (p=0.473), and adl scale scoring margin (p=0.743). after psm analysis, sublobar resection and lobectomy showed different lymphadenectomy (p=0.000), number of postoperative chest drainage is the main concern which both surgeons and patients focused on. our results showed that total chest drainage had a relation with age (correlation coefficient 0.380, p=0.000), hospitalization costs (correlation coefficient 0.320, p=0.001), number of lymph nodes removed (correlation coefficient 0.505, p=0.000), duration of operation (correlation coefficient 0.489, p=0.000), postoperative albumin level (correlation coefficient −0.228, p=0.017) and postoperative nlr (correlation coefficient 0.339, p=0.000). related results are shown in figure 1 . postoperative nlr had a positive relation with total chest drainage (correlation coefficient 0.346, p=0.000), length of postoperative hospital stay (correlation coefficient 0.358, p=0.000), postoperative d-dimer level (correlation coefficient 0.189, p=0.050), and perioperative adl scale scoring margin (correlation coefficient 0.282, p=0.003). (figure 2) in the past 20 years, thoracic minimally invasive surgery (mis) has continuously pushed the limit, and enhanced recovery after surgery (eras) has gradually gained popularity. to reduce surgical damage and improve the quality of life of patients have gradually become the needs of patients and the goals of surgeons. there has been evidence that patients with vats gained noninferior long-term survival when compared with patients with thoracotomy for stage i non-small cell lung cancer (nsclc). 6 compared with thoracotomy, mis was associated with shorter hospital stays, lower pain levels and fewer complications. [7] [8] [9] [10] in recent years, the proportion of vats in our center has increased, and our patients have recovered well. but there is no convincing evidence about whether different lung resections by means of vats really have the same minimally invasive effects on patients. sublobar resection, including segmentectomy and wedge resection, has gradually emerged, because patients could obtain good lung function retention, 11 and in patients with early-stage nsclc, segmentectomy seemed to provide a superior recovery in quality of life compared with lobectomy. 12 but whether vats segmentectomy could preserve lung function better than vats lobectomy in patients with poor lung function remains unclear. 13 suzuki et al suggested that no functional advantage for segmentectomy was observed during long-term follow-up, possibly due to compensatory lung growth after lobectomy. 14 in particular, complex lung segment resection might have limited recovery of residual lung function, resulting in the same loss of lung function as lobectomy. 15 with regard to cancer prognosis, patients with early-stage lung cancer undergoing segmentectomy showed a similar survival compared with patients undergoing lobectomy. [16] [17] [18] [19] but it is undeniable that there is difference in lymph node dissection between segmentectomy and lobectomy.17, 20 our study supported this opinion. some studies revealed that, segmentectomy was associated with similar complications compared with lobectomy, except for air leak. 21, 22 our study suggested that patients showed similar clinical outcomes in postoperative complications, postoperative admission to icu, adl scale scores margin, and readmission within 30 days among different lung resections. postoperative inflammatory response can affect patients' immunity. the evaluation of inflammatory response should also be considered in mis. many studies have suggested that nlr, as an indicator of inflammatory response, could also affect the tumor microenvironment, thereby affecting tumor prognosis and cytokines released by the tumor microenvironment could also cause neutrophil recruitment. 23, 24 the results of this study showed that perioperative nlr was an important indicator, which was related to total chest drainage, length of postoperative hospital stays, postoperative d-dimer level and perioperative adl scale scoring margin. no significant difference was found in nlr between different lung resections, although statistical difference in lm was significant. consequently, we thought different lung resections we performed might share similar inflammatory response and they had the same minimally invasive effect on patients. in addition, based on the results of this study, we hypothesized that nlr could be used to detect the clinical outcome of patients to some extent. above all, our results showed that there were differences in chest drainage duration and total chest drainage between different lung resections, but the differences in cost, length of postoperative hospital stay, blood test results, complications in the short-term were not significant. although there were statistically differences in total chest drainage and chest drainage duration between different lung resections, they were not significantly different from the overall mean, and the inflammatory response shared a similar level between different lung resections. all the lung resections we performed were safe and feasible. patients underwent different lung resections shared similar shortterm outcomes. therefore, for the choice of lung resection, we believe that the length of hospital stays and complications cannot be the reasons for sublobar resection. how to exactly measure the trauma stress and inflammatory response of different lung resections are still what we need to tackle. whether the so-called mis can produce minimally invasive effect on patients still needs continuous improvement by surgeons. for cancer treatment, radical tumor resection, as the primary principle, should be considered firstly, while patient's quality of life and secondary resectability of pulmonary function (srpf) should also be weighed. here, we define srpf that, for patients with multiple primary lung cancer, the surgeon should try to preserve as much normal lung tissue as possible to ensure that the second primary cancer on the same side would be removable in the future, which also this study has several limitations. being a single-center retrospective review means it has all the biases based on this study type. oncological outcomes and long-term effects were unclear. prognosis for different lung resections may vary and large-scale, multi-center trials are needed. sublobar resection, including wedge resection and segmentectomy, were as safe and feasible as lobectomy in our study. patients underwent different lung resections shared similar short-term outcomes regarding medical expense, complications, and surgical damage. to some extent, postoperative nlr level could be used to detect the clinical outcomes of patients in critical condition. indications of lung resection should be standardized to ensure that srpf is the main purpose of sublobar resection. long-term oncology comparison between different resections is essential for further instruction. this study was reviewed and approved by the institutional review board of pumch. the authors are accountable for all aspects of the work. patients were informed of treatment and collection of data. written informed consent was obtained from all eligible patients. to the doctors, nurses, and all workers on the front line for their devotion to the control of novel coronavirus epidemic. there is no funding to report. the authors report no conflicts of interest in this work. cancer management and research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. submit your manuscript here: https://www.dovepress.com/cancer-management-and-research-journal global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries cancer statistics, 2020 differences in postoperative changes in pulmonary functions following segmentectomy compared with lobectomy a national analysis of longterm survival following thoracoscopic versus open lobectomy for stage i non-small-cell lung cancer video-assisted thoracic surgery versus open lobectomy for lung cancer: a secondary analysis of data from the american college of surgeons oncology group z0030 randomized clinical trial thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensitymatched analysis from the sts database video-assisted thoracoscopic surgery versus open lobectomy for primary non-small-cell lung cancer: a propensity-matched analysis of outcome from the european society of thoracic surgeon database postoperative pain and quality of life after lobectomy via video-assisted thoracoscopic surgery or anterolateral thoracotomy for early stage lung cancer: a randomised controlled trial evaluation of the residual lung function after thoracoscopic segmentectomy compared with lobectomy perioperative course and quality of life in a prospective randomized multicenter phase iii trial, comparing standard lobectomy versus anatomical segmentectomy in patients with non-small cell lung cancer up to 2 cm, stage ia (7th edition of tnm staging system) lung segmentectomy: does it offer a real functional benefit over lobectomy? does segmentectomy really preserve the pulmonary function better than lobectomy for patients with early-stage lung cancer? surg today systemic and regional pulmonary function after segmentectomy comparison of segmentectomy and lobectomy in stage ia adenocarcinomas segmentectomy is equivalent to lobectomy in hypermetabolic clinical stage ia lung adenocarcinomas lobectomy does not confer survival advantage over segmentectomy for non-small cell lung cancer with unsuspected nodal disease comparison of perioperative and oncological outcomes between video-assisted segmentectomy and lobectomy for patients with clinical stage ia non-small cell lung cancer: a propensity score matching study long-term results for clinical stage ia lung cancer: comparing lobectomy and sublobar resection comparison of pulmonary segmentectomy and lobectomy: safety results of a randomized trial comparison of postoperative complications between segmentectomy and lobectomy by video-assisted thoracic surgery: a multicenter study neutrophils in the tumor microenvironment neutrophil-to-lymphocyte ratio and risk of lung cancer mortality in a low-risk population: a cohort study we would like to give our sincere thanks to nurses in our center for their nursing services. and we want to pay tribute key: cord-343842-2klytw6c authors: takamura, shiki title: persistence in temporary lung niches: a survival strategy of lung-resident memory cd8(+) t cells date: 2017-07-01 journal: viral immunol doi: 10.1089/vim.2017.0016 sha: doc_id: 343842 cord_uid: 2klytw6c respiratory virus infections, such as those mediated by influenza virus, parainfluenza virus, respiratory syncytial virus (rsv), severe acute respiratory syndrome coronavirus (sars-cov), rhinovirus, and adenovirus, are responsible for substantial morbidity and mortality, especially in children and older adults. furthermore, the potential emergence of highly pathogenic strains of influenza virus poses a significant public health threat. thus, the development of vaccines capable of eliciting long-lasting protective immunity to those pathogens is a major public health priority. cd8(+) tissue-resident memory t (t(rm)) cells are a newly defined population that resides permanently in the nonlymphoid tissues including the lung. these cells are capable of providing local protection immediately after infection, thereby promoting rapid host recovery. recent studies have offered new insights into the anatomical niches that harbor lung cd8(+) t(rm) cells, and also identified the requirement and limitations of t(rm) maintenance. however, it remains controversial whether lung cd8(+) t(rm) cells are continuously replenished by new cells from the circulation or permanently lodged in this site. a better understanding of how lung cd8(+) t(rm) cells are generated and maintained and the tissue-specific factors that drive local t(rm) formation is required for optimal vaccine development. this review focuses on recent advance in our understanding of cd8(+) t(rm) cell establishment and maintenance in the lung, and describes how those processes are uniquely regulated in this tissue. m emory t cells have been divided into two distinct subsets based on their distinct migratory properties (107) . central memory t (t cm ) cells express lymph nodehoming receptors l-selectin (cd62l) and cc-chemokine receptor 7 (ccr7), and preferentially circulate between lymph nodes and blood. effector memory t (t em ) cells lack the expressions of these receptors and circulate between the blood and nonlymphoid barrier tissues such as the skin, lung, intestines, and female reproductive tract. upon secondary infection, t em cells exhibit immediate effector functions at the site of infection, while t cm cells undergo extensive expansion in the draining lymph nodes before migrating to the site of infection and eliminating virus-infected cells (106) . it has recently emerged that memory t cells in the nonlymphoid tissues, which had previously been classified as a circulating t em population, include a noncirculating cell population that resides permanently within the peripheral tissues. these cells have been termed tissue-resident memory t (t rm ) cells (34) and comprise the majority of memory t cells in the nonlymphoid tissues that confer immediate protection against peripheral infection (119) . low levels of t em cells also transit the peripheral tissues and contribute to local protection (83) . more recent studies have revealed that t rm cells are present in wide variety of lymphoid and nonlymphoid tissues, including brain, salivary glands, thymus, spleen, lymph nodes, liver, kidneys, pancreas, heart, and dorsal root ganglia (96) . the generation and maintenance of t rm cells in each of these tissues differ significantly, indicating a major role for tissue-specific instruction (53) . therefore, there is a need to identify the unique signals underlying each tissue microenvironment and the molecular mechanisms that instruct t rm formation. during primary respiratory virus infections, antigenspecific cd8 + t cells are crucial to the elimination of virusinfected cells and in the case of influenza viruses, cross reactive cd8 + t cell-mediated immunity can provide protection against different viral strains (heterosubtypic immunity) (21) . thus, understanding the mechanisms by which cd8 + t rm cells are established in the lung has important implications for vaccine development. following resolution of respiratory virus infections, cd8 + t rm cells persist in at least two distinct compartments of the lung: the lung interstitium/parenchyma and the lung airways (44) . cd8 + t rm cells in the lung interstitium/parenchyma are mainly found as confluent peribronchiolar cell infiltrates in the interstitium and alveolar spaces (123) . note that the term ''lung parenchyma'' indicates a part of lung involved in gas exchange, such as the alveoli, alveolar ducts, and respiratory bronchioles, but does not include the lung interstitium. in contrast, cd8 + t rm cells in the lung airways are localized primarily in the epithelial layers of the upper respiratory tract and can be easily isolated by bronchoalveolar lavage (bal) (25, 47, 48) . both t rm populations confer rapid protection against secondary infection (37, 91, 135) , and the number of antigen-specific cd8 + t rm cells in those tissues correlates with the efficacy of protection. importantly, however, the molecular and cellular mechanisms underlying their recruitment, differentiation, maintenance, and recall differ significantly (44) . thus, the precise discrimination of those populations is necessary to comprehensively understand cd8 + t cell-mediated antiviral immunity in the lung. this review will focus primarily on influenza and parainfluenza virus infections and discuss recent insights into the course of cd8 + t rm cell establishment in the lung interstitium/ parenchyma and airways, from initial priming, to tissue migration, local differentiation, and maintenance. cd8 + t cell priming following a respiratory virus infection occurs in the lung-draining mediastinal lymph nodes (mln) after lung-resident antigen-presenting cells (apcs) have transported viral antigens to that site. lymph noderesident cd8a + conventional dendritic cells (dcs) that acquire antigens from migrant respiratory dcs were initially thought to be pivotal in the initiation of antiviral cd8 + t cell responses (10) . it has recently become apparent, however, that two subsets of respiratory dcs transport viral antigens from the lung and prime naive cd8 + t cells in the mln (60) . moreover, those two dc subsets exhibit distinct t cell stimulatory functions and also regulate the tempo of migration to the mln upon infection, resulting in the generation of distinct memory cd8 + t cell subsets in both lymphoid and nonlymphoid tissues (11, 35) . in brief, cd103 + respiratory dcs that reside between/below epithelial cells possess the specialized ability to uptake apoptotic cellassociated antigens (e.g., virus-infected cells) and crosspresent them on the mhc class i molecules (20, 43, 46) . upon infection, migration of antigen-laden cd103 + respiratory dcs to the mln dominates the early stage of infection (2-4 days) (36, 60) . these cd103 + respiratory dcs express high levels of the costimulatory molecule cd24, which has been decorated with high mobility group box 1 (hmgb1), a damage-associated molecular pattern (damp) released from dying cells, thereby providing strong stimulatory signals to cd8 + t cells through its ligand, receptor for advanced glycan end-products (rage) (61) . cd8 + t cells activated by these cd103 + respiratory dcs proliferate vigorously and become potent effector cells that preferentially home back to the lung to eliminate virally infected cells (36, 52) . since entry into the peripheral tissues is necessary for subsequent differentiation into cd8 + t rm cells, priming with cd103 + respiratory dcs is potentially the primary factor controlling the development of t rm cells. in contrast, cd11b hi respiratory dcs transport and cross-present viral antigens in the mln at later time points during the infection (5-7 days) (7, 60, 94) . those include cells that originally reside in the lung interstitium and new emigrants to the lung in response to inflammation (e.g., monocyte-derived dcs) (35) . cd11b hi respiratory dcs uniquely upregulate cd70, the ligand for cd27, in response, in part, to thymic stromal lymphopoietin (tslp) secreted by virus-infected lung epithelial cells (136) , and are thus capable of providing costimulatory signals to cd8 + t cells (7) . several lines of evidence support the notion that cd11b hi respiratory dcs contribute less to antiviral cd8 + t cell immunity than cd103 + respiratory dcs because only the selective loss of cd103 + respiratory dcs leads to a severe reduction in the antigen-specific cd8 + t cell responses (36, 46, 60, 100) . the weaker stimulatory potential of cd11b hi respiratory dcs presumably explains their characteristics: preferential generation of the memory cd8 + t cell population that persists in the secondary lymphoid organs, rather than generation of fully differentiated effector cd8 + t cells (61) . thus, the distinct functionality of respiratory dc subsets critically impacts the memory cd8 + t cell heterogeneity. a study by mikhak et al. has suggested that lung dcs are capable of imprinting cd4 + t cell homing to the lung through selective upregulation of ccr4 (93) . however, since lung dcs from naive animals that are expanded by injection of fms-like tyrosine kinase 3 ligand (flt3l) have been demonstrated in this study, it is unclear which dc populations contributed to the lung imprinting. considering the migratory property of activated t cells to the lung, it is likely that dcs purified from flt3l-treated lung tissues largely contain lungresident cd103 + dcs, but not cd11b hi dcs. it is interesting to speculate whether lung imprinting signals could also affect cd8 + t cells. new definitions provided by intravascular staining intravascular (i.v.) staining of cells in the bloodstream by i.v. injection with specific antibodies before harvesting the cells has revolutionized the analysis of lung t rm (5, 6) . since the lung is a highly vascularized organ, it became apparent that a majority of cd8 + t cells purified from the lung tissues in earlier studies were contaminants from the blood. in fact, >95% of total cd8 + t cells in naive animals and *50% of antigen-specific memory cd8 + t cells in mice that had recovered from an influenza virus infection were found to be derived from the lung vasculature (6, 126) . note that because the lung airways are segregated from the blood vessels, i.v. staining has essentially no impact on the cells in these tissues. however, a careful reinterpretation of previously published data regarding cells in the lung interstitium/parenchyma analyzed without i.v. staining is required. for instance, it has been reported that antigen-specific cd8 + t cells generated by intraperitoneal infections were efficiently recruited to the interstitium/parenchyma, but not to the lung airways when the effector t cell numbers peak (9-11 days) even in the absence of progressive infection or inflammation in the lung (122) . furthermore, parabiosis experiments in which pairs of mice are surgically joined revealed that significant numbers of memory cd8 + t cells could also be recruited to the lung as up to half of the cells in this organ were replaced by circulatory cd8 + t cell populations (63, 84) . by using i.v. staining, those conclusions have been revised. first, although effector cd8 + t cells migrate into the interstitium of normal lungs more efficiently than naive cd8 + t cells (33) , the extent is much less than that observed in the presence of infection/inflammation in the lung (123) . second, the migration of circulating memory cd8 + t cells to the lung under steady-state conditions is also relatively limited, as the ratio of new immigrants to resident cells never exceeds 20% (123) . on the basis of these new findings, we propose that the migration of effector as well as naive /memory cd8 + t cells into the noninflamed lung be termed as ''basal recruitment'' and be distinguished from ''active recruitment'': migration of antigen-specific effector cd8 + t cells to the lung in response to inflammation in the tissues (fig. 1) . strict discrimination between basal and active recruitment is important because tissue-derived instructive factors (e.g., antigen and inflammatory stimuli) that dictate t rm differentiation differs significantly depending on how the cells were recruited. influence of chemokines on the active and basal recruitment of cd8 + t cells to the lung unlike skin and intestines, where specialized adhesion molecules and chemokine receptors regulate selective migration of t cells to those tissues (e.g., integrin a4b7 and fig. 1. compartmentalization of cd8 + t rm cells and cd8 + t em cells in the lung. memory cd8 + t cells in the lung consists of a major (*80%) population of t rm cells and a minor (*20%) population of t em cells. during the acute phase of a respiratory virus infection, effector cd8 + t cells are recruited to the lung (active recruitment) and acquire tissuederived instructions necessary for differentiation into terminal effector cells. cd8 + t rm precursors are recruited to the site of tissue damage during later stages of the infection and receive instructive signals from several factors (such as local antigen and tgf-b) before differentiating into t rm cells. cd8 + t rm niches (ramds) are created as a consequence of tissue remodeling and provide temporal spaces for the maintenance of cd8 + t rm cells. cd8 + t rm cells in the ramds are maintained in a cd69-independent manner due to spatial separation from the lymphatics. because cd8 + t rm cells in the lung airways are short-lived, this population may be maintained by the continual recruitment of cells from the cd8 + t rm pool in the lung interstitium/parenchyma. cd8 + t em cells are recruited to the uninfected lung interstitium during steady state (basal recruitment). those cells are segregated from the cd8 + t rm niches and residual antigen-presenting cells in the lung, thereby causing them to exit from this tissue through the lymph in response to s1p-induced chemotactic signal. some cells are activated by antigen-independent stimulus in the interstitium and transiently express cd69. cd69-mediated inhibition of s1p 1 leads to temporal retention of cd8 + t em cells in the interstitium, which potentially enable subsequent recruitment of cells to the lung airways. ramds, repair-associated memory depots; s1p, sphingosine 1-phosphate; t em , effector memory t; tgf-b, transforming growth factor-b; t rm , tissue-resident memory t. ccr9 for the intestines, and cutaneous lymphocyteassociated antigen [cla], ccr4, and ccr10 for the skin, respectively), molecules that specifically regulate t cell trafficking to the lung have not yet been reported. instead, general factors such as lymphocyte function-associated antigen-1 (lfa-1) (125) and inflammatory chemokine receptors ccr5 and cxc chemokine receptor 3 (cxcr3) have been shown to be involved (134) . ccr5 ligands are constitutively expressed in the normal lung and regulate the basal recruitment of ccr5 + effector cd8 + t cells to the interstitium (33) . upon respiratory virus infection, the expression of ccr5 binding chemokines as well as cxcr3 binding chemokines is upregulated in the lung (64, 134) , and various cell types are involved in the secretion of these chemokines, including epithelial cells, dcs, macrophages, endothelial cells, and mast cells (22, 99, 124) . ccr5 is also transiently expressed on the surface of antigen-experienced cd8 + t cells in the circulation shortly after respiratory virus infection (peaking at day 2 postinfection), and this upregulation is probably induced by proinflammatory cytokines (64) . since only limited numbers of virally primed antigen-specific cd8 + t cells exist at this time point, ccr5-mediated active recruitment of cells to the lung airways is antigen independent (23). this influx (3-5 days) is a part of acute response during respiratory virus infections (44) , and antigen-nonspecific memory cd8 + t cells recruited to the lung airways provide ''innate'' protection (64) . nevertheless, the lack of ccr5 alone has essentially no impact on the active recruitment of expanded antigen-specific effector cd8 + t cells to the lung (5-10 days) (30, 66) , suggesting the redundancy of signals through various inflammatory chemokine receptors in this process. in contrast to ccr5, cxcr3 is expressed in a large fraction of antigen-specific effector as well as memory cd8 + t cells, and plays a major role in the active recruitment of those cells to the inflamed lung (30, 77, 111) . after entry into the lung, cxcr3 ligands guide effector cd8 + t cells to the sites of infection/inflammation in the lung and accelerate effector maturation (66) . some cells maintain the expression of cxcr3 and are preferentially recruited to the vicinity of virus-infected epithelial cells and airway lumen, while cells that receive signals from il-12 and il-15 downregulate cxcr3 and are retained in the peribronchioarterial area where they provide a ''second wave'' of protection (1). interestingly, lim et al. have demonstrated that neutrophils that infiltrated the lung during the early phase of infection (around day 4) leave long-lasting trails of cxcl12 that guide and accelerate the migration of effector cd8 + t cells to the lung airways in a cxcr4-dependent manner (76) . thus, cxcr3 and cxcr4 cooperatively regulate the active recruitment of cells into the inflamed lung. cxcr3 also contributes to the basal recruitment of memory cd8 + t cells to the lung airways in the absence of any infection in the lung (116) . this feature may reflect, in part, the superior functionality of cxcr3 hi memory cd8 + t cells in mounting recall responses against respiratory virus infection (45) . it has also been reported that cxcr6 + memory cd8 + t cells accumulate in the lung following intranasal, but not intradermal delivery of antigen (72, 73) . in fact, the expression of cxcl16 is strongly enhanced in response to inflammatory stimuli, thereby accelerating the active re-cruitment of effector t cells into inflamed tissues (2, 38, 85) . interestingly, this chemokine is constitutively expressed in the lung (18, 127) . these observations strongly suggested that cxcr6 contributes to both active and basal recruitment of cd8 + t cells to the lung. it is well known that sphingosine 1-phosphate (s1p) plays a key role in the egress of lymphocytes from the lymph nodes and thymus (4, 86) , and recent studies indicate that this is also the case for the nonlymphoid tissues (71, 78, 115, 123) . s1p is present at high levels in the blood and lymph, which results in the continuous downregulation of its receptor s1p receptor-1 (s1p 1 ) on circulating t cells (102, 110) . upon tissue entry, cd8 + t cells instantly recover surface expression of s1p 1 due to the relatively low concentration of s1p in the parenchymal tissues (15) and are thereby subjected to opposite chemotaxis toward the lymphatics (78, 115) . hence, a balance between the s1p-mediated exit signal and the chemokine-mediated migratory signals controls the direction of t cell migration, which potentially reflects the differential dynamics between t em and t rm cells within the peripheral tissues. the t cell expression of s1p 1 is regulated by at least two distinct mechanisms: local cytokine-induced transcriptional downregulation of s1pr1, which encodes s1p 1 (115) , and the activation maker cd69-mediated posttranscriptional antagonism (8, 114) . under the basal recruitment (in the absence of strong chemokine signaling in the tissues), forced expression of s1p 1 or genetic deletion of cd69 results in the significant loss of tissue-circulating cd8 + t cells as well as cd8 + t rm precursors in the nonlymphoid tissues, including the lung (78, 115, 123) , which is likely due to the accelerated tissue egress mediated by s1p 1 . importantly, although pharmacological inhibition of s1p 1 by the agonist fty720 leads to transient accumulation of cd69 -cd8 + t cells in the lung, this was reversed when fty720 treatment was discontinued (123) . these findings suggest that t cells are committed to leave tissues during steady-state conditions, unless s1p 1 expression is inhibited. since recruitment of cd8 + t cells into the lung does not lead to the downregulation of klf2 or its downstream target s1pr1 (123), local reactivation and subsequent upregulation of cd69 are likely crucial for the temporal retention of tissue-circulating cd8 + t cells in the lung under the basal recruitment conditions (fig. 1) . in contrast, we have demonstrated that this is not the case during the acute phase of infection where the lack of cd69 has a little impact on the active recruitment of effector cd8 + t cells to the lung unless a cd69-intact wildtype competitor t cell is present (123) . thus, during the acute phase of infection, inflammation-induced chemotactic signaling overrides the s1p-mediated exit signal, which may explain the appearance of cd8 + t cells specific for unrelated antigens in the lung (23, 123) . ccr7 is also known to regulate the egress of effector cd8 + cells from the lung interstitium through the lymph (12, 19, 56) . this is consistent with the fact that activated respiratory dcs migrate to the mln in a ccr7-dependent manner (39, 42) . as is the case with s1p 1 , antigen recognition in the lung leads to downregulation of ccr7 on effector cd8 + t cells, suggesting that s1p 1 and ccr7 cooperatively accelerate the egress of antigen-specific cd8 + t cells from the lung. it is also noteworthy that antigen-dependent selective retention of effector cd8 + t cells may prevent overt pathogenesis by decreasing the number of bystander t cells at the site of infection (56) . however, as most ccr7 should be already downregulated when expanded effector cd8 + t cells leave the mln and all of previous data were analyzed in the presence of large proportions of cell contaminants in the blood, analysis using i.v. staining would be required to precisely understand the role of this chemokine receptor. integrin-mediated retention in the specific microenvironment of the lung in addition to cd69, high levels of integrin a1b1 (vla-1: very late antigen-1) and aeb7 (detected by cd49a and cd103, respectively) expression are unique hallmarks of cd8 + t rm cells in the lung compared to circulatory memory cd8 + t cell populations in the lymphoid and nonlymphoid tissues, including t em cells in the lung (123) . vla-1 preferentially binds to type iv and type i collagen (40, 58, 128) . type iv collagen constitutes the major structural component of basement membranes of the vascular endothelium and airway epithelium, while type i collagen is widely distributed in the lung interstitium (104) . in contrast, integrin aeb7 binds to e-cadherin, which forms adherens junctions between lung airway epithelial cells (98) . analyses using blockade antibodies or knockout mice have revealed that both vla-1 and integrin aeb7 are not required for the active recruitment of cd8 + t cells to the lung (74, 103) . instead, these integrins fine-tune the distribution of migrant cd8 + t cells within the lung. for example, vla-1 promotes distribution of cells in close proximity to the basement membranes of the blood vessels as well as the airways (103, 104) . the binding of vla-1 to type iv collagen, in combination with signaling through tumor necrotic factor (tnf) receptor ii, also protects effector cd8 + t cells in the airways from apoptosis during the acute phase of infection (105) . interestingly, compared to effector cd8 + t cells, effector cd4 + t cells recruited to the lung exhibit lower vla-1, but higher vla-2 (integrin a2b1 detected by cd49b), which prefer type i, but not type iv collagen, allowing cd4 + t cells to localize preferentially in the lung interstitium, but not in the airways (104) . integrin aeb7 also promotes retention of effector cd8 + t cells in the lung airways (51, 74) . it is important to note, however, that such integrin-mediated retention signals are likely redundant or supportive as the lack of one of those integrins does not lead to the significant loss of effector cd8 + t cells in the lung airways (74, 103) . it is currently unclear what signals are responsible for the upregulation of vla-1 on cd8 + t cells. antigen-specific cd8 + t cells in nonlymphoid tissues are mostly vla-1 + , indicating the universal role of this integrin in the retention of cells that are ''basally'' recruited to the nonlymphoid tissues (103) . interestingly, large numbers of vla-1 + cd8 + t cells accumulate in the lung following respiratory, but not systemic infections/vaccinations (108, 123) . furthermore, local inflammation in the presence of cognate antigen, but not inflammation alone, enables the conversion of circulating cd8 + t cells into vla-1 + cd8 + t rm cells in the lung (123) , suggesting the impact of local reactivation on the upregulation or maintenance of vla-1 expression on actively recruited cd8 + t cells to the inflamed nonlymphoid tissues. in support of this, cd49a expression defines a marker of cd8 + t rm cells having a highly cytolytic potential (13) . however, additional experiments are required to precisely resolve the question where and when upregulation of vla-1 occurs. on the other hand, transforming growth factor-b (tgf-b) signaling is known to be necessary for the upregulation of integrin aeb7 as lack of this signal results in the complete loss of cd103 + cd8 + t cells in the lung (51, 133) . cd4 + t cell-derived interferon-c (ifn-c) potentially contributes to the secretion of tgf-b in the lung, thereby helping cd8 + t cell retention in the airways by inducing the expression of cd103 (70) . based on these data, the upregulation of integrins in the lung and the consequential regulation of tissue distribution of the effector t cells could also be considered a consequence of local tissuederived instruction that promotes t rm differentiation. it is well established that effector cd8 + t cells recruited to the lung subsequently encounter respiratory dc subsets that present cognate antigen. this ''second hit'' with cognate antigen at the site of infection improves the cell capacity to secrete ifn-c (88) and induces additional rounds of proliferation (87) . in addition to stimulating the cd8 + t cells, respiratory dcs trans-present il-15 to prevent rapid apoptosis of the cells (89) . while these antigen stimulation processes regulate terminal effector differentiation, several studies have indicated that local antigen recognition also serves as a primary tissue-derived instructive factor requisite for effective t rm differentiation (9, 59, 90, 97, 123, 133) . in the case of surface and mucosal tissues, such as skin and vagina, cd8 + t rm cells can be generated independent of cognate antigen. for example, forced recruitment of cells to the epithelial tissues by antigen-independent local inflammation or topical chemokine administration results in the establishment of cd8 + t rm cells (81, 113) , a method known as ''prime-pull.'' furthermore, basal levels of cd8 + t rm cells can be deposited in multiple nonlymphoid tissues even after systemic infection in the absence of any local antigen presentation or inflammation (115, 119) . it should be noted that such bystander deposition of cd8 + t rm cells does not occur in the lung. as we have recently demonstrated, cd8 + t cells actively recruited to the lung by antigen-independent inflammation completely disappear after resolution of inflammation in the lung (123) . in contrast, the combination of local inflammation and cognate antigen successfully promotes lung cd8 + t rm cell development (123) . thus, the lung is a unique tissue where local antigen is required for the establishment of cd8 + t rm cells. this appears to be based on the structural differences between lung and other surface/mucosal tissues, which will be discussed in detail later. despite the fact that cd8 + t rm cells can be established in the skin independent of cognate antigen, there is a significant increase in the t rm formation when antigen is present (59, 97) , indicating that although local antigen recognition is not necessary for the establishment of t rm in all tissues, it nevertheless enhances t rm deposition. hence, a second hit with cognate antigen in the 442 takamura peripheral tissues plays a key role not only for terminal effector differentiation but also function as pivotal tissue instruction for t rm differentiation. it is not understood how signals elicited by the local antigen can induce either terminal effector differentiation or t rm development. how is this decision checkpoint regulated? there are at least four potential explanations. first, the decision to become a terminal effector or t rm cell following local reactivation may be cell-intrinsically precommitted before recruitment, perhaps reflecting whether the cells originated from short-lived effector cells (slecs) or memory precursor effector cells (mpecs) (57, 80) . second, as previously described, a division of labor between respiratory dc subsets may also take place even within the lung, as cd103 + dcs, but not cd11b hi dcs, preferentially drive cd103 expression upon cd8 + t cell activation (133) . third, fate decisions between terminal effector and t rm may be determined by temporal deviation of reactivation (16) . for example, in the case of cd4 + t cells, mckinstry et al. have shown that late antigen recognition, which is necessary for memory formation, occurred at days 5-8 postinfection, and have termed this time window as the ''memory check point'' (90) . whereas a second hit for terminal effector differentiation may occur a little earlier, as cognate antigen-presenting respiratory dcs were transferred intranasally on day 3 and analyzed by day 5 (88) . actually, this third hypothesis is mainly attributed to the fourth hypothesis in which effector versus memory fate decision may be regulated by the strength of cd8 + t cell activation. both, the levels of antigen presentation (17) as well as the levels of the ''third signal'' induced by inflammatory cytokines (14) influence the activation status. such antigenic as well as inflammatory signals may be redundant at early phases of infection, which bias cd8 + t cell differentiation toward terminal effector cells, while weak signaling at later time points preferentially promotes memory differentiation (16) . distinct microdistribution of cd8 + t cells also influences differential acquisition of the activation signals. for example, cxcr3 hi cells receive stronger stimulatory signals and preferentially become terminal effectors, while lack of this receptor puts cells away from the core of inflammatory microenvironment, and ultimately promotes memory differentiation (50, 66, 69) . all those factors must cooperatively regulate cd8 + t cell fate following late antigen recognition. there is strong evidence that cd8 + t rm precursors receive bona fide tcr signaling in the peripheral tissues since the cells exhibit high levels of nur77 expression (9, 59) . however, the molecular mechanisms by which tcr signalinginduced events elicit t rm formation remain unclear. cd4 + t cells produce il-2 in response to late antigen recognition, and autocrine il-2 signaling at the memory check point improves memory cd4 t cell survival in the spleen, mln, and lung, (90) suggesting it as a potential mechanism. another observation is that inhibition of the mammalian target of rapamycin (mtor) during the early phase of infection selectively impairs the formation of cd8 + t rm cells in the small intestine, while simultaneously enhancing memory generation in the spleen (118) , suggesting that mtor expression in response to local reactivation may play a role in the t rm differentiation by modulating the metabolic status. clearly, a great deal more study is required to understand how local antigen restimulation optimizes cd8 + t rm formation. in addition to late antigen recognition, local cytokine signaling is also crucial for the formation of cd8 + t rm cells in the lung. tgf-b is produced by a wide variety of cell types in the lung, including alveolar macrophages, neutrophils, activated alveolar epithelial cells and endothelial cells (31) . although tgf-b is known as a profibrotic cytokine and its overproduction is critically associated with pulmonary fibrosis (31), influenza virus-infected animals recover without the acquisition of lung fibrosis (68, 123) , suggesting that tgfb production as well as its activation is rather stable during the course of infection. this is true despite the fact that some strains of influenza virus surface protein can activate latent tgf-b in the lung (109) . local tgf-b signaling does not require sma-and mad-related protein 4 (smad4) (51) , and plays a key role in the downregulation of t-box transcription factors eomes and t-bet in effector cd8 + t cells, both of which are required for effector to t rm transition (82) . upon skin cd8 + t rm differentiation, eomes expression is fully extinguished, while t-box expression remains at a low level, which sustains cd122 expression (il-15 receptor b-chain) and enables to receive il-15-medaited survival signal (82) . as t-bet imposes repression of cd103, the downregulation of t-bet reciprocally leads to the expression of integrin aeb7. although the requirement of tgf-b in t rm differentiation seems universal for all nonlymphoid tissues, il-15 may not be essential for the establishment of cd8 + t rm cells in the lung. in fact, il-15 production is increased especially in cells purified from the airways and also the lung tissues following influenza virus infection, and lack of il-15 results in transient reduction in the number of effector cd8 + t cells in the airways at the early phase of infection (7-12 days) (130) . however, such reduction is no longer observed at the memory phase of infection without any treatment (131) , indicating that the lack of il-15 essentially has no impact on the ultimate generation of cd8 + t rm cells in the lung. at later time points during an acute virus infection (around day 7), effector cd8 + t cells recruited to the lung produce a significant amount of il-10 in response to cd4 + t cell-derived il-2 and innate lymphoid cell-derived il-27 (101, 120, 121) . cd8 + t cell production of il-10 is correlated with its terminal maturation and is vital in preventing excess inflammation in the lung (121) . since il-10 induces activation of the signal transducer and activator of transcription 3 (stat3) that promotes memory cd8 + t cell differentiation, it is tempting to speculate that il-10 produced by terminal effector cd8 + t cells at the memory check point may impact neighbor as well as late-comer cd8 + t rm precursors, and promote memory maturation in the lung. in the case of skin, cd8 + t rm precursors recruited to the skin persist in an epidermal niche that is originally occupied by dendritic epidermal cd t cells (detcs). this results in their lifelong persistence (137) . because normal lung tissues do not exhibit such preformed niches to displace, additional ''space'' is required for the cells to inhabit. it has long been believed that lung cd8 + t rm cells are maintained in the ectopic lymphoid tissues developed in response to respiratory virus infections, such as inducible bronchus-associated lymphoid tissues (ibalt) (95) . however, our group has demonstrated that such structures are primarily populated with cd4 + t cells as well as b cells, but relatively few numbers of cd8 + t cells (123) . rather, cd8 + t rm cells are enriched specifically in niches created at the site of tissue regeneration after injury, which are termed as repair-associated memory depots (ramds) (123) . histologically, ramds represent confluent foci of peribronchiolar lymphocytic infiltrates with diffuse thickening of alveolar walls in surrounding area. thus, the niches exist primarily in the lung interstitium with partial extension to the lung parenchyma. the appearance of cytokeratin-expressing cell aggregates, known as krt pots, is a unique hallmark of ramds. krt pots comprise distal airway stem cells that begin to emerge in the lung around day 7 postinfection, proliferate vigorously, and subsequently differentiate and reconstruct the damaged lung tissues (68, 129, 140) . however, it is unclear whether those cells directly impact the differentiation of cd8 + t rm cells. cd8 + t rm cells in the ramds do not form a specific organized structure and are simply sequestered in this site, while cd4 + t rm cells in the ibalt typically form clusters and surround b cell follicles (123) . such distinct distributions between cd8 + and cd4 + t rm cells in the lung clearly reflect their division of labor upon recall, in which cd8 + t rm cells exert their function as cytotoxic t lymphocytes (ctls) at the damaged site, while cd4 + t rm cells and b cells need to interact with each other in the ibalt for sustained germinal center formation (3). there is also rigid compartmentalization between lung cd8 + t rm cells and cd8 + t em cells that circulate between the lung and blood. for instance, cd8 + t em cells in the lung are widely, but sparsely distributed in the unaffected lung interstitium, and never involved in the ramds unless de novo niches are newly created (123) . as described previously, cd8 + t em cells exit lung tissues mainly through s1p-induced chemotaxis to the lymph. in contrast, inhibition of s1p 1 is no longer required for the retention of cd8 + t rm cells in the ramds due, in part, to limited access to s1p gradient in this microenvironment (123) . importantly, not only tissue-circulating cd8 + t em cells but also effector cd8 + t cells are incapable of being involved in the ramds later than the peak of cd8 + t cell response in the lung (around day 10, which also reflects the peak of tissue damage) (123) . because administration of cognate antigen in combination with the prime-pull strategy enables de novo creation of the ramd and subsequent establishment of cd8 + t rm cells in the lung (123) , the availability of cognate apcs in the ramds likely restricts the numbers of cd8 + t cells deposited. indeed, there is a competition among antigen-specific effector cd8 + t cells to interact with cognate apcs in the inflamed microenvironment (97) . interestingly, this competition occurs even between effector cd8 + t cells with distinct specificities when epitopes specific for those effectors are presented on the same dcs, thereby shaping the local repertoire (97) . such conventional-as well as cross-competition of local antigens may explain the selective deposition of cd8 + t rm cells expressing high-affinity tcrs (32) . a revised theory on continual recruitment and permanent deposition a primary definition of t rm cells is that they are maintained in the lymphoid and nonlymphoid tissues without recirculation. this definition clearly applies to lung airway memory cd8 + t cells because, once recruited, those cells do not return to the circulation or the lung interstitium (47) . however, maintenance of this population differs significantly from that of cd8 + t rm cells in other mucosal tissues. for example, although lung airway memory cd8 + t cells are not highly apoptotic, it has demonstrated that the half-life of this population is *2 weeks (24). such a short lifespan is likely due to the biophysical effects of the harsh airway environment: cells are being cleared by phagocytic cells or removed through mucociliary clearance. furthermore, airway memory cd8 + t cells do not proliferate (47) . based on these findings, a concept emerged that memory cd8 + t cells in the airways are continuously replaced by new cells recruited from the circulation as a process of memory t cell maintenance. this was confirmed by the continuous appearance of lfa-1 + cells in the airways even in the situation that memory cd8 + t cells downregulate lfa-1 within 48 h after entry into the airways (24) . importantly, however, our recent findings have confirmed that only a limited number of cells in the lung airways are continually replaced by cells from the circulation. in brief, ratios of memory cd8 + t cells recruited from the circulation to the airways peaked at only *20% by 2 weeks after parabiotic surgery, and this ratio was maintained for up to 7 weeks (123) . thus, while *20% of memory cd8 + t cells in the airways are new recruits, *80% are obviously segregated from blood-born memory cd8 + t cells, just like t rm cells in other mucosal tissues. this raised a question whether the latter cell population can survive for long in the harsh airway environment. we speculate that continual recruitment is also essential for *80% of memory cd8 + t cells in the airways. if this is the case, their source may be cd8 + t rm pools in the lung interstitium/parenchyma, but not memory cd8 + t cells in the circulation (fig. 1) . as is the case with cells in the lung airways, memory cd8 + t cells in the lung interstitium/parenchyma consist of at least two distinct memory t cell subpopulations: *80% bona fide t rm cells present in the ramds and *20% t em cells present the lung interstitium (123) (fig. 1) . in the steady state in the lung, there is a balance between basal recruitment-mediated influx and s1p-mediated efflux through the lymph that maintains the flat ratio of cd8 + t em cells (fig. 1) . we hypothesize that a small fraction of cells are activated by antigen-independent inflammatory stimuli, potentially due to exposure with airborne contaminants. these cells upregulate cd69 expression, causing them to transiently persist in the interstitium, and some of them are then recruited to the airways through a process of basal recruitment (fig. 1) . the mechanisms by which cd8 + t rm cells in the ramds are maintained remain unclear with possibilities, including homeostatic proliferation or prolonged longevity. there is evidence that cd8 + t rm cells in the lung interstitium/ parenchyma and airways are maintained independent of homeostatic cytokine il-15 (131) . this is consistent with the relatively lower level of expression of il-15 receptor b on memory cd8 + t cells in the lung compared to those in the spleen (112) . nevertheless, these findings do not exclude a possibility that other factors may drive homeostatic turnover of cd8 + t rm cells in the lung ramds, such as residual antigen-induced reactivation. although numerous questions remain in this field, the discovery of specific niches in the lung interstitium/parenchyma has substantial implications in 444 takamura understanding the factors regulating the maintenance of cd8 + t rm cells in the lung. niches and residual antigen: factors that potentially restrict the numbers of cd8 + t rm cells maintained in the lung following a respiratory virus infection, the number of antigen-specific cd8 + t cells in the lung peaks on day 10 and then declines dramatically as infection subsides. this is followed by the establishment of cd8 + t rm cells at around 1 month postinfection. the absolute number of cd8 + t rm cells in the lung wanes over time, which results in a decrease in the protective efficacy of these cells against secondary infection with a homologous virus (67) . for example, cd8 + t rm cell-mediated protective immunity is essentially lost at 4-6 months postinfection (135) . this contrasts with the situation in the skin where cd8 + t rm cells can persist up to 1 year (137) . the shorter lifespan of lung cd8 + t rm cells could be explained by our recent findings regarding the niche-dependent maintenance of cd8 + t rm cells in the lung (123). as described above, cd8 + t rm cells are predominantly accumulated in the ramds: disorganized peribronchiolar foci that are temporarily created at the site of tissue damage. in fact, peribronchiolar foci still remain in the lung at a month postinfection, despite the fact that inflammatory responses have largely abated at this time point. as tissue regeneration proceeds, the size of the ramds shrinks over time and tends to disappear several months postinfection. thus, we suggest that the decrease in numbers of cd8 + t rm cells in the lung for the first couple of months simply depends on the size of the ramds. we also suggest that an organized lymphoid structure like ibalt persists for longer periods and low numbers of cd8 + t rm cells persist in the ibalt following disappearance of ramds. this hypothesis is based on the idea that lung tissues do not initially have preexisted niches in which t cells can persist. thus, the progressive loss of temporarily created ''spaces'' significantly restricts the long-term maintenance of cd8 + t rm cells. as discussed above, cognate antigen that remains in the ramds is also a potential factor regulating the number of cd8 + t rm cells in the lung. in fact, viral antigen can be detected in the peribronchiolar lymphocytic infiltrates (62) as well as bronchial epithelial cells (41) at least a month postinfection. furthermore, cd8 + t rm cells, but not t em cells, express cd69 as well as pd-1, indicative of recent activation (123, 135) . those observations suggest that residual antigen presentation is limited in the ramds, but not in the unaffected lung interstitium. thus, the reduced cd8 + t rm persistence in the ramds is also potentially caused by reduction in the level of residual antigen presentation. importantly, despite the fact that pd-1 impairs the protective efficacy of memory cd8 + t cells in the lung (28, 92) , accumulating evidence suggests that these cells never succumb to functional exhaustion (25, 44) . thus, the level of residual antigen presentation must be lower than that exhibited during a typical chronic infection. in line with this, pd-1 as well as other potential inhibitory molecules may act to prevent excessive immunopathology (26, 27, 29) by maintaining the cells in a quiescent state (49) . furthermore, reactivation of cd8 + t rm cells in the lung leads to sustained expression of interferon-induced transmembrane protein (ifitm3), which is involved in conferring resistance against subsequent virus infection (132) . hence, in contrast to chronic infection, the repeated acquisition of weak cognate signals may be beneficial rather than harmful for the maintenance of cd8 + t rm cells in the lung. a remaining question is how apcs avoid being eliminated by antigen-specific cd8 + t rm cells. it is tempting to hypothesize that pd-1-mediated partial inhibition may play a role in this escape without inducing the global exhaustion. it has also demonstrated that residual antigen presentation persists in the mln for several months after acute respiratory virus infections (54, 55, 62, 75, 122, 138) . because memory cd8 + t cells in the mln and the lung airways exhibit similar activated phenotypes, it has been suggested that reactivation by residual antigen in the mln induces the migration of memory cd8 + t cells from the mln to the lung airways, resulting in the continual influx of cells to the airways (138) . in other words, reactivation in the mln induces phonotypic changes in memory t cells from lymph node-surveying t cm to peripheral tissue homing t em cells (122, 138) . however, it should be noted that the basal levels of continual recruitment of cells to the airways also occur in the absence of residual antigen (65) . furthermore, memory cd8 + t cells in the circulation gradually lose the expressions of blimp-1 and hobit (117), key transcription factors regulating tissue retention (79) . as a result, the efficacy of continual (basal) recruitment to the airways wanes over time (117) . one should also be mindful of the fact that signature markers of t rm cells, such as cd69 and cd103, could be upregulated on lung-circulating blood-born cd8 + t em cells in certain tissue environments (65) or basal levels of tnf secreted in the lung (117) . however, it is still unclear whether t em -derived cd69 + cd103 + cells acquire certain tissue residency. (117, 123) thus, a more precise analysis of migratory property is required to define cd8 + t rm cells in the lung. nevertheless, lung-circulating cd8 + t em cell populations should not be neglected as those populations could be majority when bona fide t rm cells disappeared (117) , and have an ability to contribute, in part, to the protective immunity upon rechallenge (116) . ultimately, a better understanding of cd8 memory in the lung is essential for the development of safe and effective vaccines capable of generating long-lasting antigen-specific memory cd8 + t cells. despite a great deal of progress in understanding cd8 + t cell memory in the lung and recent success in generating lung cd8 + t rm cells by vaccination (37, 133, 139) , our identification of specific niches for cd8 + t rm cells in the lung and other data raises a fundamental possibility that lung cd8 + t rm cells are by necessity short lived (e.g., several months). this is primarily due to the lack of preexisting cd8 + t rm niches in the lung and the shortlived nature of these niches. furthermore, peribronchiolar foci fill alveolar spaces, thereby reducing the efficacy of gas exchange, indicating that a risk (tissue damage and resultant functional impairment) is unavoidable to create niches for lung cd8 + t rm cells. thus, long-term maintenance of cd8 + t rm cells in the lung and repeated tissue damage are two sides of the same coin, and future study should be focused on the balance between protective efficacy and immune pathology when considering the vaccines that target cd8 + t rm cells in the lung. tissue-specific programming of memory cd8 t cell subsets impacts protection against lethal respiratory virus infection the transmembrane cxc-chemokine ligand 16 is induced by ifngamma and tnf-alpha and shed by the activity of the disintegrin-like metalloproteinase adam10 distinct germinal center selection at local sites shapes memory b cell response to viral escape expression of the sphingosine 1-phosphate receptor, s1p1, on t-cells controls thymic emigration intravascular staining for discrimination of vascular and tissue leukocytes cutting edge: intravascular staining redefines lung cd8 t cell responses temporal changes in dendritic cell subsets, cross-priming and costimulation via cd70 control cd8(+) t cell responses to influenza cd69 suppresses sphingosine 1-phosophate receptor-1 (s1p1) function through interaction with membrane helix 4 shortlived antigen recognition but not viral infection at a defined checkpoint programs effector cd4 t cells to become protective memory distinct migrating and nonmigrating dendritic cell populations are involved in mhc class i-restricted antigen presentation after lung infection with virus regulating the adaptive immune response to respiratory virus infection chemokine receptor ccr7 guides t cell exit from peripheral tissues and entry into afferent lymphatics cd49a expression defines tissue-resident cd8 + t cells poised for cytotoxic function in human skin inflammatory cytokines as a third signal for t cell activation sphingosine-1-phosphate and lymphocyte egress from lymphoid organs cutting edge: latecomer cd8 t cells are imprinted with a unique differentiation program tcr signaling in t cell memory the chemokine cxcl16 is highly and constitutively expressed by human bronchial epithelial cells chemokine receptor ccr7 required for t lymphocyte exit from peripheral tissues cd103 + pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen balancing immune protection and immune pathology by cd8(+) t-cell responses to influenza infection mast cells expedite control of pulmonary murine cytomegalovirus infection by enhancing the recruitment of protective cd8 t cells to the lungs nonspecific recruitment of memory cd8 + t cells to the lung airways during respiratory virus infections memory t cell populations in the lung airways are maintained by continual recruitment cutting edge: effector memory cd8 + t cells in the lung airways retain the potential to mediate recall responses viral acute lower respiratory infections impair cd8 + t cells through pd-1 acute viral respiratory infection rapidly induces a cd8 + t cell exhaustion-like phenotype programmed death-1 impairs secondary effector lung cd8(+) t cells during respiratory virus reinfection multiple inhibitory pathways contribute to lung cd8 + t cell impairment and protect against immunopathology during acute viral respiratory infection cxcr3-deficiency protects influenza-infected ccr5-deficient mice from mortality the impact of tgf-beta on lung fibrosis: from targeting to biomarkers cutting edge: resident memory cd8 t cells express highaffinity tcrs preferential migration of effector cd8 + t cells into the interstitium of the normal lung memory t cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus division of labor between dendritic cell subsets of the lung clearance of influenza virus from the lung depends on migratory langerin+cd11b-but not plasmacytoid dendritic cells a distinct lunginterstitium-resident memory cd8(+) t cell subset confers enhanced protection to lower respiratory tract infection a disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of cxc chemokine ligand 16 controls for lung dendritic cell maturation and migration during respiratory viral infection analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell-collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type i long-term survival of influenza virus infected club cells drives immunopathology cd4 + and cd8 + t cells exhibit differential requirements for ccr7-mediated antigen transport during influenza infection crosspresenting cd103 + dendritic cells are protected from influenza virus infection t-cell memory and recall responses to respiratory virus infections activation phenotype, rather than central-or effector-memory phenotype, predicts the recall efficacy of memory cd8+ t cells lung cd103 + dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto mhc class i for presentation to cd8 t cells long-term maintenance of virus-specific effector memory cd8 + t cells in the lung airways depends on proliferation activated antigen-specific cd8 + t cells persist in the lungs following recovery from respiratory virus infections programs for the persistence, vigilance and control of human cd8 + lungresident memory t cells expression of chemokine receptor cxcr3 on t cells affects the balance between effector and memory cd8 t-cell generation smad4 promotes differentiation of effector and circulating memory cd8 t cells but is dispensable for tissue-resident memory cd8 t cells optimal generation of tissue-resident but not circulating memory t cells during viral infection requires crosspriming by dngr-1+ dendritic cells tissue instruction for migration and retention of trm cells unexpected prolonged presentation of influenza antigens promotes cd4 t cell memory generation persistent depots of influenza antigen fail to induce a cytotoxic cd8 t cell response tissue exit: a novel control point in the accumulation of antigen-specific cd8 t cells in the influenza a virus-infected lung inflammation directs memory precursor and short-lived effector cd8(+) t cell fates via the graded expression of t-bet transcription factor interaction of type iv collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1 local antigen in nonlymphoid tissue promotes resident memory cd8 + t cell formation during viral infection respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic cd8 + t cell responses distinct dendritic cell subsets dictate the fate decision between effector and memory cd8(+) t cell differentiation by a cd24-dependent mechanism antigen persistence and the control of local t cell memory by migrant respiratory dendritic cells after acute virus infection dynamics of blood-borne cd8 memory t cell migration in vivo the chemokine receptor ccr5 plays a key role in the early memory cd8 + t cell response to respiratory virus infections cutting edge: antigen is not required for the activation and maintenance of virus-specific memory cd8 + t cells in the lung airways inflammatory chemokine receptors regulate cd8(+) t cell contraction and memory generation following infection immunity to respiratory viruses distal airway stem cells yield alveoli in vitro and during lung regeneration following h1n1 influenza infection chemokine receptor cxcr3 facilitates cd8(+) t cell differentiation into short-lived effector cells leading to memory degeneration cd4 + t cell help guides formation of cd103+ lung-resident memory cd8 + t cells during influenza viral infection the sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of peripheral tissue t lymphocytes into afferent lymphatics chemokine gene expression in lung cd8 t cells correlates with protective immunity in mice immunized intra-nasally with adenovirus-85a cxcr6 is a marker for protective antigen-specific cells in the lungs after intranasal immunization against mycobacterium tuberculosis environmental and antigen receptor-derived signals support sustained surveillance of the lungs by pathogen-specific cytotoxic t lymphocytes prolonged antigen presentation by immune complexbinding dendritic cells programs the proliferative capacity of memory cd8 t cells neutrophil trails guide influenza-specific cd8(+) t cells in the airways cxcl10/ cxcr3-mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and cd8(+) t cell efficacy cutting edge: cd69 interference with sphingosine-1-phosphate receptor function regulates peripheral t cell retention hobit and blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes the developmental pathway for cd103(+)cd8+ tissue-resident memory t cells of skin long-lived epithelial immunity by tissue-resident memory t (trm) cells in the absence of persisting local antigen presentation t-box transcription factors combine with the cytokines tgf-beta and il-15 to control tissue-resident memory t cell fate hidden memories: frontline memory t cells and early pathogen interception activated primary and memory cd8 t cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin a transmembrane cxc chemokine is a ligand for hiv-coreceptor bonzo lymphocyte egress from thymus and peripheral lymphoid organs is dependent on s1p receptor 1 cutting edge: contribution of lung-resident t cell proliferation to the overall magnitude of the antigen-specific cd8 t cell response in the lungs following murine influenza virus infection protective influenza-specific cd8 t cell responses require interactions with dendritic cells in the lungs il-15 transpresentation by pulmonary dendritic cells promotes effector cd8 t cell survival during influenza virus infection effector cd4 t-cell transition to memory requires late cognate interactions that induce autocrine il-2 airway-resident memory cd8 t cells provide antigenspecific protection against respiratory virus challenge through rapid ifn-gamma production local blockade of epithelial pdl-1 in the airways enhances t cell function and viral clearance during influenza virus infection lung dendritic cells imprint t cell lung homing and promote lung immunity through the chemokine receptor ccr4 unique type i interferon responses determine the functional fate of migratory lung dendritic cells during influenza virus infection role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity tissue-resident memory t cells: local specialists in immune defence antigen-dependent competition shapes the local repertoire of tissue-resident memory cd8+ t cells e-cadherin: gatekeeper of airway mucosa and allergic sensitization the host immune response in respiratory virus infection: balancing virus clearance and immunopathology pi3kgamma is critical for dendritic cell-mediated cd8 + t cell priming and viral clearance during influenza virus infection ifngammaproducing, virus-specific cd8 + effector cells acquire the ability to produce il-10 as a result of entry into the infected lung environment promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate the collagen binding alpha1beta1 integrin vla-1 regulates cd8 t cell-mediated immune protection against heterologous influenza infection collagen distribution and expression of collagen-binding alpha1beta1 (vla-1) and alpha2beta1 (vla-2) integrins on cd4 and cd8 t cells during influenza infection the alpha1beta1 integrin and tnf receptor ii protect airway cd8 + effector t cells from apoptosis during influenza infection central memory and effector memory t cell subsets: function, generation, and maintenance two subsets of memory t lymphocytes with distinct homing potentials and effector functions mucosal imprinting of vaccine-induced cd8 + t cells is crucial to inhibit the growth of mucosal tumors influenza virus neuraminidase activates latent transforming growth factor beta lymphocyte sequestration through s1p lyase inhibition and disruption of s1p gradients inhibiting cxcr3-dependent cd8 + t cell trafficking enhances tolerance induction in a mouse model of lung rejection loss of il-7r and il-15r expression is associated with disappearance of memory t cells in respiratory tract following influenza infection a vaccine strategy that protects against genital herpes by establishing local memory t cells cd69 acts downstream of interferon-alpha/beta to inhibit s1p1 and lymphocyte egress from lymphoid organs transcriptional downregulation of s1pr1 is required for the establishment of resident memory cd8 + t cells lung airway-surveilling cxcr3(hi) memory cd8(+) t cells are critical for protection against influenza a virus dynamics of influenza-induced lung-resident memory t cells underlie waning heterosubtypic immunity cutting edge: generation of effector cells that localize to mucosal tissues and form resident memory cd8 t cells is controlled by mtor quantifying memory cd8 t cells reveals regionalization of immunosurveillance cd4 + t cell help and innate-derived il-27 induce blimp-1-dependent il-10 production by antiviral ctls effector t cells control lung inflammation during acute influenza virus infection by producing il-10 the route of priming influences the ability of respiratory virusspecific memory cd8 + t cells to be activated by residual antigen specific niches for lung-resident memory cd8 + t cells at the site of tissue regeneration enable cd69-independent maintenance endothelial cells are central orchestrators of cytokine amplification during influenza virus infection lfa-1 is required for retention of effector cd8 t cells in mouse lungs lung niches for the generation and maintenance of tissue-resident memory t cells an alternatively spliced cxcl16 isoform expressed by dendritic cells is a secreted chemoattractant for cxcr6+ cells characterization of a type iv collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins lineage-negative progenitors mobilize to regenerate lung epithelium after major injury a role for il-15 in the migration of effector cd8 t cells to the lung airways following influenza infection cutting edge: il-15-independent maintenance of mucosally generated memory cd8 t cells enhanced survival of lung tissue-resident memory cd8(+) t cells during infection with influenza virus due to selective expression of ifitm3 antibodytargeted vaccination to lung dendritic cells generates tissue-resident memory cd8 t cells that are highly protective against influenza virus infection chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza a virus infection in mice lung-resident memory cd8 t cells (trm) are indispensable for optimal cross-protection against pulmonary virus infection tslp promotes influenza-specific cd8+ t-cell responses by augmenting local inflammatory dendritic cell function persistence of skin-resident memory t cells within an epidermal niche residual antigen presentation after influenza virus infection affects cd8 t cell activation and migration vaccine-generated lung tissue-resident memory t cells provide heterosubtypic protection to influenza infection p63(+)krt5(+) distal airway stem cells are essential for lung regeneration we thank dr. tomoya katakai (niigata university) for helpful discussions. this work is supported by grant-in-aid for young scientists (a) 24689043, and grant-in-aid for scientific research (c) 16k08850 from ministry of education, culture, sports, science and technology of japan, and grants from takeda no competing financial interests exist. key: cord-268729-n7slf5tx authors: wissinger, e l; saldana, j; didierlaurent, a; hussell, t title: manipulation of acute inflammatory lung disease date: 2008-05-07 journal: mucosal immunol doi: 10.1038/mi.2008.16 sha: doc_id: 268729 cord_uid: n7slf5tx inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the “threshold of ignorance” may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. supplementary information: the online version of this article (doi:10.1038/mi.2008.16) contains supplementary material, which is available to authorized users. the mucosal immune system must maintain composure in the presence of an onslaught of antigenic and potentially pathogenic material. exposed to the outside world with, in most cases, only a single epithelial cell barrier protecting them, our mucosal surfaces have developed a sophisticated system of immune exclusion, ignorance and tolerance. the best characterized of these are described in the gastrointestinal tract. an understanding of immunity in the respiratory tract has lagged behind that of the gut, and although numerous key components have emerged, the sequence of events from initial inhalation to immune pathology in the lower respiratory tract is still unclear. despite best efforts to maintain immune homeostasis, respiratory inflammatory disease is common and significantly life threatening. this review will highlight mechanisms that maintain lung immune homeostasis and current therapeutic efforts to contain infection-induced exaggerated acute inflammation once it occurs. the respiratory tract includes the nasopharyngeal cavity, trachea and larynx, bronchi, bronchioles, and finally the alveoli. organized lymphoid tissue is embedded in some, but importantly not all, of these stages in the respiratory tree. similarly, draining lymph nodes are associated with only a few of these sites. the cellular composition, requirements for activation, and expansion dynamics of respiratory tract associated lymph nodes are virtually similar to any other lymph node and will therefore not be discussed in detail here. we will focus on the regulation (or de-regulation) of immune cells embedded in the respiratory tract itself. considering the total surface area of the respiratory tract constitutive, embedded organized lymphoid tissue is actually quite rare ( figure 1 ). organized structured lymphoid tissue exists in the nasal cavity of rodents (nasal associated lymphoid tissue, nalt) as paired lymphoid structures at the entrance to the pharyngeal duct, but identical structures in man remain elusive (for a review, see reference bienenstock and mcdermott 1 ). organized lymphoid follicles are observed in post-mortem specimens extracted from 150 children that contain occasional germinal centers, which are associated with lymphocytes in the overlying nasal epithelium and the presence of high endothelial venules. however, in adults such lymphoid tissue is disseminated across the whole nasal mucosa, 2 and is analogous to the less well-organized diffuse lymphoid tissue (termed d-nalt) lining the nasal passages of mice. 3 in man, diffuse nalt develops after birth, likely in response to antigen, and b-and t-cell responses parallel those that occur in lymph nodes. the waldeyer ' s ring comprising the nasopharyngeal (upper midline in nasopharynx, adenoids), paired tubal (around openings of auditory tube), paired palatine (either side of the oropharynx), and lingual (under the mucosa of the posterior third of the tongue) tonsil(s) are thought of as analogous structures to nalt, but are located outside of the respiratory tract and probably also contribute to gastrointestinal immunity. experiments with mice show that, unlike peripheral lymphoid organs, nalt develops independently of lymphotoxin-. however, its structure and function are perturbed in lymphotoxin--knockout mice, possibly due manipulation of acute inflammatory lung disease el wissinger 1 , j saldana 1 , a didierlaurent 1 , 2 and t hussell 1 inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the " threshold of ignorance " may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. to impaired expression of cxcl13, c -c chemokine ligand19 (ccl19), and ccl21, which are crucial for the recruitment and placement of lymphocytes and dendritic cells (dcs). 4 the only other organized lymphoid structure described to date located within the respiratory tract is bronchus-associated lymphoid tissue (balt) (reviewed by bienenstock and mcdermott 1 ). whether it routinely contributes to primary immune responses or maintenance of t-and b-cell memory in the respiratory tract is not known. 5,6 however, a recent study in mice lacking peripheral lymphoid organs suggests that balt can initiate anti-influenza immunity and provide sufficient t cells to mediate protection against a second infection. 7 humoral immune responses elicited by balt are primarily mediated by immunoglobulin a (iga) and igg produced both locally and by balt-derived b cells that traffic to distant mucosal sites. 8, 9 similarly located t-cell responses have been noted. on the basis of these findings, balt can be thought of as functionally analogous to mucosal lymphoid aggregates in the intestine. present in up to 40 % of children and adolescents (to age 20), balt is rare in the lungs of healthy adults. 10,11 although originally described at the bifurcations of the bronchi, immediately beneath the epithelium, 12,13 in the absence of antigen balt is rare 14 and may be controlled or limited by regulatory t cells. 15 inflammation in the lung is associated with balt neogenesis and is described in a variety of pulmonary (moyron-quiroz et al 7 and references therein) and non-pulmonary 16 inflammatory conditions. homeostatic chemokines, including ccl19 and ccl21, in mice are required for development of such inducible balt (ibalt). 4 the extent of ibalt appears to depend on the level of inflammation in the local microenvironment, and suggests that it is developed when required. mice lacking oxidoreductases, that protect from oxidative stress, display heightened cellularity and inflammatory cytokines and ibalt is more prevalent. 17 whether it remains associated with larger airways and persists long after resolution of inflammation is still uncertain. intriguingly, mice lacking peripheral lymph nodes and spleen, but retaining ibalt, clear influenza infection (albeit slower) and survive higher doses of virus than do immune-competent mice. such lymphotoxin-knockout mice show slower generation of influenza-specific t cells that eventually reach wild-type levels, similar to antibody isotype switching to igg and t-cell cytokine production and effector function. this indicates that immune responses generated in ibalt although slower are protective and potentially less pathologenic. 7 this may represent a qualitative difference between local and peripherally derived immune cells or simply reflect the reduced magnitude of immunity when ibalt is the only inducing immune compartment. regardless, in the case of lung immunity, secondary lymphoid tissues are not essential for the maintenance of immunological memory, since a pulmonary infection with influenza virus is handled equally as efficiently in their absence. 18 to date no studies have shown that organized embedded lymphoid tissue such as ibalt and nalt contribute directly to inflammatory pathology in the lung. they may initiate production of immune t and b cells that then track to less organized lung immune compartments, 7,19 -22 but their neogenesis (ibalt) or continued presence (nalt), per se , is not associated with pathology. instead, pathological lung inflammation is attributed to those compartments that lack organized lymphoid structures, the airways and lung parenchyma. this could also be said of inflammation in the gut; peyer ' s patches and mesenteric lymph nodes may not be directly associated with pathology, whereas the lamina propria is. what the lamina propria and lung parenchyma have in common is a loose scattering of non-organized immune cells and a vast surface area of potentially non-professional antigen-presenting cells (apcs); the epithelium. the epithelium expresses constitutive mhc class i and, when inflamed, mhc class ii and b7 molecules. 23, 24 it can, therefore, process and present antigen and activate t cells, but can it turn them off? we believe this non-professionalism and lack of immune-cell organization leads to immune dysregulation. during inflammation, the mediastinal lymph nodes and ibalt expand in an organized and precisely compartmentalized manner. although low frequencies of antigen-specific cells can be observed in these sites, 25 the ratio of immune-cell subsets does not significantly change. contraction of lung-associated lymph nodes is also well controlled; again cell proportions are retained. in lung compartments devoid of organized lymphoid tissue however, immune cells are recruited in droves by a chemotactic gradient 26 -28 from infected epithelium and / or tissue resident and alveolar macrophages. once in the lung parenchyma or the airways, they do not form structures analogous to ibalt or lymph nodes, and it is here that pathological damage occurs, toll-like receptors ligands dominate, and inflammatory cytokines are produced by the infiltrate in abundance. unlike the lung-associated lymph nodes, the airways and lung parenchyma, therefore, experience dramatic shifts in their cellular composition. this is illustrated graphically in figure 2 . the approximate cellular composition in nalt, airways, and lung is shown in homeostasis and at the peak of respiratory infections by three very different pathogens, influenza virus, the bacterium streptococcus pneumoniae , and the fungus cryptococcus neoformans . obviously at other stages of the infection, slightly different cells will dominate, but only the peak of inflammation is presented for clarity. for example, natural killer cells dominate in the airways at days 3 -4 of a viral infection. 29 at its peak of activity, the influenzainfected airway and lung is dominated by cd8 + and cd4 + t cells, 25,30 -32 whereas during s. pneumoniae infection, macrophages, neutrophils, and t cells are more abundant. 33, 34 c. neoformans in c57bl / 6 mice induces an eosinophil-dominated response in the lung and airways. 35 -37 this infiltrate in the air spaces and lung for all pathogens is dramatically different to the same sites in homeostasis that contain few lymphoid cells but a prominent macrophage population. 38 -40 note that the nalt, despite being infected with all three pathogens, does not substantially alter the proportion of immune-cell subsets present, and the same is also true for the relatively non-infected lung draining lymph nodes. this lack of control and excessive response is only observed in a minority, but when it occurs it is life threatening. for most of us, respiratory pathogens are cleared by non-inflammatory means, including iga that does not fix complement well, but in dimeric form agglutinates and physically excludes antigen by a process known as immune exclusion. 41, 42 what, therefore, goes wrong in a minority? to address some of these questions, we need to understand how immune homeostasis is maintained in health in these non-organized lung compartments (reviewed by holt 43 ) and what pathways contribute to immune pathology. epithelial cells contribute a multitude of strategies to maintain lung immune homeostasis (for a review, see reference holt 43 ). in addition to barrier function, they secrete a variety of antimicrobial substances (surfactant protein c, mucins, and antimicrobial peptides), affect airway smooth-muscle, dc, and memory t-cell activation via nitric oxide production; 44, 45 assist in cell recruitment via production of cytokines and chemokines; 46, 47 and prolong cell survival by secreting stimulating factors such as granulocyte -macrophage colony-stimulating factor. 48, 49 raz and co-workers 50,51 highlight an interesting pathway critical for maintaining alveolar macrophage homeostasis, involving integrin v 6 that localizes these cells next to epithelial expressed transforming growth factor-(tgf-). this may explain why these cells are refractory to migration to the draining lymph nodes. 52 for inflammation to proceed, this inhibitory pathway must be overcome, which is mediated by a toll-like receptor-induced conformational change of macrophages, disruption of tgf-signaling, and reduced integrin expression. we often assume that innate immunity is inactive in the absence of antigen. however, the work of raz et al . clearly shows that active suppression is required for homeostasis. this is also observed in mice lacking components of nadph oxidase 53 that have heightened basal levels of airway macrophage activation due to loss of feedback inhibition. active suppressive mechanisms, therefore, set a " threshold of ignorance " . those that succumb continually to inflammatory lung disease may, therefore, have dysregulated homeostatic pathways or the threshold, which antigen must exceed to induce inflammation, set too low. in the cases of tgf--mediated suppression of alveolar macrophages, homeostasis is overcome by cleavage of the integrin tethering it to the respiratory epithelium. homeostasis is restored when macrophage-released matrix metalloproteinases transform latent tgf-into its active form. 51 it may, therefore, be possible to harness these pathways artificially to dampen inflammatory lung disease with the caveat that pathogen clearance may be affected by such a global antiinflammatory strategy. 54 macrophages, particularly in the airways, have long been known to have an immune-suppressive phenotype. renewal is achieved primarily via local cell proliferation; recruitment via ccl2:ccr2 does occur, although such cells may take days to mature into the classical immune-suppressive phenotype. 55 in addition to shielding the immune system from inhaled antigens, 56 they display poor phagocytic activity 57 and tend not to migrate well to draining lymph nodes. macrophages held in homeostasis also affect other cell types that may otherwise be proinflammatory within the respiratory tract (for a review, see reference holt 43, 58 ) . dc migration to the draining lymph nodes is enhanced upon macrophage depletion, 52 and t-cell-mediated inflammatory disease ensues to antigens that would otherwise have been ignored, 59 most likely due to the usual direct suppressive influence that alveolar macrophages have on dc function. 39 both myeloid and plasmacytoid dcs (pdcs) are present within the lung, both increase and are recruited rapidly during inflammation, and are attracted by chemokines and cytokines produced by epithelial cells and alveolar macrophages. 43,60 -62 myeloid dc responses are similar to counterparts found elsewhere in the body. pdcs, however, also appear to play a tolerogenic role in the respiratory tract. they have poor apc activity 63 (but once activated enhance cd8 + t-cell responses in vivo 64 and possibly cd4 + t cells at distant sites 65 ), promote inhalation tolerance, 66 and can protect from development of allergic airway disease (reviewed by de heer et al , 66 hammad and lambrecht, 67 and lambrecht 68 ). during acute respiratory viral infections, pdcs perform dual functions of promoting viral clearance by secretion of type i interferon (ifn), and limit inflammation by induction of interleukin-10 (il-10) (reviewed by grayson and holtzman 69 ). their role in limiting lung inflammation can be clearly seen in respiratory syncytial virus (rsv)infected mice where pdc depletion leads to increased viral replication and enhanced immunopathology in the lungs 70, 71 . dcs encounter antigen predominantly in the lung parenchyma, although microbial sampling, via dendrite projections through the epithelial cells into the airway lumen, may induce activation, maturation, and migration, arming them to support potent t-cell responses. 72 quite what returns these cells to homeostasis is unknown but may involve the level of toll-like receptor signals and / or the influence of surface expressed inhibitory receptors such as cd200r. 73 airway epithelial cells may also control dc activity via tgf-analogous to alveolar macrophages, 51 although this has not been proven. whether dcs actually transmigrate into the airspaces is still controversial. 74 -76 ccr2 assists their transit across the endothelium and ccr6 their subsequent migration into the airway. 74 the recently described cd103 + ( e 7), cd207 + , cd11b lo dcs express all of the requisite chemokine receptors to draw them into the airways, 77 but their presence in this compartment has not been proven. clearly, harnessing the homeostatic pathways described above may help to resolve ongoing inflammation. however, it is equally likely that immune cell types and pathways brought into the lung with inflammatory cells during inflammation also provide a therapeutic opportunity. it would not be possible to cover all of the pathways attempted to limit lung inflammation. we will, therefore, restrict our analysis to acute infectious events (i.e., not asthma) and to targeted therapeutic strategies (i.e., not global anti-inflammatories such as corticosteroids or experiments in gene-deleted animals). immune-mediated pathology can be manipulated at any stage of its generation. from a clinical perspective, however, after onset of identifiable symptoms would be the most beneficial. for this reason it is often resolution of inflammation that is targeted, which can include modulation of cell survival, successive waves of recruitment, and ongoing innate immunity. it is still unclear whether immune excess in the respiratory tract stems from over-exuberant recruitment, proliferation within the airspaces, and / or accumulation in the absence of clearance of innate and adaptive immune cells. evidence suggests that the airspaces may not support efficient t-cell proliferation 78 -80 despite memory t cells acquiring bromodeoxyuridine staining at the same rate as secondary lymphoid organs. 25, 80 it is likely that the inflammatory cytokine and chemokine cascade that ensues upon infection 81 -86 will prolong immunecell survival, but at the same time enhance their recruitment, either directly or by altering vascular or epithelial permeability. 87, 88 an inflammatory environment is also associated with the highest expression of " late co-stimulatory molecules " on t cells that prevent activation-induced cell death and rely on cognate ligands expressed on apcs for signalling. ox40 (cd134) is one such late co-stimulator that is restricted to recently activated t cells, whereas the ligand (ox40l) is expressed on a number of cell types, predominantly apcs. 89 stimulation through ox40 promotes cd4 and cd8 t-cell survival, clonal expansion, 90 inhibition of regulatory t cells, 91 and enhanced immunity to a variety of pathogens. 36,92 -94 however, during acute influenza virus infection of mice, transient blockade of ox40 is more beneficial; alleviating illness and pathology to influenza, without review compromising pathogen clearance or immunological memory. 95 a permanent absence of multiple late co-stimulators, however, can compromise immunological memory. 96 -98 manipulation of one late co-stimulatory pathway, therefore, leaves others intact to seed the memory t-cell pool. 97 this suggests that a full compliment of late co-stimulators may actually be prolonging t-cell survival and contributing to pathology. inflammatory cytokines, such as those abundantly expressed in the airways during infection, are known to increase ox40l on apcs. 99 due to lack of immune organization in the airways, late co-stimulatory molecules and their ligands may be downregulated too late to avoid bystander tissue damage. the observation of ox40l on inflamed endothelium highlights the difficulty in separating the effects of immune modulators on cell survival vs. cell recruitment, 100, 101 especially when ligation of ox40l on endothelial cells induces secretion of chemokines. 101,102 4-1bb, like ox40, is restricted to late-effector t cells and its absence or blockade impairs cd8 + t-cell responses to influenza, 103 although the effect on associated respiratory pathology is not yet known. care must be taken, however, since not all inducible co-stimulators result in a beneficial outcome when blocked. during lung influenza infection, icos blockade impairs respiratory t cells to such an extent that the virus escapes clearance. 32 this is similar to treatment of influenza-infected mice with a ctla4-ig fusion protein (that blocks cd28 binding to b7 molecules) 104 and cd40l-knockout mice infected with c. neoformans 105 (where macrophage antimicrobial strategies are impaired). it is likely that targeting cd27 may also produce untoward side effects, since it is expressed on resting na ï ve and memory t cells and is crucial for the formation cd8 + t-cell responses. 96, 106 perhaps the defining line for therapeutic potential should be placed between those late co-stimulators that absolutely depend on t-cell receptor and constitutive cd28 signaling for their expression and those that can appear in a bystander fashion in the presence of inflammatory cytokines. the likely effect of co-stimulatory blockade and the site where manipulation may have the most effect is shown in figure 3 . in addition to late co-stimulatory molecules, a number of other pathways affect the longevity of lung inflammation during acute infection. whether apoptosis is beneficial or harmful depends on the specific infection and the dominant cell type that mediates its clearance. 107 as a general rule, apoptosis favors the host in chronic and acute intracellular bacterial and viral infections (as the process clears the pathogen), but is detrimental for extracellular bacteria. 107 for example, during infection of rats with pneumocystis , alveolar macrophage apoptosis delays clearance of the organism that can be improved by administering caspase-9 inhibitors. 108 similarly, apoptosis of airway epithelial cells via fas / fas ligand is essential to prevent dissemination of pseudomonas aeruginosa . 109 however, during influenza infection it may be more advantageous to reduce cell survival, especially in the case of tnf producing cd8 + t cells. tnf receptor-ii and very late antigen--1 synergize to protect cd8 t cells in the influenza virus infected airways from apoptosis, 110 whereas engagement of qa-1b by cd94 / nkg2a transmits a negative signal that limits immune pathology. 111 it may, therefore, be possible to resolve t-cell inflammation before bystander tissue damage occurs by blocking or enhancing these surface receptors once the viral load has reduced. this would only be useful, however, if the strategy specifically targeted a defined cell population, since apoptosis of airway epithelial cells and leukocytes may be linked to the pathology observed in those infected with highly pathogenic avian influenza. 112, 113 furthermore, apoptosis leading to systemic lymphopenia, 114 observed during influenza infection, may assist virus propagation and survival. other respiratory pathogens, such as cytomegalovirus and parainfluenza virus, use antiapoptotic strategies to prolong survival of the cells they infect. 115 chlamydia blocks apoptosis by affecting the release of cytochrome c from mitochondria. 116 the extracellular pathogens pseudomonas cepacia figure 3 the location of the dominant site of action of co-stimulatory molecule blockade during acute lung infection. this model assumes that antigen specific t cells are primed in the lung-associated lymph nodes (e.g., mediastinal). as such blockade of cd28 on t cells using the b7 competitor ctla4:ig will affect t-cell priming in organized lymphoid tissue ( a ). the same is also likely to be true for icos that is induced rapidly after t-cell receptor and cd28 signalling. by contrast, ox40 and 4-1bb are expressed at very low levels in the lung-associated lymph nodes but upregulated in the lung parenchyma and airways, most likely due to re-recognition of antigen in situ and / or the inflammatory cytokine environment. blockade will, therefore, impact these sites rather than the lymph nodes ( b ). note that ox40 and 4-1bb blockade may also affect primed t-cell migration, as their respective ligands are present on inflamed endothelium ( c ). in the airways, the dominant effect will be to allow activation-induced cell death to progress; in the parenchyma, however, it may be a combination of activation-induced cell death ( d ) and migration. and s. pneumoniae cause apoptosis of neutrophils and airway epithelial cells ( table 1 ) , 117 respectively, to aid survival. modulation of apoptosis is, therefore, complicated; what may benefit the host for one infection would compromise it to another. the recruitment and trafficking of leukocytes in response to inflammation is a tightly regulated process that can tip the balance between protection from infection and immune mediated damage in the lung. in brief, it involves slow rolling (concomitant to the activation of leukocytes), an increase in integrin expression and avidity to regulate the rolling arrest, adhesion strengthening as well as spreading, and intravas-cular crawling culminating in paracellular or trans-cellular migration. 118, 119 since excessive cell recruitment is a feature of many acute lung infections, targeting specific molecules involved in any of the above-mentioned events may be beneficial. blocking monoclonal antibodies against the integrin very late antigen-4 (natalizumab) 120 and lymphocyte function-associated antigen-1 (efalizumab) 121 are currently used to treat inflammatory autoimmune disorders and crohn ' s disease, but are also immunosuppressive and have not been studied in the context of acute respiratory infection. 122 the potential benefit of blocking chemokines or their receptors using competitive blockers or antagonistic compounds is well described (reviewed by glass et al 123 ransohoff 124 ). secreted chemokines bind glucosaminoglycans on endothelial cells, forming chemoattractant gradients that direct cells to inflammatory sites, 125 and are classified into constitutive homeostatic and inflammatory (requiring a proinflammatory stimulus such as ifn-, tnf, or microbial products) chemokines. 126 chemokine receptors are transmembrane g-protein-coupled molecules that trigger a signal transduction event resulting in activation and firm adhesion of the migrating cell. 127 the interactions between chemokines and their receptors are functionally redundant; many chemokines bind the same receptor and one chemokine can bind several receptors. as such it can be difficult to design reliable therapeutics that disrupt the interaction of one particular chemokine with its receptor. 128 therapeutic administration of antibodies that block macrophage inflammatory protein-2 during influenza infection reduces neutrophil recruitment by 49 % and improves lung pathology without altering viral clearance. 129 however, this strategy requires testing in co-infection models, since neutrophils are critical for clearance of most respiratory bacteria that commonly cause secondary pneumonia in the presence of influenza virus. 130, 131 rantes (ccl5) is another potential target produced by respiratory epithelial cells during a variety of viral infections. rantes induces ccr1-, ccr3-, and ccr5-expressing t cell, eosinophil, and monocyte recruitment to the lung. 132 during rsv infection, ccl5 is expressed by infected epithelia and resident macrophages and secreted into the airway during the first 48 h of infection. production is then taken over by newly recruited t cells. blocking this chemokine with a competitive inhibitor (met-rantes) 133 during a primary rsv infection reduces lung immunopathology. however, heightened cell recruitment occurs during homologous rsv re-challenge, suggesting that manipulation during the first infection severely compromises immunological memory. 134 the same strategy has been tested in a mouse model of pneumonia virus infection. this highly lethal mouse pathogen induces a disease closely resembling severe human rsv infection in man, that is abrogated by co-administration of the antiviral agent rivabirin and met-rantes. 135 in contrast to inhibition of chemokines during virus-induced lung inflammation, some infections may require their administration. p. aeruginosa lung infection causes airway neutrophil infiltration that rapidly apoptose and become toxic. administration of recombinant monocyte chemoattractant protein-1 / ccl2 recruits and activates lung macrophages that clear apoptotic neutrophils before they cause pathology. 136 ifninducible protein 10 (cxcl10) is critical not only for clearance of klebsiella pneumoniae , 137 but is also produced during lung viral infection where neutralization may be beneficial. 138 many chemokine receptors are also increased during respiratory viral infection; cxcr3, for example, is upregulated during murine gammaherpes virus b8 infection, and its absence delays viral clearance. 139 antagonism of ccr1 reduces mortality of pneumovirus infection of mice, 140 and absence of ccr1 also prevents the rsv-induced exacerbation of asthma. 141 relatively little has been accomplished in this area, especially therapeutically, however, due to the paucity of reagents available for chemokine-receptor blockade. another method for altering lymphocyte migration is to manipulate sphingosine 1-phosphate receptors that are required for egress of lymphocytes from the thymus and peripheral lymph nodes, and impact on vascular permeability. fty720 is a novel synthetic immunosuppressive drug that inhibits lymphocyte emigration from lymphoid organs by binding and activating sphingosine 1-phosphate receptors. 142 sphingosine 1-phosphate is known to play a role in endotoxin-induced lung injury by affecting endothelial barrier function, 143 and is currently in trial for a variety of inflammatory disorders, including transplantation, 144 but is yet to be tested in acute lung infection. modulation of lung innate immunity represents another potential therapeutic target. however, once again, few studies have tested manipulating it after the onset of symptoms during acute respiratory infection; most examine the impact of an innate immunity-associated molecule using gene-depleted animals or prior to infection. manipulation of innate immunity may seem to be akin to " shutting the stable door after the horse has bolted, " since innate immunity is activated first and drives subsequent adaptive immune responses. however, each epithelial cell or macrophage infected sends off the next wave of an immune response. at the time of clinical presentation, therefore, both innate and adaptive immunity will be in full swing. manipulating innate immunity at this stage will assist the resolution process, but whether inhibition or activation is required will depend on the pathogen. one way to temper innate immunity is to mature the lung microenvironment or instill probiotic microbes that would compete with the survival of pathogenic microorganisms. oral administration of lactobacillus casei during lung s. pneumoniae infection is protective, resulting in more rapid clearance, a shorter period of septicemia, and decreased s. pneumoniae load in the lungs. this benefit is attributed to increased neutrophils, myeloperoxidase, and il-10 that limits lung tissue damage and is likely mediated by migration of mature apcs from the gut to the lung that are better equipped for bacterial clearance. 145 prior infection in the lung also has a beneficial effect on some subsequent acute respiratory infections through modification or maturation of the microenvironment. 146 -151 prior influenza infection, for example, reduces subsequent toll-like receptor responsiveness of alveolar macrophages for prolonged periods of time. 152 administration of microbial products, such as cpg dna or a modified bacterial labile toxin (ltk63), also protects against an array of subsequent respiratory pathogens, 153,154 as do chronic or acute infections in distant sites. 155 -157 the ability of pathogen-derived proteins to modify acute respiratory infections, however, is yet to be tested therapeutically. resveratrol, a polyphenolic compound found in red wine, inhibits nuclear factor-b activation, decreases mortality and pro-inflammatory cytokines (tnf, il-1 , and il-6) to serratia marcescens pneumonia in rats. though this strategy increases neutrophil numbers, they resolve more rapidly. 158 similarly, review an acidic polysaccharide compound from cordyceps militaris , an insect-borne fungus, has anti-viral properties in a murine influenza infection model. 159 intranasal administration of the fungal polysaccharide decreases mortality and influenza viral titers, while increasing lung pro-inflammatory cytokines. in vitro , influenza infected macrophage cell lines treated with resveratrol display enhanced inducible nitric oxide synthase and nitric oxide (no), suggesting that this compound may function through non-specific stimulation of alveolar macrophages in vivo . 159 enhanced innate and adaptive immunity and reduced microbial load is also observed upon therapeutic administration of retinoic acid in mice infected with mycobacterium tuberculosis . 160 the increased numbers of macrophages, natural killer and t cells, and increased expression of ifn-, tnf, inducible nitric oxide synthase, il-10, and cxcl10 may also benefit other acute respiratory bacteria or fungi, though during viral infection it would be predicted to be detrimental. acute respiratory infections are yet to be examined. a critical pathway for clearance of pathogens and infected lung epithelial cells is via no and reactive oxygen and nitrogen species. no contributes to host defence and mediates both proand anti-inflammatory effects (reviewed in 161, 162 ) . as with many of the therapeutic treatments discussed, the timing and extent of modulation of no and associated free radicals is critical. while there is no clear consensus in the literature, there are a few examples of successful intervention in this pathway. in rats with p. aeruginosa pneumonia, treatment with inhaled no post-infection improves bacterial clearance through direct bactericidal effects, increased recruitment of neutrophils to the airways 163 or enhanced endothelial permeability. 164 during k. pneumoniae infection of rats, inhaled no also suppresses bacterial replication and decreases lung intercellular adhesion molecule-1 expression, myeloperoxidase activity, tnf levels, and nuclear factor b activity. thus, no has many paracrine effects on inflammatory cells, but also promotes bacterial clearance. 165 during viral infection, the impact of no manipulation is less clear. inhibition of no during rsv infection reduces pulmonary inflammation and bystander tissue damage, but viral replication increases. 166 similar therapeutic strategies have been employed to reduce local concentration of reactive oxygen species and reactive nitrogen species, which are produced by infected lung epithelium and macrophages. during rsv infection of mice, administration of the antioxidant, butylated hydroxyanisole, decreases illness scores, weight loss, and lung neutrophil recruitment. again a multi-factorial attenuation of inflammation occurs. 167 the same is observed in influenza-infected mice treated with a free-radical scavenger, manganese superoxide dismutase, within 48 -96 h of infection. this enzyme has potent anti-inflammatory properties, resulting in less lung consolidation and improved arterial oxygen saturation, presumably as a result of decreased tissue damage. 168 reduced tissue damage also occurs in mice lacking superoxide dismutase or treated therapeutically with a manganic porphyrin that scavenges reactive oxygen species. 53 therefore, inhibition of reactive oxygen species / reactive nitrogen species and no is beneficial for acute respiratory viral infection, but likely to be detrimental for concurrent respiratory bacteria. modulation of immune-receptor signaling is in its infancy with regards to acute respiratory infection and may be limited by (a) the toxicity / safety profile of available drugs, (b) formulation challenges for in vivo delivery, (c) a lack of specificity due to shared receptor associations, and (d) problematic pharmacokinetics (sustained blockage will be detrimental for protection against infection and drugs may have to be delivered locally to prevent systemic effects). the only truly therapeutic manipulation, to date, is abrogation of airway fluid clearance during rsv infection, caused by interaction of uridine triphosphate with purinergic receptors, by post-infection administration of an active metabolite of leflunomide, a77-1726. leflunomide restores airway fluid clearance, providing symptomatic relief and reduced lung inflammation and hypoxemia, without impairing viral replication or clearance. 169 although signaling molecules associated with pattern recognition and cytokine receptors are well described, little is known about the complexity of innate pathways induced by a whole pathogen, especially in the lungs. 170 controlling the signaling pathways leading to exuberant inflammation is of major interest, but a balance needs to be struck to maintain host defence against infection. a feasible approach might be by treating patients at the peak of inflammation where immune mediators are in excess and using drugs with a short half-life. this is a promising strategy, but, to our knowledge, no drugs targeting signaling components are reported efficacious in human lung infection, although they are under development. 171 a comprehensive review of relevant inhibitors of inflammatory signaling pathways can be found elsewhere. 172 receptors recognizing pathogens such as toll-like receptors or nod-like receptors, but also tnf and il-1 , which are often involved in amplification of the inflammatory response, are potent activators of nf-b. 173 nf-b induction is a key factor triggering inflammation in the influenza-infected epithelium. 174 mice treated with a cell-permeable peptide that reduces nf-b levels, via an effect on i-kappa-b kinase beta, reduces pulmonary rsv-induced inflammation. 175 again, this strategy may negatively affect respiratory bacteria, since intra-tracheal adenovirus delivery of a dominant nf-b inhibitor impairs clearance of respiratory p. aeruginosa , 176 despite the reduced inflammation observed with purified bacterial products. 177, 178 peroxisome proliferator-activated receptor-is a nuclear receptor involved in the stress response during lung injury and attenuates inflammatory responses by inhibiting nf-b. 179, 180 many steroids target peroxisome proliferator-activated receptor-and show encouraging results in models of lung inflammation. 172 a series of natural and synthetic ligands for these receptors have been developed and rsv-specific responses in human lung epithelial cell lines are reduced by some of these agonists. 181 mitogen-activated protein kinase p38 and c-jun-n-terminal kinase pathways may also provide future suitable targets. activation of the p38 pathway is often associated with induction of nf-b (in toll-like receptor responses, for example) and is thought to maintain inflammatory responses by stabilizing cytokine mrna. 182 inhibiting the mitogen-activated protein kinase pathway may, therefore, favor termination of inflammation. inhibitors of p38 reduce the epithelial disruption caused by rsv and bordetella pertussis . 183, 184 again, inhibition of this pathway, to our knowledge, although tested in asthma, 185 has not been tested in lung infection models. another approach to reduce pathogen-induced immunopathology during acute infection is to target signaling molecules involved in cell migration. downstream of chemokine receptors is the phosphatidylinositol 3-kinase, which activates protein kinase c and rho gtpases. 186 phosphatidylinositol 3-kinase inhibitors reduce the recruitment of neutrophils and t cells in vivo . 187 -189 although this treatment is efficient in alleviating chronic inflammation such as asthma, 190 it is not validated for acute microbial infection. in addition, use of phosphatidylinositol 3-kinase inhibitors may interfere with development of the innate response to bacteria. 191 with the identification of each new cytokine, a series of papers describing their manipulation in models of acute infection has followed. all of them cannot be detailed here due to space constraint, but our discussion can be limited to those that have been tested therapeutically in acute lung infection models. the first, type-i ifn, plays such an important role in limiting viral replication that they have developed strategies to avoid it. during rna virus lung infection ifn-is produced predominantly by alveolar macrophages (or, to a lesser extent, pdcs) whose depletion impairs viral clearance. 192 dosing with ifn-and a double-stranded rna ifn-inducer, 4 h after sars coronaviruas infection, reduces lung viral titers. 193 similarly, rsv or human metapneumovirus-infected balb / c mice, treated intranasally with recombinant ifn-, have reduced lung viral titers and inflammatory disease as compared with untreated controls. 194 prophylactic treatment of sars coronavirus-infected macaques with pegylated ifn-significantly reduces viral load and pulmonary damage; post-exposure treatment is effective, although producing intermediate results. 195 recombinant ifn-, therefore, appears beneficial for reducing viral replication and associated pathology when administered early after infection. the influence of concomitant bacterial infection requires examination. other early innate cytokines important in respiratory viral and bacterial infections include il-1, il-12, and tnf. their blockade or promotion, however, is complicated by the fact that respiratory bacteria tend to require them for clearance. for example, rhinovirus induces il-1-receptor antagonist, il-1ra, from airway epithelial cells, which facilitates resolution of inflammation. 196 however, il-1ra enhances bacterial outgrowth in the lungs of mice with pneumococcal pneumonia, without the benefit of reducing the host response. 197 therefore, this pathway is predicted to be good for one lung infection but bad for another. blockade of il-12 is another example where neutralization benefits the severity of lung viral infection, 198, 199 but impairs clearance of lung histoplasma capsulatum 200 and legionella pneumophila 201 infections. the list continues with neutralization of tnf, which benefits immune pathology induced by influenza and rsv infection, 81, 202 but not respiratory bacteria or fungi, 203, 204 especially if treatment is prolonged (although this will depend on the precise strategy used 205 ). webster and coworkers reported recently that absence of tnf and il-6 is of no benefit during murine influenza h5n1 infection. 206 however, up to 50 % survival was observed in some experiments and the gene-depleted animals used may harbor other developmental abnormalities. viruses induce local production of ifn-by t and non-t cells in the respiratory tract, and its neutralization not only reduces local lung cellularity and systemic humoral responses to influenza virus infection in mice, 207 but may also delay viral clearance. 208 ifn-is also required for clearance of s. pneumoniae . 209 it would appear that the new cytokine on the block, il-17, may also present opposing effects in viral and bacterial lung infection. suitable reagents are yet to be developed for il-17 neutralization, but in vivo blockade of il-23p19 alone, or in combination with il-23 / il-12p40 (required for th17 development), significantly reduces mycoplasma pneumoniae -induced il-17 and subsequent bacterial clearance, possibly via reduced neutrophil activity. 210 k. pneumoniae clearance also depends on il-17, 211 but the influence of il-17 or il-23 neutralization on respiratory viral infection is unknown. administration of immune suppressive cytokines has been considered for infection induced lung inflammatory disease and encountered similar problems. il-10 neutralization increases survival of mice infected with k. pneumoniae . 212 in contrast, influenza induces indoleamine 2,3-dioxygenase and il-10 production, which may limit lung inflammation. however, treatment with an indoleamine 2,3-dioxygenase inhibitor (that would reduce il-10) induces a 20-fold reduction in lung s. pneumoniae load. 213 intranasal il-10 treatment of rsv-infected mice reduces lung nuclear factor b dna-binding activity, chemokine gene expression, and airway inflammation. 175 similarly, administration of tgf--encoding plasmid reduces inflammation to viral and fungal lung pathogens, but, without exception, prevents their clearance. 54 although a plethora of strategies have been used to modulate lung inflammation during acute infection, few are tested therapeutically after the onset of clinical symptoms, and even less are tested in models of common co-existing lung pathogens. hundreds of immune modulators are, therefore, beneficial during influenza infection, but what of the bacteria that sometimes accompany them? would immune therapeutics work best in combination with antibiotics? equally, selection of immune modulators requires precision in determining exactly what the patient is infected with. in the absence of this knowledge, we may apply a beneficial strategy to one supposed infection, but create an altogether different type of problem. several gaps remain in our knowledge of how immune homeostasis is maintained in the respiratory tract, inflammatory pathways that overcome review them, and the precise effector / memory phenotype of immune cells within the airways and lung parenchyma. controversy also still surrounds the potential of " acute " respiratory infections to persist, since detection of pathogen genome is common, but few studies have been able to demonstrate classical reactivation long after the primary infection. should persistence exist then, depending on the nature of the persisting organism, immune modulators may cause their reactivation. the development of sensitive tools for pathogen detection, and elucidation of specific gene expression patterns in patients with acute infection, 214 mean that future use of targeted immune modulators is not impossible as long as we are able to strike a balance between immune pathology and immune defence. inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis balt development and augmentation of hyperoxic lung injury in mice defi cient in nqo1 and nqo2 . free radic persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs role of cxc chemokine ligand 13, cc chemokine ligand (ccl) 19, and ccl21 in the organization and function of nasal-associated lymphoid tissue antigenspecifi c cd8(+) t cells persist in the upper respiratory tract following infl uenza virus infection antibody-forming cells in the nasal-associated lymphoid tissue during primary infl uenza virus infection anti-tnp-forming cells in bronchus-associated lymphoid tissue (balt) and paratracheal lymph node (ptln) of the rat after intratracheal priming and boosting with tnp-klh induction of mhc class ii antigens on rat bronchial epithelial cells by interferon-gamma and its effect on antigen presentation luminal antigens access late endosomes of intestinal epithelial cells enriched in mhc i and mhc ii molecules: in vivo study in crohn's ileitis frequency, specifi city, and sites of expansion of cd8+ t cells during primary pulmonary infl uenza virus infection memory t cell recruitment to the lung airways chemokines in acute respiratory distress syndrome chemokines and their receptors guiding t lymphocyte recruitment in lung infl ammation intracellular interferon-gamma expression in natural killer cells precedes lung cd8+ t cell recruitment during respiratory syncytial virus infection t cell responses to infl uenza virus infection: effector and memory cells effector cd4+ and cd8+ t-cell mechanisms in the control of respiratory virus infections a critical role for icos co-stimulation in immune containment of pulmonary infl uenza virus infection accumulation of gamma/delta t cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection evidence for the involvement of lung-specifi c gammadelta t cell subsets in local responses to streptococcus pneumoniae infection immunity to a pulmonary cryptococcus neoformans infection requires both cd4+ and cd8+ t cells ox40 ligation on activated t cells enhances the control of cryptococcus neoformans and reduces pulmonary eosinophilia il-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary cryptococcus neoformans infection in genetically susceptible mice (c57bl/6) the prolonged life-span of alveolar macrophages downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages th1 and th2 cytokine induction in pulmonary t-cells during infection with respiratory syncytial virus induction of secretory immunity and memory at mucosal surfaces iga receptors in health and disease regulation of immunological homeostasis in the respiratory tract inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor human bronchial epithelium controls th2 responses by th1-induced, nitric oxide-mediated stat5 dephosphorylation: implications for the pathogenesis of asthma differential recognition of tlr-dependent microbial ligands in human bronchial epithelial cells impact of bronchial epithelium on dendritic cell migration and function: modulation by the bacterial motif kpompa bronchial epithelial cell-derived cytokines (g-csf and gm-csf) promote the survival of peripheral blood neutrophils in vitro diesel exhaust particle-exposed human bronchial epithelial cells induce dendritic cell maturation organ-specifi c regulation of innate immunity induction of a homeostatic circuit in lung tissue by microbial compounds modulation of dendritic cell traffi cking to and from the airways an absence of reactive oxygen species improves the resolution of lung infl uenza infection tgf-beta prevents eosinophilic lung disease but impairs pathogen clearance cytokine modulation of the immunosuppressive phenotype of pulmonary alveolar macrophage populations sequestration of inhaled particulate antigens by lung phagocytes. a mechanism for the effective inhibition of pulmonary cell-mediated immunity inhibitory activity of unstimulated alveolar macrophages on t-lymphocyte blastogenic response alveolar macrophage in the driver's seat alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice identifi cation and characterization of human pulmonary dendritic cells mouse respiratory tract dendritic cell subsets and the immunological fate of inhaled antigens origin and steady-state turnover of class ii mhc-bearing dendritic cells in the epithelium of the conducting airways different roles for human lung dendritic cell subsets in pulmonary immune defense mechanisms murine plasmacytoid dendritic cells induce effector/ memory cd8+ t-cell responses in vivo after viral stimulation organ-dependent in vivo priming of naive cd4+, but not cd8+, t cells by plasmacytoid dendritic cells dendritic cell subsets and immune regulation in the lung recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic infl ammation dendritic cells and the regulation of the allergic immune response emerging role of dendritic cells in respiratory viral infection plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus plasmacytoid dendritic cells limit viral replication, pulmonary infl ammation, and airway hyperresponsiveness in respiratory syncytial virus infection lung dendritic cell migration cd200 and membrane protein interactions in the control of myeloid cells ccr2 and ccr6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen chemokine receptor ccr2 but not ccr5 or ccr6 mediates the increase in pulmonary dendritic cells during allergic airway infl ammation specifi c migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes a major lung cd103 (alphae)-beta7 integrin-positive epithelial dendritic cell population expressing langerin and tight junction proteins long-term maintenance of virus-specifi c effector memory cd8+ t cells in the lung airways depends on proliferation differential t cell function and fate in lymph node and nonlymphoid tissues memory t cell populations in the lung airways are maintained by continual recruitment tumor necrosis factor as a mediator of infl ammation in infl uenza a viral pneumonia cascade of fever production in mice infected with infl uenza virus tnf soluble receptor and antiserum against tnf enhance lipopolysaccharide fever in mice sickness behavior in mice defi cient in interleukin-6 during turpentine abscess and infl uenza pneumonitis the role of mip-1 alpha in infl ammation and hematopoiesis prolonged production of tnf-alpha exacerbates illness during respiratory syncytial virus infection effects of infl ammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration role of tnf-alpha in lung tight junction alteration in mouse model of acute lung infl ammation tnf/tnfr family members in costimulation of t cell responses costimulation of cd8 t cell responses by ox40 ox40 ligand shuts down il-10-producing regulatory t cells evaluation of ox40 ligand as a costimulator of human antiviral memory cd8 t cell responses: comparison with b7.1 and 4-1bbl immunotherapy with ox40l-fc or anti-ctla-4 enhances local tissue responses and killing of leishmania donovani 4-1bb and ox40 stimulation enhance cd8 and cd4 t-cell responses to a dna prime, poxvirus boost vaccine a critical role for ox40 in t cell-mediated immunopathology during lung viral infection during viral infection of the respiratory tract, cd27, 4-1bb, and ox40 collectively determine formation of cd8+ memory t cells and their capacity for secondary expansion 4-1bb and ox40 act independently to facilitate robust cd8 and cd4 recall responses cooperation between 4-1bb and icos in the immune response to infl uenza virus revealed by studies of cd28/icos-defi cient mice ox40 ligand expressed by dcs costimulates nkt and cd4+ th cell antitumor immunity in mice role of ox40 signals in coordinating cd4 t cell selection, migration, and cytokine differentiation in t helper (th)1 and th2 cells signaling of gp34 (ox40 ligand) induces vascular endothelial cells to produce a cc chemokine rantes/ccl5 therapeutic targeting of the effector t-cell co-stimulatory molecule ox40 temporal segregation of 4-1bb versus cd28-mediated costimulation: 4-1bb ligand infl uences t cell numbers late in the primary response and regulates the size of the t cell memory response following infl uenza infection differential requirement for cd80 and cd80/cd86-dependent costimulation in the lung immune response to an infl uenza virus infection disruption of cd40/cd40l interaction infl uences the course of cryptococcus neoformans infection cd27 promotes survival of activated t cells and complements cd28 in generation and establishment of the effector t cell pool lung infections: role of apoptosis in host defense and pathogenesis of disease suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia cd95/cd95 ligand interactions on epithelial cells in host defense to pseudomonas aeruginosa the alpha1beta1 integrin and tnf receptor ii protect airway cd8+ effector t cells from apoptosis during infl uenza infection cutting edge: engagement of nkg2a on cd8+ effector t cells limits immunopathology in infl uenza pneumonia apoptosis and pathogenesis of avian infl uenza a (h5n1) virus in humans avian infl uenza a/hk/483/97(h5n1) ns1 protein induces apoptosis in human airway epithelial cells depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent infl uenza a (h5n1) virus isolated from humans the role of interleukin-10 in the inhibition of t-cell proliferation and apoptosis mediated by parainfl uenza virus type 3 inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation aspects on the interaction of streptococcus pneumoniae and haemophilus infl uenzae with human respiratory tract mucosa getting to the site of infl ammation: the leukocyte adhesion cascade updated lymphocyte homing and homeostasis pharmacological properties, toxicology and scientifi c rationale for the use of natalizumab (tysabri) in infl ammatory diseases anti-adhesion antibodies efalizumab, a humanized anti-cd11a monoclonal antibody fatalities in natalizumab treatment -a ' no go ' for leukocyte recirculation approaches? chemokine regulation of infl ammation during acute viral infection the many roles of chemokines and chemokine receptors in infl ammation strategies for chemokine antagonists as therapeutics chemokines: role in immune cell traffi c intracellular signalling controlling integrin activation in lymphocytes multiple chemotactic factors: fi ne control or redundancy? therapeutic effect of anti-macrophage infl ammatory protein 2 antibody on infl uenza virus-induced pneumonia in mice interactions between infl uenza and bacterial respiratory pathogens: implications for pandemic preparedness how do viral infections predispose patients to bacterial infections? selective attraction of monocytes and t lymphocytes of the memory phenotype by cytokine rantes extension of recombinant human rantes by the retention of the initiating methionine produces a potent antagonist role of ccl5 (rantes) in viral lung disease the pneumonia virus of mice infection model for severe respiratory syncytial virus infection: identifying novel targets for therapeutic intervention essential contribution of monocyte chemoattractant protein-1/c -c chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia interferon-inducible protein 10, but not monokine induced by gamma interferon, promotes protective type 1 immunity in murine klebsiella pneumoniae pneumonia differential chemokine expression following respiratory virus infection refl ects th1-or th2-biased immunopathology role of cxcr3 in the immune response to murine gammaherpesvirus 68 functional antagonism of chemokine receptor ccr1 reduces mortality in acute pneumovirus infection in vivo respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon ccr1-associated immune responses lymphocyte egress from thymus and peripheral lymphoid organs is dependent on s1p receptor 1 novel therapies for microvascular permeability in sepsis phenotypic and functional differences between lymphocytes from nalt and nasal passages of mice lactobacillus casei administration reduces lung injuries in a streptococcus pneumoniae infection in mice memory of mice and men: cd8+ t-cell cross-reactivity and heterologous immunity induction, exacerbation and inhibition of allergic and autoimmune diseases by infection infl uenza virus lung infection protects from respiratory syncytial virus-induced immunopathology memory cd8+ t cells in heterologous antiviral immunity and immunopathology in the lung specifi c history of heterologous virus infections determines antiviral immunity and immunopathology in the lung prior exposure to live mycobacterium bovis bcg decreases cryptococcus neoformans -induced lung eosinophilia in a gamma interferon-dependent manner sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory infl uenza infection stimulation via toll-like receptor 9 reduces cryptococcus neoformans -induced pulmonary infl ammation in an il-12-dependent manner innate imprinting by the modifi ed heat-labile toxin of escherichia coli (ltk63) provides generic protection against lung infectious disease infections and allergy -helminths, hygiene and host immune regulation amelioration of infl uenza-induced pathology in mice by coinfection with trichinella spiralis colonic bacterial infection abrogates eosinophilic pulmonary disease resveratrol ameliorates serratia marcescens -induced acute pneumonia in rats in vivo anti-infl uenza virus activity of an immunomodulatory acidic polysaccharide isolated from cordyceps militaris grown on germinated soybeans retinoic acid therapy attenuates the severity of tuberculosis while altering lymphocyte and macrophage numbers and cytokine expression in rats infected with mycobacterium tuberculosis nonspecifi c defence mechanism: the role of nitric oxide infl ammatory cells and oxygen radicals benefi cial effects of nitric oxide inhalation on pulmonary bacterial clearance inhaled nitric oxide increases endothelial permeability in pseudomonas aeruginosa pneumonia anti-infl ammatory effects of inhaled nitric oxide are optimized at lower oxygen concentration in experimental klebsiella pneumoniae pneumonia immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung infl ammation inhibitory effects of recombinant manganese superoxide dismutase on infl uenza virus infections in mice post-infection a77-1726 blocks pathophysiologic sequelae of respiratory syncytial virus infection complexities of targeting innate immunity to treat infection nuclear factor kappa b is a promising therapeutic target in infl ammatory lung disease targeting signal transduction as a strategy to treat infl ammatory diseases signaling pathways downstream of patternrecognition receptors and their cross talk the ikappab kinase is a key factor in triggering infl uenza a virus-induced infl ammatory cytokine production in airway epithelial cells ikappab kinase is a critical regulator of chemokine expression and lung infl ammation in respiratory syncytial virus infection targeted immunomodulation of the nf-kappab pathway in airway epithelium impacts host defense against pseudomonas aeruginosa airway epithelium controls lung infl ammation and injury through the nf-kappa b pathway duration and intensity of nf-kappab activity determine the severity of endotoxin-induced acute lung injury peroxisome proliferator-activated receptor-gamma is a new therapeutic target in sepsis and infl ammation involvement of ppar nuclear receptors in tissue injury and wound repair peroxisome proliferator-activated receptor-gamma agonists inhibit respiratory syncytial virus-induced review expression of intercellular adhesion molecule-1 in human lung epithelial cells the many paths to p38 mitogen-activated protein kinase activation in the immune system mapk and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption critical involvement of p38 map kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice chemoattractant receptor signaling and the control of lymphocyte migration airway infl ammation: chemokine-induced neutrophilia and the class i phosphoinositide 3-kinases tissue-and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo regulation of phosphatidylinositol 3-kinase by polyisoprenyl phosphates in neutrophil-mediated tissue injury blockade of infl ammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-tat importance of phosphoinositide 3-kinase gamma in the host defense against pneumococcal infection alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques rhinovirus regulation of il-1 receptor antagonist in vivo and in vitro : a potential mechanism of symptom resolution interleukin-1 receptor antagonist transiently impairs antibacterial defense but not survival in murine pneumococcal pneumonia interleukin 12 administration enhances th1 activity but delays recovery from infl uenza a virus infection in mice il-12 treatment attenuates th2 and b cell responses but does not improve vaccine-enhanced lung illness jr interleukin-12 neutralization alters lung infl ammation and leukocyte expression of cd80, cd86, and major histocompatibility complex class ii in mice infected with histoplasma capsulatum in vivo regulation of replicative legionella pneumophila lung infection by endogenous interleukin-12 inhibition of tumor necrosis factor reduces the severity of virus-specifi c lung immunopathology the role played by tumor necrosis factor during localized and systemic infection with streptococcus pneumoniae transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells neutralization of tumor necrosis factor (tnf) by antibody but not tnf receptor fusion molecule exacerbates chronic murine tuberculosis inhibition of the cytokine response does not protect against lethal h5n1 infl uenza infection in vivo blockade of gamma interferon affects the infl uenza virus-induced humoral and the local cellular immune response in lung tissue administration of anti-ifn-gamma antibody to b2-microglobulindefi cient mice delays infl uenza virus clearance but does not switch the response to a t helper cell 2 phenotype role of interferon-gamma in valpha14+ natural killer t cell-mediated host defense against streptococcus pneumoniae infection in murine lungs il-23-dependent il-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory mycoplasma pneumoniae infection cutting edge: roles of toll-like receptor 4 and il-23 in il-17 expression in response to klebsiella pneumoniae infection neutralization of il-10 increases survival in a murine model of klebsiella pneumonia infl uenza-induced expression of indoleamine 2,3-dioxygenase enhances interleukin-10 production and bacterial outgrowth during secondary pneumococcal pneumonia gene expression patterns in blood leukocytes discriminate patients with acute infections comparison of murine nasal-associated lymphoid tissue and peyer's patches isolation and characterization of mouse nasalassociated lymphoid tissue role of type 1 t helper cells in the resolution of acute streptococcus pneumoniae sinusitis: a mouse model key: cord-274474-u2fdicgz authors: majumder, joydeb; minko, tamara title: targeted nanotherapeutics for respiratory diseases: cancer, fibrosis, and coronavirus date: 2020-10-13 journal: adv ther (weinh) doi: 10.1002/adtp.202000203 sha: doc_id: 274474 cord_uid: u2fdicgz systemic delivery of therapeutics for treatment of lung diseases has several limitations including poor organ distribution of delivered payload with relatively low accumulation of active substances in the lungs and severe adverse side effects. in contrast, nanocarrier based therapeutics provide a broad range of opportunities due to their ability to encapsulate substances with different aqueous solubility, transport distinct types of cargo, target therapeutics specifically to the deceased organ, cell, or cellular organelle limiting adverse side effects and increasing the efficacy of therapy. moreover, many nanotherapeutics can be delivered by inhalation locally to the lungs avoiding systemic circulation. in addition, nanoscale based delivery systems can be multifunctional, simultaneously carrying out several tasks including diagnostics, treatment and suppression of cellular resistance to the treatment. nanoscale delivery systems improve the clinical efficacy of conventional therapeutics allowing new approaches for the treatment of respiratory diseases which are difficult to treat or possess intrinsic or acquired resistance to treatment. the present review summarizes recent advances in the development of nanocarrier based therapeutics for local and targeted delivery of drugs, nucleic acids and imaging agents for diagnostics and treatment of various diseases such as cancer, cystic fibrosis, and coronavirus. lung diseases are one of the main causes of death among both men and women worldwide. the mortality rates for lung diseases have been increasing by each year. [1, 2] therefore, methods of developing new therapeutic solutions as well as improving the current therapies for the common lung diseases such as asthma, cystic fibrosis, chronic obstructive pulmonary disease, lung cancer, and coronavirus infections remain the main focus in the fields of targeted drug delivery. the widely utilized conventional drug delivery methods usually induce adverse side effects. [3, 4] recent development of nanoscale-based systems opens a door for a doi: 10.1002/adtp.202000203 better delivery of therapeutics by addressing the limitations of conventional therapy. nanocarrier-based drug delivery systems can increase bioavailability of poorly water-soluble therapeutics and address other barriers and shortcomings of traditional drugs. [5] while majority of the marketed drugs are poorly water soluble, which limits their administration at high doses, [6, 7] nanoscale-based drug delivery systems were found to improve solubility and increase therapeutic efficacy of free non-bound drugs. [8, 9] similarly, biomacromolecular therapeutics such as nucleic acids (dna, small interfering rna, antisense oligonucleotides, etc.) are usually degraded in the biological fluids and difficult to deliver at their target site. [10, 11] however, delivery of nucleic acids via nanocarrier based systems increased their stability and concentration at the target site. [12] [13] [14] [15] [16] moreover, nanoscale drug delivery systems can be administered via different routes, such as intravenous, [17, 18] oral, [19, 20] and inhalation [15, 16, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] routes. furthermore, nanoscale delivery systems are less toxic and immunogenic than the traditional viral vector-based gene delivery systems. [31] [32] [33] because of such handful advantages, researchers around the world have been applied significant efforts in recent years to develop various nanosized carriers for targeted delivery of therapeutics. over the decade, several nanocarrierbased therapeutics were also approved by fda for clinical application. [34, 35] thus, the recent development of wide spectrum nanoscale systems introduced a new way in diagnosis, treatment and prevention of diseases. previously, we formulated main requirements and basic concept of effective drug and nucleic acid delivery systems for effective treatment of diseases including cancer. [36] to enhance treatment effectiveness, the advanced system should provide for a 1) protected delivery of active components in order to prevent their degradation during its journey to targeted cells; 2) targeted transport specifically to the site of action with the aim of limiting adverse side effects of treatment upon healthy organs, tissues, and cells; 3) modulation of pump drug resistance with the purpose to prevent drug efflux from the diseased cells; 4) suppression of nonpump resistance in order to overcome other resistance mechanisms non related to drug efflux pumps; and 5) controlled release of active components in a predefined desired manner. [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] these main requirements concretized for cancer treatment and general composition of advanced proapoptotic drug delivery system are shown in figure 1 . in this review, we summarize recent reports on the development of various nanotherapeutics including nanocarrierbased drugs and nucleic acids for the treatment of lung diseases with emphasis on lung cancer, idiopathic pulmonary, and cystic fibrosis and recent coronavirus infections. [15, 16, 23, 24, [46] [47] [48] [49] [50] targeted drug delivery of therapeutics is aimed at transporting of the administered active component predominately at a desired site of action limiting its accumulation in healthy organs and tissues. this goal can be achieved by both passive and active targeting of drugs. [37, [52] [53] [54] in passive targeting, high molecular weight substances are accumulated in targeted cells because of specific pathophysiological characteristics of the diseased cells and surrounding microenvironment. for instance, passive targeting to solid tumors is dependent on the enhanced permeability of vessels that supply blood, oxygen, and nutrients to the tumor and limited lymphatic drainage from the tumor environment. this phenomenon was termed as the enhanced permeability and retention (epr) effect. [55] a schematic representation of the epr effect is displayed in figure 2 . however, the efficiency of passive targeting is limited and the conditions responsible for the epr effect is not attributed for all diseased tissues which often develop specific mechanisms for resisting traditional treatment approaches. [56] on the other hand, active targeting is achieved mainly by decorating the surface of the nanocarriers with targeting moieties such as antibodies, [57] proteins, [58] peptides, [59] aptamers, [60] [61] [62] lectins, carbohydrates, and glycoproteins, [63] small molecules, [64] [65] [66] [67] [68] etc. which have strong affinity to their cellular binding partners such as tumor figure 2 . a schematic representation of the enhanced permeability and retention (epr) effect. the leaky vasculature and dysfunctional lymphatics of tumors allow the preferential accumulation and retention of high molecular weight nanoparticles in solid tumors. reproduced with permission. [51] copyright 213, hindawi. antigens, cell surface receptors, tumor vasculature. [69] such active targeting nanocarriers are designed to increase the accumulation of therapeutics at their site of actions with limiting exposure to other healthy organs thereby reducing the risk of adverse side effects. [70] [71] [72] [73] [74] [75] nanocarrier-based passive and active targeting of cancerous cells and their advantages are presented in figure 3 . several receptor proteins (such as receptors specific for folate, lhrh, transferrin, etc.) are often overexpressed in tumor cells. a schematic demonstration of various overexpressed cell surface receptors in lung under pathological conditions is . passive and active targeting cancer cells. passive targeting depends on the enhance permeability and retention (epr) effect. active targeting may be achieved by enabling the uptake of nanotherapeutics by receptor mediated endocytosis. for instance, nanoparticles may be decorated with a ligand targeting receptors (or other molecules) overexpressed on the plasma membrane of cancer cells. outlined in figure 4 . western blot analysis revealed the presence of the targeting receptor proteins such as folate receptor alpha (fra), epidermal growth factor receptor (egfr), integrin etc. in different lung cancer cells as shown in the right panel of figure 4 . folic acid, transferrin etc. were widely explored as affinity ligands for targeting many tumors cells. [76] [77] [78] for example, recently, researcher developed a gold nanocarrier loaded with drug aurimmune cyt-6091 and functionalized with tumor necrosis factor alpha (tnf-) using polyethylene glycol (peg) linker for treating lung cancer. tnf-was used as both targeting and therapeutic agent. [79] bind-014 is another nanocarrier based therapeutics which was investigated in phase ii clinical trial for patients with non-small cell lung cancer. [80] bind-014 is a polylactic acid (pla) based nanocarrier system wherein the anticancer drug docetaxel was entrapped. the surface of this system was coated with peg and targeting ligands against prostate-specific membrane antigen (psma) which is usually abundant in prostate cancer cells as well as in the non-prostate cancers, such as nsclc. [81] figure 5a represented a schematic illustration of bind-014 composed of a hydrophobic pla polymeric core and a hydrophilic peg corona decorated with small-molecule targeting ligands, and an encapsulated anticancer drug docetaxel. [82] the ct scans acquired from a patient suffering from a primary cholangiocarcinoma revealed regression of the lung metastases after two cycles of bind-014 treatment ( figure 5b ). [83] these results indicated that bind-014 was clinically active and nontoxic for nsclc. lung cancer is the prime cause of cancer death worldwide. chemotherapy, radiation therapy and their combination with surgery are the current therapeutic options for all type of lung cancers. [84] in most cases of lung cancer chemotherapy, drugs are often administered intravenously, and they can circulate throughout the body affecting both normal and cancer cells. over the last two decades, various nanoparticles (such as metal-based nanoparticles, lipid-based nanoparticles, polymeric nanoparticles, etc.) were explored for targeted therapeutic delivery and diagnostic applications (or combination of both in one theranostic system). [51, 85, 86] however, the translational application of naked metallic nanoparticles (as imaging contrast agents) is limited due to their toxicity. [87, 88] therefore, lipid and polymer-based nanoparticles have received more attention of the researchers for drug delivery and theranostics applications. in this review, we will summarize recent reports on the development of lipid and polymer based nanocarriers for targeted delivery of drugs and nucleic acids for the treatment of lung cancer. drugs can be either encapsulated physically or bonded chemically through linker with the nanocarriers and can be delivered to almost all organs because of their small size and ease of penetration of many biological barriers. over the years, a broad range of nanocarriers were evaluated for targeted delivery of several anticancer drugs for lung cancers. few examples of lipid-based nanoparticles as well as polymeric nanoparticles for targeted drug delivery applications have been summarized below. lipid-based nanoparticles: lipid-based nanoparticles possess unique benefits necessary for drug delivery application. lipidbased nanoparticles have an advantage of being the least toxic among other nanocarriers with a substantial progress in the fields of drug and nucleic acid delivery using lipid-based nanoassemblies. [89] here, we have summarized recent reports of several lipid-based nanosystems including liposome, nanostructured lipid carriers (nlcs), and micelles and their application as targeted drug delivery systems. liposomes: liposome is a type of lipid-based nanoparticle with a bilayer structure comprised of phospholipids, phosphatidylcholine, cholesterol, etc. (figure 6 ). liposome can incorporate lipid-soluble drugs in its lipid bilayer structure as well as encapsulate water-soluble drugs in its inner aqueous core. [90] liposomes were widely investigated for drug delivery applications because of their hydrophobic and hydrophilic drug loading capability as well as their biocompatibility properties. [16, 23, 26, 27, 44, [91] [92] [93] [94] [95] doxil is the first liposomal drug which got fda approval as an anticancer nanotherapeutics in 1995. [96] over the last two decades, researchers have explored various liposomal formulations (such as temperature-sensitive liposomes, [97] cationic liposomes, [98] and archaeosomes, [99] etc.) for drug and gene delivery applications ( figure 6 ). for example, song et al. developed a multifunctional liposome based complex system for in vivo treatment of nsclc. the authors loaded an anticancer drug epirubicin inside the aqueous core of the liposome and an anti-metastatic drug honokiol into the lipid bilayer of the formulation. the surface of the liposome was further conjugated with a somatostatin targeting peptide octreotide which can binds to somatostatin receptors overexpressed in cancer microenvironment and facilitate targeted drug delivery. this complex liposomal system showed improved in vivo anticancer activity. [100] cisplatin is one of the widely used drugs for the treatment of lung cancer. however, it showed nephrotoxicity in patient with high doses. [101] devarajan et al. reported a liposomal formulation of cisplatin (namely the impact of the nanotherapeutic bind-014, a polymeric nanodrug carrier, encapsulating docetaxel, and targeting prostate specific membrane antigen on human drug metastases from a primary cholangiocarcinoma. a) a schematic representation of bind-014 composed of a biodegradable and hydrophobic pla polymeric core and a hydrophilic peg corona decorated with small-molecule targeting ligands, and an encapsulated anticancer drug (docetaxel). adapted with permission. [82] copyright 2013, dovepress. b) representative axial images from contrast-enhanced ct scans obtained from a patient with lung metastases at baseline and at day 42 after two treatment cycles of bind-014. red circles indicate locations of metastatic lesions observed in the baseline ct scan. adapted with permission. [83] copyright 2012, american association for the advancement of science. lipoplatin) which showed less nephrotoxicity when compared with free non-bound cisplatin. [102] paclitaxel is another anticancer drug which was widely used for the treatment of various cancers including lung cancer. in a phase i clinical trial in nsclc patients, a liposomal formulation of paclitaxel showed enhanced therapeutic efficacy. [103] in a recent study, researchers prepared a liposomal formulation containing both cisplatin and paclitaxel for the treatment of lung cancer. in a phase iii trial, this liposomal formulation displayed improved therapeutic activity and reduced nephrotoxicity in nsclc patients. [104] in the last decade, researchers have developed a special type of liposomesarchaeosomes, which are made with ether lipids unique to the domain of archaeobacteria. [105] achaean-type lipids consist of archaeol (diether) and/or caldarchaeol (tetraether) core structures. the membrane of this type of liposomes is made with both conventional phospholipids and bipolar lipids ( figure 6 ). archaeosomes can be made using standard procedures used for liposome preparation including a sonication of hydrated film, extrusion, and detergent dialysis. in contrast to conventional liposomes, archaeosomes remain stable at acidic ph, high temperature and pressure, resist oxidative degradation and can be sterilized by autoclaving. a stability of archaeosomes at a wide physiological (or lower) temperature range opens a possibility of encapsulating thermally stable compounds. however, the uptake of archaeosomes by phagocytic cells can be up to 50-fold greater when compared with conventional liposomes, which may be considered as their disadvantage. nanostructured lipid carriers: nlcs are another widely used lipid-based nanocarrier for targeted drug and gene delivery applications. nlcs are composed of biodegradable and biocompatible lipids and usually prepared by mixing a liquid lipid mixture containing unsaturated lipid or oils to a solid lipid ( figure 6 ). among the lipid-based nanocarrier, nlcs have the advantages of ease of manufacturing processes, drug protection during the storage, low toxicity, biodegradation, etc. these and other benefits of nlcs made them a promising therapeutic delivery system in recent years. [15, 25, 29, 106] here, we have summarized few examples of nlc-based therapeutic nanomedicines for drug and gene delivery. for instance, guo et al. [107] prepared a dual drug paclitaxel (ptx) and 5-demethylnobiletin (dmn) loaded and cetuximab (cet) functionalized cet-ptx-dmn-nlcs for the combination therapy of lung cancer. the treatment of the cet-ptx-dmn-nlc inhibited the growth of the lung cancer cells as compared to the single ptc-nlc and dmn-nlc treatments. the authors also observed remarkable inhibition of in vivo lung tumor for the treatment of this dual drug containing cet-ptx-dmn-nlc. [107] wang et al. [108] developed a dual drug loaded nlc system for the treatment of lung cancer. this dual drug loaded ptx/dox nlc displayed 3ã� higher activity as compared to that of single drug ptc-nlc and dox-nlc treatment as revealed in cytotoxicity assay in ncl-h460 cells. also, in vivo study of this dual drug nlc on a non-small cell lung cancer mice model showed improved the anticancer activity. [108] micelles: micelles consist of lipid molecules arranged in a spherical form in polar solvents (e.g., water). polar groups of lipids form an outer shell of the nanoparticles in polar solvent system while lipid hydrophobic tails create an inner core of micelles. in contrast to liposomes, micelles usually have a single hydrocarbon chain ( figure 6 ). in 2007, kim et al. prepared nanosized micelles (genexol-pm) loaded with the anticancer drug paclitaxel for the treatment of nsclc. [109] this nanotherapeutic was found to deliver higher paclitaxel dose with reduced drug toxicity as well as exhibited significant antitumor activity in the treatment of advanced nsclc. a series of nanocarrier systems which included liposomes, micelles, quantum dots, mesoporous silica nanoparticles, dendrimers, and peg polymers were prepared and examined in our laboratory in order to find out the suitable nanocarrier for local and inhalation delivery of anticancer drugs to the lungs. we investigated organ distribution and retention of all these nanocarriers in the lung and observed higher accumulation of liposomes and micelles based nanocarriers in lungs as compared to that of mesoporous silica nanoparticles, quantum dots, and dendrimers. [26] we found a significant enhancement of anticancer activity of doxorubicin when it was delivered to mice bearing lung tumor by inhalation by liposome-based system. this study revealed that lipid-based nanocarriers such as liposomes with higher accumulation of drug in lungs and longer retention time were more suitable and effective than non-lipidbased nanocarriers in treating lung cancer by inhalation. [26] polymeric nanoparticles: polymeric nanoparticles are usually prepared by self-assembly of various block-copolymers with alternate hydrophobic unit between blocks. various biodegradable polymers such as poly (lactic-co-glycolic) acid (plga), polycaprolactone, poly (lactic acid) (pla), chitosan etc. were widely used for the preparation of polymeric nanoparticles mainly because of their biocompatibility and controlled release properties. while core-shell of such polymeric nanoparticles can encapsulate hydrophobic drugs, the surface of the polymeric nanoparticles can be modified for receptor targeted drug delivery. [110, 111] in the past two decades, many polymeric nanoparticles have been investigated for treatment of different diseases including lung cancers and enhancing efficacy of anticancer drugs. [112] in a recent report, hu et al. [113] developed paclitaxel encapsulated polycaprolactone/poly (ethylene glycol)/polycaprolactone nanoparticles for the combined treatment of lung cancer with chronomodulated chemotherapy. this combination treatment showed better inhibition of tumor growth in vivo. wang et al. [114] developed a new strategy for delivering drug loaded polymeric nanoparticles at the disease site using mesenchymal stem cells (msc) as carrier. docetaxel was encapsulated in the nanoparticles and tested in vivo. inhibition of the tumor growth was observed in the animal's experiment, which also revealed the translocation of nanoparticles from msc to cancer cells. researchers developed peg modified and taxane encapsulated polylactic acid figure 7 . glutathione stimuli responsive organic plga-ss-peg nanocarriers for targeted delivery of anticancer agent homoharringtonine (hht) to lung cancer cells. a) core-shell structured nanoparticles were synthesized using the solvent evaporation approach of oil-in-water. the plga nanomedicine release was triggered by reduced glutathione (gsh) overexpressed in tumor cells after receptor mediated endocytosis. b) in vivo antitumor efficacy of plga nanomedicine. treatment with plga-ss-peg nanoparticles reduced the tumor volume significantly when compared with non-treated and treated with free non-bound hht groups. adapted with permission. [48] copyright 2017, springer. nanoparticles for lung cancer treatment. the authors observed significant improvement of the efficacy of chemoradiation therapy in an a549 lung tumor xenograft model. [115] in another report, investigators developed a polymeric nanoparticle system comprised of block copolymers of peg and polylactic acid and encapsulated paclitaxel and cisplatin for treatment of lung cancer. [116] tseng et al. prepared gelatin based polymeric nanoparticles conjugated with biotinylated egf (begf) motif for egfrtargeted drug delivery. [117] the authors observed enhanced cellular uptake of this polymeric nanoparticle in egfr overexpressing cancer cell lines such as lung cancer cells. zhang et al. [118] studied glutathione stimuli responsive organic plga-ss-peg nanocarriers for targeted delivery of anticancer agent homoharringtonine (hht) to lung cancer cells. a relatively high level of glutathione reduced (gsh) in cancer cells caused disulfide-bond (ss) breakage in a reductive manner and release of hht inside cancer cells. this nanocarrier showed a reasonable biocompatibility and was further decorated with an epidermal growth factor receptor (egfr) aptamer as a targeting moiety. after endocytosis of this nanosystem by lung cancer cells, the high level of glutathione in tumor cells stimulated the release of the loaded drug. finally, this multifunctional and stimuli responsive nanocomplex inhibited the growth of human lung cancer cells and displayed better therapeutic efficacy when compared with the free non-bound anticancer agent (figure 7) . combined lipid-polymer nanoparticles: in addition of nanocarriers prepared mainly with lipids or polymers, complex anisotropic nanoparticles containing lipid and polymeric structures (as well as polymers with different properties) were prepared. [119] [120] [121] [122] [123] [124] as pierre-gilles de gennes pointed in his 1991 nobel prize lecture, [125] similarly to the ancient roman god of gates janus who was portrayed with two faces-one facing the past, and one facing the future, janus particles also have two distinct parts with antagonistic properties (figure 8 ). one such janus structure with two faces (lipid and polymeric) was tested in our laboratory for inhalation lung delivery of a mixture of lipoand hydrophilic drugs namely curcumin and doxorubicin. [28] these janus particles were synthesized from binary mixture of biodegradable and biocompatible materials and evaluated for cytotoxicity and genotoxicity. the inhibition of lung tumor growth by the combination treatment was significantly higher when compared with either free drugs or nanoparticles containing only one drug. this study showed that such janus particle could be explored for the simultaneous co-delivery of hydrophilic and hydrophobic drugs. carbon nanomaterials: carbon nanomaterials are a new class of nanosized materials comprised of sp2 hybridized carbon atoms with hexagonal structure. common carbon nanomaterials include 0d fullerenes, 1d carbon nanotubes (cnts), and 2d graphene such as graphene oxide. [126] among the carbon figure 8 . suppression of lung tumor growth in mice treated by inhalation with janus nanoparticles containing anticancer drug(s). a) ancient roman god of gates janus who was portrayed with two faces (photo by a. kokorin on behance). b) representative optical, c) scanning electron and d) fluorescence microscope images of anisotropic biodegradable biphasic polymer/lipid janus nanoparticles. polymeric phase of nanoparticles was labeled with fitc (green fluorescence); lipid phase was labelled with dir (red fluorescence). e) representative optical and f) magnetic resonance images of untreated (control) and treated mice four weeks after tumor instillation. g) changes in lung tumor volume after beginning of the treatment with nanoparticles containing doxorubicin (dox), curcumin (cur), and both drugs. mice were treated twice per week. means â± sd are shown. reproduced with permission. [28] copyright 2014, acs publications. nanomaterials, cnts have received significant attention in the past two decades for their high surface area, biocompatibility and drug-loading capacity which made them suitable for wide range of applications such as drug delivery, tissue engineering, biosensors, cosmetic products etc. [127, 128] . there are mainly three types cnts namely single wall-, double wall-and multi wall-carbon nanotubes with diameter up to 100 nm and lengths up to microns size. [129, 130] surface of these cnts can be functionalized with hydrophobic and hydrophilic drugs for their targeted delivery applications. carbon nanomaterials such as cnts were explored as an attractive systems in targeted drug delivery application. in 2017, kim et al. developed peg-coated carbon nanotube system loaded with small molecule bcl-2 inhibitor abt-737 for its targeted delivery to lung cancer cells. [131] the authors investigated cellular uptake, apoptosis, and cytotoxicity this peg-cnt-abt737 nanotube system in lung cancer a549 cells and observed bcl-2-mediated apoptosis of lung cancer cells. the peg-cnt-abt737 system also excreted improved cytotoxic activity in a549 cells when compared with treatment by free non-bound abt737. the drug loaded nanotubes represented an effective system for inducing bcl-2-mediated apoptosis in lung cancer cells. in 2019, cirillo et al. reported a ph-responsive nanohybrid system comprised of multi-walled carbon nanotubes and chitosan for delivery of methotrexate to lung cancer cells. [132] this chitosan coated cs-mwcnt nanohybrid system displayed its ph-responsive behavior and showed faster and higher release of the drug methotrexate in acidic (ph 5.0) versus neutral (ph 7.4) environments. such a nanohybrid system showed reduced drug toxicity in normal lung mrc-5 cells while it exerted anticancer activity in lung cancer h1299 cells. mesoporous silica nanoparticles: nanosized silica particles also known as mesoporous silica nanoparticles (msns) have been investigated in the past two decades for various drug and gene delivery application. because of large pore size, high surface area, good chemical stability, biocompatibility, and ease of surface modification with targeting ligands, msns based nanomaterials were extensively studied for various therapeutic delivery applications. [6, 30, 131, 133, 134] for example, wang et al. designed a nanosized drug delivery system containing anticancer drug paclitaxel into the core-shell of mesoporous silica nanoparticle (pac-csmsn) for the treatment of lung cancer. [135] this csmsns formulation improved the adsorption of the poorly water-soluble drug paclitaxel. the authors found pac-csmsn system was more effective in promoting cell apoptosis in a549 lung cancer cells than the free drug. this pac-csmsn system was administered for three consecutive days in animals and no indication of inflammation was observed in the lung biopsy. all these results indicated that such pac-csmsn system has the potential for inhalation delivery of paclitaxel for the treatment of lung cancer. 2018, jing-hua sun et al. prepared another msns system for codelivery of a photosensitizer chlorin e6 (ce6) and a drug doxorubicin (dox) for both photodynamic therapy and chemotherapy of lung cancer. [136] the anticancer drug doxorubicin was encapsulated into the pores of msns system while ce6 was conjugated with the msns through covalent bonding. treatment with these dox@msns-ce6 hybrid nanoparticles increased the level of cellular reactive oxygen species and exerted synergistic therapeutic effect in lung cancer a549 cells when compared with treatment by each individual component. gold nanoparticles: gold nanoparticles (aunps) are one of the extensively used inorganic nanocarriers for various biomedical applications including drug and gene delivery. because of high atomic number, stable nature and surface plasmon resonance properties, aunps can serve as stable contrast agents for photothermal therapy and medical imaging. [137, 138] moreover, aunps possess unique physiochemical characteristics such as high biocompatibility and low-toxicity as well as aunps are non-immunogenic which made gold nanoparticles as an attractive nanocarriers for various biomedical applications. [139] [140] [141] in 2014, qian et al. conjugated cetuximab (c225), a targeting agent for epidermal growth factor receptor (egfr) with aunps for the treatment of egfr positive non-small cell lung cancer (nsclc). [142] this c225-aunps inhibited proliferation and migration of a549 cells and accelerated apoptosis in a549 cells as compared to treatment with free c225 alone. the activity of c225-aunps was higher in a549 cells with higher egfr expression than in h1299 cells with low egfr expression. treatment of nude mice bearing tumor xenografts with c225-aunps showed significant suppression of tumor size. such egfrtargeted aunps system can be a promising strategy for targeted delivery of therapeutics in egfr positive nsclc cells. in 2018, ramalingam et al. conjugated doxorubicin on the surface of gold nanoparticles through polyvinylpyrrolidone linker for the treatment of human lung cancer cells. [143] these dox-pvp-au nanoparticles inhibited the growth of human lung cancer cells more effectively than both the pvp-aunps and free drug. treatment with these nanoparticles induced early and late apoptosis as well as upregulated expression of tumor suppressor genes in the human lung cancer cells. this dox-pvp-au nanoparticle system represents a promising drug delivery approach for lung cancer therapy. cell based drug carriers: biocompatibility, biodegradability, and cytotoxicity of synthetic nanocarriers represent a substantial problem. moreover, exogenous carriers potentially may induce immune responses. consequently, drug carriers prepared from human live cells or their derivatives attract a considerable attention in recent years. such carriers demonstrate native targeting mechanisms and controlled release of the encapsulated drug molecules. several types of human cells have been considered for a targeted drug delivery for treatment of cancers and variety of other pathological conditions, such as cardiovascular and inflammatory diseases. [144, 145] major characteristics of different cell types used for drug delivery are presenting in the figure 9 . erythrocytes are usually used for a systemic drug delivery and do not possess intrinsic tropism. in contrast, platelets, neutrophils, adipose cells, macrophages, and stem cells can be used for targeted delivery of therapeutics because of their native tropism to tumors, circulating tumor cells, sites with inflammation, and hypoxic conditions as well as microorganisms. [145] a strategy of drug loading into human cells used for therapeutic delivery is selected based on the nature and properties of drugs, loading capacity and release mechanisms. therapeutic agents can be loaded into cellular cytoplasm of carrier cells, attached to their surface by a membrane insertion, nonspecific noncovalent or targeted interaction as well as covalent coupling methods. the release of payload may be continuous (e.g., after slow hydrolysis of a prodrug and exocytosis) or triggered by various internal in vivo signals (glucose, hormones, cytokines, and other biomolecules, ph, and changes in cell shape) or external stimuli (light, ultrasound, magnetic field, temperature, etc.). [145] a comprehensive list of cell based carriers designed for intraperitoneal, intratracheal, intravenous, subcutaneous and left anterior descending delivery of therapeutics developed during the last decade is presented in the open access review by lutz et al. [144] gene therapy has become a promising therapeutic option in recent time for lung cancer. several different nanocarriers (such as dendrimer, micelles, gold nanoparticles, liposomes, lipid nanoparticles, auroliposome etc.) were explored as carriers of nucleic acids with effective results. [14, 15, 23, 25, 27, 29, 42, 43, 45, 92, 134, [146] [147] [148] [149] examples of carriers used for the delivery of nucleic acids are presented in figure 10 . nucleic acids used as gene therapeutics are negatively charged because they are composed of few, several or many nucleotides with phosphate backbones carried one negative charge per residue. [150] consequently, they often delivered as conjugates formed with positively charged (cationic) carriers. such conjugation not only protects gene material from degradation in the blood stream and improves pharmacokinetics of the resulting complex, but also neutralizes positive charge of highly toxic anionic carriers limiting their cyto-and genotoxicity. [151] however, an encapsulation inside nanocarriers or direct conjugation of native or modified nucleic acids via different (preferably cleaved inside targeted cells, e.g. sâ��s) bonds are also used to form a stable system for an effective gene delivery. small chunks of nucleic acids can be modified to decrease their negative charge and encapsulated inside nanocarriers. in our laboratory, the dna backbone of all bases in antisense oligonucleotides (aso) was pethoxy modified in order to make the entire aso neutral and increase their incorporation efficacy into liposomes. [149] such modification also enhanced nuclease resistance of aso. liposomal aso were successfully used to suppress pump and nonpump resistance of cancer cells. [27, 41, 43, 91] it should be stressed, that treatment of cancer with nucleic acids alone (e.g., sirna, antisense oligonucleotides) in most cases demonstrate a pretty limited anticancer effect. however, a combination of anticancer drug(s) with nucleic acids targeted to the drug efflux pumps, antiapoptotic, and other cancer cell defensive proteins/mrnas is expected to substantially enhance anticancer efficacy of both anticancer drugs and nucleic acids. such a concept of advanced proapoptotic anticancer delivery system was first developed and tested in the laboratory of professor minko at rutgers university almost 20 years ago. [36, 37] possible structures of such multifunctional nanoparticles are presented in figure 1 . in several such systems, figure 9 . various cell types employed for drug delivery. redrawn from. [144, 145] where nucleic acids (sirna and antisense oligonucleotides) were used as suppressors of drug efflux pumps (pump drug resistance) and antiapoptotic cellular defense (non-pump resistance). [54, 91] lipid-based nanoparticles: lipid-based nanoparticles such as liposomes, nlcs, and various polymeric nanoparticles are widely used to protect and deliver nucleic acids ( figure 10) . few examples of both liposome and nlc based nucleic acid delivery systems are discussed below. liposomes: gopalan et al. prepared dotap/cholesterol based nanocarrier system for direct delivery of tumor suppressive gene at the tumor site. this system was effective and nonimmunogenic. [152] in an early study, our group prepared a multicomponent liposomal delivery system for improving anticancer activity of doxorubicin against multidrug-resistant human non-small-cell lung cancer cells. this multi-component liposomal system was included doxorubicin as an anticancer drug, antisense oligonucleotide (aso) as a suppressor of pump resistance for mrp1 mrna and another aso as a suppressor of nonpump resistance for bcl2 mrna. [27, 41, 43, 92, 149] antisense oligonucleotides were p-ethoxy modified to decrease their charge, enhance nuclease resistance, and increase incorporation efficacy into liposomes. we reported successful intracellular delivery of both doxorubicin and asos to lung cancer cells. also, this liposomal treatment increased anticancer efficacy of doxorubicin and inhibited synthesis of both mrp1 and bcl2 proteins. this multicomponent liposomal system displayed 10-fold higher cytotoxicity as compared to both free and liposomal doxorubicin treatment against the resistant lung cancer cells and could be used for the enhancement of anticancer activity of doxorubicin against multidrug-resistant lung cancer cells. in a similar study, we used a complex liposomal drug delivery system containing anticancer drug doxorubicin and both mrp1 and bcl2 targeting antisense oligonucleotides for inhalation treatment in lung cancer cells. [27] while empty liposome, free antisense oligonucleotides and their combination treatment showed almost no influence on viability of lung cancer cells; liposome targeted to both mrp1 mrna and bcl2 mrna significantly inhibited the growth of the lung cancer cells. we evaluated this complex liposomal system on an orthotopic murine model of human lung cancer and the results revealed its higher chemotherapeutic efficacy with lower side effects as compared to that observed for individual treatment of each component. [27] nanostructured lipid arriers: garbuzenko et al. reported a multi-functional nanostructured lipid carrier (lhrh-nlc-sirnas-tax) composed of an anticancer drug paclitaxel (tax), a peptide analog targeted to luteinizing hormone-releasing hormone (lhrh) receptor and a pool of sirnas as inhibitors of different types of egfr-tks. this lhrh-nlc-sirnas-tax nanoparticle system was investigated in various human lung cancer cell lines and in vivo on an orthotopic nsclc mouse model and displayed good organ distribution, stability, solubility, and improved anticancer activity when compared with free individual drugs and non-targeted therapy. [15, 25, 29] han et al. developed a multifunctional blc system for delivery of anticancer drug doxorubicin (dox) and green fluorescent protein plasmid (pegfp) dna as a prototypical nucleic acid in lung cancer cells. the authors prepared dox and pegfp encapsulated nlc and modified its surface with transferrin-targeting motif. transferrin-modified and dox and pegfp co-encapsulated nlc system showed higher in vitro and in vivo transfection of plasmid dna than the other control treatment. these results also indicated that such multifunctional nlc system could be an effective method for both drug and gene delivery for the treatment of lung cancer. [153] in another work, han et al. prepared an nlc system for delivery of plasmid-containing enhanced green fluorescence protein (pegfp) in lung cancer cells. the authors prepared the pegfp-loaded nlc and decorated its surface with transferrin (tf) targeting ligands. this tf-nlc/pegfp showed higher transfection efficiency as observed in in vitro and in vivo studies than the non-targeted nlc/pegfp-suggesting this nlc system could be a promising vehicle for gene therapy in lung cancer. [154] polymeric and hybrid nanoparticles: chitosan-based polymeric nanoparticles were widely used for the delivery of nucleic acids. because of ionizable sidechain amino groups in chitosan, it has cationic nature which made chitosan polymer a good vehicle for the delivery of anionic agents such as sirna, dna etc. okamoto et al. developed a chitosan-based nanoparticle for delivery of pcmv-luc gene into the lung cancer cells. [155] nafee et al. prepared a chitosan based nanocarrier system loaded with an antisense oligonucleotide such as 2-o-methyl-rna for the treatment of lung cancer. the authors evaluated the inhibitory function of telomerase after treating the nanocarrier in lung cancer cells and observed 50% reduction of the telomerase activity in a549 lung cancer cells. these chitosan nanoparticles were safe and effective for the treatment of lung cancer. [156] dhananjay et al. developed a polymeric nanoparticle system comprised of pei-peg copolymer for the delivery of akt1 shrna in lung cancer cells. [157] both service and internally anionic polypropylenimine tetrahexacontaamine (ppi) and polyamidoamine (pamam) dendrimers were successfully used for the delivery of nucleic acids into cancer cells. [158] [159] [160] it was found that dendrimers and additional caging of resulting nanoplexes protected nucleic acids from degradation and effectively delivered genetic material inside cancer cells. delivered sirna demonstrated high intracellular activity and effectively knocked down gene expression and synthesis of targeted proteins. carbon nanomaterials: carbon nanomaterials such as cnts decorated with positively charged polymers were investigated as gene carrier in recent years for specific delivery of nucleic acids. [161, 162] podesta et al. prepared amino-functionalized mwcnt system for delivery of sirna and tested it using an animal xenograft and orthotopic breast tumor models. [137] treatment with this mwnct-sirna complex significantly delayed the growth of tumor and increased the survival of the tumor bearing animals. varkouhi et al. developed mwcnt systems decorated with cationic pei (cnt-pei) and cnt-pyridinium for the delivery of sirna to lung cancer cells. [163] both these cnts displayed cytotoxicity effect and gene silencing activity in h1299 human lung cancer cells, while non functionalized cnts did not show any such effects. mesoporous silica nanoparticles: mesoporous silica nanoparticles have been investigated as a delivery vehicle of various cargo molecules including nucleic acid therapeutics. for example, dilnawaz et al. developed msn based system for co-delivery of anticancer drug doxorubicin and sirna in lung cancer cells. [164] this combinational treatment enhanced in vitro cellular uptake, cytotoxic effect in a549 lung cancer cells. in 2020, song yinxue et al. developed a complex msn system comprised of a polyphenolic drug myricetin (myr), sirna specific to multidrug resistance protein (mrp-1) and a targeting ligand folic acid (fa) in order to improve delivery efficiency of myr in nsclc cells. [165] this targeted myr-mrp-1/msn-fa nanoparticles showed significant cellular uptake and reduced viability of a549, nci-h1299 lung cancer cells when compared with free drug and other controls. in vivo results revealed that this system was more effective in suppressing the tumor growth and it might be an attractive therapeutic strategy for the treatment of nsclc. gold nanoparticles: gold nanoparticles possess good biodistribution, physiological stability and low cytotoxicity which made them an attractive vehicle for delivery of various payloads including large biomolecules such as nucleic acids. [166] over the years, researchers have explored both non-covalent and covalent conjugation of the nucleic acids such as sirna, oligonucleotides etc. on the surface of aunps for their effective transportation to the target cells. for instances, conde et al. developed a peg modified gold nanoparticle system by conjugating of rgd peptide and cmyc sirna on the surface of gold nanoparticles and tested this system on mice bearing cmt/167 lung carcinoma tumors. [167] the authors observed downregulation of the c-myc oncogene and significant inhibition of lung tumor growth after the treatment with si-rna/rgd aunps. recently, an innovative hybrid formulation (so-called auroliposomes) consisting of liposomes loaded with 20-nm gold nanoparticles (aunps) was developed and used for the sirna delivery ( figure 10 ). it was found that auroliposomes modulated the intracellular uptake and silencing efficacy leading to the enhanced suppression of tumor growth in vivo when compared with conventional liposomes. [168] nanocarriers have the potential to enhance the diagnosis of diseases. recently, nanobased materials and methods have emerged as novel diagnostic tools for several diseases. over the years, various nanocarriers were explored for the delivery of imaging (or both imaging and therapeutic) agents for diagnosis of many diseases including lung cancer. [169] [170] [171] for example, researchers designed folic acid functionalized dendrimers containing gold nanocarrier as cancer-targeted imaging probes for computed tomography (ct) imaging of lung cancer cells. [172] ct imaging after nanoparticle uptake revealed the presence of these gold nanocarriers in the lysosomes of lung adenocarcinoma cells. in another study, researchers developed ultrasmall (3.0 â± 0.1 nm) gadolinium containing nanoparticles (so called ultra-small rigid platforms or usrps) for enhancing ultrashort echo time (276 ms) proton mri of the lung. [173] these nanoparticles were prepared using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (dota) as a chelator and was delivered by the intratracheal instillation. the authors observed the substantial (>250%) enhancement of mri signal in the lungs for almost 2.5 h after instilling the solution of the nanoparticles. erten et al. prepared a dextran core-based stealth pegylated liposomes containing anticancer drug doxorubicin, iron oxide as an mri contrast agent and boron dipyrromethene (bodipy) fluorescence stain for imaging theranostics applications. [174] the authors observed strong ability of these liposomal nanoparticles of enhancing both types of imaging in the in vivo murine model of lewis lung cancer. in another report, lowery et al. labeled a tumor targeted doxorubicin loaded liposomes with alexa fluor 750 for imaging of lung tumor. [175] an hvggssv peptide with a selective binding to irradiated tumors was used as a targeting moiety in order to deliver the anticancer drug and imaging agent specifically to irradiated tumors limiting their accumulation in the normal tissues. these liposomes (100 nm) contained maleimide and amine functionalized peg chains for the conjugation of the cysteine containing peptide and the n-(succinyl)-fluorophore, respectively. doxorubicin in theranostic liposomes was loaded by the ph gradient. the authors studied these fluorophore labeled irradiated tumor targeted liposomes in murine model of lewis lung cancer. they found that such a radiationguided tumor-targeted delivery of liposomes enhanced the delivery of the fluorophore and anticancer drug specifically to irradiated tumors in the lungs, effectively induced cell death and limited cell proliferation within lung tumors finally inducing a delay in tumor growth and destruction of tumor blood vessels and increase of apoptosis in lung tumor cells. hvggssv targeting peptide also increase the accumulation of an entire system in irradiated tumors enhancing imaging quality. cystic fibrosis is an inherited disorder, caused by mutations of the cystic fibrosis transmembrane conductance regulator (cftr) gene. over-production of mucous in the lungs causes airway obstruction resulting in infectious diseases such as cystic fibrosis (cf). [176] typically, heterogeneous and large molecular weight oligomeric gel-forming mucin glycoprotein are produced in cf. gene therapy is the mainstay therapy to inhibit the mutation of cftr protein. gene therapies involve delivery of sirna, dna etc. into cells to rescue the function of the defective cftr gene. usually, viral and non-viral vectors are employed to transfer correct copies of cftr gene in the effected cells in lungs. [177] because of small size, nanocarriers have emerged as an effective vehicle for delivery of gene through the mucus barriers. nanoscale carriers can be used as vectors for gene therapy due to their less immunogenic and good gene transport capacity. [178] nanocarrier based non-viral vectors are easy to prepare as compared to that of viral vectors. [179] in recent years, researchers attempted to develop various nucleic acid based nanocarrier to affect mutation of cftr gene to change the composition of mucin as well as to minimize the mucin production. [180, 181] in an early research, konstan et al. developed a dna nanoparticle for the treatment of cystic fibrosis and observed effective transfer of vector gene. [182] in order to overcome the mucus barrier, recently, suk et al. prepared a densely peg-coated dna nanoparticle system, which can penetrate extracorporeal human cystic fibrosis to deliver its payload. [183] this nanocarrier displayed better gene transfer after intranasal administration to mice as compared to other carriers. minko et al. prepared a liposomal--tocopherol (lat) formulation for the treatment of hypoxic lung injury in rats. the authors evaluated antioxidant and antiapoptotic activity of this lat in rats with severe hypoxia and observed significant antihypoxic effects. [93] it was found that, treatment with lat of rats under severe hypoxic conditions (breathing of 6% of oxygen within two hours) normalized lung phospholipid composition, inhibited lipid peroxidation, suppressed genes responsible for the development of lung damage and improved breathing pattern. finally, such a treatment two-times decreased the mortality of the animals under severe hypoxic conditions. lately in our lab, a similar liposomal system for inhalation delivery of prostaglandin e2 (pge2) was developed for treatment of pulmonary fibrosis. [16] this liposomal system was evaluated for local delivery of pge2 using a standard bleomycin-induced murine model of idiopathic pulmonary fibrosis. the results revealed that liposomes were accumulated in higher amount in lungs after inhalation delivery when compared with intravenous administration. besides, this inhalation treatment reduced fibrotic injury in the lung tissues. these data probed that the inhalation administration of liposomal form of pge2 can be an effective therapy for cystic fibrosis in the lungs. to further improve inhalation treatment of idiopathic pulmonary fibrosis (ipf) by liposomal pge2, sirnas targeted to major proteins responsible for the lung damage under ipf (mmp3, ccl12, and hif1a) were added to the nlc based nanoparticles containing pge2 and tested on the similar experimental model of lung fibrosis using inhalation delivery. [23] this enhanced advance system was more effective in the treatment of ipf when compared with sirna and pge2 delivered separately. another combination of drugs in one nlc-based nanoparticle system was recently tested for the treatment of lung manifestation of cystic fibrosis (cf). [24] the system included lumacaftor for the correction of correct p.phe508del mutation (the loss of phenylalanine at position 508) and cftr potentiator ivacaftor for increasing the open probability of cftr channels. this system was tested in vitro using cf cells and in vivo on homozygote/homozygote bi-transgenic mice with spontaneously developed cf. the system was delivered in vivo by inhalation. the results showed a high efficacy of the proposed treatment of the lung manifestation of cf. wang et al. prepared rapamycin and azithromycin loaded polymeric nanocarrier via nanoprecipitation method. [184] nanocomposite microparticles (ncmp) were formulated from this nanoparticle for the inhalation delivery of antibiotics in the form of dry powder aerosols. these, nanocomposite microparticles displayed aerosol dispersion characteristics indicating their deposit in the lungs. viral infections in respiratory systems such as in lungs have become a worldwide public health threat in recent years. several emerging positive-stranded rna coronaviruses [185, 186] such as severe acute respiratory syndrome coronavirus (sars-cov), [187] [188] [189] middle east respiratory syndrome coronavirus (mers-cov) [191] etc. not only threatened public health, but also caused international epidemics in the past two decades. recent outbreak of coronavirus infection caused by the severe acute respiratory syndrome-coronavirus-2 pathogen has seriously threatened public health all over the world. the taxonomic name "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) given by the international committee on taxonomy of viruses (ictv) became official to refer to this virus strain. on february 11, 2020 the world health organization (who) officially named the "coronavirus disease 2019" as "covid-19". [192] the genome of sars-cov-2 is a 29 903 bp with single-stranded rna (ss-rna). the complete genome sequence of sars-cov-2 is available in the national center for biotechnology1 (ncbi) database, with id nc_04 5512. [193, 194] covid-19 is characterized by severe respiratory disease along with mild to high fever, cough, and shortness of breath. covid-19 has been considered as an emerging disease and on march 11, 2020 the outbreak of this disease has been declared as global pandemic by the who. [195, 196] this virus has been found to spread from person to person mainly through respiratory droplets, cough, sneeze, etc. [197] causing severe acute respiratory distress syndrome (ards). as of september 20, 2020, this virus has already infected more than thirty million people and caused 950000 deaths with billions of people are at risk around the world. [198] despite repeated outbreaks of sars-cov in 2003 and mers-cov in 2012, no potent vaccines and anti-viral drugs are commercially available against these viral infections-mainly due to the fact that the outbreaks of these viruses were rapidly contained and did not reappear. [199] therefore, there are no effective treatment for the ongoing pandemic of covid-19, a close subtype of sars-cov. [200] because of constant emergence of new viruses including current sars-cov-2 infection, there is an urgent need for the development of potent and broad-spectrum vaccines and antiviral drugs for effective control of viral diseases. since the first report of sars-cov-2 infection in late december in 2019, both researchers and clinicians have been attempted clinical trials of several known antiviral drugs, their combination as well as development of vaccine in patients with confirmed covid-19 disease. this review is mainly focused for summarizing recent developments of nanotherapeutics for respiratory diseases including sars-cov, mers-cov, and covid-19. therefore, other types of therapeutic and diagnostic methods such as small molecule antiviral therapeutics, anti-sars-cov-2 antibody treatments, convalescent plasma therapy etc. [201] [202] [203] [204] which have been discussed elsewhere are out of the scope of this review. briefly, we will summarize recent innovation of nanobased diagnostics such as nanoparticle-based pcr and anti-body test as well as nanoparticle-based therapeutic approaches for covid-19. diagnostic tests are essential not only for monitoring every stage of a disease, but also to identify new patients with that illnessespecially for an outbreak of viral disease. typical diagnosis methods for viral diseases include nucleic acid detection of the viral genome in clinical samples. currently, covid-19 has been diagnosed by real-time polymerase chain reaction (rt-pcr) test for the detection of viral genome, serological, and immunological assays for the detection of anti-sars-cov-2 antibody in patient samples as well as chest computed tomography (ct) imaging for screening abnormal observations in chest scans. [205, 206] however, most of these methods are laborious and time-consuming processes. therefore, there is an urgent need for developing timeeconomic, easily performed and point-of-contact test for the detection of this virus. because of similar size and shape of sars-cov-2 virus with the synthetic nanoparticles, researchers have attempted to develop nanoparticle based diagnostic methods for covid19 . for examples, huang et al. developed a rapid, easily operated and cost-effective detection of the igm antibody produced in serum sample of patient with covid-19. [207] the authors prepared a colloidal gold nanoparticle-based lateral-flow (aunp-lf) system composed of various low-cost inorganic nanomaterials. the aunp-lf strip was developed for the sample test by coating an analytical membrane with the sars-cov-2 nucleoprotein followed by conjugating anti-human igm antibody. this method can detect the sars-cov-2 virus in 15 min using only 20 âµl of serum sample of the patient. the authors have figure 11 . schematic representation for the selective naked-eye detection of sars-cov-2 rna mediated by the suitably designed aso-capped gold nanoparticles. this naked eye detection method involves the isolation of the viral rna from the clinical swab sample from covid-19 patient and then incubation of the viral rna samples with aso capped gold nanoparticles for 5 min. at the end, rnase h is added in the viral composite of aso-capped gold nanoparticles and the resulting mixture is incubated for 5 min at 65 c to get the visual precipitate. reproduced with permission. [208] copyright 2020, acs publications. evaluated the specificity of this detection method against the results of widely used tr-pcr's test. this aunp-lf assay has a great potential for large-scale and fast detection of covid-19 disease specially during this pandemic period. [207] in another report, moitra et al. developed gold nanoparticle based colorimetric assay for naked-eye detection of sars-cov-2 virus present in patient samples. [208] the authors decorated the gold nanoparticles (aunps) with thiol-modified antisense oligonucleotides (asos) which are specific for n-gene (nucleocapsid phosphoprotein) of sars-cov-2 virus. the use of rnaseh in this detection helps to cleave the rna-dna hybrid resulting in the visually detectable precipitation from the experimental solution. this aunp system can detect the presence of sars-cov-2 stain in the isolated rna samples within 10 min. thus, this method can be a very promising for visual detection of covid-19 positive patient without the use of typical instrumental procedures as outlined in figure 11 . [208] there are no clinically approved therapeutics by the u.s. food and drug administration (fda) to prevent or treat the covid-19 disease. however, clinical trials of many known antiviral drugs are ongoing in patient with confirmed covid-19 disease. [209, 210] for instance, a newly developed antiviral drug remdesivir previously effective against ebola virus diseases which interferes with viral rna polymerase (rdrp) and arrests viral replication [211] was repurposed for the treatment of covid-19 infections. [212] human trials showed promising clinical improvements in â��70% of patients. [213] recent studies showed that the sars-cov-2 virus has similar size range (50-150 nm) and spherical shape like the synthetic nanoparticles. [214] therefore, nanosized spherical therapeutics can be promising to detect and neutralize coronaviruses as previously evaluated for sars-cov, mers-cov etc. various nanoparticle based therapeutic approaches have been discussed here for mers-cov, sars-cov, and covid-19 diseases. for instance, huang et al. prepared gold nanorod complex of heptad repeat 1 (hr1) peptide inhibitors for middle east respiratory syndrome coronavirus (mers-cov) disease. [215] this gold nanorod complex was biocompatible and metabolically stable and displayed 10-fold higher inhibition of membrane fusion between host cells and mers-cov via hr1/hr2-mechanism as compared to that of the free inhibitor treatment. this the gold nanorod based anti-viral system showed a great promise in treating mers-cov infection. lin et al. prepared a virus-like hollow nanoparticle comprised of biodegradable polymer and a viral antigen along with an adjuvant. [216] this plasmid like nanoparticle was capable of delivering of both antigens and stimulator of interferon genes agonist adjuvant to induce potentiation to the immune cells. the authors observed that this nanoparticle-based mers-cov vaccine was effective against a lethal dose of mers-cov infection as compared to control treatment in a mers-cov-permissive transgenic mouse model. the potency of this nanoparticle-based vaccine for mers-cov was demonstrated, and this study provides a new outline for developing nanocarrier based vaccine for viral pathogen. loczechin et al. prepared seven different carbon quantum dots (cqds) and investigated their anti-viral activity against the human coronavirus hcov-229e infections. [217] these cqds displayed a concentration-dependent virus inactivation and cqd produced from 4-aminophenylboronic acid showed better activity with ec 50 5.2 â± 0.7 âµgâ·ml â��1 . the mechanistic studies revealed that interaction of the surface functional groups of the cqds with entry receptors of the hcov-229e virus resulted in inhibition of the infection. these results suggested such cqds systems might be explored for developing anti-viral therapeutics for other coronavirus infections (figure 12) . [217] polymeric nanoparticles consisting of poly (ethylene glycol)block-poly(lactide-coglycolide) (peg-plga) were developed for delivery of diphyllin, a novel vacuolar atpase blocker for its antiviral activity against the feline coronavirus infection. [218] treatment with these nanoparticles significantly reduced toxicity and enhanced antiviral effect of diphyllin. these nanoparticles were well tolerated as revealed in animal study in mice following high-dose intravenous administration. the results of the study indicated that such diphyllin nanoparticles could be explored as effective host-targeted antiviral therapeutics for other coronavirus infections. coleman et al. developed a novel strategy for preparing spike nanoparticles which in combination with adjuvants produced high titer anti-bodies in mice against both the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) infections. [219] the results showed that these spike nanoparticles were able to neutralize the antibody responses in mice-suggesting a step towards nanovaccine development. [219] recently, fabric material-based face mask containing hydrophilic absorbent layers and hydrophobic barrier layers was constructed. [220] this fabric masks were found to show equivalent or better filtration and adsorption of nanoparticle like aerosols than the commercial n95 respirators. the aerosols were composed of fluorescent labeled virus like nanoparticles for tracking their transmission through the fabric masks. the authors evaluated 70 different combinations of common fabric materials using forced convection air flux with pulsed aerosols. this fabric masks can be used to protect from the inhalation of such viruses. in an early study, li et. al reported that treat-ment based on rna interference (rnai) exhibited antiviral immunity in mammals. [221] in 2018, sohrab s. et al. designed and developed a series of lipid and polymeric nanoparticles for delivery of therapeutic sirna for the treatment of mers-cov infection. [222] since sars-cov-2 is a single strand rna virus like other coronaviruses, therefore inhibiting the life cycle of sars-cov-2 viruses via silencing their viral mrna in the host cells by rna interference might be an effective therapy for covid-19. [223] each group of people received second vaccination after 28 days of the first vaccination. systematic side effects such as headache, fatigue, pain at the injection site etc. were observed in half the participants particularly after second vaccination with higher dose treatment. initial results revealed that treatment of mrna-1273 induced immune responses in all participants and the antibody responses were higher for the higher dose treatment participants. recently, corbett et al. studied this mrna-1273 vaccine in nonhuman primates and observed that treatment with this vaccine candidate increased neutralizing antibody levels that were higher than in human convalescent-phase serum sample. [223] the mrna-1273 vaccine is now under further evaluation for covid-19. bnt162b1 is another nanoparticle encapsulated mrna vaccine candidate that encodes receptor binding domain (rbd) of spike glycoprotein of sars-cov-2. recently, mulligan et al. conducted a placebo-controlled phase 1/2 clinical trial of nucleosidemodified mrna vaccine which was formulated in a lipid-based nanoparticle system for targeting rbd of spike glycoprotein of sars-cov-2 virus (clinical trial identifier: nct04368728). [223] the authors performed a randomized and placebo-controlled trial of bnt162b1 vaccine in 45 healthy adults. there were 12 participants for each of the dose level 10, 30, and 100 âµg of the vaccination and nine participants in placebo with bnt162b1 increased the sars-cov-2 neutralizing titers with dose level in the serum sample. thus, nanosized therapeutics such as nanoformulation of anti-viral drugs, nanovaccines, etc. can be promising options for the diagnosis and treatment of such viral infection. as summarized in this review, the past two decades have witnessed substantial amount of work in the development and applications of nanocarrier based systems for targeted delivery of drugs, gene, imaging agents etc. as well as nanoparticle-based diagnostics for various respiratory diseases. several preclinical and clinical investigations revealed that nanocarrier-based systems address many limitations of conventional therapy not only by site-specific delivery of therapeutics at the lung tissue, but also reducing the drug availability into other organs thereby reducing adverse side effects. besides, nanocarrier based systems demonstrated sustain and control release of the therapeutics than the burst release observed in systematic delivery of therapeutics in lungs. moreover, nanosized carriers have a potential to overcome the mucus barrier and poor lung penetration associated with various respiratory diseases. similarly, recent development of various nanoparticle-based detection methods for coronavirus infection showed a great promise in the development of time-economic diagnosis of covid-19 disease. while such nanoscale systems promise new therapeutic options for respiratory diseases, still the most challenging task is their safety assessment. preparation of an appropriate size of nanoparticles in each batch of synthesis is also challenging. many such challenges need to be overcome in order to translate the nanotherapeutics into clinical practice. though nanotechnology can find a way for further application of nanotherapeutics against the covid-19 diseases, still more research needs to be conducted for evaluation of nanosized therapeutics for covid-19. it has been considered that the effective vaccine against covid-19 will be available in 12-18 months. therefore, early diagnosis, effective treatment etc. are essentials to mitigate the spread of this infection before any clinically approved vaccine comes in the market. finally, the following should be mentioned. because the major result of covid-19 infections is acute respiratory distress syndrome, the discussed nanomedicines for treatment lung hypoxia and fibrosis potentially can be used for the treatment of covid-19 in combination with other antiviral actions. such pilot investigations have been recently initiated in our laboratory. european lung white book theranostics sub-cell drug discovery today molekulare virologie theranostics 2020 this work was supported in part by grants (r01 ca238871 and r01 ca209818) from the national institutes of health (nih). some figures were created with biorender.com and using servier medical art images, which are licensed under a creative commons attribution 3.0 unported license (https://smart.servier.com). key: cord-301935-0qjo94ty authors: varma, ratna; soleas, john p.; waddell, thomas k.; karoubi, golnaz; mcguigan, alison p. title: current strategies and opportunities to manufacture cells for modeling human lungs date: 2020-08-22 journal: adv drug deliv rev doi: 10.1016/j.addr.2020.08.005 sha: doc_id: 301935 cord_uid: 0qjo94ty chronic lung diseases remain major healthcare burdens, for which the only curative treatment is lung transplantation. in vitro human models are promising platforms for identifying and testing novel compounds to potentially decrease this burden. directed differentiation of pluripotent stem cells is an important strategy to generate lung cells to create such models. current lung directed differentiation protocols are limited as they do not 1) recapitulate the diversity of respiratory epithelium, 2) generate consistent or sufficient cell numbers for drug discovery platforms, and 3) establish the histologic tissue-level organization critical for modeling lung function. in this review, we describe how lung development has formed the basis for directed differentiation protocols, and discuss the utility of available protocols for lung epithelial cell generation and drug development. we further highlight tissue engineering strategies for manipulating biophysical signals during directed differentiation such that future protocols can recapitulate both chemical and physical cues present during lung development. end-stage lung disease is the third leading cause of morbidity and mortality worldwide events that occur, that differentiation models attempt to mimic, and highlight how human lung embryology has served as the blueprint to create the common pathway of lung directed differentiation protocols. we then discuss the evolution of directed differentiation protocols to find opportunities for creating specific populations of airway and lung epithelia through targeted manipulation of key signaling pathways in 2d and 3d models. we further describe how these models have been used to recapitulate different airway and lung diseases. finally, we discuss how tissue engineering and biophysical cues using biomaterials can be utilized during lung directed differentiation to mimic patterning cues present in development to augment current differentiation protocols. directed differentiation protocols have been designed to mimic in vivo human lung development [39] . indeed, in vitro models of lung development have provided unique insight into human lung development [40] . as human lung development has been described at great length in earlier reviews, [41, 42] , we provide a brief overview as follows (schematically represented in figure 1 ). during early embryogenesis (at 14 days post fertilization), a process called gastrulation begins with the appearance of a structure called primitive streak, through which cells migrate to form the primary embryonic germ layers (definitive endoderm, mesoderm, and ectoderm) [43] [44] [45] . definitive endoderm expands, thereby forming the primitive gut tube comprised of three endodermal regions: foregut, midgut, and hindgut [46, 47] . this is when lung development begins, at approximately four weeks into embryonic life, with the outgrowth of foregut endoderm [48, 49] and continues through eight years of post-natal life [50] . there are five stages to lung development: 1. embryonic (weeks 4-7): the future lung buds emerge from the ventral side of the primitive foregut endoderm into the surrounding mesenchyme and develop into embryonic lung buds with early trachea and bronchi [51] [52] [53] [54] [55] . 3 . canalicular (weeks [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] : development of the respiratory, or gas-exchanging, airways is initiated, primitive alveoli form, and the future distal epithelium begins to thin as distal epithelial markers are expressed [41, 57] . 4 . saccular (weeks [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] : emergence of sac-shaped distal airways, which develop crests with muscle and elastin to create indentations. these distal airways extend to form alveoli by 29 weeks [58] . the developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange and further differentiation of alveolar epithelial cells (aec) i and ii takes place. septation leads to an increase in surface area of the gas exchanging portion of the developing lung and prepares the fetus to breath air during this stage [50, 59] . during the embryonic period, early lung is genetically defined by the expression of transcription factor nk2 homeobox 1 (nkx2.1) and sry-box 2 (sox2) [60] [61] [62] . during human lung development, it has been found that the lung buds and branches given off during the pseudoglandular period are mostly sox2 + sox9 + [17, 62, 63] . both sox2 and sox9 are individual markers of the early proximal or distal lineage, respectively [60, [64] [65] [66] . over the course of the canalicular and saccular periods of development (weeks , these double positive populations downregulate one sox protein and maintain expression of the other as these cells mature towards proximal or distal lineages [62] . the proximal airway (closer to the mouth) is comprised of a pseudostratified columnar epithelium that is responsible for the conducting airway function: debris and pathogen removal (ciliated cells), mucus production (goblet cells), prevention of airway inflammation (club cells), and humidification of air as it passes through to the distal lung compartment [67] [68] [69] . the squamous distal epithelium, j o u r n a l p r e -p r o o f composed of alveolar epithelial cells (aec i and ii), facilitates the respiratory function of the lung as air in the epithelial compartment is brought into close apposition to blood from the pulmonary vasculature; it also secretes surfactants, which play an immunologic role and decrease the surface tension present at the air-liquid interface, thereby preventing alveolar collapse [70] . in humans, a number of cell types are found in the proximal airway, each identified with specific markers ( table 1) [71, 72] . one mechanism by which lung epithelia begin to mature is based on chemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [73] . key players including fibroblast growth factors (fgfs) [64, [74] [75] [76] [77] [78] [79] [80] , wnts [81] [82] [83] [84] , and bone morphogenetic proteins (bmps) [85] [86] [87] [88] [89] [90] are known to induce the differentiation of early lung progenitors in a controlled manner. for example, in mouse, it has been found that fgf10 plays a role in bud outgrowth [77] and drives lung progenitors towards a distal fate [78, 79] through canonical wnt signaling [64, 81, 91] . proximal epithelia develop because they are located further away from distally located fgf reservoirs in the mesenchyme, in a mechanism that appears dependent on concentration gradients [64] . bmp4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventral (back to front) and proximodistal (top to bottom) patterning in the nascent lung [88] . bmp4 is also present at high levels in distal bud tips and epithelia including aec ii cells [88, 89] , however, its inhibition promotes a proximal fate and, along with bmp2 inhibition, ciliated cell development [87, 88, 90] . j o u r n a l p r e -p r o o f while the cell fate of early proximal and distal lineages is directed through chemical signals, the lung epithelium itself undergoes marked changes in architecture, a process known as branching morphogenesis [79, 92] . from the simple tube of the anterior foregut endoderm to the complex tubular structure of the adult, a highly stereotyped mechanism of branching morphogenesis facilitates the outgrowth, division, placement, and structure of lung airway [42] . branching morphogenesis of the lung is driven by three simple and iteratively used processes: domain branching, planar and orthogonal bifurcation [93] . the first form of branching is domain branching: along a primary branch, buds form in a linear and sequential fashion, from proximal to distal. the next form of branching is planar bifurcation, in which the tip of the forming tube bifurcates to create two new tips, which subsequently elongate and bifurcate again, creating four tips. the last process of branching is known as orthogonal bifurcation. in this process, the initial planar bifurcation is followed by a rotation around the planar axis which creates two new tips through bifurcation. a critical gene in this process, sprouty, has been found to attenuate erk1/2 signaling, thereby altering the orientation of cell division and future tube elongation [94] . other critical genes and regulatory networks associated with fgf signaling also contribute to controlling the periodicity of the branched network [95] . although elements such as domain specification, bifurcation, rotation and branch generation remain largely undetermined [93, 96] , new technologies involving high-resolution live imaging, tension sensing, and force-mapping are opening paths to further explore and explain the branching morphogenesis phenomenon [97] . the early structure of the lung gives rise to a striking architectural separation of future sox2 + proximal lineages and sox9 + distal lineages, at least in mice [98] . the diameter of tube generated during branching morphogenesis in the pseudoglandular and canalicular stages has a small degree of variance within each stage as measured from electron micrograph sources of fetal human tissue [99] . this suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on the developing epithelia. once the basic organ structure has formed, the lung continues to be exposed to mechanical cues as it continues to mature. in several cases, these cues have been shown to be essential for correct organ function. in utero, the fetal lung is a secretory organ that only converts to an absorptive one, to prepare for breathing after birth, through a change in the activity of j o u r n a l p r e -p r o o f chloride and sodium channels late in development. fetal lung secretions result in a static fluid pressure of around 2.5 cmh 2 o in the developing terminal sacs of the fetus, which propels branching morphogenesis outwards into the developing thoracic cavity [100, 101] . lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequently results in the creation of smaller than normal lungs (pulmonary hypoplasia) [102] , highlighting the importance of this mechanical pressure during lung development. in addition, cyclic strain is generated from fetal breathing movements (fbm) in utero that prime the airway for use after birth. fbm are detectable from the tenth week of pregnancy and begin as infrequent and erratic activity with long quiescent periods. as development continues, these quiescent periods decrease and sustained periods of fetal breathing occur. these breathing movements vary with the fetal sleep cycle and can be chemically tuned [57] , and alter the volume of terminal sacs by around 5% [100, 103] , again highlighting the importance of mechanical signals influencing lung development. finally, a novel fgf10/fgfr2-dependent tensional mechanism has been shown by which distal epithelial cells in the lung accumulate motor proteins at the apex of the cell, thereby becoming resistant to compression from increasing fluid pressure within the tube lumen. cells under this tension are more likely to become aec ii cells, while those under compression become aec i cells [102] . interestingly, while the above examples highlight the importance of specific mechanical signals in the growth, development, and differentiation of the lung, psc directed differentiation protocols of the lung are primarily based on mimicking the sequential chemical changes that occur during lung development. 26, 62, 63] , reveals that sox2 + sox9 + progenitors are common in the developing lung buds and that branch tips of the pseudoglandular staged lung give rise to both proximal and distal epithelia [63] . moreover, specific protein markers have been found to differ in both timing and location of expression between human and mouse models: pro-spc in mouse is expressed early and throughout the developing mouse epithelium [117, 118] , while in human, pro-spc is rarely detected early in development and is only robustly found later in distal epithelia [63] . these examples highlight that, while there are similarities, development and patterning of mouse and human lungs is different, and these differences require human models to be fully appreciated. human psc protocols have generally followed the same differentiation chronology as that of mouse directed differentiation, wherein definitive endoderm, anterior foregut endoderm, progenitors. in all cases, these lung progenitors are then either sorted or directly guided towards proximal or distal progeny in 2d or 3d culture systems. ideally, products of directed differentiation protocols should mimic the cell proportions present in human airways and lungs (table 1) , however current protocols have not progressed that far. while these protocols continue to be refined, the percentage of select cell populations generated from these protocols have been summarized in table 2 . protocols to create proximal lung epithelia have focused on the production of the four major cells types present: ciliated, goblet, club, and basal cells (see table 1 for a summary of markers for each cell type). motivation for creating proximal epithelia in the field has primarily been to develop patient-specific cystic fibrosis (cf) models [11, 24, 27] and/or to produce epithelia with multi-ciliated cell populations for protocol validation [30, 33] . a shift towards human psc-derived cf models has been critical as mouse models do not accurately represent j o u r n a l p r e -p r o o f journal pre-proof cf disease progression and phenotypes seen in humans [128] [129] [130] . as such, the first evidence of human psc proximalization using cf patient-derived pscs was shown by mou non-lung hepatic cells (serpina1 + , sox9 + , apoa2 + , afp + ) [27] . multi-ciliated cells were only observed within spheroids when notch signaling was inhibited, at the expense of downregulated scgb1a1 expression, or when spheroid cells were further cultured in ali conditions. other mature epithelial populations including club and goblet cells were not assessed post ali culture. it is unclear whether 2d or 3d culture systems resulted in more representative proximal populations, although it is worth noting that the 3d spheroids could be manipulated to produce a variety of proximal epithelia ranging from progenitor to differentiated populations. the most recent approach, described by de overall, the aforementioned studies differed drastically from each other with regards to the timing and chemical modulation of each phase of differentiation towards proximal epithelia, and consequently produced variable results. while it is evident that 3d culture augments maturation, no protocol to date has been able to efficiently produce all functional epithelial populations present in the airway in proportions representative of those in vivo. furthermore, these studies have not thoroughly elucidated the mechanisms of proximal patterning. barring the application of fgf18 [11] (known to enhance proximal programming [133] ), protocols have adopted growth factors based on trial and error without understanding why, for example, fgf10 signaling (which is known to favor distal lung development) promotes production of proximal progenitors [11, 27, 30, 33, 37, 119] . as such, the quest for obtaining mature airway progenitors, such as ngfr + cells, comes at the cost of elongated protocol lengths, heterogenous maturation levels of resulting populations, and missed opportunities for understanding why these populations do not result in a histologically appropriate epithelium [29] . it is apparent that the timing of signaling molecule delivery as well as the competence of subjected cell populations to respond to a given signaling molecule are of extreme importance. the spatiotemporal dynamics of cell signaling are non-linear, are more complex in vivo, and are not fully appreciated in the latter stages of current directed differentiation protocols. this may explain the incongruence amongst different protocols, primarily those assessing the effects of gsk3î² and wnt signaling [27, 29, 30] , all of which targeted populations at non-comparable protocol stages. therefore, a deeper analysis is required to appropriately explain and mimic these dynamics in vitro. furthermore, recreating these spatiotemporal signaling patterns during directed differentiation protocols may potentially require repurposing molecular delivery tools from other fields such as drug delivery and tissue engineering [134] [135] [136] . interestingly, most existing protocols have been skewed towards generating multi-ciliated cells at the expense of goblet and club cells by subjecting airway progenitors to notch inhibition, which is known to decrease goblet cell populations [137, 138] . goblet cells, in j o u r n a l p r e -p r o o f addition to club cells, have recently been discovered as a source for generating multi-ciliated cells in primary airway epithelia [139] . club cells play a key role in epithelial injury, wherein they de-differentiate into basal cells in the absence of basal cells such that they can give rise to ciliated and club cell populations to repair a denuded epithelium [140] . therefore, in the future it will be critical to identify protocols to create psc-derived cultures containing these cell types, and not just multi-ciliated cells, in order to fully capture the dynamics of airway injury and repair for drug screening. overall, based on current progress, the konishi et al. and mccauley et al. protocols are considered the most relevant for generating functional airway epithelia. the alveolar space in the distal lung is comprised of two epithelial cell types: aec i and aec ii (see table 1 for specific markers of each cell type meanwhile, progenitor-like sox9 + spc + and sox9 + hopx + clusters were prominently present with minimal mature spc + (5%) and hopx + (4%) populations. further refinement of this protocol bifurcated proximal "human lung" and distal "bud tip progenitor" organoid development by culturing foregut spheroids in fgf10 with 1% serum or fgf7, chir, and ra in serum-free media, respectively [121] . after 65 days of culture, the "human lung" organoids expressed p63, foxj1, and mesenchymal markers with no sign of mature epithelial features; some spc and hopx staining was also observed. only after an 8 week-long in vivo implantation did mature ciliated actub + cells appear. "bud tip progenitor" organoids also contained heterogenous muc5ac + , hopx + , spb + , and spc + cells after 120 days. however, when seeded into naphthalene-injured mouse airways, they gave rise to actub + and muc5ac + cells. in general, this protocol diverged to produced lung organoids with heterogeneous populations of either predominantly proximal or distal epithelia, which required prolonged culture or in vivo implantation for maturation (limited in this case). a key aspect of the "human lung" organoids was their inclusion of a mesenchymal population to study epithelialmesenchymal crosstalk during lung development. implantation to promote maturation (incomplete in this case). all described directed differentiation protocols for distal epithelia utilized a 3d culture approach in some format, however only those that established lung specification in 2d culture prior to a 3d transition demonstrated promising results. these protocols do not completely depend on spontaneous organoid assembly, are highly responsive to fine tuning with morphogens, and can therefore provide better insight into the cellular responses in lung development to generate therapeutic strategies accordingly. although the "human lung" organoid and "lung bud progenitor" organoid-based protocols may be useful for studying complex cellular interactions during disease progression or furthermore, the employment of notch inhibition during the "preconditioning" phase may have played a role in promoting both aec i and aec ii populations, and therefore the aforementioned signaling pathways need to be collectively assessed. inspiration can be sought from a recent study which described a computational modeling approach, based on single cell rna sequencing, that predicted the optimal time point for chir withdrawal for maintaining a nkx2.1 + spc + lung fate; its findings were further supported by empirical studies [35] . employment of such techniques will prove essential for understanding fate choice and developing customized target lung populations. the study of airway and lung diseases is limited by animal models as they do not recapitulate human disease phenotypes and progression adequately. for example, existing mouse j o u r n a l p r e -p r o o f models of cystic fibrosis (cf) vary greatly in their ability to represent relevant organ pathologies and are deficient in developing spontaneous lung disease observed in humans [141] [142] [143] . similarly, pulmonary fibrosis is most commonly studied in the bleomycin-induced lung fibrosis mouse model, which results in faster disease progression, eventual resolution of disease phenotype over time, and is obscured by the wide range of bleomycin doses administered for induction of injury [144] . studies exploiting psc-derived lung culture models to explore the effect of drugs in human lung cells are therefore beginning to emerge. culture models of proximal airway epithelia have been applied for drug discovery primarily in the context of cf. cf is an autosomal recessive genetic disorder caused by mutations in the epithelial chloride channel gene, cftr, which consequently leads to accumulation of excess mucous and compromised mucociliary clearance [145] , affecting multiple organs. cf phenotypes have been studied widely in primary or psc-derived intestinal [146] [147] [148] , rectal [149] [150] [151] , pancreatic [152] , and airway models [11, 27, 33, 153, 154] . in the context of airway models, ali culture has been the gold-standard for studying primary airway epithelia derived from cf patients. in concordance with this method, wong et al. and developed an il-11 antibody which reversed late-stage lung fibrosis by significantly decreasing ecm deposition in an animal model [162] . evaluation of this il-11 antibody using j o u r n a l p r e -p r o o f psc-derived lung organoid models can provide better insight into their applicability for human disease. distal lung organoids have also been applied to model respiratory viral infections caused by respiratory syncytial virus (rsv), human parainfluenza virus type 3 (hpiv3) [163, 164] , as well as the measles virus (mev). chen and colleagues infected lbos with rsv, resulting in characteristic luminal shedding of epithelia [26] , which leads to small airway obstruction and consequent bronchiolitis in clinical settings [165] . interestingly, sach et al. demonstrated that prior incubation with palivizumab (an antibody that prevents rsv from fusing with cells) prevented rvs from replicating in primary airway organoids [154] , which would be valuable to assess in psc-derived lung or airway organoids. meanwhile, hpiv3 infected aec ii in lbos and temporally reached peak infection similar to that in primary alveolar epithelia [164] , confirming clinical data. hpiv3 infection did not result in either epithelial shedding or syncytium formation in the lbos as did rsv [26] and mev [164] infections, respectively, again confirming clinical phenotypes. this showed the ability of lung organoid models to not only demonstrate virus-specific infection, but also to recapitulate phenotypes observed in the clinic. another condition modeled by alveolar or lung organoids is spb deficiency, a lethal neonatal autosomal recessive disease which necessitates lung transplantation for patient survival. while there has been great progress in the establishment and maturation of lung epithelia from psc populations, a number of limitations have emerged that will require optimization and augmentation of current protocols to create better developmental and disease models, and specific cell populations: a. lack of control over which populations are produced -understanding or recapitulation of signaling pathways beyond proximodistal patterning is currently limited, as the ratio of aec ii versus aec i cells; or club cells versus goblet cells cannot be reliably predicted. furthermore, while development of reporter lines and identification of surface markers for sorting have [32, 34] helped the advancement of distal lung protocols (for aec ii cells specifically) in the last few years [28, 36] , such techniques are limited in current proximal airway protocols [168] . f. minimal recapitulation of physiological conditions -current alveolar organoids are beginning to represent relevant cell types, however, they are embedded in matrigel and grown in submerged culture. they, therefore, fail to provide an ali environment, which is critical to in vivo functionality. as in proximal protocols, cell products often need to be dissociated and regrown on transwells, which allow ali culture by exposing cells to media basally and air apically, for further assessment. g. high cost -associated with the growth factor and small molecules required for chemically directed stem cell differentiation, and the expertise required to reliably create lung epithelia with these protocols. the use of commercially available 2d based endoderm differentiation kits have greatly decreased the level of expertise required to achieve this early stage of differentiation. based on current advances, we have made recommendations for directed differentiation protocols that generate proximal and distal epithelia in figure 2 . the timing of chemical signals present during lung development has been well mimicked in current differentiation protocols. the lung, however, develops in response to chemical signals within a highly dynamic mechanical environment of cyclic strain, pressure and a complex branching tubular architecture [169] . indeed, it is well established that mechanical cues can impact progenitor cell fate [102, [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] and emerging evidence suggests that the mechanical environment can be manipulated to produce predictable fate choices in stem and progenitor cells [181] [182] [183] [184] . for example, the importance of biophysical manipulation associated with tissue has been exploited only to a limited extent to augment and guide directed differentiation protocols to address some of the above limitations [170, [185] [186] [187] . organoid cultures, for instance, allow self-assembly of tissue-like structures and enable further maturation of proximal and distal epithelia [12, 26, 28, 119] . in this section of the review, we highlight tools from tissue engineering that have been used to manipulate tissue structure and the resulting biophysical signals experienced during stem cell differentiation in both 2d and 3d (figure 3 and table 3 ). furthermore, we explore the opportunity to utilize such tools to engineer mechanical signaling as a strategy to augment and refine existing chemical differentiation protocols. note, we do not consider the use of simply culturing differentiating cells on substrates or in 3d hydrogels with variable mechanical stiffness but point the reader to excellent reviews on this subject [188, 189] . micropatterning of the culture surface is one strategy that has been used to manipulate the physical organization of 2d stem cell colonies and the resulting mechanical environment individual cells experience within the cell sheet. micropatterning entails deposition of j o u r n a l p r e -p r o o f extracellular matrix (ecm) protein islands with highly specific shapes and sizes on non-adhesive surfaces, via micro-contact printing (âµcp) [190] [191] [192] [193] [194] or soft lithography [195] [196] [197] . individual cells or cell populations are thereby restricted to the area of the surface where the adhesive protein islands are present. the shape of the island, therefore, geometrically constrains the shape of single and groups of cells in 2d, which determines the pattern of adhesive attachments between the cells and the underlying surface, and hence the mechanical state of the cells [198] . chen and colleagues presented early evidence that geometric constraint affects cell fate by demonstrating that cell growth and apoptosis are directly related to ecm pattern size through its control of cell spreading [194, 199] . not only is the size of the ecm pattern important, but also the shape it holds, specifically in relation to its aspect ratio and subcellular curvature. as shown by kilian et al., despite the presence of equipotential differentiation signals, osteogenic differentiation of heterogeneous mesenchymal stem cells (mscs) was promoted by increasing the ecm pattern aspect ratio at the single cell level [181] . further, in a pentagon-shaped design, the curvature of the lines connecting vertices was varied from convex to concave and was shown to guide cell differentiation choice from adipogenic to osteogenic, respectively, by manipulating subcellular myosin ii polarization, tension, and integrin localization. evidently, such micropatterned islands not only exert control over the growth and survival of cells, but also enable manipulation of cell differentiation through changes in intracellular tension. for example, by probing tension at the cell-boundary interface through confined 2d geometries, lee et al. found that patterned melanoma cancer cells occupying larger arc angles, or smaller magnitudes of curvature, expressed higher cancer stem cell markers [195] . furthermore, these markers were preferentially found at the edge of the micropattern, a consequence of perimeter tension acting through the p38-map kinase pathway. interestingly, once removed from the defined geometric environment, the cells lost their activated cancer phenotype. these tools could provide an excellent platform for subtly manipulating self-organization of psc populations to understand and influence their differentiation. while not in the context of augmenting directed differentiation specifically, the use of micropatterning has been applied to explore pluripotency and fate choice during early development. based on a previous finding that human psc differentiation is dependent on colony size [200] , nazareth et al. developed a high throughput âµcp platform, with optimized j o u r n a l p r e -p r o o f colony size, and probed early cell fates (pluripotent, neuroectoderm, primitive streak, and extraembryonic) in response to different media conditions and developmental factors [190] . such micropatterned surfaces were also used by warmflash and colleagues to investigate embryonic germ layer patterning of human pscs [201] . they found that bmp4 treatment results in spatially segregated regions that delineate ectoderm, primitive streak and trophoblast-like tissues within the patterned colonies. this pattern was shown to be mediated by the colony edge, as opposed to colony size, with bmp4 signalling progressively being restricted to the edge of the colony. these findings were further confirmed by tewary et al., who explained this effect to be caused by the emergence of a phosphorylated smad1 gradient. the establishment of this gradient is initially controlled via inhibition by noggin, followed by a restriction of bmp4 sensitivity to the edge of the colony due to re-localization of bmp receptors throughout the rest of the colony [202] . such micropatterned platforms have also been used to map fate choices made during mouse [203] and human [204] gastrulation events, and therefore are a powerful tool for elucidating fate choice during lung development. it is not clear however, how the mechanical state of the cells within these micropatterned islands impacts chemical cue secretion, and hence the local gradients of chemical signals that result in patterning of cell fate in these studies. another method to manipulate the physical organization and biophysical cues in a differentiating cell sheet is through substrate texture [205] . cellular behaviours including proliferation, adhesion, and differentiation have been linked to underlying substrate topographical cues [205] [206] [207] [208] [209] [210] . these cues are recognized by cellular protrusions called filopodia and lamellipodia, through integrin receptors and focal adhesions [211] [212] [213] [214] , which in turn dynamically modify their shape and exert protrusive forces [215] [216] [217] [218] [219] . in this section, we will provide key examples of substrate topographies, as well as grooves, based on their relevance for lung epithelial organization. these topographies can be microfabricated using various techniques, such as etching, photolithography, soft lithography, and stereolithography, that are scalable, precise, and provide high fidelity [220] . j o u r n a l p r e -p r o o f stem cell fate choices have been shown to respond to topographical features [221, 222] . for example, viswanathan et al. assessed the ability of different topographies to mimic sinusoidal epidermal undulation to induce in vivo-like biophysical cues. their screened undulating topography created î²1 integrin patterning that is reminiscent of the human dermis and more differentiated cells were found localized to the troughs of their pattern in a highly repeatable fashion. these findings suggest that replicating the physical organization of the dermal microenvironment promotes tissue-level organization and alters the positioning of the epidermal stem cells towards the in vivo state [223] . this group further applied a screening platform called topochip [224] , that incorporated features of varying sizes, roundedness, and distribution density, to assess human psc proliferation and pluripotency in the absence of ecm coatings [225] . topographies that ranked high in the screen not only supported psc proliferation, but also allowed maintenance of oct4 and sox2-expressing pluripotent colonies. in conjunction with computational modeling, this platform was able to predict topographical features conducive to maintaining psc pluripotency, thus demonstrating great promise for exploring how to use tissue organization patterning to control cell fate. application of this platform for probing keratinocyte differentiation revealed that differentiation is linked to changes in cell morphology, which is influenced by substrate topography, and mediated by rho kinase activity [226] . based on these studies, it is evident that application of biophysical cues alone can impact differentiation. high-throughput technologies like topochip could be used in the future to understand and mimic cell fates of proximal or distal lung epithelia. towards this goal of recapitulating the physiological morphology of distal lung alveoli, we recently developed largersized topographical features, specifically hemispherical cavities, that enabled seeding of multiple cells and further allowed maintenance of primary aec i and aec ii cells [227] . the ability of this platform to promote psc differentiation towards these cell types has yet to be explored, however. grooved topographical cues specifically, through their ability to modulate cytoskeletal alignment and cellular shape, have also demonstrated great promise in guiding cell fate [228, j o u r n a l p r e -p r o o f 229]. in the context of neural differentiation, ankam and colleagues generated a multiarchitectural chip (marc), that incorporated a range of isotropic and anisotropic topographies at both micro and nano scales, to differentiate pscs towards neural progeny without the use of embryoid bodies. anisotropic nanoscale grooves (250 nm) promoted neuronal differentiation with cell alignment and elongation, and isotropic pillars enhanced astrocyte differentiation with cellular branching within 7 days of culture. meanwhile, conventional culture protocols were unable to induce these populations without additional culture steps and/or prolonged culture up to 30 days [230] . neural differentiation on nanogrooves was attributed to actomyosin contractility via vinculin-associated focal adhesions [231] . marc further enabled investigation of nuclear morphology and histone methylation [232] , thereby exemplifying that such platforms can allow exploration of the mechanism of biophysical cues translating to dna modulation during differentiation. the influence of groove topography on differentiation has been highlighted in other contexts as well. abagnale et al. developed a micro-grooved chip, incorporating systematic variation of groove widths and ridges, to study msc differentiation towards adipogenic and osteogenic progeny. while wider ridges led to higher adipogenic differentiation with formation of fat droplets, thinner ridges enhanced osteogenic differentiation with calcium phosphate precipitation [233] . interestingly, groove width had minimal impact on favouring differentiation towards either lineage. the ridge-mediated differentiation effect was associated with cell morphology and focal adhesion formation wherein wider ridges resulted in rounder cell morphology with many large focal adhesions, as compared to thinner ridges leading to cellular elongation with fewer and smaller sized focal adhesions. nano-scale groove topography was also shown to alter the spatial conformation of psc colonies by elongating them, consequently affecting cell fate [234] . this effect was particularly potent at the colony edges and controlled by separate and differential localization of yap and taz during psc maintenance and differentiation. in general, grooved topography can provide great insights into psc fate choice and potentially expedite differentiation protocols. the marc platform illustrates the importance of combining biophysical and biochemical cues, especially as exposure to topography induced a higher yield of functional and mature progeny within a short time frame as compared to flat j o u r n a l p r e -p r o o f substrates or standard directed differentiation protocols [230, 235] . this is of extreme importance as current directed differentiation protocols for airway and lung epithelia require longer than 60 days of culture to achieve functional cell types [12, 27, 28, 30, 36, 119] . although the inclusion of topography has not been investigated for promoting lung differentiation, we have explored grooved substrates for aligning airway epithelia during differentiation to achieve coordinated unidirectional ciliary beating [236] . our unpublished data demonstrates that while primary human basal-derived epithelia lose their alignment on grooved topography over time, epithelia generated from human psc-derived airway progenitors maintain their alignment throughout differentiation in ali culture. while 2d cues have enabled the community to clearly demonstrate the capability of biophysical cues to manipulate cell fate, 2d approaches are limited in their biological applicability as most physiologic physical and mechanical cues occur in 3d environments [237] . [186] . further, these microchambers were validated for use in developmental drug toxicity screening, through which they were able to represent thalidomide embryopathy by exhibiting diminished contractility, beating frequency, and size of cardiac chambers. geometric constraint was also applied to cerebral organoids by lancaster and colleagues to minimize variability associated with neural induction efficiency. this entailed addition of a j o u r n a l p r e -p r o o f physical cue in the form of polymer microfibres, around which the organoid self-organized [170] . in conjunction with an established chemical protocol, these microfibres served to pattern developing organoids leading to recognizable neuronal features including a cortical plate, radial units, along with organized radial neuronal migration in a reproducible manner. this addition of a simple physical cue substantially increased the patterning and organization of brain organoids compared to those derived from protocols only relying on biochemical cues. another strategy to control cell and tissue geometry in 3d is the use of micromoulding to create defined mechanical microenvironments which in turn alter cell and tissue level organization and differentiation. this approach was first developed by nelson et al. using 3d collagen moulds to study branching morphogenesis of mammary epithelial cells [238, 239] . seeded mammary epithelia conformed to the 3d architectures, forming hollow tubules, and demonstrated predictable branching patterns according to mould geometry and presence of inhibitory morphogens. this technique was further used to understand the mechanism of cellular rearrangement in mammary ducts [240] and exhibit that mechanical stress gradients control the pattern of branching morphogenesis [241] . inspired by this method and its applicability for studying lung branching morphogenesis, we developed tubular constructs of physiologically relevant diameters to guide self-assembly of lung progenitors [242] . using this approach, we demonstrated that specification of these bipotent sox2 + sox9 + lung progenitors was dependent on geometry, wherein tubes of 100 âµm diameter led to a distal sox9 + fate, while 400 âµm diameter tubes remained in a sox2 + sox9 + lung progenitor state. the mechanism of this effect was dependent on canonical wnt signalling, and due to differences in cellular tension induced by patterning the progenitor cells into a 3d tube structure. while the addition of mechanical cues influences fate choice, its role in inducing cell functionality, especially at the organoid level, needs to be elucidated. currently, there is scant evidence of lung directed differentiation being manipulated in a 3d context [183, 242, 243] . beyond our exploration of patterning early lung fates through micromoulding, dye et al. have recently applied tissue engineering techniques during lung organoid formation. in their case, foregut endoderm spheroids cultured on highly degradable synthetic polymers demonstrated enhanced ability to differentiate into proximal airway epithelia after in vivo implantation [183] . evidently, the field of lung directed differentiation is in its nascent stages for using biophysical in the future, human psc-derived lung tissue models have the potential to enable exploration of infection, disease and regeneration mechanisms of action to impact drug discovery and drug development, and further inform patient-specific drug selection. while lung models remain in their infancy, the investment necessary to translate such models into practical use is worthwhile given that they offer a number of key advantages over primary cells or mouse models. firstly, psc-derived platforms enable modeling of human disease. another major advantage of psc-derived cells, specifically in the context of lung, is the potential to directly associate specific patient genetics and cell phenotypes with clinical conditions, as is underway in the field of lung cancer [244, 245] . this will avoid complications associated with prior exposure of primary cell donors to a plethora of environmental (such as smoking) and pharmaceutical stimuli. furthermore, establishing models specifically from psc sources potentially enables generation of the large number of cells and cell types necessary for personalized disease modeling. a number of challenges exist however, to translate these models into widespread use for drug discovery and development. one major challenge in the field, highlighted in this review, is the standardization of robust cell manufacturing protocols. lung epithelial models require not only large cell numbers, but also the correct proportion of cell types. additionally, for a variety of functional read-outs, these cell types must be appropriately spatially organized. therefore, standardized protocols are needed to both manufacture lung cells and assemble these cells into reproducible and clinically representative "lung tissues" at the scales required for screening. this will be essential to enable the generation of in vitro lung test tissues with sufficiently low batch-to-batch and within-batch variation for screening with high reproducibility. towards this challenge, methods will continue to emerge to control psc differentiation into the different proximal and distal lung cell types. for example, in this review, we have highlighted the emerging evidence that an opportunity exists to further improve differentiation control and disease modeling by mimicking mechanical cues experienced during development. beyond the strategies described in previous sections, this concept could be expanded further in the future to mimic additional aspects of lung development. for example, the developing lung is exposed to variations in oxygenation [246, 247] , which is known to impact cell fate choices [248] [249] [250] , therefore the use of optimized oxygenation levels could be an attractive and easily scalable strategy to further refine directed differentiation culture protocols. the developing lung is also subject to various other physical cues at the organ-scale including 1) pressure from amniotic fluid, which serves to expand the nascent alveolar compartment; 2) fetal breathing movements that provide a stretch-based physical cue which serves as a maturation signal; and 3) pulsatile flow from the extensive vascular network present throughout the organ. techniques that mimic these forces to control lung cell fate are emerging. these include the use of shear to produce relatively homogenous populations of aec i and ii [251] , the use of cyclic mechanical stretch [252] , and the use of patterned hydrogels to enable perfusion of lung-type structures [253] . scaling some of these complex mechanical setups to enable large scale efficient manufacturing, however, could be a challenge. the challenge of assembling lung cells into reproducible arrays of "lung tissues" is starting to be addressed by emerging high-throughput techniques that seek to purify specific cell populations [254] [255] [256] that could be later mixed in controlled ratios to generate precise tissue compositions. bioprinting [257] [258] [259] and cellular assembly efforts, including organoid fabrication through dna programming [260] , could also potentially prove useful to enabling complex tissue assembly in a manner that is adequately scalable and reproducible for screening. establishing such "ground-truth" benchmarking data sets to validate the ability of in vitro models to distinguish both positive and negative hits will be absolutely critical to establish confidence in lung culture models and to ensure wider spread community adoption and impact. j o u r n a l p r e -p r o o f table 3 . highlights of mechanical cues influencing cell fate. *high throughput not shown in the paper but could be easily developed. mortality in the united states chronic respiratory diseases global mortality trends, treatment guidelines, life style modifications, and air pollution: preliminary analysis global and regional trends in copd mortality collaborators, global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study outcomes after lung transplantation pharmacologically-induced mitotic synchrony in airway epithelial cells as a mechanism of action of anti-inflammatory drugs toxicological comparison of cigarette smoke and e-cigarette aerosol using a 3d in vitro human respiratory model mechanical forces induce an asthma gene signature in healthy airway epithelial cells small airway-ona-chip enables analysis of human lung inflammation and drug responses in vitro a human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional cftr protein long-term expansion of alveolar stem cells derived from human ips cells in organoids limitations of animal studies for predicting toxicity in clinical trials: part 2: potential alternatives to the use of animals in preclinical trials optimization of normal human bronchial epithelial (nhbe) cell 3d cultures for in vitro lung model studies, sci rep cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies mst1/msp as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas regeneration of the entire human epidermis using transgenic stem cells human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of acta2, sox2, and sox9 introduction to stem cell therapy a pure population of lung alveolar epithelial type ii cells derived from human embryonic stem cells derivation of distal airway epithelium from human embryonic stem cells generation of lung epithelial-like tissue from human embryonic stem cells efficient derivation of purified lung and thyroid progenitors from embryonic stem cells generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells generation of multipotent lung and airway progenitors from mouse escs and patient-specific cystic fibrosis ipscs a three-dimensional model of human lung development and disease from pluripotent stem cells efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of wnt signaling glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3d culture directed induction of functional multi-ciliated cells in proximal airway epithelial spheroids from human pluripotent stem cells in vitro generation of human pluripotent stem cell derived lung organoids generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells prospective isolation of nkx2-1-expressing human lung progenitors derived from pluripotent stem cells trajectories define lineage plasticity windows during differentiation of human psc-derived distal lung progenitors derivation of self-renewing lung alveolar epithelial type ii cells from human pluripotent stem cells single-cell transcriptomic profiling of pluripotent stem cell-derived scgb3a2+ airway epithelium the cellular and physiological basis for lung repair and regeneration: past, present, and future stem cells: a recapitulation of development understanding human lung development through in vitro model systems lung organogenesis lung development: orchestrating the generation and regeneration of a complex organ adapting the 14-day rule for embryo research to encompass evolving technologies human embryo research and the 14-day rule establishment and organization of germ layers in the gastrulating mouse embryo wnt signaling: implications in endoderm development and pancreas organogenesis morphogenesis and maturation of the embryonic and postnatal intestine patterning and plasticity in development of the respiratory lineage regulation of early lung morphogenesis: questions, facts and controversies langman's medical embryology developmental biology of the pulmonary circulation early endoderm development in vertebrates: lineage differentiation and morphogenetic function foregut separation and tracheo-oesophageal malformations: the role of tracheal outgrowth, dorso-ventral patterning and programmed cell death origin, differentiation, and maturation of human pulmonary veins transformation of the aortic-arch system during the development of the human embryo development of the intrasegmental bronchial tree: the pattern of branching and development of cartilage at various stages of intra-uterine life avery's diseases of the newborn airway and blood vessel interaction during lung development transcriptional control of lung morphogenesis multiple roles for sox2 in the developing and adult mouse trachea sox2 is important for two crucial processes in lung development: branching morphogenesis and epithelial cell differentiation vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term selfrenewing organoids localized fgf10 expression is not required for lung branching morphogenesis but prevents differentiation of epithelial progenitors the id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells sox9 plays multiple roles in the lung epithelium during branching morphogenesis airway epithelial barrier function regulates the pathogenesis of allergic asthma mechanisms of cilia-driven transport in the airways in the absence of mucus tissue engineering airway mucosa: a systematic review alveolar epithelial cells: master regulators of lung homeostasis lung organoids: current uses and future promise plasticity in the lung: making and breaking cell identity wnt and fgf mediated epithelial-mesenchymal crosstalk during lung development, developmental dynamics : an official publication of the different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung fgf10 maintains distal lung bud epithelium and excessive signaling leads to progenitor state arrest, distalization, and goblet cell metaplasia targeted expression of a dominant negative fgf receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung fibroblast growth factor 10 (fgf10) and branching morphogenesis in the embryonic mouse lung fgf-10 is a chemotactic factor for distal epithelial buds during lung development fgf-10 induces sp-c and bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium fibroblast growth factor interactions in the developing lung beta-catenin maintains lung epithelial progenitors after lung specification wnt signalling in lung development and diseases hyperactive wnt signaling changes the developmental potential of embryonic lung endoderm wnt/beta-catenin signaling acts upstream of n-myc, bmp4, and fgf signaling to regulate proximal-distal patterning in the lung smad1 and its target gene wif1 coordinate bmp and wnt signaling activities to regulate fetal lung development activation of the canonical bone morphogenetic protein (bmp) pathway during lung morphogenesis and adult lung tissue repair bmp signalling controls the construction of vertebrate mucociliary epithelia bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development niche-mediated bmp/smad signaling regulates lung alveolar stem cell proliferation and differentiation evidence from normal expression and targeted misexpression that bone morphogenetic protein (bmp-4) plays a role in mouse embryonic lung morphogenesis a comprehensive analysis of fibroblast growth factor receptor 2b signaling on epithelial tip progenitor cells during early mouse lung branching morphogenesis bmp4 and fgf10 play opposing roles during lung bud morphogenesis the branching programme of mouse lung development control of mitotic spindle angle by the ras-regulated erk1/2 pathway determines lung tube shape fgf-regulated etv transcription factors control fgf-shh feedback loop in lung branching mathematical approaches of branching morphogenesis, front genet patterned cell and matrix dynamics in branching morphogenesis two nested developmental waves demarcate a compartment boundary in the mouse lung an electron microscope study of the fetal development of human lung the effects of mechanical forces on fetal lung growth lamb fetal pulmonary fluid. i. validation and significance of method for determination of volume and volume change the strength of mechanical forces determines the differentiation of alveolar epithelial cells fetal pulmonary development: the role of respiratory movements embryonic stem cells generate airway epithelial tissue efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations efficient differentiation of human embryonic stem cells to definitive endoderm bmp-4 is required for hepatic specification of mouse embryonic stem cellderived definitive endoderm induction and monitoring of definitive and visceral endoderm differentiation of mouse es cells retinoic acid synthesis and signaling during early organogenesis distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis the in vivo genetic program of murine primordial lung epithelial progenitors epithelial progenitor cells in lung development, maintenance, repair, and disease preparing for the first breath: genetic and cellular mechanisms in lung development epithelial progenitor cells in lung development, maintenance, repair, and disease airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling the hippo pathway effector yap controls patterning and differentiation of airway epithelial progenitors lung epithelial tip progenitors integrate glucocorticoid-and stat3-mediated signals to control progeny fate embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages derivation of epithelial-only airway organoids from human pluripotent stem cells pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung-versus thyroid-lineage specification generation of lung organoids from human pluripotent stem cells in vitro smoking-dependent distal-to-proximal repatterning of the adult human small airway epithelium cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface the airway goblet cell number and proliferation of clara cells in normal human airway epithelium number and proliferation of basal and parabasal cells in normal human airway epithelium morphometric characteristics of cells in the alveolar region of mammalian lungs cystic fibrosis mouse models defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis an animal model for cystic fibrosis made by gene targeting basal cells as stem cells of the mouse trachea and human airway epithelium notch-dependent differentiation of adult airway basal stem cells fibroblast growth factor 18 influences proximal programming during lung morphogenesis microparticle-mediated sequestration of cell-secreted proteins to modulate chondrocytic differentiation a microparticle approach to morphogen delivery within pluripotent stem cell aggregates homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules activation of notch1 or notch3 signaling skews human airway basal cell differentiation toward a secretory pathway submersion and hypoxia inhibit ciliated cell differentiation in a notch-dependent manner novel dynamics of human mucociliary differentiation revealed by single-cell rna sequencing of nasal epithelial cultures dedifferentiation of committed epithelial cells into stem cells in vivo animal models in the pathophysiology of cystic fibrosis airway disease phenotypes in animal models of cystic fibrosis comparative biology of cystic fibrosis animal models exploring animal models that resemble idiopathic pulmonary fibrosis personalized medicine for cystic fibrosis: establishing human model systems ipsc-derived intestinal organoids from cystic fibrosis patients acquire cftr activity upon talen-mediated repair of the p.f508del mutation generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells stem cell-derived organoids to model gastrointestinal facets of cystic fibrosis rectal organoids enable personalized treatment of cystic fibrosis characterizing responses to cftr-modulating drugs using rectal organoids derived from subjects with cystic fibrosis a functional cftr assay using primary cystic fibrosis intestinal organoids patient-derived pancreas-on-achip to model cystic fibrosis-related disorders human nasal epithelial organoids for therapeutic development in cystic fibrosis long-term expanding human airway organoids for disease modeling idiopathic pulmonary fibrosis acute exacerbation of idiopathic pulmonary fibrosis: lessons learned from acute respiratory distress syndrome? hermansky-pudlak syndrome in vitro disease modeling of hermansky-pudlak syndrome type 2 using human induced pluripotent stem cell-derived alveolar organoids modeling of fibrotic lung disease using 3d organoids derived from human pluripotent stem cells interleukin-11 is a therapeutic target in idiopathic pulmonary fibrosis revisiting respiratory syncytial virus's interaction with host immunity, towards novel therapeutics authentic modeling of human respiratory virus infection in human pluripotent stem cell-derived lung organoids respiratory syncytial virus infection (rsv) reversal of surfactant protein b deficiency in patient specific human induced pluripotent stem cell derived lung organoids by emerging therapies for idiopathic pulmonary fibrosis, a progressive age-related disease generation of a nkx2.1 -p63 double transgenic knock-in reporter cell line from human induced pluripotent stem cells (mhhi006-a-4) invited review: mechanochemical signal transduction in the fetal lung guided self-organization and cortical plate formation in human brain organoids tissue mechanics orchestrate wnt-dependent human embryonic stem cell differentiation pulmonary epithelial cell differentiation in the nitrofen-induced congenital diaphragmatic hernia impaired alveolar epithelial cell differentiation in the hypoplastic lung in nitrofen-induced congenital diaphragmatic hernia responsive culture platform to examine the influence of microenvironmental geometry on cell function in 3d vitro model alveoli from photodegradable microsphere templates control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by rho and cytoskeletal tension, developmental dynamics : an official publication of the polarized cellular mechano-response system for maintaining radial size in developing epithelial tubes mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung coordination of receptor tyrosine kinase signaling and interfacial tension dynamics drives radial intercalation and tube elongation coordinated directional outgrowth and pattern formation by integration of wnt5a and fgf signaling in planar cell polarity geometric cues for directing the differentiation of mesenchymal stem cells human lung organoids develop into adult airway-like structures directed by physicochemical biomaterial properties induction of lung-like cells from mouse embryonic stem cells by decellularized lung matrix mechanically induced development and maturation of human intestinal organoids in vivo self-organizing human cardiac microchambers mediated by geometric confinement self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche mechanical forces direct stem cell behaviour in development and regeneration materials as stem cell regulators high-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias patterning mouse and human embryonic stem cells using micro-contact printing simple approach to micropattern cells on common culture substrates by tuning substrate wettability nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells geometric control of cell life and death interfacial geometry dictates cancer cell tumorigenicity soft lithography in biology and biochemistry patterning proteins and cells using soft lithography cell shape, cytoskeletal tension, and rhoa regulate stem cell lineage commitment using self-assembled monolayers to pattern ecm proteins and cells on substrates niche-mediated control of human embryonic stem cell self-renewal and differentiation a method to recapitulate early embryonic spatial patterning in human embryonic stem cells a stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning mapping cell migrations and fates in a gastruloid model to the human primitive streak nanostructured scaffold as a determinant of stem cell fate a graphene-based platform for induced pluripotent stem cells culture and differentiation nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions stem cell responses to plasma surface modified electrospun polyurethane scaffolds nanotopographic influence on the in vitro behavior of induced pluripotent stem cells the relationship between substrate topography and stem cell differentiation in the musculoskeletal system environmental sensing through focal adhesions nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells the effect of substrate microtopography on focal adhesion maturation and actin organization via the rhoa/rock pathway extracellular-matrix tethering regulates stem-cell fate investigating filopodia sensing using arrays of defined nano-pits down to 35 nm diameter in size investigating the limits of filopodial sensing: a brief report using sem to image the interaction between 10 nm high nano-topography and fibroblast filopodia properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components filopodia: molecular architecture and cellular functions how filopodia pull: what we know about the mechanics and dynamics of filopodia engineering microscale topographies to control the cell-substrate interface stem cell fate dictated solely by altered nanotube dimension the control of human mesenchymal cell differentiation using nanoscale symmetry and disorder mimicking the topography of the epidermal-dermal interface with elastomer substrates an algorithm-based topographical biomaterials library to instruct cell fate scalable topographies to support proliferation and oct4 expression by human induced pluripotent stem cells micro-scaled topographies direct differentiation of human epidermal stem cells design of biomimetic substrates for long-term maintenance of alveolar epithelial cells the sequence of alignment of microtubules, focal contacts and actin filaments in fibroblasts spreading on smooth and grooved titanium substrata cell guidance by ultrafine topography in vitro substrate topography and size determine the fate of human embryonic stem cells to neuronal or glial lineage actomyosin contractility plays a role in map2 expression during nanotopography-directed neuronal differentiation of human embryonic stem cells temporal changes in nucleus morphology, lamin a/c and histone methylation during nanotopography-induced neuronal differentiation of stem cells surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages surface topography guides morphology and spatial patterning of induced pluripotent stem cell colonies sequential application of discrete topographical patterns enhances derivation of functional mesencephalic dopaminergic neurons from human induced pluripotent stem cells topographically grooved gel inserts for aligning epithelial cells during air-liquid-interface culture deconstructing the third dimension: how 3d culture microenvironments alter cellular cues tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression self-organization of engineered epithelial tubules by differential cellular motility endogenous patterns of mechanical stress are required for branching morphogenesis assembly of lung progenitors into developmentally-inspired geometry drives differentiation via cellular tension development of a three-dimensional bioengineering technology to generate lung tissue for personalized disease modeling somatic mutations drive distinct imaging phenotypes in lung cancer phenotype-genotype correlation in multiple primary lung cancer patients in china the fetal circulation, pathophysiology of hypoxemic respiratory failure and pulmonary hypertension in neonates, and the role of oxygen therapy perinatal oxygen in the developing lung local lung hypoxia determines epithelial fate decisions during alveolar regeneration influence of prenatal hypoxia and postnatal hyperoxia on morphologic lung maturation in mice hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor mechanical stretch induces fetal type ii cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway multivascular networks and functional intravascular topologies within biocompatible hydrogels ultrahighthroughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells high-throughput genome-wide phenotypic screening via immunomagnetic cell sorting situ expansion, differentiation and electromechanical coupling of human cardiac muscle in a 3d bioprinted 3d bioprinting pluripotent stem cell derived neural tissues using a novel fibrin bioink containing drug releasing microspheres biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels programmed synthesis of three-dimensional tissues a single-cell atlas of the airway epithelium reveals the cftr-rich pulmonary ionocyte cellnet: network biology applied to stem cell engineering single cell transcriptional archetypes of airway inflammation in cystic fibrosis single-cell rna sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis transcriptional regulatory model of fibrosis progression in the human lung defined threedimensional microenvironments boost induction of pluripotency graphical abstract key: cord-337789-pabaoiqs authors: oprinca, george-călin; muja, lilioara-alexandra title: postmortem examination of three sars-cov-2-positive autopsies including histopathologic and immunohistochemical analysis date: 2020-08-27 journal: int j legal med doi: 10.1007/s00414-020-02406-w sha: doc_id: 337789 cord_uid: pabaoiqs this paper describes three autopsy cases with postmortem diagnosis of sars-cov-2 infection, with detailed macroscopic examination as well as advanced microscopic studies of organ tissues collected using hematoxylin-eosin stains and immunohistochemical markers. two of the cases were admitted briefly in the county clinical emergency hospital of sibiu, and one was found deceased at his home address. all three autopsies were completed at the county morgue, in the covid-19 restricted area, using complete protective equipment. the lungs of the patients seemed to be the center organ of invasion and pathogenesis of the novel coronavirus with diffuse areas of condensation, subpleural retraction zones but with different aspect of the classic bacterial bronchopneumonia. microscopic evaluation revealed viral cytopathic effect of type ii pneumocytes with a couple of cells that presented cytoplasmic and nuclear inclusions and who tend to form clusters mimicking multinucleated giant cells. hyaline membranes and destruction of the alveolar wall as well as microthrombi formation within the small blood vessels were constantly found in almost all our three cases. the spleen had sustained white pulp atrophy with absence of lymphoid follicles. there were no microscopic signs of viral infection on the myocardium or the other organs. infections with coronaviruses are widespread in humans and animals. human pathogens include causative agents of rhinitis-like infections but also the virus of the "severe acute respiratory syndrome" (sars), which first erupted in china in 2002 and the novel coronavirus sars-cov-2 with the outbreak starting in december 2019 in china [1] . the genome of covs is a single-stranded positive-sense rna (+ ssrna) (~30 kb) with 5′-cap structure and 3′-poly-a tail. the genomic rna is used as template to directly translate polyprotein 1a/1ab (pp1a/pp1ab), which encodes nonstructural proteins (nsps) to form the replication-transcription complex (rtc) in double-membrane vesicles (dmvs) [2] . the covid-19 global pandemic has put to the test not only governments worldwide but also healthcare professionals from many countries, in trying to find a way to stop the spread of the disease, to find a cure for those infected, and most of all, in finding a vaccine. all these developments can be accomplished only with the help of intense medical research. a great number of clinical studies have been published so far, but only few autopsy reports have been published. only few histopathological and immunohistochemical studies have been released despite the length of time, more than 7 months, passed since the outbreak in wuhan, hubei province of china. in order to fully understand the pathogenesis and physio-pathological manifestations of sars-cov-2 infections, we must first break through the unknown morphological aspects of every tissue and organ that has succumbed to the novel coronavirus, find specific or non-specific modifications within the cells and tissues, and clearly define the response of the organism on a microscopic level. after that, we can draw a map of how exactly the virus interacts with the human organism, a george-călin oprinca and lilioara-alexandra muja contributed equally to this work. particularly important step in finding a definitive treatment for individuals affected by the virus. we feel it is particularly important for the future management of covid-19 patients that more autopsy reports like this one should be published with no hesitations. this paper describes three autopsy cases with unknown cause of death, with full macroscopic examination as well as histopathologic and immunohistochemical analysis of collected organ tissues, including the lung from which reverse transcription polymerase chain reaction (rt-pcr) tests were made to determine sars-cov-2 infection. all three cases were found positive postmortem. we ran one full autopsy on a 27-yearold male found dead at his home and other two thoracoabdominopelvic autopsies. the first one was a 79-year-old woman admitted briefly to the clinical emergency hospital of sibiu county, under the suspicion of covid-19 infection but with death occurring before a pcr test could be performed. the second thoraco-abdominopelvic autopsy of a 70-year-old male was admitted at the same hospital. he presented with superior digestive hemorrhage but with no suspicion of infection at that time, since no respiratory symptoms or fever were present and there was no known history of contact with covid-19-positive individuals. we performed a background check of admissions to hospital on all three cases, with detailed research of their medical records and with a thorough analysis of their complete laboratory and radiological findings. we prepared a total of approximately 40 microscopic hematoxylin-eosin slides; 10 immunohistochemical slides including cd3, cd5, cd20, and cd30 as well as cytokeratins (ck-ae1-ae3, ck-mnf116, and ck7) with a total of 250 microscopic pictures from different pathological sites of tissue and cellular changes for research purpose. all three autopsies were done in sibiu county morgue, in the covid-19 restricted area, using complete protective equipment. the first case presented is of a 79-year-old female patient with a history of hypertension, ischemic cardiomyopathy, and atrial fibrillation, who was hospitalized 2 months prior to death for choledochal preampular intraluminal obstruction, which was managed through endoscopic retrograde cholangiopancreatography (ercp). thirteen days after discharge, she presented to the emergency department with symptoms such as vomiting, abdominal pain, fatigue, loss of appetite, and productive cough. her chest radiography showed bilateral perihilar congestive processes. she was diagnosed with acute pneumopathy and discharged from hospital with prescribed medication. after other 2 days, the patient was readmitted with a new range of symptoms such as fatigue, difficulty in breathing, and fever. a repeated chest x-ray pointed out that the congestive processes had an aggravated evolutionary aspect. notable laboratory findings are relative clotting disorder, acute renal failure, increased white blood cell count with neutrophilia, and lymphocytopenia (table 1) . despite comprehensive treatment, including antibiotics, corticosteroids, and assisted oxygenation, the patient's condition deteriorated, and she presented asystole without response to resuscitation, so death was declared. subsequent clinical information confirmed that her husband, who was as well hospitalized, was found to be infected with sars-cov-2. no specific pathological findings were observed upon external examination of the body. the cranial cavity was not opened. the thoracic cavity was clean, and the pleura was soft, smooth, and glassy. multiple dark grey lymphadenopathies were discovered in the mediastinum as well as in the hilum of both the lungs, between 3 and 20 mm in diameter. the external aspect of the lungs was of purple dark coloration with signs of diffuse congestion. the surface had small subpleural retraction zones, and by palpation, the lungs had a more elastic consistency with lack of crepitations. immediately after dissection of the lung, massive dark deoxygenated blood leaked from the small blood vessels, and a frothy pink fluid was discharged upon compression. the pulmonary parenchyma had a dark brown appearance with diffuse areas of condensation. the heart was increased in size, the right atrium and ventricle were dilated, and the left ventricle suffered moderate to severe eccentric hypertrophy. the myocardium had a relatively normal aspect only with loosely focal fibrotic striations. coronary atherosclerosis was present but with preserved luminal permeability. hepatomegaly with a "nutmeg" appearance of the liver was found, suggesting chronic passive congestion. (table 2 ) lung architecture was modified with diffuse alveolar damage consistent with acute lung injury. the intra-alveolar septa were ruptured, with scattered lymphocytes; there was destruction of the alveolar wall with desquamation of the pneumocytes and formation of hyaline membranes, and fibrin deposits were present in the alveolar spaces. there was type ii pneumocyte hyperplasia. the most important aspect we discovered was the focal type ii pneumocytes that underwent cytopathic changes. there was marked enlargement (cytomegaly) of these cells with enlarged irregular nucleus with single-centered, prominent, nucleolus, and round to oval inclusions situated at the periphery of the nucleus and surrounded by clear halo. the cytoplasm was rich, eosinophilic, and lacked viral inclusions. in some areas, these cells tend to form clusters and imitate multinucleated giant cells and are defined as multinucleated giant cell-like pneumocyte aggregates. upon further examination of the lung parenchyma, we came across focal areas of rich polymorphonuclear infiltration, composed of mainly neutrophils, which translates as focal areas of bacterial superposition. also, we must mention that there were areas of intra-alveolar proteinaceous exudate, capillary congestion, and amylaceous bodies present in examined sections ( fig. 1 and table 3 ). upon microscopic examination of the heart, we concluded that there were no specific pathological changes indicating viral myocarditis. there was a mild to moderate perivascular edema and vascular congestion. areas of small contraction band-like lesions were seen, and there were only a few scattered lymphocytes between the myocardial fibers. microscopic examination of the liver revealed a preserved lobular architecture with some degree of centrilobular vascular congestion. the hepatocytes had intracellular large clear droplets defined as macrovesicular steatosis. there was periportal fibrosis with focal porto-portal and porto-central bridging fibrosis and a mild to moderate lymphocytic infiltrate contained within the portal tract, most probably secondary to obstructive biliary disease. the biliary ducts were of a normal architecture. the renal parenchyma studied had relatively unspecific changes. there were some glomeruli loss in the renal cortex most probably due to age, and we identified the presence of focal microthrombi formation within the glomerular capillary lumen, but no other specific morphologic abnormality was evident upon studying the glomeruli architecture. the proximal convoluted tubules had suffered loss of cytoplasmic and nuclear detail (rarefaction) and retraction of tubule epithelial cells from basement membranes, most probably due to the autopsy being performed 24 h later after death. we identified only small areas of acute tubular injury with coagulative necrosis of focal tubular epithelial cells. there was passive congestion within the medullary blood vessels. no inflammatory cells could be identified in the sampled specimen that was examined, and there were no viral cytopathic changes within the renal cells. microscopy of the spleen showed autolytic changes but also disorganization of spleen compartments translated by marked congestion with white pulp atrophy and with the absence of lymphoid follicles. the remaining lymphoid tissue was found around the central arteries. the capsule was intact (fig. 2) . on the lung samples from this case, we managed to perform immunohistochemistry studies, to be able to understand exactly which type of cells was involved in the pathogenesis of sars-cov-2. pancytokeratin panels (ck-ae1-ae3; ck-mnf116) most importantly found positivity within hyaline membranes, corroborating the fact that these membranes formed by destruction of the epithelial lining, and they had no fibrin origin. cytokeratin 7 (ck7) was reactive in pneumocytes that underwent viral cytopathic effect, including in clusters of giant celllike pneumocytes aggregates. we also found that ck7 fig. 1 microscopic aspect of lung (case 1)-hematoxylin-eosin (× 100, × 400). a (arrow) (× 400); b (arrow head) (× 400) type ii pneumocyte hyperplasia with viral cytopathic effect forming clusters and imitating multinucleated giant cells. c (× 100) hyaline membranes (black triangle) and scattered lymphocytes within the alveolar septum (red triangle). d (× 400) focal area of neutrophilic inflammatory infiltrate immunostain was forming cytoplasmic halos of nonreactivity in a small number of cytopathic cells revealing possible cytoplasmic viral inclusions. focal areas of lymphocytic infiltrates described earlier were reactive to cd3 and cd5, and only scattered lymphocytes were found positive to cd20 in the examined lung tissue. cd23 and cd30 were found negative in every sample examined (fig. 3) . the second case was a male patient, 27 years of age who was found dead in his house. before the autopsy was performed, we checked his medical history, and we found out that he requested an ambulance 6 days before his death. on that occasion, he was examined in the hospital's emergency department for dry cough and shortness of breath. however, there was no history of recent travel or known exposure to sick contacts; thus he was not tested at that time for sars-cov-2. he was discharged from the emergency unit with recommendation of further evaluation at the pulmonary disease department; however, he was found dead 6 days later. searching further through his medical history, we found out that 1 year ago, he had two hospitalizations for bronchopneumonia, acute respiratory failure, hypercapnic respiratory acidosis, septal deviation, ethmoidal sinusitis, and sleep apnea syndrome (which remained in observation). both times he was admitted to hospital in the past, he was in a coma. two months prior to his death, he had another admission with influenza virus infection. he underwent treatment, and he gradually recovered. it is important to mention that he suffered from class 1 obesity and he was a smoker. external examination of the body revealed the absence of trauma and no specific signs that could have indicated a violent cause of death. mild edema and vascular congestion and no ischemic or hemorrhagic lesions were found upon cross section of the cerebral hemispheres. the thoracic cavity lacked any fluid accumulations. the visceral pleura was clean, smooth, and glassy with the presence of groups of small brown to red papule-like lesions on the subpleural surface of both the lungs accompanied by small to medium foci of subpleural retraction zones of the pulmonary parenchyma. the lungs were heavy and congested with lack of crepitations upon palpation. cross section revealed a fleshy dark red parenchyma of elastic consistency with dirty dark deoxygenated liquid blood that leaked from the blood vessels, and a moderate frothy pink fluid was discharged upon compression (fig. 4) . there was a mild dilatation of the right atrial and ventricular cavity. no other pathological findings were observed upon macroscopic examination of the other organs (tables 2). microscopic examination of the pulmonary tissue revealed large areas of alveolar damage with destruction of the alveolar wall lining and intra-alveolar septa, marked vascular congestion, accompanied by intra-alveolar hemorrhage. in the alveolar space, we found large numbers of hemosiderin-laden macrophages, detached pneumocytes, and a rich intra-alveolar eosinophilic homogenous material suggesting marked pulmonary edema. the detached type ii pneumocytes within the alveolar space were enlarged with a round to oval structure, a big irregular nucleus with prominent nucleolus, and coarse clumped chromatin at the edge of the nucleus. sometimes binucleation was present. all these changes suggest viral cytopathic effect. these cytopathic cells tend to form groups like in the other case, mimicking multinucleated giant cells. focal areas of mild pneumocyte hyperplasia were observed. there was only focal presence of hyaline membranes. foci of lymphocytic inflammatory infiltration could be seen with an affinity for the perivascular spaces and around the small to medium size bronchi. in some areas, the lymphocytes tend to invade the vascular wall but with no fibrinoid necrosis formation, suggesting a mild vasculitic reaction. the epithelium of the bronchi was detached and eroded, and there was mild glandular hyperplasia within the bronchus walls. the blood vessels were markedly dilated with hematic content, the walls of these vessels were fibrotic as well as the perivascular space with fibrotic trabecula extending from the spaces to the intra-alveolar septum. in some areas of the affected lung, there was a presence of necrosis, with cellular debris and neutrophilic inflammatory response. small blood vessels in these areas are microthrombotic but with no signs of leukocytoclastic vasculitis upon the small, medium, or large blood vessels of the examined lung tissues (table 3 and fig. 5 ). microscopic examination of the myocardium revealed areas of small vessel thrombosis but with no histological signs of ischemia. there was marked vascular congestion, mild edema between the muscle fibers, and no inflammatory infiltrate in the examined specimens. the myocardial architecture was normal beside scattered areas of myocardial fibers that tended to form contraction bands, most likely due to acute circulatory disorders originated immediately before death. the thickness of the arteries was normal with preserved luminal permeability and no sign of atherosclerosis or myocarditis (fig. 6 ). the deceased was a 70-year-old man with a complex medical history of hypertension, ischemic cardiomyopathy, chronic heart failure (nyha iii), mitral insufficiency, tricuspid insufficiency, atrial fibrillation, aortocoronary bypass (in 2008), chronic renal failure, generalized atherosclerosis, chronic rhinitis, and chronic pharyngitis. two months prior to his death, he was prescribed with anticoagulant drugs for his cardiovascular diseases. one day prior to death, he was admitted to hospital for melena and hematochezia, having blood pressure 94/39 mmhg, and sao2 = 75%. laboratory specimens drawn on that day detected a hypocoagulatory state, acute renal failure, increased level of c-reactive protein, increased white blood cell count with neutrophilia, severe hemorrhagic anemia translated by decreased red blood cell count, and extremely low level of hemoglobin (3.8 g/dl) ( table 1 ). due to his condition, he presented cardiac arrest without response to resuscitation, so death was declared. there was no history of recent travel or known exposure to sars-cov-2-positive contacts. no specific pathological findings were observed upon external examination of the body. the cranial cavity was not opened. the examination of the thoracic cavity revealed about 250 ml of clear serous fluid in the pleural space, on each side. the inferior lobes of each lung and part of the middle lobe of the right lung had a fleshy appearance, but with no increase in volume, the surface of the lobes was smooth and glassy. upon palpation, the consistency was more elastic with areas that lack crepitations. there was compensatory emphysema at the level of the bilateral superior lobes. dark deoxygenated liquid blood leaked upon dissection of the lung parenchyma, and a mild frothy pink fluid was discharged upon compression. the sectioned surface had small areas of a more intense reddish color between stretched areas of dark purple color. dilatation of the right atrium and right ventricle and left ventricular hypertrophy was observed. the coronary arteries suffered extreme atherosclerosis, with marked narrowing of the arterial lumen. upon dissection we found areas of myocardial fibrosis secondary to an old myocardial infarction. the gastric mucosa was hyperemic with small areas of diffuse hemorrhage. moderate amount of hematic content was found within fig. 4 macroscopic aspect of lung (case 2). a (arrows) subpleural parenchymal retraction zones. b (arrow heads) groups of small brown to red papule-like lesions on the subpleural surface of both lungs. c fleshy dark red parenchyma of elastic consistency with dirty dark deoxygenated liquid blood that leaks from the blood vessels the intestinal lumen, but upon examination of the mucosa, there were no signs of ulcers or hemorrhagic tumors. the liver was increased in volume with diffuse fibrosis and regenerative nodules. the spleen was mildly decreased in volume (table 2) . in this case, the pulmonary architecture was relatively preserved, with lack of diffuse lung injury and hyaline membrane formation. there was marked vascular congestion with the presence of numerous dilated blood vessels with hematic content, focal areas where the intra-alveolar walls were ruptured and focal areas of pulmonary edema. detachment of the alveolar lining with scattered pneumocytes throughout the alveolar space and mild to moderate type ii pneumocyte hyperplasia were also a constant finding. these pneumocytes had an increased volume with rich eosinophilic cytoplasm, a round to oval sometimes irregular nucleus with round prominent nucleolus and course, clumped chromatin, mainly at the edge of the nucleus. sometimes binucleation was present. these type ii pneumocytes had suffered viral cytopathic effect, similar with the other two cases. there was a mild to moderate lymphocytic inflammatory reaction, more evident in the perivascular region, with some lymphocytes invading the vascular wall but without fibrinoid necrosis. there were areas of fibrosis within the intra-alveolar septum and atelectasis zones. some small blood vessels had clumped red blood cells in the lumen, forming microthrombi (table 3 and fig. 7) . the architecture of the renal parenchyma was preserved. the glomeruli were shrunken, and the bowman space was widened. there was focal vacuolar degeneration in the cytoplasm of some proximal convoluted tubule cells and cellular debris in the luminal space, consistent with a degree of acute tubular necrosis, most probably by renal hypoperfusion. there is also marked peritubular congestion more accentuated in the medullary parenchyma. there is diffuse disruption of the normal hepatic architecture with regenerative nodules of hepatocytes, fig. 6 microscopic aspect of myocardium (case 2)-hematoxylin eosin (× 400). small vessel thrombosis (star) fig. 5 microscopic aspect of lung (case 2)-hematoxylin-eosin (× 100, × 400). a (× 100) destruction of alveolar walls with marked congestion, intra-alveolar hemorrhage, and diffuse desquamation of pneumocytes. b (arrows) (× 400) moderate perivascular lymphocytic inflammatory response with invasion of vascular walls. c (× 400) groups of hemosiderinladen macrophages (arrow heads) with microthrombi formation within a small blood vessel (star). d (× 400) numerous pneumocytes who underwent viral cytopathic effect, one binucleated (triangle) surrounded by fibrous connective tissue that bridges between portal tracts, consistent with chronic liver disease. within this collagenous tissue, there were scattered lymphocytes and a proliferation of bile ducts. microscopy of the spleen like in our first case showed an abnormal architecture with marked congestion, red pulp necrosis and white pulp atrophy, with absence of lymphoid follicles. the residual lymphoid tissue was found around the central arteries. the capsule was intact. in 2 out of 3 cases, there was no known positive covid-19 contact and no recent travel history to or from countries with high number of infections in that period of time. in case 1, it was known that the husband was found positive during his one admission, but the chronological order of the infections could not be determined. in two out of these three cases, the cause of death was a result of direct lung injury due to viral pneumonia, in contrast to our third case where the cause of death was due to a massive diffuse intestinal hemorrhage after an anticoagulant drug overdose in a patient with undiagnosed hepatic cirrhosis. for the two cases with hospital admissions prior to their death, the laboratory findings were consistent with an inflammatory response due to infection. complete blood count showed leukocytosis with mild neutrophilia and mild lymphocytopenia, with a c-reactive protein of 307.17 mg/l in our first case and a c-reactive protein of 14.12 mg/l in our last case. the lungs were heavy, congested with an elastic consistency, and lack of crepitations upon palpation in cases 1 and 2, the same cases in which the direct cause of death was due to acute lung injury from sars-cov-2 infection. in case number 3, the inferior lobes of each lung and part of the middle lobe of the right lung had a fleshy appearance, but with no increase in volume. upon cross section, the lung tissues examined had a fleshy dark red or dark brown appearance, with patchy consolidations and with focal areas of normal parenchyma, more or less depending on the severity of pulmonary damage. pulmonary congestion and edema were also a constant finding. in our first case, we found multiple dark grey lymphadenopathies up to 20 mm in diameter, at the level of the bilateral pulmonary hilum and mediastinum. we found several important microscopic lesions of the lungs, which helped us draw a clearer picture of the pathology of covid19 and help us better understand the pathogenesis and pathophysiology of the new viral disease. the presence of alveolar wall destruction with detachment of the surface epithelial lining with various degree of type ii pneumocyte hyperplasia confirms that impaired gas exchange by loss of alveolar-capillary membrane can still be considered an important pathophysiological mechanism, in which sars-cov-2 produces acute respiratory distress syndrome (ards). these findings were backed up by previews studies [2] [3] [4] [5] [6] [7] . the consistent presence of pneumocytes increased in volume, with big round to oval, sometimes irregular nucleus, prominent central or peripheral nucleolus and clumped, course chromatin, with some cells that present binucleation or small, pyknotic, intense basophilic nuclear inclusions situated to the periphery of the nucleus, can be interpreted as a viral cytopathic effect, thus confirming the viral origin of the pulmonary infection. pernazza a. et al. described similar findings of reactive hyperplasia and focal pneumocytes with nuclear inclusions [8] , as well as suess c. et al. who found patchy and severe type ii pneumocyte hyperplasia with atypically enlarged pneumocytes with large nuclei, amphophilic granular cytoplasm, and prominent nucleoli showing viral cytopathiclike changes [7] . in all three cases investigated, we discovered areas where these cells tend to form clusters and cohere one to another, fig. 7 microscopic aspect of lung (case 3)-hematoxylin-eosin (× 100, × 400). a (arrow) (× 400) mild to moderate perivascular lymphocytic infiltrate with some lymphocytes with tendency of invading the vascular wall. b (× 400) viral cytopathic changes within type ii pneumocytes, one with binucleation (arrow head). c (stars) (× 100) microthrombi formation within the small blood vessels of the lung forming giant cell-like syncytial aggregates, which can be mistaken with multinucleated giant cells of histiocytic origin. like in our study, zhe xu*, lei shi* et al. described these cells as multinucleated syncytial cells with atypical enlarged pneumocytes characterized by large nuclei, amphophilic granular cytoplasm, and prominent nucleoli that were identified in the intra-alveolar spaces, showing viral cytopathic-like changes and no obvious intracytoplasmic or intra-nuclear viral inclusions [9] , as well as previews studies in the literature [3] . after immunohistochemical studies, we confirmed that indeed these cells are of pneumocytic origin with cytokeratin positivity. hyaline membranes, specific of diffuse alveolar damage, were found more prominent in our first case and only focally on our second case. the presence of microthrombi in the lung parenchyma was found constantly, and in our 27-year-old who died at home (case 2), we found these microthrombi even in some areas of the myocardium. the presence of microthrombi was described in many other case reports suggesting an important pattern of sars-cov-2 pathogenesis [4, 5, 7] . we have put the problem that our pulmonary pathological findings were, in some degree, caused by ventilator-induced lung injury, but we concluded that this was not the case of our three patients because, to our knowledge, only our first patient was put on a ventilator but for a short period of time right before death. mild vasculitic reaction consistent with invasion of pulmonary vascular wall by lymphocytes but with no presence of fibrinoid necrosis was found upon two out of three cases (case 2 and case 3). this finding is consistent with a form of endotheliitis, discussed in detail by varga z. et al. who found viral inclusion structures upon studying under an electron microscope endothelial cells of a patient's kidney who was confirmed with sars-cov-2 infection. histopathology described accumulation of inflammatory cells associated with endothelium, as well as apoptotic bodies, in the heart, small bowel, and lung [10] . other studies confirmed the same findings as lymphocytic capillaritis within the lung and viral endotheliitis of myocardial vessels [11] . beside the lungs, we could not find specific signs of vasculitis, perivasculitis, and capillaritis, and we concluded that our inflammatory response around the vessels of the other organs was a perivascular inflammatory infiltrate. the first two cases presented focal neutrophil infiltrate in the lung tissues examined. other publications described more or less the same microscopic lesions [4, 7, 12, 13] . the myocardium tissues presented small areas of contraction bands and scattered lymphocytes; no signs of myocarditis were present. the same conclusion was expressed by sharon e. fox et al. [14] . dettmeyer et al. confirmed the presence of lymphocytic myocarditis in the right ventricle of a sars-cov-2-positive case. microscopy of the kidney revealed focal areas of acute tubular necrosis upon the proximal convoluted tubules overlaying postmortem changes. other studies revealed the same injury upon the proximal tubules with loss of brush border and nonisometric vacuolization, which partially may be caused by direct virulence of sars-cov-2 [15] . some authors described microscopic alterations consistent with collapsing glomerulonephritis [16, 17] ; however, this was not present in our specimens. in case 1, small focal microthrombosis of the glomerular capillaries was found upon microscopic examination. immunohistochemical studies done to define our type of lymphocytic inflammatory infiltrate in our lung cut sections established that mostly cd3, cd5-positive t lymphocytes, and only scattered cd20-positive b lymphocytes were found during examination, consistent with recent literature [4, 8] . conflict of interest the authors declare that they have no conflict of interest. human rights statements and informed consent all procedures performed in studies involving human participants were in accordance with the ethical standards and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. all personal data were anonymized in order to protect the identity of our patients. autopsy results including histology may be used anonymously for scientific purpose. animal rights this article does not contain any studies with animal subjects performed by any of the authors. pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 püschel k (2020) evidence for systematic autopsies in covid-19 positive deceased: case report of the first german investigated covid-19 death autopsy in suspected covid-19 cases gross and histopathological pulmonary findings in a covid-19 associated death during self-isolation early histologic findings of pulmonary sars-cov-2 infection detected in surgical specimen pathological findings of covid-19 associated with acute respiratory distress syndrome moch h (2020) endothelial cell infection and endotheliitis in covid-19 histopathological findings following sars-cov-2 infection with and without therapy -report of 3 autopsies. rechtsmedizin 30 dying with sars-cov-2 infection-an autopsy study of the first consecutive inside the lungs of covid-19 disease vander heide rs (2020) pulmonary and cardiac pathology in covid-19: the first autopsy series from new orleans renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china covid-19-associated collapsing glomerulopathy: an emerging entity acute kidney injury due to collapsing glomerulopathy following covid-19 infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-338070-y8zi8iz9 authors: liu, wei; ren, xiaojuan; wang, qian; zhang, yan; du, junfeng title: pharmacological inhibition of poly (adp-ribose) polymerase by olaparib ameliorates influenza-virus-induced pneumonia in mice date: 2020-08-31 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-04020-5 sha: doc_id: 338070 cord_uid: y8zi8iz9 treatments against influenza a viruses (iav) have to be updated regularly due to antigenic drift and drug resistance. poly (adp-ribose) polymerases (parps) are considered effective therapeutic targets of acute lung inflammatory injury. this study aimed to explore the effects of parp-1 inhibitor olaparib on iav-induced lung injury and the underlying mechanisms. male wild-type c57bl/6 mice were intranasally infected with iav strain h1n1 to mimic pneumonia experimentally. olaparib at different doses was intraperitoneally injected 2 days before and 5 consecutive days after virus stimulation. on day 6 post-infection, lung tissues as well as bronchoalveolar lavage fluid (balf) were sampled for histological and biochemical analyses. olaparib increased the survival rate of iav mice dose-dependently. olaparib remarkably reduced iav mrna expression, myeloperoxidase (mpo) level, and inflammatory cell infiltration in iav lungs. moreover, olaparib significantly reduced the level of interleukin (il)-1β, tumor necrosis factor (tnf)-α, interferon (ifn)-γ, il-6, and il-4 and increased il-10 in iav lungs. also, olaparib efficiently reduced il-6, monocyte chemotactic protein (mcp)-1, granulocyte colony-stimulating factor (g-csf), tnf-α, chemokine (c–x–c motif) ligand (cxcl)1, cxcl10, chemokine (c–c motif) ligand (ccl)3, and regulated on activation, normal t cell expressed and secreted (rantes) release in iav balf. olaparib decreased parylated protein content and p65, iκbα phosphorylation in iav lung tissues. this study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production possibly via modulation of parp-1/nf-κb axis. electronic supplementary material: the online version of this article (10.1007/s10096-020-04020-5) contains supplementary material, which is available to authorized users. influenza is an acute infectious disease affecting respiratory tracts accompanied with different clinical manifestations ranging from wild to lethal. influenza causes seasonal, unpredictable epidemics and it is now one of the major public health concerns worldwide [1, 2] . according to the report of the world health organization (who) posted online in 2018, only influenza a virus (iav) has caused pandemics up to now, and most human influenza cases are due to the infection of two iav strains, h1n1 and h3n2. iav have laid heavy burdens on global population and economy these years [3] . although vaccine inoculation and antiviral drug administration have been proved to be effective ways to control influenza, epidemics occur sometimes as a result of antigenic drift, which urges the development of novel anti-influenza drugs [4] . poly (adp-ribose) polymerases (parps) family is composed of 18 members which are involved in the bioprocesses, including dna repair, cell cycle regulation, transcription, and so on [5, 6] . abnormal expression of parps is correlated with necrotic cell death, cancer, and some inflammatory disorders [7] . parp-1 activation has been regarded as one of the critic mechanisms underlying lung inflammation in the context of lipopolysaccharide (lps) and elastase stimulations experimentally [8, 9] . evidences indicated the abnormal increased expression of parp-1 in non-pulmonary cells, alveolar epithelial cells, and lung tissue after iav infection [10, 11] , suggesting its potential as the target for the treatment of iav infection. one of the parp inhibitors, olaparib, is reported to ameliorate acute lung injury induced by elastase and lps [9, 12] . yet the role of olaparib on iav-induced lung injury has rarely been reported. this study aimed to explore the effects of parp inhibition by olaparib on iav infection. animals specific-pathogen-free 8-to 9-week-old male wild-type (wt) c57bl/6 mice, weighing 25 to 30 g, were purchased from the nanjing model animal center and kept at 25°c in a 12-h light/ dark cycle, with free access to food and water. animals were allowed to acclimate to the housing environment for 1 week before experimental procedures. all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. the a/font monmouth/47(h1n1, fm1) mouse-adapted influenza virus (chinese center for disease control and prevention) was first plaque purified in the madin-darby canine kidney mdck cells, followed by replication in 9-day-old chicken embryos. the virus pool was pretitrated in mice before further studies in order to determine a suitable challenge dose. the murine model of viral pneumonia was constructed by intranasal infection with h1n1 according to previously described [13] . briefly, ketamine (50 mg/kg weight) and pentobarbital (30 mg/kg weight) were intraperitoneally injected to induce the mild anesthesia in mice. next, a 50-μl influenza virus in ice-cooled pbs, containing fifteen 50% mouse lethal challenge doses (mld50), was infected intranasally. mice were kept on a 37°c thermal insulation blanket for 20 min to recover. the day of virus infection was defined as experimental day 1 and 0-days post-infection. two grouping methods were applied in this study. for the part of survival rate analysis, mice were randomly divided into 5 groups, that is, the influenza a virus (iav) group (h1n1 virus + normal saline), the ola groups (h1n1 virus + 2.5, 5, 10 mg/kg olaparib), and the positive control group (h1n1 virus + 10 mg/kg oseltamivir). for the part of biochemical detection, mice were divided into 3 groups, the control group (normal saline), the iav group (h1n1 virus + normal saline), and the iav + ola group (h1n1 virus + 10 mg/kg olaparib). olaparib (selleck chemicals, houston, tx, usa) at different doses was intraperitoneally injected 2 days before and 5 consecutive days after h1n1 virus challenge. the day of virus challenge was defined as experimental day 1. oseltamivir (hoffmann-la roche, basel, switzerland) was administered in a similar manner to olaparib. after anesthetization, mice were infected intranasally with 50-μl influenza virus in ice-cooled pbs, containing five mld50, at experimental day 1 (0 days post-infection). then mice in different groups were followed for 15 consecutive days post-infection with the number of deaths being monitored daily. the severity of pulmonary edema induced by h1n1 virus challenge was quantified by lung indexing. five days after virus infection, the body weight of mice was measured using an electronic analytical balance. then lungs were dissected, washed in pre-cooled pbs, and weighed. the lung index was calculated by the following formula: lung weight/body weight × 100%. at day 6 post-infection, lungs were dissected and fixed in 4% paraformaldehyde for further histopathological analysis. paraffin sections in 5-μm thickness were stained with hematoxylin and eosin (h&e) and examined using a light microscope. the severity of the lung injury was assessed by scoring the h1n1-induced lung histopathological changes using the scoring system previously described [14] . in brief, the grading was conducted in a blinded manner with the grader unaware of the concrete group or mice being reviewed. the scores of 0 to 4 were defined to represent normal, mild, severe, and very severe lung injury, respectively. in concrete, 0 was valued for normal lung, 1 for lower than 25%, 2 for 25-50%, 3 for 50-75%, and 4 for higher than 75% lung involvement, respectively. at day 6 post-infection, lung homogenates and balf were sampled from different groups of mice. for lung homogenates, the lungs were dissected and homogenized in ice-cold ripa lysis (sigma) supplemented with protease inhibitor (sigma) on ice for 2 h. after 4°c centrifugations at 4000 g for 20 min, supernatants were collected and stored at − 80°c for further biochemical analysis, including myeloperoxidase (mpo), cytokines, and targeted protein levels. total protein content of lung homogenates was determined using a bca assay kit (beyotime, shanghai, china). for balf sampling, bronchoalveolar lavage was conducted according to previously described by yashiro m et al. [15] . briefly, after anesthesia, the left main bronchus was ligated and the right lung was quickly lavaged twice by 1 ml of cold sterile pbs. then the collected balf was centrifuged at 2000 rpm for 10 min at 4°c, and the supernatant was stored at − 80°c for measurement of cytokine levels. mpo activity was assessed by 3,3′,5,5'-tetramethylbenzidine (tmb, sigma-aldrich, st. louis, mo, usa) method according to previously described [16] . in brief, equal amount of sample (10 μl) was sequentially mixed with 0.75-mm hydrogen peroxide (80 μl), 2.9-mm tmb dissolved in 14.5% dimethyl sulfoxide (110 μl), and 150-mm sodium phosphate buffer (ph 5.4) in a 96-well plate for 5-min incubation at 37°c. then 2-m sulfuric acid (sigma) was added to stop the reaction and absorbance at 450 nm was measured to evaluate mpo activity in each sample. the mpo levels were expressed as pmol mpo per milligram of lung tissue (pmol/mg protein). cytokines and chemokines in lung homogenate and balf samples were measured using commercial assay kits according to the recommended protocols. interleukin-1 β (il-1β), interleukin-4 (il-4), interleukin-6 (il-6), interleukin-10 (il-10), interferon-γ (ifn-γ), and tumor necrosis factor-α (tnf-α) were measured by enzyme-linked immunosorbent assay (elisa) kits (r&d system, minneapolis, mn, usa). monocyte chemotactic protein-1 (mcp-1), granulocyte colony-stimulating factor (g-csf), chemokine (c-x-c motif) ligand 1 (cxcl1), chemokine (c-x-c motif) ligand 10 (cxcl10), chemokine (c-c motif) ligand 3 (ccl3), and regulated on activation, normal t cell expressed and secreted (rantes) were measured using a mouse multi-cytokine/chemokine magnetic bead panel (millipore, billerica, ma, usa) and analyzed on a luminex 100 system (luminex, austin, tx, usa). the level of target factors was expressed as pg/mg protein for lung homogenate detection and pg/ml for balf detection. real-time quantitative polymerase chain reaction (rt-qpcr) viral load was determined by rt-qpcr according to previously described by li y et al. [17] with slight modifications. the total rna of isolated lung tissues at day 6 post-infection was extracted using trizol method (life technologies, carlsbad, ca, usa) according to the manufacturer's instructions. then equal amount of rna for each sample was transcribed into complementary dna (cdna) using the first strand cdna synthesis kit (takara, dalian, china) following the instructions. quantitative pcr (qpcr) was performed using a sybr green suit (takara) to determine the mrna expression levels of target genes, iav m gene, and gapdh, in an abi real-time pcr system (applied biosystems, new york, ny, usa), with the following amplification procedures: 1-min preincubation at 98°c, 40 cycles of 98°c for 10 s, 56°c for 20 s, and 72°c for 30 s. each sample was performed in triplicate in one single technical repetition. the primer sequences used in this study were as follows: iav m, 5'-aatggtgcaggcgatagag-3′ (forward) and 5'-tacttgcggcaacaacgagag-3′ (reverse); gapdh, 5'-tgaggtcaatgaaggggtcg-3′ (forward) and 5'-tgaggtcaatgaaggggtcg-3′ (reverse). the relative quantitation of iav was calculated using the comparative 2 −δδct method. gapdh was used as the inner control. the parylated protein content in the lung tissues was analyzed by western blot. briefly, − 80°c stored lung homogenate samples were thawed on ice. for each sample, 25-μg protein was separated through 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (millipore), followed by 1-h block in 5% nonfat milk at room temperature. then the membranes were incubated at 4°c overnight with primary antibodies against par (1:1000, abcam, cambridge, uk), phospho-p65 (p-p65, 1:2000, abcam), p65 (1:2000, abcam), phospho-iκbα (p-iκbα, 1:1500, abcam), iκbα (1:1500, abcam), or β-actin (1:2000, santa cruz, ca, usa), respectively. after the 2-h incubation with peroxidase-conjugated secondary antibodies (abcam) at room temperature, the enhanced chemiluminescence kit (millipore) was used to detect the proteins of interest in the uvp biospectrum imaging system (biospectrum, ca, usa). βactin served as the inner control. each experiment was performed for at least three times. data were expressed as mean ± sd for each assay. statistical analysis was conducted by a one-way analysis of variance (anova) test using the spss software (chicago, il, usa). p < 0.05 was considered to be statistically significant. for the body weight change analysis, the two-way anova followed by tukey's multiple comparison test was used. this study aimed to investigate whether olaparib (chemical structure in fig. 1a ) possessed protective effects against influenza virus challenge. from day 3 post-infection, the iavinfected mice began to exhibit clinical symptoms, and on day 6 post-infection, the symptoms got worse, which included but not limited to the following, such as weight loss (fig. s1) , inactivity, rapid shallow breathing, and poor appetite, indicating the successful construction of viral pneumonia model experimentally. figure 1b showed that the iav-only mice began to die on day 4 post-infection until day 12 post-infection. the positive control, oseltamivir, improved the survival rate of infected mice significantly compared with those in the iavonly group. to the expectations, mice in the olaparib group showed higher survival rate compared with that in the iav group in a dose-dependent manner, indicating that olaparib could powerfully protect against influenza virus challenge in by h&e staining and the quantitative analysis of histological changes in the lung tissues (e) (n = 8 for each group). data were presented as mean ± sd. ## p < 0.01 and ### p < 0.001 compared with the control. * p < 0.05 and ** p < 0.01 compared with iav mice. additionally, olaparib-treated iav mice exhibited lower weight loss compared with untreated ones, suggesting it might possess lighter side effects (fig. s1) . also, the dose of 10 mg/kg olaparib was chosen for further investigation as a result of the highest survival rate among all olaparib groups. as olaparib treatment evidently reduced the virus-induced mortality, pathological changes were further explored to assess the influences of olaparib on the lung injury severity. in comparison with the control group, the lung index of mice in the iav group was significantly higher than that in the control group, and olaparib treatment remarkably reduced the lung index, suggesting that olaparib could alleviate the pulmonary edema induced by iva infection (fig. 2a) . as shown in fig. 2b , higher mrna expression of iav was detected in iav lungs, while no virus was detected in the control lung tissues, indicating the direct relationship between iav infection and pathological manifestations of mice model. unsurprisingly, less iav was detected in the lungs of olaparib-treated iav group, indicating the antiviral effect of this drug functions in the iav model mice. virus infection always correlated with leukocyte filtration to the target organs. we next determined the mpo levels and the marker of leucocytes, in lung tissues. figure 2c showed that iav infection elevated the mpo levels compared with that in the control group, whereas olaparib evidently reduced mpo levels, illustrating that olaparib reduced leucocyte infiltration to the lungs of iav mice. morphologic analysis was performed using h&e staining of lung tissues. as shown in fig. 2d , extensive inflammatory cell infiltration, especially around bronchioles, was presented in iav lungs, signifying lung edema might lead to the breathing difficulty in infected mice. and olaparib treatment attenuated the pathological abnormalities in iav lungs. lung histopathological grading numerically pointed that olaparib rectified the lung injury caused by iav infection (fig. 2e) . virus-infected pneumonia always comes along with abnormal release of inflammatory cytokines. next the level of inflammatory cytokines in lung homogenate was detected to investigate whether olaparib could influence the release of inflammatory cytokines in lung tissue. as shown in fig. 3a , b, c, d, e, compared with those in the control group, pro-inflammatory cytokines, il-1β, tnf-α, ifn-γ, il-6, and il-4, were significantly elevated, and anti-inflammatory cytokine, il-10, was remarkably descended in the iav group, while olaparib treatment obviously rectified the abnormal release of the above inflammatory cytokines, meaning that olaparib might possess anti-inflammatory effect in murine lung tissue under the iav context. the detection of cytokine/chemokine in balf samples at day 6 post-infection showed that il-6, mcp-1, g-csf, tnf-α, cxcl1, cxcl10, ccl3, and rantes were remarkably increased in the iav group compared with those in the control group, while olaparib treatment significantly reduced the abnormal increased levels of the above cytokine/chemokines, which was similar with the results obtained from lung tissue (fig. 4a-h) . to explore the mechanisms underlying the protective effect of olaparib against iav-induced injury to murine lungs, western blot was performed to detect the parps, the marker of apoptosis. iav infection increased the parylated protein content in lung homogenate samples compared with the control group, while parp inhibitor olaparib significantly reduced the parylated protein content in iav-infected samples (fig. 5a and c) . nf-κb genes are reported to be the targets of olaparib in the context of lps stimulation to the lungs [12] . as shown in fig. 5b , d, and e, iav remarkably elevated the p-p65 and p-iκbα protein expression in lung homogenates compared with those of the control group, while olaparib obviously decreased the abnormal increase of the two proteins, suggesting that olaparib inhibits the activation of nf-κb signaling pathway in the condition of iav infection. and there were no significant differences of the total expressions of p65 and ikba among different groups (fig. 5b) . the highly transmissible human pathogen iav mainly attacks human respiratory epithelium with its hemagglutinin binding with sialic acid to initiate endocytosis [18] . severe symptoms after iav infection are commonly found in the infant, elderly, pregnant, as well as the immunocompromised populations [19] [20] [21] [22] . iav can cause serious pandemics in a short time, with shocking and heart-wrenching facts in history, e.g., the 1918 great influenza killed almost 1/30 of the global population only in 1.5 years. also iav is capable of generating new strains adapted to human beings. that is the very reason that influenza vaccines have to be updated regularly. also antiviral drugs have to be replaced as a result of the increasing drug resistance developed by viable iav subtypes. thus, it is urgent to explore novel anti-iav drugs to enrich the available antiviral drug bank. this study investigated for the first time the possible role of olaparib on iav infection in a murine model and found that parp-1 inhibitor olaparib remarkably relieved iav-induced lung injury and lung inflammation possibly via inhibition of parp, as well as p65, and iκbα phosphorylation. parp has been indicated to be the target of treatment strategy against cancers and inflammatory disorders [23, 24] . the pro-inflammatory effect of parp-1 has been highlighted in a variety of non-pulmonary and pulmonary inflammatory diseases, including arthritis, allergic encephalomyelitis, asthma, acute lung injury (ali), and so on [25] [26] [27] . parp-1 is activated and involved in the lungs of allergen-induced asthma animal models via modulating immune cell recruitment, airway modeling, as well as cytokine production, mainly th2 cytokines [27] [28] [29] . parp-1 is reported to play a critical role in lps-induced and mechanical ventilation-induced ali in mice [30, 31] . hence parp-1 might be a convincing target for the prevention and treatment of lung inflammatory injury. genetic as well as pharmacological measures modulating parp-1 activity were proved to be effective to alleviate the lung inflammation and injury experimentally from the aspects of reducing neutrophil infiltration and macrophage accumulation [32] , blocking the activation of nf-κb and ap-1 [33] . additionally, parp-1 inhibition can relieve the secondary kidney injury induced by ali [34] . as a potent parp inhibitor, olaparib has already been approved to be used in cancer patients for clinical trials with acceptable toxicity. olaparib downregulates nf-κb-related genes including tnf-α and il-1β, decreases neutrophil, and alleviates edema of lpsstimulated murine lungs [12] . our study found that olaparib possesses protective effects against iav-induced lung inflammation, suggesting that this drug might exert non-specific anti-inflammatory roles. as the outbreak of severe coronavirus infection since december 2019, it is urgent to explore the mechanisms of virus-induced damage to lung tissues or other organs, as well as develop more effective antiviral drugs or therapies to prevent related damages to patients. our study western blot analysis of parylated protein content (a) and p-p65, p65, p-iκbα, and iκbα (b) in lung samples from each experimental group. the relative expressions were normalized to control (c, d, e). data are presented as mean ± sd. ## p < 0.01 and ### p < 0.001 compared with the control. ** p < 0.01 and *** p < 0.001 compared with iav explored the protective effects of olaparib in experimental virus-induced pneumonia, suggesting its possible application in viral pneumonia treatment in the future. there are some shortcomings in our study. we did not perform toxicity analysis about the influence of olaparib on normal physiology of mice. the relationship between parp-1/nf-κb and iav-induced lung inflammation as well as the concrete mechanisms was not well illustrated. related experiments would be performed in the future study. the present study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production, possibly via modulation of parp-1/nf-κb axis. this study indicates the non-specific anti-inflammatory effect of olaparib in lung disorders and would shine light on the development for treatment against pneumonia infected by viable iav subtypes. conflict of interest the authors declare that they have no competing interests. ethical approval all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. informed consent not applicable. the genomic and epidemiological dynamics of human influenza a virus global circulation patterns of seasonal influenza viruses vary with antigenic drift rationale and opportunities in estimating the economic burden of seasonal influenza across countries using a standardized who tool and manual variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies poly (adp-ribose) polymerases in double-strand break repair: focus on parp1, parp2 and parp3 role of poly (adp-ribose) polymerase in cell-cycle checkpoint mechanisms following gamma-irradiation the functional role of poly (adpribose) polymerase 1 as novel coactivator of nf-kappab in inflammatory disorders activation of poly(adp-ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation parp-1 inhibition ameliorates elastase induced lung inflammation and emphysema in mice infection of human retinal pigment epithelial cells with influenza a viruses erucic acid from isatis indigotica fort. suppresses influenza a virus replication and inflammation in vitro and in vivo through modulation of nf-kappab and p38 mapk pathway parp inhibitor, olaparib ameliorates acute lung and kidney injury upon intratracheal administration of lps in mice combined effect of anti-high-mobility group box-1 monoclonal antibody and peramivir against influenza a virus-induced pneumonia in mice critical role for cxcr2 and cxcr2 ligands during the pathogenesis of ventilator-induced lung injury redox-active protein thioredoxin-1 administration ameliorates influenza a virus (h1n1)-induced acute lung injury in mice measuring myeloperoxidase activity in biological samples oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice influenza virus amino acid changes in ha and determinants of pathogenicity associated with influenza virus a h1n1pdm09 during the winter seasons impact of pregnancy on intra-host genetic diversity of influenza a viruses in hospitalised women: a retrospective cohort study more than just a common cold: endemic coronaviruses oc43, hku1, nl63, and 229e associated with severe acute respiratory infection and fatality cases among healthy adults influenza virus: small molecule therapeutics and mechanisms of antiviral resistance targeted therapy: ariel3 -broad benefit of parp inhibitors in ovarian cancer targeted therapy for cancer using parp inhibitors signaling mechanism of poly (adpribose) polymerase-1 (parp-1) in inflammatory diseases beyond dna repair, the immunological role of parp-1 and its siblings poly (adp-ribose) polymerase-1 in lung inflammatory disorders: a review gene knockout or pharmacological inhibition of poly (adp-ribose) polymerase-1 prevents lung inflammation in a murine model of asthma inhibition of poly (adp-ribose) polymerase prevents allergen-induced asthma-like reaction in sensitized guinea pigs lipopolysaccharide activates erk-parp-1-rela pathway and promotes nuclear factor-kappab transcription in murine macrophages inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates ventilator-induced lung injury effects of parp-1 deficiency on airway inflammatory cell recruitment in response to lps or tnf: differential effects on cxcr2 ligands and duffy antigen receptor for chemokines parp-1 inhibitor, dpq, attenuates lps-induced acute lung injury through inhibiting nf-kappab-mediated inflammatory response inhibition of poly (adenosine diphosphate-ribose) polymerase attenuates lung-kidney crosstalk induced by intratracheal lipopolysaccharide instillation in rats publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-336782-0zkb39v1 authors: fraile gutiérrez, v.; ayuela azcárate, j. m.; pérez torres, d.; zapata, l.; rodríguez yakushev, a. l.; ochagavía calvo, a. title: narrative review of ultrasound in the management of the critically ill patient with sars-cov-2 infection (covid-19): clinical applications in intensive care medicine date: 2020-11-02 journal: nan doi: 10.1016/j.medine.2020.10.002 sha: doc_id: 336782 cord_uid: 0zkb39v1 the clinical picture of sars-cov-2 infection (covid-19) is characterized in its more severe form, by an acute respiratory failure which can worsen to pneumonia and acute respiratory distress syndrome (ards), and get complicated with thrombotic events and heart dysfunction. therefore, admission to the intensive care unit (icu) is common. ultrasound, which has become an everyday tool in the icu, can be very useful during covid-19 pandemic, since it provides the clinician with information which can be interpreted and integrated within a global assessment during the physical examination. a description of some of the potential applications of ultrasound is depicted in this document, in order to supply the physicians taking care of these patients with a adapted guide to the intensive care setting. some of its applications since icu admission include verification of the correct position of the endotracheal tube, contribution to safe cannulation of lines, and identification of complications and thrombotic events. furthermore, pleural and lung ultrasound can be an alternative diagnostic test to assess the degree of involvement of the lung parenchyma by means of the evaluation of specific ultrasound patterns, identification of pleural effusions and barotrauma. echocardiography provides information of heart involvement, detects cor pulmonale and shock states. the disease caused by sars-cov-2 (covid-19) is characterized by pneumonia clinical presentation with fever and cough accompanied by multifocal nodular (round or oval) ground-glass opacities in the lungs that can progress to acute respiratory distress syndrome (ards) and requires admission to an intensive care medicine service (icms) in a high percentage of patients. 1 on the other hand, infection due to sars-cov-2 can trigger thrombotic phenomena and severe deterioration of other organs like the heart. ultrasound can be a very useful tool during the management of the covid-19 pandemic because it provides real-time non-invasive bedside images of patients admitted to intensive care units (icu). 2---4 also, it increases safety in invasive procedures. in this context, the information obtained through different ultrasound imaging modalities can be interpreted and integrated in the patient's complete general evaluation while being examined. recently, several articles have been published on the role of ultrasound in the assessment of pulmonary and cardiovascular functions in patients with sars-cov-2. 5---8 the committee of ultrasound practice accreditaion of the spanish society of intensive and critical care medicine and coronaryunits (semicyuc) has elaborated this document with some of the multiple applications of ultrasound as a guide for doctors who treat patients with covid-19, especially during icu admission (table 1) . the arrival of portable ultrasound machines has made them available for use in most icmss. also, the existence of wire-less ultrasound transducers or probes connected to a tablet or smartphone connected through usb, although of lower image quality compared to bigger machines, allows us to obtain ultrasound images from patients with covid-19. they bring more advantages compared to portable consoles like smaller sizes, lighter weight, they are easy to carry, power up fast, have a certain degree of asepsis, and can be easily covered with 1-use only disposable plastic cases. all of it reduces the risk of contamination and nosocomial infection, while facilitates cleaning and sterilization. 7, 9 the use of dedicated portable ultrasound transducers specifically for patients with covid-19 is advised with special precautions of asepsis and sterility to reduce cross infections. covering the keyboard and the probe is advised too. 10 the ultrasound machine should be cleaned following the recommendations established to fight the current pandemic 11, 12 (appendix b annex 1 of the digital supplementary data [dsd] ). the airway management through orotracheal intubation (oti) and while on mechanical ventilation (mv) is the usual procedure for the management of patients with severe acute respiratory failure (arf) due to sars-cov-2. oti is a technique that can release aerosols, which is why it should be performed by a properly trained professional through videolaryngoscopy preferably and with the recommended personal protection equipment (ppe). 13 confirming the correct positioning of the orotracheal tube (ott) after the oti is essential. it is often based on the capnography and physical examination (direct visualization of the passage of the ott through the vocal cords, symmetric elevation of both hemothoraces, lack of noise at epigastral level, auscultation of vesicular murmur, and condensation of the tube with ventilation). 14--16 the suboptimal access to resources like capnography and the inconvenience associated with the ppe when performing a complete physical examination make the ultrasound a very useful imaging modality in this context. the tracheal ultrasound has proven to be non-inferior to capnography to rule out esophageal intubation. 17 for this purpose, it is necessary to use a linear or convex transducer and acquire the cross-sectional view from the anterior region of the neck, in the mid-line above the sternal notch. in this plane, the trachea looks like a hyperechogenic inverted u-shaped line generated by the mucosa-air interface and a posterior comet-tail artifact (fig. 1a) . the correct intubation of the trachea does not cause any changes in the image described since no new interface between the patient's airway and the ott is generated (both contain air). also, because the ott is located behind the artifact generated by the trachea (which would limit its visualization). on the echocardiography, the accidental intubation of the esophagus generates a new interface between the esophageal mucosa and the air inside the ott giving rise to a second comet-tail artifact that originates at the deepest level of the trachea and is lateral to it (fig. 1b) . the main advantage of this type of study is that ventilation does not need to be started to detect the incorrect position of the ott, thus avoiding the possibility of aspiration of gastric content; its limitations are the impossibility to see the ott directly from inside the airway and the patient's possible anatomical alterations. pleural ultrasonography provides direct anatomical information on the expansion of the lungs and entry of air into the lungs. 18 it can replace auscultation and chest x-rays as the method to rule out accidental selective bronchial intubation (asbi). for this purpose, it is required to use a linear or convex probe to examine, at least, the upper and lower points of the blue protocol in both hemithoraces. 19 in these planes, on the 2d ultrasound, the pleura appears as a horizontal hyperechogenic line with movement generated by the slide of the parietal pleural surface over the visceral pleura with respiratory movements called lung sliding or lung displacement sign. 20 on m-mode it can also be registered statically by cutting the pleural line producing an image called seashore sign. the presence of both signs in each hemithorax is indicative of bipulmonary insufflation (fig. 1c) . the consequence of asbi is the lack of airing of the contralateral lung that can be found by the absence of lung sliding and a typical «bar code» pattern or stratosphere sign on m-mode (fig. 1d) . the transformation of a unilateral bar code pattern into a normal lung sliding pattern associated with the partial removal of the ott is indicative that the initial position of the tube corresponds to an asbi of contralateral lung (fig. 7) . the main advantages of this kind of examination are the possibility of showing the airing of the lung without having to use the stethoscope and its immediacy compared to the chest x-ray. the main limitation is the existence of differential diagnoses of the stratosphere sign (lack of ventilation, pneumothorax, tumors, etc.). • it allows the clinician to obtain bedside images, reduces exposure for the health personnel, and minimizes the risks involved in the transfer of patients • it allows the discrimination of low-risk patients from high-risk patients who may require second-level radiological assessments • it depends on the experience and training of the physician performing the test. also, it requires a structured training process. • it is superior to the simple x-ray for the detection of pneumothorax, pleural effusion, pneumonia, interstitial syndrome, and for the differential diagnosis of acute dyspnea • in the thoracic ultrasound, the clinical signs are the determinant factor regarding the interpretation of the data obtained. the signs are not very specific, but they are highly sensitive • it has proven its utility for the identification of different b-line patterns and to distinguish cardiogenic pulmonary edema from ards • it is an imaging modality for superficial structures so if the pathological process does not reach the pleura nothing will show • portable devices are easier to sterilize because their surface is smaller compared to other radiological devices • both the ultrasound probe and the transducer can be a reservoir for pathogens and a source of contamination • it avoids radiation and serial assessments can be performed because it allows an almost continuous monitorization to detect early signs of lung damage pu, pulmonary ultrasound. in the covid-19 pandemic, the main trait of critically ill patients is pneumonia progressing towards ards with a characteristic diagnostic pattern on the ct scan. 1 assessing patients with arf using the blue protocol (bedside lung ultrasound in emergency) is one of the well-known and consolidated applications of pulmonary ultrasound. 19 although the medical literature available on the use of pulmonary ultrasound (pu) to assess patients with suspected sars-cov-2 infection and its potential applications is still scarce, some data that show the good correlation between pu findings and thoracic ct scans. 6 although the ct scan is the recommended imaging modality, the difficulties associated with moving the patient, the high risk of spreading the microorganism during the transfer, and the later disinfection of the radiology rooms make pu a valid diagnostic imaging modality for the bedside assessment of the degree of pulmonary damage through the analysis of specific ultrasound patterns. 21 the pu should not be used separately but complement the echocardiography in the assessment of several conditions of the critically ill patient. 22, 23 the advantages and limitations of pu are shown on table 2 . the examination is performed in the supine decubitus position by dividing each hemothorax in quadrants. 24 the articles that study the utility of assessing different ultrasound patterns of airing of the lungs to obtain a score analyze 6 areas in each hemithorax outlined by 3 longitudinal lines at sternal level (long axis of the clavicle towards the diaphragm), anterior and posterior axillary as anatomical references outlining 3 different areas: anterior (1 and 2), lateral (3 and 4), and posterior (5 and 6). by drawing a cross-sectional line at nipple level these areas are divided into upper and lower regions. this is how 6 different examination regions are established so they can be studied through an evolutionary analysis. actually, a recent pilot study has proven that it is non-inferior to the protocols that use more quadrants. 25 pleural effusion (pe) and the presence of consolidation are analyzed on region 6 or equivalent to the plaps point (posterolateral alveolar and/or pleural syndrome) of the blue protocol 24 (appendix b annex 3 of dsd). the patterns of airing of each pulmonar region are ( fig. 2 ): • pattern a: typical pattern of normal airing. pleural line with preserved sliding, presence of well spaced a-lines and ≤ 2 b-lines ( fig. 2a and figure b ). • pattern b1: presence of more than 2 well spaced, diffuse b-lines in different thoracic regions called septal rockets (fig. 2c ). • pattern b2: confluent b-lines separated between them by ≤ 3 mm (ground-glass rockets) due to a more severe loss of aired lung (fig. 2d ). • pattern c: lung consolidation suggestive of significant loss of airing of the lung due to the accumulation of fluid and/or cells in the alveoli. the consolidation can be found anywhere in the hemithorax but applying the transducer to the plas point detects 95% of the cases ( fig. 2e and figure f ). patterns a, b1, and b2 include the presence of lung sliding (appendix b annex 3 of the dsd). in the case of patients with covid-19 the typical findings are based on disease progression. the first signs on the pu show an irregular distribution of focal b-lines, later confluent, that spread towards several regions of the pulmonary surface. disease progression is represented by the appearance of small subpleural consolidations with a thickened and irregular pleural line and areas associated with white lung, regions with pattern a patches, and anomalous areas without massive pe. disease progression will show a consolidation pattern, especially in dependent regions of the body with or without air bronchogram and its increasing spread across the pulmonary surface, indicative of progression to arf that may require mv. the air/fluid correlation determines the ultrasound appearance of the lung and provides different patterns based on the degree of aired parenchyma, which will allow us to assign a different score to each region under study. pu can be useful to monitor the progression of the disease using the lung ultrasound score (lus). 26, 27 this scoring system uses a model to explore 12 regions (appendix b annex 4 of the dsd). the score is based on the worst ultrasound pattern registered in each of the 12 regions studied by assigning the following values: pattern a = 0 points; pattern b1 = 1 point; pattern b2 = 2 points; pattern c = 3 points. the overall score is the sum of the points assigned to each region (from 0 that would be indicative of a totally aired lung to a maximum of 36, that would be describing a totally condensed lung). during the natural progression of infection due to sars-cov-2 at the icu setting, the patients' infiltrates often improve on the chest x-ray. however, the pu will still show persistent pathological findings with pleural line thickening and abundant b-lines that may be predictive of a difficult mv disconnection or weaning failure 27 (fig. 3) . the lus score tells us about the aired pulmonary mass and provides clinical and prognostic information. that is why the lus can be used to predict what patients may need to be admitted to the er after presenting with fever and dyspnea. also, to detect patients in whom mv weaning may fail while at the icu. further studies are needed to validate this scoring system in patients with pneumonia due to sars-cov-2. other pulmonary conditions, although rare in the early stages of pneumonia due to sars-cov-2 can appear during the natural progression of the disease such as pneumothorax or pleural effusion. detecting pneumothoraces is important to assess mv-or iatrogenic-induced barotrauma after recruitment maneuvers of central venous cannulation (cvc). the pu has been confirmed as an alternative imaging modality to the chest x-ray in the diagnosis of pneumothorax with a 98% specificity and a 75% sensitivity. 28, 29 although a systematic study can be conducted in unstable patients in the supine decubitus position and since air tends to occupy the thorax anterior side, the anterior regions should be examined first. a linear or micro-convex transducer is enough to assess the pleural line and the lung artefacts (fig. 4) . three signs rule out the presence of pneumothorax 20,30 : • the presence of lung sliding is indicative that both pleural layers are in contact and rules out the presence of pneumothorax (npv, 100%) in the space where the transducer is placed. however, its absence is not confirmed by its low specificity. • seeing 1 b-line only is indicative that both layers are attached, which rules out the presence of pneumotho-rax with a 100% npv. extra care should be paid here to distinguish b-lines from e-lines (vertical hyperechoic lines originated at the thoracic wall soft tissues in the presence of subcutaneous emphysema) (fig. 4a ). • presence of lung pulse: the rhythmic movement of the pleural line in synchrony with the heart beats in the absence of lung sliding is indicative that both the parietal and visceral pleuras are in contact. nonetheless, regional ventilation is impaired (eg, atelectasis) because the air between both pleural layers is blocking its transmission. sequential diagnosis is achieved with 2 signs (fig. 8 ). • lung sliding abolished with the presence of a-lines (profile a' in the blue protocol) (fig. 4b ). • find the lung point between the collapsed lung and the pneumothorax collection of air (lung point) ( fig. 4c and d) . it is a dynamic sign indicative of an impaired normal and abolished sliding during 2d ventilation or by the succession of normal images (seashore sign) during inspiration and horizontal lines (strathosphere sign or bar code sign) durante exhalation on m-mode. it is a specific sign with a 66% sensitivity and a 100% specificity to diagnose pneumothorax. 30 the more lateral and lower the lung point is to the thoracic wall, the more it has spread. a very posterior or absent lung point is indicative of a massive pneumothorax with complete lung atelectasis. 31 pleural effusion (pe) is a rare complication of covid-19. we should not forget that the international sonsensus conference on lung ultrasound establishes that «for the detection pe, the up is more accurate than the x-ray in the supine position and almost as accurate as the ct scan» (level a). 32 pe is often gravity dependent and accumulates in dependent regions of the thorax. that is why examination should start close to the diaphragm with a convex probe being the plaps point the location where it is better detected. pe appears as an often anechoic, echo-free space between the parietal and visceral layers of the pleura and always above the diaphragm. 33 the latter allows us to distinguish pe from ascites. the appearance of pe should be interpreted within the clinical context. its appearance should not be assessed separately in the therapeutic decision-making process since an anechoic pe can be indicative of both an exudate and a transudate 34 (appendix b annex 5 of the dsd). one of the most common findings in severe disease due to sars-cov-2 is a profoundly impaired hemostatic function whose occurrence has prognostic implications. over two thirds of the patients who die of this disease meet the diagnostic criteria of disseminated intravascular coagulation (dic) proposed by the isth (international society on thrombosis and haemostasis). however, its incidence rate among survivros is < 1%. 35 the predominant component of an impaired hemostatic function is not bleeding diathesis but prothrombotic state associated with venous thromboembolic phenomena and microvascular thrombosis with high levels of d-dimer and fibrinogen. in critically ill patients, these phenomena are associated with a higher rate of thrombosis in intravascular devices and vascular occlusive phenomena. 35, 36 vascular ultrasound identifies certain thrombotic phenomena and allows us to start a therapeutic strategy for control and prevention, which eventually may improve prognosis. 37 for its study it is necessary to use a highfrequency linear probe (7−15 mhz) to explore the venous territory with special attention to cvc insertion areas and complete the study of the venous blood vessels of both legs all the way up to the popliteal vein. it has been estimated that up to 90% of all embolisms occurred in patients with pulmonary thromboembolisms come from the lower extremity proximal veins. thrombi appear as echogenic material inside the blood vessel. however, immature thrombi may not be echogenic and there are times when they are partially compressible due to their gelatinous consitency. therefore, the main diagnostic criterion is the lack of vessel compressibility and not the visualization of a blood clot. the use of vascular ultrasound increases safety and effectiveness during cvc insertion compared to techniques based on anatomical references. 38---40 before starting the puncture, vascular accesses should be explored to determine which is the most suitable one and detect vascular anatomical variables. 41 in cases of venous thrombosis and/or anatomical impairment, the access vein should be changed 42 (fig. 5) . the size of the blood vessel that is going to be catheterized is important and, in ideal conditions, the external diameter of the catheter should correspond, at most, to a third of the internal diameter of the vein (for example, a 4-fr catheter requires a 4 mm-wide vein). the risk of thrombosis increases when the caliber of the catheter is wider than recommended. 43 it is necessary to keep the measures of asepsis including the use of sterile gel and a protective cover for the probe to reduce the risk of catheter related bacteremia. two approaches are advised here: ultrasound-guided, the ultrasound is performed prior to the puncture to study the target vessel, analyze its size, depth, patency, and vascular puncture using the seldinger technique. this technique visualizes the tip of the needle while inside the blood vessel during the procedure and the advance of the metal guidewire and later ultrasound-guided placement of the catheter: after confirming the size of the blood vessel and lack of inner thrombi, the puncture is performed without seeing the needle with the ultrasound transducer 39 (appendix b annex 6 of the dsd). cardiovascular and cardiac damage due to sars-cov-2 seems to be associated with a higher mortality rate. myocardial lesion is confirmed through higher biomarker levels of myocardial damage (troponin i or t or natriuretic peptides) that has been reported with higher rates (from 7% to 28%), 44---48 and is associated with more icms admissions, greater need for mv, coagulopathy, acute kidney injury, and a higher mortality rate. 44 the mechanism of myocardial damage is a complex one, but the direct lesion mediated by the angiotensinconverting enzyme 2 (ace 2), hypoxia, and the possibility of a cytokine inflammatory response have been postulated in its pathogenesis. 49, 50 there are 2 different types of myocardial lesions: • subacute: it consists of the progressive increase of the biomarker levels of myocardial damage and inflammation (d-dimer, ferritin, lactate, etc.) with slow progression and gradual impairment of organ functions probably associated with a lesion due to cytokine storm or hemophagocytic lymphohistiocytosis that eventually leads to the death of the patient 2/3 weeks after symptom onset. • acute: damage of sudden onset with poor progression that causes sudden myocardial dysfunction within a few hours. it is consistent with myocarditis or stress cardiomyopathy. with the actual evidence base available this second pattern is apparently rare. 51---53 the echocardiography, both basic and advanced, is essential for the management of patients with sars-cov-2 infection with shock or myocardial damage and for the detection of acute cor pulmonale (acp) ( table 3) . it is essential to know the interactions between the lung and the heart of patients on mv as well as the interac-tions between these patients and vasoactive and inotropic drugs and systems of extracorporeal membrane oxygenation (ecmo). 54, 55 echocardiographic studies should be conducted according to the international recommendations on heart quantification. 56 in light of the high volume of patients due to the current pandemic it is important to minimize the time devoted to each study and perform targeted echocardiographies to answer easy questions. 57 in patients with high biomarker levels of myocardial damage, the differential diagnosis of acute myocardial infarction (ami), myocarditis or pulmonary thromboembolism (pte) should be performed. left ventricular systolic function (lvsf) is the most studied element in echocardiographies and is an essential aspect of echocardiographic studies. keeping a proper cardiac output not only depends on cardiac contractility, but also on preload, afterload, heart rate, and a proper segmental contraction synchrony. the effect of all of these factors should be taken into consideration when interpreting echocardiographic findings. left ventricular ejection fraction (lvef) is often estimated using the biplane method. however, qualitative or semiquantitative visual assessments have proven a good correlation with the standard method in critically ill patients due to the worse definition of endocardial border in this population, which is why it can be a valid alternative. 2, 58 we should mention that lvef is mostly influenced by left ventricular (lv) contractility and afterload. 59 therefore, it does not adequately show the contractile capabilities of the left ventricle in an acute situation, but the way the cavity adapts itself to loading conditions, which can have very important dynamic changes in states of shock. 60 the disease due to sars-cov-2 often causes damage to segmental contractility. when these alterations are identified in the theoretical territory of a coronary artery the possibility of acute ischemia associated with a type-1 ami should be suspected, especially if it is associated with consistent electrocardiographic changes and high biomarker levels of myocardial damage. however, studies published during the pandemic show a higher rate of type-2 amis. when these alterations are diffuse or not consistent with the territory of theoretical distribution of a coronary artery, alternative diagnoses like myocarditis or stress cardiomyopathy should be suspected especially in unstable patients. 44, 50, 61 myocarditis is an entity often associated with virus infections and has a wide constellation of clinical presentations that go from mild clinical signs to cardiogenic shocks or ventricular arrhytmias that may eventually lead to the death of the patient. the sars-cov-2 coronavirus is capable of damaging the myocardium through direct or indirect action mediated by the inflammatory response it triggers. this clinical status should be suspected in patients with electrocardiographic alterations and high levels of cardiac biomarkers. although the gold standard for diagnosis purposes is endomyocardial biopsy, in the routine clinical practice it is established based on the cardiac magnetic resonance (cmr) imaging findings. however, given the potential risks involved in the transfer of critically ill patients to undergo an mri for diagnostic purposes and the technical difficulties of mv, the echocardiography is a not a very reliable imaging modality in many patients. it can help diagnose fulminant myocarditis after visualization of the acute thickening of ventricular walls due to interstitial edema and segmental contractility alterations that trigger a reduced lvef and, occasionally, left ventricular dilatation, right ventricular (rv) dysfunction, and pericardial effusion without signs of hemodynamic compromise. 57 stress cardiomyopathy, apical ballooning syndrome or the tako-tsubo syndrome is a clinical syndrome often triggered by a stressor (such as sars-cov-2 infection). it is characterized by transient left ventricular dysfunction due to segmental contractility regional alterations associated with electrocardiographic changes and high levels of cardiac biomarkers. 62 up to 4 different patterns of segmental alterations have been reported. however, the most common of all is hypokinesia or akinesia of medial and apical segments with compensatory hypercontractility of basal segments that give the left ventricle a typical balloon appearance. the differential diagnosis of the least common patterns with acute coronary syndrome or acute myocarditis requires performing a cardiac magnetic resonance (cmr) imaging. 63 although the echocardiography does not provide continuous information on the patient's hemodynamic status, it is an excellent tool to characterize the state of shock, choose the best therapeutic option, and assess the response to it. 4 the european guidelines on hemodynamic monitoring in critically ill patients 64 recommend performing an echocardiography as the best way to study cardiac function. also, they establish the quantification of the left ventricular outflow tract velocity time integral (lvot vti) as the only parameter to estimate the systolic volume of these patients by omitting the estimate of the lvot area and monitoring its changes after therapeutic measures like the administration of fluids, inotropic, and vasopressor drugs 60, 64 (appendix b annex 7 of the dsd). in the case of the overall left ventricular systolic function measusing the lvot vti and the qualitative visual assessment of lvef is advised. other parameters more difficult to obtain, time-consuming or associated with greater limitations are ill-advised. 60 in patients on ecmo support, it is necessary to perform an echocardiography to assess the cardiac function prior to cannulation in order to choose the most suitable modality: veno-venous or veno-arterial. 65---67 use of echocardiography for the detection and management of acute cor pulmonale the acute cor pulmonale (acp) is defined as a right ventricular failure due to primary pulmonary disorder that triggers a sudden increase of pulmonary vascular resistances. 54 the leading causes of acp in patients admitted to an icms are ards and pte. 68 the echocardiographic findings that define acp are 69 : • right ventricular dilatation • paradoxical septal motion • dilatation of the inferior vena cava (ivc) • pulmonary hypertension • right ventricular systolic dysfunction the acp is a common cause of circulatory failure in ards and can be associated with a higher mortality rate in its more severe forms. 70 the detection of acp leads to the use of ventilatory strategies «with protective cardiopulmonary parameters» that should control the main determinants of an increased right ventricular afterload: proper control of plateau pressures (< 27 cmh 2 o), and distension (< 18 cmh 2 o), optimization of oxygenation, and correction of hypercapnia. 71, 72 assessing the right ventricular size the assessment of the right ventricle should start in the parasternal long-axis view (plax) of the left ventricle that shows the size of the rv and its relation to the lv. the parasternal short-axis view (psax) at papillary muscle or mitral valve level shows the rv hugging the lv in a crescent shape. when the rv is dilated, the anterolateral wall is difficult to see and there is clockwise rotation of the cavity. the apical 4-chamber (a4c) view provides the best information on the size of the rv because we can compare it to the lv. but for this to happen the a4c view needs to be focused on the rv to make sure there is no foreshortening phenomenon of the rv and the left ventricular outflow tract cannot be seen (appendix b annex 8 of the dsd). 73 although standard measures of the rv can be taken with specific cuts, the correlation between both ventricles is often used to establish the right ventricular dilatation 69 : • rv/lv < 0.6 → normal size of the rv. • rv/lv = 0.6---1 → mild dilatation of the rv. • rv/lv > 1 → severe dilatation of the rv. interventricular septum (ivs) is a structure that both ventricles share although functionally it is part of the lv in algorithm for emergency airway management in patietns with coronavirus though comprehensive management including visual inspection and ultrasound use. before starting intubation, it will be necessary to confirm the presence of bilateral lung sliding. after preliminary verification, the oti will be initiated through the implementation of the difficult airway management algorithm of every center. here the videolaryngoscopy as the first device is advised. the direct visualization of the passage of the ott through the vocal cords facilitates the start of ventilation, the identification of visual signs of a proper intubation, and the adjustment of the position of the ott using the bilateral pleural ultrasound. being unable to see the passage of the ott through the vocal cords rules out the possibility of esophageal intubation before starting ventilation to avoid the pulmonary aspiration of gastric content. v. fraile gutiérrez et al. normal conditions. the ivs is often convex towards the rv and concave towards the lv and keeps this morphology during the entire cardiac cycle. however, in the acp, the ivs is flattened during the entire cardiac cycle giving it its characteristic d-shape. the ifc is studied in the subcostal plane. in the acp we may find a dilated ifc (> 21 mm) with scarce respiratory collapse. its size and collapsibility are the ultrasound method used to assess pressure in the right atrium. the assessment of pulmonary hypertension using the echocardiography is performed by estimating pulmonary artery systolic pressure (pasp) using the tricuspid regurgitation pressure gradient (appendix b annex 9 of the dsd). when the acoustic window is limited or suboptimal, a better signal can be acquired through the iv administration of 10 ml of agitated saline solution. the dap can be measured through a cvc or estimated from the size and respirophasic variation of the ifc. in acute situations of pressure overload the rv cannot adapt fast enough and the pressures found in these cases are often relatively low (< 60 mmhg). when the pasp cannot be estimated using the method already described, the pulmonary artery acceleration time (paat) can be used (appendix b annex 9 of the dsd). in order to quantify the right ventricular systolic function, the recommendations established by the international guidelines should be followed. 56, 74 the parameters most commonly used are the tricuspid annular plane systolic excursion (tapse) and the tricuspid annular peak systolic velocity (tapsv) mediated by the tissue doppler imaging (st wave). these parameters are easy to obtain, reproducible, and have a good correlation with other more complex methods to study the right ventricular systolic function 54 (appendix b annex 10 of the dsd). a large number of patients with covid-19 develop ards and need mv in the decubitus pone position to treat arf over long periods of time. during this time, many patients require an echocardiographic study for different reasons. however, placing patients in the supine decubitus position just to undergo this test can worsen their respiratory failure. the alternatives are using a transesophageal transducer or performing a transthoracic echocardiography (tte) in the prone decubitus position. to that end, the patient needs to be placed in the swimmer position with his left upper extremity above his head. the patient's left shoulder needs to be elevated on a pillow and the transducer placed at 5th left intercostal space level in the midclavicular line to obtain all the measures associated with the apical plane. 75 (fig. 6 ). none reported. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.medine.2020.10.002. a novel coronavirus from patients with pneumonia in china consensus document on ultrasound training in intensive care medicine. care process, use of the technique and acquisition of professional skills hemodynamic monitoring in the critically patient. recomendations of the cardiological intensive care and cpr working group of the spanish society of intensive care and coronary units point-of-care lung ultrasound in patients with covid-19 -a narrative review chinese critical care ultrasound study g. findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic covid-19 outbreak: less stethoscope, more ultrasound lung ultrasound and b-lines quantification inaccuracy: b sure to have the right solution point-of-care lung ultrasound findings in novel coronavirus disease-19 pnemoniae: a case report and potential applications during covid-19 outbreak persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents covid-19 lo que el técnico tiene que saber products with emerging viral pathogens and human coronavirus claims for use against sars-cov-2 surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) verification of endotracheal tube placement the assessment of four different methods to verify tracheal tube placement in the critical care setting comparison of three different methods to confirm tracheal tube placement in emergency intubation ) for confirming endotracheal tube placement during emergency intubation confirmation of endotracheal tube placement after intubation using the ultrasound sliding lung sign blue-protocol and falls-protocol: two applications of lung ultrasound in the critically ill a bedside ultrasound sign ruling out pneumothorax in the critically ill. lung sliding thoracic ultrasonography: a narrative review chest ultrasound in acute respiratory distress syndrome global and regional diagnostic accuracy of lung ultrasound compared to ct in patients with acute respiratory distress syndrome relevance of lung ultrasound in the diagnosis of acute respiratory failure: the blue protocol six versus eight and twenty-eight scan sites for b-line assessment: differences in examination time and findings bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment ultrasound assessment of lung aeration loss during a successful weaning trial predicts postextubation distress* diagnosis of pneumothorax by radiography and ultrasonography: a meta-analysis pleural ultrasonography versus chest radiography for the diagnosis of pneumothorax: review of the literature and meta-analysis thelung point: an ultrasound sign specific to pneumothorax sonographic diagnosis of pneumothorax focused abdominal sonogram for trauma: the learning curve of nonradiologist clinicians in detecting hemoperitoneum pleural ultrasonography whole body ultrasonography in the critically ill abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy real-time ultrasound-guided catheterisation of the internal jugular vein: a prospective comparison with the landmark technique in critical care patients guidelines for performing ultrasound guided vascular cannulation: recommendations of the american society of echocardiography and the society of cardiovascular anesthesiologists guidelines on the use of ultrasound guidance for vascular access anatomical variations in the internal jugular veins of cancer patients affecting central venous access. anatomical variation of the internal jugular vein international evidence-based recommendations on ultrasound-guided vascular access espen guidelines on parenteral nutrition: central venous catheters (access, care, diagnosis and therapy of complications) association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinicolaboratory study of 25 fatal cases of covid-19 in wuhan cardiac involvement in a patient with coronavirus disease 2019 (covid-19) potential effects of coronaviruses on the cardiovascular system: a review covid-19 and the cardiovascular system coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin first case of covid-19 complicated with fulminant myocarditis: a case report and insights the right ventricle in ards a decade of progress in critical care echocardiography: a narrative review recommendations for cardiac chamber quantification by echocardiography in adults: an update from the american society of echocardiography and the european association of cardiovascular imaging expert opinion of the working group on echocardiography of the polish cardiac society on performing echocardiographic examinations during covid-19 pandemic visually estimated ejection fraction by two dimensional and triplane echocardiography is closely correlated with quantitative ejection fraction by real-time three dimensional echocardiography beyond ejection fraction: an integrative approach for assessment of cardiac structure and function in heart failure pearls and pitfalls in comprehensive critical care echocardiography clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study international expert consensus document on takotsubo syndrome (part ii): diagnostic workup, outcome, and management international expert consensus document on takotsubo syndrome (part i): clinical characteristics, diagnostic criteria, and pathophysiology consensus on circulatory shock and hemodynamic monitoring. task force of the european society of intensive care medicine position paper for the organization of ecmo programs for cardiac failure in adults echocardiography: do we need new standards for ecmo patients? recomendaciones deḧacerÿno hacerën el tratamiento de los pacientes críticos ante la pandemia por coronavirus causante de covid-19 de los grupos de trabajo de la sociedad española de medicina intensiva echo-doppler demonstration of acute cor pulmonale at the bedside in the medical intensive care unit acute cor pulmonale predictors of mortality in acute respiratory distress syndrome. focus on the role of right heart catheterization acute respiratory distress syndrome: the heart side of the moon management of right heart failure in the intensive care unit guidelines for the echocardiographic assessment of the right heart in adults: a report from the american society of echocardiography endorsed by the european association of echocardiography, a registered branch of the european society of cardiology, and the canadian society of echocardiography contemporary management of acute right ventricular failure: a statement from the heart failure association and the working group on pulmonary circulation and right ventricular function of the european society of cardiology transthoracic cardiac ultrasound in prone position: a technique variation description key: cord-309722-04pp3lv0 authors: qiu, yingshan; lam, jenny k. w.; leung, susan w. s.; liang, wanling title: delivery of rnai therapeutics to the airways—from bench to bedside date: 2016-09-20 journal: molecules doi: 10.3390/molecules21091249 sha: doc_id: 309722 cord_uid: 04pp3lv0 rna interference (rnai) is a potent and specific post-transcriptional gene silencing process. since its discovery, tremendous efforts have been made to translate rnai technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). similar to other nucleic acid-based therapeutics, the major hurdle of rnai therapy is delivery. pulmonary delivery is a promising approach of delivering rnai therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. it is a non-invasive route of administration that is generally well accepted by patients. however, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. understanding these barriers is essential for the development an effective rna delivery system. in this review, the different barriers to pulmonary drug delivery are introduced. the potential of rnai molecules as new class of therapeutics, and the latest preclinical and clinical studies of using rnai therapeutics in different respiratory conditions are discussed in details. we hope this review can provide some useful insights for moving inhaled rnai therapeutics from bench to bedside. lung diseases are among the leading causes of death worldwide. current treatment of lung diseases such as lung cancer [1] , respiratory infections [2, 3] , inflammatory diseases [4, 5] and pulmonary fibrosis [6] have limited efficacy. almost two decades ago, double-stranded rnas (dsrnas) were discovered to play an important role in regulating gene functions by a sequence-specific post-transcriptional gene silencing mechanism called rna interference (rnai) [7] . the therapeutic potential of rnai was soon realized. it presents a new and powerful approach to treat or prevent many diseases including respiratory disorders by modulating gene expression [8] [9] [10] [11] [12] [13] . rnai can be mediated by various types of rna molecules, including long dsrna, short interfering rna (sirna), short hairpin rna (shrna) and microrna (mirna). the mechanism of rnai is illustrated in figure 1 , and the general properties of different types of rnai molecules are summarized in table 1 . long dsrnas (around 500-1000 nucleotides in length) have been employed to study gene functions. after the exogenous dsrna is introduced into the cytoplasm of the cells, it is cleaved by rnase iii enzyme dicer into short dsrna called sirna. the sirna which is 21-23 nucleotides in length is loaded into a protein complex called the rna-induced silencing complex (risc). the sirna is then unwound, and the sense strand (also known as the passenger strand) of the sirna is degraded, whereas the remaining antisense strand (also known as the guide strand) guides the activated rics to the target messenger rna (mrna) through full complementary binding. the mrna is then cleaved by argonaute 2 (ago2) in the risc, leading to the silencing of the target gene [14, 15] . since long dsrna is known to trigger immunostimulatory response through the activation of dicer-related antiviral pathways and induction of type 1 interferon (ifn) [16] , it is less suitable for therapeutic use. in contrast, synthetic sirna is a more promising gene silencing mediator because of the lower risk of immune response. it is also the most widely investigated rnai molecule for therapeutic applications, with over 26 clinical trial studies being initiated since 2004 [17] . shrna is a sequence of rna transcribed in the nucleus of the cells from a dna vector by either rna polymerase ii or iii. the primary transcript is called primary shrna (pri-shrna), which contains a hairpin like stem-loop structure. the pri-shrna is processed into a 50-70 nucleotides long loop-stem precursor shrna (pre-shrna) by a protein complex containing the rnase iii nuclease drosha and the dsrna binding domain protein dgcr8. it is then transported to the cytoplasm through a specialized nuclear membrane protein, exportin-5 (exp5). the loop sequence of the pre-shrna is cleaved by the dicer to form a double-stranded sirna. this endogenously produced sirna is loaded into risc and induce rnai through the similar process as the synthetic sirna [18, 19] . since shrna expression unit can be incorporated into viral vectors and continuously synthesized by the host cell, it can induce long-lasting gene silencing effect. the risc-loading process of shrna is about ten times more efficient than sirna, implicating that lower dose of shrna is required to maintain the therapeutic efficacy with less off-target effect [20] . however, the shrna approach is a dna-based strategy depending on the expression of shrna encoding gene which often requires viral vectors. from the delivery perspective, the introduction of synthetic sirna to the cytoplasm is a more straightforward way to induce rnai. the use of viral vectors for delivery poses safety concerns in its therapeutic applications [15] . mirna is a naturally occurring non-coding rna that plays a key role in regulating gene expression. primary mirna (pri-mirna) is transcribed by rna polymerase ii from endogenous mirna gene in the nucleus. the hairpin-containing pri-mirna is structurally similar to the pri-shrna, therefore the maturation pathway of mirna is also similar to shrna [18] . the pri-mrna is converted by drosha/dcgr8 complex into precursor mirna (pre-mirna), which contains 70-100 nucleotides with interspersed mismatches and adopts a loop structure. the pre-mirna is subsequently transported to the cytoplasm by exp5, and is processed by the dicer into mature mirna of 18-25 nucleotides in length [21] . as opposed to sirna, the antisense strand of mirna is only partially complementary to the target mrna, leading to gene silencing via translational repression and/or mrna deadenylation. position 2 to 7 of the 5 end of the mirna is the "seed sequence", which is essential for target recognition, and the mirna binding sites of mrna are located in the 3 untranslated region (utr) [22] . there are two major approaches of mirna-based therapeutics: (i) mirna inhibition [23, 24] , which suppresses the action of the endogenous mirna by antisense oligonucleotide (antimir); and (ii) mirna replacement [25, 26] , which introduces synthetic mirna (mirna mimic) to restore the functions of the endogenous mirna. only the latter is discussed in this review. compared to sirna-based therapeutics, mirna has the potential to target multiple genes due to the imperfect binding to the target mrna [27] . rnai therapeutics offer several advantages over the traditional small molecules and protein-based drugs. they could virtually target any genes with high selectivity, including those "undruggable" targets. the design and synthesis of rnai molecules are relatively simple because they do not need a cellular expression system, complex protein purification, or refolding schemes [28] . compared to the antisense oligonucleotides, rnai is more potent and specific [29] . despite its huge therapeutic potential, delivery remains to be a major barrier to the clinical application of rnai therapeutics [30, 31] . for the treatment of respiratory disorders, pulmonary route of administration can deliver rnai molecules directly to the site of action, thereby lowering the dose required while minimizing systemic adverse effects. the rnai molecules can also avoid the rapid clearance by serum nuclease in the bloodstream. moreover, inhalation is a non-invasive route of administration that is easily accepted by patients compared to parenteral administration, leading to better patient compliance [14, 32] . however, pulmonary drug delivery is a challenge with several key biological barriers, which are highlighted in figure 2 . rnai therapeutics offer several advantages over the traditional small molecules and protein-based drugs. they could virtually target any genes with high selectivity, including those "undruggable" targets. the design and synthesis of rnai molecules are relatively simple because they do not need a cellular expression system, complex protein purification, or refolding schemes [28] . compared to the antisense oligonucleotides, rnai is more potent and specific [29] . despite its huge therapeutic potential, delivery remains to be a major barrier to the clinical application of rnai therapeutics [30, 31] . for the treatment of respiratory disorders, pulmonary route of administration can deliver rnai molecules directly to the site of action, thereby lowering the dose required while minimizing systemic adverse effects. the rnai molecules can also avoid the rapid clearance by serum nuclease in the bloodstream. moreover, inhalation is a non-invasive route of administration that is easily accepted by patients compared to parenteral administration, leading to better patient compliance [14, 32] . however, pulmonary drug delivery is a challenge with several key biological barriers, which are highlighted in figure 2 . human respiratory tract is divided into the conducting region (nasal cavity, pharynx, trachea, bronchi and bronchioles) and the respiratory region (respiratory bronchioles and alveoli). the former is the pathway for gases conduction whereas the latter is the site of gaseous exchange [14] . the extensive branched structure of the airways with varying length and diameter presents the primary hurdle in pulmonary drug delivery. the mechanisms of inhaled particle deposition in the lungs include inertial impaction, gravitational sedimentation, diffusion, interception and electrostatic precipitation [33] . as illustrated in figure 3 , the first three mechanisms are the major mechanisms which are greatly influenced by the aerodynamic size of the particles [34] . the optimal particle size for lung deposition is between 1 and 5 µm [35, 36] . larger particles are likely to be impacted and trapped on the upper airway wall at bifurcations while the small particles from 0.1 to 1 µm are easily exhaled during normal breathing. smaller particles under 100 nm may be successfully deposited in the alveolar space because of the increasing diffusional mobility [37] . however, aerosols under 100 nm are difficult to produce, which make their application less favorable. human respiratory tract is divided into the conducting region (nasal cavity, pharynx, trachea, bronchi and bronchioles) and the respiratory region (respiratory bronchioles and alveoli). the former is the pathway for gases conduction whereas the latter is the site of gaseous exchange [14] . the extensive branched structure of the airways with varying length and diameter presents the primary hurdle in pulmonary drug delivery. the mechanisms of inhaled particle deposition in the lungs include inertial impaction, gravitational sedimentation, diffusion, interception and electrostatic precipitation [33] . as illustrated in figure 3 , the first three mechanisms are the major mechanisms which are greatly influenced by the aerodynamic size of the particles [34] . the optimal particle size for lung deposition is between 1 and 5 µm [35, 36] . larger particles are likely to be impacted and trapped on the upper airway wall at bifurcations while the small particles from 0.1 to 1 µm are easily exhaled during normal breathing. smaller particles under 100 nm may be successfully deposited in the alveolar space because of the increasing diffusional mobility [37] . however, aerosols under 100 nm are difficult to produce, which make their application less favorable. the presence of fluid layers in the airways, including the mucus and the pulmonary surfactant, also creates a barrier to the delivery of drug molecules to the lungs. mucus is a gel of three-dimensional network structure. it acts as a sieve that filters out large molecules (>500 nm) [38] . mucins, which are the major components of mucus, contain the hydrophilic glycosylated residues rich in serine and threonine and the hydrophobic non-glycosylated cysteine-rich domains. this characteristic allows electrostatic, hydrophilic and hydrophobic interactions between mucus and drug particles [39] . since mucus is continuously produced, shed and replaced, and together with the mucociliary clearance action, the fast turnover rate of mucus leads to the rapid clearance of the entrapped drug molecules, preventing them from reaching the epithelium [40, 41] . moreover, cough clearance that occurs when the mucus reaches the throat is another removal mechanism of rnai molecules from the airways. on the other hand, the role of pulmonary surfactant in nucleic acid delivery is controversial. pulmonary surfactant is composed of approximately 90% lipids and 10% proteins [42] . it has been suggested that the lipids and proteins in the lung surfactant interact with the non-viral cationic lipid-based delivery system, leading to the premature release of nucleic acids [43, 44] . conversely, polymer-based delivery systems were found to be more compatible with pulmonary surfactant. commercial pulmonary surfactant was employed in the preparation of some polymeric nanoparticles to facilitate the cellular uptake of sirna to improve gene silencing effect [45, 46] . apart from the physical barriers, the rnai molecules may undergo enzymatic degradation following pulmonary delivery. rna is extremely susceptible to nuclease activity. naked sirna has a short half-life of less than 15 min in serum [47] . due to the lack of serum in the lungs, the half-life of unmodified sirna in the airways can reach up to three hours in mouse [48] . rnai molecules may also be taken up and removed by alveolar macrophages upon lung deposition. the alveolar macrophages are responsible for engulfing and digesting foreign particles in the lower airways via phagocytosis. unless the target of rnai molecules is located inside the macrophages, such as mycobacterium tuberculosis which is the causative agent of tuberculosis that typically reside in the alveolar macrophages [49] , otherwise the rnai molecules are subjected to degradation inside the macrophages before reaching their target sites. the presence of fluid layers in the airways, including the mucus and the pulmonary surfactant, also creates a barrier to the delivery of drug molecules to the lungs. mucus is a gel of three-dimensional network structure. it acts as a sieve that filters out large molecules (>500 nm) [38] . mucins, which are the major components of mucus, contain the hydrophilic glycosylated residues rich in serine and threonine and the hydrophobic non-glycosylated cysteine-rich domains. this characteristic allows electrostatic, hydrophilic and hydrophobic interactions between mucus and drug particles [39] . since mucus is continuously produced, shed and replaced, and together with the mucociliary clearance action, the fast turnover rate of mucus leads to the rapid clearance of the entrapped drug molecules, preventing them from reaching the epithelium [40, 41] . moreover, cough clearance that occurs when the mucus reaches the throat is another removal mechanism of rnai molecules from the airways. on the other hand, the role of pulmonary surfactant in nucleic acid delivery is controversial. pulmonary surfactant is composed of approximately 90% lipids and 10% proteins [42] . it has been suggested that the lipids and proteins in the lung surfactant interact with the non-viral cationic lipid-based delivery system, leading to the premature release of nucleic acids [43, 44] . conversely, polymer-based delivery systems were found to be more compatible with pulmonary surfactant. commercial pulmonary surfactant was employed in the preparation of some polymeric nanoparticles to facilitate the cellular uptake of sirna to improve gene silencing effect [45, 46] . apart from the physical barriers, the rnai molecules may undergo enzymatic degradation following pulmonary delivery. rna is extremely susceptible to nuclease activity. naked sirna has a short half-life of less than 15 min in serum [47] . due to the lack of serum in the lungs, the half-life of unmodified sirna in the airways can reach up to three hours in mouse [48] . rnai molecules may also be taken up and removed by alveolar macrophages upon lung deposition. the alveolar macrophages are responsible for engulfing and digesting foreign particles in the lower airways via phagocytosis. unless the target of rnai molecules is located inside the macrophages, such as mycobacterium tuberculosis which is the causative agent of tuberculosis that typically reside in the alveolar macrophages [49] , otherwise the rnai molecules are subjected to degradation inside the macrophages before reaching their target sites. once the rnai molecules overcome the aforementioned extracellular barriers and reach the target cells, they need to overcome a set of intracellular barriers. the ultimate site of actions depends on the types of rnai molecules. for synthetic sirna and mirna mimic, they need to reach the cytoplasm where the risc locates, whereas dna encoding shrna or mirna need to enter the nucleus in order for transcription to take place. surface adhesion is the first step of cellular entry. there are various endocytic mechanisms involved in the cellular uptake of macromolecules, which subsequently influence their intracellular trafficking and delivery efficiency [50] [51] [52] [53] [54] [55] . the clathrin-dependent endocytosis is the most common route of cellular entry for macromolecules [56] . following cell entry, the rnai molecules are entrapped in the early endosomes where progressive acidification occurs. the late endosomes then fuse with the lysosomes, which contain hydrolases that degrade the rnai molecules [56, 57] . the rnai molecules must escape from endosome at early stage to exert their biological effect. strategies of endosomal escape have been reviewed in other literatures [58] [59] [60] [61] . for shrna or mirna vectors, nuclear entry is an additional barrier which is particularly challenging due to the presence of nuclear membranes that are impermeable to most substances except small non-polar molecules [62] . non-viral vectors are inefficient in delivering dna into the nucleus. some viruses are evolved to transport their genome into the nucleus; hence, they become attractive dna carriers to improve transduction efficiency. however, poor safety profile, high production cost and the risk of immunogenicity [63] limited the applications of viral vector mediated rnai to laboratory tool. a delivery vector or carrier is generally necessary to overcome the aforementioned barriers by promoting cellular uptake and offering protection to the rnai molecules. rnai delivery system can be categorized into viral and non-viral vectors according to their nature, and these delivery systems have been extensively reviewed [63] [64] [65] [66] [67] . the viral vectors refer to the of use of viruses to deliver genetic materials to the cells. they are extremely efficient in transducing cells and providing either transient or long-term gene expression, depending on the type of viruses employed. most of the in vivo studies used the adenoviruses, adeno-associated virus (aav) and retroviruses to express the plasmid dna encoding shrna or mirna to induce rnai [68] . the most attractive property of viral vectors is that they have the ability to access the nucleus of the cells. thus, they have high efficacy to express the rna and subsequently regulate the gene expression [69] [70] [71] [72] . however, the clinical application of viral vectors is limited by the toxicity, insertional mutagenesis (associated with retroviruses) and immunogenicity (associated with adenoviruses) [63] . in addition, the smaller size of avv limits the amount of therapeutic gene that can be inserted. compared to viral vectors, non-viral vectors generally have better safety profile and lower production cost. they have been investigated for the pulmonary delivery of the rnai molecules. lipid-based, polymer-based and peptide-based are three major non-viral delivery systems [73] . the lipid-based vectors are the most commonly used vectors for rnai delivery. they include cationic liposomes, solid lipid nanoparticles, solid nanostructured lipid carriers, lipidoids and ph-responsive lipids [74] . many commercially available transfection agents (i.e., lipofectamine, oligofectamine, transit-tko and dharmafect) are lipid-based systems [75] [76] [77] [78] . despite the promising transfection efficacy, the major challenges of using the lipids are the immune response and cytotoxicity [79] . in addition to the lipids, biocompatible and biodegradable natural polymers such as chitosan and dextran, as well as synthetic polymers such as poly lactic-co-glycolic acid (plga), polyethylenimine (pei) and pamam dendrimer are used for the preparation of polymeric nanoparticles for rna delivery [80] [81] [82] [83] [84] [85] . the polymers have relatively low toxicity compared to the lipid-based vectors and are more versatile for chemical modification. for peptide-based delivery systems, peptides such poly(l-lysine) (pll), cell penetrating peptides (cpps) and ph-responsive peptides have been investigated for the delivery of the rnai molecules [86] [87] [88] [89] [90] . different types of peptides mediate cellular transfection via different mechanisms such as facilitating cellular uptake and promoting endosomal escape. peptides can be used to carry the rnai molecules alone or act as a functional component in other carriers. currently, efficient peptides with low toxicity and high transfection efficacy are still under development. it is noted that for pulmonary delivery, the reduced nuclease activity in the lungs makes successful delivery of unmodified naked rna become possible [91, 92] . however, the mechanism of how naked sirna could overcome the extracellular and intracellular barrier following pulmonary administration still remains to be understood. the therapeutic potential of rnai-based therapy targeting respiratory disorders has been widely explored in the past decade. in the following sections, the pre-clinical and clinical studies that involved the delivery of rnai molecules to the airways for the treatment of lung diseases are discussed. lung cancer is a leading cause of cancer death in the world. the mainstay of treatment is chemotherapy, which is indicated for around 70% of newly diagnosed patients with local and advanced metastatic disease [93] . other treatment options include surgery and radiotherapy [94] . angiogenesis inhibitors [95] and epidermal growth factor receptor (egfr) inhibitors [96, 97] are new medications licensed for the treatment of lung cancer. despite various treatment options, the prognosis of lung cancer is poor. the major problems of chemotherapy are toxicity, lack of specificity and drug resistance [98] . in recent years, pulmonary delivery of rnai molecules is being explored as a new treatment for lung cancer [99] . genes that are associated with tumor cell growth and drug resistance are potential targets. table 2 summarizes some recent in vivo studies of pulmonary rnai delivery for lung cancer treatment. genes that are essential for tumor growth and progression, such as akt1 (rac-alpha serine/threonine-protein kinase b) and c-myc, are evaluated as rnai targets. akt1 is an important mediator of cell growth, proliferation and survival of non-small cell lung cancer (nsclc). activation of akt1 pathway is observed in most nsclc patients. it is found to reduce chemo-and radiation-induced apoptosis to extend the survival of tumor cells [100, 101] . sorbitol diacrylatepolyethylenimine (sda-pei) was employed to deliver shrna targeting akt1 (shakt1) and cdna encoding programmed cell death protein 4 (pdcd4) in a dual expression vector via pulmonary route to murine lung cancer model. simultaneous akt1 inhibition and pdcd4 overexpression could induce potent anticancer effect. the total tumor number and the tumor size larger than 1 mm in the lung were significantly reduced after eight doses [101] . glycerol triacrylate-spermine (gt-spe) polyspermine [102] and lentivirus [103] were also employed as carriers of shrna targeting akt1 signaling pathway for pulmonary delivery. although repeated aerosol delivery of lentiviral-based shrna can achieve potent knockdown of the target, inhalation is a procedure that generates large number of viral particles. there are safety concerns regarding the use of viral vectors for inhalation, including the potential mutagenicity, immunogenicity and the risk of aerosol-transmitted infections [104] . therefore, aerosol delivery of rnai molecules using viral vectors must go through vigorous biosafety evaluation for clinical use. notes: aimp2-dx2, aminoacyl-trna synthetases (ars)-interacting multifunctional protein 2 lacking exon 2; akt1, rac-alpha serine/threonine-protein kinase b; bcl-2, b-cell lymphoma 2; dox: doxorubicin; egfp, enhanced green fluorescence protein; gpt-spe: glycerol propoxylate triacrylate and spermine; gt-spe, glycerol triacrylate-spermine; mrp1, multidrug resistance protein 1; npt2b: sodium-dependent phosphate co-transporter 2b; pdcd4: programmed cell death protein 4; pei: polyethylenimine; rgd, arginine-glycine-aspartic acid peptide; rpn2, ribophorin ii; nps, nanoparticles; sda-pei, sorbitol diacrylatepolyethylenimine; tax, paclitaxel. c-myc oncogene is a downstream conduit for most oncogenic signals. it is necessary for the growth control, differentiation and apoptosis. abnormal expression of c-myc is associated with many tumors. de-activation of myc is efficacious for lung tumor therapy [111] . conde et al. delivered sirna targeting c-myc to the lung of mouse using rgd-surface-modified gold nanoparticles (aunps) by intratracheal instillation. rgd peptide (arg-gly-asp) is a cell adhesion peptide responsible to mediate cell adhesion and proliferation by binding to the integrin avb3 receptor family, a specific marker for angiogenesis and up-regulated in the endothelium. this strategy successfully suppressed c-myc expression level and tumor cell proliferation, resulting in approximately 80% increase in survival of orthograft mouse tumor models [107] . genes associated with mdr were also evaluated as target of rnai, such as ribophorin ii (rpn2) [106] , b-cell lymphoma 2 (bcl-2) [112] and multidrug resistance protein 1 (mrp1) [110] . rpn2 is known to be associated with the apoptosis in docetaxel-resistant human breast cancer cells [113] , and its involvement in nsclc was investigated recently [114] . a novel rnai molecule (pnkrna) targeting rpn2 was developed and delivered to mice bearing lung cancer by intrapulmonary delivery. this new class of rnai agent has a unique helical structure containing a central stem and two loops, and is claimed to be stable against nuclease degradation. inhibition of lung tumor growth was observed without any signs of toxicity after treatment. the antitumor effect was triggered by pnkrna targeting rpn2 without the use of other anticancer drugs. it could be attributed to the multiple activities of rpn2, which is anti-apoptotic and involved in the regulation of tumor survival [106] . however, the mechanism of how the naked the pnkrna molecules overcame the extracellular and intracellular barriers to initiate rnai was not elucidated. besides the inhibition of mdr associated proteins alone, the co-delivery of anticancer drugs with rnai therapeutics is as an alternative approach to tackle mdr in various types of cancers [112, [115] [116] [117] . combination therapy offers the potential advantages of synergistic activity, overcoming the drug resistance and reducing the side effects of chemotherapy. bcl-2 is a main player of nonpump cellular resistance [118] and mrp1 is responsible for drug efflux in cancer cells [119] . they were investigated in the combination approach. taratula et al. prepared a lipid-based nanoparticles containing sirna targeting bcl-2 and mrp1, as well as anticancer drugs (doxorubicin or paclitaxel). the nanoparticles were delivered to the lung of tumor mouse model using a nose only exposure chamber for inhalation. almost complete disappearance of lung tumor was observed in animals treated with paclitaxel plus the sirnas. in contrast, treatment with paclitaxel alone only slowed down the growth of the tumor [110] . xu et al. developed a polyethylenimine (pei)-based delivery system that simultaneously delivered sirna targeting bcl-2 and doxorubicin to the lungs of mice with metastatic lung cancer. synergistic antitumor efficacy was achieved compared with the delivery of doxorubicin or sirna alone [105] . these studies indicated that suppression of resistance gene by rnai molecules via inhalation could restore the sensitivity of chemotherapeutic drugs, leading to the death of tumor cells. respiratory infection can be caused by a wide range of microorganisms in the airways including bacteria and viruses [120] . it is one of the most common reasons for hospitalization among adults [121] . due to the rise of antimicrobial resistance, many infectious diseases including respiratory infections have become difficult to treat. rnai therapeutics appears to be an attractive approach to fight against infections [122] . the pre-clinical studies of rnai therapeutics to combat against respiratory infections are summarized in table 3 . since rnai was first reported to inhibit respiratory syncytial virus (rsv) in 2001 [131] , an increasing number of studies have been initiated to explore the potential of rnai technology to treat other respiratory viral infections including influenza [86, 127, 132, 133] and severe acute respiratory syndrome (sars) [134] . rnai molecules offer several advantages as antiviral therapeutics. they can specifically target the conserved region of mrna sequence in the viral genome, making it effective against mutated viral strains. in addition, rnai molecules can be designed rapidly to target viral genes, shortening the lead time of developing new antiviral agents, which is particularly useful in response to an infection outbreak. to achieve effective antiviral effect, the viral targets must be essential for the pathogenesis of viral infection and/or replication cycle. it is also desirable that the target genes share similar conserved sequence among different strains to achieve broad viral inhibition. furthermore, the viral gene should be substantially different with human gene to minimize any undesirable side effects. rsv infection is one of the most common infections in children [2, 135] . in adult, rsv infection usually leads to self-limited upper respiratory illness [136] . however, severe rsv infection has been observed in elderly patients with lung diseases or in immunocompromised patients [137] . current management of rsv includes bronchodilators, corticosteroids, antibiotics and supportive care [2] , but their efficacy is not satisfactory. to date, there is no licensed vaccine available to prevent this disease [138] . there is an unmet need to develop effective rsv therapeutics and vaccines in both the pediatric and adult population. in 2005, bitko et al. reported the inhibition of rsv and parainfluenza virus (piv) replication by sirna targeting viral phosphoprotein (p protein), a crucial subunit of the viral rna-dependent rna polymerase. since rsv replicate primarily in the superficial layer of the respiratory epithelium, local delivery of rnai molecules to the lungs is a rational approach to inhibit rsv replication [139] . in the study, transit-tko was employed as transfection agent and the sirna was administered to the lungs of balb/c mice by intranasal instillation 4 h before viral challenge. the rsv and piv titer was reduced by over 90% five days after infection. the use of naked sirna also showed substantial inhibition of infection, with approximately 70%-80% efficiency achieved as compared to complexed sirna. interestingly, the administration of sirna before and concomitant with rsv infection was more effective in reducing viral load than treatment after infection [123] . the prophylactic regimen may not be a clinically attractive approach. to achieve optimal therapeutic benefit, fast detection and early intervention are crucial for the prognosis of rsv infection. it is desirable to initiate the antiviral rnai therapy as quickly as possible once rsv infection is confirmed in clinical practice. p protein may not be an ideal target as it is limited by its specificity to a particular viral strain [124] . another sirna, aln-rsv01, was designed to target the highly conserved region of the mrna encoding viral nucleocapsid protein (n protein). n protein is a core protein in the rna polymerase and plays a key role in the replication cycle of rsv [140] . after intranasal administration of aln-rsv01 to the lungs of mouse at 4 h prior to viral infection, 2.5-to 3.0-log-unit reductions in viral load was observed in comparison with the mismatch sirna control. for the treatment regimen, comparable antiviral efficacy was achieved in multiple daily doses of aln-rsv01 at day 1, 2 and 3 post-infection [124] . the phase i clinical study of aln-rsv01 was reported in 2008 [139] . intranasal administration of aln-rsv01 was well tolerated and the side effect profile was similar to placebo. later in 2010, the antiviral effect of aln-rsv01 was evaluated in a phase ii trial. aln-rsv01 was administered by nasal spray daily to healthy adults for two days before and for three days after rsv inoculation. the group treated with aln-rsv01 had a significantly lower number of rsv infection compared to the placebo group [140] . another phase ii b clinical trial of aln-rsv01 in rsv-infected lung transplant patients was also completed [141] . the primary endpoint of the study was to evaluate the effect of aln-rsv01 on the incidence of new or progressive bronchiolitis obliterans syndrome (bos) in rsv-infected lung transplant patients. even though the study marginally missed the primary endpoint statistically (p = 0.058), inhaled aln-rsv01 treatment induced a clinically meaningful reduction in the incidence of bos, reinforcing the potential of inhaled rnai as new antiviral therapeutics. seasonal influenza viruses, especially influenza type a virus, cause annual epidemics that led to millions of death worldwide [142] . it is one of the major public health problems. two categories of antiviral drugs are currently approved to treat influenza, including m 2 ion-channel protein inhibitors (adamantanes) and neuraminidase inhibitors (zanamivir and oseltamivir). however, some influenza a virus strains have shown to be resistant to these drugs [143, 144] , making the development of new antiviral drug urgently needed. a number of studies were carried out to investigate the use of rnai to inhibit viral gene expression and protect cells from influenza infection. the most commonly investigated targets are the nucleocapsid protein (np), polymerase acidic protein (pa) and polymerase basic protein (pb). these proteins are indispensable to influenza viral replication. more importantly, they are highly conserved across different subtypes of influenza virus strains [132] . the first animal study applying rnai against influenza was reported by ge et al. [145] . sirna targeting np or pa were complexed with pei. the complexes were injected intravenously to the mice 3 h before or 5 h after influenza virus (pr8 h1n1) infection. the antiviral effect of sirna targeting np was dose-dependent and combination of sirnas targeting np and pa enhanced the effect. in the same study, shrna targeting np or pb was also investigated. the dna was mixed with infasurf, a commercial available pulmonary surfactant. after intranasal instillation, a significant reduction of virus titer was detected 24 h after infection although the effect was less prominent compared with intravenous injection of pei complexes. infasurf may play a role as nucleic acid carrier. little is known about the interaction between dna and pulmonary surfactant in the airspace. it could be possible that the dna interacted with the charged lipid components in the pulmonary surfactant leading to the efficient spread and absorption in the airways [146] . another study reported the administration of sirna targeting np and/or pa in lipid carrier to influenza-infected mice by hydrodynamic injection and intranasal administration. the combined sirna delivery method effectively protected animals against lethal challenge of highly pathogenic avian influenza a viruses, including the h5n1, h7n7 and h9n2 subtypes [126] . since then, the potential of sirna for the treatment of influenza virus infection was continuously evaluated in vitro [133, [147] [148] [149] and in vivo [127] [128] [129] . however, the exploration of rnai for the treatment of influenza appears to reach the bottleneck. it could be partly attributed to the criticism of the antiviral effect induced by sirna, which was due to innate immune response rather than viral inhibition through rnai. development of chemically modified rnai molecules with minimal immunostimulatory effect could be helpful to delineate the mechanism of the antiviral effects [150] . tuberculosis (tb) is a bacterial lung infection caused by mycobacterium tuberculosis (mtb). because of the emergence of multidrug-resistant (mdr) and extensively drug-resistant (xdr) tb, the slow development of new anti-tb agents and the inefficiency of vaccination, tb is still a major global health problem. who reported that 9.6 million people suffered from tb in 2014 and 1.5 million die annually because of the disease [151] . a new and effective tb therapeutics is highly sought after. the approach of rnai-based therapeutics against tb aims to modulate the gene expression of the host instead of the bacillus because bacteria do not contain the requisite machinery for rnai. currently, rnai has been investigated for tb treatment in two directions: (i) targeting the host factors that are essential for the survival of mycobacteria; and (ii) modulating the host immune response to facilitate the elimination of mycobacterium [49] . autophagy is a host defense mechanism against mtb. boosting autophagy could be an effective strategy to promote the eradication of the mycobacteria. hence, a number of host factors that are involved in the regulation of autophagy were explored as the target of rnai, including bfl-1/a1 [152] , rap22a [153] , ras homologue enriched in brain (rheb) [154] and uv radiation resistance-associated gene (uvrag) [155] . down-regulation of these factors promoted autophagosome formation and reduced the bacterial growth. however, the existing studies of using rnai to suppress host factors are preliminary and in vivo data is lacking. moreover, it was recently reported that autophagy pathway may not be essential for restricting mtb growth [156] . further investigations are required to understand the relationship between mtb infection and autophagy pathway, and to better position the approach of rnai for the control of tuberculosis infection. another strategy is to modulate host immune response to improve the antimicrobial ability of the host [49] . one of the key characteristics of tb infection is the formation of granuloma, which is a compact, organized aggregate of immune cells. granuloma is previously considered as a host-protective structure to sequester the infecting mycobacteria, but recent work revealed that the pathogens could take advantage of granuloma formation to facilitate their proliferation and dissemination in the host [157] , as it provides a hospitable environment for mycobacteria to survive and replicate over a long period of time. there is a complicated chemokines and cytokines network involved in the granulomatous response. rnai can be used to target immunosuppressive cytokines in order to inhibit the growth of mtb. among them, tumor necrosis factor (tnf)-α and ifn-γ are critical in activating macrophages and triggering the formation of granuloma [158] . xcl1 (lymphotactin) is a chemokine produced by activated cd8 + t cells during chronic tb infection. it was suggested that xcl1 regulates ifn-γ production by cd4 + t cells and contribute to the stability of the granuloma [159] . when a single dose of sirna targeting xcl1 was delivered to the lungs of mice that were chronically infected with tb, the expression of xcl1 was effectively inhibited. the suppression of xcl1 expression in the lungs was associated with the decreasing number of t lymphocytes, reduction in the ifn-γ response, disorganized granulomatous lesions and higher fibrosis [130] . although the inhibition effect elicited by sirna was transient and did not provide significant therapeutic benefit, this proof-of-concept study demonstrated that the local-environment and immunopathology of the lungs can be modulated to favor host response via pulmonary delivery of sirna. other targets of rnai-based immunotherapy include the transforming growth factor β (tgf-β) and interleukin 10 (il-10) [13] . both of them are found to be elevated in tb patients, and their high level of expression is connected with tb reactivation [160] . during chronic infection, these cytokines restrain inflammation through affecting helper t cell 1 (t h 1) response and macrophage activation, leading to substantial reduction of their antimicrobial activity [161] . in that study, sirna targeting tgf-β1 was administered through the intrapulmonary route to il-10 knockout mice at 60 days post-infection. tgf-β1 gene silencing resulted in the increased expression of the antimicrobial mediators nitric oxide (no) and inducible nitric oxide synthase (inos), and significantly reduced the bacterial load for at least four weeks after treatment. a synergistic effect at improving the antimicrobial activity in the lungs was observed when both il-10 and tgf-β1 were targeted simultaneously. although the inhibition of bacterial load in this study was modest, it showed that sirna immunotherapy is a feasible approach to enhance the host antimicrobial capacity. moreover, mdr and xdr tb pose huge challenges for the current chemotherapeutic agents. combined immunotherapy targeting immunosuppressive cytokines and chemotherapy to target drug resistance bacteria population is a direction that is worth future investigation. respiratory inflammatory diseases impose substantial healthcare burden worldwide as many of the patients are inadequately controlled by current therapies [4, 5, 162] . these diseases are characterized by inflammation in the respiratory tract, which is manifested with increased expression of inflammatory cytokines and chemokines [163, 164] . the common lung inflammatory diseases include asthma, chronic obstructive pulmonary disease (copd) and acute lung injury (ali). table 4 lists the major in vivo studies that examine the effects of rnai molecules in respiratory inflammatory disease. notes: ahr, airway hyperresponsiveness; ali, acute lung injury; balf, bronchoalveolar lavage fluid; cd86, cluster of differentiation 86; c-kit, a stem cell factor receptor; dcs, dendritic cells; hmgb1a, high mobility group box-1 a peptide; ifu, infectious unit; lps, lipopolysaccharide; mpl, myeloproliferative leukemia virus oncogene; ova, ovalbumin; r3v6, an arginine-rich peptide; rip2, receptor-interacting protein 2; rsv, respiratory syncytial virus; s1plyase, sphingosine-1-phosphate lyase, socs, suppressors of cytokine signaling protein 3; stat6, signal transducer and activator of transcription factor 6; syk, spleen tyrosine kinase; tf-pei, transferrin polyethylenimine; t h 2, t helper 2 cells; tnf-α, tumor necrosis factor-α; vegfr, vascular endothelial growth factor. asthma is characterized by the variable and reversible airflow obstruction, airway hyperresponsiveness (ahr), mucus hypersecretion and chronic inflammation [176] . the most common form of asthma is allergic asthma, which is triggered by environmental stimuli such as house dust and seasonal pollen [177] . according to who, it is estimated that around 334 million peoples have asthma worldwide [178] . the mainstay treatments of asthma are corticosteroids, β 2 -adrenergic receptor agonists and leukotriene receptor antagonists, either alone or in combination. although these medications can manage the conditions in most of the patients, there are still challenges of poor patient compliance, intolerability and long term adverse effects [179] . for persistent allergic asthmatic patients, omalizumab which is an anti-immunoglobulin e (ige) monoclonal antibody can be used, but the high cost and parenteral administration make it less favorable [180] . alternative anti-inflammatory drugs are needed. many inflammatory mediators are identified to be involved in the pathogenesis of asthma and they are investigated as potential targets of rnai therapy. these include cytokines/chemokines [181] , transcription factors [168] , tyrosine kinases/tyrosine kinase receptors [172] and co-stimulatory molecules [182] . in allergic asthma, there is predominant activation of cd4 + t helper 2 (t h 2) cell-driven inflammatory response [183] . t cell activation and t h 2 cytokine expression are increased in the airways of atopic asthma patients after exposure to allergen [184] . t h 2-type cytokines, interleukin-4 (il-4), il-5 and il-13, are the main players in the pathology of allergic airway inflammation, contributing to allergic sensitization, ige production, eosinophil infiltration and ahr respectively [183, 184] . clinical studies that examine the therapeutic potential of using monoclonal antibodies (mabs) to neutralize the bioactivity of il-4 [185] , il-13 [186, 187] and il-5 [188] or target their receptors are in progress. similarly, rnai technology can be employed to target these cytokines [169, 181] . targeting suppressor of cytokine signaling (socs) 3 is an alternative approach [12] . socs proteins are negative regulators of cytokine signaling pathway, which are involved in t h 2 cells mediated allergic response via controlling the balance between t h 2 and t h 1 cells [189] . the expression of socs3 is a pathological marker of allergic disease. after sirna targeting, socs3 was intranasally administered to the lungs of chronic asthmatic mouse model [12] , the silencing of socs3 down-regulated the expression of t h 2 cell associated cytokines, il-4, il-5 and il-13, leading to substantial reduction of airway inflammation, ahr as well as ige production. reduction of mucus secretion and collagen deposition was also detected in the airways. besides t h 2 cell, other t cells subsets, such as t h 9 and t h 17 cells, also contribute the inflammatory response in asthma by releasing il-9 and il-17a. these molecules may also be the potential targets for rnai therapy [183] . furthermore, the combination of inflammatory and antiviral sirnas is a prospective therapy for patients with virus-induced exacerbation of severe uncontrolled asthma, in view of the report that respiratory viral infection is related to 80% of asthma exacerbation in children and adults [190] . the therapeutic potential of targeting il-4 and p protein of rsv simultaneously by sirnas was examined in a mouse model of rsv-induced asthma exacerbation [181] . the intranasal delivery of the two sirnas was able to: (i) significantly suppress il-4 mrna expression and rsv replication in the lungs; (ii) reduce eosinophil and neutrophil infiltration in bronchoalveolar lavage fluid (balf); (iii) inhibit ahr and inflammation; and (iv) increase interferon (ifn)-γ expression. many transcription factors including nuclear factor-κb (nf-κb) [168] , signal transducer and activator of transcription factor 6 (stat6) [166] and gata-binding protein 3 (gata3) [170] are involved in the production t h 2 cytokine in the airways of patients with asthma. they were investigated as targets for rnai in the management of asthmatic inflammation. nf-κb plays a key role in the expression of many pro-inflammatory proteins [191] . inhibition of nf-κb pathway in the lungs was reported to be beneficial in asthma model [176, 192] . one approach to inhibit the activation of nf-κb signaling pathway is to target receptor-interacting protein 2 (rip2), which is a transcriptional product and an activator of nf-κb. suppression of rip2 expression was achieved in mice by intratracheal delivery of sirna, resulting in the inhibition of ovalbumin (ova)-induced cytokine release, inflammatory cell infiltration, mucus hypersecretion and serum ige level. this result suggested that rip2 could be a novel therapeutic target for the treatment of asthma [168] . consistent with this finding, suppression of stat6 [10] and gata3 [170, 193] in murine model of allergen-induced asthma by rnai significantly inhibited the expression of downstream t h 2 cytokines. these studies support the hypothesis of using rnai molecules targeting transcription factors to manage airway inflammation and ahr of asthma. tyrosine kinases, which are broadly classified into receptor and non-receptor types, play an important role in activating the transcription factors for inflammatory gene expression upon activation by the inflammatory signals [194, 195] . the key receptor tyrosine kinases involved in the inflammatory response in asthma include epidermal growth factor receptor (egfr), c-kit (a stem cell factor receptor) and vascular endothelial growth factor receptor (vegfr), while the non-receptor forms are spleen tyrosine kinase (syk), src and janus kinase (jak) [196] . syk is a pivotal intracellular signaling molecule involved in the activation of several pro-inflammatory transcription factors [197] . inhibition of syk expression by antisense oligonucleotides has proven effective in suppression of tracheal contraction and pulmonary inflammation in animal models of ova-induced asthma [198] . when naked sirna targeting syk was administered to the lungs of allergen-induced asthma mice model via nasal instillation, the recruitment of inflammatory cells in the balf was reduced [194] . similarly, intranasal administration of sirna targeting c-kit also induced a promising result in reducing mucus secretion and airway inflammation in experimental asthma mouse model [173] . moreover, clinical studies have been performed on a sirna targeting syk (excellair™ developed by zabecor pharmaceuticals, usa) for the treatment of asthma. in the phase i study, 75% of asthma patients receiving excellair™ by inhalation for 21 consecutive days showed improvement in breathing or reduced the usage of rescue inhaler compared to placebo. the drug was well tolerated by patients without eliciting serious side effects. a phase ii clinical trial excellair™ has been initiated in 2009; the results are not yet available during the preparation of this review [199, 200] . targeting dendritic cells (dcs) in the airways by rnai is another strategy for the treatment of asthma. dcs are antigen presenting cells that could be found in the airways [5, 201] . they are actively involved in the differentiation of t h 2 cells and hence contribute to the progress of airway inflammation [202] . dcs express co-stimulatory molecules, cluster of differentiation (cd) 80 and 86, on the surface. the binding of cd80/cd86 with cd28 and cytotoxic t lymphocyte-associated antigen-4 (ctla-4) molecules on t cells is necessary for priming naive t cells into t h 2 cells [203] . blocking cd80 and/or cd86 activity with pharmacological inhibitors can inhibit t cell activation and alleviate ahr and pulmonary inflammation in mice exposed to aerosolized allergen challenge [204] . likewise, intratracheal administration of sirna targeting cd86 significantly reduced its expression in the airway mucosa of mouse model of allergic asthma. cd86 sirna treatment also contributed to the amelioration of ova-induced airway eosinophilia, hyperresponsiveness, the elevations of ige in the sera and the production of inflammatory cytokines in balf [171] . copd is a persistent lung disease characterized by irreversible chronic airflow limitation. the disease is usually progressive and associated with an increased chronic inflammatory response in the lungs [205, 206] . it is the result of long term exposure to air pollutants and usually associated with cigarette smoking [205] . it affects around 10% of the population in the world. it is the fourth leading cause of death in most of the industrialized countries and expected to become the third by 2020 [207] . three classes of medications used to manage copd are bronchodilators (including β 2 -adrenergic receptor and anticholinergic drugs), corticosteroids and phosphodiesterase-4 inhibitor. they are effective in relieving the symptoms of copd, but none of them can reverse the progressive deterioration in lung function or cure the disease [4, 205] . it is desirable to develop a novel therapy that can simultaneously control the symptoms and improve the long-term prognosis of the disease. despite similar clinical features, the mechanisms of underlying airway inflammation in asthma and copd are different with distinct patterns of inflammatory cells and mediators being involved [163] . the mechanisms and the mediators that drive the progression of chronic inflammation and emphysema in copd are far from fully understood [208] . the major signaling factors involved in the pathogenesis of copd include tnf-α, ccl2, cxcl8, tgf-β and vegf [206] . many of the cytokines and chemokines secreted in the airways of copd patients are modulated by the nf-κb pathway [163] . therefore, rnai targeting nf-κb pathway can be a strategy for the management of copd [176, 209] . current research work on rnai therapy for copd remains mainly in the in vitro stage, likely due to the insufficient understanding of the highly complex mechanisms underlying the pathology of the disease [120] . moreover, the degree of airway inflammatory response is determined both by genetic and epigenetic factors. since multiple inflammatory mediators are involved in the disease process, blocking a single type of molecule is unlikely to exert significant clinical impact [206] . mirna has the advantage of targeting multiple genes simultaneously. it is involved in the regulation of the inflammatory process of copd by modulating the transcription and translation of genes. differentiated expression of several mirnas, like let-7c, mir-125b, mir-15b [210] and mir-199a-5p [211] were identified in copd patients compared to subjects without copd. modulation the expression of multiple genes using mirna could be a direction of rnai therapy for the treatment of copd. ali is a severe form of diffuse lung disease that is commonly caused by acute systemic inflammatory disorders, such as sepsis, pneumonia, trauma and pancreatitis [11, 212] . it is a progressive disease, which can lead to acute respiratory distress syndrome (ards) or more critically, multiple organ failure and even death [175] . its pathological changes include the release of pro-inflammatory cytokines and chemokines, and the destruction of the epithelium-capillary interface; the latter can facilitate the extravasation of protein-rich fluid and the infiltration of neutrophils, thereby exacerbating the condition [213, 214] . since the molecular mechanism underlying the pathology of ali is not well understood, there is no pathophysiologic-driven therapeutic approach available [162] . rnai targeting the essential inflammatory mediators could offer protection against the progression of the disease. in an in vivo study, systemic delivery of sirna targeting tnf-α significantly reduced the expression of tnf-α in the lungs and lung injury in a shock-induced ali mouse model [175] . the findings suggested that pulmonary vascular cells, but not the epithelial cells, contribute to the release of tnf-α leading to lung injury following a systemic inflammatory insult. although these findings are contradictory to the others who suggest that pulmonary epithelial cells are the major players for causing lung injury induced by a systemic disease [215, 216] , nevertheless, they provide evidence that support the use of sirna targeting tnf-α in the management of ali. increase microvascular permeability is a major cause of amplification of the inflammatory responses in ali, leading to the progression to severe pathological condition [214] . the integrity of pulmonary and systemic endothelial barrier is maintained by the bioactive lipid, sphingosine-1phosphate (s1p), which can be irreversibly degraded by an enzyme called sphingosine-1-phosphate lyase (s1plyase) [214] . therefore, suppressing endothelial s1plyase expression by sirna is a potential therapeutic approach to maintain the integrity of the endothelial barrier [217] . nanoparticles containing sirna targeting s1plyase and recombinant high mobility group box-1 a peptide (hmgb1a) were delivered to the lungs of lps-induced ali mouse model by intratracheal instillation. hmgb1a peptide is an antagonist of the pro-inflammatory cytokine hmgb1 and provides additional anti-inflammatory effect in ali. an arginine-rich r3v6 peptide was used as a carrier. s1plyase level was significantly inhibited in the balf, leading to a significantly reduction of inflammatory cytokine (tnf-α and il-6) and inflammatory response in the lungs of the treated animal [11] . although several in vivo studies demonstrated promising results of using rnai therapeutics for the management of respiratory inflammatory diseases, there are several concerns that need to be taken into consideration with this approach. the distribution of naked sirna following intranasal and intratracheal administration was investigated. it was shown that the sirna molecules were predominantly accumulated in the peribronchial epithelial cells within 24 h post-administration [12] . since the inflammatory mechanism of diseases like asthma appears to be driven by the activated t h 2 lymphocytes [218] , it is highly desirable for rnai molecules to target the activated t cells in the airways to achieve therapeutic benefit. however, specific targeting to t cells is extremely difficult [174] . effective t cells transfection in vitro was achieved by electroporation [219] , an approach that is not clinically feasible. recently, a ligand-polymer conjugate sirna delivery system, transferrin-polyethylenimine (tf-pei), has been developed to target t cells in the lungs [174] . the rational of such a design was based on the increased expression of transferrin receptors on t cells after activation. biodistribution study showed that tf-pei polyplexes can selectively deliver the fluorescently labeled sirna to the activated t cells in a murine asthma model. however, the therapeutic effect of the system was not evaluated. therefore, targeting delivery of rnai therapeutics to specific cell types associated with the disease is a field that certainly required further exploration. most of the key signaling molecules involved in the pathogenesis of respiratory inflammatory disorders have multiple functions and participate in normal physiological activities to maintain homeostasis. short-term regimen (up to four days) was employed in the majority of the animal studies. the duration of treatment may not be sufficient to observe any long-term effect. since many inflammatory diseases are chronic disorders, long-term therapy is often required to control the disease. therefore, it is crucial to evaluate the long-term effect of any rnai therapeutics. inhaled corticosteroid is commonly used to control the symptoms of lung inflammatory disorders. steroid treatment reduces airway inflammation, partially through down-regulation of the key effectors molecules, such as c-kit [220] and gata3 [221] . for this reason, it is necessary to determine the effect of concurrent medications on the expression of targeting molecules before initiating the rnai therapy. given that lung inflammatory diseases are heterogeneous diseases that are categorized into different subtypes according to the inflammatory profiles and responses to treatment, it is desirable to identify the patients' profile in order to maximize the benefit of rnai therapy. pulmonary fibrosis is a respiratory disorder characterized by the progressive and irreversible destruction of lung architecture, which ultimately leads to organ malfunction, disruption of gas exchange and even death [222] . it is initiated by lung tissue damage resulting from autoimmune disorders or exposure to external irritants. the damage in turn triggers inflammatory reactions for tissue repair in the attempt to restore the normal tissue architecture and functions [222] . under pathological conditions, extracellular matrix (ecm) components, which are released by the activated myofibroblasts during the wound healing process, are excessively produced and accumulate at the site of tissue injury, resulting in fibrosis [223] . there are many forms of pulmonary fibrosis. all of them are lethal diseases with high mortality rate but with very limited therapeutic options [223] . thus pulmonary fibrosis is a lung disease with huge unmet medical need. rnai has been exploited to suppress the production of key molecules involved in the fibrotic process, such as tgf-β [92, 224] , amphiregulin (ar) and connective transforming growth factor (ctgf) [8, 225] (table 5) . clinical trials examining the effect of neutralized antibodies to block the activity of tgf-β are currently ongoing [14] . since lung epithelial cells are the key cell types in the pathogenesis of pulmonary fibrosis, local administration of rnai targeting tgf-β to the lungs can ensure high local concentration of rnai molecules to produce therapeutic effects. intratracheal delivery of sirna targeting tgf-β1 could effectively inhibit pulmonary fibrosis, improved lung function, and prolonged survival in human tgf-β1 transgenic mice [224] . in another in vivo study, intranasal delivery of mirna-326 mimic, which was designed to suppresses the expression of tgf-β and other profibrotic genes, significantly reduced tgf-β1 expression level and further alleviated the fibrotic feature in the bleomycin-induced pulmonary fibrosis [92] . the results suggest the potential of targeting tgf-β or its signaling pathway using rnai technology for the management of lung fibrosis. notes: ar, amphiregulin; ccl2, chemokine (c-c motif) ligand 2/monocyte chemoattractant protein-1; ctgf, connective tissue growth factor; pdgf, platelet-derived growth factor; pdmaema, poly(dimethylamino)ethylmethacrylate; pmapeg: poly(methylether-methacrylate-ethyleneglycol); samirna, self-assembled micelle interfering rna; tgf-β, transforming growth factor β. one complication of using tgf-β as the molecular target of rnai is related to its physiological role in maintaining immune and cellular homeostasis. it has a role in tumor suppression as suggested by the finding that specific inhibition of tgf-β in mouse stromal fibroblasts results in spontaneous carcinoma formation in the adjacent epithelium [226] . as a result, targeting the downstream effectors of tgf-β may offer the therapeutic benefit of inhibiting the tgf-β-mediated fibrotic response while preserving the major physiological function of the profibrotic cytokine [227] . ctgf is a key molecule that regulates fibroblast mitosis and promotes collagen deposition in pulmonary fibrosis induced by tgf-β. a non-viral delivery system consists of poly(dimethylamino)ethylmethacrylate (pdmaema) and its copolymer with poly(methylether-methacrylate-ethyleneglycol) [pmapeg] was used to incorporate and form complexes with sirna targeting ctgf. after intratracheal administration, significant reduction of ctgf expression, collagen deposition and inflammatory cytokines (il-6, tnf-α and tgf-β) production were observed in bleomycin-induced lung fibrosis animal model. more importantly, the survival rate of the animals treated with sirna targeting ctgf was significantly increased in comparison with the group treated with scrambled sirna [225] . another downstream mediator of tgf-β is amphiregulin (ar), which is an epidermal growth factor receptor (egfr) ligand primarily induced by tgf-β1 [228] . sirnas targeting ar and ctgf were delivered to the lung of pulmonary fibrosis mouse model via either intratracheal or intravenous administration. to improve the efficiency and cell-specific delivery, a modified self-assembled micelle interfering rna nanoparticles (samirna) was developed. the nanoparticles contain hydrophilic polymer and hydrophobic lipid on each ends of sirna and can spontaneously form micelle in solution. after administration of ar or ctgf samirnas, the expression of ar and ctgf, and collagen accumulation in the lung of pulmonary fibrosis model were significantly reduced compared to control samirna [8] . rnai technology has been under rapid development for the last decade. the therapeutic potential of rnai molecules in airway diseases has been demonstrated in several clinical studies (table 6) , and they could be the solution to some unmet medical needs. no rnai therapeutics has gained approval by regulatory authority at the time this manuscript was prepared. while delivery is generally considered as the major obstacle to rnai therapeutics development, it is interesting to notice that sirnas or mirna mimics do not need a carrier to generate therapeutic effects following pulmonary delivery. the cellular uptake mechanism of naked rna in the airways is not clear, but this is certainly an attractive feature that allows simple formulation without the need of delivery vectors. chemically modified sirna or mirna is necessary to improve the stability of the rna molecules. on the other hand, the delivery of shrna or mirna encoding plasmids requires viral vectors to ensure efficient transduction and the subsequent expression of rnai molecules. however, the use of viral vectors could raise safety concerns for clinical applications, especially when they are used for pulmonary delivery. the employment of liposomal or polymer based nanoparticle delivery technology could be a promising approach. whether the nanoparticles or naked rna are used, it is important to evaluate the pharmacokinetic profile of each delivery system to ensure the rnai therapeutics are localized at the site of action but not quickly distributed to other organs in the body. in terms of formulation of rnai therapeutics, dry powder aerosol is preferred over the liquid aerosol due to higher stability and the dry powder inhalers are easier to operate with better lung deposition [229] . this would be the direction for rnai formulation development. among the different types of rnai molecules for therapeutic development, sirna has made significant progress, with a couple of clinical studies investigate the pulmonary delivery of sirna to treat respiratory diseases. there are also a growing number of clinical pipelines involving the use of sirna and other rnai molecules. several key questions are raised that need to be answered. for infectious diseases, as demonstrated in several in vivo studies, it appears that rnai is more effective in prophylactic regimen than in therapeutic regimen. how can we improve the therapeutic efficacy of rnai in combating infections to make it clinically relevant? for inflammatory diseases, long-term therapy is expected. how can we minimize the undesirable effects when cytokines/chemokines are being targeted? when the respiratory functions are compromised in patients with lung disease, is pulmonary delivery a suitable route for administration? as complicated signaling pathway is involved in the pathogenesis of different lung disorders, a thorough investigation of the underlying pathological mechanisms of the disease is a prerequisite for identifying the target of rnai. despite the challenges and obstacles, we believe that the pulmonary delivery of rnai molecules targeting airway diseases is promising. a systematic review of the clinical effectiveness of first-line chemotherapy for adult patients with locally advanced or metastatic non-small cell lung cancer respiratory syncytial virus-a comprehensive review novel drugs against tuberculosis: a clinician's perspective the copd pipeline asthma: pathogenesis and novel drugs for treatment a review of current and novel therapies for idiopathic pulmonary fibrosis potent and specific genetic interference by double-stranded rna in caenorhabditis elegans self-assembled micelle interfering rna for effective and safe targeting of dysregulated genes in pulmonary fibrosis histological quantification of gene silencing by intratracheal administration of dry powdered small-interfering rna/chitosan complexes in the murine lung development of pre-clinical models for evaluating the therapeutic potential of candidate sirna targeting stat6 combined delivery of hmgb-1 box a peptide and s1plyase sirna in animal models of acute lung injury gene silencing of socs3 by sirna intranasal delivery inhibits asthma phenotype in mice local pulmonary immunotherapy with sirna targeting tgfβ1 enhances antimicrobial capacity in mycobacterium tuberculosis infected mice pulmonary delivery of therapeutic sirna rnai therapeutics: principles, prospects and challenges immune responses to dsrna: implications for gene silencing technologies preclinical and clinical development of sirna-based therapeutics in vivo delivery aspects of mirna, shrna and sirna subcellular fate and off-target effects of sirna, shrna, and mirna shrna: similarities and differences sirna versus mirna as therapeutics for gene silencing regulation of microrna biogenesis systemic delivery of anti-mirna for suppression of triple negative breast cancer utilizing rna nanotechnology lipid nanoparticle delivery of a microrna-145 inhibitor improves experimental pulmonary hypertension microrna-495 mimics delivery inhibits lung tumor progression developing an effective therapeutic by delivery of synthetic microrna-520e in lung cancer treatment development of small rna delivery systems for lung cancer therapy a protocol for designing sirnas with high functionality and specificity comparison of antisense oligonucleotides and sirnas in cell culture and in vivo rna interference (rnai)-based therapeutics: delivering on the promise? rna-based drugs and vaccines polyethylenimines for rnai-mediated gene targeting in vivo and sirna delivery to the lung nanoparticle-mediated pulmonary drug delivery: a review influence of particle size on regional lung deposition-what evidence is there? in vivo, in vitro and ex vivo models to assess pulmonary absorption and disposition of inhaled therapeutics for systemic delivery inhaling medicines: delivering drugs to the body through the lungs kissel, t. sirna delivery to the lung: what's new? airway mucus function and dysfunction structure and function of the polymeric mucins in airways mucus regulation of mammalian ciliary beating mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues composition, structure and mechanical properties define performance of pulmonary surfactant membranes and films pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro the influence of natural pulmonary surfactant on the efficacy of sirna-loaded dextran nanogels cellular uptake mechanism and knockdown activity of sirna-loaded biodegradable deapa-pva-g-plga nanoparticles bio-inspired pulmonary surfactant-modified nanogels: a promising sirna delivery system inhibition of rnase a family enzymes prevents degradation and loss of silencing activity of sirnas in serum biokinetic studies of non-complexed sirna versus nano-sized pei f25-lmw/sirna polyplexes following intratracheal instillation into mice potential and development of inhaled rnai therapeutics for the treatment of pulmonary tuberculosis clathrin-independent pathways of endocytosis targeting of nanoparticles to the clathrin-mediated endocytic pathway dna internalized via caveolae requires microtubule-dependent, rab7-independent transport to the late endocytic pathway for delivery to the nucleus high density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/dna polyplexes on the cellular processing of non-viral nanomedicines for nucleic acid delivery: mechanisms and methods gateways for the intracellular access of nanocarriers: a review of receptor-mediated endocytosis mechanisms and of strategies in receptor targeting image-based analysis of lipid nanoparticle-mediated sirna delivery, intracellular trafficking and endosomal escape breaking down the barriers: sirna delivery and endosome escape enhancing endosomal escape for nanoparticle mediated sirna delivery endosomal escape pathways for delivery of biologicals endosomal escape pathways for non-viral nucleic acid delivery systems. in molecular regulation of endocytosis nuclear entry of nonviral vectors pulmonary administration of small interfering rna: the route to go? aerosol delivery of sirna to the lungs. part 2: nanocarrier-based delivery systems in vivo gene delivery by nonviral vectors: overcoming hurdles & quest delivery materials for sirna therapeutics non-viral vectors for gene-based therapy viral vector-mediated rna interference sustained mirna-mediated knockdown of mutant aat with simultaneous augmentation of wild-type aat has minimal effect on global liver mirna profiles construction of sh-rps6 lentivirus vectors and its effect on proliferation in lung adenocarcinoma a549 cell lines. sichuan da xue xue bao yi xue ban influence of suppression of epstein-barr virus-encoded latent membrane protein 1 by raav vector mediated rna interference on metastatic ability of nasopharyngeal cancer cells in vivo pseudotyped adeno-associated virus 2/9-delivered ccl11 shrna alleviates lung inflammation in an allergen-sensitized mouse model. hum aerosol delivery of sirna to the lungs. part 1: rationale for gene delivery systems lipid-based vectors for sirna delivery attenuation of fibrosis in vitro and in vivo with sparc sirna advanced transfection with lipofectamine 2000 reagent: primary neurons, sirna, and high-throughput applications small interfering rna (sirna) targeting vegf effectively inhibits ocular neovascularization in a mouse model antibody-directed cell-type-specific delivery of sirna toxicity of cationic lipids and cationic polymers in gene delivery recent developments in nucleic acid delivery with polyethylenimines polyethylenimines for sirna and mirna delivery in vivo chitosan-coated plga nanoparticles for dna/rna delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides tat-bmps-pamam conjugates enhance therapeutic effect of small interference rna on u251 glioma cells in vitro and in vivo biodegradable dextran nanogels for rna interference: focusing on endosomal escape and intracellular sirna delivery rna interference in vitro and in vivo using a chitosan/sirna nanoparticle system formulation of ph responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids peptides used in the delivery of small noncoding rna intracellular delivery of molecular cargo using cell-penetrating peptides and the combination strategies polyester analogue of poly-l-lysine as a soluble poly-l-lysine functionalized large pore cubic mesostructured silica nanoparticles as biocompatible carriers for gene delivery intratracheal administration of small interfering rna targeting fas reduces lung ischemia-reperfusion injury microrna-326 regulates profibrotic functions of transforming growth factor-β in pulmonary fibrosis rnai therapeutic platforms for lung diseases metastatic non-small-cell lung cancer (nsclc): esmo clinical practice guidelines for diagnosis, treatment and follow-up fda drug approval summary: bevacizumab (avastin ® ) plus carboplatin and paclitaxel as first-line treatment of advanced/metastatic recurrent nonsquamous non-small cell lung cancer approval summary: erlotinib maintenance therapy of advanced/metastatic non-small cell lung cancer (nsclc) fda drug approval summary: gefitinib (zd1839)(iressa ® ) tablets nanomedicine as an emerging platform for metastatic lung cancer therapy aerosol gene delivery using viral vectors and cationic carriers for in vivo lung cancer therapy akt/protein kinase b is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation dual expression of shakt1 and pdcd4 suppresses lung tumorigenesis in k-ras la1 mice suppression of lung cancer progression by biocompatible glycerol triacrylate-spermine-mediated delivery of shakt1 lentivirus-aimp2-dx2 shrna suppresses cell proliferation by regulating akt1 signaling pathway in the lungs of aimp2+/-mice factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises pulmonary codelivery of doxorubicin and sirna by ph-sensitive nanoparticles for therapy of metastatic lung cancer a novel platform to enable inhaled naked rnai medicine for lung cancer in vivo tumor targeting via nanoparticle-mediated therapeutic sirna coupled to inflammatory response in lung cancer mouse models knockdown of the sodium-dependent phosphate co-transporter 2b (npt2b) suppresses lung tumorigenesis gene silencing in a mouse lung metastasis model by an inhalable dry small interfering rna powder prepared using the supercritical carbon dioxide technique nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and sirna modelling myc inhibition as a cancer therapy co-delivery of doxorubicin and bcl-2 sirna by mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug-resistant cancer cells rpn2 gene confers docetaxel resistance in breast cancer prognostic and therapeutic impact of rpn2-mediated tumor malignancy in non-small-cell lung cancer co-delivery of chemosensitizing sirna and an anticancer agent via multiple monocomplexation-induced hydrophobic association polypeptide cationic micelles mediated co-delivery of docetaxel and sirna for synergistic tumor therapy co-delivery of sirnas and anti-cancer drugs using layered double hydroxide nanoparticles bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy multidrug resistance proteins mrp3, mrp1, and mrp2 in lung cancer correlation of protein levels with drug response and messenger rna levels sirna-based therapies for pulmonary diseases bacterial complications of respiratory tract viral illness: a comprehensive evaluation rna interference-based therapeutics: new strategies to fight infectious disease inhibition of respiratory viruses by nasally administered sirna rna interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung effective small interfering rnas targeting matrix and nucleocapsid protein gene inhibit influenza a virus replication in cells and mice rna interference of avian influenza virus h5n1 by inhibiting viral mrna with sirna expression plasmids intrapulmonary delivery of xcl1-targeting small interfering rna in mice chronically infected with mycobacterium tuberculosis phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering rna and its application in the reverse genetics of wild type negative-strand rna viruses use of sirnas to prevent and treat influenza virus infection expression of a single sirna against a conserved region of np gene strongly inhibits in vitro replication of different influenza a virus strains of avian and swine origin using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis respiratory syncytial virus infection in adults respiratory syncytial virus infection in elderly and high-risk adults strategic priorities for respiratory syncytial virus (rsv) vaccine development. vaccine evaluation of the safety, tolerability and pharmacokinetics of aln-rsv01, a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus complete results of our aln-rsv01 phase iib study influenza a viruses: new research developments adamantane-resistant influenza a viruses in the world (1902-2013): frequency and distribution of m2 gene mutations influenza virus resistance to neuraminidase inhibitors rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription barrier or carrier? pulmonary surfactant and drug delivery targeted small interfering rna-immunoliposomes as a promising therapeutic agent against highly pathogenic avian influenza a (h5n1) virus infection small interfering rna targeting m2 gene induces effective and long term inhibition of influenza a virus replication inhalable dry powder formulations of sirna and ph-responsive peptides with antiviral activity against h1n1 influenza virus misinterpreting the therapeutic effects of small interfering rna caused by immune stimulation. hum who world health organization: tuberculosis-global tb programme core functions bfl-1/a1 acts as a negative regulator of autophagy in mycobacteria infected macrophages higher order rab programming in phagolysosome biogenesis microrna-155 promotes autophagy to eliminate intracellular mycobacteria by targeting rheb microrna-125a inhibits autophagy activation and antimicrobial responses during mycobacterial infection tuberculosis: autophagy is not the answer revisiting the role of the granuloma in tuberculosis the tuberculous granuloma: an unsuccessful host defence mechanism providing a safety shelter for the bacteria? xcl1 (lymphotactin) chemokine produced by activated cd8 t cells during the chronic stage of infection with mycobacterium tuberculosis negatively affects production of ifn-γ by cd4 t cells and participates in granuloma stability enhanced production of tgf-beta by blood monocytes from patients with active tuberculosis and presence of tgf-beta in tuberculous granulomatous lung lesions down-modulation of lung immune responses by interleukin-10 and transforming growth factor β (tgf-β) and analysis of tgf-β receptors i and ii in active tuberculosis pathogenesis of indirect (secondary) acute lung injury immunology of asthma and chronic obstructive pulmonary disease acute lung injury small interfering rnas targeted to interleukin-4 and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. hum small interfering rna against transcription factor stat6 inhibits allergic airway inflammation and hyperreactivity in mice vegf controls lung t h 2 inflammation via the mir-1-mpl (myeloproliferative leukemia virus oncogene)-p-selectin axis receptor-interacting protein 2 gene silencing attenuates allergic airway inflammation let-7 microrna-mediated regulation of il-13 and allergic airway inflammation lentiviral-mediated gata-3 rnai decreases allergic airway inflammation and hyperresponsiveness small interfering rna against cd86 during allergen challenge blocks experimental allergic asthma effect of locally administered syk sirna on allergen-induced arthritis and asthma intranasal sirna targeting c-kit reduces airway inflammation in experimental allergic asthma targeted delivery of sirna to activated t cells via transferrin-polyethylenimine (tf-pei) as a potential therapy of asthma the role and source of tumor necrosis factor-α in hemorrhage-induced priming for septic lung injury targeting the nf-κb pathway in asthma and chronic obstructive pulmonary disease asthma: defining of the persistent adult phenotypes international ers/ats guidelines on definition, evaluation and treatment of severe asthma investigating the value of omalizumab in the treatment of severe persistent allergic asthma: a systematic review of cost-effectiveness studies sirnas targeted to il-4 and rsv reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. hum pulmonary delivery of sirna via polymeric vectors as therapies of asthma functions of t cells in asthma: more than just th2 cells the role of t lymphocytes in the pathogenesis of asthma dupilumab in persistent asthma with elevated eosinophil levels a phase 1, randomized, placebo-controlled, dose-escalation study of an anti-il-13 monoclonal antibody in healthy subjects and mild asthmatics efficacy and safety of tralokinumab in patients with severe uncontrolled asthma: a randomised, double-blind, placebo-controlled, phase 2b trial benralizumab, an anti-interleukin 5 receptor α monoclonal antibody, versus placebo for uncontrolled eosinophilic asthma: a phase 2b randomised dose-ranging study suppressor of cytokine signaling 3 (socs3) in th2 cells evokes th2 cytokines, ige, and eosinophilia. curr role of viral respiratory infections in asthma and asthma exacerbations nf-κb: a key role in inflammatory diseases persistent activation of nuclear factor-κb signaling pathway in severe uncontrolled asthma treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of gata-3 expression inhibitors of the tyrosine kinase signaling cascade for asthma tyrosine kinase inhibitors: a new approach for asthma the potential use of tyrosine kinase inhibitors in severe asthma syk activation in dendritic cells is essential for airway hyperresponsiveness and inflammation inhibition of allergic inflammation in the airways using aerosolized antisense to syk kinase clinical status of duplex rna current progress of sirna/shrna therapeutics in clinical trials dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma essential role of dendritic cell cd80/cd86 costimulation in the induction, but not reactivation, of t h 2 effector responses in a mouse model of asthma tracking and treating activated t cells essential role for both cd80 and cd86 costimulation, but not cd40 interactions, in allergen-induced th2 cytokine production from asthmatic bronchial tissue: role for αβ, but not γδ, t cells global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: gold executive summary cellular and molecular mechanisms of chronic obstructive pulmonary disease immunologic aspects of chronic obstructive pulmonary disease recent advances in pre-clinical mouse models of copd regulation of airway muc5ac expression by il-1β and il-17a; the nf-κb paradigm gene expression networks in copd: microrna and mrna regulation microrna-199a-5p is associated with hypoxia-inducible factor-1α expression in lungs from patients with copd acute lung injury review the acute respiratory distress syndrome the acute respiratory distress syndrome silencing of fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis tnf-induced death signaling triggers alveolar epithelial dysfunction in acute lung injury protection of lps-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression innate and adaptive immune responses in asthma high-efficiency transfection of primary human and mouse t lymphocytes using rna electroporation stem cell factor and its receptor c-kit as targets for inflammatory diseases suppression of gata-3 nuclear import and phosphorylation: a novel mechanism of corticosteroid action in allergic disease integrating mechanisms of pulmonary fibrosis pulmonary fibrosis: pathogenesis, etiology and regulation development and preclinical efficacy of novel transforming growth factor-β1 short interfering rnas for pulmonary fibrosis noncovalenly pegylated ctgf sirna/pdmaema complex for pulmonary treatment of bleomycin-induced lung fibrosis tgf-ß signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia modifiers of tgf-β1 effector function as novel therapeutic targets of pulmonary fibrosis amphiregulin, an epidermal growth factor receptor ligand, plays an essential role in the pathogenesis of transforming growth factor-β-induced pulmonary fibrosis dry powder formulation of plasmid dna and sirna for inhalation the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses rna interference therapy in lung transplant patients infected with respiratory syncytial virus the authors declare no conflict of interest. key: cord-300124-voyjcjzw authors: soldati, gino; smargiassi, andrea; inchingolo, riccardo; demi, libertario title: reply to colorimetric triage for patients with covid‐19 date: 2020-08-27 journal: j ultrasound med doi: 10.1002/jum.15460 sha: doc_id: 300124 cord_uid: voyjcjzw nan to the editor: we thank dr antúnez montes for his interesting proposal to link the colorimetric triage for patients with covid-19 based on both pocus findings and clinical parameters and our lus scoring system and acquisition protocol together in a joint classification. 1 indeed, lus can be very useful to widen the medical examination and intercept peripheral changes in covid-19 pneumonias. although lus has low specificity, by detecting and assessing lung involvement in suspected or confirmed cases, it can support clinical suspicions and potentially play a role in managing decisions. in our scoring system, 1 pathologic lus findings in patients with covid-19 patients can be classified as follows: score 0: the pleural line is regular. horizontal reverberant artifacts and mirror effects are present. score 1: the pleural line has slight alterations with sporadic vertical artifacts. score 2: the pleural line has relevant alterations. there is a predominance of vertical artifacts. smallto-large subpleural consolidations are present. score 3: the pleural line is irregular and cobbled. dense and extended white lung, with or without large consolidations, is present. the above-described scoring system is linked to progressive and variegated alterations of peripheral lung tissue. [1] [2] [3] [4] [5] these alterations can be due to, eg, diffuse alveolar damage with alveolar-interstitial exudation, alveolar shrinking and collapse with microatelectasis, subpleural consolidations caused by inflammation phenomena, and, at least in part, ischemic lesions from coagulation disorders. 6 when multiple areas are labeled with a high score, clinical conditions are likely to be worse. therefore, the idea to link the scoring system to the colorimetric triage is interesting and may add more information during the first medical approach. in a pandemic context, patients admitted to the ed with nonspecific respiratory symptoms but suspected of covid-19 should undergo an early lus examination performed according to a standardized acquisition protocol and scoring system. 1 patients should be kept isolated and hospitalized while waiting for virologic tests in cases with lus findings suggestive of pulmonary involvement compatible with covid-19 pneumonia: ie, the presence of areas with a score of 2 or 3 concurrently with altered intrapulmonary gas exchanges (yellow and red colorimetric triage). next, a high-resolution computed tomographic scan of the chest should be performed with or without administration of an iodinated contrast agent, depending on each clinical case and according to local protocols. 7 differently, patients with good clinical conditions (ie, patients for whom most of the examined areas are associated with score of 0, few with a score of 1, and who have conserved intrapulmonary gas exchanges) may be considered at low risk (green colorimetric triage) and can be monitored even at home according to local protocols and kept isolated while waiting for virologic tests. 7 in conclusion, we would like add a consideration on the counting of vertical artifacts. 8 since the imaging frequency, bandwidth, and focal point position can influence the appearance of vertical artifacts, 3, [9] [10] [11] and the subjective nature of the counting operation itself introduces another strong degree of variability, we advise the authors not to rely on counting vertical artifacts in the generation of the colorimetric triage. proposal for international standardization of the use of lung ultrasound for patients with covid-19: a simple, quantitative, reproducible method artifactual lung ultrasonography: it is a matter of traps, order, and disorder physical mechanisms providing clinical information from ultrasound lung images: hypotheses and early confirmations the role of ultrasound lung artifacts in the diagnosis of respiratory diseases lung ultrasonography may provide an indirect estimation of lung porosity and airspace geometry contrast-enhanced ultrasound in patients with covid-19: pneumonia, acute respiratory distress syndrome, or something else lung ultrasonography for early management of patients with respiratory symptoms during covid-19 pandemic routine use of point-of-care lung ultrasound during the covid-19 pandemic ultrasonography in lung pathologies: new perspectives time for new international evidence-based recommendations for point-of-care lung ultrasound determination of a potential quantitative measure of the state of the lung using lung ultrasound spectroscopy key: cord-016280-d47e3art authors: friedberg, joseph s.; kucharczuk, john c. title: pleura: anatomy, physiology, and disorders date: 2008 journal: surgery doi: 10.1007/978-0-387-68113-9_75 sha: doc_id: 16280 cord_uid: d47e3art disorders of the pleura and pleural space reflect some of the oldest diseases encountered in surgical history. hippocrates described the symptoms of empyema 2400 years ago: “empyema may be recognized by the following symptoms: in the first place the fever is constant, less during the day and greater at night, and copious sweats supervene. there is a desire to cough and the patient expectorates nothing worth mentioning.” he also described an open drainage procedure: “when the fifteenth day after rupture has appeared, prepare a warm bath, set him upon a stool, which is not wobbly, someone should hold his hands, then shake him by the shoulders and listen to see on which side a noise is heard. and right at this place, preferably on the left, make an incision, then it produces death more rarely.”1,2 beyond providing less-wobbly stools, few advances were made for more than 2000 years that allowed surgeons to routinely enter the pleural cavity, the fear being a potentially fatal pneumothorax. with the advent of positive pressure ventilation in the early 1900s, pneumothorax was no longer a prohibitive risk, and the era of surgical intervention in the pleural cavity had begun.3 embryologically, the pleural cavity is created during a month, starting in the third week of gestation. initially, the lateral plate forms two layers, the splanchnopleura and the soma topleura, which subsequently develop into the visceral and parietal pleura, respectively. eventually, it is the visceral pleura that surrounds the lung and the parietal pleura that lines the remainder of the chest cavity. the different embryological origins of the visceral and parietal pleura are responsible for the separate vascular, lymphatic, and neural supplies of these two structures as seen in the adult. by the end of the seventh week of gestation, the diaphragm has separated the thoracic cavity from the peritoneal cavity, and by the third month of gestation the two pleural cavities have expanded sufficiently to encase the pericardium. 4 in the adult, both pleural surfaces are approximately 30 to 40l1m thick and are composed of a single layer of mesothelial cells with an underlying layer of connective tissue. depending on their location, the mesothelial cells may be flat, cuboidal, or columnar. mesothelial cells characteristically have numerous microvilli that playa role in phagocytosis as well as contributing to the lubricious nature of the pleural surfaces. surfactant molecules, produced by the mesothelium, line the pleural surfaces and, secondary to similar electrical charge, repulse each other and facilitate sliding, analogous to the lubrication achieved with graphite. it is these apposing layers of mesothelial cells that form the potential space of the pleural cavity and that glide over each other during respiration. the connective tissue layer contains the neurovascular and lymphatic supply of the pleura. there are certain important differences in this layer between the visceral and parietal pleura. for the visceral pleura, the connective tissue layer is functionally continuous with the fibroelastic network of the lung itself. functionally, it is this relationship that prevents the visceral pleura from being surgically separated from the surface of a normal lung. pathological disruption of this connection, however, may result in subpleural air collections known as blebs. s the connective tissue layer for the parietal pleura may also be tightly adherent to the underlying structures, as is characteristic of the diaphragmatic pleura. around the skeletal portion of the thorax, however, the pleura is bound to the underlying tissue by another connective tissue layer called the endothoracic fascia, which forms a natural cleavage plane. it is this plane that the surgeon develops when performing an "extrapleural" dissection. the blood supply to the visceral pleura in humans is thought to reflect that of the lung itself, with a dual arterial supply from both the pulmonary and bronchial arteries and singular venous drainage into the pulmonary veins. the blood supply to the parietal pleura is from systemic arteries only and drains, predominantly, into peribronchial and intercostal veins, but it may also drain directly into the azygous vein and vena cava. the visceral pleura is innervated by vagal and sympathetic fibers but has no somatic innervation and is therefore insensate. the parietal pleura is also innervated with sympathetic and parasympathetic fibers, but it is also somatically innervated. thus, the parietal pleura is capable of sensing and transmitting the sensation of pain. "pleurisy" from inflammation and pain from chest tubes, during insertion and subsequently as well, are attributable to the somatic intervention of the parietal pleura. there are also differences in the lymphatic drainage between the two pleural layers. the visceral pleura drains through a lymphatic network into the pulmonary lymphatics, which eventually flow toward the pulmonary hilum. this lymphatic system is richer in the lower lobes than the upper lobes. the parietal pleural lymphatics drain to different locations. the mediastinal pleura drains to the mediastinal and tracheobronchial nodes. the chest wall drains anteriorly to the internal thoracic chain and posteriorly toward the intercostal nodes near the heads of the ribs. the diaphragmatic pleura drains to the parasternal, middle phrenic, and posterior mediastinal lymph nodes. there are also transdiaphragmatic lymphatic communications that allow some degree of lymphatic flow from the peritoneum to the pleural space. the parietal pleura also differs from the visceral pleura by virtue of the presence of kampmeier foci and stomata. kampmeier foci are collections of activated mesothelial and lymphoreticular cells, centered about a lymphatic core, that augment the pleura defensive capabilities. they are concentrated in the lower mediastinal region of the parietal pleura. 6 ,7 stomata are 2-to 6-~m pores that communicate directly with the parietal pleural lymphatics. during inspiration, these pores have the capacity to stretch, and their architecture is such that they form functional one-way valves. thus, they provide for a very effective system for draining both fluid and particles, including both red blood cells and macrophages. it is the presence of these pores on the parietal pleural surface that makes it predominantly, if not exclusively, responsible for clearance of cells and particulate matter from the pleural space. it should be noted that although stomata are well studied and characterized in sheep and other mammals, the definitive presence of stomata in humans is less well established. in each hemithorax, the visceral pleura is a continuous surface that completely envelops the entire lung, including the fissures. at the pulmonary hilum, it continues on as the parietal pleura to line the mediastinum, chest wall, diaphragm, and cupola of the chest cavity. in humans, the pleural cavities are completely separate, coming into contact with each other for a short distance behind the upper half of the body of the sternum (fig. 74.1 ). it is this pleural separation of the right and left chest cavities that prevents bilateral pneumothoraces from occurring as the result of a unilateral chest injury. at the costophrenic and costomediastinal sinuses, the parietal pleural folds back on itself, providing a potential space into which the lungs can expand during inspiration. 8 superiorly, the pleura extends above the bony thorax into the base of the neck (fig. 74.2 ). this fact explains why pneumothorax may complicate internal jugular central line placement as well as subclavian central line placement. anteriorly, the pleura extends to the sixth rib, to the ninth rib laterally, and to the twelfth rib posteriorly ( fig. 74.2 ). in the living patient, the lung can fill the entire posterior recess. in a review of 100 chest radiographs, 80% of patients were found to have lung present at or below the level of the 12th rib, and in 18% it was seen at the level of the first lumbar vertebra. 9 these external landmarks of the pleural space are of practical clinical significance, particularly when evaluating a patient with penetrating trauma. the pulmonary ligament is a double fold of the mediastinal pleura that tapers down from the root of the lung, where it is in continuity with the visceral pleura, to the caudal mediastinum. this ligament is one of the structures that must be divided to perform a pneumonectomy or lower lobectomy. it is also routinely divided to its superior border, the inferior pulmonary vein, when attempting to provide mobility to the lower lobe after resecting the upper or middle lobes. the lymph nodes within the pulmonary ligament are the level 9 nodes, which are n2 lymph nodes, and should be routinely harvested when performing a resection for lung cancer. the pleura has both mechanical and physiological functions. it transmits negative pressure from the thorax to the lung, thereby opposing the lung's natural elastic recoil and maintaining pulmonary expansion. during respiration, this function is performed in an environment of very low friction, thereby allowing the lungs to glide smoothly over the internal thoracic surfaces as they expand and contract. the pleura also controls the environment of the chest cavity by maintaining fluid homeostasis, preventing or removing air collections and keeping the space sterile. under normal conditions, the pleural cavity is a potential space with a thickness ranging from 10 to 20f..lm. the lung is maintained in an expanded state by the maintenance of negative pressure in the pleural space; this allows the expandable chest cavity to overcome the opposing forces exerted by the natural elastic recoil of the lung. the resting pressure in the pleural space, when the lung is at its functional residual capacity, is slightly negative at -2 to -5 cm h20. when measured in an upright posture, there is more negative pressure in the apex of the chest than at the diaphragm, likely a gravitational effect. the negative pressure continues to increase through inspiration, with the pressure ranging from -25 to -35 cm h20 at the vital capacity. disorders that decrease the compliance of the lung or increase airway resistance further increase the negative pressure in the pleural space with inspiration. 5 ,9 initially, it would seem curious that gas is not drawn out of solution into the pleural space by the negative pressure in the space. it is the lower partial pressure of gases on the venous side of the pleural circulation, as opposed to the arterial side, that prevents spontaneous pneumothorax from occurring under normal conditions. this difference in partial pressures between the two sides of the circulation is mainly a result of oxygen absorption. unless the pressure in the pleural space decreases to significantly less than -socm h20, the sum total of the forces under normal conditions favors absorption of gas out of the pleural space. this diffusion gradient also accounts for reabsorption of gas that is introduced into the pleural space. the clearance rate of gases introduced into the pleural space is dependent on the concentrations of those gases with respect to their partial pressures in the pleural circulation. as the partial pressure of nitrogen is the greatest in the air we breathe, it therefore constitutes the highest partial pressure of the gases that form a pneumothorax. nitrogen also has the highest partial pressure of the gases in our circulation; this can be decreased by altering the composition of inspired gases. thus, administration of supplemental oxygen decreases the partial pressure of nitrogen in the bloodstream and thereby increases the nitrogen pressure gradient between the circulation and the pneumothorax, favoring more rapid reabsorption of the trapped gas. this relationship serves as the rationale for placing a patient on supplemental oxygen to facilitate reabsorption of a pneumothorax that is not being externally evacuated. 10 under normal conditions, the pleural space contains very little fluid, estimated at approximately 0.3 ml/kg. the fluid is generally hypooncotic, with a protein content of approximately 1 g/dl. the mechanisms of fluid production and reabsorption are complicated and not completely understood. numerous forces interact from both the parietal and visceral pleura, including their respective hydrostatic and on co tic pressures. respiratory movement and gravity are both thought to have roles in maintaining the fluid dynamics of the pleural cavity. the predominant factor, however, is thought to be the uptake of fluid into the parietal pleural lymphatics. these lymphatics tend to be concentrated in the dependent portions of the chest cavity. under normal conditions, this flow rate has been estimated at 0.1 to o.lsml/kg/ h. the lymphatic flow rate has the capacity to increase and has been estimated to reach as high as 30ml/h, approximately 700ml/day in an average-size individual. when the dynamics of this equilibrium are unbalanced beyond the rate at which the lymphatics are able to compensate, pleural effusion accumulates. 7 ,9,11 it is interesting to note that the exact purpose of the pleura is still not fully understood. empirically, it is clear that the pleura maintains fluid and gas homeostasis, mechanically couples the lungs to the bellows mechanism for respiration, and maintains sterility in its described space. to accomplish this, however, it is not clear how important it is to have the configuration of two opposing layers with a small amount of intervening fluid. it has been observed, for instance, that there is little change in pulmonary function tests in patients before and after fusion of the pleural space. 12 it is interesting to note that some mammals do not have a pleural space, similar to patients who have undergone pleurodesis, but that these animals clearly function normally. thus, the exact necessity for having two pleural membranes defining a potential space remains somewhat of a mystery. there are a large number of pleural disorders. the majority lead to symptoms as a result of mechanical compression of the lung, although many may be asymptomatic or may present with constitutional symptoms or pain. in most cases, the pathology results from the presence of something in the pleural space, which as previously described, is normally a potential space. therefore, in an effort to organize this large number of disorders, they are grouped according to what abnormal phase of material is occupying the pleural space, that is, gas, liquid, or solid. when gas enters the pleural space, it is referred to as a pneumothorax. when liquid enters the pleural space, it may sometimes be broadly referred to as an exudative or transudative effusion but is frequently classified according to the type of liquid, such as hemothorax, chylothorax, or empyema. last, solid masses may occupy the pleural space. most benign masses are pleural plaques, but there are also rare benign tumors of the pleura, some of which may reach enormous size. malignant masses of the pleura are usually cancers that have metastasized to the pleura, but there are also some rare primary tumors, most commonly mesothelioma. sometimes, the groups may overlap, with more than one abnormal phase of material filling the pleural space. such examples include air and blood, a hemopneumothorax, after trauma or air and pus, a hydropneumothorax, which may be seen with empyemas resulting from a bronchopleural fistula. frequently, however, there is a predominant, if not sole, etiology for the abnormal accumulation; thus, the following sections review disorders of the pleura according to the state of matter that is abnormally occupying the spacegas, liquid, or solid. pneumothorax is defined as air in the pleural space. it may occur traumatically, iatrogenically, or spontaneously. spontaneous pneumothorax may be subclassified as primary or secondary, with primary spontaneous pneumothorax arising in an otherwise healthy patient and secondary spontaneous pneumothorax arising as a complication in a patient with known underlying pulmonary disease. essentially any pneumothorax resulting from pleural disruption can present as a tension pneumothorax. this condition represents a true emergency and is discussed separately. pneumothorax presentation and diagnosis pneumothorax may cause pain or dyspnea, or it may be asymptomatic, depending on its size and the underlying pulmonary function of the patient. physical findings may range from none to the classic findings seen with a tension pneumothorax: contralateral tracheal deviation, ipsilateral absent breath sounds, and percussive hyperresonance. electrocardiographic changes may be present, including diminished voltage, right-axis deviation, or t-wave changes that may mimic a subendocardial myocardial infarction. '3 except for tension pneumothorax, most cases require an upright chest radiograph to establish the diagnosis. as the pneumothorax occupies a greater proportion of the chest cavity at expiration than inspiration, the former is more sensitive for detecting the diagnostic pleural line. a computed tomographic (ct) scan of the chest is the most sensitive test and may demonstrate a small amount of air in the pleural space that is not visible on the plain radiograph. for all pneumothoraces, the common goal is removal of air from the pleural space. depending on the etiology, however, prevention of recurrence may also be an objective of the treatment. options for treatment range from observation to thoracotomy. selection of the appropriate modality depends on a number of factors, including, but not limited to, presentation, previous history, comorbidities, need for positive pressure ventilation, associated effusion, and even the patient's lifestyle. in addition, the size of the pneumothorax can also play a significant role in determining the appropriate treatment. the following sections review the basic technique and indications for the different treatment options that are available and give specific recommendations for different pneumothoraces to be described in the following section. observation is generally reserved for patients who are asymptomatic and are diagnosed with a small primary spontaneous pneumothorax or a simple iatrogenic pneumothorax. in such situations, the patient is followed with serial radiographs to ensure that the pneumothorax is decreasing in size. when a patient is breathing room air, gas is absorbed from the pleural cavity at approximately 1.25% of the pleural volume/day, approximately 50 to 70ml/day.'4 supplemental oxygen, by mechanisms reviewed in the physiology section, can increase this rate up to 4.2 %/day.is as it is a minimal intervention, it is reasonable to place all hospitalized patients on supplemental oxygen if they are being observed for a pneumothorax. there are several factors to weigh when considering observation alone for a patient with a pneumothorax. the first is that deaths have been reported in patients with pneumothorax who were being observed. development of umecognized tension pneumothorax was believed to have played a role in these cases. 16 this fact highlights the selectivity and judgment required to simply follow these patients, particularly on an outpatient basis. another consideration is that a lung that has not fully expanded by 2 weeks is at risk for fibrous peel deposition and subsequent entrapment. correction of this situation commits the patient to a surgical procedure that might have been avoided by initial evacuation of the pneumothorax. it is recommended that observation be considered only for patients with a simple pneumothorax whose size involves 15% or less of the volume of the chesty if the pneumothorax has not resolved within 1 to 2 weeks, intervention to achieve full expansion should be instituted. another factor to consider, particularly with primary spontaneous pneumothorax, is that observation alone does nothing to decrease the chance of recurrence. last, in this age of economic constraints, it may be more cost-effective to definitively treat a pneumothorax on presentation. simple aspiration can be considered in the case of a simple pneumothorax in which there is no suspicion of an ongoing air leak and the patient is not on positive pressure ventilation. some authors believe that in select situations aspiration is the treatment of choice." the goal of aspiration is to remove air from the pleural space. it conveys no protection from an ongoing leak or recurrence in the future . the procedure is performed in a manner similar to that used for decompressing a tension pneumothorax. after sterilely preparing the skin and infiltrating with a local anesthetic, a 16-or l8-gauge intravenous catheter is placed into the pleural space in the midclavicular line over the superior surface of the second rib. the needle is then withdrawn, and the catheter is connected to a short length of intravenous tubing capped with a threeway stopcock. a 60-ml syringe is then used to aspirate air from the chest cavity . when air can no longer be aspirated, the catheter is withdrawn, and the first chest x-ray is obtained. if 4 l of air are aspirated and no resistance is met, there is an ongoing air leak, and a chest tube should be placed . percutaneous tube thoracostomy is a good option for a simple pneumothorax. many consider this the procedure of choice for simple pneumothoraces. cited advantages are therapeutic and cost-effectiveness as well as less trauma compared to standard tube thoracostomy. depending on the size and etiology of the pneumothorax, success rates for these catheters are reported in the 85% to 90% range ." the catheters range in size from 9 to 16 french and are placed using a catheter-overneedle or seldinger technique. the kits (e.g., arrow pneumothorax kit, arrow international, reading, pa) are usually equipped with all the necessary supplies to insert the catheters and an adapter such that the catheter can be connected to a heimlich valve or a standard suction device such as pleur-evac (dsp worldwide, fall river, mal. thes e tubes are limited by their size and would be a poor choice for a patient with a large air leak . the principal factor in determining the flow rate through a tube is the diameter of the tube . thus, a patient with a massive air leak, especially on positive pressure ventilation, should have a standard chest tube placed . as a general guide, it takes at least a 28-french tube to accom-modate approximately l5l/min of flow at -locm h 20 suction." a standard chest tube should be placed for failure of a percutaneous tube, a pneumothorax associated with significant fluid collection, or a pneumothorax for which the leak is expected to overwhelm a small-caliber tube, more likely in the setting of positive pressure ventilation. such tubes are generally placed under local anesthesia and sedation, employing sterile technique. apical tubes, as employed for drainage of air, are best placed in the mid-or anterior axillary line in the third or fourth intercostal space. it is generally recommended to tunnel the tube subcutaneous up one interspace before entering the pleural cavity. the tunnel serves two purposes. first, it forms a flap valve that helps prevent entrance of air into the chest after the tube is removed. second, the tunnel allows the surgeon to control the direction of the tube, anteriorly when placed for air or posteriorly when placed to drain fluid . the tube can then be placed for passive or active drainage. passive drainage may be achieved by connecting the tube to a heimlich flutter valve or waterseal on a pleur-evac. for active drainage, most surgeons use a threebottle system ( fig. 74 .31, generally unified as a commercially available unit such as pleur-evac. active drainage expedites and facilitates full expansion of the lung . although also reported with passive drainage, the rare complication of reexpans ion pulmonary edema appears to be more common with active suction. 21 ,22 generally viewed as a "floor procedure," chest tube placement should be given all the consideration of a major operation. although it can be performed with minimal discomfort, utilizing intravenous sedation and strategic local anesthesia, a chest tube placed by an inexperienced operator without expert supervision can be a horrific experience for a patient. in addition to the discomfort, chest tube placement may be accompanied by a number of complications including empyema, lung injury and bleeding, and death." therefore, coagulation profiles and immunocompetency should be taken into consideration for all patients considered for this procedure . intravenous analgesia, a short-acting benzodiazepine, or both should be used for an elective chest tube placement. proper use of local anesthesia is critical for patient comfort during the procedure. the amount of local anesthetic that can be administered is limited by toxicity. a small amount of anesthetic is injected for the skin incision, and the remainder is accurately injected along the course of the tunnel that will be created to place the tube. this step allows the surgeon both to locate the superior surface of the rib and, by aspirating, to identify the parietal pleura. a small bolus of anesthesia can be injected at the level of the parietal pleura, and if adequate time is allowed after injection of the pleura, the discomfort of the tube placement can be limited to "pressure" and not sharp pain. as with all procedures under local anesthesia, it is also important to prepare the patient for any anticipated sensations, particularly the entrance into the pleural space as well as the possibility for triggering severe coughing if a collapsed lung is rapidly reexpanded. with the exception of those patients with a large open pneumothorax, essentially all patients considered for surgical treatment for a pneumothorax already have a chest tube in place. with a progressive air leak, tension physiology is imminent once positive pressure ventilation is instituted unless there is a pathway for egress of air from the pleural space or intubation is performed directly with a double-lumen endotracheal tube such that the leaking lung can be immediately isolated. specific surgical procedures are discussed under the appropriate sections, but it is worth noting that there are two surgical approaches available, video-assisted thoracoscopic surgery (vats) or standard thoracotomy. general indications for surgical intervention for a pneumothorax are failure of less-invasive therapy or occurrence of the pneumothorax in the context of additional indications for chest exploration. a vats approach can be employed, at least initially, for most pneumothoraces. examples of contraindications to a vats approach are a pneumothorax secondary to an esophageal perforation, major airway disruption, or a pneumothorax accompanied by significant ongoing bleeding or concomitant trauma to other thoracic organs. pneumothoraces can be broadly grouped as spontaneous or traumatic. spontaneous pneumothoraces can be further subclassified as primary or secondary, with primary arising in patients with no known underlying pulmonary disease and secondary spontaneous pneumothoraces arising as a complication of known underlying pulmonary disease. traumatic pneumothoraces can be subclassified as those that are the result of blunt or penetrating trauma to the chest or those that are iatrogenically induced secondary to an invasive procedure or barotrauma from positive pressure ventilation. in this chapter, the former are referred to as traumatic pneumothoraces and the latter as iatrogenic pneumothoraces. primary spontaneous pneumothorax most commonly occurs in tall young men but may occur in anyone at any age. the peak incidence has been reported to occur for both men and women 25 to 34 years old. it is approximately six times more common in men than women. it is thought that the final common pathway for most primary spontaneous pneumothoraces is rupture of subpleural blebs. it is also thought that inflammation of the distal airways plays a significant role in the pathogenesis of this disorder. the lack of communication between these blebs and the distal airways, and hence the inability to rapidly decompress, may explain the increased incidence of primary spontaneous pneumothorax associated with significant drops in atmospheric pressure. the role of inflammation may explain why spontaneous pneumothorax is much more common in smokers. in fact, there appears to be a dose-response relationship, with light smokers (fewer than 13 cigarettes/day) running a risk 7 times that of nonsmokers and those smoking more than 22 cigarettes/day at least 100 times more likely to have a spontaneous pneumothorax than nonsmokers." it is generally accepted that recurrent spontaneous pneumothoraces become increasingly likely with each successive occurrence. the exact statistics vary, but it is estimated that the risk of recurrence in the absence of aggressive preventive measures is in the range of 25% after the first recurrence and 50% after the second recurrence.p the chance of recurrence is very much related to the treatment undertaken for the initial spontaneous pneumothorax. cessation of smoking also decreases the risk of recurrence. selection of treatment remains an area of controversy. a consensus statement was released to provide guidance in treatment selection." observation alone should be selected for treatment on the initial presentation of a small, asymptomatic primary pneumothorax in a patient without any associated comorbidities. it should be reserved for patients who have no barotrauma risks and have ready access to medical help. this treatment may be particularly appropriate for patients who present with heavy cigarette abuse and are willing to stop smoking. aspiration is another option, but it confers no significant protection from recurrence. once the decision has been made to violate the pleura for aspiration, it is probably worthwhile leaving a catheter through which air can be continuously aspirated and subsequent pleurodesis can be performed. if a large air leak is anticipated or if there is significant effusion associated with the pneumothorax, then a standard 28-french chest tube should be placed. neither technique, without pleurodesis, seems to convey significant protection from recurrence. surgical treatment remains the gold standard in preventing recurrence of spontaneous pneumothorax. some of the indications for surgical treatment of a spontaneous pneumothorax include a second pneumothorax (ipsilateral recurrence or a new pneumothorax on the contralateral side); tension physiology; synchronous bilateral pneumothoraces; associated hemothorax (likely secondary to a tom adhesion and complicating approximately 5% of spontaneous pneumothoraces); failure of tube thoracostomy; and lifestyle factors. surgery should be considered if a leak persists for more than 48 h. lifestyle issues that are accepted indications for surgical therapy at the initial presentation of a primary spontaneous pneumothorax include occupational exposure to barotrauma (scuba diving or flying nonpressurized high-altitude aircraft) and poor accessibility to medical care. the surgical procedure for spontaneous pneumothorax is resection of the blebs that are usually present, most commonly located in the apex of the upper lobe or the superior segment of the lower lobe. resection of the blebs is performed with a pulmonary stapling device. most surgeons also perform a mechanical pleurodesis of the pleura, utilizing an abrasive material such as an electrocautery scratch pad, or perform parietal pleurectomy. currently, vats is considered the preferred surgical approach . a standard triad of video ports can be employed in most cases ( fig. 74.4 ). use of a 30°thoracoscope facilitates visualization at the apex of the chest. the thoracoscope is introduced through the inferior port with a grasping device, and the thoracoscopic stapler is introduced through the two superior ports. a sponge stick or folded electrocautery scratch pad can be used for the pleurodesis, with or without a parietal pleurectomy of the bony hemithorax. at the conclusion of the operation, a single 28-french chest tube , with additional ports cut through the radioopaque line , can be placed posteriorly to the apex of the chest. a rongeur is useful for cutting extra holes in chest tubes. if the surgeon is more comfortable placing an anterior and posterior chest tube, two video ports can be created low in the chest at the future chest tube sites, and a single port can be placed in line with a potential thoracotomy incision for grasping and stapling. in skilled hands, the entire lung can be well visualized and mobilized for stapler application, mechanical pleurodesis, and if necessary, pleurectomy ( fig. 74.5 ). for most of these cases, thoracotomy offers little advantage with respect to visualization or performance of the bleb resections, pleurectomy, and pleurodesis. secondary spontaneous pneumothorax is a more serious condition than primary spontaneous pneumothorax because of its occurrence in patients who likely have significantly less pulmonary reserve than the typical patient presenting with a primary spontaneous pneumothorax. as opposed to primary spontaneous pneumothoraces, secondary spontaneous pneumothoraces are associated with a significant mortality." historically, the most common cause of secondary spontaneous pneumothorax has been chronic obstructive pulmonary disease (copd). there are many other causes of secondary spontaneous pneumothorax, including, but not limited to, cystic fibrosis, asthma, cancer, many types of infection, sarcoid, collagen vascular diseases, and catamenial pneoumothorax. the principal factor affecting choice of treatment in these patients is the nature of the underlying pulmonary disease. if the patient is symptomatic, which is far more likely with this patient population, there is no role for observation. furthermore, an increase in the pneumothorax could possibly place the patient's life in jeopardy. therefore, observation of a secondary spontaneous pneumothorax is not recommended. tension physiology is frequently unnecessary to cause clinical decompensation in these patients. any consideration of employing positive pressure ventilation in a patient with a secondary pneumothorax should serve as an indication for thoracostomy tube placement. earnest consideration should be given to sclerosis for prevention in most of these cases. an important exception is the patient who is awaiting lung transplantation because adhesions resulting from sclerosis can significantly complicate explantation of the native lung at the time of transplantation. another consideration is the nature of the underlying pulmonary disease. if the pneumothorax occurs in the setting of a disease, such as certain malignancies or infections, it may not be possible to staple the lung or to achieve total lung expansion . in these cases, particularly if the patient is terminally ill, consideration should be given to sending the patient home with a chest tube and a heimlich valve if this provides adequate palliation. iatrogenic pneumothorax may be the most common cause of pneumothorax." the most common causes include transthoracic needle biopsy, central line placement, thoracentesis, figure 74.5. intraoperative photograph taken through the videothoracoscope demonstrates an apical bleb (within the grasping forceps iin a patient presenting with a recurrentprimaryspontaneous pneumothorax. the bleb was then resected with a thoracoscopic stapling device, introduced through the third port incision. subsequently, a parietalpleurectomy of the entire chest wall pleura and a mechanical pleurodesis of the mediastinal and diaphragmatic pleura were performed. a single chest tube was then inserted through the lowest incision, which had been used for the videothoracoscope duringthe procedure. transbronchial pulmonary biopsy, and positive pressure ventilation. the management of an iatrogenic pneumothorax must take into account a number of factors, including the etiology, symptoms, and size of the pneumothorax and ventilatory status of the patient. it is logical to consider those patients with pneumothorax secondary to positive pressure ventilation, barotrauma, separately from those who developed pneumothorax secondary to violation of the visceral pleura. the development of a pneumothorax as a result of barotrauma is an indication for immediate placement of a standard chest tube. this indication is also true for a procedure-induced pneumothorax in a ventilated patient because positive pressure ventilation can rapidly lead to a tension pneumothorax. the clinician should always consider pneumothorax as a cause for instability in a ventilated patient who recently underwent thoracentesis or central line placement. the majority of iatrogenic pneumothoraces are procedure induced. these pneumothoraces differ from spontaneous pneumothoraces in that the patient is not at an increased risk for recurrence. for small, asymptomatic pneumothoraces, observation is appropriate, and thoracostomy tube placement and sclerosis are not indicated. for larger pneumothoraces or symptomatic pneumothoraces in ambulatory patients, simple aspiration or temporary placement of a small percutaneous catheter is the preferred approach of many clinicians. any closed pneumothorax arising from visceral pleural disruption has the potential to develop into a tension pneumothorax. tension pneumothorax occurs when air accumulates in the pleural space in excess of intrapleural pressure and actively compresses the ipsilateral lung. this tension physiology will eventually lead to contralateral mediastinal shift and, in addition to pulmonary embarrassment, can severely limit venous return and compromise cardiac output. untreated, tension pneumothorax may lead to cardiopulmonary arrest and for this reason is a life-threatening emergency. tension pneumothorax is believed to occur when a pleural disruption forms a functional one-way valve allowing air to escape from the lung but not reenter. such physiology is occasionally well tolerated in a healthy adult and can await chest tube placement under urgent, but controlled, conditions. if, however, the patient is in distress and the diagnosis is suspected, placement of a 16-or is-gauge intravenous catheter over the second rib, in the midclavicular line, will convert the tension pneumothorax to an open pneumothorax. after decompression has been achieved, a chest tube can then be placed in the usual manner. this procedure should always be performed immediately in any patient who is decompensating and for whom tension pneumothorax is in the differential diagnosis. it is a mistake to wait for a confirmatory chest xray in such a situation. although pneumothorax is a disorder of air that has entered into the pleural space, blebs and bullae are also disorders of abnormal air collections, but still contained within the lung. only blebs can be considered a true pleural disorder. blebs arise when air escapes from the pulmonary parenchyma and is trapped in the visceral pleura. simply stated, blebs are subpleural collections of air. they are usually small, less than 2 em, and tend to occur at the apex of the upper lobe or the apex of the superior segment. the significance of blebs is uncertain. by themselves, it is doubtful that they cause any significant effect on pulmonary function. their primary clinical significance lies in the fact that they appear to be involved in the pathogenesis of spontaneous pneumothorax. no specific treatment is indicated for the finding of pulmonary blebs in the absence of pneumothorax. cessation of smoking, as always, is recommended. bullae are air collections measuring at least 1em, but may become so large that they occupy the greater part of the hemithorax. as opposed to blebs, bullae are formed by destruction and coalescence of alveoli. they may demonstrate trabeculated lumens formed by the residual structural elements from the lung parenchyma they replaced. blebs may be an incidental finding in patients with otherwise normal lungs, but bullae are likely to be associated with some form of pulmonary disease, most likely emphysema. bullous disease is also frequently asymptomatic. in such cases, cessation of smoking and annual chest x-rays are sufficient. in the setting of known underlying lung disease, treatment of that disorder is the priority. pneumothorax, infection, or hemoptysis can prompt surgical intervention. surgical intervention can also be indicated for compression of normal lung tissue to improve pulmonary function. generally, very good results can be anticipated if the bulla is occupying more than 500/0 of the hemithorax, compressing well-perfused parenchyma.29 pleural effusions are a very common disorder encountered by the clinician. there are many potential causes of effusions (table 74 .1). occasionally, it is possible to deduce the etiology in the context of the patient's chest radiograph and concurrent morbidities. frequently, however, the fluid must be sampled to yield a diagnosis. there is a normal composition of pleural fluid (table 74. 2) and a host of tests that can be performed on the fluid in pursuit of a diagnosis (table 74. 3). once the fluid is sampled, it will fall into one of two categories, transudative or exudative, and 990/0 of exudative effusions will demonstrate at least one of the following characteristics: pleural fluid protein/serum protein ratio greater than 0.5, pleural fluid lactate dehydrogenase (ldh)/serum ldh greater than 0.6, or pleural fluid ldh more than twothirds of the upper normal limit for serum ldh. 30 transudative effusions result from a perturbation in the hydrostatic or oncotic forces that affect fluid formation and turnover in the pleural space, as described in the physiology section. this imbalance results in fluid accumulation in the pleural space. for transudative effusions, the goal is to drain the effusion for symptomatic relief, if necessary, but to focus on the systemic disease. exudative effusions result from diseases that involve the pleura and may be broadly grouped as benign or malignant. the treatment of an exudative effusion is disease specific. pleural effusions may be asymptomatic or may cause the patient to present with shortness of breath, secondary to compression of pulmonary parenchyma, as well as other symptoms. a nonspecific sign that is compatible with an effusion is the presence of a nonproductive cough. if the disorder causing the effusion has provoked an inflammatory response in the parietal pleura, the patient may complain of pain with respiration, known as pleuritic chest pain. restricted chest wall movement or change in the contour of the hemithorax may be evident, depending on the nature of the effusion and its effect on pleural pressure. if the effusion is unilateral and massive, the trachea may deviate to the contralateral side. absence of vocal fremitus, dullness to percussion, and decreased breath sounds are all characteristic apf/s, pleural fluid-to-serum ratio. biu, concentration in international units. findings on physical examination. if the pleura is inflamed, there may be an audible rub. a rub is likely to precede a significant effusion that will separate the roughened pleural surfaces and diminish or resolve the rub. in the case of a hydropneumothorax, there may be an audible ii splash," as originally described by hippocrates. plain radiographs of the chest remain the most common test obtained to evaluate a suspected effusion. if the effusion is free flowing, the lateral costophrenic angle may be blunted on an upright posteroanterior radiograph. the lateral radiograph is more sensitive than the posteroanterior view, but neither is as sensitive as the lateral decubitus projection. additional studies that are commonly used to obtain more information about a suspected effusion include ultrasound and ct scan. ultrasound is helpful in distinguishing pleural thickening from pleural fluid and determining if an effusion is complex or simple, and it has the advantage of portability. it can be used to help direct the clinician to the best area to perform a thoracentesis at the bedside. ct scans give the most information with respect to exact location of an effusion and may be particularly helpful in distinguishing effusion from pleural disease or parenchymal disease. if the clinical scenario warrants diagnosis of the effusion, then the next step is to perform a thoracentesis to obtain a specimen for analysis and, possibly, to drain the effusion for relief of symptoms. the decision to perform such a procedure should be taken seriously for the complications can be significant and include pneumothorax, hemothorax, and conversion of a sterile effusion into an empyema. thus, it is important to make sure the patient is not coagulopathic, and if there is any question regarding the appropriate site to insert the needle, a bedside ultrasound should be performed. sterile technique must be observed. the procedure is most easily accomplished with the patient in the sitting position and leaning over a bedside table that has been padded with one or two pillows. for a diagnostic tap, a long 25-gauge needle can be used to infiltrate with lidocaine and can be used as a finder needle. a 22-gauge needle is then used to perform the aspiration. if a therapeutic tap is indicated, a similar technique is employed, except that a catheter is placed into the chest cavity and connected via tubing to a three-way stopcock, which in tum is connected to a syringe or a vacuum bottle. a number of commercial kits are available for this purpose. regardless of the technique, it is generally recommended that not more than 1l of fluid should be aspirated at one time as this increases the chances of developing reexpansion pulmonary edema. the exact etiology of this syndrome is not fully understood and may be accompanied by 200/0 mortality." treatment for reexpansion edema is supportive care. if the effusion is transudative, then it is most likely secondary to congestive heart failure, hepatic insufficiency, or renal insufficiency. pleural effusions secondary to congestive failure are the most common transudative effusions.f most of these effusions are bilateral. the presence of a unilateral effusion or bilateral effusions of significantly different sizes does not exclude this diagnosis, but would be unusual. the disorder is thought to result from increased pressure at the pulmonary capillary level secondary to left heart failure. the treatment is the same as for other transudative effusions and is directed at the underlying cause, in this case, congestive failure. if the etiology is unclear or if the effusion remains unchanged after the congestive heart failure has improved, a diagnostic thoracentesis should be performed. occasionally, it is necessary to perform a therapeutic tap for symptomatic relief. approximately 5% of patients with hepatic cirrhosis will develop pleural effusions as a result of their disease." twothirds of cirrhotic pleural effusions are right sided. usually, the patient will have ascites in addition to the pleural effusion. the pleural effusion is thought to result from a one-way communication and fluid flow from the peritoneum, across the diaphragm, to the pleural space. the treatment should be directed at the liver failure and ascites, with diuresis and salt restriction as the initial steps in management. decompression of the portal circulation, percutaneously or surgically, may be indicated to treat the underlying disease. if the pleural effusion is unresponsive to these measures, or if pulmonary symptoms necessitate intervention, the options include drainage and pleurodesis. sometimes surgical intervention is indicated, combining closure of a demonstrated peritonealpleural communication with pleurodesis, this has been accomplished using vats techniques as well as thoracotomy.34,35 shunting ascites to the venous system is another option; however, these patients are generally poor surgical candidates unless their liver failure is corrected. it is best to avoid placement of a chest tube for drainage of a pleural effusion secondary to cirrhosis as the high drainage rates from decompression of ascites across the diaphragm can make these tubes difficult to remove. nephrotic syndrome is another disorder associated with pleural transudates. these effusions tend to be bilateral and result from decreased plasma oncotic pressure. again, treatment should be aimed at the primary disorder. in severely symptomatic patients, drainage and sclerosis can be considered. other conditions that may provoke a transudative effusion include, but are not limited to, pulmonary embolism, superior vena cava obstruction, peritoneal dialysis, myxedema, glomerulonephritis, meigs syndrome, and sarcoidosis. exudative effusions can be broadly grouped into benign and malignant effusions. the malignant effusions arise most commonly from metastatic disease but can also herald the presence of a primary malignancy of the pleura. the benign causes of exudative effusion include a long list of conditions, including, but not limited to, infectious diseases, pulmonary embolism, collagen vascular diseases, drug-induced disorders, bleeding, chyle leak, subdiaphragmatic infections, pancreatitis, and esophageal perforation. if the cause is not obvious, then a thoracentesis should be the next step in diagnosing the etiology of the effusion. the following sections discuss the conditions most likely to be encountered by the surgeon. malignant effusions represent one of the most common indications for chest tube placement. the tumors most frequently associated with a pleural effusion include lung cancer, breast cancer, ovarian cancer, and lymphoma. dyspnea from pulmo if the patient's lung does not expand, or if pleurodesis has failed, then chronic drainage becomes the next option. the options include internal or external drainage. for external drainage, the patient may undergo repeat therapeutic thoracenteses or placement of a long-term drainage catheter. intermittent thoracentesis may be the best for a patient who is minimally symptomatic or has a very short life expectancy. indwelling catheter placement for intermittent drainage proa large number of agents are available for pleural sclerosis. talc is the most popular chemical sclerosant used. a large metaanalysis reviewing 36 randomized controlled trials including over 1499 patients concluded that: "the currently available evidence supports the need for chemical sclerosants for successful pleurodesis, the use of talc as the sclerosant of choice, and thoracoscopic pleurodesis as the preferred technique for pleurodesis based on efficacy. there was no evidence for an increase in mortality following talc pleurodesis. ,, 40 doxycycline is the cheapest of the sclerosing agents. it is also the one that most commonly causes significant discomfort. it is administered dissolved in 50 to 100ml sterile saline and 200mg lidocaine via chest tube. bleomycin generally causes little discomfort but is the most expensive agent. it is administered dissolved in 100ml of sterile saline via chest tube. although generally well tolerated, intrapleural bleomycin is absorbed systemically and is therefore not recommended for patients who are receiving chemotherapy, are immunosuppressed, or have renal failure. support can be found in the literature for using any of these agents in nearly any situation. a rational approach is to use talc for pleurodesis of malignant effusion and doxycycline for benign indications if the patient can safely tolerate significant sedation with intravenous narcotics and benzodiazepines or if the patient already has an epidural catheter in place. even with instillation of intrapleural lidocaine, patients will commonly describe doxycycline pleurodesis as one of the most painful experiences of their lives. an argument can be made to avoid the use of talc when there is potential contamination of the pleural space, either primarily or secondarily by some other site of active infection. talc, as a permanent foreign body, can serve as nidus for infection and result in chronic empyema. in a patient with a malignant effusion, in whom there is concern for possible contamination of the pleural space, bleomycin pleurodesis would be a reasonable option. if the lung expands completely when fluid has been drained, then pleurodesis is an option. if the lung does not expand, pleural apposition cannot occur, and injection of a sclerosant will not work. in fact, the sclerosant may further hinder the absorptive mechanisms of the pleura, thereby making the effusion worse. the literature does not support the belief that the pleural drainage must be less than is0ml/day to achieve effective sclerosis. equal results and greater cost-effectiveness appear to occur if the sclerosis is performed as soon as the lung is fully expanded, regardless of the volume of drainage." there is also no support for the time-honored tradition of "rolling" the patient to achieve even distribution of the sclerosing agent. a prospective, randomized study utilizing instillation of 99mtc-sestamibi-labeled talc suspension has demonstrated that the dispersion of talc suspension and the overall success rate in patients with malignant effusions is not influenced by the position of the patient." nary compression is the most common symptom produced by a malignant effusion. malignant effusions are exudative and frequently sanguinous in appearance. the diagnosis can frequently be established by cytological demonstration of cancer cells in the fluid, although up to 400/0 of effusions yield nondiagnostic cytology." thus, if malignancy is suspected and the fluid cytology is nondiagnostic, a pleural biopsy should be considered. the approach of choice is vats if surgery is required to establish a diagnosis. depending on their surgical risk, patients may be well served by going to the operating room early in their course for diagnosis and drainage. under general anesthesia, a single chest tube incision can be created through which the effusion can be drained and the thoracoscope introduced for examination and photodocumentation of the pleural cavity. pleural biopsies can then be performed through the same incision by sliding the camera port out of the incision onto the proximal scope and sliding a biopsy forceps alongside the scope, through the same incision. a s-mm 30°thoracoscope is particularly helpful for this procedure as it allows the surgeon to look over the surfaces of the chest cavity and to move the scope off to the side, which facilitates manipulation of the biopsy forceps. if the lung demonstrates the ability to fully expand and a malignant diagnosis is confirmed, intraoperative talc poudrage can be considered. all this can be accomplished through a single 10-to is-mm incision. once the etiology of the malignant effusion is established, a treatment strategy can be formulated." surgical debulking of metastatic pleural tumor is generally not part of the treatment algorithm outside an experimental protocol. most patients are relegated to chemotherapy, radiation therapy, or palliative measures directed at preventing further fluid accumulation. some tumors, such as small cell lung cancer, breast cancer, ovarian cancer, and lymphoma, may respond well to chemotherapy, including resolution of the pleural effusion. mediastinal radiation therapy may also be indicated in treatment of the patient's tumor, especially if the tumor has involved the thoracic duct and resulted in a chylothorax. if the patient is not receiving treatment for the underlying malignancy or reaccumulates the effusion in spite of treatment, an alternative strategy must be considered if the effusion is causing symptoms. the first choice is pleurodesis, with other options for failure of this technique. vides good palliation." commercial kits such as the pleurex catheter (denver biomedical, denver co) are available. another option would be placement of a pleuroperitoneal shunt for internal drainage of the pleural space and decompression of the lung. internal drainage can be accomplished by implanting a shunt, such as the denver shunt (denver biomaterials), which has a pumping chamber that the patient can press to transfer fluid across the negative pressure gradient from the pleural cavity to the peritoneal cavity. there are a number of downsides to this option. placement requires an operation, usually under general anesthesia; a small percentage of the shunts will obstruct; and the patient must actively pump the shunt to transfer fluid. thoracotomy with decortication, in the presence of a malignant effusion, is rarely indicated. although highly variable, the average survival of a patient with a malignant effusion from lung cancer is on the order of 4 months, whereas for breast or ovarian cancer it may be more in the range of 7 to 9 months.f thus, recovery from such an operation is likely to result in decreased quality of life for a significant portion of the patient's remaining time. effusions associated with pneumonia (parapneumonic effusions) are the most common cause of benign exudative effusions. they result from visceral pleural inflammation that alters the normal fluid balance of the pleural space. these effusions may initially be sterile, but if the parenchymal infection spreads to the effusion, an empyema results. there is a continuum that reflects the natural history of untreated parapneumonic effusions, from a thin, clear sterile collection to an infected fibrous peel encasing the lung. the first stage is the exudative stage, characterized by fluid exuding from the lung into the pleural space, likely from the pulmonary interstitial space. this stage should resolve with antibiotic therapy and generally does not require drainage. normal ph and glucose with a low ldh and white blood cell count are characteristic of the fluid at this stage. untreated, the effusion is likely to progress to the [ibtopurulent stage, characterized by increased fluid that is heavily laden with white blood cells, microorganisms, and cellular debris. fibrin is deposited on the pleural surfaces, and the stage is set for pulmonary entrapment. at this point, the fluid ph and glucose level fall, and the ldh rises. chest tube drainage is indicated but becomes more difficult as the effusion loculates with fibrinous septae. the final stage is the organizational stage, during which fibroblasts grow into the effusion, laying down a thick fibrous peel that encases the lung and results in entrapment. the remaining effusion is thick and infected and may necessitate through the chest wall or into the lung. the presentation of a parapneumonic effusion or empyema depends, to a certain extent, on the organism causing the infection. for aerobic organisms, the presence of the effusion has little impact on the clinical picture, which is that of a bacterial pneumonia: fever, chest pain, and a productive cough. an anaerobic infection, frequently as a result of aspiration, is more likely to present in a subacute manner. a patient with an anaerobic empyema may have symptoms for more than a week before seeking medical help, and significant weight loss may be a chief component of their presentation. true empyema thoracis is simply defined as pus in the pleural space, a clear indication for drainage. for a small simple parapneumonic effusion in a patient being treated with and responding to appropriate antibiotics, there is no indication for drainage. the issue is how to identify the effusion that is not yet frankly purulent but will require drainage to resolve. if the patient with pneumonia continues to have a large or increasing effusion, then a thoracentesis should be performed. the fluid from the tap should be sent for analysis of glucose, ph, ldh, amylase, protein, complete blood count with differential, gram stain, aerobic/anaerobic bacterial cultures, and if indicated, special microorganism cultures and stains. if malignancy is suspected, cytology should also be sent. as seen in table 74 .5 from the american college of chest physicians parapneumonic effusions panel evidence-based guideline," the risk outcome can be stratified and used to determine whether drainage is warranted. these remain, however, only guidelines, and sound clinical judgment is essential. once the decision has been made to drain the fluid collection, a number of options are available: aspiration, chest tube drainage, vats drainage, limited thoracotomy and open drainage, or full thoracotomy with drainage and decortication. for diagnosis and initial treatment of a free-flowing pleural effusion, aspiration is an appropriate initial step. if the clinical situation mandates further drainage, then the clinician has several options. if the effusion is free flowing, then placement of a standard chest tube is a reasonable option. if the effusion is loculated, ultrasound guidance may be helpful either for marking the ideal location for thoracentesis or thoracostomy tube placement or for placement of a percutaneous drainage catheter. the use of intrapleural streptokinase or urokinase has been advocated if drainage fails due to loculations." in several well-constructed studies, however, it has been shown that enzymatic treatment will increase the volume of chest tube output but not affect the clinical course. the role of these agents for treatment of empyema remains undefined." there are several surgical options for patients with an empyema. the goals of surgical therapy are to establish drainage and, depending on the situation, to eliminate space in the pleural cavity. space elimination can be accomplished by decortication to allow the lung to expand, collapsing the chest wall with a thoracoplasty, or transposing muscle flaps to fill the space. a critical component is always to establish drainage. the least-invasive option is to explore the chest cavity thoracoscopically, disrupt loculations, debride the visceral pleura, and strategically place chest tubes. frequently, it is possible to accomplish this procedure utilizing the patient's existing chest tube sites as video ports. this option is most likely to be successful if performed in the exudative or early fibrinopurulent stages." once in the organizational stage, the lung is encased in a fibrous peel that most often requires an open thoracotomy to adequately remove. if the patient is able to tolerate such a procedure, then this represents the most effective treatment of the problem. the goal in such a situation is to drain the infection and obliterate any space with reexpanded lung 1'h is the preferred pleural fluid chemistry test, and ph must be determined using a blood gas analyzer. if a blood gas analyzer is not available, pleural fluid glucose should be used (po glucose> 60mgjdl; pi glucose < 60mgjdl). the panel cautions that the clinical utility and decision thresholds for ph and glucose have not been well established. "clinical experience indicates that effusions of this size do not require thoracentesis for evaluation but will resolve. tissue. if a portion of the lung has already been removed or if the infection has rendered portions nonviable, mandating resection, then space may become an issue. the favored option is to transpose muscle flaps into the chest cavity to obliterate any residual space that exists after the lung has been decorticated and reexpanded. the commonly used muscle flaps are serratus, latissimus, and pectoralis. omentum is also a good option. a good approach in these cases is to enter the chest through a vertically oriented muscle-sparing thoracotomy such that both serratus and latissimus are spared and can be harvested if necessary. after a drainage procedure, the clinician is faced with the management of the chest tubes. the classic treatment of a chest tube placed into an empyema is to leave it to closed suction drainage for 2 to 3 weeks. thereafter, the tubes are taken off suction and converted to open drainage, slowly withdrawing them over the course of several more weeks. another option is to leave the tubes in place for approximately 1 week on suction. if the lung is fully expanded, drainage is minimal «soml/day), and the patient has no further signs of infection, then the tubes may be removed. the critical point is that the lung must be fully expanded. cases in which this strategy is safe and effective usually demonstrate full expansion of the lung on the chest x-ray and "walling off" of the chest tube, characterized by essentially no drainage and lack of respiratory variation in the waterseal chamber of the pleur-evac. for patients with chronic empyema or empyema with bronchopleural fistula or patients unable to tolerate thoracotomy, an open drainage procedure may represent the best option. these procedures involve localizing the most dependent portion of the empyema cavity and resecting a portion of the overlying rib. the cavity is then entered, the pleural space debrided, and if possible, the lung decorticated. depending on the size of the cavity, it may close spontaneously or may require reconstruction, usually with a muscle flap. in an elderly or infirm patient, the cavity may be left open. given enough time, even cavities of substantial size will commonly close. almost any organism can cause an infection associated with a pleural effusion. tuberculosis may cause a pleural effusion that tends to be unilateral and of moderate size. they can be difficult to diagnose based on chemical and microbiological evaluation of the pleural fluid but usually demonstrate granulomatous pleuritis on closed pleural biopsy if the diagnosis is in doubt. the effusion usually responds to appropriate antibiotic therapy and, unless symptomatic or part of a mixed empyema, usually does not require drainage or surgery. viral effusions usually elude diagnosis and are self-limited. human immunodeficiency virus (hiv) does not appear to cause pleural effusions, but patients with hiv are more likely to develop pleural complications associated with a bacterial pneumonia. effusions may accompany any of a number of fungal pulmonary infections. the primary treatment is appropriate antibiotic therapy and, depending on the infection, drainage. of note, aspergillus empyemas are almost always associated with a bronchopleural fistula, or a history of previous treatment of tuberculosis with artificial pneumothorax, and almost always require surgical evacuation as part of their treatment. although relatively uncommon in the united states, the clinician should be aware that pleural effusions frequently accompany a number of parasitic infections. again, appropriate drug therapy is essential. of note, rupture of pleural or hepatic cysts into the pleural space can present with acute symptoms and, as in the case of echinococcus, may represent an indication for urgent thoracotomy to debride and drain the pleural space and to drain the original cyst. pleural effusions may accompany pulmonary emboli in 300/0 to 500/0 of cases. although the majority of these effusions are exudative, approximately one-quarter may be transudative. this is likely to be determined by the relative contribution to the effusion by the two mechanisms thought to be primarily responsible for the effusion. transudative effusions are thought to exude from the parietal pleura secondary to right heart failure. exudative effusions are thought to arise from the visceral pleural secondary to release of local factors from the emboli that increase capillary permeability. the treatment for pulmonary emboli with effusion is the same as for pulmonary emboli without effusion, and as always, the key factor is to consider the diagnosis in a patient with any of the symptoms suggestive of pulmonary embolus. inflammation or malignancy below the diaphragm can cause exudative pleural effusions as well as transudative effusions secondary to hepatic or renal dysfunction, as discussed in the transudative effusion section. acute pancreatitis generally leads to a left-sided effusion, likely as a result of transdiaphragmatic transfer of exudative ascites arising from pancreatic inflammation. the fluid almost always has an elevated amylase, and that amylase is frequently higher than the serum amylase. the fluid generally resolves with resolution of the pancreatic inflammation. pancreatic abscess can also cause a pleural effusion; again, the treatment is the usual treatment of a pancreatic abscess. a pancreatic pseudocyst can decompress into the pleural space, forming a pancreaticopleural fistula. these effusions tend to be large, usually left sided, very high in amylase, and usually accompanied by chest, not abdominal, symptoms. this finding is thought to be secondary to decompression of the pseudocyst into the thorax. treatment of this disorder is conservative, the same as the initial treatment of any pancreatic pseudocyst. the role of drainage of the effusion remains controversial, and the clinician should be aware of the risk of infection and the fact that the drainage is likely to be massive and to reaccumulate rapidly until the fistula has closed. should conservative treatment fail, a percutaneous or surgical drainage procedure should be planned. subphrenic abscess from any number of intraabdominal sources can lead to an exudative effusion. the effusions rarely are culture positive and tend to have a very high white cell count, yet the ph is usually above 7.20, and the glucose is usually greater than 60mgjdl. treatment of the effusion should be symptomatic as the approach is treatment of the abscess and its underlying cause. the effusion usually resolves with these measures. the clinician is advised always to consider esophageal perforation in the diagnosis of a pleural effusion, particularly after instrumentation of the esophagus or retching. iatrogenic injury accounts for two-thirds of these injuries, and the patient frequently complains of chest pain. the condition usually presents with a left-sided effusion, but it may be either side or bilateral and accompanied by, or replaced with, a pneumothorax. the fluid is almost always high in amylase from saliva that has leaked into the pleural space. the mortality rate is high, up to 600/0, which underscores the imperative of prompt diagnosis. if the diagnosis is being entertained, a contrast swallow study should be obtained. treatment depends on how early the disruption is diagnosed and ranges from primary repair to esophageal exclusion. pleural drainage, cessation of ongoing pleural soilage, nutritional support and antibiotics are necessary components of the treatment strategy. chylothorax is an exudative effusion caused by disruption of the lymphatics in the chest, most commonly the thoracic duct, and subsequent drainage of chyle into the pleural space. the initial presentation of a chylothorax is determined by the size of effusion and its mechanical effects within the hemithorax. once a chest tube is in place, the symptoms are determined by the persistence of the drainage. the longer the drainage continues, the more dangerous it becomes, with the consequences being dehydration, nutritional depletion, and immunocompromise." more than 50% of chylothoraces are secondary to ductal obstruction and disruption by tumor, with lymphoma accounting for 75% of these cases. approximately 250/0 of chyle leaks are traumatic, with iatrogenic trauma the most common. of the iatrogenic causes, esophageal resection is the leading cause and is more common with transhiatal esophagectomies than transthoracic esophagectomies. the diagnosis is established by analysis of the fluid. although classically thought of as milky in appearance, chyle may appear serosanguinous, particularly in the fasting state. a triglyceride level in the fluid greater than 110mgjdl is highly suggestive of chyle, whereas a level below 50mgjdl essentially excludes the diagnosis of chylothorax. intermediate values require a lipoprotein analysis to prove the presence of chylomicrons to establish the diagnosis." some believe that the most reliable test is to give an oral challenge of cream and to observe the tube drainage for gross changes. the treatment of chylothorax remains controversial; some authors advocate a generous period of conservative treatment, while others recommend early intervention. all agree that a prolonged, high-output leak can be devastating. conservative therapy involves pleural drainage and total parenteral nutrition. if the leak is secondary to a malignancy, then chemotherapy or radiation therapy may be the treatment of choice. for other chylothoraces, surgical intervention is indicated when conservative measures have failed. the timing of such intervention is debatable, but most authors agree it is unwise to wait more than 1 week in the setting of an unremitting leak. it is important not to wait until the patient is immunocompromised and nutritionally depleted before deciding to operate as this would unnecessarily increase the risk of the surgery. at the time of surgery, heavy cream is administered via nasogastric tube immediately after intubation; this makes the duct more visible and may also identify the area of leakage. a number of surgical options are available, including parietal pleurectomy, direct ligation of the leak, and mass ligation of the duct. mass ligation offers at least an 800/0 chance of resolving the leak." many advocate performing a ligation of the duct on the right side, just as it emerges from the diaphragm, regardless of the side of the chylothorax. this ligation has traditionally been performed through a small thoracotomy incision in the sixth or seventh interspace. this procedure is figure 74.6. intraoperative photograph through a video thoracoscope shows probe under the thoracic duct, which has been dissected free at the level of the diaphragm on the right side. note thoracic duct, aorta, lung, and diaphragm . this duct was clipped and ligated via this vats(video-assisted thoracoscopic surgery] approach through three lo-mm incisions that resulted in immediate and complete resolution of the patient's chyle leak. readily accomplished thoracoscopically, utilizing three 1.0-to 1.s-cm port incisions (fig. 74.6 ). this procedure seems to offer less morbidity and equal efficacy to the open procedure. if the expertise exists , occlusion of the duct by interventional radiologists may be an option and would be the intermediate step to consider between failure of conservative therapy and surgical ligation of the duct . should all other measures fail, or if the right chest is hostile, the duct can be approached transabdominally in the aortic hiatus. the overwhelming majority of hemothoraces are caused by trauma, including iatrogenic trauma. there are other, significantly less common, causes, including bleeding from metastatic tumors involving the pleura, hemorrhage during anticoagulation therapy for pulmonary emboli, and catamenial hemothorax. the potential consequences of an undrained hemothorax include conversion to an empyema, provocation of a pleural effusion, and conversion to a fibrothorax with lung entrapment. the initial treatment of any hemothorax should be pleural drainage with a large-bore (32-or 36-french) chest tube. if the tube becomes clogged or is inadequate, additional tubes should be strategically placed . up to 4% of hemothoraces will become infected. this complication is more common in patients admitted in shock, with gross contamination of the pleura at the time of injury, with prolonged chest tube drainage, and with concomitant abdominal injuries. the pleural effusion associated with hemothoraces may occur after the blood has been evacuated and the chest tubes are removed . if the fluid is infected, it should be treated accordingly. if not , these effusions tend to be self-limited and require no further intervention. use of vats is safe and effective in evacuating blood from the chest cavity. in a prospective randomized trial, early vats drainage of retained hemothorax was found to decrease duration of tube drainage, hospital stay, and cost ." the earlier clot evacuation is attempted, the more likely vats evacuation will be completely successful. in addition, if intervention is insti-tuted early, the entire operation can frequently be performed through the existing chest tube incisions, occasionally requiring one additional videoscopic port incision. collagen vascular disorders may be associated with pleural effusions. in each case, the treatment of the effusion is symptomatic, with the primary therapy treatment of the underlying disorder. the two most common diseases are rheumatoid arthritis and systemic lupus erythematous. rheumatoid disease may involve the pulmonary parenchyma as well as the pleura. there are numerous other causes of exudative effusions. these include but are not limited to cardiac surgery, lung transplantation, asbestos exposure, dressler's syndrome, meigs syndrome, yellow nail syndrome, sarcoid, postpartum state, trapped lung, radiation exposure, ovarian hyperstimulation, amyloidosis, acute respiratory distress syndrome (ards), electrical bums, and uremia. a limited number of benign and malignant solid disorders affect the pleura. the most common benign conditions are fibrothorax, pleural plaques, diffuse pleural thickening, and benign fibrous tumors of the pleura. the most common malignancies are tumors metastatic to the pleura. the primary malignancy of the mesothelium is mesothelioma, and numerous cancers, such as liposarcoma and fibrosarcoma, may arise from any of the elements forming the connective tissue layer of the pleura. fibrothorax results from deposition of a thick fibrous layer along the pleural surface . this layer may cause entrapment of the lung as well as contraction and immobility of the skeletal hemithorax. the most common causes of fibrothorax are hemothorax, tuberculosis, and bacterial pneumonia. the treatment of fibrothorax is decortication, which is generally a major operation requiring a full thoracotomy. patients who are being considered for decortication should be low-risk surgical candidates who are symptomatic from their restriction and have pulmonary parenchyma that is anticipated to expand on release. generally, the indication is significant pulmonary compromise in a patient whose fibrothorax is stable or has been worsening for at least several months (fig. 74.7) . the pathogenesis of pleural plaques remains unclear. they are thought to be predominantly caused by asbestos, perhaps secondary to release of local factors in response to the foreign body after macrophage phagocytosis. pleural plaques are hard, raised, discrete areas involving the parietal pleura, particularly in the lateral, posterior portion of the hemithorax; 80% of pleural plaques that are due to asbestos are bilateral, with the majority of unilateral plaques thought to be secondary to other causes. pleural plaques do not appear to be predecessors of mesothelioma.f diffuse pleural thickening, like pleural plaques, appears to be predominantly related to asbestos exposure. other causes, such as drug reaction, hemothorax, and tuberculosis a figure 74.7. anteroposterior radiograph of the chest (ai demonstrates scoliosis and volume loss resulting from a flbrothorax caused by a tuberculous empyema . computed tomographic cross section (b) demonstrates the thick parietal pleural peel entrapping healthy lung have been reported. unlike pleural plaques, however, diffuse pleural thickening affects the visceral pleura. its exact etiology also remains unclear, but it is thought that inflammatory factors are likely to play a major role, particularly in the setting of a resolving asbestos-induced pleural effusion. there appears to be an initial decrease in pulmonary function associated with diffuse pleural thickening that tends to remain stable over time. again, there is no specific treatment recommended, just routine surveillance. the benign fibrous tumor of the pleura is rare and primary. these tumors arise from the visceral pleura, are not associated with asbestos, and are frequently discovered incidentally on chest x-ray. these tumors may reach enormous size, thereby causing symptoms by virtue of compression of other structures ( fig. 74.8) . surgery is the treatment of choice and is almost always curative.52 the most common malignancies of the pleura are metastatic, predominantly from lung, breast, or colon primaries (fig. 74.9 ). there are rare primary sarcomas arising from the connective tissue elements of the pleura, but the most common primary malignancy of the pleura is mesothelioma. essentially all malignancies of the pleura portend a grim prognosis as a result of either their conferment of metastatic status or the recalcitrance of the vast majority of de novo pleural malignancies to current treatments. mesothelioma is a rare tumor, with only 1500 to 3000 cases per year in the united states, but is thought to be on the rise in both the united states and intemationally. this is thought to be primarily related to the 20-to so-year lag between asbestos exposure, with which there is a clear causal link, and the relatively recent or lax restrictions on the use of asbestos.p'" another epidemiological factor of great concern is the link between the simian vacuolating virus 40 (sv40) and mesothelioma. the virus has been demonstrated in human mesotheliomas and is capable of inducing meso-b parenchyma. notice contraction and overlapping of ribs in both radiographs. the patient underwent a decortication with dramatic improvement of her scoliosis and pulm onary funct ion. thelioma by itself in animal models . it has been identified as a contaminant in millions of vaccines administered in the united states and may be a predisposing factor to mesothelioma formation in humans." pleural mesothelioma is almost always unilateral and is diagnosed by pleural biopsy. from the time of diagnosis, the median survival is 4 to 12 months. the tumor usually presents with dyspnea secondary to a pleural effusion but may also present with chest pain or constitutional symptoms. the cancer tends to line the chest cavity as a thick, plaque-like mass, fusing the two pleural layers and invaginating between the lobes of the lung (fig. 74.10 ). it progresses inexorably in a locoregional manner, invading lung, diaphragm, pericardium, and chest wall. contrary to popular belief, the disease also has the capacity to metastasize, but the lethality of the locoregional disease tends to preclude the clinical manifestation of any metastatic disease unless the natural history of the cancer is altered by a successfullocoregional treatment. although a number of staging systems exist, none has been universally adopted, although most incorporate the usual characteristics of the tnm system. this is likely a reflection of the small number of patients and even smaller number of centers investigating this disease. a staging system should accurately stratify prognosis, and this aspect of a staging system continues to evade researchers. as an example, cell type of mesothelioma (epithelial, sarcomatous, or mixed) is not currently part of any staging system, although some of the best results to date have been reported in patients restricted to the epithelial cell type, with 39% 5-year survival for patients treated with surgery, radiation, and chemotherapy." as a result of this deficiency in macroscopic staging, researchers are looking toward innovative technologies, like genetic profiling, in an effort to better determine prognosis and which patients might benefit from aggressive treatment protocols." there remains no accepted treatment for mesothelioma. because of the tumor's diffuse nature, complex anatomic presentation, and inherent recalcitrance to most treatment this patient presented with compla ints of bilateral lower-extremity swelling that proved secondary to inferior vena cava compression. photograph (e) shows the resected mass that arose from the visceral pleura of the right middle lobe and that was readily separated from the remainder of the lung with a small wedge resection of the affected area. modalities, it is an extremely difficult cancer to treat. because of encasement of the lung and potential pulmonary toxicity, there is no role for radiation as single-modality definitive treatment. radiation can be used with curative intent, as an adjuvant treatment combined with surgery in which the lung has been removed, or in a palliative manner for localized areas of symptomatic invasion. mesothelioma has proven to be one of the most chemoresistant tumors that oncologists face. cytotoxic agents have rarely yielded response rates greater than 30% . there have, however, been some encouraging results with newer chemotherapeutic agents , especially antimetabolites, which have shown response rates as high as 45% when used in combination with platinum-based conventional chemotherapy." to date, the treatments that have met with the greatest measure of success for pleural mesothelioma are those that combine surgery, for debulking, and other modalities to address the residual disease that almost certainly exists, even after the most /i complete" resection. the adjuvant treatments that have been performed, some intraoperatively, include radiation, photodynamic therapy, and hyperthermic chemotherapy intracorporeal lavage. systemic chemotherapy or immunotherapy have been used in adjuvant and neoadjuvant capacities.50 almost all surgery-based multimodality strategies that are performed with curative intent mandate removal of the lung as part of the debulking and preparation for the adjuvant treatments. some strategies, such as employment of photodynamic therapy for killing residual local disease after surgical debulking, may permit the option of performing lungsparing procedures." many cancers can metastasize to the pleural space, but one of the most frequently encountered is non-small cell lung cancer (nsclc), as either bulky disease or a malignancy.t -" in the absence of distant metastases, the presence of pleural dissemination confers an international system for staging lung cancer stage of iiib based on the pleural disease being considered a t4 tumor." the median survival for patients with this subset of stage iiib nsclc has been reported from 2 months to greater than 1 year but is generally within the 6-to 9-month range.64-66 in fact, the survival rate for this subset of stage iiib patients is so poor that it has been suggested that this disease should be upstaged to stage iv because of the similar survival rates." the standard of care for patients with nsclc with pleural dissemination remains palliative chemotherapy, assuming the patient is able to tolerate this treatment, and some intervention to palliate effusion if it is symptomatic. radiotherapy is rarely administered to these patients because of the pulmonary toxicity from hemithoracic radiotherapy; surgery has been shown to have little impact on survival for these patients and is accompanied by a local failur e rate as high as 90% . there are anecdotal reports of long-term survivors after surgery, but it is safe to say that there should be no role for surgery outside of some type of multimodality protocol approach as with mesothelioma.p:" two recent studies have reported encouraging results for surgery-based multimodality approaches to this disease but require validation in larger studies. in one study, the investigators operated on 22 patients, performing extrapleural pneumonectomies on all 22, but in 11 they also performed intraoperative hyperthermic chemotherapy perfusion. all patients were staged as mo, and the groups were evenly split with respect to nodal status, with the majority being no(7/11 and 8/11) and the remainder being n1 (2/11 and 1/11) or n2 (2/11 and 2/11) in the study and control groups, respectively. all patients received adjuvant chemotherapy. the group of evenly matched patients who did not receive intraoperative b (b) is a section through an extrapleural pneumonectomy specimen of a resected mesothelioma showing the same pathology as seen on the scan . hyperthermic chemotherapy perfusion had a median survival of 6 months. the group who received intraoperative hyperthermic chemotherapy perfusion had a median survival of 20 months. another recent study employed systemic chemotherapy, in a neoadjuvant capacity, and intraoperative photodynamic therapy to address microscopic locoregional disease after surgical debulking. the median survival for all 22 patients enrolled was 22 months from the time of surgery, with 12 of the 20 patients having pneumonectomy; the remainder had parenchymal sparing anatomic resections ranging from bilobectomy to segementectomy. in 18 of the 20 patients having intraoperative photodynamic therapy, n2 disease was present." both of these studies emphasize the importance of clinical trials that utilize a multimodality approach to cancers involving the pleura and the importance of a component to specifically address the residual microscopic disease that remains after even the most aggressive surgical resection. the medical works of hippocrates classic descriptions of disease eloesser 1. milestones in chest surgery embryology and anatomy of the pleura thoracic surgery exchanges through the pleura distribution of lymphatic stomata on the pleural surface of the thoracic cavity and the surface topography of the pleural mesothelium in the golden hamster respiratory physiology: the essentials baltimore: lippincott, williams and wilkins oxygen therapy for spontaneous pneumothorax the rate of formation and lymphatic removal of fluid in pleural effusions the impact of pleurodesis in malignant effusion on respiratory function the electrocardiographic manifestations of spontaneous left pneumothorax spontaneous pneumothorax and its treatment noninvasive treatment of pneumothorax with oxygen inhalation civilian spontaneous pneumothorax. treatment options and long-term results management of spontaneous pneumothorax spontaneous pneumothorax. comparison of thoracic drainage vs. immediate or delayed needle aspiration treatment of pneumothoraces utilizing small caliber chest tubes the performance of four pleural drainage systems in an animal model of bronchopleural fistula focal reexpansion pulmonary edema after drainage of large pleural effusions: clinical evidence suggesting hypoxic injury to the lung as the cause of edema reexpansion pulmonary edema complications in cardiothoracic surgery smoking and the increased risk of contracting spontaneous pneumothorax treatment of spontaneous pneumothorax: a more aggressive approach? management of spontaneous pneumothorax secondary spontaneous pneumothorax significance of iatrogenic pneumothoraces pulmonary diseases and disorders pleural effusions: the diagnostic separation of transudates and exudates focal reexpansion pulmonary edema after drainage of large pleural effusions: clinical evidence suggesting hypoxic injury to the lung as the cause of edema transudative effusions hepatic hydrothorax outcome analysis of cirrhotic patients undergoing chest tube placement recurrent pleural effusion complication liver cirrhosis cytologically proved malignant pleural effusions: distribution of transudates and exudates management of malignancyassociated pleural effusion: current and future treatment strategies efficacy of short term versus long term tube thoracostomy drainage before tetracycline pleurodesis in the treatment of malignant pleural effusions distribution of talc suspension during treatment of malignant pleural effusion with talc pleurodesis pleurodesis for malignant pleural effusions (cochrane review) outpatient management of malignant pleural effusion by a chronic indwelling catheter survival and talc pleurodesis in metastatic pleural carcinoma, revisited. report of 125 cases medical and surgical treatment of parapneumonic effusions: an evidence-based guideline for the american college of chest physicians parapneumonic effusions panel intrapleural streptokinase for empyema and complicated parapneumonic effusions ongoing search for effective intrapleural therapy for empyema: is streptokinase the answer? video-assisted thoracoscopic surgery for fibrinopurulent pleural empyema in 67 patients the lipoprotein profile of chylous and nonchylous pleural effusions chylothorax: an assessment of current surgical management early evacuation of traumatic retained hemothoraces using thoracoscopy: a prospective, randomized trial pleural diseases. baltimore: lippincott williams and wilkins solitary fibrous tumor of the pleura the role of alimta in the treatment of malignant pleural mesothelioma: an overview of preclinical and clinical trials malignant mesothelioma-the uk experience malignant pleural mesothelioma new developments about the association of sv40 with human mesothelioma multimodality management of malignant pleural mesothelioma gene expression profiles predict survival and progression of pleural mesothelioma chemotherapy for malignant pleural mesothelioma: past results and recent developments chemotherapy for malignant pleural mesothelioma: past results and recent developments phase it trial of pleural photodynamic therapy and surgery for patients with non-smallcell lung cancer with pleural spread recursive partitioning analysis of 1999 radiation therapy oncology group (rtog) patients with locally-advanced non-small-cell lung cancer (la-nsclc): identification of five groups with different survival prognostic value of pleural effusion in patients with non-small cell lung cancer revisions in the international system for staging lung cancer indications for pleurectomy in malignant effusion neoadjuvant chemotherapy and operations in the treatment of lung cancer with pleural effusion malignant pleural effusion in non-small cell lung cancer-time for a stage revision multimodality treatment programs for malignant pleural mesothelioma using highdose hemithorax irradiation extrapleural pneumonectomy for lung cancer with carcinomatous pleuritis prognosis of resected non-small cell lung cancer patients with carcinomatous pleuritis of minimal disease implications of staging in lung cancer intraoperative intrapleural hypotonic cisplatin treatment for carcinomatous pleuritis pleuropneumonectomy and postoperative adjuvant chemotherapy for carcinomatous pleuritis in primary lung cancer: a case report of long-term survival comparison of pleuropneumonectomy and limited surgery for lung cancer with pleural dissemination phase it trial of pleural photodynamic therapy and surgery for patients with non-smallcell lung cancer with pleural spread gray's anatomy minimal access surgery in oncology chemical pleurodesis for malignant pleural effusion key: cord-306266-8qdrshz3 authors: scully, crispian title: respiratory medicine date: 2014-06-25 journal: scully's medical problems in dentistry doi: 10.1016/b978-0-7020-5401-3.00015-1 sha: doc_id: 306266 cord_uid: 8qdrshz3 ●. upper respiratory infections are commonplace, especially in young people, and are often contagious; ●. lower respiratory infections are often contagious and some are potentially fatal; ●. asthma is common and may be life-threatening; ●. chronic obstructive pulmonary disease is common and disabling; ●. tuberculosis worldwide is an important infection, affecting people with hiv/aids or malnutrition particularly; ●. lung cancer is common and usually has a poor prognosis. • upper respiratory infections are commonplace, especially in young people, and are often contagious the respiratory tract consists of the upper respiratory tract (urtnose, paranasal sinuses, pharynx and larynx; discussed in ch. 14) and the lower respiratory tract (lrt): the respiratory airways (trachea, bronchi and bronchioles) and lungs (respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli), discussed in this chapter. protective mechanisms in the respiratory tracts include a mucociliary lining. particles or pathogens are trapped in the mucus and driven by ciliary action (the ciliary elevator) to the pharynx. mucociliary trans port declines with age but any effect on clinical infection has not been proved. lymphoid tissues of the waldeyer ring (adenoids, palatine and lingual tonsils) are important in developing an immune response to pathogens. however, the best respiratory defence mechanism is the cough reflex, the components of which include cough receptors, affer ent nerves, the cough centre, and efferent nerves and effector muscles. impairment of any of these -as may be seen in older patients or those with conditions associated with lowered consciousness (e.g. sedative use and neurological disease) -can weaken protection. dysphagia or impaired oesophageal motility may exacerbate the tendency to aspi rate foreign material. the alveolar defence mechanisms include mac rophages, immunocytes, surfactant, phospholipids, immunoglobulin g (igg), ige, secretory iga, complement components and factor b; many immune defects manifest with recurrent respiratory infections. lung function is vital to gas exchange -the blood absorbs oxygen and releases carbon dioxide. normal gas exchange requires adequate alveolar ventilation, normal ventilation/blood flow relationships and adequate alveolar-capillary membrane surface area. breathing (ven tilation) depends on respiratory drive, which reacts to the respiratory load. this process requires work and results in gas exchange. oxygen is transported in combination with haemoglobin in erythro cytes and a small amount dissolved in plasma. the oxyhaemoglobin dissociation curve is sigmoidal; once the oxygen saturation falls below 95%, the amount of o 2 transported to the tissues and brain falls rapidly. high temperatures, acidosis, raised co 2 and raised 2,3 diphosphoglycerate (2,3dpg) levels encourage oxygen offloading, whereas fetal haemoglobin and carboxyhaemoglobin have the con trary effect. chronic hypoxaemia (e.g. at high altitudes) stimulates release of erythropoietin from the kidneys, with a rise in red cell pro duction, and raised 2,3dpg. athletes have abused erythropoietin to gain competitive advantage (ch. 33). the most common lrt disorders are asthma and chronic obstructive pulmonary disease (copd). respiratory disorders are common, and are often caused or aggravated by tobacco smoking. they may significantly affect general anaesthesia (ga) and conscious sedation (cs), since they are often a contraindica tion to use of benzodiazepines, opioids, ga agents and other respira tory depressants. impaired gas exchange leads to laboured breathing and can cause significant incapacity. features include cough, sputum production, wheeze, dyspnoea, chest pain, cyanosis, fingerclubbing ( fig. 15.1) , use of accessory muscles of respiration with indrawing of the intercostal spaces (hyperinflation), and abnormalities in chest shape, movements, respiratory rate and breath sounds. cough may be a feature of any respiratory problem but, if chronic, may herald serious disease -for example, copd, cancer or infec tion such as tuberculosis. mucoid or mucopurulent sputum is often a feature ( fig. 15 .2); purulent sputum indicates acute bronchitis, bronchiectasis or lung abscess. blood (haemoptysis) or bloodstained sputum, though common in acute infections (especially in preexisting copd), bronchiectasis and pulmonary embolism, may herald an even more serious condition -for example, possibly one due to carcinoma or tuberculosis. wheezing is caused by airways obstruction and is a typical sign of asthma or copd. breathlessness (dyspnoea) is distress ing, and may be caused by respiratory or cardiovascular disease, or by anaemia, and is particularly ominous if it persists at rest. excessive resistive load, such as in asthma, copd and cystic fibro sis, impairs airflow. elastic load increases because of, for example, interstitial fibrosis, muscle paralysis and obesity. diagnosis of respiratory disorders is from the clinical features sup ported by imaging (especially chest radiography). spiral computed tomography (ct) can now scan the lungs in a quick 20-30second breathhold and therefore, instead of producing a stack of individual ct slices, which may be misaligned due to patient movement or breathing in between slices, provides highresolution three dimensional images. respiratory function tests can measure individual components of the respiratory process. spirometry is the basic screening test for assess ing mechanical load problems, the quantification involving determi nation of the vital capacity (vc) -slow vital capacity (svc) and/or forced vital capacity (fvc) -and the speed of maximal expiratory flow (mef; fig. 15 .3). in health, about 75% of a normalsized vc is expelled in 1 second (fev 1 ). the peak flow meter, which measures the peak expiratory flow rate (pefr; the earliest portion of forced expiration), is a simple measure of airflow obstruction, when the fev 1 is a much smaller fraction of the vc. in lung restriction, the diminished vc can be mostly expelled in about 1 second. serial meas urements (e.g. in asthma) provide valuable information about disease progress. the reversibility of airways obstruction is usually assessed by spirometry before and after use of a bronchodilator agent. arterial blood gas analysis yields considerable information about gas exchange efficiency. arterial hypoxaemia in adults is defined as pao 2 below 10.7 kpa breathing room air, although it is not usually treated as clinically important unless below 8 kpa, when oxygen saturation will be 90% or less (table 15 .1). arterial carbon dioxide tension (paco 2 ) is used as an inversely pro portional index of 'effective' alveolar ventilation. hence, a high paco 2 is taken to indicate poor alveolar ventilation. alveolar hypoventila tion (raised paco 2 ) with a normal ph probably represents a primary ventilatory change present long enough for renal mechanisms to compensate, as in chronic ventilatory failure. ventilation/blood flow relationships are most simply assessed by considering the size of the difference between the amounts of oxygen and carbon dioxide in the blood and in the air; the differences are small if the lungs are work ing efficiently. disparity between ventilation/blood flow ratios results in abnormally wide differences -and then alveolar-arterial po 2 and arterial-alveolar pco 2 gradients will be abnormal. alveolar capillary surface area is assessed by measuring the uptake of carbon monoxide -usually abnormal in diffuse interstitial inflam matory and fibrotic processes and in emphysema. assessing bronchial reactivity and the exercise response can help evaluate breathlessness. simple exercise testing provides information about overall fitness and the appropriateness of cardiorespiratory responses. radionuclide lung scanning, blood gas analysis and sputum cytology or culture are sometimes needed in addition. management can include oxygen administration by mask or nasal cannula (figs 15.4 and 15.5) . lrt disorders can cause significant incapacity and are often a con traindication to ga, and even to cs. asthma is common, affecting 2-5% of the overall population; it is on the increase, particularly in childhood, with a frequency of up to 20% in some highincome countries. asthma usually begins in childhood or early adult life; about half the patients with asthma develop it before age 10 years. bronchial hyperreactivity causes reversible airway obstruction from smooth muscle constriction (bronchospasm), mucosal oedema and mucus hypersecretion. there are two main types, extrinsic (allergic) and intrinsic asthma (table 15 .2). extrinsic (allergic) asthma, the main childhood type, may be pre cipitated by allergens in animal dander, feathers or hair, drugs (e.g. nonsteroidal antiinflammatory drugs [nsaids] and some antibiot ics), food (e.g. eggs, fish, fruit, milk, nuts), house dust (mite allergens) or moulds. patients frequently have or develop other allergic diseases, such as eczema, hay fever and drug sensitivities. extrinsic asthma is associated with ige overproduction on allergen exposure, and release of mast cell mediators (histamine, leukotrienes, prostaglandins, bradykinin and platelet activating factor), which cause bronchospasm and oedema. about 75% of asthmatic children lose their asthma or improve by adulthood. intrinsic asthma is usually of adult onset and not aller gic, but appears rather to be related to mast cell instability and airway hyperresponsivity. triggers include emotional stress, gastro oesophageal reflux or vagally mediated responses. either type of asthma can be triggered by: infections (especially viral, mycoplasmal or fungal); irritating fumes (e.g. traffic or cigarette smoke); exercise (possibly due to cold air); weather changes; emotional stress; foods (e.g. nuts, shellfish, strawberries or milk) or additives (such as tartrazine); and drugs (e.g. aspirin and other nsaids, beta blockers and angiotensinconverting enzyme inhibitors [aceis]). in wellcontrolled patients with asthma, clinical features may be absent. during an asthmatic episode, symptoms may include dysp noea, cough and paroxysmal expiratory wheeziness with laboured expiration. the frequency and severity of attacks vary widely between individuals (table 15 .3). patients may become distressed, anxious and tachycardic, have reduced chest expansion and be using accessory respiratory muscles to increase their ventilatory effort. nasal polyps are common, especially in aspirinsensitive asthmatics. children with asthma initially suffer from repeated 'colds' with cough, malaise and fever, often at night. asthma is typically diagnosed when the patient has more than one of the following -wheeze, cough, difficulty breathing and chest tightness -particularly if these are frequent and recurrent; are worse at night and in the early morning; occur in response to, or are worse after, exercise or other triggers, such as exposure to pets, cold or damp air, or with emotions or laughter; or occur without an association with colds. there is often: ■ a personal history of atopic disorder ■ a family history of atopic disorder and/or asthma ■ widespread wheeze, heard on chest auscultation ■ a history of improvement in symptoms or lung function in response to adequate therapy. a prolonged asthmatic attack, which is refractory to treatment, may lead to lifethreatening status asthmaticus (persisting for more than 24 hours). failure of the patient to complete a sentence, indrawing of the intercostal muscles, a rapid pulse, a silent chest and signs of exhaustion are suggestive of impending respiratory arrest. diagnosis of asthma is from the clinical history and presentation, based on recognizing a characteristic pattern of episodic symptoms in the absence of an alternative explanation. investigations include a chest radiograph (to exclude other diagnoses, such as a pneumo thorax), spirometry (serial pefr), skin tests and blood examination (usually eosinophilia, raised total ige and specific ige antibody concentrations, which may help identify allergens). occasionally, a histamine or methacholine challenge is used if the diagnosis is unclear. in children with an intermediate probability of asthma, who can perform spirometry and have evidence of airways obstruction, assess the change in fev 1 or pefr in response to an inhaled bronchodilator (reversibility) and/or the response to a trial of treatment for a speci fied period; if there is significant reversibility, or if a treatment trial is beneficial, a diagnosis of asthma is probable. management includes patient education, smoking cessation advice, avoidance of identifiable irritants and allergens, and use of drugs. home use of peak flow meters allows patients to monitor progress and detect any deterioration that may require urgent modification of treatment. treatment should be based on the amount by which peak flow is reduced (a pefr diary should be kept). drugs used for asthma management (table 15 .4) include oxygen, shortacting β 2 agonists (sabas; such as salbutamol), corticosteroids, leukotriene receptor antagonists and omalizumab (a recombinant humanized monoclonal antiige antibody that reduces the antigen specific ige). inhaled longacting β 2 agonists (labas) may be needed ( fig. 15.6 ). deaths from asthma are usually a result of failure to recognize dete rioration or reluctance to use corticosteroids. other factors that have been studied include: ■ air pollution -there is an association between air pollution and aggravation of existing asthma ■ allergen avoidance -there is no consistent evidence of benefit ■ breast-feeding -there is evidence of a protective effect in relation to early asthma ■ electrolytes -there is no consistent evidence of benefit ■ fish oils and fatty acid -there is no consistent evidence of benefit ■ house dust mites -measures to reduce the numbers of house dust mites do not affect asthma severity ■ immunotherapy -allergenspecific immunotherapy is beneficial in allergic asthma ■ microbial exposure -there is insufficient evidence to indicate that the use of probiotics in pregnancy reduces the incidence of childhood asthma ■ modified milk formulae -there is no consistent evidence of benefit pets -there are no controlled trials on the benefits of removing pets from the home ■ tobacco -exposure to cigarette smoke adversely affects quality of life, lung function, need for rescue medications and longterm control with inhaled steroids. there is an association between maternal smoking and an increased risk of infant wheeze ■ weight reduction -there is an association between increasing body mass index and symptoms of asthma. elective dental care should be deferred in severe asthmatics until they are in a better phase; this can be advised by the patient's general practitioner. asthmatic patients should be asked to bring their usual medica tion with them when coming for dental treatment. local anaesthe sia (la) is best used; occasional patients may react to the sulphites present as preservatives in vasoconstrictorcontaining la, so it may be better, where possible, to avoid solutions containing vasoconstric tor. adrenaline (epinephrine) may theoretically enhance the risk of arrhythmias with betaagonists and is contraindicated in patients using theophylline, as it may precipitate arrhythmias. relative analgesia with nitrous oxide and oxygen is preferable to intravenous sedation and gives more immediate control. sedatives in general are better avoided as, in an acute asthmatic attack, even ben zodiazepines can precipitate respiratory failure. ga is best avoided, as it may be complicated by hypoxia and hyper capnia, which can cause pulmonary oedema even if cardiac function is normal, and cardiac failure if there is cardiac disease. the risk of post operative lung collapse or pneumothorax is also increased. halothane or, better, enflurane, isoflurane, desflurane and sevoflurane are the preferred anaesthetics, but ketamine may be useful in children. allergy to penicillin may be more frequent in asthmatics. drugs to be avoided, since they may precipitate an asthmatic attack (see later), include those listed in box 15.1. acute asthmatic attacks may also occasionally be precipitated by anxiety; it is important to attempt to lessen fear of dental treatment by gentle handling and reassurance. even routine dental treatment can trigger a clinically significant decline in lung function in approximately 15% of asthmatics. acute asthmatic attacks are usually selflimiting or respond to the patient's usual medication, such as a betaagonist inhaler, but status asthmaticus is a potentially fatal emergency (ch. 1). there may be complications caused by the antiasthmatic drugs (table 15 .5). gastrooesophageal reflux is not uncommon, with occasional tooth erosion. periodontal inflammation is greater in asthmatics than in those without respiratory disease. persons using steroid inhalers may develop oropharyngeal candidosis or, occasionally, angina bullosa haemorrhagica. guidelines on the management of asthma may be found at: http://www.sign.ac.uk/guidelines/fulltext/101/index.html, http:// www.nice.org.uk/guidance/qualitystandards/indevelopment/asthma. jsp and http://www.britthoracic.org.uk/portals/0/guidelines/ asthmaguidelines/qrg101%202011.pdf (all accessed 30 september 2013). churg-strauss syndrome (css) is a rare, potentially fatal, systemic vasculitis similar to polyarteritis nodosa (pan), characterized by severe asthmalike attacks with peripheral eosinophilia, and intravas cular and extravascular granuloma formation with eosinophil infiltra tion and skin lesions in 70%. cardiopulmonary involvement is the main cause of death. css is diagnosed if at least 4 of the 6 criteria listed in box 15.2 are positive. the 5year survival of untreated css is 25%. combination treatment with cyclophosphamide and prednisolone (prednisone) provides a 5year survival of 50%. management problems relating to patients with css may include res piratory impairment and corticosteroid treatment (ch. 6). chronic obstructive pulmonary disease (copd; chronic obstructive airways disease, coad) is a common, chronic, slowly progressive, irre versible disease (most frequently a combination of chronic bronchitis and emphysema), characterized by breathlessness and wheeze (airways obstruction), cough and sputum. chronic bronchitis is defined as the excessive production of mucus and persistent cough with sputum production, daily for more than 3 months in a year over more than 2 consecutive years. it leads to production of excessive, viscous mucus, which is ineffectively cleared from the airway, obstructs and stag nates, and becomes infected, usually with streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae. patchy areas of alveolar collapse can result. emphysema is dilatation of air spaces dis tal to the terminal bronchioles with destruction of alveoli, reducing the alveolar surface area available for respiratory exchange. copd is now the preferred term for conditions with airflow obstruction because of a combination of airway and parenchymal damage; patients were previ ously diagnosed as having chronic bronchitis or emphysema. copd is characterized by airflow obstruction -defined as an fev 1 / fvc ratio reduced to less than 0.7. if fev 1 is 80% or more, a diagno sis of copd should only be made if there are respiratory symptoms (e.g. dyspnoea or cough). the airflow obstruction is not fully revers ible, does not change significantly over months, and is usually progres sive in the long term. the most important causes of copd include cigarette smoking, environmental pollution, dusts, chemicals or occupational exposures to various substances. exposure to smoke from home cooking or heating fuels may contribute. deficiency of the antiproteolytic enzyme alpha1antitrypsin is a rare cause of emphysema. there is often significant airflow obstruction before the person is aware of it and so copd typically remains undiagnosed until patients are in their fifties. differentiation from asthma is important (table 15 .6). a diagnosis of copd should be considered in patients over the age of 35 who have a risk factor (e.g. smoking) and exertional breath lessness, chronic cough, regular sputum production, frequent winter 'bronchitis' or wheeze. clinical judgment is based on history, physical examination, confirmation of airflow obstruction using spirometry (postbronchodilator spirometry) and assessment of the severity of dyspnoea (tables 15.7 and 15.8). copd is characterized by breathlessness and wheeze (airways obstruction), cough and an early morning mucoid sputum production. to investigate symptoms that seem disproportionate to spirometric impairment progressive dyspnoea, low oxygen saturation, carbon dioxide accumu lation (hypercapnia) and metabolic acidosis mean that patients may ultimately become dyspnoeic at rest ('respiratory cripples'), especially when recumbent (orthopnoea), and eventually develop respiratory failure, pulmonary hypertension, right ventricular hypertrophy and rightsided heart failure (cor pulmonale). two clinical patterns of copd are recognized: ■ 'pink puffers' -patients with emphysema who manage to maintain normal blood gases by hyperventilation, and are always breathless but not cyanosed; rather they are pink from vasodilatation ■ 'blue bloaters' -patients with chronic bronchitis who lose their co 2 drive, fail to maintain adequate ventilation and become both hypercapnic and hypoxic with central cyanosis, cor pulmonale and oedema (for these patients, the respiratory drive is from the low po 2 and thus oxygen administration is contraindicated) (table 15 .9). the diagnosis of copd is based upon clinical history and presen tation. investigations include a chest radiograph (which may show hyperinflated lung fields with loss of vascular markings); arterial blood gases (which should be measured if pulse oximetry shows oxygen satu ration less than 92%); spirometry; and lung function tests. fev1 is reduced in all cases (fev 1 of less than 40% signifies severe copd) and the flow-volume curve shows a typical pattern, with reduced flow rates at mid and lowerlung volumes. a ratio of fev 1 :fvc of less than 70% confirms airways obstruction. patients with copd and their family should be educated about the disease, and about required lifestyle changes and medication. nondrug therapy includes: stopping smoking (nicotine replacement therapy or bupropion may help); exercise by pulmonary rehabilitationof proven benefit; weight loss (improves exercise tolerance); and vaccination (pneumococcal and influenza vaccines). drug therapy includes shortacting bronchodilators (anticholinergic drugs [ipra tropium bromide]) and β 2 agonists (salbutamol) to treat the reversible component of airway disease; corticosteroids (inhaled or systemic); and antibiotics (amoxicillin, trimethoprim or tetracycline). mucolytics, such as carbocisteine, reduce acute exacerbations by almost onethird. longterm oxygen therapy (ltot) reduces mortality. people with stable copd who remain breathless or have exacerba tions, despite using shortacting bronchodilators, should be offered the following as maintenance therapy: ■ if fev 1 is 50% of predicted or more: use either a longacting β 2 agonist (laba) or longacting muscarinic antagonist (lama). ■ if fev 1 is less than 50% predicted: either a laba with an inhaled corticosteroid (ics) in a combination inhaler, or a lama. offer a lama in addition to a laba plus ics to people with copd who remain breathless or have exacerbations, despite taking laba plus ics, irrespective of their fev 1 . provide pulmonary rehabilitation for all who need it; noninvasive ventilation (niv) is the treatment of choice for persistent hyper capnic ventilatory failure during exacerbations not responding to medical therapy. the frequency of exacerbations should be reduced by appropriate use of inhaled corticosteroids and bronchodilators, and vaccinations. bronchodilators (shortacting β 2 agonists [saba] and shortacting muscarinic antagonists [sama]) should be the initial empirical treat ment for the relief of breathlessness and exercise limitation. ics have potential adverse effects (including nonfatal pneumonia) in people with copd. offer a oncedaily lama in preference to fourtimes daily sama to people with stable copd who remain breathless or have exacerbations, despite using shortacting bronchodilators as required, and in whom a decision has been made to commence regular maintenance bronchodilator therapy with a muscarinic antagonist (see above). most patients -whatever their age -are able to acquire and main tain an adequate inhaler technique. bronchodilators are usually best administered using a handheld inhaler device (including a spacer device if appropriate). patients with distressing or disabling dyspnoea, despite maximal therapy using inhalers, should be considered for nebulizer therapy. they should be offered a choice between a face mask and a mouth piece to administer their nebulized therapy, unless the drug specifically requires a mouthpiece (for example, anticholinergic drugs). some patients with advanced copd may require maintenance oral corticosteroids when these cannot be withdrawn following an exacer bation. these individuals should be monitored for the development of osteoporosis and given appropriate prophylaxis. theophylline should only be used after a trial of saba and laba, and only to those who are unable to use inhaled therapy, as there is a need to monitor plasma levels and interactions. the dose of theo phylline prescribed should be reduced at the time of an exacerbation if macrolide or fluoroquinolone antibiotics (or other drugs known to interact) are given. there is insufficient evidence to recommend prophylactic antibiotic therapy in the management of stable copd. mucolytic drug therapy should be considered in patients with a chronic cough productive of sputum. if patients remain symptomatic on monotherapy, their treatment should be intensified by combining therapies from different drug classes, such as: ■ β 2 agonist and theophylline ■ anticholinergic and theophylline. inappropriate oxygen therapy in people with copd may depress respiration. ltot is indicated in patients with copd who have a pao 2 of less than 7.3 kpa when sta ble, or a pao 2 greater than 7.3 kpa and less than 8 kpa when stable, and one of: secondary polycythaemia, nocturnal hypoxaemia (oxygen saturation of arterial blood [sao 2 ] of less than 90% for more than 30% of the time), peripheral oedema or pulmonary hypertension. to reap the benefits of ltot, patients should breathe supplemental oxygen for at least 15 hours per day. to ensure that all those eligible for ltot are identified, pulse oximetry should be available in all health care settings. the assessment of patients for ltot should comprise the measurement of arterial blood gases on two occasions at least 3 weeks apart in patients who have a confident diagnosis of copd, who are receiving optimum medical management and whose copd is stable. patients should be warned about the risks of fire and explosion and told not to smoke when using oxygen. ambulatory oxygen therapy should be considered in patients on ltot who wish to continue oxygen therapy outside the home, and who have exercise desaturation, are shown to have an improvement in exercise capacity and/or dyspnoea with oxy gen, and are motivated to use oxygen. adequately treated patients with chronic hypercapnic respiratory failure who have required assisted ventilation during an exacerbation, or who are hypercapnic or acidotic on ltot, should be referred to a specialist centre for consideration of longterm niv. advanced emphysema is occasionally treated with sur gery -excision of large acquired bullae or, rarely, lung transplantation. patients with copd who need dental care can be classified as follows: ■ patients at low risk -experience dyspnoea on effort but have normal blood gas levels. these patients can receive all dental treatment with minor modifications. ■ patients at moderate risk -experience dyspnoea on effort, are chronically treated with bronchodilators or recently with corticosteroids, and pao 2 lowered. a medical consultation is advised to determine the level of control of the disease before any dental treatment. ■ patients at high risk -have symptomatic copd that may be end stage and poorly responsive to treatment. with these patients, a medical consultation is essential before any dental treatment is carried out. patients with copd are best treated in an upright position at midmorning or early afternoon, since they may become increasingly dyspnoeic if laid supine. it may be difficult to use a rubber dam, as some patients are mouthbreathers and not able to tolerate the additional obstruction. la is preferred for dental treatment, but bilateral mandibular or palatal injections should be avoided. patients with copd should be given relative analgesia only if absolutely necessary, and only in hospital after full preoperative assessment. cs with diazepam and midazolam should not be used, as benzodiazepines are respiratory depressants. patients should be given ga only if absolutely necessary, and intravenous barbiturates are contraindicated. secretions reduce airway patency and, if lightly anaesthetized, the patient may cough and contaminate other areas of the lung. postoperative respiratory complications are more prevalent in patients with preexisting lung diseases, especially after prolonged operations and if there has been no preoperative preparation. the most important single factor in preoperative care is cessation of smok ing for at least 1 week preoperatively. respiratory infections must also be eradicated; sputum should first be sent for culture and sensitivity, but antimicrobials such as amoxicillin should be started without await ing results. the medical management of copd should be optimized prior to surgery. the ultimate clinical decision about whether or not to proceed with surgery should rest with a consultant anaesthetist and consultant surgeon, taking account of comorbidities, functional status of the patient and necessity for the surgery. composite assessment tools, such as the american society of anesthesiologists (asa) scoring system, and not just lung function, are the best criteria for the assessment of patients with copd before surgery. those taking corticosteroids should be treated with appropri ate precautions (ch. 6). interactions of theophylline with other drugs, such as adrenaline (epinephrine), erythromycin, clindamycin, azithro mycin, clarithromycin or ciprofloxacin, may result in dangerously high levels of theophylline. ipratropium can cause dry mouth. guidelines for the management of copd may be found at: http:// publications.nice.org.uk/chronicobstructivepulmonarydisease cg101 (accessed 30 september 2013). respiratory viruses usually spread by touch or airborne transmission and the very small particles (2-0.2 micrometres) can avoid the upper respiratory tract defences and the mucociliary elevator to reach the lung alveoli. a range of viruses can cause lower respiratory tract infections (lrtis ; table 15 .10). some viruses (e.g. influenza and respiratory syncytial) can spread from the upper to the lower respira tory tract via infection of the respiratory epithelium and can lead to bacterial superinfection and pneumonitis (pneumonia). mycoplasmal (atypical) pneumonia and tuberculosis (tb) may be direct infections. epidemics of a potentially fatal severe acute respiratory syndrome (sars) have been caused by a coronavirus that originated in china and spread worldwide; h5n1 bird influenza also arose as an epidemic; and a similar epidemic, but of swine influenza (h1n1), emanated from mexico (see later). bacterial infections, such as pneumonia or lung abscess, can also result from material aspirated into the lungs, and are usually unilat eral. those who aspirate more than others have, as a result, more frequent lrti and this is seen in alcohol and other drug abusers, as well as comatose patients. exogenous penetration and contamination of the lung can result from trauma (e.g. a stab wound or road traffic accident) or surgery. entamoeba histolytica can occasionally cause pneumonia -by direct extension from an amoebic liver abscess (table 15 .11). patients with endocarditis, or septic pelvic or jugular thrombo phlebitis, may experience lrti acquired haematogenously and then it is often bilateral. immunocompromised persons (e.g. those with human immunode ficiency virus/acquired immune deficiency syndrome [hiv/aids] and transplant recipients) and people with bronchiectasis or cystic fibrosis are also susceptible to respiratory infections by a range of opportun istic microbes. pneumocystis jiroveci (p. carinii), for example, is a com mon cause of potentially fatal pneumonia in immunocompromised patients -especially those with hiv/aids (chs 20 and 21). clinical features of lrti vary according to the part of the respiratory tract mainly affected: ■ bronchiolitis causes rapid respiration, wheezing, fever and dyspnoea -but is restricted mainly to infants. ■ bronchitis causes cough, wheezing and sometimes dyspnoea. ■ pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. antimicrobial therapy is indicated, particularly for pneumonia. antivirals have not been highly effective. oxygen may be needed. pneumococcal vaccine is indicated for older people. the majority of lrtis are severe illnesses, and are contraindications to all but emergency dental treatment. ga is hazardous and absolutely contraindicated. dental treatment should be deferred until recovery, or be limited to pain relief. influenza is mainly a communitybased infection transmitted in house holds and communities. healthcareassociated influenza infections can arise in any healthcare setting, most commonly when influenza is also circulating in the community. influenza is a contagious disease caused by influenza virus types a, b or c. type a has two main subtypes (h1n1 and h3n2); it causes most of the widespread influenza epidemics and can occasionally be fatal. type b viruses generally cause regional outbreaks of moderate severity, and type c viruses are of minor significance. a person can spread influenza starting 1 day before they feel sick and for another 3-7 days after symptoms start. influenza can be pre vented or ameliorated by vaccination each autumn; this is especially indicated for older people and those with cardiorespiratory disease. influenza attacks virtually the whole respiratory tract; symptoms appear suddenly after 1-4 days and include fever, sore throat, nasal congestion, headache, tiredness, dry cough and muscle pains (myalgia). most people recover in 1-2 weeks but infection can be lifethreatening, mainly because primary influenzal viral pneumonia can lead to sec ondary bacterial pneumonia or can exacerbate underlying conditions (e.g. pulmonary or cardiac disease). the old and very young, and those with chronic disorders, are more likely to suffer complications, such as pneumonia, bronchitis, sinusitis or otitis media. influenza has also been followed by depression, encephalopathy, myocarditis, myositis, pericarditis, reye syndrome and transverse myelitis. rest, maintenance of fluid intake, analgesics, antipyretics, and avoid ance of alcohol and tobacco help relieve symptoms. aspirin must never be given to children under the age of 16 years who have 'flulike symptoms, and particularly fever, as this can cause reye syndrome. zanamivir (an antiviral that works against influenza types a and b) can shorten the symptoms by approximately 1 day, if treatment is started during the first 2 days of illness. other antiviral drugs include amantadine, oseltamivir and rimantadine; they may be helpful but their use is restricted mainly to immunocompromised persons, since they can cause adverse effects. influenza can be a severe contagious illness so all but emergency den tal treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. influenza type a subtype h5n1 can cause an illness known as 'avian influenza' or 'bird 'flu' in birds, humans and many other animal spe cies. hpai a(h5n1) -'highly pathogenic avian influenza virus of type a of subtype h5n1' -is the causative agent and is enzootic in many bird populations, especially in southeast asia. it has spread globally and resulted in the deaths of over 100 people and the slaughter of mil lions of chickens. a vaccine that could provide protection (prepandrix) has been cleared for use in the european union. h5n7 is a more recent emergent infection, similar in many respects. swine influenza is common in pigs in the midwestern united states, mexico, canada, south america, europe (including the uk, sweden and italy), kenya, china, taiwan, japan and other parts of eastern asia. transmission of swine influenza virus from pigs to humans is not com mon, but can produce symptoms similar to those of influenza. a 2009 outbreak in humans ('swine 'flu') was due to an apparently new strain of h1n1 arising from a reassortment produced from strains of human, avian and swine viruses. it can pass from human to human. antiviral agents such as oseltamivir may help. vaccines are now available. an outbreak of a lifethreatening febrile respiratory infection appeared in 2003, originating from guangdong, china, and was named severe acute respiratory syndrome (sars). caused by a newly recognized coronavirus (sarsassociated coronavirus, sarscov), sars spread via close contact to many countries across the world. according to the world health organization, 8437 people worldwide became sick with sars during the course of the first recognized outbreak and 813 died. the incubation period of 2-7 days is followed by a high fever (above 38.0°c), malaise, headache and myalgia. some people also experience mild upper respiratory symptoms and, after 2-7 days, lower respiratory signs -a dry cough and dyspnoea, potentially progressing to hypox aemia. sars can cause a pneumonia with a mortality approaching 10%, particularly in older or immunocompromised people. artificial ventilation has been needed in 10-20% of cases. antiviral agents, such as oseltamivir or ribavirin, may help. inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as attenuated vaccines, are under development. sars is a severe illness, and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. for all contact with suspect sars patients, careful hand hygiene is important, including handwashing with soap and water; if hands are not visibly soiled, alcoholbased handrubs may be used as an alternative to handwashing. if a suspected sars patient is admitted to hospital, infection control personnel should be notified immediately. infection control measures (www.cdc.gov/ncidod/hip/iso lat/isolat.htm; accessed 30 september 2013) should include standard precautions (e.g. hand hygiene): healthcare personnel should wear eye protection for all patient contact; contact precautions (e.g. gown and gloves for contact with the patient or their environment); and airborne precautions (e.g. an isolation room with negative pressure relative to the surrounding area and use of an n95 filtering disposable respirator for persons entering the room). pneumonia is classed as 'primary' if it occurs in a previously healthy individual, and is usually lobar; it is called 'secondary' if it follows some other disorder, such as previous viral respiratory infections, aspir ation of foreign material, lung disease (bronchiectasis or carcinoma), depressed immunity (e.g. alcoholism or immunosuppression), or aspir ation of oral bacteria ( pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. complications can include lung abscess or empyema (pus in pleural cavity). it is important to avoid alcohol and tobacco, but use analgesics and antipyretics to relieve the symptoms. broadspectrum antimicrobi als given promptly and empirically usually include a macrolide (azithromycin, clarithromycin or erythromycin), quinolone (moxiflox acin, gatifloxacin or levofloxacin), or doxycycline for outpatients. for in patients, cefuroxime or ceftriaxone plus a macrolide is used. prophylaxis includes immunization against influenza and pneumococci. pneumonia is a severe illness and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. ventilatorassociated pneumonia (vap) is discussed later. legionellosis is a bacterial respiratory infection caused by one of the family legionellaceae, gramnegative aerobic bacilli, ubiquitous in water and soil but particularly preferring warm aquatic environments. the term legionnaire's disease was coined as a result of an outbreak of the previously unrecognized respiratory disease in an american legion meeting in philadelphia in 1976, but it is now recognized worldwide, many infections being contracted during travel abroad, particularly to spain, turkey and some other mediterranean areas. legionella bacteria can be found in natural freshwater environments, usually in insufficient numbers to cause disease. legionella grow best in warm water, as in hot tubs, cooling towers, hot water tanks, large plumbing systems, or the airconditioning systems of large buildings. though there are over 30 legionellaceae, most infections are caused by legionella pneumophila. disease is contracted by inhalation of contaminated mist or vapour, mainly (approximately 46%) through aerosolization of infected water in airconditioning systems, hotwater systems, humidifiers, nebulizers, showers and spa pools. outbreaks have mostly been linked to aerosol sources in the community, cruise ships and hotels, with the most likely sources being whirlpool spas, air conditioning units in large buildings, potable (drinking) water systems, and water used for bathing. risk factors include: ■ exposure to: recent travel with an overnight stay outside of the home (outbreaks of travelassociated legionellosis are infrequently identified but more than 20% of cases are thought to be associated with recent travel) whirlpool spas recent repairs or maintenance work on domestic plumbing ■ systemic illhealth: alcohol use chronic kidney disease diabetes immune defects liver disease malignancy smoking. illness mainly affects males over 45, smokers, heavy drinkers, older people and the immunocompromised. also vulnerable are travellers, especially middleaged and older tourists, and conference or business groups, possibly because of tiredness or age. many young people have been exposed to infection and become seropositive, but remained healthy. there is no evidence of persontoperson transmission of legionellosis. legionellosis manifests as one of two clinical syndromes (table 15 .14). legionnaire's disease is typically a lobular type of pneumonia, which can be fatal but is fortunately rare; infection can range from discrete patches of inflammation and consolidation to involvement of whole lobes. pontiac fever is milder and usually subsides rapidly, often with out treatment. people who should be tested for legionnaire's disease include those with pneumonia in the following groups: because legionella is commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation. diagnosis can be by rapid urine molecular testing for l. pneumophila antigen, and culture of respiratory secretions on selective media. sensitivity and specificity of the diagnostic tests are shown in table 15 .15. pontiac fever is a selflimited illness; most cases recover within 1 week and few benefit from antibiotic treatment. overall mortality in legionnaire's disease may be as high as 10%, and over 25% in older people and up to 80% in the immunocompromised. erythromycin is standard treatment; cephalosporin is an alternative. legionella species are present in roughly twothirds of potable water samples collected from domestic and institutional taps and drinking fountains, and from a similar percentage of dental units, but water from these dental units often has higher bacterial concentrations (ch. 31). there are reports of legionella infections in dental unit water lines, and antibodies and occasionally frank infection demonstrated in dental staff; at least one patient appears to have contracted and died from infection emanating from a dental practice. prevention is crucial, involving (ch. general aspects tuberculosis (tb) , an infection caused by mycobacteria, affects approxi mately onethird of the world's population (1.5 billion people); it is a major global health problem, some 2 million people dying from it annu ally. tb disproportionately affects the poorest persons in both high income and developing countries. in highincome countries, most human tb arises from mycobacterium tuberculosis, transmitted from person to person through the air. tb usually affects the lungs initially (pulmonary tb) but can also involve brain, kidneys, spine and other parts. from victorian times to about the second world war, mycobacterium bovis infection from infected cows' milk (bovine or btb) was a major cause of morbidity and mortality; it was clinically and pathologically indistin guishable from infection caused by m. tuberculosis. cattletesting and a slaughter programme became compulsory in 1950 and, by the 1980s, the incidence of tb in cattle had been substantially reduced. tuberculosis from m. bovis in cows' milk was virtually eliminated in highincome countries by the tuberculin testing of cattle and pasteurization of milk. in the developing world, many cattle still have tb, and btb is still seen. btb has also increased in highincome countries over the last two dec ades and an infection rate of up to 38% in badgers -and transmission to cattle -may explain this. tb is not spread by touch or by drinking glasses, dishes, sheets or clothing. it is usually transmitted by infected sputum, typically from close contacts such as family members, but is unlikely to be transmit ted between normal social contacts. tb can present an occupational risk to healthcare professionals, including dental staff. one outbreak of drugresistant tb in new york involved at least 357 patients, most of whom contracted tb in one of 11 hospitals; nearly 90% of the patients were also hivpositive, and most were young males of hispanic or african heritage. tb has been transmitted between pas sengers during longhaul airline flights. the risk of transmitting tb though air circulation is now low because the highefficiency particu late air (hepa) filters on newer commercial aircraft are of the same type as those used in hospital respiratory isolation rooms; indeed, the number of times air is cleaned each hour exceeds the recommendation for hospital isolation rooms. subsaharan africa has the highest rates of active tb per capita, driven primarily by the hiv epidemic. the absolute number of cases is highest in asia, with india and china having the great est burden of disease globally. in the usa and most western european countries, the majority of cases occur in foreignborn residents and recent immigrants from countries in which tubercu losis is endemic. immunocompromised people -such as diabetics and severely immuno deficient patients, like those with hiv/aids (about 30% of south africans with hiv/aids also have tb) -and patients in prisons or institutions are at risk. tb also mainly affects medically neglected persons, such as vagrants, alcoholics, intravenous drug abusers or older homeless people. the main groups at increased risk for infection therefore include people who are resourcepoor or immunoincompetent, especially: tb in developing countries is particularly widespread and is increasing, the highest rises in incidence being in southeast asia, subsaharan africa and eastern europe. in highincome countries, the incidence is also rising, probably because of worsening social deprivation, homelessness, immigration, hiv infection and intra venous drug abuse. it is now as common in london as in the devel oping world, and is seen especially in immigrants, such as those from the indian subcontinent, africa and south asia. this increase appears to be a result of the development of tb disease in individu als who may have been infected for some time and of new infections acquired in the uk, or as a result of travel to other countries where tb is common. london accounted for the highest proportion of cases in the uk in 2011 (39%), followed by the west midlands region (11%); 74% of these were born outside the uk and mainly originated from south asia and subsaharan africa. in 2011, there was a rise in the number of tb cases compared to 2010, as well as an increase in drug resistance. more information on tb, including statistics, can be found at: http:// www.hpa.org.uk/publications/infectiousdiseases/tuberculosis/ and http://www.tbfacts.org/tbstatistics.html (both accessed 30 september 2013). initial infection with tb is usually subclinical. about 10% of those infected develop overt disease; of these, half will manifest within 5 years (primary tb), while the remainder will develop postprimary disease. inhaled mycobacteria may cause subpleural lesions (primary lesion) and lesions in the regional lymph nodes (primary complex). body defences usually localize the mycobacteria, though these remain viable; infected persons are not obviously ill and are unlikely to know they are infected (latent ; table 15 .16). latent tb infection (ltbi) usu ally becomes active only after many years, if body defences become weakened (box 15.3). however, active tb can develop shortly after mycobacteria enter the body, if body defences are impaired such as in ageing, drug or alcohol abuse, or hiv/aids. also, in massive infec tions, acute active tb can result, typically causing a chronic productive cough, haemoptysis, weight loss, night sweats and fever. erythema nodosum may be associated. extrapulmonary tb is less common; it may appear as glandular involvement in the neck or elsewhere, and is less infectious than pulmonary tb. lymph node tb may lead to lymphadenopathy, caseation of the nodes and pressure symptoms -for example, on the bronchi. postprimary tb follows reactivation of an old primary pulmonary lesion and results in features ranging from a chronic fibrotic lesion to fulminating tuberculous pneumonia. the pulmonary lesions may extend and lead to a pleural effusion. reactivation or progression of primary tb may also result in widespread haematogenous dissemina tion of mycobacteria -'miliary tb'. multiple lesions may involve the central nervous system, bones, joints, and cardiovascular, gastrointes tinal and genitourinary systems. clinical presentation in tb is thus variable, depending on the extent of spread and the organs involved. as it frequently passes unrecog nized for so long, the mortality is high. similar illnesses to tb may also be caused by atypical (nontuber culous) mycobacteria, such as m. avium complex (mac; see below). the diagnosis of tb is suggested by the history and confirmed by physical examination, a massively raised erythrocyte sedimentation rate (esr), positive tuberculin skin tests (tsts; mantoux or heaf test for a delayed hypersensitivity reaction to protein from m. tuberculosis [purified protein derivative; ppd]) and chest imaging. hypersensitivity develops with 2-8 weeks of infection and can be detected by conversion of the tst from negative to positive, but tsts are neither 100% sensi tive nor specific. a positive mantoux reaction indicates previous immu nization (bcg; bacille calmette-guérin -live attenuated m. bovis) or current infection -not necessarily disease. chest radiography may show scarring and hilar lymphadenopathy. computed tomography (ct) may show areas of calcification or highlight a tuberculous abscess. smears and culture of sputum, blood, laryngeal swabs, bronchoalveolar lavage, gastric aspirates or pleural fluid may be tested for mycobacteria. polymerase chain reaction (pcr) techniques have greatly acceler ated the diagnosis and speciation, though ziehl-neelsen, auramine or rhodamine microbial stains are still used. the mycobacteria growth indicator tube (mgit) system gives results as early as 3-14 days. blood assay for m. tuberculosis (bamt) may be positive by interferongamma release assay (igra). some 15% of people over 65 years have a positive igra. the igra can be used in place of (but not in addition to) tst. igras measure the immune reactivity to m. tuberculosis. white blood cells from most persons that have been infected with m. tuberculosis will release interferongamma (ifnγ) when mixed with m. tuberculosis antigens. a positive test result sug gests that m. tuberculosis infection is likely; a negative result suggests that infection is unlikely. latent infection (ltbi) can be diagnosed with either a tuberculin skin test or an igra (more specific). igra gives a result within 24 hours and should be used biological therapy is given, such as for rheumatoid arthritis or inflammatory bowel disease. prior bcg vacci nation does not cause a falsepositive igra test result. more informa tion on the igra is available at: http://www.cdc.gov/tb/publications/ factsheets/testing/igra.htm (accessed 30 september 2013). active tb is diagnosed by sputum microscopy and culture in liquid medium with subsequent drugsusceptibility testing. nucleic acid people who should be tested for tb include those who have symp toms, those who have had close daytoday contact with active tb disease (family member, friend or coworker), those who have hiv infection or aids, those with lowered immunity, those who are required to for employment or school, and those about to be treated with biological agents. the top priority of tb control programmes is to identify and give complete treatment to all patients with active disease. tb is a notifi able disease and contact tracing is an important aspect of limiting spread. treatment with antibiotics is indicated for people who are sick with tb, those infected but not sick, and those who are close contacts of infectious tb cases. treatment for 'symptomatic sputumpositive' patients, which should be instituted as soon as possible, is combination chemotherapy, usually isoniazid plus rifampicin plus pyrazinamide or ethambutol for 2 months, with continuation of daily isoniazid and rifampicin for a further 4 months. treatment for 'asymptomatic' patients who are believed to have been infected by contacts, but are not unwell, includes isoniazid for 6 months or isoniazid and rifampicin for 3 months. rifapentine is a longacting rifampicin used once weekly. fluoroquinolones (moxifloxacin) may also act against tb. there may be resistance to one or more than one antibiotic. currently, given the potential risk of drugresistant tb being present, treatment is usually started with isoniazid, rifampicin, pyrazinamide and ethambutol (or a quinolone such as gatifloxacin or moxifloxacin) for 2 months, then isoniazid and rifampicin for 4 months. all antituberculous drugs (table 15 .17) have potentially serious adverse effects and require careful monitoring. if patient compliance is considered to be poor, directly observed therapy (dot), where drugs are dispensed by and taken in the presence of a healthcare profes sional, may be indicated. new drugs are on the horizon. immunization using bcg is advocated for schoolchildren, highrisk individuals and healthcare professionals -although its efficacy has been questioned. new vaccines are in development. chemoprophylaxis with isoniazid and rifampicin is indicated in a number of situations (box 15.4) . tb can become resistant to the drugs used to treat it particularly when the drugs are misused or mismanaged. this may occur, for example, when: in some developing countries, approximately 10% of cases are multi ple antibioticresistant; this is termed multidrugresistant tuberculosis (mdrtb); in the uk, only a small minority currently fall into this category but the number of cases is increasing. mdrtb is defined as resistance to rifampicin and isoniazid; it may be atypical in presenta tion and the infection disseminates. more than 4% of people with tb worldwide have mdrtb, and eastern europe has a high prevalence. mdrtb is seen mainly in people with hiv/aids and in hiv/aids and in africans. bedaquiline, is a new antitubercular agent the first active agent against tuberculosis to be registered since 1963. extensively drugresistant tuberculosis (xdrtb) is a rare type of mdrtb, not only resistant to isoniazid and rifampin, but also to any fluoroquinolone and at least one of three injectable secondline drugs (i.e. amikacin, kanamycin, or capreomycin). xdrtb is of special concern for immunocompromised people (e.g. with hiv/aids), who are more likely to develop tb, and have a higher risk of death if they do develop it. xdrtb is most often encountered in people from eastern europe, russia and africa. it has been transmitted in healthcare facilities and is now seen worldwide. it is essentially untreatable, though capreomycin has been used effectively to treat mdrtb in hivpositive individuals. totally drugresistant tb was reported initially in 2007-2009 in india, iran and italy; it is spreading, despite denials, and is most disquieting. chronic ulcers, usually on the tongue dorsum, are the main oral manifestation of tb. they result from coughing of infected sputum from pulmonary tb, including in hivinfected persons with tb, but are rare and such cases (usually middleaged males) may result from neglect of symptoms or default from treatment. occasionally, the diagnosis is made from biopsy of an ulcer after granulomas are seen microscopically. acidfast bacilli are rarely seen in oral biopsies, even with the help of special stains, so unfixed material should also be sent for culture if possible. tuberculous cervical lymphadenopathy is the next most common form of the infection and is particularly com mon among those from south asia. most tb lymphadenitis is pain less, with several enlarged, matted nodes, but systemic symptoms are present only in a minority and only about 15% have pulmonary mani festations on radiography (fig. 15.7) . diagnosis relies on tuberculin testing, which can be positive in both tuberculous and non tuberculous mycobacterial cervical lymphadenitis. any person with lymphadenop athy and recent conversion from a negative to positive tuberculin test should be suspected of having mycobacterial infection, and this should prompt biopsy (e.g. fineneedle aspiration biopsy) for culture or histo logical confirmation. pcr will improve diagnosis, as culture must wait 4-8 weeks for a result. oral complications of antitubercular therapy are rare, but rifabutin and rifampicin can cause red saliva. pulmonary tb is of high infectivity, as shown by cases of tuber culous infection of extraction sockets and cervical lymphadenitis in 15 patients treated by an infected member of staff at a dental clinic. dental staff who themselves were hivpositive, working in a dental clinic for hivinfected persons in new york, have died from tb con tracted occupationally. transmission of mdrtb between two dental workers may have occurred in an hiv dental clinic. infection control is thus important, so staff with tb are usually precluded from their occupation until treated. management of a patient with tb depends upon the level of poten tial infectivity (table 15.18) . patients with open pulmonary tb are con tagious, and dental treatment is thus best deferred until the infection has been treated. treatment with appropriate drugs for 2 weeks drasti cally reduces the infectivity of patients with pulmonary tb. if patients with open pulmonary tb must be given dental treatment, special pre cautions should be used to prevent the release of mycobacteria into the air, to remove any that are present and to stop their inhalation by other persons. reduction of splatter and aerosols, by minimizing cough ing and avoiding ultrasonic instruments, and use of a rubber dam, are important. improved ventilation, ultraviolet germicidal light, new masks and personal respirators, and other personal protective devices, such as hepa filters, are indicated ( fig. 15.8) . mycobacteria are very resistant to disinfectants, so that heat sterilization must be used. la is safe and satisfactory. relative analgesia is contraindicated because of the risk of contamination of the apparatus. ga is also contraindicated for dental treatment because of the risk of contamina tion of the anaesthetic apparatus and because of impaired pulmonary function. aminoglycosides, such as streptomycin, enhance the activity of some neuromuscular blocking drugs and in large doses may alone cause a myasthenic syndrome. possible drug interactions are shown in table 15 .19. other factors, such as alcoholism or intravenous drug use (ch. 34), hepatitis (ch. 9) or hiv disease (ch. 20), may also influence dental management. mycobacteria other than tuberculosis (mott) are widely distributed in water, soil, animals and humans, and rarely cause disease. severe mott infections have been seen, however, in individuals predisposed because of defects in the interleukin12 (il12) and interferongamma (ifngamma) pathways. mycobacterium abscessus, a bacterium found in water, soil and dust, has been known to contaminate medications and products, including medical devices. healthcareassociated m. abscessus can cause a vari ety of infections, usually of the skin, but it can also cause lung infec tions in persons with various chronic lung diseases and is increasingly recognized as an opportunistic pathogen in cystic fibrosis (cf) patients persontoperson transmission of atypical mycobacteria is not important in acquisition of infection, except for skin infections. on rare occasions, mott skin infections have followed tattooing with contaminated tattoo inks. many people become infected with and har bour mott in their respiratory secretions without any symptoms or evidence of disease. individuals with respiratory disease from mott do not readily infect others and, therefore, do not need to be isolated. mott are generally not infectious to others. infection with m. abscessus is usually caused by injections of con taminated substances or by invasive medical procedures employing contaminated equipment or material. infection can also occur after accidental injury where the wound is contaminated by soil. there is very little risk of transmission from person to person. mac complex, m. scrofulaceum and m. kansasii are possible causes of tuberculous cervical lymphadenitis. mac may also infect the lungs (similar to tb), skin or lymph nodes. lung disease is also caused occasionally by m. kansasii, mainly in middleaged and older persons with underlying chronic lung conditions. m. fortuitum and m. chelonae may cause skin and wound infections and abscesses, frequently associated with trauma or surgery. m. marinum may cause 'swimming pool granuloma', a nodular lesion that may ulcerate, usually on an extremity. m. ulcerans may produce chronic ulcerative skin lesions, usually of an extremity. m. abscessus skin infections present with swollen and/ or painful areas that are usually red, warm and tender to the touch, and which can also develop into boils or pustules. other features of m. abscessus infection are fever, chills, muscle aches and malaise. cervical lymphadenitis due to mac, m. scrofulaceum and m. kansasii may affect otherwise healthy young children, most commonly pre school females who have unilateral cervical lymphadenopathy, typically in the submandibular or jugulodigastric nodes, and they may form a 'cold abscess'. mott is the usual cause in children under 12 years but tb is more common in older patients. absence of fever or tuber culosis, a positive tuberculin test and failed response to conventional antimicrobials are highly suggestive of mott, but definitive diagnosis is by smear, culture or pcr of biopsy material obtained by fineneedle aspiration or removal of nodes. treatment is based on results of laboratory testing, which should identify the appropriate antibiotic. preventive treatment of close contacts of persons with disease caused by mott is not needed. most mott are resistant to standard antitubercular medication and, though it is possible that clarithromycin or clofazimine may have some effect, excision of affected nodes is the usual recommended therapy. water from dental units may contain mott species; mycobacterial proliferation in biofilms may explain the extent of this contamination (ch. 31). aspiration syndromes are conditions in which foreign substances are inhaled into the lungs and which can have consequences ranging from asphyxia to infection and lung abscess. dental restorations or frag ments of teeth, plaque, gastric contents and other materials may be aspirated, especially if material enters the pharynx, and particularly if the cough reflex is impaired for any reason. most commonly, aspiration syndromes involve oral or gastric contents associated with gastrooesophageal reflux disease (gord), swallowing dysfunction (ch. 7), neurological disorders and structural abnormalities, such as a pharyngeal pouch. cricopharyngeal dys function involves cricopharyngeal muscle spasm or achalasia of the superior oesophageal sphincter, and can be seen in infants who have a normal sucking reflex but have incoordination during swallowing, pos sibly secondary to delayed development or cerebral palsy. anatomical disorders, such as cleft palate, pharyngeal pouch, oesophageal atresia, tracheooesophageal fistula, duodenal obstruction or malrotation, and motility disorders, such as achalasia, may have an aspiration risk. infirm older patients are also at risk of aspiration, especially if they are bedbound or have neurological disorders. isolated superior laryngeal nerve damage, vocal cord paralysis, cerebral palsy, muscular dystrophy and riley-day syndrome (familial dysautonomia) are all associated with increased risk of aspiration. ventilatorassociated pneumonia (vap), as defined by the centers for disease control and prevention (cdc), is present when the chest radiograph shows new or progressive infiltrate, consolidation, cavitation or pleural effusion in conjunction with either new onset of purulent sputum or change in character of sputum, and an organism isolated from blood, or the isolation of an aetiological agent from a specimen obtained via suction aspiration through an endotracheal or tracheostomy tube. the major route for acquiring endemic vap is oropharyngeal colo nization by endogenous flora or by exogenously acquired pathogens from intensive care units. vap is the most commonly reported health careacquired infection in patients receiving mechanical ventilation, with prevalence rates consistently in the 10-20% range. mortality rates in vap are at least double those in patients without vap, ranging from 24% to 85% when the infection is caused by a multidrugresistant gramnegative pathogen. the healthcare infection control practices advisory committee of the cdc has developed guidelines for the prevention of vap. these include strategies aimed at preventing aspiration of contaminated oral or gastric material (e.g. raising the head of the bed and draining subglottic secretions), and interventions to alter bacterial coloniza tion of stomach (e.g. stress ulcer prophylaxis and selective digestive decontamination) and mouth. oral hygiene, suctioning and the provi sion of moisture to lips and oral mucosa, plus toothbrushing, may be important in prevention of vap. there are also strategies for manag ing ventilator circuits (e.g. replacement of ventilator circuits, use of closed rather than open suction, and use of heat moisture exchange as opposed to heated circuit technology). lung abscess is a localized infection leading to cavitation and necro sis. while some cases result from aspiration of foreign material, most develop from pneumonia caused by infection with staph. aureus or klebsiella pneumoniae. bronchial obstruction by carcinoma is another important cause. symptoms resemble those of suppurative pneumonia. there is a risk of infection spreading locally or leading, via septicaemia, to a brain abscess. diagnosis rests mainly on the chest radiograph, which may sometimes show cavitation or a fluid level. antimicrobial chemotherapy, postural drainage and relief by bronchoscopy of any obstruction are indicated. a wellrecognized cause of lung abscess is inhalation of a tooth or fragment, a restoration or rarely, an endodontic instrument. when undertaking endodontics or cementing restorations, such as inlays or crowns, a rubber dam or other protective device should always be used to avoid the danger of inhalation. lung abscesses may also result from aspiration of oral bacteria, particularly anaerobes, especially in infirm older patients or those who are intubated. the other main dangers in dentistry are with ga, particularly if an inadequate throat pack has been used. patients who inhale tooth frag ments or dental instruments must have chest radiographs (lateral and posteroanterior) and, if necessary, bronchoscopy. loeffler syndrome appears to be an allergic reaction, usually to the parasitic worm ascaris lumbricoides, or drugs such as sulphonamides. it manifests with pulmonary infiltrates (and abnormal chest radio graph) and eosinophilia (eosinophilic pneumonia). the disease usually clears spontaneously. sarcoidosis, so named because skin lesions resembled a sarcoma, is a multisystem granulomatous disorder, seen most commonly in young adult females in northern europe, especially in people of african heritage. the aetiology is unclear but propionibacterium acnes and p. granulosum have been implicated and associations have been reported with exposure to inorganic particles, insecticides, moulds and occupations such as firefighting and metalworking. serum sam ples contain antibodies directed against mycobacterium tuberculosis antigens. sarcoidosis is associated with hladrb1 and dqb1, and a butyrophilinlike 2 (btnl2) gene on chromosome 6. thelper 1 (th1) cells release il2 and ifnγ, and augment macrophage tumour necrosis factor alpha (tnfα) release. cd25 regulatory t cells cause a limited impairment of cellmediated immune responses (partial anergy) but no obvious special susceptibility to infection. sarcoidosis affects the thorax in 90%, but has protean manifestations and can involve virtually any tissue (table 15 .20). sarcoid most typi cally causes löfgren syndrome (fever, bilateral hilar lymphadenopathy, arthralgia and erythema nodosum, especially around the ankles; figs 15.9 and 15.10). other common presentations may include pulmonary infiltration and impaired respiratory efficiency, with cough and dyspnoea in severe cases, or acute uveitis, which can progress to blindness. susceptibility to lymphomas has been suggested but not confirmed. because of its vague and protean manifestations, sarcoidosis is under diagnosed. in the presence of suggestive clinical features, helpful investigations include: chest radiography (enlarged hilar lymph nodes); raised serum angiotensinconverting enzyme (sace ; table 15 .21) in acute disease (this is insensitive, nonspecific and a poor guide to therapy); positive gallium67 citrate or gadolinium or positron emis sion tomography (pet) scans; labial salivary gland or transbronchial biopsy (for histological evidence of noncaseating epithelioid cell granulomas) -except in löfgren syndrome, which is a classical clini cal diagnosis. 18 fdeoxyglucose pet is helpful in identifying sites for biopsy. nonspecific findings may include mild anaemia, leukopenia, eosinophilia, hypergammaglobulinaemia, raised esr and low serum albumin. hypercalcaemia is common because of extrarenal produc tion of active vitamin d and can result in renal damage. alkaline phosphatase, 5'nucleotidase, lysozyme and adenosine deaminase levels are raised in hepatic sarcoidosis. evidence of impaired delayed hypersensitivity reactions to some antigens may be useful. kveim skin tests are not now used. half the patients with sarcoidosis remit within 3 years and about 66% remit by 10 years. patients with only minor symptoms usually need no treatment but corticosteroids, sometimes with azathioprine, methotrexate, tetracyclines, hydroxychloroquine, infliximab or etaner cept, are given if there is active organ disease (ocular disease, progres sive lung disease, hypercalcaemia or cerebral involvement). biopsy of the minor salivary glands frequently shows noncaseating granulomas and association with other features of sarcoidosis, par ticularly hilar lymphadenopathy. this is an important diagnostic find ing that may obviate more invasive procedures. sarcoidosis can involve any of the oral tissues but has a predilection for salivary glands. asymptomatic swelling of the parotid glands or cervical nodes, and less frequently the lips, may accompany systemic disease. superficial or deepseated red submucosal nodules may develop intraorally and on the lips. nontender, wellcircumscribed, brownishred or violaceous nodules with superficial ulceration have also been reported. the oral and lip lesions may occasionally precede systemic involvement. there is enlargement of the major salivary glands in about 6% of cases; some have xerostomia, and the association of salivary and lacri mal gland enlargement with fever and uveitis is known as uveoparotid fever (heerfordt syndrome). salivary swelling may also be seen with out other features of heerfordt syndrome. the salivary gland swellings usually resolve on treatment of sarcoidosis but this may take up to 3 years. facial palsy and other cranial neuropathies may be seen. there is also an association with sjögren syndrome, when ssa and ssb serum autoantibodies are found. rarely there is an association of thyroiditis with addison disease, sjögren syndrome and sarcoidosis (tass syndrome). there is a group of patients who have histologi cal features of sarcoid in one or more sites in the mouth, such as the gingivae, but no systemic manifestations. a few of these patients may ultimately develop other more or less systematized disease but the majority probably have isolated lesions. such cases, where no exog enous cause for the granulomatous reaction can be found, are regarded as having 'sarcoidlike' reactions (orofacial granulomatosis) and treat ment is unnecessary. however, patients should be kept under observa tion for as long as possible. management of patients with systemic sarcoidosis may include con sideration of respiratory impairment, uveitis and visual impairment, renal disease, jaundice or corticosteroid treatment. la is safe and satisfactory. cs is contraindicated if there is any res piratory impairment. ga should only be given in hospital. lung cancer is the most common cancer in highincome countries in males and most frequently affects adult urban cigarettesmokers. bronchogenic carcinoma accounts for 95% of all primary lung cancer and has also become increasingly common in women (because of increased tobacco use), to the extent that the mortality rate for the two sexes has become almost equal. metastases from cancers elsewhere are also frequently found in the lungs. recurrent cough, haemoptysis, dyspnoea, chest pain and recurrent chest infections are the predominant features. local infiltration may cause pleural effusion, lesions of the cervical sympathetic chain (horner syndrome), brachial neuritis, recurrent laryngeal nerve palsy or obstruction of the superior vena cava with facial cyanosis and oedema (superior vena cava syndrome). there are many nonmetastatic extrapulmonary effects of bron chogenic (or other) carcinomas -for example, weight loss, anorexia, fingerclubbing, neuromyopathies, thromboses (thrombophlebitis migrans), muscle weakness, various skin manifestations and ectopic hormone production (of antidiuretic hormone, adrenocorticotropic hormone, parathyroid hormone and thyroidstimulating hormone). metastases from bronchogenic cancer are common and typically form in the brain (which may manifest with headache, epilepsy, hemi plegia or visual disturbances), liver (hepatomegaly, jaundice or ascites) or bone (pain, swelling or pathological fracture). the diagnosis is based on history and physical examination, supported by radiography, ct and magnetic resonance imaging (mri), sputum cytology, bronchoscopy and biopsy. spiral ct appears to detect tumours at an early stage. the overall 5year survival rate is only 8%. radiotherapy is the most common treatment. only some 25% of patients are suitable for surgery but, even then, the 5year survival is only about 25%. chemotherapy has been disappointing, except in smallcell carcinomas. dental treatment under la should be uncomplicated. cs should preferably be avoided. ga is a matter for specialist management in hospital, as patients often have impaired respiratory function, espe cially after lobectomy or pneumonectomy. this, along with any muscle weakness (myasthenic syndrome, eaton-lambert syndrome) that can make the patient unduly sensitive to the action of muscle relaxants, makes ga hazardous. oral cancer may be associated with lung cancer, and vice versa, or develop at a later stage (ch. 22). such synchronous or metachronous primary tumours must always be ruled out. metastases can occasionally affect the orofacial region and cause enlargement of the lower cervical lymph nodes, epulislike softtissue swellings or labial hypoaesthesia or paraesthesia in the jaw. soft palate pigmentation is a rare early oral manifestation. lung cancer is a fairly common cause of death in dental techni cians, but it is unknown whether this is due to smoking alone or to dust inhalation. cystic fibrosis (cf) is one of the most common fatal hereditary dis orders. inherited as an autosomal recessive trait, with an incidence of about 1 in 2000 births, it is the most common inherited error of metabolism and is seen mainly in people of european descent. the gene responsible is on chromosome 7q. cf is caused by defects in the cystic fibrosis transmembrane conductance regulator (cftr), a protein that appears to be part of a cyclic adenosine monophosphate (camp)regulated chloride channel, regulating cl − and na + transport across epithelial membranes, and ion channels and intracellular fluid flow in sweat, digestive and mucus glands. the basic defect in cf is abnormal chloride ion transport across the cell membrane of nearly all exocrine glands. the blockage of salt and water movement into and out of cells results in the cells that line the lungs, pancreas and other organs producing abnormally thick, sticky mucus that can obstruct the airways and various glands, especially in the respiratory tract and pancreas. involved glands (lungs, pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary and sweat glands) may become obstructed by this viscid or solid eosino philic material. recurrent respiratory infections result in a persistent productive cough and bronchiectasis, with the lungs becoming infected with a variety of organisms including staph. aureus, haemophilus influenzae, pseudomonas aeruginosa, strep. pneumoniae, burkholderia cepacia, and sometimes mycoses or mycobacteria. mycobacterium abscessus is a nontuberculous mycobacterium increasingly recognized as an opportunistic pathogen in cf patients. viral infections, such as mea sles, can have severe sequelae. pancreatic duct obstruction leads to pancreatic insufficiency, with malabsorption and bulky, frequent, foulsmelling, fatty stools. gallstones, diabetes, cirrhosis and pancreatitis may result. sinusitis is very common. growth is frequently stunted. the mutations can also cause con genital bilateral absence of the vas deferens, so fertility is impaired in most males with cf. in women, fertility may be impaired by viscid cervical secretions, but many women have carried pregnancies to term. most patients have a high concentration of sodium in their sweat (also reflected in the saliva); a sweat test showing sodium and chloride values of more than 60 mmol/l is considered positive, between 40 and 60 mmol/l equivocal, and less than 40 mmol/l negative. physiotherapy and postural drainage are crucially important. clearance of sputum is helped by water aerosols and bronchodila tors (terbutaline or salbutamol), but mucolytics such as carbocisteine, methyl cysteine and dornase alfa are of questionable effectiveness. treatment with ivacaftor, a cftr potentiator, improves chloride transport through the ion channel. amoxicillin and flucloxacillin are effective prophylactic antimicrobi als and may be given by aerosol. vaccination against measles, whoop ing cough and influenza is important. a low fat intake, adequate vitamins and oral pancreatic enzyme replacement (pancreatin) are also necessary. doublelung or heart-lung transplantation may eventually become necessary. sinusitis is very common; most cf patients have recurrent sinusitis and nasal polyps. the major salivary glands may enlarge and hyposali vation sometimes occurs. the lowfat, highcarbohydrate diet and dry mouth may predispose to caries. enamel hypoplasia and black stain may be seen, and both dental development and eruption are delayed. tetracycline staining of the teeth was common but should rarely be seen now. pancreatin may cause oral ulceration if held in the mouth. la is satisfactory but cs is usually contraindicated because of poor respiratory function. ga is contraindicated if respiratory function is poor. lung disease, such as bronchiectasis, liver disease and diabetes, may complicate treatment. bronchiectasis is dilatation and distortion of the bronchi. causes include: ■ congenital defects, which should be considered in all patients include cystic fibrosis, kartagener syndrome, alpha1antitrypsin deficiency, collagen defects (e.g. marfan syndrome) there is no identifiable underlying cause in about 50% of adults and 25% of children. the damaged and dilated bronchi lose their ciliated epithelium and therefore mucus tends to pool, causing recurrent lrtis, typically with strep. pneumoniae, haemophilus influenzae or pseudomonas aeruginosa. overproduction of sputum, which is purulent during exacerbations, a cough (especially during exercise or when lying down) and finger clubbing are typical features, with recurrent episodes of bronchitis, pneumonia and pleurisy. haemoptysis is not uncommon. in advanced bronchiectasis, chest pain, dyspnoea, cyanosis and respiratory failure may develop. complications may include cerebral abscess and amyloid disease. chest radiography and pulmonary function tests are required. high resolution ct (hrct) is useful. postural drainage is important. antimicrobials, such as amoxicillin, cephalosporins or ciprofloxacin, are given for acute exacerbations and for longterm maintenance treatment. ga should be avoided where possible and is contraindicated in acute phases. workers exposed to airborne particles may develop pulmonary disease (pneumoconiosis), which ranges from benign (e.g. siderosis) to malig nant, as in mesothelioma from asbestosis (see appendix 15.1), but any pneumoconiosis can cause significant incapacity. ga may be contraindicated; the physician should be contacted before treatment. berylliosis may be a hazard in some dental technical laboratories, when lung cancer is more frequent. respiratory complications following surgical operations under ga include segmental or lobar pulmonary collapse and infection. they are more common after abdominal surgery or if there is preexistent respiratory disease or smoking (see also ch. 3), and can be signifi cantly reduced by smoking cessation, preoperative physiotherapy and bronchodilators, such as salbutamol. if postoperative pulmonary infection develops, sputum should be sent for culture, and physiotherapy and antibiotics should be given. the common microbial causes are strep. pneumoniae and haemophilus influenza; in this case, suitable antibiotics include amoxicillin and erythromycin. hospital infections may include other microorganisms, such as mrsa, klebsiella, pseudomonas and other gramnegative bacteria. inhalation (aspiration) of gastric contents can cause pulmonary oedema and may be fatal (mendelson syndrome); it is most likely if a ga is given to a patient who has a stomach that is not empty, has a hiatus hernia or is in the last trimester of pregnancy. prevention is by ensuring the stomach is empty preoperatively; if it is not, an anaes thetist should pass an endotracheal tube. antacids or an h2receptor blocker, such as cimetidine or ranitidine, may be given by mouth pre operatively to lower gastric acidity. if gastric contents are aspirated, the pharynx and larynx must be carefully sucked out. systemic corticosteroids have been recommended but probably do not reduce the mortality. respiratory distress in premature infants may be caused by immaturity of surfactantproducing cells, when the alveoli fail to expand fully; this necessitates endotracheal intubation for many weeks. it may, in turn, result in midface hypoplasia, palatal grooving or clefting, or defects in the primary dentition. the same oral effects may be seen with prolonged use of orogastric feeding tubes. the degree to which subsequent growth corrects these deformations is currently unknown, though the palatal grooves typically regress by the age of 2 years. using soft endotracheal tubes does not obviate this problem and, at present, the best means of avoiding palatal grooving appears to be the use of an intraoral acrylic plate to stabilize the tube and protect the palate. acute respiratory distress syndrome (ards) is a sequel to several types of pulmonary injury and some infections, including those with oral viridans streptococci. patients with endstage pulmonary disease are considered for potential transplantation, usually using a lung from a braindead organ donor. a combination of ciclosporin, azathioprine and glucocorticoids is usu ally given for lifelong immunosuppression to prevent a tcell, alloim mune rejection response. inhaled nitric oxide modulates pulmonary vascular tone via smooth muscle relaxation and can improve ventilation/perfusion matching and oxygenation in diseased lungs. early graft failure following lung transplantation has been described by various investi gators as reimplantation oedema, reperfusion oedema, primary graft failure or allograft dysfunction. pathologically, this entity is diffuse alveolar damage. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. cardiopulmonary transplantation (heart and lung transplantation) is the simultaneous surgical replacement of the heart and lungs in patients with endstage cardiac and pulmonary disease, with organs from a cadaveric donor. all transplant recipients require lifelong immunosuppression to pre vent a tcell, alloimmune rejection response. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. national institutes of health: national institute of allergy and infectious diseases healthcare infection control practices advisory committee guideline for the prevention of healthcare associated pneumonia nosocomial pneumonia: state of the science extensively drugresistant tuberculosis as a cause of death in patients coinfected with tuberculosis and hiv in a rural area of south africa a review of the possible role of oral and dental colonization on the occurrence of health careassociated pneumonia: underappreciated risk and a call for interventions reducing ventilatorassociated pneumonia through advanced oraldental care: a 48month study apic infection control and applied epidemiology: principles and practice sepp. ventilatorassociated pneumonia and oral care: a successful quality improvement project guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings a randomized trial of dental brushing for preventing ventilatorassociated pneumonia pneumonia associated with a dental unit waterline the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention aspects of human disease 32 chronic obstructive pulmonary disease (copd) aspects of human disease 31 in vitro antibacterial activities of oral care products against ventilatorassociated pneumonia pathogens the impact of a simple, lowcost oral care protocol on ventilatorassociated pneumonia rates in a surgical intensive care unit intermittent suction of oral secretions before each positional change may reduce ventilatorassociated pneumonia: a pilot study current trends and newer concepts on diagnosis, management and prevention of respiratory tract infections key: cord-289103-6i7wf41w authors: mcelyea, christine; do, christopher; killu, keith title: lung ultrasound artifacts in covid-19 patients date: 2020-08-25 journal: j ultrasound doi: 10.1007/s40477-020-00526-y sha: doc_id: 289103 cord_uid: 6i7wf41w lung ultrasound is an essential tool in critical care, made more so by the enhanced precautions associated with the covid-19 pandemic. here we describe 2 cases of multiple, small shred signs seen on ultrasound of covid-19 patients. lung ultrasound is an essential tool in identifying lung disease and most practitioners will be able to identify pathology not visible on chest x-ray [1] . as of the summer of 2020, there are more than 12.5 million reported cases of covid-19 caused by the coronavirus 2 (sars-cov-2) causing a pandemic that has presented many challenges in the traditional approach to patients with hypoxemia and shortness of breath or respiratory failure. the disease has affected over 188 countries and reported deaths are over 500,000 so far across the globe [2, 3] . traditional radiologic imaging for patients who present with suspected pneumonia, including chest x-ray or ct chest is not routinely recommended to limit healthcare worker exposure. using bedside ultrasound, with the appropriate precautions, can help the practitioner significantly in identifying the lung pathology [4] . studies describing lung ultrasound findings have been increasing over the last 25 years, with more recent articles describing the use of ultrasound in a standardized fashion to identify lung disease in covid-19 era [5] . we present the case of a 64-year-old woman with a past medical history of developmental delay with associated dementia and schizoaffective disorder, and recurrent urinary tract infections who was sent to the emergency department from her skilled nursing facility for fevers of 38.7 °c. initial complete blood count demonstrated leukocytosis to 14.5 with 8% bands. a ct of the abdomen and pelvis demonstrated bladder wall thickening concerning for cystitis; she was admitted and treated with ceftriaxone for presumed urinary tract infection. by day 4 of admission, her fevers had failed to remit, and a ct of the chest was obtained which demonstrated bilateral, patchy ground-glass opacities. a sars cov-2 pcr was sent and returned positive on day 7 of admission at which time she was started on azithromycin and hydroxychloroquine. she developed worsening hypoxic respiratory failure and was intubated on day 13 of admission and transferred to our facility for further care. of note, her nursing home roommate was also admitted to our icu with the novel coronavirus. lung ultrasound was used in addition to the earlier conventional modalities of chest x-ray and ct scan. she received one dose of tocilizumab without significant improvement in her respiratory status and after further decline was transitioned to comfort-focused measures by her family and passed away. a 76-year-old female with a past medical history of type 2 diabetes who presented with 2 weeks of body aches, fevers, cough, and progressive shortness of breath; with several family members experiencing similar symptoms at home. in the emergency department, she was noted to be tachypneic with pulse oximetry as low as the mid-50 s. a chest x-ray demonstrated bibasilar infiltrates. she was initially placed on a non-rebreather mask at 15 l per minute, then high flow nasal cannula with oxygen saturation improving to 90%. a sars cov 2 pcr was sent, subsequently returning positive, and she was admitted to the progressive care unit where her respiratory status remained tenuous. several goals of care meetings were held with the patient and her care teamshe initially endorsed do not resuscitate and do not intubate status, given this, in combination with worsening hypoxia, she was given one dose of 400 mg iv tocilizumab on day 2 of admission. by day 4 of admission, her high flow nasal cannula had increased to 60 l per minute of flow with fio2 95% and oxygen saturation in the 80 s. at this time, she requested alteration of her code status to full care-she then transferred to the intensive care unit, underwent endotracheal intubation, and was placed on mechanical ventilation. ultrasound exam of her lungs was performed on the day of intubation. lung ultrasound for both patients was performed using a fujifilm sonosite x-port machine (seattle) with a linear transducer with a frequency of 13 mhz. the b mode was used with a depth dependent on the patient's chest wall size, ranging between 3.6 and 4.4 cm, with optimal gain at 0.5 and the mechanical index was set low at 0.5. full contact and droplet precautions and barriers were applied. both patients were in a supine position with the head of the bed at 30° angle. anterior lung fields were examined to cover most lung zones [6] . eight (8) in patient a, the findings described were noted in the upper anterior lung zones bilaterally, while a chest x-ray obtained concurrently demonstrated infiltrate in bilateral lower lateral lung zones. in the right and left lateral lower zones, there was atelectasis noted by ultrasound. in patient b, there was a consolidation of both lower lung zones corresponding to the areas examined by ultrasound showing shred signs, with the pebbles appearance scattered on the upper anterior lung zones fig. 2 . some of the findings listed above were described by recent articles using ultrasound in covid-19 patients [7] . in this article, we are listing the findings with a more detailed description and emphasis on possible early changes and their characters that could be unique for this disease. covid-19 patients can present in different stages with different signs, symptoms, and radiological criteria [8] . it is estimated that over 17% of infected individuals, who present with mild to moderate disease may not show any radiological signs on chest x-ray [9] . application of bedside lung pocus is well established, and many studies have shown [1] . detecting lung consolidation and interstitial lung disease can be achieved effectively using lung us [10] . using bedside ultrasound to help manage patients with covid-19 is becoming more prevalent and effective. we presented two cases of covid-19 with acute respiratory failure, one with nearnormal chest x-ray and the other with bilateral infiltrates. using bedside lung pocus helped us identify the severity of the disease, as well as confirm the clinical diagnosis of a pneumonic process. unable to perform daily cxr, and or cat scans of the chest due to the infectious process and limitation of isolation and avoiding excessive exposure, lung pocus can be performed initially and early in the disease process when it can detect abnormalities not detected by cxr, and can assist in daily management as well. we noted some findings that could be specific for the disease or representing a continuum of alveolar interstitial lung disease. the thickening of the pleural line was noted with skip areas of normal pleura. shred signs tended to develop in multiple fashion as small aggregates, affecting mainly anterior upper zones, and they were not seen as the usual bacterial lobar pneumonia, involving larger areas of consolidations. areas resembling pebbles were noted on the ultrasound and described above, need to be investigated further, its significance could represent the loss of integrity to lung tissue secondary to poor blood flow distribution as hypothesized by some researchers [11] , and our hypothesis also supported by previous older and some recent literature [12, 13] , most probably representing the first manifestation of the known shred sign as an evolving pneumonic process at different stages since it is close to areas where the shred signs were developing, fig. 3 . we were able to capture these early changes since ultrasound was used earlier in covid patients as we suspect pneumonia in those patients even with a normal cxr as inpatient a, representing part of the early interstitial and parenchymal lung disease and the early manifestation and development of shred sign, not described in previous literature. this may help investigators and clinicians identify patients with early pneumonia without other radiological evidence. using higher frequency transducer helped us identify these findings, which sometimes aren't visible, using the traditional phased array transducer with the usual 12-15 cm depth. the advantages of ultrasound in this pandemic are the ease of use, the reproducibility of the testing, and limiting radiation exposure, infection, and transmission of disease. other advantages are clear in limited-resource areas as well as in pregnant women when limiting radiation exposure [14, 15] . limitations to our exams were not including the posterior lower lobes in the back. this was not done to limit the study time and decrease exposure. other inherent limitations include the possibility of cross-contamination of other individuals if cleaning and isolation precautions are not followed properly, and operator variability. we recommend using a dedicated ultrasound machine to perform these exams. further research and studies need to be done to verify these findings and investigate the changes noted in our ultrasound exam. . 3 illustration of the pathophysiology of the different ultrasound signs including the pebbles resembling artifacts. note areas of normal lung parenchyma and air-filled alveoli with poor transmission to ultrasound, reflecting and scattering the waves (arrows) as bright pebbles like artifacts. also, areas of lung tissue with early buildup and accumulation of alveolar secretions, mucous and inflammatory cells, leading to transmission of the ultrasound beam and its refraction to deeper tissues (arrow heads). thickening of the interstitial layer with fluid accumulation can lead to the reverberation of the ultrasound beam between the pleural line and the interlobular septa, leading to the development of the comet tail, or b lines on the ultrasound screen comparative performance of pulmonary ultrasound, chest radiograph, and ct among patients with acute respiratory failure covid-19) treatment guidelines. nih. gov point-of-care lung ultrasound findings in novel coronavirus disease-19 pnemoniae: a case report and potential applications during covid-19 outbreak proposal for international standardization of the use of lung ultrasound for patients with covid-19: a simple, quantitative, reproducible method united states is there a role for lung ultrasound during the covid-19 pandemic? a novel coronavirus from patients with pneumonia in china clinical characteristics of covid-19 in china. reply relevance of lung ultrasound in the diagnosis of acute respiratory failure: the blue protocol covid-19 does not lead to a "typical" acute respiratory distress syndrome usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water the role of ultrasound lung artifacts in the diagnosis of respiratory diseases effectiveness of rapid lung ultrasound training program for gynecologists and obstetricians managing pregnant women with suspected covid-19 social consequences of covid-19 in a low resource setting in sierra leone, west africa key: cord-306076-ygfnkgqp authors: fujita, yu; takeshita, fumitaka; kuwano, kazuyoshi; ochiya, takahiro title: rnai therapeutic platforms for lung diseases date: 2013-02-06 journal: pharmaceuticals (basel) doi: 10.3390/ph6020223 sha: doc_id: 306076 cord_uid: ygfnkgqp rna interference (rnai) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. the induction of rnai relies on small silencing rnas, which affect specific messenger rna (mrna) degradation. two types of small rna molecules, i.e. small interfering rnas (sirnas) and micrornas (mirnas), are central to rnai. drug discovery studies and novel treatments of sirnas are currently targeting a wide range of diseases, including various viral infections and cancers. lung diseases in general are attractive targets for sirna therapeutics because of their lethality and prevalence. in addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. recently, increasing evidence indicates that mirnas play an important role in lung abnormalities, such as inflammation and oncogenesis. therefore, mirnas are being targeted for therapeutic purposes. in this review, we present strategies for rnai delivery and discuss the current state-of-the-art rnai-based therapeutics for various lung diseases. traditional surgery, bivas-benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. endotracheal applications are currently being used by many practitioners in the pulmonary field [22, 34] ; this is useful for studying pulmonary drug delivery in mice. however, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) endotracheal route: sirnas are sprayed directly from the mouth into the lungs using a microsprayer ® aerolizer (penn-century, philadelphia, pa, usa) and a laryngoscope. it is important to maintain a clear view of the trachea during the procedure. intranasal delivery is another common method of pulmonary drug application in animal studies. in many studies, in vivo success has been demonstrated in delivering sirnas to the lungs intranasally [22, 35, 36 ]. an experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. although the success in delivering sirnas intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of sirnas. phase ii clinical trials have been initiated for the treatment of respiratory syncytial virus (rsv) infection, making use of intranasal application of naked chemically modified sirna molecules that target viral gene products [17, 38] (see section 3.1.1. for details). intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. therefore, sirnas administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. in fact, it has been reported that intranasal application of unformulated sirnas resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [31] . the intranasal method is suitable for some lung diseases, such as upper respiratory infection by rsv, and it also has potential for systemic delivery rather than pulmonary delivery of sirnas. therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. careful choice of efficient delivery in response to the condition of lung diseases is necessary. the use of aerosols to deliver medication to the lungs has a long history. administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. there are several inhalation devices available for the delivery of drugs into the lungs. metered dose inhalers (mdis) and dry powder inhalers (dpis) are the most common modes of inhaled delivery. mdis are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (copd), and a spacer is an external device that is attached to an mdi to allow for better drug delivery by enhanced actuation and inhalation coordination. for most mdis, the propellant is one or more gases called chlorofluorocarbons (cfcs). although cfcs in drugs are safe for patients to inhale, they are harmful to the environment. therefore, further development of inhalable sirnas may not be the best way forward. dpis are devices that deliver medication to the lungs in the form of dry powder. the use of dpis has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [39] and low-molecular-weight heparin [40] ; thus, it could be a better device for delivering sirnas to the lungs. the advantages of dpis are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using sirnas have relied on intratracheal or intranasal delivery. the reason could be the difficulty in formulating inhalable sirnas and maintaining the stability during the delivery process. a suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. the use of spray-drying as a technique for engineering dry powder formulations of sirna nanoparticles, which might enable the local delivery of biologically active sirna directly to the lung tissue, has been demonstrated [24, 25] . in the future, the technique is desirable to estimate the in vivo study on sirna therapy for inhalation. in the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. there are two main barriers to efficient pulmonary sirna delivery to the cells of the lung. the first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [41, 42] (figure 2) . a remarkable feature of the respiratory tract is its high degree of branching. airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. all of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. it is generally acknowledged that the critical factor for efficient sirna delivery depends on the properties of rnai drug particles in terms of size, charge, shape, velocity and density. for efficient pulmonary sirna delivery, the particles must be deposited in the lower respiratory tract. deposition in the airway is affected by the particle size and patient's pulmonary function. a particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract [23] . in addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the sirna deposition to the diseased area of the respiratory tract. besides, the extracellular barriers to sirna delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. at the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. during infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [43] . moreover, asthma and copd are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [44] . the airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. figure 2 . extracellular barriers to pulmonary sirna delivery. the anatomical feature of the respiratory tract is its high degree of branching. the mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. the deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. in the alveolar, clara cells and type ii alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. the surfactants act as the main barrier for sirna delivery because they reduce the transfection efficiency. in addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. the particles taken up into the macrophages are subsequently degraded inside the cells. these factors present major barriers to targeted pulmonary delivery. the second is the airway cell membrance and its intracellular barriers ( figure 3 ). for efficient gene silencing in the lungs, sirnas must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. once the sirnas reach the target cells, they must be trafficked into the cytoplasm and taken up by argonaute (ago)2/rna-induced silencing complex (risc), which degrades mrnas and, subsequently, suppresses the sequence-specific gene expression. for efficient endocytosis to occur, particles should be under 150 nm in size. particles within this size range could also avoid macrophage uptake and delayed lung clearance [45] . the physicochemical properties of sirnas also play a significant role in crossing the biological membrane. despite their small size, the negative charge and chemical degradability of sirna molecules prevent them from readily crossing biological membranes. therefore, efficient sirna delivery approaches need to overcome this limitation by facilitating cellular uptake. one of the main functions of a delivery vector is to facilitate the cellular uptake of sirnas [46] . the electrostatic complexation of sirna molecules with cationic lipids and polymers helps to mask their net negative charge. the positively charged sirna carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [47] . after cellular internalization, the sirna carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. to avoid lysosomal degradation, sirnas must escape from the endosome into the cytoplasm, where they can associate with the rnai machinery. endosomal escape is a major barrier for efficient sirna delivery [48, 49] . the endosomal entrapment and lysosomal degradation of sirna and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. an ideal delivery agent should protect sirnas from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. multiple approaches for the delivery of sirnas have been reported, ranging from the relatively simple direct administration of saline-formulated sirnas to lipid-based and polymer-based nanoparticle approaches and sirna conjugation and complexation approaches [50] . the negative charge and chemical degradability of sirnas under physiologically relevant conditions make its delivery a major challenge. accordingly, the delivery of sirnas usually requires a vector or carriers for their transfection into the target cells. in general, both viral and non-viral vectors are being assessed for sirna delivery to lung cells. some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [51] and to induce rnai in a range of animal tissues [52] . recently, guo et al. showed that lentivirus-mediated sirna was used to specifically knock down the expression of nuclear protein 1 (nupr1) in vivo, which resulted in inhibited tumor growth [53] . however, viral-based delivery has several disadvantages. the immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [54] . recent well-documented cases, such as the death of jesse gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [55] . in addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [56] . . intracellular barriers to pulmonary sirna delivery. barriers to cellular internalization are dependent on the surface properties of sirna and carriers (e.g., charge and size). after sirnas are successfully taken into the target cells by endocytosis, the main barriers for delivering sirnas to its site of action are the endosomal entrapment and lysosomal degradation of sirna and carriers. to direct target-gene silencing, the sirnas need to escape from the endosome into the cytoplasm, where they associate with the ago2/rna-induced silencing complex (risc) to direct the cleavage of mrnas bearing complementary binding sites. as an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of sirnas to the lungs due to their reduced toxicity [57] . ongoing research into the transfection of primary cells and whole organisms with sirna using non-viral transfection agents has produced some promising results. lipid-based delivery vectors are successfully used to deliver sirna in vitro and in vivo [58] . cationic lipids are composed of positively charged head, a linker and hydrophobic. in general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. many commercial sirna transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-dharmfect [30] , oligofectamine [59] , lipofectamine [60] and transit-tko [35] . similarly, cationic polymers have also been assessed for sirna delivery to lung cells. cationic polymer polyethylenimine (pei) is widely used for sirna delivery [13, 61] . pei is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. on the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [62, 63] . although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective sirna delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. an important point for sirna-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [64, 65] . interestingly, some studies have shown that it was possible to administer "naked sirnas" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [66] . the term "naked sirnas" refers to the delivery of sirnas without any delivery vectors. naked sirnas are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. thus, some studies of systemic delivery of naked sirnas have failed to achieve the downregulation of the targeted gene [67, 68] . in contrast, there have also been some successes of locally delivering naked sirnas to the lungs [15, 16, 20, 31] . a few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked sirnas [16, 35] . indeed, in one clinical trial, the delivery of naked sirnas for the treatment of rsv has been used [17, 38] . this successful evidence can be because that naked sirnas for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. although it is unclear how the naked sirnas cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked sirnas (aln-rsv01) that were administered intranasally. this explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. the active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. naked sirna delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. nevertheless, the advantage of naked sirnas over delivery vectors in the treatment of lung diseases is controversial [69, 70] . further in vivo investigations about both naked sirnas and non-viral vectors are required. lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. the high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. above all, copd is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 [71] . therefore, decisive action is needed to stem the rising health and economic burden this represents. chronic lung diseases, such as copd and asthma, are disorders of the airways largely related to the presence of persistent inflammation. the approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. this was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. corticosteroids are still an important therapeutic intervention. however, they are used with limitations in copd and moderate to severe asthma. likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. treatment of lung disease using cell-specific targeting as well as rnai techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. pulmonary applications of sirna in in vivo conditions are frequently studied and often result in clinical trials [57, 72] . the findings of recent clinical studies of pulmonary rnai therapeutics are discussed. since the discovery of rnai, the therapeutic potential of sirnas has been rapidly recognized. in 2004, the first human clinical trial of rnai-based therapy was initiated for the treatment of age-related macular degeneration with a sirna targeting vegf-receptor 1 delivered intravitreally [73] . many studies have been conducted over the past few years that involve the delivery of sirnas to the lungs for the treatment of various lung diseases. delivery to the lungs will be most important to moving sirna technology into the clinic. a number of sirna-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and rsv infection. table 1 is a summary of clinical trials of sirna-based therapeutics [74] . sirna shows potential for the treatment of various pulmonary viral infections, and it has been reported that sirna-based therapeutics can also be used in the treatment of influenza [13] , parainfluenza virus [35] , severe acute respiratory syndrome (sars) [14] , and rsv [35] . above all, rsv is the most promising therapeutic target of sirnas. rsv is a common cause of serious respiratory infections in infants and children. it also produces significant morbidity and mortality in adult immunocompromised or elderly populations [75] . an rsv vaccine is not available, and the only approved antiviral therapy for rsv is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. thus, a safe and efficacious rsv therapy has long been awaited for both pediatric and adult patients. rnai-based therapy has shown promising effects in murine models of rsv infection [35] . the sirna, aln-rsv01, is directed against the mrna encoding the n-protein of rsv that exhibits specific in vitro and in vivo anti-rsv activity. it is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. aln-rsv01 has undergone complete phase i intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [38] . additionally, aln-rsv01 has been evaluated in a randomized, double-blind, placebo-controlled phase ii trial in lung transplant patients with rsv respiratory tract infection [76] . the administration of aln-rsv01 to rsv infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. based on these results, a larger multinational, randomized, double-blind phase iib trial of aln-rsv01 has been initiated in lung transplant patients to confirm and extend these findings. cancer is a major target of rnai-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by rnai. lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. rnai-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. the major advantage of rnai therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. the simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving sirnas and mirnas. there have already been significant improvements in sirnas for primary or metastatic lung cancer treatment by targeting oncogenes such as akt1 [9] , wilms tumor 1 (wt1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (igf-1r) [77] , nupr1 [53] and ezh2 [78] . some of these studies have successfully shown the efficacy of rnai-based therapy through intrapulmonary administration of sirnas with non-viral vectors. although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with sirnas is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. there are currently some clinical trials in progress estimating the safety and efficacy of sirna-based drugs for cancer treatment. atu027, a sirna-lipoplex targeted against protein kinase n3 (pkn3), prevented lung metastasis in a phase i trial of various cancer models [79] . pkn3 is a downstream effector of the phosphoinositide 3-kinase (pi3k) signaling pathway [80] , which regulates diverse cellular responses, including development, growth, and survival [81] . recently, pkn3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with atu027 in cultured primary endothelial cells showed an essential role of pkn3 for endothelial tube formation and migration [79] . atu027 can be considered as a potential sirna for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. inflammatory lung disease, also called copd, includes a wide range of lung ailments. these related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. they are influenced by a combination of environmental, genetic, and epigenetic components [82] . copd is a chronic inflammatory disease of the airways. this disease is hallmarked by airflow that is not fully reversible. systemic and local airway inflammation has been implicated in the pathogenesis of copd [83] . copd is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. smoking might cause cells to senesce due to dna damage through increased cell turnover, which in turn leads to accelerated telomere shortening [84] . lately, a lot of studies have investigated the role of cellular senescence in the development and progression of copd [85] . although several medication classes, including inhaled corticosteroids, are used for copd treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. current interventions that have been shown to improve mortality in copd are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. many people are developing copd, and the cause of this condition is complicated and not thoroughly understood. one key factor is genetic susceptibility. some studies have shown a large genetic contribution to the variability in pulmonary function and copd [86, 87] . polymorphisms in multiple genes have been reported to be associated with copd [87] , such as transcription factor [e.g. nuclear factor-kappa b (nfκb)] [88] , extracellular matrix (e.g., matrix metalloproteinase-12 (mmp-12)) [89, 90] , cytokines [e.g. tumor necrosis factor (tnf)-α] [91] , chemokines [e.g. interleukins (il)-8, il-8 receptor and chemokine receptor (ccr)1] [92, 93] , and apoptosis (e.g., caspase-3 and vascular endothelial growth factor (vegf)) [94, 95] . many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. although several new treatments that target the inflammatory process are now in clinical development, such as tnf-α inhibitors and i-kappab kinase complex 2 (ikk2) inhibitors [96, 97] , clinical trials with sirnas have never been performed in copd. the delay of drug development for copd might be due to the relatively recent emergence of research addressing the molecular basis of copd. furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of copd and to develop monitoring techniques to support the development of rnai therapies. currently, no available treatments reduce the progression of copd or suppress the inflammation in small airways and lung parenchyma. the rnai-based approach for the key molecules also has potential implications for the treatment of copd. asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. the world health organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease [98] . inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. however, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. many target genes have been identified that participate in the pathogenesis of asthma. the most promising targets include genes coding for cytokines (il-4, il5, and il-13), cytokine and chemokine receptors (il-4 receptor and ccr3), and tyrosine kinases [spleen tyrosine kinase (syk) and lck/yes-related novel tyrosine kinase (lyn)], as well as for transcription factors [signal transducers and activators of transcription 1 (stat1), stat6, gata3, and nfκb] that are involved in asthma [19, 99, 100] . the genes that have been assessed as sirna targets for the treatment of asthma in preclinical models are reported [101] . currently, in a clinical trial for asthma, excellair tm (zabecor, bala cynwyd, pa, usa), a sirna that targets syk, is being used. the kinase is involved in signaling from a b cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. it has been reported that antisense oligonucleotides administered by aerosol were potent to decrease syk expression, mediator release from alveolar macrophages, and syk-dependent pulmonary inflammation [102] . moreover, inhibition of inflammatory mediators was shown in a study using sirna targeting syk in airway epithelial cells [103] . following the successful results of the company's phase i clinical trial, a phase ii trial for its asthma drug candidate excellair tm has already been initiated. some of the current treatments for asthma and other inflammatory conditions, such as tnf-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. in contrast, sirna targeting syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. overall, recent progress of sirnas to the lungs has also improved the therapeutic feasibility of rnai for inflammatory lung diseases. the rapid progress will put sirna-based therapeutics on a fast track to the clinic. mirnas are small endogenous noncoding rnas that regulate gene expression by repressing translation or promoting the degradation of their target mrna. mirnas regulate gene expression by binding to the 3′ untranslated region (utr) of their target mrnas and mediating mrna degradation or translational inhibition. in the human genome, transcripts of approximately 60% of all mrnas are estimated to be targeted by mirnas [104] . according to their function, mirnas play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [105] . a growing number of mirnas have been shown to be involved in different lung diseases. this evidence makes mirnas a promising technology for current and future therapeutic development. we discuss the role of some mirnas in various lung diseases as well as the possible future of these discoveries in clinical applications. table 2 shows the summary of mirnas in therapeutic development. at this point, a mirna-based therapy has already entered a phase ii clinical trial. there is evidence that upregulation or downregulation of mirnas is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. many studies have focused on the role of mirnas in inflammatory lung diseases, such as copd [116, 117] , pulmonary fibrosis [118] [119] [120] [121] , and asthma [122] [123] [124] [125] (table 3) . [130] [117, 129] the pathogenesis of copd is attributed to not only chronic inflammation in the airways but also systemic inflammation [131] . cigarette smoking is the main risk factor for the development of copd. smoking has been shown to cause biological change in the gene expression of the lungs [132] , and there are some reports about smoking-related mirnas [117, 129, 130] . however, there are few reports that focus on the mirnas related to the pathogenesis of this disease with systemic inflammatory components. recent study on pulmonary fibroblasts of copd patients presents less expression of mir-146a after stimulation with proinflammatory cytokines when compared with non-copd subjects with similar smoking histories [127] . the downregulation of mir-146a resulted in a prolonged mrna half-life of cyclooxygenase-2, thus increasing prostaglandin e2 in fibroblasts from copd subjects. moreover, ezzie et al. researched the difference of mirna profiles expressed in the lungs of smokers with and without copd. they concluded that mir-223 and mir-1274a were the most affected mirnas in subjects with copd [126] . yet, copd is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [133] . a better understanding of the complexity of the disease and potential clinical relevance of the identified mirnas is needed. pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. this disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (tgf-β) and other cytokines produced at the lesion of active fibrosis [128] . recently, it was reported that mirnas may play an important regulatory role in the pulmonary fibrotic change in the lungs. the downregulation of let-7d in idiopathic pulmonary fibrosis (ipf) resulted in increased collagen deposition and alveolar septal thickening [119] . in addition, liu et al. reported that the oncogenic mir-21 was found to be upregulated in ipf patients and in the murine lungs with bleomycin-induced fibrosis [118] . although these mirnas may be potential therapeutic targets because their expression is related to the regulation of tgf-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. pulmonary fibrosis is also a complicated illness that can have many different causes. focus on the role of mirnas in asthma has recently increased. asthma is an inflammatory disease of the airway that is characterized by an abnormal response of t helper-2 (th2)-type cd4+t lymphocytes against inhaled allergens [134] . in a different asthmatic mouse model, there was an observed increase in the expression of mir-21 in the lungs [123] . this report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of il-12, favoring the th2 lymphocyte response. a toll-like receptor 4 (tlr4)-induced th2 lymphocyte induces high expression of mir-126, and selective blockade of mir-126 suppressed the asthmatic phenotype [124] . in addition, airway remodeling is a characteristic feature of asthma and has important functional implications. rodriguez et al. have shown that mir-155 is related to the development of inflammatory infiltration into the lung and airway remodeling [122] . thus, some studies present a functional connection between mirna expression and asthma pathogenesis and suggest that targeting mirnas in the airways may lead to anti-inflammatory treatments for allergic asthma. despite the evidence from experimental models, the expression profiling of mirnas in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in mirna expression [135] . further investigations about the role of mirnas related to asthma pathogenesis are required. although the basic evidence of mirna biology is still providing new insights, applications of mirna-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [136] . one reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. the presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. understanding the role that mirnas play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. given the significant roles that mirnas play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of mirna-based therapies, including restoring functions of tumor suppressive mirnas or inhibiting oncogenic mirnas. the development of mirna-based therapies for lung cancer is growing prosperously with the help of new rnai technologies. compared to sirna-based therapies, which are already in clinical trials, mirnas are less toxic and have the potential to target multiple genes. the difficulty associated with mirna delivery is mainly equal to that of sirnas. the critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [137] . there are two strategies as the therapeutic applications of mirnas for lung cancer [138] . one strategy is mirna replacement therapy, which involves the re-introduction of a tumor suppressor mirna mimic to restore a loss of the function. mirna mimics are synthetic rna duplexes designed to mimic the endogenous functions of mirnas with chemical modifications for stability and cellular uptake. the concept of mirna replacement therapy is most exemplified by the let-7 mirna. let-7 is a tumor-suppressor mirna in non-small-cell lung cancer that inversely correlates with the expression of the ras oncoprotein, a key cancer gene [139] . intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that mirna replacement therapy is indeed promising [106, 140, 141] . another mirna that shows the value of mirna replacement is provided by mir-34a [107, 142] . local and/or systemic delivery of a synthetic mir-34a mimic led to accumulation of mir-34a in the tumor tissue and inhibition of lung tumor growth. lately, ling et al. also showed that tumor suppressor mir-22 exhibited anti-lung cancer activity through post-transcriptional regulation of erbb3 [143] . thus, therapeutic mirna mimics have a powerful potential by attacking multiple genes relevant to several diseases. however, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of mirna mimics will lead to an accumulation of exogenous mirnas in normal cells [138] . although the assumptions are well founded, there is still insufficient evidence for toxicity caused by mirna mimics. indeed, several in vivo studies failed to reveal side effects caused by the mirna mimics and suggested that delivery of mirna mimics to normal tissues was well tolerated [107, 141] . it will be important to research mirna mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. the second strategy is directed toward a gain of function and aims to inhibit oncomirs by using anti-mirnas. chemical modifications, such as 2'-o-methyl-group and locked nucleic acid (lna), would increase oligo stability against nucleases [144] . antisense oligonucleotides contained in these modifications are termed antagomirs or "lna-antimirs" [144, 145] . they are oligonucleotides with sequences complementary to the endogenous mirna and inhibit the specific mirna function. an lna-antimir against mir-122 has been shown to effectively silence mir-122 in non-human primates [145] , and the findings support the potential of these compounds as a new class of therapeutics. moreover, it has also been reported that anti-mir-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth [146] . relative to studies on mirna mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. this illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to rnase, and pharmacologic properties. therefore, inhibition of mirna function by chemically modified antimir oligonucleotides has become an important and widely used approach. recent data from the first phase ii study in patients with chronic hcv infection treated with the lna-modified antimir-122 showed that this compound was well tolerated and provided continuing viral suppression. an increasing number of studies have examined the therapeutic potential of mirnas. recently, the evidence of roles for mirnas in determining drug resistance has emerged [147] . cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. in this situation, combining mirna mimics or antimir with chemotherapy may potentiate the efficacy of the cancer treatment in the future. in addition, mirnas related with cancer stem cells may significantly broaden the field of mirna-based therapy and suggest that mirnas can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [108, 148] . hence, the main challenge is the successful delivery and chemical modifications of the therapeutic mirnas to the target tissue without harming normal tissues. rnai-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. one of the greatest challenges in rnai-based therapy continues to be the delivery method of the therapeutic sirnas and mirnas to the target cells. pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. a realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. the advancement of pulmonary sirna delivery to the clinic illustrates that rnai-based therapy holds a central place in the future treatment of lung diseases. on the other hand, mirnas have the opportunity to target multiple genes in a fine-tuned manner, and the mirna-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. in particular, anti-mirna therapy by chemically modified antimir oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. increased evidence has indicated that mirnas fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. further understanding of the detailed mechanisms of rnai-based therapy and investigations of more effective delivery methods are required for future development. these novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients. strategies for silencing human disease using rna interference rnai therapeutics: a potential new class of pharmaceutical drugs small interfering rna inhibits hepatitis b virus replication in mice rnai suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia confirming the rnai-mediated mechanism of action of sirna-based cancer therapeutics in mice chemical modification of sirna sirna and isrna: two edges of one sword targeted delivery of antisense oligodeoxynucleotide and small interference rna into lung cancer cells poly(ester amine)-mediated, aerosol-delivered akt1 small interfering rna suppresses lung tumorigenesis poly(beta-amino ester) as a carrier for si/shrna delivery in lung cancer cells inhibition of non-small cell lung cancer cell proliferation and tumor growth by vector-based small interfering rnas targeting her2/neu rodriguez-padilla, c. wt1 gene silencing by aerosol delivery of pei-rnai complexes inhibits b16-f10 lung metastases growth inhibition of influenza virus production in virus-infected mice by rna interference using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque intrapulmonary delivery of xcl1-targeting small interfering rna in mice chronically infected with mycobacterium tuberculosis effective treatment of respiratory alphaherpesvirus infection using rna interference a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus rna interference: new therapeutics in allergic diseases emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease suppression of plasminogen activator inhibitor-1 by rna interference attenuates pulmonary fibrosis development and preclinical efficacy of novel transforming growth factor-beta1 short interfering rnas for pulmonary fibrosis pulmonary delivery of therapeutic sirna the lung as a route for systemic delivery of therapeutic proteins and peptides spray drying of sirna-containing plga nanoparticles intended for inhalation design of an inhalable dry powder formulation of dotap-modified plga nanoparticles loaded with sirna silencing of fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis in vivo gene silencing (with sirna) of pulmonary expression of mip-2 versus kc results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury nonviral sirna delivery to the lung: investigation of peg-pei polyplexes and their in vivo performance intratracheal versus intravenous liposomal delivery of sirna, antisense oligonucleotides and anticancer drug attenuation of fibrosis in vitro and in vivo with sparc sirna rnai-mediated suppression of constitutive pulmonary gene expression by small interfering rna in mice intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations non-invasive pulmonary aerosol delivery in mice by the endotracheal route pulmonary vaccine delivery inhibition of respiratory viruses by nasally administered sirna noninvasive delivery of small inhibitory rna and other reagents to pulmonary alveoli in mice immunological and toxicological implications of short-term studies in animals of pharmaceutical aerosol delivery to the lungs: relevance to humans evaluation of the safety, tolerability and pharmacokinetics of aln-rsv01, a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) clinical evaluation of inhaled insulin inhalable lactose-based dry powder formulations of low molecular weight heparin extracellular barriers in respiratory gene therapy nanodelivery in airway diseases: challenges and therapeutic applications expression of respiratory mucins in fatal status asthmaticus and mild asthma remodeling in asthma and chronic obstructive lung disease polymeric nanocarriers for drug delivery to the lung nonviral delivery of synthetic sirnas in vivo rapid crossing of the pulmonary endothelial barrier by polyethylenimine/dna complexes delivery of sirna therapeutics: barriers and carriers cellular sirna delivery using cell-penetrating peptides modified for endosomal escape delivering silence: advancements in developing sirna therapeutics gene silencing by adenovirus-delivered sirna effect of adenovirus-mediated rna interference on endogenous micrornas in a mouse model of multidrug resistance protein 2 gene silencing lentivirus-mediated rnai knockdown of nupr1 inhibits human nonsmall cell lung cancer growth in vitro and in vivo cancer gene therapy: challenges and opportunities us gene therapy in crisis rnai-dependent and -independent antiviral phenotypes of chromosomally integrated shrna clones: role of vasp in respiratory syncytial virus growth non-viral sirna delivery to the lung lipid-based systemic delivery of sirna protection against lethal influenza virus challenge by rna interference in vivo fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by pi3-kinase and nf-kappa b full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung cationic liposome-mediated delivery of sirnas in adult mice lipidic systems for in vivo sirna delivery expression profiling reveals off-target gene regulation by rnai induction of an interferon response by rnai vectors in mammalian cells lack of interferon response in animals to naked sirnas rnai-mediated gene-targeting through systemic application of polyethylenimine (pei)-complexed sirna in vivo systemic delivery of rafsirna using cationic cardiolipin liposomes silences raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer pulmonary gene silencing in transgenic egfp mice using aerosolised chitosan/sirna nanoparticles a combinatorial library of lipid-like materials for delivery of rnai therapeutics immunologic aspects of chronic obstructive pulmonary disease drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics suppression of ocular neovascularization with sirna targeting vegf receptor 1 the us national institutes of health respiratory syncytial virus infection in elderly and high-risk adults rna interference therapy in lung transplant patients infected with respiratory syncytial virus down-regulation of igf-ir using small, interfering, hairpin rna (sirna) inhibits growth of human lung cancer cell line a549 in vitro and in nude mice ezh2 silencing with rnai enhances irradiation-induced inhibition of human lung cancer growth in vitro and in vivo atu027, a liposomal small interfering rna formulation targeting protein kinase n3, inhibits cancer progression pkn3 is required for malignant prostate cell growth downstream of activated pi 3-kinase cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer the stability of mrna influences the temporal order of the induction of genes encoding inflammatory molecules inhaled and systemic corticosteroids in chronic obstructive pulmonary disease cigarette smoke induces senescence in alveolar epithelial cells senescence in chronic obstructive pulmonary disease identifying targets for copd treatment through gene expression analyses targeting the nf-kappab pathway in asthma and chronic obstructive pulmonary disease neutrophil elastase contributes to cigarette smoke-induced emphysema in mice pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease tumor necrosis factor-alpha gene polymorphism in chronic bronchitis characterization of cigarette smoke-induced inflammatory and mucus hypersecretory changes in rat lung and the role of cxcr2 ligands in mediating this effect comprehensive gene expression profiling of rat lung reveals distinct acute and chronic responses to cigarette smoke inhalation alveolar wall apoptosis causes lung destruction and emphysematous changes inhibition of vegf receptors causes lung cell apoptosis and emphysema copd: current therapeutic interventions and future approaches p38 mapk inhibitors, ikk2 inhibitors, and tnfalpha inhibitors in copd chronic respiratory disease-asthma sirna as a therapy for asthma targeting allergic airway diseases by sirna: an option for the future? antisense-and rna interference-based therapeutic strategies in allergy aerosolized syk antisense suppresses syk expression, mediator release from macrophages, and pulmonary inflammation syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells many roads to maturity: microrna biogenesis pathways and their regulation micrornas in lung diseases regression of murine lung tumors by the let-7 microrna development of a lung cancer therapeutic based on the tumor suppressor microrna-34 the microrna mir-34a inhibits prostate cancer stem cells and metastasis by directly repressing cd44 dysregulation of micrornas after myocardial infarction reveals a role of mir-29 in cardiac fibrosis therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection control of stress-dependent cardiac growth and gene expression by a microrna mir-15 family regulates postnatal mitotic arrest of cardiomyocytes microrna-195 promotes palmitate-induced apoptosis in cardiomyocytes by down-regulating sirt1 microrna-206 delays als progression and promotes regeneration of neuromuscular synapses in mice defective erythroid differentiation in mir-451 mutant mice mediated by 14-3-3zeta downregulation of the serum response factor/mir-1 axis in the quadriceps of patients with copd microrna expression in induced sputum of smokers and patients with chronic obstructive pulmonary disease mir-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis inhibition and role of let-7d in idiopathic pulmonary fibrosis mir-29 is a major regulator of genes associated with pulmonary fibrosis participation of mir-200 in pulmonary fibrosis requirement of bic/microrna-155 for normal immune function microrna-21 is up-regulated in allergic airway inflammation and regulates il-12p35 expression antagonism of microrna-126 suppresses the effector function of th2 cells and the development of allergic airways disease down-regulation of mir-133a contributes to up-regulation of rhoa in bronchial smooth muscle cells gene expression networks in copd: microrna and mrna regulation reduced mir-146a increases prostaglandin e(2)in chronic obstructive pulmonary disease fibroblasts identification of keratinocyte growth factor as a target of microrna-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions downregulation of microrna expression in the lungs of rats exposed to cigarette smoke micrornas as modulators of smoking-induced gene expression changes in human airway epithelium copd as a lung disease with systemic consequences--clinical impact, mechanisms, and potential for early intervention transcriptomic studies of the airway field of injury associated with smoking-related lung disease copd, a multicomponent disease: implications for management asthma and allergic inflammation microrna expression profiling in mild asthmatic human airways and effect of corticosteroid therapy micrornas in inflammatory lung disease--master regulators or target practice? artificial micrornas as sirna shuttles: improved safety as compared to shrnas in vitro and in vivo the promise of microrna replacement therapy ras is regulated by the let-7 microrna family the let-7a microrna protects from growth of lung carcinoma by suppression of k-ras and c-myc in nude mice the let-7 microrna reduces tumor growth in mouse models of lung cancer nanoparticles modified with tumor-targeting scfv deliver sirna and mirna for cancer therapy tumor suppressor mir-22 suppresses lung cancer cell progression through post-transcriptional regulation of erbb3 silencing of micrornas in vivo with "antagomirs lna-mediated microrna silencing in non-human primates regression of a549 lung cancer tumors by anti-mir-150 vector role of microrna in anticancer drug resistance microrna mir-34 inhibits human pancreatic cancer tumor-initiating cells this work was supported in part by a grant-in-aid for the third-term the authors declare that there are not conflicts of interest to report. key: cord-284332-p4c1fneh authors: bosma, karen j.; taneja, ravi; lewis, james f. title: pharmacotherapy for prevention and treatment of acute respiratory distress syndrome: current and experimental approaches date: 2012-09-19 journal: drugs doi: 10.2165/10898570-000000000-00000 sha: doc_id: 284332 cord_uid: p4c1fneh the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, β(2)-adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col-3), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, b 2 -adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col-3), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. acute lung injury (ali) and the acute respiratory distress syndrome (ards) arise from direct or indirect injury to the lungs, and results in a life-threatening form of respiratory failure. ali/ards is both common and serious: 6.5-8.5% of patients admitted to an intensive care unit (icu) will be diagnosed with ali or ards, [1] [2] [3] and approximately one-quarter to one-half of these patients will succumb to this disease process. [1, [4] [5] [6] over the past 40 years, ards has been the focus of extensive basic science and clinical research, although no single pharmacotherapy has been shown to reduce mortality in a large, randomized, controlled, multicentre trial of adult patients. the reasons for this are manifold, and include issues of dosing, route of administration and timing of the various interventions tested. more importantly, however, may be the nature of the disorder itself: the diagnosis of ards envelops a heterogeneous group of patients with varying causes and pathophysiological mechanisms at work. the notion that a therapeutic agent that can successfully alter a single biological target in an animal model of ali will reduce mortality in all patients with ards may be unrealistic. nonetheless, there is reason for hope on the scientific horizon. recent advances have been made in our understanding of the pathophysiological mechanisms underlying ali/ards, leading to the identification of potential novel targets for pharmacological intervention. some therapies are best aimed at preventing the development of ards, while others treat the syndrome as it unfolds or aid in its resolution. the challenge lays in identifying the subgroup of patients most likely to benefit from such focused therapy. this paper reviews the current experimental and existing approaches to managing ards, highlighting the pathophysiological basis for their use and potential for future clinical development. ali may occur following a direct insult to the pulmonary system such as aspiration of gastric contents, bacterial pneumonia or viral pneumonitis (e.g. h1n1 influenza virus), or an indirect insult such as the systemic inflammatory response associated with pancreatitis, sepsis or multiple trauma. table i shows common direct and indirect causes of ali/ards. whether this 'first hit' to the lung is direct or indirect, a pulmonary inflammatory response may occur, which often is adaptive and self-limited. however, when coupled with repeated 'hits' to the lung from insults such as injurious mechanical ventilation or other secondary processes such as hypotension, a cycle of intense inflammation and worsening pulmonary injury ensues. the 'multiple hit' theory of ards progression also provides a framework for studying the disease process (figure 1). clinically, ali manifests as bilateral airspace disease observed on chest radiograph and hypoxaemia, such that the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao 2 /fio 2 ) is greatly reduced. according to the 1994 american european consensus conference (aecc) definition, a chest radiograph consistent with pulmonary oedema and a pao 2 /fio 2 ratio <300 is sufficient to diagnose ali in the setting of an inciting pulmonary insult and the absence of congestive heart failure. the aforementioned criteria but with a pao 2 /fio 2 ratio <200 is classified as ards. [8] although differentiated by the aecc definition, ali and ards are often grouped together for the purpose of clinical trial enrolment and are treated as a single entity throughout this review. although not all patients follow the same clinical course, progression of ali/ards may be considered along a pathophysiological timeline of early, mid and late phases, with considerable overlap between these phases. table ii summarizes the pathogenetic mechanisms at work during each phase, linking each biological pathway to a potential drug therapy. a general overview of the pathophysiology of ards is provided here, with more detailed descriptions of the specific biologic pathways discussed in sections 3.1-3.3 as they pertain to each potential pharmacological therapy. the early phase, within the first 72 hours of the inciting lung injury, is characterized by inflammatory damage to the alveolar-capillary barrier. this results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. this inflammation-induced pulmonary oedema disrupts normal gas exchange and increases the work of breathing, leading to respiratory failure and the need for mechanical ventilation. mechanical ventilation itself may cause secondary insult to the already inflamed oedematous alveoli. during each tidal breath induced by mechanical ventilation, unstable alveoli undergo cyclical collapse and shearing open, termed 'atelectrauma'. furthermore, the non-collapsed alveolar units may receive a greater proportion of the delivered tidal volume, leading to damage due to overdistention or 'volutrauma'. further breakdown of the endothelial-epithelial barrier may occur with atelectrauma and volutrauma, along with the release of local proinflammatory mediators which further b see text for details. aa = arachidonic acid; arb = angiotensin receptor antagonist (blocker); fa = fatty acid; gm-csf = granulocyte macrophage colonystimulating factor; hne = human neutrophil elastase; icam-1 = intercellular adhesion molecule-1; il-8 = interleukin-8; mmps = matrix metalloproteinases; nf-kb = nuclear factor-kb; paf = platelet-activating factor; ppar-g = peroxisome proliferator activated receptor-g; rhpaf = recombinant human paf; tnfa = tumour necrosis factor-a. propagate this cycle of ventilator-exacerbated lung injury. [7] as inflammation ensues, neutrophils are recruited to the lung. damaged endothelial cells exhibit increased activity of the transcription factor nuclear factor-kb (nf-kb), which upregulates the surface expression of intercellular adhesion molecule (icam)-1. icam-1 mediates leukocyte adhesion and migration across the endothelium to the alveolar epithelium. activated neutrophils release proteases, such as matrix metalloproteinases (mmp) and neutrophil elastase (ne), which further damage the alveolarcapillary membrane. [9] activated neutrophils also contain high levels of arachidonic acid, [10] which is metabolized into leukotrienes, prostaglandins and thromboxanes. leukotrienes attract more neutrophils, prostaglandins are proinflammatory mediators, and thromboxanes play a role in vasoconstriction and platelet and leukocyte aggregation. neutrophil recruitment and activation may be an adaptive physiological response to injury, or may incite a vicious cycle of inflammation and further damage. [9] at this stage, patients may recover from the initial insult, with clearance of the pulmonary oedema and restoration of the barrier between capillary endothelial and alveolar epithelial cells, or may progress to the exudative or mid phase of ards. it is not fully understood why two patients exposed to the same insult may have completely different clinical courses; however, genetic factors, [11] co-morbid illnesses such as diabetes mellitus and alcohol addiction, [12] nutritional status, medications and exposure to further insults are all likely to play a role. understanding the host and environmental factors that place a patient at high risk of progressing to the exudative phase of ards will facilitate identification of targets for earlier intervention. the exudative or subacute phase typically occurs over the 3-7 days following the initial insult. pathologically, this mid phase is characterized by formation of intra-alveolar hyaline membranes rich in plasma proteins, fibrin and cellular debris. [13] a biopsy of the lungs at this stage will show diffuse alveolar damage and, clinically, the lungs have poor compliance with ongoing gas exchange problems including hypoxaemia and elevated dead space fraction. the inflammatory milieu within the alveoli, coupled with the cyclical opening, stretching and collapsing of alveoli via mechanical ventilation, initiates a number of pathogenic pathways in concert or in series. these include disruption of surfactant function and metabolism, ongoing neutrophil recruitment and activation, along with increased expression and release of inflammatory mediators, imbalance of oxidant and antioxidant activity, and activation of complement and coagulation cascades. each of these pathways is further discussed to provide context for the drugs or therapies aimed at ameliorating these various mechanisms (see section 3). interestingly, only a minority of patients will succumb to severe hypoxaemia or hypercarbia, as the major source of mortality is not the pulmonary injury per se, but rather the occurrence of multiple organ failure. in this setting, the injured lung may represent a rich source of inflammatory mediators that could contribute to the development of multi-organ failure. for example, stress failure and necrosis of the endothelial-epithelial barrier may allow various inflammatory mediators, bacteria and endotoxins to quickly spread from the lungs into the systemic circulation. indeed, it is this de-compartmentalization of inflammatory mediators from the lungs into the circulation that is felt to lead to cell apoptosis in distal organs, [14] and ultimately multiple organ dysfunction syndrome (mods) [ figure 2 ]. [15] once mods develops, disease is often irreversible and mortality may increase significantly to 60-98%, the latter occurring when three or more organs are involved for a period of more than 7 days. [15] [16] [17] thus, a key to developing novel therapies that will reduce mortality in ards will be identification of the cellular and molecular mechanisms by which ards leads to mods. survivors of the first week of ali/ards may enter the late phase of the disorder, known as the fibroproliferative phase. during days 8-28, the exudates and hyaline membranes become organized, and fibrosis may become apparent. type ii alveolar cells proliferate and line the alveolar walls, fibroblasts migrate and differentiate into myofibroblasts in the interstitial and alveolar spaces, and a collagen-rich extracellular matrix is laid down in the interstitium. [13] alveoli may be destroyed, pulmonary vascular area may be reduced and chronic inflammation is generally present. patients in the fibroproliferative phase of ards may slowly recover, or may fail to wean from mechanical ventilation and succumb to complications of a lengthy critical illness or pre-existing co-morbid illnesses. pharmaceutical interventions for late ards must interrupt the fibrosing alveolitis and aid in resolution, remodelling and repair of injured lungs. [13] often, therapies that might be beneficial during the early phase of lung injury are started too late in the course of the disease, when fibrosis is already established, muting their potential efficacy. when tested specifically for the late fibroproliferative phase of ards, anti-inflammatory therapies have yielded disappointing results. basic science research examining mechanisms of idiopathic pulmonary fibrosis may illuminate therapeutic pathways for fibroproliferative ards, but further work is required in this area. although no pharmacological therapies have been proven to reduce mortality in large, randomized controlled trials (rcts) involving adult patients, it appears that improvements in supportive care have reduced mortality to some extent. for example, mortality estimates ranged progression of acute respiratory distress syndrome (ards) to multi-organ failure (mof). initially, inflammatory damage to the alveolar-capillary barrier results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. there, the proteinaceous fluid interferes with the function and metabolism of the endogenous surfactant system. coupled with this, neutrophils that infiltrate lungs are subsequently activated and represent an important source of inflammatory mediators and oxygen free radicals, inducing further epithelial and endothelial cell damage and an altered host immune response. newly secreted mediators and/or spillover of inflammatory mediators from the lung into the systemic circulation ultimately contribute to the development of mof. inflammatory mediators released from organs such as the liver, heart and kidney return to the lung via the systemic circulation and may contribute to further pulmonary inflammation. thus, each new insult to the pulmonary system accelerates the acute lung injury cycle (reproduced from bosma et al. [7] [2007], with permission). from 55% to 65% as reported in the literature in the 1980s and early 1990s [18] [19] [20] [21] to more recent estimates of 32-46% in observational epidemiological studies [1, 2] and 25-31% in large clinical trials. [5, 6] although this mortality reduction may in part reflect differences in diagnostic criteria used post publication of the 1994 aecc definition, undoubtedly the largest impact has been the move to more 'protective' strategies of mechanical ventilation. in 2000, the national institutes of healthsponsored ards network (ardsnet) trial involving low tidal volume ventilation was published, and now constitutes the standard of care for patients with ali and ards. this trial compared a traditional tidal volume of 12 ml/kg with a lower tidal volume of 6 ml/kg in 861 patients and reported a mortality reduction from 40% in the control arm to 31% in the treatment arm. [5] these results definitively ended the debate fuelled by three previous inconclusive smaller trials regarding lower versus conventional tidal volumes. in terms of furthering ali/ards research, several lessons have been learned from this landmark study. first, ardsnet was set up to conduct well designed, large, phase iii studies with a concerted effort to optimize patient enrolment through involvement of many centres in an organized and cohesive group. [22] this enabled a study sufficiently powered to realize a mortality difference to be conducted within a reasonable timeframe, and pointed the way for other similarly structured ards research networks to become established. second, the treatment arm was associated with lower oxygenation values than the conventional arm, highlighting the potential danger of relying on oxygenation or other physiological parameters as surrogates for mortality. third, this study demonstrated that a nonpharmacological intervention could alter mortality, indicating that future rcts need to be carefully standardized in all aspects of supportive care in both treatment and control arms. one potential caveat ensuing from this study has been the assumption that any additional proven therapy would reduce mortality across a population as heterogeneous and diverse as that enrolled in the ardsnet low tidal volume trial. this approach may be misguided, as subsequent studies have demonstrated differences between patients with direct and indirect causes of ali/ards in responsiveness to specific therapies. [23, 24] research is ongoing to determine whether newer modes of mechanical ventilation, such as high-frequency oscillation (hfo), can further improve outcomes in ards relative to the ardsnet low tidal volume strategy. [25] in addition, other aspects of supportive care have been evaluated in large clinical trials, some conducted by ardsnet, and have proven effective in reducing morbidity associated with critical illness. these include cautious fluid management, [26, 27] adequate nutrition, [28] prevention of ventilator associated pneumonia, [29] [30] [31] [32] prophylaxis for deep venous thrombosis [33] and gastric ulcers, [34] weaning of sedation and mechanical ventilation as early as possible, [35] and physiotherapy and rehabilitation. [36] a recent review of all patients enrolled in ardsnet studies between 1996 and 2005 showed that these advancements in critical care (aside from lower tidal volume ventilation) are likely responsible for the improved survival in ali/ards patients in clinical trials noted over the last decade. [37] additional modalities used as 'rescue therapies' for the ards patient at risk of succumbing to severe hypoxaemia or respiratory acidosis have also been tested, including nitric oxide, prone positioning, hfo and extracorporeal membrane oxygenation (ecmo). nitric oxide [38] and prone positioning [39, 40] have not been shown to reduce mortality or duration of mechanical ventilation in patients with ali/ards, and are therefore not recommended for routine use. however, combined together, these therapies may provide a sustained improvement in oxygenation for patients with severe hypoxaemia and a mortality benefit for patients who are failing conventional mechanical ventilation strategies. [39] [40] [41] a clinical trial of hfo for routine care of patients with ards is currently underway, but existing evidence supports its use as salvage therapy if instituted early for patients failing conventional ventilation, [42] and may have additive benefits when combined with nitric oxide and prone positioning. [43] finally, ecmo has recently been studied in the cesar trial (see table iii for a list of trial acronyms). [44] this study showed that transferring adult patients with severe but potentially reversible respiratory failure, whose murray score exceeds 3.0 or who have a ph of <7.20 on optimum conventional management, to a centre with an ecmo-based management protocol, significantly improved survival without severe disability. recent evidence suggests ecmo is also useful for rescue therapy for adults with severe ards due to h1n1-influenza a virus infection. [45] pharmacological treatments for ali/ards may be employed prior to the onset of ards or in the early, mid or late phases of ards (table iv) . accordingly, their purpose may be to prevent ali in those at risk, mitigate the pathogenic mechanisms responsible for the cycle of lung injury and systemic inflammation in established ards, or aid in lung healing and repair. some therapies, such as corticosteroids, have been studied for prevention of ards, treatment of early ards and treatment of late ards, and are discussed within each context. the concept that ards may be prevented in those at high risk after an inciting insult is not new, but is one that is garnering greater attention in the scientific literature in recent years. since no pharmacological agent has proven effective in treating established ards in adults, attention has turned to prophylactic treatment to prevent the development of ards in those at highest risk. of course, any pharmacotherapy that is initiated prior to the diagnosis of disease must have a very high benefit to risk ratio and be cost effective. as such, it should have the following attributes: (i) be low risk, without serious adverse effects; (ii) be easily and widely applicable; and (iii) be relatively inexpensive. drug classes studied for ards prevention include imidazoles (e.g. ketoconazole), ace inhibitors, thiazolidinediones (e.g. rosiglitazone), chemically modified tetracycline derivatives, antioxidants, and corticosteroids and other immunomodulating agents. over 20 years ago, the first clinical trial examining prophylactic use of ketoconazole to prevent ards in patients at risk was published. [46] the rationale for using ketoconazole, an antifungal drug with anti-inflammatory properties, was as follows. as mentioned in section 1, patients with ards have increased levels of arachidonic acid metabolites in their bronchoalveolar fluid. [10, 87] metabolism of arachidonic acid leads to the production of leukotrienes, prostaglandins and thromboxanes. thromboxane a 2 is a potent vasoconstrictor, and is involved with platelet and leukocyte aggregation, while leukotrienes act as powerful chemokines to attract neutrophils. ketoconazole is an antifungal agent of the imidazole class which selectively blocks thromboxane synthetase. ketoconazole also inhibits 5-lipoxygenase, the enzyme necessary to generate leukotrienes, and inhibits procoagulant activity. [55] in addition to showing promise in preclinical animal studies, when given prophylactically to patients at risk of developing ards, ketoconazole has been shown to reduce the incidence of severe ards in three small trials. a 1988 study of 71 patients admitted to a surgical icu showed that in the group treated prophylactically with oral ketoconazole 200 mg/day, 2 of 35 patients (6%) ultimately developed ards, whereas 11 of 36 (31%) patients in the control group developed ards (p < 0.01). [46] similar results followed in a 1993 study of 54 patients with septic shock admitted to a surgical icu, where the incidence of ards in the group treated with ketoconazole 400 mg/day was 15% (4 of 26 patients) compared with 64% (18 of 28 patients) in the control group (p = 0.002), and mortality was 15% versus 39%, respectively (p = 0.05). [47] although both of these studies were conducted prior to the 1994 aecc definition, ards was strictly defined in the aforementioned studies, including a pao 2 /fio 2 ratio <150 or intrapulmonary shunt >20% in patients requiring mechanical ventilation and who had diffuse infiltrates on chest radiograph without clinical evidence of heart failure as pulmonary arterial occlusion pressures were <18 mmhg. building on the results of these two studies, sinuff and colleagues [48] developed practice guidelines for prophylactic ketoconazole use, and tested the implementation and efficacy of these guidelines in two icus (one control and one active comparator). they reported a significantly decreased incidence of ards in the icu population receiving ketoconazole prophylaxis, although mortality was equivalent within the two units. [48] in 2000, ardsnet published the karma study evaluating oral ketoconazole versus placebo for patients within 36 hours of an established diagnosis of ali or ards according to the 1994 aecc definition. [55] the study was stopped early after enrolment of 234 patients for failing to show a difference in mortality or ventilator-free days. of note, this study was designed to look at early treatment of ali/ards rather than prevention of ards in patients at risk, and therefore did not necessarily negate the findings of the three previous smaller studies. furthermore, a problem identified in the karma study was that even though blood ketoconazole concentrations were adequate, urinary metabolites of thromboxane were not affected, raising the possibility that the proper dose to achieve an anti-inflammatory effect was not given. however, since the karma f mortality reduction in subgroup of patients with ards, septic shock and relative adrenal insufficiency. g no mortality reduction in larger study, n = 180 (lasrs). il-8 = interleukin-8; ma = meta-analysis; mc = multicentre; ppar-c = peroxisome proliferator activated receptor-g; rhpaf = recombinant human platelet-activating factor; rsp-c = recombinant surfactant protein-c; sc = single-centre. study showed no difference in mortality, widely considered the most important endpoint to achieve, further research on ketoconazole for ali/ards ceased. [55] additionally, ketoconazole has numerous drug interactions and requires an acidic milieu to be absorbed via the enteral route, making routine use in the icu complicated. further research should examine whether other drugs in the imidazole class given intravenously have similar anti-inflammatory properties, and also establish the inflammatory dose-response curve for ali/ards. in addition, although the concept that prevention of ards will definitely lead to decreased mortality is intuitive, this still has to be proven in large multicentre clinical trials. the authors are unaware of any studies being conducted in this area presently. angiotensin-converting enzyme (ace) is produced in the lungs and is responsible for converting angiotensin i into angiotensin ii, a peptide active in vasoconstriction and sodiumfluid balance to maintain blood pressure homeostasis. ace inhibitors and angiotensin ii receptor antagonists (blockers; arbs) are classes of drugs commonly used to treat hypertension, and prevent progression of diabetic nephropathy in patients with diabetes. ace inhibitors also help to preserve vascular structure and function, by exerting a protective effect on endothelial cells. endothelial cell damage is the catalyst for the inflammatory and coagulation cascades activated in ali/ards. thus, the protection of endothelial cells offered by ace inhibitors may have a beneficial role in ards. [49] studies in transgenic mice have shown that ace, angiotensin ii and angiotensin ii receptor type 1a may promote lung injury, whereas ace2, a close homologue of ace, and angiotensin ii receptor type 2 may protect against severe lung dysfunction in models of ards. [88] the ace inhibitor captopril has been shown to prevent severe lung injury in an oleic acid-induced model in rats. in this model, captopril reduced expression of icam-1 in lung tissue, indicating a protective effect on endothelial cells, diminished activity of tissue plasminogen activator, involved in coagulation, and blocked nf-kb, the major signal transduction pathway that regulates the expression of multiple early-response genes related to inflammation. [49] in humans, two small cohort studies have demonstrated that polymorphism of the ace gene increases susceptibility to the development of ards and its outcome. [89, 90] two additional studies, published only in abstract form to date, have examined the association between ace inhibitor use and ards. a retrospective cohort study of 1423 adult critically ill patients found that 5.5% of patients developed ards after hospital admission, and that preexisting, long-term use of an ace inhibitor or arb was associated with decreased risk of ards development, after adjusting for predisposing conditions (odds ratio [or] 0.49; 95% ci 0.25, 0.94; p = 0.03). [91] the second abstract, a case-control study nested within a prospective cohort of 1553 critically ill patients at risk for ards, reported that patients on ace inhibitors had a lower prevalence of respiratory failure on admission to icu, but not lower incidence of ards after adjusting for confounders on multivariate analysis. however, among patients who developed ards, ace inhibitor use was associated with lower mortality (adjusted hazard ratio 0.66; 95% ci 0.45, 0.99). [92] the associations observed in these clinical studies is consistent with preclinical animal data, but requires further research prior to being applicable clinically. [90] peroxisome proliferator activated receptors (ppars) are ligand-activated transcription factors related to thyroid hormone, steroid and retinoid receptors. [50] there are three isoforms: ppar-g, ppar-a and ppar-b/d. ppar-g plays a central role in glucose homeostasis. thiazolidinediones, a class of oral antidiabetic drugs, are synthetic ligands for ppar-g. synthetic ppar-g agonists also have anti-inflammatory properties, inhibiting proinflammatory cytokine production and macrophage activation in vitro. [93, 94] this action is mediated in part by antagonizing the activity of transcription factor nf-kb. when activated, nf-kb induces overexpression of inflammatory cytokines such as tumour necrosis factor (tnf)-a, which in turn induces upregulation of icam-1 expression, as well as recruitment and activation of immune cells. icam-1, expressed on the surface of endothelial cells, mediates leukocyte adhesion and migration through endothelium into tissues. the anti-inflammatory properties of thiazolidinediones have been demonstrated in vivo in murine models of inflammatory bowel disease [95] and rheumatoid arthritis. [96] rosiglitazone is the most potent selective ppar-g of the thiazolidinediones. prophylactic administration of rosiglitazone has been shown to attenuate ali in an animal model of pancreatitis-associated ali. [50] in this study, rosiglitazone was dissolved and given intravenously to rats 30 minutes prior to induction of acute pancreatitis by sodium taurocholate. compared with control group rats with acute pancreatitis and its associated lung injury, prophylactic administration of rosiglitazone resulted in a significantly lower histological pulmonary injury score, reduced pulmonary expression of tnfa and icam-1 messenger rna, and decreased lung tissue myeloperoxidase activity, a measure of neutrophil infiltration in the lung. [50] this suggests that prophylactic rosiglitazone mitigates the ali associated with acute pancreatitis by its anti-inflammatory effect. unfortunately, the safety of rosiglitazone has recently been questioned due to its augmentation of sodium and water retention, leading to increased incidence of congestive heart failure in diabetic patients placed on this drug long-term. [97, 98] thus, further animal studies are needed to confirm the effects of rosiglitazone in acute pancreatitis and evaluate potential complications related to its use, prior to proceeding to human studies. during the early phase of lung injury, neutrophils are recruited into the pulmonary vasculature and activated to release proteases, such as mmps and ne, which damage the alveolarcapillary membrane, [51] resulting in further release of inflammatory mediators. a single laboratory in the state university of new york (new york, ny, usa) has demonstrated in various animal models that blocking the proteases ne, mmp-2 and mmp-9 with a unique modified tetracycline can prevent the increased pulmonary vascular permeability that ultimately leads to ards. the same group has developed a 'two-hit' porcine model of sepsis plus gut ischaemiareperfusion injury that parallels the insidious onset of sepsis-induced ards in humans. in this model, anaesthetized yorkshire pigs undergo cross-clamping of the superior mesenteric artery for 30 minutes to induce intestinal ischaemia, followed by intraperitoneal placement of a faecal blood clot. pigs are then awakened, extubated and taken to an animal icu for 48 hours of continuous observation, where they receive intravenous fluids, broad-spectrum antibacterials and pain control medications. when the pao 2 /fio 2 ratio falls below 250, pigs are anaesthetized and placed back on mechanical ventilation with tidal volumes of 10 ml/kg. in this model, they demonstrated that prophylactic administration of a synthetic, nonantimicrobial derivative of tetracycline called incyclinide (col-3; collagenex pharmaceuticals), prevented the development of both ards and septic shock. [51] incyclinide has not yet been tested in any human studies of ards prevention; however, the complex model developed by this group contains all the elements of a clinically relevant animal model and, therefore, these results show potential for phase ii studies. oxidative stress is associated with development of ards and mods via direct tissue injury. nathens and colleagues [52] examined the effect of antioxidant supplementation using atocopherol and ascorbic acid in critically ill surgical patients. in a prospective rct of 595 surgical icu patients (mainly victims of trauma), they found antioxidants did not reduce the risk of developing ards, but did decrease the risk of developing mods, and shortened duration of mechanical ventilation and length of icu stay. [52] antioxidants supplementation and nutritional strategies are now being studied for critically ill patients with early signs of mods, [99] but not specifically for ards prevention. antioxidants and nutrition have also been studied for treatment of ards, and are further discussed in this context in section 3.2.3. given that excessive and protracted inflammation is the overriding principle responsible for the various pathophysiological mechanisms leading to ards, broad and potent anti-inflammatory drugs, such as corticosteroids, would seem to be a rational choice for prevention. four rcts, published between 1985 and 1988, have examined the use of corticosteroids to prevent the onset of ards in patients at risk. a recent meta-analysis of these studies demonstrated that preventive corticosteroids may actually increase the risk of developing ards in critically ill adults. [53] furthermore, the meta-analysis suggested a weakly increased risk of death associated with preventive corticosteroid therapy in those patients who ultimately developed ards. thus, corticosteroid therapy is not recommended for preventing ards in those at risk. corticosteroid therapy has also been extensively studied for the treatment of established disease in the early and late phases, and is discussed further in these contexts (see the corticosteroids subsection of section 3.2.5 and section 3.3.1). platelet-activating factor (paf) is a potent proinflammatory mediator that is degraded by the enzyme paf acetylhydrolase. recombinant human paf acetylhydrolase (rhpaf-ah; epafipase) was studied in a phase iib rct to prevent ards in septic patients. [54] 127 patients with severe sepsis were randomized to receive rhpaf-ah 1 mg/kg, rhpaf-ah 5 mg/kg or placebo. the incidence of ards was not different amongst the three groups, but 28-day all-cause mortality was significantly decreased in the 1 mg/kg treatment group compared with placebo (21% vs 44%; p = 0.03). therefore, although rhpaf-ah does not appear to be an effective prophylactic treatment for ards, it may hold promise for treatment of severe sepsis. the majority of research to date has focused on treating ards once the diagnosis is established. although many studies are designed to treat 'early ards', with randomization occurring within 48 hours of diagnosis, these studies also likely capture many patients in the exudative phase of ards with intra-alveolar hyaline membranes and histological diffuse alveolar damage at the time of enrolment. this problem arises in part because the diagnostic criteria for ards are subjective and lack sensitivity and specificity when compared with pathological diagnosis. [100] thus, timing an intervention at a certain point after 'diagnosis' could result in the patient receiving treatment in the early, mid or even late pathophysiological stage of ali/ards. some more recent studies are now targeting time after intubation rather than time after diagnosis to achieve more uniform timing of intervention. however, since the acute and exudative phases occur along a continuum and are not generally distinguished clinically, therapies targeting these phases will be considered concomitantly. therapies currently under investigation for early and/or exudative ards include those targeting the disrupted surfactant system, oxidative stress and antioxidant activity, neutrophil recruitment and activation, expression and release of inflammatory mediators, activation of the coagulation cascade, and microvascular injury and leak. treatment of the overall inflammatory response with agents such as corticosteroids has also been studied and is discussed. finally, the only drugs specifically targeting resolution of the alveolar oedema of the acute phase are b 2 -adrenergic receptor agonists (b 2 -agonists). clearance of alveolar oedema depends on the balance between oedema formation and reabsorption. the rate of fluid reabsorption depends on the active transport of sodium and electrolytes; water follows in the direction of the transported electrolytes. the active transport of salt and water occurs via epithelial sodium channels induced via na + /k + adenosine triphosphatase (atpase). [101] b 2 -agonists are thought to increase alveolar fluid clearance via two possible mechanisms: (i) increasing the levels of intracellular cyclic adenosine monophosphate, which in turn upregulates na + /k + atpase, causing increased sodium transport across alveolar type ii cells; and (ii) reducing alveolar-capillary permeability, thereby decreasing oedema formation. preliminary animal and ex vivo studies demonstrated the potential of b 2 -agonists to accelerate the rate of alveolar fluid clearance. [102, 103] a small, single-centre rct randomized 40 patients with ali/ards to receive intravenous salbutamol (albuterol) 15 mg/kg/h or placebo for 7 days. [56] the primary endpoint of balti-1 was extravascular lung water measured by the singleindicator transpulmonary thermodilution system (picco ò ; pulsion medical systems) at day 7. patients in the salbutamol group had lower extravascular lung water and plateau pressures, although oxygenation did not differ between the treatment and placebo groups. this latter finding was perhaps due to the vasodilatory effects of b 2 -agonists contributing to shunting of oxygen in the capillary bed. there was no difference in 28-day mortality or ventilator-free days, although the study was not sufficiently powered to detect a difference in these endpoints. [56] funded by the medical research council, the same investigators in the uk are now conducting balti-2, using the same intravenous salbutamol protocol as in balti-1, but powered to detect clinically important outcomes. [104] it will be interesting to determine if the physiological benefits observed in balti-1 confer a reduction in 28-day allcause mortality in balti-2. aerosolized b 2 -agonists have fewer systemic adverse effects than intravenous preparations. the national heart, lung and blood institute (nhlbi), in conjunction with ardsnet, conducted a study of an aerosolized b 2 -agonist, the alta study. [105] the study was stopped for futility at the first interim analysis after enrolling 279 patients. [57] there was no difference in the primary outcome of ventilator-free days to day 28. this study may have been negative for the following reasons: (i) delivery of nebulized drug to lung injury sites may have been suboptimal, as was the case with aerosolized surfactant; and/or (ii) less severely ill patients with ali (rather than ards with more severe hypoxaemia) may retain adequate alveolar fluid clearance without the need for upregulation with b 2 -agonists. sixty-day mortality in the alta study was 19.7% compared with a 28-day mortality of 60% in the severely ill group of patients who received physiological benefit from intravenous salbutamol in balti-1. [106] exogenous surfactant administration has been very successful in treating and preventing neonatal respiratory distress syndrome (nrds). given the physiological and pathological similarities between nrds and ards, exogenous surfactant therapy has been under investigation for treatment of ali/ards for over a decade. although clinical trial results have been largely disappointing, recent studies show promise. the strong scientific rationale for targeting the disrupted surfactant system, as well as lessons learnt from previous trials, therefore merit further attention. endogenous surfactant is composed of 90% lipids (mainly phosphatidylcholine and phosphatidylglycerol) and 10% proteins. the role of endogenous surfactant in the healthy lung is to decrease surface tension and thereby prevent alveolar collapse. in addition, surfactant plays a role in suppressing inflammation and scavenging free oxygen radicals. four apoproteins have been identified, termed surfactant protein (sp)-a, -b, -c and -d. whereas the presence of either or both of the hydrophobic surfactant proteins sp-b and -c are important for the biophysical function of surfactant, the hydrophilic proteins sp-a and -d perform the various host defence roles, including modulation of leukocytes, enhancement of the function of phagocytic cells [107] and regulation of the host's immune system. [108, 109] in ali, disruption of the endogenous surfactant system occurs by a number of mechanisms: injury to alveolar type ii cells results in abnormal synthesis and secretion of surfactant, serum proteins that leak into the airspace interfere with surfactant function, serine endopeptidase and phospholipase a 2 cause degradation of surfactant, and, finally, mechanical ventilation, particularly with high tidal volumes, causes conversion of functional surfactant aggregate forms into dysfunctional forms. without optimal surfactant function, there is high surface tension at the alveolar surface in a non-uniform pattern within the lung leading to alveolar instability and collapse. the presence of bacteria within the airspace may also release and activate endotoxins, a process that is augmented in the presence of an abnormal surfactant system. based on the functional importance of the endogenous surfactant system in the normal lung and, more importantly, the consequences of an altered surfactant system in ali/ards, there is good rationale to consider exogenous surfactant administration as a therapeutic intervention in these patients. [109] in 1996, a phase iii, double-blind rct tested an aerosolized, synthetic surfactant called exosurf ò (glaxo wellcome) in 725 patients with sepsis-induced ards. [58] this study showed no significant difference in overall survival, duration of mechanical ventilation or oxygenation between the treatment groups and standard care. it was postulated that this lack of efficacy was due to a low level of alveolar deposition of the aerosolized preparation and/or due to the absence of surfactant proteins in the preparation. [23] currently, this surfactant preparation is not being evaluated for patients with ali/ards and is no longer marketed in the us. shortly afterwards, a smaller, open-label phase ii clinical trial evaluated tracheal instillation of a liquid bolus of the natural bovine extract surfactant, survanta ò (ross laboratories), in patients with severe ards. [59] there was a trend toward decreased mortality in the group of patients receiving up to four doses of phospholipids 100 mg/kg surfactant compared with the patients in the control group (18.8% vs 43.8%; p = 0.075), and no safety concerns were identified. however, survanta ò contains only very small amounts of sp-b. coupled with concerns regarding resource limitations, no further clinical trials of this exogenous surfactant preparation for adults with ards have been performed. recognizing the importance of surfactantspecific proteins brought progress to clinical surfactant research. in 2004, results were published for two phase iii clinical trials evaluating effect of a liquid, recombinant sp-c (rsp-c) surfactant, venticute ò (nycomed), instilled intratracheally in patients with established ards. [23] the two studies enrolled a total of 448 patients within 24 hours of diagnosis of ards and were powered to show a difference in ventilator-free days. although oxygenation was significantly better during the 24-hour treatment period in the surfactant group, there were no significant differences noted in the number of ventilator-free days or in 28-day survival. [23] a post hoc analysis demonstrated that patients with 'direct' causes of ards (i.e. pneumonia, witnessed aspiration of gastric contents or both) had a mortality benefit with surfactant treatment compared with standard care. a followup meta-analysis pooling results of five multicentre studies of rsp-c confirmed this finding: the subgroup of patients with severe ards due to pneumonia or aspiration had decreased mortality when treated with rsp-c (26.3% vs 39.3% in the usual care group; p = 0.018). [24] subsequently, a prospective phase iii rct evaluating effect of venticute ò in 1200 patients with pneumonia or aspiration of gastric contents was conducted. the study was terminated at 800 patients due to futility. neither these results nor the potential reasons for futility have been published to date. [60] calfactant (infasurf ò , ony inc.) is a modified natural surfactant produced by extracting the phospholipids, neutral lipids and surfactantspecific proteins sp-b and sp-c from newborn calf lungs. in in vivo animal lung studies, calfactant has shown greater surface activity than exosurf ò and survanta ò , [110] [111] [112] [113] and the highest level of resistance to inactivation due to its high ratio of protein sp-b to phospholipids. [114] [115] [116] from 2000 to 2003, calfactant was used in a multicentre study of ali/ards in the paediatric population 1 week (full-term infants) to 21 years of age. overall, calfactant significantly improved oxygenation and reduced mortality (19% vs 36%; p = 0.03), although the greatest impact was observed in the subgroup of patients with direct ali/ards while calfactant had little effect in patients with indirect ali or ards. [61] indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. it is of note, however, that this study differs from other adult studies in that the majority of paediatric patients had direct causes of ards and the most common cause of death was respiratory failure, whereas adult studies have included a larger proportion of patients with indirect causes, such as sepsis, wherein the most common cause of death is multi-organ failure. based on those encouraging results, pneuma pharmaceuticals began conducting a large phase iii multicentre rct of calfactant for direct ards (origin of ards must be infectious pneumonia, aspiration, near drowning, smoke inhalation without pulmonary burn or inhaled industrial gas) in adults and children. a total of 880 patients in two consecutive studies of patients under 12 and over 12 years of age was planned. however, after the first interim analysis in january 2010, the paediatric arm of the study was stopped for futility due to an unexpectedly low mortality rate. recruitment in the adult arm (ages 12-85 years) is continuing as the interim analysis did not reveal futility or any safety concerns (wilson d, university of virginia health sciences center, charlottesville, va, usa, personal communication). [117] since reactive oxygen species also contribute to the tissue damage incurred in ali, antioxidant therapies have also been investigated as therapeutic options for established disease. n-acetylcysteine (nac) is a commercially available antioxidant approved for the treatment of paracetamol (acetaminophen) toxicity. nac is a precursor for glutathione, an antioxidant present in normal lungs and deficient in bronchoalveolar lavage fluid from ali/ards patients. additionally, because of its thiol group, nac can scavenge reactive oxygen species such as hydrogen peroxide and superoxide anion. in an rct of 46 patients, nac and oxothiazolidine carboxylate (procysteine ò , clintec technologies inc.), another glutathione precursor, were studied for their combined effect in ali/ards but failed to reduce mortality compared with placebo, [62] negating promising results of three prior small studies. [63] [64] [65] interestingly, recent evidence suggests that genetic diversity may explain variable responsiveness to nac. glutathione-s-transferases (gsts) are enzymes from a complex, multigene family with important roles in oxidative stress pathways. a study by moradi and co-workers [118] demonstrated that deletion of specific gst gene polymorphisms correlated with mortality and that treatment with nac significantly lowered mortality in these subgroups of patients. these results suggest that patients with gst gene deletions are more vulnerable to oxidative stress contributing to ards and may be in greater need of antioxidant therapy. [118] antioxidant supplementation to enteral nutrition rich in omega-3 fatty acids has also been investigated for patients with ali/ards. while the rationale for nutritional antioxidants such as vitamins e and c is to reduce the oxidative stress present in ali, the purpose of the omega-3 fatty acids is to reduce production of proinflammatory mediators. eicosanoids, such as prostaglandins, thromboxanes and leukotrienes, derived from omega-3 fatty acids are generally much less proinflammatory than those derived from omega-6 fatty acids. since omega-6 fatty acids compete with omega-3 fatty acids for the same rate-limiting enzymes in the production of eicosanoids, diets with a high proportion of omega-6 fats are thought to be proinflammatory and prothrombotic. examples of polyunsaturated omega-3 fatty acids are a-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. [119] a phase ii study enrolling 98 patients with ali compared an antioxidant enteral feeding formula containing eicosapentaenoic acid, g-linolenic acid and antioxidant vitamins with placebo, and observed improved oxygenation, reduced pulmonary inflammation, fewer days of mechanical ventilation and fewer non-pulmonary organ failures in the treatment arm, although there was no difference in mortality between this approach and the control group. [66] ardsnet proceeded to conduct the omega study, a phase iii study examining efficacy of omega-3 and antioxidant supplementation to enteral nutrition. the study was stopped for futility, but results have not yet been published. [67, 68] several therapies aimed at modulating neutrophil activity have been studied. to understand why previous clinical trials have been negative and highlight potential targets for novel therapies, it is important to understand the role of neutrophils in propagating lung injury and mods. polymorphonuclear neutrophils (pmns) form the first line of defence against invading pathogens, and neutropenia or defective neutrophil function predisposes the host to increased morbidity. extensive clinical and experimental data support the role of the activated neutrophil in the pathogenesis of organ injury in sepsis. the lung is particularly vulnerable. postmortem studies of patients with ards show massive pulmonary accumulation of neutrophils, with the highest counts in non-survivors. [120] the pathological impact of neutrophils may be due to their activation, transmigration or delayed apoptosis. however, neutrophil-independent mechanisms of ali must also exist, since ards has been described in neutropenic patients. neutrophil kinetics in the pulmonary circulation differ substantially from that of microvascular beds in the systemic circulation. the pulmonary circulation harbours a large intravascular reservoir of leukocytes, mainly neutrophils, referred to as the 'marginated pool'. [121] this marginated pool may equal or even exceed the pool of circulating neutrophils and exchanges with the latter as an ongoing phenomenon. thus, it is important to appreciate that circulating neutrophils, when isolated for experimental analysis, may not represent the characteristics of the entire population of neutrophils in the bloodstream. intravital microscopic studies have revealed that, in contrast to the systemic circulation where neutrophil sequestration is almost exclusively confined to the venular compartment, the major site of neutrophil retention in the lung is the alveolar capillary bed. [122] neutrophil activation can also lead to cytoskeletal changes that reduce cell deformability and slow their transit time through the alveolar capillaries. since one of the earliest manifestations of ards is accumulation of large numbers of neutrophils in the alveolar capillaries, it is possible that the accumulation of neutrophils may initiate selective capillary blockade and arteriovenous shunting leading to hypoxia seen in ards. activated neutrophils also produce human ne (hne), a protease capable of producing tissue damage by means of its degradation of elastin, fibronectin, laminin, collagen and proteoglycans. normally, protease inhibitors impede ne, but in the setting of an overwhelming inflammatory response, neutrophils generate reactive oxidants that inactivate endogenous protease inhibitors, leaving the activity of hne unchecked. this may lead to increased pulmonary inflammation and endothelial cell permeability. [9] sivelestat (elaspol ò , ono pharmaceuticals) is a competitive inhibitor of ne. it was launched in japan after a phase iii study demonstrated reduced icu stay and improved pulmonary function in patients with ali associated with the systemic inflammatory response syndrome (sirs). [70] however, the strive study [69] was terminated early after randomizing 492 patients from 105 sites in six countries, when the data and safety monitoring board found a trend to increased mortality at 180 days. final analysis revealed no difference in 28-day all-cause mortality (26% in both groups) or number of ventilator-free days between the treatment group and controls. epi-hne-4 or depelestat (debiopharm s.a.) is another hne inhibitor currently under development for treatment of inflammatory pulmonary diseases, including ali. in a repeated lung injury rat model depelestat administration afforded a significant protective effect on lung compliance and alveolar inflammation at day 14 compared with the control group. [71] a phase ii study examining safety and efficacy of intravenous depelestat for patients with ards has been completed, but results have not yet been published. [123] neutrophil transmigration neutrophil margination allows for a molecular interaction between the cell surfaces of the neutrophil and endothelial cell to occur. subsequently, as a consequence of cell surface integrins and their ligands, neutrophils undergo adhesion with endothelial cells. following adherence, neutrophils must pass through the endothelial monolayer, interstitial tissue and alveolar epithelium to reach the alveolar space. passage of large numbers of activated neutrophils can cause epithelial damage, sloughing and increased permeability both due to mechanical force exerted by neutrophil pseudopodia as well as due to release of toxic substances such as proteinases (e.g. elastases, cationic peptides, defensins, oxidants and mmps). [9] while there are conflicting reports on the effects of elastase on increased epithelial permeability, cationic peptides such as defensins can cause both epithelial and endothelial cell injury. defensin levels have been found to be greatly elevated in patients with ards and their levels correlate with the severity of lung injury. [124] neutralizing its effects could be important in the management of ards. ongoing research is examining if defensins can be used to identify patients with ali at an early stage. [125] delayed apoptosis of neutrophils once egressed into the extravascular space, neutrophils cannot return to the circulation and their elimination is dependant upon their clearance by apoptosis and subsequent recognition and elimination by macrophages and other phagocytic cells. normally, neutrophils are terminally differentiated cells with a terminal half-life of 5-6 hours in vivo. upon completion of their lifespan, neutrophils institute a programme of cell death known as 'apoptosis' and are then removed from the circulation by the liver and spleen. apoptosis, as opposed to necrosis, is believed to be crucial for resolution of inflammation as it does not result in loss of cell membrane integrity and bystander tissue damage by release of intracellular enzymes, proteases and reactive oxygen species. [126] expression of neutrophil apoptosis is delayed in ards. [127] this is not an unexpected finding, especially since pmn apoptosis is delayed in other critically ill patients with sepsis, trauma and burns. [128, 129] apoptosis of neutrophils may be an important consequence in determining the extent of lung injury. for example, it has been shown that the induction of neutrophil apoptosis by the administration of dead escherichia coli prior to reperfusion resulted in significant improvement in lung injury. [130] induction of neutrophil apoptosis in the alveolar space has the potential for resolution of inflammation in ards, and can be carried out in a number of ways that could include multiple strategies such as ligation of fas, activation of proapoptotic caspases and modulation of mitogen-activated protein kinases or transcription factors such as nf-kb. hastening neutrophil apoptosis in the alveolar space may also decrease the probability of secondary necrosis and further tissue damage in ards. it is intriguing that no significant differences were found between the expression of neutrophil apoptosis in patients at risk and those with established ards, nor did the extent of apoptotic inhibition correlate with overall outcome in ards. [131] therefore, while it is well established that ards is associated with accumulation of large numbers of neutrophils in alveolar spaces, their contribution to the severity of ards in humans remains uncertain. in summary, targeting neutrophil responses in ards may have therapeutic potential. however, as has been learnt from various ali and sepsis trials in the past, simple strategies to control dysregulated neutrophil behaviour may not be effective. rather, key stages of neutrophil function and kinetics may need to be identified in different clinical phases of ards, and selective immunomodulation strategies may need to be identified for individual patients. in addition to modulation of neutrophil function, there are other facets of the immune and inflammatory response currently under investigation as potential therapeutic targets for treatment of ards. these include modulation of macrophage activity with granulocyte macrophage colony-stimulating factor (gm-csf), inhibition of inflammatory mediators and broad suppression of the inflammatory response with corticosteroids. although most prostaglandins are proinflammatory mediators, prostaglandin e 1 (pge 1 ) has potential beneficial effects in ali, specifically due to its ability to modulate neutrophil activation. however, exogenous pge 1 is associated with several adverse effects and patient intolerance due to haemodynamic instability has been observed. tlc-c-53 (ventusô; the liposome company) is a liposomal dispersion of pge 1 . the development of pge 1 in liposomal form may potentiate its role in neutrophil downregulation, improve peripheral delivery of the drug to the lung and decrease systemic adverse effects, thus providing a good rationale for testing in humans. [73] a phase iii trial of 350 patients with ards randomized to intravenous tlc-c-53 at escalating doses for 7 days versus placebo found no difference in duration of mechanical ventilation or 28-day mortality between the treatment and control groups, although treatment was associated with accelerated improvement in oxygenation. [72] however, more than 50% of patients required a dose reduction due to hypotension or hypoxaemia. interestingly, those patients who tolerated and received at least 85% of the full dose had a shorter duration of mechanical ventilation. a subsequent multicentre phase iii trial of tlc-c-53 in 102 ards patients [73] demonstrated no differences in time to liberation from the ventilator or 28-day mortality; the trend to shorter duration of hypoxaemia in the treatment group failed to reach statistical significance. gm-csf has been shown to stimulate phagocytosis and oxidative functions of host defence neutrophils, monocytes and macrophages. [74] in addition to its systemic actions, gm-csf may also influence pulmonary host defence by modulating alveolar macrophage function and surfactant metabolism. as noted, apoptosis of neutrophils is an important mechanism by which these cells are cleared from inflamed lung regions, thereby facilitating resolution of inflammation. although both granulocyte colony-stimulating factor and gm-csf are thought to inhibit neutrophil apoptosis, in animal models of lung injury, gm-csf has been shown to help restore capillary barrier integrity, [132] preserve alveolar epithelial function and improve alveolar fluid clearance. [133] a pilot study of 45 patients with ards undergoing serial bronchoalveolar lavage found that patients who survived ards had higher concentrations of gm-csf in the bronchoalveolar lavage fluid on day 1 than patients who died. [134] the authors speculated that gm-csf might improve survival by prolonging the neutrophil lifespan in the alveoli and/or inducing proliferation of alveolar macrophages, thereby improving host defence and reducing infectious complications in this setting. in a phase ii trial, molgramostim (schering-plough), a recombinant human gm-csf, was given intravenously at a low dose (3 mg/kg) for 5 days to ten patients with severe sepsis and sepsis-related pulmonary dysfunction (defined as a pao 2 /fio 2 ratio of <287 with a pulmonary infiltrate on chest radiograph). [74] the primary outcome was 30-day survival, and secondary outcomes included oxygenation, occurrence of ards and degree of organ dysfunction at day 5. there was no difference in 30-day survival between the treatment and placebo groups, but oxygenation improved in the gm-csf group. ards was present in four of ten patients in the gm-csf group on study entry, but resolved in two of these patients by day 5, whereas in the placebo group ards was present in three patients on study entry and five patients on day 5. organ dysfunction was similar between the two groups, with no change between study entry and day 5. from july 2004 to july 2009, the nhlbi enrolled patients who had been diagnosed with ali/ards for at least 3 days into a phase ii rct of recombinant gm-csf (sargramostim [leukine ò ], genzyme corporation) versus placebo. [75] the primary outcome was the number of ventilator-free days during days 1-28. secondary outcomes included measures of lung epithelial cell integrity, alveolar macrophage function, changes in severity of respiratory gas exchange, non-respiratory organ failure and incidence of ventilator-associated pneumonia. this study has been completed, but results have not yet been published. [75] cytokine inhibitors cytokines are glycoproteins that act as messengers to cell surface receptors to promote or diminish the inflammatory cascade. specific cytokines are observed in high amounts in the bronchoalveolar lavage fluid of patients with ards, and are thought to play an important role in propagating lung injury. unsaturated phosphatidic acid plays an important role in intracellular signalling leading to neutrophil accumulation within the lungs, as well as proinflammatory cytokine expression and cell membrane oxidation, all of which leads to lung tissue damage. [135] lisofylline (cell therapeutics) is a cytokine inhibitor that impedes synthesis of phosphatidic acid-1a and, therefore, was thought to hold potential for treatment of ards. however, ardsnet stopped a phase ii/iii trial, the larma study, for futility after the first interim analysis failed to demonstrate any difference in 28-day mortality, ventilator-free days, organ failures or levels of circulating free fatty acids. [76] interleukin (il)-8 is another potent chemoattractant for neutrophils, observed in high levels in patients with early ards [136] and associated with increased mortality. [137] anti-il-8 monoclonal antibody has been shown to reduce pulmonary oedema and neutrophil accumulation in animal models of ards [77, 78] but has not yet been tested in humans. finally, tnfa has long been recognized as an important proinflammatory cytokine in ards, but more recent evidence suggests that it actually plays a dichotomous role in both contributing to permeability oedema but also increasing alveolar fluid clearance capacity. monoclonal anti-tnfa antibodies have been tested in patients with sepsis with disappointing results. [138] given its dual role in alveolar oedema formation and resorbtion, a more sophisticated approach than simply blocking all tnfa activity is likely to be required in ards. studies examining the efficacy of corticosteroids for acute exudative ards have shown conflicting results. in 1987, bernard et al. [80] published results of a study of 99 patients with ards randomized to high-dose pulse methylprednisolone (30 mg/kg every 6 hours for 24 hours) or placebo. there was no difference in 45-day mortality (60% vs 63%; p = nonsignificant) but the confidence intervals were wide, suggesting that the study may have been underpowered to detect a small difference in a population with heterogenous outcomes. in 2007, meduri and colleagues [79] published their results of 91 patients with severe early ards (<72 hours) from five hospitals randomized to methylprednisolone 1 mg/kg/day for 28 days versus placebo. they found corticosteroids significantly reduced icu mortality (21% vs 43%; p = 0.03), duration of mechanical ventilation and length of icu stay. [79] annane et al. [81] published a post hoc analysis of 177 ards patients enrolled in an rct of low-dose corticosteroids in septic shock. patients in the treatment group received hydrocortisone 50 mg every 6 hours plus fludrocortisone 50 mg/day for 7 days. although there was no mortality difference for ards patients overall, ards patients with relative adrenal insufficiency and septic shock had significantly reduced mortality when treated with low-dose hydrocortisone (53% vs 75% in the placebo group; p = 0.01). [81] the use of corticosteroids for acute exudative ards remains controversial, although the evidence is more definitive for corticosteroid treatment initiated late for fibroproliferative ards (see section 3.3.1). a study examining low doses of corticosteroids as adjuvant therapy for lung injury associated with h1n1 influenza virus (cortiflu) is planned. [139] 3.2.6 activated protein c microvascular injury and coagulation play critical roles in the pathogenesis of ali. plasma protein c levels are decreased in patients with ali, and are associated with higher mortality and fewer ventilator-free days. [82] recombinant human activated protein c (rhapc; drotrecogin alfa; eli lilly) was tested in a phase iii clinical trial of patients and demonstrated a significant mortality reduction from 30% to 24% in patients with severe sepsis. [140] a phase ii study was sponsored by the nhlbi to determine if drotrecogin alfa increased ventilator-free days in patients with ali (patients with severe sepsis were excluded). the study was terminated by the data safety monitoring board. although drotrecogin alfa significantly increased plasma protein c levels and decreased pulmonary dead space fraction, there was no significant difference in the number of ventilator-free days or in 60-day mortality (5 of 38 vs 5 of 37 patients, respectively; p = 1.0). [82] 3.2.7 hmg-coa reductase inhibitors (statins) hmg-coa reductase inhibitors, commonly known as statins, have recently been proposed as a treatment for ali/ards. the rationale for this is based on animal models suggesting that statins can attenuate organ dysfunction by reducing vascular leak and inflammation. [84] a prospective cohort study in ireland showed a nonsignificant trend towards lower odds of death in ards patients receiving a statin during their icu admission (or 0.27, 95% ci 0.06, 1.21; p = 0.09). [83] however, a recently published retrospective cohort study from the mayo clinic (rochester, mn, usa) showed no difference in mortality or organ dysfunction in ards patients treated with statins. [84] stip is currently enrolling patients admitted to an icu with respiratory distress and a pao 2 /fio 2 ratio <300 due to the h1n1 pandemic strain of influenza. [141] patients in this trial will be randomized to receive rosuvastatin 20 mg/day or placebo for 21 days. since this is a specific subpopulation of patients with ali, findings from this study may not be generalizable to other ali subgroups. the sails trial (also rosuvastatin 20 mg/day vs placebo) is also planned but not yet open for recruitment. [142] patients who survive the early and exudative phases of ards generally enter a period from week 1 to 3 consisting of fibroproliferation and organization of exudative debris within the airspace. this fibroproliferative relatively 'late' phase either slowly resolves or progresses to fibrosis. during this phase, patients are at risk of dying from other complications such as mods, or may fail to wean from mechanical ventilation due to severely impaired respiratory muscle and lung function. those who successfully wean off mechanical ventilation may have residual pulmonary fibrosis and reduced exercise capacity. for resolution to occur, removal of inflammatory cells, cellular debris, and soluble and insoluble proteins needs to take place. as noted in section 3.2.4, apoptosis of neutrophils facilitates resolution of inflammation. monocyte and macrophage phagocytic clearance of apoptotic cells appears to be an important mechanism by which neutrophils are cleared from inflamed lung regions. soluble proteins are likely to be primarily removed via paracellular diffusion, but removal of insoluble proteins appears to depend on the function of alveolar macrophages. mechanisms involved in remodelling of hyaline membranes and restoration of a functional alveolar-capillary barrier are incompletely understood at present, but therapeutic interventions aimed at modulation of phagocytosis/apoptosis are being evaluated. to date, far less research has targeted this later phase of the disease, as most trials have focused on earlier preventative processes. fibroproliferative ards is characterized by ongoing inflammation. in addition to being tested for prevention of ards, and treatment of the early and mid exudative phases, corticosteroids have also been tested for efficacy in reversing the fibrosing alveolitis of the late phase of ards. a study by meduri and colleagues [86] examined the effect of prolonged methylprednisolone therapy (2 mg/kg/day for 32 days) on 24 patients with severe ards that was unresolved after 7 days of respiratory failure. although this study demonstrated a significant hospital mortality benefit (2 of 16 patients [12%] in the corticosteroid group died vs 5 of 8 [62%] in the placebo group), the significance of these findings was controversial for two reasons: the calculated sample size to demonstrate a 30% absolute difference in mortality was 99 patients but the study was terminated early after enrolment of 24 patients, and the mortality in the placebo group was slightly higher than anticipated. [86] to shed further light on this issue, ardsnet specifically designed a study to focus on the late fibrotic stage of the disease, called lasrs. [85] this study examined the role of corticosteroids in 180 patients in the late phase (>7 days from onset) of persistent ards. methylprednisolone, dosed at 2 mg/kg/day for 14 days followed by tapering doses until day 25, was compared with placebo. there was no difference in 60-or 180-day mortality rates. methylprednisolone improved oxygenation, respiratory system compliance and blood pressure, resulting in an increased number of ventilator-free and shock-free days; however, a higher rate of neuromuscular weakness and, if initiated more than 14 days after the onset of ards, a significant increased mortality was observed in the methylprednisolone group. therefore, despite the improvement in cardiopulmonary physiology, methylprednisolone does not improve overall mortality in ards and is not recommended for treatment of late ards. given these results, the convincing lack of efficacy for prevention of ali prior to diagnosis and the lack of evidence of benefit in the early phase, corticosteroids cannot be recommended for routine treatment of ali/ards at any stage, at this time. furthermore, it may prove to be exceedingly difficult to determine which individual patient might benefit from corticosteroids and at what specific point to intervene. clearly, the current status of treatment options for patients with ali/ards is suboptimal. at this time, the clinical management of patients with ali/ards involves supportive therapy only. this primarily includes low stretch or 'lung protective' mechanical ventilation, conservative fluid management and adequate nutritional support. although the term 'supportive' may sound somewhat discouraging, these are important observations, not only because they impact on the outcome of patients with ali/ards but also because they should be embraced and implemented as 'standard care' for this patient population. furthermore, any new therapy being tested should be compared with optimal 'standard care'. other methods proposed to offer greater protection to the lungs while providing mechanical support to respiration include hfo and ecmo. studies into these modes are ongoing. although supportive therapies have reduced mortality, there is still significant need for improvements. previous studies have provided important insight into the pathophysiology of ali/ards. research is ongoing into therapies to prevent ali/ards in those at risk, treat it early in its course or aid in its resolution. each of these goals is associated with specific challenges. demonstrating that a prophylactic intervention reduces mortality, morbidity and is cost effective is challenging at best. this is most likely to occur when the risk of acquiring the disease is high, the outcome of the disease is uniformly devastating and treatment for the disease is nonexistent. for some critically ill patients at risk for ards, this may be the case. however, the diagnosis of ali/ards encompasses a very heterogeneous population, with incompletely understood risk factors and non-uniform, diverse outcomes. the greatest likelihood of success for prophylactic therapy will come when we have further delineated the subgroups at highest risk of dying from ali/ards and have accurate diagnostic tests to identify these patients. for ali/ards, specifically targeting the pathogenic mechanisms responsible for the increased risk of death in these patient subgroups would theoretically be high yield. basic science research identifying genetic polymorphisms of patients with highest mortality or greatest need for specific therapies shows great promise in this regard, but is not yet clinically applicable. until then, validating biomarkers and clinical indicators for poor prognosis in ali/ards should remain a primary research goal. finding therapies to treat ards in its late fibroproliferative phase is also in great need. too often patients survive the early and mid phase of ards only to succumb to complications in the late phase or undergo withdrawal of life support as they are unable to be weaned from mechanical ventilation. research into mechanisms of idiopathic pulmonary fibrosis may help identify common pathways to target for therapy. to date, the majority of research has focused on treating ali/ards in its earlier stages, in the hope that the disease process may be reversed prior to the patient entering the fibroproliferative phase. progress in finding therapies to treat established ards has been slow and hampered by a long series of negative clinical trials. however, there are several lessons to be learned from these rcts. first, a 'one-size-fits-all' approach has not worked for pharmacotherapy for ards. in this sense, the syndrome of ali/ards may be likened to cancer. cancer as a broad term signifies the uncontrolled replication of abnormal cells, but there are specific chemotherapeutic treatments for specific types of cancer, depending on its origin. some treatments may be effective for more than one type of cancer, but not for other types, and the magnitude of the benefit might vary according to the type and stage of disease. oncologists would not design a trial enrolling all patients with differing types of cancer and expect to find a single drug that shows a survival benefit. yet, that is what has been attempted with several large ards trials. recent studies have demonstrated that direct ards is likely to respond differently than indirect ards, and in fact within these broad categories, pathogenesis may differ. therefore, different therapies may need to be developed for specific aetiologies such as sepsis-related ards, sirs-related ards and various direct causes of ards. second, a well designed negative rct does not necessarily mean that the therapy tested should be abandoned. it means that the therapy is likely to not be appropriate for widespread application. however, just because a drug does not work for every ards patient does not necessarily mean it should not be used for anyone with ards. for example, there is no evidence for treating all patients with acute ards with corticosteroids, but there is evidence that treating ards patients with relative adrenal insufficiency and septic shock with low doses of hydrocortisone is likely to be beneficial. similarly, nitric oxide should not routinely be applied to all patients with ali/ards, but may be useful in refractory hypoxaemia, particularly in conjunction with other ventilation rescue strategies. third, a negative rct should potentially lead to further research so that we can gain further insight as to why the therapy failed to yield a clinical benefit. thomas edison, when asked why he pursued his quest to invent a functional and practical light bulb after innumerable failed attempts, is reported to have replied, ''i have not failed. i've just found 10 000 ways that won't work''. ards research should take us from bench to bedside and back to the bench again. basic science can help us understand basic mechanisms of disease, discover why a therapy failed, then provide new ideas to apply to the clinical realm. rcts are necessary to prove benefit and quantify risk prior to changing clinical practice. since we are in urgent need of therapies to treat ali/ ards, it is necessary that rcts continue to advance our clinical care. however, these rcts need to be well founded in basic biology and physiology research, and focused on specific hypotheses regarding mechanisms of disease. continuing to conduct large clinical trials on heterogeneous patients with ali/ards from multiple aetiologies will not only prove ineffective but also add enormous cost to the healthcare system. the most significant and promising finding from an rct to date is that calfactant, the natural bovine surfactant rich in sp-b and -c, reduces mortality in ali from 36% in the control arm to 19% in the paediatric population. indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. the ongoing cards study will attempt to reproduce that finding in adults with direct causes of ards. [117] this trial is continuing enrolment after the first interim analysis, with a target completion date of march 2011. great gains have been made in providing supportive management to patients with ali/ards. ongoing and future research efforts will provide important insights into the complex pathophysiologies involved and may provide further rationale for patient-specific therapies and/or combination therapies targeting the various mechanisms contributing to this disorder. understanding who is at greatest risk of succumbing to ali/ards and establishing the optimal time to intervene will be essential to improving mortality for this syndrome. epidemiology and outcome of acute lung injury in european intensive care units: results from the alive study incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three australian states characteristics and outcomes in adult patients receiving mechanical ventilation: a 28-day international study a simple clinical predictive index for objective estimates of mortality in acute lung injury the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome emerging therapies for treatment of acute lung injury and acute respiratory distress syndrome the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination transepithelial migration of neutrophils: mechanisms and implications for acute lung injury arachidonic acid remodeling in human inflammatory cells migrating to the lung in vivo recent advances in genetic predisposition to clinical acute lung injury chronic alcohol abuse is associated with an increased incidence of acute respiratory distress syndrome and severity of multiple organ dysfunction in patients with septic shock pharmacotherapy of acute lung injury and acute respiratory distress syndrome injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome does programmed cell death (apoptosis) play a role in the development of multiple organ dysfunction in critically ill patients? a review and a theoretical framework an overview of mortality risk prediction in sepsis multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome a multicenter registry of patients with acute respiratory distress syndrome: physiology and outcome adult respiratory distress syndrome: risk with common predispositions causes of mortality in patients with the adult respiratory distress syndrome improved survival of patients with acute respiratory distress syndrome (ards): 1983-1993 about the ards network effect of recombinant surfactant protein c-based surfactant on the acute respiratory distress syndrome a search for subgroups of patients with ards who may benefit from surfactant replacement therapy the oscillation for ards treated early (oscillate) trial pilot study review of a large clinical series: association of cumulative fluid balance on outcome in acute lung injury. a retrospective review of the ardsnet tidal volume study cohort pulmonaryartery versus central venous catheter to guide treatment of acute lung injury1 the use of an inflammation-modulating diet in patients with acute lung injury or acute respiratory distress syndrome: a metaanalysis of outcome data efficacy of an expanded ventilator bundle for the reduction of ventilatorassociated pneumonia in the medical intensive care unit continuous aspiration of subglottic secretions in the prevention of ventilatorassociated pneumonia in the postoperative period of major heart surgery oral decontamination for prevention of pneumonia in mechanically ventilated adults: systematic review and meta-analysis supine body position as a risk factor for nosocomial pneumonia in mechanically ventilated patients: a randomised trial thromboprophylaxis in medicalsurgical critically ill patients stress ulcer prophylaxis in critically ill patients: resolving discordant meta-analyses efficacy and safety of a paired sedation and ventilator weaning protocol for mechanically ventilated patients in intensive care (awakening and breathing controlled trial): a randomised controlled trial effects of physical training on functional status in patients with prolonged mechanical ventilation recent trends in acute lung injury mortality: 1996-2005 effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and meta-analysis effect of prone positioning in patients with acute respiratory distress syndrome: a metaanalysis effect of mechanical ventilation in the prone position on clinical outcomes in patients with acute hypoxemic respiratory failure: a systematic review and meta-analysis1 hemodynamic and gas exchange response to inhaled nitric oxide and prone positioning in acute respiratory distress syndrome patients high-frequency oscillatory ventilation in adults: the toronto experience high-frequency oscillatory ventilation for adult patients with ards efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial extracorporeal membrane oxygenation for 2009 influenza a (h1n1) acute respiratory distress syndrome ketoconazole prevents acute respiratory failure in critically ill surgical patients a double-blind, prospective, randomized trial of ketoconazole, a thromboxane synthetase inhibitor, in the prophylaxis of the adult respiratory distress syndrome development, implementation, and evaluation of a ketoconazole practice guideline for ards prophylaxis angiotensin-converting enzyme inhibitor captopril prevents oleic acid-induced severe acute lung injury in rats rosiglitazone attenuates the severity of sodium taurocholate-induced acute pancreatitis and pancreatitis-associated lung injury chemically modified tetracycline prevents the development of septic shock and acute respiratory distress syndrome in a clinically applicable porcine model randomized, prospective trial of antioxidant supplementation in critically ill surgical patients corticosteroids in the prevention and treatment of acute respiratory distress syndrome (ards) in adults: meta-analysis recombinant platelet-activating factor acetylhydrolase to prevent acute respiratory distress syndrome and mortality in severe sepsis: phase iib, multicenter, randomized, placebo-controlled, clinical trial ketoconazole for early treatment of acute lung injury and acute respiratory distress syndrome: a randomized controlled trial the beta-agonist lung injury trial (balti): a randomized placebo-controlled clinical trial randomized, placebo-controlled trial of an aerosolized beta-2 adrenergic agonist (albuterol) for the treatment of acute lung injury aerosolized surfactant in adults with sepsis-induced acute respiratory distress syndrome: exosurf acute respiratory distress syndrome sepsis study group bovine surfactant therapy for patients with acute respiratory distress syndrome venticute in patients with pneumonia or aspiration of gastric contents and intubation/ventilation/oxygenation impairment (by2001/m1-007) [clinicaltrials.gov identifier nct00074906 effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial a trial of antioxidants n-acetylcysteine and procysteine in ards: the antioxidant in ards study group n-acetylcysteine enhances recovery from acute lung injury in man: a randomized, double-blind, placebo-controlled clinical study glutathione (gsh) repletion by n-acetylcysteine (nac) in patients with the adult respiratory distress syndrome (ards) antioxidant treatment with n-acetylcysteine during adult respiratory distress syndrome: a prospective, randomized, placebocontrolled study effect of enteral feeding with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome: enteral nutrition in ards study group omega study: information update early versus delayed enteral feeding and omega-3 fatty acid/antioxidant supplementation for treating people with acute lung injuryj or acute respiratory distress syndrome (the eden-omega study neutrophil elastase inhibition in acute lung injury: results of the strive study a phase iii clinical study of neutrophil elastase inhibitor ono-5046 na in sirs patients beneficial effect of an inhibitor of leukocyte elastase (epi-hne-4) in presence of repeated lung injuries liposomal prostaglandin e1 (tlc c-53) in acute respiratory distress syndrome: a controlled, randomized, double-blind, multicenter clinical trial. tlc c-53 ards study group a multi-centre, doubleblind, placebo-controlled study of liposomal prostaglandin e1 (tlc c-53) in patients with acute respiratory distress syndrome a randomized phase ii trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction a randomized trial of gm-csf in patients with ali/ards [clinicaltrials.gov identifier nct00201409 national institutes of health. randomized, placebo-controlled trial of lisofylline for early treatment of acute lung injury and acute respiratory distress syndrome acid aspiration-induced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms humanized monoclonal antibody against the chemokine cxcl-8 (il-8) effectively prevents acute lung injury methylprednisolone infusion in early severe ards: results of a randomized controlled trial high-dose corticosteroids in patients with the adult respiratory distress syndrome effect of low doses of corticosteroids in septic shock patients with or without early acute respiratory distress syndrome randomized clinical trial of activated protein c for the treatment of acute lung injury acute lung injury and the acute respiratory distress syndrome in ireland: a prospective audit of epidemiology and management statin administration did not influence the progression of lung injury or associated organ failures in a cohort of patients with acute lung injury efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial prostacyclin and thromboxane a2 formation is increased in human sepsis syndrome: effects of cyclooxygenase inhibition angiotensin-converting enzyme 2 protects from severe acute lung injury angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome chronic use of angiotensin pathway inhibitors is associated with a decreased risk of acute respiratory distress syndrome use of ace inhibitors and development and outcome in ards the peroxisome proliferator-activated receptor-g is a negative regulator of macrophage activation ppar-gamma agonists inhibit production of monocyte inflammatory cytokines a novel therapy for colitis utilizing ppar-gamma ligands to inhibit the epithelial inflammatory response ppar y ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice thiazolidinediones in type 2 diabetes: a cardiology perspective long-term risk of cardiovascular events with rosiglitazone: a meta-analysis trial of glutamine and antioxidant supplementation in critically ill patients (redoxs) [clinicaltrials.gov identifier nct00133978 comparison of clinical criteria for the acute respiratory distress syndrome with autopsy findings lung epithelial fluid transport and the resolution of pulmonary edema alveolar fluid clearance in the resected human lung upregulation of alveolar epithelial active na+ transport is dependent on beta2-adrenergic receptor signaling balti-2 (beta agonist lung injury trial-2) drug study of albuterol to treat acute lung injury (alta) [clinicaltrials.gov identifier nct00434993]. us national institutes of health, clinicaltrials.gov a role for b2 agonists in ards: the question remains unanswered surfactant proteins a and d and pulmonary host defense the future of surfactant therapy during ali/ards the role of exogenous surfactant in the treatment of acute lung injury a mutation in the surfactant protein b gene responsible for fatal neonatal respiratory disease in multiple kindreds target disruption of the surfactant protein b gene disrupts surfactant hemeostasis, causing respiratory failure in newborn mice differential activity and lack of synergy of lung surfactant proteins sp b and sp c in interactions with phospholipids surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants is outcome from ards related to the severity of respiratory failure? calfactant for direct acute respiratory distress syndrome (cards) the role of glutathione-s-transferase polymorphisms on clinical outcome of ali/ards patient treated with n-acetylcysteine enteral omega-3 and acute respiratory distress syndrome evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome the marginated pool physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries safety and efficacy study of depelestat in acute respiratory distress syndrome (ards) patients [clinicaltrials.gov identifier nct00455767 high concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome biological markers to identify early sepsis and acute lung injury programmed cell death (apoptosis) and the resolution of systemic inflammation the role of apoptosis in acute lung injury delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity inhibition of apoptosis in polymorphonuclear neutrophils from burn patients a novel therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo neutrophil apoptosis in the acute respiratory distress syndrome granulocyte/ macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis regulators of endothelial and epithelial barrier integrity and function in acute lung injury low doses corticosteroids as adjuvant therapy for the treatment of severe h1n1 flu (cortiflu) safety and dose relationship of recombinant human activated protein c for coagulopathy in severe sepsis us national institutes of health, clinicaltrials.gov us national institutes of health, clinicaltrials.gov assistant professor, department of medicine, university hospital, 339 windermere road the authors thank jeanette mikulic for her assistance with preparation of the manuscript. no sources of funding were used to assist in the preparation of this review. the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord-308071-1bk3xuwf authors: lang, christian; jaksch, peter; hoda, mir alireza; lang, györgy; staudinger, thomas; tschernko, edda; zapletal, bernhard; geleff, silvana; prosch, helmut; gawish, riem; knapp, sylvia; robak, oliver; thalhammer, florian; indra, alexander; koestenberger, markus; strassl, robert; klikovits, thomas; ali, kamran; fischer, gottfried; klepetko, walter; hoetzenecker, konrad; schellongowski, peter title: lung transplantation for covid-19-associated acute respiratory distress syndrome in a pcr-positive patient date: 2020-08-25 journal: lancet respir med doi: 10.1016/s2213-2600(20)30361-1 sha: doc_id: 308071 cord_uid: 1bk3xuwf nan most patients with covid-19 have a mild or asymptomatic disease course; however, about 10% require admission to an intensive care unit (icu) because of acute respiratory distress syndrome (ards). 1, 2 mortality rates of up to 60% have been reported for this subgroup. 3, 4 lung transplantation remains the ultimate treatment option for various chronic end-stage lung diseases. in addition, it can be considered as a salvage therapy for carefully selected patients who have severe treatment-refractory ards. 5 however, wide uncertainty exists as to whether lung transplantation could have a place in the treatment of severe covid-19 and, if so, what the optimal timing for such a treatment should be. herein, we report the first case of lung transplantation for a patient with a persistently positive severe acute respiratory syndrome coronavirus 2 (sars-cov-2) real-time rt-pcr test result. on march 21, 2020, at a time when the first covid-19 cases were reported in austria, a 44-year-old woman was admitted to klinikum klagenfurt am wörthersee (klagenfurt, austria) with symptoms of fever and cough, and tested positive for sars-cov-2 by nasopharyngeal swab real-time rt-pcr on the same day (day 0). her medical history was unremarkable, except that she had mild psoriatic arthritis, which did not require any systemic treatment, and a diagnosis of idiopathic cd4 lymphocytopenia without any clinical relevance. on day 6 after the first positive sars-cov-2 test, the patient's respiratory condition worsened, requiring transfer to the icu and subsequent intubation. therapy was further escalated by femorofemoral venovenous extracorporeal membrane oxygenation (ecmo), initiated on day 13. administration of immunoglobulins, tocilizumab, and lopinavir, and use of prone positioning did not improve the patient's condition. on day 20, the patient developed spontaneous bleeding from the right thoracic cavity, which required surgical opening of her chest and haematoma evacuation. a small, self-limiting intracerebral bleed in the left temporal lobe did not require any intervention. during this episode of bleeding, the patient required multiple transfusions. a final treatment attempt with convalescent plasma therapy (day 32) was initiated, which offered no benefit, and the patient was subsequently transferred to the medical university of vienna (vienna, austria) on day 48 as a possible candidate for lung transplantation. on arrival, the patient required moderate doses of vasopressors (0·3 µg/kg per min norepinephrine), presented with normal kidney function, and had a bilirubin level of 48 µmol/l. partial pressure of arterial oxygen was about 70 mm hg while ventilated with airway pressure release ventilation of 25 mbar over 8 mbar positive end-expiratory pressure and a fractional concentration of oxygen in inspired air of 1·0. with these settings, her tidal volume was only around 50 ml and she was fully dependent on the venovenous ecmo support (blood flow 3·8 l/min and sweep gas flow 6 l/min). subsequent reduction of the sedation levels led to spontaneous opening of her eyes. when turning the patient, repeated episodes of desaturation accompanied by bradycardia occurred and the ecmo cannulation had to be changed to a femorojugular configuration using larger cannulas (25f/21f). the changes to the cannulation resulted in stabilisation of the patient; however, ecmo run with regular physiotherapy while the patient was awake to avoid further deconditioning, as previously described in the setting of bridge to transplant, 6, 7 was not feasible. 6,7 a complete investigation was initiated to consider the possibility of lung transplantation. pulmonary ct angiography revealed complete consolidation of the lungs with large necrotic areas and air inclusions, raising high suspicion of bacterial superinfection. additionally, there was almost no perfusion in large parts of the lower lobes, suggesting extensive infarctions of the peripheral parts of the lung parenchyma and thrombosis of small and medium-sized pulmonary arteries (figure 1). ct of the cns showed a reduction in the size of the lesion corresponding to the previously reported small left-temporal haemorrhage. on day 52, an increase of acute-phase proteins and a positive candida albicans blood culture were noted. furthermore, bilirubin increased to 170 µmol/l, so a superinfection of the necrotic lung with subsequent sepsis-associated liver dysfunction was assumed (appendix p 4). during the whole course of treatment from day 0, rt-pcr testing for sars-cov-2 was repeat edly positive, both in nasopharyngeal swabs and in bronchoalveolar lavage samples (figure 2). however, real-time rt-pcr cycle threshold (ct) values were exceptionally high, with some values greater than 33, suggesting that these positive results could have derived from nucleic acid segments of residual virus without actual infectivity. 8 to better define the presence of an active sars-cov-2 infection, vero cell cultures were used, 9 which turned out to be negative after 7 days of culture (three passages). analyses for pre-existing antibodies showed high levels of class i and class ii hla antibodies, probably due to the previous administration of multiple blood products, including convalescent plasma. cytological analysis of bronchoalveolar lavage fluid exposed predominantly necrotic material, and flow cytometric analysis showed the absence of alveolar macrophages but high abundance of immature neutrophils and cell debris (appendix p 2). these findings further corroborated the profound damage to the lungs. on the basis of all these examinations, a comprehensive interdisciplinary discussion on the direction of treatment was held on day 52, which resulted in a consensus that the lungs of the patient had no potential for recovery. consequently, the alternatives of terminating the treatment or proceeding to lung transplantation were discussed, which resulted in the final decision in favour of transplantation. this decision was based on the following considerations: (1) virus culture was negative and real-time rt-pcr ct values were high; (2) it was more than 5 weeks since the start of the sars-cov-2 infection; (3) no alternative treatment options were available; (4) the case was a single-organ failure in a young patient; (5) it was a preseptic condition originating from the lungs; and (6) there were no other obvious barriers for long-term recovery. as the patient could not consent to the procedure, next-of-kin consent was obtained from her spouse. she was put on the waiting list on day 52 and was granted a lung allocation score of 49·3. daily immunoabsorption therapy was initiated, with the aim of achieving desensitisation of the patient (ie, removal of preformed antibodies from the circulation) before transplantation (appendix p 4). on day 58, a suitable donor organ became available and a sequential bilateral lung transplantation was performed. intraoperatively, a central venoarterial ecmo circuit was installed and the venovenous ecmo was kept running in parallel with a reduced flow of 1 l/min. without clear anatomical borders, only extrapleural mobilisation of the lungs was possible. the implantation was equally challenging because of the remarkably fragile tissue quality of the recipient's bronchus and vessels, most likely due to a spread of the infectious process (ie, inflammatory changes resulting from the previous infection) to these tissues. meticulous haemostasis was performed; nevertheless, a total of 30 units of packed red blood cells and five units of platelets were required to establish coagulation. at the end of the procedure, signs of incipient reperfusion oedema were observed, so the central venoarterial ecmo support was switched to prolonged peripheral femorofemoral venoarterial ecmo and venovenous ecmo was removed. 10 the patient was transferred to the icu in a stable condition and was put into prone position to relieve the lower lobes of the lungs. reopening of the chest was indicated on postoperative day 1 for haematoma evacuation. thereafter, the patient quickly recovered, the venoarterial ecmo system could be removed on postoperative day 3 and primary graft dysfunction grading at 72 h was 0. standard triple immunosuppression was initiated, including tacrolimus, mycophenolate mofetil, and steroids. as the patient was highly presensitised, six additional treatment cycles of immunoabsorption were done and antithymocyte globulin was administered. as expected, the crossmatch between donor and recipient was positive; however, in serial blood samples taken after transplantation, class ii donor-specific antibodies were substantially reduced and class i donorspecific antibodies were completely absent (appendix p 3). similar to most recipients bridged to their transplant for a long time, the further postoperative course of the patient was characterised by slow recovery. from day 72 after the initial positive sars-cov-2 test (postoperative day 14), the patient was regularly mobilised to sitting at the edge of the bed, and on day 83 (postoperative day 25), the patient was able to speak via a tracheal multifunction cannula. she was able to be transferred to a non-icu ward on day 121 (postoperative day 63), was able to walk with some assistance, and was recovering from neuromuscular deconditioning. at this stage, no functional impairments related to the cerebral bleeding were apparent. cellular analysis of bronchoalveolar lavage samples confirmed regular recovery with mainly viable cells, an increasing abundance of alveolar notably, real-time rt-pcr for sars-cov-2 was performed regularly after the transplantation and remained positive in samples from nasopharyngeal swabs and bronchoalveolar lavage until postoperative day 10, but was negative thereafter. in addition, a second vero cell culture from a bronchoalveolar lavage sample retrieved on postoperative day 7 was negative and thus confirmed absence of infectivity in the patient. pathological examination of the explanted lungs showed large zones of necrosis almost completely taking up both lower lobes and large areas of the upper lobes. a large proportion of alveoli were destroyed and replaced by granulation tissue, corresponding to massive diffuse alveolar damage. throughout all lobes, remnants of substantial widespread thromboembolism were present (figure 1). to our knowledge, available evidence for lung transplant ation in covid-19 is limited to two preliminary reports from china, suggesting that this treatment might be an option for sars-cov-2 pcr-negative patients. 11, 12 the case we present here extends the reports from china by showing that lung transplantation can be done in patients with positive rt-pcr results, provided that vero cell cultures confirm non-infectivity. of the cases reported so far through the scientific literature and the media, ours seems to represent the first successful lung transplantation of a patient with covid-19 outside of china. according to who guidelines, laboratory confirmation of sars-cov-2 is defined as positive rt-pcr of a nasal or pharyngeal swab. 13 however, an increasing body of evidence shows that pcr positivity can persist for several days or even weeks beyond virus infectivity. 14 a study by bullard and colleagues 9 showed that sars-cov-2 infectivity was restricted mainly to patients with a duration of symptoms shorter than 8 days and low rt-pcr ct values (<24). although vero cell cultures are not yet universally available, they are considered the gold standard to establish virus infectivity. at day 144, the patient remained well. despite the success of this case, it is important to emphasise that lung transplantation is an option for only a small proportion of patients with covid-19. many patients with covid-19 who are admitted to the icu are older than the acceptable age limit for the procedure or have other comorbidities that might preclude them from lung transplantation. furthermore, it has been shown that even patients with severe covid-19-related ards have some potential to recover. 15 as donor organs are a scarce resource, it is important to consider lung transplantation only for patients with irreversibly damaged lungs. in our case, a complete absence of pulmonary gas exchange after 5 weeks of venovenous ecmo, extensive signs of necrosis, and thrombotic occlusions of peripheral vessels on ct angiography, as well as highly abnormal bronchoalveolar lavage cytology, suggested that the patient was a good candidate for transplantation. this judgment was later confirmed by pathological examination of the explanted lungs. the critical status of a patient with such extensive lung damage, the permanent risk of bacterial superinfection, and the imminent muscle loss are strong arguments to consider lung transplantation early, certainly before an already complex situation becomes completely unmanage able. in our opinion, it is therefore highly unlikely that a strategy of rehabilitation on ecmo first and lung transplantation at a later point would have resulted in an acceptable clinical outcome. furthermore, it is important to make full use of such short-lived opportunities for critically ill patients, and sars-cov-2 rt-pcr positivity alone should not exclude them from lung transplantation when it is the only remaining potentially successful therapy. this case report shows that lung transplantation should be added to the armamentarium of therapies for patients with covid-19-related ards. the criteria applied herein for patient selection and timing of lung transplantation need to be validated in future studies. clinical features of patients infected with 2019 novel coronavirus in wuhan, china covid-19 and italy: what next? clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study characteristics and outcomes of 21 critically ill patients with covid-19 in washington state lung transplantation as a therapeutic option in acute respiratory distress syndrome twenty-year experience with extracorporeal life support as bridge to lung transplantation extracorporeal life support as a bridge to lung transplantation-experience of a high-volume transplant center temporal dynamics in viral shedding and transmissibility of covid-19 predicting infectious sars-cov-2 from diagnostic samples bilateral lung transplantation on intraoperative extracorporeal membrane oxygenator: an observational study lung transplantation as therapeutic option in acute respiratory distress syndrome for coronavirus disease 2019-related pulmonary fibrosis lung transplantation for elderly patients with end-stage covid-19 pneumonia covid-19: why test? who to test? how to test? virological assessment of hospitalized patients with covid-2019 extracorporeal membrane oxygenation for critically ill patients with covid-19-related acute respiratory distress syndrome: worth the effort? cl, pj, ps, wk, and kh drafted the report. mah, gl, tk, ka, ts, et, bz, or, and mk were involved in the treatment of the patient. sg, hp, rg, and sk acquired, analysed, and interpreted pathology specimens, radiology images, or cytology specimens. ft, ai, and rs analysed and interpreted virology data. gf interpreted donor-specific antibody tests. all authors were responsible for critical revision of the manuscript and approved the final version before submission. we declare no competing interests. key: cord-317993-012hx4kc authors: movia, dania; prina-mello, adriele title: preclinical development of orally inhaled drugs (oids)—are animal models predictive or shall we move towards in vitro non-animal models? date: 2020-07-24 journal: animals (basel) doi: 10.3390/ani10081259 sha: doc_id: 317993 cord_uid: 012hx4kc simple summary: this commentary focuses on the methods currently available to test the efficacy and safety of new orally inhaled drugs for the treatment of uncurable respiratory diseases, such as chronic obstructive pulmonary disease (copd), cystic fibrosis or lung cancer, prior to entering human experimentation. the key question that the authors try to address in this manuscript is whether there is value in using and refining current animal models for this pre-clinical testing, or whether these should be relinquished in favor of new, more human-relevant non-animal methods. abstract: respiratory diseases constitute a huge burden in our society, and the global respiratory drug market currently grows at an annual rate between 4% and 6%. inhalation is the preferred administration method for treating respiratory diseases, as it: (i) delivers the drug directly at the site of action, resulting in a rapid onset; (ii) is painless, thus improving patients’ compliance; and (iii) avoids first-pass metabolism reducing systemic side effects. inhalation occurs through the mouth, with the drug generally exerting its therapeutic action in the lungs. in the most recent years, orally inhaled drugs (oids) have found application also in the treatment of systemic diseases. oids development, however, currently suffers of an overall attrition rate of around 70%, meaning that seven out of 10 new drug candidates fail to reach the clinic. our commentary focuses on the reasons behind the poor oids translation into clinical products for the treatment of respiratory and systemic diseases, with particular emphasis on the parameters affecting the predictive value of animal preclinical tests. we then review the current advances in overcoming the limitation of animal animal-based studies through the development and adoption of in vitro, cell-based new approach methodologies (nams). respiratory diseases constitute a huge burden in our society. it has been calculated that, worldwide, around 235 million people are living with asthma [1], 251 million with chronic obstructive pulmonary disease (copd) [2] , and more than 70,000 people with cystic fibrosis [3] . furthermore, 3 million people are affected by idiopathic pulmonary fibrosis (ipf) [4] , and 10 million people contract tuberculosis (tb) annually [5] . in addition to this, lung cancer continues to be the leading cause of cancer death worldwide, accounting for 1.8 million deaths in 2018 [6] ; whereas, pneumonia still constitutes the inhalation is the preferred administration method for treating respiratory diseases [13] , as: (i) it delivers the drug directly at the site of action, resulting in a rapid therapeutic onset with considerably lower drug doses, (ii) it is painless and minimally invasive thus improving patients' compliance, and (iii) it avoids first-pass metabolism, providing optimal pharmacokinetic conditions for drug absorption and reducing systemic side effects [14] [15] [16] . it should be noted here, inhalation differs from intranasal administration for the drug portal-of-entry (poe) and targeted site of action. intranasal drugs are sprayed into the nostrils, producing a local effect in the nasal mucosa; whereas, inhalation occurs through the mouth, with the oids, also referred to as orally inhaled drug products (oips), having their efficacy in the lungs. notably, attempts have been made to develop oids that exert their therapeutic action outside the lung, for the treatment of systemic diseases [17] . the latter include, for example, migraine headaches, treated with aerosols of ergotamine or hydroxyergotamine, and type 1/type 2 diabetes, for which inhaled insulin products have been developed (e.g., exubera-withdrawn in 2008 due to poor revenue-and afrezza-the uptake of which has also been impacted by socio-economic issues). oid therapeutic categories currently approved for the clinical treatment of respiratory diseases include drugs for the treatment of asthma and copd, such as β2 adrenergic agonists (e.g., albuterol, formoterol) and muscarinic antagonists (e.g., ipratropium, tiotropium) inducing bronchodilation, or glucocorticosteroids (e.g., fluticasone and budesonide) reducing inflammation. oids for the treatment of cystic fibrosis are also available for clinical use, with most of them falling into the therapeutic categories of mucolytics (e.g., saline and acetyl choline), aiming at thinning the mucus for facilitating its clearance from the patient's lungs. alternatively, leukocyte dnase, reducing inflammation, and antimicrobial agents (e.g., tobramycin), treating the bacterial infection characteristic of this disease, are also administered as oids. various devices can be used to administer oids to patients, including dry-powder inhalers (dpis), pressurized metered-dose inhalers (pmdis) and nebulizers. these devices have been extensively discussed in several recent works [18] [19] [20] [21] [22] [23] [24] [25] . briefly, dpis deliver powder particles carrying the drug; pmdis and nebulizers generate liquid droplets containing the drug. to be effective, an inhalation device must be easy to use and forgiving of poor patient's compliance, while providing reproducible effective dosing. thus, a through characterization of the performance of the inhalation device is required at regulatory level, when developing an oid. such characterization is based on in vitro, ex vivo and in vivo (on human volunteers) tests, as extensively described in the scientific literature [26] [27] [28] [29] [30] [31] [32] [33] animal models are not used in the characterization of the efficiency and reproducibility of inhalation delivery devices. this is due to the fact that, dpis and pmdis are breath-actuated and therefore not compatible with animal exposure; whereas for nebulizers modifications are needed in line with the animal model adopted. thus, our manuscript, which focuses on the potential reduction and replacement of animals studies in oid development, does not discuss the impact of inhalers' performance on the effectiveness of inhalation therapies [34] , a current challenge discussed in detail elsewhere [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . despite the major advantages over i.v. administration of drugs, inhalation therapy encounters several obstacles in achieving an effective therapeutic dose for the successful treatment of respiratory and/or systemic diseases. below, we describe the journey of an oid once administered and the human-specific features that, in the authors' opinion, strongly impact on the current low translation rate of oids, as these are poorly replicated in the current preclinical models. when an oid is administered to a patient, its liquid or powder aerosol enters the human respiratory system via the oropharynx. oid deposition in the oropharynx is invariably wasteful, reducing the oid dose reaching the lungs. this indeed constitutes the first feature to keep into account for developing an effective inhalation therapy [45] . rodent models cannot reproduce this feature, as they are obliged nose-breathers. however, other animal models (e.g., dogs) can be used to overcome the limitations posed by rodents. also, oid deposition in the oropharynx must be minimized in clinics to avoid severe side-effects in the patients. side-effects can be due to both local and systemic toxicity, as oids accumulating in the mouth and throat enter the body through swallowing. achieving an optimal oid deposition pattern in the patients' lung is the second feature to keep into account for an effective inhalation therapy [46] . to reach its site of action and/or absorption, the oid needs to pass through the so-called extrathoracic (or et) region of the larynx, enter the tracheobronchial region and reach the small and/or peripheral (alveoli) airways. drug absorption and translocation into the blood flow can in fact occur from all parts of the lung, but it occurs more readily in the alveoli [47] , where there is a large surface area and a relatively thin layer of epithelial and endothelial cells separating the inhaled drug from the blood flow. the oid journey within the complex, branched structure of the human lung is influenced by two main parameters of the particles/droplets carrying the drug [48] : (i) velocity [49] ; and (ii) aerodynamic size distribution (the so-called apsd) [13] . both parameters strongly impact on the drug deposition pattern and, subsequently, on the effectiveness of the inhalation therapy. velocity is defined by the delivery system employed in the oid administration. generally, high velocity results in increased deposition in the oropharynx and tracheobronchial regions; whereas, low velocity generates a peripheral deposition pattern [13] . it goes without saying that oids cannot reach those part of the respiratory tract where velocity is null, i.e., those parts of the lung that are not ventilated. this is particularly relevant to consider when developing oids against respiratory diseases [50] , which are characterized by the partial or full obstruction of the respiratory tract (e.g., asthma, copd, cystic fibrosis and lung cancer). combination of drugs where bronchodilators or mucolytics are used in a synergistic manner with other drug therapies, can be used to modulate oid velocity and increase the efficacy of the inhalation therapy. in parallel, the deposition mechanism of the aerosol particle/droplets in the bronchial tree changes depending on their apsd [13] . droplets/particles with large aerodynamic size deposit by impaction or interception mechanisms in the oropharynx or just beyond the trachea bifurcation. the smaller droplets/particles deposit in the smaller airways by sedimentation, subject to gravity. among those, the droplets/particles with aerodynamic size below 3 µm further move to the alveoli by diffusion or brownian motion. it should be noted here, droplets/particles deposition follows stokes' law [45] . the consequence is that, since most of the droplets/particles are near spherical, their aerodynamic size can be small despite being geometrically large. this happens when particles/droplets have low density, which is determined by the composition of the oid formulation. oid deposition pattern is currently evaluated in in vitro, cell-free experiments, achieving good predictive value [51] . once the oid deposits on the airways, removal mechanisms, such as mucociliary clearance in the conducting airways and macrophage clearance in the alveolar space, can be responsible for the drug elimination and/or degradation [52] , thus hindering the local efficacy and/or the systemic absorption of the oid. mucociliary clearance is the upward movement of mucus driven by beating cilia towards the pharynx, where mucus is subsequently swallowed and pass into the gastrointestinal tract [53] . in macrophage clearance, the oid is phagocytosed by alveolar macrophages and cleared by transport to the lung-draining lymph nodes [54, 55] . compared with mucociliary clearance, macrophage clearance is far slower [56] and, therefore, its action is typically assumed to be negligible for oids, unless the drug is known to be degraded by alveolar macrophages [57] . absorptive drug clearance is yet another clearance mechanism by which an oid is cleared from the lung through the blood circulation, a mechanism that is heavily dependent on perfusion. perfusion levels, however, vary between the different lung regions. in the alveoli, perfusion levels are the highest and drugs have a very short half-life; by contrast, in the tracheobronchial region, the perfusion rate is lower, thus offering a longer drug bioavailability [58] . removal mechanisms constitute the third feature to keep into account for developing an effective inhalation therapy. as described in detail in section 2.1.1, this feature is species-specific [59] and, therefore, human-specific removal mechanisms are not replicated by animal models. notably, human-specific removal mechanisms can be reproduced by in vitro, cell-based nams [60] [61] [62] [63] , as discussed in detail in section 2.2. to exert local or systemic efficacy, oid dissolution and absorption are indeed necessary [64] . the thickness and constitution of the pulmonary lining fluid, which can be modified by lung diseased states [65] , influence oid dissolution and, subsequently, absorption [66] , constituting the fourth feature to keep into account for developing an effective inhalation therapy. while the mucus layer (produced by goblet cells in the bronchial region) acts as a physical barrier, surfactants produced by alveolar cells in the peripheral airways reduce surface tension and facilitate drug dissolution [13] . noteworthy, oid dissolution rates strongly depend on disease-specific airway characteristics (e.g., copd is characterized by a thick mucus, hindering oid efficacy), which are not replicated by conventional preclinical models. noteworthy, in vitro, cell-based nams have the potential to reproduce the disease-specific composition of pulmonary lining fluid [67] . finally, the multicellular composition of the lung is the fifth feature to keep into account for developing an effective inhalation therapy, by playing an important role in defining oid delivery efficiency. for example, mast cells have protective functions against inhaled drugs; dendritic cells, together with macrophages, are the first line of defense of the lung immune system, sampling for and removing constantly any exogenous material such as drugs. clara cells are involved in oid metabolism. interestingly, the human lung has relatively low metabolic activity as compared to the gastro-intestinal tract or the liver [68, 69] . this constitutes a distinct advantage for inhalation therapy over oral drug administration. however, protease activity is generally increased in lung diseases as a result of chronic inflammation (e.g., enhanced activity of cytochrome p450 in patients affected by lung cancer [70, 71] or copd [72] ); this can indeed reduce the biopersistence and bioavailability of some oids (e.g., insulin [73] ). protection against metabolic activity has been achieved in inhalation therapy by drug encapsulation into carriers (e.g., liposomes [74] [75] [76] [77] [78] ). animal models and humans differ in the metabolism and distribution/types of cell populations lining the airways. for example, it has been shown that the average number of cells per alveolus for rats versus humans is: 21 vs. 1,481 for endothelial cells, 13 vs. 106 for interstitial cells, 6 vs. 67 for epithelial type ii cells, 4 vs. 40 for epithelial type i cells, and 1.4 vs. 12 for alveolar macrophages [79] . this has important clinical implications during the oid development. notably, the human-specific composition and metabolism of the lung can indeed be replicated more closely by adopting in vitro, cell-based nams, as described in the following sections. based on the multiple mechanisms and processes described above, it is evident that oid development is not an easy task. overall, a sound understanding of the features involved in the oid journey is necessary to use the most predictive preclinical models to overcome the complex, intrinsic challenges associated with inhalation therapy. interestingly, such challenges have certainly not hindered the interest of the pharmaceutical industry in inhalation therapy. based on a search carried out by the authors in july 2020, 2542 inhalation clinical trials for new, combination, and existing products, encompassing 666 drug interventions, 1111 different conditions and 115 rare diseases, have been logged on clinicaltrials.gov in the last four years (search terms: interventional studies; inhalation; start date from 01/01/2016 to 31/12/2020). to put this into context, a total of 97,744 interventional studies, comprising 2867 drug interventions, have been registered on clinicaltrials.gov in the same time period. consequently, inhalation clinical trials make for the 2.6% of the total number of interventional studies registered in the time period under consideration (2016-2020), and 23.2% of the total drug interventions examined. it is important to observe that more than half of these inhalation studies are for systemic conditions, thus demonstrating an interest that expands beyond the domain of respiratory diseases. preclinical studies of new oid candidates generally start from compound profiling in high-throughput in vitro studies [80] . compounds with promising efficacy results progress to in vivo studies. three preclinical animal-based studies are currently required by regulatory authorities before approving the request of clinical study for a novel oid. these are: (i) the range finding study, (ii) the repeat dose study, and (iii) the carcinogenicity study. other specialized studies can be necessary, such as safety pharmacology studies, reproductive studies, and neonatal/juvenile studies for pediatric oids. animal-based inhalation studies are carried out mainly in rats, mice or rabbits by exposure in restraint tubes [81] . dogs and primates can also be used for testing oids in more realistic settings, via facemasks or helmets [82] . although high-throughput cell-based assays can provide insightful information at the early stages of preclinical development, the cell models used fall short in recapitulating the complex interactions between different cell types and tissues/organs occurring in human. conventional in vitro models are in fact formed by one cell type grown as a flat, two-dimensional culture; thus, they are a simplistic representation of the human lung tissue [83] . furthermore, many in vitro assays use transformed cell lines that exhibit gene and protein expression that strongly differ from their primary counterpart [83] . on the other hand, various uncertainties characterize the animal-based preclinical studies currently required for regulatory purposes. the first level of uncertainty is associated with the type of devices used to administer the oid to the animal. while clinical nebulizers can be used in the preclinical environment (upon small modifications), dpis and pmdis cannot be employed to expose animal models at the preclinical screening level, as these devices are breath actuated. to overcome this issue, specialized equipment is used to expose the animal to an aerosol in a restrained environment. aerosol of powders is achieved via, for example, rotating brush generators or wright dust feed. an algorithm-based extrapolation [84] is then applied to define dose ranges to be used in clinical trials. the delivered dose is calculated as the amount of oid per unit of body weight that is presented to the animal. due to the two parameters (velocity and aerodynamic size distribution) affecting oid deposition patterns in the lungs, as discussed in the section above, and to the species of the animal model used, the deposited dose is only a fraction of the delivered dose. the fda assumes 100% deposition in humans, 10% in rats and 25% in dogs or non-human primates, irrespective of any information that has been produced by the submitting company [85] . this indeed generates uncertainties when calculating clinical overages. the second level of uncertainty in in vivo studies is posed by the animal model itself [86] . for example, rodents are obligate nose breathers; this strongly influences how inhaled compounds deposit in the respiratory tract. this and other interspecies differences have been extensively discussed by the authors in a recent perspective [59] . preclinical studies during oid development requires a clear understanding of such interspecies differences and their impact on the screening outcomes in terms of oid efficacy, toxicity and recovery from adverse effects. although not required at regulatory level, disease animal models are also used in preclinical research, particularly in the oncological field, as proof of concept for demonstrating oid efficacy. the authors have performed a literature search on pubmed using the searching terms "(inhaled drug) and (in vivo) and (efficacy)". the search results showed that, in the last five years, 116 articles used disease animal models to test the efficacy of oids. however, animal use as disease models needs to be viewed cautiously. in animal models, disease features are reproduced by applying exogeneous stimuli (e.g., allergens, irritant gas exposures, cigarette smoke, etc.) [87] . this modelling process is however incomplete, as the use of single stimuli does not mimic the disease etiology and chronicity observed in patients. the next section of this commentary focuses on this specific aspect, complementing the authors' previous publication [59] and further discussing if and how new approach methodologies (nams) could become useful in the attempt to overcome the limitations of current animal models and increase oid translation rate. for completeness, it should be mentioned here that the abbreviation "nams" is often used in toxicology to refer broadly to any non-animal technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment. examples of nams include non-mammalian model systems, (e.g., caenorhabditis elegans [88] [89] [90] , drosophila melanogaster [91] [92] [93] , zebrafish [94] [95] [96] and dictyostelium [97] ) and computational (in silico) approaches [98] , which indeed offer opportunities for mimicking human respiratory diseases in a predictive manner. however, the scope of the nams considered in our commentary includes only in vitro, non-animal cell models for the testing of oids. based on the most recent advances in tissue-engineering technologies, in vitro cell-based nams for screening the efficacy of oids can be classified in three main categories [99] : (i) tissue-mimetic lung cultures grown at the air-liquid interface (ali); (ii) lung organoids; and (iii) lung-on-chip. ali cultures mimic one of the main properties of the lung epithelium, i.e., the direct contact with the gas phase (air). this provides a tissue-mimetic environment that makes it possible for airway epithelial cells to proliferate and differentiate in vitro into a pseudostratified, ciliated epithelium that produces mucus. thus, ali cultures provide an excellent method for testing oid dissolution and absorption, while enabling testing of the drug in its aerosol form. whitcutt et al., were among the first research groups to report mucociliary differentiation in ali cultures [100] . today, ali cultures are known to be particularly useful in understanding the mechanisms of respiratory diseases, including the cell-cell and cell-extracellular matrix interactions during airways remodeling [101] [102] [103] . also, they can replicate some of the key features that need to be kept into account when developing an inhalation therapy, namely (i) the constitution and thickness of the pulmonary lining fluid [67] and (ii) mucociliary clearance [60] [61] [62] . for example, ali cultures have been used to model the effects of smoke exposure on epithelial cells [104] and the authors have created a complex, diseased ali culture model capable of reproducing the chemoresistance mechanisms observed in patients affected by non-small-cell lung cancer [105, 106] . also, culturing human airway epithelial cells isolated from patients, makes it possible to conduct patient-specific research and drug-screening, for example in cystic fibrosis, asthma and copd [107] [108] [109] [110] . with the aim of further increasing the predictive value of this in vitro nam, ali co-cultures have also been developed. in ali co-cultures, the lung cell populations are mixed or partially separated, depending on the experimental set-up. in general, the immune cells are cultured in direct contact with the epithelial cells; whereas, fibroblasts and endothelial cells are separated from the epithelial cells by the transwell permeable membrane. cell separation is due to the relative difference in the culturing conditions of the various cell types and the consequent need to separate them. this constitutes one of the main limitations of ali models, as separated cells cannot establish physical (cell-to-cell) interactions as per in vivo conditions. this indeed affects the detected responses during oid preclinical testing. the second type of in vitro, cell-based nams currently available for oid testing are lung organoids. these are grown from human induced pluripotent stem cells (ipscs) cultured within a natural or synthetic extracellular matrix to form three-dimensional (3d), hollow cell spheroids of basal, ciliated and secretory cells [111] . through differentiation and self-organization of the ipscs, an in vitro culture with lung tissue-specific morphogenetic and histological properties is formed [112] . to date, several organoids representative of the various human lung regions [39] and assessing a variety of pulmonary diseases [39, 113, 114] have been developed. in the context of oid preclinical testing, lung organoids can be used for modeling respiratory diseases and, therefore, as a platform for screening the efficacy of inhalation therapies [115, 116] . indeed, technical limitations are inherent with the use of lung organoids. lungs are in fact subjected to mechanical deformation during breathing cycles, a deformation that is currently hard to model in organoids. furthermore, there is still a lack of established in vitro lung organoids with a functional representation of the vasculature network. most importantly, lung organoids lack an important feature for oid testing, i.e., the direct contact of epithelial cells with the air. as mentioned above, lung organoids are spherical cultures. they present an interiorized lumen, with epithelial cells facing inwards rather than outwards; this makes drug administration extremely difficult and reduces the application of organoids in the screening of oid absorption. microfluidic technologies allow to add further complexity and functionality to the in vitro ali models described above. the so-called "lung-on-chip" is a microfluidic-based in vitro system in which lung epithelial cells are grown on one side of a membrane, and stromal cells on the other surface. liquid and air are circulated through the system to mimic air and blood flow in the lung. the applications of lung-on-chip range from basic research to drug discovery [117] , where the oid can be introduced in the air flow as per in vivo conditions. probably the most famous example of this in vitro, cell-based nam is the breathing lung-on-chip developed by huh and co-workers at the wyss institute of harvard university (usa), capable of reproducing both the physiological and pathological responses of the human lung, a rudimentary circulatory system and the mechanical stress associated with breathing [118] [119] [120] . the immediate application of lung-on-chip has been for toxicity testing [121, 122] ; more recently, this model has been exploited for improving understanding of the complex lung disease processes and their responses to therapeutics [123] [124] [125] , with applications extending even to the most recent need of a fast drug discovery for covid-19 treatment [126] . lung-on-chip systems allow, in fact, the in vitro creation of highly tissue-mimetic lung disease models [127, 128] , thus allowing, for example, to model the human response and the effects of existing and novel therapeutics when the lung is infected by the influenza virus or by viral pseudoparticles expressing spike protein of sars-cov-2, the virus responsible for covid-19 development [126] . the clear advantage of lung-on-chip systems over ali cultures or lung organoids is the possibility of mimicking the pulmonary mechanical stretch during in-and exhalation, while replicating the air-blood barrier for studying oid absorption. furthermore, lung-on-chip models allow evaluating the impact of the mucociliary clearance mechanism overcoming the lack of directionality in cilia beating function characteristic of fully-differentiated in vitro ali models [63] . nevertheless, the lung-on-chip models share some of the limitations of ali cultures, i.e., the impairment of physical crosstalk among different cell types. in fact, even in the most recent and advanced developments in "tumor-on-a-chip" cell culture technology, successfully used to create in vitro human orthotopic models of non-small-cell lung cancer [129] , the lung cancer cells (cultured under ali conditions) are physically separated from the lung endothelial cells by a porous, permeable membrane [130] . it is noteworthy to mention that, in the respiratory disease field, two additional categories of in vitro, cell-based nams exists, although these have not been used for oid testing to date. the first category is constituted by explant or ex vivo cultures, namely isolated perfused lungs and precision cut lung slices. these are better representations of the in vivo situation than any of the previous three nam types mentioned above. the use of ex vivo cultures in oid testing is however hindered by the hurdles associated with their manipulation, and by donor-specific differences that make the oid screening outcomes often not significant or difficult to interpret [131] . the second category includes the engineered, reconstructed lung organs [132] . these are formed from several cell types co-cultured within scaffolds that aim at replicating the composition and architecture of the human lung acellular stroma [133] . mechanical or biochemical stimuli can be added to tailor the properties of the scaffold and increase the similarity to the lung stroma in vivo. the first engineered lung organ was built from a decellularized lung matrix used as scaffold [134] . more recently, 3d bioprinting techniques have been used to produce the lung organs in vitro. for 3d bioprinting, cells are combined with bioactive hydrogels composed of synthetic (e.g., polyethylene glycol, pluronic) or natural (collagen, chitosan, fibrin, gelatin, matrigel, alginate) polymers [135] . the use of reconstructed lung organs in oid preclinical screening is currently hampered by the low throughput of these methods. to summarize, in this commentary we have presented an overview of the in vitro, cell-based nam systems that, to date, have been successfully employed to fill the technological gap that is believed to hindering the effective oid translation from the lab bench to the clinic. in the past, oid failure at clinical trial stage was mainly due to poor pharmacokinetics and bioavailability. today, these are rarely a cause of failure, as the pharmaceutical industry greatly invested in the development and application of much more accurate prediction and modelling approaches. lack of efficacy is now the most common cause of oid attrition [11] ; this appears to be associated to the fact that preclinical animal models are poorly representative of human respiratory diseases [136] . improved in vitro non-animal methods could provide a more human-relevant predictive value so that compounds would fail earlier in their course of development [137] . furthermore, we have provided a brief overview of those in vitro, cell-based nams that, in the future, we believe they could be adapted towards oid testing. although in vitro, cell-based nams still have limitations, the advantages associated with their use is evident and future efforts should aim at validating these systems for regulatory acceptance [59] . in the development of oids, we should therefore invest in moving away from animal studies. in the last decades, significant funding and precious time have been spent on developing animal models, despite the known species differences that make the results obtained from such models often unreliable when translated to humans. as dr. francois busquet and colleagues from the center for alternatives to animal testing-europe state for covid-19, human-relevant approaches offer crucial advantages of speed and "much more robust and exacting data than any animal experiment could deliver" [138] . in this instance, we believe it is important to highlight that directive 2010/63/eu on the protection of animals used for scientific purposes aims non only at reducing but at the "full replacement of procedures on live animals for scientific and educational purposes, as soon as it is scientifically possible to do so" [139] . consistently with this aim, in 2016 the netherlands has been the first eu member state to present a roadmap for phasing out animal testing in the safety research on chemical substances, food ingredients, pesticides and medicines (including veterinary medicines) [140] . the recent advances in tissue engineering, microfluidic and organ-on-chip technologies are providing researchers with tools for the development of human-relevant, in vitro nams. thus, it is essential now that the respiratory disease research community embraces these tools, bringing them forward towards regulatory validation. chronic obstructive pulmonary disease (copd) global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review world cancer report-cancer research for cancer prevention who. coronavirus disease (covid-2019) situation reports. available online global respiratory drugs market to 2023-a changing therapeutic landscape as key patents expire and biologics, targeted therapies and cftr modulators for asthma and cystic fibrosis treatment emerge as market growth drivers global respiratory drugs market barriers to new drug development in respiratory disease the r&d cost of a new medicine inhaled therapy in respiratory disease: the complex interplay of pulmonary kinetic processes heuze-vourc'h, n. in a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration heuze-vourc'h, n. inhalation of immuno-therapeutics/-prophylactics to fight respiratory tract infections: an appropriate drug at the right place! front pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications will pulmonary drug delivery for systemic application ever fulfill its rich promise? recent advances in aerosolised drug delivery 100 years of drug delivery to the lungs understanding dry powder inhalers: key technical and patient preference attributes delivery technologies for orally inhaled products: an update inhalation devices, delivery systems, and patient technique inhalation devices: from basic science to practical use, innovative vs generic products optimizing drug delivery in copd: the role of inhaler devices inhalation therapy devices for the treatment of obstructive lung diseases: the history of inhalers towards the ideal inhaler drug delivery devices: issues in drug development developing ways to evaluate in the laboratory how inhalation devices will be used by patients and care-givers: the need for clinically appropriate testing metered dose inhaler (mdi) and dry powder inhaler (dpi) products-quality considerations guidance for industry inhaled formulation and device selection: bridging the gap between preclinical species and first-in-human studies in vitro testing for orally inhaled products: developments in science-based regulatory approaches pulmonary drug delivery. part ii: the role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications in vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals validation of a general in vitro approach for prediction of total lung deposition in healthy adults for pharmaceutical inhalation products biological obstacles for identifying in vitro-in vivo correlations of orally inhaled formulations patient education and adherence to aerosol therapy trying, but failing" -the role of inhaler technique and mode of delivery in respiratory medication adherence the role of inhalation delivery devices in copd: perspectives of patients and health care providers matching inhaler devices with patients: the role of the primary care physician organoids as a model system for studying human lung development and disease device use errors with soft mist inhalers: a global systematic literature review and meta-analysis irregular and ineffective: a quantitative observational study of the time and technique of inhaler use problems with inhaler use: a call for improved clinician and patient education inhalers: to switch or not to switch? that is the question inhaler competence in asthma: common errors, barriers to use and recommended solutions particle transport and deposition: basic physics of particle kinetics pulmonary drug delivery: from generating aerosols to overcoming biological barriers-therapeutic possibilities and technological challenges inhaling medicines: delivering drugs to the body through the lungs guideline on the pharmaceutical quality of inhalation and nasal products drug delivery to the small airways the impact of pulmonary diseases on the fate of inhaled medicines-a review models of deposition, pharmacokinetics, and intersubject variability in respiratory drug delivery overcoming lung clearance mechanisms for controlled release drug delivery mucociliary clearance in the airways challenges for inhaled drug discovery and development: induced alveolar macrophage responses alveolar macrophages transport pathogens to lung draining lymph nodes report no 125-deposition, retention and dosimetry of inhaled radioactive substances pharmacometric models for characterizing the pharmacokinetics of orally inhaled drugs systems pharmacology approach for prediction of pulmonary and systemic pharmacokinetics and receptor occupancy of inhaled drugs in vitro alternatives to acute inhalation toxicity studies in animal models-a perspective air liquid interface culture can alter ciliary beat pattern in epithelium from primary ciliary dyskinesia patients characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling responses of well-differentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to burkholderia cenocepacia infection mucociliary defense: emerging cellular, molecular, and animal models pulmonary drug metabolism, clearance, and absorption pulmonary surfactants and their role in pathophysiology of lung disorders measurements of deposition, lung surface area and lung fluid for simulation of inhaled compounds human cellular models for the investigation of lung inflammation and mucus production in cystic fibrosis expression and localization of cyp3a4 and cyp3a5 in human lung expression and regulation of xenobiotic-metabolizing cytochrome p450 (cyp) enzymes in human lung smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome p450ia in lung cancer patients cytochrome p450-mediated pulmonary metabolism of carcinogens: regulation and cross-talk in lung carcinogenesis expression of cytochrome p450 mrnas in type ii alveolar cells from subjects with chronic obstructive pulmonary disease proteolytic enzymes as a limitation for pulmonary absorption of insulin: in vitro and in vivo investigations development of liposomal ciprofloxacin to treat lung infections liposomal formulations for inhalation the rationale and evidence for use of inhaled antibiotics to control pseudomonas aeruginosa infection in non-cystic fibrosis bronchiectasis liposomes for pulmonary drug delivery: the role of formulation and inhalation device design amikacin liposome inhalation suspension for treatment-refractory lung disease caused by mycobacterium avium complex (convert). a prospective, open-label, randomized study lower respiratory-tract structure of laboratory-animals and humans-dosimetry implications in vitro cell culture models for evaluating controlled release pulmonary drug delivery in vivo animal models for controlled-release pulmonary drug delivery preclinical models for pulmonary drug delivery reconstituted 2d cell and tissue models association of inhalation toxicologists (ait) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals toxicologic testing of inhaled pharmaceutical aerosols species comparison of drug absorption from the lung after aerosol inhalation or intratracheal injection translational models of lung disease modeling molecular and cellular aspects of human disease using the nematode caenorhabditis elegans modeling human diseases in caenorhabditis elegans elegans as a model organism for in vivo screening in cancer: effects of human c-met in lung cancer affect c. elegans vulva phenotypes a drosophila model of cigarette smoke induced copd identifies nrf2 signaling as an expedient target for intervention drosophila in asthma research a drosophila asthma model-what the fly tells us about inflammatory diseases of the lung zebrafish: model for the study of inflammation and the innate immune response to infectious diseases using in vivo zebrafish models to understand the biochemical basis of neutrophilic respiratory disease modeling inflammation in the zebrafish: how a fish can help us understand lung disease what can dictyostelium bring to the study of pseudomonas infections? semin heuze-vourc'h, n. innovative preclinical models for pulmonary drug delivery research options for modeling the respiratory system: inserts, scaffolds and microfluidic chips a biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells adapting the electrospinning process to provide three unique environments for a tri-layered in vitro model of the airway wall use of porous membranes in tissue barrier and co-culture models a novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture ali multilayered co-cultures mimic biochemical mechanisms of the cancer cell-fibroblast cross-talk involved in nsclc multidrug resistance multilayered cultures of nsclc cells grown at the air-liquid interface allow the efficacy testing of inhaled anti-cancer drugs antibacterial defense of human airway epithelial cells from chronic obstructive pulmonary disease patients induced by acute exposure to nontypeable haemophilus influenzae: modulation by cigarette smoke altered generation of ciliated cells in chronic obstructive pulmonary disease primary epithelial cell models for cystic fibrosis research asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus regeneration of the lung: lung stem cells and the development of lung mimicking devices in vitro generation of human pluripotent stem cell derived lung organoids a three-dimensional model of human lung development and disease from pluripotent stem cells modelling cryptosporidium infection in human small intestinal and lung organoids use of three-dimensional organoids and lung-on-a-chip methods to study lung development, regeneration and disease organoids as a powerful model for respiratory diseases lung-on-a-chip technologies for disease modeling and drug development reconstituting organ-level lung functions on a chip a human breathing lung-on-a-chip a human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice a lung/liver-on-a-chip platform for acute and chronic toxicity studies a 3d human lung-on-a-chip model for nanotoxicity testing multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development microphysiological lung models to evaluate the safety of new pharmaceutical modalities: a biopharmaceutical perspective impaired wound healing of alveolar lung epithelial cells in a breathing lung-on-a-chip human organs-on-chips as tools for repurposing approved drugs as potential influenza and covid19 therapeutics in viral pandemics biomimetic human lung-on-a-chip for modeling disease investigation small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro human organ chip models recapitulate orthotopic lung cancer growth, therapeutic responses, and tumor dormancy in vitro microengineered cancer-on-a-chip platforms to study the metastatic microenvironment bridging the gap between science and clinical efficacy: physiology, imaging, and modeling of aerosols in the lung modeling the lung: design and development of tissue engineered macro-and micro-physiologic lung models for research use tissue-informed engineering strategies for modeling human pulmonary diseases three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration 3d in vitro/ex vivo systems animal models of asthma: value, limitations and opportunities for alternative approaches human tissue models for a human disease: what are the barriers? thorax harnessing the power of novel animal-free test methods for the development of covid-19 drugs and vaccines on the protection of animals used for scientific purposes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank moreno carrer for the technical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-313431-swkcdvx8 authors: becerra-diaz, mireya; song, mason; heller, nicola title: androgen and androgen receptors as regulators of monocyte and macrophage biology in the healthy and diseased lung date: 2020-08-07 journal: front immunol doi: 10.3389/fimmu.2020.01698 sha: doc_id: 313431 cord_uid: swkcdvx8 androgens, the predominant male sex hormones, drive the development and maintenance of male characteristics by binding to androgen receptor (ar). as androgens are systemically distributed throughout the whole organism, they affect many tissues and cell types in addition to those in male sexual organs. it is now clear that the immune system is a target of androgen action. in the lungs, many immune cells express ars and are responsive to androgens. in this review, we describe the effects of androgens and ars on lung myeloid immune cells—monocytes and macrophages—as they relate to health and disease. in particular, we highlight the effect of androgens on lung diseases, such as asthma, chronic obstructive pulmonary disease and lung fibrosis. we also discuss the therapeutic use of androgens and how circulating androgens correlate with lung disease. in addition to human studies, we also discuss how mouse models have helped to uncover the effect of androgens on monocytes and macrophages in lung disease. although the role of estrogen and other female hormones has been broadly analyzed in the literature, we focus on the new perspectives of androgens as modulators of the immune system that target myeloid cells during lung inflammation. the immune system is essential for maintaining homeostasis within tissues and organs and protecting them against threats, such as harmful pathogens or cancerous transformation (1) . it comprises both innate and adaptive components. the innate immune system is made up of the innate lymphoid (innate lymphoid cells [ilcs] , natural killer cells [nks] , and lymphoid tissue inducers [lti] ) and innate myeloid subsets (2, 3) . the innate immune system consists of a network of immune cells and molecules that provide rapid, first-line defense against pathogens. in contrast, the adaptive immune response, made up of b and t lymphocytes (4), takes days or even weeks to become established (5) . innate immune cells express pattern recognition receptors that recognize unique and conserved pathogen-associated molecular patterns such as lipopolysaccharide (lps), viral ssrna, and fungal β-glucan (6) . b and t cells have evolved to recognize a finer repertoire of self-and nonself-antigens that facilitate pathogen-specific actions, immunologic memory generation, and host immune homeostasis regulation (4) . to accomplish this, the adaptive immune response involves a tightly regulated interplay between t and b lymphocytes and antigen-presenting cells of the myeloid lineage, such as dendritic cells (dcs), monocytes, and macrophages (4) . myeloid cells arise from the bone marrow. the type and magnitude of the immune response is influenced by biological sex and age (7) , and therefore differs between males and females. sex differences in the function of the immune system arise from both genetic (chromosomal) sex differences and differences mediated by the action of male and female sex hormones. because the concentration of sex hormones changes over the lifespan and throughout the course of the menstrual cycle in women, the function of the immune system also changes during different stages of life. innate myeloid immune cells, like other cell types, express sex hormone receptors and are responsive to sex hormones (8) . sex hormones are synthesized from cholesterol through a defined enzymatic cascade, predominately in the gonads and the adrenal glands (9) . sex hormones are also produced in other tissues, including the brain, placenta, mammary glands, liver, and adipose tissue (9) (10) (11) . in addition to driving sexual development of egg and sperm production, sex hormones are responsible for the development of male and female secondary sexual characteristics, like breast development and growth of facial hair, that occur during puberty (12) . androgens include testosterone, dihydrotestosterone (dht), androstenedione, androstenediol, and dehydroepiandrosterone (dhea), with dht being the most potent (13) . the concentration of androgens in circulation is about seven-fold higher in adult men than in adult women (14, 15) . estradiol and progesterone are the predominant female sex hormones (16) synthesized by the ovaries and adrenal glands. both male and female sex hormones are bound to the plasma proteins, albumin and sex hormone binding globulin (shbg), and only a small percentage exists as free hormone (1-2%). thus, the bioavailability of sex hormones is regulated by their biosynthesis and also the amount of albumin and shbg. importantly, sex hormones mediate not only anatomic differences between women and men but also direct sex differences in immune responses, leading to different risks for immunologic diseases (17) . overall, women have a greater risk for autoimmune diseases (such as systemic sclerosis and systemic lupus erythematosus) (18) , whereas men are more likely to die of infectious and parasitic diseases (19) . moreover, men have a greater risk of non-reproductive cancers (20) (21) (22) . both gender and sex are important mediators of these and other health and disease differences observed between men and women. while gender refers to the array of socially constructed roles, attitudes, personality traits, and behaviors, sex represents a biological characteristic of an individual (23) , including the hormonal milieu and chromosome complement (22) . in general, estrogens are considered to have proinflammatory properties and androgens are thought to have anti-inflammatory properties (24) . in the united states (25) and worldwide (26) , relevant evidence highlights important epidemiologic sex differences in incidence, susceptibility, and severity of a number of diseases that affect the respiratory tract. in this review, we will focus on how male sex hormones, the androgens, modulate the response of myeloid cells in the lung and how this modulation impacts the outcome of different diseases of the lung. biological sex mediates differences in the incidence and pathophysiology of lung diseases. these differences arise from sex differences in the structure and function of the lung itself, and also in the immune cells that populate the lung and are recruited to it during inflammation. before birth, the female lung has several structural advantages over the male lung. surfactant is produced earlier, and, although the female lung is smaller, it has more alveoli per unit area. neonatal females have higher expiratory flow rates than do male neonates when corrected for size. thus, male sex is a major risk factor for the development of respiratory distress syndrome, bronchopulmonary dysplasia in neonates (27) (28) (29) (30) , and asthma in childhood (31, 32) . in addition to the contribution of structural differences of the lung between the sexes, sex differences in lung function and lung diseases are also dependent on the action of sex hormones. we have summarized some broad concepts that define how testosterone and estrogen affect lung macrophage function and how this may contribute to the outcome of particular lung diseases in figure 1 . as testosterone rises after puberty, the immunosuppressive effects of this hormone on protective immune responses to infectious diseases in males can worsen pulmonary disease. this would be exemplified by tuberculosis or influenza. some of these effects are a result of androgen effects on critical inflammatory macrophage functions although the effects on the adaptive immune system also have a significant contribution to the overall outcome. thus, testosterone appears to play a key immunoregulatory role in lung macrophages. testosterone's immunoregulatory properties also appear to be dependent on the amount of cellular expression of ar and on the concentration of the hormone. low concentrations of testosterone have been noted in patients with asthma, copd, and tuberculosis. low testosterone may also be linked to insufficient control of tissue-damaging inflammatory responses seen in copd and pulmonary fibrosis. estrogen tends to promote wound healing responses in macrophages. dysregulation of wound healing responses and overactive tissue remodeling macrophages in the lung could be broadly used to describe the th2 response in allergic asthma, which is worse in women. cancer could also be considered an aberrant wound healing response driven by m2-like tumor associated macrophages. we have highlighted here how sex hormones contribute to changes in lung macrophage function that contribute to lung disease. however, it should be pointed out that not every sex difference in lung disease is due to direct effects on macrophages but on the broader coordinated immune response as a whole. figure 1 | sex differences in lung diseases discussed in this review and how they may be connected to the effects of androgens (and estrogens) on inflammatory macrophages in the lung. asthma in boys than in girls. with the onset of puberty, male and female sex hormones and their effects on the structural cells of the lung and on the immune system contribute to the incidence of asthma (33, 34) . the incidence and severity of asthma are greater in adult women than in adult men (35, 36) and greater in female than in male mice (37, 38) . female sex hormones, such as estrogen, appear to worsen asthma, although a straightforward correlation between amount of female sex hormone and asthma symptoms has not been concluded. androgens have multiple immunoregulatory and bronchodilatory functions and may contribute to, or be biomarkers for, better lung function in men (39) . accordingly, serum testosterone is low in men with moderate to severe asthma (39) (40) (41) . in one study, each 25 ng/dl increase in serum testosterone correlated with a 3% (95% ci, 1%-4%; p = 0.002) decrease in the likelihood of having asthma (42) . on the other hand, high concentrations of testosterone and cyclic amp in sputum of asthmatic women during the luteal phase of the menstrual cycle were thought to play a role in premenstrual exacerbations (43) . the idea that sex hormones may be a causal factor in asthma was significantly strengthened by a recent study of 7,615 adults that quantified serum sex hormones and asthma outcomes (44) . that study showed that low testosterone in both women and men was associated with an increased incidence of asthma. the other interesting finding was that higher testosterone was protective against asthma in obese women. obesity is a risk factor for asthma (45) (46) (47) . therefore, how high body mass index (bmi) and circulating sex hormones together affect asthma requires further investigation. another androgen, dehydroepiandrosterone (dhea), also known as androstenolone, is an endogenous steroid hormone and one of the most abundant circulating steroids in humans. it is a precursor for the synthesis of both testosterone and estrogen. dhea is sulfated at the c3β position into dhea-s by the action of the sulfotransferase enzymes sult2a1 and sult1e1 in the adrenal glands. the amount of dhea-s in the circulation is ∼250-300 times those of dhea. dhea became of interest to the asthma field because women with severe asthma had very low concentrations of dhea-s (48) and dhea-s concentration correlated with lung function (33) . interestingly, dhea-s is suppressed by oral or inhaled glucocorticoids, the mainstay therapy for asthma (49) . human dhea peaks at around age 20 and then follows an age-dependent decline until they reach prepubertal concentrations. reduced secretion of dhea with age has been related to a number of age-associated conditions. replacement of dhea has been considered as a possible therapeutic that could activate protective responses in an aging immune system. dhea is known to downregulate th2-inflammatory cytokines while upregulating il-2 synthesis (50, 51) in concanavalin a-stimulated peripheral blood mononuclear cells from adult males with atopic dermatitis (52, 53) . thus, it was hypothesized that it would be a useful treatment for atopic diseases including asthma and the results of the clinical trials for dhea in asthma patients show promise. the results are discussed in a later section titled "effects of androgen exposure on monocytes, macrophages in humans with lung disease". sex differences also have been reported in chronic obstructive pulmonary disease (copd), a heterogeneous, chronic, and progressive respiratory disorder that includes chronic bronchitis and emphysema (54) . chronic exposure of the airways to insults, such as cigarette smoke, leads to epithelial cell injury, destruction of pulmonary capillary vasculature, acceleration of epithelial cell senescence, and airway remodeling. the loss of lung compliance ultimately leads to copd (55, 56) . copd was previously thought to affect mostly elderly men, primarily because of the higher prevalence of smoking in men. however, as smoking rates increased in women, the number of copd cases in women exceeded that of men (57) . these differences are not only based on gender, as women develop more severe copd with earlyonset disease (<60 years) and have greater susceptibility to copd with lower tobacco exposure (58) . moreover, increasing age in female smokers leads to a faster annual decline in forced expiratory volume in the first second when compared to that of male smokers, even when they smoke fewer cigarettes (59) . similarly, pulmonary fibrosis is another lung disease that manifests sex differences (60) , with men being more affected than women (61, 62) . it is characterized by destruction of the pulmonary parenchyma and deposition of extracellular matrix with alterations in phenotype of both fibroblasts and alveolar epithelial cells (63) . the lungs are also the target of respiratory viruses such as influenza a ("flu"), respiratory syncytial virus, and coronaviruses, such as severe acute respiratory syndrome and the middle east respiratory syndrome. the viruses infect the airway epithelial cells and cause damage to the epithelial barrier by themselves or as a result of the immune response to the viral infection. sex differences have been noted in the immune response to influenza a virus and to the influenza vaccine. in general, women have a more robust protective immune response to influenza virus and vaccine than do men. although this elevated response is helpful in clearing virus, women of reproductive age also experience higher mortality and hospitalizations (64) (65) (66) (67) (68) , possibly from collateral tissue damage to the lungs. the vigorous immune response in women also means that women experience more adverse events after vaccination (69) . indeed, a systems biology approach identified that high testosterone was correlated with a blunted response to the flu vaccine in men (24) . as testosterone wanes in elderly men, mortality increases (70) . since the male immune response to the virus is also less robust, the incidence of seasonal flu is generally higher in men than in women in developed countries, according to the world health organization (71). it is not yet known how fluctuations in sex hormones across the menstrual cycle and lifespan affect the immune system's response to the influenza virus in humans. mouse studies have revealed that estrogen is protective at high, but not low, concentrations (72, 73) . on the other hand, testosterone replacement in gonadectomized or aged male mice enhanced survival rates (74) . despite these findings in mouse models, studies examining the effect of sex hormones on cellular and molecular mechanisms in human immune cells during influenza infection are lacking. like influenza infection, tuberculosis (tb), a lung disease caused by mycobacterium tuberculosis, exhibits notable sex differences in the number of cases worldwide, with men being almost twice as frequently affected than women (75, 76) . both sex and gender differences impact the incidence of tb. although tb affects less women than men in adulthood (75) , women in their economically active years (15-59 years old) have a higher tb incidence compared to women in other age groups (77) . this indicates that factors associated with gender, such as exposure to the bacteria, are important in this disease. however, because male predominance does not occur in children, this suggests that biological factors such as male sex hormones also play a significant role (75) . this is supported by a study of medically castrated men who experienced a significantly smaller proportion of death from tb, 8 .1% compared to 20.6% in intact men (78) . understanding how androgens lead to the greater susceptibility of men to tb is critical, as tb is still one of the leading fatal infectious diseases worldwide and may also may favor the development of other diseases, such as lung cancer (79) . lung cancer is a very complex disease that depends on a number of variants such as sex, gender, race, and socioeconomic status (80) . the development of lung cancer is also related to environmental factors, such as pollution due to industrialization and urbanization (81) . an additional gender-associated risk factor, significantly linked to developing lung cancer, is cigarette smoking (80) . historically, more men develop lung cancer and suffer lung cancer-associated deaths compared to women (80) . however, the incidence of lung cancer has changed notably in both women and men. in men, lung cancer incidence started to increase in the 1920s and started to decrease in the early 1990s, while in women, the mortality rates and incidence began to rise in the 1960s (80) . changes in smoking habits in the last several decades with a rise in the number of women who smoke correlate with an increase in the incidence of lung cancer in this demographic group (80) . smoking is definitely a key factor in the development of lung cancer; however, recent studies show a higher incidence of lung cancer in young women compared to young men (82, 83) , even when the prevalence of cigarette smoking among young women has approached but not exceeded that among men (84) . this suggests that the higher incidence of lung cancer in women is not explained simply by gender differences in smoking habits: a deeper analysis of differences mediated by sex, such as greater sensitivity to tobacco smoke in women is warranted (85, 86) . furthermore, men and women develop different specific types of lung cancer. malignant mesothelioma is more common in men, while women develop more adenocarcinoma (87), particularly non-small cell lung cancer (nsclc) (88) . women have a superior survival rate for lung cancer compared to men (89) . tumor-associated macrophages are critical in tumor progression yet how androgens influence macrophage behavior in lung cancer and in responses to treatment must be addressed more deeply to develop better therapies and increase survival rates in men. the lungs are a primary interface with the external environment. the delicate structures needed for gas exchange make them susceptible to damage from invading pathogens and toxic molecules. some insults to the lung can lead to the development of chronic conditions such as allergic asthma. as a protective mechanism, alveolar macrophages clear the air space of infectious, toxic, or allergenic particles to maintain homeostasis in the alveoli. thus, alveolar macrophages have a dual function as inflammatory cells, phagocytosing and killing inhaled bacteria or viruses, and also as controllers of the inflammatory immune response, minimizing alveolar damage. resident alveolar macrophages are seeded embryonically from yolk sac and fetal liver monocytes (90) (91) (92) . in asthma and other lung diseases, recruited alveolar macrophages derived from blood monocytes can turn into pathogenic cells, worsening the condition (93, 94) . mouse alveolar macrophages are characterized by high surface expression of siglec f and produce tgfβ. tgfβ both supports am development (95) and their maintenance of immune homeostasis by induction of tregs and suppression of b and t cell proliferation. another important function of am is the clearance of surfactant. am from male and female mice respond differently to surfactant protein a (sp-a) (96, 97) . sp-a acts as an opsonin and is important in clearance of pathogens. sex differences in am responses to surfactant could affect bacterial clearance and regulate the production of proinflammatory mediators. the molecular mechanisms that mediate these differences and how sex hormones change this important am function is an open question. in the human lung, there appears to be more diversity in the subtypes of lung macrophages compared to mice. the main determinant of the frequency of subtypes of macrophages in humans appears to be their anatomical location within the lung. am are the predominant immune cells in the lung airways (bronchi and bronchioalveolar space). flow cytometric panels have employed hla-dr, cd163, cd169, and cd206 to differentiate between am, im and monocytes. human am were identified as large, highly autofluorescent cd14-cd16+ cells that also express cd206, cd169, and marco (98, 99) . there appear to be two populations of am distinguished by either high or low expression of cd163. more recent approaches to characterize the macrophage populations in the lung involve single-cell transcriptomic analysis (100, 101) . although macrophages show a large variation in the transcriptional phenotype, expression of marco, ccl18, apoc1, apoe, pparg, and mrc1 was found in macrophages from healthy donors (100, 101), while chi3l1, marcks, il1rn, pla2g7, mmp9, and spp1 were highly expressed in macrophages from pulmonary fibrosis patients (101) . thus, a second contributor to diversity is likely the activation state of the cells. there are no data that describe sex differences in human am responses and the effect of sex hormones on these cells. from our mouse and human mdm studies, we would predict that androgens augment the immune homeostatic functions of these cells in the male lung. further work is still needed to standardize characterization of the different subpopulations of human lung macrophage populations and their role in maintaining healthy lung function and in disease. interstitial macrophages (ims), are another macrophage population found in the lung. they are a minor population of monocyte-derived macrophages (102) , which comprise 30-40% of lung macrophages (103) and are localized in the lung parenchyma (104) . ims contribute to maintaining homeostasis through the spontaneous release of il-10, a cytokine that dampens inflammation (105) . ims can prevent the development of aberrant type 2 allergic responses triggered by inhaled allergens (104) and have been related to reduction of asthma (106, 107) . different subpopulations of ims have been found in the lung; however, their characterization has not arrived at a consensus due to difficulties in their identification and isolation. in the mouse lung, different subpopulations of ims have been described based on the expression of surface markers. one report described three different subpopulations of ims based on the differential expression of proinflammatory cytokines, chemokine ligands, mhc-ii, cd11c, cd206, and lyve-1 (108); other group identified two subpopulations, based on similar markers but including cx3cr1 (109) . moreover, ims subpopulations can be also described based on the different anatomic locations these cells populate inside the mouse lung parenchyma (110) . further work is needed to better characterize and define the different im populations, as the different subtypes may have different functions during the inflammatory process. smaller in size than their am counterparts, human ims express more of the monocytic marker cd14 than am, perhaps suggesting their monocytic origin, and have lower expression of cd169 than human am. the responses of im to androgen will depend on their expression of ar which has not been measured. this will be a challenge due to difficulties in clearly identifying this population (and its subpopulations) from the monocytic, am and other myeloid populations in the lung. monocytes are produced in the bone marrow along with a number of other myeloid cells. myeloid cells originate from common pluripotent hematopoietic stem cells and represent the major subset of white cells in circulation (111) . these cells comprise basophils, neutrophils, eosinophils, dcs, monocytes, and macrophages, among others (112) . monocytes are released into circulation, then blood monocytes are recruited into inflamed tissue and can mature into macrophages or dendritic cells. there are two main subsets of mouse monocytes, "classical" or ly6c high monocytes that originate directly from ly6c + precursors, and "non-classical" or ly6c low monocytes that derive from ly6c high monocytes (113). the origin of ly6c low monocytes was demonstrated by sunderkotter, et al. by tracking the maturation of dii-labeled ly-6c high monocytes into dii-labeled ly6c low monocytes (114) . this process depends on the transcription factor nr4a1, which regulates the development and survival of ly6c low monocytes (113). these two monocyte subsets mirror the human cd14 + classical and cd16 + non-classical monocyte populations, respectively (115) . ly6c high monocytes highly express the chemokine receptor cc-chemokine receptor 2 (ccr2), whereas ly6c low monocytes highly express cx3cr1 (116) . importantly, ccr2 expression is required for ly6c + monocyte egress from the bone marrow into the circulation and entry into noninflamed and inflamed tissues (117) (118) (119) from the blood (120). as monocytes migrate into tissue, they mature into macrophages developing unique, tissue-dependent morphology and functions (121) . they lose expression of ly6c and gain expression of mhc class ii, becoming more efficient antigen-presenting cells (122) . some authors have proposed the concept of "tissue monocytes, " which are monocytes that can enter non-lymphoid organs without obligatory differentiation into macrophages. therefore, monocytes are much more than simply precursors for macrophages. in human lungs, monocytes, which can be both beneficial and pathogenic in a variety of pulmonary diseases (123) , are present at steady state (124) . multiple-color cytometric analysis on cells obtained from different anatomical locations of the lung of healthy subjects (non-smokers with normal lung function and absence of disease or infection) revealed that while intermediate monocytes (cd14 + cd16 + ) are more frequent in the airways, classical monocytes (cd14 + cd16 − ) are more frequent in blood (124) . moreover, the different monocyte subsets produced tnfα to different degrees upon stimulation with tlr ligands (3,4, and 7/8). thus, the anatomic location where samples are obtained should be considered and reported when working with human bronchoscopies, as this may alter the type and abundance of monocytes and macrophages found. accurate identification of monocytes in the lung compartments in humans has been a challenge because monocytic "contamination" from the blood vessels (125, 126) . overcoming this challenge, desch et al. performed a flow cytometric phenotyping study and identified two additional lung monocyte populations by analyzing lungs obtained from donors who died of non-pulmonary causes (127) . cd14 + cd206 − cd1c − cd1a − intravascular monocytes were similar to cd14 + blood monocytes and cd14 + cd206 + cd1c − cd1a − monocytes were described as tissue "monocytes." these studies highlight that we are just at the beginning of understanding the complexity of lung monocyte subtypes and their functions depending on the inflammatory state of the lung. other myeloid populations, like dcs, occupy the lung parenchyma at steady state, and their relative numbers change during inflammation. we refer readers to previous excellent reviews in this journal that cover the importance of dcs in immune responses in the lung and how they are affected by sex differences. therefore, we will not discuss dcs here (2, (128) (129) (130) (131) (132) . polarization is a very important effector characteristic observed in monocytes and macrophages. polarization refers to the change in phenotype and function of monocytes and macrophages as they are exposed to different inflammatory milieus or factors in the tissue microenvironment. to understand the effects of the differing inflammatory or tissue environments on monocyte-macrophage phenotype and function, researchers have used cytokines and other factors in vitro to mimic different inflammatory and tissue microenvironments. monocytes and macrophages stimulated with interferon-γ, lps, tnfα, interleukin (il)-12, and granulocyte-macrophage colonystimulating factor promote a pro-inflammatory macrophage phenotype denoted as m1 polarization. the activation state was also known as "classical" activation. m1-polarized macrophages mediate immunity to intracellular infections, such as viruses and bacteria, and they are generally considered tumoricidal (133) (134) (135) (136) . m1 macrophages accomplish these functions by inducing production of nitric oxide, reactive nitrogen intermediates, reactive oxygen species, and hydrogen peroxide (137) (138) (139) . in contrast, activation of macrophages with il-4 or il-13, as in extracellular parasitic infections and allergic reactions, leads to m2 polarization or "alternative" activation of macrophages (140) . m2 macrophages produce inflammatory mediators and chemokines, such as chitinase-like proteins (141), il-13 (142) , ccl17, ccl18, ccl22, and ccl24, which activate th2 cells and promote eosinophil infiltration into the lungs (143, 144) . in allergic asthma, a th2-inflammatory response to inhaled allergens drives lung macrophages toward an m2 phenotype. increased number and percent of m2 macrophages have been correlated with asthma severity and a decline in lung function in humans and mouse models (145) (146) (147) . similarly, m2 macrophages are the predominant subset seen in pulmonary fibrosis and are responsible for fibrogenesis (148) . during copd, the number of macrophages in airways, lung parenchyma, bronchoalveolar lavage fluid, and sputum increases (149, 150) . this increase may occur as a result of enhanced monocyte recruitment from circulation in response to chemokines such as ccl2 and cxc-chemokine ligand-1, which are increased in the sputum and bronchoalveolar lavage fluid of patients with copd (151) . unlike in allergic asthma and pulmonary fibrosis, macrophages in copd are polarized toward an m1 profile (152) . in addition to affecting men and women differently, another commonality of copd is that macrophages both in the alveolar space and in lung tissue present an altered activation phenotype. different concentrations of cytokines (tnf-α, il-1β, il-6, il-10, il-12) and chemokines (ccl2, ccl5, ccl7, ccl13, ccl22, il-8, cxcl9, and cxcl10) are found comparing smokers to healthy subjects (153) (154) (155) (156) (157) (158) (159) (160) (161) . thus the external provoking stimulus uniquely shapes macrophage phenotype and function. while the m1/m2 designations are useful for in vitro studies with stimulation with defined cytokines, the in vivo phenotype of macrophages exists on a spectrum somewhere in between these two well-defined opposing phenotypes or does not fit the paradigm at all. for example, m1 and m2 markers can exist simultaneously within the same cell in some cases (162) (163) (164) . the key factors dictating the macrophage phenotype or activation state are the stage of the immune response and the soluble factors and interactions in a particular tissue microenvironment. for example, the lung environment is rich in gm-csf, tgfβ, and pparγ and is critical for development of mature ams after birth in both mice (90, 91, (165) (166) (167) (168) (169) and humans (170) (171) (172) (173) (174) (175) . furthermore, interactions between cd200 on type ii alveolar epithelial cells and cd200r on the surface of the am deliver regulatory signals to the am to prevent proinflammatory signaling and macrophage activation (176) . thus, macrophage nomenclature has evolved as our understanding of the phenotypes and functions of different types of tissue resident macrophages, recruited monocytes and monocyte-derived macrophages advances. in-depth studies of the effects of androgens and other sex hormones on tissue macrophage plasticity and phenotype have yet to be carried out. because androgens are lipophilic steroid hormones, they can easily diffuse across cell membranes without the need for receptor-mediated import (8) . androgens in circulation are found mostly bound to sex hormone-binding globulin and albumin (8) . free (unbound) steroid sex hormones can signal through two different mechanisms: the classical ar, located in the intracellular compartment, and the membrane, or nonclassical, ar (8) . androgen binding to classical and nonclassical ars mediates genomic and non-genomic androgen effects, respectively (177) . upon androgen binding, the classical ar undergoes a conformational change and dissociates from heat-shock and other chaperone proteins. an androgen-ar complex is formed that translocates to the nucleus, dimerizes, and binds to androgen responsive elements that modulate the transcription of target genes (178) . importantly, it has been reported that the androgen-ar complex can also mediate nongenomic changes (179) by causing calcium flux and by activating second messenger pathways including erk, akt, and mapk, at least in cell lines (179) (180) (181) . whereas, genomic modulation may need hours or days (182), non-genomic modulation can occur within seconds to minutes after androgen exposure, does not involve the complex binding to dna, and therefore does not affect transcription of target genes (177) . dhea has no known unique receptor and is not a direct ar agonist. it affects immune function but, because it can interact with other sex hormones, it has been difficult to establish its mechanisms of action. most studies of androgen-ar complex-mediated gene expression have been carried out in the context of male reproductive tissue in prostate cancer (pca) (183) (184) (185) . as previously discussed, immune cells are responsive to sex hormones, and almost all immune cells express sex hormone receptors (8) . mouse monocytes, macrophages (186) , and dcs (187) express both classical and non-classical ars although the vast majority studies do not specifically dissect the role of the two types of ar on the outcomes being measured in the study. because recent literature has described how sex steroids modulate the functions of dcs (2, 128, 129), we will not discuss it here. we will focus on the importance of androgen-ar regulation of monocyte and macrophage function and how androgen-ars modulate monocytes and macrophages in lung diseases. androgen receptor expression in mouse and human monocytes and macrophages is summarized in table 1 . in general, the expression of the mrna and protein for classical ar has been assessed, often by non-quantitative means, and non-classical ars have not been measured. we have summarized the outcomes of many studies on mouse and human monocyte-macrophages responses in the presence of androgens in figure 2 . in general, monocyte-macrophage exposure to androgen results in a reduction of pro-inflammatory responses (boxed and shaded in green). it is possible that the reduction in inflammation by androgen may be due to ar suppression of estrogen/erα-driven pro-inflammatory responses. ar was demonstrated to inhibit erα activity by binding eres in breast cancer cells (201) . whether this indirect mechanism accounts for the broad immunosuppressive effects of androgens in normal untransformed immune cells is not known. in keeping with reduced pro-inflammatory responses, we found that androgen enhanced il-4-induced m2 polarization of bone marrow derived and alveolar macrophages in vitro and macrophage-specific deficiency of ar diminished m2 polarization of lung macrophages in vivo (188) . in some cases, however, inflammatory responses are increased by androgens (boxed and shaded in red). the different responses may be due to different types of tissue macrophages or experimental system. monocyte-macrophage responses are dependent on the concentration of the hormone, expression of ar, and upon the inducing stimuli to which the macrophage is exposed. the majority of in vitro studies examining the effects of androgens on monocytes and macrophages have not clearly acknowledged or separated the effect of androgen on membrane ars and nonclassical ar signaling from that of classical ars. therefore, we have to assume that the studies described in the section below are a result of classical ar activity unless explicitly investigated or stated. determining how non-classical ar signaling and androgen-independent activation of ar affects monocyte and macrophage function is a gap in our knowledge that must be addressed in future studies. androgens modulate the expression of proinflammatory molecules such as tnfα in mouse monocytes and macrophages. in 2009, lai et al. (192) demonstrated that lps-induced production of tnfα was decreased in bmm lacking classic ars. moreover, they found that ar, in the presence of dht, induced tnf-α promoter activity (192) . on the other hand, several reports have suggested the contrary. in one study that used splenic macrophages from midline laparotomy trauma-hemorrhaged mice, dht suppressed tnf-α production from lps-stimulated cells (202) . this effect was also observed in the mouse macrophage cell line j774 (203) , in which testosterone inhibited tnf-α production. in addition, testosterone also decreased expression of the proinflammatory molecule nitric oxide in response to lps in the mouse macrophage cell lines raw 264.7 (204) and j774 (203) , but it enhanced the expression of il-10 in the latter. other molecules important in monocyte-macrophage functions are also affected by androgens. for example, the expression of ccr2 was enhanced in mouse monocytes by androgens and thereby enhanced chemotaxis (192) . however, suppressing ar with sirna in prostate cells increased macrophage recruitment via ccl2 upregulation, which might promote prostate cancer (205) . phagocytosis was increased by testosterone in rat peritoneal macrophages at 10 −12 m but not at concentrations lower or higher than 10 −12 m (206). cytotoxicity of raw macrophages to the mouse prostate cancer cell line, tramp c2, was enhanced by dht alone (193) . this was attributed to enhanced expression of the m1 polarization markers, trail and tnf-α, in the macrophages. testosterone (100, 200 , and 400 nm) induced apoptosis in mouse bmm through fas-fasl (207) and activation of caspase 3, 8 and poly (adp-ribose) polymerase (208) . in terms of m2 polarization of macrophages, we showed recently that in vitro exposure of bmm to dht prior to il-4 stimulation enhanced chi3l3 and arg1 gene expression, as well as production of ym1 (188) . androgen amplified the m2 phenotype by increasing il-4-mediated m2 polarization. our results were similar to those found in response to il-4 in the raw cell line (209) . this enhanced m2 macrophage polarization correlated with decreased tlr4 expression and sensitivity to a tlr4-specific ligand observed in testosterone-treated raw cells (210) . taken together, these observations suggest that androgens and ars can either promote or suppress inflammatory properties of mouse macrophages, depending on the external environmental conditions, ar expression, and concentration of hormone. overall, androgens are more likely to reduce polarization of m1 macrophages. this could represent an important mechanism by which inflammatory pathways are downregulated in males. the opposite effects seen in different inflammatory contexts highlight the need for a deeper and broader study of the androgen/armediated modulation of monocytes and macrophages, as these cells participate in both the initial and late phases of immune responses in a variety of diseases. most of the studies analyzing the role of ar have focused on prostate cancer, primarily in transformed cell lines (211-213) but macrophages are vital in cancer development and metastasis (205) . furthermore, it is important to consider that opposing effects could result from differential activation of either classical or non-classical (arindependent) effects (195, 214) which have been rarely studied to date. androgens affect a number of key monocyte and macrophage functions. studies of androgen receptor function in human monocytes and macrophages have focused primarily on the roles of male sex and sex hormones in promoting atherosclerotic foam cell formation (196) and inhibiting cutaneous wound healing (186, 215) . foam cells are a type of macrophage localized in the blood vessel walls where they engorge cholesterol (216). foam cells exhibit enhanced inflammatory cytokine secretion and cause atheroma, contributing to cardiovascular disease (216, 217). the effect of androgen on monocytes and macrophages in other immune-mediated human diseases where monocytes and macrophages play a role has been neglected. the degree of ar expression in monocytes and macrophages is likely the primary determinant of responsiveness, although most studies examining responses to androgens do not quantify ar expression (see table 1 ). the expression or action of androgens on non-classical ars in human monocytes and macrophages has yet to be examined carefully. most studies assume that the outcomes that are measured are a result of the activity of classical ar. sex differences in ar content may also play a role in responsiveness. this fact highlights the importance of considering the sex of cells in all in vitro studies to accurately assess how sex hormones affect the responses of monocytes and macrophages. apoptosis was significantly greater in human thp-1 cells cultured for 7 days with 10 nm testosterone than in control cells or cells treated with estradiol (e2), owing to a reduction in proliferating cell nuclear antigen, induction of poly-adp ribose polymerase-cleaved, an increase in iκb-α, and a decrease in phosphorylated iκb-α (218) . e2, in contrast, promoted cell survival. other studies noted concentration-and time-dependent regulation of apoptosis in thp-1 cells, with an increase in the proto-oncogene bax and fas (219) . androgen exposure inhibited proliferation of the human monoblastic leukemia cell line u937, depending on the concentration and time of exposure (220) . cell cycle arrest occurred at the g2/m phase, although another study measured no effect of testosterone on pma-differentiated u937 cells (221) . how testosterone regulates apoptosis and survival of untransformed primary human monocytes and mdms has not been well-studied. toxicity was observed when monocytes were differentiated into macrophages over 8 days in the presence of 0.1 mg/ml androgen, but not at lower concentrations of the hormone (222) . testosterone reduced the viability of monocytes from a healthy control and a patient with systemic lupus erythematosus in a concentration-dependent fashion (223, 224) . these two studies highlight the importance of concentration in studies of sex hormones. an additional example is the finding that e2 enhances tnf-α secretion from antigen-stimulated t-cells at low concentrations and inhibits secretion at high concentrations (225) . il-1β-induced nf-κb activation is also inhibited at high but not at low e2 concentrations (226) . hence, it is important to carry out in vitro studies of sex hormone responses over a wide range of physiologic concentrations of sex hormones. in general, androgens have a suppressive effect on proinflammatory cytokine expression in monocytes and mdms. this finding is consistent with the idea that the immune system of females produces cytokines in response to pathogens and insults more robustly than that of males. monocyte or mdm expression of tnf-α, il-1β, il-6, and il-8 is reduced in the presence of testosterone (227) (228) (229) . many studies in this field have relied on human cell lines, such as thp-1 and u937, with or without pma-induced differentiation into macrophages, and differentiated hl-60 cells, although primary monocytes and mdms have been used in a few cases (224, 230) . another immunoregulatory function of testosterone is the upregulation and secretion of c1 inhibitor (c1inh) from monocytes (231) . c1inh is a 105 kda plasma protein whose main function is inhibition of the complement system to prevent spontaneous activation. thus, testosterone keeps complement activation in check. another mechanism by which testosterone limits inflammation is by decreasing the generation of reactive oxygen species generation from differentiated hl-60 cells. interestingly, the production of reactive oxygen species in response to zymosan, but not lps, was inhibited by testosterone (228) . in terms of allergic immune responses, metabolism of arachidonic acid into inflammatory leukotrienes (lts) via the 5-lipoxygenase (5-lo) pathway is sex-dependent in human monocytes. pergola et al. (232) reported that primary human peripheral blood monocytes from women synthesize more 5-lo product than do the same cells from men. 5α-dht (10 nm) suppressed lt synthesis in female cells to the levels observed in males. erk activation by androgens reduced phospholipase d activity in monocytes and impaired 5-lo product formation by reducing active diacylglycerides. the other branch of arachidonic acid metabolism is the cyclooxygenase (cox) pathway, which generates prostaglandins. prostaglandin e2 (pge2), one of the most abundant cox products produced by the airway epithelium and smooth muscle (233, 234) , can either stimulate or suppress immune cell function. testosterone reduced pge2 production in monocytes obtained from heparinized peripheral blood of healthy adults and incubated for 24 h with lps (235) . a few studies have examined the effect of dhea on human monocytes and macrophages. in the presence of lps, dhea induced il-6 and tnf-α production by primary human monocytes and il-8 and tnf-α production by thp-1 cells (236) . in these experiments, dhea counteracted the effects of cortisol and the glucocorticoid receptor on lps-induced il-6 and tnf-α by inducing expression of the scaffolding protein rack1 (receptor for activated c kinase 1) in thp-1 cells and primary human monocytes (237) . rack1 is involved in multiple signal transduction cascades, including the mapk, protein kinase c, and src signaling pathways. rack1 shuttles proteins around the cell, anchors proteins at particular locations, and is involved in cell migration (238) . in contrast, dhea added to alveolar macrophages lavaged from 11 non-smoking asbestos workers significantly reduced superoxide anion release in vitro (239) , consistent with its role in dampening th2inflammation (240) . therefore, the effect of dhea on monocytes and macrophages may be stimulus-dependent and needs more in-depth investigation. the formation of foam cells (lipid-filled macrophages) is generally associated with the pathogenesis of cardiovascular diseases, such as atherosclerosis. however, foam cells are also found in patients with silicosis (241) and other fibrotic lung diseases (242) and in tuberculosis. alveolar macrophages take up extracellular and intracellular lipids in response to inhaled silica, vaping products (243) , and mycobacterium tuberculosis (244) . furthermore, the metabolism of fatty acids by macrophages by β-oxidation for sustained energy production is a key feature of the functional phenotype of macrophages with a pro-resolving, tissue reparative (m2) phenotype. therefore, we have included how androgens modulate foam cell formation and lipid handling in macrophages as part of this discussion. macrophages from men and those exposed to testosterone favor the processes of lipid handling and foam cell formation, supporting evidence that atherosclerosis is a male-dominant disease when age is taken into account (245) . atherosclerotic plaques composed of a number of different immune cells form in blood vessel walls. in advanced stages of atherosclerosis, macrophages in plaques take up oxidized low-density lipoprotein (ldl), creating foam cells. eventually, cholesterol crystals accumulate, trigger inflammation and plaque rupture. the role of sex in the inflammatory events of atherosclerosis has been reviewed elsewhere (246) . in vitro studies have sought to ascertain how testosterone promotes these processes by utilizing primary mdms. in mdms from healthy men, androgen treatment was shown to upregulate genes involved with lipoprotein processing, transporter proteins, cell-surface adhesion, and other pathways, but none of these genes were upregulated in female macrophages (247) . the marked sex specificity of androgen effects on human macrophage gene expression is most likely related to sex differences in mdm ar expression. similarly, treatment of mdms with modified and native ldl led to changes in expression of mrnas involved in homeostatic regulation of lipid metabolism, depending on the sex of macrophage donors (248) . functionally, androgen-treated mdms from men but not women accumulate cholesteryl esters (196) . male macrophages exhibit increased rates of lysosomal acetylated ldl degradation and upregulated expression of scavenger receptor class b type i (249), increasing high-density lipoprotein (3)-induced cholesterol efflux. the expression of ar in monocytes/macrophages also upregulates lectin-type oxidized ldl receptor 1 molecules that are involved in foam cell formation (198) . however, corcoran et al. (250) observed no effect of testosterone on cholesterol content or efflux from mdms of healthy male and postmenopausal female donors (age 50-70 years). because their study used healthy donors, it is possible that the absence of other health-related factors, such as smoking, poor health, and genetic risk factors for coronary heart disease in the healthy blood donors may have produced these results. chemotaxis of thp-1 cells was diminished when androgen receptor was knocked down by sirna suggesting a role for ar in migration of monocytes (198) . the authors identified tnf-α as a key ar-regulated molecule important in monocyte migration. in contrast, a handful of studies have tested the effect of testosterone on primary human monocyte phagocytosis and migration, but no effect was found (222, (251) (252) (253) . testosterone did not change the cytotoxic capacity of monocytes from male donors (age range 18-40 years) to lyse red cells sensitized with igg antibodies (254) . most studies that have used mouse models to investigate sex differences in lung diseases have focused on the role of estrogen and estrogen receptors (255) (256) (257) . the importance of androgen and ars in lung disease has been poorly studied. earlier studies were directed at modulating monocyte and macrophage functions unconnected to ar function, as 15 years ago it was believed that mouse macrophages did not express classical ars (189) . nevertheless, recent studies have examined sex differences in mouse models of allergic asthma, copd, and influenza. we and others have reported sex differences in mouse models of allergic lung inflammation (37, 38, 188, 255) . some of the observed differences have been clearly attributed to the effect of androgens. we showed that dht reconstitution of castrated male mice reduced overall lung inflammation (188) . a reduction of total serum ige and total immune cell recruitment to the lungs, specifically eosinophils, revealed the regulatory effect of androgens on several cell types. however, the unexpected enhancement of the production of the canonical m2 macrophage marker involved in eosinophil recruitment (258, 259) , ym1, by dht in alveolar macrophages (188) showed that androgens have a regulatory or an activating effect depending on the cell type. we demonstrated that deletion of classical ars on monocytes and macrophages (ar flox lysmcre mice) resulted in reduced inflammation (less eosinophil recruitment to the alveolar space), along with less mucus production and lung cell infiltrate, despite no differences in serum testosterone level between arsufficient and ar flox lysmcre mice (188) . this finding indicates the importance of androgens as modulators of m2 macrophage polarization and the critical role of these cells in allergic lung inflammation. other recent studies have shown that testosterone has an anti-inflammatory role in a mouse model of allergic lung inflammation induced by house dust mite but focused on other cell types in lung, such as th2 (260) and ilc2 cells (261, 262) . similarly, high concentrations of androgens in circulation have been related to a decrease in the expression of tnfα and other proinflammatory cytokines, such as il-6 and il-1β, in rodent macrophage cell models and in human monocytes (203, 223, 224, 230, 263) . how androgen and ars impact functions on ims still needs to be studied. at the time this review was written, no reports on ar expression in ims were found. however, we hypothesize that as ims are derived from blood monocytes (102) , but once in the tissue they develop an intermediate size and phenotype between monocytes and am (103, 264) , their expression of ar could be somewhere in between. therefore, androgen and ars could regulate the functions and activation of these cells. this requires further study, as ims are a constitutive macrophage population in the lung, and may play a role mediating sex differences in lung diseases. mouse models have also shown that sex differences affect copd. in 2016, tam et al. (265) reported that smokeinduced copd is characterized by small airway remodeling in female but not male mice and that ovariectomy before smoke exposure ameliorates the disease. another study focusing on α-1 antitrypsin deficiency, the leading genetic cause of emphysema, also uncovered a higher susceptibility of female mice for this condition (266) . however, these studies did not determine if androgens mediate resistance to copd, or if the key to the observed sex differences is ovarian sex hormones. thus, the role that androgens play in copd and copd models remains unclear. mouse studies that have focused on sex differences in influenza showed that at moderate influenza virus a (iav) loads, morbidity, mortality, and the associated inflammatory response is greater in female than in male mice, but that mortality is similar at higher loads (72, 267, 268) . the role of sex hormones was well-addressed in these studies. high levels of estrogen in estrogen-reconstituted female mice protected against lethal iav doses (72), whereas the lower estrogen levels in intact females were associated with greater inflammatory responses and increased morbidity after infection. similar observations were made after progesterone replacement (269) . in males, a decrease in androgen levels after castration increased morbidity and pathology upon iav infection, but replacement of testosterone or dht reduced morbidity, mortality, and inflammation (72, 74) . these findings suggest that although estrogen may be protective or detrimental, depending on concentration, androgens may suppress inflammation in a broader way. gonadectomy studies in mice have been used to uncover the role of androgens in tb. similar to observations in castrated men, castrated male mice that displayed greater pro-inflammatory responses in the lung (more tnf-α, ifn γ, il-12, inos, and il-17) than intact males. ifn-γ-activated macrophages (m1 macrophages) control of tb infection in both human and mouse (270) . ovary removal in females did not impact susceptibility to tb (271) , suggesting that testosterone is responsible for male susceptibility to tb. we previously reported that dht enhances m2 macrophage polarization through ar (188) . therefore, we speculate that the greater male susceptibility to tb could be at least in part mediated by enhanced m2 responses that are poorly protective and decrease protective proinflammatory macrophage responses. formal studies to address this idea as well as how androgen effects on other key immune cell players in tb are needed. how androgens affect monocyte and macrophage biology in lung cancer models in mice has not been well-studied. monocytes and macrophages are important cellular players in tumorigenesis. tumor-associated macrophages (tams) can be classified into two phenotypes that are either pro-inflammatory and tumoricidal (m1-like) or promote tumor growth and suppress anti-tumor immune responses (m2-like) (272) (273) (274) . as mentioned previously, sex hormones augment m2 macrophage polarization, thus, play an important role in lung carcinogenesis. the greater overall incidence of lung cancer in men could be explained by an enhanced m2 polarization by androgens (188) . on the other hand, estrogen has been shown to induce tumor angiogenesis (275) . estrogen signaling though the camp, mapk, and akt pathways with the consequent phosphorylation of erk and egfr signaling, along with the enhanced expression of cmyc and cyclin d, results in nsclc cell proliferation (276) . mouse models must therefore address the role of androgens on monocytes and macrophage function in the establishment and progression of lung cancer in male and female animals. few studies have examined the effect of sex hormones on peripheral blood monocytes and lung macrophages from men and women with asthma or the other lung diseases we have discussed here. in women with asthma, dominance of m2 macrophages in airways and lung tissue has been documented (277) and a connection between female sex and female sex hormones surmised. there is a paucity of literature regarding how introducing or depleting exogenous sex hormones (such as in female-to-male transgender individuals receiving testosterone supplementation or women with estrogen blockade) affects the function of blood monocytes and lung macrophages in men and women with asthma. most studies correlate concentrations of sex hormones with either inflammatory markers, such as cytokines or chemokines in serum and other fluids, or with lung function measurements. we will summarize below the small number of studies in which androgen concentrations were manipulated in humans and the effects on monocyte or macrophage function. hypogonadism in men refers to a deficiency in testosterone production from the testes that results from testicular, hypothalamic, or pituitary abnormalities. klinefelter's syndrome in men, which is a result of additional x-chromosomes (e.g., xxy), is the most common cause of hypogonadism. testosterone replacement therapy is the primary treatment option to restore physiologic testosterone levels, typically in the range of 300 to 800 ng/dl. in general, exogenously administered testosterone has a suppressive effect on the proinflammatory immune response from monocytes. for example, spontaneous production of proinflammatory cytokines (il-1β, il-6, and tnfα) ex vivo was reduced or completely absent in the monocytes and dcs from men with type-2 diabetes who had partial androgen deficiency and were treated for 12 months with testosterone replacement. this suppression was maintained for 3 more months after testosterone withdrawal (278) . testosterone replacement therapy also is associated with a reduction or complete abrogation of spontaneous ex vivo production of inflammatory cytokines by antigen-presenting cells (279) . on the other hand, the circulating monocytes from hypogonadal men treated with testosterone replacement therapy exhibited significantly upregulated expression of cd107b at baseline compared to monocytes from healthy controls. this was also seen after stimulation with cpg oligodeoxynucleotides to mimic bacterial dna exposure (280) . membrane expression of cd107b, also known as lysosome-associated membrane protein (lamp)2, is indicative of release of lysosome and/or phagolysosome contents into the extracellular medium, a mechanism that may be involved in killing and/or digesting target cells. these data suggest that testosterone increases the inflammatory function of these cells, an effect that would contrast with its typical role as an immunosuppressant. the immune system of individuals with klinefelter's syndrome provides unique insight into the genetic contribution of the x-chromosome and that of diminished testosterone to sex differences in different diseases. men with klinefelter's syndrome have an increased risk of developing autoimmune diseases, particularly those that are typically female-dominant, such as rheumatoid arthritis and systemic lupus erythematosus (281) . as might be predicted due to the negative effect of lower concentration of testosterone on lung function, men with klinefelter's syndrome are more likely to be diagnosed with pulmonary diseases, such as copd and pneumonia (282) . asthma is also reported in these individuals (283) (284) (285) and it was successfully controlled with long-acting β-agonists and oral testosterone replacement in one case report (283) . at the cellular level, however, cytokine production in stimulated whole blood from klinefelter's men was similar to that of women (286) . these data suggest that the effect of the additional x-chromosome was more dominant than the reduction in circulating androgen in klinefelter's men. in the same study, however, purified monocytes showed the opposite response: cytokine production from the monocytes of healthy and klinefelter's men was similar and less robust than that from the monocytes of women. this observation led to the opposite conclusion-that androgen plays a more important role in monocyte cytokine production than does chromosomal complement. pcos is a disease characterized by hyperandrogenism, amenorrhea, and polycystic ovaries. the cystic folliclesovarian theca cells-produce testosterone that causes significant elevations in serum concentrations of testosterone, androstenedione, dhea, and dhea-s. in women with pcos, serum testosterone is in the range of 45-150 ng/dl (2-5 nmol/l) (287), compared with a range of 20-60 ng/dl in healthy, ovulatory women (288) . this endocrinopathy is associated with metabolic disorders, such as dyslipidemia, insulin resistance, metabolic syndrome, and cardiovascular complications. immune function is impaired in women with pcos, leading to increased secretion of autoantibodies and increased risk of type 1 diabetes, asthma, and thyroid disease (289) . because androgens downregulate the inflammatory responses that contribute to asthma, one might hypothesize that women with pcos would have less asthma. however, htet al. (290) found that asthma prevalence was 15.2% in women with pcos compared to only 10.6% in women without pcos (p = 0.004). women both with and without pcos who had asthma tended to have a higher bmi than those without asthma (290) . after multivariable analysis, the authors concluded that both pcos and high bmi were independently associated with asthma (291) . it is therefore possible that testosterone contributes to the chronic inflammatory state that accompanies high bmi and that the metabolic dysfunction overpowers the protective effects of testosterone on asthma development. few cellular and molecular studies have endeavored to uncover mechanisms that explain the association between asthma and pcos. at the cellular level, circulating monocytes from women with pcos expressed the receptor for advanced glycation endproducts (rage) more strongly than monocytes from healthy control women (292) . ages are involved in the pathogenesis of a number of chronic lung diseases, ranging from cystic fibrosis to asthma. rage can also bind other alarmins, such as the s100a8/a9 heterodimer (calprotectin) or the high-mobility group box (hmgb)1 protein. both of these ligands have been implicated in the pathogenesis of allergic asthma (291, 293) , as they induce cell proliferation or apoptosis, inflammation, collagen synthesis, and cell migration in many different cell types. the concentration of age proteins and testosterone correlated positively, even after controlling for bmi and other metabolic function tests (292) . taken together these two studies suggest that monocytes from women with pcos would be more responsive to rage ligands. this heightened responsiveness could promote cellular inflammatory responses that contribute to asthma pathogenesis. studies are needed to examine how the increased testosterone in women with pcos affects circulating monocytes and lung macrophages to increase asthma prevalence in this group. testosterone has been administered therapeutically for asthma. in an early study, asthmatic women were given testosterone either daily for 5 days over 2 weeks or daily for 3 days over 2 or more weeks. although the number of participants in the study was small, 88% saw improved symptoms, with 47% reporting no asthma attacks up to 3 months later (294, 295) . no studies have examined the effect of exogeneous testosterone administration on blood monocytes or lung macrophages in men and women with asthma. testosterone deficiency is also present in patients with copd (296) (297) (298) (299) (300) (301) (302) (303) (304) . in a clinical study of exercise and testosterone injection in men with copd, the interventions did not significantly alter pulmonary function or blood gas variables (305) . on the other hand, a retrospective study of two large cohorts of men who commenced testosterone replacement therapy within 12 months of a copd diagnosis showed a 4.2-9.1% decrease in hospitalizations, dependent on age (306) . more work is needed to understand how testosterone and its signaling pathways can be harnessed to alleviate lung disease without affecting reproductive systems or having unwanted metabolic effects. asthmatic patients have decreased serum concentration of dhea and dhea-s (307) (308) (309) . therefore, some clinical trials have tested whether dhea-s supplementation reduces asthma. men and women with poorly controlled moderate-to-severe asthma were given nebulized dhea-s for 6 weeks. this treatment led to a statistically significant improvement in the asthma control questionnaire (acq) and trends toward better asthma symptom scores and more symptom-free days and nights (310) . oral dhea for 2 weeks improved lung function in asthmatic women with low dhea-s < 200 µg/dl (48) . however, neither of these clinical studies examined the cellular component of the disease pre-or post-intervention. dhea and dhea-s are also lower in patients with copd than in healthy controls, and copd leads to pulmonary hypertension (ph). dhea supplementation improved the 6-min walk test, pulmonary hemodynamics, and the diffusing capacity of the lungs for carbon monoxide of patients with ph-copd (311, 312) . the therapeutic potential of dhea is currently being investigated in 24 patients with ph in the ediphy (effects of dhea in pulmonary hypertension) trial. however, outcome measures of this trial do not include examination of the immune cells or the effect of dhea treatment on those cells. analysis of immune cell function would add important cellular mechanistic insight to these types of trials and help uncover some of the widespread effects of this hormone on the immune system. modulation of monocyte and macrophage function mediated by the interaction of androgen and ar has been examined mostly by correlative studies in humans following lifespan changes in sex hormones or using hormonal manipulation in mouse models of lung disease. most human-based reports are merely descriptive or correlative and do not consider variables such as age, bmi, and phase of the menstrual cycle as key modulators of circulating sex hormone concentrations. taking these factors into account should be encouraged if we are to gain a better understanding of the impact of sex hormones in health and disease. analyses of the function of immune cells from male and female healthy controls and patients with lung diseases are needed to unlock how sex hormones alter the biology of the innate and adaptive immune response. studying the role of sex hormones as modulators of the immune system is complex because they interact with other hormonal systems and with one another, and because of the nearly ubiquitous expression of sex hormone receptors in most cells of the body. males and females have all types of sex steroids, although in different circulating concentrations. in humans, changes in the concentration of sex steroids have implications for lung health and may contribute to disease by affecting the function of the immune system. female sex hormones have been more widely studied as immune system modulators than have androgens. more focus in the future must be directed to how androgens affect the immune system and the interaction between male and female sex steroids in immune function. historically, animal models have used only males as study subjects, leaving females aside out of concern for the variability in results introduced by sexually mature adult females with active estrous cycles. as a result, biomedical and preclinical research has neglected to reflect more than 50% of the world's population. this omission had some notable negative consequences: eight of ten drugs withdrawn by the fda between 1997 and 2000 had significant health risks to women (313) . it was not until 2016 that the nih addressed this oversight with its requirement to include sex as a biological variable in all research studies (314) . the practices of using only male animals, not clearly reporting the sex (and age) of the animals used, and mixing male and female results have obscured a proper understanding of how sex and sex hormones influence normal biology and that of disease states. moreover, many reports comparing sex as a variable lack strict controls on culture conditions in vitro, which can alter the results. for example, if investigators fail to appreciate that animal serum or ph indicators, such as phenol red, may act as a source of steroids or sex hormone receptor agonists and do not clearly report their use, the interpretation and reproducibility of the experiments can be diminished. we strongly advocate for the use of hormone-free serum or animal serum replacements (for human cell studies) and use of culture medium that does not contain sex steroid receptor agonists. moreover, rigorous experimentation should include careful and detailed reporting of cell culture conditions, donor sex and age for cell studies, accurate age and sex in animal work (adherence to arrive guidelines), and separate male and female results. here, we have highlighted the importance of sex hormones as modulators of monocytes and macrophages and the important role of these innate immune cells in lung diseases where sex differences are apparent. these cells are part of a larger response that includes the adaptive immune system as well as the structural cells of the lung that are all affected by the action of sex steroids. as such, how innate cells like monocytes and macrophages shape the pulmonary immune response and how they resolve lung inflammation differently in the male and female lung and in the presence of different sex steroids needs intensive study. uncovering the cellular and molecular mechanisms will be crucial for finding new ways to treat different lung diseases depending on the sex of the patient. mb-d, ms, and nh wrote and revised the manuscript, interpreted the literature, approved and are accountable for all aspects of the final version. all authors contributed to the article and approved the submitted version. funding nih (nhlbi) r01 hl124477 (to nh). dept of defense cdmrp w81xwh-16-1-0509 (to nh). overview of the immune response androgen-induced immunosuppression innate lymphoid cells: 10 years on adaptive immunity immunological memory: lessons from the past and a look to the future immunity to fungal infections human immune system variation androgen-dependent immune modulation in parasitic infection the steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals extra-gonadal sites of estrogen biosynthesis and function human reproductive biology clinical biochemistry of dihydrotestosterone serum testosterone in women as measured by an automated immunoassay and a ria harmonized reference ranges for circulating testosterone levels in men of four cohort studies in the united states and europe estrogen-and progesterone-mediated structural neuroplasticity in women: evidence from neuroimaging sex affects immunity the x chromosome in immune functions: when a chromosome makes the difference ecology and evolution. sex differences in mortality rate sex disparities in cancer mortality and survival gender differences in cancer susceptibility: an inadequately addressed issue sex differences in immune responses available online at systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination american lung association: lung disease data women and lung disease. sex differences and global health disparities epidemiology of hyaline membrane disease in the united states: analysis of national mortality statistics factors affecting the sex differential in neonatal mortality: the role of respiratory distress syndrome very low birth weight outcomes of the national institute of child health and human development neonatal research network preterm male infants need more initial respiratory and circulatory support than female infants gender differences in asthma in childhood and adolescence effects of endogenous sex hormones on lung function and symptom control in adolescents with asthma asthma, the sex difference gender differences in adult-onset asthma: results from the swiss sapaldia cohort study epidemiology of asthma in children and adults. front pediatr airway inflammation and remodeling in two mouse models of asthma: comparison of males and females female mice are more susceptible to the development of allergic airway inflammation than male mice higher serum testosterone and dihydrotestosterone, but not oestradiol, are independently associated with favourable indices of lung function in community-dwelling men the serum testosterone level of patients with bronchial asthma treated with corticosteroids and untreated mechanical stress is communicated between different cell types to elicit matrix remodeling elevated testosterone is associated with decreased likelihood of current asthma regardless of gender effects of sex hormones on bronchial reactivity during the menstrual cycle sex steroid hormones and asthma in a nationwide study of us adults high-fat diet-induced obesity worsens th2 immune response and immunopathologic characteristics in murine model of eosinophilic oesophagitis diet and metabolism in the evolution of asthma and obesity obesity and severe asthma dehydroepiandrosterone supplementation may benefit women with asthma who have low androgen levels: a pilot study serum dehydroepiandrosterone sulfate concentration as an indicator of adrenocortical suppression during inhaled steroid therapy in adult asthmatic patients dehydroepiandrosterone enhances il2 production and cytotoxic effector function of human t cells preliminary studies on the effect of dehydroepiandrosterone (dhea) on both constitutive and phytohaemagglutinin (pha)-inducible il-6 and il-2 mrna expression and cytokine production in human spleen mononuclear cell suspensions in vitro gender difference, sex hormones, and immediate type hypersensitivity reactions dehydroepiandrosterone modulation of lipopolysaccharide-stimulated monocyte cytotoxicity chronic obstructive pulmonary disease: a palliative medicine review of the disease, its therapies and drug interactions mechanism of lung injury caused by pm10 and ultrafine particles with special reference to copd cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview global burden of copd: risk factors, prevalence, and future trends gender differences in copd: are women more susceptible to smoking effects than men? female smokers beyond the perimenopausal period are at increased risk of chronic obstructive pulmonary disease: a systematic review and meta-analysis sex differences in physiological progression of idiopathic pulmonary fibrosis incidence and mortality of idiopathic pulmonary fibrosis and sarcoidosis in the uk mortality from pulmonary fibrosis increased in the united states from 1992 to the pathogenesis of pulmonary fibrosis: a moving target excess pneumonia-influenza mortality by age and sex in three major influenza a2 epidemics sex-and age-related differences in morbidity rates of 2009 pandemic influenza a h1n1 virus of swine origin in japan critically ill patients with 2009 influenza a(h1n1) infection in canada epidemiologic and clinical features among patients hospitalized in wisconsin with 2009 h1n1 influenza a virus infections high mortality from respiratory failure secondary to swineorigin influenza a (h1n1) in south africa is there a difference in the immune response, efficacy, effectiveness and safety of seasonal influenza vaccine in males and females? -a systematic review lower mortality from h1n1 influenza in older argentineans: men more affected elevated 17beta-estradiol protects females from influenza a virus pathogenesis by suppressing inflammatory responses 17β-estradiol protects females against influenza by recruiting neutrophils and increasing virus-specific cd8 t cell responses in the lungs age and testosterone mediate influenza pathogenesis in male mice sexual inequality in tuberculosis sex differences in tuberculosis gender and tuberculosis control: towards a strategy for research and action biological differences between the sexes and susceptibility to tuberculosis tuberculosis and lung cancer lung cancer statistics epidemiology of lung cancer patterns in lung cancer incidence rates and trends by histologic type in the united states us lung cancer trends by histologic type higher lung cancer incidence in young women than young men in the united states sex differences in lung cancer susceptibility: a review differences in lung cancer risk between men and women: examination of the evidence incidence trends and gender differences in malignant mesothelioma in new south wales sex differences in cancer mechanisms the estrogen pathway as a modulator of response to immunotherapy alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf induction of the nuclear receptor ppar-γ by the cytokine gm-csf is critical for the differentiation of fetal monocytes into alveolar macrophages yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span the cytokine tgf-β promotes the development and homeostasis of alveolar macrophages sex differences in the response of the alveolar macrophage proteome to treatment with exogenous surfactant protein-a in vivo rescue of alveolar macrophages from sp-a knockout mice with exogenous sp-a nearly restores a wild type intracellular proteome; actin involvement flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues flow cytometry reveals similarities between lung macrophages in humans and mice a cellular census of human lungs identifies novel cell states in health and in asthma single-cell transcriptomic analysis of human lung provides insights into the pathobiology of pulmonary fibrosis exposure to bacterial cpg dna protects from airway allergic inflammation by expanding regulatory lung interstitial macrophages developmental origin of lung macrophage diversity lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice il-10-producing lung interstitial macrophages prevent neutrophilic asthma the microbial environment and its influence on asthma prevention in early life hay fever, hygiene, and household size three unique interstitial macrophages in the murine lung at steady state two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung purification and characterization of mouse hematopoietic stem cells role of lipid rafts in hematopoietic stem cells homing, mobilization, hibernation, and differentiation subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response delivery of mir-146a to ly6c(high) monocytes inhibits pathogenic bone erosion in inflammatory arthritis blood monocytes consist of two principal subsets with distinct migratory properties minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes critical roles for ccr2 and mcp-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor ccr2 impaired monocyte migration and reduced type 1 (th1) cytokine responses in c-c chemokine receptor 2 knockout mice homeostasis in the mononuclear phagocyte system monocyte differentiation and antigen-presenting functions ccr2+ monocytederived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality dendritic cells and monocytes with distinct inflammatory responses reside in lung mucosa of healthy humans retention of leukocytes in capillaries: role of cell size and deformability intravascular staining for discrimination of vascular and tissue leukocytes flow cytometric analysis of mononuclear phagocytes in nondiseased human lung and lung-draining lymph nodes estrogen receptor-dependent regulation of dendritic cell development and function. front immunol hormonal modulation of dendritic cells differentiation, maturation and function: implications for the initiation and progress of systemic autoimmunity sex hormones regulate innate immune cells and promote sex differences in respiratory virus infection the development and function of lungresident macrophages and dendritic cells dendritic cells in lung immunopathology identification of interferongamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity macrophage polarization: different gene signatures in m1(lps+) vs classically and m2(lps-) vs. alternatively activated macrophages the chemokine system in diverse forms of macrophage activation and polarization myeloid colony-stimulating factors as regulators of macrophage polarization inhibition of nitric oxide (no) production in lipopolysaccharide (lps)-activated murine macrophage raw 264.7 cells by the norsesterterpene peroxide, epimuqubilin a macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm macrophage plasticity and polarization: in vivo veritas interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation a chitinaselike protein in the lung and circulation of patients with severe asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease alternatively activated macrophages and airway disease broncho-alveolar macrophages express chemokines associated with leukocyte migration in a mouse model of asthma alternative macrophage activation is increased in asthma phenotypic characterization of lung macrophages in asthmatic patients: overexpression of ccl17 more alternative activation of macrophages in lungs of asthmatic patients chop deficiency protects mice against bleomycin-induced pulmonary fibrosis by attenuating m2 macrophage production the nature of small-airway obstruction in chronic obstructive pulmonary disease cellular and molecular mechanisms of asthma and copd increased levels of the chemokines groα and mcp-1 in sputum samples from patients with copd identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease impaired innate immune alveolar macrophage response and the predilection for copd exacerbations tobacco smoking inhibits expression of proinflammatory cytokines and activation of il-1r-associated kinase, p38, and nf-kappab in alveolar macrophages stimulated with tlr2 and tlr4 agonists carbonylation caused by cigarette smoke extract is associated with defective macrophage immunity effects of cigarette smoke on toll-like receptor (tlr) activation of chronic obstructive pulmonary disease (copd) macrophages chemokine expression by small sputum macrophages in copd cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine differential gene expression analysis in human monocyte-derived macrophages: impact of cigarette smoke on host defence cigarette smoke exposure attenuates cytokine production by mouse alveolar macrophages dysregulated functions of lung macrophage populations in copd a circulating cell population showing both m1 and m2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement polarization of human monocyte-derived cells with vitamin d promotes control of mycobacterium tuberculosis infection involvement of granulocyte-macrophage colonystimulating factor in pulmonary homeostasis gm-csf regulates alveolar macrophage differentiation and innate immunity in the lung through targeted ppar{gamma} deficiency in alveolar macrophages disrupts surfactant catabolism systemic analysis of ppargamma in mouse macrophage populations reveals marked diversity in expression with critical roles in resolution of inflammation and airway immunity granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology peroxisome proliferator-activated receptor-gamma is deficient in alveolar macrophages from patients with alveolar proteinosis idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor familial pulmonary alveolar proteinosis caused by mutations in csf2ra pulmonary alveolar proteinosis caused by deletion of the gm-csfrα gene in the x chromosome pseudoautosomal region 1 human gm-csf autoantibodies and reproduction of pulmonary alveolar proteinosis a critical function for cd200 in lung immune homeostasis and the severity of influenza infection androgen receptor structure, function and biology: from bench to bedside molecular biology of the androgen receptor: from molecular understanding to the clinic testosterone stimulates intracellular calcium release and mitogen-activated protein kinases via a g protein-coupled receptor in skeletal muscle cells nongenomic androgen activation of phosphatidylinositol 3-kinase/akt signaling pathway in mc3t3-e1 osteoblasts androgens promote maturation and signaling in mouse oocytes independent of transcription: a release of inhibition model for mammalian oocyte meiosis androgens are effective bronchodilators with anti-inflammatory properties: a potential alternative for asthma therapy androgen receptor: structure, role in prostate cancer and drug discovery androgen receptor splice variants and prostate cancer: from bench to bedside androgen receptor-dependent and -independent mechanisms driving prostate cancer progression: opportunities for therapeutic targeting from multiple angles androgen receptor-mediated inhibition of cutaneous wound healing foxo3, estrogen receptor alpha, and androgen receptor impact tumor growth rate and infiltration of dendritic cell subsets differentially between male and female mice androgen and androgen receptor as enhancers of m2 macrophage polarization in allergic lung inflammation modulation of leishmania donovani infection and cell viability by testosterone in bone marrow-derived macrophages: signaling via surface binding sites sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury androgen receptor deficiency in monocytes/macrophages does not alter adiposity or glucose homeostasis in male mice monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local tnf-alpha expression dihydrotestosterone increases cytotoxic activity of macrophages on prostate cancer cells via trail macrophage may responses to androgen via its receptor testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages androgen receptor expression is greater in macrophages from male than from female donors. a sex difference with implications for atherogenesis oral contraceptives modify dna methylation and monocyte-derived macrophage function new therapy via targeting androgen receptor in monocytes/macrophages to battle atherosclerosis effect of testosterone on expression of androgen receptor in human monocytic cell line thp-1 androgen and estrogen receptors are present in primary cultures of human synovial macrophages androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer androgenmediated modulation of macrophage function after trauma-hemorrhage: central role of 5alpha-dihydrotestosterone sex hormones modulate inflammatory mediators produced by macrophages testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages targeting the androgen receptor with sirna promotes prostate cancer metastasis through enhanced macrophage recruitment via ccl2/ccr2-induced stat3 activation steroid sex hormones and macrophage function: regulation of chemiluminescence and phagocytosis upregulation of fas and fasl expression in testosterone-induced apoptosis of macrophages testosterone induces apoptosis via fas/fasl-dependent pathway in bone marrowderived macrophages testosterone regulates 3t3-l1 pre-adipocyte differentiation and epididymal fat accumulation in mice through modulating macrophage polarization testosterone reduces macrophage expression in the mouse of toll-like receptor 4, a trigger for inflammation and innate immunity inhibition of lncap prostate cancer cells by means of androgen receptor antisense oligonucleotides linking prostate cancer cell ar heterogeneity to distinct castration and enzalutamide responses role of androgen receptor in progression of lncap prostate cancer cells from g1 to s phase non-classical actions of testosterone: an update 216. randolph gj. mechanisms that regulate macrophage burden in atherosclerosis the role of lipids and lipoproteins in atherosclerosis sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line modulation of cell growth and apoptosis by sex hormones in cultured monocytic thp-1 cells effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, u937 estrogen regulates cytokine production and apoptosis in pmadifferentiated, macrophage-like u937 cells the effect of steroids on differentiation and function of cultured, mononuclear cells testosterone suppresses anti-dna antibody production in peripheral blood mononuclear cells from patients with systemic lupus erythematosus testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells effect of estradiol on cytokine secretion by proteolipid protein-specific t cell clones isolated from multiple sclerosis patients and normal control subjects the role of cbp in estrogen receptor cross-talk with nuclear factor-kappab in hepg2 cells estradiol and testosterone influence the mrna expression and the time course of inflammatory cytokines in activated human monocytic cell line immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated hl-60 cells, granulocytes, and monocytes inflammation-mediated abrogation of androgen signaling: an in vitro model of prostate cell inflammation sex hormone modulation of proinflammatory cytokine and c-reactive protein expression in macrophages from older men and postmenopausal women hormonal regulation of complement biosynthesis in human cell lines-i. androgens and gamma-interferon stimulate the biosynthesis and gene expression of c1 inhibitor in human cell lines u937 and hepg2 testosterone suppresses phospholipase d, causing sex differences in leukotriene biosynthesis in human monocytes cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells production of pge2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors effects of sex hormones on production of prostaglandin e2 by human peripheral monocytes activation of human monocytes by lps and dhea role of androgens in dhea-induced rack1 expression and cytokine modulation in monocytes rack1, a multifaceted scaffolding protein: structure and function dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis effects of dehydroepiandrosterone on th2 cytokine production in peripheral blood mononuclear cells from asthmatics lipid uptake by alveolar macrophages drives fibrotic responses to silica dust regulation of macrophage foam cell formation during nitrogen mustard (nm)-induced pulmonary fibrosis by lung lipids lipid-laden macrophages as biomarkers of vaping-associated lung injury storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific sex differences in the prevalence of, and trends in, cardiovascular risk factors, treatment, and control in the united states sex differences in inflammation during atherosclerosis androgens up-regulate atherosclerosis-related genes in macrophages from males but not females: molecular insights into gender differences in atherosclerosis effect of sex hormones on levels of mrnas coding for proteins involved in lipid metabolism in macrophages testosterone up-regulates scavenger receptor bi and stimulates cholesterol efflux from macrophages lamon-fava s. the effect of 17beta-estradiol on cholesterol content in human macrophages is influenced by the lipoprotein milieu comparative effect of the calcium antagonist verapamil and the synthetic steroids gestrinone and danazol on human monocyte phagocytosis in vitro physiological concentration of 17 beta-estradiol inhibits chemotaxis of human monocytes in response to monocyte chemotactic protein 1 effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes male-female differences in the cytotoxic activity of human monocytes in vitro estrogen signaling contributes to sex differences in macrophage polarization during asthma oral exposure to low dose bisphenol a aggravates allergic airway inflammation in mice sex differences and sex steroids in lung health and disease arginase-1 and ym1 are markers for murine, but not human, alternatively activated myeloid cells eosinophilic crystalline pneumonia as a major cause of death in 129s4/svjae mice testosterone decreases house dust mite-induced type 2 and il-17a-mediated airway inflammation testosterone attenuates group 2 innate lymphoid cell-mediated airway inflammation androgen signaling negatively controls group 2 innate lymphoid cells steroid sex hormones regulate the release of tumor necrosis factor by macrophages morphometric comparisons of rat alveolar macrophages, pulmonary interstitial macrophages, and blood monocytes sex differences in airway remodeling in a mouse model of chronic obstructive pulmonary disease sex-specific differences in emphysema using a murine antisense oligonucleotide model of α-1 antitrypsin deficiency antibody responses and cross protection against lethal influenza a viruses differ between the sexes in c57bl/6 mice sex differences in h7n9 influenza a virus pathogenesis progesterone-based therapy protects against influenza by promoting lung repair and recovery in females lymphocyte-dependent inhibition of growth of virulent mycobacterium tuberculosis h37rv within human monocytes: requirement for cd4+ t cells in purified protein derivativepositive, but not in purified protein derivative-negative subjects the influence of sex steroid hormones in the immunopathology of experimental pulmonary tuberculosis tumor-associated macrophages of the m2 phenotype contribute to progression in gastric cancer with peritoneal dissemination targeting macrophages: therapeutic approaches in cancer perivascular m2 macrophages stimulate tumor relapse after chemotherapy combining the multitargeted tyrosine kinase inhibitor vandetanib with the antiestrogen fulvestrant enhances its antitumor effect in non-small cell lung cancer regulation of endogenous gene expression in human non-small cell lung cancer cells by estrogen receptor ligands human asthma is characterized by more irf5+ m1 and cd206+ m2 macrophages and less il-10+ m2-like macrophages around airways compared with healthy airways persistence of androgenic effects on the production of proinflammatory cytokines by circulating antigen-presenting cells after withdrawal of testosterone treatment in aging type 2 diabetic men with partial androgen deficiency androgen-replacement therapy depresses the ex vivo production of inflammatory cytokines by circulating antigen-presenting cells in aging type-2 diabetic men with partial androgen deficiency testosterone replacement therapy in hypogonadal men is associated with increased expression of lamp-2 (cd107b) by circulating monocytes and dendritic cells klinefelter syndrome: integrating genetics, neuropsychology, and endocrinology morbidity in klinefelter syndrome: a danish register study based on hospital discharge diagnoses klinefelter syndrome and bronchial asthma: is there any relationship between the low testosterone levels and asthma exacerbations? a case of klinefelter's syndrome with refractory asthma, diabetes mellitus and rib fracture klinefelter's syndrome with asthma the number of x chromosomes influences inflammatory cytokine production following toll-like receptor stimulation serum free testosterone in polycystic ovary syndrome measured with a new reference method total testosterone assays in women with polycystic ovary syndrome: precision and correlation with hirsutism medical comorbidity in polycystic ovary syndrome with special focus on cardiometabolic, autoimmune, hepatic and cancer diseases: an updated review asthma in reproductive-aged women with polycystic ovary syndrome and association with obesity emerging role of hmgb1 in lung diseases: friend or foe increased levels of serum advanced glycation end-products in women with polycystic ovary syndrome s100a8/a9: a mediator of severe asthma pathogenesis and morbidity? can testosterone therapy in bronchial asthma testosterone and asthma sex hormone alterations and systemic inflammation in chronic obstructive pulmonary disease the endocrinologic changes in critically ill chronic obstructive pulmonary disease patients reduced pulmonary function is associated with lower levels of endogenous total and free testosterone. the tromso study hypogonadism, quadriceps weakness, and exercise intolerance in chronic obstructive pulmonary disease testosterone levels in men with chronic obstructive pulmonary disease with or without glucocorticoid therapy hypogonadism in men with chronic obstructive pulmonary disease: prevalence and quality of life androgens and chronic obstructive pulmonary disease hypogonadism in elderly men-what to do until the evidence comes effects of testosterone and resistance training in men with chronic obstructive pulmonary disease testosterone replacement therapy and hospitalization rates in men with copd decreased adrenal sex steroid levels in the absence of glucocorticoid suppression in postmenopausal asthmatic women adrenocortical function in bronchial asthma dehydroepiandrosterone sulphate concentrations in asthmatic patients: pilot study nebulized dehydroepiandrosterone-3-sulfate improves asthma control in the moderate-to-severe asthma results of a 6-week, randomized, double-blind, placebo-controlled study dhea) improves pulmonary hypertension in chronic obstructive pulmonary disease (copd): a pilot study association of adrenal hormone metabolites and mortality over a 6-year follow-up in copd patients with acute exacerbation sex as an important biological variable in biomedical research sex as a biological variable: a 5-year progress report and call to action key: cord-252810-rko3e5va authors: basil, maria c.; katzen, jeremy; engler, anna e.; guo, minzhe; herriges, michael j.; kathiriya, jaymin j.; windmueller, rebecca; ysasi, alexandra b.; zacharias, william j.; chapman, hal a.; kotton, darrell n.; rock, jason r.; snoeck, hans-willem; vunjak-novakovic, gordana; whitsett, jeffrey a.; morrisey, edward e. title: the cellular and physiological basis for lung repair and regeneration: past, present, and future date: 2020-04-02 journal: cell stem cell doi: 10.1016/j.stem.2020.03.009 sha: doc_id: 252810 cord_uid: rko3e5va the respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. in this review and as members of the nih/nhlbi-supported progenitor cell translational consortium, we discuss key aspects of lung repair and regeneration. we focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. we also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues. the respiratory system is organized into multiple integrated compartments comprising multiple tissues that perform gas exchange between the blood and the external environment. the various anatomical regions of the respiratory tract are populated by numerous types of unique epithelial, vascular, mesenchymal, and immune cells critical for the functioning of each particular compartment. historically, the development of the respiratory system has been thought to involve several discrete morphogenetic steps including lineage specification, branching morphogenesis, sacculation, and alveologenesis (morrisey and hogan, 2010) . while these steps were previously conceived of in terms of distinct temporal stages of development, more recent evidence has suggested that there is overlap between these stages and particular events such as cell specification and commitment, which are now thought to occur very early and coincident with the basic patterning of the respiratory airway tree (frank et al., 2019) . the branched network of airways and gas exchange surfaces co-develops with the cardiovascular system to bring both organ systems into intimate proximity for full functionality. more details on these important developmental events can be found in several recent reviews (herriges and morrisey, 2014; hines and sun, 2014; morrisey and hogan, 2010; nikoli c et al., 2018; whitsett et al., 2019; zepp and morrisey, 2019) . the culmination of these events is the generation of an extensive surface area for efficient gas exchange that in the human lung comprises approximately 70 m 2 . this review will focus on how the mature respiratory system maintains its normal homeostatic structure and function and how it responds to injury and regenerates itself. we will explore the cellular constituents of the two major compartments in the lungs-the gas exchange alveoli and the conducting airways including the trachea-and describe established and emerging techniques to explore human lung regeneration. the lung alveolus is composed of multiple epithelial, endothelial, and mesenchymal cell types (figure 1 ). in addition to these resident cell types, the alveolus also is inhabited by several immune cell lineages, including alveolar macrophages, interstitial macrophages, and dendritic cells and several recent datasets have shown this diversity of cells at single-cell resolution in both animals and humans (guo et al., 2019; travaglini et al., 2019; vieira braga et al., 2019) . emerging data suggest there is some degree of inter-cellular communication between the lineages in this niche, but our understanding of the crosstalk among alveolar cell lineages during homeostasis or regeneration remains poor. the alveolar compartment remains largely quiescent in the uninjured lung, and most cells within this niche exhibit a relatively slow turnover. after lung injury, multiple alveolar cell types are able to proliferate, and when repair is effective both alveolar structure and function are restored. this ability to react to injury involves both activation of self-renewal as well as differentiation into more mature cell lineages. the self-renewal and differentiation of various lung epithelial cells are modulated by a growing list of cell types that includes neighboring epithelial cells, mesenchymal cells, airway smooth muscle, neurons and neuroendocrine cells, endothelium, and various leukocyte populations (barkauskas et al., 2013; cao et al., 2017; lechner et al., 2017; lee et al., 2017; rafii et al., 2015; zepp et al., 2017) . these studies have highlighted recurrent themes regarding the signals that can drive alveolar epithelial regeneration, including wnt signaling. alveolar epithelial response to injury. there are two primary lineages in the alveolar epithelium: the alveolar epithelial type 1 (at1) and type 2 (at2) cells (figure 1 ). at1 cells cover 95% of the alveolar surface area and are closely juxtaposed with the capillary plexus. at2s are responsible for generating pulmonary surfactant, which is essential for reducing the surface tension of the alveolar surface area to prevent the lungs from collapsing upon every breath. at1 and at2 cells are specified very early in lung development and at2 cells do not appreciably generate figure 1 . alveolar cell lineages involved in lung repair and regeneration (a) the human distal airways connect with the alveolar niche through a transitional respiratory airway (also called the respiratory bronchiole or rb) region. the rb is lined with a simple but poorly characterized cuboidal epithelium while the more intermediate airways exhibit a pseudostratified epithelium containing secretory, goblet, and ciliated cells that may exhibit as yet distinct heterogeneity. of note, basal cells are found in human intermediate and respiratory airways. (b) mice do not have respiratory bronchioles and transition from the intermediate airways, which exhibit a pseudostratified nature but lack basal cells, into the alveolar region. the distal badj region in the mouse lung, which is not found in the human lung, contains the basc population. the architecture and cell lineages found in both the mouse and human lungs are very similar and contain both at1 and at2 epithelial lineages as well as various mesenchymal lineages and vascular endothelial cells. (c) the various cell types found in the distal airways and alveolus of the human and mouse lung. at1 cells during early postnatal lung growth (frank et al., 2016 (frank et al., , 2019 . however, in adult mice, at2 cells can act as both a selfrenewing stem cell like population and regenerating at1 cells after injury (barkauskas et al., 2013) . a sublineage within the at2 cell population that expresses the transcriptional target of wnt signaling, axin2, has been shown to play a dominant role in repairing the lung alveolus after acute injury (nabhan et al., 2018; zacharias et al., 2018) . these cells, which have been called alveolar epithelial progenitors or aeps, preferentially re-enter the cell cycle after injury, self-renew, and regenerate mature at1 and at2 cells. aeps appear primed to enter the cell cycle and they respond robustly to wnt and fgf7 signaling (zacharias et al., 2018) . in alveolar development, wnt-responsive at2 cells selfrenew in the presence of wnt signaling and differentiate in its absence (frank et al., 2016) , but it is not yet known whether these same signals drive renewal versus differentiation during regeneration. a recent report also shows that aeps promote metastasis in models of lung cancer (laughney et al., 2020) . aeps have been identified in the human lung and are responsible for generating the majority of at2 cell growth in human alveolar organoids (zacharias et al., 2018) . in contrast to at2s and aeps, the at1 cell contribution to alveolar epithelial regeneration is thought to be very limited (jain et al., 2015) . following the surgical removal of lung tissue in adult mice and other model organisms, new alveoli are formed to compensate for lost alveolar surface area (buhain and brody, 1973) . this compensatory lung growth after partial pneumonectomy is commonly used as a ''sterile'' model of lung regeneration. not surprisingly, this regenerative response involves the coordinated actions of nearly every cell type in the lung including epithelial cells, endothelial cells, mesenchymal cells, and leukocytes (chen et al., 2012; jain et al., 2015; lechner et al., 2017; rafii et al., 2015) . in this model, a small number of cells expressing hopx + , a marker for the at1 lineage, were found to both proliferate and, in rare instances, give rise to sftpc + at2 cells (jain et al., 2015) . a recent study revealed heterogeneity within the at1 cell population where expression of igfbp2 marked the most mature at1 cell subtype that lacks differentiation capacity following pneumonectomy . although cells expressing at1 markers proliferate during compensatory lung growth after partial pneumonectomy, whether bona fide at1 cells are able to contribute to repair after acute lung injury is largely unknown. alveolar endothelial response to injury. effective gas exchange is dependent upon at1 cell and pulmonary capillary endothelial cell (pcec) proximity, and successful alveolar regeneration requires re-establishment of this spatial relationship. following lung injury in rodents, there is rapid proliferation in the microvasculature, with expansion of resident microvascular endothelial progenitor cells . these cells are marked by cd34 and cd309, and in organoid culture demonstrate significant vasculogenic capacity. while both the mechanisms of pcec regeneration and cellular identities within this compartment are incompletely understood, endothelial cells expressing sox17 have recently been shown to play a role in endothelial regeneration after endotoxic-induced vascular injury (liu et al., 2019a) . additionally, pcecs enhance alveologenesis following injury, with pcec-derived vegfr2 and fgfr1 mediating epithelial proliferation (ding et al., 2011) . this signaling is thought to be through mmp14, and possibly co-mediated through platelet activation of endothelial sdf-1 receptors (rafii et al., 2015) . significant additional heterogeneity is thought to exist within the distal lung endothelium, with differential vasculogenic capacities and crosstalk with the epithelium , and this has recently been revealed in single-cell transcriptomic studies of the homeostatic lung and its response to acute injury (niethamer et al., 2020) . this study demonstrated the presence of multiple microvascular endothelial subsets including one which expresses high levels of car4 and cd34. this study also showed that a population of proliferating endothelial cells emerges soon after influenza injury and single-cell informatic trajectory analysis suggests that these cells arise from multiple endothelial subsets. future work is needed to define the functional role for these endothelial subsets in both normal alveolar homeostasis and the response to injury. alveolar mesenchymal response to injury. the lung alveolus is also home to a complex mixture of mesenchymal cell types, many of which are in close physical association with both alveolar epithelial and endothelial cells and play an active role in alveolar epithelial regeneration. pioneering electron microscopy studies demonstrated direct and extensive contacts between lung fibroblasts and at2 cells (sirianni et al., 2003; walker et al., 1995) . mesenchymal cells expressing the platelet-derived growth factor alpha (pdgfra) are often found in close association with at2 cells (barkauskas et al., 2013; green et al., 2016; zepp et al., 2017) . the first functional evidence of trophic interactions between these populations was the observation that pdgfra + fibroblasts support the growth and differentiation of at2s in an alveolar organoid co-culture assay (barkauskas et al., 2013) . alveolar organoids have been recently used to provide functional evidence that multiple signaling pathways originate in pdgfra + cells to influence at2 cell self-renewal and differentiation into at1 cells including fgf7, bmp, and il6 (chung et al., 2018; green et al., 2016; zepp et al., 2017) . as molecular techniques have evolved, an emerging literature has revealed molecular and functional heterogeneity of lung alveolar mesenchymal cells. in particular, accumulating data demonstrate the previously underappreciated heterogeneity of cells within the pdgfra + population of fibroblasts in the adult (green et al., 2016; zepp et al., 2017) . single-cell transcriptome analysis combined with spatial distance mapping recently demonstrated that one subpopulation of fibroblasts, defined by the co-expression of axin2 and pdgfra, is localized particularly close to at2 cells and provides signals including il6, fgfs, and bmp antagonists that promote the selfrenewal and differentiation of at2s (zepp et al., 2017) . this fibroblast population has been named the mesenchymal alveolar niche cell or manc and these cells are thought to have a critical role in homeostatic alveolar regeneration following injury (zepp et al., 2017) . immune response to alveolar injury. resident and circulating leukocytes are thought to also play a critical role in alveolar repair and regeneration, with elegant in vivo and in vitro studies demonstrating that ''inflammatory'' cytokines have direct effects on the proliferation and differentiation of both airway and alveolar epithelial cells (danahay et al., 2015; katsura et al., 2019; kuperman et al., 2002; tadokoro et al., 2014; xie et al., 2018) . however, our understanding of the interactions between alveolar epithelial cells and resident or circulating leukocytes is in its infancy. macrophages, the primary resident immune cell of the alveolus, begin to populate the lung during embryonic lung development (tan and krasnow, 2016) . lps stimulation of these early resident macrophages leads to impaired branching morphogenesis, attributed to changes in integrin, bmp, and wnt signaling (blackwell et al., 2011) . whether unstimulated resident macrophages have a normal role in branching morphogenesis or the differentiation of alveolar epithelial cells remains to be elucidated. however, there are multiple lines of evidence indicating that leukocytes play important roles in adult alveolar regeneration, mediated at least in part through bidirectional intercellular communication with alveolar epithelial cells. for example, following either chemical or infectious lung injury, resident alveolar macrophages can stimulate epithelial proliferation through the production of wnt ligands (hung et al., 2019) . a subset of alveolar macrophages can also act as memory macrophages and help guide the rapid activation of multiple chemokines including those that stimulate neutrophils . compensatory lung growth after partial pneumonectomy provides a relatively simple model to study pro-regenerative epithelial-immune interactions without the confounding effects of infection and inflammation. this model was used to demonstrate that platelets can initiate a regenerative cascade by secreting sdf1 after pneumonectomy. this stimulates capillary endothelial cells to express mmp14, releasing egf ligands from the extracellular matrix and subsequently promoting at2 cell proliferation and differentiation (rafii et al., 2015) . following partial pneumonectomy, local production of the chemokine ccl2 leads to the recruitment of ccr2 + monocytes to the lung (lechner et al., 2017) . these monocytes and resident macrophages can be polarized by il13 that is secreted by type 2 innate lymphoid cells, and loss of this axis impairs optimal compensatory lung growth after pneumonectomy. data from an in vitro co-culture assay suggest that macrophages can directly modulate at2 cell survival and self-renewal (lechner et al., 2017) . recruited ccr2 + monocytes have also been implicated in dysplastic alveolar repair following both bleomycininduced lung injury and other models of lung fibrosis (misharin et al., 2017; venosa et al., 2019) . these studies highlight the context-dependent roles of ccr2 + monocytes in both normal and abnormal lung regeneration. in infectious and more destructive lung injury models, it can be difficult to discern the inflammatory roles of leukocytes from regenerative roles, if such a distinction exists. nevertheless, data from a growing number of contexts have shown that resident and recruited immune cells are essential for resolution of lung injury. for example, the depletion of macrophages during the resolution phase of bleomycin-induced lung injury prolonged the fibrotic response and impaired resolution (gibbons et al., 2011) . this effect was attributed to a decrease in the clearance of accumulated extracellular matrix but could also involve disrupted communication with epithelial progenitor cells or other populations. other immune populations are activated or recruited to the alveolar niche following lung injury. we have nascent understandings of the inflammatory cellular diversity and intercellular communications that determine normal versus abnormal lung regeneration in response to lung injury. understanding this diversity and improving model systems, including more precise animal models and organoid and lung-on-a-chip models that incorporate immune cells, will allow us to study the contributions of immune cell communications that drive lung repair (gkatzis et al., 2018) . maintenance and regeneration of the proximal airway epithelium. the proximal airways in mice and humans are exposed to frequent insults from the environment and serve as the first line of immune and toxin defense in the lung, warming and filtering the air as it passes to more distal regions. much of what is understood about airway regeneration comes from studies of the mouse trachea, which most closely resembles the structure of human proximal airways. there are many parallels between the murine trachea and the human intrapulmonary airways, with the most relevant for this discussion being the presence of basal cells. in mice, basal cells reside in the trachea and proximal main stem bronchi; however, in humans this population extends for several airway generations (figures 1 and 2) . murine intrapulmonary airways are not pseudostratified and do not contain basal cells which are the primary stem cell population in human airways. thus, intrapulmonary mouse airways should not be used as a model system for the study of human airways. the pseudostratified upper airway and tracheal epithelium exhibits very slow turnover during health but several of the mature lineages are capable of re-entering the cell cycle to replenish loss of neighboring cells and maintain an epithelial barrier. basal cells in the proximal airways are the major stem cell population that selfrenew and when necessary give rise to multiple cell types such as secretory, goblet, and multi-ciliated cells (figure 2 ; hegab et al., 2012; hong et al., 2004; rock et al., 2009 rock et al., , 2011 . this process is critical for both maintenance and cellular regeneration after significant injury and is controlled by notch signaling (mori et al., 2015; rock et al., 2011; ruiz garcã­a et al., 2019; stupnikov et al., 2019) . although it was once thought that basal cells were a rather homogeneous population, recent findings reveal more complexity and have demonstrated their early origins in development . careful lineage tracing of cytokeratin 5 (krt5) basal cells over time indicated that at least two populations of basal cells exist in the upper airway, with one acting more as a self-renewing stem cell, and the other committed to luminal differentiation (watson et al., 2015) . consistent with this paradigm, depending on enhanced notch2 signaling or c-myb expression, basal stem cells directly give rise to secretory cells or multi-ciliated cells, respectively (pardo-saganta et al., 2015) . recent studies have uncovered additional complexity within the pseudostratified airway epithelium of mouse trachea and human large airway, with the description of new, rare cell types including cftr-rich ionocytes plasschaert et al., 2018) . multiple studies using lineage tracing analysis combined with single-cell transcriptomic work confirmed that the krt5 basal cell population was capable of giving rise to all the observed cell types within the airway, including the newly identified ionocyte, and other rare epithelial cell subsets such as the tuft cell, which is not normally present in uninjured mouse airways mori et al., 2015; rane et al., 2019; rock et al., 2011; ruiz garcã­a et al., 2019) . moreover, tuft cells, also known as solitary chemosensory cells, ectopically emerge after influenza injury in the mouse and may play a role in post-injury dysplastic remodeling of the lung (rane et al., 2019) . some of these lineage relationships in mice can be modeled in vitro with primary human cells in culture, highlighting the shared regenerative potential across species (rock et al., 2009) . while basal cells are a main driver of regeneration after airway injury, other cell types have been shown to contribute as facultative progenitors. lineage tracing analysis of airway scgb1a1 + cells revealed that secretory cells proliferate to help maintain the club cell population ( figure 2 ; rawlins et al., 2009; van keymeulen and blanpain, 2012) and are a major source of multi-ciliated cells in normal airways, particularly in the more distal murine airways where basal cells are not typically found. in addition, subsets of secretory cells expressing upk3a in mice, so-called variant-club cells, have been shown to be localized near neuroendocrine bodies, suggesting a possible niche, and are capable of giving rise to both secretory and ciliated cells in development; however, a role in the response to injury is not yet clear (guha et al., 2012 (guha et al., , 2017 . additionally, neuroendocrine (ne) cells also can function as facultative progenitors after airway injury, interact with immune lineages during expansion, and may harbor sublineages with enhanced progenitor capacity in ne bodies (branchfield et al., 2016; garg et al., 2019; ouadah et al., 2019) . in extreme situations, mature secretory/club cells (defined by scgb1a1 expression) have been reported to dedifferentiate in the setting of marked basal cell loss, and contribute to the basal stem cell pool, although the homeostatic or physiologic role of this in injury is not clear (tata et al., 2013) . in addition, a novel pathway of basal cell repletion from the submucosal gland was also reported by two different groups using lineage tracing and multiple airway injury models (lynch et al., 2018; tata et al., 2018) . these investigators found migration of glandular myoepithelial cells into the airways with subsequent differentiation to mature airway lineages including basal cells. a number of distinct, small populations of progenitor cell types have been reported to contribute to regeneration of distal airway epithelia after injury in mice, which lacks basal cells (barkauskas et al., 2013; bertoncello and mcqualter, 2010; chen, 2017; perl et al., 2005) . one of the first was a variant club/ secretory cell (v-club cells) defined by its location near neuroendocrine bodies and low cytochrome cyp2f2 expression, making these cells resistant to naphthalene injury and thus a source of regenerative cells after this injury (giangreco et al., 2009; hong et al., 2001) . more recently upk3a was identified as a unique marker for v-club cells. a upk3a creer lineage trace (guha et al., 2017) demonstrated that these cells give rise to club cells and ciliated cells during homeostasis and after naphthalene airway injury, as predicted from prior studies (giangreco et al., 2009; hong et al., 2001; volckaert et al., 2011) . although seemingly limited, in these studies some vclub/secretory cells were also noted to differentiate into at2 cells during bleomycin-induced alveolar injury, implying a capacity for mobilization and distal migration. a second small population of stem/progenitor cells was identified at the branch point between distal murine airways and alveolus and was termed bronchoalveolar stem cells (bascs). bascs were initially characterized by dual expression of the secretory cell marker scgb1a1 and the at2 marker sftpc by immunostaining and their localization at the bronchioalveolar duct junction (badj) (kim et al., 2005) . stem cell antigen-1 (sca1) was also proposed as a marker for these cells and has been used in flow cytometry to purify these cells for in vitro culture studies (raiser and kim, 2009 structure of the pseudostratified large airways and trachea of the mouse lung showing submucosal glands which are lined with both myoepithelial cells and other luminal secretory lineages. the smaller airways of the mouse lung contain various luminal secretory and ciliated epithelial lineages as shown. some of these including the bascs and the h2-k1 high secretory cell subtypes have been proposed to generate alveolar epithelium after severe injury. after naphthalene injury and peri-junctional at2 alveolar cells after bleomycin injury or influenza injury (liu et al., 2019b; salwig et al., 2019) . one of the limitations of these studies is the use of scgb1a1 expression to mark bascs, as this gene and protein are known to be expressed in a subset of at2 cells, which could confound such lineage tracing . while the genetic depletion of the basc population resulted in a delay of the murine regenerative response to injury, full regeneration was eventually observed, suggesting that bascs are not absolutely required for lung regeneration. an additional distal airway stem/progenitor cell population with regenerative potential was identified descriptively as lineage-negative epithelial progenitors (lneps), also referred to as distal airway stem cells (dascs). these cells are distinct from v-club/secretory cells and bascs. lneps were originally defined as distal airway integrin b4 + /cd200 + cells without discernible mature lineage markers by protein immunostaining (vaughan et al., 2015) . these cells are also sox2 + , as expected for an airway epithelial cell. subsequently it became apparent that lneps/dascs are composed of both trp63 + cells and trp63 ã� cells. the trp63 + cells appear to be holdovers from embryonic trp63 + basal cells and some 20% of these cells could be traced with the scgb1a1 creer mouse line . although true basal cells are not normally found in the distal mouse airway, the rare krt5 + /trp63 + lneps/dascs expand and mobilize after influenza injury to generate collections (or pods) of krt5 + basal-like cells throughout the heavily damaged areas of influenza-injured mice (vaughan et al., 2015; xi et al., 2017; zuo et al., 2015) . this mobilization is initially protective to the mouse but ultimately is a dysplastic response as krt5 + basal cells have limited potential to differentiate into at2 cells and the mouse becomes permanently burdened with airway-like cystic structures throughout the alveolar compartment. appearance of these permanent cystic structures was linked with activated notch signaling, and blockade of hypoxemia response via deletion of hypoxia inducible factor 1 (hif1a) in airway cells promotes the contribution of airway cells to regeneration of at2s and subsequent improvement in oxygen saturation (xi et al., 2017) . contribution of distal airway progenitors to alveolar repair. although endogenous at2s represent the primary regenerative responders to various alveolar insults and regenerate the overwhelming number of new at2 and at1 cells after limited alveolar damage, several lines of evidence implicate activation of distal airway epithelial cells as an alternative source of alveolar epithelial cells following certain types of severe lung injury (barkauskas et al., 2013; chapman et al., 2011; xi et al., 2017) . recently, rare mhc high (h2-k1 high ) club cell-like progenitors have been described within the larger scgb1a1 lineage-traced population of all lung cells. these cells have proliferative capacity, appear to give rise to at2 and at1 cells following bleomycin-induced lung injury, and can be purified by flow cytometry using anti-h2-k1 antibodies (kathiriya et al., 2020) . h2-k1 high cells are a subpopulation of b4/cd200 cells, express low or no mature lung epithelial lineage markers (e.g., scgb1a1 or sftpc) at the protein level, and represent $5% of all scgb1a1 creer -labeled lung cells, but exhibit clonogenic potential to generate these broader populations in cell culture outgrowth assays (kathiriya et al., 2020) . h2-k1 high cells were found to survive and differentiate into at2 and at1 cells in vivo after intra-airway transplanta-tion into bleomycin-injured mice. these cells were also found to be specifically targeted by h1n1 pr8 influenza virus, consistent with a prior report showing b4 + /cd200 + cells as a primary target of the viral injury (quantius et al., 2016) . the alveolar epithelial differentiation capacity of h2-k1 high progenitors after bleomycin injury contrasts sharply with the differentiation repertoire of the rare trp63 + cells which predominantly gives rise to dysplastic krt5 + pods, but virtually no alveolar epithelial cells (ray et al., 2016; vaughan et al., 2015; xi et al., 2017) . additional studies combining injury models with modern cell lineage tracking techniques and single-cell analysis are needed to clarify the injuryspecific activation of airway cells and their relative contributions toward alveolar regeneration. importantly, whether bascs, lneps, and h2-k1 high progenitors represent similar or overlapping populations of cells remains unclear. future studies will need to directly compare these distal airway cells to the resident at2 and aep cell populations, to more fully assess their ability to repair and regenerate the alveolar niche after acute injury and in chronic diseases. contribution of the mesenchyme to airway regeneration. a series of complex and interconnected interactions are employed to maintain distal airway epithelium during quiescence and after injury within the local niche. the local niche is comprised of the extracellular matrix (ecm) and several mesenchymal cell types. specifically, the mesenchyme plays a critical role in function of distal airway epithelium by providing a number of signaling cues that ultimately determines the stem cell response during injury. much of what is known about the interplay in adults between the mesenchyme and the epithelium has been derived from studying lung development. these developmental signaling pathways include wnt/b-catenin, fibroblast growth factor 10 and its receptor fgfr2 (fgf10/fgfr), retinoic acid, and sonic hedgehog (shh) members of transforming growth factor b (tgfb) superfamily including bone morphogenetic proteins (bmp), hippo/yes-associated protein (yap), and notch signaling. for example, airway smooth muscle cells promote epithelial repair through the production of fgf10 volckaert et al., 2011 ) and a population of pdgfra + fibroblasts promote the differentiation of multi-ciliated cells through the production of il6 (tadokoro et al., 2014) . in addition to signaling to the airway epithelium during regeneration, the airway mesenchyme can undergo a phenotypic change following injury that drives abnormal regeneration. a recently identified mesenchymal subpopulation identified by axin2 expression but lacking pdgfra expression, the axin2 + myogenic progenitor or amp, becomes activated after naphthalene airway injury, begins to express acta2, and contributes the airway fibrosis in the naphthalene model (zepp et al., 2017) . a more thorough review on these pathways and their cell-specific functions can be found elsewhere (kotton and morrisey, 2014; leach and morrisey, 2018; lee and rawlins, 2018; zepp and morrisey, 2019) . the human lung contains many structural and anatomic differences which make it unique from its murine counterpart. the murine lung has a 6,000-fold smaller tidal volume, an 8,000 times smaller surface area, and roughly half the generations of airways compared to its human counterpart (irvin and bates, 2003; knust et al., 2009; thurlbeck, 1967 ). this significant difference in organ size poses distinct challenges for gas distribution, immune function, and gas exchange. these differences highlight the critical variances that need to be accounted for as the field moves from bench to bedside. the alveolus is one of the most architecturally conserved regions between the murine and human lung (figure 1 ). conservation of the cellular populations between mouse and human alveoli have been demonstrated through multiple techniques and include the above-described at1 and at2 epithelial lineages and the aep sublineage. furthermore, the regenerative capacity of the alveolus appears to be retained across species, as discussed above. the cellular anatomy of trachea and the most proximal airways in mice and the large airways in humans is very similar. the cells of the murine trachea and their progenitor and regeneration capacity appear to be closely aligned with in vitro models of human proximal airway regeneration. however, the distal conducting airway anatomy of the mouse is quite different from humans. in mice, the intrapulmonary airways are almost completely devoid of cartilaginous rings, bronchial blood support, submucosal glands, and pseudostratified epithelium, all of which is in direct contrast to their human counterparts. furthermore, murine conducting airways terminate directly into alveolar sacs at the site of the badj. humans do not have a badj, and instead have a distinct distal airway compartment which includes the respiratory bronchioles. these distal respiratory bronchioles are interdigitated with alveolarlike structures that leads into the larger alveolar compartment. there is no analogous counterpart to the human respiratory bronchioles in mice. terminal and respiratory bronchioles in humans contain krt5 + basal cells that are generally absent in the distal airways of mice. to date, a basc-like cell population has not been found in humans, perhaps due to the lack of the corresponding badj anatomical niche. thus, the cellular origins of distal airway repair and the origin of airway-based stem/progenitors mobilized during alveolar injury in humans are likely different from that of mice. given the significant differences between the human and mouse lung, especially in the distal airways, greater focus is needed to study the human lung, both through descriptive assessment using new techniques including single-cell analysis as well as more sophisticated assays including organoids, ex vivo lung explants, and pluripotent stem cell-derived human lung lineages. idiopathic pulmonary fibrosis (ipf) is the most common adult interstitial lung disease (ild), a class of pulmonary diseases pathologically defined by interstitial fibrosis, inflammation, or the combination of fibrosis and inflammation (lederer and martinez, 2018) . a recognized theory of ipf pathogenesis places the initial site of ''micro-injury'' in the alveolar space with the at2 cell holding an important position in disease development. recent transcriptional interrogation of the distal epithelium in ipf identified activation of cell stress and senescence pathways, and murine modeling of at2 cell dysfunction from expression of either mutant sftpc, loss of telomere function, and increased mechanical tension have provided in vivo proof of concept that disruption of at2 cell homeostasis is a driver of lung fibrosis (katzen et an emerging hypothesis of ipf pathogenesis is that the dysfunctional at2 cell loses its facultative progenitor capacity creating a regenerative void for lung repair. in support of this hypothesis, a cardinal feature of the pathobiology of ipf is bronchiolization, a term coined decades ago to codify the observation by chest pathologists that epithelial cells with airway markers accumulate in the fibrotic regions of human lungs, appearing to extend the junctional region between distal airways and remaining alveoli (chilosi et al., 2002) . micro-honeycomb cysts, another cardinal feature of fibrotic remodeling in humans, are mainly lined by epithelial cells with airway markers, including basal, goblet, and ciliated markers (seibold et al., 2013) . the realization that distal airway stem/progenitors robustly mobilize in mice to occupy alveolar surfaces suggests a parallel process in humans. while potentially protective against loss of tissue integrity, the progenitors arising from distal airways are also subject to signals that direct their differentiation to airway rather than alveolar phenotypes. these include hypoxia and notch signaling (chen, 2017) . once differentiated to airways cells, differentiation to normal at2 or at1 cells may be very difficult and inefficient, potentially accounting for the dysplastic structures that dominate the pathobiology of lung fibrosis (kumar et al., 2011; vaughan et al., 2015) . in this paradigm, the dysplastic epithelial cysts accumulating progressively in fibrotic human lungs represent remnants of failed repair, as discussed above. future efforts to both better understand the development of at2 cell progenitor dysfunction and to minimize the pathway of airway differentiation of activated progenitors within alveoli of humans could be therapeutic. chronic obstructive pulmonary disease chronic obstructive pulmonary disease (copd) is a leading cause of morbidity and mortality worldwide, and prevalence continues to increase across the globe with the continued rise in cigarette use and toxic biomasses (burney et al., 2015) . in contrast to ipf, which is characterized by robust cellular production at the site of injury resulting in a dysplastic, fibrotic pulmonary parenchyma, the cardinal feature of copd in the alveolar space is cellular loss and alveolar simplification, known as emphysema. mechanistic studies in copd, however, have failed to generate novel therapeutics aimed at actual lung regeneration. while much of the work on the deleterious effects of toxin exposures on airway cells has been done in bronchial or upper airway cells, careful anatomic studies have suggested that the site of obstruction is localized to the distal airways and in particular the respiratory airways (mcdonough et al., 2011; koo et al., 2018) . the cellular structure and composition of the distal airway region of the human respiratory system, in particular the respiratory bronchioles, is poorly understood. the final endpoint of emphysema, however, is marked by loss of the alveolar epithelium, which can be studied in murine models. studies have pointed to an increase in senescence in at2 cells and the associated endothelium as a mechanism for the development of copd in certain patient populations and the increased prevalence of the disease with aging (gao et al., 2017) . together with the presence of increased senescence in lung fibroblasts from patients with copd, these studies may suggest an exhausted phenotype in a final common pathway downstream of repeated alveolar repair in the setting of recurring toxic exposure (mâ�¬ uller et al., 2006) . furthermore, defective alveolar epithelial repair has been associated with reduced wnt signaling in the copd microenvironment, and this defective response has been suggested to be downstream of a shift in canonical to noncanonical wnt signaling in the presence of toxic stimuli such as cigarette smoke (baarsma et al., 2017) . whether this change in wnt signaling is associated with an aberrant or absent response by the wnt-responsive aep sublineage in the at2 cell population, remains unclear. more work is needed to understand how toxic injuries incite distal epithelial cell responses in copd, in order to be able to develop therapies aimed at bona fide repair of the alveolus, and therefore clinical improvement. deriving lung epithelium de novo via directed differentiation of escs/ipscs the fact that most types of primary lung epithelial cells have been difficult to propagate as stable phenotypes in cell culture has raised significant hurdles for performing basic mechanistic studies in vitro for human lung lineages. a potential solution to these challenges has emerged with the discovery of techniques to differentiate pluripotent stem cells (pscs) in vitro into a diversity of lung lineages. embryonic stem cells (escs) and their engineered equivalents-induced pluripotent stem cells (ipscs)represent the gold standards of pscs, and a rapidly emerging literature has gradually led to successful methods for controlling their differentiation in cell culture. directed differentiation of pluripotent stem cells. the in vitro differentiation of either escs or ipscs into specific tissue lineages can be guided by adding combinations of growth factors or small molecules to media at specific times during culture (figure 3) , in order to recapitulate the signaling pathways that regulate in vivo organ development (murry and keller, 2008) . the process of recapitulating development in vitro to sequentially pattern pluripotent stem cells toward desired fates is termed ''directed differentiation'' and has been successfully applied for deriving multiple cell types from escs/ipscs, including lung epithelia . to date most of the cell types produced from esc/ipsc have an immature phenotype and are not yet ready for clinical applications. initial attempts at deriving lung epithelium from pscs were inefficient, stochastic, used incompletely defined media, or relied on the presence of drug-resistance genes (coraux et al., 2005; van haute et al., 2009; wang et al., 2007) . the difficulty in part was due to a lack of information regarding normal lung development in vivo. as basic mechanisms were discovered that regulate the formation of definitive endoderm, an ability to derive lung epithelia from pscs in vitro via sequential differentiation into definitive endoderm and subsequent patterning into foregut endoderm followed (green et al., 2011; kubo et al., 2004) . the most successful of these strategies to date has involved the exogenous addition of growth factors and inhibitors at specific times and concentrations to mimic the progressive cell signaling between the endoderm and mesoderm that specify definitive endoderm, pattern the endoderm anteriorly and then ventrally, ultimately inducing nkx2-1 + respiratory progenitors that differentiate into lung epithelium (green et al., 2011; longmire et al., 2012; mou et al., 2012; rankin et al., 2016) . a variety of studies to date indicate that the key step of initial specification of the lung epithelial lineage from foregut endodermal precursors generated from pscs in vitro can be monitored by assessing the kinetics and efficiency of expression of nkx2-1 or reporters for this locus (gotoh et al., 2014; green et al., 2011; longmire et al., 2012; mou et al., 2012; rankin et al., 2016) . these studies indicate that lineage specification requires the precise temporal activation of wnt, bmp, and ra signaling (huang et al., 2014; rankin et al., 2016; serra et al., 2017) . although a high degree of agreement exists on strategies to generate lung-specified anterior foregut endoderm (afe), the generation of mature lineages is less straightforward. similar to other organs and tissues, the generation of fully mature cells remains challenging and is a major limitation to the application of human psc-derived cells for disease modeling and regenerative medicine (lancaster and knoblich, 2014) . nevertheless, substantial progress has been made in recent years and some guiding principles have emerged. in initial studies, differentiation of psc-derived lung progenitors was performed in adherent 2d the key signaling factors and the main stages of the in vitro derivation of lung epithelial cells are provided. following lung specification, late withdrawal of chir gives rise to mature at2 cells (dark red). early withdrawal of chir gives rise to airway progenitors (light blue) or a mixture of mature airway cells (dark blue) and at2 cells, depending on the protocol used. cultures, both in mouse (longmire et al., 2012; mou et al., 2012) and human (firth et al., 2014; huang et al., 2014 huang et al., , 2015 mou et al., 2012) . this led to cultures containing a mixture of cells of undetermined maturity, but including cells expressing markers of at2 cells. however, expression of sftpc, one of the most specific markers of at2 cells, was sparse. culture of the same cells on thin slices of human decellularized lung matrix induced strong expression of sftpc, suggesting that sfptc expression and hence emergence of at2 cells was facilitated by a 3d environment (huang et al., 2014) . furthermore, cultures in 3d conditions, typically matrigel, induced sftpc expression accompanied by emergence of a mature at2 program that includes production of surfactant proteins and phospholipid (gotoh et al., 2014; jacob et al., 2017; yamamoto et al., 2017) . culture systems have now been developed where at2 cells can be propagated as spheroids in 3d matrigel cultures in presence and absence of feeders consisting of pulmonary fibroblasts yamamoto et al., 2017) . similarly, airway progenitors generated from developmental lung progenitors specified to an anterior fate can be grown as epithelial spheres containing ciliated and secretory cells in 3d cultures (konishi et al., 2016; mccauley et al., 2017) . single-cell rna sequencing profiles of these spheres (mccauley et al., 2018) has confirmed the derivation of airway lineages, but has also raised a cautionary note as non-lung endodermal lineages (e.g., hepatic or gut epithelia) also appear within these spheres over extensive culture periods, representing an ongoing challenge to the field. further efforts have resulted in the generation of more complex lung organoids from pscs. lung organoids grown in matrigel droplets can generate airway structures after xenografting into mice, while the bud tip organoids could only generate small pockets of ciliated cells after transplantation into naphthaleneinjured lungs of immunodeficient mice (dye et al., 2015 (dye et al., , 2016 miller et al., 2018 miller et al., , 2019 . in a second model, psc-derived anterior foregut endoderm cells were grown in suspension culture and as spheres containing respiratory endoderm, and the endodermal cells expressed sonic hedgehog (shh), while the mesenchymal component expressed shh targets. these organoids were termed lung bud organoids . most of the cell types in these psc lung organoid assays have been shown to consist of fetal stage cells, again illustrating the challenges associated with achieving full maturation dye et al., 2015 dye et al., , 2016 miller et al., 2018 miller et al., , 2019 . the origin of the mesenchymal cells that appear to co-develop along with endoderm in these psc lung organoid cultures to date remains unclear. the second emerging principle arising from experience with ipsc models is a need to better understand the temporal developmental signaling pathways required for lung lineage specification. for example, bmp inhibition is required for afe specification (green et al., 2011) . however, consistent with mouse genetic models, subsequent bmp agonism promotes lung fate from afe (huang et al., 2014; longmire et al., 2012 ). an important component for the differentiation of at2 cells is dexamethasone, 8-bromo-camp, and isobutylmethylxanthine (dci), fgf7 or 10, and gsk3b agonism using the small molecule chir9902 (huang et al., 2014 longmire et al., 2012; serra et al., 2017; yamamoto et al., 2017; miller et al., 2018) . withdrawal of gsk3b inhibition, which results in reduced wnt signaling, appears to induce a proximal fate in lung progenitors . conversely, maintenance of chir9902 in 2d cultures and subsequent plating in 3d cultures in the presence of chir9902 yielded at2 spheroids that could be replated and expanded for many passages . interestingly, full morphological maturation accompanied by reduced proliferation of at2 cells could be accomplished by withdrawal of gsk3b inhibition in the 3d cultures . in some 3d models, withdrawal of chir9902 led to both proximal and distal maturation, with the generation of ngfr + postnatal basal cells, at2 cells, and cells expressing markers of at1 cells (de carvalho et al., 2019) . gsk3b inhibition was also required to generate renewing distal tip progenitors (miller et al., 2018) . together, these studies indicate that further analysis of the temporal activation and repression of wnt signaling in driving lung endoderm fate is needed. importantly, gsk3b integrates multiple inputs and affects a wide variety of signaling pathways and cellular process, and some of its effects may be independent of wnt signaling (patel and woodgett, 2017) . additional signaling pathways including notch firth et al., 2014; mccauley et al., 2017; konishi et al., 2016) and retinoic acid are important for promoting lung endoderm cell fate (miller et al., 2018) . a third principle guiding effective directed differentiation of pscs into lung epithelium is the proper initial establishment of lung progenitor fate. the purity of the psc-derived lung progenitors, which can vary from line to line, remains a problem . one solution is the use of reporters, such as fluorochrome constructs targeted to nkx2.1, sftpc, or scgb3a2 loci, which allow flow cytometric purification of desired cell types (gotoh et al., 2014; hawkins et al., 2017; mccauley et al., 2017 mccauley et al., , 2018 serra et al., 2017) . these cell lineage-specific reporters have been useful in defining the differentiation strategies of various cell types in the lung. however, a drawback of reporter lines is that this strategy is unlikely to be approved for the purification of human cells for clinical therapies. a second solution is the use of surface markers to enrich for lung endoderm progenitors such as carboxypeptidase m or cd47 hi cd26 lo cells (gotoh et al., 2014; hawkins et al., 2017; korogi et al., 2019) . lung disease modeling via pluripotent stem cells. one of the major applications for ipsc-derived lung epithelial lineages is providing an in vitro platform for studying human pulmonary diseases. early applications of ipscs attempted to model cystic fibrosis (cf), a disease caused by mutations in the anion channel protein, cftr (cystic fibrosis transmembrane conductance regulator), which leads to progressive lung damage due to impaired mucociliary clearance, inflammation, and recurrent respiratory infections. wong et al. (2012) developed the first ipscderived model of cf using a 2d system in which cf-ipscderived epithelial cells exhibited lower cftr expression and reduced anion transport in response to forskolin, a known cftr activator. a subsequent 2d airway model was published by firth et al. (2014) , in which the authors demonstrated the presence of functional cftr channels, with anion currents that could be modulated by both forskolin and a cftr inhibitor. crispr correction of a cftr mutation in ipscs by two separate groups then demonstrated an in vitro rescue of channel function (crane et al., 2015; firth et al., 2015) . finally, recent work from mccauley et al. (2017) used ipsc-derived airway epithelial spheres to demonstrate that crispr correction of cftr mutants can rescue defects in forskolin-induced organoid swelling, providing an easily visualized read-out for future high throughput screens of airway cftr function. more recent applications of ipsc-derived lung models have focused on a broad diversity of genetic lung diseases, lung cancer, or platforms for the study of viral respiratory infections. for example, 2d differentiation of hpsc-derived lung progenitors allowed modeling influenza susceptibility in ipscs from a patient with a genetic deficiency in irf7 (ciancanelli et al., 2015) and from a patient with tlr3 mutation (lim et al., 2019) . the importance of these findings lies in the fact that they showed that not only interferon production by immune cells was relevant to the increased susceptibility of these patients to lethal influenza infection, but that interferon production within distal lung epithelial cells is likely involved as well. in branching 3d organoids, the pathological changes associated with respiratory-syncytial virus-associated bronchiolitis could be reproduced and infection with clinical isolates of parainfluenza virus could be modeled (porotto et al., 2019) . in 2d cultures, through genetic manipulation of rb and p53 expression in notch-inhibited epithelium enriched in neuroendocrine cells, a human in vitro model for small cell lung cancer was developed . for modeling genetic diseases affecting the distal lung, distal specification and generation of expandable at2 cells in 3d allowed modeling of sftpb deficiency, which was corrected by crispr/cas9-mediated gene editing . recessive mutations in some genes implicated in hermansky-pudlak syndrome (hps) cause hps-associated interstitial pneumonia (hpsip), a clinical entity similar to idiopathic pulmonary fibrosis (lederer and martinez, 2018) . introduction of hps mutations associated with hpsip in escs promoted fibrotic changes in branching lung organoids , while deletion of hps8, which is not associated with hpsip, did not . further studies revealed an essential role for il11 in the fibrotic process, suggesting that il11 is a potential therapeutic target in this intractable disease . additional work using at2 cells generated from patient-specific ipscs profiled the at2 cell dysfunction that results from hps1 mutations (korogi et al., 2019) . many false starts and claims of engraftment of exogenous cells into injured murine lung tissue have confused the field for the past 20 years, with an extensive literature emerging during the controversial claims of bone marrow plasticity during the 1990s (reviewed in kotton, 2012) . more recently a few groups have begun to publish more convincing evidence in support of the capacity of epithelial stem/progenitor cells, of both mouse and human origin, to survive in injured mouse lungs after intratracheal or intravenous delivery (farrow et al., 2018; miller et al., 2018; nichane et al., 2017; rosen et al., 2015; vaughan et al., 2015) . there is also recent evidence of more mature at2 cells of both mouse and human origin surviving transplantation into injured mouse lungs (hillel-karniel et al., 2020; weiner et al., 2019) . whether these cells are functionally integrated into host lung tissue, are long-lived, and retain expression of a complete lung transcriptomic program are still open questions that will need to be addressed before these cells can be referred to as ''engrafted.'' for example, only one study to date has attempted to profile the transcriptomes of grafted cells at single-cell resolu-tion (nichane et al., 2017) . the use of lineage tracing techniques and clonality studies are notably absent from most prior reports, raising uncertainty about the cellular origins of the source cells and preventing assessment of the stem cell properties of transplanted cells. importantly, physiologic improvement of injured mouse lungs after cell transplantation as described in some of the above reports might be explained by paracrine effects or transient secondary reparative effects on recipient lung tissue rather than by direct, durable replacement of the epithelium with functional cells. in addition to alveolar cell therapy, a recent study demonstrated that it is possible to correct cf mutations using primary airway basal cells, which could be used for engraftment into patients (vaidyanathan et al., 2020) . many additional challenges remain in the translation of experimental cell therapy in mice to humans with major lung injury, including the issue of allogeneic rejection, possibly circumvented by the development of autologous, syngeneic ipscderived stem/progenitors if these were to have engraftable potential mccauley et al., 2017; miller et al., 2018) . the limitations listed above have also resulted in a dearth of information regarding the mechanisms or microenvironmental niche interactions that might regulate homing, survival, proliferation, or functional activation of potentially engrafted cells. more sophisticated methods are needed to quantify the contributions of exogenously delivered cells, relative to either endogenous cells or competing alternative source cells. additional studies are needed to resolve the ongoing debate over which of many candidate lung cell populations, from resident progenitors or ipsc derivatives to common mature airway or alveolar cells, might have the best relative potential to reconstitute an injured epithelium after transplantation. the extensive cellular heterogeneity within the respiratory system and the lack of insight into the phenotype differences in these purported cell lineages make single-cell genomics a very useful tool in the study of lung repair and regeneration. this spatial diversity in architecture and cellular heterogeneity reinforces the importance of sampling at multiple regions along the respiratory airway axis to obtain a true cell atlas. advances in scrna-seq techniques allow for prediction of cell state, origins, and trajectories without requiring permanent genetic labeling of cells. new tools enabling data analysis, visualization, and interpretation are being developed at a rapid pace and are readily available as ''open source'' tools. likewise, data web portals provide the research community with open access to rich diversity of rna, protein, and lipidomic and imaging data related to the lung. examples of this include the lung gene expression atlas and breath databases (https://research.cchmc.org/ pbge/lunggens/mainportal.html and https://www.lungmap.net) (du et al., 2017) . while time series of single-cell transcriptomic data are most useful in identifying progenitor cells and their trajectories, these processes can be identified in pseudotime using data derived from a single time point when large numbers of cells are in transition; for example, during organ formation, tissue repair, or during active disease. likewise, single-cell transcriptomic data generated with embryonic stem cells or induced pluripotent stem cells as they differentiate into distinct lung cell types have provided new insights into the identity of lung cell progenitors and the regulatory networks controlling cell fate decisions hawkins et al., 2017; mccauley et al., 2018; nikoli c et al., 2018) . trajectory inference algorithms, or pseudotime analysis, reconstruct continuous cell state transitions from ''snapshots'' of single-cell transcriptomic data, providing insights into gene expression kinetics and regulatory dynamics of biological processes (gerber et al., 2018; guo et al., 2017; hawkins et al., 2017) . wanderlust (bendall et al., 2014) , monocle 1 (trapnell et al., 2014) , waterfall (shin et al., 2015) , and tscan (ji and ji, 2016) are among the early cell trajectory inference algorithms using scrna-seq data. the general analytical workflow of a pseudotime analysis algorithms includes (1) modeling individual cells as data points in high-dimensional gene expression space, where each dimension is defined by the expression of a gene; (2) projecting cells onto a lower-dimensional space using a dimension reduction method, e.g., principal component analysis, which finds a smaller set of new dimensions, each being a combination of original input genes, to represent major variances in the original gene expression space, reduce noise, and enable data visualization; (3) determining cell trajectories in the reduced dimensional space; (4) ordering single cells by progress along the cell trajectories; and (5) identifying gene expression patterns along cell orderings. with the evolution of single-cell technologies and exponential growth in numbers of cells collected from single-cell experiments (svensson et al., 2018) , scrna-seq datasets have captured complex cell trajectories that often contain multiple cell type decision branches. monocle 2 (qiu et al., 2017) , wishbone (setty et al., 2016) , dpt (haghverdi et al., 2016) , slice (guo et al., 2017) , slingshot (street et al., 2018) , and urd (farrell et al., 2018) can be used to infer tree-structured cell trajectories from scrna-seq data, reconstructing branched cell transitional paths from a progenitor toward multiple cell fates. to infer a robust tree-structured cell lineage model, slingshot (street et al., 2018) combines the use of multiple techniques recently developed for processing highly noisy scrna-seq data and allows users to specify initial and terminal cells to supervise parts of the tree construction, which might lead to more accurate dataspecific lineage tree inference. monocle 2 (qiu et al., 2017 ) is a comprehensive computational pipeline for scrna-seq analysis, including gene expression modeling, preprocessing, cell clustering, differentiation expression analysis, and cell trajectory inference analysis. recently developed methods, including paga (wolf et al., 2019) and monocle 3 (cao et al., 2019) , can be used to infer more complex, non-tree-based cell trajectories. together with a number of implementation improvements in computational efficiency and memory usage, paga and monocle 3 can be applied to infer complex lineage relations from large scrna-seq datasets with more than 1 million cells, for example inferring the lineage relations of cells from a whole adult animal (plass et al., 2018) or the reconstruction of 10 disjointed major cell trajectories from $1.5 million mouse embryonic cells during organogenesis (cao et al., 2019) . most of the trajectory inference methods pseudo-temporally order cells purely based on transcriptomic similarity without estimating cell states (e.g., differentiation states). they require the use of external knowledge, such as time information, cell identity, marker gene expression, or user inputs, to determine the start and end points and the directions of inferred cell trajectories. stemid (grâ�¬ un et al., 2016) , slice (guo et al., 2017) , and rna velocity (la manno et al., 2018) provide the capability to estimate cell differentiation states, predicting progenitor cells and directions of cell differentiation transitions. stemid (grâ�¬ un et al., 2016) and slice (guo et al., 2017) both exploit the concept of entropy for predicting cell differentiation states from single-cell transcriptomic profiles. rna velocity showed that scrna-seq data can provide not only static information on mrna abundance of each gene in a single cell but also dynamic information on how the expression of each gene in a single cell is changing through annotating the ratio of unspliced and spliced transcripts of each gene in each cell, predicting the future state of each cell (la manno et al., 2018) . single-cell experiments can be performed at multiple time points relevant to biological processes, such as lung development, regeneration, or disease progression, generating singlecell time-course data. trajectory inference methods, including stitch (wagner et al., 2018) and waddington-ot (schiebinger et al., 2019) , utilize the time of collection to supervise the analysis and improve the accuracy of pseudotime analysis using timecourse scrna-seq data (wagner et al., 2018) . since data collected at multiple time points can be influenced by technical variations in cell isolation, library generation, and sequencing, stitch (wagner et al., 2018) analyzed time-course scrnaseq data by first constructing a single-cell graph from each time point and then joining pairs of graphs from adjacent time points by connecting similar cells (wagner et al., 2018) . waddington-ot, on the other hand, directly estimates a transitional probability between any two cells from consecutive time points in a time-course scrna-seq data without explicitly inferring a lineage topology (schiebinger et al., 2019) . given a set of cells at a time point, waddington-ot predicts the most likely ancestor cells in earlier time points and descent cells in later time points based on the derived cell-cell transition probabilities, reconstructing a cell transitional path across time points. importantly, these cellular trajectory models require experimental validation either in the form of cell-type-specific genetic lineage tracing in mice or the use of cellular barcoding strategies in non-murine systems such as was performed in a recent study to predict the differentiation of lung epithelial progenitors from pluripotent stem cells (hurley et al., 2020) . single-cell analysis has been used extensively to assess both mouse and human lung development and disease. improved scrna-seq methods carefully coupled with cell-type-specific lineage tracing has uncovered new developmental and regenerative origins for multiple lung cell lineages. the early specification of at1 and at2 cells, coincident with such early and basic tissue patterning processes such as branching morphogenesis, and the heterogeneity of embryonic alveolar epithelial progenitors, was recently uncovered using scrna-seq methods (frank et al., 2019; guo et al., 2019) . the heterogeneity within the adult lung mesenchyme was recently described and revealed the importance of the mesenchymal niche in alveolar homeostasis and regeneration (guo et al., 2019; lee et al., 2017; zepp et al., 2017) . human lung scrna-seq has been reported in both ''normal'' lungs and in diseased lungs such as idiopathic pulmonary fibrosis (reyfman et al., 2019; xu et al., 2016) . scrna-seq was also used to identify the ionocyte, which is thought to play a key role in cystic fibrosis plasschaert et al., 2018) . there are multiple consortia that are working to establish a human lung cell atlas including the nih-supported lungmap and the chan-zuckerberg-supported human cell atlas. one of the major hurdles in using single-cell techniques to map the human lung is the high level of cell heterogeneity that exists in the organ, which means that isolation procedures including both upstream and downstream processing are going to be highly variable depending on the site and research group involved. these issues are revealed in many experiments that lack significant representation of fragile cells such as at1 cells and over-representation of immune cells. in the absence of procedures and protocols that generate highly reproducible isolation of all known cell types, both qualitatively and quantitatively, across most if not all lung samples, the resulting datasets are unlikely to provide an accurate cell atlas. moreover, the respiratory system is highly complex and contains many spatially restricted compartments and niches which need to be uniquely sampled, possibly using different techniques for each region. thus, much work remains to be done to ensure that such single-cell mapping efforts can reproducibly isolate and characterize all resident and immune cells within the human respiratory system. bioengineering of the lung has made remarkable progress in the recent years, motivated by strong clinical needs and enabled by advances in tissue engineering and stem cell biology. as many as 25 million people suffer from end-stage lung disease in the united states alone, with $400,000 patients dying each year, a third of these from nonmalignant diseases (optn, 2012; morrisey and hogan, 2010; petersen et al., 2010) . worldwide, lung disease remains the third leading cause of death (murphy et al., 2018; rabe et al., 2007) . notably, most lung diseases affect epithelium, including the acute respiratory distress syndrome (ards), chronic obstructive pulmonary disease (copd), emphysema, cystic fibrosis (cf), and pulmonary fibrosis. lung transplantation, the only definitive treatment option for end-stage lung disease, remains hampered by a severe shortage of donor organs. in addition, a majority of donor lungs are deemed unacceptable for transplantation at the time of receipt (ware et al., 2002) , making lung the least utilized solid organ and necessitating the use of extracorporeal membrane oxygenation (ecmo) as a bridge to transplant for critically ill patients (fuehner et al., 2012; javidfar and bacchetta, 2012; klein et al., 2010; pomfret et al., 2008; ware et al., 2002) . to overcome this crisis, there are major efforts to increase the number of transplantable lungs, including (1) criteria expansion, i.e., accepting older donors, lungs donated following cardiac death (bittle et al., 2013; elgharably et al., 2015; van raemdonck et al., 2009 ), (2) ex vivo lung perfusion (evlp) to recover marginally unacceptable donor lungs (cypel and keshavjee, 2015) , (3) bioengineering of functional lungs by populating decellularized lungs or scaffolds with epithelial and vascular cells (ott et al., 2010; petersen et al., 2010; rosen et al., 2015; wagner et al., 2013; wobma and vunjak-novakovic, 2016) , and (4) utilization of xenogeneic lungs from swine or non-human primates (cooper et al., 2012; laird et al., 2016) . thus far, the number of lung transplantations remains steady, resulting in the increasing waitlist mortality (valapour et al., 2015) . new strategies are being developed to increase the numbers of lungs for transplantation by recovering marginal quality donor lungs and developing physio-logical ex vivo platforms for modeling lung disease. the enormous complexity of the lung, with its hierarchical architecture, more than 40 cell types, and a very large area ($70 m 2 ) for gas exchange, presents major challenges to lung recovery (crapo et al., 1982; massaro and massaro, 1996; weibel, 1973) . finally, generation of humanized whole lungs through stem cell complementation in other species could lead to a new source of transplantable lungs . ex vivo lung perfusion (evlp) and cross-circulation with a living host. among current approaches, evlp holds the strongest potential for immediate clinical impact by recovering marginally unacceptable donor lungs, which has been already implemented and is being further evaluated in clinical trials (popov et al., 2015) . notably, many of the conditions that render donor lungs unacceptable for transplantation (e.g., aspiration, infection, pulmonary contusions) could be reversible. however, conventional methods of donor lung preservation that involve cold static ischemia preclude endogenous repair and recovery (guibert et al., 2011; pinezich and vunjak-novakovic, 2019) . the field of ex vivo lung perfusion (evlp) is now addressing this limitation by providing initially unacceptable donor lungs with physiologic conditions of normothermia, perfusion, and ventilation, to recover function outside the body to a level acceptable for transplantation (makdisi et al., 2017; tane et al., 2017) . since the introduction of evlp by steen and colleagues in 2001 (steen et al., 2001) , evlp platforms have successfully demonstrated short-term support and recovery of marginal quality donor lungs in pre-clinical and clinical settings (cypel et al., 2011; warnecke et al., 2018) . a major limitation of evlp is the lack of whole-body homeostasis that requires renal, hepatic, pancreatic, and neurohormonal functions. the current durations of clinical usage of evlp systems are too short for advanced therapeutic interventions (e.g., immunomodulation, cell therapy) (warnecke et al., 2012) . moreover, current evlp systems cannot recover the majority of unusable donor lungs, due to the lack of appropriate physiologic milieu for endogenous repair. using a clinically relevant swine model, a cross-circulation platform has been established with the recipient support enabling 36 h of normothermic perfusion for the maintenance and recovery of injured lungs . in recent studies, the duration of lung support on cross-circulation has been extended to 4 days (hozain et al., 2019) . cross-circulation is actually an old technique that has been used in the past to support patients suffering from a critical but potentially reversible illness given sufficient time for recovery (e.g., hepatic insufficiency, uremia, eclampsia) (burnell et al., 1965 (burnell et al., , 1967 eschbach et al., 1964 ) by a healthy individual. for extracorporeal lungs, cross-circulation was used to extend the duration of lung maintenance ex vivo from hours to days, by providing metabolic clearance and systemic factors to the perfused and ventilated lung (figure 4 ; o'neill et al., 2017) . this method allowed time for multiscale therapeutic interventions, with the aid of real-time theranostic (therapeutic + diagnostic) imaging (kim et al., 2015b . by the end of cross-circulation support, the lungs that were severely damaged by ischemia or gastric aspiration exceeded transplantation criteria, and the recipients tolerated the procedure without significant changes in physiologic parameters. these findings suggest that cross-circulation could enable extended support necessary for gene and cell therapies of the extracorporeal lungs. lung bioengineering by complete decellularization and recellularization of the lung parenchyma and vasculature. the niklason and ott laboratories have pioneered a methodology that involves removal of all cells from the lung and subsequent infusion of epithelial cells into parenchymal region and endothelial cells into the vascular region of the lung (ott et al., 2010; petersen et al., 2010) . however, these bioengineered lungs fail shortly upon transplant, due to incomplete regeneration and leaky vasculature causing alveolar edema and thrombosis (ott et al., 2010; petersen et al., 2010 petersen et al., , 2011 petersen et al., , 2012 . while this highly innovative approach has advanced through many meritorious studies, three limitations remain: (1) the inability to restore gas exchange, (2) the inability to maintain vascular function upon transplantation, and (3) the need for very high cell numbers (billions) to repopulate the lung (petersen et al., 2010; song et al., 2011) . targeted treatment of lung epithelium with the preservation of lung vasculature. in principle, the limitations of full decellularization/recellularization of the lung could be overcome if acutely injured donor lungs are treated to replace only the epithelium in the most injured regions of the lung, while preserving the integrity of the lung vasculature. because acute injury tends to affect some regions of the lung and not the entire lung (raghavendran et al., 2011) , a targeted treatment could help preserve much of the existing lung function and facilitate regeneration of the injured regions. an alternate approach to lung bioengineering has been developed that is based on removing damaged epithelial cells from distal lungs while maintaining the integrity of the basement membrane, surrounding lung cells and matrix, and the functionality of lung vasculature. denuded regions in the lung airway are repopulated with epithelial progenitors. the maintenance of an intact vascular network was considered critical for maintaining the blood-gas barrier as well as for supporting survival of newly delivered cells. this airway-specific approach was first demonstrated in the rat model, resulting in vascularized lung grafts that supported the attachment and growth of human adult pulmonary cells and stem-cell-derived alveolar progenitor cells . targeted decellularization/recellularization of the lung epithelium was achieved by introducing soluble reagents (such as the solutions for cell removal, suspensions of therapeutic cells) in micro-volume liquid plugs to the targeted branches of the pulmonary airway: upper airways, small airways (bronchioles), or the most distal lung (alveoli). liquid plugs (only <1 ml in volume) were instilled into the upper airway and pushed into a specific more distal airway to form liquid film covering lung epithelium, using programmed ventilation of the lung in conjunction with radiation-free transpleural imaging (kim et al., 2015a) . combination of long-term lung support ex vivo with the targeted treatment of lung epithelium. cell replacement therapy offers compelling prospects for the treatment of injured lungs, if the extracellular matrix (including the basement membranes) could be preserved. a new methodology for lung regeneration is now under development through an integrated use of three components: (1) extended duration of lung support ex vivo (several days) by cross-circulation with a living host, (2) targeted treatment of the injured regions of the lung (from conducting airways to distal regions), with the preservation of the surrounding lung parenchyma, and (3) selective removal and replacement of lung epithelium with the preservation of lung vasculature (for immediate blood supply for lung survival and function) (figure 4) . the same issues regarding whether newly delivered or replaced cells merit use of the controversial term, ''engraftment,'' (indicating true, durable integration of functional cells) apply to this novel approach as discussed above (see cell-based therapy for the respiratory system) and have not yet been addressed. the utility of this approach was demonstrated in clinical-scale swine models for recovering lungs unsuitable for transplant due to acute injury by ischemia and gastric aspiration (guenthart et al., 2019) ( figure 5 ). in both cases, the lung epithelium was removed from injured regions while preserving the surrounding cells, matrix, and intact lung vasculature , and the denuded epithelial regions were repopulated with pulmonary progenitor cells. in one study, the injured lungs were maintained on cross-circulation support without decline in lung function and were subjected to therapeutic interventions that enabled cellular regeneration and improved function over the course of 36 h (guenthart et al., 2019) . regeneration of severely damaged lungs to the levels necessary for meeting the transplantation criteria would help significantly increase the pool of donor lungs available for transplant, for example by recovering lungs rejected because of gastric aspiration damage. on cross-circulation, the injured lungs recovered their mechanical compliance to 68% of that of uninjured control lungs, corresponding to a 6-fold improvement relatively to the levels measured post-injury. a lung injury scoring rubric that has been developed in these studies (guenthart et al., 2019; o'neill et al., 2017) was also used to assess the extent of lung injury over 4 days of lung maintenance and recovery (hozain et al., 2019) . the lung injury scores decreased across all categories, with the greatest improvements observed in alveolar cells functionality, lung edema, and cell apoptosis. the recovered lungs displayed regeneration of cell surface markers in the pulmonary epithelium, endothelium, tight and gap junctions, recovery of subcellular structures (e.g., intracellular vesicles), and cellular function and integrity (guenthart et al., 2019) . throughout the duration of extracorporeal support, the recipient tolerated cross-circulation with no significant changes in physiologic parameters. after 36 h of the procedure, the lungs injured by ischemia or gastric aspiration exceeded transplantation criteria, as evidenced by the gross appearance, thermography, and p-v loops that were consistent with recovered lungs (figure 5 ). future studies may lead to interventional cross-circulation being used for the expansion of donor lung pools through the salvage of severely damaged lungs. the field of lung regeneration has advanced immensely in the last decade. the emergence of cell-type-specific lineage tracing coupled with better injury models has allowed investigators to begin to directly link the behavior among the various cell lineages in the lung and their responses to acute injury. new methods for generating human lung cells using psc technologies, coupled with better imaging modalities and evolving assays such as human lung organoids and precision cut lung slices, are allowing for the direct use of human lung tissue to experimentally and mechanistically assess the response to acute injuries and chronic diseases. the recent advent of single-cell genomics and advanced bioengineering techniques allow not only for the exquisite definition of all cell types within the human and mouse lung but provides clinically relevant approaches to regenerate unusable human lungs for transplant. finally, the ongoing covid-19 pandemic highlights the need for a better understanding of lung regeneration in the face of overwhelming acute viral injury. given that lung disease remains the third leading cause of morbidity and mortality in the world, all of these advances and many more will be needed to develop better therapeutic approaches for the many patients in need. lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity noncanonical wnt-5a signaling impairs endogenous lung repair in copd type 2 alveolar cells are stem cells in adult lung single-cell trajectory detection uncovers progression and regulatory coordination in human b cell development endogenous lung stem cells: what is their potential for use in regenerative medicine? the use of lung donors older than 55 years: a review of the united network of organ sharing database nf-kb signaling in fetal lung macrophages disrupts airway morphogenesis pulmonary neuroendocrine cells function as airway sensors to control lung immune response compensatory growth of the lung following pneumonectomy observations on cross circulation in man acute hepatic coma treated by cross-circulation or exchange transfusion global and regional trends in copd mortality targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis the single-cell transcriptional landscape of mammalian organogenesis integrin a6b4 identifies an adult distal lung epithelial population with regenerative potential in mice origin and regulation of a lung repair kit dynamic regulation of platelet-derived growth factor receptor a expression in alveolar fibroblasts during realveolarization a three-dimensional model of human lung development and disease from pluripotent stem cells generation of pulmonary neuroendocrine cells and sclc-like tumors from human embryonic stem cells abnormal re-epithelialization and lung remodeling in idiopathic pulmonary fibrosis: the role of del-tan-p63 niche-mediated bmp/smad signaling regulates lung alveolar stem cell proliferation and differentiation infectious disease. life-threatening influenza and impaired interferon amplification in human irf7 deficiency correction and restored function of the cftr gene in cystic fibrosis induced pluripotent stem cells cell number and cell characteristics of the normal human lung extending the donor pool: rehabilitation of poor organs normothermic ex vivo lung perfusion in clinical lung transplantation notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cellderived lung progenitors in 3d culture endothelialderived angiocrine signals induce and sustain regenerative lung alveolarization functional vascularized lung grafts for lung bioengineering lung gene expression analysis (lgea): an integrative web portal for comprehensive gene expression data analysis in lung development in vitro generation of human pluripotent stem cell derived lung organoids a bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids expanding the donor pool: donation after cardiac death a technique for repetitive and long-term human cross circulation single-cell reconstruction of developmental trajectories during zebrafish embryogenesis epithelial disruption: a new paradigm enabling human airway stem cell transplantation generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells functional gene correction for cystic fibrosis in lung epithelial cells generated from patient ipscs emergence of a wave of wnt signaling that regulates lung alveologenesis by controlling epithelial self-renewal and differentiation early lineage specification defines alveolar epithelial ontogeny in the murine lung extracorporeal membrane oxygenation in awake patients as bridge to lung transplantation b2-microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence consider the lung as a sensory organ: a tip from pulmonary neuroendocrine cells single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration stem cells are dispensable for lung homeostasis but restore airways after injury ly6chi monocytes direct alternatively activated profibrotic macrophage regulation of lung fibrosis use of three-dimensional organoids and lung-on-a-chip methods to study lung development, regeneration and disease generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells prospective isolation of nkx2-1-expressing human lung progenitors derived from pluripotent stem cells repair and regeneration of tracheal surface epithelium and submucosal glands in a mouse model of hypoxic-ischemic injury lung development: orchestrating the generation and regeneration of a complex organ multi-lineage lung regeneration by stem cell transplantation across major genetic barriers tissue crosstalk in lung development clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion in vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations multiday maintenance of extracorporeal lungs using cross-circulation with conscious swine efficient generation of lung and airway epithelial cells from human pluripotent stem cells the in vitro generation of lung and airway progenitor cells from human pluripotent stem cells macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a trefoil factor 2-dependent mechanism reconstructed single-cell fate trajectories define lineage plasticity windows during differentiation of human psc-derived distal lung progenitors measuring the lung function in the mouse: the challenge of size differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells plasticity of hopx(+) type i alveolar cells to regenerate type ii cells in the lung bridge to lung transplantation with extracorporeal membrane oxygenation support tscan: pseudo-time reconstruction and evaluation in single-cell rna-seq analysis distinct airway epithelial stem cells hide among club cells but mobilize to promote alveolar regeneration il-1 and tnfa contribute to the inflammatory niche to enhance alveolar regeneration an sftpc brichos mutant links epithelial er stress and spontaneous lung fibrosis identification of bronchioalveolar stem cells in normal lung and lung cancer targeted delivery of liquid microvolumes into the lung rapid retraction of microvolume aqueous plugs traveling in a wettable capillary controlled delivery and minimally invasive imaging of stem cells in the lung organ donation and utilization in the united states stereological estimates of alveolar number and size and capillary length and surface area in mice lungs directed induction of functional multi-ciliated cells in proximal airway epithelial spheroids from human pluripotent stem cells small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study in vitro disease modeling of hermansky-pudlak syndrome type 2 using human induced pluripotent stem cell-derived alveolar organoids next-generation regeneration: the hope and hype of lung stem cell research lung regeneration: mechanisms, applications and emerging stem cell populations development of definitive endoderm from embryonic stem cells in culture distal airway stem cells yield alveoli in vitro and during lung regeneration following h1n1 influenza infection direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma rna velocity of single cells lung xenotransplantation: a review organogenesis in a dish: modeling development and disease using organoid technologies regenerative lineages and immune-mediated pruning in lung cancer metastasis repairing the lungs one breath at a time: how dedicated or facultative are you? recruited monocytes and type 2 immunity promote lung regeneration following pneumonectomy idiopathic pulmonary fibrosis developmental mechanisms and adult stem cells for therapeutic lung regeneration anatomically and functionally distinct lung mesenchymal populations marked by lgr5 and lgr6 severe influenza pneumonitis in children with inherited tlr3 deficiency sox17 is required for endothelial regeneration following inflammation-induced vascular injury lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction efficient derivation of purified lung and thyroid progenitors from embryonic stem cells submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium ex vivo lung perfusion review of a revolutionary technology formation of pulmonary alveoli and gas-exchange surface area: quantitation and regulation efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of wnt signaling single-cell transcriptomic profiling of pluripotent stem cell-derived scgb3a2+ small-airway obstruction and emphysema in chronic obstructive pulmonary disease in vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells generation of lung organoids from human pluripotent stem cells in vitro monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span a revised airway epithelial hierarchy includes cftr-expressing ionocytes notch3-jagged signaling controls the pool of undifferentiated airway progenitors generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells preparing for the first breath: genetic and cellular mechanisms in lung development generation of multipotent lung and airway progenitors from mouse escs and patient-specific cystic fibrosis ipscs lung fibroblasts from patients with emphysema show markers of senescence in vitro mortality in the united states differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development single-cell wnt signaling niches maintain stemness of alveolar type 2 cells telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis isolation and 3d expansion of multipotent sox9 + mouse lung progenitors defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury expression of mutant sftpc in murine alveolar epithelia drives spontaneous lung fibrosis cross-circulation for extracorporeal support and recovery of the lung organ procurement and transplantation network and scientific registry of transplant recipients 2010 data report regeneration and orthotopic transplantation of a bioartificial lung rare pulmonary neuroendocrine cells are stem cells regulated by rb, p53, and notch injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations glycogen synthase kinase 3: a kinase for all pathways? conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli tissue-engineered lungs for in vivo implantation bioreactor for the long-term culture of lung tissue matrix composition and mechanics of decellularized lung scaffolds bioengineering approaches to organ preservation ex vivo cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics a single-cell atlas of the airway epithelium reveals the cftr-rich pulmonary ionocyte solving the organ shortage crisis: the 7th annual american society of transplant surgeons' state-of-the-art winter symposium ex vivo lung perfusion -state of the art in lung donor pool expansion authentic modeling of human respiratory virus infection in human pluripotent stem cell-derived lung organoids reversed graph embedding resolves complex single-cell trajectories influenza virus infects epithelial stem/progenitor cells of the distal lung: impact on fgfr2b-driven epithelial repair global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: gold executive summary platelet-derived sdf-1 primes the pulmonary capillary vascular niche to drive lung alveolar regeneration aspiration-induced lung injury commentary: sca-1 and cells of the lung: a matter of different sorts development of solitary chemosensory cells in the distal lung after severe influenza injury a retinoic acid-hedgehog cascade coordinates mesoderm-inducing signals and endoderm competence during lung specification the role of scgb1a1+ clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium rare sox2 + airway progenitor cells generate krt5 + cells that repopulate damaged alveolar parenchyma following influenza virus infection single-cell transcriptomic analysis of human lung provides insights into the pathobiology of pulmonary fibrosis basal cells as stem cells of the mouse trachea and human airway epithelium notch-dependent differentiation of adult airway basal stem cells preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice novel dynamics of human mucociliary differentiation revealed by single-cell rna sequencing of nasal epithelial cultures bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia in vivo optimal-transport analysis of single-cell gene expression identifies developmental trajectories in reprogramming the idiopathic pulmonary fibrosis honeycomb cyst contains a mucocilary pseudostratified epithelium pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung-versus thyroid-lineage specification wishbone identifies bifurcating developmental trajectories from single-cell data single-cell rna-seq with waterfall reveals molecular cascades underlying adult neurogenesis human alveolar wall fibroblasts directly link epithelial type 2 cells to capillary endothelium enhanced in vivo function of bioartificial lungs in rats transplantation of lungs from a non-heart-beating donor lung vascular cell heterogeneity: endothelium, smooth muscle, and fibroblasts slingshot: cell lineage and pseudotime inference for single-cell transcriptomics modeling of fibrotic lung disease using 3d organoids derived from human pluripotent stem cells jagged and delta-like ligands control distinct events during airway progenitor cell differentiation exponential scaling of single-cell rna-seq in the past decade il-6/stat3 promotes regeneration of airway ciliated cells from basal stem cells developmental origin of lung macrophage diversity ex vivo lung perfusion: a key tool for translational science in the lungs dedifferentiation of committed epithelial cells into stem cells in vivo myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury the internal surface area of nonemphysematous lungs the dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells a molecular cell atlas of the human lung from single cell rna sequencing high-efficiency, selection-free gene repair in airway stem cells from cystic fibrosis patients rescues cftr function in differentiated epithelia annual data report: lung generation of lung epithelial-like tissue from human embryonic stem cells tracing epithelial stem cells during development, homeostasis, and repair lung donor selection and management lineage-negative progenitors mobilize to regenerate lung epithelium after major injury epithelial expression of an interstitial lung disease-associated mutation in surfactant protein-c modulates recruitment and activation of key myeloid cell populations in mice a cellular census of human lungs identifies novel cell states in health and in asthma parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury can stem cells be used to generate new lungs? ex vivo lung bioengineering with decellularized whole lung scaffolds single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo neutrophil migration through preexisting holes in the basal laminae of alveolar capillaries and epithelium during streptococcal pneumonia a pure population of lung alveolar epithelial type ii cells derived from human embryonic stem cells pulmonary alveolar type i cell population consists of two distinct subtypes that differ in cell fate assessment of lungs rejected for transplantation and implications for donor selection normothermic perfusion of donor lungs for preservation and assessment with the organ care system lung before bilateral transplantation: a pilot study of 12 patients normothermic ex-vivo preservation with the portable organ care system lung device for bilateral lung transplantation (inspire): a randomised, open-label, non-inferiority, phase 3 study clonal dynamics reveal two distinct populations of basal cells in slow-turnover airway epithelium morphological basis of alveolar-capillary gas exchange mesenchyme-free expansion and transplantation of adult alveolar progenitor cells: steps toward cell-based regenerative therapies building and regenerating the lung cell by cell tissue engineering and regenerative medicine 2015: a year in review paga: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional cftr protein progressive pulmonary fibrosis is caused by elevated mechanical tension on alveolar stem cells local lung hypoxia determines epithelial fate decisions during alveolar regeneration single-cell deconvolution of fibroblast heterogeneity in mouse pulmonary fibrosis single-cell rna sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis long-term expansion of alveolar stem cells derived from human ips cells in organoids spatial-temporal lineage restrictions of embryonic p63 + progenitors establish distinct stem cell pools in adult airways induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor cellular crosstalk in the development and regeneration of the respiratory system distinct mesenchymal lineages and niches promote epithelial self-renewal and myofibrogenesis in the lung p63(+)krt5(+) distal airway stem cells are essential for lung regeneration the following authors were supported by funds from the national institutes of key: cord-335597-anrzcsrt authors: nan title: 44. jahrestagung der österreichischen gesellschaft für pneumologie date: 2020-10-26 journal: wien klin wochenschr doi: 10.1007/s00508-020-01745-3 sha: doc_id: 335597 cord_uid: anrzcsrt nan patientencharakteristik, anamnese, symptome: eine 40-jährige patientin wurde nach plötzlichem auftreten von dyspnoe, tachypnoe und rechtsthorakalen schmerzen mit ausstrahlung in den rechten oberbauch vorstellig. die anamnese bezüglich traumas oder chronischen vorerkrankungen war unauffällig. diagnostik und diagnose: die blutgasanalyse zeigte eine hypoxie. laborchemisch fand sich lediglich eine isolierte, milde leukozytose. die thoraxauskultation ergab rechtsseitig verminderte atemgeräusche. die computertomografie des thorax wies einen rechtsseitigen hinteren zwerchfelldefekt mit intrathorakaler hernierung des kolon ascendens und rechtsseitigen pneumothorax mit mediastinalshift nach links aus. bildgebend bestand mithin der verdacht auf eine kolonperforation und eine bochdalek-hernie. differentialdiagnostik: obwohl bochdalek-hernien bei erwachsenen selten sind, müssen sie differentialdiagnostisch bei patienten mit dyspnoe berücksichtigt werden. eine intrathorakale kontamination nach bakterieller translokation oder hohlorganperforation kann zur entwicklung von pleuraempyemen führen. die sorgfältige intraoperative lavage und drainage der thoraxhöhle hat deshalb in diesen fällen große bedeutung. therapie: es erfolgte eine notfalllaparotomie, welche die diagnose bestätigte. ein 2 cm langer zwerchfelldefekt mit inkarzeriertem und perforiertem kolon ascendens wurde erkannt. unter erweiterung der bruchpforte konnte das kolon nach intraabdominell reponiert werden. aufgrund von ischämischen veränderungen sowie der kolonperforation wurde eine rechtsseitige hemikolektomie mit anlage einer seit-zu-seit ileotransversostomie erforderlich. nach ausgedehnter abdomineller und transdiaphragmaler thorakaler lavage wurde eine bülaudrainage platziert, zwerchfelldefekt und bauchdecke wurden mittels naht verschlossen. am 13. postoperativen tag wurde aufgrund eines rechtsseitigen pleuraempyems nach diagnostischer vats eine anterolaterale thorakotomie mit anschließender pleuradekortikation zur sanierung erforderlich. die patientin erholte sich letztlich gut und wurde am 12. postoperativen tag nach hause entlassen. patientencharakteristik, anamnese, symptome: im juli 2020 wird eine 62-jährige patientin vom niedergelassenen pneumologen an unsere abteilung überwiesen. die aufnahme erfolgt aufgrund einer vor ca. zwei wochen suspizierten pneumonie, welche mit amoxicillin-clavulansäure behandelt wurde. im aufnahmegespräch berichtet die patientin über deutliche abgeschlagenheit und belastungsdyspnoe (stiegen steigen) bei bisher altersentsprechend sehr guter leistungsfähigkeit. in der anamnese sind eine atypische pneumonie vor einem jahr, eine primär biliäre cholangitis (ed 2019 im rahmen der pneumonie) und eine substituierte hypothyreose auffällig. in den von uns initial erhobenen laborbefunden zeigen sich moderat erhöhte entzündungsparameter (crp 5,79 mg/ dl, leukozyten 15,1 g/l), woraufhin die antibiotische therapie auf azithromycin 500 mg umgestellt wird. eine durchgeführte computertomographie des thorax zeigt bipulmonale fleckige milchglastrübungen, peribronchovaskuläre verdichtungen mit teils positivem pneumobronchogramm und hiläre sowie mediastinale lymphadenopathie. zur abklärung der dementsprechend im raum stehenden differenzialdiagnosen einer sarkoidose, vaskulitis bzw. eines lymphoms werden eine bronchoskopie mit transbronchialer lymphknotenbiopsie (lk11l, lk11r, lk7 und lk4r) und detaillierte laborchemische untersuchungen durchgeführt. diagnostik und diagnose: im verlauf kommt es zu einer verschlechterung der nierenfunktion (creatinin 1,77 mg/dl) und einer proteinurie (1,4 g/l), ansteigenden entzündungsparametern (crp, leukozyten, bsg) und einem positiven ergebnis für mpo-anca (27 iu/ml). die histopathologische aufarbeitung der lymphknotenbiopsien zeigt ausgeprägte reaktive zell-und kernveränderungen ohne hinweis für eine infiltration mit malignen zellen. die erhobenen befunde erhärten den verdacht auf eine mpa-anca-assoziierte vaskulitis mit pulmonaler und renaler beteiligung. die patientin wird an die universitätsklinik für nephrologie transferiert, wo komplikationslos eine nierenbiopsie durchgeführt wird. hierbei zeigt sich das histologische bild einer pauci-immunen glomerulonephritis mit halbmondbildungen und schlingennekrosen in 50 % der glomeruli, vereinbar mit der diagnose einer mpo-ancaassoziierte vaskulitis. therapie: nach entsprechender aufklärung der patientin beginnen wir mit der therapie mit rituximab 1000 mg (aktuell zwei gaben erhalten, dritte gabe geplant) sowie methylpred-therapie: im rahmen einer rechtsseitigen thorakotomie erfolgte eine offene wedge-resektion des oberlappen sowie mittellappens rechts, wobei sich intraoperativ histologisch der verdacht einer lymphominfiltration ergab. die fistulierung zur speiseröhre wurde mittels vena azygos-patch gedeckt und übernäht. eine protektive endoluminale vac-anlage konnte bereits am 4. postoperativen tag entfernt werden. histologisch bestätige sich ein aggressives non-hodgkin-lymphom der b-zellreihe, speziell eines diffusen großzelligen b-zell-lymphoms (zentroblastisch-polymorph), nicht-keimzentrumstyp nach hans-klassifikator. von onkologischer seite wurde einer therapie mittels r-chop initiiert bei postoperativ chirurgisch unauffälligem status. allergie mit biss background: lung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (clad). the mechanisms leading to clad remain elusive due to insufficient understanding of the complex post-transplant adaptation processes. here, we aimed to better understand the processes preceding clad, and investigate their association with future changes in allograft function. methods: we performed an exploratory cohort study in 78 patients, including broncho-alveolar lavage samples from lung donors and recipients (after transplantation). we analyzed the alveolar microbiome using 16s rrna sequencing, the cellular composition using flow-cytometry, and conducted metabolome and lipidome profiling. results: we established distinct temporal dynamics for each of the analyzed data sets. comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. we further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. a decline in forced expiratory volume during the first second (fev1) is a major characteristic of lung allograft dysfunction in transplant recipients. by using a machine learning approach, we could accurately predict future changes in fev1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power. conclusions: broncho-alveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. the lung microbiome can predict future changes in lung function with high precision. über eine anterolaterale thorakotomie rechts im 6. icr inklusive adäquate onkologische lymphadenektomie position 4 rechts bis 11i the authors marked with an asterisk (*) are the corresponding authors. abstracts ögp conclusions: patients with copd are insufficiently evaluated for cad due to overlapping symptoms. current cad risk scores for stable chest pain appear inappropriate for patients with copd. background: oncologic patients are regarded the population most at risk of developing a severe course of covid-19 due to the fact that malignant diseases and chemotherapy often weaken the immune system. in the face of the ongoing sars-cov-2 pandemic, how particular patients deal with this infection remains an important question. in the period between the 15th and 26th of april 2020, a total of 1227 patients were tested in one of seven oncologic outpatient clinics for sars-cov-2, regardless of symptoms, employing rt-qpcr using bgi real-time fluorescent rt-pcr kit for detecting 2019-ncov2 on applied bioscience abi7500 instruments. results: of 1227 patients, seventy-eight (6.4 %) were tested positive of sars-cov-2. only one of the patients who tested positive developed a severe form of covid-19 with pneumonia (curb-65 score of 2), and two patients showed mild symptoms. fourteen out of 75 asymptomatic but positively tested patients received chemotherapy or chemo-immunotherapy according to their regular therapy algorithm (+/-4 weeks of sars-cov-2 test), and 48 of 78 (61.5 %) positive tested patients received glucocorticoids as co-medication. none of the asymptomatic p02 current symptom-based risk scores for stable coronary artery disease evaluation are not applicable in copd patients background: cardiovascular diseases are arguably the most important comorbidity in patients with chronic obstructive pulmonary disease (copd). despite an increased prevalence of coronary artery disease (cad) in copd patients, there are no dedicated diagnostic recommendations. we investigated whether copd patients receive adequate primary evaluation of cad despite overlapping symptoms. methods: 302 patients with copd, who underwent invasive coronary angiography (ica), were retrospectively matched (for age, bmi and cardiovascular risk factors) with 302 patients without functional lung diseases. quality and onset of symptoms prior to ica were documented and individual patients' pre-test probabilities according to esc guidelines were calculated. endpoints were delay of ica referral after symptom onset and clinical outcome, defined as subsequent revascularization. results: mean delay between symptom onset and ica was 19.9 ± 22.0 months in copd patients compared to 8.3 ± 12.7 months in the control group (p < 0.0001). copd patients had a lower rate of typical chest pain (25.2 % vs. 38.1 %, p = 0.0009), and dyspnoea only (18.2 % vs. 26.8 %, p = 0.015). sub-analysis of gold grades revealed an incremental delay with increasing copd severity: gold 1: 16.1 ± 17.3 months; gold 2: 17.6 ± 22.1 months; gold 3: 20.1 ± 21.3 months and gold 4: 24.2 ± 23.4 months. furthermore, the revascularization rate increased with higher pre-test probability for the control group, but not for patients with copd gold 1-4. abstracts background: chronic thromboembolic pulmonary hypertension (cteph) is characterized by severe pulmonary artery hypertension and presence of sleep-disordered breathing (sdb) with associated hypoxemia which could further contribute to the severity of hypertension adversely affecting the outcome. limited data are available on the prevalence of sdb in cteph and so far, the effect of balloon pulmonary angioplasty (bpa) on sdb has not been evaluated. we hypothesized that subjects with cteph have a high prevalence of sdb, both obstructive and central sleep apnea with associated hypoxemia, which could improve with bpa. methods: 33 consecutive patients with cteph underwent treatment-naïve and post-bpa polygraphy (nasal-pressure-sensor, thermistor, thoracoabdominal-excursion-sensors, pulse oximeter; alice pdx, philipps®) and hemodynamic and echocardiographic assessments. results: before bpa, prevalence of sdb (defined as an apnea-hypopnea index (ahi) >5 per hour) was 75 %: 8 patients infected patients showed unexpected complications due to the sars-cov-2 infection during the cancer treatment. conclusions: these data clearly contrast the view that patients with an oncologic disease are particularly vulnerable to sars-cov-2 and suggest that compromising therapies could be continued or started despite the ongoing pandemic. moreover, the relatively low appearance of symptoms due to covid-19 among patients on chemotherapy and other immunosuppressive co-medication like glucocorticoids indicate that suppressing the response capacity of the immune system reduces disease severity. background: for the further crisis management of the corona pandemic and the socio-economic impact on society, a strategy that allows selective isolation measures is particularly important. so far, it has been assumed that patients suffering from covid-19 develop antibodies that provide immunity and are thus protected from a reinfection with sars-cov-2. this also forms the basis of the assumption that rapid vaccine development will lead to rapid control of the pandemic. in the present study, we analyzed the antibody development of 77 oncology patients 14 days after positive rt-qpcr testing for sars cov2. methods: rt-qpcr and anti-sars-cov2-antibody methods from bgi (mgieasy magnetic beads virus dna/rna extraction kit) and roche (elecsys anti-sars-cov-2 immunoassay) were used, respectively, according to the manufacturers' specifications. results: surprisingly, in only 6 of 77 individuals with a confirmed history of covid-19 antibody development was detected. despite of multiple testing, these patients did not develop antibodies in subsequent tests. conclusions: first analyses indicate that patients may benefit from inpatient pr after hospitalization due to covid-19. symptoms of dyspnea, cough, depression, and anxiety decreased significantly over the course of the pr, whereas quality of life significantly increased. pr could therefore play an important role in dealing with the pandemic. follow-up assessments three and six months after the pr are currently ongoing. background: fatigue is among the most common symptoms in covid-19 patients and about 50 % still suffer from persistent fatigue 2 months later [1] . some studies discuss a possible link to obstructive sleep apnea syndrome (osas) [2] , however, no studies have been published in covid-19 patients after discharge. therefore, we examined covid-19 patients for the presence of osas within 4 weeks after acute hospital discharge during inpatient pulmonary rehabilitation. methods: from may until july 2020 we screened all eligible covid-19 patients for osas using polygraphy. if the screening revealed an apnea-hypopnea index (ahi) of ≥ 15, further diagnostics using polysomnography were conducted. furthermore, we assessed the sleepiness using the epworth sleepiness scale (ess) and the body-mass-index (bmi). results: 37 patients were eligible for the study, of which 24 were willing to participate. mean age was 56.4 (range: 33-83), 24 % were female, and mean bmi was 31.8 (range: 22.2-53.3). only four patients had an ahi <5, whereas eleven fulfilled the criteria for an at least moderately severe osas (ahi ≥15). the ahi was significantly correlated with the bmi (r = 0.44, p < 0.05) but not with the ess (r = -0.09, p = 0.71). a positive airway pressure therapy was indicated in eight patients (33 %); five agreed to the therapy (20 %). conclusions: according to our data, rates of osas are extraordinarily high in patients after hospitalization due to covid-19. this could be an explanation for frequently mentioned symptoms of tiredness and attention deficit. yet, further studies are needed to examine a possible causal association and correlations to other common symptoms, such as cognitive impairment or sleep disturbances. (24 %) without sdb, 18 (54 %) with predominantly obstructive sleep apnea (osa; ahi = 26), and 7 (21 %) with predominantly central sleep apnea (csa; ahi = 41). osa was associated with male-gender, obesity and overnight fluid-shifts, whereas csa with worse right ventricular end-diastolic diameter. patients with sdb had significantly higher oxygen-desaturation index (odi) and tendency for worse desaturation than those without sdb. after bpa, mean ahi and odi decreased by 45 % (p = 0.004) and 28 % (p = 0.001). in osa patients, ahi decreased from 26 to 19 (p = 0.088) and in csa patients from 41 to 21 (p = 0.011). along with improvement in sdb, nocturnal desaturation decreased (time-below-90 % from 50 % to 43 % of time-in-bed, p = 0.037). conclusions: this is the first study of the effects of bpa on sdb in cteph. we found high prevalence of sdb, both osa and csa, in consecutive subjects underdoing bpa, and report that bpa significantly improved sleep-disordered breathing and nocturnal desaturations. future randomized controlled trials are needed to determine if effective treatment of sdb improves central hemodynamics, morbidity and mortality of patients with cteph. pulmonary rehabilitation following covid-19 -first short-term results regarding symptoms, quality of life, and psychological burden of disease background: even though many studies have been published on covid-19 within the last months, little is known about the results of pulmonary rehabilitation (pr) following a severe infection. therefore, the current study examines the changes of wellbeing through inpatient pr after covid-19. methods: we surveyed patients at the beginning (t1) and the end (t2) of inpatient pr following hospitalization due to covid-19. we assessed respiratory symptoms (dyspnea, cough, and phlegm expectoration) and pain with symptom rating scales, fatigue with the brief fatigue inventory (bfi), quality of life with the euroqol-questionnaire (eq-5d-sl), and symptoms of depression and anxiety with the patient health questionnaire (phq-d). results: from the beginning of may until the end of june 2020, 31 patients were eligible, of which 25 patients could be included in the study and completed all t1 and t2 assessments (mean age: 58.1; 38.9 % female; 52.8 % after invasive ventilation). at t1 the participants were heavily burdened and dyspnea on exertion was by far the most common and burdensome impairment. over the course of the pr (mean treatment duration: 26 days; range: 21-35 days), the data revealed improvements in all mentioned outcomes. dependent samples t-tests revealed statistical significance in all variables, except for pain (p = 0.058) and phlegm expectoration (p = 0.258). effect sizes ranged from small (d = 0.48, p < 0.01 for dyspnea at rest) to large abstracts background: patients with repaired congenital diaphragmatic hernia (cdh) often suffer from obstructive airway disease. nitrogen multiple breath washout (n2-mbw) is a sensitive method to detect ventilation inhomogeneity and peripheral airway pathology with higher sensitivity than conventional spirometry. we set out to obtain detailed information about peripheral airway pathology by n2-mbw in addition to conventional lung function testing. methods: we prospectively compared school-aged children following cdh repair and healthy controls using spirometry, body plethysmography and n2-mbw. group analyses were made using t-test and mann-whitney-u test, as appropriate. matching criteria included age, gender and level of physical activity. results: 13 (median [iqr] age 10 [8-13] years, f:m = 5:8) former patients and 11 matched healthy controls (9 [7-12] years, f:m = 4:7) were included. mean lung clearance index (lci) was highly similar in both groups (7.4 vs. 7.4; p = 0.893). slope of conducting airways (scond) was significantly higher (0.029 vs. 0.017; p = 0.032) in cdh patients. fev1 (88 vs. 101 %pred; p = 0.009), mef25 (72 vs. 108 %pred; p = 0.009), mef50 (78 vs. 109 %pred; p = 0.002) and fef25-75 (66 vs. 95 %pred; p = 0.003) were significantly lower in cdh patients. rv (133 vs. 85 %pred; p = 0.001), rv/tlc ratio (39 vs. 24 %pred; p = 0.001) and airway resistance (reff ) (146 vs. 103 %pred; p = 0.003) were significantly higher in cdh patients, whereas there was no significant difference in tlc (99 vs. 106 %pred; p = 0.230) and fvc (100 vs. 102 %pred; p = 0.698). three cdh patients had lci and eight scond values above the upper limit of normal (healthy controls: two and three, respectively). according to conventional lung function testing, 7/13 former patients showed an obstructive, none a restrictive pattern and six had normal lung function. fev1% correlated significantly positively with mef25%, mef50% and fef25-75 % and negatively with rv/tlc ratio. conclusions: we found significant airway obstruction in both central and peripheral airways and hyperinflation in patients with congenital diaphragmatic hernia compared to healthy controls. central sleep apnea in pacing-induced cardiomyopathy background: sleep disordered breathing, in particular central sleep apnea (csa) is common in heart failure patients, but its role in pacing induced cardiomyopathy has not been studied yet. in this study entitled upgrade, we set out to evaluate the effect on sleep architecture and sleep disordered breathing in picm patients receiving biventricular pacing. methods: presence of csa was assessed by single-night polysomnography (psg) in 54 picm patients within one month after left ventricular lead implantation (with biventricular stimulation still not activated). csa was diagnosed in half of patients (n = 27). patients with moderate or severe csa were randomized to cardiac resynchronisation therapy (crt) versus right ventricular pacing (rvp) in a double-blinded cross-over design and re-scheduled for a follow up psg 3-5 months, after repeated assessment of sleep and crossing-over another psg was conducted 3-5 months later. results: crt led to a significant increase in left ventricular ejection fraction and significant reduction in left ventricular end systolic volumes and n-terminal pro brain natriuretic peptide plasma levels, whereas no significant effect was observed with ongoing rvp. csa was significantly improved after 3.9 (3.2-4.4) months of crt: apnea hypopnea index (ahi) decreased from 39.1 (32.1-54.0) events per hour at baseline to 22.2/h (10.9-36.7) by crt (p < 0.001). central apnea index decreased from 27.1/h (17.7-36.1) at baseline to 6.8/h (1.1-14.4) after crt activation (p < 0.001). ongoing rvp yielded only a minor improvement in ahi and central apnea index. pre-existent csa did not affect structural response rate and had no impact on mid-term follow up (median 2.8 years). conclusions: csa is highly prevalent in patients with picm. crt upgrading significantly improves csa leading to a similar outcome in picm patients without pre-existent csa. upgrade is an investigator-initiated independent clinical trial, supported by the önb jubiläumsfondsprojekt nr. 15974. this study was further supported by an unlimited scientific grant from the boston scientific investigator sponsored research , named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was observed in wuhan (china) for the first time and subsequently spread rapidly across the globe. the lung is the virus' primary target organ, but many other organs are affected, too. in consequence, therapy focuses on both, pulmonary and systemic symptoms. at present, there is no established pharmacological treatment for covid-19 available, however, many studies are currently on their way. treatment is based on local (i. e. inhalation) and systemic therapy, often in ventilated patients. prerequisites for inhalation therapy are measures to prevent infection of health care personnel, use of adequate systems for drug administration and compounds suitable for pulmonary delivery. we reviewed publications on covid-19 treatment for strategies for save inhalation therapy of ventilated/non-ventilated patients and compounds used in clinical studies. strategies for inhalation administration differ in respect of disease severity and use of personnel protective equipment is essential. in mild-disease patients, asthma/copd treatment is preferred by pmdis/dpis, if necessary. jet/mesh nebulizers can be used with mouth pieces/nasal cannulas (no face masks to avoid aerosol spread) and one-way filters/valves. in ventilated patients, mesh nebulizers with filters should be used. physical therapy/suctioning should not be combined with aerosol therapy (avoidance aerosol spread). numerous compounds/biomolecules are under study for inhalation treatment of covid-19, e. g. interferons (with/without systemic administration of antivirals, such as ribavirin, lopinavir, ritonavir), sargramostim (gm-csf), aviptadil (synthetic vasoactive intestinal polypeptide), das181 (recombinant sialidase), pul042 (immunostimulant, tlr 2/6/9 agonist), budesonide, nitrogen oxide and hydrogen. in summary, inhalation therapy is important for treatment of pulmonary symptoms of covid-19. strategies for pulmonary drug delivery differ in respect to disease severity (e. g. mild symptom patients vs. ventilated patients). various pharmacological compounds/biomolecules are under study. however, up to now there is no established inhalation treatment of covid-19. ual basis. recent studies show that hs can improve health even in cf babies. methods: the surveys provide information about acceptance, reason and frequency of 3-7 % hs application using a jet or vibrating membrane nebuliser (vmn; eflow®rapid) in children ≤ 6 years. the online questionnaires address healthcare professionals in paediatric cf centres in the uk and german speaking countries (dach). in the uk 21 and in dach 17 paediatric cf centres participated in the survey. from both regions, a total of 54 healthcare professionals responsible for almost 1900 cf children ≤ 6 years of age responded. results: secretolysis by hs in children ≤ 6y is rated excellent, very good, or good by 81.1 % in uk and by 100 % in dach. the tolerability is reported excellent to good by 67.1 % (uk)/100 % (dach). in both surveys approx. 90 % of caregivers declare to use vmn in cf patients ≤ 6 y and less than 25 % in infants ≤ 2 y. caregivers satisfaction regarding the ease of inhalation with vmn in infants ≤ 2 y is rated excellent to good by 70.5 % (uk)/59 % (dach) and in the group 2-6y by 100 % (uk and dach). conclusions: the results of the survey reveal that in children ≤ 6 years nebulized hs for secretolytic therapy is general practice and well tolerated by this age group. they are consistent with the latest recommendations in the official cf guidelines for children, which confirm the benefits of using hs. in addition, the data also reflect the trend in the uk, where the annual report of the uk cf registry refers that the use of hs in young children is increasing every year. expression patterns of heat shock protein 90 in patients with thymic epithelial tumors regarding the world health organization classification background: thymic epithelial tumors (tets) are rare malignancies with unique association to the paraneoplastic syndrome myasthenia gravis (mg). heat shock protein 90 (hsp90) harbour great potential as cancer biomarker and hsp90 inhibitors approach clinical cancer therapy. methods: to investigate hsp90 tissue expression patterns, we analysed tumor tissues of completely resected tet patients (n = 101; 78 thymomas and 23 thymic carcinomas (tcs)), regular thymic tissue of six mg patients, and four patients without mg, background: pulmonary hemodynamics during exercise may help to identify early pulmonary vascular disease in systemic sclerosis (ssc). whether they are of prognostic relevance in this subset of patients is unknown. we tested the association between pulmonary heamodynamics at rest and peak exercise with all-cause mortality in patients with ssc. methods: all ssc patients with resting mpap < 25 mm hg and at least 1-year follow-up data who underwent symptomlimited exercise right heart catheterization between april 2005 and december 2018, were analyzed. age-adjusted cox-regression analyses were performed to assess the association between pulmonary hemodynamics and mortality. 76]) turned out as age-independent predictors of mortality. in contrast, resting pulmonary hemodynamics (mpap, pulmonary arterial wedge pressure, co, pvr and tpr) were not associated with age-adjusted mortality. conclusions: in this study assessing the prognostic relevance of pulmonary exercise hemodynamics in patients with systemic sclerosis, pvr and tpr at peak exercise as well as mpap/co-slope and tpg/co-slope turned out as age-independent predictors of all-cause mortality. clinical survey with hypertonic saline (3-7%) and the eflow®rapid in cf children below 6 years of age mnt: 100 % a: 60 % ab: 7 % b1: 11.1 % and b2: 11.5 b3: 100 % tc: 95.7 %). we detected hsp90 expression in centroblasts, but not centrocytes, of germinal centres in 100 % of mg patients with fth. all lymphoid follicles of myasthenic patients expressed hsp90 protein. hassall's corpuscles showed no hsp90 expression in every tissue sample. we did not detect thymic hsp90 expression in four patients with regular thymic morphology or five patients with tth. conclusions: hsp90 expression data propose high potential for hsp90 as an additional immunohistochemical marker for mnt, who-b3 thymoma, and tc or as a possible candidate molecule for targeted therapy. caution is warranted in tet patients with mg overexpressing hsp90. later-line treatment with lorlatinib in alkand ros1-rearrangement-positive nsclc: a retrospective, multicenter analysis background: anti-fibrotic medication is effective in progressive fibrosing interstitial lung diseases (ild), but a subgroup of fibrotic ild patients also benefits from immunomodulatory therapies. additional to high-resolution computed tomography (hrct), blood and broncho-alveolar lavage (bal) biomarkers could help to identify such phenotypes. methods: hrct of 127 subsequent single-center ildboard patients (mean age 65 (standard deviation 14) years, 65 % male), were evaluated for radiological findings considered noninflammatory (reticulation including honeycombing (ret), traction bronchiectasis (tbr), emphysema (emp)) or active inflammatory (consolidations (con), ground glass opacities (ggo), noduli (ndl), mosaic attenuation (mos)) in 6 distinct lung regions. each resulting score was further graded as minimal (0-1 regions involved), medium (2-4) and extensive (5-6). associations between blood and bal biomarkers and radiological finding scores were evaluated using spearman correlation coefficients, kruskal-wallis tests were used for significance testing between the graded subgroups. results: blood neutrophil, lymphocyte and eosinophil fraction, neutrophil-lymphocyte ratio (nlr) and bal lymphocyte fraction consistently showed opposite correlations for inflammatory versus non-inflammatory hrct finding scores. blood lymphocyte fraction significantly differed by the graded extent of ggo (p = 0.032) and con (p = 0.027), eosinophil count by tbr (p = 0.006) and nlr by con (p = 0.009). c-reactive protein significantly related to ggo (p = 0.023) and con (p = 0.004), while ldh showed multiple significant positive associations with ret (p = 0.01), tbr (p < 0.001), ggo (p = 0.049) and mos (p = 0.027). in bal fluid, lymphocyte fraction had a significant interaction with ggo (p = 0.017). conclusions: biomarkers from peripheral blood and bal may have the potential to differentiate predominantly noninflammatory or fibrotic from active inflammatory radiological ild patterns. the german severe asthma registry: obesity is associated with asthma parameters abstracts (sgrq-c) was used. furthermore, we asked, independent from each other, the patient as well as the treating physician to estimate the global health status of the patient (excellent, good, fair, poor, very poor). inclusion criteria were a physician's diagnosis of copd and age ≥ 40 years. subjects with a history of lung surgery, lung cancer or copd exacerbation within the last four weeks were excluded. results: 67 pulmonologists and 6 general practitioners participated and enrolled 1,175 copd patients. of those 248 patients did not fulfill the gold criteria for copd (fev1/ fvc ≥ 0.7) and 77 were excluded due to missing data. finally, 850 patients (62.8 % men; mean age 66.2 ± 0.3 (se) years; mean fev1%pred. 51.5 ± 0.6 (se)) were analyzed. in 48.7 % of study participants, patients and physicians disagreed on the global health status. in 29.7 % it was estimated better by the physician than by the patient (overrated patients), and in 19.0 % it was underrated. multivariate regression analysis indicated that overrated patients had a statistically significant better lung function (fev1), less exacerbations and a lower total sgrq-c score compared to underrated patients. conclusions: in stable copd outpatients, treated by pulmonologists and general practitioners, the global health status, most likely indicating the burden of copd, tends to be overestimated by physicians in patients with milder airway obstruction and less exacerbations and underestimated in patients with more severe airway obstruction and frequent exacerbations. this discordant perception of global health might severely affect treatment options. lowering of mean pulmonary artery pressure is a prognostic marker in pulmonary hypertension background: treprostinil (tre), a prostacyclin analog, is effective for the treatment of pulmonary arterial hypertension and non-operable chronic thromboembolic pulmonary hypertension (cteph). we hypothesized that a greater change of hemodynamics is of prognostic value. therefore, we evaluated effects of first-line subcutaneous (sc) tre in patients with severe pulmonary hypertension (ph) and analyzed the prognostic value of hemodynamic response at 1 year on treatment. methods: data was prospectively collected from patients with pre-capillary ph in who functional class iii or iv, mean right atrial pressure of 10 mm hg, and/or cardiac index ≤ 2.2 liters/min/m 2 . patients received first-line sctre. dose adjustments were performed individually according to clinical symptoms and side effects. results: between 1999 and 2019 138 patients were treated with first-line sctre. all patients were classified as non-lowrisk at baseline (6mwd > 440, who functional class i or ii, right atrial pressure < 8 mm hg and cardiac index ≥ 2.5 l/min/m 2 ). 18 (13 %) patients underwent double lung transplantation, and 59 (42.8 %) died of any cause. overall survival rates at 1, 5, 10, and 15 years were 91 %, 57 %, 31 % and 29 %. the strongest predictor of outcome was change in mpap after one year of sctre. change in mpap -18.4 ± 7.9 mm hg (p = 0.012) was associated with the best subsequent survival of 12.7 ± 1.5 years. obesity is a risk factor for asthma severity. this study aims to evaluate associations of obesity with severe asthma parameters in the german severe asthma registry including 1065 patients (49 ± 17 yrs, 58 % female, bmi 27 ± 6 kg/m², 26 % obese (bmi ≥ 30 kg/m²)), 107 treated with anti-ige-and 237 with anti-il5(r) antibodies. the same proportion of obese patients received biologics as non-obese patients, but obese patients were more frequently on lama therapy (60 % vs. 51 % p = 0.01), had more exacerbations (4.5 ± 4/y vs. 3.6 ± 3/y, p = 0.003), worse quality of life and more often uncontrolled asthma (88 % vs. 78 %, p < 0.001), as reflected by worse acq (3.2 ± 1.4 vs. 2.8 ± 1.5), act (12 ± 5 vs. 15 ± 5) and maqlq scores (3.5 ± 1.2 vs. 4.0 ± 1.3; all p < 0.001). obesity was associated with less blood eosinophils (37 % vs. 53 %, p < 0.001), more neutrophils (6.2 ± 2.8g/l vs. 5.7 ± 2.8g/l, p = 0.02) and lower lung function parameters: fev1 (1.8 ± 0.7 l vs. 2 ± 0.8 l, p = 0.01) and fvc (2.8 ± 1 l vs. 3.1 ± 1 l, p < 0.001). non-obese patients were more often non-smokers (42 % in non-obese vs. 27 % in obese, p < 0.001). conclusions: in our severe asthma cohort, obesity represents a specific phenotype of severe asthma that is significantly associated with exacerbations, worse quality of life, lower blood eosinophil numbers, as well as lower fev1, and fvc. discordant perception of global health between copd outpatients and their physicians -real world data from the clara project of pulmonary exercise hemodynamics with all-cause mortality in patients with normal or mildly elevated pulmonary arterial pressure (pap) at rest. methods: patients with unexplained dyspnea and/or suspected pvd undergoing exercise right heart catheter (rhc) at our ph-clinic were retrospectively analysed. exercise rhc was performed in case of a resting mpap < 25 mm hg. in a first step, dichotomized resting-, submaximal-and maximal exercise hemodynamic variables were analysed using multivariate cox regression, adjusted for sex and age, to identify prognostic cut-offs. best cut-off for each variable was defined as the cut-off score with the smallest p-value. in a second step, the relevance of cut-offs, derived from the first model, was assessed using a multivariate model also accounting for age, sex, cardiopulmonary comorbidities, smoking history, and pulmonary resting hemodynamics. results: 207 patients were included (69 % female, age: 62 ± 13 yr, mpap: 18 ± 4 mm hg). median observational-time was 4.3 yr (iqr: 2.0-8.5) with n = 40 (19 %) mortality events. mpap/ cardiac ouput (co)-slope, transpulmonary gradient (tpg)/coslope and pulmonary arterial wedge pressure (pawp)/co-slope turned out as sex and age independent predictors of mortality. best cut-offs were found at 7.5 mm hg/l/min (mpap/coslope), 3.9 mm hg/l/min (tpg/co-slope) and 6.0 mm hg/l/ min (pawp/co-slope). in the second model, correcting for age, sex, cardiopulmonary comorbidities, smoking history and pulmonary resting hemodynamics, mpap/co-slope (hr: 2.84, 95 %ci: 1.22-6.59; p = 0.015), tpg/co-slope (hr: 2.60, 95 %ci: 1.17-5.82; p = 0.020) and pawp/co-slope (hr: 4.92, 95 %ci: 1.95-12.44; p = 0.001) remained significant predictors of allcause mortality. conclusions: in patients with normal or mildly elevated pap at rest, pulmonary pressure/co-slopes are predictors of allcause mortality, independent from age, sex, cardiopulmonary comorbidities and resting pulmonary hemodynamics. of the annual average of 197 500 insured patients 6.2 to 9.6 % claimed an ao medication. distribution of the age groups 0-6, 7-17, 18-56 and > 56 years was 4.0, 12.1, 39.4 and 44.6 %, respectively. based on diagnoses (hospital and sick leave data), age (< 56 years) and drug patterns an asthma cohort was selected (48 % of patients with ao). annual relative prevalence of selected drug groups is presented in fig. 1 . from 2013 to 2014 the reduction in reliever drugs is associated with an increase in controller medication, in particular, combinations of inhaled corticosteroids (ics) and formoterol (f). subgroup analyses show that this pattern is consistent in differently defined asthma cohorts, not present in a copd cohort and more marked in asthma patients seen by a respiratory specialist. 2013 two large studies on the single inhaler treatment (sit) concept were presented and in 2014 sit was introduced in the gina report. conclusions: change in asthma treatment recommendation was effectively translated into practice in burgenland. pulmonary pressure/flow slopes during exercise as independent predictors of mortality in patients at risk for pulmonary hypertension philipp douschan* 1,2 , vasile foris 1,2 , alexander avian 3 , teresa sassmann 1,2 , horst olschewski 1,2 , gabor kovacs 1,2 background: patients with early pulmonary vascular disease (pvd) typically show an abnormal hemodynamic response to exercise. however, it is unknown whether pulmonary exercise hemodynamics are of prognostic relevance in patients with early pvd, independent from pulmonary resting hemodynamics. the aim of this study was to assess the association fig. 1 | p24 abstracts of extrafine beclomethasone-dipropionate, formoterol-fumarate and glycopyrronium (bdp/ff/ g, trimbow® 87/5/9 µg) in patients with copd. methods: a prospective, multicenter nis was conducted over 52 weeks in pulmonary and general practices in austria in 2018/19. eligible patients with copd had an indication for treatment with bdp/ff/g according to the summary of product characteristics. in addition to tolerability, lung function, exacerbation rate, symptom scores and copd assessment test (cat) were recorded. results: 265 patients (male 66 %, mean age 67 years) with moderate to very severe airflow limitation (gold grade 2-4: 96.2 %) and persistent symptoms (gold b: 62.3 %, gold d: 34 %) according to the 2018 gold report were included. by end of 52 weeks, lung function parameters (fev1, fev1 %, and fvc; p < 0.001) and symptoms (cough, sputum and shortness of breath; p < 0.001) improved significantly compared to baseline. a clinically-relevant improvement from baseline in cat score was observed at week 12 and persisted at week 52 in gold b (from 22.1 to 15.3 points; p < 0.001) and gold d (from 25.5 to 16.6 points; p < 0.001) patients. a significant reduction of moderate and severe exacerbations over the study period was also observed (57.4 % and 27.3 % respectively; p < 0.001). by end of 52 weeks, 93.7 % continued on the treatment. there were 21 adverse reactions reported, of which 16 were non-serious (e. g. oral mycosis) and five were serious, but none of which were deemed drug-related. conclusions: results of this study support the tolerability and effectiveness of bdp/ff/g in patients with copd in a realworld setting. patients treated with extrafine bdp/ff/g experienced an improvement in lung function, symptom control and reduction in exacerbations. tests for diagnostics of covid-19 -principles and approvals of commercially available tests rüdiger siekmeier* 1 , tanja grammer 2 , winfried märz 2,3,4 in december 2019 an unknown viral infection was firstly described in a local fish and wild animal market in wuhan/ china which was identified as a novel coronavirus infection by the chinese center for disease control and prevention (ccdc) on jan. 7th 2020 and announced as 2019-new coronavirus disease (2019-ncov, now covid-19) by the world health organization (who) on feb. 11th 2020. rapidly spreading across the globe up to begin of august 2020 at least 18 million of infections and 650000 deaths were reported worldwide. therefore, there was an urgent need of laboratory tests. in our analysis we looked for commercially available covid-19 tests. at july 31th 2020 at least 280 commercially available tests were described (https:// www.360dx.com/coronavirus-test-tracker-launched-covid-19-tests). of these, 193 are based on pcr methods (mostly pcr, qpcr) (with federal drug agency (fda; very most) or center of disease control (cdc; few) emergency use authorization background: patients with acute exacerbations of copd do not only suffer from physical symptoms but also from psychological distress and stress. as pharmacological interventions showed only limited effectiveness in targeting the latter, a need for alternative treatment options emerges. in other chronic conditions, mindfulness interventions are effective in reducing psychological distress and stress. however, research on mindfulness interventions in copd is still scarce and not focusing on exacerbations. therefore, the present study reviewed the existing literature and explored the acceptability, feasibility, and implementation of mindfulness interventions focusing on exacerbations in copd patients. methods: firstly, literature examining mindfulness interventions in copd patients was reviewed. secondly, a qualitative and explorative study using semi-structured interviews was conducted. the sample consisted of 10 copd patients (60 % women; m = 74.40 years, sd = 8.30) hospitalised after an acute exacerbation. data were analysed using thematic analysis. results: the literature review yielded eight studies, providing preliminary evidence for the feasibility and effectiveness of mindfulness interventions in copd patients. the qualitative analysis revealed five main findings: (1) patients express an openness and need for new treatment approaches. (2) mindfulness is difficult to differentiate from other mind-body concepts. (3) implementation conditions are crucial for patient's interest. (4) limitations of the application of interventions must be considered. (5) not interested patients differ from interested ones. conclusions: hospitalized copd patients showed a strong interest in new treatment approaches like mindfulness interventions. focusing on mindfulness interventions during exacerbations seem acceptable and feasible. future studies investigating those are needed and should consider implementation conditions, patients' needs and physical limitations. background: systemic sclerosis is a chronic autoimmune disease characterized by inflammation and tissue remodelling. increases in the expression and of the ap-1 transcription factor fra-2 has been shown in the skin of these patients. in mice ectopic overexpression of fra-2 leads to a systemic sclerosis phenotype, strongly affecting the skin and lung. fra-2 transgenic mice show pronounced pulmonary inflammation, vascular remodelling and lung fibrosis. although, the role of several immune cells such as macrophages, b and t lymphocytes has been investigated, the contribution of innate lymphoid cells (ilc) to disease pathogenesis remains elusive. the focus of this study was to determine the development and function of ilc and their role in scleroderma. methods: multi-colour flow cytometry was used to evaluate the inflammatory cell landscape in fra-2 transgenic mice in a time dependent manner. primary cells were isolated and functional assays e. g. apoptosis (caspase activation) and proliferation (ki67 staining and cell counts) were performed in vitro. results: pronounced changes in the inflammatory profile of the lung were observed in a time dependent manner, with increased numbers of t cells and eosinophils and reduced ilc. similar changes were also reflected in the blood, spleen and liver. isolated ilc exhibited decreased proliferation and functional activity. importantly, reduced numbers and function of ilc was already observed before the first signs of lung fibrosis, as assessed by collagen deposition and lung function measurements. conclusions: this early dysregulation suggests that ilc play an important role in the development of lung fibrosis in scleroderma and restoration of ilc could prevent the progression of the disease. (eua)/with ce-mark/with eua and ce-mark: 161(12 pending)/60/28) serving as gold standard for virus diagnostics after sampling of nasal/throat swabs in acute infection or other molecular methods (isothermal amplification (4/0/0), crispr (2/0/0), sequencing (2/0/0) and others (1/0/0). 3 more tests (1/1/1) are based on immunological antigen detection of virus peptides after sampling of nasal/throat swabs in acute infection which are typically poct tests based e. g. on immunofluorescence-based lateral flow technology or chromatographic digital immunoassay providing results in a few minutes. 71 tests (50(16 pending, 1 revoked)/34/14) allow measurement of immunoglobulines igm, iga and igg alone/in combination in blood samples and provide information on the immune status after convalescence. analytical principles of these are different and some (e. g. lateral flow assays) serve for rapid diagnostics. in summary, number and quality of tests rapidly increased. recent development is based on regulatory guidelines (e. g. https://www.gov.uk/government/publications/how-testsand-testing-kits-for-coronavirus-covid-19-work) and includes also combined tests for discrimination against other diseases (e. g. influenza). serum tumor maker dynamics as predictive biomarkers in nsclc chemo-immunotherapy and mono-immunotherapy maintenance -a retrospective cohort study department of pulmonology, johannes kepler university linz, linz, austria objectives: to evaluate serum tumor markers (stm) as biomarkers for treatment, monitoring and prognosis in advanced non-small cell lung cancer (nsclc) treated with chemoimmunotherapy. methods: patients having received platinum-based doublet chemotherapy (cht) and pd-1/pd-l1-directed immune checkpoint inhibitor (ici) combination therapy were retrospectively followed. carcinoembryonic antigen (cea), carbohydrate antigen 19-9 (ca19-9), cytokeratin-19 fragments (cyfra 21-1) and neuron specific enolase (nse) were routinely measured at nsclc diagnosis. the marker with the highest relative elevation was defined "leading stm", its change was assessed between cht-ici initiation as well as first mono-ici maintenance therapy and the respective subsequent restaging. corresponding computed tomography (ct) evaluations were analyzed according to response evaluation criteria in solid tumors (recist). for both cht-ici and ici-maintenance phase, stm and recist response were evaluated regarding progression-free (pfs) and overall survival (os) in kaplan-meier analyses. results: among 80 cht-ici patients (41 % women, mean age 63 years), median pfs was 5 months (m; 4,9) and median os was 15 m (10,/). pfs was significantly (p = 0.042) longer, when stm concomitantly decreased (9 m (5,12; n = 41)) vs. 5 m (3,6; n = 16). in the 54 (67.5 %) patients who received mono-ici maintenance, stm decrease was associated with significantly (p < 0.001) longer pfs (16 m (7,/; n = 16) vs. 3.5 m (2,6; n = 22)). median os was not reached in most subgroups in both treatment phases. patients with radiologically stable or progressive disease and concomitant stm decrease vs. increase had similar pfs in the cht-ici setting (4 m (3,7; n = 16) vs. 4.5 (2,6; n = 14)), but longer pfs in the mono-ici maintenance setting (13 m (7,16; n = 10) vs. 3 m (2,4; n = 17)). employing a retrospective approach, we collected and analyzed data of all patients with advanced non-small-cell lung cancer who received ici monotherapy with atezolizumab, nivolumab or pembrolizumab at the kepler university hospital linz between may 2015 and december 2019. kaplan-meier analyses were used to evaluate pfs and os. uni-and multivariate cox-regression analyses were calculated to show the impact of influencing variables. results: of 228 patients, 166 persons died (72.8 %). regarding to the 136 male patients, 99 died (72.8 %). for female patients it was 69 out of 92 (72.8 %). kaplan-meier analyses showed no significant difference for pfs (median length 3,5 months, p = 0.273) or os (median length 10 months, p = 0.592) between men and women. with regards to gender related predictors of outcome like pd-l1 expression or ecog-score, we observed considerable differences: pd-l1 expression could be shown a significant predictor for pfs and ecog status predicted os in men. however, we could not verify any significant predictors for female patients. conclusions: in our retrospective research covering 228 participants, we could not verify the higher chance of survival among male patients, frequently mentioned in previous studies. the finding that we could not verify any significant predictors for female patients shows the necessity for further research in that field especially in women. background: endothelial cells (ec) represent a key cell type in the homeostatic regulation of vascular and lung function, including vasoreactivity, coagulation, immune processes and barrier function. disturbances in ec function have been associated with development and progression of pulmonary hypertension, both in its idiopathic form or associated with interstitial lung disease. however, it is not clear whether these functional changes are associated with altered ec composition. methods: we performed single cell rna sequencing on pulmonary arteries isolated from donors and pulmonary hypertension patients. bioinformatics analysis was conducted to gain unbiased insight into ec heterogeneity at the single cell level. multiplex immunofluorescence staining was combined with confocal imaging of lung tissue samples to assess the spatial heterogeneity of pulmonary artery ec. results: our data revealed that ec in adult human pulmonary arteries are composed of three major populations. each population was characterized by enrichment in a specific set of biological processes determining their distinct functional roles. background: systemic sclerosis (ssc) is an autoimmune disorder leading to fibrosis of skin and other internal organs. one ssc hallmark is severe vasculopathy with impaired vascular permeability and tone as an early disease manifestation. pulmonary complications are the main cause of mortality in patients, but treatment options are still limited. pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis (ipf), however its effectiveness in ssc-pf is still unknown. here, we investigated the effectiveness of pirfenidone in a preclinical ssc-pf model, the fra-2 transgenic (tg) mouse. methods: fra-2 tg and wild-type (wt) control mice received either standard or pirfenidone supplemented diet. pulmonary function testing, fibrosis quantification, inflammatory cell profiling of bronchoalveolar lavage (bal) and lung tissue as well as transcriptome analysis of lung homogenates was performed. using in vitro electric cell impedance sensing measurements pirfenidone effects on endothelial cell permeability were analysed. results: compared to wt mice, tg mice had decreased lung function and elevated levels of inflammatory cells in bal and lung. pirfenidone exacerbated this phenotype further by increasing collagen deposition and worsening lung function. these functional and structural changes were associated with significantly higher lung tissue and bal inflammation, characterized by predominant eosinophilic infiltration in pirfenidone-treated tg mice. of note, pirfenidone did not alter lung function, collagen deposition or inflammation in wt mice. transcriptomic profiling indicated the activation of inflammatory cell recruitment and extravasation pathways with significant downregulation of the endothelial cell barrier protein ve-cadherin. further, pirfenidone led to decreased resistance of pulmonary microvascular endothelial cell monolayers in vitro. conclusions: pirfenidone was associated with significant deterioration of lung function and elevated inflammatory infiltrates in the lungs of ssc mice. our study shows that this effect might be due to disturbances of endothelial cell integrity upon pirfenidone treatment especially in diseases with a predisposed vasculature such as ssc. background: there is a growing interest in metabolic profiling of pulmonary arterial hypertension (pah) due to current findings suggesting significant metabolic changes causing pulmonary arterial remodeling and linking pah to insulin resistance. such findings may have major impact on future diagnostic and therapeutic strategies for pah. however, most of the studies have enrolled patients with severe disease whose reduced physical activity may have a profound effect on insulin sensitivity. we aimed to directly measure insulin sensitivity in ipah patients by applying the gold standard method botnia-clamp. methods: we assessed insulin sensitivity in five non-diabetic, normal weight patients with severe idiopathic pah and preserved physical activity in comparison to their age-, sex-, and body composition matched non-diabetic healthy controls. for assessing insulin sensitivity, the hyperinsulinemic-euglycemic (botnia) clamp was performed in a simultaneous pairwise matched-control manner. results: in this study we detected no indication of insulin resistance in patients characterized by manifest ipah but no major limitations in their daily physical activity. both ipah and control groups displayed normal efficacy of glycemic control. the botnia clamp measurements showed no differences in insulin response or insulin sensitivity in any of the ipah patients when compared to their healthy controls and also the comparison of the groups showed no significant differences. in ipah, the whole-body glucose disposal capacity in response to insulin infusion showed the same characteristics as in healthy controls. conclusions: this study does not support insulin resistance to be a primary cause of pulmonary vascular remodeling in ipah. multiplex imaging analysis confirmed in situ relative frequencies of ec populations and revealed their characteristic spatial distribution throughout the pulmonary artery tree. arteries in pulmonary hypertension patients displayed altered composition of ec population characterized by the diminished presence of one cell cluster. conclusions: in this study, we have revealed ec heterogeneity in the human pulmonary arteries at a single cell resolution and uncovered evidence for their distinct functional specification. cellular therapy aimed at restoration of the affected cluster or expression of their key genes could serve as a potential therapeutic option in treatment of pulmonary hypertension. clinical relevance of exercise hemodynamics and right ventricular function in copd patients asthma may have consulted the internet for self-medication advice. methods: on july 01 2020, we queried google trends for the terms "coronavirus asthma", "-copd", "-hypertension", "-diabetes" and "-cancer", all representing pre-existing conditions constituting a major risk for covid-19. when further exploring the health-seeking behavior of patients affected by asthma and/ or copd during the covid-19 outbreak, we focused on those therapeutic approaches with the highest rsv world-wide and thus comparable. results: we observed highest rsv for "coronavirus asthma" followed by "coronavirus diabetes" and "coronavirus cancer", "coronavirus hypertension" ranked fourth together with "coronavirus copd". paralleling the world-wide covid-19 outbreak, highest rsv was seen for the topics "salbutamol", "montelukast", "ipratropium bromide", "beclometasone", and "flucticasone propionate", encompassing mainly relievers, followed by inhaled corticosteroids (ics). conclusions: despite other risk factors like hypertension having been largely debated in the media, our analysis revealed highest search volumes for asthma. considering the gina guidelines in which the authors explicitly state that asthma treatment should no longer be based solely on short-acting bronchodilators, our data clearly indicates a fail in reaching asthma patients with respective fundamental changes in therapy. even more alarming is the high search volume for montelukast, since the fda released a boxed warning for montelukast in march 2020 because of serious neuropsychiatric side-effects. our findings emphasize the urgent need of spreading guidelines and respective updates in a timely manner more intensively in order to reach the general public-especially in a world with an ongoing, potentially life-threatening pandemic. is there a difference in local disease control between a vats and thoracotomy approach? most studies suggest a similar outcome, but nodal upstaging as a quality parameter is frequently reported to be higher in thoracotomy patients. if more positive lymph nodes are missed by vats, pn0 in these patients should result in a higher failure rate of local disease control. in this study we analyze the difference of vats to open thoracotomy regarding above mentioned parameters. methods: the institutional database was queried. exclusion criteria were pathologic nodal positive status, metastatic disease, tumour size >4 cm, adjuvant/neoadjuvant therapy. 422 patients were included. the vats cohort included 350 patients, the thoracotomy cohort 72 patients. results: a vats approach in patients with pathologic n0 disease did not show a significantly higher rate for local or lymph node recurrence compared to thoracotomy (12.9 % vs. 19.4 %; p = 0.142). there was no difference in disease-free and overall survival comparing the two groups. comparing the location of recurrence, thoracotomy patients showed a significantly higher rate of metastatic disease (3.04 % vs. 11.54 %; p = 0.014), beneficial effects of multidisciplinary rehabilitation in post-acute covid-19 tionnaire related to covid-19 specific symptoms was filled out by all participants. results: 12,419 subjects participated (5,984 lead participants, 6,440 household members). the projected number of cases according to age and sex for vienna is 21,504 cases (1.13 %). the cumulative number of positive tested cases in vienna until may 20th was 3,020. hence, the projected number is 5.5 to 9.1 times larger than the observed cases. the relative risk of seropositivity by age was highest for children aged 6-9 years [rr 1.21 (ci 0.37-4.01)] and lowest for subjects 65 years and older [rr 0.47 (ci 0.21-1.03)]. half of the infected subjects developed no or mild symptoms. in a multivariate analysis (fig. 1) taste and smell disturbances were most strongly related to sars-cov-2-specific antibody positivity. the infection probability within households with one confirmed sars-cov-2-specific antibody-positive person was 31 %, about 30 times higher than the general ambulatory infection risk. conclusions: prevalence rates in vienna are low (1.13 %) with the highest seroprevalence in young children and lowest in older (≥65 years) inhabitants. taste and smell disturbances are very prevalent in covid-19 infected persons and can guide clinicians in diagnosis-and decision making of covid-19. distribution and prognostic significance of gluconeogenic and glycolytic phenotypes in nonsmall cell lung cancer background: skin prick test (spt) is a minimal invasive diagnostic test, identifying type-i-sensitization, which is associated with symptoms as wheezing, atopic dermatitis and rhinitis. prevalence data vary between countries from 37.7 %-68.6 %. data about the prevalence in austria are scarce. moreover, associated factors for positive spt have only been investigated in specific age-(e. g. children or adults only) and subgroups (e.g asthmatics).therefore, our aim was to investigate the prevalence of positive spt in a general austrian population, to define associated factors and compare the prevalence and associated factors between childhood and adulthood. methods: data was obtained from the lead study, an observational, population-based cohort. we included 11.283 participants with a valid spt and analyzed two age groups separately: childhood (6-18 yrs; n = 1439) and adulthood (19-82 yrs, n = 9844). multivariate regression model was used to identify factors associated with positive spt including socioeconomic status, allergic and/or respiratory diseases, lung function, body composition, lifestyle habits, smoking exposure, pet exposure, and family history. results: in our study the overall prevalence of a positive spt is 37.6 % and is higher in male compared to female in all age pins (fig. 1) . house dust mite and grasses mix are the most prevalent allergens. factors positively associated with positive spt in childhood are doctor's diagnosed allergy or asthma and diagnosed parental allergy; in adulthood are doctor's diagnosed allergy or asthma, diagnosed parental allergy, and high socioeconomic status. smoking (current, former and secondhand) is ally, glycolytic and gluconeogenic gene expression was inferred from the cancer genome atlas (tcga) datasets. results: pck2 was preferentially expressed in the lung adenocarcinoma subtype, while glut1 expression was higher in squamous cell carcinoma. glut1 and pck2 were inversely correlated, glut1 showing preferential expression in larger tumors while pck2 was highest in smaller tumors. however, a mixed phenotype showing the presence of both, glycolytic and gluconeogenic cancer cells was frequent. in lung adenocarcinoma, pck2 expression was associated with significantly improved overall survival compared to glycolytic or mixed tumors, while the opposite was found for glut1. pck1/2 expression was enhanced in metastases compared to primary tumors. the metabolic tumor microenvironment and the 3-dimensional context play an important role in modulating both pathways, since pck2 expression preferentially occurred at the tumor margin and hypoxia differentially regulated glycolysis and gluconeogenesis in nsclc cells in vitro. conclusions: glycolysis and gluconeogenesis are activated in nsclc in a tumor size and oxygenation dependent manner and show a differential correlation with outcome. the frequent co-activation of gluconeogenesis and glycolysis in nsclc should be considered in potential future therapeutic strategies targeting cancer cell metabolism. abstracts assays showed significantly reduced migration of rgs5-/-neutrophils towards chemokines with preserved intra-cellular calcium signaling. importantly, the attenuated neutrophil migration was associated with activated rhoa, suggesting rhoa as a predominant negative regulator of neutrophil transmigration. conclusions: our findings demonstrate the efficacy of silenced rgs5 for suppressing neutrophilic hyperinflammation in two different animal models. the specific effects of rgs5 loss might provide an option for a novel therapeutic intervention in inflammatory lung diseases with recurrent exacerbations, without compromising infection defense mechanisms. endothelial dysfunction following enhanced tmem16a activity in human pulmonary arteries background: endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (pah). ion channelome changes have long been connected to vascular remodelling in pah, yet only recently the focus shifted towards ca2+-activated cl-channels (cacc). the most prominent member of the cacc tmem16a has been shown to contribute to the pathogenesis of idiopathic pah (ipah) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (paecs) and in the development of endothelial dysfunction remains underrepresented. methods: using healthy donor (n = 3) and ipah (n = 3) lungs, we analysed the expression of tmem16a in primary human paecs. ipah was mimicked with selective adenoviral overexpression encoding tmem16a tagged with mcherry. tmem16a activity was investigated by patch clamp. live cell ca2+ imaging was applied to detect changes in ca2+ homeostasis. paec proliferation, apoptosis, tube-formation, wound healing assay, no production and measurements of paecs metabolic state addressed functional consequences of increased tmem16a activity. the role of endothelial tmem16a in the tone of pulmonary arteries ex vivo was investigated by wire myography. results: here we report enhanced tmem16a activity in ipah paecs. upon tmem16a overexpression in healthy primary human paecs in vitro and in human pulmonary arteries negatively associated with positive spt in adulthood, but not in childhood. conclusions: our study in an austrian general population identified that 1. positive spt is highly prevalent, 2. is more prevalent in male, 3. the main allergens observed are grasses mix and house dust mite, and 4. there is a difference in factors associated with spt in childhood vs adulthood. neutrophil recruitment in the acute inflammatory phase of interstitial lung disease is determined by rgs5 methods: data was obtained from the austrian lead study, an observational, population-based cohort study. adults aged 25-82 years with valid lf and metabolic data, including waist circumference (wc) for central obesity, and dxa scan for vat (n = 9.157) were included in this analysis. lf was assessed by spirometry pre-and post-bronchodilation (bd). abnormal lf was defined as fev1 pre bd<lln (gli) and further divided into obstructive (fev1 pre<lln + fev1/fvc post<lln) and restrictive (fev1 pre<lln + fev1/fvc post≥lln + fvc post<lln) pattern. regression models were performed analysing the association between mets components (idf 2006) and abnormal, obstructive, and restrictive lf pattern (corrected for age, sex, smoking status). results: fig. 1 shows odds ratios of mets components and vat associated with impaired lf pattern. increased vat was associated with both, obstructive (or 1.5 [1.3-1.8]) and restrictive (or 2.4 [1.8-3.0]) lf pattern, while increased wc was not. conclusions: vat showed a consistent association with lf impairment independent of the type of impairment, in contrast to the different components of the mets. in december 2019 an unknown viral infection was firstly described in a local fish/animal market in wuhan/china. it was identified as a novel coronavirus infection by the chinese center for disease control and prevention (ccdc) on jan. 7th and announced as 2019-new coronavirus disease (2019-ncov, now covid-19) by the world health organization (who) on feb. 11th 2020. the disease rapidly spreads across the globe (begin of august 2020 at least 18 million of infections and 650000 deaths reported worldwide) and until now no specific treatment has been described. therefore, measures for disease prevention (e. g. vaccines, rapid testing, control of viral transmission by masks/face shields, gloves, digital monitoring/social ex vivo, we demonstrate the functional consequences of the augmented tmem16a activity: alterations of ca2+ dynamics and enos activity as well as decreased no production, paecs proliferation, wound healing, tube-formation and acetylcholine-mediated relaxation of human pulmonary arteries. we propose, that erk1/2 pathway is specifically affected by elevated tmem16a activity, leading to these pathological changes. conclusions: with this work we introduce increased tmem16a activity in the outer cell membrane of human paecs important for the development of endothelial dysfunction in ipah. metabolic syndrome and visceral adipose tissue (vat) are associated with obstructive and restrictive lung function impairment background: malignant pleural mesothelioma (mpm) is a highly aggressive tumor with dismal outcome and thus, investigating reproducible pretreatment parameters to assess benefit from treatment modalities and to estimate survival is of outmost importance. this study aims to identify potential serum prognostic biomarkers in a large retrospective cohort of a high-volume institution. methods: we performed a retrospective analysis of the clinical records, serum parameters at diagnosis, and longterm outcome of patients with pathologically diagnosed mpm between 1994 and 2019. previously published prognostic serum biomarkers were tested and validated and, in consequence, the prognostic value of an inflammatory serum marker score (vmis-vienna mesothelioma inflammation score) was investigated. results: in total, 195 patients (147 male, 75.4 %) were included. median age was 67 years (iqr 58-74). median overall survival (os) for all cases was 14 months (95 % ci; 11.43-17.17). survival was 56 % at 1 year, 17 % at 3 years, and 10 % at 5 years. median os and disease-free survival (dfs) of patients undergoing multimodality treatment including surgery was 23 months (95 % ci; 19.64-26.70) and 16 months (95 % ci; 12.72-18.68), respectively. the optimal cut-off values for pretreatment neutrophil-to-lymphocyte ratio (nlr), serum fibrinogen and crp were 4.05, 537.5 mg/dl and 0.865 mg/dl, respectively. univariate analysis revealed that age (p = 0.007), ecog status (p = 0.032), tumor stage (p = 0.003), histologic subtype (p = 0.003), type of treatment (p < 0.001), nlr (p < 0.001), fibrinogen (p < 0.001) and crp (p < 0.001) were significantly associated with os. accordingly, the vmis (consisting of pretreatment nlr, fibrinogen and crp) was highly associated with os (score 0 vs. 1, hr 1.55, p = 0.040; score 0 vs 2, hr 2.97, p < 0.001). in multivariate analysis, vmis (p = 0.003), histologic subtype (p = 0.002), and the type of treatment (p = 0.001) were independently associated with os. conclusions: vmis was found to be an independent prognostic score in mpm helping to stratify patients into different treatment groups. methods: we performed a retrospective analysis of all inpatients aged <18 years, who were admitted to our department between january 2010 and december 2019 for suspected or confirmed tuberculosis (tb) infection, for the presence of otb. results: we were able to identify four paediatric cases with otb (cohort: 527 inpatients, 0.76 %) (fig. 2) . three patients suffered from posterior ( fig. 1) and one from anterior uveitis associated with tb. two patients were diagnosed with pulmonary ultra wide-field fundus photography of the right eye shows two (one at the temporal inferior and one at the nasal superior vessel arcades) yellowish choroidal granulomas with ill-defined borders and substrantial elevation. there seems to be a serous retinal detachment around the granulomas (case #4) abstracts tb after being referred by the ophthalmologist, the remaining showed ocular involvement in the setting of disseminated tb. all patients were treated with systemic corticosteroids and anti-tuberculosis therapy (att). patients received topical steroids additionally, one of whom (case #2) also received steroids intravitreally once for persisting macular oedema. visual acuity remained normal in two patients. conclusions: despite a higher incidence of extrapulmonary tb in the paediatric age group in general (2), otb was uncommon in our cohort. when disseminated disease is suspected or ocular symptoms are noted in paediatric tb patients, a detailed ophthalmological examination is imperative. lung transplantation for acute respiratory distress syndrome patients: a single center experience background: acute respiratory distress syndrome (ards) is a rapidly progressive lung disease with a high mortality rate. although lung transplantation (ltx) remains the only lifesaving option for certain end-stage pulmonary diseases, ards as indication for ltx is discussed controversially and only limited data are available. for example, in the eurotransplant region, only 17 patients transplanted for ards are documented. since we have followed a liberal approach of accepting ards patients for ltx, we reviewed retrospectively the outcome of this particular patient cohort in our center. methods: from 08/98 to 03/19 at total of 10 patients transplanted for ards were identified. demographic and clinical data of these patients were collected and analysed. results: eight/10 patients were listed and transplanted while on extracorporeal membrane oxygenation (ecmo). the remaining 2 patients could be initially successfully weaned from ecmo but remained respirator dependent. the median mechanical ventilation time and median length of ecmo was 33.5 (3.5-62.3) and 29.5 (28-38) days before ltx. primary graft dysfunction (pgd) grades at 72 hrs were 40 % (n = 4), two patients were classified as pgd ungradable (due to prophylactically prolonged ecmo). length of icu and hospital stay was 38 (24.5-52) and 57 (43.5-94) days. the 30-day mortality was 10 % and 1-year survival rate was calculated as 55.5 % (fig. 1) . one patient developed chronic lung allograft dysfunction (clad) and received re-transplantation 12 years after the primary procedure. the median follow up time was 82 (49.9-1065) days. conclusions: lung transplantation is feasible in carefully selected ards patients. given the lack of alternative treatment options it provides acceptable long-term results. investigating prognosis in malignant pleural mesothelioma: the vienna mesothelioma inflammation score (vmis) including pretreatment neutrophil-to-lymphocyte ratio, fibrinogen, and crp is independently associated with overall survival background: malignant pleural mesothelioma (mpm) is a highly aggressive tumor with dismal outcome and thus, investigating reproducible pretreatment parameters to assess benefit from treatment modalities and to estimate survival is of outmost importance. this study aims to identify potential serum prognostic biomarkers in a large retrospective cohort of a high-volume institution. methods: we performed a retrospective analysis of the clinical records, serum parameters at diagnosis, and longterm outcome of patients with pathologically diagnosed mpm between 1994 and 2019. previously published prognostic serum biomarkers were tested and validated and, in consequence, the prognostic value of an inflammatory serum marker score (vmis-vienna mesothelioma inflammation score) was investigated. results: in total, 195 patients (147 male, 75.4 %) were included. median age was 67 years (iqr 58-74). median overall survival (os) for all cases was 14 months (95 % ci; 11.43-17.17). survival was 56 % at 1 year, 17 % at 3 years, and 10 % at 5 years. median os and disease free survival (dfs) of patients undergoing multimodality treatment including surgery was 23 months (95 % ci; 19.64-26.70) and 16 months (95 % ci; 12.72-18.68), respectively. the optimal cut-off values for pretreatment neutrophil-to-lymphocyte ratio (nlr), serum fibrinogen and crp were 4.05, 537.5 mg/dl and 0.865 mg/dl, respectively. univariate analysis revealed that age (p = 0.007), ecog status (p = 0.032), tumor stage (p = 0.003), histologic subtype (p = 0.003), type of treatment (p < 0.001), nlr (p < 0.001), fibrinogen (p < 0.001) and crp (p < 0.001) were significantly associated with os. accordingly, the vmis (consisting of pretreatment nlr, fibrinogen and crp) was highly associated with os (score 0 vs. 1, hr 1.55, fig. 1 | v01 survival curve from 1998-2019 (n = 10) abstracts 1 3 628 ögp p = 0.040; score 0 vs 2, hr 2.97, p < 0.001). in multivariate analysis, vmis (p = 0.003), histologic subtype (p = 0.002), and the type of treatment (p = 0.001) were independently associated with os. conclusions: vmis was found to be an independent prognostic score in mpm helping to stratify patients into different treatment groups. surgical management and risk analysis of primary malignant pulmonary and chest wall sarcomasretrospective results from a high-volume center katharina sinn 1 , berta mosleh 1 , richard hritcu 1 , györgy lang 1 , jose matilla 1 , konrad hoetzenecker 1 , shahrokh taghavi 1 , walter klepetko 1 , thomas klikovits 1 , mir alireza hoda 1 ögp background: postoperative pain management after videoassisted thoracoscopic surgery (vats) is still a heavily debated topic, which may affect patients' pain, need of opioids, and postoperative length of stay (los). this study aims to analyse the effects of an additional intercostal catheter (icc) in comparison to a single shot intraoperative intercostal nerve block (ssinb) alone. methods: all patients receiving an anatomic vats resection from 06/2019-03/2020 were analysed. icc placement at our department was initiated in september 2019. patients with misplacement of the icc were analysed in the ssinb group. exclusion criteria for analysis was a prolonged los because of postoperative complications not related to pain-management protocol (one hiatal hernia with oesophageal perforation, one hematologic comorbidity requiring further workup). the icc cohort included 28 patients, the ssinb cohort 39 patients. results: pain scores on the first postoperative day, after chest drain removal and highest pain score measured did not differ between the two groups (p = 0.472/0.487/0.242). in four patients (12.1 %), icc showed a primary non-function. the overall amount of opioids (piritramide, 3.750 mg vs. 12.500 mg; p = 0.001) as well as the duration of opioid usage (0.50 days vs. 1.36 days; p = 0.001) was significantly less in the icc cohort. the icc cohort showed a significantly shorter postoperative los of 5.18 (mean, range 4-7) days in comparison to 6.95 (mean, range 4-16) days (p = 0.001). chest drain duration was significantly shorter in the icc cohort (2.54 days vs. 3.79 days; p = 0.013). there was no difference in comorbidities (coronary artery disease, diabetes mellitus, copd; p = 0.841/0.724/0.152), age, gender or postoperative complications (p = 0.672/0.806/0.740). conclusions: pain management with icc reduces the amount of opioids and number of days with opioids patients' require to achieve sufficient analgesia while at the same time reduces the postoperative los and chest drain duration. in conclusion, icc is an effective tool in postoperative pain management. analysis of pain management after anatomic vats resection in austrian thoracic surgery departments persistent symptoms in patients after acute covid-19 covid-19 and obstructive sleep apnea sars-cov-2-specific antibody seroprevalence in a general population -the viennese lead covid-19 study background: pulmonary hypertension (ph) is a frequent complication of copd and is associated with poor survival. however, pulmonary and right ventricular hemodynamics and their association with physical capacity in copd patients with out-of-proportion dyspnea is unknown. in this study we hypothesized that copd patients show abnormal hemodynamics during exercise, associated with impaired exercise capacity.methods: out of outpatients with unexplained dyspnea, copd patients with mpap < 25 mm hg and age and sex matched controls without copd and mpap < 25 mm hg, who underwent right heart catheterization at rest and during symptom-limited ergometer exercise, were compared.results: we analyzed 26 copd patients (66 ± 11 yr, fev1: 56 ± 25 %) and 26 controls (66 ± 10 yr, fev1: 96 ± 22 %). in copd, resting mpap and pvr were slightly elevated (median: 21 (iqr:18-23) vs. 17 (14-20) mm hg, p = 0.022 and 2.5 (1.9-3.0) vs. 1.9 (1.5-2.4) wu, p = 0.020). at peak exercise, mpap and pvr were markedly elevated (47 (40-52) vs. 38 (32-44) mm hg, p = 0.015 and 3.1 (2.2-3.7) vs. 1.7 (1.1-2.9) wu, p = 0.028), whereas pulmonary vascular compliance (pvc) and right ventricular output reserve (0.9 (0.5-1.2) vs. 1.3 (0.7-1.8) mm hg, p = 0.020 and (1.9 (1.6-2.6) vs. 3.0 (2.0-4.4) ml/mm hg, p = 0.045) were significantly reduced. mpap/co-slope (6.9 (5.5-10.9) vs. 3.7 (2.4-7.4) mm hg/l/min, p = 0.007) and transpulmonary gradient (tpg)/co-slope (3.5 (1.6-4.5) vs. 1.6 (0.9-3.5) mm hg/l/ min, p = 0.048) were significantly steeper in copd. compared to controls, copd patients had worse exercise performance (peak oxygen uptake (vo2) (65 (55-82) vs. 95 (87-102) %predicted, p < 0.001). moreover, peak vo2 was significantly correlated with peak exercise mpap (p = 0.001, r = -0.540), pvr (p = 0.001, r = -0.568), pvc (p = 0.008, r = 0.495), pulmonary arterial stiffness index (p < 0.001, r = -0.591), rv output reserve (p = 0.008, r = 0.456), mpap/co-slope (p = 0.006, r = -0.491) and tpg/coslope (p = 0.013, r = -0.429).conclusions: in copd, pulmonary and right ventricular hemodynamics are markedly changed as compared to patients without copd. these findings are strongly associated with exer-abstracts backgroud: the corona virus disease 2019 (covid-19) pandemic increases the demand for post-acute care in patients after a severe disease course. various longterm sequelae are expected and physical and rehabilitation medicine (prm) is challenged to support the recovery both physically and mentally. the aim of this study was to explore dysfunctions and outcome of covid-19 survivors after early post-acute rehabilitation.methods: this study analysed the rehabilitative outcomes of a subgroup of patients included in a prospective observational multicenter study to investigate long-term sequelae in patients hospitalized for sars-cov-2 infection.results: a total of 23 subjects discharged after severe to critical covid-19 infection underwent an individualized, multiprofessional treatment plan. despite a high proportion of patients requiring mechanical ventilation with consequent symptoms of post intensive care syndrome, indices of performance status and pulmonary parameters improved significantly.conclusions: covid-19 survivors show varying degrees of physical and psychological dysfunctions in the early period of recovery after hospital discharge. in this phase, patients strikingly benefit from multidiscinplinary inpatient rehabilitation, but the course of residual impairments remains unclear. assessing self-medication for obstructive airway disease during covid-19 using google trends background: inhibition of glycolysis has been considered as a therapeutic approach in aggressive cancers including lung cancer. abbreviated gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (pepck), was recently discovered to partially circumvent the need for glycolysis in lung cancer cells. however, the interplay of glycolysis and gluconeogenesis in lung cancer is still poorly understood.methods: we analyzed the expression of glut1, the prime glucose transporter, and of pck1 and pck2, the cytoplasmic and mitochondrial isoforms of pepck, in 450 samples of nonsmall cell lung cancer (nsclc) and in 54 nsclc metastases using tissue microarrays and whole tumor sections. spatial distribution was assessed by automated image analysis. addition-most likely due to a longer follow-up time. other clinical factors did not differ between groups.conclusions: vats lobectomy does not result in a higher rate of local disease recurrence, suggesting adequate lymph node dissection with this approach. other factors than the surgical technique might be responsible for nodal upstaging, therefore nodal upstaging should no longer be used as a quality marker for lymph node dissection.background: acute exacerbation of interstitial lung disease (ild) is associated with a poor prognosis and high mortality. the regulator of g protein signaling5 (rgs5) is present in the lung as well as in neutrophils. however, the potential of rgs5 to limit neutrophilic hyperinflammation and thus disease progression in acute exacerbation has not been explored so far.methods: to investigate the functional relevance of rgs5 invivo, we examined the acute inflammatory bleomycin response phase of pulmonary fibrosis utilizing rgs5 knock out (rgs5-/-) and wild type (wt) mice at age 12-16 weeks (equivalent to adult age in humans). the lipopolysaccharide (lps)-induced acute lung injury model was used as a second model. the in-vivo studies were extended with analysis of the chemokine levels in the lung, the migratory behavior of the neutrophils obtained from wt and rgs5-/-animals and the downstream pathways in-vitro.results: in the acute phase of pulmonary fibrosis, rgs5-/mice had preserved lung function as compared with wt mice which showed significant combined ventilatory disorders. analysis of specific immune cell subpopulations in bronchoalveolar lavage fluid (balf) and lung tissue showed a significant attenuation in the total cell number in the balf of rgs5-/-animals, predominantly made up of neutrophils and lowered myeloperoxidase (mpo) enzymatic activity in lung tissue. a similar picture was seen in the lps lung injury model. our in-vitro abstracts background: cystic fibrosis (cf) leads to airway colonisation with pathogens. the significance of colonisation with fungi is not entirely clear, and epidemiological data regarding colonisation with fungi are somewhat contradictory. candida albicans and aspergillus fumigatus have been most frequently reported, with colonisation rates between 38-78 % and 4-42 %, respectively.methods: we analysed results of sputum cultures from paediatric and adult patients of our centre taken between 2009 and 2019. modified culture methods had been used for fungal cultures. to determine chronic colonisation we applied modified leeds criteria.results: 8,941 sputum specimens from 148 patients were analysed. of 39,868 isolated pathogens, 8,919 (22.4 %) were fungi cultivated from 5,070/8,941 specimens (56.7 %). 144/148 (97.3 %) patients were at least colonised once. colonisation rates with fungi did not change significantly between 2009 (76/91; 83.5 %) and 2019 (94/108; 87.0 %). some species seem to be associated with chronic colonisation while others do not (fig. 1) .conclusions: almost all patients showed colonisation with one or more fungi. the proportions of patients already colonised in childhood and over many years were very high. some species were detected at much higher rates than previously reported. these differences are more likely due to culture methods and the long observation period than to differences in cohorts. adequate culturing methods are crucial to study the impact of fungal-colonisation in cf patients.contact tracing (apps)) are of great importance. we analysed a rapidly increasing number of publications investigating virus transmission by aerosols and the role of masks/shields for prevention. in brief, infectious aerosols are produced by coughing, sneezing, speaking and even normal breathing of infected individuals demonstrating the role of the lung as an "aerosol generator". in aerosol the virus is stable for 3 hours and coughing/sneezing in contrast to normal breathing can transport aerosol for a distance of about 6 meters. half lifetimes of viable virus are 1 h (aerosol, copper-surface), 3 h (cardboard) and 7 h (plastic-surface) underlining the need of masks/shields and adequate disinfection. a major number of professional masks/ face-shields were developed, e. g. n95 mask, surgical mask, face-fitting mask, mit face shield differing strongly in respect to their properties for filtering and infection prevention; however, comparative head-to-head studies are missing. in summary, masks/face shields play an important role for prevention of covid-19 infection. however, there are differences in respect to type of the masks/face shields and their indication for use, e. g. general population, health care workers and the use should be combined with other methods of disease prevention. airway colonisation with fungi in cystic fibrosis patients results: 35 patients with a median age of 46 years were identified. the most common histology was ewing-sarcoma (n = 8, 23 %). 19 (54 %) patients underwent neoadjuvant therapy (49 % cht, 2.9 % rt, 2.9 % immunotherapy). median tumor size at diagnosis was 10.6 cm (range, 1 to 35 cm). extended resection was necessary in 54 % (n = 19), intraoperative use of ecmo in 1 patient. postoperative complications were observed in 7 patients (20 %). adjuvant therapy was administered in 17 (49 %) patients (5.7 % cht, 8.6 % rt, 26 % chrt). 90-day mortality was 2.9 %. there was no significant difference in overall survival (os) or disease free survival (dfs) between ps and cws (p = 0.240 and p = 0.348) or in type of histology (ewing sarcoma vs others, p = 0.708 and p = 0.122). 5-year os and dfs were 68 % and 51 %, median os was not reached. median dfs was 79.33 months. patients requiring extended resection (> 1 involved anatomical structures) had a trend for impaired os (hr 0.171, p = 0.060) and significantly worse dfs (hr 0.19, p = 0.003).conclusions: radical resection of primary malignant ps and cws offers good long-term outcome with low complication rate despite extended resections. however, extent of disease and subsequent necessity for extended resection is an unfavorable factor for long-term survival. background: the ests eurolung scores were established to predict postoperative morbidity and mortality in patients undergoing anatomic lung resections. since its introduction, the eurolung scores have been updated once and an easy-touse and free-of-charge smart phone app has been created. so far, the scores have not been validated in other patient cohorts. herein we aimed to elaborate the accuracy of the various eurolung scores in our vats cohort. methods: the eurolung scores were calculated for a consecutive cohort of 729 patients scheduled for vats lobectomy. postoperative complications, as defined and used by the eurolung scores, were then analyzed in this prospectively maintained database.results: overall, the observed complication rate was 10.7 % in the vats lobectomy database. the eurolung1 predicted a mean risk of morbidity of 21.7 % with a weak eta correlation (η) (eurolung1: η = 0.192; 2016 parsimonious eurolung1: η = 0.167; 2019 parsimonious eurolung1: η = 0.174). a better coherence was observed with the parsimonious eurolung1 (2016; 11.8 %) and the current parsimonious eurolung1 (2019; 11.5 %). binary logistic regression analysis of the included parameters showed that extended resections and ppofev1% were associated with increased complications in the eurolung1 scores. 30-day mortality was 0.8 % (predicted mortality according to eurolung2: 1.4 %, parsimonious eurolung2: 1.11 %) and was associated with ppofev1% for both scores and coronary artery disease for the eurolung2 score only. the eurolung2 showed a larger area under the roc curve than the parsimonious eurolung2 (0.59 vs. 0.57). again, only a very weak eta correlation between predicted and observed mortality was found for the eurolung2 (η = 0.002) and the parsimonious eurolung2 (2016) (η < 0.001).conclusions: even though predicted and observed morbidity/mortality rates were comparable in our cohort the scores were not useful to predict the individual risk in this vats cohort. therefore, the scores should not be used to permit or refuse surgical therapy. initial experience with intercostal catheter for postoperative pain management in vats background: postoperative pain and its management influences patients' rehabilitation, postoperative complications and quality of life. despite its impact there are no uniform guidelines. different centers seem to use various strategies for pain management. in this study we aim to analyze pain management regimens after vats lobectomy used in austrian thoracic surgery units with a special interest in opioid usage and strategies to avoid opioids.methods: a questionnaire was designed to assess the current use of regional anesthesia, post-operative pain medication and characteristics of individual pain management regimens. the questionnaire was sent to all thoracic surgery units in austria, with nine out of twelve departments returning them.results: pain management varied between all centers. all departments use regional anesthesia perioperatively. four out of 9 centers use epidural analgesia or an intercostal catheter for postoperative regional anesthesia in at least 50 % of patients. two departments follow an opioid restrictive regimen, 5 depending on visual analogue scale (vas) and two administer opioids on a fixed schedule. three out of 9 departments use nsaids on a fixed schedule. the most used medication is metamizole (8 out of 9 centers; 6 on a fixed schedule, two depending on vas) followed by piritramid (6 out of 9 centers; none as a fixed prescription). all centers reported that their regimen is standardized (with 8 centers basing it on an in-house standard) and all assessed their patients pain scores on a regular basis.conclusions: there is no standardized postoperative pain management regimen after anatomic vats resections. there seems to be a trend towards prolonged postoperative regional anesthesia to reduce opioid consumption in some centers. a further prospective study is in preparation to evaluate the feasibility of opioid-free postoperative pain management and its impact on quality of life. perioperative mortality and morbidity following pneumonectomy for severe inflammatory disease of the lung background: some cases of severe pulmonary inflammation are not amenable to conservative treatment. if pneumonectomy is required in highly septic and instable patients the inherent risk of the procedure increases further. in a retrospective study we analysed these patients comparing them to elective pneumonectomy in patients with malignant disease.methods: during the last 15 years 163 patients (age: 60.2 +/-11.3 years; males: 124, females 39) underwent pneumonectomy. 41 of these cases had underlying severe inflammatory disease (central necrotizing abscess or pulmonary gangrene with accompanying empyema) whereas 122 had resection for malignant tumours. abstracts results: the inflammatory group was significantly younger (56.4 +/-13.4 vs. 61.5 +/-9.8 years; p = 0.01) and had a significantly lower bmi (22.8 +/-6 vs. 25.6 +/-4.6; p = 0.003) than the malignant group. there were no differences concerning cigarette or alcohol consumption or copd, coronary artery disease or peripheral arterial occlusive disease. both the rate of severe perioperative complications (30.3 % vs. 41.4 %) and of perioperative death (3.28 % vs. 34.1 %) were significantly higher in patients with inflammatory disease (p = 0.000). in spite of the high perioperative mortality rate of pneumonectomy in inflammatory disease, 5-years survival rate (38.5 % vs. 26.8 %) showed no statistically significant difference between the two groups.conclusions: though sometimes required as a life-saving procedure in severe inflammation of the lung, pneumonectomy in such conditions carries a high perioperative morbidity and mortality. if the first 6 months after pneumonectomy are survived however, the prognosis of this subgroup is fair. problem at the pneumonectomy stump. salvage by myoplastic closure background: for primary closure when material is lacking and for the treatment of bronchial stump dehiscence following pneumonectomy a variety of methods such as pericardial or omental flap as well as myoplastic techniques have been advocated. we present our experience with myoplasty for closure of the main bronchus stump.methods: retrospective analysis of 163 pneumonectomies within the last 15 years (age: 60.2 +/-11.3 years; males: 124, females 39). in 9 patients (5.5 %) problems at the bronchial stump were present (6 on the right and 3 on the left side), thereof 3 primary impossibilities of direct closure and 6 secondary dehiscences.results: in one case dehiscence occurred one day after pneumonectomy, in the remaining 4 patients dehiscence became evident after a mean of 17.2 days (6-30 days). the 3 impossibilities of direct closure of the stump derived from necrosis and fistula following bifurcational stenting in lung cancer, from lack of viable bronchial tissue during completion pneumonectomy following left-sided sleeve resection and from bronchial necrosis in aspergillosis. closure of the stump was done by pedicled pectoralis major flap in 6, by pedicled diaphragmatic flap in 2 and by pedicled sternoleidomastoideus flap in one patient. if deemed necessary, second-look procedures and/or thoracostomy and vac were additionally used. one patient did not survive his septic condition and died within 30 days (11.1 %). the overall 5 years survival rate was 50 %.conclusions: pedicled myoplastic flaps provide reliable closure even in detrimental cases of dehiscence of the main bronchus stump.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-315085-rucfowvv authors: sekulic, miroslav; harper, holly; nezami, behtash g; shen, daniel l; sekulic, simona pichler; koeth, aaron t; harding, clifford v; gilmore, hannah; sadri, navid title: molecular detection of sars-cov-2 infection in ffpe samples and histopathologic findings in fatal sars-cov-2 cases date: 2020-05-26 journal: am j clin pathol doi: 10.1093/ajcp/aqaa091 sha: doc_id: 315085 cord_uid: rucfowvv objectives: to report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) positive individuals. methods: postmortem examination was completed following centers for disease control and prevention public guidelines. numerous formalin-fixed paraffin-embedded (ffpe) tissue types from each case were surveyed for sars-cov-2 rna by quantitative reverse transcription polymerase chain reaction (qrt-pcr). sars-cov-2 viral genome was sequenced by next-generation sequencing (ngs) from ffpe lung tissue blocks. results: postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. viral rna was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qrt-pcr method. rna sequencing using ngs in case 1 revealed mutations most consistent with western european clade a2a with orf1a l3606f mutation. conclusions: sars-cov-2 testing and viral sequencing can be performed from ffpe tissue. detection and sequencing of sars-cov-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus’s pathophysiologic effects on human health. used viral culture, electron microscopy, in situ hybridization, or immunohistochemical staining to detect viral involvement in different organs. in this study we report postmortem findings and detection and sequencing of sars-cov-2 viral rna from formalin-fixed paraffinembedded (ffpe) samples of multiple organs collected in 2 patients with antemortem detection of sars-cov-2. the decedents underwent postmortem examination following centers for disease control and prevention (cdc) public guidelines for the collection of specimens and were performed without the examination of brain or spinal cord. the lungs from case 1 were infused with formalin under gravity via the primary/main bronchi and allowed to fix for 24 hours in a container of formalin before dissection and sampling of sections. the lungs of case 2 were dissected fresh, and sampling of sections was done at the same time. for both autopsies the following organ tissues were sampled: lungs (all lung lobes), main bronchi, trachea, heart (left ventricle anterior/lateral/posterior, right ventricle, and interventricular septum), hilar and peribronchial lymph nodes, spleen, esophagus, stomach, small intestine, large intestine, liver (left and right aspects), pancreas, bilateral adrenal glands, bilateral kidneys, urinary bladder, skin, and skeletal muscle (psoas). representative tissue samples were submitted and in standard manner formalin-fixed and paraffin-embedded. paraffin tissue sections were stained with h&e and examined under light microscopy. rna was extracted from 3 unstained slides (0.5 μm thick) using maxwell rsc rna ffpe kit (promega) in elution volume of 30 μl. the cdc 2019-novel coronavirus real-time rt-pcr diagnostic panel assay was adapted as previously described 5 using taqpath 1-step rt-qpcr mastermix (thermo fisher), 2019-ncov n1 and n2 primer/probe sets, and control rnase p primer /probe set (idt). cycle threshold values less than 40 were considered positive. lung tissue samples from 2 non-sars-cov-2 patients with dad were run as controls and were negative. leftover extracted rna used for pcr was used for sequencing with an ampliseq sars-cov-2 panel. seven μl of rna was converted to cdna using superscript vilo iii (thermo fisher). library preparation was performed using ampliseq sars-cov-2 panel set and ampliseq plus library kit (thermo fisher). templating was performed with ion chef kit and sequencing was performed on ion s5 prime with io 530 chip kit (thermo fisher). alignment to sars-cov-2 reference (genbank id: mn908947) and variant calling was performed with ion torrent suite 5.10. variants calls were further confirmed by manual inspection using integrative genomics browser (broad institute). only variants with allele frequency greater than 90% were called. genomic epidemiology classification was performed using nextstrain.org on april 27, 2020. 6 the decedent was an 81-year-old man admitted from an assisted living facility with acute respiratory failure, fever, and positive nasopharyngeal swab sars-cov-2 test. eight days prior to admission the patient was noted to have developed an afebrile cough, initially thought to be associated with congestive heart failure. six days later, the patient was found to be febrile (38.7°c), and the following day he required 2 l/ min of oxygen. empirical management for concern of respiratory tract infection was initiated, and an outpatient nasopharyngeal swab collection for sars-cov-2 testing was performed. once the patient's sars-cov-2 testing returned positive he was immediately admitted the same day. the patient's medical history was otherwise notable for dementia, radiologic evidence of a left lung mass (managed with hospice care), coronary artery disease (status post coronary artery bypass grafting), atrial fibrillation (biventricular pacemaker implanted), congestive heart failure, peripheral artery disease (status post iliac stenting), diabetes mellitus, hypertension, dyslipidemia, chronic kidney disease, gout, smoking, cerebrovascular accidents, and urinary tract infections. surgical history was further notable for carotid endarterectomy, left inguinal hernia repair, and cataract surgery. laboratory testing around the time of admission was notable for cbc with pancytopenia, serum creatinine of 1.55 mg/dl, blood urea nitrogen of 37 mg/dl, serum glucose of 104 mg/dl, and brain natriuretic peptide of 1,156 pg/ml. blood and urine bacterial cultures showed no growth. infectious disease testing was negative for legionella, streptococcus pneumoniae, and hiv. body temperature was 37.8°c. pitting edema around the ankles was noted on examination. chest roentgenogram on the day of admission demonstrated diffuse patchy opacities in the right lung and subtle patchy opacities in the left lower lung ❚image 1❚. chest computed tomography (ct) on the same date revealed multifocal peripheral and central ground-glass opacities throughout the bilateral lungs, a pulmonary mass in the medial aspect of the left lower lobe, small left-sided pleural effusion, moderate cardiomegaly, and calcifications of the coronary arteries and thoracic aorta. after admission, the patient was initially given 2 l/ min of oxygen via nasal cannula but began to require increasing oxygen support up to 6 l/min. the patient was also noted to have evidence of encephalopathy compounding baseline dementia. based upon the patient's overall poor functional/physiologic status, underlying malignancy, comorbidities, and superimposed infection, it was determined that the patient's prognosis would be poor even with further management and as such, the patient was provided comfort measures. the patient was declared dead 5 days after admission, and an autopsy sine brain and spinal cord was permitted by the family. the autopsy was performed 29 hours after death. the postmortem gross examination of the lungs after 24 hours of inflation and submersion fixation with formalin revealed congested parenchyma superimposed on emphysematous changes, and the left lower lobe contained a mass lesion (4.5 cm in greatest dimension) ❚image 2❚. before fixation, the right lung weighed 1,200 g, and the left lung weighed 1,040 g. microscopically, the lungs revealed involvement of all lung lobes by acute/exudative phase of dad characterized by hyaline membrane formation, scattered squamous metaplasia of distal airways (alveoli, alveolar ducts, and respiratory bronchioles), and background emphysematous changes (image 2). bronchial and tracheal sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium. sars-cov-2 rna was detected in section of lung parenchyma, bronchi, and lymph node ❚table 1❚. the left lower lobe mass lesion was morphologically and immunophenotypically most consistent with large cell carcinoma, and there was evidence of metastatic involvement of ipsilateral hilar and peribronchial lymph nodes. other organs that revealed significant findings that contributed to the patient's overall physiologic status and terminal decline included the kidneys and heart. the kidneys showed evidence of acute tubular injury on a background of moderately advanced chronic changes of the renal parenchyma. the heart was enlarged (620 g) and showed changes associated with chronic ischemic heart disease: severe stenosis of native coronary arteries (left anterior descending, left circumflex, and right main coronary arteries), patent graft vessels, and moderately extensive replacement-type interstitial fibrosis. aside from the aforementioned findings, the gross and microscopic examinations of remaining organs/tissue did not reveal significant findings. lower levels of sars-cov-2 rna were detected in sections of spleen, heart, intestine, liver, and skeletal muscle. sars-cov-2 rna was not detected in samples of esophagus, stomach, kidney, adrenal, and skin samples ( table 1) . sequencing of the extracted viral rna from the lung showed that the virus derived from claude a2a with g11083t (orf1a l3606f) mutation ❚table 2❚. in summary, the cause of death was sars-cov-2 infection occurring in the setting of metastatic carcinoma, diabetes, and chronic ischemic cardiomyopathy, leading to respiratory failure. ❚image 1❚ (case 1) chest roentgenogram performed on the day of admission revealed diffuse patchy opacities in the right lung and subtle patchy opacities in the left lower lung (a), and computed tomography showed multifocal peripheral and central ground-glass opacities throughout the bilateral lungs, a pulmonary mass in the medial aspect of the left lower lobe, small left-sided pleural effusion, moderate cardiomegaly, and calcifications of the coronary arteries and thoracic aorta (b; the pulmonary mass and coronary artery calcifications are not present in the illustrated coronal slice). postmortem gross examination of the right lung showed congested parenchyma superimposed on emphysematous changes (c; bar scale equals 1 cm; pink areas represent incomplete formalin fixation). the second decedent was a 54-year-old man with a history of hypertension and type 2 diabetes mellitus. he presented to a community hospital with an acute episode of shortness of breath, which worsened with exertion, and a generally dry cough of 2 days' duration. the patient denied having fever, headache, malaise, and gastrointestinal symptoms. he reported having never smoked. in the emergency department, his vital signs were notable for tachycardia (heart rate of 118 beats/min) and oxygen saturation of 76%. he was afebrile. physical examination was notable for unlabored breathing with diminished, yet clear, breath sounds bilaterally, tachycardia, and good peripheral pulses. a chest roentgenogram showed diffuse bilateral airspace opacities with some areas of consolidation of the lower lungs, concerning for pneumonia ❚image 3a❚. the patient was admitted to the intensive care unit (icu) with acute hypoxic respiratory failure, requiring 15 l/min of oxygen via nonrebreather mask. initial labs showed an elevated d-dimer, and although there was some concern for pulmonary embolism, a ct scan was not pursued due to underlying renal function issues (elevated serum creatinine). instead, empiric anticoagulation was initiated (heparin bridged to enoxaparin sodium). a nucleic amplification test performed on a nasopharyngeal swab sample came back positive for sars-cov-2 later the same night. tests for influenza and respiratory syncytial virus were negative. on day 1 after admission, ❚image 2❚ (case 1) postmortem microscopic examination of the lungs showed diffuse alveolar damage characterized by hyaline membrane formation (a, ×100) and scattered squamous metaplasia of distal airways (b, ×100) on a background of emphysematous changes. bronchial sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium (c, ×100), and the left lower lobe lung mass lesion was consistent with large cell carcinoma (d, ×100). the patient was consented and enrolled in a trial to study the utility of the antiviral medication remdesivir in treating sars-cov-2-infected patients. he received his first dose of the 10-day course the same day. over the next few days, the patient's renal function showed improvement, but he still remained hypoxic despite escalation in oxygen therapy. a repeated chest roentgenogram performed 4 days after admission showed increased opacities bilaterally with a more consolidative appearance at the right lung base. blood and urine cultures grew coagulase-negative staphylococcus and enterococcus faecalis, respectively, and the patient was started on antibiotics (vancomycin and piperacillin/tazobactam). the patient's wbc count also increased significantly over time, with a shift to increased absolute neutrophil counts and immature precursors in the peripheral blood. lymphopenia was present beginning on day 2 of admission. a respiratory culture had no growth of bacteria or fungi. serial chest roentgenograms showed initial worsening of his lung disease to more severe diffuse interstitial infiltrates ❚image 3b❚ with subsequent stabilization of his imaging. he had increasing oxygen requirements beginning on day 9 after admission, and, despite appropriate therapy, continued to have low oxygen saturation. ❚image 2❚ (cont) the kidneys were notable for acute tubular injury on a background of moderately extensive chronic changes of the renal parenchyma (e, ×100). the heart showed sequelae of coronary artery atherosclerosis and chronic ischemic injury with replacement-type interstitial fibrosis (f, ×20). laboratory assessments around this time were notable for rapidly rising serum creatinine and liver enzymes. as a result of continued oxygen requirements, he was intubated on day 10 and also started on propofol with subsequent drop in blood pressure. intravenous fluid resuscitation and pressor medications were started. the patient's oxygen saturation still showed no improvement, so a cisatracurium besylate drip was started and the patient was transferred to our institution for further management. while a central line was being placed, the patient became bradycardic with a heart rate of 30 beats/min. an arterial blood gas test at the time showed respiratory and metabolic acidemia. despite further medication, the patient's heart rate continued to decrease to approximately 20 beats/min without a measurable blood pressure. the patient entered pulseless electrical activity arrest and resuscitative measures were initiated, with return of spontaneous circulation in approximately 2 min. the patient was evaluated for possible extracorporeal membrane oxygenation; however, he was deemed to be a poor candidate by the shock team. ventilator settings at the time were as follows: positive end-expiratory pressure, 15 cm h 2 o; fraction of expired o 2 , 100%; and respiratory rate, 20 breaths/min. despite continued icu support, the patient died 10 days after admission to the hospital and 12 days after first reported onset of symptoms. the autopsy was limited to examination of the chest and abdomen only, per the family's request. the autopsy was performed 39 hours after death. at postmortem examination, external findings were primarily limited to those of medical interventions such as intubation and central line placement. the decedent was overweight, bordering on obese, with a body mass index of 29.9 kg/m 2 . in situ examination was significant for bilateral serosanguineous pleural effusions of 300 ml within each hemithorax. grossly, bilateral lungs were strikingly heavy (right lung, 2,050 g; left lung, 1,100 g) with a congested appearance. sectioning revealed a diffuse and relatively uniform firmness to the parenchyma ❚image 3c❚. there were no focal lesions. histologically, sections from all lobes of the lungs showed varying stages of dad with some areas demonstrating prominent hyaline membrane formation and significant pneumocyte hyperplasia (acute/exudative stage) ❚image 4a❚ and others exhibiting patchy to diffuse intra-alveolar fibroblastic proliferation and interstitial edema (organizing stage) ❚image 4b❚. areas of marked intra-alveolar acute inflammation were also present focally involving all lobes except the left lower lobe, diagnostic of acute bronchopneumonia ❚image 4d❚. varying degrees of pulmonary edema, clusters of multinucleated giant cells ❚image 4c❚, and foci of squamous metaplasia were also noted scattered throughout the lung parenchyma. of note, despite extensive sampling and microscopic review, microthrombi were not identified. ❚image 3❚ (case 2) chest roentgenogram performed on the day of admission revealed diffuse bilateral airspace opacities with some areas of consolidation in the lower lungs, consistent with pneumonia (a). chest roentgenogram performed on day 10 of hospital admission (1 day prior to death) showed extensive multifocal patchy infiltrates throughout the lungs bilaterally (b). postmortem gross examination of bilateral lung showed congested and firm parenchyma (c). submitted sections of the trachea ❚image 4e❚ and proximal bronchi showed no significant abnormalities. additional significant findings noted at autopsy included evidence of diabetic glomerulosclerosis and acute tubular necrosis within bilateral kidneys ❚image 4f❚, an enlarged heart (560 g) with left ventricular hypertrophy, mild calcified atherosclerotic coronary artery disease, liver (2,300 g) and spleen (270 g) were enlarged secondary to acute congestion, and necrotizing granulomata within a right peribronchial lymph node and the spleen. a grocott methenamine silver stain performed on the section of lymph node revealed yeast organisms most consistent with incidental histoplasma capsulatum. sars-cov-2 rna was detected in sections of lung parenchyma, bronchi, lymph node, and spleen ( table 1) . the presence of sars-cov-2 rna was not detected in the heart, esophagus, stomach, intestines, liver, skeletal muscle, kidney, adrenal gland, or skin in case 2 (table 1) . attempts at viral genome sequencing did not produce adequate coverage for successful genomic evaluation in this case, possibly due to low viral titers resulting from remdesivir administration. in summary of these findings, the cause of death was sars-cov-2 infection occurring in the setting of diabetes and underlying cardiovascular disease leading to respiratory and subsequent multiorgan system failure. ❚image 4❚ (case 2) postmortem microscopic examination of the lungs revealed varying stages of diffuse alveolar damage characterized by extensive hyaline membrane formation (acute/exudative stage) (a, ×100) and intra-alveolar fibrin deposition (organizing stage) (b, ×100). scattered multinucleated giant cells were also seen (c, ×400). the patient also had significant superimposed bronchopneumonia (d, ×200). here we report the detection of sars-cov-2 rna in a wide range of organs similar to that reported by an investigation of cases from washington state. 7 this is the first study to our knowledge to demonstrate sars-cov-2 rna detection in several major organs and to sequence sars-cov-2 genome from postmortem ffpe sections. sars-cov-2 and sars-cov use spike (s) proteins to bind to the host cell via angiotensin converting enzyme 2 (ace2) as an entry receptor leading to the internalization of the complex by the host cell. [8] [9] [10] [11] ace2, a counterregulatory component of the renin angiotensin aldosterone system, is mainly localized in the heart, kidney, endothelium, and testis, but is also expressed at low levels in many other tissues such as the brain, intestines, and lung. 11 in this study, we detected sars-cov-2 rna in a number of ace2 expressing tissues including lungs, heart, and intestines. we were also able to detect viral rna from ffpe samples from upper airways, lymph node, spleen, and liver, which mirrors the finding of prior reports with analysis from fresh tissue. 7, 12 although virus rna was detected in extrapulmonary tissues, there was no histomorphologic evidence of acute pathologic changes attributable to the virus itself or an inflammatory response. although relatively few reports documenting postmortem pathologic findings in patients with sars-cov-2 have been published to date, specific patterns of injury have emerged. as is expected in the clinical setting of pneumonia and ards, the lungs have been found to be the most consistently and severely affected. several reports have demonstrated features of dad, primarily in the acute or exudative phase in the majority of autopsies performed. 2, 3, 8, 13 these have been characterized by intra-alveolar fibrin with associated hyaline membrane formation, markedly reactive pneumocytes, and variable squamous metaplasia. fewer cases showed more advanced changes associated with dad, including intra-alveolar fibroblastic proliferation and interstitial edema. 8, 13 additional findings that have been frequently noted include multinucleated giant cells, 3, 8, 13 acute inflammation in the form of bronchopneumonia and/or acute bronchiolitis, and relative paucity of chronic inflammatory cells. 2, 8, 11 both of our cases exhibited features of the acute phase of dad, while case 2 showed more prominent organization. in case 1, these features were superimposed on a background of chronic emphysematous changes and in the setting of concomitant large cell carcinoma of the left lung with associated ipsilateral nodal metastasis. additional pulmonary findings in case 2 included significant superimposed acute bronchopneumonia and several clusters of multinucleated giant cells. both patients also demonstrated histopathologic features of acute renal injury superimposed on a background of moderate chronic changes to the renal parenchyma, in keeping with their histories of diabetes mellitus and hypertension. case 2 exhibited classic features of nodular diabetic glomerulosclerosis. both patients also demonstrated congestive splenomegaly and ❚image 4❚ (cont) tracheal sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium (e, ×100). the kidneys were notable for acute tubular necrosis on a background of diffuse and nodular diabetic glomerulosclerosis (f, ×200). sinusoidal congestion of the liver. while these features have also been mentioned in other reports, 2,13 they may simply be related to terminal changes in these acutely ill patients rather than a direct result of the viral infection. also in keeping with the reported literature, the additional pathologic changes identified in our cases were associated with the patients' underlying chronic medical conditions and did not seem to be specifically related to sars-cov-2 infection. for example, case 1 showed advanced cardiovascular disease as evidenced by cardiomegaly, severe atherosclerotic coronary artery disease, and remote ischemic changes within the myocardium. cardiomegaly without significant coronary artery disease or evidence of ischemic injury was seen in case 2. neither heart showed features of lymphocytic myocarditis, which has been reported as a possible consequence of sars-cov-2 infection. 8 additionally, although several anecdotal reports regarding hypercoagulability have also begun circulating, histologic documentation of microthrombi has only rarely been reported in the published literature. 2 we were unable to identify any microthrombi in our 2 patients. gross and histopathologic examination of the upper respiratory tract (main bronchi and trachea), pancreas, adrenal glands, gastrointestinal tract tissues, urinary bladder, skin, and skeletal muscle in our cases showed no significant changes. other postmortem ancillary studies have been performed on sars-cov-2 patients. localization studies of sars-cov-2 by immunofluorescence revealed prominent expression on alveolar epithelial cells, including damaged, desquamated cells within the alveolar space. 4 ultrastructural examination by electron microscopy was able to locate viral particles in type ii pneumocytes, upper airway and intestinal mucosal epithelial cells, and proximal tubular epithelial cells. 7 tian et al 12 performed reverse transcriptase polymerase chain reaction (rt-pcr) for sars-cov-2 on postmortem ffpe core biopsies collected from 4 patients and reported detection of sars-cov-2 rna in heart, lung, and liver in at least 1 patient in their cohort. in our study we were able to consistently detect the virus in lung and lymph node samples, while bronchus samples also contained viral rna, but at lower relative concentration compared to lung tissue. it has been postulated that moderate levels of rna in lymph nodes reflect viral presence in leukocytes that may serve as a route for the virus to disseminate from airways or lung parenchyma to other organs. 7 similarly, in an autopsy study performed on macaques on day 4 post infection, the highest sars-cov-2 rna levels were detected in lungs, using quantitative rt-pcr. viral rna was also detected in the lymph nodes in 3 out of 4 animals. 13 monitoring the spread of virus in a pandemic is critical in disease control. shared sars-cov-2 genotyping efforts and resources like the global initiative on sharing all influenza data (gisaid, https://www.gisaid.org), nextstrain (https://nextstrain.org), and johns hopkins university dashboard (https://coronavirus.jhu.edu/map. html) have enabled dynamic tracking of the evolution of the pandemic, enhanced monitoring of infection patterns, and have allowed better estimations of the affected population size in communities. 14 like other rna viruses, the rna-dependent rna polymerase of sars-cov-2 lacks proofreading capability, leading to an accumulation of mutations in its genome over time. mutations in the critical proteins, including the s protein, rna polymerase, rna primase, and nucleoprotein, have been discovered. 15, 16 these mutations are not equally distributed across the genome and generate hotspots that can be linked to specific geographic location of the outbreak. knowledge and monitoring of these mutation sites is critical in designing a vaccine for preventing the infection as well as therapies for treatment of sars-cov-2. 14, 17 further studies showing the effect of these mutations on pathogenicity and pathobiology of the virus are needed. sequencing of viral rna from ffpe lung tissue from the case 1 autopsy showed mutations most consistent with a subset of the western european clade a2a (c3037t, c14408t, a23403g), 16 with mutations enriched in new york state a2a cases (c1059t and g25563t). 15 the viral rna from our cases also had g11083t mutation that to date has been described in only 2% (13/648) of the a2a clade that contain c1059t mutation. it has been suggested that the g11083t mutation is more common in regions (italy and brazil) with higher fatality rates. 18 the g11083 mutation was described in the first case of sars-cov-2 in italy. 19 a study examining the transmission and evolution of sars-cov-2 in cruise quarantine suggests that the g11083t mutation can be transmitted via rna recombination. 20 linkage disequilibrium analysis suggests that rna recombination with a g11083t mutation may contribute to the increase of mutations among the viral progeny. 20 in this case we did observe other mutations not extensively described in previous genomic sequencing efforts, including point mutations c9515t, c23378t, and in-frame deletion 12137_12142del (table 2 ). more studies are needed to determine if g11083t, or other mutations detected in this case, may increase the fitness of the carrier virus as a benefit allele in the future. the challenge that pathologists and the medical community at large face is determining if deaths in individuals who present with the clinical signs and symptoms of sars-cov-2 infection are truly infected with the virus. it is not standard for all autopsies to take swabs or put aside fresh tissue for potential china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china covid-19 autopsies pathological findings of covid-19 associated with acute respiratory distress syndrome histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 comparison of abbott id now, diasorin simplexa, and cdc fda eua methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from individuals diagnosed with covid-19 nextstrain: real-time tracking of pathogen evolution histopathology and ultrastructural findings of fatal covid-19 infections structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model phylogenetic network analysis of sars-cov-2 genomes host, viral, and environmental transcriptome profiles of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) ajcp / original article the role of phylogenetic analysis in clarifying the infection source of a covid-19 patient evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission decoding the lethal effect of sars-cov-2 (novel coronavirus) strains from global perspective: molecular pathogenesis and evolutionary divergence molecular characterization of sars-cov-2 from the first case of covid-19 in italy faster de novo mutation of sars-cov-2 in shipboard quarantine molecular testing for sars-cov-2 detection, whereas production of ffpe tissue blocks is standard for most autopsies that require histologic examination. the ability to detect sars-cov-2 rna from ffpe tissue may assist in cases in which there are clinical, or even histologic, findings that overlap findings found in sars-cov-2 infection. ffpe blocks are stored on many autopsies in many places from the period preceding the recognized onset of community-acquired sars-cov-2 infection. recent reports suggest that sars-cov-2 infection has been detected in cases that preceded the previously recognized onset of community transmission in the united states. our results suggest that sequencing of ffpe materials could contribute to a greater understanding of the early onset and spread of sars-cov-2 infection in the united states or other countries.possible limitations for molecular testing on autopsy samples include sampling bias if virus involvement is focal. this can be addressed by obtaining multiple samples from each organ. our results showed widespread positivity of samples throughout sites in the lung and airways. this study supports sampling of upper and lower airways, immune organs (lymph node and spleen), and to a lesser degree the intestine, heart, and liver. another concern is postmortem tissue autolysis and rna degradation. further studies are required to determine the stability of viral rna in different organs after death. finally, since the virus may affect each organ differently, individual organs may be expected to have different viral loads at any given time in the course of the disease. however, the clinical correlation of such findings is yet to be determined.in conclusion, the findings of our postmortem examinations support major histopathologic features previously reported in the literature, the most striking of which is dad. changes in other organs, like the kidneys and heart, are likely secondary or related to underlying diseases. as might be expected given this pattern of injury, sars-cov-2 rna is more easily detected in lung and upper airway tissue when compared to other organs. interestingly, our study also documents detection in hematopoietic and lymphoid organs (lymph node and spleen) and may lend credence to the theory that leukocytes could serve as a route for dissemination of the virus from airways to other organs. 8 we propose that detection of sars-cov-2 rna from postmortem ffpe tissues from multiple sites can aid in tracking the spread of the novel coronavirus and in gaining a better understanding of its pathophysiologic effects on human health. additionally, the retrospective sequencing of archived ffpe material may allow for more definitive determination of the rate of prevalence of sars-cov-2 infection and therefore providing more accurate epidemiologic data on the virus's course and entry into a given population. key: cord-334528-xenq90xj authors: chen, hsing i title: acute lung injury and acute respiratory distress syndrome: experimental and clinical investigations date: 2011-03-17 journal: j geriatr cardiol doi: 10.3724/sp.j.1263.2011.00044 sha: doc_id: 334528 cord_uid: xenq90xj acute lung injury (ali) or acute respiratory distress syndrome (ards) can be associated with various disorders. recent investigation has involved clinical studies in collaboration with clinical investigators and pathologists on the pathogenetic mechanisms of ali or ards caused by various disorders. this literature review includes a brief historical retrospective of ali/ards, the neurogenic pulmonary edema due to head injury, the long-term experimental studies and clinical investigations from our laboratory, the detrimental role of no, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ali/ards. acute lung injury (ali) or acute respiratory distress syndrome (ards) is a serious clinical problem with high mortality. [1] in animals and humans, ali can be induced by various causes such as brain injury, [1] [2] [3] [4] enterovirus, [5, 6] japanese b encephalitis, [7] and coronavirus. [8, 9] the risk factors for ards included septicemia, acid aspiration, infection, traumatic injury, fat embolism, ischemia/ reperfusion, and other caused. [1, 6, 8, [10] [11] [12] [13] [14] [15] [16] [17] [18] our cardiopulmonary laboratory has carried out experimental studies and clinical investigations on ali and ards since 1973. [1] [2] [3] 17, 19, 20] the purposes of this review article are: (1) to describe in brief the historical perspective of ards and ali; (2) to draw attention of an important clinical issue of neurogenic ali; (3) to present the experimental studies and clinical investigations from our laboratory from 1973 to 2009; (4) to elucidate the functional role of nitric oxide (no) and other mediators involved in the pathogenesis of ards/ali; (5) to define the risk factors for ards and ali; and (6) to discuss the pathogenetic mechanisms and therapeutic regimen for ards/ali. ali or pulmonary embolism (pe) has been reported in humans and animals with intracranial disorders such as head trauma, brain tumor, intracranial hypertension or cerebral compression. early studies in our laboratory demonstrated that acute pe of hemorrhagic and fulminant type occurred accompanying severe hypertension and bradycardia (cushing responses) in rats following cerebral compression (cc) or intracranial hypertension (ich). the lung pathology was characterized by intravascular congestion and disruption of pulmonary large and small vessels leading to severe alveolar hemorrhage (alveolar flooding). these changes was prevented by spinal transection, sympathectomy and sympathoadrenergic blocking agents, but was not affected by decerebration, adrenalectomy, vagotomy and atropine. these results suggest that sympathetic nervous system is pivotal in the neurogenic pe. brain areas above the medulla oblongata and parasympathetic nervous system play little role. [2] a series of studies was carried out to elucidate the hemodynamic events involved in the neurogenic pe. in anesthetized rats, we measured the aortic and pulmonary blood flow and used techniques of right and left heart bypass. the imbalance in the right and left ventricular output was characterized by a rapid and dramatic decline in aortic flow accompanying a gradual decrease in pulmonary arterial flow. in rats with a right heart bypass, ich produced severe pulmonary hypertension and pe. in the left heart-bypassed rats, ich induced systemic hypertension, http://www.jgc301.com; jgc@mail.sciencep.com | journal of geriatric cardiology whereas no significant changes occurred in the lungs. [4] in anesthetized dogs with a total heart bypass preparation, ich produced constriction of the systemic and pulmonary resistance and capacitance vessels. [21] [22] [23] [24] the implications of these findings are: (1) central sympathetic activation elicits increase in the systemic and pulmonary vascular resistance associated with decreases in vascular capacity in both circulations; (2) the major cause of volume and pressure loading in the pulmonary circulation is acute left ventricular failure resulting in a marked decrease in aortic flow; and (3) systemic venous constriction causes a shift of blood from the systemic to the pulmonary circulation ( figure 1) . a schematic representation summarizes the neural and hemodynamic consequence caused by cerebral compression (figure 2 ). spectral analysis of the aortic flow and pressure wave was employed to evaluate the hemodynamics of steady and pulsatile components. in anesthetized dogs, ich caused significant increases in characteristic impedance, pulse wave reflection and total peripheral resistance with decrease in arterial compliance and cardiac output. the ventricular work was elevated. [25] clinical study in patients with head injury of various severities, analysis of the heart rate variability with frequency analysis revealed increased low frequency percentage, and low to high frequency ratio with decrease in high frequency. the findings indicate augmented sympathetic and attenuated parasympathetic drive. these autonomic functional changes were related to the severity of brain-stem damage. [26] these two studies further support the contention that central sympathetic activation is involved in the cushing pressor response and consequent hemodynamic and autonomic alterations. in 1990s, my associates and i were interested in the study of chest disorders. we developed an isolated perfused rat's lung in situ preparation ( figure 3 ). previous method involved removing the isolated lungs from the body and placing the organ on a force-displacement transducer to record the changes in lung weight and these procedures were rather complicated and unstable. our in situ preparation does not require removing the lungs. instead, the isolated lungs were left in situ. the whole rat was placed in a scale platform to measure the change in body weight (bw). since the lungs are completely isolated from the body, the changes in bw reflect the lung weight (lw) changes. the preparation can be accomplished in 15 min. we used a digital-analogue converter to transfer the weight change from the scale platform to a recorder. the lw thus could be continuously monitored during the experiment. in this model, we can obtain the lung weight gain, lw/bw ratio, the changes in pulmonary arterial, capillary and venous pressures, the microvascular permeability (capillary filtration coefficient, k fc ), protein concentration in bronchoalveolar lavage (pcbal), dye leakage, and exhaled nitric oxide (no). the concentration of nitrate/nitrite, methyl guanidine (an index for hydroxyl radical), proinflammatory cytokines [tumor necrosis factor α (tnf α ) and interleukin-1 β (il-1 β )] and other factors in the lung perfusate can also be detected. early animal experimentations investigated the pathogenesis, modulators and mediators involved in the ali induced by phorphol, air embolism, platelets, hypoxia, ischemia/reperfusion, endotoxin [lipopolysaccharide (lps)]. the major finding is that cyclooxygenase products of arachidonic acid, thromboxane a 2 in particular is involved in the ali and pulmonary hypertension caused by phorbol, platelets and air embolism. [27, 28] furthermore, we found that l-arginine and inhaled no enhanced the lung injury caused by air embolism, while blockade of no synthase (nos) with n ω -nitro-l-arginine methyl ester (l-name) attenuated the ali. [28] the result suggests that no is also involved. during the summers from 20012003, we encountered a total of 48 children suffering from hand, foot, and mouth figure 2 . isolated and perfused lung in situ preparation. the system consists of a perfusion pump with heat exchanger and a venous reservoir. the rat is artificially ventilated. pulmonary arterial pressure (pap) and venous pressure (pvp) are monitored with transducers. the whole rat is placed on a balance platform to record the body weight change. since the lung is isolated from the whole body, the change in body weight reflects the lung weight change. disease. [6] chest radiography on admission revealed clear lung. however, 21 out of 48 cases developed severe dyspnea, hyperglycemia, leukocytosis, and decreased blood oxygen tension. arterial pressure (ap) and heart rate (hr) fluctuation ensued. spectral analysis of the ap and hr variabilities showed elevation in sympathetic activity at the onset of respiratory stress. thereafter, parasympathetic drive increased with declines in ap and hr. these children died within 4 h after the onset of ards. before death, chest radiography revealed severe lung infiltration. similar to japanese b encephalitis, destruction of the medullary depressor area caused initial sympathetic activation. reversetranscriptase polymerase chain reaction (rt-pcr) found marked inos mrna expression in the lung parenchyma, suggesting inos may also be involved in the pathogenesis of ards in patients with enterovirus 71 infection. furthermore, we have reported ards in patients with leptospirosis. [18] in leptospirosis-induced ards, histochemical stain demonstrated spirochetes bacteria in the alveolar space. the pathology included alveolar hemorrhage, myocarditis, portal inflammation and interstitial nephritis. antigen retrieval immunohistochemical stain disclosed inos expression in the alveolar type 1 cells, myocardium, hepatocytes and renal tubules. spectral analysis of ap and hr variabilities indicated decreased sympathetic drive with increased parasympathetic activity. the changes in autonomic functions led to severe hypotension and bradycardia. biochemical determinations suggested multiple organ damage. the pathogenesis of lung and other organ injury might also involve inos and no production. [18, 29] in subjects with scrub typhus, orientia tsutsugamushi infection caused alveolar injury. marked inos expression was found in the alveolar macrophages with increase in plasma nitrate/nitrite, suggesting that no production from the alveolar macrophages accounts for the ali. [30] the victim from rabies was a woman bitten by a wild dog. in addition to sign of hydrophobia, hypoxia, hypercapnia, hyperglycemia and increased plasma nitrate/nitrite were observed. the woman died of alveolar hemorrhage shortly after admission. [31] recently, we encountered five cases with long-term malignancy. these subjects displayed signs of respiratory distress following an episode of hypercalcemia. two cases died of ards after the plasma calcium was increased above 6 mmol/l. search of literatures revealed that holmes et al. [32] reported a patient who died of ards following a hypercalcemia crisis caused by a parathyroid adenoma. we conducted animal experiments in whole rodent and isolated perfused rat's lungs. our results indicated that hypercalcemia (calcium concentration > 5 mmol/l) caused severe ali in conscious rats and isolated lungs. immunohistochemical staining showed inos activity in the alveolar macrophages and epithelial cells. reversetranscriptase polymerase chain reaction (rt-pcr) found marked increase in inos mrna expression in lung parenchyma. hypercalcemia also increased nitrate/nitrite, methyl guanidine, proinflammatory cytokines and procalcitonin. pretreatment with calcitonin or l-n 6 (1iminoethyl)-lysine (l-nil, an inos inhibitor) attenuated the hypercalcemia-induced changes. we proposed that hypercalcemia produced a sepsis-like syndrome. the ali caused by hypercalcemia may involve no and inos. [33, 34] in addition to the aforementioned animal experimentations and clinical observations that no production through the inos may be involved in the lung injury due to various causes, our research team demonstrated that endotoxemia produced in anesthetized rats by intravenous administration of lipopolysaccharide (lps, endotoxin) provoked systemic hypotension, endothelial damage and ali accompanied by increased plasma nitrate/nitrite and expression of inos mrna, tnf α and il-1 β . the lps-induced changes were abolished by nonspecific and specific inos inhibitors such as n ω -monomethyl-l-arginine (l-nmma), l-name, aminoguanine and dexamethosone. [35] this study suggested that no/inos, tnf α and il-1 β were involved in the endotoxemia-induced ali. generation of no from the activated neutrophil caused alveolar injury from smoke inhalation. [36] experiments in many laboratories using specific inos inhibitors and/or inos-knockout animals have supported the contention that no/inos is responsible for the oxidative stress and endothelial damage in the ards/ali caused by endotoxin, ozone exposure, carrageenan treatment, hypoxia, acute hyperoxia, bleomaycin administration, acid aspiration and other causes. [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] our laboratory further provided evidence to suggest that the no/inos system is involved in the pathogenesis of ali caused by air embolism, [47] fat embolism, [48] [49] [50] ischemia/ reperfusion, [51] [52] [53] oleic acid [54] and phorbol myristate acetate. [55] in these recent studies, various insults caused increase in nitrate/nitrite in plasma or lung perfusate, upregulation of inos mrna in lung parenchyma accompanied with elevation of proinflammatory cytokines such as tnf α , il-1 β and il-6. lin et al. [56] have suggested that an increase in inos mrna triggers the release of proinflammatory cytokines in septic and conscious rats. the inflammatory responses results in multiple organ damage including ali. inhibition of inos with s-methylisothiourea (smt) or l-nil attenuated the inflammatory changes, release of no and cytokines and prevented the organ dysfunction and ali. [52] in animal experiments and clinical investigations, the risk factors causing ali/ards include head injury, intracranial hypertension, [2] [3] [4] [57] [58] [59] [60] [61] [62] sepsis, [12, 17, 35, 37, 39, 42, 44, [63] [64] [65] [66] and infections. [6] [7] [8] [10] [11] [12] 17, 18, [29] [30] [31] 67] pulmonary embolic disorders journal of geriatric cardiology | jgc@mail.sciencep.com; http://www.jgc301.com such as fat and air embolism are less common causes. [7, 15, 28, 47, [68] [69] [70] ischemia/reperfusion lung injury may develop as a consequence of several pulmonary disorders such as pulmonary artery thromboendarterectomy, thrombolysis after pulmonary embolism and lung transplantation. [13, [51] [52] [53] [71] [72] [73] [74] gastric aspiration occurs frequently in surgical patients under anesthesia and other causes such as blunt thoracic trauma, impaired glottis competency, and pregnancy. [73, 75, 76] it is one of the major causes of acute respiratory syndrome (ards). [77, 78] intratracheal instillation of hydrochloric acid (hci) or gastric particles has been employed as experimental model of acute lung injury (ali). [16, [79] [80] [81] in addition, amphetamine, phorbal myristate acetate, oleic acid have been employed for the induction of ali. [82] [83] [84] [85] [86] phorbol myristate acetate (pma, 12-o-tetradecanoyl-phorbol-13-acetate), an ester derivative from croton oil has been used to induce ali. [65, 83, 86, 87] experiments in vivo and in vitro have demonstrated that pma is a strong neutrophil activator. [87] [88] [89] [90] activation and recruitment of neutrophil that lead to release of neutrophil elastase and other mediators may play an initial role in the pathogenesis of ali. [91, 92] the oleic acid-induced ali has several clinical implications. first, the blood level of oleic acid was significantly elevated in patients with ards. [93, 94] second, the proportion of oleic acid incorporated into surfactant phospholipids was also increased in patients with ards and sepsis. [95, 96] these observations have provided evidence to suggest that serum level of oleic acid as a prediction or prognostic factor for ards. [84, 93] early studies focused on the potential toxic effects of high oxygen fractions on inspired air. [97] ventilator-induced ali was attributed to the deleterious effects on capillary stress due to alveolar overdistension. cyclic opening and closing of atelectatic alveoli during mechanical ventilation might cause lung injury and enhance the injured alveoli. recent evidence indicated that over distension coupled with repeated collapse and reopening of alveoli initiated an inflammatory cascade of proinflammatory cytokines release. [68, [98] [99] [100] in spite of the risk factors and causes, the pathophysiology of ards/ali has generally considered to be initiated by formation of alveolar edema (even hemorrhage) that is enriched with protein, inflammatory cells or red blood cells. after damage of alveolar-capillary barrier, impairment of gas exchange occurs, with decrease in lung compliance and increases in dispersion of ventilation and perfusion and intrapulmonary shunt. hypoxia, reduction in arterial oxygen partial pressure to fraction of oxygen in inspired air pao 2 /fio 2 , hypercapnia ensued despite ventilation with high oxygen. [1, 2, 67, 68, 101, 102] in addition to the potential toxic effects of no and free radicals, certain chemokines, cytokines, neutrophil elastase, myeloperoxidase and malondialdehyde have been shown to be associated with several types of ards/ali. [50, 54, 55, 91, [103] [104] [105] the balance between proinflammatory and anti-inflammatory mediators is regulated by transcriptional factors mainly nuclear factor-κ b (nf-κ b). [106] pulmonary fluid clearance and ion transport are important factors to determine the extent of lung edema. regulator factors include cystic fibrosis transmembrane conductance regulators, sodium-and potassium-activated adenosine triphophatase (na + -k + -atpase), protein kinases, aclenylate cyclase, and cyclic adenosine monophosphate (camp). [12, 29, 107, 108] the treatment of ards/ali is difficult and complex. several review articles and monographs have addressed the issue of possible therapeutic regimen. the modalities include extracorporeal membrane oxygenation, prone position, mechanical ventilation with appropriate tidal volume and respiratory pressure, fluid and hemodynamic management and permissive hypercapnic acidosis. [68, 100, [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] other pharmacological treatments are anti-inflammatory and/or antimicrobial agents to control infection and to abrogate sepsis, adequate nutrition, surfactant therapy, inhalation of no and other vasodilators, glucocorticoids and other nonsteroid anti-inflammatory drugs, agents that accelerate lung water resolution and ion transports. [68, 102, [120] [121] [122] [123] [124] although most animal experimentations on these pharmacological options showed favorable results, the effectiveness and outcomes in clinical studies or trials were conflicting. beta agonists to facilitate water removal and ion transport have been shown to be promising. these agents may also stimulate secretion of surfactant and have no serious side effects. there were several reports on the pharmacological and molecular actions of beta agonists, surfactant and vascular endothelial growth factor and related molecules as well as angiotensin-converting enzyme (ace). [107, 125, 126] in addition to the experimental studies and clinical investigations on the pathogenesis of ali/ards, our laboratory has carried out several experimentations on the therapeutic regimen for this serious disorder. in conscious rats, regular exercise training attenuates septic responses such as systemic hypotension, increases in plasma nitrate/nitrite, methyl guanidine, blood urea nitrogen, creatinine, amylase, lipase, asparate aminotransferase, alanine aminotransferase, creatine phosphokinase, lactic dehydrogenase, tnf α, and il β . exercise training also abrogates the cardiac, hepatic and pulmonary injuries caused by endotoxemia. [124] insulin exerts anti-inflammatory effects on the ali and associated biochemical changes following intravenous administration of lipopolysaccharide (lps). [127] propofol (2,6-diisopropylphenol) has been commonly used for sedation in critically ill patients. [128] this anesthetic has rapid onset, short duration and rapid elimination. [129] propofol protects the anesthetized rats from ali caused by endotoxin. [65] in conscious rats, oleic acid results in sepsis-like responses including ali, inflammatory reactions and increased in neutrophil-derived factors (neutrophil elastase, myeloperoxidase and malondialdehyde), nitrate/nitrite, methyl guanidine, inflammatory cytokines. it depresses the sodium-and potassium-activated atpase, but upregulates the inos mrna expression. pretreatment and posttreatment with propofol alleviates or reverses the oleic acid-induced lung pathology and associated biochemical changes. [54] pentobarbital, an anesthetic agent commonly used in experimental studies and a hypnotic for patients improves the pulmonary and other organ functions following lps administration. it also increases the survival rate. [15] a later study by yang et al. [130] further revealed that pentobarbital suppressed the expression of tumor necrosis factor α , which might result from decrease in the activities of nuclear factor-κβ and activator protein 1 and reduction in expression of p38 mitogen-activated protein kinase. in vivo examination of cytotoxic effects of lps disclosed that lps caused multiple organ dysfunctions. these changes were attenuated by pentobarbital. pentobarbital also reduced the cell aptosis caused by deforoxamine-induced hypoxia. nicotinamide or niacinamide (compound of soluble b complex) abrogates the ali caused by ischemic/reperfusion or endotoxin by mechanism through inhibition on poly (adp-ribose) synthase or permerase cytoxic enzyme and subsequent suppression of inos, no, free radicals and proinflammatory cytokines with restoration of adenosine triphosphate atp. [48, 53] n-acetylcysteine, an antioxidant and cytoprotective agent with scavenging action on reactive oxygen species and inhibitory effects on proinflammatory cytokines ameliorated organ dysfunctions due to sepsis in conscious rats. [131, 132] in a similar endotoxin-induced ali model, we found that n-acetylcysteine improved the lps-induced systemic hypotension and leukocytopenia. it also reduced the extent of ali, as evidenced by reductions in lung weight changes, exhaled no and lung pathology. in addition, n-acetylcysteine diminished the lps-induced increases in nitrate/nitrite, tnf α , and il β [64] in isolated lungs, n-acetylcysteine attenuated the ali caused by phorbol myristate acetate. [86] in a recent study, we reported that posttreatment with n-acetylcysteine prevented the ali caused by fat embolism. [50] our series of experimental studies provided results in favor of n-acetylcysteine. the conflicting results and practice guidelines from clinical studies in the recommendation of n-acetylcysteine in critically ill patients [133, 134] were commented and analyzed by molnár. [135] the clinical application of results from animal studies requires further investigations. ards or ali is a serious clinical problem with high mortality. the risk factors leading to ali/ards include head injury, intracranial disorders, sepsis and infections. pulmonary embolic disorders such as fat and air embolism are less common causes. ischemia/reperfusion lung injury may develop as a consequence of several pulmonary disorders such as lung transplantation. gastric aspiration occurs frequently in several conditions such as anesthesia, trauma and pregnancy. the ventilator-induced ali has been attributed to the deleterious effects on capillary stress due to alveolar overdistension. in experimental studies, phorbol myristate acetate and oleic acid have been employed to induce ali. the pathogenesis of ards/ali is complex. experimental studies and clinical investigations from our and other laboratories have indicated the detrimental role of nitric no through inducible no synthase (inos). activation and recruitment of neutrophils that lead to release of neutrophil elastase, myeloperoxidase, malondialdehyde and proinflammatory cytokines may play an initial role in the pathogenesis of ali/ards. the possible therapeutic regimen for ali/ards include extracorporeal membrane oxygenation, prone position, fluid and hemodynamic management and permissive hypercapnic acidosis etc. other pharmacological treatments are antiinflammatory and/or antimicrobial agents, inhalation of no, glucocorticoids, surfactant therapy and agents that facilitate lung water resolution and ion transports. adrenergic beta agonists are able to accelerate lung fluid and ion removal and to stimulate surfactant secretion. there are reports on the actions of vascular endothelial growth factor and related molecules as well as angiotensin-converting enzyme. our laboratory has reported experimental studies on the effectiveness of several regimen for ali/ards. in conscious rats, regular exercise training alleviates the endotoxin-journal of geriatric cardiology | jgc@mail.sciencep.com; http://www.jgc301.com induced ali. propofol and n-acetylcysteine exert protective effect on the ali causes by endotoxin, oleic acid and phorbol myristate acetate. we have also provided evidence that insulin possesses anti-inflammatory effect. pentobarbital is capable of reducing the endotoxin-induced ali and associated changes. in addition, nicotinamide or niacinamide (soluble b complex) abrogates the ali caused by ischemia/ reperfusion or endotoxemia. these nonpharmacological and pharmacological therapeutic strategies require further investigations for clinical application. acute respiratory distress syndrome pulmonary edema and hemorrhage resulting from cerebral compression pulmonary edema and hemorrhage as a consequence of systemic vasoconstriction centrogenic pulmonary hemorrhagic edema induced by cerebral compression in rats. mechanism of volume and pressure loading in the pulmonary circulation clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease mechanism of fulminant pulmonary edema caused by enterovirus 71 acute pulmonary oedema: rare causes and possible mechanisms a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome pulmonary edema in meningococcal septicemia associated with reduced epithelial chloride transport static inflation attenuates ischemia/reperfusion injury in an isolated rat lung in situ endotoxin-induced acute lung injury and organ dysfunction are attenuated by pentobarbital anaesthesia clinical and pathological features of fat embolism with acute respiratory distress syndrome effects of nitric oxide synthase inhibitor on acid aspiration-induced lung injury in rats nitric oxide in the cardiovascular and pulmonary circulation--a brief review of literatures and historical landmarks pathophysiological mechanism of lung injury in patients with leptospirosis nitric oxide in systemic and pulmonary hypertension neural and hemodynamic mechanisms of neurogenic pulmonary edema a scintiphotographic study of pulmonary edema and hemorrhage induced by cerebral compression and norepinephrine participation of regional sympathetic outflows in the centrogenic pulmonary pathology the cushing responses in the systemic and pulmonary circulation: the role of adrenal glands, bronchial circulation and pulmonary innervation systemic and pulmonary hemodynamic responses to intracranial hypertension effects of intracranial hypertension on steady and pulsatile haemodynamics in dogs sympathetic and parasympathetic activities evaluated by heart-rate variability in head injury of various severities cyclooxygenase pathway mediates lung injury induced by phorbol and platelets air embolism-induced lung injury in isolated rat lungs the involvement of nitric oxide and beta-adrenergic pathway signalling in pulmonary oedema and fluid clearance pulmonary pathology in patients associated with scrub typhus acute respiratory distress syndrome associated with rabies pulmonary oedema in hypercalcaemic crisis acute respiratory distress syndrome associated with hypercalcemia without parathyroid disorders the detrimental role of inducible nitric oxide synthase in the pulmonary edema caused by hypercalcemia in conscious rats and isolated lungs effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats role of neutrophils and nitric oxide in lung alveolar injury from smoke inhalation endogenous nitric oxide and the pulmonary microvasculature in healthy sheep and during systemic inflammation role of inducible nitric oxide synthase in endotoxin-induced acute lung injury effect of aminoguanidine on lung fluid filtration after endotoxin in awake sheep nitric oxide synthase inhibitors attenuate ozone-induced airway inflammation in guinea pigs. possible role of interleukin-8 inducible nitric oxide synthase-knockout mice exhibit resistance to pleurisy and lung injury caused by carrageenan role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis effects of acute hypoxia and lipopolysaccharide on nitric oxide synthase-2 expression in acute lung injury pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase proinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury inhibition or knock out of inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury nitric oxide modulates air embolism-induced lung injury in rats with normotension and hypertension niacinamide abrogates the organ dysfunction and acute lung injury caused by endotoxin nitric oxide mediates acute lung injury caused by fat embolism in isolated rat's lungs n-acetylcysteine attenuates acute lung injury induced by fat embolism nitric oxide mediates lung injury induced by ischemia-reperfusion in rats inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats nicotinamide abrogates acute lung injury caused by ischaemia/reperfusion protective effects of propofol on acute lung injury induced by oleic acid in conscious rats the involvement of nitric oxide, nitric oxide synthase, neutrophil elastase, myeloperoxidase and proinflammatory cytokines in the acute lung injury caused by phorbol myristate acetate inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats edema and congestion of the lungs from intracranial hemorrhage pulmonary oedema and intracranial lesions increased intracranial pressure and pulmonary edema. 1. clinical study of 11 patients the hemodynamic response of dogs and monkeys to increased intracranial pressure mechanisms of neurogenic pulmonary edema hemodynamic study of acute neurogenic pulmonary edema in children the lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock n-acetylcysteine abrogates acute lung injury induced by endotoxin propofol exerts protective effects on the acute lung injury induced by endotoxin in rats physiological and chemical indicators for early and late stages of sepsis in conscious rats clinical year in review iii: asthma, lung transplantation, cystic fibrosis, acute respiratory distress syndrome the acute respiratory distress syndrome unravelling the fat embolism syndrome pulmonary embolism pulmonary reimplantation response in single-lung transplantation reperfusion pulmonary edema after pulmonary artery thromboendarterectomy aspiration pneumonitis and aspiration pneumonia reperfusion pulmonary edema after thrombolytic therapy of massive pulmonary embolism aspiration during anaesthesia: a computer-aided study of 185,358 anaesthetics clinical significance of pulmonary aspiration during the perioperative period improved survival of patients with acute respiratory distress syndrome (ards): 1983-1993 acute lung injury in the medical icu: comorbid conditions, age, etiology, and hospital outcome inhibition of acid-induced lung injury by hyperosmolar sucrose in rats surfactant alterations in acute inflammatory lung injury from aspiration of acid and gastric particulates evaluation of exogenous surfactant in hcl-induced lung injury free radicals mediate amphetamine-induced acute pulmonary edema in isolated rat lung effects of an endogenous nitric oxide synthase inhibitor on phorbol myristate acetate-induced acute lung injury in rats oleic acid inhibits alveolar fluid reabsorption: a role in acute respiratory distress syndrome? comparative effects of vaporized perfluorohexane and partial liquid ventilation in oleic acid-induced lung injury n-acetylcysteine attenuates the acute lung injury caused by phorbol myristate acetate in isolated rat lungs pentoxifylline rescue preserves lung function in isolated canine lungs injured with phorbol myristate acetate a novel oral neutrophil elastase inhibitor (ono-6818) inhibits human neutrophil elastase-induced emphysema in rats pretreatment with fk506 improves survival rate and gas exchange in canine model of acute lung injury cepharanthin, an alkaloid from stephania cepharantha, inhibits increased pulmonary vascular permeability in an ovine model of sepsis neutrophils and acute lung injury neutrophils mediate acute lung injury in rabbits: role of neutrophil elastase an increase in serum c18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome plasma fatty acid changes and increased lipid peroxidation in patients with adult respiratory distress syndrome alteration of fatty acid profiles in different pulmonary surfactant phospholipids in acute respiratory distress syndrome and severe pneumonia bronchoscopic administration of bovine natural surfactant in ards and septic shock: impact on biophysical and biochemical surfactant properties pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project. i. light microscopy ventilator-induced lung injury cellular stress failure in ventilator-injured lungs acute respiratory distress syndrome: a historical perspective the acute respiratory distress syndrome acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management leukocyte elastase: physiological functions and role in acute lung injury roles of neutrophil elastase and superoxide anion in leukotriene b4-induced lung injury in rabbit chemokines in acute respiratory distress syndrome transcriptional mechanisms of acute lung injury alveolar epithelial fluid transport in acute lung injury: new insights mechanisms of pulmonary edema clearance perspective on lung injury and recruitment: a skeptical look at the opening and collapse story critical care in ajrccm alveolar recruitment in acute lung injury prone position augments recruitment and prevents alveolar overinflation in acute lung injury hypercapnic acidosis attenuates endotoxin-induced acute lung injury protective effects of hypercapnic acidosis on ventilator-induced lung injury permissive hypercapnia: role in protective lung ventilatory strategies hypercapnia via reduced rate and tidal volume contributes to lipopolysaccharide-induced lung injury permissive hypercapnia impairs pulmonary gas exchange in the acute respiratory distress syndrome nonventilatory treatments for acute lung injury and ards ventilatory management of acute lung injury and acute respiratory distress syndrome acute respiratory distress syndrome: pharmacological treatment options in development pathophysiology and pharmacological treatment of pulmonary hypertension in acute respiratory distress syndrome inhaled nitric oxide therapy in adults exercise training attenuates septic responses in conscious rats vascular endothelial growth factor and related molecules in acute lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure insulin attenuates endotoxin-induced acute lung injury in conscious rats comparison of propofol and midazolam for sedation in critically ill patients an update of its use in anaesthesia and conscious sedation the reduction of tumor necrosis factor-alpha release and tissue damage by pentobarbital in the experimental endotoxemia model n-acetylcysteine ameliorates lipopolysaccharide-induced organ damage in conscious rats post-treatment with n-acetylcysteine ameliorates endotoxin shock-induced organ damage in conscious rats n-acetylcysteine treatment to prevent the progression of multisystem organ failure: a prospective, randomized, placebo-controlled study antioxidant supplementation in sepsis and systemic inflammatory response syndrome n-acetylcysteine as the magic bullet: too good to be true experimental studies and clinical investigations were supported in part by grants from the "national science council". the grant no. this fiscal year is nsc99-2320-b-320-010-my3. the author is grateful to ms. s. y. huang for the assistance in typing an editing. i appreciate the long-term coworkers involved this and other studies in my laboratory. key: cord-257114-pxmflm2c authors: burguete, sergio r.; maselli, diego j.; fernandez, juan f.; levine, stephanie m. title: lung transplant infection date: 2012-12-26 journal: respirology doi: 10.1111/j.1440-1843.2012.02196.x sha: doc_id: 257114 cord_uid: pxmflm2c lung transplantation has become an accepted therapeutic procedure for the treatment of end‐stage pulmonary parenchymal and vascular disease. despite improved survival rates over the decades, lung transplant recipients have lower survival rates than other solid organ transplant recipients. the morbidity and mortality following lung transplantation is largely due to infection‐ and rejection‐related complications. this article will review the common infections that develop in the lung transplant recipient, including the general risk factors for infection in this population, and the most frequent bacterial, viral, fungal and other less frequent opportunistic infections. the epidemiology, diagnosis, prophylaxis, treatment and outcomes for the different microbial pathogens will be reviewed. the effects of infection on lung transplant rejection will also be discussed. significant progress has been made since the first human lung transplant (lt) in 1963, and although survival after transplantation was initially plagued by issues of rejection, the advent of immunosuppression ushered in a new era in transplantation science and made long-term survival a possibility. with this success came the dilemma of post-transplant infectious complications, which, to this day, remain a significant contributor to overall morbidity and mortality in the lung transplant recipient (ltr). of all solid organ transplants, lungs are the most prone to infection, and this is likely due to several factors unique to the lung allograft. apart from constant exposure to the outside environment, the lungs are exposed to the colonized native airway and have been stripped of their usual mechanisms of defence including the cough reflex, bronchial circulation and lymphatic drainage. these factors, coupled with the induction of an immunosuppressed state collaborate to produce an environment that is ripe for the development of infection. apart from direct injury, infection leads to several complications that may then have an effect on overall survival including the development of both acute and chronic rejection with eventual graft failure. the immune modulating effects of some pathogens, such as cytomegalovirus (cmv), can also augment the risk of developing other infections further leading to increased morbidity. 1 a thorough and comprehensive screening and management approach must be undertaken to optimize the survival of these patients and minimize the risk of infectious complications. we present a review of the major infectious complications following lt as well as recent recommendations for the evaluation and management of these entities. the respiratory tract is the most common area of infection after lt, and bacterial pneumonia is the most common infectious complication. cmv is the second most common complication, and its occurrence is much higher than in other solid organ recipients. 2 it appears that the critical period for infections after lt is within the first 90 days. in a recent epidemiological study in which 51 ltr were followed for a mean of 38.2 months, 75% of infectious episodes occurred within the first year after transplantation, and nearly half (42%) occurred within the first 3 months. 3 bacterial disease accounted for the largest proportion of infections (48%) followed by viral, fungal and mycobacterial disease (35%, 13% and 4%, respectively). in the early post-lt period (days to 1 month), nosocomial organisms account for the majority of infections. following this period and for the next several months, at a time when immunosuppression is at the highest level, opportunistic organisms such as cmv and fungi account for the majority of infections. in the late post-transplant period, community-acquired bacterial and viral infections develop, although infection with health care-associated organisms remains common (fig. 1) . it is within the first year that infection makes the biggest impact on mortality. according to the registry of the international society for heart and lung transplantation, infection is listed as the leading cause of mortality, accounting for 31% of deaths within the first year after transplant. 5 thereafter, infection is a close second to bronchiolitis obliterans syndrome (bos) as a cause of death. recently, it has been increasingly recognized that infection may both predispose the airways to the development of bos and increase the mortality of those with bos, thus still contributing significantly to this mortality. 6 the lungs are unique organs in that they are constantly exposed to antigens from both the environment (inhaled antigens) and the bloodstream (blood-borne antigens). the upper airways and pulmonary tissue have defence mechanisms composed of physical barriers and cellular components. physical barriers include hairs in the nasal cavity, mucus secretions, cilia and turbulent airflow generated by the nasal cavity that prevent pathogens from reaching the lower airways. despite these barriers, pathogens may still reach and infect the pulmonary tissue. there are several risk factors that make ltr more vulnerable to infection (table 1) . immediately postsurgery, ltr may have disruption of normal physical barriers and are at risk of aspiration and infection (e.g. use of nasogastric and endotracheal tubes). 7, 8 there are also other important changes that happen postsurgery. first, during the surgical procedure of lt, there is a complete disruption of the bronchial circulation, and this may cause a loss of epithelium integrity, ciliary function and mucus production. 9 these effects are transient because of the development of collateral circulation but remain at risk of infection during the initial stages. [9] [10] [11] second, denervation of the allograft may suppress the cough reflex and promote bronchial hyperresponsiveness. 2 third, the lymphatic drainage of the allograft is also severed promoting stasis and oedema in the bronchial tissues impairing normal healing. 2 fourth, stenosis or necrosis may occur at the site of the bronchial anastomosis, which may in turn facilitate colonization and invasion by opportunistic pathogens and decrease the clearance of secretions beyond the anastomosis. 12 at the cellular level, the ltr is vulnerable to infection due to the immunosuppression regimen used to prevent rejection affecting multiple inflammatory cellular lines and cytokines. the regimen consists of induction agents (medications used immediately post-transplant) and maintenance agents for prolonged use. because immunosuppression is needed indefinitely, ltr has a life-long increased risk for opportunistic pathogens to proliferate and cause significant complications. the maintenance immunosuppression regimen consists typically of a calcineurin inhibitor, an antimetabolite and corticosteroids. 13, 14 the calcineurin inhibitors used in lt are cyclosporine a and tacrolimus. cyclosporine a binds to cyclophylin preventing the activation of the nuclear factor of activated t-lymphocytes (t cells) by calcineurin. tacrolimus binds to fk-binding protein 12 inhibiting calcineurin and preventing the activation of the nuclear factor of activated t cells. 13, 15 by reducing the activation of nuclear factor of activated t cells, both drugs reduce the production of interleukin-2 limiting the clonal expansion of activated t cells (fig. 2) . 16 azathioprine and mycophenolate mofetil (mmf) are the commonly used antimetobolites after lt. azathioprine, a derivative of 6-mercaptopurine, inhibits both ribonucleic acid and deoxyribonucleic acid production, reducing the proliferation of both t cells and b-lymphocytes. mmf is a prodrug of mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase (fig. 2 ). this enzyme is responsible for the synthesis of guanine nucleotides, which both t cells and b-lymphocytes are critically dependent of. 17 other maintenance agents that have been used less frequently to maintain immunosuppression include sirolimus and everolimus. sirolimus binds to the fk-binding protein 12 and through the mammalian target of rapamycin pathway prevents the synthesis of deoxyribonucleic acid and proteins by t cells (fig. 2) . 18 through an independent mechanism, sirolimus also affects b-lymphocytes and decreases cytokine and antigen production. 19 everolimus reduces the mammalian target of rapamycin kinase activity, inhibiting the downstream pathways of proliferation and activation of t cells. 20 finally, through the alteration of gene transcription factors, corticosteroids can exert a wide variety of immunosuppressive effects: interruption of antigen presentation, changes in the production of cytokines and alteration in the proliferative responses of various cell lines. 21 the use of induction agents after lt varies among centres. these agents include okt3, antithymocyte globulin (atg), alemtuzumab and basiliximab. okt3 is a murine monoclonal antibody that inactivates the t cell receptor-cd3 complex preventing the activation of circulating t cells with a partial sparing of t regulatory cells. atg is a polyclonal antibody directed against lymphocytes. it depletes circulating lymphocytes through complement-mediated lysis and destruction by the reticuloendothelial system after opsonization. 13 basiliximab is a chimeric monoclonal antibody that targets the a subunit of the interleukin-2 receptor inhibiting the differentiation and proliferation of t cells. 22, 23 alemtuzumab is a murine monoclonal antibody that targets cd52. this receptor is present in macrophages, monocytes, b-lymphocytes and t cells among other inflammatory cells. the binding of cd52 causes complementmediated cytolysis and activation of pathways leading to apoptosis. 13 the use of okt3 is now significantly limited due to an increase risk of infection. [24] [25] [26] [27] for this reason, most centres have elected to use atg, basiliximab or alemtuzumab, in combination with corticosteroids for induction of immunosuppression after lt. 28 evaluation of large series of solid organ recipients has shown that this combination prevents graft rejection and improves survival. 29 atg does not increase the rate of infections in transplant recipients and has been associated with a survival benefit. 30, 31 basiliximab compared with atg does not increase the risk of infection and was safer than okt3 in heart and ltr. 22, 23, 26, 32 alemtuzumab was recently shown to improve survival compared with atg. 33 despite these positive outcomes, the immunosuppression is more profound during induction, and patients should be monitored closely for infection during this period. despite the removal of both lungs during bilateral procedures, residual colonization and/or infection can remain in the thoracic cavity, the bloodstream, the upper airways or the sinuses. those patients with cystic fibrosis (cf) present the highest risk for recipient-harboured infection due to the frequent colonization and infection with multiresistant microorganisms including bacteria (gram-negative rods and gram-positive cocci) and fungi. resistant gramnegative organisms pose perhaps the greatest risk, and some studies suggest an association between pretransplant colonizing organisms from patients with suppurative lung disease and pneumonias following lt. 34 the majority of recent data suggests that patients colonized with multi-drug-resistant pseudomonas appear to have acceptable outcomes, including survival following lt, and should not be excluded on that criterion alone. 35, 36 in contrast, a former subspecies of pseudomonas, now subspeciated as burkholderia cenocepacia due to its unique resistance patterns, can pose significant problems in transplant recipients. there have now been at least nine distinct genotypic variants (genomovars) identified in the burkholderia cenocepacia complex. 37 colonization with burkholderia cenocepacia complex (genomovar 3) can result in significant morbidity and mortality post-transplant and should be considered a strong relative contraindication to lt, 38, 39 although isolated reports of successful outcomes have been reported. 40 in one study of 75 patients, 38 there was a significant difference in 1-year-survival between those patients not infected (92%) and those colonized with a non-burkholderia cenocepacia strain (89%) compared with those colonized with burkholderia cenocepacia (29%). similar results of variable survival rates based on burkholderia cenocepacia species have been found in other studies. 37, 39 because of these overwhelming data, the majority of transplant centres will not transplant colonized or infected patients with this organism. when evaluating the potential lt donor, routine screening is done to prevent transmission of donorharboured infection to the recipient. 41 donor screening includes routine serology for viral infection including cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, among others. in addition, the potential donor lungs are evaluated radiographically and bronchoscopically. despite these measures, infection may still occur. to potentially pre-empt the development of donortransmitted infection at the time of the transplant procedure, a culture swab or wash, or a portion of the donor bronchus is sent for culture. in contrast with some older studies, 42, 43 more recent data suggest that recovery of an organism from the donor lung, a schematic overview of the mechanisms of action of medications used for immunosuppression. il-2 is required for the activation of the mtor pathway and progression of the t cell cycle. both csa and tacrolimus reduce the activation of nfat, which in turn results in a decreased production of il-2. basiliximab is a monoclonal antibody that inhibits the il-2 receptor. sirolimus and everolimus inhibit the mtor pathway through inhibition of specific enzymes. alemtuzumab targets protein cd52 causing t cell dysfunction. both mmf and aza disrupt key elements of the deoxyribonucleic acid synthesis affecting the progression of the t cell cycle. aza, azathioprine; csa, cyclosporine a; fkbp12, fk-binding protein 12; impdh,inosine-50-monophosphate dehydrogenase; il-2, interleukin-2; il-2r, il-2 receptor; mmf, mycophenolate mofetil; mtor, mammalian target of rapamycin; nfat, nuclear factor of activated t-lymphocytes. including a positive gram stain, or subsequent growth in culture does not always translate into infection and/or poor outcomes in the recipient. 34, 44, 45 in one study of 80 ltr, the investigators noted that organisms were grown from 57% or 89% of donors for a total number of isolates of 149. 44 of these, most isolates were staphylococci or streptococci. post-transplant pneumonias were found in 41% of recipients in this study; however, pseudomonas, and not gram-positive organisms, was the most prevalent causative organism. the results of this study and others 45 suggest that the presence of organisms in the donor does not necessarily predict post-transplant pneumonia, and perhaps this donor criterion should be re-evaluated. despite these suggestions and because empirical bacterial prophylaxis was used in the majority of these studies, the general practice is to routinely initiate prophylactic, broad-spectrum antibiotics (regimens are discussed later) and then narrow the antibiotic therapy based on donor isolates. 41 any patient with suppurative lung disease, such as cf or bronchiectasis, being considered for lt will receive a bilateral procedure with attempts at avoiding infection from a remaining native lung. however, in those diagnoses where a single lt may be performed, such as chronic obstructive pulmonary disease or interstitial lung disease, the native lung may harbour infectious organisms that can infect the new graft, particularly when the patient is subjected to immunosuppression. alternatively, the native lung can develop severe infection leading to sepsis and further compromise. although attempts at avoiding this risk are undertaken by routine pretransplant screening, examples of infection that can be harboured in the native lung include bacteria, fungi (perhaps contained in a mycetoma) or non-tuberculous mycobacteria (ntm). 46 as part of the initial pretransplant evaluation, all potential transplant recipients should undergo careful screening for infection. although there may be some variation between transplant centres, routine screening includes serological measurement for cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, and screening for latent tuberculous infection. the results obtained from this screening are used to assess the patient's overall candidacy for lt (e.g. human immunodeficiency virus is generally an exclusion) and also to stratify the patient for screening and prophylaxis in the post-lt period (e.g. cmv and epstein-barr virus). recommendations for recipient and donor presolid organ transplant screening are published from the american society of transplantation. 41 pneumonias comprise the most common cause of infection following lt, and bacterial pathogens remain the most common cause of all pneumonias. 34, 47 in a multicentre, prospective study from spain, with a median follow-up of 180 days, 85 episodes of pneumonia were documented in 236 ltr for an incidence of 72 episodes/100 lt years. 47 of these, bacteria were the most common pathogen accounting for 82.7% of the pneumonias. bacterial pneumonia is most common in the early post-transplant period (1-30 days) usually due to infection with health care-associated and nosocomial organisms (fig. 1 ). in the spanish study, 40 of 85 of pneumonias (44%) occurred in the first 30 days following transplant. nearly 3/4 of all bacterial pneumonias (72%) were due to gram-negative organisms-most commonly pseudomonas (incidence 118.6 episodes per 1000 ltr/year). staphylococcus aureus and acinetobacter infections were the second most common bacterial isolates (each with an incidence of 67.8 episodes/1000 ltr/year). the median time to development of gram-negative pneumonia was 31 days with a range of 3-394 days. grampositive cocci-related pneumonias also occurred in the early post-transplant period at a median of 35.5 days (range 2-486 days) post-transplant. other bacterial isolates from this and other studies span the spectrum of health care-acquired infectious organisms. similarly, p. aeruginosa was found to be the most common isolate accounting for 33.3%, staphylococcus aureus comprised 26.8%, and aspergillus 16%. 34 pneumonia is also seen in the late post-transplant period. throughout the lifespan of the ltr, ongoing contact with hospital settings, both outpatient and inpatient, and frequent antibiotic exposure commonly result in infections with health careassociated, often resistant, pathogens. communityacquired pneumonias can also develop in the late post-transplant period. 48 in a single-centre study, 14 out of 220 ltr (6.4%) developed invasive pneumococcal infection (pneumonia and/or sepsis) at a median of 1.3 years after transplantation (incidence rate: 22.7 cases per 1000 person-years). routine vaccination for pneumococcus with the pneumococcal polysaccharide vaccine is recommended both before and every 5 years following lt. 41 in general, the approach to suspected pneumonia at any time period post-transplant includes sputum, blood cultures and often bronchoscopy with bron-choalveolar lavage (bal), sterile brush and sometimes biopsy. the role of new biomarkers such as procalcitonin for diagnosis or follow-up has not been well established in the ltr. due to the high incidence of early post-transplant pneumonia, whether derived from the recipient, donor or nosocomially acquired, broad-spectrum postoperative prophylaxis is routinely used. prophylaxis in the post-transplant period varies by centre but typically includes a third generation cephalosporin and vancomycin and is then tailored to the results of donor and recipient cultures, or as clinically indicated for 7-10 days. prophylactic antibiotic treatment should be extended to 14 days for known pretransplant recipient colonization. for specific prophylactic regimens for viral and fungal pathogens, see later. treatment of bacterial pneumonia includes standard regimens as outlined by the american thoracic society and infectious disease society of america treatment for health care-acquired pneumonia. 49 in the setting of known prior colonization or infection, initial antibiotic selection may be based on prior culture and sensitivity results. typical antibiotics used should include coverage for gram-negative (including pseudomonas) and gram-positive (including staphylococcus aureus) pathogens. in general, 8-14 days of therapy is recommended. in the case of resistant organisms, inhaled aminoglycosides may also be added to the treatment regimen. pneumonia has significant impact on overall posttransplant survival and the eventual complication of chronic rejection. in the spanish study, attributable 1-year survival was reduced in those patients developing pneumonia of any aetiology (29.5% mortality) versus those without pneumonia (14% mortality), although bacterial pneumonia alone was not separated out in this analysis. these authors also found that the probability of survival during the first year of follow-up was significantly higher in the multivariate analysis in lt recipients who did not have a pneumonia episode compared with those that had at least one episode of pneumonia. 47 in the bonde et al. study, pneumonia was found to be an independent predictor of overall mortality. 44 viral infection after lt is common and classified into disease caused by cmv or caused by other community-acquired respiratory viruses (carv). a recent study showed that a viral pathogen was responsible for 25 of 71 infectious episodes in a cohort of ltr, with cmv accounting for 68% of those cases. additionally, the majority of cmv episodes occurred within the first 3 months following lt, while the majority of the later infections were due to influenza and occurred after 1 year (fig. 1) . 3 among the opportunistic infections following lt, cmv is the most prevalent and most important despite significant advances in both diagnosis and management. as well as contributing directly to both morbidity and mortality, mounting evidence suggests a relationship between cmv pneumonitis and chronic rejection in the form of bos and decreased survival despite treatment. 50 cmv seropositivity can range from 30% to 97% in the general population, and after infection, the patient will harbour the virus for life. of all solid organ transplants, ltr has the highest risk of developing cmv disease. 51 the incidence of cmv infection has been reported to range from 30% to 86% in post-ltr, with a mortality of 2-12%. 52 this increased incidence is thought to be due partly to the high viral load of cmv transmitted in the lymphatics of the lung compared with other solid organs, as well as the high level of immunosuppression required for lung allograft. the most important risk factor for the development of cmv infection is the donor-positive/recipientnegative serostatus of a transplant patient, as these patients will lack immunity to cmv. the lowest risk occurs in donor-negative/recipient-negative patients. 51 other important risk factors include type and intensity of both induction and maintenance immunosuppression, concurrent infections, rejection and host factors such as age or comorbities. 51, 52 there is almost a symbiotic relationship between rejection and cmv infection. both of these individual processes induce a cytokine cascade that in essence promotes the development of the other. tumour necrosis factor-alpha, a key signal in the reactivation of cmv from latency, is released during allograft rejection, thereby facilitating the onset of viral replication and subsequent infection. conversely, infection of the vascular endothelium and smooth muscle by cmv leads to an upregulation of adhesion molecules promoting an increase in the quantity of inflammatory cells in the graft and subsequent development of rejection. additionally, molecular mimicry and the production of anti-endothelial antibodies with cmv may also play a role in the development of rejection. 52 cmv serology of both donor and recipient must be checked prior to transplant. 53 there is an important distinction between cmv infection and disease. infection is defined as 'cmv replication regardless of symptoms', while disease is defined as 'evidence of cmv infection with attributable symptoms', such as 'a viral syndrome with fever and/or malaise, leukopenia, thrombocytopenia or as tissue invasive disease'. 51, 54 recent technologies have effected a shift in the diagnosis of cmv infection and disease. the previous method of diagnosis, pp65 antigen detection, has been replaced by quantitative nucleic acid-based amplification testing via polymerase chain reaction (pcr) for the recognition of viraemia by most centres, with 85% of institutions using this method for monitoring and diagnosis. 55 there are no universally accepted viral load cut-offs for positive and negative results, and that reported values may be dissimilar between different laboratories. despite this, current guidelines on the management of cmv in solid organ transplant patients do not clearly favour one test over the other and cite both as acceptable options for diagnosis. additionally, viral culture of blood or urine has a limited role for diagnosis and is not routinely recommended. 53 most recently, tests for cell-mediated immunity against cmv have shown promise for predicting risk of developing disease. lisboa and colleagues demonstrated that cell-mediated immunity to cmv, as shown by a cd8+ t cell response assay, was associated with decreased risk of developing disease in patients with detectable low-level viraemia. twenty four of 26 patients (92.3%) with a positive interferon-gamma release assay were able to clear their viraemia without disease compared with 5 of 11 (45.5%) in patients with a negative cell-mediated immunity at onset (p = 0.004). 56 in a similar study, the same group was able to show that a negative assay was associated with a higher chance of developing late-onset cmv after prophylaxis. in their study, cmv disease occurred in 2/38 (5.3%) patients with a detectable interferongamma response versus 16/70 (22.9%) patients with a negative response (p = 0.038). 57 there are two accepted approaches to the prevention of disease from cmv, universal prophylaxis and preemptive therapy, and although there are no randomized trials comparing one strategy versus the other in ltr, most centres favour the former or may sometimes employ both. 55 the first, universal prophylaxis, involves administration of antivirals to all transplant patients deemed to be at high risk by serostatus. the second, pre-emptive therapy, is comprised of monitoring at-risk patients for viral replication and administering antivirals at a predetermined level of replication in the hopes of treating patients prior to the onset of disease. a cochrane review comparing prophylaxis in different groups of solid organ transplant patients with antivirals versus placebo or no treatment showed a significant reduction in disease (relative risk 0.42), infection (relative risk 0.61), mortality from cmv disease (relative risk 0.26) and allcause mortality (relative risk 0.63). interestingly, the review also found a decrease in the risk of developing herpes-simplex virus, varicella-zoster virus and bacterial infections. 58 prophylaxis may not only be beneficial in decreasing direct morbidity and mortality from cmv disease but may also have secondary effects by decreasing the morbidity and mortality of both acute and chronic rejection. the cochrane review mentioned earlier failed to show a difference in acute rejection episodes, but other small studies have shown statistically significant differences in ltr specifically and it is generally believed that prevention of cmv decreases the risk for acute rejection. [58] [59] [60] the data for bos are more encouraging. a recent study by chmiel and colleagues was able to show a 23% absolute risk reduction of developing bos in a group of ltr on cmv prophylaxis as compared with a historical cohort that was not prophylaxed and a 35% absolute risk reduction compared with data in the literature (p = 0.002). 1 most centres provide prophylaxis for a period of 3-6 months after transplantation; however, the optimal duration of prophylaxis has not been well established and is currently under debate. 55 the guidelines recommend a minimum of 6 months for ltr. 53 recent data suggest that this window of prophylaxis should possibly be extended, especially for donor-positive/ recipient-negative patients. palmer and colleagues report the first randomized, placebo-controlled trial showing a decrease in the risk of cmv disease with extended prophylaxis. in this study, 136 ltr who completed 3 months of valganciclovir prophylaxis were randomized to an additional 9 months of valganciclovir versus placebo. the risk of cmv disease was reduced (32% vs 4%; p < 0.001) in the extendedcourse group versus the short-course group. there were also statistically significant reductions in cmv infection (64% vs 10%; p < 0.001) and disease severity as measured by viral load with extended treatment. acute rejection episodes, opportunistic infections, adverse events and cmv ul97 ganciclovir-resistance mutations were similar between both groups. 61 the international consensus guidelines list valganciclovir and ganciclovir (oral or intravenous (iv)) as the antivirals of choice for the prevention of cmv disease and state that cmv immunoglobulin may also be used in combination with these two, but there are limited data to support its use. 53 although foscarnet was commonly used in the past for cmv disease, the significant risk of nephrotoxicity with concomitant calcineurin-inhibitor use has made it fall out of favour for the relatively safer agents ganciclovir and valganciclovir. 55 and, although the recommendation for treatment of severe disease is still iv ganciclovir, the results of the valcyte in cmv disease treatment of solid organ recipients trial have made valganciclovir a viable choice in the treatment of less severe cmv. 53 the in cmv disease treatment of solid organ recipients trial randomized 321 solid organ transplant recipients with non-life-threatening cmv disease to either oral valganciclovir or iv ganciclovir. valganciclovir demonstrated non-inferiority in regard to clinical resolution of disease as well as eradication of viraemia in both the intent-to-treat and the per-protocol arms of the study. 62 the current guidelines recommend oral valganciclovir at twice-daily dosing or iv ganciclovir for the treatment of nonsevere cmv disease. as there are no efficacy data for valganciclovir in severe or life-threatening disease, iv ganciclovir is still the 'gold standard' for those patients. in both groups, serial monitoring of viraemia should occur optimally at 1-week intervals, and treatment should be continued for a minimum of 2 weeks and until viral eradication has been documented with two consecutive tests. the use of secondary prophylaxis is generally recommended for 1-3 months after treatment of disease. 53 infection with a carv is common after lt, and with the development of new diagnostic techniques, the incidence quoted in older literature is likely underestimated. a study of ltr undergoing serial surveillance and diagnostic bal over a 3-year period showed that a respiratory virus was isolated in 51.6% of patients on at least one bal sample. rhinovirus was the most common pathogen isolated, followed by parainfluenza, coronavirus, influenza, metapneumovirus and respiratory syncytial virus (rsv). 63, 64 carv is being increasingly recognized as contributors to significant morbidity in immunocompromised hosts and can cause severe and life-threatening pneumonitis. additionally, there appears to be evidence that infection with these organisms can also lead to a decrease in graft survival. a retrospective cohort study of 259 ltr followed over 5 years showed a significantly increased risk of developing bos or death from bos in the group that was diagnosed with a carv infection. 65 given the paucity of effective antiviral treatment for most of these viruses, early diagnosis is essential for both treatment and to minimize spread among other immunocompromised patients. with the exception of influenza and rsv, for which treatments exist, supportive care and a reduction in immunosuppression remain the cornerstones of care for the treatment of carv. a complete listing of all the viruses that commonly affect ltr would be beyond the scope of this article so we will focus on those that have the most clinical bearing, namely influenza, rsv, human metapneumovirus and parainfluenza. as it typically does not cause respiratory tract disease, we will not discuss epstein-barr virus, except to mention its known association with post-transplant lymphoproliferative disorder after lt. infection of normal hosts with influenza most commonly causes a self-limited disease with upper respiratory symptoms, myalgias and fever; however, infection in ltr appears to be associated with increased risk of lower respiratory tract involvement by either a primary viral or a concomitant bacterial superinfection. this was illustrated in a small series of ltr admitted for influenza where all appeared to have pulmonary parenchymal involvement on imaging and by bal as well as in another series by vilchez and colleagues, where 7 of 15 patients with influenza were found to have pulmonary infiltrates, 5 of which were attributed to a primary viral pneumonia after bal. 66, 67 novel h1n1 influenza appears to have similar clinical features, although there appears to be an increased rate of gastrointestinal symptoms such as nausea and diarrhoea; which may be prominent. 68 due to the increased severity of disease, all ltr and their household contacts should receive annual influenza vaccination for prevention of disease. 69 diagnosis is essential, and efforts should be made to establish the type, as specific therapy will depend on resistance patterns. 69 diagnosis of seasonal influenza is made by rapid antigen detection of nasopharyngeal swabs, but this method appears to be unsatisfactory for detection of novel h1n1 and molecular real-time pcr methods are currently approved for use when swine flu is suspected. 70 in addition to supportive care and isolation, treatment involves the use of the antiviral agents amantadine and rimantidine for susceptible influenza a strains, and zanamavir and oseltamivir for both influenza a and b strains. due to the variation in circulating strains from year to year, it is important to stay abreast of the current recommendations from the centers for disease control and prevention 71 for appropriate treatment. 72 in addition, given the prolonged viral shedding, the typical treatment course of 5 days may be insufficient in ltr, and prolonged therapy may be required. some experts advocate treating influenza even if symptom onset is greater than 48 h and treating until viral replication ceases. 73 treatment of novel h1n1 is limited by the resistance of the strain to the m2 inhibitors: amandatine and rimantidine. as such, current guidelines recommend treatment with oseltamivir or perhaps even zanamavir if resistance is suspected to this agent. iv or higher dose therapy is recommended for critically ill patients, and immunosuppression should be decreased. 63, 64 rsv by the age of 2, virtually, all children have been infected with rsv, although reinfection can occur throughout life, and early acquisition after transplant or with augmented immunosuppression is a risk factor for severe disease. 72 as with influenza, infection can vary from a self-limited upper respiratory illness to severe pneumonia and occurs through inhalation of infectious droplets and contact with fomites, making isolation precautions paramount for prevention. there are currently no available vaccines for rsv and no recommended therapies for prevention. due to a lack of data for effective antiviral treatment, the only universally accepted recommendations for therapy are supportive care and a reduction of immunosuppression. 72 ribavirin, which has shown in vitro activity against rsv, is approved for treatment of lower tract disease by showing benefit in stem cell recipients. 74, 75 there are otherwise no controlled studies showing efficacy with the use of inhaled ribavirin in transplant patients. despite this, inhaled ribavirin remains the most commonly used treatment for rsv with one report showing a multidrug regimen of ribavirin, steroids, rsv-iv immunoglobulin and palivizumab to be safe, effective and associated with stability of lung function. 76 two small case series have shown promise for parenteral and oral ribavirin in ltr. 77, 78 an optimal treatment strategy for disease due to rsv is yet to be determined, and further studies are needed to better delineate effective agents that can safely be used in the lt setting. like rsv, human metapneumovirus and parainfluenza are members of the paramyxovirus family and present similarly to rsv. although typically they are milder than rsv, they have been shown to cause severe disease and have also been associated with both acute rejection and bos. 67, 79, 80 real-time pcr is the diagnostic modality of choice, and a diagnosis should be pursued, as clinical features alone are not specific enough to distinguish between the carv. supportive care remains the mainstay of treatment although inhaled ribavirin appears to be increasingly used for the treatment of these pathogens in patients with lower respiratory tract involvement despite a lack of controlled trials. furthermore, some experts also consider the use of iv immunoglobulin with significant disease for both parainfluenza and human metapneumovirus. 72, 80 fungal infections are a common complication after lt with an estimated incidence of 15-35% and an overall mortality of 80%. 81 complications at the site of the anastomosis (i.e. stenosis or necrosis) create the ideal environment for these infections to thrive. other risk factors include the immunomodulatory effect of coexistent infections (i.e. viral) and neutropenia. [82] [83] [84] as previously mentioned, transmission of infection from donor to host after lt can occur, or the native lung may serve as a reservoir of fungal organisms during single lt. 85 this is particularly important in chronic obstructive pulmonary disease patients in whom the lung surfaces are irregular and may have colonized bullae. 84 pretransplant fungal colonization is common, especially in patients with cf and chronic obstructive pulmonary disease, and it has been associated with post-transplant fungal infection and bos, 86 although not all colonized patients develop active/invasive infection. 83 the most common fungal pathogens in ltr are candida and aspergillus species, while zygomycetes, scedosporium, fusarium, cryptococcus species, histoplasmosis and coccidiomycosis occur less commonly. in general, these infections are more prevalent during the first few months after transplantation and, in some cases such as with cryptococcus species, histoplasmosis or coccidiomyocosis, can present as a reac-tivation of a latent infection. fungal infections can manifest as invasive disease with a reported 1-year cumulative incidence of 8.6% in ltr. 87 similarly, disseminated disease, post-transplant empyema, and airway and anastomotic infection have been reported. aspergillus species are the most common cause of invasive fungal infection after lt with an incidence of 32%. 84 more than half the cases occur within the first six months following lt, 84 (fig. 1 ) and more often involve ltr than other solid organ recipients. 88 several species have been described as pathogenic: aspergillus terreus, aspergillus flavus, aspergillus fumigatus and aspergillus niger. among these species, aspergillus fumigatus remains the most common cause of invasive disease. 89 the majority of aspergillus isolates in sputum or bal represent colonization (23%), and only a fraction of these will develop invasive disease (<10%), which carries a high mortality. 69, 90, 91 in ltr, the risk of invasive pulmonary aspergillosis rises with airway colonization by aspergillus species. 84, 89, 92 colonization is found in up to 50% of patients with cf. despite higher colonization compared with other populations, these patients have lower risk of invasive aspergillosis, but a higher risk for aspergillus tracheobronchitis. 93 in addition to colonization, airway ischaemia and bos have also been implicated as risk factors for invasive aspergillosis. 84, 89, 92 disseminated disease has been reported with an incidence of 22%, occurring as reactivation from an occult focus and/or as a new post-transplant infection. 84 other less common manifestations, such as mediastinal masses, skin, softtissue, sinus, orbit, central nervous system, sternal wound and chest wall infections, have also been described. 89, 91 diagnosis there are limited data on the role of minimally invasive tests such galactomannan, pcr and 1,3-b-dglucan assay for the diagnosis of invasive aspergillosis in ltr. 94,95 1,3-b-d-glucan, a cell component of all fungi, has been used in the diagnosis of multiple invasive fungal infections, but unfortunately, the role in ltr has limitations. 96 diagnosis of invasive aspergillosis may require aggressive procedures (i.e. biopsy) to verify tissue involvement; however, this is not always possible, and often, the diagnosis is reached on evaluation of computed tomography chest findings and fungal staining/culture from bronchoscopy (i.e. bal). the radiological findings of invasive aspergillosis include consolidations, nodules, cavitary lesions and mass-like opacities, often with a 'halo sign'. 84 in cases where the diagnosis is not possible with a less invasive approach, a biopsy with fungal stain/culture and histopathology may be required. once the diagnosis of invasive pulmonary aspergillosis is made, computed tomography or magnetic resonance of the central nervous system is suggested to rule out disseminated disease. over the years, the use of antifungal prophylaxis has decreased the overall risk of aspergillosis. despite this, the risk of late infection after discontinuation of prophylaxis or even while using it is still present. 97 the treatment of pretransplant colonization has not been shown to reduce the incidence of post-transplant aspergillosis, but invasive disease in the pretransplant setting should be treated. 90 recent data has shown the superiority of voriconazole compared with amphotericin b deoxycholate in patients with invasive pulmonary aspergillosis, but solid organ transplant patients were poorly represented in the study. 98 a major concern with the use of voriconazole in ltr is the interaction with most of the immunosuppressants used in this population. tacrolimus, sirolimus and cyclosporine can potentially increase the serum concentrations of voriconazole. for this reason, close monitoring of drug levels is needed. other options for the treatment of invasive aspergillosis are posaconazole and itraconazole, but their roles as first-line agents are not well established. the echinocandins (caspofungin, micafungin and anidulafungin) have shown some in vitro activity against aspergillus species, but their utility as firstline antifungals for this infection has not been studied either. the evidence for combined therapy with two or more agents as initial therapy is limited and not recommended. despite several alternatives, voriconazole remains the standard therapy for invasive aspergillosis along with reduction of immunosuppression. 99 voriconazole levels should be monitored carefully, especially in cf patients where serum concentrations can be variable. 99, 100 in general, target trough levels should range between 1 and 5 mg/ml. duration is typically recommended for a minimum of 12 months and depends on clinical and radiographical improvement. finally, surgical resection might be indicated when there is progression of disease despite optimal antifungal therapy, life-threatening haemoptysis, sinus infection or lesions in the proximity of great vessels, pericardium or in the brain. 82 severe candidal infections can appear within weeks to months after transplant, especially in the presence of heavier donor or recipient colonization. 91 typically candida infections occur within the first 30 days after lt and appear to be the second most common cause of invasive fungal infection in ltr. 69 candidaemia usually occurs during the first 4 weeks and is often related to the intensive care unit stay and the surgical procedure; however, parenchymal lung infection is rare. 101 mortality for invasive candidal infections, excluding anastomotic infections, has been estimated at more than 50%. 102 cultures are essential for the diagnosis of candidal infection in ltr. identification of species and susceptibilities need to be obtained as intrinsic resistance and dose-dependent susceptibility has been reported in different candida species. 103 other methods such as b-d-glucan have not reached significant accuracy for clinical use, 104 while others such as pcr are still experimental. candida species are commonly found in the oropharynx and can potentially colonize the airway. their presence in respiratory secretions may make it difficult to differentiate between invasive infection and colonization. invasive lung infection with candida is very infrequent even in the lt recipient colonized with candida. 97 clinical suspicion, culture results and direct bronchoscopic findings should guide any decision for treatment of candidal infections. echinocandins and liposomal amphotericin b are the first-line agents for empirical therapy of suspected candidal infection. 69 this is especially true in ltr who are at risk of developing severe candidal disease. fluconazole has been put forward as an empirical agent as well but is frequently reserved for patients with mild-to-moderate disease, nonneutropenic and at low risk for candida glabrata and candida krusei, for which it has less activity. empirical therapy should then be adjusted based on susceptibilities. for candida albicans infections, fluconazole and echinocandins have been effective, but in widespread disease, amphotericin b might be considered. finally, the duration of therapy varies among patients and with the degree and severity of infection. in candidaemia, treatment can extend up to 2 weeks but may be even longer in cases of more invasive disease. 69 histoplasmosis, coccidioidomycosis and rarely, blastomycosis are endemic mycoses that can potentially cause infection in transplant recipients. when present in this population, pulmonary and disseminated disease can occur with a high mortality. 105 these are especially important in endemic areas of the united states such as the midwest for histoplasmosis and the southwest for coccidiomycosis. 106 histoplasmosis can present in the early or late posttransplant period as a consequence of reactivation of a latent infection, new exposure or donor-derived infection. 106 the diagnosis can be delayed, but in ltr, urinary antigen appears to be a better diagnostic tool than the fungal antibody serologies. 106 the presence of fever without a clear source should raise clinical suspicion for disseminated histoplasmosis in any transplant patient, especially when pancytopenia and absence of pulmonary manifestations are present. in patients whose explanted lung is found to have histoplasmosis, antifungal prophylaxis after transplant seems effective at preventing reactivation of this infection. 106 there is no clear consensus about the duration of prophylaxis, and 18 months has been reported to be effective. 106 coccidioidomycosis is typically acquired when patients are exposed to the desert soil of the southwestern united states and northern mexico. the most common mechanism of infection in lt recipients is reactivation, but donor-derived transmission has also been reported. 107 patients in whom there is evidence of prior coccidioidomycosis, either radiographically or serologically, may require lifelong antifungal prophylaxis after transplant. 91 cryptococcus infections can present in solid organ transplant recipients as a pulmonary or extrapulmonary process. 108 the incidence of cryptococcus infection in ltr has been estimated around 2% and has been commonly associated with exposure to pigeons and other birds. 90 interestingly, ltr may be less likely to have a positive cryptococcal antigen test in the setting of isolated pulmonary cryptococcosis. 38, 108 an immunosuppressive regimen containing a calcineurin inhibitor has been associated with decreased mortality possibly due to synergistic effects between calcineurin inhibitors and antifungal agents use to treat cryptococcus. 109 however, a recent study has reported the occurrence of an immune reconstitution syndrome-like illness in some transplant patients after the initiation of antifungal therapy for cryptococcal infection. 110 zygomycotic infections appear to be escalating in frequency in immunosuppressed patients, and this trend has been partially attributed to the increasing use of voriconazole for therapy and prophylaxis. 111 this infection is characterized by vascular invasion of affected tissues with subsequent infarction and necrosis. in ltr, it can manifest as bronchial anastomotic or parenchymal infection with a mortality of 87% in the latter. 112, 113 its management includes the combination of surgical debridement and antifungal agents. in the united states, 80% of transplant centres use antifungal prophylaxis, 114 and approximately 81% perform pretransplant surveillance for fungal colonization. 115 despite this, there is still no general consensus regarding the most appropriate prophylactic strategy in the peritransplant window. although there are no randomized trials evaluating their efficacy, several antifungal agents have been used for prophylaxis in ltr. for universal prophylaxis, voriconazole, itraconazole and amphotericin b are commonly used, while targeted prophylaxis with fluconazole (candida), voriconazole and itraconazole (aspergillus) are used based on the results of surveillance bronchoscopy. 114 in general, the choice for antifungal prophylaxis depends, in part, on the presence of specific risk factors such as colonization with aspergillus, presence of airway stents or ischaemia, single lung transplantation, cmv infection, hypogammaglobulinaemia or treatment of acute rejection. 69 despite a lack of controlled trials, several studies suggest potential prevention of invasive aspergillosis with the use of either compound of amphotericin b. 116, 117 inhaled amphotericin b has lower systemic toxicity, better delivery to the site of fungal exposure and a lower likelihood of resistance when compared with systemic antifungal therapy. 116, 118, 119 the data regarding voriconazole for prophylaxis in ltr is promising, especially given the excellent bioavailability, broad antifungal coverage and good drug levels achieved in lung tissue. 120, 121 unfortunately, the numerous drug interactions with some of the immunosuppressants, and its potential adverse effects may preclude its use as a first-line prophylactic agent. itraconazole has clinical effectiveness similar to the combination of voriconazole and inhaled amphotericin b and may have lower hepatotoxicity when compared with voriconazole. 114 duration of antifungal prophylaxis varies from centre to centre. the use of voriconazole or itraconazole for 3-6 months with or without amphotericin b has been shown to decrease the incidence of aspergillus infection after transplantation. 88 the use of inhaled amphotericin b is typically for 2 weeks or is discontinued at the moment of discharge. in cases where pretransplant fungal colonization is present, patients may be treated for several weeks before lt and continued for up to 3 months after transplantation. because ltr is at high risk for fungal infections, antifungal prophylaxis should be started in most patients after lt with careful consideration of sideeffects and interactions to improve outcomes and be guided by cultures from donor, graft and recipient. mycobacterial infection after lt is rare. previously, most of these infections were secondary to mycobacterium tuberculosis. 122 more recently, data have shown an increase in the incidence of ntm, particularly mycobacterium abscessus, ranging between 3% and 9%. 123, 124 chalermskulrat et al., reported higher isolation of ntm in end-stage cf patients undergoing pre-lt evaluation (19.7%) than in post-lt cf patients (13.7%). 124 colonization, especially when m. abscessus was isolated, was associated with an increased risk for invasive mycobacterial infection in cf patients. 124 over the last 10 years, multiple cases of m. abcessus in lt recipients have been reported with pleuropulmonary and disseminated disease. [125] [126] [127] in addition, there is an increase in both mortality and disseminated disease associated with m. abcessus in solid organ transplant recipients. 128 on the other hand, m. avium complex and other ntm infections are less common, and their impact on morbidity and mortality is less severe compared with m. abcessus. 129 if during the pretransplant evaluation, the clinical presentation and radiographical findings are suggestive of ntm infection, diagnostic testing and therapy should be considered before transplantation. in the cf population, the presence of ntm should not preclude lt, but careful monitoring for recurrence after transplant should be performed. 124 the diagnostic criteria of the american thoracic society and infectious disease society of america apply to pre-and post-ltr (symptoms, radiological findings and microbiology). 130 similarly, the antimicrobial therapy recommended in the ntm guidelines is applicable to ltr. 130 therapy for mycobacterial infection in the immunosuppressed patient can be problematic particularly due to drug interactions and increased toxicity. nevertheless, these infections can be controlled, and some patients achieve an appropriate response and cure. anastomotic tracheobronchitis is a unique form of pulmonary infection 131 that usually develops in the first 6 weeks to 3 months following lt. during the transplant procedure, the bronchial circulation is not reanastomosed, and thus, the bronchial anastomosis must receive collateral blood flow from the pulmonary circulation, is subject to ischaemia and may be susceptible to infection. this diagnosis is easily confirmed with bronchoscopic examination revealing purulence, ulcerations, pseudomembranes, necrotic material, dehiscence and sometimes narrowing at the site of the anastomoses, and histological and culture results. the organisms most commonly causing tracheobronchitis in this setting are bacteria-(pseudomonas, staphylococcus) and fungi aspergillus (an incidence of 32% and 20%, respectively) and candida. 84, 132, 133 treatment includes appropriate antibacterial and/or antifungal antimicrobials. the treatment of airway anastomotic infections with fungi is with a combination of both systemic and sometimes inhaled antifungal agents. 134, 135 for aspergillosis, the combination of voriconazole and nebulized amphotericin b along with reduction of immunosuppression has been advocated. 99, 134 duration of therapy for tracheobronchitis is usually determined by resolution under bronchoscopic surveillance. late sequelae may include stenosis and or stricture requiring intervention with balloon dilation or occasionally endobronchial stent placement. a study demonstrated a decrease in 5-year survival in single ltr who developed bronchial anastomosis fungal infections. 132 other types of bacterial infection described in ltr include those of the pleural space, blood stream and wounds, with organisms often isolated in the nosocomial setting, and clostridium difficile. pneumocystis jiroveci pneumonia (pjp) occurs exclusively in immunosuppressed states. the risk of infection is higher during the first 6 months after lt due to the degree of immunosuppression during this period. 136 cmv infection is also an independent risk factor for pjp. 137 despite this, pjp remains a rare complication after lt. 138 the low rate of infection is due to the use of prophylaxis with trimethoprimsulfamethoxazole as a first-line agent, and dapsone, pentamidine and atovaquone as alternatives. 139, 140 trimethoprim-sulfamethoxazole has been shown to have better tolerance, potentially treat a wider range of infections, and has fewer side-effects. 139 there is controversy regarding the duration of prophylaxis after transplant. a study revealed that the rate of pjp did not decline after 1 year of transplantation, suggesting that prophylaxis should be continued beyond this period. 141 ltr should receive at least 6 months of prophylaxis post-transplant, and if tolerated, adequately, it should be continued indefinitely. in those patients in whom prophylaxis has been discontinued, it should be resumed if the patient develops acute or chronic rejection requiring augmented immunosuppression. the standard therapy for pjp is trimethoprim-sulfamethoxazole in combination with corticosteroids. as previously noted, mmf is used frequently as part of the immunosuppression regimen after lt. interestingly, this medication has shown antimicrobial properties against several pathogens including pneumocysitis spp. 142, 143 in three comparative studies, none of a total of 1152 transplant patients who received mmf developed pjp compared with an infection rate of 1.8% in a similar group that did not receive mmf. [144] [145] [146] the mechanism for these effects remains unknown, but it is likely that mmf may benefit ltr by two different mechanisms. in lt, nocardia remains an important pathogen with a frequency of 0.6-2.1% and a directly attributable mortality of up to 30%. 147 it is important to note that some of these patients (60-100%) were on treatment with prophylactic trimethoprim-sulfamethoxazole, a medication to which nocardia is classically susceptible to, underscoring the resistance of some strains to prophylaxis therapy. 147 the treatment for nocardia is trimethoprim-sulfamethoxazole, but resistance has been documented and other alternatives have been used successfully: imipenem, amikacin, third generation cephalosporins, minocycline, moxifloxacin, linezolid and dapsone. 148 despite the relatively low frequency of nocardia in lt, because of the high risk of mortality and the ability to mimic other infections, clinicians must have awareness of this pathogen to improve an early diagnosis to initiate appropriate therapy. chronic rejection following lt is manifested pathologically by bronchiolitis obliterans and clinically by worsening obstructive dysfunction on pulmonary function, the bos. bos is the rate-limiting factor in long-term survival following lt, and up to 50% of ltr will develop bos. 5, 149 the aetiology remains unclear, although acute rejection is one of the identified risk factors. emerging evidence continues to point towards infectious aetiologies as important factors in the pathogenesis of bos. several different viral, bacterial and fungal pathogens have been implicated in this process. 150, 151 these findings are critical regarding the understanding the mechanisms of rejection and possible therapies to prevent it. cmv was the first pathogen linked to the development of bos. cmv pneumonitis is associated not only with bos but also with decreased survival despite treatment. 50 furthermore, there has been an absolute risk reduction in the development of bos with the use of cmv prophylaxis, supporting the evidence that this virus may play an important role in the pathogenesis of rejection. 1 carv infections, including rsv, human metapneumovirus and parainfluenza virus, were also identified as a significant risk factor for developing bos. 65, 67, 79, 80 bacterial colonization and infection may be a contributing risk factor to the development of bos. [152] [153] [154] [155] because macrolides are felt to slow the progression of bos, it has been postulated that this response is due to the potential treatment of a chronic infection with mycoplasma pneumoniae or chlamydia pneumoniae, 154, 156 although macrolide immunomodulation also plays an important role. it has been shown that a positive serology and pcr testing for chlamydia pneumoniae on bal samples increases the rate of bos and early mortality. 157, 158 supporting this theory further, a study recently demonstrated that macrolides can prevent the development of bos. 153 fungal pathogens have been also associated with the development of bos. 159 fungal pneumonitis and aspergillus colonization have been identified as independent risk factors for bos and mortality related to rejection. 151, 159, 160 moreover, the combination of lateonset aspergillosis and chronic allograft dysfunction was a risk factor for poorer survival. 132 despite several advances in surgical technique, immunosuppression and prophylaxis, infection continues to remain an important cause of death and disease in the ltr. although there are non-modifiable factors that are innate to the patient or to the nature of the procedure, there are several modifiable factors that can be recognized and changed so as to optimize the patient's chances for survival and further extend life. prompt recognition and treatment of these factors is paramount for appropriate management. prophylaxis strategies continue to evolve and show promise for several of the infectious agents. avoidance of these infectious complications may not only lead to a decrease in the direct consequences of infection but also to a reduction in the subsequent causes of ultimate graft failure including both acute and chronic rejection. antimicrobial resistance is a growing problem, and although newer antimicrobials will likely be of benefit, especially against viral and fungal pathogens, prevention of these diseases remains the best approach. careful consideration and further research are needed regarding the mechanisms by which infection and subsequent inflammation alters the immunoregulatory machinery of the host and subsequently leads to the development failure of the allograft. factors that are important in evaluating an infectious episode include time after transplant, immunosuppression, cmv serostatus, prophylaxis regimen and treatment for acute rejection. 3 given that outcomes appear to be improved with early recognition and treatment of disease, all practitioners must always maintain a high index of suspicion caring for these patients. ganciclovir/valganciclovir prophylaxis decreases cytomegalovirus-related events and bronchiolitis obliterans syndrome after lung transplantation epidemiology and management of infections after lung transplantation early and late infections in lung transplantation patients the registry of the international society for heart and lung transplantation: twenty-eighth adult lung and heart-lung transplant report-2011 bronchiolitis obliterans syndrome development in lung transplantation patients risk factors for icu-acquired pneumonia effect of nasogastric tubes on the nose and maxillary sinus pulmonary infection defense after lung transplantation: does airway ischemia play a role? airway epithelium of transplanted lungs with and without direct bronchial artery revascularization bronchial transsection and reanastomosis in pigs with and without bronchial arterial circulation anastomotic airway complications after lung transplantation immunosuppression for lung transplantation current trends in immunosuppression for lung transplantation conventional and novel approaches to immunosuppression use of cyclosporine in lung transplantation clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients rapamycin blocks cell cycle progression of activated t cells prior to events characteristic of the middle to late g1 phase of the cycle rapamycin in transplantation: a review of the evidence clinical pharmacokinetics of everolimus corticosteroid effects on cell signalling basiliximab in lung transplantation: preliminary experience basiliximab versus rabbit anti-thymocyte globulin for induction therapy in patients after heart transplantation risk/benefit ratio of perioperative okt3 in cardiac transplantation risk factors for early, cumulative, and fatal infections after heart transplantation: a multiinstitutional study a randomized multicenter comparison of basiliximab and muromonab (okt3) in heart transplantation: simcor study induction therapy in lung transplantation: a prospective, controlled clinical trial comparing okt3, anti-thymocyte globulin, and daclizumab anonymous organ procurement and transplantation network (optn), scientific registry of transplant recipients (srtr) annual data report. department of health and human services, health resources and services administration, healthcare systems bureau, division of transplantation induction immunosuppression improves long-term graft and patient outcome in organ transplantation: an analysis of united network for organ sharing registry data rabbit antithymocyte globulin as induction immunotherapy in pediatric heart transplantation the impact of induction on survival after lung transplantation: an analysis of the international society for heart and lung transplantation registry basiliximab as an alternative to antithymocyte globulin for early immunosuppression in lung transplantation five-year outcomes with alemtuzumab induction after lung transplantation bacterial and fungal pneumonias after lung transplantation the impact of panresistant bacterial pathogens on survival after lung transplantation in cystic fibrosis: results from a single large referral centre survival of lung transplant patients with cystic fibrosis harboring panresistant bacteria other than burkholderia cepacia, compared with patients harboring sensitive bacteria impact of burkholderia infection on lung transplantation in cystic fibrosis survival after lung transplantation of cystic fibrosis patients infected with burkholderia cepacia complex clinical outcome following lung transplantation in patients with cystic fibrosis colonised with burkholderia cepacia complex: results from two french centres survival of burkholderia cepacia sepsis following lung transplantation in recipients with cystic fibrosis disease community of practice. screening of donor and recipient prior to solid organ transplantation bacterial colonization of the donor lower airways is a predictor of poor outcome in lung transplantation a review of lung transplant donor acceptability criteria impact of donor lung organisms on post-lung transplant pneumonia a positive donor gram stain does not predict outcome following lung transplantation native lung complications in single-lung transplant recipients and the role of pneumonectomy pneumonia after lung transplantation in the resitra cohort: a multicenter prospective study invasive pneumococcal infections in adult lung transplant recipients infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcareassociated pneumonia cytomegalovirus pneumonitis is a risk for bronchiolitis obliterans syndrome in lung transplantation cytomegalovirus in solid organ transplant recipients cytomegalovirus and lung transplantation international consensus guidelines on the management of cytomegalovirus in solid organ transplantation working group on infectious disease monitoring. american society of transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation an international survey of cytomegalovirus management practices in lung transplantation clinical utility of cytomegalovirus cell-mediated immunity in transplant recipients with cytomegalovirus viremia cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients cytomegalovirus prevention in high-risk lung transplant recipients: comparison of 3-vs 12-month valganciclovir therapy update and review: state-of-the-art management of cytomegalovirus infection and disease following thoracic organ transplantation extended valganciclovir prophylaxis to prevent cytomegalovirus after lung transplantation: a randomized, controlled trial oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients guidance on novel influenza a/h1n1 in solid organ transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients the novel 2009 h1n1 influenza virus pandemic: unique considerations for programs in cardiothoracic transplantation common infections in the lung transplant recipient rapid-test sensitivity for novel swine-origin influenza a (h1n1) virus in humans pneumonia and influenza death rates-united states, 1979-1994 diseases community of practice. rna respiratory viral infections in solid organ transplant recipients respiratory viral infections in transplant recipients community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodeficiency virus infection ribavirin therapy in bone marrow transplant recipients with viral respiratory tract infections a multi-drug regimen for respiratory syncytial virus and parainfluenza virus infections in adult lung and heart-lung transplant recipients intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation efficacy of oral ribavirin in lung transplant patients with respiratory syncytial virus lower respiratory tract infection viral infections in lung transplant recipients incidence and morbidity of human metapneumovirus and other community-acquired respiratory viruses in lung transplant recipients fungal infections after lung transplantation diseases community of practice. invasive aspergillosis in solid organ transplant recipients increased mortality after pulmonary fungal infection within the first year after pediatric lung transplantation aspergillus infections after lung transplantation: clinical differences in type of transplant and implications for management donor-to-host transmission of bacterial and fungal infections in lung transplantation assessment of infection risks prior to lung transplantation invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (tran-snet) the incidence of invasive aspergillosis among solid organ transplant recipients and implications for prophylaxis in lung transplants aspergillosis in lung transplantation: incidence, risk factors, and prophylactic strategies infections after lung transplantation antifungal prophylaxis in lung transplantation aspergillus infections in transplant recipients aspergillus infection in lung transplant recipients with cystic fibrosis: risk factors and outcomes comparison to other types of transplant recipients aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients molecular detection and species-specific identification of medically important aspergillus species by real-time pcr in experimental invasive pulmonary aspergillosis the (1,3){beta}-dglucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation fungi and molds following lung transplantation voriconazole versus amphotericin b for primary therapy of invasive aspergillosis treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america voriconazole pharmacokinetic variability in cystic fibrosis lung transplant patients significance of blood stream infection after lung transplantation: analysis in 176 consecutive patients fungal infections after lung transplantation diseases community of practice. candida in solid organ transplant recipients approach to the diagnosis of invasive aspergillosis and candidiasis histoplasmosis in solid organ transplant recipients: 10 years of experience at a large transplant center in an endemic area posttransplantation disseminated coccidioidomycosis acquired from donor lungs cryptococcosis in solid organ transplant recipients calcineurin inhibitor agents interact synergistically with antifungal agents in vitro against cryptococcus neoformans isolates: correlation with outcome in solid organ transplant recipients with cryptococcosis an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients emerging invasive zygomycosis in a tertiary care center: epidemiology and associated risk factors fungal infections in lung transplant recipients mucormycosis of the bronchial anastomosis: a case of successful medical treatment and historic review antifungal prophylaxis with voriconazole or itraconazole in lung transplant recipients: hepatotoxicity and effectiveness a survey of antifungal management in lung transplantation nebulized liposomal amphotericin b prophylaxis for aspergillus infection in lung transplantation: pharmacokinetics and safety feasibility, tolerability, and outcomes of nebulized liposomal amphotericin b for aspergillus infection prevention in lung transplantation safety of aerosolized amphotericin b lipid complex in lung transplant recipients invasive fungal infections in lung transplantation: role of aerosolised amphotericin b voriconazole prophylaxis in lung transplant recipients intrapulmonary penetration of voriconazole in patients receiving an oral prophylactic regimen tuberculosis in transplanted lungs the spectrum of mycobacterial infection after lung transplantation non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation mycobacterium abscessus chest wall and pulmonary infection in a cystic fibrosis lung transplant recipient lung transplantation in patients with cystic fibrosis and mycobacterium abscessus infection mycobacterium abscessus infections in lung transplant recipients: the international experience mycobacterium abscessus infection in solid organ transplant recipients: report of three cases and review of the literature nontuberculous mycobacterial infection in hematopoietic stem cell and solid organ transplant recipients an official ats/ idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases airway complications and management after lung transplantation: ischemia, dehiscence, and stenosis aspergillus infections in lung transplant recipients: risk factors and outcome pre-emptive therapy with azoles in lung transplant patients. geneva lung transplantation group candidal anastomotic infection in lung transplant recipients: successful treatment with a combination of systemic and inhaled antifungal agents anastomotic infections in lung transplant recipients prevention of infection due to pneumocystis spp. in human immunodeficiency virus-negative immunocompromised patients rejection treatment and cytomegalovirus infection as risk factors for pneumocystis carinii pneumonia in renal transplant recipients the impact of invasive fungal diseases on survival after lung transplantation antimicrobial prophylaxis regimens following transplantation pneumocystis pneumonia should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? mycophenolate mofetil: effects on cellular immune subsets, infectious complications, and antimicrobial activity novel anti-pneumocystis carinii effects of the immunosuppressant mycophenolate mofetil in contrast to provocative effects of tacrolimus, sirolimus, and dexamethasone rs-61443 (mycophenolate mofetil). a multicenter study for refractory kidney transplant rejection anonymous mycophenolate mofetil for the treatment of refractory, acute, cellular renal transplant rejection the tricontinental mycophenolate mofetil renal transplantation study group. anonymous a blinded, randomized clinical trial of mycophenolate mofetil for the prevention of acute rejection in cadaveric renal transplantation nocardia infection in lung transplant recipients pulmonary nocardiosis: risk factors, clinical features, diagnosis and prognosis report of the ishlt working group on primary lung graft dysfunction part ii: definition. a consensus statement of the international society for heart and lung transplantation bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome pseudomonas aeruginosa colonization of the allograft after lung transplantation and the risk of bronchiolitis obliterans syndrome a randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation long-term azithromycin therapy for bronchiolitis obliterans syndrome: divide and conquer? impact of graft colonization with gram-negative bacteria after lung transplantation on the development of bronchiolitis obliterans syndrome in recipients with cystic fibrosis azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation chlamydia pneumoniae infection after lung transplantation chlamydia pneumoniae serology in donors and recipients and the risk of bronchiolitis obliterans syndrome after lung transplantation aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome infections in lung allograft recipients: ganciclovir era key: cord-277455-r69j2tnw authors: lim, jun hyeok; ryu, jeong-seon; cho, sang yong; kim, hyun-jung; jeon, sang hoon; kim, jung soo; nam, hae-seong; cho, jae hwa; kwak, seung min; lee, hong lyeol title: small-cell lung cancer presenting as fatal pulmonary hemorrhage date: 2018-03-21 journal: open med (wars) doi: 10.1515/med-2018-0009 sha: doc_id: 277455 cord_uid: r69j2tnw small-cell lung cancer (sclc) is a lung cancer histological subtype unusual in its favorable response to cytotoxic chemotherapy. life-threatening manifestations at presentation are rarely reported and should be an important clinical concern. we report a case of a 63-year-old man presenting with rapid-onset refractory severe thrombocytopenia, development of massive hemoptysis, and death from respiratory failure. this case provides clinicians a reference for this unusual presentation and carries clinical implications for managing sclc patients. small-cell lung cancer (sclc) represents 15-20% of all lung cancers [1] . sclc differs from non-small-cell lung cancer in its rapid tumor doubling time, high growth fraction, early development of widespread metastasis, and better response to platinum doublets chemotherapy. thus chemotherapy is a treatment mainstay, even in poor eastern cooperative oncology group (ecog) performance status [2, 3] . bone marrow involvement or paraneoplastic syndrome is common in patients with sclc [4] . hematologic abnormalities such as anemia, leukopenia, and thrombocytopenia are reported to be occasionally accompanied by bone marrow metastasis or paraneoplastic phenomenon [5, 6] . however, complications such as fatal hemorrhage are rarely reported. the clinical presentation can make diagnosis or treatment difficult. herein, we report an sclc patient who presented with rapid-onset, refractory severe thrombocytopenia and development of fatal pulmonary hemorrhage. a 63-year-old man visited an outpatient clinic complaining of cough and dyspnea (borg scale 4). he was a current smoker of 20 pack-years and denied histories of taking any medications or illness including cardiovascular, allergic, rheumatologic or respiratory diseases. a complete blood count revealed values within normal range, except for a lower value of platelet count, 91,000/mm 3 . increased haziness on the lower lobe of the right lung was noted on his chest radiography. when he returned after 10 days, he was admitted with blood-tinged sputum and aggravated dyspnea (borg scale 6). his ecog performance status was two. he was afebrile. an arterial blood gas study revealed ph 7.44, paco 2 37.5 mmhg, pao 2 77.6 mmhg, hco 3 25 mmol/l, and spo 2 95% on room air. complete blood count results were as follows: leukocytes 6,270/mm 3 (neutrophil 61.2%, lymphocyte 27.5%, monocyte 3.9%, eosinophil 3.9%, and basophil 0.7%), hemoglobin 10.6 g/dl, hematocrit 29.6%, and platelets 11,000/mm 3 . the serum lactate dehydrogenase level was 1,324 iu/l; c-reactive protein, 6.40 mg/dl. hepatic and renal function testing were within normal range. prothrombin time, activated partial thromboplastin time, and d-dimer were within normal range as well. a 1.6 cm sized mass in the lower lobe of the right lung and multiple lymphadenopathies in mediastinal and right supraclavicular areas were noted on chest ct scan ( fig. 1) . a peripheral blood smear revealed leukoerythroblastosis with nucleated erythrocyte, left shifted neutrophils. anti-platelet antibody and anti-neutrophil cytoplasmic antibody were negative. anti-nuclear antibody was within pulmonary hemorrhage at sclc presentation normal range (1:20). because of the risk of bleeding due to severe thrombocytopenia, a bronchoscopic examination was not feasible and was postponed. a bone marrow examination was not performed because the patient was unable to maintain prone position due to dyspnea. platelet concentrates and packed red blood cells were started, given daily, and dexamethasone, 40 mg was intravenously administered for four days. however, his platelet count remained stationary (fig. 2) . on the fifth day after admission, cytological examination of his sputum yielded a diagnosis of sclc. metastatic lesions were not observed on brain mri and bone scintigraphy. on the seventh day, massive hemoptysis (≥ 200 ml per day) abruptly occurred and his dyspnea was rapidly aggravated to 8 of borg scale. a chest ct scan revealed diffuse ground glass opacities and consolidation in both lung fields. tranexamic acid and empirical broad-spectrum antibiotics including piperacillin-tazobactam, levofloxacin were initiated intravenously. sputum gram stain and culture for bacteria and fungus revealed no organism. sputum culture for adenovirus, parainfluenza virus, rhinovirus, respiratory syncytial virus, metapneumovirus, coronavirus, bocavirus, and enterovirus were negative. antibody studies for mycoplasma pneumoniae and rickettsia were negative, as was an antigen study for streptococcus pneumoniae. his arterial blood gas study showed ph of 7.49, paco 2 of 28.7 mmhg, pao 2 of 58.3 mmhg, hco 3 22 mmol/l, and spo 2 91 % on oxygen supplied via reservoir mask flow rate 15l/min. on the tenth day, he was intubated and ventilated mechanically. he rapidly deteriorated and died of respiratory failure on the twelfth day. his family did not want to have a postmortem examination, against physician's recommendation. the research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the helsinki declaration, and has been approved by the authors' institutional review board or equivalent committee. informed consent: written informed consent was obtained from the patient for publication of this manuscript and any accompanying images. to our knowledge, this is the first report describing an sclc patient presenting with fatal pulmonary hemorrhage due to refractory thrombocytopenia. sclc is the most aggressive histological subtype of lung cancer. however, favorable response is expected in 60% to 70% of patients with extensive-stage disease when systemic chemotherapy is given [3, 4] . life-threatening manifestations rarely present in sclc patients. when such manifestations do appear, as in the present case, the rapid-onset, unusual presentation may delay diagnosis or hesitation in deciding treatment, affecting survival adversely [7] . therefore, prompt diagnosis and treatment is a critical issue [8] . hematological abnormalities including anemia, leukopenia, and thrombocytopenia are commonly observed due to the toxicity of anti-cancer therapy. however, hematologic abnormalities resulting from bone marrow metastasis and paraneoplastic phenomenon are not as common as those resulting from anti-cancer therapy [5, 6] . severe thrombocytopenia developed rapidly and was refractory to platelet transfusion and dexamethasone administration. in addition, tests for anti-platelet antibody and anti-neutrophil cytoplasmic antibody were negative and anti-nuclear antibody was within normal range. these findings indicate that autoimmune disease was not likely to be the cause of thrombocytopenia [9] . bone marrow examination is a diagnostic process for evaluating hematological abnormality or staging the disease. however, it rarely changes the stage [10] and bone marrow as an isolated metastatic site is found in fewer than 5% of sclc patients [3] . leukoerythroblastic reaction on peripheral blood smear is suggestive of bone marrow metastasis in the present case [11, 12] . although bronchoscopy may be considered for localization or treatment in massive hemoptysis, it is contraindicated in a patient with severe thrombocytopenia [13] . a diagnostic and treatment plan should be determined after taking into account the benefit and risk on an individual patient basis. it is challenging to do in every possible case. the clinical presentation of the current case is unfamiliar to clinicians and rapid-onset, life threatening. however, prompt treatment with systemic chemo-therapy should have been considered because he was considered fit for systemic chemotherapy by virtue of his younger age, acceptable ecog performance status, and absence of comorbid diseases. cancer statistics national comprehensive cancer network. nccn clinical practice guidelines in oncology small cell lung cancer-version 2 small-cell lung cancer thrombocytopenia in solid tumors bone marrow involvement in small cell lung cancer: prognostic significance and correlation with hematological and biochemical parameters delays in the diagnosis and treatment of lung cancer lactic acidosis with small cell carcinoma. rapid response to chemotherapy paraneoplastic autoimmune thrombocytopenia in solid tumors is bone marrow examination in small-cell lung cancer really necessary? leukoerythroblastosis and cancer frequency, prognosis, and physiopathologic significance systemic malignancies as a cause of unexpected microangiopathic hemolytic anemia and thrombocytopenia british thoracic society guideline for diagnostic flexible bronchoscopy in adults: accredited by nice acknowledgements: this work was supported by grants (hi15c0554 and hi16c0286) from the korea health technology r&d project, ministry of health and welfare, republic of korea. the authors have no conflicts of interest to declare. key: cord-315948-o4uj3l8r authors: kim, se yong; kim, se jin; yoon, doran; hong, seung wook; park, sehhoon; ock, chan-young title: a case of statin-induced interstitial pneumonitis due to rosuvastatin date: 2015-06-30 journal: tuberc respir dis (seoul) doi: 10.4046/trd.2015.78.3.281 sha: doc_id: 315948 cord_uid: o4uj3l8r statins lower the hyperlipidemia and reduce the incidence of cardiovascular events and related mortality. a 60-year-old man who was diagnosed with a transient ischemic attack was started on acetyl-l-carnitine, cilostazol, and rosuvastatin. after rosuvastatin treatment for 4 weeks, the patient presented with sudden onset fever, cough, and dyspnea. his symptoms were aggravated despite empirical antibiotic treatment. all infectious pathogens were excluded based on results of culture and polymerase chain reaction of the bronchoscopic wash specimens. chest radiography showed diffuse ground-glass opacities in both lungs, along with several subpleural ground-glass opacity nodules; and a foamy alveolar macrophage appearance was confirmed on bronchoalveolar lavage. we suspected rosuvastatin-induced lung injury, discontinued rosuvastatin and initiated prednisolone 1 mg/kg tapered over 2weeks. after initiating steroid therapy, his symptoms and radiologic findings significantly improved. we suggest that clinicians should be aware of the potential for rosuvastatin-induced lung injury. statins, hydroxymethylglutaryl coa reductase inhibitors, are known to lower the plasma low-density lipoprotein (ldl) cholesterol level and reduce the incidence of cardiovascular events and mortality 1, 2 . currently available statins include lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, tuberc respir dis 2015;78:281-285 www.e-trd.org old wallpapers at the time. after the cause removed, it had not recurred over 2 years. he had also been administered metformin 500 mg daily for 16 months for diabetes mellitus. for a transient ischemic attack secondary to vertebral artery stenosis, he was hospitalized and started on acetyl-l-carnitine, cilostazol, and rosuvastatin according to the following schedule: acetyl-l-carnitine, 500 mg, once daily for 48 days prior to this hospitalization; cilostazol, 100 mg, twice daily; and rosuvastatin, 5 mg, once daily for 27 days prior to this hospitalization. while continuing those medications, he developed a fever and cough 3 days prior to this hospital visit. although he was prescribed an antibiotic (augmentin) for 3 days at a local clinic, his dyspnea, cough, and fever gradually increased. he was referred to our hospital for proper evaluation and management. he had no known drug allergies. upon admission, the patient's vital signs were as follows: blood pressure, 116/77 mm hg; pulse, 114 beats per minute; respiratory rate, 26 breaths per minute; and body temperature, 36.9 o c. on physical examination, a coarse breath sound with fine crackles but without wheezing was heard upon auscultation of both lungs. the patient' s laboratory test results were as follows: hemoglobin, 14.1 g/dl; white blood cell count, 8,770 cells/μl (neutrophils, 62.6%; lymphocytes, 26.0%; monocytes, 7.2%; eosinophils, 3.9%; and basophils, 0.3%); and platelet count, 391,000 cells/μl. the patient' s serum biochemistry test results revealed the following: aspartate aminotransferase, 35 iu/l; alanine aminotransferase, 35 iu/l; total bilirubin, 0.4 mg/dl; alkaline phosphatase, 311 iu/l; total protein, 7.6 g/dl; albumin, 4.0 g/dl; blood urea nitrogen, 26.0 mg/ dl; creatinine, 0.84 mg/dl; total cholesterol, 106 mg/dl; creactive protein, 1.06 mg/dl; and ldl cholesterol, 56 mg/dl. his coagulation profile was within normal limits. a urinalysis with microscopy was clear. an arterial blood gas analysis of the fraction of inspired oxygen (fio 2 ) was conducted with the patient breathing room air and revealed the following: ph, 7.46; partial pressure of oxygen (pao 2 ), 54.6 mm hg; partial pressure of carbon dioxide (paco 2 ), 37.4 mm hg; bicarbonate (hco 3 − ), 26.0 meq/l; and saturation level of oxygen (sao 2 ), 85.9%. a chest radiograph showed a subtle, diffuse groundglass opacity in both lower lung fields and subsegmental atelectasis in the left lower lobe. a chest computed tomography scan revealed diffuse ill-defined ground-glass opacities with septal line thickening bilaterally, combined with several subpleural ground-glass opacity nodules ( figure 1) . a pulmonary function test revealed the following: forced expiratory volume in 1 second (fev 1 )/forced vital capacity (fvc) ratio, 102; figure 1 . (a-e) chest radiography showed subtle diffuse ground glass opacity in both lower lobe field and subsegmental atelectasis in left lower lobe field on admission. a computed tomography scan of the chest showed diffuse ill-defined ground glass opacity with septal line thickening in bilateral lung fields combined with several subpleural ground glass opacity nodules on admission. www.e-trd.org fev 1 , 2.8 l (94%); fvc, 3.47 l (92%); and diffuse capacity for carbon monoxide (dlco), 57%. based on his past medical history, clinical symptoms, and laboratory findings, a diagnosis of dild was suspected. consequently, bronchoscopy with bronchoalveolar lavage (bal) was performed. there were no endobronchial lesions revealed with bronchoscopy. the bal fluid results were as follows: white blood cell count, 480 cells/μl (neutrophils, 9%; lymphocytes, 30%; macrophages, 57%; eosinophils, 4%; and basophils, 0%); and red blood cell count, 30 cells/μl. microbiological culture and polymerase chain reaction of the bronchoscopic wash specimens were negative for mycobacterium tuberculosis, pneumococcus and respiratory virus. the real-time polymerase chain reaction of bronchial washing specimens was performed as following: mycoplasma, chlamydia, legionella, bordetella, haemophilus pneumonia, adenovirus, rhinovirus, coronavirus, influenza virus a and b, parainfluenza virus, respiratory syncytial virus a and b, bocavirus, metapneumovirus. the results were all negative. additionally, the urine antigen and immunologic study were performed to exclude others pathogen. the results revealed the following: streptococcal pneumonia and legionella urinary antigen, negative; mycoplasma pneumoniae igg/igm, negative; and chlamydia pneumoniae igg (17.65, positive; normal range, <9 index), igm (4.62, negative; normal range, <9 index). according to the cytology, the majority of the alveolar macrophages had a foamy appearance (figure 2 ). to make a differential diagnosis of connective disease or allergic disease, immunologic studies and multiple allergosorbent test system (mast) were performed as follow: all allergens of mast, class 0 (0.00-0.34 iu/ml); rheumatoid factor, negative; c3 complement, 135.00 mg/dl (normal range, 90-180 mg/ dl); c4 complement, 42.20 mg/dl (normal range, 10-40 mg/ dl); anti-nuclear antibodies and anti-neutrophil cytoplasmic antibodies, negative. considering the previous reports and past drug history, rosuvastatin was strongly suspected as the cause of the patient' s dild. thus, rosuvastatin was discontinued, and steroid therapy (prednisolone, 1 mg/kg of body weight) was initiated. after the steroid therapy, the patient' s symptoms improved. the prednisolone dose was gradually tapered over 2 weeks. two weeks later, the patient was in complete remission according to a chest radiograph, and the previously noted diffuse ground-glass opacities in both lung fields had disappeared (figure 3 ). statins are indicated for the treatment of hyperlipidemia and the prevention of cardiovascular events for patients with multiple risk factors. although statins appear to be safe and well tolerated clinically, statins have a low frequency of side effects including hepatotoxicity, myotoxicity, and proteinuria 2,3 . however, adverse reactions involving the respiratory system are uncommon. several pulmonary toxicity cases secondary to statins have been reported [4] [5] [6] . the prevalence of statininduced interstitial pneumonitis (sili) that has been mainly associated with simvastatin, fluvastatin, and atorvastatin was high 7 . rosuvastatin-induced interstitial pneumonitis has been rarely reported. to our knowledge, just 3 cases in taiwan and 1 case in the united states have been reported 7, 8 . in addition, rosuvastatin-induced interstitial lung disease has not been reported in korea, to our knowledge. a definite mechanism for sili is not yet known. however, some authors proposed that the potential toxicity mechanism might be mediated by intracellular lipid metabolism, mitochondrial metabolism, and immunologic response, as well as some genetic or other predisposing factors 7 . diagnosis of sili generally depends on the relationship between causative agents and clinical symptoms, as well as radiologic findings and the exclusion of infectious diseases, because of its unclear mechanism. although the histopathological findings are also non-diagnostic, based on prior reports and studies, the possibility of diagnosis is significantly increased if the alveolar macrophages have a foamy appearance, as confirmed by cytology, along with the patient' s clinical history 8 . furthermore, some reports suggested that the most prominent feature of dild on bal fluid was a lymphocytic alveolitis either pure or associated with neutrophil and/or eosinophilic alveolitis along with an imbalance in t lymphocyte phenotype 9 . in our case, infectious diseases were excluded based on the negative microbial culture results. in addition, the patient's symptoms and bilateral infiltration began after starting rosuvastatin that was revealed by chest radiology. the alveolar macrophages finally had a foamy appearance that was revealed by cytology. the alveolar macrophages finally had a foamy appearance that was revealed by cytology with increased lymphocytes and eosinophils on cellular profile of bal fluid. thereafter, after discontinuing rosuvastatin and initiating steroid therapy, the patient' s symptoms and radiologic findings had improved. therefore, to our knowledge, our patient was the first diagnosed with rosuvastatin-induced interstitial lung disease in korea. in comparing with the previous cases in taiwan and the united states, clinical course, radiologic findings and cytology were similar to our case. and they also did not perform a lung biopsy. however, increasing lymphocytes and eosinophils along with the predominance of macrophage on cellular profile of bal fluid was revealed only in our patient. we believe a foamy macrophage appearance could help in the diagnosis of sili, coupled with the clinical symptoms and radiologic findings, and clinicians should consider the potential for rosuvastatin-induced lung injury. a-e) day 14 after initiation of steroid therapy, chest radiography showed disappearance of the previously noted diffuse groundglass opacities in both lung fields. there were no other remarkable findings. www.e-trd.org of statins: implications for cardiovascular event reduction safety of statins: focus on clinical pharmacokinetics and drug interactions the safety of rosuvastatin as used in common clinical practice: a postmarketing analysis dermatomyositis with lung involvement in a patient treated with simvastatin polymyalgia, hypersensitivity pneumonitis and other reactions in patients receiving hmg-coa reductase inhibitors: a report of ten cases a case of interstitial lung disease with atorvastatin (tahor) and a review of the literature about these effects observed under statins statins and interstitial lung disease: a systematic review of the literature and of food and drug administration adverse event reports statin-induced lung injury: diagnostic clue and outcome drug-induced interstitial lung disease: mechanisms and best diagnostic approaches no potential conflict of interest relevant to this article was reported. key: cord-290677-3gdcyrrz authors: de virgiliis, francesco; di giovanni, simone title: lung innervation in the eye of a cytokine storm: neuroimmune interactions and covid-19 date: 2020-08-25 journal: nat rev neurol doi: 10.1038/s41582-020-0402-y sha: doc_id: 290677 cord_uid: 3gdcyrrz covid-19 is an infectious disease caused by the coronavirus sars-cov-2, which was first reported in wuhan, china, in december 2019 and has caused a global pandemic. acute respiratory distress syndrome (ards) is a common feature of severe forms of covid-19 and can lead to respiratory failure, especially in older individuals. the increasing recognition of the neurotropic potential of sars-cov-2 has sparked interest in the role of the nervous system in respiratory failure in people with covid-19. however, the neuroimmune interactions in the lung in the context of ards are poorly understood. in this perspectives article, we propose the concept of the neuroimmune unit as a critical determinant of lung function in the context of covid-19, inflammatory conditions and ageing, focusing particularly on the involvement of the vagus nerve. we discuss approaches such as neurostimulation and pharmacological neuromodulation to reduce tissue inflammation with the aim of preventing respiratory failure. nl63 and hku1, cause mild respiratory diseases. however, the worldwide spread of two previously unrecognized viruses, the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), revealed to the world the lethal potential of human coronaviruses 1 . another highly pathogenic coronavirus, which has been named sars-cov-2 and causes a disease known as covid-19, was reported in december 2019 in wuhan, china 2 . this novel virus caused a national outbreak of severe pneumonia in china and has rapidly spread around the world owing to its high infectivity, causing a global pandemic. sars-cov-2 can cause acute, highly lethal pneumonia with clinical symptoms similar to those reported for sars-cov and mers-cov 1 . a substantial proportion of patients who are admitted to intensive care units worsen in a short period of time and die from respiratory failure 1 . to infection. interestingly, ace2 is expressed in nuclei involved in the central regulation of cardiovascular and respiratory functions, such as the paraventricular nucleus, the nucleus of the tractus solitarius (nts) and the rostral ventrolateral medulla, as well as in the motor cortex and raphe 17 . in this perspective, we briefly summarize our current knowledge regarding the immune response to sars-cov-2. we then consider how lung afferent and efferent innervation might crosstalk with the immune system to modulate lung function, thereby critically affecting clinical outcomes following sars-cov-2 infection. the respiratory tract is a major barrier tissue and is often the first point of contact and entry for harmful substances and pathogens, including viruses. alveolar epithelial cells are important cellular targets for sars-covs and the avian h5n1 virus 18, 19 , which elicit a prompt immune response. while attempting to eliminate the virus, this response can compromise respiratory function owing to excessive tissue inflammation, leading to acute respiratory distress syndrome (ards). the first immune cell types to encounter viral antigens in the respiratory tract are the alveolar and interstitial macrophages, which are capable of eliminating many different pathogens from the lungs by phagocytosis. in addition, interstitial macrophages seem to be involved in dampening the immune response in loco after influenza virus infection 20, 21 . dendritic cells are the primary antigen-presenting cells (apcs), and resident mature mhcii hi cd11c hi respiratory dendritic cells are crucial for the induction of adaptive immune responses. plasmacytoid dendritic cells are also recognized as major producers of type i interferons during infection and can transport antigens from the infected lung to the draining lymph nodes, where they serve as potent apcs for the induction of virus-specific t cells 22, 23 . following exposure to apcs in the draining lymph nodes, virus-specific naive t cells undergo a stepwise process of activation, proliferation and differentiation to become effector t cells, which can migrate to the site of infection and mediate antiviral immune responses. virus-specific memory t cells are antigen-experienced importantly, coronavirus infections are not always confined to the respiratory tract, and clinical studies have revealed a degree of symptomatic heterogeneity in people with covid-19, with anosmia being a frequent occurrence 3 and severe neurological symptoms being reported in some cases [4] [5] [6] [7] . these observations suggest that a range of different tissues and cell types, including the nervous system 3, 8 , are affected by the virus. the entry of sars-cov-2 into human host cells is mediated mainly by the transmembrane proteins angiotensin-converting enzyme 2 (ace2) and transmembrane protease serine 2 (tmprss2), which are highly expressed in the airway epithelium, lung parenchyma, vascular endothelium, heart, kidney and small intestine in humans [9] [10] [11] . most notably, both ace2 and tmprss2 are expressed in the olfactory epithelium and lower respiratory tract, which are densely innervated 12, 13 . these barrier tissues are postulated to be key invasion sites for sars-cov-2 and might be involved in nervous system infection. ace2 is widely expressed throughout the nervous system [14] [15] [16] , making neural cells susceptible cells that have acquired the capacity to rapidly implement effector functions and exert antiviral activity. three main antiviral effector mechanisms have been identified: lysis of infected cells following exocytosis of granules containing perforin and granzyme from cytotoxic t cells, possibly with the cooperation of b cells; tumour necrosis factor (tnf) receptor family-dependent apoptosis of infected cells and phagocytosis following recognition of these cells by antibodies; and production of pro-inflammatory mediators by t cells in response to encountering virus-infected cells. immune responses to sars-cov-2 clinically, the immune responses induced by sars-cov-2 infection consist of two phases. during the initial incubation and in non-severe stages, a specific adaptive immune response is produced to eliminate the virus and abort disease progression. if the protective immune response is impaired, the virus is allowed to replicate and can cause widespread tissue damage. the lungs seem to be the main target, although other organs with high ace2 expression, such as the intestine, heart, brain and kidney, might also be vulnerable 11, 24 . the second phase is usually associated with increased severity of the disease and is characterized by potentially life-threatening lung inflammation 25 and the appearance of systemic symptoms such as high fever and severe muscle pain. the damaged cells in the lungs elicit a strong innate immune response, which seems to be largely mediated by pro-inflammatory macrophages and granulocytes 26, 27 . this response is thought to be responsible for the cytokine release syndrome or 'cytokine storm' that is believed to contribute to ards 25 . the idea that the cytokine storm is mediated by leukocytes other than t or b cells is supported by observations of lymphocytopenia and sometimes atrophy of the lymphatic tissues, such as lymph nodes and spleen, in patients with severe covid-19 (refs 24,26,28-30 ). in line with observations in lethal cases of sars 31 and mers 32 , patients infected with sars-cov-2 show increased numbers of neutrophils and macrophages in their airways and blood 28, 33 . levels of inflammatory cytokines such as il-1, tnf and il-6 can be high in the lungs of patients with covid-19 (refs 1,34 ); therefore, blocking one or more of these cytokines could potentially benefit these individuals by reducing the severity of the cytokine storm and ards. initial attempts to improve covid-19 outcomes by blocking il-6 have shown some promise 35 , although the complexity of the immune response to sars-cov-2 might limit the efficacy of this approach. a recent study found that in individuals with severe covid-19, plasma levels of il-6 were generally above the normal range but lower than the median values typically reported in ards 36 , suggesting that the inflammatory and cytokine profiles induced by sars-cov-2 infection are more complex than previously thought, and that the role of cytokine dysregulation following sars-cov-2 infection requires further clarification. elevated levels of cytokines might have an impact on lung function even when below the cytokine storm threshold because of the unique inflammatory profile associated with covid-19. in addition, levels of specific cytokines might be elevated predominantly in the lung alveolar microenvironment, contributing to respiratory distress. interestingly, with age, macrophage activity declines and pro-inflammatory cytokine levels increase 37 ; moreover, naive t cell production and effector memory t cell competency decline 38, 39 . together, these phenomena could contribute to worsening of the severity and outcomes of sars-cov-2 infection in older individuals, as discussed in more detail below. the possible role of lung innervation in the regulation of the immune response to sars-cov-2 is an important area of investigation. the lungs are highly innervated, receiving sensory, sympathetic and parasympathetic fibres ( fig. 1 ). sympathetic innervation of the lungs originates from sympathetic ganglia that arise from the upper thoracic segments of the spinal cord and provide noradrenergic innervation to bronchial blood vessels and submucosal glands. the tenth cranial nerve, or vagus nerve, supplies all parasympathetic and most of the sensory nerve fibres to the airways, although some more minor sensory innervation originates from the t1-t6 dorsal root ganglia. the vagus nerve divides into the superior laryngeal and recurrent laryngeal nerves, which carry sensory fibres originating in the jugular and nodose ganglia and parasympathetic cholinergic fibres originating in the petrosal ganglion to the trachea and main bronchi. smaller branches of the vagus nerve innervate the rest of the lower respiratory tract 40, 41 . stimulation of parasympathetic pathways induces bronchoconstriction, mucus secretion and bronchial vasodilation, mediated by muscarinic m3 receptors 42, 43 . muscarinic m2 receptors are present on presynaptic cholinergic nerve terminals 44 , and activation of these receptors inhibits acetylcholine release and might serve to limit cholinergic bronchoconstriction. interestingly, parainfluenza virus has been shown to decrease m2 muscarinic receptor function on parasympathetic nerves 45 , and loss of m2 receptor-dependent negative feedback increases acetylcholine release onto airway smooth muscle 46 . expression of m2 and m3 receptors by lung macrophages has been described 47 , suggesting that acetylcholine could directly influence lung macrophage activity and, thus, inflammation. in addition, cytokines such as ifnγ, tnf and il-1β seem to influence both the expression and the activity of m2 receptors [48] [49] [50] . these cytokines are released in response to viral infection and their levels are increased in the lungs of patients infected with sars-cov or sars-cov-2 (refs 1,51,52 ). in line with these findings, virus-induced airway hyperresponsiveness in humans seems to be mediated by the vagus nerve 53 , raising the possibility that the dyspnoea and respiratory failure observed in patients with severe covid-19 is exacerbated by neuroimmune crosstalk in the lungs. most of the afferent sensory fibres arising from the airways run along the vagus nerve and project to the nts in the medulla. a small population of sensory fibres that innervate the lungs, known as the chemosensitive c-fibres, contain neuropeptides, including calcitonin gene-related peptide (cgrp) and the tachykinins substance p and neurokinin a. substance p and neurokinin a are potent inducers of airway smooth muscle contraction, vasodilation, bronchial oedema and mucus hypersecretion, which are all typical symptoms of inflammatory airway diseases. a recently discovered class of vagal throat-innervating p2ry1 + neurons has been shown to be involved in protective reflexes elicited by various airway irritants 54 . although their precise mechanism of action remains elusive, these sensory neurons seem to detect cell mechanical responses mediated by airway stretch receptors such as piezo2 and initiate defensive reflexes that ensure airway integrity 55 . dysfunction of laryngeal neurons, which is life-threatening and can cause pulmonary aspiration, dysphagia and choking, might contribute to the respiratory distress observed in patients with covid-19. interestingly, www.nature.com/nrneurol altered activation of vagal fibres can lead to many of the symptoms of allergic diseases, such as asthma and rhinitis, and of chronic obstructive pulmonary disease (copd), including sneezing, coughing, mucus hypersecretion and bronchoconstriction 42, 56 . another class of sensors that are present in the respiratory tract are the neuroepithelial cell bodies (nebs). nebs contain several bioactive mediators, including bombesin, cgrp, 5-hydroxytryptamine and atp 57 . they form compact structures located within the airway mucosa with their apical surface exposed to the airway lumen and are innervated mostly by vagal afferent fibres 58 . nebs are multimodal sensors that respond to a range of stimuli in the airway lumen, including hypoxia, hypercapnia, mechanical stretch, nicotine and nociceptive irritants 59, 60 . hyperplasia of nebs has been reported in a number of paediatric and adult lung diseases, including viral infections, many of which are associated with symptoms of dyspnoea on presentation 57, 61 . the effects of neb-derived cgrp include bronchoconstriction, vasodilation and modulation of inflammatory responses in the lung 62 . bombesin, the principal peptide in human nebs, might also be involved in modulating respiratory function and immune responses in the lungs either by directly inducing bronchoconstriction or by signalling to inflammatory cells 63, 64 . importantly, in a number of studies the vagus nerve has been found to have an immunomodulatory (but mainly anti-inflammatory) function [65] [66] [67] [68] . the immunomodulatory effects seem to be mediated by three different pathways. the first pathway is the neuroendocrine immune axis that connects the cns with the intestinal immune system, where pro-inflammatory cytokines (il-1β, il-6 and tnf) released from the intestinal mucosa activate vagal afferents that terminate in the nts 65 . the second pathway is the splenic sympathetic anti-inflammatory pathway, in which the vagus nerve stimulates the splenic sympathetic nerve. in this pathway, noradrenaline released at the distal end of the splenic nerve activates β2 adrenergic receptors of splenic lymphocytes, which release acetylcholine. in turn, the vagus nerve originates in the brainstem and provides most of the sensory and all of the parasympathetic nerve fibres that innervate the airways, via the jugular and nodose ganglia (sensory) and petrosal ganglion (parasympathetic). a minority of sensory fibres in the lungs originate in the t1-t6 dorsal root ganglia and run in spinal nerves along with sympathetic fibres originating in the thoracic ganglia. the insets show the principal neurotransmitters originating from the fibres that innervate the airways and the target receptors of these neurotransmitters. 5-ht, 5-hydroxytryptamine; ach, acetylcholine; cgrp, calcitonin gene-related peptide; machr, muscarinic ach receptor; nachr, nicotinic ach receptor; no, nitric oxide; sk, substance k; sp, substance p. acetylcholine inhibits the release of tnf by spleen macrophages through α7 nicotinic acetylcholine receptors (α7nachrs). the third pathway is the cholinergic anti-inflammatory pathway mediated through vagal efferent fibres that synapse onto enteric neurons, which in turn release acetylcholine at the synaptic junction with macrophages 65, 69 . as lung macrophages have been shown to express m2 and m3 receptors 47 , acetylcholine release in the airways is likely to contribute to immune modulation and cytokine release in the lung microenvironment. nerve-and airway-associated macro phages (nams) are a newly discovered interstitial tissue-resident macrophage subset found in close association with neuronal projections in the airways 21 . these cells show distinct transcriptional signatures, including cd169 + cd11c − and mhcii hi , as well as high expression of genes involved in communication with nerve cells, such as c1q and cx3cr1. ural et al. showed that nams proliferated robustly following infection with influenza virus in wild-type mice. however, in mice genetically depleted of nams, infection induced an exaggerated immune response with excessive production of inflammatory cytokines and immune cell infiltration into tissues 21 . alveolar macrophages are responsible for direct clearance of viruses, whereas nams seem to be involved in moderating inflammation in the lungs during viral infection, in part by functioning as apcs, in addition to playing a possible role in neuroimmune communication. a plausible hypothesis is that these nams act in concert with neuronal cells to control inflammation, and that malfunctioning of this system in older or immunocompromised people could contribute to the cytokine storm and ards in patients with severe covid-19 or other respiratory virus infections. taken together, the findings described in this section indicate that vagus nerve activity could modulate systemic and local inflammation, probably by acting on tissue-resident immune cells, such as nams in the lungs. we have termed this functional association between immune and nerve cells the neuroimmune unit (niu), which is schematically summarized in fig. 2 . the morbidity due to infectious diseases such as respiratory syncytial virus infection, viral influenza and pneumococcal pneumonia is higher in older individuals than in the younger population, suggesting that the ability to resist infectious diseases and mount protective immune responses declines with age 70 . in addition, ageing is associated with enhanced homeostatic activation of the innate immune system, marked by elevated levels of tissue and circulating pro-inflammatory cytokines such as ifnγ, il-1β, il-6 and tnf 71 surveillance as well as an increase in the production of pro-inflammatory cytokines. following viral infection, these cytokines are further produced locally by immune cells, but their levels are potentially reduced by vagus nerve activity, leading to resolution of inflammation. with ageing, decreases in vagal immunomodulatory function and the immune cell response to pathogens, combined with increased levels of pro-inflammatory cytokines, could contribute to the induction of a cytokine storm, leading to respiratory failure and death. machr, muscarinic ach receptor; tnf, tumour necrosis factor. www.nature.com/nrneurol resulting in macrophages with reduced phagocytosis and nitrite burst activity in the presence of a markedly increased inflammatory cytokine response 72 . modulation of autophagy might preserve macrophage function during ageing, therefore reducing the morbidity and mortality associated with inflammageing 73 . the term 'immunosenescence' refers to the functional decay of immunity with age, with thymic involution being an early sign. the hallmarks of immunosenescence include an inverted cd4 to cd8 ratio, loss of naive t cells, increased numbers of terminally differentiated t cells and oligoclonal expansion of virus-specific t cells 38, 74 . the activation of natural killer (nk) cells is substantially enhanced in older individuals 75 , and latent cytomegalovirus infection has been shown to contribute to the immunosenescence of t and nk cells 76, 77 . importantly, senescent cd8 + cd28 − effector t cells lack perforin, rendering them inefficient as antigen-specific killers during viral infection 78, 79 . however, in certain diseases such as copd and rheumatoid arthritis, cd8 + cd28 − cells have been reported to express increased levels of the cytotoxic mediators perforin and granzyme b and the pro-inflammatory cytokines ifnγ and tnf, and can cause substantial tissue damage in an antigen non-specific manner 80 . ageing is also associated with changes in autonomic nervous system function, characterized by increases in sympathetic and decreases in parasympathetic nervous activity 81, 82 . a study of heart physiology showed that vagal control of heart rate and the response of the vagus nerve to stimulation decrease with age 83 . furthermore, the responsiveness of muscarinic receptors in the heart, but not in the peripheral vasculature, was shown to decline with age 83 . in line with these findings, vagus nerve stimulation (vns) can ameliorate age-related alterations in the cardiac baroreflex, heart rate and autonomic responses, and is associated with improved cardiopulmonary outcomes 84, 85 . ageing is associated with alterations in neuronal activity in multiple systems, and changes in neuropeptide release in the lung mucosa could lead to aberrant pulmonary function and immunological modulation. age-related disruption of the crosstalk between the nervous and immune systems might lead to impaired immunity, clearance and contractility of the airways and could potentially contribute to the cytokine storm and ards in people with severe covid-19. the high infectivity of sars-cov-2 and the severe morbidity associated with sars-cov-2 infection, along with our limited understanding of the biology of the virus and a lack of effective treatments or vaccines for covid-19, has made the covid-19 outbreak a global emergency. the principal lethal threats for patients with covid-19 are ards and respiratory failure. these complications of sars-cov-2 infection are predominantly observed in older 86 and immunocompromised people 86 and are mainly due to hyperreactivity and maladaptive responses of the immune system, which is unable to mount a balanced inflammatory response and instead produces a sustained, ineffective and often detrimental reaction known as a cytokine storm. age-related increases in pro-inflammatory cytokine levels and decreases in the efficiency of immune effector cells, such as macrophages or t cells, could make older and immunocompromised people particularly vulnerable to the virus. treatments that target immune cells or pro-inflammatory cytokines have been tested with some degree of success, but the role of neuroimmune crosstalk in the context of infectious and inflammatory diseases, including sars-cov-2 infection, has been largely overlooked. nevertheless, strong evidence exists of a mutual association between nerve and immune cells, with aberrant activity of one cell type affecting the other. in the context of sars-cov-2 infection, specific tissueresident macrophages that are involved in modulating inflammation following viral infection are in close contact with vagal fibres innervating the lungs, and this 'neuroimmune synapse' could be one of the keys to controlling aberrant inflammation in patients with severe covid-19. in addition, in several studies neurological symptoms have been observed in patients with covid-19 (refs 4,5,13,87 ), suggesting a possible role for the nervous system in disease progression. a feasible clinical approach to covid-19 could involve the modulation of the pulmonary niu by vns, using stimulators that are currently available to treat certain forms of pharmacoresistant epilepsy. vns has been shown to reduce lipopolysaccharide-induced expression of pro-inflammatory cytokines such as il-1β, il-6 and tnf in the brain and spleen 68 . as the vagus nerve extensively innervates the lungs, vns could potentially exert anti-inflammatory actions on the respiratory tract. vagal activity has a crucial role in lung physiology by controlling airway smooth muscle tone, vasodilation, mucus secretion and mucociliary clearance, mainly through muscarinic receptor activity 88 . in healthy individuals, muscarinic acetylcholine receptor (machr) physiology is in homeostasis; however, following sars-cov-2 infection, physiological responses may be disrupted, leading to a maladaptive inflammatory response. this disruption could be especially marked in older and immunocompromised individuals, in whom the vagal tone is typically altered and the ageing-dependent inflammatory response to viral infection is poorly controlled. the exploration of vns as a possible treatment for covid-19 is currently limited by the complexity of vagus nerve anatomy and a lack of knowledge of the optimum stimulation parameters. development of a tailored neuromodulatory approach would require a detailed investigation of vagus nerve anatomy and activity during lung inflammation ( fig. 3 ). it is known that the release of acetylcholine by vagal terminations in the airways could be important not only for smooth muscle contraction 89 , mucus secretion 90, 91 and vasodilation in pulmonary vessels 92 but also to modulate lung macrophage activity and inflammatory responses. a functional role for machrs in immune responses has been demonstrated in lung mast cells and alveolar macrophages. for instance, histamine release from mast cells is blocked by acetylcholine and other machr agonists in human bronchi 93, 94 . in support of a macrophage-dependent mechanism, m3 receptor antagonists prevent acetylcholine-induced release of chemotactic cytokines from macrophages 95 . in addition to muscarinic-dependent inflammation, nachrs have been suggested to serve as co-receptors for sars-cov-2 (ref. 96 ). the nachr pathways that respond to vns can modulate inflammation via α7nachr 69 , which is expressed by macrophages. agonists of α7nachr, including nicotine, have been proven to reduce macrophage-dependent cytokine production and inflammation in animal models of pancreatitis 97 and peritonitis 98 . thus, nachr modulation by sars-cov-2 might account for the hyperinflammatory features that are observed in a subgroup of patients with covid-19. together, these observations suggest that the use of clinically suitable acetylcholine receptor agonists or antagonists could help dampen the inflammatory response in the lung microenvironment in the context of sars-cov-2 infection. further studies are needed to clarify the role and importance of neuroimmune crosstalk both in patients with covid-19 and in animal models of covid-19 ( fig. 3 ). the use of animal models could help uncover the cellular and molecular mechanisms that underlie the mutual relationships between specific nerve fibres and selected immune cell subsets. in experimental models, chemogenetic and optogenetic techniques can be used to manipulate neuronal activity by targeting specific nerve fibres to test how they modulate immunity. selective immune cell depletion or immunomodulation could provide further insights into neuroimmune communication. in the clinic, the neuroimmune synapses could be visualized by electron microscopy and further analysed by immunohistochemistry in human biopsy or post-mortem tissues. human sputum or blood samples, as well as lung parenchyma obtained by biopsy, could also be tested for the presence of specific cytokines and cell types. the presence of ace2 on neurons makes them susceptible to sars-cov-2 infection, which could lead to neuronal damage and aberrant signalling. afferent and efferent vagal fibres are functionally and molecularly distinct as they convey information in opposite directions, release specific neurotransmitters and express unique combinations of receptors that could communicate with different immune cell subtypes. these fibres comprise a sensory-motor feedback loop that regulates physiological respiratory functions, the disruption of which by viral infection could cause respiratory dysfunction. the vagus nerve could also provide an entry point for the virus to the cns, including areas of the brain that control respiration, such us the nts. the nts is involved in the control of several critical functions besides respiration that might be affected following infection, including cardiac rhythm and food intake regulation. therefore, vagal infection may result not only in respiratory distress but also in other symptoms such as cardiac dysfunction or loss of appetite. these hypotheses could be tested experimentally using chemogenetic and optogenetic techniques to selectively activate or inhibit afferent or efferent vagal tracts and examine whether this modulation affects immune-dependent and immune-independent respiratory, cardiac and other autonomic functions in health and disease. in line with our niu model, therapeutic targeting of transient receptor potential cation channel subfamily v member 1 (trpv1)-expressing nerve fibres in the lungs, using the trpv1 agonist resiniferatoxin, has been suggested to modulate inflammatory and immune signalling activity, leading to reduced mortality and better overall outcomes in people with covid-19 (ref. 99 ). although this approach seems promising, several potential downsides need to be considered. trpv1 is expressed only by a small subset of chemoreceptor fibres (the c-fibres), but the majority of afferent fibres innervating pulmonary structures are carried by vagal or dorsal root ganglion fibres from the upper thoracic segments. in addition, resiniferatoxin is an ultra-potent agonist that binds to and forces the opening of the trpv1 channel, leading to a sustained increase in intracellular calcium levels 100 . this, in turns, disrupts mitochondrial metabolism and results in permanent neurolysis, making this drug potentially toxic for nerve cells and pulmonary neuroimmune homeostasis. another potentially interesting unexplored target is the neuropeptide cgrp. cgrp antagonism could limit bronchoconstriction and vasodilation and limit the inflammatory response 62 , and the recent development and commercialization of anti-cgrp and anti-cgrp receptor monoclonal antibodies for the treatment of migraine might enable fast repurposing for ards. similarly, neurokinin a receptor antagonists, which antagonize the binding of both neurokinin a and the closely related substance p, have been developed for clinical use against nausea and vomiting following surgery and chemotherapy 101, 102 , and could be repurposed to counteract lung inflammation and bronchoconstriction in people with covid-19. nausea and vomiting, which are controlled by the area postrema, nts and dorsal motor nucleus of the vagus, have also been reported in patients with covid-19 and could potentially be treated with neurokinin a receptor antagonists. additional drugs that specifically modulate the niu, such as bombesin antagonists, might be developed to improve clinical outcomes in patients with covid-19 and could also be tested in a broader range of neuroimmune diseases. in conclusion, the integrated niu model that we have proposed suggests novel strategies for the development of targeted and effective treatments that could ameliorate the symptoms and mitigate the life-threatening consequences of sars-cov-2 infection. a strategic roadmap for further research and therapeutic development is provided in fig. 3 . in the long term, this model might also provide a new perspective for the discovery of effective therapies for a broad range of inflammatory and infectious diseases and the discovery of potentially novel biological phenomena. fig. 3 | studying the neuroimmune unit: a roadmap. the diagram depicts a stepwise roadmap to increase our understanding of the neuroimmune unit and the potential discovery of pharmacological, genetic and physiological treatments that could be used in the clinic. cgrp, calcitonin gene-related peptide. www.nature.com/nrneurol clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin cov-2: olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection concomitant neurological symptoms observed in a patient diagnosed with coronavirus disease 2019 neurologic manifestations of hospitalized patients with coronavirus disease involvement of the nervous system in sars-cov-2 infection psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and meta-analysis with comparison to the covid-19 pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the central nervous system tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell rna-seq novel coronavirus disease (covid-19) and central nervous system complications: what neurologist need to know brain angiotensin-converting enzymes: role of angiotensin-converting enzyme 2 in processing angiotensin ii in mice anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice angiotensin-converting enzyme 2 in the rostral ventrolateral medulla regulates cholinergic signaling and cardiovascular and sympathetic responses in hypertensive rats angiotensin-converting enzyme 2: central regulator for cardiovascular function avian flu: influenza virus receptors in the human airway h5n1 virus attachment to lower respiratory tract a critical function for cd200 in lung immune homeostasis and the severity of influenza infection identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties regulating the adaptive immune response to respiratory virus infection respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic cd8+ t cell responses covid-19 infection: the perspectives on immune responses pathological findings of covid-19 associated with acute respiratory distress syndrome immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing covid-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome a new coronavirus associated with human respiratory disease in china dysregulation of immune response in patients with covid-19 in wuhan, china clinical and immunological features of severe and moderate coronavirus disease 2019 immunopathogenesis of coronavirus infections: implications for sars middle east respiratory syndrome clinical features of 85 fatal cases of covid-19 from wuhan. a retrospective observational study hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis il-6 signalling pathway inactivation with siltuximab in patients with covid-19 respiratory failure: an observational cohort study is a "cytokine storm" relevant to covid-19? ageing and the immune system: focus on macrophages age related human t cell subset evolution and senescence age is not just a number: naive t cells increase their ability to persist in the circulation over time innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves the sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry chronic obstructive pulmonary disease overview of neural mechanisms in asthma evidence for prejunctional muscarinic autoreceptors in human and guinea pig trachea parainfluenza virus infection damages inhibitory m2 muscarinic receptors on pulmonary parasympathetic nerves in the guinea-pig dysfunction of m2-muscarinic receptors in pulmonary parasympathetic nerves after antigen challenge expression of muscarinic receptors by human macrophages interleukin-1β mediates virus-induced m2 muscarinic receptor dysfunction and airway hyperreactivity virus-and interferon-induced loss of inhibitory m2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons ifnγ increases m2 muscarinic receptor expression in cultured sympathetic neurons severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection an airway protection program revealed by sweeping genetic control of vagal afferents piezo2 senses airway stretch and mediates lung inflation-induced apnoea mechanisms in copd: differences from asthma pulmonary neuroendocrine cell system in health and disease functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors pulmonary neuroendocrine cells. their secretory products and their potential roles in health and chronic lung disease in infancy functional facets of the pulmonary neuroendocrine system the role of virus-specific immunoglobulin e in airway hyperresponsiveness calcitonin gene-related peptide as inflammatory mediator bombesin-related peptides and their receptors: recent advances in their role in physiology and disease states immunomodulatory functions of the diffuse neuroendocrine system: implications for bronchopulmonary dysplasia the vagus nerve in the neuro-immune axis: implications in the pathology of the gastrointestinal tract anti-inflammatory effects of abdominal vagus nerve stimulation on experimental intestinal inflammation the vagus nerve and the inflammatory reflex -linking immunity and metabolism electric stimulation of the vagus nerve reduced mouse neuroinflammation induced by lipopolysaccharide α7 nicotinic acetylcholine receptor (α7nachr) expression in bone marrowderived non-t cells is required for the inflammatory reflex influenza-and respiratory syncytial virus-associated mortality and hospitalisations age-associated increased interleukin-6 gene expression, late-life diseases, and frailty autophagy controls acquisition of aging features in macrophages pulmonary macrophages: key players in the innate defence of the airways ageing and life-long maintenance of t-cell subsets in the face of latent persistent infections the impact of ageing on natural killer cell function and potential consequences for health in older adults aging and the immune system: the impact of immunosenescence on viral infection, immunity and vaccine immunogenicity immunosenescence: implications for response to infection and vaccination in older people hiv-specific cd8 + t cells exhibit reduced and differentially regulated cytolytic activity in lymphoid tissue harnessing cd8 + t cells under hiv antiretroviral therapy steroid resistant cd8 + cd28 null nkt-like pro-inflammatory cytotoxic cells in chronic obstructive pulmonary disease effect of aging on gender differences in neural control of heart rate aging of the autonomic nervous system and possible improvements in autonomic activity using somatic afferent stimulation changes in vagal activity and response to muscarinic receptor agonists with age effects of transcutaneous vagus nerve stimulation in individuals aged 55 years or above: potential benefits of daily stimulation vagus nerve and vagus nerve stimulation, a comprehensive review: part i sars-cov-2 and covid-19 in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes the international european academy of neurology survey on neurological symptoms in patients with covid-19 infection muscarinic receptor antagonists: effects on pulmonary function sympathetic versus parasympathetic nervous regulation of airways in dogs nervous control of mucin secretion into human bronchi on muscarinic control of neurogenic mucus secretion in ferret trachea parasympathetic nervous control of tracheal vascular resistance in the dog acetylcholine via muscarinic receptors inhibits histamine release from human isolated bronchi muscarinic control of histamine release from airways. inhibitory m1-receptors in human bronchi but absence in rat trachea glucocorticoids mediate reduction of epithelial acetylcholine content in the airways of rats and humans a nicotinic hypothesis for covid-19 with preventive and therapeutic implications the vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice the cholinergic antiinflammatory pathway regulates the host response during septic peritonitis the role of afferent pulmonary innervation in poor prognosis of acute respiratory distress syndrome in covid-19 patients and proposed use of resiniferatoxin (rtx) to improve patient outcomes in advanced disease state: a review deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control human substance p receptor binding mode of the antagonist drug aprepitant by nmr and crystallography aprepitant: a review of its use in the prevention of nausea and vomiting the authors thank p. muraro, m. botto and r. reynolds for their valuable critical insight and suggestions. both authors contributed to all aspects of the article. the authors declare no competing interests. nature reviews neurology thanks j. sellner, f. chigr and other, anonymous, reviewer(s) for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-290233-5skk3nj4 authors: wang, k.; kang, s.; tian, r.; zhang, x.; zhang, x.; wang, y. title: imaging manifestations and diagnostic value of chest ct of coronavirus disease 2019 (covid-19) in the xiaogan area date: 2020-03-23 journal: clin radiol doi: 10.1016/j.crad.2020.03.004 sha: doc_id: 290233 cord_uid: 5skk3nj4 aim: to report the epidemiological, clinical, and radiological characteristics of patients with covid-19 in xiaogan, hubei, china. materials and methods: the complete clinical and imaging data of 114 confirmed covid-19 patients treated in xiaogan hospital were analysed retrospectively. data were gathered regarding the presence of chest computed tomography (ct) abnormalities; the distribution, morphology, density, location, and stage of abnormal shadows on chest ct; and observing the correlation between the severity of chest infection and lymphocyte ratio and blood oxygen saturation (spo(2)) in patients. results: chest ct revealed abnormal lung shadows in 110 patients. regarding lesion distribution, multi-lobe lesions in both lungs were present in most patients (80 cases; 72.7%). lesions most frequently involved both the peripheral zone and the central zone (62 cases; 56.4%). regarding lesion morphology, 56 cases (50.1%) demonstrated patchy shadows that were partially fused into large areas. thirty cases showed ground-glass opacity (27.3%), 30 cases showed the consolidation change (27.3%), and the remaining 50 cases showed both types of changes (45.4%). the progressing stage was the most common stage (54 cases; 49.1%). ct results showed a negative correlation with spo(2) and lymphocyte numbers (p<0.05), with r-values of −0.446 and −0.780, respectively. conclusion: spiral ct is a sensitive examination method, which can be applied to make an early diagnosis and for evaluation of progression, with a diagnostic sensitivity and accuracy better than that of nucleic acid detection. coronaviruses are single-stranded positive-strand rna viruses belonging to the genus nestiviridae, coronaviridae, and orthokinovirus subfamily. 1, 2 at present, epidemiological and clinical characteristics and imaging data of coronavirus disease 2019 (covid-19) are lacking. this study analysed the epidemiological and clinical characteristics and imaging data of 114 patients diagnosed with covid-19 admitted to xiaogan hospital, xiaogan, hubei, china, to describe the lung imaging manifestations and disease development in patients with covid-19, further explore the correlations between imaging manifestations and clinical data, and clarify the role of chest computed tomography (ct) imaging examination in the diagnosis and follow-up of this disease. the aim of the present study was to inform the global community about the emergence of this novel coronavirus and its ct imaging characteristics and clinical characteristics. the study comprised 114 patients with confirmed covid-19 treated at xiaogan hospital from 25 january 2020, to 9 february 2020, with complete medical records. the study was approved by the hospital's ethics committee and written informed consent was obtained from patients involved before enrolment when data were collected retrospectively. all patients underwent chest ct examination at the time of admission. a ge discovery ct 750 hd ct system was used for spiral ct of the entire lungs with a tube voltage of 120 kv, tube current of 320 ma, field of view (fov) of 500 mm, collimator width of 0.5e1.5 mm, and layer thickness and layer spacing of 1e5 mm. for severe and critical cases, the pitch was increased appropriately (1e1.5 pitch) to reduce both the scanning time and respiratory motion artefacts. epidemiological data, symptoms, and physical signs at the time of admission, laboratory test results, and the ct results at admission of all diagnosed covid-19 infected patients were analysed retrospectively. the data were obtained through the inpatient medical record system (his) and image storage and transmission system (pacs). in cases where electronic records were unavailable or epidemiological and symptomatic data were unclear, data were obtained through telephone communication with the doctor in charge or the patient and the patient's family. spss 19.0 software was used for statistical analysis. data fitting a normal distribution were represented using the mean (standard deviation [sd]), and data with a nonnormal distribution were represented using the median value (interquartile range [iqr] ). count data were represented using case numbers and percentages. spearman's test was used to analyse the correlation between measurement data and ordered classification variables. of the 114 cases, 90 (78.9%) patients had a history of residence or travel in wuhan, or a history of contact with people from wuhan, including one medical personnel who was infected at work. the remaining 24 cases (21.1%) had no clear epidemiological history. there were 58 males (50.9%) and 56 females (49.1%). one-hundred and eleven patients (93.9%) had fever in this study, including 91 cases (79.8%) with cough, nine cases (7.9%) with sputum, six cases (5.3%) with sore throat, 27 cases (23.7%) with chest tightness, 27 cases (23.7%) with dyspnoea, and three cases (2.7%) with diarrhoea. the complaints of 107 patients (93.9%) included the above multiple symptoms, and 18 patients (15.8%) had fever, cough, and dyspnoea, who constituted the largest subgroup. sixty patients (52.6%) had underlying diseases, of whom 33 patients (28.9%) had hypertension, seven patients (6.1%) had cardiovascular diseases, five patients (4.4%) had digestive diseases, 15 patients (13.2%) had endocrine system diseases, one patient (0.9%) had neural system disease, one patient (0.9%) had a malignant tumour, and five patients (4.4%) had respiratory diseases (table 1 ). all patients underwent chest ct imaging examination. three patients (2.6%) showed no obvious abnormalities in the lungs at the time of initial diagnosis, and one patient (0.9%) showed insufficient dilation of the right middle and lower lung, no obvious abnormalities in the lung lobes, and moderate effusion in the right thoracic cavity. the chest ct imaging manifestations of the remaining 110 cases (96.5%) are described below (table 2 ). the lung field was divided into the peripheral zone and the central zone according to the halfway point of the distance from the chest wall of the same layer to the hilum of the lung. the zone near the lung hilum is the central zone, and the zone far from the hilum is the peripheral zone. lesions located between the two zones or covering both zones were recorded as involving both zones. in the present study, the lung lesions of 48 cases (43.6%) were located in the peripheral zone and lesions of 62 cases (56.4%) involved both the peripheral and the central zones (fig 1) . regarding the distribution of the lesions in the lung lobes in the first chest ct, 10 cases involved one lobe of one lung (9.1%), six cases involved multiple lobes of one lung (5.5%), 80 cases involved multiple lobes of both lungs (72.7%), four cases involved bilateral lower lungs (3.6%), and 10 cases involved bilateral middle and lower lungs (9.1%). the lesions were patchy and partially fused in 56 cases (50.1%), the lesions were spherical in 28 cases (25.5%), and the remaining 26 cases (14.5%) showed both types of lesions (fig 2) . according to the level of pneumonia infiltration density on the ct images, the lesions were divided into groundglass opacity (ggo) or consolidation. ggo is defined as a reduction in local transparency of the lung tissue, light shadow with uneven density, and visible lung texture inside the lesion, which is usually seen on lung windows and not on mediastinal windows, or with a shadow size significantly smaller than revealed on the lung window. the density of consolidation change is higher than that of ggo, with even density and no visible lung texture inside the lesion, it may merge into a large area as inflammation progresses, and some lesions show the air bronchogram sign. in this group of patients, 30 cases showed ggo (27.3%), 30 cases showed the consolidation change (27.3%), and the remaining 50 cases showed both types of changes (45.4%). according to the lesion characteristics described above, early stage manifestations were defined as mainly subpleural patchy, lumpy segment, or sub-segment ggo; the progressing stage manifestations were defined as ggo and consolidation involving multiple lobes of both lungs; and severe stage manifestations were defined as diffuse lesions of both lungs, a few patients might manifest the typical white lung sign, and the air bronchogram sign was common. in this study, 30 patients were in an early stage (27.3%), 54 were in a progressing stage (49.1%), and 26 were in a severe stage (23.6%). sixteen patients underwent 2e4 ct examinations in the outpatient and inpatient department, with an interval of 4e19 days. twelve patients (75%) showed disease progression, manifesting enlarged lesions, and/or denser lesions and thickened cord-like shadow. the patients were divided into mild, moderate, and severe disease progression based on the extent of the lesion enlargement and the degree of consolidation, including four mild cases (25%), five moderate cases (31.2%), and zero severe cases (0%). some patients with moderate progression showed the air bronchogram sign in lesions. four (25.0%) cases showed slight mitigation of the lesions, demonstrating a slight reduction of the lesion size of <5% (fig 3) . there were specific signs seen at ct comprising (1) the batwing sign: a large symmetrical shadow around the bilateral lung hila, which looks like bat or butterfly wings; (2) the white lung sign: also known as "blizzard lung", showing diffusely distributed flocculent, nodular, and flaky high-density shadows in both lungs, which partially merge and consolidate; (3) the rosa roxburghii sign: demonstrating semi-round ggo distributed in the periphery zone of lung lobes, which look like the rose, rosa roxburghii; (4) the gypsum sign: manifesting as patchy consolidation in the lung lobes with different densities. among these signs, an isolated batwing sign or multiple rosa roxburghii signs are characteristic ct manifestations of the disease (fig 4) . the range of spo 2 under the natural breathing conditions of the patients in this study was 70e99%. spearman's correlation statistical analysis was performed on spo 2 and ct staging with results of p<0.05 and r¼e0.446, suggesting a weak negative correlation between the two. the lymphocyte numbers of the patients were estimated from the routine blood test data at the time of admission or initial diagnosis. spearman's correlation statistical analysis was performed on lymphocyte numbers and ct staging with results of p<0.05 and r¼à0.780, suggesting a moderate negative correlation between the two. ct staging and hypersensitive c-reactive protein hypersensitive c-reactive protein level of the patients was estimated from the routine blood test data at the time of admission or initial diagnosis. spearman's correlation statistical analysis was performed on c-reactive protein level and ct staging with results of p>0.05 indicating no correlation between the two. patient 1, a 52-year-old woman with a history of hypothyroidism, was admitted on 21 january 2020, complaining of fever of 5 days duration with 38.6 c as the highest body temperature. the patient denied a history of contact with the wuhan south china seafood market and people from wuhan. spo 2 at admission was 98%, hypersensitive creactive protein was 3.18 mg/l, and the lymphocyte number was 2.16â10 9 /l. the first real-time reverse transcription polymerase chain reaction (rt-pcr) of her respiratory specimen for viral nucleic acid was negative. ct showed multiple scattered ggo shadows in the periphery zone of both lungs with a typical rosa roxburghii sign, suggesting a high possibility of viral pneumonia in both lungs. the second real-time rt-pcr of respiratory specimen for viral nucleic acid was positive. patient 2, a 56-year-old man whose wife was diagnosed with covid-19, did not have any discomfort or abnormal clinical indicators. chest ct examination was performed during the quarantine period, and the result showed two ggo nodule shadows in both lower lungs, suggesting a high possibility of viral pneumonia. realtime rt-pcr of his respiratory specimens for viral nucleic acid was negative three times. the patient considered highly suspicious for infection and was monitored. the fourth real-time rt-pcr of his respiratory specimen for viral nucleic acid was positive, and the patient was diagnosed with an infection. patient 3, a 43-year-old woman, complained of fever for 1 day with the highest body temperature of 38.5 c. the patient denied a history of contact with the wuhan south china seafood market and people from wuhan. the spo 2 at admission was 99%, hypersensitive c-reactive protein was 2.44 mg/l, and her lymphocyte count was 2.88â10 9 /l. chest ct showed no significant abnormalities in either lung. the first real-time rt-pcr of respiratory specimen detecting viral nucleic acid was positive, and the patient was diagnosed with covid-19. covid-19 can spread explosively in local areas or worldwide. the latency period of this disease is 1e14 days, mostly 3e7 days. 3, 4 the clinical manifestations are mainly fever and fatigue, but some patients may also be asymptomatic virus carriers, with mild-onset symptoms and no fever. the lesion density of lung inflammation is very low, mostly demonstrating cloud-like changes and ggo. the lesions are relatively localised, with patchy, sub-segmental, or segmental distribution. they are mainly distributed underneath the pleura with uneven density. these ct manifestations reflect the pathological process of lung injury in patients with viral pneumonia 3,4 and the distribution of most of the lesions are in the peripheral zone of the lungs, peripheral bronchus, and alveoli. this distribution may be related to the infection mode of viral pneumoniadrespiratory droplet transmission. with the progression of the disease, the number of lesions increases and expands to involve multiple lung lobes, and the lesions become denser. ggo coexists with consolidation or cord shadows. if viral pneumonia has not been effectively inhibited clinically, it progresses to the severe stage, demonstrating diffuse lesions in both lungs, and white lung with air bronchogram sign in a few cases. chest ct examination has the characteristics of short examination time and high-density resolution. under the current situation, chest ct can provide examinations to a wide range of patients who are suspected, confirmed, and under observation as one of the main diagnostic methods of covid-19 and even as the initial examination on admission. the diagnostic value of chest ct mainly lies in the detection of lesions (even early lesions that are easily missed at radiography), characterisation of lesions, and assessment of severity to facilitate further clinical classification and treatment. the current clinical situation is complicated. among the patients in the present study, the following conditions were observed: (1) the patient had a fever, no epidemiological history, and abnormal manifestations in the lungs on ct, while the first nucleic acid test was negative, and the second test was positive; (2) the patient had symptoms, a history of exposure, and abnormal manifestations in the lungs on ct, while the first two nucleic acid tests were negative, and the third was positive; (3) the nucleic acid test was positive; however, the patient was asymptomatic, had an epidemiological history, and ct showed no abnormal manifestations in the lungs. three days later, the ct examination showed abnormal findings in the lungs. from the above analysis, it is evident that in this specific period and under the current clinical situation, patients with an abnormal chest ct examination should be quarantined, and the patient's epidemiological history should be investigated. after consideration of other test results, and excluding influenza and mycoplasma infection, at least one or more sars-cov-2 nucleic acid tests are required; however, some studies have shown that the accuracy rate of the ct examination in the diagnosis of sars-cov-2 infection was only 76.4%. only three cases (2.6%) in this study showed no apparent abnormalities in the lungs at the initial ct examination, and the overall accuracy rate of ct examination in the present study was 97.3%, which is much higher than the accuracy reported in the literature. the reason may be that the present study is a retrospective analysis and, in order to summarise the ct imaging characteristics better, the selected cases were all confirmed cases. regarding follow-up, ct can dynamically observe the changes in lung lesions, provide objective and fair data to monitor disease progression or improvement, and assess the changes of lung lesions, such as shrinkage, expansion, or absorption, dissipation or densification, and fibre strand formation. second, ct is easy to operate and highly reproducible. the same scan plane of multiple ct examinations can be compared and observed through the pacs system, which will demonstrate an evolving process of disease progression. lymphocyte count and spo 2 , as commonly used clinical indicators, can reflect the degree of inflammation absorption and the severity of the disease and indicate the prognosis of the disease. a negative correlation was found between the ct stage and spo 2 and lymphocyte number were analysed in the present study; that is, the higher the ct stage, the lower the spo 2 and the lower the lymphocyte number. there was no correlation between ct stage and hypersensitive c-reactive protein. therefore, the changes of spo 2 and lymphocyte numbers can reflect the progress of ct imaging to a certain extent, although the correlation was not significant. therefore, the most intuitive index for monitoring the absorption of inflammatory lesions in the lungs is chest ct. spo 2 and the lymphocyte number can be used as reference indexes, while hypersensitive c-reactive protein has no reference value. in conclusion, currently, the diagnosis of covid-19 requires comprehensive consideration of exposure history, clinical manifestations, laboratory tests, and imaging examinations. 5e8 according to the latest version of the covid-19 diagnosis and treatment plan issued by the ministry of health, lung ct is the main diagnostic method for covid-19 suspected cases and one of the leading observation indicators for judging whether a patient can be discharged. therefore, lung ct has an irreplaceable role in the screening and diagnosis of covid-19, monitoring disease progression, and assessing whether the patient can be discharged. the following recommendations are proposed based on the results of this study: (1) each patient with typical clinical symptoms should undergo a lung ct examination. patients with typical lung signs should be included in the clinically suspected cases for quarantine. for patients without visible lung signs but with clinical symptoms, lung ct should be rechecked in 3e5 days. (2) positive lung ct results instead of the positive nucleic acid test should be used as a criterion for admission to the quarantine ward. (3) hospitalised patients should undergo ct examinations every 5e7 days, and a low-dose scan should be employed. in summary, clinicians and radiologists should recognise the importance of chest ct in the diagnosis and treatment of covid-19, be familiar with the features and diagnostic points of the chest imaging of covid-19, and strengthen communication within the radiology community, which is especially crucial in the battle against covid-19. outbreak of pneumonia of unknow etiology in wuhan china: the mystery and the miracle epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing pre-and posttreatment chest ct findings: 2019 novel coronavirus (2019-ncov) pneumonia ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan initial ct findings and temporal changes in patients with the novel coronavirus pneumonia (2019-ncov): a study of 63 patients in wuhan, china the authors declare no conflict of interest. key: cord-265606-c1zo47sw authors: feng, zhe-min; zhuang, zhen-jie; he, wen-bo; ding, jian-ping; yang, wen-jun; chen, xue-yuan title: lung cancer with diffuse ground-glass shadow in two lungs and respiratory failure date: 2016-08-05 journal: chin med j (engl) doi: 10.4103/0366-6999.186632 sha: doc_id: 265606 cord_uid: c1zo47sw nan pulmonary ground-glass shadow is a common clinical imaging manifestation shared by many pulmonary diseases such as interstitial pneumonia, pulmonary fungal infection, parasitic infection, viral pneumonia, and heart failure. some of the lung cancers, especially lung adenocarcinoma, can also present ground-glass-like nodules. the early diagnosis and differential diagnosis of the ground-glass shadow are very important for lung cancer. even though there are a lot of studies in this field in recent years, diffuse and uniform ground-glass opacity is rarely reported in lung adenocarcinoma. in this study, a case of lung adenocarcinoma complicated with respiratory failure is reported to show diffuse uniform ground-glass shadow in the chest computed tomography (ct). in addition, we discuss this case in the context of related literation hoping clinical and imaging doctors could be aware of this in clinical practice. a 56-year-old female patient was admitted to hangzhou normal university affiliated hospital on november 12, 2014. she had fever for 1 week with the highest temperature reaching 39°c, coughed white sputum with no blood or yellowish stuff, and complained chest tightness and dyspnea when severe coughing. she felt nausea and even vomit in the course but no coffee-like objects were vomited. she was treated with intravenous cefuroxime and levofloxacin in outpatient service for 3 days without expected improvement. she had been diagnosed as type 2 diabetes in the past and had been given metformin and acarbose treatment. clozapine and trihexyphenidyl and other drugs were daily administrated orally to treat schizophrenia diagnosed previously. she has no smoking and drinking history, no omophagia fish, and shrimp history. no obvious abnormalities were found in the regular physical examination and chest x-ray scan [ figure 1a ] performed a half year before. physical examination showed body temperature of 38.2°c, pulse rate of 89/min, respiratory rate of 22 breaths/min, blood pressure of 125/70 mmhg (1 mmhg = 0.133 kpa), and pulse oxygen saturation of 85% (concentration of oxygen inhalation: 21%). her consciousness was clear,with poor spirit and mild cyanosis. no enlarged superficial lymph nodes were found and widely moist rales could be heard in two lungs. her heart rate was 89 beats/min with no pathological murmur. the abdomen is soft, no lower limbs dropsy. on auxiliary examination, chest ct scan [ figure 1b -1e, november 12, 2014] found diffused uniform ground-glass shadow in two lungs with no enlarged mediastinal lymph nodes. on admission, preliminary diagnosis revealed: (1) diffuse lung disease of unknown origin, respiratory failure, (2) type 2 diabetes, and (3) schizophrenia. after admission, the patient presented high fever and pulmonary diffuse exudate with respiratory failure. considering previous history of type 2 diabetes, we suspected pulmonary infection with unknown pathogen. oxygen therapy and hypoglycemic therapy were given. in addition, 4.5 g of piperacillin-tazobactam by intravenous drip bid and 0.4 g of moxifloxacin once a day (qd) combined with anti-infection and cough expectorant were given to relieve symptoms and simultaneously improve the relevant inspection. laboratory examination: routine blood test + high-sensitive c-reactive protein (hs-crp), leukocyte 19.67 × 10 9 /l, percentage of neutrophils 91.0%, 6.1% of lymphocyte percentage and 0 of eosinophil cell percentage, red-cell count 4.65 × 10 12 /l and 130 g/l of hemoglobin, platelet count of 216 × 10 9 /l, hs-crp 30.99 mg/l. arterial blood gas analysis: ph 7.31, partial pressure of carbon dioxide in artery 27.10 mmhg, arterial partial pressure of oxygen 57.90 mmhg, arterial oxygen saturation 87.10%. b-type natriuretic peptide: 65.30 pg/ml. allergen determination: total immunoglobulin e 11.50 iu/ml, no specific allergen was detected. procalcitonin <0.5 ng/ml. antinuclear antibodies, double-stranded-dna, anti-sjogren syndrome a antibody, anti-sjogren syndrome b antibody and other autoantibodies were all negative. perinuclear antineutrophil cytoplasmic antibody, cytoplasmic antineutrophil cytoplasmic antibodies and myeloperoxidase antibody was negative. tumor screening: α-fetoprotein -3.61 ng/ml, carcinoembryonic antigen (cea) -2.5 ng/ml, carbohydrate antigen (ca) 199-25 ku/l, ca 125-14 u/ml, all were in the normal range. interferon gamma release test for tuberculosis infection: negative. 1,3-beta-glucan test: 300.2 pg/ml (reference value <100.5 pg/ml); galactomannan test: negative. nucleic acid detection of influenza a virus (throat swab): negative; human immunodeficiency virus antibody: negative, urine cytomegalovirus dna detection: negative. acid fast bacilli is negative in 3 smear tests. sputum cytology found no tumor cells. electrocardiograph: sinus rhythm. cough, shortness of breath gradually worsened after 3 days treatment with piperacillin-tazobactam combined with moxifloxacin. body temperature had been fluctuating between 37°c and 38°c since the admission. the patient was administered with 0.2 g voriconazole by intravenous drip q12h and 40 mg methylprednisolone by intravenous qd to inhibit fungal infections and inflammation for 4 days. intermittent noninvasive ventilation was given as needed. on the 5 th day of admission, electronic bronchoscopy was performed and showed that the trachea and bronchial lumen was smooth and the mucosa was congested. a huge amount of white foam sputum was found in the tracheal cavity to be constantly emitted after suction; no stenosis and neoformation, no cheese-like necrosis tissue was observed. oxygen saturation increased from 70% to 80% to above 93% after foamy sputum was suctioned. transbronchial lung biopsy (tblb) was performed in the right lower lung and tblb pathology reported adenocarcinoma [ figure 1f ]. epidermal growth factor (egfr) gene and anaplastic lymphoma kinase-echinoderm microtubule-associated protein-like (alk-eml4) fusion gene were further analyzed, and no mutation was found. the patient was given antitumor chinese traditional medicine, anti-infection treatment, and nutritional support together with noninvasive ventilation and other treatment to relieve symptom. the patient died of respiratory failure on the 20 th day after admission. diffuse pulmonary effusion in the two lungs in imaging examination is very common. in clinical practice, the chest ct scan showed ground-glass-like change due to diffuse exudation in two lungs often suggests special pathogen infection (such as hematogenous pulmonary tuberculosis, pulmonary fungal infection, and viral pneumonia), exogenous allergic pulmonary inflammation, interstitial pneumonia, acute pulmonary edema, etc. for this kind of imaging pattern, infectious and noninfectious diseases are two aspects routinely considered in diagnosis and differential diagnosis. the hematogenous pulmonary tuberculosis is characterized by toxic symptoms of tuberculosis infection such as fever, night sweats, cough, and expectoration. in the chest x-ray, small nodular shadows with special pattern in size, distribution, and uniformity of the density are found in the two lungs in the acute stage. the diameter of the nodules is usually in the range of 1-3 mm, and the density and the distribution are even. while in subacute and chronic hematogenous pulmonary tuberculosis, nodules are mainly located in the middle and upper lung, with the diameter varies in a wider range of 3-7 mm. the density and distribution of these nodules are uneven, and some are with the patches and cord-like exudation. the pulmonary shadow occurs 1-3 weeks behind the clinical symptoms, and most of the lesions are fused into two pulmonary diffuse shadows. viral pneumonia is inflammation caused by viral invasion of lung parenchyma and interstitial. the chest ct mainly manifests features of the interstitial pneumonia such as increased lung texture, the ground-glass-like shadow, and small patch or the like. severe viral pneumonias, such as highly pathogenic avian influenza virus infection, severe acute respiratory syndrome virus infection, often progress rapidly into dangerous condition. cytomegalovirus pneumonia, frequently happen to immunocompromised patients, shows imaging of cloud and mist effusion in two lungs. examination of the virus dna or rna in the blood, body fluids and secretions of the viral pneumonia patients, and serum virus-specific antibodies can help diagnosis. pneumocystis carinii pneumonia is prone to immune function defect patients. diffuse ground-glass opacity is the most distinguished imaging features. with the progress of this disease, the shadow expands rapidly from hilus, then developed alveolar consolidation. finding of hydatid in specific staining such as giemsa staining, methylene amine blue staining, and gomori daya hexamine silver staining in sputum smear, bronchoalveolar lavage fluid smear, or tblb specimen can be used as the basis of diagnosis. detection of serum antibody and complement binding test can also provide evidence for diagnosis. acute allergic pulmonary inflammation is a lung disease caused by immune response induced by exposure to organic dust antigen in susceptible individuals. ct scan shows ground-glass shadow, diffusedly located in both lungs or mainly in lower lung. micronodules can be seen sometimes, with blurred edge and less than 3 mm in diameter. symptoms can be relieved after antigen removal. acute interstitial pneumonia is acute onset, manifested as glass-like change with fuzzy hair, or a wide range of distribution of the linear, reticular, small nodules, or even the consolidated shadow. the chest x-ray performed half a year ago before the patient was admitted to hospital was reviewed and it showed clear lung fields with no obvious abnormalities. at the time of admission, the chest ct showed diffuse exudation, uniform ground-glass shadow, which is more obvious in the right lung, visible air bronchogram sign in the right lower lobe of the lung. considering the patient is with acute onset, fever and chills, and elevated leukocyte counts and hs-crp inflammatory index while tumor marker test were within normal range, we first suspected infectious diseases on admission based on type 2 diabetes histories. through examination, the pulmonary tuberculosis, pulmonary spore bacteria pneumonia, cytomegalovirus pneumonia, human infection with highly pathogenic avian influenza, and other diseases such as severe viral pneumonia were excluded. congest heart failure and acute pulmonary edema are also excluded. therefore, pathologic analysis is very important to disease diagnosis and treatment. the types of lung cancer from different anatomic sites also differ. the central type of lung cancer is mainly squamous cell carcinoma and small cell carcinoma while peripheral lung cancer is mostly adenocarcinoma. it was reported that the pathological types of lung cancer with diffuse exudation in chest ct were adenocarcinoma, [1] which mainly occurred in clara cells, type ii alveolar epithelial cells, and mucous cells. in diffuse lung adenocarcinoma, cancer cells usually grow invasively along alveolar and alveolar ducts, spread along the lymphatic vessels, or metastasis to bilateral lungs by the way of lymph and blood. [2, 3] as a result, diffuse pulmonary adenocarcinoma in the x-ray manifests pulmonary diffuse nodular or patchy shadow. in the chest ct [4] examination, it shows multiple or diffuse nodules or patchy exudation shadow. nodules are miliary, occasionally acinar with size of 3-5 mm in diameter, fuzzy edge could be found in some lesions. the nodules in the lungs are distributed randomly, asymmetrically, and unevenly with various densities. patchy shadow commonly shows multiplely and diffusedly. these lesions often merge with each other, involving a segment or the entire lung. the patient was diagnosed as lung adenocarcinoma by tblb examination. for the pathological diagnosis of diffuse pulmonary disease, percutaneous lung puncture and tblb is a common clinical biopsy method. compared with percutaneous lung puncture, tblb has the advantages of simplicity, fast, and safeness. in clinical work, safe, effective method of biopsy should be selected according to the severity of the patient's condition, imaging performance, general situation, and other factors. the positive rate of tblb in diffuse lung adenocarcinoma was about 58.3%. [5] the pathological diagnosis of diffuse lung adenocarcinoma patients was more advanced, no surgical opportunity. [6] genes analysis of egfr, alk-eml4 fusion gene, k-ras, [7] etc., should be routinely performed. the patient with corresponding mutation might benefit more from egfr tyrosine kinase inhibitor, new angiogenesis inhibitors, and other targeted drug treatment. the remarkable diffuse, uniform grinding glass-like shadow is different from the nodular or patchy shadow of majority diffuse lung adenocarcinoma, but rarely reported. moreover, with fever, increased inflammation index, normal cea, and other tumor marker test together with diabetes history, it is prone to be misdiagnosed as fungal infection, cytomegalovirus pneumonia, interstitial pneumonia, allergic pneumonia, congest heart failure, and other diseases. an interesting phenomenon was the right lung formed an "s"-shaped curve in the chest x-ray scan because the patient prefers to lie on the right side in her illness [ figure 1g ]. visible large white frothy sputum observed in bronchoscopy suggested that the diffuse ground-glass shadow was relevant to the infiltration of cancer cells and hypersecretion infiltrates. because of the hypersecretion traits of cancer cells, a large number of secretions gathered in the lung, causing diffuse infiltration, exudation. with the change of body position, "s"-shaped curve appeared in the chest x-ray due to the gravity effect of liquid. in summary, the diffuse, uniform ground-glass shadow as the main imaging features of lung cancer in chest ct is very rare. clinicians should be aware of this possibility. in clinical practice, we should follow the correct diagnostic method and strengthen differential diagnosis from pulmonary tuberculosis, fungal infection, viral pneumonia, interstitial pneumonia, heart failure and so on. the formation of diffuse ground-glass shadow was related with infiltration hypersecretion of cancer cell. nil. there are no conflicts of interest. the bronchioloalveolar carcinoma and peripheral adenocarcinoma spectrum of diseases bronchioloalveolar carcinoma: an exceptional cause of diffuse lung disease in a patient with acute myeloid leukemia evolving concepts in the pathology and computed tomography imaging of lung adenocarcinoma and bronchioloalveolar carcinoma aspects of bronchioloalveolar carcinoma and of adenocarcinoma with a bronchioloalveolar component: ct findings a case of diffuse pneumonic type of mucinous adenocarcinoma treated with reduction surgery kras(g12d) and nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung key: cord-280374-yj0r4rwt authors: jain, richa; gupta, kirti; bhatia, anmol; bansal, arun; bansal, deepak title: hepatic sinusoidal-obstruction syndrome and busulfan-induced lung injury in a post-autologous stem cell transplant recipient date: 2018-01-04 journal: indian pediatr doi: 10.1007/s13312-017-1172-5 sha: doc_id: 280374 cord_uid: yj0r4rwt veno-occlusive disease of the liver is mostly encountered as a complication of hematopoietic stem cell transplantation with myeloablative regimens with an incidence estimated to be 13.7%. it is clinically characterized by tender hepatomegaly, jaundice, weight gain and ascites. strong clinical suspicion and an early recognition of clinical signs are essential to establish the diagnosis and institute effective regimen. another complication of cytotoxic drugs given for cancers, is development of busulfan-induced lung injury. a strong index of suspicion is needed for its diagnosis, especially in setting where opportunistic fungal and viral infections manifest similarly. we illustrate the clinical and autopsy finings in a 2½-year-old boy who received autologous stem-cell transplantation following resection of stage iv neuroblastoma. he subsequently developed both hepatic veno-occlusive disease and busulfan-induced lung injury. the autopsy findings are remarkable for their rarity. volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome increased efforts, hypoxia (saturation on room air: 90%; increasing to 95% on 40% fio2). tachycardia was present (heart rate 132/minute); however, circulatory parameters were normal (bp 98/54 mm hg, normal capillary refill time and pulse pressure, warm extremities). pallor was present, with no evidence of skin or mucosal bleeding. systemic examination was consistent with pneumonitis as the patient had tachypnea, bilateral equal air entry, and presence of coarse crepitations in bilateral lung fields. abdominal examination showed dark brown pigmentation over abdomen with no tenderness, guarding or rigidity. hepatomegaly was present with liver palpable 3 cm below costal margin (span 8 cm). spleen was not palpable. there was no free fluid. cardiac and neurological examinations were essentially normal. the index case was managed as a case of pneumonitis post auto-sct. respiratory support was initially provided with nasal prongs-continuous positive airway pressure. intravenous antibiotics were started cefoperazone-sulbactam, amikacin and azithromycin. due to rapidly progressive respiratory distress, child was transferred to pediatric intensive care unit where mechanical ventilation was provided. there was single episode of fever on the day of admission (38.7 o c) with a subsequent afebrile period throughout the hospital stay. progressive respiratory distress worsened into acute respiratory distress syndrome (ards). ventilation strategy was modified accordingly. on day 63, there was development of hypotensive shock, initially responding to fluid boluses. on day 65, the shock necessitated inotropic support (dopamine, adrenaline, noradrenaline and pre-terminally, and vasopressin). there was development of left sided pneumothorax followed by cardiac arrest on the same day. the patient could be revived and pneumothorax was drained. multi-organ dysfunction developed with acute kidney injury (onset day 63), requiring peritoneal dialysis. significant transaminitis with elevated bilirubin levels was documented. antibiotics were changed to vancomycin and meropenam on day 63; azithromycin was continued. intravenous co-trimoxazole and gancyclovir were added in therapeutic, renal modified doses. platelet concentrates were transfused to maintain a platelet count above 20 × 10 6 /mm 3 . there was development of refractory shock on day 67. the child suffered another cardiac arrest on day 68, and could not be revived. the fig. 1a-d) . with these chest radiographs, possible etiologies considered were infective, including fungal pneumonia and pcj pneumonia, cmv disease and miliary tuberculosis (tb). non-infective etiologies under consideration included pulmonary graft-versus host disease (gvhd) and pulmonary veno-occlusive disease (vod). this is a case of neuroblastoma, stage iv, day 68 post auto-sct, presenting with fever, pneumonia, hypoxia, and investigations showing polymorphic leucocytosis with deranged liver function tests (lft). in a posttransplant patient, complications can be divided according to the duration subsequent to transplant. in the initial 30 days, there is presence of neutropenia; between 30-100 days is the early post-engraftment phase and beyond 100 days is late post-engraftment phase. the index case developed symptoms in the early post engraftment phase. common complications seen in early post-engraftment phase can be divided into infective and non-infective. infective etiologies include cmv which can explain both pneumonia and hepatitis. it is a common pathogen causing disease 3 weeks post sct. india is an endemic country for cmv. in the index case, eta demonstrated polymerase chain reaction (pcr) positivity volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome for cmv along with radiological findings which were supportive of the diagnosis. however, the child deteriorated despite administration of gancyclovir from day 2 onwards, which is unusual. typically blood pcr is positive in such cases, though not mandatory for diagnosis of cmv pneumonia. fungal infections are the next possibility, supported by the presence of candida in eta on two occasions. presence of normal neutrophil count and a normal serum galactomannan are odd points. galactomannan <0.5 has shown a good negative predictive value for aspergillus infection [2] . other viral infections that are important in post-sct scenario include respiratory syncytial virus (rsv), para-influenza virus, influenza, metapneumovirus, and coronavirus. multiorgan failure and lymphopenia is common in these patients. patients with rsv often require ventilation. associated co-infection with fungus, especially aspergillus can be seen. in the absence of investigations directed towards the myriad respiratory viruses, it is difficult to rule in or rule out these infections. tuberculosis should be considered in an immunocompromised patient in an endemic country; however, the rapid onset of disease, absence of a contact and negative evaluation make it unlikely. in our case other bacterial infections typically seen in an immunocompromised child are also unlikely in view of sterile cultures, complete absence of fever and normal creactive protein (crp).though this clinical presentation can be caused by infection with pcj, it is an uncommon infection. other atypical infections like nocardia and cryptococcus are rarer still. the non-infective etiologies causing respiratory symptoms in a post-transplant setting can be pulmonary gvhd, idiopathic pneumonia syndrome (ips), bronchiolitis obliterans syndrome (bos), cryptogenic organising pneumonia (cop) and sos. ips is a very common disease in this situation, but is typically seen post allo-sct and hepatitis is not an associated feature. on-going hepatic sos is unlikely as there was no weight gain or tender hepatomegaly. gvhd and bos are also typically diseases seen in allo-sct setting. pulmonary sos is very rare and normal echo findings negate this possibility. the clinical presentation is consistent with cop, though it is more common in females undergoing allogenic transplant. the final diagnosis is neuroblastoma stage iv, day + 68 post auto-sct (bu-mel) with pneumonitis, ards and multi-organ failure; likely etiology being fungal pneumonia or cmv pneumonia and hepatitis secondary to ischemia with underlying sos. pediatric hemato-oncologist 1: ips occurs post sct day+60 to 80. this child had typical bilateral basilar infiltrates and hypoxia. moreover, ips has a relationship with use of busulfan and pre-existing sos. presence of cmv positivity in eta is of questionable significance as it is a common organism. histopathological evidence from lung biopsy is essential to prove cmv pneumonia. liver dysfunction in the form of transaminitis was likely due to shock and ischemia. pediatric hemato-oncologist 2: bacterial and fungal infections cannot be excluded despite absence of fever, several sterile cultures and continued normal values of crp, though less likely. however, both cmv and pcj infections are possible with normal crp. absence of adventitious lung sounds at initial presentation, along with presence of hypoxia may be a pointer towards pcj pneumonia. immunocompromised state, lymphopenia and the fact that the child was not on pcj prophylaxis are important here. moreover, cmv is ubiquitous in our volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome pediatric population, and in pediatric oncology patients, we have seen a near 100% seropositivity. reactivation of cmv can occur at any point of time in these patients. important non-infective possibilities are ips and cryptogenic pneumonia. pulmonary sos is quite unlikely given the normal echo findings. adult hematologist: immune reconstitution posttransplant takes typically 6 to 12 months. this child was immunocompromised. adenovirus infection can be considered. it can be rarely seen in association with hepato-pulmonary syndrome. the excised tumor on histology was categorized as differentiating neuroblastoma ( fig. 2a-c) . autopsy revealed normal serous cavities. liver weighed 290 g and revealed irregular areas of sinusoidal congestion, confluent at places with necrosis of adjoining parenchyma involving both right and left lobe (fig. 2d) . no thrombi were identified in right and left hepatic vein or inferior vena cava. microscopically, areas of centrizonal congestion were identified (fig. 2e) . furthermore, the dominant pathology was seen in the central vein and terminal hepatic venule (thv). there was varying degree of obliterative changes with subendothelial fibrosis and laying down of reticulin fibres and collection of extracellular matrix in subintimal zone. at places, the thv was completely obliterated with wipe out of centrizonal hepatocytes while the periportal hepatocytes were preserved (web fig. 1a-e) . in other regions, extravasated rbcs and areas of hemorrhage were noted in centri-zonal regions. besides acute obliterative changes, subacute changes in form of deposition of collagen around the hepatic venule and collection of hemosiderin laden macrophages were also noted. loss of hepatic parenchyma resulted in approximation of central veins structures (web fig. 1a) . both lungs weighed 245g with dull pleura. microscopy revealed features of busulfan-induced lung injury with marked prominence of type ii pneumocytes; many of them demonstrated nuclear atypia and hyperchromasia. marked thickening of interstitium with fibrosis was also noted (web fig. 2a-b) . other regions showed patchy acute bronchopneumonia and alveolar haemorrhages. features of pulmonary arteriopathy were also noted with prominence of intra-acinar arterioles. there were no features of vod in the pulmonary veins. an occasional focus of septic emboli with candida infiltration into parenchyma was noted. no cmv inclusions were noted in lungs. pcr carried out on lung tissue for adenovirus, rsv and metapneumovirus were negative. acute ulcers with candida infiltration were noted in stomach and small intestine. candida had disseminated to heart causing mural endocarditis, myocardial abscess and tiny (2-4 mm) vegetations on left atrial wall (webfig. 2 c-f). both tricuspid and mitral valves were normal. dissemination with formation of fungal abscesses were also detected in psoas muscle and omental fat. subsequent to septic emboli, infarcts were detected in right kidney (upper pole) with thrombi within the branches of renal vessels and spleen. right kidney also revealed features of acute tubular necrosis in noninfracted regions. no residual tumor was detected in lymph nodes, thymus and bone marrow. the autopsy diagnosis is concluded as follows: in a known case-of neuroblastoma, undifferentiated (adrenal) post-autologous stem-cell transplant: • features of busulfan-induced lung injury with organizing bronchopneumonia and pulmonary arterial hypertension; • veno-occlusive disease in liver. • fungal (candida) ulcers in git with extensive dissemination to heart (mural endocarditis and myocardial abscess), lungs, skeletal muscle and omental fat producing embolic infarcts in right kidney and spleen. • no residual disease in bone marrow. hepatic sinusoidal obstruction syndrome (sos) is an obliterative venulitis of thv which occurs as a result of cytoreductive therapy prior to hematopoietic stem cell transplantation (hsct), ingestion of pyrrolizidine alkaloids, or radiation therapy [3] [4] [5] [6] . the primary pathogenetic event is the endothelial injury of sinusoids and small hepatic veins. following which, there is deposition of fibrin-related aggregates and oedema in the subendothelial zone [3] . accumulation of these aggregates and entrapment of fluid and cellular debris progressively occlude the hepatic venous flow and leads to post-sinusoidal intrahepatic hypertension. this is accompanied by necrosis of perivenular hepatocytes. histologically, acute, sub-acute and chronic forms of sos have been described depending upon collagenization and fibrosis of terminal hepatic venule. incidence of sos varies from 0-70%, as it depends on the conditioning regimen used as well as upon the patient's risk factors [4] [5] [6] . sos occurs more often after allo-than after auto-hsct (8 v/s 3%, respectively), suggesting a role of immune reactions in this disorder [7] . few independent studies have documented increase in circulating levels of plasminogen activator inhibitor-1 (pai-1), a molecule released by the endothelial cells, in patients developing sos [8, 9] . increased pai-1 levels might be of clinical utility in challenging clinical situations in patients with hyperbilirubinemia occurring after hsct. it forms one of the therapeutic targets for defibrotide, which reduces circulating pai-1 levels along with other actions. other endothelial markers, like intercellular adhesion molecule-1 (icam-1), e-selectin, von willebrand factor (vwf), and thrombomodulin may also be helpful in early identification of patients at risk of sos who may benefit from early introduction to therapies [10] . diagnosis of sos is based on constellation of signs and symptoms and serum bilirubin levels. hepatic sos is clinically characterized by jaundice caused mainly by conjugated hyperbilirubinemia, tender hepatomegaly, fluid accumulation manifested as rapid weight gain and ascites [4] . most commonly used diagnostic criteria for sos includes the seattle criteria [11] , the modified seattle criteria [1] , and the baltimore criteria [12] . because of its high incidence and mortality, prophylaxis for hepatic sos is widely practiced, using different regimens in different centres. when hepatic sos is established, specific therapy is usually given in addition to general supportive care, especially in moderate or severe cases. hepatic sos is a formidable challenge both for patients undergoing stem cell transplantation and for their physicians. the second pathology in this child which significantly contributed to his downhill course was busulfan induced lung injury. intriguingly, in the present clinical setting, busulfan induced lung injury remains an diagnosis of exclusion, particularly with respect to considering usual and atypical infections. its clinical presentation includes a spectrum ranging from acute, rapidly progressive respiratory distress to chronic, interstitial lung disease with insidious onset [13, 14] . the pathophysiology of drug-induced lung injury is not fully understood but direct toxicity of the drug to parenchymal cells, cell-mediated immune reactions and release of cytokines are believed to contribute to the lung injury. the pathologic findings consist of mainly diffuse interstitial pneumonitis, organizing alveolitis and cellular atypia within type ii pneumocytes. the injury pattern with busulfan is diffuse alveolar damage (dad) either in acute exudative phase with alveolar and interstitial oedema and hyaline membranes; or late reparative phase, which is characterized by proliferation of type ii pneumocytes and interstitial fibrosis [15] . marked atypia of the type ii pneumocytes is a morphological clue in favour of busulfan induced lung injury in contrast to organizing bacterial pneumonia. moreover, pcr for cmv is helpful in excluding viral pneumonia. the prevalence of drug-induced pulmonary toxicity is increasing, and more than 100 drugs are now known to cause lung injury. because this lung injury can be progressive and fatal, early recognition is important. the diagnosis of pulmonary drug toxicity should be considered in any patient with a history of drug therapy who presents with new or progressive respiratory complaints. the superadded fungal ulcer which developed preterminally with extensive dissemination to heart causing mural endocarditis and myocardial abscess eventually led to the demise of the child. hepatic sos contributes considerably to transplantation-related morbidity and mortality. recognition of this disease in the post-transplantation setting remains a challenge in the absence of specific diagnostic features as many other more common conditions can mimic it. a high index of suspicion is needed to identify patients with sos. while hepatic sos and busulfan induced lung injury are commonly reported as isolated findings following autologous sct, the coexistence of these are extremely rare and have not been documented in the literature thus far. the present case adds observational data to the existing literature and highlights the importance of keeping high index of suspicion for these two entities in patients following hsct, and early institution of effective therapy. veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of 355 patients serum galactomannan assay for the diagnosis of invasive aspergillosis in children with haematological malignancies sinusoidal obstruction syndrome (hepatic veno-occlusivedisease) hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation sinusoidal obstruction syndrome vascular disorders of the liver. american association for the study liver diseases incidence and outcome of hepatic venoocclusive disease after blood or marrow transplantation: a prospective cohort study of the european group for blood and marrow transplantation. european group for blood and marrow transplantation chronic leukemia working party the relevance of plasminogen activator inhibitor 1 (pai-1) as a marker for the diagnosis of hepatic veno-occlusive disease in patients after bone marrow transplantation endothelial dysfunction after bone marrow transplantation: increase of soluble thrombomodulin and pai-1 in patients with multiple transplant-related complications prediction of veno-occlusive disease using biomarkers of endothelial injury venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors venoocclusive disease of the liver following bone marrow transplantation busulphan lung in childhood lung function 5 yrs after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide interstitial pneumopathies caused by busulfan. histologic, developmental and bronchoalveolar lavage analysis of 3 cases key: cord-287622-xnksvy21 authors: carpagnano, giovanna e; lacedonia, donato; palladino, grazia pia; logrieco, giuseppe; crisetti, elisabetta; susca, antonia; logrieco, antonio; foschino-barbaro, maria p title: aspergillus spp. colonization in exhaled breath condensate of lung cancer patients from puglia region of italy date: 2014-02-18 journal: bmc pulm med doi: 10.1186/1471-2466-14-22 sha: doc_id: 287622 cord_uid: xnksvy21 background: airways of lung cancer patients are often colonized by fungi. some of these colonizing fungi, under particular conditions, produce cancerogenic mycotoxins. given the recent interest in the infective origin of lung cancer, with this preliminary study we aim to give our small contribution to this field of research by analysing the fungal microbiome of the exhaled breath condensate of lung cancer patients from puglia, a region of italy. methods: we enrolled 43 lung cancer patients and 21 healthy subjects that underwent exhaled breath condensate and bronchial brushing collection. the fungal incidence and nature of sample collected were analysed by using a selected media for aspergillus species. results: for the first time we were able to analyse the fungal microbioma of the exhaled breath condensate. 27.9% of lung cancer patients showed a presence of aspergillus niger, or a. ochraceus or penicillium ssp. while none of the healthy subjects did so. conclusion: the results confirmed the high percentage of fungal colonization of the airways of lung cancer patients from puglia, suggesting the need to conduct further analyses in this field in order to evaluate the exact pathogenetic role of these fungi in lung cancer as well as to propose efficient, empirical therapy. lung airways are perpetually exposed to inhaled particulate materials that include pollens, viruses, bacterial and fungal spores [1] . while many of these particles are innocuous, some spores have the potential to germinate and cause invasive lung diseases [1] . a recent theory suggests that infections could also trigger lung cancerogenesis [2] . this hypothesis has been formulated mainly for viruses, although it has been not excluded that also bacteria and fungi could be involved. if it is possible that the infections could cause lung cancer, it is sure that the lungs of subjects with cancer are particularly susceptible to infection that significantly compromises the patient's prognosis [2] . the spectrum of pulmonary infections depends on the underlying immunologic deficit or deficits, whereas fungal infections are common if neutropenia persists [3] . lung cancer is also characterized by airways inflammation, that usually follows cigarette smoking which is likely to play a role in cancer transformation [4, 5] . the inflammatory status also further compromises host lungs, promoting tissue invasion and systemic dissemination of the infection [4, 5] . the main fungus that could play a role in lung cancer transformation by colonizing the airways is the aspergillus, a mold that forms conidia which, owing to their small size, can bypass mucociliary clearance mechanisms and are inhaled into terminal airways and phagocytosed by alveolar macrophages (amøs) [6] [7] [8] . toxigenic aspergillus species are common in the mediterranean environment, colonizing different crops, including maize, grapes and dried fruits [9] [10] [11] and are known to be able to produce mycotoxins, such as aflatoxin, ochratoxin a and fumonisins [12] , when host plants are stressed by extreme temperature or moisture conditions, poor soil fertility or insect damage. despite the potential clinical and therapeutic consequences of the hypotheses of lung cancerogenesis triggered by fungal infection (and particularly by aspergillus) and notwithstanding the prevalent rates of mold colonization in lung cancer patients significantly increased in the last decade [13] , no comprehensive studies are available on this field. the few studies present in literature have described airways' fungal colonization in lung cancer patients or as a one-off report or simply express this as the consequence of the immunodepressive status characteristic of tumours [14] [15] [16] [17] . however, several studies are available supporting the viral origin of lung cancer [2, 18] . among these studies, one emerging from our group recently analysed viral colonization in the exhaled breath condensate (ebc) of non-small cell lung cancer (nsclc) patients, involving a sample from airways that is completely non-invasive and apparently suitable for microbiological studies ( [2] , unpublished data). we support the potential of this sample as it contains volatile, soluble and omic markers, most of which have already been demonstrated to be useful in the non-invasive diagnosis of several lung diseases, and particularly of lung cancer, which still results in many fatalities, mainly because it is always diagnosed at an advanced stage, as there are no noninvasive screening tools providing a key for its early detection and improving a person's chances of survival. with this study we want to give a preliminary contribution to this field of research, giving a view of the incidence and nature of fungal colonisations in lung cancer patients from puglia and correlating eventual positivity with anthropometric, clinical and oncologic data. thus, for the first time to our knowledge, we analysed the fungal microbiome in the ebc, comparing results with paired bronchial brushing. characteristics of the patients 64 consecutive patients with a suspicion of lung cancer who consented to the study were enrolled at the unit of thoracic surgery, casa di cura la madonnina, bari and at the department of respiratory disease, foggia university (table 1) . written informed consent was obtained from all the subjects upon approval of the study by the ethics committees of the university of foggia. all the patients were enrolled in the study before pathological diagnosis. all of them also underwent standard staging procedures consisting in a physical examination, serum chemistry analysis, brain, chest and abdomen ct scans, and a radionuclide bone scan. in such cases the definitive diagnosis of malignancy derived from a positive cytohistology of the samples is obtained broncoscopically. following the histological analysis carried out on specimens, 21 subjects turned out to be negative and were considered controls. in the remaining 43 subjects, the suspicions of lung cancer were confirmed. squamous cell carcinoma was diagnosed in 36 (83.7%) subjects, whereas 7 (16.2%) subjects were found to be affected by adenocarcinoma. overall the nsclc patients 16 (37.2%) were classified as stage i, 5 (11.6%) as stage ii, 12 (27.9%) as stage iii and 10 (23.2%) as stage iv. all the subjects underwent ebc and bronchial brushing collection (the latter was carried out during bronchoscopy). all subjects underwent a fungal investigation in these samples. type and frequency of these colonisations were analyzed. information on their smoking habit was acquired at the time of diagnosis. twenty-five of the nsclc patients were current/ ex-smokers (58.1%, 43.3 ± 25.2 pack/year), of whom 8 ex-smokers (32%) had quitted smoking 9.65 ± 8.5 years previously, whereas 18 were non-smokers. a detailed history relating to their family history of lung cancer or any other cancer was collected in a pre-tested proform. bronchial brushing specimens were collected in an icecold phosphate-buffered saline solution during fiberoptic bronchoscopy. the sample was transferred on freshly prepared dichloran rose-bengal chloramphenicol agar (drbc, oxoid) medium. the plates were incubated at 25°c for 7 days and examined daily. ebc collection and processing 1 ml of ebc in one setting from each patient at the time of diagnosis. the ebc was collected by using a condenser (eco-screen jaeger, wurzburg, germany). the condensate was transferred on freshly prepared dichloran rose-bengal chloramphenicol agar (drbc, oxoid) medium. the plates were incubated at 25°c for 7 days and examined daily. aspergillus colonies were subcultured for subsequent identification to species level. species names were determined by following the taxonomic keys of klich [18] . culture were incubated for 7 days on cya (czapek yeast extract agar) at 25°c and 37°c, cz (czapekdox agar) and mea (malt extract agar) at 25°c to enable morphological features exploited as diagnostic characters. penicillium colonies were identified only at genus level. in order to assess the association between categorical variables such as sex, smoking habit or fungi positivity in the brushing or in the ebc and positivity to lung cancer or between histological subtypes or tnm stage and mold positivity, the chi square test (or fisher exact's test, when necessary) were calculated. t-student was used for independent samples in order to assess the differences in continuous variables (age, number of pack/years smoked) between lung cancer patients and controls. a p value of < 0.05 was considered statistically significant. lung cancer cases and healthy subjects enrolled in the present study numbered 43 and 21, respectively. demographic and clinical data of study subjects are summarized in table 1 . we were able to detect a fungal colonization by aspergillus niger, aspergillus ochraceus and penicillium ssp in the ebc. 12 (27.9%) of nsclc patients, and 0 (0%) controls turned out to be fungi colonized in the ebc: 5 by aspergillus niger (11.6%), 3 by aspergillus ochraceus (6%), 4 by penicillum spp. (9.3%). fungi positivity in the ebc was always confirmed in paired bronchial brushing. the ebc was found to have a similar sensibility compared to the bronchial brushing for fungi detection. when analyzing lung cancer patients no difference was found according to sex, age, histotype, stage, smoking habit, pack years, time since quitting smoking in subjects with fungal colonization (p > 0.05). this was a preliminary study that aimed to analyse the incidence and nature of fungal colonization in lung cancer patients from region puglia of italy. for the first time to our knowledge, we tested fungal microbioma of exhaled breath condensate and paired bronchial brushing of patients with diagnosed lung cancer and of healthy subjects and in 27.9% of lung cancer patients we detected the presence of mold (aspergillus niger, a. ochraceus and penicillium ssp), but not in any of the healthy subjects. the fungal positivity in airways didn't correlate with the oncologic data. it has been suggested that the fungal colonization could contribute to or trigger -as do other virus infections (hpv, cmv etc) -pathophysiologic processes associated with lung cancer [19] . however, while there is evidence of the involvement of specific fungal species in asthma, chronic obstructive pulmonary disease (copd) [20, 21] and cystic fibrosis (cf) [21] [22] [23] [24] , little is known of the airway fungal microbiota in the pathogenesis of lung cancer [19] . given the nascent of airway microbiome research in lung cancer and some recent efforts to advance these areas, this article has analysed fungal microbiota in the ebc and bronchial aspiration of lung cancer patients from region puglia of italy [25] [26] [27] [28] [29] . there are only a few studies that have investigated fungal colonization in the airways of lung cancer patients [14] using bronchial aspirates [14] , bronchoalveolar lavage [30] , sputum [15] or lung tissue [17] . all these studies have described fungal positivity as a one-off report or as the result of the immunodepression. bearing in mind that identifying other risk factors (more than the smoking habit) could have socio-economic consequences for lung cancer, offering a useful tool for prevention programs that are still in a state of bankruptcy, we have designed this preliminary study with the aim of analysing the possible fungal involvement in lung cancerogenesis. in consideration of the usefulness of using noninvasive methods to analyse the airways of these patients feeling the brunt of several diagnostic tests and often of surgery, we tested the fungal microbiome for the first time in the ebc. our group previously demonstrated the possibility to analyse viral colonization in this sample of patients affected by lung cancer and supported the recent theory on a possible infective aetiology of lung tumours. in this study we sought to direct our efforts to the identification of the new cancerogenic microrganisms in the ebc, toward which the screening programs could be directed, by testing fungal microbiome. we found a positivity for aspergillus niger and a. ochraceus in samples from lung cancer patients, which are species of fungi that may release cancerogenic mycotoxins such as ocratoxin a e fumonisins. these findings led us to suggest that also these microorganisms and their toxic secondary metabolites may play a role in lung cancer development, although further studies are needed to support this hypothesis. furthermore, we observed that fungal positivity in this sample was always the same as had been observed in paired bronchial brushing. the overlap of molds in ebc and bronchial brushing proved that the ebc contains identical information on airway colonisation as previously demonstrated for hpv [2] , cmv, ebv (unpublished data) and somatic dna alterations specific to lung cancer [31] , but also further supported the high sensibility and specificity of this sample, confirming its potential usefulness in oncological clinical practice. we were unable to find any correlation between fungi positivity and sex, age, histotype, stage, smoking habit of subjects studied that would help us in the definition of the pathogenetic role of fungi identified in lung cancer patients. however, the number of patients enrolled in this study was low and justified our results, which we intend to verify on a larger population. another recognized limit of the study was not to have tested the presence of fungi in paired plasma, something that might help us to better interpret the origin of fungi in the airways of patients with lung cancer and also its significance. further very important analyses should be addressed also to analyze fungal genotypes isolated, in order to asses their ability to produce toxins, and above all, to evaluate the effective presence in human fluids or in lung tumor tissue of mycotoxins potentially produced by aspergillus species isolated. this was just a preliminary study that will be followed by a genomic and epigenomic characterization and mycotoxin analysis, which we hope will help us in defining the association between this mold, lung cancerogenesis and the pathogenetic mechanisms involved. we further are planning to better study whether the fungi colonization is the cause or consequence of lung cancer investigating the immunologic status of the fungi-positive patients through the analysis of the density of infiltrating inflammatory cells in surgical tissue samples and the dosage of the plasmatic immunoglobin concentration. in conclusion, it can be stated that we have demonstrated that the exhaled breath condensate is a suitable sample, even for the fungal colonization of airways. furthemore, we found that 28% of lung cancer patients from region puglia of italy showed apergillus niger, aspergillus ochraceus and penicillium spp in their airways, opening up future perspectives in this field. with this study we couldn't verify whether fungal colonization is the cause or consequence of lung cancer but we can offer a broader view on the infective status of airways of lung cancer patients. in consideration of the potential of our results, we support any future studies in this emerging field that could open up the possibility of microbiota manipulation as a novel therapeutic strategy for screening, treatment or management of lung cancer. abbreviation ebc: exhaled breath condensate. there aren't financial disclosures from any of the authors. authors' contributions gec designed the present study and drafted the manuscript. dl enrolled patients. gpp carried out bronchial brushing analyses. gl enrolled patients. ec enrolled patients. as participated in fungal culture and analysis. al participated in fungal analysis and designed the present study. mpfb reviewed the manuscript. all authors read and approved the final manuscript. author details 1 institute of respiratory disease, department of medical and occupational sciences, university of foggia, foggia, italy. 2 university of bari, bari, italy. 3 institute of sciences of food production, research national council, bari, italy. aspergillus fumigatus triggers inflammatory responses by stage-specific b-glucan display hpv in exhaled breath condensate of lung cancer patients the spectrum of pulmonary infections in cancer patients could exhaled ferritin and sod be used as markers for lung cancer and prognosis prediction purposes? exhaled matrix metalloproteinase-9 in lung cancer aspergillus fumigatus and aspergillosis selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to aspergillus. observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes phagocytosis and intracellular fate of aspergillus fumigatus conidia in alveolar macrophages biodiversity of complexes of mycotoxigenic funal species associted with fusarium ear rot of maize and aspergillus rot of grape reduction of mycotoxins and toxigenic fungi in the mediterranean basin maize chain mycotoxin risks and toxigenic fungi in date, prune and dried apricot among mediterranean crops mycotoxin and toxigenic fungi molecular detection of microorganisms in distal airways of patients undergoing lung cancer surgery successful pneumonectomy for invasive pulmonary aspergillosis and advanced non-small cell-lung cancer bronchogenic carcinoma and secondary aspergillosis-common yet unexplored: evaluation of the role of bronchoalveolar lavage-polymerase chain reaction and some nonvalidated serologic methods to establish early diagnosis non-small cell lung cancer coexisting with pulmonary aspergilloma identification of common aspergillus species the emerging relationship between the airway microbiota and chronic respiratory disease: clinical implications pneumocystis infection and the pathogenesis of chronic obstructive pulmonary disease airway obstruction is increased in pneumocystiscolonized human immunodeficiency virus-infected outpatients effect of aspergillus fumigatus and candida albicans on pro-inflammatory response in cystic fibrosis epithelium characteristics of pathogenic fungi and antifungal therapy in cystic fibrosis detection of hyphomycetes in the upper respiratory tract of patients with cystic fibrosis characterization of the oral fungal microbiome (mycobiome) in healthy individuals pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions microarray-based detection and genotyping of viral pathogens viruses in the faecal microbiota of monozygotic twins and their mothers cancer, febrile neutropenia and pulmonary images: findings in bronchoalveolar lavage in children 3p microsatellite signature in exhaled breath condensate and tumor tissue of patients with lung cancer submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord-017030-tzuyo6tx authors: henao-martínez, andrés f.; montoya, josé g. title: infections in heart, lung, and heart-lung transplantation date: 2018-12-08 journal: principles and practice of transplant infectious diseases doi: 10.1007/978-1-4939-9034-4_2 sha: doc_id: 17030 cord_uid: tzuyo6tx half a century has passed since the first orthotopic heart transplant took place. surgical innovations allowed for heart, lung, and heart-lung transplantation to save lives of patients with incurable chronic cardiopulmonary conditions. the complexity of the surgical interventions, chronic host health conditions, and antirejection immunosuppressive medications makes infectious complications common. infections have remained one of the main barriers for successful transplantation and a source of significant morbidity and mortality. recognition of infections and its management in this setting require outstanding clinical skills since transplant recipients may not exhibit classic signs or symptoms of disease, and laboratory work has some pitfalls. the prevention, identification, and management of infectious diseases complications in this population are a priority to undertake to improve the medical outcomes of transplantation. herein, we reviewed the historical aspects, epidemiology, and prophylaxis of infections in heart, lung, and heart-lung transplantation. we also discuss the most prevalent organisms affecting the host and the organ systems involved. louder! louder!" dr. john gibbon jr. used for the first time in 1953 a heart-lung respirator to keep a patient alive while performing heart surgery. dr. norman shumway at stanford developed and perfected the first surgical technique leading to heart transplantation surgery. after dr. christian barnard's first orthotopic heart transplant in december 1967, and dr. shumway first heart transplant in the united states in january 1968, heart transplantation became a standard therapeutic option for life-threatening congestive failure and started to be performed in the hundreds over the next following years at different centers. heart transplant surgery faced complications due in part to rejection and infection. however, the development of more selective immunosuppressive therapy and improvements in prevention, detection, and treatment of infections allowed for heart transplant surgery to increase rapidly worldwide. four thousand and ninety six heart (3529 adults) transplants were reported to the international society of heart and lung transplant registry (ishl) in 2011 [1] . the landscape of infection affecting heart transplant patients has been shaped by different factors: (a) implementation of more selective calcineurin-based immunosuppressive protocols, (b) lessened immunosuppressive induction regimens, (c) the institution of antimicrobial prophylaxis resulting in a significant decrease or delay in the emergence of major infections episodes including p. jirovecii (pcp), nocardia spp., listeria spp., toxoplasma gondii, cytomegalovirus, toxoplasmosis, cytomegalovirus (cmv), herpes simplex virus (cmv), varicella zoster virus (vzv), and invasive fungal infections, (d) introduction of novel diagnostic technology facilitating earlier recognition and treatment of infections, (e) expansion in the criteria to select donors and recipients to include various scenarios dealing with hbv, hcv, and hiv infections [2] , and (f) shift toward predominantly grampositive bacterial infections and multiresistant bacteria in recent years [3] [4] [5] . a stanford team lead by dr. bruce reitz performed a lung transplantation as a combined heart-lung transplant procedure in 1981 [6] . shortly after, thoracic surgeons optimized the single-and double-lung transplant procedures. improvement of surgical techniques, especially bronchial anastomosis and evolution of flush perfusion lung preservation, decreased the perioperative bronchial complications substantially. similarly to heart transplantation, improvements in immunosuppressive regimens, antimicrobial prophylaxis, and graft preservation led to enhancement in survival among lung transplant recipients. in contrast to cardiac, lung transplantation has faced the challenge of infections unique to the transplant of this organ. mold infections of the anastomotic site, host versus graft disease, and serious infections with mycobacterium abscessus, chlamydia spp., bronchiolitis, and burkholderia cepacia complex are among infectious complications rarely observed in other transplant patients [7] . transplantation of thoracic organs has improved the quality of life and prevented the death of thousands of individuals worldwide. graft survival and life expectancy have been markedly improved in these patients due to the introduction of more optimal immunosuppression, antimicrobial prophylaxis, and diagnostic technology allowing the earlier diagnosis and treatment of infection and rejection. finally, further control of infection is likely to result from implementation of new approaches to assess the net state of immunosuppression in these patients. infection was recognized as a major threat to thoracic transplantation from the early inception days [8] . there are several factors predisposing thoracic transplant recipients to infections: (a) factors present before transplantation: age, presence of comorbidities (e.g., chronic kidney disease, diabetes mellitus, cancer, etc.), nutrition status, latent infections, colonization with healthcare-associated organisms, and occult community-acquired infections; (b) factors during the surgery: duration of the transplant procedure, graft injury including ischemic time, colonization or latent infection of the graft, surgical instrumentation (e.g., mechanical ventilation, invasive devices such as catheters, drains, foley catheters, etc.), icu stay, and need for re-interventions; and (c) factors present after transplant: degree of immunosuppression, cmv infection, and rejections ( a total of 4096 heart transplants were performed in 2011. heart transplant recipients have an average age of 54 years and are predominantly man (76%). they have a significant history of smoking (46%) and hypertension (45%) and have cardiomyopathy (54%) followed by coronary artery disease (37%) as the leading causes of transplant [1] . the historical (pediatric and adult transplants between 1982 and 2011) 1-year, 5-year, and 10-year survival rates are 81%, 69%, and 50%, respectively. overall median survival is 11 years, but it increases up to 13 years for those surviving the first year after transplantation. although not associated with increased posttransplant mortality, infections before transplant can affect up to 25% of heart transplant candidates. being bronchitis and soft tissue infections, the more commonly present [9] . despite no major changes in the distribution of causes of death since 1994, infections remained a predominant factor of mortality during the first 3 years after transplant. it contributes with up to almost 20% of causes of death [3] . the global incidence of infections in heart transplant ranges between 30% and 60% and the associated mortality between 4% and 15% [10] . the incidence of infection measured as major infectious episodes per patient has steadily declined from 2.83 in the early 1970s to 0.81 in the early 2000s [3, 8, 11] . the most frequent type of infection is bacterial (44%), followed by viral (42%), fungal including pneumocystis jirovecii (14%), and protozoa (0.6%). unfavorable functional outcomes are observed in patients who developed infections in the first year of transplant, mainly associated with bloodstream, cmv, and lung infections [12] . pulmonary and central nervous system (cns) infections are independent predictors of mortality among heart transplant recipients. reactivation of latent parasitic infections residing in extra-cardiac tissues in the host or transmitted in the transplanted heart is an important consideration. the classic example is the reactivation of trypanosoma cruzi. chagas disease is a vectorborne illness transmitted by triatomine bugs, and it is endemic in latin america. the ethnicity or origin of either the donor or the recipient from these regions should raise the concern for possible reactivation. chagas reactivation was documented in 38.8% of cases in a cohort of brazilian heart transplant recipients, where chagas cardiomyopathy was the second most common indication for transplant (34.9%) [13] . chagas can also reactivate from the transplanted heart procured from a seropositive donor and transplanted into a seronegative recipient. although with a substantial decreased on its prevalence in the most recent eras, toxoplasmosis is another important consideration in this setting. similarly to chagas, toxoplasma gondii-also with a predilection to invade the myocardium-can be transmitted by reactivation of quiescent cysts in the recipient or the transplanted heart [14] . by 2011, 3640 adults received lung transplantation, the highest reported number of procedures up to that date, driven mainly by the increase of double-lung transplants. doublelung transplant is indicated for septic lung diseases (e.g., cystic fibrosis). around 66% of recipients were aged 45-65 years old. the most frequent indications for transplant were copd (34%), followed by interstitial lung disease (ild) (24%), bronchiectasis associated with cystic fibrosis (cf) (17%), and α1at deficiency-related copd (6%) [15] . the overall (from 1994 to 2011) 1-year, 5-year, and 10-year survival rates among lung recipients are 79%, 53%, and 31%, respectively. overall median survival is 5.6 years. lung transplants from cmv seronegative donors have better survival rates than from cmv seropositive donor. thirty-day mortality was led by graft failure (24.7%) and non-cmv infections (19.6%). during the remainder of the year, non-cmv infections were the leading cause of death (35.6%). infection is still prominent as the cause of death following the first year of transplant after bronchiolitis obliterans syndrome (bos)/chronic lung rejection or graft failure [15] . other infections complications historically present among the ten primary causes of death within the first year include sepsis, pneumonia, and fungal infections [16] . high lung allocation score (las) at the time of transplantation is associated with a lower 1-year survival and higher rates of infections among lung transplant recipients [17] . sixty-three adult heart-lung transplantations were reported to the ishl registry in 2011. sixty-six percent of recipients were in the group range from 18 to 49 years old. sixty-three percent of the indications were for congenital heart disease and idiopathic pulmonary arterial hypertension. heartlung transplant for cf was higher in europe and other centers compared to north american. when compared to lung only transplants, short-term survival was worse, but long-term survival was better for the heart-lung transplant recipients. their 1-year, 5-year, and 10-year survival rates were 63%, 44%, and 31%, respectively. the median survival was 3.3 years and 10 years for those surviving the first year. similarly, they have graft failure (27%), technical complications (21.9%), and non-cmv infections (17.8%) as leading causes of death during the first 30 days posttransplant. non-cmv infections (35.1%) were the top cause of death after 1 month and within 1 year of transplant. after the first year, bos/late graft failure and non-cmv infections were the predominant causes of death [15] . among other risk factors for mortality in lung transplantation are cystic fibrosis, nosocomial infections, and mechanical ventilation before transplant [18] . infections in lung transplant recipients are predominantly bacterial (48%), viral (35%), fungal (13%), and mycobacterial (4%) [19] . in 60%, the infection site is pulmonary. risk factors for infection vary by the type of organism. mechanical ventilation (mv) for >5 days immediately following transplant surgery and isolation of staphylococcus aureus (sa) from airway cultures in the recipient were considered risk factors for invasive sa infections in a retrospective study of patients with lung and heart-lung transplants [20] . likewise, risk factors for the development of healthcare-associated infections with gram-negative organisms, aspergillus, legionella, and mrsa (methicillin-resistant staphylococcus aureus), include prolonging mv, renal failure, use of atg (antithymocyte globulin), and recurrent rejections episodes [21] . additionally, α-1-antitrypsin deficiency and repeat transplantation are also risk factors for nosocomial infections. mycobacterium tuberculosis transmission from lung donors with latent infection has been documented in highly endemic areas [22] . colonization with mdr organisms (pseudomonas aeruginosa, burkholderia, acinetobacter, nontuberculous mycobacteria (ntm), and scedosporium) before transplantespecially important in cf patients-can predict the development of challenging infections to treat after transplant [23] . patients should undergo a comprehensive evaluation of potential infectious complications associated with transplantation. a detailed medical history including previous vaccinations, history of past infections, exposures (geographical, occupational, animal, etc.), travel, and foreign-born status among others should be obtained. clinicians shuold perform routine serologies for the detection of pathogen-specific igg for cmv, hsv, ebv (vca), vzv, hepatitis b (hbsag, hbsab, hbcab), hiv, hepatitis c, and syphilis. toxoplasma igg should also be performed in heart and heart-lung transplant candidates. additionally, we recommend to obtain ua, urine culture, cxr, and tuberculin skin test (tst), or a quantiferon assay. in lung and heart-lung transplant candidates, sputum should be cultured for bacterial, fungal, and afb studies. some centers advocate the screening of patients for colonization with mdr (multidrug resistant) bacteria such as mrsa and vre (vancomycin resistant enterococci), which it may have an impact on the type of antibacterial prophylaxis used preoperatively or the empirical antibiotics should sepsis develop in the immediate postoperative period. in potential lung recipients, previous respiratory colonization with mdr pseudomonas, especially in cf patients, should not exclude them from transplant [24] . on the other hand, if colonization with b. cenocepacia (genomovar iii) in cf is present transplant is relatively contraindicated [25, 26] . checking for endemic fungi such as coccidioides immitis or for the parasites trypanosoma cruzi, strongyloides stercoralis, and leishmania spp. is indicated in the presence of the appropriate risk factors [27] [28] [29] [30] [31] . histoplasma capsulatum has reactivated during immunosuppressive therapy [32] . infections after solid organ transplantation (sot) are rare and attributable to transmission from the donor [33] . furthermore, latent histoplasmosis can be present with negative serologies and treatment after transplant carries a good outcome. therefore the role of screening for histoplasmosis is of questionable significance [34] . the type of evaluation may change if the donor is alive or deceased depending on the available time to collect the samples. similarly to recipients, donors should undertake a comprehensive assessment including a complete history, assessment of risk factors, exposures, immunizations, and previous or current infections. donors should be screened for hiv, hepatitis b/c, syphilis, and tuberculosis. furthermore, we recommend to obtain serologies for cmv, ebv, hsv, vzv, and toxoplasma gondii, and for htlv-1/ htlv-2 in endemic areas. in high-risk donors, the use of nucleic acid amplification tests (naat) for hbv, hcv, and hiv should be considered. additionally, blood cultures to document an occult bacteremia are recommended. in lung transplant donors, we recommend obtaining respiratory cultures through bronchoscopy to detect colonizing organisms and target them to prevent invasive infections in the donor. culturing the media of the allograft during acquisition or processing have been advocated to reduce the risk of mycotic aneurysms among kidney transplant recipients, which may apply to other sot [35] . screening of donors for endemic mycosis is not well established. on the other hand, heart transplant donors should be screened for chagas if the donor was born in latin america [29] . finally, it is important to highlight the increase recognition of emerging, unusual viral infections such as west nile virus, lymphocytic choriomeningitis virus, rabies, and different human coronaviruses [34, 36] . testing for those organisms should be done based on individual assessments. immunization should be optimized before transplantation since the recipient will have better chances to mount an adequate immune response [37] . the advisory committee on immunization practices (acip) [38] and the guidelines for immunizations in solid organ transplantation [39] recommend inactivated influenza vaccine annually. tetanus, diphtheria, and acellular pertussis (tdap) should be administered to all adults who have not previously received tdap or have an unknown status. varicella vaccination with two doses in patients without evidence of immunity or a single dose of zoster vaccination, inactivated polio vaccine, hepatitis a/b, hpv (three series through 26 years of age), and meningococcal and pneumococcal vaccines should be administered [38] . it is remarkably important to vaccinate all household members as well. bcg and rabies vaccines can be considered under some extenuating or exposure-related indications. see table 2 .3. education of the patient and the family members is a cornerstone to establishing effective preventive measures. emphasis should be enforced about hand hygiene and food handling. additionally, potential sources of bacteria, fungi (e.g., aspergillus), and toxoplasmosis such as plants and flowers, cleaning pet's litter or cages, eating uncooked meat, acquiring new pets, construction areas, farming, barnyard activities, and smoking marihuana should be avoided. if those recreational or occupational exposures are unavoidable; appropriate gear, such gloves, must be worn. education about possible community exposures is also important. close contacts with persons with fevers or rash potentially infected with vzv, herpes zoster, or influenza should be circumvented as well. patients should cook all meals thoroughly, wash all fruits and vegetables, and shun all unpasteurized products. safe sex practices are recommended. if any foreign travel is planned, seeking evaluation in a specialized travel clinic is advisable. guidelines for the management of surgical antimicrobial prophylaxis list cefazolin (2 g, 3 g for patients with weight >120 kg every 4 h) as the recommended regimen for heart, lung, and heartlung transplantation surgery. clindamycin (900 mg every 6 h) or vancomycin (15 mg/kg) can be substituted as alternative agents in beta-lactam allergic patients [40, 41] . this recommendation can be adjusted individually, based on local hospital surveillance data or previous knowledge of colonizing organisms (e.g., addition of aztreonam, gentamicin, or a single-quinolone dose). however, the widespread use of quinolones may increase the resurgence of antimicrobial resistance. the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients. recommendation to continue antibacterial prophylaxis until chest and mediastinal tubes are removed lacks sufficient evidence. redosing will depend on the procedure duration and associated blood loss. the recipient does not need treatment if a localized infection was present in the donor, except during meningitis where concomitant bacteremia often coexist. in meningitis and bacteremia, it is prudent to treat the recipient for 2-4 weeks [34] . indications for antifungal prophylaxis in heart transplant recipients are not clear. a systemic review showed no benefit of antifungal therapy to prevent invasive fungal infections in transplants recipients other than liver [42] . although a prospective cohort of heart transplant recipients showed targeted prophylaxis-an echinocandin for a median of 30 days with the presence of at least one risk factor for invasive aspergillosis (ia) (reoperation, cytomegalovirus disease, posttransplantation hemodialysis, and another patient with ia in the program 2 months before or after the procedure)-was highly effective and safe in preventing ia episodes [43] , no consensus exists for universal antifungal prophylaxis in heart transplant recipients. most centers have adopted antifungal prophylaxis including inhaled amphotericin b, oral itraconazole, or iv targeted echinocandin prophylaxis. in lung and lung-heart transplant recipients, fungal prophylaxis should be considered, especially if pretransplantation respiratory cultures either from the donor lung or recipient airways shows aspergillus or candida. one approach is to use inhaled amphotericin b (50 or 100 mg in extubated or intubated patients, respectively) daily until 4 days after transplant and then weekly until hospital discharge in patients with no known colonization [44, 45] . if a mold has been isolated, voriconazole is recommended up to 4 months after transplant. although evidence and efficacy need to be confirmed, combination antifungal prophylaxis therapies is used at some centers [46] . pneumocystis jiroveci prophylaxis is done with trimethoprimsulfamethoxazole (tmp-smx) for 6 months, up to 1 year. some centers extend the pjp prophylaxis to lifelong. tmp-smx also confers protection against toxoplasma, nocardia, and listeria species infections. alternatively, dapsone, inhaled pentamidine, or atovaquone can be used in patients with a history of sulfa allergy. tmp-smx is recommended at many centers for lifelong in toxoplasmosis seronegative recipients of seropositive cardiac donors (toxoplasma d+/r−) [11] . cmv prevention is recommended to all d+/r− and r+ patients. there are two common strategies for cmv prevention: antiviral prophylaxis and preemptive therapy. both approaches possess similar success rate and their advantages and disadvantages [47] . guidelines recommend valganciclovir or intravenous ganciclovir as the preferred antivirals. oral ganciclovir is an option in heart transplant patients, although it possesses a low oral bioavailability and therefore the theoretical risk of increased resistance. often, cmv immune globulin is used as an adjunctive agent. in heart recipients, prophylaxis is recommended for 3-6 months in d+/r− and 3 months in r+. in lung and heart-lung recipients, the duration of prophylaxis is 12 months and 6-12 months in d+/r− and r+ recipients, respectively [48] . in d−/r− patients, otherwise not receiving cmv active agents, antiviral prophylaxis against other herpes viruses, such as hsv and vzv, should be considered. use of oral cmx001 (oral liposomal formulation of cidofovir) in hematopoietic-cell transplants reduced cmv-related events and may have a potential role in preventing cmv in other transplant settings [49] . refer to table 2 .4 for a list of prophylaxis recommendations. this period is characterized more commonly for nosocomial, bacterial infections. thus, the bacterial organisms present are often mdr (e.g., vre, mrsa). in heart transplant recipients, skin and soft tissue infections (ssti), surgical site infection, and mediastinitis are of concern during this period. likewise, lung and lung-heart transplant recipients may develop infections related to previous respiratory colonization (pseudomonas, aspergillus). other significant infections include aspiration pneumonitis, healthcare-and ventilatorassociated pneumonia, catheter-related bloodstream infections (crbsi), nosocomial utis, and clostridium difficile colitis. donor-derived infections during this period can be present and will include hsv, lymphocytic choriomeningitis virus (lcmv), rhabdovirus (rabies), west nile virus (wnv), and hiv. toxoplasma gondii and trypanosoma cruzi are also serious donor-derived infections in heart transplant recipients that can develop within the first 6 months posttransplantation [50] . during this period, reactivation of latent infections usually occurs. hence, bacterial infections such as those caused by nocardia asteroides, listeria monocytogenes, and mycobacteria tuberculosis typically occur. additionally, fungal infections by aspergillus spp., cryptococcus neoformans, and p. jiroveci and parasitic by toxoplasma gondii, leishmania spp., strongyloides, and trypanosoma cruzi can also be seen. viral infections present during this period include herpesviruses (hsv, vzv, cmv, and ebv) and adenovirus. development of infections after 6 months are predominantly community-acquired pneumonia and urinary tract infections. other diseases include aspergillus and mucor species, nocardia, rhodococcus, and late viral infections including cmv, hepatitis b and c, jc polyomavirus infection, posttransplant lymphoproliferative disorder (ptld), hsv encephalitis, and viral community-acquired infections (e.g., coronavirus, west nile virus, influenza). it is important to recognize transplant recipients as a patient population with increased susceptibility to infections and the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients b doses of valganciclovir, ganciclovir, and other antibiotics may require adjustment for renal function have a low threshold to perform diagnostic workup in the presence of any concerning signs or symptoms. infections monitoring is also done in a structured way when preemptive therapy for cmv is in place (as opposed to universal prophylaxis). protocols vary by the transplant center but, usually, implies a weekly cmv pcr or pp65 ag monitoring [51] . likewise, monitoring of cell-mediated immunity (cmi) using a quantiferon-cmv assay may be useful predicting late-onset cmv disease once cmv prophylaxis has been stopped [52] . cmi also have been monitored for ebv using an enzyme-linked immunospot assay [53] . immunoglobulin g (igg), c3, igg2 levels, and nk cell counts have been proposed as an attempt to identify the risk of infection in heart transplant recipients within the first year [54] . significant drug-drug interactions exist among antimicrobial and immunosuppressive agents. patient medication list should be reviewed carefully. ctp3a4 strong inducers such as nafcillin reduce tacrolimus serum concentrations. in contrast, azoles such as fluconazole can result in increased levels of tacrolimus or cyclosporine. for voriconazole, the dose of tacrolimus needs to be reduced by two-thirds [55] and the cyclosporine dose by 50% [56] . rifamycins can have an opposite drug-drug interaction by decreasing the concentrations of prednisone, cyclosporine, tacrolimus, sirolimus, and mycophenolate mofetil (mmf) [57, 58] . likewise, tacrolimus administration along with quinolones may cause qt prolongation [59] . in heart transplant patients, bacterial infections have similar clinical manifestations commonly observed in other patient populations. however, clinical signs may be subtle or absent (e.g., afebrile). they are the most frequent type of infections in this setting, reaching up to 50% of all infections [3] . the most common are pulmonary infections followed by bacteremias, mediastinal, and skin infections. staphylococcus aureus-predominantly methicillin-resistant-can cause ssti, ventilator-associated pneumonia, mediastinitis, crbsi, other forms of bacteremia, and osteomyelitis. in contrast, coagulase-negative staphylococcus is more commonly associated with crbsi. among gram-negative bacteria, pseudomonas aeruginosa is common, usually of pulmonary origin. escherichia coli is the primary causal organism of utis. extended-spectrum β-lactamase (esbl)producing klebsiella pneumoniae, escherichia coli, klebsiella oxytoca, and citrobacter freundii are also found in 2.2% of heart transplant recipients [60] . nocardia species are well recognized as an opportunistic pathogen in this setting. although relatively rare in heart transplant recipients (frequency <1%), nocardia is only second in frequency in heart transplant after lung transplant recipients [61] [62] [63] . pertinent-independent risk factors associated with the development of this infection in sot include high-dose steroids, history of cmv disease, and high levels of calcineurin inhibitors [62] . with the almost universal prophylaxis with tmp-smx, nocardia infection is less common and often present late, usually after 1 year posttransplant [63] . when they occurred, they affect the lung predominantly, which is the port of entry for disseminated infections and cns invasion. also, it can cause skin nodules and abscesses. listeria monocytogenes can also be seen in heart transplant recipients and can count for a significant proportion of the bacterial meningitis cases in this setting [64] . additionally, myocarditis and myocardial abscesses with this organism have also been documented [65] . mycobacterium tuberculosis and nontuberculous mycobacteria (ntm), although, documented to occur in heart transplantation, are rare in the united states [66, 67] . however, it is important to recognize that the development of tuberculosis (tb) can be more prevalent in some endemic regions and often present with extrapulmonary involvement [68, 69] . legionellosis and rhodococcus equi with mainly pulmonary manifestations (pneumonia, pulmonary infiltrates, or cavitation) are another significant infections among heart transplant recipients [70] . fungal infections excluding pcp represent around 4.0% of all the infections. from them, invasive mold infections (imi) are a significant contribution to morbidity and mortality among heart transplant recipients. the incidence in this population can reach 10 per 1000 person-years, and its associated mortality is approximately 17% [71] . aspergillus represents up to 65% of all imi. its median time of onset is about 46 days, although late presentation (>90 days) has been more recently recognized associated with receipt of sirolimus in conjunction with tacrolimus for refractory rejection or cardiac allograft vasculopathy [72] . the most common clinical presentation for aspergillosis includes fever, cough, and single or multiple pulmonary nodules [73] . extrapulmonary manifestations include spondylodiscitis, infective endocarditis, mediastinitis, endophthalmitis, and brain and cutaneous abscesses [74] [75] [76] [77] [78] . dissemination tends to affect the cns in a good proportion of the cases. mucormycosis is the second most frequent mold affecting heart transplant recipients. mucor, along with other non-aspergillus molds (e.g., scedosporium, ochroconis gallopava), are associated with disseminated infections, cns involvement, and poorer outcomes [79, 80] . pneumocystis jiroveci (pcp)-although with a marked reduction in inci-dence with the introduction of universal prophylaxis-is still a significant pathogen and cases may occur late after heart transplant. cryptococcosis, although infrequent among sot patients, has its higher incidence in heart transplant recipients [81] . usually, its manifestations present late and affect the lungs and the cns predominantly. histoplasmosis and coccidioidomycosis occurred typically in the first year after transplant. antigenuria was the most sensitive diagnostic test in sot for histoplasmosis [82] . finally, candida infections are an important cause of morbidity and mortality as well. rate of colonization is higher than in the general population [83] . candida most commonly causes an oral mucosa infection. although there has been a decline of invasive infections over time, these do occur and typically in the form of bloodstream infections secondary to catheter-related infections, tracheobronchitis, or disseminated disease [84] . additionally, other confined end-organ injuries such as endophthalmitis and esophagitis can also be seen. cmv infection is of critical importance among sot. in heart transplant recipients, cmv has been inconsistently associated with cardiac allograft vasculopathy [85] . furthermore, cmv leads to upregulation of pro-inflammatory cytokines, increase procoagulant response, left ventricular dysfunction, allograft rejection, and an increase of opportunistic infections [86] . the greatest risk for developing cmv disease is cmv-negative recipients of cmvpositive organs (d+/r−), followed by d+/r+ and d−/r+. a clinical report estimated that the rate of infections in heart transplant ranges between 9% and 35%, and disease is present in around 25% of patients [87] . the clinical manifestations are not unique to heart transplant recipients and include a cmv syndrome (fevers, myalgias, arthralgias, malaise, leukopenia, and thrombocytopenia). cmvassociated end-organ injury in this setting includes most frequently pneumonitis and gastrointestinal disease [10] . other manifestations comprise myelosuppression, hepatitis, and pancreatitis. in contrast to the high frequency observed in aids patients, chorioretinitis in heart transplant patients is relatively rare [87] . guidelines on cmv diagnosis and managements are discussed in more detail in chap. 55 and also have been published elsewhere [88] . other herpes viruses are of important consideration as well. ebv-associated t-cell ptlds are more frequent in heart transplant recipients (0.4%) than in other sot patients [89] . ptld is a significant contributor to morbidity and mortality in the pediatric heart transplant population [90] . human t-lymphotropic virus type i (htlv1), human herpes virus (hhv)-6, hhv-7, and hhv-8 might play a role in ebv(−) t-cell ptlds as well. herpes viruses can manifest, as in other hosts, as mucocutaneous lesions for hsv, herpes zoster for vzv, infectious mononucleosis in the case of ebv, kaposi sarcoma for hhv-8, and encephalitis for hhv-6/7. hepatitis, colitis, pneumonitis, and gastrointestinal disease have also been attributed to dissemination with certain herpes viruses. herpes viruses can present with disseminated skin lesions (with or without vesicle formation) and fever of unknown origin. adenovirus has been associated with rejection, ventricular dysfunction, coronary vasculopathy, and the need for retransplantation. the current standard treatment for adenovirus is cidofovir, but outcomes are not optimal [91] . chronic hepatitis without an identifiable cause should prompt testing for hepatitis e virus (hev). chronic hev infection leads to the rapid development of fibrosis. hev testing should be done with rna pcr due to a delay in the antibody response. we recommend decreased immunosuppression and ribavirin therapy for 3 months [92, 93] . other less common manifestation that should be considered under the correct epidemiologic risk factors include htlv-1/ htlv-2-associated myelopathy, rabies, lymphocytic choriomeningitis virus, subacute measles encephalitis, mumps (associated parotitis, orchitis, vestibular neuritis, and allograft involvement), dengue virus, orf virus, human coronavirus, and influenza [36] . cardiac transplant itself is one the predictors for development of toxoplasmosis [94] . other associated risk factors include negative serum status before transplant, diagnosis of cytomegalovirus (cmv) infection, and high-dose prednisone. toxoplasmosis can be transmitted by the donor heart (d+/r−, especially during the first 3 months) or can reactivate from the recipient (>3 months). most of the infections developed during the first 6 months posttransplant and are predominantly primary infections. about 22% of infected patients had a disseminated infection carrying an estimated 17% mortality. toxoplasmosis can manifest otherwise with myocarditis, encephalitis, pneumonitis, or chorioretinitis. diagnosis requires identification of tissue cysts surrounded by an abnormal inflammatory response, detection of toxoplasma dna in body fluids by pcr, or positive toxoplasma-specific immunohistochemistry in affected organs. posttransplant serological tests are not helpful for diagnosis and may be misleading since results may change or not regardless of the presence of toxoplasmosis [95] . the preferred treatment regimen is a combination of pyrimethamine with sulfadiazine [96] . advanced chagasic cardiomyopathy is a primary indication for heart transplantation in some centers [13] . trypanosoma cruzi, the causal organism of chagas disease, can be transmitted up to 75% of the time from infected heart donors (d+/r−) [97] . additionally, chagas disease can reactivate from the donor once immunosuppression is in place (r+). the reactivation rate can range between 22% and 90% in recipients with chronic chagasic cardiomyopathy undergoing heart transplant [98] [99] [100] . additional risk factors for reactivation include rejection episodes, neoplasms, and use of mmf [98] . the mean onset of symptoms is approximately 112 days [101] . once manifested, chagas can present with nonspecific symptoms such as fever, malaise, anorexia, hepatosplenomegaly, and lymphadenopathy. myocarditis, pericarditis, and encephalitis are also seen. reactivation can mimic rejection and exhibits congestive heart failure, av block and skin manifestations such as nodules and panniculitis. increased eosinophil count and anemia can be indirect indicators of reactivation [102] . diagnosis is made with the visualization of circulating trypomastigotes in peripheral blood. additionally, blood and tissue pcr can be used. tissue amastigotes can be seen in biopsy h&e preparations (fig. 2.1) . finally, serologies are a crucial aspect in the diagnosis especially if seroconversion have been documented. in asymptomatic individuals, when the diagnosis of chagas has been established in the donor, monitoring should be instituted with weekly blood t. cruzi pcr and microscopy [29] . preferred antitrypanosomal therapy consists on benznidazole. nifurtimox is an alternative treatment option. posaconazole has anti-parasitic activity but carries high failure rates [103, 104] . gi disease with isospora (cystoisospora) belli, cryptosporidium, cyclospora, and microsporidia has been reported to affect sot recipients. microsporidiosis can manifest with disseminated disease: fever, keratoconjunctivitis, cns involvement, cholangitis, cough, and thoracic/ abdominal pain [94] . other rare parasitic infections affecting heart transplants include leishmaniasis, strongyloidiasis, and free-living amoebas [94, 105] . the rate of surgical site infections (ssi)-sternal wound infections-in patients receiving antimicrobial prophylaxis ranged from 5.8% to 8.8% following heart transplant procedures [41] . heart transplantation itself is an independent risk factor for ssis. other risk factors include age, prophylaxis with ciprofloxacin alone, positive wire cultures, female gender, previous left ventricular assist device (vad) placement, bmi >30 kg/m 2 , previous cardiac procedures, and inotropic support for hemodynamic instability [41, 106] . similarly to other hosts, staphylococcus species are the predominant organism causing sstis. mrsa can reach up to 21% of the cases. gram-positive organisms: vre (e. faecalis), coagulase-negative staphylococci, and other enterococcus species are other etiologic agents. candida and selected gram negatives such as enterobacteriaceae, p. aeruginosa, and stenotrophomonas maltophilia can cause ssis as well [107] . sternal osteomyelitis often complicates deep ssi. additionally, sternal wound infections by ntm and fungi such as aspergillus and scedosporium have been documented [108, 109] . herpes zoster is also an important consideration and source of morbidity. herpes zoster (hz) is found as a complication in 19-22% of the patients with a median time of presentation ranging from 0.73 to 2.10 years [64, 110] . close to half may develop postherpetic neuralgia. multi-dermatome involvement, zoster ophthalmicus, and meningoencephalitis are also described. exposure to mmf is an independent risk factor. conversely, cmv prophylaxis reduces the risk for hz. bloodstream infections (bsis) are a risk factor for mortality among heart transplant recipients. likewise, sot recipient status is an independent risk factor for developing bacteremia [111] . in heart transplant recipients; the rate of bsi ranged between 16% and 24%. the median onset is about 51-191 days, and the sources are in order of frequency: lower respiratory tract, urinary tract, and crbsi. gram-negative bacteria were more commonly isolated. they are in order of appearance e. coli, p. aeruginosa, and k. pneumoniae. more common grampositive bacteria were s. aureus, s. epidermidis, e. faecalis, and l. monocytogenes. directly attributable mortality is 12.2%. among the identifiable independent risk factors to develop bsi are hemodialysis, prolonged intensive care unit stay, and viral infections [112, 113] . infective endocarditis (ie) is seen more frequently among heart transplant recipients than in the general population. with ie occurred, it most commonly involves the mitral and tricuspid valves and staphylococcus aureus and aspergillus are the main etiologic organisms. the main predisposing factors in this setting are believed to be the frequent use of vascular indwelling catheters and the frequency of endomyocardial biopsies [114] . staphylococcus aureus bacteremia in heart transplant recipients ranges from 10% to 38% [11, 115] . the sources of sa bacteremia in sot are crbsi (30%), pneumonia (24%), wound (14%), endocarditis (10%), intra-abdominal infections (9%), bone and joint (7%), cardiac devices (3%), uti (1%), and ssti (1%) [115] . immediately following heart transplant and during the 1st month, patients are more susceptible to develop pneumonia, most of which are healthcare or ventilator associated and therefore caused by nosocomial organisms such as mrsa, pseudomonas aeruginosa, and other gram negatives including acinetobacter and esbl-enterobacteriaceas. pneumonia is one the major contributors to mortality in the early postoperative period. pneumonia-related mortality approaches 15% [116] . after the 1st month, interstitial pneumonia and pneumonitis can develop, and the differential includes herpesviruses (hsv, cmv, vzv) and respiratory syncytial virus (rsv), toxoplasma gondii and pneumocystis jiroveci. pulmonary nodules with or without cavitation can be caused by fungi such as coccidioidomycosis, aspergillosis, mucormycosis, cryptococcosis; bacterial including actinomycosis, tuberculosis, atypical mycobacterial infections, nocardia, rhodococcus equi, and gramnegative bacilli; and noninfectious causes like pulmonary infarction or lymphoproliferative disorders [117, 118] . pulmonary nodules are seen in about 10% of the patients, and the median detection time is about 66 days. the associated symptoms are fever and cough. the most frequent etiology is aspergillus followed by nocardia, and rhodococcus. cmv is an exceedingly rare cause of pulmonary nodules. the diagnostic approach with the higher yield is transthoracic fine needle aspiration followed by bronchoalveolar lavage and transtracheal aspiration [118] . communityacquired pneumonia caused by streptococcus pneumonia, legionella spp., mycoplasma, and influenza is another source of morbidity [10] . mediastinitis is a common complication in this setting. in patients receiving antimicrobial prophylaxis, mediastinitis develops in 3-7% of the patients [107, 119] . a ct scan is usually necessary to determine the extension of the infection. mrsa staphylococcus epidermidis, gram-negative bacteria, and aspergillus fumigatus are frequently found as the causal organisms [120] . antimicrobial therapy should be accompanied by aggressive surgical debridement [121] . there are not distinctive abdominal-pelvic complications among heart transplant recipients. clostridium difficile is a common hospital-related cause of diarrhea associated with the use of antimicrobials. other etiology for diarrhea second-ary to acute gastroenteritis can present in a protracted way in this setting. listeria infection can present as a febrile gastroenteritis illness as well. nontyphoid salmonella infection has been described to complicate the early postoperative period in a center in taiwan [122] . acute cholecystitis can affect heart transplant recipients advocating to have a low threshold to use ultrasound as a screening method [123] . acute pancreatitis with abscess formation has also been described [124] . as pointed above, hepatitis e can present with persistently abnormal liver tests. although less frequent than in kidney transplant recipients, urinary tract infections are an important cause of morbidity. utis are predisposed by foley catheters. the organisms most commonly involved are gram-negative bacteria, enterococcus, and candida. polyomavirus nephropathy by bk virus has been described in heart transplant recipients and might be a contributor to chronic kidney disease [125] . the need for urgent transplantation and multiple transfusions are independently associated with infectious, neurologic complications. its overall mortality can reach 12% [64] . donor-derived meningoencephalitides affecting heart transplant recipients usually manifest within the first 30 days. these infections include west nile virus, arenaviruses (e.g., lcmv), and rabies. wnv can manifest with a guillain-barré-like axonopathy with cerebrospinal fluid (csf) pleocytosis. in addition to meningitis or encephalitis, ataxia, myelitis, optic neuritis, polyradiculitis, and seizures can also be observed [126] . wnv can be also acquired by the recipient in the community or through blood transfusions and present at a later time [127] . other infectious forms of meningitis and encephalitis that can present after the 1st month include listeriosis, streptococcus pneumoniae, trypanosoma cruzi, toxoplasma, hhv-6, and disseminated herpes virus infections (cmv, vzv, hsv, and ebv) [128] [129] [130] . the absence of appropriate primary prophylaxis or monitoring increases their risk. aspergillus causes the majority of brain abscess. additionally toxoplasma, tuberculosis, listeria spp., cryptococcus neoformans, scedosporium spp., and nocardia can also be causative agents [129] . concomitant pulmonary involvement is common, particularly for those whose portal of entry is the respiratory tract. progressive multifocal leukoencephalopathy (pml), a demyelinating disease caused by the reactivation of jc virus, has a usual median onset of 27 months. it carries a marked high case fatality rate and a median survival of 6.4 months in sot [131] . the use of rituximab as an antirejection treatment seems to confer an increased risk for pml [132] . htlv-1-associated myelopathy (ham) has been described as well in sot. bacterial infections are the most common type of infections among lung and lung-heart transplant recipients. the anatomic site most frequently affected is the respiratory tract, usually manifested with pneumonia, sinusitis, or tracheobronchitis. previous colonization, healthcare associated, and procedures related are the primary sources. for patients with cystic fibrosis (cf), knowledge of previous colonization results may provide some diagnostic and therapeutic advantages. pseudomonas aeruginosa is a predominant colonizing pathogen in cf. however, acinetobacter baumannii, burkholderia species, stenotrophomonas maltophilia, achromobacter xylosoxidans, ntm, pandorea, and ralstonia are also observed [23] . furthermore, pathogens that are known to cause nosocomial pneumonia during the 1st month include staphylococcus aureus, pseudomonas aeruginosa, other gram negatives (klebsiella pneumoniae, enterobacter cloacae, serratia marcescens, escherichia coli, acinetobacter species), and anaerobes. gram-positive bacteria are a common source of infections making up to 40% of them [133] . the most common sites affected were the respiratory tract, followed by bacteremia, skin, wound, and catheter related. the pathogens more frequently identified are staphylococcus species (77%), enterococcus species (12%), streptococcus species (6%), pneumococcus (4%), and eubacterium lentum (1%). staphylococcus aureus infection can develop up to 20% of lung recipients. sa commonly causes pneumonia, followed by tracheobronchitis, bacteremia, intrathoracic infections, and sstis [20] . streptococcus pneumoniae is community acquired and present with pneumonia, usually after 6 months posttransplant. pseudomonas aeruginosa has high rates of colonization (up to 40%) and disease (30%) [134] . other significant bacterial infections that may present after the 1st month are mycobacterium tuberculosis, ntm, nocardia, rhodococcus, and legionella. isolation of ntm in lung transplant recipients without evidence of disease is not associated with increased mortality [135] . nocardiosis can occur in about 2% of the lung transplant recipients. the median time of onset ranges from 14.3 to 34.1 months [136, 137] . nocardia asteroides, n. farcinica, n. nova, and n. brasiliensis have been reported. n. farcinica appears to carry worse outcomes. this infection can present as a breakthrough in the presence of trimethoprim-sulfamethoxazole for p. jiroveci prophylaxis, although the isolates may remain susceptible. mortality has been reported to range between 18% and 40%. the native lung is more frequently affected in single-lung transplant recipients. nodules are the more prevalent radio-graphic finding. extrapulmonary involvement affecting the skin and brain can be seen. hypogammaglobulinemia and neutropenia seem to confer additional risk factors for nocardiosis in this setting [137] . fungal infections are frequent complications in lung and lung-heart transplant. they present in about 15-35% and carry an overall mortality close to 60% [138] . aspergillus and candida are the most frequent causative agents. other important fungi include cryptococcus spp., mucormycosis, endemic fungi (histoplasma, coccidioides, and blastomyces spp.), scedosporium spp., fusarium spp., and dematiaceous molds. candida infections are prominent during the 1st month after transplantation. it can be one of the most common causes of bsi in this setting [139] . although colonization of the upper airways and gastrointestinal tract is common, candida additionally can cause mucocutaneous disease, tracheobronchitis, anastomosis site infections, crbsi, and disseminated disease. aspergillus spp. lead as the cause of invasive fungal infections. its attack rate of infection is almost ten times compared to that in other sot patients (estimated incidence of 6% among lung transplant recipients) [140, 141] . a. fumigatus is the most common species, but a. terreus, a. flavus, and a. niger have been described as well. the main predisposing risk factors in this setting are intense immunosuppression, previous colonization with aspergillus spp., airway ischemia, and bos. single-lung transplant possesses the greatest risk to developing an invasive aspergillus infection carrying a higher mortality than double-lung and heart-lung transplant recipients. single-lung recipients are usually older and more likely to have copd as the indication for transplantation [140] . aspergillus infections can present as tracheobronchitis, pneumonia, or disseminated disease. extrapulmonary involvement includes sinusitis, cns or orbits infections, and vertebral osteomyelitis. aids in the diagnosis can include surveillance bronchoscopies (bronchoalveolar lavage stain and culture; biopsy), chest ct and serum/bal galactomannan, beta-d-glucan, and pcr. the presence of pulmonary nodular lesions in invasive infections can carry better outcomes [142] . voriconazole is the treatment of choice. it is important to note that immune reconstitution inflammatory syndrome (iris) can develop at a median of 56 days in 7% of treated lung transplant recipients [143] . in aspergillus tracheobronchitis, nebulized amphotericin b and debridement of the bronchial anastomosis are important adjuvant measures to systemic antifungal therapy [144, 145] . pneumocystis jirovecii pneumonia manifests from 1 to 6 months. its incidence has been reduced dramatically with universal tmp/smx prophylaxis. cryptococcosis with a rate of 2% in lung transplant recipients presents with pulmonary involvement, but dissemination with meningitis can occur. furthermore, cryptococcus skin manifestations like cellulitis and cryptococcus-associated iris have been documented [146, 147] . viral infections are a common cause of morbidity among lung transplant recipients. the most common viruses are (1) cmv among the herpes viruses and (2) community-acquired respiratory viruses. as in other sot recipients, the higher risk to develop cmv infection is among d+/r−, followed by d+/r+, d−/r+, and d−/r−. this last scenario carries less than 5% of risk [48, 148] . lung transplant recipients possess higher risk for cmv than other sot with an estimated incidence of 30-86% [87] . the lung is considered a primary reservoir for cmv latency, and abundant lymphocytic tissue surrounds the transplanted organ. additionally, the use of antilymphocyte antibodies to treat rejection or for immunosuppression and other herpesviruses infections are additional risk factors for cmv disease [149] . interferon (ifn)-γ (+874t/t) polymorphism increases ifn levels and may be a predisposition for cmv disease [150] . cmv is significantly associated with bos, which reduces survival after the first year posttransplant [151] . cmv disease is most commonly manifested by pneumonitis or viral syndrome and less frequently with gastrointestinal disease. among lung transplant recipients, ganciclovir-resistant cmv carries an increased morbidity and mortality [152] . infections with community-acquired respiratory viruses ranged from 7.7% to 64%. these infections are associated with increased risk to develop pneumonia, graft dysfunction manifested by lung function loss, bos, high calcineurin inhibitor blood levels, and increase mortality [153] [154] [155] . these viruses include influenza, parainfluenza, respiratory syncytial virus (rsv), coronaviruses, human rhinovirus, adenovirus, human metapneumoviruses, and bocaviruses. the hospitalization rates are higher for influenza and parainfluenza (50% and 17%, respectively) [154] . symptoms are usually nonspecific. diagnosis often requires detection of viral nucleoprotein antigens in nasopharyngeal swabs or bronchoalveolar lavage (bal) by enzyme immunoassay or fluorescent antibody or the amplification of nucleic acid by pcr. ribavirin may possess activity against paramyxoviruses (rsv, metapneumovirus, and parainfluenza). ribavirin is administered inhaled, orally, or intravenously. oseltamivir or zanamivir is the treatment choice of influenza a or b [156] . adamantanes (amantadine and rimantadine) are not active against influenza b, and there is a marked increase resistance among influenza a strains [156] . similarly to other sot recipients, dna viruses like non-cmv herpesviruses (hsv-1,-2), vzv, hhv-6,-7,-8, and ebv are a source of significant morbidity including but not limited to cmv-negative viral syndrome, rash, pneumonitis, hepatitis, and encephalitis [157] . lastly, polyomavirus such as bk virus (bkv), jc virus (jcv), and simian virus 40 (sv40)-although fre-quently encountered in lung transplant recipients with an unclear causality-may cause worsening renal function or survival [158] . ptld is also a well-recognized complication. a trend toward late ptld presentation (>1 year) has been documented where b symptoms are more predominant as well as extra-graft involvement [159] . as other immunosuppressive states, certain parasitic infections can complicate lung and heart-lung transplants recipients. it is critical to elicit a detailed history and geographic risk factors to determine the risk of acquisition and the potential etiologic agent. toxoplasmosis can result from primary infection or reactivation of previous latent infections. toxoplasmosis can develop in patients with negative epidemiological history for cat ownership or consumption of undercooked meat. in patients with primary toxoplasmosis, nonspecific symptoms such as fever, lymphadenopathy, or organ injury may be present. reactivation can cause encephalitis with or without space-occupying brain lesions, seizures, chorioretinitis, fever of unknown origin, pneumonitis, myocarditis, and rash. although cases of the lung fluke, paragonimus westermani have not been reported in lung transplantation, it can be a potential threat in endemic areas where this organism is endemic. other parasites that can target the lung in immunosuppressive states include echinococcus, schistosoma, and strongyloides stercoralis [160] . strongyloidiasis can present as hyperinfection syndrome [161] . leishmania, although infrequently seen, has been reported among lung and lung-heart recipients [30] . free-living amoebas can affect this population as well. amoebic granulomatous dermatitis and disseminated infection presenting with ulcerative skin lesions, respiratory failure, and seizures have been described in lung transplant recipients [162, 163] . finally, alimentary protozoa, including cryptosporidium, which present with diarrhea and may elevate tacrolimus levels [164] , and microsporidia, which present with unusual manifestations like myositis or granulomatous interstitial nephritis, affects lung transplant recipients [165, 166] . the overall rate of ssis is about 13% with a significant proportion of infections being organ or space occupying (72%), deep incisional (17%), and superficial (10%) [18, 41] . independent risk factors to develop ssi are diabetes, female donor, prolonged ischemic time, and the number of red blood cells transfusion during the perioperative period [167] . ssis are associated with a 35% mortality within the first year of transplantation. the most common organisms found to cause ssi or mediastinitis are p. aeruginosa, candida species, s. aureus (including mrsa), enterococcus, coagulasenegative staphylococci, burkholderia cepacia, e. coli, proteus mirabilis, serratia marcescens, acinetobacter baumannii, enterobacter cloacae, and klebsiella species. there is a correlation in up to 33% of the patients' ssi causative organisms with previous pathogens colonizing recipients' native lungs at the time of the transplant [167] . the median onset is 25 days after lung transplant [167] . although rare, ntm can cause ssi infections among lung transplant recipients. the most frequently encountered are mycobacterium avium complex followed by mycobacterium abscessus and mycobacterium gordonae. ntm ssi infections can be complicated by progressive disseminated disease or requirement of lifelong suppressive therapy [135] . other organisms such as mycoplasma hominis and lactobacillus spp. have also been described. deep infections can affect up to 5% of the patients. sternal osteomyelitis can reach up to 6% of these deep infections. causative organisms for sternal osteomyelitis include pseudomonas aeruginosa, serratia marcescens, and scedosporium. non-sternal osteomyelitis affecting the calcaneus bone has complicated a disseminated infection with aspergillus fumigatus [168] . bloodstream infections (bsis) occur with an estimated rate of 25% among lung transplant recipients. a major proportion of bsis occur in the early posttransplant period. bsis infections are significantly associated with worse survival [139, 169] . the most common organisms encountered are staphylococcus aureus, pseudomonas aeruginosa, and candida [139] . pseudomonas aeruginosa bsi-predominantly present during the transplant hospitalization period and more commonly affecting cf patients-is followed in frequency by burkholderia cepacia and candida albicans. conversely, staphylococcus aureus was the predominant organism after transplantation discharge. in an estimated 70% of bsi, the source was pulmonary, followed in frequency by crbsi, gastrointestinal infection, peritonitis, and uti. a pulmonary source of bacteremia in sot often develops into septic shock [170] . although unusual, cases of aspergillus fumigatus endocarditis have been described following lung transplantation [171] . often patients had cf as the underlying lung disease and a median of 8 ± 6 months presentation. this complication carries a high mortality and often requires a combination of antifungal therapy with valvular replacement surgery. infectious complications related to the chest cavity include mediastinitis, cardiac (pericarditis and myocarditis), lung parenchyma infections (nodular infiltrates, cavitation, or pneumonia), bronchial anastomosis infections, and pleural space infections (bronchopleural fistula and empyema). empyema followed by mediastinitis and pericarditis, in addition to surgical wound infections and sternal osteomyelitis, is the most frequent deep ssi complications affecting the chest cavity. empyema presents in around of 3.6% of cases. it occurs during the first 6 months after transplantation (median 46 ± 39 days) carrying an estimated mortality of 28.6% [172] . most common organisms found are staphylococcus spp., e. coli, enterobacter spp., klebsiella spp., mycoplasma hominis, vre, and candida. furthermore, mycobacterium abscessus was isolated as a rare causative agent of empyema as well [173] . the degree of immunosuppression, reduced renal function, previous sternotomy, and re-exploration due to bleeding are listed as potential risk factors for mediastinitis [119] . there is an increased prevalence of mediastinitis caused by gram negatives and fungi among lung transplant recipients. causative organisms for mediastinitis are similar to ssi and are listed above. infectious pericarditis can be present up to 6% of the patients (isolated organisms include mssa, mycoplasma hominis, and scedosporium prolificans) [167, 174, 175] . due to their high fatal rate, fungal bronchial anastomotic infections are critical to recognize. pneumonia is believed to affect around 21% of lung recipients and 40% of heart-lung recipients. nosocomial organisms cause early pneumonia as in other posttransplant settings. the donor's lung seems to be the primary source for pneumonic infections, although the recipients' upper airways or sinuses are also potential sources. preoperative colonization with gram-negative rods and colonized infected donor bronchus or perfusate are recognized risk factors for pneumonia. likewise, pretransplantation colonizing microorganisms from suppurative lung disease are associated with pneumonia development posttransplant [176] . the most common causal organisms are pseudomonas aeruginosa, staphylococcus aureus, and aspergillus spp. other pathogens include bacteria such as b. cepacia, enterobacter species, s. maltophilia, klebsiella species, s. epidermidis, and e. coli, and fungi such as fusarium spp., cryptococcus neoformans, and paracoccidioides brasiliensis [176] . after the 1st month, pneumonia can present as local infiltrates, diffuse interstitial infiltrates, and nodules with or without cavitation. this type of presentation may aid in the possible causative microorganism. the list of potential pathogens is extensive and includes in addition to the already mentioned nocardia, chlamydia pneumonia, legionella, tb, ntm, pneumocystis jirovecii, rhodococcus, herpesviruses (cmv, hsv, and vzv), respiratory viruses, endemic fungi (e.g., histoplasmosis), mucormycosis, and scedosporium spp. [177] [178] [179] . similarly to other sot, common infectious complications affecting the gastrointestinal or genitourinary tract include clostridium difficile colitis and utis. intra-abdominal com-plication carries an overall increase mortality [180] . frequent gi symptoms presenting posttransplant are diarrhea which can affect almost 30% of lung transplant recipients and abdominal pain. abdominal pain should prompt further investigation for potential intra-abdominal causes. in the pediatric population, the possibility of ptld should be investigated since it carries a high mortality [181] . other described infectious intra-abdominal complications include digestive perforation (seen in 6%) [182] , retroperitoneal abscesses, cholecystitis, perianal abscesses, esophagitis, pancreatitis, pancreatic abscesses, hepatitis, diverticulitis, appendicitis, cmv colitis, megacolon, and colon rupture [180, 183, 184] . in developing countries, persistently abnormal liver enzymes should prompt testing for hev. hev rna should be used for screening. oral ribavirin seems to be safe and effective in this setting [185] . cns symptoms developing during the 1st month following lung or heart-lung transplantation should trigger the concern for donor-derived viral infections. lcmv often is accompanied by csf normal to low glucose, marked elevated protein, and mild pleocytosis [36] . although with unclear benefit, ribavirin has been used. donor-transmitted rabies is an uncommon but neurologic devastating complication that occurs within the first 30 days of transplant. lung transplantation has been described as a potential causal mechanism [186] . other organisms known to cause meningitis in lung transplant recipients are cryptococcus, tuberculosis, wnv, and herpesviruses [187, 188] . diagnosis of wnv in this setting requires nuclear acid amplification due to the unreliability of serologic testing. scedosporium apiospermum infections often cause dissemination including cns abscesses in addition to pulmonary involvement among lung transplant recipients [189] . it is important to differentiate from other molds, since amphotericin b is ineffective against scedosporium spp. in severe cases or refractory disease without an appropriate surgical debridement, the addition of terbinafine to voriconazole may prove to be useful [190] . other recognized organisms causing occupying brain lesions are fusarium, nocardia, aspergillus, toxoplasmosis, cryptococcus neoformans, listeria, and cladophialophora bantiana [191] [192] [193] . pml, a late manifestation, can be associated with intensified immunosuppression or rituximab. cidofovir followed by mirtazapine can be considered as a form of therapy for pml. infections in heart, lung, and heart-lung transplant recipients are a complex, dynamic, and evolving process. many factors such as demographics, timing, type of transplant, anatomy, and microbiology, among others, interplay in the development of these fatal complications. pertinent recognition and treatment of these infections improve transplantation outcomes. the registry of the international society for heart and lung transplantation: thirtieth official adult heart transplant report-2013; focus theme: age heart transplantation during active infective endocarditis: case report and review of the literature changing trends in infectious disease in heart transplantation clinical epidemiology of the global expansion of klebsiella pneumoniae carbapenemases infections caused by gram-positive bacteria: a review of the global challenge heart-lung transplantation lung transplantation for patients with cystic fibrosis and burkholderia cepacia complex infection: a single-center experience infectious complications after cardiac transplantation in man epidemiology and clinical impact of infection in patients awaiting heart transplantation infection in heart transplantation infectious complications among 620 consecutive heart transplant patients at stanford university medical center effect of infectious diseases on outcome after heart transplant infections in heart transplant recipients in brazil: the challenge of chagas' disease diagnosis and management of infectious diseases in cardiothoracic transplantation and mechanical circulatory support the registry of the international society for heart and lung transplantation: thirtieth adult lung and heart-lung transplant report-2013; focus theme: age thoracic transplantation in the united states: an analysis of unos registry data high lung allocation score is associated with increased morbidity and mortality following transplantation post-operative nosocomial infections after lung and heart transplantation early and late infections in lung transplantation patients staphylococcus aureus infections in the early period after lung transplantation: epidemiology, risk factors, and outcomes epidemiology and management of infections after lung transplantation mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management impact of multidrug-resistant organisms on patients considered for lung transplantation the effects of panresistant bacteria in cystic fibrosis patients on lung transplant outcome survival after lung transplantation of cystic fibrosis patients infected with burkholderia cepacia complex clinical outcome following lung transplantation in patients with cystic fibrosis colonised with burkholderia cepacia complex: results from two french centres expanded infectious diseases screening program for hispanic transplant candidates coccidioidomycosis and lung transplantation screening and treatment of chagas disease in organ transplant recipients in the united states: recommendations from the chagas in transplant working group visceral leishmaniasis in lung transplantation strongyloidiasis in transplant patients reactivation histoplasmosis after treatment with anti-tumor necrosis factor alpha in a patient from a nonendemic area assessment of infection risks prior to lung transplantation screening of donor and recipient prior to solid organ transplantation outcome of renal transplantation in eight patients with candida sp. contamination of preservation fluid rare and emerging viral infections in transplant recipients immune responsiveness and protective immunity after transplantation advisory committee on immunization practices (acip) recommended immunization schedule for adults aged 19 years and older -united states idsa clinical practice guideline for vaccination of the immunocompromised host prophylaxis strategies for solid-organ transplantation clinical practice guidelines for antimicrobial prophylaxis in surgery antifungal agents for preventing fungal infections in solid organ transplant recipients targeted antifungal prophylaxis in heart transplant recipients comparative safety of amphotericin b lipid complex and amphotericin b deoxycholate as aerosolized antifungal prophylaxis in lung-transplant recipients safety of aerosolized amphotericin b lipid complex in lung transplant recipients antifungal prophylaxis in lung transplantation cytomegalovirus in solid organ transplantation: epidemiology, prevention, and treatment cytomegalovirus in solid organ transplantation cmx001 to prevent cytomegalovirus disease in hematopoietic-cell transplantation infection in solid-organ transplant recipients cytomegalovirus in solid organ transplant recipients cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients evaluation of epstein-barr virus-specific immunologic response in solid organ transplant recipients with an enzyme-linked immunospot assay decreased levels of serum complement c3 and natural killer cells add to the predictive value of total immunoglobulin g for severe infection in heart transplant recipients drug interaction between voriconazole and calcineurin inhibitors in allogeneic hematopoietic stem cell transplant recipients effect of voriconazole on the pharmacokinetics of cyclosporine in renal transplant patients drug interaction between mycophenolate mofetil and rifampin: possible induction of uridine diphosphate-glucuronosyltransferase interaction of rifampin and glucocorticoids. adverse effect on renal allograft function drug-induced long qt syndrome extended spectrum beta-lactamase-producing enterobacteriaceae infection in heart and lung transplant recipients and in mechanical circulatory support recipients nocardia infection in heart transplant recipients risk factors, clinical characteristics, and outcome of nocardia infection in organ transplant recipients: a matched case-control study pulmonary nocardiosis in heart transplant recipients: treatment and outcome infectious and non-infectious neurologic complications in heart transplant recipients listerial myocarditis in cardiac transplantation tuberculosis in heart transplant recipients infections due to nontuberculous mycobacteria in kidney, heart, and liver transplant recipients clinical features and outcomes of tuberculosis in solid organ transplant recipients tuberculosis after heart transplantation: twenty years of experience in a single center in taiwan legionellosis in heart transplant recipients epidemiology, outcomes, and mortality predictors of invasive mold infections among transplant recipients: a 10-year, single-center experience late-onset invasive aspergillosis in organ transplant recipients in the current era invasive aspergillosis in the setting of cardiac transplantation cutaneous aspergillosis: a report of six cases aspergillus endophthalmitis following orthotopic heart transplant aspergillus fumigatus endocarditis of the mitral valve in a heart transplant recipient: a case report mediastinitis caused by aspergillus fumigatus with ruptured aortic pseudoaneurysm in a heart transplant recipient: case study aspergillus spondylodiscitis: successful conservative treatment in 9 cases opportunistic mycelial fungal infections in organ transplant recipients: emerging importance of non-aspergillus mycelial fungi disseminated ochroconis gallopava infection in a heart transplant patient cryptococcosis in organ transplant recipients: an overview histoplasmosis after solid organ transplant presence of candida spp. in the oral cavity of heart transplantation patients trends in invasive disease due to candida species following heart and lung transplantation cardiac allograft vasculopathy: current knowledge and future direction cytomegalovirus-associated allograft rejection in heart transplant patients update and review: state-of-the-art management of cytomegalovirus infection and disease following thoracic organ transplantation updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation clinicopathological characteristics of posttransplant lymphoproliferative disorders of t-cell origin: single-center series of nine cases and meta-analysis of 147 reported cases post-transplant lymphoproliferative disorder in pediatric heart transplant recipients adenovirus infections in heart transplantation clinical implications of chronic hepatitis e virus infection in heart transplant recipients ribavirin for chronic hepatitis e virus infection in transplant recipients parasitic infections in solid organ transplant recipients primary and reactivated toxoplasma infection in patients with cardiac transplants. clinical spectrum and problems in diagnosis in a defined population toxoplasmosis in heart transplant recipients donor-derived trypanosoma cruzi infection in solid organ recipients in the united states risk factors for chagas' disease reactivation after heart transplantation a systematic review of studies on heart transplantation for patients with end-stage chagas' heart disease heart transplantation in 107 cases of chagas' disease chagas disease in the immunosuppressed host eosinophil blood count and anemia are associated with trypanosoma cruzi infection reactivation in chagas' heart transplant recipients immunosuppression and chagas disease: a management challenge randomized trial of posaconazole and benznidazole for chronic chagas' disease leishmaniasis in a heart transplant patient incisional surgical infection in heart transplantation incidence, treatment strategies and outcome of deep sternal wound infection after orthotopic heart transplantation scedosporium apiospermum pneumonia and sternal wound infection in a heart transplant recipient aspergillosis with aspergillus osteomyelitis and diskitis after heart transplantation: surgical and medical management incidence and risk factors for herpes zoster following heart transplantation population-based study of the epidemiology and the risk factors for pseudomonas aeruginosa bloodstream infection bloodstream infections among heart transplant recipients bloodstream infection in heart transplant recipients: 12-year experience at a university hospital in taiwan infective endocarditis following orthotopic heart transplantation: 10 cases and a review of the literature staphylococcus aureus bacteremia in solid organ transplant recipients: evidence for improved survival when compared with nontransplant patients pulmonary complications in heart transplant recipients pulmonary nocardiosis in a heart transplant patient: case report and review of the literature lung nodular lesions in heart transplant recipients mediastinitis in heart and lung transplantation: 15 years experience bacterial mediastinitis after heart transplantation: clinical presentation, risk factors and treatment surgical treatment of mediastinitis after cardiac transplantation nontyphoid salmonella infection in heart transplant recipients cholelithiasis in heart transplant patients survival following rupture of a pancreatic abscess in a heart transplant recipient polyomavirus nephropathy in native kidneys of nonrenal transplant recipients west nile virus infection after cardiac transplantation community-acquired west nile virus infection in solid-organ transplant recipients trypanosoma cruzi infection reactivation manifested by encephalitis in a chagas heart transplant recipient central nervous system infections in heart transplant recipients listeria meningitis in transplant recipients progressive multifocal leukoencephalopathy in transplant recipients progressive multifocal leukoencephalopathy in a heart transplant recipient following rituximab therapy for antibody-mediated rejection clinical spectrum of gram-positive infections in lung transplantation frequency of pseudomonas aeruginosa colonizations/infections in lung transplant recipients non-tuberculous mycobacterial infection among lung transplant recipients: a 15-year cohort study nocardia infection in lung transplant recipients challenges in the diagnosis and management of nocardia infections in lung transplant recipients fungal infections after lung transplantation significance of blood stream infection after lung transplantation: analysis in 176 consecutive patients aspergillus infections after lung transplantation: clinical differences in type of transplant and implications for management the incidence of invasive aspergillosis among solid organ transplant recipients and implications for prophylaxis in lung transplants risk factors and outcomes in lung transplant recipients with nodular invasive pulmonary aspergillosis immune reconstitution syndrome-like entity in lung transplant recipients with invasive aspergillosis anastomotic infections in lung transplant recipients treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america cellulitis caused by cryptococcus neoformans in a lung transplant recipient an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients cytomegalovirus in transplantation -challenging the status quo cytomegalovirus and lung transplantation a polymorphism linked to elevated levels of interferon-gamma is associated with an increased risk of cytomegalovirus disease among caucasian lung transplant recipients at a single center cytomegalovirus replication within the lung allograft is associated with bronchiolitis obliterans syndrome ganciclovir-resistant cytomegalovirus (cmv) infections among lung transplant recipients are associated with poor outcomes despite treatment with foscarnet-containing regimens community-acquired respiratory viral infections in lung transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study viral infections in lung transplant recipients antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) dna viral infections complicating lung transplantation polyomavirus infection and its impact on renal function and long-term outcomes after lung transplantation posttransplantation lymphoproliferative disorder in lung transplant recipients: a 15-year single institution experience parasitic lung infections detection and treatment of strongyloides hyperinfection syndrome following lung transplantation a case of successful treatment of cutaneous acanthamoeba infection in a lung transplant recipient disseminated acanthamoebiasis after lung transplantation cryptosporidium enteritis in solid organ transplant recipients: multicenter retrospective evaluation of 10 cases reveals an association with elevated tacrolimus concentrations myositis due to the microsporidian anncaliia (brachiola) algerae in a lung transplant recipient key diagnostic features of granulomatous interstitial nephritis due to encephalitozoon cuniculi in a lung transplant recipient epidemiology and outcomes of deep surgical site infections following lung transplantation aspergillus fumigatus empyema, arthritis, and calcaneal osteomyelitis in a lung transplant patient successfully treated with posaconazole epidemiology of bloodstream infections in the first year after pediatric lung transplantation bacteremia and septic shock after solid-organ transplantation aspergillus endocarditis in lung transplant recipients: case report and literature review empyema complicating successful lung transplantation mycobacterium abscessus empyema in a lung transplant recipient scedosporium prolificans pericarditis and mycotic aortic aneurysm in a lung transplant recipient receiving voriconazole prophylaxis mycoplasma hominis pericarditis in a lung transplant recipient: review of the literature about an uncommon but important cardiothoracic pathogen bacterial and fungal pneumonias after lung transplantation mold infections in lung transplant recipients nosocomial legionellosis in three heart-lung transplant patients: case reports and environmental observations chlamydia pneumoniae infection after lung transplantation intraabdominal complications after lung transplantation abdominal involvement in pediatric heart and lung transplant recipients with posttransplant lymphoproliferative disease increases the risk of mortality prevalence and management of gastrointestinal complications in lung transplant patients: mitos study group the spectrum of colonic complications in a lung transplant population gastrointestinal complications in heart and in heart-lung transplant patients chronic hepatitis e infection in lung transplant recipients management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus tuberculous meningitis in a lung transplanted patient impact of rituximab-associated b-cell defects on west nile virus meningoencephalitis in solid organ transplant recipients scedosporium apiospermum (pseudoallescheria boydii) infection in lung transplant recipients combination antifungal therapy in the treatment of scedosporium apiospermum central nervous system infections cladophialophora bantiana brain abscess in a solid-organ transplant recipient: case report and review of the literature disseminated fusarium infection with brain abscesses in a lung transplant recipient central nervous system infections in heart and heart-lung transplant recipients no funding agencies had any role in the preparation, review, or approval of this paper. the views expressed in this paper are those of the authors and do not necessarily represent the views of the university of colorado denver or stanford university. no conflict of interests was reported by andrés f. henao-martínez and josé g. montoya. key: cord-328266-bjs6ywlf authors: gunasekaran, muthukumar; bansal, sandhya; ravichandran, ranjithkumar; sharma, monal; perincheri, sudhir; rodriguez, francisco; hachem, ramsey; fisher, cynthia e.; limaye, ajit p.; omar, ashraf; smith, michael a.; bremner, ross m.; mohanakumar, thalachallour title: respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection date: 2020-04-30 journal: the journal of heart and lung transplantation doi: 10.1016/j.healun.2019.12.009 sha: doc_id: 328266 cord_uid: bjs6ywlf background respiratory viral infections can increase the risk of chronic lung allograft dysfunction after lung transplantation, but the mechanisms are unknown. in this study, we determined whether symptomatic respiratory viral infections after lung transplantation induce circulating exosomes that contain lung-associated self-antigens and assessed whether these exosomes activate immune responses to self-antigens. methods serum samples were collected from lung transplant recipients with symptomatic lowerand upper-tract respiratory viral infections and from non-symptomatic stable recipients. exosomes were isolated via ultracentrifugation; purity was determined using sucrose cushion; and presence of lung self-antigens, 20s proteasome, and viral antigens for rhinovirus, coronavirus, and respiratory syncytial virus were determined using immunoblot. mice were immunized with circulating exosomes from each group and resulting differential immune responses and lung histology were analyzed. results exosomes containing self-antigens, 20s proteasome, and viral antigens were detected at significantly higher levels (p < 0.05) in serum of recipients with symptomatic respiratory viral infections (n = 35) as compared with stable controls (n = 32). mice immunized with exosomes from recipients with respiratory viral infections developed immune responses to self-antigens, fibrosis, small airway occlusion, and significant cellular infiltration; mice immunized with exosomes from controls did not (p < 0.05). conclusions circulating exosomes isolated from lung transplant recipients diagnosed with respiratory viral infections contained lung self-antigens, viral antigens, and 20s proteasome and elicited immune responses to lung self-antigens that resulted in development of chronic lung allograft dysfunction in immunized mice. exosomes; graft rejection; respiratory viral infection; lung transplantation; chronic rejection; antigens; antibodies lung allograft failure from chronic lung allograft dysfunction (clad) is the leading cause of death beyond the first year after lung transplant (ltx). roughly 70% of ltx recipients (ltxrs) with clad have bronchiolitis obliterans syndrome (bos) 1 and include both obstructive and restrictive phenotypes. 2 the term restrictive allograft syndrome was introduced by sato et al 3 and was diagnosed in 30% of bilateral ltx patients with clad. the diagnosis was based on finding a restrictive ventilatory defect and had radiographic findings of interstitial opacities with 41% having upper zone involvement. previously reported risk factors for clad include acute rejection, 4−6 cytomegalovirus (cmv) pneumonitis, 7 antibodies (abs) to donor human leukocyte antigen (hla), 8, 9 abs to non-hla lung-associated self-antigens (sags), 10−12 primary graft dysfunction, 13 and respiratory viral infections (rvis). 14−19 the immunologic mechanisms that underlie the development of clad remain unknown, and therapy for established clad is generally ineffective. rvi after ltx has been associated with increased risk of clad. 15, 17, 18 fisher et al 17 conducted a large retrospective study that used systematic definitions, adjudicated assignment of clad by blinded reviewers, and highly sensitive and specific molecular diagnosis of rvi and found a strong and independent association between symptomatic rvi and clad 17 ; other studies have also found an association between rvi and clad. 15,18−20 potential mechanisms for rvi-induced clad pathogenesis were not assessed. we recently demonstrated that ltxrs with acute and chronic rejection have circulating exosomes that contain donor-mismatched hla, lung sags, and immunoregulatory microrna; exosomes from stable ltxrs do not have these same features. 21 a study by dieud e et al 22 demonstrated that the presence of 20s proteasome in exosomes increases their immunogenicity. in this study, we tested the hypothesis that rvi-induced allograft injury may induce circulating exosomes that contain donor hla, sags, and viral antigens, which may activate donor-specific immune responses and increase the risk of clad. we performed a retrospective case-control study of 35 adult ltxrs diagnosed with symptomatic upper-and/or lower-tract rvi (cases) and 32 adult ltxrs who had no rvi diagnosis (controls). patients were eligible for the study if they had undergone ltx at barnes-jewish hospital, washington university, st. louis, missouri, between 2011 and 2015, or at norton thoracic institute, st. joseph's hospital, phoenix, arizona between 2016 and 2018, and had stored serum available. baseline patient demographics, transplant details, and laboratory data were collected from patient charts. all patients were followed up for at least 6 years, with clinical and laboratory information collected. the end-point of bos was diagnosed according to the guidelines from the international society for heart and lung transplantation. 23 rvi testing was performed when indicated for compatible signs and symptoms. only patients with symptomatic rvi were included. rvi was diagnosed using the biofire filmarray pcr (biom erieux, marcy-l' etoile, france), which detects 17 types of respiratory viruses, including adenovirus, coronavirus (types hku1, nl63, 229e, oc43), human metapneumovirus, human rhinovirus/enterovirus, influenza (a, a/h1, a/h3, a/h1-2009, b), parainfluenza 1-4, and respiratory syncytial virus (rsv). both upper (nasopharyngeal swabs) and lower (bronchoalveolar wash or lavage) specimens were included. patients were considered to have a lower-tract infection if they had a positive lower-tract specimen or upper respiratory specimen along with either lower respiratory symptoms (cough, wheezing) or decline in forced expiratory volume. ltxrs in the control group had no evidence of symptomatic rvi during the period of serum collection. post-transplant immunosuppression comprised a triple immunosuppressive regimen of tacrolimus or cyclosporine, mycophenolate mofetil or azathioprine, and prednisone. this study was approved by the institutional review boards at washington university and st. joseph's hospital. all laboratory analyses were performed by personnel blinded to clinical outcomes, and all clinical end-points were adjudicated by personnel who were blinded to laboratory results. determination of abs to lung sags by enzymelinked immunosorbent assay (elisa) elisa was used to analyze serum samples from ltxrs diagnosed with rvi and from stable controls for measuring abs to two sags, collagen-v (col-v) and k-alpha-1 tubulin (ka1t) detailed in our previous publication. 9 in addition to lung sags, we used a kidneyassociated sag, collagen-iv (col-iv) (meridian, a33125h), as a control. samples were considered positive if the values were greater than the mean + 2 standard deviations of the healthy controls' values. ab concentration was calculated using a standard curve from known concentrations of col-v and ka1t abs (bd pharmingen 550513, sanjose, ca). exosomes were isolated from serum samples of ltxrs with rvi and from stable controls by ultracentrifugation as previously described. 21, 24, 25 exosome purity was validated using the sucrose cushion method. 21, 26 the presence of the exosome-specific markers cd9 (312102, biolegend, san diego, ca) and alix (634502, biolegend) was assessed using immunoblot. determination of lung sags, 20s proteasome, and viral antigens using immunoblot immunoblot was used to detect sags, 20s proteasome, and viral antigens in exosomes from ltxrs diagnosed with rvi and from stable controls. total exosome protein (3 mg) was resolved in polyacrylamide gel electrophoresis, and the proteins were transferred into a polyvinylidene difluoride membrane. the membrane was blocked with 5% non-fat milk in 1x phosphate buffered saline and was probed with exosome-specific marker cd9 (312102, biolegend), col-v (ab7046, abcam, cambridge, united kingdom), and ka1t (sc-12462-r, santa cruz biotechnology, dallas, tx). 20s proteasome subunit a3 (sc-58414, santa cruz biotechnology), rhinovirus vp3 (ma5-18249, thermo fisher scientific, waltham, ma), coronavirus (nb100-64754, novus biologicals, littleton, co), and rsv glycoprotein g (7950-0980, bio-rad laboratories, hercules, ca) were used as primary abs; secondary abs conjugated with horseradish peroxidase (hrp) were used specific to primary ab. the blots were washed with pbs tween (thermo fisher scientific), developed using chemiluminescent hrp substrate (wbkls0500, milliporesigma, burlington, ma), and exposed using odyssey clx imaging system (li-cor biosciences, lincoln, ne). the band intensity of target protein was quantified using imagej software and normalized with cd9. immunization of c57bl/6 mice with exosomes from ltxrs diagnosed with rvi and stable controls exosomes (10 mg/100 ml) isolated from ltxrs with rvi or from stable controls were used for immunization of c57bl/6 mice ( days 1, 7, 18, and 25) . prior experiments have demonstrated that injury to the native lungs is required for abs to lung sags to cause lesions. 27 therefore, 0.1 m hydrochloric acid was administered intrabronchially on both groups on day 0 before immunization with exosomes. serum samples collected on days 10 and 30 were used to detect abs to col-v and ka1t by elisa. on day 30, the mice were killed and splenocytes isolated to enumerate sag-specific cytokines producing cells by enzyme-linked immunospot assay (elispot). detection of abs to lung sags in serum samples from mice using elisa serum samples from mice immunized with exosomes of ltxrs diagnosed with rvi and from stable controls were used to measure abs against col-v and ka1t using elisa as described previously. 21, 27 to detect murine abs, we used goat−anti-mouse conjugated with hrp (1:10,000) as secondary ab. the plates were developed with chemiluminescent reagent and the reactions were stopped with 0.1 n hydrochloric acid. the optical density of each well was measured at a wavelength of 420 nm. serum concentration of abs to lung sag was calculated using the standard curve obtained with known concentration of abs to sags. samples were considered positive if the values were greater than the mean + 2 standard deviations of the healthy controls' values. splenocytes were isolated from mice immunized with exosomes of ltxrs diagnosed with rvi and of stable controls. elispot was performed as described previously. 28 cytokine-producing cells were analyzed, and the spots were enumerated and subtracted from experimental control wells and reported as spots per million. lungs from mice immunized with exosomes from ltxrs with rvi and from stable ltxrs were histologically analyzed to detect lesions and cellular infiltration by hematoxylin and eosin and trichrome staining, as described previously. 28 lungs were fixed in 10% formaldehyde and embedded in paraffin blocks. sections 4 to 5 mm thick were cut and mounted on slides (leica, wetzlar, germany) for hematoxylin and eosin and trichrome staining. images were obtained on a leica microscope at £ 40 and morphometric analysis was performed using aperio imagescope software (leica). five different areas were examined for fibroproliferation, epithelial abnormalities, and cellular infiltration. slides were scanned and whole slide images were analyzed using aperio image scope (https://www.leicabiosystems.com/digitalpathology/manage/aperio-imagescope/) and imagej software (https://imagej.nih.gov/ij/). for analysis of infiltrates, manual annotation of areas with prominent as well as mild or no infiltrate was performed on whole slide images, and the fraction of the total tissue area with prominent infiltrate was determined using image scope. for evaluation of fibrosis, whole slide images of lung sections stained with trichrome stain were exported as tiff files. color deconvolution of the tiff files was performed in imagej using a color deconvolution plugin (https://imagej.net/colour_deconvolu tion). the extent of blue-staining collagenous fibrosis was then determined using standard tools available in the imagej suite. data analysis was performed using prism 6 software from graphpad, inc. the ab levels for lung sags, optical density of exosomes containing lung sags, and viral antigens between rvi ltxrs and controls were compared using mann−whitney or two-tailed student's t-test, as indicated. statistical data in each cohort was expressed as mean § standard error. p-values < 0.05 were considered statistically significant in each comparative analysis. the mean optical density of exosomes containing lung sags and viral antigens was calculated after normalization with exosome-specific marker cd9 and comparative analysis was performed using mann−whitney u test. patient demographics, age, sex, ethnicity, and hla-mismatch status were not significantly different between groups (table 1) . acute cellular rejection (acr) occurred after rvi in 2 (a1, acr) patients and in 1 (a1, acr) stable ltxr control. acute antibody-mediated rejection occurred in 5 patients diagnosed with rvi and in none of the stable ltxrs. donorspecific antibodies developed during follow-up in 8 figure 2 ). exosomes from patients diagnosed with rsv were analyzed for the presence of sags and rsv glycoprotein g by immunoblot. viral antigens were seen in 4 of 10 patients with rsv infection; no stable ltxrs had viral antigens ( figure 3a) . furthermore, significantly increased levels of sags and rsv antigens (mean optical intensity: were demonstrated in ltxrs diagnosed with rsv compared with stable ltxrs (figure 3b ). immunoblot results showed that coronavirus antigens were detected in exosomes of 5 of 12 patients diagnosed with coronavirus compared with no stable ltxrs (figure 3a ). levels of sags (mean optical intensity: col-v, 1.37 § 0.19 vs 0.7 § 0.14, p = 0.015; ka1t, 1.2 § 0.25 vs 0.21 § 0.08, p = 0.003; coronavirus, 3.78 § 1.05 vs 0.83 § 0.27, p = 0.0217) were significantly higher in exosomes from ltxrs with coronavirus than in stable ltxrs (figure 3b ). twelve patients with rhinovirus infection and 10 stable ltxrs were selected to detect exosomes containing sags and rhinovirus antigens. patients diagnosed with rhinovirus (6/10) showed rhinovirus antigens, but stable ltxrs did not (figure 3a) . the mean optical density of exosomes containing sags (mean optical intensity: col-v, 2.54 § 0.6 vs 0.92 § 0.2, p = 0.028; ka1t, 9.32 § 2.4 vs 1.78 § 0.86, p = 0.015; rhinovirus, 5.35 § 1.63 vs 1.14 § 0.16, p = 0.030) was significantly higher in exosomes isolated from ltxrs diagnosed with rhinovirus compared with stable ltxrs (figure 3b ). to determine 20s proteasome in exosomes isolated from stable ltxrs (n = 4) and ltxrs diagnosed with rvi (n = 5), we performed immunoblot using abs to the a3 subunit of 20s proteasome. we found significantly higher levels of 20s proteasome a3 subunit in exosomes isolated from ltxrs diagnosed with rvi compared with stable ltxrs (mean optical density, ltxrs with rvi vs stable ltxrs: 1.74 § 0.6 vs 0.37 § 0.35, p = 0.0317). alix served as an exosome-specific marker and loading control ( figure 4 ). figure 6 ). exosomes containing lung-associated sags in ltxrs diagnosed with rvi and stable ltxrs. exosomes isolated from serum samples of ltxrs with rvi (n = 34) and stable ltxrs (n = 30) were analyzed for the presence of lung-associated sags (col-v and ka1t) by immunoblot. the mean relative optical densities of col-v (1.9 § 0.2 vs 0.73 § 0.09, p = 0.0003) and ka1t (4.06 § 1.09 vs 0.83 § 0.31, p = 0.009) were significantly higher in ltxrs with rvi than stable ltxrs. optical density was measured using imagej software and the od value of sags were calculated in ltxrs with rvi and stable ltxrs after normalization with cd9 od value. cd9 also served as loading control and exosome-specific markers. the presence of lung sags in the exosomes was compared between the cohorts using mann−whitney test. asterisk indicates statistically significant. col-v, collagen-v; ka1t, k-alpha-1 tubulin; ltxr, lung transplant recipient; od, optical density; rvi, respiratory viral infection; sag, self-antigen. lungs harvested from mice immunized with exosomes from ltxrs with rvi and stable ltxrs were subjected to histopathological analysis. mice immunized with exosomes from ltxrs diagnosed with rvi showed inflammatory cells in bronchioles and vessels. notably, lesions involving bronchioles, cellular infiltration, and increased fibrosis were also observed ( figure 7) . in contrast, no significant differences in cellular infiltration and lesions were evident in the mice immunized with exosomes from stable ltxrs (figure 7a) . lung-associated sags and viral antigens were demonstrable in exosomes isolated from patients with rvi. exosomes isolated from serum samples of patients with rvi and from stable ltxrs were used to detect the presence of lung-associated sags and viral antigens using immunoblot. the results showed a significant increase in lung-associated antigens and viral antigens. (a) rsv (n = 10), coronavirus (n = 12), and rhinovirus (n = 12) in respective patients with viral infection compared with stable ltxrs (n = 30). (b) graphical representation shows the optical density of lung-associated sags and viral antigens measured in rvi and stable ltxrs using imagej software. optical density of lung sags and viral antigens were normalized with exosomes specific marker cd9. the presence of lung sags and viral antigens in the exosomes was compared between the cohorts using mann−whitney test. asterisk indicates statistically significant. col-v, collagen-v; cv, coronavirus; ka1t, k-alpha-1 tubulin; ltxr, lung transplant recipient; rsv, respiratory syncytial virus; rv, rhinovirus; rvi, respiratory viral infection; sag, self-antigen. exosomes containing 20s proteasome core in ltxrs with rvi and stable ltxrs. circulatory exosomes isolated from ltxrs with rvi (n = 5) and stable ltxrs (n = 4) were used to detect the presence of 20s proteasome subunit a3 using immunoblot. (a) the exosomes isolated from patients with rvi showed a significant increase in 20s proteasome compared with exosomes from stable ltxrs (mean optical density: 1.74 § 0.6 vs 0.37 § 0.35, p = 0.0317). alix served as loading control and exosome-specific marker. (b) graphical representation shows optical intensity of 20s proteasome a3 subunit abundance in ltxrs with viral infection and stable ltxrs. the presence of 20s proteasome was compared between stable ltxrs and ltxrs with rvi using student's t-test. ltxr, lung transplant recipient; rvi, respiratory viral infection. these results demonstrate that circulating exosomes from ltxrs diagnosed with rvi-induced cellular infiltration and alveolar lesions in the lungs of mice. furthermore, histopathological analysis demonstrates interstitial fibrosis, which after human lung transplant is similar to the pathology seen in restrictive allograft syndrome. the morphometric data (figure 7b ) are given for the representative images. studies have demonstrated an association between rvi and clad. [15] [16] [17] [18] 20 fisher et al 16, 17 applied molecular diagnostic methods to test for rvi in a large cohort of ltxrs. they not only found high rates of rvi but also demonstrated an independent association between rvi and clad 17 and suggested further study to characterize the viral determinants and to define the mechanisms by which rvi increases the risk for clad. rvi after ltx has been shown to dysregulate the regulatory t cells, indicating that rvi can lead to dysregulation of tolerance to sags, leading to induction of immune responses to sags and increasing the risk of clad. 14 studies by our group and others showed that abs to lung sags have been shown to develop and correlate with development of clad in ltxrs. 11, 12, 29 pre-existing abs to sags have also been reported to increase the incidence of primary graft dysfunction, to induce proinflammatory cytokines, and to increase development of donor-specific antibodies and clad after ltx. 29 we recently demonstrated that ltxrs diagnosed with acute and chronic rejection have circulating exosomes that express mismatched donor hla and sags. we proposed that the exosomes originating from transplanted lungs may contribute to the immune pathogenesis of clad after ltx. 21 based on these findings, we postulated that symptomatic rvi may induce exosomes containing sags from the transplanted organ, and that persistence of circulating exosomes with sags can lead to immune responses resulting in increased risk of clad. in this study, we determined the development of abs to sags in ltxrs diagnosed with rvi. our results, presented in figure 1 , demonstrated that ab titers to sags were significantly higher in patients diagnosed with rvi than in stable ltxrs. this demonstrates that rvi can induce a humoral immune response to sags. circulating exosomes isolated from ltxrs diagnosed with bos express mismatched donor hla and sags (col-v and ka1t), confirming their source as the lung allograft and suggesting that exosomes can induce immune responses to alloantigens and sags, increasing the risk for clad. 21 walker et al 30 demonstrated that exosomes released from cmv-infected lung endothelial cells of ltxrs induces cd4 t-cell responses to cmv antigens. furthermore, a human nasopharyngeal cell line transfected with epstein-barr virus (ebv) has been shown to release exosomes containing viral peptide latent membrane protein 1 and fibroblast growth factor 2. 31 the exosomes isolated from ebv-transformed b cells contain ebv viral antigen glycoprotein 350, which can specifically bind to b cells. 32 these findings support our hypothesis that rvi has the potential to induce exosomes containing lung sags and viral antigens from the transplant recipient with rvi. our results demonstrate that circulating exosomes isolated from patients diagnosed with symptomatic rvi had not only sags but also viral antigens. in this study, we selected patients diagnosed with rsv, coronavirus, and rhinovirus and demonstrated that exosomes isolated from these patients contained specific viral antigens along with sags. therefore, exosomes are induced following viral infection that contain viral antigens and sags. preliminary analysis of serial circulating exosomes containing viral antigens demonstrated that in 3 of 5 ltxrs, there was a transient presence of circulating exosomes. in contrast, 2 of 5 ltxrs with rvi had persistence of circulating exosomes with lung sags and viral antigens. this interesting finding needs to be confirmed to determine the role of circulating exosomes in inducing immune responses leading to clad. it is likely that the exosomes with viral antigenic epitopes can activate cross-reactive t cells, which can play a role in the pathogenesis of clad after ltx. 33, 34 we demonstrated that exosomes isolated from symptomatic patients with rvi contained increased sags and viral antigens; if further studies identify a useful threshold exosomes from ltxrs with rvi induce a humoral immune response to lung sags. serum samples collected on days 10 and 30 from c57bl/6 mice immunized with exosomes isolated from ltxrs with rvi (n = 5) and from stable ltxrs (n = 5) were utilized to measure abs to lung sags by elisa. serum samples collected on day 30 from mice immunized with exosomes from ltxrs with rvi showed significantly increased abs to sags (col-v, 28.1 § 4.0 vs 45.9 § 6.5, p = 0.04; ka1t, 230.4 § 77.1 vs 604.6 § 140, p = 0.04) when compared with mice injected with exosomes from stable ltxrs. the antibody development was compared between the cohorts using student's t-test. asterisk indicates statistically significant. ab, antibody; col-v, collagen-v; elisa, enzyme-linked immunosorbent assay; ka1t, k-alpha-1 tubulin; ltxr, lung transplant recipient; rvi, respiratory viral infection; sag, self-antigen. concentration, this could potentially serve as a biomarker for clad. a study by dieud e et al 22 demonstrated that exosomes isolated from endothelial cells contained 20s proteasome and therefore increased the immunogenic potential to the kidneyassociated sag perlecan. intravenous injection of exosomes in c57bl/6 mice led to humoral immune responses to perlecan, suggesting that presence of kidney sags, along with 20s proteasome, increases the immunogenicity of the exosomes. 22 to demonstrate that exosomes containing sags along with viral antigens can be immunogenic, we isolated exosomes from ltxrs with rvi and stable ltxrs and immunized into c57bl/6 mice. mice immunized with exosomes from rvi developed increased levels of abs to sags than mice immunized with exosomes from stable ltxrs. additionally, mice immunized with exosomes from ltxrs with rvi showed increased interferon gamma− and il-17−producing cells and reduced il-10−producing cells compared with mice injected with exosomes from stable ltxrs. these results confirm that exosomes containing sags, viral antigens, and 20s proteasome exosomes from ltxrs with rvi induce cytokine-producing t cells to lung sags. spleens were collected on day 30 from c57bl/6 mice immunized with exosomes of ltxrs with rvi (n = 5) and from stable ltxrs (n = 5) were used to measure cytokine-producing t cells against lung sags by elispot. mice immunized with exosomes of rvi showed significant increase in t cells producing il-17 and interferon gamma to sags. mice injected with exosomes isolated from stable ltxrs showed increased frequency of il-10−producing t cells compared with mice immunized with rvi exosomes. the cytokine levels were compared between the cohorts using mann−whitney test. asterisks indicate statistically significant. elispot, enzyme-linked immunospot assay; il, interleukin; ltxr, lung transplant recipient; rvi, respiratory viral infection; sags, self-antigens; ** statistically significant. are immunogenic and can induce abs to sags and alter t-cell cytokine responses, which can lead to clad. our study is limited in that exosomes isolated from patients diagnosed with rvi were not analyzed in mice models of obliterative airway diseases following lung transplantation. therefore, we cannot definitively conclude that exosomes from patients with rvi can increase the incidence of clad development. the sample size used in the mouse model was small and we used pooled exosomes for immunization. therefore, the role of individual viruses in inducing exosomes that are immunogenic cannot be concluded from the studies presented. we have shown that serum samples collected from ltxrs with rvi had increased abs to lung sags and exosomes containing lung sags and viral antigens compared with stable ltxrs. however, viral rna in the exosomes and its role in immune activation needs to be determined in future studies. another limitation is that the role of individual rvi viruses to induce exosomes that can increase the risk for clad were not determined because of the limited availability of retrospectively collected samples. our study, however, demonstrated that exosomes derived from ltxrs with rviinduced interstitial fibrosis and inflammatory cell infiltration by adoptive transfer of exosomes (gain of function) in a mice model, which suggests that rvi exosomes are sufficient to induce lesions in mice with similarities to the pathology seen in restrictive allograft syndrome in human ltxrs. we further demonstrated increased humoral and cellular immune responses to lung sags in mice immunized with exosomes from ltxrs with rvi compared with mice immunized with exosomes from stable ltxrs. based on these, we proposed that rvi-induced exosomes containing lung sags and viral antigens can augment humoral and cellular immune responses to lung sags and alloantigens, increasing the risk of clad. these results strongly suggest a biologically plausible mechanistic link between rvi induction and release of circulating exosomes with sags and the development of clad, which should be assessed in a large prospective cohort. fibrosis and cellular infiltration were demonstrable in mice injected with exosomes isolated from ltxrs with rvi. mice were killed on day 30 and their lungs were collected and analyzed using hematoxylin and eosin and trichrome staining. (a) interstitial and inflammatory infiltrates and fibrosis was more prominent in mice injected with exosomes from ltxrs with rvi compared with mice injected with exosomes from stable ltxrs. images were obtained on a leica microscope at £ 40 and morphometric analysis was performed using aperio imagescope software (leica). (b) the morphometric data are given for the representative images. ltxr, lung transplant recipient; rvi, respiratory viral infection. lung transplantation bronchoscopic monitoring after lung transplantation restrictive allograft syndrome (ras): a novel form of chronic lung allograft dysfunction acute antibody-mediated rejection after lung transplantation revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection pathologic interpretation of transbronchial biopsy for acute rejection of lung allograft is highly variable cytomegalovirus infection in lung transplant recipients human leukocyte antigen mismatches predispose to the severity of bronchiolitis obliterans syndrome after lung transplantation alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts antibodies to self-antigens predispose to primary lung allograft dysfunction and chronic rejection de novo production of k-alpha1 tubulin-specific antibodies: role in chronic lung allograft rejection antibodies to k-alpha 1 tubulin and collagen v are associated with chronic rejection after lung transplantation impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome respiratory virus-induced dysregulation of t-regulatory cells leads to chronic rejection respiratory viruses and chronic rejection in lung transplant recipients respiratory virus infections and chronic lung allograft dysfunction: assessment of virology determinants symptomatic respiratory virus infection and chronic lung allograft dysfunction respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death the impact of viral respiratory tract infections on long-term morbidity and mortality following lung transplantation: a retrospective cohort study using a multiplex pcr panel adenovirus infection in the lung results in graft failure after lung transplantation donor-derived exosomes with lung self-antigens in human lung allograft rejection the 20s proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection a new classification system for chronic lung allograft dysfunction circulating exosomes with distinct properties during chronic lung allograft rejection exosomes expressing the self-antigens myosin and vimentin play an important role in syngeneic cardiac transplant rejection induced by antibodies to cardiac myosin isolation and characterization of exosomes from cell culture supernatants and biological fluids immune response to tissue-restricted self-antigens induces airway inflammation and fibrosis following murine lung transplantation antibodies to mhc class i induce autoimmunity: role in the pathogenesis of chronic rejection pre-transplant antibodies to kalpha1 tubulin and collagen-v in lung transplantation: clinical correlations cytomegalovirus-infected human endothelial cells can stimulate allogeneic cd4+ memory t cells by releasing antigenic exosomes epstein-barr virus latent membrane protein 1 promotes concentration in multivesicular bodies of fibroblast growth factor 2 and its release through exosomes exosomes containing glycoprotein 350 released by ebv-transformed b cells selectively target b cells through cd21 and block ebv infection in vitro the carrying pigeons of the cell: exosomes and their role in infectious diseases caused by human pathogens exosomes exploit the virus entry machinery and pathway to transmit ifn-alpha-induced antiviral activity the authors have no conflicts of interest to disclose.the authors acknowledge billie glasscock and clare sonntag for their assistance in preparing and submitting this manuscript.this study was supported by national institutes of health grants ai123034, hl056643, and hl092514 (tm). key: cord-016374-38fk66zb authors: shi, yuxin; lu, puxuan; yang, yuxin; hu, chunhong; jin, haiying; kong, lili title: human infected h7n9 avian influenza date: 2016-06-23 journal: radiology of influenza doi: 10.1007/978-94-024-0908-6_11 sha: doc_id: 16374 cord_uid: 38fk66zb human infected h7n9 avian influenza is an acute respiratory infectious disease caused by subtype h7n9 of avian influenza virus. since the first case was reported in yangzi river delta of china in feb. 2013, the total cases with definitive diagnosis had been up to 451 cases, including 176 cases of death, by dec. 31, 2014, with a mortality rate of roughly 39 %. human infected h7n9 avian infl uenza is an acute respiratory infectious disease caused by subtype h7n9 of avian infl uenza virus. since the fi rst case was reported in yangzi river delta of china in feb. 2013, the total cases with defi nitive diagnosis had been up to 451 cases, including 176 cases of death, by dec. 31, 2014, with a mortality rate of roughly 39 %. currently, subtype h7n9 of avian infl uenza virus has been isolated from the secretions and excretions of poultry, which is highly homologous with human infected h7n9 avian infl uenza virus. the source of its infection may be poultry carrying h7n9 avian infl uenza virus. and no defi -nite evidence has been found supporting interpersonal transmission of h7n9 avian infl uenza virus, but its limited continual transmission from person to person has not been excluded. human is infected via respiratory tract. and close contact to excretions or secretions of infected poultry and direct contact to the virus can also cause its infection. individuals who have a history of contact to poultry within 1 week prior to onset are high risk population, such as those engaging in poultry raising, selling, slaughtering and processing. the patients commonly experience infl uenza like symptoms, such as fever, cough with a little sputum, accompanying headache, muscle soreness and general malaise. in severe cases, the condition rapidly progresses, commonly into severe pneumonia within 5-7 days and the body temperature mostly above 39 °c, with dyspnea and accompanying bloody sputum. the condition may rapidly develop into acute respiratory distress syndrome (ards), sepsis, septic shock, and even multiple organs failure. some patients may also develop mediastinal emphysema and pleural effusion. and its incubation period generally lasts for no more than 7 days. clinically, human infected h7n9 avian infl uenza can be categorized into 3 types, namely asymptomatic (latent infection), slight and severe. in the hospital we work in, 18 patients were hospitalized, with 1 case of slight infection and 17 cases of severe infection. the patient with slight infection only experienced fl ue like symptoms, such as fever, but no lung abnormality. however, the 17 patients with severe infection experienced severe pneumonia, with the conditions meeting the diagnostic criteria of severe pneumonia induced by h7n9 avian infl uenza virus issued by national health and family planning commission. the clinical manifestations of severe pneumonia induced by human infected h7n9 avian infl uenza are as the following: the early lesions are mainly located in lower lobe of one lung, predominantly with a segmental distribution. radiologically, they are ground glass opacity, interlobular septal thickening and acinar nodules. the pulmonary lesions progresses rapidly from small fl akes of opacity to large consolidations, from one segment to multiple segments, from one lung to both lungs, and from lower lobes to the whole lungs. within 24-48 h, the pulmonary lesions may spread rapidly to involve multiple lobes of both lungs. in severe cases, the pulmonary lesions show a significant increase (≥50 %) within several hours. the lesions develop rapidly from ground glass opacity to consolidation opacity, and the density of consolidation opacity increases, with occurrence of serous effusion. the pulmonary lesions commonly reach their peak within 5-0 days, with observable lung air sac at this time. for the cases with no complication, if prompt anti-viral therapy administered, the lesions begin to be absorbed slowly after their reaching the peak, fi rstly the ground glass opacity, and then consolidations and the lesions in the central area adjacent to the hilum. the consolidations show decreasing density to sparse and the consolidated lung tissues gradually infl ate. during the whole process of absorption, the lesions in middle and upper lung lobes are absorbed earlier than the lesions in dorsal lower lung lobes and the subpleural lesions. the early lesions that emerge during the early and progressive stages are absorbed late, and the lesions that emerge late are fi rstly absorbed. radiologically, pulmonary fi brosis is demonstrated as small patches, fi brous cords like, grid like, and small ground glass opacity in subpleural areas of both lungs and/or doral segments of both lower lungs. meanwhile, subpleural paraseptal emphysema, scar type emphysema, subpleual lung bullae and limited bronchiectasis are revealed. a 53-year-old man of han nationality complained of intermittent fever for 13 days and diffi culty breathing for 4 days that aggravated as well as unconsciousness for 2 h. after ineffective treatment in another hospital, he was transferred to our hospital on aug. 16, 2014. laboratory tests showed nucleic acid of h7n9 avian infl uenza virus positive, with increased neutrophils and decreased lymphocytes. blood biochemistry revealed increased levels of γ-glutamyl transpeptidase, lactate dehydrogenase and α-hydrobutyrate dehydrogenase. during hospitalization for treatment, he received bed-side chest dr radiography per day (totally 8 bed-side chest dr fi lms and 3 chest ct scans) to assess his conditions. after emergency rescuing, he was cured and discharged on sep. 5, 2014. after that, he received 2 follow-up chest ct scans for re-examination. [radiological demonstration] fig. 11 .1 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza virus, severe pneumonia, and ards. [discussion] pneumonia induced by human infected h7n9 avian infl uenza is an acute respiratory infectious disease caused by subtype h7n9 of avian infl uenza virus and it occurs in any age group of people. before onset, the patient usually has a history of exposure to an environment contaminated by avian infl uenza virus, and the common clinical manifestations include cough, expectoration, and fever. the condition progresses rapidly in seriously ill patients, with occurrence of severe pneumonia within 3-7 days after onset. the body temperature mostly persists above 39 °c with dyspnea, which rapidly further progresses into acute respiratory distress syndrome (ards), sepsis, septic shock, and even multiple organs failure. pneumonia induced by human infected h7n9 avian infl uenza is radiologically characterized by the following: 1. morphologically, the lesions are pulmonary consolidation, ground glass opacity and interstitial changes. 2. the lesions are diffusely distributed in both lungs in severe cases, which are more serious in lower lungs. 3. the lesions show rapid progression, with emergence or increase of consolidations during the progressive stage. 4. the primary ground glass opacity in lungs shows obvious consolidation inside within a short period of time. otherwise, the range with primary consolidations enlarges with increased density. 5. the lesions are migratory, with absorption of primary lesions within a short period of time and newly emerging lesions in other parts of lungs, which are radiologically characteristic. 6. the lesions show slow absorption, with signs of pulmonary fi brosis after cured. 7. chest ct demonstrations are inconsistent to the clinical symptoms because the absorption of lesions lags behind. re-examination by chest ct scan show slight absorption of the lung lesions after the clinical symptoms are obviously improved. pneumonia induced by human infected h7n9 avian infl uenza should be differentiated from the following diseases: its occurrence is related to prevalence of infl uenza, with rapid progression. radiologically, the lesions are mainly confi ned and segmental alveolar consolidation, which may further develop into diffuse lesions with air bronchogram. by chest x-ray, it shares commonalities with pneumonia induced by h7n9 avian infl uenza, and their differential diagnosis is challenging. and its fi nal defi nitive diagnosis should be based on the laboratory tests. it is more common in children whose chest x-ray demonstrations include increased, thickened and blurry lung markings. the lesions distribute extensively, fig. 11 .1 chest x-ray demonstrated consolidation and ground glass opacity in the right upper lung and the left lower lung, with uneven density, poorly defi ned boundary, and inner air bronchogram ( a ). re-examination after 4 days demonstrated consolidation and ground glass opacity in the right upper lung and the left lower lung, with decreased density and smaller lesion range ( b ). re-examination after 6 days showed consolidation and ground glass opacity in the right upper lung and the left lower lung, with further decreased density and smaller lesion range ( c ). re-examination after 12 days revealed consolidation and ground glass opacity in the right upper lung and the left lower lung, with further decreased density and smaller lesion range; and partially interstitial fi brosis ( d ). re-examination by chest ct scan after 36 days demonstrated multiple small patches of dense opacity in both lungs and in lateral part of lungs, predominantly in the right upper lung and the left lower lung, and partially interstitial fi brosis ( e , f ). re-examination by chest ct scan after 5 months demonstrated obviously decreased small patches of dense opacity in both lungs, with rare fi brous cords like opacity ( g ) which are characterized by many lung markings, many lesions of emphysema, many integrated lesions and many large lesions, but rare round lesions, rare lesions of lung bullae and rare pleural effusion as well as consistency between radiological signs and clinical symptoms. 3. severe acute respiratory syndrome (sars) the patients with sars have defi nite epidemiological history of contact. by chest x-ray, it is demonstrated with diversifying lesions, among which cotton like exudation is the most common; multi-focus granuloma and false cavity are possibly its specifi c signs. a 65-year-old man of han chinese, experienced cough, expectoration, obvious increase of the body temperature, dyspnea, tachypnea, chest distress and shortness of breath. by ct scan in another hospital, infl ammation of both lungs and bilateral pleural effusion were revealed. he was transferred into our hospital on dec. 12, 2014. laboratory tests showed the nucleic acid of h7n9 avian infl uenza virus positive; eutrophils within normal range, decreased lymphocytes count; increased levels of γ-glutamyl transpeptidase, lactate dehydrogenase, α-hydrobutyrate dehydrogenase, serum lipoprotein, and c reactive protein; increased creatinine level that exceeded the normal range as well as increased levels of creatinine and cystatin c. bedside chest dr radiography per day (totally 5 chest dr fi lms) in our hospital was ordered to monitor the lesion changes. death fi nally occurred on dec. 16, 2014 due to multiple organs failure after ineffective emergency rescuing. [radiological demonstration] fig. 11 .2 [diagnosis] critical case of human infected h7n9 avian infl uenza, severe pneumonia, ards, and multiple organs dysfunction. [discussion] in this case, the patient was radiologically demonstrated with diffuse small patches of dense opacity in both lungs that are ground glass like as well as integration of lesions and enlarged range with lesions along with progression of the condition. for this case, the condition should be differentiated from highly pathogenic h5n1 avian infl uenza, seasonal infl uenza (including type a infl uenza h1n1), bacterial pneumonia, sars, novel coronaviral pneumonia, adenoviral pneumonia, chlamydia pneumonia, and mycoplasma pneumonia. the defi nitive diagnosis is mainly based on etiological examinations. a b fig. 11. 2 chest x-ray demonstrated diffuse small patches of dense opacity in both lungs that are ground glass like ( a ). re-examination after 1 day showed diffuse small patches of dense opacity in both lungs that are ground glass like, with no obvious change compared to the previous chest x-ray ( b ). re-examination after 3 days revealed diffuse small patches of dense opacity in both lungs that are ground glass like, some of which are integrated; enlarged range with lesions in the left lung and the right upper lung; increased density of the opacity ( c ). re-examination after 4 days demonstrated diffuse small patches of dense opacity in both lungs that are ground glass like, with further aggravation ( d ) c d case 3 a 60-year-old man was hospitalized due to fever for 4 days and cough for 1 day. by physical examinations, his body temperature was 37.6 °c, with clear sound by lung percussion, clear breathing sounds by auscultation with no obvious dry and moist rales. laboratory tests revealed wbc count 6.19 × 10 9 /l, gr% 77.11 %, mono% 12.4 %, lymphocytes ratio 10.32 %, hs-crp 85 mg/l, and esr 9 mm/h. serological test for m. pneumoniae was negative and blood biochemistry showed no abnormity. chest ct indicated large high-density opacity with uneven density and poorly defi ned boundary in the lingual segment of left upper lung lobe. and the diagnosis was considered to be pneumonia of the left lung. after treatment for 2 days, the patient still experienced fever and was transferred into a large hospital for treatment. after that, the patient showed persistent fever with a body temperature of 37.6 °c. bedside chest x-ray after 10 days demonstrated increased and blurry lung markings and multiple cotton like and fl akes of opacity in both lungs. bedside color doppler ultrasonography showed liquid opacity in the right thoracic cavity and pericardium. and the patient experienced continuous decrease of oxygen saturation to around 60 % and his blood pressure decreased to around 70/50 mmhg. at that night, throat swabs by professionals from cdc showed positive and the diagnosis was defi ned to be human infection of h7n9 avian infl uenza virus. the patient was then transferred to the local hospital specialized in infectious diseases, and death occurred 1 week later. the patient reported a history of close contacts to feces of birds and chickens. [radiological demonstration] fig. 11 .3 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza. [discussion] human infected h7n9 avian infl uenza is a type of type a infl uenza, with the fi rst 3 cases reported in shanghai and anhui province of china in mar. 2013. human infected h7n9 avian infl uenza virus is a newly emerging recombinant virus, which infects human via respiratory tract after contact to poultry with the infection. it has an incubation period of no more than 7 days. and the patients commonly experience fl u like symptoms, such as fever, cough, expectoration of a little sputum as well as accompanying headache, muscle soreness and general malaise. in severe cases, the condition progresses rapidly into severe pneumonia, mostly with a body temperature of above 39 °c, dyspnea and possibly accompanying bloody sputum. severe pneumonia may further progresses rapidly into ards, mediastinal emphysema, sepsis, shock, consciousness disturbance, and acute renal damage. by chest radiology, the disease is characterized by primary lesions in one lung that gradually involve both lungs along with progression of the condition. in addition, the multiple lesions in both lungs show a multi-segmental and multi-lobar distribution. for this case, the patient had a history of close contacts to feces of birds, and the disease is radiologically characterized by infl ammation of lobes in one lung at its early stage. along with the progression of the condition, both lungs are rapidly involved and ards fi nally occurred. the diagnosis was defi ned based on the fi nding of throat swabs to be a typical case of human infected h7n9 avian infl uenza. for this case, the condition should be differentiated from other types of pneumonia. compared to other types of pneumonia, the patient in this case showed no obvious characteristic symptoms and physical signs and the defi nitive diagnosis is mainly based on the etiological examination. a 20-year-old woman complained of fever with the highest body temperature of 39 °c, headache, cough, expectoration of rust colored sputum, and systemic malaise for 1 day. laboratory test showed wbc 3.8 × 10 9 /l. she denied a history of contact to poultry. [radiological demonstration] fig. 11 .4 [diagnosis] severe pneumonia induced by human infected h7n9 avian infl uenza. [discussion] for this case, the chest radiology demonstrated: 1. in the early stage, chest x-ray demonstrated ground glass opacity in one lung that showed lobar distribution with no opacity across the interlobar fi ssure. 2. the lesions were predominantly ground glass opacity that rapidly progressed to involve both lungs, with diffuse distribution and enlarged range with lesions. the ggo was shown to develop into consolidations with air bronchogram inside. 3. after positive treatment, the lesions were gradually absorbed and improved, with smaller range with lesions, decreased density as well as evolvement into fi brous cords like, grid like and honeycomb like opacity. based on these radiological fi ndings, we know that pneumonia induced by human infected h7n9 avian infl uenza resembles to other types of viral pneumonia, with concurrent parenchymal and interstitial involvements. however, pneumonia induced by human infected h7n9 avian infl uenza shows more obvious alveolar exudation in its early stage that overlaps lung interstitium. after partial absorption of alveolar exudation, fi brous cords like, grid like and honeycomb like opacity are radiologically demonstrated. in this case, the condition should be differentiated from bacterial pneumonia and pneumonia induced by h1n1 infl uenza. by radiology, bacterial pneumonia is characterized by increased and blurry lung markings and nonspecifi c patchy opacity in both lungs, which is more common in bilateral middle and lower lung fi elds as well as middle and medial parts of both lungs. clinically, wbc count increases. pneumonia induced by h1n1 infl uenza shows early signs of ggo lesions in subpleural area and around bronchi. for this case, the differential diagnosis is challenging and should be based on virological detection. case 5 a 79-year-old woman experienced nasal obstruction, runny nose, fever with the highest body temperature of 39 °c, cough and expectoration. she denied a recent history of contact to poultry and denied a history of contact to patient with infl uenza. she was detected with nucleic acid of h7n9 avian infl uenza virus positive. sputum culture revealed a. baumanii positive (++++) and aspergillosis positive. she had a medical history of cholestatic hepatocirrhosis as well as coronary heart disease and atrial fi brillation for 10 years. and she had medical history of right femoral neck fracture 4 months ago due to traumatic injury, which was treated by internal fi xation and replacement. currently, she was bedridden for a long period of time, and death fi nally occurred. [radiological demonstration] fig. 11 .5 [diagnosis] pneumonia induced by h7n9 avian infl uenza, mixed lung infection by aspergillus and a. baumanii. [discussion] recently, literature reports indicated that the susceptible populations to pneumonia induced by human infected h7n9 avian infl uenza include the elderly, those with underlying disease or compromised immunity. by ct scans, the most common sign is ground glass opacity, followed by consolidation, air bronchogram and interlobular septal thickening. the condition may be complicated by pleural effusion or air containing cyst. the lesions of the disease show a wide range, commonly involving 3 or more lung lobes. qingle et al. reported a group of cases, with no formation of cavity. for this case, the patient was an elderly, with medical histories of hepatocirrhosis, femoral neck fracture, long-term bedridden and compromised immunity. by sputum culture, aspergillus was positive, indicating a complication of pulmonary aspergillosis, which is most likely the reason for intrapulmonary nodules and cavities. in addition, she also showed an infec-tion of a. baumanii and death fi nally occurred. all the above clinical symptoms and radiological signs indicated that pneumonia induced by h7n9 avian infl uenza may be further complicated by fungal and bacterial infection, particularly in the elderly patients with poor condition of health and compromised immunity. a b c d fig. 11 .5 bedside chest x-ray demonstrated decreased transparency of both lungs, with diffuse large high density opacity that was more obvious in the right lung than in the left lung ( a ). ct scan showed diffuse ground glass opacity, grid like opacity, and fl akes of high density opacity in both lungs; nodular opacity in the left upper lung lobe and in the right middle lung lobe; halo signs around the nodules in the left upper lung lobe ( c was the 1 mm reconstructed image of the same layer as b ) ( be ). re-examination by ct scan after 2 days showed no improvement of the lung lesions; cavity within the nodules in the left upper lung; halo sign around the nodules; obviously enlarged nodules in the right middle lung lobe ( f , g ) [brief case history] a 67-year-old man experienced fever, cough, and expectoration of a little whitish sputum, with the highest body temperature of 38.5 °c. he has no history of physical examination and other histories of underlying diseases. the nucleic acid test for h7n9 was positive. [radiological demonstration] fig. 11 .6 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza. [discussion] the most common sign of pneumonia induced by human infected h7n9 avian infl uenza by ct scan is ground glass opacity, followed by consolidation, air bronchogram and interlobular septal thickening, possibly with pleural effusion. in some cases, air containing cyst can be observed, with a large area of involvement, commonly 3 or even more lung lobes. during the treatment, the condition may repeatedly improve and deteriorate, with the lung lesions fi rstly absorbed and then progress that is rapid in some cases. in some patients with good physical constitution and early diagnosis, the condition can be obviously improved after several days treatment, just like the present case. by ct scan, pneumonia induced by human infected h7n9 avian infl uenza is demonstrated with no specifi c signs, resembling to other viral pneumonia. and the fi nal diagnosis should be based on clinical manifestations and laboratory tests. radiological examinations play important role in guiding clinical treatment, monitoring disease progression, assessing therapeutic effi cacy, and predicting the prognosis. feng et al. scored the radiological demonstrations of pneumonia induced by human infected h7n9 avian infl uenza, which showed that a high score indicates a high mortality rate. the air containing cyst in the right lower lung showed no obvious changes ( g ). re-examination after 1 month, the lesions in lungs were obviously absorbed, still with fl akes and grid like high density opacity that predominantly under the pleura ( i as the 1 mm reconstructed image of the same layer of h ) ( h , i ). bilateral pleural effusion was completely absorbed. ( j ) the air containing cyst in the right lower lung was enlarged ( k ) g h i k j fig. 11 .6 (continued) case 7 a 33-year-old man, working as a cook, was hospitalized due to the chief complaints of chest distress for 5 days that deteriorated, with fever, cough and expectoration for 3 days. about 5 days ago, the patient caught a cold and began to experience chest distress that deteriorated after physical activities, and the body temperature within normal limits. about 3 days ago, his condition deteriorated, with inability of supine during nights, fever, cough, expectoration and a body temperature of 38.5 °c. by physical examination on admission, he was in a state of stupor, shortness of breath, heart rate 115/min, dullness by percussion of the right lung, coarse breathing sound of both lungs, dry rales of both lungs that predominantly right lung. chest x-ray demonstrated multiple patches of opacity in both lungs that predominantly in the right lung. by laboratory tests, wbc 6.07 × 10 9 /l, ly% 19.92 %, ck 1431u/l, ldh 988u/l, crp 58.8 mg/l, cd3 + cd8 + 36.6 %, cd3 − cd56 + 15.9 %. within 2-5 days after onset, the condition deteriorated rapidly, with occurrence of severe pneumonia and multiple organs dysfunction. in addition to the laboratory fi nding of h7n9 nucleic acid positive, he was diagnosed with human infected h7n9 avian infl uenza. after therapies of anti-viral therapy, anti-infl ammatory therapy, the use of hormones and mechanical ventilation as well as symptomatic therapies for multiple organs dysfunction, his condition was obviously improved. ten days after his hospitalization, tracheal incubation was removed, consecutive nucleic acid tests for 3 times showed negative, and the indeces for routine blood test, heart function, liver function and kidney function were within normal limits. he was then discharge after hospitalization for 28 days. six months later, he began to experience superior tracheal stenosis, which was treated by tracheal stent implantation. [imaging manifestations] fig. 11 .7 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza. [discussion] the diagnostic evidence for this case is as the following: 1. the patient reported a history of contact to secretions and excretions of poultry. and his condition developed rapidly, with the occurrence of severe pneumonia, multiple organs dysfunction, persistent increase of the body temperature since d 5 after onset. by laboratory tests, he showed no increase of wbc count but decrease of lymphocyte count, and h7n9 nucleic acid positive. 2. by chest x-ray and ct scan, he showed multiple fl akes of ground glass like and consolidation opacities in both lungs with air bronchogram. the lesions were in both lower lungs, predominantly the right lung, that progress rapidly and distribute extensively. 3. after 10 days treatment by anti-viral therapy, use of hormones and mechanical ventilation, the lung lesions were absorbed and improved. consecutive nucleic acid test for h7n9 for 3 times showed negative. radiological evidence for the diagnosis is as the following: 1. the saliva acid of poultry, specifi cally binding to h7n9 avian infl uenza virus is mainly located in the lower respiratory tract, namely the lungs, but none in the upper respiratory tract. human infection of h7n9 avian infl uenza virus directly induces pneumonia, with radiological signs of pneumonia early after onset. 2. early after onset, the lesions may be confi ned within one lung, the most commonly the right lung. if both lungs involved, the lesions distribute in multiple segments and multiple lung lobes that are predominantly and more serious in the right lung. in addition, the lesions in both lower lungs are more serious than in both upper lungs, which is possibly related to the posture for radiology and the anatomy of trachea. radiologically, both the parenchymal and interstitial lesions are demonstrated as large consolidation, interlobular septal thickening, and sporadic ground glass opacity, with air bronchogram. 3. during the progressive stage, human infected h7n9 avian infl uenza virus invades the lung tissues to induce the cytokine storm due to the absence of immunity to the new virus. therefore, systematic infl ammatory responses are triggered, with subsequent occurrence of acute respiratory distress syndrome (ards), shock and multiple organs failure. radiologically, the lesions in lungs rapidly develop to involve multiple segments and lobes. due to diffuse alveolar damages, chest x-ray and ct scan demonstrate extensively distributed ground glass opacity and consolidation opacity in both lungs, with air bronchogram, predominantly in both lower lungs or the right upper lung. in some cases, subpleural line and/or interlobular septal thickening is demonstrated; and in some other cases, pleural effusion. pneumothorax and mediastinal lymphadenectasis may also be demonstrated. 4. during the convalescent stage, the lung lesions are demonstrated with slow absorption, shrinkage, and lightening in color after active anti-viral therapy and chest x-ray showed multiple fl akes of consolidation in both lungs that was predominantly in the right lung ( b ). on d 19 after onset, the respiratory symptoms were improved. chest x-ray showed absorption and decrease of the consolidation ( c ). reexamination 2 months after onset, the patient was cured and discharged. ct scan showed fi brous cords like opacity in a small quantity in both lower lungs ( d ). reexamination 6 months after onset, the patient began to experience chest distress again. coronary reconstruction following tracheal ct scan showed superior tracheal stenosis, possibly related to tracheal intubation ( e ). chest distress was immediately alleviated after tracheal stent implantation ( human infected h7n9 avian infl uenza should be differentiated from the following diseases. its early radiological signs include predominant ggo surrounding bronchovascular tree or in the subpleural area. and the lesions progress into extensive alveolar consolidation. both pathologically and clinically, the changes are mild, with occurrence of death due to ards in rare critically ill patients. during the early stage of human infected h7n9 avian infl uenza, singular or multiple exudative lesions in multiple segments or lobes rapidly progress into ards and even multiple organs dysfunction. the diagnosis can be defi ned based on nucleic acid test of the virus. radiologically, both lungs are demonstrated with diffuse ggo and extensive consolidation. the lesions progress rapidly, mostly distribute in the lungs periphery and mainly involve the subpleural area. the interstitial lesions of sars are more obvious than those of human infected h7n9 avian infl uenza, with interlobular septal thickening in paving-stones sign by hrct. in the cases of lobar pneumonia, both wbc and neutrophil counts show signifi cant increase. the lesions commonly involve one lung, more commonly the right lung, and multiple lobes involvement is rare. during its consolidation period, the lesions are commonly dense with air bronchogram. however, human infected h7n9 avian infl uenza is characterized by wbc count within normal limits and decreased lymphocyte count. the lesions rapidly develop into consolidations in multiple segments and lobes as well as ards. thoracic bacterial infection may occur during treatment due to poor immunity of the patient, tracheal intubation and the use of hormones in a large quantity. case 8 a 56-year-old man complained of fever (with the highest body temperature of 39.5 °c), sore throat and muscle soreness for 6 days with no known causes. his condition was hardly improved after anti-viral and symptomatic therapies, with occurrence of cough and expectoration for 2 days as well as chest distress, shortness of breath and consciousness disturbance for 1 day. arterial blood gas analysis indicated respiratory acidosis and carbon dioxide retention. he reported no history of contact to poultry but a medical history of thymoma and its surgical removal. [radiological demonstration] fig. 11 .8 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza; severe pneumonia; type ii respiration failure. [discussion] after onset of clinical symptoms, the patient was diagnosed with ordinary infl uenza and corresponding therapy was given, with no obvious therapeutic effi cacy. he paid no attention to his condition until its deterioration and received anti-viral therapy and respiratory supportive therapy. the initial radiological examination showed lobar and segmental consolidation of one lung, which resembles to those in the consolidation period of lobar pneumonia. after 6 days, the pulmonary lesions obviously progressed and were diffusely distributed in both lungs. in the following radiological examinations, the lesions deteriorated again, with gradual deterioration following slight absorption of the lesions and subsequent slow absorption. based on clinical manifestations and laboratory tests, it was speculated that it may be related to occurrence of mixed infection in the late stage of the disease. long-term following up observation indicated that the lesions are subject to interstitial fi brosis after their absorption. in this case, differential diagnosis should be made from lobar pneumonia during its early stage. but it is challenging because both diseases show extremely similar radiological signs. however, in the patients with lobar pneumonia, clinical symptoms and signs can be obviously improved after administration of antibiotics and symptomatic therapy. but in this case, active therapies showed no obvious therapeutic effi cacy, and the condition of the patient aggravated. such outcomes should be paid focused attention and its etiological evidence should be traced to change therapeutic regime. ct scan showed consolidation in the left upper lung lobe, with air bronchogram inside ( b ). on d 13 after onset, bedside chest x-ray showed diffuse fl akes of consolidation and ground glass opacity in both lungs ( c ). on d 18 after onset, bedside chest x-ray showed absorption of the lesions in the right lower lung but aggravating lesions in the left lower lung ( d ). on d 28 after onset, ct scan showed large consolidation in both lungs and aggravated consolida-tion in the right lower lung, with interlobular septal thickening ( e ). on d 31 after onset, bedside chest x-ray demonstrated absorption of the lesions in the left lung in grid like and honeycomb like changes ( f ). on d 32 after onset, ct scan showed interstitial thickening predominantly in the left upper lung, slightly decreased density of consolidation in the left lower lung, and obvious consolidation in the right lung ( g ). by reexamination 18 months after the onset, ct scan showed a small quantity of residual fi brous cords like opacity, indicating fi brosis ( a 38-year-old man complained of fever with no known causes with the highest body temperature of 39.4 °c since dec. 9, 2013, accompanying paroxysmal cough and expectoration of yellowish white thick sputum in a small quantity. he paid a clinic visit in a local hospital. but on dec. 12, 2013, he was hospitalized due to aggravation of the condition. chest x-ray showed large consolidation in the left lower lung and sporadic patches of opacity in the right lung. one day after treatment, these symptoms aggravated. blood gas analysis showed type i respiratory failure. after that, the patient was transferred into icu. by physical examination on admission, his body temperature 36.1 °c, heart rate 116/min, breathing rate 36/min, and blood pressure 116/67 mmhg. he respirated with the assistance of ventilators and showed no response to calling. conjunctiva slightly swollen in both eyes, with no congestion, no yellowish sclera, equal size of bilateral pupils in a diameter of about 2 mm that are sensitive to light. pharynx congested, bilateral tonsils no swelling, no special abnormality in heart examination. fremitus weakened at the left chest, no sensation of pleural friction; dullness of the left lower lung by percussion; coarse breathing sounds of both lungs that was relatively weaker in the left lung; fi ne moist rales in both lungs. he reported a history of buying chicken in a market and cooking it on nov. 20, 2013. routine blood test showed wbc 3.48 × 10 9 /l, gr% 76.1 %. blood biochemistry revealed ck 3319u/l, ldh 882u/l, ckmb 32.5u/l, k + 2.83 mmol/l, na + 133 nmol/l, troponin and bnp within normal limits, crp 26.71 mg/l, alt 136u/l, ast 470u/l, alb 27 g/l. blood gas analysis showed ph 7.48, po 2 47 mmhg, pco 2 25.4 mmhg, and be -5 mmol/l. cd4 t cell count was 42/μl. by etiological examination, his tracheal aspirates showed h7n9 nucleic acid positive. [radiological demonstration] fig. 11 .9 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza. [discussion] for this case, the symptoms and signs are in line with the diagnostic criteria of severe pneumonia induced by human infected h7n9 avian infl uenza established by the national health and family planning commission of china. at the early stage, the patient showed pulmonary consolidation, with complication of pleural effusion. and radiologically, the lesions are characterized by the following: 1. on d 3 after onset, plain chest x-ray showed poorly defi ned patches of opacity in the left middle and lower lung fi elds as well as left pleural effusion; ground glass opacity in the right middle lung fi eld. chest ct scan on the same day demonstrated patches of ground glass opacity in the right middle lung lobe and the left upper lung; large consolidation in the left lower lung lobe, with air bronchogram inside. 2. on d 14 after onset, the lesion of lung consolidation was obviously absorbed, with patches of ggo in the right upper and middle lung and the left upper lung, predominantly in the subpleural areas; the lesion of consolidation in the left lower lung was absorbed compared to previous ct scan fi ndings. and the air opacity in the anterior mediastinum and the anterior thoracic wall is induced by invasive ventilation. 3. reexamination by chest ct scan on d 39 after onset revealed almost absorbed ggo in both lungs and consolidation in the left lower lung; but observable lesions of pulmonary interstitial fibrosis, such as small ggo in lower lungs and interlobular septal thickening. in addition, the gas opacity in the anterior mediastinum and anterior thoracic wall was also absorbed. pneumonia induced by h7n9 avian infl uenza should be differentiated from other emerging infectious diseases, such as viral pneumonia induced by h5n1 avian infl uenza virus and type a h1n1 infl uenza virus as well as sars. radiologically, they share commonalities of ggo and consolidation, with rapid change and progression. early lesions of pneumonia induced by h7n9 avian infl uenza are predominantly located in both lower lungs, which is not characteristic of pneumonia induced by h5n1 avian infl uenza virus or type a h1n1 infl uenza virus and sars. therefore, its differential diagnosis based on radiology is challenging, and clinical diagnosis should be defi ned based on epidemiological investigation and etiological examination. a 55-year-old man experienced frequent paroxysmal cough and expectoration of a little yellowish white thick sputum since jan. 5, 2014. his condition was diagnosed as hypertension due to a blood pressure of 154/96 mmhg and he received medication to lower his blood pressure. several days later, he was hospitalized on jan. 10, 2014 due to the aggravating condition. chest x-ray revealed bronchiectasis in the left lower lung with accompanying infection, which was then considered as lung infection. he was then admitted to the respiratory department for treatment, but his cough was not improved by anti-infection therapy. on jan. 13, 2014, he still experienced fever, with the highest body temperature of 38 °c. an immediate blood gas analysis indicated respiratory failure, and he was transferred to icu for tracheal intubation and assisted breathing via ventilator. bedside chest x-ray revealed extensive infection in both lungs and possible bronchiectasis in left lower lung, indicating signifi cant progression compared to the condition on jan. 10, 2014. routine blood test showed wbc count 2.61 × 10 9 /l, gr% 70.9 %, rbc count 5.0 × 10 12 /l, hgb 158 g/l, plt 71 × 10 9 /l. blood gas analysis revealed ph 7.45, po 2 50 mmhg, and pco 2 24 mmhg. on jan. 13, 2014, laboratory test by cdc showed reported alveolar lavage fl uid positive to h7n9 avian infl uenza virus, and peripheral cd4 t cell count 66/μl. [radiological demonstration] fig. 11 .10 [diagnosis] pneumonia induced by human infected h7n9 avian infl uenza. [discussion] in this case, the patient was an elderly man with no defi nitive history of contact to live poultry. the onset symptoms include cough and expectoration, and laboratory tests showed decreased peripheral wbc count, decreased platelet count, and decreased blood potassium level. on d 5 after onset, chest x-ray revealed infection in the left lower lung. the patient experienced repeated fever and the condition rapidly progressed into respiratory failure, multiple organs failure and infectious shock. 11 .10 on d 5 after onset, chest x-ray showed poorly defi ned patches of high density opacity in the left lower lung fi eld; overlapping of some lesions with heart shadow ( a ). on d 8 after onset, chest x-ray showed fl akes of ground glass opacity in both lungs, and consolidation in the bilateral middle and lower lung fi elds ( b ). on d 9 after onset, chest ct scan showed multiple large high density opacity and ground glass opacity in both lower lung lobes, predominantly in the left lower lung lobe ( c ). large consolidation was revealed in both lower lungs, with air bronchogram and multiple cystic translucent areas ( d ). on d 14, chest ct scan showed unevenly distributed ground glass opacity in the bilateral upper and middle lung lobes, consolidation in the left upper lung with multiple small translucent areas with no wall ( e ). both lower lungs were revealed with patches of ggo like high density opacity and the left lower lung was shown with large consolidation and round translucent opacity in different sizes inside. they were mainly distributed in the left subpleural area and the dorsal lungs. compared to the chest ct scan fi lm 5 days ago, the lesions were absorbed ( f ). on d 209, the left lung was shown with unevenly distributed ground glass opacity; the left subpleural area and anterior mediastinum were shown with lung bullae ( h ). lung bullae were sporadic in both lower lung lobes ( h ) a c e f d b on d 5 after onset, chest x-ray indicated infection in the left lower lung and on d 8 after onset, the lesions spread to involve both lungs. on d 9 after onset, chest ct scan revealed large consolidation and ground glass opacity in each lobe of both lungs, with multiple small translucent areas inside. these radiological signs indicated rapid progression of the lesions, which are rarely observed in patients with bacterial and common viral pneumonia. this case is radiologically characterized by the followings: 1. the lung lesions are mainly consolidation and ggo, which emerge early after onset. and the lesions are mainly distributed in the subpleural area of both lower lung lobes. air bronchogram is observable in the consolidation opacity. 2. the lesions progress rapidly but are absorbed slowly. the lesions are fi rstly confi ned within one lung, but rapidly spread to involve lobes and segments of both lungs. the condition rapidly develops into respiratory failure. with the use of various therapies, the lung lesions are gradually absorbed, with slower absorption of interstitial lesions than parenchymal lesions. g h fig. 11 .10 (continued) case 11 a 39-year-old woman experienced cough and expectoration of yellowish sputum with no known causes since jan. 15, 2014. her condition was not improved after treatment in a local hospital on jan. 17. and she was then hospitalized in a superior hospital due to a diagnosis of lung infection. physical examinations showed a body temperature of 38 °c, heart rate 95/min, respiration rate 19/min and blood pressure 123/73 mmhg. she was well conscious in good spirits, no shortness of breath, and cooperative to the physical examinations. by palpation, the swallow lymph nodes no enlarged, and 3 depression sign negative (−). pharynx congested, bilateral tonsils no swollen with no pus spots, and oral cavity no koplik's spot. the chest no malformation, bilateral breathing mobility equal, breathing sounds of both lung coarse, moist rales in the right lower lung, and no pleural friction sounds. the heart beat rhythm regular, and no murmurs by auscultation. the abdomen was soft, liver and spleen unpalpable under ribs, the whole abdomen no tenderness and rebound, and bowel sound normal. both lower limbs no swollen. she denied medical histories of other diseases but experienced vomiting, nausea and pruritus of right lower leg after oral intake of cephaloridine 1 day ago. she also denied a history of contact to live poultry, but reported a history of eating chickens 6 days ago. routine blood test showed wbc count 5.2 × 10 9 /l, gr% 63 %, and ly% 30.1 %. blood gas analysis revealed ph 7.47, pco 2 31 mmhg, po 2 67 mmhg, na + 134 mmol/l, k + 2.7 mmol/l, and ca 2+ 0.91 mmol/l. sputum test showed nucleic acid of h7n9 avian infl uenza virus positive. [radiological demonstration] fig. 11 .11 [diagnosis] pneumonia induced by h7n9 avian infl uenza. [discussion] this case is radiologically characterized by the followings: 1. on d 2 after onset, chest plain x-ray showed small fl ake of high density opacity at the cardiodiaphragmatic angle of right lower lung fi eld, indicating emergence of lesions in lower lung in the early stage of pneumonia induced by human infected h7n9 avian infl uenza. on d 3 after onset, chest x-ray further proved fl ake of ggo in the posterior basilar segment of right lower lung lobe, and opacity with mixed ggo density with parenchymal density. 2. on d 6 after onset, chest ct scan demonstrated rapid progression of the lesions in both lungs, with large consolidation in the right lower lung lobe and sporadic poorly defi ned patches of opacity in the right middle lung lobe and each basilar segment of the left lower lung lobe. within 72 h, the progression of lesions was more than 50 %, which was in line with the diagnostic criteria of severe pneumonia induced by human infected h7n9 avian infl uenza. 3. at the peak of lesion progression, the lesions began to be absorbed after effective therapy was given, but the convalescent stage lasted for a relatively long period of time. on d 31 after onset, chest ct scan showed absorption of most lesions but still residual cords like opacity and small ggo in the subpleural area of posterior basilar segment in the right lower lung lobe. fig. 11 .11 on d 2 after onset, anterior-posterior and lateral x-ray showed poorly defi ned small fl ake of high density opacity near the cardiodiaphragmatic angle in the right lower lung fi eld. and lateral x-ray demonstrated poorly defi ned small fl ake of opacity in the overlapping area of posterior costophrenic angle and thoracic vertebrae in the right lower lung fi eld ( a , b ). on d 3 after onset, chest ct scan showed fl ake of ground glass opacity in the posterior basilar segment of right lower lung lobe ( c ). the right lower lung lobe was revealed with large fl ake of opacity with mixed ggo density and parenchymal density ( d ). on d 6 after onset, chest ct scan demonstrated large consolidation in the posterior basilar segment of right lower lung ( e ). the right middle and lower lung was revealed with large consolidation with air bronchogram inside. the left lower lung was revealed with fl ake of consolidation and ground glass like high density opacity. compared to the chest ct scan fi lm 3 days ago, the lesions signifi cantly progressed ( f ). on d 31 after onset, chest ct scan demonstrated absorbed consolidation opacity in the right lower lung lobe at the bronchial bifurcating level ( g ). the right lower lung lobe was revealed with ggo like opacity and interstitial fi brous cords like opacity near diaphragmatic dome. the left lingual lung lobe was revealed with ggo like opacity and interstitial fi brous cords like opacity ( swine-origin infl uenza a(h1n1)viral infection: radiographic and ct fi ndings radiological demonstrations of lung lesions and their dynamic changes in human infected h7n9 avian infl uenza human infections with the emerging avian infl uenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome association of radiologic fi ndings with mortality in patients with avian infl uenza h7n9 pneumonia human infection with a novel avian-origin infl uenza (h7n9) virus ct scan demonstrations and their relationship with virus load and cd4 t cell count in pneumonia induced by human infected h7n9 avian infl uenza radiological data analysis of pneumonia induced by human infected h7n9 avian infl uenza: report of 12 cases recent understandings and insights about the emerging infectious disease: human infected h7n9 avian infl uenza chest radiological demonstrations of human infected h7n9 avian infl uenza chest radiological data of human infected h7n9 avian infl uenza: report of 13 cases imaging diagnosis of emerging infectious diseases. beijing: people's medical publishing house radiological demonstrations of severe pneumonia induced by human infected h7n9 avian infl uenza and their dynamic changes bedside chest radiography of novel influenza a (h7n9) virus infections and follow-up findings after short-time treatment emerging h7n9 infl uenza a (novel reassortant avian origin) pneumonia: radiologic fi ndings clinical and radiological features of human infected h7n9 avian infl uenza primary radiological data analysis about pneumonia induced by human infected h7n9 avian infl uenza chest imaging of h7n9 subtype of human avian infl uenza chest radiological demonstrations of pneumonia induced by h7n9 avian infl uenza virus radiological demonstrations of severe pneumonia induced by h7n9 avian infl uenza virus human infected avian infl uenza key: cord-016009-qa7bcsbu authors: starkel, julie l.; stapke, christina; stanley-o’malley, abigail; noland, diana title: respiratory date: 2019-10-07 journal: integrative and functional medical nutrition therapy doi: 10.1007/978-3-030-30730-1_51 sha: doc_id: 16009 cord_uid: qa7bcsbu lung disease rivals the position for the top cause of death worldwide. causes and pathology of the myriad lung diseases are varied, yet nutrition can either affect the outcome or support treatment in the majority of cases. this chapter explores the modifiable risk factors, from lifestyle changes to dietary intake to specific nutrients, anti-nutrients, and toxins helpful for the nutritionist or dietitian working with lung disease patients. general lung health is discussed, and three major disease states are explored in detail, including alpha-1 antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. although all lung diseases have diverse causes, many integrative and functional medical nutrition therapies are available and are not being utilized in practice today. this chapter begins the path toward better nutrition education for the integrative and functional medicine professional. anti nutrients and inhibitors of lung physiology -942 51 lung disease is far more prevalent worldwide than commonly thought. in fact, death from chronic lung disease is increasing, and as of 2017, chronic obstructive pulmonary disease (copd) has become the third leading cause of death in the united states in the past decade, disproportionately affecting the elderly [1] . another lung disease, asthma, affects 1 in 13, or about 25 million americans, according to the centers for disease control and prevention and the national center for health statistics [2] . this is 7.6% of adults, more women than men, and 8.4% of children. asthma is the leading chronic disease in children [3] . this disease has been increasing since the early 1980s in all age, sex, and racial groups. in europe, lung disease represents 15% of all deaths -the fourth leading cause. according to the world health organization (who), in 2008, 9.5 million people died from acute or chronic lung disease, representing one sixth of the global total deaths [4] . worldwide, four respiratory disease categories appear in the top ten leading causes of death in 2010 [5] . specifically, copd was the third leading cause of death, followed by lower respiratory infections as the fourth, lung cancer as the fifth, and tuberculosis as the tenth [4] . the major risk factor is smoking, leading to 50% of all lung disease-related deaths in europe, where smoking is more prevalent (28% prevalence) than in the united states (15% prevalence) by nearly twofold [6, 7] . lung cancer, particularly non-small-cell lung cancer (nsclc) subtype, is the leading cause of cancer-related death worldwide [8] . added together, lung disease rivals the position for the top cause of death. throughout the life cycle, diet and lifestyle are important modifiable risk factors in the development, progression, and management of obstructive lung diseases, such as asthma and copd [9] , as well as restrictive lung diseases such as pulmonary fibrosis and sarcoidosis. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. as with many diseases, maintaining a healthy lifestyle, including sufficient sleep, low stress, regular exercise, a whole foods diet rich in phytonutrients from plants (fruits and vegetables), and potential anti-inflammatory supplements, is beneficial in supporting the body during these difficult diseases. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. high inflammatory foods should be avoided, such as fried foods and foods disproportionately high in carbohydrates, sugar, alcohol, and excessive protein. a healthier suggestion would be a diet with more than half of all food consumed as vegetables, about one third as protein, and the remainder (one sixth) as other foods, such as fruits, dairy, grains, or starches. some dietary supplements may also be recommended for their anti-inflammatory benefits, which will be discussed later in the chapter. as human life expectancy increases, we can expect to see more chronic disease. the world health organization estimates that by 2030, chronic lung disease will account for 20% (one fifth) of all deaths [10] , up from one sixth in 2008. despite these growing numbers, relatively little human nutrition research exists for respiratory health, compared to other, less prevalent, diseases. investigators in the areas of aging and lung biology suggest some hope, using genetics and animal models, as well as epidemiological research, to further the general medical approach to lung disease. the pulmonary system is composed of the upper and lower respiratory tracts. air flows in through the nose or mouth, past the frontal and maxillary sinuses, down the pharynx (throat), past the larynx (voice box), and then down the trachea. this makes up the upper respiratory tract. once past the trachea, the air divides into the left and right bronchi, which supply the left and right lungs, each divided into five sections called lobes. the bronchi then divide into smaller bronchioles, at the end of which are air sacs called the alveoli. this makes up the lower respiratory tract [11] (. fig. 51 .1). the diaphragm is the central muscle that is used for breathing. the intercostal muscles, located between the ribs, and the abdominal muscles are helpful for breathing out when the breath becomes labored, such as during exercise. the neck muscles and the muscles in the collarbone area help with breathing when the other muscles are compromised or impaired. in some neurological diseases, such as amyotrophic lateral sclerosis (als) [see 7 chap. 51, newton], nerve damage from the brain to breathing muscles can result in impaired movement of these muscles and thus impaired breathing. in certain cases, such as in lung cancer when a lobectomy, removal of part of the lung, is required, there is an expected decrease in short-and long-term pulmonary function and oxygenation. however, respiratory muscle strength may be preserved [13] . in a pneumonectomy, removal of the entire lung, dramatic changes in thoracic anatomy take place, such as elevation of the hemidiaphragm, hyperinflation of the remaining lung, and influx of fluid into the postpneumonectomy space [14, 15] . there are phagocytic macrophages on the cellular surface of the alveoli, type i epithelial cells and type ii epithelial cells. phagocytic macrophages destroy inhaled bacteria and serve an important role in suppressing or activating the immune response to antigens and pathogens, similar to dendritic cells discussed below. macrophage function has been shown to be inhibited by cigarette smoke [16] . alveolar macrophages also secrete enzymes, arachidonic acid metabolites, growth factors, immune response components, cytokines, and lymphocytes [17] . type i cells are responsible for maintaining the structure of the alveolar wall, whereas type ii cells and clara cells are responsible for the production of pulmonary surfactant (composed of 85-90% lipid and 10-15% protein as lecithin and myelin), which is essential for lung function. the surfactant reduces surface tension, facilitating easier stretching and collapsing of alveoli during respiration [18] . diseases associated with inadequate surfactant production are acute/adult respiratory distress syndrome (ards) and infant respiratory distress syndrome (irds) [19] . irds is seen in premature babies born prior to 32 weeks of gestation due to immature development of pulmonary surfactant, which only begins to develop around the 20th week of gestation [18] . dipalmitoylphosphatidylcholine, phosphatidylglycerol, and cholesterol compose the lipid portion of the surfactant, where apoproteins and proteins found in blood plasma compose the protein portion [18, 20] . the importance of cholesterol is minimized in today's medical community. those with higher levels of cholesterol tend to have more in their fatty cell membranes which resist pathogenesis at a cellular level. low cholesterol predicts a greater risk of dying from gastrointestinal, neoplastic, or respiratory diseases. it occupies 30-40% of our cell membranes, enhances the mechanical strength of the membrane, and reduces permeability [21] . it suppresses main-phase transition of the lipid bilayer [22] . collagen, a fibrous protein, along with elastin and proteoglycans, is a fundamental component of the connective tissue that composes the lungs, and collagen is present in the blood vessels, bronchi, and alveolar interstitium [23] . connective tissue in the lung is key for the passive diffusion of oxygen and carbon dioxide that characterizes alveolar-capillary gas [18] . collagen homeostasis is vital to maintaining respiratory function, where collagen production and degradation are balanced. dysregulated collagen homeostasis that favors collagen production over degradation can lead to pulmonary fibrosis and compromised lung function [24] . some key nutrients to consider for collagen synthesis and crosslinking to maintain connective tissue integrity are vitamin c, vitamin b6, iron, copper, zinc [25] , riboflavin, thiamin, and pantothenic acid [11] . the airways of the respiratory system (with the exception of parts of the nose and mouth) have cilia, special hairs coated with mucus that trap pathogens and other particles that enter with the air that is inhaled. cilia are responsible for triggering this mucus upward toward the pharynx where these particles or bacteria can be coughed out or swallowed. mucus present in the lungs can also trap inhaled particles such as viruses, bacteria, and smoke particulates [11, 12] . along the lining of the respiratory tract, there are several types of cells that are involved in immune response, such as secretory cells (i.e., goblet cells and clara cells) and mast cells. ciliated epithelium and mucus secreted by glands present on airways, goblet cells, and the secretory products of clara cells serve an important mechanism for lung protection. however, excessive goblet cells or hypertrophy of mucous glands may result in increased viscosity of mucus seen in pathologies like bronchitis [16] . ciliary function is also impaired by cigarette smoke [16] . dendritic cells are also found in the airway lining from the trachea to the alveoli. immature dendritic cells phagocytize bacteria or other antigens, where they then mature and travel to lymphoid tissues to communicate with the immune system. this delivery of antigens can promote tolerance of the antigen by releasing anti-inflammatory cytokines. conversely, this delivery can also trigger the opposite response if the antigen is recognized as a pathogen, where t lymphocytes are activated and inflammatory cytokines are released [16] . one potential cause of infections in the upper respiratory tract or bronchial tubes, such as bronchitis, or deep in the lungs, such as pneumonia, is when cilia become damaged and do not trap inhaled germs and particles as effectively. in diseases such as cystic fibrosis, thick mucus secretions can accumulate in the airways and lungs, making it hard to clear and thus increasing risk for infection. in asthma, specific inhaled particles can trigger a reaction causing the airways to narrow, restricting breathing [12] . surface enzymes and factors can also be found in the lining of the airways that compose the majority of the innate immune system of the respiratory tract. these include: 5 lysozymes: found in leukocytes with bactericidal properties 5 lactoferrin: a bacteriostatic agent (inhibits bacterial reproduction) synthesized by lymphocytes and glandular mucosal cells 5 alpha-1 antitrypsin: an antiprotease to protect lung tissue from excessive enzymatic activity 5 interferon: an antiviral substance that may be produced by lymphocytes and macrophages 5 complement: participates as a cofactor in antigenantibody reactions [16] gas exchange takes place in the alveoli so oxygen can enter the body to support metabolic function and the carbon dioxide product from these functions can be removed. this is accomplished through millions of capillaries in the alveoli. these capillaries in the alveoli then connect to arteries and veins that move blood throughout the body. the pulmonary artery supplies carbon dioxide-rich blood to these capillaries within the alveoli to remove carbon dioxide, and the oxygen-rich blood then gets delivered to the heart through the pulmonary vein. the lungs also serve the vital function of maintaining acid-base balance through changes in minute ventilation. these changes affect the ph of the blood by either retaining or excreting carbon dioxide [11] . poor physiologic management of co 2 and bicarbonate can lead to the conditions of respiratory acidosis and respiratory alkalosis. respiratory acidosis is characterized by higher blood concentrations of co 2 and h + , caused by hypoventilation or decreased rate of breathing. hypoventilation can have acute or chronic etiologies, resulting from copd, interstitial lung diseases, respiratory muscle fatigue (i.e., extended asthma attack), or mechanical abnormalities (i.e., deformities). respiratory alkalosis is characterized by lower blood concentrations of co 2 and h + due to hyperventilation, or increased rate of breathing. possible causes of hyperventilation can also be chronic or acute, such as pneumonia and fever, increased stress and anxiety, liver disease, stroke or meningitis, pregnancy, overuse of aspirin and/or caffeine, excessive mechanical ventilation, or increases in altitude [18] . a pulse oximeter tool can be used to measure the percentage of oxygenated hemoglobin in an individual's blood to determine their overall respiratory status. typically, oxygen saturations of 92% or less are indicative of central hypoxia [26] . pulse oximetry is especially useful for assessing individuals with asthma and copd [26] . oral health must also be considered as a contributing factor to respiratory health [27] . for example, in patients affected with periodontal disease, 1 mm of dental plaque could contain around 10 9 of bacteria. one potential mechanism of this connection is aspiration of bacteria from the oropharynx into the upper or lower respiratory tracts, leading to their adherence to the alveolar and bronchial lining, potentially colonizing respiratory ducts and causing respiratory infections. in addition, cytokines and enzymes associated with inflammation of periodontal tissues can be transferred into the lungs, potentially triggering or exacerbating lung infections [27] (. fig. 51 . a systematic review done in 2013 examined oral health in the elderly and its association with risk of aspiration pneumonia. this review suggested that maintaining oral health, such as brushing after each meal, cleaning dentures once per day, and professional oral healthcare, potentially reduced the amount of potential respiratory pathogens that resulted in lower incidence of aspiration pneumonia [28] . several other systematic reviews have found that adequate oral hygiene plays an important role in preventing pneumonia, particularly in clinical settings where there is increased risk for hospital-acquired pneumonia (hap) and ventilatorassociated pneumonia (vap), as well as in older populations [29] . in addition, associations have been made between copd and the risk of periodontitis, although systematic reviews have established that these associations are preliminary and further studies are needed [29] . another important consideration in respiratory health is orofacial development and structure. anatomical obstructions at the level of the nose and pharynx, such as those caused by allergic rhinitis and hypertrophy of the tonsils, pose an increased risk for obstructive sleep apnea syndrome and respiratory infections due to lack of airflow through the upper respiratory system [30] . it has been established that the lung has a microbiome of its own that may have a large impact on health and disease [31] . the fungal microbiome, or mycobiome, may also have a significant impact on respiratory health, although more research is needed to determine definitive associations [31] . dysbiosis may occur in the lungs with a bacterial infection. a few specific bacterial strains have been studied, and one, in particular, pseudomonas aeruginosa, seems to grow in inflammatory conditions. it then seems to encode inflammatory components causing further inflammation. anti-inflammatory nutrients could help stop the cycle, and vitamin d use has some research supporting this. recurrent bacterial respiratory infections may damage lungs and lead to worse outcomes in future lung disease [32] . an increased interest in research of the relationship of the airway and gut microbiome is indicating potentially positive results regarding the use of probiotics in pediatric populations that may aid in asthma prevention and intervention [33, 34] . the gut-lung axis has also been established, where the microbiomes of the lung and gut have been immunologically linked and are thought to have an impact on respiratory disease [35, 36] . the autophagy mechanism within our microenvironment provides a constant "cleanup" system to recycle cell debris from microscopic biowaste generated by dynamic cellular biochemistry [37] . enzymes such as neutrophil elastase function like garbage disposals recycling waste molecules. alpha-1 antitrypsin is a thermostat-like control factor that signals the proteolytic enzymes to stop and protect healthy tissue from being affected. antiproteases in the lung, such as alpha-1 antitrypsin, are required to prevent the overactivity of neutrophil elastase to prevent the degradation of healthy lung tissue. those with the genetic mutations of a1at deficiency are at disadvantage, and subsequent lung tissue damage can occur promoting lung diseases like copd, asthma, bronchitis, and emphysema. key components of lung structure are elastin and collagen, which provide support for the bronchioles and clusters of alveoli (acini). the key enzyme present in these cells is neutrophil elastase, which is responsible for the destruction of respiratory bacteria. protease and antiprotease imbalance in the lung resulting in emphysema can be caused by alpha-1 antitrypsin deficiency and nicotine in cigarette smoke or polluted inhalant exposure [18] . ifmnt approaches to the a1at-deficient patient assess for nutrient insufficiencies for some of the important connective tissue, collagen, and elastin system key nutrients: vitamin c, vitamin d, biotin, balanced fatty acids, and gut microbiome. when insufficiencies or deficiencies are identified, appropriate food and dietary supplementation interventions can be recommended. it should be noted that if an individual is identified with a1at deficiency genotype, the status of liver health should also be assessed, as a1at pathophysiology can express in liver cirrhosis. more recent studies of respiratory disease [38] have revealed the relationship with bacterial or viral infections exacerbating the individual's genotype eliciting expression of the associated diseases. one of the most recognized inherited conditions of altered autophagy mechanisms is alpha-1 antitrypsin deficiency, with 80-100 genetic variants affecting severity of lung expression. low levels of circulating a1at allow potentially harmful enzymes like neutrophil elastase to remain in the lungs unchecked. low levels of a1at, and the consequent proliferation of neutrophil elastase, leave lung tissue vulnerable to destruction, resulting in a decline in lung function. there are several categories of lung disease and many diseases within those categories (. table 51 .1). some micronutrients and phytonutrients have important antioxidant and methyl-donating properties important for the lungs and therefore have great role in a nutritional approach to lung health. iron's interaction with the lungs is essential. it carries oxygen from the lungs to the peripheral parts of the body, as well as carbon dioxide back to the lungs to be exhaled. however, too little or too much iron can pose a problem for the lungs. before iron administration, it is important to rule out hemochromatosis, or iron overload, for an individual. iron-deficiency anemia often presents in many chronic diseases including those of the lung, such as copd, lung cancer, and ipf [57] . increased mortality, decreased quality of life, increased hospital admissions, and cost of treatment have been reported for those with chronic disease and low iron [58] . anemia of chronic disease (acd) is usually at the root of this. acd is often the result of inflammation. inflammatory proteins, including il-6, stimulate the production of hepcidin in the liver, which inhibits absorption and increases storage of iron resulting in a functional iron deficiency. typical iron markers, such as transferrin saturation, total iron binding capacity (tibc), and ferritin, are also affected by inflammation and are less useful markers in chronic disease. soluble transferrin receptor (stfr) seems to be a lesser known marker that is less affected by inflammation [59] . because of the difficulty with iron absorption, intravenous iron is often used to replete deficiencies. as iron is a pro-oxidant, researchers studied any negative repercussions. there does not seem to be any increased oxidative stress with intravenous iron, but glutathione, the body's endogenous super antioxidant, does seem to decrease, likely in response to the pro-oxidative activity of iron. in a recent study, administration with vitamin e was seen to eliminate these negative effects [57] . excessive iron can also be problematic for lung health for those with the genetic mutation for hemochromatosis (hfe). disorders of iron overload are increasingly being recognized as risk factors for most of the chronic diseases like cardiovascular, alzheimer's, and cancer [60] . high iron can catalyze the formation of highly reactive hydroxyl radicals, oxidative stress, and programmed cell death. in the instance of lung cancer and other cancers affecting the lungs, tumors sequester iron for their own growth, usually leaving the patient with iron-deficiency anemia. in fact, 90% of cancer patients undergoing chemotherapy are iron deficient. inflammation also plays a role in iron homeostasis. the pro-inflammatory cytokines cascade down to affect the proteins that regulate . chronic obstructive pulmonary disease (copd) disease that restricts airflow through either inflammation of the lining of the bronchial tubes or destruction of alveoli increased risk of emphysema if genetic variant of alpha-1 antitrypsin deficiency and smoking or exposed to high levels of air pollution [11] bronchiectasis a disorder of the airways that leads to airway dilation and destruction, chronic sputum production, and a tendency toward recurrent infection [39] bronchiolitis airway injury that can be caused by infections, irritants, toxic fumes, drug exposures, pneumonitis (typically viral), organ transplants, connective tissue disorders, vasculitis, or other insults [40] dyspnea shortness of breath or difficulty breathing [11] emphysema thinning and destruction of the alveoli, resulting in decreased oxygen transfer into the bloodstream and shortness of breath. increased risk of emphysema if genetic variant of alpha-1 antitrypsin deficiency and smoking or exposed to high levels of air pollution [11] alpha-1 antitrypsin deficiency a deficiency of a1at, a protein produced in the liver that protects the lungs from excessive neutrophil elastase, an autophagic enzyme. a1at may also accumulate in liver and cause liver disease [55] obstructive asbestosis fibrotic lung disease resulting from extensive inhalation of asbestos fibers [42] desquamative interstitial pneumonitis (dip) form of idiopathic interstitial pneumonia that is more common in cigarette smokers but may be seen in nonsmokers, in patients with underlying connective tissue diseases or those exposed to inorganic dust/particles [43] sarcoidosis immune-mediated systemic disorder that is characterized by granuloma formation of the lung parenchyma and the skin [44] restrictive pathophysiologyneuromuscular weakness amyotrophic lateral sclerosis (als) progressive neurological disease that affects the motor neurons of the nervous system [11] guillain-barre syndrome progressive immune system attack on the peripheral nerves, usually following an infectious illness such as a respiratory infection. may eventually cause respiratory distress syndrome [11] restrictive pathophysiologychest wall/pleural disease kyphoscoliosis kyphoscoliosis: a deformity of the thoracic cage that results in restriction of the lungs and impairs pulmonary function [45] ankylosing spondylitis autoimmune inflammatory disorder characterized by inflammation of the axial skeleton and peripheral joints [46] chronic pleural effusions chronic accumulation of fluid between the two outer membranes surrounding the lungs [11] pulmonary vascular disease pulmonary embolism blood clot that typically originates from thrombi in the deep venous system of the legs and travels to the lungs pulmonary arterial hypertension (pah) progressive disorder of primary pulmonary arterial vasculopathy characterized by a mean pulmonary arterial pressure >25 mm hg at rest (>30 mmhg during exercise) [48] iron homeostasis [61] . iron can also impair cytokine secretion, which can leave those with an iron overload much more susceptible to infection, increasing the morbidity and mortality of infectious diseases, including those of the lung [59] . oxidative stress may contribute to injury of lung tissue, causing further fibrosing in those lung diseases with that characteristic. allele variants in the genes associated with iron homeostasis (c282y, s65c, and h63d hfe) are significantly more common in those with idiopathic pulmonary fibrosis (ipf) than those without ipf (40.4% ipf patients vs 22.4% non-ipf) and are associated with higher irondependent oxygen radical generation [62] . iron is implicated in lung pathology. monitoring iron status and using supplements or diet to aid the body in increasing or decreasing the iron load are imperative for the nutritionist working with lung disease patients. choosing a good non-constipating form of iron is important, such as iron glycinate. the b vitamins are also important to monitor for lung health. vitamin b6 and its bioactive form, p-5-p, are typically known to protect dna from mutation or damage [63] . however, there is mixed evidence on its role for lung cancer. some research has shown that it is helpful for lung cancer patients as it is important for apoptosis when using chemotherapy, because it sensitizes cancer cells to apoptosis [63] . however, research in 2017 showed that adult male smokers taking greater than 20 mg vitamin b6/day for long periods tended to have a greater risk for lung cancer. many variables, including genetic variants, form of b6, and the status of other co-nutrients may be at play [64] . other studies showed that men in the top quintile of vitamin b6 serum concentration had about one half the risk of lung cancer, and specifically, vitamin b6 and folate were inversely associated with risk of lung cancer [65] . . squamous cell (epidermoid) carcinoma about 25-30% of all lung cancers. these start in early versions of squamous cells, which are flat cells that line the inside of the airways in the lungs. often linked to a history of smoking and tend to be found in the central part of the lungs, near the bronchus [50] large cell (undifferentiated) carcinoma about 10-15% of lung cancers. it can appear in any part of the lung and tends to grow and spread quickly. a subtype of large cell carcinoma, known as large cell neuroendocrine carcinoma, is a fast-growing cancer that is very similar to small-cell lung cancer [51] small-cell lung cancer (sclc) about 10-15% of lung cancers are sclc. typically start in the cells lining the bronchi and parts of the lung such as the bronchioles or alveoli [52] infectious diseases pneumonia inflammation of the lungs, usually caused by bacteria, viruses, or fungi [11] bronchitis inflammation and eventual scarring of the lining of the bronchial tubes accompanied by restricted airflow, excessive mucus production, and persistent cough [11] tracheitis bacterial infection that can develop in the trachea [53] infant respiratory distress syndrome also known as hyaline membrane disease (hmd) or respiratory distress syndrome, this condition affects the alveolar ducts and terminal bronchioles in which the hyaline membrane is a fibrinous material composed of blood and cellular debris, caused by the absence of proper surfactant production due to an immature or poorly developed lung [54] upper respiratory infection (uri) acute infections involving the nose, sinuses, pharynx, larynx, trachea, and bronchi, referred to as the common cold [11] bronchopulmonary dysplasia (bpd) chronic lung disorder which may affect infants who have been exposed to high levels of oxygen therapy and ventilator support [11] other cystic fibrosis disease characterized by abnormally thick mucus secretions from the epithelial surfaces of many organ systems, including the respiratory tract, the gastrointestinal tract, the liver, the genitourinary system, and the sweat glands [11] acute lung injury clinical and radiographic changes in lung function associated with critical illness (acute respiratory distress syndrome is most severe form) [11] respiratory because of disagreement in research, particularly with smokers or former smokers, using food first for b vitamins may be a prudent way forward. good sources of vitamin b6 are fish, chickpeas, chicken, potatoes, turkey, bananas, ground beef, and winter squash. pyridoxal kinase (pdxk) is the enzyme that converts pyridoxine and other vitamin b6 precursors to its bioactive form of p-5-p. dysfunction of this enzyme is a good prognostic for lung cancer and other lung diseases. mthfr 1298aa genotype is associated with a higher risk of lung cancer in women but not in men. the mthfr 677tt genotype was associated with a significantly decreased risk of lung cancer in women but not in men. in contrast, the mthfr c677t and a1298c polymorphisms interacted with smoking status in men but not in women [66] . methylation gene testing is imperative to understand the patient's status. some studies suggest that a higher intake of riboflavin (vitamin b2) may protect against lung cancer in smokers [67] . folate deficiency was also associated with asthma and attacks of shortness of breath [8] . correcting acidosis may preserve muscle mass in diseases where wasting is an issue, such as copd or ipf. for those receiving chemotherapy, a higher ph (more alkaline status) is helpful for muscle mass protection. high alkaline diets contain more fruits and vegetables, and those supply more magnesium, which is needed to activate vitamin d. as discussed below, vitamin d is extremely helpful for lung health. sleep quality involves maintaining adequate 7-8 hours with good sleep hygiene (see 7 chap. 34). good rem cycling, feeling refreshed upon awakening, and other characteristics of good sleep play significant roles in maintaining healthy acid-base balance. dietary intake of the minerals magnesium, potassium, sodium, chloride, and calcium promotes the balance of acidbase microenvironment. after exposure and tissue retention of toxic minerals and metals, these substances can contribute to perturbations in the acid-base metabolic milieu. some conditions reduce oxygen intake and should be addressed. one of the most common oxygen-impairing conditions is sleep apnea, altered sleep with random halting of breathing during sleep that is often accompanied by snoring. other limiting conditions are respiratory diseases like copd, a1at deficiency, asthma, cystic fibrosis, etc. vitamin a is an important antioxidant and a general umbrella term for several fat-soluble retinoids, including retinol, retinal, and retinyl esters. there are also other substances that are provitamin a carotenoids or precursors to vitamin a. two forms are found in foods, the preformed forms of retinol or retinyl esters, which are found in dairy, fish, caviar, and meats (especially liver), and the provitamin a carotenoids, including the most important and common provitamin a carotenoid, beta-carotene, as well as others including alpha-carotenes and cryptoxanthin, which are found in plant-based foods. our bodies must convert these two forms within our cells to retinal and retinoic acid, the active forms of vitamin a in the body. new studies of the gene, β-carotene 15,15′-monooxygenase (bcmo1), which is responsible for the enzymatic conversion of β-carotene to vitamin a, are revealing that individuals with heterozygous or homozygous bcmo1 snps have 30-60% less efficient conversion than those with normal gene function (see 7 chap. 17) [68] . other carotenoids found in food, such as lycopene, lutein, and zeaxanthin, are not converted to vitamin a but have other antioxidant benefits in the body. most vitamin a is stored in the liver as retinyl esters, and deficiency is not visible until these stores are nearly depleted. vitamin a's role as an antioxidant helps the lungs in several ways, including maintaining alveolar epithelium cells and preventing development of respiratory tract infections. most of the developed world's population does not have a risk of deficiency due to sufficient vitamin a intake. however, most people with cystic fibrosis have pancreatic insufficiency, which reduces the ability to absorb fat and therefore the fat-soluble vitamins a, d, e, and k. according to a study in 2002, between 15% and 40% of people with cystic fibrosis had a vitamin d deficiency, also a fat-soluble vitamin. with the addition of pancreatic replacement treatments, better nutrition, and vitamin a supplementation, deficiency has become rare. however, improved vitamin a status has not been thoroughly studied as of 2018, and therefore it is largely unknown if an improved vitamin a status has any effect on cystic fibrosis [69] . vitamin a deficiency has been shown to be associated with emphysema in rats. smoke exposure significantly decreases vitamin a concentration in lung tissue, significantly more in those with copd [70] . retinoic acid seems to play a beneficial role in the treatment of ipf. a review showed that in all studies, retinoic acid decreased fibrosing, the formation of collagen, and reduced the expression of alpha-smooth muscle actin (alpha-sma), all hallmarks of ipf [71] . it is important to not take large doses of vitamin a if one is in a malnourished state as it can cause toxicity and should be monitored with blood testing of vitamin a retinol. nourish the body with all foods and all nutrients slowly. the non-provitamin a carotenoids have also shown some benefit. lycopene, found in high amounts in guavas, watermelon, tomatoes, papaya, grapefruit, sweet red peppers, asparagus, purple cabbage, mangos, and carrots, slowed forced expiratory volume (fev) decline in former smokers [70] . vitamin d's importance with lung health cannot be understated. vitamin d deficiency, or even insufficiency, is linked to accelerated decline in lung function, increased inflammation, and reduced immunity in chronic lung diseases. vitamin d has a role in the regulation of inflammation, immunity, cellular proliferation, senescence, differentiation, and apoptosis. sufficient vitamin d levels are correlated with better asthma control, better immune response related to respiratory infections, and reduced severity of exacerbations with copd and asthma when exposed to inflammation-causing pathogenic activity [72] . vitamin d is obtained through sunlight on the skin (without sunscreen) and very few dietary sources. therefore, supplementation is generally recommended. higher vitamin d levels are shown to be protective in many lung disease states. sufficient levels improve treatment response with medications and reduce asthma severity [68] . with infectious diseases of the lung, higher vitamin d concentrations are shown to have a protective action [6] . vitamin d has a protective effect on lungs of smokers, and higher levels of vitamin d inhibit the pro-fibrotic phenotype of lung fibroblasts and epithelial cells. current data suggest an inverse association between serum vitamin d and lung cancer risk, and vitamin d deficiency at 16-20 weeks' gestation is associated with impaired lung function and asthma at 6 years of age [73] . lower levels of vitamin d are associated with an increased risk for respiratory infections, cystic fibrosis, chronic obstructive pulmonary disease, and interstitial lung disease [74] . vitamin c is an important antioxidant that helps decrease oxidative damage in the body, including in lung tissue. it is also essential for lipid metabolism. it is present in the airway surface liquid and creates an interface between the epithelial cells and the external environment. vitamin c is a cofactor in collagen synthesis, which can aid in repair of bronchial and alveolar tissue when damaged. it also provides beneficial control of lipid peroxidation of cellular membranes, including those surrounding as well as those within intracellular organelles. vitamin c has some of the best lung protective capabilities, according to current research. vitamin c may also diminish oxidative attack on nonlipid nuclear material and is an antioxidant component of plasma and extracellular fluids surrounding the lungs. it is an antioxidant that not only fights oxidative stress but also reduces oxidized vitamin e and glutathione, allowing them to become active as antioxidants again. vitamin c is antiinflammatory and is helpful in all inflammatory states of the lung, even allergies. there are many ways in which vitamin c, along with its antioxidant partners, glutathione, vitamin e, vitamin a, and plant-based phytonutrients, affects lung health. it is well established that increased levels of vitamin c in the diet improve health outcomes for smokers and their offspring, as smoking depletes vitamin c [75, 76] . vitamin c is also helpful in fighting infectious diseases such as respiratory infections and pneumonia, copd regardless of smoking status, asthma, and lung cancer [77] . specifically, in certain lung cancers, vitamin c, along with other nutrients such as lysine, proline, epigallocatechin gallate, and zinc, can inhibit the proliferation of certain carcinoma lines and induce apoptosis, as well as inhibit lung cancer metastasis [78] . even in lung transplants, vitamin c is helpful against oxidative stress by reducing glutathione and lowering lipid peroxidation, along with vitamins a and e [79, 80] . the literature suggests these benefits can be achieved at 500-3000 mg/day. check iron status before administering vitamin c supplementation as vitamin c doubles iron absorption from foods. vitamin e's primary role is as an antioxidant, breaking free radical chain damage and preventing peroxidation of lipid molecules. this vitamin also is promising with regard to beneficial effects on lung function preservation. oxidative stress and inflammation are key features in many lung diseases; therefore nutrients with antioxidant capacity can be useful. a few studies suggest that alpha-tocopherol found in sunflower and olive oils has a beneficial effect on fev (forced expiratory volume), whereas gamma-tocopherol found in canola, soybean, and corn oils has a negative effect on fev [81] . however, from these authors' perspective, this is likely due to the source and type of the oils, which can be inflammatory, rather than the form of vitamin e. for example, a recent study showed that gamma-tocopherol was protective in allergic asthma [82] . in addition, sufficient levels of vitamin e, in the alpha-tocopherol form, were found to reduce susceptibility of the elderly to acquiring pneumonia. some of the positive effects of vitamin e are synergistic with vitamin c [83] . phytonutrients have been found to have two effects with respect to lung disease: one is a symptom-improving pattern, and the other is a rate-reducing pattern [84] . idiopathic pulmonary fibrosis (ipf) is largely characterized by reduced antioxidant and increased inflammatory action. recent literature is showing the ability of certain flavonoids, in particular quercetin, to reduce inflammation and act as a strong antioxidant countering the pro-oxidant environment of ipf. quercetin is recognized as the most potent ros scavenger. taken together with glutathione, the impact is even greater, and it seems to help improve the antioxidant and inflammatory status more for those with ipf than non-diseased controls [85] . curcumin has been shown to slow or limit fibrosing in murine studies related to lung, liver, or kidney fibrosing [86] [87] [88] [89] . it has also been shown to attenuate metastatic melanoma in the lungs when delivered in a nanoparticle [90] . the potential for curcumin is interesting and hopeful. fisetin and fenugreek have also been studied as useful phytonutrients that help combat inflammation in lungs [91, 92] . fisetin is found in apples, strawberries, persimmons, cucumbers, and onions, among many other fruits and vegetables. fenugreek is a plant used frequently in south and central asian cooking, where both the seeds and leaves are used. there are now supplements available for both of these phytonutrients. this is a reminder to eat a primarily plantbased diet when combating inflammation and to broaden our palates to include healthy foods and ingredients from other cultures than our own. lastly, the powerful antioxidant cannabidiol (cbd), from the cannabis and closely related hemp plants, is a powerful shield against oxidative stress, prevalent in lung disease [93] . the research is not robust regarding lung function and minerals, and most has been done with regard to cystic fibrosis where bone density is associated with general nutritional status, including minerals. there have also been many studies trying to determine a correlation between mineral status and copd, where, again, the research shows that mineral status is not predictive but overall nutrient status may fall if not monitored. in contrast, one study in japan showed an inverse association between dietary calcium and the risk for copd [94] . in an nih-aarp diet and health study, magnesium, iron, selenium, zinc, and copper intakes, both dietary and supplemental, were studied with respect to lung cancer. mineral supplementation did not affect lung cancer risk, yet dietary intake of calcium, along with vitamin d, and iron reduced the risk, and dietary intake of magnesium increased risk [95] . boron has been shown to be protective against lung cancer, along with other nutrients, at levels of 3 mg/day [96] . there is some research showing that selenium is helpful, particularly for smokers, for improved fev. higher magnesium status is correlated to better fev but is not yet seen as an association. this may be due to magnesium's role as the vitamin d activator. there have been a few studies showing increased copper levels are related to decreased fev. some recent research has also shown that dietary zinc and iron are associated with reduced lung cancer, but the same was not seen with calcium, copper, magnesium, or selenium [97] . low mineral bone density is prevalent at a higher rate among cystic fibrosis patients, and therefore supplementation with vitamin d, vitamin k2, magnesium, calcium, and the trace minerals can be helpful [98] . alpha-lipoic acid (ala) is a powerful antioxidant endogenously produced in the human body from foods such as yeast, organ meats, spinach, broccoli, and potatoes and is both water-and fat-soluble. ala, along with n-acetyl cysteine (nac), glycine, and vitamin c, is an important precursor to glutathione, which is a powerful endogenous antioxidant and the primary antioxidant in the lungs. ala has been shown to be anti-inflammatory in lung tissue in those with acute lung injury, and the proposed action is via inhibition of the nf-kappab signaling pathway [99] . ala has also been shown to downregulate some cancerpromoting actions prevalent in lung cancer, likely by this same pathway [100] . it also may alleviate nicotine-induced lung oxidative stress [101] . n-acetyl cysteine (nac), another precursor to glutathione, is a powerful antioxidant on its own as well. in relation to the lungs, nac helps the clearance of mucus in the lungs by pulmonary cilia. this has been shown to be effective at 400-600 mg/day in divided doses [102] . there is significant research on nac and lung health, showing improvement with nearly all lung issues, including nearly 40 studies showing improvement for bronchitis [103] , infectious diseases by reducing the bacterial count [104] , smokers, and people with asthma and copd, through both its antioxidant effects and by reducing the viscosity of sputum and mucus. at an oral dose of 1800 mg/day, the mean glutathione concentration in lung tissue increased by 49% on one study [105] . there are additional studies showing improvement for those with copd, asthma, cystic fibrosis, pulmonary fibrosis, and symptoms related to allergies or other infections. the dose that has been studied and has been shown to be most useful is 600 mg twice daily and more effective if nebulized [106, 107] . both ala and nac supplementation should be accompanied by vitamin b6 and the complex of b vitamins to prevent an elevation in liver enzymes (. fig. 51 .3). there are several specialty labs that conduct micronutrient analysis and functional testing, such as genova diagnostics and spectracell. these tests can be useful for evaluating levels of individual nutrients as they function in the body, rather than just in serum, which is not an accurate indicator of tissue or functional status. patients suffering from copd, interstitial lung disease, and other diseases tend to have muscle and weight loss related to respiratory acidosis, and increasing weight and muscle mass helps with quality of life. respiratory acidosis occurs with co 2 buildup where the lungs are no longer able to effectively exchange o 2 and co 2 . nutritional supplementation should attempt to reduce metabolic co 2 production. fat metabolism produces less co 2 than carbohydrate metabolism, so emphasizing a higher fat, lower carbohydrate diet can be helpful [110] . in general, a high intake of omega-6 fatty acids is associated with poorer forced expiratory volume (fev) in patients with lung disease because of their pro-inflammatory nature. however, a complete fatty acid panel or a red blood cell membrane fatty acid test would reveal more details about the status of an individual's omega-6 pathway. certain omega-6s and the work of their corresponding metabolizing enzymes such as elongase and delta-5 or delta-6-desaturase may allow healthful omega-6s (linoleic (la), gamma-linolenic (gla), lipoxins [111] , prostaglandin 1 series metabolites) to flow down an anti-inflammatory pathway instead. important cofactors for this pathway are vitamin b2, vitamin b3, vitamin b5, vitamin b6, biotin, vitamin c, zinc, and magnesium. lipid metabolism dysregulation is understood to be part of the pathogenesis of idiopathic pulmonary fibrosis. in ipf, free fatty acids play a role in the proliferation of fibroblasts. certain fats, in particular palmitic acid, oleic acid, and linoleic acid, are elevated in the lungs of those with ipf, whereas stearic acid is low. stearic acid is found in meat, poultry, fish, grain products, and milk and milk products. the palmitic, oleic, and linoleic acids enhance the tgf-ß1-induced expression of α-smooth muscle actin (sma) and collagen type 1 in mrc-5 cells, which can lead to fibrosis. stearic acid inhibits the levels of these fibrosing cells. stearic acid also improves the thrombogenic and atherogenic risk factor profiles [112] . in one study on patients with copd, omega-3 fatty acids were found to reduce inflammation in bacterial infections of the lungs without suppressing the ability to clear the bacteria. those taking epa, dha, ala, and gla had improved exercise capacity and had lower risk of developing copd [113] . although results have been mixed over the years possibly due to doses used in studies, a recent 2018 prospective study showed that pufas (omega-3s) from fish help prevent lung cancer and can be part of treatment during lung cancer. in general, the strongest evidence for improved lung function and slowing decline is with the epa and dha forms of omega-3 fatty acids [114] . because of toxicity issues in fish, increasing quality supplements vs fish intake may be more prudent. protein is essential for all lung conditions, and lack of it can result in poorer pulmonary function, decreased exercise capacity, and increased risk exacerbations. since many lung diseases have oxidative stress as a characteristic, it can cause protein carbonylation which may negatively affect dna expression and lipid membranes. nutritional supplementation with added protein and healthy carbohydrates can increase body weight and muscle strength and improve quality of life. those with copd, interstitial lung diseases, and others that affect oxygen absorption and co 2 exhalation have greater levels of hypoxia and sometimes respiratory acidosis, which exacerbates the loss of muscle through oxidative stress and inflammation. supplementation of free essential amino acids versus complete proteins has been shown to help prevent muscle wasting among copd patients. muscle-building exercise is often prescribed for those with copd and interstitial lung diseases [115] . supplemental l-carnitine at 2-6 g/day for 1-2 weeks increased the capacity of copd patients to rehabilitate and build muscle and helped inspiratory muscle strength. carbohydrates should be monitored for sufficient but not excessive levels. more co 2 is produced with the utilization of carbs versus fats for energy. therefore, with gas exchange being an issue with most lung disorders, a slightly higher fat and lower carbohydrate diet may be indicated. it is worth mentioning fiber for a moment, as it is mostly delivered in carbohydrate-rich foods. there is evidence that consuming whole fruits and vegetables higher in dietary fiber is associated with reduced severity of asthma and copd [116] . a diet that derives its carbohydrates from vegetables and fruits rather than from processed carbohydrates such as grains, breads, pasta, or added sugars will deliver fewer carbohydrate grams. glutathione (gsh), a tripeptide composed of cysteine, glutamine, and glycine and produced from methionine, is in every cell in the body. it is the most powerful and abundant endogenous antioxidant in the airway epithelial lining and is responsible for detoxification of electrophilic compounds, the scavenging of free radicals, and modulation of cellular processes such as dna synthesis and repair, differentiation, apoptosis, and immune function [117] . it is also a heavy metal chelator. it is more effective than some other antioxidants because it is intracellular and extracellular. in isolated type ii alveolar epithelial cells, extracellular glutathione inhibits hyperoxia-induced injury, inhibits pro-inflammatory cytokine release, and promotes cell growth. it is obviously very important to maintaining lung function as this is the inflammatory process that begins lung cell or tissue damage, as mentioned above. the highest levels of glutathione concentrations in the body are in the lungs, liver, and brain. gsh depletion leads to activation of nf-kb (pro-inflammatory signaling) and increased pro-inflammatory gene transcription and cytokine release from histone deacetylase suppression in epithelial cells. total and reduced gsh concentrations are much lower in people with ards, pulmonary fibrosis, and hypersensitivity pneumonitis than observed in healthy adults. alterations in alveolar and lung gsh metabolism are widely recognized as a central feature of many inflammatory lung diseases such as idiopathic pulmonary fibrosis, acute respiratory distress syndrome, cystic fibrosis, and asthma [118] . we make glutathione in the body with cysteine and methionine, and it is difficult to take exogenously because digestion can destroy it. the precursors of cysteine (essential), glutamine, and glycine and cofactors (vitamin c, vitamin e, vitamins b1, b2, b6, and b12, folate (b9), minerals selenium, magnesium, and zinc, and alpha-lipoic acid, see below) are therefore recommended so that the body can produce it on its own. the two enzymes necessary to produce it, gamma-glutamylcysteine synthetase and glutathione synthetase, must also be functioning well. we also recycle glutathione if the precursors and cofactors are available. cysteine is usually the most rate-limiting precursor, and many people supplement with n-acetylcysteine to provide the body with this nutrient. although glutathione is produced in every cell of the body, the greatest production is in the liver, so focusing on liver health is important to maintain good glutathione production. production declines with age and with lung disease, as well as other conditions. there are very few foods containing glutathione; they are raw or very rare meat, especially liver, unpasteurized milk and other unpasteurized dairy products, and freshly picked fruits and vegetables, such as avocado and asparagus. however, as mentioned earlier, it may be destroyed during digestion. glutathione contains sulfur molecules, which may be why foods high in sulfur help to boost its natural production in the body. these foods include: 5 cruciferous vegetables, such as broccoli, cauliflower, brussels sprouts, and bok choy 5 allium vegetables, such as garlic and onions 5 eggs 5 nuts 5 legumes 5 lean protein, such as fish and chicken other foods and herbs that help to naturally boost glutathione levels include: 5 milk thistle (a liver-regenerating herb) 5 flaxseed 5 guso seaweed 5 whey glutathione is also negatively affected by insomnia. getting enough rest on a regular basis can help increase levels. addressing a drop in glutathione for lung health involves maintaining good levels of the precursors and cofactors mentioned above. a good way to bring in the less abundant amino acid cysteine is to take n-acetylcysteine (nac). doses of 400-600 mg were more effective than placebo in reducing symptoms [117] . supplemental selenium can also help with glutathione production. glutathione supplementation has also become more effective. there are several forms, from capsules to topical liposomal, which have shown good absorption. inhaled gsh has good research for use in cystic fibrosis (cf), chronic otitis media with effusion (ome), hiv seropositive individuals, idiopathic pulmonary fibrosis (ipf), and chronic rhinitis. it is not recommended for asthma due to significant side effects, and additional evidence is needed to determine if use with emphysema is recommended although theoretically it should be useful. it is also not recommended to use inhaled gsh during cancer chemotherapy treatment as it may interfere with the medication's actions. the mechanism of action of inhaled glutathione is limited to the upper airways and lungs and does not seem to affect serum levels. before considering inhaled gsh treatment, the patient should undergo urine sulfite sensitivity testing using a readily available special test strip called "em-quant 10013 sulfite test. " if positive, inhaled gsh should not be used as bronchoconstriction may occur. the recommended dose is 600-5000 mg per day, depending on response, and whether inhaled gsh is considered safe. efficacy should be tested using a baseline pulmonary function test and a follow-up test after a prescribed time later [119] (. fig. 51 .4, 7 box 51.1). there are also serum tests for glutathione levels. these cofactors are vitamin c; vitamin e; vitamins b1, b2, b6, and b12; folate (b9); minerals selenium, magnesium, and zinc; and alpha-lipoic acid. what do the glutathione cofactors do that makes them so important? 5 direct cysteine toward glutathione production and increase cellular uptake of cysteine 5 help form the glutathione molecule out of the three precursor amino acids 5 help recycle glutathione from its oxidized gssg form back to its reduced (active) gsh form 5 help maintain glutathione levels and keep the gssg-gsh ratio balanced 5 recycle each other, improving overall antioxidant activity 5 stimulate the activity of the whole glutathione enzymatic system co10 is a fat-soluble compound produced endogenously and also available through food and supplementation. it is required in the production of atp, is a powerful antioxidant, and therefore is helpful against oxidative stress, an important issue in lung disease. coq10 achieves its strong effects through a set of different mechanisms. it influences genes through its epigenetic effect to reduce inflammation, helps with the immune system, and even reduces aging by reducing systemic oxidative stress and mitochondrial aging [120] . lungs are the most susceptible organ to oxidant damage because they interact directly with oxygen. therefore, it makes sense that antioxidants, and those that especially affect the lungs, are helpful in tissue and lung cell preservation [121] . coq10 levels are significantly lower in those with copd and asthma with insignificant amounts of research on the levels of coq10 with other lung issues. it has been shown that supplementing patients with coq10 resulted in measurable benefits. in one study, patients with copd using steroids to reduce inflammation were able to reduce their steroid dosage when using coq10 [122] . in another study, benefits were shown for copd patients during exercise, measuring performance, tissue oxygenation, and heart rate at a low dosage of 90 mg/day [123] . the levels of coq10 in the blood have been shown to indicate the degree of systemic oxidative stress, which implies it could be used as a marker to assess copd [121] . several studies confirm the beneficial role of coq10 in decreasing oxidative stress, cardiovascular risk, and modulating inflammation during aging. dosage levels of 1200 mg/ day of coq10 have been shown to be therapeutic. however, in the reduced, more absorbable form, ubiquinol, 400 mg/ day, was shown to be as effective. there is a wide range of toxins and anti-nutrients that can significantly impact the respiratory system. this can occur through acute or chronic exposure to these agents. the earth's air is the source of oxygen, and the lungs provide access to that oxygen to support life. the human need for oxygen is precarious because humans can only survive for about 6 minutes without the precious gas. from about 1760 to sometime between 1820 and 1840 in europe and the united states, the ramp-up of new industrial revolution manufacturing processes opened a new era of increasing chemical and heavy metal atmospheric contamination. these pollutants can enter the body through breathing the polluted air. the more concentrated atmospheric pollutant densities cluster around areas of dense population. the dirty air provides a serious direct threat to those with respiratory diseases. an integrative and functional approach to assessing an individual with respiratory disease needs to include consideration of potential environmental contributors to the etiology of a condition. . table 51 .2 lists environmental pollutants that are known to promote lung pathology. a 2016 study published in the canadian respiratory journal examined exhaled fractional nitric oxide (feno)an indicator of inflammation in the lungs -in school children at three different schools located three different distances from a large steel mill [127] . steel processing is known to be a source of ambient iron, nickel, lead, copper, vanadium, and zinc. the study found statistically significant differences in feno between the two closer schools compared to the farthest school from the mill, indicating potential increased lung inflammation caused by heavy metals and/or air pollutants [127] . although acute metal toxicity is possible, chronic, low-grade exposure is more common and may contribute to respiratory complications and disease. an individual's ability to vitamin c -as an antioxidant, it assists glutathione in this function and has been shown scientifically to raise glutathione levels short term; it is recycled by glutathione from its oxidized state back to its active state, thus strengthening antioxidant defenses; vitamin c also recycles vitamin e and alpha-lipoic acid vitamin e -as an antioxidant it also assists glutathione in eliminating free radicals much like vitamin c; it is also required for the proper functioning of glutathione enzymes; it recycles vitamin c and alpha-lipoic acid b vitamins -vitamins b1 and b2 maintain glutathione and its enzymes in their active forms; vitamin b2 participates in the formation of a glutathione molecule; vitamin b6 influences glutathione synthesis indirectly as it is important for the proper functioning of amino acids including gsh precursors; vitamin b6 increases the amount of magnesium (a vital cofactor) that can enter cells; folate (b9) pushes cysteine toward glutathione production rather than homocysteine production; folate and vitamin b12 work together in amino acid metabolism and protein synthesis. you can read more vitamin b12 deficiency and its effect on immune health at 7 http://www. immunehealthscience. com/vitamin-b12-deficiency. html selenium -part of the enzyme glutathione peroxidase (gpx). glutathione peroxidases, also known as selenoproteins, are a family of antioxidant enzymes that speed up the reaction between glutathione and free radicals magnesium -required for the proper functioning of the enzyme gamma-glutamyl transpeptidase (ggt) involved in the synthesis of glutathione zinc -zinc deficiency reduces glutathione levels, especially in red blood cells. however, zinc levels above normal have pro-oxidant properties and reduce glutathione too alpha-lipoic acid -an antioxidant produced by the body; it has been scientifically proven to enhance and maintain glutathione levels by stimulating enzymes involved in the synthesis of glutathione; it also helps increase the cellular uptake of cysteine, the crucial building block of glutathione; in addition, alpha-lipoic acid recycles vitamins c and e based on data from ref. [124] eliminate these metals via detoxification in conjunction with gastrointestinal health and other factors can serve as important factors in whether or not these metals accumulate in the body. chronic arsenic exposure may be linked to respiratory complications [128] . chronic arsenic ingestion via contaminated drinking water may be connected to respiratory symptoms such as chronic cough, shortness of breath, blood in sputum, and abnormal breath sounds [129] . arsenic can also be ingested through foods such as rice and rice products, shellfish, and seaweeds, which have been shown to have high levels of inorganic arsenic (more toxic than organic arsenic found in fish) [120] . however, ingested inorganic arsenic is typically biotransformed and excreted in the urine [130] . that said, altered biotransformation has been observed depending on an individual's age, gender, nutritional status, and genetic polymorphisms responsible for the biotransformation of inorganic arsenic [130] . chronic inhalation versus ingestion may result in irritation of the throat and respiratory tract [131] . individuals most affected by arsenic exposure are children, nursing children, and infants of exposed pregnant mothers [132] . acute inhalation of cadmium may lead to dyspnea and coughing [133] . long-term exposure to cadmium has been reported to contribute to emphysema, dyspnea, and inflammation of the nose, pharynx, and larynx [123] . individuals most affected by cadmium toxicity are those with occupations with cadmium exposure, such as those who work in certain types of factories, women, due to higher intestinal absorption because of low iron stores, and residents of asia due to high intake of rice grown in contaminated soil [134] . the 2013 us national health and nutrition examination survey (nhanes) demonstrated an association between obstructive lung disease and serum lead and cadmium concentrations in the blood, where cadmium was shown to partially mediate the association between smoking and obstructive lung disease [135] . in the 2015 korean nhanes, obstructive lung function was found to be associated with higher serum blood levels of cadmium and lead as well [136] . the specific mechanism of heavy metal burden and its effects on respiratory health must be further investigated. although testing and treatment of heavy metal burden have its limitations, it is worth considering as heavy metal accumulation can wreak havoc on the body. an example of heavy metal testing that can be used in practice is urine provocation testing with a chelating agent, such as fda-approved dmsa. eliminating heavy metals from the body can be potentially harmful and requires careful monitoring and guidance by an experienced healthcare professional. air pollutants that are used as indicators of air quality are carbon monoxide, lead, nitrogen dioxide, ozone, particles, and sulfur dioxide [137] . air pollution has been shown to have adverse effects on human health [138] . a 2017 systematic review and meta-analysis done in china showed an association between respiratory disease and ambient nitrogen dioxide, which is increased through fuel combustion, industrial production, and fuel exhaust [129] . diesel exhaust particles in particular have been associated with an increase in cytokines such as il-2, il-6, and ige in nasal mucosa [139] . nitrogen dioxide in particular can potentially contribute to respiratory disease as it is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [138] . in another study performed in england, air concentration of nitrogen dioxide was significantly associated with respiratory hospital admissions [140] . other pollutants, such as fine particulate matter and ozone, have been shown to significantly affect respiratory function in copd patients [141] . increased ozone exposure has also been associated with increased airway inflammation and respiratory symptoms along with decreased respiratory function in children [142] . optimization of nutrition and antioxidant status is essential to combating the potential health effects of air pollutants. . [143] . it would be reasonable to assume having adequate stores and ability to utilize these nutrients may protect against other insults to the respiratory system as discussed in this section through their anti-inflammatory properties. acute and chronic exposure to certain chemicals can also pose a risk to respiratory health. obtaining a full occupational and social history when assessing individuals is important in order to identify any potential exposure to chemicals. one of the most well-known and common toxic chemical exposures that affects respiratory health is cigarette smoke. smoking cigarettes has been identified as a main cause of copd [144] . increased oxidative stress from inhaling cigarette smoke appears to activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)-1, il-6, and il-8 and tumor necrosis factor-ɑ (tnf-ɑ) [144] . it also appears to reduce anti-inflammatory cytokines such as il-10 [145] . electronic cigarettes, or e-cigs, have been increasing in popularity in recent years and are marketed as a better alternative to tobacco cigarettes. however, recent evidence suggests that the vapor and associated chemicals produced by e-cigs may be harmful to the respiratory system, although further research is needed to determine the mechanism [146, 147] . exposure to metalworking fluid aerosols has been associated with asthma, hypersensitivity pneumonitis, impaired lung function, allergic alveolitis, and sinusitis [148] . a 2015 review also identified an association between occupational exposure to pesticides and increased risk of asthma and chronic bronchitis [149] . there are many chemicals that are toxic when inhaled. for example, inhalation of chlorine is toxic to the lungs, where low doses can cause airway injury and high doses can cause both airway and alveolar injury [150] . these injuries can manifest as dyspnea, hypoxemia, pulmonary edema, and pneumonitis [150] . high doses of carbon dioxide, such as that released from dry ice, can also induce respiratory failure. stress may also play a role in respiratory health and the body's ability to combat insults imposed on the respiratory system. from a physiological standpoint, it is worth noting that acute stress via activation of the sympathetic nervous system increases ventilation through the production of glucocorticoids [139] . repeated acute stress may also affect growth and repair mechanisms [139] . chronic biological stress in the form of infections can also be inflammatory and negatively affect the immune system and may affect an individual's susceptibility to respiratory complications. see the chronic infections and respiratory health section on page # below for further information on this association. however, appropriate amounts of physical stress, such as in the form of exercise, can be beneficial to respiratory health. some research has indicated a benefit of aerobic exercise to respiratory muscle strength in cystic fibrosis patients [151] . chronic stress can be defined as recurrent acute stress or inability to moderate acute stress responses [139] . this can be in the form of physical or emotional stress. chronic stress and negative emotions such as depression, anxiety, and anger may be linked to endocrine and immune processes [152] . immunoglobulin e (ige) and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [139] . it has been hypothesized that increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [139] . another connection between emotions and respiratory health is acknowledged in east asian medicine, noting the association between the lungs and feelings of sadness, grief, and anxiety [153] (. table 51 .3). asthma is a chronic inflammatory lung disease, triggered by either an ige allergic reaction or nonallergic factors, and results in reversible airway obstruction and inflammation of the airway [11] . it is characterized by recurrent episodes of wheezing, breathlessness, coughing, and chest tightness [11] . severe asthma or asthma that is chronic or poorly controlled may lead to airway and lung remodeling that involves deposition of fibrotic tissue which leads to constriction of the bronchi [18] . although the exact mechanisms have not yet been identified, compromised nutritional status, such as deficiencies in selenium, zinc, and vitamins a, c, d, and e, has been connected to asthma [155] . the pathophysiology of asthma, nutrition considerations, genotypic characteristics, and lifestyle influences will be discussed in this section. there are numerous potential triggers to the development and/or exacerbation of asthma which can be summarized in 7 box 51.2. the various causes of asthma have led to the classification of several different subtypes and endotypes of asthma in hopes of choosing more targeted treatments. the pathophysiology of asthma is complex and not fully understood, due in part to its heterogeneous nature, which necessitates its organization into individual phenotypes and endotypes. this organization is important to be able to utilize targeted treatments by identifying the root causes of the symptoms. however, more research is needed to more clearly identify the specific pathological mechanisms of each phenotype and particular treatment responses [156] . two of the most common asthma phenotypes are allergic and nonallergic asthma [147] ; allergic is characterized by increased th2 immunity (th2 high) and nonallergic defined by varying mechanisms depending on the trigger (th2 low) [157] (see also 7 chap. 19) . allergic asthma involves the ingestion of typically harmless environmental triggers (listed in . table 51 .3) by antigenpresenting cells in the bronchi, which interact with immature helper t cells that, in turn, trigger an unwarranted allergic response [18] . this reaction occurs from repeated exposure to a trigger and is referred to as the type 1 hypersensitivity response [18] . this increased th2 immunity upregulates eosinophilic inflammation, tissue damage, airway hyperresponsiveness, and bronchoconstriction [113] . mast cell activation disorders, which is characterized by diseases and conditions related to mast cell mediators and the activation of mast cells, must also be considered when addressing allergic asthma [158] . in contrast, nonallergic asthma can be caused by other factors such as anxiety, exercise, stress, dry air, cold air, viruses, hyperventilation, smoke, or other irritants [11] . . inhaled cadmium (cd) is deposited in the alveoli where it is then absorbed into the bloodstream cd is transported to erythrocytes or bound to albumin, where it is then taken up by the liver to form a complex with metallothionein (mt) cd interferes with the absorption of zinc and competes for the same enzyme binding sites enzymatic activity of zinc-dependent enzymes reduces preferential binding of cd to mt can cause zinc deficiency altered biotransformation and excretion of ingested arsenic via contaminated water are linked to respiratory complications chronic inhalation of arsenic may result in irritation of respiratory tract diesel exhaust particles in particular have been associated with increase in cytokines such as il-2, il-6, and ige in nasal mucosa [139] nitrogen dioxide is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [102] rising pollen and mold counts [154] increasing ozone [154] chemicals increased oxidative stress from inhaling cigarette smoke may activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)-1, il-6, il-8, tumor necrosis factor-ɑ(tnf-ɑ) cigarette smoke may reduce anti-inflammatory cytokines such as il-10 [145] stress repeated acute stress may also affect growth and repair mechanisms [139] ige and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [139] increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [139] ( individuals suffering from nonallergic asthma will tend to be less responsive to th2-targeted treatments due to a differing immune response at play [157] . some of the additional proposed phenotypes are eosinophilic, exacerbation-prone, exercise-induced, fixed obstruction/airflow limitation, poorly steroid-responsive, and adult-onset obesity-related [159] . several of the proposed endotypes are summarized in . the american partnership for eosinophilic disorders defines eosinophilic asthma as a type of asthma characterized by especially high levels of eosinophils, more commonly developed later in adulthood, although may occur in some children [160] . many with eosinophilic asthma do not have underlying allergies or history of allergic conditions such as eczema, food allergy, and hay fever, which are thought to be seen more in people with allergic asthma [160] . in contrast to allergic asthma, the cause of eosinophilic asthma is still unknown. histamine intolerance must also be considered in assessing the root cause of asthma. ingesting histamine-rich foods and beverages such as bananas, grapes, strawberries, citrus fruits, tomatoes, nuts, chocolate, pineapples, fish, spinach, fermented foods, and beverages [161] has been shown to provoke a histamine response that may result in asthma exacerbations, among many other potential signs and symptoms [162] . disruptions in redox, or oxidation/reduction, reactions in addition to hindered antioxidant defense have been . usually not responsive to glucocorticoids [159] based on data from ref. [157] found to be a risk factor for asthma severity and development [163] . the levels of glutathione, one of the lung's most predominant antioxidants in both reduced and unreduced forms, are thought to be important for lung homeostasis and tied to asthma [163] . more research is needed to determine the exact differences in the pathophysiologies of the various subtypes of asthma in order to develop more targeted treatments. minerals such as zinc, selenium, copper, and manganese may serve as cofactors to major enzymes with antioxidant activity in the lung, such as superoxide dismutase, catalase, and glutathione peroxidase [164] . asthma has been associated with decreased activity of these enzymes [165] . low selenium intake has been associated with multiple chronic diseases including asthma [163] . selenium serves as a cofactor to glutathione peroxidase, an enzyme with antioxidant activity in the lung that is responsible for maintaining gsh/gssg redox balance [163] . imbalance between oxidants and antioxidants seems to serve an important role in asthma. levels of nonenzymatic antioxidants glutathione, ascorbic acid, alpha-tocopherol, lycopene, and beta-carotene, in addition to antioxidant enzymes superoxide dismutase (sod) and glutathione peroxidase, were significantly lower in asthmatic children compared to healthy controls [165] . the amino acids glycine and glutamine, which are important in glutathione synthesis, were also found to be significantly lower in children with asthma [165] . dha has also been found to be abundant in airway mucosa, where it is decreased in individuals with asthma and cystic fibrosis [166] . magnesium is known to elicit the relaxation of bronchial smooth muscle, decrease responsiveness to histamine, have an anti-inflammatory effect, and decrease the susceptibility of animals to developing anaphylactic reactions [25] . it is estimated that two-thirds of the population in the western world is not consuming the recommended daily allowance of magnesium [167] . magnesium can be used intravenously as an effective treatment of acute asthma attacks. one double-blind controlled trial that used 1.2 g of magnesium sulfate when patients did not respond to treatment with beta-agonists found decreased likelihood of hospitalization and improved lung function [168] . magnesium sulfate as an adjunct therapy with bronchodilators and steroids has also been shown to have a benefit in children with moderate to severe asthma [168] . although the exact mechanism is not yet known, magnesium is thought to increase glutathione concentrations in the lung [169] . more research is needed to determine additional associations between specific nutrients and asthma. however, optimization of the nutrients discussed in this section has the potential to reduce the severity and/or progression of asthma (. fig. 51.5) . asthma has a strong genetic component, with more than 100 genes associated with it in varying degrees across many populations [18] . more recent potential genetic associations include filaggrin, which encodes for the epithelial barrier; ormdl3, which encodes transmembrane protein; beta-2 adrenergic receptor gene, expressed throughout smooth muscle and epithelial cells of the lung; and interleukin-4 receptor gene, which has a variant associated with elevated ige [171] . a 2011 systematic review and meta-analysis showed that deficiencies in selenium, zinc, vitamins a, c, d, and e, and low fruit and vegetable intake could be associated with the development of asthma [155] . although this data is tenuous due to lack of randomized controlled trials, it does give some indication of the relationship between nutrition status and dietary patterns with respect to asthma development. more research needs to be done to isolate the impact of these nutrients and dietary patterns on asthma prevention and development. a 2015 review conducted by berthon and wood noted the protective effects of the mediterranean diet for allergic respiratory diseases as evidenced by epidemiological studies. this diet emphasizes minimally processed plant foods in the form of fruit, vegetables, cereals, beans, breads, nuts, seeds, and olive oil and low to moderate intake of dairy, poultry, fish, and wine, as well as low intake of red meat [172] . this association was the strongest in children, where the mediterranean diet had a protective effect on atopy, wheezing, and asthma symptoms [172] . however, there is less data available to support this pattern in adults. the same review noted an association between the "western" diet, which emphasizes refined grains, red and cured meats, french fries, sweets and desserts, and highfat dairy products and increased risk of asthma in children [172] . a meta-analysis and systematic review done in 2014 showed a reduction of risk in childhood wheezing with high fruit and vegetable intake and also showed negative association between fruit and vegetable intake and asthma risk in adults and children [173] . in contrast, food allergy has been especially linked with allergic asthma in children [161] . a study examining food allergy in asthmatic children identified higher serum levels of ige in asthmatic children compared to healthy controls, where all asthmatic children in the study were also identified as having a positive skin prick test (spt) to various food allergens [174] . a study done on 322 children under the age of 1 diagnosed with asthma, with or without allergic rhinitis, was placed on a meat-based formula of carrots, beef, broccoli, and apricots for 6 weeks. it was found that 61% had nearly complete resolution of symptoms [25] . this same study also found that the most common food triggers were milk, egg, chocolate, soy, legumes, and grains [25] . while food allergy as a cause of asthma is more common in children, hidden food allergy has been reported to be the root cause of asthma in around 40% of adults [25] . improvement in respiratory symptoms was also seen in a small study of adults given an antigen-free elemental diet in a hospital setting [25] . removal of food triggers has also been linked to improvement in exercise-induced asthma [25] . identifying food allergies can be a complicated process because many of the testing methodologies such as skin prick tests (spts) and blood tests can yield false-positive results for up to 50-60% of cases, according to the food allergy research & education organization [175] . a food elimination diet and/or oral food challenge can be a powerful tool in determining food allergy specific to asthma symptoms, where a dietitian or nutritionist in conjunction with physician and/or allergist can serve an important role through this process to support the individual. oxidative stress may play a key role in the development of asthma, which can also be true for the development of chronic diseases such as cardiovascular disease, diabetes, and cancer [117] . it has been shown that obesity may be a risk factor for people with and without allergy and may worsen pre-existing asthma [159] . individuals with asthma are twice as likely to have gastroesophageal reflux disease (gerd) than people who do not have asthma, especially those resistant to treatment [159] . celiac disease and asthma have also been linked. an italian cohort study was done that showed a significant association between treated asthma and celiac disease, where antibiotic exposure in the first year of life was controlled for and not found to contribute to this association [176] . it has also been found that individuals with celiac disease following a glutenfree diet experienced improvement in asthma symptoms [25] . it is well-known that toxic exposure to particulate matter, airborne pollutants, or cigarette smoke can trigger asthma symptoms [165] . more specifically, a dose-dependent relationship between cigarette smoke exposure and rates of asthma has been shown [165] . traffic density and asthma exacerbations have also been clearly demonstrated [165] . certain medications may also serve as triggers to asthma. aspirin-exacerbated respiratory disease (aerd) is considered another asthma subtype caused by nonsteroidal anti-inflammatory drugs (nsaids) and is characterized by asthma, chronic rhinosinusitis, and acute respiratory reactions [159] . in addition, overuse of antibiotics in childhood has been linked to asthma [18] , indicating a connection between the microbiome and asthma development. allergic bronchopulmonary mycosis (abpm) noted in . table 51 .4 is caused by a hypersensitivity reaction to fungal colonization of the airways [159] . this is typically caused by the fungus aspergillus fumigatus. without treatment, this may lead to fixed airflow obstruction and bronchiectasis [159] . the progression of asthma is complex and multifaceted, from preconception through childhood and adulthood. research suggests that early life events are largely predictive for regulatory mechanisms within the pulmonary immune system [177] . for example, prenatal exposure to a farming environment, one rich in microbial compounds, is thought to influence innate immune patterning in the mother which may affect the development of the neonatal immune system [177] . this influence in immune patterning can be seen through higher expression of toll-like receptors 2 and 4 and cd14 on peripheral blood cells, which implies possible desensitization to allergens in children [178] . t regulatory cells, which serve an important role in immune regulation and are thought to play an important role in asthma by suppressing the th2 inflammatory response to harmless air particles, have been shown to be impaired in the cord blood of neonates at hereditary risk for allergy [179] . in the 2017 study performed by singh et al. looking at serum ige and cutaneous sensitivity to food allergens in asthmatic children here was a negative correlation of total ige and duration of breastfeeding, indicating a connection between breastfeeding and the immune response [174] . additionally, reduced maternal intake of vitamins d and e and zinc during pregnancy has been associated with increased asthma symptoms in children [180, 181] . vitamin d has been associated with the maintenance and/or development of the t regulatory cells stated earlier in mice; however more research is needed to determine a definitive association in humans [177] . a clinical trial performed on non-smoking asthmatic patients showed higher vitamin d levels were associated with greater lung function; furthermore, supplementation with vitamin d showed improved treatment response to glucocorticoids [182] . vitamin d may also directly increase the antiinflammatory cytokine, interleukin (il)-10 and also enhance steroid-induced il-10 production (see . fig. 51.6) [177] . more research is needed to determine the exact mechanism of vitamin d in asthma and respiratory disease. beta-agonists, combined with corticosteroids, serve as the primary conventional therapy [183] . typically, a short-acting beta-agonist will first be prescribed to manage symptoms as needed, where low-dose inhaled corticosteroids may also be prescribed [156] . if symptoms persist, it is recommended to evaluate problems such as adherence to use, inhaler technique, or persistent allergen exposure and comorbidities [156] . once these are ruled out, the step-up treatment is a combination of an inhaled corticosteroid with a long-acting beta-agonist [156] . a summary of other conventional treatments and their mechanisms can be found in . table 51 .5 below. unfortunately, conventional methods for the treatment of asthma may have harmful side effects. for example, the use of . fig. 51.6 effect of asthma treatments on regulatory pathways. (reprinted from lloyd and hawrylowicz [177] . with permission from elsevier) systemic glucocorticoids may lead to immunosuppression, cataracts, and osteoporosis, where long-acting beta-agonists have the potential of increasing asthma exacerbation risk and death [25] . beta-agonist desensitization is thought to be one of the reasons for increasing asthma exacerbation risk and death [184] . related to several subtypes of asthma and their differing pathophysiologies, it is important to first determine the subtype before deciding on treatment. for example, in an individual with allergic asthma, this could be a potentially simple fix once the allergen that exacerbates symptoms is identified. a more conventional approach may involve starting the individual on an inhaled corticosteroid or an ige antagonist (i.e., omalizumab) [159] , rather than identifying the root cause of the patient's symptoms. while medications may be warranted until the trigger is identified, finding the underlying causes may not be common practice in many conventional settings. in contrast, the ifmnt assessment takes a much deeper dive into identifying triggers and any nutrient insufficiencies, inflammation or immune dysregulation, biochemical individuality, lifestyle, energy dysfunction, toxic load, sleep, and stress issues are taken into account. with this information, the practitioner can make more targeted dietary, lifestyle, and supplement recommendations to obtain sustained resolution of symptoms by treating the root cause (. table 51 a 26-year-old female presented with a complaint of reactive airway disease, which was diagnosed as asthma and had been prescribed inhalers. she reported that she felt like she had difficulty breathing most of her life, especially when exercising. however, her condition was not severe enough to seek help until she was 25 years of age. she reported a lot of stress during this time related to applying for a postgraduate training position. she also reported 1 year prior to diagnosis developing new allergic symptoms. her past medical history was significant for conditions related to airways, including chronic sinus infections, strep throat, bronchitis, and recurrent pneumonia. she could not remember the last time she felt well but assumed it was sometime as a young child. her nutrition and health goals were to breathe better and to not have to rely on inhalers. the following data was collected on her initial visit. . methyl xanthine found in tea used less commonly due to side effects relaxes airways due to inhibition of phosphodiesterases; acts as a functional antagonist in airway smooth muscle [171] based on data from ref. [18] . table 51 .6 summary of an integrative and functional medical nutrition therapy assessment adequacy of nutrient-dense foods to begin to assess nutritional status organic or nonorganic to assess toxic load and nutrient intake food preparation and processing to assess nutrient content and identify potential contaminants (e.g., plastic endocrine disruptors) assess food sensitivities or intolerances to identify potential triggers microbiome status: assess comprehensive digestive stool analysis for microbiology and fermented food intake; history of antibiotics or microbiota agonists (medications, toxins, stress, etc.) toxin intake via plastics or inhalation and skin absorption which may affect immune response assess flavonoids intake as they are antioxidant and anti-inflammatory compounds with mast cell inhibitory action; adequacy may reduce airway reactivity consider celiac disease and gluten intake as potential inflammatory antigens mineral assess and restore zinc, selenium, magnesium, manganese, iron, and iodine status to normal reference. caution to not supplement or intake of food sources higher than reference antioxidants assess and restore antioxidant balance; vitamins a, c, d, and e and glutathione assess quercetin intake (leafy vegetables, broccoli, red onions, peppers, apples, grapes, black and green tea, red wine) as it may act as mast-cell stabilizing agent inhibiting release of histamine, tnf-alpha release, formation of prostaglandin d2, reducing interleukin production consider supplementation of quercetin if quercetin intake is low [185] protein status assess and restore to support connective tissue and immune status ensure adequate glutamine and glycine intake oils/lipid/fatty acids assess fatty acid balance as dha important in lung tissue integrity assess adequate serum cholesterol and fat intake to support lipid bilayer important for cellular function in lung (epithelial cells, surfactant production, etc.) methylation assess methylation status and detoxification capacity of toxins related to asthma exacerbation; important assessment biomarkers suggested: mcv/mch, homocysteine, methylmalonic acid, rbc folate, genomic methylation snps inflammation/immune dysregulation assess asthma biomarkers to help identify root cause (see . fig. 51 extreme exhaustion, depression, add, anxiety (accompanied by panic attacks), constipation, pain in legs, neuropathy in feet (numbness and tingling), rapid heartbeat, and a very severe rash on feet known as chilblains. 5 utis -recurrent as a child. 5 poor immune function (frequent infections). 5 antibiotic use (very frequent from childhood into adulthood). 5 sinus infections, strep throat, and bronchitis -she had recurrent sinus infections and strep throat about once a year every year and often this would lead to bronchitis, she could not remember if she had these issues before middle school. 5 depression, anxiety, add. 5 acne. 5 peptic ulcers. 5 yeast infections -multiple throughout college. 5 eczema. 5 two recent episodes of pneumonia the last episode resulted in her asthma diagnosis. 5 asthma. evaluate exposure to fungus to identify allergic bronchopulmonary mycosis assess individual's medication history, considering short-and long-term use of conventional treatments evaluate exposure to particulate matter, airborne pollutants, cigarette smoke, or toxic metals such as cadmium and arsenic sleep and stress assess sleep adequacy (7-9 hours with 5-hour rem sleep) and quality (good sleep hygiene with little light/ sound/emf disturbance) to support detoxification of toxins that may worsen respiratory status and aid in repair of damaged lung tissue . periostin plays a role in the pathogenesis of allergic diseases, including asthma, as it is associated as a downstream molecule of the cytokine, il-13. periostin is used as a biomarker for type 2 immunity and can be used to determine the potential effectiveness of medications used to treat asthma, such as anti-ige antibodies and anti-il-13 antibodies. asthmatic patients with high serum periostin tend to be aspirin intolerant, eosinophilic, late asthma onset, and have a high nitric oxide fraction. high periostin can also indicate a reduced response to inhaled corticosteroids [186, 224] . periostin in the right panel is stained brown and is localized in the thickened basement membrane in asthmatic patients. (reprinted from izuhara et al. [186] . with permission from the korean academy of asthma, allergy and clinical immunology) for several weeks, led to being immobile for almost 2 weeks 5 8th-12th grade: was often sick (strep, sinus infections, bronchitis); described it as being constantly sick from fall through winter every year; also developed eating disorder during this time; had severe menstrual cramps (induced vomiting) accompanied by acne, which led to being put on birth control at age 17 as a precursor to accutane (never prescribed); chronic constipation starting during this time university 5 freshman -sophomore year: eating disorder was most severe during this time. 5 first semester of freshman year: developed digestion issues, after eating certain foods (especially mexican or salsas), stomach would become distended, experienced pain, and often would result in vomiting. pain so severe during finals week she was admitted to er with no diagnosis. ct scan revealed possible peptic ulcers. 5 junior-senior year: depression, anxiety, and inability to focus were most severe during this time which resulted in missing a lot of class and struggling as a student; suffered multiple panic attacks; gained a lot of weight (from 120 to 180 lb); end of senior year became engaged to be married -moved to dallas, tx. 5 lived in dallas for 6 months, continued to experience depression and anxiety and weight gain, and moved back to home state 5 initially started running (~2 miles a day) and experiencing inability to breathe, diagnosed with pneumonia, prescribed inhaler to help with running; other symptoms: eczema around the eyes and neck (after running outside), pain in calves, numbness and poor circulation in feet (pulse not detected by several health professionals), and development of chilblain rash (very painful, itching, lasts about 3-4 weeks from development to resolution); increased running -ran a half-marathon. visited pcp and several specialists for help with chilblain rash with no resolution or diagnosis; lost a lot of weight (from 170 to 140 lb). 5 ongoing increased depression, anxiety, and inability to focus; pcp rx cymbalta (depression and anxiety); cymbalta discontinued after ~2 months (did not tolerate side effects), continued psychological therapy for several months; chilblain rash continued. stopped running long distances. gained weight back (from 140 to 170 lb); subsequently saw blog for integrative rd and followed suggestion to eliminate gluten and focusing on whole foods diet. 5 chilblains and eczema began to resolve while following integrative rd recommendations of gluten-free diet with some improvements. however, difficulty breathing got worse, and diagnosis of asthma was made with fast-acting inhaler used for exercise; as time progressed, breathing continued to worsen, led to daily inhaler use. weight at this time is still at around 170 lb. high dairy diet (consumed dairy products at most meals and snacks), consumed three smaller meals with three snacks in between 5 meals and snacks balanced with protein, fat, and carbs, with carbs coming from fruits and vegetables and fat mainly from full fat cheese, greek yogurt, and butter 5 mostly nonorganic produce and commercially raised meats digestion, assimilation, and elimination 5 hx of peptic ulcers and chronic constipation (bm ~1-2 times a month) 5 bms currently at about 2 × per week on encounter utilization, cellular, and molecular (mapdom) 5 hx of likely gluten sensitivity. 5 presented symptoms of possible dairy sensitivity (bloating, acne, asthma). 5 evidence for compromised intestinal barrier. 5 minerals: infrequent bms could indicate low fiber or low mineral status (mg); when bms do occur, they are hard and dry (low mg); severe menstrual cramps (low mg); labs showed low k and na, on yaz birth control (low zinc and low b vitamins). 5 antioxidants: consumed adequate fruits and vegetables each day. 5 protein: has some evidence of poor/slowed wound healing as evidenced by sore on leg that has not completely healed after a year; cuts that take months to heal. 5 d and fat-soluble a, e, and k vitamins -hx of poor immune function (low d), vdr +/+ (low d and possibly a). 5 oils/fatty acids: high omega-6/omega-3 ratio, higher intake of damaged fats, very low intake of omega-3. 5 methylation: symptoms of depression, anxiety, add combined with mthfr c677t snp and on yaz (low b6 and folate). 5 eicosanoid fatty acids status -suspect issues with pge1 series pathway to control inflammation due to following signs and symptoms: allergies, autoimmune condition (asthma), peptic ulcers, eczema, and severe menstrual cramps 5 immune function -suspect gut dysbiosis due to following s&s: poor immune function, yeast infections, hx frequent antibiotic use, cyst, and constipation body composition 5 genetic makeup that indicated prone to gluten and dairy sensitivity, low vitamin d status, and impairment in methylation 5 broad spectrum probiotic + fermented foods 5 bioactive b complex (includes 50 mg p5p b6 and 800 mcg 5 thf) 5 260 mg gla evening primrose oil and zinc 3. aim to eat three larger meals a day, allowing space in between of ~ 5 hours; increase omega-3 intake by adding in small fatty fish, such as sardines or anchovies, once per week and taking fish oil; decrease omega-6 intake, switch from conventionally raised meats to organic, pasture-raised; and replace fat in diet from dairy with coconut sources, more nuts, and avocados. 5 patient presented ~6 months after the initial visit (september 2015). her breathing had improved immensely. she was able to stop taking her albuterol inhaler before exercise, recently stopped daily inhaler. 5 after dairy-free diet for 3 months, reintroduced dairy (cheese, butter, yogurt). asthmatic symptoms returned about 2-3 days after the addition of each. noticed the more dairy consumed, the worse her symptoms became. 5 at time of appointment, diet whole foods, gluten-free, and dairy-free. weight loss 10 lb within the first month of going dairy-free, continued to lose some weight. when reintroduced dairy symptoms of bloating and increase in weight, which resolved returning to dairyfree diet. 5 bms are regular now at ~ 2 × daily. this patient case followed some common patterns in the development of chronic disease and the comorbidities that are common, especially autoimmune conditions like asthma. the first is the genetic susceptibility of the individual; several snps are prone to dairy sensitivity. second, significant evidence for gut dysbiosis, promoted compromised gut barrier, can contribute to the development of dairy sensitivity. third is the exposure to dairy protein antigen. diet history evidenced trigger for asthmatic condition. additionally, inflammation, immune dysfunction, and methylation issues present. signs and symptoms significant for decrease in pge1 series anti-inflammatory pathways. low dietary omega-3s potential contributor to asthma. immune dysfunction evidenced by extensive history of infection-antibiotic use. genomic snp mthfr c667t gene, which indicated a greater need for folate. the use of yaz birth control and symptoms of depression, anxiety, and add known further to deplete b6 and folate. the diet and supplements recommended targeted control of inflammation, restore gut ecology, promote proper methylation, and replete nutrient insufficiencies. results from 6-month follow-up showed successful outcome in helping improve breathing and wean her off of inhalers. this case is an example of the ifmnt approach able to address the complexity of the whole patient story and bring the metabolic priorities into a manageable intervention program for the individual. one study found that the composition of the nasopharyngeal microbiota in children was linked to the frequency of upper respiratory tract infections and acute sinusitis [189] . a study that intranasally inoculated mice with lactobacillus fermentum reduced the amount of s. pneumoniae in the respiratory tract and increased the number of macrophages in the lung and lymphocytes in the trachea [189] . these findings may indicate a benefit of manipulating the upper respiratory tract microbiota with orally or nasally administered probiotics in the prevention and/or treatment of upper respiratory tract infections. allergic bronchopulmonary mycosis (abpm) is caused by a hypersensitivity reaction to fungal colonization of the airways. this is typically caused by the fungus aspergillus fumigatus. without treatment this may lead to fixed airflow obstruction and bronchiectasis [159] . guillain-barre syndrome (gbs) is a rare neurological disorder in which the body's immune system attacks the peripheral nervous system, known as the network of nerves located outside of the brain and spinal cord [190] . it is often preceded by a bacterial or viral infection. there are several potential mechanisms in which these infections trigger gbs. if an individual contracts a campylobacter jejuni bacterial infection, antibodies made to fight this infection can attack axons in motor nerves, which can potentially cause paralysis and respiratory failure [190] . campylobacter can be ingested via contaminated food or other exposures [190] . pérez-guzmán 2005 states that hypocholesterolemia is common among tuberculosis patients and suggests that cholesterol should be used as a complementary measure in antitubercular treatment [8] . alpha-1 antitrypsin (a1at) deficiency is an underrecognized disease in the united states, with around documented 100,000 people suffering from it, according to the alpha-1 foundation. this deficiency is inherited through autosomal codominant transmission, meaning affected individuals have inherited an abnormal aat gene from each parent [191] . individuals with this deficient allele present with aat levels at less than 35% to low-end normal levels [191] . however, it is also possible for individuals with a variant of this allele to be asymptomatic given different environmental conditions or lifestyle factors, such as refraining from smoking to reduce lung disease development risk [191] (7 box 51.4). a1at deficiency most often manifests in the lungs as chronic obstructive pulmonary disease (copd) (i.e., emphysema or bronchiectasis or "genetic copd"). a1at deficiency is often undiagnosed because people with genetic copd experience the same symptoms as people with copd, such as [191] : 5 shortness of breath 5 wheezing the only way you will know for sure if you have genetic copd due to alpha-1 is to get tested. ________ a1at deficiency can manifest in the liver as cirrhosis. symptoms related to the liver 5 unexplained liver disease or elevated liver enzymes 5 eyes and skin turning yellow (jaundice) 5 swelling of the abdomen (ascites) or legs 5 vomiting blood (from enlarged veins in the esophagus or stomach) a1at expresses sometimes in the skin as panniculitis [191] . panniculitis typically appears as raised red spots on the skin, which may break down and give off an oily discharge. while panniculitis spots (called nodules) may appear anywhere on the body, the most common places are the thighs, buttocks, and areas subject to injury or pressure. normal genotype m m 5 most common abnormal genes are called s and z 5 abnormal variant combinations: 5 zz (highest risk) 5 sz (lower risk increasing if smoker, inhalant pollutants) 5 mz (lower risk of carrying an a1at gene variant; considered "carriers") 5 alpha-1 is the most commonly known genetic risk factor for emphysema 5 up to 3% of all people diagnosed with copd may have undetected alpha-1 5 alpha-1 can also lead to liver disease. the most serious liver diseases are cirrhosis and liver cancer 5 the world health organization (who), american thoracic society (ats), and the european respiratory society (ers) recommend that everyone with copd be tested for alpha-1 alpha-1 is a progressive disease that benefits from early detection. it can cause serious lung diseases, such as copd and emphysema when undiagnosed. in some cases, alpha-1 can also cause liver disease [225] symptoms related to the lung [225] : 5 shortness of breath 5 wheezing 5 chronic bronchitis, which is cough and sputum (phlegm) production that lasts for a long time 5 recurring chest colds 5 less exercise tolerance 5 year-round allergies 5 bronchiectasis the alpha-1 antitrypsin (a1at) protein protects the body, especially fragile lung tissues, from the damaging effects of a powerful enzyme called neutrophil elastase that is released from white blood cells. in a1at deficiency, a genetic mutation reduces levels of the protective protein in the bloodstream. a1at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. smoking, which can inhibit what little a1at protein an affected person does have, increases the risk of lung disease. alpha-1 antitrypsin deficiency is completely determined by mutations in a single gene. the severity of symptoms is mostly a function of which mutations a person has and how many copies. however, smoking can greatly increase the risk of lung disease due to aat mutations. 23andme reports data only for the pi * m, pi * s, and pi * z versions of the gene that encodes aat. keep in mind that it is possible to have another mutation that causes this condition that is not included in this report [192] . a1at deficiency is a genetic disorder that reduces circulating levels of a protein that protects the lungs by trapping a1at in the liver, where the protein is produced, and prevents a1at from entering circulation. a1at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. when a disease-causing mutation is fairly common, as the pi * s and pi * z mutations are in europeans, it suggests that the mutation actually conferred an evolutionary advantage at one time. some researchers have suggested that several thousand years ago when the pi * z and pi * s mutations first arose, these versions of the gene for a1at gave people a survival advantage by creating an environment in their lungs that helped fight off infections. the scientists theorize that the antimicrobial benefits of the aat mutations outweighed the cost of an increased risk of copd and liver disease in the era before antibiotics were available [193] . in contrast to lung disease, manifestation of liver disease related to a1at can be referred to as a "toxic gain of function, " due to accumulation of mutant a1at protein rather than protease deficiency within the liver [144] . when taken together, fibrotic lung diseases are the leading cause of mortality worldwide. under the umbrella of interstitial lung disease (ild), pulmonary fibrosis (pf) is the most common. any ild that involves scarring of the lungs falls in the pulmonary fibrosis category. pulmonary fibrosis is the scarring of lungs, which destroys tissue over time, making it impossible to transfer oxygen from inhaled air into the bloodstream. there are more than 200 different diseases under the pulmonary fibrosis umbrella. because pf is often misdiagnosed or goes undiagnosed, there is not an accurate count of those with these diseases. however, it is estimated that as many as 1 in 200 adults over 60, or 200,000 people in the united states, are affected [184] . there are more than 50,000 deaths from ipf every year in the united states. more people die each year from idiopathic pulmonary fibrosis than from breast cancer [194] . there are other forms of interstitial lung disease including the newly identified pleuroparenchymal fibroelastosis, cryptogenic organizing pneumonia (cop), desquamative interstitial pneumonitis, nonspecific interstitial pneumonitis, hypersensitivity pneumonitis, acute interstitial pneumonitis, interstitial pneumonia, sarcoidosis, and asbestosis [195] . symptoms include cough and dyspnea, restrictive pulmonary function tests with impaired gas exchange, and progressive lung scarring. the disease progresses with an initiation of inflammation. fibrosing starts with the action of transforming growth factor-β (tgf-β)-dependent differentiation of fibroblasts to myofibroblasts, which then express α-sma (smooth muscle actin) [196] . after the tgf-β-dependent differentiation of fibroblasts to myofibroblasts, which express α-sma, there is sustained, excessive deposition of collagen by the myofibroblasts in the lung interstitium leading to the progressive lung damage in patients with pf [185] . research published in 2011 supported the idea that dysfunctional type ii aecs (alveolar epithelial cells) facilitate lung fibrosis through increased susceptibility to injury, leading to excessive and dysregulated remodeling [197] . the disease seems to progress in steps, and inflammation is not typically present continuously, except during certain periodic episodes of deterioration (. fig. 51.8 ). there are five main categories of pf causes: drug-induced, radiation-induced, environmental, autoimmune, and occupational. of these five, four have identifiable causes. some of the autoimmune diseases that can lead to pf are rheumatoid arthritis, scleroderma, sjogren's syndrome, polymyositis, dermatomyositis, and antisynthetase syndrome. idiopathic pulmonary fibrosis (ilp) is defined as pf with an unknown cause, including a genetic cause for some families [see . fig. 51.9 ]. the symptoms of ilp are a dry, hacking cough, shortness of breath, fatigue, chest discomfort, loss of appetite, and unexplained weight loss, all caused by the fibrosing of the lungs. diagnosis can be difficult, and pf is often misdiagnosed as copd or other more common lung diseases. in addition, in the recent past, path to a true diagnosis was invasive. since damage to the lungs, even through a diagnostic biopsy, can trigger further lung damage or a period of fibrosis, many physicians or patients are cautious with a biopsy approach to diagnosis. since the current treatments are limited, one must evaluate whether defining the exact form of pf is necessary for treatment and follow-up. difficulty breathing, crackling sounds while breathing, and low oxygen levels are the first indicators. clubbed fingernails may also be a symptom. high-resolution ct scans are performed, which can show scarring. the pulmonologist will ask many questions and order more blood tests to try to distinguish between the 200 forms of pf. the future is pointing to molecular endotyping as a more accurate way to diagnose. molecular endotyping includes genetic, metabolic, transcriptional, and environmental factors to help determine the pathophysiology [199] . genetic research has been progressing for a couple decades with illuminating results. there are more than a dozen genetic variants that have been associated with this family of diseases. researchers now believe at least 20% of idiopathic pulmonary fibrosis (ipf) patients with multiple family members suffering from ipf have some common familial genetic variants, which may allow researchers to eventually drop the term idiopathic and further define various forms or categories, with differing progression or outcome. the name given to this version of interstitial pneumonias is familial interstitial pneumonia (fip) [200] [see . currently two categories of genetic focus have been defined: those genes related to telomere biology (shorter telomeres) and those related to surfactant protein processing. the genes related to shorter telomeres are tert, terc, htr, dkc1, and rtel1. more mutations have been found in the tert gene, which encodes the protein component of telomerase, than any other gene. further research may allow targeted therapies to affect the genetic expression associated with the development of ipf [201, 202] . a common variant within the promoter of the muc5b gene is the most replicated single-nucleotide polymorphism related to familial and sporadic forms of ipf as well as early radiographic findings of ipf [203] (. figs. 51.9 and 51.10). wound contraction and re-epithelialization . fig. 51.8 the cellular and molecular mechanisms of fibrosis in multiple organs. the cellular and molecular mechanisms of fibrosis in multiple organs. once an injury occurs in an organ, epithelial and/or endothelial cells are impaired, which results in the release of chemokines and growth factors, including il-13 and tgf-b1. macrophages and monocytes are recruited and activated, both of which further release cytokines and chemokines and further induce fibroblast activation. activated fibroblasts transform into a-sma-expressing myofibroblasts and migrate into the wound along the fibrin lattice. ecm is excessively accumulated, and some parenchymal cells (hepatic stellate cells in the liver, tubular epithelial cells in the kidney, alveolar epithelial cells in the lung, or cardiomyocytes in the heart) are further differentiated into myofibroblasts or fibroblasts by the stimulation of cytokines and chemokines, especially for tgf-b1. after the inflammatory phase, two events occur. one is the regeneration of injured tissues followed by wound contraction and reepithelialization. in contrast, once chronic injury, inflammation, and necrosis occur, myofibroblasts are perpetually activated, and excessive ecm is deposited, finally resulting in fibrosis formation. ctgf, connective tissue growth factor; ecm, extracellular matrix; egf, epidermal growth factor; emt, epithelial-mesenchymal transition; hsc, hepatic stellate cell; il, interleukin; mmp, matrix metalloproteinase; tgf, transforming growth factor; timp, tissue inhibitors of metalloproteinase. (reprinted from chen et al. [198] . with permission from elsevier) conventional treatment is typically palliative. the american thoracic society recognizes that supplemental oxygen and transplantation are the only suggested treatments for ipf. supplemental oxygen is prescribed, and the need for oxygen increases over the progression of the disease. keeping the oxygen saturation level over 90% (normal is in the upper 90s) is ideal and is how healthcare providers determine the level of supplemental oxygen to be used. cardiovascular exercise, in this case called pulmonary rehabilitation, is recommended to maintain as much use of the lungs as possible. infrequently, nutrition and counseling are recommended and are placed into the category of symptom management. nutrition can have a significant role in the management of this disease, but little implementation exists in some of the proposed protocols. there are currently two medications available in the united states with minor impact on the disease progression: nintedanib (commonly called ofev) and pirfenidone (esbriet). histopathological quantification showed similar amounts of dense collagen fibrosis, fibroblast foci, and alveolar macrophages in untreated or pirfenidone-or nintedanibtreated ipf patients [204] . both have significant side effects, including fatigue and gi issues, and patients may have to evaluate their quality of life versus length of life. other antiinflammatories or immune-suppressing medications used are corticosteroids, mycophenolate mofetil/mycophenolic acid (cellcept®), or azathioprine (imuran®). immunesuppressing drugs may be harmful for those with short telomeres, and researchers are exploring this potentially contradictory recommendation [205] . lung transplantation is a final effort. about is half of all transplants. with the prevalence of this disease closer to 200,000, this is a small fraction of those with the disease. some of those with the transplant go on to live productive lives, while others develop pf again, in the transplanted lungs. overall, there is a shorter life expectancy in those with pf, because of telomere shortening. bone marrow or immune response abnormalities have been found in some ipf cases before and after lung transplantation, which increases the associated morbidity. as stated above, inflammation occurs at the beginning and throughout the progression of all fibrosing diseases, including those of the lungs. therefore, reducing inflammation is one wise strategy to slow fibrosing. there are several nutrients that can help slow or reverse the inflammation involved in the fibrosing process. the following two-part diagram shows where in the fibrosing pathogenesis each phytonutrient acts [198] (. fig. 51 .11). a few of those compounds are discussed in more detail here. curcumin, the active constituent in the common spice turmeric, has been shown to reduce fibrotic activity in several studies. in mice, curcumin inhibited collagen secretion of ipf fibroblasts. it affects the signaling of tgf-β, in a dosespecific manner, resulting in reduced expression of α-sma, which is responsible for inappropriate fibrosing. this was shown in vitro and in vivo in mice, with intraperitoneal, but not oral, administration. at the time of the study, oral ingestion of curcumin was not adequately absorbed into plasma, and there was greater than ten times plasma concentration of curcumin following an intraperitoneal injection [88] . however, some new oral products on the market are showing greater absorption. the results of this study suggest more research into curcumin, including improved delivery into patients. for example, some delivery options may include nebulized curcumin directly into the lungs, binding it to highly absorbable agents for oral use or liposome-encapsulated curcumin suitable for intravenous use (already shown to be effective in an animal model). according to manufacturers of curcumin products, some are more readily absorbed than others. one study on fibrosing suggested that a dose of around 2200 mg curcumin split into three doses taken with meals including pepper (bioperine) achieved doses that were sufficient to exert the desired therapeutic effect. research into using quercetin also has some promising results in slowing the progression of ipf. quercetin reversed lung fibrosing in mice and reversed the disease progression normally caused by typical pulmonary senescence markers [206] . it is worth mentioning that n-acetylcysteine (nac), a long-used therapeutic agent for breaking down mucus in the lungs, has not been found to be effective in those with ipf. in fact, due to its acidic nature, it has even been shown to be harmful when used in the inhaled form [207] . several of the drugs being developed have a natural product as a model or foundation. until a drug or gene therapy is developed that stops or reverses this disease, it may make sense for the patient to focus on anti-inflammation and reducing myofibroblast activation, the extracellular matrix (ecm) accumulation, and the epithelial-mesenchymal transition (emt) process. the phytochemicals listed in . fig. 51 .11 would be good ones to investigate. with the recent identification of genes associated with ild, a call for gene-related therapies both related to telomere lengthening and connective tissue disease has been initiated, and this type of therapy, as with any disease, could be personalized [208] . one recent study looked at various biomarker values as a more precise way of diagnosing. the biomarker molecules were classified according to their involvement into alveolar epithelial cell injury, fibroproliferation, and matrix remodeling as well as immune regulation. furthermore, genetic variants of tollip, muc-5b, and other genes associated with a differential response to treatment and with the development and/or the prognosis of ipf were identified. research into personalized medicine for treatment is starting [209] . although controversial, because of the lack of research on interpretation of the results, telomere length testing is available directly to consumers and through healthcare . fig. 51.11 antifibrosis therapy. the molecular mechanisms and therapeutic targets of natural products against fibrosis. a tgf-b exerts a profibrotic effect through smad-dependent [target (1)] and smadindependent pathways [target (2) ]. in the smad-dependent pathway, tgf-b1 directly phosphorylates and activates the downstream mediator smad2 and smad3 through tgf-b receptor i, and then smad2 and smad3 bind smad4, which forms a complex that moves into the nucleus and initiates gene transcription. smad7, transcribed by smad3, is a negative regulator of tgf-b/smad signaling, and the imbalance between smad3 and smad7 contributes to fibrosis. pi3k, erk, and p38 mapk are downstream mediators of the smad-independent tgf-b pathway. pparg [target (3)] could inhibit tgf-b to reduce fibrosis, while ctgf [target (4)], a matricellular protein, contributes to wound healing and virtually all fibrotic pathology. additionally, gas6 contributes to fibrosis through the tam receptor, which further activates the pi3k/akt pathway. similarly, lpa triggers fibrosis through the lpa1 receptor [target (5) ] that stimulates b-catenin to induce fibrogenesis. the activation of the hedgehog pathway [target (6) ] induces the transcriptional activity of gli to express target genes, which have an important role in interstitial fibrosis, undergoing myofibroblast transformation and proliferation. il pathway [target (7) ] stimulates nf-kb [target (8) ] to activate tgf-b to induce fibrogenesis, while nrf2 [target (9) ] antagonizes nf-kb activity to protect against fibrosis. b the chemical structures of isolated compounds and their therapeutic targets are presented. ctgf, connective tissue growth factor; il, interleukin; lrp, low-density lipoprotein receptor-related protein; ri, transforming growth factor-b receptor i; rii, transforming growth factor-b receptor ii; sara, smad anchor for receptor activation; stat, signal transducer and activator of transcription; tcf, t-cell factor; tgf, transforming growth factor. (reprinted from chen et al. [198] practitioners. there are a few different methods: quantitative polymerase chain reaction, or qpcr, which has a 20% variability rate, and flow cytometry and fluorescent in situ hybridization, or flow-fish, which has a 5% variability rate. most research labs use flow-fish for research. telomere length is a hot topic in research, the antiaging industry, and with popular health blogs. shorter-thanaverage telomeres have also been linked to heart disease and heart failure [163, 210, 211] , cancer [212] , diabetes [213] , and osteoporosis [214] . research has shown ways to slow telomere shortening. some include reducing stress, meditation, practicing loving kindness (a technique encouraging compassion) [215] , reducing exposure to air pollution and toxins [216] , cardiovascular exercise [217] , and a healthy fat and high vegetable diet [218, 219] . one study showed that 45 minutes of cardiovascular exercise three times per week resulted in longer telomeres representing 10 years of biological age, similar to those of marathon runners, compared to those who didn't exercise much or at all [220] . intermittent fasting, which reduces oxidative stress and keeps weight in check, has exploded in the scientific literature as a way to increase longevity and slow telomere shortening [221, 222] . nicotinamide adenine dinucleotide (nad+) supplements may also help maintain telomere length by activating sirtuins, the antiaging enzymes; parps, which are involved in dna repair; and cd38, which plays a role in insulin production. another supplement, cycloastragenol, derived from the herb astragalus, has also been shown to activate telomerase in mice. an ingredient called ta-65 has been derived and is used in supplements [223] . overall, a healthy lifestyle and diet seem to delay the shortening of telomeres. with relation to pf, the gene mutations involved in telomere shortening may or may not be influenced by the above interventions. more research is needed for this. pulmonary fibrosis is a devastating disease with no management or a known cure. the integrative and functional medicine nutritionist can help her/his patient by managing weight, encouraging a healthy diet full of anti-inflammatory foods and encouraging a healthy lifestyle with exercise and stress reduction. there is some promising research into natural supplement use to target the different areas of progression within the disease process and some ongoing drug and gene therapy development to follow. the prevalence of lung disease in the united states and worldwide is growing and will continue to grow rapidly with the deterioration of earth's atmosphere, which is caused by pollutants such as industrial and construction toxins and volcanic and wildfire particulates. poor maternal, childhood, and adult nutrition from micronutrient-poor diets resulting in nutrient insufficiencies, not necessarily nutrient deficiencies, is also contributing to increased lung disease diagnoses or poorer results during treatment [226, 227] . lifestyle choices and habits also play a role in the development of many of the lung diseases in today's world, such as smoking or vaping, which uses chemicals that are poorly studied to date. other lung diseases have their roots in genetics. some key processes drive many lung diseases, with the inflammatory process being the most important, according to current literature. nutrition can be of great help with inflammation, using a diet rich in whole foods providing micronutrients and phytonutrients. understanding genetics is also key to unraveling the causes and potential future treatments for many lung diseases. those patients with both genetic and environmental determinants, such as in those who smoke and have genes associated with copd, are at the greatest risk [228] . despite the prevalence of lung disease, there is a general lack of nutrition knowledge among practitioners, including familiarity with the research about the use of nutrition for prevention, slowing disease progression, or as a treatment of lung disease. historically, nutrition has been used in a supportive role, primarily monitoring macronutrients to prevent weight loss, muscle atrophy, and acid/alkaline balance. although this is extremely important, more attention needs to be directed toward emphasizing micronutrients and phytonutrients. research is strong regarding the benefits of vitamins, minerals, and pre-and probiotics, and indeed, some integrative and functional practitioners are using vitamin and mineral nutritional therapy in oral, intramuscular, and intravenous applications, when allowed, in practice. a newer area of research is around nutraceuticals, including targeted vitamins, minerals, and plantderived constituents concentrated to therapeutic doses. some exciting research around the use of curcumin and quercetin, for example, has been shown to dampen inflammation to the point of disrupting the disease process (see above). the expanding knowledge of the microbiome is identifying the importance of the lung and airway microbiome in respiratory health. more research, and indeed more education for nutritionists around the existing research, is needed to fully understand the best opportunities for the use of nutrition in the treatment or prevention of lung disease. the intersection of aging biology and the pathobiology of lung diseases: a joint nhlbi/nia workshop chronic respiratory disease | gateway to health communication | cdc global status report on noncommunicable diseases the top 10 leading causes of death in the united states vitamin d status has a linear association with seasonal infections and lung function in british adults respiratory health and disease in europe: the new european lung white book impact of dna repair, folate and glutathione gene polymorphisms on risk of non-small cell lung cancer nutrition and respiratory health-feature review measuring the global burden of disease and epidemiological transitions: 2002-2030 nutrition therapy & pathophysiology decrease in pulmonary function and oxygenation after lung resection sequelae and complications of pneumonectomy surgery for tumor recurrence in a pneumonectomy space function and structure of the respiratory system function and structure of the respiratory system the respiratory system chapter 2. function and structure of the respiratory system cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer the effect of membrane lipid composition on the formation of lipid ultrananodomains physical studies of cholesterolphospholipid interactions lung collagen composition and synthesis. characterization and changes with age always cleave up your mess: targeting collagen degradation to treat tissue fibrosis concord: fritz perlberg publishing the use (or otherwise) of pulse in general practice the two-way association of periodontal infection with systemic disorders: an overview oral health care and aspiration pneumonia in frail older people: a systematic literature review periodontal systemic associations: review of the evidence association between oral habits, mouth breathing and malocclusion the microbiome and the lung vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data probiotics supplementation in children with asthma: a systematic review and meta-analysis probiotics in asthma and allergy prevention gut-lung axis: the microbial contributions and clinical implications the gut-lung axis in respiratory disease the role of autophagy in allergic inflammation: a new target for severe asthma the lung microbiome in moderate and severe chronic obstructive pulmonary disease learn about bronchiectasis american lung association learn about bronchiolitis american lung association idiopathic pulmonary fibrosis | national heart, lung, and blood institute (nhlbi) american lung association desquamative interstitial pneumonia american lung association kyphoscoliosis -an overview diagnosis and treatment of inflammatory joint disease. hip pelvis pulmonary arterial hypertension (pah) lung carcinoid tumor what is small cell lung cancer? cell lung cancer bacterial tracheitis alpha-1 foundation | alpha1. org obstructive sleep apnea (osa) in adults anemia in chronic obstructive pulmonary disease and the potential role of iron deficiency disruption of iron homeostasis and lung disease iron homeostasis in health and disease out of balance -systemic iron homeostasis in iron-related disorders inflammation and neurodegeneration laboratory, chronic diseases research center (cedoc), nova medical school (nms)/faculdade de ciências médicas hfe gene variants and iron-induced oxygen radical generation in idiopathic pulmonary fibrosis prognostic impact of vitamin b6 in lung cancer effects of vitamin b6 metabolism on oncogenesis, tumor progression and therapeutic responses association of the b-vitamins pyridoxal 5′-phosphate (b6), b12, and folate with lung cancer risk in older men sex differences in risk of lung cancer associated with methylene-tetrahydrofolate reductase polymorphisms dietary intake of b vitamins and methionine and risk of lung cancer two common single nucleotide polymorphisms in the gene encoding β-carotene 15,15′-monoxygenase alter β-carotene metabolism in female volunteers consensus report on nutrition for pediatric patients with cystic fibrosis potential micronutrients and phytochemicals against the pathogenesis of chronic obstructive pulmonary disease and lung cancer the controversial role of retinoic acid in fibrotic diseases: analysis of involved signaling pathways vitamin d and susceptibility of chronic lung diseases: role of epigenetics vitamin d deficiency at 16 to 20 weeks' gestation is associated with impaired lung function and asthma at 6 years of age vitamin d and lung disease dietary vitamin c intake protects against copd: the korea national health and nutrition examination survey in 2012 smokers with adequate vitamin c intake show a preferable pulmonary function test vitamin c mitigates oxidative stress and tumor necrosis factor-alpha in severe community-acquired pneumonia and lps-induced macrophages inhibitory effect of a mixture containing vitamin c, lysine, proline, epigallocatechin gallate, zinc and alpha-1-antitrypsin on lung carcinogenesis induced by benzo(a) pyrene in mice thoracic transplantation: oxidative stress and nutritional intakes in lung patients with bronchiolitis obliterans syndrome dual role of vitamin c utilization in no2-induced oxidative stress in lung tissues of mice two faces of vitamin e in the lung vitamin e isoform γ-tocotrienol downregulates house dust mite-induced asthma serum tocopherol levels and vitamin e intake are associated with lung function in the normative aging study dietary nutrients associated with preservation of lung function in hispanic and non-hispanic white smokers from new mexico the disturbed redox-balance in pulmonary fibrosis is modulated by the plant flavonoid quercetin curcumin inhibits transforming growth factor β induced differentiation of mouse lung fibroblasts to myofibroblasts directory of open access journals curcumin attenuates radiationinduced inflammation and fibrosis in rat lungs curcumin effect on bleomycininduced pulmonary fibrosis in mus musculus antifibrotic effects of curcumin are associated with overexpression of cathepsins k and l in bleomycin treated mice and human fibroblasts orally administered chitosan-coated polycaprolactone nanoparticles containing curcumin attenuate metastatic melanoma in the lungs effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: decisive role of bax, nrf2, nf-κb, muc5ac, tnf-α and il-1β evaluating the ameliorative potential of plant flavonoids and their nanocomposites in bleomycin induced idiopathic pulmonary fibrosis cannabidiol and (-)delta9-tetrahydrocannabinol are neuroprotective antioxidants dietary intake of six minerals in relation to the risk of chronic obstructive pulmonary disease mineral intake and lung cancer risk in the nih-american association of retired persons diet and health study nothing boring about boron dietary mineral intake and lung cancer risk: the rotterdam study bone mineral density, lung function, vitamin d and body composition in children and adolescents with cystic fibrosis: a multicenter study the alpha-lipoic acid derivative dhlhzn: a new therapeutic agent for acute lung injury in vivo suppression of a cancer stem-like phenotype mediated by alpha-lipoic acid in human lung cancer cells through down-regulation of β-catenin and oct-4 influence of alpha-lipoic acid on nicotine-induced lung and liver damage in experimental rats effect of n-acetylcysteine in subjects with slow pulmonary mucociliary clearance the effect of oral n-acetylcysteine in chronic bronchitis: a quantitative systematic review the intrabronchial microbial flora in chronic bronchitis patients: a target for n-acetylcysteine therapy? the effect of oral n-acetylcysteine on lung glutathione levels in idiopathic pulmonary fibrosis attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term n-acetylcysteine treatment high-dose oral n-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis national institutes of health potential micronutrients and phytochemicals against the pathogenesis of chronic obstructive pulmonary disease and lung cancer the relation between dietary intake of individual fatty acids, fev1 and respiratory disease in dutch adults lipoxins: nature's way to resolve inflammation the role of free fatty acids in idiopathic pulmonary fibrosis association between ω3 and ω6 fatty acid intakes and serum inflammatory markers in copd a prospective study of dietary polyunsaturated fatty acids intake and lung cancer risk effectiveness of essential amino acid supplementation in stimulating whole body net protein anabolism is comparable between copd patients and healthy older adults whole fruits and fruit fiber emerging health effects glutathione redox control of asthma: from molecular mechanisms to therapeutic opportunities is there any relationship between plasma antioxidant capacity and lung function in smokers and in patients with chronic obstructive pulmonary disease? the treatment of pulmonary diseases and respiratoryrelated conditions with inhaled (nebulized or aerosolized) glutathione. evid based modifications of plasma proteome in long-lived rats fed on a coenzyme q10-supplemented diet increased oxidative stress in patients with chronic obstructive pulmonary disease (copd) as measured by redox status of plasma coenzyme q10. pathophysiology coenzyme q10 supplementation reduces corticosteroids dosage in patients with bronchial asthma effects of coenzymeq 10 administration on pulmonary function and exercise performance in patients with chronic lung diseases glutathione cofactors. available at geology and health: closing the gap pulmonary fluorosis: a review comparison of the fractional exhaled nitric oxide levels in adolescents at three schools located three different distances from a large steel mill inorganic arsenic and respiratory health, from early life exposure to sex-specific effects: a systematic review the broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem chronic arsenic toxicity & human health human exposure to dietary inorganic arsenic and other arsenic species: state of knowledge, gaps and uncertainties toxic substances portal -arsenic dynamed plus record no. t114977, chronic arsenic poisoning toxicological aspects of cadmium and occupational health activities to prevent workplace exposure in japan: a narrative review current status of cadmium as an environmental health problem serum heavy metals and obstructive lung disease: results from the national health and nutrition examination survey relationship between blood levels of heavy metals and lung function based on the korean national health and nutrition examination survey iv-v department of the environment and energy systematic review and meta-analysis of the association between ambient nitrogen dioxide and respiratory disease in china a framework for examining social stress and susceptibility to air pollution in respiratory health quantifying the impact of current and future concentrations of air pollutants on respiratory disease risk in england short-and long-term effects of ambient ozone and fine particulate matter on the respiratory health of chronic obstructive pulmonary disease subjects weekly personal ozone exposure and respiratory health in a panel of greek school children. environ health perspect nutritional solutions to reduce risks of negative health impacts of air pollution cigarette smoking and inflammation revisited effects of tobacco smoke on immunity, inflammation and autoimmunity electronic cigarette, effective or harmful for quitting smoking and respiratory health: a quantitative review papers review: the toxicity of e-cigarettes and children's respiratory health respiratory health and allergies from chemical exposures among machining industry workers in selangor, malaysia occupational pesticide exposures and respiratory health persistent effects of chlorine inhalation on respiratory health respiratory muscle function in patients with cystic fibrosis. pediatr pulmonol emotions, morbidity, and mortality: new perspectives from psychoneuroimmunology understanding mindbody interaction from the perspective of east asian medicine. evid based complement alternat med climate change and respiratory disease: european respiratory society position statement nutrients and foods for the primary prevention of asthma and allergy: systematic review and meta-analysis global strategy for asthma management and prevention. glob initiat asthma tests for assessing asthma pediatric expression of mast cell activation disorders asthma phenotypes and endotypes: an evolving paradigm for classification a histamine-free diet is helpful for treatment of adult patients with chronic spontaneous urticaria serum diamine oxidase activity in patients with histamine intolerance leukocyte telomere length and cardiovascular disease in the cardiovascular health study regulation of antioxidant enzymes in lung after oxidant injury oxidative stress in asthma resolution of acute inflammation in the lung the alkaline diet: is there evidence that an alkaline ph diet benefits health? magnesium in prevention and therapy effects of magnesium supplementation on the glutathione redox system in atopic asthmatic children diet and asthma: vitamins and methyl donors drugs for asthma dietary inflammatory index is related to asthma risk, lung function and systemic inflammation in asthma fruit and vegetable intake and risk of wheezing and asthma: a systematic review and meta-analysis correlation of cutaneous sensitivity and cytokine response in children with asthma coeliac disease and asthma association in children: the role of antibiotic consumption regulatory t cells in asthma prenatal farm exposure is related to the expression of receptors of the innate immunity and to atopic sensitization in school-age children characterization of regulatory t cells in urban newborns is vitamin d deficiency to blame for the asthma epidemic? maternal food consumption during pregnancy and the longitudinal development of childhood asthma vitamin d levels, lung function, and steroid response in adult asthma camp regulation of airway smooth muscle function quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of plcβ and pde4 using periostin as a biomarker in the treatment of asthma biomarkers in asthma | aaaai developmental bisphenol a exposure modulates immune-related diseases nasopharyngeal microbiota composition of children is related to the frequency of upper respiratory infection and acute sinusitis disorders/patient-caregiver-education/fact-sheets/guillain-barré-syndrome-fact-sheet clinical manifestations, diagnosis, and natural history of alpha-1 antitrypsin deficiency let's talk about alpha-1 antitrypsin deficiency alpha1-antitrypsin deficiency what is pulmonary fibrosis | pulmonary fibrosis foundation home page -ipf foundation ipf foundation a clinical evaluation of baofeikang granule in combined pulmonary fibrosis and emphysema treatment -pulmonary fibrosis foundation endoplasmic reticulum stress enhances fibrotic remodeling in the lungs natural products as a source for antifibrosis therapy idiopathic pulmonary fibrosis: phenotypes and comorbidities the genetic basis of idiopathic pulmonary fibrosis telomerase mutations in families with idiopathic pulmonary fibrosis telomerase and idiopathic pulmonary fibrosis genetics of pulmonary fibrosis wolters pj. histopathological and molecular analysis of idiopathic pulmonary fibrosis lungs from patients treated with pirfenidone or nintedanib telomere shortening is behind the harm of immunosuppressive therapy in idiopathic pulmonary fibrosis quercetin enhances ligand-induced apoptosis in senescent idiopathic pulmonary fibrosis fibroblasts and reduces lung fibrosis in vivo application of n-acetylcysteine in pulmonary disorders. in: the therapeutic use of n-acetylcysteine (nac) in medicine integrating genomics into management of fibrotic interstitial lung disease existing and emerging biomarkers for disease progression in idiopathic pulmonary fibrosis association of shorter mean telomere length with risk of incident myocardial infarction: a prospective, nested case-control approach telomere length of circulating leukocytes is decreased in patients with chronic heart failure de vivo i. telomere length, cigarette smoking, and bladder cancer risk in men and women monocyte telomere shortening and oxidative dna damage in type 2 diabetes telomere length in leukocytes correlates with bone mineral density and is shorter in women with osteoporosis loving-kindness meditation practice associated with longer telomeres in women association between leukocyte telomere shortening and exposure to traffic pollution: a cross-sectional study on traffic officers and indoor office workers physical exercise prevents cellular senescence in circulating leukocytes and in the vessel wall association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease telomere length, oxidative damage, antioxidants and breast cancer risk physical activity and telomere length: impact of aging and potential mechanisms of action protein restriction, epigenetic diet, intermittent fasting as new approaches for preventing age-associated diseases fasting mimicking diet is very relevant for health and longevity cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management periostin: an emerging biomarker for allergic diseases zemaira® | alpha-1 antitrypsin deficiency diagnosis assessment of quality of life in children suffered from asthma identifying maternal conditions affecting altered embryologic development. neonatal advanced practice nursing: a case-based learning approach susceptibility for cigarette smoke-induced damp release and damp-induced inflammation in copd key: cord-016235-2lhrkmrv authors: roden, anja c.; tazelaar, henry d. title: lung date: 2010-05-17 journal: pathology of solid organ transplantation doi: 10.1007/978-3-540-79343-4_7 sha: doc_id: 16235 cord_uid: 2lhrkmrv experiments with animals in the 1940 and 1950s demonstrated that lung transplantation was technically possible [33]. in 1963, dr. james hardy performed the first human lung transplantation. the recipient survived 18 days, ultimately succumbing to renal failure and malnutrition [58]. from 1963 through 1978, multiple attempts at lung transplantation failed because of rejection and complications at the bronchial anastomosis. in the 1980s, improvements in immunosuppression, especially the introduction of cyclosporin a, and enhanced surgical techniques led to renewed interest in organ transplantation. in 1981, a 45-year-old-woman received the first successful heart–lung transplantation for idiopathic pulmonary arterial hypertension (ipah) [106]. she survived 5 years after the procedure. two years later the first successful single lung transplantation for idiopathic pulmonary fibrosis (ipf) [128] was reported, and in 1986 the first double lung transplantation for emphysema [25] was performed. experiments with animals in the 1940 and 1950s demonstrated that lung transplantation was technically possible [33] . in 1963, dr. james hardy performed the first human lung transplantation. the recipient survived 18 days, ultimately succumbing to renal failure and malnutrition [58] . from 1963 through 1978, multiple attempts at lung transplantation failed because of rejection and complications at the bronchial anastomosis. in the 1980s, improvements in immunosuppression, especially the introduction of cyclosporin a, and enhanced surgical techniques led to renewed interest in organ transplantation. in 1981, a 45-year-old-woman received the first successful heart-lung transplantation for idiopathic pulmonary arterial hypertension (ipah) [106] . she survived 5 years after the procedure. two years later the first successful single lung transplantation for idiopathic pulmonary fibrosis (ipf) [128] was reported, and in 1986 the first double lung transplantation for emphysema [25] was performed. over the following years, the number of lung transplants rapidly increased, and the operation became an accepted treatment for an end-stage lung disease. today, there are four major surgical approaches to lung transplantation: single and bilateral lung transplantation (blt), heart-lung transplantation, and transplantation of lobes of lungs from living donors. in 2007, 2,708 lung transplantation procedures were reported worldwide to the registry of the international society for heart and lung transplantation (ishlt) in adults, the highest number for any year until then [21] . in the same year, 93 lung transplantations were reported in children, the majority in adolescents (12-17 years old) [6] . although the number of single lung transplantations has been relatively stable, blts have continuously increased within the past 15 years. in fact, in 2007, blt was the most common lung transplantation procedure performed with 69% of all lung transplantation procedures, largely due to transplantation for cystic fibrosis and chronic obstructive lung disease/ emphysema which made up for 26.6 and 25.7% of all blts between 1995 and 2008 [21] . the mean age of transplant recipients has consistently increased since 1989 rising to an all time high of 50.8 years in 2008 [21] . the most common indications for lung transplantation in adults are chronic obstructive pulmonary disease (copd)/emphysema, ipf, cystic fibrosis and alpha-1 antitrypsin deficiency emphysema (aat) (see table 7 .1) [21] . indications for pediatric lung transplantation vary by age (see table 7 .1). in children over 5 years old, cystic fibrosis is the most common indication [6] , followed by ipah. in contrast, in infants and preschool children, lung transplantations are usually performed for ipah, congenital heart disease, idiopathic interstitial pneumonitis, and surfactant protein deficiency. well-selected patients with systemic diseases such as sarcoidosis, lymphangioleiomyomatosis, and pulmonary langerhans' cell histiocytosis have also had satisfactory results after lung transplantation [27, 71, 91, 99, 119] as have selected patients with scleroderma [84, 110, 113] . multiple cases of incidental t1n0m0 or even stage iiia non-small cell carcinoma in the excised native lungs of transplant recipients have been reported [14, 30, 124] . although one patient with stage iiia poorly differentiated squamous cell carcinoma died 6 months after transplantation of a neoplastic thromboembolus, patients with t1n0m0 carcinoma are generally free of recurrence. currently, only patients with near end-stage lung disease and a limited life expectancy should be considered for lung transplantation [95] . however, since lung transplantation is a rapidly evolving field, there are no hard and fast rules about who may be transplanted. when choosing a transplantation procedure, several issues are considered including the shortage of organ donors, the original disease, and the center's experience with graft and patient survival. general guidelines for the selection of the procedure have been proposed [36] and are based on the nature of the underlying lung disease. while blts are mandatory for cystic fibrosis [6, 21] ipf idiopathic pulmonary fibrosis; aat alpha1-antitrypsin deficiency; ipah idiopathic pulmonary arterial hypertension; lam lymphangioleiomyomatosis; ob obliterative bronchiolitis [35] , this procedure has also become more popular for indications such as aat, copd, ipf, and ipah. singlelung transplantation is usually performed in patients with restrictive fibrotic lung disease, eisenmenger syndrome with reparable cardiac anomaly, and older patients with copd. heart-lung transplantation is considered in patients with eisenmenger syndrome with irreparable cardiac defect, pulmonary hypertension with cor pulmonale, or end-stage lung disease with concurrent severe cardiac disease [83, 89] . transplantation of lobes from living donors is a recently developed technique involving bilateral implantation of the lower lobes usually from two blood group-compatible living donors. the procedure has been performed in patients with cystic fibrosis, although the indications have been recently broadened. the functional and survival outcomes are similar to those achieved with conventional transplantation of cadaveric lungs. donation of a lobe decreases the donor's lung volume by an average of approximately 15%, which is not associated with long-term functional limitation. other factors of the recipient that must be taken into consideration on an individual basis include ventilator dependence, previous cardiothoracic surgery, and preexisting medical conditions (e.g., hypertension, diabetes mellitus, osteoporosis) since posttransplantation medical regimen can worsen these illnesses. severe coronary artery disease is a contraindication to lung transplantation. however, coronary artery bypass grafting at the same time as lung transplantation has been performed with a reasonably good outcome in some centers, although less invasive preoperative interventions, such as percutaneous transluminal coronary angioplasty and stenting, are preferred. although the donor selection criteria may vary amongst centers, generally acceptable donor criteria include age of donor <65 years for lung transplantation and <45 years for heart-lung transplantation. in 2008, the average donor age was 35.5 years [21] . other donor criteria include the absence of severe chest trauma or infection, no prolonged cardiac arrest (heart-lung transplantation only), minimal pulmonary secretions, negative screens for hiv, hepatitis c, and hepatitis b and blood type (abo) compatibility. a close match of lung size between donor and recipient, pao 2 > 300 mmhg on 100% fraction of inspired oxygen (fio 2 ), clear chest radiograph and no history of malignant neoplasms are also required. most transplant centers will use lungs from a cytomegalovirus (cmv)-positive donor for transplantation into a cmv-negative donor given an adequate postoperative cmv prophylaxis. with the current techniques, satisfactory graft function can be obtained after an ischemic interval of as long as 6-8 h. for pulmonary preservation, systemic heparinization of the donor and hypothermic flush perfusion of the allograft are most commonly used in clinical practice. most flush solutions are administered at a temperature of 4°c, while topical cooling is carried out by filling the pleural cavity with iced crystalloid solution. the harvested lungs are then immersed in crystalloid solution, packed in ice, and transported at a temperature of 1-4°c. the infusion and transport is performed during active ventilation and static inflation with o2, respectively. acute and chronic alloreactive injury to the donor lung affects both the vasculature and the airways [123] . usually, rejection is evaluated on transbronchial biopsies (see below sect. 7.3). on only rare occasions, wedge biopsies are performed. other specimens might include explants for retransplant or autopsy specimens. acute rejection is characterized by perivascular mononuclear cell infiltrates, which may be accompanied by sub-endothelial chronic inflammation (e.g., endotheliitis or intimitis), and also by lymphocytic bronchiolitis. in contrast, chronic rejection is manifest by fibrous scarring, involving the bronchioles and sometimes associated with accelerated fibrointimal changes affecting pulmonary arteries and veins. the presence of presumed irreversible dense eosinophilic hyaline fibrosis in airways and vessels remains the key histologic discriminator between acute and chronic rejection of lung. the histologic changes are divided into grades based on intensity of the cellular infiltrate, and the presence and absence of fibrosis. hyperacute rejection occurs within minutes to a few hours after the newly transplanted organ begins to be perfused. it is a type ii hypersensitivity reaction, mediated by preexisting antibodies to abo blood groups, human leukocyte antigens (hla) class i, or other antigens on graft vascular endothelial cells. preexisting antibodies can result from previous pregnancies, blood transfusions, or a previous transplant. antibody binding provokes complement and cytokine activation leading to endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. the outcome is usually fatal. in the lungs, hyperacute rejection grossly presents by edema and cyanosis of the graft. histologically, platelet thrombi, neutrophilic infiltration, fibrin thrombi, necrosis of vessel wall, and morphologic features of diffuse alveolar damage (dad) are observed [29] . although hyperacute rejection is a well-known complication in kidney and heart transplantations, in lung transplantation it appears to be rather rare with only five cases reported. one patient reported presented with severe hypoxia, high fever, hemodynamic instability and developing acute renal failure 1 h after completion of the anastomoses [29] . chest radiograph displayed a completely opacified left lung, with homogenous infiltrates. bronchoscopy revealed abundant pink frothy fluid draining from the allograft. mean pulmonary artery pressure increased to 29 mmhg. the patient died 24 h later. at autopsy, the vascular and bronchial anastomoses appeared patent without signs of injury. the transplanted lung showed red hepatization and a firm consistency. microscopically, signs of acute lung injury were evident. although a pretransplant panel-reactive antibody (pra) was negative, flowcytometry revealed 56 and 45% reactivity against hla class i and ii, respectively with anti-a2 detected among the preformed antibodies. three other reported patients with hyperacute rejection died within 4 h to 13 days after transplantation [11, 19, 43, 116] . although in three of the five reported patients pretransplant pras were negative, crossmatch was positive in all cases with anti-a2 the most common identified antibody. collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative pras may occur and pretransplantation cross match is not often possible [29] . acute rejection is the host's response to the recognition of the graft as foreign. most patients develop at least one episode of acute rejection within the first 3 weeks following transplantation, typically in the first 5-10 days, with 36% of patients experiencing at least one episode in the first year [21] . obliterative bronchiolitis (ob) is the most common late cause of mortality and morbidity after lung transplantation occurring in 28% by 2.5 years and 74% by 10 years in patients who survive at least 14 days [21] . it also has a significant negative impact on quality of life parameters. risks for acute rejection include hla mismatching, type of immunosuppression, infection, and recipient factors. it is generally thought that the intensity of host alloimmune response is related to recipient recognition of differences with the donor hla antigens and that this process drives acute lung allograft rejection. a higher degree of mismatch increases the risk of acute rejection [101, 115, 141] . however, this effect is not consistent across all hla loci or studies. mismatches at the hla-dr, hla-b [115] , and hla-a [101] loci, as well as a combination of all three loci [141] , appear important. in addition, the ishlt registry has not found a correlation between hla mismatching and survival [130] . thus, while hla mismatching between donor and recipient likely contributes to the immunologic basis for acute rejection, it is difficult to discern if a mismatch at a particular locus or if different degrees of mismatch significantly alter the overall risk for acute rejection. viral infections have been thought to modulate the immune system and heighten alloreactivity. indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus (rsv), rhinovirus, coronavirus, and parainfluenzavirus [44, 73, 137] . although cmv is considered a potential risk factor for ob, studies directly linking cmv infections or cmv prophylaxis strategies with acute rejection have been inconsistent [118] . in one study, chlamydia pneumoniae infection was linked to the development of acute rejection and ob [50] . several host genetic characteristics have been suggested to modulate acute lung rejection. for instance a genotype leading to increased il1-production may protect against acute rejection [147] and a multidrug-resistant genotype (mdr1 c3435t) appears to predispose to persistent acute rejection resistant to immunosuppressive treatment [148] . the effect of age on acute rejection appears to be bimodal, with the lowest incidence of acute rejection in infancy (<age 2) [61] and increased risk during childhood as compared with adulthood [117] . the incidence of acute rejection in older lung transplant recipients (age 65 and higher) does not seem to change [32] ; in fact, an increased rate of infections in older lung transplant recipients is thought to contribute to an increased mortality detected at one center arguing for reduced immunosuppression in these patients [55] . the clinical course of acute rejection can be variable. patients with acute rejection might present with dyspnea, fever, leukocytosis, and a widened alveolararterial oxygen gradient. higher-grade rejection appears to cause more severe symptoms and can lead to acute respiratory distress [32] . in patients with rejection, pulmonary function testing may show a decrease in forced expiratory volume in 1 s (fev 1 ) and vital capacity (vc). although spirometry has a sensitivity of greater than 60% for detecting infection or rejection grade a2 and higher, it cannot differentiate between the two [133] . the usefulness of spirometry is diminished in single lung transplant recipients, as the contralateral native lung dysfunction confounds the pulmonary function test results [7] . pulmonary function testing should therefore be used only as an adjunct to clinical evaluation. although in approximately half of the cases of acute rejection, the findings on chest radiograph are normal, chest radiographs may reveal ill-defined perihilar and lower lobe opacities, along with septal lines and pleural effusions. findings on ct scan might include ground-glass opacities, septal thickening, volume loss, nodules and consolidations, and pleural effusions. infiltrates observed on imaging studies during the first week after lung transplantation usually are caused by reimplantation response (i.e., reperfusion edema). persistent infiltrates beyond the first week suggest acute rejection or infection. however, although early small studies attempted to demonstrate the usefulness of chest x-rays and chest ct scans in the diagnosis of rejection, more recent data show very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes [51] . exhaled nitric oxide (no) is also an attractive marker of lung injury; it has been correlated with lymphocytic bronchiolitis [31] and acute rejection [120] . furthermore, in a study of inert gas single breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection [133] . although lung transplantation has come of age, the development of ob remains the biggest hurdle preventing long-term survival in many patients [136] . because of its low sensitivity (28%) and specificity (75%), ob remains difficult to prove pathologically with transbronchial biopsy [24, 72] . as a consequence, a clinical definition, called bronchiolitis obliterans syndrome (bos) was proposed [24] . this is based on pulmonary function criteria, initially fev 1 evolution, and more recently, mid-expiratory flow rate (fef ) [38] . the onset of bos-symptoms is usually insidious, with progressive exertional dyspnea, often accompanied by cough, which may be dry or productive. the incidence of bos is highest after the first year following lung transplantation. however, the risk of bos increases to 60-80% 5-10 years after the lung transplantation procedure. it remains the leading cause of morbidity and death after lung transplantation, accounting for about 19-29% late mortality and some 35% of patients affected by the condition 5 years after transplantation [21] . as discussed above, an acute immunologic event (acute rejection) may trigger the onset of ob [63] . however, nonalloimmune injury such as a respiratory tract infection (cmv or non-cmv viral infection) is increasingly recognized as also having an important impact on the development of ob [41, 46] . in fact, cmv pneumonitis affects over 20% of lung transplant recipients. despite treatment, it increases the risk for ob and death. early detection of ob in a preclinical stage is ideal so that aggressive attempts can be made to prevent a fully developed syndrome. however, to date, no particular marker to indicate ob, either from the peripheral blood or bronchoalveolar lavage (bal) fluid, has predicted a risk for this disease. an association between chronic aspiration and ob after heart-lung transplantation has also been observed [105] , and there is a high frequency of gastroesophageal reflux with resultant aspiration following lung transplantation [28, 57, 97, 105, 108, 143] . for instance, one study described gastrointestinal complications including gastroesophageal reflux in 51% of patients who underwent lung transplantation [80] . other than leading to an increase in acute rejection by exacerbating the alloimmune response, aspiration of gastric contents might act independently of the alloimmune response to promote allograft injury and the development of ob. experiments in rats have further substantiated the association between chronic aspiration and ob [79] . despite the common clinical impression that lymphocytic pleural effusions are a hallmark of acute rejection, published data are inconclusive [65] . collectively, although the presentation of the patient and several ancillary studies might suggest a transplant rejection process, tissue diagnosis is necessary for definitive diagnosis. in 1990, the ishlt sponsored the lung rejection study group (lrsg), a workshop to develop a "working formulation" for the diagnosis of lung rejection by transbronchial biopsy [9] . the proposed grading scheme found wide acceptance. due to developments in the field and experience with its use, lrsg has remet twice since to assess the merits of the grading scheme. the revisions were published in 1996 [145] and 2007 [123] , respectively. the grading scheme is strictly pathologic and does not consider any clinical parameters. due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. the most recent classification of lung allograft biopsies is the 2007 ishlt revised consensus classification of allograft rejection [123] (see table 7 .2). this classification was revised from the 1996 ishlt consensus classification. there were no significant changes in the grading of acute rejection in the latest classification, but it was acknowledged that minimal acute rejection is not solely a high power diagnosis but can also be recognized at low power in an adequately alveolated biopsy. in the 1996 grading system, small airways rejection was graded in a four-tiered system, b1 through b4, respectively (see table 7 .2). the 2007 classification consolidated those into low grade and high grade small airways inflammation to make the evaluation easier. the "r" behind b1 and b2 denotes the revised 2007 classification. chronic airways rejection is not divided into active and inactive anymore but only into present and not present. there are no differences in the classification of chronic vascular rejection between the 1996 and 2007 classification. r denotes revised an attempt should be made to accurately distinguish the grade of rejection since treatment is largely dependent on the histologic grade. furthermore, an effort should be made to review the report of the previous biopsy and to comment on whether rejection is ongoing or resolving. although in general, patients with grade a1 biopsies will not receive increased immunosuppression, surveillance might be intensified and the time to the next biopsy shortened. in contrast, a2 biopsy patients will receive an increase in immunosuppression. grade a3 and a4 patients will be treated similar and receive more immunosuppression. inter-and intraobserver variability in grading can impact treatment and outcome [16, 22] . studies have evaluated the inter-and intraobserver variability of the 1996 grading scheme. two studies found relatively good interobserver agreements for the a-grades (kappa of 0.65 and 0.73) [16, 22] , but this could not be replicated in another study in which the kappa was 0.47 in spite of dichotomization of the a-grades to a0/a1 vs. a2-4 [122] . intraobserver agreement for acute rejection has been found to be good: kappa values of 0.65 and 0.795 [16, 122] . infection can complicate the diagnosis of acute rejection. viral infection in particular can cause mononuclear inflammation [126] . in addition, alveolar damage with macrophage and fibrin accumulation was found to be present in 80% of transbronchial biopsies in the first 6 months after transplantation, further increasing interobserver pathologist discordance. therefore, in general, the lrsg recommends grading rejection only after the exclusion of infection. in contrast to the a-grade, the interobserver variability for grading airways inflammation (b-grades) was only fair with kappas of app 0.3 [16, 122] . for this reason, the lrsg has now simplified b-grading to two possible grades. this nomenclature is to be used for grading of noncartilagenous small airways only after rigorous exclusion of infection [123] . eosinophils can accompany acute lung rejection and are recognized in the 2007 ishlt classification in high grade rejection (grades a3/a4) [123] . the presence of eosinophils has been suggested as a risk factor for bos in small single-center studies [114] . furthermore, high proportions of b cells have been shown to accompany steroid-resistant rejection in lung transplant patients [146] . the mechanisms by which b cells contribute to refractory rejection are not entirely clear, although they may reflect ongoing humoral rejection, perhaps explaining the diminished responsiveness to standard immunosuppression. mast cells have been identified in acute rejection biopsies of increasing a-grade but their role has not been elucidated [144] . rejection and chronic airways inflammation should be distinguished from bronchiolar associated lymphoid tissue (balt). balt is found in the vicinity of airways, usually contains black anthracotic pigment and presents as a rather nodular collection of chronic inflammatory cells which does not surround a vessel. acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis. with progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. the majority of the mononuclear cells in acute rejection are t cells, although a few studies have described increased populations of b cells or eosinophils [104, 123, 146] . in grade a0 normal pulmonary parenchyma is present. scattered infrequent blood vessels, particularly venules, in the alveolated lung parenchyma are surrounded by a relatively thin chronic mononuclear infiltrate (see fig. 7 .1). the lymphocytic rim is rather small and does not spill into the adjacent interstitium. endotheliitis and eosinophils are absent. although previous classification systems commented on a certain number of lymphocyte layers, the current ishlt classification only requires a complete rim of vessels by lymphocytes. in adequately alveolated and artifact-free specimens, the lymphocytic infiltrates might be detected at low magnification. although in mild acute rejection the perivascular infiltrate of lymphocytes is essentially confined to the perivascular adventitia without infiltrating the interstitium, there are more layers of lymphocytes surrounding the vessel and lymphocytes might focally, minimally spill into the adjacent interstitium (see fig. 7 .2). more frequent perivascular mononuclear infiltrates are seen surrounding venules and arterioles. they are readily recognizable at low magnification. these infiltrates usually consist of a mixture of small round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages, and eosinophils. there is frequently subendothelial infiltration by mononuclear cells which may be associated with hyperplastic or regenerative changes in the endothelium, i.e., endotheliitis. concurrent lymphocytic bronchiolitis may be seen in association with mild acute rejection. grade a3 acute rejection shows easily recognizable cuffs of venules and arterioles by dense perivascular mononuclear cell infiltrates which are commonly associated with endotheliitis (see fig. 7 .3). eosino phils a b and even occasional neutrophils are common. this grade is defined by the extension of the inflammatory cell infiltrate into perivascular and peribronchiolar alveolar septa which broadens the interalveolar septa and airspaces are associated with collections of intraalveolar macrophages in the zones of septal infiltration. type ii pneumocyte hyperplasia and histologic features of acute lung injury may become apparent. in severe rejection there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endotheliitis (see fig. 7 .4). this may be associated with intra-alveolar necrotic epithelial cells, macrophages, eosinophils, hemorrhage, and neutrophils and usually some evidence of acute lung injury in form of organizing pneumonia or hyaline membranes. parenchymal necrosis, infarction, or necrotizing vasculitis might be identified; however, these features are more evident on surgical rather than transbronchial lung biopsies. it should be noted that a paradoxical diminution of perivascular infiltrates can occur as cells extend into alveolar septa and spaces where they are admixed with macrophages. this grade can sometimes be difficult to distinguish from an infectious process, harvest/reperfusion injury, or drug toxicity, however, the presence of perivascular inflammation is helpful in establishing the diagnosis. this grade applies only to small airways such as terminal or respiratory bronchioles. bronchi, if present, should be described separately. it is important to mention in the pathology report whether or not small airways are present. the r behind grades 1 and 2 denotes the revised 2007 version. the small airways appear unremarkable without evidence of bronchiolar inflammation. low grade inflammation is characterized by lymphocytes within the submucosa of the bronchioles (see fig. 7 .5). the lymphocytic infiltrates can be infrequent and scattered or forming a circumferential band, however, intra-epithelial lymphocytic infiltration is not present. although occasional eosinophils may be seen within the submucosa, there is no evidence of epithelial damage, neutrophils, necrosis, ulceration, or significant amount of nuclear debris. this grade combines and replaces the 1996 working formulation b1 and b2 grades. in high grade small airways inflammation there is marked lymphocytic infiltrate of the airway epithelium and airway wall. the mononuclear cells in the submucosa appear larger and activated with greater numbers of eosinophils and plasmacytoid cells. in addition, there is evidence of epithelial damage including necrosis, metaplasia, and marked intra-epithelial lymphocytic infiltration. in its most severe form, high grade airways inflammation is associated with epithelial ulceration, fibrino-purulent exudate, cellular debris, and neutrophils. it is important to exclude an infectious process. in this grade the changes are ungradeable due to sampling problems, infection, tangential cutting, artifact etc. the working formulation for pulmonary rejection has equated ob with one type of chronic rejection, the c-grade. the term as used in the consensus classification is restricted to submucosal and intraluminal scarring of membranous and respiratory bronchioles. when large tissue sections of lung are examined, the process of ob is pan-lobar but patchy. the small airways appear similar in size to the accompanying artery with a ragged inner surface. fibrosis is not present. narrowing of the airways due to fibrosis in the airway wall is seen. the fibrosis is usually eccentric (see fig. 7 .6). ob may be manifest by a histopathologic spectrum of changes depending on the acuteness of the process, the degree of organization and the amount of accompanying inflammation. in the acute phase there is usually loose myxoid granulation tissue with variable numbers of inflammatory cells filling or partially obstructing the airway lumen, resembling "organizing pneumonia." in later phases, ob may consist of eccentric, occasionally confluent plaques of dense hyalinized collagen applied to the wall of bronchioles. metaplastic squamous or cuboidal epithelium may cover these bronchiolar scars. in other airways a slit-like lumen may remain as a result of a confluent submucosal scar or intraluminal polyps of scar tissue. capillaries supplying these intraluminal masses of collagen are occasionally prominent. in the most severe cases the bronchiolar lumen can be entirely occluded by dense scar tissue and be recognizable only with the aid of an elastic stain, its location adjacent to an artery, and by the presence of residual circumferential smooth muscle. in the later phases, inflammation may be minimal. usually, the scarring process is confined exclusively to respiratory bronchioles and terminal bronchioles, although it may occasionally involve adjacent alveoli. the pulmonary arteries appear of a similar size as the accompanying airway. the intima is slender, the media not thickened. chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. wedge biopsies, explants, or autopsy material may reveal it. in the typical case, pulmonary arteries and more often veins are thickened by fibrointimal connective tissue (see fig. 7.7) . also, thickening is usually concentric. chronic vascular rejection may be patchy and typically involves smaller vascular arteries and veins. in the early stages of chronic vascular rejection, the intimal proliferation occurs on the luminal aspect of an intact elastic lamella. subsequently, the internal elastica may become fragmented and discontinuous. occasionally the underlying muscular wall becomes thinned. in approximately half of the reported cases a concurrent endovasculitis has been observed. the process is similar in pulmonary veins, although the intimal deposits may be less cellular and more waxy, eosinophilic, and sclerotic. chronic vascular rejection should be distinguished from recanalizing thrombi. unlike the situation with heart transplant recipients, chronic vascular rejection in lung transplants has not resulted in graft loss; however, some patients develop pulmonary hypertension particularly those with bos [92, 111] . mimickers of severe acute rejection include conditions that might present with acute lung injury or dad. these conditions include infection, drug toxicity, antibody mediated rejection (amr), or harvest/reperfusion injury. therefore, careful slide review for perivascular inflammation should be performed, stains for microorganisms including gomori-grocott methenamine silver stain (gms) and acid fast bacilli (afb) might be added and careful search for viral inclusions should be undertaken. although perivascular mononuclear infiltrates are helpful to identify rejection, these are not entirely specific for acute rejection and many other conditions may simulate or mimic alloreactive lung injury [126] . differential diagnostic considerations include cmv pneumonitis, pneumocystis jiroveci pneumonia and posttransplantation lymphoproliferative disease (ptld). further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases. amr or humoral rejection is well established in other solid organ transplantation. it was originally recognized in kidney transplant patients who presented with acute allograft rejection, antidonor antibodies, and poor prognosis [125] . amr is thought to be due to circulating antibodies that are either preformed because of pregnancy, blood transfusion, or previous organ transplantation or arise de novo after transplantation due to hla-mismatch. although amr is an increasingly recognized entity in lung transplantation, it is still under investigation. early observations were based on the phenomenon of hyperacute rejection, where preexisting donor-specific antibodies lead to complement activation and rapid graft loss. later it has been recognized that lung transplant recipients also can develop antibodies after transplantation to the allograft that might lead to amr. evidence suggests that amr occurs to donor major histocompatibility (mhc) antigens, although other endothelial and epithelial antigens expressed in the lung may become antibody targets as well. the recent development of very sensitive and specific solid phase flowcytometry and luminex-based methodologies has allowed for accurate detection of antibody specificities in sensitized recipients and it has become clear that more patients than previously expected present with preformed anti-hla antibodies. immune stimulation by prior infections or autoimmunity might contribute to the development of antibodies to allomhc in those patients with no identifiable risk factors. these preexisting antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection) or accelerated humoral rejection and bos [23] . furthermore, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) [48] , persistent rejection, increased bos [96] or worse overall survival [56] in patients with anti-hla antibodies. this effect is apparent both with pretransplant hla sensitization and with the development of de novo anti-hla donorspecific antibodies after transplantation [96] . about 10-15% of lung transplant recipients are presensitized to hla antigens [3] . even though "unacceptable antigens" are avoided during the virtual crossmatch, patients with positive pretransplant pra are at higher risk for posttransplant complications. their posttransplant pra can stay stable or increase via generation of either donor-specific or nondonor-specific anti-hla antibodies. similarly, patients that had negative pra screening tests before transplantation can develop de novo nondonor-specific or donor-specific anti-hla antibodies after transplantation. the mechanisms by which antibody promotes lung allograft injury remain poorly understood. antibody binding to allomhc or other endothelial or epithelial targets in the lung could lead to activation of the complement cascade with complement deposits leading to endothelial cell injury, production of proinflammmatory molecules, and recruitment of inflammatory cells. com plement independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation [23] . as demonstrated by in vitro studies, anti-hla antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors [64] , potentially contributing to the generation of obliterative airway lesions. recent studies have attempted to evaluate immunoglobulins (ig) and complement deposits in the subendothelial space. septal capillary deposits of igs and complement products such as c1q, c3d, c4d, and c5b-9 have been described in association with anti-hla antibodies [62, 90] as well as allograft dysfunction and bos [82, 139] . the concept of specific histopathologic features associated with humoral rejection remains controversial in lung transplantation. recent studies question the relation between complement or ig staining and allograft rejection [112, 138] . others demonstrate that c3d and c4d staining can occur in lung transplant recipients with nonalloimmune lung injury such as infection and primary graft dysfunction (pgd) with no evidence of anti-hla antibodies [139] . differences in staining techniques between different laboratories may further explain some of the inconsistencies in the published data. the 2007 ishlt revised consensus classification did not agree upon any histopathologic features that might be specific for amr in lung [123] . although capillary injury/small vessel intimitis might raise the suspicion of amr, these are nonspecific findings that can also occur in severe acute rejection, infection, or harvest/reperfusion injury. furthermore, although often the term "capillaritis" is mentioned, ishlt recommends to use the term "capillary injury" which allows for a broader spectrum of morphological changes of the vessels including intimitis, whole wall thickness inflammation, neutrophilic infiltration (which should be distinguished from neutrophilic margination), thrombosis, and necrosis. furthermore, signs of dad and intra-alveolar hemorrhage can occur with amr although again, they are not specific. given the lack of specific histologic findings of amr in lung transplantation, a multidisciplinary approach to diagnosis is recommended that includes the following: (1) the presence of circulating antibodies (hla antibodies, antiendothelial and antiepithelial antibodies), (2) focal or diffuse c4d deposition (see fig. 7 .8), (3) histologic features of acute lung injury or hemorrhage (dad, capillary injury with neutrophils and nuclear debris) and (4) clinical signs of graft dysfunction. if amr is clinically, immunopathologically, or histologically suspected, one might perform immunostains for c3d, c4d, cd68, and cd31. however, these stains are extrapolates from kidney and heart transplantation and although there have been several studies advocating their use [47, 62, 123, 139] , there are no large scale studies validating their use. c4d has been studied extensively in kidney and heart transplant and has been identified as a useful adjunct in the diagnosis of amr in these organs. however, in lung that is not the case. one of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemistry (ihc) as well as immunofluorescence (if). often, c4d binds to elastic laminas or shows other nonspecific binding. staining is commonly only focal and therefore sensitivity and specificity have not been established yet given the limited sample size of transbronchial biopsies. only linear, continuous subendothelial staining of capillaries, arterioles, and/or venules count as positive by ihc. also, c4d is not specific to amr but also can be seen in infection, harvest/ reperfusion injury, or even in severe acute rejection, basically in any process that is associated with complement activation. there is no ihslt recommendation at this time regarding to the coexistence of amr and acute rejection. although clinical evaluation of the patient may suggest the possibility of rejection, tissue evaluation is necessary for a definitive diagnosis. initially, investigators were hesitant to routinely utilize transbronchial biopsy to monitor the graft as it was thought that the amount of tissue obtained would be too small to make a definitive diagnosis. there was also concern about passing a bronchoscope over the anastomosis. however, transbronchial biopsy is now a routine procedure and has become the gold standard to evaluate the graft for acute and chronic rejection, infection, and possible recurrent disease since currently, no surrogate markers have been sufficiently validated as means to reproducibly identify patients with acute rejection. specifically, there is no serological marker to indicate rejection is occurring. after lung transplantation the total bal fluid cell count is constantly increased even in periods with no evidence of infection or rejection [127] . in the early posttransplantation period (first 4 weeks), there is a dominance of neutrophils in the bal fluid (up to 25-50% of total cell count) until about 3 months later when the cell count normalizes [127] . acute rejection has been associated with elevated cd8+ t cells, activated cd4+ t cells, a trend toward increased nk t cells, increased b cells, and decreased nk cells in the bal [53] . nevertheless, no study has proven the bal cellular composition to be adequately sensitive or specific in the discrimination of rejection from infection [107] . small studies have found a correlation between acute rejection and elevation of il17 [134] , il15 [10] , and ifn gamma in the bal [10] . recent advantages in genomics offer the potential for more specific means of diagnosing rejection in lung transplantation. a pilot study of gene expression in the bal of lung transplant recipients found that gene expression signatures related to t-lymphocyte function, cytotoxic cd8 activity, and neutrophil degranulation correlate with acute rejection [98] . however, these studies have no clinical use at the present time. recently published studies in heart transplantation describe the use of peripheral blood gene expression profiling to identify future risk of cardiac allograft rejection [88] . a similar study is now underway in lung transplantation, known as the lung allograft rejection gene expression observational (largo) study. preliminary data from almost 900 patients, similar to the cargo results, show differential gene expression in the lymphocyte priming and neutrophil homeostasis pathways for a0 vs. ³a2 acute lung rejection [66] . such testing may hold promise for a noninvasive technique to monitor the status of the transplanted organs. another study described marked serum elevations of hepatocyte growth factor (hgf) in lung transplant . the high power view shows capillaritis in a case of amr characterized by focally thickened interstitium due to neutrophils and nuclear debris with capillary destruction. the insert reveals c4d immunoperoxidase stain (see arrow) focally lining the endothelium of capillaries and small vessels (magnification × 400, insert × 600) recipients with acute rejection. however, smaller elevations in hgf also occurred with lung infection, and additional studies are needed to validate specificity and sensitivity of hgf for acute rejection [1] . in conclusion, although there are a few promising noninvasive techniques, these are early studies that need to be confirmed in larger studies before being considered for widespread clinical use. therefore, microscopic tissue evaluation remains the gold standard for diagnosing rejection. bronchoscopy and biopsy postlung transplantation are generally performed if there is any clinical suspicion of rejection, infection, recurrent disease, or ptld. in addition, many transplant centers now follow protocol biopsies. however, the pros and cons of surveillance bronchoscopies with biopsy are still debated and therefore, there are no guidelines established in regards to the timing of postlung transplantation surveillance bronchoscopy and biopsy. one institution reports surveillance biopsies in pediatric lung transplant recipients at 1 week and 1, 3, 6, and 12 months posttransplantation [8] . the rational for surveillance biopsies includes the occurrence of clinically silent acute rejection, inadequate surrogate markers for acute rejection, and the relatively low risk of the bronchoscopy procedure. the yield for acute rejection by transbronchial biopsies was reported 6.1-31% and 25% or greater in studies performing surveillance transbronchial biopsies [17, 60] , clinically indicated and follow-up bronchoscopies [18] . grade a2 and higher acute rejection has been found in a relatively high percentage of asymptomatic patients, ranging from 22 to 39% [54, 131] . silent acute rejection appears most common within the first 3 months of transplantation (24.8% at 0-3 months; 16.7% at 3-12 months; 2.7% after 1 year) [87] . the rate of unsuspected but clinically significant infection was highest between 3 and 12 months posttransplantation but a relatively high rate (18.9%) was also detected after 1 year. one small study questioned the benefits of performing surveillance bronchoscopy with biopsy, suggesting that the benefits do not outweigh the procedural risks [132] . in this study, changes in management based on transbronchial biopsy and bal results were significantly higher in the clinically indicated group (65%) compared with the surveillance group (13%) and were mostly related to infection. interestingly, no silent acute rejection episode requiring treatment was detected in the surveillance group of this study. collectively, these findings may reflect differences in programs and operators. although it has been shown that transbronchial biopsy is often the only mean to reveal silent acute rejection of the allograft, definitive evidence that treatment of such episodes have a positive impact on survival or prevention of bos has yet to be demonstrated. however, based on the link between acute rejection and development of bos, surveillance transbronchial biopsies in asymptomatic lung transplant recipients has become common practice in many large lung transplantation centers because evidence suggests that patients who have multiple episodes of low grade (a1) lesions within the first 12 months posttransplantation develop early onset bos. however, there were no differences in overall survival in patients with and without a1 acute rejection [59] . there is also evidence that a1 rejection increases the risk of higher-grade subsequent rejections (³a2) [13, 34] . the finding of a solitary perivascular monocytic infiltrate was followed by worsening acute rejection in four untreated patients in one study, while the treatment of such a solitary infiltrate in nine patients resulted in improvement of the rejection score [67] . the presence of severe lymphocytic bronchiolitis also has been associated with increased risk of bos and death after lung transplantation, independent of the presence of acute rejection [49] . a study [49] in which surveillance transbronchial biopsies were performed at 3, 6, 9, and 12 weeks posttransplantation, at the time of symptoms, and for follow-up of acute rejection or cmv pneumonia showed that patients who develop acute small airways rejection within the first year after transplantation are at risk of development of bos at 1.76, 3.3, and 5.5 years after detection of b3/ b4 lesion (by 1996 ishlt criteria, see table 7 .2), b2 lesion or b0/b1 lesion, respectively. this study strongly suggests that the main utility of surveillance biopsies may be to use lymphocytic bronchiolitis as a surrogate predictor of long-term outcome. however, the impact of therapy for lymphocytic bronchiolitis has yet to be assessed. although complications related to bronchoscopy occur, including a small risk of severe complications, the majority of cases have a favorable outcome. adverse events reported with bronchoscopy in lung transplant recipients include transient hypoxemia, bleeding, pneumothorax, arrhythmia, and anesthesia related complications [74, 87] . bronchoscopy may have contributed to faster deterioration of one patient's already critical condition; however, in the posttransplantation setting, -bronchoscopy has no reported mortality [18, 60] , although there are anecdotal reports of death related to transbronchial biopsy. the 2007 ishlt revised consensus classification of acute allograft rejection [123] requires the evaluation of at least five pieces of well-expanded alveolated parenchyma. however, the bronchoscopist may need to submit more than five pieces to provide this minimum number. further biopsies may improve the detection of ob, although there is no specific number of small airways required by the ishlt consensus classification to exclude this diagnosis. gentle agitation in formalin to open up the alveoli may improve the histologic appearance of the fragments. histologic examination should include three levels from the paraffin block for hematoxylin and eosin (h&e) staining [123] . connective tissue stains such as trichrome or verhoeff-van gieson (vvg) stain to evaluate airways for the presence of submucosal fibrosis are essential for the diagnosis of ob, and atherosclerosis. silver stains such as gms can be performed for fungi, including pneumocystis, but have not been routinely mandated by the 2007 ishlt revised consensus classification, given the numerous microbiologic, serologic, and molecular techniques available for the diagnosis of opportunistic infections. bal may be performed at the time of biopsy and is useful for the exclusion of infection, but currently has no clinical role in the diagnosis of acute rejection (see above). infection is the leading cause of death in lung transplant recipients. factors that increase a patient's susceptibility to infection after transplantation include immunosuppression, reduced mucociliary clearance, decreased cough reflex resulting from denervation, and interruption of lymphatic drainage. bacterial pneumonias are the most common infections following lung transplantation [26] and occur in more than 35% of patients during the first year after transplantation (highest incidence is during the first month posttransplantation) (see fig. 7 .9). furthermore, bacterial pneumonia remains a major infectious complication throughout the patient's life. the donor lung is affected most often. gram-negative organisms are most common, especially enterobacter and pseudomonas. bronchitis secondary to pseudomonas species or staphylococcus aureus infection also is observed. bacterial pneumonia typically manifests radiographically as a lobar or multilobar consolidation. viral pneumonias develop in approximately 11% of patients who have undergone lung transplantation. they occur at any time following transplantation. cmv is the second most common cause of pneumonia in patients who have received lung transplants, and it is the most common opportunistic infection (35-60% of opportunistic infection) [26] . it represents the most significant viral infection, and usually occurs 1-4 months after transplantation. primary infection is the most serious form and is observed in 50-100% of seronegative patients who received a graft from a seropositive donor. in patients who are seropositive, secondary cmv infection develops from reactivation of latent disease following the institution of immunosuppressive therapy or from infection with a different strain of cmv. infected patients may be asymptomatic or may develop a fulminant pneumonia, possibly with extrathoracic findings such as retinitis, hepatitis, and gastritis. presenting symptoms include dyspnea, fever, and cough. the most common finding on chest radiographs in patients with cmv infection is diffuse parenchymal haziness. ct scan findings include areas of ground-glass attenuation; reticulation; multiple, small, ill-defined 1-to 3-mm nodules; and, even less commonly, areas of dense consolidation. the diagnosis of cmv pneumonia can be made by bronchoscopy with lavage and biopsy. cytopathic changes associated with cmv infection include cytomegaly, multiple small basophilic cytoplasmic inclusions, and a large nuclear inclusion surrounded by a halo with thickened nuclear membrane (see fig. 7 .10). prophylactic therapy with acyclovir and immune globulin has not reduced the incidence of cmv infections in patients who have undergone transplant procedures. a less common cause of viral infection includes the herpes simplex virus (hsv) infection. patients with hsv infection present with fever, cough, and dyspnea, but they demonstrate symptomatic improvement after therapy with intravenous acyclovir. radiographic findings may be absent or may demonstrate diffuse groundglass opacities. histopathologically, the classic hsv inclusion consists of a dense, eosinophilic mass within the nucleus that is surrounded by a clear halo and peripherally marginated, beaded nuclear chromatin. hsv may form multinucleated cells. opportunistic fungal infections are less common than viral infections, but they are associated with higher mortality. fungal pneumonias usually occur 10-60 days following transplantation and more commonly involve the transplanted lung. however, they also can involve the native lung in single lung transplantation cases, especially in patients with copd. ct imaging studies most commonly reveal a combination of nodules (multiple, variable sizes, irregular margins), consolidation, and ground-glass opacification. pleural effusions are also common (63% of cases). gms will help to identify fungal organisms. however, even in the absence of identifiable organisms on gms, infection might be considered and cultures and serology should be attempted. locally invasive or disseminated aspergillus infection accounts for 2-33% of posttransplantation infections and 4-7% of deaths in patients who undergo lung transplantation. aspergillus infection most commonly is characterized by local invasion of a necrotic bronchial anastomosis (i.e., ulcerative aspergillus infection, when involving the lung parenchyma, tends to cavitate and has an upper-lobe predominance. histopathologically, the hyphae of aspergillus sp are septated and branching. they appear uniform and grow in parallel fashion with septae at regular intervals. the branching is dichotomous and usually at 45°angle. inhaled amphotericin b is often used in the immediate posttransplantation period to help eliminate this complication [26] . patients are also discharged on voriconazole as daily prophylaxis for the first year. patients who have undergone lung transplant procedures have an increased susceptibility to p jiroveci infection, but prophylaxis with trimethoprim-sulfamethoxazole is effective in preventing the infection (incidence is nearly 0%). without prophylaxis, the incidence of p jiroveci infection approaches 90%. histologically, cysts of p jiroveci are round to oval, measure 5-7 mm in diameter, and often have very prominent grooves or folds. p jiroveci pneumonia classically consists of an intra-alveolar foamy exudate in which the organisms appear as small "bubbles" in a background of proteinaceous exudate, although a variety of other reactions can occur as well [129] . the incidence of ptld is significantly higher after thoracic organ transplantation compared with any other solid-organ transplant [94] . they develop in 4-10% of lung transplant recipients, as opposed to an approximate 2% incidence in other solid organ recipients, with 60% of the ptlds presenting in the allograft. in adults, ptlds are the most common neoplasms at 1 year posttransplantation (peak, 3-4 months posttransplantation) [21] . in children [6] , lymphomas are by far the most common posttransplantation malignancy at any time. in fact, ptld is a major cause of morbidity and mortality in pediatric lung transplant recipients [12] . risk factors for ptld after solid organ transplantation include patient's age, type of organ transplanted, ebv infection status of host before transplantation, and the immunosuppressive regimen [4, 77, 93, 140] . ptlds are a clinically, morphologically, and molecularly heterogeneous spectrum of lymphoproliferative disorders, ranging from early, ebv-driven, polyclonal, polymorphic proliferations (infectious, "mononucleosis-like") to ebv-positive or ebv-negative monomorphic tumors [15, 69, 70, 100] . ptlds most commonly are associated with ebv infection of b-cells either by reactivation of latent virus or primary ebv, most commonly acquired from donor organs [12] . a recent study showed [37] that five of seven lung-transplanted children who developed ptld were ebv-negative recipients who received ebv-positive lungs. in a series [81] of 988 heart and/or lung transplant recipients, 17 ptlds were identified. amongst the 17 ptld cases, there were two b-cell monoclonal polymorphic ptlds and 15 b-cell monomorphic ptlds (see fig. 7 .11) (13 diffuse large b-cell lymphomas (dlbcl) and two burkitt lymphomas). ebv was detected in 9 of the 17 patients. all cases showed monoclonal igv gene rearrangements; igv somatic hypermutation was found in 88% of cases, indicating a prevalent origin from germinal center-experienced b cells. given the association between ebv infection and ptld, elevation in ebv viral load measured in peripheral blood has been proposed as a marker for the development of ptld [109] . in addition, monitoring the degree of immunosuppression after diagnosis of ptld with immune function assays and immunosuppressive drug levels may aid clinicians in assessing the risk for ptld and monitoring treatment after diagnosis [45] . t-cell ptlds tend to occur later and tend not to be associated with ebv infection. t-cell ptlds are associated with a worse prognosis than b-cell ptld. patients with ptlds may be asymptomatic, or they may have nonspecific complaints such as fever, weight loss, dyspnea, and lethargy. solitary or multiple pulmonary nodules ranging in size from 0.1 to 5 cm are the most common pulmonary manifestation of patients with ptlds. mediastinal and hilar adenopathy also can be observed in 22-50% of cases. patients who present with a solitary pulmonary nodule have a better overall prognosis. however, most ptlds have a rapid onset, a predilection for extranodal sites, and an aggressive clinical behavior with poor outcome [15, 69, 70, 78, 100] . as a result of the high mortality associated with ptld, treatment often poses a challenge. the most common treatment of ptld has included decreasing immunosuppression in combination with surgical resection, antiviral agents, radiation, and chemotherapy [45, 103, 109] . the reduction or withdrawal of immunosuppressive therapy may result in a partial or complete regression of the ptld [69] . a higher risk of graft rejection associated with ptld treatment is one reason for the need for early detection of the disease [45, 102, 103, 109] . in adults, malignancy remains a common complication after lung transplantation [21] . among lung transplantation survivors, 3.5, 12.6, and 28.1% have a malignancy at 1, 5, and 10 years posttransplantation, respectively. although lymphoproliferative disorders are most common at 1 year posttransplantation, nonmelanoma skin malignancy is the leading malignancy at 5 and 10 years posttransplantation. in children [6] , the incidence of posttransplantation malignancy is higher than in adults early after transplantation with 5.9% of children developing them within the first year after transplantation. at 5 years posttransplantation, 13.1% were found to have a malignancy. at any time posttransplantation, lymphomas are by far the most common posttransplantation malignancy in children. the risk for cancer is higher following lung transplantation than after kidney transplantation [110, 121] . lung transplant patients have unique characteristics, as their baseline genetic and environmental background, and immunosuppressive regimens are different from patients receiving kidney transplant, all of which may contribute to the increased risk [2] . besides nonmelanoma skin cancer and lymphoproliferative disorders, other malignancies repor ted to be associated with solid organ transplantation include: kaposi's sarcoma, anogenital cancers, oral cavity malignancies, esophageal and urinary bladder cancer, hepatocellular carcinoma, and sarcomas [110, 121] . acute graft vs. host disease (gvhd) is an uncommon and usually fatal complication of lung transplantation for which no effective therapy exists. among the ten reported patients, eight had grade 3 to 4 acute gvhd and died within 208 days. there is one recent case report [42] of a patient with grade 3 to 4 acute gvhd after blt who was successfully treated with highdose corticosteroids after basiliximab and extracorporeal photopheresis were unsuccessful. after 26 days the patient had developed low grade fever, chills, and pruritic maculopapular rash on the chest that spread to the proximal extremities in the next 4 days. liver function tests were elevated. a skin biopsy showed dermal inflammation, patchy loss of basal layer with vacuolar degeneration, apoptosis, and cell necrosis suggesting gvhd. a few days later, profuse, green, watery diarrhea developed with abdominal pain and paralytic ileus. colonoscopy showed ulceration of mucosa and destruction of intestinal crypts, features compatible with gvhd. there was leucopenia, severe anemia, and deterioration of liver function. the diagnosis was confirmed by chimerism study that revealed a recipient/donor lymphocyte ratio of 50:50. a second case of acute gvhd after lung transplantation was successfully treated with corticosteroids [5] . this patient presented with mild symptoms (pruritic rash and blurry vision) suggesting low-grade gvhd. the diagnosis was confirmed when a small dose of prednisone resulted in the resolution of gvhd within 3 weeks. lung harvest/reperfusion (ischemia/reperfusion, i/r) injury after transplantation remains the most common cause of early posttransplantation respiratory failure and manifests typically during the first 72 h after transplant [75] . reported rates are as great as 41% [52] . the 30-day mortality of patients with i/r injury is about 40%, compared with 7% in patients without i/r injury [86] . the clinical equivalent to i/r injury is pgd. the ishlt working group on pgd proposed a four-tiered grading scheme of pgd based on pao 2 /fio 2 (p/f) ratio and radiological chest infiltrates assessed at time points up to 72 h: t (0-within 6 h of reperfusion, 24, 48, and 72 h) [20] . i/r injury usually presents with the immediate impairment in lung function after transplantation accompanied by rapid development of pulmonary edema, increased pulmonary vascular resistance, and decreased airway compliance. patients with i/r injury require prolonged mechanical ventilation with greater hospital stays and are at an increased risk of multiorgan failure. lung i/r injury has long-term consequences and is a risk factor for late graft failure (ob) [39, 40] . i/r injury can occur due to prolonged ischemia during transplantation. ischemic injury to the pulmonary vascular endothelium increases permeability and results in pulmonary edema. histologically, i/r injury presents as acute lung injury pattern including dad (see fig. 7 .12). perivascular infiltrates are usually not present and distinguish it from acute rejection. however, infection can present similarly and needs to be excluded with stains, cultures, and serology. the pathophysiology of lung i/r injury remains incompletely understood. the lungs are particularly susceptible to i/r injury, likely owing to the rich vascularity and relatively large surface area over which blood-borne components interact with the endothelium. the mechanisms of i/r injury are diverse and include generation of reactive oxygen species (ros), leukocyte activation/recruitment, complement and platelet activation, abnormalities in pulmonary vascular tone, and increased procoagulant activity. the production of proinflammatory cytokines is increased considerably in the lung after i/r. expression of cytokines in the lung after i/r may not only cause immediate tissue injury, but may predispose the lung allograft to rejection. several studies suggest that lung i/r injury is biphasic, with a distinct, acute injury characterized by macrophage activation followed by a later, neutrophildependent injury [39] . although recurrent disease in the allograft has been reported in some of the transplantation cases, in general, this has not been clinically significant. diseases for which recurrences in the allograft have been reported include giant cell interstitial pneumonia, sarcoidosis, lymphangioleiomyomatosis, langerhans' cell histiocytosis, allergic bronchopulmonary aspergillosis, desquamative interstitial pneumonia, bronchioloalveolar carcinoma, alveolar proteinosis, and diffuse pan bronchiolitis. morphological features are identical to the disease in nontransplanted lung. bronchial dehiscence is the most common anastomotic airway complication in the early postoperative period. it occurs in 2-3% of cases. ischemia at the anastomotic site is the major factor in the development of this complication. dehiscence probably is best assessed by bronchoscopy; however, ct scans typically demonstrate the presence of extraluminal gas, which is 100% sensitive and 72% specific for dehiscence. patients with telescoping anastomoses also may develop small anastomotic diverticula, which appear as smooth rounded air collections at the inferior-medial aspect of the anastomosis. anastomotic stricture occurs in approximately 10% of cases, and the risk for stenosis may be increased with a telescoping anastomosis. stenoses often manifest with progressive airflow obstruction that can be difficult to differentiate from other causes, such as acute rejection or bos. stricture probably is best evaluated by bronchoscopy; however, ct scans often demonstrate the area of narrowing. treatment is stenting, typically with an expandable metallic stent. more recently, balloon dilatation has obviated the need for stents in some centers. app 43% of lung transplantations between 1995 and 2008 were single lung transplants (slt) [21] . copd, aat, and ipf accounted for app 85% of the indications for slts [21] . advantages of slt over blt include a technically easier procedure, shorter surgery time, and the ability to transplant two patients from one donor. however, there are disadvantages to slt. for instance, survival rates for slt and blt recipients are different, diverging most obviously in later years after transplantation. 5-year survival for slt was 46% compared with 54% for blt [130] . other disadvantages of slt include less pulmonary reserve, and the propensity for complications related to the residual native lung. native lung complications have previously been reported in 13.8-50% of patients. they may be associated with significant morbidity and mortality [85, 135] and may partly explain why outcomes with slt are inferior to those of blt. a recent study [68] reported the occurrence of pneumothoraces, malignancy, aspergilloma, pneumonia, bronchopleural fistulas, and pulmonary embolism. some forms of advanced lung disease, including copd and ipf, are associated with increased risk for lung cancer, a complication that can arise in the native lung after transplantation [76, 142] . patients also often have structural damage to the native lung from their underlying disease process, which can increase the risk of other complications such as aspergilloma and pneumothoraces. the median time from transplantation to major native lung complication was 1.28 years (range, 0.04-5.1 years). in one study [68] , 8 of 18 patients with native lung complications died thereof. median posttransplant survival was lower in slt recipient with significant native lung complications (3.2 vs. 5.3 years, p = 0.004). native lung pneumonectomy was performed in 11 patients. in patients with native lung complications there was a trend toward improved posttransplant survival in patients who underwent native lung pneumonectomy compared with those who did not undergo pneumonectomy. however, there was no difference in survival between patients who underwent native lung pneumonectomy to those who had no native lung complication. the majority of patients with ob also have severe bronchiectasis. by specimen bronchograms, the bronchial tree shows alternating areas of dilatation and constriction. microscopically, the bronchiectasis may be associated with areas of mucous plugging, goblet cell hyperplasia, squamous metaplasia, denudation of the bronchial epithelium, submucosal scarring, and acute and chronic inflammation of the bronchial wall. occasionally foreign body giant cells are present, probably representing a manifestation of aspiration. obliteration of the terminal respiratory bronchioles is often observed distal to these areas. the bronchiectasis of lung allografts is probably the result of several factors including immune-related injury, infection, mucostasis, aspiration, and loss of innervation. despite advances in operative management, lung preservation, critical care, and immunosuppression, longterm survival in lung transplantation remains limited. in fact, outcomes for lung transplantation are the worst of any solid organ transplant [75] . the main obstacles to present day lung transplantation involve: (1) lack of donor organs, (2) i/r injury, (3) acute rejection, and (4) development of ob. the increased susceptibility of the lung to injury, infection, and constant environmental exposure with local innate immune activation likely contributes to the high rates of rejection. the ishlt registry reports a 1-year survival rate of 78% and 5-year survival rate of 52% [21] . mortality is highest in the first year, which consistently decreases across subsequent time periods (see table 7 .3). in the first 30 days, graft failure, non-cmv infections, cardiovascular complications, and technical problems account for most of the mortality. after the first year, bos and non-cmv infections were the predominant causes of death. by 5 years, malignancies and cardiovascular causes account for almost 17% of reported causes of death. however, compared with data beginning in 1988, overall survival has consistently improved by era. the improvement in survival is largely due to improvement in the 1 year survival. long term survival has improved as well, although to a seemingly lesser degree. evidence suggests that age, pretransplant diagnosis, and donor cmv status play important prognostic roles. for instance, the survival half-life for patients older than 65 years of age was 3.2 years compared with 6.3 years for those aged 35-49. overall survival rates at 3 months after transplantation are lowest for ipf (86%) and ipah (78%) and highest for cf (91%) and copd (91%), most likely due to differences in early complications, including pgd [21] . cmv seropositivity of the donor is associated with worse survival; however, the underlying reasons for this association are not entirely clear. inducing a state of immune suppression is the key to successful clinical lung transplantation. the immunosuppressive regimens used for lung transplantation are based on successful protocols that have evolved for renal and heart transplantation. posttransplantation morbidities (see table 7 .4) present at 5 years are those commonly caused or exacerbated by immunosuppressive medicines, including hypertension, renal dysfunction, and dyslipidemia. bos and malignancies are other common posttransplantation morbidities. for instance, bos had developed in 28% of patients by 2.5 years after transplantation and in 74% by 10 years. however, most surviving patients reported no activity limitations at 1, 3, 5, and 10 years (>80% at each time point). furthermore, 13% of survivors reported at least one malignancy at 5 years after transplantation, and 28% were affected by malignancies at 10 years. survival after pediatric lung transplantation is similar to that reported in adults with a median survival of 4.5 years for the period 1990-june 2007. but, results are clearly improving [6] . one and 5-year survival rates for pediatric recipients transplanted in the most ob obliterative bronchiolitis recent era (2002-6/2007) are 83 and 50%, respectively, compared with 67 and 43% for recipients transplanted between1988 and 1994. graft failure, technical issues, cardiovascular failure, and infection are the most common causes of pediatric death in the early posttransplant period whereas infection, graft failure and bos are the most common causes of late death. the prevalence of bos steadily increases with time posttransplantation. as expected, the cumulative incidence of malignancy also increases with time after transplantation, with lymphoproliferative disorders making up the great majority of reported malignancies in children. despite the complications, the functional status of the great majority of long-term pediatric survivors is very good, with 84% of 5-year survivors reporting no limitations in activity. a total of 57 pediatric retransplant procedures were reported between january 1994 and june 2008. the majority of these procedure were performed >12 months after the initial transplantation. survival over this period was slightly poorer than for primary transplantations, being 41% at 5 years. prediction of lung-transplant rejection by hepatocyte growth factor development of malignancy following lung transplantation utility of peritransplant and rescue intravenous immunoglobulin and extracorporeal immunoadsorption in lung transplant recipients sensitized to hla antigens posttransplant lymphoproliferative disease in thoracic organ transplant patients: ten years of cyclosporine-based immunosuppression graft-vs.-host disease in lung and other solid organ transplant recipients registry of the international society for heart and lung transplantation: twelfth official pediatric lung and heart/lung transplantation report-2009 limitations of spirometry in detecting rejection after singlelung transplantation surveillance bronchoscopy in children during the first year after lung transplantation: is it worth it? a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: lung rejection study group. the international society for heart transplantation elevation of interleukin-15 protein expression in bronchoalveolar fluid in acute lung allograft rejection hyperacute rejection in single lung transplantation-case report of successful management by means of plasmapheresis and antithymocyte globulin treatment posttransplantation lymphoproliferative disorders in pediatric thoracic organ recipients interstitial inflammatory lesions of the pulmonary allograft: a retrospective analysis of 2697 transbronchial biopsies comparison between referral diagnosis of patients requiring transplantation and pathologic diagnosis of native lungs the morphologic and molecular genetic categories of posttransplantation lymphoproliferative disorders are clinically relevant reliability for grading acute rejection and airway inflammation after lung transplantation yield of surveillance bronchoscopy for acute rejection and lymphocytic bronchitis/bronchiolitis after lung transplantation diagnostic yield and therapeutic impact of flexible bronchoscopy in lung transplant recipients hyperacute rejection of a pulmonary allograft. immediate clinical and pathologic findings report of the ishlt working group on primary lung graft dysfunction part ii: definition. a consensus statement of the international society for heart and lung transplantation the registry of the international society for heart and lung transplantation: twenty-sixth official adult lung and heart-lung transplantation report-2009 analysis of the different histologic lesions observed in transbronchial biopsy for the diagnosis of acute rejection. clinicopathologic correlations during the first 6 months after lung transplantation antibody-mediated organallograft rejection a working formulation for the standardization of nomenclature and for clinical staging of chronic dysfunction in lung allografts. international society for heart and lung transplantation doublelung transplant for advanced chronic obstructive lung disease infectious complications in pulmonary allograft recipients lung transplantation for pulmonary langerhans' cell histiocytosis: a multicenter analysis improved lung allograft function after fundoplication in patients with gastroesophageal reflux disease undergoing lung transplantation hyperacute rejection after single lung transplantation: a case report management of lung transplant recipients with bronchogenic carcinoma in the native lung inhaled corticosteroids and the treatment of lymphocytic bronchiolitis following lung transplantation are symptom reports useful for differentiating between acute rejection and pulmonary infection after lung transplantation? experimental transplantation of vital organs pattern and predictors of early rejection after lung transplantation improved results of lung transplantation for patients with cystic fibrosis lung transplantation posttransplant lymphoproliferative disease in pediatric lung transplant recipients: recent advances in monitoring bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response ischemiareperfusion injury after lung transplantation increases risk of late bronchiolitis obliterans syndrome non-immune acute graft injury after lung transplantation and the risk of subsequent bronchiolitis obliterans syndrome (bos) severe acute graft versus host disease after lung transplant: report of a case successfully treated with high dose corticosteroids hyperacute rejection following lung transplantation influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome use of an immune function assay to monitor immunosuppression for treatment of post-transplant lymphoproliferative disorder risk factors for the development of obliterative bronchiolitis after lung transplantation anti-human leukocyte antigen antibodies, vascular c4d deposition and increased soluble c4d in broncho-alveolar lavage of lung allografts hlaspecific antibodies are associated with high-grade and persistent-recurrent lung allograft acute rejection severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation chlamydia pneumoniae infection after lung transplantation acute rejection following lung transplantation: limitations in accuracy of thin-section ct for diagnosis update of early respiratory failure in the lung transplant recipient bronchoalveolar immunologic profile of acute human lung transplant allograft rejection the importance of bronchoscopy with transbronchial biopsy and bronchoalveolar lavage in the management of lung transplant recipients the effect of recipient's age on lung transplant outcome pretransplant panel reactive antibody in lung transplant recipients is associated with significantly worse post-transplant survival in a multicenter study gastroesophageal reflux disease in lung transplant recipients lung homotransplantation in man association of minimal rejection in lung transplant recipients with obliterative bronchiolitis prospective analysis of 1, 235 transbronchial lung biopsies in lung transplant recipients rejection is reduced in thoracic organ recipients when transplanted in the first year of life c4d deposition in lung allografts is associated with circulating anti-hla alloantibody acute and chronic onset of bronchiolitis obliterans syndrome (bos): are they different entities? anti-hla class i antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome pleural effusion from acute lung rejection refining the identification of discriminatory genes for rejection in lung transplantation: the largo study significance of a solitary perivascular mononuclear infiltrate in lung allograft recipients with mild acute cellular rejection native lung complications in single-lung transplant recipients and the role of pneumonectomy immunodeficiency-associated lymphoproliferative disorders correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders the us experience with lung transplantation for pulmonary lymphangioleiomyomatosis the diagnosis of obliterative bronchiolitis after heart-lung and lung transplantation: low yield of transbronchial lung biopsy clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant lung transplantation at duke university medical center pulmonary inflammation after lung transplantation the incidence of cancer in patients with idiopathic pulmonary fibrosis and sarcoidosis in the uk posttransplant lymphoproliferative disorders not associated with epstein-barr virus: a distinct entity? identification of prognostic factors in 61 patients with posttransplantation lymphoproliferative disorders chronic aspiration of gastric fluid induces the development of obliterative bronchiolitis in rat lung transplants gi complications after orthotopic lung transplantation b-cell posttransplant lymphoproliferative disorders in heart and/or lungs recipients: clinical and molecular-histogenetic study of 17 cases from a single institution c3d and the septal microvasculature as a predictor of chronic lung allograft dysfunction selection and evaluation of recipients for heart-lung and lung transplantation outcomes of lung transplantation in patients with scleroderma complications (excluding hyperinflation) involving the native lung after single-lung transplantation: incidence, radiologic features, and clinical importance evolving strategies in lung transplantation for emphysema surveillance bronchoscopy in lung transplant recipients: risk versus benefit clinical implications and longitudinal alteration of peripheral blood transcriptional signals indicative of future cardiac allograft rejection lung transplantation: a decade of experience acute humoral rejection of human lung allografts and elevation of c4d in bronchoalveolar lavage fluid lung transplantation for end-stage pulmonary sarcoidosis: outcome in a series of seven consecutive patients pulmonary hypertension in patients with bronchiolitis obliterans syndrome listed for retransplantation epstein-barr virus-negative post-transplant lymphoproliferative disorders: a distinct entity? lymphomas after solid organ transplantation: a collaborative transplant study report general overview of lung transplantation and review of organ allocation development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome gastroesophageal reflux as a reversible cause of allograft dysfunction after lung transplantation bronchoalveolar lavage cell gene expression in acute lung rejection: development of a diagnostic classifier lung transplantation for lymphangioleiomyomatosis the changing pattern of posttransplant malignancies does human leukocyte antigen matching influence the outcome of lung transplantation? an analysis of 3, 549 lung transplantations lung retransplantation after ptld: a single center experience and review of literature of ptld in lung transplant recipients posttransplant lymphoproliferative disorder: incidence, presentation, and response to treatment in lung transplant recipients alemtuzumab in the treatment of refractory acute rejection and bronchiolitis obliterans syndrome after human lung transplantation importance of chronic aspiration in recipients of heart-lung transplants heart-lung transplantation: successful therapy for patients with pulmonary vascular disease clinical utility of bronchoalveolar lavage cell phenotype analyses in the postoperative monitoring of lung transplant recipients gastroesophageal reflux as cause of obliterative bronchiolitis: a case report epstein-barr virus polymerase chain reaction and serology in pediatric post-transplant lymphoproliferative disorder: three-year experience lung transplantation and systemic sclerosis pulmonary hypertension associated with lung transplantation obliterative bronchiolitis and vascular remodeling of the allograft humoral (antibody-mediated) rejection in lung transplantation lung transplantation in scleroderma compared with idiopathic pulmonary fibrosis and idiopathic pulmonary arterial hypertension eosinophilic granulocytes and interleukin-6 level in bronchoalveolar lavage fluid are associated with the development of obliterative bronchiolitis after lung transplantation mismatches at the hla-dr and hla-b loci are risk factors for acute rejection after lung transplantation susceptibility of lung transplants to preformed donor-specific hla antibodies as detected by flow cytometry paediatric incidence of acute rejection and obliterative bronchiolitis: a comparison with adults risk factors for bronchiolitis obliterans: a systematic review of recent publications lung transplantation in patients with scleroderma: case series, review of the literature, and criteria for transplantation exhaled nitric oxide in human lung transplantation: a noninvasive marker of acute rejection severe organ involvement in systemic sclerosis with diffuse scleroderma interpretation of transbronchial lung biopsies from lung transplant recipients: inter-and intraobserver agreement revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection stage i adenocarcinoma presenting in the pneumonectomy specimen at the time of single lung transplantation national conference to assess antibody-mediated rejection in solid organ transplantation perivascular inflammation in pulmonary infections: implications for the diagnosis of lung rejection bronchoalveolar lavage in lung transplantation. state of the art toronto lung transplant group: unilateral lung transplantation for pulmonary fibrosis surgical pathology of pulmonary infections registry of the international society for heart and lung transplantation: twenty-fourth official adult lung and heart-lung transplantation report-2007 the role of transbronchial lung biopsy in the treatment of lung transplant recipients: an analysis of 200 consecutive procedures singleinstitution study evaluating the utility of surveillance bronchoscopy after lung transplantation role of pulmonary function in the detection of allograft dysfunction after heart-lung transplantation the role of interleukin-17 during acute rejection after lung transplantation complications in the native lung after single lung transplantation obliterative bronchiolitis following lung transplantation: from old to new concepts? parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function c4d staining of pulmonary allograft biopsies: an immunoperoxidase study c3d and c4d deposition early after lung transplantation increased frequency of posttransplant lymphomas in patients treated with cyclosporine, azathioprine, and prednisone influence of human leukocyte antigen matching on long-term outcome after lung transplantation alpha1-antitrypsin deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer risk lung transplantation exacerbates gastroesophageal reflux disease the potential role of mast cells in lung allograft rejection revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group can immunohistological analysis of transbronchial biopsy specimens predict responder status in early acute rejection of lung allografts? interleukin-10 production genotype protects against acute persistent rejection after lung transplantation the impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients key: cord-258362-6qk2brax authors: chang, a.b.; byrnes, c.a.; everard, m.l. title: diagnosing and preventing chronic suppurative lung disease (csld) and bronchiectasis() date: 2010-12-04 journal: paediatr respir rev doi: 10.1016/j.prrv.2010.10.008 sha: doc_id: 258362 cord_uid: 6qk2brax current diagnostic labelling of childhood bronchiectasis by radiology has substantial limitations. these include the requirement for two high resolution computerised tomography [hrct] scans (with associated adversity of radiation) if criteria is adhered to, adoption of radiological criteria for children from adult data, relatively high occurrence of false negative, and to a smaller extent false positive, in conventional hrct scans when compared to multi-detector ct scans, determination of irreversible airway dilatation, and absence of normative data on broncho-arterial ratio in children. a paradigm presenting a spectrum related to airway bacteria, with associated degradation and inflammation products causing airway damage if untreated, entails protracted bacterial bronchitis (at the mild end) to irreversible airway dilatation with cystic formation as determined by hrct (at the severe end of the spectrum). increasing evidence suggests that progression of airway damage can be limited by intensive treatment, even in those predestined to have bronchiectasis (eg immune deficiency). treatment is aimed at achieving a cure in those at the milder end of the spectrum to limiting further deterioration in those with severe ‘irreversible’ radiological bronchiectasis. when a child has chronic productive cough, digital clubbing, chest wall deformity, adventitial chest signs, and dilated airways on high resolution computerised tomography chest (hrct) scan, almost all clinicians would recognise that the child has bronchiectasis. however, in our current era with improved clinical care, access to health and diagnostic modalities most children are paediatric respiratory reviews 12 (2011) 97-103 the reader will be able: to discuss the limitation of diagnosis of childhood bronchiectasis on current radiological criteria. to define the diagnoses of protracted bacterial bronchitis and chronic suppurative lung disease and describe their relationship to bronchiectasis. to examine likely precursors of chronic suppurative lung disease to illustrate the merits of early and intense treatment of children with symptoms of bronchiectasis. to discuss the limited evidence for early and intense treatment to prevent advancement of disease in children with chronic suppurative lung disease and bronchiectasis. diagnosed at an earlier stage in disease progression and most do not have the this classical presentation with radiological changes of severe bronchiectasis as described many decades ago. 1, 2 the vicious circle hypothesis, currently accepted as the most coherent explanation of bronchiectasis formation in the absence of interstitial lung disease, suggests that impaired host defence leads to bacterial (and likely biofilm colonisation) of conducting airways. this induces inflammation resulting in damage to the airways and further impairment of mucociliary clearance. in many cases of bronchiectasis, a period of many months, years or even decades may precede the development of sufficient damage to appear as classical 'bronchiectasis' on a hrct scan. the rate of damage progression is likely dependent on a number of factors: whether there is an underlying problem such as cystic fibrosis (cf), the extent (and type) of the pathogenic colonisation/infection, 3 the frequency of exacerbations, 4 access to healthcare, and the efficacy of, and adherence to, therapeutic interventions. in cystic fibrosis, for example, the appearance of bronchiectasis on hrct scans has been greatly delayed with more aggressive anti-microbial therapy and strategies designed to enhance mucociliary clearance. it is increasingly recognised that many (if not most) severe chronic lung can be largely prevented and attention has focused on possible interventions in childhood. [5] [6] [7] it is highly likely that some, if not most, cases of non-cf bronchiectasis can be prevented through intervention designed to improve airway clearance and eliminate bacteria (with associated inflammatory 'soup' and possibly biofilms) from the airways and that this approach would be most successful if initiated in childhood. 8, 9 in this paper, we discuss the limitations of current diagnostic criteria, precursors of bronchiectasis and the evidence (albeit limited) on why children with protracted bronchitis, suppurative lung disease and bronchiectasis require vigilant medical follow-up and appropriate therapies. as noted above, the current paradigm suggests that there is a spectrum of disease associated with the presence and/or persistence of pathogenic bacteria in the lower airways. in these children the cough is associated with bacterial infection and/or colonisation of the conducting airways. this ranges from being transient (after a viral lower respiratory tract infection) through to the patient with severe bronchiectasis described previously. a common thread is the presence of bacteria suggesting that this is a spectrum of disease in which the manifestations can vary markedly between and within individuals over time. prior to the 'diagnosis' of 'idiopathic bronchiectasis' being made on the basis of a hrct scan, the patient (children and adults) may have been labelled as having recurrent viral infections, asthma, and/or chronic bronchitis. [10] [11] [12] yet it is likely the same disease process that caused them to be 'chesty' (with wet cough) from childhood. in two relatively large series (n = 103-150), most (60-80%) adults newly diagnosed with bronchiectasis (on hrct) have had chronic wet cough or productive cough since childhood. 13, 14 unfortunately the subject of how bronchiectasis evolves has received relatively little attention over the past 40 years. the high case numbers of bronchiectasis described in the 1940 s and 1950 s 2 with a seeming resurgence described from the 1990 s [15] [16] [17] suggests two possibilities. either it was under-recognised in the intervening years, when there was a co-existing strong focus on asthma, or that the widespread use of oral antibiotics in early childhood to treat 'viral respiratory tract infections' also treated developing persistent bacterial bronchitis (pbb) and/or pneumonia. eradicating bacteria prior to the formation of biofilms is theoretically easier than after the biofilms have become established and in the same way many pneumonias were probably inadvertently treated so many cases of bacterial bronchitis were probably inadvertently prevented. we speculate that the reappearance of an old disease may in part be the unintended consequence of an appropriate attempt to reduce antibiotic prescribing for acute viral respiratory tract infections. the terminology used is also confusing as it suggests that there are different diseases rather than a spectrum of clinical phenotypes with a common underlying problem. the use of some of the diagnostic terms is discussed in next section and has been summarised in a previous review. 9 classically, bronchiectasis is a radiological or pathological diagnosis characterised by irreversible bronchial dilation. currently this is most commonly diagnosed by hrct which has replaced bronchograms. the key features of bronchiectasis on hrct scans are (a) one or more 'dilated' bronchi defined as the internal luminal diameters of the airways exceeding the diameter of the adjacent vessel, (b) non-tapering of the bronchi and (c) presence of visible bronchi adjacent to the mediastinal pleura or within the outer 1-2 cm of the lung fields. however, bronchiectasis (radiological diagnosis) may be reported by radiologists in patients with interstitial lung diseases (such as pulmonary fibrosis) where traction on the airways causes bronchial dilation. traction bronchiectasis in the absence of chronic productive or wet cough will not be considered further in this paper. in children, cough is wet rather than productive, as young children usually do not expectorate, 9 and following treatment the cough often temporarily resolves. 4 csld describes a clinical syndrome where there are symptoms of chronic endobronchial suppuration with or without hrct evidence of radiological bronchiectasis. the presenting symptoms are identical to bronchiectasis; a prolonged moist or productive cough, exertional dyspnoea, features of reactive airway disease, growth failure, recurrent chest infections and/or wet cough responsive to antibiotics. physical signs include clubbing, chest wall deformity, adventitious sounds and/or hyperinflation. 1 haemoptysis is rare in children. however, absence of symptoms (other than wet cough) and signs do not reliably exclude either bronchiectasis or csld. lung abscess and empyema (previously included as csld) have distinct radiological characteristics, are not discussed here and in our era should not be considered as within the csld category. most children have a productive or wet cough for several years before a diagnosis is made. 1 pathobiological studies [18] [19] [20] and clinical observations suggest many patients have bronchitis initially that, untreated, gradually evolves into bronchiectasis. 9, 10 the entity of pbb has been described in children where a wet cough completely resolves following antibiotic treatment. 9,10 many of these children were previously misdiagnosed with asthma and had responded poorly to asthma therapies. in some settings these children would have been classified as having 'difficult or severe asthma'. 9,10 this is also likely relevant to adults. from a recent study, 40% of newly referred adults (and who had a ct scan) with 'difficult asthma' were found to have bronchiectasis. 11 the definitions of bronchiectasis, csld and pbb have limitations as their associated symptoms and signs overlap and lack specificity. whether these conditions are different or reflect part of a spectrum of disease severity remains undetermined. 9 however, we believe absolute reliance on a radiology-based definition is also unsatisfactory for the following reasons: 1. it is unknown when the radiological changes consistent with bronchiectasis occur in the context of a patient with symptoms of csld/bronchiectasis. adult-based studies have shown that bronchography (the previous gold standard for diagnosis of bronchiectasis) is superior to hrct scans especially in mild disease. 21, 22 furthermore recent studies have shown that contiguous 1-mm slices are superior in diagnostic terms compared to conventional hrct images (1 mm slice every 10 mm). 23 , 24 hill et al reported that the contiguous 1-mm slices protocol (using multidetector ct scan) demonstrated 40 extra lobes with bronchiectasis not identified on conventional hrct in 53 adults. 23 false negative results are more likely to occur when the disease is mild and localised. 21 as children are likely to have less severe bronchiectasis than adults, it is possible that the hrct in a sub-group of children with clinical symptoms of bronchiectasis may still have insufficient sensitivity to detect early signs of this disorder. in addition it is more difficult to obtain appropriate full inspiratory scans in young children. 2. a significant number of children have clinical characteristics of bronchiectasis but their hrct scans do not meet the criteria for radiological bronchiectasis (figure 1 ). hrct findings of bronchiectasis were derived from adult studies and may not be applicable to children as airways and morphologic changes in the lung occur with maturation and aging. 25, 26 one of the key hrct signs of bronchiectasis is increased broncho-arterial ratio (defined as the diameter of the bronchial lumen divided by the diameter of its accompanying artery) of > 1-1.5. however this ratio is influenced by age 27 and we argue that a lower bronchoarterial ratio should be used in children to diagnose bronchiectasis. extrapolation of this line suggests that the cut-off should be at 0.4-0.5 in infancy. indeed the broncho-arterial ratio in young children (0-5 years) without csld symptoms was 0.49 to 0.58. 28 a study based on multi-detector ct chest scans in 41 children (aged < 18 years) without pulmonary symptoms described that broncho-arterial ratio of ! 0.8 should be considered abnormal. 29 3. to fulfil the criteria of 'irreversible dilatation' at least two hrct scans are required. performing more than one hrct scan purely for diagnostic reasons (as opposed to required for management reasons) in some settings is highly impractical and unnecessarily increases cancer risk from cts in children and adolescents. 30 4. hrct scans performed in different clinical states, such as during an acute pulmonary exacerbation, immediately following treatment or when clinically stable, may yield different results. while hrct scans are ideally performed in a 'non-acute state', this is sometimes difficult to define. a 'non-exacerbation state' is not necessarily the same as post-treatment state. clinicians have realised that this is a significant limitation. the liverpool group described bronchial dilatation resolving completely in 6 of 21 children with radiologically defined bronchiectasis when hrct scans were repeated immediately following intensive medical therapy. 31 we and others have observed that radiological 'bronchiectasis' can resolve following appropriate treatment though this is unlikely in more advanced distortion of the conducting airways. also, field eluded to this pre-bronchiectatic state back in the 1940's. 32 the above reasons had lead some clinicians, particularly paediatricians, to use the term csld. 9,33 clinically these conditions overlap and the eventual diagnosis is evident only with further investigations and time. while the principles of managing all three conditions are the same, there are few published intervention studies, especially for csld. until further evidence is available, we believe recognising the continuum of a pathogenic process is important given the: (i) spectrum of disease, (ii) increasing evidence that early diagnosis and treatment improves outcomes and reduces pulmonary decline, 8, 34 (iii) difficulties surrounding robust definitions outlined above, and (iv) increasing recognition that 'bronchiectasis' may be reversible, at least in the milder cases. these issues and diagnostic categories were raised in a previous review 9 and guidelines. 35, 36 similarly, the categories were recently endorsed by the thoracic society of australia and new zealand and the australian lung foundation in their position statement. 37 this places the focus on the unifying driver of symptoms and progressive damage which is infection (or colonisation) of the airways with pathogenic bacteria (with likely biofilm formation) [ ( ) t d $ f i g ] figure 1 . hrct of the lower lobes of a child aged 3 years and 7 months during routine investigation for a chronic wet cough that was found to be related to common variable immunodeficiency. the hrct shows borderline bronchiectasis. most respiratory paediatricians considered bronchiectasis was present in this hrct scan but two paediatric radiologists reported absence of abnormal airway dilatation (and thus absence of bronchiectasis). following treatment (immunoglobulin replacement, intravenous antibiotics and airway clearance), the cough cleared after 2 weeks. child is currently well (now aged 5.5 years), cough free and has no abnormality spirometry. and places the clinical manifestation in this context. while there is currently no evidence on the detection of biofilm in the airways (other than for cf and diffuse panbronchiolitis), 38 it is however likely important in the pathophysiology of chronic lower airway infections and contributes to the difficulty in eradicating infection once established. in the mucosa of ears (which is arguably relevant to lower airways), the importance of biofilms in chronic otitis media has been described. 39 in this paradigm, bronchiectasis is viewed as a radiological or pathological sign resulting from a long standing process in the same way that a myocardial infarction is an event in some patients with ischemic heart disease. as with ischaemic heart disease the paradigm of persistent bacterial bronchitis is a description of a process which can have a clinical expression from asymptomatic, minimal symptoms to life threatening severe impairment of function or premature death. 40 moreover with appropriate intervention progression can be arrested or even reversed. 34, 41 precursors of bronchiectasis and lung function abnormality aetiological associations and risk factors for bronchiectasis are discussed by kapur and karadag in this series. 42 pbb and csld as precursors were briefly discussed above and in other publications. 9,10,43 other potential precursors of airway dysfunction, csld and bronchiectasis are further briefly discussed below. while low birth weight and pre-existing small lungs are predictors of future lung function parameters, there is increasing evidence that early events in life are equally important determinants of adult pulmonary dysfunction in human 5, 44, 45 and animal studies. 46, 47 harding et al showed that later size and structure of lungs in sheep born preterm (but not requiring respiratory support) were dependent on post-natal growth rather than being low-weight at birth. 46, 47 this is plausible as lung growth continues at least through the first two years of life and, in the lung parenchyma, this occurs by increasing alveolar number as opposed to increasing alveolar size. 48 events such as respiratory infections and persistent neutrophilic inflammation (eg pbb 49 when not treated) during this critical period of lung development may lead to long term pulmonary effects. reviews on the biology of persistent airway neutrophilia and its potential damaging effects are available elsewhere. 50, 51 in recent years, large epidemiological data from the copd literature have shown that in addition to tobacco smoking, the antecedence of these chronic respiratory diseases occurs in childhood. 5, 45 while bronchiectasis is not copd, both diseases share some common features when advanced (chronic productive cough, fixed airflow limitation). two studies 52,53 have also described the high prevalence (29-50%) of bronchiectasis in adults with copd. whether or not these adults had bronchiectasis before being labelled as having copd is unknown ie the temporal relationship between the two diseases is unknown. similarly there is a substantial overlap between bronchiectasis with asthma 11 and copd. 54 in bronchiectasis-specific literature, recurrent hospitalisations for respiratory infections and prematurity have been shown to be independent significant risk factors for the later development of bronchiectasis in children. 55 as it would appear that childhood respiratory disease and risk factors are relevant for the development of adult respiratory disease, epidemiological studies documenting this relationship are reviewed below. nine years follow-up data on community based samples of 20-44-yr-old subjects from 29 centres that participated in the 'european community respiratory health survey' described that significant respiratory infections in the first 5-years of life were associated with a lower fev 1 (adjusted difference of -144mls, 95%ci -211 to -78 for hospitalised episode) even when excluding 'ever asthmatics' and 'current wheezers'. 44 dharmage and colleagues 44 further documented that the impact of early infections was more significant in subjects exposed to maternal or active smoking. from the same cohort, svanes et al 5 reported that early life disadvantage (maternal and paternal asthma, severe respiratory infections before aged 5-years, maternal smoking) were significantly associated with adult development of copd. longitudinal data from the newcastle thousand families cohort likewise described that childhood respiratory infections contributed significantly to future adult lung function. 56 other important factors from this study 56 were birth weight, breast feeding (for >4 weeks), asthma and smoking. a study from scotland 45 described that respiratory disease in early life was associated with a higher risk in adulthood of chronic productive cough, dyspnoea and doctor diagnosis of asthma, bronchitis or emphysema (adjusted odds ratios ranging from 1.40 to 6.95 for these outcomes). these recent epidemiological studies support older data showing that childhood pneumonia was related to poor lung function in adults with no history of wheeze. 57 while recall bias is always an issue in the design of studies described here, the findings from several large studies are at least consistent and supported by animal work 19 and bronchiectasis-specific literature. 55 can csld and bronchiectasis be prevented? bronchiectasis causes an accelerated lung function decline and premature death in adults. 3 in indigenous australian adults (a group that generally receive sub-optimal treatment) the mortality of a hospital-based cohort of 61 adults (mean age of 42 + sd 15-yrs) was 11.5% within 12-months. 40 elsewhere, mortality rates in adults with bronchiectasis vary widely from a 4-yr survival of 58% (turkey), 75% survival at 8á8-yrs (finland) to 81% survival at 14-yrs (scotland). 3 in the past, surgical interventions to reduce the severe symptoms of bronchiectasis were common practice. currently lobectomies or pneumonectomies for children with bronchiectasis are rarely appropriate in affluent countries 37 but remain a common and important treatment option in less affluent countries. 58 the fact that the natural history of bronchiectasis and mortality has altered with improvements in health and the environment suggests that with the implementation of other preventative factors, the progression of bronchiectasis could be ameliorated in the majority of children. furthermore, there is evidence demonstrating: (a) the effect that exacerbations and/or delayed treatment is associated with lung function decline, (b) children at risk of bronchiectasis can have normal lungs with early diagnosis and appropriate management, and (c) appropriate treatment reduces exacerbations of bronchiectasis. (a) exacerbations and/or delayed treatment is associated with lung function decline, increased mortality risk is associated with the degree of lung function impairment. 3 determinants of accelerated lung function decline in adults with bronchiectasis are frequency of hospitalised exacerbations, increased systemic inflammatory markers and colonization with p. aeruginosa. 59 available longitudinal fev 1 data in children with non-cf bronchiectasis are inconsistent but support that early and intensive treatment improves lung function in children with reduced fev 1 at diagnosis and prevents deterioration in the following 2-5 year period. 34, 41 one londonbased retrospective study (31 children over 4-yrs) found that with intensive treatment lung function improved but did not necessarily normalize. 34 in contrast, an auckland (new zealand) study (44 children over 4.5yrs) described a decline of fev 1 of 1.9% per year. 60 a brisbane (australia) retrospective study (52 children over 3-yrs, 25 over 5-yrs) found that children with normal lung function at diagnosis maintained normal lung function at 5-yrs, but those with low lung function at diagnosis were likely to have low lung function at 5-yrs, although improved. 41 the brisbane study also found that the only significant predictor of fev 1 decline (over 3yrs) was frequency of hospitalized exacerbations. 41 with each exacerbation, the fev 1 %predicted decreased by 1.95% (p = 0.048) adjusted for time. 41 the other two published cohorts 34,60 did not examine exacerbations as a determinant of lung function decline. thus, interventions that reduce exacerbations are likely to be important for later adult lung dysfunction. furthermore, adult data has shown that recurrent exacerbations is one of the strongest predictors of poor qol. 61 data on asthma exacerbations in child and adulthood mirrors these findings. o'byrne and colleagues described that exacerbations requiring hospitalisation or emergency treatment were associated with accelerated lung function (fev 1 ) decline in subjects not on preventative therapy. 62 it could be argued that children with exacerbations were predestined to have lung function decline anyway, however what evidence there is suggests that children at risk of development of csld should receive appropriate therapies 37 to improve immediate symptoms and minimise future lung dysfunction. (b) people at risk of bronchiectasis can have normal lung function with early appropriate management. there is increasing evidence that intensive treatment of children either at risk of, or who have, bronchiectasis prevents poor lung function in adulthood. 8, 34, 41, 63, 64 in two heterogeneous cohort studies, 34, 41 primary immunodeficiency (as the prescribed aetiology) did not relate to any marker of bronchiectasis severity or to future lung function decline. with respect to primary ciliary dyskinesia [pcd] and primary immunodeficiency, three groups 8,63,64 described that delayed diagnosis was associated with more severe disease. a large (n = 182) australian study of adults newly diagnosed with bronchiectasis has shown that the decline in fev 1 correlates (r = 0.51) with the duration of chronic wet cough, 13 the most common symptom. 36 for each additional year of cough, fev 1 %predicted declined 0.51% in non-smokers. 13 in children with 'right middle lobe syndrome', priftis and colleagues 65 found a positive correlation to duration of the deterioration of symptoms prior to presentation with development of bronchiectasis (p = 0.03). duration of symptoms also correlated with an unfavorable clinical outcome. 65 appropriate therapy improves prognosis 63 and reduces respiratory exacerbations. 66 the frequency of exacerbations is higher in those with more severe disease 67 and unmanaged csld/bronchiectasis. 66 in a turkish study of 111 children, 'intensive medical treatment' (prompt antibiotic use, physiotherapy, bronchodilators) reduced exacerbation rates from 6.6 ae sd 4 to 2.9 ae 2.9 per year. 66 exacerbation frequency per year is directly related to bronchial wall thickening on hrct scans 67 and severe bronchial wall thickness was the most adverse prognostic determinant in a study using serial chest hrct scans. 68 while there is little randomised controlled trial (rct) data on therapies that reduce exacerbations, 69 available data in adults suggest that standard therapies used for csld and bronchiectasis (eg pneumococcal vaccination, 70 antibiotics 71,72 ) reduce respiratory exacerbations. a cohort study 71 and one short term (6-mo) rct 72 described improvement in lung function and reduction in exacerbation frequency by 50-67% when regular azithromycin was given to adults with bronchiectasis. whether this reduction is related to azithromycin's anti-microbial effect or the immune-modulatory influences remain to be defined. despite the known importance of exacerbations in most chronic respiratory diseases (e.g. asthma, 62,73 copd 74 ) data are scarce for the triggers, definitions, associated clinical features and evidence for treatment of bronchiectasis in both children and adults. 69, 75, 76 a review of exacerbations in people with bronchiectasis is available elsewhere. 69 whether viruses and other non-bacterial respiratory pathogens such as mycoplasma and chlamydia spp. trigger bronchiectasis exacerbations has never been examined. a brisbane retrospective study found that 34% of exacerbations were preceded by an upper respiratory illness. 4 however, a systematic study utilising modern, sensitive molecular techniques and inclusive of the more recently described viruses (eg human metapneumovirus, human coronaviruses (hcov nl63 and hku1), human bocaviruses and polyomaviruses, rhinovirus c) have not been undertaken. clearly this is required as one of the first steps in understanding triggers of respiratory exacerbations in children with bronchiectasis. recent studies have indicated that in a significant proportion of children with chronic cough referred to secondary care, the cough is attributable to infection (or colonisation) of the airways with pathogenic respiratory organisms. the clinical manifestations of this bacterial bronchitis are variable being influenced by factors such as age, extent of infection and the degree of damage to the airways. symptoms may be limited to a cough, typically a moist cough (protracted bacterial bronchitis), while in older children and those with significant damage to the conducting airways (bronchiectasis), expectoration of sputum and more persistent cough occurs. absent or ineffective treatment regimes lead to progressive damage that can ultimately lead to severe pulmonary impairment. many unanswered questions remain. in this article we have highlighted the current limitations of dependence on radiological diagnoses of childhood bronchiectasis. we also presented the evidence why early diagnosis, active and close monitoring, and intensive treatment are advocated in children with protracted bronchitis, csld and bronchiectasis. clearly further studies are required to delineate appropriate diagnostic labelling, pathogenesis questions as well as clinical trials that address prevention and treatment issues. the direct measurement of neutrophilic inflammation in the airways of children. prospective studies determining factors governing lung function decline and exacerbations. the role of viruses and chlamydia species in respiratory exacerbations and persistence of symptoms. to determine whether chronic nasopharyngeal carriage of pathogenic bacteria contribute to csld or bronchiectasis. the roles of bacteria viral-bacteria interaction, biofilms and other non respiratory bacteria in the pathophysiology of non-cf bronchiectasis. evidence based studies examining the role of early therapy in the prevention and/or progression of bronchiectasis. childhood bronchiectasis diagnosis by radiological techniques in children were extrapolated from adult studies and have substantial limitations. early diagnosis and appropriate management likely prevent disease progression. protracted bronchitis and chronic suppurative lung disease (csld) are likely precursors of bronchiectasis, if left untreated. children with conditions at risk of bronchiectasis should be vigilantly monitored and appropriately treated when wet cough is present to reduce the likelihood of developing csld and bronchiectasis. non-cf bronchiectasisclinical and hrct evaluation bronchiectasis in childhood: i. clinical survey of 160 cases mortality in bronchiectasis: a longterm study assessing the factors influencing survival exacerbations in non cystic fibrosis bronchiectasis: clinical features and investigations early life origins of chronic obstructive pulmonary disease cough in the pediatric population copd: a pediatric disease bronchiectasis secondary to primary immunodeficiency in children: longitudinal changes in structure and function state of the art -chronic wet cough: protracted bronchitis, chronic suppurative lung disease and bronchiectasis outcomes in children treated for persistent bacterial bronchitis qualitative analysis of high resolution computed tomography scans in severe asthma evaluation and outcome of young children with chronic cough phenotypes of adult bronchiectasis: onset of productive cough in childhood and adulthood an investigation into causative factors in patients with bronchiectasis prevalence and economic burden of bronchiectasis trends and burden of bronchiectasis-associated hospitalizations: usa new zealand national incidence of bronchiectasis ''too high'' for a developed country the biology of bacterial colonization and invasion of the respiratory mucosa criteria for development of animal models of diseases of the respiratory system: the comparative approach in respiratory disease model development studies of pneumonia in childhood: iv. bronchiectasis and fibrosis of the lung ct/bronchographic correlations in bronchiectasis high resolution ct and bronchography in the assessment of bronchiectasis comparison between conventional interrupted high-resolution ct and volume multidetector ct acquisition in the assessment of bronchiectasis conventional high-resolution ct versus helical high-resolution mdct in the detection of bronchiectasis composition changes in human tracheal cartilage in growth and aging, including changes in proteoglycan structure mechanical properties of human tracheal cartilage bronchoarterial ratio and bronchial wall thickness on high-resolution ct in asymptomatic subjects: correlation with age and smoking structural airway abnormalities in infants and young children with cystic fibrosis bronchoarterial ratio on high resolution ct scan of the chest in children without pulmonary pathology-need to redefine bronchial dilatation estimating cancer risks from pediatric ct: going from the qualitative to the quantitative reversible bronchial dilatation in children: comparison of serial high-resolution computer tomography scans of the lungs bronchiectasis in childhood: ii. aetiology and pathogenesis, including a survey of 272 cases of doubtful irreversible bronchiectasis aetiology of chronic suppurative lung disease non-cystic fibrosis bronchiectasis in childhood: longitudinal growth and lung function bronchiectasis in indigenous children in remote australian communities. a position statement management of bronchiectasis and chronic suppurative lung disease (csld) in indigenous children and adults from rural and remote australian communities bronchiectasis and chronic suppurative lung disease (csld) in children and adults in australian and new zealand: thoracic society of australia and new zealand and australian lung foundation position statement airway biofilms: implications for pathogenesis and therapy of respiratory tract infections direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media bronchiectasis in central australia: a young face to an old disease longitudinal growth and lung function in pediatric non-cf bronchiectasis -what influences lung function stability? differences and similarities in non-cf be between developing and affluent countries utility of signs and symptoms of chronic cough in predicting specific cause in children do childhood respiratory infections continue to influence adult respiratory morbidity? medical history of respiratory disease in early life relates to morbidity and mortality in adulthood lung parenchyma at maturity is influenced by postnatal growth but not by moderate preterm birth in sheep postnatal growth rate, but not mild preterm birth, influences airway structure in adult sheep challenged with house dust mite growth of the lung parenchyma early in life prospective assessment of protracted bacterial bronchitis: airway inflammation and innate immune activation advances in neutrophil biology: clinical implications neutrophils in chronic inflammatory airway diseases: can we target them and how? bronchiectasis, exacerbation indices, and inflammation in chronic obstructive pulmonary disease physiological and radiological characterisation of patients diagnosed with chronic obstructive pulmonary disease in primary care genetics of asthma and copd. similar results for different phenotypes a hospital-based case-control study of bronchiectasis in indigenous children in central australia lifecourse predictors of adult respiratory function: results from the newcastle thousand families study effect of pneumonia and whooping cough in childhood on adult lung function risk factors affecting outcome and morbidity in the surgical management of bronchiectasis factors associated with lung function decline in adult patients with stable non-cystic fibrosis bronchiectasis longitudinal pulmonary function of childhood bronchiectasis and comparison with cystic fibrosis quality-of-life determinants in patients with clinically stable bronchiectasis the effects of inhaled budesonide on lung function in smokers and nonsmokers with mild persistent asthma longitudinal study of lung function in a cohort of primary ciliary dyskinesia pulmonary complications in primary hypogammaglobulinemia: a survey by high resolution ct scan the role of timely intervention in middle lobe syndrome in children non-cystic-fibrosis bronchiectasis in children: a persisting problem in developing countries high-resolution ct quantification of bronchiectasis: clinical and functional correlation a comparison of serial computed tomography and functional change in bronchiectasis non-cystic fibrosis bronchiectasis exacerbations pneumococcal vaccines for children and adults with bronchiectasis effects of long-term low-dose azithromycin in patients with non-cf bronchiectasis the disease-modifying effects of twice-weekly oral azithromycin in patients with bronchiectasis statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice management and prevention of chronic obstructive pulmonary disease exacerbations: a state of the art review procalcitonin in stable and unstable patients with bronchiectasis quality of life and inflammation in exacerbations of bronchiectasis key: cord-016211-8j8n9enn authors: lu, puxuan; zhao, qingxia title: highly pathogenic avian influenza date: 2015-04-30 journal: radiology of infectious diseases: volume 1 doi: 10.1007/978-94-017-9882-2_18 sha: doc_id: 16211 cord_uid: 8j8n9enn highly pathogenic avian influenza is an acute respiratory infectious disease caused by some viral strains of avian influenza virus a. its severity is highly diverse ranging from common cold-like symptoms to septicemia, shock, multiple organ failure, reye syndrome, pulmonary hemorrhage, and other complications leading to death. according to the laws, human infection of highly pathogenic avian influenza has been legally listed as class b infectious diseases in china. and it has been stipulated that it should be managed according to class a infectious diseases in china. h5n1 is categorized into the genus of infl uenzavirus a , the family of orthomyxoviridae , and the order of mononegavirales , which is a segmented negative singlestranded rna virus (-ssrna virus). by electron microscopy, it is demonstrated with a typical sphere shape and a diameter of 80-120 nm. its nucleocapsid is spirally symmetric with envelope. the isolated strain is initially fi lament-like with a diameter of about 20 nm and a length of 300-3,000 nm. under an electron microscope, hemagglutinin protrudes into a homotrimer composed of three non-covalent combined protein molecules, while neuraminidase is demonstrated as a mushroom-like homotetramer with a head and a stem. the virus is composed of an envelope, matrix protein, and viral core, consecutively from external to internal of the virus. h7n9 is a subtype of avian infl uenza virus that is categorized into the family of orthomyxoviridae . its envelope is covered by two types of surface glycoproteins, hemagglutinin (h) and neuraminidase (n) . h can be further divided into 15 subtypes, while n 9 subtypes. all human infl uenza viruses can cause avian infl uenza, but not all avian infl uenza viruses can result in human infl uenza. among all avian infl uenza viruses, h5, h7, h9, and h3 can infect humans, and h5 is highly pathogenic. infl uenza virus can be categorized into 135 subtypes by hxnx, including avian infl uenza virus subtype h7n9. it prevailed in birds with no infection of humans. and its biological properties, pathogenicity, and transmissibility largely remain unknown. h7n9 is a new recombinant virus with its genes from avian infl uenza virus h9n2. currently, it has been found with mutation of pb2 gene at the site of 701. laboratory experiments have demonstrated that the mutated virus is highly pathogenic to canidae. mutation of 627 amino acid site is also possible. therefore, avian infl uenza virus h7n9 cannot be transmitted from person to person but only from pets or living poultry to person. ha on the envelope of infl uenza virus is an adhesion protein. in a binding way of neu5acα2-6gal, it can bind to the cell membrane at the human respiratory mucosa. the highly pathogenic avian infl uenza h5n1 virus can break through the barrier of species to acquire the capability of binding to receptor of human host cells. such a change in binding to receptor is the key for human infection of avian infl uenza virus. since the avian infl uenza virus h5n1 was fi rstly isolated from chickens in 1995, multiple outbreaks of avian infl uenza has occurred. on may 9, 1997, a strain of avian infl uenza virus was isolated from a young child aged 3 years in hong kong, which was etiologically defi ned as avian infl uenza virus. in the same year in hong kong, 18 cases were defi ned as human infection of avian infl uenza, with six cases of death. this was the fi rst report about direct human infection of avian infl uenza, arousing worldwide shock and attention. in mid-february 2003, two residents in hong kong were infected by avian infl uenza virus h5n1 with one case of death. since december 2003, outbreaks of avian infl uenza in poultry have been consecutively reported in korea, japan, vietnam, cambodia, the philippines, chinese taiwan, and some provinces of mainland china. outbreaks of human infection of highly pathogenic avian infl uenza virus h5n1 have been continually reported in vietnam, thailand, indonesia, china, and other asian countries since late 2003. by january 1, 2012, a total of 583 cases of human infected highly pathogenic avian infl uenza virus h5n1 have been reported across the world, with 344 cases of death and a mortality rate of over 50 %. on april 5, 2013, human infected avian infl uenza virus h7n9 was fi rstly reported in eastern china. avian infl uenza virus h7n9 has strong pathogenicity with a high mortality rate. by 4 pm on august 10, 2013, a total of 135 defi nitely diagnosed cases, including 1 in chinese taiwan, have been reported in china, with 44 cases of death and a mortality rate of 32.6 %. avian infl uenza virus commonly exists in many domestic poultries, such as turkeys, chickens, guineafowls, geese, ducks, coturnixs, and parrots. the virus might be carried in their secretion and excretion, feathers, organs, and eggs. avian infl uenza virus h5n1 can infect some wild poultries. wild water poultries, especially those with asymptomatic infection, play an important role in the natural transmission of the viruses. wild and water poultries like geese, terns, wild ducks, peafowls, and seagulls, especially migrating water poultries, are more likely to transmit avian infl uenza virus via excretions carrying the viruses. birds such as swallows, chukars, bar-headed geese, ravens, sparrows, and gray herons can be the source of its infection. migration of birds plays a signifi cant role in spreading avian infl uenza viruses. the virus can infect humans via inhaling virus-containing droplets and droplet core into the respiratory tract. by such a way of inhaling, the viral particle is inhaled into the human body to cause the disease. avian infl uenza virus (aiv) also spreads along with air. direct contact of human to diseased poultry or asymptomatic infected poultry and their secretions or excretions is a route of transmission. in addition, inhaling of viral particles in contaminated environment is another route of transmission. subsequently, the viral particles adhere to the respiratory tract to cause the disease. indirect or direct contact is also the route of transmission. close contacts of human to h5n1-infected poultry or their feces can cause the infection. direct contact and indirect contact of possibly contaminated utensils can self-inoculate the virus into the upper respiratory tract, mucosa of eye conjunctiva, and skin wound. in addition, avian infl uenza virus can also be transmitted via the alimentary tract, skin wound, the conjunctiva, aerosol, blood, vertical transmission, laboratory and transportation transmission, as well as nosocomial infection. it is speculated that human is infected by avian infl uenza virus mainly via direct contact to the diseased chickens and birds, namely, from poultry to person. it is also speculated that pigs are fi rstly infected by viruses carried by secretion and excretion of diseased poultry. and then human is infected via contacts to secretion, excretion, blood, skin, and fur of the diseased pigs, namely, from poultry to person via pig. the second way of transmission needs further epidemiological studies to verify. currently, the evidence for human infection of avian infl uenza virus h5n1 supports transmission from poultry to person. and the transmission from environment to person is possible, while the transmission from person to person has not been fully proved. it is generally acknowledged that human is not susceptible to avian infl uenza. although human infection of avian infl uenza has broken out in many regions, the case of human infection of avian infl uenza has been rarely reported. based on the data of the outbreaks, any age group can be infected and children aged under 12 years occupy a high percentage, but with mild symptoms. human infection of avian infl uenza virus has no signifi cant gender difference. invasion of avian infl uenza virus to the human respiratory epithelium can cause fl u-like symptoms, viremia, and viral pneumonia. in some serious cases, the patients may die from respiratory failure or multiple organ failure. following pathological autopsy of a death case from avian infl uenza in hong kong, china, it was discovered that pathological changes at the lungs are typical viral interstitial infl ammatory changes, necrosis, and fi brous proliferation. the pathological changes of organs are characterized by diffuse phagocytosis of erythrocytes, leukocytes, and platelets by macrophages, which is known as reactive hemophagocytic syndrome. such a syndrome is the result of cytokinemia caused by the release of multiple cytokines into blood triggered by infection of avian infl uenza virus. after invasion of highly pathogenic avian infl uenza virus into the human body, it replicates and multiplies in large quantities within cells to cause structural and functional damages of infected cells and consequent apoptosis. at the same time, a large quantity of specifi c proteins of highly pathogenic avian infl uenza virus acts as a superantigen to activate the immune system and to keep it to be highly active. and large quantities of infl ammatory cytokines are released to damage normal human cells and their structure. statistical analysis has demonstrated that 60-70 % human infection of avian infl uenza is severe, which may clinically develop into acute lung injury (ali) or acute respiratory distress syndrome (ards). pathological studies have demonstrated that the early changes of pulmonary tissues include pulmonary edema and the formation of hyaline membrane, being consistent with the early manifestations of ali or ards. at the advanced stage, the pathological changes include alveolar injury, erythrocytes and fi brous exudates, hyperplasia of interstitial fi broblasts, and deposition of collagen fi bers. the parenchymal cells at the lung, liver, kidney, gastrointestinal tract, and adrenal glands are subject to retrogressive degeneration and necrosis. clinical evidence can also be found consistent to these changes, indicating the occurrence of complicating multiple organ dysfunction syndrome (mods). in some cases, the conditions progress into multiple organ failure (mof) with an extremely high mortality rate. the possible reasons are as the following: (1) the direct destructive effects of infection of avi on capillary endothelium; (2) functional increase of vascular permeability mediated by cytokines and infl ammatory mediators such as metabolites of arachidonic acid, prostaglandin, leukotriene, and thromboxane; and (3) structural increase of vascular permeability due to injuries of capillary endothelial cells and basement membrane mediated by infl ammatory cells, endotoxin produced by gram-negative bacillus, fi brin and its degradation products, complements, polymorphonuclear granulocytes, platelets, free fatty acid, bradykinin, proteolytic enzymes, lysosomal enzymes, inhaling of high concentration oxygen, and formation of microthrombus. the infl ammatory cells pass through infi ltrative lung tissue space and release infl ammatory mediators to exacerbate lung injury. the injuries of capillary endothelial cells lead to increased permeability and increased fl uid overfl ow. meanwhile, the lymphatic drainage fails to increase correspondingly, leading to fl uid retention. therefore, interstitial and alveolar edema occurs. in addition, increased permeability of capillary endothelium increases the level of protein in the interstitial fl uid to be close to the level of protein in the plasma. the decreased osmotic pressure of plasma within vascular vessels exacerbates interstitial edema. in addition to alveolar collapse as well as increased interstitial negative pressure, edema is further exacerbated. in the cases with acute lung injury, the infl ammatory cells and mediators can directly cause structural and functional damages to type i and type ii alveolar epithelial cells, destructed basement membrane, and leakage of tissue fl uid, protein, and infl ammatory cells into the alveolus. thus, alveolar edema occurs. damaged type ii alveolar epithelial cells can cause a reduced production of surfactants within the alveolus, impaired sodium icon transportation, as well as weakened or lost function of repairing type i alveolar epithelial cells and synthesizing anti-injury cytokines. all of these changes further exacerbate pulmonary edema and dysfunctional ventilation. increased ventilation at the early stage its etiological factors include emotional tension, pain caused by trauma, and hypoxemia. hypoxemia is the main etiological factor. in the cases with ali or ards, due to lung tissue space edema and alveolar edema, the alveolus is subject to hyperplasia and hypertrophy of alveolar epithelium as well as formation of alveolar hyaline membrane. the gas exchange between the alveolus and capillary is impaired, with disproportion of ventilation and blood fl ow. consequently, serious hypoxemia occurs. its etiological factors include as follows: (1) paravascular interstitial edema and the following decreased interstitial negative pressure increase the risk of small airway collapse and consequent atelectasis; (2) pulmonary edema decreases production of surfactant in the alveolus and its activity, which further lead to shrinkage or collapse of the alveolus; and (3) pulmonary vascular congestion and increased pulmonary blood volume. pulmonary compliance refers to change of lung capacity caused by change of unit pressure. in the cases with acute pulmonary injury due to infection of avian infl uenza, their functional residual capacity decreases, with pulmonary interstitial congestion and edema as well as decreased surfactants. therefore, the pulmonary compliance is subject to decrease. therefore, oxygen demand in breathing signifi cantly increases, with shallow and rapid breathing and reduced tidal volume. the effective alveolar ventilation decreases to aggravate hypoxia. at the advanced stage, pulmonary interstitial fi brosis occurs to further decrease pulmonary compliance. hemodynamic change at lung due to hypoxia, the blood fl ow is rapid, with shortened time for blood passing through the alveolus. meanwhile, hyperplasia of the alveolar capillary membrane prolongs the time needed for gas exchange. therefore, venous blood passing through the alveolus fails to be suffi ciently oxygenated, resulting in return of a certain quantity of mixed venous blood into the left heart. blood shunt refers to the percentage of venous blood in the arterial blood, which is normally below 3 %. however, in the cases with acute lung injury or ards, disproportional ventilation and blood fl ow causes insuffi cient gas exchange in the alveolus. and the venous blood circulating in the capillaries fails to be suffi ciently oxygenated. therefore, obviously increased venous blood is mixed in the arterial blood to fl ow back to the left heart. in some cases, the blood shunt may increase even to 30 %. in the early stage after onset, mixed alkalosis may occur in severe patients due to hyperventilation. in the advanced stage, a large quantity of anaerobic metabolites gains their access into blood fl ow due to severe hypoxia to cause severe metabolic acidosis. in the terminal stage, respiratory failure leads to respiratory acidosis. therefore, acid-base imbalance in the cases of acute lung injury or ards evolves from transient metabolic or mixed alkalosis at the early stage to mixed acidosis at the advanced stage. human infection of avian infl uenza commonly causes systemic infl ammatory response syndrome (sirs), which further progresses into mods. in severe cases, multiple organ failure occurs at the terminal stage. primary pathological changes of human infection of highly pathogenic avian infl uenza include severe pulmonary lesions, toxic changes of immune organs and other organs, and secondary infection. the causes of death include: (1) progressive respiratory failure caused by diffuse alveolar injury; (2) multiple organ functional impairment of the liver, kidney, and heart; and (3) compromised immunity and secondary infection. by naked eye observation, the lung is subject to swelling, increased weight, obvious consolidation that is more serious at the lower lung lobe, and dark reddish in color. mild adhesion can be found between the lung and parietal pleura. on the section of lung, blood stasis and edema are obvious, with exudates of light reddish foamy bloody fl uid. the trachea and bronchus are subject to mucosal congestion. in their canals, there are light reddish foamy secretions. in the thoracic cavity, a small quantity of light yellowish fl uid can be observed. at the early stage, diffuse alveolar injury can be observed at both lungs, characterized by acute diffuse exudation. the alveolar cavity is fi lled with exudates of light reddish fl uid and infl ammatory cells in different quantities, mostly lymphocytes, monocytes, plasma cells, and phagocytes, but rarely neutrophils. in the alveolus, there are also shedding, degenerative, and necrotic alveolar epithelial cells. in some alveoli, hemorrhage, cellulose, and hyaline membrane can be observed. in addition, hyperplasia of type ii alveolar epithelium can also be observed. under an electron microscope, there are injury of alveolar epithelial cells, protein-like fragments in the alveolar cavity, as well as necrotic and apoptotic epithelial cells, lymphocytes, and histiocytes. the alveolar wall is severely damaged, and lysed erythrocytes can be observed in the alveolar cavity. nuclear margination is observable in some residual alveolar epithelial cells. there are also expansion of rough endoplasmic reticulum, swelling of mitochondria, and vacuole. the lymphocytes are subject to decrease in quantity and scattering distribution. the lymph sinus is dilated, possibly with focal necrosis. the histiocytes are subject to proliferation, with phagocytosis of erythrocytes and lymphocytes. the spleen is subject to slight swelling and smooth and dark reddish surface. under a microscope, the fi ndings include blood stasis, edema, expanded red pulp, and atrophic white pulp. around the white pulp, atypical lymphocytes can be found. in addition, infi ltration of small quantities of infl ammatory cells can be observed in the splenic sinus, with histiocytosis and phagocytosis of blood cells. in the bone marrow, reactive histiocytosis and phagocytosis of blood cells can be observed. human infection of avian infl uenza might have a longer incubation period than other human infl uenza, which generally lasts for 1-3 days, commonly 1-7 days but 21 days in some cases. the interval between the familial onset is generally 2-5 days, with a maximum of 8-21 days. the duration of incubation period is related to the virus pathogenicity, the quantity of invading viruses, route of infection, and the immunity of infected person. human infection of avian infl uenza commonly occurs in winters and springs. it has an acute onset and rapid progress. within 1 week after onset, the conditions may rapidly progress and deteriorate into acute lung injury, acute respiratory distress syndrome, pulmonary hemorrhage, pleural effusion, pancytopenia, multiple organ failure, shock, reye syndrome, and secondary bacterial infection and septicemia. death may occur due to these complications. severe cases are commonly found in adults with a past history of good health, with higher fever persisting for a long period of time. nearly all patients with pneumonia receive artifi cial ventilation, and it is often complicated by arsd and mods, with a high mortality rate. almost all patients with avian infl uenza experience fever, mostly with a body temperature of mostly above 38 °c and rarely 41 °c. the fever types are diverse, but continued fever, remittent fever, and irregular fever are the most common. the patients with mild avian infl uenza experience fever for 1-7 days, mostly 3-4 days. with the conditions improved, the body temperature gradually returns to normal. however, the body temperature of patients with severe avian infl uenza might rise to above 39 °c within 2-3 days, sometimes even 41 °c. the high fever is persistent. some patients might experience persistent high fever but gradually drop of the body temperature to normal, with improved toxic symptoms. the patients with avian infl uenza experience serious systemic toxic symptoms in the early stage after onset, such as headache, fatigue, general muscular soreness, and general upset. according to the clinical data of 59 cases of avian infl uenza in hong kong of china, thailand, vietnam, especially ho chi minh city, and cambodia, 28.1 % (9/32) of the cases develop headache and 28.9 % of these cases develop general muscle soreness, costalgia, and general upset. but according to clinical data in china, 45 % of the cases develop headache, 40 % muscular pain, 80 % fatigue, and 60 % aversion to cold. symptoms of the respiratory system include respiratory catarrh symptoms, cough and expectoration, dyspnea, cyanosis, acute respiratory distress syndrome, pulmonary hemorrhage, pleuritis, and pleural effusion. the respiratory catarrh symptoms include rhinorrhea and nasal obstruction and pharyngalgia in some cases. most patients with avian infl uenza develop cough and expectoration at day 3 after onset. cough is paroxysmal and violent, with a small quantity of white or yellowish white mucous sputum. sometimes, the sputum is bloody. dyspnea commonly occurs at day 6 after onset, mostly mixed dyspnea characterized by diffi culty exhaling and inhaling as well as accelerated respiratory rate. the patients with avian infl uenza may develop cyanosis at the lips and skin mucosa. the extensive lung lesions cause decreased proportion of ventilation to blood fl ow, with consequent occurrence of hypoxemia. as a result, increased hemoglobin reduction in capillaries during the systemic circulation causes the occurrence of cyanosis. acute respiratory distress syndrome (ards) is characterized by progressive dyspnea, with a respiratory rate of above 20 times per minute. the rate is progressively accelerated, which may reach up to 60 times per minute. the patients experience cyanosis, irritation, restlessness, and consciousness disturbance. refractory hypoxemia may occur. due to excessive ventilation induced by obvious hypoxemia, paco 2 is subject to decrease, contributing to respiratory alkalosis. the patients with severe avian infl uenza can develop pulmonary hemorrhage due to diffuse alveolar injury and diffuse intravascular blood coagulation. the clinical manifestations include cough-up typical bloody sputum. in severe cases of human infection by avian infl uenza, the patients develop pleuritis and pleural effusion at the middle or advanced stage, mostly 6-12 days after the onset. most patients have rough breathing sound, with accompanying bronchial breathing sound. the breathing sound at the affected lung is weak, with rare moist or wheezing rales but no pleural friction. some patients can develop digestive symptoms at the early stage, including poor appetite, nausea, vomiting, abdominal pain, abdominal distention, diarrhea, and watery stool. some patients experience subjective palpitation, often accompanied by precordial upset and chest distress. in severe cases, the patients experience rapid heart rate and decreased blood pressure, which rapidly develop into shock. in rare cases, the patients might be hospitalized due to hypotension and shock. some patients may experience neurological symptoms, such as headache, irritation, vomiting, convulsion, and lethargy. in rare severe cases, the patients experience initial symptom of serious diarrhea, with following occurrence of convulsion and coma. death may occur in such patients. multiple organ failure includes respiratory failure, heart failure, renal insuffi ciency or failure, liver dysfunction, and disseminated intravascular coagulation. the typical course of human infection with avian infl uenza can be divided into three stages: the early stage, the progressive stage, and the convalescent stage. the early stage refers to the initial 1-4 days after the onset. it is characterized by an acute onset with fever as the fi rst symptom and a body temperature of above 38 °c. other symptoms might also occur, including respiratory symptoms like cough with sputum as well as headache, fatigue, general muscle soreness, costalgia, and general upset. the progressive stage usually begins at day 5 after the onset and lasts for about 16 days, which may be longer in rare cases. compared to the patients with sars, the progressive stage of human infection with avian infl uenza is longer. after the progressive stage, the conditions gradually develop into the convalescent stage, which is usually 22 days after the onset. the patients experience gradual alleviation and absence of toxic symptoms. the body temperature returns to normal and the lung lesions are gradually absorbed and improved. more than 50 % of the patients can be discharged after their convalescent stage lasting for about 2 weeks. based on the severity of clinical symptoms, human infection of avian infl uenza can be divided into mild, common, severe, atypical, and asymptomatic. the patients experience mild respiratory symptoms like mild dry cough, no obvious cough, no tachypnea, and no dyspnea. the liver function shows no obvious abnormality. radiological examination demonstrates no sign of pneumonia. and the prognosis is good. the patients experience typical symptoms of human infection with avian infl uenza, including fever with a body temperature above 38 °c, headache, general pain, fatigue, dry throat, and poor appetite. the patients may also experience respiratory symptoms including cough with bloody sputum, even shortness of breath and cyanosis. by auscultation, low breathing sound at both lungs can be heard with moist or wheezing rales. x-ray demonstrates shadows at the lungs. the peripheral leukocyte count is normal or slightly decreased and there is no hypoxemia by blood gas analysis. the patients experience no serious complications such as ards or multiple organ failure. clinically, severe type is common. the conditions develop rapidly with dramatic deterioration and a high mortality rate. the conditions of severe type may rapidly progress into acute lung injury or ards, leading to respiratory failure. multiple system dysfunction or failure commonly occurs to complicate the conditions. secondary infection of multiple systems at multiple locations by multiple pathogens may also occur. most patients of this type die from progressive respiratory failure or multiple organ failure. the patients with one of the following conditions can be defi ned as the severe type: 1. dyspnea with breathing rate during rest being at least 30/ min, accompanied by one of the following conditions: first, x-ray demonstrates multilobar lesions or anterior-posterior x-ray demonstrates a total area of lesions accounting for over one-third of both lungs. second, the total area of lesions increases by above 50 % within 48 h and by anterior-posterior x-ray accounts for over one-fourth of both lungs. 2. obvious hypoxemia occurs with an oxygenation index below 300 mmhg (1 mmhg = 0.133 kpa). 3. shock or multiple organ dysfunction syndrome develops. 4. invasion of the virus to the central nervous system causes viral encephalitis. 5. reye syndrome occurs in children. 6. serious secondary bacterial infection occurs, especially septicemia or septic shock. atypical type refers to rare patients with mild symptoms, no fever or only mild upset, and no sign of pneumonia. the patients experience no obvious respiratory symptoms, with favorable prognosis. epidemiological studies have demonstrated that rare patients develop no onset of the disease despite of a history of close contact to patient with avian infl uenza or diseased poultry and positive h5n1 antibody. clinically, the patients show no obvious symptoms and signs. it has been reported by who on january 11, 2006, that two children in turkey were detected with positive avian infl uenza virus antibody but show no corresponding symptoms. during the outbreak of avian infl uenza in 1997 in hong kong of china, a nurse with close contacts to patients with avian infl uenza showed positive h5n1 virus antibody. h7n9 is a subtype of avian infl uenza virus. the patients with its infection commonly show fl u-like symptoms, such as fever and cough with a small quantity of sputum, and accompanying headache, muscle soreness, and general upset. in severe cases, the conditions progress rapidly, characterized by severe pneumonia, a body temperature persistently above 39 °c, dyspnea, and bloody sputum. the disease can rapidly progress into acute respiratory distress syndrome, mediastinal emphysema, sepsis, shock, consciousness disturbance, and acute renal injury. pathogenic avian infl uenza-related complication in china, the patients with avian infl uenza often develop ards 8-11 days after the onset. the typical symptoms include progressive dyspnea and even respiratory distress. with the progress of conditions, the patients develop cyanosis, irritation, restlessness, extensive interstitial infi ltration in both lungs, and accompanying dilation of umbilical vein, pleural reaction, or a small quantity of effusion. the condition may further develop into multiple organ failure. at the early stage of human infection with avian infl uenza, the pulmonary interstitium may be involved. but in most patients, the interstitial lesions can be gradually absorbed. in extremely rare severe patients, after pulmonary infl ammation lesions are absorbed at the convalescent stage, pulmonary interstitial fi brosis or hyperplasia still remains. the severity of pulmonary fi brosis is related to the severity of pulmonary lesions, age, obviously compromised immunity, and existence of basic disease. in most cases, pulmonary fi brosis can be healed 6 months after discharge from hospital. in rare severe cases, pulmonary fi brosis may persist a longer period of time. the patients with avian infl uenza show different degrees of infl ammatory exudates at lung tissue. respiratory bronchiolitis obliterans causes breakage of alveolar elastic fi ber, which further develops into pneumothorax or bronchopleural fi stula. in the cases complicated by secondary lung infection, purulent changes are found to aggravate lung injury. in some patients with prior basic lung disease, such as chronic obstructive lung disease and congenial lung diseases, pneumothorax is more likely to occur. the patients of severe type need treatment of noninvasive or invasive ventilation, which is more likely to induce pneumothorax. the occurrence of mediastinal emphysema and subcutaneous emphysema in patients with avian infl uenza commonly follows the use of respirator. during the progress of human infection with avian infl uenza, the conditions are more likely to be complicated by infections, especially bacterial pneumonia. secondary bacterial pneumonia is the main cause of death in patients with avian infl uenza. the most common pathogenic bacteria include streptococcus pneumoniae , staphylococcus aureus , or haemophilus infl uenzae . infection of mixed bacteria may occur. the common pathogenic fungi include candida albicans and aspergillus . and its incidence rate is related to gender, age, existence of basic disease, nutrition, length of hospitalization, and the use of glucocorticoids. according to literature reports, 60-70 % of human infection with avian infl uenza is complicated by liver dysfunction, with slightly increase of alt and ast. however, in china, almost 100 % of human infection with avian infl uenza shows liver dysfunction, with slight to moderate increase of transaminase. in some cases, the patients may show moderate jaundice. liver dysfunction mostly occurs 2-3 weeks after the onset. however, liver function failure has not yet been reported in cases with human infection of avian infl uenza. some patients with avian infl uenza might develop cardiomyopathy with different severity at different stages, which is clinically characterized by chest distress, precordial upset or dull pain, palpitation, and shortness of breath. about 20-40 % of the patients experience bradycardia, slight to moderate increase of myocardial enzymes cpk and ldh, and ecg abnormality. in some serious cases, the patients experience rapid heart rate and decreased blood pressure, which may develop into shock. some severe cases with human infection of avian infl uenza might develop rapid heart rate, nodal tachycardia, and acute heart failure at day 10-18 after the onset. death occurs in such patients due to heart failure or peripheral circulatory failure. a large amount of proteinuria (>3 g/l) occurs in some severe patients with avian human infl uenza in the early stage of onset. at the same time, they might also develop oliguria without hematuria, symptoms caused by urinary irritation, and abnormal number of serum creatinine as well as urea nitrogen. in other cases in the early stage of onset, decreased proportion of urine, polyuria, and erythrocytes as well as casts in the urine can also be found. in some patients with avian infl uenza, the conditions may be complicated by myositis, characterized by remarkable tenderness of the involved muscle and swelling muscle with no elasticity. the most commonly involved muscle is at the lower limbs. some severe cases might also develop myoglobinuria due to rhabdomyolysis, leading to renal failure. reye syndrome is one of the common complications in children with avian infl uenza, with a high mortality rate. it is clinically characterized by nausea and vomiting, followed by symptoms of involved central nervous system, such as lethargy, coma, or delirium. there are usually no localized neurological signs, with jaundice due to hepatomegaly. gene detection has simple operational procedures but with high sensitivity, specifi city, and accuracy. the detection result can be rapidly harvested. and it represents the orientation of virus detection for avian infl uenza virus. quantitative rt-pcr is the best way to detect avian infl uenza virus at the early stage of infection, with results obtained within 4-6 h. pharyngeal swab can carry more viruses than nasal swab, with a higher positive rate. but negative fi nding by just once detection cannot exclude the possibility of virus infection of avian infl uenza h5n1 virus. repeated detections are recommended to defi ne the diagnosis. isolation of avian infl uenza virus from respiratory specimens such as nasopharyngeal secretion and tracheal aspiration is the classical way to defi ne the diagnosis of human infection by avian infl uenza. double sera should be collected at the early or convalescent stage. hemagglutination inhibition test, complement fi xation test, or enzyme-linked immunosorbent assay (elisa) can be performed to detect the antibody of avian infl uenza virus. an at least four times increase of the antibody titer is an indicator for retrospective diagnosis. x-ray, ct scanning, and mr imaging are important ways for the diagnosis of human infection by avian infl uenza and its complication, differential diagnosis, therapeutic assessment, and prognosis analysis. according to the criteria for clinical diagnosis of human infection by avian infl uenza, radiological fi nding of infi ltration shadow at the lungs is important for early diagnosis of human infection by avian infl uenza. consecutive x-rays can demonstrate the dynamic changes of lesions, which is the important way to assess the progress of the conditions, the therapeutic effect, and the prognosis. the radiological demonstrations should be analyzed based on the stages and clinical types. at the early stage, commonly at day 1-4 after the onset, the imaging demonstrations are characterized by focal fl akes or patches of shadow due to focal consolidation at the lungs. about 90 % of patients with avian infl uenza within 7 days after the onset demonstrate by ct scanning singular or multiple small fl akes of shadow with low-density and poorly defi ned boundary. most of the shadow is singular with irregular shape. in some cases, pulmonary markings are subject to increase and thickness with predominately peripheral distribution. in the large fl ake of consolidation shadow, air bronchus sign is demonstrated. a small quantity of effusion can be demonstrated in the pleural cavity. most patients experience aggravation of the conditions 14 days after the onset. initially, the small fl akes of shadows may turn into large fl ake of multiple or diffuse lesions, which develop from unilateral occurrence to bilateral occurrence, from singular lung fi eld to multiple lung fi elds. in severe cases, obvious changes can be demonstrated within 1-2 days after the onset. the severe cases develop diffuse infi ltrative lesions at unilateral lung or bilateral lungs in large fl akes of ground-glass opacity and pulmonary consolidation shadow, with inner air bronchus sign. with the progress of conditions, diffuse consolidation shadows are demonstrated at the lungs, possibly with white lung sign at both lungs. the lesions of pneumonia in the cases of human infected avian infl uenza are gradually absorbed within 15-30 days, and the lesions of most patients can be completely absorbed. however, in rare cases, the lesions are partially absorbed with development of fi brosis or proliferation of pulmonary interstitial tissues. obvious proliferation of pulmonary interstitial tissues may occur 30-40 days after the onset, fi rstly occurring as thickening of interlobular septum and intralobular interstitium as well as subpleural arc shape linear shadow. the fl akes of shadow at the lungs shrink with increased density, with following occurrence of high-density cord-like or honeycomb-like shadow. in some serious cases, pulmonary interstitial proliferation causes shrinkage of lung volume and shift of mediastinum towards the affected lung. pulmonary interstitial proliferation may extensively exist at the lungs, characterized by thickening of interlobular septum, intralobular septum, and interstitium as well as subpleural arch shape linear shadow. pulmonary interstitial fi brosis is characterized by honeycomb-like shadow and referred bronchiectasis. after the conditions remain stable, the lesions begin to be absorbed, with decreased range and decreased density. in some cases, despite no abnormal fi ndings by x-ray, ct scanning still demonstrates light ground-glass opacity, which may remain for a long period of time. therefore, regular ct scanning is recommended to demonstrate lesions that fail to be demonstrated by x-ray. chest x-ray demonstrations of human infected avian infl uenza are characterized by their rapid change, which is also an important difference from common pneumonia and other atypical pneumonia. at the early and progressive stages, the lung lesions are subject to rapid changes during a short period of time (the shortest period being 12 h), with expansion, perfusion, and migration of the lesions. the shape, range, and location of the lesions may also be subject to changes. the absorption of lesions generally occurs 14 days after the onset, but in rare mild type of cases, it may occur at day 7 after the onset, with decreased range and density of lesions. for those with favorable therapeutic effect, the large fl akes of shadow at the lungs can be signifi cantly changed within 1 day. ards is the main cause of death in patients with avian infl uenza. in severe cases, diffuse alveolar consolidation and ground-glass opacity can be demonstrated at the lungs. preliminary observations demonstrate that in the cases of death extensive pulmonary consolidation and white lung sign are commonly demonstrated during the progressive stage. a boy aged 6 years complained of fever and cough for 15 days, which aggravated with accompanying chest distress, shortness of breath, headache, and muscle soreness for 1 week. he lived in a region with deaths of diseased chicken and ducks and he had a history of intake of diseased chicken and duck. real-time pcr of pharyngeal swab and rt-pcr demonstrated positive nucleic acids of avian infl uenza virus h5n1. his mother died from respiratory failure on the day when the boy experienced the onset 7 days after her complaint of high fever and cough. the cases complicated by pneumonia are radiologically demonstrated with fl akes of shadow at the lungs. in severe cases, the conditions progress rapidly, with ground-glass opacity, pulmonary consolidation shadow, and accompanying small quantity of pleural effusion (figs. 18.6 , 18.7 , 18.8 , and 18.9 ). in the cases with ards, the lesions are extensively distributed. a female patient aged 67 years developed fever with a body temperature fl uctuating between 38.7 and 39.0 °c for about 1 week after she returned home from zhejiang, china. she had no remarkable cough with sputum. by routine blood test, wbc 3.68 × 10 9 /l and n 58.1 %. by routine urine microscopy, erythrocytes 15-20/hp. at a local hospital, she was suspected to have viral upper respiratory infection and was then treated with anti-viral oral medication. after positive therapy, her temperature still fl uctuated around 39 °c, and she went to the emergency department of our hospital on march 27, 2013. by auscultation, breathing sound at both lungs is rough with a few moist rales. chest ct scanning indicated consolidation shadow at the right upper lung lobe, which was suspected to be infl ammatory disease. by routine blood test, wbc 5.35 × 10 9 /l and n 68.2 %, and by routine urine microscopy erythrocytes 16-20/hp as well as normal bun and creatinine. the patient was immediately administered anti-infection and symptomatic supportive treatment. after these therapies, her body temperature failed to return to normal and reached to the highest temperature of 39.5 °c on march 29. she then developed chest distress and cough with rare foamy sputum that is diffi cult to be expectorated. meanwhile, hypoxemia occurred. thus, the administered antibiotic was upgraded to tienam to fi ght against the suspected infection. bipap and methylprednisolone were also administered to facilitate ventilation, anti-infl ammation, and bronchial dilation. after the active therapy, the high body temperature slightly decreased but hypoxemia gradually aggravated. by auscultation, breathing sound at both lungs is rough, with moist rales at the right upper a male patient aged 56 years complained of fever for 7 days as well as cough with sputum and chest distress for 3 days. lung. x-ray indicated extensive infl ammation at both lungs and lobar pneumonia at the right upper lung. she was immediately offered simv + psv via tracheal intubation and treated with additional medicine including norvancomycin and acyclovir to fi ght against the infection and virus. her oxygen saturation fl uctuated between 75 and 80 %, indicating severe conditions. and she was transferred to icu due to suspected diagnosis of severe pneumonia and respiratory failure. by physical examination, t 37.3 °c, p 66/min, r 20/min, and bp 171/84 mmhg. she was unconscious and had cyanosis at lips and weakened breathing movement at the right side. percussion demonstrated dullness at the right upper lung while clear sound at the other lung fi elds. the breathing sound was rough at both lungs with moist rales, particularly at the right upper lung. on april 1 after her hospitalization, operational procedures were performed to exclude the possibility of human infected avian infl uenza but demonstrated positive avian infl uenza (h7n9) by local cdc. after consultation of experts, the therapies were modifi ed. on april 2013, she underwent tracheotomy but still treated with mechanical ventilation by a respirator. on april 13, 2013, spo 2 decreased to 65 % with fl uctuating blood pressure and a minimum of 79/43 mmhg. her highest body temperature was 40.3. treated by modifi ed therapies, the heart rate and blood pressure once returned to normal, but fi nally clinical death was declared after emergency rescuing. on april 1, 2013, laboratory tests were performed. by routine blood test, wbc 7.70 × 10 9 /l, gr 90.4 %, ly 5.8 %, hgb 131 g/l, and plt 162 × 10 9 /l. by blood gas analysis, ph 7.41, po 2 6.88 kpa, pco 2 6.22 kpa, and sao 2 80.5 %. by blood biochemistry, urea 9.5 mmol/l, cr 57 μmol/l, ua 216 μmol/l, k 3.86 mmol/l, na 128 mmol/l, cl 95 mmol/l, ca 1.98 mmol/l, p 1.51 mmol/l, and co 2 32.3 mmol/l. on april 13, 2013, by routine blood test, wbc 9.65 × 10 9 /l, gr 93.4 %, ly 2.5 %, hgb 79 g/l, and plt 79 × 10 9 /l. by blood gas analysis, ph 7.14, po 2 10.45 kpa, pco 2 10.00 kpa, and sao 2 85.3 %. by blood biochemistry, k 6.20 mmol/l, na 146 mmol/l, cl 90 mmol/l, ca 1.88 mmol/l, p 3.88 mmol/l, and co 2 28.4 mmol/l. by detection of infl ammatory indicators, pct 0.14 ng/ml and crp 43 mg/l. apparent pulmonary interstitial hyperplasia fi rstly causes interlobular septal hyperplasia, intralobular interstitial hyperplasia, and subpleural arch shape linear shadow. the fl akes of shadows at the lungs shrink with increased density, with gradual development of strips and honeycomblike high-density shadow at the lungs. severe pulmonary interstitial hyperplasia causes reduced lung volume and shift of mediastinum towards the affected lung. pulmonary interstitial hyperplasia may be extensively found at the lungs, characterized by thickened interlobular septum, thickened intralobular interstitium, and subpleural arch shape line. otherwise, it can be demonstrated as local irregular high-density patches of and cord-like shadows. intrapulmonary honeycomb-like shadow and referred bronchiectasis are indicators of pulmonary interstitial fi brosis. bacterial infection x-ray or ct scanning demonstrates fl akes of and mass-like shadows at the lungs. fungal infection x-ray and ct scanning demonstrate diversifi ed lesions. they may be scattering small nodular shadows at the lungs or patches of shadows at the middle and lower lung fi elds. otherwise, the lesions are demonstrated as mass-like or cavity-like shadows or fused lesions into large fl akes of shadows in a large range. pneumothorax is manifested as shedding of visceral pleura away from the chest wall. by x-ray, it is demonstrated as hairlike linear shadow parallel to the chest wall and no lung markings exterior to the linear shadow. ct scanning demonstrates transparent areas without lung markings at the peripheral thoracic cavity as well as compression and insuffi cient expansion of the lung. demonstrations of mediastinal emphysema by x-ray include vertical gas strip between the heart shadow and the paratracheal soft tissue shadow and linear shadow parallel to the mediastinum form by elevated mediastinal pleura supported by a thin layer of gas. lateral x-ray demonstrates that the thymus and vascular shadows in the anterior mediastinum are surrounded by gas. flow of gas at the tangent line in involved subcutaneous soft tissue is demonstrated as cystic or strips of gas containing shadow, with the skin being abnormally elevated and thickened. in the cases with gas gathering at the surface of the pectoralis major, characteristic strips of transparent shadows resembling to fan-shaped distributed muscular fi bers are demonstrated at the upper lung fi elds. ct scanning demonstrates gas density linear shadow around the mediastinum and shift of mediastinal pleura towards the lung fi eld. gas in the mediastinum fl ows along the cervical fascia space to the neck and thoracic subcutaneous tissue, thus producing subcutaneous gas density shadow. the diagnosis can be defi ned based on the contact history, clinical manifestations, and laboratory fi ndings. the contact history plays a critical role in the diagnosis of human infected avian infl uenza. large quantities of viruses are excreted along with saliva, nasal secretions, and feces from the infected poultry. direct contact to infected poultry or contact to the utensils contaminated by their feces or secretions is believed to be the main route for spreading avian infl uenza virus. the following epidemiological data facilitates the diagnosis of human infected avian infl uenza: 1. one week prior to the onset, the patients visited the epidemic focus. 2. one week prior to the onset, the patients had close contact to secretions or excretions from infected poultry. 3. one weeks prior to the onset, the patients lived nearby an area with the cases of avian infl uenza or traveled at an epidemic region. 4. the patients have a history of close contact to patients with avian infl uenza. 5. some patients may have no defi ned epidemiological history of avian infl uenza. for those with no direct epidemiological data, the patients should be carefully inquired about contact history to water contaminated by avian infl uenza. 6. human infection of avian infl uenza spreads from chicken, duck, goose, and other poultries, especially chickens. therefore, outbreak of avian infl uenza, especially in chickens, is prior to the outbreak of its human infection. this is an important clue and basis for the diagnosis of human infected avian infl uenza. the incubation period of human infected avian infl uenza generally lasts for 1-7 days, commonly 2-4 days but may be as long as 8 days. the interval of its occurrence in family members is about 2-5 days, maximally 8-17 days but may be as long as 21 days. different subtypes of avian infl uenza virus can cause variant clinical symptoms after they infect human. avian infl uenza h5n1 has an acute onset with its early symptoms resembling to common infl uenza. it is characterized by fever persisting for 1-14 days with a body temperature over 39 °c and maximally 41 °c and accompanying rhinorrhea, nasal obstruction, cough, sore throat, headache, muscle soreness, and general upset. in some cases, the patients may develop digestive symptoms including nausea, abdominal pain, diarrhea, and watery stool. persistent high fever may occur in severe patients, with rapid progress of the conditions into apparent pneumonia, acute lung injury, and acute respiratory distress syndrome. the total wbc count is normal or lower than normal, especially with decreased absolute lymphocyte count. the platelet count is normal or is subject to slight to moderate decrease. the decreases of leukocytes, platelets, and lymphocytes, particularly the decrease of lymphocytes, are related to the severity of clinical symptoms and the mortality rate. respiratory specimens from patients are collected to detect the antigens of nucleocapsid protein (np) or matrix protein (m1) or subtype h by immunofl uorescence assay or enzymelinked immunosorbent assay (elisa). avian infl uenza virus can be isolated from the respiratory specimen from patients. the serum-specifi c antibody of positive avian infl uenza virus like h5n1 or at least four times increase of antibody titer of avian infl uenza virus subtype strains in paired serum from the early and convalescent stages facilitates diagnosis. the severe type is demonstrated with: 1. diffuse distribution of lung lesions, commonly with large fl ake of or multiple patches of fused shadows at most of unilateral lung or multiple lobes and segment of bilateral lungs. the diffuse lesions can be demonstrated at the early stage, which persist for a long period of time. 2. rapid progress of the lesions, with signifi cant development of the lesions with a short period of time. the focal lesions at the early stage rapidly expand to large fl ake of diffuse shadow. the density of shadow changes also rapidly, with rapid mutual transformation between groundglass opacity and consolidation. 3. the conditions rapidly develop into acute respiratory distress syndrome, with demonstrations of extensive highdensity shadow at both lungs. the patients commonly experience an acute onset with symptoms of aversion to cold, high fever, headache, dizziness, general soreness, fatigue, and other toxic symptoms. the patients may also experience respiratory symptoms such as sore throat, dry cough, rhinorrhea, and lachrymation. in rare cases, the patients experience poor appetite, abdominal pain, abdominal distension, vomiting, diarrhea, and other digestive symptoms. by peripheral blood test, the total wbc count is normal or lower. infl uenza virus can be successfully isolated from the nasopharyngeal secretion of patients. an at least four times increase of antibody titer against infl uenza virus can be detected in serum collected at convalescent and acute stages. direct detection of the infl uenza virus antigen in the epithelial cells at the respiratory tract is positive, and the antigen of infl uenza virus after one generation reproduction by sensitive cells is positive. infl uenza virus pneumonia commonly occurs in infants and young children, the elderly and the weak, and patients with chronic disease or compromised immunity. its incubation period is short, commonly lasting for 1-3 days. typical infl uenza is commonly characterized by acute onset with sudden symptoms of systemic toxic symptoms, such as aversion to cold, chills, high fever, general soreness, severe headache, fl ushed face, congested conjunctiva, and weakness. but the nasal and pharyngeal symptoms are mild or unremarkable. the respiratory symptoms include gradually exacerbated cough with blood-tinged or bloody sputum, shortness of breath, and cyanosis. the fever commonly persists for a short period of time. severe infl uenza virus pneumonia is demonstrated with obvious pulmonary lesions, commonly with diffuse bubbling and wheezing. by laboratory test, decreased leukocytes, leftward shift of neutrophil nuclei, and relatively increased lymphocytes can be found. radiological examinations of the chest demonstrate scattering cotton-wool-like lesions at both lungs or lesions of interstitial pneumonia. etiological test can provide direct evidence to defi nite the diagnosis of infl uenza, but with no value for the early diagnosis. throat lavage fl uid for virus isolation is inoculated into chick embryo allantois. agglutination test is then performed with the throat lavage and chick erythrocytes. however, agglutination inhibition test for chick erythrocytes should be simultaneously performed to exclude nonspecifi c agglutination. such a test has a positive rate of 26-50 %. the serum antibody detection, commonly by agglutination inhibition test of erythrocytes or complement fi xation test, can be performed simultaneously, with a positive rate of 95 %. contrast detection should be performed at the early stage and 2 weeks after the onset, and an at least four times increase of the antibody titer has diagnostic value. 18.9.2.1 epidemiological history 1. the patient has a history of close contact to patients with sars, or he/she is a member of the possibly infected population, or defi nite evidence has found supporting his/ her transmission to others. 2. one weeks prior to the onset, the patients visited or lived at the region with cases of sars. the disease has an acute onset, with fever as its initial symptom and a body temperature of above 38 °c as well as occasional aversion to cold. the patients may also experience headache, joint pain, muscle soreness, fatigue, and diarrhea. although upper respiratory catarrh symptoms are commonly absent, cough and chest distress may develop, particularly dry cough with a little sputum and occasional blood-tinged sputum. in severe cases, the patients experience rapid respiration, shortness of breath, or apparent respiratory distress. the lung lesions are not remarkable, possibly with rare moist rales or pulmonary consolidation. the peripheral wbc count is normal or decreases and the lymphocyte count commonly drops. the lungs are demonstrated with fl akes and patches of infi ltrative shadows or grid-like shadow. some patients show rapid progress of the lesions, with large fl akes of shadows commonly at both lungs. and the shadow is absorbed slowly. the pulmonary shadow demonstrated by chest x-ray might be inconsistent to the clinical symptoms and signs. autopsy and pathological analysis of human death cases from highly pathogenic avian infl uenza a (h5n1) infl uenza a (h5n1): will it be the next pandemic infl uenza? are we ready avian infl uenza a (h5n1) infection in humans the evolution of h5n1 infl uenza viruses in ducks in southern china induction of proinfl ammatory cytokines in human macrophages by infl uenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? fatal outcome of human infl uenza a (h5n1) is associated with high viral load and hypercytokinemia two clusters of human infection with infl uenza a/h5n1 virus in the republic of azerbaijan h5n1 infl uenza: a protean pandemic threat current studies on human infection of avian infl uenza chest x-ray demonstrations of viral pneumonia caused by avian infl uenza a (h5n1) in children review and implication of prevention again human infection of avian infl uenza in hong kong strategies for the diagnosis and treatment of human infection of avian infl uenza the fi rst clinical report on human infection of avian infl uenza a (h5n1) in shenzhen, china chest x-ray imaging of patients with sars chest x-ray demonstrations of sars radiological demonstrations of viral pneumonia caused by human infection of highly pathogenic avian infl uenza a (h5n1) the fi rst case report of pneumonia due to human infection of avian infl uenza a (h5n1) in mainland china avian infl uenza a (h5n1) infection in eastern turkey in 2006 re-emergence of fatal human infl uenza a subtype h5n1 disease evaluation of a genetically modifi ed reassortant h5n1 infl uenza a virus vaccine candidate generated by plasmid-based reverse genetics h5n1 virus attachment to lower respiratory tract antibacterial medication is commonly ineffective to treat sars. pulmonary candidiasis is characterized by white foamy thick sputum or cheese-like sputum with fermented stinky odor. detection of sulfur granules in the sputum facilitates the diagnosis of pulmonary actinomycosis. the accompanying wheezing at the lungs and increased eosinophils in the blood are helpful for the diagnosis of allergic bronchopulmonary aspergillosis (abpa). pulmonary aspergilloma has characteristic signs by chest x-ray. allergic bronchopulmonary aspergillosis (abpa) is demonstrated with migratory infi ltrative lesions at lobes or segments. otherwise, abpa can be demonstrated with segmental or lobar atelectasis due to obstructed bronchus by mucus but no shift of interlobar fi ssure. invasive pulmonary aspergillosis at the early stage may be characterized by localized or multiple infi ltrative lesions or nodular lesions at both lungs. the lesions commonly rapidly expand to integrate into consolidation or cavity. the diagnosis of pulmonary candidiasis can be defi ned based on positive fi ndings of candida albicans by sputum culture for three consecutive times, fi nding of fungal threads by smear, or proved pathogenicity by animal inoculation. the fi ndings of fl exuous hyphae by smear of bronchofi broscopic extracts or growth of aspergillus by culture can defi ne the diagnosis of pulmonary aspergillosis. when sulfur granules are observed in sputum or tissues of fi stula wall, the diagnosis of pulmonary actinomycosis can be defi ned. otherwise, the fi nding of pathogenic microorganism after anaerobic culture and the diagnosis of pulmonary actinomycosis can also be defi ned. 1. the patients have no defi nitive history of close physical contacts or inhaling of respiratory droplets. hiv/aids or in patients with hiv/aids. 3. the patients experience no typical symptoms, such as acute fever, headache, muscle soreness, joint pain, fatigue, and other infl uenza-like symptoms. 4. chest x-ray demonstrates slow change of lung shadows.the lung lesions commonly undergo a series of pathological changes including exudation, infi ltration, integration, consolidation, and interstitial fi brosis. at the early stage, symmetric miliary alveolar effusion shadows are demonstrated at both lungs. the lesions further develop into ground-glass opacity. at the middle stage, fl akes of or consolidation shadows are demonstrated, which are actually infi ltration and integration. after timely and appropriate intervention, the pulmonary shadow can be gradually absorbed, possibly with residual cord-like shadows of pulmonary interstitial fi brosis. otherwise, the patients may die from respiratory failure or multiple organ failure. 5. antibody against hiv is positive. human infected avian infl uenza (h7n9) should be differentiated from highly pathogenic avian infl uenza (h5n1), seasonal infl uenza (including infl uenza a h1n1), bacterial pneumonia, sars, novel coronavirus pneumonia, adenovirus pneumonia, chlamydia pneumonia, and mycoplasma pneumonia. and the differential diagnosis is mainly based on etiological test. key: cord-034469-ew90eef4 authors: dos santos rocha, andre; fodor, gergely h.; kassai, miklos; degrugilliers, loic; bayat, sam; petak, ferenc; habre, walid title: physiologically variable ventilation reduces regional lung inflammation in a pediatric model of acute respiratory distress syndrome date: 2020-10-31 journal: respir res doi: 10.1186/s12931-020-01559-x sha: doc_id: 34469 cord_uid: ew90eef4 background: benefits of variable mechanical ventilation based on the physiological breathing pattern have been observed both in healthy and injured lungs. these benefits have not been characterized in pediatric models and the effect of this ventilation mode on regional distribution of lung inflammation also remains controversial. here, we compare structural, molecular and functional outcomes reflecting regional inflammation between pvv and conventional pressure-controlled ventilation (pcv) in a pediatric model of healthy lungs and acute respiratory distress syndrome (ards). methods: new-zealand white rabbit pups (n = 36, 670 ± 20 g [half-width 95% confidence interval]), with healthy lungs or after induction of ards, were randomized to five hours of mechanical ventilation with pcv or pvv. regional lung aeration, inflammation and perfusion were assessed using x-ray computed tomography, positron-emission tomography and single-photon emission computed tomography, respectively. ventilation parameters, blood gases and respiratory tissue elastance were recorded hourly. results: mechanical ventilation worsened respiratory elastance in healthy and ards animals ventilated with pcv (11 ± 8%, 6 ± 3%, p < 0.04), however, this trend was improved by pvv (1 ± 4%, − 6 ± 2%). animals receiving pvv presented reduced inflammation as assessed by lung normalized [(18)f]fluorodeoxyglucose uptake in healthy (1.49 ± 0.62 standardized uptake value, suv) and ards animals (1.86 ± 0.47 suv) compared to pcv (2.33 ± 0.775 and 2.28 ± 0.3 suv, respectively, p < 0.05), particularly in the well and poorly aerated lung zones. no benefit of pvv could be detected on regional blood perfusion or blood gas parameters. conclusions: variable ventilation based on a physiological respiratory pattern, compared to conventional pressure-controlled ventilation, reduced global and regional inflammation in both healthy and injured lungs of juvenile rabbits. acute respiratory distress syndrome (ards), characterized by the acute onset of severe hypoxic respiratory failure, remains a prevalent and often lethal condition in intensive care [1] . although mechanical ventilation is a crucial life-saving treatment for ards, there is a considerable body of evidence indicating that prolonged positive-pressure ventilation can initiate, perpetuate or aggravate injury to lung tissue [2, 3] . the resulting exaggerated mechanical stress, along with the monotonous alveolar opening and closing, exerts shear stress and increased strain in the lung tissue [4] , conditions that contribute to ventilator-induced lung injury (vili). while various modalities of mechanical ventilation have been proposed to reduce vili [5] [6] [7] [8] , protective ventilation with monotonous tidal volume (vt) may not be the only rational strategy. in recent years, it has been advocated that mechanical ventilation reproducing the natural variability of breathing is better than conventional modes [9, 10] . variable ventilation has been shown to be beneficial for gas exchange and respiratory mechanics in various animal models with healthy [11] [12] [13] or injured lungs, including ards [14] [15] [16] [17] . we have previously established a variable ventilation modality using pre-recorded breathing patterns of healthy animals [18] . this physiologically variable ventilation (pvv) is characterized by breath-to-breath variability of vt and respiratory rate, in contrast to the monotonous conventional ventilation modes. recent interest in variable ventilation stems from the need to reduce cyclic alveolar reopening during mechanical ventilation, especially in injured lungs, to avoid development or propagation of lung inflammation, atelectasis and subsequent hypoxemia [19] . whereas some studies demonstrated the beneficial effect of introducing variability into lung recruitment [20, 21] , and others reported improvement in global respiratory mechanical and functional parameters [11] [12] [13] [14] [15] [16] [17] [18] , there is still a lack of detailed knowledge about the pathophysiological background related to the functional and regional behavior of the lung during variable ventilation. moreover, the potential of pvv in the context of pediatric ards has not been characterized. to investigate the effect of pvv, lung functional and structural changes were compared to those obtained with conventional monotonous ventilation in normal lungs and ards, in a pediatric model. global respiratory parameters were measured to characterize the overall lung condition. regional lung aeration, pulmonary perfusion and inflammation were assessed by functional imaging using positron-emission tomography (pet) and single-photon emission computed tomography (spect) combined with x-ray computed tomography (ct). a more detailed description of the methods can be found in additional file 1. new zealand white rabbit pups of both sexes, aged 4 to 5 weeks, were included in the present study (mean weight: 630 g, 370-860 g). this age can be approximated to an equivalent human age of 6 to 8 months [22] . rabbits underwent tracheostomy and continuous intravenous (iv) anesthesia using propofol (10 mg/kg/h), fentanyl (5 µg/ kg/h), midazolam (0.2 mg/kg/h) and atracurium (0.6 mg/ kg/h). the protocol of the study is depicted in fig. 1 . under baseline (bl) conditions, pressure-controlled ventilation was applied, using a positive end-expiratory pressure (peep) of 6 cmh 2 o, a fraction of inspired oxygen (fio 2 ) of 0.4, a vt of 8 ml/kg and a respiratory rate to achieve normocapnia (end-tidal co 2 of 5.5-6%). arterial and central venous blood gas analyses and respiratory mechanical measurements were performed at bl. subsequently, animals were randomized for the absence (ctrl) or presence (ards) of lung injury. mild ards, according to the berlin definition [23] , was induced by combination of intravenous lipopolysaccharide (20 µg/ kg) and injurious ventilation (vt = 40 ml/kg, 0 cmh 2 o peep, fio 2 = 1.0) with a target range of partial pressure of arterial oxygen (pao 2 )/fio 2 ratio of 250-300 mmhg. when the target range of pao 2 /fio 2 was reached, animals were further randomized for the ventilation mode: five-hour mechanical ventilation (vt = 8 ml/ kg, peep = 6 cmh 2 o) was applied using either pressure-controlled ventilation (pcv) or pvv. fio 2 was adjusted according to pao 2 /fio 2 : using fio 2 = 0.4 above 250 mmhg; fio 2 = 0.6 between 200-250 mmhg; fio 2 = 0.8 between 100-200 mmhg, and fio 2 = 0.9 in the case that pao 2 decreased below 100 mmhg. arterial blood gas and respiratory mechanics were measured hourly (t1-t5). after 5 h (t5), in vivo lung imaging was performed under continuous application of the ventilation mode. subsequently, animals were euthanized with iv sodium thiopental (100 mg/kg). bronchoalveolar lavage was performed ex vivo in the right lung, and the left lung was extracted for histological analysis. respiratory mechanical parameters were assessed by the wave-tube method of the forced oscillation technique, as detailed previously [14] . the constant-phase model [24] was fitted to the spectra to separate airway and tissue compartments of the respiratory system. airway resistance (raw), tissue damping (g) and tissue elastance (h) were estimated from the fits. a commercially available pediatric ventilator (servo-i, maquet critical care, solna, sweden) was used with special firmware. the applied variable pattern was the reproduction of physiological breathing in rabbit pups, obtained using unconstrained whole-body plethysmography. structural imaging of the respiratory system was acquired using ct. regional lung perfusion was assessed though spect imaging using 99m tc-labeled iv albumin macroaggregates. regional distribution of inflammatory activity was assessed using pet imaging of fluorodeoxyglucose ( 18 f-fdg) [25] . lung radiodensity was expressed in mean pixel value (mpv), while pet and spect activity were expressed as standardized uptake value (suv) normalized for voxelwise fraction of lung tissue [26] . ct images were segmented to well aerated, poorly aerated and non-aerated zones, based on radiodensity, as well as to ventral and dorsal halves. these segmented zones were considered when analyzing pet and spect images. cell and cytokine content of the bronchoalveolar lavage fluid (balf) was analyzed as detailed previously [18] . a histological lung injury score was determined according to the american thoracic society guidelines [27] . tracheal pressure, airflow, arterial pressure, central venous pressure (cvp) and electrocardiogram were digitized and continuously recorded. mean arterial pressure (map) and heart rate (hr) were assessed from these curves. the primary outcomes of the present study were defined as respiratory mechanical parameters (raw, tissue damping and elastance), arterial blood gas parameters (lactate, ph, pao 2 /fio 2 and paco 2 ) and imaging parameters. secondary outcomes were hemodynamic and ventilation parameters, cytokine levels and lung injury histological indices. data are presented as mean ± half-width of 95% confidence interval. normality of the data was assessed for each variable with the shapiro-wilk test. in case of a failed normality test, the variable was log-transformed. repeated measures analyses of variance (anova) using linear mixed-effect model fits by a restricted maximum likelihood (reml) method were applied to calculate statistical significances followed by dunnett or holm-sidak post-hoc tests, using a significance level of p < 0.05, and all p values two-sided. forty-four rabbits were randomized into one of four experimental groups. eight rabbits were excluded from the analysis due to vital issues precluding the 5 h of ventilation (pneumothorax, n = 7; hemorrhage, n = 1). therefore, 36 rabbits were included in the final analyses, with zrs: impedance of respiratory system; bg: blood gas; peep: positive end-expiratory pressure; lps iv: lipopolysaccharide intravenous; pcv: pressure-controlled ventilation; pvv: physiological variable ventilation; ctrl: control; ards: acute respiratory distress syndrome the following distribution: 12 rabbits were included in the pcv-ards group, 10 rabbits in pvv-ards, 7 rabbits in pcv-ctrl and 7 rabbits in pvv-ctrl. parameters characterizing respiratory mechanics obtained prior to initiating the 5-h ventilation are displayed in additional file 1: table s1 . changes in respiratory mechanical parameters relative to those obtained immediately after the induction of lung injury are displayed in fig. 2 . applying pcv for 5 h led to significant increases in tissue elastance (t1-t5, p < 0.01) in the control animals and in raw in the ards model (t1-t5, p < 0.03). conversely, ventilating the lungs with pvv resulted in a significant decrease in tissue damping in control animals (t1-t5, p < 0.01), whereas no change in respiratory mechanics was detected in the ards model. comparison of the two ventilation modes revealed significantly lower relative changes with pvv in tissue damping for the control animals (t4-t5, p < 0.03) and tissue elastance for the ards model (t1-t5, p < 0.01). figure 3 depicts the blood gas parameters during the 5-h ventilation. inducing lung injury led to significant impairment of the blood oxygenation index (pao 2 /fio 2 ), confirming the presence of mild to moderate ards, according to the berlin definition [23] . further drift in pao 2 /fio 2 was observed in the pvv-ards group that resulted in statistically significant decreases after the monotonous ventilation with pcv had no effect on the blood gas parameters in the control animals, whereas a systematic decrease in ph and plasma lactate concentration was observed in the ards groups (t1-t5, p < 0.001). applying variable ventilation for 5 h in the control group had no systematic effect on gas exchange, whereas higher paco 2 levels (t1-t5, p < 0.05) were associated with significantly diminished ph and elevated lactate in animals with ards (t1-t5, p < 0.01). representative ct, pet and spect images with the corresponding regional aeration maps in control and ards conditions are shown in fig. 4 . more heterogeneous lung structure, as indicated by heterogeneous regional distribution of 18 f-fdg uptake and 99m tc-labeled albumin macroaggregates, was observed in the presence of ards. the pet uptake values calculated for the total lung and at regional levels are summarized for the study groups in the left panels of fig. 5 . when averaging the entire lung, significantly lower mean 18 f-fdg uptake was evidenced for the lungs in the animals ventilated with pvv, regardless of the presence of lung injury. this difference was also detected at the regional level in rabbits with healthy lungs ventilated with pvv (p < 0.04). characterizing the differences in 18 f-fdg uptake among the various aeration zones, defined by ct density, revealed the highest activity in the well aerated zones, with 2 to threefold differences compared to the non-aerated zones (p < 0.01, well aerated vs. poorly aerated or non-aerated). likewise, ventral (non-dependent) regions presented significantly higher 18 f-fdg uptake compared to dorsal (dependent) regions in both ventilation modes. furthermore, significantly decreased mean 18 f-fdg uptake was observed in the control animals ventilated with pvv compared to those with pcv (p < 0.01). no evidence for a difference in spect activity was detected between the protocol groups (fig. 5 , right panels). however, regional perfusion was significantly and consistently higher in the well aerated zones and the dorsal zones of the lung, without differences between the experimental groups. the detailed results on secondary outcomes (hemodynamic and ventilation parameters, cytokine levels and lung injury histological indices) can be found in additional file 1. in the presence of ards, significantly higher driving pressure was required to maintain the same minute ventilation than in healthy animals (p < 0.01, ards vs. ctrl, additional file 1: figure s2 ). in the ctrl group, a progressive reduction in driving pressure was observed with pvv (p < 0.01 vs. t0, additional file 1: figure s2) , which was not observed in animals ventilated with pcv. no differences were detected between the two ventilation modes in regards of the hemodynamic parameters (additional file 1: figure s4 ), lung injury score (additional file 1: table s2 ), cytokine and cell content of balf (additional file 1: table s3 ). in the present study, a combined approach consisting of lung functional and structural assessment was used to investigate differences in the global and regional effects of pvv and the conventional monotonous pressure-controlled mode in a pediatric model of normal lungs and ards. the use of pvv decreased pulmonary inflammation, as assessed by 18 f-fdg uptake, independent of lung condition. the decreased lung inflammation observed with pvv was also detected as an improvement in respiratory tissue elastance. neither the use of pcv nor pvv affected blood gas and lung morphology indices. respiratory system mechanical parameters obtained in bl conditions or following induction of lung injury exhibited excellent agreement with previous data from the same species with similar weight range [14] [15] [16] 28] . furthermore, the time course of the respiratory mechanical parameters over 5 h of ventilation in the control groups is in accordance with that observed previously in an experimental model using adult rabbits [18] . since increases in tissue damping and elastance reflect lung volume loss and stiffening of the lung tissue [29, 30] , the lack of an increase of elastance in the pvv-ctrl group suggests that lung derecruitment did not occur, and this conclusion is also supported by the lower inspiratory driving pressure achieved in this group. moreover, the significant differences in elastance between the pcv-ards and pvv-ards groups observed after the 5-h ventilation suggest a protective effect of the variability on the conservation of lung volume in the presence of ards. studies using models of mild-to-moderate lung injury have found similar beneficial effects on respiratory mechanics for variable ventilation [15, 31] , and this protective effect was not observed in the presence of more severe ards [14] . global and regional lung metabolic activity were measured by 18 f-fdg uptake, a reliable biomarker of inflammation in the lung [32] . this marker is indicative of neutrophil activation in acute lung injury and ards [33] [34] [35] . previous studies have shown that voxelwise ratio of lung parenchyma and air content influences 18 f-fdg fig. 5 left panels depict pulmonary inflammation characterized by pet imaging normalized to the tissue fraction. right panels show pulmonary circulation characterized by spect imaging, normalized to tissue fraction. upper panels represent mean pet and spect intensities averaged for the entire lung. middle panels demonstrate the regional distribution based on aeration zones. bottom panels represent the regional distribution based on the dependent (dorsal) and non-dependent (ventral) zones. suv: standardized uptake value; pcv: pressure-controlled ventilation; pvv: physiological variable ventilation; ards: presence of lung injury; ctrl: absence of lung injury. *p < 0.05 vs. well-aerated or vs. dorsal, † p < 0.05 vs. pcv uptake quantification, requiring normalization for the tissue fraction [26, 36] , which was performed in the current study. after 5 h of ventilation, we observed significantly lower indices of global and regional lung inflammation in the animals ventilated with pvv. specifically, a significantly higher inflammatory activity characterized the well aerated and non-dependent lung zones, both in control and injured groups. this finding is consistent with results from previous experiments studying injured lungs, in which lung inflammation assessed by 18 f-fdg uptake was correlated with regional strain [37, 38] . the significantly lower inflammation associated with pvv may be explained by the fact that the variability of the delivered vt contributes to tidal recruitment [12, 15] , therefore reducing strain in the open, aerated zones. it is worth noting that pvv exerts the most beneficial effect in the well and poorly aerated zones under both control and ards conditions (fig. 5) . conversely, the collapsed non-aerated zones were obviously unaffected by ventilation modes since these units were not subjected to strain. these findings further confirm the importance of focusing on regional ventilation when assessing the benefit of ventilation strategies. spect imaging confirms differences in regional distribution of lung perfusion when it is related to aeration zones. however, the lower blood perfusion in the ventral lung regions as compared to the dorsal zones can be attributed to the gravity effect and/or to the blood shift to the dorsal zones as a consequence of positive pressure and lung overdistension. the beneficial effects of pvv on respiratory mechanics and lung inflammation were not reflected in changes in blood gas parameters. the lack of improvement in oxygenation may be related in part to the more severe hypoxemia in this group, which required a higher fio 2 (65% vs 55% in groups pvv-ards and pcv-ards, respectively). moreover, the increase in lactate levels suggest the development of metabolic acidosis in both groups of ards animals, which may be the consequence of inadequate tissue oxygen delivery. moreover, the timespan of the experiment (5 h) may be too short to detect effects on gas exchange. we may hypothesize that the more prominent inflammation observed in the pcv groups would build up and potentially cause gas exchange problems over the course of days. the presence of ards was evident in the elevated lung injury score compared to control groups. in agreement with previous studies, lung injury score did not differ between the ventilation modes [14, 39] . the discrepancy between the functional and structural findings may be explained by the faster onset of functional changes, compared to the relatively longer time needed for morphological changes to become apparent. lung inflammation quantified using balf cell counts and pro-inflammatory cytokines, unlike in vivo imaging, did not reveal differences between the ventilation modes. in vivo imaging gives a more comprehensive measure of pulmonary inflammation at the early phase of ards, as it demonstrates the alveolar as well as the interstitial compartments of the lung. additionally, 18 f-fdg uptake reflects the acute metabolic activation of neutrophils and captures lung inflammation without barrier disruption, opposite to balf neutrophils and cytokines, providing a more rapid assessment of inflammatory processes. in this context, it is worth noting that the control groups also showed increased inflammation and lung injury indices (balf cytokines and histological injury score). these findings suggest that, despite the use of protective ventilation in the control groups, prolonged mechanical ventilation triggered the development of lung inflammation. this could potentially explain the lack of significant difference in normalized 18 f-fdg uptake between control and ards lungs. the similarity in the values of systemic hemodynamic parameters observed for the experimental groups is expected from the similarity in the overall lung perfusion as assessed by spect imaging. however, the significantly higher regional perfusion measured in the dependent zones can be attributed to the physiological distribution of lung perfusion that occurs in supine position [40] and is enhanced under positive pressure ventilation [41] . considering the regional aeration of lung tissue, the significantly lower perfusion observed in the poorly and non-aerated zones can be explained by the hypoxic pulmonary vasoconstriction mechanism [42] . there are some methodological aspects of the present study that warrant consideration. in this study we used a cone beam ct [43] . this device uses less radiation and creates higher resolution images than the regular fan beam ct; however, it produces more scatter artefacts, which can alter the measured values [44, 45] . due to technical limitations, breath gating was not performed in any of the acquisitions; therefore, basal lung areas had artefacts due to motion of the abdominal organs during breathing. the lung volume containing these artefacts was similar however, among rabbits. the animal model to induce ards calls for some considerations as well. the components of the model were chosen to mimic the various pathophysiological aspects of ards observed in humans. namely, intravenous lps contributes to the inflammatory component of the disease and it has also been described to induce surfactant dysfunction [46] . injurious ventilation using high vt combined with no peep contributes to development of volume-and barotrauma due to the supraphysiologic tidal volumes and respiratory pressures, whereas the absence of peep promotes tidal closures and exerts shear stress on the lung tissues [47] . the use of an fio 2 of 1.0 during this injurious ventilation period facilitates lung volume loss and development of ventilation heterogeneities [48] . while the surfactant dysfunction can restore to some extent during the 5-h timeframe of the experimental protocol, the functional and morphological damage is still present in the lungs, supported by the marked and highly significant changes observed between the control and ards groups regardless of the ventilation mode applied. measurements of respiratory mechanical parameters also warrant some considerations. while raw is mainly specific to the flow resistance of the conducting airways [49] , the tissue parameters damping and elastance include not only pulmonary components but are also influenced by other structures of the total respiratory system, mainly the chest wall [49] . previous literature attributed a chest wall contribution of approximately 30-50% to these parameters [50] and since the chest wall contribution is not expected to change after lung injury and mechanical ventilation [51] , the observed changes are interpreted as being mainly of pulmonary origin. therefore, the corresponding changes registered in tissue damping and elastance are predictably underestimating the real pulmonary changes. our data demonstrate the beneficial effect of variable ventilation based on a physiological breathing pattern in healthy lungs and in mild to moderate ards, in an experimental pediatric model. this positive effect was detected in the absence of deterioration in respiratory tissue elastance and in decreased regional lung inflammation measured by pet imaging. ventilation for five hours with physiologically variable ventilation provided better protection on aerated lung zones than with monotonous pressure-controlled ventilation. while further studies in humans might be needed, our results suggest that the application of a physiological breathing pattern as the driving signal of mechanical ventilation may have a better lung protective ability than conventional modes in scenarios where prolonged mechanical ventilation is required. supplementary information accompanies this paper at https ://doi. org/10.1186/s1293 1-020-01559 -x. additional file 1. supplementary information on methods and ancillary results ( figures s1-s4 and tables s1-s3). epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries ventilator-induced lung injury: from the bench to the bedside ventilator-induced lung injury ventilator-induced lung injury: in vivo and in vitro mechanisms effect of positive end-expiratory pressure on regional ventilation distribution during mechanical ventilation after surfactant depletion recurrent recruitment manoeuvres improve lung mechanics and minimize lung injury during mechanical ventilation of healthy mice acute respiratory distress syndrome: advances in diagnosis and treatment fifty years of research in ards. vt selection in acute respiratory distress syndrome life-support system benefits from noise when mechanical ventilation mimics nature variable ventilation induces endogenous surfactant release in normal guinea pigs biologically variable ventilation prevents deterioration of gas exchange during prolonged anaesthesia variable ventilation associated with recruitment maneuver minimizes tissue damage and pulmonary inflammation in anesthetized lung-healthy rats variable ventilation is equally effective as conventional pressure control ventilation for optimizing lung function in a rabbit model of ards variable tidal volume ventilation improves lung mechanics and gas exchange in a rodent model of acute lung injury comparison of variable and conventional ventilation in a sheep saline lavage lung injury model variable ventilation improves pulmonary function and reduces lung damage without increasing bacterial translocation in a rat model of experimental pneumonia comparison between neurally-assisted, controlled, and physiologically variable ventilation in healthy rabbits mechanisms of ventilator-induced lung injury in healthy lungs quantitative computed tomography in porcine lung injury with variable versus conventional ventilation: recruitment and surfactant replacement resolution of pulmonary edema with variable mechanical ventilation in a porcine model of acute lung injury rabbits and men: relating their ages acute respiratory distress syndrome: the berlin definition input impedance and peripheral inhomogeneity of dog lungs pet/ct in nononcological lung diseases: current applications and future perspectives improved correction for the tissue fraction effect in lung pet/ct imaging an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals the protective effects of volatile anesthestics against the bronchoconstriction induced by an allergic reaction in sensitized rabbit pups airway inhomogeneities contribute to apparent lung tissue mechanics during constriction comparison of static end-expiratory and effective lung volumes for gas exchange in healthy and surfactant-depleted lungs variable tidal volumes improve lung protective ventilation strategies in experimental lung injury in vivo assessment of lung inflammatory cell activity in patients with copd and asthma effects of positive end-expiratory pressure and spontaneous breathing activity on regional lung inflammation in experimental acute respiratory distress syndrome micro-autoradiographic assessment of cell types contributing to 2-deoxy-2-[(18)f]fluoro-d-glucose uptake during ventilator-induced and endotoxemic lung injury positive end-expiratory pressure above lower inflection point minimizes influx of activated neutrophils into lung the importance of correction for tissue fraction effects in lung pet: preliminary findings does regional lung strain correlate with regional inflammation in acute respiratory distress syndrome during nonprotective ventilation? an experimental porcine study effect of prone position on regional shunt, aeration, and perfusion in experimental acute lung injury periodic fluctuation of tidal volumes further improves variable ventilation in experimental acute respiratory distress syndrome the influence of gravity on regional lung blood flow in humans: spect in the upright and head-down posture v/q distribution and correlation to atelectasis in anesthetized paralyzed humans hypoxic pulmonary vasoconstriction development and validation of two phantoms for quality control in cone-beam ct image quality improvement in cone-beam ct using the super-resolution technique dental cone beam ct: a review • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? choose bmc dynamic mechanical interactions between neighboring airspaces determine cyclic opening and closure in injured lung high inspired oxygen fraction impairs lung volume and ventilation heterogeneity in healthy children: a double-blind randomised controlled trial repeated measurements of airway and parenchymal mechanics in rats by using low-frequency oscillations different contributions from lungs and chest wall to respiratory mechanics in mice, rats, and rabbits lung and chest wall impedances in the dog in normal range of breathing: effects of pulmonary edema publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank aurélie baudat, xavier belin and sylvie roulet for their technical assistance in animal handling, surgical preparation and sample processing. the authors thank didier colin, stéphane germain and frédéric bois for their technical support in imaging acquisition. the authors also thank matts wallin and magnus hallback for their excellent technical support in the application of the variable pattern. authors' contributions adsr: study design, experimental work, data collection, data analyses and article drafting. ghf: study design, experimental work, data collection, data analyses and article drafting. mk: data collection and data analyses. ld: data collection and data analyses. sb: study design, interpretation of results and article drafting. fp: study design, data analyses, interpretation of results and article drafting. wh: study design, data analyses, interpretation of the results and article drafting. all authors read and approved the final manuscript. this work was supported by the swiss national science foundation [32003b_169334]. ghf received a research grant of the european society of anaesthesiology and sb received a grant from alliance campus rhodanien. key: cord-253891-d1ei287l authors: geddes, duncan title: the history of respiratory disease management date: 2016-04-23 journal: medicine (abingdon) doi: 10.1016/j.mpmed.2016.03.006 sha: doc_id: 253891 cord_uid: d1ei287l lung diseases have shifted from infections – tuberculosis, pneumonia – to diseases of dirty air – chronic obstructive pulmonary disease, asthma and lung cancer. new diseases have emerged from industrial pollution and hiv, while better imaging has revealed others previously unrecognized. scientific advances in microbiology, imaging and clinical measurement have improved diagnosis and allowed better targeted treatment. advances in treatment have been dramatic, the most important being drugs (antibiotics, cortisone, β(2)-adrenoreceptor agonists), ventilatory support (from iron lung to nasal positive-pressure ventilation), inhaled therapy (metered dose inhalers, nebulizers) and lung surgery (resections, video-assisted thoracoscopic surgery, transplantation). delivery of care has shifted from sanatoria for the rich but nothing at all for the poor, to hospitals and universal coverage. generalists have turned into super-specialists and doctors have been joined by growing numbers of professions allied to medicine (pams). management of lung disease has vastly improved but the impact of disease remains. the lungs are the most exposed of the internal organs and throughout the past 150 years have occupied centre stage for disability and death. the diseases have changed: some fade, some emerge and others come and go, but all through this period our knowledge has grown. we understand these breathing bags better than ever before, we can see into them in ways unimagined in the 19th century and we have a growing armamentarium to fight their disorders. some key events are listed as a historical timeline of respiratory medicine (table 1) . change is sometimes real and sometimes where the spotlight falls. lung diseases surge and sometimes seem to go. pneumonia finished off sir francis bacon in 1626 as he stuffed a chicken full of snow, rene descartes in 1650 as he taught freezing philosophy lessons to queen christina of sweden and leo tolstoy in 1910 in a lonely railway station. tuberculosis (tb) gave john keats a far from easeful death in 1821 but provided violetta in 1853 with the best last act of all in la traviata. and then these two captains of the men of death drifted away. antibiotics cured them and left the theatre empty for the diseases of dirty air. foul smoke inhalation lifted lung cancer from zero to hero, promoted chronic obstructive pulmonary disease (copd) from the wings to centre stage, and gave small cameo parts to interstitial lung diseases. the air became opaque with coal dust underground, while up above asbestos and other factory chemicals were inhaled, lingered in the lungs and slowly scarred them. new chapters were written into books of lung pathology, new x-ray patterns described. asthma annoyed william of orange and charles dickens but was never a 19th-century celebrity. however, 100 years later, with the help of bill clinton and david beckham, it became a star; allergy replaced psychology as the cause, and big pharma smelt and dealt in gold. sleep apnoea moved on from dickens' fat boy snoring in the background to crest the wave of obesity and became the fastest growing branch of lung disease. and then, just as the audience was beginning to forget them, came the triumphal re-entry of the infections. tb, declining long before the discovery of streptomycin e but only in the countries of the rich e returned. of course, it had never really left at all, surging on among the malnourished and overcrowded in poorer countries. drug-resistant bacteria emerged and tb deaths rose again. pneumonia, too, had never gone away. osler in the 1920s named it the 'old man's friend' and may have been right for donald bradman in 2001 or ronald reagan in 2005. but pneumonia was no friend to the increasing numbers of immunocompromised individuals, their defences weakened by malnutrition (george orwell in 'how the poor die'), cancer treatments or, from 1981, hiv (freddie mercury in 1991). other viruses hit the headlines. spanish flu killed 50 million people in 1918, and since then smaller flu epidemics, severe acute respiratory syndrome and middle eastern respiratory syndrome have shown that, in spite of vaccination, viruses are still very effective killers. in 1850, eyes, hands, nose and ears e minimally aided by a stethoscope e were all the doctor had. he could see pallor, cyanosis or a flapping tremor but did not understand them. he could feel chest movements and tap out different tones with over the past 150 years: c infections have declined but returned while asthma, chronic obstructive pulmonary disease and lung cancer have surged c scientific advances, especially in imaging and microbiology, have improved diagnosis c new targeted treatments with antibiotics, corticosteroids, ventilatory support and lung surgery have revolutionized management c delivery of care has shifted from inefficient remedies for the rich to specialized treatment for all duncan geddes md frcp cbe is an honorary consultant at the royal brompton hospital, london and professor of respiratory medicine at imperial college, london, uk. his main clinical and research interests are cystic fibrosis, lung cancer and emphysema. he was president of the british thoracic society and chairman of asthma uk. competing interests: none declared. elegant percussive fingers but had no idea of what was going on inside. he could smell anaerobes (new-mown hay with an arri ere gout of stale faeces) and he could wield a stethoscope as an instrument of erudition rather than knowledge. his drug treatments were herbs e poppy, willow bark, stramonium, wormwood e and his surgical skills were few. tb was treated with rest, varying diets and lifestyle changes. love and sex in particular had to be avoided e difficult as in sanatoria there was little else to do. acute infections and fevers were cupped, bled or scarified, partly in order to remove bad humours and partly to structure time while waiting for nature to kill or cure. did the doctor do more harm than good? no one knew. then came the age of scientific discovery. three key advances were pasteur's germ theory of disease, roentgen's discovery of the 20th century saw a cascade of scientific discoveries, but much more important was development and application of the earlier discoveries. microbiologists gave us precise diagnosis and the tools to develop vaccines and antibiotics. radiologists showed what was really going on, culminating in computed tomography scanning (1970), with its ever-greater resolution rivalling histopathology (figure 1 ). public health improved with housing laws, school meals, free milk, mass mini x-ray vans and programmes of vaccination. lung function testing developed more slowly, taking 100 years to move from hutchison's lung volumes to tiffeneau's flow rates. this was followed by a major academic push in the 1950s to understand gas exchange, with attempts to make carbon monoxide transfer and body plethysmography clinically useful. more important, however, were blood gas measurements. today spirometry is encouraged in primary care, with occasional benefits over peak flow meters, but simple oximeters have become an essential tool of lung medicine. endoscopy took off slowly from the first laryngoscopy in 1849 to rigid bronchoscopy (1904), toughed out with either no anaesthesia at all or, for wimps, cocaine spray. all changed in 1972 with japanese fibreoptics; by 1980, diagnostic bronchoscopy was being carried out by physicians, not surgeons. science and luck precede new treatments by 10e50 years. the three main types e drugs, devices and surgery e hit the healthcare market in completely different ways. new drugs get the most attention as they are launched with a fanfare, they can be prescribed as soon as they are approved, and all doctors can use them. patients report instant improvement and the makers make money. device development is slower but at least as important. specialists start to use them and slowly the word goes round. slower still and much more invisible to patients and primary care are advances in surgery. three out of thousands of new chemical drugs revolutionized lung treatment. antibiotics came first and deserve first prize. synthetic sulphonamides in the 1930s were toxic and unsuitable for systemic use. moulds had been used to treat infections for centuries before fleming investigated penicillin in 1928 (poland, france, italy, belgium and costa rica can all claim to have got there first); florey and pfizer were essential in taking it on to manufacture in 1942. then came streptomycin (1944), tetracycline (1945), choramphenicol (1947), erythromycin (1949), isoniazid (1952) and rifampicin (1957) . the transition from watching an infection to treating it was dramatic, and it must have been a wonderful era to be a doctor. close behind came the miracle of cortisone. in 1949, patients with rheumatoid arthritis picked up their beds and walked, and the wonder drug began to cure all known diseases; recreational use led to cortisone parties in new york. by 1955, the benefits and adverse effects had been quickly established clinical observation e controlled trials take longer. asthma attacks responded to treatment, and with inhaled corticosteroids came long-term control. third prize goes to the b-adrenoreceptor agonists, first isoprenaline, then salbutamol and finally the long-acting badrenoreceptor agonists. selective b 2 -adrenoreceptor agonists are among the world's best-selling drugs for asthma, copd, sporting performance and beef production. again, there are thousands of these; some assist ventilation, some get drugs in or mucus out, but two types dominate the field: ventilators and inhalers. the simplest positive-pressure support devices, bag and mask, work, but endotracheal tube and intensive care are better. drinker's iron lungs (1927) countered respiratory paralysis from polio, other negativepressure devices e turtles, ponchos and cuirasses e being less effective. in the 1980s, non-invasive positive-pressure ventilation took off, and devices have been improving ever since (figure 2) , keeping hundreds of thousands of people alive who would, 100 years ago, have died. linked to this are oxygen delivery devices; cylinders, liquid oxygen reservoirs and cumbersome concentrators have been replaced by neat portable versions weighing less than 2 kg. old inhalers were inefficient: steam with pleasant-smelling herbs is soothing and still enjoyed today but delivers little to the lungs. nebulizers started in the 1870s with a squeeze-bulb version, but the particle size needed for penetration into the lungs was not understood until 1950s. the first efficient metereddose inhaler, based on perfume spray technology, was launched in 1955 and has lasted well. dry powder inhaler research started in 1930s; the aerohaler ò (1949) did not work, the rotahaler ò (1960s) and spinhaler ò (1970s) worked to some degree, but the 1990s turbohaler and diskhaler were good and there are now over 30 different versions. big pharma did all the research as it was rather too hum-drum for academia. it takes many small steps to climb a mountain. key were endotracheal intubation (1904), anaesthetic gases (chloroform 1847, halothane 1951), antisepsis (1870s), blood groups and safe transfusion (1901), antibiotics (1940s) and, most important of all, brave pioneers. each new operation builds on the experience of others; artificial pneumothorax (1830s) moved on to thoracoplasty (1880s) and plombage (1920s). the first survival from pneumonectomy was in 1931; the next 16 patients died. eventually, surgery for tb turned into lung cancer resection in the 1950s. drainage of pleural fluid was routine from 1850 (axel munte describes draining an empyema in a pet ape during a home visit in the story of san michele in 1929). thoracoscopy started in 1910 and advanced in the 1970s with fibreoptics. it has since developed into video-assisted thoracoscopic surgery for investigation of pleural disease, lung biopsy, treatment of pneumothorax and simple resections. lung transplantation opened new and wonderful prospects for endstage lung disease with the first successful operations in the 1970s and has now become routine, albeit limited by scarcity of donor organs. thoracic surgery led on to heart surgery, and by the 1960s there was a danger that lung surgery would be left behind. however, in the 1970s, lung surgery developed separately and has flourished. organizational change can be as important as technical. delivery of care lung medicine was a major part of the general doctor's workload in the 19th century until the 1850s sanatorium movement e well the rack e and this specialization led on to chest clinics. these were alongside but separate from hospitals and endured professional snobbery. general physicians looked down on chest physicians ('chest e yes, physician e never') before the systems were integrated in the 1960s. today, the lung specialist is often the best general physician. tb nurses morphed, after a brief spell in the wilderness, into respiratory nurses with their own training programmes. some work in primary care and some are specialist nurses in hospitals and have been joined by respiratory therapists, specialist physiotherapists, lung function measurers, psychologists and the rest. the room can now be very full as a single patient faces hoards of multi-disciplinary professionals sharing out the management. in spite of all this change lung disease, a major killer 150 years ago, remains a major killer today. the sanitary condition of the labouring population the rack. vallencourt books london: everyman's library the story of san michele, new edition. london: john murray in: shooting an elephant; and other essays key: cord-292862-ezrkg0dc authors: myerson, jacob w.; patel, priyal n.; habibi, nahal; walsh, landis r.; lee, yi-wei; luther, david c.; ferguson, laura t.; zaleski, michael h.; zamora, marco e.; marcos-contreras, oscar a.; glassman, patrick m.; johnston, ian; hood, elizabeth d.; shuvaeva, tea; gregory, jason v.; kiseleva, raisa y.; nong, jia; rubey, kathryn m.; greineder, colin f.; mitragotri, samir; worthen, george s.; rotello, vincent m.; lahann, joerg; muzykantov, vladimir r.; brenner, jacob s. title: supramolecular organization predicts protein nanoparticle delivery to neutrophils for acute lung inflammation diagnosis and treatment date: 2020-04-18 journal: biorxiv doi: 10.1101/2020.04.15.037564 sha: doc_id: 292862 cord_uid: ezrkg0dc acute lung inflammation has severe morbidity, as seen in covid-19 patients. lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries (“margination”). we sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. we designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. we found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. specifically, nanoparticles with agglutinated protein (naps) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. we validated this finding by engineering protein-conjugated liposomes that recapitulate nap targeting to neutrophils in inflamed lungs. we show that naps can diagnose acute lung injury in spect imaging and that nap-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. finally, we demonstrate that ischemic ex vivo human lungs selectively take up naps, illustrating translational potential. this work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and naps have significant potential in detecting and treating respiratory conditions arising from injury or infections. the covid-19 pandemic tragically illustrates the dangers of acute inflammation and infection of the lungs, for both individuals and societies. acute alveolar inflammation causes the clinical syndrome known as acute respiratory distress syndrome (ards), in which inflammation prevents the lungs from oxygenating the blood. severe ards is the cause of death in most covid-19 mortality and was a major cause of death in the 1918 influenza epidemic, but ards is common even outside of epidemics, affecting ~200,000 american patients per year with a ~35-50% mortality rate. 1-6 ards is caused not just by viral infections, but also by sepsis, pneumonia (viral and bacterial), aspiration, and trauma. 3, 4 largely because ards patients have poor tolerance of drug side effects, no pharmacological strategy has succeeded as an ards treatment. 5, [7] [8] [9] therefore, there is an urgent need to develop drug delivery strategies that specifically target inflamed alveoli in ards and minimize systemic side effects. neutrophils are "first responder" cells in acute inflammation, rapidly adhering and activating in large numbers in inflamed vessels and forming populations of "marginated" neutrophils along the vascular lumen. [10] [11] [12] [13] [14] [15] [16] neutrophils can be activated by a variety of initiating factors, including pathogen-and damage-associated molecular patterns such as bacterial lipopolysaccharides (lps). 17, 18 after acute inflammatory insults, neutrophils marginate in most organs, but by far most avidly in the lung capillaries. 6, 14, 15, 19, 20 neutrophils are therefore key cell types in most forms of ards. in ards, marginated neutrophils can secrete tissue-damaging substances (proteases, reactive oxygen species) and extravasate into the alveoli, leading to disruption of the endothelial barrier and accumulation of neutrophils and edematous fluid in the air space of the lungs ( figure 1a ). 6, 15, 16, [19] [20] [21] targeted nanoparticle delivery to marginated neutrophils could provide an ards treatment with minimal side effects, but specific delivery to marginated neutrophils remains an open challenge. antibodies against markers such as ly6g have achieved targeting to neutrophils in mice, but also deplete populations of circulating neutrophils. [22] [23] [24] [25] additionally, while ly6g readily marks neutrophils in mice, there is no analogous specific and ubiquitous marker on human neutrophils. 22 therefore, antibody targeting strategies have not been widely adopted for targeted drug delivery to these cells. 24 as another route to neutrophil targeting, two previous studies noted that activated neutrophils take up denatured and agglutinated bovine albumin, concluding that denatured protein was critical in neutrophil-particle interactions. 26, 27 nanoparticle structural properties such as shape, size, and deformability can define unique targeting behaviors. [28] [29] [30] [31] [32] here, we screened a diverse panel of nanoparticles to determine the nanostructural properties that predict uptake in pulmonary marginated neutrophils during acute inflammation. as a high-throughput animal model for ards, we administered lps to mice, causing a massive increase in pulmonary marginated neutrophils. we show that two initial leads in our screen, lysozyme-dextran nanogels (ldngs) and crosslinked albumin nanoparticles (anps), selectively home to marginated neutrophils in inflamed lungs, but not naïve lungs. in our subsequent screen of over 20 diverse nanoparticles, we find that 13 protein nanoparticles, all defined by agglutination of protein in amorphous nanostructures (nanoparticles with agglutinated proteins, naps), but not by denatured protein, have specificity for lps-inflamed lungs. in contrast to naps, we demonstrate that three symmetric protein nanostructures (viruses/nanocages) have biodistributions unaffected by lps injury. we show that polystyrene nanoparticles and five liposome formulations do not accumulate in injured lungs, indicating that nanostructures that are not based on protein are not intrinsically drawn to marginated neutrophils in acute inflammation. we then engineered liposomes (the most clinically relevant nanoparticle drug carriers) as naps, through conjugation to protein modified with hydrophobic cyclooctynes, encouraging protein agglutination on the liposome surface by hydrophobic interactions. we thus show that supramolecular organization of proteins, rather than chemical composition, best predicts uptake in marginated neutrophils in acutely inflamed lungs. we then demonstrate proof of concept for naps as diagnostic and therapeutic tools for ards. we show; a) 111 in-labeled naps provide diagnostic imaging contrast that distinguishes inflammatory lung injury from cardiogenic pulmonary edema; b) napliposomes can significantly ameliorate edema in a mouse model of severe ards; c) naps, but not crystalline protein nanostructures, accumulate in ex vivo human lungs rejected for transplant due to ards-like conditions. collectively, our results will demonstrate that supramolecular organization of protein, namely protein agglutination, predicts strong, intrinsic nanoparticle tropism for marginated neutrophils. this finding indicates that naps, encompassing a wide range of nanoparticles based on or incorporating protein, have biodistributions that are responsive to inflammation. naps could be useful beyond ards, since marginated neutrophils play a pathogenic role in a diverse array of inflammatory diseases, including infections, heart attack, and stroke. [10] [11] [12] [13] [14] but our findings provide a clear path forward for using naps to improve diagnosis and treatment of ards. to quantify the increase in pulmonary marginated neutrophils after inflammatory lung injury, radiolabeled clone 1a8 anti-ly6g antibody (specific for mouse neutrophils) was administered intravenously (iv) to determine the location and concentration of neutrophils in mice. iv injection of lps subjected mice to a model of mild ards. accumulation of anti-ly6g antibody in the lungs was dramatically affected by iv lps, with 20.81% of injected antibody adhering in lps-injured lungs, compared to 2.82% of injected antibody in naïve control lungs ( figure 1b) . agreeing with previous studies addressing the role of neutrophils in systemic inflammation, biodistributions of anti-ly6g antibody indicated that systemic lps injury profoundly increased the concentration of neutrophils in the lungs. 6, 10, 14, 18 single cell suspensions prepared from mouse lungs were probed by flow cytometry to further characterize pulmonary neutrophils in naïve mice and in mice following lps-induced inflammation. to identify intravascular populations of leukocytes, mice received iv fluorescent cd45 antibody five minutes prior to sacrifice. single cell suspensions prepared from iv cd45-stained lungs were then stained with anti-ly6g antibody to identify neutrophils. a second stain of single cell suspensions with cd45 antibody indicated the total population of leukocytes in the lungs, distinct from the intravascular population indicated by iv cd45. flow cytometry showed greater concentrations of neutrophils in lps-injured lungs, compared to naïve lungs ( figure 1c , counts above horizontal threshold indicate positive staining for neutrophils, figure 1d , rightmost peak indicates positive staining for neutrophils). comparison of ly6g stain to total cd45-positive cells indicated 53.5% of leukocytes in the lungs were ly6g-positive after lps injury, compared to 5.6% in the naïve control ( figure 1d , center panel). comparison of ly6g stain to iv cd45 stain indicated that the majority of neutrophils were intravascular, in both naïve and lpsinjured mice. in naïve mice, 85.3% of neutrophils were intravascular and in lps-injured mice, 96.2% of neutrophils were intravascular ( figure 1d , right panel). the presence of large populations of intravascular neutrophils following inflammatory injury is consistent with previously published observations. 6, 10, 14, 18, 33 histological analysis confirmed results obtained with flow cytometry and radiolabeled anti-ly6g biodistributions. staining of lung sections indicated increased concentration of neutrophils in the lungs following iv lps injury ( figure 1e , left panels). co-registration of neutrophil staining with tissue autofluorescence (indicating tissue architecture) broadly supported the finding that pulmonary neutrophils reside in the vasculature ( figure 1e , right panels). previous work has traced the neutrophil response to bacteria in the lungs, determining that pulmonary neutrophils pursue and engulf active bacteria following either intravenous infection or infection of the airspace in the lungs. 18, 34, 35 we injected heat-inactivated, oxidized, and fixed e. coli in naïve and iv-lps-injured mice. with the bacteria stripped of their functional behavior, e. coli did not accumulate in the lungs of naïve control mice (1.47% of initial dose in the lungs, blue bars in figure 1f ). however, pre-treatment with lps to recapitulate the inflammatory response to infection led to enhanced accumulation of the deactivated e. coli in the lungs (7.69% of initial dose in the lungs, red bars in figure 1f ). with e. coli structure maintained but e. coli function removed, the inactivated bacteria were taken up more avidly in lungs primed by an inflammatory injury. in order to identify nanostructural parameters that correlate with nanoparticle uptake in inflamed lungs, we conducted an in vivo screen of a diverse array of nanoparticle drug carriers. the screen was based on the method used above for tracing inactivated bacteria: inject radiolabeled nanoparticles into mice and measure biodistributions, comparing naïve with iv-lps mice. to validate that the radiotracing screen would measure uptake in pulmonary marginated neutrophils, we more fully characterized the in vivo behavior of two early hits in the screen. lysozyme-dextran nanogels (ldngs, ngs) and poly(ethylene)glycol (peg)crosslinked albumin nps have been characterized as targeted drug delivery agents in previous work. [36] [37] [38] here, ldngs (136.4±3.6 nm diameter, 0.10±0.02 pdi, supplementary figure 1a ) and peg-crosslinked human albumin nps (317.8±3.6 nm diameter, 0.14±0.05 pdi, supplementary figure 1b) were administered in naïve and iv-lps-injured mice. neither np was functionalized with antibodies or other affinity tags. the protein component of each particle was labeled with 125 i for tracing in biodistributions, and assessed 30 minutes after iv administration of nps. both absolute ldng lung uptake and ratio of lung uptake to liver uptake registered a ~25-fold increase between naïve control and lps-injured animals (figure 2a , supplementary table 1) . specificity for lps-injured lungs was recapitulated with peg-crosslinked human albumin nps. albumin nps accumulated in naïve lungs at 6.34% injected dose per gram organ weight (%id/g), and in lps-injured lungs at 87.62 %id/g, accounting for a 14-fold increase in lung uptake after intravenous lps insult ( figure 2b , supplementary table 1) . single cell suspensions were prepared from lungs after administration of fluorescent ldngs or peg-crosslinked albumin nps. flow cytometric analysis of cells prepared from lungs after np administration enabled identification of cell types with which nps associated. firstly, the total number of cells containing ldngs or albumin nps increased between naïve and lps-injured lungs. in naïve control lungs, 2. ly6g stain for neutrophils indicated that the bulk of ldng and albumin np accumulation in lps-injured lungs could be accounted for by uptake in neutrophils. in figure 2c and 2d, counts above the horizontal threshold indicate neutrophils and counts to the right of the vertical threshold indicate cells containing ldngs ( figure 2c ) or albumin nps ( figure 2d ). in iv-lps-injured lungs, ldng and albumin np uptake was dominated by neutrophils ( figure 2c , figure 2d , upper right quadrants indicate nppositive neutrophils). in lps-injured lungs, the majority of neutrophils, >70% of cells, contained significant quantities of nanoparticles, compared to <20% in naïve lungs. likewise, the majority of nanoparticle uptake in the lungs (>70%) was accounted for by nanoparticle uptake in neutrophils ( figure 2e , 2f, 2g, 2h, supplementary table 2) . for np uptake not accounted for by neutrophils, cd45 staining indicated that the remaining np uptake was attributable to other leukocytes. co-localization of albumin np fluorescence with cd45 stain showed that 91.9% of albumin np uptake was localized to leukocytes in naïve lungs and 97.8% of albumin np uptake was localized to leukocytes in injured lungs (supplementary figure 3c, supplementary figure 3d ). for ldngs, localization to neutrophils in injured lungs was confirmed via histology. ly6g staining of lps-injured lung sections confirmed colocalization of fluorescent nanogels with neutrophils in the lung vasculature ( figure 2i ). slices in confocal images of lung sections indicated that ldngs were inside neutrophils ( figure 2j ). intravital imaging of injured lungs allowed real-time visualization of ldng uptake in leukocytes in injured lungs. ldng fluorescent signal accumulated over 30 minutes and reliably colocalized with cd45 staining for leukocytes ( figure 2k , supplementary movie 1). ldng pharmacokinetics were evaluated in naïve and iv-lps-injured mice (supplementary figure 4) . in both naïve and injured mice, bare ldngs were rapidly cleared from the blood with a distribution half-life of ~3 minutes. in naïve mice, transient retention of ldngs in the lungs (25.91 %id/g at five minutes after injection) leveled off over one hour. in iv-lps-treated mice, ldng concentration in the lungs reached a peak value at 30 minutes after injection, as measured either by absolute levels of lung uptake or by lungs:blood localization ratio. ldng biodistributions were also assessed in mice undergoing alternative forms of lps-induced inflammation. intratracheal (it) instillation of lps led to concentration of ldngs in the lungs at 81.31 %id/g. liver and spleen ldng uptake was also reduced following it lps injury, leading to a 45-fold increase in the lungs:liver ldng localization ratio induced by it lps injury (supplementary figure 5) . as with iv lps injury, it lps administration leads to neutrophil-mediated vascular injury focused in the lungs. 14 mice were administered lps via footpad injection to provide a model of systemic inflammation originating in lymphatic drainage. 39 ldng uptake in the lungs and in the legs was enhanced by footpad lps administration. at 6 hours after footpad lps administration, ldngs concentrated in the lungs at 59.29 %id/g, an 11-fold increase over naïve. at 24 hours, ldngs concentrated in the lungs at 202.64 %id/g (supplementary figure 6a) . total ldng accumulation in the legs accounted for 0.85% of initial dose (%id) in naïve mice, 2.65 %id in mice 6 hours after footpad lps injection, and 8.34 %id at 24 hours after footpad injection (supplementary figure 6b) , indicating ldngs can concentrate in inflamed vasculature outside the lungs. previous work has indicated that nps based on denatured albumin accumulate in neutrophils in inflamed lungs and at sites of acute vascular injury, whereas nps coated with native albumin do not. 26, 27 we have characterized lysozyme-dextran nanogels and crosslinked human albumin nps with circular dichroism (cd) spectroscopy to compare secondary structure of proteins in the nps to secondary structure of the native component proteins (supplementary figure 7a-b) . identical cd spectra were recorded for ldngs vs. lysozyme and for albumin nps vs. human albumin. deconvolution of the cd spectra via neural network algorithm trained against a library of cd spectra for known structures verified that secondary structure composition of lysozyme and albumin was unchanged by incorporation of the proteins in the nps. 40 free protein and protein nps were also probed with 8-anilino-1naphthalenesulfonic acid (ansa), previously established as a tool for determining the extent to which hydrophobic domains are exposed on proteins. 41 consistent with known structures of the two proteins, ansa staining indicated few available hydrophobic domains on lysozyme and substantial hydrophobic exposure on albumin (supplementary figure 7c -d, blue curves). ldngs had increased hydrophobic accessibility vs. native lysozyme whereas albumin nps had reduced hydrophobic accessibility compared with native albumin. therefore, our data indicate that lysozyme and albumin are not denatured in ldngs and albumin nps, but the nps composed of the two proteins present a balance of hydrophobic and hydrophilic surfaces differing from the native proteins. the previous section demonstrates that two different nanoparticles based on protein, shown not to be denatured in cd spectroscopy studies, have uptake in lpsinflamed lungs driven by uptake in marginated neutrophils. we next undertook a broader study considering how aspects of np structure including size, composition, surface chemistry, and structural organization impact np uptake in lps-injured lungs. as examples of different types of protein nps, variants of ldngs (representing nps based on hydrophobic interactions between proteins), crosslinked protein nps, and nps based on electrostatic interactions between proteins were traced in naïve control and iv-lps-injured mice. as examples of nps based on site-specific protein interactions (rather than site-indiscriminate interactions leading to crosslinking, gelation, or chargebased protein nps), we also traced viruses and ferritin nanocages in naïve and lpstreated mice. liposomes and polystyrene nps were studied as examples of lipid and polymeric nanostructures. nanoparticles based on hydrophobic protein interactions ldng size was varied by modifying lysozyme-dextran composition of the nps and ph at which particles were formed. 42 figure 2 , all sizes of ldngs accumulated in lps-injured lungs at higher concentrations than in naïve lungs, with accumulation in injured lungs reaching ~20% of initial dose for all types of ldngs (supplementary table 3 ). variations in size and composition of ldngs therefore did not affect ldng specificity for lps-injured lungs. expanding on data with peg-nhs ester-crosslinked human serum albumin particles, we varied the geometry and protein composition of nps based on peg-nhs protein crosslinking. human serum albumin nanorods (aspect ratio 3:1), bovine serum albumin nps (317. table 4 ). lysozyme nps accumulated in naïve lungs at a uniquely high concentration of 137.47 %id/g, compared to 170.92 %id/g in inflamed lungs. degree of uptake in injured lungs, along with injured vs. naïve contrast, did vary with protein np composition. however, acute inflammatory injury resulted in a minimum three-fold increase in lung uptake for all examined crosslinked protein nps, excluding crosslinked lysozyme, which still accumulated in injured lungs at a high concentration (25.64% of initial dose). we traced recently-developed poly(glutamate) tagged green fluorescent protein (e-gfp) nps, representing a third class of protein np based on electrostatic interactions between proteins and carrier polymer or metallic particles. 43 negatively-charged e-gfp was paired to arginine-presenting gold nanoparticles (89.0±1.6 nm diameter, pdi 0.14±0.04) or to poly(oxanorborneneimide) (poni) functionalized with guanidino and tyrosyl side chains (158.9±6.2 nm diameter, pdi 0.17±0.03) (supplementary figure 1d) . for biodistribution experiments with poni/e-gfp hybrid nps, tyrosine-bearing poni was labeled with 131 i and e-gfp was labeled with 125 i, allowing simultaneous tracing of each component of the hybrid nps. the two e-gfp nps, with structure based on charge interactions, had specificity for iv lps-injured lungs. comparing uptake in lps-injured lungs to naïve lungs, we observe an lps:naïve ratio of 2.37 for poni/e-gfp nps as traced by the poni component, 2.57 for poni/e-gfp nps as traced by the e-gfp component, and 2.79 for au/e-gfp nps ( figure 3c , supplementary figure 10 ). poni/e-gfp particles, specifically, accumulated in lpsinjured lungs at 26.77% initial dose as measured by poni tracing and 27.24% initial dose as measured by gfp tracing, indicating effective co-delivery in the inflamed organ. acute inflammatory injury therefore resulted in a two-to three-fold increase in pulmonary uptake of nps constructed via electrostatic protein interactions. nanoparticles based on symmetric protein organization adeno-associated virus (aav), adenovirus, and horse spleen ferritin nanocages were employed as examples of protein-based nps with highly symmetrical structure (see supplementary figure 1d for dls confirmation of structure). [44] [45] [46] for each of these highly ordered protein nps, iv lps injury had no significant effect on biodistribution and levels of uptake in the injured lungs were minimal ( figure 3d table 5 ). therefore, highly ordered protein nps traced in our studies did not have tropism for the lungs after acute inflammatory injury. liposomes and polystyrene nps were studied as example nps that are not structurally based on proteins. dota chelate-containing lipids were incorporated into bare liposomes, allowing labeling with 111 in tracer for biodistribution studies. carboxylate polystyrene nps were coupled to trace amounts of 125 i-labeled igg via edci-mediated carboxy-amine coupling. liposomes had a diameter of 103.6±8.7 nm (pdi 0.09±0.01) and igg-polystyrene nps had a diameter of 230.5±2.8 nm (pdi 0.14±0.01) (supplementary figure 1c -d). liposomes accumulated in inflamed lungs at a concentration of 16.89 %id/g, accounting for no significant change against naïve lungs. lps injury actually induced a reduction in the lungs:liver metric, from 0.20 for naïve mice to 0.15 for lps-injured mice. polystyrene nps accumulated in inflamed lungs at 11.67 %id/g (1.75% initial dose), so iv lps injury did in fact induce increased levels of np uptake in the lungs, from a concentration of 2.40 %id/g in the naïve lungs ( figure 3e, supplementary figure 12 ). however, neither bare liposomes nor polystyrene nps were drawn to lps-injured lungs in significant concentrations. significantly, isolated proteins did not home to lps-inflamed lungs themselves. we traced radiolabeled albumin, lysozyme, and transferrin in naïve control and iv lpsinjured mice (supplementary figure 13 , supplementary table 6 ). in injured mice, albumin, lysozyme, and transferrin localized to the lungs at low concentrations and no significant differences were recorded when comparing naïve to lps-injured lung uptake. the data presented in figure 3 and supplementary figures 8-13 indicate that a variety of protein-based nanostructures have tropism for acute inflammatory injury in the lungs. nps based on agglutination of proteins in non-site-specific interactions (naps, figure 3a -c, supplementary figures 8-10 ) all exhibited either significant increases in lung uptake after lps injury or high levels of lung uptake in both naïve control and lpsinjured animals. nanostructures based on highly symmetrical protein organization had no specific tropism for inflamed lungs ( figure 3d ). representative nanostructures not based on proteins, bare liposomes and polystyrene beads, did not home to inflamed lungs ( figure 3e ). we next engineered naps from liposomes, a nanoparticle shown above to have no intrinsic neutrophil tropism. our methods for engineering nap-like liposomes serve to validate the finding that supramolecular organization of protein in nanoparticles predicts neutrophil tropism. liposomes were functionalized with rat igg conjugated via sata-maleimide chemistry (sata-igg liposomes) or via recently demonstrated copper-free click chemistry methods. 47 briefly, click chemistry methods entailed nhs-ester conjugation of an excess of strained alkyne (dibenzocyclooctyne, dbco) to igg, followed by reaction of the dbco-functionalized igg with liposomes containing peg-azide-terminated lipids (dbco-igg liposomes, figure 4a ). dbco-igg liposomes had a diameter of 128.3±4.3 nm and a pdi of 0.17±0.03 and sata-igg liposomes had a diameter of 178.8±6.9 nm and a pdi of 0.23±0.03 (supplementary figure 1c) . in mice subjected to iv-lps, sata-igg liposomes accumulated in the lungs at a concentration of 22.26 %id/g ( figure 4b , yellow bars). dbco-igg liposomes, by contrast, concentrated in the lungs at 117.16 %id/g, corresponding to 17.57% of initial dose and roughly matching the accumulation of 130nm ldngs in the inflamed lungs ( figure 4b , brown bars). for comparison, bare liposomes, as in figure 3e , concentrated in the inflamed lungs at 16.89 %id/g ( figure 4b , green bars). for dbco-igg liposomes, the inflamed vs. naïve lung uptake accounted for a twelve-fold change. dbco-igg liposomes specifically accumulated in injured lungs, whereas sata-igg liposomes and bare liposomes did not (supplementary figure 14 , supplementary table 7) . it lps instillation also led to elevated concentrations of dbco-igg liposomes in the lungs. biodistributions of the dbco-igg liposomes indicated a pulmonary concentration of 145.89 %id/g at 1 hour after it lps, 160.13 %id/g at 2 hours after it lps, and 127.78 %id/g at 6 hours after it lps (supplementary figure 15 ). even at early time points after direct pulmonary lps insult, dbco-igg liposomes accumulated in the inflamed lungs. results in figure 4b were obtained by introducing a 20-fold molar excess of nhs-ester-dbco to rat igg before dbco-igg conjugation to liposomes (dbco(20x)-igg liposomes). optical density quantification of dbco indicated ~14 dbco per igg following reaction of dbco and igg at 20:1 molar ratio (supplementary figure 16) . to test the hypothesis that dbco functions as a tag that modifies dbco-igg liposomes for neutrophil affinity in settings of inflammation, we varied the concentration of dbco on igg prepared for conjugation to azide liposomes. dbco was added to igg at 10-fold, five-fold, and 2.5-fold molar excesses. a 10-fold molar excess resulted in ~6 dbco per igg, a 5-fold molar excess resulted in ~3 dbco per igg, and a 2.5-fold molar excess resulted in ~2 dbco per igg (supplementary figure 16) . igg with different dbco loading concentrations was conjugated to azide liposomes. dbco-igg liposomes had similar sizes across all dbco concentrations (supplementary figure 1c) , with diameters of ~130 nm and pdis < 0.20. the different types of dbco-igg liposomes were each traced in iv-lps injured mice. titrating the quantity of dbco on dbco-igg liposomes indicated that liposome accumulation in the lungs of injured mice was dependent on dbco concentration on the liposome surface. concentration of dbco-igg liposomes in inflamed lungs attenuated with decreasing dbco concentration on igg (supplementary table 8 , figure 4c ). therefore, only igg with high concentrations of dbco served as a tag for modifying the surface of liposomes for specificity to pulmonary injury. flow cytometry verified the specificity of dbco-igg liposomes for neutrophils in injured lungs ( figure 4d -e). as with ldngs and albumin nps in figure 2c -h, single cell suspensions were prepared from lps-inflamed and naïve control lungs after circulation of fluorescent dbco-igg liposomes. confirming the results of biodistribution studies, 4.90% of cells were liposome-positive in naïve lungs, compared to 33.92% of all cells in lps-inflamed lungs (supplementary figure 17a-b) . dbco-igg liposomes predominantly accumulated in pulmonary neutrophils after iv lps. there were more neutrophils in the injured lungs and a greater fraction of neutrophils took up dbco-igg liposomes in the injured lungs, as compared to the naïve control ( figure 4d -e). approximately one half of neutrophils in iv lps-injured lungs contained liposomes. dbco-igg liposomes were also highly specific for neutrophils in inflamed lungs, with ~90% of liposome-positive cells in the injured lungs being neutrophils (supplementary table 9 ). the remaining dbco-igg liposome uptake in the lungs was accounted for by other cd45-positive cells (supplementary figure 17c -e). 99.0% of liposome uptake colocalized with cd45-positive cells in lps-injured lungs and 98.7% of liposome uptake in the naïve lungs was associated with cd45-positive cells. accordingly, less than 1% of liposome uptake was associated with endothelial cells (supplementary figure 17f -g). dbco(20x)-igg itself did not have specificity for inflamed lungs (supplementary figure 18 ). uptake of dbco(20x)-igg in naïve and injured lungs was statistically identical and the biodistribution of the modified igg resembled published results with unmodified igg. 48 these results verify that dbco-igg modifies the structure of immunoliposomes, but does not function as a standard affinity tag by acting as a surface motif with intrinsic affinity for neutrophils. indeed, cd spectroscopic and ansa structural characterization of dbcomodified igg and dbco-igg liposomes resembled results obtained for ldngs and crosslinked albumin nps. igg secondary structure, as assessed by cd spectroscopy, was unchanged by dbco modification (supplementary figure 19a) . deconvolution of cd spectra via neural network algorithm indicated identical structural compositions for dbco(20x)-igg, dbco(10x)-igg, dbco(5x)-igg, dbco(2.5x)-igg, and unmodified igg, showing that igg was not denatured by conjugation to dbco. ansa was used to probe accessible hydrophobic domains on dbco(20x)-igg and dbco(20x)-igg liposomes (supplementary figure 19b) . ansa fluorescence indicated more hydrophobic domains available on dbco(20x)-igg liposomes than on dbco(20x)-igg itself, resembling results for lysozyme and ldngs. therefore, addition of a hydrophobic moiety to protein on the surface of liposomes led to uptake of the liposomes in pulmonary marginated neutrophils after inflammatory insult. this result indicates that hydrophobic interactions between proteins on the surface of functionalized liposomes, like the protein interactions in naps, predict liposome tropism for marginated neutrophils in inflamed lungs. including nps from our four classes of protein-based nps, two non-protein nps (bare liposomes and polystyrene nps), and five types of igg-coated liposomes, we traced 23 nanoparticles in naïve and inflamed mice. direct assessment of naïve-toinflamed shifts in lung uptake led us to identify 13 naps with specificity for inflamed lungs. to verify this assessment and derive additional patterns in the broader data set, we undertook linear discriminant and principal components analyses of the biodistribution data for our 23 nanoparticles, along with three isolated proteins. grouping the 23 nanoparticles and three proteins according to the classes defined in figure 3 and supplementary figures 8-13 , we completed a linear discriminant analysis of the naïve-to-inflamed shift for particle retention in the lungs, blood, liver, and spleen (supplementary figure 20a) . data for particle uptake in each organ was normalized by subtracting and then dividing by the mean uptake over all particles. the first two eigenvectors, dominated by splenic uptake and a combination of liver and lung uptake, respectively, accounted for 96% of variation in the data. the resulting projection of the data along the first two linear discriminant analysis eigenvectors was analyzed by k-means clustering to confirm the classes of nanoparticle with specificity for the inflamed lungs (supplementary figure 20b) . indeed, division of the data into two clusters supported the delineation of the 13 nanoparticles with specificity for inflamed lungs. naps, nanoparticles based on protein gelation, crosslinking, and charge association, all aligned in one cluster. as an exception, dbco(20x)-igg liposomes were considered as a unique class of particle and the linear discriminant analysis indicated that the inflammation-specific liposomes had in vivo behavior resembling that of ldngs or poni-gfp nanoparticles. this analysis of the liposome biodistributions supports the classification of dbco(20x)-igg liposomes as naps. igg-coated polystyrene nanoparticles and dbco(10x)-igg liposomes were part of the k-means cluster without inflammation specificity, but data for these two particles resided close to the voronoi boundary distinguishing the two clusters. principal component analysis comparing normalized nanoparticle uptake in inflamed lungs to normalized retention in liver, spleen, and blood provided a reductive metric to compare the distinct in vivo behavior of nanoparticles in the classes identified by linear discriminant analysis. most variation in the biodistribution data was accounted for by an eigenvector closely aligned to variation in pulmonary uptake (supplementary figure 21a) . data was projected along that first eigenvector and magnitude of the projection was determined for each nanoparticle (supplementary figure 21b) . first eigenvector projection values were then grouped according to the classes examined above via linear discriminant analysis. only the classes in the inflammation-specific kmeans cluster had positive average first eigenvector projections. all other particle classes had average first eigenvector projections indistinguishable from isolated protein (supplementary figure 21b) . principal component and linear discriminant analyses of our compiled biodistributions confirmed; a) identification of naps as nanoparticles with distinct tropism for inflamed lungs and; b) alignment of dbco(20x)-igg liposome in vivo behavior with that of other naps. computerized tomography (ct) imaging is a standard diagnostic tool for ards. ct images can identify the presence of edematous fluid in the lungs, but ct cannot distinguish between the two major types of pulmonary edema: non-inflammatory cardiogenic pulmonary edema (cpe) and ards-associated edema. 49 we sought to use naps to distinguish inflammatory lung injury from cpe in diagnostic imaging experiments. we induced cpe in mice via prolonged iv propranolol infusion. 50 edema was confirmed via ct imaging of inflated lungs ex vivo and in situ. three-dimensional reconstructions of chest ct images were partitioned to distinguish airspace and lowdensity tissue, as in normal lungs (white, yellow, and light orange signal in figure 5a ), from high-density tissue and edema (red and black/transparent signal in figure 5a ). quantification of ct attenuation and gaps in the reconstructed three-dimensional lung images indicated profuse edema in lungs afflicted with model cardiogenic pulmonary edema ( figure 5a 200 nm ldngs were traced in mice with induced cardiogenic pulmonary edema. ldngs accumulated in the edematous lungs at 14.52 %id/g concentration, statistically indistinguishable from lung uptake in naïve mice and an order of magnitude lower than the level of lung uptake in mice treated with iv lps ( figure 5c ). naïve and iv lps-injured mice were dosed with ldngs labeled with 111 in via chelate conjugation to lysozyme. 111 in uptake in naïve and lps-injured lungs was visualized with ex vivo spect-ct imaging to indicate capacity of ldngs for imagingbased diagnosis of inflammatory lung injury ( figure 5d ). 111 in signa was colocalized with anatomical ct images for reconstructions in figure 5d . 111 in spect signal was detectable in lps-injured lungs, but 111 in spect signal was at background level in naïve lungs (supplementary movies 4 and 5). reduced spect signal in the liver of lps-injured mice, in agreement with biodistribution data, was also evident in coregistration of spect imaging with full body skeletal ct imaging (supplementary movies 6 and 7). therefore, naps with tropism for marginated neutrophils have the ability to detect and assess ards-like inflammation via spect-ct imaging. since those same naps do not accumulate in lungs afflicted with cpe, naps have potential for differential diagnosis of acute lung inflammation against cpe. in recent work, we demonstrated that human donor lungs rejected for transplant due to ards-like phenotypes can be perfused with nanoparticle solutions. 51 these perfusion experiments evaluate the tendency of nanoparticles to distribute to human lungs ex vivo. we used this perfusion method to evaluate nap retention in inflamed human lungs. first, fluorescent ldngs were added to single cell suspensions prepared from human lungs. 5 µg, 10 µg, or 50 µg of ldngs were incubated with 6x10 5 cells in suspension for 1 hour at room temperature. after three washes to remove unbound ldngs, cells were stained for cd45 and analyzed with flow cytometry ( figure 7a -b). the majority of ldng uptake in the single cell suspensions was attributable to cd45positive cells. ldngs accumulated in the human leukocytes, extracted from inflamed lungs, in a dose-dependent manner, with 35.1% of leukocytes containing ldngs at a loading dose of 50 µg. therefore, our prototype nap was retained in leukocytes from human lungs. to test ldng tropism for inflamed intact human lungs, fluorescent or 125 i-labeled ldngs were infused via arterial catheter into ex vivo human lungs excluded from transplant. immediately prior to ldng administration, tissue dye was infused via the same arterial catheter to stain regions of the lungs directly perfused by the catheterized branch of the pulmonary artery ( figure 7c ). after infusion of ldngs, phosphate buffered saline infusion was used to rinse away unbound particles. perfused regions of the lungs were dissected and divided into ~1g segments, then sorted into regions deemed to have high, medium, or low levels of tissue dye staining. for lungs receiving fluorescent ldngs, well-perfused and poorly-perfused regions were selected for sectioning and fluorescent imaging. fluorescent signal from ldngs was clearly detectable in sections of well-perfused tissue, but not poorly-perfused tissue ( figure 7d ). in experiments with 125 i-labeled ldngs, 131 i-labeled ferritin was concurrently infused (i.e. a mix of ferritin and ldngs was infused) as an internal control particle shown to have no tropism for injured mouse lungs. with ldngs and ferritin infused into the same lungs via the same branch of the pulmonary artery, ldngs retained in the lungs at 52.15% initial dose and ferritin retained at 9.27% initial dose ( figure 7e ). ldng accumulation in human lungs was focused in regions of the lungs with high levels of perfusion stain, with concentrations of 4.66 %id/g in the "high" perfusion regions, compared to 0.44 %id/g in the "medium" perfusion regions. ferritin accumulation was more diffuse, with 0.47 %id/g in the "high" perfusion regions, compared to 0.35 %id/g in the "medium" perfusion regions (supplementary figure 21) . ldngs, a prototype nap shown to home to neutrophils in acutely inflamed mouse lungs, specifically accumulated in perfused regions of inflamed human lungs, but ferritin nanocages, a particle with no tropism for neutrophils, concentrated at much lower levels in injured human lungs. our data thus indicate that nap tropism for neutrophils in inflamed mouse lungs may be recapitulated in human lungs. previous studies indicate that nanoparticles can interfere with neutrophil adhesion in inflamed vasculature. 52 we designed studies to evaluate whether or not naps mitigate the neutrophil-mediated effects of lung inflammation. namely, we administered ldngs, dbco(20x)-igg liposomes, or bare liposomes in mice subjected to model ards and determined whether or not the nanoparticles prevented lung edema induced by inflammation. mice were treated with nebulized lps as a high-throughput model for severe ards. to evaluate physiological effects of the model injury, bronchoalveolar lavage (bal) fluid was harvested from mice at 24 hours after exposure to lps. in three separate experiments, nebulized lps induced elevated concentrations of neutrophils, cd45-positive cells, and protein in the bal fluid. in naïve mice, cd45-positive cells concentrated at 1.42x10 4 cells per ml bal and neutrophils concentrated at 1.11x10 4 cells per ml bal. after lps injury, cd45-positive cells and neutrophils concentrated at 6.97x10 5 and 6.96x10 5 cells per ml bal, respectively. in naïve mice, protein concentrated in the bal fluid at 0.12 mg/ml and in lps-injured mice, protein concentrated in the bal at 0.36 mg/ml ( figure 6 , white and grey bars). vascular disruption after nebulized lps treatment thus led to accumulation of protein-rich edema in the alveolar space. dbco(20x)-igg liposomes, ldngs, and bare liposomes were compared for effects on vascular permeability in model ards. nps were administered as an iv bolus (20 mg per kg body weight) two hours after nebulized lps administration. as in untreated mice, bal fluid was harvested and analyzed at 24 hours after exposure to nebulized lps. bare liposomes or ldngs did not have significant effects on vascular injury induced by nebulized lps, as measured by either leukocyte or protein concentration in bal fluid ( figure 6 , red and green bars). dbco(20x)-igg liposomes, however, had a significant salient effect on both protein leakage and cellular infiltration in the bal ( figure 6 , brown bars). with dbco(20x)-igg liposomes administered two hours after nebulized lps, cd45-positive cells and neutrophils in bal were reduced to concentrations of 3.04x10 5 and 3.48x10 5 cells per ml, respectively. protein concentration in the bal was reduced to 0.21 mg/ml by dbco(20x)-igg liposome treatment. as measured by protection against cellular or protein leakage, relative to untreated mice, dbco(20x)-igg liposomes provided 59.6% protection against leukocyte leakage, 49.7% protection against neutrophil leakage, and 67.4% protection against protein leakage. dbco(20x)-igg liposomes, without any drug, altered the course of inflammatory lung injury to limit protein and leukocyte edema in the alveoli. our results with dbco(20x)-igg liposomes indicate that some naps can interfere with neutrophil extravasation into the alveoli and thus limit edema following inflammatory injury. however, our results with ldngs show that tropism for marginated neutrophils is not alone sufficient to limit the neutrophil-mediated effects of inflammatory lung injury. neutrophils concentrate in the pulmonary vasculature during either systemic or pulmonary inflammation. 10, 11, 14, 17, 18 these marginated neutrophils can recognize and engulf bacteria. 17, 18, 35 therefore, neutrophils surveil the vasculature for potentially pathogenic foreign species, with the pulmonary vasculature serving as a "surveillance hub" in the case of systemic or pulmonary infection and inflammation. 10, 17, 18, 35 our results with e. coli are noteworthy in this context: when e. coli are stripped of functional properties by heat treatment, oxidation, and fixation, but maintain their structure, uptake of the bacteria in the lungs only occurs after systemically prompting neutrophils with an inflammatory signal, lps. inflammation thus leads to pulmonary uptake of the e. colishaped particles. in large part, the overall outcome of this study is an accounting of nanoparticle structural properties that lead to recognition by "surveilling" neutrophils in the inflamed lungs, analogously to e. coli recognition by pulmonary neutrophils. including different liposomal formulations, 23 nanoparticles were screened in our biodistribution studies comparing pulmonary nanoparticle uptake in naïve and lps-inflamed mice. thirteen different nanoparticles exhibited specificity for inflamed lungs over naïve lungs, with flow cytometry data indicating that at least three of those nanoparticle species specifically and avidly gather in neutrophils. the thirteen nanoparticles with specificity for the inflamed lungs have a range of properties. seven different proteins were used in the inflammation-specific particles. the particles have sizes ranging from ~75 nm to ~350 nm, include both spheres and rods, and have a range of zeta potentials. however, our analyses classify the inflammation-specific nanoparticles as; 1) nanoparticles with structure based on hydrophobic interactions between proteins; 2) nanoparticles with structure based on non-site-specific protein crosslinking; 3) nanoparticles based on charge interactions between proteins. put broadly, these three classes can all be grouped as structures based on protein agglutination, without regard for site-specific interactions or symmetry in the resulting protein superstructure. we define the term nanoparticles with agglutinated proteins (naps) to indicate that particles with tropism for pulmonary marginated neutrophils during inflammation share commonalities in supramolecular organization. we identify naps as a broad class, rather than a single particle type. accordingly, we have presented diverse nap designs, implying a diversity of potential nap-based strategies for targeted treatment and diagnosis of ards and other inflammatory disorders in which marginated neutrophils play a role (e.g. local infections or thrombotic disorders). 10, 12, 13, 17, 18 the diversity of naps will allow versatile options for engineering neutrophil-specific drug delivery strategies to accommodate different pathologies. in contrast to naps, three particles (adenovirus, aavs, and ferritin) characterized by highly symmetric arrangement of protein subunits into a protein superstructure [44] [45] [46] did not accumulate in the inflamed neutrophil-rich lungs. these three particles have evolved structures that lead to prolonged circulation or evasion of innate immunity in mammals. [53] [54] [55] [56] it is conceivable that neutrophils more effectively recognize less patterned and more variable protein arrangements that may better parallel the wide variety of structures presented by the staggering diversity of microbes against which neutrophils defend. 20, 35 to support our conclusions regarding supramolecular organization and neutrophil tropism, we re-engineered liposomes, particles with no intrinsic neutrophil tropism, to behave like naps. protein arrangement on the surface of dbco-igg liposomes was predicted to recapitulate protein agglutination seen in naps based on hydrophobic interactions. introduction of dbco to igg entails conjugation of a highly hydrophobic moiety 57 to hydrophilic residues on the igg. replacing dbco with the less hydrophobic modifying group used in sata-maleimide conjugation 58 abrogates the inflammation specificity observed with dbco-igg liposomes. likewise, titrating down the amount of dbco on the igg, thus limiting the hydrophobic groups on the protein, also ratchets down the targeting behavior of the dbco-igg liposomes. our data therefore points towards hydrophobic interactions between proteins on the liposome surface being a determinant in liposome uptake in neutrophils in the inflamed lungs. essentially, the dbco-igg liposomes may reproduce the hydrophobic interaction structural motif seen in naps produced by protein gelation (i.e. ldngs). nap-liposomes may be particularly attractive for future clinical translatability. liposomes are prominent among fda-approved nanoparticle drug carriers. 59 further, even without cargo drugs, nap-liposomes conferred significant therapeutic effects in a mouse model of severe ards. ldngs, despite high levels of uptake in inflamed lungs, did not have the same therapeutic effect as the nap-liposomes. this result suggests that the composition of the liposomes may be important for their therapeutic effect. among possible mechanisms for the therapeutic effect, we note that lipid rafts are major signaling hubs in neutrophils. 60, 61 the lipid content of the nap-liposomes (particularly the cholesterol content) may modulate neutrophil lipid rafts dependent on cholesterol. we have also observed that neutrophil content in the inflamed alveoli is markedly reduced by nap-liposomes. in this context, we note published work demonstrating that certain nanoparticles, in a still undetermined manner dependent on particle composition, can drive redistribution of neutrophils from the lungs to the liver. 52 as a major corollary, our findings indicate many protein-based or proteinincorporating nanoparticles developed for therapeutic applications may accumulate in inflamed lungs, even when those nanoparticles were designed to accumulate elsewhere. the variety of protein nanostructures accumulating in inflamed lungs in our data includes particles that have been investigated as targeted drug delivery vehicles where marginated neutrophils are not the intended site of accumulation. 36, 38, 47, 48, 62 the patterns in our data indicate that future studies may reveal additional nanoparticles that accumulate in the lungs following inflammatory insult. this study therefore serves as evidence that inflammatory challenges may prompt profound off-target changes in the biodistributions of nanomaterials, including dramatic shunting of nanoparticles and any associated drug payload to the lungs. the nanoparticle targeting profiles documented in naïve or, for instance, tumor model studies may be overturned by, for instance, bacterial infection in a patient receiving the nanoparticle. in conclusion, supramolecular organization in nanoparticle structure predicts nanoparticle uptake in pulmonary marginated neutrophils during acute inflammation. specifically, nanoparticles with agglutinated protein (naps) accumulate in marginated neutrophils, while nanoparticles with more symmetric protein organization do not. nap tropism for neutrophils allowed us to develop naps as diagnostics and therapeutics for ards, and even to demonstrate nap uptake in inflamed human lungs. future work may more deeply explore therapeutic effects of naps in ards and other diseases in which neutrophils play key roles. this study also obviates future testing of supramolecular organization as a variable in in vivo behavior of nanoparticles, including screens of tropism for other pathologies and cell types. these studies could in turn guide engineering of new particles with intrinsic cell tropisms, as with our engineering of nap-liposomes with neutrophil tropism. these "targeting" behaviors, requiring no affinity moieties, may apply to a wide variety of nanomaterials. but our current findings with neutrophil-tropic naps indicate that many protein-based and protein-coated nanoparticles could be untapped resources for treatment and diagnosis of devastating inflammatory disorders like ards. lysozyme-dextran nanogels (ldngs) were synthesized as previously described. 37 ,42 70 kda rhodamine-dextran or fitc-dextran (sigma) and lysozyme from hen egg white (sigma) were dissolved in deionized and filtered water at a 1:1 or 2:1 mol:mol ratio, and ph was adjusted to 7.1 before lyophilizing the solution. for maillard reaction between lysozyme and dextran, the lyophilized product was heated for 18 hours at 60°c, with 80% humidity maintained via saturated kbr solution in the heating vessel. dextran-lysozyme conjugates were dissolved in deionized and filtered water to a concentration of 5 mg/ml, and ph was adjusted to 10.70 or 11.35. solutions were stirred at 80°c for 30 minutes. diameter of ldngs was evaluated with dynamic light scattering (dls, malvern) after heat gelation. particle suspensions were stored at 4°c. crosslinked protein nanoparticles and nanorods were prepared using previously reported electrohydrodynamic jetting techniques. 63 the protein nanoparticles were prepared using bovine serum albumin, human serum albumin, human lysozyme, human transferrin, or human hemoglobin (all proteins were purchased from sigma). protein nanorods were prepared using chemically modified human serum albumin. for electrohydrodynamic jetting, protein solutions were prepared by dissolving the protein of interest at a 7.5 w/v% (or 2.5 w/v% for protein nanorods) concentration in a solvent mixture of di water and ethylene glycol with 4:1 (v/v) ratio. the homobifunctional amine-reactive crosslinker, o,o′-bis[2-(n-succinimidylsuccinylamino)ethyl]polyethylene glycol with molecular weight of 2kda (nhs-peg-nhs, sigma) was mixed with the protein solution at 10 w/w%. protein nanoparticles were kept at 37°c for 7 days for completion of the crosslinking reaction. the as-prepared protein nanoparticles were collected in pbs buffer and their size distribution was analyzed using dynamic light scatting (dls, malvern). glutamic acid residues (e20-tag) were inserted at the c-terminus of enhanced green fluorescent protein (egfp) through restriction cloning and site-directed mutagenesis as previously reported. 64 proteins were expressed in an e. coli bl21 strain using standard protein expression protocol. briefly, protein expression was carried out in 2xyt media with an induction condition of 1 mm iptg and 18 °c for 16 h. at this point, the cells were harvested, and the pellets were lysed using 1% triton-x-100 (30 min, 37 °c)/dnase-i treatment (10 minutes). proteins were purified using hispur cobalt columns. after elution, proteins were preserved in pbs buffer. the purity of native proteins was determined using 8% sds−page gel. polymers (poni) were synthesized by ring-opening metathesis polymerization using third generation grubbs' catalyst as previously described. 65 in brief, solutions in dichloromethane of guanidium functionalized monomer and grubbs' catalyst were placed under freeze thawing cycles for degassing. after warming the solutions to room temperature, the degassed monomer solutions were administrated to degassed catalyst solutions and allowed to stir for 30 minutes. the polymerization reaction was terminated by the addition of excess ethyl vinyl ether. the reaction mixture was further stirred for another 30 min. the resultant polymers were precipitated from excess hexane or diethyl ether anhydrous, filtered, washed and dried under vacuum to yield a light-yellow powder. polymers were characterized by 1 h nmr and gel permeation chromatography (gpc) to assess chemical compositions and molecular weight distributions, respectively. subsequent to deprotection of boc functionalities, polymer was dissolved in the dcm with the addition of tfa at 1:1 ratio. the reaction was allowed to stir for 4 hours and dried under vacuum. excess tfa was removed by azeotropic distillation with methanol. afterwards, the resultant polymers were re-dissolved in dcm and precipitated in anhydrous diethyl ether, filtered, washed and dried. polymers were then dissolved in water and transferred to biotech ce dialysis tubing membranes with a 3000 g/mol cutoff and dialyzed against ro water (2−3 days). the polymers were then lyophilized dried to yield a light white powder. poni polymer/e-tag protein nanocomposites (ppncs) were prepared in polypropylene microcentrifuge tubes (fisher) through a simple mixing procedure. 0.5625 nmol of 54 kda poni was incubated with 0.45 nmol of egfp at room temperature for 10 minutes prior to dilution to 200 µl in sterile pbs and subsequent injection. similarly, 0.9 nmol of arginine-tagged gold nanoparticles, prepared as described, 43 were combined with 0.45 nmol of egfp to prepare egfp/gold nanoparticle complexes. azide-functionalized liposomes were prepared by thin film hydration techniques, as previously described. 47 the lipid film was composed of 58 mol% dppc (1,2dipalmitoyl-sn-glycero-3-phosphocholine), 40 mol% cholesterol, and 2 mol% azide-peg2000-dspe (all lipids from avanti). 0.5 mol% top fluor pc (1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine) was added to prepare fluorescent liposomes. 0.2 mol% dtpa-pe (1,2-distearoyl-sn-glycero-3phosphoethanolamine-n-diethylenetriaminepentaacetic acid) was added to prepare liposomes with capacity for radiolabeling with 111 in. lipid solutions in chloroform, at a total lipid concentration of 20 mm, were dried under nitrogen gas, then lyophilized for 2 hours to remove residual solvent. dried lipid films were hydrated with dulbecco's phosphate buffered saline (pbs). lipid suspensions were passed through 3 freezethaw cycles using liquid n2/50°c water bath then extruded through 200 nm cutoff tracketched polycarbonate filters in 10 cycles. dls assessed particle size after extrusion and after each subsequent particle modification. liposome concentration following extrusion was assessed with nanosight nanoparticle tracking analysis (malvern). for conjugation to liposomes, rat igg was modified with dibenzylcyclooctyne-peg4-nhs ester (dbco, jena bioscience). igg solutions (pbs) were adjusted to ph 8.3 with 1 m nahco3 buffer and reacted with dbco for 1 hour at room temperature at molar ratios of 2.5:1, 5:1, 10:1, or 20:1 dbco:igg. unreacted dbco was removed after reaction via centrifugal filtration against 10 kda cutoff filters (amicon [def] . dbcomodified igg was incubated with azide liposomes at 200 igg per liposome overnight at room temperature. unreacted antibody was removed via size exclusion chromatography, and purified liposomes were concentrated to original volume against centrifugal filters (amicon). maleimide liposomes were also prepared via lipid film hydration. 66 lipid films comprised 54% dppc, 40% cholesterol, and 6% mpb-pe (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-n-[4-(p-maleimidophenyl) butyramide]), with lipids prepared, dried, resuspended, and extruded as described above for azide liposomes. igg was prepared for conjugation to maleimide liposomes by one-hour reaction of 10 sata (n-succinimidyl s-acetylthioacetate) per igg at room temperature in 0.5 mm edta in pbs. unreacted sata was removed from igg by passage through 7 kda cutoff gel filtration columns. sata-conjugated igg was deprotected by one-hour room temperature incubation in 0.05 m hydroxylamine in 2.5 mm edta in pbs. excess hydroxylamine was removed and buffer was exchanged for 0.5 mm edta in pbs via 7 kda cutoff gel filtration column. sata-conjugated and deprotected igg was added to liposomes at 200 igg per liposomes for overnight reaction at 4°c. excess igg was removed by size exclusion column purification, as above for azide liposomes. 150 nm carboxylate nanoparticles (phosphorex) were exchanged into 50 mm mes buffer at ph 5.2 via gel filtration column. n-hydroxysulfosuccinimide (sulfo-nhs) was added to the particles at 0.275 mg/ml, prior to incubation for 3 minutes at room temperature. edci was then added to the particles at 0.1 mg/ml, prior to incubation for 15 minutes at room temperature. igg was added to the particle mixture at 200 igg per nanoparticle, prior to incubation for 3 hours at room temperature while vortexing. for radiotracing, 125 i-labeled igg was added to the reaction at 5% of total igg mass. the igg/particle mixture was diluted with 10-fold volume excess of ph 5.2 mes buffer and the diluted mixture was centrifuged at 12000xg for 3 minutes. supernatant was discarded and pbs with 0.05% bsa was added at desired volume before resuspending the particles via sonication probe sonication (three pulses, 30% amplitude). particle size was assessed via dls after resuspension, and particles were used immediately after dls assessment. top10 e. coli were grown overnight in terrific broth with ampicillin. bacteria were heat-inactivated by 20-minute incubation at 60°c, then fixed by overnight incubation in 4% paraformaldehyde. after fixation, bacteria were pelleted by centrifugation at 1000xg for 10 minutes. pelleted bacteria were washed three times in pbs, prior to resuspension by pipetting. bacterial concentration was verified by optical density at 600 nm, prior to radiolabeling as described for nanoparticles below. bacteria were administered in mice (7.5x10 7 colony forming units in a 100 µl suspension per mouse). protein, horse spleen ferritin nanocages (sigma), or adeno-associated virus (empty capsids, serotype 8) were prepared in pbs at concentrations between 1 and 2 mg/ml in volumes between 100 and 200 µl. films of oxidizing agent were prepared in borosilicate tubes by drying 300 µl of 0.5 mg/ml iodogen (perkin-elmer, chloroform solution) under nitrogen gas. alternatively, iodobeads (perkin-elmer) were added to borosilicate tubes (one per reaction). protein solutions were added to coated or beadcontaining tubes, before addition of na 125/131 i at 25 µci per 100 µg of protein. protein was incubated with radioiodine at room temperature for 5 minutes under parafilm in a ventilated hood. iodide-protein reacottions were terminated by purifying protein solutions through a 7 kda cutoff gel column (zeba). additional passages through gel filtration columns or against centrifugal filters (amicon, 10 kda cutoff) were employed to remove free iodine, assuring that >95% of radioactivity was associated with protein. lysozyme-dextran nanogels, crosslinked protein nanoparticles, e. coli, or adenovirus were similarly iodinated. at least 100 µl of particle suspension was added to a borosilicate tube containing two iodobeads, prior to addition of 100 µci of na 125 i per 100 µl of suspension. particles were incubated with radioiodine and iodobeads for 30 minutes at room temperature, with gentle shaking every 10 minutes. to remove free iodine, particle suspensions were moved to a centrifuge tube, diluted in ~1 ml of buffer and centrifuged to pellet the particles (16000xg/30 minutes for nanogels, 16000xg/30 minutes for crosslinked protein particles, 10000xg/30 minutes for adenovirus, and 1000xg/10 minutes for e. coli). supernatant was removed and wash/centrifugation cycles were repeated to assure >95% of radioactivity was associated with particles. particles were resuspended by probe sonication (three pulses, 30% amplitude) for nanogels or crosslinked protein nanoparticles or pipetting for adenovirus or e. coli. nanoparticle labeling with 111 in 111 in labeling of nanoparticles followed previously described methods, with adaptation for new particles. 47 all radiolabeling chelation reactions were performed using metal free conditions to prevent contaminating metals from interfering with chelation of 111 in by dtpa or dota. metals were removed from buffers using chelex 100 metal affinity resin (biorad, laboratories, hercules ca). lysozyme-dextran nanogels were prepared for chelation to 111 in by conjugation to s-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-scn-bn-dota, macrocyclics). nanogels were moved to metal free ph 8.3 1 m nahco3 buffer by three-fold centrifugation (16000xg for 15 minutes) and pellet washing with metal free buffer. p-scn-bn-dota was added to nanogels at 1:25 mass:mass ratio, prior to reaction for 30 minutes at room temperature. free p-scn-bn-dota was removed by three-fold centrifugal filtration against 10 kda cutoff centrifugal filters, with resuspension of nanogels in metal-free ph 4 citrate buffer after each centrifugation. dota-conjugated nanogels or dtpa-containing liposomes in ph 4 citrate buffer were combined with 111 incl3 for one-hour chelation at 37°c. nanoparticle/ 111 incl3 mixtures were treated with free dtpa (1 mm final concentration) to remove 111 in not incorporated in nanoparticles. efficiency of 111 in incorporation in nanoparticles was assessed by thin film chromatography (aluminum/silica strips, sigma) with 10 µm edta mobile phase. chromatography strips were divided between origin and mobile front and the two portions of the strip were analyzed in a gamma counter to assess nanoparticleassociated (origin) vs. free (mobile front) 111 in. free 111 in was separated from nanoparticles by centrifugal filtration and nanoparticles were resuspended in pbs (liposomes) or saline (nanogels). for spect/ct imaging experiments (see spect/ct imaging methods below) with nanogels, 80 µci of 111 in-labeled nanogels, used within one day 111 in labeling as described above, were administered to each mouse. for tracing 111 in-labeled liposomes in biodistribution studies, liposomes were labeled with 50 µci 111 in per µmol of lipid. nanoparticle or protein biodistributions were tested by injecting radiolabeled nanoparticles or protein (suspended to 100 µl in pbs or 0.9% saline at a dose of 2.5 mg/kg with tracer quantities of radiolabeled material) in c57bl/6 male mice from jackson laboratories. biodistributions in naïve mice were compared to biodistributions in several injury models. biodistribution data were collected at 30 minutes after nanoparticle or protein injection, unless otherwise stated, as in pharmacokinetics studies. briefly, blood was collected by vena cava draw and mice were sacrificed via terminal exsanguination and cervical dislocation. organs were harvested and rinsed in saline, and blood and organs were examined for nanoparticle or protein retention in a gamma counter (perkin-elmer). nanoparticle or protein retention in harvested organs was compared to measured radioactivity in injected doses. for calculations of nanoparticle or protein concentration in organs, quantity of retained radioactivity was normalized to organ weights. mice subject to intravenous lps injury were anesthetized with 3% isoflurane before administration of lps from e. coli strain b4 at 2 mg/kg in 100 µl pbs via retroorbital injection. after five hours, mice were anesthetized with ketamine-xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration) and administered radiolabeled nanoparticles or protein via jugular vein injection to determine biodistributions as described above. for mice subject to intratracheal (it) lps injury, b4 lps was administered to mice (anesthetized with ketamine/xylazine) at 1 mg/kg in 50 µl of pbs via tracheal catheter, followed by 100 µl of air. 66 biodistributions of lysozyme-dextran nanogels in it-lps-injured mice were assessed as above 16 hours after lps administration. biodistributions of liposomes in it-lps-injured mice were assessed at 1, 2, or 6 hours after lps administration. mice subject to footpad lps administration were provided b4 lps at 1 mg/kg in 50 µl pbs via footpad injection. biodistributions of lysozyme-dextran nanogels were obtained at 6 or 24 hours after footpad lps administration. lysozyme-dextran nanogel biodistributions were also traced in a mouse model of cardiogenic pulmonary edema. 50 to establish edema, mice were anesthetized with ketamine/xylazine and administered propranolol in saline (3 µg/ml) via jugular vein catheter at 83 µl/min over 120 minutes. lysozyme-dextran nanogel biodistributions were subsequently assessed as above. single cell suspensions were prepared from lungs for flow cytometric analysis of cell type composition of the lungs and/or nanoparticle distribution among different cell types in the lungs. c57bl/6 male mice were anesthetized with ketamine/xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration) prior to installation of tracheal catheter secured by suture. after sacrifice by terminal exsanguination via the vena cava, lungs were perfused by right ventricle injection of ~10 ml of cold pbs. the lungs were then infused via the tracheal catheter with 1 ml of a digestive enzyme solution consisting of 5 u/ml dispase, 2.5 mg/ml collagenase type i, and 1 mg/ml of dnase i in cold pbs. immediately after infusion, the trachea was sutured shut while removing the tracheal catheter. the lungs with intact trachea were removed via thoracotomy and kept on ice prior to manual disaggregation. disaggregated lung tissue was aspirated in 2 ml of digestive enzyme solution and incubated at 37°c for 45 minutes, with vortexing every 10 minutes. after addition of 1 ml of fetal calf serum, tissue suspensions were strained through 100 µm filters and centrifuged at 400xg for 5 minutes. after removal of supernatant, the pelleted material was resuspended in 10 ml of cold ack lysing buffer. the resulting suspensions were strained through 40 µm filter and incubated for 10 minutes on ice. the suspensions were centrifuged at 400xg for 5 minutes and the resulting pellets were rinsed in 10 ml of facs buffer (2% fetal calf serum and 1 mm edta in pbs). after centrifugation at 400xg for 5 minutes, the rinsed cell pellets were resuspended in 2% pfa in 1 ml facs buffer for 10 minutes incubation. the fixed cell suspensions were centrifuged at 400xg for 5 minutes and resuspended in 1 ml of facs buffer. for analysis of intravascular leukocyte populations in naïve and inflamed lungs, mice received an intravenous injection of fitc-conjugated anti-cd45 antibody five minutes prior to sacrifice and preparation of single cell suspensions as described above. populations of intravascular vs. extravascular leukocytes were assessed by subsequent stain of fixed cell suspensions with percp-conjugated anti-cd45 antibody and/or apcconjugated clone 1a8 anti-ly6g antibody. to accomplish staining of fixed cells, 100 µl aliquots of the cell suspensions described above were pelleted at 400xg for 5 minutes, then resuspended in labeled antibody diluted in facs buffer (1:150 dilution for apcconjugated anti-ly6g antibody and 1:500 dilution for percp-conjugated anti-cd45 antibody). samples were incubated with staining antibodies for 20 minutes at room temperature in the dark, diluted with 1 ml of facs buffer, and pelleted at 400xg for 5 minutes. stained pellets were resuspended in 200 µl of facs buffer prior to immediate flow cytometric analysis on a bd accuri flow cytometer. all flow cytometry data was gated to remove debris and exclude doublets. control samples with no stain, obtained from naïve and iv-lps-injured mice, established gates for negative/positive staining with fitc, percp, and apc. single stain controls allowed automatic generation of compensation matrices in fcs express software. comparison of percp anti-cd45 signal with fitc anti-cd45 signal indicated intravascular vs. extravascular leukocytes. comparison of apc anti-ly6g signal with fitc anti-cd45 signal indicated intravascular vs. extravascular neutrophils, with percp and apc co-staining verifying identification of cells as neutrophils. similar staining and analysis protocols enabled identification of nanoparticle distribution among different cell types in the lungs. to enable fluorescent tracing, lysozyme-dextran nanogels contained fitc-dextran, dbco-igg liposomes contained green fluorescent top fluor pc lipid, and crosslinked albumin nanoparticles were labeled with nhs ester alexa fluor 488. alexa fluor 488 labeling of albumin nanoparticles was accomplished by incubation of the nhs ester fluorophore with nanoparticles at 1:25 mass:mass fluorophore:nanoparticle ratio for two hours on ice. excess fluorophore was removed from nanoparticles by 3-fold centrifugation at 16000xg for 15 minutes followed by washing with pbs. nanoparticles were administered at 2.5 mg/kg via jugular vein injection and circulated for 30 minutes, prior to preparation of single cell suspensions from lungs as above. fixed single cell suspensions were stained with apc-conjugated anti-ly6g or percp-conjugated anti-cd45 as above. additional suspensions were stained with 1:150 dilution of apc-conjugated anti-cd31, in lieu of anti-ly6g, to identify endothelial cells. association of nanoparticles with cell types was identified by coincidence of green fluorescent signal with anti-cd45, anti-ly6g, or anti-cd31 signal. as described previously, 47 thirty minutes after injection of 80 µci of 111 in-labeled nanogels, anesthetized mice were sacrificed by cervical dislocation. mice were placed into a milabs u-spect (utrecht, netherlands) scanner bed. a region covering the entire body was scanned for 90 min using listmode acquisition. the animal was then moved, while maintaining position, to a milabs u-ct (utrecht, netherlands) for a fullbody ct scan using default acquisition parameters (240 µa, 50 kvp, 75 ms exposure, 0.75° step with 480 projections). for naïve mice and mice imaged after cardiogenic pulmonary edema, ct data was acquired as above without spect data. the spect data was reconstructed using reconstruction software provided by the manufacturer, with 400 µm voxels. the ct data were reconstructed using reconstruction software provided by the manufacturer, with 100 µm voxels. spect and ct data, in nifti format, were opened with imagej software (fiji package). background signal was removed from spect images by thresholding limits determined by applying renyi entropic filtering, as implemented in imagej, to a spect image slice containing ngassociated 111 in in the liver. background-subtracted pseudo-color spect images were overlayed on ct images and axial slices depicting lungs were selected for display, with ct thresholding set to emphasize negative contrast in the airspace of the lungs. imagej's built-in 3d modeling plugin was used to co-register background-subtracted pseudo-color spect images with ct images in three-dimensional reconstructions. ct image thresholding was set in the 3d modeling tool to depict skeletal structure alongside spect signal. for three-dimensional reconstructions of lung ct images, thresholding was set, as above, for contrast emphasizing the airspace of the lungs, with thresholding values standardized between different ct images (i.e. identical values were used for naïve and edematous lungs). images were cropped in a cylinder to exclude the airspace outside of the animal, then contrast was inverted, allowing airspace to register bright ct signal and denser tissue to register as dark background. three-dimensional reconstructions of the lung ct data, and co-registrations of spect data with lung ct data, were generated as above with imagej's 3d plugin applied to ct data cropped and partitioned for lung contrast. quantification of ct attenuation employed imagej's measurement tool iteratively over axial slices, with measurement fields of view manually set to contain lungs and exclude surrounding tissue. mice were exposed to nebulized lps in a 'whole-body' exposure chamber, with separate compartments for each mouse (mpc-3 aero; braintree scientific). to maintain adequate hydration, mice were injected with 1ml of sterile saline warmed to 37°c, intraperitoneally, immediately before exposure to lps. lps (l2630-100mg, sigma aldrich) was reconstituted in pbs to 10 mg/ml and stored at -80°c until use. immediately before nebulization, lps was thawed and diluted to 5 mg/ml with pbs. lps was aerosolized via a jet nebulizer connected to the exposure chamber (neb-med h, braintree scientific, inc.). 5 ml of 5 mg/ml lps was used induce the injury. nebulization was performed until all liquid was nebulized (~20 minutes). liposomes or saline sham were administered via retro-orbital injections of 100 µl of suspension (25 mg/kg liposome dose) at 2 hours after lps exposure. mice were anesthetized with 3% isoflurane to facilitate injections. bronchoalveolar lavage (bal) fluid was collected 24 hours after lps exposure, as previously described. 66 briefly, mice were anesthetized with ketamine-xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration). the trachea was isolated and a tracheostomy was performed with a 22-gauge catheter. the mice were euthanized via exsanguination. 0.8 ml of cold bal buffer (0.5 mm edta in pbs) was injected into the lungs over ~1min via the tracheostomy and then aspirated from the lungs over ~1min. injections/aspirations were performed three times for a total of 2.4ml of fluid added to the lungs. recovery bal fluid typically amounted to ~2.0ml. bal samples were centrifuged at 300xg for 4 minutes. the supernatant was collected and stored at -80°c for further analysis. protein concentration was measured using bio-rad dc protein assay, per manufacturer's instructions. the cell pellet was fixed for flow cytometry as follows. 333 µl of 1.6% pfa in pbs was added to each sample. samples were incubated in the dark at room temperature for 10 minutes, then 1 ml of bal buffer was added. samples were centrifuged at 400xg for 3min, the supernatant was aspirated, and 1 ml of facs buffer (2% fetal calf serum and 1 mm edta in pbs) was added. at this point, samples were stored at 4°c for up to 1 week prior to flow cytometry analysis. to stain for flow cytometry, samples were centrifuged at 300xg for 4 min, the supernatant was aspirated, and 100 µl of staining buffer was added. staining buffer used was a 1:1000 dilution of stock antibody solution (apc anti-mouse cd45; alexa fluor 488 anti-mouse ly6g, biolegend) into facs buffer. samples were incubated with staining antibody for 30 minutes at room temperature in the dark. to terminate staining, 1 ml of facs buffer was added, samples were centrifuged at 300xg for 4 minutes, and supernatant was aspirated. cells were resuspended in 900 µl of facs buffer and immediately analyzed via flow cytometry. flow cytometric analysis was completed with a bd accuri flow cytometer as follows: sample volume was set to 100 µl and flow rate was set to 'fast'. unstained and single-stained controls were used to set gates. forward scatter (pulse area) vs. side scatter (pulse area) plots were used to gate out non-cellular debris. forward scatter (pulse area) vs. forward scatter (pulse height) plots were used to gate out doublets. the appropriate fluorescent channels were used to determine stained vs. unstained cells. the gates were placed using unstained control samples. single-stain controls were tested and showed there was no overlap/bleed-through between the fluorophores. final analysis indicated the quantity of leukocytes (cd45-positive cells) and neutrophils (ly6g-positive cells) in bal samples. human lungs were obtained after organ harvest from transplant donors whose lungs were in advance deemed unsuitable for transplantation. the lungs were harvested by the organ procurement team and kept at 4°c until the experiment, which was done within 24 hours of organ harvest. the lungs were inflated with low pressure oxygen and oxygen flow was maintained at 0.8 l/min to maintain gentle inflation. pulmonary artery subsegmental branches were endovascularly cannulated, then tested for retrograde flow by perfusing for 5 minutes with steen solution containing a small amount of green tissue dye at 25 cm h2o pressure. the pulmonary veins through which efflux of perfusate emerged were noted, allowing collection of solutions after passage through the lungs. a 2 ml mixture of 125 i-labeled lysozyme-dextran nanogels and 131 ilabeled ferritin nanocages were injected through the arterial catheter. ~100ml of 3% bsa in pbs was passed through the same catheter to rinse unbound nanoparticles. a solution of green tissue dye was subsequently injected through the same catheter. the cannulated lung lobe was dissected into ~1 g segments, which were evaluated for density of tissue dye staining. segments were weighed, divided into 'high', 'medium', 'low', and 'null' levels of dye staining, and measured for 131 i and 125 i signal in a gamma counter. for experiments with cell suspensions derived from human lungs (chosen for research use according to the above standards), single cell suspensions were generously provided by the laboratory of edward e. morrisey at the university of pennsylvania. aliquots of 600,000 cells were pelleted at 400xg for 5 minutes and resuspended in 100 µl pbs containing different quantities of lysozyme-dextran nanogels synthesized with fitc-labeled dextran. cells and nanogels were incubated at room temperature for 30 minutes before two-fold pelleting at 400xg with 1 ml pbs washes. cells were re-suspended in 200 µl facs buffer for staining with apcconjugated anti-human cd45, applied by 20-minute incubation with a 1:500 dilution of the antibody stock. cells were pelleted at 400xg for 5 minutes and resuspended in 200 µl pbs for immediate analysis with flow cytometry (bd accuri). negative/positive nanogel or anti-cd45 signal was established by comparison to unstained cells. singlestained controls indicated no spectral overlap between fitc-nanogel fluorescence and anti-cd45 apc fluorescence. proteins were prepared in deionized and filtered water at concentrations of 0.155 mg/ml for human albumin, 0.2 mg/ml for hen lysozyme, and 0.48 mg/ml for igg. crosslinked albumin nanoparticles, lysozyme-dextran nanogels, and igg-coated liposome suspensions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the concentrations of the corresponding protein solutions. protein and nanoparticle solutions were analyzed in quartz cuvettes with 10 mm path length in an aviv circular dichroism spectrometer. the instrument was equilibrated in nitrogen at 25°c for 30 minutes prior to use and samples were analyzed with sweeps between 185 and 285 nm in 1 nm increments. each data point was obtained after a 0.333 s settling time, with a 2 s averaging time. cdnn 40 software deconvolved cd data (expressed in millidegrees) via neural network algorithm assessing alignment of spectra with library-determined spectra for helices, antiparallel sheets, parallel sheets, beta turns, and random coil. 8-anilino-1-naphthalenesulfonic acid (ansa) at 0.06 mg/ml was mixed with lysozyme, human albumin, or igg at 1.5 mg/ml in pbs. for nanoparticle analysis, nanoparticle solutions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the 1.5 mg/ml concentration of the protein solutions. protein or nanoparticles and ansa were reacted at room temperature for 30 minutes. excess ansa was removed from solutions by 3 centrifugations against 3 kda cutoff centrifugal filters (amicon). after resuspension to original volume, ansa-stained protein/nanoparticle solutions/suspension were examined for fluorescence (excitation 375 nm, emission 400-600 nm) and absorbance (240-540 nm) maxima corresponding to ansa. for imaging neutrophil content in naïve and iv-lps-injured lungs, mice were intravenously injected with rat anti-mouse anti-ly6g antibody (clone 1a8) and sacrificed 30 minutes later. lungs were embedded in m1 medium, flash frozen, and sectioned in 10 µm slices. sections were stained with percp-conjugated anti-rat secondary antibody and neutrophil-associated fluorescence was observed with epifluorescence microscopy. similar procedures enabled histological imaging of lysozyme-dextran nanogels in iv-lps-injured lungs. nanogels synthesized with rhodamine-dextran were administered intravenously in injured mice 30 minutes prior to sacrifice. lungs were sectioned as above and stained with clone 1a8 anti-ly6g antibody, followed by briliant violetconjugated anti-rat secondary antibody. sections of human lungs were obtained after ex vivo administration (see nanoparticle administration in human lungs above) of lysozyme-dextran nanogels synthesized with rhodamine dextran. regions of tissue delineated as perfused and nonperfused, as determined by arterial administration of tissue dye as above, were harvested, embedded in m1 medium, flash frozen, and sectioned in 10 µm slices. epifluorescence imaging indicated rhodamine fluorescence from nanogels, coregistered to autofluorescence indicating tissue architecture. a mouse was anesthetized with ketamine/xylazine five hours after intravenous administration of 2 mg/kg b4 lps. a jugular vein catheter was fixed in place for injection of lysozyme-dextran nanogels, anti-cd45 antibody, and fluorescent dextran during imaging. in preparation for exposure of the lungs, a patch of skin on the back of the mouse, around the juncture between the ribcage and the diaphragm, was denuded. while the mouse was maintained on mechanical ventilation, an incision at the juncture between the ribs and the diaphragm, towards the posterior, exposed a portion of the lungs. a coverslip affixed to a rubber o-ring was sealed to the incision by vacuum. the exposed portion of the mouse lung was placed in focus under the objective by locating autofluorescence signal in the "fitc" channel. with 100 ms exposure, fluorescent images from channels corresponding to violet, green, near red, and far red fluorescence were sequentially acquired. a mixture of rhodamine-dextran nanogels (2.5 mg/kg), brilliant violet-conjugated anti-cd45 antibody (0.8 mg/kg), and alexa fluor 647 labeled 70 kda dextran (40 mg/kg) for vascular contrast was administered via jugular vein catheter and images were recorded for 30 minutes. images were recorded in slidebook software and opened in imagej (fiji distribution) for composition in movies with coregistration of the four fluorescent channels. all animal studies were carried out in strict accordance with guide for the care and use of laboratory animals as adopted by national institute of health and approved by university of pennsylvania institutional animal care and use committee (iacuc). male c57bl/6j mice, 6-8 weeks old, were purchased from jackson laboratories. mice were maintained at 22-26°c and on a 12/12 hour dark/light cycle with food and water ad libitum. ex vivo human lungs were donated from an organ procurement agency, gift of life, after determination the lungs were not suitable for transplantation into a recipient, and therefore would have been discarded if they were not used for our study. gift of life obtained the relevant permissions for research use of the discarded lungs, and in conjunction with the university of pennsylvania's institutional review board ensured that all relevant ethical standards were met. error bars indicate standard error of the mean throughout. significance was determined through paired t-test for comparison of two samples and anova for group comparisons. linear discriminant analysis and principal components analysis were completed in gnu octave scripts (adapted from https://www.bytefish.de/blog/pca_lda_with_gnu_octave/, and made available in full in the supplementary materials). findings in this study contributed to united states provisional patent application number 62/943469. raw imaging, flow cytometry, gamma counter, and spectroscopy data supporting the findings of this study are available from the corresponding author upon reasonable request. all other data supporting the findings of this study are available within the paper and its supplementary information files. covid-19 in critically ill patients in the seattle region -case series the 1918 influenza pandemic: insights for the 21 st century lung safe investigators; esicm trials group. epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries incidence and outcomes of acute lung injury. b. engl nanomedicine for the treatment of acute respiratory distress syndrome. the 2016 ats bear cage award-winning proposal the mercurial nature of neutrophils: still an enigma in ards? endothelial nanomedicine for the treatment of pulmonary disease balti-2 study investigators. effect of intravenous β-2 agonist treatment on clinical outcomes in acute respiratory distress syndrome (balti-2): a multicentre, randomised controlled trial national heart, lung, and blood institute acute respiratory distress syndrome (ards) clinical trials network. randomized, placebo-controlled clinical trial of an aerosolized β2-agonist for treatment of acute lung injury neutrophil function in inflammation and inflammatory diseases paradoxical roles of the neutrophil in sepsis: protective and deleterious targeting neutrophils in ischemic stroke: translational insights from experimental studies neutrophil function in ischemic heart disease contribution of neutrophils to acute lung injury neutrophils kinetics in health and disease neutrophil-endothelial cell interactions in the lung the multifaceted functions of neutrophils neutrophils in the activation and regulation of innate and adaptive immunity what drives neutrophils to the alveoli in ards? pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome neutrophil margination, sequestration, and emigration in the lungs of l-selectin-deficient mice ly6 family proteins in neutrophil biology use of ly6g-specific monoclonal antibody to deplete neutrophils in mice neutrophil targeted nano-drug delivery system for chronic obstructive lung diseases therapeutic targeting of neutrophil granulocytes in inflammatory liver disease prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils neutrophil-mediated delivery of therapeutic nanoparticles across blood vessel barrier for treatment of inflammation and infection non-affinity factors modulating vascular targeting of nano-and microcarriers physical approaches to biomaterial design impact of particle elasticity on particle-based drug delivery systems factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles cell-mediated delivery of nanoparticles: taking advantage of circulatory cells to target nanoparticles neutrophil sequestration and migration in localized pulmonary inflammation. capillary localization and migration across the interalveolar septum neutrophil recruitment to the lungs during bacterial pneumonia the lung is a host defense niche for immediate neutrophilmediated vascular protection icam-1 targeted nanogels loaded with dexamethasone alleviate pulmonary inflammation flexible nanoparticles reach sterically obscured endothelial targets inaccessible to rigid nanoparticles long-circulating janus nanoparticles made by electrohydrodynamic co-jetting for systemic drug delivery applications the transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo quantitative analysis of protein far uv circular dichroism spectra by neural networks selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel lysozyme-dextran core-shell nanogels prepared via a green process in vivo editing of macrophages through systemic delivery of crispr-cas9-ribonucleoprotein-nanoparticle nanoassemblies adeno-associated virus structural biology as a tool in vector development structure of human adenovirus cisplatin encapsulation within a ferritin nanocage: a high-resolution crystallographic study vascular targeting of radiolabeled liposomes with bio-orthogonally conjugated ligands: single chain fragments provide higher specificity than antibodies targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin acute respiratory distress syndrome: diagnosis and management novel role for cftr in fluid absorption from the distal airspaces of the lung red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude neutrophil-particle interactions in blood circulation drive particle clearance and alter neutrophil responses in acute inflammation the tlr9-myd88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice adeno-associated viral vectors at the frontier between tolerance and immunity serum ferritin: past, present and future facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries a new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay doxil®--the first fda-approved nano-drug: lessons learned lipid rafts regulate lipopolysaccharide-induced activation of cdc42 and inflammatory functions of the human neutrophil alterations in membrane cholesterol cause mobilization of lipid rafts from specific granules and prime human neutrophils for enhanced adherence-dependent oxidant production generation of targeted adenoassociated virus (aav) vectors for human gene therapy biphasic janus particles with nanoscale anisotropy direct cytosolic delivery of crispr/cas9-ribonucleoprotein for efficient gene editing direct cytosolic delivery of proteins through coengineering of proteins and polymeric delivery vehicles antioxidant protection by pecam-targeted delivery of a novel nadph-oxidase inhibitor to the endothelium in vitro and in vivo red: anti-ly6g stain. green: tissue autofluorescence. (f) biodistributions of heat-inactivated, fixed, and 125 ilabeled e. coli in naïve (n=4) and iv-lps-injured (n=4) mice tissue autofluorescence). (k) single frame from real-time intravital imaging of ldng (red) uptake in leukocytes (green) in iv-lps-inflamed pulmonary vasculature (blue, alexa fluor 647-dextran) biodistributions in iv-lps-injured mice for azide-functionalized liposomes conjugated to igg loaded with 2.5, 5, 10, and 20 dbco molecules per igg (bars further to the right correspond to more dbco per igg). (d) mouse lungs flow cytometry data indicating ly6g anti-neutrophil staining density vs. levels of dbco(20x)-igg liposome uptake. (e) flow cytometry data verifying increased dbco(20x)-igg liposome uptake in and specificity for neutrophils following lps insult (inset: verification of increased concentration of neutrophils in the lungs following lps key: cord-265658-wjqezs0v authors: carranza-rosales, pilar; carranza-torres, irma edith; guzmán-delgado, nancy elena; lozano-garza, gerardo; villarreal-treviño, licet; molina-torres, carmen; villarreal, javier vargas; vera-cabrera, lucio; castro-garza, jorge title: modeling tuberculosis pathogenesis through ex vivo lung tissue infection date: 2017-09-12 journal: tuberculosis (edinb) doi: 10.1016/j.tube.2017.09.002 sha: doc_id: 265658 cord_uid: wjqezs0v tuberculosis (tb) is one of the top 10 causes of death worldwide. several in vitro and in vivo experimental models have been used to study tb pathogenesis and induction of immune response during mycobacterium tuberculosis infection. precision cut lung tissue slices (pclts) is an experimental model, in which all the usual cell types of the organ are found, the tissue architecture and the interactions amongst the different cells are maintained. pclts in good physiological conditions, monitored by mtt assay and histology, were infected with either virulent mycobacterium tuberculosis strain h37rv or the tb vaccine strain mycobacterium bovis bcg. histological analysis showed that bacilli infecting lung tissue slices were observed in the alveolar septa, alveolar light spaces, near to type ii pneumocytes, and inside macrophages. mycobacterial infection of pclts induced tnf-α production, which is consistent with previous m. tuberculosis in vitro and in vivo studies. this is the first report of using pclts as a system to study m. tuberculosis infection. the pclts model provides a useful tool to evaluate the innate immune responses and other aspects during the early stages of mycobacterial infection. mycobacterium tuberculosis, the primary cause of human tuberculosis (tb), has infected one third of all humanity, and it is a leading cause of death by a single infectious agent [1] . diverse approaches have been used to study tb pathogenesis. discovery of mechanisms of adherence, infection routes, secreted or contact-dependent bacterial factors, evasion of immune response, signaling, and dissemination in tissues have been possible using in vitro and in vivo models. such studies can reveal useful information to aid in the development of products or strategies to prevent or treat tb. both, in vitro as well as in vivo experimental systems have advantages and limitations. each has become complementary to the other to dissect the pathogenic mechanisms of microorganisms and host immune response. an alternative model, bridging in vivo and in vitro, is an ex vivo model that uses original tissue explants: the precision cut lung tissue slices (pclts). in this system, all of the typical cell types of the organ are found, the tissue architecture and interactions between the different cells are maintained, and metabolic and transport functions are preserved [2] . lung tissue slices have been used for toxicity studies [3] , biotransformation [4] , metabolism of xenobiotics [5] , and to study infectious agents like coronavirus [6] , retrovirus [7] , influenza and parainfluenza viral strains [2,8e12] , viruses of the bovine respiratory disease complex [13, 14] , respiratory syncytial virus (rsv), and only one bacteria chlamydophila pneumonia [15] . these reports show that lung tissue slices can be used in modeling different aspects of infection processes by various pathogens. in the current study, we have optimized pclts for use as an alternative model to study mycobacterial infection in lung tissue. 2.1. bacterial strains and growth conditions m. tuberculosis strain h37rv and m. bovis bcg were cultured in middlebrook 7h9 broth supplemented with oleic acid/albumin/ dextrose/catalase (oadc), grown to mid-logarithmic phase, and stored at à70 c until needed. bacterial titers for each batch of frozen stocks were determined in triplicate by colony-forming units (cfu) counting on middlebrook 7h10 agar. before infection experiments, aliquots of bacterial stocks were thawed at 37 c, mixed vigorously by vortexing, and diluted in tissue-culture medium to provide inocula to achieve the multiplicity of infection (moi) indicated. moi was confirmed from these preparations by cfu counting on middlebrook 7h10 agar for each experiment. pclts were prepared from 18 to 20 weeks old male balb/c mice (mus musculus) (harlan mexico). mice were euthanized by cervical dislocation following institutional and international guidelines for humanitarian care of animals used in experimental work. then pleural cavity was exposed under aseptic conditions and trachea was cannulated to infiltrate the lungs with 0.7% low-gelling temperature agarose in rpmi 1640 medium at 37 c. tissues were allowed to cool with ice to obtain an appropriately-firm consistency to cut the slices and the lungs were excised and immersed in sterile krebs-henseleit (kb) buffer (ph 7.4 at 4 c). five millimeter diameter cores of lung tissue were obtained and then the cores were cut with a brendel vitron tissue slicer (vitron, tucson, az, usa) in oxygenated kb buffer (4 c, 95:5% o 2 :co 2 ) into 350e400 mm thick tissue slices. pclts were transferred into 24-well microplates (one per well) with 1 ml of dmem/f12 medium supplemented with 10% bovine fetal serum and 25 mm glucose. pclts were incubated at 37 c and 5% co 2 in an orbital shaker (~40 rpm). after 1 h, medium was changed every 30 min to remove agarose. viability of pclts was determined by tetrazolium dye mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in 24 well plates [16] . od 570nm values were read in a microplate reader (synergy ht, biotek, winooski, vt, usa). viability of pclts was 95% at 72 h (data not shown). immediately after the last wash to remove agarose, the pclts were infected with 1.8 â 10 6 bacterial inoculum in 300 ml applied on top of the slices. infected lung slices were incubated stationary for 1 h to facilitate adherence and infection. then, 700 ml of complete dmem/f12 medium was added to each well and the microplates were incubated at 37 c and 5% co2 in an orbital shaker (~40 rpm) for different infection periods. cfu were determined by plating in triplicate on middlebrook 7h10 agar at each time point. after the indicated infection times, pclts were fixed with buffered formalin (10% in pbs) at 4 c for 24 h to inactivate the mycobacteria [17] . a different microplate was used for each time point to avoid formaldehyde vapors from affecting other slice cultures. infected and control slices were embedded in paraffin using an automated tissue processor (citadel 1000, shandon, pennsylvania, usa). sections of 5 mm thickness were obtained from the embedded tissues using a microtome (vitron inc, tucson, az). the sections were stained with hematoxylin & eosin (h&e) or ziehl-neelsen (z-n) dyes and visualized under a conventional light microscope. at each time point, uninfected and infected pclts were transferred into rnalater ® solution, kept at 4 c for 12 h, and then stored at à20 c. rna was extracted from all the samples at the same time using the rneasy ® protect mini kit (qiagen) following manufacturer instructions. quantity and purity of rna were determined using a nanodrop spectrophotometer nd-1000 (thermo scientific, massachusetts, usa). integrity of rna was visualized in agarose gels (1.5%) under denaturing conditions. reverse transcription was performed using the improm-ii reverse transcription system from promega ® (madison, wisconsin, usa) with random hexamers following manufacturer instructions. the generated cdna was stored at à20 c until used. quantitative pcr assays for tnf-a were carried on by using taqmanâ ® gene expression assays labeled with fam™ (dye/mgb probe) from applied biosystems. as endogenous gene expression control, the murine gapdh gene labeled with vic (dye/mgb probe) was used. each reaction mix contained 12.5 ml of taqman ® universal pcr master mix (2â), 1.25 ml of taqman ® gene expression assay (20â) mix, 3 ml of cdna (100 ng/ml) and 11.7 ml of nucleasefree water for a final volume of 25 ml. pcr was run in a 7500 real time pcr system from applied biosystems using the following parameters: (1) 50 c for 2 min, (2) 95 c for 10 min, and (3) 95 c for 15 s, then 60 c for 1 min; step 3 was repeated 40 times. cycle threshold (c t ) data were exported to excel to calculate expression levels comparing infected vs infected tissue lung slices at the same time point by ddc t method using the following equation: then, an anova analysis using the dunnett's method (p < 0.05) for comparison of means was performed to determine significance of values at the different time points. histopathological analysis of balb/c mouse pclts by h & e staining showed that uninfected lung tissue slices maintained normal tissue architecture with no evidence of tissue damage up to 5 days of culture (fig. 1) . in infected pclts, at 24 h after infection, there was an increase in lymphocytes in areas where bacilli were located (fig. 2) . mycobacteria infected pclts showed a mild inflammatory response characterized by the presence of lymphocytes, macrophages and multinucleated cells. fig. 3 show pclts sections infected with m. tuberculosis h37rv or m. bovis bcg at different time points (0e48 h). m. bovis bcg bacilli were mainly located as free bacilli in the alveolar spaces and sometimes near type ii pneumocytes. m. tuberculosis bacilli were located nearer to alveolar septa and near or in contact with type ii pneumocytes. mycobacteria-infected pclts showed bacilli in the alveolar spaces with a tendency to group in the alveolar septum, near to or in contact with epithelial cells, but no bacteria were seen within pneumocytes. meanwhile, intracellular bacteria were observed inside macrophages (fig. 4) . presence of multinucleated giant cells and inflammatory cell aggregates, characteristic of a granulomatous disease like tuberculosis, were also frequently seen (fig. 4) . table 1 shows the cfu of both m. tuberculosis h37rv and m. bovis bcg in plcts at different time points. there were not statically differences in number of cfu per slice. comparison of tnf-a induction by mycobacteria infection by the ddct method indicated that tnf-a expression was higher in lung tissue slices infected with m. bovis bcg than in infected lung slices infected with m. tuberculosis. the peak of expression was at 6 h with m. tuberculosis producing an increase of 2.62 fold and m. bovis bcg increasing 3.38 fold (fig. 5) . results of dunnett's analysis of tnf-a gene expression are statistically significant for m. bovis bcg at 2, 6, 12 and 24 h and m. tuberculosis at 6 h compared to control values (fig. 5 ). despite all of the recent m. tuberculosis studies, there remain many unknown mechanisms associated with the tuberculosis pathogenesis due to the complex interactions between mycobacteria and host cells. several in vivo and in vitro models have been useful to obtain information on different aspects of tuberculosis disease initiation and progression. both types of models have advantages and weaknesses and in the end, they have become complementary to each other as sources of valuable data on m. tuberculosis virulence factors as well as on host immune responses to the pathogen. here, we present an alternative and complementary ex vivo model using murine precision cut lung tissue slices to study m. tuberculosis infection. in this system all the usual cell types of the lung are present, the tissue architecture is maintained, as are the interactions between the different cells, and also important, the metabolic and transport functions are preserved. additionally, at present it is virtually impossible to stop using animals in research and in a support for the moral and ethical issues handling experimental animals, this model reduces the number of animals used by experiment. the main target organ for m. tuberculosis infection is the lung; therefore, respiratory tissue is the obvious first choice to evaluate pclts to study m. tuberculosis interactions with lung cells. harford and hamlin [18] , prepared murine tissue slices by hand and reported the lung slices as an infection model for influenza virus, confirming the good physiological condition of the tissue by the presence of active ciliary movement. the first report on precision cut lung slices was a study published by ebsen et al. [15] , in which lung slices were infected with chlamydophila pneumoniae and respiratory syncytial virus. by using morphological analyses, these authors demonstrated that bacterial and viral infections can be successfully performed in this system. pclts studies, the model showed reproducibility and the pclts had good physiological conditions during the time of the assay [7, 12, 15] . recently, pclts from sheep were infected with jaagsiekte sheep retrovirus, the causative agent of ovine pulmonary adenocarcinoma. the virus was able to infect cells, produce new infectious virions, and induce cell proliferation. also, infected lung slice cells expressed markers of type ii pneumocytes and phosphorylated akt and erk1/2, resembling the phenotype of natural and experimentally-derived opa in sheep [7] . a critical factor on pclts model is how the tissue is maintained in good physiological conditions to perform the experiments. previous work in our lab showed that murine pclts are viable up to 96 h after obtaining, as monitored by mtt assay and histological studies [19] ; all the experiments in this study were done during the first 24 h after infection assuring that pclts were suitable to work. umachandran and colleagues [20] , showed that in their conditions rat pclts were metabolically viable for 8 h, a short time compared with our murine pclts; however, differences may be due to how the tissues were obtained and incubated. moreover, the rat slice thickness mean was 600 nm, which may limit gas exchange and adequate nutrient flow into the tissue compared to the 450 mm thickness of our slices. one of the main goals in this work was the optimization to obtain, culture and establish reproducible m. tuberculosis infections. previously, arriaga et al., [21] detected presence of mycobacterial dna by pcr in situ in macrophages, fibroblasts, [15] in mice pclts. migration of immune cells is a signal that pclts remain immunologically active. henjakovic et al. [27] reported that immunoactive substances such as lipopolysaccharide (lps), macrophage-activating lipopeptide-2 (malp-2), interferon-g (ifng), and dexamethasone induced the characteristic responses to these substances in pclts. these facts support this model as suitable to test immune responses against pathogens. further studies on the phenotype of the immune cells in pclts will determine what kind of cells interact directly with mycobacterium. one advantage of this system is the possibility to add exogenous specific immune cell types to the pclts and analyze the role of each cell linage during the challenge with microbes. therefore, to analyze the ability of pclts to respond immunologically we tested the tnf-a induction on pclts infected with mycobacteria. m. bovis bcg induced a higher amount of tnf-a than m. tuberculosis h37rv, measured by qrt-pcr. peaks of expression were at 6 h after infection with an increase of 2.62 and 3.38 fold for m. tuberculosis and m. bovis bcg, respectively. differences in expression are not due to differential growth of strains as differences in the number of cfu per slice were not statistically significant as shown in table 1 , but due to differential tnf-a induction by the infecting strains. these results agree with those reported by wong et al. [28] where they found that virulent strains produced a lower induction of tnf-a than non-virulent strains. these findings suggest that virulent strains may inhibit cellular mechanism to evade innate immune response. an example is the suppression of il-12 production and negative regulation of apoptosis in host macrophages [29e31] which would allow mycobacteria to survive inside the cells protected from immune response, helping further tissue dissemination. in a report of ex vivo human lung tissue infected with m. tuberculosis, m. avium and m. abscessus, ganbat et al. [32] , found macrophages, neutrophils, monocytes, and pneumocytes-ii infected with mycobacteria and nuclear alterations resulting in cell death depending on the mycobacterial species used. this system provides a similarity to the original lung microenvironment with its native cell population, orientation, and structural integrity. in comparison, our model provide homogenous tissue size, favoring gas exchange, nutrient availability, and allowing viability of tissue up to 96 h. also, mycobacterial inoculum used in our experiments is lower and distribution of bacteria is easier due to the size of the pclts. morphological changes and the induction of tnf-a of pclts experimentally infected with mycobacteria suggest this system as an alternative model to study different aspects of tuberculosis pathogenesis under controlled conditions in a closest way to what occurs in vivo. also, the number of animals used by this approach is lower than typical in vivo models. the possible use of pclts to study immune responses, virulence factor action, or even to test therapeutic agents are some facets to explore in future research. world health organization mouse lung slices: an ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses the use of human lung slices in toxicology the early allergic response in small airways of human precision-cut lung slices precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues differential sensitivity of well-differentiated avian respiratory epithelial cells to infection by different strains of infectious bronchitis virus jaagsiekte sheep retrovirus infection of lung slice cultures treating viral exacerbations of chronic obstructive pulmonary disease: insights from a mouse model of cigarette smoke and h1n1 influenza infection replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses in vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses innate immune response to a h3n2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices three viruses of the bovine respiratory disease complex apply different strategies to initiate infection infection of differentiated airway epithelial cells from caprine lungs by viruses of the bovine respiratory disease complex infection of murine precision cut lung slices (pcls) with respiratory syncytial virus (rsv) and chalmydophila pneumoniae using the krumdieck technique biaxial distension of precision-cut lung slices use of a colorimetric assay to measure differences in cytotoxicity of mycobacterium tuberculosis strains effect of influenza virus on cilia and epithelial cells in the bronchi of mice rebanadas de tejidos: un modelo alternativo en investigaci on biom edica (undergraduate thesis) metabolic and structural viability of precision-cut rat lung slices in culture immunological and pathological comparative analysis between experimental latent tuberculous infection and progressive pulmonary tuberculosis persistence of dna from mycobacterium tuberculosis in superficially normal lung tissue during latent infection demonstration of spread by mycobacterium tuberculosis bacilli in a549 epithelial cell monolayers internalization of mycobacterium tuberculosis by macropinocytosis in non-phagocytic cells internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial a549 cells the potential role of lung epithelial cells and beta-defensins in experimental latent tuberculosis ex vivo testing of immune responses in precision-cut lung slices molecular characterization of clinical isolates of mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages mycobacterial lipoarabinomannans: modulators of dendritic cell function and the apoptotic response mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defense mechanism mycobacterium tuberculosis subverts innate immunity to evade specific effectors mycobacteria infect different cell types in the human lung and cause species dependent cellular changes in infected cells we would like to thank russell k. karls for the critical review of the manuscript. this work was supported by paicyt-uanl cn 1534-07, fis-imss 2005/1/i/070 fis/imss/prot/008, inmunocanei-conacyt 253053. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord-280857-0o1ikwks authors: goligher, ewan c.; jonkman, annemijn h.; dianti, jose; vaporidi, katerina; beitler, jeremy r.; patel, bhakti k.; yoshida, takeshi; jaber, samir; dres, martin; mauri, tommaso; bellani, giacomo; demoule, alexandre; brochard, laurent; heunks, leo title: clinical strategies for implementing lung and diaphragm-protective ventilation: avoiding insufficient and excessive effort date: 2020-11-02 journal: intensive care med doi: 10.1007/s00134-020-06288-9 sha: doc_id: 280857 cord_uid: 0o1ikwks mechanical ventilation may have adverse effects on both the lung and the diaphragm. injury to the lung is mediated by excessive mechanical stress and strain, whereas the diaphragm develops atrophy as a consequence of low respiratory effort and injury in case of excessive effort. the lung and diaphragm-protective mechanical ventilation approach aims to protect both organs simultaneously whenever possible. this review summarizes practical strategies for achieving lung and diaphragm-protective targets at the bedside, focusing on inspiratory and expiratory ventilator settings, monitoring of inspiratory effort or respiratory drive, management of dyssynchrony, and sedation considerations. a number of potential future adjunctive strategies including extracorporeal co(2) removal, partial neuromuscular blockade, and neuromuscular stimulation are also discussed. while clinical trials to confirm the benefit of these approaches are awaited, clinicians should become familiar with assessing and managing patients’ respiratory effort, based on existing physiological principles. to protect the lung and the diaphragm, ventilation and sedation might be applied to avoid excessively weak or very strong respiratory efforts and patient-ventilator dysynchrony. lung and diaphragm-protective mechanical ventilation is a novel approach that aims to limit side effects of mechanical ventilation in critically ill patients. this approach integrates the principles of lung-protective ventilation with the new concept of diaphragm-protective ventilation in an effort to simultaneously protect both organs. the approach centers on optimizing patient respiratory effort to avoid lung and diaphragm injury while maintaining acceptable respiratory homeostasis. ultimately, the goal of the approach is to reduce the duration of mechanical ventilation, enhance survival, accelerate recovery, and prevent long-term disability in patients with acute respiratory failure. (ventilator-induced lung injury, vili), patient breathing effort (patient self-inflicted lung injury, p-sili), or both together are generating the forces applied to the lung [2] . bedside measures of stress are available (changes in transpulmonary pressure, driving pressure), but not for measuring the resulting strain, making it challenging to appropriately individualize mechanical ventilation settings to maximize lung protection. furthermore, even if global stress can be measured quite precisely using transpulmonary pressure calculated from airway and esophageal pressure, the effect of gravity on the edematous lung makes the distribution of collapse and aeration very uneven between the dependent and non-dependent lung regions; therefore, global indices do not reflect regional stress or strain. to minimize total stress and strain, dependent regions (usually prone to atelectasis) often require recruitment while non-dependent regions (usually well ventilated) require relief of overdistension. during invasive ventilation, tidal volume (v t ) is routinely scaled to predicted body weight (pbw), which correlates with lung volume in healthy subjects. this correlation is much less accurate in patients with acute respiratory distress syndrome (ards) because of alveolar flooding and atelectasis, resulting in a "baby lung" much smaller than the predicted lung volume [3, 4] . using the driving pressure to scale tidal volume to respiratory system compliance (crs, v t /crs = airway driving pressure, δpaw) is particularly attractive because crs is affected by the aerated lung size and could, therefore, better reflect the global strain (v t / baby lung). driving pressure correlates with ards outcomes among patients with the same v t /pbw [5] and may be useful to guide tidal ventilation, although its role remains to be tested in a prospective trial. it should be acknowledged that static airway pressure is not a very reliable marker of lung stress (both at end-inspiration and end-expiration), because it reflects contributions from both the lung and chest wall (two pressures acting in series). obese patients are an example where high intrathoracic pressure (and therefore higher plateau pressure) exist because of the weight imposed by the chest wall [6] . lung stress is preferably measured as transpulmonary pressure (p l ), which allows to quantify the contribution of the lung and chest wall to changes in airway pressure. the respiratory muscle pump drives alveolar ventilation and is composed of a number of skeletal muscles acting in a highly organized fashion. the diaphragm is the primary muscle of inspiration and the lateral abdominal wall muscles are the most prominent expiratory muscles [7] . mechanical ventilation is employed to unload the respiratory muscle pump and limit the consequences of high breathing effort (e.g., dyspnea sensation, respiratory failure, possible respiratory muscle injury). however, mechanical ventilation delivered as the predominant breathing source can also lead to diaphragm atrophy and injury with a substantial deleterious impact on patient outcome [8] . clinical studies demonstrate that after 24 h of mechanical ventilation, 64% of patients exhibit diaphragm weakness [9] and at the time of weaning, diaphragm weakness is present in up to 80% of patients with weaning difficulties [10] . while many factors contribute to diaphragm weakness in the critically ill [11] , both excessive and insufficient respiratory muscle unloading rapidly result in deleterious changes in diaphragm structure and function [11] . low respiratory muscle effort, due to ventilator over-assist or sedation, may result in muscle atrophy, while high effort has been associated with load-induced injury (fig. 1) . in a landmark study, levine et al. demonstrated the development of diaphragm disuse atrophy in brain dead patients on controlled mechanical ventilation [12] and subsequent studies confirmed the presence of time-dependent fiber atrophy in the diaphragm of ventilated patients [13, 14] . in line with these findings, ultrasound studies demonstrated that low diaphragm effort during mechanical ventilation is associated with time-dependent development of atrophy [15] and that atrophy is associated with poor outcomes [8] . it may be hypothesized that patients are at risk of developing load-induced diaphragm injury, as suggested by the presence of fiber injury, sarcomeric disruption, inflammation and contractile dysfunction in biopsies [13] and acute increases in diaphragm thickness on ultrasound [15] this hypothesis requires further confirmation. taken together, these considerations suggest that the diaphragm might be protected by titrating ventilation and sedation to restore early diaphragm activity while avoiding excess respiratory effort. the various lines of physiological and clinical evidence suggesting that a respiratory effort level similar to that of resting quiet breathing is probably optimal for both lung and diaphragm protection were recently summarized elsewhere [16] . this review explains the principles of lung and diaphragm-protective mechanical ventilation. the overall aim of this approach is to limit the adverse effects of mechanical ventilation on the lung and the diaphragm at the same time. this requires understanding of the pathophysiology of ventilator-induced lung injury, critical illness-associated diaphragm weakness and especially respiratory drive. we discuss clinical applicable techniques to monitor lung and diaphragm function, and how to use these techniques to optimize ventilator settings and sedation. future techniques that allow to control respiratory drive are discussed. to implement lung and diaphragm-protective mechanical ventilation, the variables that mediate injury, principally lung stress and respiratory effort, should be monitored. the available monitoring techniques, their advantages and disadvantages, and proposed specific targets are summarized in table 1 . airway driving pressure, δpaw (i.e., plateau pressure-peep tot ), is a measure that aims to estimate global tidal lung stress [5] . δpaw can be measured either during controlled or assisted ventilation by manual or automated short end-inspiratory and end-expiratory occlusions [17] [18] [19] importantly, δpaw is determined by transpulmonary driving pressure (δp l ) and driving pressure across the chest wall (δp cw ); thus changes in chest wall elastance affect δpaw, without affecting lung stress [20] . because pendelluft and regional variations in lung stress are "dynamic" phenomena that cannot be detected under static conditions, the risk of excess regional lung stress during assisted breathing may be more accurately estimated by dynamic δp l (δp l,dyn , peak p l -end-expiratory p l ) rather than by static measures like δpaw [21, 22] . esophageal pressure (pes) monitoring, as an estimate of pleural pressure, can provide information about both the predisposition to end-expiratory collapse and atelectasis (end-expiratory p l ) and alveolar overdistension within the baby lung (elastance-derived plateau p l ) [23] . monitoring and controlling respiratory muscle effort are major challenges in implementing lung and diaphragm-protective mechanical ventilation. the gold standard to quantify global respiratory muscle effort is the esophageal pressure-time product (ptp), while the ptp of the transdiaphragmatic pressure (pdi, i.e., difference between gastric pressure (pga) and pes) during inspiration provides a measure of diaphragmatic effort [24] . the amplitude of pes or pdi during tidal breathing provides a simple estimate of the pressure generated by all respiratory muscles (pes), or the diaphragm (pdi), whereas the expiratory increase in pga reflects expiratory muscle activity. the diaphragm electrical activity (eadi) is the most precise surrogate of respiratory drive and correlates with indices of effort [25] but with considerable variability between patients. also, values for peak eadi in young healthy subjects during tidal breathing may vary between 4 and 29 μv [26] . nevertheless, changes in eadi are useful to monitor changes in patient's respiratory drive and effort, especially to identify patients at risk for ventilator over-assistance. finally, pes or eadi can complement ventilator waveform analysis to facilitate the identification of patient-ventilator dyssynchronies. other less invasive techniques are available to monitor patient breathing efforts during mechanical ventilation at the bedside. airway occlusion pressure (p 0.1 ), the deflection in paw during the first 0.1 s of an inspiratory effort against an occluded airway, is an estimate of the respiratory drive and can be used to detect both very low and high effort [27] . the maximum deflection of paw during a whole breath occlusion (δpocc) has been recently shown to accurately detect excessive respiratory muscle pressure (pmus) or δp l,dyn ; this maneuver can also be used to assess different forms of patient-ventilator dyssynchrony [28, 29] . ultrasound can be used to visualize and quantify the thickening of the diaphragm during inspiration in the zone of apposition (thickening fraction, tfdi) [30] . tfdi provides an index of diaphragmatic contractility and correlates reasonably well with inspiratory effort (δpes) and eadi [31] . in conclusion, although estimation of pleural pressure using an esophageal balloon appears to be the preferred technique to quantify lung stress and respiratory effort, the technique is currently not widely implemented; moreover, the potential impact on patient outcome remains to be determined in clinical studies. we suggest routine monitoring of tidal volume, inspiratory plateau pressures and airway driving pressure to limit lung injury, and p0.1 to monitor respiratory drive and prevent inadequate effort (table 1) . several strategies can be used to facilitate lung and diaphragm protective ventilation, including modulation of ventilator inspiratory and expiratory assist, drugs that modify respiratory drive and/or effort, extracorporeal co 2 removal (ecco 2 r) and electrical stimulation of the respiratory muscles, as shown in fig. 2 . here, we will briefly discuss these different strategies. a lung and diaphragm-protective ventilation approach aims to minimize lung stress and strain while limiting diaphragm atrophy and injury. to achieve these goals, inspiratory ventilator settings can be adjusted to (1) modulate the patient's inspiratory effort, (2) minimize the dynamic lung stress, and (3) prevent or correct patient-ventilator dyssynchrony or any form of mismatch between needs and support. titrating the inspiratory ventilator settings to optimize respiratory effort requires a thorough understanding of the control of breathing under mechanical ventilation [32, 33] , acknowledging that the control of breathing system responds to changes in ventilatory demands by modifying inspiratory effort (and thus tidal volume) to a greater extent than respiratory rate [34] . therefore, the inspiratory ventilator settings will affect the inspiratory effort by modifying the delivered tidal volume, and thus, in spontaneously breathing patients, increasing pressure or volume assist will increase the delivered tidal volume and reduce the inspiratory effort (as respiratory drive depends mainly on the chemoreflex control of arterial ph). excessive assist, resulting in a tidal volume that is higher than the patient's demands, may almost abolish the patient's the inspiratory effort, and as such promote diaphragmatic atrophy. however, increasing inspiratory support may not attenuate inspiratory effort in the presence of high respiratory drive due to stimuli other than arterial ph/paco 2 , such as pain, anxiety, or stimulation of peripheral lung receptors by lung edema or inflammation [32] . in such case, transpulmonary pressure (and hence dynamic lung stress) may progressively increase with increasing inspiratory support. increasing fio 2 to increase pao 2 and reduce the hypoxic stimulus to breathe may alleviate increased respiratory drive in some patients (hyperoxemia is not required to achieve this effect) [35] . in a volume-targeted mode, the patient's effort will be modified mainly by the set tidal volume and the flow delivery profile (flow pattern and peak flow). in pressuretargeted modes, the delivered tidal volume, and thus the patient's inspiratory effort, is influenced by the set inspiratory pressure, rise time and cycling-off criterion, and of course the mechanical properties of the respiratory system [36] . irrespective of the mode of assist, the delivered tidal volume and respiratory effort will together determine global and regional lung stress, depending on the mechanical properties of the respiratory system [37] . neurally adjusted ventilatory assist (nava) delivers inspiratory assist proportional to the electrical activity of the diaphragm [38] . increasing inspiratory assist will reduce diaphragm electrical activity (and vice versa) over a wide range of respiratory demand, and consequently tidal volume remains relatively stable over a wide range of assist [39] . in theory, pulmonary reflex mechanisms prevent patients from spontaneously inspiring large tidal volumes and nava may therefore facilitate lung-protective ventilation. also, diaphragm inactivity due to overassistance is unlikely in nava, as low diaphragm activity will immediately reduce inspiratory assist. future studies should confirm the role of nava in lung and diaphragmprotective ventilation, but recent randomized trials suggest clinical benefit of nava (reduced time on the ventilator) compared to pressure support mode [40, 41] . the expiratory ventilator setting (i.e., positive end-expiratory pressure, peep) has been traditionally adjusted to optimize oxygenation and/or lung mechanics [42, 43] . a higher peep ventilation strategy (of which there are several, generally resulting in 15 ± 4 cmh 2 o) is currently recommended over lower peep (approximately 9 ± 3 cmh 2 o) in moderate and severe ards [44] . in the presence of spontaneous breathing during mechanical ventilation, a higher peep strategy offers several additional potential advantages to facilitate lung and diaphragm-protective ventilation (fig. 1) . first, in patients with significant lung recruitability, peep reduces the amount of atelectatic 'solid-like' lung and, therefore, can achieve a more homogeneous distribution of the tidal pleural pressure swing (∆ppl) over the whole lung surface following a diaphragmatic contraction. the even distribution of inspiratory dynamic stress can diminish injurious asymmetric inflation associated with spontaneous effort (i.e., pendelluft), reducing regional lung stress in dependent lung regions [45] . second, by increasing endexpiratory lung volume, forcing the diaphragm to operate at a shorter length and thereby impairing diaphragm neuromuscular coupling [46, 47] , increased peep can attenuate the force generated by diaphragmatic contraction [48] . indeed, several clinical studies provide indirect evidence to suggest that higher peep may render spontaneous effort less injurious in patients with acute respiratory failure before intubation [49] , in patients with ards [45, 50] , and in pediatric patients with lung injury [51] . on the other hand, preliminary experimental evidence suggests that if the diaphragm is maintained at a shorter length during acute mechanical ventilation, the diaphragm muscle fibers could adapt to the reduced length by absorbing sarcomeres in series (i.e., longitudinal atrophy) [52] . this may result in fibers overstretching with the release of peep during a t-tube weaning trial or after extubation. the possibility of diaphragm weakness resulting from excess peep should therefore be borne in mind. patient-ventilator dyssynchronies may cause lung and/or diaphragm injury by increasing dynamic lung stress and/ or injurious diaphragmatic contractions, respectively. dyssynchronies may occur during inspiration (flow starvation, short cycles, prolonged insufflation and reverse triggering), during expiration (auto-triggering, ineffective effort) or both during inspiration and expiration (reverse triggering and double triggering). we will briefly discuss dyssynchronies most relevant for lung and diaphragmprotective ventilation; for more extensive discussion of dyssynchronies we refer to other reviews [53] . reverse triggering, a diaphragmatic contraction triggered by mechanical inflation, is common in fully sedated patients (in whom drive is abolished) [54] . reverse triggering can induce breath stacking resulting in excessive tidal volumes and high dynamic lung stress [55] , and it may create eccentric diaphragm loading conditions with resultant muscle injury [56] . when necessary to avoid breath stacking, reverse triggering can be abolished by neuromuscular blocking agents. alternatively, the development of reverse triggering may indicate that sedation should be stopped to allow the patient to take control of ventilation. in patients with relatively high respiratory drive and a low respiratory system time constant, the neural inspiration time may exceed the mechanical inflation (premature cycling). in such cases, the contraction of the inspiratory muscles continues during mechanical expiration and the diaphragm is forced to contract while lengthening (eccentric contraction). in volume-targeted modes, unmet high demands appear as 'flow-starvation' , a downward curvature of inspiratory paw, and the patient may experience dyspnea and distress, which can be resolved by increasing inspiratory flow rate using a decelerating flow pattern. strong inspiratory efforts may result in double-triggering, breath stacking and, therefore, delivery of high tidal volumes. a better match of mechanical and neural inspiratory time can be achieved by increasing ventilator inspiratory time and using a decelerating flow pattern in volume-assist control mode, by decreasing the cycling-off criterion in pressure support mode, or using proportional modes of assist. importantly, in patients with high respiratory drive, modification of inspiratory time may not suffice to resolve dyssynchrony. increasing the level of assist to match the patient's demands should be considered, but, if that results in an injurious high ventilation, other means to decrease the patient's respiratory drive, such as sedation, may be required. another dyssynchrony occurring in patients with absent or low respiratory drive is auto-triggering, i.e., the delivery of a ventilator-assisted breath in the absence of patient effort. auto-triggering due to strong cardiac oscillations transmitted to the paw or airflow signal is more likely to occur when the respiratory system time constant is low, such as in ards. air leaks and moisture in the ventilator circuit are also common causes of auto-triggering. ineffective triggering (or ineffective efforts) develops when a patient's effort fails to trigger a ventilator-delivered breath. ineffective triggering is generally the consequence of weak inspiratory efforts, either from low respiratory drive due to sedation, metabolic alkalosis or excessive ventilatory assist, or because of diaphragm weakness. when the respiratory system time constant is high, (i.e., obstructive lung disease), ventilator over-assistance results in delayed cycling, dynamic hyperinflation, and increased intrinsic peep, predisposing to ineffective triggering. decreasing the level of assist can therefore alleviate ineffective efforts [57] . over-assistance in assisted ventilation can also induce apneas during sleep. interestingly, several studies have demonstrated that nava improves patient-ventilator interaction, especially reducing the risks of ineffective efforts and over-assist [39, 58] . whether the reduced duration of mechanical ventilation reported in some nava trials [40, 41] results from improved patient-ventilator interaction remains to be investigated. sedation can facilitate lung and diaphragm-protective ventilation by ameliorating, when present, excessive respiratory effort. complete suppression of respiratory drive and effort with sedation can also contribute to diaphragm disuse atrophy. a judicious approach to sedation is key and monitoring of respiratory drive and effort may be helpful in selecting the sedation strategy that facilitates lung and diaphragm-protective ventilation. before administering sedation to address excessive respiratory drive or ventilator dyssynchrony, ventilator settings should be adjusted and other factors increasing respiratory drive such as metabolic acidosis or pain should be addressed. relying on sedation alone to enhance patient-ventilator interaction without addressing these issues can paradoxically exacerbate dyssynchrony, prolong mechanical ventilation, and exacerbate diaphragm dysfunction [59] . recent clinical practice guidelines have recommended an "analgesia-first approach" to minimize the risk of excessive sedation as opioids during mechanical ventilation were associated with less dyssynchrony and depressed consciousness in comparison to sedative-based approaches [60] . nevertheless, when elevated respiratory drive cannot otherwise be resolved, sedatives can attenuate the ventilatory response to hypoxemia and hypercapnia and cortical input to the respiratory centres [33] (table 2) . propofol and benzodiazepines are gamma-aminobutyric acid (gaba) agonists known to cause respiratory depression, primarily by reducing the amplitude of respiratory effort [61] [62] [63] . because benzodiazepines are associated with a high risk of delirium and prolonged mechanical ventilation [64] , propofol is the preferred sedative of choice for controlling high respiratory drive. because propofol or benzodiazepines reduce the amplitude of inspiratory effort, ineffective triggering may develop as sedation depth increases [61] . inhalational sedation offers a potential alternative for controlling respiratory effort though clinical experience is limited to date [65] . to avoid excessive sedation, strategies aimed at active titration of sedatives or daily interruption of sedation should be employed and respiratory drive and effort should be monitored closely. for patients without excessive breathing effort (table 2) , a multimodal analgesia approach that minimizes opiate use is recommended to avoid diaphragm inactivity. dexmedetomidine is a selective alpha-2 agonist which, in contrast to propofol and benzodiazepines, provides sedation, anxiolysis, and analgesia without respiratory depression [66] . this property makes it an interesting drug of choice to preserve awareness and diaphragm contractility and at the same time limiting excess delirium risk in agitated patients without elevated respiratory drive. the prone position has been used for decades in early ards to improve oxygenation and over time an appreciation for the lung-protective benefit of prone positioning has emerged [67] . as the amount of lung tissue is larger in dorsal lung regions, gravitational forces generate more dependent atelectasis in the supine position compared to prone position. therefore, ventilation-perfusion matching is improved in the prone position and, more importantly, the energy applied to the lung by mechanical ventilation is distributed among more (non-atelectatic) alveoli, reducing lung stress. this is the putative basis for the observed mortality benefit of prone positioning in patients with ards [68] . the mechanistic benefits of prone positioning may also apply under assisted ventilation with spontaneous breathing, because the lung recruitment accrued by prone positioning may attenuate 'solid-like' lung behaviour and reduce effort-dependent regional lung stress. prone positioning improves oxygenation in spontaneously breathing patients with covid-19 pneumonia [69] ; it is possible that prone positioning could also reduce the risk of patient self-inflicted lung injury [70] . thus, prone positioning might facilitate safe spontaneous breathing and diaphragm-protective ventilation as well as lung protection. eliminating co 2 is the primary purpose of alveolar ventilation. ecco 2 r reduces the ventilatory demands, decreasing the respiratory effort, and thus may ameliorate dynamic lung stress. ecco 2 r is feasible and effective in reducing tidal volume, driving pressure, and mechanical power in patients with ards [71] . in spontaneously breathing patients, ecco 2 r can dampen respiratory drive and effort [72] , theoretically reducing the requirement for ventilatory support or sedation to control respiratory effort. karagiannidis et al. showed that increasing sweep gas flow, increasing co 2 elimination, in ards patients undergoing extracorporeal membrane oxygenation (ecmo) reduced respiratory drive, estimated by eadi [73] . mauri et al. [7] also showed that higher ecco 2 r support reduced p 0.1 , respiratory muscle effort, and transpulmonary pressure in spontaneously breathing patients recovering from severe ards [74] . pilot clinical studies have explored the extreme possibility of extubating severe ards patients early after intubation by means of ecco 2 r: preliminary results were encouraging but they also recognized the need to identify the subgroup of patients with a high probability of success [75] [76] [77] . despite the appeal and physiological rationale of this strategy, there are relevant limitations. first, in some patients, non-chemoreceptive stimuli (pain, agitation, discomfort, metabolic acidosis, lung mechanical stimuli) may predominate and high respiratory drive may persist despite ecco 2 r [78] . second, ecco 2 r requires full anticoagulation and the risk of bleeding is not insubstantial [79] . third, the application of ecco 2 r may exacerbate hypoxemia by various mechanisms [80] . complete neuromuscular blockade may increase the risk for diaphragm disuse atrophy and increases sedation requirements. low-dose neuromuscular blockers ("partial neuromuscular blockade") is an interesting compromise between total paralysis and strenuous breathing efforts, particularly when respiratory effort does inadequately respond to titration of ventilatory support or sedation. the feasibility of partial neuromuscular blockade has been evaluated in a proof of concept study in patients with moderate ards and high respiratory drive on partially supported modes [81] . titration of rocuronium decreased tidal volume from approximately 9 ml/ kg to approximately 6 ml/kg while maintaining pdi at approximately 5 cmh 2 o (within the physiological range for diaphragm activity in healthy subjects). these preliminary findings suggest that partial neuromuscular blockade could be a feasible approach to achieving lung and diaphragm-protective ventilation targets in patients with high respiratory effort. importantly, partial neuromuscular blockade does not reduce respiratory drive, but only the mechanical consequences of high drive. this dissociation between central drive and respiratory muscle mechanical output may result in dyspnea [33] ; adequate relief of dyspnea and distress must be ensured by judicious application of sedatives and opioids. future clinical studies should confirm the safety and efficacy of prolonged partial neuromuscular blockade in ventilated patients. neuromuscular stimulation ("pacing") uses electrical currents to generate muscle contraction in the absence of volitional efforts, making it an attractive intervention in incapacitated critically ill patients. there is growing interest in neuromuscular stimulation as a novel strategy to preserve or restore respiratory muscle activity and, in turn, to prevent or treat icu-acquired diaphragm weakness. in addition, by inducing diaphragm contractions, neuromuscular stimulation may improve lung aeration of dependent lung regions [82] . pacing must be synchronized with the ventilator and potentially injurious inspiratory efforts must be avoided. there is as yet no clinical evidence of benefit from diaphragm pacing in icu patients. direct stimulation of the phrenic nerves by surgically implanted electrodes has been employed to restore spontaneous ventilation in patients with high-level spinal cord injury and central hypoventilation syndrome [83] . the feasibility of direct pacing using temporary implanted electrodes for the prevention of diaphragm dysfunction is currently under investigation in cardiac surgery patients identified to be at risk for prolonged mechanical ventilation (nct04309123). preclinical work showed that this technique could reduce the development of diaphragm type ii fiber atrophy [84, 85] . recently, reynolds et al. presented a first-in-human series of temporary transvenous phrenic nerve pacing in surgical patients and showed that this technology delivered safe and effective diaphragm contractions [86] . this strategy is currently being studied as potential intervention for improving diaphragm strength in difficult-to-wean patients (nct03096639). the role of transvenous phrenic nerve pacing for the prevention of diaphragm disuse atrophy remains to be investigated. neuromuscular stimulation strategies targeting the expiratory muscles of icu patients are less well studied. this is surprising, as stimulation of the expiratory abdominal wall muscles can be employed noninvasively via surface electrodes placed over the abdominal wall. feasibility of a breath-synchronized expiratory muscle stimulation technique during the early phase of mechanical ventilation was recently demonstrated with promising results [87] and its efficacy is under investigation (nct03453944). clinicians caring for mechanically ventilated patients are generally well aware of the risk of causing barotrauma, volutrauma, and atelectrauma. given the mounting evidence of clinically important diaphragm atrophy and injury, consideration must also be given to protecting the diaphragm. based on the foregoing discussion about ventilation and sedation, a basic algorithm and approach to lung and diaphragm-protective ventilation is presented in fig. 3 . clinical trials testing new ventilation algorithms and sedation strategies targeted at optimizing respiratory effort are required to confirm the benefit of the lung and diaphragm-protective approach outlined in this paper. the benefit of adjunctive strategies such as ecco 2 r, partial neuromuscular blockade and phrenic nerve stimulation requires further evaluation, in particular to identify the subpopulations of patients most likely to benefit from these more costly and invasive interventions. for the present, we encourage clinicians to incorporate routine monitoring of respiratory drive and effort in their clinical practice and to adjust the ventilator to achieve a physiological level of effort where possible while carefully attending to the effect on lung stress. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by-nc/4.0/. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. received: 2 september 2020 accepted: 8 october 2020 fig. 3 clinical-physiological pathway for achieving lung and diaphragm-protective ventilation targets. it should be stressed that at each step clinical evaluation of the patient, including signs of high breathing effort, agitation, and over-sedation is of major importance and should be interpreted together with clinical-physiological measurements as outlined in this pathway. δp: change in airway pressure during inspiration; p 0.1 : decrease in airway pressure during the first 100 ms of inspiratory effort against an occluded airway; paco 2 : arterial carbon dioxide tension; peep: positive endexpiratory pressure; pes: esophageal pressure; p l : transpulmonary pressure; pocc: airway pressure deflection during a whole breath occlusion; rr: respiratory rate; v t : tidal volume ventilator-induced lung injury mechanical ventilation to minimize progression of lung injury in acute respiratory failure lung stress and strain during mechanical ventilation for acute respiratory distress syndrome volume delivered during recruitment maneuver predicts lung stress in acute respiratory distress syndrome driving pressure and survival in the acute respiratory distress syndrome prevalence of complete airway closure according to body mass index in acute respiratory distress syndrome: pooled cohort analysis expiratory muscle dysfunction in critically ill patients: towards improved understanding mechanical ventilation-induced diaphragm atrophy strongly impacts clinical outcomes diaphragm dysfunction on admission to the intensive care unit. prevalence, risk factors, and prognostic impact-a prospective study coexistence and impact of limb muscle and diaphragm weakness at time of liberation from mechanical ventilation in medical intensive care unit patients critical illnessassociated diaphragm weakness rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans diaphragm muscle fiber weakness and ubiquitin-proteasome activation in critically ill patients rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans evolution of diaphragm thickness during mechanical ventilation. impact of inspiratory effort lung and diaphragm-protective ventilation driving pressure is associated with outcome during assisted ventilation in acute respiratory distress syndrome driving pressure during proportional assist ventilation: an observational study airway pressure morphology and respiratory muscle activity during end-inspiratory occlusions in pressure support ventilation esophageal and transpulmonary pressure in the clinical setting: meaning, usefulness and perspectives spontaneous effort causes occult pendelluft during mechanical ventilation understanding spontaneous vs. ventilator breaths: impact and monitoring esophageal manometry and regional transpulmonary pressure in lung injury monitoring of the respiratory muscles in the critically ill estimation of patient's inspiratory effort from the electrical activity of the diaphragm information conveyed by electrical diaphragmatic activity during unstressed, stressed and assisted spontaneous breathing: a physiological study airway occlusion pressure as an estimate of respiratory drive and inspiratory effort during assisted ventilation monitoring patient-ventilator interaction by an end-expiratory occlusion maneuver a novel non-invasive method to detect excessively high respiratory effort and dynamic transpulmonary driving pressure during mechanical ventilation respiratory muscle ultrasonography: methodology, basic and advanced principles and clinical applications in icu and ed patients-a narrative review measuring diaphragm thickness with ultrasound in mechanically ventilated patients: feasibility, reproducibility and validity physiology of the respiratory drive in icu patients: implications for diagnosis and treatment respiratory drive in critically ill patients. pathophysiology and clinical implications the injurious effects of elevated or nonelevated respiratory rate during mechanical ventilation effects of short-term oxygenation changes on acute lung injury patients undergoing pressure support ventilation effects of pressure ramp slope values on the work of breathing during pressure support ventilation in restrictive patients volume-controlled ventilation does not prevent injurious inflation during spontaneous effort neural control of mechanical ventilation in respiratory failure comparison between neurally adjusted ventilatory assist and pressure support ventilation levels in terms of respiratory effort neurally adjusted ventilatory assist versus pressure support ventilation in difficult weaning: a randomized trial neurally adjusted ventilatory assist in acute respiratory failure: a randomized controlled trial fifty years of research in ards. setting positive end-expiratory pressure in acute respiratory distress syndrome optimum end-expiratory airway pressure in patients with acute pulmonary failure an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome high positive end-expiratory pressure renders spontaneous effort noninjurious mechanics of the human diaphragm during voluntary contraction: statics diaphragmatic neuromechanical coupling and mechanisms of hypercapnia during inspiratory loading mechanics of the diaphragm effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial high positive end-expiratory pressure allows extubation of an obese patient pendelluft detection using electrical impedance tomography in an infant. keep those images in mind positive end-expiratory pressure ventilation induces longitudinal atrophy in diaphragm fibers mechanical ventilation: state of the art mechanical ventilationinduced reverse-triggered breaths: a frequently unrecognized form of neuromechanical coupling quantifying unintended exposure to high tidal volumes from breath stacking dyssynchrony in ards: the breathe criteria diaphragmatic myotrauma: a mediator of prolonged ventilation and poor patient outcomes in acute respiratory failure asynchrony consequences and management neurally adjusted ventilatory assist as an alternative to pressure support ventilation in adults: a french multicentre randomized trial observational study of patient-ventilator asynchrony and relationship to sedation level clinical practice guidelines for the prevention and management of pain, agitation/sedation, delirium, immobility, and sleep disruption in adult patients in the icu effects of propofol on patient-ventilator synchrony and interaction during pressure support ventilation and neurally adjusted ventilatory assist effects of propofol on respiratory drive and patient-ventilator synchrony during pressure support ventilation in postoperative patients: a prospective study respiratory depression by midazolam and diazepam lorazepam is an independent risk factor for transitioning to delirium in intensive care unit patients inhalational volatile-based sedation for covid-19 pneumonia and ards effects of intravenous dexmedetomidine in humans. i. sedation, ventilation, and metabolic rate prone positioning in acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome use of prone positioning in nonintubated patients with covid-19 and hypoxemic acute respiratory failure is the prone position helpful during spontaneous breathing in patients with covid-19? determinants of the effect of extracorporeal carbon dioxide removal in the supernova trial: implications for trial design control of breathing using an extracorporeal membrane lung autoregulation of ventilation with neurally adjusted ventilatory assist on extracorporeal lung support control of respiratory drive and effort in extracorporeal membrane oxygenation patients recovering from severe acute respiratory distress syndrome spontaneous breathing during extracorporeal membrane oxygenation in acute respiratory failure extracorporeal membrane oxygenation instead of invasive mechanical ventilation in patients with acute respiratory distress syndrome extracorporeal membrane oxygenation can successfully support patients with severe acute respiratory distress syndrome in lieu of mechanical ventilation spontaneous breathing patterns during maximum extracorporeal co2 removal in subjects with early severe ards european society of intensive care medicine trials group and the "strategy of ultra-protective lung ventilation with extracorporeal co2 removal for new-onset moderate to severe ards" (supernova) investigators (2019) feasibility and safety of extracorporeal co2 removal to enhance protective ventilation in acute respiratory distress syndrome: the supernova study understanding hypoxemia on ecco2r: back to the alveolar gas equation partial neuromuscular blockade during partial ventilatory support in sedated patients with high tidal volumes effects of anesthesia and paralysis on diaphragmatic mechanics in man diaphragm pacing: the state of the art can phrenic stimulation protect the diaphragm from mechanical ventilation-induced damage? mitigation of ventilatorinduced diaphragm atrophy by transvenous phrenic nerve stimulation diaphragm activation in ventilated patients using a novel transvenous phrenic nerve pacing catheter abdominal functional electrical stimulation to assist ventilator weaning in critical illness: a double-blinded, randomised, sham-controlled pilot study key: cord-323566-jck799zq authors: cheung, oi-yee; graziano, paolo; smith, maxwell l. title: acute lung injury date: 2017-11-05 journal: practical pulmonary pathology: a diagnostic approach doi: 10.1016/b978-0-323-44284-8.00006-5 sha: doc_id: 323566 cord_uid: jck799zq a wide variety of insults can produce acute lung damage, inclusive of those that injure the lungs directly. the clinical syndrome of acute onset respiratory distress, dyspnea, and bilateral infiltrates is referred to as acute respiratory distress syndrome. the histologic counterpart of acute respiratory distress syndrome is diffuse alveolar damage, classically characterized by hyaline membranes. other histologic features of acute lung injury include intraalveolar fibrin, organization, interstitial edema, and reactive pneumocytes. diffuse alveolar damage and other histologic features of acute lung injury are nonspecific as to etiology, and once identified require the pathologist to search the biopsy for further features that may help identify a specific etiology. this chapter reviews the temporal sequence of acute lung injury and explores the large variety of specific etiologic causes with emphasis on helpful histologic features to identify. resultant endothelial and alveolar epithelial cell injury is attended by fluid and cellular exudation. subsequent reparative fibroblastic proliferation is accompanied by type ii pneumocyte hyperplasia. 4, 10 the microscopic appearance depends on the time interval between insult and biopsy and on the severity and extent of the injury. 2 dad is the usual pathologic manifestation of ards and is the best-characterized prototype of acute lung injury. from studies of ards, the pathologic changes appear to proceed consistently through discrete but overlapping phases ( fig. 6 .1)-an early exudative (acute) phase ( fig. 6.2a and b) , a subacute proliferative (organizing) phase ( fig. 6.2c) , and a late fibrotic phase ( fig. 6. 3). 2, 4, 5, 9, 11 the exudative phase is most prominent in the first week of injury. the earliest changes include interstitial and intraalveolar edema with variable amounts of hemorrhage and fibrin deposition ( fig. 6.4 ). hyaline membranes (fig. 6 .5), the histologic hallmark of the exudative phase of ards, are most prominent at 3 to 7 days after injury (eslide 6.1). minimal interstitial mononuclear inflammatory infiltrates ( fig. 6.6 ) and fibrin thrombi in small pulmonary arteries (fig. 6 .7) also are seen. type ii pneumocyte hyperplasia ( fig. 6.8 ) begins by the end of this phase and persists through the proliferative phase. the reactive type ii pneumocytes may demonstrate marked nuclear atypia, with numerous mitotic figures (fig. 6.9 ). the proliferative phase begins at 1 week after the injury and is characterized by fibroblastic proliferation, seen mainly within the interstitium but also focally in the alveolar spaces ( fig. 6.10 ). the fibrosis consists of loose aggregates of fibroblasts admixed with scattered inflammatory cells, reminiscent of organizing pneumonia acute interstitial pneumonia (hammanin experimental ards, the exact time of injury is known, and the entire lung proceeds through the phases at the same time. in a patient who develops diffuse alveolar damage from any cause, the acute lung injury may begin in different areas at different times, so a biopsy specimen may demonstrate injury at various phases in this sequence. ( in ards the inciting event is frequently extrathoracic, and lung injury is therefore superimposed on normal preexisting structure. a b figure 6 .7 acute respiratory distress syndrome: fibrin thrombi in arteries. acute lung injury results in local conditions that lead to arterial thrombosis. thrombi in various stages of organization may be seen (larger pulmonary artery in part a, smaller pulmonary artery in part b). ( fig. 6 .11); collagen deposition is minimal. reactive type ii pneumocytes persist. immature squamous metaplasia may occur ( fig. 6 .12) in and around terminal bronchioles. the degree of cytologic atypia in this squamous epithelium can be so severe as to mimic malignancy ( fig. 6 .13). the hyaline membranes are mostly resorbed by the late proliferative stage, but a few remnants may be observed along alveolar septa. some cases of dad resolve completely, with few residual morphologic effects, but in other cases, fibrosis may progress to extensive structural remodeling and honeycomb lung. as might be expected, a review of outcomes for 109 survivors of ards revealed persistent functional disability at 1 year after discharge from intensive care. 12 by definition, ards has a known inciting event. the foregoing description is based on a model of ards due to oxygen toxicity, wherein the evolution of histopathologic abnormalities can be studied over a defined time period. 2, 5 in practice, lung biopsy most often is performed in patients without a known cause or specific time of onset of injury. moreover, with some causes of acute lung injury, the damage evolves over a protracted period of time, or the lung may be injured in repetitive fashion (e.g., with drug toxicity). in such circumstances, the pathologic changes do not necessarily progress sequentially through defined stages as in ards, so both acute and organizing phases may be encountered in the same biopsy specimen. the basic histopathologic elements of acute lung injury are presented in box 6.2. acute fibrinous and organizing pneumonia (afop) is a histologic pattern of acute lung injury with a clinical presentation similar to that of classic dad, in terms of both potential etiologic disorders and outcome. it differs from dad in that hyaline membranes are absent. the dominant feature is intraalveolar fibrin balls or aggregates, typically in a patchy distribution. organizing pneumonia in the form of luminal loose fibroblastic tissue is present surrounding the fibrin (eslide 6.2). the alveolar septa adjacent to areas of fibrin deposition show a variety of changes similar to those of dad, such as septal edema, type ii pneumocyte hyperplasia, and acute and chronic inflammatory infiltrates. the intervening lung shows minimal histologic changes. afop may represent a fibrinous variant of dad. in some patients, both dad and afop disease patterns may be present simultaneously. 13, 14 specific causes of acute lung injury infection infection is one of the most common causes of acute lung injury. if the lung injury pattern is accompanied by a significant increase in neutrophils, areas of necrosis, viral cytopathic effect, and/or granulomas, infection should lead the differential diagnosis. among infectious organisms, viruses most consistently produce dad. 2, 5 occasionally, fungi (e.g., pneumocystis) and bacteria (e.g., legionella) also can cause infections manifesting as dad. some of the organisms that are well known to cause acute lung injury with characteristic histopathologic changes are discussed next. considerable structural remodeling may take place after ards as these atelectatic spaces fuse to form consolidated areas of lung parenchyma at the microscopic level. influenza is a common cause of viral pneumonia. the histopathology ranges from mild organizing acute lung injury (resembling organizing pneumonia) in nonfatal cases to severe dad with necrotizing tracheobronchitis ( fig. 6 .14) in fatal cases. 15, 16 specific viral cytopathic effects are not identifiable by light microscopy. on ultrastructural examination, intranuclear fibrillary inclusions may be seen in epithelial and endothelial cells. 17 the coronavirus responsible for severe acute respiratory syndrome produces the acute lung injury associated with this disorder. 13, [18] [19] [20] both dad and afop patterns have been identified in affected patients. on ultrastructural examination, involved lung tissue revealed numerous to moderate numbers of cytoplasmic viral particles in pneumocytes, many within membrane-bound vesicles. [21] [22] [23] the virus particles were spherical and enveloped, with spikelike projections on the surface and coarse clumps of electron-dense material in the center. most had sizes ranging from 60 to 95 nm in diameter, but some were as large as 180 nm. measles virus produces a mild pneumonia in the normal host but can cause serious pneumonia in immunocompromised children. adenovirus is an important cause of lower respiratory tract disease in children, 29, 30 although adults (particularly those who are immunocompromised) 31 and military recruits also are occasionally affected. 32 the lung shows necrotizing bronchitis, or bronchiolitis, accompanied by dad. the pathologic changes are more severe in bronchi, bronchioles, and peribronchiolar regions ( fig. 6.16a ). two types of inclusions can be observed in lung epithelial cells: an eosinophilic intranuclear inclusion with a halo usually is less conspicuous than the more readily identifiable "smudge cells" (see fig. 6 .16b). these latter cells are larger than normal and entirely basophilic, with no defined inclusion or halo evident by light microscopy. 29 on ultrastructural examination, smudge cell inclusions are represented by arrays of hexagonal particles. 33 herpes simplex virus is mainly a cause of respiratory infection in the immunocompromised host. two patterns of infection are recognized: airway spread resulting in necrotizing tracheobronchitis ( fig. 6 .17) and bronchitis and bronchiolitis, and dad. 24 the characteristic histologic feature is the presence of multinucleated giant cells (fig. 6 .15a) with characteristic eosinophilic intranuclear and intracytoplasmic inclusions. [24] [25] [26] [27] [28] these cells are found in the alveolar spaces and within alveolar septa (fig. 6.15b ). viral inclusions are seen on ultrastructural examination as tightly packed tubules. 28 interstitial (alveolar septal) edema fibroblastic proliferation in alveolar septa alveolar edema alveolar fibrin and cellular debris, with or without hyaline membranes reactive type ii pneumocytes blood-borne dissemination producing miliary necrotic parenchymal nodules. dad and hemorrhage can occur in both forms. 34, 35 characteristic inclusions may be seen in bronchial and alveolar epithelial cells ( fig. 6 .18). the more obvious type is an intranuclear eosinophilic inclusion surrounded by clear halo (cowdry a inclusion), and the other is represented by a basophilic to amphophilic ground-glass nucleus (cowdry b inclusion). rounded viral particles with double membranes are seen under the electron microscope. 34, 35 varicella-zoster virus causes disease predominantly in children and is the agent of chickenpox. 36 pulmonary complications of chickenpox are rare in children with normal immunity (accounting for less than 1% of the cases). by contrast, pneumonia develops in 15% of adults with chickenpox; immunocompetent and immunocompromised persons are equally affected. 32, 36 the histopathologic picture in varicella pneumonia ( fig. 6.19 ) is similar to that in herpes simplex. although identical intranuclear inclusions are reported to occur, 32, 36 these can be considerably more difficult to identify in chickenpox pneumonia. cytomegalovirus is an important cause of symptomatic pneumonia in immunocompromised persons, especially those who have received bone marrow or solid organ transplants, and in patients with human immunodeficiency virus infection. [37] [38] [39] the histopathologic findings range from little or no inflammatory response to hemorrhagic nodules with necrosis ( fig. 6 .20a) and dad. 37 the diagnostic histopathologic b a with many organisms (see fig. 6 .22b). 44, 45 however, in the mildly immunocompromised patient this feature is not observed or the pathologic changes may be subtle. in such cases, several "atypical" manifestations have been described. 43, 45, 46 dad is the most dramatic of these atypical presentations ( fig. 6 .23a), with the organisms present within hyaline membranes ( fig. 6 .23b) and in isolated intraalveolar fibrin deposits. 46 the grocott methenamine silver (gms) method is routinely used to stain the organisms, which typically are seen in small groups and clusters (figs. 6.22b and 6.23b). 43, 45, 46 bacterial infection common bacterial pneumonias rarely cause dad; however, this lung injury pattern has been described in legionnaires' disease, mycoplasma pneumonia, and rickettsial infection. [47] [48] [49] [50] [51] pattern, seen in endothelial cells, macrophages, and epithelial cells, consists of cellular enlargement, a prominent intranuclear inclusion, and an intracytoplasmic basophilic inclusion ( fig. 6 .20b). 37 hantavirus is a rare cause of acute lung injury. [40] [41] [42] the infection produces alveolar edema, hyaline membranes, and atypical interstitial mononuclear inflammatory infiltrates (fig. 6.21 ). [40] [41] [42] spherical membrane-bound viral particles have been found in the cytoplasm of endothelial cells by electron microscopy. pneumocystis jiroveci (previously known as pneumocystis carinii) is the most common fungus to cause dad. [43] [44] [45] the histopathology of pneumocystis infection in the setting of profound immunodeficiency is one of frothy intraalveolar exudates ( [afb] stains or gms or warthin-starry silver stain, etc.) on every lung biopsy specimen exhibiting dad. systemic connective tissue disorders are a well-known cause of diffuse lung disease. [52] [53] [54] [55] [56] [57] [58] [59] in some cases, lung involvement may be the first manifestation of the systemic disease, even without identifiable serologic evidence. 57 histologic clues that suggest the acute lung injury is secondary to connective tissue disease include associated bronchiolitis (especially if it is follicular bronchiolitis), pleuritis, capillaritis, hemorrhage, and legionella is a fastidious gram-negative bacillus that causes acute respiratory infection in older adults and immunodeficient individuals. 47, 48, 51 the histopathologic pattern is that of a pyogenic necrotizing bronchopneumonia ( fig. 6 .24a) affecting the respiratory bronchioles, alveolar ducts, and adjacent alveolar spaces. dad is common. 47, 48, 51 the rod-shaped organisms (fig. 6 .24b) can be identified by dieterle silver stain. 51 of note, in immunocompromised patients, any type of infection can cause dad, with pneumocystis pneumonia being the most common. 28 for this reason, it is essential to use special stains (acid-fast bacilli and small vessel vasculitis ( fig. 6 .25b), and pulmonary edema also may be observed. 52, 57, 60 immunofluorescence studies demonstrate immune complexes in lung parenchyma, and both immune complexes and tubuloreticular inclusions may be seen on ultrastructural examination. 57, 58, 60 rheumatoid arthritis a significant percentage of patients with rheumatoid arthritis have lung disease. 53, 54, [61] [62] [63] [64] many different morphologic patterns of lung disease in rheumatoid arthritis have been described, 54, 57, 59 with the rheumatoid nodule being the most specific. acute lung injury has been reported ( fig. 6.26 ), referred to as acute interstitial pneumonia in some publications 65 and as dad in others. 54 a cellular lymphoplasmacytic infiltrate. acute lung injury has been reported to occur in the following connective tissue diseases. pulmonary involvement in systemic lupus erythematosus (sle) may manifest as pleural disease, acute or chronic diffuse inflammatory lung disease, airway disease, or vascular disease (vasculitis and thromboembolic lesions). acute lupus pneumonitis (alp) is a form of fulminant interstitial disease (fig. 6 .25a) with a high mortality rate. 52 patients present with severe dyspnea, tachypnea, fever, and arterial hypoxemia. alp represents the first manifestation of sle in approximately 50% of affected persons. 52, 58 the most common histopathologic feature of this acute disease is dad (eslide 6.3). alveolar hemorrhage, with capillaritis b a polymyositis/dermatomyositis, a systemic connective tissue disorder, is well known to be associated with interstitial lung disease. 55, 56 three main clinical presentations are recognized: (1) acute fulminant respiratory distress resembling the so-called hamman-rich syndrome, (2) slowly progressive dyspnea, and (3) an asymptomatic form with abnormalities on radiologic and pulmonary function studies. 59 three major histopathologic patterns have been observed: dad (fig. 6 .27a), organizing pneumonia ( fig. 6.27b) , and chronic fibrosis (fig. 6.27c )-the so-called usual interstitial pneumonia (uip) pattern. 66 the rapidly progressive clinical presentation is associated with a dad histopathologic pattern on lung biopsy studies and carries the worst prognosis. 56 dad associated with scleroderma and mixed connective disease also has been described. 57, 67 many patients with connective tissue disease receive drug therapy during the course of their illness. a large number of drugs, including cytotoxic agents used for immunosuppression, are known to cause dad. in addition, as a desired result of therapy, patients may be immunosuppressed, making the exclusion of infection a high priority in the case of acute clinical lung disease. drugs can produce a wide range of pathologic lung manifestations, and the causative agents are numerous. [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] the spectrum of drug-induced lung disease runs the entire gamut from dad to fibrosis. between these two extremes, subacute clinical manifestations may include organizing pneumonia, chronic interstitial pneumonia, eosinophilic pneumonia, obliterative bronchiolitis, pulmonary hemorrhage, pulmonary edema, pulmonary hypertension, venoocclusive disease, and granulomatous interstitial pneumonia. 78, 82, 83 dad is a common and dramatic manifestation of pulmonary drug toxicity. 78 many drugs are known to cause dad. 82 a few of the more common ones are discussed next. (drug-related lung disease is also discussed in chapter 8.) as a generalization, marked cytologic atypia and numerous foamy macrophages in the airspaces are histologic harbingers of possible drug reaction. dad frequently is caused by cytotoxic drugs, and the commonly implicated ones include bleomycin (fig. 6 .28), busulfan ( fig. 6.29) , and carmustine. 5, 78, 82 patients usually present with dyspnea, cough, and diffuse pulmonary infiltrates. [84] [85] [86] [87] [88] the histologic pattern most commonly is one of nonspecific acute lung injury with hyaline membranes, but some changes may be present to at least suggest a causative agent. for example, the presence of acute lung injury with associated atypical type ii pneumocytes with markedly enlarged pleomorphic nuclei 89 and prominent nucleoli (see fig. 6 .29) is characteristic for busulfan-induced pulmonary toxicity, and, on ultrastructural examination, intranuclear tubular structures have been found in type ii pneumocytes in association with administration of busulfan and bleomycin. [89] [90] [91] [92] in most cases, the possibility that a drug is the cause of dad can only be inferred from the clinical history. considerations in the differential diagnosis typically include other treatment-related injury or complication of therapy (e.g., concomitant irradiation or infection). for example, oxygen therapy is a well-recognized cause of dad (fig. 6 .30) and also may exacerbate bleomycin-induced lung injury. 93 methotrexate (fig. 6 .31) is another commonly used cytotoxic drug that can cause acute and organizing dad. 94 methotrexate also produces other distinctive patterns, such as granulomatous interstitial pneumonia (see chapter 8) that is seldom seen in association with other commonly used chemotherapeutic agents. to complicate matters further, methotrexate also is used in the treatment of rheumatoid arthritis, a disease known to produce dad independently as one of its pulmonary manifestations. 57, 62 epidermal growth factor receptor tyrosine kinase inhibitors have been reported to be associated with dad. 95, 96 the increasing use of targeted therapy drugs in cancer patients warrants a notice of this category as a potential cause. amiodarone is a highly effective antiarrhythmic drug that is increasingly recognized as a cause of pulmonary toxicity. 77,97-101 because patients taking amiodarone have known cardiac disease, the clinical presentation often is complicated, with several superimposed processes potentially affecting the lungs in various ways. clinical and radiologic considerations typically include congestive heart failure, pulmonary emboli, and acute lung injury from other causes. 77, 101 distinctive features may be present on chest computed tomography scans. 77 the lung biopsy commonly shows acute and organizing lung injury (fig. 6 .32a and eslide 6.4). other patterns include chronic interstitial pneumonitis with fibrosis and organizing pneumonia. 99 characteristically, type ii pneumocytes and alveolar macrophages show finely vacuolated cytoplasm in response to amiodarone therapy (see fig. 6 .32b), but these changes alone are not evidence of toxicity because they also may be seen in patients taking amiodarone who do not have evidence of lung toxicity. 97 methotrexate and gold, common agents for treatment of rheumatoid arthritis, are frequently implicated in lung toxicity. methotrexate is discussed earlier in this chapter. organizing dad (fig. 6.33 ) and chronic interstitial pneumonia are commonly described pulmonary manifestations of so-called gold toxicity. 74, 76, 102 acute eosinophilic pneumonia acute eosinophilic pneumonia was first described in 1989 103 and is characterized by acute respiratory failure, fever of days' to weeks' duration, diffuse pulmonary infiltrates on radiologic studies, and eosinophilia in bronchoalveolar lavage fluid or lung biopsy specimens in the absence of infection, atopy, and asthma. 104 peripheral eosinophilia frequently is described but is not a consistent finding at initial presentation. 103, 105 acute eosinophilic pneumonia is easily confused with acute interstitial pneumonia because both manifest as acute respiratory distress without an obvious underlying cause. 104 histologically, the disease is characterized by acute and organizing lung injury showing classic features (fig. 6.34 ) of (1) alveolar septal edema, (2) eosinophilic airspace macrophages, (3) tissue and airspace eosinophils in variable numbers, and (4) marked reactive atypia of alveolar type ii cells (eslide 6.5). intraalveolar fibroblastic proliferation (patchy organizing pneumonia) and inflammatory cells are present to a variable degree. hyaline membranes and organizing intraalveolar fibrin also may be present (fig. 6.35) . the most significant feature is the presence of interstitial and alveolar eosinophils. infiltration of small blood vessels by eosinophils also may be seen. it is important of special stains applied to tissue sections or cytologic preparations (e.g., afb, gms, or warthin-starry silver stain) also is essential to rule out infectious organisms in this setting. so-called pulmonary hemorrhage syndromes may feature the histopathologic changes of acute lung injury, 112 in addition to the characteristic alveolar hemorrhage and hemosiderin-laden macrophages. in some patients, dad may be the dominant histopathologic pattern. 113 in a study by lombard et al. in patients with goodpasture syndrome, all showed acute lung injury ranging in distribution from focal to diffuse lung involvement. 113 histopathologic examination demonstrated typical acute and organizing dad, with widened and edematous alveolar septa, fibroblastic proliferation, reactive type ii pneumocytes, and, rarely, even hyaline membranes (figs. 6.37 and 6.38). alveolar hemorrhage, either focal or diffuse, was present in all cases. capillaritis, an important finding indicating true alveolar hemorrhage, 112 also was seen, as evidenced by marked septal neutrophilic infiltration. capillaritis was absent in one case for which dad was the dominant histopathologic pattern. microscopic polyangiitis can manifest as an acute interstitial pneumonia both clinically and histopathologically. affected patients have vasculitis as the known cause of acute lung injury. 114 alveolar hemorrhage with arteritis, capillaritis ( fig. 6.38) , and venulitis may be seen in some cases. 114 polyarteritis nodosa and vasculitis associated with systemic connective tissue disease (notably sle and rheumatoid arthritis) can also show acute lung injury with alveolar hemorrhage as the dominant histopathologic finding. 57, 115 cryoglobulinemia is a rare cause of acute lung injury and alveolar hemorrhage. [116] [117] [118] radiation can produce both acute and chronic damage to the lung, manifesting as acute radiation pneumonitis and chronic progressive fibrosis, respectively. 119 the effect is dependent on radiation dosage, total time of irradiation, and tissue volume irradiated. concomitant chemotherapy and infections, which in themselves are causes of dad, may potentiate the effect of radiation injury. 5, 79, 120, 121 acute radiation pneumonitis manifests 1 to 2 months after radiation therapy. 5, 121 with traditional external beam radiation the pneumonitis is typically confined to the radiation field. however, more diffuse radiation pneumonitis can be seen following yttrium 90-impregnated microsphere chemoembolization for nonoperable hepatic tumors. 122 clinical findings include dyspnea, cough, pleuritic pain, fever, and chest infiltrates. the lung biopsy specimen shows acute and organizing dad. 119, 121 markedly atypical type ii pneumocytes with enlarged hyperchromatic nuclei and vacuolated cytoplasm constitute a hallmark of the disease (fig. 6.39a) , and increased numbers of alveolar macrophages are seen. foamy cells are present in the intima and media of pulmonary blood vessels in some cases, and thrombosis ( fig. 6.39b) , with or without transmural fibrinoid necrosis, is common. 79, [123] [124] [125] disease presenting as classic acute respiratory distress syndrome by definition, ards must be associated with an identifiable inciting event. the histopathologic pattern is that of classic dad. the histopathologic changes should be consistent with those expected for the time interval from the onset of clinical disease (see later). in many cases the ards may be caused by a combination of factors, each potentiating the other. 4 for the purposes of illustration, a few thoroughly studied causes are discussed next. to distinguish acute eosinophilic pneumonia from other causes of dad because patients typically benefit from systemic corticosteroid treatment, with prompt recovery. however, before initiation of immunosuppressive therapy, infection should be rigorously excluded by culture and special stains because parasitic and fungal infections also can manifest as tissue eosinophilia. treatment with steroids prior to the biopsy can make the number of eosinophils less impressive. acute interstitial pneumonia, also commonly referred to as hamman-rich syndrome, is a fulminant lung disease of unknown etiology occurring in previously healthy patients. [107] [108] [109] acute interstitial pneumonia is one of the major idiopathic interstitial pneumonias included in the most recent classification scheme for diffuse interstitial pneumonia. 110 patients usually report a prodromal illness simulating viral infection of the upper respiratory tract, followed by rapidly progressive respiratory failure. the mortality rate is high, with death occurring weeks or months after the acute onset. 107, 109 the classic histopathologic pattern is that of acute and organizing dad, 107,109 with septal edema and hyaline membranes in the early phase and septal fibroblastic proliferation with reactive type ii pneumocytes prominent in the organizing phase. in practice, a combination of acute and organizing changes ( fig. 6 .36) often is seen in the lung at the time of biopsy. 111 a variable degree of airspace organization, mononuclear inflammatory infiltrates, thrombi in small pulmonary arteries, and reparative peribronchiolar squamous metaplasia also are seen in most cases. because acute interstitial pneumonia is idiopathic, other specific causes of acute lung injury must be excluded before making this diagnosis. considerations in the differential diagnosis include infection, connective tissue disease, acute exacerbation of idiopathic pulmonary fibrosis (ipf), drug effect, and other causes of dad. 111 most cases of dad are not acute interstitial pneumonia, and detailed clinical information, radiologic findings (localized vs. diffuse disease), serologic data, and microbiologic results will often point to or rule out a specific etiologic condition. use figure 6 .36 acute interstitial pneumonia (aip). idiopathic aip may take the form of every possible morphologic manifestation of acute respiratory distress syndrome, depending on the timing of biopsy relative to the onset of symptoms. here, a classic pattern of diffuse alveolar damage (dad) with hyaline membranes of variable cellularity is seen (midproliferative phase). interstitial fibroblastic proliferation may be more or less prominent from case to case and should not serve as a qualifying morphologic finding for the diagnosis. aip is nothing more than dad of unknown causation. oxygen is a well-known cause of ards and a useful model for all types of dad. 4, 126, 127 oxygen toxicity also is important in that it is widely used in the care of patients, often in the setting of other injuries that can potentially cause ards, such as sepsis, shock, and trauma. exposure to high concentrations of oxygen for prolonged periods can lead to characteristic pulmonary damage. in 1958 pratt first noted pulmonary changes due to high concentrations of inspired oxygen. 128 in 1967 nash et al. described the sequential histopathologic changes of this injury, 126 later reemphasized by pratt. 127 in neonates receiving oxygen for hyaline membrane disease, bronchopulmonary dysplasia was reported to occur. 129 as might be expected, the features of hyaline membrane disease in neonates and oxygen-induced dad in adults are indistinguishable (see fig. 6 .30). other inhalants such as chlorine gas, mercury vapor, carbon dioxide in high concentrations, and nitrogen mustard all have been reported to cause ards. 2,4,5 massive extrapulmonary trauma and shock first became recognized as causes of unexplained respiratory failure during the wars of the second half of the 20th century. a variety of names were assigned to this wartime condition, including shock lung, congestive atelectasis, traumatic wet lung, da nang lung, respiratory insufficiency syndrome, posttraumatic pulmonary insufficiency, and progressive pulmonary consolidation. 2 it which can be performed even on autopsy specimens. other ingested toxins (e.g., kerosene, rapeseed oil) also have been reported to cause ards. 5 pathologist approach to the differential diagnosis of acute lung injury the histologic spectrum encountered in acute lung injury is broad. very early cases may look nearly normal with only mild interstitial and alveolar edema. other more advanced cases are clearly abnormal with fibrin, inflammation, and organization. the basic elements of the acute injury pattern include interstitial edema, alveolar edema, fibrin, hyaline membranes, reactive pneumocytes, and organization (see box 6.2). acute lung injury is a pathologic pattern and by itself is a nonspecific finding. from a practical perspective, after an acute lung injury pattern is became clear that shock of any cause (e.g., hypovolemia due to hemorrhage, cardiogenic shock, sepsis) could cause ards, and that in most cases, a number of factors come into play. in the typical presentation, dyspnea of rapid onset is accompanied by development of diffuse chest infiltrates several hours to days after an episode of shock. after ards begins, the mortality rate is high. 1,2,130 paraquat is a potent herbicide that causes the release of hydrogen peroxide and superoxide free radicals, resulting in damage to cell membranes. [131] [132] [133] oropharyngitis is the initial sign of poisoning, followed by impaired renal and liver function. approximately 5 days later, ards develops. the histopathologic pattern in most cases is one of organizing dad (fig. 6.40 ). the diagnosis is confirmed by tissue analysis for paraquat, b a raise consideration of immunologically mediated pulmonary hemorrhage. 112 care must be taken not to interpret the pigmented macrophages seen in the lungs of cigarette smokers as evidence of hemorrhage. 135 the hemosiderin in macrophages related to true hemorrhage in the lung (from any cause) is globular, often slightly refractile, and golden-brown in color. 57, [112] [113] [114] presence of atypical cells. viral infections often produce cytopathic effects, including intracellular inclusions (see chapter 7) . examples of intracellular inclusions are the cowdry a and b inclusions seen in herpesvirus infection, cytomegaly with intranuclear and intracytoplasmic inclusions of cytomegalovirus, the multinucleated giant cells of measles virus and respiratory syncytial virus, and the smudged cells of adenovirus infection. 33, 37, 38, 136, 137 chemotherapeutic drugs such as busulfan and bleomycin often are associated with markedly atypical type ii pneumocytes, which may have enlarged pleomorphic nuclei and prominent nucleoli. 90, 91 markedly atypical type ii pneumocytes that may be suggestive of a viropathic effect also are seen in radiation pneumonitis. 79, 124, 125 presence of foamy cells. alveolar lining cells with vacuolated cytoplasm accompanied by intraalveolar foamy macrophages are characteristic features seen in patients taking amiodarone, and amiodarone toxicity may lead to acute lung injury changes. [97] [98] [99] 101 in some cases of radiation pneumonitis, foam cells are seen in the intima and media of blood vessels. 79, 125 presence of foreign material. foreign material in the spaces in the form of vegetable matter or other food elements is indicative of aspiration. massive aspiration events may cause dad. other foreign material, such as radiation impregnated beads may also be encountered. presence of advanced interstitial fibrosis. clinical ipf is associated with the changes of uip on pathologic examination (see chapter 8), with advanced lung remodeling. of interest, ipf undergoes episodic exacerbation, and on occasion such exacerbation may be overwhelming, with resultant dad. 138 it is prudent to examine lung biopsy sections for the presence of dense fibrosis with structural remodeling (microscopic honeycombing) in cases of dad, to identify the rare case of ipf that manifests for the first time as an acute episode of exacerbation. because the morphologic manifestations of acute diffuse lung disease may be relatively stereotypical, clinicopathologic correlation is often helpful in arriving at a specific diagnosis. a summary of the more important history and laboratory data pertinent to this correlation is presented in box 6.3. identified, careful search for the following additional features often help to narrow the list of possible causes (summarized in immune status acuity of onset radiologic distribution and character of abnormalities history of inciting event (e.g., shock) history of lung disease (e.g., usual interstitial pneumonia with current acute exacerbation) history of systemic disease (e.g., connective tissue disease, heart disease) history of medication use or drug abuse history of other recent treatment (e.g., radiotherapy for malignancy) results of serologic studies: erythrocyte sedimentation rate determination, assays for autoimmune antibodies (e.g., ana, rf, anca, scl-70, jo-1) results of microbiology studies one of the first questions to be addressed is whether or not a known inciting event was identified clinically (i.e., is this ards?). next, the results of any sampling procedures to identify infection should be checked, along with application of special stains to the tissue sections, to exclude infection. finally, data regarding related disease, such as infection, autoimmune disease, underlying lung disease, are needed. for example, if the patient is immunosuppressed, infection should always be the leading consideration in the differential diagnosis. another point to keep in mind is that patients with certain diseases may be taking medications with the potential to cause dad (e.g., amiodarone for cardiac arrhythmia). moreover, laboratory studies may reveal antibodies related to connective tissue disease (e.g., antineutrophil antibody, rheumatoid factor, jo-1, scl-70, antifibrillarin, anti-mpp10, ss-a, ss-b). regarding the pathologist's role and responsibility in biopsy cases of acute lung injury, use of special stains for organisms (at a minimum, methenamine silver and acid-fast stains) is indicated. additional stains (auramine-rhodamine, dieterle or warthin-starry silver stain, immunohistochemical stains for specific organisms, or molecular probes) may be used, especially in patients known to be immunocompromised from any cause. the pathology in immunocompromised patients may not show necrosis, neutrophils, or granulomas, all features favoring an infectious etiology. self-assessment questions and cases related to this chapter can be found online at expertconsult.com. acute and fibrinous organizing pneumonia (eslide 6.2) a. history-a 55-year-old female presented with acute onset dyspnea. her past medical history was significant for rheumatoid arthritis for which she had recently begun methotrexate. imaging studies show bilateral ground-glass infiltrates in upper and lower lobes. a surgical lung biopsy was performed. b. pathologic findings-from scanning magnification, the lung architecture appears preserved without significant fibrosis. at higher power there is an extensive airspace filling process. many airspaces are filled with fibrin and scattered inflammatory cells. in other areas there is light pink material suggestive of edema. finally, some early fibroblastic polyps of organization are present. the interstitium shows diffuse alveolar damage with hyaline membranes (eslide 6.1) a. history-a 49-year-old male without significant past medical history presented to the emergency room with acute shortness of breath and cough. a week prior he participated in a half marathon without difficulty. he was taking no medications and had no exposures. his oxygen saturation was 82% on room air. he progressed to respiratory failure after being admitted to the intensive care unit. a surgical lung biopsy was performed. b. pathologic findings-from scanning magnification the biopsy shows preserved lung parenchyma without significant scarring. however, there is a diffuse process that gives the biopsy a "pink" appearance from low power. at higher power, the histologic features of diffuse alveolar damage (dad) are recognized including alveolar wall edema, reactive type-ii pneumocytes, and hyaline membranes. a few foci of organization are also present. a significant inflammatory cell infiltrate is not recognized. there is no pleuritis, hemosiderosis, granulomas, or necrosis. c. diagnosis-diffuse alveolar damage. d. discussion-features of acute lung injury are readily apparent, and the numerous hyaline membranes support a diagnosis of diffuse alveolar hemorrhage. the biopsy is negative for numerous eosinophils, foamy macrophages, alveolar hemorrhage, foreign material, neutrophils, necrosis, and granulomas. therefore the histology does not suggest a particular etiology on this case. acid-fast and fungal stains were negative. extensive serologic screening studies were negative, and cultures are negative to date. because the additional work-up is negative, this case is best categorized as acute respiratory distress syndrome. lung pathology of fatal severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong fatal measles (rubeola) pneumonia in adults isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash persistence of measles virus and depression of antibody formation in patients with giant-cell pneumonia after measles measles and its relationship to giant cell pneumonia (hecht pneumonia) katzenstein and askin's surgical pathology of non-neoplastic lung disease histopathology of fatal adenovirus infection of the respiratory tract in young children bronchiolitis obliterans, bronchiectasis and other sequelae of adenovirus type 21 infection in young children adenovirus infection in the immunocompromised patient viral infections of the respiratory tract fatal adenovirus pneumonia in a newborn identified by electron microscopy and in-situ hybridization herpes simplex virus pneumonia: clinical, virologic, and pathologic features in 20 patients herpes simplex virus infection of the adult lower respiratory tract varicella infection and pneumonia among adults cytomegalovirus pulmonary disease cytomegalovirus pneumonia in bone marrow transplant recipients: miliary and diffuse patterns cytomegalovirus after allogeneic bone marrow transplantation hantavirus pulmonary syndrome is distinguishable from acute interstitial pneumonia hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. the hantavirus study group hantavirus pulmonary syndrome in the united states. a new pathological description of a disease caused by a new agent lung biopsy in pneumocystis carinii pneumonia. a histopathologic study of typical and atypical features the diagnosis of pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome using subsegmental bronchoalveolar lavage disseminated pneumocystis carinii infection in a patient with acquired immunodeficiency syndrome pneumocystis infection masquerading as diffuse alveolar damage: a potential source of diagnostic error legionnaires' disease. pathological and historical aspects of a new disease legionnaires' disease pneumonia: histopathologic features and comparison with microbial and chemical pneumonias open lung biopsy in mycoplasma pneumoniae pneumonia adult respiratory distress syndrome in q fever the pathology of the legionella pneumonias. a review of 74 cases and the literature pulmonary manifestations of systemic lupus erythematosus: review of twelve cases of acute lupus pneumonitis pulmonary involvement in the collagen vascular diseases lung biopsy in rheumatoid arthritis 41st aspen lung conference: overview. chest pulmonary pathology of acute respiratory distress syndrome acute respiratory distress in adults diffuse alveolar damage-the role of oxygen, shock and related factors acute lung injury patterns: diffuse alveolar damage and bronchiolitis obliteransorganizing pneumonia mortality rates for patients with acute lung injury/ards have decreased over time the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination acute respiratory distress syndrome: the berlin definition adult respiratory distress syndrome the pulmonary physician in critical care 6: the pathogenesis of ali/ards atlas of pulmonary surgical pathology one-year outcomes in survivors of the acute respiratory distress syndrome pulmonary pathology of severe acute respiratory syndrome in toronto fatal acute fibrinous and organizing pneumonia in an infant: the histopathologic variability of acute respiratory distress syndrome clinicopathology study of 33 fatal cases of asian influenza pathologic features of lung biopsy specimens from influenza pneumonia cases intranuclear fibrillary inclusions in influenza pneumonia the spectrum of pathological changes in severe acute respiratory syndrome (sars) lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity oxygen-exacerbated bleomycin pulmonary toxicity methotrexate pneumonitis: review of the literature and histopathological findings in nine patients severe acute interstitial pneumonia and gefitinib fatal interstitial lung disease after erlotinib for non-small cell lung cancer amiodarone-associated pulmonary toxicity: a clinical and pathologic study of eleven cases amiodarone pulmonary toxicity. clinical, radiologic, and pathologic correlations amiodarone lung: pathologic findings in clinically toxic patients amiodarone pulmonary toxicity. recognition and pathogenesis (part i) acute amiodarone-induced lung toxicity alveolar-interstitial pneumopathy after gold-salts compounds administration, requiring mechanical ventilation acute eosinophilic pneumonia as a reversible cause of noninfectious respiratory failure acute eosinophilic pneumonia: histopathologic findings in nine patients a clinical study of idiopathic eosinophilic pneumonia acute eosinophilic pneumonia: a review of 12 cases acute diffuse interstitial fibrosis of the lungs acute interstitial pneumonia. a clinicopathologic, ultrastructural, and cell kinetic study hamman-rich syndrome revisited an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias acute interstitial pneumonia pathologic approach to pulmonary hemorrhage surgical pathology of the lung in anti-basement membrane antibody-associated goodpasture syndrome six cases of microscopic polyarteritis exhibiting acute interstitial pneumonia the lung in polyarteritis nodosa: a pathologic study of 10 cases alveolar hemorrhage in cryoglobulinemia-an indicator of poor prognosis lung involvement in essential mixed cryoglobulinemia pulmonary hemorrhage in cryoglobulinemia radiation injury in surgical pathology. part i enhanced pulmonary toxicity with bleomycin and radiotherapy in oat cell lung cancer diffuse alveolar damage. surgical pathology of diffuse infiltrative lung disease. orlando: grune and stratton incidence of radiation pneumonitis after hepatic intra-arterial radiotherapy with yttrium-90 microspheres assuming uniform lung distribution pulmonary effects of radiation therapy radiation effects in the lung pulmonary lesions associated with oxygen therapy and artificial ventilation pathology of pulmonary oxygen toxicity pulmonary capillary proliferation induced by oxygen pulmonary disease following respirator therapy of hyalinemembrane disease. bronchopulmonary dysplasia pulmonary disease in polymyositis/ dermatomyositis: a clinicopathological analysis of 65 autopsy cases interstitial lung disease in polymyositis and dermatomyositis. clinical features and prognosis as correlated with histologic findings pulmonary pathology in patients with systemic autoimmune disease interstitial lung disease in systemic lupus erythematosus interstitial lung disease in collagen vascular disease microangiitis in lupus-induced pulmonary hemorrhage pulmonary lesions and rheumatoid arthritis pulmonary involvement in patients with rheumatoid arthritis open lung biopsy of patients with rheumatoid arthritis potential pathogenesis and clinical aspects of pulmonary fibrosis associated with rheumatoid arthritis rapidly fatal pulmonary fibrosis: the accelerated variation of interstitial pneumonitis polymyositis-dermatomyositis-associated interstitial lung disease organizing diffuse alveolar damage associated with progressive systemic sclerosis pulmonary disease complicating intermittent therapy with methotrexate desquamative interstitial pneumonia following chronic nitrofurantoin therapy pulmonary changes induced by amphophilic drugs large-dose bleomycin therapy and pulmonary toxicity. a possible role of prior radiotherapy pulmonary disease induced by drugs fibrosing alveolitis, bronchiolitis obliterans and sulfasalazine therapy pulmonary injury induced by gold salts treatment chemotherapy-induced eosinophilic pneumonia. relation to bleomycin gold-induced pneumonitis drug-induced pulmonary disease. an update pulmonary drug toxicity: radiologic and pathologic manifestations radiation-induced and chemotherapy-induced pulmonary injury pulmonary toxicity syndrome following cdep (cyclophosphamide, dexamethasone, etoposide, cisplatin) chemotherapy drug-induced lung injury katzenstein and askin's surgical pathology of non-neoplastic lung disease drug-induced lung disease: high-resolution ct and histological findings drug-induced pulmonary disease (parts 1 and 2) drug-induced interstitial lung disease drug-induced lung disease drug-induced infiltrative lung disease drug-induced lung disease the effect of busulfan on human epithelia effect of long-term administration of busulfan respiratory bronchiolitis-associated interstitial lung disease and its relationship to desquamative interstitial pneumonia the respiratory syncytial virus and its role in acute bronchiolitis morphology of adenovirus type-3 infection of human respiratory epithelial cells in vitro acute exacerbation in idiopathic pulmonary fibrosis. analysis of clinical and pathologic findings in three cases improved survival of patients with acute respiratory distress syndrome (ards): 1983-1993 paraquat and the lung pulmonary ultrastructure of the late aspects of human paraquat poisoning paraquat poisoning. an update acute respiratory distress syndrome in mycoplasma pneumonia: a case report and review can show a bronchovascular distribution c. is associated with mechanical ventilation d. shows microcysts mantled by giant cells e. all of the above answer: e 8. peripheral cysts in hypoplastic lung tissue have been associated with: a. cri du chat syndrome b. holoprosencephaly c. beckwith-wiedemann syndrome d. down syndrome e. cornelia de lange syndrome answer: d 9 acinar pulmonary dysplasia: a. features cystic change and enlargement of all lobes b. accounts for one of the most common surgical specimens in pediatric lung pathology hyperplasia: a. refers to an increased number of alveoli relative to the corresponding conducting airways which of the following is not in the macroscopic differential diagnosis of cystic lung lesions in children? a. adenomatoid malformation b. intralobar sequestration c. congenital lobar overinflation d. lymphangioleiomyomatosis e. pneumatocele answer: d 3. pulmonary sequestration is characterized by: a. communication with second-order bronchial lumina b. solely systemic vascular supply c. exclusive extralobar localization d. densely apposed, atelectatic airspaces e. multifocal aggregates of eosinophils answer: b 4. extralobar pulmonary sequestrations may occasionally contain which one of the following heterotopic tissues? a. bone b. glial nodules c. hepatoid anlage d. striated muscle e. enteric-type epithelium answer: d 5. congenital malformations of the pulmonary airways: a. are most often seen in stillborns or newborns b. represent malformations of each bronchopulmonary segment c. may be difficult to subclassify in fetal lungs d. must be distinguished from pleuropulmonary blastoma e. all of the above answer: e 6. which one of the following tissues may have implications for future lung pathology, if it is present in a congenital malformation of the pulmonary airways? a. striated muscle b. cartilage c. mucinous epithelium d. embryonic-type mesenchymal tissue e. lymphoid aggregates answer: c 146.e2 19. which one of the following storage disorders does not usually involve the lung parenchyma? a. niemann-pick disease b. gaucher disease c obliterative bronchiolitis in children can be associated with all of the following except: a. adenovirus b. influenza c. stevens-johnson syndrome d. paragonimiasis e. graft-versus-host disease answer: d acute eosinophilic pneumonia (eslide 6.5) a. history-a previously healthy 29-year-old female presented to the emergency room with acute-onset shortness of breath and cough. she was initially evaluated and admitted to the medicine floor for presumed pneumonia. however, she quickly deteriorated and was transferred to the medical intensive care unit and required intubation. imaging studies showed bilateral ground-glass opacities without lobar distribution. additional history obtained from the patient's roommate revealed the patient was recently treated with sulfamethoxazole and trimethoprim for a urinary tract infection. b. pathologic findings-the overall architecture of the lung appears intact, but there is a diffuse acute lung injury pattern including alveolar wall edema, airspace fibrin, organization, and scattered hyaline membranes. pneumocytes show marked reactive atypia. there are numerous eosinophils in the airspaces, embedded within the fibrin, and within the interstitium. numerous airspace macrophages are also present. no necrosis or granulomas are identified. c. diagnosis-acute eosinophilic pneumonia. d. there are four key histologic features in acute eosinophilic pneumonia, all of which are satisfied in this case. i. alveolar septal edema ii. eosinophilic airspace macrophages iii. tissue and airspace eosinophils iv. reactive atypia of type-ii pneumocytes there is a differential diagnosis for the acute eosinophilic pneumonia pattern of injury including drug reaction, infection, connective tissue disease, smoking related, and idiopathic. rigorous exclusion of infection is imperative and requires both infectious stains on the tissue blocks and culture studies. recognition of this injury pattern is of particular importance as these patients typically respond dramatically to high-dose steroids and have a better prognosis than that of diffuse alveolar damage. in this patient the exposure to a sulfa drug in the days prior to presentation was the likely etiology. she was treated with steroids, dramatically improved, and was discharged in 4 days. amiodarone-induced diffuse alveolar damage (eslide 6.4) a. history-a 71-year-old male presented to the emergency room with acute shortness of breath first noted the evening prior. his past history was significant for a deceased donor renal transplant 10 days prior to presentation for end-stage renal disease secondary to diabetes. he also had a history of hypertension and atrial fibrillation. imaging studies showed bilateral ground-glass opacities in the upper and lower lobes. b. pathologic findings-from scanning magnification there is preserved architecture without significant fibrosis. there is diffuse alveolar wall thickening, mostly by edema. overlying pneumocytes show reactive epithelial changes. numerous hyaline membranes and focal fibrin in airspaces are present. some airspaces are filled with numerous macrophages showing finely vacuolated cytoplasm. some acute lupus pneumonitis (eslide 6.3)a. history-a 34-year-old african-american female presented with the emergency room with cough and shortness of breath. upon further questioning, she reported some blood-tinged sputum. the patient was febrile, and chest imaging studies showed bilateral ground-glass infiltrates without lobar distribution. serologic studies revealed an elevated erythrocyte sedimentation rate and c-reactive protein and positive antinuclear antibodies and anti-double-stranded dna antibodies. a surgical lung biopsy was performed. b. pathologic findings-the biopsy shows preserved lung architecture with a diffuse abnormality from scanning magnification. there is extensive alveolar wall edema with numerous foci of hyaline membranes. patchy organization is present, along with a relatively diffuse lymphoplasmacytic interstitial infiltrate. c. diagnosis-acute lupus pneumonitis. d. discussion-based on the histologic features alone, this biopsy is diagnostic of diffuse alveolar damage. however, the clinical history is required to arrive are a more specific diagnosis of acute lupus pneumonitis. the biopsy does show a mild increase in lymphoplasmacytic interstitial inflammation that would be unusual for most cases of idiopathic acute respiratory distress syndrome.edema and a mixed lymphoplasmacytic infiltrate. no hemorrhage, necrosis, or hyaline membranes are present. c. diagnosis-acute fibrinous and organizing pneumonia (afop). d. discussion-afop presents in the same fashion as diffuse alveolar damage (dad) and the differential diagnosis for afop and dad is the same, including drug reaction, toxin exposure, connective tissue disease, infection, and as an idiopathic reaction. they both represent forms of acute lung injury. in this case the degree of lymphoplasmacytic inflammation in the interstitium raises the possibility of a background connective tissue disease. additional history revealed she had recently cut her methotrexate dose in half to save money. she had also recently experienced inflammatory flares in her joints. all of these factors support a diagnosis of afop related to rheumatoid arthritis. a definitive etiology for afop is identified in a minority of patients.pneumocytes show similar cytoplasmic vacuolization. there is no necrosis, neutrophils, or hemorrhage. c. diagnosis-diffuse alveolar damage (dad) with foamy macrophages.a drug reaction leads the differential diagnosis. d. discussion-based on the presence of the patchy but marked cytoplasmic vacuolization in the macrophages and pneumocytes, a drug reaction is the most likely etiology for the dad pattern. in particular, amiodarone is a commonly used drug that causes this cytoplasmic vacuolization, even in the absence of associated lung injury. this was communicated to the clinical services who identified the patient was indeed taking amiodarone, even on the day of transplant. amiodarone-induced lung injury is associated with prolonged use of the drug and with an inciting event (such as a major operation). this patient had been on amiodarone for several years. following clinicopathologic correlation, this case is best diagnosed as amiodarone-induced dad. the patient was treated with pulse high-dose steroids and eventually had a full recovery. key: cord-310840-h49dx92d authors: eslamy, hedieh k.; newman, beverley title: pneumonia in normal and immunocompromised children: an overview and update date: 2011-09-30 journal: radiologic clinics of north america doi: 10.1016/j.rcl.2011.06.007 sha: doc_id: 310840 cord_uid: h49dx92d pneumonia is an infection of the lung parenchyma caused by a wide variety of organisms in pediatric patients. the role of imaging is to detect the presence of pneumonia, and determine its location and extent, exclude other thoracic causes of respiratory symptoms, and show complications such as effusion/empyema and suppurative lung changes. the overarching goal of this article is to review cause, role of imaging, imaging techniques, and the spectrum of acute and chronic pneumonias in children. pneumonia in the neonate and immunocompromised host is also discussed. normal and immunocompromised children: an overview and update hedieh k. eslamy, md, beverley newman, md* pneumonia is an infection of the lower respiratory tract, involving the lung parenchyma. the world health organization estimates that there are 150.7 million cases of pulmonary infection each year in children younger than 5 years, with as many as 20 million cases severe enough to require hospital admission. 1 in north america and europe, the annual incidence of pneumonia in children younger than 5 years is estimated to be 34 to 40 cases per 1000, and decreases to 7 cases per 1000 in adolescents 12 to 15 years of age. 2, 3 the mortality in pediatric patients caused by pneumonia in developed countries is currently low (<1 per 1000 per year). 3 however, pneumonia is still the number one cause of childhood mortality in developing countries. 1, 4 the overarching goal of this article is to review cause, current role of imaging, imaging techniques, and the spectrum of acute and chronic pneumonias in children. pneumonia in the neonate and immunocompromised host is also discussed. infectious agents causing pneumonia in children include viruses, bacteria, mycobacteria, mycoplasmas, fungi, protozoa, and helminths. etiologic diagnoses of pneumonia are not so easy to determine or so accurate as is sometimes implied. in addition, proof of the cause of pneumonia is not obtained in most cases. there is a great deal of overlap in the radiographic appearance of pneumonias caused by different organisms. imaging is usually poor at predicting the broad category (eg, bacterial vs viral) of infectious agent, let alone the specific agent. preexisting lung disease may not only predispose to pulmonary infection but also modify the appearance of pulmonary consolidation. furthermore, because the lungs can respond to a diverse disease processes in only a limited number of ways, it is common for the radiographic features of both acute and chronic infectious pneumonia to overlap considerably with many noninfectious lung diseases. such noninfectious lung diseases are identified as pneumonia mimics in this article. viral pneumonia is rare in the neonatal period, because of conferred maternal antibody protection, whereas bacterial pneumonia is most frequently caused by pathogens acquired during labor and delivery, and is more prevalent in premature babies. with decreasing maternal antibody levels, viral pneumonia occurs at a peak between 2 months to 2 years of age. bacterial infections become relatively more common in older children from 2 years to 18 years of age. 5 the lung response to an infective antigen seems to be more agespecific than antigen-dependent (ie, bacteria vs viral). therefore, lobar and alveolar lung opacities are more common in older children and are more frequently caused by bacterial infections, whereas interstitial opacities are seen in all age groups, and are relatively nonspecific as to the type of causative organism. 6, 7 the role of imaging, including chest radiographs, ultrasound (us) and computed tomography (ct), is to detect the presence of pneumonia, determine its location and extent, exclude other thoracic causes of respiratory symptoms, and show complications such as parapneumonic effusion/ empyema and suppurative lung complications. 5 although magnetic resonance (mr) imaging is not routinely used for evaluating pneumonia in children, it is a promising imaging modality particularly for children with chronic lung conditions who require repeat imaging studies. frontal and lateral chest radiographs are the mainstay, and often the only, imaging needed in pediatric pulmonary infection. this imaging can be supplemented with other views such as lateral decubitus or other imaging modalities as the circumstances warrant. decubitus views are not useful when an entire hemithorax is opacified because layering fluid cannot be identified without any adjacent air. the main use of us is to identify, quantify, and characterize a parapneumonic effusion/empyema, as well as provide image guidance for drainage and identify residual collections after treatment. 8, 9 operator availability and expertise are important factors in making us a useful tool for evaluating pulmonary infection. although intrapulmonary fluid-filled cavities and even lung abscesses within consolidated lung can be identified on us, ct provides a more global view of the disease process. ct is often used to further evaluate: (1) suppurative lung complications and to differentiate these from parapneumonic effusion/empyema; (2) patients with recurrent or chronic pneumonia and concern for an underlying lesion; and (3) immunocompromised children with noncontributory or confusing chest radiographs and clinical findings that could be secondary to lung infection. 5 close attention to ct technique is crucial for imaging evaluation of pneumonia in pediatric patients. ct with low radiation dose technique should be carefully performed in all cases. eighty to 120 kvp with weight-based low milliampereseconds coupled with radiation dose modulation techniques is appropriate in most children for evaluation of pneumonia. multiple ct image acquisitions are usually not needed and the scan field of view should be tailored to the area of interest (especially if following a specific lesion serially over time) to further decrease the overall radiation dose. 10 occasionally, it may be useful to acquire additional expiratory scans to assess air trapping, which is an early imaging finding associated with small airway disease. in this situation, often at least 1 or both ct acquisitions can be obtained using a high-resolution ct (hrct) gap technique. to obtain optimal ct imaging at peak inspiration and close to expiratory residual volume, controlled ventilation (cvict) in infants and young children ( 5 years old) or spirometer-controlled ct in older children may be needed. 11 young children have little intrinsic tissue contrast. therefore, intravenous contrast is almost always needed for ct imaging of infection especially if mediastinal delineation is required. the exception is when hrct is used only for evaluating lung parenchymal and airway disease. breath-holding is usually desirable but can be adapted on a case-by-case basis depending on the needs of the study and the ability of the child to cooperate. however, for the study to be interpretable, gross patient motion should be absent. sedation or anesthesia may be required in infants, young children, or children with intellectual disability. delays between induction of anesthesia and scanning need to be minimized to prevent the potential for lung atelectasis with anesthesia. the anesthesiologist needs to pay close attention to techniques for preventing atelectasis or recruiting lung before the ct imaging. 12 peltola and colleagues 13 recently published their experience with mr imaging of lung infections in children using free-breathing t2-weighted, short tau inversion recovery, and t1-weighted with fat saturation precontrast and postcontrast sequences. their study showed that lung parenchymal, pleural, and lymph node inflammatory abnormalities can be characterized by mr imaging in children with lung infection. therefore, mr imaging might potentially be used to further evaluate suspected, acute complications of pneumonia. 13 children with chronic lung conditions and recurrent infection, such as cystic fibrosis, who are often subjected to substantial radiation exposure from repeated ct studies, would benefit the most from mr imaging evaluation of the lungs instead of ct. although mr imaging may not provide as much detail compared with ct especially with early, small or subtle changes ( fig. 1) , there are promising indications of a role for mr imaging in pulmonary infection. [13] [14] [15] there are several different descriptions of basic patterns of lung diseases on chest radiographs. in this review article, we adopt the one described by hansell and colleagues. 16 almost all of these are seen as part of the spectrum of infectious lung disease ( table 1) . pneumonia and bronchiolitis are both common in infants and have overlapping clinical and imaging features. many studies, particularly those in the developing world, use the term acute lower respiratory tract illness and make no attempt to differentiate pneumonia from bronchiolitis. 17 bronchiolitis occurs in children less than 2 years of age, who typically present with cough, coryza, and wheezing. bronchiolitis is a major cause of morbidity and mortality in infants. 18 respiratory syncytial virus (rsv) is the most common cause 2) . such imaging findings are related to diffuse airway inflammation and partial (air trapping) or complete (atelectasis) airway obstruction. 19 similar changes are seen in older children (>2 years of age) with bronchitis although the features of diffuse small airway obstruction are less common in these older children with larger airways. pneumonia can be divided into several syndromes based on clinical presentation, imaging appearance, underlying predisposition, and cause. pneumonia syndromes that are discussed in this article include acute focal pneumonia, atypical pneumonia, miliary or nodular pneumonia, progressive or fulminant pneumonia, aspiration pneumonias, pulmonary infiltrates with eosinophilia (pie), and chronic or recurrent pneumonia. 20 neonatal pneumonia is briefly highlighted separately. pneumonia in immunosuppressed individuals is included in the general discussion of pneumonia syndromes and then specifically reviewed with regard to the different infections associated with various types of immunodeficiency. acute and chronic complications of pneumonia are also reviewed. characteristics that are typical for acute focal pneumonia include fever more than 38.8 c (102 f), a toxic appearance, and a focal opacity on chest radiographs. pleuritic chest pain in lower-lobe pneumonia is sometimes referred to the abdomen and may be mistaken clinically for an acute abdominal condition. acute focal pneumonia is most often caused by bacterial infection with streptococcus pneumonia. other causes of acute focal pneumonia are summarized in box 1. the chest radiograph of acute focal pneumonia usually shows a dense, typically more peripheral airspace opacity, which may appear segmental, lobar, or spherical ( figs. 3 and 4) . [21] [22] [23] in a febrile child with a spherical density on a chest radiograph, the most likely diagnosis is a round pneumonia but the possibility of an underlying neoplasm may be considered. round pneumonias tend to be solitary, have well-defined borders, and are often located in the perihilar region or posteriorly in the lungs. the radiograph should be carefully scrutinized for features of consolidation such as air bronchograms as opposed to those of a mass such as vascular/airway displacement or bony erosion. a second view such as a lateral radiograph may be helpful because a round pneumonia is often less masslike in appearance on an orthogonal view. this is one of the few scenarios in which radiologic follow-up after about 2 weeks may be useful to document interval resolution of acute pneumonia. 22 acute respiratory distress may be secondary to an intrathoracic mass causing airway or lung compression, especially when there is complete opacification of a hemithorax on radiographs (fig. 5) . intrapulmonary masses including both benign and malignant entities may present clinically with acute superinfection. in addition, other conditions or anatomic variants may be mistaken for pneumonia when a chest radiograph is obtained in a child with a fever and respiratory symptoms. atypical features in pneumonia include prominent extrapulmonary features (eg, headache, sore throat, and pharyngeal exudates), minimal or disparate chest signs on physical examination, subacute onset, nonfocal lung opacity on chest radiographs, lack of clinical response to antibiotics, lack of substantial leukocytosis, and a slow disease course. common infectious causes of atypical pneumonia are summarized in box 2. on chest radiographs, the pulmonary opacity is seen as either airspace, reticular (linear), or bandlike opacities in a nonfocal, patchy, or mottled distribution, with various degrees of density, usually without a single dense area of consolidation (fig. 6 ). most patients with atypical pneumonia can be classified into one of the following subgroups or a combination of two of them based on findings on chest radiographs: 20 acute interstitial pneumonia chest radiographs show a patchy, nonfocal reticular pattern. causes of acute interstitial pneumonia include self-limited viral infections and other pathogens. subacute minimal patchy pneumonia chest radiographs show 1 or more patches of minimal foci of airspace opacity. the most common causes of subacute minimal patchy pneumonia are mycoplasma pneumoniae, chlamydia pneumoniae, and adenoviruses. chest radiographs show a dense focal airspace opacity that is segmental or subsegmental. most of the other features of acute focal pneumonia are absent. tuberculosis needs to be excluded in these patients. most children exposed to mycobacterium tuberculosis do not develop active disease but can have latent foci that may reactivate at a later date particularly if they become immunosuppressed or debilitated. primary infection of mycobacterium tuberculosis is more likely in infants with local spread from the initial parenchymal/lymph node complex to form larger single or multifocal parenchymal lesions, typically with prominent hilar and mediastinal lymph node involvement ( fig. 7) and occasional pleural or pericardial disease. the primary focus as well as involved nodes may cavitate with liquefaction of the caseous material and ultimately calcification (see fig. 7 ). enlarged lymph nodes may encroach on adjacent bronchi and cause bronchial narrowing with resultant air trapping or collapse in the distal lung (fig. 8) . distant spread to other organs may occur either via lymphatics or hematogenously (including military lung involvement). 24, 25 infections with more than 1 organism may cause the atypical pneumonia pattern, resulting in confusing persistence of the illness or prominent findings in another organ system. an example of this situation is influenza infection with superimposed typical or atypical pneumonia (see fig. 6b ). the more common mimics that simulate the appearance of atypical pneumonia syndromes are summarized in box 3. miliary or nodular pneumonia is characterized by chest radiographic findings of multiple miliary or larger nodular opacities. miliary pneumonia in pediatric patients is seen most commonly in tuberculous and fungal infections (fig. 9 ). nodular pneumonia (including reticular and reticulonodular patterns) in pediatric patients is seen in septic emboli, viral pneumonia, lymphocytic interstitial pneumonia associated with epstein-barr virus (ebv) infection with underlying human immunodeficiency virus (hiv) infection, and some fungal and bacterial infections (box 4; figs. 10 and 11). 25, 26 septic pulmonary emboli usually occur secondary to a focal staphylococcus aureus infection (eg, right-sided bacterial endocarditis, septic thrombophlebitis, osteomyelitis, soft tissue infection, or urinary tract infection). the pulmonary nodules in septic emboli may cavitate (see fig. 11 ). 27 mimics of the pattern of miliary or nodular pneumonia are summarized in box 5. pneumonia is deemed progressive when it becomes radiologically and clinically worse despite antibiotic therapy that should be effective against the presumed cause. in this situation, the cause is often nonbacterial pathogens and mimics should also be carefully considered. fulminant pneumonia is defined as a severe bilateral pneumonia with an unusually rapid progression clinically or radiologically, over 24 to 48 hours after initial presentation. a common cause of progressive or fulminant pneumonia is the influenza virus during an epidemic. uncommon infectious causes of this pattern and mimics are summarized in boxes 6 and 7, respectively ( fig. 12 ). 20 aspiration pneumonia refers to the pulmonary consequences of abnormal entry of fluid, particulate matter, or endogenous secretions into the lower airways. aspirated material can be relatively inert, toxic, or oropharyngeal secretions. the most commonly aspirated materials in children include oropharyngeal secretions, gastric contents, water, hydrocarbon, lipid, and foreign bodies. radiographic pulmonary opacities related to aspiration may have an upper rather than lower lobe distribution when the child aspirates in the supine position. bacterial aspiration pneumonia is an infectious process caused by the inhalation of oropharyngeal secretions that are colonized by pathogenic bacteria. the basic defect leading to bacterial aspiration pneumonia is failure of the normal oropharyngeal defense mechanisms. the patient typically has a depressed state of consciousness, abnormal swallowing, a neuromuscular defect that prevents adequate coughing, or an abnormal connection between the airway and esophagus (such as an h-type tracheoesophageal fistula). acute lung aspiration (mendelson syndrome) is an acute chemical injury caused by inhalation of gastric contents. in neurologically normal children, gastric aspiration usually occurs as a complication of anesthesia. the diagnosis of acute aspiration is mainly clinical and usually involves witnessed inhalation of vomitus or tracheal suctioning of gastric contents. 28, 29 chronic lung aspiration (cla) is repeated passage of food, gastric reflux, or saliva into the subglottic airways that causes chronic or recurrent respiratory symptoms. cla may present with chronic cough, wheeze, noisy breathing, choking during feeding, recurrent episodes of pneumonia or bronchitis, and failure to thrive. chronic aspiration often results in progressive lung disease, recurrent pneumonia, chronic airway inflammation, bronchiectasis, and respiratory failure. it is a major cause of death in children with severe neurologic disorders (fig. 13) . pulmonary aspiration may occur as a result of swallowing dysfunction, gastroesophageal reflux, and inability to adequately protect the airway from oral secretions or a combination of these. anatomic conditions that predispose to aspiration lung disease include esophageal stricture or obstruction (eg, vascular ring, foreign body, achalasia), cleft palate, tracheoesophageal fistula (fig. 14) , laryngeal cleft, and bronchobiliary fistula. 28, 29 aspiration related to near-drowning occurs when fluid enters the lungs without being prevented by laryngospasm. it typically manifests as pulmonary edema radiographically. 30 in a recent series of 83 children, secondary infections from aspiration related to near-drowning were rare. 31 hydrocarbon pneumonia is an acute, intense chemical pneumonitis after unintentional aspiration of volatile hydrocarbon compounds. most cases of hydrocarbon pneumonia occur in children. chest radiographs typically show bilateral, scattered pulmonary densities with middle and lower zone predominance. such densities may become confluent and progress to acute respiratory distress syndrome (ards) and respiratory failure. they typically worsen over the first 72 hours and then clear over the next few days. however, occasionally radiographic changes may take weeks to months to be cleared. obstructive emphysema, pneumatoceles, subsegmental, or segmental atelectasis may also be seen. 32 lipoid pneumonia is a rare form of pneumonia caused by inhalation or aspiration of a fatty substance. oral administration of various oils is a common cultural practice, including mineral oil, olive oil, shark liver oil, cod liver oil, coconut oil, and ghee. such oily materials can readily slide into the airway even in normal infants and young children without eliciting a cough reflex and are poorly removed by cilia. lipoid pneumonias are typified by mild, subacute, or chronic clinical findings with accompanying marked radiographic changes. chest radiographs of children with lipoid pneumonia typically show bilateral parahilar illdefined, airspace opacities. in a series of 7 pediatric patients, ct showed dense consolidation surrounded by ground-glass opacity with a geographic lobular distribution. 33 within the dense consolidations, areas with relatively low attenuation were identified in only 1 patient. therefore, low-density consolidation in the posterior lungs is an infrequent ct finding in the diagnosis of lipoid pneumonia in children (fig. 15 ). interlobular septal thickening in areas of ground-glass opacity (ie, crazy paving pattern) has also been described in children with lipoid pneumonia. 33 lipoid pneumonias may be complicated by superimposed infection especially with atypical mycobacteria. slow recovery usually takes place with cessation of the oil administration. there may be residual scarring/fibrosis especially with animal rather than vegetable oils. 34, 35 foreign body aspiration can also result in pneumonia. accidental aspiration of both organic and nonorganic foreign bodies is a cause of childhood morbidity and mortality, requiring prompt recognition and early treatment to minimize the potentially serious and sometimes fatal consequence. eating is the most common circumstance during which it occurs, with small food items being the most common foreign bodies aspirated. coughing, choking, acute dyspnea, and sudden onset of wheezing are the most common symptoms. clinical signs of foreign body aspiration have low positive predictive values. chest radiographs are the initial imaging modality for patients with clinically suspected tracheobronchial aspiration of a foreign body. chest radiographs may show air trapping, atelectasis, a radiopaque foreign body (rare), or be normal (fig. 16) . 36 when the routine inspiratory chest radiograph is unhelpful or confusing, inspiratory and expiratory radiographs (in a cooperative child) or bilateral decubitus views (in a younger child unable to follow breathing instruction) are useful in confirming focal or unilateral air trapping. in selected cases, ct (possibly integrated with virtual bronchoscopy) may be considered to exclude a foreign body. ct evaluation may avoid bronchoscopy or provide the exact location and postobstructive complications of the foreign body before bronchoscopy. 37 an underlying chronic unrecognized airway foreign body should be considered among other causes of recurrent or chronic pneumonia, particularly in the pediatric population (see fig. 16 ). pie syndrome comprises a group of heterogeneous disorders having the common findings of lung disease and eosinophilia in the peripheral blood, bronchoalveolar lavage fluid, or pulmonary interstitium. pie syndrome is rare in children. a subclassification for the pie syndromes in children is summarized in box 8. 38, 39 infectious causes of pie syndrome are uncommon and include chlamydia trachomatis (especially in infants less than 3 months of age), allergic bronchopulmonary aspergillosis (in asthmatics and cystic fibrosis), parasitic larvae in lungs (toxocara, ascaris, and others), and fungi (eg, cryptococcus, candida species). 20 the radiographic findings in pie syndromes tend to be nonspecific. chest radiographs may show interstitial, alveolar, or mixed (interstitial and alveolar) infiltrates, which tend to be bilateral and diffuse. certain pie syndromes may be associated with more specific findings. the classic radiographic appearance of chronic eosinophilic pneumonia is characterized by peripheral infiltrates with sparing of the central lung zones. this radiographic appearance has been described as the "photographic negative of pulmonary edema." 40 bronchiectasis with mucoid impaction is generally present on chest radiographs or ct in patients with allergic bronchopulmonary aspergillosis (fig. 17) . 41 acute eosinophilic pneumonia is frequently associated with small bilateral pleural effusions. imaging is often helpful in determining the extent of disease, localizing the potential sites for lung biopsy, and in assessing response to therapy once treatment has begun. 38 chronic pneumonia is defined as a pulmonary opacity that does not improve within 1 month. it is best classified from the anatomic pattern, as focal, interstitial, with hilar lymphadenopathy, or with cysts, cavities, or spherical masses. spherical masses, with or without cavitations, are often features of an infectious cause. infectious causes and mimics of this pattern are summarized in boxes 9 and 10 (see fig. 16 ; figs. [18] [19] [20] . 42 obstructive atelectasis may both mimic and predispose to chronic pneumonia. it may have many underlying causes, including foreign body, mucoid impaction, narrowed bronchus, and extrinsic bronchial compression by cardiovascular anomalies, lymphadenopathy, tumor, or postpneumonic inflammatory changes. anomalies of the lung, mediastinum, and diaphragm that may mimic an acute or chronic pneumonia pattern include atypical thymus, diaphragmatic eventration and hernia, tracheal bronchus, lung hypoplasia, and congenital bronchopulmonary malformations (bpms). 20 several of these lesions, such as the bpms, predispose to recurrent or chronic infection but differentiating an infected from uninfected lesion may be difficult or impossible on imaging. sometimes having previous imaging for comparison is helpful in terms of features such as the new presence of fluid in a previously air-filled cavity or perilesional consolidation. recurrent pneumonia is defined as more than 1 episode within a 1-year period or more than 3 episodes in a lifetime. many children with a chronic pulmonary lesion (especially a congenital anomaly) are believed to have recurrent pneumonias if chest radiographs are taken only during a febrile illness. recurrent pneumonias may be either focal or interstitial (linear). underlying abnormalities that may predispose to recurrent focal pneumonia include chronic aspiration (see section on aspiration pneumonia), congenital heart disease, bronchopulmonary foregut malformations (including bpms with enteric-respiratory tract fistula), airway abnormalities (foreign body, stenosis, bronchiectasis, cystic fibrosis, immotile cilia disease), paralysis or eventration of the diaphragm, and congenital, acquired, and iatrogenic immune deficiencies. 43, 44 recurrent interstitial pneumonia may be secondary to asthma, hypersensitivity pneumonitis, or pneumonias in children with aids (including pneumocystis jiroveci pneumonia, lymphoid interstitial pneumonitis, and recurrent streptococcus pneumoniae infection) (see fig. 10 ). 20 neonatal lung infections can be generally classified into 3 types depending on the initial source of neonatal infection: transplacental, perinatal, and postnatal (including nosocomial) infections. transplacental infections enter the fetus hematogenously via the umbilical cord. most infants affected with transplacental infections typically recurrent aspiration caused by tracheoesophageal fistula without esophageal atresia in a 6day-old girl who presented with recurrent episodes of apnea, cyanosis, and choking desaturations. barium esophagram shows an oblique connection (red arrow) coursing anterosuperiorly from the esophagus to the trachea at the level of the thoracic inlet. contrast has opacified the central tracheobronchial airways. inferior to the fistula, there is a focal mild narrowing of the esophagus (blue arrow), raising concern for a congenital esophageal stricture. manifest systemic and multiorgan disease rather than a primary lung infection. the most common transplacental infection is caused by cmv, which manifests as a diffuse reticulonodular pattern. 45 other less common, transplacentally acquired pneumonias include rubella, syphilis, listeria monocytogenes, and tuberculosis. perinatal infections can be acquired via ascending infection from the vaginal tract (most commonly group b streptococcus or escherichia coli), transvaginally during the birth process, or nosocomially in the neonatal period. 46 radiographic findings in neonatal pneumonia are nonspecific in differentiating between various etiologic pathogens, as well as differentiating pneumonia from other causes of respiratory distress (eg, transient tachypnea of the newborn, surfactant deficiency disease, and meconium aspiration). the most common radiographic manifestation of neonatal pneumonia is bilateral coarse perihilar reticular densities with possible scattered airspace opacities (fig. 21) . solitary lobar consolidations are uncommon. 47 there is an association between group b streptococcal pneumonia and an ipsilateral diaphragmatic hernia. 48 chest radiographs in group b streptococcal sepsis can mimic the diffuse ground-glass opacity seen in surfactant deficiency disease. however, the presence of this finding in a full-term infant or the presence of cardiomegaly or pleural effusions may help to differentiate group b streptococcal infection from surfactant deficiency disease. 49 pneumonia caused by chlamydia trachomatis occurs in about 10% of infants born to women who carry this organism in their genital tract and becomes symptomatic more than 2 to 3 weeks after birth. chlamydia pneumonia is characterized by hyperinflation and bilateral diffuse reticular perihilar densities that are disparate with relatively mild clinical symptoms. 50 concomitant conjunctivitis, which used to be a useful clue to the cause, is prevented by the routine instillation of antibacterial eye drops at birth. neonates with chlamydia pneumonia frequently have accompanying eosinophilia. bordetella pertussis has recently resurfaced to produce epidemics of infection, probably related to waning community immunity. the clinical presentation of pertussis in the newborn may lack some features (characteristic whooping cough and fever) typical of the disease in older children. the clinical presentation of the most severely affected newborns may be dominated by marked respiratory distress, cyanosis, and apnea. mortality caused by pertussis usually results from secondary pneumonia, encephalopathy, cardiac failure, or pulmonary hypertension. a suggested mechanism for pulmonary hypertension that may develop in newborns with bordetella pertussis infection is formation of leukocyte thrombi in pulmonary venules secondary to hyperleukocytosis. 51, 52 the classic radiographic appearance in pertussis is the shaggy heart with diffuse peribronchial cuffing related to airway inflammation. however, chest radiographic findings such as hyperaeration, atelectasis, segmental consolidation, and lymphadenopathy are usually nonspecific. recurrent pulmonary infection, with bacterial, viral, or occasionally fungal pathogens, are frequent problems in neonates undergoing prolonged hospitalization and complex treatments, especially in premature infants with chronic lung disease. radiographic alterations caused by infection may be subtle when superimposed on chronic lung changes. 47 pneumonia is a common disease in the immunocompromised host. immunocompromise may be congenital (congenital immunodeficiencies), acquired (hiv/aids, malnutrition) or iatrogenic (during chemotherapy for cancer or after tissue transplantation). immunodeficient states can result in: (1) humoral immunodeficiency (hypogammaglobulinemia, functional b-lymphocyte deficiency accompanying hiv infection); (2) cellular immunodeficiency (severe malnutrition, late stages of aids, some congenital immunodeficiencies such as digeorge syndrome); and (3) neutrophil dysfunction and neutropenia (chronic granulomatous disease, pure neutropenia). iatrogenic immunodeficiencies may be a combination of neutropenia or neutrophil dysfunction, innate or drug-induced defective lymphocyte function, and drug-induced breaks in the oral and intestinal mucosal barriers. 53 the causes of pneumonia in the immunocompromised host consist not only of the same agents that cause pneumonia in the normal host but also of several opportunistic agents depending on the type and severity of immunodeficiency as well as temporal pattern after chemotherapy or transplant. in an immunocompromised child with a noncontributory chest radiograph and clinical findings that could be attributed to a lung infection, chest ct is often required for evaluation of a possible lung infection. in this situation, there are 4 major advantages of chest ct over chest radiographs. first, the presence, pattern, and extent of the disease process are better visualized. second, more than 1 pattern of abnormality may be detected, suggesting dual pathologic entities. third, invasive diagnostic procedures (eg, bronchoscopy or needle aspiration) can be more precisely planned. fourth, ct also allows for increased sensitivity in assessment of the response to treatment. 54, 55 although the radiographic or ct appearance might not be specific for a pathogen, knowledge of the clinical setting in combination with the type and severity of immunodeficiency and imaging pattern may narrow the differential diagnosis. a commonly encountered clinical issue is the possibility of fungal infection in immunocompromised children. the hallmark ct finding of fungal infections is the presence of pulmonary nodules. such pulmonary nodules are often clustered peripherally and can show poorly defined margins, cavitation, or a surrounding halo of ground-glass opacity (ct halo sign) (fig. 22) . the ct halo sign is a nonspecific finding and represents either hemorrhage around a nodule or neoplastic or inflammatory infiltration of the lung parenchyma. in the immunocompromised host, the ct halo sign can be seen most commonly with fungal infections (eg, aspergillosis, mucormycosis, or candida) but also in viral infections (eg, cmv infection, herpes infection), organizing pneumonia, and pulmonary hemorrhage. 54, [56] [57] [58] [59] [60] [61] microorganisms associated with severe pneumonia in immunodeficiency states are summarized in box 11. lip is a form of subacute pneumonia seen in several states of immunologic dysregulation, particularly in children with hiv infection. lip is characterized by micronodules of proliferating lymphoid tissue associated with infection by ebv. chest radiographs in lip show a characteristic diffuse, bilateral nodular, or reticulonodular pattern 19 . wegener granulomatosis mimicking chronic cavitary pneumonia in a 15-year-old male who presented with hemoptysis and respiratory distress. initial chest radiograph showed bilateral confluent airspace opacities secondary to diffuse pulmonary hemorrhage (not shown). contrast-enhanced ct obtained 9 days after initial presentation shows multifocal consolidation with cavitation (arrow), ground-glass opacities, and a right pneumothorax. (see fig. 10 ). other recognized associated imaging findings of lip include consolidation, mediastinal adenopathy, and bronchiectasis. 26, 58, 62 radiologic features in common hiv-associated infections in pediatric patients are summarized in table 2 . in pediatric patients with iatrogenic deficiencies of the immune system, pneumonia is commonly caused by opportunistic bacteria and fungi, acquired nosocomially or from resident mucosal flora (fig. 23) . in addition, after solid-organ transplantation although total immunoglobulin levels are normal, children and adolescents may be susceptible to encapsulated bacteria (eg, streptococcus pneumoniae, haemophilus influenzae). viruses that commonly cause pneumonia in healthy hosts (eg, rsv, influenza, parainfluenza viruses, human axial contrast-enhanced ct shows multiple bilateral pulmonary nodules, some have rims of groundglass opacity (arrow), which is also known as the ct halo sign. microorganisms associated with severe pneumonia in immunodeficiency states in pediatric patients metapneumovirus, and adenovirus) display greater virulence in both children and adults after solid-organ or human stem cell transplantation, particularly when cellular immunity is profoundly suppressed. in the posttransplant setting, ebv can cause progressive pulmonary disease in the form of posttransplantation lymphoproliferative disease. 53 in 2 studies of hrct in bone marrow transplant recipients, the most useful distinguishing feature was the presence of large nodules and visualization of the halo sign, suggestive of fungal infection. 63, 64 after solid-organ transplantation, nosocomially acquired bacteria predominate as a cause of pneumonia in the first month. later, viruses, especially cmv and adenovirus, as well as listeria, nocardia, and aspergillus, may be the cause. after more than 6 months after solid-organ transplantation, community-associated bacterial pneumonia becomes more common. 53 a variety of noninfectious pulmonary processes can present with acute or subacute clinical findings mimicking pulmonary infection. 54 these findings include alveolar hemorrhage, pulmonary edema, drug reaction, idiopathic interstitial pneumonia, benign and malignant lymphoproliferative disorders, constrictive bronchiolitis, bronchiolitis obliterans with organizing pneumonia, and chronic graft-versus-host disease. the ct findings of many of these entities are nonspecific. 54, [57] [58] [59] [60] [61] abbreviations: alveolar, focal of diffuse alveolar pattern; interstitial, focal or diffuse interstitial pattern; ln, lymphadenopathy; 1 and 11, refer to relative frequency of radiographic finding with each organism/disease. acute complications of pneumonia can be categorized as suppurative lung parenchymal complications and pleural complications. suppurative lung parenchymal complications span a spectrum of abnormalities and include cavitary necrosis, lung abscess, pneumatocele, bronchopleural fistula (bpf), and pulmonary gangrene. the name given to the suppurative process depends on several factors including the severity and distribution of the process, condition of the adjacent lung parenchyma, and temporal relationship with disease resolution. 65, 66 cavitary necrosis cavitary necrosis represents a dominant area of lung necrosis associated with a variable number of thin-walled cysts. characteristic findings on ct for cavitary necrosis include loss of normal lung architecture, poor parenchymal enhancement, loss of the lung-pleural margin, and multiple thin-walled fluid-filled or air-filled cavities (fig. 24) . cavitary necrosis is seen earlier in chest us or ct compared with chest radiography because these cavities need to be filled with air to be visible on chest radiographs. such cavities filled with air are accomplished only after communication with bronchial airways. most pediatric patients can be managed successfully with conservative treatment. follow-up chest radiographs typically show complete or near-complete resolution of the cavitary necrosis (see fig. 24 ). 65, 66 lung abscess lung abscess is a severe complication of pneumonia in children, mostly occurring in the presence of predisposing factors, such as congenital or acquired lung abnormalities, or immunodeficiency. lung abscess represents a dominant focus of suppuration surrounded by a well-formed fibrous wall. the predominant pathogens isolated from primary lung abscesses in children include streptococcal species, staphylococcus aureus and klebsiella pneumoniae. children with a lung abscess have a significantly better prognosis than adults with the same condition. 67 lung abscesses are uncommon in immunocompetent children. on contrast-enhanced ct, a lung abscess appears as a fluid-filled or airfilled cavity with a thick definable, enhancing wall (fig. 25) . 65, 66, 68 although lung abscess in children has been managed successfully for many years with prolonged courses of intravenous antibiotics, the evolution of interventional radiology has seen the accelerated use of percutaneously placed pigtail drainage catheters using us and ct guidance. 67 pneumatocele pneumatocele is a term given to thin, smoothwalled air-filled cysts seen at imaging and may represent a later or less severe stage of resolving or healing lung necrosis (see fig. 24 ). pneumatoceles are most often caused by severe lung infection from staphylococcal pneumonia. however, they may be seen with other bacterial infections including streptococcus pneumonia and after hydrocarbon aspiration. on ct, thin-walled small or large cysts containing air with or without fluid are identified. the wall of a pneumatocele does not enhance. the surrounding lung may be opacified but does not typically show findings of lung necrosis. 65, 66 pneumatoceles usually resolve spontaneously over time although pneumatoceles may be atypically persistent in children with hyper-ige syndrome. large pneumatoceles containing fluid can be a source of ongoing infection and may occasionally require drainage. bronchopleural fistula bpf is defined as a communication between the lung parenchyma or airways and the pleural space. central bpfs (ie, main or lobar bronchi communicating with the pleural cavity) most often develop after traumatic injury to large airways or leak from the bronchial stump after pneumonectomy or lobectomy. the main causes for peripheral bpfs (ie, segmental or more distal airways or lung parenchyma communicating with the pleural cavity) are necrotizing pulmonary infection (ie, cavitary necrosis), trauma, lung surgery, and malignancy. 69, 70 presence of air in the pleural space before aspiration or drainage attempts is suggestive of either a peripheral bpf or infection with a gas-producing organism. multidetector ct with thin-section axial and multiplanar reformation images may show a fistulous tract between the pleural space and peripheral airway or lung parenchyma in peripherally located bpfs (fig. 26) . 70 pulmonary gangrene pulmonary gangrene is a rare complication of severe lung infection with devitalization of lung parenchyma and secondary infection. 71 the primary feature that distinguishes pulmonary gangrene from necrotizing pneumonia and lung abscess is the extent of necrosis and the fact that thrombosis of large vessels plays a prominent role in the pathogenesis. chest imaging shows lobar consolidation with bulging fissures and is followed by tissue breakdown to form many small cavities, which subsequently coalesce into a single large cavity occupying the entire lobe. such a large cavity is filled with fluid and irregular pieces of sloughed lung parenchyma. however, these findings are not invariably present. surgical resection of necrotic tissue is often necessary for proper management of children with pulmonary gangrene. [71] [72] [73] [74] the differential diagnosis of suppurative lung complications includes an underlying cystic axial contrast-enhanced t1-weighted mr image of the brain shows a rim-enhancing brain abscess in the right occipital lobe. the patient underwent right lower lobectomy and craniotomy for resection of these lesions. congenital bpm that has become secondarily infected. prior and follow-up imaging may aid in the distinction. the presence of large, welldefined cysts early in the course of the illness or a systemic arterial supply to the lung may be helpful in suggesting an underlying bpm, although a chronic inflammatory process in the lower lobe can acquire some systemic vascular supply from diaphragmatic vessels. pleural complications from acute pneumonia include parapneumonic effusion and empyema. parapneumonic effusion is defined as a pleural effusion in the setting of a known pneumonia. it may be simple or complicated based on the absence or presence of the infecting organism within the pleural space, respectively. 75 empyema is defined as thick purulent pleural effusion. it may be free-flowing or loculated. progression of a pleural effusion to empyema occurs through 3 stages: exudative, fibrinopurulent, and organization. parapneumonic effusions complicate pneumonia in 36% to 56% of cases in pediatric patients. 76 empyema complicates an estimated 0.6% of all childhood pneumonias. 77 chest radiographs can often detect a parapneumonic collection, although some fluid, especially in a subpulmonic location, may not be visible and is often seen better on a decubitus film. in cases with complete or almost complete opacification of a hemithorax with or without contralateral mediastinal shift, additional erect or decubitus views are unhelpful in defining the quantity or nature of the pleural fluid. us is most helpful in this situation because it can readily distinguish a parapneumonic collection from extensive consolidation or an underlying mass. the us determination of the echogenicity of the pleural collection (anechoic or echogenic) and showing fibrin strands, septations, loculations, or fibrinous pleural rind is helpful in determining appropriate therapy (see fig. 26 ). treatment options for parapneumonic effusions/ empyemas include antibiotics alone, simple tube drainage, chest drain insertion with fibrinolytics, or surgery (eg, video-assisted thoracoscopic surgery or open thoracotomy with decortication). although imaging techniques are used as a guideline, they do not always accurately stage empyema, predict outcome, or guide decisions regarding surgical versus medical management. 75 ct provides a more global overview of pleural and pulmonary abnormality from acute pneumonia, but is poor at differentiating parapneumonic effusion from empyema in pediatric patients. findings on ct, in patients with parapneumonic effusion/ empyema, include: (1) enhancement and thickening of visceral and parietal pleura; (2) thickening and increased density of extrapleural subcostal tissues; and (3) increased attenuation of extrapleural subcostal fat. 78 loculation can be inferred by the presence of a lenticular fluid collection or nondependent air. septations are usually not appreciated on ct (see fig. 26 ). pleuropulmonary infection may occasionally spread to involve the chest wall, including soft tissues and adjacent bones. mycobacterium tuberculosis, aspergillus, and actinomyces are the most common organisms in this scenario. chronic complications or consequences of pneumonia include parenchymal scarring, bronchial wall thickening, bronchiectasis, a predisposition to asthma, constrictive bronchiolitis, fibrothorax and a trapped lung, fibrosing mediastinitis, constrictive pericarditis, and pleural thickening. for practical purposes, bronchiectasis, constrictive bronchiolitis, fibrothorax and trapped lung, and fibrosing mediastinitis are discussed in the following sections. bronchiectasis is defined by the presence of permanent and abnormal dilation of the bronchi. this condition usually occurs in the context of chronic airway infection causing inflammation. bronchiectasis is nearly always diagnosed using hrct. the main diagnostic features of bronchiectasis on hrct are: (1) internal diameter of a bronchus that is wider than its adjacent pulmonary artery; (2) failure of the bronchus to taper peripherally; and (3) visualization of bronchi in the outer 1 to 2 cm of the lung zones (see fig. 17 ; figs. 27 and 28). a wide variety of factors predisposing to the development of bronchiectasis have been identified, including hereditary (cystic fibrosis, ciliary dyskinesia), infective, immunodeficiency (antibody deficiency), obstructive (intrabronchial foreign body), and systemic causes. causes most commonly associated with bronchiectasis are childhood infections, including pneumonia, pertussis, complicated measles, and tuberculosis (eg, mycobacterium tuberculosis and mycobacterium avium complex). [79] [80] [81] constrictive bronchiolitis (bronchiolitis obliterans) constrictive bronchiolitis (bronchiolitis obliterans) is characterized by the presence of concentric narrowing or obliteration of the bronchioles caused by submucosal and peribronchiolar fibrosis. a common cause of constrictive bronchiolitis is previous childhood infection, resulting in the so-called swyer-james syndrome, identifiable as asymmetric hyperlucent lung on chest radiographs. whereas the process may appear unilateral on chest radiographs, there is usually bilateral but asymmetric abnormality on ct (see fig. 28 ). central bronchiectasis and a characteristic mosaic appearance with patchy expiratory air trapping are seen on hrct. causes and associations of constrictive bronchiolitis include previous infections (viral including adenovirus, rsv, influenza, parainfluenza; mycoplasma and pertussis), collagen vascular diseases, previous transplant, toxic fume exposure, ingested toxins, drugs, and cryptogenic constrictive bronchiolitis. 82 pleural fibrosis can result from a variety of inflammatory processes (box 12). the development of pleural fibrosis follows severe pleural inflammation, which is usually associated with an exudative pleural effusion. fibrothorax and trapped lung are 2 uncommon consequences of pleural fibrosis (see fig. 27 ). 83 fibrothorax represents the most severe form of pleural fibrosis. with a fibrothorax, there is dense fibrosis of the visceral and parietal pleural surfaces, leading to fusion of these membranes, contracture of the involved hemithorax (and ipsilateral mediastinal shift), and reduced mobility of the lung and thoracic cage (see fig. 27 ). decortication is the only potentially effective treatment of fibrothorax in patients with severe respiratory compromise. 83, 84 a trapped lung is characterized by the inability of the lung to expand and fill the thoracic cavity because of a restrictive, fibrous, visceral pleural peel (see fig. 27 ). restriction of lung parenchymal expansion and subsequent negative pressure in the pleural space result in filling of the pleural space with pleural fluid (usually a transudate). the diagnosis of a trapped lung implies chronicity, stability over time, and a purely mechanical cause for the persistence of a fluid-filled pleural space. patients with a trapped lung usually do not experience improvement in dyspnea after thoracentesis. in symptomatic patients, decortication should be considered. the underlying lung parenchyma should be assessed before decortication. if the trapped lung is severely diseased and fibrotic, decortication is unlikely to result in lung reexpansion and the procedure does not provide symptomatic benefit. in contrast, lung entrapment is the result of an active inflammatory process or malignancy in the pleural space, leading to a restricted pleural space. pleural fluid from lung entrapment is an exudate, and symptoms in patients with lung entrapment typically improve after thoracentesis. 83, 85 fibrosing mediastinitis fibrosing mediastinitis is a rare condition characterized by proliferation of fibrous tissue within the mediastinum. symptoms are related to compression of the central airways, superior vena cava, pulmonary veins, pulmonary arteries, and esophagus. the most common cause of this disorder is fungal infection, especially histoplasma capsulatum in the united states. 86 pneumonia is an infection of the lung parenchyma caused by a wide variety of organisms in pediatric patients. imaging evaluation plays an important role in children with pneumonia by detecting the presence of pneumonia and determining its location and extent, excluding other thoracic causes of respiratory symptoms, and showing complications such as effusion/empyema and suppurative lung changes. clear understanding of the underlying potential cause, current role of imaging, proper imaging techniques, and characteristic imaging appearances of acute and chronic pneumonias can guide optimal management of pediatric patients with pneumonia. who child health epidemiology reference group. global estimate of the incidence of clinical pneumonia among children under five years of age pneumonia: an eleven-year study in a pediatric practice incidence of community-acquired pneumonia in the population of four municipalities in eastern finland pneumonia: the leading killer of children what imaging should we perform for the diagnosis and management of pulmonary infections? differentiation of bacterial and viral pneumonia in children aetiology of community-acquired pneumonia in children treated in hospital ultrasound of the chest in children (mediastinum excluded) us in the diagnosis of pediatric chest diseases multidetector ct in children: current concepts and dose reduction strategies computed tomography scanning techniques for the evaluation of cystic fibrosis lung disease increased inspiratory pressure for reduction of atelectasis in children anesthetized for ct scan magnetic resonance imaging of lung infections in children follow-up of acute pulmonary complications in cystic fibrosis by magnetic resonance imaging the role of advanced imaging techniques in cystic fibrosis follow-up: is there a place for mri? basic patterns in lung disease the epidemiology of acute respiratory tract infection in young children: comparison of findings from several developing countries susceptibility to bronchiolitis in infants the chest radiograph in acute bronchiolitis moffet's pediatric infectious diseases: a problem-oriented approach the many radiologic facies of pneumococcal pneumonia round pneumonia: imaging findings in a large series of children review of new and newly discovered respiratory tract viruses in children. pediatr emerg care tuberculosis in children: an update tuberculosis in children: an update chest radiographic features of lymphocytic interstitial pneumonitis in hiv-infected children clinical and radiographic spectrum of septic pulmonary embolism aspiration lung disease recurrent pneumonia in children: clinical profile and underlying causes chest radiographs of near-drowned children respiratory complications of accidental drownings in children hydrocarbon pneumonitis squalene aspiration pneumonia in children: radiographic and ct findings as the first clue to diagnosis lipoid pneumonia in children following aspiration of mineral oil used in the treatment of constipation: highresolution ct findings in 17 patients lipoid pneumonia presenting as non resolving community acquired pneumonia: a case report foreign body inhalation in children: an update utilization of low dose multidetector ct and virtual bronchoscopy in children with suspected foreign body aspiration pulmonary infiltrates with eosinophilia syndromes in children parasitic pulmonary eosinophilia peripheral opacities in chronic eosinophilic pneumonia: the photographic negative of pulmonary edema eosinophilic lung diseases: state of the art juvenile laryngeal papillomatosis with pulmonary parenchymal spread. case report and review of the literature recurrent pneumonia in children: a case report and approach to diagnosis assessment of the child with recurrent chest infections radiological imaging of the neonatal chest intra-amniotic infection and premature rupture of the membranes imaging of medical disease of the newborn lung neonatal radiology. acquired diaphragmatic hernia with group b streptococcal pneumonia radiographic findings in early onset neonatal group b streptococcal septicemia chlamydia trachomatis in children pertussis with severe pulmonary hypertension in a newborn with good outcome-case report malignant pertussis in the pediatric intensive care unit principles and practice of pediatric infectious diseases revised reprint fungal pulmonary infections after bone marrow transplantation: evaluation with radiography and ct investigation and management of a child who is immunocompromised and neutropoenic with pulmonary infiltrates ct halo sign: the spectrum of pulmonary diseases thoracic mycoses from opportunistic fungi: radiologic-pathologic correlation thoracic disease in children with aids childhood leukemia: diagnostic accuracy of bedside chest radiography for severe pulmonary complications acute lung disease in the immunocompromised host: ct and pathologic examination findings cytomegalovirus pneumonia in transplant patients: ct findings pulmonary infections in hiv-positive children pulmonary infections after bone marrow transplantation: high-resolution ct findings in 111 patients pulmonary infections following bone marrow transplantation: high-resolution ct findings in 35 paediatric patients pneumonia in children: decreased parenchymal contrast enhancement-ct sign of intense illness and impending cavitary necrosis cavitary necrosis complicating pneumonia in children: sequential findings on chest radiography lung abscess in children lung abscess in a child with mycoplasma pneumoniae infection peripheral bronchopleural fistula: ct evaluation in 20 patients with pneumonia, empyema, or postoperative air leak multi-detector row computed tomographic evaluation of bronchopleural fistula: correlation with clinical, bronchoscopic, and surgical findings gangrene of the lung: treatment in two stages massive pulmonary gangrene pulmonary gangrene. a complication of bacterial pneumonia pulmonary gangrene complicating bacterial pneumonia imaging of parapneumonic pleural effusions and empyema in children therapy of parapneumonic effusions in children: videoassisted thoracoscopic surgery versus conventional thoracostomy drainage management of empyema in children ct appearance of parapneumonic effusions in children: findings are not specific for empyema an investigation into causative factors in patients with bronchiectasis the pathophysiology of bronchiectasis bronchiectasis: an update bronchiolar disorders: a clinical-radiological diagnostic algorithm pleural fibrosis physiological evaluation of results of pulmonary decortication trapped lung sclerosing mediastinitis mimicking anterior mediastinal tumor key: cord-284974-e7vl774c authors: filipovic, n.; saveljic, i.; hamada, k.; tsuda, a. title: abrupt deterioration of covid-19 patients and spreading of sars cov-2 virions in the lungs date: 2020-11-02 journal: ann biomed eng doi: 10.1007/s10439-020-02676-w sha: doc_id: 284974 cord_uid: e7vl774c a unique feature of covid-19 interstitial pneumonia is an abrupt progression to respiratory failure. our calculation shows that this abrupt deteriorate may be caused by a sudden shift in the spread of virus-laden bioaerosols through the airways to many different regions of the lungs from the initial site of infection. electronic supplementary material: the online version of this article (10.1007/s10439-020-02676-w) contains supplementary material, which is available to authorized users. on the cdc website 1 it says ''clinicians should be aware of the potential for some patients (of to rapidly deteriorate one week after illness onset''. the concept of ''cytokine storm'' 4 is discussed to explain this rapid deterioration. but why does this happen several days after the initial infection? while many mechanisms possibly contribute to spread of the infection, such as lymphatic circulation, we hypothesize that a major driver of the abrupt deterioration several days after the initial infection is caused by a sudden shift in the spread of virus-laden bioaerosols through the airways to many different regions of the lungs from the initial site of infection. clinically, severe sars cov-2 infections exhibit multi-region patchy patterns of ground-glass opacity (ggo) throughout the lungs detected by computed tomography (ct). 3 this ct presentation is a unique characteristic of covid-19 and is consistent with our hypothesis. to explore this possibility, we have made a computer simulation. suppose some part of the lung is initially infected (figure 1a ). pulmonary edema develops locally and a meniscus with virus-laden fluid may be formed. during breathing, the meniscus is stretched and can rupture, releasing a number of small droplets called bioaerosols. 2 the bioaerosols move toward the airway opening with the expired air; while most of the bioaerosols exit the body, some of them remain suspended in the airways. in the next inhalation, some of the exited particles may reenter the lungs with little deposition due to the low level of diffusion. the particles, which remain in the lung, are likely to retrace their path back to the original position (figure 1b) . as the disease progresses, however, the distribution of airflow in the lungs changes. the infected part of the lungs contributes to a decreased volume of airflow, altering the airflow dynamics, and, at the same time, an increased production of virus-laden bioaerosols. this shift of airflow pattern distributes virus-laden bioaersols to other, even distant, parts of the lungs (figure 1c) . if multiple areas of the lungs are simultaneously infected, an overwhelming innate immune response may occur resulting in a widely-distributed ''cytokine storm''. considering the negligible (but non-zero) diffusivity of bioaerosols, the observation that the deterioration occurs several days after the initial infection. our hypothesis suggests that targeting therapies to slow primary ggo lesion growth and to control airflow patterns could slow intra-lung viral spreading and help prevent the rapid respiratory deterioration observed in severe covid-19 patients. figure. (a) suppose some part of the lung is initially infected by inhaled sars cov-2 virus (in this example, we assume that the left upper lobe is initially infected). as a result, near the lesion, virus-laden meniscus may be formed and a rupture of those menisci may form a number of virus-laden small bioaerosols (shown as red dots). (b) those bioaerosols move toward the airway opening with the expired air. while most of them exit from the body, some of them may remain suspended in the airways. some of particles, which exit the body, may reenter the lungs in the next inhalation and move around the lungs, but deposition of those reentered particles may be minuscule due to their low diffusivity. some of the particles, which remain suspended in the airways retrace back to their original position due to the negligible (but no zero) diffusivity. (c) however, as the disease progresses, the production of virus-laden bioaerosols increases, the tissues available for gas exchange become smaller and smaller as the infected lesion becomes larger and larger. this may dramatically alter the distribution of airflow in the lungs, resulting in a substantial spread the infection to other parts of the lungs. this may cause cytokine storm. a dramatic change of flow distribution in the lungs can be seen as a significant alteration cross sectional map pattern from figs. 1a, to 1c. cdc coronavirus disease propagation and breakup of liquid menisci and aerosol generation in small airways covid-19): a systematic review of imaging findings in 919 patients into the eye of the cytokine storm. microbiol key: cord-006653-fy0yg0xh authors: popper, helmut h. title: interstitial lung diseases—can pathologists arrive at an etiology-based diagnosis? a critical update date: 2012-12-07 journal: virchows arch doi: 10.1007/s00428-012-1305-0 sha: doc_id: 6653 cord_uid: fy0yg0xh interstitial lung diseases (ild) encompass a group of diseases with a wide range of etiologies and a variety of tissue reactions within the lung. in many instances, a careful evaluation of the tissue reactions will result in a specific diagnosis or at least in a narrow range of differentials, which will assist the clinician to arrive at a definite diagnosis, when combining our interpretation with the clinical presentation of the patient and high-resolution computed tomography. in this review, we will exclude granulomatous pneumonias as well as vascular diseases (primary arterial pulmonary hypertension and vasculitis); however, pulmonary hypertension as a complication of interstitial processes will be mentioned. few entities of pneumoconiosis presenting as an interstitial process will be included, whereas those with granulomatous reactions will be excluded. drug reactions will be touched on within interstitial pneumonias, but will not be a major focus. in contrast to the present-day preferred descriptive pattern recognition, it is the author’s strong belief that pathologists should always try to dig out the etiology from a tissue specimen and not being satisfied with just a pattern description. it is the difference of sorting tissue reactions into boxes by their main pattern, without recognizing minor or minute reactions, which sometimes will guide one to the correct etiology-oriented interpretation. in the author’s personal perspective, tissue reactions can even be sorted by their timeliness, and therefore, ordered by the time of appearance, providing an insight into the pathogenesis and course of a disease. also, underlying immune mechanisms will be discussed briefly as far as they are essential to understand the disease. electronic supplementary material: the online version of this article (doi:10.1007/s00428-012-1305-0) contains supplementary material, which is available to authorized users. interstitial lung diseases (ild) are characterized by a diffuse infiltration of both lungs, usually evaluated by highresolution computed tomography (hrct) scan. the types of infiltrating cells are not predefined, so this can be inflammatory as well as tumor cells. tumorous infiltration will be excluded, since there are too many entities, not only carcinomas but also lymphomas and certain sarcomas such as angiosarcomas. therefore, our main focus will be on interstitial pneumonias (ip). many classifications on ip have been published over the last four decades, and our understanding of these processes has improved stepwise and resulted in a refined schema, which can be used to sort these diseases accordingly. however, there are still many problems, making the use of this classification problematic to pathologists, not specifically dealing with lung pathology. originally, liebow [1] proposed a classification based on morphological descriptions, with the following entities: usual interstitial pneumonia (uip), bronchiolitis obliterans-interstitial pneumonia (bip), diffuse alveolar damage (dad, also acute interstitial pneumonia [aip] , clinically corresponding to electronic supplementary material the online version of this article (doi:10.1007/s00428-012-1305-0) contains supplementary material, which is available to authorized users. acute respiratory distress syndrome [ards]), lymphocytic interstitial pneumonia (lip), desquamative interstitial pneumonia (dip), and giant cell interstitial pneumonia (gip). katzenstein's updates from 1993 and 1998 [2, 3] were the next major step, adding nonspecific interstitial pneumonia (nsip) to the list of uip, dip, bip, and aip/dad and removing lip and gip because an etiology could be assigned to them. later on, muller and colby showed a radiologic-pathologic correlation and used the previously created name bronchiolitis obliterans-organizing pneumonia (boop) [4, 5] instead of bip. when these entities were combined with clinical data, it was apparent that there was a major difference between uip and the "rest": patients with uip had a worse prognosis and most of them died within 5 years after diagnosis [6] . another intention to separate idiopathic interstitial pneumonias (iip) from those with known cause was to provide prognostic and therapeutic information to the clinicians: no response of patients with uip/idiopathic pulmonary fibrosis (ipf) towards corticosteroids and immunosuppressive drugs and dismal prognosis, whereas responsiveness of patients with nsip to corticosteroids and immunosuppressive drugs and a better prognosis. the next step happened, when uip and the fibrosing variant of nsip were compared to each other, showing that the initial difference vanished especially when evaluated for 10 years survival [6] . then, ip were classified into idiopathic and those with known etiology: dip and respiratory bronchiolitis with or without interstitial lung disease (rbild) were excluded from idiopathic because, in both entities, cigarette smoking was identified as the main cause of the disorder. lip was also skipped, probably because of a clearly defined etiology in almost all cases, either lymphoma, allergic, or autoimmune diseases. gip was skipped, since it either is induced by hard metal inhalation or viral infection (measles, respiratory syncytial virus [rsv] , and others) [7, 8] . what makes the present-day classification complicated is the combination of radiology, pathology, and pulmonology, resulting in provisional diagnoses or divergent names for pathology and clinics. in addition, the final diagnosis needs to be discussed between clinicians, radiologists and pathologists. this also changed the general view: many clinicians suppose they are the only ones being able to make the diagnosis of ipf, and pathologists in the classification committee have accepted it. there are examples which support such a perspective: organizing pneumonia (op) has a wide variety of etiologic causes, and the idiopathic form cryptogenic organizing pneumonia (cop) needs exclusion of all other causes, which on several occasions can be done by pathologists, but in other cases only by combining morphology with clinical information. furthermore, radiology has gained a major impact on the diagnosis of ild, which resulted in decreasing numbers of patients for whom a pathologic diagnosis is required. it cannot be neglected that pathologists have also contributed to this situation: there are colleagues who are happy to make the diagnosis of, e.g., uip and do not further look for features which would allow the differentiation between uip/ipf versus other causes of uip, e.g., collagen vascular diseases (cvd). based on recommendations from a joint committee established by the european respiratory society (ers) and american thoracic society (ats) pathologists, radiologists, and pulmonologists proposed a new classification and also a diagnostic algorithm for ild [9, 10] (table 1 ; fig. 1 ). in this review, i will discuss iip following the schema shown in fig. 1 . when discussing ip with known etiology, i will group these diseases according to their etiologic basis. therefore, some of the morphologic patterns of idiopathic ip will be recapitulated. finally, ild with various etiologies will be discussed at the end of this review. granulomatous pneumonias including extrinsic allergic alveolitis/hypersensitivity pneumonia (eaa/hp) will be excluded because this alone would fill a review on its own. however, in some of the diseases to be discussed, granulomas do appear and, therefore, will be explained briefly. this review is based on the personal experience of the author being responsible for the diagnostic workup at the medical university of graz, but also acting as a consultant for many central european hospitals, which allowed me to set up a huge lung and pleura biobank. in addition, several published articles and reviews in this area including published classifications by the ats/ers joint committees on ild are critically reviewed. usual interstitial pneumonia/idiopathic pulmonary fibrosis uip/ipf is a chronic progressive fibrosing disease of the lung, which leads to death of the patient usually within 5-10 years after the diagnosis is made. it affects predominantly patients in their fourth to fifth decade of life; however, lesions may occur much earlier and remain undetected until acute interstitial pneumonia (aip) diffuse alveolar damage (dad) they will cause impaired lung function by their increasing number-uip/ipf is seen more often in younger-aged patients, probably due to increased awareness. characteristically, lesions are found in both lower lobes with a predominance of subpleural regions. the involvement of both lobes is most often symmetrical. by hrct, different features can be seen: fibrotic and scar lesions, ground glass areas, and honeycombing. cystic lesions, consolidations, and scars are found on gross cut surface. the pleura usually show multiple retractions, giving the surface a cobblestone appearance, but pleuritis is not seen. the histological hallmarks are fibroblastic foci, scars and diffuse fibrosis, honeycomb areas, and uninvolved areas in between (figs. 2 and 27; suppl. fig. 28 ). the cause and the etiology of ipf/uip are not well understood. there is a working hypothesis, which can explain some of the features. the disease starts with an as yet unidentified epithelial injury causing apoptosis of pneumocytes [11] [12] [13] [14] . inflammatory signals released by the dying pneumocytes cause transformation and proliferation of fibroblasts and myofibroblasts in a myxoid stroma and repair [15] (so-called fibroblastic focus). genetic abnormalities may underlie these apoptotic responses: in the recent years, research in familial forms of ipf has highlighted the importance of surfactant apoproteins in maintaining homeostasis between injury and repair and that mutations in the surfactant apoprotein c gene might be causally related to the development of familial ipf [16] . in these familial ipf, mutations in genes encoding surfactant apoprotein c and a2 increases endoplasmic stress reactions in pneumocytes type ii, and in addition, mutations in the telomerase genes tert and terc are responsible for telomere shortening, probably decreasing the pool of peripheral lung stem cells and thus impairing repair and regeneration [17] . this later defects are also found in sporadic ipf cases. therefore, inhalation of any kind of toxic material from the environment might cause an overwhelming oxygen stress reaction leading to increased apoptosis of pneumocytes and impaired regeneration [18] . this fits quite well into the epidemiology of ipf patients: the majority are smokers, and some have a history of environmental dust exposure [19, 20] . there is also evidence of epithelialmesenchymal transition (emt) of pneumocytes into myofibroblasts (fig. 3) , and also scattered bone marrow-derived mesenchymal stem cells seem to move into these foci [21] [22] [23] . these foci undergo maturation with collagen deposition, and finally, the process results in fibrosis of alveolar septa and bronchiolar walls [13] . this in turn causes obstruction of the terminal airways resulting in cystic destruction of the remaining peripheral lobules, giving rise to honeycombing and remodeling of the lung parenchyma [14, 24, 25] . the process develops stepwise, which means there are lung lobules not affected yet looking normal, whereas others are destroyed or even completely lost to fibrosis and scarring. this is meant by the term "timely heterogeneity." in the author's experience, a diagnosis of uip/ipf can be established in some cases even without clinical information when the following features are given: fibroblastic foci, timely heterogeneity (involved and uninvolved peripheral lobules), cystic and fibrotic destruction resulting in honeycombing, and most importantly, the absence of inflammatory infiltrates in areas of fibroblastic foci, absence of granulomas, or features of other interstitial inflammation. let us briefly characterize the main morphologic features, since this still causes confusion and misunderstanding: the fibroblastic focus lies within the walls of alveolar and interlobular septa, as well as bronchioles. they do not project into the alveolar lumen. in early stages, they are composed of myofibroblasts and fibroblasts in an immature myxoid matrix. this matrix will stain for immature collagen and reticulin fibers. the overlaying surface is either denuded (no pneumocytes) or can show pneumocyte regeneration with a lot of reactive changes of the nuclei, even epithelial giant cells can be present (figs. 2 and 27a ). when the focus get's older, mature collagen appears and the cells look more like fibrocytes. the overlaying epithelium looks reactive and usually has a type ii or bronchiolar cell appearance. the honeycomb lesion was originally defined by radiologists as a single or multicystic lesion within a fibrotic lung area [26] . given the differences in resolution between hrct and histology, there is a substantial difference in size between the two. pathologically, a so-called honeycomb lesion is a cystic lung lesion involving a secondary lobule. this lobule has lost most of the peripheral alveoli, shows a cystic central area composed of bronchioles and centroacinar structures, covered by a cuboidal and cylindrical epithelium, resembling bronchiolar epithelium and transformed pneumocytes type ii (figs. 2b and 4). in some cases, a pseudostratified squamous-looking epithelium can be present. the cyst walls are fibrotic and often merge with scarred lung tissue or large fibrotic areas involving sometimes a subsegment of the lung. within the lumen, mucus can accumulate, and in late stage, this can be the starting point for secondary infection and bronchopneumonia, causing death of the patient. i prefer the term lobular cystic lung remodeling, instead of honeycombing, because of the size differences between hrct and microscopy (suppl. fig. 28 ). the areas of fibrosis and scarring and the uninvolved lung tissue (heterogeneity) do not need an explanation. but what about inflammation? from what we understand presently, ipf/uip is not an immune-driven or classic emt in a fibroblastic focus: immunohistochemistry using double stain for ttf1 (brown, nuclear) and smooth muscle actin (red, cytoplasmic) in the same cells. magnification, ×200 fig. 4 cystic remodeling of lobules, the histologic correlate to ct scan of honeycombing; bar, 20 μm; compare the size of the cystic lesion to a ct lesion inflammatory disease. therefore, we do not expect inflammatory cells within the fibroblastic foci. if lymphocytes appear in numbers (>10/hpf) within a fibroblast focus, this should raise the possibility of an underlying immune reaction. the appearance of granulocytes within these foci should prompt the search of remnants of hyaline membranes because this may represent organizing dad. the ats/ers recommends that a panel of experts composed of pulmonologists, radiologists, and pathologists (clinical-radiological-pathological [crp]) should make the diagnosis of ipf. the clinical presentation and course, the hrct picture, and the pathologic pattern of uip should be combined. in some cases, the diagnosis of ipf can be based on clinical and ct findings alone. whenever pathologic evaluation is involved, a diagnosis of uip is mandatory for the diagnosis of ipf. acute exacerbation of uip/ipf is clinically characterized by rapid worsening of the patient's symptoms, with severe hypoxia most often requiring mechanical ventilation and oxygen supply. many patients will die under this condition. histologically, two types of acute exacerbations can be seen when examining autopsy cases: secondary infection with infectious pneumonia in the background of uip or multiple fibroblastic foci and severe fibrosis leaving not much lung parenchyma for ventilation. in these latter cases, there is usually severe lung edema present. if a viral infection is present, the histological pattern is dad [27] overlaying uip; if bacterial or fungal infection causes exacerbation, a purulent bronchopneumonia is found. besides, in ipf, a uip pattern can occur in many other diseases, such as autoimmune diseases, allergic diseases, toxic inhalation, drug-induced pneumonias, and many more. this still causes a lot of confusion because the term uip is not used uniformly: some authors use uip strictly in the sense of ipf, others do not care about etiology and simply diagnose uip as a pattern, and a third group discerns uip and uip-like tissue reactions. the same happens with clinicians: most think that a uip diagnosis already means ipf and are confused to learn that uip can present in chronic eaa as well as drug reactions for example. we will discuss these later on. nsip is a diffuse ip, characterized by loose lymphocytic, macrophagocytic, and histiocytic cell infiltration within alveolar septa combined with mild fibrosis. there is no timely heterogeneity, meaning that the lesions seem to have appeared at the same time. hyperplasia of the bronchus-associated lymphoid tissue (balt) is usually not present [28, 29] . the lung architecture is preserved in contrast to uip, and cystic destruction is absent. two forms are discerned, which in some cases might represent timely sequences of the disease: the cellular and fibrotic types (figs. 5 and 6). both behave differently; the cellular type has a better prognosis, whereas the fibrotic variant is more close to uip [6] . in the etiologic background, nsip is most often associated with autoimmune diseases, especially with cvd [30] [31] [32] [33] [34] . an association with druginduced pneumonia and also with allergic diseases such as eaa/hp has also been reported [35, 36] . only those cases without an identifiable etiology are labeled as idiopathic nsip. however, the morphologic pattern is identical; therefore, in most instances, idiopathic nsip remains a clinical diagnosis. clinically, nsip shows diffuse infiltrations, corresponding to ground glass opacities on hrct. symptoms as in the other ild are quite unspecific. many patients with nsip will respond to corticosteroid and/or immunosuppressive drug treatment, and also spontaneous resolution of the disease has been reported [37, 38] . cop is a diagnosis of exclusion, based on the morphology of op (formerly boop). on hrct, op/cop shows a pattern with combinations of ground glass opacities and consolidations and the almost diagnostic tree-in-bud pattern, sometimes also reticulonodular pattern [39] . in rare cases, the consolidation can mimic a tumor [40] . histologically, the hallmark of op is an intra-alveolar granulation tissue, the so-called masson body (figs. 7 and 27). it consists of proliferating fibroblasts and myofibroblasts with inflammatory cells like neutrophils, lymphocytes, histiocytes, and macrophages. few hemosiderin-laden macrophages are often present. the granulation tissue can start from the wall of bronchi, bronchioli, and alveoli. there is usually a defect of the epithelial layer and also the basal lamina. fibroblasts and myofibroblasts grow into the defect; however, in contrast to normal repair, the granulation tissue does not stop but continuously grows into the airspaces, filling these completely or incompletely. in later stages, pneumocytes will grow over these granulation tissue plugs and, therefore, a slit-like airspace can be formed (fig. 8 ) [40] . the amount of inflammatory cells within the granulation tissue depends on the cause of op. the morphologic pattern of op has a very wide range of etiologies. it can occur as a post-infectious process, in inactive or resolving stages of autoimmune disease/cvd and vasculitis, in toxin inhalation, in drug-induced lung diseases, in chronic inflammatory bowel diseases, or idiopathic, which is cop [41] [42] [43] [44] . in some cases of op, the etiologic cause can be determined, for example, by viral inclusion bodies in postviral op ( fig. 9 ) or by endothelial cell reactions in druginduced op. in some cases, an additional pathologic tissue reaction besides op can also point to the underlying etiology. so what are the diagnostic features? & granulation tissue growing into bronchi, bronchioles and alveoli, usually with remnants of inflammatory cells. & fibrotic occlusion of whole lobules or remaining slitlike spaces covered by pneumocytes. & a mixture of inflammatory cells within these granulation tissue plugs depending on the cause of previous damage. if looking for the etiology, one should also closely investigate the small blood vessels and the regenerating pneumocytes: viral inclusion bodies might be still visible, scattered neutrophilic granulocytes can be found in the granulation tissue in cases of bacterial or fungal infection, and eosinophils might be seen pointing to a previous drug-induced pneumonia. in virus-induced pneumonias, another feature can be found, even after several months: single transformed pneumocytes showing atypical nuclei and a homogenously stained smudged chromatin pattern (suppl. fig. 29 ). in drug-induced and metabolic as well as in autoimmune diseases, the vascular walls can show various structural changes, making an etiology-based diagnosis probable: eccentric vasculopathy with scattered lymphocytes and without endothelial damage might point to deposition of idiotypic-anti-idiotypic immune complexes (without complement activation; fig. 15 ; suppl. fig. 30 ), endothelial damage with fibrosis and repair can point towards druginduced damage (suppl. fig. 29 ). cop as a crp diagnosis is a diagnosis of exclusion: if all possibly underlying diseases are excluded, cop can be diagnosed. this has some importance, since cop responds well to corticosteroid treatment. clinically, aip (also ards) is characterized by acute onset of severe hypoxia, with the radiological appearance of white lung. histologically, there is edema and fibrinous exudate, widened edematous alveolar septa. later on, hyaline membranes are formed (dad). inflammatory infiltrates are usually scarce. depending on the cause of dad, neutrophilic and/or eosinophilic granulocytes can be found in bacterial, toxic, or drug-induced dad or scattered lymphocytes are seen in viral and rickettsial infections [45, 46] (fig. 9 ). inflammatory infiltrates may be even absent such as in various kinds of shock. rarely, cases of "idiopathic aip" have been reported. probably, some of these cases represent cases of undiagnosed systemic lupus erythematosus (sle) or drug toxicity. in the author's experience, in all cases sent for consultation and primarily labeled as idiopathic dad, an etiology could finally be established. so it might be questioned, if idiopathic dad does exist. hamman and rich described an ip with fulminant course, leading to death in their six cases within 6 months. in the authors' description, there was no hyaline membrane mentioned, but a proliferation of fibroblasts. since the tissues from these cases were all lost, this disease cannot be reconstructed and remains an enigma [47] . the sequence of events in dad is largely dependent on the cause: toxic metabolites of drugs or released collagenase and elastase from necrotizing pancreatitis will cause endothelial damage, followed by leakage of the small peripheral blood vessels. this causes edema, followed by pneumocyte cell death (hypoxia). serum proteins will pass into the alveolar lumina, coagulate there, and by the breathing movements are compressed into hyaline membranes. in case of airborne disease, e.g., infection or inhaled toxins, pneumocytes type i die followed by type ii. the basement membrane is either preserved or destroyed (especially in viral infection). this again causes leakage of capillaries, edema with/ without bleeding, protein extravasation into the alveoli, and finally, formation of hyaline membranes. the lethality of dad is still high despite improvements which have been made in the past decade. in some cases, the progression of the disease might be blocked by antiprotease treatment [48] . in more recent time, extracorporeal oxygenation or nitric oxide treatment has shown some benefit. if the patient survives the acute phase, dad will be organized, which is essentially an op-type of lesion, but is most often labeled as organizing dad: granulation tissue grows into the alveoli and hyaline membranes are incorporated into the plugs. they can be demonstrated several weeks after the initial injury. if a tissue biopsy or an autopsy specimen is available early on in the course of the disease, the etiology might be elucidated. in viral infection, inclusion bodies can be seen, later followed by atypical proliferation and transformation of pneumocytes type ii. in contrast to atypical pneumocyte hyperplasia (aah), these atypical cells are single, do not form a continuous layer along the alveolar wall, and usually show bizarre nuclei and nucleoli. rickettsial infection results in less pronounced proliferation of pneumocytes. in shock-and drug-induced dad, the endothelia will undergo apoptosis and necrosis, and fibrin cloths might be seen in capillaries. in these cases, the alveolar septa are widened and edematous. inflammatory cells are scarce or absent. in later stages of drug-induced pneumonia, scattered eosinophils are encountered-their function being completely unknown in these cases. what are the characteristics of dad? & edematous fluid accumulation in alveoli and in the interstitium (depending on the time course), & fibrin cloths in alveoli with/without hyaline membranes, & scarce inflammatory infiltrates (neutrophils and/or lymphocytes, etiology-dependent), & minor diagnostic but etiologically important features are damage of pneumocytes, endothelial cells, fibrin thrombi in small blood vessels, and regeneration±atypia. lip almost vanished from the literature in the last 5 years. the major problem is the separation from nsip. when nsip was described, it was never clearly separated from lip [49] . when comparing my own cases and reports from the literature, it becomes evident that differences do exist: in lip, the lymphocytic and plasmocytic infiltration is dense, hyperplasia of the balt is common, and within lymph follicles, germinal centers are usually present [49] . the infiltration in lip is more diffuse, architectural distortion is common, and scarring does occur. histiocytic and monocytic cellular infiltrations are much less pronounced compared to nsip. lymphoepithelial lesions do occur similar to lymphomas, in some entities aggressively infiltrating and destroying the epithelium and in other cases no epithelial disruption does occur. in contrast to nsip, the architecture of the peripheral lung is remodeled, especially in later stages (figs. 10 and 11). the organization phase is often characterized by op. within the etiologic spectrum, similar diseases are found as in nsip: autoimmune diseases especially cvd, allergic diseases such as eaa (acute and subacute), allergic drug reactions, and in children, different types of immunodeficiency (t cell defect, nk cell defect). the most important differential diagnosis, however, are extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (malt/balt) type and lymphomatoid granulomatosis type i. in all cases, the clonality has to be evaluated and a lymphoma needs to be excluded by proof of multiclonality. also, it is important to exclude posttransplant lymphoproliferative disease [50] , which can present in a similar pattern (large lymphoid cells usually epstein-barr virus-positive). however, it should be taken into account that some of the autoimmune diseases have a high propensity of developing non-hodgkin lymphomas later in the course [51] . within the autoimmune diseases, sjøgren's disease (sjs) most often presents with the lip pattern [33, 52] . what are the characteristics of lip? & diffuse dense lymphoplasmocytic infiltrates in alveolar septa and bronchial/bronchiolar walls. in some cases, the lymphocytic infiltration can form concentric rows encasing capillaries and venules; & hyperplasia of balt with well-formed follicular centers; & focal fibrosis and scarring with distortion of the peripheral lung architecture; & lymphoepithelial lesions; & eccentric sclerosis of vessels walls with narrowing of lumina. this is usually a sign of deposition of immune complexes in the vessel walls and should prompt the search for diseases associated with the production of autoantibodies, such as sjs and systemic sclerosis (ssc). in autoimmune-induced ild, many different factors come together: a wide variety of immune reactions can cause a wide variety of tissue reactions, for example, circulating autoantibodies either capable or devoid of complement activation, circulating immune complexes including large insoluble immune complexes formed by idiotypic-antiidiotypic antibody networks, activation of coagulation, metabolism of proinflammatory substances, involvement of different types of leukocytes, and not the least, drugs given for the relieve of symptoms. these drugs themselves can cause toxic or inflammatory side effects. each of the different diseases will induce a different reaction pattern, and this pattern will be modified during the course of the disease. therefore, we cannot expect a single reaction or pattern, but a complex picture composed of new and old lesions, resolving lesions, and acute exacerbations of the disease. it will always be of help to know the mechanisms and underlying pathogenesis of each disease to interpret the histological picture in its presented form. since we have already extensively discussed the different ip, we will focus now on the modifications induced by the autoimmune diseases. rheumatoid lung disease ip in rheumatoid arthritis (rha) is not uncommon; however, it is often complicated by additional drug reactions, which can look like rha-induced pneumonia. most often, nsip is associated with this disease [34, 53] , but these reported studies have in common a selection bias: they looked up the incidence of rha in cases presenting with nsip. the diversity of reaction patterns is much better reflected in studies looking up patients with rha and lung involvement [54] . uip was more common in this study. in our own experience (stacher et al., submitted), most often, a mixture of reaction patterns occurred, such as uip combined with dense lymphocytic infiltrates or lip and uip combined with op or nsip. if features of dad occur, one should discuss drug reactions with the clinicians because many immunosuppressive drugs given in rha and other cvd can cause dad [45, 46, 55, 56] . not uncommonly, granulomas are present too. these are most often foreign body-type granulomas with giant cells, sometimes classical rheumatoid granulomas with palisading histiocytes, and rarely epitheloid cell granulomas [55] . in classical rheumatoid granulomas, it is essential to exclude infectious organisms, since patients receiving immunosuppressive drugs like methotrexate and leflunomide are prone to acquire infections [56] [57] [58] (figs. 12 and 13). this is even more important with the new "biologicals." the etiology of rha is still an enigma. genetic variants for several immune regulators such as toll receptors and interleukins may form the basis for the susceptibility to adversely react against antigens and immune complexes trapped in cartilages and by that induce an inflammatory reaction, resulting in cartilage damage [59, 60] . regulatory t cells either deficient or functionally impaired might also play an important role in rha. in addition to the genetic basis for the disease, the autoimmune reaction might be triggered by streptococcal infections, and in that respect, probably mimicry of proteins of the organism might come into play. in acute lupus manifestation, which is more often autopsied and rarely seen as surgical material, hemorrhagic pneumonia, infarcts, or dad or all mixed are found [61] [62] [63] . most probably, the extent of any of these reactions depend on the extent of intravascular death of neutrophils attacked by lupus autoantibodies: low numbers of dying neutrophils might release less toxic enzymes and, therefore, cause focal endothelial cell death, interstitial edema, proteins leaking out into alveolar spaces, and finally, dad with hyaline membrane formation. in case of massive neutrophilic cell death, there might be massive leakage of vessel walls and hemorrhage will occur. in later stages, dad will be organized, so op is another feature found in systemic lupus. since the disease affects the coagulation cascade, lung infarction is a common feature of sle. i personally have seen, in addition, perivascular amyloidosis in a classical active sle case. the occurrence of pulmonary hypertension is less well understood [64, 65] . the etiology of sle is also not well understood. autoantibodies directed against granulocytic enzymes have been shown to be secondary effects and not the cause of sle. in a recent study in familial forms of sle, a null mutation in the dnase1l3 gene has been found. this finding confirms the critical role of impaired clearance of degraded dna in sle as a probable cause [66] , a finding also seen in adult sle [67] . however, given the wide range of autoantibodies found in sle, there might be much more autoimmune mechanisms involved than anticipated [68, 69] . ssc usually presents with a mixture of tissue reactions, dependent on the immune phenomena present at the time of biopsy. most often, a uip or nsip pattern is found, accompanied by lymphocytic infiltration of lip type and a hyperplasia of balt (suppl. fig. 31 ). germinal centers are less common compared to sjøgren's syndrome. ill-formed granulomas composed of histiocytic and/or epitheloid cells can be seen (fig. 14) . the distribution pattern of the ip is irregular, involving peripheral as well as midzone areas of the lung. this is clinically helpful in separating it from uip/ ipf. another feature helpful in the diagnosis is a vasculopathy. medium-and small-sized arteries show a thickening of the intima and media. within the thickened vessel wall, there is a myxoid change of the matrix. a few lymphocytes can be seen, however, no endothelial necrosis or any other sign of vasculitis. these changes can possibly be best interpreted as a consequence of immune complex deposition (fig. 15 ). functionally, these vascular changes will cause pulmonary hypertension, which is common in ssc [70, 71] . genetic studies provided some new insights into ssc. interleukins, especially il-8, has an impact on lung fibrosis in ssc patients [72] . also, transforming growth factor-beta (tgf-β) and connective tissue growth factor (ctgf) received attention as essential factors in the pathogenesis of ssc. ctgf mrna expression was observed in the fibrotic lesions, serum ctgf concentrations were significantly elevated, and correlated with skin sclerosis and lung fibrosis. in an animal model, tgf-β induced subcutaneous fibrosis and subsequent ctgf application caused persistent fibrosis. based on these data, the authors of this study hypothesized that tgf-β induces fibrosis in the early stage, whereas ctgf acts to maintain tissue fibrosis [73] . in another study, fibrillin has been investigated. it has been demonstrated that caveolin-1 and fibrillin-1 influence storage and regulation of tgf-β and other cytokines and fibrillin-1 mutations might be responsible for a congenital form of scleroderma called stiff skin syndrome [74, 75] . in addition to tissueresident fibroblasts, bone marrow-derived fibroblasts and endothelial and epithelial cells undergoing emt are also under the control of fibrillin. gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of ssc fibroblasts [75] . the impaired expression of the nuclear orphan receptor ppar-γ in ssc seems to play an important role in causing uncontrolled progression of fibrosis through impaired control of fibroblast activation and differentiation [76] . dermatomyositis rarely affects the lung. if lung involvement is present, various forms and combinations of pneumonias can occur. uip and nsip are the most common alterations; however, histiocytic and ill-formed epitheloid cell granulomas can be encountered in some cases in my personal experience. lymphocytic infiltrates are quite common, most often exceeding what is seen in nsip and better matched by lip. vasculopathy is rare. the serosa is usually involved too; however, this is also common in other cvd. polyserositis in contrast to the other cvd cannot be diagnosed on tissues, since these serosal infiltrations are unspecific and occur in a multitude of diseases. sjøgren's disease sjs is another multisystemic collagen vascular or autoimmune disease. it affects predominantly the mucosa of salivary and lacrimal glands, but can also similarly affect the lung. the main finding is an aggressive lymphocytic infiltration into the epithelial lining of bronchi and bronchioles and a diffuse infiltration of the alveolar walls (lip). in the bronchi/bronchioles, lymphoepithelial lesions occur, similar to what is seen in marginal zone lymphomas. the epithelial layer is disrupted, which later on is repaired and can present as op (fig. 10) . the lymphocytic infiltration is polyclonal and composed of t and b lymphocytes. lymphollicles are well formed and will show activated germinal centers. other types of ip can be associated to lip, even uip can occur, usually in the form of uip-lip mixed pattern. as in ssc, interleukins play an important role in sjs too. il-12 overexpressing transgenic mice developed bronchial and alveolar abnormalities such as lymphocytic infiltrates around the bronchi, cell proliferation in the alveolar septa, and increased interstitial and alveolar macrophages, strikingly similar to those found in the lungs of sjøgren's patients. there were also fourfold higher numbers of natural killer cells. a new mouse model highlights the role of il-12 in the initiation of sjøgren's syndrome [77] . rangel-moreno studied the hyperplasia of balt in patients with pulmonary involvement in rha and sjøgren's syndrome. increased expression of cxcl13 and ccl21, as well as b cell-activating factor of the tnf family (baff), icos ligand, and lymphotoxin, correlated with balt hyperplasia. the presence of balt hyperplasia correlated also with tissue damage in the lungs of these patients [78] . in a recent investigation, genes related to one of the major symptoms of sjøgren's syndrome have been identified, namely, the immunological attack of salivary and lacrimal glands resulting in the loss of acinar cell tissue and function, leading to stomatitis sicca and keratoconjunctivitis sicca. one gene lying on chromosome 1 (autoimmune exocrinopathy 2 [aec2]) and the second on chromosome 3 (autoimmune exocrinopathy 1 [aec1]) have been shown to be necessary and sufficient to replicate sjs-like disease in c57bl/6 mice. aec2 lies distal to the centromere. this chromosomal region contains several sets of genes known to correlate with various immunopathological features of sjs. one gene in particular, tumor necrosis factor (ligand) superfamily member 4 (or ox40 ligand), encoding a product whose biological functions correlate with both physiological homeostasis and immune regulations, could be a potential candidate sjs susceptibility gene [79] . although many open questions remain, this mouse model opens the way to better understand this autoimmune disease and also will serve to study the mechanisms, which are responsible for the common development into malt lymphomas. mixed cvd cannot be diagnosed with certainty. the combination of features of two different cvd makes it almost impossible to come up with an etiologic suggestion or proposal. depending on the features of the single cvd, morphologic mixtures can be found. for example, mixed sjøgren-lupus cvd can either have dominant features of sjs or systemic lupus [80] . however, general features suggestive of cvd are usually present: a combination of different features of ip with lymphocytic infiltrations, hemorrhage, etc. so when to think about lung affected by autoimmune diseases? & any combination of ip; & any kind of ip with a high proportion of lymphocytes; & any combination of an ip with other inflammatory reactions not fitting within ips, such as a combination of uip/ nsip or lip with epitheloid or histiocytic granulomas; & any kind of ip with unusual vasculopathy (not sclerosis!) and/or alveolar hemorrhage. goodpasture's syndrome is an autoimmune disease not related to cvd. the cause has been identified recently: by mimicry against bacterial proteins, cross-reacting autoantibodies are formed, which bind to the α-3 chain of collagen iv causing disruption and hemorrhage by complement activation (most often the alternate pathway) [81, 82] . there are still unexplained features such as why collagen iv in glomerular, alveolar, and alveolar capillary basement membranes is attacked, but not those in other organs. the major finding is alveolar hemorrhage without infiltrating leukocytes. later on, macrophages with hemosiderin can be seen within alveoli and septa (fig. 16) . depending on the duration of the disease, the final result is septal fibrosis [83] . other autoimmune diseases affecting the lung ip can be encountered in behcet disease and kikuchi disease, whereas in whipple's disease, granulomatous pneumonia with histiocytes and macrophages is the prominent feature, similar to what is seen in the small bowel [84] . igg4-related sclerosis is an autoimmune disease involving many organs such as the pancreas, mediastinum, and retroperitoneal space. hypergammaglobulinemia and deposition of igg4 is seen in affected tissues. in the lung, the disease usually presents with extensive lymphoplasmocytic infiltration (suppl. fig. 32 ), very often simulating inflammatory pseudotumor (plasmocytic variant); however, in this disease, increased numbers of igg4-positive plasma cells are found (≥25 % of igg-positive cells), not seen in inflammatory pseudotumor (myofibroblastic tumor) [85] . this inflammation typically results in sclerosis of the lung tissue [86] . phospholipid autoantibody-mediated lung disease is another rare disease not only affecting the lung but also other organs [87] . in the affected lung, the major finding is alveolar hemorrhage or hemorrhagic pneumonia and dad (suppl. fig. 33 ), followed by an op in a later stage. the cause is unknown, and the morphologic findings are not specific. the disease needs the proof of phospholipid autoantibodies in the serum. airway-centered interstitial fibrosis (acif) is a new, recently described ild mainly affecting patients with a history of environmental exposure to toxic or allergic substances. also, cocaine abuse was found in one [88] . the morphology is characterized by fibrosis along the small bronchi extending into the peripheral lung following a lobular distribution. in some cases, fibroblastic foci can occur, however, always associated with this distribution pattern. cystic lung remodeling is absent; instead, a whole lobule or subsegment is destroyed by fibrosis. metaplastic epithelium is common in the affected lobules and also hyperplasia of smooth muscle cells (muscular cirrhosis; fig. 17 ). the disease rapidly progresses, and in the reported series, almost half of the patients died of disease. corticosteroid treatment was effective in some patients. here, we will focus on several diseases with quite different morphological features, but all of them related to cigarette smoke exposure, most often in young-aged heavy smokers. combinations of these diseases are currently more often seen, probably due to the increased use of hrct followed by lung biopsy because of the wide variety of differential diagnosis, even including malignant disease. langerhans cell histiocytosis (lhch, histiocytosis x, eosinophilic granuloma) is caused by excessive inhalation of tobacco smoke. it occurs predominantly in young-aged people. it has been postulated that tobacco plant antigens still present within tobacco smoke (incomplete combustion) might cause this accumulation and proliferation of langerhans cells, which are part of the antigen-presenting reticulum cell population [89] [90] [91] . so the continuous exposure of langerhans cells to plant proteins in susceptible persons might cause proliferation of these cells to keep with the increasing amount of antigens. langerhans cells proliferate within bronchial mucosa as well as in the peripheral lung. in bronchi, this proliferation causes necrosis of the mucosa, occlusion of the lumen, and finally, scar tissue [92] (fig. 18 ; suppl. fig. 34 ). langerhans cell proliferation is accompanied by an infiltration of eosinophils, hence the old name eosinophilic granuloma. these eosinophils are attracted by cytokines such as interleukin 4 secreted by the langerhans cells [93] . eosinophilic granulocytes might also act cytotoxic in cooperation with langerhans cells releasing eosinophilic basic proteins and destroy the epithelium. the granulomas undergo regression especially in patients with smoking cessation. the resulting scar has a star-like appearance and is surrounded by bronchiolectasis and emphysema blebs. on ct scan, this results in a characteristic picture (starry sky). an underlying genetic abnormality has recently been identified [94] [95] [96] . mutations in the braf oncogene has resulted that lhch is now regarded as a tumor. however, several issues remain to be solved before this view can be accepted: lung cases are most often induced by smoking and, in many patients, will undergo regression in case of smoking cessation, which is unlikely in a tumor [91] . braf mutations were not found in all cases. on the other hand, there exists a tumorous form, characterized by a multiorgan involvement, seen in children and young adults and not related to smoking. so probably, we might be confronted with two different diseases. so far, the reactive form cannot be discerned from the tumor form other than by involvement of at least two organ systems [97, 98] . further investigations hopefully will increase our knowledge about this disease. in the differential diagnosis, lhch has to be separated from other histiocytosis or reticulum cell proliferations by their positive staining for cd1a and langerin [99, 100] , whereas the positivity for s100 protein is not specific. respiratory bronchiolitis-interstitial lung disease rbild is a common disease in heavy smokers. rb can be seen in many cigarette smokers with lung carcinoma, if the lung tissue adjacent to the tumor is sampled and investigated. rb can also be seen combined with lhch [101] . an accumulation of alveolar macrophages within the respiratory bronchioles and the adjacent centrilobular region of the lung lobules characterize rbild. the macrophages usually contain dirty brownishyellow fine granular pigment (fig. 19) . ultrastructurally, this pigment represents phagolysosomes filled with tobacco waste [102] [103] [104] . functionally, this macrophage accumulation obstructs the terminal bronchioles and impairs airflow, resulting in distension of alveoli and eventually rupture of septa. some authors use the diagnosis of rb only in those cases, where no other pathology is present, others this author included follow the morphology and accept rb diagnosis also in heavy smokers with lung carcinoma in whom rb is also present. the argument is that rb is a disease of smokers, and there is no good argument why smokers are not allowed to have more than one disease, e.g., rb, carcinoma, lhch. however, this results in different statistical figures: if rb diagnosis is only accepted presenting as a singular disease, it is rare; if diagnosed by its morphological features regardless of other smoking induced diseases, it is a common disease, present in many patients with lung cancer. rb and rbild in my opinion are subsequent stages of the same disease. in the early stages, accumulation of alveolar macrophages is concentrated within bronchioles. if tobacco smoke exposure goes on, more and more areas of the centroacinar region of alveoli are occupied by these cells, resulting in radiological ground glass opacities, now clinically called rbild. the term dip was created by liebow in 1965 [105] , long before immunohistochemistry was invented. liebow misinterpreted the cells accumulating within the alveoli as pneumocytes type ii, therefore the term desquamative. by immunohistochemistry, these cells were identified as macrophages [92, 106, 107] . therefore, the name macrophagocytic pneumonia would have been more appropriate. by definition, dip is characterized by an accumulation of pigmented smoker macrophages within alveoli, completely obscuring the peripheral airspaces. no infiltration of bronchioles is present (suppl. fig. 35 ). fibrosis of alveolar septa, if present, is mild. dip can radiologically simulate a tumor with ground glass opacity, not uncommonly misdiagnosed as adenocarcinoma in situ [101] . smoking-induced interstitial fibrosis (srif) and rbassociated ild might represent the same diseases characterized by rb and a paucicellular eosinophilic collagenous thickening of alveolar septa with a subpleural distribution [108, 109] . in some areas, the disease resembles fibrotic nsip, but the typical association with tobacco smoking points to this underlying etiology. in looking up several cases of rb, we also recognized similar reactions as described by s. yousem and a.l. katzenstein, but in addition, also cases showing fibroblastic foci associated with emphysema blebs and fibrosis (fig. 20) . in these cases, also rb could be seen in different areas. in contrast to uip, there were no honeycomb lesions and almost all lobules showed changes of centrilobular emphysema. some of these patients were clinically diagnosed as having chronic obstructive pulmonary disease (copd); in others, the lesions were found incidentally because of pneumothorax. so this might represent another form of smoking-induced lung fibrosis, probably resulting from the release of toxic enzymes from macrophages and subsequent alveolar septa destruction and repair. acute interstitial pneumonia/diffuse alveolar damage many different agents can cause aip/dad. classical ones are viral infections, but toxic inhalation, drug reactions, and all variants of shock reactions will also present with this morphologic picture. the features have been described above, so we need to focus only on specific changes pointing to a specific etiology. in viral infection, the most characteristic feature is the viral inclusion body, which can present either as nice large inclusion bodies (cytomegalovirus [cmv], respiratory syncytial virus [rsv]) or by red-violet-stained nucleic acids forming illdefined speckles in nuclei and/or cytoplasm (adenovirus) [27] . typically, the infected cell shows enlargement, an atypical large bizarre nucleus, and an accentuated nuclear membrane due to increased nucleic acid traffic induced by the virus. these cellular features can last for several months. in contrast to preneoplastic lesions in viral infection, the atypical cells are single cells being grouped together with otherwise normal-looking pneumocytes. giant cell interstitial pneumonia (see also under pneumoconiosis) gip has a quite narrow etiologic spectrum either being caused by hard metal dust or by viral infection. the former will be discussed later. several viruses can cause gip, the classical one being measles virus. however, in contrast to pneumoconiosis in infections, the giant cells are mixed epithelial as well as macrophagocytic. the epithelial giant cells (hecht cells) are transformed pneumocytes type ii in whom nuclear division was not followed by cell division, giving rise to multinucleation [110] . the additional features are identical to dad as described above. especially within the epithelial cells, viral inclusion bodies can be found (suppl. fig. 36 ). besides measles, rsv can also present, with this picture predominantly in children [111] . we have already described op under the idiopathic ilds, so we have only to focus on other causes of op. op can have a great variety of causes. in many cases, this is a post-infectious organization of a purulent bacterial bronchopneumonia, when for several reasons the exudate could not be completely degraded and, therefore, has to be organized by granulation tissue. also, in viral infections, organizing dad is morphologically identical to op, as discussed previously. in other cases, op is a form of organization of an autoimmune disease, usually in the inactive phase. the resolution phase of toxic inhalation is usually in the form of op, and drug-induced lung toxicity is also often organized the same way (suppl. fig. 29 ). chronic pneumonia of infancy originally, surfactant-related ip with alveolar proteinosis were included into chronic pneumonia of infancy (cpi); however, since the different causes of alveolar proteinosis were discovered, it has been excluded. therefore, cpi has been reduced to those pediatric interstitial diseases with unknown cause. it is now quite rare. cpi is characterized by an infiltration of lymphocytes and macrophages/histiocytes in the alveolar septa, accumulation of debris within the alveoli, and hyperplasia of type ii pneumocytes (suppl. fig. 37 ), all causing thickening of the septa and impaired gas exchange. cpi predominantly occurs in newborn or small children [112, 113] . in many instances, a careful investigation of the biopsies might uncover underlying infectious diseases, such as wilson-mikity syndrome and infections caused by respirotropic viruses, chlamydiae, or uroplasms [114] ; another cause might be gastroesophageal reflux [115] . in rare instances, a metabolic disease, interstitial glycogenosis, might be the cause of cpi [116, 117] . however, it should be taken into account that, although the clinical symptoms in affected children are severe, the density of the inflammatory cells is much less compared to pneumonias in adults. surfactant-related interstitial pneumonias-alveolar proteinosis alveolar proteinosis occurs in two forms, either as a genetically inherited disease or as an acquired disease of adults [118] [119] [120] [121] [122] . it is characterized by an accumulation of surfactant lipids and proteins within the alveoli, in severe cases completely obstructing the peripheral parenchyma and causing severe dyspnea. morphologically, there is eosinophilic material within the alveoli sometimes mixed with debris and, in some cases, accompanied by an inflammatory infiltration of the alveolar walls. in the inherited form, the underlying genetic defect is either a mutation in the surfactant apoprotein genes b or c or a mutation in the transporter protein gene abca3 [123, 124] . this results in the production of immature surfactant or an accumulation of surfactant within giant lamellar bodies. this material is secreted but does not function as mature surfactant. the result is alveolar collapse, followed by apoptosis of pneumocytes, infiltration of inflammatory cells (lymphocytes, macrophages), and sometimes also formation of hyaline membranes or fibrin cloths within alveoli. the histological picture simulates but never completely imitates irds (suppl. fig. 38 ). in the acquired form (predominantly adults), different defects of the degradation cascade of surfactant do occur. the most common is a deficiency of granulocyte macrophage colony-stimulating factor (gm-csf) caused by autoantibodies against this protein [118, 120, 121, 125] . gm-csf is necessary for the uptake and subsequent degradation of surfactant by alveolar macrophages. the hallmark is an accumulation of surfactant material within alveoli. there is usually no inflammatory infiltrate present (fig. 21a) . the diagnosis can be even easily made by bronchoalveolar lavage (bal): the recovered fluid looks milky. in later stages, the disease can get chronic. in addition to the accumulation of surfactant lipids and proteins, there is a diffuse interstitial fibrosis, which can cause death of the patient (fig. 21b) . sometimes, alveolar proteinosis can be induced by tuberculosis, acute silicosis, and other diseases; the mechanisms in these are still unclear. although some authors prefer to include idiopathic neuroendocrine hyperplasia of infancy (inhi) into pediatric ild, i do not follow this line because inhi does not present with diffuse interstitial infiltrations but rather with an increase of neuroendocrine cells within bronchi and bronchioles and less pronounced neuroendocrine bodies in the periphery [126] . idiopathic pulmonary hemosiderosis is an interstitial disease usually found in children, but also young adults. the characteristic clinical feature is recurrent alveolar hemorrhage and morphologically diffuse lymphocytic and histiocytic/macrophage infiltrations in both lungs. the etiology is largely unknown. many patients develop iron deficiency anemia due to recurrent bleeding. there is usually a high mortality rate. a degradation of elastic fibers followed by an incrustation by iron-containing proteins (prussian blue-positive) can be seen on histologic examination and is a diagnostic feature. usually, foreign body giant cells are found in the areas of ongoing destruction of the elastic fibers ingesting the fibers together with the iron coat [127] (suppl. fig. 39 ). in later stages, interstitial fibrosis results. vasculitis, granulomatous inflammation, or immunoglobulin deposits are absent. corticosteroids alone or in combination with other immunosuppressive agents may be effective for either exacerbations or maintenance therapy of idiopathic pulmonary hemosiderosis [128] . lymphangioleiomyomatosis lymphangioleiomyomatosis (lam) is an inherited disease, based on the dysfunction of tsc genes. tsc1 coding for hamartin and tsc2 coding for tuberin are both required for controlling the expression and regulation of mtor and the mtor complexes torc1/2. both proteins form a complex, which activates camp kinase, which subsequently inactivates mtor. in lam, tsc2 is more often mutated then tsc1 [129] [130] [131] [132] . tuberin is thus not functional and does not form heterodimers with hamartin, and torc1/2 is constantly activated. tsc mutation can be a germline mutation as in tuberous sclerosis syndrome or mutated somatically, called by some authors as "form fruste" [133] . the second allele is most often mutated somatically. the type of mutation also dictates the extent of syndromes and tumors associated with this mutation. patients carrying the germline mutation present with genetic instability causing several somatic mutations and, therefore, in addition to lam, can present with different other benign tumors. previously, lung transplantation was one of the few choices of treatment; since the discovery of the function of both tsc proteins, a clinical trial with anti-mtor therapy has been started and seems to be effective [134] [135] [136] . interestingly, in rare cases, with lung transplantation, a recurrence of the disease occurred from circulating lam cells, repopulating the transplanted lungs [137] [138] [139] [140] [141] . lam is characterized by a proliferation of immature myoblasts in the periphery of the lung and in lymph nodes. in addition, perivascular epitheloid cells (pec) also proliferate (probably derived from pericytic stem cells). the proportion of myoblasts and pec can vary considerably: in some cases of lam, pec are numerous, in other cases scarce. the cells together form microscopic nodules in the bronchiolar mucosa, along lymphatics and arteries, and in alveolar septa, causing lymphatic obstruction and rupture (chylothorax) and also bronchial obstruction and cystic lung destruction. these myoblasts show immature myofilaments, are not ordered in parallel as normal smooth muscle cells, and also have a more epitheloid appearance. nuclei are round and larger than in regular smooth muscle cells. most importantly, these cells proliferate in locations where no muscle cell proliferation occurs (suppl. fig. 40 ). by immunohistochemistry, the myoblasts express smooth muscle actin, and a few also desmin, whereas the pec express hmb45 [133, 142] . hermansky-pudlak syndrome is an autosomal recessive disease, which results in the inability of several cells to form intracellular vesicles. this can affect the ability to form melanosomes (resulting in albinism), platelet granules (inducing bleeding diathesis), and also phagolysosomes in macrophages and lamellar bodies in type ii pneumocytes [143] . to date, seven forms are known, but only two of them (types i and iv) constantly affect the lung, causing phagocytosis defects with accumulation of macrophages, lamellar body defects in type ii pneumocytes with disturbed surfactant release and macrophage foam cell changes, inflammation, and finally, lung fibrosis [144] [145] [146] [147] . the characteristic morphologic changes are hyperplasia of pneumocytes type ii, giant lamellar bodies (large pas-positive cytoplasmic granules), accumulation of foamy macrophages within the alveoli, lymphocytic interstitial infiltration and lung fibrosis, sometimes as op with intra-alveolar granulation tissue, and also as interstitial fibrosis with myofibroblastic foci, identical to those in uip ( fig. 22 ; suppl. fig. 41 ). even both types of fibrosis op and uip can be present concomitantly in one patient. in contrast to uip/ipf, honeycomb lesions are scarce, and usually, the lung is diffusely involved, leaving not much uninvolved tissue in between (no timely heterogeneity). in contrast to uip/ipf, the involvement of the lung is not symmetric, fibrosis can occur peripherally as well as centrally, and is always patchy. ultrastructurally, there are no well-formed lamellar bodies within pneumocytes type ii. within macrophages, lysosomes are enlarged and irregularly contoured, while phagolysosomes are absent (no fusion of lysosomes with phagosomes). erdheim-chester disease is a rare systemic histiocytosis (non-langerhans dendritic cells) that may present with pulmonary symptoms. the condition seems to be nonfamilial and typically affects middle-aged adults. radiographic and pathologic changes in the long bones are diagnostic, but may mimic multisystemic lhch [148, 149] . patients often present with extraskeletal manifestations. advanced pulmonary lesions are associated with extensive fibrosis that may lead to cardiorespiratory failure [150] . in rare instances, there are diffuse dense infiltrations by histiocytes accompanied by lymphocytes and plasma cells. these histiocytes are negative for langerhans cell markers (cd1a, langerin) and markers for follicular and interdigitating dendritic cells (cd35, cd83), but can be positive for s100 protein (much less intensive staining compare to interdigitating dendritic cells) and are usually positive for cd68, cd163, and lysozyme (suppl. fig. 42 ). mutation within the braf gene has been found in few cases (personal communication). histiocytes can express pdgfrα and pdgfrβ, which might be used as therapeutic targets. diffuse panbronchiolitis (dpb) was first described in patients from southeast asia. it is characterized by an accumulation of macrophages within bronchioles and a lymphocytic infiltration within the bronchiolar walls. hyperplasia of balt does occur and follicle centers might be present, usually related to recurrent infections [151] . in contrast to lip, the infiltration is always concentrated along bronchi and bronchioles. dpb might be difficult to separate from other forms of bronchiolitis. however, there are some features which are of help: in dpb, the obstructive lesions are confined to the respiratory bronchioli; chronic parasinusitis is common; follicular bronchiolitis is a common finding [152] (fig. 23) . the cause is a phenotypic variation in the hla system involving hla-b54, hla-a11, and hla-drb5*010/020 [153] , which leads to susceptibility to otherwise nonpathogenic bacterial infection in immunocompetent children. children with this hla type need to be treated with erythromycin every time a respiratory tract infection occurs. in nontreated children, the disease will cause secondary destruction of the peripheral lung with cyst formation and fibrosis. initially, dpb was mainly identified in children of asian descent; however, this disease has also been diagnosed in caucasians [154] . but it should be mentioned that the diagnosis should be confirmed by morphologic analysis because of similarity with other forms of bronchiolitis, seen by hrct [103, 155] . amyloidosis is a metabolic disease characterized by the deposition of amyloid in lung tissues. amyloidosis is presently regarded as a protein misfolding disease [156, 157] . normally, four proteins are folded into a complex with the help of chaperones. many different forms of amyloid proteins have been identified. misfolding can occur in several different compositions of amyloid [158] . most common are amyloid a and p; however, microglobulin 2β, transthyretin, and others can also be seen in pulmonary amyloidosis. the reason for amyloid deposition can be a tumor; one of the best-known examples is plasmocytoma. in this case, amyloid is formed out of immunoglobulins secreted by the tumor cells. chronic inflammation such as cvd can also cause amyloid deposition [159, 160] . amyloid deposition can occur as a tumor-like deposit, either along the bronchial tree or within the lung periphery (nodular form), or it can be diffuse perivascular and interstitial (suppl. fig. 43 ). amyloid stains red by eosin stain, orange by the congo red stain, or can be demonstrated by immunohistochemistry using specific antibodies for the different components [161] . if a congo red stain is applied, the tissue section should be examined under polarized light, where the stain exhibits a green birefringence. amyloid deposits very often will cause a tissue reaction with foreign body giant cells forming granulomas, and also calcification and metaplastic bone formation can occur. the major differential diagnosis to amyloidosis is igg4-mediated fibrosis and hyalinosis; however, the dense lymphoplasmocytic infiltrations will lead to the correct diagnosis. we will not discuss tracheobronchopathia chondroosteoplastica because this is a localized process in the trachea and large bronchi. although pulmonary ossification would also qualify to be included into metabolic diseases, it is usually a very focal disease and thus does not fit into diffuse interstitial diseases. however, diffuse metabolic calcification and microlithiasis are diffuse interstitial processes and fall herein. metabolic/metastatic pulmonary calcification diffuse calcification of alveolar septa can occur in patients with hyperparathyroidism and hypercalcemia [162] [163] [164] [165] . most often, the kidneys and the stomach, which functionally are also involved in ion exchange, can be affected. in the lungs, calcium deposits are seen along the alveolar septa, forming a network completely outlining the septa (fig. 24 ). there is no inflammatory reaction towards the calcium deposits in contrast to microlithiasis. treatment needs to be focused on the underlying disease; no specific treatment for the calcification does exist. microlithiasis is a diffuse lung disease characterized by a deposition of microliths in alveolar septa and lumina with a foreign body giant cell reaction (fig. 25) . the giant cells phagocytose the microliths. large foreign body cell granulomas can be formed. the microliths usually show a center which can be calcified. the etiology was unknown until recently: first, familial cases were described [166] , and finally, a candidate gene, slc34a2, that encodes a type iib sodium phosphate cotransporter was found to be mutated in all six patients being investigated. slc34a2 is specifically expressed in type ii pneumocytes, and the mutation abolishes the normal protein function [167, 168] . there exist no specific therapy; in severe cases, lung transplantation can be considered [169, 170] . microlithiasis should be clearly separated from lung tissue with alveoliths, i.e., concentric lamellae of proteins around a crystalline center, lying within alveolar lumina without any tissue reaction. eosinophilic lung diseases all together are rare. lhch has already been discussed. we will not discuss bronchial asthma, although it can present with severe eosinophilic infiltrations along the bronchi and bronchioles, but much less in the alveoli. we also exclude churg-strauss syndrome because it would require a discussion of systemic vasculitis. allergic bronchopulmonary mycosis (formerly allergic bronchopulmonary aspergillosis) is not in our focus, since the majority of cases present as a specific form of bronchitis (mucoid impaction) and less frequently as bronchocentric granulomatosis. there are rare forms of allergic bronchopulmonary mycosis presenting as eosinophilic pneumonia, however, not showing any specific feature other then the forms discussed below. eosinophilia is most often induced by the release of interleukins 4 and 5 [171, 172] . it can vary quite remarkably in the different diseases, less pronounced in lhch, whereas massive in eosinophilic pneumonias. by bal, a tentative diagnosis can be made: eosinophil counts in bal usually are between 5 and 20 % in lhch, whereas in the eosinophilic pneumonias, it usually exceeds 30 %. in parasitic diseases, sometimes the parasites (larvae) might be seen in bal. acute eosinophilic pneumonia (aep) is characterized by an acute onset with dyspnea and diffuse infiltrations by eosinophils in both lungs. blood eosinophilia can be present, especially in loeff ler's syndrome and in parasitic infections. on histology, the alveoli and the alveolar and bronchiolar walls are stuffed by eosinophils and macrophages. etiologically, parasitic infection or hypersensitivity reaction for drugs is the cause; however, an idiopathic form also does exist. an infection by helminthes will usually cause mild symptoms, whereas in filarial infections (tropic eosinophilia), the symptoms can be severe (high fever, cough, wheezing, peripheral eosinophilia; suppl. fig. 44 ) [173, 174] . in very rare cases, an allergic reaction for fragments of fungi can present with aep (allergic bronchopulmonary mycosis). many drugs can cause eosinophilia and pneumonia such as beclomethasone, bleomycin, carbamazepine, chlorpromazine, chlorpropamide, cromolyn, dilantin, gold salts, nonsteroidal anti-inflammatory drugs, naproxen, nitrofurantoin, penicillin, phenothiazine, propylthiouracil, phenylbutazone, sulfonamide, tetracycline, and clarithromycin, so a proper investigation of treatment protocols is advised (more details are found on www.pneumotox.com). loeffler's syndrome is a self-limiting condition, resolving usually within 1 month. on ct scan, migratory pulmonary infiltrates can be seen, accompanied by a peripheral blood eosinophilia. in some cases, specific causes such as chronic eosinophilic leukemia can be identified; however, in other cases, no underlying etiology can be identified. in acute idiopathic eosinophilic pneumonia, there is acute febrile illness usually of 1 week duration, with myalgia, chest pain, and hypoxemic respiratory failure. on ct scan, alveolar and interstitial infiltrates are seen; in bal, eosinophils increase over 25 %, however, there is no blood eosinophilia. patients rapidly respond to steroid therapy. no cause can be identified. chronic eosinophilic pneumonia is a serious disease that requires a specific treatment. most often affected are middleaged patients, sometimes also young atopic women (suppl. fig. 45 ). the disease starts with an insidious onset with progressive respiratory symptoms. a history of asthma is present in 50-60 % of cases. some cases can be attributed to drug toxicity (ampicillin, bleomycin, nitrofurantoin, penicillin, streptomycin, tetracycline, and others); in others, hypersensitivity to fungi has been shown. some cases represent chronic infection with parasites. single cases have been described where chronic eosinophilic pneumonia was associated with cocaine or nickel carbonyl vapor inhalation. the characteristic histologic picture shows dense eosinophilic infiltrations accompanied by macrophages and lymphocytes. there can be eosinophilic abscesses. fibroblast proliferations can be seen focally, which finally results in interstitial fibrosis. hypereosinophilic syndrome is a rare disease of unknown cause [175] . it is characterized by increased eosinophilic infiltrations in multiple organs (mainly in the heart and central nervous system, rarely in the lungs). fatal cases have been reported mainly caused by restrictive cardiomyopathy. environmentally induced diffuse interstitial lung diseases (excluding conventional pneumoconiosis) there are two forms of gip, one caused by infection such as measles virus (see above), while the other form caused by inhalation of hard metal dust. hard metal is an alloy composed of cobalt, chromium, tungsten, titanium, and a variety of other metals. in experiments, it has been shown that cobalt is the most toxic compound within this alloy, can easily dissolve out, and will subsequently induce giant cell formation of macrophages [176] [177] [178] (fig. 26) . following the deposition of metal dust the disease starts with respiratory bronchiolitis; giant cells loaded with compound from this alloy are formed early on. the macrophage accumulation spreads further to the periphery, and finally macrophages and giant cells can be found in alveoli, septa, and peribronchiolar. also dad can be seen in some cases. the disease can present with acute illness, shortness of breath, and severe hypoxia. if the patient survives the acute onset, peribronchial/peribronchiolar fibrosis and fibrosis of alveolar septa results [178] [179] [180] . large scars are usually absent. fig. 26 gip in hard metal disease. note the accumulation of foreign body giant cells containing a dirty brown-black material, in this case identified as tungsten and titanium. many other components of the hard metal (cobalt, nickel, and chromium) alloy can dissolve easily and are, therefore, dissolved by fixation and embedding procedures, so mainly tungsten and titanium compounds remain and can be detected by energy-dispersive x-ray spectroscopy. h&e; magnification, ×400 asbestosis asbestosis is another diffuse fibrosing lung disease, which can present with uip or op morphology. in the early phase, it starts with bronchiolitis and peribronchiolar pneumonia. later on, fibrosis with uip morphology can be the predominant feature. in contrast to uip/ipf, the fibrosis is focal, asymmetric in distribution, and early on can involve central portions of the lung. the diagnostic clue is the demonstration of asbestos bodies together with lung fibrosis. drug-induced ild present with a variety of tissue reaction, most of them already discussed in previous chapters. a common presentation is dad as an acute reaction, often followed by organizing dad, where hyaline membranes can still be recognized, and finally, ending as op and lung fibrosis. other drugs induce nsip-like tissue reactions. the major problem in interpreting drug reaction in the lung is our limited understanding of drug metabolism. some drugs will induce toxic injury of endothelial cells, thus the blood barrier is leaking, and proteins can enter the interstitium and finally the alveolar lumen. here, these proteins will form complexes and, by the action of respiration, hyaline membranes will form. in addition, the exudate from the capillaries will cause a transient edema and this is followed by hypoxia affecting the pneumocytes. this damage will contribute to dad development. later on, op can result. other drugs act on the immune system, forming fig. 27 comparison of different types of fibroblast/ myofibroblast proliferations; a, b fibroblastic focus in uip by h&e and movat stains; by movat stain, the immature collagen fibers are stained green, whereas mature collagen (usually collagen 1) stains yellow. c early granulation tissue in op, here bronchiolitis obliterans, d later stage of op; note the proliferating immature blood vessels, quite characteristic in these early phase op. different types of fibrosis in emphysemaassociated lung fibrosis: e scarring with entrapped emphysema blebs and f fibroblastic focus within the wall of an emphysema bleb. magnification; bars, 50, 50, 50, 50, 200 , and 50 μm, respectively immune complexes either because immunogenic by itself or by complexing with endogenous proteins like a hapten. in these cases, an nsip or lip pattern can result. in these cases, scattered eosinophils are regularly found. granulomas are rarely formed, usually pointing to an underlying immune mechanism. since our understanding is so limited and no systematic experimental investigation has been performed, we still need to rely on databases, summarizing all described drug reactions in a systematic way (www.pneumotox.com) (fig. 27 and suppl. fig. 28 ) [45, [181] [182] [183] [184] [185] [186] [187] . the interstitial pneumonias. frontiers of pulmonary radiology pathophysiologic, roentgenographic and radioisotopic considerations idiopathic interstitial pneumonia: classification and diagnosis idiopathic pulmonary fibrosis: clinical relevance of pathologic classification radiologic findings are strongly associated with a pathologic diagnosis of usual interstitial pneumonia nonspecific interstitial pneumonia: correlation between thin-section ct findings and pathologic subgroups in 55 patients idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia chronic granulomatous disease of childhood: respiratory cytology giant cell interstitial pneumonia in a hard-metal worker. cytologic, histologic and analytical electron microscopic investigation european international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. ats/ ers joint statement adopted by the ats board of directors an official ats/ers/jrs/alat statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management the pathogenesis of idiopathic pulmonary fibrosis compartmentalized expression of c-flip in lung tissues of patients with idiopathic pulmonary fibrosis usual interstitial pneumonia pathogenetic mechanisms in usual interstitial pneumonia/idiopathic pulmonary fibrosis transcription factor gata-6 is expressed in quiescent myofibroblasts in idiopathic pulmonary fibrosis nonspecific interstitial pneumonia and usual interstitial pneumonia with mutation in surfactant protein c in familial pulmonary fibrosis idiopathic pulmonary fibrosis: update on genetic discoveries reactive oxygen species are required for maintenance and differentiation of primary lung fibroblasts in idiopathic pulmonary fibrosis prognostic factors for idiopathic pulmonary fibrosis: clinical, physiologic, pathologic, and molecular aspects is idiopathic pulmonary fibrosis an environmental disease? circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis migratory marker expression in fibroblast foci of idiopathic pulmonary fibrosis repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis tnf-alpha sensitizes normal and fibrotic human lung fibroblasts to fas-induced apoptosis extracellular superoxide dismutase has a highly specific localization in idiopathic pulmonary fibrosis/usual interstitial pneumonia diagnosing fibrotic lung disease: when is high-resolution computed tomography sufficient to make a diagnosis of idiopathic pulmonary fibrosis? viral infection in acute exacerbation of idiopathic pulmonary fibrosis prognostic significance of histopathologic subsets in idiopathic pulmonary fibrosis nonspecific interstitial pneumonia. individualization of a clinicopathologic entity in a series of 12 patients nonspecific interstitial pneumonia associated with polymyositis and dermatomyositis: serial high-resolution ct findings and functional correlation histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome the major histopathologic pattern of pulmonary fibrosis in scleroderma is nonspecific interstitial pneumonia variations in histological patterns of interstitial pneumonia between connective tissue disorders and their relationship to prognosis nonspecific interstitial pneumonia pattern as pulmonary involvement of rheumatoid arthritis the pathogenesis of chronic hypersensitivity pneumonitis in common with idiopathic pulmonary fibrosis: expression of apoptotic markers hypersensitivity pneumonitis associated with leflunomide therapy a histologic pattern of nonspecific interstitial pneumonia is associated with a better prognosis than usual interstitial pneumonia in patients with cryptogenic fibrosing alveolitis differences in clinical features and prognosis of interstitial lung diseases between polymyositis and dermatomyositis collagen vascular disease-related lung disease: high-resolution computed tomography findings based on the pathologic classification bronchiolitis obliterans. organizing pneumonia the lung in inflammatory bowel disease noninfectious lung pathology in patients with crohn's disease ultrastructural evidence of alveolar epithelial injury in idiopathic bronchiolitis obliteransorganizing pneumonia bronchiolitis obliterans-organizing pneumonia (boop)-like variant of wegener's granulomatosis. a clinicopathologic study of 16 cases lymphomatoid granulomatosis and diffuse alveolar damage associated with methotrexate therapy in a patient with rheumatoid arthritis diffuse alveolar damage: uncommon manifestation of pulmonary involvement in patients with connective tissue diseases fulminating diffuse interstitial fibrosis of the lungs the gastric juice aspiration syndrome (mendelson syndrome). aspects of pathogenesis and treatment in the pig facts and controversies in the classification of idiopathic interstitial pneumonias epstein-barr virus-associated pneumonia in patients with posttransplant lymphoproliferative disease after hematopoietic stem cell transplantation lymphoproliferation in autoimmunity and sjogren's syndrome interstitial lung disease in primary sjogren syndrome idiopathic non-specific interstitial pneumonia: as an "autoimmune interstitial pneumonia" histopathologic pattern and clinical features of rheumatoid arthritis-associated interstitial lung disease rheumatoid arthritis with extensive lung lesions lethal pneumonitis under leflunomide therapy the treatment of inflammatory arthritis with methotrexate in clinical practice: treatment duration and incidence of adverse drug reactions lethal acute respiratory distress syndrome during anti-tnf-alpha therapy for rheumatoid arthritis stem cell and lung cancer development: blaming the wnt, hh and notch signalling pathway the pathogenesis of rheumatoid arthritis the lung in systemic lupus erythematosus. analysis of the pathologic changes in 120 patients diffuse alveolar hemorrhage in lupus nephritis pulmonary involvement of systemic lupus erythematosus: analysis of 90 necropsies fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated with connective tissue diseases a retrospective study of pulmonary infarction in patients with systemic lupus erythematosus from southern taiwan suppression of stat3 activity sensitizes gefitinibresistant non small cell lung cancer cells dnasei in pathogenesis of systemic lupus erythematosus mutational hotspots in the mitochondrial genome of lung cancer autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in sle patients pulmonary arterial hypertension complicating connective tissue diseases interstitial vascularity in fibrosing alveolitis interleukin-8. differential expression in lone fibrosing alveolitis and systemic sclerosis hypothesis: pathogenesis of systemic sclerosis scleroderma-like properties of skin from caveolin-1-deficient mice: implications for new treatment strategies in patients with fibrosis and systemic sclerosis fibrosis in systemic sclerosis: emerging concepts and implications for targeted therapy fibrosis in systemic sclerosis: emerging concepts and implications for targeted therapy il-12 overexpression in mice as a model for sjogren lung disease inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis identification of possible candidate genes regulating sjogren's syndrome-associated autoimmunity: a potential role for tnfsf4 in autoimmune exocrinopathy the respiratory system in connective tissue disorders t cell epitope mimicry in antiglomerular basement membrane disease zebrafish to humans: evolution of the alpha3-chain of type iv collagen and emergence of the autoimmune epitopes associated with goodpasture syndrome diffuse alveolar hemorrhage syndromes unusual systemic disorders associated with interstitial lung disease igg4-positive plasma cells in inflammatory pseudotumor (plasma cell granuloma) of the lung igg4 related sclerosing disease with multiple organ involvements and response to corticosteroid treatment spectrum of vascular pathology affecting patients with the antiphospholipid syndrome airway-centered interstitial fibrosis: a distinct form of aggressive diffuse lung disease neutrophil priming by cigarette smoke condensate and a tobacco antiidiotypic antibody decreased tobacco-glycoprotein-induced lymphocyte proliferation in vitro in pulmonary eosinophilic granuloma eosinophilic granuloma of lung. clinical aspects of primary histiocytosis in the adult pulmonary langerhans cell histiocytosis: emerging concepts in pathobiology, radiology, and clinical evolution of disease differential in situ cytokine profiles of langerhans-like cells and t cells in langerhans cell histiocytosis: abundant expression of cytokines relevant to disease and treatment recurrent braf mutations in langerhans cell histiocytosis recent advances in the understanding of langerhans cell histiocytosis langerhans cell histiocytosis and erdheim-chester disease langerhans cell histiocytosis research. past, present, and future langerhans cells histiocytosis in one family gene expression during differentiation of human dendritic cells from cord blood cd34 stem cells coexpression of cd1a, langerin and birbeck's granules in langerhans cell histiocytoses (lch) in children: ultrastructural and immunocytochemical studies the overlap between respiratory bronchiolitis and desquamative interstitial pneumonia in pulmonary langerhans cell histiocytosis: high-resolution ct, histologic, and functional correlations respiratory bronchiolitis: a clinicopathologic study in current smokers, exsmokers, and never-smokers bronchiolitis, an update smokingrelated interstitial lung diseases: a concise review desquamative interstitial pneumonia morphology and pathogenesis of desquamative interstitial pneumonitis desquamative interstitial pneumonitis. the intra-alveolar cells are macrophages clinically occult interstitial fibrosis in smokers: classification and significance of a surprisingly common finding in lobectomy specimens respiratory bronchiolitis-associated interstitial lung disease with fibrosis is a lesion distinct from fibrotic nonspecific interstitial pneumonia: a proposal cell-cell fusion induced by measles virus amplifies the type i interferon response giant cell pneumonia due to respiratory syncytial virus. occurrence in severe combined immunodeficiency syndrome chronic pneumonitis of infancy. a unique form of interstitial lung disease occurring in early childhood usual interstitial pneumonitis in infancy. clinical and pathologic evaluation cytomegalovirus associated neonatal pneumonia and wilson-mikity syndrome: a causal relationship? recurrent pulmonary disease in children: a complication of gastroesophageal reflux pulmonary interstitial glycogenosis in the setting of lung growth abnormality: radiographic and pathologic correlation diffuse lung disease in infancy: a proposed classification applied to 259 diagnostic biopsies autoantibodies against granulocyte macrophage colony-stimulating factor are diagnostic for pulmonary alveolar proteinosis pulmonary alveolar proteinosis aerosolized gm-csf ameliorates pulmonary alveolar proteinosis in gm-csf-deficient mice gm-csf regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense mutation of sftpc in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease genetic disorders of surfactant dysfunction genetic disorders influencing lung formation and function at birth elevated il-10 inhibits gm-csf synthesis in pulmonary alveolar proteinosis persistent tachypnea of infancy is associated with neuroendocrine cell hyperplasia idiopathic pulmonary haemosiderosis revisited eight years follow-up of a case with idiopathic pulmonary hemosiderosis after corticosteroid therapy mutational analysis of the tuberous sclerosis gene tsc2 in patients with pulmonary lymphangioleiomyomatosis mutations in the tuberous sclerosis complex gene tsc2 are a cause of sporadic pulmonary lymphangioleiomyomatosis chromosome typing in lymphangioleiomyomatosis of the lung with and without tuberous sclerosis evidence that lymphangiomyomatosis is caused by tsc2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis european respiratory society guidelines for the diagnosis and management of lymphangioleiomyomatosis multicenter phase 2 trial of sirolimus for tuberous sclerosis: kidney angiomyolipomas and other tumors regress and vegf-d levels decrease sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial efficacy and safety of sirolimus in lymphangioleiomyomatosis recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis lymphangioleiomyomatosis: recurrence after single lung transplantation molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis lymphangioleiomyomatosis: recurrence after lung transplantation lymphangiomyomatosis recurrence in the allograft after single-lung transplantation transbronchial biopsy in lymphangiomyomatosis of the lung. hmb45 for diagnosis molecular genetics of the hermansky-pudlak and chediak-higashi syndromes hermansky-pudlak syndrome type 4 (hps-4): clinical and molecular characteristics hermansky-pudlak syndrome type 1: gene organization, novel mutations, and clinical-molecular review of non-puerto rican cases the molecular machinery for the biogenesis of lysosome-related organelles: lessons from hermansky-pudlak syndrome hermansky-pudlak syndrome: a disease of protein trafficking and organelle function erdheim-chester disease with multisystem involvement in a 4-year-old erdheim-chester disease: clinical, radiologic, and histopathologic findings in five patients with interstitial lung disease pulmonary pathology of erdheim-chester disease diffuse panbronchiolitis comparative clinicopathology of obliterative bronchiolitis and diffuse panbronchiolitis association of hla genes with diffuse panbronchiolitis in chinese patients diffuse panbronchiolitis: not just an asian disease: australian case series and review of the literature clinical and histopathological findings in two turkish children with follicular bronchiolitis common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution aspects on human amyloid forms and their fibril polypeptides sjogren's syndrome with multiple bullae and pulmonary nodular amyloidosis pulmonary amyloidosis and unusual lung involvement in sle immunohistochemical classification of amyloid in surgical pathology revisited calcium deposition with or without bone formation in the lung metabolic lung disease: imaging and histopathologic findings metastatic pulmonary calcification in chronic renal failure metastatic pulmonary calcification in a dialysis patient: case report and a review familial occurrence of pulmonary alveolar microlithiasis in 3 siblings mutations in slc34a2 cause pulmonary alveolar microlithiasis and are possibly associated with testicular microlithiasis mutations in the slc34a2 gene are associated with pulmonary alveolar microlithiasis pulmonary alveolar microlithiasis: report on 576 cases published in the literature pulmonary alveolar microlithiasis expression of adhesion molecules in allergic lung diseases il-5 and eosinophilia acute eosinophilic pneumonia: a case report and review of the literature parasitic pulmonary eosinophilia the idiopathic hypereosinophilic syndrome comparative study of the acute lung toxicity of pure cobalt powder and cobalt-tungsten carbide mixture in rat experimental research into the pathogenesis of cobalt/hard metal lung disease pathologic spectrum and lung dust burden in giant cell interstitial pneumonia (hard metal disease/cobalt pneumonitis): review of 100 cases metal-induced diffuse lung disease is giant cell interstitial pneumonitis synonymous with hard metal lung disease? oxidant-induced lung injury in anticancer therapy naphthalene-induced respiratory tract toxicity: metabolic mechanisms of toxicity pulmonary metabolism of foreign compounds: its role in metabolic activation lung injury: cell-specific bioactivation/deactivation of circulating pneumotoxins cryptogenic organizing pneumonitis during oxaliplatin chemotherapy for colorectal cancer: case report fatal pulmonary toxicity after a single dose of cyclophosphamide pathology of drug-induced lung disease key: cord-016659-26zz8kaw authors: chen, feng; ren, meiji; li, hongjun title: influenza date: 2016-06-23 journal: radiology of influenza doi: 10.1007/978-94-024-0908-6_8 sha: doc_id: 16659 cord_uid: 26zz8kaw influenza, abbreviated as flu, is an acute respiratory infectious disease caused by influenza virus, which is mainly spread along with droplets with strong infectivity. the influenza virus may cause epidemics or pandemics of influenza and its incidence ranks the first among legally listed infectious diseases. the prevalence of influenza peaks in autumns and winters, with short illness course and self limitation. however, influenza can be complicated by pneumonia or other serious complications that may cause death in populations of infants, young children, the elderly, those with underlying heart and lung disease and those with compromised immunity. influenza, abbreviated as flu, is an acute respiratory infectious disease caused by influenza virus, which is mainly spread along with droplets with strong infectivity. the influenza virus may cause epidemics or pandemics of influenza and its incidence ranks the first among legally listed infectious diseases. the prevalence of influenza peaks in autumns and winters, with short illness course and self limitation. however, influenza can be complicated by pneumonia or other serious complications that may cause death in populations of infants, young children, the elderly, those with underlying heart and lung disease and those with compromised immunity. in the year of 1971, who officially issued the nomenclature system of influenza virus, and the influenza virus is divided into 3 types based on the antigenic properties of the virus nuclear protein since then, type a, type b and type c. influenza virus is an rna virus, which is the most stable in an environment with a ph value of 6.5-7.9 and is intolerant to high temperature. it loses its pathogenicity after heated to a temperature of 56 °c for several minutes, and can be inactivated at a temperature of 100 °c for 1 min. in an environment with a low temperature, the virus is more stable, being capable of surviving for more than 1 months at a temperature of 4 °c, and more than 5 months at a temperature of −70 °c. the influenza virus is sensitive to dryness, ultraviolet radiation, and commonly used disinfectants such as ethanol and iodophor. the patients with influenza and persons with asymptomatic infection are the main sources of its infection. the infectivity persists from the terminal incubation period to the terminal acute period after onset, and the infectivity is the strongest in the initial 2-3 days after onset. influenza virus exists in the respiratory secretions of the patients or persons with asymptomatic infection and spreads via airborne transmission. by talking, coughing or sneezing, the virus spreads into air along with droplets or aerosols, and causes infection after their being inhaled into susceptible individuals. the virus can also spread via direct or indirect contacts to the mucosa in the oral cavity, nasal cavity, and eyes. populations are generally susceptible to influenza, which is not related to the gender and occupation. after infection, individuals acquire certain immunity. among influenza virus a, b and c as well as different subtypes of influenza a virus, no cross immunity exists. and influenza virus can be repeatedly infected. after infection, the acquired immunity only persists for a short period of time. despite of antibodies in the blood, the person can be infected by the same virus again. the incubation period of influenza lasts for about 13 days, and several hours in some cases. its onset is sudden and acute and the patients mainly experience systemic toxic symptoms but inapparant respiratory symptoms. according to the clini-f. chen • m. ren • h. li (*) beijing youan hospital, capital medical university, beijing, china e-mail: lihongjun00113@126.com cal manifestations, influenza can be divided into the following types. the simplex type is the most common, and is often characterized by sudden onset of aversion to cold and high fever with a body temperature of up to 39-40 °c. fever is the most important initial sign, often accompanied by headache, systemic muscle and joint soreness and pain, fatigue, poor appetite, and other toxic symptoms. some patients may experience such symptoms as photophobia and tears. other symptoms, such as nasal obstruction, runny nose, sore throat, voice hoarseness and other respiratory symptoms also show at the onset. pneumonia type may be secondary to the simplex type or occurs as primary influenza virus pneumonia, which is caused by spread of influenza virus from upper respiratory tract to lower respiratory tract. the pneumonia type commonly occurs in the elderly, children, patients with underlying heart and/or lung disease, pregnancies, and individuals with compromised immunity. the main manifestations include persistent high fever, difficulty breathing, cynosis, severe cough, expectoration of foamy mucous sputum or purulent sputum, expectoration of sputum with blood. the toxic type of influenza is extremely rare in clinical practice, which is caused by invasion of influenza virus into the central nervous system and cardiovascular system, with manifestations of toxic symptoms. clinically, the patients experience symptoms of encephalitis or meningitis, with high fever, coma, delirium, convulsions, and even meningeal irritation sign and diffuse intravascular coagulation that indicate serious condition. the gastrointestinal type of influenza is common in children, and is mainly characterized by nausea, vomiting, diarrhea, and abdominal pain. chest x-ray demonstrates mainly interstitial pneumonia and bronchial pneumonia, initially with poorly defined thickening of the lung markings, predominantly both lower lung field significantly; increased density of the lung markings resembling to ggo. during the progressive stage, the lung fields are demonstrated with grid like opacity and network like nodular opacity, with the nodules smaller than 5 mm. such signs may be concurrently shown with thickened and blurry lung markings, with a distribution in both lower lung fields and around the hilum. in the late stage, cystic changes are shown in different sizes due to bronchiolar inflammatory occlusion in honeycomb like lungs, in addition to shrinkage of lungs, elevated diaphragm and shift of interlobar fissure. ct scan demonstrates small nodular opacity, ggo, tree-bud sign and mosaic like perfusion as well as interlobular septal thickening, subpleural line, adjacent pleural thickening, and pleural effusion. chest x-ray shows alveolar pneumonia (lobar pneumonia) or bronchial pneumonia (lobular pneumonia). alveolar pneumonia is mainly demonstrated as lobar consolidation opacity with high homogeneous density or consolidation opacity with high homogeneous density occupying part of lung lobe, possibly with air bronchogram. due to the different location of the lung lesions, the radiological demonstrations are accordingly different, with one or multiple lung lobes involved. bronchial pneumonia is demonstrated as thickened lung markings, with poorly defined nodular opacity in diameters of 6-8 mm or poorly defined flakes of opacity. large poorly defined patch of opacity with uneven density is the result of overlapping lesions of lobular alveolitis. bronchial obstruction by mucus is demonstrated as lobular atelectasis or focal emphysema in the diseased area. bronchiolar occlusion may cause a small triangular shaped lesion of atelectasis. the lesions are commonly located in the medial parts of both lower lung fields, with more lesions in the posterior lung lobe than in the anterior lung lobe. and the lesions distribute along bronchi, with smooth air flow in the segmental and lobar bronchi. terminal bronchiolar mucosa may be subject to congestion, edema and inflammatory exudation to cause obstructive emphysema, which is demonstrated as increased transparency of both lungs, thoracic extension, widened intercostal space, lowered and flat diaphragmatic muscle. ct scan demonstrates consolidation with uniform shape, lobar distribution and inner air bronchogram as well as poorly defined nodular and patches of opacity in different sizes that distributes along bronchial bundles. in addition, lobular pulmonary atelectasis and focal emphysema are also shown by ct scan. enhanced lung markings. the lesions are commonly located in both lower lung fields and around the hilum. chest ct scan demonstrates small nodular opacity in lungs, ggo, tree-buds sign and mosaic like perfusion, interlobular septal thickening, subpleural line, adjacent pleural thickening and pleural effusion. by radiology, the condition of this case was mild, present difficulty for the diagnosis, which depended on rich experience of the radiological clinician. in this case, influenza virus pneumonia manifested as interstitial pneumonia should be differentiated from other viral pneumonia, such as hand-foot-mouth virus pneumonia and measles virus pneumonia. the initial lesions of hand-foot-mouth virus pneumonia are mainly interstitial changes with an extensive distribution and possible involvement of each lung lobe. the lesions commonly distribute bilaterally, with enhanced and deranged lung markings in both lungs as well as common grid like and cords like opacity. along with the progression of the lesions, chest x-ray demonstrates changes of the lesions, with peripheral spread of the lobular lesions along bronchi and inflammatory consolidation of alveoli and adjacent lung tissue. the lesion may also invade alveolar duct, alveolar sac and alveoli in the lung lobule to cause lobular inflammatory exudation, demonstrated as small patches of increased density opacity confined within lung lobe or segment. there are more exudation opacities in the upper lung lobes than in the lower lung lobes and more in the right lung lobes than in the left lung lobes. chest x-ray demonstrates measles virus pneumonia as flakes or diffuse ground glass opacity and/or thickened bronchovascular bundles. ct scan demonstrates poorly defined centrilobular nodules, ground glass opacities, interlobular septal thickening as well as lobular or segmental consolidations. adenovirus pneumonia is demonstrated as thickened and blurry lung markings as well as small nodular opacities along lung markings in the middle and medial parts of bilateral middle and lower lung fields, possibly with fused lesions. the radiological demonstrations of influenza virus pneumonia resemble to those of other viral pneumonia, and the radiological diagnosis of the primary disease is, therefore, challenging. the diagnosis can be defined based on laboratory tests. a 7-years-old boy complained of fever and cough for 2 days, with the highest body temperature of 37.7 °c. laboratory tests revealed wbc count 4.21 × 10 9 /l, and nucleic acid of influenza virus positive. [radiological demonstration] fig. 8 .1 this is a case of influenza virus pneumonia, demonstrated by chest x-ray as interstitial inflammation in both lungs. chest x-ray demonstrates primary influenza virus pneumonia mainly as interstitial pneumonia and bronchial pneumonia, early with poorly defined but enhanced lung markings, predominantly in bilateral lower lung fields. in addition, the lung markings show an increased density, resembling to ggo. during the progressive stage, the lung fields are demonstrated with reticular opacity and reticular nodular opacity, with nodules smaller than 5 mm. such opacities may be concurrently demonstrated with poorly defined but the disease should be mainly differentiated from measles virus pneumonia, pulmonary alveolar pneumonia and allergic pneumonia. chest x-ray demonstrates measles virus pneumonia as flakes or diffusely distributed ggo and/or thickened bronchovascular bundles. by ct scan, measles virus pneumonia is demonstrated as poorly defined centrilobular nodules, ground glass opacity, interlobular septal thickening as well as lobular or segmental consolidation opacities. bacterial pneumonia is mainly demonstrated as alveolar pneumonia or bronchial pneumonia. alveolar pneumonia is mainly demonstrated as lobar consolidation opacity with high uniform density or consolidation opacity occupying part of lung lobe, with air bronchogram inside. bronchial pneumonia is demonstrated as thickened lung markings, with poorly defined nodular opacity or poorly defined flakes of opacity. allergic pneumonitis is a non-asthmatic allergic lung disease caused by a group of different allergens. chest x-ray may show no abnormalities or diffuse interstitial fibrosis, commonly with bilateral patches or nodular infiltration, thickening of the bronchial lung markings or small acinar like changes. ct scan shows thickened bronchovascular thickening, poorly defined small patches of and ground glass opacities along the bronchovascular bundles. the demonstrations by ct scan are irregular, possibly with inconsistencies between radiological findings and clinical symptoms. unlike bacterial pneumonia, the radiological signs of viral pneumonia may be inconsistent with the clinical symptoms. therefore, radiological diagnosis of primary virus pneumonia is challenging and the diagnosis can be defined based on the laboratory tests. a 6-years-old boy complained of fever and cough with skin rashes for 6 days, with the highest body temperature of 38.9 °c. laboratory tests revealed wbc count 2.26 × 10 9 /l, pco 2 42.6 mmhg, and po 2 86.5 mmhg; the nucleic acid of influenza virus positive. [radiological demonstration] fig. 8.2 [diagnosis] influenza virus pneumonia. [discussion] this case of influenza virus pneumonia is typically as virus pneumonia. chest x-ray demonstrates primary influenza virus pneumonia as interstitial pneumonia and bronchial pneumonia, with initial radiological signs of enhanced but poorly defined lung markings, predominantly in bilateral lower lungs. the lung markings also show increased density resembling to ground glass opacity. during the progressive stage, the lung fields are demonstrated with reticular and reticular nodular opacities, with nodules smaller than 5 mm. such opacities can be concurrently demonstrated with enhanced but poorly defined lung markings. the lesions commonly distribute in both lower lung fields and around the hilum. the demonstrations by ct scan are diversifying and overlapping, including small nodules in lungs, tree-buds sign with sporadic and centrilobular distribution, ground glass opacity with lobar distribution, diffuse ground glass opacities accompanied by thick interstitial change as well as interlobular septal thickening, subpleural line, adjacent pleural thickening, and pleural effusion. the radiological findings are in consistency with the histopathological demonstrations. a 7-years-old boy complained of fever and cough for 2 days, with aversion to cold and the highest temperature of 39.4 °c. laboratory tests revealed wbc count 15.4 × 10 9 /l; the nucleic acid of influenza virus positive. [radiological demonstration] fig. 8.3 [diagnosis] influenza complicated by bacterial pneumonia. [discussion] this is a case of influenza complicated by bacterial pneumonia, with typical signs of lobar pneumonia. lobar pneumonia is commonly caused by streptococcus pneumoniae, with sudden and acute onset and a short course of illness. chest x-ray demonstrates bacterial pneumonia as alveolar pneumonia (lobar pneumonia) or bronchial pneumonia (lobular pneumonia). alveolar pneumonia is mainly demonstrated as lobar consolidation with high uniform density or consolidation with high uniform density occupying a part lung lobe, with air bronchogram inside. the lesions at different sites show different radiological signs, with lesions involving one lung lobe or multiple lung lobes. bronchial pneumonia is demonstrated as thickened lung markings, with poorly defined nodular or flakes of opacity in a diameter of 6-8 mm. and the large poorly defined patches of opacity with uneven density is actually overlapping opacities of multiple lobular alveolitis. occlusion of bronchi by mucus is demonstrated as lobular atelectasis or focal emphysema, while occlusion of bronchiole causes radiological sign of a small triangle shaped lung atelectasis. the lesions are commonly located in the medial parts of both lower lung fields, with more lesions in the posterior lung lobe than in the anterior lung lobe, which distribute along bronchial branches with smooth air flow in the segmental and lobar bronchi. congestion, edema and inflammatory exudation of terminal bronchiolar mucosa may cause obstructive emphysema, which is demonstrated as increased transparency of both lung fields, extended thorax, widened intercostal space, and lowered flat diaphragm. ct scan demonstrates uniform shaped consolidations with lobar distribution, with air bronchogram inside, and poorly defined nodular and patches of opacity in different sizes along bronchical bundle as well as lobular atelectasis or focal emphysema. it should be mainly differentiated from viral pneumonia, klebsiella pneumonia and mycoplasma pneumonia. chest x-ray demonstrates viral pneumonia mainly as interstitial pneumonia and bronchial pneumonia. during its early stage, chest x-ray demonstrates enhanced but poorly defined lung markings, predominantly in the both lower lung fields, with increased density like ggo. during the progressive stage, the lung fields are demonstrated with reticular opacity and reticular nodules, which mainly distribute in both lower lung fields and around the hilum, with a diameter of less than 5 mm. during the advanced stage, bronchiolar inflammatory occlusion causes cystic changes in different sizes, with honeycomb like lung. the lung is demonstrated with shrinkage, elevated diaphragm and shift of interlobar fissure. ct scan demonstrates small nodules, ground glass opacity, tree-buds sign and mosaic like perfusion. klebsiella pneumonia is an acute lung inflammation caused by klebsiella pneumoniae, which commonly occurs in populations of those with chronic alcoholism or malnutrition and the elderly. chest x-ray demonstrations can be classified into 3 types: increased lung markings type; lobular type or diffuse pneumonia type; and lobar consolidation type or lung abscess type. compared to chest x-ray, ct scan can more favorably display the lesions. in its early stage, klebsiellar pneumonia is demonstrated with lobular sporadic distribution of patches or irregular dense opacities, which involve multiple lung segments and fuse rapidly to show lobar consolidation in the right upper lung lobe. due to the thick exudated fluid from the lesion, the interlobar fissure is demonstrated to drop. the lesions are susceptible to necrosis, followed by formation of lung abscess, which is commonly multiple small cavities with a diameter of less than 2 cm. the healing proce ss of these cavities is long, commonly with residual extensive fibrosis. mycoplasma pneumonia is an acute respiratory infection and pneumonia caused by mycoplasma pneumonia, with common occurrence in both children and adults. most of the patients show cold agglutination test positive. in its early stage, chest x-ray demonstrates increased poorly defined lung markings and blurry cloud like or homogenous opacities, commonly in the middle and lower lung fields. such opacities adjacent to the hilum are dense, and its density gradually lightens along with its distance from the hilum, with poorly defined boundary and involvement of partial lung lobe. mycoplasma pneumonia with lobar lesion can not be differentiated from lobar pneumonia induced by other pathogenic bacteria. chest ct scan mainly shows ground glass like opacity in lungs, nodular or small patches of consolidation with air cavity, thickened bronchovascular bundle, buds-in-tree sign, large consolidation as well as accompanying mediastinal lymphadenectasis and pleural effusion. a 22-years-old woman, pregnant for 38 weeks, complained of fever and aversion to cold for 2 days with the highest body temperature of 39.1 °c. laboratory test revealed wbc count 19.48 × 10 9 /l, pco 2 33.8 mmhg, po 2 43.6 mmhg, sao 2 80.9 %; the nucleic acid of influenza virus positive. [radiological demonstration] fig. 8 .4 [diagnosis] influenza complicated by ards. [discussion] different populations with influenza, show different clinical manifestations, and the special populations include children, the elderly, the pregnancy and those with compromised immunity. in the middle or late stage of pregnancy, women, after infected by influenza virus, experience the symptoms of fever and cough, with vulnerability to pneumonia. the condition rapidly progresses into dyspnea, hypoxemia and even ards, with outcomes of miscarriage, premature delivery, fetal distress and intrauterine fetal death. in addition, it may induce aggravation of the underlying diseases, with occurrence of death in severe cases. in this case, the patient was a young woman during her pregnancy, who was diagnosed with influenza complicated by ards and death occurred after active treatment due to multiple organs failure. ards is the typical manifestation of advanced stage acute lung damage, which is basically diffuse capillary damage of lung with increased permeability due to intrapulmonary or extrapulmonary serious disease. its pathological changes include pulmonary edema, hyaline membrane formation and pulmonary atelectasis, with clinical manifestations of acute respiratory failure syndrome characterized by progressive respiratory distress and intractable hypoxemia. the radiological abnormalities of ards are related to leakage of edema fluid containing a large amount of protein and its filling into the alveolar cavity after damage to the alveolar epithelium or diffuse damage to alveolar wall. and its staging by radiology is closely related to the pathological changes, including the exudative stage, the proliferative stage and the fibrosis stage, with intercorrelationship and overlapping. chest x-ray commonly demonstrates diffuse opacity in both lungs, and detectable lesions of the underlying disease, e.g. severe pneumonia induced by a variety of pathogens. ct scan demonstrates uneven distribution of the lesions: (1) with almost no abnormality in the gravity independent region (e.g. supine, anterior thoracic cavity); (2) with ggo in the anterior and middle thoracic cavity; and (3) with consolidation in the gravity dependent region. chest x-ray demonstrated thickened lung markings in both lungs, decreased transparency of the right lower lung field, and poorly defined flakes of opacities in the right lower lung (a). ct scan demon-strated wedge shaped consolidation opacity in the right lower lung lobe, with its sharp end pointing to the hilum, air bronchogram inside and surrounding poorly defined small patches of opacities (b) in the cases with no capillary damage in lung, the patches of opacities evenly distribute in both lungs, with no gravity dependent lesions and no gravity dependent changes of the lesions. such a phenomenon facilitates its differential diagnosis from other lung infections. in the advanced stage of ards, radiology demonstrates twisted and stretching of the bronchi, shrinkage of lung segment or lobe, grid like opacity, cords like opacity, honeycomb like opacity, and even honeycomb like lung in severe cases. it should be mainly differentiated from viral pneumonia, bacterial pneumonia and pulmonary edema. chest x-ray demonstrates viral pneumonia mainly as interstitial pneumonia and bronchial pneumonia. in its early stage, chest x-ray demonstrates enhanced but poorly defined lung markings, predominantly both lower lung fields, with increased density like ggo. in its progressive stage, the lung fields are demonstrated with reticular opacity or reticular nodules and the nodules commonly distribute in the both lower lung fields and around the hilum, with a diameter of less than 5 mm. it its advanced stage, bronchiolar inflammatory occlusion causes cystic changes in different sizes to show honeycomb like lung, with shrinkage of lung, elevated diaphragm and shift of interlobar fissure. ct scan demonstrates small nodular opacity, ground glass opacity, tree-buds sign and mosaic like perfusion in lungs. chest x-ray demonstrates bacterial pneumonia mainly as alveolar pneumonia or bronchial pneumonia. alveolar pneumonia is demonstrated as lobar consolidation with high uniform density or consolidation with high uniform density occupying part of lung lobe, with air bronchogram inside and one or multiple lung lobes involved. bronchial pneumonia is demonstrated as thickened lung markings, poorly defined nodular or flakes of opacity in a diameter of 6-8 mm. bronchial occlusion by mucus can be demonstrated as lobular atelectasis or focal emphysema in the diseased area. the lesions are commonly located in the medial part of both lower lung fields, with more in the posterior lung lobe than in the anterior lung lobe, that distribute along bronchial branches. and the segmental and lobar bronchi show smooth air flow. ct scan demonstrates consolidations with uniform shape and lobar distribution, with air bronchogram inside, and poorly defined nodular or patches of opacities of different sizes along bronchial bundles as well as lobular atelectasis and focal emphysema. pulmonary edema and acute or chronic systolic or diastolic heart dysfunction due to various etiological factors can lead to increased pressure in the pulmonary vein and pulmonary capillaries as well as pulmonary congestion. the liquid firstly accumulates in the perivascular sheath in lungs and interlobular space to cause pulmonary interstitial edema, which then flow into the alveolar cavity to cause pulmonary parenchyma edema. chest x-ray demonstrates interstitial edema as thickened, deranged and re-ranged lung markings, thickened but poorly defined vascular markings in both upper lung fields, enlarged and dense hilar opacities in both lungs, thickened and dilated but poorly defined vascular markings in the middle and medial parts of both lung fields. however, chest x-ray demonstrates interstitial edema with fine vascular markings in peripheral lung field and well defined peripheral lung field. its further progression can be demonstrated with flakes of opacity in both lungs with butterfly wing like shape and concentric distribution. chest x-ray demonstrates alveolar edema as initially poorly defined flakes of opacities in different sizes with sporadic distribution in both lungs. along with its progression, chest x-ray demonstrates large flakes of high density opacity after fusion, which extends from the hilum to the peripheral lung with gradually light density, in typical butterfly wing like sign. a b fig. 8.4 chest x-ray demonstrated decreased transparency of both lung fields and large consolidation opacity in both lungs, with air bronchogram inside (a). reexamination after treatment for 2 days showed that absence of lung markings in both lungs, further decreased transparency of both lung fields, and diffuse high density in both lungs (b) diagnostic criteria of acute lung injury and acute respiratory distress syndrome what has computed tomography taught us about the acute respiratory distress syndrome? imaging diagnosis of interstitial pneumonia chest x-ray demonstrations of pediatric hand-footmouth disease complicated by pneumonia imaging of communityacquired pneumonia: roles of imaging examinations, imaging diagnosis of specific pathogens and discrimination from noninfectious diseases pneumonia: high-resolution ct findings in 114 patients key: cord-034406-i1hbx3pz authors: matthews, abigail a.; ee, pui lai rachel; ge, ruowen title: developing inhaled protein therapeutics for lung diseases date: 2020-10-30 journal: mol biomed doi: 10.1186/s43556-020-00014-z sha: doc_id: 34406 cord_uid: i1hbx3pz biologic therapeutics such as protein/polypeptide drugs are conventionally administered systemically via intravenous injection for the treatment of diseases including lung diseases, although this approach leads to low target site accumulation and the potential risk for systemic side effects. in comparison, topical delivery of protein drugs to the lung via inhalation is deemed to be a more effective approach for lung diseases, as proteins would directly reach the target in the lung while exhibiting poor diffusion into the systemic circulation, leading to higher lung drug retention and efficacy while minimising toxicity to other organs. this review examines the important considerations and challenges in designing an inhaled protein therapeutics for local lung delivery: the choice of inhalation device, structural changes affecting drug deposition in diseased lungs, clearance mechanisms affecting an inhaled protein drug’s lung accumulation, protein stability, and immunogenicity. possible approaches to overcoming these issues will also be discussed. biological drugs are revolutionising the treatment and management of many serious illnesses including cancer, autoimmune disorders, and rare genetic diseases, with about a third of all new drug approvals by the food and drug administration (fda) consisting of biological drugs [1] . however, over the past decades, the development of inhaled therapeutics for the treatment of respiratory diseases has largely been focused on small molecules (corticosteroids, β2 agonists, and muscarinic antagonists), with only one inhaled protein biologic drug pulmozyme® being approved by the fda to date [2] . in the treatment of lung diseases via inhalation therapy, biological drugs such as proteins/polypeptides offer many advantages over small molecule drugs. proteins delivered via the pulmonary route could accumulate in the lungs while having a poor ability to traverse the airblood barrier due to their large molecular weight. this would result in higher target site accumulation (airway epithelial cells, alveolar macrophages, neutrophils etc) and minimise systemic toxicity, as compared to small molecule drugs that would pass easily into the systemic circulation after reaching the lungs [2] [3] [4] . in addition, protein therapeutics display higher potencies (picomolar to femtomolar range) than small molecules (nanomolar range), as well as highly specific receptor binding to reduce off-target effects [5] . notably, although peptide drugs (< 5 kda or < 40-50 amino acids in length) share some of the characteristics with protein/polypeptide drugs such as both are composed of amino acids linked via peptide bond and both having high target specificity, most of peptide drugs are much smaller in sizes, conferring them with some distinct differences including less enzyme stability, higher tissue penetration ability etc. in addition, many peptide drugs harbour chemical modifications in the form of peptidomimetics and/or cyclization, and some have direct cell membrane penetrating ability. these peptide drugs are not covered in this review. protein therapeutics are conventionally administered via the systemic route, although this has proven to be an inefficient approach for drug delivery to the lung, not mentioning the additional danger of exposing the rest of the body vulnerable to toxicity [4, 6] . for instance, monoclonal antibodies (mabs) are found at higher levels (500-10,000 times more) in serum than in bronchoalveolar lavage (bal) fluid following intravenous administration, a trend that has been demonstrated in all species [4] . by the same token, protein therapeutics delivered via the airways also pass poorly from the lungs into the systemic circulation. for example, only low amounts of anti-vegf-a g6-31 mab (5.1%) and cetuximab (11%), an anti-egfr mab, were present in the serum after aerosol delivery in mouse models of lung cancer [7, 8] . this means that high concentrations of the protein drug can be attained in the lung via pulmonary delivery, suggesting that lower doses of inhaled protein can have an equivalent or even superior therapeutic effect for lung diseases when compared to the higher doses that would be needed from systemic administration [9] . indeed, it was reported that the nebulised effective dose of avidinox-anchored biotinylated cetuximab was 1/25, 000 of the intravenous effective dose in a mouse model of advanced metastatic lung cancer [10] . although higher pulmonary levels of protein therapeutics can be achieved through inhalation compared to systemic administration, this could be offset by the short residence time of proteins in the lung compared to plasma. proteins and antibodies are mostly cleared from the lungs within 24 h, while plasma half-lives of full-length antibodies following intravenous injection can reach 3 weeks and more [11] . nevertheless, there are strategies that can be employed to increase the local residence time of protein therapeutics in the lungs, and these will be discussed in detail later on in this review. besides improving pharmacokinetic and toxicity profiles of protein therapeutics, the inhalation route is non-invasive and allows for self-administration, which could improve patient compliance [4, 5, 12] . in 1993, pulmozyme® (dornase alfa/deoxyribonuclease i), was introduced for the treatment of cystic fibrosis [9] . it has been almost three decades since then, and no other inhaled protein therapeutics for topical treatment of a lung disease has reached the market, despite the aforementioned advantages that inhaled proteins possess. presently, and to the best of our knowledge, ten inhaled protein therapeutics are being assessed in clinical trials for the treatment of a range of lung diseases including asthma, cystic fibrosis, lung cancer, copd and covid-19 (table 1) [13] [14] [15] [16] [17] [18] . there are also protein therapeutics such as mabs that are in the preclinical stages [19, 20] . in order to drive more of these therapies into clinical development and eventually to the market, it is crucial to take into account the challenges unique to the development of these agents into potential treatments so that they may be utilised successfully for pulmonary delivery. in this review, we will discuss the challenges in developing an inhaled protein therapeutic for lung diseases, as well as approaches that could help to circumvent these issues. the focus of this review is on the pulmonary delivery of protein drugs for local action in the lung, however, examples of inhaled proteins for systemic action will be mentioned wherever relevant. challenges and considerations in the development of protein therapeutics for local lung delivery choice and limitations of drug delivery device there are mainly three classes of inhalation devices namely dry powder inhalers (dpis), nebulisers, and metered dose inhalers (mdis). dpis deliver drug as a solid aerosol, and powder formulations possess inherent stability and shelf life benefits [4, 21] . however, the temperature and shear stress during the manufacturing processes needed to produce powders (e.g. freeze drying, spray drying) could lead to protein degradation [21] . dpis have been used for the marketed inhalable insulin formulations exubera® (approved in 2006, but was discontinued after 1 year due to large device size, high pricing, and safety concerns) and afrezza® (still commercially available) [22] . moreover, dpis have shown promising results in studies assessing their use for inhaled protein formulations. for example, weers et al. (2019) showed that dry powder formulations of csj117 (antithymic stromal lymphopoietin (tslp) mab fragment) could achieve a total lung dose (tld) of about 95% of the delivered dose (dd) with the use of particle engineering techniques such as via the introduction of surface corrugation through the addition of trileucine [23] . nebulisers (jet, ultrasonic, and mesh) generate aerosol droplets from a liquid solution of the drug [4] . the first and only inhaled protein formulation approved for pulmonary delivery to date, pulmozyme®, is administered via jet nebuliser. nebulised formulations are less expensive to produce and test, because the manufacturing process for these formulations does not include extra drying steps. nevertheless, prolonged storage of proteins in liquid solutions can lead to protein instability through degradation pathways (i.e. deamidation and hydrolysis), temperature and ph changes, and aggregation (through agitation of the aqueous carrier) [21] . furthermore, across all nebuliser types, the process of nebulisation exposes the protein to physical stresses such as shearing forces and heat, as well as the large air-liquid interface (ali) that could alter protein conformation and/or structure through denaturation, chemical modifications (oxidation, deamidation), and aggregation [4, 15] . device-specific limitations such as the shear forces generated by jet nebulisers, and the temperature increases that occur in ultrasonic nebulisers, can also lead to protein degradation. jet and ultrasonic nebulisers actually recycle 99% of the primary aerosol, and a molecule would typically be subjected to 10-15 cycles of nebulisation before leaving the nebuliser as a secondary aerosol [24] . this subjects the molecules to high shear stress in these devices, resulting in the denaturation of proteins, with the extent of protein denaturation and degradation varies depending on the characteristics of the individual protein [25] . for example, for jet nebulizer delivery of ldh and urease, there is a log-linear degradation with a fraction of protein degraded with every recirculation [26, 27] . in contrast, igg and g-csf has a rapid initial decline in native proteins in the first 5-10 min [26, 28] . for ultrasonic nebulizers, heating resulting from ultrasonic radiation in addition to aerosol recirculation generated various protein denaturation and degradation in different proteins [25] . for example, the degradation of ldh with ultrasonic nebulizer presented a sigmoidal progression instead, indicating different denaturation process and factors involved from jet nebulizers [29] . by comparison, vibrating mesh nebulisers employ single-pass technology, ensuring that there is no recirculation of droplets into the reservoir; they do not alter solution temperature, and produce less shear forces inside the drug reservoir during nebulisation, making them more suitable for the delivery of protein therapeutics [24] [25] [26] [27] [28] [29] [30] [31] . in fact, several studies have reported that jet and ultrasonic nebulisers produce lower levels of activity, lower amounts of protein monomers (because of partial degradation), and more aggregates (with or without excipients). on the other hand, mesh nebulisers appear to maintain protein integrity to a greater extent than other nebulisers [32] . safe aerosolisation with mesh nebulisers has already been demonstrated in several studies of labile drugs including proteins and mabs [33] [34] [35] [36] [37] [38] . the detailed designs and comparisons of various types of nebulizers have been reviewed previously and we will not elaborate further here [25, [30] [31] [32] 39] . recently, nanoengineered particles using metal-phenolic networks (mpns) with highly defined physical properties have been used to encapsulate both small molecule and macromolecules including proteins for pulmonary delivery via nebulisation. intratracheal nebulization delivery of fitc-labelled bovine serum albumin (bsa, 65 kda) in mice demonstrated that these capsules are biocompatible and biodegradable, showing > 85% of the capsules in the lung after 20 h, while only < 4% remaining after 30 days without causing obvious lung inflammation or toxicity. although still in early stage of development, these mpn particles may revolutionize the nebulization delivery of protein drugs and provide a more protected environment for effective pulmonary delivery [40] . moreover, new generation nebulisers are being developed such as surface acoustic wave (saw) nebulisers and the more recent hydra (hybrid resonant acoustics) nebulisers that provide new platforms for inhaled drug delivery. saw use surface waves to generate aerosols which can preserve macromolecule integrity that has been shown to be efficient in aerosolising proteins [41] . hydra uses a hybrid combination of surface and bulk sound waves to generate the aerosol droplets, and can overcome the low nebulisation rate of saw nebulisers and conventional nebulisers, while also avoiding the potential damage to proteins due to high shear (jet nebulisation) or cavitation (ultrasonic nebulisation). the first human lung deposition study using a prototype hydra nebuliser has been reported recently, indicating successful lung deposition of a radiolabelled small molecule [42] . it is probable that hydra nebulisers may be developed for pulmonary protein drug delivery. mdis deliver drug through an aerosol burst, and allow for the controlled delivery of specific amounts of drug to the lungs [21, 22] . however, as with nebulisers, the use of aqueous solutions is not ideal for protein storage [22] . there are also concerns that the hydrofluoroalkane (hfa) propellants used in mdis could denature proteins [21, 43] . despite this concern, there are examples of studies showing that proteins can remain stable in hfa-containing mdi formulations. quinn et al. (1999) utilised raman spectroscopy to analyse the secondary conformations of lysozyme in the hfa propellants tetrafluoroethane (hfa 134a) and heptafluoropropane (hfa 227), demonstrating that structural integrity of lysozyme was preserved in both hfas, and that there is potential for proteins to be developed as mdi formulations without compromising their conformational stability [44] . moreover, liao et al. (2005) demonstrated that spray-dried lysozyme and catalase that were stabilised with excipients (sugars and/or 80% polyvinyl alcohol) and then stored in hfa 134a at room temperature for 6 months showed retention of biological activity [45] . when choosing an inhalation device, it is important to be cognisant of the fact that not all devices in the same category are equivalent. for instance, although it is generally accepted that there is minimal heating of the drug reservoir in vibrating mesh nebulisers, and that heating occurs to a lesser degree than in ultrasonic nebulisers, considerable temperature increases have been reported in some brands of vibrating mesh nebulisers (pari eflow®, akita 2 apixneb®, and aeroneb go), with temperatures of up to 40°c being reached towards the end of nebulisation [24] . therefore, it is essential to choose the inhalation device carefully, bearing in mind that the best device type is the one that confers the most stability to the protein drug formulation. soft mist inhaler (smi), which also generate aerosols from liquid, is the newest type of inhaler which does not use any propellent. as only one medicine respimat uses smi, its suitability for protein drug delivery is not clear. another factor to consider is the aerodynamic diameter of aerosol particles, which is critical to control where the particles will be deposited in the respiratory track after inhalation. to be therapeutically effective, the drug containing particles need to be deposited into the correct location within the respiratory track. for example, for therapeutics for copd, drugs need to be delivered to the deep lung (the alveolar space) for which it requires the aerodynamic diameter of the particles to be between 1 and 5 μm. larger size particles will generally be deposited in the oropharyngeal region and be ingested, while small particles < 1 μm may be exhaled during the next breathing cycle. thus, suitable aerosol particle sizes need to be selected for precise drug delivery into the lung to enhance drug efficacy while simultaneously reducing harmful side effects [5] . the respiratory tract comprises of a series of branching airways, which can be categorised into two parts: the conducting zone and the respiratory zone. the conducting zone consists of the trachea, bronchi, bronchioles, and terminal bronchioles. the airway wall in the conducting zone is too thick for diffusion and this region does not contain alveoli. as such, no gas exchange takes place here, and the purposes of the conducting zone include transmitting air to the respiratory zone, as well as to warm, moisture and cleaning the inspired air. the respiratory zone consists of respiratory bronchioles, alveolar ducts, and alveolar sacs, and facilitates gas exchange between the air and the bloodstream. alveoli can occasionally be found in the walls of the respiratory bronchioles, and are abundant in the alveolar ducts and alveolar sacs [45, 46] . given the branched structure of the lungs, it is not only important to achieve high deposition rates, but also to obtain an appropriate deposition pattern for the respiratory disease in question i.e. the protein therapeutic would not only need to reach the lung, but would also need to reach the correct target site within the lung. for instance, a therapeutic for asthma would have to reach the large airway, as asthma mainly affects the bronchi, while a drug for emphysema in copd would need to go deeper and reach the small airways of the lung because emphysema affects the alveolar region [47] . the amount and pattern of lung deposition is not only affected by the device and the characteristics of the inhaled drug (particle size and physicochemical properties of the formulation), but also by factors that are influenced by the specific disease state, including breathing patterns, lung geometry (i.e. airway diameter, number of alveoli) and structure, and nasal, oral, and pharyngeal anatomy [45] . these factors need to be considered, and if possible, alterations to the drug formulation can be made to address these issues. for example, in certain lung pathologies (for instance cystic fibrosis, copd, and chronic sinusitis), the airway mucus becomes thicker. it has been reported that the thickness of the mucus layer ranges from 2 to 30 μm in normal lungs to more than 260 μm in cystic fibrosis and other obstructive airway diseases [48] . this presents a physical barrier that the protein drug would need to penetrate to reach its target site in the lung and exert its effects. the addition of anti-adhesive molecules (e.g. polyethylene glycol, peg) in the formulation may help to promote the translocation of the protein drug through the thickened mucus, although it should be noted that the adhesive properties of peg depend on peg molecular weight (mw) [12] . while high mw pegs display mucoadhesive properties, low mw pegs are able to prevent mucoadhesion, with pegs of mw up to 40 kda able to provide effective mucus penetration [49] [50] [51] . notably, as macromolecules, proteins have a relatively poor ability to penetrate the epithelial layer to reach the deep parenchyma lung. however, depending on the molecular weight and aerosol characteristics, a portion of the proteins would be able to reach the abluminal side of the epithelium or the air-blood interface in the thin alveolar wall, triggering local or even systemic immune signalling that may provide beneficial therapeutic effect in some cases [15] . a good lung deposition pattern would be worthless if the protein therapeutic cannot withstand the lung's clearance mechanisms. inhaled proteins would be subjected to clearance by three mechanisms. the first clearance mechanism is mucociliary clearance (mcc), which is the coordinated beating of cilia lining the nasal cavity, trachea, and bronchi, in order to move the mucus towards the larynx/pharynx, thereby pushing dust, microorganisms, and insoluble particles that are trapped in the mucus out of the lungs and into the upper airways to eventually be swallowed [46] . the surface lining of the airways in normal lungs consists of an aqueous layer adjacent to the epithelium and a surfactant containing film layer at the air-liquid interface. the peri-ciliary aqueous layer has a relatively low viscosity, while the surfactant film layer is more viscous. the surfactant film plays an important role in the displacement of airway particles towards the epithelium where they will be immersed and retained. the extent of particle immersion depends on the surface tension of the film. the lower the surface tension, the greater the immersion of particles into the aqueous layer adjacent to the epithelium [52, 53] . it is possible that some protein monomers could quickly reach the stagnant aqueous layer and not be subjected to mcc. on the other hand, some protein monomers would become aggregated during the inhalation delivery process and the aggregates may stay with the surfactant film layer at the air-liquid interface for some time for mcc to take effect. anti-adhesive formulations (achieved by using lower mw pegs for example) could be used to circumvent mcc clearance of inhaled therapeutics, thus increasing their lung accumulation. the mucus-penetrating ability of such pegs has already been demonstrated in multiple studies [12, [54] [55] [56] [57] [58] [59] [60] [61] . the second clearance mechanism is macrophage uptake, which is the primary clearance mechanism in the alveoli. proteins are taken up by alveolar macrophages in the deep lung via pinocytosis, and the uptake of particles is size dependent [5, 12, 62] . large proteins (≥ 40 kda) would have more time to be engulfed by alveolar macrophages by virtue of their slower transport and absorption across the alveolo-capillary barrier, while small proteins and peptides (≤ 25 kda) are absorbed rapidly from the airspaces and thus, may not be impacted by alveolar macrophage uptake as much. in essence, pinocytosis by alveolar macrophages could become significant for macromolecules with mw > 40 kda [62] . the use of excipients can help to reduce clearance of large proteins by alveolar macrophages. for example, koussoroplis et al. (2014) showed that pegylation conferred increased residence time to antibody fragments anti-il-17a f (ab ′)2 and anti-il-13 fab′ (unconjugated f (ab′)2 was 98 kda and unconjugated fab′ was 47 kda), and that the effect was due to mucoadhesion as well as evasion of alveolar macrophage uptake [11] . protein pegylation may potentially also alter its deposition pattern in the respiratory track, due to changes in molecular weight, hydrophilicity etc. however, systemic analyses of the effect of pegylation on protein deposition pattern in the respiratory track are still needed in order to know if a consistent deposition pattern and behaviour can be reached based on how pegylation is achieved. the third clearance mechanism is absorption into the systemic circulation. after deposition in the alveolar region, aerosol drug particles may dissolve in pulmonary epithelial lining fluid if the drug is water-soluble, and become available for systemic absorption and clearance [46] . for the purpose of topical lung treatment, the goal would be to minimise systemic absorption, which is greatly influenced by protein mw. the bioavailability of a protein after absorption from the lung decreases as protein mw increases. small peptides are absorbed rapidly from the lungs with 20-50% of the bioavailability for subcutaneous injection [63] . proteins with mw of 6-50 kda exhibit moderate absorption, with bioavailability ranging from 10 to 40%, although it should be noted that pulmonary absorption studies in animals may lead to an overestimation of bioavailability. for example, systemic bioavailability after aerosol administration in animals for growth hormone (gh) and interferon α (ifnα) was 45% and 70% respectively, compared to only 3-10% in humans [63, 64] . large mw antibodies (~150 kda) are not significantly absorbed across the lung, and bioavailability is negligible (<< 10%) unless an active transport system is included [63] . apart from high protein mw, the presence of obstructive lung diseases (e.g. asthma, copd, cystic fibrosis) can also reduce systemic absorption and bioavailability of proteins and other drugs [46] . for example, henry et al. (2003) reported that healthy subjects had significantly higher area under the curve (auc) and mean maximum concentration (c max ) after insulin inhalation than asthma patients, indicating that less insulin was absorbed into the systemic circulation in asthma patients [65] . in addition, diderichsen et al. (2013) reported that the c max of an inhaled long acting β 2 agonist (pf-00610355) was found to be reduced by 31% and 52% for copd and asthma patients respectively, compared to healthy volunteers [66] . additional possibility for the lower systemic absorption of drugs in lung disease patients could be due to altered drug deposition pattern in the diseased lung, for which further studies are needed. regardless the underlying mechanisms, effective local lung retention of protein drugs may not be a major issue for the successful inhalation treatment of obstructive lung diseases, since the protein can be relatively well retained in the lungs. inhaled protein therapeutics may undergo various degradation mechanisms during production, processing and/or storage. these degradation pathways may be physical (denaturation and non-covalent aggregation) or chemical (mainly covalent aggregation, deamidation, oxidation and/or glycation). denaturation is the result of physical stresses including low/high temperatures, high salt concentrations, organic solvents, and air/water or ice/water interfaces. removal of the stressor may be spontaneously reversible (for some single domain proteins), but is usually irreversible for most of the larger multi-domain proteins [67] . surface-induced aggregation is one of the common mechanisms of non-covalent aggregation, and one example of when it occurs is during the process of nebulisation [15, 67] . as most proteins are amphiphilic and surface active, they have a tendency towards adsorption at the ali. upon adsorption, conformation changes may occur, exposing hydrophobic residues to the interface to avoid contact with water, thus leading to aggregation and unfolding, which are the main factors contributing to protein instability [67, 68] . chemical degradation of proteins (deamidation, oxidation, glycation) may also cause aggregation (either covalent or non-covalent) [67] . aggregation has been extensively studied but chemical modifications have not, despite having implications on biological activity and immunogenicity [4, 15] . aggregation can result from both physical and chemical pathways; therefore, it is useful to also evaluate chemical changes in inhaled proteins. most studies assessing stability of inhaled protein formulations focus on formation of aggregates, while studies that also examine chemical changes are few and far between. one study did, however, consider chemical changes when evaluating the technical feasibility of delivering dornase alfa using perforated vibrating membrane devices for nebulisation. in this study, besides detecting protein aggregates, stability was also evaluated by measuring the percent deamidation of dornase alfa at asn 74 (the main chemical change for the protein), which was shown to be inversely proportional to dornase alfa potency [35] . another study looked at methionine 59 oxidation [met(o)] of nebulised insulin-like growth factor-1 (igf-i), and how that correlated with aggregate formation and bioactivity. highly aggregated samples displayed a complete loss of bioactivity, while samples with complete oxidation but minimal aggregation showed partial retention of bioactivity. limited met(o) formation and no aggregation was observed following delivery with air-jet or vibrating mesh nebulisers [36] . bandi et al. (2019) conducted a study to compare the effects of deamidation and oxidation on interferon alpha-2a (ifna2a), as deamidation of asparagine and glutamine residues, and oxidation of methionine residues are two of the most common chemical alterations that occur in pharmaceutical proteins that could compromise their efficacy and safety [68] . these findings revealed that deamidation destabilised ifna2a and enhanced its tendency to aggregate under stressful conditions, and reduced its function to a greater extent than oxidation. this is the first study that quantitatively compared the effects between deamidation and oxidation of a therapeutic protein [68] . it would be a good strategy to conduct such studies early on in the development of therapeutic protein candidates in order to identify the chemical modifications that a particular protein would be susceptible to, and to test out various excipients that could resolve specific stability issues. the protein therapeutic also needs to remain stable after reaching the lung, which can be challenging due to the high numbers of serine proteases and aminopeptidases present in the lung mucosa [69] [70] [71] [72] . these proteases could degrade protein drugs even before they reach their target sites within the lung. the use of appropriate excipients in the formulation such as peg, could help to enhance protein resistance to proteolysis by these lung proteases. for example, zhang et al. (2014) evaluated the stability of fibronectin (fn) preferentially pegylated at lysine residues using different mw pegs [2 kda (peg2), 5 kda (peg5) or 10 kda (peg10)] against the protease α-chymotrypsin. they showed that pegylation protected fn from proteolysis and that peg mw positively correlated with proteolytic stability (i.e. after 30 min of proteolysis, 4%, 34%, 43% and 65% of the starting amounts of native fn, fn-peg2, fn-peg5 and fn-peg10 respectively were remaining) [73] . one must also be aware of the possibility of protein aggregates forming in the lungs. lasagna-reeves et al. [74] demonstrated that mice exposed to inhaled insulin (exubera®) in a chamber twice daily for 1 week developed amyloid aggregates of insulin in both the proximal and distal airways, as well as the lung parenchyma (epithelium and muscle layer of the bronchi, bronchioles, and in the alveolar lining cells). the formation of insulin aggregates coincided with a significant decrease in respiratory flow rates, and also with caspase-9 activation. previous studies investigating the link between changes in pulmonary function and inhaled insulin use focused on formation of anti-insulin antibodies, or pulmonary inflammation and subsequent airway remodelling, but none of the published works before this looked at insulin aggregation in the lungs as a contributor to pulmonary dysfunction after inhaled insulin use [74] . indeed, exu-bera® was reported to cause cough, dyspnea, increased sputum and epistaxis [75] . this example highlights the possibility of inhaled proteins forming aggregates in the lungs, and thus the need for toxicity testing and safety studies examining this possibility to be done early on in the development of an inhaled protein candidate, during preclinical studies. in addition, proteins and other macromolecules have the potential to induce immunogenicity, with the production of anti-drug antibodies (adas) as the main immune response [5] . the development of adas in patients can alter pharmacokinetics, drastically reduce efficacy, and can also lead to severe adverse events or even lethal consequences [76] . immunogenicity is also linked to protein stability, as the presence of aggregates can render the protein immunogenic. as aggregates are typically composed of denatured molecules, they would exhibit no or decreased activity, but at the same time, aggregates are usually immunogenic leading to adas with important clinical implications [4] . aggregates are believed to be recognised and processed via non-specific uptake by antigen presenting cells and specific uptake by b cells. they may unmask neo−/cryptic/repetitive epitopes, and these differences may influence the mechanism by which they activate the immune system [76] . currently, only a few excipients have been approved by the fda for inhalation due to a dearth of toxicological studies for inhaled excipients [15, 67] . there is also very limited number of excipients that are approved by fda for biologics, rendering formulators limited choices to improve protein formulations when excipients are searched on the fda's inactive ingredient database guidance. furthermore, very few novel excipients have been investigated for biologic products; most are cyclodextrin-based excipients [77] . as such, there is a need for more extensive toxicity testing to identify novel excipients for pulmonary delivery. for excipients already known to increase protein stability, a trial and error approach needs to be taken in determining their suitability for a particular protein formulation, as an excipient may work for one protein but not for another for various reasons including sequence differences [15] . excipients that are commonly used in liquid formulations (nebulisers and mdis) include buffering or ph adjusting agents, and surfactants, and those that are commonly used in dry powder formulations (dpis) include sugars, polyols, and amino acids [67, 78] . buffering or ph adjusting agents such as sodium chloride, sodium citrate, hydrochloric acid, sodium hydroxide, and citric acid, are added to maintain the ph of the formulation. it is important to choose the right buffering agent at an appropriate concentration, as most proteins in solution only remain stable within a narrow ph range. different buffer systems and concentrations can also affect the aggregation pattern of proteins [67] . kim et al. [79] analysed the stability of a fusion protein, etanercept (marketed enbrel®), with changing ph and buffer concentrations. increasing the ph of etanercept from ph 6.6 to 8.6 resulted in a decrease in protein size and increase in aggregation. under high buffer concentrations (30 mm tris buffer), changes in protein size was reduced and irreversible aggregation was not observed, while in lower buffer concentrations (10 mm tris buffer), larger aggregates (~1 μm) were observed across the ph range [79] . surfactants (polysorbates, sorbitan esters, oleic acid, and soy lecithin) are frequently used to prevent aggregate formation, and they work by displacing protein molecules from the ali [46, 62] . polysorbates are the most commonly used surfactants, and are already being used to preclude aggregation in formulations of intravenously administered antibodies [34] . polysorbate 80 has been reported to lead to stabilisation in various inhaled protein formulations including those for granulocyte-colony stimulating factor (g-csf), lactate dehydrogenase (ldh), tissue plasminogen activator (t-pa) and aviscumine (recombinant mistletoe lectin) [68] . the ability of polysorbates and other surfactants to stabilise a protein and hinder aggregate formation is contingent on the protein-to-surfactant ratio. respaud et al. [34] examined the effects of various antibody and surfactant (polysorbate 20) concentrations to optimise the protein-to-surfactant ratio for a nebulised antibody formulation. the authors determined that high concentrations of either surfactant or protein could minimise the formation of medium and large-sized aggregates, without significantly affecting the volume mean diameter (vmd) of the aerosol cloud, ensuring suitability for inhalation. therefore, including surfactants and raising protein concentration to enhance the stability of inhaled protein formulations is a viable strategy, although it should be noted that this approach needs to be evaluated and optimised for each drug and device pairing being developed into an inhaled protein formulation [34] . sugars (sucrose, trehalose, raffinose and lactose) and polyols (mannitol) stabilise proteins through the preferential hydration of proteins via steric repulsion of sugar/ polyol molecules from the native protein [4, 68] . lactose is often used as a drug carrier in dpis, however, it may not be suitable for proteins because it is a reducing sugar, and it could interact with amino groups in proteins (maillard reaction) [67] . on the other hand, non-reducing sugars such as sucrose, trehalose and raffinose would not undergo the maillard reaction with proteins, and thus could be used as alternatives to lactose [81] . sellers et al. [82] demonstrated that sucrose could help to improve the stability of a dry powder formulation of ldh. supercritical fluid (scf) drying of ldh without excipients lead to irreversible loss of activity (only 15% recovered after rehydration). inclusion of 10% (w/w) sucrose during dehydration lead to an increase in activity recovered (to~60%), and there was almost complete retention of activity when polysorbate 20 was added in addition to sucrose [82] . trehalose and raffinose are currently not approved for any administration routes, but have been evaluated in experimental studies with promising results. for instance, ógáin et al. [81] incorporated lysozyme into nanoporous microparticles of trehalose and raffinose. lysozyme showed good retention of specific activity after storage for 12 weeks at either 4°c (98.2 ± 7.1% for lysozyme:trehalose and 99.1 ± 7.1% for lyzosyme:raffinose) or 25°c (92.5 ± 7.1% for lysozyme:trehalose and 90.8 ± 7.1% for lyzosyme:raffinose) [81] . mannitol was used as an excipient in the formulation of exubera® (table 2 ) [80] . small amino acids (histidine, arginine, alanine, glycine, lysine, isoleucine) are also used as stabilisers, and they work by the "water substitution mechanism" in which the amino acids hydrogen bond with the protein during drying to preserve the native protein structure in the dried state [4, 83] . ajmera and scherlieβ [84] screened different amino acids and their combinations for their ability to stabilize catalase during spray drying. when various ratios of arginine, glycine and histidine were mixed with catalase, some formulations were able to maintain close to 100% catalase activity [84] . despite encouraging results in studies such as this one, there is a lack of data on the local toxicity of the various amino acids following inhalation, which could limit their use. however, as they are endogenous substances, they may not present major safety issues for local lung delivery [67, 85] . the polyol, peg, could be used for both liquid and powder formulations of inhaled proteins. small mw pegs (< 10 kda) are often used as excipients in oral, intravenous and nasal formulations. larger pegs (up to 40 kda) may be used in pegylated biopharmaceuticals, and safety testing for these formulations are done during development on a case-by-case basis [86] . pegylation is a commonly used method to enhance solubility and stability, as well as to decrease immunogenicity of bioactive drugs including but not limited to proteins, peptides, antibody fragments, and enzymes, and is achieved by the covalent or noncovalent conjugation of peg to the biomolecule [87, 88] . pegylation can also help to reduce clearance and increase lung accumulation and residence time of inhaled protein therapeutics. for instance, conjugation of a peg chain to two antibody fragments (anti-il-17a f (ab′)2 and anti-il-13 fab′) increased their levels in mouse lungs following intranasal administration. fortyeight hours post-administration, levels of unconjugated antibody fragments in the lungs had dropped to 10% and 14% of the original deposited dose of f (ab′)2 and fab′ respectively, while this value was 40% for both pegylated fragments [11] . furthermore, conjugation of a peg chain to an anti-il-17a fab' antibody fragment increased pulmonary retention in all three species tested (mice, rats, and rabbits) following intratracheal administration. unconjugated fragments were cleared from the lungs within 24 h while large amounts of pegylated fragments still remained for up to 48 h [89] . the two biggest challenges in developing particle systems for pulmonary drug delivery are to maintain colloidal stability during aerosolisation and to achieve high delivery efficacy. encapsulation of proteins in carriers could provide multiple benefits such as protection from enzymatic degradation and specific targeting to the site of action through targeting ligands [5] . furthermore, carriers may also be used to provide sustained drug release, accumulating in the lungs and releasing therapeutic levels of the protein drug over extended periods of time. this would enhance efficacy while averting peaks in local drug concentrations that could cause pulmonary toxicity [90] . proteins, including insulin, calcitonin, and igg, have already been loaded into various carriers such as microparticles, liposomes, and solid lipid nanoparticles [90, 91] . indeed, afrezza® uses techno-sphere® technology, in which fumaryl diketopiperazine (fdkp), an excipient added into the formulation, selfassembles into microspheres, entrapping the insulin. upon reaching the alveolar zone of the lung, the tech-nosphere® particles rapidly dissolve in the ph-neutral environment and release the insulin for systemic absorption [75] . although this approach has not been extensively explored for topical lung delivery of proteins, and more work needs to be done on the use of carriers for the purpose of systemic delivery of proteins through the lungs, some promising results have been reported that support further development of this approach. tawfeek et al. [92] encapsulated a model mucinolytic enzyme, αchymotrypsin (which is very sensitive to unfolding and formulation conditions), in a novel biodegradable pegco-polyester microparticle carrier. the encapsulated αchymotrypsin exhibited retention of enzymatic activity and the results indicated suitability of the carrier for potential use in the delivery of macromolecules as dpi formulations for the treatment of lung diseases [92] . in another study by osman et al. [93] , various surface modifications were made to dnase i loaded microparticles using different excipients in order to provide higher lung deposition, enzyme stability and biological activity. surface modifying the microparticles with polyglutamic acid (pga) or dextran was found to provide high inhalation indices (emitted fraction (ef), respirable particle fraction (rp), and effective inhalation index (ei)) and increased mucolytic activity in cystic fibrosis sputum. this could be explained by the resulting surfaces of the particles after modification with pga (rough dented surfaces) or dextran (dimpled surfaces). compared to spherical particles with similar physical properties, corrugated particles have surface asperities that could reduce the true contact area between particles, decreasing powder cohesiveness and enhancing aerosol performance [93] . advancements in drug-loaded capsules for pulmonary delivery have been made in both inhalable dry powder or liquid drug formulations [94] [95] [96] . for dry powder drug particles, precise control of the particle size has been reported using the particle replication in nonwetting templates (print) technology [97, 98] . for control of aerodynamic particle size in liquid aerosols such as in nebulized liquid formulation, the recently reported mpns have presented promising possibilities. mpnbased drug-loaded capsules with highly defined physical properties can be generated for both macromolecular protein drugs and small molecule chemical drugs [54] . these new developments may transform inhalation drug delivery in the near future. one drawback with the use of carriers is their rapid uptake by alveolar macrophages [99, 100] . phagocytosis of carriers by alveolar macrophages can result in fast clearance and reduced residence time, limiting the therapeutic efficacy of the carrier-associated drug. this would be an issue for the treatment of chronic lung diseases such as asthma and copd, where the goal of using a carrier system would be to achieve controlled and continuous drug release over an extended period of time. however, various formulation design strategies may be employed to reduce the uptake of particulate carriers by alveolar macrophages including modulation of particle size, shape, surface charge and surface coating [101] . studies on the use of various polymer coatings demonstrate reduced alveolar macrophage uptake of coated carriers. for example, jones et al. [102] showed that respirable microspheres coated with dipalmitoyl phosphatidylcholine (dppc; a major component of lung surfactant) were able to significantly reduce phagocytic uptake by nr8383 in cultured alveolar macrophages compared to uncoated microspheres. the uptake of dppc coated microspheres was found to be only 24.1 ± 7.86%, 31.9 ± 3.74% or 36.6 ± 3.66%, of the uptake of uncoated microspheres for ratios of 5, 10 or excess microspheres per nr8383 cell respectively [102] . furthermore, shen et al. [103] demonstrated that surface coating of hydrogel nano-and microparticles with peg showed significantly reduced uptake by alveolar macrophages both in vitro (in mh-s cells) and in vivo (in mice) compared to unpegylated particles of the respective size. at 24 h post-dose, the fold difference between pegylated and unpegylated 80 × 320 nm, 1.5 μm, and 6 μm particles in bronchioalveolar lavage fluid (balf), was 1.5, 3.4 and 3.7 respectively [103] . on the other hand, drug-loaded particles may be advantageous for anti-tuberculosis drugs as efficient uptake of drugs into alveolar macrophages could potentially enhance the drug's efficacy to kill the parasitic mycobacterium tuberculosis that hide inside the cells [104] . if the usage of a carrier is to be included in the protein formulation, it should be noted that the formulation (i.e. combination of protein and carrier) would need to be optimised together with the choice of device, as the chosen carrier may not work well with all inhalation device types. for instance, liposomes may be delivered to the lungs either by dry powder inhalation or nebulisation of a liposome suspension. however, nebulised solutions of liposomes may cause instability as nebulisation has been reported to disrupt liposomal structure, leading to the release of loaded drug. these issues can be avoided with the use of dry powders of liposomes instead [90] . hence, although this review has presented a general overview for the various aspects of protein formulation design (such as choice of device, excipients), it is important to test out the formulation and device together to determine which combination works best. this review analyses the various obstacles that an inhaled protein drug would need to overcome in order to reach the lungs and exert its therapeutic effects. these obstacles include the physical and chemical stresses experienced by the protein during production/storage/ aerosolisation, the need to overcome mucociliary clearance and physical barriers arising from disease conditions in order to reach target sites within the lung, and the need to remain stable in spite of the presence of abundant proteases, and to evade clearance by alveolar macrophages after reaching the lungs (fig. 1) . all of these threats to the integrity of the protein need to be carefully considered, so that pre-emptive measures can be taken while designing the protein formulation to ensure its therapeutic efficacy. nevertheless, although the information provided here may serve as general considerations in developing pulmonary protein therapeutics, empirical testing of the formulation together with the device should still be performed to determine the best combination for a particular protein. several key areas will require further investigation in order to support the development of more successful inhaled protein therapies, and maintaining the stability of the inhaled protein is of paramount importance. firstly, more studies could look at other instability issues beyond protein aggregation. in depth studies on the specific chemical modifications that a protein would be susceptible to, such as the one conducted by bandi et al. (2019) , could be carried out on therapeutic protein candidates so that they may be developed into stable and effective treatments [68] . moreover, the scarcity of fdaapproved excipients for inhaled therapeutics further limits drug developers, and expanding this list through increased toxicological testing of new excipients would provide more options for formulation design. finally, innovative approaches such as the use of novel carrier systems should be employed for the purpose of topical lung delivery, as carrier systems could greatly enhance the stability and pharmacokinetic profile of proteins. these approaches would greatly benefit the field of pulmonary drug delivery, and will ultimately allow more inhaled protein therapeutics to reach the clinic. funding support for this work is provided by the grant moe2017-t2-2-122 awarded to ruowen ge from singapore ministry of education, republic of singapore. capturing the benefits of competition for patients current approaches to the discovery of novel inhaled medicines inhaled protein/peptide-based therapies for respiratory disease nebulization as a delivery method for mabs in respiratory diseases carriers for the targeted delivery of aerosolized macromolecules for pulmonary pathologies inhalation of immuno-therapeutics/ −prophylactics to fight respiratory tract infections: an appropriate drug at the right place! front immunol vegf neutralizing aerosol therapy in primary pulmonary adenocarcinoma with kras activating-mutations the airways, a novel route for delivering monoclonal antibodies to treat lung tumors inhaled therapy in respiratory disease: the complex interplay of pulmonary kinetic processes efficacy of aerosol therapy of lung cancer correlates with egfr paralysis induced by avidinox-anchored biotinylated cetuximab pegylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract repurposing of gamma interferon via inhalation delivery 2 phase ii clinical trial results of alidornase alfa for the treatment of cystic fibrosis protalix biotherapeutics announces phase ii clinical trial results for alidornase alfa in cystic fibrosis presented at the 40th european cystic fibrosis society conference. protalix biotherapeutics designing inhaled protein therapeutics for topical lung delivery: what are the next steps? inhaled gm-csf in a pulmonary alveolar proteinosis patient refractory to plasmapheresis combined with multiple whole lung lavages synairgen doses first patient in covid-19 trial ansun biopharma enrolls first patient in proof of concept trial of das181 for the treatment of covid-19 direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies in a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration challenges and future prospects for the delivery of biologics: oral mucosal, pulmonary, and transdermal routes non-invasive delivery strategies for biologics idealhalers versus realhalers: is it possible to bypass deposition in the upper respiratory tract? that's cool! -nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer protein stability in pulmonary drug delivery via nebulization protein nebulization: i. stability of lactate dehydrogenase and recombinant granulocyte-colony stimulating factor to air-jet nebulization stability of urease during aerosolization protein nebulization some factors associated with the ultrasonic nebulization of proteins nebulizers for drug delivery to the lungs the function and performance of aqueous aerosol devices for inhalation therapy devices for improved delivery of nebulized pharmaceutical aerosols to the lungs vibrating mesh nebulisation of pro-antimicrobial peptides for use in cystic fibrosis effect of formulation on the stability and aerosol performance of a nebulized antibody a technical feasibility study of dornase alfa delivery with eflow® vibrating membrane nebulizers: aerosol characteristics and physicochemical stability insulin-like growth factor-i aerosol formulations for pulmonary delivery development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin effective nebulization of interferon-γ using a novel vibrating mesh factors to consider when selecting a nebulizer for a new inhaled drug product development program engineering of nebulized metal-phenolic capsules for controlled pulmonary deposition pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform in vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (hydra) technology prospects of formulating proteins/peptides as aerosols for pulmonary drug delivery protein conformational stability in the hydrofluoroalkane propellants tetrafluoroethane and heptafluoropropane analysed by fourier transform raman spectroscopy computational modeling of lung deposition of inhaled particles in chronic obstructive pulmonary disease (copd) patients: identification of gaps in knowledge and data the impact of pulmonary diseases on the fate of inhaled medicines-a review drug delivery for traditional and emerging airway models. organs-on-a-chip nanodelivery in airway diseases: challenges and therapeutic applications addressing the peg mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier nanoparticles coated with high molecular weight peg penetrate mucus and provide uniform vaginal and colorectal distribution in vivo surfactant displaces particles toward the epithelium in airways and alveoli pulmonary surfactant: surface properties and function of alveolar and airway surfactant transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy protein nanocages that penetrate airway mucus and tumor tissue use of single-site-functionalized peg dendrons to prepare gene vectors that penetrate human mucus barriers rapid transport of large polymeric nanoparticles in fresh undiluted human mucus highly compacted biodegradable dna nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy pegylated enhanced cell penetrating peptide nanoparticles for lung gene therapy lung gene therapy with highly compacted dna nanoparticles that overcome the mucus barrier the penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles preclinical models for pulmonary drug delivery formulation technology to repurpose drugs for inhalation delivery will pulmonary drug delivery for systemic application ever fulfill its rich promise? inhaled insulin using the aerx insulin diabetes management system in healthy and asthmatic subjects characterizing systemic exposure of inhaled drugs: application to the long-acting β2-agonist pf-00610355 inhaled proteins: challenges and perspectives 2d nmr analysis of the effect of asparagine deamidation versus methionine oxidation on the structure, stability, aggregation, and function of a therapeutic protein respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants role of proteases in lung disease: a brief overview role of proteases in chronic obstructive pulmonary disease proteases and antiproteases in chronic neutrophilic lung disease -relevance to drug discovery pegylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity inhaled insulin forms toxic pulmonary amyloid aggregates technosphere®: an inhalation system for pulmonary delivery of biopharmaceuticals immunogenicity of different stressed igg monoclonal antibody formulations in immune tolerant transgenic mice the effects of substituted cyclodextrins on the colloidal and conformational stability of selected proteins formulation strategy and use of excipients in pulmonary drug delivery effects of ph and buffer concentration on the thermal stability of etanercept using dsc and dls practical, regulatory and clinical considerations for development of inhalation drug products particle engineering of materials for oral inhalation by dry powder inhalers. i-particles of sugar excipients (trehalose and raffinose) for protein delivery dry powders of stable protein formulations from aqueous solutions prepared using supercritical co2-assisted aerosolization mechanisms of protein stabilization in the solid state stabilisation of proteins via mixtures of amino acids during spray drying amorphous powders for inhalation drug delivery pegylation, an approach for improving the pulmonary delivery of biopharmaceuticals what is the future of pegylated therapies? from synthesis to characterization of site-selective pegylated proteins pegylation prolongs the pulmonary retention of an anti-il-17a fab' antibody fragment after pulmonary delivery in three different species delivery strategies for sustained drug release in the lungs multifunctional nanocarriers for lung drug delivery dry powder inhalation of macromolecules using novel peg-copolyester microparticle carriers inhalable dnase i microparticles engineered with biologically active excipients inhaled nano-and microparticles for drug delivery nanoparticles for drug delivery to the lungs advanced therapeutic strategies for chronic lung disease using nanoparticle-based drug delivery formulation of high-performance dry powder aerosols for pulmonary protein delivery microfabricated engineered particle systems for respiratory drug delivery and other pharmaceutical applications polymeric nanoparticles in development for treatment of pulmonary infectious diseases update on macrophage clearance of inhaled micro-and nanoparticles particle engineering to enhance or lessen particle uptake by alveolar macrophages and to influence the therapeutic outcome the inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages distribution and cellular uptake of pegylated polymeric particles in the lung towards cell-specific targeted delivery drug delivery for tuberculosis: is inhaled therapy the key to success? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-018001-ris02bff authors: garrido, guillermo; dhillon, gundeep s. title: medical course and complications after lung transplantation date: 2018-06-23 journal: psychosocial care of end-stage organ disease and transplant patients doi: 10.1007/978-3-319-94914-7_26 sha: doc_id: 18001 cord_uid: ris02bff lung transplant prolongs life and improves quality of life in patients with end-stage lung disease. however, survival of lung transplant recipients is shorter compared to patients with other solid organ transplants, due to many unique features of the lung allograft. patients can develop a multitude of noninfectious (e.g., primary graft dysfunction, pulmonary embolism, rejection, acute and chronic, renal insufficiency, malignancies) and infectious (i.e., bacterial, fungal, and viral) complications and require complex multidisciplinary care. this chapter discusses medical course and complications that patients might experience after lung transplantation. the lungs normally have a dual blood supply, consisting of 1) large pulmonary arteries that provide desaturated blood under low pressure for alveolar gas exchange and 2) smaller bronchial arteries that provide oxygenated blood under systemic pressure for nutrition and oxygenation of the bronchi and lung tissue. as the only solid organ transplant that does not undergo primary systemic (i.e., bronchial) arterial revascularization at the time of surgery, lung transplants rely on the deoxygenated pulmonary arterial circulation and are especially vulnerable to the effects of injury and ischemia [4] . it has been hypothesized that the absence of the bronchial system in the lung allograft increases susceptibility to microvascular injury and chronic airway ischemia, which may be implicated in the genesis of chronic rejection and other complications [5] . similarly, the native lymphatics and the neural supply to lung allografts are disrupted at the time of trans-plantation. the impact of these disruptions on lung transplant outcomes remains unclear, though it is possible that these changes lead to higher susceptibility to the development of pulmonary edema and infections, worse airway clearance, and ineffective cough [6] . lastly, the lung allografts have higher exposure to immunogenic compounds, as compared to other organs, by ventilation. the ongoing exposure to various inhaled injurious agents may also predispose lung allografts to develop chronic rejection. there is a vast array of complications from lung transplantation. broadly these complications can be divided into noninfectious and infectious complications and have been summarized in table 26 .1. these complications arise at different times in the postoperative period [7] . the understanding of timing of various complications post-lung transplant can lead to early recognition and management of these complications. epithelium, and alveolar macrophages. the interaction between these cells leads to release of cytokines, reactive oxygen intermediates, and proteolytic enzymes leading to graft dysfunction [9] . the severity of pgd falls along a spectrum, ranging from mild dysfunction to severe lung injury. pgd can affect 10-25% of transplanted patients, and the 30-day mortality can be as high as 50%. furthermore, severe pgd after lung transplantation has been associated with development of subsequent chronic rejection and graft dysfunction [10] . the management of pgd is largely supportive and includes lung-protective ventilation strategies (low tidal volume, high positive end-expiratory pressure), judicious fluid management, inhaled nitric oxide or other inhaled pulmonary vasodilators to improve oxygenation, and extracorporeal life support (ecls) for the most severe cases. re-transplantation is an option for highly selected cases, but it is generally not recommended due to suboptimal outcomes [11] . lung transplant recipients are at increased risk of vte. the risk factors include major surgery status, hypercoagulable state, high dose of corticosteroids, immobility, and indwelling vascular access. the reported incidences of pulmonary embolism (pe) and deep venous thrombosis (dvts) postlung transplantation are approximately 5-15% and 20-45%, respectively [12] . the pulmonary embolism in setting of limited pulmonary reserve due to pgd, postoperative atelectasis, and single-lung transplantation can have catastrophic consequences, thus underscoring the need for early and appropriate vte prophylaxis after lung transplantation [13] . the diagnosis can be made with computed tomography (ct) pulmonary angiography, ventilation-perfusion scan, or by documentation of dvt by doppler ultrasonography. the treatment is the same as for vtes in general, although the risk of postoperative bleeding needs to be weighed against the risk of pe. the choice of anticoagulant is based on kidney function, periprocedural reversibility of anticoagulant effect, and drug interactions, with unfractionated heparin, low-molecular-weight heparin, and/or warfarin being by far the most common agents used. in case of ongoing bleeding or high risk of bleeding, inferior vena cava filters can be used as a temporizing measure. inadvertent injury to various intrathoracic nerves during lung transplantation is a well-recognized and common complication. the most commonly affected structures are the phrenic and vagus nerves. the reported rates of phrenic nerve injury have ranged from 3% to 9% in lung transplant cases. this rate can be as high as 40% in combined heart-lung transplantation [14, 15] . diaphragmatic dysfunction as a consequence of phrenic nerve injury can present clinically with dyspnea, hypoventilation and hypercapnia, and hypoxemia or as difficult wean from the ventilator. diaphragmatic paralysis can lead to increased length of stay and ventilator dependence. diagnosis can be confirmed by documenting paradoxical movement of affected diaphragm during quiet and deep breathing, using fluoroscopy or ultrasound visualization. the vagal nerve injury post-lung transplantation can lead to gastroparesis with associated risk of gastroesophageal reflux (gerd) and aspiration events. these in turn can place lung allograft at risk for recurrent infections, bronchiectasis, and possibly chronic allograft dysfunction [16] [17] [18] . common symptoms of gastroparesis include early satiety, decreased appetite, abdominal pain, and bloating. a diagnosis is usually made by a nuclear medicine gastric emptying study. the potential management strategies include minimizing transit delaying medications (e.g., opioids), the use of pro-motility agents, placement of post-pyloric feeding tubes, botulinum toxin injection to the pylorus, and surgical fundoplication in conjunction with pyloroplasty [17] . the pleural complications in early post-lung transplantation period include pleural effusions, hemothorax, pneumothorax, empyema, chylothorax, and interpleural communication. these complications usually arise as a result of the pleural disruption from the surgery itself, though rejection and immunosuppressive regimens may also play a role. the risk factors for the development of pleural complications include previous thoracic surgery, pleural adhesions, and donor-recipient size mismatch [19, 20] . pleural effusions are extremely common in the early postlung transplant period. the reported incidence has been 100% in some series [19, 20] . all patients have chest tubes in place immediately post-operation to allow lung re-expansion, pleural air, and fluid drainage. the increased amount of pleural fluid post-lung transplantation is related to capillary leak due to allograft ischemia reperfusion, fluid overload, bleeding, and surgical interruption of allograft lymphatics at the time of explantation [19, 20] . late pleural effusions can be a consequence of infection, acute rejection, trapped lung physiology from pleural fibrosis, or malignancy [21, 22] . in general, all pleural effusions need to be evaluated to rule out complicated effusions such as hemothorax, empyema, and chylothorax. these entities have all been associated with negative patient outcomes and are treated with a range of medical and surgical procedures depending on the condition and severity. for example, a chylothorax might necessitate mechanical interruption of thoracic duct, or hemothorax may need thoracotomy for control of bleeding. pneumothoraxes are common after lung transplantation. they can result from donor-recipient size mismatch, bronchopleural fistulas that occur secondary to operative injury or bronchial anastomoses dehiscence, or as a consequence of transbronchial biopsies performed in the course of allograft evaluation. small and stable pneumothoraxes after lung transplantation can be managed by watchful waiting, though larger or symptomatic pneumothorax may require chest tube drainage. an inadequately drained, hemodynamically significant pneumothorax can be a medical emergency necessitating urgent drainage [23, 24] . in patients who have undergone sequential bilateral lung transplantation (bslt) or heart-lung transplantation (hlt), interpleural communication due to surgical severance of the pleural recesses that separate the left and right pleural spaces can develop. this entails that pleural issues in these patients must be managed aggressively as pneumothoraxes can be bilateral and life threatening, and empyema can spread quickly. vascular anastomotic complications can arise either early or late in the post-transplant course and can have very severe adverse consequences. pulmonary artery stenosis can be secondary to mechanical kinking, disruption, or narrowing of the anastomosis, sometimes due to the particulars of donor anatomy or due to thrombosis [25] . the clinical picture is usually consistent with pulmonary hypertension and right ventricular failure. diagnosis can be made through pulmonary angiography and can be managed with interventions such as balloon dilation and stent deployment. occasionally, patients may require surgery for definitive management of the stenosis. pulmonary vein occlusion post-lung transplantation is a rare but serious complication. the commonest cause of pulmonary vein occlusion is the development of thrombosis at the anastomotic junction of the pulmonary veins and the left atrium, though inadvertent narrowing or ligation of pulmonary veins has also been reported. the potential clinical consequences include hypoxic respiratory failure, pulmonary edema, and cardio-embolic events. this entity should be included in the differential diagnosis of a patient with acute pulmonary edema post-lung transplantation. diagnosis is usually made by transesophageal echocardiography or ct angiography [26, 27] . the airway complications after lung transplantation can be classified by time of occurrence. early anastomotic complications, usually within 1 month of transplantation, include infection, dehiscence, and necrosis at the anastomotic sites. later complications include bronchopleural, bronchovascular and bronchomediastinal fistulae, excessive granulation tissue, bronchomalacia, and airway stenosis. airway anastomotic complications do not seem to be associated with decreased survival; however, they do negatively impact quality of life and significantly increase healthcare resource utilization [28] . the risk factors for airway anastomotic complications include colonization with burkholderia cepacia and aspergillus fumigatus, pgd, acute rejection, prolonged mechanical ventilation, and sirolimus use prior to anastomotic healing [29, 30] . bronchial necrosis and dehiscence occur 1-2 weeks after transplant. they can present with dyspnea, difficulty weaning from the ventilator, persistent air leak on the water seal, pneumomediastinum, and subcutaneous emphysema and infection, with symptoms ranging from mild to severe. depending on the severity, management can range from observation and antibiotics to minimally invasive or surgical repair. bronchial stenosis is the narrowing of the airway lumen, usually at the site of the anastomosis. patients can present with wheezing, cough, post-obstructive pneumonias, decline in pulmonary function tests (pfts), and stridor. the bronchial narrowing can also present distal to the anastomosis causing lobar lobe collapse. this syndrome occurs 2-6 months post-transplant but can present as late as 12 months. treatment options include close monitoring, bronchial dilatation with or without stent placement, and re-transplantation [31] . allograft rejection is a major cause of morbidity and mortality post-lung transplantation. at least a third of patients are reported to have acute rejection in the first year after transplant. acute rejection in itself seldom leads to mortality, but it is a main risk factor for the development of chronic rejection. the chronic rejection of lung allograft is the major hurdle to long-term survival after transplantation. despite the use of potent and novel immunosuppressive regimens, the incidence of chronic rejection and long-term survival post-transplant has remained essentially unchanged over the last two decades [1, 32] . acute cellular rejection (acr) is the most common kind of acute lung transplant rejection and is mediated by t lymphocytes. symptoms and signs of acr include dyspnea, cough, fever, and hypoxia. high-grade rejection may be associated with respiratory failure. mild acr can be asymptomatic and frequently detected on surveillance pulmonary function testing and/or transbronchial biopsies. current imaging modalities are not diagnostic but may reveal useful findings such as infiltrates and ground-glass opacities [32, 33] . flexible bronchoscopy with transbronchial biopsies is the gold standard for diagnosis. histologically, acr is characterized by the presence of perivascular and/or peribronchiolar (grade b) lymphocytes in the absence of infectious etiologies [32, 34, 35] . risk factors for acr include the number of hla mismatches between donor and recipient, although it is unclear which specific hlas have more impact. other reported risk factors are age, with older patients having more rejection, immunosuppressive regimen used (tacrolimus regimens reject less), other genetic factors such as il-10 production, and documented gerd. acr has also been documented following infections with certain viruses, such as rhinovirus, parainfluenza virus, influenza virus, human metapneumovirus, coronavirus, and respiratory syncytial virus. the treatment for acr is not uniform, and high-quality randomized controlled trials are lacking. there is wide agreement that severe cases of acr must be treated, but there is variability among transplant centers on whether to treat milder cases. the mainstay of therapy is high-dose corticosteroids. in cases that are refractory or recurrent, usually the immunosuppressive regimen gets intensified or altered, and medications such as anti-thymocyte globulin (atg), antiinterleukin 2-receptor (il-2r) antagonists, muromonab-cd3 (okt3), and alemtuzumab (anti-cd52 monoclonal antibody), among others, can be used [36, 37] . antibody-mediated rejection (amr) is believed to be mediated by donor-specific antibodies (dsa) against human leukocyte antigens (hla) and other donor antigens. these antibodies may have been present in the recipient prior to transplant, although most appear to develop after transplantation. amr is described as the combination of the following: donor-specific anti-hla antibodies, evidence of complement deposition in allograft biopsies, histologic tissue injury, and clinical allograft dysfunction [38] . once the aforementioned antibodies bind their receptors in the graft, they are capable of binding complement, specifically c1q. this can trigger complement-mediated cell destruction and inflammation. the development of de novo anti-hla antibodies is associated with poor prognosis [39, 40] . the mainstay of amr management involves depletion and/or neutralization of anti-hla antibodies by plasma exchange or intravenous immunoglobulin (ivig), followed by rituximab infusion. rituximab is an anti-cd-20 chimeric antibody that targets b-cell function and can decrease production of antibodies. in cases of refractory amr, newer agents such as bortezomib (anti-proteasome 26s) and the anticomplement antibody eculizumab have been tried with limited success. successful clearance of anti-hla antibodies has been associated with decreased risk of development of chronic rejection following amr [32] . the term chronic lung allograft dysfunction (clad) encompasses pathologies that lead to chronic dysfunction of lung allograft. clad is predominantly a consequence of chronic rejection and is a major hurdle to long-term survival. the two major phenotypes of clad include (i) bronchiolitis obliterans syndrome (bos) and (ii) restrictive allograft syndrome (ras) [41, 42] . bos is the predominant form of clad and is the number one cause of death after 1 year of transplantation. it is reported to occur in up to 76% of lung transplant recipients at 10 years post-transplant, and it is a major cause of morbidity, negative impact in quality of life, and increased costs. bos is defined by a sustained (>3 weeks) decline in the forced expiratory volume in the first second of expiration (fev1); provided alternative causes of pulmonary dysfunction have been excluded. at the tissue level, the hallmark of bos is obliterative bronchiolitis (ob), which is an inflammatory/fibrotic process affecting the small non-cartilaginous airways (membranous and respiratory bronchioles) characterized by subepithelial fibrosis causing partial or complete luminal occlusion [43, 44] . risk factors include prior episodes of acute rejection, cytomegalovirus infection (cmv), community-acquired respiratory viruses (carv) infection, history of pgd, isolation of aspergillus fumigatus and pseudomonas aeruginosa, the presence of gerd, and other immune-mediated factors [44] . the diagnosis can be made conditionally without histopathology (bos) or definitively with histopathology (bo). transbronchial biopsy is an insensitive method for detecting bo, and the clinical use of bos is the favored method for diagnosis and monitoring. the treatment of bos is disappointing in terms of outcomes; often success is measured in slowing the decline or stabilizing it. beyond augmentation of immunosuppression, azithromycin, extracorporeal photopheresis, montelukast, methotrexate, aerosolized cyclosporine, alemtuzumab, and total lymphoid irradiation have been used with limited success [44, 45] . ras has been more recently described and occurs in less than a third of patients with clad. these patients present with predominant restriction, and the survival is worse as compared to patients with bos. the median survival postdiagnosis is 8 months. ct scan shows interstitial opacities, ground-glass opacities, upper lobe-dominant fibrosis, and honeycombing. the only identified risk factor for the development of ras is late-onset diffuse alveolar damage (dad), occurring later than 3 months after lung transplant. there is no proven treatment for this condition, and re-transplantation remains technically challenging [46, 47] . lung transplant and associated immunosuppression are an established risk factor for development of cancer [48] . the commonest malignancy post-lung transplant is the squamous cell cancer of the skin. the single-lung transplant recipients are at higher risk of development of lung cancer in their native lungs. this increased risk is in part related to the increased risk of cancer due to underlying disease (e.g., emphysema, idiopathic pulmonary fibrosis) [49, 50] . similarly, the transplant recipients with cystic fibrosis remain at an elevated risk for development of gastrointestinal malignancies [49] . it is imperative that transplant recipients adhere to age-appropriate health screening after transplant. additionally, all lung transplant recipients should undergo skin cancer screening annually. the risk is especially high for of viral infection associated malignancies such as lymphoma, kaposi sarcoma, and anogenital cancers [49] . post-transplant lymphoproliferative disorders (ptld) encompass an array of diseases involving clonal expansion of b lymphocytes, ranging from polyclonal benign disorders to aggressive malignant lymphomas. the reported incidence of non-hodgkin lymphoma post-lung transplant has been as high as 28 cases/100,000 person-years [49] . there is a significant association between ptld and epstein-barr virus (ebv) infection, especially in patients who acquire infection the novo after being transplanted. ptld is managed by reducing the intensity of immunosuppression if possible, with specific chemotherapy for more severe and refractory cases. hyperammonemia affects 1-4% of the lung transplant population; it is a rare but potentially fatal complication. it can be secondary to systemic infection with mycoplasma hominis and ureaplasma, which break down urea as an energy source, generating ammonia as a waste product. this likely represents a donor-derived infection and can respond to early appropriate antibiotic treatment [51] . postoperative liver dysfunction and urea-cycle enzyme deficiencies can also cause hyperammonemia. diabetes mellitus (dm) is common in lung transplant recipients, with 25-30% of patients developing it in the first year post-transplant and up to 40% at 5 years. the use of glucocorticoids, calcineurin inhibitors, obesity, and advanced age is a significant risk factor for the development of dm. the development of dm in lung transplant recipients is associated with decreased survival. a close and judicious glycemic control is indicated in this patient population [52, 53] . patients who undergo lung transplantation have multiple risk factors to develop acute kidney injury (aki) post-transplant, including decreased renal perfusion before, during, and/or after surgery, drug toxicities, and systemic infections. aki affects as many as 70% of patients with approximately 8% patients requiring renal replacement therapy (rrt). the postoperative renal failure necessitating the use of rrt is associated with increased risk of early mortality [54, 55] . by 3 years, 25% of surviving lung transplant recipients develop severe renal dysfunction (serum creatinine >2.5 mg/ dl), and that percentage rises to 40% at 10-year mark [1] . the risk factors for development of chronic kidney disease (ckd) include older age, dm, hypertension, smoking history, and use of nephrotoxic drugs. ckd is also associated with higher mortality in lung transplant recipients [56] . recipients of lung transplant are at risk for development of osteopenia and osteoporosis due to multiple factors such as malnutrition, immobility, chronic corticosteroid use, calcineurin inhibitor use (e.g., tacrolimus), and other comorbidities. the strategies to prevent and reverse bone losses after transplant need to be proactively implemented. treatment includes adequate supplementation of calcium, vitamin d, use of bisphosphonates, enhancing physical activity, and minimizing contributing medications, if possible [57, 58] . dyslipidemia is also very common in lung transplant recipients, as high as 59%, and it may be related to the aforementioned metabolic risk factors. treatment usually entails lifestyle modifications and cholesterol lowering medications. there are multiple cardiac complications after lung transplantation, both short and long term. atrial dysrhythmias are very frequent in the early postoperative period, likely related to stress of major surgery, catecholamine surge, medication side effects, and mechanical stresses related to vascular anastomoses. the reported incidence has been as high as 25-35% [59, 60] . these arrhythmias are usually managed with medications aimed at rate and rhythm control. hemodynamically significant and/or refractory arrhythmias may require electric cardioversion. atrial dysrhythmias are associated with increased length of hospital stay and increased mortality [59, 60] . over the long term, lung transplant recipients are at increased risk for developing coronary artery disease (cad). as they progress into long-term survival, these patients have cumulative impact from risk factors previously discussed in this chapter, namely, dm, dyslipidemia, ckd, hypertension, chronic corticosteroid use, and other immunosuppressive medication. these risk factors should be carefully managed to decrease the impact of cad and related complications, with a combination of lifestyle modifications and specific medical therapies [61] . lung transplant recipients experience a decrease in skeletal muscle strength and function, including respiratory and limb muscles. this is likely related to reduced activity postoperatively and deconditioning, corticosteroid-induced myopathy, critical illness-related weakness (neuropathy/myopathy), and in the case of the diaphragm, phrenic nerve injury. this issue seems to be consistent in lung transplant recipients and independent of pre-transplant diagnosis and surgery type. muscle weakness, deconditioning, and sarcopenia are associated with adverse outcomes and decrease in quality of life. aggressive rehabilitation is standard and important in the post-transplant care [62, 63] . lung transplant recipients are at an increased risk for acquiring infections due to the immunosuppressed state, constant environmental pathogen exposure, decreased cough reflex, impaired mucociliary clearance, and lymphatic disruption. infectious complications are responsible for about a quarter of post-transplant deaths [64] . pneumonias are the most significant bacterial infection in lung transplant recipients, and the highest risk is in the first 30 days post-transplant. in the early period, they are more likely to be caused by hospital-acquired organisms, which tend to be more virulent and more resistant to antibiotics. the patients with cystic fibrosis are frequently colonized by multidrug-resistant organisms and are at increased risk of pneumonia post-transplant. in later stages, community-acquired organisms become more prevalent. moreover, throughout the post-transplant period, the patients are susceptible to numerous opportunistic infections [65] . other commonly encountered bacterial infections in this patient population include pleural space infections, blood stream infections (bsis), and soft tissue infections. the bsis and empyema carry a high risk of morbidity and mortality [66, 67] . pseudomonas aeruginosa, burkholderia cepacia, staphylococcus aureus (including methicillin-resistant), and other gram-negative organisms are common causes of serious infections in post-lung transplant period. these organisms have high rates of antibiotic resistance and are associated with worse outcomes [68] [69] [70] . streptococcus pneumoniae is the most common cause of communityacquired pneumonia, and immunosuppressed patients have increased risk of disseminated infection [71] . clostridium difficile associated diarrhea is a major complication in hospitalized, immunosuppressed and debilitated patients and is associated with increased hospital length of stay and mortality [72] . molds are common fungal entities affecting lung allografts. aspergillus spp. are the most common and have a predilection for the respiratory tract [73] . lung transplants have the highest incidence of invasive aspergillosis among solid organ transplant recipients, and it is the most common invasive fungal infection in lung transplant. aspergillus is ubiquitous in the environment and is acquired by inhalation. there are three main described presentations: invasive pulmonary disease, tracheobronchial aspergillosis, and disseminated disease, all of which are associated with varying degrees of increased mortality. other implicated molds include fusarium, scedosporium, and mucormycosis. these infections are difficult to treat and are associated with poor clinical outcomes [73] . candida spp. are another common pathogen in lung transplant setting. oral candidiasis is the most common manifestation of this infection. however, candida infections can also manifest as candidemia, empyema, surgical wound infection, and disseminated disease. serious candida infections have been associated with increased mortality, though rates have been declining over time [74] . other fungal infections in this patient population include opportunistic infections, such as pneumocystis jiroveci and cryptococcus, as well as endemic fungi, such as histoplasma capsulatum, coccidioides immitis, and blastomyces dermatitidis [75, 76] . viral infections contribute to morbidity and mortality from acute infection and have been associated with an increased risk of rejection, chronic allograft dysfunction, lymphoproliferative and other neoplastic diseases, and other extra pulmonary organ damage [77] . cytomegalovirus (cmv) is the most significant viral infection occurring in solid organ transplant recipients and is the second most common infection, after bacterial pneumonia. cmv infection can range from latent infection, to asymptomatic viremia, to cmv disease manifested with clinical symptoms and end-organ involvement. severity of disease may range from mild to life threatening. when there is organ damage, affected organs can include the lungs, pancreas, intestines, retina, kidney, liver, and brain. cmv disease is associated with increased mortality [77, 78] . other notable dna viruses from the herpesviridae family include epstein-barr virus (ebv), which is associated with increased risk of ptld and other malignancies, herpes simplex virus (hsv) 1 and 2, varicella-zoster virus (vzv), and human herpesvirus 6, 7, and 8 [77] . community-acquired respiratory viruses, including influenza, are a major source of respiratory symptoms and mor-bidity after lung transplantation. these infections may also be associated with development of chronic allograft dysfunction [79] . currently, the median survival for all adult lung transplant recipients is 6 years [1] . bilateral lung recipients appear to have a better median survival compared to single-lung recipients (7 versus 4.5 years) [1] . overall lung transplantation confers clinically meaningful and statistically significant improvements in health-related quality of life (hrqol). greater than 80% of lung transplant recipients report no activity limitations [80] . the care of lung transplant recipients is multidisciplinary, labor intensive, and comprehensive. it includes management of immunosuppression regimen, opportunistic infection prophylaxis, prevention and management of various comorbidities, and complications. a typical medication regimen consists of three classes of immunosuppression drugs (i.e., calcineurin inhibitor, cell-cycle inhibitor, and corticosteroids), as well as opportunistic infection prophylaxis against pneumocystis jiroveci, other fungal infections, and cmv. in early postoperative period and after hospital discharge, the recipients are closely monitored in outpatient setting. typical clinic visits include thorough medication reconciliation, clinical exam, pulmonary function testing, chest radiographs, and laboratory examinations. the role of surveillance bronchoscopies with transbronchial biopsies in monitoring of lung allograft remains unclear. while lung transplantation improves survival and quality of life in patients with end-stage lung disease, it is associated with multitude of noninfectious and infectious complications. lung transplant recipients have one of the shortest survival rates among other solid organ recipients, due to some unique characteristics of the lung allograft, including its unique blood supply and risk for ischemia, disruption of the native lymphatics and the neural supply during the transplant surgery, and exposure to immunogenic entities via ventilation. among noninfectious complications, pgd, vte, and rejection are the most important ones. clad affects most patients long term and remains a significant clinical concern and contributor to early mortality in lung transplant recipients. lung transplant recipients are also at increased risk for a variety of malignancies, due to their underlying disease, comorbidities, and immunosuppressed status; thus they require vigilant monitoring and screening for cancer. infectious complications (i.e., bacterial, fungal, viral) are also important contributors to morbidity and mortality, with bacterial pneumonias and cmv most commonly seen. patients require multidisciplinary and intensive follow-up and aftercare, ongoing vigilance, early recognition and treatment, and open and frequent communication between recipients, caregivers, and healthcare team providers. the registry of the international society for heart and lung transplantation: thirtieth adult lung and heart-lung transplant report--2013; focus theme: age long-term health status and quality of life outcomes of lung transplant recipients the registry of the international society for heart and lung transplantation: thirty-fourth adult heart transplantation report-2017; focus theme: allograft ischemic time every allograft needs a silver lining lung transplant airway hypoxia: a diathesis to fibrosis? a critical role for airway microvessels in lung transplantation pulmonary complications of lung transplantation report of the ishlt working group on primary lung graft dysfunction, part i: definition and grading-a 2016 consensus group statement of the international society for heart and lung transplantation report of the ishlt working group on primary lung graft dysfunction part iii: mechanisms: a 2016 consensus group statement of the international society for heart and lung transplantation report of the international society for heart and lung transplantation working group on primary lung graft dysfunction, part ii: epidemiology, risk factors, and outcomesa 2016 consensus group statement of the international society for heart and lung transplantation report of the ishlt working group on primary lung graft dysfunction part iv: prevention and treatment: a 2016 consensus group statement of the international society for heart and lung transplantation venous thromboembolic complications of lung transplantation: a contemporary single-institution review pulmonary embolectomy after single-lung transplantation diaphragmatic paralysis: a complication of lung transplantation leuven lung transplant g. phrenic nerve dysfunction after heart-lung and lung transplantation post-surgical and obstructive gastroparesis gastroparesis is common after lung transplantation and may be ameliorated by botulinum toxin-a injection of the pylorus upper gastrointestinal dysmotility in heart-lung transplant recipients acute and chronic pleural complications in lung transplantation pleural space complications associated with lung transplantation pleural effusion from acute lung rejection mesothelioma after lung transplantation frequency and management of pneumothoraces in heart-lung transplant recipients shifting pneumothorax after heart-lung transplantation endovascular management of early lung transplant-related anastomotic pulmonary artery stenosis four-year prospective study of pulmonary venous thrombosis after lung transplantation pulmonary venous obstruction after lung transplantation. diagnostic advantages of transesophageal echocardiography primary graft dysfunction and other selected complications of lung transplantation: a single-center experience of 983 patients airway complications and management after lung transplantation: ischemia, dehiscence, and stenosis airway complications after lung transplantation: treatment and long-term outcome segmental nonanastomotic bronchial stenosis after lung transplantation acute cellular and antibody-mediated allograft rejection are symptom reports useful for differentiating between acute rejection and pulmonary infection after lung transplantation? heart lung the role of transbronchial lung biopsy in the treatment of lung transplant recipients. an analysis of 200 consecutive procedures revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection acute allograft rejection: cellular and humoral processes transplant/immunology network of the american college of chest p. a survey of clinical practice of lung transplantation in north america antibody-mediated rejection of the lung: a consensus report of the international society for heart and lung transplantation acute antibody-mediated rejection after lung transplantation acute antibody-mediated rejection after lung transplantation chronic lung allograft dysfunction phenotypes and treatment update on chronic lung allograft dysfunction bronchiolitis obliterans syndrome: the achilles' heel of lung transplantation an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome therapy options for chronic lung allograft dysfunction-bronchiolitis obliterans syndrome following first-line immunosuppressive strategies: a systematic review neutrophilic reversible allograft dysfunction (nrad) and restrictive allograft syndrome (ras) restrictive allograft syndrome (ras): a novel form of chronic lung allograft dysfunction comparison of the incidence of malignancy in recipients of different types of organ: a uk registry audit spectrum of cancer risk among us solid organ transplant recipients bronchogenic carcinoma complicating lung transplantation disseminated ureaplasma infection as a cause of fatal hyperammonemia in humans risk factors for development of new-onset diabetes mellitus after transplant in adult lung transplant recipients prevalence and predictors of diabetes after lung transplantation: a prospective, longitudinal study short-term and long-term outcomes of acute kidney injury after lung transplantation incidence and outcomes of acute kidney injury following orthotopic lung transplantation: a population-based cohort study chronic kidney disease after lung transplantation: incidence, risk factors, and treatment osteoporosis and fractures after solid organ transplantation: a nationwide population-based cohort study bone loss and fracture after lung transplantation contemporary analysis of incidence of post-operative atrial fibrillation, its predictors, and association with clinical outcomes in lung transplantation atrial arrhythmias after lung transplant: underlying mechanisms, risk factors, and prognosis new-onset cardiovascular risk factors in lung transplant recipients skeletal muscle force and functional exercise tolerance before and after lung transplantation: a cohort study maximal exercise capacity and peripheral skeletal muscle function following lung transplantation pneumonia after lung transplantation in the resitra cohort: a multicenter prospective study nocardia infections in solid organ transplantation significance of blood stream infection after lung transplantation: analysis in 176 consecutive patients empyema complicating successful lung transplantation multidrug-resistant gram-negative bacteria infections in solid organ transplantation the impact of pan-resistant bacterial pathogens on survival after lung transplantation in cystic fibrosis: results from a single large referral centre methicillinresistant, vancomycin-intermediate and vancomycin-resistant staphylococcus aureus infections in solid organ transplantation invasive pneumococcal infections in adult lung transplant recipients clostridium difficile in solid organ transplant recipients mold infections in lung transplant recipients fungal infections in lung transplant recipients endemic fungal infections in solid organ transplantation cryptococcus neoformans infection in organ transplant recipients: variables influencing clinical characteristics and outcome dna viral infections complicating lung transplantation cytomegalovirus and lung transplantation community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study quality of life in lung transplantation key: cord-027684-5tpgyjzt authors: protić, alen; bura, matej; juričić, kazimir title: a 23-year-old man with left lung atelectasis treated with a targeted segmental recruitment maneuver: a case report date: 2020-06-24 journal: j med case rep doi: 10.1186/s13256-020-02409-6 sha: doc_id: 27684 cord_uid: 5tpgyjzt background: lung atelectasis are nonventilated parts of lung tissue and occur as a result of the collapse of the pulmonary parenchyma (alveoli). various therapeutic procedures for inflating the collapsed pulmonary parenchyma, such as bronchial aspiration and/or standard recruitment maneuvers, are not always successful. case presentation: we report a case of a 23-year-old croatian man with a parapharyngeal abscess on the left side of the neck with spreading of infection in the mediastinum and left side of the thorax and consequent major atelectasis of the left lung. the patient was mechanically ventilated. we decided to apply a new method in which a pulmonary artery catheter was placed (guided by bronchoscope) on the entrance to the lower left bronchus. the pulmonary artery catheter balloon was inflated to achieve bronchial closure. using another respirator, we ventilated the affected lobe separately with continuously high pressure of 30 cmh(2)o. after 30 minutes, we removed the pulmonary artery catheter from the lower left bronchus and placed it in the upper left bronchus and repeated the procedure. our method allowed a significantly longer duration (30 minutes) of continuously high pressure of 30 cmh(2)o separately to only one of the total of five lobes of the lungs while the other four lobes were simultaneously ventilated continuously with protective ventilation mode. conclusion: use of a pulmonary artery catheter and two respirators in our patient’s case proved to be a successful method for recruiting the atelectatic lung while maintaining protective ventilation of the lung segments without atelectasis. lung atelectasis are nonventilated parts of lung tissue and occur as a result of the collapse of the pulmonary parenchyma (alveoli). usually, it appears in the lower and posterior portions of the lung due to a long duration in supine position, which can enhance an inflammatory process in the lung and result in pneumonia [1] . lung atelectasis in clinical practice can be treated with bronchoaspiration and continuous positive airway pressure (cpap) during spontaneous breathing or positive end-expiratory pressure (peep) during mechanical ventilation [2] . both methods have an effect on the entire pulmonary parenchyma, including nonatelectatic parts of the lung. by increasing the basic peep level to 10 or 12 cmh 2 o in mechanically ventilated patients, trying to prevent atelectasis, the side effects of overstretching unaffected alveoli and increasing intrathoracic pressure also occur. barotrauma and volutrauma of the nonatelectatic lung parenchyma may result, as well as hemodynamic instability due to decreased cardiac output [3, 4] . a more invasive approach to resolving lung tissue atelectasis in the last 15 years has been the recruitment maneuver (inspiratory hold) for 30 to 40 seconds with a positive pressure of 30 to 40 cmh 2 o, where open alveoli are inflated more extensively and the effect on closed alveoli is questionable. case presentation a 23-year-old croatian man without any significant past medical history was admitted to our intensive care unit (icu) due to a parapharyngeal abscess on the left side of the neck with spreading of infection in the mediastinum and the left side of the thorax. urgent surgery was performed with incision of the parapharyngeal abscess, neck dissection, and left side thoracotomy with incision and drainage of the mediastinum and thorax. in the postoperative period in the icu, the patient was sedated and mechanically ventilated with antibiotic therapy according to microbiological findings (blood, urine, bronchoalveolar lavage, and tissue sample taken during surgery). after the first surgery, streptococcus mitis was isolated from the parapharyngeal abscessus. in the second surgery, staphylococcus sp. was isolated from the mediastinal and neck swab wounds. from the beginning (upon admission to the icu), the patient was treated with meropenem and linezolid. during the second week of treatment, he started to develop a nosocomial infection of the lung caused by multiresistant pseudomonas aeruginosa, which was treated with ceftolozane/tazobactam 3 × 3 g intravenously. in the next 10 days, control computed tomographic (ct) scans of the neck and chest showed progression of mediastinal infiltrates and the formation of organized pleural effusion on the left side that required additional surgery. due to atelectasis of the left lung that persisted during the second week of treatment in the icu, bronchoscopy with bronchoaspiration and recruitment maneuvers were performed several times. residual pleural effusions were drained several times before and after weaning procedures with a smallbore pleural catheter and seldinger technique. on the 30th day of the patient's illness, sedation was stopped, and the patient awoke promptly. he was hemodynamically stable and ready for weaning, which was successfully done in the next 24 hours. when the patient was extubated and started to breathe spontaneously, we enhanced his active physical therapy in combination with a cough assist device (coughassist e70; philips respironics, hamburg, germany). after the weaning procedure, the patient was fully conscious and hemodynamically stabile with blood pressure 115/70 mmhg, heart rate of 86 beats/minute, and body temperature of 36.7°c. the patient was spontaneously breathing with oxygen saturation (spo 2 ) of 94%, fraction of inspired oxygen (fio 2 ) of 50%, partial pressure of oxygen (pao 2 ) of 9.19 kpa, carbon dioxide pressure (pco 2 ) of 5.7 kpa, and pao 2 / fio 2 ratio of 138, but almost no rising of the left side of the thorax and no breath sounds on the same side. we performed lung ultrasound, which showed atelectasis of the major part of the left lower lobe and the posterior part of the upper lobe on the 34th day of the patient's stay in the icu. we used electrical impedance tomography (dräger pulmovista 500; dräger, lübeck, germany) as additional diagnostic support for the ultrasound, which confirmed reduced air entrance in the major part of the left lung. finally, according to local protocol, we performed a ct scan of the chest, which confirmed previous findings of complete atelectasis of the left lower lobe and major atelectasis of the left upper lobe (fig. 1) . we decided to apply a new method whereby we used different experiences of single-lung ventilation respecting all basic principles of various types of mechanical ventilation. our trial, which we named "targeted segmental recruitment," was one of the last attempts to avoid the proposed life-threatening surgical reduction of the lung parenchyma in our young patient. his acute physiology and chronic health evaluation ii (apache ii) score upon admission was 7, and his sequential organ failure assessment (sofa) score upon admission was 6. on the day before we performed the segmental recruitment maneuver, his apache ii score was 12, and his sofa score was 6. his procalcitonin level on the day before segmental recruitment maneuver was 0.171 μg/l. the patient was analgosedated, intubated with the univent 8.5-mm tube (tube with integrated endobronchial blocker; vitaid, lewiston, ny, usa), and mechanically ventilated. we removed the endobronchial blocker and replaced it with a pulmonary artery (pa) catheter. with the help of a bronchoscope, using a loop through the working channel of the bronchoscope, the pa catheter was placed in the entrance to the lower left bronchus, and the pa catheter balloon was inflated to achieve bronchial closure. using the appropriate connectors, we connected the pa catheter to the second ventilator (dräger evita 2) and applied 30 cmh 2 o of pressure of the 30% oxygenated air in cpap ventilation mode for 30 minutes. the decision for the pressure of 30 cmh 2 o was made on the basis of the fact that pressures higher than 35 cmh 2 o are associated with barotrauma and the clinical appearance of pneumothorax [5] . the right lung and the upper lobe of the left lung were ventilated the entire time by controlled mechanical ventilation with protective ventilation parameters using the dräger evita xl ventilator. after 30 minutes, we removed the pa catheter from the lower left bronchus and placed it in the upper left bronchus with the described procedure and repeated the cpap maneuver. again, the right lung and the lower lobe of the left lung were simultaneously ventilated by controlled mechanical ventilation with protective ventilation parameters. within the next 12 hours of the performed procedure, the patient was awakened and extubated. after the targeted segmental recruitment, he was breathing spontaneously with improved clinical parameters as well as better rising of the left side of the thorax with audible respiratory sounds on the left side. the pulmovista 500 monitoring was applied continuously, and better ventilation of the left lung was noted in the first hours after the procedure. in the next 48 hours, we applied the coughassist device to the patient, and he was encouraged to cough. after 48 hours (36th day of treatment), a ct scan of the thorax was performed, indicating significantly better ventilation of the left lung (fig. 2) . during and after the procedure, spo 2 , end-tidal carbon dioxide (etco 2 ), invasive blood pressure, and pulse were monitored. the patient did not have any significant decrease in spo 2 or increase in etco 2 , nor did he have hemodynamic instability or changes in heart rhythm. before and after the procedure, arterial blood gas analyses were performed as part of routine laboratory processing ( table 1 ). the patient was discharged to home 49 days after admission, and he was mobile on his own with stabile hemodynamic and respiratory status and without signs of infection. the collapse of the pulmonary parenchyma is a problem that became more visible when diagnostic methods such as ct scanning and ultrasound became more commonly used in clinical practice. in the past few decades, many good ideas have found useful application, such as increasing the basic peep level, prone positioning of the patient, recruitment maneuver with inspiratory hold, and others. the recruitment maneuver has become standard practice in many icus all over the world, despite a well-known effect being overstretching of unaffected alveoli with a questionable effect on collapsed alveoli. a study that used a recruiting maneuver in patients with pneumonia combined with acute respiratory distress syndrome or the need for vasopressors showed increased mortality, which confirmed the potential harm of recruiting maneuvers [6] . unilateral recruitment using an endobronchial blocker has been described in the literature as a successful method of treating atelectasis [7] . the endobronchial blocker was used to protect the left lung while recruitment of the right lung atelectasis was applied for 3 minutes. in this case report, step-forward was done regarding duration of the recruitment maneuver, despite the fact that healthy lung was not ventilated in that period. in our clinical practice, we often use the cpap recruitment maneuver, usually with 40 cmh 2 o pressure for 40 seconds if the patient does not have any hemodynamic instability. if hemodynamic instability is present, we shorten the period of inflation to 20 seconds. our patient was a healthy person with no comorbidities and showed no emphysematous or other changes besides pneumonic infiltration in the lung parenchyma, which can cause barotrauma to the lungs. in the literature, recruitment maneuvers with pressure greater than 30 cmh 2 o are often described. in cases of chronic changes in the lung, we will surely reduce the time of the inflation. after the extubation, we usually encourage secretion removal with aggressive physical therapy and the coughassist device. bronchoaspiration before extubation is our routine procedure in these patients. therefore, we concluded that we had a patient who had undergone many repeated therapeutic procedures, all with the purpose of inflating the left lung (bronchial aspiration, standard recruitment maneuvers, thoracic drainage), and surgical lung resection became an option for treatment. therapeutic options according to the new idea of targeted segmental recruitment as a lifesaving procedure were discussed with the patient's mother, and she signed the informed consent. our method allowed a significantly longer duration (30 minutes) of continuously high pressure of 30 cmh 2 o separately to only one of the five total lobes of the lungs, which meant significantly less increase in the total intrathoracic pressure in comparison with a classic recruitment maneuver. the targeted segmental recruitment procedure significantly reduces the risk of barotrauma of healthy alveoli as well as the negative effect on the hemodynamic stability of patients compared with conventional recruitment maneuvers. targeted segmental recruitment with the help of a pa catheter and the use of two respirators in our patient's case proved to be a successful method for recruiting the atelectatic lung while maintaining protective ventilation of the lung segments without atelectasis. abbreviations: crp c-reactive protein, fio 2 fraction of inspired oxygen, pao 2 partial pressure of arterial oxygen, paco 2 carbon dioxide pressure, spo 2 oxygen saturation types and mechanisms of pulmonary atelectasis recruitment maneuvers in acute respiratory distress syndrome hemodynamics effects of recruitment maneuver ventilator-induced lung injury relationship between ventilatory settings and barotrauma in the acute respiratory distress syndrome heterogeneous effects of alveolar recruitment in acute respiratory distress syndrome: a machine learning reanalysis of the alveolar recruitment for acute respiratory distress syndrome trial a new treatment modality for unilateral atelectasis: recruitment maneuver with endobronchial blocker publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. abbreviations apache ii: acute physiology and chronic health evaluation ii; cpap: continuous positive airway pressure; ct: computed tomographic; etco 2 : end-tidal carbon dioxide; icu: intensive care unit; pa: pulmonary artery; peep: positive end-expiratory pressure; sofa: sequential organ failure assessment authors' contributions ap came up with the idea for the lifesaving procedure explained in this case report. ap performed the procedure and was a major contributor in writing the manuscript. kj helped to carry out the process and to develop the idea. mb analyzed and interpreted the patient data and was a contributor to the writing of the manuscript. all authors read and approved the final manuscript.authors' information ap is a professor in the school of medicine, university of rijeka, and head of the anesthesiology and icu clinic, university hospital rijeka, rijeka, croatia. kj is head of the icu in the anesthesiology and icu clinic. mb is a resident in the anesthesiology and icu clinic. no financial support and sponsorship. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. all data generated or analyzed during this study are included in this published article. this manuscript is a case report about a lifesaving procedure. because it involves human data, the patient signed a consent form for medical information/data about himself to be published. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests.received: 2 march 2020 accepted: 25 may 2020 key: cord-030130-n1x6gcn2 authors: hurtado, daniel e.; erranz, benjamín; lillo, felipe; sarabia-vallejos, mauricio; iturrieta, pablo; morales, felipe; blaha, katherine; medina, tania; diaz, franco; cruces, pablo title: progression of regional lung strain and heterogeneity in lung injury: assessing the evolution under spontaneous breathing and mechanical ventilation date: 2020-08-06 journal: ann intensive care doi: 10.1186/s13613-020-00725-0 sha: doc_id: 30130 cord_uid: n1x6gcn2 background: protective mechanical ventilation (mv) aims at limiting global lung deformation and has been associated with better clinical outcomes in acute respiratory distress syndrome (ards) patients. in ards lungs without mv support, the mechanisms and evolution of lung tissue deformation remain understudied. in this work, we quantify the progression and heterogeneity of regional strain in injured lungs under spontaneous breathing and under mv. methods: lung injury was induced by lung lavage in murine subjects, followed by 3 h of spontaneous breathing (sb-group) or 3 h of low v(t) mechanical ventilation (mv-group). micro-ct images were acquired in all subjects at the beginning and at the end of the ventilation stage following induction of lung injury. regional strain, strain progression and strain heterogeneity were computed from image-based biomechanical analysis. three-dimensional regional strain maps were constructed, from which a region-of-interest (roi) analysis was performed for the regional strain, the strain progression, and the strain heterogeneity. results: after 3 h of ventilation, regional strain levels were significantly higher in 43.7% of the rois in the sb-group. significant increase in regional strain was found in 1.2% of the rois in the mv-group. progression of regional strain was found in 100% of the rois in the sb-group, whereas the mv-group displayed strain progression in 1.2% of the rois. progression in regional strain heterogeneity was found in 23.4% of the rois in the sb-group, while the mv-group resulted in 4.7% of the rois showing significant changes. deformation progression is concurrent with an increase of non-aerated compartment in sb-group (from 13.3% ± 1.6% to 37.5% ± 3.1%), being higher in ventral regions of the lung. conclusions: spontaneous breathing in lung injury promotes regional strain and strain heterogeneity progression. in contrast, low v(t) mv prevents regional strain and heterogeneity progression in injured lungs. supraphysiological levels of pulmonary tissue deformation during mechanical ventilation (mv), measured in terms of relative volume change, are associated with worse outcomes during acute respiratory failure [1] [2] [3] . tissue deformation can be expressed as global strain, can be defined as the ratio of tidal volume (v t ) over the endexpiratory lung volume (eelv) for the lung. global strain has been used to determine safe thresholds of v t to prevent secondary lung injury (i.e., ventilator-induced lung injury) [1] . however, lung damage can occur in patients under mv, even if they are ventilated within the defined global safety limits. lung deformation heterogeneity and regional overdistention have been proposed as promotor mechanisms in these conditions. a spatial correlation between areas of increased regional strain and areas of tissue inflammation has been found in a high global strain model, demonstrating the potential role of regional biomechanical behaviors in the progression of lung injury [2] . considering these findings, a better understanding of the spatiotemporal progression of regional strain and heterogeneity may be the key to avoid progression of damage to the lungs during respiratory failure [3] . clinicians are now concerned by the impact of spontaneous breathing with or without mv during acute respiratory failure. experimental and clinical studies have demonstrated that vigorous spontaneous breathing overlapping with mv may worsen lung injury [4] [5] [6] [7] . however, in non-intubated subjects with lung injury and spontaneous unregulated ventilatory efforts, regional forces generated by the respiratory muscles may lead to injurious effects on a regional level and induce the progression of the lung injury, a phenomenon known as patient selfinflicted lung injury [8] . with these thoughts in mind, we designed this study of experimental lung injury to understand how regional lung inflation patterns evolved in time in spontaneousbreathing subjects and in under controlled low v t mv. we hypothesize that regional deformation in lung injury progresses in time in spontaneous-breathing lungs, whereas it remains uniform in subjects under controlled mv. the study protocol was approved by the universidad andres bello bioethics committee. sprague-dawley rats (sex paired) were considered for this study. the rats were maintained in humidity, light, and temperature-controlled environment inside a dedicated animal research facility. food and water were provided ad libitum. after inhalatory induction with 2% isoflurane (aesica queenborough ltd., uk), the rats were anesthetized by an intraperitoneal injection of ketamine (30 mg −1 kg −1 , drag pharma invetec s.a.) plus xylazine (5 mg −1 kg −1 , alfasan, woerden-holland). after tracheal instillation with 1% lidocaine (drag pharma, santiago, chile), tracheal intubation was performed with a 16g bd angiocath ® catheter (becton-dickinson infusion therapy systems inc., utah, usa). for intubation, an adequate level of anesthesia was assumed if the pedal reflex was absent. otherwise, a second ketamine (15 mg −1 kg −1 ) plus xylazine (5 mg −1 kg −1 ) dose was administered. lung injury was induced by saline lavage, as previously reported [9] , but we adapted the model to maintain a group without mv during the observation period after lung injury. in short, each animal was placed in supine decubitus after intubation, and one instillation of 7.5 ml −1 kg −1 of warm normal saline was flushed in the airway. the residual fluid was suctioned from the airway (surfactant depletion). after lavage, the animals were stabilized for an average time of 10 min with a volumecontrolled mv strategy, using a v t of 6 ml kg −1 , positive end-expiratory pressure (peep) of 5 cmh 2 o, i:e ratio 1:2, respiratory rate (rr) of 90 breaths per minute and an inspiratory fraction of oxygen (fio 2 ) of 1, which was delivered by a ventelite ® small animal ventilator (holliston, ma, usa). block randomization was used to assign the animals to the spontaneous breathing group (sb-group) and the low v t controlled mv group (mv-group) after induction of lung injury and stabilization. extubation in the sb-group was performed after stabilization when spontaneous respiratory effort was detected during a gradual lowering of respiratory frequency; then, animals were supported with a fio 2 of 1 in the oxygen chamber (somnosuite ® for rats, kent scientific, torrington, ct, usa). for the mvgroup, mv was performed using the following parameters: v t 6 ml kg −1 , peep 5 cmh 2 o, i:e ratio 1:2, rr of 90 breaths per minute and fio 2 . all subjects were placed in a prone position for the rest of the study. rectal temperature, electrocardiogram, rr, and oxygen saturation (spo2) were monitored and recorded using the small animal physiological monitoring system (holliston, ma, usa). body temperature was maintained at 38 ± 1 °c through the controlled heating surface of the system. after 15 min of clinical stability in both groups, the vital signs were registered, and the first set of images was acquired. then, the subjects were observed and monitored for 3 h. after this period, a second set of images was obtained. during the observation phase, dissociative anesthesia was adjusted to suppress motor activity in the sb-group and to suppress the respiratory effort and ventilatory asynchrony in the mv-group. initially, the sbgroup consisted of nine subjects, and the mv-group consisted of six subjects. at the end of the study, the animals were killed by intravenous administration of a lethal dose of thiopental (50 mg/kg, richmond laboratories, buenos aires, argentina). a schematic depicting the experimental protocol can be found in additional file 1: figure s1 . micro-computed tomography (micro-ct) images were acquired using a commercial skyscan 1278 in vivo micro-ct scanner (bruker microct, kontich, belgium). images at end-of-expiration (ee) and end-of-inspiration (ei) were acquired at the beginning (t1) and the end (t3) of the ventilation stage. the scanner includes a physiological monitoring system to track the breathing of the subject in order to deliver time-resolved four-dimensional (4d) image sequences. in the sb-group, respiratory gating based on the thorax movement was employed to reduce the effect of motion artifacts [10] , while in the mv-group, the acquisition was controlled by the mechanical ventilator cycles. scans were performed using a source voltage of 70 kv and a source current of 140 μa. the isotropic voxel resolution was 100 μm. the retrospectively synchronized "listmode" scan was performed with an exposure time of 40 ms, a scan rotation of 360° and a step of 0.75°. the entire scanning procedure took approximately 16 min. micro-ct images were then postprocessed using the software provided by bruker (nrecon, tsort, data-viewer, and ctan) to increase the signal-to-noise ratio and to enhance the contrast. ring artifact and hardening filters were employed to improve the quality of the acquired images. median and unsharp mask filters were applied to reduce the noise and enhance the definition of boundaries in the images. lung images were segmented using the active-contour method implemented in the itk-snap software (university of pennsylvania, philadelphia, usa) [11] . manual corrections were performed when necessary, and the resulting segmented images were carefully checked by independent clinical experts to assure anatomically correct structures. two masks for ee and ei images were generated during the segmentation step. a first mask, denoted as whole-lung mask, considered the whole-lung domain. a second mask, denoted as aerated-lung mask, only considered lung regions that belonged to compartments classified as poorly aerated, normal, and hyperaerated, according to their values of hounsfield units and the ranges defined for these compartments that have been reported in the literature [12] [13] [14] [15] . non-aerated regions were excluded from this second mask. the aerated-lung mask was then used to compute the end-of-inspiration lung volume (eilv) and end-of-expiration lung volume (eelv). tidal volume was defined as v t = eilv − eelv. the global strain was calculated as v t /eelv, and minute ventilation was determined as v min = rr × v t . the distribution of aeration compartments was computed from the whole-lung masked images, dividing the lung in four compartments according to the hu intensity, as described above. aeration distribution was reported as stacked bar charts using the percentage of the total lung volume that each compartment occupied. besides, for the sb-group, the aeration distribution of the dorsal region and of the ventral region was computed from dividing the lung image into two subregions of equal volume along the dorsal-ventral direction. the image-based biomechanical analysis was performed following the approach introduced by our group in previous publications [16, 17] . in brief, the niftyreg library [18] was employed to perform image registration between aerated-lung masks of ee and ei to obtain the displacements between the expiratory and inspiratory states of the lung. a 3d tetrahedral finite-element mesh was created from the aerated-lung mask at ei for each lung of all subjects. the displacement of the mesh from ee to ei allowed for the calculation of local volumetric strain. the biomechanical approach used in this work has been summarized in non-technical terms elsewhere [2] . additional file 2: figure s2 shows a schematic diagram of the sequential steps performed for obtaining the 3d regional lung strain maps, which are indicative of local parenchymal stretching [19, 20] . to allow for regional comparison between groups, lungs in each subject were divided into ten segments with approximately equal volumes along the apical-basal (ab) direction and into ten segments along the dorsal-ventral (dv) direction. by intersecting all ab and dv segments, we constructed a matrix of 10 × 10 regions of interest (rois) that are independent of one another. during this procedure, some ab and dv segments did not intersect, and therefore some of the rois were void. weighted mean and standard deviation values of regional volumetric strain were computed for each roi, where the sample includes tetrahedra contained in each roi, and weighting is performed according to each tetrahedron volume. the time evolution of the regional volumetric strain at each roi was studied by means of the regional strain progression index (spi), defined for each roi as spi = (1 + roimean strain at t3)/(1 + roi-mean strain at t1). we note that spi is a relative measure of deformation progression. an spi = 1 implies no evolution of regional strain, an spi > 1 is related to temporal progression (amplification) of regional strain, and spi < 1 implies a reduction of regional strain over time. to evaluate the dispersion of regional strain in an roi, we defined the regional strain heterogeneity index (shi) as the coefficient of variation of the roi strain distribution, which is expressed in terms of volumetric change, i.e., shi = (1 + roi standard deviation)/(1 + roi-mean). a wilcoxon signed-rank test was employed to assess intra-group differences in time for global physiologic parameters such as global strain, spo 2 , eelv, v t , rr, and v min , as well as those parameters in the lung aeration compartments. the time progression of regional deformation in each roi was studied by means of the wilcoxon signed-rank test to assess absolute differences in regional strain between t1 and t3. relative differences in spi were studied by means of a mann-whitney u-test to detect if spi was different from 1.0. the feltz-miller asymptotic test for the equality of coefficients of variation from k populations [21] was employed to independently detect differences of shi between t1 and t3 for each roi. values are expressed as the mean ± sem. all calculations were done using the software for statistical computing r version 3.5.3 (http://www.r-proje ct.org/). surfactant depletion resulted in severe respiratory failure during the stabilization phase, with an s/f ratio 85 ± 3 mmhg for all subjects. statistical and image analysis was carried out using five subjects in the sb-group and five subjects in the mv-group due to the following considerations: (i) mortality in the sb-group was high, with three out of nine subjects dying before completing the observation period and image acquisition; and (ii) the ct images acquired in one animal of each group displayed a notorious alteration of the thoracic-abdominal region, preventing a reliable analysis. the group weights were 271 ± 7 g and 303 ± 15 g for the sb-group and the mv-group, respectively. table 1 reports the physiologic parameters (spo 2 , rr, v t , v min , eelv) and global strain for both groups under study. additional file 3: table s1 shows the individual physiologic data for both groups. no significant changes were detected between t1 to t3 in any of the groups. the distribution of lung aeration is shown in fig. 1 . there were significant changes in time in lung tissue aeration in the sb-group, decreasing normal lung tissue and increasing non-aerated tissue at t3 compared to t1. there were no significant differences in mv-group. additional file 4: figure s3 shows the dorsal and ventral distributions of aeration compartments in the sb-group at t1 and t3. when analyzing the changes in aeration in the sb-group, we found an increase of 29.9% in the non-aerated ventral region and an increase of 17.7% in the nonaerated dorsal region. additional file 5: figure s4 shows ct images at ee and ei for t1 and t3 in sb-group. at ee, we observe the collapse progression over time, particularly at the ventral regions. at ei, we observe a progression of aeration in the basal-dorsal region. roi array maps reporting roi-mean regional volumetric strain for the sb-group and mv-group at t1 and t3 are shown in fig. 2 (see fig. 2a for a sketch depicting the apical-basal and ventral-dorsal directions). when comparing rois in t1 and t3, there was a significant table 1 physiologic data for the experimental groups at the beginning and at the end of the ventilation stage, either for sb-group or mv-group eelv, v min , v t , and global strain were obtained from image analysis of μ-ct images no significant changes were detected between t1 to t3 in any of the groups regional strain in the sb-group at t1. c regional strain in the mv-group at t1. d regional strain in the sb-group at t3. e regional strain in the mv-group at t3. significant within-subject differences are denoted by * (p < 0.05) increase in regional volumetric strain in 38 out of 87 rois (43.7%), which were predominantly located in the basal-dorsal quadrant (fig. 2b, d) . in contrast, in the mv-group, only 1 out of 84 rois (1.2%) was found to be significantly different between t1 and t3 (fig. 2c, e) . additional file 6: figure s5 shows the 3d regional volumetric strain maps for representative subjects of the sbgroup and mv-group at t1 and t3. when analyzing volumetric regional strain over time, all rois in the sb-group (91 out of 91) had a significant spi greater than 1, showing progression (fig. 3a) . a spatially homogeneous progression trend is therefore observed for the whole lung in this group. in contrast, in 83 out of 84 rois of the mv-group, spi was not different than 1, meaning an absence of progression of volumetric strain. roi array maps reporting the roi-mean spi for the sb-group and mv-group are shown in fig. 3 . heterogeneity of regional deformation was assessed through the shi, for which roi arrays are reported in fig. 4 . in the sb-group, we found a significant increase in time in shi in 24 out of 91 rois (26.4%) (fig. 4a, c) . in the mv-group (fig. 4b, d) , only 4 out of 84 rois resulted in a significant increase of shi between t1 and t3 (4.7%). in this work, we studied the lung regional strain distribution, heterogeneity, and deformation progression in subjects spontaneously breathing and subjects on controlled low-v t mv in a murine lung-injury model. we found that a significant progression in regional volumetric strain and heterogeneity was observed after 3 h of spontaneous breathing in injured lungs. changes in lung regional strain during spontaneous breathing were concurrent with the tomographic progression of the nonaerated-tissue compartment of the lung and a reduction of the normal-tissue compartment, in accordance with de-recruitment phenomenon, with collapse progression being higher in ventral regions of the lung. in contrast, the mv-group had limited progression of the regional strain and heterogeneity at the end of the study. a key finding of our study is that regional strain significantly progressed in the sb-group, but did not result in major changes in the mv-group. we note that in the sb-group, global strain increased in roughly 50% from t1 to t3, but due to intra-group variability and the small sample size, this mean increase did not result in significant differences. while this situation is a limitation of the study, it also highlights the high sensitivity of regional deformation analysis in detecting strain progression when compared to global strain. another interesting finding is that strain progression was more substantial in the dorsal and basal regions in sb-group. this observation is supported by the fact that the progression of the lung collapse is stronger in the ventral areas than in the dorsal areas. since collapsed tissue is not expected to deform, it is the dorsal region the one expected to deform the most, which is confirmed by our regional strain analysis. these results suggest the deformation mechanisms associated with the contraction of the diaphragm are relevant to regional deformation. the caudal movement of the diaphragm is relative to its initial relationship with costal insertion, so the capacity to generate force in the caudal direction increases proportionally to the reduction of eelv, as in severe lung injury [22] . these findings are in agreement with the experiment of yoshida and coworkers [23] , where spontaneous breathing was beneficial in subjects with lung injury under mv when its severity was mild. the opposite effects occurred when the lung injury was severe, in which spontaneous breathing amplified the injury, thus increasing transpulmonary pressures, atelectasis, cyclic collapse, and histological signs of damage. similar to the findings of yoshida et al. in mechanically ventilated subjects with severe lung injury, we found more lung damage in sb-group. the paradox of spontaneous breathing and lung damage can be explained by the solid-like behavior of the injured lungs. contraction of the diaphragm generates non-uniform fluctuations of pleural pressure across the lung surface, producing an unsuspected overstretch in dependent regions and displacement of alveolar gas to non-dependent regions of the lungs (i.e., pendelluft) [23] . in our study, the progression of non-aerated tissue in the sb-group may have intensified these phenomena, resulting in an imperfect elastic anisotropic inflation and amplifying the damage in the poorly aerated compartment of the lungs [4] . another finding in our study was the progression of the heterogeneity of deformation of the lung, measured in terms of regional shi, in the sb-group. this observation suggests that sustained vigorous spontaneous ventilatory efforts might promote the progression of deformation heterogeneity in subjects with severe lung injury. in contrast, subjects on controlled mv showed fewer rois with progression of shi. inhomogeneity of ventilation has been proposed as a promoter of lung injury associated with ventilatory support. lung injury promoters are responsible for the amplification of damage injured lungs, even when mv parameters are within standard safety limits (non-harmful). the concept of stress raisers has been introduced to explain the amplification of damage in areas of high inhomogeneity [24, 25] , linking the biological response in the lung parenchyma to the regional deformation in localized areas of the lung. the regional analysis showing inhomogeneity in the sb-group suggests that injurious patterns of ventilation in subjects without mv (spontaneous breathing), such as tidal recruitment, anisotropic inflation, and pendelluft phenomena, among others, can be associated with progression of injury, although the method we used cannot accurately characterize them. these findings take on particular translational relevance because regional differences in tissue aeration have been related to stress raisers and in patient mortality [26] . it is important to note that regional stress is related to regional strain by means of constitutive relations (regional elastance); since regional strain can be directly estimated from image-based biomechanical analysis, it may serve as a better predictor of regional stress [27] . it has been proposed that hyperventilation, due to vigorous diaphragm contraction, can amplify the lung injury. surprisingly, we did not find significant changes in v t in sb-group over time. with these observations, an important question arises: if strain increases in spontaneously breathing subjects, why v t does not change? several mechanisms might explain this conundrum. first, high respiratory drive progressively induces higher inspiratory flow over time. in the inhomogeneous lung, as sbgroup, fast alveolar units received more air, whereas the slow ones got deaerated, similar to other observations in subjects under mv [28, 29] . also, high peak inspiratory flow in spontaneous breathing (deaccelerating pattern) increases the damage because the viscoelastic adaptation of the lung parenchyma does not have enough time to dissipate harmful forces [30] . second, the hering-breuer reflex is a mechanism that can limit v t during spontaneous breathing in subjects with a high respiratory drive. third, vigorous breathing efforts in subjects with lung injury cannot be adjusted or regulated, even with appropriate sedation [23] . a larger amplitude of the diaphragm caudal movement generates an excessive negative intrathoracic and interstitial pressure, contributing to venous return and formation of edema [31] . concurrent with the progression of regional strain and heterogeneity, the sb-group displayed an increase in the non-aerated compartment and a reduction of the normally aerated compartment, which is associated with alveolar collapse. this result is in agreement with observations in subjects with acute lung injury breathing spontaneously under mv [4] , and the ones described by mascheroni et al. in an experimental ovine study. the authors observed a severe deterioration of pulmonary function after 3.5-13 h of pharmacologically induced hyperventilation in spontaneously breathing animals without lung disease. mv and pseudoparalysis prevented these alterations. this study confirms that vigorous spontaneous ventilation can affect the lung, and controlled mv can prevent or attenuate the damage of the lung in this setting [32] . the development of hydrostatic lung edema in the sb-group might be a possible interpretation of these findings. in this group, a higher negative pleural pressure caused by more substantial spontaneous breathing efforts increases the transvascular alveolar pressure, which in turn results in augmented lung perfusion and finally, in edema [33] [34] [35] [36] [37] . also, we need to consider that lung inflammation in regions exposed to high strain might lead to an increase in permeability; thus, they are more susceptible to edema. our work suffers from certain limitations that should be improved in future experiments. operational restrictions and scanning-time demand imposed by the micro-ct scanner did not allow for the use of invasive monitoring. it took over 15 min for a full scan, and inside the scanning chamber, the spontaneous respiratory effort could not be monitored using esophageal pressure monitoring; monitoring gas exchange was also not possible. these restrictions prevented us from quantifying the parameters of global lung mechanics-oxygenation and ventilation-which are needed to classify the severity of the lung injury as well as the intensity of the respiratory effort. we note that subjects in the sb-group were also under anesthesia, which may modify the respiratory pattern. in addition, it is important to mention that our strain measurements in the mv-group are related to dynamic strain and do not account for the deformations that may occur due to the use of peep volume, which we believe are small compared to the dynamic strain. a technical limitation concerned with the image registration and biomechanical analysis is the fact that currently, the regional strain can only be computed in aerated regions of the lung. in particular, regional strain in the non-aerated areas was not calculated. this technical limitation does not allow us to conclude regarding the deformation of collapsed areas of the lung. despite these limitations, we highlight the unique character of this experimental design to study "patient self-inflicted lung injury" (p-sili). we measured the regional strain and heterogeneity in spontaneously breathing subjects in the whole lung. future studies should include a regional analysis of inflammation and atelectasis that could be spatially related to the different deformation measures proposed in this study to confirm correlations between regional deformation, tissue inflammation and edema, and their progression over time. spontaneous breathing can induce progression of lung injury by many mechanisms, a phenomenon known as p-sili. we identified a progression of regional deformation and heterogeneity in injured lungs under spontaneous breathing, but not in low v t mv subjects. this topic has profound implications in translational research, as patients with acute respiratory insufficiency can spontaneously breathe for extended periods before starting appropriate mv support, and also during unsuccessful weaning [38, 39] . understanding the mechanisms involved in the progression of lung damage and its main determinants-heterogeneity and stress raisers, among others-will better support the decision to start or hold off mv support, thus balancing risks and benefits and potentially improving the clinical outcome. low v t mv is a strategy that can attenuate stress raising phenomena, thus reducing the maldistribution of regional strain dictated by lung heterogeneity [26] . future studies need to assess whether other modalities of respiratory support, such as noninvasive mv and high-flow nasal cannulas, can attenuate the progression of lung injury and regional volumetric lung strain. lung stress and strain during mechanical ventilation: any safe threshold? does regional lung strain correlate with regional inflammation in acute respiratory distress syndrome during nonprotective ventilation? an experimental porcine study deterioration of regional lung strain and inflammation during early lung injury the comparison of spontaneous breathing and muscle paralysis in two different severities of experimental lung injury impact of spontaneous breathing during mechanical ventilation in acute respiratory distress syndrome the role of spontaneous effort during mechanical ventilation : normal lung versus injured lung neuromuscular blockers in early acute respiratory distress syndrome mechanical ventilation to minimize progression of lung injury in acute respiratory failure semiautomatic segmentation of longitudinal computed tomography images in a rat model of lung injury by surfactant depletion mapping regional strain in anesthetised healthy subjects during spontaneous ventilation userguided 3d active contour segmentation of anatomical structures: significantly improved efficiency and reliability longitudinal micro-ct as an outcome measure of interstitial lung disease in tnftransgenic mice positive end-expiratory pressure at minimal respiratory elastance represents the best compromise between mechanical stress and lung aeration in oleic acid induced lung injury spontaneous breathing improves lung aeration in oleic acidinduced lung injury a scanographic assessment of pulmonary morphology in acute lung injury improving the accuracy of registration-based biomechanical analysis: a finite element approach to lung regional strain quantification spatial patterns and frequency distributions of regional deformation in the healthy human lung fast free-form deformation using graphics processing units registration-based lung mechanical analysis of chronic obstructive pulmonary disease (copd) using a supervised machine learning framework effect of local tidal lung strain on inflammation in normal and lipopolysaccharide-exposed sheep an asymptotic test for the equality of coefficients of variation from k populations actions of the respiratory muscles spontaneous effort causes occult pendelluft during mechanical ventilation stress distribution in lungs: a model of pulmonary elasticity the role of three-dimensionality and alveolar pressure in the distribution and amplification of alveolar stresses lung inhomogeneity in patients with acute respiratory distress syndrome ventilator-induced lung injury: the anatomical and physiological framework role of strain rate in the pathogenesis of ventilator-induced lung edema effects of inspiratory flow on lung stress, pendelluft, and ventilation heterogeneity in ards: a physiological study the future of mechanical ventilation: lessons from the present and the past spontaneous breathing: a double-edged sword to handle with care acute respiratory failure following pharmacologically induced hyperventilation: an experimental animal study esophageal and transpulmonary pressure in the clinical setting: meaning, usefulness and perspectives changes in transmural central venous pressure in man during hyperventilation exacerbation of acute pulmonary edema during assisted mechanical ventilation using a low-tidal volume, lung-protective ventilator strategy micromechanics of alveolar edema do spontaneous and mechanical breathing have similar effects on average transpulmonary and alveolar pressure? a clinical crossover study treatment of acute hypoxemic non-hypercapnic respiratory insufficiency with continuous positive airway pressure delivered by a face mask: a randomized controlled trial acute respiratory distress syndrome: predictors of noninvasive ventilation failure and intensive care unit mortality in clinical practice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors acknowledge the financial support of the supplementary information accompanies this paper at https ://doi. org/10.1186/s1361 3-020-00725 -0.additional file 1: figure s1 . schematic of the experimental protocol.additional file 2: figure s2 . schematic of the image acquisition process and image-based biomechanical analysis employed to construct threedimensional lung regional strain maps.additional file 3: table s1 . individual physiologic data for both groups. figure s4 . ct images of a representative subject in the sb-group showing the regional progression of lung collapse at the end of expiration (ee) and aeration at the end of inspiration (ei): (i) subject at t1 during ee, (ii) subject at t3 during ee, (iii) subject at t1 during ei, (iv) subject at t3 during ei.additional file 6: figure s5 . regional volumetric strain maps for representative subjects of the sb-group (top row) and the mv-group (bottom row) at t1 and t3. progression of regional strain and heterogeneity in time is observed for the sb-subject, which reaches volumetric strain levels of up to 80%. regional strain distribution remains uniform and homogeneous in the mv subject. this work received funding from the consejo nacional de desarrollo científico y tecnológico conicyt chile through grants fondecyt regular 1160631, fondecyt regular 1180832, and fondef idea id17i10301, and from the millekey: cord-006760-mgrxo21j authors: lee, james c.; diamond, joshua m.; christie, jason d. title: critical care management of the lung transplant recipient date: 2012-06-22 journal: curr respir care rep doi: 10.1007/s13665-012-0018-9 sha: doc_id: 6760 cord_uid: mgrxo21j lung transplantation provides the prospect of improved survival and quality of life for patients with end stage lung and pulmonary vascular diseases. given the severity of illness of such patients at the time of surgery, lung transplant recipients require particular attention in the immediate post-operative period to ensure optimal short-term and long-term outcomes. the management of such patients involves active involvement of a multidisciplinary team versed in common post-operative complications. this review provides an overview of such complications as they pertain to the practitioners caring for post-operative lung transplant recipients. causes and treatment of conditions affecting early morbidity and mortality in lung transplant recipients will be detailed, including primary graft dysfunction, cardiovascular and surgical complications, and immunologic and infectious issues. additionally, lung donor management issues and bridging the critically ill potential lung transplant recipient to transplantation will be discussed. since the modern era of lung transplantation began in 1983 with the first series of successful human lung transplants [1], there have been remarkable advances in this potentially lifesaving procedure for thousands of patients with end-stage lung and pulmonary-vascular diseases. however, the overall survival rates of lung transplant recipients in comparison to other solid organ transplant recipients is lagging, due in part to the unique technical, immunogenic, and infectious aspects of transplanting human lungs [2] . in more recent eras, survival has improved, largely due to improvements affecting the early post-transplant period [2] . despite these improvements, early morbidity and mortality remain important limiting factors for long term success; therefore, early recognition and management of problems that arise before and after lung transplantation in the intensive care unit setting are key to the long term success of the recipient. this review aims to summarize the most important aspects of the critical care management of the lung transplant recipient in the peri-operative time period [3] [4] [5] [6] . donor management in the icu the continued relative lack of supply of organs in contrast to the increasing demand for lung transplantation has spurred interest in expanding the traditionally accepted definition of the "ideal" lung donor, whose criteria of age <55, pao 2 >300, minimal smoking history, and clear chest x-rays have contributed to lung acceptance rates of less than 20 % [7] . one avenue to expand the pool beyond this seemingly restrictive definition is the use of "extended donors" with liberalized selection criteria. some transplant centers have shown that the use of these donors have comparable shortterm outcomes to "ideal donors." other centers have described prolonged icu stays and increased early mortality with the use of donor lungs with infiltrates and/or purulent secretions [8] [9] [10] [11] [12] . aggressive donor management by the team caring for a potential lung donor may result in the improvement of the function of "extended" donor lungs closer to the range of "ideal" organ and thus increasing lung donor conversion rates [13] [14] [15] . a protocol-based approach for the management of potential organ donors, and particularly the ventilator management of potential lung donors, is an effective way to standardize variation in practice styles in the community as well as improve donor conversion rates. the university of texas at san antonio showed that with protocols designed to incorporate standardized lung recruitment maneuvers, aggressive donor fluid management, and aspiration-reduction precautions, rates of lung procurement can be significantly increased. of 98 actual donors during a 4 year protocol period, 54 % were lungs from patients initially considered poor donors [16] . a similar experience in quebec showed that simple lung recruitment protocols can be instituted safely and effectively to increase procurement rates and organ availability, of particular importance in large geographic areas with limited donors [17] . education of intensivists on care of the brain dead patient is key, as proper management of such patients may affect both procurement rates as well as lead to improved immediate post-transplant outcomes. reviewed recently by naik and angel [18• ], brain death elicits hemodynamic instability, activation of inflammatory pathways, and endocrine dysfunction that can profoundly impacts the quality and function of the donated lungs. in conjunction with an active local donor procurement organization, active donor management is necessary to treat these homeostatic derangements. mascia et al. showed in a survey of 15 icus in italy, that there is a clear tendency towards maintaining potentially injurious ventilatory management strategies and not performing recruitment maneuvers after the pronouncement of brain death [19] . this same group also recently demonstrated beneficial effects of employing lung protective ventilatory strategies (tidal volume 6-8 mg/kg predicted body weight, peep 8-10 cm h 2 o) on potential lung donors in a randomized controlled trial compared to conventional ventilatory parameters (tidal volume 10-12 mg/kg predicted body weight, peep 3-5 cm h 2 o) [20] . of 118 patients enrolled into the study, 54 % of donors from the lung protective ventilator strategy group went on to donate lungs vs. 27 % of conventional ventilatory strategy group. six month outcomes of lung recipients from both groups did not differ [20] . the management of the predisposing advanced lung diseases in lung transplant candidates who become acutely ill while awaiting lung transplantation can pose a challenge to the critical care practitioner. given the sometimes unpredictable nature of donor availability, the icu care of such patients has the potential to be prolonged, during which time-sensitive issues such as nutritional status, functional capacity, and infection avoidance in an effort to maintain listing eligibility become the focus of care. since the institution of the lung allocation score (las) in 2005 in the u.s. [21] , the concept of net survival benefit as a balance of risk of death on the waitlist vs. chance of survival at 1 year has driven organ allocation, often assigning the highest scores to patients who are acutely ill and mechanically ventilated. traditionally, requirement for mechanical ventilation had been viewed as a contraindication for active listing at most lung transplant centers due to the fear for poor outcomes. as described by mason et al., after querying the united network for organ sharing for lung transplantation from october 1987 through january 2008, these fears are not unfounded [22••] . the authors showed that of 15,934 transplants performed, 586 patients were on mechanical ventilation and 51 were on extracorporeal membrane oxygenation (ecmo) at the time of transplantation, both factors that contribute to the highest las scores. survival rates at 1, 6, 12, and 24 months were significantly worse in both mechanical ventilation and ecmo supported; patients; for example, 1 year survival was 72 % for the 51 ecmo bridged patients vs. 93 % for the unsupported patients. those patients that received mechanical ventilation tended to be younger, have higher oxygen requirement, poorer renal function, and diagnoses other than emphysema such as cystic fibrosis. of note, the increase in mortality seen in patients with pre-operative mechanical ventilation or ecmo support seemed to be limited to the early time period after lung transplant; patients who required aggressive support pretransplant who survived the first 6 months had comparable long-term survival to those not requiring pre-transplant support [22••] . therefore, these historical administrative data suggest that improvements in the pre-operative morbidity of these procedures, such as reducing sedation, paralytics, or immobility in the pre-operative critical-ill patients, could lead to reasonable long-term outcomes. in recent years, pre-operative life support of the potential recipient has evolved. the concept of "bridging to transplantation" involves the use of mechanical support systems to sustain a patient in respiratory failure until the lung transplant can be performed, often with concurrent aggressive rehabilitation and physical therapy if at all possible [23••, 24] . similar to advances in mechanical circulatory support in heart transplantation, technical advances in the redesign of circulatory pumps, membrane oxygenators, and venous catheters has now made less invasive ecmo support feasible without immobilizing or paralyzing the patient in most cases. smaller, bilumen catheters, introduced into the jugular vein and the inferior and superior vena cava to drain venous blood and simultaneously provide oxygenated blood into the right atrium [25] , may potentially allow patients to be awake, nonventilated, and ambulatory during ecmo support. as this field is rapidly evolving, further research will need to be done on selection of appropriate patients [26, 27•, 28-31] . the immediate post-operative period in the icu remains the most critical for the lung transplant recipient, requiring continuous hemodynamic monitoring, often maximal ventilatory support, and close observation of chest tube output for evidence of bleeding or other surgical complications. aggressive peri-operative antibiotic coverage is employed, often tailored to pre-transplant culture data, with consideration of induction immunosuppression. often, newly instituted transplant medications have the potential for unforeseen side effects on the kidneys, central nervous system, and other organs. the following sections highlight the most important critical care issues in the post-operative lung transplant recipient. a comprehensive list of peri-operative complications is listed in table 1 . the various etiologies of respiratory failure following lung transplantation have been reviewed [32••, 33, 34] and will also be addressed in sections below. the most frequent and significant cause of early mortality after lung transplantation is primary graft dysfunction (pgd), a form of injury to the allograft resulting in large part from ischemia-reperfusion injury from the transplant process itself. pgd affects up to 30 % of all lung transplants, and it leads to prolonged mechanical ventilation and icu length of stay, poor functional outcomes, and an increased risk of bronchiolitis obliterans syndrome (bos) [35, 36] . in its most severe form, pgd presents as diffuse alveolar infiltrates in the allograft in the absence of cardiogenic pulmonary edema, infection, or cellular rejection that can lead to refractory hypoxia. several clinical risk factors for pgd have been described, to which malnutrition the icu physician should be attuned in order to assess the possibility of pgd in the critically ill lung transplant recipient. these include donor characteristics such as female gender, african-american race, extremes of donor age, elevated pulmonary arterial systolic pressure at the time of transplant, obesity and pre-existing diagnoses of pulmonary arterial hypertension and idiopathic pulmonary fibrosis [37] [38] [39] [40] . surgical and intra-operative risk factors for pgd include blood product administration, single transplant procedure and use of cardiopulmonary bypass [41] [42] [43] [44] [45] . as most prior studies are hampered by small numbers, several of these risk factors have been inconsistently reported. ongoing multi-centered prospective studies are underway to better understand the clinical risk factors for severe pgd. treatment of pgd is supportive. other potentially reversible etiologies (table 1) should be ruled-out utilizing the information available to the icu physician such as pulmonary arterial catheter measurements, cvp, radiographs, bronchoscopy, and echocardiography. mechanical ventilator support should be continued while simultaneously avoiding excessive colloid or crystalloid administration. diuresis should be initiated with blood pressure support if needed, as the lung parenchyma is damaged with evidence of capillary leak [46] . theoretical benefits of lung protective ventilator strategies (low stretch, high peep) are extrapolated from the ards literature. as a rescue therapy, pressurecontrolled ventilation modes may be preferentially utilized to minimize barotrauma and airway/anastomosis complications. inhaled nitric oxide, while not proven to be effective in preventing pgd [47] , may have benefit in improving oxygenation, reducing mean pulmonary arterial pressure, and increasing mean systemic arterial pressure in the first 6-8 h after transplant [48] . ventilator management of pgd in single lung transplants with copd can be challenging. acute hyperinflation and significant v/q mismatch can occur, perhaps necessitating dual-lumen independent lung ventilation which can be logistically challenging for the icu staff. in severe and refractory cases, ecmo has been applied in those pgd cases not responsive to traditional mechanical ventilation. in 2009 the university of pittsburgh published their experience with ecmo in heart-lung and lung transplant recipients over a 15 year period. of 763 patients, 7.6 % required ecmo, instituted within the first 7 days after transplant; 39 of 58 patients were successfully weaned off ecmo. thirty day-, 1 year-, and 5 year-survival in this group was 80 %, 59 %, and 33 % respectively [49•] . in this severely ill population, it has been shown that late institution of ecmo, or inability to wean off ecmo, has led to near universal poor outcomes [49•, 50] . most recently, hartwig et al. have investigated whether the use of venovenous ecmo and improvements in icu technology have affected outcomes. at a center where venovenous ecmo was the routine treatment for severe pgd, over a 9 year period of time, 28 of 498 patients required ecmo. patients were able to be weaned from ecmo 96 % of the time, and survival was better than in previous reports: 82 %, 64 %, and 49 % at 30 day, 1 year and 5 years, respectively. while encouraging, the authors did notice worse allograft function in ecmo survivors at 3 years [51••] . this study illustrates that with evolving technology and increased experience, venovenous ecmo may be successfully utilized in very select cases of profound respiratory failure following lung transplantation. the lung transplant recipient with elevated pulmonary arterial pressures at the time of transplant or an underlying diagnosis of pulmonary arterial hypertension requires particularly close attention immediately after lung transplantation. the proper care of such patients begins prior to surgery, as the anesthesiologist should be vigilant to avoid sudden rises in pulmonary vascular resistance and subsequent right heart failure [52•]. intra-operative transesophageal echocardiography can be a useful tool to evaluate right ventricular function. pulmonary vasodilators such as inhaled nitric oxide, milrinone, and inhaled prostacyclin can reduce right ventricular afterload and expedite recovery of the rv in the post-operative state [52• ]. most transplant recipients will require vasopressors during the surgical procedure, and it is not uncommon to return to the icu with vasopressors being administered with the expectation of quick weaning of such agents. fluid management should be aimed at maintaining cardiac output but also minimizing pulmonary edema with active use of pulmonary arterial catheter measurements or echocardiography if available. arrhythmias after lung transplant are typically supraventricular in origin and are common, ranging between 34 % and 74 %. older patients seem particularly at risk for this complication [4] . in a recent review of 200 lung transplant recipients, atrial fibrillation occurred in 39 % of patients within 14 days after surgery, with a mean onset at 3.8 +/-3 days. mean icu stay and hospital stays are lengthened when atrial arrhythmias are experienced [53] . in the icu, hemodynamically significant arrhythmias should be treated aggressively with cardioversion when indicated; otherwise, medical management will usually suffice. if these issues persist, consideration should be given to antiarrhythmic administration such as amiodarone, as well as initiation of anticoagulation. when bleeding complications are concurrent, this can be problematic. the propensity for intra-operative bleeding in lung transplant recipients can often be anticipated prior to the surgical procedure, with proper precautions taken. recipients with an underlying history of heart disease with coronary stents in place may chronically be on antiplatelet agents such as clopidogrel, which will increase the risk of bleeding substantially. additionally, patients with severe pulmonary hypertension may be on warfarin therapy that requires reversal. the explantation of native lungs can also lead to substantial bleeding; scarred lung parenchyma may be fibrotic and adherent to pleural surfaces, or inflamed and associated with chronic foci of infection such as in sarcoidosis or cystic fibrosis patients. other infections such as aspergillomas with reactive pleural involvement sometimes pose a prohibitive risk for bleeding during the explantation of native lungs and can lead to operative demise if significant. in the post-operative setting, bleeding risk must be monitored through serial laboratory studies, chest tube drainage measurements, and radiographs. rapidly enlarging effusions or "white out" of a lung field may indicate a significant pleural bleed, which may not be appreciated based on recorded output alone should the chest tube malfunction or be improperly positioned. differences in size matching present special challenges for management of the lung transplant recipient. lung transplant recipients with fibrotic lung diseases will tend to have smaller thoracic cavities for their height, and because of this, there may be difficulties finding properly size-matched donors. donor lungs may be volume reduced intraoperatively using linear stapling, though potential complications from this type of procedure include air leaks and bronchopleural fistula formation [5] . if lungs are too big for the chest cavity in the immediate postoperative period, the team may choose to delay chest closure if the median sternotomy approach is used, for instance. in the post-operative state, patients with open chests require specialized nursing attention and broadened antibiotic and antifungal coverage. size mismatches of donor lungs that are too small for a thoracic cavity may lead to persistent pleural effusions and high chest tube output. in these situations, chest wall remodeling may occur over time or the recipient may be left with chronic post-operative effusions. vascular anastomotic complications can lead to severe and sudden compromise in the lung transplant recipient. fortunately, these are rare, but may carry high mortality. pulmonary arterial stenosis or thrombus formation typically presents with hypotension and evidence of right heart failure. pulmonary venous thrombosis, usually in proximity to the pulmonary vein-left atrial anastomosis typically presents with hypotension and either lobar or diffuse pulmonary edema with refractory hypoxemia (fig. 1) [6] . because of the rarity of these conditions, diagnosis can be difficult and requires a high index of suspicion. urgent transesophageal echocardiography should be performed at the bedside for patients with a rapid change of course for diagnosis before potential surgical intervention. thrombolysis is a high-risk intervention that can be considered for pulmonary vein thrombosis [54] ; however, management usually involves surgical re-exploration. in the immediate post-operative state, the bronchial anastomoses are prone to complications due to the bronchial circulation being sacrificed during the transplant procedure. this relative ischemia may then be exacerbated by intra-or post-operative hypotension or other hemodynamic fluctuations, making the anastomosis susceptible to necrosis, dehiscence, and infection. frank bronchial dehiscence is rare, on the order of 1 %; partial dehiscence can be addressed with the temporary placement of self-expanding wire stents to encourage granulation tissue growth and healing [32••, 55, 56] . in most lung transplant programs, it is the general practice to sacrifice the bronchial arterial supply when implanting the newly transplanted lung. in spite of concerns that bronchial artery revascularization (bar) prolongs ischemic time and increases operative risk of bleeding, centers who routinely employ bar argue for potential benefits of fewer airway complications and reduced bos risk [57] [58] [59] [60] . before bar can be advocated for widespread use, extension of these techniques to a broader range of centers with consistent surgical competency needs to be addressed. hyperacute and acute rejection hyperacute rejection is a distinct and rare form of lung rejection and is described mostly in case reports [61] [62] [63] [64] [65] [66] . it is characterized by an early and rapid onset, minutes to hours after reperfusion, and is the result of preformed recipient antibodies causing profound allograft dysfunction via mechanisms such as complement activation from abo incompatibility or unrecognized significant anti-hla antibodies to the donor. clinically, one sees pink frothy sputum, profound hypoxemia, and pathologically a coagulopathy with fibrin and platelet thrombi formation within minutes to hours of reimplantation. the first case report appeared in 1996 as described by frost et al. and illustrates the typical presentation: the patient described was a single lung recipient who tolerated a few hours of hyperacute rejection [65] . the patient had a history of two pregnancies, no blood transfusions, and a calculated pra was approximately 33 %. three hours after implantation a donor specific class i antibody to b8 was identified. the patient underwent treatment with plasmapheresis, cytoxan, and ultimately the allograft was removed and the patient relisted for re-transplant. the recipient died 10 days later before another donor could be identified [65] . other case reports detail patient survival after suspected hyperacute rejection with similarly aggressive immunosuppression regimens [61] . although traditionally thought not to occur in the days following transplantation, acute cellular rejection can be seen as early as a week after transplant, and it can make treatment of other icu complications difficult. for instance, during treatment of profound infections in critically ill lung transplant recipients, targeted immunosuppression levels may be lowered or agents stopped altogether in efforts to allow the patient to fend off the current infection. beyond the initial hospitalization, acute cellular rejection is a common occurrence especially in the first year post-transplant, monitored with surveillance bronchoscopy with transbronchial biopsies. the initiation of several immunosuppressive agents in the early post-operative period not only predisposes the transplant recipient to infectious complications, but can cause transient renal dysfunction that may be exacerbated by other concurrent medical complications. the calcineurin inhibitors tacrolimus and cyclosporine are the main culprits for acute renal dysfunction. these agents induce vasoconstriction of the afferent renal arteriole leading to reduction of renal blood flow and glomerular filtration rate. if the critically ill lung transplant recipient experiences peri-operative hypotension, aggressive diuresis for pgd, and is on numerous potentially other nephrotoxic medications, renal dysfunction may be prolonged and severe, leading to serious long-term complications. in a series of 219 lung and heartlung transplant recipients surviving at least 6 months, 91.3 % had a decrease in kidney function, and end stage renal disease occurred in 7.3 % at a median duration of 28 months [67] . infectious complications are a frequent and important cause of morbidity and mortality in the post-operative lung transplant recipient. in addition to the relatively high levels of immunosuppression required by lung transplant recipients, the lungs are unique when compared to other solid organ transplants in that they are continually exposed to the external environment, thereby putting the allografts at risk for many more potential infectious insults. this section will focus on the infectious issues surrounding the care of the lung transplant recipient in the immediate post-operative time period. pre-transplant culture data are vitally important when caring for lung transplant recipients in the icu. ideally patients with underlying suppurative lung diseases such as bronchiectasis or cystic fibrosis will have recent culture data with which to guide immediate antibiotic therapy choices in the post-operative period. organisms such as multi-drug resistant pseudomonas species, methicillin resistant staph aureus, rapidly-growing nontuberculous mycobacteria (ntmb), and fungal organisms will directly impact peri-operative antibacterial and antifungal choices and will likely affect treatment duration as well. in patients with cystic fibrosis, the sinuses and upper respiratory tract may be a reservoir for ongoing infections and therefore aggressive antibiosis and prolonged therapy is often necessary. cultures taken intra-operatively, from bronchoscopy performed after bronchial anastomoses are completed, as well as pleural and chest wall cultures can be very useful as well. the former provide up-to-date sampling of the potential donor flora, which can be used in conjunction with cultures obtained from the donor site to help guide antibiotic therapy. chest cavity cultures can be helpful in recipients with structurally abnormal lungs (e.g. cavitary lesions) or parenchymal pulmonary nodules that may be suspicious for chronic infections such as aspergillus species or ntmb. culture data from the organ donor may potentially affect post-transplant care in the icu. as lung donors are ventilator-dependent, tracheal aspirate cultures are routinely performed, as well as blood and urine cultures. however, such information may not be readily available at the time of transplant, so any significant change in postoperative course or concern for progressing infection in the recipient should prompt an investigation into the results of donor cultures. empiric broad spectrum perioperative antibiotic prophylaxis is often employed, but the decision to continue such treatment is on a case-bycase basis, often impacted by information derived from donor culture results. viral infections in the post-operative state are rare, but conceivably can either be transmitted via the donor or result from an early or subclinical respiratory virus in the recipient at the time of surgery and induction immunosuppression. recipients may have been exposed to community acquired viruses such as respiratory syncytial virus, adenovirus, parainfluenza, and influenza, which may become clinically apparent in the peri-operative period as fulminant respiratory or systemic infections. in contrast, although cmv is a commonly seen viral pathogen in post-transplant patients, overwhelming cmv infection is rare in the immediate post-operative state in the modern era. most centers will institute cmv prophylaxis of varying duration depending on the cmv status of the donor and recipient. due to the wide variety of common and opportunistic infections to which the lung transplant population is susceptible, it is often prudent for the icu practitioner to employ the expertise of transplant infectious disease specialists to help manage such cases. in addition, the presence of a dedicated transplant pharmacist as part of the multidisciplinary team is helpful in monitoring for significant medication interactions that affect serum drug levels and for side effects such as nephrotoxicity. the care of the lung transplant recipient in the immediate post-operative period is a complex undertaking that requires a multidisciplinary team led by the icu practitioner working in conjunction with the transplant medical and surgical teams. the lung transplant recipient is at risk for several categories of complications. with donor supply shortages and increasing numbers of patients awaiting transplant, the scenario of employing more extended criteria lungs in increasingly critically ill recipients at the time of transplant is becoming more likely. great care must be taken to reduce the impact of immediate post-operative morbidity on long term outcomes in this population. disclosure no potential conflicts of interest relevant to this article were reported. lung transplantation for pulmonary fibrosis. toronto lung transplant group the registry of the international society for heart and lung transplantation: twentyeighth adult lung and heart-lung transplant report management of the patient undergoing lung transplantation: an intensive care perspective critical care aspects of lung transplantation lung transplantation: donor and recipient critical care aspects perioperative management in lung transplantation a review of lung transplant donor acceptability criteria outcomes of extended donor lung recipients after lung transplantation successful transplantation of marginally acceptable thoracic organs extended donor lungs: 11 years experience in a consecutive series marginal donor lungs: a reassessment liberalization of donor criteria in lung and heart-lung transplantation availability of lungs for transplantation: exploring the real potential of the donor pool lung donor selection and management donor selection and management impact of a lung transplantation donor-management protocol on lung donation and recipient outcomes pulmonary recruitment protocol for organ donors: a new strategy to improve the rate of lung utilization special issues in the management and selection of the donor for lung transplantation ventilatory and hemodynamic management of potential organ donors: an observational survey effect of a lung protective strategy for organ donors on eligibility and availability of lungs for transplantation: a randomized controlled trial lung allocation in the united states large registry review of united states experience with lung transplantation of patients on mechanical respiratory support bridges to lung transplantation active rehabilitation and physical therapy during extracorporeal membrane oxygenation while awaiting lung transplantation: a practical approach use of bicaval dual-lumen catheter for adult venovenous extracorporeal membrane oxygenation extracorporeal membrane oxygenation as a bridge to lung transplant: midterm outcomes extracorporeal membrane oxygenation in awake patients as bridge to lung transplantation ambulatory venovenous extracorporeal respiratory support as a bridge for cystic fibrosis patients to emergent lung transplantation primary lung transplantation after bridge with extracorporeal membrane oxygenation: a plea for a shift in our paradigms for indications double lumen bi-cava cannula for veno-venous extracorporeal membrane oxygenation as bridge to lung transplantation in non-intubated patient successful lung retransplantation after extended use of extracorporeal membrane oxygenation as a bridge good recent general overview of pulmonary complications following lung transplantation respiratory failure after lung transplantation update of early respiratory failure in the lung transplant recipient primary graft dysfunction primary graft dysfunction: definition, risk factors, short-and long-term outcomes clinical risk factors for primary graft failure following lung transplantation effect of donor age and ischemic time on intermediate survival and morbidity after lung transplantation risk factors for primary graft dysfunction after lung transplantation risk factors for early primary graft dysfunction after lung transplantation: a registry study effect of cardiopulmonary bypass on early graft dysfunction in clinical lung transplantation plasma levels of receptor for advanced glycation end products, blood transfusion, and risk of primary graft dysfunction cardiopulmonary bypass is associated with early allograft dysfunction but not death after double-lung transplantation early lung allograft function in twin recipients from the same donor: risk factor analysis lung transplant for interstitial lung disease: outcomes for single versus bilateral lung transplantation report of the ishlt working group on primary lung graft dysfunction part vi: treatment a randomized trial of inhaled nitric oxide to prevent ischemia-reperfusion injury after lung transplantation effects of inhaled nitric oxide following lung transplantation extracorporeal membrane oxygenation for primary graft dysfunction after lung transplantation: long-term survival institution of extracorporeal membrane oxygenation late after lung transplantation -a futile exercise? improved survival but marginal allograft function in patients treated with extracorporeal membrane oxygenation after lung transplantation anesthetic management for lung transplantation atrial fibrillation, atrial flutter, or both after pulmonary transplantation systemic recombinant tissue plasminogen activator lysis for left atrial thrombus formation after single-lung retransplantation endobronchial stent placement for the management of airway complications after lung transplantation short-term deployment of self-expanding metallic stents facilitates healing of bronchial dehiscence lung transplant airway hypoxia: a diathesis to fibrosis? airway complications after lung transplantation can be avoided without bronchial artery revascularization bronchial blood supply after lung transplantation without bronchial artery revascularization bronchial artery revascularization in lung transplantation: techniques, experience, and outcomes hyperacute rejection in single lung transplantation-case report of successful management by means of plasmapheresis and antithymocyte globulin treatment fulminant hyperacute rejection after unilateral lung transplantation hyperacute rejection of a pulmonary allograft. immediate clinical and pathologic findings hyperacute rejection after single lung transplantation: a case report hyperacute rejection following lung transplantation hyperacute rejection after lung transplantation caused by undetected low-titer anti-hla antibodies predictors of renal function following lung or heart-lung transplantation key: cord-260729-b12v3c8c authors: de lang, anna; baas, tracey; teal, thomas; leijten, lonneke m; rain, brandon; osterhaus, albert d; haagmans, bart l; katze, michael g title: functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov–infected macaques date: 2007-08-10 journal: plos pathog doi: 10.1371/journal.ppat.0030112 sha: doc_id: 260729 cord_uid: b12v3c8c the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. using functional genomics, we analyzed early host responses to sars-cov infection in the lungs of adolescent cynomolgus macaques (macaca fascicularis) that show lung pathology similar to that observed in human adults with sars. analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (il)-6, il-8, and ip-10, which corresponds to the host response seen in acute respiratory distress syndrome. as opposed to many in vitro experiments, sars-cov induced a wide range of type i interferons (ifns) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our studies emphasize that the induction of early ifn signaling may be critical to confer protection against sars-cov infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars. infection with sars-cov causes lower respiratory tract disease with clinical symptoms that include fever, malaise, and lymphopenia [1] . approximately 20%-30% of sars patients require management in intensive care units, and the overall fatality rate has approached 10%. interestingly, children seem to be relatively resistant to sars, but the reason for this restriction is not known [2] [3] [4] . the clinical course of sars follows three phases [5, 6] . in the first phase, there is active viral replication and patients experience systemic symptoms. in the second phase, virus levels start to decrease while antibodies, which are effective in controlling infection, increase. however, pneumonia and immunopathological injury also develop in this phase. ultimately, in the third phase, fatal cases of sars progress to severe pneumonia and acute respiratory distress syndrome (ards), characterized by the presence of diffuse alveolar damage (dad) [1, 7] . it has been hypothesized that the pathological changes are caused by a disproportional immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl10 (ip-10), ccl2 (mcp-1), interleukin (il)-6, il-8, il-12, il-1b, and interferon (ifn)-c [8] [9] [10] [11] [12] [13] . these in vivo data have been confirmed with in vitro experiments, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types [14] [15] [16] [17] [18] [19] . in contrast, production of type i ifns seems to be inhibited or delayed by sars-cov in vitro [14] [15] [16] [17] [18] [20] [21] [22] . moreover, no ifn-a or ifn-b has been detected in the sera of sars patients or in lungs of sars-cov-infected mice [23] [24] [25] . recent in vitro studies demonstrated that type i ifn inhibition or delay may be orchestrated by sars-cov proteins orf 3b, orf 6, and n [26] . the inhibition of ifn production would benefit sars-cov replication, since pretreatment of cells with ifn before sars-cov infection efficiently prevents replication in these cells [21, [27] [28] [29] [30] . furthermore, prophylactic treatment of macaques with pegylated ifn-a reduces sars-cov replication in the lungs [31] . although ifn production was absent in clinical samples, gene and protein expression profiles in these patients were likely impacted by clinical treatments and concurrent preexisting disease. in addition, most if not all virus-host response information is from clinical blood/sera samples that were taken relatively late during infection-little is known about what happens early during infection. animal studies are of great value to decipher the host's initial innate immune response, without confounding clinical treatment (steroid and mechanical ventilation) or underlying co-morbidity. in order to elucidate early host responses during the acute phase of sars-cov infection, we infected cynomolgus macaques with sars-cov and used macaque-specific microarrays and real-time (rt)-pcr techniques to study host gene expression profiles. adolescent cynomolgus macaques infected with sars-cov develop dad similar to sars patients, but clear most of the virus in the lungs by day 6 [7] . because sars-cov replicates predominantly in the lower respiratory tract of macaques, the virus infects a range of cells, including type 1 and type 2 pneumocytes, that are different from those analyzed in vitro. the ability to simultaneously examine virus replication and host response gene expression profiles in the lungs of these animals during the acute phase of sars offers the opportunity to further unravel the pathogenesis of sars. six cynomolgus macaques were inoculated with sars-cov strain hku-39849 and lung tissues were collected at day 1 (n ¼ 2, 1a and 1b) or day 4 (n ¼ 4, 4a-4d). no lesions or clinical symptoms were detected on day 1 after sars-cov infection, whereas on day 4, three out of four monkeys were lethargic, with one of these animals showing mildly labored breathing. pathological changes at day 4 post infection included dad, characterized by flooding of the alveoli with edema fluid, infiltration of neutrophils, damage to the alveolar and bronchial epithelia, and occasional type 2 pneumocyte hyperplasia, as described earlier [31] . four mock-infected animals were included in the study to serve as a reference for host response without viral challenge and to examine outbred inter-animal variation. our previous experience with a/ texas/36/91 influenza virus demonstrated that viral mrna was detected in representative samples of the lung rather than throughout the whole lung [32] . based on this experience, the level of infection in separate lung samples was evaluated using rt-pcr. sars-cov mrna was detected in all animals, and 13 pieces out of the total of 16 lung pieces from infected animals contained high levels of virus, while the three remaining pieces of lung contained very low levels of virus (;3-4 logs lower, figure 1a) . no viral rna could be detected in the samples from the mock-infected animals. for gene expression experiments, lung samples from sars-cov-infected animals were compared to a reference lung sample from mockinfected animals. the three samples with lower virus levels (1a-low, 4a-low, and 4d-low) were analyzed individually so as not to dilute the gene expression of pooled pulmonary samples with higher sars-cov levels and also to potentially further define pulmonary infection. samples from animals with high viral mrna levels showed greater gene expression changes (;2,000 genes day 1, ;800 genes day 4) compared to samples from animals with low levels of viral mrna (;400 genes), indicating a response of lung tissue to the virus ( figure 1b) . additionally, the two day 1 animals showed higher numbers of differentially expressed genes than the day 4 animals. in contrast, gene expression analysis of the separate mock samples revealed limited differentially expressed genes. in order to examine how gene expression would be influenced by presence of virus, timing after inoculation, and individual animal variation, global expression profiling was performed. hierarchical clustering methods were used to order rows (genes) and columns (samples) to identify groups of genes or samples with similar expression patterns [33, 34] . these data were plotted as a heat map in which each matrix entry represents a gene expression value (figure 2a ). red corresponds to higher gene expression than that of the controls; green corresponds to lower gene expression. this analysis yielded 2,050 genes with day 1 samples on one side of the heat map and day 4 samples on the other side of the heat map, indicating an influence of timing after inoculation. there are two major roots to the hierarchical dendrogram, with the larger root composed of all the day 1 samples and the three day 4 samples with the highest virus levels. the smaller root is composed of the remaining day 4 samples with the lowest sars-cov levels. although transcriptional profiling shows some variation when comparing samples from the same animal, the underlying gene expression is similar with a reduction in fold change in the ''low'' samples. these comparisons suggest that both individual animal variation and the ''asynchronous'' nature of the infection in the animals' lungs are factors involved in determining transcription of cellular genes. to validate that the host response from infected animals comprises a stronger transcriptional profile than individual variation from mock-infected animals, differential gene expression patterns in the separate mock samples were investigated, but only 38 genes were differentially expressed ( figure 2b ). these results suggest that underlying basal levels of gene transcription do not confound expression levels after infection. even in a basal state, some low-level lung-to-lung variations were identified within the same animal but not enough to disrupt segregation of lung pieces based on mock-infected animals. severe acute respiratory syndrome coronavirus (sars-cov) infection causes a progressive atypical pneumonia. in typical cases, largely confined to adult and elderly individuals, acute respiratory distress syndrome develops, and admission to an intensive care unit is required. although these complications can be fatal, most sars patients recover, suggesting that protective immune responses are operational. in this study, we simultaneously examined virus replication and host-response gene expression profiles in macaque lungs during the acute phase of sars to gain more insight into the early events that take place after sars-cov infection. we show that a strong host response is induced in the lungs of sars-cov-infected macaques, illustrated by the induction of several pathogenic cytokines and chemokines. interestingly, antiviral pathways are activated as well, demonstrated by the presence of phosphorylated signal transducer and activator of transcription 1 (stat1) transcription factors throughout the lung, but not in sars-cov-infected cells. a subset of cells was shown to produce interferon-b, a cytokine involved in the resistance to many viral infections and able to activate stat1. activation of this antiviral pathway upon sars-cov infection may be an important escape route of the host to withstand the devastating effects of sars-cov. different time points after inoculation, a venn diagram was generated with each set (circle) holding to the parameters of an absolute fold change . 2 and p , 0.0001 in at least two animals ( figure 3a ). the day 1 set contained 1,278 genes and the day 4 set contained 950 genes. when examining host responses that were similar throughout the course of the infection, the intersection of the day 1 and day 4 sets indicates that 597 genes show shared responses. the heat map of these 597 genes is shown in figure 3b . if more stringent criteria were used to find common responses in all six animals, using the 1,278 genes from the day 1 set and the 129 genes that are differentially expressed in all day 4 animals, a subset of 97 genes was identified. this subset included ifnstimulated genes (isgs), like ifits, mx1, gbp1, and g1p2, and also various chemokines and cytokines, such as cxcl10 (ip-10), ccl2 (mcp-1), il-6, and il-8 ( figure s1 ). these same cytokines and chemokines have been reported to be upregulated in human sars cases [9] [10] [11] [12] . this set also included cathepsin l (ctsl), which has been shown to be required for sars-cov entry into a cell [35] . even though only 97 genes were commonly regulated in all animals, indicated with blue bars in figure 3b , the heat map highlights that the other 500 genes show similar expression trends. both sets of commonresponse genes showed similar functionality: cellular growth and proliferation, cell death, cellular movement, immune response, and cell-to-cell signaling. next, we analyzed genes that were differentially expressed exclusively on either day 1 or day 4 in order to find signature gene expression patterns for each day. genes identified as unique responses at day 1 (681 genes) and at day 4 (353 genes) in the venn diagram showed unique functionality ( figure 3c ). the gene expression profile at day 1 shows a prominent innate host response to viral infection; top functional categories on day 1 are the immune response, the hematological system, and the immune and lymphatic system. genes like ifn-c, ccl4 (mip-1-b), csf3, il1a, and tnf are included in these categories. at day 4, a smaller panel of unique differentially expressed genes that play a role in cell cycle, cellular assembly, and dna repair were identified like ccnb2, ccne1, cdca5, cenpa, chaf1a, and prc1. in order to investigate genes that are most strongly regulated after sars-cov infection, genes included in the venn diagram ( figure 3a ) that also held to an absolute fold change . 5 were queried ( figure s2 ). from this set, genes that were involved in the immune response and lung repair processes were used to generate a heat map ( figure 4) . a number of genes that have been reported to be up-regulated in sars patient sera, such as ccl2 (mcp-1), cxcl10 (ip-10), il-6, and il-8, were strongly (;20-fold) induced in all animals. many cell cycle and matrix genes indicative of tissue repair processes were also highly differentially expressed at day 4 (e.g., anln, areg, cdc2, cdkn3, cks2, fosl1, and kif2c). likewise, tissue factor pathway inhibitor 2 (tfpi2), an anticoagulant, was strongly up-regulated during infection in all animals (averaging ;20-fold), as well as plscr1, ser-pine1 (pai1), and thbs1, all genes involved in procoagulation and platelet activation, were induced. concomitant expression of tfpi2 with these pro-coagulation genes might function as an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. surprisingly, expression of diverse ifn-a genes and expression of ifn-b was up-regulated ;10to 20-fold in the day 1 samples. furthermore, ifn-c, a type ii ifn, was efficiently transcribed on day 1 after sars-cov infection (;5-fold). other genes associated with the induction of ifns like ddx58 (rig-i), irf-7, and signal transducer and activator of transcription 1 (stat1), were also highly induced (;8fold). up-regulation of type i ifns in these sars-cov infected macaques is remarkable, since sars-cov inhibits ifn production in many in vitro studies. we did not detect induced ifn-b mrna expression using ma104 cells or caco2 cells and the sars-cov-hku virus (unpublished data). not only ifns, but also several ifn-responsive genes (e.g., g1p2, gbp1/2, ifi/ifits, mx1/2, isg20, and oas1/2/l) were highly transcribed, showing a persistent activation of the innate immune response. furthermore, suppressor of cytokine signaling 1(socs1) is induced at the onset of infection, presumably to establish negative feedback to attenuate cytokine signaling. of note, ifit1 (isg56/ifi56), often used to gauge ifn induction, was up-regulated an average of ;13fold. to further explore some of the pathogenic and antiviral pathways that are induced after sars-cov infection, we investigated the transcription of various cytokines, chemokines, ifns, isgs, and transcription factors involved in the jak/stat pathway. as can be seen in figure 5a , a wide range of chemokines and cytokines are differentially expressed after sars-cov infection in macaque lungs, especially on day 1 after infection. besides previously mentioned chemokines, we detected monocyte chemotactic protein genes like ccl8 (mcp-2) and ccl7 (mcp-3), but also ccl11 (eotaxin), a chemotactic protein for eosinophils. in the samples with low sars-cov mrna levels, the induction of chemokines is less evident, suggesting that the presence of these molecules is restricted to areas in the lung where virus is present. furthermore, sars-cov-infected macaques showed a stronger induction of ifns (14 unique genes) and isgs (20 unique genes) on day 1 than day 4 and when virus was present at high levels. note that besides ifn-a, ifn-b, and ifn-c, the ifn-ks (il-29, il-28a, il-28b), which are type i ifns, were induced in samples with high sars-cov levels. in the absence of viral rna, no ifns, but interestingly, a number of isgs (17 unique genes) were detected, suggesting paracrine stimulation ( figure 5b ). differential expression of a selection of strongly upregulated genes, cxcl10 (ip-10), il-6, il-8, and ifn-b, was confirmed using rt-pcr ( figure 6 ). in accordance with the microarray data, the rt-pcr data showed that cxcl10 (ip-10), il-6, il-8, and ifn-b were all expressed at levels that were approximately 100 times higher in the sars-cov-infected animals at day 1 than in the uninfected control animals and were still elevated on day 4 after infection. as can be seen in figure 6 , the induction of ifn-b was strongly correlated to the presence of virus (r spearman ¼ 0.88, p , 0.0001). for cxcl10 (ip-10), il-6, and il-8 the correlation is less evident, which is not surprising since these cytokines can be induced by other factors than the virus itself. in order to visualize the host response in the lungs of sars-cov-infected macaques, ifn-b production and translocation of phosphorylated stat1 was studied using immunohistochemistry. in the lungs of the sars-cov-infected macaques, a modest number of cells stained positive for ifnb at day 1 post infection, whereas no ifn-b-positive cells could be detected in mock-infected macaques ( figure 7a -7c). notably, most of the cells that stained positive for ifn-b were located very close to blood vessels, but not in the alveoli where most sars-cov antigen-positive cells (mainly type 2 pneumocytes at 1 day post infection) are located. to examine whether the ifns that are produced in the lungs of these sars-cov-infected macaques are biologically active and able to induce stat1 phosphorylation and translocation, lung sections of the infected macaques were stained with antibodies against phosphorylated stat1. as shown in figure 7d and 7e, no phosphorylated stat1 could be detected in the lungs of pbs-infected macaques, while in the lungs of sars-cov-infected macaques, cells with phosphorylated stat1 in their nucleus were abundantly present. subsequently, the same pieces of lung from sars. genes were included if they met the criteria of a 2-fold change or more (p 0.0001). a two-of-nine strategy allowed samples to cluster together if profile similarities existed based on timing of inoculation (n ¼ 2 samples for day 1). (b) the number after pbs refers to the animal (i.e., pbs 1), while the number after the dash refers to the lung piece (i.e., pbs 1-1). thirty-eight genes are displayed with an absolute fold change . 2 and p ,0.0001 in at least two animal samples. up-regulated genes are indicated in bold underline. only one gene, hla-dqa1, was down-regulated . 5. no up-regulated genes met these criteria in mock-infected animals. separate mock samples (i.e., pbs 1-1) were compared to the total mock pool. doi:10.1371/journal.ppat.0030112.g002 cov-infected macaques at day 1 were double stained for phosphorlylated stat1 and sars-cov (figure 7 f) . notably, phosphorylated stat1 was not detected in the nucleus of sars-cov-infected cells (type 2 pneumocytes), while cells directly adjacent to these sars-cov-infected cells stained for phosphorylated stat1 in many, but not all, foci containing sars-cov-positive cells. thus, type i ifns are produced in the lungs of sars-cov-infected macaques, and are able to activate the jak/stat pathway. however, translocation of stat1 does not occur in sars-cov-infected pneumocytes. although recent studies indicate that the sars-cov orf6 protein is able to inhibit nuclear translocation of stat1 in vitro, this was not demonstrated in experiments using infectious sars-cov [26] . in order to assess whether sars-cov inhibits phosphorylation and translocation of stat1, ma104 cells were infected with sars-cov for 24 h and then either fixed directly or treated with type i ifn. cells infected with sars-cov, but not treated with ifn, stained positive for sars-cov (unpublished data), but lacked staining for phosphorylated stat1, indicating that sars-cov or other soluble mediators are not able to induce stat1 phosphorylation ( figure 8 ). after treatment of the ma104 cells with ifn, phosphorylated stat1 could be detected in the nucleus of most cells, but not in the nucleus of sars-cov-infected cells (figure 8 ). this demonstrates that sars-cov inhibits the translocation of phosphorylated stat1 to the nucleus, confirming our in vivo data. besides inhibiting translocation of phosphorylated stat1, sars-cov also seems to reduce stat1 phosphorylation, as the majority of sars-covinfected cells contained low levels of phosphorylated stat1 in their cytoplasm. pathogenic viruses escape the antiviral action of the ifn system by inhibiting both ifn production and signaling pathways. here, we report that even though production and signaling of type i ifns is inhibited by sars-cov in vitro as well as in sars-cov-infected cells in vivo, high levels of type i ifns are induced in the lungs of sars-cov-infected macaques. these ifns are able to activate stat1, followed by the transcription of numerous isgs. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our results emphasize the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars-cov infection in cynomolgus macaques. to our knowledge, this study represents the first functional genomics investigation of sars-cov infection in cynomolgus macaques. all experimental animals showed signs of infection because viral mrna could be detected in random samples from the lung, indicating that the virus had spread throughout the whole lung at the time of necropsy. furthermore, pathological examination of sars-cov-infected macaques at day 4 post infection revealed multifocal dad [31] . unlike 10% of humans with sars, which are mainly restricted to the elderly, adult macaques used in this study do not succumb to sars-cov infection. however, the sars-cov-induced pathology in these macaques likely resembles the pathological changes seen in the majority of human sars patients that recover from the disease. although none of the current animal models has fully reproduced all features of sars, the most important aspects of this disease are observed in experimentally infected macaques, providing valuable insights into the initial innate immune response after infection without confounding clinical treatment or underlying co-morbidity. using macaque-specific microarrays, we were able to observe that with early infection, high levels of viral mrna corresponded to a strong cellular host response. this strong host response is dominated by genes involved in the immune response and includes a wide range of genes corresponding with what is seen in human ards. during the acute phase of human ards, activated neutrophils and macrophages enter the alveoli and produce a number of cytokines and chemokines such as il-6, il-8, and cxcl10 (ip-10) [36] , as were found in the lungs of our sars-cov-infected macaques. researchers have postulated that these genes also predict adverse sars patient outcome [37] . during the chronic phase of human ards, type 2 pneumocytes start to proliferate and differentiate in order to repair the damaged lung. at day 4, [14, 38] . we also detected a strong presence of genes involved in the coagulation pathway, including tfpi2, serpine1, and timp1. the idea of a pro-coagulation profile mimics the clinical-pathological observations of sars patients that showed unusually disseminated small vessel thromboses in the lungs [5, 39] . additionally, cathepsin l was up-regulated in all sars-cov-infected macaques. induction of this gene after sars-cov infection is quite interesting because cathepsin l is an endosomal protease that is necessary for sars-cov to infect a cell [35] . remarkably, sars-cov infection in macaques leads to a strong transcription of ifns. not only ifn-a, ifn-b, and ifnk (all type i ifns), but also ifn-c, a type ii ifn, were all highly up-regulated, especially on day 1 after infection. the expression of ifn-b, which strongly correlated to the amount of virus present, continued throughout day 4 and was confirmed using immunohistochemistry; ifn-b-positive cells could be detected in the lungs of the sars-cov-infected macaques. the induction of ifn-b in these sars-covinfected macaques is surprising, because several reports have shown that sars-cov inhibits or delays type i ifn production in a number of cell types [14] [15] [16] [17] [18] 20, 22] . for example, sars-cov blocks a step in the activation of irf-3, a transcription factor that is required for ifn-b induction [21] . in addition, the sars-cov proteins orf3b, orf6, and nucleocapsid have been shown to function as ifn antagonists, as has the sars-cov nsp1 gene that prevents the production of sendai virus-induced ifn-b in 293 cells [26, 40] . interestingly, it was recently shown that plasmacytoid dendritic cells (pdcs) are able to produce ifn-a and ifn-b after sars-cov infection, while conventional dcs did not produce these type i ifns [41] . pdcs are known for their ability to produce very high amounts of ifn-a and ifn-b and are considered firstline sentinels in immune surveillance in the lung [42] [43] [44] [45] [46] . we speculate that the ifn-b-producing cells detected in the lungs of sars-cov-infected macaques are pdcs. future studies may address the nature of these ifn-producing cells once technical difficulties in detecting pdcs in macaque tissues have been tackled. these studies may also shed light on whether decreasing numbers of pdcs observed in clinical blood samples from human sars patients are caused by sequestering of pdcs by the lungs, destruction of pdcs by sars-cov, or destruction or suppression of pdcs by steroid treatment [47] . when ifns are produced, they bind to their receptors on the cell membrane, after which stat1, a key member of the jak/stat pathway, is phosphorylated and subsequently translocated to the nucleus, followed by the production of a wide range of ifn-stimulated genes. in vitro, sars-cov inhibited translocation of stat1 to the nucleus, and phosphorylation of stat1 was strongly reduced. however, the inhibition of stat1 phosphorylation was not absolute because cells with low levels of phosphorylated stat1 in their cytoplasm were also detected. in accordance with our data, kopecky-bromberg et al. recently showed that the sars-cov protein orf6 is able to inhibit stat1 translocation [26] . this strategy is not unique to sars. other viruses have been shown to be able to block signaling of ifns by affecting phosphorylation and/or translocation of the stat proteins. for example, measles virus v protein inhibits translocation of stat1, but does not affect phosphorylation, whereas measles virus p protein blocks both of these processes [48] . other paramyxoviruses, like rinderpest virus, nipah virus, hendra virus, and mumps virus, as well as flaviviruses like west nile virus and japanese encephalitis virus, are able to block activation of stat1 and stat2 [49] [50] [51] [52] . inhibition of stat1 phosphorylation is not always complete. for example, sendai virus suppresses tyrosine phosphorylation of stat1 during the early stages of infection, but this block becomes leaky after a couple of hours with phosphorylated stat1 accumulating in the cytoplasm [53] . in contrast to these in vitro data, we observed phosphorylated stat1 in the nuclei of numerous cells in the lungs of sars-cov-infected macaques, indicating that these cells had been activated by the ifns produced in the lung. however, phosphorylated stat1 was not detected in sars-covinfected cells. the observations made in this study indicate that sars-cov-infected macaques produce ifns in response to virus infection and are further capable of activating the stat1 pathway in cells surrounding the sars-cov-infected cells. the importance of ifns in controlling sars-cov infection has been suggested in several animal studies. mice clear sars-cov in the absence of nk cells, t cells, or b cells, suggesting that innate immune responses are sufficient to limit sars-cov infection in these animals [23] . indeed, stat1 knock out mice, which are resistant to the effects of ifns, to some extent show a worsening of pulmonary disease and an increase in viral replication in the lungs compared to normal mice after infection with sars-cov [54] . although ifn treatment was not conducted in sars-cov infection mouse studies, prophylactic treatment of macaques with pegylated ifn-a protects type 1 pneumocytes from infection with sars-cov [31] . in addition, potent antiviral activity is observed in vitro when cells are treated with ifns before they are infected with sars-cov [27, 29, 30] . although we cannot determine the effect of neutralizing ifn-b in sars-covinfected animals, based on the experiments utilizing recombinant ifns in these animals, we postulate that type i ifns are partly responsible for the relatively mild clinical symptoms that are seen in sars-cov-infected macaques. in addition, a recent study again demonstrated the importance of ifns in viral infections, as macaques infected with the highly pathogenic and fatal 1918 influenza virus showed limited induction of type i ifns (only ifna4 reached fold changes . 5) and delayed induction of isgs, while macaques infected with the low-pathogenic k173 influenza virus showed a strong induction of these antiviral molecules early during infection [55] . notably, ifn-b was not up-regulated (absolute fold change , 2) in any of the influenza virusinfected animals, even in those animals that recovered, unlike sars-cov-infected macaques that showed a very strong presence of ifn-b. in conclusion, our study demonstrates that cynomolgus macaques can be infected with sars-cov, as indicated by presence of viral mrna at different locations throughout the lung at day 1 and day 4, with gross pathology becoming noticeable at day 4. furthermore, we show that infection of cynomolgus macaques with sars-cov leads to a strong immune response, including the induction of various cytokines and chemokines, resembling the host response seen in human sars patients. strikingly, despite the fact that sars-cov infection blocks the production of ifns in vitro, type i ifns are strongly induced in the lungs of sars-covinfected macaques. the production of ifn early during infection leads to widespread activation of stat1 and the production of isgs. this suggests that, although sars-cov blocks ifn signaling in infected cells, locally produced ifns are capable of activating non-infected cells and possibly can prevent infection of these cells. thus, sars-cov infection in macaques leads to the differential activation of both pathogenic and antiviral signaling pathways in vivo, and the outcome may be determined by the relative contribution of these signaling pathways. were infected intratracheally with 1 3 10 6 tcid 50 sars-cov (hku-39849) as described earlier [31] . virus stocks were generated in vero e6 cells that were defective in ifn production. two animals were euthanized on day 1 after infection and four animals were euthanized on day 4. in addition, four animals were mock (pbs) infected and euthanized on day 4, serving as a negative control group. one lung from each monkey was fixed in 10% formalin for histopathology and immunohistochemistry while the other was used for real-time pcr and microarrays. lung samples were randomly excised from three different lung areas (cranial, medial, caudal) and stored in rnalater (ambion, http://www.ambion.com/). sixteen pieces of lung were taken from the sars-cov-infected animals, two to three pieces of lung per animal. twelve pieces of lung were taken from the mock-infected animals, three pieces of lung per animal. individual lung samples in rnalater were transferred to trizol reagent (invitrogen, http:// www.invitrogen.com/), homogenized using polytron pt2100 tissue grinders (kinematica, http://www.kinematica.ch), and then processed to extract rna. all experiments were executed under a biosafety level 3, and approval for animal experiments was obtained from the institutional animal welfare committee. oligonucleotide microarray analysis. infected macaque lung samples were co-hybridized with a reference mock-infected macaque lung sample on macaque oligonucleotide arrays containing 131 viral probes, corresponding to 26 viruses, and 22,559 rhesus probes, corresponding to ;18,000 rhesus genes. the reference mock-infected sample was created by pooling equal mass quantities of total rna extracted from the 12 individual lung pieces from mock-infected animals. an agilent 2100 bioanalyzer was used to check the purity of the total rna prior to crna probe production with the agilent low rna input fluorescent linear amplification kit (agilent technologies, http://www.agilent.com/). arrays were scanned with an agilent dna microarray scanner, and image analysis was performed using agilent feature extractor software (agilent technologies). each microarray experiment was done with two technical replicates using dye reversal [56] . all data were entered into a custom-designed database (expression array manager) and analyzed with resolver 4.0 (rosetta biosoftware, http://www.rosettabio.com/) and spotfire deci-sionsite for functional genomics (spotfire, http://www.spotfire.com/). in our data analysis, genes were selected to be included for transcriptional profile based on two criteria: a greater than 99.99% probability of being differentially expressed (p 0.0001) and an expression level change of 2-fold or greater. ingenuity pathway analysis (ingenuity systems, http://www.ingenuity.com/) was used to functionally annotate genes according to biological processes and canonical pathways. in accordance with proposed miame standards, primary data are available in the public domain through expression array manager at http://expression.microslu.washington.edu/ expression/index.html [57] . quantitative real-time rt-pcr. rt-pcr was performed to detect sars-cov mrna and to validate cellular gene expression changes as detected with microarrays. each reaction was run in triplicate using taqman 2x pcr universal master mix (applied biosystems, http:// www.appliedbiosystems.com/) with primers and probe specific for the sars-cov nucleoprotein gene [7] , or for macaque cellular genes (sequences shown in table 1 ). differences in gene expression are represented as the fold change in gene expression relative to a calibrator and normalized to a reference, using the 2 àddct method [58] . gapdh (glyceraldehydes-3-phosphate dehydrogenase) or 18s rrna were used as endogenous controls to normalize quantification of the target gene. the samples from the mock-infected macaques were used as a calibrator. immunohistochemistry. formalin-fixed, paraffin-embedded lung samples from sars-cov-infected and mock-infected macaques were stained for sars-cov, phosphorylated stat1, and ifn-b using mouse-anti-sars-nucleocapsid (clone ncap4, mouse igg2b; imgenex, http://www.imgenex.com/), mouse-anti-phospho-stat1 (clone st1p-11a5, mouse igg2a-j; zymed laboratories, http://www. invitrogen.com/), and rabbit-anti -ifn-b (chemicon, http://www. chemicon.com/), respectively. after deparaffinization, antigen retrieval was performed using a citrate buffer for the sars-cov and stat1 staining. no antigen retrieval was performed when staining for ifn-b. goat-anti-mouse igg2a hrp, goat-anti-mouse igg2b ap (southern biotech, http://www.southernbiotech.com/), and anti-rabbit igg-hrp (dako, http://www.dako.com/) were used as secondary antibodies. signals were developed with fast red and dab (sigma, http://www.sigmaaldrich.com/) and counterstained with mayer's hematoxylin. in vitro sars-cov and stat1 staining. ma104 cells (african green monkey foetal kidney cells, ecacc) were cultured in eagle's minimal essential medium (emem; cambrex, http://www.cambrex. com/) supplemented with 2 mm glutamine, 1% non-essential amino acids and 10% foetal bovine serum. cells were seeded in 96-well plates and infected with sars-cov (moi 0.5), and 24 h after infection, selected wells were treated with universal type i ifn (5,000 u/ml, sigma) for 30 min at 37 8c. subsequently, cells were fixed with 10% neutral-buffered formalin and treated with 70% ethanol. sars-cov-infected cells were visualized using purified human igg from a convalescent sars patient (csl), followed by staining with an antibody to human igg, linked to alexa fluor 594 (invitrogen). phosphorylated stat1 was visualized using mouse-anti-phospho-stat1 (zymed), followed by staining with a fitc-linked antibody to mouse igg. lung pathology of fatal severe acute respiratory syndrome severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update clinical presentations and outcome of severe acute respiratory syndrome in children clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study the severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome an interferongamma-related cytokine storm in sars patients expression profile of immune response genes in patients with severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome characterization of cytokine/chemokine profiles of severe acute respiratory syndrome a probable role for ifn-gamma in the development of a lung immunopathology in sars comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229e cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells a human in vitro model system for investigating genome-wide host responses to sars coronavirus infection inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro severe acute respiratory syndrome coronavirus fails to activate cytokinemediated innate immune responses in cultured human monocyte-derived dendritic cells inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists treatment of sars with human interferons potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics hybrid hierarchical clustering with applications to microarray data analyzing microarray data using cluster analysis inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry the acute respiratory distress syndrome early enhanced expression of interferon-inducible protein-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclincyclin-dependent kinase complex and blocks s phase progression in mammalian cells lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation control of coronavirus infection through plasmacytoid dendritic-cellderived type i interferon the nature of the principal type 1 interferon-producing cells in human blood different roles for human lung dendritic cell subsets in pulmonary immune defense mechanisms plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon plasmacytoid dendritic cells in immunity characterization of myeloid and plasmacytoid dendritic cells in human lung longitudinal alteration of circulating dendritic cell subsets and its correlation with steroid treatment in patients with severe acute respiratory syndrome tyrosine 110 in the measles virus phosphoprotein is required to block stat1 phosphorylation inhibition of interferon signaling by the new york 99 strain and kunjin subtype of west nile virus involves blockage of stat1 and stat2 activation by nonstructural proteins mumps virus v protein antagonizes interferon without the complete degradation of stat1 blocking of interferoninduced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism rinderpest virus blocks type i and type ii interferon action: role of structural and nonstructural proteins sendai virus c protein impairs both phosphorylation and dephosphorylation processes of stat1 resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus statistical design and the analysis of gene expression microarray data minimum information about a microarray experiment (miame)-toward standards for microarray data analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method we thank s. smits for her assistance with sars-cov infections and rna isolations. author contributions. adl, tb, ado, blh, and mgk conceived and designed the experiments. adl, tb, tt, lml, and br performed the experiments. adl, tb, and blh analyzed the data. adl, tb, ado, blh, and mgk wrote the paper.funding. this work was supported by the us national institutes of health, r01 grant hl080621-01a1, and by the european union, grant sp-22-ct-2004-511060.competing interests. the authors have declared that no competing interests exist. key: cord-010994-1ynel55w authors: abe, kyoko; horiguchi, takashi; enzan, keiji; masaki, yoko; nishikawa, toshiaki; kimura, tetsu title: nicorandil, a k(atp) channel opener, attenuates ischemia–reperfusion injury in isolated rat lungs date: 2020-02-21 journal: lung doi: 10.1007/s00408-020-00339-0 sha: doc_id: 10994 cord_uid: 1ynel55w purpose: nicorandil is a hybrid between nitrates and k(atp) channel opener activators. the aim of this study was to evaluate the nicorandil’s effects on ischemia–reperfusion (ir) lung injury and examine the mechanism of its effects. methods: isolated rat lungs were divided into 6 groups. in the sham group, the lungs were perfused and ventilated for 150 min. in the ir group, after perfusion and ventilation for 30 min, they were interrupted (ischemia) for 60 min, and then resumed for 60 min. in the nicorandil (n) + ir group, nicorandil 6 mg was added before ischemia (nicorandil concentration was 75 µg ml(−1)). in the glibenclamide + n + ir group, the l-name (n(ω)-nitro-l-arginine methyl ester) + n + ir group and odq (1h-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) + n + ir group, glibenclamide 3 µm, l-name 100 µm, and odq 30 µm were added 5 min before nicorandil administration, respectively. we measured the coefficient of filtration (kfc) of the lungs, total pulmonary vascular resistance, and the wet-to-dry lung weight ratio (ww/dw ratio). results: kfc was significantly increased after 60 min reperfusion compared with baseline in the ir group, but no change in the sham group. an increase in kfc was inhibited in the n + ir group compared with the ir group (0.92 ± 0.28 vs. 2.82 ± 0.68 ml min(−1) mmhg(−1) 100 g(−1); p < 0.01). also, nicorandil attenuated ww/dw ratio was compared with ir group (8.3 ± 0.41 vs. 10.9 ± 2.5; p < 0.05). nicorandil’s inhibitory effect was blocked by glibenclamide and odq (p < 0.01), but not by l-name. conclusions: nicorandil attenuated ir injury in isolated rat lungs. this protective effect appears to involve its activation as k(atp) channel opener as well as that of the sgc-cgmp pathway. ischemia-reperfusion (ir) injury in patients undergoing lung transplant surgery remains a problematic and unresolved issue. ir injury is known to cause primary graft dysfunction, also known as primary graft failure, and to be associated with increased short-and long-term mortality after lung transplantation [1, 2] . however, the underlying mechanisms, especially how and where endothelial cells are impaired during and after ir lung injury, remain unclear. nicorandil is a hybrid compound with nitrate and k atp channel opener properties [3] that has shown protective effects against coronary events in patients with stable angina [4] . although the protective effects of nicorandil on ir injury may be because of its k atp channel opener properties [5] , its effects on microvascular permeability both during and after ir lung injury have yet to be evaluated. in addition, nicorandil's nitrate properties have yet to be investigated in patients with ir lung injury. therefore, the objective of the present study was to investigate whether nicorandil reduces the risk of ir injury in an isolated buffer perfused rat lung model, and whether nicorandil's beneficial effects are a result of its k atp channel opener properties and nitric oxide production, or are attributable to activation of the soluble guanylyl cyclase (sgc)/ cyclic guanosine monophosphate (cgmp) pathway. the present experimental protocol was approved by the animal experiment committee of the akita university graduate school of medicine in akita, japan, and was performed in accordance with the relevant aspects of the arrve guidelines. first, male sprague-dawley rats ranging in weight from 300-350 g were anesthetized intraperitoneally with sodium pentobarbital (30 mg/kg). following local anesthesia with 1.0% lidocaine, tracheostomy and sternotomy were performed, and the lungs were mechanically ventilated using a ventilator (ugo basile muromachi kikai co., ltd., tokyo, japan) with room air at a rate of 50 breaths/min, a tidal volume of 2.5 ml, and a positive end-expiratory pressure of 2 cmh 2 o. following a median sternotomy, heparin (200 u) was injected into the right ventricle and allowed to circulate for approximately 3 min. next, the pulmonary artery (pa) was cannulated through the right ventricle, and the snare around the pa was tightened to isolate the lung inflow. similarly, the left atrium (la) was cannulated though the left ventricle and tightened. next, the lungs were ventilated with the same gas mixture of 21% o 2 , 5% co 2 , and balanced nitrogen, and then perfused with a bicarbonate-buffered physiological salt solution containing 119 mm nacl, 4.7 mm kcl, 1.17 mm mgso 4 , 22.6 mm nahco 3 , 1.18 mm kh 2 po 4 , and 3.2 mm cacl 2 . we then added 100 mg dextrose, 20 mu insulin, and 5 g bovine serum albumin (sigma-aldrich, co., st. louis, mo, usa) to each 100 ml of this stock solution. the perfusate ph was adjusted to 7.35-7.45 through the addition of sodium bicarbonate. next, to monitor weight changes, the heart and lungs were removed en bloc and placed on an electronic balance (gx-400; a and d, tokyo, japan) in a humidified chamber with a set temperature of 37 ºc (fig. 1) . we then discarded the first 50 ml of perfusate, which contained large amounts of residual blood cells and plasma, and used 80 ml of perfusate for recirculation. we monitored and recorded pulmonary arterial pressure (ppa) and pulmonary venous pressure (ppv) continuously using calibrated pressure transducers (baxter, il, usa) and a multichannel recorder (rm 6000; nihon kohden, tokyo, japan), respectively. next, a flow probe (ff-030t; nihon kohden) connected to an electromagnetic flowmeter (mfv2100; nihon kohden) was placed in the perfusion circuit for continuous monitoring of blood flow (q). ppa was adjusted by maintaining the flow at 0.04 ml/g body weight/min through the adjustment of the arterial reservoir level at the beginning of the experiment. consequently, the driving pressure was kept constant throughout the experiment. the zero level for pressures was set at the position of the right atrium. total pulmonary vascular resistance (pvr) (rt) was calculated using the following formula: the change in lung weight induced by the elevation of venous pressure was used to determine the index of . the pump continuously circles the perfusate from the venous reservoir to the arterial reservoir to maintain a constant driving pressure. ad analog-to-digital, la left atrium, pa pulmonary artery, paw airway pressure, ppa pulmonary arterial pressure, ppv pulmonary venous pressure microvascular permeability to water (kfc). the measurement of kfc in isolated lungs has been described in detail [6, 7] . during ventilation and lung perfusion, the venous and arterial reservoirs were rapidly elevated by 6 cmh 2 o for 7 min, and the gain in lung weight, which consisted of two phases-rapid weight gain owing to changes in blood volume, and a slower, more prolonged phase of weight gain owing to transcapillary filtration-was recorded. next, the logarithm of the rate of lung weight gain (∆w/∆t) was plotted as a function of time. the ∆w/∆t was then analyzed during a 3-to 6-min interval of elevated reservoirs. the initial rate of fluid filtration [(∆w/∆t)t = 0] was calculated by extrapolating ∆w/∆t at time 0. finally, we calculated kfc by dividing ∆w/∆t at time 0 [(∆w/∆t) t = 0] by changes in pulmonary capillary pressure (ppc), and normalized using the baseline lung wet weight (ww) and expressed in ml min −1 mmhg −1 100 g −1 lung tissue. to determine ppc, the arterial and venous lines were simultaneously occluded for more than 5 s, and pulmonary arterial and venous pressures were converged to a specific level. change in ppc was calculated as the difference between that measured before versus at 7 min after elevation of the reservoir level. although ventilation was discontinued during ppc measurement, a constant flow of mixed gas (2 l/min) was administered at 2 cmh 2 o of airway pressure. at the end of the experiment, baseline lung ww was estimated by subtracting the weight of extrapulmonary tissues from the total weight of the lungs and extrapulmonary tissues, which was measured before perfusion. the lungs were used to estimate the tissue wet weight to dry weight (ww/dw) ratio. after recording the ww of the lung tissue, the lungs were placed in a drying oven at 60 ºc for 2 weeks and reweighed [6] . the lung ww/dw ratio was calculated using the following formula: (ww -dw)/dw [6] . after the start of perfusion, the isolated perfused rat lungs were observed for 30 min to establish an isogravimetric state (fig. 2) . the lung preparations were then classified into six experimental groups. in the sham group (n = 6), the lungs were continuously perfused and ventilated for 150 min without ir, whereas in the ir group (n = 6), ventilation and perfusion were interrupted (ischemia) after equilibration and the lungs were maintained in the humidified chamber for 60 min, while the airway was kept open. the lungs were reperfused and reventilated for 60 min after ischemia. meanwhile, in the nicorandil (n) + ir group (n = 6), nicorandil (6 mg) was added to the perfusate (nicorandil concentration in the perfusate was 75 µg/ml) followed by ischemia for 60 min; the preparation was then perfused for 60 min. in the glibenclamide (gli) + n + ir group (n = 6), glibenclamide (3 µm) was added to the perfusate 5 min before nicorandil administration, followed by ischemia for 60 min; the preparation was then perfused for 60 min. in the n ω -nitro-l-arginine methyl ester (l-name) + n + ir (n = 6) and 1h[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (odq) + n + ir (n = 6) groups, a similar protocol was used, except that glibenclamide was replaced with l-name (100 µm), an inhibitor of endogenous no synthesis, and odq (30 µm), a sgc inhibitor. values are expressed as means ± standard deviation. we did not calculate a priori statistical power. the sample size was chosen based on related studies with a similar design [6, 8] . within-group differences were analyzed using one-way repeated measures analysis of variance (anova) (statview 5.0; abacus concepts, berkeley, ca, usa). multiple samples of the same time interval were analyzed using one-way ischemia indicates no ventilation without perfusion, while reperfusion restoration of ventilation and perfusion anova, followed by a bonferroni-dunn test for post hoc comparisons. p values less than 0.05 were considered statistically significant. we used 11, 12, 11, 10, 10, and 12 rats in the sham, ir, n + ir, gli + n + ir, l-name + n + ir, and odq + ir groups to obtain 6 samples in each group resulting from the failure of surgical technique or pa/la tube positioning. no significant differences were observed in ph, pco 2 , po 2 or base excess of perfusate just before ischemia between any groups (table 1) . changes in pvr tended to increase 60 min after ir in all groups compared with baseline; however, none of these differences was significant (table 2 ). in addition, no significant differences were found between any of the groups at baseline or 60 min after ir. baseline kfc values were similar in all groups. in the ir group, the kfc after 60 min of reperfusion was significantly increased compared with baseline (0.81 ± 0.25 vs. 2.82 ± 0.68 ml min −1 mmhg −1 100 g −1 , p < 0.01), whereas no changes were seen in kfc in the sham group (0.87 ± 0.25 vs. 1.35 ± 0.37 ml min −1 mmhg −1 100 g −1 , no significant difference). compared with the ir group, administration of nicorandil significantly attenuated the increase of ir-induced kfc after 60 min of reperfusion (0.92 ± 0.28 vs. 2.82 ± 0.68 ml min −1 mmhg −1 100 g −1 , p < 0.01). nicorandil's inhibitory effect on the increase of ir-induced kfc was blocked by glibenclamide (0.85 ± 0.26 vs. 2.81 ± 0.75 ml min −1 mmhg −1 100 g −1 ; p < 0.01) and odq (0.76 ± 0.77 vs. 2.43 ± 0.89 ml min −1 mmhg −1 100 g −1 ; p < 0.01), but not by l-name (1.02 ± 0.28 vs. 1.07 ± 0.32 ml min −1 mmhg −1 100 g −1 ; no significant difference) (fig. 3) . in the ir group, the lung ww/dw ratio increased after ir. by contrast, the lung ww/dw ratio remained unchanged after ir in the n + ir and l-name + n + ir groups, similar to the sham group. however, compared with the n + ir group, significant increases in the lung ww/dw ratio were observed in the gli + n + ir and odq + n + ir groups (fig. 4 ). the major findings of the present study are as follows: (1) nicorandil administration before ischemia ameliorated increases in pulmonary microvascular permeability after ir in isolated rat lung preparations; (2) the protective effects of nicorandil against ir lung injury was blocked by glibenclamide as a k atp channel blocker and odq as a sgc inhibitor. these findings indicate that the salutary effects of nicorandil involve at least two mechanisms, the effect of the k atp channel opener and that of the activation of the sgc-cgmp pathway. no significant differences were found in pvr between groups at baseline or at 30 and 60 min after ir. according to the experimental findings by liu et al. [6] , isoflurane-sevoflurane administration before ischemia attenuated ir injury without a significant pvr change in isolated rat lungs which was similar to the results in the present study. meanwhile, functional vascular impairment from exposure to ir in the rat lung is likely to be restricted to the endothelial cell layer by the inhibition of the pulmonary vasodilator response to endothelium-dependent pulmonary vasodilators such as histamine and acetylcholine [9] . however, ir has been shown not to affect the response to sodium nitroprusside, an endothelium-independent pulmonary vasodilator [9] . these findings suggest that functional pulmonary vascular injury stemming from exposure to ir seems is restricted to the endothelial cell layer. in the present study, nicorandil attenuated the increase in microvascular permeability after ir in rat lungs, and that effect was blocked by glibenclamide. because glibenclamide, a k atp channel blocker, did not affect the increase in microvascular permeability produced by ir in rat lungs [8] when administered without nicorandil, the activation of k atp channels by nicorandil is likely to have attenuated the increase in pulmonary microvascular permeability after ir. the activation of k atp channels in the lung by administration of cromakalim could help protect against and even reverse the endothelial damage associated with reperfusion injury after ischemia [8] . the protective effect of a k atp channel opener appears to be due to blockade of superoxide anion production by leukocytes or endothelial cells because the opening of the k atp channel inhibited oxygen-derived free radical production by neutrophils [10] . in addition, our results showed that administration of odq as a sgc inhibitor before ischemia blocked the salutary effect of nicorandil. since odq has been demonstrated to exert no effect on kfc with and without ir in the rat lung [11] , nicorandil also seems to exert salutary effects on ir lung injury through increasing intracellular cgmp by activating sgc as another mechanism [3] . based on these considerations, the beneficial effects of nicorandil could be due the activation of the sgc-cgmp pathway. indeed, nicorandil, because of its chemical structure, is considered a nitrate generator and a no donor. since sgc is a no receptor, several reports have shown that no donors reduced ir injury through activating sgc [12, 13] . during ir injury, endogenous no and cgmp levels in tissues or organs were rapidly reduced [14] . in fact, the administration of no to non-heart beating rat lungs during warm ir was associated with reduced ir injury and increased cgmp levels [13] . irinduced pulmonary microvascular leak, which requires the activation of sgc, was also attenuated by inhaled no [11] . therefore, in the present study, the administration of nicorandil may have increased cgmp levels by the activation of sgc. another study reported that nicorandil elevated cgmp levels, but not no generation, which was confirmed by ozone chemiluminescence reactions using human or rat liver microsomes (p450-rich fractions) with the addition of nadph [15] . however, elevated cgmp levels in examined tissues were not inhibited by the no trapping agent, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide [16] . in the present study, it is possible that the administration of nicorandil increased cgmp due to the direct activation of sgc. the administration of l-name before ischemia did not attenuate the protective effect of nicorandil. this result may indicate that enhanced endothelial-derived no fig. 4 changes in lung wet-to-dry weight ratio. data are mean ± sd (n = 6 per group). *p < 0.01 versus sham group. # p < 0.01 versus ir group activity by nicorandil does not account for the increase in pulmonary permeability under ir lung injury. the higher antiplatelet aggregation activity of endothelial cells caused by nicorandil was reduced when endothelial cells were treated with l-name and exposed to a hypoxia-reoxygenation condition [17] , suggesting that the anti-aggregation activity of endothelial no was enhanced by nicorandil. considered together with the current results, the enhancing effect of nicorandil in endothelial-derived no activity on ir injury may be involved only when blood cell components exist. the findings of the present study may be transferrable not only to lung transplantation but also to thoracic surgery with long-term cardiopulmonary bypass or lung injury with ischemia-reperfusion. however, blood flow in the bronchial arteries is often maintained during cardiopulmonary bypass unlike lung transplantation. this study did have some limitations. first, we used bicarbonate-buffered physiologic salt solution instead of blood as a perfusate. blood components, especially neutrophils, play an important rule on ir injury. the interaction between neutrophils and endothelial cells releases various proteases and causes cell injury. to exclude these effects and examine nicorandil's effects on endothelial cells, we used bicarbonate-buffered physiologic salt solution. second, we did not measure no generation. therefore, whether no generation was involved in the mechanism of nicorandil's protective effect in our present study remains unclear. however, using ozone chemiluminescence detection methods, nicorandil has been shown not to generate no [15] . hence, the administration of nicorandil is likely to increase cgmp because of the direct activation of sgc. third, the dose of nicorandil used in the present study was higher than the clinical dosage. yamashita et al. used about tenfold of the clinical nicorandil dose and demonstrated that nicorandil ameliorated the ir injury of a lung allograft in a canine lung transplantation model [5] . in addition, furuya et al. [17] . demonstrated that 300 µm of nicorandil improved postischemic cardiac function in a rat heart-lung reperfusion model [16] . it is, therefore, possible that rats are less sensible than humans to k atp channels. in summary, nicorandil attenuated ir injury in isolated rat lungs. this protective effect was blocked by glibenclamide, a k atp channel blocker, and odq, a sgc inhibitor, but not by l-name. these results suggest that the protective effects of nicorandil after ir lung injury can be explained by the activation of a k atp channel opener as well as that of the sgc-cgmp pathway. impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome nicorandil as a hybrid between nitrates and potassium channel activators effect of nicorandil on coronary events in patients with stable angina: the impact of nicorandil in angina (iona) randomised trial nicorandil, a potent adenosine triphosphate-sensitive potassium-channel opener, ameliorates lung allograft reperfusion injury isoflurane-sevoflurane adminstration before ischemia attenuates ischemia-reperfusion-induced injury in isolated rat lungs estimation of the filtration coefficient of pulmonary exchange vessels atp-sensitive k + channels are not involved in ischemia-reperfusion lung endothelial injury preconditioning modulates pulmonary endothelial dysfunction following ischemia-reperfusion injury in the rat lung: role of potassium channels emd 52692 (bimakalim), a new potassium channel opener, attenuates luminol-enhanced chemiluminescence and superoxide anion radical formation by zymosanactivated polymorphonuclear leukocytes inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak nitroglycerin reperfusion reduces ischemia-reperfusion injury in non-heart-beating donor lungs inhaled nitric oxide reduces ischemia-reperfusion injury in rat lungs from non-heart-beating donors cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway nicorandil elevates tissue cgmp levels in a nitric-oxideindependent manner nicorandil enhances the effect of endothelial nitric oxide under hypoxia-reoxygenation: role of the katp channel effects of nicorandil on myocardial function and metabolism in the post-ischaemic reperfused heart with or without inhalation anaesthetics we are grateful to yoshitsugu tobe (laboratory technician, department of anesthesia and intensive care medicine, akita university graduate school of medicine, akita city, akita, japan) for their technical assistance in performing the animal experiments.authors' contributions ka: this author helped design and conduct the study, collect the data, perform the analysis, and write the manuscript. th: this author helped design the study and write the manuscript. ke: this author helped design the study and revise the manuscript. ym: this author helped conduct the study and revise the manuscript. tn, tk: this author helped review the original study data and revise the manuscript.funding this work was supported by grants in aid for scientific research from japan society for the promotion of science (no. 19591775). conflict of interest the authors declare that they have no conflict of interest. key: cord-011337-cyku17s8 authors: hsu, fushun; how, cheng-hung; huang, shang-ran; chen, yi-tsun; chen, jin-shing; hsin, ho-tsung title: locating stridor caused by tumor compression by using a multichannel electronic stethoscope: a case report date: 2020-05-09 journal: j clin monit comput doi: 10.1007/s10877-020-00517-8 sha: doc_id: 11337 cord_uid: cyku17s8 a 67-year-old male patient with chronic obstructive pulmonary disease was admitted to a hospital in northern taiwan for progressive dyspnea and productive cough with an enlarged left upper lobe tumor (5.3 × 6.8 × 3.9 cm(3)). previous chest auscultation on outpatient visits had yielded diffuse wheezes. a localized stridor (fundamental frequency of 125 hz) was captured using a multichannel electronic stethoscope comprising four microelectromechanical system microphones. an energy-based localization algorithm was used to successfully locate the sound source of the stridor caused by tumor compression. the results of the algorithm were compatible with the findings obtained from computed tomography and bronchoscopy (mean radius = 9.40 mm and radial standard deviation = 14.97 mm). we demonstrated a potential diagnostic aid for pulmonary diseases through sound-source localization technology based on respiratory monitoring. the proposed technique can facilitate detection when advanced imaging tools are not immediately available. continuing effort on the development of more precise estimation is warranted. chest auscultation has had long played a useful role in physical examination well before its widespread use after the invention of stethoscope 200 years ago [1] . respiratory sounds are auscultated from the trachea (tracheal sounds), larger bronchial airways (bronchial sounds), or lungs (vesicular sounds). tracheal and bronchial sounds are heard clearly during both inspiratory and expiratory phases, whereas vesicular sounds can be heard clearly only during the inspiratory and early expiratory phases [2, 3] . pulmonary pathologies are suspected when the frequency or intensity of the respiratory sounds change or when common adventitious (abnormal) sounds are identified [1, 3] . adventitious sounds can be classified into two groups according to their duration. the duration of continuous adventitious sounds (cass) is mostly larger than 250 ms, and the duration of discontinuous adventitious sounds (dass) is less than 25 ms [3] . cass can be further classified into subtypes such as wheeze, stridor, and rhonchus depending on their continuity, duration, phase (timing), pitch, quality, cause, and associated diseases [3] . immediate management of abnormalities detected by timely and adequate auscultation of cass related to airway narrowing or obstruction can prevent severe consequences or even death. moreover, the identification of cass and their source through auscultation enables clinicians to make clinical judgments. conventional auscultation has several drawbacks and limitations. first, conventional auscultation cannot be used fushun hsu and cheng-hung how have equally contributed to this work. continuously. [3] . furthermore, the interpretation of the auscultation results is dependent on the practitioner's hearing ability, training, and expertise. interpretations can vary considerably, and agreement rate among practitioners is usually fair [4] [5] [6] . recent innovation in computerized sound analysis has increased the clinical value of auscultation by the precision improvement in sound recording and acoustic indexes. [7] moreover, computerized analysis involving microphone arrays can be used for locating the source of adventitious sounds; however, this method has not been widely discussed. acoustic source localization technologies have been reported extensively in the literature over the past twenty years. these technologies are categorized into the following three types according to the type of physical measurements: (1) received signal strength or energy; (2) time of arrival (toa) or time difference of arrival (tdoa); and (3) direction of arrival (doa) [8, 9] . meng and xiao reviewed various energy-based formulations, including centralized, sequentially distributed, and fully distributed algorithms, and their corresponding problem-solving approaches to obtain single or multiple source locations [9] . cobos et al. provided problem formulations and solutions for the toa, tdoa and doa models [8] . since 2000, many studies have adopted acoustic source localization technologies to locate lung sounds. kompis et al. proposed a method to locate lung sound and derive an acoustic image [10] . salehin et al. used eigen-basis decomposition to locate lung sound with a circular array of sensors [11] . mckee and goubran compared an energy-based model (identifying the location of maximum power) with a toa model for sound localization in the human thorax [12] . ozer et al. [13] and henry et al. [14] have used the boundary element method to model the acoustic properties of the lung and locate the sound source in the lung. unlike the aforementioned acoustic models, sen, saraclar and kahya used an independent component analysis (ica) and a center of weights approach to determine the location of crackles (a type of das) [15] . charleston-villalobos used ica and a time variant autoregressive model to derive a heat map of crackle locations [16] . adventitious lung sound localization is potentially beneficial in diagnosing pulmonary diseases because the location of the sound source can indicate the anatomical region that is most likely affected. in the present study, an energybased localization method was used to locate a persistent stridor resulting from tumor compression. a multichannel electronic stethoscope was used in the method. a 67-year-old male chain smoker was admitted to a hospital in northern taiwan because of an enlarged left upper lobe (lul) lung mass and a 4-month history of shortness of breath and productive cough. a year prior to the current admission, the patient underwent thoracoscopic wedge resection and pleurodesis for left spontaneous pneumothorax. chest auscultation on outpatient visits yielded diffuse wheezes. treatment for the patient's chronic obstructive pulmonary disease (copd) did not alleviate his progressive symptoms. chest x-ray revealed no pneumothorax recurrence; however, an enlarged lul lung mass was observed. chest computed tomography (ct) performed 2 months prior to the current admission revealed an irregular left lingular lung mass measuring 5.3 × 6.8 × 3.9 cm 3 with a halo sign (fig. 1a) . the patient did not experience chest pain, hemoptysis, and marked weight loss since the symptoms began. lung malignancy was suspected. the patient was left hemiplegic due to a stroke 1 year prior to the current admission. his past medical history included hypertension, diabetes mellitus, copd, asthma, and copd overlap syndrome. at the time of admission, the patient was experiencing mild to moderate respiratory distress. stridor was heard at multiple sites with a conventional stethoscope but was more pronounced over the left middle lung field. no fever was observed but the complete blood count indicated leukocytosis. the patient's chest x-ray revealed a left upper lung mass with obstructive pneumonitis. appropriate medical treatment, including oxygen support and empirical antibiotics, was provided. the pathology report of the patient obtained through ct-guided biopsy 1 week after admission indicated extensive necrosis, which was compatible with the ulcerative and necrotic tissues observed during bronchoscopy performed 2 days later. tumor necrosis could not be excluded. the patient contracted right lower lobe pneumonia that soon progressed to acute respiratory failure. adult respiratory distress syndrome subsequently developed, and the patient was intubated 2 weeks after admission. five weeks after admission, the second bronchoscopy revealed dynamic collapse of the narrowed left inferior bronchial orifice during coughing and exertion with a widened left second carina (fig. 1c) . suspected tumor infiltration at the left upper division caused lingular orifice occlusion (fig. 1d) ; however, the cytology report indicated mainly reactive mesothelial cells and inflammatory cells. although the patient was weaned from mechanical ventilator support and extubated 6 weeks after admission, left upper lung atelectasis persisted. the patient refused further aggressive treatment and passed away a few days later. we recorded the chest sounds of the patient when the patient was under mechanical ventilation support. the recording device was an experimental multichannel stethoscope comprising four acoustic sensor patches, in each of which there was a microelectromechanical system (mems) microphone, spa1687lr5h-1 (knowles, illinois, usa). the four sensor patches were attached on the second and fifth intercostal spaces along the left and right midclavicular lines (fig. 1a) . the sensitivity of the microphone is -38 dbv/pa. the acoustic signal first passed through an amplifier, opa2376aidgkr (texas instruments, texas, usa), with a gain of 18 db, and then an analog to digital converter, ads8867idgs (texas instruments, texas, usa), before subsequent analysis. the sampling rate was 4,000 hz, and the bit depth was 16 bits. the received time signals of the four sensors were obtained as spectrograms by using the short-time fourier transform (ft) with a hamming window filter (window size = 512, overlapped ratio = 0.85) (fig. 2a-d) . an experienced physician first manually drew regions of interest (rois) on the spectrograms obtained from sensor 4, which contained the stridor (white rectangles in the fig. 2d ), because the most prominent sound was recorded by sensor 4. then, the rois with the same frequency boundaries and temporal boundaries as the previously outlined rois were automatically a tumor with dimensions of 5.3 × 6.8 × 3.9 cm 3 at the left upper lung (red arrow). chest sounds were recorded using four sensor patches on the second and fifth intercostal spaces along the left and right midclavicular lines (four small red circles). the source location was estimated according to 30 successive stridor signals and displayed as a visual cue (red shaded round area). the center and radius (29.94 mm) of the visual cue were the mean radius value and twice the radial standard deviation of the estimated location, respectively. b the left lung is not observed in the 3d illustration of the intra-thoracic air-way created using a region-growing-based method. orifices leading to the left lung (red arrow: left superior lobar bronchus; black arrow: lingular bronchus; cyan arrow: left inferior lobar bronchus) may be occluded or narrowed to ensure that the seeds are prevented from growing into the region. c bronchoscopy performed during intensive care indicated widening of the left second carina, which implied external compression in the vicinity. d bronchoscopy also revealed mucosa infiltration, with a cobble-stone appearance over the lul bronchus. the lingular orifice was completely occluded by mucus and blood clots defined on the other spectrograms (white dashed rectangles in the fig. 2a-c) . the locations of all the rois were confirmed through visual inspection. after determining the rois, we transformed the audio signals located between the temporal boundaries in each roi into a power spectrum by using the fast ft. figure 3 (a-d) displays the spectrum of the signals within the same time period bounded by one of the rois derived from sensor 1 to sensor 4. the energy peak of the stridor appeared between the frequency boundaries of the rois in the spectrum (red lines in fig. 3a-d) . the peak value of the stridor derived from the four sensors can be used to locate the source of the sound according to an energy-ratio least-squares method described in the following section. a simple energy-based localization model with a least-square optimization [17] was used to locate each stridor. assuming that n sensors are used to receive isotropic acoustic signals propagated from a single-point sound source, the signal energy measured on the ith sensor at time point t, which is denoted as y i (t), can be expressed as follows: in eq. (1), i(t) is the energy at 1 m from the source; η i is the propagation delay from the source to the ith sensor; θ(t) is the source location at time t, r i is the location of ith sensor; g i is the gain factor of the ith acoustic sensor; α is an energy decay factor; s i (t) is the energy intensity at the sensor location; and ε i (t) is a term that depends on the modeling errors of the parameters g i , r i , and α as well as the additive noise of y i (t). we calculated the average energy over the time window [t − t/2, t + t/2] by using the following expression: (2) where x i is the signal intensity measured by the ith sensor and l is the time interval. if t is comparatively large, η i can be neglected. in addition, we assumed that the source and the sensors were stationary. in this case, eq. (2) can be expressed as follows: where d i denotes the distance between the source and the ith sensor. empirically, the decay exponent α is close to 2 when the sound propagation medium is air without reverberation. however, determining the precise value of the decay exponent is difficult because of the material heterogeneity in the thoracic cage and reverberation. in this report, we simply assumed that α = 2. several approaches have been reported to solve eq. (3) and obtain the source location. in the present study, the computing ratios of the energy peaks of the stridor were obtained using a pair of sensors. the energy ratio k ij of a pair of sensors can be expressed as follows: when 0 < k ij < 1, the coordinates of the sound source θ that satisfy eq. (4) must reside on the circumference of a d-dimensional localizing hypersphere described by the following equation: the center c ij and radius ρ ij of the aforementioned hypersphere associated with sensors i and j are expressed as follows: the source of the stridor was estimated according to 30 successive stridor signals. the mean radius and radial standard deviation were 9.40 and 14.97 mm, respectively. a visual cue was displayed as a circular area (red shaded area in fig. 1a ) whose center and radius were the mean value and twice the radial standard deviation (29.94 mm) of the estimated location, respectively. a three-dimensional (3d) illustration of the airway (fig. 1b) was created using a region-growing-based method [18] , with starting seed points placed inside the trachea according to the volumetric ct images. the absence of the left lung in the images implied nonpatency or narrowing of the airway leading to the left lung, which was compatible with the findings obtained from bronchoscopy performed during admission (fig. 1c, d) . chest auscultation with a conventional stethoscope is subjective and depends mainly on the experience of the medical personnel performing the examination. in the current case, diffuse wheezes were heard on chest auscultation and was taken for granted because the patient had underlying copd. because of the limitation of human ears and ambient noise in the environment, identification of the focal stridor hidden underneath was difficult until image study indicated obstructive pneumonitis. adventitious sounds can be used to study cardiopulmonary conditions. the acoustic properties of sound can be used to investigate the pathological condition of patients. stridor is typically described as a musical sound caused by the turbulent airflow resulting from narrowing or obstruction in the upper respiratory tract, and it has a fundamental frequency of approximately 500 hz [1] . in adults, stridor can indicate various pathological conditions, such as airway occlusion, narrowing, or compression, which require immediate assessment and management. such sounds are recognizable because of the collapse of extrathoracic airways [14] . when stridor is detected in the upper respiratory tract, it can be easily distinguished from wheeze because it is more clearly heard during inhalation rather than during exhalation. however, the focal stridor derived from tumor compression of the branching bronchus in this case is fig. 4 illustration of six localizing hyperspheres (colored circles) derived from four acoustic sensors (blue stars) and the estimated center of the source location (red star) more likely to be incorrectly identified as wheeze because of its ambiguous presentation during the expiratory phase and past medical history. by using advanced computerized sound analysis and locating technology, we observed that the sound predominantly occurred in the inspiratory phase on the spectrogram. the sound source was closer to the central airway than to the peripheral region. therefore, a focal stridor with a relatively low frequency (fundamental frequency: 125 hz) cannot be easily missed in the proposed system. we successfully located the sound source to be a circular area (mean radius = 9.40 mm and radial standard deviation = 14.97 mm) by using a simple energy decay model (fig. 1a) . since the exact location of the sound source is not obtainable, the trueness [19] of our proposed method is not reported. however, the estimated sound source covers the area including the orifice of the inferior bronchus, which is the proposed origin of the stridor. this result is in agreement with the bronchoscopy results of total occlusion in the left lingular orifice and dynamic collapse of the left inferior bronchial orifice presumably because of tumor compression. the other in vivo studies [12, 15, 16] do not report the trueness because of the unobtainable location of sound source. on the other hand, the acoustic imaging system proposed by kompis et al. cannot resolve the differences below about 20 mm on the gelatin phantom [10] . according to this standard, our proposed method performs adequately. to create a model that can more accurately locate the origin of adventitious sounds, additional acoustic parameters, such as the sound transmission path, attenuation factors of different tissue types, heterogeneity of tissues, tension of the vibration membrane of the sensor, sound leakage from the space between the skin and sound collector, and noise, should be considered. henry and royston proposed such a boundary element approach that models the airway tree structure, lung parenchyma and surrounding chest wall, and its accuracy can reach 2.6 mm to 9.8 mm in different simulated scenarios [14] . however, to build a boundary element model requires a ct scan to obtain the geometry of the subject's thorax beforehand. the absence of the left lung in 3d illustration (fig. 1b ) indicated compromised airflow in the left lung. however, the actual patency of the airways leading into left lung parenchyma cannot be determined from the diagram. the partial volume effect [20] occurred because of the poor longitudinal resolution (0.58 × 0.58 × 5 mm) of the ct images. the voxel value of the narrowed bronchial lumen was contaminated by the hounsfield unit of the surrounding tissues; therefore, the seeds could not grow into distal segments. conclusively, we demonstrated the importance and feasibility of the analysis and source localization of adventitious sounds by using a multichannel electronic stethoscope in clinic. the presented method is useful in locating an adventitious sound source at the lobular level through chest auscultation. the information regarding the sound location facilitates clinical diagnosis and treatment strategies in a noninvasive and radiation-free manner. if limited imaging modalities are available, a portable device equipped with sound-source localization software can be potentially used for diagnosis, especially when the clinical condition is progressing rapidly and critically. more effort is warranted in developing refined models in order to accommodate use in clinical practice. fundamentals of lung auscultation auscultation of the respiratory system automatic adventitious respiratory sound analysis: a systematic review real-world evaluation of the eko electronic teleauscultation system inter-rater agreement of auscultation, palpable fremitus, and ventilator waveform sawtooth patterns between clinicians wheeze detection in the pediatric intensive care unit: comparison among physician, nurses, respiratory therapists, and a computerized respiratory sound monitor computerized lung sound analysis as diagnostic aid for the detection of abnormal lung sounds: a systematic review and meta-analysis a survey of sound source localization methods in wireless acoustic sensor networks energy-based acoustic source localization methods: a survey acoustic imaging of the human chest localizing lung sounds: eigen basis decomposition for localizing sources within a circular array of sensors sound localization in the human thorax boundary element model for simulating sound propagation and source localization within the lungs localization of adventitious respiratory sounds acoustic mapping of the lung based on source localization of adventitious respiratory sound components adventitious lung sounds imaging by ica-tvar scheme energy-based collaborative source localization using acoustic microsensor array medical image segmentation using 3d seeded region growing understanding the meaning of accuracy, trueness and precision computed tomography: principles, design, artifacts, and recent advances acknowledgements we thank heroic faith medical science corporation ltd. for providing a multichannel stethoscope. this manuscript was edited by wallace academic editing.funding this was a self-funded research. ethical approval this study was reviewed and approved by the research ethics review committee of far eastern memorial hospital (case number: 107052-f). the research was conducted in accordance with the 1964 helsinki declaration and its later amendments or comparable ethical standards.informed consent informed consent was obtained from the patient's daughter. figure 4 illustrates the six localizing hyperspheres related to four sensors and one sound source. when k ij →1, the solution of eq. (4) forms a hyperplane between d i and d j , which can be expressed as follows:according to a study [17] , when . the gradient of j(r) can be expressed as follows:a gradient descent method can then be used to obtain the solution of θ by minimizing the objective function in eq. (8).signal processing, source localization, and visual cue display were achieved on an asus f302u laptop (cpu: i5-6200 u, 2.4 ghz; os: win10 64-bit; ram: 8 gb; graphics card: nvidia geforce 920mx) by using a platform developed through matlab 2019a (the mathworks inc., massachusetts, usa). key: cord-283078-vz98pp4h authors: zakaria, dina mohamed; zahran, noha mahmoud; arafa, samia abdel aziz; mehanna, radwa ali; abdel-moneim, rehab ahmed title: histological and physiological studies of the effect of bone marrow-derived mesenchymal stem cells on bleomycin induced lung fibrosis in adult albino rats date: 2020-10-22 journal: tissue eng regen med doi: 10.1007/s13770-020-00294-0 sha: doc_id: 283078 cord_uid: vz98pp4h background: lung fibrosis is considered as an end stage for many lung diseases including lung inflammatory disease, autoimmune diseases and malignancy. there are limited therapeutic options with bad prognostic outcome. the aim of this study was to explore the effect of mesenchymal stem cells (mscs) derived from bone marrow on bleomycin (blm) induced lung fibrosis in albino rats. methods: 30 adult female albino rats were distributed randomly into 4 groups; negative control group, bleomycin induced lung fibrosis group, lung fibrosis treated with bone marrow-mscs (bm-mscs) and lung fibrosis treated with cell free media. lung fibrosis was induced with a single dose of intratracheal instillation of blm. bm-mscs or cell free media were injected intravenously 28 days after induction and rats were sacrificed after another 28 days for assessment. minute respiratory volume (mrv), forced vital capacity (fvc) and forced expiratory volume 1 (fev1) were recorded using spirometer (power lab data acquisition system). histological assessment was performed by light microscopic examination of h&e, and masson’s trichrome stained sections and was further supported by morphometric studies. in addition, electron microscopic examination to assess ultra-structural changes was done. confocal laser microscopy and pcr were used as tools to ensure mscs homing in the lung. results: induction of lung fibrosis was confirmed by histological examination, which revealed disorganized lung architecture, thickened inter-alveolar septa due excessive collagen deposition together with inflammatory cellular infiltration. moreover, pneumocytes depicted variable degenerative changes. reduction in mrv, fvc and fev1 were recorded. bm-mscs treatment showed marked structural improvement with minimal cellular infiltration and collagen deposition and hence restored lung architecture, together with lung functions. conclusion: mscs are promising potential therapy for lung fibrosis that could restore the normal structure and function of blm induced lung fibrosis. electronic supplementary material: the online version of this article (10.1007/s13770-020-00294-0) contains supplementary material, which is available to authorized users. lung fibrosis is a progressive fatal disease that affects many patients worldwide. it is the end-stage of many lung diseases. the main histopathological characteristic features of lung fibrosis are excessive deposition of extracellular matrix, accretion of fibroblasts, collapsing of alveoli and loss of normal lung architecture [1] [2] [3] . causes of lung fibrosis include lung inflammatory disease, autoimmune diseases, hypersensitivity pneumonitis, drug-induced interstitial lung disease, infection, and malignancy. however, idiopathic lung fibrosis (ipf) is considered to be one of the most common and aggressive forms of lung fibrosis with high mortality and morbidity. it develops without an identified underlying cause and terminates with severe affection of lung functions. there are many risk factors for the development of ipf, the most important of which is cigarette smoking, where the history of cigarette smoking has been associated with sporadic and familial cases. moreover, ipf was suggested to be linked to chronic exposure to air pollutants and recurrent pulmonary infection, whether bacterial or viral [4, 5] . adding to the burden is the global spread of the covid-19 pandemic caused by sars-cov2 virus and the shown evidence of scarring on some patients' lungs during or after recovering from the illness. yet still, there is no evidence whether this scarring is progressive or not [6] . unfortunately, once lung scarring occurs, it cannot be reversed, so there is no cure for lung fibrosis, whatever the cause. only limited therapeutic options are available, making lung fibrosis a life-threatening disease. surgical therapy is the last option where lung transplantation has been the only effective treatment proved [7, 8] . recently, regenerative medicine has become a remarkably valuable therapeutic strategy in different diseases, where it presents a promising modality that can replace organ transplantation with all its hazards. owing to their unique properties, stem cells turned out to be the main pillar in regenerative medicine due to its controlled yet unlimited self-renewal capacity, and its differentiation ability into different specific cell lines. adult mesenchymal stem cells (mscs) isolated from the bone marrow, umbilical cord, and adipose tissue are the most commonly used [9] . mscs have potent regenerative, angiogenic, anti-apoptotic, immune-modulatory, and anti-inflammatory properties, besides their multilineage capacity, which make them able to differentiate into a wide range of cell lines and thus have a great therapeutic potential for different diseases. this is besides their accessibility, homing, and engraftment at the site of injury and their ability to modify the microenvironmental factors at the engraftment site by the molecules and vesicles they secret known as extracellular vesicles [10, 11] . bleomycin (blm) is a chemotherapeutic agent that is used widely in the treatment of many malignancies as classical hodgkin lymphoma, melanoma, ovarian carcinoma, and testicular neoplasms [12] [13] [14] . however, it has serious side effects on the lung. the use of blm is limited by the development of pulmonary fibrosis in 3-5% of patients receiving this chemotherapeutic agent [15] . it is distributed mainly in the liver and spleen but also lungs. the kidney and heart receive a considerable amount of blm as well. however, blm selectively affects the lung because it lacks the enzyme bleomycin hydrolase that hydrolyzes the l-aminoalanine moiety of blm. this step prevents the metabolite of blm from binding metals such as iron, thus triggering an oxidative stress reaction [16] . as such, it has been the agent of choice for the induction of lung fibrosis model. in such context, the current study, assessed the effect of stem cell therapy using mscs for treatment of blm induced lung fibrosis model. the study was conducted on 35 sprague-dawley albino rats. five male rats aged 3 weeks (27-32 g) were used for bone marrow isolation and thirty female rats weighing 150-200 g 6-8 weeks of age were used in the experimental groups. rats were allowed to acclimate for 2 weeks before the experiment and were housed under a 14-10 h lightdark cycle with food and water provided ad libitum. experiments were conducted in accordance with the approved guidelines set by the research ethics committee of alexandria faculty of medicine, egypt. stem cell processing was carried out in the center of excellence for research in regenerative medicine and its application (cerrma), alexandria faculty of medicine. ethical approval was attained from the medical ethics committee of alexandria faculty of medicine (irb no: 00012098-fwa no: 00018699). the 30 female rats were divided randomly into 2 groups; control group, n = 10, which was further subdivided into a negative control group (cg) and a positive control group, in the latter lung fibrosis was induced for optimization of model and named fibrotic group (fg). these groups were given an intratracheal injection of saline or a single dose of bleomycin (5 mg/kg body weight, in 0.4 ml of saline) respectively. vial of bleomycin powder was dissolved in 5 ml saline. animals were held in 'upright position by an assistant and the neck was pulled backward. a syringe filled with blm was fitted to an orogastric tube, and then was pushed gently against the soft palate to reach the trachea. induction of lung fibrosis was assessed 28 days after bleomycin injection [17] . experimental group (where lung fibrosis was induced) eg n = 20, then it was subdivided into; bone marrow-derived mesenchymal stem cells treated group (bm-mscsg) where rats were injected with a single dose of passage 3 (p3) bm-mscs in complete media, and cell free media treated group (cfg) where rats were injected once with cell free complete media, intravenously 28 days after induction of fibrosis. histological assessment was done after another 28 days from treatment and was further supported by physiological assessment of lung functions. the bleomycin used in the current study was commercially purchased as bleocel 15 iu injection: bleomycin sulfate powder for solution for injection/infusion manufactured by celon labs. five male rats were sacrificed by overdose anesthesia (100 mg\kg phenobarbital) under a class ii biosafety cabinet, for the collection of bone marrow. the femur and tibia were dissected bilaterally and all excess tissue was removed. dissected intact bones were soaked in 70% ethanol in a sterile petri dish for 2 min for disinfection, then washed with phosphate buffer saline pbs (pbs, lonza, bornem, belgium) and finally placed in culture medium in a sterile petri dish for flushing the bone marrow. the proximal and the distal ends of both tibiae and femurs were trimmed and the bone marrow was flushed by inserting a 23-gauge needle attached to a 5 ml syringe with complete culture medium (ccm) [18] [19] [20] . bone marrow cells were cultured in complete media (low glucose dulbecco's modified eagle medium (lg-dmem) (1.0 g/l glucose, sigma-aldirch, st. louis, mo, usa) supplemented with 10% fetal bovine serum (fbs, sigma-aldirch), 1% l-glutamine (lonza) and 1%penicillin/streptomycin (pen/strept, lonza) and incubated in a co2 incubator at 37°c in a humidified atmosphere with 5% co2. after 48 h the medium was discarded, and the cells were washed with pbs and fed with a fresh complete medium. during the proliferation period, the medium was replaced twice weekly. cells were split after reaching 70-80% confluence using 0.025% (w/v) trypsin/edta (thermo fisher scientific, waltham, ma, usa) in a ratio 1:3 [21] [22] [23] . mscs at passage three p3 were used in the current experiment. follow-up of cultured cells was done using inverted phase contrast microscope equipped with the digital camera. (olympus ckx41sf, tokyo, japan) [22] . the colony-forming potential of the cultured cells at passage 3 was tested. in this assay, 100 cells were plated on a six-well plate in complete media and incubated for 14 days. the cells were fixed and stained for 30 min at room temperature in 3.0% crystal violet solution (sigma aldrich) in 100% methanol. the number of colonies for each well was counted and the plating efficiency or-cfu potential was calculated as the number of colonies formed/ number of cells plated 9 100. all visible colonies were counted; the number of colonies displaying five or more cells was scored under the phase-contrast inverted microscope. a cfu potential of over 40% was considered to be optimal for mscs' culture [24] . cells were characterized using fluorescent-labeled monoclonal antibodies (mab) for cd90 and cd 45 markers. trypsinized cells at passages 3 were incubated at room temperature for 30 min in the dark, with monoclonal phycoerythrin (pe)-conjugated antibody for cd45 (abcam, ab23396, cambridge, uk) and monoclonal allophycocyanin-conjugated antibody for cd90 (anti-thy1.1) (abcam, ab225). immunofluorescence on cells was analyzed using becton-dickinson, facs caliber flow cytometer equipped with cell quest software [25] . a count of 2 9 10 6 bm-mscs in 1 ml complete media or 1 ml of complete media without cells were injected intravenously in the tail vein of bm-mscs treated and cell free media treated groups respectively [26] [27] [28] . before injection of bm-mscs, the cells' cytoplasmic membranes were labeled with fluorescent probe (chloromethyl -benzamide octadecyl indocarbocyanines (cm-dii)) (molecular probes, thermo fisher scientific). labeled cells were viewed under confocal laser microscopy (leica microsystems, dmi8, wetzlar, germany) 72 h after injection in the lung tissue of 2 rats [29] . the lung tissues were processed for identification of male bm-mscs which were injected into female rats through identification of y chromosome; using real-time quantitative polymerase chain reaction [30, 31] . detection of sry dna was performed using the following primers; forward (5-catcgaagggttaaagtgcca-3) and reverse (5-atagtgtgtag-gttgttgtcc-3) [32, 33] . real-time pcr amplification, data acquisition, and analysis were carried out using the real-time detection system software (applied biosystems 7500, foster city, ca, usa). at the end of the study, 28 days after treatment, all rats were sacrificed and both lungs were dissected, then each lung was divided into two pieces. one piece was fixed in 10% neutral-buffered formalin, then processed to obtain (6 um) thin sections. some sections were routinely stained with h&e and others with masson's trichrome for light microscopic examination using, (olympus bx41) equipped with spot digital camera (olympus dp20). histomorphometric study was done, using nih fijió program (nih, bethesda, md, usa), where the area percentage of collagen fibers in masson' trichrome stained sections inter-alveolar septal thickness and alveolar surface area in h & e stained sections, were measured in five randomly selected sections for each item. data was presented as mean ± standard deviation (sd) of randomly selected ten fields/section (n = 5/group) [34] . the second piece was cut into small pieces (1/2-1 mm3) and immediately fixed in 3% phosphate buffered glutaraldehyde ph 7.4, then processed to obtain ultra-thin sections for transmission electron microscope examination, tem (jem-100 cx electron microscope, jeol, tokyo, japan) [35] [36] [37] . data were analyzed using ibm spss software package version 20.0. (armonk, ny, usa: ibm corp). qualitative data were described as number and percent. quantitative data were described as the mean ± standard deviation. the studied groups were compared using a 2-sided -test and one way anova with post hoc test (tukey's) for pairwise comparison. the p value was judged at the 5% level [38] . the cell cultures were monitored daily using contrast phase inverted light microscope. in primary culture p0, cells were small and rounded, they started to be spindle in shape after 48 h of culture, displaying a heterogeneous population and reached 70-80% confluency in approximately 7-9 day. with passaging, cell growth tended to be accelerated and morphology changed gradually exhibiting large, flattened and spindle shaped cells. mitotic rounded cells appeared demonstrating proliferation. at p3 the culture represented a homogenously fibroblastic like cell monolayer ( fig. 1a -c). after 5-7 days of incubation, cells gradually proliferated into small colonies, which increased after 2 weeks of culture to form larger colonies. two weeks post seeding, colonies were made obvious with crystal violet staining (fig. 1d ). the colony-forming assay showed that each well with 100 cells gave 90% ± 1.06 of colonies after 14 days. facs analysis for bm-mscs passage 3 showed that 98.04% of the cultured cells expressed the mesenchymal multipotent cd90 surface marker, while they were negative for the cd45 hematopoietic marker (fig. 1e ). labeled bm-mscs were tracked in lung tissue 72 h after injection. bm-mscs were seen in the alveolar pneumocytes type 1 under the laser scanning confocal microscope tissue eng regen med (fig. 1f ). rq-pcr results showed that sry gene was expressed in female rats injected by male bm-mscs. the expression of sry gene in bm-mscsg was 3.10 folds in comparison to cg (supplementary table s1 ). fg had shown a significant decrease in mrv, fvc and fev1 by 72%, 26% and 25% respectively as compared to control group p b 0.05. also, a significant decrease was seen in cfg 71%, 25% and 24% regarding mrv, fvc and fev1 respectively in comparison to fg. treatment with bm-mscs caused a significant increase in measured parameters compared to cg and cfg reaching control values, where mrv increased by 75%, fvc and fev1 increased by 25% compared to fg and cfg p b 0.05. fev1-fvc (%) showed normal ratio with no significant difference between all groups (supplementary table s2 , fig. 2a,b ). normal architecture of lung tissue was observed by using light microscope in lung sections of cg, where thin interalveolar septa and patent alveoli were noticed. pneumocytes type i with typical flattened nuclei and type ii with rounded nuclei bulging into alveolar lumen, were observed lining the alveoli. bronchioles appeared patent and lined with simple columnar epithelium (fig. 3a, b) . in fg, extensive degenerative changes and distortion of lung architecture were observed in a patchy pattern, where most of alveoli appeared collapsed. in addition, markedly thickened inter-alveolar septa with extensive cellular infiltration were observed in most areas of the lung tissue. blood vessels revealed evident congestion. as regards the bronchioles, massive lymphocytic infiltration around bronchiolar wall was noticed, together with evident tissue eng regen med bronchiolar epithelial desquamation, which was seen in lumen (fig. 3c, d) . marked amelioration of degenerative signs with evident restoration of lung architecture was seen in bm-mscs treated group when compared to fg, where most of the alveoli appeared patent with thin inter-alveolar septa. focal areas of limited thickening of inter-alveolar septa were still noticed. almost normal appearance of bronchioles was also observed. however, limited remnant blood vessel congestion was still seen (fig. 4a, b) . examination of lung sections in rats that received cell free media cfg, revealed excessive thickening of the inter-alveolar septa associated with evident cellular infiltration. most of the alveoli appeared with collapsed lumina, while some other alveoli appeared with narrowing of their lumina. diffuse extravasation of rbcs was also noticed (fig. 4c, d) . marked increase in trichrome green stained areas, indicating massive collagen deposition was noticed, within inter-alveolar septa, around blood vessels and bronchioles in fg in comparison to cg. in cg normal pattern of trichrome stained areas, thus reflecting normal collagen distribution within the inter-alveolar septa was demonstrated (fig. 5a, b) . examination of the lung sections of bm-mscssg revealed limited and focal distribution of green colored trichrome stained areas within the inter-alveolar septa as compared to cfg, which appeared to be more diffuse and extensive within the inter-alveolar septa, hence reflecting remarkable decrease in collagen deposition in bm-mscssg (fig. 5c, d) . the morphometric analysis of table s3 , fig. 2c ). the alveolar surface area measurement, to assess the degree of alveolar collapse, showed that there was a significant decrease in surface area of alveoli in fc versus the cg with mean of 236.8 ± 35.12 vs. 3659.1 ± 832.5 respectively (p \ 0.001). on the other hand, bm-mscsg showed a significant increase in the surface area of alveoli 4095.2 ± 454.8 in comparison to fg and cfg. the alveolar surface area of bm-mscsg reached that of the cg with no significant difference between them (p \ 0.001). (supplementary table s3, fig. 2d ). the inter-alveolar septal thickness was also measured to evaluate the degree of interruption of air-blood barrier that occurred as a consequence of collagen deposition. bm-mscsg showed significant decrease of inter-alveolar septal thickness in comparison to group fg and cfg showing thickness of 0.88 ± 0.31, 108.5 ± 23.3 and 97.31 ± 6.80 respectively. yet, there was not a significant difference between bm-mscsg and cg 4.60 ± 0.37. (supplementary table s3 , fig. 2e ). cg depicted patent alveoli, where pneumocyte type ii was observed with its characteristic, apical microvillus boarder and abundant lamellar bodies within the cytoplasm. pneumocyte type i was observed with its basal lamina fused with those endothelial cells of the neighboring blood capillary. endothelial lining of blood capillaries showed normal regular outline with normal euchromatic nucleus (fig. 6a, b) . on the other hand, variable degenerative changes were encountered ultra-structurally in fg, where, some type ii pneumocytes exhibited vacuolated lamellar bodies with loss of lamellar arrangement. luminar surface of some other type ii cells depicted decreased and irregular microvilli. in addition, nuclear changes were noticed in some pneumocytes that showed irregular nuclear membrane together with increased condensed chromatin (fig. 6c, d) . apoptotic bodies from type ii cells were also observed in some areas (fig. 6d) . inter-alveolar septa revealed prominent fibroblasts together with evident collagen deposition. the alveolar lumen showed marked narrowing and even obliteration in some areas. as regards type i pneumocytes, they were less frequently encountered than type ii pneumocyte. alveolar macrophages and extravasated rbcs were also seen in the inter-alveolar septum. examination of endothelial lining of inter-alveolar capillaries revealed irregular outline of the nucleus with excessive marginal clumping of chromatin (fig. 6e, f) . examination of lung sections of rats in bm-mscg, revealed marked improvement in lung ultra-structure. type i & ii pneumocytes showed apparently normal ultrastructure with euchromatic nuclei. type ii pneumocytes revealed most of lamellar bodies with their typical lamellar pattern, together with enhanced exocytosis into alveolar lumen (fig. 7a) . prominent microvilli were apparent on their apical border. endothelium of blood capillaries depicted normal appearance with regular nuclear outline and intact junctional folds. inter-alveolar septum showed minimal deposition of collagen fibers (fig. 7c, d) . whereas, in cfg extensive degenerative changes were marked inter-alveolar septa thickening with cellular infiltration is seen (i). diffuse extravasation of rbcs is also noticed (asterisks). (h&e stain, a 9 100, b 9 400, c 9 100, d 9 400) tissue eng regen med encountered in type i & type ii pneumocytes, with disorganization of lamellar bodies. detachment and sloughing of degenerated type i pneumocytes into alveolar lumen was noticed in this group, together with alveolar macrophages. apoptotic bodies and rarified cells were also seen. moreover, excessive collagen deposition was observed within inter-alveolar septum (fig. 7e, f ). most etiologies that affect the lung and lead to lung fibrosis, show a direct effect on the capacity of lung renewal leading to eventual permanent lung fibrosis [39] . the use of stem cells could thus be an important potential promising option in lung tissue regeneration to trigger a paradigm shift from traditional supportive therapy to effective therapy [40] [41] [42] [43] . in such a context, it was the scope of the current study to assess the ameliorative effect of mscs on blm induced pulmonary fibrosis in rat models. moreover, combining histological assessment with physiological tests for lung functions, presents a novel approach for a more accurate evaluation of lung fibrosis and assessment of the efficacy of the therapeutic strategy. in this study, pulmonary fibrosis was induced by blm referring to established data from previous research work [44] [45] [46] [47] . there is a range of potential routes of blm administration in rodents, including intravenous (iv), subcutaneous (sc), intranasal, and intratracheal (it), the latter has been by far the most preferred mode of administration due to its direct toxic and sustained effect on the lung tissue. futhermore, it induces a classical picture of lung fibrosis, thus providing a valid model for experimental therapeutic studies [48] . safety of the used vehicle with blm (saline) was confirmed histologically, where the examination of lung tissue of cg revealed normal histological features. in consequence, the different histological changes that were encountered in blm treated group fg could be referred to the impact of blm toxicity. though histological alteration resulting from blm administration is the gold standard confirmatory method tissue eng regen med for developing lung fibrosis yet, lung function tests were further assessed and results showed a significant reduction in mrv, fvc, and fev1 in fg and cfg compared to normal control rats denoting the presence of restrictive lung disease. blm cytotoxicity encloses oxidative stress as one of the mechanisms of the induced lung tissue injury [49, 50] , where blm can bind fe(ii) forming a complex, which is subsequently oxidized to fe(iii) in presence of o2, resulting in the reduction of oxygen to free radicals and production of reactive oxygen species (ros), such as, o2 , hydroxyl radicals, and fe(iii). then, this bleomycin complex binds to the dna helix through a nucleophilic bond, resulting in dna strand breaks. in addition, membrane lipid peroxidation and subsequent membrane damage occur. histological examination of h & e stained sections from the blm induced fibrosis group fg, revealed evident distortion of lung architecture, where marked thickening of the inter-alveolar septa and collapsed alveoli were noticed in a patchy distribution over much of the lung tissue. induced lung fibrosis was further confirmed by histomorphometric analysis of trichrome stained sections that showed a significant increase in the percentage area of collagen deposition in the induced fibrosis group in comparison to the control group. this can be attributed to the effect of blm, where it stimulates the alveolar macrophages for the production of inflammatory and profibrotic notice massive collagen (cl) deposition together with marked thickening of inter-alveolar septum. an inter-alveolar macrophage (m) is also seen. (a 9 1500, b 9 5000, c 9 1500, d 9 5000, e and f 9 3000) tissue eng regen med cytokines such as interleukin-1(il-1), macrophage inflammatory protein-1, and others. nevertheless, blm induces type ii pneumocyte hyperplasia that unlike normal type ii cells, contributes to the secretion of some of these cytokines. such cytokines lead to further proliferation and activation of fibroblasts with subsequently increased collagen deposition and eventually alveolar collapse [51, 52] . moreover, some of these cytokines act as chemoattractants thus leading to inflammatory cell recruitment and cellular infiltration [53, 54] . this came in agreement with the present study, where massive cellular infiltration was also noticed within the inter-alveolar septa and peribronchiolar. moreover, the electron microscopic results came in accordance with the light microscopic results, where marked collagen fibers deposition was observed within the inter-alveolar septa, together with evident fibroblasts, electron microscopic results of the blm induced fibrosis group, revealed variable degenerative changes, where type i pneumocytes were infrequently encountered, unlike type ii pneumocytes that were more frequently seen. this can be attributed to the direct toxic effect of blm on type i pneumocyte [55, 56] . in addition, variable degenerative changes depicted by type ii pneumocytes can be referred to membrane lipid peroxidation effect induced by the generated free oxygen radicals. furthermore, endothelial cells of inter-alveolar blood capillaries revealed an irregular outline of the nucleus with excessive marginal clumping of chromatin. in accordance, allawzi a et stated that blm increases the vascular another type ii cell (a2) shows excessive cytoplasmic vacuolation (v). several apoptotic bodies are observed (arrow). notice marked collagen (cl) deposition within inter-alveolar septum. (al); alveolar lumen. f a type ii pneumocyte (a1) shows excessive cytoplasmic vacuolation. a type i pneumocyte (arrow) appears rarified and detached from its basement membrane. apparent narrowing of alveolar lumen is noticed (asterisk). (a 9 4000, b 9 5000, c 9 3000 d 9 2500, e 9 2000, f 9 1200) tissue eng regen med permeability of pulmonary capillaries with subsequent initial congestion of the lung. this can explain vascular congestion that was observed histologically in lung sections of fibrosis group. then, this increased permeability contributes to the inflammatory state that eventually ends with fibrosis [50, 57] . there is certainly loads of good evidence to suggest that lung fibrosis is a vicious circle where the initial injury promotes fibrosis through impaired signal regulation. this can occur from alveolar epithelial cell dysfunction with enhanced integrin production and activation, intensified mesenchymal-epithelial interactions by disrupted cell membrane activity or epithelial mesenchymal transition (emt). another hypothesis is that fibrogenesis is driven by dysregulated fibroblast/myo-fibroblast responses to epithelial injury [58, 59] . the origin of myofibroblasts in lung fibrosis has been questioned for many years and epithelial mesenchymal transition (emt) as a source of these cells has been suggested. nevertheless, lineage tracing in transgenic mice indicates that the contribution of epithelial cells that have undergone emt could be insignificant for the fibroblast population. rather, recent findings indicate that emt facilitates a pro-fibrotic microenvironment by inducing local fibroblasts to be triggered by the paracrine effect of some factors produced by alveolar epithelial cells [60, 61] . nevertheless, increased collagen production seems to be more likely to be related to blm reaction involving increased permeability of protein influx and inflammatory cells. as such, fibroblasts may be exposed to quantities of mediators found in the bloodstream, produced by resident cells of lung or those that enter lung tissues [62] . after binding to of blm complex to dna and fe(ii) oxidation into fe(iii), a nucleophilic bond occurs at the dna desoxyribose c4' position. this will lead eventually to a break in the dna strand. furthermore, the free radicals produced by this process, leading to enhanced dna breaks that ultimately lead to cell death. this can explain the nuclear changes encountered ultra-structurally, such as shrunken irregular nuclei with increased clumped chromatin [15, 63] . moreover, fe(ii) regeneration provides the ternary complex blm-fe(ii)-o2 with catalytic activity, where it is assumed that every blm molecule can produce from 8 to 10 dna breaks [15, 46, 63, 64] . thus, enhancing more the cellular degenerative changes. apoptotic bodies that were encountered ultra-structurally, can be correlated to blm direct toxic effect on the dna, where it may cause single-(ssb) or double-stranded (dsb) dna breaks and can induce cell death by pseudo-apoptosis-similar to apoptosis but characterized by rapid dna fragmentation-or mitotic cell death [65, 66] . these degenerative changes had an impact on lung functions as was confirmed by the results of lung function tests where minute respiratory volume (mrv), forced expiratory volume (fev1), and forced vital capacity (fvc) showed marked reduction in fg and cfg. restrictive lung diseases including fibrosis reduce the fvc and fev1 proportionally thus the fev1/fvc ratio is either maintained normal or may be increased if the fvc is decreased more as compared to fev1. fev1/fvc ratio in this study was indifferent between groups, though fev1 and fvc were significantly decreased in fg and cfg in comparison to cg and bm-mscsg denoting the existence of a restrictive lung abnormality in the two former groups. in the current study, the homing of the male donor cells was confirmed in the injured lung of female recipients by pcr for the sry gene which is found on the y chromosome, and by the presence of labeled mscs in the lung tissue visualized by confocal microscope shortly after mscs injection. homed bm-mscs attenuated lung fibrosis significantly, as was evident on histological examination of lung sections from the msc's treated group, where marked decreased deposition of collagen within the inter-alveolar septa was observed. most of the alveoli appeared patent with thin inter-alveolar septa. histomorphometric results of trichrome stained sections further confirmed such an ameliorative effect of stem cell therapy, where there was a significant decrease in the percentage area of collagen deposition and an increase in the alveolar surface area as compared to the blm induced fibrosis group and the group that received cell free media. it is worth mentioning that collagen deposition was significantly decreased in the msc's treated group in comparison to the control group. such a finding can be referred to the significant therapeutic potential of bm-msc's thus overcoming even mild injurious effects of pollutants in the air, which might affect lung tissue of control group rats. this came in accordance with the ultra-structural results that showed evident restoration of ultra-structural features of pneumocytes types i and & ii. endothelium of blood capillaries had a normal appearance with a regular nuclear outline and intact junctional folds. the inter-alveolar septum showed minimal deposition of collagen fibers. together with previous studies, it can be concluded based on the findings of the present study that the administration of and mscs can mitigate lung fibrosis. those results agreed with lung function results that showed great improvement in mrv, fev1, and fvc where they all increased reaching normal values with no significant difference with cg, denoting the restoration of the lung ventilatory functions [67] . the present evidence relies on soluble mediators released by mscs such as il-1 receptor antagonist, il-10, keratinocyte growth factor, hepatocyte growth factor, angiopoietin 1, and transforming growth factor-b appear to play a significant role in the repair of acute and fibrotic injuries [68, 69] . these cytokines are characterized by being anti-inflammatory and anti-fibrotic. it has been established that il-10 suppresses the functions of macrophages and neutrophils [70] . among key functions of (tgf)-b1 are regulation of inflammatory and proliferation processes in addition to controlling cell growth, cell proliferation, cell differentiation, and apoptosis [71] . furthermore, mscs can promote the polarization of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype through the production of immunosuppressive molecules and metabolites, also, mscs could directly dissolve fibrosis through the secretion of matrix metalloproteinase, an enzyme that is capable of degrading the extracellular matrix [72] . adding to this regenerative power of mscs that depends on its paracrine effect, these cells could still differentiate to the injured cell phenotype and thus restore the structure and function of injured tissue. the histological examination of lung tissue in cfc further confirmed that the restoration of lung structure and function in bm-msc treated group was due to the regenerative power of mscs, as cfg showed massive collapsing of alveoli together with increased septal thickness and evident cellular infiltration. such results prove that free cell media did not have any therapeutic effect; nevertheless, instead, there was a prolonged exposure to blm injurious effect on lung tissue without an opposing therapeutic option, where histological features of fibrosis and degeneration were even more pronounced than that seen in the fibrotic untreated group. in such a context, mscs proved to be a therapeutic strategy with remarkable ameliorative potential in the induced lung fibrosis model in the current study. in conclusion, the blm experimental model proved to be a reproducible model to mimic lung fibrosis. the methods implemented in the present work confirmed that the intravenous injection is an ultimate approach ensuring engraftment of transplanted mscs. the applied techniques for morphological and functional assessment proved the efficiency of transplanted bm-mscs in restoring the lung architecture and functions in the blm experimentally induced fibrosis. the established results on the experimental level should enhance clinical trials that would lead to the application of mscs as a therapeutic strategy for treatment and preventing the progression of lung fibrosis. attenuation of bleomycin-induced pulmonary fibrosis in rats by flavocoxid treatment time for a change: is idiopathic pulmonary fibrosis still idiopathic and only fibrotic? idiopathic pulmonary fibrosis: effects and optimal management of comorbidities air pollution exposure is associated with lower lung function, but not changes in lung function, in patients with idiopathic pulmonary fibrosis tobacco smoking and risk for idiopathic pulmonary fibrosis: a prospective cohort study in uk biobank pulmonary fibrosis secondary to covid-19: a call to arms? symptom prevalence of patients with fibrotic interstitial lung disease: a systematic literature review general overview of lung transplantation and review of organ allocation introduction to stem cell therapy dormancy in the stem cell niche the ground state of embryonic stem cell self-renewal bleomycin use in the treatment of hodgkin lymphoma (hl): toxicity and outcomes in the modern era ovarian immature teratoma associated with pregnancy electrochemotherapy with bleomycin and cisplatin enhances cytotoxicity in primary and metastatic uveal melanoma cell lines in vitro mouse models of bleomycin-induced pulmonary fibrosis dli induced by nonmolecular target antineoplastic drugs: what are the characteristics of dli in nonmolecular target antineoplastic drugs? in: drug-induced lung injury a reliable method for intratracheal instillation of materials to the entire lung in rats isolation of adipose and bone marrow mesenchymal stem cells using cd29 and cd90 modifies their capacity for osteogenic and adipogenic differentiation characterization of mesenchymal stem cells derived from rat bone marrow and adipose tissue: a comparative study isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs an improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow in vitro cultivation technology of rat bone marrow mesenchymal stem cells expression of inflammatory cytokines in mesenchymal stromal cells is sensitive to culture conditions and simple cell manipulations colony forming unit assays immunophenotypic characterization of ovine mesenchymal stem cells estimation of cell number by hemocytometry counting. cold spring harb protoc mesenchymal stromal cells in animal bleomycin pulmonary fibrosis models: a systematic review the possible therapeutic role of mesenchymal stem cells in amiodarone-induced lung injury in adult male albino rats distribution of the cm-dil-labeled human umbilical cord vein mesenchymal stem cells migrated to the cyclophosphamide-injured ovaries in c57bl/6 mice engraftment assessment in human and mouse liver tissue after sex-mismatched liver cell transplantation by real-time quantitative pcr for y chromosome sequences mesenchymal stem cells inhibited development of lung cancer induced by chemical carcinogens in a rat model role of bone marrow mesenchymal stem cells in the treatment of ccl4 induced liver fibrosis in albino rats: a histological and immunohistochemical study melatonin maximizes the therapeutic potential of non-preconditioned mscs in a den-induced rat model of hcc light microscope and slide preparation. carleton's histological technique specimen staining and contrast methods for transmission electron microscopy fixation, dehydration and embedding of biological specimens. practical methods in electron microscopy. part i electron microscopy 2: practical procedures. theory and practice of histological techniques encyclopedia of statistical sciences lung ageing and copd: is there a role for ageing in abnormal tissue repair? paracrine factors from mesenchymal stem cells attenuate epithelial injury and lung fibrosis modified mesenchymal stem cells using mirna transfection could modify lung fibrosis progression translational research in acute lung injury and pulmonary fibrosis: modified mesenchymal stem cells using mirna transduction alter lung injury in a bleomycin model mesenchymal stem cell exosomes ameliorate experimental idiopathic pulmonary fibrosis by modulating alveolar macrophage phenotype and monocyte recruitment respiratory function and quantitative measurement of fibrosis in a rat model of bleomycin-induced lung fibrosis ilc2 promotes bleomycin-induced pulmonary fibrosis though il-33/ st2 signaling pathway the bleomycin model of pulmonary fibrosis modeling pulmonary fibrosis with bleomycin administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by ute-mri and histology oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis oxidative toxicology of bleomycin: role of the extracellular redox environment bleomycininduced lung injury il-37 attenuates lung fibrosis by inducing autophagy and regulating tgf-b1 production in mice asiatic acid ameliorates pulmonary fibrosis induced by bleomycin (blm) via suppressing pro-fibrotic and inflammatory signaling pathways macrophages: friend or foe in idiopathic pulmonary fibrosis? pulmonary pathology of ards: diffuse alveolar damage pulmonary manifestations of acute lung injury: more than just diffuse alveolar damage building a consensus regarding the nature and origin of mesenchymal stem cells cells and cellular interactions in the pathogenesis of idiopathic pulmonary fibrosis proteomic analysis of altered extracellular matrix turnover in bleomycin-induced pulmonary fibrosis epithelial-mesenchymal transition contributes to pulmonary fibrosis via aberrant epithelial/fibroblastic cross-talk mechanisms of bleomycin-induced lung damage antitumor antibiotics: bleomycin, enediynes, and mitomycin bleomycin: new perspectives on the mechanism of action bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized the ratio of single-to double-strand dna breaks and their absolute values determine cell death pathway pulmonary function tests mesenchymal stem cell therapy and lung diseases lung stem and progenitor cells secretion of immunoregulatory cytokines by mesenchymal stem cells anti-inflammatory and pro-inflammatory roles of tgf-b, il-10, and il-22 in immunity and autoimmunity suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow acknowledgements authors would like to acknowledge the center of excellence for research in regenerative medicine and its applications, cerrma (stdf funded), for providing the adequate facilities and environment for the processing and characterization of the bm-mscs under completely sterile conditions. conflict of interest the authors declare that they have no conflicts of interest.ethical statement all experiments were approved by the medical ethics committee of alexandria faculty of medicine (irb no: 00012098-fwa no: 00018699). key: cord-335382-fk4um9nw authors: farver, carol f.; zander, dani s. title: molecular basis of pulmonary disease date: 2012-08-10 journal: molecular pathology doi: 10.1016/b978-0-12-374419-7.00018-4 sha: doc_id: 335382 cord_uid: fk4um9nw pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible; therefore, lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. this chapter presents a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. it suggests that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible and so lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. what follows is a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. we believe that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. lung cancer is a major cause of morbidity and mortality throughout the world. the most recent estimates available from the surveillance, epidemiology, and end results (seer) program of the national cancer institute are that in 2007 over 213,000 people in the united states were diagnosed with cancer of the lung and bronchus, and over 160,000 will have died due to this disease [1] . however, in the past decade incidence and mortality rates have begun to move in a more positive direction, particularly in men. overall, men show a decline in lung cancer incidence, while in women, although lung cancer rates grew from 1975 through 1998, they stabilized from 1998 through 2004 [2] . similarly, cancer death rates due to lung cancer have declined for men and have slowed for women. although, for women, lung cancer death rates have increased since 1975, the rate of increase has slowed to 0.2% annually from 1995 to 2004 [2] . these trends parallel changes in the prevalence of tobacco smoking, the most important risk factor for development of lung cancer. given the tremendous societal and individual impacts of this disease, it is not surprising that the molecular biology of lung cancer is a major focus of investigation. elucidation of the molecular pathogenesis of these neoplasms has progressed significantly, offering insights into new, targeted therapies, and predictors of prognosis and therapeutic responsiveness. recognition of precursor lesions for some types of lung cancers has been facilitated by our expanded understanding of early molecular changes involved in carcinogenesis. the world health organization (who) classification scheme is the most widely used system for classification of these neoplasms (table 18 .1) [3] . although there are numerous histologic types and subtypes of lung cancers, most of the common malignant epithelial tumors can be grouped into the categories of nonsmall cell lung cancers (nsclcs) and small cell carcinomas (sclcs). nsclcs include adenocarcinomas (acs), squamous cell carcinomas (sqccs), large cell carcinomas, adenosquamous carcinomas, and sarcomatoid carcinomas. sclcs include cases of pure and combined small cell carcinoma. common pulmonary symptoms associated with these tumors include cough, shortness of breath, chest pain or tightness, and hemoptysis (coughing up blood). since some tumors cause airway obstruction, they predispose to pneumonia, which can be an important clue to the existence of a tumor in some patients. constitutional symptoms can include fever, weight loss, and malaise. some neoplasms will declare themselves with symptoms related to local invasion of adjacent structures such as chest wall, nerves, superior vena cava, esophagus, or heart. sclcs are known for early and widespread metastasis and are therefore particularly prone to being discovered through presentations as metastases in distant sites. some tumors are discovered due to pathophysiologic changes triggered by the release of soluble substances from tumor cells. endocrine syndromes due to elaboration of hormones are well recognized, and include cushing syndrome, syndrome of inappropriate antidiuretic hormone, hypercalcemia, carcinoid syndrome, gynecomastia, and others. hypercoagulability commonly occurs with lung cancers, leading to manifestations of venous thrombosis, nonbacterial thrombotic endocarditis, and disseminated intravascular coagulation. hematologic changes can include anemia, granulocytosis, eosinophilia, and other abnormalities. other paraneoplastic syndromes such as clubbing of the fingers, myasthenic syndromes, dermatomyositis/polymyositis, and transverse myelitis are noted in subsets of patients. when lung cancer is suspected, evaluation of the patient includes a thorough clinical, radiologic, and laboratory assessment, with collection of tissue or cytology samples to establish a pathologic diagnosis of malignancy and to classify the tumor type. fiberoptic bronchoscopy is often performed to collect samples for diagnosis. sample types can include transbronchial and endobronchial biopsies, bronchial brushings, bronchial washings, bronchoalveolar lavage samples, and transbronchial needle aspirates. submission of sputum samples for cytologic malignant epithelial tumors examination can provide a diagnosis in some cases, particularly for centrally located tumors such as sqcc and sclc. tumors arising in a peripheral location can also be sampled, in many cases, by fine needle aspiration or core needle biopsy performed under radiologic guidance. if a pleural effusion is present in combination with a lung parenchymal tumor, analysis of the pleural fluid cytology often allows one to establish a diagnosis. pleural biopsy, mediastinoscopy with biopsy, and wedge biopsy can also be performed, depending on the clinical and radiologic findings. for tumors with apparent distant metastasis, biopsy of the metastasis focus can both establish a pathologic diagnosis and determine the stage of the tumor. the prognosis of lung cancers is closely related to tumor stage. for nsclcs, the american joint commission on cancer tnm staging system is widely used (table 18. 2) [4] , and for sclcs, disease is classified as limited (restricted to one hemithorax) or extensive. overall, for lung cancers, the 5-year survival is 13.4% for men and 17.9% for women [5] . an important factor leading to this relatively poor survival is the late stage at which many lung cancers are diagnosed. information from the seer database, from 1996-2003, indicates that 16%, 35%, 42%, and 7% of patients were diagnosed with localized, regional, distant, or unstaged disease, respectively [5] . the corresponding 5-year survival rates are 49.0%, 15.3%, 2.8%, and 8.7%, and 10year survival rates are 37.8%, 10.3%, 1.6%, and 5.1% [5] . for patients with nsclcs, treatment depends on stage and comorbid conditions [6] . surgical resection is the preferred approach to treatment of localized nsclcs, provided there is no medical contraindication to operative intervention. lobectomy or more extensive resection (depending on tumor extent) is usually recommended rather than lesser surgeries, unless other comorbid conditions preclude these procedures. tumor 3 cm in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus t2 tumor with any of the following features of size or extent: > 3 cm in greatest dimension, involves main bronchus ! 2 cm distal to the carina, invades visceral pleura, associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung t3 tumor of any size that directly invades the chest wall, diaphragm, mediastinal pleura, parietal pericardium; or lies < 2 cm distal to the carina but without involvement of the carina; or is associated with atelectasis or obstructive pneumonitis of the entire lung t4 tumor of any size that invades the mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or has separate tumor nodule(s) in same lobe; or is associated with a malignant pleural effusion. regional lymph nodes (n) nx regional lymph nodes cannot be assessed n0 no regional lymph node metastasis n1 metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, including intrapulmonary nodes involved by direct extension of the primary tumor n2 metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s) n3 metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene or supraclavicular lymph node(s). mx distant metastasis cannot be assessed m0 no distant metastasis m1 distant metastasis; includes separate tumor nodule(s) in a different lobe. occult t0 n0 m0 stage 0 tis n0 m0 stage ia t1 n0 m0 stage ib t2 n0 m0 stage iia t1 n1 m0 stage iib t2 n1 m0 t3 n0 m0 stage iiia t1 n2 m0 t2 n2 m0 t3 n1 m0 t3 n2 m0 stage iiib any t n3 m0 t4 any intraoperative mediastinal lymph node sampling or dissection is also recommended for accurate pathologic staging and determination of therapy. subsets of patients also benefit from chemotherapy and radiotherapy. for more advanced nsclc and for sclc, chemotherapy and radiotherapy are the primary treatment modalities [6] . rare patients with limited-stage sclcs can be considered for surgical resection with curative intent. development of lung cancer occurs with multiple, complex, stepwise genetic and epigenetic changes involving allelic losses, chromosomal instability and imbalance, mutations in tumor suppressor genes (tsgs) and dominant oncogenes, epigenetic gene silencing through promoter hypermethylation, and aberrant expression of genes participating in control of cell proliferation and apoptosis [7] . there are similarities as well as type-specific differences in the molecular alterations between nsclcs and sclcs, and between sqccs and acs [8] [9] [10] . oncogenes that play a part in the pathogenesis of lung cancer include myc, k-ras (predominantly acs), cyclin d1, bcl2, and erbb family genes such as egfr (epidermal growth factor receptor) (predominantly acs) and her2/neu (predominantly acs) [11, 12] . also, lung cancers often display abnormalities involving tsgs including tp53, rb, p16 ink4a , and new candidate tsgs on the short arm of chromosome 3 (dutt1, fhit, rasff1a, fus-1, bap-1) [11, 13] . as research advances, these lists continue to grow, and as knowledge has expanded about the roles of these genes in carcinogenesis and tumor behavior, new targeted therapeutic agents have been designed to treat this disease ( figure 18 .1 and table 18 .3) [14] . many other agents are under investigation. in cancers, chromosomal regions harboring tsgs and oncogenes are often deleted or amplified. allele loss involving loci in 3p14-23 is a consistent feature of lung cancer pathogenesis [15, 16] . wistuba et al. reported allelic losses of 3p, often multiple and discontinuous, in 96% of the lung cancers studied and in 78% of the precursor lesions [15] . larger segments of allelic loss were noted in most sclcs (91%) and sqccs (95%) than in acs (71%) and preneoplastic/preinvasive lesions [15] . there was allelic loss in the 600-kb 3p21.3 deletion region in 77% of the lung cancers; 70% of the normal or reneoplastic/preinvasive lesions associated with lung cancers; and 49% of the normal, mildly abnormal, or preneoplastic/ preinvasive lesions found in smokers without lung cancer, but no loss was seen in the samples from people who had never smoked [15] . 8p21-23 deletions are also frequent and early events in the pathogenesis of lung carcinomas [17] , and other common alterations include loh at 13q, 17q, 18q, and 22p [16] . allelic losses that are more frequent in sqccs than acs include deletions at 17p13 (tp53), 13q14 (rb), 9p21 (p16 ink4a ), 8p21-23, and several regions of 3p [11, 15, 17, 18] . a recent study utilizing a bacterial artificial chromosome array to perform high-resolution whole genome profiling of sqcc and ac cell lines showed that regions of frequent amplification shared by both types of tumors included 5p; chromosome 7, 8q, 11q13, 19q, and 20q; and common regions of deletion included 3p, 4q, 9p, 10p, 10q; chromosome 18; and chromosome 21 [10] . however, acs appeared to have higher frequencies of deletion of chromosome 6; 8p, 9q, 15q; and chromosome 16 than sqccs, and possess small regions of amplification on chromosomes 12 and 14 not seen in sqccs. chromosome arms 2q and 13q were frequently deleted in ac but amplified in sqcc cell lines. both types of tumors showed deletion of chromosome arm 17p, but it was more frequent in the sqcc cell lines, while amplification of chromosome 17p was more frequent in acs. amplification of chromosome 3q was common to both types of tumors but showed frequent alteration at 3q23-3q26 in the sqcc lines and at 3q22 in the ac lines. inactivation of recessive oncogenes is believed to occur through a two-stage process. it has been suggested that the first allelic inactivation occurs, often via a point mutation, and the second allele is later inactivated by a chromosomal deletion, translocation or other alteration such as methylation of the gene promoter region [19] . inactivating mutations in the tsg tp53, which encodes the p53 protein, are the most frequent mutations in lung cancers. these mutations are found in up to 50% of nsclcs and over 70% of sclcs, and are largely attributable to direct dna damage from cigarette smoke carcinogens [20] . tp53 mutational patterns show a prevalence of g to t transversions in 30% of smokers' lung cancers versus only 12% of lung cancers in nonsmokers [20] . p53 protein is a transcription factor and a key regulator of cell cycle progression; cellular signals induced by dna damage, oncogene expression, or other stimuli trigger p53dependent responses including initiating cell cycle arrest, apoptosis, differentiation, and dna repair [21] . loss of p53 function in tumor cells can result in inappropriate progression through the dysregulated cell cycle checkpoints and permits the inappropriate survival of genetically damaged cells [22] . the p16 ink4a -cyclin d1-cdk4-rb pathway, which plays a central role in controlling the g1 to s phase transition of the cell cycle, is another important tumor suppressor pathway that is often disrupted in lung cancers. it interfaces with the p53 pathway through p14 arf and p21 waf/cip1 . thirty percent to 70% of nsclcs contain mutations of p16 ink4a , including homozygous deletion or point mutations and epigenetic alterations, leading to p16 ink4a inactivation [22] . almost 90% of sclcs and smaller numbers of nsclcs, on the other hand, display loss of rb expression [23] , and mutational mechanisms usually responsible include deletion, nonsense mutations, and splicing abnormalities that lead to truncated rb protein [22] . p16 ink4a leads to hypophosphorylation of the rb protein, which causes arrest of cells in the g1 phase. the active, hypophosphorylated form of rb regulates other cellular proteins including the transcription factors e2f1, e2f2, and e2f3, which are essential for progression through the g1/s phase transition. loss of p16 ink4a protein or increased complexes of cyclin d-cdk4-6 or cyclin e-cdk2 lead to hyperphosphorylation of rb with resultant evasion of cell cycle arrest and progression into s phase [21, 23] . cell cycle progression is inhibited by p21 waf/cip1 through its inhibition of the cyclin complexes. the 10%-30% of nsclcs lacking detectable alterations in p16 ink4a and rb may have abnormalities of cyclin d1 and cdk4, which cause inactivation of the rb pathway [22] . figure 18 .2 provides an overview of the p53 and retinoblastoma (rb) pathways, showing the complex interactions between the components [21] . epigenetic alterations (hypermethylation of the 5 0 cpg island) of tsgs are also frequent occurrences during pulmonary carcinogenesis, and methylation profiles of nsclcs show relationships to smoke exposure, histologic type, and geography. methylation rates of p16 ink4a and apc and the mean methylation index (mi) (a reflection of the overall methylation status) in current or former smokers were significantly higher than in never smokers; the mean mi of tumors was highest in current smokers; methylation rates of apc, cdh13, and rarbeta were significantly higher in acs than in sqccs; methylation rates of mgmt and gstp1 in cases from the united states and australia significantly exceeded those from japanese and taiwanese cases; and no significant gender-related differences in methylation patterns were found [24] . proto-oncogene activation and growth factor signaling are important in pulmonary carcinogenesis. the tyrosine kinase epidermal growth factor receptor (egfr) is frequently mutated in nsclcs, particularly in acs, and the mutational status is important in determining response to tyrosine kinase inhibitors. a related pathway, the phosphoinositide 3-kinase (pi3k)/akt/mammalian target of rapamycin (mtor) pathway, is frequently deregulated in pulmonary carcinogenesis. as reviewed by marinov et al., this pathway has been reported to mediate the effects of several tyrosine kinase receptors, including egfr, c-met, c-kit, and igf-ir, on proliferation and survival in nsclc and sclc [25] . clinical trials are ongoing, investigating the efficacy of the mtor inhibitor rapamycin and its analogues on lung cancer [26] . her2/neu is another related receptor tyrosine kinase that is upregulated in approximately 20%-30% of nsclcs [27, 28] , but unlike the situation with her2/neu-positive breast cancers, treatment with anti-her2/neu antibody (trastuzumab) does not seem to yield comparable benefits for nsclc when used alone or in combination with chemotherapy [28, 29] . point mutations of ras family proto-oncogenes (most often at k-ras codons 12, 13, or 61) are detected in 20%-30% of lung acs and 15%-50% of all nsclcs [22] . although farnesyl transferase inhibitors prevent ras signaling, these agents have not shown significant activity as single-agent therapy in untreated nsclc or relapsed sclc [30] . myc family genes (myc, mycn, and mycl), which play roles in cell cycle regulation, proliferation, and dna synthesis, are more frequently activated in sclcs than in nsclcs, either by gene amplification or by transcriptional dysregulation [22] . vascular endothelial growth factor (vegf) is a homodimeric glycoprotein that is overexpressed in many lung cancers and directly stimulates endothelial cell proliferation, promotes endothelial cell survival in newly formed vessels, and induces proteases involved in the degradation of the extracellular matrix needed for endothelial cell migration [31] . its angiogenic effects are mediated by three receptors: vegfr-1, vegfr-2, and vegfr-3; ligand binding leads to tyrosine kinase activation and activation of the signaling pathways required for angiogenesis [31] . monoclonal antibodies to vegf (bevacizumab) and tyrosine kinase inhibitors to vegfrs have been developed and show promise for treatment of nsclc. a phase iii trial of bevacizumab showed significantly improved overall and progression-free survival when this agent was used in combination with standard first-line chemotherapy in patients with advanced nsclc, and several smallmolecule vegfr tyrosine kinase inhibitors have yielded favorable results in phase i and ii trials in nsclc [32] . micrornas are a recently discovered class of nonprotein-coding, endogenous, small rnas which regulate gene expression by translational repression, mrna cleavage, and mrna decay initiated by mirna-guided rapid deadenylation [33] . some micrornas such as let-7 have been suggested to play roles in carcinogenesis by functioning as oncogenes or tumor suppressors, negatively regulating tsgs and/or genes that control cell differentiation or apoptosis [33] . investigations of the therapeutic potential of micrornas are also under way. in the 2004 version of the who classification scheme, ac is defined as "a malignant epithelial tumour with glandular differentiation or mucin production, showing acinar, papillary, bronchioloalveolar or solid with mucin growth patterns or a mixture of these patterns" [34] . ac has become the most frequent histologic type of lung cancer in parts of the world. it occurs primarily in smokers, but represents the most common type of lung cancer in people who have never smoked and in women. a small subset of these tumors arise in patients with localized scars or diffuse fibrosing lung diseases such as asbestosis and interstitial pneumonia associated with scleroderma [35] . these neoplasms usually arise in the periphery of the lung, and are more likely to invade the pleura and chest wall than other histologic types of lung cancers. radiologic studies can show one or more nodules, ground-glass opacities, or mixed solid and ground-glass lesions. on gross examination, the neoplasms are often solitary gray-white nodules or masses, sometimes with necrosis or cavitation, which pucker the overlying pleura. mucin-producing tumors can have a glistening, gelatinous appearance. other presentations include a pattern of consolidation resembling pneumonia (usually bronchioloalveolar carcinoma) ( figure 18 .3), multiple nodules, diffuse interstitial widening due to lymphangitic spread, endobronchial lesions with submucosal infiltration, and diffuse visceral pleural infiltration and thickening resembling mesothelioma. common histologic patterns displayed by acs include acinar ( figure 18 chapter 18 molecular basis of pulmonary disease mixtures of these patterns are very frequent. less common histologic subtypes include fetal ac, mucinous (colloid) ac, mucinous cystadenocarcinoma, signet ring ac, and clear cell ac [34] . acs usually exhibit differentiation toward clara cells or type ii pneumocytes or, less often, goblet cells. they manifest a range of differentiation extending from very well-differentiated tumors with extensive gland formation and little cytoatypia, to poorly differentiated, solid tumors that cannot be categorized as acs unless one orders a mucin stain (figure 18.7) . however, most examples include readily identifiable glands. invasiveness is reflected by the presence of neoplastic glands that infiltrate through stroma or pleura, stimulating a fibroblastic (desmoplastic) response ( figure 18.4) , or by cells in the lumens of blood vessels or lymphatics. in recent years, atypical adenomatous hyperplasia (aah) has been recognized as a precursor lesion for peripheral pulmonary acs. this lesion is defined as "a localized proliferation of mild to moderately atypical cells lining involved alveoli and, sometimes, respiratory bronchioles, resulting in focal lesions in peripheral part iv molecular pathology of human disease alveolated lung, usually less than 5 mm in diameter and generally in the absence of underlying interstitial inflammation and fibrosis" (figure 18 .8) [36] . aah exists on a histologic continuum with bronchioloalveolar carcinoma (bac), which is defined as an in situ (noninvasive) form of ac, in which the neoplastic cells grow along alveolar septa (lepidic growth) without invasion of stroma or vasculature ( figure 18 .5, figure 18 .6) [34] . most bacs exceed 1 cm in diameter and consist of cells with greater degrees of cytoatypia than aah. although aah is found in approximately 3% of patients without lung cancer at autopsy [37] , it has been reported in 9%-21% of lung resection specimens with all types of primary lung cancer and 16%-35% of lung resection specimens with ac [36] . the progenitor cell for bac and aah is believed to be an epithelial cell located at the junction between the terminal bronchiole and alveolus, termed the bronchioalveolar stem cell [38] . a recently published large-scale study of primary lung acs, using dense single nucleotide polymorphism arrays, described 57 significantly recurrent copy-number alterations in these tumors (table 18 .4) [12] . twenty-six of 39 autosomal chromosome arms showed consistent large-scale copy-number gain or loss, and 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions, were found. although some of the alterations involved regions known to harbor a proto-oncogene or tsg, these genes remain to be identified in some of the other regions affected. amplification of chromosome 14q13.3 was the most common event noted, found in 12% of samples. this region includes nkx2-1, which encodes a lineage-specific transcription factor (thyroid transcription factor-1 [ttf-1]) that activates transcription of target genes including the surfactant proteins, and may be an important proto-oncogene involved in a significant fraction of lung acs. immunohistochemical staining for ttf-1 can be performed to detect expression of this factor in most lung adenocarcinomas, aiding in the determination of the lung as the site of origin of the tumor (figure 18 .9). additional work using small interfering rna (sirna)mediated knockdown of this gene in lung cancer cell lines with amplification led to reductions in tumor cell proliferation, through both decreased cell cycle progression and increased apoptosis, suggesting that gene amplification and overexpression contribute to lung cancer cell proliferation rates and survival [39] . egfr and k-ras mutations are mutually exclusive mutational events in ac of the lung, which suggests the existence of two independent oncogenic pathways [40, 41] . egfr is a receptor tyrosine kinase whose activation by ligand binding leads to activation of cell signaling pathways such as ras/mitogen-activated protein kinase (mapk) and phosphatidylinositol-3-kinase, which in turn propagates signals for proliferation, blocking of apoptosis, differentiation, motility, invasion, and adhesion [21] . tumor-acquired mutations in the tyrosine kinase domain of egfr, often associated with gene amplification, have been found in approximately 5%-10% of nsclcs in the united states, and are associated with ac histology, never-smoker status, east asian ethnicity, and female gender [14, 40, 42] . egfr mutations are frequently in-frame deletions in exon 19, single missense mutations in exon 21, or in-frame duplications/insertions in exon 20, and occasional missense mutations and double mutations can also be detected [40, 43] . egfr mutation has an inverse correlation with methylation of the p16 ink4a gene and sparc (secreted protein acidic and rich in cysteine), an extracellular ca2ã¾-binding glycoprotein associated with the regulation of cell adhesion and growth [41] . egfr status is an important predictor of response to egfr kinase inhibitors: patients with egfr mutations are most likely to have a significant response to egfr tyrosine kinase inhibitor therapy, and egfr amplification and protein overexpression have been reported to correlate with survival after egfr tyrosine kinase inhibitor therapy [14, 44] . k-ras is a member of the ras family of proteins, which function as signal transducers between cell membrane-based growth factor signaling and the mapk pathways [21] . k-ras mutations are associated with smoking, male gender, and poorly differentiated tumors [43] . her2 (also known as egfr2 or erbb2), a member of the egfr family of receptor tyrosine kinases, is mutated in less than 2% of nsclc, and does not occur in tumors with egfr or k-ras mutation [45] . the her2 mutations are in-frame insertions in exon 20 and are significantly more frequent in acs (2.8%), never smokers (3.2%), asian ethnicity (3.9%), and women (3.6%), similar to egfr mutations [45] . alterations in dna methylation appear to be important epigenetic changes in cancer, contributing to chromosomal instability through global hypomethylation, and aberrant gene expression through alterations in the methylation levels at promoter cpg islands [46] . this lesion, which has been defined as a precursor lesion for peripheral pulmonary adenocarcinomas, consists of a wellcircumscribed nodule measuring several millimeters in diameter, in which alveolar septa are lined by mildly moderate atypical cells. epigenetic differences exist between egfr-mediated and k-ras-mediated tumorigenesis, and may interact with the genetic changes. a recent study showed that the probability of having egfr mutation was significantly lower among those with p16 ink4a and cdh13 methylation than in those without, and the methylation index was significantly lower in egfr mutant cases than in wild-type. in contrast, k-ras mutation was significantly higher in p16 ink4a methylated cases than in unmethylated cases, and the methylation index was higher in k-ras mutant cases than in wild-type [47] . sqcc is defined as "a malignant epithelial tumour showing keratinization and/or intercellular bridges that arises from bronchial epithelium," in the who classification scheme [48] . it is a common histologic type of nsclc that is closely linked to cigarette smoking. in most patients, this tumor arises in a mainstem, lobar, or segmental bronchus, producing a central mass on imaging known tumor suppressor genes and proto-oncogenes defined as found in either cosmic30, cgp census31, or other evidence; if there is more than one known proto-oncogene in the region, only one is listed (priority for listing is, in order: known lung adenocarcinoma mutation; known lung cancer mutation; other known mutation (by cosmic frequency); listing in cgp census). @myc is near, but not within, the peak region. ksingle gene deletions previously seen, this study provides new mutations as well. part iv molecular pathology of human disease studies. many of these tumors have an endobronchial component that can cause airway obstruction, leading to postobstructive pneumonia, atelectasis, or bronchiectasis. not infrequently, it is the pneumonia that prompts evaluation of the patient and leads to discovery of the tumor. less often, sqccs develop in the periphery of the lung. gross examination reveals a tan or gray mass that usually arises in a large bronchus and often includes an endobronchial component (figure 18 .10, figure 18 .11). partial or complete airway obstruction can be associated with changes of pneumonia, bronchitis, abscess, bronchiectasis, or atelectasis. necrosis and cavitation are very common in these tumors. involvement of hilar lymph nodes by tan-gray tumor can be visible in some resected specimens. microscopically, the key features of this tumor are its keratinization, sometimes with formation of keratin pearls, and intercellular bridges ( figure 18 .12). as is true of acs, the degree of differentiation of this tumor varies from very well differentiated cases, in which there are abundant keratinization and intercellular bridges and little cytoatypia, to very poorly differentiated cases, in which keratinization and intercellular bridges can be quite inconspicuous and the tumor consists of sheets of large atypical cells with marked cytoatypia and frequent mitoses. however, most cases fall more toward the middle of the spectrum. invasiveness is reflected by the presence of irregular nests and sheets of cells that infiltrate through tissues, stimulating a fibroblastic response, or by cells inside vascular or lymphatic spaces. invasive sqccs are often accompanied by sqcc in situ and dysplasia, their precursor lesions. these lesions arise in the bronchi and may be contiguous with the invasive tumor or exist as one or more separate foci. these precursor lesions can also be observed without coexisting invasive carcinoma. like sqcc, tobacco smoking is the main predisposing factor for sqcc in situ and dysplasia. unlike invasive sqcc, however, these lesions are not invasive-they do not extend through the basement membrane of the bronchial epithelium. grossly, they may be invisible or appear as flat, tan or red discolorations of the bronchial mucosa, or tan wart-like excrescences. microscopically, these lesions encompass a chapter 18 molecular basis of pulmonary disease range of squamous changes that include alterations in the thickness of the bronchial epithelium, the maturational progress of squamous differentiation, cell size, and nuclear characteristics ( figure 18 .13, figure 18 .14) [11, 49] . as dysplasia increases from mild to moderate to severe, the epithelium thickens, and maturation is increasingly impaired. the basilar zone expands with epithelial cell crowding, the intermediate zone shrinks, and there is reduced flattening of the superficial squamous cells. cell size, pleomorphism, and anisocytosis usually increase, and there is coarsening of the chromatin and appearance of nucleoli, nuclear angulations, and folding. in carcinoma in situ, although the epithelium may or may not be thickened and the cell size may be small, medium, or large, there is minimal or no maturation from the base to the superficial aspect, and the atypical nuclear features are present throughout the entire thickness of the epithelium. mitoses appear in the lower third (mild or moderate dysplasia), lower two-thirds (severe dysplasia), or throughout the full thickness of the epithelium (carcinoma in situ). basal cells in the bronchial epithelium are believed to represent the progenitor cells for invasive sqcc, and the sequence of events leading to sqcc is believed to include basal cell hyperplasia, squamous metaplasia, squamous dysplasia, carcinoma in situ, and invasive sqcc (figure 18 .14) [11, [49] [50] [51] . regression of lesions preceding invasive sqcc can occur, particularly the earlier lesions [52] . however, severe dysplasia and carcinoma in situ are associated with a significantly increased probability of developing invasive sqcc in patients followed over time with surveillance bronchoscopy [53] . wistuba and colleagues evaluated sqccs and precursor lesions for loss of heterozygosity (loh) at 10 chromosomal regions (3p12, 3p14.2, 3p14.1-21.3, 3p21, 3p22-24, 3p25, 5q22, 9p21, 13q14 rb, and 17p13 tp53) part iv molecular pathology of human disease frequently deleted in lung cancer and found multiple, sequentially occurring allele-specific molecular changes in separate, apparently clonally independent foci, early in the pathogenesis of sqccs of the lung, suggesting a field cancerization effect [11, 18] . they observed clones of cells with allelic loss at one or more regions in 31% percent of histologically normal epithelium and 42% of specimens with hyperplasia or metaplasia; increasing frequency of loh within clones with increasing histopathologic lesional severity; the most frequent and earliest regions of allelic loss at 3p21, 3p22-24, 3p25, and 9p21; increasing size of the 3p deletions with progressive histologic changes; and tp53 allelic loss in many histologically advanced lesions (dysplasia and cis) [18] . an overview of the sequential molecular events leading to invasive sqcc is shown in figure 18 .14 [11] . large cell carcinoma is an undifferentiated nsclc without light microscopic evidence of squamous or glandular differentiation, although squamous or glandular features may be detectable by ultrastructural examination (figure 18 .15) [54] . histologic subtypes of large cell carcinoma include large cell neuroendocrine carcinoma (lcnec), combined lcnec, basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, and large cell carcinoma with rhabdoid phenotype [54] . clinical signs and symptoms resemble those of other types of nsclc. most tumors develop as peripheral lung masses, except for basaloid carcinomas, which usually form centrally located masses. histologically, large cell carcinomas consist of sheets and nests of large cells with vesicular nuclei, prominent nucleoli, and moderate or abundant amounts of cytoplasm. lcnecs demonstrate neuroendocrine architectural features and immunohistochemical or ultrastructural evidence of neuroendocrine differentiation. basaloid carcinomas display nests of small, monomorphic, rounded or fusiform tumor cells with little cytoplasm, numerous mitoses, comedo-type necrosis, and hyaline or mucoid stromal degeneration. clear cell carcinoma consists of large tumor cells with clear cytoplasm. precursor lesions are not currently recognized for any of the subtypes of large cell carcinoma. however, basaloid carcinoma is associated with squamous dysplasia in about one-third of cases [54] . large cell carcinomas are poorly differentiated carcinomas that can demonstrate features of ac (most frequent), sqcc, or neuroendocrine differentiation when examined by immunohistochemistry, electron microscopy, or molecular methods [55] . these tumors often demonstrate losses of 1p, 1q, 3p, 6q, 7q, and 17p, and gains of 5q and 7p, more closely resembling acs than other histologic types of lung cancer [56] . common molecular abnormalities include tp53 mutation, c-myc amplification, and p16 promoter hypermethylation, while k-ras mutation is less common [55] . egfr tyrosine kinase domain mutation is not characteristic of large cell carcinomas, and egfrviii (deletion mutations in the extracellular domain of egfr) is uncommon [57, 58] . the major categories of pulmonary neuroendocrine (ne) neoplasms include small cell carcinoma (sclc), large cell neuroendocrine carcinoma (lcnec), typical carcinoid, and atypical carcinoid. sclc and lcnec are high-grade carcinomas, typical carcinoid is a low-grade malignant neoplasm, and atypical carcinoid occupies an intermediate position in the spectrum of biologic aggressiveness. in one large series, the 5-year and 10-year survival rates for typical carcinoid were 87% and 87%, 56% and 35% for atypical carcinoid, 27% and 9% for lcnec, and 9% and 5% for sclc, respectively [59] . by light microscopy, these tumors display ne architectural features including organoid nesting, a trabecular arrangement, rosette formation, and palisading. these patterns are more prominent in carcinoids than in lcnecs and may or may not be visible in individual sclcs. typical carcinoids contain fewer than 2 mitoses per 2 mm 2 (10 hpf) and lack necrosis ( figure 18 .16), while atypical carcinoids show 2-10 mitoses per 2 mm 2 (10 hpf) or necrosis, which is often punctate [60] . sclc consists of small, undifferentiated tumor cells with scant cytoplasm and finely granular chromatin and absent or inconspicuous nucleoli ( figure 18 .17). nuclear molding is characteristic, necrosis is common, and the mitotic rate is typically high, with a mean of over 60 mitoses per 2 mm 2 [61] . combined differences also exist in the characteristics of patients with carcinoids, as compared to patients with sclc and lcnec. patients with carcinoids are typically younger and less likely to smoke than those with sclcs and lcnecs, the vast majority of whom have a current or previous history of tobacco smoking [62, 63] . rare patients with carcinoids have the multiple endocrine neoplasia 1 (men1) syndrome, an association that is not seen with sclcs and lcnecs. in addition, an association with diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (dipnech) has been noted for carcinoids but not for sclcs and lcnecs, leading to classification of dipnech as a preinvasive lesion in the most recent version of the who classification scheme [64] . dipnech is a diffuse proliferation of single cells, small nodules (ne bodies), and linear proliferations of pulmonary ne cells that may reside in the bronchial and/or bronchiolar epithelia ( figure 18 . 19) , and may be accompanied by extraluminal proliferations part iv molecular pathology of human disease (tumorlets and carcinoids) [64] . however, morphologically identifiable precursor lesions for sclc and lcnec have not been established. molecular markers of pulmonary ne tumors include chromogranin a, synaptophysin (figure 18.20) , and n-cam (cd56). these markers are expressed by all categories of ne tumors, with higher frequencies observed in the carcinoids and atypical carcinoids than in small cell and large cell neuroendocrine carcinomas. gastrin-releasing peptide, calcitonin, other peptide hormones, the insulinoma-associated 1 (insm1) promotor and the human achaete-scute homolog-1 (hash1) gene have also been reported as overexpressed by these tumors [65, 66] . thyroid transcription factor-1 (ttf-1) is expressed by 80%-90% of sclcs, 30%-50% of lcnecs, and 0%-70% of carcinoids [67] [68] [69] [70] . sclcs [71] [72] [73] [74] [75] [76] . more than 90% of sclcs and sqccs demonstrate large, often discontinuous segments of allelic loss on chromosome 3p, in areas encompassing multiple candidate tumor suppressor genes, including some of those listed previously [15, 75] . atypical carcinoids show a higher frequency of loh at 3p, 13q, 9p21, and 17p than typical carcinoids, but not as high as the high-grade ne tumors [77] . some typical and atypical carcinoids possess mutations of the multiple endocrine neoplasia 1 (men1) gene on chromosome 11q13 or loh at this locus [78] , while these abnormalities occur with lower frequencies in sclcs and lcnecs, supporting separate pathways of tumorigenesis [79] . men1 encodes for the nuclear protein menin, which is believed to play several roles in tumorigenesis by linking transcription factor function to histone-modification pathways, in part through interacting with the activator-protein-1 family transcription factor jund, modifying it from an oncoprotein into a tumor suppressor protein [80] . oncogenes frequently amplified in sclcs include myc (8q24), mycn (2p24), and mycl1 (1p34), and additional amplified genes that represent candidate oncogenes include the antiapoptotic genes tnfrsf4 (1p36), dad1 (14q11), bcl2l1 (20q11), and bcl2l2 (14q11) [76] . the myc proteins are transcription factors that are important in cell cycle regulation, proliferation, and dna synthesis, and can induce p14 arf , leading to apoptosis through p53 if cellular conditions do not favor proliferation [21] . tsgs are inactivated in the majority of sclcs. eighty percent to 90% of sclcs demonstrate tp53 mutations, as compared to more than 50% of nsclcs, fewer atypical carcinoids, and virtually no typically carcinoids [74, 81] . most of the tp53 mutations in sclcs are missense point mutations that result in a stabilized p53 mutant protein which can be easily detected by immunohistochemistry [71] . p53 protein overexpression occurs frequently in high-grade ne carcinomas, but is unusual in typical carcinoids and intermediate in atypical carcinoids [82, 83] . dysregulation of p53 produces downstream effects on bcl-2 and bax. antiapoptotic bcl-2 predominates over proapoptotic bax in the high-grade ne carcinomas, while the reverse is true for carcinoids [82] . lcnecs resemble sclcs in their high rates of tp53 mutation and predominance of bcl-2 expression over bax expression [84] . alterations compromising the p16 ink4a /cyclin d1/rb pathway of g1 arrest are consistent in high-grade pulmonary ne carcinomas (92%), primarily through loss of rb protein, but are less frequent in atypical carcinoids (59%) and are uncommon in typical carcinoids [23] . mutations in the rb1 gene exist in many sclcs, with associated loss of function of the gene product [71, 74, 85] . in another study, 89% of the ne carcinomas (excluding carcinoids) versus 13% of the non-ne carcinomas exhibited loh and loss of rb-protein expression [86] . the hypophosphorylated form of rb protein functions as a cell cycle regulator for g1 arrest; cyclin d1 overexpression and p16 ink4a loss produce persistent hyperphosphorylation of rb with consequent evasion of cell cycle arrest [23] . recent data also suggest that in sclcs, overexpression of mdm2 (a transcriptional target of p53) or p14 arf loss leads to evasion of cell cycle arrest through the p53 and rb pathway ( figure 18 .2) [71] . the transcription factor e2f-1 appears to play a role in cellular proliferation by activating genes required for s phase entry. e2f-1 product is overexpressed in 92% of sclcs and 50% of lcnecs, and is significantly associated with a high ki67 index and bcl-2:bax ratio >1 [87] . a mediator of the proteasomal degradation of e2f-1, the s phase kinase-associated protein 2 (skp2) f-box protein accumulates in high-grade ne carcinomas (86%), and its overexpression has been associated with advanced stage and nodal metastasis in pulmonary ne tumors [88] . in the high-grade ne tumors, skp2 appears to interact with e2f-1 and stimulate its transcriptional activity toward the cyclin e promoter [87, 88] . telomeres play an important role in the protection of chromosomes against degradation. telomerases, the enzymes that synthesize telomeric dna strands, serve to counterbalance losses of dna during cell divisions. high telomerase activity has been noted in over 80% of sclcs and lcnecs [89] [90] [91] versus 14% or fewer typical carcinoids [91, 92] . expression of human telomerase mrna component (hterc) and human telomerase reverse transcriptase (htert) mrna were reported, respectively, in 58% and 74% of typical carcinoids; and in 100% and 100% of atypical carcinoids, lcnecs and sclcs, and telomere length alterations in lcnecs and sclcs were greater than in typical carcinoids [92] . aberrant methylation of cytosine-guanine (cpg) islands in promoter regions of malignant cells is an important mechanism for silencing of tsgs (epigenetic inactivation). methylation of dna involves the transfer of a methyl group, by a dna methyltransferase, to the cytosine of a cpg dinucleotide [93] . rassf1a is a potential tsg that undergoes epigenetic inactivation in virtually all sclcs and a majority of nsclcs through hypermethylation of its promoter region [94, 95] . ne tumors have lower frequencies of methylation of p16, apc, and cdh13 (h-cadherin) than nsclcs [95] . sclcs have higher frequencies of methylation of rassf1a, cdh1 (e-cadherin), and rarb than carcinoids [95] . promoter methylation of casp8, which encodes the apoptosis-inducing cysteine protease caspase 8, was also found in 35% of sclcs, 18% of carcinoids, and no nsclcs, suggesting that casp8 may function as a tsg in ne lung tumors [96] . although histologically defined precursors for sclc are lacking, a higher incidence of genetic abnormalities is found in the normal or hyperplasic airway epithelium of patients with sclc than nsclc [97] . by extension, it has been suggested that sclc may arise directly from histologically normal or mildly abnormal epithelium, rather than evolving through a sequence of recognizable histologic intermediary changes [11] . relatively little is known about molecular abnormalities in precursors of carcinoids. although carcinoids have been viewed as arising from tumorlets, 11q13 (int-2) allelic imbalance is significantly more common in carcinoids (73%) than in tumorlets (9%), and may represent an early event in carcinoid tumor formation [98] . the int-2 gene lies in close proximity to men1, a tumor suppressor gene frequently mutated in ne tumors [98] . the molecular pathology of dipnech remains to be elucidated. mesenchymal neoplasms included in the who classification scheme (table 18 .1) encompass a spectrum of malignant and benign proliferations that show differentiation along multiple lineages. overall, these tumors are much less common in the lung than are epithelial neoplasms. information about molecular pathogenesis has emerged for some of the mesenchymal neoplasms. pulmonary inflammatory myofibroblastic tumor (imt) is a lesion composed of myofibroblastic cells, collagen, and inflammatory cells that primarily occurs in individuals less than 40 years of age, and is the most common endobronchial mesenchymal lesion in childhood ( figure 18 .21) [99] . synovial sarcoma is usually a soft tissue malignancy, but uncommonly arises in the pleura or the lung and often takes an aggressive course [100] . pulmonary hamartomas are benign neoplasms consisting of mixtures of cartilage, fat, connective tissue, and smooth muscle, which present as coin lesions on chest radiographs and are excised in order to rule out a malignancy ( figure 18 .22). many imts demonstrate clonal abnormalities with rearrangements of chromosome 2p23 and the anaplastic lymphoma kinase (alk) gene [101] . the rearrangements involve fusion of tropomyosin (tpm) n-terminal coiled-coil domains to the alk c-terminal kinase domain, producing two alk fusion genes, tpm4-alk and tpm3-alk, which encode oncoproteins with constitutive kinase activity [102] . like their soft tissue counterparts, more than 90% of pulmonary and pleural synovial sarcomas demonstrate a chromosomal translocation t(x;18) (syt-ssx) [103, 104] . detection of this translocation can be very helpful for confirming the diagnosis of synovial sarcoma in this unusual location. most pulmonary hamartomas show abnormalities of chromosomal bands 6p21, 12q14-15, or other regions [105] , corresponding to mutations of high-mobility group (hmg) proteins, a family of nonhistone chromatin-associated proteins that serve an important role in regulating chromatin architecture and gene expression [106] . malignant mesothelioma (mm) is an uncommon, aggressive tumor arising from mesothelial cells on serosal surfaces, primarily the pleura and peritoneum, and less often the pericardium or tunica vaginalis. the most important risk factor for mm is exposure to the subset of asbestos fibers known as amphiboles (crocidolite and amosite) [107] . the incidence of this tumor in the united states peaked in the early to mid-1990s, and appears to be declining, likely related to decreases in the use of amphiboles since their peak period of importation in the 1960s [107] . these tumors are characterized by long latency periods between asbestos exposure and clinical presentation of the tumor, with a mean of 30-40 years [108] . radiation, a nonasbestos fiber known as erionite, and potentially other processes associated with pleural scarring have also been implicated in the causation of smaller numbers of cases of malignant mesothelioma [108] , and a role for simian virus 40 (sv40) in the genesis of this tumor has been suggested by some, but remains controversial [109, 110] . pleural mm most commonly arises in males over the age of 60. presenting features typically include a hemorrhagic pleural effusion associated with shortness of breath and chest wall pain. weight loss and malaise are common. by the time the tumor is discovered, patients usually have extensive involvement of the pleural surfaces. with progression, the tumor typically invades the lung, chest wall, and diaphragm. lymph node metastasis can cause superior vena caval obstruction, and cardiac tamponade, subcutaneous nodules, and contralateral lung involvement can also occur. from the time of diagnosis, the median survival is 12 months [110] . treatment may include surgery, chemotherapy, radiotherapy, immunotherapy, or other treatments, often in combination [110] . the intent of surgery is usually palliative. whether extrapleural pneumonectomy with chemotherapy and radiotherapy can lead to cure is unclear [111] . new agents are currently under investigation for their potential to improve the life expectancy and quality of life in patients with this aggressive malignancy. gross pathologic features of mm include pleural nodules which grow and coalesce to fill the pleural cavity and form a thick rind around the lung. a firm tan appearance is common, and occasionally the tumor can have a gelatinous consistency (figure 18 .23). extension along the interlobar fissures and invasion into the adjacent lung, diaphragm, and chest wall are characteristic. further spread can occur into the pericardial cavity and around other mediastinal structures, and distant metastases can also develop. histologically, mm manifests a wide variety of histologic patterns. the major histologic categories include epithelioid mesothelioma, sarcomatoid mesothelioma, desmoplastic mesothelioma, and biphasic mesothelioma [108] . epithelioid mesothelioma consists of round, ovoid, or polygonal cells with eosinophilic cytoplasm and nuclei that are usually round with little cytoatypia (figure 18 .24). these cells most often form sheets, tubulopapillary structures, or gland-like arrangements, and some tumors can have a myxoid appearance due to production of large amounts of hyaluronate. sarcomatoid mesothelioma is composed of malignant-appearing spindle cells occasionally accompanied by mature sarcomatous components (osteosarcoma, chondrosarcoma, others). desmoplastic mesothelioma can be a diagnostic challenge due to its frequently bland appearance and resemblance to organizing pleuritis. it consists of variably atypical spindle cells in a dense collagenous matrix ( figure 18 .25). helpful features for separating figure 18 .23 malignant mesothelioma. the tan/white tumor involves the entire pleura surrounding and compressing the underlying parenchyma, which appears congested but relatively unremarkable. chapter 18 molecular basis of pulmonary disease this tumor from organizing pleuritis include invasion of chest wall muscle or adipose tissue and necrosis. biphasic mesotheliomas include both epithelioid and sarcomatoid elements, each comprising at least 10% of the tumor [108] . pathologic diagnosis of mm has been greatly assisted by the expanded availability of antibodies for use in immunohistochemistry [112] . mesothelial differentiation can be supported by immunoreactivity with cytokeratin 5/6, calretinin ( figure 18 .26), hbme-1, d2-40, and other antibodies. histologic distinction of epithelioid mesotheliomas from metastatic acs is a common need in practice, and a panel approach using calretinin and cytokeratin 5/6, with other antibodies reactive with acs (cea, moc-31, ber-ep4, leu m1, b72.3, and others) will usually be successful. electron microscopy can also be helpful in difficult cases by demonstrating long thin microvilli in many mms with an epithelioid component. pan-cytokeratin staining is helpful for supporting a diagnosis of sarcomatoid or desmoplastic mm as opposed to sarcoma, since most (but not all) sarcomas will not stain for pan-cytokeratin. other mesothelial and mesenchymal markers can also be useful for assisting in the differentiation of mm from histologically similar sarcomas. precursor lesions for mm have not been clearly defined from a histologic standpoint, although it is likely that an in situ stage exists [108] . the term atypical mesothelial hyperplasia has been recommended for surface (noninvasive) proliferations of mesothelial cells of uncertain malignant potential [108] . exposure to asbestos fibers is believed to trigger the pathobiological changes leading to the majority of mms. currently, it is believed that asbestos may act as an initiator (genetically) and promoter (epigenetically) in the development of mms [113] . the degree to which tumorigenesis results from direct interactions of the fibers with the mesothelial cells, or through other mechanisms involving oxidative stress (or both), is unresolved [113, 114] . multiple chromosomal alterations are often noted in mms, and inactivation of tsgs plays an important part in the pathogenesis of mm [113] . a variety of genetic abnormalities have been reported including deletions of 1p21-22, 3p21, 4p, 4q, 6q, 9p21, 13q13-14, 14q, and proximal 15q, monosomy 22, and gains of 1q, 5p, 7p, 8q22-24, and 15q22part iv molecular pathology of human disease 25 [108, 115] . the most common genetic abnormality in mm is a deletion in 9p21 encompassing the cdkn2a locus encoding the tumor suppressors p16 ink4a and p14 arf , which participate in the p53 and rb pathways and inhibit cell cycle progression ( figure 18 .2) [113, 116] . recent studies have shown that sv40 large t antigen (present in some mms) inactivates the tsg products rb and p53, raising the possibility that asbestos and sv40 could act as co-carcinogens in mm and suggesting that perturbations of rb-and p53-dependent growth-regulatory pathways may be involved in the pathogenesis of mm [115] . other common findings include inactivating mutations with allelic loss in the tsg neurofibromin 2 (nf2), found at chromosome 22q12 [117] , and inactivation of cdkn2a/p14 arf and gpc3 (another tsg) by promoter methylation [108] . loss of cdkn2a/ p14 arf also results in mdm2-mediated inactivation of p53 [116] . however, in mms, unlike many other epithelial tumors, mutations in the tp53, rb, and ras genes are rare [118] . the wnt signal transduction pathway is also abnormally activated in mms and appears to play a role in pathogenesis [119] . activation of the pathway leads to accumulation of b-catenin in the cytoplasm and its translocation to the nucleus. interactions with tcf/ lef transcription factors promote expression of multiple genes including c-myc and cyclin d. the mechanism of activation does not appear to involve mutations in the b-catenin gene, but may instead involve more upstream components of the pathway, such as the disheveled proteins [119] . recent evidence also suggests that the phosphatidylinositol 3-kinase (pi3-k/akt) pathway is frequently activated in mms, and that inhibition of this pathway can increase sensitivity to a chemotherapeutic agent [120] . the wilms' tumor gene (wt1) is also expressed in most mms, but its role in the pathogenesis of mm is unclear [114] . finally, egfr signaling in mms has recently become a focus of greater attention, and there are some data showing that the egfr is an early cell membrane target of asbestos fibers and is linked to activation of the mapk cascade [113] . unfortunately, a phase ii clinical trial of gefitinib treatment in patients with mms did not show effectiveness, despite egfr overexpression in over 97% of cases [121] . another study found that common egfr mutations conferring sensitivity to gefitinib are not prevalent in human malignant mesothelioma [122] . further investigation continues into new, potentially efficacious agents for the treatment of mm. non-neoplastic pulmonary pathology comprises inflammatory and fibrosing diseases of the conducting airways, alveoli, vessels, and lymphoid tissue. this pathology may be localized or diffuse, may either have an obvious etiology or be idiopathic, and may cause injury that is reparable or irreparable. most importantly, an understanding of non-neoplastic lung pathology plays a vital role in the clinical management of these diseases. this section covers the major types of obstructive and interstitial diseases, the vascular lesions, the pneumonias, the occupational diseases, the major histiocytic conditions, and the most common developmental anomalies. this list does not include all of the non-neoplastic diseases that can affect the lung, but it represents those that are responsible for the majority of illness. also, the conditions highlighted within each of these categories are those about which we best understand the molecular biology of the disease mechanisms. obstructive lung diseases are characterized by a reduction in airflow due to airway narrowing. this airflow reduction occurs, in general, by two basic mechanisms: (i) inflammation and injury of the airway, resulting in obstruction by mucous and cellular debris within and around the airway lumen; and (ii) destruction of the elastin fibers of the alveolar walls, causing loss of elastic recoil and subsequent premature collapse of the airway during the expiratory phase of respiration. there are four major obstructive lung diseases: asthma, emphysema, chronic bronchitis, and bronchiectasis. asthma is a chronic inflammatory disease of the airways that affects more than 150 million people worldwide. the prevalence of disabling asthma has increased over 200% since 1969, ranging from as low as 1% in rural ethiopia to over 20% among children in parts of central and south america [123] . in the united states, asthma affects approximately 8%-10% of the population and is the leading cause of hospitalization among children less than 15 years of age [123] . clinically, the disease is defined as a generalized obstruction of airflow with a reversibility that can occur spontaneously or with therapy. it is characterized by recurrent wheezing, cough, or shortness of breath resulting from airway hyperactivity and mucus hypersecretion. the hyperresponsiveness is a result of acute bronchospasm and can be elicited for diagnostic purposes using histamine or methacholine challenges. the key feature of these symptoms is that they are variable-worse at night or in the early morning, and in some people worse after exercise. it has previously been assumed that these symptoms are separated by intervals of normal physiology. however, evidence is now accumulating that asthma can cause progressive lung impairment due to chronic morphologic changes in the airways. the treatment strategies for this complex disease are myriad. in atopic individuals, allergen avoidance should be the primary therapy. for example, in children, reducing exposure to house dust mites early in life decreases sensitization and the incidence of disease. for those who do develop the disease, avoidance of allergens later in life improves symptom control. established treatments for asthma flairs include inhaled corticosteroids, and short-acting and long-acting b2-adrenoceptor agonists. phosphodiesterase (pde) inhibitors such as theophylline have been used for decades to treat asthmatic bronchoconstriction, but both cardiac and central nervous systems side effects have limited their use. newer pde inhibitors without side effects include non-xanthine drugs such as rofumilast. the pathologic changes to the airways in asthma are very similar to those seen in chronic bronchitis. they consist of a thickened basement membrane with epithelial desquamation, goblet cell hyperplasia, and subepithelial elastin deposition. in the wall of the airway, smooth muscle hypertrophy and submucosal gland hyperplasia are also present ( figure 18 .27). in acute asthma exacerbations, a transmural chronic inflammatory infiltrate with variable amounts of eosinophilia may be present, resulting in epithelial injury and desquamation that can become quite pronounced. one sees clumps of degenerating epithelial cells mixed with mucin in the lumen airway. these aggregates of degenerating cells are referred to as creola bodies and can be seen in expectorated mucin from these patients. also present in these sputum samples are charcot-leyden crystals, rhomboid-shaped structures that represent breakdown products from eosinophil cytoplasmic granules ( figure 18 .28). the changes seen in the walls of these airways represent long-term airway remodeling caused by prolonged inflammation. this remodeling may play a role in the pathophysiology of asthma. the amount of airway remodeling is highly variable from patient to patient, but remodeling has been found even in patients with mild asthma. currently, the effect of the treatment on this chronic pathology is unclear [124] . the pathogenesis of asthma is complex, and most likely involves both genetic and environmental components. most experts now see it as a disease in which an insult initiates a series of events in a genetically susceptible host. no single gene accounts for the familial component of this disease. genetic analysis of these patients reveals a prevalence of specific hla alleles, polymorphisms of fc erib, il-4, and cd14 [125, 126] . asthma can be classified using a number of different schema. most commonly, asthma is divided into two categories: atopic (allergic) and nonatopic (nonallergic). atopic asthma results from an allergic sensitization usually early in life and has its onset in early childhood. nonatopic asthma is late-onset and, though the immunopathology has not been as well studied, probably has similar mechanisms to atopic asthma. although this nosology is convenient for purposes of understanding the mechanisms of the disease, most patients manifest a combination of these two categories with overlapping symptoms. th0 pathogenetic mechanisms of both types encompass a variety of cells and their products. these include airway epithelium, smooth muscle cells, fibroblasts, mast cells, eosinophils, and t-cells. the asthma response includes two phases: an early response comprising an acute bronchospastic event within 15-30 minutes after exposure, and a late response that peaks approximately 4-6 hours and that can have prolonged effects. if one wants to understand this complex response, it is best to divide it into three components: (i) a type 1 hypersensitivity response, (ii) acute and chronic inflammation, and (iii) bronchial hyperactivity. type 1 hypersensitivity in general, human asthma is associated with a predominance of type 2 helper cells with a cd4ã¾ phenotype. these th2-type cells result from the uptake and processing of viral, allergen, and environmental triggers that initiate the episode. the processing includes the presentation of these triggers by the airway dendritic cells to naive t-cells (th0), resulting in their differentiation into populations of th1 and th2. the th2 differentiation is a result of il-10 release by the dendritic cells, and the th2 cells then part iv molecular pathology of human disease further propagate the inflammatory reaction in two ways. first, they release a variety of cytokines such as il-4, il-5, and il-13 that mediate a wide variety of responses. il-4 and il-13 stimulate b-cells and plasma cells to produce ige, which, in turn, stimulates mast cell maturation and the release of multiple mediators, including histamine and leukotrienes. second, these th2 cells secrete il-5 that, together with il-4, also stimulates mast cells to secrete histamine, tryptase, chymase, and the cysteinyl leukotrienes causing the bronchoconstrictor response that occurs rapidly after the exposure to the allergen. il-5 from these lymphocytes also recruits eosinophils to the airways and stimulates the release of the contents of their granules, including eosinophil cationic protein (ecp), major basic protein (mbp), eosinophil peroxidase, and eosinophil-derived neurotoxin. these compounds not only induce the bronchial wall hyperactivity but are also responsible for the increased vascular permeability that produces the transmural edema in the airways. the cells can differentiate into th1 cells as a result of il-12 produced by dendritic cells. these th1 cells produce interferon-gamma (ifn-g), il-2, and lymphotoxin, which play a role in macrophage activation in delayedtype hypersensitivity reactions as seen in diseases such as rheumatoid arthritis and tuberculosis [123] . these th1 cells are predominantly responsible for defense against intracellular organisms and are more prominent in normal airways and in airways of patients with emphysema than in asthmatics. however, in severe forms of asthma, th1 cells are recruited and have the capacity to secrete tumor necrosis factor (tnf)-a and ifn-g, which may lead to the tissue-damaging immune response one sees in these airways (figure 18 .29) [127, 128] . acute and chronic inflammation the role of acute and chronic inflammatory cells, including eosinophils, mast cells, macrophages, and lymphocytes, in asthma is evident in the abundance of these cells in airways, sputum, and bronchoalveolar samples from patients with this disease. the number of eosinophils in the airways correlates with the severity of asthma and the amount of bronchial hyperresponsiveness. proteins released by these cells including ecp, mcp, and eosinophil-derived neurotoxin cause at least some of the epithelial damage seen in the active form of asthma. neutrophils are prominent in the more acute exacerbations of asthma and are probably recruited to these airways by il-8, a potent neutrophil chemoattractant released by airway epithelial cells [123] . these cells also release proteases, reactive oxygen species (ros), and other proinflammatory mediators that, in addition to the epithelial damage, also contribute to the airway destruction and remodeling that occurs in the more chronic forms of this disease. the susceptibility of the epithelium in asthma to this oxidant injury may be increased due to decreased antioxidants such as superoxide dismutase in these lungs [129] . finally, mast cells are activated to release an abundance of mediators through the binding of ige to fceri, high-affinity receptors on their surface. allergens bind to ige molecules and induce a cross-linking of these molecules, leading to activation of the mast cell and release of a number of mediators, most notably histamine, tryptase, and various leukotrienes, including leukotriene d 4 (ltd 4 ), and interact with the smooth muscle to induce contraction and the acute bronchospastic response [130] . allergen bronchial hyperactivity the cornerstone of asthma is the hyperactive response of the airway smooth muscle. the mechanism by which this occurs combines neural pathways and inflammatory pathways. as stated, the inflammatory component of this response comes predominantly from the mast cells. the major neural pathway involved is the nonadrenergic noncholinergic (nanc) system. although cholinergic pathways are responsible for maintaining the airway smooth muscle tone, it is the nanc system that releases bronchoactive tachykinins (substance p and neurokinin a) that bind to nk2 receptors on the smooth muscle and cause the constriction that characterizes the acute asthmatic response [123] . in addition to these acute mechanisms, the airway also undergoes structural alterations to its formed elements. in the mucosa, these changes include goblet cell hyperplasia and basement membrane thickening. within the submucosa and airway wall, increased deposition of collagen and elastic fibers results in fibrosis and elastosis, and both the smooth muscle cells and the submucosal glands undergo hypertrophy and hyperplasia. these irreversible changes are a consequence of chronic inflammatory insults on the airways through mechanisms that include release of fibrosing mediators such tgfb and mitogenic mediators such as epidermal and fibroblast growth factors (egf, fgf). the exact mechanisms by which this occurs are not clearly defined, but the similarity of these factors with those involved in branching morphogenesis of the developing lung has led to a focus on the effect of inflammation on the interaction of the epithelium with the underlying mesenchymal cells [128] . the term chronic obstructive pulmonary disease (copd) applies to emphysema, chronic bronchitis, and bronchiectasis, those diseases in which airflow limitation is usually progressive, but, unlike asthma, not fully reversible [131] . the prevalence of copd worldwide is estimated at 9%-10% in adults over the age of 40 [132] . though there are different forms of copd with different etiologies, the clinical manifestations of the most common forms of the disease are the same. these include a progressive decline in lung function, usually measured as decreased forced expiratory flow in 1 second (fev1), a chronic cough, and dyspnea. emphysema and chronic bronchitis are the most common diseases of copd and are the result of cigarette smoking. as such, they usually exist together in most smokers. chronic bronchitis is defined clinically as a persistent cough with sputum production for at least 3 months in at least 2 consecutive years without any other identifiable cause. patients with chronic bronchitis typically have copious sputum with a prominent cough, more commonly get infections, and typically experience hypercapnia and severe hypoxemia, giving rise to the clinical moniker blue bloater. emphysema is the destruction and permanent enlargement of the air spaces distal to the terminal bronchioles without obvious fibrosis [133] . these patients have only a slight cough, while the overinflation of the lungs is severe, inspiring the term pink puffers. the pathologic features of copd are best understood if one considers the whole of copd as a spectrum of pathology that consists of emphysematous tissue destruction, airway inflammation, remodeling, and obstruction [134] . the lungs of patients with copd usually contain all of these features, but in varying proportions. the pathologic features of chronic bronchitis include mucosal pathology that consists of epithelial inflammation, injury, and regenerative epithelial changes of squamous and goblet cell metaplasia. in addition, the submucosa shows changes of remodeling with smooth muscle hypertrophy and submucosal gland hyperplasia. these changes are responsible for the copious secretions characteristic of this clinical disease, although studies have reported no consistent relationship between these pathologic features of the large airways and the airflow obstruction [135] . the pathology definition of emphysema is an abnormal, permanent enlargement of the airspaces distal to the terminal bronchioles accompanied by destruction of the alveolar walls without fibrosis [133] . the four major pathologic patterns of emphysema are defined by the location of this destruction. these include centriacinar, panacinar, paraseptal, and irregular emphysema. the first two of these are responsible for the overwhelming majority of the clinical disease. centriacinar emphysema (sometimes referred to as centrilobular) represents 95% of the cases and is a result of destruction of alveoli at the proximal and central areas of the pulmonary acinus, including the respiratory bronchioles ( figure 18 .30). it predominantly affects the upper lobes the remaining two types of emphysema, paraseptal and irregular, are rarely associated with clinical disease. in paraseptal emphysema, the damage is to the distal acinus, the area that abuts the pleura at the margins of the lobules. damage in this area may cause spontaneous pneumothoraces, typically in young, thin men [136] . irregular emphysema is tissue destruction and alveolar enlargement that occurs adjacent to scarring, secondary to the enhanced inflammation in the area. though this is a common finding in a scarred lung, it is of little if any clinical significance to the patient. though the emphysema in these lungs plays the dominant role in causing the obstruction, small airway pathology is also present. respiratory bronchiolitis refers to the inflammatory changes found in the distal airways of smokers. these consist of pigmented macrophages filling the lumen and the peribronchiolar airspaces and mild chronic inflammation and fibrosis around the bronchioles (figure 18 .33). the pigment in these macrophages represents the inhaled particulate matter of the cigarette smoke that has been phagocytized by these cells. the macrophages in turn release proteases, which destroy the elastic fibers in the surrounding area, resulting in the loss of elastic recoil and the obstructive symptoms. in general, copd is a result of inflammation of the large airways that produces the airway remodeling characteristic of chronic bronchitis as well as inflammation of the smaller airways that results in the destruction of the adjacent tissue and consequent emphysema. the predominant inflammatory cells involved in this process are the alveolar macrophages, neutrophils, and lymphocytes. the main theories of the pathogenesis of copd support the interaction of airway inflammation with two main systems in the lung: the protease-antiprotease system and the oxidant-antioxidant system. these systems help to protect the lung from the many irritants that enter the lung via the large pulmonary surface area that interfaces with the environment. in the protease-antiprotease system, proteases are produced by a number of cells, including epithelial cells and inflammatory cells that degrade the underlying lung matrix. the most important proteases in the lung are the neutrophil elastases, part of the serine protease family, and the metalloproteinases (mmps) produced predominantly by macrophages. these proteases can be secreted in response to invasion by environmental irritants, most notably infectious agents such as bacteria. in this setting, their role is to enzymatically degrade the organism. however, proteases can also be secreted by both inflammatory and epithelial cells in a normal lung to repair and maintain the underlying lung matrix proteins [137] . to protect the lung from unwanted destruction by these enzymes, the liver secretes antiproteases that circulate in the bloodstream to the lung and inhibit the action of the proteases. in addition, macrophages that secrete mmps also secrete tissue inhibitors of metalloproteinases (timps). a delicate balance of proteases and antiproteases is needed to maintain the integrity of the lung structure. an imbalance that results in a relative excess of proteases (either by overproduction of proteases or underproduction of their inhibitors) leads to tissue destruction and the formation of emphysema. this imbalance occurs in different ways in the two major types of emphysema: centriacinar and panacinar. in centriacinar emphysema, caused primarily by cigarette smoking, there is an overproduction of proteases primarily due to the stimulatory effect of chemicals within the smoke on the neutrophils and macrophages. though the exact mechanism is not completely understood, most studies support that nicotine from the cigarette smoke acts as a chemoattractant, and ros also contained in the smoke, stimulate an increased release of neutrophil elastases and mmps from activated macrophages, leading to the destruction of the elastin in the alveolar spaces [137] . this inflammatory cell activation may come about through the activation of the transcription factor nfkb that leads to tnfa production [132] . in addition, the elastin peptides themselves may attract additional inflammatory cells to further increase the protease secretion and exacerbate the matrix destruction [137] . unlike centriacinar emphysema, panacinar emphysema is most commonly caused by a genetic deficiency of antiproteases, usually due to alpha-1 anti-trypsin (aat) deficiency, a condition that affects approximately 60,000 people in the united states [138] . aat deficiency is due to a defect in the gene that encodes the protein aat, a glycoprotein produced by hepatocytes and the main inhibitor of neutrophil elastase. the affected gene is the serpina1 gene (formerly known as p1), located on the long arm of chromosome 14 (14q31-32.3). the genetic mutations that occur have been categorized into four groups: base substitution, in-frame deletions, frame-shift mutations, and exon deletions. these mutations usually result in misfolding, polymerization, and retention of the aberrant protein within the hepatocytes, leading to decreased circulating levels. aat deficiency is an autosomal codominant disease with over 100 allelic variants, of which the m alleles (m1-m6) are the most common; these alleles produce normal serum levels of a lessactive protein [139] . individuals who manifest the lung disease are usually homozygous for the alleles z or s (zz and ss phenotype) or heterozygous for the 2 m alleles (mz, or sz phenotype) [139] . an aat concentration in plasma of less than 40% of normal confers a risk for emphysema [140] . in individuals with the zz genotype, the activity of aat is approximately one-fifth of normal [141] . the second system in the lung involved in the pathogenesis of emphysema is the oxidant-antioxidant system. as in the protease system, the lung is protected from oxidative stress in the form of ros by antioxidants produced by cells in the lung. ros in the lung include oxygen ions, free radicals, and peroxides. the major antioxidants in the airways are enzymes including catalase, superoxide dismutase (sod), glutathione peroxidase, glutathione s-transferase, xanthine oxidase, and thioredoxin, as well as nonenzymatic antioxidants including glutathione, ascorbate, urate, and bilirubin [142] . the balance of oxidants and antioxidants in the lung prevents damage by ros. however, cigarette smoke increases the production of ros by neutrophils, eosinophils, macrophages, and epithelial cells [143] . evidence that damage to the lung epithelium and matrix is a direct result of ros includes the presence of exhaled h 2 o 2 and 8-isoprostane, decreased plasma antioxidants, and increased plasma and tissue levels of oxidized proteins, including various lipid peroxidation products. in addition to this direct effect, ros may also induce a proinflammatory response that recruits more inflammatory cells to the lung. in animal models, cigarette smoke induces the expression of proinflammatory cytokines such as il-6, il-8, tnfa, and il-1 from macrophages, epithelial cells, and fibroblasts, perhaps through activation of the transcription factor nfkb [144, 145] (figure 18.34) . finally, there is some evidence that cigarette smoke further disturbs the oxidant-antioxidant balance in the lung by depleting antioxidants such as ascorbate and glutathione [132] . bronchiectasis represents the permanent remodeling and dilatation of the large airways of the lung most commonly due to chronic inflammation and recurrent pneumonia. these infections usually occur because airway secretions and entrapped organisms cannot be effectively cleared. this pathology dictates the clinical features of the disease, which include chronic cough with copious secretions and a history of recurrent pneumonia. the five major causes of bronchiectasis are infection, obstruction, impaired mucociliary defenses, impaired systemic immune defenses, and congenital. these may produce either a localized or diffuse form of the disease. localized bronchiectasis is usually due to obstruction of airways by mass lesions or scars from previous injury or infection. diffuse bronchiectasis can result from defects in systemic immune defenses in which either innate or adaptive immunity may be impaired. diseases due to the former include chronic granulomatous disease (cgd), and diseases due to the latter include agammaglobulinemia/hypogammaglobulinemia and severe combined immune deficiencies. defects in the mucociliary defense mechanism that is responsible for physically clearing organisms from the lung may also cause diffuse bronchiectasis. these include ciliary dyskinesias that result in cilia with aberrant ultrastructure and cystic fibrosis (cf). congenital forms of bronchiectasis are rare but do exist. the most common include mounier-kuhn's syndrome and williams-campbell syndrome, the former causing enlargement of the trachea and major bronchi due to loss of bronchial cartilage, and the latter causing diffuse bronchiectasis of the major airways probably due to a genetic defect in the connective tissue [146, 147] . the pathology of bronchiectasis is most dramatically seen at the gross level. one can see dilated airways containing copious amounts of infected secretions and mucous plugs localized either to a segment of the lung or diffusely involving the entire lung as in cystic fibrosis (figure 18.35) . microscopic features include chronic inflammatory changes similar to those of chronic bronchitis but with ulceration of the mucosa and submucosa leading to destruction of the smooth muscle, and elastic in the airway wall and the characteristic dilatation and fibrosis. these enlarged airways contain mucous plugs comprising mucin and abundant degenerating inflammatory cells, a result of infections that establish themselves in these airways following the loss of the mucociliary defense mechanism. bacteria may be found in these plugs, most notably p. aeruginosa. the pathogenetic mechanism of bronchiectasis is complex and depends on the underlying etiology. in general, the initial damage to the bronchial epithelium is due to aberrant mucin (cystic fibrosis), dysfunctional cilia (ciliary dyskinesias), and ineffective immune surveillance (defects in innate and antibody-mediated immunity), leading to a cycle of tissue injury, repair, and remodeling that ultimately destroys the normal airway. the initial event in this cycle usually involves dysfunction of the mucociliary mechanism that inhibits the expulsion from the lungs of organisms and other foreign substances that invade the airways. this may be due to defects in the cilia or the mucin. ciliary defects are found in primary ciliary dyskinesia, a genetically heterogeneous disorder, usually inherited as an autosomal recessive trait that produces immotile cilia with clinical manifestations in the lungs, sinuses, middle ear, male fertility, and organ lateralization [148] . over 250 proteins make up the axoneme of the cilia, but mutations in 2 genes, dnai1 and dnah5, which encode for proteins in the outer dynein arms, most frequently cause this disorder [149] . in cf the main defect affects the mucin. in patients with this autosomal recessive condition, there is a low volume of airway surface liquid (asl) causing sticky mucin that inhibits normal ciliary motion and effective mucociliary clearance of organisms. this is due to a defect in the cystic fibrosis transmembrane conductance regulator (cftr) gene, located on chromosome 7 that encodes a camp-activated channel which regulates the flow of chloride ions in and out of cells and intracellular vacuoles, helping to maintain the osmolality of the mucin. this protein is present predominantly on the apical membrane of the airway epithelial cells, though it is also involved in considerable subapical, intracellular trafficking and recycling during the course of its maturation within these cells. this genetic disease manifests in multiple other organs that depend on chloride ion transport to maintain normal secretions, including the pancreas, intestine, liver, reproductive organs, and sweat glands [150] . the genetic mutations in cf influence the cftr trafficking in the distal compartments of the protein secretary pathway, and various genetic mutations produce different clinical phenotypes of the disease. over 1600 mutations of the cftr gene have been found. however, only four of these mutations occur at a frequency of greater than 1%. these mutations are grouped into five classes according to their functional deficit: group i, cftr is not synthesized; group ii, cftr is inadequately processed; group iii, cftr is not regulated; group iv, cftr shows abnormal conductance; group v, cftr has partially defective production or processing. approximately 70% of cf patients are in group ii and have the same mutation, f508d cftr, a deletion of phenylalanine at codon 508 [154] . in these patients, most of the cftr protein is misfolded and undergoes premature degradation within the endoplasmic reticulum, though a small amount of the cftr protein is present on the apical membrane and does function normally. cf patients may have a combination of genetic mutations from any of the five groups. however, those patients with the most severe disease involving both the lungs and pancreas usually carry at least two mutations from group i, ii, or iii [151] . systemic immune deficiencies cause bronchiectasis through the establishment of persistent infection and inflammation. there are four major categories of immune deficiencies. the first category consists of a number of genetic diseases that cause either agammaglobulinemia or hypogammaglobulinemia. these include xlinked agammaglobulinemia (xla) and common variable immunodeficiency (cvi). xla is caused by a mutation of the bruton's tyrosine kinase (btk) gene that results in the virtual absence of all immunoglobulin isotypes and of circulating b lymphocytes. in cvi there is a marked reduction in igg and iga and/or igm, associated with defective antibody response to protein and polysaccharide antigens. as expected, both of these diseases increase susceptibility to infections from encapsulated bacteria. the second category of immune deficiency is hyper-ige syndrome, a disease with markedly elevated serum ige levels that is characterized by recurrent staphylococcal infections. the third category is chronic granulomatous disease (cgd), a genetically heterogeneous group of disorders that have a defective phagocytic respiratory burst and superoxide production, inhibiting the ability to kill staphylococcus spp. and fungi such as aspergillus spp. finally, severe combined immune deficiency (scid) comprises a group of disorders with abnormal t-cell development and b-cell and/or natural killer cell maturation and function, predisposing these patients to pneumocystis jiroveci and viral infections [152] . after the initial insult, the subsequent steps in the development of bronchiectasis include destruction of the epithelial cells and bronchial wall connective tissue matrix by the proteases and ros secreted by the neutrophils. this proinflammatory milieu is produced by multiple factors. first, infections can persist in these lungs due to defective host immune systems and mechanisms certain organisms have developed to evade these immune defenses. for example, pseudomonas aeruginosa, changes from a nonmucoid to a mucoid variant and also releases virulence factors to protect against phagocytosis [153] . second, in the case of cystic fibrosis, neutrophils are directly recruited by proinflammatory cytokines, such as interleukin-8 (il-8), released from the bronchial epithelial cells as a result of the defective cgft protein [154] . finally, the necrotic cellular debris and other breakdown products act as chemoattractants that recruit more inflammatory cells to the airway wall, further exacerbating the damage. the final phase of the repair and remodeling begins when macrophages invade and recruit fibroblasts that secrete collagen, leading to the fibrosis seen in the pathology. however, in the absence of effective airway clearance mechanisms, these ectatic airways remain a reservoir of infection that continues the cycle of inflammation and tissue destruction. the idiopathic interstitial pneumonias (iips) comprise a group of diffuse infiltrative pulmonary diseases with a similar clinical presentation characterized by dyspnea, restrictive physiology, and bilateral interstitial infiltrates on chest radiography [155] . pathologically, these diseases have characteristic patterns of tissue injury with chronic inflammation and varying amounts of fibrosis. by recognizing these patterns, a pathologist can classify each of these entities and predict prognosis. however, the pathologist cannot establish the etiology, since these pathologic patterns can be seen in multiple clinical settings. the pathologic classification of these diseases, originally defined by liebow and carrington in 1969 [156] , has undergone important revisions over the past 35 years with the latest revision by the american thoracic society/european respiratory society in 2003 [157] . the best known and most prevalent entity of the iips is idiopathic pulmonary fibrosis (ipf), which is known pathologically as usual interstitial pneumonia (uip). uip is a histologic pattern characterized by patchy areas of chronic lymphocytic inflammation with organizing and collagenous type fibrosis. these patients usually present with gradually increasing shortness of breath and a nonproductive cough after having had symptoms for many months or even years. imaging studies usually reveal bilateral, basilar disease with a reticular pattern [155] . therapy begins with corticosteroids, advancing to more cytotoxic drugs such as methotrexate and cytoxan, but most current therapies are not effective in stopping the progression of the disease. the current estimates are that 20/100,000 males and 13/100,000 females have the disease, most of whom progress to respiratory failure and death within 5 years [158] . the pathology is characterized by a leading edge of chronic inflammation with fibroblastic foci that begin in different areas of the lung at different times. these processes produce a variegated pattern of fibrosis, usually referred to as a temporally heterogenous pattern of injury [159] . because it occurs predominantly in the periphery of the lung involving the subpleura and interlobular septae, the gross picture is one of more advanced peripheral and basilar disease (figure 18.36) . the progression from inflammation to fibrosis includes interstitial widening, epithelial injury and sloughing, fibroblastic infiltration, and organizing fibrosis within the characteristic fibroblastic foci. deposition of collagen by fibroblasts occurs in the latter stages of repair. the presence of the abundant collagen produces stiff lungs that are unable to clear the airway secretions, leading to recurrent inflammation of the bronchiolar epithelium with eventual fibrosis and breakdown of the airway structure. this remodeling produces mucousfilled ectatic spaces giving rise to the gross picture of honeycomb spaces, which is seen in the advanced pathology ( figure 18 .36) [160] . theories of the pathogenesis of ipf have evolved over the past decade. early theories favored a primary inflammatory process, while current theories favor the concept that the fibrosis of the lung proceeds independently of inflammatory events and develops from aberrant epithelial and epithelial-mesenchymal responses to injury to the alveolar epithelial cells (aecs) [161] . the aecs consist of two populations: the type 1 pneumocytes and the type 2 pneumocytes. in normal lungs, type 1 pneumocytes line 95% of the alveolar wall, and type 2 pneumocytes line the remaining 5%. however, in lung injury, the type 1 cells, which are exquisitely fragile, undergo cell death, and the type 2 pneumocytes serve as progenitor cells to regenerate the alveolar epithelium [162] . though some studies have suggested that repopulation of the type 2 cells depends on circulating stem cells, this concept remains to be fully proven. according to current concepts, the injury and/or apoptosis of the aecs initiates a cascade of cellular events that produce the scarring in these lungs. studies of aecs in lungs from patients with ipf have shown ultrastructural evidence of cell injury and apoptosis as well as expression of proapoptotic proteins. further, inhibition of this apoptosis by blocking a variety of proapoptotic mechanisms such the fas-fas ligand pathway, angiotensin, and tnfa production, and caspase activation can stop the progression of this fibrosis [163] . the result of the aec injury is the migration, proliferation, and activation of the fibroblasts and myofibroblasts that leads to the formation of the characteristic fibroblastic foci of the uip pathology and the deposition and accumulation of collagen and elastic fibers in the alveoli (figure 18.37 ). this unique pathology may be a result of the increased production of profibrotic factors such as transforming growth factor-a (tgfa) and tgfb, fibroblastic growth factor-2, insulin-like growth factor-1, and platelet-derived growth factor. an alternative pathway might involve overproduction of inhibitors of matrix degradation such as timps (tissue inhibitors of matrix production) [164] . in support of the former mechanism, fibroblasts isolated from the lungs of ipf patients exhibit a profibrotic secretory phenotype [165] . multiple factors, such as environmental particulates, drug or chemical exposures, and viruses may trigger the initial epithelial injury, but genetic factors also play a role. approximately 2%-20% of patients with ipf have a family history of the disease with an inheritance pattern of autosomal dominance with variable penetrance. two genetic mutations have been implicated in this familial form of ipf. one large kindred has been reported with a mutation in the gene encoding surfactant protein c, and six probands have been a b reported with heterozygous mutations in genes htert or htr, encoding telomerase reverse transcriptase and telomerase rna, respectively, resulting in mutant telomerase and short telomeres [166] . adult respiratory distress syndrome (ards) represents a constellation of clinical, radiologic, and physiologic features in patients with acute respiratory failure that can occur after a variety of insults. ards is defined by clinical criteria that include a rapid onset of severe hypoxemia that is refractory to oxygen therapy, the presence of abnormal chest radiographs with evidence of bilateral alveolar filling and collapse, increased pulmonary artery occlusion pressure, and a resistance to improved oxygenation regardless of mechanical ventilation therapy [167] . treatment of ards includes eliminating the underlying cause, protective ventilation strategies that improve oxygenation, and supportive treatment that may include administration of corticosteroids. the pathology of ards is diffuse alveolar damage (dad), whose histologic picture is one of inflammation and fibrosis that diffusely involves all of the structures of the alveolus and is similar throughout the affected areas of the lung [168] . dad is divided into three major phases that follow each other chronologically after the original insult. these are exudative, proliferative, and fibrotic dad. the initial injury primarily involves the epithelium of the alveolar wall and the endothelium in the capillary, causing the destruction and sloughing of the type 1 pneumocytes into the alveolar space and a breakdown of the tight junctions of the endothelium. in combination, these two events result in the loss of the epithelial-endothelial barrier of the alveolus and leakage of plasma from the capillary into the alveolar space. this flooding of the airspace with fluid markedly decreases oxygen exchange and causes the hypoxia that these patients experience. in addition, acute inflammatory changes of the endothelium also cause thrombi to form in vessels, adding to a decreased amount of blood circulating through the lung and further compromising gas exchange. as air is brought into the alveoli, the positive pressure within the alveolar space forces the plasma against the alveolar wall, producing a membranous morphology referred to as hyalin membranes characteristic of the first phase of dad, referred to as exudative dad (figure 18.38) . this initial injury is followed by a sequence of events that represent the lung's efforts to repair itself. first, type 2 pneumocytes undergo hyperplasia and re-epithelialize the alveolar wall after the loss of the type 1 cells. this re-establishes the epithelial barrier and, because these cells secrete surfactant, results in increased surfactant production, which lowers the surface tension of the alveolus and inhibits its collapse. because of the increased numbers of type 2 pneumocytes, this is known as the proliferative phase of dad (figure 18.38) . in the final phase of dad, fibrotic dad, fibroblasts migrate in from the adjacent interstitium to the alveolar space and produce organizing and irreversible fibrosis within both the alveolar space and the interstitium. in addition to this mechanism, fibrosis may also occur in those areas where alveolar walls collapse when surfactant is decreased during the initial insult. the histopathologic picture during this fibrotic phase is one of thickened alveolar septa, intra-alveolar granulation tissue, microcyst formation, and areas of irregular alveolar scarring. in rare cases, these microcysts progress to large cysts, an adult equivalent of bronchopulmonary dysplasia. the cellular events of dad are complex and incompletely understood. in general, the disease can be broken down into two phases. in the first, a large influx of neutrophils and plasma enter the alveolar space. the role the neutrophils play in the initial cellular injury and death is unclear, but it is known that they are necessary for this injury to occur. in addition, clinical studies have shown that within the peripheral blood and bronchoalveolar lavages (bal) of these patients, neutrophils are present along with a myriad of proinflammatory cytokines, such as il-8, il-1, and tgfa, all of which are capable of recruiting them to the lung. also present in these fluids are mediators that recruit fibroblasts such as tgfb. all of these mediators are probably the result of upregulation of nfkb, a proinflammatory transcription factor, in alveolar macrophages. the adherence of neutrophils to the capillary endothelium in the lung occurs through adhesion molecules such as selectin, integrin, and immunoglobulins. neutrophil adherence and subsequent transmigration through the endothelium of the lung capillaries may cause some endothelial damage. however, most speculate that ros and reactive nitrogen chapter 18 molecular basis of pulmonary disease species (rns) secreted by the neutrophils modulate the majority of this injury [169] . this is supported by the finding that patients with ards have products of oxidative damage such as hydrogen peroxide (h 2 o 2 ) in the exhaled breath and myeloperoxidase and oxidized aat in the bal. the cell injury and death of the type 1 pneumocytes most likely occurs via two mechanisms: lipopolysaccharide (lps)-induced caspase-dependent apoptosis and hyperoxia-induced cell death through apoptosis and nonapoptotic mechanisms [170] . in the former, lps, an immunogenic component of the outer membrane of gram-negative bacteria, may trigger innate immune and inflammatory responses via toll-like receptors that bind fas-associated death domain protein and caspase-9, leading to epithelial cell death. in hyperoxia-induced cell death, hyperoxia may induce the expression of angiopoietin 2 (ang2) in lung epithelial cells. ang2 is an angiogenic growth factor that can activate caspase pathways and lead to apoptotic cell death [170] . cell death in ards is not limited to these mechanisms, and further study of many of pathways by which this can occur is needed. lymphangioleiomyomatosis (lam) is a rare systemic disease of women, usually in their reproductive years (average age of 35 years), that is characterized by a proliferation of abnormal smooth muscle cells giving rise to cysts in the lungs, abnormalities in the lymphatics, and abdominal tumors, most notably in the kidneys. in addition to sporadic cases (denoted as s-lam), lam also affects 30% of women with tuberous sclerosis (denoted as tsc-lam), a genetic disorder with variable penetrance associated with seizures, brain tumors, and cognitive impairment [171, 172] . global estimates indicate that tsc-lam may be as much as 5-fold to 10-fold more prevalent than s-lam, though at least some suggest that tsc-lam may have a milder clinical course than s-lam [172] . clinically, lam patients usually present with increasing shortness of breath on exertion, obstructive symptoms, spontaneous pneumothoraces, and chylous effusions or with abdominal masses consisting of either angiomyolipomas and/or lymphangiomyomas. chest imaging studies characteristically reveal hyperinflation with flattened diaphragms and thin-walled cystic changes. mortality at 10 years from the onset of symptoms is 10%-20% [173] . lam appears as small, thin-walled cysts (0.5-5.0 cm) randomly throughout both lungs [174] (figure 18.39) . microscopically, lam lungs contain a diffuse infiltration of smooth muscle cells, predominantly around lymphatics, veins, and venules. most notably, one finds smooth muscle cells in the subpleural with hemosiderin-laden macrophages in the adjacent field, and the macrophages are also seen on bronchoalveolar lavage specimens from these patients. the hemosiderin pigment in these lungs is thought to be secondary to microhemorrhages from the obstruction of the veins ( figure 18 .40) [175] . the smooth muscle cells in lam react to antibodies to hmb-45, a premelanosomal protein. other melanosome-like structures are also found in lam cells, suggesting that these cells have characteristics of both smooth muscle and melanosomes [176] . the lesional cells in lam are smooth muscle-like with both spindled and epithelioid morphology [177] . these cells are the same in both s-lam and tsc-lam part iv molecular pathology of human disease and are a clonal population although they lack other features of malignancy [178] . molecular studies reveal that the abnormal lam cell proliferation is caused by mutations in one of two genes linked to tuberous sclerosis: tuberous sclerosis complex 1 or 2 (tsc1 or tsc2). these two genes control cell growth and differentiation through the akt/mammalian target of rapamycin (mtor) signaling pathway [172] . in this pathway, a growth factor receptor (such as insulin or pdgf receptors) becomes phosphorylated when an appropriate ligand binds, resulting in activation of downstream effectors and ultimately akt. the gene products of tsc1 and tsc2 are hamartin and tuberin, which act as dimers to maintain rheb (a member of the ras family) in a gdp-loaded state via statins, acting as a break to the akt/mtor pathway, thereby retarding protein synthesis and cell growth. in lam cells, loss-of-function mutations in these two genes remove this inhibition, leading to enhanced rheb activation, mtor activation (with raptor), and subsequent phosphorylation of downstream molecules which result in uncontrolled cell growth, angiogenesis, and damage to the lung tissue ( figure 18 .41) [179] . the abnormal proliferation of lam cells is thought to damage the lung through overproduction of matrix metalloproteinases (mmps), which degrade the connective tissue of the lung architecture, destroy the alveolar integrity, and result in cyst formation with air trapping [179] . these destructive capabilities of the lam cells are enhanced by their secretion of the angiogenic factor vegf-c, which is thought to cause the proliferation of lymphatic channels throughout the lung [179] . sarcoidosis is a multisystemic disease that involves the lung in over 90% of the cases [180] . it is most common in the 20-40-year age group and among females. in the united states, african americans are more commonly affected than caucasians [181] . the clinical picture of sarcoidosis is variable, but most patients present with systemic symptoms including fatigue, weight loss, and fever. the most common finding on chest imaging studies is bilateral hilar lymph node enlargement and reticular, reticulonodular, and focal alveolar opacities within the lung parenchyma [182] . pulmonary sarcoidosis is characterized by granulomas which consist of activated histiocytes, called epithelioid histiocytes that form nodules ranging in size from 15-20 microns (figure 18 .42) [183] . unlike infectious granulomas that usually contain areas of central necrosis, the granulomas in pulmonary sarcoidosis are predominantly non-necrotizing [184] . also, the granulomas in sarcoidosis follow a distribution along the lymphatics, which includes the area in the subpleural, along the interlobular septae and around the bronchovascular area containing the bronchiole and branch of the pulmonary artery (figure 18 .42). the granulomas occur much more commonly in the upper lobes, leading to the predominant upper lobe fibrosis and bronchiectasis that can be seen in longstanding sarcoidosis [185] . despite over 50 years of research on sarcoidosis, the etiology remains unknown. most agree that the disease is probably a result of environmental triggers acting on a genetically susceptible host [186, 187] . a genetic basis of sarcoidosis has been suggested by studies that demonstrate familial clustering and racial variation [188, 189] . further, complex inheritance patterns for the disease suggest that more than one gene may be involved [190] . several genes of the major histocompatibility complex (mhc) region of the genome have been implicated. most are clustered on the short arm of chromosome 6 that encompasses the human leukocyte antigen (hla) domain. the hla class i mhc molecules associated with sarcoidosis are the hla-b7 and hla-b8 class i alleles [191, 192] . hla class ii molecules implicated in susceptibility include the hla-dr alleles [193, 194] . genes other than mhc genes thought to regulate the susceptibility to sarcoidosis include those for chemokines such as macrophage inflammatory protein-1a and rantes (ccr5 and ccr3) [195, 196] . environmental factors that have been implicated are those that are aerosolized. therefore, these environmental agents have a mode of entry into the lungs and can cause granulomas in the lung, similar to sarcoidosis. these factors can be divided into two major categories, which include infectious and noninfectious agents. the mycobacteria have been the most extensively studied organisms. however, their role in this disease remains controversial due to the difficulty in identifying them by either culture or histochemical stains in sarcoid tissue. recently, molecular techniques have been able to demonstrate mycobacterial nucleic acid in sarcoid tissue [197, 198] . however, even studies using this technology have not produced consistent results, and the role of these organisms in the disease requires further study. the immune response in sarcoidosis has two major features: (i) the initial event leading to granuloma formation and (ii) the progression of this granulomatous response to either resolution or fibrosis [199] . the formation of the granulomas, triggered by activation of tcells and antigen-presenting dendritic histiocytes, results in a release of proinflammatory cytokines and chemokines, and recruitment, activation, and proliferation of mononuclear cells, predominantly t-cells. these activated t-cells are predominantly cd4-expressing t-helper (th) cells, which release ifn-g and il-2. alveolar macrophages at the site release tnfa, il-12, il-16, and other growth factors. this results in the granuloma formation and alveolitis, the characteristic morphologic features of the disease [200] . the second phase of this immunologic response that leads to either resolution of the disease or persistence of the granulomas and fibrosis is less well characterized. ongoing granuloma formation and inflammation may be a result of the persistent presence of antigens, the excessive synthesis of chemotactic factors, or the part iv molecular pathology of human disease persistence of the mononuclear cells within the granulomas. importantly, the role of the t-cells in these granulomas is to secrete cytokines that attract, stimulate, and ultimately deactivate the fibroblasts that are responsible for the fibrosis that is seen in the chronic disease. the balance between the profibrotic mediators such as tgfb, insulin-like growth factor-i, platelet-derived growth factor (pdgf), and the antifibrotic mediators, such as ifn-g, probably dictates the natural history of sarcoidosis in the lung [201] . genes involved in macrophage-derived cytokines, chemokines, and mediators of fibrosis are all possible candidates for the underlying genetic cause of this complicated disease. pulmonary alveolar proteinosis (pap) is a rare disease of the lungs characterized by accumulation of surfactant in the alveolar spaces. the names alveolar proteinosis, lipoproteinosis, or perhaps most accurately phospholipoproteinosis, apply equally to this entity. pap takes three forms clinically: (i) congenital (2%), (ii) secondary (5%-10%), and (iii) idiopathic or primary (88%-93%) [202] [203] [204] . pap arises in previously healthy adults with the median age at diagnosis of approximately 40 years and a male-to-female ratio of 2.7:1. the clinical presentation is variable and usually includes an insidious onset of slowly progressive dyspnea, a dry cough, and other symptoms of respiratory distress, including fatigue and clubbing. however, almost one-third of patients are asymptomatic and are found clinically by abnormal chest x-rays [205, 206] . the secondary form of pap can be found in patients with environmental exposures, including fine silica, aluminum, titanium dioxide, and kaolin dust [206] . also, secondary pap may be found in patients with malignancies, most commonly hematologic malignancies such as myelogenous leukemia [207, 208] . chest imaging studies in both the idiopathic and secondary forms most commonly show fine, diffuse, feathery nodular infiltrates, centered in the hilar areas, sparing the peripheral regions [206] . on chest computerized tomographs, the infiltrates may have a geometric-type shape, sometimes referred to as crazy paving [209] . the most prominent microscopic feature of both idiopathic and secondary pap is the filling of the alveoli with finely granular period acid-schiff-positive diastaseresistant (pasd) acellular material (figure 18 chapter 18 molecular basis of pulmonary disease material consists of phospholipids (90%); surfactant proteins a, b, c, and d (10%); and carbohydrate (<1%) [210] . alveolar macrophages (ams) with prominent foamy cytoplasm are commonly seen, while alveolar septa are remarkably normal in appearance. in some alveolar spaces there are denser, more solid clumps of pas-d-positive material. definitive pathologic differences between the idiopathic and secondary forms of pap have not been well documented [211, 212] . the etiologies of the two adult forms of pap have been well studied with the most known about the idiopathic variant. theories of the pathogenesis of this form have focused on the abnormal accumulation of the surfactant-like material within the alveolar spaces. since the regulation of surfactant levels in the alveoli depends on appropriate synthesis, recycling, and catabolism, the two opposing hypotheses have included overproduction versus decreased degradation of this material. in normal hosts, surfactant is essential to maintaining the low surface tension needed for proper alveolar inflation and gas exchange. the critical role of maintaining the proper composition and amount of surfactant in the alveoli is performed by two cell types: type 2 pneumocytes and alveolar macrophages [213] . the type 2 pneumocytes synthesize surfactant in the endoplasmic reticulum and golgi, and store it as lamellar bodies [213] , which are then delivered to and fuse with the apical plasma membrane, secreting the surfactant into the airways [214] . catabolism of surfactant is carried out by type 2 pneumocytes and ams. in pap, most evidence suggests that the clearance of surfactant by the am is decreased [203, 215] . the first clue as to the underlying mechanism for this defect in am function came in 1994 when studies revealed that knockout mice deficient in granulocytemacrophage colony-stimulating factor (gm-csf) develop lung lesions similar to those in patients with pap [216] . this rather serendipitous finding prompted explorations centered on the am and the effect diminished gm-csf might have on its cellular functions. subsequent studies from humans with pap revealed an autoimmune mechanism by which a circulating neutralizing antibody to gm-csf blocked its binding to the gm-csf receptor, depressing the effect of gm-csf on the ams [217] [218] [219] . neutralizing antibodies to gm-csf have most often been identified in the idiopathic variant of pap. however, recently these antibodies have also been reported in patients with secondary pap [220] . genes that control many functions in the am are controlled by signaling pathways initiated by gm-csf binding to the am. one pathway is mediated through a transcription factor pu.1 that controls genes involved in surfactant degradation, among other bactericidal functions [221, 222] . another transcription factor, peroxisome-proliferator-activated receptor g (pparg), is also part of a pathway activated by gm-csf. pparg controls the expression of genes involved in intracellular lipid metabolism. ams from patients with pap have a deficiency of this transcription factor, which is correctable by gm-csf therapy [223] . overall, the lack of gm-csf-initiated signaling in ams from patients with pap leads to inhibition of both pparg and pu.1 pathways. this results in decreased surfactant catabolism, intracellular lipid metabolism, and the accumulation of surfactant in the alveoli (figure 18 .44). pulmonary hypertension consists of a group of distinct diseases whose pathology is characterized by abnormal destruction, repair, remodeling, and proliferation of all compartments of the pulmonary vascular tree, including arteries, arterioles, capillaries, and veins. the classification of these diseases has undergone a number of revisions. the most recent revision (in 2003) groups these diseases based on both their pathologic and clinical characteristics [224] . there are five major disease categories in the current classification system: (i) pulmonary arterial hypertension (pah); (ii) pulmonary hypertension with left heart disease; (iii) pulmonary hypertension associated with lung disease and/or hypoxemia; (iv) pulmonary hypertension due to chronic thrombotic and/or embolic disease; and (v) miscellaneous causes, including sarcoidosis, histiocytosis x, and lymphangioleiomyomatosis. the clinical course of most patients with pulmonary hypertension begins with exertional dyspnea, and progresses through chest pain, syncope, increased mean pulmonary artery pressures and, eventually, right heart failure. the rate of this clinical progression varies among patients, from a few months to many years [225] . treatment of these diseases focuses on blocking the mediators involved in the pathogenesis of the diseases. however, current therapies rarely prevent progression of the disease, and lung transplantation provides the only hope for long-term survival. the major group of this classification, pah, can be subdivided into familial pah, idiopathic pah, pah associated with other conditions (such as connective tissue diseases, hiv, congenital heart disease), and pah secondary to drugs and toxins (such as anorexigens, cocaine, and amphetamines). in these diseases, the primary pathology is localized predominantly in the small pulmonary arteries and arterioles. however, two other diseases in this group, pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis, involve predominantly other components of the pulmonary vasculature, the veins, and the capillaries, respectively. the pathologic changes seen in the pulmonary vessels of these patients primarily reflect injury to and repair of the endothelium. early pathologic changes include medial hypertrophy and intimal fibrosis that narrows and obliterates the vessel lumen. these are followed by remodeling and revascularization, producing a proliferation of abnormal endothelial-lined spaces. these structures are known as plexogenic lesions and are the pathognomonic feature of pah (figure 18.45) . in the most severe pathologic lesions, these abnormal vascular structures become dilated or angiomatoid-like and may develop features of a necrotizing vasculitis with transmural inflammation and fibrinoid necrosis. though the exact pathogenetic mechanism of pah remains unknown, research over the past 10 years has begun to offer some clues. the familial form of pah, with a 2:1 female-to-male prevalence, has an autosomal dominance inheritance pattern with low penetrance. the genetic basis for this has been found to be germline mutations in the gene encoding the bone morphogenetic protein receptor type 2 (bmpr2). these mutations account for approximately 60%-70% of familial pah and 10%-25% of patients with sporadic pah [233] . approximately 140 bmpr2 mutations have been identified in familial pah, each resulting in a loss of receptor function, either through alteration in transcription of the gene through missense, nonsense, or frameshift alterations in the codon or by rna spicing mistakes [226] . the mechanism by which a single mutation to the bmpr2 gene induces vascular smooth muscle proliferation and decreased apoptosis that is not completely understood, but it most likely involves defects in the bmpr2 signaling pathway. bmpr2 is a receptor for a family cytokines (bmps) that are members of the tgfb superfamily of proteins that play a role in the growth and regulation of many cells, including those of the pulmonary vasculature. in the vascular smooth muscle cells of the lung, tgfb signaling causes a proliferation of smooth muscle in pulmonary arterioles, while bmpr2 signaling causes an inhibition of the proliferation of these cells, favoring an apoptotic environment. the bmpr2 signaling occurs through an activation of a receptor complex (bmpr1 and bmpr2) that leads to phosphorylation and activation of a number cytoplasmic mediators, most notably the smad proteins (mothers against decapentaplegic). these smad proteins, especially the smad 1, smad 5, and smad 8 complex with smad 4, translocate to the nucleus where they target gene transcription that induces an antiproliferative effect in the cell. in familial pah, the bmrpr2 gene mutation may lead to insufficient protein product and subsequent decreased protein function, in this case decreased bmpr2 receptor function, decreased smad protein activation, and decreased antiproliferative effects in the vascular smooth muscle cells. the imbalance between the proproliferative effects of the tgfbs and the antiproliferative effects of the bmps results in the formation of the vascular lesions of pah (figure 18 .46) [227, 228] . despite these advances, questions regarding the pathogenesis of pah remain. most notably, why do only 10%-20% of patients with the mutation develop clinical disease? some speculate that genes confer susceptibility but a second hit is required to develop the clinical disease, such as modifier genes or environmental triggers, perhaps drugs or viral infections [227, 229] pulmonary vasculitides present as diffuse pulmonary hemorrhage and are usually caused by one of three major pulmonary vasculitis syndromes: wegener's granulomatosis, churg-strauss syndrome, and microscopic polyangiitis. all three diseases have similar clinical presentations and considerable overlap in their pathologic features as small vessel systemic vasculitides that affect the lung as well as other organs, most notably the kidney. wegener's granulomatosis (wg) is an unusual disease that affects the upper and lower respiratory tract and the kidneys. it usually presents between 40 and 60 years of age and is slightly more common in men than women. the clinical presentation depends on the affected organ, but when the lung is involved, hemoptysis is the major presenting symptom. chest imaging studies may show a variety of patterns, most commonly bilateral ground glass opacities with masses, usually in the lower lobes that may cavitate. immunologic testing of peripheral blood or end organ tissue can be helpful in revealing characteristic immunofluorescent staining patterns for antineutrophilic cytoplasmic antibody (anca), an antibody that targets two substances: proteinase 3 (pr3) and myeloperoxidase (mpo). when present in either the blood or the tissue, the pattern of immunofluorescent staining can be cytoplasmic (canca) or perinuclear (panca). the former pattern is more commonly seen in wegener's granulomatosis, and the latter is more commonly seen in microscopic polyangiitis and churg-strauss syndrome (css). css is a systemic disorder defined by the presence of asthma, peripheral blood eosinophilia, and systemic vasculitis. similar to wg, it usually presents between 40 and 60 years of age, and a clinical diagnosis requires a history of asthma, a peripheral blood eosinophilia, neuropathy, an abnormal chest imaging study, and sinusitis. other organs involved include the heart, the central nervous system, kidneys (though less commonly than wg), gastrointestinal tract, and skin. chest imaging usually shows patchy, multifocal infiltrates; masses and cavitation are rare. laboratory tests reveal positive panca tests in 70% of patients. microscopic polyangiitis (mpa) is similar to both wg and css in that it is a systemic vasculitis that involves the lung and usually presents in the fourth or fifth decade of life. the clinical onset is usually sudden with fever, weight loss, myalgias, and arthralgias. the kidney is the main organ involved, and mpa is the most common cause of pulmonary-renal syndrome. lung involvement occurs in approximately 50% of the patients, and skin and upper respiratory tract are other common sites. similar to wg and css, anca testing is helpful with positive panca in 80% of patients. chest imaging usually shows bilateral infiltrates without masses, similar to css. treatment for all three diseases is immunosuppression with glucocorticoids or cyclophosphamide, and all three usually respond well, although wg has a greater relapse rate after treatment than either css or mpa [230] . the pathology of wg, css, and mpa have overlapping features of an acute and chronic vasculitis that involves medium-and small-sized vessels in the lung. the inflammatory cell infiltrate that destroys the blood vessels is both lymphocytic and neutrophilic, and areas of fibrinoid necrosis are seen. however, in wg, there are characteristic areas of microabscesses that lead to masses of geographic necrosis with basophilia. scattered multinucleated giant cells are present, but no wellformed granulomas are seen. this helps to distinguish it from other vasculitides and infection (figure 18.47) . similarly, the pathology of css has distinguishing features, with the early pathology characterized by an eosinophilic pneumonia with areas of loosely formed granulomas with central necrosis containing degenerating eosinophils (figure 18 .48). the infiltrate is predominantly eosinophils, but neutrophils, lymphocytes, and plasma cells are also present. capillaritis can be seen in wg, csg, and mpa, and all three have hemosiderin deposition present, both within alveolar macrophages and deposited in the connective tissue of the interstitium and the vessel walls. the pathogenesis of these three pulmonary hemorrhage syndromes is similar to the mechanisms of these diseases in the kidney. in general, these diseases in the lung and the kidney represent immune-mediated these lesions are thought to be the early form of the larger areas of geographic necrosis that produces the mass-like nodules found in these lungs. chapter 18 molecular basis of pulmonary disease necrotizing vasculitides that have few or no immune deposits in the vessels but exhibit the presence of anca autoantibodies to myeloperoxidase (mpo) and proteinase 3 (pr3), the components of primary granules of neutrophils. mpa and css are primarily diseases of mpo antibodies, and wg is primarily a disease of pr3 antibodies. the mechanism by which the ancas are induced is not known but may be part of an autoimmune response to environmental exposures early in life. these autoantibodies then inflict damage on the vessels through a mechanism that is not yet completely understood. one theory suggests that circulating ancas bind to pr3 and mpo on the surface of neutrophils and initiate a respiratory burst, degranulation, and apoptosis. ros and proteases are released and inflict endothelial and tissue damage on the adjacent vessel. the anca binding may also induce the release of proinflammatory cytokines and chemokines such as il-1 and tnfa that further contribute to the vascular inflammation. the second theory postulates that circulating immune complexes of excess anca antigen (mpo or pr3) and anca autoantibodies attach to the vascular endothelium and activate complement that results in the chemotaxis and adhesion of inflammatory cells, causing these cells to undergo a respiratory burst and, as in the first theory, release of ros and proteases that cause the vascular endothelial damage. in both theories, it is important to remember that mpo and pr3 are also present in monocytes and that anca autoantibodies may be involved with monocytes in similar ways to release inflammatory mediators [231] . infectious diseases of the lung are a common cause of pulmonary disease given the constant exposure of the lungs to the environment. various organisms are capable of causing these infections, including common viruses and bacteria, as well as more uncommon fungi, parasites, and protozoa. the diagnosis of the specific etiologic agent can be challenging given that most have similar clinical features and many are difficult to identify in the lung tissue. this brief overview of the defense mechanisms the lung uses to protect itself will serve to introduce the pathology of these lung infections. the lung has multiple anatomic mechanisms by which it defends itself against invasion by various pathogens. first, the upper nasal cavities and respiratory tract serve as anatomic barriers to inhaled organisms. the ciliated epithelium and torturous cavities of the sinuses screen large organisms (typically larger than 10 microns). for those particles that venture further down the respiratory tract, the cough reflex that the upper trachea elicits serves to expel them up and out. second, the mucociliary tree of the upper respiratory tract captures organisms that evade these two mechanisms. the bronchial epithelium contains cilia of up to 20 microns in length that extend into the air surface liquid (asl). the asl is a bilayer of 50-100 microns in thickness consisting of a low-viscosity or watery lower layer that is covered by a high-viscosity or gel upper layer secreted by adjacent goblet cells. this sticky upper layer serves to trap organisms, and the coordinated beating of the cilia moves these entrapped invaders up this mucociliary escalator to the larynx, where they can be expectorated. present in the secretions of the large airways and within the surfactant lining the alveolar walls are soluble mediators secreted by various cells. these mediators include lysozyme and lactoferrin, which lyse bacteria and inhibit their growth; the defensins and cathelicidins, small peptides both with microbicidal properties; and surfactant proteins a and d at the alveolar level, which bind to microorganism and enhance phagocytosis and also have direct bactericidal activity [232] . the major cells of the innate immune response of the lung are the alveolar macrophages (am) and the polymorphonuclear leukocytes (pmn). neutrophils phagocytize and destroy bacteria such as s. aureus, s. pneumoniae, and h. influenzae through a respiratory burst that generates nadph oxidase-dependent ros. in some instances, ams may ingest but not kill an organism. this occurs with such organisms as mycobacterium spp., nocardia spp., and legionella spp. because of the ability of these organisms to continue to replicate within part iv molecular pathology of human disease the am, cell-mediated immunity is required for their complete elimination. patients with defects in nadph oxidase are especially prone to respiratory infections by such organisms as s. aureus, nocardia spp. and aspergillus spp. bronchial epithelial cells are important in innate immunity through secretion of cytokines and molecules including il-1, il-5, il-6, il-8, and granulocytemacrophage colony-stimulating factor (gm-csf). these molecules attract macrophages as well as neutrophils and other inflammatory cells to the area to enhance the inflammatory response to the organism [233] . bronchial epithelial cells also serve an important role in recognizing pathogens through patternrecognition receptors (prrs). natural killer (nk) cells are involved in the innate immune response with surface receptors that recognize cells infected with viruses such as rsv, influenza, parainfluenza, and rhinovirus. the nk cells release ifn-g, which recruit other immune cells to add to the antiviral response. dendritic cells are tissue histiocytes positioned around the airways and lymphatics in the lung that recognize pathogens and their antigens and trigger the proliferation and amplification of antigen-specific tcells. this immune response bridges the innate immune response to the adaptive immune responses and is especially important in fungal infections. this mechanism is mediated through toll-like receptors (tlrs) that are able to distinguish pathogens from self-components by triggering cytokine production through nfkb and ap-1 and expressing co-stimulatory molecules necessary for this t-cell activation [234] . for those organisms that evade the basic, innate immunity of the lung, there are adaptive immune mechanisms that encompass both humoral and cellular immune mechanisms. humoral immunity is an important defense against encapsulated bacteria, most notably s. pneumoniae, and for other pyogenic bacteria such as h. influenzae, and staphylococci spp., and resolution of these infections requires the production of igg antibodies to the organisms. cellular immunity is especially important against such respiratory viral infections as influenza, rsv, cmv, varicella, and also against opportunistic infections. these viruses induce a cd4ã¾ and cd8ã¾ t-cell response that clears the lung of these viruses within 8-10 days post infection. granulomas are a common inflammatory response to both pathogens and foreign material. the most notable granulomatous infections in the lung are due to mycobacteria and fungal organisms. activation of cd4ã¾ t-cells by these organisms leads to proliferation and differentiation of these cd4ã¾ t-cells into t-helper-1 cells. the release of ifn-g by the th-1 cells activates lung macrophages to form epithelioid macrophages that have an increased ability to kill the microorganisms and express surface molecules that promote cell-to-cell fusion into giant cells. in addition, activation of these macrophages results in the release of numerous cytokines including ifn-g and tnfa. in patients who are deficient in cd4ã¾ t-cells or ifn-g, granuloma formation is very poor, altering the pathologic picture of these infections. this effect is most obvious in the nontuberculous mycobacterial infections, which have numerous patterns of injury depending on the immune status of their host. pneumonias can be broadly categorized into one of five major clinicopathologic categories, including (i) community-acquired pneumonias (acute and atypical), (ii) nosocomial pneumonias, (iii) aspiration pneumonias and lung abscess, (iv) chronic pneumonias, and (v) pneumonias in immunocompromised hosts. each type presents with a characteristic clinical pattern and may be caused by any of several pathogens so that treatment is many times empiric. the first category comprises community-acquired pneumonias (cap). these represent the majority of the lung infections that receive medical treatment, usually on an outpatient basis, with low (<1%) mortality. patients hospitalized for these infections typically have other comorbidities. the responsible organisms include respiratory syncytial virus (rsv); rhinovirus, parainfluenza, and influenza virus; bacteria, including mycoplasma pneumoniae and rickettsia; and most notably chlamydia pneumonia. chlamydia causes what is termed atypical pneumonia with a clinical course characterized by a progressive onset of fever without chills, a dry cough, and chest imaging that reveals focal infiltrates. acute or typical cap presents abruptly with high fever, chills, productive cough, and radiographs with lobar or segmental consolidation. the most common pathogens are streptococcus pneumoniae, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. the second category, nosocomial pneumonias, consists of infections acquired within the hospital or from healthcare associated facilities. these infections are usually found in patients with predisposing risk factors and are a major source of morbidity and mortality, with some studies reporting a mortality range of 20%-50%. the most common risk factors include respiratory ventilation, artificial airways, nasogastric tubes, supine positioning, and medications that alter gastric emptying. the responsible organisms include klebsiella spp., legionella spp., staphylococcus aureus, and pseudomonas aeruginosa. the third category includes aspiration pneumonias and lung abscesses. these infections occur in the setting of patients with aberrant swallow or gag reflexes that allow gastric or oral contents into the airways. the organisms where necrosis and cavity formation occurs include s. aureus, k. pneumoniae, the anaerobic oral flora, and mycobacteria. clinically, these infections may have an acute course with fever and dyspnea or a more insidious course, many times with patients first presenting with lung cavities, empyemas, or necrotizing pneumonias. the fourth category, chronic pneumonias, includes indolent infections that cause a localized mass-like lesion in an otherwise healthy host. nocardia and actinomyces spp. are the most common pathogens, but mycobacteria and fungi may also cause these pneumonias. the fifth category includes pneumonias that occur in the setting of an immunocompromised patient. these include a number of organisms that otherwise would not act as pathogens such as the viruses cmv and hsv, the fungi aspergillosis and pneumocystis pneumonia, and the bacterium mycobacterium avium complex. streptococcus pneumoniae streptococcus pneumoniae, a gram-positive diplococcus also known as pneumococcus or diplococcus pneumonia, is a common cause of bacterial pneumonia in infants and elderly patients, alcoholics, diabetics, and patients with immunosuppression. this pneumonia usually presents abruptly with chills, a cough with rust-colored sputum and pleuritis, with high fevers, tachycardia, and tachypnea. the characteristic gross pathology is a lobar pneumonia that progresses from a red acute phase to a gray organizing phase. a fibrinous pleuritis is common, which eventually organizes to entrap the lung parenchyma in a fibrous capsule [235] . the microscopic examination reveals abundant fibrin, neutrophils, and extravasated red blood cells within the alveolar space and congested capillaries. hemophilus influenzae hemophilus influenzae is a gramnegative bacillus that inhabits the upper respiratory tract and can cause otitis media, epiglottitis, and meningitis, and usually enters the lung through aspiration or hematogenous spread. six serotypes are defined based on their capsular antigens, with type b the most common cause of pneumonias. this type of pneumonia is most commonly found in children or in the elderly with underlying chronic lung disease such as emphysema, cystic fibrosis, bronchiectasis, in patients with hiv infection, or in alcoholics. this bacterial pneumonia is usually preceded by a viral or mycoplasma infection that damages the mucociliary elements in the airways and allows for colonization by h. influenzae. the symptoms include fever; a productive, purulent cough; and myalgias. the incidence of this pneumonia as a common community-acquired pneumonia in children is quite low due to the advent of effective vaccines. however, it is increasing in incidence as a nosocomial infection [236] . like pneumococcal pneumonia, the pathology of h. influenzae pneumonia is in a lobar distribution with a neutrophilic-rich infiltrate and a pleural effusion. necrosis and empyema may occur but are uncommon. staphylococcus aureus staphylococcal pneumonia is caused by staphylococcus aureus, gram-positive cocci that usually spread to the lung through the blood from other infected sites, most often the skin. though a common community pathogen, it is found twice as frequently in pneumonias in hospitalized patients. it often attacks the elderly and patients with cf and arises as a co-infection with influenza viral pneumonia. the clinical course is characterized by high fevers, chills, a cough with purulent bloody sputum, and rapidly progressing dyspnea. the gross pathology commonly reveals an acute bronchopneumonia pattern (figure 18 .49) that may evolve into a necrotizing cavity with congested red/purple lungs and airways that contain a bloody fluid and thick mucoid secretions. the histologic pattern is characterized by a bronchopneumonia that spreads distally from the small airways into to the alveolar spaces (figure 18 .50) to form abscesses that connect with the pleural surface and may result in empyemas. the treatment of this organism has become increasingly problematic due to antibioticresistant strains, most notably methicillin-resistant s. aureus. legionella pneumophila legionella are gram-negative bacilli found predominantly in aquatic habitats such as lakes, rivers, and ponds. standing pools of water from humidifiers and other water outlets may be other sources. approximately 50% of air conditioners contain these bacilli. though 15 serogroups of legionella have been identified, 3 cause the overwhelming majority of human pneumonia. the clinical disease takes two forms: (i) legionnaires' disease, named after the outbreak of pneumonia at the 1976 american legion convention in philadelphia; and (ii) pontiac fever, a self-limiting flu-like disease with nonspecific symptoms. legionella pneumonia presents as a severe infection of the lung with chills and rigors with a nonproductive cough. it can progress rapidly to systemic symptoms of nausea, vomiting, and diarrhea and can lead to renal failure and death without immediate antibiotic therapy. the infected lungs are remarkably red and congested and appear to be distended with fluid. the microscopic picture reveals fibrinopurulent exudates that fill the alveolar space mixed with a necrotic, cellular infiltrate of degenerating neutrophils and monocytes (figure 18 .51). hyaline membranes may form in the periphery of the lesions, and pleural effusions consisting of fibrinoserous exudates are common. pseudomonas aeruginosa pseudomonas aeruginosa is a gram-negative bacillus that is found throughout the environment and in 50% of the airways of hospitalized patients. it usually enters the body through a disruption of the epithelial surface by cuts, burns, or therapeutic devices such as mechanical ventilators or intravascular catheters. pneumonias caused by this organism are usually found in intensive care units of hospitals and burn units, in patients with underlying chronic lung diseases including cystic fibrosis, emphysema, and in patients with prolonged hospitalization. the pathology is necrosis with a bronchopneumonia pattern that usually consists of an area of congestion and hemorrhage that is surrounded by a halo of tan/white consolidation (figure 18.52) . a necrotizing vasculitis with abundant organisms in vessel walls can be seen, and cavitation is common ( figure 18 .53). in treated lungs, healed cavities or pneumatoceles may appear as smooth-walled fibrous cysts. other gram-negative bacilli gram-negative bacilli such as klebsiella pneumoniae, acinetobacter, and various enterobacteriaceae spp. are common nosocomial pathogens. similar to p. aeruginosa, these pathogens colonize the oropharynx and are usually introduced into the lung by inhalation or aspiration of oral contents. the most notable of these is friedlander's pneumonia caused by k. pneumoniae, the most common cause of gramnegative bacterial pneumonia. this typically occurs in men over 40 years of age, usually in the setting of alcoholism, diabetes mellitus, or chronic lung disease. these patients produce large amounts of thick, bloody sputum, a product of the viscous mucopolysaccharide capsule of the organism, and present with severe systemic symptoms of hypotension and generalized weakness. the pathology of these pneumonias is similar to pseudomonas pneumonia with marked cavitation and abundant organisms on microscopic examination. nocardia spp. nocardiosis of the lung is caused by nocardia asteroides, a gram-positive rod found in the soil or organic matter. this infection is most common in immunocompromised adult patients and can be seen in the setting of pulmonary alveolar proteinosis, chronic lung diseases, and mycobacterial and other granulomatous diseases that affect the lung. its clinical course is indolent and usually begins 1-2 weeks before the patient presents for medical therapy. cough is common, often with thick, purulent sputum. in the immunocompromised setting, fever, chills, dyspnea, and hemoptysis are common, and weight loss may occur as the disease progresses. the pathology is remarkable for suppurative abscess formation with multiple cavities filled with green, thick pus. the inflammatory infiltrate consists of neutrophils, macrophages, and abundant necrotic debris with epithelioid histiocytes and giant cells within the wall of the cavity (figure 18 .54). empyema and pleura involvement occur in the majority of cases. mycoplasma and rickettsia pneumonias mycoplasma pneumoniae pneumonia is among the most common infections of the lower respiratory tract and usually occurs in small epidemics in closed populations. it often presents with atypical features of a progressive onset, fever without chills, a dry cough, diffuse crackles on physical examination, and chest imaging studies that reveal patchy interstitial infiltrates. the pathologic features are a result of the attachment of the organisms to the bronchiolar epithelium where they cause epithelial injury and ulcerations through secretion of peroxide [237] . in cases of severe infection, diffuse alveolar damage may be present. chlamydial pneumonia chlamydia spp. causes pneumonia in a variety of clinical settings. chlamydia trachomatis is an infection found predominantly in the postnatal period, chlamydia psittaci is the result of direct transmission from infected birds, including parakeets, parrots, and pigeons. chlamydia pneumoniae is the most common of the three and is a frequent cause of community-acquired pneumonia. it typically causes a very mild or asymptomatic infection with fever, sore throat, and nonproductive cough. the course of this infection may be severe in the elderly. chest imaging studies show alveolar infiltrates, and pleural effusions are present in the majority of cases. the pathology has not been well defined since the infection is usually self-limited. however, in experimental animal models there is a neutrophilic response in the early stages, and an interstitial, peribronchiolar, and perivascular infiltrate of lymphocytes, macrophages, and plasma cells in the latter stages of the infection. mycobacteria, a major cause of lung infections, are nonmotile, aerobic, catalase-producing, acid-fast bacilli. clinically significant lung infections can be caused by m. tuberculosis and by a group of nontuberculous mycobacteria (ntm). the latter group consists of over 100 species, of which three cause the overwhelming majority of pulmonary disease. these are m. avium-intracellulare (m. avium complex), m. kansasii, and m. fortuitum-chelonae. throughout history, tuberculosis (infection with m. tuberculosis) was the major disease caused by these organisms and was responsible for worldwide morbidity and mortality. however, over the past two decades lung diseases caused by ntm have become much more common and now represent the majority of the pulmonary mycobacterial disease. mycobacterium tuberculosis pulmonary tuberculosis is spread by interpersonal contact through aerosolized droplets. once in the alveoli, the bacteria cause a cell-mediated inflammatory response that is capable of inducing granuloma formation and necrosis. as in all infections, the extent of the disease is a function of the host's immune response. the most susceptible part iv molecular pathology of human disease patients are those with certain conditions that include immunosuppression, diabetes, malignancy, renal failure, among others. clinically, an infected patient has a productive cough, fever, and weight loss, and may develop hemoptysis as the cavitation progresses and erodes into the pulmonary vessels. extensive involvement of the lung can produce significant dyspnea and pleuritic chest pain. the pathology of tuberculosis is primarily that of granuloma formation and acute pneumonia. the granulomas are predominantly necrotizing, and the pneumonia usually contains abundant fibrin and neutrophils that fill the alveolar spaces. the gross lesions are referred to as caseous or cheese-like, because of the amount of necrosis present. this caseous material can extend into airways and is commonly coughed up during the active disease. in chronic forms of the disease, the area can undergo fibrosis and involute into a firm, hard scar. there are three major clinicopathologic variants of the disease: (i) primary tuberculosis, (ii) postprimary or reactivation tuberculosis, and (iii) progressive fibrocavitary disease. primary tuberculosis. in this form of the disease, the initial site of infection can be anywhere in the lung, but is usually in the lower lobe or anterior segment of the upper lobe, the areas that receive the most ventilation. the lesion usually consists of a dense consolidation with acute pneumonia and necrotizing granulomas. cavitation may occur, especially in the setting of immunocompromised hosts. from these foci, the organisms may spread through the lymphatics to elsewhere in the lung, the hilar lymph nodes, and the bloodstream, and lay dormant for long periods of time. the combination of the primary site of infection and the involved hilar lymph nodes is known as a ghon complex [238] . postprimary tuberculosis. this form of tuberculosis represents reactivation of old, scarred primary lesions long after the initial insult. the lesion can occur anywhere in the lung where the bacteria from the primary lesion have spread, but is usually apical. it consists of a focus or organizing pneumonia and fibrosis with central caseation. in an active lesion, the typical parenchymal pattern is an acute pneumonia with cavitation that expands to include the surrounding lung with aggregates of granulomas. the controversy surrounding this lesion arises as some evidence suggests that these lesions represent exogenous reinfection. the pathology of reactivation or reinfection may be indistinguishable, although reactivation tuberculosis may appear to arise out of a fibrotic, calcified chronic lesion [239] . progressive fibrocavitary disease. this form of the disease may arise out of either primary or postprimary tuberculosis. however, the latter is the more common scenario. the cavities that develop in this form of the disease begin as a slowly progressive, necrotizing pneumonia with abundant granulomas (figure 18 .55). the active disease may spread through the airways, causing ulceration, necrosis, and fibrosis of the surrounding bronchi and bronchioles. the extension of the disease in this way depends on the host, and patients with depressed immune systems can have large areas of the lung involved with massive pulmonary necrosis. usually, a fibrous capsule develops in the area of the cavitation, although inspissated necrotic material into the adjacent airways remains a continuous source of inflammation that can lead to reinfection and ongoing scarring [240] . nontuberculous mycobacteria the nontuberculous mycobacteria (ntm) are ubiquitous inhabitants of our environment, isolated from soil, fresh and brackish water, house dust, birds, animals, and food, and are increasingly important in causing pulmonary disease. there are currently more than 100 ntm species known. those organisms thought to be pathogenic to the lung include the clinical presentation of these lung infections can vary from minimally symptomatic small lesions discovered by routine radiography to sudden hemoptysis from advanced disease with severe cavitation (table 18 .5). the two most characteristic lesions are those of diffuse infiltrates in an immunocompromised patient, seen most commonly in the hiv-positive population and an viruses most pulmonary infections are due to viruses from four major groups: influenza, parainfluenza, respiratory syncytial virus (rsv), and adenovirus (table 18 .6) [241] . the clinical presentations of these infections have some common features, including insidious onset, nonproductive cough, fever, and chest pain. chest imaging studies usually reveal bilateral, multifocal infiltrates, most without evidence of cavitation or pleural involvement. these infections are mild, self-limiting, and require no more than supportive therapy except in immunocompromised hosts, where the clinical course can be much more serious. also, immunocompromised patients are susceptible to other viruses such as herpesvirus and cytomegalovirus pneumonias, which are not common pathogens in normal hosts [242] . since the 1980s, a subset of pulmonary viral infections has emerged with a much more aggressive clinical course, most notably sars, coronavirus, and hantavirus. these viruses present with systemic symptoms of headache, myalgias, and weakness followed by a deteriorating clinical course with respiratory distress, shock and, in over 50% of the cases, death [243, 244] . therapy for most respiratory viral infections is supportive, although antivirals are available for some viruses, mostly used in the setting of immunocompromised patients. ribavirin, a guanosine analogue, is the main antiviral used for rsv; m2 inhibitors or adamantanes (amantadine and rimantadine) are used against influenza a and neuraminidase inhibitors (oseltamivir and zanamivir) are used against both influenza a and b [245] . cytomegalovirus is treated with ganciclovir, foscarnet, or cidofovir, while herpesvirus is treated with acyclovir [241] . the pathologic patterns of injury for most viruses are similar, making morphologic distinctions among them difficult. however, some characteristic patterns emerge, most notably in those viruses that cause cytopathic changes. influenza, adenovirus, sars, coronavirus, and hantavirus all cause an acute lung injury pattern with diffuse alveolar damage, and in the case of the latter two viruses, evidence of hemorrhage and edema. influenza and adenovirus will also cause a necrotizing bronchiolitis due to their preferential infection of bronchial epithelial cells. finally, some viral infections can be distinguished by their characteristic cytopathic inclusions. adenovirus can be identified by characteristic smudge cells that present in advanced stages of the disease and represent adenovirus particles in the nucleus of an infected cell (figure 18 .56). cytomegalovirus has both nuclear owl's eye inclusions, as well as cytoplasmic inclusions (figure 18 .57). herpesvirus has glassy intranuclear inclusions and can also have multinucleation (figure 18 .58). fungi are larger and more complex than bacteria, and their patterns of injury in the lung are different and in general more destructive. these pathogens are common in our environment and enter the lungs through inhalation. though many fungi are capable of causing pulmonary disease, most only inhabit the lung as colonizers. those of most concern for causing clinical disease include the endemic fungi of north america-histoplasma capsulatum, blastomyces dermatitidis, and coccidioides immitis-and two fungi that are commonly seen in immunocompromised hosts-aspergillus fumigatus and pneumocystis jiroveci. histoplasma capsulatum histoplasma capsulatum is a dimorphic fungus most prevalent in the middle portion of the united states from the great lakes to tennessee. the fungus is present in soil that has been contaminated with guano and other debris by nesting birds, most commonly blackbirds and chickens, and by bats. the organism lives in the environment as spores or conidia and germinates to form hyphae. these structures divide to create the yeast forms, which, when inhaled, induce granuloma formation in the lung. approximately 75% of people have skin tests that are positive for exposure to h. capsulatum, but most exposures do not cause clinical disease. disease typically occurs in people exposed to large amounts of organisms, such as construction workers who move large volumes of dirt or spelunkers who venture into bat-ridden caves. the acute disease has flu-like symptoms which are self-limiting. healed disease may leave behind calcified granulomas in the lung that appear as buckshot on chest imaging studies. the most chronic forms of this disease may slowly progress, giving rise to cavitating and fibrous lesions. in the immunocompromised host, disseminating histoplasmosis can be seen, although reactivation is uncommon [246] . the pathology reveals characteristic necrotizing granulomas distributed around the airways (figure 18 .59), which contain silver-positive yeast forms of 2-4 microns. these granulomas may resolve into scarred nodules, which can calcify and produce the characteristic chest images. cavities may form in the apices with progression of the disease, and the disseminated form of the disease has an abundance of organisms both within macrophages in the lung and throughout many organs in the body. blastomyces dermatitidis blastomyces dermatitidis is also endemic to the middle united states, including the ohio and mississippi river valleys. it is found in wooded terrain, usually during the wet seasons, putting campers and outdoorsmen at risk. the clinical disease takes two forms, cutaneous and systemic, the latter beginning in the lungs through inhalation. the acute pulmonary infection takes a nonspecific form with fever, malaise, and chest pain. imaging studies may show either infiltrates or a mass-like infiltrate. thus, blastomyces infection may mimic other diseases, and the diagnosis may be delayed. some patients go on to chronic disease with cavitation or progressive pulmonary blastomycosis, which manifests as acute respiratory distress syndrome, cavitary lesions, and a poor prognosis [247] . the pathology of blastomyces infection is similar to histoplasmosis with necrotizing granulomas. however, the lesions are larger, showing more neutrophilic necrosis. the organisms are also larger (8-15 microns), with prominent broad-based budding, and are apparent on routine hematoxylin and eosin staining (figure 18 .60). coccidioides immitis coccidioides immitis is found in the semi-arid desert climate of the southwestern united states. the organisms are inhaled as spores, causing an acute disease characterized by fever, chills, chest pain, dyspnea, and hemoptysis. chest imaging studies typically show consolidation and cavitation, and hilar lymphadenopathy is common. reactivation and dissemination are possible in patients with previous infection, whether or not they are immunocompromised patients [248] . the pathology of pulmonary coccidioidomycosis is neutrophilic, suppurative, and granulomatous. the organisms appear as large spherules containing endospores, visible on silver stains. the spherules are 30-100 microns in diameter and the endospores that are released into the surrounding tissue proceed to mature into new spherules (figure 18.61) . as in histoplasmosis, cavitating lesions may have hyphal forms that begin to germinate. aspergillus fumigatus aspergilli are asexual mycelial fungi that are ubiquitous in the environment as airborne aspergillus spores. they are weak pathogens that produce invasive infections predominantly in immunocompromised hosts or in those with significant chronic lung diseases. in tissue, aspergilli form septate hyphae, 3-6 microns in diameter, with characteristic acute-angle, dichotomous branching (figure 18.62) . these organisms affect the lung in three major ways: (i) saprophytic growth in bronchi or pre-existent cavities; (ii) as an allergic or hypersensitivity reaction, predominantly in asthmatics; and (iii) invasive aspergillosis in immunocompromised hosts [249, 250] . as a saprophyte, aspergillus produces surface growths or minute masses of hyphae, usually in bronchiectatic cavities, emphysematous bullae, or scars from previous lung diseases such as tuberculosis or sarcoidosis. the pathology is usually that of a fibrous-walled cavity containing degenerating hyphae (figure 18 .63). in this setting, hyphae do not invade into the lung tissue, but surface erosion of a vascularized cavity may cause hemoptysis. aspergillus causes an immunologic response resulting in mucoid impaction or eosinophilic pneumonia in asthmatics, an entity known as allergic bronchopulmonary aspergillosis (abpa). pathologically, one sees mucoid plugs and superficial erosions of the airways with histiocytic inflammation, with only rare hyphal fragments present. the final form of the disease, invasive pulmonary aspergillosis, is found in severely immunocompromised, neutropenic patients. the hyphae, which disseminate through the blood, invade the blood vessels causing thrombosis, hemorrhage, and infarction to form typical targetoid lesions. this form of the disease has a poor prognosis despite aggressive antifungal therapy. pneumocystis jiroveci the taxonomy of pneumocystis jiroveci (formerly pneumocystis carinii) has changed over the past decade. previously thought to be a protozoan based on the histological characteristics of its trophozoite and cyst life forms, it has recently been placed in the fungal kingdom after ribosomal rna was found to have sequences compatible with the ascomycetous fungi [251] . the inability to culture pneumocystis jiroveci has slowed the understanding of this organism. animal models have helped in defining the antigenic and genotypic differences among the various pneumocystis organisms, which has led to the proposal for species-specific strains, with p. jiroveci found in human infections [252] . the molecular methods used for the typing these species examine a number of gene loci. most importantly, sequence analysis of the thymidylate synthase (ts) and superoxide dismutase (soda) gene loci, the epsp synthase domain of the multifunctional arom gene, and the mitochondrial small subunit ribosomal rna (mtssu rrna) locus have been used to distinguish the various pneumocystis species that infect different mammalian hosts [253] . clinically, p. jiroveci causes disease predominantly in the immunocompromised setting. pneumocystis pneumonia (pcp) has been found during recent times most commonly in the aids population, but prior to this epidemic, it was found in malnourished infants and other severely immunocompromised hosts. because this organism has not been cultured, the diagnosis of pcp continues to be challenging. the clinical characteristics are nonspecific and vary with the patient's immune status. in the hiv population, patients typically develop a subacute onset of progressive dyspnea, a nonproductive cough, malaise, and a low-grade fever. in the non-hiv population, the presentation is more acute, with fulminant respiratory failure associated with cough and fever, and usually requiring mechanical ventilation [254] . chest imaging studies typically show bilateral, symmetric, fine reticular interstitial infiltrates involving the perihilar area, which spread to involve the entire lung. figure 18 .62 aspergillosis. aspergillus fumigatus grows within necrotizing cavities of the lung as branching septated fungal hyphae, as seen on this grocott methenamine silver stain. figure 18 .63 aspergilloma. fungal hyphae from aspergillus fumigatus can colonize chronically inflamed lungs with cavities and may grow to form fungal balls with a dark, green color that are treated by surgical resection, as seen in this case of a lobectomy specimen. treatment is usually with trimethoprim/sulfamethoxazole and intravenous pentamidine. survival is 50%-95% even in severely immunocompromised patients. the life cycle of p. jiroveci consists of three stages: trophozoite, cyst, and sporozoite. the trophozoite form, which adheres to the type 1 epithelium, replicates and enlarges through three precyst stages before maturing into a cyst form that is found in the alveolar space. sporozoites develop within immature cysts through meiosis and mitosis. the mature cyst contains eight haploid sporozoites. the rupture of the cyst wall releases sporozoites into the surrounding environment where they mature into trophozoites. the pathology of the infection is predominantly due to the interaction of the organism with the epithelium. the attachment of the organism to the lung epithelium is via glycoprotein a present on the surface of the organism. the binding of the organism to the type 1 cell occurs via surface receptors on the type 1 cell that include macrophage mannose receptors. these interact with glycoprotein a and activate pathways in the organism that induce genes encoding for pathways that induce mating and proliferation responses, and for the formation of pheromone receptors, transcription factors, and heterotrimeric g-protein subunits [263] . in addition to these genetic effects, the cyst wall contains chitins, polymers, and other substances, in particular, 1,3-glucan, that maintain its integrity and induce the inflammatory response of the host. the 1,3-glucan in the wall of the organism stimulates the release by the macrophages of reactive oxidant species and the generation of potent proinflammatory cytokines, such as tnfa, which bind to the organism and exert a toxic effect. once inside the macrophage, the organism is incorporated into the phagolysosome and degraded. tnfa also directly recruits other inflammatory cells including neutrophils, lymphocytes, and circulating monocytes, and induces the release of il-8 and ifn-g that recruit and activate inflammatory cells [255] . in aggregate, the recruitment of these inflammatory cells and the mediators they release is responsible for the damage to the lung epithelium and endothelium that is seen in this disease [255] . the pathology of pcp has typical and atypical variants. typically, the lung contains a dense interstitial plasma cell pneumonia that expands alveolar walls. the epithelium consists predominantly of type 2 pneumocytes, and the alveolar spaces contain an eosinophilic, frothy exudate, which contains fine, hemoxylin-stained dots that represent a thickening in the cyst wall (figure 18.64) . in this form of the disease, the organisms are abundant and the diagnosis can usually be made by bronchoalveolar lavage. atypical pathologic variants include a necrotizing variant that has a pattern similar to the typical form with exudative alveolar infiltrates, but which undergoes necrosis and cavity formation. these cavities heal into fibrous-walled cysts, similar in gross appearance to those found in pseudomonas pneumonia. a third variant has wellformed granulomas involving the airways, a pattern common to histoplasmosis and tuberculosis. in this form, the organisms are rare and very difficult to find, even with tissue organismal stains. in general, the pathologic pattern of injury depends on the host's immune status, with the typical pathology found in severely immunocompromised hosts as the aids population and the atypical forms found in hosts with immune systems that are less compromised. pulmonary langerhans cell histiocytosis (plch) and erdheim-chester disease are histiocytic diseases that primarily affect the lung. other histiocytic diseases may affect the lung, such as niemann-pick disease, gaucher disease, hermansky-pudlak and rosai-dorfman disease, but these are not considered primarily lung histiocytic diseases. pulmonary langerhans' cell histiocytosis (plch) is a disease of the dendritic histiocytes of the lung referred to as langerhans' cells (lcs). this disease is part of a group of diseases that are characterized by a proliferation of langerhans cells in organs throughout the body that range from a malignant systemic disease as is seen in children [256] to the pulmonary variant that is seen in adolescents and adults. plch is usually the result of inflammatory or neoplastic stimuli in lungs of smokers or in lungs involved by certain neoplasms [257] . chest radiographs from patients with plch usually reveal bilateral nodules, predominantly in the upper lobes, which are worrisome for metastatic disease. treatment involves smoking cessation and steroid therapy. typically, the disease undergoes spontaneous regression. approximately 15%-20% of patients will progress to irreversible end-stage fibrosis [258] . the pathology of plch consists of airway-based lesions with a proliferation of lcs. the early cellular lesions contain a mixture of cells including langerhans' cells, lymphocytes, plasma cells, and eosinophils ( figure 18 .65). though it was previously referred to as eosinophilic granuloma, eosinophils are not the major cell type present, and the lesion is, at best, a loosely formed granuloma. immunohistochemistry reveals the lcs to be diffusely, strongly immunoreactive to s-100 protein and cd1a. ultrastructural analysis reveals intracytoplasmic organelles called birbeck granules, a normal constituent of langerhans' cells, in greater numbers in plch [259] . the pathogenetic mechanisms of plch focus on defects in the homeostasis of dendritic cells (dcs) in the lungs of smokers and the role tobacco smoke may play in stimulating the proliferation of these cells [260] . some studies suggest that stimulation of alveolar macrophages by chemicals in smoke results in secretion of such cytokines as gm-csf, tgfb, and tnfa [261] . in transgenic mice, accumulation of dcs around airways may be a result of excess gm-csf [262] . other theories suggest that cigarette smoke stimulates the secretion of bombesin-like peptide by the neuroendocrine cells in the bronchiolar epithelium and leads to a similar stimulation of alveolar macrophages and a cytokine milieu that promotes the proinflammatory proliferative changes [262] . not all smokers get plch, leading to the suggestion that only smokers with an underlying genetic susceptibility will develop the disease. studies have established that in some cases the lcs in plch are clonal, suggesting that cellular abnormalities must play some part in the pathogenesis of the diseases [263] . to support this, studies have shown genetic mutations and allelic loss of tumor suppressor genes in smokers with plch [264] . the mechanisms by which this proliferation of lcs leads to the destruction of the bronchiolar epithelium and the other observed pathology are unclear. lcs in normal lungs have little ability to interact with t-cells or act as effective antigen-presenting cells, but the lcs of plch have a mature immunophenotype, expressing b7-1 and b7-2, the co-stimulatory molecules needed for lymphostimulatory activity [265] . whether this more mature immune phenotype leads to an unregulated immune response and destruction of the bronchial epithelial cells is not known. however, some studies have shown that bronchiolar epithelial cells may induce the expression of this mature phenotype by secreting cytokines in response to environmental stimulants such as cigarette smoke or viral infections, or by the development of hyperplastic or dysplastic lesions that express new foreign antigens [265] . erdheim-chester disease (ecd) is a systemic non-langerhans' cell histiocytosis of adults that most commonly involves the long bones. involvement of other organs, including the lung, has been reported. lung involvement occurs in approximately 20%-35% of the cases, and the patients usually present with cough, dyspnea, rhonchi, and pleuritic pain. radiographically, the lungs reveal infiltrates in a lymphatic distribution, predominantly upper lobe, with prominent interstitial septal markings that can mimic sarcoidosis [266] [267] [268] [269] [270] [271] [272] . pulmonary involvement by ecd may have an unfavorable prognosis, and the fibrosis that ensues is one of the most frequently reported causes of death [266, 273] . the treatment of ecd is variable with corticosteroids, chemotherapy, surgical resection, and radiation therapy reported [273] . non-langerhans' cell histiocytes of dendritic cell phenotype are the main cells present in this disease. this infiltrate contains foamy histiocytes with scattered giant cells, a scant number of lymphocytes or plasma cells, and some fibroblasts. the histiocytes express cd68 (macrophage antigen) and factor xiiia (dendritic cell antigen), but express s-100 protein weakly or not at all, and do not express cd1a. ultrastructural analysis reveals phagolysosomes, but no birbeck granules are present [273] . this infiltrate that involves the lung is usually present in the pleura and subpleura, within the interlobular septa and around the bronchovascular structures. the remainder of the lung parenchyma is unremarkable, though fibrosis and paracicatricial emphysema can appear in the late stages of the disease [266] . the etiology of ecd is not known, but this rare disease has been established as primarily a macrophage disorder [274] . these histiocytes have abundant phagolysosomes and express the antigen cd1a and are consistent with a phagocytic cell, most likely closely related to alveolar macrophages. the peripheral monocytosis and the proinflammatory cytokine profile that is found in these patients might suggest that the histiocytic infiltrate is a result of systemic monocytic activation and invasion of circulating monocytes into the tissues throughout the body [275] . recently, an ecd patient was successfully treated by an agent toxic to monocytes, supporting the theory that these cells play a part in the disease [275] . alternatively, end organ cytokine production by local inflammatory cells resulting in proliferation and differentiation of resident immature histiocyte populations may produce a similar picture. another interesting observation is that erdheim-chester has been reported to occur in patients with langerhans' cell histiocytosis [276] , which may suggest that this is a disease where macrophages transition between two different phenotypes along the differentiation spectrum of tissue dendritic cells [276] . whether this is a benign or malignant proliferation has not been established. of 5 patients studied, clonality has been demonstrated in 3 by polymerasechain reaction [277] . environmental exposures are a major cause of lung disease and can cause a wide spectrum of both acute and chronic pathology. many organic and inorganic materials can cause lung damage, and because of their similar patterns of injury and long latent periods, it can be difficult to isolate the exact offending agent without a thorough clinical history. the two occupational lung diseases presented here-asbestosis and silicosis-represent pneumoconiosis, which are defined as diseases which result in diffuse parenchymal lung injury due to inhaled inorganic material. both have many pathologic patterns of injury that depend on the amount and length of time of exposure, and both can also cause neoplastic diseases of the lung. asbestos fibers are naturally occurring silicates that are commonly used in construction materials such as cement and insulation and in many textiles. they can be separated into two groups based on their mineralogic characteristics. serpentine fibers, named as such because they are long and curly, include chrysotile asbestos. amphibole fibers, more straight and rodlike, include predominantly amosite and crocidolite asbestos. in the united states most of the asbestos is chrysotile. the amphiboles are more pathogenic and are responsible for most of the neoplastic and non-neoplastic pulmonary diseases associated with asbestos exposure. by definition, asbestosis is bilateral diffuse interstitial fibrosis of the lungs that can be attributed to asbestos exposure. the disease, which mostly affects textile and construction workers, is usually the result of direct exposure over 15-20 years. the latency to clinical disease is inversely proportional to the level of exposure. the symptoms are a gradual onset of shortness of breath, a cough with dry rales at the bases on inspiration, and digital clubbing. in the early disease, the chest x-ray shows basilar disease that begins predominantly as thickening of the subpleural, but progresses as infiltrates and fibrosis that involve the middle zone, eventually leading to thickening of the airways and traction bronchiectasis. the apex of the lung is usually spared. the clinical findings are nonspecific and have considerable overlap with uip, so the diagnosis is usually made only when a history of significant exposure is discovered. the gross picture includes a bilateral lower lobe gray/tan fibrosis with honeycomb changes in late disease. microscopically, asbestosis can cause many patterns of injury in the lung, but the most common is collagenous deposition in the areas of the lymphatics where the fibers are in the highest concentration. these areas include the subpleural, interlobular septae, and around the bronchovascular areas that contain a bronchiole and a branch of the pulmonary artery. hyalinized pleural plaques are a common manifestation of asbestos exposure but are not specific for asbestos and can be found in the absence of pulmonary parenchymal disease. eventually, the fibrosis involves the alveoli beyond the bronchioles and causes distortion of the lung architecture to form remodeled, dilated airspaces similar to those seen in uip. distinguishing this fibrosis from other forms of fibrosing lung disease can be difficult, but the presence of ferruginous bodies, asbestos fibers coated by iron, proteins, and a mucopolysaccharide coat are indicative of significant asbestos exposures and support this diagnosis (figure 18 .66) [278] . figure 18.66 asbestosis. this cytopathologic preparation from a bronchoalveolar lavage specimen illustrates an asbestos fiber coated by an iron-protein-mucopolysaccharide substance and appears as a golden brown, beaded structure known as a ferruginous body. silicosis results from chronic, high-dose exposure to crystalline silica, which consists of silicon and oxygen with trace amounts of other elements, usually iron. the most common silica is quartz, which is present in large amounts in such rocks as granite, shale, and sandstone and is among the more fibrogenic of all silica types. thus, occupations most at risk for silicosis include sandblasting, quarrying, stone dressing, and foundry work where exposure to quartz is high. the disease takes three major clinical and pathologic forms that have different clinical characteristics. simple or nodule silicosis is marked by the presence of fine nodules 1 cm, on chest imaging studies, usually in the upper lobes. patients with this condition are typically asymptomatic, with normal respiratory physiology. the pathology in these lungs reveals discrete, hard nodules that have a green/gray color, centered either on the small airways or in the subpleura. microscopically, these nodules have an early stellate shape that eventually transforms to a more whorled appearance with dustladen macrophages scattered throughout it. polarized light examination reveals weakly birefringent material. complicated pneumoconiosis represents similar pathologic findings only with larger and more circumscribed nodules, which coalesce into a large upper lobe mass, a condition known as progressive, massive fibrosis ( figure 18.67) . these patients are symptomatic with a productive cough and mixed pulmonary function tests with a reduced diffusing capacity as the fibrosis increases. diffuse interstitial fibrosis may occur; however, unlike asbestosis, this pattern is found in pneumoconiosis. when complicated pneumoconiosis is found with rheumatoid nodules in the setting of a patient with rheumatoid arthritis, this is known as caplan's syndrome. the pathogenesis of both asbestosis and silicosis depends upon inflammation and fibrosis caused by the inhaled fibers. in humans, the amount of fiber needed to cause fibrosis varies from person to person. this may be related to a difference in fiber deposition based on the size of the lungs or to the efficacy with which the lung clears these fibers [256] . some studies have also suggested that fiber length determines the amount of pathology. however, this association has not been confirmed in humans for either asbestosis or silicosis. in both diseases, it is known that other factors increase the risk of developing disease. for example, smokers exhibit worse disease than nonsmokers with similar exposures to asbestosis. the mechanism for this effect is unclear, although speculation centers on the inhibition of fiber clearance in smokers. also, it is known that smoking enhances the uptake of fibers by pulmonary epithelial cells and in this way may increase the fibrogenic and inflammatory cytokine production by these cells. the cellular mechanisms by which both asbestos and silica fibers induce the inflammation and fibrosis are mediated predominantly through alveolar macrophages. in the case of silica, it is known that the uptake of these fibers into the alveolar macrophages is by way of a scavenger receptor expressed on the surface of the cell known as marco (macrophage receptor with a collagenous structure). once inside the cells, the fibers activate the release of ros that can lead to cellular and molecular damage through a number of pathways. first, ros can directly cause lipid peroxidation, membrane damage, and dna damage. second, silicainduced free radicals can trigger phosphorylation of cellular proliferation pathways through mitogen-activated protein kinases (mapks), extracellular signal regulated kinases (erks), and p38. these pathways are also involved in the proliferation of fibroblasts in asbestosis and of mesothelial and epithelial cells in the neoplastic diseases associated with the inhalation of these fibers [279] . in addition, these fibers can activate proinflammatory pathways controlled by such transcription factors as nuclear nfkb and activator protein 1 (ap-1). these pathways result in the activation of the early response genes c-fos and c-jun and the release of proinflammatory cytokines such as il-1 as well as fibrogenic factors such tnfa [280] . tnfa plays a prominent role in both diseases, and its regulation has been studied in animal models exposed to silica. it is now known that a transcription factor labeled nuclear factor of activated t-cells (nfat) plays a key role in the regulation of tnfa. figure 18 .67 complicated pneumoconiosis/ progressive massive fibrosis. this sagittal cut section of lung reveals a large gray/black mass that extends from the apex to include the majority of the lung. the patient had a long history as a coal mine worker, and the microscopic sections revealed abundant anthracotic pigment and scarring in this area. binding sites for nfat have been found in the promoter region of the tnfa gene. the mediation of silica-induced tnfa transcription is probably via o 2-but not h 2 o 2 [280, 281] . atresia of the lung represents a premature closure of the airway at any level of the bronchial tree including the lobar, segmental, or subsegmental airways. clinically, these children usually present between 10 and 20 years of age for symptoms of dyspnea, wheezing, recurrent pneumonias, or for incidental findings on a chest imaging study. these lesions are more common in the proximal segmental bronchi, right more often than left. when atresia is associated with anomalies of the vascular supply to the affected airway, the lesion represents a separate, aberrant segment of lung known as a sequestration, either intralobar or extralobar type. the pathology of bronchial atresias and sequestrations represents sequelae of chronic inflammation due to the accumulation of secretions in these blind-end airways. these features consist of cystically dilated airways with mucus and parenchymal fibrosis with honeycomb changes. in intralobar sequestrations (ils), the anomalous vessel is a muscular artery that enters through the pleura from an aortic source, usually from the thoracic area. ils are separate, isolated areas of lung invested with the normal visceral pleura without bronchial or arterial connections (figure 18 .68). extralobar sequestrations (els) are pyramid-shaped accessory pieces of lung that have their own pleura with an artery from the lung but without airway connections. the category of congenital pulmonary cystic diseases represents the majority of congenital pulmonary disease and includes foregut cysts and cystic adenomatoid malformations. foregut cysts include bronchogenic, esophageal, and thymic cysts that form from defects in the foregut branching. clinically, these cysts are usually incidental findings on chest imaging studies, but they can present with complications due to infection or hemorrhage. pathologic features of these cysts include subtle differences that are usually only apparent after microscopic examination. grossly, these cysts usually arise proximally either within the mediastinum (over 50%) or in the proximal regions of the lungs, right more commonly than left, along the esophagus, and rarely within the lung parenchyma or below the diaphragm [282] . microscopically, each cyst contains a simple cuboidal or columnar epithelium, ciliated or nonciliated, that may undergo squamous metaplasia. distinguishing among the three types of cysts requires the presence of other elements. bronchogenic cysts have submucosal glands and/or hyaline cartilage within their walls, and thymic cysts may contain residual thymus. congenital cystic adenomatoid malformations (ccam), now more commonly referred to as congenital pulmonary airway malformations (cpam), are segments of lung with immature airways and alveolar parenchyma. these are usually classified by their predominant cyst size into types 0-4. type 1 cysts, which contain a main large cyst of up to 10 cm, are the most common. these cysts are distinguished from foregut cysts upon the recognition in the cpam of immature alveolar duct-like structures connecting to the surrounding lung parenchyma. this type of cpam is also notable, as it is known to undergo malignant transformation, usually to mucinous bronchioloalveolar cell adenocarcinomas. these anomalies arise due to defects during the various stages of development and are best considered within these developmental stages. the embryonic stage occurs within the first 3-7 weeks of life when the ventral wall of the foregut separates into the trachea and esophagus and branches to form the left and right lungs. the splanchnic mesenchyme that surrounds this foregut forms the vascular and connective tissues of the lungs. defects in this phase result in complete lack of lung development known as pulmonary agenesis and incomplete separation of the trachea and esophagus, causing tracheal-esophageal atresias and fistulas. the pseudoglandular stage, between weeks 7-17 of development, is a time of rapid development of the conducting airways including the bronchi and bronchioles and the expansion of the peripheral lung into the acinar buds. the mesenchymal tissue figure 18 .68 intralobar sequestration. the tan and white mass involving this left lower lobectomy specimen represents chronic pneumonia and fibrosis in the sequestered area of the lung. the dilated airways are features of an endstage fibrosis that is commonly found in this entity. part iv molecular pathology of human disease that surrounds these buds begins to thin, becomes vascularized, and forms the cartilage that surrounds the more proximal branching airways. during the canalicular (week [17] [18] [19] [20] [21] [22] [23] [24] , saccular (weeks [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] , and alveolar (weeks 36 to maturity) stages of development, the acinar buds continue to expand, and the mesenchyme surrounding this continues to thin. during the canalicular stage, the pulmonary vascular bed begins to organize, the distance between the blood in the vascular spaces and the air in the alveoli narrows, and the respiratory epithelium begins to form. the gas exchange unit of the alveolus becomes functional during the saccular stage with further differentiation of the respiratory epithelium to include clara cells, ciliated and nonciliated cells, and type 2 cells with the first production of surfactant occurring during this period. this gas exchange unit continues to mature during the alveolar stage with the growth and septation of the alveoli. this process continues postnatally through 6-8 years of age. the different types of cpams arise at different stages of development. cpams 0, 1, and 2 are a result of defects during the early embryonic and pseudoglandular stages of development, producing pathology with features of primitive alveolar buds and immature and abnormal airway cartilage structures. cpams 3 and 4 result from abnormal formation of the more distal airways and pulmonary parenchyma during the canalicular, saccular, and alveolar phases, causing pathology with immature alveolar, or alveolar simplification with enlarged alveoli [283] . various genetic defects in the pathways that control lung morphogenesis have been associated with these congenital lung diseases. two major transcription factors are responsible for the normal branching morphogenesis. the first, thyroid transcription factor-1 (ttf-1), is a member of the nkx2.1 family of hemeodomain-containing transcription factors. this factor plays a role in the lung epithelial-specific gene expression and proper lung bud development in the embryonic stage, as well as in the maturation of the respiratory epithelium. the second major factor is somatic hedgehog (shh)/gli, expressed by endodermally derived cells and required for branching morphogenesis. the development of the lung bud from the foregut endoderm depends on the appropriate expression of these lung-specific genes at the correct time in development. in the presence of genetic defects, aberrant lung development may occur. for example, mutations of various types in the shh/gli gene have been found to cause tracheoesophageal fistulas, anomalous pulmonary vasculature, and aberrant airway branching. also, deletions in the ttf-1 gene are associated with tracheoesophageal fistulas and a variety of forms of lung dysgenesis [284] . finally, factors present in the surrounding mesenchyme play a role in inducing the proper development of the pulmonary endoderm. a prominent mesenchymal factor in this process is fibroblast growth factor (fgf), which modulates both the proximal and distal lung branching morphogenesis. deletions in this gene may cause lung agenesis and tracheal malformations [284] . surfactant dysfunction disorders represent a heterogenous group of inherited disorders of surfactant metabolism, found predominantly in infants and children. pulmonary surfactant includes both phospholipids and surfactant proteins, designated surfactant proteins a, b, c, and d (sp-a, sp-b, sp-c, sp-d), synthesized and secreted by type 2 cells beginning in the canalicular stage of lung development. damage to type 2 cells during this time period can lead to acquired surfactant deficiencies. however, more commonly these deficiencies are the result of genetic defects of the surfactant proteins themselves. the major diseases are caused by genetic defects in the surfactant protein b (sftpb, chromosome 2p12-p11.2); surfactant protein c (sftpc, chromosome 8p21); and adenosine triphosphate (apt)-binding cassette transporter subfamily a member 3 (abca3, chromosome 16p13.3). defects in sftpb and abca3 have an autosomal recessive inheritance pattern, and defects in sftpc have an autosomal dominant pattern. sp-b deficiency is the most common. it presents at birth with a rapidly progressive respiratory failure and chest imaging studies showing diffuse ground glass infiltrates. the gross pathology in these lungs consists of heavy, red, and congested parenchyma with microscopic features that range from a pap-like pattern to a chronic pneumonitis of infancy (cpi) pattern. in sp-b deficiency, the pap pattern predominates with a histologic picture of cuboidal alveolar epithelium and eosinophilic pas-positive material within the alveolar spaces that appears with disease progression. in the late stages of the disease, the alveolar wall thickens with a chronic inflammatory infiltrate and fibroblasts. this alveolar proteinosis-type pattern of injury can be confirmed with immunohistochemical studies that establish the absence of sp-b within this surfactant-like material. diseases due to abca3 or sftpc deficiency may present within a week of birth or years later; the former has a poor prognosis, but the latter has a more variable prognosis with some patients surviving into adulthood. indeed, sp-c mutations have also been recognized in some families as a cause of interstitial pneumonia and pulmonary fibrosis in adults [285] . the pathology of sp-c deficiency has more cpi features and less proteinosis. in contrast, abca deficiency can have either pap or cpi features, with the former present early in the disease and the latter present in more chronically affected lungs [286] . the sp-b gene (sftpb) is approximately 10 kb in length and is located on chromosome 2. there are over 30 recessive loss-of-function mutations associated with the sftpb gene. however, the most common mutation is a gaa substitution for c in codon 121, found in about 70% of the cases. the lack of sp-b leads to an abnormal proportion of phosphatidylglycerol and an accumulation of a pro-sp-c peptide, leading to the alveolar proteinosis-like pathology. sp-c protein deficiency is due to a defect in the sftpc gene localized to human chromosome 8. there are approximately 35 dominantly expressed mutations in sftpc that result in acute and chronic lung disease. approximately 55% of them arise spontaneously, and the remainder are inherited. the most common mutation is a threonine substitution for isoleucine in codon 73 (i73t), found in 25% of the cases, including both sporadic and inherited disease [287] . this mutation leads to a misfolding of the sp-c protein, which inhibits its progression through the intracellular secretory pathway, usually within the golgi apparatus or the endoplasmic reticulum [288] . the absence of sp-c within the alveolar space causes severe lung disease in mouse models. infants with documented mutated prosp-c protein, the larger primary translation product from which sp-c is proteolytically cleaved, can have respiratory distress syndrome (rds) or cpi. in older individuals, pathologic patterns observed in the lungs with these mutations include nonspecific interstitial pneumonitis (nsip) and uip. in this affected adult population, the pathology and age of disease presentation vary even within familial cohorts, suggesting the involvement of a second hit, perhaps an environmental factor [289] . the abca3 protein is a member of the family of atpdependent transporters, which includes the cftr, and is expressed in epithelial cells. mutation in this gene results in severe respiratory failure that is refractory to surfactant replacement. the cellular basis for the lack of surfactant in patients with this genetic mutation is not known. the presence of abnormal lamellar bodies within the type 2 cells by ultrastructural analysis suggests a disruption in the normal surfactant synthesis and packaging in this disease. there is some evidence that this gene contains promoters that share elements consistent with their activation by the transcription factors ttf-1 and foxa7, and deletions in either or both of these genes may play a role in this disease [289] . annual report to the nation on the status of cancer pathology and genetics of tumours of the lung, pleura ajcc cancer staging manual varying), national cancer institute, dccps, surveillance research program, cancer statistics branch, released diagnosis and management of lung cancer executive summary: accp evidence-based clinical practice guidelines genetics of preneoplasia: lessons from lung cancer allelotyping demonstrates common and distinct patterns of chromosomal loss in human lung cancer types genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering high resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array cgh lung cancer preneoplasia characterizing the cancer genome in lung adenocarcinoma focus on lung cancer new molecularly targeted therapies for lung cancer high resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints genetic and molecular alterations allelic losses at chromosome 8p21-23 are early and frequent events in the pathogenesis of lung cancer sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma hereditary cancers disclose a class of cancer genes tobacco smoke carcinogens, dna damage and p53 mutations in smoking-associated cancers molecular oncogenesis of lung cancer molecular genetics of lung cancer the p16/cyclin d1/rb pathway in neuroendocrine tumors of the lung smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer targeting mtor signaling in lung cancer the potential role of mtor inhibitors in non-small cell lung cancer her-2/neu expression in archival non-small cell lung carcinomas using fdaapproved hercep test epidermal growth factor family of receptors in preneoplasia and lung cancer: perspectives for targeted therapies lack of trastuzumab activity in nonsmall cell lung carcinoma with overexpression of erb-b2: 39810: a phase ii trial of cancer and farnesyl transferase inhibitors for patients with lung cancer angiogenesis inhibitors in the treatment of lung cancer antiangiogenic therapy in nonsmall cell lung cancer micrornas as oncogenes and tumor suppressors pathology and genetics of tumours of the lung, pleura, thymus and heart lung cancer associated with several connective tissue diseases: with a review of literature atypical adenomatous hyperplasia atypical adenomatous hyperplasia of the lung in autopsy cases identification of bronchioalveolar stem cells in normal lung and lung cancer genomic profiling identifies titf1 as a lineage-specific oncogene amplified in lung cancer clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers exclusive mutation in epidermal growth factor receptor gene, her-2, and kras, and synchronous methylation of nonsmall cell lung cancer somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers distinct epidermal growth factor receptor and kras mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features combination of egfr gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib somatic mutations of the her2 kinase domain in lung adenocarcinomas the role of dna methylation in the development and progression of lung adenocarcinoma mutational and epigenetic evidence for independent pathways for lung adenocarcinomas arising in smokers and never smokers squamous cell carcinoma squamous dysplasia and carcinoma in situ lung cancer and lung stem cells: strange bedfellows? follow-up of bronchial precancerous lesions and carcinoma in situ using fluorescence endoscopy the natural course of preneoplastic lesions in bronchial epithelium surveillance for the detection of early lung cancer in patients with bronchial dysplasia large cell carcinoma molecular pathology of large cell carcinoma and its precursors karyotypic characterization of bronchial large cell carcinomas abnormalities of epidermal growth factor receptor in lung squamous-cell carcinomas, adenosquamous carcinomas, and large-cell carcinomas: tyrosine kinase domain mutations are not rare in tumors with an adenocarcinoma component egfr mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid the concept of pulmonary neuroendocrine tumours small cell carcinoma pulmonary carcinoid: presentation, diagnosis, and outcome in 142 cases in israel and review of 640 cases from the literature pulmonary neuroendocrine tumors: incidence and prognosis of histological subtypes. a population-based study in denmark diffuse idiopathic pulmonary neuroendocrine cell hyperplasia the insulinoma-associated 1: a novel promoter for targeted cancer gene therapy for small-cell lung cancer hash1 expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors ttf-1 expression is specific for lung primary in typical and atypical carcinoids: ttf-1-positive carcinoids are predominantly in peripheral location usefulness of cdx2 and ttf-1 in differentiating gastrointestinal from pulmonary carcinoids expression of thyroid transcription factor-1 in the spectrum of neuroendocrine cell lung proliferations with special interest in carcinoids cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors small cell carcinoma chromosomal imbalances in human lung cancer small-cell lung cancer is characterized by a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q and 17p neuro-endocrine tumours of the lung. a review of relevant pathological and molecular data tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of myc family gene amplification genetic changes in the spectrum of neuroendocrine lung tumors identification of men1 gene mutations in sporadic carcinoid tumors of the lung men1 gene mutation analysis of high-grade neuroendocrine lung carcinoma mechanisms of disease: multiple endocrine neoplasia type 1-relation to chromatin modifications and transcription regulation molecular genetics of small cell lung carcinoma apoptosis-related factors p53, bcl2, and bax in neuroendocrine lung tumors analysis of p53, k-ras-2, and c-raf-1 in pulmonary neuroendocrine tumors. correlation with histological subtype and clinical outcome neuroendocrine carcinomas and precursors rb and cyclin dependent kinase pathways: defining a distinction between rb and p16 loss in lung cancer loss of heterozygosity at the rb locus correlates with loss of rb protein in primary malignant neuro-endocrine lung carcinomas distinct pattern of e2f1 expression in human lung tumours: e2f1 is upregulated in small cell lung carcinoma e2f-1, skp2 and cyclin e oncoproteins are upregulated and directly correlated in high-grade neuroendocrine lung tumors telomerase activity in small-cell and non-small-cell lung cancers differential expression of telomerase reverse transcriptase (htert) in lung tumours lack of telomerase activity in lung carcinoids is dependent on human telomerase reverse transcriptase transcription and alternative splicing and is associated with long telomeres telomere length, telomerase activity, and expressions of human telomerase mrna component (hterc) and human telomerase reverse transcriptase (htert) mrna in pulmonary neuroendocrine tumors genetics of neuroendocrine and carcinoid tumours epigenetic inactivation of rassf1a in lung and breast cancers and malignant phenotype suppression dna methylation profiles of lung tumors differential inactivation of caspase-8 in lung cancers molecular changes in the bronchial epithelium of patients with small cell lung cancer 11q13 allelic imbalance discriminates pulmonary carcinoids from tumorlets. a microdissection-based genotyping approach useful in clinical practice inflammatory myofibroblastic tumour mesenchymal tumours alk1 and p80 expression and chromosomal rearrangements involving 2p23 in inflammatory myofibroblastic tumor tpm3-alk and tpm4-alk oncogenes in inflammatory myofibroblastic tumors primary intrathoracic synovial sarcoma: a clinicopathologic study of 40 t(x;18)-positive cases from the french sarcoma group and the mesopath group primary pulmonary and mediastinal synovial sarcoma: a clinicopathologic study of 60 cases and comparison with five prior series a high frequency of tumors with rearrangements of genes of the hmgi(y) family in a series of 191 pulmonary chondroid hamartomas chromosomal translocations in benign tumors: the hmgi proteins changing trends in us mesothelioma incidence pathology and genetics of tumours of the lung, pleura, thymus and heart sv40 and the pathogenesis of mesothelioma advances in malignant mesothelioma surgical management of malignant pleural mesothelioma: a systematic review and evidence summary application of immunohistochemistry to the diagnosis of malignant mesothelioma cellular and molecular parameters of mesothelioma diffuse malignant mesothelioma: genetic pathways and mechanisms of oncogenesis of asbestos and other agents that cause mesotheliomas asbestos, chromosomal deletions, and tumor suppressor gene alterations in human malignant mesothelioma cytogenetic and molecular genetic changes in malignant mesothelioma neurofibromatosis type 2 (nf2) gene is somatically mutated in mesothelioma but not in lung cancer advances in the molecular biology of malignant mesothelioma update on the molecular biology of malignant mesothelioma human mesothelioma cells exhibit tumor cell-specific differences in phosphatidylinositol 3-kinase/akt activity that predict the efficacy of onconase gefitinib in patients with malignant mesothelioma: a phase ii study by the cancer and leukemia group b common egfr mutations conferring sensitivity to gefitinib in lung adenocarcinoma are not prevalent in human malignant mesothelioma new insights into the pathogenesis of asthma placebo-controlled immunopathologic study of four months of inhaled corticosteroids in asthma the pharmacogenetics of asthma: an update genetics and the variability of treatment response in asthma immunology of asthma and chronic obstructive pulmonary disease pathogensis of asthma superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity the role of the mast cell in the pathophysiology of asthma global initiative for chronic obstructive lung disease (gold): global strategy for the diagnosis mocopd mechanisms of cigarette smokeinduced copd: insights from animal models the definition of emphysema: report of the national heart, lung and blood institute division of lung diseases workshop frontiers in emphysema research advances in the pathology of copd. histopath pathology of chronic airflow obstruction molecular pathogenesis of emphysema alpha 1-antitrypsin pi-types in 965 copd patients alpha-1-antitrypsin deficiency: pathogenesis, clinical presentation, diagnosis, and treatment alpha-1-antitrypsin deficiency: current concepts z-type alpha 1-antitrypsin is less competent than m1-type alpha 1-antitrypsin as an inhibitor of neutrophil elastase mediators of chronic obstructive pulmonary disease oxidative stress and lung inflammation in airways disease oxidative stress and redox regulation of lung inflammation of copd genetics of chronic obstructive pulmonary disease congenital bronchiectasis due to deficiency of bronchial cartilage (williams-campbell syndrome): a case report tracheobronchomegaly-the mounier-kuhn syndrome: report of two cases and review of the literature a human syndrome caused by immotile cilia genetic causes of bronchiectasis: primary ciliary dyskinesia cystic fibrosis identification of the cystic fibrosis gene: cloning and characterization of complementary dna genetic causes of bronchiectasis: primary immune deficiencies and the lung emerging and unusual gram-negative infections in cystic fibrosis altered respiratory epithelial cell cytokine production in cystic fibrosis imaging of idiopathic interstitial pneumonias the interstitial pneumonias and by the ers executive committee the genetic approach in pulmonary fibrosis idiopathic pulmonary fibrosis: clinical relevance of pathologic classification pathology of advanced interstitial diseases: pulmonary fibrosis, sarcoidosis, histiocytosis x, autoimmune pulmonary disease, lymphangioleiomyomatosis idiopathic pulmonary fibrosis: multiple causes and multiple mechanisms? role of epithelial cells in idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis: new insights into pathogenesis aberrant wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ telomerase mutations in families with idiopathic pulmonary fibrosis correlative study of adult respiratory distress syndrome by light, scanning, and transmission electron microscopy pulmonary pathology of the adult respiratory distress syndrome roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome acute lung injury and cell death: how many ways can cells die? lymphangioleiomyomatosis: clinical course in 32 patients lymphangioleiomyomatosis: a clinical update pulmonary lymphangiomyomatosis: correlation of ct with radiographic and functional findings clinical and molecular insights into lymphangioleiomyomatosis. sarc vasc dif lung dis markers of cell proliferation and expression of melanosomal antigen in lymphangioleiomyomatosis smooth muscle-like cells in pulmonary lymphangioleiomyomatosis enter line ht. lymphangiomyoma, a benign lesion of chyliferous lymphatics synonymous with lymphangiopericytoma molecular pathogenesis of lymphangioleiomyomatosis: lessons learned from orphans current concepts of the pathogenesis of sarcoidosis pulmonary sarcoidosis the morbid anatomy of sarcoidosis sarcoidosis: a clinicopathologic review of three hundred cases, including twenty-two autospies pathology of sarcoidosis what is sarcoidosis? gene-environment interactions in sarcoidosis: challenge and opportunity epidemiology, demographics and genetics of sarcoidosis familial aggregation of sarcoidosis. a case-control etiologic study of sarcoidosis (access) genetic basis of remitting sarcoidosis: triumph of the trimolecular complex? increased hla-b7 antigen frequency in south carolina blacks in association with sarcoidosis human leukocyte antigen class i alleles and the disease course in sarcoidosis patients hla and sarcoidosis in the japanese analysis of restriction fragment length polymorphism for the hla-dr gene in japanese familial sarcoidosis is linked to the major results from a genome-wide search for predisposing genes in sarcoidosis a search for mycobacterial dna in granulomatous tissues from patients with sarcoidosis using the polymerase chain reaction molecular analysis of sarcoidosis and control tissues for mycobacteria dna difficult treatment issues in sarcoidosis pulmonary fibrosis of sarcoidosis. new approaches, old ideas pulmonary alveolar proteinosis pulmonary alveolar proteinosis granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis pulmonary alveolar phospholipoproteinosis: experience with 34 cases and a review pulmonary alveolar proteinosis: recent advances unsuspected pulmonary alveolar proteinosis complicating acute myelogenous leukemia alveolar proteinosis as a consequence of immunosuppression. a hypothesis based on clinical and pathologic observations crazy-paving appearance at thin-section ct: spectrum of disease and pathologic findings surfactant proteins in pulmonary alveolar proteinosis in adults pulmonary alveolar proteinosis. staining for surfactant apoprotein in alveolar proteinosis and in conditions simulating it surfactant apoprotein in nonmalignant pulmonary disorders regulation of surfactant secretion in alveolar type ii cells gm-csf regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology pulmonary alveolar proteinosis is a disease of decreased availability of gm-csf rather than an intrinsic cellular defect autoantibodies against granulocyte-macrophage colony-stimulating factors are diagnostic for pulmonary alveolar proteinosis idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in japan 1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor gm-csf regulates alveolar macrophage differentiation and innate immunity in the lung through pu peroxisome proliferatoractivated receptor-gamma is deficient in alveolar macrophages from patients with alveolar proteinosis clinical classification of pulmonary hypertension narrative review: the enigma of pulmonary art genetics and mediators in pulmonary arterial hypertension primary pulmonary hypertension update in pulmonary arterial hypertension recent advances in the diagnosis of churg-strauss syndrome new insight into the pathogenesis of vasculitis associated with antineutrophil cytoplasmic autoantibodies defensins: antimicrobial peptides of innate immunity innate immunity in the lung: how epithelial cells fight against respiratory pathogens strategic targets of essential hostpathogen interactions bacterial infections worldwide haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates mycoplasma pneumoniae and its role as a human pathogen the clinical spectrum of primary tuberculosis in adults. confusion with reinfection in the pathogenesis of chronic tuberulosis exogenous reinfection as a cause of recurrent tuberculosis after curative treatment the natural history of the tuberculous pulmonary lesion nonbacterial pneumonia pulmonary infections in transplantation pathology retrospective diagnosis of hanta-virus pulmonary syndrome analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis viral respiratory tract infections in transplant patinets histoplasmosis and blastomycosis epidemiological and clinical featues of pulmonary blastomycosis current concepts: coccidioidomycosis pathology of infectious diseases the histologic spectrum of necrotizing forms of pulmonary aspergillosis ribosomal rna sequence shows pneumocystis carinii to be a member of the fungi a new name (pneumocystis jiroveci) for pneumocystis from humans strain typing methods and molecular epidemiology of pneumocystis pneumonia advances in the biology, pathogenesis and identification of pneumocystis pneumonia pneumocystis pneumonia mechanisms in the pathogenesis of asbestosis and silicosis cells of the dendritic cell lineage in human lung carcinomas and pulmonary histiocytosis s pulmonary langerhans' cell histiocytosis surface phenotype of langerhans cells and lymphocytes in granulomatous lesions from patients with pulmonary histiocytosis x pulmonary dendritic cells aberrant chemokine receptor expression and chemokine production by langerhans cells underlies the pathogenesis of langerhans cell histiocytosis transgenic expression of granulocyte-macrophage colony-stimulating factor induces the differentiation and activation of a novel dendritic cell population in the lung pulmonary langerhans cell histiocytosis: molecular analysis of clonality genotypic analysis of pulmonary langerhans cell histiocytosis adult pulmonary langerhans cell histiocytosis erdheim-chester disease: clinical, radiologic, and histopathologic findings in five patients with interstitial lung disease erdheim-chester disease. clinical and radiologic characteristics of 59 cases erdheim-chester disease with prominent pulmonary involvement associated with eosinophilic granuloma of mandibular bone diffuse interstitial pneumonia revealing erdheim-chester's disease erdheim-chester disease erdheim-chester disease: a rare multisystem histiocytic disorder associated with interstitial lung disease fulminant multisystem non-langerhans cell histiocytic proliferation with hemophagocytosis: a variant form of erdheim-chester disease pulmonary pathology of erdheim-chester disease erdheim-chester disease: a primary macrophage cell disorder treatment of erdheim-chester disease with cladribine: a rational approach pulmonary involvement with erdheim-chester disease: radiographic and ct findings chester-erdheim disease: a neoplastic disorder the pathology of asbestos-associated disease of the lung and pleural cavities: diagnostic critera cellular and molecular mechanisms of asbestos-induced fibrosis essential role of ros-mediated nfat activation in tnf-alpha induction by crystalline silica exposure cell-type-specific regulation of the human tumor necrosis factor alpha gene in b cells and t cells by nfatp and atf-2/jun bronchogenic cysts: clinicopathological presentation and treatment cystic lesions of the lung in children: classification and controversies transcriptional control of lung morphogenesis nonspecific interstitial pneumonia and usual interstitial pneumonia with mutation in surfactant protein c in familial pulmonary fibrosis abca3 mutations associated with pediatric interstitial lung disease a common mutation in the surfactant protein c gene associated with lung disease genetic disorders of surfactant proteins genetic disorders of surfactant homeostasis pathology and genetics of tumours of the lung, pleura key: cord-023306-3gdfo6vd authors: nan title: tsanz oral abstracts date: 2010-03-01 journal: respirology doi: 10.1111/j.1440-1843.2010.01735.x sha: doc_id: 23306 cord_uid: 3gdfo6vd nan introduction very little is known about adult health of survivors of extreme preterm birth. the aim of this study was to assess the burden of respiratory symptoms in a cohort of young adults born prematurely compared to sibling controls. method one hundred and fifty six children born prematurely (26-33 weeks gestation) at the mater hospital brisbane between [1989] [1990] were mailed questionnaires to assess their respiratory symptoms using a modified version of the european community health survey. term-born siblings were invited to act as controls. results thirty six responses were received (23%). the studied cohort consisted of 36 cases (64% female) and 17 controls (59% female). the median age was 19 years (18 -21) in the cases and 18 years (16 -27) in the control group (p = ns). shortness of breath (sob) was reported in 25% of the preterm cases, but nil in the control group (p = 0.044). there was a higher incidence of day/night cough (50% vs. 13%, p = 0.0135) and morning cough (37% vs. 0%, p = 0.0045) in the preterm cases compared to controls. the preterm cases were also more likely to experience a chest infection before the age of five (38% vs. 13% of controls, p = 0.046). sob was unrelated to a history of asthma, atopy, exposure to smoke or domestic animals as there was no difference between controls and cases. conclusion a higher incidence of sob was reported in young adults who were born prematurely compared to sibling matched controls, and this appears to be unrelated to asthma and atopy. further subjects will be enrolled interstate to assess predictors of respiratory symptoms in early adulthood. background currently all protocols for checking patient readiness for oral intake post fibre optic bronchoscopy (fob) are non-evidence based. there is a need to establish the shortest safe time to implement resumption of oral intake following administration of local anaesthesia for various reasons. we examined return times for the gag reflex and swallow response. method a prospective study of 100 consecutive patients presenting for a fob, age 18-86, were assessed for optimum time to check the gag and swallow reflex. the gag was checked pre and post fob by the touch method (tickle back of throat), swallow was checked post fob with a sip of water at various times. results after 1 hour 67% and 90% of patients had a gag reflex (n = 82) and swallow response (n = 100) respectively. this increased to 88% and 96% at 1½ hours, 95% and 99% at 2 hours, 100% and 100% at 2½ hours. the amount of sedation or length of procedure did not correlate with the return of the gag reflex or swallow response. none of the patients who swallowed with the gag still absent coughed or aspirated. eighteen patients did not have a gag reflex pre fob. conclusion the gag reflex and swallow response are separate. data shows it is possible to swallow safely without a gag reflex. the time to safe swallow is much shorter than recommended in current protocols. however, we consider it safer to allow oral intake when both reflexes are present, if the gag is present pre fob. otherwise the patient should wait for 1½ hours post fob as 88% have a returned gag reflex, if swallow response is also back. nomination none. introduction home monitoring in copd may identify acute exacerbations earlier, enabling prompt treatment, thus improving morbidity and mortality. reactance (x rs ), measured by forced oscillation technique has a potential role in home monitoring. the aim of this study was to determine within-and between-day repeatability of resistance (r rs ), x rs , and spirometry in stable copd subjects. methods ten copd subjects underwent seven consecutive home visits consisting of three measures of fot (1 minute recording) and spirometry before and after 200 mcg ventolin via spacer. results subject characteristics mean (sd) -age 74.7 years (7.43), smoking history 48.6 pack years (17.3), post-bd (post-bronchodilator) fev1 51.1% predicted (16.3), fev1/fvc ratio 0.4 (0.11). repeatability measures are reported as intra-class correlation coefficient (icc) and sd of within subject variance (sw). introduction chronic obstructive pulmonary disease (copd) is the single most important risk factor for lung cancer (affecting 50-90% of those diagnosed). considerable overlap exists between smokers who develop copd and/or lung cancer, suggesting involvement of shared pathogenic pathways (inflammation, matrix remodeling and cell death). in a prospective study of high and low risk smokers we have combined snps from gwas and candidate gene studies to develop a susceptibility score for lung cancer (lcss). methods seven hundred and twenty eight high risk individuals (chronic smokers, >40years old, >20 pack years, spirometric confirmed copd), and 484 smokers without copd were recruited and followed for a mean of 5 years. cohorts were matched for smoking history, ethnicity, gender and age, thereby excluding confounding from these variables. all volunteers completed spirometry, modified ats respiratory questionnaire and gave blood for dna. iplex and taqman systems were used to genotype the snp panel. cases are in the high risk (copd) cohort (6% over 5 years, mean score = 6.3) and 11 (21%) from the low risk cohort, normal lung function (2% over 5 years, mean score 3.4). the healthy unaffected smokers' mean score was 2.3. this prospective study confirms the risk status assigned by the lcss with 41 (6%) vs. 11 (2%) lung cancers over the 5 year follow up (or = 2.6 (%% ci 1.3-5.4, p = 0.005).the performance characteristics of the lcss reported here, confirm its utility, in correctly identifying smokers at greatest lung cancer risk. conclusion in this prospective study, we show that the lcss identifies those at greatest risk of lung cancer who might benefit from aggressive preventive strategies such as cessation and chemoprevention. the author is not aware of any conflict of interest. malignant mesothelioma (mm) remains an incurable cancer and its global incidence is rising rapidly. alternative therapeutic strategies are therefore required. bacterial products have been trialled in an effort to enhance local immunity and have demonstrated tumouricidal activity. staphylococcal enterotoxins (se) are classic models of superantigens that have potent mitogenic activity on t cells and demonstrated anti-tumour effects in several cancer models. intrapleural delivery of staphylococcal enterotoxin c (sec) has been used in china for many years as a pleurodesing agent. however, it is unknown whether sec actually kills cancer cells. in this study, we examined the efficacy of sec in the treatment of mm. sec was added at various concentrations (0-10 ng/ml) to several human and murine mm cell lines and a human benign mesothelial cell line in vitro. dose dependant cytotoxicity was observed in all cell lines resulting in a significant reduction in viability at higher doses (10 ng/ml) when using trypan blue exclusion and wst-1 assays (0.001 < p < 0.05). in an effort to elucidate the mechanism of action of sec, annexin v staining and flow cytometry were used to measure apoptosis. results demonstrated a significant increase in apoptosis in mm cells when treated with sec compared to untreated controls (0.001 < p < 0.05). on the contrary, benign mesothelial cells appeared to be resistant to the apoptotic effects of sec at equivalent concentrations (p > 0.05). elisa based assays were used to examine cytokine profiles in culture supernatants of sec treated mm cells and benign mesothelial cells. levels of the pro-inflammatory cytokine il-8 decreased in sec treated mm cells (0.001 < p < 0.01) compared to a significant increase in levels observed in benign mesothelial cells (0.01 < p < 0.05). these results suggest that sec kills mm cells in vitro with some specificity and its activity against mm in vivo warrants investigation. conflict of interest no. purpose we examined age trends in the distribution of stage at diagnosis in patients presenting with non-small cell lung cancer (nsclc) at tertiary hospitals. methods we used the queensland integrated lung cancer outcomes project (qilcop), a clinical registry which collects information on about 40% of all lung cancer patients in queensland, to analyse the distribution of clinical (tnm) stage among 3,283 patients diagnosed with nsclc between 2000 and 2005. differences in stage distribution across age were analysed using tests of proportions and multivariable logistic regression with stage as the dependent variable and other demographic characteristics as covariates. results the median age at diagnosis of patients in the study was 68 years (range 26-95) and 68% were males. the overall proportions of stages i, ii, iii, and iv were, respectively, 30%, 9%, 29%, and 32%. the percentage of stage i disease increased with age (p < 0.001), from 19% in those younger than 55 years to 26%, 31%, and 37% in those aged 55-64, 65-75, and 75 years or older, respectively. age differences in stage distribution remained significant in multivariable analysis controlling for gender, rural residence, and socioeconomic status. among the other characteristics, only gender differences in stage was significant, with stage i cancer being more common in women compared to men (34% vs. 29%, p = 0.02). australia has the world's highest incidence of mesothelioma, a disease which has no proven effective therapy. the median survival of less than 12 months and five year survival of 5% have not changed in two decades. radical resection has a high attrition rate with most cases recurring locally, and few patients have durable responses to chemotherapy. symptoms are related to local disease which compresses the lung and causes severe chest pain from enlarging tumour masses. aim to improve local control of mesothelioma by high dose radiotherapy using advanced technologies that precisely define the active tumour and reduce toxicity to normal tissues. methods all patients had 18 fdg pet scans co-registered with a simulation ct scan to define the target volume, and follow-up pet scans were analysed to assess the residual total glycolytic volumes (tgv) after radiotherapy. acute and long term toxicities were assessed. results between 2003 and 2009 thirty patients with incompletely resected pleural mesothelioma were treated with radiation doses of 30 to 60 gy to part or all of one hemithorax. all patients who received chemotherapy had progressed prior to radiotherapy. in 2006 we introduced a program using a new technique called intensity-modulated radiotherapy (imrt). tgvs reduced by 60% after radiotherapy, median survival was extended to 24 months and there were no major radiation toxicities, the most common being grade two pneumonitis. relapses were frequent on extended follow-up, the majority in areas outside the radiotherapy field. conclusions imrt is effective in maximising local control in mesothelioma patients who have had extrapleural pneumonectomies, and for selected patients with an intact lung. toxicities are manageable and locoregional control is very good. high dose radiotherapy is recommended for most mesothelioma patients for long term palliation and control of locoregional progression. introduction mortality benefits for ldct screening are not yet known. the queensland lung cancer screening study is screening up to 750 high risk volunteers based on the nlst/acrin protocol. aims observational cohort study to assess: disease detection rate; lung nodule work-up; cost; quality of life issues; smoking cessation; biomarker collection feasibility. methods recruitment via local advertisement and press release. major inclusion criteria: age 60-74 years; smoking history ‡30 pack years; fit for surgery. volunteers have one prevalence and two incidence scans and follow-up for three more years. ldct parameters: phillips brilliance 64 slice multidetector scanner; low-dose protocol; 0.9mm slice width. scan reporting: two radiologists independently; independent cad reading (phillips brilliance software); final report is agreed by consensus. results see table 1 -to be updated. magnetic resonance imaging (mri) is a useful modality for assessing chronic thromboembolic pulmonary hypertension (cteph) before pulmonary endarterectomy (pea). cardiac mri provides more accurate right ventricular (rv) data than echocardiography. mr angiography demonstrates vascular changes reliably to a segmental level and mr perfusion shows disease distribution. aim to examine the relationship between changes in mri parameters with clinical and haemodynamic outcomes post-pea. methods rv end-diastolic volume (rvedv), rv ejection fraction (rvef), vessel abnormalities and lobar perfusion defects were determined with mri before and after peas performed during [2004] [2005] [2006] [2007] . changes in new york heart association (nyha) functional class, six minute walk distance (6mwd), mean pulmonary artery pressure (mpap) and cardiac output (co) were collected retrospectively from patient charts. results nineteen patients assessed pre-pea were of mean ± sd age 57 ± 12 years, nyha class 2.8 ± 0.7, 6mwd 414 ± 103 metre and mpap 42±10 mmhg. immediately post-pea, mpap fell by 13 ± 7 mmhg which was related to changes in rvedv of 26 ± 15% (r 2 = 0.38, p = 0.006). at 6-12 months post-pea, 6mwd improvement (115 ± 99 metre) was related to changes in rvef of 28±49% (r 2 = 0.40, p = 0.007) but angiographic changes had a weak relationship with nyha class shift (r 2 = 0.22, p = 0.034). perfusion generally improved after pea relating weakly with rvedv (r 2 = 0.3, p = 0.032) but not clinical outcomes. conclusions this study shows that improvements in mri parameters (rv data more than angiographic findings) after pea correspond to clinical and haemodynamic outcomes. we have demonstrated a useful imaging test for monitoring patients post-pea with no radiation exposure. purpose allergic reactions to antibiotics are very common in cystic fibrosis (cf) patients and can complicate treatment in patients with multi-or pan-resistant bacterial species. we hypothesised that post-transplant immunosuppression may reduce the requirement for desensitisation. methods and materials a retrospective review was performed in june 2009 to detect prescribing practices and any changes in allergy patterns before and following lung transplantation in cf. since antibiotic desensitisation has not been used post-transplant at our institution, our aim was to review our experience with antibiotic rechallenge, without desensitisation, in the post-transplant setting. results fifty eight cf patients, 34 (58%) female, aged 34 years, range 15-50 years, 3 heart-lung, 3 heart-lung liver have undergone transplantation at our institution. 36 (62%) had a pre-transplant history of ige (angioedema -8%) or non-ige (e.g. rash, nausea, arthralgia or liver dysfunction -92%) mediated reactions to at least one antibiotic (29 % penicillin, 21 % cephalosporin, 13 % carbapenem, 9% aztreonam and 28% others). desensitization to antibiotics with non ige mediated reaction was attempted in 9 out of the 36 patients pre-transplant and successful on all occasions. in 27 other patients, alternative antibiotics were selected and desensitisation was not required. after transplantation, 18 (50%) patients with non ige mediated reactions were rechallenged without desensitisation on 25 occasions. no life-threatening reactions were observed. only two episodes required antibiotic cessation and both recovered without incident. new onset of adverse reaction to iv colistin and voriconazole occurred in two patients following transplantation. conclusions cautious antibiotic rechallenge can be successfully achieved without desensitisation in the majority of patients who have had non ige mediated allergic phenomena to the same compound prior to transplantation. idiopathic pulmonary fibrosis (ipf) is characterised by marked collagen deposition. the receptor subunit gp130 has been associated with the progression of fibrosis. the interleukin (il)-6 family of cytokines all require gp130 to initiate signal transduction to activate either the extracellular regulated kinase (erk) or signal transducer and activator of transcription (stat) pathways. the il-6 family of cytokines consist of il-6, il-11, oncostatin m (osm), leukaemia inhibitory factor (lif), cardiotrophin-1 (ct-1), ciliary neurotrophic factor (cntf), il-27 and il-31. previous studies performed in our laboratory have demonstrated that exaggerated gp130-stat3 signalling is fundamental to the development of bleomycin-induced lung fibrosis in a murine model. we hypothesise that pulmonary fibrosis is mediated by il-6 family cytokine/gp130-stat1/3 signalling. the aim of the current study was to identify which of the il-6 family cytokines are important in the development of bleomycin-induced pulmonary fibrosis. bleomycin or control saline was administered intranasally to individual il-6 knockout (il-6 -/-) mice and dual il-6 and il-11 a-receptor knockout (il-6 -/-;il-11ar -/-) mice. collagen production was examined by histology and hplc in lung tissue 30 days post treatment. no significant increase in collagen was observed in bleomycin treated il-6 -/or il-6 -/-;il-11ar -/mice implicating a role for il-6 in the development of pulmonary fibrosis. interestingly, the histology of il-6 -/-;il-11ar -/mice displayed marked emphysema which was not observed in individual il-6 -/mice suggesting this is an il-11-mediated response. the role of il-6 family cytokines in proliferation, myofibroblast differentiation and collagen expression were examined using fibroblasts isolated from wildtype (wt) and genetically engineered mice containing point mutations to prevent gp130-erk1/2 signalling (gp130 757f ) or gp130-stat1/3 signalling (gp130 stat ). overall, there was no significant increase in proliferation 48 hours post cytokine stimulation as assessed by wst-1 reagent. il-6 and il-11 did not stimulate a-sma or collagen expression above control, measured by real time pcr. in conclusion, increasing evidence suggests that il-6 plays an important role in the development of bleomycininduced pulmonary fibrosis but this does not appear to be induced by direct effects of il-6 on fibroblast proliferation, differentiation or collagen production. mannose binding lectin (mbl) is a key mediator of innate immunity and efferocytosis (clearance of apoptotic cells) and is thus important in protecting against tissue damage. reduced mbl is implicated in airways disease including infection, copd and bos, however, 'normal' plasma levels of mbl are highly variable due genetic polymorphisms complicating correlation with disease processes. we have previously shown reduced mbl and defective efferocytosis in bal from patients with post-transplant bos, but there are conflicting reports of the link between low plasma mbl levels, increased complement activation and graft rejection. to compare mbl levels in the peripheral blood and bal compartments, we investigated mbl in paired plasma and bal from 46 lung transplant recipients (10 stable; 32 stable with infection, 7 with lymphocytic bronchiolitis and 10 with bos) and in plasma from 10 and bal from 17 controls. in plasma, mbl levels were highly variable. no significant differences were noted among the transplant groups although levels were significantly reduced in all transplant patients vs. controls. there was no correlation between mbl and time post-transplant or pre-transplant diagnosis. in bal, mbl levels were less variable and significantly reduced in patients with bos (mbl ng/ml: controls: 6.2 ± 1; stable 8.6 ± 3.8; stable infected 10.7 ± 2.7; bos 2.2 ± 1.7). interestingly, in all patients with lb, mbl levels were very low in both plasma (mean 35.8 ng/ml) and bal (3 ng/ml). low levels of mbl in the airway may play a role in reduced efferocytosis, leading to tissue damage and airways disease post-transplant. in normal subjects, external dead space with exercise is associated with a slower deeper breathing pattern compared at the same ventilation. with simulated lung restriction and constant intensity exercise, we tested the hypothesis that dead space combined with reduced exercise intensity to match ventilation, would alter pattern of breathing, reduce inspiratory reserve volume (irv) and thus increase dyspnea. methods eleven healthy male subjects, aged 28(8) (sd) years completed separate visits with (a) no restriction and (b) chest wall strapping to reduce fvc by 30 (7) introduction glossopharyngeal breathing (gpb) is used by competitive breath-hold divers to increase lung gas content above tlc to improve performance. this occurs by both lung expansion and gas compression. whilst gpb is known to induce hypotension and tachycardia, little is known about the changes that occur to both the pulmonary circulation and the structural integrity of the thorax. the aim of this study was to investigate these changes within an elite cohort. methods six male breath-hold divers were studied. exhaled vc was measured before and after gpb. subjects were studied in the supine position at baseline tlc and after maximal gpb above tlc at least 72 hours apart. tc 99 m labelled macro aggregated albumin was injected and a computed tomography (ct) of the thorax was performed during breath-hold. dynamic and single photon emission ct (spect) images were generated and analysed by two blinded nuclear medicine physicians for perfusion intensity (wilcoxon signed rank test) and dynamic regional blood flow. a paired t-test was used to assess physiological parameters. registered ct images were used to determine structural change in the thorax. results five subjects increased exhaled vc with gpb [mean (sd)] by 1.4 (0. 3) l (p < 0.001). there was a reduction in perfusion intensity following gpb in the anterior (p < 0.03) and inferior (p < 0.01) lung segments. there was no change in the timing of blood flow. 69% of the increase in expired lung volume above baseline tlc was via thoracic expansion (0.97 (0.3) l (p < 0.05)) with a caudal displacement of the diaphragm. one subject who was not proficient at gpb had no change in exhaled volume, ct appearance or lung perfusion. background we have developed a sensitive method to determine conductance-lung volume (conductance profile) and distensibility-lung volume (distensibility profile) relationships using the forced oscillation technique (fot). using this method, we aimed to assess the effect of a short-acting bronchodilator (bd) on these profiles in asthma. methods twenty two asthmatics and 20 healthy controls completed distensibility measurements (fot) and lung function tests before and after bd. the conductance and distensibility profiles were described continuously and determined at specific lung volumes, residual volume (rv), frc, tlc and midway between frc and tlc (mid). results following administration of bd: the conductance profile in the asthma group was shifted upwards, and the distensibility profile was altered such that significant increases in distensibility were observed at rv (p < 0.001) and frc (p < 0.01), but not at mid or tlc. in contrast, no changes were seen in the conductance or distensibility profiles in the control group. post-bd distensibility in asthma remained reduced compared to controls. conclusion using a sensitive method for determining conductance and distensibility profiles, we found that both conductance and distensibility are reduced in asthma across a range from low to high lung volumes. both these profiles are altered in asthma after bd but not in controls. we propose that in asthma the remaining deficit in distensibility after bd will provide unique insight into to altered airway mechanical function due to airway remodelling. cigarette smoke exposure is a major risk factor in susceptibility to serious respiratory infections, particularly in children. although smoke exposure is known to alter immunity to infection, the underlying molecular mechanisms are not well understood. aim identify regulatory mechanisms that drive impaired macrophage function. methods the mh-s alveolar macrophage cell line was exposed to a short 15 minute pulse of cigarette smoke extract (cse) prior to challenge with lps or fitc-e.coli. results cse blocked phagocytosis of e.coli and inhibited lps activation of canonical and alternative tlr4 pathways. both nfjb translocation and transactivation pathways were compromised as cse inhibited ijba degradation and p65 phosphorylation. cse also blocked ap-1 activity by inhibiting p38, but not jnk or erk1/2. we next excluded lps tolerance mechanisms involving receptor internalisation or induction of negative regulators. as free radical species are abundant in cse we investigated their role using the potent scavenger, reduced glutathione (gsh). since gsh restored all responses, we screened a panel of oxidative/nitrosative stress markers and identified carbonylation as the only cse inducible marker. oxyblot analysis confirmed that cse potently introduced carbonyl groups to many proteins (30-100 kda range) in a dose and time dependent manner that inversely correlated with tnf-a expression. cse treated macrophages also displayed heavily carbonylated pseudopodia that was reversed by gsh as determined by immunocytochemistry (icc). conclusion macrophage sensing and ingestion of pathogen is compromised by protein carbonylation of the outer membrane where phagocytic receptors cluster and also penetrates cytoplasmic regions where signalling moieties reside. therefore, targeting single pathways will not restore macrophage function due to the global nature of cse mediated carbonylation. support nhmrc. background asthma shows varying levels of resistance to effective treatment by glucocorticoids. in studies on tgfb-induced epithelial mesenchymal cell transition (emt) using the a549 type ii human epithelial cell line, the regulatory effect of the glucocorticoid, dexamethasone (dex, 0.1-1000 nm) on interleukin-1a (il-1a)-induced interleukin-8 (il-8) generation was markedly reduced in the presence of tgf-b 400 pm (n = 7; p < 0.05). aim our studies were designed to characterise the mechanism of the glucocorticoid resistance. results the resistance induced by tgf-b was: concentration dependent (4-400 pm); a glucocorticoid class effect as it also occurred with budesonide; independent of emt: it was observed within 4 hours, whereas emt requires 3 days; also observed in the central airway epithelial cell line, beas-2b and passaged, primary bronchial epithelial (nhbe) cells. treatment of a549 cells with sb431542, a tgfb receptor type i kinase inhibitor, restored the dex inhibitory effect on il-8 release (from control 17 ± 7% to 45 ± 7%, inhibition in sb431542 1 lm, n = 9; p < 0.05). in a549 cells transfected with a glucocorticoid response element (gre)-driven reporter gene, tgfb (40 pm) inhibited the gre response to dex (0.1-1000 nm) by more than 80%. in addition, tgf-b impaired dex regulation of the gre-dependent gene, ijb. pge2 plays a protective role in asthma by inhibiting airway inflammation. it is predominantly produced by epithelial cells in response to pro-inflammatory stimuli and acts as an autocrine and paracrine mediator. prostanoids have been shown to regulate expression of enzymes involved in their metabolism, as well as expression of their receptors and that regulation is tissue-and cell-specific. despite its importance, however, mechanisms underlying the regulation of expression of enzymes involved in pge2 metabolism and its receptors in human lung epithelial cells have remained elusive. therefore, we hypothesised that pge2 regulates expression of pge2 synthase 1 (pges1) and its receptors (e prostanoid (ep) 1-4) in human airway epithelial cells. methods real time rt pcr and facs analysis were used to assess mrna and protein expression, respectively in human airway epithelial cells 16 hbe before and after pge2 stimulation. results pge2 up-regulates pges1 in time and concentration dependant manner. in addition, ep receptors (ep1, ep2 and ep4) were up-regulated following pge2 stimulation at mrna level. however, these receptors show different dynamics in expression. while ep1 reaches peak in mrna expression at 6 hour, peak expression for ep2 and ep4 is at 12 hour post stimulation. monocyte derived dendritic cells (dcs) have been recognised for their potential role in immune responses and their functional relevance with regard to adaptive immune responses although more detailed knowledge of dc biology in human airways is required. the objective of this study was to modify the monocyte derived dcs from peripheral blood and direct the cells via exposure to pro-inflammatory conditions as seen in copd, with a view to identifying novel targets for cellular therapy. we characterised monocyte-derived dcs in culture and evaluated the effects of human rhinovirus infected bronchial epithelial cells, pi:c (polyinosine-polycytidytic acid) and bc (bacterial extract) on directing dc differentiation and maturation in culture. we found an impaired adaptive immune response, and in particular, an impaired cd8 effector cell function in copd patients. our dc culture results showed that both mhc-i and mhc-ii expression on dcs from copd were significantly down regulated compare to healthy controls, which could affect mhc restricted ag presentation, and lead to a failure to activate responder t cells. furthermore, we tested the capability of monocyte-precursors to differentiate into functional dcs. only a very small percentage of cultured monocytes from patients with copd was capable of differentiating into mature dcs, compared with healthy controls. during dc activation, there was up-regulation of co-stimulatory (cd80/86) and maturation markers (mhcs), enabling dc to activate naïve t cells in mixed lymphocyte culture both in copd patients and healthy controls. our preliminary data indicated that this activation leads to the generation of effector t cells, further study is needed. defective dc activation of t cells may underlie poor t cell responsiveness in copd in response to inflammation and may, in part, determine the response to therapy. our data suggest a promising role in vitro for pharmacologic treatment as a means of generating functional dcs and will further stimulate speculation regarding their potential clinical application. support nhmrc australia. anxiety and depression are the two most common and the least treated comorbidities associated with chronic obstructive pulmonary disease (copd). they have been found to have a statistically significant and independent adverse association with mortality, longer length of hospital stay, persistent smoking and worse physical and social function. evidence for various interventions to overcome these symptoms in copd is limited or inconclusive. methods this is a cochrane review. all randomised controlled trials (rcts) and cross over trials (cots) dealing with pharmacological and/or psychological interventions for anxiety and/or depression in adults with copd were considered. search was performed via various medical search engines and cochrane database register. duration of follow-up in the studies was generalised as short-term (0-3 months), medium-term (3-6 months) and long-term (6-12 months). the data was analysed using the fixed effect model and difference in symptom control by interventions as mean difference (md) or the standardised mean difference (smd) depending upon the heterogeneity of various scales used. results eight rcts/cots were included. pharmacotherapy showed nil significant effect to control anxiety (smd of -0.33, p value 0.16, n = 76), however, showed significant benefit in controlling depression symptoms (smd -0.57, p value 0.02, n = 76) in copd over short-term. on the other hand, cbt was found to be beneficial in controlling both anxiety (md -5.0, p value 0.04, n = 30) as well as depression (md -7.30, p value 0.02, n = 30) in copd over short term. conclusion cbt is superior as compared to pharmacotherapy over short-term to control anxiety and depression in copd. background the emergence of clonal pa and associated risk of cross-infection is a major cause for concern in cf centres in several countries, including australia. two clonal pa strains (aes1 and aes2) have been detected in several eastern australian cf centres, but the overall prevalence of these and other strains throughout australia remains unknown. methods a cross-sectional study involving 18 cf centres (8 paediatric, 9 adult, 1 combined) was performed. in total, 838 sputum-producing children and adults with documented pa infection provided two sputum samples, 6 months apart, and 3 pa isolates from each sample were genotyped by reppcr-based cluster analysis. results collectively, aes1 and/or aes2 strains were identified in 44% of pa infected cf patients and found in all participating cf centres. several other minor clonal strains were also identified in many centres. only 25% of patients were infected with unique (non-clonal) pa strains and 8% of patients were infected with more than one clonal strain. conclusion clonal pa strains are common in australian patients with cf. there is marked variation in prevalence of both major and minor clonal strains between cf centres and within states. longitudinal analysis of the clinical impact of clonal pa infection is urgently required so as to allow an evidence-based approach to patient management and infection control. reduced glutathione (gsh), a major component of anti-oxidant defence, is transported into the lung via cftr. gsh protects against myeloperoxidase (mpo)-induced oxidative stress by undergoing oxidation to gssg and gsa. excess mpo induces chlorination of tyrosine (3-cl-tyr) via the production of hypochlorous acid. we undertook a cross-sectional survey of young children with cf participating in our early surveillance program that includes annual bronchoalveolar lavage (bal) and chest ct scan. markers of neutrophilic inflammation and of the gsh system were determined in bal and the presence of bronchiectasis (b) and air trapping (at) determined on chest ct. 188 samples from children with cf (mean age 3.35 years) and nine samples from children without cf (mean age 6.13 years) undergoing investigation for chronic respiratory symptoms (ncf) were studied. cf samples had more neutrophils that ncf samples (geometric mean 42.6 vs. 3.0 · 10 3 /ml fluid retrieved), more mpo (239.4 vs. 0.31 ng/ml, p < 0.001), lower levels of gsh (1393.7 vs. 3285.1 nm, p < 0.001) and a trend for a lower gsh:gssg (46.2 vs. 95.2, p = 0.07). within the cf samples levels of mpo correlated with gssg (p = 0.01), and gsa (p < 0.001) and 3-cl-try correlated with gssg (p = 0.005) and gsa (p < 0.001) indicating neutrophilic inflammation exceeding anti-oxidant defence capability. however, after controlling for age and the presence of free neutrophil elastase, there were no relationships between gsh, gssg or gsa and either b or at. while our data demonstrate gsh deficiency and defective anti-oxidant defence, these are not related to structural lung disease. longitudinal studies will be required to determine the impact of gsh deficiency in the initiation and progression of lung disease in cf. support cfft, inc (usa); nhmrc, acfrt, mcri and pmh foundation (aust). introduction there are no australasian guidelines for the detection and eradication of pseudomonas aeruginosa in preschool children with cf. the optimal eradication regimen for preschool children remains uncertain. aims to develop australasian guidelines for p. aeruginosa detection and eradication in preschool children with cf based on a national multi-centre randomized control trial. methods an electronic web-based questionnaire was sent to every tertiary paediatric cf centre in australasia to determine current detection and eradication practices. results all eleven centres completed the survey. a combination of positive oropharyngeal culture (opc) and confirmatory bronchoalveolar lavage (bal) culture was the most common method of p. aeruginosa detection (55%), with surveillance frequencies varying; annually (n = 3, 27%), every clinic visit (3, 27%) and clinically indicated (4, 36.4%). eradication treatment was instigated on one positive culture (opc n = 5, bal culture n = 5 centres) for p. aeruginosa at any bacterial density (50%) or bacterial density ‡105 cfu/ml (40% of centres). eradication regimens varied between centres with most (81%) using intravenous antibiotics either alone (n = -1); in combination with 3-12 months nebulised antibiotics (n = 3); or with 1-3 months nebulised and oral antibiotics (n = 5). choice of regimen was influenced by clinical status in three centres. two centres used combinations of inhaled and oral antibiotics alone. failure to eradicate resulted in a change in treatment in 64% of centres. inhaled tobramycin 80 mg (28 days and 12 months) was considered the least acceptable regimen for preschool children in terms of efficacy and burden of care with most favouring more aggressive iv treatments. conclusion this survey supports a call for evidenced based australasian guidelines for the detection and eradication of p. aeruginosa in preschool children. comparative trials of the most favoured eradication regimens would enhance this process. pseudomonas aeruginosa is the most important respiratory pathogen in cystic fibrosis (cf). although unproven, it is generally thought to be acquired from the environment. however, molecular typing studies indicate person-to-person transmission by some clonal strains may also occur. clonal p.aeruginosa strains have not been compared previously with isolates collected from non-cf patients, animals or the environment. aim to determine sequence-based clonality of p.aeruginosa isolates collected from several different ecological niches. methods mlst and sequence type (st) analysis was performed on 519 isolates collected from cf patients (n = 216), non-cf patients (n = 116), animals (n = 107), and the natural environment (n = 80). cf isolates included each of the major and minor clonal australian strains and a range of unique strains isolated from patients residing in se qld. non-cf, animal and environmental isolates were collected from the same region. results of the 282 individual strains detected, 51 (18.1%) were found in more than one niche. overall, 29 unique and minor clonal cf strains were detected in at least one other niche; including 10 cf strains found in the environment. to date, none of the three major qld cf clonal strains have been detected in another niche. conclusions in cf, environmental exposure to p aeruginosa seems important for acquiring unique and minor clonal strains. finding that the three major clonal strains were confined to cf patients further suggests person-to-person transmission is occurring and/or strain associated adaptation to the cf lung. support nhmrc, acfrt, tpch foundation. background clostridium difficile colitis remains a rare but potentially life threatening complication in cf patients particularly in the post lung transplant setting. aim (1) to review the incidence of c. difficile colitis among non-transplant and post-lung transplant cf patients attending our centre. (2) to identify the clinical features in cf patients presenting with cdad. method retrospective study on c. difficile toxin status in all fecal samples collected in cf patients between 2000 to 2009 was reviewed. patients with positive c. difficile toxin were identified as index cases, those with negative samples were selected as control cases. results two hundred and twenty-three fecal samples were collected from 74 cf patients. nineteen cdad cases were identified in 15 patients including 13 non-transplant and two post-transplant patients. thirteen had mild colitis and two had fulminant colitis. incidence density of cdad in non-transplanted cf patients (2.8/100 000 patient-days) is comparable to the transplanted group (two episodes/ 100 000 patient-days). time to diagnose cdad among post-transplant patients is shorter than non-transplant patients (1 vs. 6 days). a significantly proportion in the cdad group had recent ciprofloxacin when comparied to controls ( 38.5% vs. 0 %, p-value: 0.0006.). a significantly higher proportion of patients in the cdad group are currently on gastric suppression therapy than control (92% vs. 59%, p-value: 0.03). conclusion the incidence density of cdad is comparable between pre and post transplant cf patients. cdad is more common among patients with prolonged ciprofloxacin treatment and concurrent gastric acid suppression. improving patient awareness by optimising patient education particularly during prescription of ciprofloxacin or gastric acid suppression treatment can prompt early presentation and management of cdad. conflict of interest no. g karmakar 1 , d milne 2 , m wilsher 1 1 green lane respiratory services, and 2 department of radiology, auckland city hospital, auckland, new zealand introduction major (massive or submissive) pulmonary embolism (pe) is a potentially lethal condition particularly if associated with cardiogenic shock. thrombolysis is accepted as standard treatment for massive pe with hypotension, but it carries substantial risk of bleeding and risk may outweigh the benefit in sub-massive pe. the objective of this study was to examine risk factors for and the outcome of major pe in this institution. methods we collected data retrospectively from all patients with mpe requiring icu admission in auckland city hospital (ach) over a 5 year period. the primary outcome variable was mortality with secondary variables including precipitating factors and morbidity. results twenty-eight subjects (12 massive pe) were identified. eight of 12 with massive pe were thrombolysed and four were treated conservatively with 30 day mortality of 50% and 25% respectively. twelve of 16 patients with sub-massive pe received thrombolysis with no mortality. significant bleeding complications were reported in four of 20 thrombolysed patients. prior surgery without dvt prophylaxis was identified as a precipitant in 10 patients. conclusions massive pe carries significant mortality irrespective of thrombolysis but such treatment appears safe in sub-massive pe. in spite of evidence of efficacy, failure to offer prophylaxis of dvt in the perioperative setting appears an ongoing risk factor for major pe. introduction there is a need to improve general understanding of the epidemiology, pathophysiology, outcome and therapies for rare (orphan) lung diseases. aims 1) to establish an electronic reporting registry of orphan lung diseases in australasia. 2) to provide a useful resource for physicians and patients. methods a website (www.arnold.org.au) was developed containing information on 30 orphan lung diseases, useful links and an on-line patient discussion forum. tsanz members were invited to participate in reporting cases electronically by a quarterly email reminder. the following data were entered: first two letters of given and family name, date of birth, postcode and whether the patient was new or old to the physician. results ethical approval in new zealand is still awaited. results twenty-three h1n1 cases of mean ± sd age 41.7 ± 7 years were confirmed from 826 known lt recipients (incidence 2.8%). cases peaked in new south wales (n = 10) and queensland (n = 6) consistent with epidemiology in the general community. clinical manifestations included allograft dysfunction in 17 of 23 (73.9%), upper respiratory tract symptoms only 26.1%, fever 52.6%, myalgias 68.4%, hypoxia 26.3% and radiological infiltrates 42.1%. mean hospitalisation was 11 ± 9.1 days. cases were diagnosed in the hospital setting (n = 16) and in the community (n = 7). other risk factors included obesity n = 7, diabetes n = 4 and malignancy n = 3. treatment consisted of oseltamivir 75 mg bd initially for 5 days in all 23 cases, extension beyond 5 days n = 14 (mean 8 ± 1.4 days) due to ongoing symptoms, steroid therapy n = 17, mechanical ventilation n = 1 and noninvasive support n = 2.5 patients (21.7%) have not returned to baseline lung function and there were four deaths (bos grade three n = 3 and grade 0 n = 1 prior to diagnosis). conclusions the incidence of h1n1 in the australian lt population was less than that estimated for the broader community but mirrored the geographical distribution. the majority experienced allograft dysfunction with most deaths recorded in those with preexisting bos grade 3. we recommend h1n1 vaccine (panvax, csl) for all lt recipients. as the only commonly transplanted organ exposed to the atmosphere, the lung allograft may be uniquely susceptible to iga deficiency. since igg deficiency is common after transplantation, we hypothesised that iga production may be similarly affected and may contribute to bos pathogenesis. methods we conducted a cross-sectional evaluation of our transplant cohort. total iga, igm, igg, and igg subclasses were measured in serum using elisa. demographic data, immunosuppression and bos status were recorded. results one hundred and twenty three patients (85% of the total cohort, aged 43.7 years (range 13.6-64.9), 49% female, 39% cf, 39% copd) were evaluated. the median iga level was 1.5 g/l (normal range 1-4 g/l). 33 patients (27%) were iga deficient, and 7 (6%) of these were pan-hypogammaglobulinaemic. iga levels were lower in patients with bos (median, iq range; 1.1, 0.8-1.7 vs. 1.6, 1.0-2.2, p = 0.006). iga deficiency was not associated with age, sex, diagnosis, transplant type or time post-transplant. the median igg level was 7.6 g/l (normal range 7-16 g/l) and 52 patients (42%) were deficient. igg levels were lower in bos (median, iq range; 6.6, 5.6-8.0 vs. 8.6, 6.3-10.5, p = 0.002) and igg deficiency was more common in copd (60%, p = 0.01). igg levels were negatively correlated with age (r 2 = 0.20, p = 0.03). multivariate logistic regression models identified iga deficiency as independently associated with bos (p = 0.04), while the association of igg deficiency with bos was explained by interaction between bos, age and copd. igm deficiency was present in 17 patients (14%) and was more common in males (76%, p < 0.01), but was not significantly different in bos. igg, iga and igm levels were not associated with immunosuppression or the use of basiliximab induction conclusions serum iga deficiency is common after transplantation and is independently associated with bos. impaired defence of mucosal surfaces because of iga deficiency may contribute to bos pathogenesis. results rv infection alone led to induction of, pstat-1, cxcl10 and release of ifn-k sirna, knockdown of mda-5 reduced cxcl10, ifn-b and pstat-1 and also reduced ifn-k. silencing of tlr-3 and rig-i alone had no effect. when combined both sirna for tlr-3 and mda-5 had no additional effect. treatment of becs with bx795 inhibited the production of type i ifn but only partially inhibited ifn-k release. blockade of p38mapk however led to substantial blocking of ifn-k release and also led to a marked reduction in the typeiifn response. conclusion we found that following infection with rv, becs type i ifn responses, pstat-1 induction as well as release of ifn-k was dependent on mda-5. blockade of tbk-1/ikk by bx795 reduced type i ifn response, but ifn-k release was only partially inhibited blockade of p38mapk resulted in suppression of both ifn-k and type ifn, suggesting this pathway is crucial in the antiviral response to rv infection. conflict of interest none. introduction pregnant women have increased susceptibility to respiratory virus infection. human rhinovirus (hrv) and influenza are the most common respiratory viruses isolated during severe exacerbations in pregnant asthmatics and are high risk factor for respiratory-related maternal and neonatal morbidity and mortality. understanding the innate and adaptive immunological processes underlying respiratory virus infection in pregnancy will lead to improved treatments, resulting in better health outcomes for both mother and baby. methods cross-sectional study of 12 pregnant asthmatics, 10 pregnant non-asthmatics, 8 non-pregnant asthmatics and 10 healthy non-pregnant women. blood mononuclear cells were cultured with hrv (rv43 or rv1b), influenza a (h3n2), phytohaemagglutinin or tlr3 and tlr7 agonists, polyinosinic:polycytidylic acid and imiquimod, respectively. protein concentrations of ifn-a, ifn-k ifn-c, il-10 and il-17 were quantified from culture supernatant by elisa and cba. results pregnant asthmatics had significantly increased il-17a and il-10 (median 19.3 pg/ml and 258.5 pg/ml, respectively) compared to healthy women (median 0 pg/ml and 27.5 pg/ml, respectively, p < 0.004). ifn-c was significantly reduced in pregnant asthmatics (median 721.8 pg/ml) compared to healthy women (174.3 pg/ml, p = 0.003). ifn-a and ifn-k were induced by hrv and influenza in response to rv43, pregnant women had decreased ifn-a production (206.5 pg/ml) compared to healthy controls (460.8 pg/ml, p = 0.002) whilst ifn-k production was significantly reduced in both pregnant asthmatics (25.7 pg/ml, p = 0.001) and pregnant non-asthmatics (0 pg/ml p < 0.0001). conclusion increased il-10 and il-17 production and reduced ifn-c, ifn-a and ifn-k are important inflammatory and anti-viral alterations during pregnancy and asthma; providing important insight into why pregnant women are more susceptible to respiratory virus infections. support nhmrc. conflict of interest no. nomination nil. the incidence of ntmld is increasing markedly in australia. it is not known why patients with ntmld are susceptible to these organisms, as most patients do not have identifiable risk factors or a documented immune defect. as cell-mediated immunity is crucial for control of mycobacterial disease, we assessed whether ntmld is associated with diminished th1 immune responses. methods our study cohort consisted of 27 patients with ntmld at different stages of treatment, 15 offspring of 12 patients and 21 unrelated healthy controls. plasma levels of cxcl10 and il-18 were assayed on all subjects by cytometric bead array or elisa. in a subset of subjects, pbmc were assessed for production of ifng, il-5, il-17 and il-10 in response to stimulation with mitogen (seb) and purified protein derivative (ppd). all data was analysed using non-parametric statistical tests. results plasma levels of both cxcl10 and il-18 were higher in ntm patients compared with unrelated controls and/or offspring (p < 0.001). cxcl10 levels were lower in patients who responded well to treatment compared to those who responded poorly (p = 0.03). compared with healthy controls, pbmc from ntm patients produced similar levels of ifng, il-5 and il-10, less il-17 (p < 0.05) in response to seb, but more il-10 in response to ppd (p < 0.01). conclusions ntmld is not associated with diminished th1 responses. elevated levels of cxcl10 indicate ongoing ifng release in vivo. ntm patients may have a bias towards il-10 production in response to mycobacterial antigens and/or harbour an intrinsic defect in th17 immunity. paracetamol is commonly used in infants as an analgesic and antipyretic. cross sectional studies have reported an association between frequent paracetamol consumption in early life and risk of childhood asthma. to date, no study has controlled for indication for use of paracetamol, or confounders such as history of respiratory infections, which is independently associated with asthma. aim to examine, in a prospective cohort study, whether frequent paracetamol exposure during early life increases the risk of childhood asthma. method six hundred and twenty infants with an atopic family history were recruited. paracetamol exposure was prospectively documented on 18 occasions to 2 years of age, including the number of days and the indication for use. an interviewer-administered questionnaire was used at 6 and 7 years to ascertain asthma in the previous 12 months. results exposure to paracetamol occurred in 97% of participants by two years of age. increasing frequency of use of paracetamol was associated with increased risk of childhood asthma (or = 1.18, 95%ci 1.00-1.39 per doubling of days of use). adjustment for frequency of respiratory infections substantially reduced the strength of these associations (aor = 1.08, 0.91-1.29). paracetamol use for non-respiratory tract infections and injury was not associated with asthma (or = 0.95, 0.81-1.12). conclusions in children with a family history of allergic disease, we found no association between early paracetamol use and risk of subsequent allergic disease when adjusted for respiratory infections, or when only paracetamol use for non-respiratory tract infections was examined. these findings do not support suggestions that paracetamol use up to age two years increases the risk of asthma. support nhmrc. aim to determine whether a water-based exercise program was effective in improving exercise capacity and quality of life in people with copd with physical co-morbidities compared to a land-based exercise program or no exercise. methods participants with copd referred to pulmonary rehabilitation and who had a physical co-morbidity were randomly allocated to one of three groups: land-based exercise, water-based exercise or a control group of no exercise. the two exercise groups trained for eight weeks, three exercise sessions per week. participants underwent measurements of respiratory function, exercise capacity and quality of life by a blinded investigator at baseline and following intervention. results of 53 participants (mean (sd) age 72 (9) knowledge of airway dimensions is critical for bronchoscopists assessing airway stenoses requiring interventions. it is also important for evaluating phenotypic features of obstructive lung diseases (old). however, real-time quantification of airway dimensions during bronchoscopy is lacking. inserted into the airways via a bronchoscope, anatomical optical coherence tomography (aoct) is a light-based imaging technique with the unique capacity to obtain such measurements. we describe the validation, research and clinical applications of bronchoscopic aoct. methods (study 1) aoct was validated in a phantom model, excised porcine airways and in 4 human subjects. (study 2) airway compliance curves were constructed and compared from aoct-based measurements in volunteers with and without old during bronchoscopy. (study 3) stenosis dimensions (length, calibre) were measured compared in patients with symptomatic airway stenosis using pre-procedure computed tomography (ct) and intra-procedure aoct (bland altman analysis) to determine interventional strategy. results in phantom and porcine airways, aoct measurements were accurate and reliable. mean ct-aoct diameter measurements differed by 0.4 ± 1.3 mm. (2) airway compliance was increased in copd (n = 9) relative to control (n = 10) and asthma (16) subjects, which were similar. (3) in 14 patients, the mean difference between ct and aoct-based stenosis measurements was 0.4 ± 8.6 mm. aoct proved more reliable when ct image quality was poor or where a delay occurred between ct and bronchoscopy. conclusions aoct provides real-time measurements of airway dimensions which are accurate and reliable and can be used for research and clinical applications. nomination ann woolcock young investigator award. support nhmrc. disclosure to declare yes. a notable feature of allergic asthma is the infiltration of mast cells into human airway smooth muscle (hasm) bundles. thus, mast cells and hasm can likely exhibit mutual functional modulation via direct cell-cell contact or through released factors. to examine this possibility, we have used a human mast cell line (hmc-1) to evaluate mast cell modulation of hasm cell function. methods hmc-1 cells were transfected with human fcria, and fcri expressing cells were flow sorted. the functional activity of these cells (hmc-1a) was examined by measurement of released cytokines via ige/antigen stimulation. hasm cells were co-cultured with hmc-1a cells, or with conditioned media derived from hmc-1a cells, to examine the impact on cytokine release. methods hmc-1a cells were activated by ige/antigen to release il-8. the co-culture of hasm cells with hmc-1a cells, induced both il-8 (n = 5, p < 0.05) and eotaxin (n = 5, p < 0.001) production from hasm cells and this effect was greatly amplified when hmc-1a cells were antigen-activated. the effect of hmc-1a co-culture could be reproduced by addition of conditioned media derived from activated hmc-1a cells. a bio-plexô cytokine array showed release of mcp-1 and mip-1b was strongly induced from hmc-1a cells upon ige/antigen stimulation. however, treatment of hasm cells with these cytokines did not elicit il-8 release. conclusions our study provides further evidence that the release of soluble mediators from activated mast cells can induce cytokine production from hasm cells. further work is currently ongoing to identify the factor/s responsible for this effect. we have previously demonstrated that gp130-mediated stat3 signaling is required for bleomycin-induced lung fibrosis in mice. to determine the role of phosphorylated stat3 (pstat3) in the development of human lung fibrosis we examined stat3 and the regulation of stat3 expression in lung tissue from idiopathic pulmonary fibrosis (ipf) patients. immunohistochemistry revealed nuclear localization of pstat3 in fibroblastic cells within fibrotic foci. suppressor of cytokine signaling-3 (socs3) is a known negative regulator of gp130-induced stat3 activation. we tested the hypothesis that reduced socs3 expression may account for elevated pstat3 in cells within the fibroblastic foci of ipf lungs. rt 2 pcr profiler analysis of the jak/stat pathway demonstrated that il-6 up-regulated socs3 mrna in both ipf and hlf. western blot analysis confirmed that socs3 expression was not aberrant in these cells, although a trend towards reduced socs3 expression was evident. our analysis identified six jak-stat pathway associated genes that were significantly altered in ipf cells following il-6 exposure for 30 minutes but of those, only il-20 was up-regulated. examination of other known regulators of stat signaling including socs1, socs2, socs4, socs5, pias1, pias3 and protein tyrosine phosphatase non-receptor type 1 (ptpn1) was performed but only socs1 expression was reduced in ipf. in summary, dysregulation of socs3 does not appear to cause high stat3 levels in ipf tissue but the potential regulation by socs1 is the subject of ongoing investigations. the phosphoinositide 3 kinase (pi3k) signal transduction pathway contributes to the airway remodelling associated with asthma; however, the precise roles of the specific pi3k isoforms are currently unknown. in this study, we investigated the roles of the class ia pi3k isoforms p110a, p110b and p110d in airway smooth muscle (asm) cells derived from asthmatic subjects and asm cells and lung fibroblasts from non-asthmatic subjects. methods cells were stimulated with transforming growth factorb (tgfb; 1 ng/ml) and/or 10% fbs in the presence or absence of specific pi3k inhibitors pik75 (p110a), tgx221 (p110b) or ic87114 (p110d) (all 0.01-1 lm) or vehicle control (dmso). fibronectin deposition, vegf and il-6 secretion were measured using elisa, mitochondrial activity was assessed by mtt assay and proliferation by bromodeoxyuridine (brdu) incorporation assay. results in non-asthmatic asm cells inhibition of p110a and p110b decreased vegf and il-6 secretion and cell proliferation (n = 11; p < 0.05), whereas in asthmatic asm cells, only inhibition of p110a (n = 4-6, p < 0.05) but not p110b (n = 4-6, p > 0.05) had an effect. furthermore, we demonstrated isoform specific roles with p110a (n = 4, p < 0.05) but not p110b (n = 3-4) or p110d (n = 3-5) modulating fibronectin deposition in asm cells and lung fibroblasts. conclusion specific pi3k isoforms have distinct roles in the regulation of inflammatory cytokines (il-6), growth factors (vegf) and extracellular matrix proteins (fibronectin) associated with airway remodelling. intrinsic differences exist in the roles of the pi3k isoforms in asthmatic asm. acute respiratory distress syndrome is characterized by inflammation and fibrosis. cellular therapies potentially restore pneumocytes and reduce inflammation. aims we evaluated the role of term human umbilical cord mesenchymal stem cells derived from wharton's jelly (umscs) and human amnion epithelial cells (haecs) in treating a bleomycin-induced model of lung injury. methods cells were administered systemically into a mouse model of acute lung injury 24 hours following intra-nasal administered bleomycin. results both haecs and umscs reduced inflammation with decreased tnf-a, il-1, il-6 and tgf-b. collagen in the lung was significantly reduced by both umscs and haecs as a possible consequence of increased degradation by matrix metalloproteinase-2 (mmp-2) and down-regulation of their endogenous inhibitors the tissue inhibitors of matrix metalloproteinases (timps) -1 and -2. umscs were detected in the lung at 2 weeks postinjection, vs. 4 weeks for haecs. in addition, umscs did not demonstrate lung differentiation while haecs developed an alveolar phenotype. conclusions both umscs and haecs, have anti-inflammatory properties and reduce fibrosis in lung injury but haecs adopt a lung phenotype support small grant monash university. nomination nil. background with improvement in clinical care and longer survival of patients with cystic fibrosis, pregnancy has become commonplace. however the impact of cystic fibrosis on maternal health and foetal outcomes requires ongoing review. methods a retrospective study of 20 pregnancies from 18 women with cystic fibrosis during the period 1995-2009 was performed. changes in lung function, body mass index, and development of gestational diabetes were recorded. foetal outcomes and maternal survival were examined and the influence of pre-pregnancy parameters on outcomes were evaluated. results mean age of pregnancy was 29.1 years with a mean pre-pregnancy fev1 of 65.6% predicted. eleven out of twenty pregnancies had a pre-pregnancy fev1 <60% predicted. during pregnancy, fev1 fell by 4.76% (ci 1.59-7.92), but recovered to baseline within 6 months post-partum. mothers gained a mean weight of 7.6 kg and gestational diabetes developed in 42.9% of women. all women delivered live births apart from one therapeutic abortion. five infants were preterm and three had low birthweight for age. four mothers either died or required lung transplantation after pregnancy on follow up. fev1 <60% predicted and body mass index <20 kg/m 2 were significant predictors of foetal complications. background carrier screening for cf has been available for many years but there is no national program for population-based screening in australia. knowledge of australian cf healthcare professionals' attitudes towards carrier screening would provide useful information about how a program could be implemented. the aim of this study was to investigate the attitudes of cf respiratory physicians and cf clinic coordinators in australia towards population-based carrier screening for cf. method a purposed designed questionnaire assessing knowledge and attitudes towards cf carrier screening was distributed to respiratory physicians and cf clinic coordinators throughout australia. results there were 111 respiratory physicians registered with the cf special interest group of tsanz and 30 cf clinic nurses identified through the cf coordinators network. seventy-five responded, 55 respiratory physicians (49.5%) and 20 coordinators (67%). forty-two (56%) respondents were in favour of population-based carrier screening for cf. sixty-four (85%) rated raising a child with cf as difficult/very difficult, 63 (84%) rated the shortened life span as a significant concern and 64 (85%) the daily treatment regimen as a significant concern. disadvantages of screening were perceived anxiety amongst carriers (n = 65, 87%) and discrimination of carriers (n = 42, 56%). respondents rated the following barriers as most important: limitations of predicting clinical outcomes (n = 47, 65%) and insufficient time and resources for providers (n = 45, 61%). fifty-four (76%) of respondents believed they had a role in the development of a cf carrier screening program. adherence to medication regimens in patients with cystic fibrosis (cf) vary substantially. direct measures of adherence using electronic monitors attached to medication bottles enable the precise recording of usage. the macrolide antibiotic, azithromycin has demonstrated clinical benefit when used in cf patients with moderate to severe impairment of lung function. aim this study compares the adherence levels of cf patients randomised to two different treatment regimens of azithromycin, measured by electronic monitoring. methods patients were prescribed the medication once (1000 mg) or three times (500 mg) a week. data were collected over 24 weeks using electronic monitoring devices. adherence measures were defined as: total adherence (total amount of medication taken divided by total medication prescribed) and total number of days adhered (number of days where prescribed doses were taken divided by number of days monitored). results the study recruited 51 participants (57% male; mean age = 33.7 sd = 8 years, mean fev1 % = 62.2, sd = 22.8). total adherence in patients prescribed the weekly regimen (m = 100.19, sd = 6.3%) were significantly different compared to the three times a week regimen (m = 89.4, sd = 17.2%, p < 0.05). no significant difference was observed in total number of days adhered. fev1% predicted negatively correlated with adherence to medication (total adherence r 2 = -0.28, p<0.05, days adhered r 2 = -0.29, p < 0.04) and to bmi (r 2 = 0.35 ,p < 0.01). positive correlation was observed between age of the participant and adherence to medication (total adherence r 2 = 0.31, p < 0.01, days adhered r 2 = 0.41, p < 0.01). conclusion participants adhered better to a once weekly regimen than a three times a week regimen. preclinical studies in non-human primates (nhp) are essential to estimate effectiveness and safety in developing gene transfer protocols to treat cystic fibrosis (cf) airway disease prior to clinical trials. lentiviral (lv) vectors can provide in vivo gene expression and persistence suited to long lasting cf airway correction 1 in mice. we have begun examination of lv gene transfer in lungs of marmosets (callithrix jacchus), a small non-human primate with lung anatomy and physiology similar to humans. methods lysophosphatidylcholine (lpc, 0.1%) pre-treatment was followed by a lv vector encoding the lacz (lv-lacz) reporter gene, pseudotyped with the vsv-g surface protein. doses were delivered into the trachea of four intubated marmosets. trachea and lungs in two animals were examined after 1 week; blood taken daily was tested for presence of vector particles. results epithelial cell lacz gene expression was present primarily in conducting airways in a patchy distribution. a transient o2 desaturation was noted in some animals after lpc administration; behavioural and physiological indices were normal postoperatively. limited patches of haemorrhage and neutrophil / mononuclear cell infiltration were deemed unremarkable by a veterinary pathologist. serum p24 lv capsid protein levels that appeared after dosing were absent after day two. conclusions these first studies indicate lpc/lv dosing procedures are well tolerated and can induce target-cell gene expression. further histological and immunological analyses are in progress. the remaining two animals will undergo longer-term assessment of the success of lentiviral lung gene transfer. background bronchiectasis and air trapping are important features of cystic fibrosis (cf) structural lung damage, however data on disease progression in young children are lacking. aim to assess longitudinal changes in ct-detected early structural lung damage in. methods subjects included 117 children (age range 0.2 to 6.9 years) who underwent 327 annual ct scans, with 210 paired scans. each ct scan consisted of three slices at endinspiration and three slices at end-expiration. the left and right upper, middle and lower zones were assessed for the presence and extent (none, less than 50%, more than 50%) of bronchiectasis and air trapping using previously described methods. infection and inflammation were assessed using bronchoalveolar lavage at the time of ct scan. results bronchiectasis was present in 37% of initial scans, persisting in 75% of subsequent scans. median extent increased from initial to subsequent scan (p = 0.000). previous psa infection and neutrophilic inflammation was associated with increased prevalence of bronchiectasis at subsequent scan (or = 2.9). air trapping was present in 67% of initial scans, persisting in 86% of subsequent scans. median extent increased from initial to subsequent scan (p = 0.005). infection and inflammation did not increase risk of air trapping, but air trapping was more common in girls (or = 2.0). bronchiectasis and air trapping commonly occurred together. the 6 minute walk test (6mwt) and incremental shuttle walk test (iswt) are commonly used to assess functional exercise capacity, prescribe the training intensity and measure the efficacy of pulmonary rehabilitation. no studies have compared these tests in patients with non-cf bronchiectasis. aims to compare peak dyspnea and heart rate (hr), and nadir oxygen saturation (spo 2 ) during the 6mwt and iswt in subjects with non-cf bronchiectasis. methods twenty-seven participants (aged 64 ± 13year, fev1 70 ± 17%pred, fvc 82 ± 16%pred) with non-cf bronchiectasis enrolled in a trial of pulmonary rehabilitation, completed two 6mwts and two iswts in random order. results the 6 minute walk distance (6mwd) and the incremental shuttle walk distance (iswd) were significantly greater on the 2nd test (both p < 0.02). the mean (95% ci) increase in the 6mwd was 22 m (9 to 35 m); 4% (2 to 7%) and in the iswd was 22 m (4 to 39 m); 6% (2 to 10%). the greatest 6mwd and iswd was 560 ± 86 m and 446 ± 151 m respectively. there was a strong relationship between the 6mwd and iswd (r = 0.89, p < 0.001). peak dyspnoea was higher for the iswt (4.2 ± 1.2 vs. 3.6 ± 1.2, p = 0.02) but there was no difference in peak hr (76 ± 11 vs. 75 ± 10% age pred maximal hr, p = 0.67) or nadir spo 2 (93.5 vs. 93.3%, p = 0.65). conclusion although peak hr was similar, the externally paced, incremental nature of the iswt may account for the higher dyspnea scores in these subjects with non-cf bronchiectasis. future research will determine the responsiveness of the iswt and 6mwt following pulmonary rehabilitation in this population. the six minute walk test (6mwt) is extensively used in clinical practice. however, the role of this test in people with dust-related lung disease remains unclear. aim the aims of the study were to investigate the relationships between exercise capacity measured by the 6mwt and the incremental peak and endurance cycle tests, the 6mwt and health-related quality of life, and the 6mwt and activity levels in people with dust-related lung disease. methods thirty male participants with asbestos related pleural disease, asbestosis or silicosis performed two 6mwts separated by 30 minutes with the better of the tests used for analysis. during the rest period, participants completed the st george's respiratory questionnaire (sgrq). on a separate day, participants performed spirometry, lung volumes, dlco and a peak and endurance cycle test. participants wore an activity monitor (sense-wear pro3) for a period of seven days. results mean (sd) age of participants was 71 (6) there was a significant correlation between 6mwt distance and peak watts (r = 0.67, p < 0.001) but not with endurance cycle time. there were significant correlations between the 6mwt and all components of the sgrq, the strongest correlation being with the 'activity' domain (r = -0.61, p < 0.001) and significant correlations between 6mwt and average daily steps (r = 0.57, p = 0.002) and average mets (r = 0.59, p < 0.001). conclusion findings suggest the 6mwt may be a useful measure of exercise capacity and may reflect daily activity in people with dust-related lung disease. the 6minute walk test (6mwt) is used to assess prognosis and evaluate exercise capacity in interstitial lung disease (ild), however the physiological load imposed by the 6mwt is unknown. this study compared cardiorespiratory responses to 6mwt and cardiopulmonary exercise testing (cpet) in ild. methods fifteen participants with ild (nine ipf), mean age 70 (standard deviation 12) years and tlco 57 (17) %predicted undertook cpet and 6mwt on the same day in random order. pulmonary oxygen uptake (vo2), ventilation (ve), carbon dioxide production (vco2), oxyhaemoglobin saturation (spo2) and heart rate were compared between the tests using a portable metabolic cart. relationships between 6 minute walk distance (6mwd) and peak cardiorespiratory responses on cpet were evaluated using correlations. results peak vo2 measured during the 6mwt was lower than during cpet (15.1(3.5) vs 17.5 (2.6) ml.kg/min, p = 0.03). oxygen consumption during 6mwt reached a mean of 87% of vo2peak achieved on cpet (95% confidence interval 76-98%vo2peak). peak ventilation, carbon dioxide production and peak heart rate were significantly lower during 6mwt, but there was no difference in nadir spo2 (90(4)% vs. 91(3)% on 6mwt and cpet respectively, p = 0.14). a higher 6mwd was associated with a higher peak work rate (r = 0.93, p < 0.001) but there were no relationships between 6mwd and peak cardiorespiratory responses on cpet. conclusions the 6mwt elicits a high but submaximal oxygen uptake in people with ild. given the poor relationship between 6mwd and peak cardiorespiratory responses elicited by cpet, the prognostic value of the 6mwt may be related to the degree of oxygen desaturation elicited by this test. results there were no differences between groups at baseline for age, gender, body mass index or respiratory function. after twelve months both groups had similar improvement in fef25-75%(mean 0.181 l/sec, 95% confidence interval 0.059 -0.303 l/sec). there was no significant effect on fev1 or fvc due to either treatment allocation or time. both groups demonstrated significant improvements in all domains of both the sgrq and lcq but there was no difference between groups. conclusion the inhalation of both isotonic (0.9%) and hypertonic (6%) saline improved small airways function and improved quality of life over 12 months, however both treatments were equally effective. introduction this project aimed to develop, implement and evaluate an innovative primary care model in community pharmacy for screening, monitoring and education of people with or at risk of sleep disorders (sd). methods a randomised control trial comparing two approaches was conducted: 1) risk assessment only (rao) or 2) risk assessment plus overnight nasal flow monitoring using the flowwizardò device (ra+). the risk assessment tool collected data on lifestyle, medical conditions, medications and included validated instruments for detecting sd. twentythree pharmacies (12 rao/ 11 ra+) recruited patients during a 4 month period. patients at significant risk for a sd were provided with information and referred to a gp. results 295 patients were recruited (rao n = 131, ra+ n = 164). of these, 24.9% (rao 19%, ra+ 29%) had an increased risk of daytime sleepiness, 27.6% (rao 28%, ra+ 31%) were at risk of significant insomnia, 43.0% (rao 37%, ra+ 47%) at risk of obstructive sleep apnea, and 36.9% (rao 34%, ra+ 38%) at risk of restless legs syndrome (rls). pharmacists recorded a total of 754 interventions and 94 patients were referred to their gp (1 of 3 screened). preliminary results showed that 126 patients (rao, n = 73, ra+, n = 53) have completed the follow-up questionnaire with 29 gp referrals being taken up and seven sd diagnosed (24% of those who took up a referral, rao, n = 5, ra+, n = 2). nine patients (41%) received a diagnosis other than a sd and 7 patients reported still awaiting sleep specialist assessment or testing. conclusion the results of this study indicate that community pharmacy is a potential site for sd screening. support the pharmacy guild of australia, investigator initiated grants. nomination nil. conflict of interest nil. jm foster 1 , l smith 1 , sz bosnic-anticevich 1 , t usherwood 1 , sm sawyer 2 , cs rand 3 , hk reddel 1 1 university of sydney, australia, 2 royal childrens hospital melbourne, australia, and 3 johns hopkins university, baltimore, usa aim to identify beliefs and behaviours associated with poor adherence which could be used to guide tailored interventions in primary care. methods patients aged ‡14 years with doctor-diagnosed asthma and a current ics/laba prescription completed questionnaires on beliefs and behaviours, side-effects, asthma control (acq), and underwent spirometry. adherence with ics/laba was measured over 6 weeks by smartinhalers which electronically recorded the time and date of each actuation. univariate and multivariate analyses of 61 questionnaire items identified predictors of adherence. results ninety-nine of 100 patients completed the study (57 female; mean ± sd fev 1 % predicted 83 ± 23; acq 0.76 ± 0.76). mean adherence was 75% ± 25 (n = 85). thirty one beliefs or behaviours were significantly associated with poor adherence (p < 0.05). factor analysis of these 31 items identified 7 themes: f1. perceived necessity; f2. safety concerns; f3. acceptance of asthma chronicity and ics/laba effectiveness; f4. advice from friends/family; f5. motivation/routine; f6. ease of use; and f7. satisfaction with asthma management. regression analysis demonstrated that 10 items in 5 themes independently predicted poor adherence (model adj. r sq. = 0.67; p < 0.001) including 'my preventer is necessary to keep my asthma under control'(f1), 'i get side effects from my steroid inhaler'(f2), 'i think i will have asthma for a long time'(f3), 'my family/ friends tell me i should use my preventer inhaler more often'(f4), 'i have a fixed daily routine for taking my asthma medications'(f5). adherence was lower for patients who attributed dental deterioration or dry eyes to their ics, but not for hoarseness. conclusions this study identified 10 key beliefs or behaviours associated with poor adherence which may be amenable to change in patient-specific primary care interventions. support asthma foundation nsw, glaxosmithkline (medications investigation of pulmonary embolism with ctpa is often performed despite low clinical risk and results in unnecessary exposure to radiation and radiocontrast as well as inefficient use of medical resources. risk stratification with a validated prediction tool (wells score) complements clinical decision making and rationalises the use of ctpa to appropriate patient groups. methods prospective assignment of wells score by requesting clinicians on a formal algorithm form was instituted in 2009. all patients being investigated for pulmonary embolism were required to have the form filled prior to performance of ctpa. patients stratified low clinical risk (wells £ 2) did not proceed to ctpa unless a senior physician override was applied. intermediate risk patients (wells 2-6) proceeded to d-dimer measurement and if above the laboratory cutoff (0.3) proceeded to imaging. all high risk patients (wells > 6) proceeded to ctpa directly. ctpa outcomes, d-dimer levels, request locations and dates were recorded. data were collected from february to august 2009. results a total of 333 patients were investigated with ctpa in this period. 65 patients (19%) did not have the wells score assigned but 268 patients (81%) had complete data. 215 (64%) request originated from the emergency department, 107 (32%) from inpatient wards and 8 (2%) and 3 (1%) from icu and outpatients respectively. the prevalence of pulmonary embolism in our study population was 13% similar to data from wells and others. 57 (21%) patients were stratified to low risk, 169 (63%) to intermediate risk and 42 (16%) to high risk. the prevalences of pulmonary embolism were 9%, 12% and 24% respectively in these risk groups, comparable with published data. when evaluated against the same period in 2008, there was an absolute reduction of 136 (30%) ctpas performed. conclusion institutional implementation of a formal clinical prediction tool into the decision making process is feasible and yields significant reduction in ctpas performed, with substantial cost savings and patient benefits. aim to conduct an rct to measure the impact of the practitioner asthma communication and education program (pace) on general practitioner (gp) management of paediatric asthma. methods gps recruited through local practice networks identified patients aged 2-14 with diagnosed asthma. gps received two 3 hour interactive workshops. results outcome data were collected from 57 intervention and 49 control gps, and 106 intervention and 107 control families. a significantly higher percent of intervention gps than control gps reported frequently providing a written asthma action plan (23.2%, p = 0.03, nnt = 4.3).intervention gps reported higher rates of giving written instructions to adjust medication (18.4%, p = 0.01, nnt = 5.4) and of providing spacers (28.7%, p = 0.01, nnt = 3.5). a significantly higher percent of intervention group children had received a written asthma action plan in the last year (15.2%, p = 0.03, nnt = 6.6). fewer intervention group children with infrequent intermittent symptoms were using regular ics (24.0%, p = 0.03, nnt = 4.2). intervention gps had higher improvements in confidence (21.5%, p = 0.03), helpfulness (20.7%, p = 0.04) and frequency of using the taught communication strategies (21.1%, p = 0.03). conclusion the pace program is the most robustly evaluated program of gp asthma education in australia. our results provide high level evidence that paediatric asthma management is improved by pace. pace may be useful for educating other health professionals involved in chronic disease management. support australian government department of health and ageing. nomination nil. conflict of interest nil. vincent siaw, karmen yai, anand rose department of respiratory medicine, flinders medical centre, bedford park, south australia, australia pulmonary embolism is a common cause for presentation to the emergency departments of tertiary hospitals. if undiagnosed it has a 30% mortality. the diagnostic algorithm includes a clinical assessment, d'dimer assays and imaging in the appropriate patient. the preferred tool in our institution for confirming a diagnosis of a pulmonary embolism is the ct pulmonary angiogram (ctpa). we decided to audit our practise of doing a clinical assessment using a standardised risk score (eg.wells score) prior to requesting a ctpa. aim to audit the use of standardised clinical risk assessments prior to requesting a ctpa when a pulmonary embolism is suspected. methods ctpa requests from the emergency department from february and march 2009 were retrieved. cases notes were screened for mention of the wells or geneva scores. individual symptoms were also studied in attempt to reconstruct the score from the notes. results of the 189 ct pulmonary angiograms performed -52 requests were from the emergency department. of these requests 15.4% (8 patients) were positive studies. systematic clinical risk assessment had been used in 23.1% (12 cases). when a systematic clinical score was performed 33.3% of the ctpa studies were positive. this was greater than when no risk score was performed (10% of ctpa studies returned positive). we aimed to compare gp and parent reports of asthma management styles from an rct of practitioner asthma communication and education (pace). methods gps recruited through local networks identified patients aged 2-14 with diagnosed asthma. intervention gps participated in two 3 hour workshops of patient education and communication techniques. results at 12 months, 106 gps (57 intervention, 49 control) and 213 parents (106 intervention, 107 control) provided data. more intervention gps (50.0%) reported checking device use (vs. 39.1%; diff = 10.9%, p = 0.33). intervention parents (53.6%) reported that gps checked their device use more frequently (vs. 47.5%; diff = 6.1%, p = 0.47). more intervention gps (83.9%) reported providing educational messages (vs. 72.3%; diff = 11.6%, p = 0.24). however, more control parents (40.4%) (vs. 35.7%; diff = -4.7%, p = 0.59) reported receiving messages. more control gps (56.5%) said they asked patients about new medication fears (vs. 52.6%; diff = -3.9%, p = 0.85), but more intervention parents (8.5%) reported being asked about this (vs. 5.6%; diff = 2.9%, p = 0.58). conclusions gp and parental reports of device use checking were consistent. reports of educational messages and communication were less consistent, though these may have been provided or used but not recognised by parents. these findings highlight that parents and their gps can have very different perceptions of some aspects of a child's asthma management. care should be taken when selecting outcome measures for clinical trials. support australian government department of health and ageing. nomination nil. conflict of interest nil. to 080 background smoking cessation interventions in outpatient settings has been clearly demonstrated to be one of the most cost effective strategies available in reducing disease burden. given the evidence of superior benefits with over nicotine replacement therapy, we aimed to evaluate its benefit in the inpatient setting for smokers admitted with acute smoking related events. methods adult patients (n = 151, 20-75 years) recruited from the respiratory, cardiology, neurology, vascular and general medical wards of the queen elizabeth hospital, lyell mcewin health service and the royal adelaide hospital were randomised to receive either vt (varenicline tartrate) plus quit sa counselling (n = 77) or quit sa counselling alone, (n = 74). results preliminary analysis shows that after three months of follow-up, smoking abstinence was achieved by 28.4% in the control and 44.2% in the intervention group, (p = 0.062). preliminary subgroup analysis indicates that cardiac patients, (n = 71) have gained the most benefit with 49.3% obtaining continuous abstinence. conclusion whilst a beneficial smoking cessation trend is evident at three months, these are only preliminary results. our recruitment target sample size is likely to provide sufficient power to identify significant differences in abstinence rates between treatment and control groups, and permit sub-group analyses of treatment effect based upon inpatient characteristics. support nil. nomination nil. introduction cigarette smoking prevalence has been in decline in australia over many decades but prevalence remains high in lower socioeconomic groups. hospital employees span the socioeconomic spectrum but there are few data on smoking prevalence from these sites. the visibility of smoking on campus conflicts with the health message that hospitals should promote but cessation services are often not provided. the queen elizabeth hospital (tqeh) has had an ongoing stop smoking service (using cost-price nrt and counselling) provided by tej since 1995 and 5-yearly surveys are conducted to assess benefits and ongoing need. methods employees of three metropolitan teaching hospitals (royal adelaide -rah, flinders medical centre -fmc and tqeh) and the alice springs hospital (ash) were sent a single page questionnaire asking about smoking status and views about smoking on campus. returns were voluntary but encouraged via a small monetary prize. tqeh was surveyed thrice (1997, 2002 and 2007) , the other hospitals were surveyed once (late 2004/ early 2005) . results almost all employees reported knowing smoking is a health hazard. most employees (smokers & non-smokers) at all hospitals, thought smoking in public view was unacceptable but support for a total ban was less than for suitable areas where smoking was allowed. tqeh smoking prevalence is much lower than the comparator hospitals where prevalence is similar to national prevalence ( introduction interleukin-17a is a cytokine released from t helper 17 (th17) cells which induces and mediates various pro-inflammatory responses. as a result, il-17a has been linked to many immune/autoimmune related diseases but its role in copd has not been explored. in the present study we investigated whether il-17a regulates cigarette smoke (cs)-induced lung inflammation. methods wild-type (wt) or mice deficient in il-17a (il-17a -/-) were placed in a perspex chamber and exposed to cs generated from nine cigs per day for 4 days. in separate experiments, cs-exposed wt mice were treated with anti-il-17a antibody. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for genomic analysis. results wt mice exposed to cs for 4 days had significantly more balf macrophages (4.3 ± 0.3(sem) · 10 5 ) and neutrophils (3.8 ± 0.3 · 10 4 ) than sham-exposed mice (1.0 ± 0.2 · 10 5 and 0, respectively) (n = 5-26, p < 0.05). however, cs-exposed il-17a -/mice had significantly fewer macrophages (2.1 ± 0.1 · 10 5 ) and neutrophils (0.6 ± 0.1 · 10 3 ) than cs-exposed wt mice (n = 5-26, p < 0.05). macrophage and neutrophil numbers in sham-exposed il-17a -/mice (1.2 ± 0.1 · 10 5 and 0.3 ± 0.2 · 10 3 ) were similar to those of sham-exposed wt mice. gene expression analysis by qpcr showed that cs-exposed il-17a -/mice had markedly reduced mcp-1, tnfa, il-17a, il-23 and mmp-12 expression compared to cs-exposed wt mice. treatment of cs-exposed mice with anti-il-17a antibody significantly reduced cs-exposed balf macrophages and neutrophils (n = 8, p < 0.05). in addition, we found that lungs of nod-scid mice deficient in t & b lymphocytes expressed il-17a in response to cs. conclusions these data show that il-17a regulates cs-induced lung inflammation and that targeting il-17a may have therapeutic utility in inflammatory lung diseases where cs plays a role. introduction in utero exposure to tobacco constituents may contribute to respiratory health problems later in childhood. glutathione s-transferases (gsts) are important in detoxification of xenobiotics. a reduction in the mother and fetus's detoxification ability due to genetic variation in gsts could expose the fetus to higher levels of toxins. objective to investigate the interactive effects of maternal smoking during pregnancy with maternal and infant gst genotypes on airway responsiveness (ar) and lung function in infancy at 1, 6 and 12 months and longitudinally throughout the first year. methods gstt1, gstp1 and gstm1 were genotyped in infants and mothers using pcr. in utero exposure to maternal smoke was evaluated by questionnaire, ar was assessed by histamine challenge and v'maxfrc was measured using the rapid thoracoabdominal compression technique. results gstt1 non-null in infants, mothers or both was associated with reduced ar at 12 months and throughout the first year and increased v'maxfrc at 6 months. maternal gstp1 val/val or ile/val was associated with increased v'maxfrc at 6 months. in infants exposed to in utero smoke, gstt1 non-null infants, mothers or both was associated with reduced ar at 1 month and throughout the first year and increased v'maxfrc throughout the first year. there were no significant associations with gstm1. conclusion gst genes may be especially important during fetal development as they may modify, through proficient detoxification, the effects of in utero maternal smoke exposure on ar and lung function in infants. funding nhmrc. conflict of interest no. introduction there are few birth cohort studies in which frequent, contemporary measures of tobacco smoke exposure have been related to lung function and airway responsiveness in later childhood. aim to examine the effects of in utero and post natal exposure to ets on lung function and airway responsiveness at age 8 years. methods children with a family history of asthma were recruited antenatally into a randomized trial of house dust mite avoidance and dietary modification 1 results a total of 220 subjects were enrolled (105 indigenous australians, 115 indonesians). in the indigenous australian setting the sgrq total score was independently associated with exacerbation frequency and lung function (% predicted fev1) whilst the symptom score was associated more strongly with ae frequency and activity score with lung function. in indonesians with ptb the total sgrq score correlated with treatment response over time as well as lung function (% predicted fvc), exercise tolerance (6mwt) and the extent of involvement on cxr. conclusions in an indigenous australian and indonesia, setting respiratory-related qol using a modified sgrq correlates with lung function, exercise performance, disease activity and treatment. these tools should be a useful addition to evaluating interventions in this setting. background epithelial mesenchymal transition is a process in which airway epithelial cells disaggregate and then migrate through the reticular basement membrane (rbm) into the lamina propria to become myofibroblasts. the aim of this study was to identify if emt is active in the airways in smokers, and whether relevant to copd. methods endobronchial biopsies (ebb) from current smokers with copd (cs; n = 17) and ex-smokers with copd (es; n = 15), smokers with normal lung function (ns; n = 16) and never-smoking controls (nc; n = 15) were stained for emt markers, s100a4 a fibroblast protein, epidermal growth factor receptor (egfr) and matrix metalloproteinase-9 (mmp-9). computer-assisted image analysis was used to quantify the expression of markers in biopsies and slides were counted by an observer blinded to subject and diagnosis. we used non-parametric statistics. results compared to nc, there was significant fragmentation of the rbm in cs, es and ns groups (p < 0.001), which was especially marked in cs and was positively related to pack years in copd subjects (r = 0.41, p = 0.02). cs, ns and es demonstrated increased staining for: basal epithelial s100a4 (p < 0.004), epithelial egfr (p < 0.001) and mmp-9 (p < 0.002) for cells in rbm 'clefts', and rbm cell s100a4 (p < 0.001) compared to nc. there was increased rbm cell s100a4 staining in cs vs. es and ns (p < 0.007). basal epithelial cells staining for s100a4 correlated negatively with airflow limitation (r = -0.49, p = 0.04) in cs, and dual staining revealed that basal s100a4 positive cells co-stained with vimentin (an additional mesenchymal marker). conclusions our findings suggest that emt is active in smokers, and is most evident in current smokers with copd, suggesting a role in copd pathogenesis. pulmonary emphysema is a major component of the chronic obstructive pulmonary disease (copd), and also predisposes affected individuals to lung cancer. emphysema can be a familial or acquired disease, with the great variation in development of disease in atrisk populations reflecting the influence of other susceptibility determinants. in this regard, the il-6 cytokine family has been linked with emphysema pathogenesis. however, studies into the definitive mechanisms by which these cytokines cause emphysema have been hampered by the absence of informative animal disease models. to address this issue, we have utilized a sophisticated animal model (gp130 f/f mice) with a subtle mutation in the il-6 cytokine family receptor gp130 which, as a consequence of abolishing binding of both shp2 and socs3, simultaneously mediates stat1/3 hyper-activation and impaired shp2-mapk and -pi3k activation. the gp130 f/f mice spontaneously develop emphysema by 6 months of age characterized by increased static compliance. lung stereology has further confirmed emphysematous changes, revealing increases in volumes of airspace and lung. among the il-6 cytokine family, il-6 expression is significantly up-regulated in the lungs of gp130 f/f mice, and genetic ablation of il-6 in gp130 f/f mice prevents the development of emphysema. notably, an increased apoptosis of alveolar cells has been identified as the underlying cellular mechanism associated with the emphysema in gp130 f/f mice. collectively, our observations identify for the first time that deregulated gp130 signalling by il-6 cause's alveolar cells to undergo apoptosis, which coincide with the pathogenesis of emphysema. furthermore, this mouse model has the enormous potential to allow us to explore common mechanistic links between copd and lung cancer. supported by the nhmrc, australia. conflict of interest no. introduction despite smoking cessation, susceptible copd patients continue to decline in lung function. understanding biological pathways and their gene ontologies would help to develop better treatments and diagnostic methods for copd. the aims of this study were to identify gene ontologies associated with mild and moderate copd by (i) profiling mrna and (ii) mirnas and their predicted targets. methods profiling was performed on total rna extracted from lung tissue of 30 copd patients undergoing resection for lung cancer. microarray platforms (operon v2 and agilent g4470 v1) were used to characterise mrnas and mirnas respectively. analysis was performed using brb array tools v8.3 and gsea. results the 30 patients were caucasian former smokers with mean (sd) age 68 (6), fev1 72 (17) % predicted, kco 70 (10)% predicted and pack years 71 (44). we identified authentic candidate genes (p < 0.01) that predicted copd progression with 70% accuracy in in-house and public datasets. genes involved in cell cycle, proliferation, development and growth were identified. increasing expression of mir-34c, a candidate mirna for emphysema progression, on lung fibroblast and epithelial cells downregulated predicted mrna targets with potential biological role in copd. conclusions we have identified multiple gene ontologies associated with copd severity. these targets have promising biological roles in copd and can be further developed as biomarkers or therapeutic targets. cigarette smoke (cs)-induced oxidative stress is known to drive the pathogenesis of copd. the antioxidant glutathione (gsh) is essential for efficient macrophage functions including phagocytosis of apoptotic cells (efferocytosis) which we have shown to be defective in copd. gsh synthesis is controlled by a cd98/xct cysteine transporter pathway. cd98 is also a ligand for galectin-3 (gal-3), a lectin important for macrophage phagocytosis and gsh synthesis. we hypothesised that targeting oxidative stress in copd by increasing gsh would increase gal-3 levels and improve efferocytosis. we investigated (a) ex vivo: oxidative stress markers (8-isoprostane; mmp9), gsh and gal-3 in bal from 20 controls and 14 current-and 13 ex-smoker copd subjects (b) in vitro: the effects of cs on alveolar macrophage production of gal-3 and gsh (c) in vivo: the effects of treatment with a gsh precursor, procysteine, on efferocytosis, gal-3 and gsh in smokeexposed mice. procysteine was administered in semi-solid mouse feed. efferocytosis was investigated in lung tissue and bal macrophages. 8-isoprostane and mmp9 were significantly increased in bal in current-and ex-smokers with copd. gsh and gal-3 were decreased in copd (gal-3, ng/ml: current 0.66 ± 0.11, ex-smoker 0.96 ± 0.26 vs. controls 3.0 ± 0.62). in vitro cs treatment decreased gal-3 expression. in vivo, cs caused decreased efferocytosis that was significantly improved by procysteine (control; smokeexposed; procysteine + smoke-exposed: bal 26.2%; 17.66%; 27.8%; tissue 35.9%; 21.6%; 34.5 %). gsh and gal-3 were also significantly increased by procysteine (gal-3, ng/ml: control 1.74 ± 0.21; smoke-exposed 0.26 ± 0.042; procysteine + smoke-exposed 1.26 ± 0.29 ng/ml). targeting oxidative stress is a viable approach to improve macrophage dysfunction in copd. support nhmrc, arc. introduction our knowledge about the effects of inhaled corticosteroids (ics) on airway remodelling in chronic obstructive pulmonary disease (copd) is limited. we have previously reported that in bronchial biopsies (bb) from copd subjects the reticular basement membrane (rbm) is fragmented and hypervascular. in this study we have examined the effects of ics on these airway remodelling changes in copd. methods in a double blind and randomised study we compared the effects of 6 months of fluticasone propionate (fp, 0.5 mg/twice daily) with placebo. bb were stained with collagen iv antibody to mark vessel endothelial basement membrane. the length of rbm splits and the number and area of vessels in the rbm were compared before and after treatment. results copd subjects were randomized 2 : 1 to receive either fp (n = 15) or placebo (n = 7). there were no differences between the groups before treatment. introduction copd is a complex disease characterised by fixed airflow obstruction and neutrophilic airway inflammation. markers of systemic inflammation such as serum amyloid a (saa) are elevated in copd. however, little is known about the relationship between airway and systemic inflammation. this study tested the hypothesis that systemic inflammation is associated with airway neutrophils in copd. methods participants with copd (n = 65, >55 years, with fev1/fvc <70 and fev1% predicted <80) and healthy controls (hc; with normal lung function n = 32 > 55 years) underwent clinical assessment, spirometry, blood collection for saa, il-6 and crp and sputum induction. sputum was processed for differential cell count and mediators. results airway proportions of neutrophils and eosinophils, levels of il-8, total mmp-9 and gene expression of il-6 were increased in participants with copd. serum il-6 (median q1-q3; (2.9 (1.7-4.9)) vs. asbestos-related lung cancers (arlc) account for 4-12% of all lung cancer, and are difficult to distinguish from non-asbestos related tumours (narlc) by clinical and histological criteria. we hypothesised that whole genome array comparative genomic hybridization (acgh) profiling could identify regions of gain and loss common and specific to asbestos-related lung cancer. methods the acgh profiling by agilent cgh 44b arrays was performed on 64 primary non-small cell lung cancers obtained from the prince charles hospital (tpch) lung tumour bank. lung cancers occurring in individuals with >=20 asbestos bodies/gram wet weight (ab/gww) of lung tissue were defined arlc and individuals with 0 ab/gww were defined narlc. genome breakpoints were called using the circular binary segmentation algorithm implemented in dnacopy. recurrent regions of amplification and deletion were identified using the genomic identification of significant targets in cancer (gistic) algorithm developed by the broad institute, controlling for false discovery rates (q < 0.05). results gistic identified recurrent copy number gains in 3q28 and narlc at q < 0.05 but none for arlc at the same threshold. to 093 introduction the relationship between asbestos exposure and asbestos related diseases (ard) such as asbestosis, lung cancer and mesothelioma are well established. less is known about asbestos exposure and non-ard respiratory diseases. aim to investigate respiratory symptoms and lung function in former workers and residents from wittenoom who have not developed an ard. methods an annual review, which includes lung function, plain chest x-ray and respiratory questionnaire, is conducted on a cohort of ex-workers and ex-residents from wittenoom. only those who had been reviewed within the previous 5 years and had not developed an ard, nor had plain chest radiographic evidence of asbestosis, were included in the analyses. the prevalence of respiratory symptoms was determined and standardised lung function z-scores calculated. predictors of symptoms and lung function were assessed using both multiple logistic and linear regression. results questionnaire data was available for 690 subjects (264 women, 264 ex-workers; mean age 61.7 ± 10.7 years), while acceptable lung function data was available for 631 subjects (249 women, 258 ex-workers). the prevalence of reported symptoms ranged between 20 and 30% for wheeze, cough, sputum, shortness of breath and bronchitis. pack years of smoking and/or being an ex-worker were the main risk factors for symptoms. standardised lung function scores (95%ci) for the total group were -0.66 (-0.75--0.57), -0.80 (-0.89--0.71) and 0.36 (0.27-0.44) for fev, fvc and fev/fvc respectively. both pack-years and cumulative asbestos exposure were independently associated with reduced fev and fvc. conclusions people previously exposed to asbestos, particularly ex-workers, have high rates of respiratory symptoms which are mostly related to smoking. reduced lung function in the cohort was associated with both smoking and cumulative asbestos exposure. conflict of interest none. introduction malignant mesothelioma (mm) is an aggressive cancer with a very poor prognosis. interactions of the components of the extracellular matrix (ecm) are now known to be important for the growth and regulation of cancer cells. tgfb is an important regulator of the ecm and in particular collagen. previous data in our laboratory has shown that blocking tgfb signaling by using tgfb antibodies inhibits collagen production and mm growth. aim to determine the signaling pathways downstream of tgfb that are important in the regulation of collagen expression in mm. methods components of the tgfb pathway were inhibited by use of chemical inhibitors and overexpression of the endogenous inhibitor smad7 in control and mm cell lines. collagen levels were measured by realtime pcr. results collagen regulation is thought to occur through the classic smad2/3 signaling pathway. our data show that smad7 overexpression inhibits tgfb-induced collagen production in normal mesothelial cells and the mesothelial cell line met-5a but not in the mm cell lines investigated. therefore, the smad2/3 pathway for collagen regulation appears to be altered in malignant mesothelioma. it was shown that smad2/3 are expressed, phosphorylated and activated by tgfb in the mm cell lines. our results indicate that nuclear import of smad4, which is responsible for the nuclear import of smad2/ 3, is altered in mm. aim to characterise impedance variability at 6 hz in asthma and its relationship to asthma severity. methods a school-based cohort of 38 non-asthmatic children, aged (mean (sd)) 9.5 (1.8) years (uptech feasibility study) were tested on two occasions 2 weeks apart. an asthma camp cohort of 22 asthmatics, aged 10.5 (1. 2) years, were tested daily for 5 days. mean resistance (rrs 6 ) and reactance (xrs 6 ) of at least three technically acceptable one minute recordings were reported. medications were not withheld. variability was assessed by intraclass correlation coefficient (icc) and within-subject standard deviation (sd w ) using first and last testing day data, and all 5 days of data for sd w severity comparison amongst asthmatics. results repeat fot measures at 6 hz were obtained in 34/38 non-asthmatic children. mean (sd) rrs 6 and xrs 6 was 6.89 (1.1) and -1.57 (0.52) for the uptech cohort, and 5.68 (1.12) and -1.27 (0.55) cmh 2 o/l/s in the asthma camp cohort respectively. rrs 6 variability was increased in asthmatics. rrs 6 variability tended to be higher in persistent vs. intermittent asthmatics but did not reach statistical significance (p = 0.07). non asthmatic (n = 34) introduction our cochrane review examining the efficacy of using feno to tailor the dose of inhaled corticosteroid showed that feno cannot be routinely recommended for clinical practice at this stage and remains uncertain. however all the 6 studies used a single feno cut-off. in this rct we determined if asthma monitoring using feno (using two different cut-offs dependent on atopy) is better than control (symptoms and fev 1 ) in preventing asthma exacerbations in children on inhaled corticosteroids. methods over 12-months, children underwent spirometry, feno, qol and asthma/ cough diary during every visit. treatment for asthma was adjusted according to pre-determined criteria taking into account atopy status and dependent on allocation group (feno or control). results about 63 children were randomised-feno group (n = 31, median age 10.2, iqr 5.75), or control group (n = 32, median age 10.1, iqr 5.69). significantly fewer children in the feno group had asthma exacerbations compared to the control group (6 vs. 15; p = 0.021) over 12-months. number needed to treat (nnt) to prevent one child from having any exacerbation in 12 months = 4 (95%ci 3, 24). parental qol improved in feno group at final visit in comparison to the qol in control group (p = 0.042). fev 1 increased in both groups over the duration of the study but there was no difference between the groups when measured at baseline (p = 0.661) and at final (p = 0.385). conclusion tailoring of asthma medications in accordance to feno levels (compared to usual management), taking into account atopy status, reduces asthma exacerbations and improves asthma qol. however both strategies equally improved fev 1 . background inhaled corticosteroids have a modest effect on improving symptom control in preschool asthmatic children. delivery of inhaled steroids with pmdi-spacers are influenced by children's proficiency in spacer technique, and adherence to prescribed medication. aim to investigate the influence of an incentive spacer (funhaler), on spacer technique, adherence to treatment, and asthma control in preschool asthmatic children. methods about 132 children aged 2-6 years, and being prescribed regular inhaled steroids in the community were randomised to receive regular inhaled fluticasone through either an aerochamber plus ò , of a funhaler ò . subjects were followed up three monthly for a year. proficiency in spacer technique was measured at each visit by measuring the amount of salbutamol inhaled from spacer onto a filter interposed between subject and spacer. adherence was monitored by smartinhaler ò electronic devices. symptoms were recorded on diary cards for a week before each study visit. results there was no difference between the funhaler group and the aerochamber group in terms of adherence to medication or measures of asthma control (p > 0.05). spacer technique was significantly better in the funhaler group in subjects younger than 4 years of age at time of randomisation (p < 0.00). there was large inter subject variation in drug dose inhaled on filter, ranging from 0 -100% (drug dose recovered from filter, as percentage of total dose recovered), and mean adherence over each 3 month period ranging from 0-100%. discussion the funhaler ò does not improve clinical outcome, but improves spacer technique in children younger than 4 years of age. the large variability in adherence and drug delivery should encourage both efforts to improve adherence, and efforts to standardise inhaled drug delivery in preschool children. 1990-1994 and 2005-2007 . about 75% of ob cases were notified within 1 year of arrival. 30 of the australianborn cases were close household contacts of an adult tb case. about 18 cases had culture confirmed disease (15 fully sensitive to first line drugs, one multidrug resistant). 70% had pulmonary and 23% had lymph node tb. about 82 cases completed the treatment, two were lost to follow-up and one died. compared to adult tb cases, children were more likely to be refugees (or 2.1 (ci 1.1-3.7)), diagnosed on contact screening (or 14.4 (7.9-25.8)), have lymphatic tb (or 2.54 (ci 1.5-4.3)), and less likely to be culture-confirmed (or .07 (ci .04-.12)). the png child visitors' cases diagnosed in queensland had a higher level of severe and culture-confirmed disease. conclusion queensland has a very low burden of childhood tb, indicating low levels of tb transmission in the community. hrgm children, especially refugees, will remain at risk due to infection acquired overseas. contact screening is an important method of diagnosing early tb, and refugee screening and preventive treatment may play a role in protecting this group. funding support nil. conflicts of interest nil. introduction in response to injury, normal and efficient epithelial repair is essential in order to maintain barrier integrity and immune function. however, aberrant repair has been suggested as a contributor to disease progression in asthma. many studies have only included subjects with atopic asthma and thus any intrinsic epithelial abnormality common to all asthmatic phenotypes is difficult to isolate. this study aimed to assess whether epithelial repair is dysregulated in asthmatic subjects and if this is common to the disease or is phenotype specific. the regulatory mechanisms promoting the cellular proliferation and migratory aspects of the repair process were also assessed. methods paediatric airway epithelial cells (paec) of atopic and non-atopic healthy and asthmatic subjects were isolated by non-bronchoscopic bronchial brushings. culture monolayers were wounded using an in-house wounding device, and the percentage of wound closure determined daily. proliferation and migration were also assessed over the course of repair using western blot. results paecs from healthy non-atopic (paec hna ) and healthy atopic (paec ha ) subjects successfully achieved full wound closure between 8-10 days. in contrast, atopic asthmatic (paec aa ) and non atopic asthmatic (paec naa ) subjects failed to fully repair and only achieved 40% wound closure by 10 days. protein analysis showed a 4-fold increase in proliferation and 2-fold increase in migratory markers during repair in paec hna . however, reduced proliferation and no migration activity were seen in paec aa. conclusion atopic and non-atopic asthmatic epithelial cells possess dysfunctional repair profiles in response to mechanical wounding. results suggest dysregulated repair is an intrinsic epithelial abnormality in asthma and this appears to be independent of phenotypic criteria or atopy. introduction refractory chronic cough is associated with increased cough sensitivity. speech pathology intervention has been shown to be an effective intervention for refractory cough but the mechanism behind the improvement is not known. this study provides objective measures of the mechanism and the number of treatments required to effect a response. methods adults with chronic cough (n = 17) were assessed before, during and after speech pathology intervention. the primary outcome measures were capsaicin cough reflex sensitivity, automated cough frequency detection and cough-related quality of life. results participants responded to the treatment with a significant improvement in coughrelated quality of life, p = 0.002, cough reflex sensitivity, c5: mean ± sd 13.2 ± 15.8 vs. c5:174.9 ± 227.3 lmol/l, p = 0.013, cough frequency cf: 72.5 ± 55.8 vs. 25 ± 27.9 coughs/hr, p = 0.009, cough threshold ct: 4.21 ± 3.83 vs. 46.9 ± 69.1 lmol/l, p = 0.009, and urge-to-cough utc: median (iqr), 5(1) vs. 1(4), p = 0.01. conclusion speech pathology management is an effective treatment for refractory chronic cough. the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, improvement in cough symptoms, laryngeal symptoms, and cough quality of life. introduction airway hyperresponsiveness (ahr) is a characteristic feature of asthma. in young asthmatics, severity of ahr is related to exhaled nitric oxide (eno), a marker of eosinophilic airway inflammation, and ventilation heterogeneity in the conducting airways (scond). with increasing age, eosinophilic inflammation decreases and ventilation heterogeneity in the very peripheral, acinar, airways worsens. aim to determine if the predictors of ahr differ in young and older asthmatics. methods about 61 young (18-46) and 41 older (50-80) asthmatic subjects underwent baseline spirometry, body plethysmography, eno, multiple breath nitrogen washout (mbnw), and methacholine (mch) challenge. ahr was expressed as dose response slope (drs = %fall fev1/lmol mch). ventilation heterogeneity of the conducting (scond) and acinar (sacin) airways were calculated from the mbnw. predictors of ahr in each group were determined by multiple linear regression. results compared to younger asthmatics, older asthmatics had lower values of eno, less severe ahr, worse acinar heterogeneity; however there were no differences in scond values. in younger asthmatics, ahr was predicted by fev1/fvc (partial r 2 = 0.29), eno (partial r 2 = 0.13) and scond (partial r 2 = 0.06) (overall r 2 = 0.48, p < 0.0001). in older asthmatics, ahr was predicted by rv % predicted (partial r 2 = 0.29), sacin (partial r 2 = 0.17) and fev1 % predicted (partial r 2 = 0.05) (overall r 2 = 0.51, p < 0.0001). conclusions the predictors of ahr are different in young and old asthmatics. in older asthmatics, eno is not a significant predictor of ahr, which may reflect the changing inflammatory profile associated with aging. the association between ahr and both rv and sacin suggests that ahr in older asthmatics is determined by abnormalities in very peripheral airways. introduction in this systematic review and meta-analysis, we sought to establish if maternal asthma is associated with an increased risk of adverse perinatal outcomes associated with size at birth and timing of birth. methods electronic databases were searched for the following terms: (asthma or wheeze) and (pregnan* or perinat* or obstet*). cohort studies published between 1975 and march 2009 were considered for inclusion. 103 articles were identified, and 40 publications involving 1,637,180 subjects met the inclusion criteria, by reporting at least one perinatal outcome in pregnant women with and without asthma. meta-analysis was conducted with subgroup analyses by study design and active asthma management. results maternal asthma was associated with an increased risk of low birth weight (relative risk [rr] 1.46, 95% confidence interval [ci] 1.22, 1.75), small for gestational age (sga, rr 1.22, ci 1.14, 1.31), very sga (rr 1.27, ci 1.18, 1.37), significantly reduced mean birth weight (weighted mean difference -93 g, ci -169, -25 g), and reduced risk of high birth weight (rr 0.84, ci 0.74, 0.95). maternal asthma was associated with an increased risk of preterm labor (rr 1.71, ci 1.14, 2.57), early preterm labor (rr 1.93, ci 1.58, 2.34) and preterm delivery (rr 1.41, ci 1.22, 1.61). the risk for preterm labor and delivery was reduced to a non-significant level in those studies reporting active management of asthma during pregnancy (rr 0.95, ci 0.73, 1.26; rr 1.07, ci 0.991, 1.26). conclusion pregnant women with asthma are at increased risk of perinatal complications which affect the baby's size and timing at birth. active asthma management may reduce the risk of preterm labor and delivery. with threats of new pandemic strains of influenza a virus and resistance to anti-virals there is a need for novel therapeutics that reduce viral replication and lung pathology. the pathology arising from pandemic influenza is due to an excessive host response characterised by a rapid, massive infiltration of inflammatory cells of the innate immune system into the airways leading to excessive reactive oxygen species (ros) production. thus, we investigated the primary enzymatic source of inflammatory cell ros, nox2-containing nadph oxidase, as a novel target against lung inflammation and pathology caused by influenza a viruses of varying virulence. wt and nox2 -/--mice were 10 4 pfu/mouse) or high virulence following infection with x-31, lungs of nox2 -/-mice displayed a significant reduction in viral titre (~40-50%), macrophages, peribronchial inflammation and mcp-1 compared to virusinfected wt mice. lung levels of il-1b were approximately 3-fold higher in nox2 -/-mice. balf macrophages, neutrophils, and t lymphocytes of nox2 -/-mice produced minimal superoxide compared to controls. the magnitude of balf and spleen influenza-specific dbnp366+ and dbpa224+ cd8 + t cells were similar in wt and nox2 -/-mice indicating that the major mechanisms of the adaptive immune response that effectively clear influenza a virus are preserved in nox2 -/-mice. in vivo administration of the nox2 inhibitor apocynin (5 mg/kg/day) significantly suppressed viral titre, airways inflammation and inflammatory cell superoxide following infection with x-31 or pr/8 strains. in conclusion, nox2 inhibition should be considered for seasonal and pandemic control of the mortality/ morbidity induced by influenza a virus, irrespective of the strain department of respiratory medicine australia, 3 cooperative research centre for asthma and airways, new south wales, australia, and 4 department of respiratory medicine therefore the aim, of this study was to examine the impact of a standard, 8-week exercise-based pr program on pal. methods about 19 subjects (65 (9) years) with copd (fev1% predicted = 58 (22)) completed pr where they undertook twice weekly exercise classes consisting of one hour of upper and lower limb strengthening exercise and aerobic exercise. pal was estimated using a multi-sensor device (sensewear, healthware bodymedia) worn for a 7 day period. an index of pal was derived by dividing total daily energy expenditure in metabolic equivalents (mets) by whole night sleeping energy expenditure (average of 3 nights sleeping). pal was measured in the week immediately prior and in the immediately following pr. results despite a significant increase in six minute walk distance (6mwd), pr resulted in no change in pal copd patients failed to increase their pal. while changes in pal may take longer to elicit i.e. the change in pal may be delayed following pr, it is possible that the current focus of pr on increasing outcomes such as 6mwd may be too narrow to elicit changes in pal little is known regarding the use of acts in patients admitted with aecopd in australia. this survey aimed to identify current practice and opinion of australian hospital physiotherapists concerning acts. methods paper-based surveys were distributed to physiotherapists of 112 'large' and 'principal referral' australian public hospitals (identified via a government health report). a response rate of 81% (n = 95 hospitals) yielded 189 surveys for analysis. results most physiotherapists (65%) prescribe acts for 60-100% of patients with ae-copd, with 90% of act treatments lasting 5-20 minutes. the techniques most frequently used for airway clearance were physical exercise (57%) and the active cycle of breathing technique (41%). the main influences on choice of act were precautions or contraindications to individual techniques (78%) and the degree of patient dyspnoea (72%). many physiotherapists (68%) prescribe acts with the aim of enhancing a patient's recovery from aecopd and 73% perceive airway clearance to be fairly or very important to the overall management of aecopd. there was mixed awareness of the evidence for acts in aecopd, with 43% of physiotherapists citing it as supportive conclusion australian physiotherapists frequently use acts for patients with aecopd and perceive their role to be important. physical exercise is the present modality of choice to achieve airway clearance acknowledgements nh&mrc, cure4cf foundation sa. conflict of interest no. aim to examine the health outcomes of 2,483 children first exposed to blue asbestos at wittenoom when they were less than 16 years of age. methods standardised mortality ratios (smr's) calculated to compare wittenoom children's mortality with the western australian population. results about 1,204 females and 1,279 males were children at wittenoom, mean age of arrival 4 years (sd 4 years); 419 (17%) were born there or moved there soon after birth. median duration of residence was 19 months (iqr 7-41 months). there were 228 deaths (75 females and 153 males) between 1950 and end of 2006. 40 deaths were from malignant mesothelioma (17% of all deaths -12 females, 28 males, -39 pleural, 1 peritoneal). among males, there was excess mortality from all causes (smr = 1.89), all cancers (smr = 3.70), mm (smr = 73), accidents, injuries and poisonings (smr = 1.54) and circulatory disease (smr = 2.21). mortality from suicide and transport accidents were also in excess but not statistically significantly increased. among females there was excess mortality from all causes (smr = 1.62), and all cancers (smr = 2.25) and mm (smr = 155). conclusion former children of wittenoom experience high cancer mortality. support nhmrc. nomination nil. conflict of interest nil. introduction blue asbestos (crocidolite) was mined and milled at wittenoom between 1943 and 1966 . tailings from the mine were distributed and used extensively throughout the town. exposure to children also occurred from the laundering of workers clothes at home. earlier work has shown a lower risk of malignant mesothelioma (mm) in children from wittenoom than in those exposed to blue asbestos as adults.a case report of a 69 year old ex-forestry worker with an 80 pack year smoking history is presented. he was referred with two distinct periods of hemoptysis, one 7 months prior to referral for which he declined investigation or follow up, and another three weeks prior to referral. on each occasion, he described two tablespoons of hemoptysis daily lasting approximately 1 month. he lives on an acreage at mt kilcoy, 94 km north of brisbane, with his wife, one goat, three dogs, one cat, 51 deer and wild birds. emphysema manifesting as gradually worsening exertional dyspnoea with wheeze, had been diagnosed years ago by his local doctor. he described symptoms of obstructive sleep apnoea including witnessed apnoea, choking arousals and loud snoring. he also reported intermittent diarrhoea for 2 years but denied weight loss or rectal blood or mucous. a ct chest showed multiple bilateral nodules of varying size the largest being 3.6 cm, and multiple low density liver lesions. the provisional diagnosis was metastatic colorectal cancer. bronchoscopy with ebus guidance did not yield the diagnosis which was eventually made by trans-thoracic needle aspiration without complication. echinococcus serology performed post procedure was >1024, consistent with echinococcus infection. this case of echinococcus disease is presented and the vectors discussed. echinococcus disease was previously prevalent in australia and new zealand, with a reduction in incidence from improved animal husbandry. with an increasing deer population in south east queensland and subsequent rising human contact, clinical awareness is necessary to avoid potential complications from biopsy and ensure cases are promptly treated rather than mistakenly diagnosed as incurable disease. introduction community-acquired pneumonia (cap) is a leading cause of mortality, morbidity and hospital admission places strain on our healthcare system. procalcitonin (pct) is a biomarker of bacterial infection which may help gauge the severity and prognosis of patients with cap. aim to examine the role of pct measurement in reducing hospital admissions, length of stay (los), and antibiotic (ab) usage in patients with cap. methods prospective, single-blinded, externally controlled study of consenting adult patients admitted with cap. pct levels were obtained on day 1 and day 3 (if indicated). the investigator evaluated clinical parameters and the pct values to determine the timing of oral ab switch and discharge. this process was used to compare with standard practice but was not actually implemented for the purpose of this study. results sixty patients were included in the study. the mean age was 66.5±21.2y and 56.3% were male. the average psi was 91 ± 40 (class iv) and the median curb-65 was 2. the mean los for this cohort was 5.32±4.56 d and the calculated los using pct guidance pathway was 3.68±2.81 d. (p = 0.00006) a multivariate analysis will be presented. conclusions our study supports the hypothesis that the incorporation of pct levels can reduce the requirement for hospital admission and los in patients with cap. a randomised prospective clinical trial is planned to help clarify these findings. support nil. introduction children in the highlands of papua new guinea (png) suffer on average 4.3 acute lower respiratory infections (alris) before age 18 months, 1/3 of which are moderate or severe. while streptococcus pneumoniae and haemophilus influenzae are the primary bacterial cause of alri in png, the role of viruses in the aetiology of alri is uncertain. aim determine identification rates of respiratory viruses in nasal samples collected from children with moderate/severe alri and healthy children aged <18 months in png. methods as part of a neonatal pneumococcal conjugate vaccine trial in the png highlands, we collected pernasal swabs from children with moderate/severe alri (n = 49) and at routine follow-up (n = 48). rt-pcr methods were used to identify a broad range of respiratory viruses. the frequency of viral detection was compared between groups of samples collected during an alri and routinely using chi-square analysis. results several viruses were detected more frequently in alri than routine samples: adenoviruses 33.3/12.5 (% of alri samples positive/% of routine samples positive) p = 0.032, influenza viruses 22.5/7.9 p = 0.023 and respiratory syncytial virus (rsv) 8.2/ 0.0 p = 0.043. human metapneumovirus and parainfluenza viruses were detected in four and three samples, with no difference between groups. human coronaviruses and human rhinoviruses (hrv) were less commonly detected in alri than in routine samples (4.2/8.3 p = 0.042 and 57.1/72.3 p = 0.178, respectively). a total of 62 different hrv strains were identified. conclusion in young children in png, viral identification rates are high, with rsv, adenoviruses and influenza viruses associated with moderate/severe alri and a large amount of genetic diversity of rhinoviruses in both sick and healthy children. introduction to increase the documentation and documented provision of an electronic asthma action plan (e-aap) to children discharged with asthma from the emergency department (ed) at chw. methods an electronic aap (e-aap) was introduced in april 2008 by a multidisciplinary team comprising representatives. at chw, aaps were available to be printed off by the intranet. to be entered into the electronic medical record (emr), the medical officer had to photocopy the completed plan which would then be scanned into the emr. evidence suggested that busy doctors, particularly in the ed, were either not providing patients with aaps on discharge or not photocopying them for the medical record. the evaluation of the e-aap consisted of a review of the documented provision of asthma action plans in the hospital wide emr (powerchart) for a year pre & post the introduction of the e-aap, a review of patients discharged from the ed with a diagnosis of asthma for similar six month periods pre and post intervention and a medical staff satisfaction survey. results the total number of plans recorded in emr increased by 816%, from 2007-2008 to 2008-2009 . the number of plans recorded for ed discharges increased significantly from 2% to 73% [p < 0.001]. the number of patients recorded as leaving the ed with a plan increased significantly from 20% to 79% (p < 0.001). the use of the e-aap in the ed is now standard of care and this is also being adopted hospital wide as more staff became familiar with its usefulness.conclusion the e-aap significantly increased the number of recorded aaps and patients discharged with a recorded aap. support nil. nomination asthma/allergy. conflict of interest no. lisa wood 1,2 , manohar garg 2 , amber wood 1,2 , peter gibson 1,2 1 centre for asthma and respiratory diseases, university of newcastle, new south wales, australia, and 2 nutraceuticals research group, university of newcastle, new south wales, australiaintroduction dietary fat activates innate immune responses, leading to an increase in systemic inflammation. however, the effect of dietary fat on airway inflammation has not been investigated. we hypothesised that a high fat intake may lead to increased airway neutrophilia in asthma. the aim of this study was to examine the effect of a high fat versus low fat food challenge on airway inflammation in asthma. methods non-obese subjects with asthma were randomized to receive a high fat/ high energy (hf) (n = 14) or low fat/ low energy (lf) (n = 16) food challenge. 16 obese subjects also received a hf challenge. subjects on the hf challenge consumed a meal containing 4480 kj, including 52% of energy (60 g) from fat. subjects on the lf challenge consumed a meal containing 840 kj, including 13% of energy (3 g) from fat. at baseline, hypertonic saline challenge and clinical assessment were performed. induced sputum samples were collected at baseline and at 4 hours. airway inflammatory markers included induced sputum total and differential cell counts, il-8 and neutrophil elastase, measured by commercial assay. tlr4 mrna expression from sputum cells was measured using rt-pcr. results at 4 hours after the food challenge, subjects on the hf challenge, had a significantly higher increase in %sputum neutrophils (16.4 (4.4 (sem)) % vs. 3.4 (4.1) %, p = 0.044) and higher fold increase in tlr4 mrna expression (2.06 (1.3-5.4 (iqr)) vs. 1.00 (0.6-1.4), p = 0.037), compared to the lf challenge. subjects on the hf challenge also had an impaired bronchodilator response, with a lower increase in fev1/fvc% at 4 hours compared to the lf challenge (1.0 (-2.0-2.6 (iqr)) % vs. 4.5 (2.7-6.8) %, p = 0.001). there were no differences in the responses of obese vs. non-obese asthmatics to the hf challenge. conclusions a high fat/ high energy challenge causes an increase in airway inflammation and suppresses bronchodilator response in asthma. strategies aimed at reducing dietary fat intake may be useful in reducing inflammation in asthma. support nhmrc project grant. introduction longitudinal fev 1 data in children with non-cystic fibrosis (non-cf) bronchiectasis is contradictory and there is no multi-factor data on the evolution of lung function and growth in this group. we longitudinally reviewed lung function and growth in children with non-cf bronchiectasis and explored biologically plausible factors associated with changes in these parameters over time.methods fifty-two children with ‡3 years of lung function data were retrospectively reviewed. changes in annual anthropometry and spirometry at year-3 and year-5 from baseline were analysed. the impact of gender, age, aetiology, baseline fev 1 , exacerbation frequency, radiological extent and period of diagnosis was evaluated. results over 3 years, the group mean fef 25-75 %predicted and bmi z-score improved by 3.01 (p = 0.04, 95%ci 0.14-5.86) and 0.089 (p = 0.01, 95%ci 0.02-0.15) per annum, respectively. fev 1 %predicted, fvc %predicted and height z-score all showed non-significant improvement. over 5 years, there was improvement in fvc %predicted (slope 1.74, p = 0.001) annually but only minor improvement in other parameters. children with immunodeficiency and those with low baseline fev 1 had significantly lower bmi at diagnosis. frequency of hospitalized exacerbation and low baseline fev 1 were the only significant predictors of change in fev 1 over 3 years. decline in fev 1 %predicted was large (but nonsignificant) for each additional year in age of diagnosis. conclusions spirometric and anthropometric parameters in children with non-cf bronchiectasis remain stable over 3-5 year follow-up period once appropriate therapy is instituted. severe exacerbations result in accelerated lung function decline. increased medical cognizance of children with chronic moist cough is needed for early diagnosis, better management and improving overall outcome in bronchiectasis. introduction it is well established that many survivors of very low birth weight (vlbw; <1500 g at birth) have impaired lung function. the aim of this study was to determine whether abnormal lung function at 25 years of age is established in childhood. a second aim was to see if abnormal lung function at 8 years of age tracks through the period of normal lung development to predict impaired maximal lung function and may be a precursor to copd in adult years. methods a cohort of vlbw (n = 210) and normal birth weight (nbw; >2500 g at birth; n = 60) has been followed for 25 years. very low birthweight participants completed spirometry and lung volumes at 8, 11, 14, 18 and 25 years of age and nbw at 14, 18 and 25 years of age. restricted maximum likelihood modeling was used for longitudinal fev 1 z-score as it allows for analysis of data from different time points that are not necessarily evenly spaced, without being affected by missing data. results about 137 vlbw children completed lung function testing at 8 years of age, 20 (14.6%) had abnormal fev 1 z-scores (defined as >2 sd's below the mean). vlbw survivors showed minimal 'catch-up' in fev 1 z-score over the 13 years of the study; those without (bronchopulmonary dysplasia) fev 1 improved 0.034 (p = 0.014) z-scores, those with bpd fev 1 improved 0.033 (p = 0.039) z-scores. vlbw with bpd survivors did not return to within normal limits. conclusions the reduced lung function in adult survivors of low birth weight is established in early childhood. while there is some improvement in growth of those with abnormal fev 1 z-scores in early childhood, those with bpd remain below two sd's from the mean, and at the age of 25 have a reduced peak lung function. introduction bronchopulmonary dysplasia (bpd) is a common complication of preterm birth. although there is evidence that individuals with a history of bpd have respiratory abnormalities in childhood, there remains a paucity of evidence regarding the outcome of the disease in adulthood. in a pilot study we recently described high resolution computed tomography (hrct) appearances of emphysema in young adults with a history of bpd. aims to describe the structural pulmonary sequelae of bronchopulmonary dysplasia in adulthood and to evaluate a scoring system originally designed for paediatric subjects. methods about 51 adult survivors of bpd underwent hrct of the chest, along with lung function testing (spirometry, lung volumes and diffusing capacity) and a respiratory health survey. the ct studies were scored by two thoracic radiologists blinded to the patient's clinical details, using a previously described system developed for children and adolescents who were born prematurely using 14 parameters. results abnormal findings were seen in all scans, the most common findings were subpleural triangular opacities (94%), linear opacities (90%), air trapping (65%) and emphysema (47%). agreement between the two observers for total score and common abnormalities varied with a linear weighted kappa value of 0.18 for linear opacities, 0.71 for triangular opacities, 0.76 for air trapping, and 0.66 for emphysema. conclusions linear and sub-triangular opacities on hrct chest are almost universal in young adults with a history of bpd. findings of emphysema and gas trapping are common and there is good interobservor agreement for these abnormalities. introduction pulmonary surfactant (ps) is synthesised by alveolar type ii epithelial cells to regulate the surface tension at the air-liquid interface of the air breathing lung. developmental maturation of ps is controlled by many factors including oxygen, glucose, catecholamines and cortisol. the intrauterine growth restricted (iugr) fetus is hypoxemic and hypoglycaemic, with elevated plasma catecholamines and cortisol. aim to determine the impact of iugr induced by chronic placental restriction via the carunclectomy model on ps maturation. methods we investigated the expression of surfactant protein (sp) -a, -b and -c and their genes in lung tissue of fetal sheep at 133 days and 141 days gestation (term, 150 ± 3 days) from control and carunclectomised merino ewes. results placentally restricted (pr) fetuses had a body weight <2 sd from the mean of control fetuses and a mean gestational pao2 <17 mmhg. pr fetuses had a reduced absolute, but not relative lung weight, decreased plasma glucose and increased plasma cortisol concentration. lung sp-a, -b and -c protein and mrna expression were reduced in pr compared with control fetuses at both ages. sp-b and -c, but not sp-a mrna expression and sp-a, but not sp-b or -c protein expression increased with gestational age. mean gestational pao2 was positively correlated with sp-a, -b and -c protein and sp-a and -c mrna expression. sp-a and -b gene expression were inversely related to plasma cortisol concentration. conclusion chronic placental restriction and hypoxemia results in an inhibition of ps maturation and thus iugr fetuses are at risk of lung complications, especially if born prematurely. support by the arc & nhmrc. nomination nil. conflict of interest nil. to 116 sue jenkins 1,2,3 , nola cecins 1,2,3 1 sir charles gairdner hospital, perth, western australia, australia, 2 curtin university of technology, perth, western australia, australia, and 3 lung institute of western australia, perth, western australia, australiaintroduction the 6mwt is widely used to assess patients with chronic lung disease (cld). anecdotal reports and studies in small numbers of patients suggest that adverse events associated with the 6mwt are rare in patients with cld. this study reports observed adverse events and predictors of oxygen desaturation during the 6mwt in patients with stable cld referred to an out-patient pulmonary rehabilitation service. methods about 741 consecutive patients completed the 6mwt in accordance with a standardised protocol that included continuous monitoring of oxygen saturation (spo 2 ) and heart rate (hr, polar). the respiratory diagnoses of the patients were chronic obstructive pulmonary disease (copd), n = 565 (76%); interstitial lung disease (ild), n = 84 (13%); bronchiectasis, n = 46 (6%) and asthma n = 39 (5%). results observed adverse events occurred in 43 tests (6%). one test was terminated when the patient reported chest pain and one patient developed persistent tachycardia (hr > 200 bpm) immediately following the test. in 35 tests (5%), the tester instructed the patient to stop walking due to profound oxygen desaturation (spo 2 <80%). six patients prematurely terminated the 6mwt due to intolerable symptoms. forty-seven per cent (n = 345) of patients demonstrated oxygen desaturation, defined as a decrease in spo 2 ‡4% to <90% during the test. pre-exercise spo 2 was a significant predictor of desaturation in the copd (1.79, 1.54 to 2.08, adjusted odds ratio [or], 95% confidence intervals) and ild (or 1.40, 1.11 to 1.77) cohorts with fev 1 also a predictor in patients with copd (or 3.02, 1.77 to 5.15). conclusions profound oxygen desaturation is the commonest adverse event observed during the 6mwt in patients with stable cld. this finding questions the american thoracic society guidelines for the 6mwt which state that oximetry is optional. introduction there is weak support for use of opiates in palliation of refractory dyspnea; respiratory depression is perceived as a major risk. we evaluated the effect of i.v. morphine on dyspnea in controlled conditions and related this to concomitant respiratory depression. methods with ethical approval, 6 healthy subjects received 0.07 mg/kg morphine sulphate or saline on separate days in a randomised controlled design. before and for 2 hours after administration, subjects performed (i) dyspnea responses (measured with a visual analog scale) to increasing p et co 2 with ventilation () constrained at resting levels (ii) unconstrainedresponses to increasing p et co 2 to assess respiratory drive. results pre morphine with constrained , all subjects tolerated an elevated p et co 2 of 50-55 mmhg; mean dyspnea rating was 56% (7(sem)). post morphine, at the same p et co 2 , mean dyspnea rating fell to 29 % (5, p < 0.05, paired t). all subjects reported reduced dyspnea at 20 minutes and this was sustained for 2 hours. no changes in dyspnea scores were seen following saline. with unconstrained, at equivalent levels of p et co 2 , morphine, but not saline, was associated with a lower in each subject for up to 2 hours; mean fell from 30 (3) to 22 (2) l/min (p < 0.05). to assess if respiratory depression could account for reduced dyspnea, scores were compared at the different p et co 2 levels that induced equivalent unconstrainedlevels with and without morphine; mean dyspnea scores were not different (54 (10) vs. 52 (10) %). conclusion a clinically moderate dose of morphine results in substantial and sustained relief of laboratory dyspnea in a small group of healthy subjects consistent with the associated degree of respiratory depression. support breathlessness research charitable trust uk; nih, usa. nomination nil. introduction pulmonary rehabilitation (pr) is a cornerstone of management for patients with chronic obstructive pulmonary disease (copd) and its efficacy is supported with level 1 evidence. despite the known benefits of pr, up to one third of those people with copd who are referred to pr choose not to participate. there is little information regarding perceived barriers to attendance in an australian health care context. methods nineteen people with copd (10 women and nine men, gold stage i-iv, age range 53-86 years) who had declined to take part in an outpatient pr program at a metropolitan teaching hospital participated in a qualitative study. semi-structured interviews were used to establish reasons for failing to attend the pr program. these interviews were transcribed verbatim and analysed using the principles of grounded theory. results three major themes were identified regarding barriers to attendance at pr. the first related to difficulties with getting there, including a lack of available transport and poor mobility. the second theme related to a lack of perceived benefit, including perceptions that pr would not improve their health or that they were currently doing enough exercise. the third major theme involved restrictions imposed by underlying medical conditions and included the influence of comorbidities and pain. minor themes that arose included competing demands, age, fatigue and program timing. conclusions in australia many patients with copd who are invited to attend pr do not perceive the program would be beneficial, feel they are too unwell to attend or have difficulty with access. further support should be offered to pr candidates and alternative methods of delivering pr to enhance uptake should be considered. introduction pulmonary rehabilitation has emerged as recommended standard care for people with chronic lung disease. however potential demand to access these services far exceeds the available resources. this study's aim was to determine if baseline measures of the bode index, dyspnea (modified medical research council questionnaire), 6 minute walk distance (6mwd), physical activity, taunton respiratory quality of life questionnaire (trq), smoking status, and frequency of hospitalisations can predict responders to pulmonary rehabilitation. methods we retrospectively evaluated all participants with a diagnosis of copd, who attended the pulmonary rehabilitation program at the prince charles hospital between 2004 and 2007. a participant was considered a responder to pulmonary rehabilitation if benefit was achieved in exercise capacity ( ‡20% increase in 6mwd) and/or quality of life ( ‡0.5 sd decrease in trq as described by cohen's effect size). prediction of responders was assessed using chi square cross tabulations and t-tests with significant measures analysed using a binary logistic regression model. results one hundred and forty-two participants (76 males, mean age 69 (9 sd) years, mean fev1 50.4 (20.3) %) who completed pulmonary rehabilitation were analysed. sixtyfive (47.8%) people were categorised as responders using the above criteria. significant mean differences were: trq 40.9 (25.2) for responders vs. 18.5 (16.3) for non-responders p < 0.001; bode index 3.4 (2.4) vs. 2.5 (1.7) p = 0.02; 6mwd 368 (123) m vs. 422 (96) m p = 0.004. the binary logistic regression model showed a higher trq score was the only factor that predicted a responder to pulmonary rehabilitation. no other measure added to the predictive power of the model. conclusion higher trq scores may be useful in predicting which participants are most likely to benefit from referral to pulmonary rehabilitation. further study is underway investigating other factors that may improve these findings. support nil. nomination nil. conflict of interest no. key: cord-016947-8f22ukjc authors: mueller-mang, christina; ringl, helmut; herold, christian title: interstitial lung diseases date: 2017-08-24 journal: multislice ct doi: 10.1007/174_2017_151 sha: doc_id: 16947 cord_uid: 8f22ukjc the term interstitial lung diseases (ild) comprises a diverse group of diseases that lead to inflammation and fibrosis of the alveoli, distal airways, and septal interstitium of the lungs. the ild consist of disorders of known cause (e.g., collagen vascular diseases, drug-related diseases) as well as disorders of unknown etiology. the latter include idiopathic interstitial pneumonias (iips), and a group of miscellaneous, rare, but nonetheless interesting, diseases. in patients with ild, mdct enriches the diagnostic armamentarium by allowing volumetric high-resolution scanning, i.e., continuous data acquisition with thin collimation and a high spatial frequency reconstruction algorithm. ct is a key method in the identification and management of patients with ild. it not only improves the detection and characterization of parenchymal abnormalities, but also increases the accuracy of diagnosis. the spectrum of morphologic characteristics that are indicative of interstitial lung disease is relatively limited and includes the linear and reticular pattern, the nodular pattern, the increased attenuation pattern (such as ground-glass opacities and consolidation), and the low attenuation pattern (such as emphysema and cystic lung diseases). in the correct clinical context, some patterns or combination of patterns, together with the anatomic distribution of the abnormality, i.e., from the lung apex to the base, or peripheral subpleural versus central bronchovascular, can lead the interpreter to a specific diagnosis. however, due to an overlap of the ct morphology between the various entities, the final diagnosis of many ild requires close cooperation between clinicians and radiologists and complementary lung biopsy is recommended in many cases. references 000 the term interstitial lung diseases (ild) comprises a diverse group of diseases that lead to inflammation and fibrosis of the alveoli, distal airways, and septal interstitium of the lungs. the ild consist of disorders of known cause (e.g., collagen vascular diseases, drug-related diseases) as well as disorders of unknown etiology. the latter include idiopathic interstitial pneumonias (iips), and a group of miscellaneous, rare, but nonetheless interesting, diseases. in patients with ild, mdct enriches the diagnostic armamentarium by allowing volumetric high-resolution scanning, i.e., continuous data acquisition with thin collimation and a high spatial frequency reconstruction algorithm. ct is a key method in the identification and management of patients with ild. it not only improves the detection and characterization of parenchymal abnormalities, but also increases the accuracy of diagnosis. the spectrum of morphologic characteristics that are indicative of interstitial lung disease is relatively limited and includes the linear and reticular pattern, the nodular pattern, the increased attenuation pattern (such as ground-glass opacities and consolidation), and the low attenuation pattern (such as emphysema and cystic lung diseases). in the correct clinical context, some patterns or combination of patterns, together with the anatomic distribution of the abnormality, i.e., from the lung apex to the base, or peripheral subpleural versus central bronchovascular, can lead the interpreter to a specific diagnosis. however, due the interstitial lung diseases (ild) are a heterogeneous group of lung disorders that result from damage to the lung by various forms of inflammation and fibrosis. by definition, ilds involve the lung interstitium that forms a fibrous skeleton for the lungs. however, many of the conditions that have been traditionally included under the heading of ilds are actually associated with extensive alterations of the alveolar and airway architecture. for this reason, the terms "diffuse infiltrative lung disease" or "diffuse parenchymal lung disease" are preferable. still, the term ilds remains in common clinical usage. ild represent more than 200 different entities, and various and often confusing classification systems are simultaneously used. one useful approach to classification is to separate the ild into diseases of known and unknown etiology ( table 1) . ild of unknown etiology (65% of all ild) can be further subdivided into the group of idiopathic interstitial pneumonias (iip), and a group comprising several rare but nevertheless interesting diseases with distinctive clinicopathologic features, such as lymphangioleiomyomatosis, langerhans cell histiocytosis, pulmonary alveolar proteinosis, and pulmonary alveolar microlithiasis. sarcoidosis has an exceptional position within the group of ilds of unknown cause, as it is relatively common and can present as a systemic disease. ct scanning is the most important noninvasive diagnostic key to the identification and characterization of ild, and aids the radiologist and the clinician in the management of patients who carry this disorder. among all noninvasive methods, it provides the highest sensitivity and specificity in the detection of ild. also, it has a higher accuracy in comparison to the clinical assessment, lung function tests and chest radiography in diagnosing a specific disorder, and adds diagnostic accuracy and confidence when added to the clinical assessment and the chest radiogram. finally, ct helps to identify the best location for lung biopsy, and provides an important basis for the follow-up of ild patients. the correct interpretation of ild requires a fundamental understanding of normal lung anatomy. the identification of the patterns of infiltration and distribution is a key to the establishment of a correct list of differential diagnoses, and sometimes to the diagnosis itself. in this sense, hrct provides an insight into lung morphology and architecture, comparable to or even beyond macroscopic pathology. the following anatomic structures and architectural components need to be considered: the peribronchovascular interstitium is also known as the axial interstitium and runs along the pulmonary arteries, the bronchial branches, and the lymphatics from the hilar regions to the lung periphery. it is not visible in healthy individuals but lymphatic diseases, such as sarcoidosis, lymphangitic carcinomatosis or pulmonary edema can lead to smooth, nodular or irregular thickening of the peribronchovascular interstitium. the secondary pulmonary lobule (spl) is the smallest anatomical unit of the lungs that can be identified on hrct scans (fig. 1 ). whereas in normal lungs, these polyhedral structures are only allusively visible in the anterior and lateral portions of the pulmonary parenchyma, they may be clearly identifiable in any region when ild are present. typical spls are irregular polyhedral units which vary in size, measuring from approximately 1-2.5 cm in diameter and incorporating up to 24 acini (webb 2006 for spls ranges from 11 to 17 mm in adults. the spl is surrounded by a mantle of connective tissue septa. a central bronchovascular bundle, consisting of the lobular bronchiole and the accompanying pulmonary artery, enters the center of the spl, where the bronchiole bifurcates into three to five terminal bronchioles. the region near the origin of the terminal bronchioles is termed the "centrilobular" region. thus, on thinsection ct images, the secondary pulmonary lobule can be divided into three components: the interlobular septa; the centrilobular region; and the lobular parenchyma. the interlobular septa extend from the pleural surface of the lung inward, and surround the spl. they consist of connective tissue, house pulmonary veins, and lymphatics and belong to the peripheral interstitial fiber system (weibel 1979) . interlobular septa are well developed in the periphery of the lungs, and in particular in the lung apex, and near the anterior, lower, mediastinal, and diaphragmatic surfaces. they are key structures to the identification of pulmonary involvement in ild, because disorders such as interstitial lung edema, sarcoidosis, or lymphangitic carcinomatosis commonly lead to thickening and consequently to better visibility of these structures. the centrilobular region contains the pulmonary artery and bronchiolar branches that supply the lobule. because lobules do not arise at a specific branching generation of the bronchial or arterial tree, it is difficult to impossible to define exactly which specific bronchus or artery supplies that secondary lobule. however, lobular bronchioles are rarely seen in normal individuals since their lumen measures approximately 1 mm in diameter, and their wall 0.15 mm, respectively. likewise, the more peripheral terminal and respiratory bronchioles cannot be resolved at ct (murata et al. 1986) . it is only in diseases of the small airways that abnormal bronchi can be visualized through thickened walls, peribronchiolar inflammation, and/or intrabronchiolar fluid and mucus accumulations. centrilobular arteries can be depicted at ct scans of normal and diseased individuals. because of the anatomic properties of the lungs, centrilobular abnormalities are best seen in the lung periphery and near the hila. the centrilobular region can be affected by vascular and small airway diseases. the lobular parenchyma consists of alveoli, the associated pulmonary capillary bed, and a fine network of connective tissue which has been termed the intralobular interstitium. these structures are too small to be directly visualized on thin-section ct, but may be indirectly assessed as they are responsible for the background density of the lung on ct scans. parenchymal background density reflects the proportions of fluid (blood and extravascular fluid), gas, and tissue. when ild causes an increase of fluid or cells within the alveoli, or thickening of the alveolar septa through cellular infiltration or fibrosis, then parenchymal background density will change in turn and ground-glass opacities may be identified at ct. conversely, a decrease in fluid, cells, and tissue (in relation to air), as seen in emphysema, causes a reduction of the parenchymal density, in comparison to the normal state. the thickening of the intralobular interstitium and the interlobular septa by fibrosis or infiltration leads to a reticular pattern. for patients with ild, the detailed visualization of the lung parenchyma and the depiction of the smallest abnormal finding is of paramount importance for any imaging approach. in order to achieve highest possible spatial resolution, a thin acquisition section thickness, a high spatial frequency reconstruction algorithm, a certain amount of radiation dose, and a small field of view are mandatory. for decades, patients with ild have traditionally been assessed with hrct (mayo et al. 1987 ). this technique uses a "step-and-shoot" or "spaced" approach, in which thin sections with 0.5-1.5 mm thickness scans are obtained at 10-20 mm intervals. the large gaps between the sections considerably lower the radiation dose applied and allows for increasing the dose for each of the thin sections. therefore, each of these sections offers a very good image quality with low noise and high spatial resolution. this high quality comes along with the disadvantage of a discontinuous ct data set with 10-20 mm gaps between the sections hampering the ability for exact comparison to prior examinations if the images are not perfectly aligned in z-direction. this "classic" hrct technique still plays a decisive role in the noninvasive investigation of patients with pulmonary disease of a diffuse distribution pattern (hansell 2001 ). with the advent of mdct, volumetric highresolution imaging has become feasible and allows for imaging of the entire lung with high spatial resolution and the generation of a continuous dataset without gaps. in addition to visualize diffuse structural changes of the lung parenchyma it adds the ability to detect and interpret sparsely distributed abnormalities of the lung parenchyma as well as focal disease which might have been missed using the classic spaced hrct approach due to the gaps between the sections. the resulting volumetric, near isotropic data sets also permit the reconstruction of high quality multiplanar images which enable for better recognition of the distribution of disease, for example, to identify and rate a possible apico-basal gradient in the lung. finally, continuous data acquisition allows the generation of thick maximum intensity projections (mip) which are helpful for the detection of micronodular disease and centrilobular abnormalities. there are also some trade-offs with volumetric high-resolution ct scanning. for most scanners, except the most recent generation, the radiation dose is five to ten times higher, and the image quality concerning noise and spatial inplane resolution is lower in comparison to the introduced sequential hrct. this image quality reduction is most apparent in the depiction of small septa, ground-glass opacities as well in increased image noise (studler et al. 2005) . its clinical significance has yet to be determined. over the last few years most vendors significantly improved their ct detector and reconstruction technology. detector quantum efficiency as well as local electronic processing and data transfer have been considerable increased allowing for better image quality compared to last detector generation or lower dose for the same image quality that was reached a few years ago. filtered back-projection (fbp) has been the standard reconstruction method over decades for all ct protocols. however, in the last years, for most of the regions of the body, the recent generations of iterative reconstruction kernels have become standard of care and outperformed fbp in terms of image quality. however, due to the requirements in terms of spatial resolution for visualizing the lung accurately, there is still a controversy in radiology, whether iterative reconstruction should be used for the lung and which strength and software algorithm is best suited. therefore, the iterative reconstruction algorithm should carefully be selected and should be applied only with moderate strength for adequate visualization of the lung parenchyma. using lower tube voltages can considerable contribute to saving radiation dose. lowering this parameter is however limited due to patient size and the ability of the x-ray tube used to compensate with higher tube currents. this can be done manually or automatically with a built-in tube voltage selection. to end up with the right number of photons, ct scanners also offer an automatic tube current modulation. these techniques allow for autoregulation of the two most important parameters of the x-ray tube. they compute the optimum photon energy depending on the size of the patient as well as the tube current necessary based on a given quality factor or reference mas of the protocol. these two techniques should always be enabled for best quality at the lowest achievable dose (prosch et al. 2013 ). in addition to these improvements, spectral shaping has been introduced as an additional dose saving technique (gordic et al. 2014) . it consists basically of a tin filter, that absorbs photons at lower energy levels before they leave the x-ray tube. this technique can be applied at tube voltages at 100 kvp or above and allows for additional dose savings as well as for reduction of beam hardening artifacts. however, it is not suitable for iodine contrast enhanced scans, because higher energy photons show less absorption by iodine then low energy photons. depending on the device used, volumetric spiral ct of the lung can be performed with dose values quite below the national reference values, if new detectors, low tube voltages or spectral shaping are applied. however, application of dedicated low dose ct has to be carried out with great care if it comes to patients with ild. whereas it is perfectly feasible to use ultralow dose for the follow-up exam of lung nodules, because nodules above 4 mm can be accurately visualized with a low number of photons, this does not apply for small septa, very small nodules and ground-glass opacities. these structures may be over-or underestimated in the low dose ct (lim et al. 2016 ). the lack of a standard and the number of different scanners with different aged technology in operation in clinical routine result in various ct protocols used for imaging ild (prosch et al. 2013) . the question whether a spaced hrct or a volumetric approach should be performed might be answered by the scanner generation and the corresponding dose efficiency of the device. as a decision support, the reference values for ct of the lung of the applicable country can be used. if a volumetric hrct (section thickness < 1.5 mm) with sufficient image quality on the particular device produces more than, e.g., between 250 and 350 dlp, than a spaced hrct might be used instead. however, nowadays the vast majority of specialized lung radiologist recommend volumetric hrct, if an adequate scanner is available. therefore, whenever a modern ct is available the suitable ct protocol for diagnosis of possible ild should offer a volumetric dataset with the following parameters: section thickness (st) â�¤ 1.5 mm, ri slightly thinner than st (ri = st*0.7), a high spatial frequency reconstruction algorithm (fbp or iterative moderate strength), dlp â�¤ the national reference value for thoracic ct (e.g., â�¤ 300 mgy/ cm), tube current: reference mas or quality factor highly dependent on the device, automatic kvp selection enabled, dose modulation enabled. low dose and ultra low dose scans should be avoided for the primary assessment. for most cases, no iodine contrast is needed. hrct pattern the linear pattern is characterized by interlobular septal thickening and outlines the usually hardly perceptible polygonal structure of the secondary pulmonary lobule. centrally, a dot-like structure is usually visible representing the centrilobular artery. the interlobular septal thickening can be smooth, like in interstitial lung edema ( fig. 2) whereas nodular interlobular septal thickening is most commonly seen in patients with sarcoidosis. in lymphangitis carcinomatosis interlobular septal thickening can be either smooth or nodular and is frequently unilateral or focal as opposed to bilateral lung involvement in interstitial edema. irregular interlobular septal thickening is typically seen in fibrotic lung disease and is usually associated with thickening of the fine interstitial network within the secondary pulmonary lobule, the so-called intralobular septa. the combination of interlobular and intralobular septal thickening represents the reticular pattern. additional signs of lung fibrosis include traction bronchiectasis and honeycombing. nodular lung diseases present with multiple nodules smaller than 1 cm. according to the distribution of these nodules in relation to the secondary pulmonary lobules three nodular patterns can be distinguished on hrct: (1) perilymphatic, (2) centrilobular, and (3) random. perilymphatic nodules are distributed along the pulmonary lymphatic spaces that are predomi-nantly found in four locations: the parahilar and the centrilobular peribronchovascular interstitium, the subpleural interstitium, and the interlobular septa. perilymphatic nodules are typically well defined and most commonly encountered in sarcoidosis, silicosis, and lymphangiosis carcinomatosa (fig. 3a) . centrilobular nodules are located in the center of the secondary pulmonary lobule next to the centrilobular peribronchovascular structures and thus can be due to vascular, lymphatic or small airway diseases, the latter being the most common cause. in centrilobular diseases the subpleural interstitium and the fissures are typically spared. centrilobular nodules can either present with ground-glass opacity or homogeneous soft tissue attenuation. ground-glass opacity nodules tend to be ill-defined and are most commonly seen in the subacute stage of hypersensitivity pneumonitis (hp) and in respiratory bronchiolitis (rb) (fig. 3b ). other less frequent causes include langerhans cell histiocytosis, pulmonary edema, and pulmonary hemorrhage. soft attenuation centrilobular nodules tend to have an asymmetric distribution and can progress to patchy consolidations. they are associated with endobronchial spread of disease, such as in bronchopneumonia, aspiration, and invasive mucinous adenocarcinoma. the tree-in-bud pattern is a subtype of a centrilobular pattern and characterized by multiple nodules that show a linear and branching distribution, resembling a budding tree. the pattern is caused by dilatation and impaction of the centrilobular bronchioles with mucus, pus, or blood. the tree-in-bud pattern is almost always caused by infection, most commonly due to bacteria or mycobacteria. in chronic disease, bronchiectasis and bronchial wall thickening may be present. extensive and diffuse tree-in-bud pattern is typically for kartagener's syndrome and cystic fibrosis. random nodules are usually sharply marginated and of soft tissue attenuation. they show a diffuse distribution with involvement of the pleural surfaces. they are caused by hematogenous dissemination and are most commonly due to metastatic disease (fig. 3c) . they may show a basilar dominance in size and number. the differential diagnosis includes miliary tuberculosis and miliary fungal infection. the lung density can be increased by either ground-glass opacity or consolidation. groundglass opacity leads to a hazy increase of lung density without obscuring the lung vessels. in consolidation the lung density is homogenously increased and the vessels are no longer visible. ground-glass opacity is a common but nonspecific pattern. in the acute clinical setting it is frequently associated with infectious diseases, pulmonary edema, and hemorrhage. it can also be the main imaging finding in acute hypersensitivity pneumonitis and acute eosinophilic pneumonia. in patients with chronic symptoms ground-glass opacity is typically seen in various subtypes of interstitial pneumonia. the combination of ground-glass opacity and consolidation is common and suggestive of organizing pneumonia, chronic eosinophilic pneumonia, and ards. less common, but nevertheless important causes of combined ground-glass opacity and consolidation are invasive adenocarcinoma and intrapulmonary lymphoma (fig. 4) . the crazy paving pattern is a term reserved for ground-glass opacities with superimposed thickening of the interlobular and intralobular septa. it has been initially described in patients with alveolar proteinosis, but is nonspecific and may be seen in infectious, neoplastic, inhalative, and hemorrhagic lung diseases.the mosaic attenuation pattern is characterized by the presence of sharply demarcated areas of various lung density. in several ilds, notably in nsip, the heterogeneous lung attenuation is due to patchy groundglass opacities next to normal lung parenchyma. in this setting, areas of higher attenuation represent the interstitial process and areas of lower attenuation represent the normal lung parenchyma. other causes of mosaic attenuation pattern are obliterative small-airways disease or occlusive vascular disease. in small airway disease bronchiolar obstruction leads air trapping, a phenomenon that is characterized by focal zones of decreased attenuation and can be enhanced by expiratory scans. it is typically seen in bronchiolitis obliterans syndrome due to chronic lung transplant rejection or infection. multifocal adenocarcinoma of the lung presenting as patchy consolidations and ground-glass opacities. in addition, bilateral pleural effusions can be seen mosaic attenuation pattern due to vascular disease is referred to as mosaic perfusion and is most commonly seen in chronic thromboembolic pulmonary hypertension (cteph). due to the regional decrease in lung perfusion, the abnormal lung is lucent and vessels appear smaller in comparison to vessels in the normal lung. a mosaic attenuation pattern that is caused by a combination of ground-glass opacities and mosaic perfusion/air trapping has been termed the headcheese sign and is most commonly found in subacute hypersensitivity pneumonitis. the low attenuation pattern can be seen in cysts, emphysema, and honeycombing. lung cysts have a thin wall and are typically greater than 1 cm in size. they can be occasionally found in patients who are otherwise normal but the presence of more than a few cysts suggests a cystic lung disease, such as lymphangioleiomyomatosis, langerhans cell histiocytosis, and lymphoid interstitial pneumonia. postinfectious pneumatoceles from pneumocystis pneumonia can also present as cystic lung disease. depending on the area of lung destruction with regard to the secondary pulmonary lobule, emphysema can be subdivided in centrilobular, panlobular, and paraseptal. as opposed to true cysts, the lucencies in centrilobular and panlobular emphysema do not have a wall. however, perifocal fibrosis or atelectasis may simulate a wall in some emphysematous regions. in addition, in paraseptal empyshema, the supleural areas of emphysema are outlined by a discrete and thin wall that corresponds to the interlobular septa. honeycombing is a typical sign of advanced fibrosis and the most important hrct feature of usual interstitial pneumonia (uip). it is characterized by multiple layers of subpleural cysts with a defined wall (1-3 mm) and a diameter between 3 and 10 mm. in uip honeycombing is predominantly found in the basal zones of both lungs, whereas in end stage fibrosis due to hypersensitivity pneumonitis, sarcoidosis or collagen vascular diseases honeycombing tends to dominate in the apical or central zones of the lung (fig. 5a-c) . as the presence of honeycombing reflects advanced fibrosis it is important to avoid pitfalls such as traction bronchiectasis, subpleural emphysema, or cystic lung diseases. in many ilds, the etiology remains unknown. most are uncommon, and some, such as alveolar microlithiasis, are exceedingly rare, but others, such as idiopathic pulmonary fibrosis and sarcoidosis, are quite common. the term idiopathic interstitial pneumonias refers to a group of seven entities with distinct histologic patterns: idiopathic pulmonary fibrosis (ipf), characterized by the pattern of usual interstitial pneumonia (uip); nonspecific interstitial pneumonia (nsip); cryptogenic organizing pneumonia (cop); respiratory bronchiolitis-associated interstitial lung disease (rb-ild); desquamative interstitial pneumonia (dip); lymphoid interstitial pneumonia (lip); and acute interstitial pneumonia (aip). in their idiopathic form, iips are rare diseases. they are, nevertheless, considered prototypes of more common secondary interstitial lung disorders, such as sarcoidosis, vasculitis, and connective tissue diseases, although they appear to follow a different and often less aggressive clinical course. the advent of hrct has had a profound impact on the imaging of iips, because the detailed delineation of the lung anatomy allows a close correlation between the histologic patterns of iips and the ct features. on the basis of ct morphology and in the correct clinical context, the radiologist can achieve an accurate diagnosis in many cases. however, due to overlap between the various entities, complementary lung biopsy is recommended in cases with indistinct imaging findings. ipf is by far the most common iip (approximately 50% of iip), and has a substantially poorer long-term survival rate than the other iips (median survival, 2-5 years) (katzenstein and myers 1998) . ipf shares nonspecific clinical symptoms, such as gradual onset of progressive dyspnea and cough, with other iip. there is a slight male predominance and patients are usually over the age of 50. typically, patients do not respond to corticosteroid treatment. in 2014 two antifibrotic drugs, nintedanib and pirfenidone were approved for treatment of ipf but survival benefit has not been established with either agent (antoniou et al. 2016) . therefore currently, the only life-prolonging therapy consists of lung transplantation (thabut et al. 2003) . while the term ipf characterizes the clinical entity, the term "usual interstitial pneumonia" is used to describe the histologic and radiologic patterns associated with ipf. the histologic and radiologic features of uip are characterized by heterogeneity with areas of normal lung alternating with patchy fibrosis. the typical computed tomographic (ct) findings in uip are predominantly basal and peripheral reticular opacities with honeycombing and traction bronchiectasis ( fig. 6a ) (mueller-mang et al. 2007 ). groundglass opacities are usually present, but limited in extent. according to the recent guidelines established by american, european, japanese and latin american societies radiologist should use the following terms to indicate the diagnostic confidence of a uip pattern (raghu et al. 2011 ): (definite) "uip" pattern, "possible uip" pattern, "inconsistent with uip" pattern. a pattern of "uip" requires the presence of following four ct findings: (1) subpleural, basal predominance, (2) reticular opacities, (3) honeycombing with or without traction bronchiectasis, and (4) absence of features listed as inconsistent with uip pattern. a "possible uip" pattern is defined by the same criteria as "uip" pattern but without honeycombing. hrct findings that are "inconsistent with uip" include (1) an upper or mid-lung predominance, (2) a peribronchovascular predominance, (3) extensive ground-glass opacities, exceeding reticulation in extent, (4) profuse micronodules (bilateral, predominantly upper lobes), (5) discrete cysts, (6) diffuse mosaic attenuation or air trapping, (7) segmental or lobar consolidation and are listed in detail in table 2 . patients with ipf may present with rapid respiratory worsening during the course of their disease. when a cause, such as pulmonary embolism, pneumothorax, or cardiac failure, cannot be defined this is termed acute exacerbation of ipf. on hrct widespread diffuse or patchy ground-glass opacities have been observed in these patients, correlating to diffuse alveolar dam-age (dad) on histopathology (fig. 6b) (kim et al. 2006 ). other complications that should be noted in patients with ipf include opportunistic pulmonary infections (e.g., pneumocystis jiroveci), pulmonary hypertension, and an increased risk of bronchial carcinoma (bouros et al. 2002) . therefore, ct scanning should involve a combination of standard volumetric ct with sequential hrct. given the clinical, radiologic, and pathologic variability of nsip, the diagnostic approach to this entity is challenging. patients with nsip are usually between 40 and 50 years old, and men and women are equally affected. compared to ipf, patients with nsip have a variable, but overall more favorable, course of disease and the majority of patients stabilize or improve on corticosteroid therapy. according to the predominance of either inflammatory cells or fibrosis, nsip is histologically subdivided into a cellular and a fibrotic subtype. cellular nsip is less common than fibrotic nsip and carries a substantially better prognosis (travis et al. 2000) . on hrct, nsip is characterized by patchy ground-glass opacities combined with irregular linear or reticular opacities (johkoh et al. 2002) (fig. 7a, b) . in fibrotic nsip traction bronchiectasis become more evident and honeycombing may be present, but is typically limited in severity and extent. it has also been referred to as microcystic honeycombing (desai et al. 2004) . in contrast to the heterogeneous lung involvement and the typical a b fig. 6 (a, b) axial ct image in a 63-year-old man with usual interstitial pneumonia (uip)/idiopathic pulmonary fibrosis (ipf) shows bilateral reticular opacities, honeycombing (black arrowheads), and traction bronchiectasis (arrow). in addition, patchy ground-glass opacities are present (white arrowheads) (a). acute exacerbation in the same patient shows marked progression of ground-glass opacities (arrowheads) (b) apico-basal gradient in uip, hrct in nsip reveals rather symmetric and homogeneous lung involvement without an obvious gradient (fig. 8) . the relative sparing of the immediate subpleural space is highly predictive of nsip and present in 20-50% of cases (fig. 9) . cop was formerly referred to as "bronchiolitis obliterans organizing pneumonia (boop)" and is characterized by the histologic pattern of organiz-ing pneumonia (op). there is no gender predilection. patients usually present between 50 and 60 years of age, and typically report a respiratory tract infection preceding their symptoms. in its idiopathic form (as cop), op is rare; however, it is frequently encountered in association with collagen vascular diseases, and in infectious and drug-induced lung diseases (cordier 2000) . on corticosteroid therapy, patient usually experience complete recovery, but relapses are common. the histologic hallmark of cop is the development of granulation tissue polyps within the alveolar ducts a b fig. 7 (a, b) axial ct image in a 61-year-old man with nsip shows bilateral subpleural irregular linear opacities (arrowhead) and ground-glass opacities (arrow) (a). follow-up ct image obtained after 6 months of corticosteroid therapy shows improvement, with partial resolution of the linear opacities and ground-glass opacities (b) fig. 8 comparison of ct features between nsip and uip. nsip (left) shows diffuse lung involvement with bilateral, peripherally located linear and reticular opacities. in uip (right) the lung abnormalities show a typical apico-basal gradient with predominance of honeycombing fig. 9 hrct shows characteristic subpleural sparing of reticular opacities (arrows) in a 67-year-old patient with nsip and alveoli, with preservation of the lung architecture. on hrct, cop is characterized by patchy peripheral or peribronchial consolidations that resemble pneumonic infiltrates and predominate in the lower lung lobes (lee et al. 1994 ) (fig. 10a, b) . frequently, air bronchograms and perifocal ground-glass opacities can be found. other common findings include sparing of the outermost subpleural area and mild cylindrical bronchiectasis. the reverse halo sign (atoll sign) is considered to be highly specific, although only seen in 20% of patients with cop. it is characterized by central ground-glass opacity surrounded by a ring or partial ring of consolidation. in addition to these typical ct features, other less specific findings can be encountered, such as irregular linear opacities, solitary focal lesions, and multiple nodules (akira et al. 1998 ). rb-ild is exclusively encountered in smokers and is thought to represent a symptomatic variant of the histologically common and incidental finding of respiratory bronchiolitis (rb). patients are usually 30-50 years old and men are affected nearly twice as often as women. after smoking cessation, prognosis is excellent. histologically, rb-ild is characterized by pigmented alveolar macrophages within the bronchioles. the typical hrct features of rb-ild are centrilobular nodules ("airspace nodules," small nodules with ground-glass opacity) that are randomly distributed or have upper lobe predominance (heyneman et al. 1999) (fig. 11a) . additional ct features are diffuse ground-glass opacities, bronchial wall thickening, and coexisting centrilobular emphysema (fig. 11b) . dip is strongly associated with cigarette smoking and is considered to represent the end of a spectrum of rb-ild. there is a male predominance and patients usually present between 30-50 years of age. most patients improve with smoking cessation and corticosteroid therapy. histologically, dip shows diffuse involvement, with filling of alveolar spaces with macrophages and desquamated alveolar cells, compared to the bronchiolocentric involvement in rb-ild. on hrct, dip is characterized by extensive and diffuse groundglass opacities with peripheral and lower lobe predominance (akira et al. 1997) (fig. 12) . the presence of small cystic spaces and irregular linear opacities are indicative of fibrotic changes. lip rarely occurs as an idiopathic disease. it is usually seen in conjunction with systemic disorders, most notably human immunodeficiency virus (hiv) infection, sjã¶gren syndrome, and variable immunodeficiency syndromes (swigris et al. 2002) . lip is more common in women than in men, and typically, patients become symptomatic in the fifth decade of life. histologically, lip a b fig. 10 axial ct image in a 75-year-old woman with cop shows bilateral, peripherally located patchy lung consolidation (arrowheads). in one of the lesions, the subpleural space is typically spared (arrow) (a). follow-up ct image obtained after 4 weeks of corticosteroid therapy shows subtotal resolution of the lung abnormalities with residual ground-glass opacities (arrowheads) (b) is characterized by diffuse interstitial cellular infiltrates that are composed of lymphocytes, plasma cells, and histiocytes. while the interstitium is expanded by these infiltrates, the alveolar airspaces are partially collapsed. the hrct findings of lip consist of bilateral, diffuse, or patchy ground-glass opacities, poorly defined centrilobular nodules, and cystic airspaces. the combination of diffuse ground-glass opacities and thickening of the interlobular septa results in the crazy paving pattern (fig. 13) . the mechanism of cyst formation has been postulated to be secondary to partial bronchiolar obstruction with air trapping due to peribronchiolar lymphocytic infiltration (desai et al. 1997 ). aip differs from the other iips in its acute course of disease, with rapid onset of dyspnea and cough, followed by respiratory failure and a high acute mortality rate of 50% or more (american thoracic society/european respiratory society 2002). aip was formerly referred to as "hamman-rich syndrome." the histological and radiological features of aip are similar to those of acute respiratory distress syndrome (ards) and can be subdivided hrct shows bilateral, patchy peripheral ground-glass opacities with a subpleural predominance and coexisting mild bronchial wall thickening. in the right lower lobe, small cystic lucencies are present lymphoid interstitial pneumonia (lip) in a 48-year-old woman with crazy paving pattern. axial ct image shows extensive ground-glass opacities and interlobular septal thickening. scattered thin-walled cysts are also present fig. 11 (a, b) rb-ild in a 44-year-old female cigarette smoker. coronal ct image shows scattered, poorly defined centrilobular nodules that are predominantly located in the upper lung lobes. note mild coexisting centrilobular emphysema (arrows). (b) axial ct image shows centrilobular nodules, patchy ground-glass opacities, and discrete zentrilobular emphysema a b into an acute or exudative phase and a late or organizing phase. ct obtained in the early phase shows extensive ground-glass opacities, sometimes in a geographic distribution (fig. 14a ). in addition, areas of consolidation can be observed in the dependent areas of the lungs. in patients who survive the acute phase of disease, ct shows fibrotic changes with architectural distortion and traction bronchiectasis, predominantly in the nondependent areas of the lung (fig. 14b ). sarcoidosis is a common systemic disorder of unknown cause characterized by the presence of noncaseating granulomas, which either can dis-solve or cause fibrosis. almost any organ can be affected, but the lungs are most frequently involved. the mean age of patients is between 20 and 40 years and there is a slight female predominance (1999) . in up to 50% of patients, sarcoidosis is incidentally discovered on radiographs. common clinical symptoms include respiratory illness, skin lesions, fatigue, and weight loss. lofgren's syndrome is a classic clinical presentation with fever, erythema nodosum, arthralgias, bihilar lymphadenopathy, and a usually benign course of disease. the diagnosis is established on the basis of clinical and radiological findings, supported by histology from transbronchial biopsy. spontaneous remissions occur in nearly twothirds of patients, but the course is chronic or progressive in 10-30% (costabel and hunninghake 1999) . the appropriate treatment depends on clinical and imaging findings and is based on corticosteroids. in patients with end-stage sarcoidosis lung transplantation has been successfully performed, but is associated with high recurrence rates of sarcoidosis (35%) (collins et al. 2001) . for the staging of sarcoidosis a system based on chest radiographs is in clinical use; stage i consists of bilateral hilar adenopathy; in stage ii sarcoidosis, patients have bilateral hilar adenopathy and diffuse parenchymal infiltration; stage iii describes parenchymal infiltration without hilar adenopathy. some authorities use a stage iv classification to indicate irreversible fibrosis. in patients with sarcoidosis, ct scans of the lung are included routinely in the diagnostic workup at initial evaluation and at follow-up. specifically, they are indicated in the setting of atypical clinical and/or chest radiograph findings, for the detection of complications of the lung disease (e.g., pulmonary fibrosis, superimposed infection, malignancy), and when chest radiographs are normal, despite clinical suspicion of the disease (costabel and hunninghake 1999) . the chest can be involved in sarcoidosis in many ways, and because of the multitude of a b fig. 14 (a, b) acute interstitial pneumonia (aip) in a 58-year-old patient. axial ct image shows bilateral ground-glass opacities in a geographic distribution (arrow). consolidation is seen in the more dependent lung (arrowheads). small coexisting bilateral pleural effusions are present (a). fibrotic changes with traction bronchiectasis (arrow) and architectural distortion in the late phase of acute aip (b) potentially different findings, sarcoidosis can be regarded as one of the great "mimickers" in thoracic radiology. the most common intrathoracic manifestation of sarcoidosis is the presence of mediastinal lymphadenopathy with usually bilateral and rather symmetric involvement of hilar lymph nodes. they can calcify in chronic disease and then show amorphous, punctate, or eggshell calcifications (fig. 15 ). in patients with sarcoidosis and parenchymal involvement nodular opacities are the predominant finding. these nodules typically range in size between 1 and 5 mm and are usually well defined. they have a perilymphatic distribution, and thus preferentially lie adjacent to the fissures and interlobular septa, along pleural surfaces, and along central peribronchovascular structures (fig. 16 ). there is a predilection for the upper lobes and the superior segments of the lower lobes of both lungs. sarcoid nodules sometimes tend to coalesce and form large parenchmyal nodules with surrounding loosely aggregated small nodules. as the shape of these coalescent granulomas resembles a galaxy, it is referred to as the sarcoid "galaxy sign" (nakatsu et al. 2002) (fig. 17) . occasionally, a single large nodule may be present in sarcoidosis and resemble bronchogenic carcinoma. ground-glass opacities are common in sarcoidosis and have been postulated to represent alveolitis in early reports; however, according to pathologic correlation ground-glass opacities in sarcoidosis are more likely to represent microgranulomas with or without perigranulomatous fibrosis (nishimura et al. 1993) . patients with predominant groundglass opacities on initial ct scan have a worse prognosis than patients with a predominant nodular pattern (murdoch and muller 1992; akira et al. 2005) . when sarcoidosis progresses to fibrosis, architectural distortion and traction bronchiectasis classically radiating from the hilum to the adjacent upper and lower lobes can be found. other common ct abnormalities in fibrotic sarcoidosis include honeycombing, cysts, and bulla formation. airway stenosis in sarcoidosis is usually due to extrinsic scarring, or to endobronchial granulomas, whereas lymphadenopathy alone is a rare cause of symptomatic airway narrowing. pneumoconiosis may simulate the appearance of sarcoidosis, but is usually easily diagnosed when correlated with clinical history. lymphangiosis carcinomatosa usually occurs in patients with a known history of malignancy and patients tend to be older than patients with sarcoidosis. also lymphangiosis carcinomatosa may be unilateral whereas sarcoidosis is typically bilateral, symmetric and upper lobe predominant. primary tuberculosis, lymphoma, and mediastinal metastases from other tumors usually present with asymmetrical nodal enlargement as opposed to the bihilar, and often symmetric hilar lymphadenopathy in stage i sarcoidosis. pulmonary langerhans cell histiocytosis (plch) (formerly called histiocytosis x) is a rare interstitial lung disease of unknown cause that primarily affects cigarette smokers under 40 years of age. most patients present with cough and dyspnea; sometimes additional systemic symptoms, such as fatigue, weight loss, and fever, are reported. smoking cessation is the most important component in the therapeutic management of plch, with stabilization or regression of clinical and radiographic features in the majority of patients. ct is very sensitive for the detection of plch and a correct diagnosis can be achieved in over 80% of cases (grenier et al. 1991 ). on ct, plch is characterized by a combination of small nodules (1-10 mm) and cysts. the cysts are thought to arise by cavitation of the nodules, have a variable wall thickness, and are often irregularly outlined (abbott et al. 2004) (fig. 18a) . usually, the lung abnormalities are most prominent in the upper lobes, with relative sparing of the lung bases near the costophrenic sulci (fig. 18b) . in later phases of the disease, nodules are less obvious and cysts are the predominant feature. in this setting, plch may mimic lymphangioleiomyomatosis, but the latter occurs almost exclusively in women, affects the lung diffusively without sparing of the lung bases, and is characterized by uniformly sized cysts. lymphangioleiomyomatosis (lam) is a rare interstitial lung disease that affects women of childbearing age exclusively. the tuberous sclerosis complex (tsc), an autosomal dominant inherited disorder, is associated with parenchymal lung changes identical to lam (pallisa et al. 2002) . histologically, lam is characterized by an abnormal proliferation of smooth muscle cells (lam cells) in the lungs and in the thoracic and retroperitoneal lymphatics. the most common fig. 17 41-year-old man with sarcoidosis. the parenchymal nodules in the right upper lobe tend to coalesce and form a large parenchymal nodule surrounded by loosely aggregated small nodules. as this resembles a galaxy it is referred to as the "sarcoid galaxy sign" initial presenting symptoms are dyspnea, spontaneous pneumothorax, and cough (johnson 1999) . the clinical course of lam is variable. normally, the disease progresses slowly with continuous deterioration of pulmonary function. ultimately, it leads to respiratory failure. because lam deteriorates with pregnancy and the use of exogeneous estrogen, several attempts at anti-estrogen therapies have been made with controversial results (taylor et al. 1990 ). lung transplantation is indicated in patients with end-stage disease. apart from the common postoperative complications of transplantation, recurrent disease in the donor lung can occur. the key findings on ct are uniformly distributed, thin-walled cysts that tend to confluate (fig. 19) . the cysts can be up to 3 cm in diameter and are equally and symmetrically distributed throughout both lungs. usually, the cyst shape is round; however, in some cases, they can be of ovoid, polygonal, or irregular shape. cyst wall thickness ranges from barely susceptible to up to 2 mm. on expiratory scans, cyst size decreases, suggesting a communication with the airway system. the lung parenchyma in between the cysts is usually inconspicuous, but, in the highly cellular forms of lam, small nodules, reticular opacification, and ground-glass attenuation can be found (aberle et al. 1990 ). pneumothorax is common in lam, and occurs in about 80% of patients within the course of the disease. about 8-14% of patients develop pulmonary hemorrhage, which presents as ground-glass opacity on hrct (lenoir et al. 1990 ). pleural chylous effusions can be found in up to 14% of patients, and are indistinguishable from protein-rich effusions of other origin on a b fig. 19 (a, b) 30-year-old woman with tuberous sclerosis complex. axial ct image shows multiple thin-walled cysts in a uniform distribution. the cysts adjacent to the upper right mediastinum tend to conflate (white arrows) (a). coronal ct image displays the uniform and bilateral distribution of the cysts throughout both lungs. the lung parenchyma between the cysts is inconspicuous (b) a b fig. 18 (a, b) pulmonary langerhans cell histiocytosis in a 26-year-old man. axial ct image demonstrates bilateral, thin-walled cysts of variable size and multiple, illdefined nodules (arrows) (a). coronal ct image better demonstrates the upper and middle lung zone predominance with relative sparing of the lung bases (b) ct. in addition, dilatation of the thoracic duct, as well as mediastinal, hilar, and retrocrural adenopathy, can be found in patients with lam. in more than 70% of patients with lam, renal angiomyolipomas can be found, which show a characteristic appearance, with negative ct values due to their fat content. in some cases, retroperitoneal cystic hypoattenuating masses indicative of lymphangioleiomyomas can be found. chylous ascites and lymphadenopathy are further extrathoracic findings in some patients (pallisa et al. 2002) . the most important differential diagnoses for lam are langerhans cell histiocytosis, and panlobular emphysema. in contrast to lam, in langerhans cell histiocytosis, the costophrenic sulci are usually spared, the cysts can be thickwalled and irregularly outlined, and nodules are predominant in the early stage of disease (bonelli et al. 1998 ). panlobular emphysema is associated with alpha-1-antiprotease deficiency. the most distinct feature of emphysema is the absence of defined walls in the areas of low attenuation, whereas cysts in lam almost invariably present with walls (johnson 1999) . eosinophilic pneumonia is divided into acute eosinophilic pneumonia (aep) and chronic eosinophilic pneumonia (cep). the pathogenesis of both forms is still unknown, but it is speculated to be a hypersensitivity reaction to an unknown antigen. however, aep has been reported after cigarette smoking, dust exposure, and smoke from fireworks. the mean age of patients with cep is 40; aep occurs at all ages. aep shows no gender predominance, whereas cep occurs more often in women. histologically, diffuse alveolar damage associated with interstitial and alveolar eosinophilia is found in aep (tazelaar et al. 1997) ; in cep, an accumulation of eosinophils and lymphocytes in the interstitium and alveoli, and sometimes interstitial fibrosis, is found. aep clinically presents as an acute febrile illness with dyspnea, pleuritic chest pain, myalgias, and respiratory failure. in aep, blood eosinophilia is often absent, but more than 25% eosinophils are found in the bronchial lavage fluid of these patients. cep has an insidious onset with fever, malaise, weight loss, and dyspnea. about 90% of these patients suffer from asthmatic symptoms. in cep, peripheral blood eosinophilia is present in more than 90% of cases, and there is an increased number of eosinophils in the bronchial lavage fluid as well (allen and davis 1994) . both aep and cep are often misdiagnosed as pneumonia, which can delay the correct diagnosis. both aep and cep show a rapid response to corticosteroids, and there usually is rapid clearing of clinical and radiographic abnormalities within several days (allen and davis 1994) . at ct, aep shows bilateral peripheral groundglass opacities, with lower lobe predominance (fig. 20) . in addition, interlobar septal thickening and thickening of the bronchovascular bundles, a b fig. 20 (a, b) acute eosinophilic pneumonia in a 37-year-old female with bal fluid eosinophilia. axial ct image obtained 5 days after onset of dyspnea shows peripherally distributed patchy areas of consolidation and ground-glass opacities accompanied by interlobular septal thickening (a). coronal ct image displays the lower lobe predominance of the infiltrates (b) as well as localized areas of consolidation, can be seen. aep is very commonly associated with pleural effusions (allen and davis 1994; johkoh et al. 2000) . the hrct findings of aep are nonspecific and may be indistinguishable from pulmonary edema, acute respiratory distress syndrome, atypical pneumonia, and hemorrhage. cep shows upper lobe predominance and peripheral nonsegmental consolidations (fig. 21) . consolidations can persist for some time, but, in the absence of treatment, they tend to migrate. consolidations are often accompanied by groundglass opacities, and a "crazy paving" appearance of the consolidations can also be seen in many cases. pleural effusions are rare in cep (mayo et al. 1989; johkoh et al. 2000) . the hrct findings of cep are very similar to those of organizing pneumonia, both presenting with peripheral, nonsegmental consolidation. however, cep has an upper lobe predominance, whereas organizing pneumonia predominantly involves the lower lobes. pulmonary alveolar proteinosis (pap) is a rare interstitial lung disease with an incidence of 3.7 per million. it is characterized by filling of the alveoli with a lipid-rich proteinaceous material (rosen et al. 1958) . three different forms of pap can be distinguished: an autosomal recessive congenital form (2%); a secondary form (10%) that is associated with various conditions, such as hematopoietic disorders (especially myelogenous leukemias), silicosis, immunodeficiency disorders, malignancies, and some infections; and an idiopathic form (90%). in idiopathic pap, several mechanisms are responsible for phospholipid accumulation in the alveoli. whether this accumulation is caused by reduced clearance or overproduction is not yet clear (prakash et al. 1987) . the median age of the patients is about 40 years, and most patients are men and have a history of smoking (ben-dov et al. 1999) . patients present with dyspnea or cough. the symptoms are usually out of proportion to the radiological findings (clinical-radiological discrepancy). in 13% of patients with pap, secondary infections with nocardia, crypococcus, or mycobacteria are observed. the treatment for pap is bronchoalveolar lavage with sterile saline, and prognosis is generally good with whole lung lavage. hrct is characterized by bilateral, symmetrical, geometric areas of ground-glass attenuation (fig. 22) . the interlobular septa are thickened, and a fine network of intralobular lines can be seen. these changes are responsible for the so-called "crazy paving" pattern. the disease does not have any preferential zonal distribution (holbert et al. 2001) . architectural distortion and bronchiectasis are absent normally; however, in a small percentage of patients, pulmonary fibrosis can be found. although the a b fig. 21 (a, b) chronic eosinophilic pneumonia in a 56-year-old man presenting with a 4 week history of cough and fever. moderate blood eosinophilia is found in laboratory workup. axial ct image shows strikingly peripheral wedge shaped airspace consolidations (a). the upper lobe predominance of the consolidations is displayed on coronal ct image (b) crazy paving pattern on hrct is suggestive of pap, this pattern can also be observed in a number of other interstitial and airspace diseases, such as pulmonary hemorrhage, pulmonary edema, hypersensitivity pneumonitis, and pulmonary adenocarcinoma (previously termed bronchioloalveolar carcinoma). the diagnosis is made by bronchoalveolar lavage and typical clinical findings. nevertheless, the gold standard in diagnosis remains open lung biopsy. pulmonary alveolar mircrolithiasis (pam) is a rare condition characterized by the formation of intraalveolar microliths (calcospherites). the pathogenesis of the micronodular calcifications is still unknown. in about 50% of cases, pulmonary alveolar microlithiasis occurs as an autosomal recessive hereditary lung disease (sosman et al. 1957) . most cases of microlithiasis are found in turkey (ucan et al. 1993) . the disease usually occurs between 30 and 50 years of age and pediatric cases are rare. in hereditary cases, there is slight female predominance. the disease is typically detected incidentally on chest films obtained for other reasons, and clinical symptoms are disproportional to the extent of radiologic findings. occasionally, patients present with stress-induced dyspnea, malaise, or fatigue. as pam progresses with the formation of tiny (0.01-3 mm) microspheres in the alveoli, it can ultimately lead to respiratory failure and cor pulmonale. in early stages, diffuse ground-glass opacifications are found throughout both lungs on ct. still, the presence of calcified micronodules is most characteristic. the distribution of the micronodules is miliary, but there is a tendency toward greater involvement of the posterior segments of the lower lobes and the anterior segments of the upper lobes (fig. 23) . due to the intra and periseptal accumulation of micronodules, interlobular septal thickening is found in almost all patients. in addition, subpleural septal thickening is frequently detected. as the disease progresses, subpleural emphysema and the formation of thin-walled subpleural cysts are pathognomonic findings in pam and might represent early lung fibrosis. the subpleural cysts are accountable for the black subpleural line on chest x-rays (korn et al. 1992) . the main differential diagnoses include miliary tuberculosis, sarcoidosis, metastatic pulmonary calcification associated with hemodialysis, silicosis, and pulmonary hemosiderosis. usually, the disease progresses very slowly, but can result in cardiac and pulmonary failure. there is no known treatment, except lung transplantation in end-stage disease. occupational and environmental lung disease comprises a wide spectrum of lung disorders caused by the inhalation or ingestion of organic and inorganic particles and chemicals. ct is very sensitive in depicting the parenchymal, as well as airway and pleural abnormalities that are associated with these diseases. hypersensitivity pneumonitis (hp), also known as exogenous allergic alveolitis (eea), is an immunologic lung disease caused by repeated exposure and sensitization to various organic and chemical antigens that lead to diffuse inflammation of the lung parenchyma. the most common diseases are farmer's lung and bird fancier's lung due to aspergillus antigen and avian proteins, respectively. based on the length and intensity of exposure and subsequent duration of illness, clinical presentations of hp are categorized as acute, subacute, and chronic progressive. in acute hp, patients present 4-12 h following heavy exposure to an inciting agent with fever, chills, and myalgias. in subacute and chronic hp, patients have an insidious onset of cough, progressive dyspnea, fatigue, and weight loss. hrct in acute hp typically shows diffuse ground-glass opacities and centrilobular nodules, most commonly in a random distribution (tomiyama et al. 2000) . however, due to the short duration of symptoms patients rarely undergo ct scanning in the acute phase. in the subacute phase, centrilobular nodules become more prominent and patchy groundglass opacities can be found. in some patients cystic lesions (3-25 mm) have been observed (franquet et al. 2003) . chronic hp is characterized by the presence of reticulation due to fibrosis superimposed on findings of subacute hp (fig. 24) . the abnormalities are usually predominantly located in the upper lobes, while the lung bases are relatively spared (silva et al. 2008 ). other common findings in chronic hp include a mosaic attenuation pattern and air trapping on expiratory imaging (small et al. 1996) . pneumoconiosis is a non-neoplastic reaction to the inhalation and accumulation of dust particles in the lung. the particles are engulfed by alveolar macrophages that release inflammatory cytokines and induce fibrotic changes. the classification of pneumoconiosis is based on chest radiographs using the international labor organization (ilo) classification scheme. the hrct features in patients with silicosis and coal worker pneumoconiosis consist of small, well-circumscribed nodules that are usually 2-5 mm in diameter and predominantly affect the upper and posterior lung zones. the nodules have a perilymphatic distribution, thus affecting the interlobular septa and the peribronchovsacular and subpleural interstitium. the nodules in silicosis tend to be larger and better defined than those in coal worker pneumoconiosis (kim et al. 2001) . occasionally, eggshell calcifications in the hilar and mediastinal lymph nodes are seen. the presence of nodules larger than 1 cm is indicative of complicated pneumoconiosis, also known as progressive massive fibrosis. these nodules coalesce and form conglomerate masses that are typically located in the upper lobe of the lung. in large lesions, cavitation may occur, which is due to either ischemic fig. 24 chronic hypersensitivity pneumonitis in a 52-year-old man related to mold exposure. axial ct image shows patchy ground-glass opacities with associated centrilobular nodules (inset: magnified view of centrilobular nodules). also note mild subpleural reticular opacities (black arrowheads) indicating fibrosis, and subtle mosaic attenuation (white arrowhead) necrosis or superinfection. in advanced disease, hilar retraction and compensatory emphysema, particularly in the lower lobes, is seen (fig. 25) . the parenchymal lung manifestations related to asbestos exposure are referred to as asbestosis and differ from the previously described "classic" pneumoconiosis. early asbestosis is characterized by subpleural linear and reticular opacities that are predominantly located in the posterior lung bases. to distinguish these abnormalities from gravity-related physiologic changes, prone scans should be included in cases of suspected asbestosis. other typical findings in asbestosis include thickened interlobular septa and centrilobular nodules. in advanced disease, ct shows bands of fibrosis, traction bronchiectasis, and honeycombing. in addition, other asbestosrelated lung abnormalities, such as pleural effusion, pleural plaques, and round atelectasis can be found. drug-induced lung injury is a common cause of acute and chronic lung disease, and most commonly occurs with cytotoxic agents, such as bleomycin, busulfan, carmustine, and cyclophosphamide (ellis et al. 2000) . the most common patterns of drug-induced lung disease include: pulmonary edema and hemorrhage; diffuse alveolar damage (dad)/acute respiratory distress syndrome (ards); organizing pneumonia (op); nonspecific interstitial pneumonia (nsip), usual interstitial pneumonia (uip); hypersensitivity pneumonitis; and eosinophilic pneumonia. in early drug-induced dad (1st week after lung injury), ct shows diffuse ground-glass opacities and consolidations, whereas, in the late phase of disease (after 1 or 2 weeks), fibrotic changes occur, such as irregular linear opacities, architectural distortion, and traction bronchiectasis. the hrct manifestations of nsip consist of scattered ground-glass opacities and irregular linear opacities (fig. 26) . the sparing of the outermost subpleural lung is highly suggestive of nsip. hrct findings in uip include reticular opacities, traction bronchiectasis and honeycombing. drug-induced op is identical to cryptogenic organizing pneumonia (cop), and manifests on ct with bilateral areas of ground-glass opacities or consolidations that are often peripheral in distribution. hypersensitivity pneumonitis usually becomes clinically apparent within hours or days after institution of drug therapy and hrct findings are similar to those caused by inhalational agents (rossi et al. 2000) with ground-glass opacities, centrilobular nodules, mosaic pattern and air trapping and pulmonary involvement can result in either acute or chronic eosinophilic pneumonia (ep). drug toxicity is a common cause of eosinophilic pneumonia and is characterized by combined lung abnormality and increased serum or tissue eosinophils. hrct in ep shows consolidation that are typically distributed peripherally and in the upper lobe. ep usually responds well to cessation of the administered drug and is exceedingly fig. 25 silicosis with progressive massive fibrosis in a 72-year-old man. coronal ct image shows a large perihilar mass in the left upper lobe. there is retraction of the hilus and marked emphysema. in addition, some scattered small nodules are present sensitive to corticosteroid therapy. within the group of noncytotoxic drugs, methotrexate and amiodarone frequently cause drug-induced lung diseases in 5-10% of patients. the most common lung injury associated with both drugs is nonspecific interstitial pneumonia. organizing pneumonia is less commonly associated with noncytotoxic drugs (fig. 27 ). radiation-induced lung injury is subdivided clinically and radiologically into an early stage, characterized by acute radiation pneumonitis, and a late stage, characterized by chronic radiation fibrosis. the degree of radiation damage to normal tissue depends particularly on total dose and the fraction of that dose, irradiated volume, individual susceptibility, preexisting lung disease, and previous or concomitant therapy. early radiation pneumonitis usually develops 1-3 months after the therapy, and the radiographic findings are typically confined to the field of radiation, resulting in a geometric shape of pulmonary opacities with a sharp demarcation line at noninvolved lung areas and disregard of anatomic boundaries. the earliest ct findings consist of subtle ground-glass opacities (fig. 28) . these hazy abnormalities can progress to patchy consolidations that sometimes also involve lung areas outside the field of radiation (davis et al. 1992) . chronic radiation fibrosis evolves within 6-24 months after radiation therapy and develops continuously from the phase of acute pneumonitis. at ct, it is characterized by the presence of reticular opacities, architectural distortion, traction bronchiectasis, and volume loss. the major differential diagnoses in radiation pneumonitis include infection, lymphangitic . 26 50-year-old woman with interstitial pneumonia (ip)/nonspecific interstitial pneumonia (nsip) following bleomycin chemotherapy for hodgkin's lymphoma. axial ct image shows irregular linear and reticular opacities (arrowheads) with subtle ground-glass opacities (arrow) in subpleural distribution carcinomatosis, and recurrence of the original malignancy. microbial infectious pneumonia is not usually confined to the field of irradiation and runs a more symptomatic clinical course than radiation pneumonitis. in lymphangitic carcinomatosis, the rapid worsening of radiographic abnormalities, with development of irregular, often nodular thickening of the interlobular septa and the bronchovascular bundles, pleural effusions, and diffuse spread to the lung, are the diagnostic clues. lung involvement is common in patients with collagen vascular diseases and may be detected with hrct before the disease has declared itself or been accurately characterized. interstitial lung disease is probably most prevalent in systemic sclerosis, but is also a common problem in rheumatoid arthritis (ra), mixed connective tissue disease, dermatomyositis/ polymyositis (dmpm), or sjã¶gren syndrome. lung involvement less frequently occurs with systemic lupus erythematosus (sle). the parenchymal manifestations of collagen vascular diseases seen at ct closely resemble those found in idiopathic interstitial pneumonias (iips) and can be classified using the same system. although the proportions of interstitial pneumonias vary, the nonspecific interstitial pneumonia (nsip) is the most frequently encountered pattern in patients with collagen vascular lung disease, especially in progressive systemic sclerosis (fig. 29 ). in keeping with the iips, the nsip pattern is characterized by subpleural reticular opacities and varying proportions of ground-glass opacities, while in patients with uip, honeycombing and tractionbronchiectasis are the dominant abnormality. the predominance of the nsip over the uip pattern might explain the more favorable prognosis in patients with interstitial pneumonia associated with collagen vascular diseases than in those with iips (kim et al. 2002) . organizing pneumonia (op) is more common in ra than in the other collagen vascular diseases and is characterized by patchy infiltrates in a peripheral distribution. lymphoid interstitial pneumonia (lip) is a typical, but rare complication in sjã¶gren syndrome in about 1% of patients during the course of their disease (swigris et al. 2002) , and hrct findings include diffuse or patchy ground-glass opacities and thin-walled perivascular cysts (fig. 30 ). in addition to the patterns of interstitial pneumonias, other parenchymal manifestations in collagen vascular diseases include alveolar hemorrhage, especially in patients with sle, and necrobiotic nodules in patients with ra, which range in size from a few millimeters to a few centimeters (remy-jardin et al. 1994) , and are usually subpleural in distribution. the increased prevalence of malignant disorders complicating the course of some disorders such as dmpm makes volumetric ct protocols mandatory in the follow-up of these patients. diffuse bleeding into the alveolar spaces most commonly occurs with immunological and hematological disorders and is clinically characterized by hemoptysis and anemia (albelda et al. axial ct image in a patient with progressive systemic sclerosis shows a mixture of fine reticular and ground-glass opacities (black arrows), associated with mild traction bronchiectasis (white arrow), consistent with a nonspecific interstitial pneumonia pattern. note esophageal dilatation (arrowheads) 1985); however, the absence of these symptoms does not rule out the diagnosis of diffuse pulmonary hemorrhage (dph). dph must be distinguished from localized pulmonary hemorrhage due to chronic bronchitis, bronchiectasis, malignancy, and infection. dph can occur in association with many collagen vascular diseases, notably systemic lupus erythematosus (sle) and vasculitis. other rare causes of dph include goodpasture syndrome and idiopathic pulmonary hemosiderosis. ct is more sensitive than chest radiograph for the detection of pulmonary hemorrhage, and shows diffuse bilateral consolidation or ground-glass opacities in the acute phase (marasco et al. 1993 ). in the subacute phase of dph, multiple small nodules associated with patchy ground-glass opacities and interlobular septal thickening have been observed (fig. 31 ). in addition, in patients with granulomatosis with polyangiitis (formerly known as wegener's granulomatosis), multiple, frequently cavitating nodules and masses, ranging from 5 to 10 cm, can be seen. ers) and the world association of sarcoidosis and other granulomatous disorders (wasog) adopted by the ats board of directors and by the ers executive committee from the archives of the afip: pulmonary langerhans cell histiocytosis lymphangiomyomatosis: ct, chest radiographic, and functional correlations serial computed tomographic evaluation in desquamative interstitial pneumonia bronchiolitis obliterans organizing pneumonia manifesting as multiple large nodules or masses long-term follow-up ct scan evaluation in patients with pulmonary sarcoidosis diffuse pulmonary hemorrhage: a review and classification eosinophilic lung diseases medical therapy in idiopathic pulmonary fibrosis pulmonary alveolar proteinosis in israel: ethnic clustering accuracy of highresolution ct in diagnosing lung diseases association of malignancy with diseases causing interstitial pulmonary changes frequency and ct findings of recurrent disease after lung transplantation organising pneumonia ats/ers/ wasog statement on sarcoidosis. sarcoidosis statement committee radiation effects on the lung: clinical features, pathology, and imaging findings benign pulmonary lymphocytic infiltration and amyloidosis: computed tomographic and pathologic features in three cases ct features of lung disease in patients with systemic sclerosis: comparison with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia drug-induced lung disease: high-resolution ct findings lung cysts in subacute hypersensitivity pneumonitis ultralow-dose chest computed tomography for pulmonary nodule detection: first performance evaluation of single energy scanning with spectral shaping chronic diffuse interstitial lung disease: diagnostic value of chest radiography and high-resolution ct high-resolution ct of diffuse lung disease: value and limitations respiratory bronchiolitis, respiratory bronchiolitis-associated interstitial lung disease, and desquamative interstitial pneumonia: different entities or part of the spectrum of the same disease process? ct features of pulmonary alveolar proteinosis eosinophilic lung diseases: diagnostic accuracy of thin-section ct in 111 patients nonspecific interstitial pneumonia: correlation between thin-section ct findings and pathologic subgroups in 55 patients rare diseases. 1. lymphangioleiomyomatosis: clinical features, management and basic mechanisms idiopathic pulmonary fibrosis: clinical relevance of pathologic classification imaging of occupational lung disease interstitial lung diseases associated with collagen vascular diseases: radiologic and histopathologic findings acute exacerbation of idiopathic pulmonary fibrosis: frequency and clinical features pulmonary alveolar microlithiasis: findings on high-resolution ct cryptogenic organizing pneumonia: ct findings in 43 patients pulmonary lymphangiomyomatosis and tuberous sclerosis: comparison of radiographic and thin-section ct findings the impact of iterative reconstruction in low-dose computed tomography on the evaluation of diffuse interstitial lung disease acute pulmonary hemorrhage. ct evaluation high-resolution ct of the lungs: an optimal approach chronic eosinophilic pneumonia: ct findings in six cases what every radiologist should know about idiopathic interstitial pneumonias centrilobular lesions of the lung: demonstration by high-resolution ct and pathologic correlation pulmonary sarcoidosis: changes on follow-up ct examination large coalescent parenchymal nodules in pulmonary sarcoidosis: "sarcoid galaxy" sign pulmonary sarcoidosis: correlation of ct and histopathologic findings lymphangioleiomyomatosis: pulmonary and abdominal findings with pathologic correlation pulmonary alveolar phospholipoproteinosis: experience with 34 cases and a review ct protocols in interstitial lung diseases-a survey among members of the european society of thoracic imaging and a review of the literature an official ats/ers/ jrs/alat statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management lung changes in rheumatoid arthritis: ct findings pulmonary alveolar proteinosis pulmonary drug toxicity: radiologic and pathologic manifestations chronic hypersensitivity pneumonitis: differentiation from idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia by using thin-section ct air-trapping in extrinsic allergic alveolitis on computed tomography the familial occurrence of pulmonary alveolar microlithiasis image quality from high-resolution ct of the lung: comparison of axial scans and of sections reconstructed from volumetric data acquired using mdct lymphoid interstitial pneumonia: a narrative review lymphangioleiomyomatosis. clinical course in 32 patients acute eosinophilic pneumonia: histopathologic findings in nine patients survival benefit of lung transplantation for patients with idiopathic pulmonary fibrosis acute parenchymal lung disease in immunocompetent patients: diagnostic accuracy of high-resolution ct idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia pulmonary alveolar microlithiasis: review of turkish reports thin-section ct of the secondary pulmonary lobule: anatomy and the image-the fleischner lecture fleischner lecture. looking into the lung: what can it tell us? key: cord-278846-nqj7ctk3 authors: ogger, patricia p.; byrne, adam j. title: macrophage metabolic reprogramming during chronic lung disease date: 2020-11-12 journal: mucosal immunol doi: 10.1038/s41385-020-00356-5 sha: doc_id: 278846 cord_uid: nqj7ctk3 airway macrophages (ams) play key roles in the maintenance of lung immune tolerance. tissue tailored, highly specialised and strategically positioned, ams are critical sentinels of lung homoeostasis. in the last decade, there has been a revolution in our understanding of how metabolism underlies key macrophage functions. while these initial observations were made during steady state or using in vitro polarised macrophages, recent studies have indicated that during many chronic lung diseases (clds), ams adapt their metabolic profile to fit their local niche. by generating reactive oxygen species (ros) for pathogen defence, utilising aerobic glycolysis to rapidly generate cytokines, and employing mitochondrial respiration to fuel inflammatory responses, ams utilise metabolic reprogramming for host defence, although these changes may also support chronic pathology. this review focuses on how metabolic alterations underlie am phenotype and function during clds. particular emphasis is given to how our new understanding of am metabolic plasticity may be exploited to develop am-focused therapies. the respiratory mucosa is a unique site, as our airways are continually exposed to particulates, viruses, bacteria, and fungi, which challenge the pulmonary immune system 1 . to maintain pulmonary homoeostasis and ensure efficient gas exchange, a complex regulatory system is in place, of which airway macrophages (ams) are a core component. ams are the most numerous immune cell type present in healthy lungs, are strategically positioned at the interface of airways and environment 2 , and critical sentinels of barrier immunity. ams form the first line of defence against inhaled particles, pathogens and antigens 3 . although inherently suppressive, ams exhibit significant functional and phenotypical specialisation, allowing efficient responses to environmental signals and rapid alterations in phenotype. increasing evidence suggests that metabolic alterations provide an additional layer of functional plasticity to am populations. activation of macrophages in vitro with a range of inflammatory stimuli, induce profound metabolic adaptations, such as the switch from oxidative phosphorylation (oxphos) to glycolysis in oxygen-sufficient conditions, similar to the "warburg effect" seen in some cancers 4 . it is now clear that how macrophages utilise energy dictates immune responses, and that manipulating cellular metabolism can alter the inflammatory response 5 . however in vivo, the unique oxygen rich environment of the airways coupled with specific local nutrient availabilities, shapes am phenotype and function. indeed, many recent studies have indicated that in chronic lung diseases (clds), such as asthma, chronic obstructive pulmonary disease (copd), cystic fibrosis (cf), idiopathic pulmonary fibrosis (ipf), and during infection such as with mycobacterium tuberculosis (mtb), there are significant alterations in am metabolic processes and that targeting these pathways could represent an exciting therapeutic approach 6, 7 . this review focuses on how metabolic adaptations underlie am phenotype and function during clds. particular emphasis is given to how our new understanding of am metabolic plasticity may be exploited to develop am-focused therapies. airway macrophages: guardians of the lung environment to maintain gas exchange, it is critical that ams sustain a naturally hyporesponsive state whilst also reserving the ability to rapidly mount effective inflammatory responses. this balance is achieved through complex am-airway epithelial cell (aec) interactions via cell surface-expressed receptors and secreted products. ams express transforming growth factor beta receptor (tgf-βr), interleukin (il)-10 receptor (il-10r), cd200 receptor (cd200r) and signal regulatory protein-α, key components mediating am: aec crosstalk and in turn, regulating am activation 8 . for example, am-aec contact decreases am phagocytosis and cytokine production in a tgf-β-dependent manner 9 . conversely, loss of the integrin αvβ6 such as through loss of contact of ams with aec upon toll-like receptor (tlr) activation leads to initiation of the am pro-inflammatory phenotype and inflammatory response 10 . ams are characterised by a distinct cellular phenotype. human ams highly express the lectin-binding transmembrane glycoprotein cd68, the adhesion molecule cd169, the integrin cd11c and mannose receptor cd206 11 (fig. 1 ). in mice, expression of the cd68 + cd206 + cd11c hi cd11b lo cell surface phenotype is conserved at steady state [12] [13] [14] , while murine ams also express the mer tyrosine kinase (mertk), sialic acid dependent adhesion molecule siglecf, hormone receptor f4/8, glycoprotein cd64 and the cd200 receptor 15 (fig. 1) . recent work in mice has indicated that many tissue resident macrophages, including those in the airways, are foetally derived and self-maintain locally with minimal contribution from circulating monocytes, during steady state conditions [16] [17] [18] [19] [20] . during murine prenatal development, foetal liver or yolk sac macrophages are the major contributing pool to am populations 21 and am colonization of the lung occurs in sequential waves in the first week of life 22 . furthermore, post birth and during maturation, circulating monocytes do not significantly contribute to lung macrophage populations at homoeostasis 18 . during pulmonary inflammatory responses however, monocytes are recruited to the lung 23, 24 and subsequently develop into am-like cells 18, 25 . thus, post-injury murine airways contain at least two ontologically distinct am populations, tissue resident ams (tr-am) which are prenatally derived and monocytederived ams (mo-ams). several groups have studied samples from lung transplant patients to investigate the origins of ams in the human lung [26] [27] [28] [29] [30] . utilizing bronchoalveolar lavage (bal) from sexmismatched lung transplant patients our group recently demonstrated that the majority of ams in human lung post-transplant are derived from peripheral classical monocytes 31 . thus, the unique airway niche combined with distinct ontological origins, age and environmental exposures results in remarkable am plasticity and adaptability [32] [33] [34] . while the recruitment of monocytes to the lungs to replenish the tr-am pool is relatively well understood in mice, the origins of ams in the human lung during healthy aging or clds requires further investigation; in particular clear markers which distinguish mo-ams and tr-am are not well established. airway macrophage metabolic phenotype at homoeostasis beyond delineation of macrophage populations based on anatomical location or ontological origin, macrophage populations are also classified according to their activation status. analogous to the th1/th2 paradigm, in vitro cultured human monocyte derived macrophages (mdms) or murine bone marrow derived macrophages (bmdms) are categorised as m1 or m2 macrophages, respectively. seminal studies have demonstrated that pro-wound healing m2 (il-4 stimulated) macrophages in vitro rely on fatty acid oxidation (fao), an intact tricarboxylic acid cycle (tca) cycle, high rates of oxphos and increased expression of arginase 1 (arg1), which catalyses the production of ornithine from arginine as precursor for collagen to facilitate wound healing 5, [35] [36] [37] . conversely, pro-inflammatory m1-macrophages rely on glycolysis and breaks in the tca cycle lead to accumulation of metabolites, many of which have signalling functions such as citrate, succinate, fumarate and α-ketoglutarate 35, 38, 39 . however, although useful in defining the range of potential macrophage responses, in vitro derived cells do not recapitulate the core aspects of am phenotypes which are shaped by the local niche 11 . as ams are highly adapted to the unique environment of the airway lumen, it is perhaps unsurprising that the metabolic state of ams is also distinct. glucose concentrations in the alveolar lumen are less than 10% of blood glucose concentrations and ams exhibit extremely low levels of glycolysis 20 ; in stark contrast to bmdms, ams do not undergo glycolytic reprogramming in response to lps 40 . consequently, ams readily engage oxphos and highly express the peroxisome proliferator-activated receptor gamma (pparγ) 41 , which regulates lipid accumulation and promotes fao to sustain oxphos. ams also play a major role in the catabolism of pulmonary surfactant, a monolayer composed mainly of phosphocholinebased lipids, phospholipids and cholesterol which lines the alveoli, lowers surface tension and prevents alveolar collapse during expiration 42 . mice lacking gm-csf and thus the am compartment, develop pulmonary alveolar proteinosis (pap), an inflammatory lung syndrome caused by the defective clearance of surfactant [43] [44] [45] . in humans, mutations in genes encoding for gm-csf receptors, result in hereditary pap as a result of progressive alveolar surfactant accumulation [46] [47] [48] [49] . am phenotype and behaviour are influenced by surfactant exposure, which has major implications for am-mediated immune responses in pulmonary tissue. there are four principle surfactant proteins (sp-a-d) and sp-a and sp-d have been shown to directly influence am functions such as cell migration, phagocytosis and activation phenotypes 42 . both sp-a and sp-d bind carbohydrates, lipids, and nucleic acids and initiate phagocytosis of inhaled pathogens and apoptotic cells 50 . furthermore, sp-a blocks the binding of tlr ligands to tlr2, tlr4 and tlr co-receptors and furthermore inhibits complement activation 51, 52 . whilst the alveoli are covered with a monolayer of surfactant, a thin layer of mucus produced by goblet cells and ciliated epithelium protects the airways. mucus serves as a barrier and facilitates clearance of microbes and pollutants. a major component of mucus are mucin glycoproteins, which may be categorized as polymerizing, nonpolymerizing and cell-surface associated. of the cell-surface associated mucins, muc1 is expressed in ams and contributes to the resolution of inflammation by decreasing phagocytic potential and pro-inflammatory cytokine production 53 . the polymerizing mucins include muc5ac and muc5b; in particular, muc5b deficiency has been linked to particle accumulation in the lung, mucus obstruction and impaired macrophage phagocytosis 54 . pro-inflammatory macrophages induce muc5b expression to aid mucociliary clearance 55 . furthermore, a single nucleotide polymorphism in the muc5b promotor has been strongly associated with the risk of developing ipf, highlighting the importance of mucins for the pulmonary environment 56 . in addition to low glucose and a lipid rich environment, the airways also have a unique distribution of amino acids and central carbon metabolites. surowiec et al. showed that whilst several glucogenic and ketogenic amino acids were present in the bronchial wash, only alanine is present in bal 57 (fig. 2 ). in addition, the central airways contained key glycolytic and oxphos metabolites such as fructose, glucose-6-phosphate, fumarate and malate as well as oxidised gluthathione (gssg, indicating oxidative stress, fig. 2) ; interestingly, these could not be detected murine ams express the lectin-binding transmembrane glycoprotein cd68, the mer tyrosine kinase (mertk), the integrin alpha x chain protein cd11c, the type i membrane glycoprotein cd200 receptor, the mannose receptor cd206, the egf-like module-containing mucin-like hormone receptor-like 1 (f4/80), the sialic acid binding lectin siglec-f and the fc receptor cd64. human ams express cd68, the adhesion molecule cd169, cd11c, cd206 as well as mhc class ii receptor hla-dr. in the periphery, suggesting either minimal secretion, high utilisation or as a result of anatomical location (i.e. close proximity to nutrient rich pulmonary capillaries) 57 . recently the lung microbiome has gained attention as a factor which modifies the pulmonary environment and directs immune responses by producing short chain fatty acids (scfa). whilst the airways and alveoli are colonised mainly by proteobacteria, bacteroidetes and firmicutes 58,59 , the nasal mucosa additionally hosts actinobacteria 60, 61 (fig. 2) . proteobacteria, bacteroidetes and firmicutes produce large amounts of scfa, including acetate, propionate and butyrate, which influence barrier function by regulating epithelial tight junctions 62 and anti-inflammatory immune responses 63 . recent advances in understanding the pulmonary microbiome during homoeostasis and clds are described in detail elsewhere 58, 59, 64 . thus, at homoeostasis ams are exposed to a unique environment, with minimal glucose availability and a distinct distribution of nutrients and scfa, which depend on anatomical location (fig. 2) . however, despite the profound influence that local substrate availabilities may exert on macrophage development, activation and function, this is an understudied area. new knowledge, which further defines especially human am substrate dependencies at homoeostasis is required in order to fully understand how local metabolic perturbations during clds may contribute to pathology. this is particularly relevant as already slight changes in nutrient availability during inflammation, such as succinate or citrate, can alter macrophage phenotypes through stabilization of hif1α, post-translational modification of proteins and production of no and ros 65 , thereby contributing to a pathological development. am metabolism during clds chronic lung diseases affect a significant proportion of the world's population, killing more than 100,000 people in the uk alone, each year 66 . persistent inflammation, impaired repair processes and pulmonary remodelling are cardinal features of clds [67] [68] [69] . there are multiple overlaps in environmental exposures driving clds, such as smoking, pollution and environmental exposures; viral infection can also exacerbate symptoms in each disease 2, 70, 71 . interestingly, am metabolic adaptation may play a central role in dictating pathology during clds and present novel therapeutic opportunities (fig. 3) . asthma. asthma is a heterogeneous disease of the airways characterized by airway remodelling, mucus production, airway hyperresponsiveness (ahr), and inflammation 72 . although most patients have good control with standard medication, a proportion experience life-threatening, severe disease 73 . ams are central to mediating type-2 inflammation against allergens and parasitic worms 2 . in vitro, macrophages respond to type-2 cytokines such as il-4 that drive an 'alternative' m2 activation phenotype, linked to wound repair and type-2 pathology 74, 75 . manipulation of am phenotype via genetic deletion of the transcription factor interferon regulatory factor 5 (irf5), a master regulator of macrophage activation 74 , promoted pulmonary remodelling, ahr and mucus secretion in mice, in an il-13 dependent manner 76 . indeed, a recent study has shown that both cd206 + am (activated by il-4 and il-13) and pro-inflammatory irf5 + am are increased in asthmatic patients 77 , highlighting the plasticity of macrophages and heterogeneity of human asthma. roles of ams during antigen induced airway inflammation include phagocytosis of apoptotic cells and eosinophils as well as triggering anti-inflammatory pathways to regulate airway hyper responsiveness, mucus secretion and matrix deposition 76 . in severe asthma however, this protective function is impaired, resulting in the loss of phagocytic ability and anti-inflammatory programme 78 , which can contribute to airway remodelling 79 . thus, ams are uniquely involved in responses to allergen and type-2 inflammation, and aberrant amphenotypes can directly influence respiratory pathology. numerous lines of evidence suggest that metabolic stress leading to the production of reactive oxygen species (ros) plays a role in asthma. increased ros have been detected in ams of asthmatic patients 80 , and contributes to lung injury 81 and proinflammatory tumour necrosis factor-alpha (tnf-α) and il-1β secretion by macrophages 82 . furthermore, heme-oxygenase-1 (ho-1), which mediates ros production in response to chemical and physical agents, is increased in ams in asthmatics 83 . in addition to these pro-inflammatory characteristics, ams show key features of a more anti-inflammatory phenotype in studies using ovalbumin to model allergic asthma. using this model, al-khami et al. show that expression of carnitine palmitoyltransferase (cpt) is increased in ams, shuttling fatty acids into the mitochondria, as well as increased gene expression of fao related genes 84 . another functional pathway that is altered in asthmatic ams and links to the underlying metabolic phenotype is the eicosanoid pathway, which is induced by th2 cytokines il-4 and il-13. eicosanoids, including prostaglandins and leukotrienes, are produced from the poly-unsaturated fatty acid arachidonic acid, which is released during asthma 85 . increased production of the eicosanoid 5-hete and leukotrienes b 4 (ltb 4 ) and e 4 (lte 4 ) has been detected in ams from asthmatic patients stimulated ex vivo 86 . this contributed to bronchial constriction and pro-inflammatory phenotype and failure to generate the anti-inflammatory eicosanoid 15-hete and prostaglandin e 2 (pge 2 ), which is associated with reduced am phagocytosis 78 . lte 4 has been shown to cause ahr in subjects with aspirin-induced asthma 87 and can be produced in ams by γ-glutamyl transpeptidase 85, 88 . il-13 furthermore induces arg1, which may further contribute to the asthmatic phenotype via metabolism of collagen precursors ornithine and proline to collagen and airway remodelling 89, 90 . several of these observed alterations have been targeted therapeutically, attempting to rewire am phenotype. these include the eicosanoid pathway, ros, glycolysis and fao. administration of the anti-inflammatory eicosanoid 15-hete inhibited leukotriene synthesis and reduced ahr in asthmatics 91 . ex vivo, the corticosteroid dexamethasone decreased levels of thromboxane b2 and ltb 4 in macrophages and asthmatic ams 92 , while prednisone decreased ltb 4 production in ams 93 . treatment with the antioxidant ad4 improved ahr and airway inflammation by decreasing ros in the ova-sensitised mouse model of allergic airway disease (aad) 94 84, 95 . together, these studies indicate that there is significant disruption of am metabolism during asthma and aad, notably via dysfunction in eicosanoid, glycolysis and fatty acid pathways. in fig. 3 altered metabolic pathways in ams drive key features of chronic lung disease. several metabolic pathways are rewired during chronic lung disease. while this response exists to clear invading pathogens and launch an inflammatory response, long-term activation of these pathways has negative implications. the glycolysis pathway supports inflammatory responses of am, while iron and metabolites produced in the tca cycle can function as bacterial substrates and contribute to pathogen survival. while fatty acid synthesis and oxidation is useful as a way of storing energy and alternative energy source during times of macrophage activation, fatty acid synthesis can also contribute to mucus production. leukotrienes contribute to the am pro-inflammatory phenotype but also cause bronchial constriction and contribute to airway remodelling in asthmatics by causing smooth muscle thickening. the amino acid arginine is a proliferator for collagen via ornithine and proline and can thereby contribute to extracellular matrix deposition. order to evaluate candidate therapies, it is crucial that studies utilise relevant preclinical models and ex vivo patient samples to understand disease. models which more closely recapitulate the complex immune response to allergens, are more likely to reveal viable targets for intervention; in particular the ovalbumin model of aad, which requires an adjuvant and a sensitization phase 96 , is a poor murine model of asthma. furthermore, our new understanding of asthma heterogeneity has allowed the development of biologics which target "type-2 high" asthma 97 ; delineation of how metabolic changes underlie distinct asthma phenotypes could lead to new treatments for other phenotypes, such as neutrophilic and paucigranulocytic asthma. copd. copd is the 5th leading cause of death in high income countries 98 , affecting over 200 million people worldwide 99 . copd is a heterogeneous disease, characterised by destruction of the parenchyma and emphysema, narrowing of the airways, remodelling and chronic inflammation driven by chronic exposure to cigarette smoke and particular matter 100 . am numbers are increased in copd bal 101 and contribute to copd pathology through numerous pathways. during copd, ams are found in areas of lung destruction and produce pro-inflammatory cytokines 102 , chemokines 100 and matrix metalloproteases (mmps) with elastolytic properties 103, 104 . at the same time, tissue inhibitor of metalloproteases (timp)-1 is decreased in ams in copd 105 and furthermore, decreased phagocytic capacity and impaired bacterial killing have been described in copd-ams [106] [107] [108] . ams of copd patients experience a high level of oxidant burden induced through cigarette smoke and subsequent increased ros production is a key feature 109 . compared to controls, copd-ams secrete increased levels of mitochondrial ros (mtros) 110 , superoxide and hydrogen peroxide 81,108 whilst glutamyl cysteine ligase for gsh synthesis is downregulated 111 . cigarette smoking also alters iron homoeostasis 112 and ams in copd show increased sequestering of iron 113 , which can furthermore contribute to ros production. bewley et al. showed recently that increased generation of mtros in copd ams results in impaired bacterial clearance 108 . this study also reported a decrease in the mitochondrial membrane potential 114 , which has recently been linked to am exposure to particulate matter 110 . this may explain the impaired phagocytic capacity of ams in copd as decreased mitochondrial membrane potential results in energy failure in the cell, proton leakage and increased mtros 115 . another study by o'beirne et al. further investigated the metabolic profile of ams from healthy smokers, non-smokers and copd patients. while all groups had similar baseline glycolysis rates, there was a decrease in coupling efficiency, maximal respiration and spare respiratory capacity in copd-ams, while proton leak was significantly increased 116 . in addition, expression of genes related to glutathione metabolism, mitochondrial transport, pyruvate metabolism, tca cycle and electron transport chain were altered in smokers and copd patients, compared to nonsmoking healthy controls 116 . other metabolic alterations in copd ams include increased expression of inducible nitric oxide synthase (inos) contributing to increased levels of nitric oxide (no) 117 and increased levels of the adenosine receptor a2br 118 , suggesting increased adenosine metabolism, which might be linked to the increased levels of hif1α in copd ams 119 . while excessive ros production through oxidant burden and iron accumulation has been identified as an important regulator of am phenotype in copd, it has only recently been linked to mitochondrial dysfunction and metabolic reprogramming. it would be interesting to follow up on these transcriptomic and metabolic alterations to understand their underlying disease driving role and to identify ways to rewire am metabolism. as corticosteroids have been found to be particularly ineffective in copd, more specific pathways involved in am function and metabolism have been investigated recently, such as the ros pathway and iron accumulation. a study by harvey et al. showed that treatment with sulforaphane in copd ams ex vivo improved bacterial clearance by activating the antioxidant and antiinflammatory nrf2 pathway 120 , while cloonan et al. found that treatment with an iron chelator or a low iron diet protected mice from cigarette smoke induced copd 121 . furthermore, procysteine, a precursor of gsh, increased am efferocytosis in a mouse model of copd 122 . overall, copd is marked by distinct iron sequestration, ros, no production and energetic dysfunction in ams; further delineation of how mitochondrial phenotype links to inflammatory processes and pathology in copd will allow the identification of molecular targets for modulating mitochondria during the disease. cystic fibrosis. cystic fibrosis (cf) is caused by mutation of the cf transmembrane conductance regulator (cftr), a chloride channel, which regulates fluid homoeostasis in mucosal surfaces. in the lung, cftr mutation and subsequent loss of function results in a reduced aqueous film covering the epithelium and mucus thickening, leading to impaired mucociliary clearance and frequent bacterial infection 123 . cf is furthermore characterised by hyper-inflammation of the lungs, airway obstruction, structural damage and progressive reduction of lung function 124 . during recurring airway inflammation, large numbers of neutrophils, macrophages and t lymphocytes infiltrate the lungs and secrete pro-inflammatory cytokines, while anti-inflammatory il-10 is reduced 125, 126 . although am numbers are increased in cf patients 127, 128 , pathogen clearance is attenuated, leading to colonisation of the airways and chronic inflammation 106, 128 . meyer et al. report a more pro-inflammatory phenotype of ams in a murine model of cf, even in the absence of infection 129 and mdms differentiated from cf patients show an increased inflammatory profile 130 , while others have shown that monocytes from cf patients had an impairment in activation upon il-13 stimulation 131 . cf-am phenotype can be heterogenous, depending on infection status and local environment. while ams from p. aeruginosa infected cf patients showed increased expression of mannose receptor cd206 and augmented arginase activity 132 , in cf sputum ams a decrease in expression of cd206 and scavenger receptor marco was detected 133 . furthermore, ams are involved in the structural damage in cf airways by secreting serine-and metalloproteases, which subsequently degrade connective tissue components 134 . the lower volume of airway surface liquid in cf airways activates ams to increase their release of mmp12, resulting in the cleavage of elastin and degradation of the airway and parenchyma 135 . in cf airways, gsh is depleted 136, 137 , while levels of iron, transferrin, haem and haemoglobin are increased 138 , resulting in high oxidative stress and ros production. ros in turn can induce tgf-β1 139 , which has recently been shown to be increased in cf-bal and ams and inhibits cftr biogenesis and cellular trafficking to the surface of epithelial cells 134 , while also contributing to airway remodelling by recruitment and differentiation of myofibroblasts 140 . however, during infection with bacteria from the burkholderia family, both mdms and ams from cf patients showed reduced superoxide production as well as decreased phosphorylation of nadph oxidase (nox) components p47 phox and p40 phox , suggesting an inherent deficit in cf-ams generating oxidative bursts for pathogen defence 141 . p. aeruginosa is one of the most common pathogens to cause recurrent pulmonary infection in cf patients and exploits the host to maintain infection by inducing production of the tca cycle metabolite itaconate in ams. itaconate exerts antimicrobial properties via inhibition of bacterial isocitrate lyase in the glyoxylate shunt 142 and to evade this mechanism p. aeruginosa has developed a way to use itaconate as an energy source 143 . similarly succinate, which is secreted in high levels during cf and especially during bacterial infection 144 , can be utilised by p. aeruginosa and s. aureus as a substrate to generate oxidative stress. changes in lipid metabolism are a hallmark of cf and increased fao, lipid turnover in cell membranes and eicosanoid production in ams have been reported. furthermore, sterol regulatoryelement binding protein (srebp), a regulator of lipid homoeostasis, has been linked to cftr loss of function 124 . this results in altered plasma and tissue fatty acid profiles, and while levels of the omega-3 fatty acid docosahexaenoic acid (dha) were unchanged in ams upon loss of cftr 145 , ex vivo treatment with dha decreased tnf-α levels 146 . furthermore, in cf ams the antiinflammatory lipoxin a 4 (lxa 4 ), which is synthesised from the fatty acid arachidonic acid, is reduced and the lxa 4 /ltb 4 ratio in cf bal is decreased 147 , while the fatty acid metabolite resolvin d1 (rvd1) has been suggested as a biomarker 148 . increased energy demand by ams in cf, either by manipulation through bacterial pathogens or to fight sustained infection, results in increased utilization of all available metabolic pathways. recently, lara-reyna et al. reported increased glycolysis, mitochondrial function, and production of tnf-α in cf macrophages is due to an alteration in the serine/threonine-protein kinase/ endoribonuclease ire1α pathway and this supports exacerbated inflammation 149 . while this study used pbmcs and monocytederived m1 macrophages from cf patients, it would be important to detect such a mechanism in ams and to target this pathway specifically. several of the above described pathways have been identified as potential drug targets in cf, however yet there are no treatments targeting ams. delivery of gsh to the lower respiratory tract improves the antioxidant barrier of cf epithelium 150 , while treatment with cysteamine and restoration of microrna 17 (mir17) and mir20 expression improves disease by restoring autophagy 151, 152 . several studies administered omega-3 fatty acids (dha/epa) to cf patients [153] [154] [155] [156] , although only one trial reported improved fev1 after 8 months treatment with dha 157 . treatment with dha in a murine model of cf decreased liver inflammation but did not improve lung morphology 158 . in conclusion, am metabolic phenotype during cf is marked by increased energy expenditure to support exacerbated inflammation and is readily exploited by bacterial pathogens, leaving ams deficient of oxidative burst capability during infection. it will be important to clarify the role of fatty acids in cf and furthermore, to target metabolic changes in ams such as increased glycolysis, oxphos and fao specifically to rewire am phenotype and prevent exploitation through bacterial pathogens. idiopathic pulmonary fibrosis (ipf). ipf is a chronic interstitial lung disease characterised by excessive extracellular matrix deposition in the lung parenchyma and has a particularly poor prognosis 159 . repetitive alveolar injury in genetically susceptible individuals causes activation of mesenchymal cells, recruitment of fibroblasts and differentiation into myofibroblasts to replace damaged alveolar epithelial cells and provide a matrix for wound healing and tissue repair 160 . during ipf, the wound healing process is dysregulated, leading to fibrotic plaque formation and excessive build-up of extracellular matrix, resulting in impaired gas exchange. ams have been identified as key contributors to the dysregulated wound healing process, by secreting large amounts of ros and tgf-β 161 . furthermore, ams can shape the extracellular matrix by secreting factors contributing to the matrix (proline, collagen) and breaking down the matrix (plasmin, mmps) [162] [163] [164] . several changes to the central carbon metabolism pathways have been identified recently in ams of ipf patients, including dysmorphic mitochondria 165 . in murine models of pulmonary fibrosis, increased glucose consumption, glycolysis and enhanced expression of key glycolytic mediators was detected 166 , while in ipf ams, expression of the pulmonary glucose transporter glut1 was increased 167 , which enabled augmented glucose uptake 166 . the increased glucose uptake via glut1 can furthermore sustain nadph production in the pentose phosphate pathway and tca cycle 168 and is therefore a key substrate for ros production via nox 169 . activation of macrophages results in the accumulation of endogenous metabolites capable of adopting immunomodulatory roles such as succinate 170 and itaconate [171] [172] [173] . recently, our laboratory identified itaconate as an endogenous anti-fibrotic in the human and murine lung. in patients with ipf, there were reduced levels of airway itaconate, and decreased expression of acod1 (which controls the synthesis of itaconate) in ams compared to healthy controls. acod1 deficiency in mice leads to more severe disease pathology and exogenous itaconate limits fibroblast activity 174 . these data indicate that am metabolites may play a key role in the pathogenesis of lung fibrosis and may be exploited for the development of anti-fibrotic therapies. ros production is a key feature of ams in ipf 175 and can occur during oxphos, by the membrane bound nox or by reaction of hydrogen peroxide with intracellular iron 176 . nox, and subsequent superoxide production, is activated by binding gtp-bound rac1 177 , which is secreted from ams in ipf 178 and can also activate the mtor signalling hub 179 . superoxide produced by nox can further react with no to form peroxynitrite (oono -), another type of ros. at the expense of nadph, no is produced in the mitochondria by inos, which is upregulated in proinflammatory macrophages 180 and in ipf-ams leading to increased levels of the cytotoxic oonoin ipf ams 181 . in the bleomycin mouse model of pulmonary fibrosis, increased levels of superoxide, no and oonowere measured in ams 182 . mtros is furthermore linked to expression of ppar-γ coactivator 1-alpha (pgc-1α), which induces metabolic reprogramming to fao and is regulated by the mitochondrial calcium uniporter (mcu), which is increased in ipf ams 183 . mcu has furthermore been shown to regulate expression of the fatty acid transporter cpt-1, which is increased in ams from ipf patients and bleomycin exposed mice 183 . while human ipf-ams have increased levels of mcu, mitochondrial calcium and expression of pgc-1a, bleomycin exposed mice utilise increased fao 166 , which is reduced in mice expressing dominant-negative mcu 183 . furthermore, these mice were protected from bleomycin induced pulmonary fibrosis. these findings highlight calcium transport and fao as pathways to target in ipf ams; however, a better understanding of the linking mechanism will be necessary. ipf ams have also been shown to be iron laden 184 , which further induces oxidative stress and ros production 185 . using rna-sequencing, lee et al. show furthermore, that macrophage activation is increased in iron laden ams in ipf, suggesting that iron accumulation plays a role in macrophage activation 185 . the proportion of ams expressing transferrin receptor (cd71), importing transferrin bound iron into the cell, are decreased in ipf ams, leading to an extracellular accumulation of transferrin. furthermore, numbers of cd71-negative macrophages are an independent predictor of survival in ipf 186 . iron metabolism is therefore likely a key pathway in ipf-ams and targeting it would be a viable option to decrease ros, oxidative stress and macrophage activation. recently, therapies targeting metabolic processes in ipf are of considerable interest. while antioxidant therapy in ipf was promising in vivo, the double-blind placebo controlled panther trial, administering either n-acetylcysteine or placebo to ipf patients for 60 weeks did not show a change in lung function parameters 187 . another arm of this study investigated the combined potential of corticosteroid prednisone, immunosuppressant azathioprine and n-acetylcysteine but was stopped prematurely due to increased mortality and adverse effects without evidence of benefit 188 . another randomized, double-blind clinical trial assessed the safety and tolerability of n-acetylcysteine in patients already receiving pirfenidone anti-fibrotic therapy. while this trial showed that n-acetylcysteine in combination with pirfenidone was safe, no change in fvc, 6-minute walk test or occurrence of adverse effects was detected 189 . another promising therapeutic avenue was the use of metformin, a potent metabolic remodelling drug often prescribed for type ii diabetes. while on a global level metformin lowers the amount of blood sugar in diabetic patients, on a cellular level metformin activates ampactivated protein kinase (ampk) leading to inhibition of tgf-β induced nox activity 190 . sato et al., have shown that metformin inhibited tgf-β induced nox activity via ampk leading to inhibition of myo-fibroblast differentiation in vitro and reduced bleomycin induced collagen deposition in vivo 191 . consistent with this, rangarajan et al. showed that metformin treatment reversed bleomycin induced pulmonary fibrosis via ampk activation, while in ipf patients ampk phosphorylation was decreased 192 . a posthoc analysis study of the effect of metformin in ipf patients however showed no change in clinically outcomes 193 , once again showing the difficulty of translating in vitro and in vivo findings into the clinic. another study investigating the nox-nrf2 imbalance as a therapeutic target showed that in vivo knockdown of nox4 and nox1/4 inhibition restored the capacity of fibrosis resolution in aged mice 194 . furthermore, treatment with nitrated fatty acids, reversed pulmonary fibrosis in a mouse model by promoting collagen uptake by ams and dedifferentiating myofibroblasts 195 . while these treatment approaches targeted metabolic changes during pulmonary fibrosis, none was specific to ams. targeting macrophage specific metabolic reprogramming, which sustains ros and tgf-β production and contributes to dysregulated wound healing in ipf would therefore be a promising approach. during respiratory tract infections, activation of pattern recognition receptors expressed by am can elicit a variety of proinflammatory host responses 2 . for example, severe coronavirus disease-19 (covid-19) associated pneumonia patients may exhibit features of systemic hyper-inflammation also known as macrophage activation syndrome or "cytokine storm" which is associated with sustained elevation of macrophage/monocytederived pro-inflammatory cytokines (e.g., il-6, il-8, tnf-α, il-1β) leading to acute respiratory distress syndrome (ards) [196] [197] [198] . using single cell approaches a recent study demonstrated that highly inflammatory, monocyte recruited ams, rather than quiescent pulmonary resident ams, predominate in the bal in covid-19 patients with severe pathology, implicating these cells in covid-19-associated ards 199 . rather than direct infection of ams, am:aec cross-talk has been identified as a major mechanism for control of many respiratory viral infections 200 and aec have been shown to be a key source of pro-inflammatory cytokines, modulating am phenotype 198, 201 . for example, rhinovirus (rv), the causative agent of the common cold, primarily infects the upper airways, however prior infection with rv attenuates subsequent am antibacterial responses 202 . although ams are susceptible to influenza a viral infection (iav), replication within ams has been shown to be minimal with the exception of several highly virulent strains [203] [204] [205] . here, we will focus on mycobacterium tuberculosis (mtb) infection, as ams are the primary infected cell type and metabolic changes in response to mtb infection are well studied. tuberculosis. tuberculosis (tb) is a contagious, chronic disease and one-third of the world's population is infected with mtb, the causative agent of tb, resulting in~2 million deaths per year (2009 world health organization report) 206 . during infection, mtb colonises ams intracellularly and disables innate intracellular defence mechanisms such as the phagolysosome and inflammasome and accesses macrophage intracellular nutrients 207 . am host defence mechanisms against mtb include production of ros and reactive nitrogen species (rns) for bacterial killing and fusing mycobacteria-containing phagosomes with lysosomes as well as autophagy and apoptosis 208 . however, virulent or multi-drug resistant strains can evade these host responses e.g. by preventing phagolysosome fusion and surviving ros/rns 209 . during mtb infection, ams shift their metabolic programme from oxphos to aerobic glycolysis, which is regulated by hif1α and interferon-gamma (ifn-γ). this metabolic shift and subsequent enhanced glycolytic flux in infected ams is crucial to control infection. mice lacking hif1α in the myeloid lineage are more susceptible to infection and show decreased cytokine and antimicrobial effector production 210 . to support this metabolic reprogramming, key glycolysis genes are upregulated in the early stages of granuloma formation in mice, supporting the shift towards aerobic glycolysis 211 206 . mtb furthermore induce ferroptosis, associated with reduced levels of gsh, superoxide and increased free iron. the ferroptosis inhibitor ferrostatin-1 (fer-1) as well as iron chelation decreased necrotic cell death of mtb-infected macrophages in vitro, while in vivo treatment with fer-1 reduced bacterial load 220 . mtb can cope in low iron environments however macrophage metabolic reprogramming during chronic lung disease pp ogger and aj byrne by downregulating their non-essential protein content via specific srna 221 . several changes in fatty acid metabolism of mtb infected ams were identified recently. compared to interstitial macrophages during mtb infection, which are reliant on glycolysis, ams utilise fa, which is induced by ppar-α 222 and have a lower burden of mtb infection 223 . to escape host defence, mtb has developed a mechanism inhibiting pathways related to autophagy, lysosomal function and fao in support of replication by inducing microrna-33 (mir-33) in the host cell. silencing of mir-33 however induced am lipid catabolism and autophagy and rescued host defence 224 . furthermore, amino acid metabolism is altered during mtb infection. in mice and macaque lungs, indoleamine 2,3-dioxygenase (ido), which is involved in tryptophan catabolism, was increased during mtb infection, while inhibition of ido in a macaque model of tb decreased bacterial burden and pathology, as tryptophan metabolites suppress host immunity 225 . while mtb relies on host lipids as energy source, existing therapies such as targeting ppar transcription factors or cholesterol synthesis have been successful mainly in animal models [226] [227] [228] [229] , whereas retrospective human studies, which investigated the effect of statins in diabetic tb patients did not show any results 230 . as mtb can also utilise iron as a substrate, another approach is to prevent iron accumulation. treatment of mtb infected human mdms and primary am with iron chelator desferrioxamine (dfx) ex vivo induced the expression of glycolytic enzymes and enhanced glycolysis, as well as il-1βα, thereby supporting host defence 231 and offers a novel therapeutic approach, which will need to be investigated in clinical trials. together, these findings highlight the distinct phenotype of ams during mtb infection, which counteracts intracellular infection through aerobic glycolysis, but is also heavily exploited by mtb bacteria feeding on host lipids and iron. targeting metabolism during chronic lung disease many potential targets have been identified recently that could rewire macrophage metabolic and phenotypic changes driving chronic lung disease. since all cells depend on oxidative phosphorylation or cytoplasmic glycolysis to synthesize atp, there is the potential for unwanted side effects by targeting specific metabolic processes. however, it is becoming increasingly apparent that it is possible to safely target metabolic pathways in patients. for example, dimethyl fumarate, a known regulator of macrophage phenotype, is a first-line-treatment for relapsingremitting multiple sclerosis 232 . indeed, metabolic processes are highly plastic with significant redundancy, modulation of these processes may have the added benefit of selectively targeting cells with high metabolic demands 233 . targeted delivery to ams may add another layer of selectivity, improving efficacy, sustained drug release and evading capture by mucus 234 . systems for inhaled am targeted drug delivery include the use of micro-and nanocarriers, including liposomes, which are phagocytosed by ams. rifampicin-loaded microspheres as a therapeutic approach for mtb have been described 235 , and have been further refined to allow a one-step assembly for rifampicin containing microspheres 236 . recently, aerosolised delivery of sirna, which posttranslationally downregulates gene expression, has been developed to target ams specifically 237 , whilst mannose coated microspheres have been developed which exploit the phagocytotic activity of ams 238 . many of these delivery vehicles have been developed to transport antibiotics targeting intracellular am bacterial infections, which are helpful for treating tb, however other drugs could be incorporated into aerosolised micro-or nano delivery systems. specifically, treatment with iron chelators, antioxidants and nitrated fatty acids has shown to rewire am phenotype and improve diverse chronic lung disease; these may be ideal candidates to develop novel, aerosolised vehicle-assisted drug delivery to ams during chronic lung disease. in the last decade enormous strides have been made regarding our understanding of how adaptations in metabolic pathways underlie macrophage phenotype and function. ams are remarkably plastic cells, orchestrating not only pathogen defence and efferocytosis, but also pulmonary tolerance and resolution. it has become increasingly clear that ams tailor their metabolic profile to fit their local niche generating ros for pathogen defence, utilising aerobic glycolysis to rapidly generate cytokines, employing the tca cycle to fuel inflammatory responses and generating metabolites with secondary signalling functions such as citrate, itaconate, succinate and fumarate. work elucidating the complexities of am metabolic alterations in the context of clds has highlighted many potential therapeutic targets (summarized in table 1 ). indeed, a lack of understanding of shared cellular mechanisms, which underlie clds has been a major obstacle in respiratory biology; identification of common am-metabolic pathways/metabolites which directly influence core features of clds would be a significant advance on the route to devising new am-directed strategies to treat pulmonary diseases which affect millions worldwide. lung homeostasis: influence of age, microbes, and the immune system pulmonary macrophages: key players in the innate defence of the airways monocytes and macrophages: developmental pathways and tissue homeostasis metabolic reprograming in macrophage polarization macrophage immunometabolism: where are we (going)? metabolic disorders in chronic lung diseases alveolar macrophage immunometabolism and lung function impairment in smoking and chronic obstructive pulmonary disease alveolar macrophages: plasticity in a tissue-specific context alveolar macrophage in the driver's seat integrin αvβ6: structure, function and role in health and disease pulmonary macrophages: a new therapeutic pathway in fibrosing lung disease? flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung identification of myeloid cell subsets in murine lungs using flow cytometry lung environment determines unique phenotype of alveolar macrophages a critical function for cd200 in lung immune homeostasis and the severity of influenza infection a lineage of myeloid cells independent of myb and hematopoietic stem cells alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages the lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation tissue-resident macrophage ontogeny and homeostasis developmental origin of lung macrophage diversity the fate and lifespan of human monocyte subsets in steady state and systemic inflammation monocyte recruitment during infection and inflammation transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages cellular chimerism of the lung after transplantation: an interphase cytogenetic study long-term persistence of human donor alveolar macrophages in lung transplant recipients long-term persistence of donor alveolar macrophages in human lung transplant recipients that influences donor specific immune responses the human alveolar macrophage direct evidence for a bone marrow origin of the alveolar macrophage in man dynamics of human monocytes and airway macrophages during healthy aging and after transplant human monocyte subsets are transcriptionally and functionally altered in aging in response to pattern recognition receptor agonists aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function agedependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults succinate is an inflammatory signal that induces il-1β through hif-1α succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization metabolic reprogramming in macrophages and dendritic cells in innate immunity tissue-resident alveolar macrophages do not rely on glycolysis for lps-induced inflammation induction of the nuclear receptor ppar-γ 3 by the cytokine gm-csf is critical for the differentiation of fetal monocytes into alveolar macrophages pulmonary surfactant protein a modulates the cellular response to smooth and rough lipopolysaccharides by interaction with cd14 gm-csf regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense gm-csf regulates alveolar macrophage differentiation and innate immunity in the lung through targeted pparγ deficiency in alveolar macrophages disrupts surfactant catabolism familial pulmonary alveolar proteinosis caused by mutations in csf2ra adult-onset hereditary pulmonary alveolar proteinosis caused by a single-base deletion in csf2rb hereditary pulmonary alveolar proteinosis caused by recessive csf2rb mutations pulmonary alveolar proteinosis caused by deletion of the gm-csfrα gene in the x chromosome pseudoautosomal region protective role of the lung collectins surfactant protein a and surfactant protein d in airway inflammation surfactant protein a regulates complement activation surfactant protein a directly interacts with tlr4 and md-2 and regulates inflammatory cellular response: importance of supratrimeric oligomerization membrane-tethered muc1 mucin counter-regulates the phagocytic activity of macrophages muc5b is required for airway defence macrophages are related to goblet cell hyperplasia and induce muc5b but not muc5ac in human bronchus epithelial cells a common muc5b promoter polymorphism and pulmonary fibrosis multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study the respiratory tract microbiome and lung inflammation: a two-way street respiratory microbiome and epithelial interactions shape immunity in the lungs the microbiome of the upper respiratory tract in health and disease the nasal cavity microbiota of healthy adults butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of ampactivated protein kinase in caco-2 cell monolayers understanding the holobiont: how microbial metabolites affect human health and shape the immune system the microbiota of the respiratory tract: gatekeeper to respiratory health immunometabolism: cellular metabolism turns immune regulator the european respiratory society. the burden of lung disease asthma-copd overlap syndrome: pathogenesis, clinical features, and therapeutic targets. 1-14 integrated genomics reveals convergent transcriptomic networks underlying chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis the pathogenesis of copd and ipf: distinct horns of the same devil? virus-induced exacerbations in asthma and copd pulmonary health effects of air pollution lung macrophages contribute to house dust mite driven airway remodeling via hif-1α type 2 innate lymphoid cells in induced sputum from children with severe asthma irf5 promotes inflammatory macrophage polarization and t h1-th17 responses local macrophage proliferation, rather than recruitment from the blood, is a signature of th2 inflammation a critical role for irf5 in regulating allergic airway inflammation human asthma is characterized by more irf5+ m1 and cd206+ m2 macrophages and less il-10+ m2-like macrophages around airways compared with healthy airways defective apoptotic cell phagocytosis attenuates prostaglandin e 2 and 15-hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages interleukin-1β causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung enhanced reactive oxygen species metabolism of airspace cells and airway inflammation follow antigen challenge in human asthma role of nadph oxidase / ros in pro-inflammatory mediatorsinduced airway and pulmonary diseases impact of oxidative stress on lung diseases up-regulation of heme oxygenase-1 in alveolar macrophages of newly diagnosed asthmatics fuelling the mechanisms of asthma: increased fatty acid oxidation in inflammatory immune cells may represent a novel therapeutic target sulfidopeptide leukotrienes contribute to human alveolar macrophage activation in asthma increased generation of the arachidonic metabolites ltb4 and 5-hete by human alveolar macrophages in patients with asthma: effect in vitro of nedocromil sodium undefined airway responsiveness to histamine and leukotriene e4 in subjects with aspirin-induced asthma synthesis and metabolism of leukotrienes in γ-glutamyl transpeptidase deficiency arginase: marker, effector, or candidate gene for asthma? the involvement of tyrosine kinases, cyclic amp/protein kinase a, and p38 mitogen-activated protein kinase in il-13-mediated arginase i induction in macrophages: its implications in il-13-inhibited nitric oxide production the effect of inhaled 15-(s)-hydroxyeicosatetraenoic acid (15-hete) on airway calibre and non-specific responsiveness in normal and asthmatic human subjects corticosteroid suppression of lipoxin a4and leukotriene b4from alveolar macrophages in severe asthma single oral dose of prednisone decreases leukotriene b4 production by alveolar macrophages from patients with nocturnal asthma but not control subjects: relationship to changes in cellular influx and fev1 a novel thiol compound, n-acetylcysteine amide, attenuates allergic airway disease by regulating activation of nf-κb and hypoxia-inducible factor-1α a potential new therapy for asthma? orchestrating house dust mite-associated allergy in the lung benralizumab: an updated treatment of eosinophilic asthma global burden of copd: risk factors, prevalence, and future trends chronic obstructive pulmonary disease: current burden and future projections new insights into the immunology of chronic obstructive pulmonary disease roles of myeloid and lymphoid cells in the pathogenesis of chronic obstructive pulmonary disease inflammatory mechanisms in patients with chronic obstructive pulmonary disease basophils trigger emphysema development in a murine model of copd through il-4-mediated generation of mmp-12-producing macrophages an immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease defective phagocytosis in airways disease the role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation impaired mitochondrial microbicidal responses in chronic obstructive pulmonary disease macrophages role of oxidants/antioxidants in smoking-induced lung diseases. free radic impairment of mitochondrial function by particulate matter (pm) and their toxic components: implications for pminduced cardiovascular and lung disease diminished immunoreactivity of γ-glutamylcysteine synthetase in the airways of smokers' lung the iron-y of iron overload and iron deficiency in chronic obstructive pulmonary disease increased iron sequestration in alveolar macrophages in chronic obtructive pulmonary disease defective bacterial phagocytosis is associated with dysfunctional mitochondria in copd macrophages mitochondrial dysfunction in macrophages: a key to defective bacterial phagocytosis in copd alveolar macrophage immunometabolism and lung function impairment in smoking and chronic obstructive pulmonary disease increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways alterations in adenosine metabolism and signaling in patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis the mif antagonist iso-1 attenuates corticosteroid-insensitive inflammation and airways hyperresponsiveness in an ozone-induced model of copd targeting nrf2 signaling improves bacterial clearance by alveolar macrophages in patients with copd and in a mouse model mitochondrial iron chelation ameliorates cigarette smokeinduced bronchitis and emphysema in mice cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine progress in understanding mucus abnormalities in cystic fibrosis airways lung inflammation in cystic fibrosis: pathogenesis and novel therapies inflammation in cystic fibrosis: an update inflammation and its genesis in cystic fibrosis alveolar macrophages and cc chemokines are increased in children with cystic fibrosis inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis azithromycin reduces exaggerated cytokine production by m1 alveolar macrophages in cystic fibrosis macrophages directly contribute to the exaggerated inflammatory response in cystic fibrosis transmembrane conductance regulator-/-mice cftr-dependent defect in alternatively-activated macrophages in cystic fibrosis characterization of macrophage activation states in patients with cystic fibrosis pivotal advance: expansion of small sputum macrophages in cf: failure to express marco and mannose receptors tgf-β1 inhibits cftr biogenesis and prevents functional rescue of δf508-cftr in primary differentiated human bronchial epithelial cells series 'matrix metalloproteinases in lung health and disease': the role of matrix metalloproteinases in cystic fibrosis lung disease glutathione and infection systemic deficiency of glutathione in cystic fibrosis iron accumulates in the lavage and explanted lungs of cystic fibrosis patients transforming growth factorβ activation in the lung: focus on fibrosis and reactive oxygen species impaired defenses of neonatal mouse alveolar macrophage with cftr deletion are modulated by glutathione and tgfβ1 human cystic fibrosis macrophages have defective calciumdependent protein kinase c activation of the nadph oxidase, an effect augmented by burkholderia cenocepacia irg1 expression in myeloid cells prevents immunopathology during m. tuberculosis infection pseudomonas aeruginosa utilizes host-derived itaconate to redirect its metabolism to promote biofilm formation pulmonary pathogens adapt to immune signaling metabolites in the airway lipid metabolism in cystic fibrosis alterations in immune response and ppar/lxr regulation in cystic fibrosis macrophages reduced 15-lipoxygenase 2 and lipoxin a4/leukotriene b4 ratio in children with cystic fibrosis pro-resolving lipid mediator resolvin d1 serves as a marker of lung disease in cystic fibrosis metabolic reprograming of cystic fibrosis macrophages via the ire1α arm of the unfolded protein response results in exacerbated inflammation glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis elevated mirc1/mir17-92 cluster expression negatively regulates autophagy and cftr (cystic fibrosis transmembrane conductance regulator) function in cf macrophages cysteamine re-establishes the clearance of pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant f508del-cftr mutation an overview of monitoring and supplementation of omega 3 fatty acids in cystic fibrosis fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis oral dha supplementation in δf508 homozygous cystic fibrosis patients bioavailability and safety of a high dose of docosahexaenoic acid triacylglycerol of algal origin in cystic fibrosis patients: a randomized, controlled study effect of an 8-month treatment with ω-3 fatty acids (eicosapentaenoic and docosahexaenoic) in patients with cystic fibrosis long-term docosahexaenoic acid therapy in a congenic murine model of cystic fibrosis idiopathic pulmonary fibrosis: guidelines for diagnosis and clinical management have advanced from consensus-based in 2000 to evidence-based in 2011 revealing the pathogenic and aging-related mechanisms of the enigmatic idiopathic pulmonary fibrosis: an integral model macrophages: friend or foe in idiopathic pulmonary fibrosis? matrix metalloproteinase: an upcoming therapeutic approach for idiopathic pulmonary fibrosis multiplex protein profiling of bronchoalveolar lavage in idiopathic pulmonary fibrosis and hypersensitivity pneumonitis metalloproteinases in idiopathic pulmonary fibrosis accumulation of damaged mitochondria in alveolar macrophages with reduced oxphos related gene expression in ipf metabolic characterization and rna profiling reveal glycolytic dependence of profibrotic phenotype of alveolar macrophages in lung fibrosis glucose transporter-1 distribution in fibrotic lung disease: association with [18f]-2-fluoro-2-deoxyglucose-pet scan uptake, inflammation, and neovascularization tracing compartmentalized nadph metabolism in the cytosol and mitochondria of mammalian cells succinate: a metabolic signal in inflammation immunoresponsive gene 1 and itaconate inhibit succinate dehydrogenase to modulate intracellular succinate levels itaconate is an anti-inflammatory metabolite that activates nrf2 via alkylation of keap1 itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation itaconate controls the severity of pulmonary fibrosis reactive oxygen species as signaling molecules in the development of lung fibrosis macrophages and iron metabolism the nox family of ros-generating nadph oxidases: physiology and pathophysiology modulation of reactive oxygen species by rac1 or catalase prevents asbestos-induced pulmonary fibrosis rac1 regulates the activity of mtorc1 and mtorc2 and controls cellular size delta-like 4 induces notch signaling in macrophages: implications for inflammation increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis production of superoxide and nitric oxide by alveolar macrophages in the bleomycin-induced interstitial pneumonia mice model mitochondrial calcium uniporter regulates pgc-1α expression to mediate metabolic reprogramming in pulmonary fibrosis iron laden macrophages in idiopathic pulmonary fibrosis: the telltale of occult alveolar hemorrhage? bronchoalveolar lavage (bal) cells in idiopathic pulmonary fibrosis express a complex pro-inflammatory, pro-repair, angiogenic activation pattern, likely associated with macrophage iron accumulation the transferrin receptor cd71 delineates functionally distinct airway macrophage subsets during idiopathic pulmonary fibrosis tollip, muc5b, and the response to n-acetylcysteine among individuals with idiopathic pulmonary fibrosis prednisone, azathioprine, and n-acetylcysteine for pulmonary fibrosis safety and tolerability of acetylcysteine and pirfenidone combination therapy in idiopathic pulmonary fibrosis: a randomised, double-blind, placebo-controlled, phase 2 trial metformin: an old dog with a new trick? metformin attenuates lung fibrosis development via nox4 suppression metformin reverses established lung fibrosis in a bleomycin model metformin does not affect clinically relevant outcomes in patients with idiopathic pulmonary fibrosis reversal of persistent fibrosis in aging by targeting nox4-nrf2 redox imbalance nitrated fatty acids reverse pulmonary fibrosis by dedifferentiating myofibroblasts and promoting collagen uptake by alveolar macrophages sars-cov-2 and viral sepsis: observations and hypotheses complex immune dysregulation in covid-19 patients with severe respiratory failure covid-19: consider cytokine storm syndromes and immunosuppression the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages h5n1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice interaction of influenza virus with mouse macrophages host defense mechanisms against influenza virus: interaction of influenza virus with murine macrophages in vitro heparin inhibits intracellular mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages pro-and anti-inflammatory cytokines in tuberculosis: a two-edged sword in tb pathogenesis mycobacterial survival strategies in the phagosome: defence against host stresses intracellular trafficking in mycobacterium tuberculosis and mycobacterium avium-infected macrophages hif-1α is an essential mediator of ifn-γ-dependent immunity to mycobacterium tuberculosis infection with mycobacterium tuberculosis induces the warburg effect in mouse lungs cutting edge: mycobacterium tuberculosis induces aerobic glycolysis in human alveolar macrophages that is required for control of intracellular bacillary replication mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes hydrogen sulfide dysregulates the immune response by suppressing central carbon metabolism to promote tuberculosis mycobacterium tuberculosis limits host glycolysis and il-1β by restriction of pfk-m via microrna-21 fumarase deficiency causes protein and metabolite succination and intoxicates mycobacterium tuberculosis an essential bifunctional enzyme in mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism the effect of the host's iron status on tuberculosis hereditary hemochromatosis results in decreased iron acquisition and growth by mycobacterium tuberculosis within human macrophages a major role for ferroptosis in mycobacterium tuberculosis-induced cell death and tissue necrosis small rna profiling in mycobacterium tuberculosis identifies mrsi as necessary for an anticipatory iron sparing response ppar-α activation mediates innate host defense through induction of tfeb and lipid catabolism growth of mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny mycobacterium tuberculosis induces the mir-33 locus to reprogram autophagy and host lipid metabolism in vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of mycobacterium tuberculosis mycobacterium bovis bacillus calmette-guérin infection induces tlr2-dependent peroxisome proliferator-activated receptor γ expression and activation: functions in inflammation, lipid metabolism, and pathogenesis mycobacterium tuberculosis activates human macrophage peroxisome proliferator-activated receptor γ linking mannose receptor recognition to regulation of immune responses liver x receptors contribute to the protective immune response against mycobacterium tuberculosis in mice statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation the effects of statin use on the development of tuberculosis among patients with diabetes mellitus desferrioxamine supports metabolic function in primary human macrophages infected with mycobacterium tuberculosis therapeutic efficacy of dimethyl fumarate in relapsingremitting multiple sclerosis associates with ros pathway in monocytes targeting immunometabolism as an anti-inflammatory strategy nanoparticle-mediated macrophage targeting -a new inhalation therapy tackling tuberculosis respirable plga microspheres containing rifampicin for the treatment of tuberculosis: screening in an infectious disease model one-step preparation of rifampicin/poly(lactic-co-glycolic acid) nanoparticle-containing mannitol microspheres using a four-fluid nozzle spray drier for inhalation therapy of tuberculosis the potential of sirna based drug delivery in respiratory disorders: recent advances and progress glycan targeted polymeric antibiotic prodrugs for alveolar macrophage infections house dust mite drives proinflammatory eicosanoid reprogramming and macrophage effector functions. allergy eur decreased expression of haem oxygenase-1 by alveolar macrophages in idiopathic pulmonary fibrosis macrophage akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis de novo fatty acid synthesis during mycobacterial infection is a prerequisite for the function of highly proliferative t cells, but not for dendritic cells or macrophages role of glutamine metabolism in host defense against mycobacterium tuberculosis infection immunometabolism within the tuberculosis granuloma: amino acids, hypoxia, and cellular respiration analyzing the impact of mycobacterium tuberculosis infection on primary human macrophages by combined exploratory and targeted metabolomics competing interests: the authors declare no competing interests.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-276732-u2d1z4ip authors: mauri, tommaso; zambelli, vanessa; cappuzzello, claudia; bellani, giacomo; dander, erica; sironi, marina; castiglioni, vittoria; doni, andrea; mantovani, alberto; biondi, andrea; garlanda, cecilia; d’amico, giovanna; pesenti, antonio title: intraperitoneal adoptive transfer of mesenchymal stem cells enhances recovery from acid aspiration acute lung injury in mice date: 2017-03-06 journal: intensive care med exp doi: 10.1186/s40635-017-0126-5 sha: doc_id: 276732 cord_uid: u2d1z4ip background: mesenchymal stem cells (mscs) might act as fine-tuners of inflammation during acute lung injury. we assessed the effects of adoptive transfer of mscs in acid aspiration acute lung injury and explored the role of long pentraxin ptx3. methods: we conducted a prospective experimental interventional study on wild-type (wt) and ptx3-deficient (ptx3(−/−)) mice. acute lung injury was induced in wt and ptx3(−/−) mice by instillation of hydrochloric acid into the right bronchus. one hour later, animals received intraperitoneal sterile phosphate-buffered saline (pbs), wt-mscs (1 × 10(6)) or ptx3(−/−)-mscs (1 × 10(6)). twenty-four hours after injury, we measured the effects of treatments on arterial blood gases, wet/dry lung weight (w/d), ct scan analysis of lung collapse, neutrophils, tnfα and cxcl1 in bronchoalveolar lavage, and plasma ptx3. d-dimer was assayed in 1 week and oh-proline in 2 weeks to track the fibrotic evolution. results: in 24 h, in comparison to pbs, wt-mscs improved oxygenation and reduced w/d and alveolar collapse. these effects were associated with decreased concentrations of alveolar neutrophils and cytokines. wt-mscs increased d-dimer concentration and decreased oh-proline levels, too. treatment with ptx3(−/−)-mscs ameliorated oxygenation, w/d, and alveolar tnfα, though to a lesser extent than wt-mscs. ptx3(−/−)-mscs did not improve lung collapse, neutrophil count, cxcl1, d-dimer, and oh-proline concentrations. the protective effects of wt-mscs were dampened by lack of endogenous ptx3, too. conclusions: in acid aspiration acute lung injury, mscs improve pulmonary function and limit fibrosis by fine-tuning inflammation. the role of ptx3 in determining mscs’ effects might merit further scrutiny. electronic supplementary material: the online version of this article (doi:10.1186/s40635-017-0126-5) contains supplementary material, which is available to authorized users. the incidence of the acute respiratory distress syndrome (ards) is elevated, and mortality in recent studies still reaches 50% for the most severe form [1] [2] [3] [4] [5] . moreover, many ards survivors develop long-term lung fibrosis, reduced respiratory function, and poor quality of life [1] . at onset, ards is characterized by severe hypoxemia and lung edema, caused by dysregulated inflammation [1, 2] . overstimulation of leukocytes, cytokine storm, and altered tissue repair are key contributors to ards severity, mortality, and long-term morbidity [3] . however, we still lack effective pharmacological therapies that fine-tune these mechanisms [4] . mesenchymal stem cells (mscs) are multi-potent cells derived from adult tissues [6] . mscs secrete multiple molecules, including anti-inflammatory cytokines, growth factors, and anti-microbial peptides, and appear as fine-tuners of host inflammation [6] . previous studies showed that mscs administration in animal models of acute lung injury increased the ability of the host to eliminate the agent, regulate neutrophil recruitment, and reverse altered lung permeability, without additional injury [7] [8] [9] [10] . in addition, intraperitoneal (i.p.) route for the administration of mscs was recently described [11] . to our knowledge, the effects of i.p. mscs have never been assessed in experimental acid aspiration acute lung injury [12] ; moreover, the effects of mscs on the fibrotic long-term evolution of acute lung injury [13] have not been described, and key molecular determinants of mscs' effects are not fully understood. in the present study, we tested in a mouse model of acid aspiration acute lung injury the effects of i.p. mscs on the early acute inflammatory reaction and on the long-term fibrotic evolution [5, 12] . moreover, we explored the role of pentraxin 3 (ptx3) in mediating mscs' effects. ptx3 is an acute-phase inflammatory mediator produced by different cell types [3, 14] that exerts protective effects in experimental acute lung injury, closely resembling those of mscs [15] . previous studies indicated that mscs produce, store, and secrete ptx3 when activated [16, 17] . the research group of dr. g. d' amico generated ptx3-deficient mscs (ptx3 −/− -mscs) [18] , which showed a significant defect in promoting tissue repair in a mice model of wound healing compared to wild-type mscs (wt-mscs) [18] . in analogy, we investigated whether ptx3 deficiency in the mscs and/or at the endogenous level might impact the ability of mscs to promote short-and longterm recovery from acid aspiration acute lung injury. the hypothesis of this study was that early treatment with mscs in a murine model of acid-induced lung injury might exert short-and long-term beneficial effects by modulation of the inflammatory response and that lack of ptx3 in mscs might reduce their efficacy. procedures involving animals and their care were conducted in conformity with the institutional guidelines complying with national and international laws and policies. the experimental protocol was submitted to the italian ministry of health and approved by the animal care unit of the university of milan-bicocca, monza, italy. mscs were isolated from female c57bl/6 wt mice and from ptx3 −/− mice by previously described procedures [18] . cryopreserved aliquots of mscs were thawed 5-7 days before the experiments, seeded at 1000-2000 cells/cm 2 , and cultured at 37°c in a 5%-co 2 atmosphere. on early morning, mscs were dethatched by trypsin and used fresh for all the experiments performed that same day. fresh mscs at passages 5 to 7 were used for the present study. recent studies showed that ptx3 −/− -mscs were similar to wt-mscs in their ability to grow spontaneously, undergo mesengenic differentiation, and express common mscs' markers [18] . as already published, ptx3 −/− -mscs drastically decreased the mitogen-induced proliferation of lymphocyte in a dose-dependent manner similarly to wt-mscs [19] . moreover, ptx3 −/− -mscs did not store or release ptx3 while they tended to produce higher levels of tumor necrosis factor-stimulated gene 6 (tsg-6) [19] compared to wt-mscs (additional file 1: figure s1 ). acid aspiration acute lung injury was induced in wt-and ptx3 −/− -mice as previously described [12] . briefly, after intubation, 1.5 ml/kg of 0.1 m hydrochloric acid was instilled into the right lung, and after 10 min, the animals were extubated and placed in an oxygenated chamber. one hour later (to reproduce possible real life clinical timing), the mice received i.p. injection of sterile phosphate-buffered saline (pbs) or 1 × 10 6 wt-mscs or 1 × 10 6 ptx3 −/− -mscs (all in equal volume of 200 μl). figure 1 shows the experimental design of the study in wt mice. the following measures were performed in all wt mice: (a) twenty-four hours after hcl instillation, the mice were sacrificed and the following analysis were performed (detailed methods are described in additional files): -arterial blood gas analysis for gas exchange -wet-to-dry ratio as index of edema -micro-ct scan to measure change over time in non-aerated lung tissue expressed as percentage of the whole lung tissue, with more negative values representing larger decrease of alveolar collapse; -histopathology examination performed according to previous study [12] evaluating alveolar serofibrinous exudate and alveolar hemorrhage -bronchoalveolar lavage for differential cell count, total protein content (with bicinchoninic acid method) and keratinocyte chemoattractant (cxcl1, previously named kc), and tumor necrosis factor-α (tnf-α) were assayed by elisa -blood withdrawal for ptx3 levels measurement in plasma (elisa assay) (b)in 1 week from lung injury d-dimer (marker of fibrinolysis) [20] and matrix metalloproteinase 13 (mmp13), an enzyme that participates in collagen degradation [21] , were detected by elisa and by western blot in lungs lysate, respectively. (c) two weeks after acid-induced lung injury, the fibrotic evolution was evaluated [22] . in particular, we performed as follows: in ptx3 −/− -mice, instead, we measured only oxygenation and wet-to-dry lung weight ratio in 24 h and oh-pro content in 2 weeks. blinded researchers performed each analysis. data are expressed as mean ± standard deviation if normally distributed and as median (interquartile range) when non-normally distributed. one-way analysis of variance (anova) or kruskal-wallis and dunnett's or dunn's post hoc tests vs. pbs group were used to assess differences between treatment effects in wt mice, as appropriate. differences in physiologic variables measured in the right vs. left lung were assessed by t test or mann-whitney u test, as appropriate. p < 0.05 was considered statistically significant. detailed methods can be found in the additional file 1 of this article. mesenchymal stem cells enhance short-and long-term recovery from experimental acid aspiration acute lung injury in 24 h, i.p. administration of wt-mscs 1 h after induction of acid aspiration acute lung injury significantly improved arterial oxygenation and decreased the alveolar-arterial oxygen gradient in wt-mice in comparison to pbs (p < 0.05 and p = 0.001, respectively) ( fig. 2a and b) , without modification of paco 2 and even in presence of slightly worse ph values (additional file 1: table s1 ). early improvement in oxygenation yielded by wt-mscs was likely obtained by reduction of lung edema: in fact, the lungs' wetto-dry ratio in 24 h was decreased by wt-mscs in comparison to pbs (p < 0.05) (fig. 2e) . similarly, micro-ct scan analysis showed that the extent of lung collapse significantly decreased between 1 and 24 h in wt mice treated by wt-mscs (p = 0.01), likely indicating decreased superimposed weight from reduced lung edema (table 1 and fig. 3 ), but not in those treated by pbs. histology performed in 24 h showed decreased disruption of lung structures in mice treated by wt-mscs in comparison to pbs (table 1) , even though this difference did not reach statistical significance. bal total protein concentrations were left unchanged by wt-mscs treatment (fig. 2f) . mice treated by wt-mscs, indeed, showed significant reduction of total cell count in bal fluid in 24 h and substantial dampening of neutrophil recruitment into the alveoli (p < 0.05 for both; fig. 2c , d) in comparison to pbs. accordingly, levels of proinflammatory cytokines (i.e., cxcl1 and tnf-α) in bal fluid were significantly reduced by wt-mscs (p < 0.05 and p < 0.01, respectively), but not by pbs (table 1) . interestingly, circulating ptx3 was reduced in wt-mice treated by wt-mscs (albeit non-significantly) and not in wt-mice treated by ptx3-deficient mscs (table 1) . in this study, we showed that treatment by i.p. wt-mscs administered 1 h after acid aspiration attenuated the evolution of fibrosis, as demonstrated by lower collagen deposition (oh-pro assay) in 2 weeks (fig. 4a ) in comparison to mice treated by pbs. in 1 week, d-dimer concentration was significantly increased in the lungs of mice treated with wt-mscs (p < 0.001, fig. 4b ) in comparison to pbs, suggesting that dampening of long-term fibrotic evolution might have followed both reduced inflammation and enhanced fibrinolysis by wt-mscs in the days after injury. in an effort to evaluate whether i.p. wt-mscs migrate systemically in mice with acid aspiration acute lung injury, we performed western blot analysis to detect gfp + wt-mscs presence in the lungs, spleen, liver, and peritoneal lavage in 24 h. additional file 1: figure s3 shows actual blots with no apparent signal of gfp + wt-mscs presence in the lungs, spleen, and liver as opposed to positive controls. in the peritoneal lavage, instead, wt-mscs were still present in 24 h but by lower intensity, probably because, as previously shown [23] , they formed aggregates and adhered to the peritoneal cavity walls. figure s1 ). in 24 h, treatment by ptx3 −/− -mscs ameliorated oxygenation only to a lesser extent than wt-mscs (fig. 2) . reduced short-term effects on oxygenation in comparison to wt-mscs were paralleled by less effective reduction of wet-to-dry lung weight ratio by ptx3 −/− -mscs (fig. 2a, b , e) and the absence of effects of ptx3deficient cells on radiological signs of regional lung collapse and edema (table 1) . histology found reduction of lung injury, albeit non-significant (table 1 ). in summary, ptx3 −/− -mscs seemed less effective than wt-mscs in limiting formation of lung edema in 24 h after acid aspiration. at variance from wt-mscs, treatment with ptx3 −/− -mscs did not reduce total cell and neutrophil count (fig. 2) as well as cxcl1 levels in the alveolar space (table 1) . thus, the more limited effectiveness of ptx3 −/− -mscs in enhancing lung recovery after acid aspiration acute lung injury might have been related to ineffective reduction of the acute inflammatory processes. moreover, ptx3 −/− -mscs could not modulate fibrinolysis in the days following injury nor impact the long-term fibrotic evolution, as demonstrated by unchanged levels of d-dimer and oh-proline in comparison to pbs (fig. 4a, b) . however, wt-and ptx3 −/− -mscs did not seem to modulate activity of mmp13 in 1 week (additional file 1: figure s2 ) to impact remodeling and fibrosis. effects of study treatments on ptx3 knockout mice with acid aspiration acute lung injury extent of lung injury was similar between wt and ptx3 −/− mice (t test in 24 h in wtmice + pbs vs. ptx3 −/− -mice + pbs: pao 2 , p = 0.151; wet-to-dry lung weight, p = 0.099). when administered to ptx3 −/− -mice: wt-mscs improved lung function and reduced fibrosis, but the difference with pbs was non-significant (additional file 1: table s3 ); ptx3 −/− -mscs induced a further non-significant reduction of the alveolararterial gradient and of wet-to-dry lung weight ratio, while pao 2 and fibrosis worsened in comparison to wt-mscs (additional file 1: table s3 ). thus, endogenous ptx3 might collaborate in the protective effects of wt-mscs from fibrosis, while it might limit their effectiveness in reducing lung edema. more results are provided in the additional files of this article. study's main findings can be summarized as follows: wt-mscs dampen short-and long-term sequelae of acid aspiration acute lung injury in mice in terms of improved oxygenation, reduced edema causing lung collapse, and reduced fibrotic evolution, likely by fine-tuning the acute inflammatory reaction and the subsequent fibrinolysis and tissue repair process; moreover, lack of ptx3 gene in mscs and in the injured host might reduce the beneficial effects of mscs. in the present study, we administered i.p. wt-mscs 1 h after intratracheal instillation of hydrochloric acid, potentially reproducing real-life treatment of ards caused by aspiration of gastric contents [1, 12] , one of the major direct causes of ards [24, 25] with a mortality rate around 35-40% and significant long-term fibrosis [1] . in 24 h from injury, we could show multiple short-term beneficial effects of wt-mscs: as previously shown [7, 26] , mscs seemed to reduce the early inflammatory reaction in the lungs and to avoid excessive response and additional damage. in our study, indeed, mscs dampened leukocyte trafficking through the alveolar-epithelial barrier as well as their activation and release of primary inflammatory cytokines. in turn, as testified by oxygenation, wet-to-dry and ct scan data, this led to decreased accumulation and/or improved clearance of lung edema and inflammatory cells in the alveolar and thirdspace compartments and to attenuated extent of alveolar collapse. however, histology did not improve after wt-mscs administration, maybe due to insufficient numerosity; similarly, protein content in bal was not reduced by mscs, but this could have followed direct extravasation after acid-induced physical disruption of the alveolar-epithelial integrity. in our model, both lungs showed physiologic alterations, thus indicating that the left lung could completely compensate for the ventilation needs of the animals (additional file 1: table s2 ) [27] . in 2 weeks from acute lung injury onset, we also showed decreased long-term collagen deposition in the lungs associated with treatment by wt-mscs. moreover, the long-term reduction of fibrosis was preceded by increased fibrinolysis in 1 week. our data, in keeping with recent literature [7, 9, 20] , seem to suggest that the beneficial effects of mscs on the fibrotic evolution of acute lung injury might include reduction of the acute-phase inflammatory reaction and reduced fibrosis in 2 weeks. moreover, decreased respiratory effort during the early phases induced by improved gas exchange could have reduced interstitial lung edema [28] and the risk of additional ventilationinduced lung injury (vili) and fibrosis [13] . in summary, it would be tempting to say that ours and the previous data indicate that mscs might be regarded as personalized modular therapies limiting short-and long-term acute lung injury severity by finetuning inflammation and tissue remodeling. however, to date, whether these hypotheses hold true and will translate in improved mortality and long-term quality of life in human ards remains to be determined. ptx3 is a marker of severity in human ards [29] , and experimental models showed that ptx3 is as key determinant of the evolution, morbidity, and mortality of acute lung injury [30, 31] . a recent study by cappuzzello and colleagues showed that while wt-mscs improved tissue repair in experimental wound healing, ptx3 −/− -mscs could not [18] . similarly, we showed that the early dampening of leukocyte migration and release of pro-inflammatory cytokines in the injured lungs by wt-mscs could not be replicated when ptx3 −/− -mscs were adopted. this likely led to poorer effects on oxygenation and wet/dry ratios and no improvement in the ct scan analysis of lung collapse as well as no decrease in inflammatory cells and acute-phase primary cytokines in the bal. the positive effect of ptx3 −/− -mscs treatment on the tnf-α levels may depend on the anti-inflammatory role of tsg6 [32, 33] , which is highly expressed in ptx3 −/− -msc (additional file 1: figure s1 ). previous studies in a mice model of bilateral acid aspiration lung injury showed that interaction between ptx3 and pselectin is crucial for regulation of leukocyte recruitment with consequences on cytokine production and lung injury [23] , and similar mechanisms might underlie lack of lung protection by ptx3 −/− -mscs. moreover, we described that long-term fibrinolysis and subsequent fibrotic evolution could not be prevented by ptx3 −/− -mscs, possibly suggesting ptx3-mediated enhancement of lung tissue repair by wt-mscs [18, 20] . on the other hand, our data indicate that the beneficial effects exerted by wt-mscs are associated with a reduction in plasma ptx3, as if improvement of lung injury preceded modification of endogenous ptx3 production. however, lack of endogenous ptx3 seemed to reduce wt-mscs effects (additional file 1: table s3 ), maybe by impairment of local cell-to-cell interaction. our results do not generate a clear hypothesis on the role of ptx3 as molecular determinant of the lung protection exerted by mscs, and further studies are warranted, maybe exploring other etiologies and time-points. in our study, we could not detect presence of wt-mscs in the liver, spleen, or lungs in 24 h, while in keeping with previous findings [34] , a signal was still present in peritoneal lavage (additional file 1: figure s3 ). on the other hand, since levels of circulating ptx3 were lower and lung fibrinolysis was increased after administration of wt-mscs, we might speculate possible migration and direct effect of mscs at the site of injury but this cannot be concluded with any confidence. in summary, our data are not definitive to elucidate whether i.p. wt-mscs act through paracrine vs. direct mechanisms. this study suffers by a number of relevant limitations: as most of the measures required sacrifice of the animals (e.g., bal), we could not assess in the same animal all the effects at different time-points but each effect was assessed in a subset of animals receiving the same injury and therapy, which might have introduced some heterogeneity. we only examined three time-points (i.e., 24 h and 1 and 2 weeks), which might have prevented us from description of other effects of wt-mscs or ptx3 −/− -mscs in acid aspiration acute lung injury. apart from resources limitation, our choice was based on previous observations on the time-course of the studied animal model [12] . we described reduced effectiveness of wt-mscs induced by lack of ptx3 only in a murine model of non-infective acid aspiration lung injury and translation of these findings to other etiologies (e.g., infective pulmonary ards) and/or to the clinical setting warrants extreme caution. while we could determine significant effects of ptx3 presence in mscs in 1 week to modulate fibrosis, the downstream effects of ptx3 presence in mscs during the early acute phase (e.g., modulation of leukocyte recruitment by binding with p-selectin) remains to be elucidated. we did not evaluate the alteration of the alveolar-capillary permeability from a more molecular point of view (such as expression of tight or adherent junction proteins), but only by measuring the lung edema following such alterations (wet-to-dry ratio and the ct scan analysis). besides the standard histological analysis, we did not perform more quantitative approach using stereological assessment of the tissue injury, and this might have limited our possibilities to describe more significant differences. this is a preliminary study: the small number of mice used and lack of other administration routes could have reduced significant differences. the experiments on ptx3 −/− mice were subsequent and separate from those on wt mice. finally, volume of fluid instilled i.p. (i.e., 200 μl) might have induced cardiovascular impairment favoring pulmonary edema. the results presented here suggest that i.p. adoptive transfer of mscs enhances shortand long-term lung recovery when cells are administered 1 h after onset of acute lung injury. ptx3, an acute-phase inflammatory mediator, might play a role in the lung protection exerted by mscs, in particular against fibrosis, but this needs further clarification. studies on the molecular mechanisms of actions of mscs as well as on the risks associated with their administration should still proceed in parallel with ongoing translational studies [35] . in particular, ptx3 genetic polymorphisms have been associated with risk of microbial infections [36] [37] [38] , and it will be important to assess whether ptx3 polymorphisms are associated with outcome in ards and in clinical trials aimed to assess the potential of mscs. the acute respiratory distress syndrome soluble receptor for advanced glycation end-products predicts impaired alveolar fluid clearance in acute respiratory distress syndrome subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials treating ards: new hope for a tough problem angiotensin-(1-7) improves oxygenation, while reducing cellular infiltrate and fibrosis in experimental acute respiratory distress syndrome human mesenchymal stem cell microvesicles for treatment of escherichia coli endotoxin-induced acute lung injury in mice stem cell therapy for acute respiratory distress syndrome: a promising future allogenic human mesenchymal stem cells for treatment of e. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung effects of intratracheal mesenchymal stromal cell therapy during recovery and resolution after ventilator-induced lung injury human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebocontrolled pilot study lung injury and recovery in a murine model of unilateral acid aspiration: functional, biochemical, and morphologic characterization mechanical ventilationassociated lung fibrosis in acute respiratory distress syndrome: a significant contributor to poor outcome alveolar pentraxin 3 as an early marker of microbiologically confirmed pneumonia: a threshold-finding prospective observational study the yin-yang of long pentraxin ptx3 in inflammation and immunity characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation proteomic analysis of tumor necrosis factor-alphainduced secretome of human adipose tissue-derived mesenchymal stem cells mesenchymal stromal cell-derived ptx3 promotes wound healing via fibrin remodeling mesenchymal stem cells: mechanisms of potential therapeutic benefit in ards and sepsis an acidic microenvironment sets the humoral pattern recognition molecule ptx3 in a tissue repair mode phenotypic overlap between mmp-13 and the plasminogen activation system during wound healing in mice inhibition of pulmonary fibrosis by the chemokine ip-10/cxcl10 intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs aspiration-induced lung injury epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice unilateral acid aspiration augments the effects of ventilator lung injury in the contralateral lung spontaneous effort during mechanical ventilation: maximal injury with less positive endexpiratory pressure elevated plasma and alveolar levels of soluble receptor for advanced glycation endproducts are associated with severity of lung dysfunction in ards patients protective effects of long pentraxin ptx3 on lung injury in a severe acute respiratory syndrome model in mice long pentraxin ptx3 deficiency worsens lps-induced acute lung injury anti-inflammatory protein tsg-6 secreted by activated mscs attenuates zymosan-induced mouse peritonitis by decreasing tlr2/nf-κb signaling in resident macrophages intravenous hmscs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein tsg-6 regulation of leukocyte recruitment by the long pentraxin ptx3 mesenchymal stem (stromal) cells for treatment of ards: a phase 1 clinical trial genetic variations affect the risk of pseudomonas aeruginosa airway colonization in cystic fibrosis patients genetic ptx3 deficiency and aspergillosis in stem-cell transplantation implementation of a pan-genomic approach to investigate holobiont-infecting microbe interaction: a case report of a leukemic patient with invasive mucormycosis we are thankful to all the personnel working in the university of milan-bicocca, san gerardo hospital and humanitas institute for the continuous support and collaboration. the datasets supporting the conclusions of this article are included within the article (and its additional file). authors' contributions tm, vz, cg, gd, and ap made substantial contributions to the conception and design of the work. all authors performed the data acquisition, analysis, and interpretation for the work. tm, vz, cc, am, ab, cg, gd, and ap drafted the work and revised it critically for important intellectual content. the final approval of the version submitted for publication was carried out by all authors. tm, vz, and ap established the accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors read and approved the final manuscript. key: cord-031033-v4yetn4f authors: martin-loeches, ignacio; dickson, robert; torres, antoni; hanberger, håkan; lipman, jeffrey; antonelli, massimo; de pascale, gennaro; bozza, fernando; vincent, jean louis; murthy, srinivas; bauer, michael; marshall, john; cilloniz, catia; bos, lieuwe d. title: the importance of airway and lung microbiome in the critically ill date: 2020-08-31 journal: crit care doi: 10.1186/s13054-020-03219-4 sha: doc_id: 31033 cord_uid: v4yetn4f during critical illness, there are a multitude of forces such as antibiotic use, mechanical ventilation, diet changes and inflammatory responses that could bring the microbiome out of balance. this so-called dysbiosis of the microbiome seems to be involved in immunological responses and may influence outcomes even in individuals who are not as vulnerable as a critically ill icu population. it is therefore probable that dysbiosis of the microbiome is a consequence of critical illness and may, subsequently, shape an inadequate response to these circumstances. bronchoscopic studies have revealed that the carina represents the densest site of bacterial dna along healthy airways, with a tapering density with further bifurcations. this likely reflects the influence of micro-aspiration as the primary route of microbial immigration in healthy adults. though bacterial dna density grows extremely sparse at smaller airways, bacterial signal is still consistently detectable in bronchoalveolar lavage fluid, likely reflecting the fact that lavage via a wedged bronchoscope samples an enormous surface area of small airways and alveoli. the dogma of lung sterility also violated numerous observations that long predated culture-independent microbiology. the body’s resident microbial consortia (gut and/or respiratory microbiota) affect normal host inflammatory and immune response mechanisms. disruptions in these host-pathogen interactions have been associated with infection and altered innate immunity. in this narrative review, we will focus on the rationale and current evidence for a pathogenic role of the lung microbiome in the exacerbation of complications of critical illness, such as acute respiratory distress syndrome and ventilator-associated pneumonia. the normal microbiota is the ecological communities of commensal, symbiotic and pathogenic microorganisms whilst the microbiome comprises all of the genetic material within a microbiota (the entire collection of microorganisms in a specific niche, such as the human gut). this can also be referred to as the metagenome of the microbiota [1, 2] . approximately 100 billion microorganisms are found in the body due to recent discoveries in molecular analysis such as next-generation sequencing (ngs) and whole metagenome shotgun sequencing (wmgs); there is an increasing body of evidence pointing towards the dysbiosis that is often defined as an 'imbalance' in the microbial community that is associated with disease [3] [4] [5] . a microbiome is shaped by multiple factors including the resident flora of the animate or inanimate vicinity and the external forces that modulate this flora [6] . it becomes a changeable reflection of diversity, and so its study can provide valuable insights into the factors that drive that diversity [7] . just as the study of global climate or the roots of language requires input from around the world, so the interpretation of the microbiome of an individual or a group of patients needs comprehensive comparative data to generate insight [8, 9] . the variability of the host microbiome-either in an individual patient over time in response to the pressures of illness [10] or in a geographically localized population in response to environmental-can yield important insight into factors that can be manipulated to improve clinical outcomes. such factors include risk of infection, emergence of resistance, spread from the environment, host susceptibility and even the resilience of the health care system [11] . in this narrative review, we will focus on the rationale and current evidence for a pathogenic role of the lung microbiome in the exacerbation of complications of critical illness, such as acute respiratory distress syndrome (ards) and ventilator-associated pneumonia (vap). though for years textbooks taught that 'the normal lung is free from bacteria', this dogma was generally repeated without citation or argument [12] . in retrospect, this claim of lung sterility was remarkable: virtually no environment on earth exists that is so extreme in temperature, ph, salinity or nutrient scarcity that microbial communities cannot be detected [13] . yet for more than a century, it was taken as fact that the warm, wet mucosa of the lower respiratory tract-mere inches below the microbial reservoir of the pharynx-is an exception to this rule [14] [15] [16] [17] [18] . each individual has a unique microbiota profile that plays many specific functions in host nutrient metabolism, maintenance of structural integrity and protection against pathogens. there is not a unique optimal microbiota composition as it can be different for each individual [19, 20] . thus, the 'revolution' in culture-independent microbiology has merely confirmed with certainty what has long been inferred indirectly: human lungs are constantly exposed to environmental bacteria. to date, more than 30 studies have used sensitive, culture-independent techniques to study lung bacteria in healthy volunteers, and none has failed to detect a distinct bacterial signal [21] . the viability of bacteria in healthy lungs has been confirmed via advanced cultivation [22] and indirectly validated via correlation with healthy alveolar immune tone in humans and mice [23, 24] . some of the confusion regarding the existence of lung microbiota reflects flawed parallels with the lower gut microbiome, which represents a wholly different ecosystem with radically different ecologic forces. whereas the gut lumen is densely populated by dense communities' bacteria, lung microbiota is scarce and associated with mucosal surfaces. whereas gut communities are relatively stable dayto-day, reflecting stable selective pressure on resident bacteria, lung communities are in constant turnover, with their identities and burdened determined by the relative balance of immigration (via microaspiration and mucosal dispersion) and elimination (via cough and mucociliary clearance). whereas the gut microbiome is nutrient-rich and characterized by intense metabolic competition amongst dense communities, the lung microenvironment is nutrient-poor, and the primary competition is between immigrating pharyngeal microbes and locally calibrated alveolar and airway host defences attempting to minimize their outgrowth [24, 25] . these ecologic differences between the lower gut and the lungs erode somewhat in conditions of acute and chronic disease: the influx of mucus and proteinrich oedema provide nutrient sources for bacteria, and once-transient bacteria become resident, shaped by selective pressure. further confusion arose via misinterpretation of clinical culture protocols, which have been optimized for detection of respiratory pathogens, not the 'background' microbiota of uninfected patients. sequencing-based studies have revealed that the normal microbiota of healthy lungs closely resembles that of the oropharynx [26] [27] [28] and, whilst commonly cultured, are routinely dismissed by clinical microbiology laboratories as 'normal oral flora'. bronchoscopic studies have revealed that the carina represents the densest site of bacterial dna along healthy airways, with a tapering density with further bifurcations [28] . this likely reflects the influence of micro-aspiration as the primary route of microbial immigration in healthy adults. though bacterial dna density grows extremely sparse at smaller airways, bacterial signal is still consistently detectable in bronchoalveolar lavage fluid, likely reflecting the fact that lavage via a wedged bronchoscope samples an enormous surface area of small airways and alveoli. bacterial communities within the lungs of healthy volunteers are relatively homogenous; the bacteria of a given individual's right middle lobe far more closely those of the same individual's left upper lobe than do other individuals' right middle lobe (i.e. intraindividual similarity is greater than interindividual similarity) [27] . how to study the lung microbiome? high densities of bacteria are always present on the skin, in the mouth, and in the upper respiratory tract. for this reason, it is important to avoid contamination with commensal bacteria from other sites when taking samples for investigation of the lower respiratory tract microbiome [29, 30] . since samples from the lower respiratory tract may have a low biomass, it increases the risk for contamination that can occur at any time from sampling to sequencing [31, 32] . the first molecular techniques used for studying the bacterial microbiome in humans were based on 16s rrna gene sequencing many years ago which is an appropriate method to assess diversity on taxonomic levels above species level. a limitation of 16s rrna gene sequencing is that whilst bacteria can normally be identified on genus and family level, species identification usually requires simultaneous evaluation of several genes [33] [34] [35] . newer technology of whole genome sequencing and metagenomics has shown better definition of the gut microbiome and what has currently been shown of the lung microbiome will also be significantly updated by these newer sequencing technologies [36] [37] [38] . an important matter is that when studying the lung microbiome, the pathogens and host response needs to be simultaneously studied by molecular methods, for instance, microbial metagenomics and transcriptomics. langelier et al. [39] performed in almost 100 patients with acute respiratory failure (arf) metagenomic nextgeneration sequencing (mngs) on endotracheal aspirates (eta) and simultaneously assessed pathogens, the airway microbiome and the host transcriptome. this study found that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome and host transcriptome represents a new tool for diagnosis in lower respiratory tract infections (lrti). the progress in molecular microbiology has developed very fast in the last years and several rapid technologies will provide biological signals taking into account the interaction of the host (e.g. via digital enzyme-linked immunosorbent assay (elisa) [40] ) and the microbes (e.g. via nanorod-pcr [41] ). another technology is microgas chromatography for the analysis of bacterial function and virulence and metabolic indices of the host response on exhaled breath [42, 43] . the field of lung microbiome is no longer limited by the speed of sequencing, processing, or measurement, but rather our ability to make sense of the highdimensional data we generate. ards is a complication of critical illness characterized by protein-rich pulmonary oedema, hypoxaemia and alveolar inflammation. alveolar inflammation, damage and subsequent oedema may be initiated by a change in pulmonary microbiome, or a change in lung microbiome may be initiated by an alveolar nutrient available after the onset of oedema [2] . even though ards is traditionally not considered to be related to microbial changes in the lung, these physiological considerations resulted in the hypothesis that pathogenic bacteria may be present in the lung of patients with ards. kyo et al. [44] analysed the lung microbiome from the bronchoalveolar lavage fluid (balf) of patients with ards found that lung bacterial burden (16s rrna gene copy numbers tended to be increased) tended to be increased, and the alpha diversity (copy numbers and relative abundance of betaproteobacteria) was significantly decreased in ards patients. in an experimental mouse model of lung injury following abdominal sepsis induced by cecal ligation and puncture, the lung microbiome was enriched with gut bacteria [3] . how did these bacteria get there? it is hypothesized that bacteria can translocate from the gut into the lymphatic system and portal circulation during critical illness [4] . if so, these changes should also be observed in patients on the icu. indeed, enrichment of gut bacteria was also observed in balf from ards patients [3] . gut bacteria and more specifically enterobacterieae enrichment in patients with ards were confirmed in a second observational cohort study [5] . both studies were performed in a selective cohort of patients with potential biases of prolonged antibiotic exposure before measurement. in a more recent study conducted in europe, patients who were treated with selective decontamination of the digestive tract (sdd) during admission at the icu, but were not treated with antibiotics prior to icu admission, validated the specific enrichment of enterobacterieae in the lungs of ards patients [45] . taken together, the current body of evidence suggests that amplification of enterobacterieae in the lung is strongly associated with ards. this association is not sufficiently explained by potential confounders such as geographical location of sampling, exposure to antibiotic therapy, amplification protocols or exact definitions of ards. the evidence for consistent dysbiosis in lung microbiome is actually stronger for ards than for most other respiratory diseases, where other microbes are enriched in different studies. however, no causal link between dysbiosis of the lung microbiome and development of lung injury has been established. this link needs to be further explored before we can conclude that lung microbiome dysbiosis is a potential target for treatment (fig. 1) . in ecological terms, pneumonia can be described as the collapse of local microbiome diversity and the emergence of a dominant pathogen [46] . several studies have therefore hypothesized that the lung changes considerably during nosocomial lower respiratory tract infections. some critically ill patients can develop pneumonia due to their clinical condition such as patients with ischaemic stroke and/or with loss of neurological control of the respiratory system. these clinical conditions can be associated to reduced airway clearance and increased bacterial translocation and therefore can develop more often respiratory infections [47] . so, the more appropriate question is 'do patients that develop pneumonia have more dysbiosis of the lung microbiome than mechanically ventilated icu patients who do not develop pneumonia'? two studies addressed this problem. the first included consecutive patients at risk for pneumonia with a duration of mechanical ventilation of more than 7 days [48] . endotracheal aspirates were performed every third day and the microbial composition was evaluated with 16s sequencing. there was a small, but significant increase in the change in beta-diversity (change in diversity of species from one environment to another) in patients who went on to develop pneumonia as compared to patients who did not develop any signs of infection and were not colonized by any bacteria according to traditional bacterial cultures. the composition of the microbiome in these patients also showed a slight enrichment of pseudomonadales. a second study conducted had a similar design and showed no difference in the change of microbiome during mechanical ventilation between patients who did and did not develop pneumonia [49] . as discussed in the accompanying editorial, the results from these studies have elegantly shown that it is time to let go of any simplistic view of vap pathogenesis [10] . one conclusion might be that lrti cannot simply be defined as a collapse of bacterial ecology as this is present also in part of the patients without pneumonia who do not show any signs of pneumonia. one could also argue that the studies did not sample the alveolar space and additional studies with balf are needed to confirm or discard these findings. furthermore, evaluation of microbial composition may be more useful in establishing the presence of a pathogen in patients who already have a clinical suspicion of pneumonia. indeed, with pre-test probability, metagenomics may provide valuable information on the pathogen causing pneumonia [11] . future studies have to consider these possibilities before we disregard the lung microbiome in nosocomial pneumonia. in the critically ill, changes in the microbiome in all habitats, including the lungs, are particularly striking. due to the devastating consequences of untreated severe infections, broad eradication is accepted as lesser evil and collateral damage on beneficial or commensal microbes is generally accepted. however, the potential long-term consequences of unwarranted side effects on the microbiome warrant a reassessment of the microbiome as a diagnostic or even therapeutic target. for example, dysbiosis of the gut microbiome itself has been described as a predictive factor for late-onset neonatal sepsis [50] suggesting that the microbiome can serve at least as a biomarker to predict ensuing nosocomial infection. moreover, albeit solid data are still missing to support interventions to restore a healthy microbiome, the strategy holds promise to impact on incidence and outcome of nosocomial infection and ensuing organ injury, including ards [51, 52] . in the light of a better understanding of off-target effects of broad-spectrum antibiotics on the microbiome, the liberal administration of antibiotics must be discussed against more sophisticated interventions to treat the bacterial infection (non-antibiotic therapies such as bacteriophages) or manipulation of the microbiome to make the residing communities more resilient (for example probiotics). in particular, the need to combine multiple anti-infective compounds in the light of diagnostic uncertainty might outweigh the benefit of early source control and explain controversial results for aggressive antibiotic strategies. for instance, in a before-andafter study hranjec et al. reported that the subgroup with least benefit from 'calculated' broad-spectrum antibiotics were patients presenting with septic shock, i.e. those in which the current paradigm would expect the highest need to initiate early anti-infective therapy [53] . thus, a holistic approach taking the microbiome into consideration carries the potential to initiate a paradigm shift in the treatment of infections in the icu. as discussed in the previous paragraphs, the lung dysbiosis seems to be common in the icu and enrichment of gut bacteria might be an important contributor to the development of lung injury and infection (fig. 2) . the relationship between gut and lung microbiome is described as the gut-lung axis [54] . because the gut microbiome can be targeted directly or indirectly with therapeutic interventions, this is an area of active study. investigations have thus far fallen into two specific pathways-first, using probiotics to help restore a premorbid microbiome, or second, to use antibiotics through an sdd approach to target specific families of organisms so as to alter the microbiome in possibly beneficial ways. further novel pharmacologic options that have direct gut microbiome modifying effects are also under development, including faecal transplantation as a possible novel treatment for microbiota dysregulation (considering the immune system during faecal microbiota transplantation for clostridioides difficile infection [55] and for the decolonization of antibioticresistant bacteria in the gut [56] ). one of the major challenges of studying the effect of these interventions is the huge variability in the gut microbiome of critically ill patients, even during the first days of icu admission [57] . furthermore, any beneficial effect of these interventions on the microbiome has yet to be assessed formally in a prospective, large-scale, randomized manner. attempting to attribute a causal impact of microbiome modifications upon clinical outcomes has been difficult to tease out as to whether changes in the microbiome are merely surrogates of some other mechanistic pathway that leads to improved clinical outcomes [58] . fundamentally, probiotics in critical illness aim to provide bacteria that may have been eradicated during the pre-and early phases of critical illness [59] . this eradication may be through administering antibiotics early in critical illness, which have been shown to greatly modify the gut microbiome [60] . alternatively, the mere onset of critical illness-be it sepsis, ards or any number of conditions, is associated with alterations of the gut fig. 2 island model for the development of lung injury based on sites of dysbiosis microbiome, which may be independent of antibiotic administration [61] . regardless, the stated goal of probiotic administration is to restore a pre-morbid microbiomeprimarily to the gut, but partially to other microbiome communities through generalized cross-talk [62] . through yet-unknown mechanisms, administering lactobacillus or bifidobacterium species through a probiotic may increase the diversity of microbial species in the gut, although more studies with rigorous outcome determinations are required [63] . in the critically ill, randomized studies and meta-analyses of randomized trials demonstrate a possible benefit of probiotic administration on the outcome of ventilator-associated pneumonia, without a difference in mortality [64, 65] , with a major challenge being a lack of standardization in dosing and composition of probiotic products [66] . larger scale studies are nearing completion and further data on the impact of microbiome modifications are forthcoming in the years ahead [59] . selective digestive decontamination, a regimen of prophylactic antibiotic administration, has been shown in small series to result in important alterations in gut microbiota, when compared with controls [67] . these changes are typically related to increasing selection for resistant organisms and decreased microbiome diversity, per a number of different metrics. given a possible benefit on patient mortality in some randomized trials [68, 69] , exploring the specific impact of this strategy on the microbiome, and related clinical outcomes, is a vital area for further study. additionally, given burgeoning evidence of crosstalk between the lung and gut microbial communities, the impact of either of these strategies on the non-gut microbiome communities in the critically ill patient remains under-investigated. given the apparent conflicting goals of sdd and probiotic administration in the critically ill as it relates to the microbiome, the role of co-administration may be difficult to conceive. however, most currently used sdd regimens are unlikely to affect the administered probiotic agent, and this may be a strategy for further investigation in targeted patients [70] . both sdd and probiotics appear to mediate their effect on patientrelated outcomes through reducing the incidence of ventilator-associated pneumonia, speaking to a crucially under-investigated relationship between the two microbiome communities and host immunology, a tantalizing area for future research. novel pharmacologic agents have also been suggested as modifiers for the gut microbiome but have yet to be formally tested in the critically ill. butyrate, a large bowel microbial fermentation product, is being investigated in pre-clinical trials as a specific modifier of gut-derived regulatory t cells [71] . administering a sialic acid analogue is being investigated as to whether it may reduce the burden of antibiotic-associated pathogens such as c. difficile by altering metabolic pathways [72] . older drugs such as metformin may have a role, with their demonstrated effects on altering the gut microbiome in patients with diabetes [73] . the lung microbiome is clearly more difficult to target than the gut microbiome due to the lack of routine administration of bacteria and bacterial products into the airways. the low biomass environment may also cause the lung microbiome to be more prone to infection induced by the introduction of, for example, probiotics. therefore, direct intervention in the lung microbiome may be sought via the alteration of regional growth conditions via the availability of nutrients or through immunomodulation. an example is the administration of macrolides in chronic obstructive pulmonary disease (copd): there is a selection for anti-inflammatory microbial metabolites and an alteration of the lung microbiome [74] . all of these possible interventions speak to the importance of achieving a better understanding of the gut-lung axis in critical illness. as this understanding evolves, the possibility of personalizing interventions for individual microbiome communities, or widespread initiation of interventions such as sdd or probiotics, would be possible. whilst patient-to-patient or staff-to-patient transmission of infection occurs within the intensive care unit, most nosocomial infections in critically ill patients arise through the invasion of normal host defences by bacteria and fungi that have become a part of an altered microbiome-either by changes in numbers or by the incorporation of species from the environment [75] . the hospital environment itself acquires a microbiome that reflects the patients that have been in it, and environmental reservoirs such as sinks, plumbing, work surfaces, and equipment can become reservoirs of resistant organisms that can infect the critically ill [76] . the inherent variability of the microbiome, therefore, provides an opportunity to study not only the individual patient, but also the forces in the environment that shape patient's outcome, and to identify specific opportunities where the persistence and transmission of pathogens can be prevented or minimized. because of the high prevalence of nosocomial infection, the environmental concentration of causative pathogens and the multiple risk factors for exposure, the icu provides a unique opportunity for intensive study of the microbiome and its role in the establishment and transmission of resistant organisms. with the emergence of new models of global acute care research collaboration through the international forum for acute care trialists (infact; www.infactglobal.org), and the launch of an infact initiative to leverage icu data to understand variability in patterns of resistance through the antimicrobial resistance in intensive care (amric) initiative. in previous years, we believed that the normal lung was free from bacteria. certainly, some features in the respiratory tract such as temperature, ph and nutrients were not beneficial for microbial growth. during critical illness, antibiotic use, mechanical ventilation, diet changes and inflammatory responses can bring the microbiome to dysbiosis. with the use of molecular techniques, we have had the opportunity to study the lung microbiome and not only in the microbial aspect but also in the responses from the host. one of the most important aspects to better determine the physiopathology of host-pathogen interaction in pulmonary complications such as ards and va-lrti is the gut-lung axis. further study of patients with disease in the respiratory tract will help us to better determine microbial diversity and constitution when comparing healthy and diseased subjects. dysbiosis and analysis of extra-pulmonary microbiome have helped to understand the complex interaction of bacterial clearance in the lung tissue and the off-target effects of broad-spectrum antibiotics on the microbiome. through therapies targeting host-pathogen interaction and the development of advance molecular testing, we will be able to have a deeper understanding in the analysis of the lung microbiome. how can we define "optimal microbiota?": a comparative review of structure and functions of microbiota of animals, fish, and plants in agriculture host-microbiota interactions in immunemediated diseases genomic approaches to studying the human microbiota defining dysbiosis and its influence on host immunity and disease the vocabulary of microbiome research: a proposal the human microbiome: our second genome factors that drive variation among gut microbial communities tools for the microbiome: nano and beyond extensive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle extreme dysbiosis of the microbiome in critical illness the influence of ethnicity and geography on human gut microbiome composition robbins pathologic basis of disease, vol. xv. philadelphia: saunders microbial life in extreme environments expériences relatives aux générations dites spontanées. comptes rendus hebdomadaires des séances de l'académie des sciences d: sciences naturelles l; 1860 studien über die aetiologie und histologie der pneumonie im kindesalter und der pneumonie im allgemeinen the relationship of sinusitis and bronchiectasis the internal surface area of the adult human lung surface area of the digestive tract -revisited what is the healthy gut microbiota composition? a changing ecosystem across age, environment, diet, and diseases a human gut microbial gene catalogue established by metagenomic sequencing the microbiome and the respiratory tract application of a neutral community model to assess structuring of the human lung microbiome enrichment of the lung microbiome with oral taxa is associated with lung inflammation of a th17 phenotype the lung microbiota of healthy mice are highly variable, cluster by environment, and reflect variation in baseline lung innate immunity the role of the microbiome in exacerbations of chronic lung diseases analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography bacterial topography of the healthy human lower respiratory tract specific ribosomal dna sequences from diverse environmental settings correlate with experimental contaminants reagent and laboratory contamination can critically impact sequence-based microbiome analyses the lung microbiome and ards. it is time to broaden the model dysbiosis in the intensive care unit: microbiome science coming to the bedside advancing our understanding of the human microbiome using qiime ribofr-seq: a novel approach to linking 16s rrna amplicon profiles to metagenomes the murine lung microbiome in relation to the intestinal and vaginal bacterial communities whole genome sequencing revealed microbiome in lung adenocarcinomas presented as ground-glass nodules classification of methanogenic bacteria by 16s ribosomal rna characterization methanogens: reevaluation of a unique biological group integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations attomolar dna detection with chiral nanorod assemblies fully automated portable comprehensive 2-dimensional gas chromatography device breathdx -molecular analysis of exhaled breath as a diagnostic test for ventilatorassociated pneumonia: protocol for a european multicentre observational study unique patterns of lower respiratory tract microbiota are associated with inflammation and hospital mortality in acute respiratory distress syndrome lung microbiota predict clinical outcomes in critically ill patients the influence of the microbiome on respiratory health aspiration pneumonia the dynamics of the pulmonary microbiome during mechanical ventilation in the intensive care unit and the association with occurrence of pneumonia identification of respiratory microbiota markers in ventilator-associated pneumonia gut microbial colonisation in premature neonates predicts neonatal sepsis critical illness and the role of the microbiome the essential role of the intestinal microbiota in facilitating acute inflammatory responses aggressive versus conservative initiation of antimicrobial treatment in critically ill surgical patients with suspected intensive-care-unit-acquired infection: a quasi-experimental, before and after observational cohort study more of the gut in the lung: how two microbiomes meet in ards considering the immune system during fecal microbiota transplantation for clostridioides difficile infection faecal microbiota transplantation for the decolonization of antibiotic-resistant bacteria in the gut: a systematic review and metaanalysis critically ill patients demonstrate large interpersonal variation in intestinal microbiota dysregulation: a pilot study collapse of the microbiome, emergence of the pathobiome, and the immunopathology of sepsis evaluating probiotics for the prevention of ventilator-associated pneumonia: a randomised placebo-controlled multicentre trial protocol and statistical analysis plan for prospect pathogen colonization of the gastrointestinal microbiome at intensive care unit admission and risk for subsequent death or infection impact of antibiotics on the gut microbiota of critically ill patients enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome alterations in fecal microbiota composition by probiotic supplementation in healthy adults: a systematic review of randomized controlled trials manipulation of the microbiome in critical illness-probiotics as a preventive measure against ventilator-associated pneumonia probiotics for preventing ventilator-associated pneumonia in mechanically ventilated patients: a meta-analysis with trial sequential analysis blurred lines: dysbiosis and probiotics in the icu effects of selective digestive decontamination (sdd) on the gut resistome decontamination strategies and bloodstream infections with antibiotic-resistant microorganisms in ventilated patients: a randomized clinical trial decontamination of the digestive tract and oropharynx in icu patients lactobacillus species: taxonomic complexity and controversial susceptibilities commensal microbe-derived butyrate induces the differentiation of colonic regulatory t cells microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug randomised, double-blind, placebo-controlled trial with azithromycin selects for antiinflammatory microbial metabolites in the emphysematous lung hospital-associated microbiota and implications for nosocomial infections investigation of a multiyear multiple critical care unit outbreak due to relatively drugsensitive acinetobacter baumannii: risk factors and attributable mortality publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. key: cord-266067-wrouqdcj authors: haywood, nathan; byler, matthew r.; zhang, aimee; roeser, mark e.; kron, irving l.; laubach, victor e. title: isolated lung perfusion in the management of acute respiratory distress syndrome date: 2020-09-17 journal: int j mol sci doi: 10.3390/ijms21186820 sha: doc_id: 266067 cord_uid: wrouqdcj acute respiratory distress syndrome (ards) is associated with high morbidity and mortality, and current management has a dramatic impact on healthcare resource utilization. while our understanding of this disease has improved, the majority of treatment strategies remain supportive in nature and are associated with continued poor outcomes. there is a dramatic need for the development and breakthrough of new methods for the treatment of ards. isolated machine lung perfusion is a promising surgical platform that has been associated with the rehabilitation of injured lungs and the induction of molecular and cellular changes in the lung, including upregulation of anti-inflammatory and regenerative pathways. initially implemented in an ex vivo fashion to evaluate marginal donor lungs prior to transplantation, recent investigations of isolated lung perfusion have shifted in vivo and are focused on the management of ards. this review presents current tenants of ards management and isolated lung perfusion, with a focus on how ex vivo lung perfusion (evlp) has paved the way for current investigations utilizing in vivo lung perfusion (ivlp) in the treatment of severe ards. acute respiratory distress syndrome (ards) is a severe, life-threatening form of acute lung injury characterized by inflammation, lung permeability, and edema [1] [2] [3] . clinical sequelae include significant hypoxia and bilateral infiltrates on chest imaging ( figure 1 ) [4] . since the original description by ashbaugh in 1967, significant advances have been made in the understanding of this disease process [2, 5] . however, this syndrome remains common, and is associated with significant morbidity and mortality [1] . diagnostic criteria of ards rely on imaging and other clinical findings. the most recent definition, the berlin definition (table 1) , deviates from the previous division of acute lung injury (ali) and ards [6] . instead, mild, moderate, and severe ards severity categories are described based on the pao 2 /fio 2 ratio. additional tenants include respiratory failure not explained by fluid overload or cardiac failure, characteristic imaging findings (figure 1 ), and origin of onset within 7 days of symptoms or known clinical insult [6] . most common etiologies of ards include sepsis, pneumonia, and aspiration, but a number of other less common risk factors exist (table 2) [1, 2] . in addition, new pathogens may emerge that manifest in ards, such as the novel coronavirus disease 2019 (covid-19) [7] . table 1 . diagnosis of acute respiratory distress syndrome: berlin criteria [6] . imaging bilateral opacities noted on either cxr or ct scan that are not otherwise explained by fluid overload or cardiac failure ventilator-induced lung injury pancreatitis trauma and burn injury blood product administration cardiopulmonary bypass ischemia-reperfusion injury following lung transplantation covid-19 infection while our understanding of this syndrome has improved over the past half century, it remains a significant burden with a large impact on both the individual and health system. a large recent international evaluation of ards in the critical care population demonstrated that 10.4% of intensive care unit (icu) patients and 23.4% of intubated icu patients had ards [1, 8] . in-hospital mortality for the severe subgroup reached as high as 46.1% [1, 8] . a recent analysis of the national inpatient sample from 2006-2014 by eworuke et al. demonstrated increasing incidence of ards and a most recent rate of 193.4 cases per 100,000 population [9] . while a downward trend in in-hospital mortality has been reported, it remained quite high over this time period, with rates for sepsis, shock, and pneumonia approximating 40% [9] . in those who physically recover, high risks of cognitive dysfunction, muscular weakness, depression, and post-traumatic stress disorder have been reported [10] . additionally, the management of patients with ards is often resource-intensive (prolonged ventilation and icu stay) resulting in significant economic impact [11] . identification and appropriate treatment of the underlying cause of ards is essential (e.g., antibiotics, resuscitation, and source control for sepsis) [1] . beyond this, the current management of identification and appropriate treatment of the underlying cause of ards is essential (e.g., antibiotics, resuscitation, and source control for sepsis) [1] . beyond this, the current management of ards is largely supportive in nature, with basic tenants including lung protective ventilation, conservative fluid management, neuromuscular blockade, and prone positioning for severe disease [1, 4] . the use of pharmacologic agents, such as inhaled vasodilators and corticosteroids, can be considered, but their use is controversial and not widely accepted [1, 4] . in severe, refractory ards, the addition of extracorporeal assistance may provide some benefit [1, 4, 12] . lung-protective ventilation with low tidal volumes may cause hypercapnia and associated respiratory acidosis. this is, to some degree, an accepted consequence, leading to a concept known as permissive hypercapnia. beneficial effects of hypercapnia have been demonstrated, including reduction in pulmonary inflammation and oxidative stress [13, 14] . however, recent evidence has emerged suggesting negative impacts of hypercapnia on lung tissue repair and alveolar fluid clearance [13, 15] . as such, extracorporeal co 2 removal with venovenous extracorporeal membrane oxygenation (ecmo) has been utilized as an alternative means of improving hypercapnia and gas exchange in severe ards [12, 13] . however, it carries the typical risks associated with extracorporeal circulation [13] . while early evidence has suggested improved outcomes in patients with severe ards managed with ecmo [16] , a recent international, randomized clinical trial evaluating the use of routine ecmo in severe ards showed no difference in mortality compared to conventional ventilatory management with ecmo as a rescue therapy [17] . for this reason, the routine use of ecmo in severe ards remains controversial. recent guidelines suggest consideration of venovenous ecmo in severe ards when pao 2 /fio 2 < 80 mmhg despite optimal ards treatment, including neuromuscular blockade, high positive end expiratory pressure (peep), and prone positioning [18] . the last half decade has produced numerous advances regarding the previously described management paradigm of ards, and outcomes have improved over this period. however, mortality remains high, with reports in severe subgroups greater than 40% [1, 8] . given this, and the continued emergence of pathogens causing severe ards, such as covid-19, development and breakthrough of new treatment modalities is crucial [7] . because current treatment strategies in ards largely rely on supportive measures to stabilize and allow for innate lung recovery over time, there is opportunity for targeted therapeutic approaches to limit disease severity and improve outcomes. isolated lung perfusion is an active area of investigation that may ultimately serve in this role. first studied in 1987, isolated lung perfusion was initially implemented in an ex vivo fashion for donor lungs prior to transplant [19] . coined ex vivo lung perfusion (evlp), it has since undergone numerous advances, leading to its current clinical use in donor lung evaluation and in the reconditioning of marginal donor lungs prior to transplant [20] . the ability of evlp to rehabilitate lungs injured in a porcine sepsis model [21] has provided the basis for a similar application-the use of isolated lung perfusion in vivo in the management of ards. here, early animal studies have demonstrated the ability of in vivo lung perfusion (ivlp) to rehabilitate sepsis-induced ards [22] . below, we review the history and current evidence for isolated lung perfusion techniques, with a focus on how evlp has provided the basis for and led to investigations into the use of ivlp for the treatment of ards. relevant works included in our review were identified from pubmed, using the following search terms: ali, ards, isolated lung perfusion, evlp, and ivlp. primary graft dysfunction (pgd) is the result of severe ischemia-reperfusion injury following lung transplant that can result in detrimental early and late outcomes [23] . surgeons are often conservative in donor lung selection, as lesser quality lungs increase the likelihood of pgd development [24] . it has been reported that only 15-20% of lungs from multiorgan donors are deemed usable for transplantation [24] [25] [26] . the unfortunate consequence of this is revealed in lung waitlist mortality-reported as high as 17.2 deaths per 100 waitlist years [27] . one strategy to extend donor lung availability is through the use of evlp. clinical application of evlp was first described by hardesty et al. in 1987 [19,26] . since this time, numerous advances have been made, many of which are due to the work of steen and colleagues, who developed a lung-specific perfusion solution (steen solution) and first utilized evlp in donation after cardiac death (dcd) lungs in 2000 [28, 29] . following this achievement, there was a body of work in the early 2000s, with additional contributions by steen et al. and wierup et al., demonstrating the ability of evlp as a clinical platform for continued assessment and rehabilitation in lungs considered unusable at the time of initial evaluation [30, 31] . the toronto lung transplant group then demonstrated the efficacy of extended-duration evlp in a landmark study published in the new england journal of medicine in 2011 [20] . here, they described 20 cases in which lungs were initially "not considered suitable for transplantation" that underwent reevaluation during a 4 h period of evlp and were ultimately successfully transplanted. the incidence of pgd within 72 h was 15% in the evlp group compared to 30% in the control group (p = 0.11), and there were no significant differences for any secondary endpoints, including ecmo, post-transplantation icu days, 30 day mortality, p/f ratio, or any severe adverse events directly attributable to evlp [20] . in the last decade, the clinical use of evlp has become more widespread, and is now used in many large lung transplant centers in north america, europe, and australia [32] . a recent multicenter prospective clinical trial (novel) examining the use of evlp has demonstrated similar early outcomes and one-year survival compared to patients that underwent transplantation with standard criteria donor lungs [33, 34] . evlp utilizes physiologic, normothermic perfusion of donor lungs [35] . this allows for a period of lung assessment during which direct examination, imaging, bronchoscopy, and blood gas analysis can be performed [26] . a general circuit for evlp is shown in figure 2 . the device is made up of two main components. the first is a ventilator to provide oxygen to the donor lungs, and the second is a circuit that drives perfusion, deoxygenates, and filters the perfusate [36] . the perfusate is deoxygenated using a gas exchange membrane with a sweep gas consisting of nitrogen, co 2 , and o 2 [23] , which is then filtered using a leukocyte filter prior to advancing into the pulmonary artery [35, 36] . the perfusate is drained from the left atrium into a reservoir prior to repeating the sequence [36] . clinical application of evlp was first described by hardesty et al. in 1987 [19,26] . since this time, numerous advances have been made, many of which are due to the work of steen and colleagues, who developed a lung-specific perfusion solution (steen solution) and first utilized evlp in donation after cardiac death (dcd) lungs in 2000 [28, 29] . following this achievement, there was a body of work in the early 2000s, with additional contributions by steen et al. and wierup et al., demonstrating the ability of evlp as a clinical platform for continued assessment and rehabilitation in lungs considered unusable at the time of initial evaluation [30, 31] . the toronto lung transplant group then demonstrated the efficacy of extended-duration evlp in a landmark study published in the new england journal of medicine in 2011 [20] . here, they described 20 cases in which lungs were initially "not considered suitable for transplantation" that underwent reevaluation during a 4 h period of evlp and were ultimately successfully transplanted. the incidence of pgd within 72 h was 15% in the evlp group compared to 30% in the control group (p = 0.11), and there were no significant differences for any secondary endpoints, including ecmo, post-transplantation icu days, 30 day mortality, p/f ratio, or any severe adverse events directly attributable to evlp [20] . in the last decade, the clinical use of evlp has become more widespread, and is now used in many large lung transplant centers in north america, europe, and australia [32] . a recent multicenter prospective clinical trial (novel) examining the use of evlp has demonstrated similar early outcomes and one-year survival compared to patients that underwent transplantation with standard criteria donor lungs [33, 34] . evlp utilizes physiologic, normothermic perfusion of donor lungs [35] . this allows for a period of lung assessment during which direct examination, imaging, bronchoscopy, and blood gas analysis can be performed [26] . a general circuit for evlp is shown in figure 2 . the device is made up of two main components. the first is a ventilator to provide oxygen to the donor lungs, and the second is a circuit that drives perfusion, deoxygenates, and filters the perfusate [36] . the perfusate is deoxygenated using a gas exchange membrane with a sweep gas consisting of nitrogen, co2, and o2 [23] , which is then filtered using a leukocyte filter prior to advancing into the pulmonary artery [35, 36] . the perfusate is drained from the left atrium into a reservoir prior to repeating the sequence [36] . while there are a number of ex vivo perfusion systems under investigation, the two most common systems used in clinical practice are the xvivo perfusion system (xps) (xps perfusion, goteborg, sweden) and the organ care system (ocs) (transmedics, andover, ma, usa) [23] . a comparison of these two systems is shown in table 3 . while both systems utilize the normothermic perfusion of donor lungs, the specific perfusate utilized is different. xps utilizes an acellular perfusate (steen solution), which contains albumin, dextran, and electrolytes, and often includes additives such as steroids and antibiotics [21, 23] . the perfusate used for ocs includes a cellular component (packed red blood cells) as well as ocs solution, which is composed of a low-potassium dextran solution and additives, such as steroids, glucose, bicarbonate, and antibiotics [23] . another distinction between these systems is that ocs is portable while xps is static [23, 38] . the ocs system integrates all components into a compact unit small enough to fit in a passenger seat in a car or plane [23] . given the portability, lungs can be instrumented onto the ocs system at the donor hospital following cold antegrade and retrograde flush. this has the theoretic benefit of minimizing cold ischemic time during transport to the recipient hospital [23, 26] . alternatively, xps is typically maintained at the recipient hospital, and lungs are instrumented onto the system upon arrival-following a period of cryopreservation [23] . arterial blood gasses are performed hourly using xps and are monitored continuously with ocs. if deemed acceptable, lungs are flushed with cold perfusate and kept cool prior to the implantation procedure [23] . evlp has undergone a transformation from an assessment and diagnostic tool to a therapeutic platform that also allows for active lung rehabilitation. this platform provides an ideal environment to deliver targeted drug therapy for lung rehabilitation, as it allows the opportunity to re-evaluate function to confirm positive treatment effect, and allows for targeted treatment of the lung, minimizing the risk of treatment side effects that may preclude systemic administration of therapeutic agents. one target of pharmacological agents in evlp has been minimizing inflammation and the reduction of pulmonary edema. numerous investigational agents have shown promise. for example, the administration of aerosolized exogenous catecholamines into the distal airspaces during evlp has been demonstrated to enhance the clearance of pulmonary edema, resulting in better graft oxygenation, pulmonary compliance, and reduced pulmonary vascular resistance [39] [40] [41] . this effect is not isolated to aerosolized delivery, as perfusion with a short-acting selective beta-2 adrenergic receptor agonist has also been associated with lower pulmonary artery pressures and better lung mechanics [42] . our laboratory and others have recently studied evlp as a platform to recondition lungs via pharmacologic treatment during ex vivo perfusion. using both murine and porcine models, we have demonstrated that the addition of a selective adenosine 2a receptor (a2ar) agonist to the evlp perfusate is associated with less pulmonary edema, lower levels of pro-inflammatory cytokines, and improved lung function [43, 44] . similarly, utilizing a porcine dcd model, our lab has demonstrated that delivery of a2ar agonist during evlp increased the likelihood of successful transplantation following prolonged periods of cold preservation [45] . the addition of a selective adenosine 2b receptor antagonist to the evlp perfusate has also been associated with improved lung function in both murine and porcine models [46, 47] . numerous other pharmacologic agents administered using the evlp platform have shown promise in mitigating the pulmonary inflammatory response, including, but not limited to, sphingosine-1-phosphate [48] , neutrophil elastase inhibitor [49] , and alpha-1-antitrypsin [50] . alpha-1-antitrypsin treatment was found to significantly reduce pulmonary edema, pulmonary cell apoptosis, and pro-inflammatory cytokine levels (il-1î± and il-8) in the perfusate [50] . similar to drug therapy, several studies have demonstrated that gene therapy coupled with evlp can repair injured lungs before transplantation. cypel et al. showed that delivery of an adenoviral vector encoding human il-10 (adhil-10), an anti-inflammatory cytokine, to human lungs improved arterial oxygen pressure and vascular resistance during evlp, concluding that delivery of adhil-10 can improve lung function [51] . yeung et al. later showed that ex vivo delivery of adhil-10 to lungs is superior to in vivo delivery, in that it leads to less vector-associated inflammation and provides superior post-transplant lung function [52] . a variety of recent studies have begun to evaluate molecular and cellular changes that occur during evlp. using a porcine model, tavasoli et al. showed that evlp resulted in reduced concentrations of nitric oxide metabolites and l-citrulline in lung tissue [53] . in addition, the ratio of l-ornithine over l-citrulline, a marker of the balance between l-arginine metabolizing enzymes, was increased in the evlp group, and expression of both arginase isoforms was increased during evlp. these data suggest that evlp induces a shift of the l-arginine balance towards arginase, leading to nitric oxide deficiency in the lung. using a rat model of evlp, lonati and colleagues described a remarkable anti-inflammatory response during evlp, including the activation of protective and anti-apoptotic pathways [54] . they also detected resolution factors in perfused, uninjured lungs, including transcripts that encode for feedback inhibitors of toll-like receptors and cytokine signaling, such as inhibitors of nuclear factor-îºb (nf-îºb) signaling iîºb (inhibitor of îºb), il-1 receptor antagonist 1, ll-1 decoy receptor, and nonfunctional interleukin 1 receptor-associated kinase-m. importantly, their data obtained in uninjured lungs was confirmed in perfused injured (dcd) lungs. these results led lonati and colleagues to conclude that the evlp molecular signature is very similar to the pattern induced by ischemic preconditioning [54] . to identify potential biomarkers during evlp, hsin et al. used a metabolomics approach in a clinical study to identify a small panel of metabolites in evlp perfusate that were highly correlated with the development of pgd after transplant [55] . in another clinical evlp biomarker study, hashimoto et al. demonstrated that levels of m30 (indicative of epithelial apoptosis) and high mobility group box 1 (hmgb-1, related to cell death and inflammation) protein in the evlp perfusate correlated with pgd after lung transplantation, and might therefore be useful biomarkers to improve donor lung assessment during evlp [56] . a recent study by elgharably et al. showed that two micrornas (mir-17 and mir-548b) were significantly upregulated in the alveolar epithelial cells of human lungs that underwent cold ischemia and evlp [57] . both mir-17 and mir-548b have expected target genes related to lung injury and share a number of mutual targets, suggesting that mir-17 and mir-548b may interact at some level in the signaling pathway and potentially provide novel therapeutic targets [57] . yeung and colleagues examined gene expression changes in human lungs during 12 h of evlp, and found that, despite increases in endothelial markers of inflammation, circulating, leukocyte, cell-specific gene expression fell during evlp [58] . these results suggest that perhaps the mechanisms underlying the benefit of evlp are nonspecific and related to innate recovery capabilities of the lung. finally, a recent study by wong et al. performed a retrospective transcriptomics analysis of dcd lungs with or without evlp, and showed that pathways associated with leukocyte function, such as phosphatidylinositol biosynthesis, phospholipase c signaling, cholesterol biosynthesis, protein targeting to vacuoles, and golgi vesicle trafficking were all downregulated in lungs after evlp [59] . these results support those of yeung et al. above [58] , which inferred that passenger leukocytes are depleted during evlp. the ability of evlp to rehabilitate injured, marginal lungs prior to transplantation has led to investigation into other forms of lung injury-namely, ards. our laboratory has demonstrated the ability of evlp to rehabilitate ards in a porcine sepsis model [21] . in this study, intravenous lipopolysaccharide (lps) was used to generate a systemic inflammatory response with associated ards [21] . lungs that were subjected to evlp with steen solution demonstrated improved oxygenation and compliance compared to the control (no evlp) [21] . this finding provided the basis for investigation of a similar in vivo technique, ivlp, in the management and rehabilitation of ards. in vivo lung perfusion (ivlp) was first investigated in the 1980s as a method for delivering high-dose chemotherapy [60, 61] . ivlp involves isolation and placement of cannulas into the pulmonary artery and veins of a single lung in vivo, so that its perfusion is removed from systemic circulation. this allows for the delivery of high dose medication to the lung parenchyma while limiting adverse systemic effects. in this way, much higher doses of chemotherapy could be used to treat lung cancer than would have otherwise been tolerated systemically. multiple clinical trials investigating the utility of ivlp in the treatment of lung cancer have shown increased survival benefit [62, 63] . these studies, coupled with studies showing that evlp can rehabilitate sepsis-induced lung injury [21] , have provided the basis and rational for investigating the use of ivlp to rehabilitate end-stage lung injury from ards. currently, investigations into the use of ivlp for the rehabilitation of ards have been limited to swine animal models, as described below. unlike evlp, which perfuses previously resected donor lungs, ivlp provides isolated lung perfusion to lungs that remain inside of a host. ivlp investigations have achieved this via a sternotomy or thoracotomy approach [22, 64] . prior to beginning the procedure, an injurious model is used to establish lung injury, which can be achieved by various protocols, including intravenous lps, surfactant washout model, intravenous oleic acid, or gastric aspiration [65, 66] . our laboratory has established a systemic lung injury model in swine using an lps infusion administered at 50 âµg/kg over 2 h to establish ards, defined as a p/f ratio less than 300 mmhg. this model creates a reproducible injury and simulates the increased capillary permeability observed in a septic response. due to the systemic, hemodynamic instability associated with this injury model, we performed ivlp via a sternotomy, in order to provide necessary cardiopulmonary support via central, venoarterial ecmo [22] . our ivlp investigations utilized perfusion of the left porcine lung, due to its optimal venous anatomy for an open approach. the left pulmonary artery and superior and inferior veins were circumferentially dissected, and cannulas were placed into the vessels, as outlined in figure 3 . these cannulas are circumferentially secured so as to isolate the left lung from systemic circulation. the cannulas are connected to an ivlp circuit that is designed similarly to that used for cardiopulmonary bypass. the circuit uses a special gas mixture and a membrane deoxygenator, which provides physiologic levels of carbon dioxide and removes oxygen from the steen perfusate. after the perfusate circulates through the lung, the lung's oxygenation and ventilation capacity are evaluated by blood gas analysis at predetermined intervals. the back pressure on the pulmonary venous drainage was maintained at between 0 to +5 mmhg by adjusting the height of the hard-shelled cardiotomy reservoir. after completion of a predetermined ivlp perfusion period (2 or 4 h), the cannulas were removed, and the lung was allowed to reperfuse back into systemic circulation. circuit includes reservoir, pump, deoxygenator, and leukocyte filter. ecmo: extracorporeal membrane oxygenation; hcu: heating-cooling unit. used with permission [22] . arrows indicate direction of perfusate flow in ivlp circuit and venous outflow/arterial inflow in ecmo circuit. multiple studies have investigated the use of ivlp for the delivery of isolated, high-dose chemotherapy to the lung. the duration of this treatment has been for 30 min [62] . however, the use of ivlp to treat ards has utilized longer treatment (perfusion) times. in 2014, dos santos and colleagues evaluated the use of prolonged ivlp in a large animal study, where they delivered ivlp for 4 h via a thoracotomy to six swine, followed by a 4 h reperfusion period [64] . here, they demonstrated that using ivlp for this duration is feasible and safe, and there was no change in lung function parameters (oxygenation and compliance) or histologic evidence of acute lung injury [64] . in 2018, our laboratory was the first to investigate the use of ivlp to rehabilitate sepsis-induced ards [22] . after undergoing an lps infusion and confirmation of ards, 4 h of left-lung ivlp with steen solution was performed in eight swine. the right lung served as an internal control, and was compared the ivlp-treated left lung. after the ivlp treatment period, the animal was decannulated from ivlp and allowed to reperfuse for 4 h. over the course of the experiment, the treated left lungs demonstrated improved oxygenation performance from baseline when compared to the right lung controls. additionally, total lung compliance was increased. the mechanism behind these improvements may be due in part to the observed decrease in levels of tumor necrosis factor alpha (tnf-î±) and interferon gamma (ifn-î³) in the treated left lungs. additionally, there was evidence of decreased pulmonary edema, demonstrated by lower wet-to-dry weight ratios in the treated left lungs. finally, there was decreased expression of the cellular adhesion molecules vascular cell adhesion molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1). these data suggest that there was decreased transmigration of leukocytes, which resulted in decreased histologic evidence of inflammation in the ivlp-treated lungs [22] . most recently, we performed a study comparing the previously used ivlp perfusion time of 4 h to 2 h of ivlp perfusion [67] . similar to our previous study, eight adult swine underwent lps infusion to induce ards, and then were randomized to either 4 h ivlp (n = 4) or 2 h ivlp (n = 4) treatment groups. the results demonstrated that 2 h of ivlp outperformed 4 h of ivlp when evaluating each circuit includes reservoir, pump, deoxygenator, and leukocyte filter. ecmo: extracorporeal membrane oxygenation; hcu: heating-cooling unit. used with permission [22] . arrows indicate direction of perfusate flow in ivlp circuit and venous outflow/arterial inflow in ecmo circuit. multiple studies have investigated the use of ivlp for the delivery of isolated, high-dose chemotherapy to the lung. the duration of this treatment has been for 30 min [62] . however, the use of ivlp to treat ards has utilized longer treatment (perfusion) times. in 2014, dos santos and colleagues evaluated the use of prolonged ivlp in a large animal study, where they delivered ivlp for 4 h via a thoracotomy to six swine, followed by a 4 h reperfusion period [64] . here, they demonstrated that using ivlp for this duration is feasible and safe, and there was no change in lung function parameters (oxygenation and compliance) or histologic evidence of acute lung injury [64] . in 2018, our laboratory was the first to investigate the use of ivlp to rehabilitate sepsis-induced ards [22] . after undergoing an lps infusion and confirmation of ards, 4 h of left-lung ivlp with steen solution was performed in eight swine. the right lung served as an internal control, and was compared the ivlp-treated left lung. after the ivlp treatment period, the animal was decannulated from ivlp and allowed to reperfuse for 4 h. over the course of the experiment, the treated left lungs demonstrated improved oxygenation performance from baseline when compared to the right lung controls. additionally, total lung compliance was increased. the mechanism behind these improvements may be due in part to the observed decrease in levels of tumor necrosis factor alpha (tnf-î±) and interferon gamma (ifn-î³) in the treated left lungs. additionally, there was evidence of decreased pulmonary edema, demonstrated by lower wet-to-dry weight ratios in the treated left lungs. finally, there was decreased expression of the cellular adhesion molecules vascular cell adhesion molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1). these data suggest that there was decreased transmigration of leukocytes, which resulted in decreased histologic evidence of inflammation in the ivlp-treated lungs [22] . most recently, we performed a study comparing the previously used ivlp perfusion time of 4 h to 2 h of ivlp perfusion [67] . similar to our previous study, eight adult swine underwent lps infusion to induce ards, and then were randomized to either 4 h ivlp (n = 4) or 2 h ivlp (n = 4) treatment groups. the results demonstrated that 2 h of ivlp outperformed 4 h of ivlp when evaluating each lung's oxygenation capacity and total lung compliance. similar to the study by mehaffey et al. [22] , this was likely due to a reduction in pulmonary edema, as indicated by improved wet-to-dry weight ratios. we also observed decreased expression of the pleiotropic cytokine il-6 [67] . this cytokine is characteristically elevated in the hyperinflammatory subphenotype of ards, and has been targeted by monoclonal antibodies in the treatment of ards associated with covid-19 [68, 69] . together, these physiologic and biochemical improvements in the treated lungs allowed more animals to successfully wean from venoarterial ecmo support in the 2 h group (three of four) than in the 4 h group (two of four). these two studies shared some limitations, such as they were performed in farm-raised animals that may have physiologic variability and were comprised of low group sizes. because we have not yet determined if the results with the lps model of ards are translatable to other etiologies of ards (e.g., pulmonary contusions, massive transfusion reaction, aspiration, etc.), the results may not be generalizable. the invasive sternotomy and thoracotomy approaches used in ivlp investigations are currently much too invasive to use in a critically ill, unstable patient. the future of ivlp in the treatment of ards will be contingent upon the advancement of catheter-based technologies that can be translated into a percutaneous platform. this will provide a minimally invasive application of ivlp in lung rehabilitation via catheters placed into peripheral vessels and threaded over a wire into the pulmonary artery and veins, instead of large surgical incisions (figure 4 ). lung's oxygenation capacity and total lung compliance. similar to the study by mehaffey et al. [22] , this was likely due to a reduction in pulmonary edema, as indicated by improved wet-to-dry weight ratios. we also observed decreased expression of the pleiotropic cytokine il-6 [67] . this cytokine is characteristically elevated in the hyperinflammatory subphenotype of ards, and has been targeted by monoclonal antibodies in the treatment of ards associated with covid-19 [68, 69] . together, these physiologic and biochemical improvements in the treated lungs allowed more animals to successfully wean from venoarterial ecmo support in the 2 h group (three of four) than in the 4 h group (two of four). these two studies shared some limitations, such as they were performed in farm-raised animals that may have physiologic variability and were comprised of low group sizes. because we have not yet determined if the results with the lps model of ards are translatable to other etiologies of ards (e.g., pulmonary contusions, massive transfusion reaction, aspiration, etc.), the results may not be generalizable. the invasive sternotomy and thoracotomy approaches used in ivlp investigations are currently much too invasive to use in a critically ill, unstable patient. the future of ivlp in the treatment of ards will be contingent upon the advancement of catheter-based technologies that can be translated into a percutaneous platform. this will provide a minimally invasive application of ivlp in lung rehabilitation via catheters placed into peripheral vessels and threaded over a wire into the pulmonary artery and veins, instead of large surgical incisions (figure 4 ). there remains a myriad of questions to be answered, in order to establish the optimal protocol for ivlp in the treatment of ards. we have demonstrated improved pulmonary function following ivlp and a 4 h reperfusion period [22, 67] . however, the optimal timing of ivlp, as well as the longterm effect of ivlp on lung function beyond 4 h of reperfusion, is yet to be determined. the early exudative phase of ards is characterized by innate inflammatory cell activation, resulting in damage to the alveolar epithelium and capillary endothelium [1, 2, 4] . while further studies are needed to determine the optimal timing of ivlp, it is likely within this early phase of ards that ivlp will there remains a myriad of questions to be answered, in order to establish the optimal protocol for ivlp in the treatment of ards. we have demonstrated improved pulmonary function following ivlp and a 4 h reperfusion period [22, 67] . however, the optimal timing of ivlp, as well as the long-term effect of ivlp on lung function beyond 4 h of reperfusion, is yet to be determined. the early exudative phase of ards is characterized by innate inflammatory cell activation, resulting in damage to the alveolar epithelium and capillary endothelium [1, 2, 4] . while further studies are needed to determine the optimal timing of ivlp, it is likely within this early phase of ards that ivlp will provide the most benefit. less damage in this phase would decrease the negative impact of innate recovery pathways that can result in lung fibrosis. limiting the progression to fibrosis is critical for recovery, as this final fibrotic phase is associated with prolonged ventilation and increased mortality [1, 4] . additionally, the rehabilitative effect of ivlp on pulmonary function in different ards models remains to be evaluated. these are important questions that warrant further investigation prior to translation into human applications. causes of ards can be grouped into two broad categories: those that cause direct lung injury and those that cause an indirect lung injury. the latter often results from the deleterious impact of a systemic inflammatory response, such as sepsis. the pathophysiology of these two categories differs, and while we have demonstrated the beneficial impact of ivlp in a sepsis model, we have yet to determine the impact in a direct lung injury model, such as aspiration or ventilator-induced lung injury. the benefit of ivlp in a sepsis model is multifactorial, but a large part is due to the protective effect of isolated lung perfusion in the setting of systemic inflammation. future use of ivlp will extend beyond simply the perfusion of steen solution to ameliorate lung injury and decrease pulmonary edema. similar to evlp, the technique of ivlp can be advanced and used as a platform for the delivery of a myriad of lung-specific drug therapies. this avenue may provide additional benefit in more direct causes of lung injury, such as gastric aspiration, pneumonia, and ventilator-induced lung injury. with administration to lung circulation that is isolated from systemic circulation, the delivery of higher-dose drug therapies than would otherwise be tolerated from systemic side effects would be possible. these therapies may include powerful antimicrobials, antivirals, immunomodulators, stem cells, genetic therapies, and any combination thereof to target the exact etiology of a patient's ards and limit the local injurious inflammatory response [70] . ards remains associated with high rates of morbidity and mortality, and advances in management will be critical to improve outcomes and decrease the drastic impact on healthcare resource utilization [1, 8, 11] . in vivo isolated lung perfusion is a promising investigational method for lung rehabilitation in severe ards. author contributions: all authors contributed to manuscript design, writing, and editing. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by several research grants from national heart, lung, and blood institute (nhlbi)/national institutes of health (nih) (t32hl007849, r01hl142110, and r01hl119218). the content is solely the responsibility of the authors and does not represent the official views of the national institutes of health. the authors declare no conflict of interest. acute respiratory distress syndrome the acute respiratory distress syndrome corticosteroids in acute lung injury: the dilemma continues current concepts of ards: a narrative review acute respiratory distress in adults acute respiratory distress syndrome clinical characteristics of coronavirus disease 2019 in china patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries national incidence rates for acute respiratory distress syndrome (ards) and ards cause-specific factors in the united states (2006-2014) recovery and outcomes after the acute respiratory distress syndrome (ards) in patients and their family caregivers acute respiratory distress syndrome: cost (early and long-term) extracorporeal membrane oxygenation for ards in adults the role of hypercapnia in acute respiratory failure protective effects of hypercapnic acidosis on ventilator-induced lung injury hypercapnic acidosis attenuates pulmonary epithelial wound repair by an nf-kappab dependent mechanism efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial extracorporeal membrane oxygenation for severe acute respiratory distress syndrome formal guidelines: management of acute respiratory distress syndrome autoperfusion of the heart and lungs for preservation during distant procurement normothermic ex vivo lung perfusion in clinical lung transplantation ex vivo lung perfusion rehabilitates sepsis-induced lung injury in vivo lung perfusion rehabilitates sepsis-induced lung injury evlp: ready for prime time? isolated lung perfusion organ donation and utilization in the united states application of ex vivo lung perfusion (evlp) in lung transplantation transplantation of lungs from a non-heart-beating donor transplantation of lungs from non-heart-beating donors after functional assessment ex vivo first human transplantation of a nonacceptable donor lung after reconditioning ex vivo ex vivo evaluation of nonacceptable donor lungs ex-vivo lung perfusion and ventilation normothermic ex vivo lung perfusion as an assessment of marginal donor lungs-the novel lung trial the novel lung trial one-year outcomes extracorporeal lung perfusion (ex-vivo lung perfusion) ex vivo lung perfusion early results in transplantation of initially rejected donor lungs after ex vivo lung perfusion: a case-control study machine perfusion of thoracic organs adrenoreceptor agonist inhalation during ex vivo lung perfusion attenuates lung injury bronchodilator inhalation during ex vivo lung perfusion improves posttransplant graft function after warm ischemia stimulation of alveolar epithelial fluid clearance in human lungs by exogenous epinephrine adrenergic agonist infusion during extracorporeal lung perfusion: effects on glucose concentration in the perfusion fluid and on lung function adenosine a 2 a agonist improves lung function during ex vivo lung perfusion ex vivo perfusion with adenosine a2a receptor agonist enhances rehabilitation of murine donor lungs after circulatory death ex vivo lung perfusion with adenosine a2a receptor agonist allows prolonged cold preservation of lungs donated after cardiac death lungs donated after circulatory death and prolonged warm ischemia are transplanted successfully after enhanced ex vivo lung perfusion using adenosine a2b receptor antagonism attenuation of pulmonary ischemia-reperfusion injury by adenosine a2b receptor antagonism increasing circulating sphingosine-1-phosphate attenuates lung injury during ex vivo lung perfusion a neutrophil elastase inhibitor improves lung function during ex vivo lung perfusion î± 1 -anti-trypsin improves function of porcine donor lungs during ex-vivo lung perfusion functional repair of human donor lungs by il-10 gene therapy ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre-and post-lung transplantation in the pig increased arginase expression and decreased nitric oxide in pig donor lungs after normothermic ex vivo lung perfusion influence of ex vivo perfusion on the biomolecular profile of rat lungs metabolic profile of ex vivo lung perfusate yields biomarkers for lung transplant outcomes higher m30 and high mobility group box 1 protein levels in ex vivo lung perfusate are associated with primary graft dysfunction after human lung transplantation human lungs airway epithelium upregulate microrna-17 and microrna-548b in response to cold ischemia and ex vivo reperfusion towards donor lung recovery-gene expression changes during ex vivo lung perfusion of human lungs potential therapeutic targets for lung repair during human ex vivo lung perfusion isolated total lung perfusion as a means to deliver organ-specific chemotherapy: long-term studies in animals isolated lung perfusion with adriamycin. a preclinical study multicenter phase ii clinical trial of isolated lung perfusion in patients with lung metastases phase ii multicenter clinical trial of pulmonary metastasectomy and isolated lung perfusion with melphalan in patients with resectable lung metastases modified in vivo lung perfusion allows for prolonged perfusion without acute lung injury overview of the pathology of three widely used animal models of acute lung injury regeneration of severely damaged lungs using an interventional cross-circulation platform two hours of protocol-driven in vivo lung perfusion improves lung function in sepsis model of acute respiratory distress syndrome thoracic breakout session 2 subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials tocilizumab treatment in covid-19: a single center experience in vivo lung perfusion as a platform for organ repair in acute respiratory distress syndrome this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-290226-rtoasm2l authors: scassellati, catia; galoforo, antonio carlo; bonvicini, cristian; esposito, ciro; ricevuti, giovanni title: ozone: a natural bioactive molecule with antioxidant property as potential new strategy in aging and in neurodegenerative disorders date: 2020-08-15 journal: ageing res rev doi: 10.1016/j.arr.2020.101138 sha: doc_id: 290226 cord_uid: rtoasm2l systems medicine is founded on a mechanism-based approach and identifies in this way specific therapeutic targets. this approach has been applied for the transcription factor nuclear factor (erythroid-derived 2)–like 2 (nrf2). nrf2 plays a central role in different pathologies including neurodegenerative disorders (nds), which are characterized by common pathogenetic features. we here present wide scientific background indicating how a natural bioactive molecule with antioxidant/anti-apoptotic and pro-autophagy properties such as the ozone (o(3)) can represent a potential new strategy to delay neurodegeneration. our hypothesis is based on different evidence demonstrating the interaction between o(3) and nrf2 system. through a meta-analytic approach, we found a significant modulation of o(3) on endogenous antioxidant-nrf2 (p < 0.00001, odd ratio (or) = 1.71 95%ci:1.17-2.25) and vitagene-nrf2 systems (p < 0.00001, or = 1.80 95%ci:1.05-2.55). o(3) activates also immune, anti-inflammatory signalling, proteasome, releases growth factors, improves blood circulation, and has antimicrobial activity, with potential effects on gut microbiota. thus, we provides a consistent rationale to implement future clinical studies to apply the oxygen-ozone (o(2)-o(3)) therapy in an early phase of aging decline, when it is still possible to intervene before to potentially develop a more severe neurodegenerative pathology. we suggest that o(3) along with other antioxidants (polyphenols, mushrooms) implicated in the same nrf2-mechanisms, can showed neurogenic potential, providing evidence as new preventive strategies in aging and in nds. life span has almost doubled in the last century (who, 2011 , wyss-coray, 2016 , and consequently aging-specific diseases are becoming prevalent (moskalev et al., 2017) . however, the pathophysiologic mechanisms underlying most of them are still poorly understood and challenges regarding treatments efficacy and costs persist. neurodegenerative diseases (nds, alzheimer's disease, ad; parkinson disease, pd; amyotrophic lateral sclerosis, als, huntington disease, hd) are the most prevalent cognitive and motor disorders of the elderly. these aging-specific diseases are characterized by the loss of homeostasis during aging, leading to low-grade stress by pathologic formation of reactive oxygen species (ros), chronic inflammation, mitochondrial dysfunction and metabolic unbalance (dugger, dickson, 2017) . in addition, these pathophenotypes are determined by abnormal aggregation of specific proteins (yanar et al., 2020) , given the connection between excessive ros accumulation and impairment in proteostasis network. despite their distinct causative factors and clinical symptoms, these diseases as well as aging have common pathogenetic features (aso et al., 2012) . this implicates potentiality in the identification of therapeutic targets on a set of disease phenotypes and physiological conditions that are mechanistically linked. thus, contrary to a hitherto linear approach that considered one disease, one medicine, to date there is a need for a new concept of therapy condensed as "several diseases, one medicine". in this way, diseases are diagnosed not only by clinical symptoms, but mainly by the underlying molecular signatures (goh et al., 2007) . based on this network medicine approach, (cuadrado et al., 2018 , cuadrado et al., 2019 reported extensive evidence about the central role playing by nuclear factor (erythroid-derived 2)-like 2 (nrf2). nrf2 is widely known and j o u r n a l p r e -p r o o f investigated as a master regulator of multiple cytoprotective responses and as a key molecular node within a cluster of a wide spectrum of diseases, including nds. moreover, nrf2 activation is impaired in aging by the involvement of microrna (zhang, h. et al., 2015 , schmidlin et al., 2019 , silva-palacios et al., 2018 . this suggests that nrf2 could represent a common therapeutic and systems medicine target, for aging and for its related disorders. nrf2 can transcriptionally modulate the cytoprotective genes belonging to the vitagene network. this network regulates endogenous cellular defense mechanisms, and involves redox sensitive genes such as members of the heat shock proteins (hsp) family (heme-oxigenase ho-1, hsp70), but also sirtuins and the thioredoxin (trx)/thioredoxin reductase (trxr1) system (calabrese, v. et al., 2010) . based on this rationale, in this review we present wide scientific background indicating how a natural bioactive molecule with antioxidant property such as the ozone (o3) can be indicated as a potential new strategy to delay neurodegeneration. this hypothesis is based on the widely demonstrated evidence regarding the interaction between o3 and nrf2 (galie et al., 2018 , siniscalco et al., 2018 , re et al., 2014 , vaillant et al., 2013 . we first describe the relevant, well known and documented molecular mechanisms related to antioxidant/anti-apoptotic/pro-autophagy processes targeted by the o3 administration via nrf2 biological pathway. secondarily, we report a list of the main stress oxidative biomarkers modulated by the o3 treatment via nrf2 and that, in turn are strongly involved in nds pathophysiology as well as in aging mechanisms. different meta-analyses have been performed to demonstrate the effect in terms of odd ratio (or) of o3 on endogenous antioxidant-nrf2 and vitagene-nrf2 systems. we thus provide scientific evidence to build a consistent rationale for apply for the first time the oxygen-ozone (o2-o3) therapy in an early phase of aging decline, when it is still possible to intervene, before to develop a potential neurodegenerative pathology. o3 is a triatomic gaseous molecule which has been using as a powerful oxidant in medicine for more than 150 years (elvis, ekta, 2011) . in nature, o3 is generated during storms due to the j o u r n a l p r e -p r o o f electrical discharges of the rays that react with atmospheric o2 to produce o3. in humans, a revolutionary discovery leaded to demonstrate that neutrophils isolated from human peripheral blood and coated with antibodies can catalyse the generation of o3 by a water oxidation pathway, leading to efficient killing of bacteria (wentworth et al., 2002 , babior et al., 2003 , lerner, eschenmoser, 2003 . in 1785, van mauren was the first identifying the distinctive odor of o3. the actual gas was later discovered by the german chemist, christian friedrich schonbein at the university of basel in switzerland on march 13th, 1839 when working with a voltaic pile in the presence of o2 (altman, 2007) . friederich noticed the emergence of a gas with an electric and pungent smell, and named it ozone, which is derived from the greek word for smell (bocci, v., 2011) . in 1860, jacques-louis soret, a swiss chemist demonstrated that the o3 was made up of three atoms of oxygen (altman, 2007) . o3 was used as first antiseptic for operating rooms and to disinfect surgical instruments in 1856, and in 1860 the first o3 water treatment plant was built in monaco to disinfect water (altman, 2007) . nikola tesla patented the first portable o3 generator in 1896 in the united states. the physicist, joachim hansler invented the first reliable o3 generator, and this was the breakthrough in the use of o3 for medical applications. this invention is considered the prelude to the ozonated autohemotherapy procedure and served as the basis for o3 therapy expansion over the last 40 years. the use of o3 in the clinical practice was introduced in the past century (wolff, 1915) . during the world war i, from 1914 to 1918, doctors used o3 to successfully treat post traumatic gangrene in german soldiers, bone fractures, inflammations, and abscesses (bocci, 2011) . due to its prophylactic proprieties, o3 was also used to prevent infections in local medical procedures and to control wound infections (merin et al., 2007) . the o2-o3 therapy is a non-invasive, non-pharmacological, no-side effect and low-cost procedure applied in medicine for the treatment of more than 50 pathological processes, whose alterations in endogenous oxidative-antioxidative balance play a crucial role. importantly, ddifferent clinical trials evidenced the effectiveness of this therapy in the treatment of degenerative j o u r n a l p r e -p r o o f disorders such as multiple sclerosis (smith et al., 2017 , delgado-roche et al., 2017 , ameli et al., 2019 , but also cardiovascular, peripheral vascular, neurological, orthopaedic, gastrointestinal and genitourinary pathologies (bocci, v., 2011 , elvis, ekta, 2011 , bocci, v., 2012 , smith et al., 2017 , braidy et al., 2018 ; fibromyalgia (moreno-fernandez et al., 2019 , tirelli et al., 2019 ; skin diseases/wound healing (fitzpatrick et al., 2018 , wang, x., 2018 ; diabetes/ulcers (martinez-sanchez et al., 2005 , guclu et al., 2016 , rosul, patskan, 2016 , ramirez-acuna et al., 2019 ; infectious diseases (smith et al., 2017 , mandhare et al., 2012 , song et al., 2018 , including the recent global pandemic disease of coronavirus disease 2019 (covid-19) (zheng et al., 2020) ; dentistry (isler et al., 2018 , khatri et al., 2015 , srikanth et al., 2013 , azarpazhooh et al., 2009 ; lung diseases (hernandez rosales et al., 2005) ; osteomyelitis (bilge et al., 2018) . the potential role of o2-o3 as an adjuvant therapy for cancer treatment has been also suggested in in vitro and animal studies as well as in isolated clinical reports (clavo et al., 2018) . at present, we have commenced a randomized double-blind clinical trial with the aim to test the efficacy of this therapy in a cognitive frailty cohort, a grant approved by the italian minister of health (rf-2016-02363298) . this pilot study will permit to validate the o2-o3 therapy in an early phase of cognitive decline, when it is still possible to intervene, before to develop a potential neurodegenerative pathology. to date, the o2-o3 therapy acquires a further prestigious significance, after the medicine nobel prize for "discovery of how cells sense oxygen" in 2019. indeed, o2 is the most vital element required for human life and it is the key to good health; o3 is o2 with an extra molecule added. the o2 availability affects genes expression of different factors (hifs, hypoxia inducible factors), leading to the activation of trophic proteins (vegf, vascular endothelial growth factor; pdgf, platelet-derived growth factor) and consequently to specific biological processes, including erythropoiesis, angiogenesis and anaerobic glucose metabolism . o3 plays a role of cellular adapter to hypoxia, because it is well known its effects in increasing the levels of vegf, j o u r n a l p r e -p r o o f pdgf, hif (curro et al., 2018 , zhang, j. et al., 2014 , re et al., 2010 , exactly as the cell does when there is no o2 available. oxidative stress is a condition where ros and nitrogen species (rns) production exceeds the cellular antioxidant defence system, leading to the imbalance between the two systems and this may contribute to the neuronal damage and the abnormal neurotransmission. it is widely known its implication in the pathogenesis and progression of nds (singh et al., 2019) . brain and mitochondria are the most involved systems due to their high sensitivity to oxidative damage caused by free radicals. oxidative damage may impair the cells in their structure and function, being cause and effect of a mitochondrial reduced activity. the damage is not confined to the brain but also evident in peripheral cells and tissues. ros and rns are also major factors in cellular senescence that leads to increase number of senescent cells in tissues on a large scale (liguori et al., 2018) . cellular senescence is a physiological mechanism that stops cellular proliferation in response to damages that occur during replication. senescent cells acquire an irreversible senescence-associated secretory phenotype (sasp), involving secretion of soluble factors (interleukins, chemokines, and growth factors), degradative enzymes like matrix metalloproteases (mmps), and insoluble proteins/extracellular matrix (ecm) components. nrf2 is a member of the cnc-basic leucine zipper (cnc-bzip) family of transcription factors. under basal condition, nrf2 binds to its repressor keap1 (kelch-like ech-associated protein 1), an adapter between nrf2 and cullin 3 protein, which leads to ubiquitination followed by proteasome degradation. this keap1-mediated degradation activity requires two reactive cysteine residues (cys273 and cys288). when o3 is administrated, it dissolves immediately in the plasma/serum and it reacts with pufa (polyunsaturated fatty acids), leading to the formation of the two fundamental messengers: hydrogen peroxide (h2o2) as a ros and 4-hydroxynonenal (4hne) as a lipid oxidation product j o u r n a l p r e -p r o o f (lop) (bocci, v. et al., 1998) (figure 1 ). ros are the early and short-acting messengers, while lops are late and long-lasting messengers. lops diffuse into all cells and inform them of a minimal oxidative stress. after the oxidative/electrophilic stress challenge (4hne, (ishii et al., 2004) , other aldehydes (levonen et al., 2004) , induced by o3 (galie et al., 2018 , siniscalco et al., 2018 , re et al., 2014 , vaillant et al., 2013 , modification of the cysteine residues of keap1 (s-hne or-s-s) inhibits ubiquitin conjugation to nrf2 by the keap1 complex (brigelius-flohe, flohe, 2011), provoking the nuclear accumulation of nrf2. once in the nucleus, nrf2 dimerizes and binds to cis-acting dna ares (antioxidant response elements) in genes such as heme oxygenase 1 (ho-1), a gene encoding enzyme that catalyses the degradation of heme in carbon monoxide (co) and free iron, and biliverdin to bilirubin. co acts as an inhibitor of another important pathway nf-κb (nuclear factor kappa b subunit 1) signalling, which leads to the decreased expression of proinflammatory cytokines, while bilirubin also acts as an important lipophilic antioxidant. furthermore, ho-1 directly inhibits the pro-inflammatory cytokines and activating the antiinflammatory cytokines, thus leads to balancing of the inflammatory process (ahmed, s. m. et al., 2017) . our research group confirmed that mild ozonisation, tested on in vitro systems, induced modulation of genes, including ho-1 (scassellati et al., 2017) . (figure 1 ). in addition, nrf2 regulates also the constitutive and inducible expression of antioxidants including, but not limited to, superoxide dismutases (sod), glutathione peroxidase (gsh-px), glutathione-s-transferase (gst), catalase (cat), nadph quinone oxidoreductase 1 (nqo1), phase ii enzymes of drug metabolism and hsps (galie et al., 2018 , bocci, v., valacchi, 2015 , pedruzzi et al., 2012 (figure 1) . a further mechanism involves casein kinase 2 (ck2), another regulator of the nrf2 activity through its phosphorylation. it has been demonstrated that o3 influenced the ck2 levels together with nrf2 phosphorylation, reducing oxidative stress and pro-inflammatory cytokines in multiple sclerosis patients (delgado-roche et al., 2017) . similarly, o3 inhibits oxidative stress through j o u r n a l p r e -p r o o f inhibition of the mitogen-activated protein kinase phosphatase (mapk) 1 signalling pathway (wang, l. et al., 2018) (figure 1a ). oxidative stress is one of the major drivers of protein misfolding that, accumulating and aggregating as insoluble inclusions can determine neurodegeneration (hohn et al., 2020 , knowles et al., 2014 . it is known that nfr2 promotes the clearance of oxidized or otherwise damaged proteins through the autophagy mechanism (tang et al., 2019) . interestingly, also o3 can modulate the degradation protein systems, not only via nrf2 pathway, but also via activation of the ampactivated protein kinase (ampk)/mammalian target of rapamycin (mtor) signaling pathway, as demonstrated in (zhao, x. et al., 2018 ) ( figure 1b ). o3 can protect against overproduction of nitric oxide (no), when no is a toxic oxidant. no can rapidly react with other free radicals such as o2 −• to generate highly reactive oxidant peroxinitrite (onoo − ) and other rns, which in turn damages the biomolecules (e.g., lipids, protein, dna/rna), playing thus a key role in chronic inflammation and neurodegeneration (massaad, 2011 , toda et al., 2009 ). it has been demonstrated that o3 downregulates inducible nitric oxide synthase (inos), which generates no (manoto et al., 2018 , smith et al., 2017 via nf-κb signalling ( figure 1c ). mitochondrial dysfunction is one of the main features of the aging process, particularly in organs requiring a high-energy source such as the heart, muscles, brain, or liver. neurons rely almost exclusively on the mitochondria, which produce the energy required for most of the cellular processes, including synaptic plasticity and neurotransmitter synthesis. mitochondrial disfunctions cause increase in ros for lowered oxidative capacity and antioxidant defence, with consequent increased oxidative damage to protein and lipids, decreased atp production and accumulation of dna damage (garcia-escudero et al., 2013 , reutzel et al., 2020 . moreover, mitochondrial j o u r n a l p r e -p r o o f bioenergetic dysfunction and release of pro-apoptotic mitochondrial proteins into the cytosol initiate a variety of cell death pathways. nrf2 transcribes several genes not only those implicated in antioxidant expression and energy regulation, but also those involved in mitochondria biogenesis: increases the mitophagy, mitochondrial levels of antioxidant enzymes, and resistance to redox regulated mitochondrial permeability transition pore opening (holmstrom et al., 2016) . multiple lines of evidence have shown that nfr2 activation is part of the retrograde response aimed at restoring mitochondrial functions after stress insults, and that the impairment of nrf2 functions is a hallmark of many mitochondrial-related disorders (shan et al., 2013) . it has been demonstrated that o3 administration can act on specific mechanisms to promote cell survival and proliferation, blocking the apoptotic processes. in particular, o3 decreases the expression of caspases 1-3-9, hifα, tumor necrosis factor-a (tnf-α), bcl-2-associated x protein (bax), poly (adp-ribose) polymerase 1 (parp-1) and p53 genes ( figure 2 ) (yong et al., 2017 , guclu et al., 2016 , wang, l. et al., 2018 . bax is located in the mitochondrial membranes and exerts pro-apoptosis effect through the mitochondrial pathway, promoting cytochrome c activation (mac nair et al., 2016) ; p53 and caspase-3 are executive molecules of apoptosis by blocking cell cycle (wang, j. et al., 2016) . enzymes such as sod, cat, and gsh-px, can regulate p53, bax and bcl-2 (bcl2 apoptosis regulator) ( figure 2 ). moreover, o3 stimulates the kreb's cycle in the mitochondria by enhancing the oxidative carboxylation of pyruvate and stimulating the production of adenosine triphosphate (atp) (guven et al., 2008) . it also causes a significant reduction of nicotinamide adenine dinucleotide (nadh), an increase of the coenzyme a levels to fuel the kreb's cycle and oxidizes cytochrome c (brigelius-flohe, flohe, 2011, elvis, ekta, 2011). o3 treatment was proven to reduce mitochondrial damage in a rat heart following ischemiareperfusion (meng et al., 2017) , as well as in a rat brain and cochlea following noise-induced j o u r n a l p r e -p r o o f hearing loss (nasezadeh et al., 2017) . moreover, in vitro, o3 increased the length of the mitochondrial cristae and the content of mitochondrial hsp70 . a list of biomarkers (29 in total) implicated in oxidative stress, in endogenous antioxidant and vitagene systems are showed in table 1 . these biomarkers have been studied and found modulated after the o2-o3 therapy in more of 150 studies performed in different in vivo (human and animal models) and in vitro samples and conditions. in table 1 , we also reported the relative functions of these biomarkers. from these 29 biomarkers, we focused, in this section, on those implicated in endogenous antioxidant-nrf2 pathway (gsh; gsh-px; glutathione reductase, gr; sod; cat; 4hne; advanced oxidation protein products, aopp in bold in table 1) . where it was possible (available studies), we performed meta-analyses for these biomarkers on human (see supplementary material). the results showed a significant increased levels of the sod-cat-gsh-px-gsh-gst-gr after o3 administration ( figure 3 , random model, z=6.15, p<0.00001, or=1.71 95%ci:1.17-2.25; even after bonferroni correction 0.05/6=0.0083). similar results were obtained even considering single markers, except for gr (z=1.04; p=0.30) and gsh (z = 0.80, p=0.42). gr has been investigated only in two studies, coming from the same authors (hernandez rosales et al., 2005) . thus, there are not enough evidence on its single real involvement. concerning gsh, diaz-luis et al., (díaz-luis et al., 2018) is the only study showing a negative effect of o3. as we followed the criteria for which the data were extracted before and after o3 treatment (see supplementary material), this study found an increased gsh levels after o3 administration, only when the authors performed the comparisons with control group of healthy subjects (in a sort of postconditioning). thus, if we eliminated this study, the results of the single meta-analysis of gsh highlighted its positive increase determined by the o3 treatment (z=2.30; p=0.02, data not shown). high heterogeneity in effect size across the studies (p < 0.00001, i 2 = 97%) was observed in these meta-analyses. this is essentially explained by the presence of different factors: the type of pathology, different concentration of o3 linked to different administration procedures and duration time treatments, age of the sample (supplementary material table 1s ). although there are contrasting results, most of the studies agree on the decrease in tissue levels of free radicals (lipid peroxidation markers, pro-oxidation biomarkers-for instance safwat et al. (safwat et al., 2014) demonstrated that o3 showed a beneficial effect on the aging reducing liver and kidney damage through its antioxidant property. o3 was efficient in j o u r n a l p r e -p r o o f elevating the reduced hepatic and renal gsh contents as well as in normalizing hepatic gsh-px activity of aged rats. moreover, o3 succeeded in attenuating the elevated hepatic and renal mda and protein carbonyls (pc) levels. another work (el-sawalhi et al., 2013) reported that o3 alleviated age-associated redox state imbalance, as evidenced by reduction of lipid and protein oxidation markers and lessening of lipofuscin deposition. moreover, o3 restored gsh levels in brain and heart tissues, and normalized gsh-px activity in the heart tissue of the aged-rats. o3 also mitigated age-associated energy failure in the heart and the hippocampus, improved cardiac cytosolic ca(2+) homeostasis and restored the attenuated na(+) , k(+) -atpase activity in the hippocampus of these rats. similarly, prophylactic administration of o3 in aged-rats normalized reduced gsh content, adenosine triphosphate/adenosine diphosphate ratio, mitochondrial sod and complex iv (cytochrome-c oxidase) activities. o3 improved glutathione redox index (gshri), complex i (nadh-ubiquinone oxidoreductase) and mitochondrial mtnos activities, and attenuated the rise mda and mitochondrial pc levels (shehata et al., 2012) . several evidence support the involvement of these biomarkers influenced by the o3 administration in the mechanisms of aging (table 1) . we prevalently focused on those implicated in the nrf2 signalling (in bold in table 1 ). it has been reported that the levels of lipid peroxidation products, reactive carbonyl compounds, such as 4hne, are increased in aging tissues (csala et al., 2015) , and this increase is positively correlated with age. impaired protein function, manifested as an increase in pc, plays a crucial role in aging processes (cabiscol et al., 2014) . with increase of pc, the spontaneous carbonyl-amino crosslinking and accumulation were mostly irreparable changes associated with aging (nowotny et al., 2014) . several findings evidenced altered levels of aopp in aging (komosinska-vassev et al., 2012 , rusanova et al., 2018 , qing et al., 2012 , silva et al., 2015 , muller et al., 2015 . a recent work although not involved in nrf2 signaling but influenced by o3 treatment, the increased oxidative damage to mitochondrial dna (mitdna) with the oh8dg (8-hydroxydeoxyguanosine) formation, represents the most common hallmark of the aging brain, marker of oxidative dna damage. the simultaneous increased oxidation of mtdna and deficiency of dna repair could enhance the lesion to mitochondrial genome, potentially causing neuronal damages (mecocci et al., 2018) . several evidence support the implication of the pro-oxidation and antioxidant defence biomarkers influenced by o3 listed in table 1 in the aetiopathogenetic mechanisms of nds. even for nds, we prevalently focused on those implicated in the nrf2 signalling (in bold in table 1 ). ad is characterized by progressive loss of cognitive and behavioral deterioration, which leads to the impairment of daily and routine activities. it is one of the most prevalent nds manifesting 45 million people worldwide. ad is characterized by the deposition of protein aggregates, extracellular amyloid plaques (a), intracellular tau () or neurofibrillary tangles, and loss of synaptic connections in specific regions of brain (schipper, 2010 , mattson, 2004 , selkoe, 2001 . the neuropathological diagnostic feature of ad is the accumulation of neurotoxic a oligomer peptides, which, along with  protein, mediates neurodegeneration, thus causing neuroinflammation, impairment in synaptic connection, cholinergic denervation, neurotransmitter imbalance, neuronal loss, and dendritic alterations. different studies indicate the relationship between a-induced oxidative imbalance and elevated levels of by-products of lipid peroxidation (e.g., 4hne, mda), protein oxidation (e.g., carbonyl), and dna/rna oxidation (e.g., oh8dg) (wang, x. et al., 2014 , zhao, y., zhao, 2013 , pratico, 2008 , mecocci et al., 2018 . these alterations were observed also in peripheral lymphocytes and lymphocyte mitochondria (for review (mecocci et al., 2018) . higher levels of pc, measured in mitochondria extracted from lymphocytes, have been observed in ad (for review (mecocci et al., 2018) . decreased levels of antioxidant enzymes like sod, cat, gsh and gssg, decreased ratio of gsh/gssg, and/or impaired expressions or activities of gsh-related enzymes have been observed in blood or brain of ad patients (singh et al., 2019 , liu et al., 2004 , kim et al., 2006 , oliveira, laurindo, 2018 . the rns such as no are also found to have a deleterious effect on neurons. indeed, rns elevation has been observed both in astrocytes as well as in neurons in an ad brain (for review (singh et al., 2019) . an increase in the expression of neuronal nnos or nos-1, cytokine-inducible inos or nos-2, and endothelial enos or nos-3 isozymes has been observed in ad astrocytes. the direct association of inos and enos with a aggregates indicating towards beta amyloid assisted in the induction of nos to produce no, which in turn leads to the formation of 3nitrotyrosine (nt) (luth et al., 2002 , luth et al., 2001 . other findings reported increased levels of ck2 in the hippocampus and temporal cortex of ad patients (rosenberger et al., 2016) and increased levels in aopp (can et al., 2013 , altunoglu et al., 2015 , compared to non-demented controls. it has been observed that ad patients show an increased oxidation of red blood cells gsh, which indicates oxidative stress in peripheral cells, and an increased level of plasma thiobarbituric acid reactive substances (tbars), which indicates a higher free radical oxidation of plasma unsaturated phospholipids (vina et al., 2005) . moreover, ho-1 has been proposed as systemic marker in early sporadic ad (schipper et al., 2000) . indeed, plasma ho-1 protein levels are significantly decreased in patients with probable sporadic ad (schipper, 2007) . the up-regulation of ho-1 in ad brain can be explained because of local oxidative stress. instead, the mechanism responsible for the downregulation of ho-1 in the blood of ad patients remains unclear, even though the existence of a ho-1 suppressor that inhibits ho-1 mrna levels in the lymphocytes in ad plasma has been proposed (maes et al., 2006) . however, the results about ho-1 plasma levels in patients with ad are controversial. a study founds no changes in the serum level of ho-1 in a big cohort of ad patients, as compared with elderly control subjects, whereas increased level were observed in pd patients, highlighting different mechanisms involved in the peripheral response to oxidative stress in the two diseases (mateo et al., 2010) . moreover, another study reports that in plasma of probable ad patients, both ho-1 and biliverdin reductase (bvr) levels are increased because of the enhanced oxidative stress. the authors suggested that plasma bvr status, more than ho-1, can represent a potential biochemical marker for the prediction of ad at the earliest stages of disease (di domenico et al., 2012) ; for review (nitti et al., 2018) . pd is the second most prevalent neurodegenerative disorder, after ad, which is characterized by the progressive degeneration of the dopaminergic neurons located in the substantia nigra (sn) pars compacta (spillantini et al., 1998) which affects movement. the main neuropathological hallmark of pd is the presence of intracellular inclusions known as lewy bodies j o u r n a l p r e -p r o o f (lbs) and neurites (lns) (forno, 1996) ; predominantly composed by misfolded and aggregated forms of the presynaptic protein α-synuclein (αsyn; a small protein with 140 amino acids abundant in presynaptic nerve terminals) (spillantini et al., 1998) . αsyn plays a role in synaptic transmission and dopamine levels adjustment. αsyn primarily affect tyrosine hydroxylase phosphorylation and activity and the expression level of dopamine transporter on the cell membrane. different evidence supported the involvement of the pro-oxidation and antioxidant defence biomarkers influenced by o3 listed in table 1 also with pd (focus on nrf2). altered levels of gsh and gssg, decreased ratio of gsh/gssg, and/or impaired expressions or activities of gsh-related enzymes have been detected in pd (liu et al., 2004) . tos and osi levels were found higher in the pd patients as compared to controls (mota et al., 2019). rns also plays major role in nitrosative stress in pd. no, produced by nnos or inos was found in large quantities in cells, as well as in the extracellular space around dopaminergic neurons (tieu et al., 2003) . it has been observed that in pd brains, no obstructs various enzymes including complex i and iv of the mitochondrial electron transport chain, hinders the function of proteins by forming s-nitrosothiols, mediates lipid peroxidation, resulting in elevated levels of ros and brain deteriorating effect. in situ hybridization and immunohistochemical studies also established the role of no in pd via postmortem brain tissue analysis, which indicates an elevated level of inos and nnos in basal ganglia structures (eve et al., 1998 , hunot et al., 1996 . onoohas been shown to inhibit the presynaptic dopamine transporter, which mediates the uptake of dopamine from the synaptic cleft to stop dopamine signalling, and to refill the dopamine vesicles. its inactivation will induce a decrease in dopamine delivery (picon-pages et al., 2019). oxidative damage in nucleic acids is likely to be a major risk factor for pd (bosco et al., 2006 , puspita et al., 2017 . oxidative dna lesions, such as 8-oxoguanine (8-oxog), accumulate in nuclear and mitochondrial genomes during aging, and such accumulation can increase dramatically in these patients (nakabeppu et al., 2007) . among the various neurodegenerative diseases, als is the most common type of motor neuron disease; it is sometimes called lou gehrig's disease, after the famous baseball player who had this condition. als is characterized by the progressive degeneration of upper and lower motor neurons in the spinal cord, cortex, and brainstem (kikuchi et al., 2002) . although for most of the increased lipid peroxidation was observed as compared with controls (oliveira, laurindo, 2018). in postmortem brain specimens of hd, a twofold increase of oh8dg in mtdna was found in the parietal and slightly less in the frontal cortex compared to controls (polidori et al., 1999) . at the core of adaptive responses at the cell and origin of biological organization is the concept of hormesis (calabrese, v. et al., 2010) . hormesis describes a process that results in ameliorating and improve cellular stress resistance, survival, and longevity in response to sub-lethal levels of stress (mattson, 2008) . generally, a favorable biological response to low exposure to any stressor is found within the hormetic zone, whereas cell damage occurs at higher doses. the hormetic dose response results from either a direct stimulation or through an overcompensation stimulatory response following disruption in homeostasis (calabrese, e. j., baldwin, 2000) . this theory is, to date a frontier area of neurobiological research, focal to understanding and developing new/complementary therapeutic approaches to nds. in this context, nrf2 is considered as a hormetic-like pathway (calabrese, v. et al., 2010) . it has widely been reported that the activation of nrf2 by several different mechanisms (calorie restriction, physical exercise, polyphenols, mushrooms) can be a way to improve life health, due to its transcriptionally modulation on the vitagene network. calabrese et al. (calabrese, v. et al., 2010) , performed an exhaustive review on this topic, and they described in detail each single element of the vitagene pathway. members of the hsp70s are, in their function as molecular chaperones, involved in folding of newly synthesized proteins and refolding of damaged or misfolded proteins, as well as in assembly and disassembly of protein complexes. trx, is a major redox control system, consisting of a 12 kda redox active protein trx, and a homodimeric selenoprotein called trxr1. trxr1 is a flavoprotein that catalyzes the nadph-dependent reduction of oxidized thioredoxin protein. it is usually located in the cytosol, but it translocates into the nucleus j o u r n a l p r e -p r o o f in response to various stimuli associated with oxidative stress, thereby playing a central role in protecting against oxidative stress. sirtuins are histone deacetylases which, in the presence of nad + as a cofactor, catalyze the deacetylation reaction of histone substrates and transcriptional regulators. sirtuins regulate different biological processes, such as apoptosis, cell differentiation, energy transduction, and glucose homeostasis. recent reviews support wide evidence on how different nutraceuticals/antioxidants can contrast aging and combat many associated pathologies, including nds (leri et al., 2020 , calabrese, e. j., 2020 . natural polyphenols (i.e. curcumin, resveratrol, flavonols present in ginkgo biloba extracts, polyphenols present abundantly in the leaves and in the ripening fruits of the olive tree, olea europaea), as well as mushrooms (hericium erinaceus, coriolus versicolor) can significantly modulated nrf2 and nrf2-dependent vitagenes expression, showing neuroprotective action. this can potentially resolves pathologies such as ad, pd and also meniere's disease, another degenerative pathology (amara et al., 2020 , trovato, siracusa, di paola, scuto, fronte et al., 2016 , trovato, siracusa, di paola, scuto, ontario et al., 2016 , trovato salinaro et al., 2018 , scuto et al., 2019 . in line with these findings, several studies demonstrated that also o3 can modulate the vitagene network expression. pharmacologically, it acts in a hormetic fashion (bocci, v. a. et al., 2011 , calabrese et al., 2013 , according an inverted v shape curve. we researched studies for metaanalyses regarding nrf2, ho-1, hsp70, trxr1 and sirtuins. whereas no studies were performed between sirtuins, trxr1 and o3, the results indicated that o3 can statistically increase the interestingly, a study reported the benefit effect of o3 on menière's disease (pawlak-osinska et al., 2004) . moreover, as reported for polyphenols and mushrooms (hsiao et al., 2016 , ferreiro et al., 2018 , oh et al., 2014 , pan et al., 2018 , hasanzadeh et al., 2020 , o3 has been found to be involved in β-catenin system (emon et al., 2017) as well as in nlrp3 (nitrogen permease regulator-like 3) inflammasome , wang, z., zhang et al., 2018 . all these evidence support that, as polyphenols and mushrooms, o3 acts in the same direction. induction of vitagenes after their supplementation/adminstration determines a maintained response to counteract intracellular pro-oxidant status, thus providing neuroprotection. preconditioning is a process whereby an initial low dose of a stressor agent upregulates adaptive mechanisms that enhance resilience against subsequent and acute stressor agents within a time-sensitive window of ∼ 10-14 days. (calabrese, e. j., 2016) . different studies demonstrated that the supplementation with coriolus versicolor (ferreiro et al., 2018 , scuto et al., 2019 , trovato salinaro et al., 2018 , trovato, siracusa, di paola, scuto, fronte et al., 2016 , and hericium herinaceus (trovato salinaro et al., 2018 , trovato, siracusa, di paola, scuto, ontario et al., 2016 , biomass and polyphenols (mao et al., 2019) can maintain the response to neutralize intracellular pro-oxidant/neuroinflammatory status, preventing different neurological conditions. same behaviour was also widely reported for o3. the term "ozone oxidative preconditioning" (ozoneop) was coined when repeated administration of o3 at nontoxic doses facilitate adaptation to oxidative stress. this occurs through mild immune system activation, enhanced release of growth factors and/or activation of metabolic pathways that help maintain redox balance (increased sod, gsh activities, decreased peroxidation). the first studies on ozoneop were conducted by barber et al., 1999 (barber et al., 1999 and leon os et al., 1998 (leon et al., 1998 . from 1998-1999, a plethora of investigations on this topic was conducted. in table 2 , we reported 65 findings, of which 55 on ozoneop, whereas 10 are the studies were on postconditioning phenomenon. we observed that ozoneop exerts a protective effect on ischemia-reperfusion injury (iri) in rat models of cochlear, hepatic, intestinal, renal, cardiac, lung and skeletal ischemia through an oxidative preconditioning mechanism that prevents the increase of the endogenous pro-oxidant and stimulates antioxidant mechanisms (table 2 ). some authors also developed an in vitro hypoxia/reoxygenation (h/r) model to simulate ozoneop, using normal rat kidney epithelial (nrk-52e) cells. this to eliminate confounding variables linked to animal models (wang, l., chen, liu, chen, weng, qiu & liu, 2014 , wang, l. et al., 2018 . interestingly, the results confirmed those obtained in in vivo animal model (table 2 ). ozoneop prevents also other different kind of injury: lipopolysaccharide (lps) injection, carbon tetrachloride, partial hepatectomy, total body irradiation, methotrexate, intraperitoneal injection of rat fecal material, sepsis, kidney and cardiac transplantation, contrast-induced nephropathy, induction of diabetes, cisplatin-induced nephrotoxicity, contrast-induced nephropathy agent, h2o2, doxorubicin, ototoxicity, noise exposure, hypothermia, lipofundin (table 2) . different methodological systems have been implemented in these studies. the several authors analysed differences in mrna gene expression levels as well as protein levels in western blot and biochemical analyses. all authors performed morphological, histopathological, immunofluorescence, and immunohistochemistry evaluations, in parallel and in concordance with molecular investigations. interestingly, in some cases, the effects observed were strongly dose and time-dependent (table 2 ). in some cases (10 in total), the studies have been performed in postconditioning, obtaining the same outcomes. león fernández et al. (leon fernandez et al., 2012) investigated the systemic redox status of patients with low back pain and neck pain, and if o3 oxidative postconditioning j o u r n a l p r e -p r o o f modified the pathological oxidative stress and protected against oxidative protein damage. in 33 patients with diagnosis of disc hernia (dh), 100% showed a severe oxidative stress. major changes in sod, total hydroperoxides, aopp, fructolysine, and mad were observed. after o3 postconditioning, there was a re-establishment of patients' cellular redox balance as well as a decrease in pain in both dh. this demonstrated that o2-o3 therapy protected against oxidation of proteins and reduced the pain. according to (cuadrado et al., 2018 , cuadrado et al., 2019 , systems medicine identifies a cluster of chronic disease pathophenotypes including nds in which nrf2 plays a fundamental role. similarly, nfr2 is strongly implicated in aging processes (zhang, h. et al., 2015 , schmidlin et al., 2019 , silva-palacios et al., 2018 . these condition/diseases share common mechanisms and results represent a first attempt to structure nrf2 as a common therapeutic and systems medicine approach. we here have presented extensively research and strength on the antioxidant activities of o3 correlated with the interaction with nrf2 (galie et al., 2018 , siniscalco et al., 2018 , re et al., 2014 , vaillant et al., 2013 , along with anti-apoptotic functions by acting on mitochondrial bax, caspases, p53 and hifα molecules (yong et al., 2017 , guclu et al., 2016 , pro-autophagy and bioenergetic activities on kreb's cycle. this paper provides a road map for mechanism-based systems medicine where o3-nfr2-vitagene network play a crucial role in the modulation of the cellular redox balance, in the reduction of the formation of ros/rns, in the change of apoptotic and autophagy mechanisms (vikram et al., 2017) . this underlines the evidence to become potential new therapeutic targets for nds, and at the sample time to reduce the aging physiological mechanisms and cognitive decline, potential risk factors to develop more severe neurodegeneration damage. challenges regarding treatments efficacy and costs still persist for nds. thus, we suggest that o2-o3 therapy could represent a useful, safe, no-invasive, no-pharmacological, economical, effective treatment for these neurodegenerative conditions. in the medical setting, this therapy employs a gas mixture of o2/o3, obtained from the modification of medical-grade o2 using j o u r n a l p r e -p r o o f certificated o3 generator device (bocci, v., 2011) . based on the basic mechanisms of action of o3 in blood, the therapeutic range of o3 has been precisely calculated and found to be 10-80 μg/ml of o3 in blood (schwartz-tapia et al., 2015) . o3 medical preparations are classified into three types: ozonized water, ozonized oil and ozonized gas, whereas different and main routes of application with relative concentrations of o3 are widely described in schwart-tapia et al., 2015 (schwartz-tapia et al., 2015 . the side effects are minimal; the world federation of ozone therapy (wfot) estimates the incidence of complications at 0.0007%. moreover, the treatment is not only perfectly tolerated but most of patients have reported a feeling of wellness and euphoria throughout the cycle. this fact explains why the compliance of the patients remains excellent throughout the years. the mechanisms of the positive effects of o3 are attributed not only to up-regulation of cellular antioxidant enzyme activity, but also to the activation of the immune and anti-inflammatory systems, modulation of nprl3 inflammasoma, action on proteasome, enhancement in the release of growth factors from platelets, improvement in blood circulation and o2 delivery to damaged tissues, and enhancement of general metabolism, along with being a potent bactericide, fungicide and virucidal with potential effect on gut microbiota (for review (scassellati et al., 2020) . consequently, these combinatorial effects could impact on cognitive and neurodegenerative domains, directly or indirectly through the mediation of gut microbiota . nrf2-are and vitagene network, but also nf-κb (nuclear factor kappa b subunit 1), nfat (nuclear factor activated t-cells), ap-1 (activated protein-1), hifα are the principal signalling pathways on which o3 exercises its effects (for review (scassellati et al., 2020) . these effects could be sharable with those involved in nds, where high inflammation and oxidant state, mitochondria dysfunctions, metabolic alterations, and slowdown in regenerative processes and immune system characterize these disorders. as reported in (smith et al., 2017) , to date systems are available and proposed to have a more precise measurement of the redox state of a patient. one system proposes simultaneously j o u r n a l p r e -p r o o f measuring different biological markers in the blood such as gsh, gsh-px, gst, sod, cat, conjugated dienes, total hydroperoxides, and tbars. using an algorithm, information can be gathered about the total antioxidant activity, total pro-oxidant activity, redox index, and grade of oxidative stress. thus, systems like this can provide insights to the correct dosage and response to o3 therapy based on oxidative stress levels seen in the patient. with the awareness that further studies are needed, this review reports substantial scientific evidence for building a rationale of using the o2-o3 therapy for delay aging processes and neurodegeneration, exploiting well documented omni various functions of o3. this therapy could represent a convenient, inexpensive monodomain intervention, working in absence of side effects that will permit to modulate the oxidant, but also immune, inflammatory, metabolic, microbiota and regenerative processes impaired in nds. there is a recent consistent upsurge of interest in complementary medicine, especially dietary supplements and foods functional in delaying the onset of age-associated nds. o3 along with other antioxidants (polyphenols, mushrooms) can open new neuroprotective strategies, and could represent therapeutic targets to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and nds. catia scassellati and antonio carlo galoforo contributed equally to this work. the authors have declared no conflict of interest. aso, e., lomoio, s., lopez-gonzalez, i., joda, l., carmona, m., fernandez-yague, n., moreno, j., juves, s., pujol, a., pamplona, r., portero-otin, m., martin, v., diaz, m. ferrer, i. 2012 . in the absence of stimuli, nrf2 (nuclear factor erythroid 2-related factor 2) binds to its repressor keap1 (kelch-like ech-associated protein), an adapter between nrf2 and cullin 3 protein, which leads to ubiquitination followed by proteasome degradation. when o3 is administrated, it dissolves various apoptotic stimuli (ischemia, reactive oxidant species, ros, ipoxia) can activate directly p53 that in turn can play a role as transcription factor and activate the expression of pro-apoptotic genes. among these, bak (bcl-2 homologous antagonist/killer) and bax (bcl-2-associated x protein) can stimulate in mitochondrial membrane the activation of cytochrome c that in turn j o u r n a l p r e -p r o o f activates apaf1 (apoptotic protease activating factor-1) and caspase 9 to close the circle to stimulate the activity of caspase 3. enzymes such as sod (superoxide dismutase), cat (catalase), and gsh-px (glutathione peroxidase), can regulate p53, bax and bcl-2. o3 administration decreases the expression of caspases 1-3-9, hypoxia-inducible factor (hifα), tumor necrosis factor-α (tnf-α), bax and p53 genes. (bid an acronym for bh3-interacting domain death agonist). ci, confidence interval; chi 2 , χ 2 test of goodness of fit; tau 2 , estimate of the between-study variance in a random-effects meta-analysis. nuclear factor nrf2, heme-oxigenase (ho-1), heat shock protein (hsp ) table 1 . list of the pro-oxidation and antioxidant defence biomarkers influenced by ozone (o3) and implicated in neurodegenerative disorders phospholipid oxidation and carotenoid supplementation in alzheimer's disease patients. free radic cardioprotective effects of ozone oxidative preconditioning in an in vivo model of ischemia/reperfusion injury in rats. scandinavian journal of clinical and laboratory investigation jid -0404375 nrf2 signaling pathway: pivotal roles in inflammation similar protective effect of ischaemic and ozone oxidative preconditionings in liver ischaemia/reperfusion injury role of protein synthesis in the protection conferred by ozoneoxidative-preconditioning in hepatic ischaemia/reperfusion effects of ozone oxidative preconditioning on nitric oxide generation and cellular redox balance in a rat model of hepatic ischaemia-reperfusion the oxygen prescription : the miracle of oxidative therapies ischemia-modified albumin and advanced oxidation protein products as potential biomarkers of protein oxidation in alzheimer's disease hericium erinaceus prevents dehp-induced mitochondrial dysfunction and apoptosis in pc12 cells mechanisms of pathophysiology of blood vessels in patients with multiple sclerosis treated with ozone therapy: a systematic review does intraperitoneal medical ozone preconditioning and treatment ameliorate the methotrexate induced nephrotoxicity in rats? association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly ozone oxidative preconditioning inhibits inflammation and apoptosis in a rat model of renal ischemia/reperfusion injury ozone oxidative preconditioning protects the rat kidney from reperfusion injury: the role of nitric oxide similarities between ozone oxidative preconditioning and ischemic preconditioning in renal ischemia/reperfusion injury human studies related to protein oxidation: protein carbonyl content as a marker of damage protein phosphatase 2a as a therapeutic target in inflammation and neurodegeneration ozone therapy as adjuvant for cancer treatment: is further research warranted? evid based low ozone concentrations promote adipogenesis in human adipose-derived adult stem cells circulating advanced oxidation protein products as oxidative stress biomarkers and progression mediators in pathological conditions related to inflammation and immune dysregulation on the role of 4-hydroxynonenal in health and disease transcription factor nrf2 as a therapeutic target for chronic diseases: a systems medicine approach therapeutic targeting of the nrf2 and keap1 partnership in chronic diseases anti-inflammatory and tissue regenerative effects of topical treatment with ozonated olive oil/vitamin e acetate in balanitis xerotica obliterans ozone-oxidative preconditioning prevents doxorubicin-induced cardiotoxicity in sprague-dawley rats ozone oxidative preconditioning prevents atherosclerosis development in new zealand white rabbits medical ozone promotes nrf2 phosphorylation reducing oxidative stress and pro-inflammatory cytokines in multiple sclerosis patients ho-1/bvr-a system analysis in plasma from probable alzheimer's disease and mild cognitive impairment subjects: a potential biochemical marker for the prediction of the disease systemic ozone therapy by rectal insufflation for immunoglobulin a deficiency pathology of neurodegenerative diseases controlled ozone therapy modulates the neurodegenerative changes in the frontal cortex of the aged albino rat modulation of age-related changes in oxidative stress markers and energy status in the rat heart and hippocampus: a significant role for ozone therapy ozone therapy: a clinical review effects of ozone on spinal cord recovery via the wnt/ &beta;-catenin pathway following spinal cord injury in rats basal ganglia neuronal nitric oxide synthase mrna expression in parkinson's disease heme oxygenase-1 protein carbonylation as a major hallmark of oxidative damage: update of analytical strategies determination of parameters of oxidative stress in vitro models of neurodegenerative diseases-a review ozone postconditioning in renal ischaemia-reperfusion model. functional and morphological evidences coriolus versicolor biomass increases dendritic arborization of newly-generated neurons in mouse hippocampal dentate gyrus ozone therapy for the treatment of chronic wounds: a systematic review neuropathology of parkinson's disease mild ozonisation activates antioxidant cell response by the keap1/nrf2 dependent pathway. free radic deconstructing mitochondrial dysfunction in alzheimer disease the human disease network glutathione redox imbalance in brain disorders effect of ozone pre-conditioning on redox activity in a rat model of septic shock toxicology mechanisms and methods the effects of ozone therapy on caspase pathways, tnf-alpha, and hif-1alpha in diabetic nephropathy effects of ozone oxidative preconditioning on radiation-induced organ damage in rats effects of ozone oxidative preconditioning on liver regeneration after partial hepatectomy in rats the efficacy of ozone therapy in experimental caustic esophageal burn effect of ozone on intestinal recovery following intestinal ischemia-reperfusion injury in a rat nitric oxide homeostasis in neurodegenerative diseases curcumin: an inflammasome silencer ozone therapy effects on biomarkers and lung function in asthma proteostasis failure in neurodegenerative diseases: focus on oxidative stress the multifaceted role of nrf2 in mitochondrial function immunomodulator 'mushroom beta glucan' induces wnt/beta catenin signalling and improves wound recovery in tilapia and rat skin: a histopathological study nitric oxide synthase and neuronal vulnerability in parkinson's disease role of nrf2 in the regulation of cd36 and stress protein expression in murine macrophages: activation by oxidatively modified ldl and 4-hydroxynonenal the effects of ozone therapy as an adjunct to the surgical treatment of peri-implantitis efficacy of comprehensive ozone therapy in diabetic foot ulcer healing protective effects of ozone oxidative postconditioning on long-term injury after renal ischemia/reperfusion in rat alterations in nitric oxide synthase in the aged cns ozone ameliorates methotrexateinduced intestinal injury in rats evaluation of effect of topical ozone therapy on salivary candidal carriage in oral candidiasis detection of n epsilon-(carboxymethyl)lysine (cml) and non-cml advanced glycation end-products in the anterior horn of amyotrophic lateral sclerosis spinal cord decreased plasma antioxidants in patients with alzheimer's disease the amyloid state and its association with protein misfolding diseases effect of hyperbaric oxygen and ozone preconditioning on oxidative/nitrosative stress induced by tourniquet ischemia/reperfusion in rat skeletal muscle effects of ozone (o(3)) therapy on cisplatin-induced ototoxicity in rats age-and gender-related alteration in plasma advanced oxidation protein products (aopp) and glycosaminoglycan (gag) concentrations in physiological ageing beneficial effects of nontoxic ozone on h(2)o(2)-induced stress and inflammation ozone preconditioning attenuates contrast-induced nephropathy in rats the hsp70/hsp90 chaperone machinery in neurodegenerative diseases ozone oxidative preconditioning is mediated by a1 adenosine receptors in a rat model of liver ischemia/ reperfusion. transplant international : official journal of the european society for ozone oxidative postconditioning reduces oxidative protein damage in patients with disc hernia ozone oxidative preconditioning: a protection against cellular damage by free radicals healthy effects of plant polyphenols: molecular mechanisms ozone in biology cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products oxidative stress, aging, and diseases glutathione metabolism during aging and in alzheimer disease expression of endothelial and inducible nos-isoforms is increased in alzheimer's disease, in app23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology aberrant expression of nos isoforms in alzheimer's disease is structurally related to nitrotyrosine formation retinal glial responses to optic nerve crush are attenuated in bax-deficient mice and modulated by purinergic signaling pathways salivary antioxidant barrier, redox status, and oxidative damage to proteins and lipids in healthy children, adults, and the elderly ozone therapy in induced endotoxemic shock. ii. the effect of ozone therapy upon selected histochemical reactions in organs of rats in endotoxemic shock characterization of alpha1-antitrypsin as a heme oxygenase-1 suppressor in alzheimer plasma human myeloperoxidase (hmpo) is expressed in neurons in the substantia nigra in parkinson's disease and in the hmpo-alpha-synuclein-a53t mouse model, correlating with increased nitration and aggregation of alpha-synuclein and exacerbation of motor impairment. free radic medical ozone therapy as a potential treatment modality for regeneration of damaged articular cartilage in osteoarthritis a meta-analysis of resveratrol protects against myocardial ischemia/reperfusion injury: evidence from small animal studies and insight into molecular mechanisms the role of hsp70 in oxi-inflamm-aging and its use as a potential biomarker of lifespan therapeutic efficacy of ozone in patients with diabetic foot neuronal and vascular oxidative stress in alzheimer's disease serum heme oxygenase-1 levels are increased in parkinson's disease but not in alzheimer's disease hormesis defined pathways towards and away from alzheimer's disease glutathione transferases and neurodegenerative diseases a long journey into aging, brain aging, and alzheimer's disease following the oxidative stress tracks biomarker development for c9orf72 repeat expansion in als relationship of advanced oxidative protein products in human saliva and plasma: age-and gender-related changes and stability during storage effect of ozone oxidative preconditioning on oxidative stress injury in a rat model of kidney transplantation. experimental and therapeutic medicine oxygen radicals, nitric oxide, and peroxynitrite: redox pathways in molecular medicine diabetic foot ulcers: current advances in antimicrobial therapies and emerging treatments myeloperoxidase: bridging the gap in neurodegeneration is ozone pre-conditioning effect linked to nrf2/epre activation pathway in vivo? a preliminary result role of ozone/oxygen in fibroblast growth factor activation. discovering the facts ozone therapy: clinical and basic evidence of its therapeutic potential cerebral mitochondrial function and cognitive performance during aging: a longitudinal study in nmri mice erythrocyte plasma membrane redox system in human aging effects of ozone oxidative preconditioning on different hepatic biomarkers of oxidative stress in endotoxic shock in mice preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis increased occurrence of protein kinase ck2 in astrocytes in alzheimer's disease pathology ozone therapy effectiveness in patients with ulcerous lesions due to diabetes mellitus new model of glutathione deficit during development: effect on lipid peroxidation in the rat brain analysis of plasma micrornas as predictors and biomarkers of aging and frailty in humans ozone ameliorates agerelated oxidative stress changes in rat liver and kidney: effects of pre-and post-ageing administration age-related changes in ampk activation: role for ampk phosphatases and inhibitory phosphorylation by upstream signaling pathways major ozonated autohemotherapy preconditioning ameliorates kidney ischemia-reperfusion injury molecular mechanisms in cognitive frailty: potential therapeutic targets for oxygen-ozone treatment effects of mild ozonisation on gene expression and nuclear domains organization in vitro do post-translational modifications influence protein aggregation in neurodegenerative diseases: a systematic review biological markers and alzheimer disease: a canadian perspective biomarker potential of heme oxygenase-1 in alzheimer's disease and mild cognitive impairment evaluation of heme oxygenase-1 as a systemic biological marker of sporadic ad the sinister face of heme oxygenase-1 in brain aging and disease redox regulation by nrf2 in aging and disease. free radic nutritional mushroom treatment in meniere's disease with coriolus versicolor: a rationale for therapeutic intervention in neuroinflammation and antineurodegeneration alzheimer's disease results from the cerebral accumulation and cytotoxicity of amyloid beta-protein frataxin deficiency leads to defects in expression of antioxidants and nrf2 expression in dorsal root ganglia of the friedreich's ataxia yg8r mouse model the potential role of ozone in ameliorating the age-related biochemical changes in male rat cerebral cortex association between advanced oxidation protein products and 5-year mortality risk among amazon riparian elderly population nrf2: molecular and epigenetic regulation during aging oxidative stress: a key modulator in neurodegenerative diseases intraperitoneal administration of oxygen/ozone to rats reduces the pancreatic damage induced by streptozotocin nrf2 and nf-b interplay in cerebrovascular and neurodegenerative disorders: molecular mechanisms and possible therapeutic approaches ozone therapy: an overview of pharmacodynamics, current research, and clinical utility aging effect on myeloperoxidase in rat kidney and its modulation by calorie restriction combined ischemic postconditioning and ozone postconditioning provides synergistic protection against renal ischemia and reperfusion injury through inhibiting pyroptosis emerging roles of ozone in skin diseases. zhong nan da xue xue bao yi xue ban oxidative stress and mitochondrial dysfunction in alzheimer's disease coriolus versicolor alleviates diabetic cardiomyopathy by inhibiting cardiac fibrosis and nlrp3 inflammasome activation aberrations in oxidative stress markers in amyotrophic lateral sclerosis: a systematic review and meta-analysis effect of ozone oxidative preconditioning on inflammation and oxidative stress injury in rat model of renal transplantation ozone protects the rat lung from ischemia-reperfusion injury by attenuating nlrp3-mediated inflammation, enhancing nrf2 antioxidant activity and inhibiting apoptosis evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation who 2011 ageing, neurodegeneration and brain rejuvenation ozone oxidative preconditioning protects the rat kidney from reperfusion injury via modulation of the tlr4-nf-kappab pathway novel biomarkers for the evaluation of aging-induced proteinopathies hypoxia and aging effect of local ozone treatment on inflammatory cytokine , growth cytokine and apoptosis molecule expression in anal fistula wound the nlrp3 inflammasome is a potential target of ozone therapy aiming to ease chronic renal inflammation in chronic kidney disease effects of ozone oxidative preconditioning on tnf-alpha release and antioxidant-prooxidant intracellular balance in mice during endotoxic shock inhibition of tumor necrosis factor-alpha release during endotoxic shock by ozone oxidative preconditioning in mice oxidative stress response and nrf2 signaling in aging. free radic increased growth factors play a role in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot ulcers ozone induces autophagy in rat chondrocytes stimulated with il-1beta through the ampk/mtor signaling pathway oxidative stress and the pathogenesis of alzheimer's disease a preliminary evaluation on the efficacy of ozone therapy in the treatment of covid-19 a control was performed with oxygen. (procaspase-1), cysteine-requiring aspartate protease-1 (caspase-1), apoptotic index, interleukin-1 beta (il-1) macroscopic and histologic view: dark and edematous tissue, inter alveolar septum, rupturing and alveolar space hemorrhage disappear mg/kg, intraperitoneal, daily for 5 days, with/without sepsis. a control was performed with oxygen. reduction: serum alanine amino transferase (alt), aspartate amino transferase (ast), creatinine (cre), thiobarbituric acid reactive substances (tbars), myeloperoxidase (mpo). increase: superoxide dismutase (sod) inducible nitric oxide synthase (inos), nitrite levels. increase dose-dependent manner: catalase (cat), glutathione peroxidase (gsh-px), superoxide dismutase (sod), glutathione (gsh) expression. morphology: recovered the majority of cells from the toxicity, regenerated cell proliferation, prevented 9.6% and 11.0% of cell loss increase dose-dependent manner: superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) mg/kg, intraperitoneally, single dose for 10 days, at the same time escherichia coli toxin (lps) (20 mg/kg). reduction: lactate dehydrogenase (ldh) (liver, kidney, lungs, heart). increase: succinate dehydrogenase (sdh) (lungs, heart), adenosine triphosphatase (atpase) (no kidney), acid phosphatase (acpase) (liver, kidney, lungs, heart), -glucuronidase (liver, kidney, lungs) histochemically detected activity of succinate dehydrogenase (sdh): extinguished enzymatic activity in central parts of the lobule and paralleled by narrowing of zone i (liver) histochemically detected activity of lactate dehydrogenase (ldh): increased activity (hepatocytes, kupffer cells, liver) histochemically detected activity of adenosine triphosphatase (atpase): decrease intensity of the reaction for atpase (liver) histochemically detected activity of acid phosphatase (acpase): lower decrease in activity (liver) this research was supported by grants from the italian ministry of health as ricerca corrente j o u r n a l p r e -p r o o f (nds) as well as in aging processes. involvement in nds involvement in aging processes 4-hne 4-hydroxynonenal: a common aldehyde byproduct of lipid peroxidation during oxidative stress. 4-hne is highly reactive and primarily produced in the brain via lipid peroxidation of arachidonic acid, a highly abundant omega-6 polyunsaturated fatty acids (pufa) component of neuronal membranes. hne may modify the atp synthase, the final step in the production of atp from electron transport chain (etc) inside mitochondria. 4-hne activates nrf2 by alkylating thiol groups of cysteine residue in keap1.( moldogazieva et al., 2019 , ayala et al., 2014 , baker et al., 2015 ( benedetti et al., 2014 , csala et al., 2015 8-ohdg 8-hydroxydeoxyguanosine (8-oxo-2'-deoxyguanosine (8-oxo-dg): oxidized derivative of deoxyguanosine. its concentrations within a cell are a measurement of oxidative stress (dna oxidation). reactive oxygen species (ros) attack guanine bases in dna easily and form 8hydroxydeoxyguanosine, which can bind to thymidine rather than cytosine; thus, the level of 8-ohdg is generally regarded as a biomarker of mutagenesis consequent to oxidative stress. , nakabeppu et al., 2007 , poulsen et al., 2014 , polidori et al., 1999 ) (mecocci et al., 2018) aopp advanced oxidation protein products: are group of oxidatively modified protein products containing dityrosine, pentosidine, and carbonylcontaining products generated by reactive oxygen species (ros) or formed via myeloperoxidase reaction during oxidative/chlorine stress. they are biomarkers of oxidant-mediated protein damage , cristani et al., 2016 ) (maciejczyk et al., 2019 , cakatay et al., 2008 , komosinska-vassev et al., 2012 , rusanova et al., 2018 , qing et al., 2012 , silva et al., 2015 , muller et al., 2015 cat catalase: it catalyzes the decomposition of hydrogen peroxide to water and oxygen. it is a scavenger enzyme of reactive oxygen species (ros), protecting the cell from oxidative damage by ros. (feitosa et al., 2018 ) (veal et al., 2018 frap ferric reducing the ability of plasma: total antioxidant capacity of plasma. (ademowo et al., 2017 ) (muller et al., 2015 , rizvi et al., 2006 fructolysine it is an amadori adduct of glucose to lysine. it is a precursor of the --j o u r n a l p r e -p r o o f 51 advanced oxidation protein products, which are induced by oxidative stress, and induces oxidative stress. glutathione reductase (or glutathione-disulfide reductase, gsr): it catalyses the reduction of glutathione disulfide (gssg) to the sulfhydryl form glutathione (gsh), which is a critical molecule in resisting oxidative stress and maintaining the reducing environment of the cell. (feitosa et al., 2018 , liu et al., 2004 , rougemont et al., 2002 ( veal et al., 2018) gsh glutathione: it is antioxidant, capable of preventing damage to important cellular components caused by reactive oxygen species (ros). it maintains cellular thiol status. (mazzetti et al., 2015 , liu et al., 2004 , gu et al., 2015 , rougemont et al., 2002 , oliveira, laurindo, 2018 ) (maciejczyk et al., 2019 , teskey et al., 2018 , oliveira, laurindo, 2018 glutathione peroxidase: it has peroxidase activity whose main biological role is to protect the organism from oxidative damage. the biochemical function is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. (mazzetti et al., 2015 , gu et al., 2015 , rougemont et al., 2002 ) (maciejczyk et al., 2019 , veal et al., 2018 gst glutathione s-transferase: it is phase ii metabolic isozyme, known for the ability to catalyze the conjugation of the reduced form of glutathione (gsh) to xenobiotic substrates for the purpose of detoxification. (mazzetti et al., 2015 , gu et al., 2015 , rougemont et al., 2002 ) (veal et al., 2018 hif-1α hypoxia-inducible factor (hif)-1alpha: is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (hif-1). it is a basic helixloop-helix pas domain containing protein and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia.(merelli et al., 2018) (yeo, 2019) ho-1 heme-oxygenase-1: it catalyzes the conversion of heme into free iron, carbon monoxide and biliverdin. it possesses two well-characterized isoforms: ho-1 and ho-2. under brain physiological conditions, the expression of ho-2 is constitutive, abundant and ubiquitous, whereas ho-1 mrna and protein are restricted to small populations of neurons and neuroglia. ho-1 is an inducible enzyme that has been shown to participate as an essential defensive mechanism for neurons exposed to oxidant challenges, being related to antioxidant defenses in certain neuropathological conditions. mda malondialdehyde: is a marker for oxidative stress. it is a reactive aldehyde produced by lipid peroxidation of polyunsaturated fatty acids. (feitosa et al., 2018 , ayala et al., 2014 ) (csala et al., 2015 , maciejczyk et al., 2019 mpo myeloperoxidase: is a peroxidase enzyme. it requires heme as a cofactor. it is expressed in neutrophil and monocyte, and is implicated in various stages of inflammatory conditions with the production of a variety of potent oxidants. (ray, katyal, 2016 , maki et al., 2019 ) (son et al., 2005) nfr2/ck2 nuclear factor erythroid 2-related factor 2: is a basic leucine zipper (bzip) protein that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. casein kinase 2: a serine/threonine-selective protein kinase implicated in cell cycle control, dna repair, regulation of the circadian rhythm, and other cellular processes. regulator of the nrf2 activity through its phosphorylation. nitric oxide: is an important cellular signaling molecule which is derived from l-arginine by nitric oxide synthase (nos). it works as a retrograde neurotransmitter in synapses, allows the brain blood flow, and has important roles in intracellular signaling in neurons from the regulation of the neuronal metabolic status to the dendritic spine growth. it is able to perform post-translational modifications in proteins by the s-nitrosylation of the thiol amino acids, which is a physiological mechanism to regulate protein function. (hannibal, 2016 , nakamura, lipton, 2020 , radi, 2018 (picon-pages et al., 2019) nitrate/nitrite: an index of no production (hannibal, 2016 , nakamura, lipton, (picon-pages et al., 2019 j o u r n a l p r e -p r o o f 53 2020, radi, 2018) nitric oxide synthase (inducible i ii, endothelial e i): it catalyzes the production of nitric oxide (no) from l-arginine. (hannibal, 2016 , nakamura, lipton, 2020 ) (jung et al., 2012) pcc/pco protein carbonyl content: catalyses the carboxylation reaction of propionyl coa in the mitochondrial matrix. (chevion et al., 2000 , fedorova et al., 2014 ) (cabiscol et al., 2014 , cakatay et al., 2008 ) pp protein phosphatase: it is a serine/threonine phosphatase. it has been found to be important in the control of glycogen metabolism, muscle contraction, cell progression, neuronal activities, splicing of rna, mitosis, cell division, apoptosis, protein synthesis, and regulation of membrane receptors and channels. reduction: malondialdehyde (mda). increase: superoxide dismutase (sod), glutathione peroxidase gsh-px. histologically: ileum: less inflammatory cell infiltration and edema, reduction in vacuolated cells in the epithelium; liver/kidney: no significant change, due probably to the cumulative prolonged effect of mtx on these tissues. (kesik et al., 2009) postconditioning : sprague dawley rats: 1, 2 mg/kg, rectal insufflations, 15 applications, once a day, ischemia/reperfusion. renal tubular epithelial cell line, nrk-52e: 20, 30, 40 μg/ml in complete medium, hypoxiareoxygenation. reduction dose-dependent manner: blood urea nitrogen (bun), creatinine (cr), malondialdehyde (mda), bcl-2-associated x (bax) and poly (adp-ribose) polymerase 1 (parp-1) expression, mapk signaling pathway. increase dose-dependent manner: superoxide dismutase (sod). histologically: ozone protected the tubular epithelium from swelling and from loss of the brush border. in vitro: reduction dose-dependent manner: mapk pathways, creb, c-fos, bcl-2associated x (bax) and poly (adp-ribose) polymerase 1 (parp-1) expression, apoptosis, malondialdehyde (mda), phosphorylation of p38, erk1/2, and jnk. increase dose-dependent manner: superoxide dismutase (sod). (wang, l. et al., 2018) postconditioning: sprague dawley rats: 2 mg/kg, rectal insufflations, 15 applications, once a day, after ischemia/reperfusion. renal tubular epithelial cell line, nrk-52e: 20, 30, 40 μg/ml in complete medium, after hypoxia-reoxygenation. morphological/immunohistochemistry: increase in collagen staining, reduction in α-sma expression. qiu, liu & zhu, 2014) postconditioning: 0.5 mg/kg, daily for the 10 days' reperfusion, after ischaemia-reperfusion. a control was performed with oxygen.reduction: serum creatinine (cr), blood urea nitrogen (bun), thiobarbituric acid reactive substances (tbars). increase: fructosamine, phospholipase a2, superoxide dismutase (sod). morphology: minimal alterations. (calunga et al., 2009) preconditioning: 1 mg/kg, rectal insufflations, 15 applications, once a day, before the kidney transplantation.reduction: serum blood urea nitrogen (bun), creatinine (cr), malondialdehyde (mda), renal allograft cell apoptosis index. increase: superoxide dismutase (sod), glutathione (gsh), catalase (cat), nuclear factor erythroid 2-related factor 2 (nrf-2), heme oxygenase 1 (ho-1). morphological/immunohistochemistry: lower levels of damage, less severe renal allograft. (qiu et al., 2017) preconditioning: 0.7 mg/kg/d, intraperitoneally, 5 days, before the induction of contrast-induced nephropathy. a control group was with oxygen.reduction: serum blood urea nitrogen (bun), creatinine (cr), serum/renal malondialdehyde (mda), total oxidant status (tos). increase: serum/renal nitric acid (no), total antioxidant status (tas). histopathologic evaluation: reduction in degeneration of tubular epithelium, dilatation of bowman capsule, necrosis in tubular epithelium, vascular congestion. (kurtoglu et al., 2015) preconditioning: 1 mg/kg, rectal insufflations, 15 applications, once a day, before ischemia/reperfusion and/or ischemic preconditioning.reduction: malondialdehyde (mda), urea nitrogen (bun), creatinine (cr), jablonski grading scale scores. increase: serum nitric acid (no), no synthase (endothelial, enos and inducible, inos) expression/protein, glutathione (gsh), superoxide dismutase (sod), glutathione peroxidase (gsh-px). histological examination/immunohistochemistry: improved renal dysfunction, histological damage, renal oxidative stress, increase presence of endothelial, enos and inducible, inos. (chen, xing, liu, zhan, zhou, zhu & chen, 2008b) preconditioning: in vitro renal tubular epithelial cell line, nrk-52e, 20, 30, 40 μg/ml in complete medium, before hypoxia/reoxygenation. reduction dose-dependent manner: 40 μg/ml apoptosis rate, malondialdehyde (mda), lactate dehydrogenase (ldh), bcl-2-associated x (bax), bcl2, poly (adp-ribose) polymerase 1 (parp-1) expression. increase dose-dependent manner: superoxide dismutase (sod). immunocytochemistry: decrease in cleaved caspase3-positive (wang, l., chen, liu, chen, weng, qiu & liu, 2014) preconditioning: 1 mg/kg, rectal insufflations, 15 applications, once a day, before ischemia/reperfusion. reduction: serum blood urea nitrogen (bun), creatinine (cr), malondialdehyde (mda), myeloperoxidase (mpo), tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), interleukin-6 (il-6), intercellular adhesion molecule (icam-1), monocyte chemoattractant protein 1 (mcp-1), toll-like receptor (tlr4), nuclear factor (nf-κb) expression/protein, caspase-3, bcl-2-associated x (bax), bcl2. morphology: decreased score in jablonski scale histology grading. (chen, xing b fau -liu, xiuheng et al., 2008) preconditioning: 1 mg/kg, rectal reduction: malondialdehyde (mda), serum blood urea nitrogen (bun), creatinine (cr), (xing et al., 2015) j o u r n a l p r e -p r o o f 56 insufflations, 15 applications, once a day, before ischemia/reperfusion. tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), interleukin-6 (il-6), intercellular adhesion molecule (icam-1), monocyte chemoattractant protein 1 (mcp-1), toll-like receptor (tlr4) and nuclear factor (nf-κb) expression/protein /immunoistochemical, caspase-3, bcl-2-associated x (bax), bcl2. morphological/immunoistochemical features: relieved tubular necrosis, medullary haemorrhage, congestion and development of proteinaceous casts, reduction in jablonski scores. preconditioning: 1 mg/kg, rectal insufflations, 15 treatments, once a day, before ischemia/reperfusion. as control was used also oxygen.reduction: serum blood urea nitrogen (bun), creatinine (cr), jablonski grading scale scores, endothelin-1. increase: serum nitric oxide (no), no synthase (endothelial, enos, inducible, inos) expression/protein, superoxide dismutase (sod), glutathione (gsh), glutathione peroxidase (gsh-px). morphology: preservation of tissue histology. (chen, xing, liu, zhan, zhou, zhu & chen, 2008a) postconditioning: 0.5 mg/kg, rectal insufflations, 10 applications, once a day, after ischemia/reperfusion. as control was used also oxygen.histopathological/morphology: no significant differences for filtration fraction and proteinuria, improvement in glomerular filtrate rate, renal plasma flow, creatinine, less overall histological damage. (fernandez iglesias et al., 2011) preconditioning:1.1 mg/kg, intraperitoneal, 5 days, before induction of diabetes. other groups were diabetic rats/insulin. preconditioning: 25 mcg/ml, intraperitoneal, 15 days, before methotrexate (20 mg/kg).reduction: malondialdehyde (mad), myeloperoxidase (mpo), tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β). increase: glutathione (gsh). histolopatologically: reduction in degeneration of glomerular structures, glomerular congestion, dilatation of bowman's space, degeneration of proximal tubuli, degeneration of distal tubuli, tubular basal membrane wrinkling, vascular congestion, interstitial edema, inflammation and cell infiltration. (aslaner et al., 2015) preconditioning: 0.36, 0.72, 1.1, 1.8, 2.5 mg/kg, rectal insufflations, 15 applications, before cisplatin-induced nephrotoxicity (6 mg/kg).reduction dose-dependent manner: creatinine (cr) (0.72, 1.1 mg/kg), thiobarbituric acidreactive substances (tbars). increase dose -dependent manner: glutathione (gsh), superoxide dismutase (sod), glutathione peroxidase (gsh-px) (0.72, 1.1 mg/kg), catalase (cat). histopathological changes: at doses of 1.8 and 2.5 mg/kg, histopathological significant improved changes in renal tissue (borrego et al., 2004) preconditioning:1 mg/kg, intraperitoneal, 6 hours before and 6 hours after contrast-induced nephropathy agent (10 ml/kg), 5 increase: total antioxidant capacity (tac), lipocalin (ngal). no alteration in creatinine. histopathological alterations: improving in renal tubular injury, hemorrhage, cast formation. preconditioning: major ozonated autohemotherapy in 5m blood rabbit, before ischemia/reperfusion. reduction: interleukin-6 (il-6), tumor necrosis factor-α (tnf-α), white blood cells, neutrophil to lymphocyte ratio (nlr), ischemia-modified albumin (ima), total oxidant status (tos), oxidative stress index (osi). increase: total antioxidant status (tas). histopathological changes: reduced the tubular brush border loss (tbbl), tubular cast (tc), tubular necrosis (tn), intertubular hemorrhage congestion (ihc), dilatation of bowman space (dbs). (sancak et al., 2016) preconditioning: 0.5 mg/kg, rectal insufflations, 15 treatments, before ischaemia/reperfusion. oxygen was used as further control.reduction: phospholipase a, fructosamine. increase: p-amino-hippurate (pah), inulin, superoxide dismutase (sod). morphology: increased renal plasma flow (rpf), glomerular filtration rate (gfr).( barber et al., 1999) preconditioning: 0.8, 2.4, 4 mg/kg, intraperitoneal, daily for 5 days, with/without sepsis. a control was performed with oxygen.reduction: serum alanine amino transferase (alt), aspartate amino transferase (ast), creatinine (cre), thiobarbituric acid reactive substances (tbars), myeloperoxidase (mpo). increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px). (rodriguez et al., 2009) preconditioning:1mg/kg, transrectal insufflations, once a day, 15 treatments, before the kidney transplant procedure.reduction: blood urea nitrogen (bun), serum creatinine (cr) (slightly), jablonski grade, serum interleukin-6 (il-6), il-18, cyclooxygenase-2 (cox-2), malonaldehyde (mda), nuclear factor nf-κbp65 and rabbit polyclonal anti-rat antibody (hmgb1) expression/protein. increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px). morphology: alleviated the morphological damages, attenuated the injury of brush border of proximal renal tubular, restrained the expression level of nf-κbp65 in renal tissue, suppressed the expression of hmgb1 in renal tissue. 150 mg/kg, intraperitoneally, single dose for 10 days, at the same time escherichia coli toxin (lps) (20 mg/kg).reduction: lactate dehydrogenase (ldh) (liver, kidney, lungs, heart). increase: succinate dehydrogenase (sdh) (lungs, heart), adenosine triphosphatase (atpase) (no kidney), acid phosphatase (acpase) (liver, kidney, lungs, heart), -glucuronidase (liver, kidney, lungs). histochemically detected activity of succinate dehydrogenase (sdh): extinguished enzymatic activity in central parts of the lobule and paralleled by narrowing of zone i (liver). histochemically detected activity of lactate dehydrogenase (ldh): increased activity (hepatocytes, kupffer cells, liver). histochemically detected activity of adenosine triphosphatase (atpase): decrease intensity of the reaction for atpase (liver). histochemically detected activity of acid phosphatase (acpase): lower decrease in (madej et al., 2007) j o u r n a l p r e -p r o o f 58 activity (liver). histochemically detectable activity of succinate dehydrogenase (sdh): the reaction in tubular epithelial cells was slightly more pronounced (kidney). histochemically detected activity of lactic dehydrogenase (ldh): less pronounced stimulation of enzyme in principal tubules and other portions of nephrons (kidney). histochemically detected activity of adenosine triphosphatase (atpase): decreased intensity of the reaction in renal glomeruli and in walls of blood vessels, particularly those of low caliper (kidney). histochemically detected activity of acid phosphatase (acpase): decreased intensity of the reaction pertained in principal tubuli and collecting duts (kidney). histochemically detected activity of succinate dehydrogenase (sdh): no more pronounced alterations (lungs). histochemically detected activity of lactate dehydrogenase (ldh): stimulation was less pronounced (lungs). histochemically detected activity of adenosine triphosphatase (atpase): no changing (lungs). histochemically detected activity of acid phosphatase (acpase): decreased activity (lungs). preconditioning: 0.2, 0.4, 1.2 mg/kg intraperitoneally, once daily, for 5 days, before lipopolysaccharide (lps) injection (30 mg/kg). dexamethasone (30 mg/kg) used as a reference drug.reduction dose-dependent manner: thiobarbituric acid reactive substances (tbars). increase dose-dependent manner: glutathione peroxidase (gpx). preconditioning: 0.2, 0.4, 1.2 mg/kg intraperitoneally, once daily, for 5 days, before lipopolysaccharide (lps) injection (0.1 mg/kg). dexamethasone (30 mg/kg) used as a reference drug.reduction dose-dependent manner: serum tumor necrosis factor (tnf)-alpha, thiobarbituric acid reactive substances (tbars). increase dose-dependent manner: glutathion-s transferase (gst), glutathione peroxidase (gsh-px). (zamora et al., 2005) preconditioning: 0.2, 0.4, 1.2 mg/kg, intraperitoneally, 0.2, 0.4 mg/kg, rectal application, once daily for five days, before lipopolysaccharide (lps)reduction dose-dependent manner: serum tumor necrosis factor (tnf)-alpha. (zamora et al., 2004) j o u r n a l p r e -p r o o f 59 injection (0.1 mg/kg). preconditioning: 50 ug/ml (4.4-5.0 ml), 15 treatments, one per day, before carbon tetrachloride (ccl4). ozone control groups were: 1. a control was with oxygen; 2. another control was ozone without ccl4.reduction: aspartic alanine transaminase (ast), phospholipase a, hepatic lipid peroxidation (tbars, thiobarbituric acid-reactive substances). increase: cholinesterase (chease), superoxide dismutases (sods), catalase (cat), calcium-dependent (ca-atpase), gluthatione (gsh), glucose-6-phosphate dehydrogenase (g6pd). morpho-metric evaluation of the hepatic damage: reduction of the damage area. (leon et al., 1998) preconditioning: 1 mg/kg, rectal insufflation, 15 treatments, one per day, before ischaemiareperfusion.reduction: aspartic alanine transaminase (ast), serum alanine aminotransferase (alt), malondialdehyde (mda) + 4-hydroxyalkenals, nitrite/nitrate (no2 -/no3 -). increase: superoxide dismutase (sod), total hydroperoxide (th), glutathione (gsh), ratio gsh/gssg. (ajamieh, h. h. et al., 2004) preconditioning; 0.7 mg/kg, intraperitoneal, daily five times, before 70% partial hepatectomy.reduction: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), tumor necrosis factor alpha (tnf-α). no alterations: interleukin-6 (il-6). histopathological examination: improve in liver weight, mitotic index, proliferating cell nuclear antigen (pcna) labeling index. (gultekin, cakmak et al., 2013) preconditioning: 0.7 mg/kg, intraperitoneal, daily five times, before total body irradiation with a single dose of 6 gy.reduction time-dependent manner: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), tumor necrosis factor alpha (tnf-α), malondialdehyde (mda). increase time-dependent manner: superoxide dismutase (sod). histopathological examination: reduction in hepatocellular degeneration, inflammation, congestion and dilatation in both sinusoids and central veins; reduced inflammatory cell infiltrate in the lamina propria; regular villous structure, abundant goblet cells in the epithelium; reduced inflammatory cell infiltrate in the lamina propria. (gultekin, bakkal et al., 2013) preconditioning: 0.5 mg/kg, intraperitoneal, daily five times, before lipopolysaccharide (lps) injection (20 mg/kg). ketamine (5 mg/kg) used as a reference drug.reduction: nuclear factor κb (nf-κb) staining. morphology/immunohistochemistry parameters: intact hepatic architecture, normal liver cell membrane integrity, little inflammatory cell infiltration (low nf-kb-positive staining). (sun, pei, 2012) preconditioning: 1 mg/kg, rectal insufflation, 15 treatments, one per day, before ischemia/reperfusion. agonist (2chloro n6 cyclo-pentyladenosine, ccpa), antagonist (8cyclopentyl-1,3-dipropylxanthine, dpcpx) of a1 subtype receptor.reduction: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), nitric oxide (no) (nitrite/nitrate (no -adenosine deaminase (ada), malondialdehyde (mad), 4-hydroxyalkenals, attenuated gssg increase, nf-kb (p65 subunit) expression, tumor necrosis factor alpha (tnf-α), heat shock protein-70 (hsp70). increase: glutathione (gsh). immunohistochemistry: remarkable preservation of the liver parenchyma architecture, prevention of the inflammatory recruitment. (león fernández et al., 2008) j o u r n a l p r e -p r o o f 60 preconditioning: 1 mg/kg, rectal insufflation, 15 treatments, one per day, before ischemia/reperfusion. cycloheximide (chx) to promote protein synthesis inhibition after ozoneop treatment.reduction: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), malondialdehyde (mad), 4-hydroxyalkenals. increase: sod (mnsod), glutathione (gsh), gsh/gssg. histological lesions: normal morphology of the acinus like sham-operated. ultrastructural analysis: normal appearance of mithocondrial, rough endoplasmatic reticulum and peroxisome, no alteration on nucleus structure. (ajamieh, h. h. et al., 2005) preconditioning: 1 mg/kg, rectal insufflation, 15 treatments, one per day, before ischemia/reperfusion and/or ischaemic preconditioning. oxygen was another control comparison.reduction: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), 5'-nt, malondialdehyde (mda), 4 hydroxyalkenals. calcium, calpain, total xanthine dehydrogenase (xdh), xanthine oxidase (xo). increase: total sylfhydryl groups. improvement in histological parameters: normal morphology of hepatic lobuli. (ajamieh, h. et al., 2002) preconditioning: 1 mg/kg, rectal insufflation, 15 treatments, one per day, before carbon tetrachloride (ccl4) (1ml/kg). an ozone control group was ozone without ccl4.reduction: uric acid, lactate, thiobarbituric acid-reactive substances (tbars). increase: hepatic glycogen, liver weight (lw)/body weight (bw) ratios, superoxide dismutase (sod), catalase (cat). histopathological findings: the permanence of glycogen deposits in hepatic cells was proved, only a minimal non-parenquimatous cell reaction co-existed around the central vein. (candelario-jalil et al., 2001) preconditioning: 0.7 mg/kg, intraperitoneal, 15 applications (once daily), before methotrexate (mtx) (6 mg/kg).reduction: malondialdehyde (mda). increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px). histologically: ileum: less inflammatory cell infiltration and edema, reduction in vacuolated cells in the epithelium; liver/kidney: no significant change, due probably to the cumulative prolonged effect of mtx on these tissues. (kesik et al., 2009) preconditioning: 10, 30, 50 μg/ml, intraperitoneal, 5 days, before sepsi induced by intraperitoneal injection of rat fecal material (0.5g per kg of animals weight) extracted from another donor rat. a control group was performed with oxygen.reduction dose-dependent manner in liver/lung: conjugated dienes (cd), thiobarbituric acid-reactive substances (tbars), total pro-oxidant activity. increase dose-dependent manner: superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), total antioxidant activity (tac). (guanche et al., 2010) preconditioning: 0.8, 2.4, 4 mg/kg, intraperitoneal, daily for 5 days, with/without sepsis. a control was with oxygen.reduction: serum alanine amino transferase (alt), aspartate amino transferase (ast), creatinine (cre), thiobarbituric acid reactive substances (tbars), myeloperoxidase (mpo). increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px). (rodriguez et al., 2009) j o u r n a l p r e -p r o o f 61 150 mg/kg, intraperitoneally, single dose for 10 days, at the same time escherichia coli toxin (lps) (20 mg/kg).reduction: lactate dehydrogenase (ldh) (liver, kidney, lungs, heart). increase: succinate dehydrogenase (sdh) (lungs, heart), adenosine triphosphatase (atpase) (no kidney), acid phosphatase (acpase) (liver, kidney, lungs, heart), -glucuronidase (liver, kidney, lungs). histochemically detected activity of succinate dehydrogenase (sdh): extinguished enzymatic activity in central parts of the lobule and paralleled by narrowing of zone i (liver). histochemically detected activity of lactate dehydrogenase (ldh): increased activity (hepatocytes, kupffer cells, liver). histochemically detected activity of adenosine triphosphatase (atpase): decrease intensity of the reaction for atpase (liver). histochemically detected activity of acid phosphatase (acpase): lower decrease in activity (liver). histochemically detectable activity of succinate dehydrogenase (sdh): the reaction in tubular epithelial cells was slightly more pronounced (kidney). histochemically detected activity of lactic dehydrogenase (ldh): less pronounced stimulation of enzyme in principal tubules and other portions of nephrons (kidney). histochemically detected activity of adenosine triphosphatase (atpase): decreased intensity of the reaction in renal glomeruli and in walls of blood vessels, particularly those of low caliper (kidney). histochemically detected activity of acid phosphatase (acpase): decreased intensity of the reaction pertained in principal tubuli and collecting duts (kidney). histochemically detected activity of succinate dehydrogenase (sdh): no more pronounced alterations (lungs). histochemically detected activity of lactate dehydrogenase (ldh): stimulation was less pronounced (lungs). histochemically detected activity of adenosine triphosphatase (atpase): no changing (lungs). histochemically detected activity of acid phosphatase (acpase): decreased activity (lungs), (madej et al., 2007) lung preconditioning: 0.7 mg/kg, intraperitoneal, 5 applications (once daily), before total body irradiation (tbi) (6 gy).reduction: malondialdehyde (mda), serum tumor necrosis factor alpha (tnf-a), interleukin-1 beta (il-1). increase: superoxide dismutase (sod). histopathological evaluation: reduction in alveolar area, interstitial congestion, and alveolar and bronchiolar hemorrhage. (bakkal et al., 2013) preconditioning: 100 μg/kg, intraperitoneal, once daily for 10 days, before ischemia/reperfusion. reduction: malondialdehyde (mda), myeloperoxidase (mpo), inflammasome (nlrp3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (asc), un-cleavable cysteine-requiring aspartate protease-1 (wang, z., zhang et al., 2018) histochemically detectable activity of succinate dehydrogenase (sdh): the reaction in tubular epithelial cells was slightly more pronounced (kidney). histochemically detected activity of lactic dehydrogenase (ldh): less pronounced stimulation of enzyme in principal tubules and other portions of nephrons (kidney). histochemically detected activity of adenosine triphosphatase (atpase): decreased intensity of the reaction in renal glomeruli and in walls of blood vessels, particularly those of low caliper (kidney). histochemically detected activity of acid phosphatase (acpase): decreased intensity of the reaction pertained in principal tubuli and collecting duts (kidney). histochemically detected activity of succinate dehydrogenase (sdh): no more pronounced alterations (lungs). histochemically detected activity of lactate dehydrogenase (ldh): stimulation was less pronounced (lungs). histochemically detected activity of adenosine triphosphatase (atpase): no changing (lungs). histochemically detected activity of acid phosphatase (acpase): decreased activity (lungs). preconditioning: rectal insufflations as five applications per week. in a group: 0.3 mg/kg/day in the first week, and 0.5 mg/kg/day in the second week. in another group, 0.6 mg/kg/day in the first week, and 1 mg/kg/day in the second week, before ischemia/reperfusion. a group was performed with oxygen.reduction dose-dependent manner: creatine kinase-mb (ck-mb), lactate, myeloperoxidase (mpo), total nitrate/nitrite (nox), thiobarbituric acid reactive substances (tbars). increase dose dependent manner: myocardial adenine nucleotides (atp, adp, amp, tan), glutathione (gsh). histological examination, ultrastructural analyses: improvement in edema in between muscle fibers, and edema within muscle fibers, good myofibrillar arrangement with only slight edema around muscle fibers, mild mitochondrial swelling with decreased matrix density and mild disruption of mitochondrial cristae and vesiculation, slight margination of chromatin near nuclear membrane. (ahmed, l. a. et al., 2012) preconditioning: 100μg/kg/day, intraperitoneally, once daily, 5 days, before ischemia/reperfusion. a control was performed with oxygen.reduction: microtubule-associated protein 1 light chain 3 (lc3bi/ii), pten-induced putative kinase 1 (pink1), cytochrome c oxidase subunit iv (cox4), caspase 3, myocardial apoptosis. increase: nuclear factor (erythroid-derived 2)-like 2 (nrf2), glutamate-cysteine ligase catalytic subunit (gclc), glutamate-cysteine ligase modifier subunit (gclm), superoxide dismutases (sods) expression. morphology: mild mitochondrial injury. validation of: 1. nuclear extracts (tata-binding protein (tbp) in nuclear extracts), 2. mitochondrial fractions separated from the cytoplasmic fraction (cytochrome c oxidase subunit iv (cox4) detectable). (meng et al., 2017) j o u r n a l p r e -p r o o f 64 preconditioning: 0.6 mg/kg, rectal insufflations, twice/week for the first 3 months, then once/week till the age of 15 months, in aged rats. a control was performed with oxygen.reduction: malondialdehyde (mda), protein carbonyls (pr co), lipofuscin, cytosolic ca 2+ (heart/hippocampus). increase: glutathione (gsh), energy status (atp, adp) (heart/hippocampus), na + , k + , atpase (hippocampus). (el-sawalhi et al., 2013) preconditioning: 50, 80 ml/kg, single (1x) or repetitive (5x) insufflation, in rat cardiac transplant model.prolonged cardiac allograft survival without any adjunctive immunosuppressive therapy, not alternated number of red blood cells, decreased number of thrombocytes, increase of white blood cells, mostly granulocytes. (stadlbauer et al., 2008) preconditioning; 0.3 mg/kg, rectal insufflation, once on alternating days for 20 sessions, before doxorubicin (2 mg/kg). the oxygen group was a further control.reduction: pro-brain natriuretic peptide (bnp), malondialdehyde (mda), advanced oxidation protein products (aopp). increase: superoxide dismutase (sod), catalase (cat). morphology: slight damage, normal morphology of cardiac fibres. 90% survival rate, reduced loss of body weight. (delgado-roche et al., 2014) 150 mg/kg, intraperitoneally, single dose for 10 days, at the same time escherichia coli toxin (lps) (20 mg/kg).reduction: lactate dehydrogenase (ldh) (liver, kidney, lungs, heart). increase: succinate dehydrogenase (sdh) (lungs, heart), adenosine triphosphatase (atpase) (no kidney), acid phosphatase (acpase) (liver, kidney, lungs, heart), -glucuronidase (liver, kidney, lungs). histochemically detected activity of succinate dehydrogenase (sdh): extinguished enzymatic activity in central parts of the lobule and paralleled by narrowing of zone i (liver). histochemically detected activity of lactate dehydrogenase (ldh): increased activity (hepatocytes, kupffer cells, liver). histochemically detected activity of adenosine triphosphatase (atpase): decrease intensity of the reaction for atpase (liver). histochemically detected activity of acid phosphatase (acpase): lower decrease in activity (liver). histochemically detectable activity of succinate dehydrogenase (sdh): the reaction in tubular epithelial cells was slightly more pronounced (kidney). histochemically detected activity of lactic dehydrogenase (ldh): less pronounced stimulation of enzyme in principal tubules and other portions of nephrons (kidney). histochemically detected activity of adenosine triphosphatase (atpase): decreased intensity of the reaction in renal glomeruli and in walls of blood vessels, particularly those of low caliper (kidney). histochemically detected activity of acid phosphatase (acpase): decreased intensity of (madej et al., 2007) j o u r n a l p r e -p r o o f 65 the reaction pertained in principal tubuli and collecting duts (kidney). histochemically detected activity of succinate dehydrogenase (sdh): no more pronounced alterations (lungs). histochemically detected activity of lactate dehydrogenase (ldh): stimulation was less pronounced (lungs). histochemically detected activity of adenosine triphosphatase (atpase): no changing (lungs). histochemically detected activity of acid phosphatase (acpase): decreased activity (lungs). preconditioning: 0.7 mg/kg, intraperitoneal, daily five times, before irradiation of 500 cgy.reduction: malondialdehyde (mda), myeloperoxidase (mpo). increase: bursting pressure values of anastomosis, hydroxyproline (hpo), superoxide dismutase (sod). histopathological evaluation: improving in anastomotic wound healing, granulation tissue development and histological changes corresponding to the local inflammatory response.(tasdoven et al., preconditioning: 0.7 mg/kg, intraperitoneal, daily five times, before total body irradiation with a single dose of 6 gy.reduction time-dependent manner: serum alanine aminotransferase (alt), aspartate aminotransferase (ast), tumor necrosis factor alpha (tnf-α), malondialdehyde (mda). increase: superoxide dismutase (sod). histopathological examination: reduction in hepatocellular degeneration, inflammation, congestion and dilatation in both sinusoids and central veins, reduced inflammatory cell infiltrate in the lamina propria, regular villous structure, abundant goblet cells in the epithelium, reduced inflammatory cell infiltrate in the lamina propria. (gultekin, cakmak et al., 2013) preconditioning: 0.7 mg/kg, intraperitoneal, 15 applications (once daily), before methotrexate (mtx) (6 mg/kg).reduction: malondialdehyde (mda). increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px). histologically: ileum: less inflammatory cell infiltration and edema, reduction in vacuolated cells in the epithelium; liver/kidney: no significant change, due probably to the cumulative prolonged effect of mtx on these tissues. (kesik et al., 2009) postconditioning: 0.7 mg/kg/day, intraperitoneally and intraluminally, laparotomy and/or ischemia/reperfusion. macroscopic appearance: increase in mucosal weight in jejunum and ileum, bowel weight in jejunum, mucosal dna and protein in jejunum and ileum, villus height and crypt depth in jejunum and ileum, crypt cell proliferation in jejunum and ileum, p-erk protein. reduction: park's injury score in jejunum and ileum, enterocyte apoptosis in jejunum and ileum, caspase 3. (haj et al., 2014) cochlear preconditioning:1 mg/kg, intraperitoneally, 7 days, before ischemia/reperfusion. reduction: apoptotic index, malondialdehyde (mda), the total oxidant score (tos). increase: superoxide dismutase (sod), glutathione peroxidase (gsh-px), total antioxidant capacity (tac), catalase (cat). histological evaluation: increased numbers of glial cells in the spiral ganglion, reduced level of vascularization. (onal et al., 2017) postconditioning: 60 ug/ml, statistically significant differences in dpoae results. (koçak et al., 2016) j o u r n a l p r e -p r o o f 66 rectal and/or intratympanic, 7 days, after cisplatin-induced ototoxicity (5-mg/kg/day). the rats were tested with distortion product otoacoustic emissions (dpoae).histopathological scoring: decreased stria vascularis damage, decreased inner-outer hair cell damage.postconditioning: 30 µg/ml, intravenous, daily administration for 14 days, at the same time with noise exposure.reduction: malondialdehyde (mda), % mitochondrial swelling, mitochondrial membrane potential (mmp), glutathione disulfide (gssg), cytochrome c (brain, cochlear). increase: glutathione (gsh), glutathione peroxidase (gsh-px), superoxide dismutase (sod) (brain, cochlear), atp. histopathological findings: prevents mitochondrial membrane potential (mmp) collapse, mitochondrial swelling, cytochrome c release.(nasezadeh et al., 2017) skeletal preconditioning: 0.7 mg/kg, intraperitoneally; 4 doses, before ischemia.reduction: malondialdehyde (mda), serum nitrite-nitrate (nox), inducible nitric oxide synthase (inos) immunostaining. increase: glutathione peroxidase (gsh-px), superoxide dismutase (sod). (koca et al., 2010) preconditioning: 0.7 mg/kg, 6 days, before ischemic period and/or hypothermia.reduction: malondialdehyde (mda), interleukin-1β (il-1β), creatinine kinase (ck), aspartate aminotransferase (ast), k + , nitric oxide (no). increase: glutathione peroxidase (gsh-px), superoxide dismutase (sod). inos immunohistochemical staining: mild intensity. (ozkan et al., 2015) pancreas preconditioning: 50 μg/kg, intraperitoneally, once a day for seven days. streptozotocin (stz) (2ml). a control was performed with oxygen.reduction: 4-hydroxynonenal (4-hne), poly(adp-ribose) polymerase-1 (parp-1), glucagon, glycemia. increase: nuclear factor nrf2, glutathione-s-transferase (gst), insulin, leptin. immunohistochemistry: reduction in tissue degeneration evidenced by the partial restoration of normal cellular population size of islets of langerhans and absence of islet damage. immunofluorescence: reduction in cell death, decreased dna damage. (siniscalco et al., 2018) postconditioning: 0.7-mg/kg, intraperitoneally, daily for 3 days. induction of acute necrotizing pancreatitis. a control was performed with oxygen.reduction: serum amylase, neopterin, lipase, aspartate aminotransferase (ast), alanine amino transferase (alt), -glutamyl transferase (gt), malondialdehyde (mad). increase: alkaline phosphatase (ap), glutathione peroxidase (gsh-px), superoxide dismutase (sod). increase in weight. lower number of infected rats. histopathologic analyses: lower degrees of necrosis and leukocyte infiltration. improving in the histological injury score.(uysal et al., 2010) postconditioning: 80 mg/kg, articular space 3 times/week (3.5 weeks) after pg/ps-induced arthritis. a control was performed with oxygen.reduction: tnfa and il-1β expression/protein, nitric oxide (no), fructolysine. increase: superoxide dismutase (sod), catalase (cat). ameliorate the join swelling, decrease of arthritis index. histological results: normal morphology. preconditioning: 1 mg/kg, rectal insufflation, 15 sessions in 5 weeks, in alternated days, 2 ml/kg of lipofundin. a control group was performed with oxygen.reduction: malondialdehyde (mda), peroxidation potential (pp), advanced oxidation protein products (aopp), nitric oxide (no). increase: glutathione (gsh). histopathology: minimal lesions in the aortas, smaller intima/media ratio.(delgadoroche et al., 2013) key: cord-000295-ft5wl70x authors: tomankova, tereza; petrek, martin; kriegova, eva title: involvement of micrornas in physiological and pathological processes in the lung date: 2010-11-23 journal: respir res doi: 10.1186/1465-9921-11-159 sha: doc_id: 295 cord_uid: ft5wl70x to date, at least 900 different microrna (mirna) genes have been discovered in the human genome. these short, single-stranded rna molecules originate from larger precursor molecules that fold to produce hairpin structures, which are subsequently processed by ribonucleases drosha/pasha and dicer to form mature mirnas. mirnas play role in the posttranscriptional regulation of about one third of human genes, mainly via degradation of target mrnas. whereas the target mrnas are often involved in the regulation of diverse physiological processes ranging from developmental timing to apoptosis, mirnas have a strong potential to regulate fundamental biological processes also in the lung compartment. however, the knowledge of the role of mirnas in physiological and pathological conditions in the lung is still limited. this review, therefore, summarizes current knowledge of the mechanism, function of mirnas and their contribution to lung development and homeostasis. besides the involvement of mirnas in pulmonary physiological conditions, there is evidence that abnormal mirna expression may lead to pathological processes and development of various pulmonary diseases. next, the review describes current state-of-art on the mirna expression profiles in smoking-related diseases including lung cancerogenesis, in immune system mediated pulmonary diseases and fibrotic processes in the lung. from the current research it is evident that mirnas may play role in the posttranscriptional regulation of key genes in human pulmonary diseases. further studies are, therefore, necessary to explore mirna expression profiles and their association with target mrnas in human pulmonary diseases. a. mirna definition, biology and function discovery of microrna (mirna) lin-4 was the first short non-coding rna discovered in 1993 as a regulator of developmental timing in caenorhabditis elegans [1] . the first non-coding rna identified in humans was let-7, which has been found involved in the control of developmental timing in humans and animals [2, 3] . soon it became evident that these short non-coding rnas are a part of much larger class of non-coding rnas and the term microrna (mirna) was introduced [4] . to date, more than 900 mirnas in homo sapiens have been identified (940 in mirbase v15). mirnas are small non-coding rnas~22 nucleotides (nt) long involved in the negative post-transcriptional gene regulation via rna interference mechanism [5, 6] . the sequences of mirnas are highly conserved among plants-microorganisms-animals, suggesting that mirnas represent a relatively old and important regulatory pathway [7] . mirnas belong to the most abundant class of human gene regulators [8] : up to a third of the human genes are regulated by mirnas [9] . mirnas are, therefore, key regulators of numerous genes in biological processes ranging from developmental timing to apoptosis [e.g. [10] [11] [12] [13] [14] ]. it has been speculated that mirnas may be associated with the regulation of almost every aspect of cell physiology [8] . mirna genes are localized in the non-coding regions or in the introns of protein-coding genes in the genomic dna. the mirna genes are much longer than biologically active, mature mirnas which originate through a multistep process [15] (figure 1 ). briefly, transcription by the rna polymerase ii leads to hundred or thousand nucleotides long primary mirna transcripts (pri-mirnas) [16] . a local stem-loop structure of pri-mirnas is then cleaved in the nucleus by the dsrna-specific ribonuclease drosha/ pasha to 70 nucleotides long precursor mirna (pre-mirna) [17] in a process known as "cropping" [18, 19] . pre-mirnas are then actively transported from the nucleus to the cytoplasm [20, 21] . in the cytoplasm, pre-mirnas are subsequently cleaved by rnase iii dicer into~22-nt mirna duplexes [17, 20] . one strand of the short-lived mirna duplex is degraded ("passenger" strand, mir*), whereas the other ("guide", mir) strand is incorporated into the rna-induced silencing complex (risc) and serves as a functional, mature mirna [8] . selection of the "guide" strand is based on the base pairing stability of both dsrna ends [22, 23] . depending on the complementarity between mirna and 3' untranslated region (utr) of target mrna there are two known mechanisms of mirnas action on mrnas: 1) target mrna degradation and 2) translational inhibition with little or no influence on mrna levels [24] (figure 2) . firstly, the deadenylation and subsequent degradation of the target mrna occurs when mirna is near-perfectly complementary with target mrna [25, 26] . a recent study proved that mrna degradation represents the major mechanism of mirna regulation [27] . the authors showed that about 84% of all protein-coding mrna targets undergo degradation while recognized by their cognate mirna [27] . secondly, the translational inhibition mirnas are transcribed by rna polymerase ii from the genomic dna as long (hundred or thousand nucleotides) primary mirna transcripts (pri-mirnas). a local stem-loop structure of pri-mirnas is then cleaved in the nucleus by the dsrna-specific ribonuclease drosha/pasha to produce a 70 nucleotides long precursor mirna (pre-mirna). pre-mirnas in form of hairpins are then actively transported from the nucleus to the cytoplasm. in the cytoplasm, pre-mirnas are subsequently cleaved by rnase iii dicer into~22-nt mirna duplexes, consisting of the "guide" (mir) strand and the "passenger" (mir*) strand. the "passenger" strand is degraded, the "guide" strand is incorporated into the rna-induced silencing complex (risc) and serves as a functional, mature mirna, acting by two different mechanisms according to the complementarity with the target mrna. adopted from kim [15] . occurs when mirna is only partially complementary to its target mrna [28] [29] [30] . in light of the recent study by guo et al [27] , this mechanism does not represent a predominant reason for reduced protein output. besides the complementarity between mirna and mrna, several other factors may influence the mirna action such as impaired processing, methylation, gene polymorphisms, gene amplification, deletion of dicer, translocations and others [31] . it is evident that single mirnas may regulate translation of numerous downstream mrnas and each mrna is likely to be regulated by several mirnas simultaneously [30, 32] . thus, identification of mirna target genes has been a great challenge [33] . numerous computational algorithms [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] were established which combined 5' seed matches, thermodynamic stability and conservation analysis in order to maximize specificity when predicting mrna targets [44] (table 1) . nevertheless, various algorithms differ in the selection of mrna targets and simultaneous application of several algorithms is, therefore, highly recommended. nowadays, many web-based applications [45] [46] [47] [48] [49] [50] [51] [52] have been developed by combining existing prediction programs with functional annotations associated to many mirna, gene, protein or biological pathway resources such as mir-base, ensembl, swiss-prot, ucsc genome browser, kegg pathway and other databases [44] (table 2) . however, because of high similarities in mirna sequences, computational algorithms may predict a large number of putative mirna binding sites on mrna targets [33] . thus, experimental validation in biological system is fundamental to complete the target prediction [44] ; the currently available methods [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] are listed in table 3 . of these, antagomir studies or immunoprecipitation of ago-bound mrnas have been specifically developed for mirna-mrna studies. antagomirs represent a novel class of chemically engineered oligonucleotides used to silence endogenous micrornas [64, 65] . immunoprecipitation is then based on the observation that each member of the argonaute (ago) protein family (catalytic components of the rna-induced silencing complex) can bind to mirnas and to partially complementary sequences in the 3'-utr of specific target mrnas. thus, using highly specific monoclonal antibodies against members of the ago protein family, ago-bound mrnas can be co-immunoprecipitated [66, 67] . the lung has a very specific mirna expression profile, highly conserved across mammalian species [68, 69] . however, the knowledge of the role of mirnas in physiological and pathological conditions in the lung compartment is still limited and it is based mainly on the studies in animal models. mirnas have been shown to be involved in 1) the lung development and homeostasis, 2) in inflammation and viral infections and 3) mirna deregulation may contribute to several pulmonary diseases ( figure 3 ). hereby, we summarize the knowledge of the involvement of mirnas in the lung and current information on their posttranscriptional regulation ongoing in the lung compartment. besides pathology we pay attention also to physiological lung because understanding mirna function in normal condition is prerequisite to description of its involvement in disease. several mirnas such as mir-155, mir-26a, let-7, mir-29, mir-15/mir-16, mir-223, mir-146a/b and the mir-17-92 cluster have been shown to be involved in homeostasis and in the lung development ( table 4 ). the pulmonary role of mir-155 was studied in murine lung, where it has been shown that mir-155 is crucially involved in the differentiation of naive t-cells into th1 and th2 cells [70, 71] . mice deficient in bic/mir-155 became immunodeficient and displayed increased lung remodelling, higher bronchoalveolar leukocytes and impaired t-and b-cell responses to inflammatory stimuli [70] . another member of mirna family, mir-26a, has been shown to be selectively expressed in the bronchial and alveolar epithelial cells in murine lung [72] . target mrna of mir-26a is the transcription factor smad1, which is involved in the regulation of bone morphogenic protein signalling during lung development and pulmonary vascular remodelling [73, 74] . thus, mir-26a might be important in controlling essential developmental and physiological events in the lung [75] . also the mir-17-92 cluster is believed to regulate the lung development because its expression is high in embryonic development and steadily declines through development into adulthood [76] . mice deficient in the mir-17-92 cluster died shortly after birth and lung hypoplasia/ventricular septal defects were demonstrated; moreover the absence of the mir-17-92 cluster let to upregulation of the pro-apoptotic protein bim and inhibition of b-cell development [77] . on the other side, the overexpression of the mir-17-92 cluster in murine models resulted in an abnormal phenotype manifested by absence of terminal air sacs, which were replaced by highly proliferative, undifferentiated pulmonary epithelium [76] . other mirnas found to be involved in the pulmonary homeostasis are members of let-7 family [78] , mir-29 [79] , mir-15 and mir-16 [80, 81] , which northern blot analysis [54] quantitative real-time pcr [55] ribonuclease protection assay [56] in situ hybridization [57] , [58] mirna mimics [59] western blot [60] immunocytochemistry [61] bead-based flow cytometry method [62] suppression of mirna expression in cells by antisense locked-nucleic acid oligonucleotides [63] antagomir assays [64] , [65] immunoprecipitation of ago-bound mrnas [66] , [67] function as tumor suppressors in lung cells. in addition, another mirna, mir-223, has been shown to be crucial for normal granulocyte development and function in the lung [82] . mir-223 mutant mice spontaneously developed neutrophilic lung inflammation with tissue destruction after endotoxin challenge [82] . two mirnas, mir-146a and mir-146b, have been shown to play central role in the negative feedback regulation of il-1β-induced inflammation; the mechanism is down-regulation of two proteins irak1 and traf6 involved in toll/interleukin-1 receptor (tir) signalling [83, 84] . also other mirnas have been shown to regulate the inflammation in mouse lung exposed to aerosolized lipopolysaccharide (lps): mir-21, -25, -27b, -100, -140, -142-3p, -181c, -187, -194, -214, -223 and -224 [72] . increase in these mirnas correlated with the downregulation of pro-inflammatory cytokine production such as tnfα [72] . the deregulation of mir-155, the mir-17-92 cluster and mir-223, mirnas involved in lung development and homeostasis, resulted in the uncontrolled lung inflammation in murine models [70, 77, 82] . based on the studies in murine models, there is evidence that mirna expression may influence also the course of pulmonary viral infections [85, 86] . mir-200a and mir-223 were detected in lethal influenza virus infection presumably contributing to the extreme mir-155 important for normal lung airway remodelling (a) [70] alteration of t-cell differentiation (a) [71] mir-26a highly expressed within bronchial and alveolar epithelial cells, important for lung development (h) [75] let-7 highly expressed in normal lung tissue, functions as a tumor suppressor in lung cells (h) [78] mir-29 functions as tumor suppressor in lung cells (h) [79] mir-15, mir-16 function as tumor suppressor genes (h) [80] , [81] mir-223 control of granulocyte development and function (a) [82] mir-146a/b central to the negative feedback regulation of il-1β-induced inflammation (h) [83] , [84] mir-200a, mir-223 contribution to the extreme virulence of the r1918 influenza virus (a) [85] mir-17 family, mir-574-5p, mir-214 upregulated at the onset of sars infection (a, h) [86] virulence of the r1918 influenza virus [85] . mir-17 family, mir-574-5p and mir-214 were upregulated at the onset of sars infection: these mirnas may help the virus to evade the host immune system and are responsible for effective transmission at the initial stage of viral infection [86] . there is evidence that upregulation or downregulation of mirnas is critical for the lung development/homeostasis and thus may contribute to development of pathological pulmonary conditions, namely to smokingrelated diseases including lung cancerogenesis, fibrosis, and other immune-mediated disorders including allergy (table 5) . recent studies have implicated the mirnas in the pathogenesis of immune system mediated pulmonary diseases. tan and colleagues [87] described that a single nucleotide polymorphism in the 3'utr of hla-g, a known asthma-susceptibility gene, disrupts the binding sites of three mirnas (mir-148a, mir-148b, mir-152) targeting this gene. thus, it is likely that the association of the hla-g gene to asthma-susceptibility may be due to the allele-specific regulation of this gene by mirnas [87] . mir-21 is a further mirna crucially involved in allergic lung inflammation. its molecular target is il-12p35, a cytokine contributing to polarization of th cells toward th2 cells [88] . mir-126 is another mirna found to be involved in the pathogenesis of allergic airways disease [89] . the blockade of mir-126 suppressed the asthmatic phenotype leading to diminished th2 responses, suppression of inflammation, reduced airways hyperresponsiveness, inhibition of eosinophil recruitment, and lower mucus hypersecretion [89] . in bronchial epithelial cells stimulated with il-4 and tnfα, let-7, mir-29a and mir-155 have been involved in the regulation of allergic inflammation [90] . multiple members of let-7 family were also found upregulated in experimental asthma model and the pro-inflammatory role of let-7 mirnas on the allergic cytokine expression was confirmed [91] . another study showed that expression of rhoa in bronchial smooth muscle cells (bsmcs), a new target for asthma therapy, is negatively regulated by mir-133a [92] . the same group later revealed that il-13 is capable of reducing the mir-133a expression in bsmcs and that the mir-133a downregulation causes an table 5 mirnas involved in pathological processes in the lung mirna function (a animal studies, h human studies) references mir-155, mir-17-92 cluster deregulation results in uncontrolled inflammation (a) [70] , [71] , [77] mir-21, mir-27b, mir-100, mir-181c, mir-223, mir-224 increased following exposure to lps (a) [72] mir-155 overexpressed in solid tumors, inhibition of tumor suppressor genes (a, h) [81] mir-223 impaired granulocyte function, regulator of granulocyte production and inflammatory response (a) [82] mir-148a/b, mir-152 allele-specific regulation of asthma susceptibility hla-g gene (h) [87] mir-21 key role in asthma (a) [88] overexpressed in solid malignancies (a, h) [103] up-regulated in bleomycin-induced fibrosis and ipf (a, h) [110] mir-126 suppression of the asthmatic phenotype by blockade of mir-126 (a) [89] downregulated in cystic fibrosis airway epithelial cells (h) [111] let-7, mir-29a, mir-155 regulation of allergic inflammation in bronchial epithelial cells (a, h) [90] let-7 pro-inflammatory effect in experimental asthma (a) [91] role in lung cancer progression (h) [99] mir-133a regulator of expression of rhoa, target for asthma therapy (a, h) [92] , [93] mir-146a reduced expression in copd fibroblasts (h) [95] mir-218, mir-15a, mir-199b, mir-125a/b, mir-294 deregulated due to smoking (a, h) [96, 97] mir-218 tumor suppressor in non-small cell lung cancer (h) [98] mir-17-92 cluster overexpressed in lung cancers (h) [102] mir-34 regulation of apoptosis in lung cancer cells (h) [105] [106] [107] mir-210 overexpressed in lung cancer (h) [108] let-7d pro-fibrotic effect in pulmonary fibrosis (a, h) [109] upregulation of rhoa, presumably resulting in an augmentation of the contraction [93] . lung cancer and chronic obstructive pulmonary disease (copd) share a common environmental risk factor in cigarette smoke exposure [94] . although extensive studies of the involvement of mirnas in lung cancer have been performed, there are only few reports focused on the role of mirnas in copd. recent study on fibroblasts from copd subjects stimulated in vitro with pro-inflammatory cytokines released less mir-146a than smokers without copd [95] . the reduced mir-146a expression resulted in prolonged mrna half-life of cyclooxygenase-2, thus increasing prostaglandin e2 in fibroblasts from copd subjects [95] . there is evidence that smoking has influence also on other mirnas. expression profiling study in the rats exposed to environmental cigarette smoke revealed 24 downregulated mirnas (especially let-7 family, mir-10, [96] . mir-294, a known inhibitor of transcriptional repressor genes, was the only mirna upregulated in smoke-exposed rats [96] . in another study, bronchial airway epithelial cells from current and never smokers differed in the expression of 28 mirnas (especially mir-218, mir-15a, mir-199b, mir-125a/b, mir-294) in comparison to smokers, whereas the majority of deregulated mirnas were downregulated in smokers [97] . similar observation was observed in lung squamous cell carcinoma, where downregulation of mir-218 was associated with a history of cigarette smoking [98] . however, the majority of mirna studies in smokingrelated diseases are focused on the role of mirnas in lung cancer. altered expression of mir-155 and let-7 has been reported in lung adenocarcinoma and expression of let-7 related to patient survival [99] . moreover, it has been shown that let-7 may also play a role in lung cancer progression [99] [100] [101] . further, increased expression of the mir-17-92 cluster has also been detected in lung cancer [102] . another mirnas involved in lung cancerogenesis are mir-21 and mir-34 families. mir-21 was shown to regulate multiple tumor/metastasis suppressor genes in lung solid tumors [103] . mir-34a/b/c have been identified to be a component of the p53 tumor suppressor network: p53 upregulates in response to dna damage the members of mir-34 family [104] , thus regulating genes involved in the cell cycle and apoptosis [105] [106] [107] . furthermore, mir-210 has been overexpressed in late stages of lung cancer, thus mediated mitochondrial alterations associated with modulation of hypoxia-inducible factor-1 activity [108] . next, mir-218 was identified as a putative tumor suppressor in non-small cell lung cancer [98] . recently, it was reported that mirnas may play pivotal regulatory role also in the fibrotic processes ongoing in the lung: the downregulation of let-7 d in idiopathic pulmonary fibrosis (ipf) resulted in the pro-fibrotic effects [109] . also, upregulation of mir-21 was reported in the lungs of ipf patients and in the murine lungs with bleomycin-induced fibrosis, whereas mir-21 expression was enhanced by pro-fibrotic tgf-β1 [110] . another disease associated with mirna change was cystic fibrosis. downregulation of mir-126 was detected in cystic fibrosis bronchial epithelial cells and its expression correlated with upregulation of tom1 mrna both in vitro and in vivo [111] . tom1, a mir-126 target, was reported to be involved in the regulation of innate immune responses through its involvement in the tlr2/4 and il-1β and tnf-α-induced signalling pathways [111] . small non-coding rnas (mirnas) play pivotal role in the posttranscriptional regulation of numerous human genes, mainly via degradation of target mrnas. there is evidence that the lung has a very specific mirna expression profile undergoing changes during the lung development. studies namely in animal models have provided evidence that mirnas participate in lung homeostasis and play pivotal role also in the control of pulmonary inflammation and viral infections. recent studies showed evidence that upregulated or downregulated expression of various mirnas play an active role in the pathogenesis of pulmonary diseases. specific mirna expression profiles were characterized for smoking related-diseases including copd and lung cancer, immune-mediated pulmonary diseases and pulmonary fibrosis. moreover, several mirnas crucial for lung development and homeostasis such as let-7, mir-155 or mir-19-72 cluster have been identified to be deregulated in pulmonary allergy, asthma or lung cancer. the knowledge of altered mirna expression profiles in diseased lung may thus offer new insights in the biology of pulmonary diseases. moreover, mirnas may represent attractive novel diagnostic biomarkers mainly due to their higher stability when compared to mrnas [112] and could potentially provide possibilities for therapeutic intervention [31, 113, 114] . ambros v: the c. elegans heterochronic gene lin-4 encodes small rnas with antisense complementarity to lin-14 a cellular function for the rna-interference enzyme dicer in the maturation of the let-7 small temporal rna control of developmental timing in animals glimpses of a tiny rna world micrornas: genomics, biogenesis, mechanism, and function micrornas: small rnas with a big role in gene regulation micro-rnas: small is plentiful advances in micrornas: implications for immunity and inflammatory diseases emerging role of micrornas in disease pathogenesis and strategies for therapeutic modulation microrna: past and present a developmental timing microrna and its target regulate life span in c. elegans mesodermally expressed drosophila microrna-1 is regulated by twist and is required in muscles during larval growth micrornas and the regulation of cell death microrna-143 regulates adipocyte differentiation microrna biogenesis: coordinated cropping and dicing microrna maturation: stepwise processing and subcellular localization the nuclear rnase iii drosha initiates microrna processing est analyses predict the existence of a population of chimeric microrna precursor-mrna transcripts expressed in normal human and mouse tissues human micrornas are processed from capped, polyadenylated transcripts that can also function as mrnas exportin-5 mediates the nuclear export of pre-micrornas and short hairpin rnas nuclear export of microrna precursors asymmetry in the assembly of the rnai enzyme complex functional sirnas and mirnas exhibit strand bias revisiting the principles of microrna target recognition and mode of action zebrafish mir-430 promotes deadenylation and clearance of maternal mrnas micrornas direct rapid deadenylation of mrna mammalian micrornas predominantly act to decrease target mrna levels posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in c. elegans the lin-4 regulatory rna controls developmental timing in caenorhabditis elegans by blocking lin-14 protein synthesis after the initiation of translation specificity of microrna target selection in translational repression integrating the micrornome into the study of lung disease microarray analysis shows that some micrornas downregulate large numbers of target mrnas experimental validation of mirna targets microrna targets in drosophila prediction of mammalian microrna targets conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets microrna target predictions across seven drosophila species and comparison to mammalian targets a combined computational-experimental approach predicts human microrna targets inference of mirna targets using evolutionary conservation and pathway analysis im: prediction of both conserved and nonconserved microrna targets in animals mitarget: microrna targetgene prediction using a support vector machine rigoutsos i: a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes fast and effective prediction of microrna/target duplexes got target? computational methods for microrna target prediction and their extension human microrna target analysis and gene ontology clustering by gomir, a novel stand-alone application mirdb: a microrna target prediction and functional annotation database with a wiki interface mirecords: an integrated resource for microrna-target interactions mirgator: an integrated system for functional annotation of micrornas huang hd: mirnamap 2.0: genomic maps of micrornas in metazoan genomes mirz: an integrated microrna expression atlas and target prediction resource microrna and mrna integrated analysis (mmia): a web tool for examining biological functions of microrna expression the database of experimentally supported targets: a functional update of tarbase microrna-155 regulates human angiotensin ii type 1 receptor expression in fibroblasts computational prediction and experimental validation of ciona intestinalis microrna genes potential mrna degradation targets of hsa-mir-200c, identified using informatics and qrt-pcr identification and characterization of micrornas from the bovine adipose tissue and mammary gland the human angiotensin ii type 1 receptor +1166 a/c polymorphism attenuates microrna-155 binding in situ detection of animal and plant micrornas hsa-mir-520 h downregulates abcg2 in pancreatic cancer cells to inhibit migration, invasion, and side populations a strategy to rapidly identify the functional targets of micrornas by combining bioinformatics and mrna cytoplasmic/nucleic ratios in culture cells widespread deregulation of microrna expression in human prostate cancer microrna expression profiles classify human cancers a cellular microrna mediates antiviral defense in human cells antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and bim silencing of micrornas in vivo with 'antagomirs' identification of human microrna targets from isolated argonaute protein complexes isolation of microrna targets by mirnp immunopurification maternally imprinted micrornas are differentially expressed during mouse and human lung development microrna expression in the aging mouse lung requirement of bic/microrna-155 for normal immune function micro-rna-155 inhibits ifn-gamma signaling in cd4+ t cells expression profiling in vivo demonstrates rapid changes in lung microrna levels following lipopolysaccharide-induced inflammation but not in the anti-inflammatory action of glucocorticoids smad1 expression and function during mouse embryonic lung branching morphogenesis bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension microrna expression profiling in mild asthmatic human airways and effect of corticosteroid therapy transgenic overexpression of the microrna mir-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells targeted deletion reveals essential and overlapping functions of the mir-17 through 92 family of mirna clusters the let-7 microrna represses cell proliferation pathways in human cells microrna-29 family reverts aberrant methylation in lung cancer by targeting dna methyltransferases 3a and 3b microrna signatures in human cancers a microrna expression signature of human solid tumors defines cancer gene targets regulation of progenitor cell proliferation and granulocyte function by microrna-223 nf-kappab-dependent induction of microrna mir-146, an inhibitor targeted to signaling proteins of innate immune responses rapid changes in microrna-146a expression negatively regulate the il-1beta-induced inflammatory response in human lung alveolar epithelial cells microrna expression and virulence in pandemic influenza virus-infected mice micrornome analysis unravels the molecular basis of sars infection in bronchoalveolar stem cells allele-specific targeting of micrornas to hla-g and risk of asthma microrna-21 is up-regulated in allergic airway inflammation and regulates il-12p35 expression antagonism of microrna-126 suppresses the effector function of th2 cells and the development of allergic airways disease coordinated changes in mrna turnover, translation, and rna processing bodies in bronchial epithelial cells following inflammatory stimulation pro-inflammatory role for let-7 micrornas in experimental asthma down-regulation of mir-133a contributes to up-regulation of rhoa in bronchial smooth muscle cells micrornas and their therapeutic potential for human diseases: mir-133a and bronchial smooth muscle hyperresponsiveness in asthma mechanisms involved in lung cancer development in copd reduced mir-146a increases prostaglandin e2 in chronic obstructive pulmonary disease fibroblasts downregulation of microrna expression in the lungs of rats exposed to cigarette smoke micrornas as modulators of smokinginduced gene expression changes in human airway epithelium microrna-218 is deleted and downregulated in lung squamous cell carcinoma unique microrna molecular profiles in lung cancer diagnosis and prognosis reduced expression of the let-7 micrornas in human lung cancers in association with shortened postoperative survival ras is regulated by the let-7 microrna family a polycistronic microrna cluster, mir-17-92, is overexpressed in human lung cancers and enhances cell proliferation microrna-21 promotes cell transformation by targeting the programmed cell death 4 gene a microrna component of the p53 tumour suppressor network differential regulation of micrornas by p53 revealed by massively parallel sequencing: mir-34a is a p53 target that induces apoptosis and g1-arrest the mir-34 family in cancer and apoptosis micrornas and lung cancer: tumors and 22-mers is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of hif-1 activity inhibition and role of let-7 d in idiopathic pulmonary fibrosis mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis greene cm: mir-126 is downregulated in cystic fibrosis airway epithelial cells and regulates tom1 expression role of micrornas in the molecular diagnosis of cancer lung microrna: from development to disease therapeutic microrna delivery suppresses tumorigenesis in a murine liver cancer model involvement of micrornas in physiological and pathological processes in the lung funding was obtained from the czech ministry of health (iga mz cr nt/ 11117-6, iga mz cr ns/10267-3, iga mz cr ns/10260-3) and in part by the internal grant agency of palacky university (iga pu project sv lf_2010_008). the authors declare no conflicting financial interests. all authors wrote and revised the manuscript, and approved the final version. the authors declare that they have no competing interests. submit your next manuscript to biomed central and take full advantage of: key: cord-018243-hyvu9nuq authors: salman, huda; cooke, kenneth r.; lazarus, hillard m. title: fibrosing alveolitis in hematologic malignancy patients undergoing hematopoietic cell transplantation date: 2010-08-19 journal: pulmonary involvement in patients with hematological malignancies doi: 10.1007/978-3-642-15742-4_42 sha: doc_id: 18243 cord_uid: hyvu9nuq although advances in antineoplastic therapy have considerably improved the survival of patients with hematological malignancies, current treatment modalities increase the risk of late complications. several forms of chronic pulmonary dysfunction due to infectious or noninfectious causes commonly occur in the months to years after chemo-radiotherapy and can be fatal or result in long-term morbidity. the judicious use of prophylactic antimicrobial agents has tipped the balance toward noninfectious etiologies. hence, while opportunistic infections still contribute to chronic lung disease, late sequelae resulting from antineoplastic therapy have been identified and reported. patients who proceed to receive hematopoietic cell transplantation (hsct) are particularly prone to developing lung complications. pulmonary dysfunction occurring after hsct may manifest with obstructive or restrictive pulmonary mechanics and may range in severity from subtle, subclinical functional changes to frank respiratory failure. insights generated using animal models suggest that the immunologic mechanisms contributing to lung inflammation after hsct may be similar to those responsible for graft-versus host disease. in sum, chronic fibrotic pulmonary dysfunction is a frequent and significant complication facing survivors of hematologic malignancies and their practitioners. the high incidence and suboptimal response to current support care and immunosuppressive therapy underscore the need for heightened awareness and continued research in this area. a 20-year-old man patient with de novo acute myelogenous leukemia (aml) was induced into complete remission with chemotherapy consisting of idarubicin and cytarabine. after consolidation with high-dose cytarabine, he later received conditioning with cyclophosphamide 60 mg/kg/day intravenously for 2 days and fractionated total body irradiation (tbi) 1,200 cgy followed by allogeneic hematopoietic cell transplant (hsct) using a hla-identical sibling donor. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus and short-course methotrexate. his clinical course was uncomplicated, but after withdrawal of immunosuppression he developed extensive chronic gvhd involving skin and liver. this complication was controlled with the re-institution of tacrolimus. surveillance pulmonary function testing completed 180 days after hsct showed evidence of mild reductions in forced expiratory volume in 1 s (fev 1 ) with preservation of forced vital capacity (fvc). follow-up study revealed significant and rapid worsening of obstructive lung disease (old) despite resolution of hepatic and skin gvhd and continued prophylaxis against viral, fungal and pneumocystis infections using acyclovir, fluconazole and trimethoprimsulfamethoxazole, respectively. reductions in pulmonary function ultimately were associated with shortness of breath and dyspnea with exertion. subsequent workup revealed ground-glass opacities with air trapping on chest computed tomography (ct) scan and evidence of progressive afo on pulmonary function testing based on reduction of fev 1 (50% of predicted normal), a ratio of fev 1 to fvc of 0.64 and a residual volume of 1.52 l (157% of predicted normal). bronchoalveolar lavage was negative for infection. video-assisted thoracoscopic biopsy of the lungs revealed changes consistent with bronchiolitis obliterans with early fibrosis. the patient continued to receive tacrolimus, and ultimately a course of oral prednisone (2 mg/kg/day) and etanercept 50 mg subcutaneous once weekly was initiated. clinical symptoms resolved and pulmonary function improved. he remains in complete remission regarding the aml. fibrosing alveolitis (fa) is a progressive and often fatal disorder characterized by sequential acute lung injury with subsequent scarring and end-stage lung disease. historically, idiopathic pulmonary fibrosis (ipf) encompassed a heterogeneous group of histologic and clinical entities arising in an idiopathic setting [1] . patients with hematologic malignancies treated with chemotherapy, radiation or hsct, such as the patient described above, commonly develop a wide variety of late and chronic pulmonary dysfunction states [2] . these complications share many of the clinical and pathologic features described in typical idiopathic fa. this spectrum of pulmonary toxicity observed during fa can be simplified by considering the time of diagnosis in relation to institution of therapy, whether the radiographic abnormalities are focal or diffuse, and by underlying histopathology. in addition, there are individual patient factors that should be considered when formulating a differential diagnosis. these include: radiotherapy delivered to the chest wall or as part • of total body irradiation (tbi) exposure to pulmonary-or cardio-toxic chemother-• apeutic agents current or prior immunosuppressant therapy • history of high-dose chemotherapy exposure prior • to autologous or allogeneic hsct history of opportunistic pulmonary infection (fun-• gal or otherwise) in the case described herein, the patient was exposed to radiation therapy in preparation for hsct and received an allogeneic graft from his hla-matched sibling. while his early posttransplant course was uncomplicated, he developed chronic gvhd of the skin and liver lung after immunosuppression was tapered. the widespread and appropriate use of prophylactic antibiotics has shifted the spectrum of pulmonary dysfunction in hsct recipients from infectious to noninfectious etiologies. this chapter will address the chronic lung complications that lead to pulmonary fibrosis and persistent organ dysfunction in each context with specific focus on hematologic malignancy patients treated using hsct. in patients with hematologic malignancies, severe lung infections frequently lead to the development of acute respiratory distress syndrome (ards). bacterial infections predominate (see table 42 .1) and arise because of severe immune suppression inherent to these disorders and their treatments. the pathology of ards involves severe alveolar epithelial cell damage, hyaline membrane formation, and festinate myofibroblast proliferation and fibrosis in the intra-alveolar spaces. affected hsct recipients who deteriorate and require intubation and mechanical ventilation for ards experience a high mortality. in one series, overall intensive care unit (icu) mortality was 74% [3] . in recent years, advancements in supportive care have resulted in significant improvement in survival [4] . however, longterm survivors continue to have residual lung dysfunction that may progress over time. in one series, autopsy evaluation revealed pulmonary fibrosis in 55% of such patients, underscoring the importance of dysregulated reparative mechanisms in the lung after an acute insult [5] . factors influencing progression to the fibro-proliferative phase of ards versus resolution and reconstitution of the normal parenchymal architecture are poorly understood. abnormal repair and remodeling may be profoundly affected by both environmental and genetic factors. in this context, mechanical ventilation may affect the macromolecules that constitute the extracellular matrix (collagen, elastin, fibronectin, laminin, proteoglycan and glycosaminoglycans) and impact the biomechanical balance within the lung parenchyma. fungal infections also may follow a chronic course of prolonged inflammation with focal or diffuse scarring ultimately resulting in significant pulmonary dysfunction. invasive aspergillosis (ia) occurs frequently in hematologic malignancy patients, particularly after an allogeneic hsct, presenting classically as angio-invasive or airway-invasive disease. angioinvasive ia is characterized histologically by invasion and occlusion of small to medium-sized pulmonary arteries by fungal hyphae. this effect leads to the formation of necrotic hemorrhagic nodules or pleuralbased, wedge-shaped hemorrhagic infarcts. the "halo sign" (nodules surrounded by areas of ground-glass attenuation) on chest ct scan strongly suggests a diagnosis of ia [6] . airway-invasive aspergillosis is characterized by the presence of organisms in the basement membrane of the bronchioles and within the airway lumen. positive yield from respiratory samples such as sputa examination or broncho-alveolar lavage (bal) is more likely in this subtype of ia than in the angio-invasive variety. clinical manifestations of acute airway-invasive aspergillosis include: acute tracheobronchitis, exudative bronchiolitis and bronchopneumonia. using high-resolution ct, the associated bronchiolitis is characterized by the presence of peri-bronchial consolidation, centri-lobular micro-nodules, and branching linear or nodular areas of ground-glass attenuation having a "tree-in-bud" appearance [7] . this form of airway-invasive aspergillosis can be associated with pseudo-membranous necrotizing tracheal involvement that can cause pneumo-mediastinum and has a high [8] . airway-invasive aspergillosis can also follow a chronic course known as chronic necrotizing aspergillosis. this condition is characterized by an indolent, granulomatous cavitary infection that may mimic reactivation of tuberculosis radiographically [9] . mortality is lower compared with the other forms of ia and often is related to the underlying disease of the patient. hematologic malignancy patients treated with chemotherapy or chest wall radiation therapy, or those who proceed to receive a hsct may develop a wide variety inflammatory noninfectious lung disorders that ultimately may lead to pulmonary fibrosis. radiation-induced lung injury first was described in 1898, soon after the development of roentgenograms [10] . in 1925 the distinction between two separate types of radiation-induced lung injury, radiation pneumonitis and radiation fibrosis, was made [11] . an entire chapter from drs. gallego and rello in this book is dedicated to radiation-related lung injury. radiation-induced lung injury results from the combination of direct cytotoxicity upon normal lung tissue and, perhaps more importantly, the development of fibrosis triggered by radiation-induced cellular signal transduction. the cytotoxic effect is largely a consequence of dna damage and death in normal lung epithelial cells. the development of fibrosis that can compromise lung function is mediated by a number of different cytokines. clinically, the most extensively studied radiation-induced cytokine is transforming growth factor beta 1 (tgf-b), which can induce fibroblast collagen deposition. a normal plasma tgf-b concentration at the conclusion of a clinical course of radiotherapy has been observed to be a predictor for the risk of pneumonitis [12] . other proinflammatory cytokines, including, but not limited to, interleukin il-6, tumor necrosis factor-alpha tnfa and il-1, are upregulated immediately after irradiation. increased il-6 plasma concentrations correlate with an increased risk of radiation-induced lung injury [13, 14] . platelet-derived growth factor (pdgf) and basic fibroblast growth factor (bfgf) are upregulated in animal models of lung irradiation injury and antedate the development of fibrosis [15] . factors affecting the development of radiation-induced lung disease are numerous and are included in table 42 .2 [16] [17] [18] [19] ; all have been reported to raise the risk of radiation pneumonitis. radiographic and bronchoscopic findings are nonspecific, and the diffusion capacity for carbon monoxide (dl) typically is depressed in patients with radiation-induced lung damage. long-term glucocorticoids may be effective in the treatment of radiationassociated lung injury in which cop is the leading pulmonary involvement; however, symptoms and radiographic abnormalities, as well as immunologically mediated lymphocytic alveolitis frequently recur with discontinuation of therapy [20, 21] . early studies suggested that pentoxifylline may have a role •• method of irradiation such as conformal radiation therapy or specialized techniques including intensity-modulated radiation therapy and stereotactic body radiation therapy [16] [17] [18] [19] in the treatment of radiation-induced fibrosis involving the skin and subcutaneous tissues as this agent also inhibits experimental bleomycin-induced pulmonary fibrosis in rats, likely via its anti-tnfa effects [22] . pentoxifylline showed a significant protective effect for both early and late lung radiotoxicity. amifostine is a pro-drug that is de-phosphorylated by alkaline phosphatase in tissues to a pharmacologically active free thiol metabolite. this drug can reduce the toxic effects of chemotherapy by acting as a scavenger of free radicals generated in tissues exposed to radiation. early evidence suggests that amifostine may decrease radiation-induced pulmonary injury without diminishing the therapeutic effect [23, 24] . captopril and other ace inhibitors also have been shown to reduce radiation-induced lung fibrosis in rats [25] , but there are no published data in humans. improvements in the perfusion and ventilation of radiation-injured lung tissue may be expected from 3 to 18 months after radiation therapy. beyond 18 months, however, further significant improvement appears unusual [26, 27] . patients with hematologic diseases are exposed to a host of traditional and newer chemotherapeutic agents that can cause lung injury at an incidence that ranges from less than 5% to as high as 60% [28, 29] . the increased complexity of multi-modality treatments and high-dose protocols designed to augment antineoplastic efficacy, particularly in the context of hsct, has increased the incidence of pulmonary complications. the diagnosis of drug-induced respiratory disease often is complex because: (1)1 patients may be exposed to several pneumo-toxic drugs concurrently or in sequence due to earlier treatment failure; (2)2 time to onset of pulmonary toxicity may be delayed, making it difficult to ascertain which agent is responsible for the pulmonary reaction; (3)3 the combination of drugs to treat malignant hematologic conditions may lead to unexpected drug interactions, producing enhanced toxicity compared with the toxicity of each agent considered separately; and (4)4 radiation therapy to the chest or tbi. other factors that play a role in the development of pulmonary toxicity include advanced age, current smoking, abrupt withdrawal of corticosteroids and the use of hsct (allogeneic vs autologous). changes in blood neutrophil counts, thrombocytopenia, coagulation deficits, volume overload or left ventricular dysfunction also can influence the spectrum and severity of pulmonary drug toxicity. in addition to overt pulmonary toxicity, subclinical drug-induced lung dysfunction often occurs in the form of reduced dlco and lung volumes or changes in cell populations in bal fluid. upon cessation of exposure to the agent, most of these changes reverse slowly in a few weeks or months. drug-induced lung injury can manifest in several patterns ( the majority of cases) , azathioprine, chlorambucil, cyclophosphamide, procarbazine and, rarely, vinca alkaloids. the onset of this condition is unpredictable; symptoms may develop a few days to years after exposure. the clinical picture includes increasing dyspnea, dry cough, high fevers and rash. the severity of illness can vary from mild to progressive respiratory failure, and associated radiographic findings may range from bilateral (usually symmetrical) interstitial or alveolar opacities to extensive consolidation with air bronchograms and volume loss [30] [31] [32] . pleural effusions and mediastinal lymph node enlargement have been reported in patients with methotrexate-induced lung injury [33, 34] . bal fluid usually shows lymphocyte predominance. a low ratio of cd4 to cd8 lymphocytes is suggestive, but not specific, for drug-induced lung disease. other bal findings include neutrophilia or a combined pattern of lymphocytosis with neutrophilia or eosinophilia [35] . appropriate stains, cultures and molecular techniques in bal fluid should be performed to exclude opportunistic infections. a lung biopsy may be required in selected cases. histopathologic features include interstitial inflammation and pulmonary granulomas. fibrosis can be present, but is generally not the dominant histopathologic feature. alveolar edema or hemorrhage may be found as a manifestation of severe methotrexate pneumonitis [34] . high-dose corticosteroids may be indicated with more advanced disease, as drug-induced nsip can lead to mortality if it is not treated promptly, but in milder cases, symptoms can subside after simple drug withdrawal [36] . although rechallenge with the drug may be safe, it is not generally recommended [37] . eosinophilic pneumonia (ep) is an unusual and unpredictable pattern of response to chemotherapeutic agents as opposed to that described following the use of some antibiotics. ep in patients with hematologic malignancies can result from treatment with fludarabine and, rarely, interferons, inhaled or parenteral pentamidine, and radiographic contrast media [33] . although methotrexate and procarbazine pneumonitis can often be associated with peripheral eosinophilia, bal and histopathologic features are not those of eps. typically, the syndrome of ep develops during or shortly after termination of treatment. a history of an allergic disorder, or repeated courses of treatment with the specific drug, may predict for a higher risk. the diseases could manifest as acute pneumonia and progress to respiratory failure [38, 39] . radiograghic findings of ep include alveolar infiltrates and the classic pattern of "photographic negative" pulmonary edema [40] . it also could cause faint ground-glass opacities, or kerley's "b" lines (dense and diffuse). ep is diagnosed by the presence of increased percentages or numbers of eosinophils in blood, bal, or lung tissue. a lung biopsy is rarely required, but discontinuance of the offending drug is essential. corticosteroid drug therapy is suggested in cases with severe involvement. the prognosis for this condition usually is good. chemotherapy-induced organizing pneumonia (op) may manifest with chest pain, dyspnea and diffuse radiographic abnormalities with [41] or without acute respiratory failure [42] , or may be discovered incidentally on chest imaging [36] . nodular op typically is seen in patients exposed to chemotherapy who develop round-shaped foci that localize mainly in lung bases, may abut the pleura and simulate metastatic nodules [43] [44] [45] . nonspecific findings are retrieved from bal, such as increases in the percentage of lymphocytes, neutrophils or eosinophils. open lung biopsy guided by the results of ct scan is the procedure of choice. the nodules correspond to sterile aggregates of mononuclear cells. histology reveals interstitial inflammation, superimposed on the dominant background of alveolar and ductal fibrosis. lung nodules with the histopathologic features of cryptogenic organizing pneumonia or of localized fibrosis can be observed after treatment with bleomycin, cyclophosphamide, vinblastine and, rarely, fludarabine [46] [47] [48] [49] . drug discontinuation and, if required, corticosteroid therapy usually are followed by improvement in most cases. organizing pneumonia (formerly boop) can be seen following hsct and is described in detail later in the chapter. diffuse alveolar damage (dad) is a serious form of pulmonary pathology that may develop in the context of drug-related lung injury. single chemotherapeutic agents (e.g., bcnu or other nitrosoureas, bleomycin, busulfan, chlorambucil, cyclophosphamide, melphalan, procarbazine, vinblastine) or multiagent cytostatic chemotherapy have been reported to cause this lung toxicity [50] . some regimens may be associated with a greater likelihood of dad than others even if they differ in one agent only. for instance, in patients with de novo-treated hodgkin's lymphoma, the substitution of gemcitabine for dacarbazine, e.g., abvg rather than abvd (doxorubicin, bleomycin, vinblastine and gemcitabine instead of dacarbazine), was associated with a 42% incidence rate of pulmonary toxicity [51] . likewise, the substitution of gemcitabine for etoposide in the dose-escalated beacopp regimen (bleomycin, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone and gemcitabine rather than etoposide) significantly escalated the likelihood of pulmonary toxicity [52] [53] [54] . concurrent administration of radiation therapy to the chest or use of tbi, supplemental oxygen and possibly colonystimulating factors (csfs) may increase the risk of dad. time to onset of dad can vary from shortly after the first administration of the offending drug to much later into the treatment course [55] . restrictive lung function patterns and hypoxemia are typical. dlco abnormalities often precede clinical symptoms. the clinical evolution of drug-induced dad varies from an isolated decrease in dlco [56, 57] or evidence of fibrosis in trans-bronchial or pulmonary biopsies [58, 59] as the only manifestation of toxicity to bilateral, interstitial and alveolar infiltrates [57, 60] . severe cases progress to an ards picture and death [61] . high-resolution ct scanning may show groundglass opacities and intra-lobular septal thickening, and the extent of changes correlates with clinical severity [62] . dysplastic pneumocytes may be retrieved by bal [63, 64] . a lung biopsy is reserved for patients with an atypical presentation or for those who do not improve with empirical antibiotic and corticosteroid treatment [65] . the main histopathologic feature of dad is consistent with hyaline membranes and fibrin deposits lining the alveolar border, dysplasia of type ii cells, free alveolar fibrin, cells and debris in alveolar spaces and various stages of interstitial edema, inflammation and organization [66] . dad may be reversible after discontinuation of drugs or after the addition of corticosteroids, or both [67] . the usual doses of oral corticosteroids may not prevent the condition from developing, but higher doses are reported to reduce the incidence [68] . the high incidence, severity and unpredictability of dad associated with chemotherapy suggest that it is reasonable to discontinue such treatment once the dlco has decreased 50% compared with pre-therapy values. although smaller decrements in dlco do not equate to toxicity and should not lead to withdrawal of chemotherapy, a precipitous decrease in the dlco indicates impending toxicity [69] . when radiation therapy is planned after the administration of chemotherapeutic agents, it is advisable to wait for any chemotherapy-induced decrease in the dlco to stabilize or show a trend toward improvement before starting radiation. finally, drug-induced pulmonary fibrosis may develop in patients receiving cytotoxic agents, such as bleomycin, busulfan, bcnu, lomustine, ccnu chlorambucil, cyclophosphamide, melphalan, vinca alkaloids, radiation therapy and tbi [70] . this entity more often is diagnosed months or years after termination of treatment. early signs of this disease are basilar or diffuse streaky opacities and volume loss. this condition can progress to honeycombing and fibrotic changes; reversal of this toxicity and the response to corticosteroids are unpredictable and often unsatisfying. histologic exam can demonstrate the characteristic dysplasia of type ii pneumocytes that reflects exposure to alkylating agents and radiation therapy. in a few patients, especially children treated for hematologic malignancies, pleural or pulmonary fibrosis may develop [71] . this process results in thoracic deformity, encasement of the lungs and severely restricts lung physiology. an accelerated variant of pulmonary fibrosis, acute interstitial pneumonia (formerly termed the hamman and rich syndrome), has been described after treatment with chlorambucil and methotrexate [72] [73] [74] . the prognosis of this condition is poor despite drug withdrawal and institution of high-dose corticosteroids. as seen in the patient description at the start of this chapter, a decline in lung function long has been identified as a significant complication in the months to years that follow allogeneic hsct. a clinical pearl from dr. bergeron in this book also very nicely describes this type of pulmonary involvement. noninfectious conditions now represent the major pulmonary causes of morbidity and mortality after hsct. idiopathic pneumonia syndrome (ips), discussed in another chapter in this book, remains one of the more common and serious pulmonary complications occurring within months after hsct. although graft-versus-host reactions may play an etiologic role, the major contributing factor is conditioning-related toxicity. among lung conditions that are more closely associated with gvhd, both bronchiolitis obliterans (brob) (onset months to years after hsct) and bronchiolitis obliterans with organizing pneumonia (cop) may lead to fa. the term cop should not be used interchangeably with bronchiolitis obliterans (bo) to describe a patient with chronic lung dysfunction after hsct, although such usage unfortunately is widespread. the two disorders differ with respect to histopathology, pulmonary function characteristics and, most importantly, response to therapy. brob is an inexorably progressive condition, whereas cop behaves similarly to idiopathic cop seen in other populations. cop after hsct usually is quite responsive to corticosteroids and in other settings may resolve spontaneously, whereas brob is not [75, 76] . organizing pneumonia also is associated with restrictive (rather than obstructive) changes on pulmonary function testing (table 42 .4). in allogeneic hsct recipients, the disparity in match between the donor graft and the recipient for the human leukocyte antigens (hlas) mediate both gvhd and graft rejection (host-versus-graft reaction). the presence of alloreactive injury to the lung attributed to gvhd is poorly defined and remains debated. in the skin, liver and intestine, gvhd produces a characteristic t-lymphocyte-mediated epithelial destruction. there are few data to support such a defined lesion with the exception of lymphocytic pneumonitis [77] . a variety of pulmonary complications have been described as manifestations of gvhd, but these associations are based primarily on the simultaneous occurrence of pulmonary abnormalities, the absence of an infectious agent and nonspecific histopathologic lesions in the setting of established gvhd in other organs. nevertheless, both acute and late-onset lung injury syndromes have shown a clinical association with gvhd, including ips, engraftment syndrome, diffuses alveolar hemorrhage, brob and cop [78] . several murine models also demonstrate pathologic lung changes in the setting of gvhd, thus supporting a mechanistic relationship between gvhd and lung injury. old and chronic afo are the most common noninfectious late pulmonary complications of allogeneic hsct. these entities are manifested on pulmonary function testing by a diminished fev1 or fev1/fvc. the incidence of these syndromes ranges from 6% to 32%, depending upon the definition of afo applied in each study [79, 80] . typically, the presentation occurs beyond the third month after hsct [81] . among patients who develop chronic gvhd, new-onset afo may develop in up to one third of the patients. in a study of 11 cases the underlying process accounting for afo was brob in 70% [82] . histologically, this process demonstrates fibrous obliteration of the lumen of respiratory and membranous bronchioles. in the absence of histopathologic evidence, new onset afo after allogeneic hsct often is referred to as "bronchiolitis obliterans syndrome" (bos). in addition to chronic gvhd, risk factors for the development of afo include increasing recipient age, pre-transplant reduction in the ratio fev1/fvc, low serum immunoglobulin levels, use of methotrexate and a history of respiratory viral infection within the first 100 days [79] . the onset typically is insidious with presenting symptoms including dry cough (60-100%), dyspnea (50-70%) and wheezing (40%), but fever is uncommon [79, 82, 83] . the chest radiograph is usually normal, but high-resolution ct scans often demonstrate evidence of expiratory air trapping, hypo-attenuation and bronchial dilation [80, [84] [85] [86] . demonstrating persistent afo using pulmonary function testing and exclusion of other causes of afo such as asthma, tobacco-related emphysema, and viral or bacterial respiratory infection establish the diagnosis. except for its utility in excluding an infectious etiology, bal is usually nonspecific [87] , and transbronchial biopsies typically are nondiagnostic due to the patchy nature of this small airway process and the limited size of samples obtained. surgical lung biopsy is rarely indicated. the etiology of new onset afo after hsct is unknown. those recognized causes in otherwise normal hosts rarely include recurrent aspiration, viral infection (influenza, adenovirus, measles) and bacterial infection (mycoplasma sp.) [88] . immunologic mechanisms inducing bronchial epithelial injury are suggested by the strong association between chronic gvhd and new onset afo. indeed, the lung epithelium may be the target of immune-mediated injury induced by donor cytotoxic t cells in chronic gvhd [89] . thus, brob after hsct may represent a manifestation of gvhd in the lung. disease progression is variable; however, the syndrome is associated with significantly increased mortality rates, and improvement in lung function is uncommon. many patients develop a progressive decline in lung function resulting in respiratory failure [79, 80] . there are no prospective studies of the treatment of new onset afo after hsct. old in the presence of chronic gvhd is managed primarily by controlling gvhd. various immunosuppressive agents have been reported to result in stabilization of lung function in 30-50%, but improvement in only 8-30% [37, 51] . in the hope that early recognition and treatment may improve outcome, routine spirometry among patients with chronic gvhd is encouraged to detect the insidious onset of this process. restrictive lung disease (rld) is defined by reductions in fvc, total lung capacity (tlc) and dlco as measured by standard pulmonary function tests (pfts). in rld, the ratio fev 1 /fvc is maintained near 100% [90, 91] . rld is common after hsct. significant decreases in fvc or tlc have been reported in as many as 25-45% of allogeneic hsct recipients by day 100. a decline in tlc or fvc after hsct (even if the absolute values for each measurement remained within the normal range) has been associated with an increase in nonrelapse mortality. tbi-containing conditioning regimens and the presence of acute gvhd have been associated with rld, in addition to obstructive lung disease [92] [93] [94] ; however, the impact of age on the development of rld is less clear. early reports suggested that the incidence of rld is lower in children compared to adults and that the incidence increases with advancing recipient age [95] . more recent studies have revealed significant rld in children receiving hsct [96] . organizing pneumonia after hsct falls under the rld pattern on liver function tests and recently was shown to be associated with prior acute and chronic gvhd. organizing pneumonia has been described in case reports as occurring after both allogeneic and syngeneic hsct; these data suggest an association of the lung lesion with chronic gvhd and intestinal ulcerations. in addition, corticosteroid therapy appeared beneficial in the resolution of the lesion. in a recent case control study, freudenberger et al. reviewed 49 cases of histologic cop [97] . the clinical features of cop in this population were similar to idiopathic and other etiologies with an association between acute and chronic gvhd and the subsequent development of cop. affected patients were more likely to have skin involvement with acute gvhd and chronic gvhd affecting the gut and oral mucosa. the causes of cop following hsct remain enigmatic, but possible etiologies include direct allo-immunologic reactions, atypical infection or atypical manifestations of ips. regardless, the clinical presentations and responses of cop are similar to other cases of idiopathic cop. the published literature contains a paucity of therapeutic trials for chronic lung injury after hsct. a study by payne and colleagues showed that the use of cyclosporine and methotrexate as gvhd prophylaxis prevented the development of old when compared to historic controls receiving prednisone and methotrexate [98] , but results of prospective, randomized trials in this setting are not available. three recently published case series have exploited the antiinflammatory effects of azithromycin to treat old in both allogeneic hsct and lung allograft recipients. each study suggested a beneficial effect of this drug on pulmonary function when administered for 12 or more weeks [99] [100] [101] . the potential role for tnfa in the pathogenesis of both old and rld suggests that agents such as etanercept may have promise, and several studies have demonstrated a potential benefit of this drug in some hsct patients with chronic lung injury [102, 103] . the immunologic mechanisms responsible for chronic, fibrotic pulmonary dysfunction after hsct remain poorly defined, in large part because of the lack of correlative data obtained from afflicted hsct recipients and the paucity of suitable sct animal models for either rld or old. chronic pulmonary disease following allogeneic hsct likely involves an initial insult to lung parenchyma followed by an ongoing inflammatory process involving the interplay between recruited donor-derived immune cells and the resident cells of the pulmonary vascular endothelium and interstitium. mechanistic insights into old following hsct have been derived from studies of lung allograft rejection. data generated from both humans and mice support the hypothesis that the development of brob in this scenario involves the secretion of inflammatory cytokines and chemokines, along with interactions between apcs and activated lymphocytes [104, 105] a tri-phasic model of chronic noninfectious lung injury after hsct has been proposed [106] . in phase i, an acute pneumonitis develops as a consequence of an allogeneic immune response, resulting in the sequential influx of lymphocytes, macrophages and neutrophils into an inflamed pulmonary parenchyma. in phase ii, a persistent inflammatory signal, in the setting of dysregulated repair mechanisms, promotes the transition from acute to chronic injury. if the inciting injurious stimulus predominantly involves bronchiolar epithelial cells, phase ii is associated with the concentric infiltration of lymphocytes and collagen deposition in the peri-bronchiolar areas resulting in the development of chronic bronchiolitis. if, however, the alveolar epithelium is the primary target, leukocyte recruitment and matrix deposition are confined primarily to the interstitial space. as chronic inflammation proceeds to phase iii, lung fibroblasts increase dramatically in number and contribute to the enhanced deposition of collagen and granulation tissue in and around bronchial structures, ultimately resulting in complete obliteration of small airways and fixed old. by contrast, fibroblast proliferation and intra-septal collagen deposition during phase iii ultimately result in interstitial thickening, septal fibrosis, significant volume loss and severe rld. clinical and experimental data suggest that the progression to a chronic, pro-fibrotic form of pulmonary toxicity involves the secretion of cytokines and chemokines [107] [108] [109] , and in this context, tnfa may be a central factor in the proposed tri-phasic model of disease. evidence for a role of tnfa in the transition from acute to chronic lung injury comes from studies using targeted over-expression of tnfa in the lungs of rodents [110] . in these models, early lung histopathology includes a lymphocytic infiltrate similar to that seen in experimental ips models [111, 112] , whereas the histologic changes associated with more prolonged exposure to tnfa show both interstitial and peribronchial inflammation that closely resemble changes seen at later time points after hsct [107, 113] . fa is characterized by sequential acute lung injury that can culminate in scarring and end-stage lung disease. despite the high success rate in treating hematologic malignancies with or without using hsct, this sequence of events continues to be a significant contributor to nonrelapse morbidity and mortality in patients with hematologic malignancies because of either the disease itself or as a result of treatment modalities employed. the pathophysiologic mechanisms contributing to the initiation and progression of disease remain poorly defined. to this end, current treatment options remain suboptimal and primarily limited to supportive care measures and antiinflammatory agents, such as corticosteroids or other immunosuppressant therapy. further research is necessary (in the form of clinical trials and pre-clinical models) to improve our understanding of fibrosing alveolits and related disorders and to ultimately design and implement targeted therapeutic strategies. idiopathic pulmonary fibrosis/cryptogenic fibrosing alveolitis sept) pulmonary complications in adult blood and marrow transplant recipients: autopsy findings jul) validation of four prognostic scores in patients with cancer admitted to brazilian intensive care units: results from a prospective multicenter study impact of recent intravenous chemotherapy on outcome in severe sepsis and septic shock patients with hematological malignancies acute interstitial pneumonitis: current understanding regarding diagnosis, pathogenesis, and natural history the ct halo sign clinical/pathologic correlations in 553 patients with primary centrilobular findings on high-resolution ct scan of the thorax the pathology of pulmonary disorders due to aspergillus spp semi-invasive" pulmonary aspergillosis: a new look at the spectrum of aspergillus infections of the lung rapport sur l'action des rayons x sur la tuberculose intrathoracic changes induced by heavy irradiation plasma transforming growth factor beta1 as a predictor of radiation pneumonitis radiation pneumonitis and early circulatory cytokine markers circulating il-6 as a predictor of radiation pneumonitis a perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis injury to the lung from cancer therapy: clinical syndromes, measurable endpoints, and potential scoring systems factors predicting radiation pneumonitis in lung cancer patients: a retrospective study factors predicting severe radiation pneumonitis in patients receiving definitive chemoradiation for lung cancer clinical radiation pneumonitis and radiographic changes after thoracic radiation therapy for lung carcinoma analysis of factors associated with radiation-induced bronchiolitis obliterans organizing pneumonia syndrome after breast-conserving therapy radiation-induced pneumonitis in the "nonirradiated" lung pentoxifylline in prevention of radiation-induced lung toxicity in patients with breast and lung cancer: a double-blind randomized trial radiotherapy or chemotherapy followed by radiotherapy with or without amifostine in locally advanced lung cancer randomized phase iii trial of radiation treatment +/− amifostine in patients with advanced-stage lung cancer radiation-induced endothelial dysfunction and fibrosis in rat lung: modification by the angiotensin converting enzyme inhibitor cl242817 pulmonary function changes after radiotherapy in non-small-cell lung cancer patients with long-term disease-free survival changes in local pulmonary injury up to 48 months after irradiation for lymphoma and breast cancer efficacy and toxicity of 12 courses of abvd chemotherapy followed by low-dose regional radiation in advanced hodgkin's disease in children: a report from the children's cancer study group high-dose cyclophosphamide, bcnu, and vp-16 (cbv) conditioning before allogeneic stem cell transplantation for patients with non-hodgkin's lymphoma drug-induced lung injury high-resolution ct of drug-induced lung disease pulmonary drug toxicity: radiologic and pathologic manifestations drug-induced pneumonitis: the role of methotrexate methotrexate pneumonitis: review of the literature and histopathological findings in nine patients spectrum of cd4 to cd8 t-cell ratios in lymphocytic alveolitis associated with methotrexate-induced pneumonitis spontaneous" resolution of two severe methotrexate-induced pneumonias successful reintroduction of methotrexate after acute pneumonitis in a patient with acute lymphoblastic leukemia relapsing acute respiratory failure induced by minocycline acute eosinophilic pneumonia associated with acute respiratory distress syndrome: case report eosinophilic lung diseases: a clinical, radiologic, and pathologic overview successful treatment of bronchiolitis obliterans organizing pneumonia with low-dose methotrexate in a patient with hodgkin's disease fulminant bronchiolitis obliterans organizing pneumonia following 2 d of treatment with hydroxyurea, interferon-alpha and oral cytarabine ocfosfate for chronic myelogenous leukemia bronchiolitis obliterans with organizing pneumonia during interferon b-1a treatment nodular form of bleomycin-related pulmonary injury in patients with osteogenic sarcoma late pulmonary changes following bleomycin administration in computed tomography. nodular fibrosis mimicking a seminoma metastasis multiple pulmonary nodules: unusual manifestation of bleomycin toxicity lung pseudometastases due to vinblastine toxicity bleomycin nodules mimicking metastatic osteogenic sarcoma multiple pulmonary nodules: an unusual presentation of fludarabine pulmonary toxicity: case report and review of literature pulmonary toxicity of antineoplastic therapy gemcitabine added to doxorubicin, bleomycin, and vinblastine for the treatment of de novo hodgkin disease: unacceptable acute pulmonary toxicity efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease severe pulmonary toxicity in patients with advanced-stage hodgkin's disease treated with a modified bleomycin, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone, and gemcitabine (beacopp) regimen is probably related to the combination of gemcitabine and bleomycin: a report of the german hodgkin's lymphoma study group severe lung toxicity with a weekly low dose chemotherapy regimen in patients with non-hodgkin's lymphoma pulmonary drug toxicity in patients with primary breast cancer treated with high-dose combination chemotherapy and autologous bone marrow transplantation cardiopulmonary toxicity after three courses of abvd and mediastinal irradiation in favorable hodgkin's disease empiric prednisone therapy for pulmonary toxic reaction after high-dose chemotherapy containing carmustine (bcnu) severe bleomycin lung toxicity: reversal with high dose corticosteroids pulmonary fibrosis induced by cyclophosphamide severe bleomycin lung toxicity: reversal with high dose corticosteroids idiopathic pneumonia syndrome after high dose chemotherapy for relapsed hodgkin's disease drug-induced lung disease: high-resolution ct and histological findings utility of bronchoalveolar lavage in the diagnosis of drug induced pulmonary toxicity severe alveolar proteinosis following chemotherapy for acute myeloid leukemia in a lung allograft recipient impact of open lung biopsy for undiagnosed pulmonary infiltrates in patients with hematological malignancies surgical pathology of non-neoplastic lung disease severe bleomycin lung toxicity: reversal with high dose corticosteroids (case report) high-dose carmustine, etoposide and cisplatinum for autologous stem cell transplantation with or without involved-field radiation for relapsed/refractory lymphoma: an effective regimen with low morbidity and mortality role of carbon monoxide diffusing capacity in the early detection of major bleomycin induced pulmonary toxicity pulmonary, pleural and thoracic changes complicating chemotherapy lung toxicity associated with cyclophosphamide use: two distinct patterns the hamman-rich syndrome following treatment of lymphoma with chlorambucil methotrexate induced diffuse interstitial pulmonary fibrosis acute interstitial pneumonitis related to gemcitabine pavletic sz bronchiolitis obliterans after allogeneic hematopoietic stem cell transplantation bronchiolitis obliterans and other late onset noninfectious pulmonary complications in hematopoietic stem cell transplantation lymphocytic pneumonitis following bone marrow transplantation in severe combined immunodeficiency the histological spectrum of pulmonary graft-versus-host disease in bone marrow transplant recipients airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation obstructive lung disease after allogeneic marrow transplantation: clinical presentation and course bronchiolitis obliterans after autologous bone marrow transplantation smallairways disease in recipients of allogeneic bone marrow transplants: an analysis of 11 cases and a review of the literature rapidly progressive air-flow obstruction in marrow transplant recipients: possible association between obliterative bronchiolitis and chronic graftversushost disease obstructive lung disease in children after allogeneic bone marrow transplantation post-transplant obstructive lung disease ("bronchiolitis obliterans"): a clinical comparative study of bone marrow and lung transplant patients late-onset noninfectious pulmonary complications after allogeneic bone marrow transplantation analysis of airflow obstruction by bronchoalveolar lavage following bone marrow transplantation: implications for pathogenesis and treatment risk factors for airflow obstruction in recipients of bone marrow transplants bronchiolitis obliterans in bone marrow transplantation and its relationship to chronic graft-v-host disease and low serum igg association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants bronchiolitis obliterans and other late onset non-infectious pulmonary complications in hematopoietic stem cell transplantation risk factors for air-flow obstruction in recipients of bone marrow transplants abnormalities of pulmonary function tests after marrow transplantation predict nonrelapse mortality pulmonary function changes 100 days and one year after bone marrow transplantation late pulmonary sequelae after childhood bone marrow transplantation the effects of bone marrow transplantation on pulmonary function in children association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants cyclosporine as possible prophylaxis for obstructive airways disease after allogeneic bone marrow transplantation azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation maintenance azithromycin therapy for bronchiolitis obliterans syndrome: results of a pilot study azithromycin in bronchiolitis obliterans complicating bone marrow transplantation: a preliminary study recombinant human tumor necrosis factor receptor fusion protein as complementary treatment for chronic graft-versus-host disease etanercept for sub-acute lung injury following allogeneic stem cell transplantation (abstract) interleukin-1 receptor antagonist as a biomarker for bronchiolitis obliterans syndrome in lung transplant recipients elevated levels of interleukin-8 and transforming growth factor-beta in bronchoalveolar lavage fluid from patients with bronchiolitis obliterans syndrome: proinflammatory role of bronchial epithelial cells lung injury following hematopoietic stem cell transplantation idiopathic pneumonia syndrome after bone marrow transplantation: the role of pretransplant radiation conditioning and local cytokine dysregulation in promoting lung inflammation and fibrosis pulmonary fibrosis: cytokines in the balance transfer of tumor necrosis factor-a to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-b1 and myofibroblasts expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. a mouse model of progressive pulmonary fibrosis an experimental model of idiopathic pneumonia syndrome after bone marrow transplantation. i. the roles of minor h antigens and endotoxin pneumopathies of the graft-versus-host reaction. alveolitis associated with an increased level of tumor necrosis factor mrna and chronic interstitial pneumonitis the histological spectrum of pulmonary graft-versus-host disease in bone marrow transplant recipients key: cord-023308-af5nihyi authors: nan title: copd sig: poster session 2 date: 2008-03-12 journal: respirology doi: 10.1111/j.1440-1843.2008.01252_6.x sha: doc_id: 23308 cord_uid: af5nihyi nan increased airway smooth muscle (asm) in asthma may be due to hyperplasia or hypertrophy of asm cells. the contribution of extracellular matrix (ecm) within asm bundles has not previously been accounted for when estimating asm cell volume. aim to estimate the mean asm cell volume in asm bundles in asthma. methods post-mortem tissues from control subjects (c n = 9); nonfatal (nfa n = 11) and fatal (fa n = 10) cases of asthma were studied. on 30 mm transverse airway sections stained with haematoxylin, the volume density (nv) of asm cell nuclei was estimated using an optical disector (¥1000). the mean cell volume (vc = 1/nv) was calculated, correcting for the volume fraction of asm (fasm) within the asm bundle (corrected vc = 1/(nv ¥ fasm)). fasm was estimated on 0.5 mm thick sections of the same airway stained with masson's trichrome. basement membrane perimeter (pbm) was used to indicate airway size. results table shows mean ϯ sd. (one-way anova) *p < 0.05 for c v fa, nfa v fa. conclusion these data suggest that although asm area is increased in asthma, mean asm cell volume is unchanged. therefore hyperplasia, not hypertrophy, of asm cells is present in both mild and severe asthma. these results were similar for both large and small airways. asthma is characterized by airway inflammation and remodelling which contribute to airway hyperresponsiveness and episodic airflow obstruction. mast cell (mc) densities are higher on the smooth muscle (asm) in asthma so their mediators may modulate other asm functions as well as cause contraction. aim to investigate the effect of mc mediators on chemokine and extracellular matrix (ecm) production by asm cells from donors with and without asthma. methods mc were isolated from the resected lung samples of 6 patients, resuspended at 10 6 cells/ml in dmem + 10% fbs and stimulated with ige/anti-ige. supernatants (sn) were collected after 2 and 24 h and the mc lysed. sub-confluent asm cells from 6 donors with and without asthma were serum deprived for 72 h before mc sn/lysates were added in dmem + 10%fbs for 48 h. il-8 and eotaxin levels in all asm sn and mc sn/lysates were measured by elisa. fibronectin and collagen iv deposition was measured in situ by immunoassay following asm cell lysis. results in asthmatic and non-asthmatic asm cells all mc sn and lysates reduced eotaxin release by up to 47% and 58%, whereas the 0-2 h mc sn significantly increased il-8 release to 178 ϯ 35.9% (p = 0.0339) and 169 ϯ 49% (p = 0.0445) of the fbs control respectively. however, only nonasthmatic asm cell il-8 release was increased by the mc 2-24 h sn (216 ϯ 85%; p = 0.0421) and cell lysates (215 ϯ 47%; p = 0.0421). the 0-2 h mc sn also increased fibronectin deposition to 143 ϯ 16% (p = 0.008) by asthmatic asm cells only. mc sn and lysates had no effect on collagen iv deposition. conclusions activated mast cell mediators differentially modulated chemokine and ecm secretion by asm cells from donors with and without asthma. thus mast cells may modulate their own recruitment to the smooth muscle and remodelling locally in the airways in asthma. supported by nhmrc. the technique of ige passive sensitization reproduces ige-related allergic responses in vitro and studies have validated this technique for investigations modelling allergic smooth muscle responses. there are no studies investigating effects of ige sensitization on rhinovirus (rv) infection. we hypothesized that rv infection is enhanced by ige sensitization, a consequence of diminished early innate immune responses. methods beas-2b epithelial cells and primary culture airway fibroblasts were sensitized with ige 24 h-7 d prior to infection with rv16. samples of tissue culture supernatant and cell lysates were collected over a 12 h period after infection for analysis. viral replication was measured by real-time rt-qpcr and viral titration and type i interferon mrna by rt-qpcr. ige receptor mrna expression was examined using rt-pcr. results initial studies to establish the model used human serum high in ige (>1000 iu/ml), this yielded inconsistent results and it was found that purified ige (1000 iu/ml) provided more reliable responses. sensitization was established after 24 h ige incubation and was comparable with up to 7 d. rt-pcr detected mrna for the ige low affinity receptor only after sensitization. following rv16 infection, vrna was increased after 24 h in ige sensitized cells (p < 0.05), but this effect varied noticeably between and within cell lines. cellular expression of ifn-b mrna increased with viral infection but in cells sensitized with ige lower levels of expression were noted (p < 0.05). conclusions ige passive sensitization enhanced rv replication in vitro but the model is constrained by significant variability between and within cell lines. the effect of sensitization on rv replication may occur through the low affinity ige receptor. activated mast cells (mc) are present in higher numbers on the airway smooth muscle (asm) in asthma compared with other inflammatory airway diseases. matrix metallo-proteinases (mmps) cleave chemokines and alter chemokine gradients by degrading the extracellular matrix and thus may modulate mc migration to the asm. aim to determine the levels of mmp-2, mmp-9 and their inhibitors, timp-1 and timp-2, secreted by asm cells from donors with and without asthma. method confluent asm cells were washed, serum-starved for 48 h and then stimulated with th1 (il-1, tnf and ifn) or th2 (il-1, il-4 and il-13) cytokines or left unstimulated. after 4 and 24 h,the sn were collected. the relative amount of pro and active forms of mmp-2 and mmp-9 in sn were determined by gelatine zymography. timp-1 and timp-2 levels in the sn were measured by elisa. results pro-and active mmp-9 were not detected. however, pro-mmp-2 levels were high in sn of asm cells from donors with (195.6 ϯ 47.2 % positive control/10 5 cells) and without (226.5 ϯ 49.2 % positive control/10 5 cells) asthma. a trend to increased active mmp-2 production by asm cells from donors with (7.3 ϯ 2.7 % positive control/10 5 cells, n = 9) compared to without (2.9 ϯ 0.7 % positive control/10 5 cells, n = 11) asthma after 24 h was not significant (p = 0.101). timp-1 and timp-2 levels respectively were high in the sn of cells from donors with (69.4 ϯ 19.6 and 21.3 ϯ 4.7 ng/10 5 cells, n = 5) and without (57.3 ϯ 13.7 and 16.6 ϯ 3.5 ng/10 5 cells, n = 5) asthma. th1 and th2 cytokine stimulation did not affect mmp or timp release. conclusions th1 and th2 cytokines did not regulate asm cell production of mmp-2, timp-1 and timp-2. altered asm mmp-2 activity is unlikely to play a role in mc chemotaxis to asm cells from donors with asthma in vitro or their presence on the asm in asthma. there has been a marked increase in the prevalence of asthma and other allergic diseases in the last few decades. one of the explanations for this is the change in our diet. one of the characteristics of the "western diet" is a high intake of both saturated and polyunsaturated fat. this prompted us to compare the effects of high fat and low fat meals on the numbers of circulating eosinophils and other leukocytes. methods we studied 12 volunteers who had allergic rhinitis and/or asthma and a peripheral eosinophil count at baseline of ն200 ¥ 10 7 /l. this was a randomized, crossover trial with participants studied on two different days. on each occasion they arrived fasting and after bloods were drawn consumed a 3000 calorie meal. one of the meals was high in saturated fat and refined carbohydrate. the other meal was low in saturated fat and high in fruit and fibre. bloods were drawn postprandially every hour for five hours. results eosinophil counts were highest in the early morning and fell over the course of the day but the decrease was less with the high fat meal (p = 0.03). over the same period of time the increase in lymphocytes (p = 0.016) was greater with the high fat meal. the high fat meal was also associated with greater increases in triglycerides (p < 0.0001) and cholesterol (0.004). conclusions in atopic individuals a high fat meal was associated with higher circulating numbers of eosinophils and lymphocytes than an isocaloric meal that was low in fat. further studies of the effect of dietary fat on eosinophilic inflammation are warranted. supported by the university of auckland research committeee. intravenous gamma globulin therapy (ivig), which is therapeutic in a variety of immune diseases, has been reported to be effective on patients with severe steroid-dependent asthma. although fcer are known to play important roles in asthma, there are few reports about the role of fcg?receptors in asthma. fcg receptor iib (fcgriib) is unique inhibitory receptor, which suppresses immune response. in this study, we evaluated the effect of ivig in allergic airway inflammation in ova-challenged mice and the mechanism of the inhibitory effects of ivig and fcgriib. method c57bl/6 mice (wt) and fcgriib deficient mice (ko) were sensitized with ovalbumin (ova) and alum and subsequently challenged with nebulized ova. before ova challenge rabbit igg was administered intravenously. the airway inflammation and effects of igg were assessed by histology, cell counts of bal fluid and airway hyperresponsiveness. result histology showed that igg treatment ameliorated the inflammation around the airway and the vessels and hypertrophy of goblet cells induced by ova challenge. the migratory activity of dcs is modulated in inflammatory diseases such as asthma. recently, we reported that immature dcs express kinin receptors and that bradykinin (bk) significantly enhances the migration of immature dc in vitro. as kinins mediate many of the pathophysiological effects associated with asthma, we hypothesized that lys-des[arg 9 ]-bk, which is produced during inflammation and acts via the b1 receptor (b1r), would inhibit migration of mature dcs. methods day 7 cultured human monocyte-derived dcs were matured with lps, tnfa +il-1b or cd40l in the absence or presence of lys-des[arg 9 ]-bk. maturation of dc was analysed by flow cytometry (facs). b1r expression was assessed by reverse-transcriptase pcr and quantitative confocal microscopy. migration of mature dc was assessed in transwell chambers with lysdes [arg 9 ]-bk and the chemokine ccl19 used as chemoattractants. results maturation of dcs was found to result in down-regulation of b1r expression to varying degrees depending upon the maturation stimulus used. mature dcs all demonstrated an ability to migrate toward lys-des[arg 9 ]-bk and ccl19. however pre-treatment with lys-des[arg 9 ]-bk decreased the migratory ability of all mature dcs to both chemoattractants. conclusions along with chemokines, lys-des[arg 9 ]-bk is likely to play a crucial role in regulating the in vivo migration of mature dc during inflammation. the production of lys-des [arg 9 ]-bk during inflammation potentially immobilizes mature dcs thereby facilitating locally-mediated immune responses within inflamed tissues. supported by the asthma foundation of western australia. introduction alternative or aberrant splicing is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms. the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to use a new and innovative approach involving the use of the genechip (r) exon array system together with real-time quantitative pcr to study asthma candidate genes in human monocyte-derived dendritic cells. asthmatic and non-asthmatic subjects provided 20 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method, and differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data indicate splice variant expression in dendritic cells from asthmatic patients is influenced by asthma severity. conclusion exon expression array analysis has generated a number of asthma candidate genes with alternative splice variants. further studies to validate these data in a replicate data set and establish the functional significance of our findings in asthma are underway. alternative or aberrant splicing occurs in more than 70% of genes and is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms 1 . the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to study one asthma candidate gene in human stimulated and unstimulated: (1) monocytes, (2) monocytederived dendritic cells and (3) lung smooth muscle cells. methods asthmatic and non-asthmatic subjects provided 40 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method. up to 50% of the monocytes were then differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. induction experiments used 1 mg/ml lps and cells were stimulated for an optimal period of 24 hrs. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data from stimulation experiments indicate splice variant production can be regulated by the inflammatory response and that this response is influenced by asthma status. conclusion preliminary experiments have confirmed the presence of an aberrant splice variant for an asthma candidate gene in the primary cells studied. further studies to confirm these data and establish the functional significance of our findings in asthma are underway. exposure to environmental factors, such as environmental tobacco smoke (ets), plays a significant role in modulating pre-existing genetic susceptibilities to diseases including asthma. the glutathione s-transferase enzymes (gsts) play an important role in the detoxification of ets. there are several gst isoforms and gstp1 codes for the gst pi isoform, which is the primary gst isoform expressed in human lung tissue. two single nucleotide polymorphisms (snps) at positions 105 and 114 have been reported in gstp1 and associated with asthma and atopy. the aim of this study was to examine the effect of these snps in combination with ets, on asthma phenotypes in a cohort of asthmatic children. children were recruited during an acute episode requiring presentation at an emergency department. genotyping using pcr-rflp was completed on 218 children and ets exposure was determined by parental questionnaire. urinary cotinine was measured in the children and was in agreement with questionnaire responses. statistical analyses were performed using spss. there were no significant associations between the genotypes and asthma severity during acute exacerbations. significant associations were found between the snps and atopy in this population with an odds ratio of 2.77 for the 105aa genotype (p = 0.029) and or of 5.47 for the 114cc genotype (p = 0.002). however, when an interaction with ets was included, the odds ratios increased to 9.02 for 105aa (p = 0.05) and 9.17 for 114cc (p = 0.020). these results suggest that there is a significant gene/environment interaction impacting on atopy in this cohort. the rage gene encodes the receptor for advanced glycation end-products (rage), a member of the immunoglobulin superfamily. rage activation by ligands, including amphoterin and s100/calgranulins, leads to prolonged nf-kb signalling and has been associated with chronic inflammation. despite high levels of rage expression in lung tissue, little research has been undertaken into the role of rage in the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. objective determine genetic associations between functional polymorphisms in the rage promoter and severe and aspirin-sensitive asthma phenotypes. methods pcr and restriction fragment length polymorphism (rflp) were used to genotype three rage promoter polymorphisms, -429t>c, -374t>a and a 63 bp deletion from -407 to -345, in a large case-control asthma population phenotyped for asthma severity, atopy and aspirin sensitivity. results no associations were identified between any of the polymorphisms and the occurrence of asthma. however, the -374a allele was linked with both severe asthma (p = 0.013) and aspirin-sensitive asthma (p < 0.001). likewise, genotypes containing the -374a allele were strongly associated with both severe asthma (or 2.10, 95% ci 1.32-3.36) and aspirin-sensitive asthma (or 3.13, 95% ci 1.45-6.77). conclusions the -374a allele of the rage gene, previously shown to lead to a 3-fold increase in promoter activity, is associated with the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. these results suggest that increased rage expression, with a concomitant increase in nf-kb signalling, may in part contribute to the inflammatory response seen in these conditions. the global prevalence of allergic diseases is rising and australia has one of the highest prevalence rates in the world. the role of early childhood infections in the development of allergic disease remains controversial. objective to examine the association between early childhood infections and the development of allergic diseases in later childhood, in high risk children. methods data were analysed from the melbourne atopic cohort study (macs) of 620 infants with 1 or more first-degree family members with atopic disease. primary risk factors assessed were otitis media, bronchitis and gastroenteritis reported in the first two years of life. outcomes were current asthma, hay fever and eczema at 6 years of age. logistic regression was used to estimate crude and adjusted odds ratios. results asthma was the most common allergic condition (25.4%, 95% ci 21.6-29.5%), followed by eczema (24.9%, 95% ci 21.1-29.0%) and hayfever (15.6%, 95% ci 12.5-19.1%). the most commonly reported infection was otitis media (58.9%, 95% ci 54.9-62.8%), then gastroenteritis (37.7%, 95% ci 33.9-41.7%) and then bronchitis (19.4%, 95% ci 16. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] .7%). all 3 types of infection within the first 2 years of life were associated with increased risk of asthma. an increased risk of asthma at 6 years was seen with otitis media (or = 1.14, 95% ci 1.02-1.3), bronchitis (or = 1.34, 95% ci 1.0-1.8) and gastroenteritis (or = 1.23, 95% ci 0.96-1.6). when the frequency of infection was examined, those who reported at least 3 episodes of gastroenteritis had a 3-4-fold increased risk and an almost 30% absolute increased risk (rd 0.34, 95% ci 0.08-0.59). conclusion these findings appear to contradict the hygiene hypothesis. the findings for gastroenteritis are novel. further examination of these associations and possible underlying mechanisms is warranted. grant support asthma foundation of victoria, nestle. background knowledge about incident cases of asthma in australia is limited because they are not routinely reported. the ability to predict the number of new cases of asthma would be helpful in allocating resources for asthma education, management and care. data on first use of medications for asthma gives an indication of the incidence of asthma. the objective of this study was to estimate the incidence rate of asthma by investigating asthma medication use in individuals. methods pharmaceutical benefits scheme (pbs) records for all prescriptions filled for inhaled corticosteroids (alone or combined formulation), cromones and leukotriene receptor antagonists from july 2002 to june 2005 were included. using a 2-year look back window, any persons who had their first prescription for any of these drugs dispensed between july 2004 and june 2005 were assumed to be incident cases. overall and age-specific incidence rates were calculated per 100 asthma-medication-free individuals. results there were 352,082 individuals who had their first asthma medication dispensed between july 2004 and june 2005, which equates to an overall incidence rate for asthma of 1.89 per 100. the incidence was higher among children aged 0-14 years (2.07) and adults aged 65 years and over (2.45) . conclusions our estimated incidence rates were consistent with those reported by others in the literature. while the pbs database was designed for administrative purposes, it can be used to estimate incidence rates for asthma. support acam is a collaborating unit of the australian institute of health and welfare and is funded by the department of health and ageing (doha). we acknowledge the pharmaceutical pricing and estimates section of doha for provision of pbs data. keywords asthma incidence, pharmaceutical benefits scheme. rosario ampon 1 , guy marks 1 , teresa to 2 , leanne poulos 1 , anne-marie waters 1 1 australian centre for asthma monitoring (acam), sydney, australia, and 2 hospital for sick children, toronto, canada background the ability to assess individual patterns of asthma medication use would have clinical relevance in targeting effective asthma education and management for this common condition. to describe longitudinal patterns of asthma medication use, we used a population-based prescription database to follow individuals from the first time they filled an asthma prescription. asthma is more commonly listed on death certificates as an associated cause of death, in people whose deaths are attributed to other causes, than as an underlying cause of death. understanding the importance of these associations would contribute towards an overall appreciation of the impact of asthma on mortality. the objective of this analysis was to estimate the prevalence of asthma as an associated cause of death when various other diseases were attributed as the underlying cause of death. background acam currently recommend 24 indicators to measure population-level asthma health and outcomes. we examined correlations among several asthma indicators covering prevalence, morbidity and mortality to try and produce a condensed set of indicators which minimized redundancy. methods seven of the 24 indicators were included in this study: prevalence of ever having doctor diagnosed asthma, prevalence of current asthma, asthma-related general practice (gp) encounters, proportion of people with asthma with an asthma action plan (aap), hospitalizations for asthma, hospital patient days for asthma, and deaths due to asthma. a correlation matrix was created for these indicators by age groups. pearson correlation coefficients ն0.7 or յ-0.7 were considered strong. results there were strong positive correlations between prevalence of ever asthma and current asthma (r = 1.0); gp visits and aap possession (r = 0.74), hospitalization (r = 0.91) and patient days (r = 0.95); and hospitalization and patient days (r = 0.90) and aap possession (r = 0.73). recent australian reports have shown that the prevalence of asthma and respiratory symptoms has decreased over the last 10-15 years. as part of a larger study investigating child health and air quality we have collected nationwide data from schoolchildren living in act, victoria, queensland, wa and sa. methods schools were selected based on proximity to air quality monitoring stations. classes from years 3 to 6 were randomly selected and all children were invited to participate. parents self completed a questionnaire that included questions about diagnosed asthma and respiratory symptoms. results a total of 1989 children provided questionnaires for analysis. the response rate varied between states and territories and ranged from 30% to 42%. the sample comprised 51.9% girls and the mean age of children was 10.2 years. ever diagnosed asthma 27.9 current asthma ('does he/she still have asthma? ') 13.8 wheeze in the past 12 months 16.1 respiratory symptoms limiting activities 11.8 missed school due to asthma or wheezing 8.8 conclusions despite the relatively low participation rate, the prevalence estimates for current asthma are similar to those reported in the national health survey 2004-05 [1] . there is no evidence of any recent increase in the prevalence of childhood asthma. methods tahs is a longitudinal population-based respiratory study of 8583 subjects which commenced in 1968 when they were 7 years of age. since the initial study another 4 follow-ups have been conducted, including the most recent follow-up when subjects were 44 years of age. lung function of the total sample was measured at baseline and in sub-samples in 3 subsequent followups. asthma was categorized as persistent, frequent or episodic when participants reported asthma symptoms in at least 3 follow-ups, in 2 follow-ups or in 1 follow-up respectively. results by age 7 years ever asthma prevalence was 16%. at age 44, 10% of those who had not reported asthma by age 7 had asthma symptoms while 75% of those who reported asthma by age 7 had no asthma symptoms. hence over all only 25% of the asthma symptoms at age 44 were attributable to asthma developed by age 7. in contrast, 91% of the persistent and frequent asthmatics had developed their asthma by age 7. persistent and frequent asthmatics had more symptoms and poorer lung function at age 7, 14 and 44 as well as more reversibility at age 44 (p < 0.05). childhood asthmatics who also had a productive cough by age 7 were more likely to have persistent asthma than those without a cough (p < 0.05). conclusions although the majority of middle-age asthma is related to postchildhood onset asthma, most severe middle-age asthma has its origin in persistent childhood disease. having productive cough in childhood may identify high risk asthmatics who require especially rigorous management in early life. one third of women experience an improvement in asthma during pregnancy, and symptoms improve in most women in the late third trimester. we hypothesized that the exacerbation rate would be reduced and that symptoms during exacerbations would be less severe in the third trimester compared to the second trimester. methods pregnant women with asthma (n = 81) were prospectively followed from recruitment (14.8 weeks (3 sd) ) to delivery at clinic visits (18, 30, 36 weeks and during exacerbation), and fortnightly phone calls. the asthma control questionnaire (acq) was administered at each contact and exacerbations classified as severe (requiring medical intervention) or mild (selfmanaged). lung function, medication use, fractional exhaled nitric oxide (feno) and full blood counts were assessed. paracetamol is commonly used in infants as an analgesic and antipyretic. it has been hypothesized that frequent paracetamol consumption may result in reduced lung capacity to cope with oxidative stress and increase risk of respiratory disease. to date, no study has examined exposure to paracetamol during infancy, when lungs are still developing, and risk of childhood asthma. method a birth cohort of 620 infants with an atopic family history was recruited. frequency of paracetamol exposure was prospectively documented up to 2 years of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results paracetamol exposure in infancy was common (97% exposed by two years of age), with some infants receiving paracetamol on up to 77 days. it has been hypothesized that mucosal immune response requires a particular micro-flora milieu in the infant's gastro-intestinal tract, and that early life antibiotic exposure may disrupt this process and increase risk of allergic disease. method a birth cohort of 620 infants with an atopic family history was recruited. exposure to oral antibiotics was prospectively documented up to 12 months of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results by one year of age, approximately 80% of infants had received at least one course of oral antibiotics. the prevalence of current asthma in childhood was approximately 30% (148/495). frequent use of antibiotics (more than 20 days exposure during first year of life) was associated with increased risk of childhood asthma (or = 2.52, 95% ci = 1.40-4.54) when compared to infant who had not been exposed. excluding infants with a diagnosis of asthma within the first two years of life, reduced this association by about 30% (or = 1.80, 95% ci = 0.90-3.57) and adjustment for gender, parental history of asthma and number of infections in the first year of life further reduced this association (or = 1.60, 95% ci = 0.79-3.22). the increased risk of childhood asthma associated with antibiotic exposure in the first year of life is, at least in part, due to confounding with early life wheeze and infections. if real, the independent effect of antibiotic exposure on risk of childhood asthma is likely to be minimal in this high risk cohort. support dairy australia, crc for asthma and airways, vichealth, nestle. the epidemiological data on asthma suggest a gender difference that varies with age. hormonal effects have been suggested as a possible explanation for these differences. the aim of this study was to examine reproductive factors and risk of asthma among the females of the tasmanian longitudinal health study (tahs). methods the tahs is a longitudinal population-based cohort study of respiratory disease which commenced in 1968 when subjects were 7 years of age. four follow-up studies have been conducted including the current most comprehensive follow-up with subjects at 44 years of age. information has now been collected on reproductive factors such as number of pregnancies, age at pregnancies, age at menarche and contraceptive pill use as well as on asthma status. reproductive factors were examined as risk factors for asthma using multiple logistic regression to adjust for all likely confounders. results a total of 2,776 women completed the most recent postal survey. of these 355 (12.8%) had current asthma, and of these women with current asthma 73.5% (261) developed asthma after childhood. on average these women were in their mid-twenties when they developed asthma (mean ϯ sd age = 26.6 ϯ 12.5 yrs). we found with increasing age at first birth an approxi-mate~30% reduced risk of current asthma in women who developed their asthma post-childhood (trend p = 0.04). we did not observe any other associations between reproductive factors and risk of asthma. conclusions our results are consistent with the hypothesis that early pregnancy may promote asthma development by altering the immune response favouring a th2 pathway. a delay in the age of first pregnancy reduces this risk of asthma. grant support nhmrc, clifford craig foundation, victorian & tasmanian asthma foundations. introduction the association between exposure to pets in early life and subsequent development of sensitization and allergic disease remains controversial. the objective of this analysis was to examine the relationship between cat exposure before birth and development of cat sensitization over time within the melbourne atopic cohort study (macs). methods the macs is a prospective longitudinal cohort study that initially recruited 620 women antenatal in melbourne from february 1990 to november 1994. detailed information on cat exposure was collected at recruitment and frequently until two years of age. skin prick test (spt) were conducted at 6, 12, 24 months and 10 years. the data were analysed by logistic regression and using generalized estimating equations (gee) for the repeated measures design. results among 620 subjects, 169 (28.8%) had a cat before birth. at 6 months, 1.9% (n = 11) of subjects were sensitized to cat and by 10 years of age 18.8% (n = 68) were sensitized. those who did not have cat before birth belong to a higher social class, and were more likely to have a father with allergic disease than those with a cat. those who developed sensitization to cat were more likely to have a paternal family history of allergic disease and more likely to be sensitized to other allergens. we did not observe any association between exposure to cat before birth and the development of sensitization to cat at 6 months (or = 0.7, 95% ci 0. 1-3.3) , 12 months (or = 1.4, 0.5-3.9), 24 months (or = 0.76, 0.2-2.5) or 10 years (or = 0.6, 0. 2-1.4) . these crosssectional results were confirmed by the gee analysis. conclusion our results fail to show an association between cat exposure before birth and development of sensitization to cat. furthermore exposure after birth in the first 18months of life was not associated with an increased or decrease risk of sensitization to cat. our results do not support either a benefit or risk associated with cat ownership and sensitization. introduction peri-natal events influence the development of asthma and atopic diseases in childhood but the current literature is contradictory on the effect of low birth weight, small for gestational age and prematurity on asthma risk. the aim of this study was to assess the relationship between these three exposures and asthma from childhood to adulthood. aim to assess the current prevalence of dda, wheeze (<12 months), atopy and ahr in children and adults in busselton. methods an age-and sex-stratified random sample of adults, selected from the electoral roll, was invited to complete a questionnaire and attend the local study centre for assessment of atopy (allergen skin tests) and ahr (methacholine). all children from participating primary and secondary schools were also invited to attend. the prevalences of dda, wheeze, atopy, ahr and "current asthma" (wheeze + ahr) were calculated. background asthma is often associated with comorbidity, however few studies have investigated comorbidities among people with this common condition. the objective of this analysis was to describe patterns of non-respiratory comorbidity among adults hospitalized with asthma in australia. methods data on hospitalizations for people aged 15 years and over with a principal diagnosis of asthma (j45, j46) were obtained from the australian institute of health and welfare's (aihw) national hospital morbidity database for the period 2005-06. patterns of comorbidity were examined by investigating additional diagnoses for non-respiratory disease according to icd-10 diseasespecific chapters. results among people aged 15 years and over hospitalized in 2005-06 with a principal diagnosis of asthma (16,566 hospitalizations; 70% female; 47% aged 35-64 years), 33% had at least one non-respiratory comorbidity. median length of stay was higher among those with at least one comorbidity (4 days) than among those with no comorbidities (2 days). among people aged 15-64 years, the most common comorbid condition was endocrine, nutritional and metabolic diseases (19%), while among those aged 65 years and over it was diseases of the circulatory system (32%). conclusions a large proportion of asthma hospitalizations in australia are associated with non-respiratory comorbidity and a longer length of stay. further, the pattern of non-respiratory comorbidity associated with asthma hospitalizations varies by age. given our rapidly ageing population, the level of comorbidity associated with asthma has implications for coordinated health care and demand on health services. support acam is a collaborating unit of the aihw and is funded by the department of health and ageing. keywords comorbidity, hospitalization, asthma. background asthma exacerbations are often triggered by viral respiratory infections, yet the influence of respiratory infections on the morbidity of acute asthma beyond the immediate period is unknown. we examined the influence of nasopharyngeal (npa) respiratory viral, chlamydia and mycoplasma detection on asthma morbidity in children presenting to the emergency department for an acute exacerbation of asthma. methods a subset (n = 78) of the 201 children enrolled for a randomized controlled trial (rct) on the efficacy of 5 vs 3 days of oral prednisolone had an npa taken at presentation. npa were examined for chlamydia, mycoplasma and respiratory viruses (enteroviruses, coronaviruses, human metapneumovirus, adenovirus, parainfluenza, influenza, rsv, rhinoviruses) by pcr. enrolled children were aged 2-16 years with recurrent wheeze and required ն600 ?g (mdi/spacer) or ն2.5 mg (nebulized) of salbutamol to reduce tachypnoea. parents filled validated diary cards for cough and asthma severity, and completed asthma qol data at enrolment and end of weeks 1 and 2. results pcr for various viruses was positive in 42 (53.8%) children, with no significant difference in the groups the children were randomized into. rhinovirus pcr was positive in the npa of 32 children, rsv in 7, hmpv in 2, adenovirus, parainfluenza, influenza a and b in one each. specimens were negative for the other micro-organisms listed above. children with a npa viral positive state were significantly (p = 0.002) younger than those with a negative state. however, there was no difference in the any of the asthma outcomes of children whose npa was positive or negative for the micro-organisms tested. conclusions in children with an acute asthma exacerbation presenting to emergency health facilities, a respiratory virus could be identified in >50% but the presence of a respiratory virus did not influence the morbidity of the asthma exacerbation at presentation or at the end of week-1 and week-2. the university of sydney, nsw 2006, and 3 royal north shore hospital, st leonards, nsw 2065 airway wall thickness measured using hrct is reported to be increased in asthmatic compared with control subjects. however, it is unknown whether wall thickness is a fixed structural characteristic of the airways or if it responds to transient changes in bronchomotor tone or airway size. aim to determine the effects of bronchomotor tone and lung volume on airway wall area measured by hrct. methods 8 patients with doctor-diagnosed asthma had partial chest hrct scans, before and after bronchodilator (bd), at frc, tlc and a volume midway between (mid-volume). airway segments were identified between branch points and matched between consecutive lung volumes both before and after bd, and also at constant lung volume before and after bd. mean lumen areas and wall areas for each airway segment at each volume were measured using automated analysis software. paired t-tests were used to determine changes due to bd and lung inflation. results 44 airways were matched before and after bd at frc. absolute airway wall area (wa) was related to airway lumen diameter (di wood smoke air pollution is of concern with respect to respiratory health due to its complex chemical composition and potential to carry air toxics into the lower respiratory system. launceston has a long history of poor winter air quality, primarily due to use of domestic wood heaters. participants in hobart had a similar prevalence of wood heater use, but hobart does not experience the same wood smoke pollution (due to differences in regional geography , asthma control and anxiety and depression were completed at baseline, immediately following (6 wks), and 3 mths after the intervention period. results clinically and statistically (p < 0.05) significant improvements in qol were observed in the exercise group at 6 wks compared to the control group. this difference was not maintained at 3 mths. 6mwd improved at 6 wks and 3 mths in the exercise group (p < 0.01), however the difference between groups was not significant. in the exercise group there was a trend towards improved asthma control and a reduction in anxiety and depression that was not observed in the control group. *p < 0.05, change at 6 wks vs baseline; home asthma monitoring is important for measuring day-to-day variation in lung function and symptoms. this approach requires the availability of complete diaries for a comprehensive assessment. we assessed the completeness of written diaries collected as part of a nation wide study of air quality and child health. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified from a cross-sectional study. these children were asked to record symptom scores and peak expiratory flows twice daily in diaries for a five week period. the diaries and peak flow devices were explained at a face-to-face meeting with parents and children. each week diaries were mailed back and parents received a phone call to encourage completion. completeness was defined as no missing responses to symptom questions or peak flow measurements in diaries from week two to week five. results data from the first 36 children (822 day records) were available for analysis. the sample included (53%) girls, mean age 10 yrs. the overall frequencies for complete records were; morning symptoms 85%, morning peak flow 85%, evening symptoms 83% and evening peak flow 82%. there was a significant trend for more complete morning peak flow records over the four weeks (cochrane-armitage trend test p < 0.007). agreement between morning and evening symptom completeness and between morning and evening peak flow completeness was fairly poor (kappa < 0.30). conclusions the completeness of symptom and peak flow records collected in this study was very high. the comprehensive follow-up protocol implemented is likely to have had an important impact on the completeness of asthma diaries. daily peak expiratory flow (pef) monitoring has been used in epidemiological studies to assess changes in lung function over time. the value of written pef diaries has been questioned because of problems with completeness and validity. this study aimed to compare stored electronic pef data and a written diary record of those data in a panel study in children with weekly reminders to aid adherence. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified in a population study. they were given electronic pef devices with a digital readout (miniwright digital, mwd, clement clarke, uk) and written symptom and peak flow diaries and instructed in their use at a meeting with parents and children. each child was asked to complete three pef manoeuvres every morning and evening for five weeks and to record these in the written diary. background previous research suggests that comorbid anxiety is associated with lower asthma-related quality of life (aqol) in adults with asthma. however, research is scant on the role of psychological interventions in these patients. aim to evaluate the effectiveness of a four-session cognitive-behavioural therapy (cbt) intervention, in improving the aqol, in participants with anxiety and asthma. method participants identified with comorbid anxiety and asthma were randomly assigned to the cbt intervention group (n = 10) and the asthma monitoring control group (n = 8) and evaluated on aqol measures, at various intervals. results nine participants, in the cbt group, completed the study. seven participants showed a clinically significant improvement in asthma-related emotional functioning (ef) and six participants in total aqol scores, at the 5-week post-intervention assessment. additionally, six participants in the cbt group indicated clinically significant improvement in ef and five participants in total aqol scores, at the 3-month follow-up assessment. only three participants in the control group completed the study. none of these participants showed any improvement in aqol scores at the 5-week or 3-month assessment. conclusion this pilot study suggests that a higher number of participants in the cbt group showed clinically significant improvement in ef and total aqol scores with higher retention rates. further research needs to confirm these findings in a larger group, identifying the elements of a successful cbt intervention and characteristics of participants who respond to the cbt intervention. gastro-oesophageal reflux disease (gord) is a risk factor for uncontrolled asthma. we conducted an update of a systematic review to assess whether treatment of gastro-oesophageal reflux in subjects with asthma improved asthma outcomes. methods randomized controlled trials (rcts) of gord treatment in adults or children that reported asthma health outcomes and had symptomatic gord were included and assessed in accordance with the standard cochrane systematic review process. subjects received pharmacological therapies compared with conservative management. results from 261 potentially relevant studies, 19 rcts were included in the review. when compared to placebo, morning peak expiratory flow did not significantly improve (change from baseline wmd 10.43, 95% ci: -9.55 to 30.42) with proton pump inhibitor treatment (n = 7 trials involving 739 participants). asthma exacerbations were not significantly less in the intervention groups compared with the control groups (odds ratio 0.66; 0.41-1.08; n = 557). conclusions while some trials reported evidence of asthma improvement with gord therapy, overall there appears to be no statistically significant evidence of a beneficial effect. it is clear that not all persons with gord and asthma will gain improved control over their asthma with gord therapy; this may be due to the heterogeneous pathophysiology of asthma. future large-scale trials would be required to demonstrate an effect on asthma exacerbations. kel and brd were supported by a cochrane airways group scholarship. background the ats/ers task force recommend the use of metered dose inhaler (mdi) and spacer for airflow limitation reversibility testing. salbutamol given via mdi & spacer has been shown to be equivalent to a nebulizer in the clinical setting. this has not been well studied in respiratory laboratory setting. aim to compare the 2 methods of reversibility testing in a laboratory setting. methods we conducted a laboratory based crossover study in a secondary hospital. patients with asthma or copd were eligible. the patients firstly underwent spirometry and reversibility testing following a standard dose of nebulized salbutamol. they were asked to return for a second set of spirometry within the same week and at the same time of day when reversibility with an mdi and spacer was recorded. we used an incremental dose of salbutamol starting from 2 puffs and up to 8 puffs. spirometry parameters were recorded 10 minutes after each intervention. the primary outcome was the percentage change in fev1 after each intervention. side effects were monitored for. results nine patients with asthma were recruited. the mean percentage change in fev1 was higher in the nebulizer group than after only 2 puffs via mdi & spacer (15.4 ϯ 7.4 vs 6.2 ϯ 8 [mean ϯ sd], p = 0.67). however, there were no differences between the 2 arms following higher doses of bronchodilator via mdi & spacer. the mean percentage change in fev1 after 4, 6 and 8 puffs were 12.6 ϯ 11.3, 15.4 ϯ 12.3, and 17.7 ϯ 13.6 respectively (p = 0.09, 0.05 and 0.07 respectively when compared to the nebulizer group). conclusion using an mdi and spacer for bronchodilator reversibility is equivalent to that of a nebulizer and should be the standard method of testing. the dose of bronchodilator needs to be at least 4 puffs as recommended by the ats/ers; however 6 puffs correlated best with a standard nebulizer route. further increments in bronchodilator dose provided little additional bronchodilatation. the study was limited by the small number of patients. asthma guidelines recommend a stepwise approach to treatment. the role of inhaled corticosteroid (ics) and long-acting beta-agonist (laba) combination therapy in asthma written action plans is not clear. objective to assess the efficacy of adjusting ics/laba combination therapy in a written action plan compared to fixed dosing in people with asthma requiring maintenance ics. methods cochrane systematic review of randomized controlled trials comparing ics/laba combination therapy in a single inhaler that is adjusted up or down according to a written action plan (wap) to comparison 1: budesonide/ formoterol given as a fixed maintenance dose (fd) (n = 9) or comparison 2: fluticasone/salmeterol fd (n = 2). results 10 parallel randomized controlled trials describing 11 interventions met the inclusion criteria. for the trials that compared wap to fd budesonide/ formoterol there were significant reductions for the wap group in exacerbations, (rr (95%ci): 0.82 (0.70 to 0.97)), severe exacerbations (rr (95%ci): 0.61 (0.37 to 0.99)) and study medications (wmd (95%ci): -1.18 (-1.23 to -1.14)) with no difference in asthma control or adverse events. the results for the two trials reporting wap budesonide/formoterol to fd fluticasone/ salmeterol were discordant and a homogenous pooled result could not be determined. of the 318 australians who died from asthma in 2005, over two thirds were over 50 years of age. this trend resulted in the national asthma council of australia (nac) calling for better management of asthma in the elderly. we designed an educational intervention using evidence based educational strategies to improve the content and style of general practice consultations for older people with asthma. methods randomized controlled trial of a multi-faceted program consisting of a group educational session, a videotaped standardized simulated patient consultation, followed by an academic detailing session. forty-two gps were randomized into an active or a control group. gps provided the names of patients who would be happy to participate in the study and the program was evaluated by patient and gp outcomes. results gps recruited into our program reported improvements in a range of clinical areas. one hundred and ten patients were recruited, their outcomes are under analysis. conclusion gps were overwhelmingly positive about participation in this trial and our intervention successfully improved the capacity and confidence of gp's to deliver care to older people with asthma. our study also developed several tools that would enable dissemination of our findings. supported by an asthma targeted in studies where direct clinical assessment is not possible, urgent health care utilization (hcu) is often used as an indirect measure of asthma control. this study aimed to identify factors predicting urgent hcu and asthma control. methods patients in nsw with a doctor diagnosis of asthma were recruited from community pharmacies, a research volunteer database, and databases of asthma foundation nsw, to complete a questionnaire about asthma. poor asthma control was defined as asthma control questionnaire (acq) score ն1.5. urgent hcu was defined as hospitalization, ed visit, or urgent doctor visit due to asthma. multiple logistic regression was used to identify predictors of poor control and urgent hcu. results questionnaires were completed by 608 adults (61% female) with a doctor diagnosis of asthma (pharmacy 260, woolcock 299, asthma foundation 87). 87% used inhaled corticosteroid (ics) ϯ long-acting b2-agonist in the last 4 wks. median age was 56 yrs (range 12-87), and 9% were current smokers. mean acq score was 1.4 (95% ci 1.3-1.5), with 40% of participants having poor asthma control (acq ն 1.5). 28% had urgent hcu for asthma in the previous year. significant independent predictors for poor asthma control were younger age, current smoking, living in more disadvantaged areas, being retired, having only primary education, and holding a concession card. predictors for urgent hcu were younger age, being in full-time employment, having only primary education, and being of non-english speaking background. neither ics use nor possession of a written asthma action plan was associated with lower risk for either poor asthma control or hcu. conclusions poor asthma control is common in nsw even in patients using inhaled corticosteroids. although urgent hcu is often used as an indirect measure of poor asthma control, it is affected by different factors, perhaps because health care utilization represents a more complex balance between need and access. bronchial challenge tests with mannitol, to measure airway hyperresponsiveness, can take up to 30 minutes and require inhalation of up to 635 mg of mannitol. our aim was to determine if positive mannitol challenges can be detected after half the maximal dose (315 mg) using the forced oscillation technique (fot) to measure response. methods 15 non-asthmatic subjects and 52 asthmatic subjects underwent standard mannitol challenge, up to 635 mg mannitol. respiratory system conductance (grs) and reactance (xrs) was measured by fot at 6 hz during 40 sec tidal breathing immediately after each dose of mannitol. fev1 was measured after fot, within 90 sec of mannitol administration. two point dose response slope (drs), was calculated for grs (drsgrs) and xrs (drsxrs) for standard tests, up to 635 mg, and for short tests by excluding data from doses above 315 mg. ability to detect a positive test, defined as pd15fev1 < 635 mg, was determined by the area under the roc curve (auc) and repeatability by intra-class correlation coefficient (icc). results 32 asthmatic and 2 non-asthmatic subjects had positive tests, with pd15 fev1 values from 9.2 to 622 mg. auc (95%ci) did not differ between standard (std) and short tests for drsgrs (p = 0.14) or drsxrs ( combined use of inhaled steroids (ics) and long acting beta-agonists (laba) have an important role in asthma management. we used data from a 2006 population sample to examine medication use in adults and children. methods all adults (18-75 years) and children (2-17 years) from within four discrete zones in northern sydney were eligible for an interview survey, as part of a study investigating health effects associated with traffic-related air pollution. the prevalence of use of short-acting beta-agonists (saba), any ics (alone or combination) and combined formulations of ics/laba in the previous three months was estimated for the study population and those with diagnosed asthma. results there were 806 children [mean (sd) age 8.7 (4.6) years and 50% female] and 2184 adults [mean (sd) age 45.6 (14.9) years and 56% female] interviewed in 1843 households, representing an overall response rate of 33%. the prevalence of ever diagnosed asthma was 16.1% in children and 17.4% in adults. medication data were missing for 301 subjects. background asthma affects 1:9 adult australians and is a leading cause of rejection for recruitment into the australian defence force (adf). within this diagnosis there is a wide spectrum of disease activity and clinical outcomes. also asthma assessment and management has improved so that many asthmatics are now fully active without any significant disruption or risk to their lives. hypothesis: there is a subgroup of asthmatics who are at very low risk from significant adverse effects from asthma and who could be considered for recruitment to the adf. aims 1. to identify the subgroup of asthmatics who could be considered for recruitment to the adf. 2. to develop an assessment process to identify this subgroup (screening). 3. to develop a process to evaluate the outcomes of any change to the recruitment standard for asthma (evaluation). methods 1. a literature review of the natural history, assessment, management and response to treatment of mild episodic and mild persistent asthma. 2. a literature review of asthma in the military. 3. a clinical review of the outcomes of known asthmatics in the adf. 4. an expert group to review the above and to develop a screening process and an evaluation of the program. the literature review identified a subgroup of asthmatics, defined as mild episodic and mild persistent, who with appropriate management, have a low risk of significant adverse asthma outcomes. they can be identified by a combination of questionnaire, spirometry and bronchial provocation testing. a screening process has been developed which allows asthmatics to be recruited with a negative mannitol or hypertonic saline challenge on 400 mg/day or less of budesonide (or equivalent) without laba. a methodology to evaluate the impact of these changes on the recruitment standard has also been developed. alexithymia is a personality trait associated with difficulty identifying and communicating emotional and physical feelings. it has been associated with poor control of asthma and near fatal asthma. the primary objectives of this study were to: (1) identify alexithymia in a cohort of australian asthma patients; (2) investigate the relationship between alexithymia and asthma control; (3) investigate the relationship between alexithymia and asthma management. methods cross sectional study of 25 moderate to severe asthma patients recruited from royal adelaide hospital outpatients. participants were either mailed the questionnaire pack or completed it after a clinic appointment. existing validated questionnaires were used. statistical analyses were performed using spss. results 11 male (44%) and 14 female (56%) patients with moderate to severe persistent asthma (mean age 44 years, sd = 11) participated. alexithymia scores ranged from 23.0 to 76.0 (x = 48.3, sd = 13.2). 12% (n = 3) of participants could be classified high alexithymia, 32% (n = 8) borderline alexithymia and 56% (n = 14) were low alexithymia. alexithymia mean scores were not statistically different across sociodemographic variables. a positive correlation/association was found between alexithymia score and asthma control score (r = 0.57, p < 0.01), quality of life (r = -0.65, p < 0.01), and adherence (p = 0.03) but not satisfaction with communication (r = -0.27, p = 0.2) or number of hospitalizations (p = 0.25). conclusions this is the first australian study to identify alexithymia among asthma patients and investigate relationship to control as well as management and communication. associations between alexithymia and asthma control were confirmed. a larger sample size is needed to determine impact of alexithymia on self-management and provision of clinical care for asthma. port hedland is impacted by iron-containing dust particles (pm10) that may activate lung cells when inhaled. furthermore, the effects of port hedland pm10 may differ from the effects of urban pm10 impacting metropolitan areas. the aim of this study was to assess the effects of port hedland pm10 on production and release of the inflammatory cytokines, il-6 and il-8, by human airway epithelial (a549) cells, and to compare these with the effects urban pm10 from metropolitan areas. methods human airway epithelial (a549) cells were exposed to pm10 collected at port hedland and at urban locations (sydney, perth). a549 cells were exposed to a range of pm10 concentrations (20-200 mg/ml) for 24 h. lipopolysaccharide (lps) and phorbol myristate acetate (pma) were used as positive controls. supernatants from cell cultures were assayed for il-6 and il-8 using specific elisa kits. rna was extracted and reverse transcribed to cdna. il-6 and il-8 mrna expression was quantified by duplex real-time pcr using taqman primer/probes. results lps stimulated a 2.7-fold increase in il-8 release and pma stimulated a 3-fold increase in il-8 release and a 30-fold increase in il-6 release. however, neither port hedland pm10 nor urban pm10 stimulated concentration dependent release of il-6 or il-8 by a549 cells. expression of il-6 or il-8 mrna was also not altered by port hedland or urban dust. cd8+ t-cells may cause airway epithelial cell apoptosis via the granzyme pathway. we have reported increased apoptosis of airway epithelial cells and increased bal t-cell expression of granzyme b in copd, and a positive correlation between the two. we hypothesized that the increased granzyme b would also be related to smoking history (pack years -pk/y), age and severity of airflow obstruction (fev1 %pred) in patients with copd. we further hypothesized that the t-cell granzyme b expression would be higher in the airway than the peripheral blood. methods we investigated t-cell intracellular granzyme b expression in blood from copd subjects (33 current and 24 ex-smokers) and 12 never-smoker controls, and bronchoalveolar lavage (bal) and bronchial brushing (intraepithelial t-cells) from a cohort of these subjects using flow cytometry. correlations between granzyme b and pk/y, age or fev1 were performed using spearman's rank correlation. granzyme b in t-cells from blood, bal and bronchial brushings were compared. results there were significant correlations between fev1 and granzyme b expression in blood and bal (blood: r -0.444, p = 0.002; bal: r -0.368, p = 0.029). there was a significant correlation between pk/y and granzyme b expression in blood (r 0.362, p = 0.002), but not in bal. there were no significant correlations between granzyme b and age. there were no significant differences in granzyme b expression in blood, bal or intra-epithelial compartments. conclusion granzyme b is expressed at similar levels in blood, bal and intra-epithelial compartments, supporting recent opinion that copd is a systemic disease. t-cell granzyme b is related to severity of airflow obstruction and smoking history in patients with copd and may be one mechanism of apoptosis leading to lung injury and airflow obstruction in copd. jc allen 1 , t schlosser, ee ramsay 1 , q ge 2 , aj ammit 1 as development of remodelled airways is correlated with deterioration of lung function, we require therapies that reduce and reverse structural changes in remodelled airways. in asthma, corticosteroids can halt some, but not all, aspects of airway remodelling. therefore, in order to aid future design of efficacious anti-remodelling agents we need a better understanding of the molecular mechanism/s underlying the development of airway remodelling and the effectiveness of corticosteroids. hyperplasia of airway smooth muscle (asm) is a feature of the remodelled airway in asthmatics. in this study we examined the effect of corticosteroids on a key regulator of g1 progressioncyclin d1. asm cells from n = 8 non-asthmatics and n = 7 asthmatics were pretreated for 1 h with vehicle or dexamethasone (0.1 mm). the temporal kinetics of cyclin d1 mrna and protein expression were measured up to 24 h after stimulation with the mitogen platelet-derived growth factor-bb (pdgf-bb). pdgf-bb induced a significant increase in cyclin d1 mrna expression in asm from non-asthmatics (2.6 ϯ 0.3-fold) and asthmatics (2.9 ϯ 0.3-fold) after 24 h stimulation. in non-asthmatics, the corticosteroid dexamethasone significantly (p < 0.05) reduced the amount of cyclin d1 mrna expressed (to 1.6 ϯ 0.2-fold). in contrast, cyclin d1 expression in asthmatics was relatively resistant to inhibition by dexamethasone; the amount of pdgf-bb-induced cyclin d1 expression in the absence or presence of dexamethasone was not significantly different ( sphingosine 1-phosphate (s1p), a bioactive sphingolipid found elevated in the airways of asthmatics, modulates myriad airway smooth muscle (asm) functions that promote inflammation and remodelling in asthma. in this study, we uncover the molecular pathway/s underlying s1p-induced secretion of il-6, and investigate if, and how, corticosteroids inhibit il-6 secretion. using cultured asm cells from non-asthmatics, we found that s1p induces il-6 secretion from asm cells via cre, but not ap-1, c/ebp or nf-kb, transcriptional regulation of il-6 gene expression. cre-dependence was supported by s1p-induced creb phosphorylation. although the corticosteroid dexamethasone reduced s1p-induced il-6 secretion in a dose-dependant manner, this inhibition appeared to occur via a pathway independent of creb/cre, suggesting the existence of a parallel pathway. as we recently discovered that the antiinflammatory actions of corticosteroids in asm can be mediated via the induction of the endogenous mitogen-activated protein kinase (mapk) inhibitor, mapk phosphatase-1 (mkp-1), we investigated whether mapk represents the parallel pathway targeted by corticosteroids. we found that s1p can induce activation of a variety of mapk, however, only p38 mapk phosphorylation was inhibited by dexamethasone; importantly, the increase in mkp-1 after corticosteroid treatment appeared to mirror the decrease in s1p-induced p38 mapk phosphorylation. furthermore, exogenous expression of mkp-1 inhibited s1pinduced il-6 secretion. taken together, these results suggest that parallel pathways exist to induce il-6 secretion (transcriptional via creb/cre and possibly post-transcriptional via p38 mapk) and serve to underscore the importance of mkp-1 upregulation as a mechanism of action of corticocosteroids in asm. angiogenesis is a hallmark feature of asthma. angiogenic promoters, such as vegf and tgfb are reported to be increased in airways of asthmatics. tumstatin, an endogenous angiogenic inhibitor, is the non-collagenous domain-1 (nc1) of the alpha3 chain of collagen iv. decreased levels of collagen iv have been reported in the airways of asthmatics. we investigated the presence of tumstatin in the airway of asthmatics and its potential role as an angiogenic inhibitor. we detected the six a chain nc1domains of col iv and the 7s domain of the a3 chain using immunohistochemistry. the level of tumstatin in serum and bal-f was measured by dot blot. western blots were used to identify the association with the rest of the collagen iv molecule. a tube formation assay using primary pulmonary endothelial cells (ppec) was performed to evaluate the role of tumstatin in the airway. the effect of intranasal tumstatin on airway hyperresponsiveness and angiogenesis was studied in an ovalbumin mouse model. tumstatin was absent in the airways of asthmatics (n = 14) while the remaining six collagen iv a chains were present. the 7s domain of the a3 chain was present in the asthmatic airway (n = 6). tumstatin was detected in both serum and bal-f samples from asthmatic volunteers (n = 10), however the level of expression was not significantly different from that in nonasthmatics (n = 7). in asthmatic serum tumstatin was part of the whole collagen iv a3 chain. tumstatin was able to inhibit ppec tube formation in a dose related manner. tumstatin inhibited angiogenesis in the mice airways and was associated with an improvement in ahr. the fact that tumstatin is absent from asthmatic airways and inhibited airway hyperresponsiveness and angiogenesis may indicate potential for therapeutic intervention in airway remodelling. this work was supported by the crc for asthma and airways and nh&mrc. introduction epithelial egfr (epidermal growth factor receptor) expression correlates with disease severity and neutrophil infiltration in asthmatic airways. acute exacerbations of asthma and copd are also associated with steroid refractory neutrophilic inflammation, with rhinoviruses being the most common trigger. .7 mg/l and il-6: 5.8 vs. 3.6 ng/l). since il-6 stimulates the acute phase response, we correlated its levels with the other markers. only crp was strongly correlated with il-6 (spearman r = 0.58, p < 0.0001), suggesting differential regulation of saa and ip10. saa discriminated between non-pathogen (n = 10) vs. pathogen-associated (n = 41) events (saa: 9.4 vs. 44.1 mg/l p = 0.005), whereas no significant change was observed in the other markers (ip-10: 139.8 vs. 170.5 ng/l, crp: 4 vs. 10 mg/l, il-6: 4.6 vs. 7.2 ng/ l). however when aecopd marker levels were stratified on the basis of pathogen type (viral = 12, bacterial = 21, viral and bacterial = 8), none of the markers were significantly altered. conclusions ip-10 is significantly elevated during an aecopd, however only saa differentiated non-pathogen from pathogen associated events. background severe persistent asthma is characterized by structural changes in the airways-airway remodelling. airway smooth muscle (asm) cells have the potential to play a key role in these processes through the release of growth factors, cytokines and extracellular matrix (ecm) proteins. we have previously studied the effects of budesonide and formoterol individually however, the effect of their combination on these characteristics of asm cells is not known. methods asm cells from asthmatic (n = 6) and nonasthmatic (n = 6) individuals were stimulated with transforming growth factor ß (tgfß) (1 ng/ml) with or without budesonide (10 -8 m) and formoterol (10 -10 and 10 -8 m) and fibronectin levels and interleukin-6 (il-6) release were measured by elisa. bronchial rings from nonasthmatic individuals (n = 2) were incubated with tgfß with or without the drugs and ecm protein expression (fibronectin and collagen i) measured using immunohistochemistry. results in nonasthmatic cells, budesonide alone induced fibronectin deposition whether tgfß was present or not. formoterol decreased fibronectin induced by tgfß and, when combined with budesonide, reversed the increase in fibronectin. a similar pattern was observed in asthmatic cells, except that budesonide did not further increase the tgfß mediated fibronectin release. as before [1] , il-6 was induced by formoterol but inhibited by budesonide. tgfßinduced il-6 was inhibited by both drugs and their combination in both cell types. in bronchial rings the presence of either drug did not affect tgfßinduced fibronectin or collagen i. severe combined immune deficiency (scid) spontaneous mutation specifically impairs differentiation of stem cells into mature lymphocytes. nod-cb17prkd scid (known as nod-scid) lacked nk cells, hence is commonly used in cell transfer experiments for transferring tissue and haematological xenografts. the aim of this study was to establish lung inflamamtory model in nod-scid strain. methods balb/c and nod-scid balb/c mice (n = 8) were exposed to cigarette smoke for 4 days, 2 and 4 weeks (9 cigarettes/day; 5 days/week). bronchoalveolar lavage fluid (balf) and lung tissue were collected for inflammatory profiling and analysis for cytokines, chemokines and protease expression and/or activity. results nod-scid have significant accumulation of macrophages in lung after 4 days, 2 and 4 weeks smoking as compared to no smoke control (p < 0.001) that was not different to balb/c (p > 0.05). nod-scid also have increased neutrophil number after 2 and 4 weeks smoking (p < 0.001). even though myeloid cell differentiation isn't affected by scid phenotype, nod-scid have one fold less neutrophil than balb/c mice (p < 0.001) that is also reflected in the reduced expression of matrix metalloproteinase-9. consistent with the known lymphopenic phenotype, nod-scid have significant but less lymphocytes recruitment as compared to balb/c mice after 4 weeks smoking (p < 0.001) despite the enhanced expression of inteferon inducible protein 10 (lymphocytes specific chemokine) in lung. both mouse strains showed the same elevation of net gelatinase and serine protease activity in lung. nodscid mice also demonstrated comparable transcriptional induction of proinflammatory cytokines (tnfa, il-6), growth factors (gm-csf, g-csf) and chemokines (mcp-1, mip-2), indicating susceptibility to smoke-induced injury. conclusions nod-scid mice are capable to mount smoke induced inflammatory response. this model may be useful to study localization and role of immunocytes, including adoptively transfer human cells in the pathogenesis of copd. supported by the nhmrc. rhinovirus (rv) is the cause of most common colds and up to 80% of asthma attacks. in our previous studies, plasminogen activator inhibitor 2 (pai-2) was expressed at high levels and was induced in vivo and in vitro by rv infection. pai-2 may have antiviral properties suggested by antiviral activity in some models, high pai-2 expression levels and further upregulation by rv infection. methods to determine whether pai-2 has antiviral activities following rv infection, o-hela, pai-2 expression-deficient cells were first transfected with pai-2 or control genes. this was followed by infection with rv and effects on viral replication were assessed by rt-qpcr for vrna and by viral titration for virus release. ifn expression was assessed by rt-qpcr. results ifn-a and -b mrna expression were induced in response to rv infection and to pai-2 expression in cells. pai-2 expression followed by rv infection elicited a synergistic response and pai-2 over-expression reduced vrna by >5 fold and viral titre by >3 log (p < 0.05). however, this effect was not specific to pai-2, as transfection of cells with control genes/plasmids reduced viral titre to a comparableextent. one of the pathological findings in idiopathic pulmonary fibrosis (ipf) is the presence on fibroblastic foci comprising cells which exhibit mesenchymal phenotypic features such as myofibroblast-like morphology, increased asma expression and collagen deposition. currently steroid treatment in ipf has shown limited efficacy. the cellular origins of these mesenchymal cells remain unclear, but evidence from other studies suggests that epithelial cells may undergo a transition to a mesenchymal cell phenotype (emt). transforming growth factor ß has been implicated in promoting this emt. in this study we have induced a morphological change in a549 cells using tgf-ß1 and assessed the influence of glucocorticoids, and the changes to the extracellular environment of the cells, on emt. methods a549 cells were grown on uncoated plastic cultures plates or those coated with monomeric or fibrillar collagen and treated with 200-500 pm tgf-ß1. the influence of the glucocorticoid, dexamethasone (dex, 1-1000 nm), or collagen type, on emt was assessed by microscopy, rt-pcr and western blotting for markers of myofibroblast phenotype. results tgf-ß1 induced an increase in mrna expression of asma (1.5 fold), collagen (7.0 fold) and fibronectin (2.0 fold). dex (100 nm) partially inhibited the expression of collagen, but had no effect on asma levels. however, dex (100 nm) reduced asma and ctgf protein levels. dex (100 nm) also prevented the tgf-ß1-induced morphological changes, regardless of ecm matrix. conclusion glucocorticoids appear to control some of the emt phenotype changes induced by tgf-ß1. however, the inability to fully inhibit these changes may contribute to the resistance of ipf to glucocorticoids. the extracellular environment may also play a role in the development of fibroblastic foci and their pharmacological responses. defective alveolar macrophage (am) phagocytic function in the airway may perpetuate inflammation via secondary necrosis of uncleared apoptotic cells in copd. we have previously reported that low-dose azithromycin improved macrophage function in vitro, although the mechanisms for this effect were not identified. we explored the possible role of the collectin pathway in the azithromycin-mediated improvement in phagocytosis as well as possible defects in this pathway in copd subjects. methods (1) mannose binding lectin (mbl), mannose receptor (mr), surfactant protein d (sp-d) were measured in copd subjects and controls. (2) the in vitro effects of addition of rhmbl, and blocking mr with a specific antibody, on am phagocytic ability were assessed. in vitro effects of azithromycin on am expression of mr were also investigated. (3) azithromycin (250 mg orally 2¥ weekly/12 weeks) was administered to 11 copd subjects. bronchoscopies were performed prior to and 12 weeks following therapy. ex vivo assessments included am phagocytic ability, levels of mbl, sp-d and mr and apoptosis of bronchial epithelial cells. results am mr expression and levels of mbl and sp-d were significantly reduced in copd subjects vs controls. azithomycin (500 ng/ml) increased mr expression by 31% in vitro. rhmbl induced a dose-dependent increase in am phagocytic ability (up to 148%). blocking mr significantly decreased am phagocytic ability by 60%. in copd patients following azithromycin therapy, we observed improved am phagcocytic ability, increased levels of mr and reduced levels of bronchial epithelial cell apoptosis. conclusions these findings strongly implicate the mr in both the defective phagocytic function of am in copd and as a target for the azithromycinmediated improvement in phagocytic ability. obstructive sleep apnea (osa) is associated with hypoxia and increased cardiovascular morbidity. t cells and monocytes play a significant role in atherogenesis via cytokine production. there have been reports of benefits of continuous positive airway pressure (cpap) therapy in osa. the purpose of this study was to characterize leucocyte inflammatory cytokine/chemokine production by t cells and monocytes in a group of osa patients and to investigate the therapeutic effects of cpap therapy. methods a comprehensive range of intracellular t-cell and monocyte proand anti-inflammatory cytokines/chemokines was investigated in peripheral blood from 5 osa patients and 5 aged-matched control subjects (with no evidence of sleep problems) using multiparameter flow cytometry. osa patients were again studied following 7 days of cpap therapy. results in osa patients there was an increase in intracellular t-cell ifng and tnfa production but no change in il-2, il-4 or tgfb compared with control. there was an increase in intracellular monocyte il-1a, il-8, tnfa, mcp-1 and mcp-3 in osa patients but no change in il-10 or il-12. following cpap therapy, t-cell ifng and tnfa production returned to 'normal' levels. however, although intracellular monocyte cytokine/chemokine production was decreased following cpap, levels were significantly elevated compared with control. conclusions osa is associated with increased intracellular proinflammatory cytokine/chemokines, many of which are increased in atherosclerotic plaques. although one week of cpap therapy resulted in amelioration of t-cell pro-inflammatory cytokines, longer cpap use or alternative therapy may be required to reduce monocyte pro-inflammatory mediators associated with atherosclerosis in patients with osa. gp130 has been associated with the progression of fibrosis especially in patients with idiopathic pulmonary fibrosis (ipf). gp130 is the common subunit of the receptor complexes for the il-6 family of cytokines including il-11 and oncostatin m (osm), where gp130-mediated signalling leads to activation of the erk or stat pathways. we have previously demonstrated exaggerated gp130-stat signalling to be fundamental to the development of pulmonary fibrosis in a murine model of bleomycin-induced lung fibrosis. the aim of this study was to elucidate the role of the il-6 cytokine family in the development of pulmonary fibrosis by identifying which il-6 family cytokines regulate fibrosis in bleomycin treated mice, and determine the effects of these cytokines on cell function. bleomycin (0.05 u/mouse) or control saline was administered intranasally to wildtype mice (wt), genetically engineered mice containing point mutations to prevent gp130 erk signalling (gp130 757f ) or gp130 stat signalling (gp130 dstat ), and duel il-6 and il-11 a-receptor knockout mice (il-6 -/-;il-11ar -/-). the effect of bleomycin on collagen production was examined in lung tissue 30 days post treatment by hplc. there was a significant increase in collagen levels in bleomycin treated wt lungs which was further increased in gp130 757f lungs. the lungs of gp130 dstat and il-6 -/-;il-11ar -/mice were protected from fibrosis suggesting that gp130-stat signalling is important in inducing lung fibrosis which may be mediated through il-6 and/or il-11. cell proliferation was examined in lung fibroblasts isolated from wt, gp130 dstat and gp130 757f mice. il-6, il-11 and osm were significantly mitogenic for gp130 dstat cells but not for wt or gp130 757f cells, reflecting different responses to the different signalling pathways. changes in cytokine profiles are currently being examined in lung tissue and serum of control and bleomycin treated mice 0-30 days after treatment. in conclusion, il-6 and il-11 are likely to play a role in bleomycin-induced fibrosis via the gp130-stat-mediated pathway, however this may not be due to regulation of proliferation induced by these cytokines. supported by the nhmrc. mimicking viral infection by application of various toll-like receptor ligands has shown clinical promise in the treatment of persistent viral infections and more recently with malignant tumours. commercially available toll-like receptor 7 ligands (tlr7l), such as those of the imidazoquinoline family have been applied clinically for the treatment of a number of conditions including basal cell carcinoma and hpv-induced genital warts. these compounds are known to retard tumour growth indirectly by promoting activation and migration of dcs, leading to a strong th1 cellular response, and directly via release of proinflammatory cytokines and promotion of tumour cell apoptosis. malignant mesothelioma (mm), an aggressive tumour with a mean survival of 9 months, is highly resistant to chemotherapy, radiotherapy and surgery and is therefore an interesting candidate for immunotherapy in the form of tlr7 ligand treatment. whilst tlr7 is known to be selectively expressed in immune cells and its relative expression low amongst other cell and tissue types in mammals, its expression on tumour cells and the consequences of such expression on tumour growth are unknown. here we describe the presence of tlr7 (mrna and protein) directly in a range of different tumours, including several murine and human mm cell lines. reactive oxygen species (ros) produced during the innate immune response are important agents of anti-pathogen defense but may also cause oxidative lung damage. glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. methods wild-type (wt) or mice deficient in glutathione peroxidase-1 (gpx-1 -/-) were placed in a perspex chamber and exposed to cigarette (cig) smoke generated from 9 cigs per day for 4 days. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for proteomic and genomic analysis. results wt mice exposed to cig smoke for 4 days had significantly more macrophages (3.1 ϯ 0.1(sem) ¥ 10 5 ) and neutrophils (4.9 ϯ 0.4 ¥ 10 5 ) than sham-exposed mice (2.2 ϯ 0.2 ¥ 10 5 and 0, respectively) (n = 6, p < 0.05). however, gpx-1mice exposed to cig smoke had significantly greater macrophages (5.4 ϯ 0.3 ¥ 10 5 ) and neutrophils (1.2 ϯ 0.1 ¥ 10 6 ) than smokeexposed wt mice (n = 6, p < 0.001). macrophage and neutrophil numbers in sham-exposed gpx-1 -/mice (1.7 ϯ 0.3 ¥ 10 5 and 0.5 ϯ 0.4 ¥ 10 3 ) were similar to those of sham-exposed wt mice (2.2 ϯ 0.2 ¥ 10 5 and 0). in addition, we found that balf of gpx1 -/mice exposed to cig smoke had an increased proteolytic burden compared with smoke-exposed wt mice as assessed by zymography and net gelatinase activity assay. conclusions these data suggest that gpx-1 protects the lung from cigarette smoke-induced inflammation and that targeting gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role. funded by nhmrc. the becs from subjects with chronic obstructive pulmonary disease (copd) are exposed to frequent infectious and inflammatory stimuli. infection with rv is known to trigger acute exacerbations and subjects with copd are particularly susceptible. we hypothesized that exposure of copd becs to these stimuli would alter their response to rv infection. methods bec were obtained by endobronchial brushing from subjects with gold stage 3 copd (n = 4, all ex-smokers), subjects with mild persistent asthma (n = 4) and healthy controls (hc, n = 4). becs were cultured and then treated with tumour necrosis factor (tnf)a 10 ng/ml or lps 100 mg/ml for 24 hrs and then infected with rv-43, rv-1b. response was measured by release of il-8, il-6 and ip-10 mrna and by elisa. virus replication measured by cell titration assay. results infection with both rv strains led to increased release of il-8 and ip-10 in all groups. exposure of hc and asthma becs to both lps and tnf led to increased release of il-8. in these becs there was no increase in release of il-8 exposed to lps and tnf and then infected with either rv. becs from subjects with copd released significantly less il-8 in response to all conditions and rv infection compared to hcs and asthma. no differences were seen in rv replication. the aim of this study was to determine opinions and attitudes to exercise from chronic obstructive pulmonary disease (copd) subjects after completion of a 12-month maintenance exercise program. methods following completion of a 12-month exercise study, which included a supervised program (intervention, n = 18) and control group (control, n = 17), copd subjects [mean age (sd): 66 (8); mean fev1 (% predicted) = 56% (19)] were asked to complete a questionnaire. the questionnaire included closedended questions using visual analogue scales (100 mm). in copd the 6 minute walk distance (6mwd) is known to increase with test repetition (familiarization) and in response to exercise training. it is unknown whether the magnitudes of these increases are related to the degree of disability of the individual patient. methods 6mwd was measured twice before and once after an 8 week out-patient exercise program in 121 patients (82 males) aged 67ϯ8.6 yrs, fev1 37ϯ15% predicted (meanϯsd) with stable copd. the changes in 6mwd following a familiarization test and following training were compared between patients grouped according to their degree of disability (defined as the pre-training 6mwd [best of 2 tests] expressed as %predicted 6mwd). *p < 0.05 gp 3 vs gp 1. conclusions before training, 6mwd increases following a familiarization test irrespective of the level of disability. the magnitude of this increase is similar in all groups when normalized for their pre-training 6mwd. following training, the increase in 6mwd is greatest in patients with the greatest disability (lowest pre-training 6mwd). in less disabled patients, the relatively smaller increase in 6mwd following training may reflect an inability to further increase stride length, thereby reducing the responsiveness of the 6mwt in this group. supported by nhmrc. endotoxin is a stimulant of the innate immune system and is a major component of cigarette smoke. smokers have evidence of increased airway neutrophils and inflammation. we hypothesized that endotoxin levels would be higher in the bronchial lavage (bl) of subjects who were former smokers and subjects with chronic obstructive pulmonary disease (copd). methods subjects were all ex-smokers for at least 5 years (n = 10, 5 copd, 5 healthy controls) or never smokers (n = 12, 6 asthma, 6 healthy controls). bl was collected and analysed for cell count and differential, culture for microbiology. the supernatant was analysed for il-8 by elisa and endotoxin by quantitative kinetic lal assay. results median endotoxin levels were significantly higher in ex-smokers 101 compared to never smokers 6.3 u/ml (p < 0.001). there were no differences between subjects with copd and hs. subjects with copd had higher median endotoxin levels (80 u/ml), compared to asthma (5.2 u/ml) and hc (6.3 u/ml, p = 0.03). there was no correlation between endotoxin levels and bl total cell count, neutrophils (%) or fev1 % predicted. there was a strong correlation with previous packet years smoked and endotoxin levels (r = 0.72, p < 0.01). conclusions bl endotoxin levels are higher in ex-smokers, including subjects with copd. despite this there is no relationship to increased neutrophilic inflammation. copd is associated with inflammation associated with ineffective repair of the injured epithelium and loss of structural integrity. we have shown that these changes may result from dysregulated 'efferocytosis' (increased apoptosis of bronchial epithelial cells and defective clearance of these cells by alveolar macrophages (am)). we have also reported that azithromycin, at subbactericidal dose, improved am phagocytic function ex vivo. methods we administered azithromycin at low dose (250 mg/ twice weekly for 12 weeks) to 10 copd subjects (7 male, age: 62 ϯ 8 yr, 5 current/ 5 ex-smokers, fev1: 63 ϯ 9% pred, fev1/fvc: 48 ϯ 9%). the study was openlabel, uncontrolled and primarily focused on objective biological responses obtained from the bronchoscopy samples taken. phagocytic ability of am (from bal), apoptosis of bronchial epithelial cells (from bronchial brushing), markers of inflammation in blood, bal and breath condensate (crp, wcc and inflammatory cytokines), health status (st. george's respiratory questionnaire), ecg and lung function were assessed pre and post-administration of azithromycin. results azithromycin significantly improved phagocytic ability of am (by 37%) and reduced bronchial epithelial cell apoptosis (by 34%). antiinflammatory effects of azithromycin included significantly reduced blood wcc and crp. there were non-significant reductions in levels of pro-inflammatory cytokines il-8, il-6 and tnf-a in blood, bal and breath condensate, and a trend for improved health status. conclusions our findings indicate a novel approach to supplement existing therapies in copd that may improve clearance of accumulated apoptotic material and reduce the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation. background the prevalence of gastro-oesophageal reflux disease (gord) across the disease spectrum in copd and bronchiectasis is not well described. the aim of this study was to determine the prevalence of symptomatic and silent gord in copd and bronchiectasis and its effect on lung function and quality of life (qol 4] ) and 18 healthy controls were recruited. the prevalence of gord in bronchiectasis was 33%; 37% in copd; 17% in controls. in copd and bronchiectasis, total nre and ri were increased in those with distal and proximal gord compared to those without gord (all p < 0.05). there was no difference in extent or severity of bronchiectasis in patients with or without gord (all p > 0.05). in copd, the relationship between proximal gord and fev1 was small to moderate (r = 0.383). sgrq symptom scores were higher in patients with bronchiectasis with increased ri (p = 0.02). increased proximal nre was associated with reduced physical (p = 0.03) and mental health (p = 0.02) in the sf-36 in copd. conclusions gord is a co-morbidity in patients with copd and bronchiectasis. the impact of gord on disease severity requires further evaluation. funding source nhmrc, the university of melbourne, monash university, physiotherapy research foundation. chronic obstructive pulmonary disease (copd) is prevalent among older people, however little is known about the influence of ageing on airway inflammation. the aim of this study was to compare airway inflammation in older people with obstructive airway disease to groups of older and younger healthy controls. methods participants (>55 years of age) with stable airway disease and incomplete reversibility (fev1% predicted <80% and fev1/fvc < 70%; copd n = 71) and healthy controls (n = 45, 35 older >55 years and 10 younger <55 years) were recruited from the respiratory ambulatory care clinic or by advertisement. participants underwent a clinical assessment, skin allergy test, hypertonic saline challenge, sputum induction and gas diffusion studies. results participants with copd had moderate airflow obstruction (mean (sd) fev1% predicted 56 (19)) and 45 (63%) were current or ex-smokers with a median (iqr) pack year history of 36 (20-54) pack years. ageing was associated with an increase in airway neutrophils (p = 0.0001). compared to older controls, participants with copd had increased airway eosinophils and lymphopenia (p = 0.004, p = 0.003 respectively), but no difference in airway neutrophils. conclusion airway neutrophilia is a feature of ageing and is not further increased in the presence of copd. copd is associated increased numbers of airway eosinophils with reduced lymphocytes which may impact on the ability of the immune system to combat infection. supported by nhmrc, the university of newcastle. chronic obstructive pulmonary disease (copd) is third leading cause of death and fourth leading cause of disease burden in australia. mechanisms involved in emphysema severity have not been fully understood. micrornas are noncoding rnas that regulate gene expression. we hypothesize that microrna expression differs between emphysema severity in copd patients. methods mirna profiling was performed using 15k agilent human oligo mirna microarrays on total rna extracted from non-tumour lung tissue from 30 copd patients undergoing resection for lung cancer. the mirnas were quantile normalized and anova was used to find differentially expressed genes. results demographic characteristics of the copd patients (mean (sd)) were age 69 (6) years, fev1 72 (17) % predicted and fev1/fvc ratio (<70%). anova identified 31 mirnas that were differentially expressed when stratified into two classes according to kco % predicted > or <75% (t-test, p < 0.05). discussion this mirna analysis has identified mirnas that may be important in emphysema severity in copd patients. further validation will be performed using qrt-pcr and mirna assays on the training set and an independent set, and target prediction and validation. t-helper type 1 (th1) and type 2 (th2) lymphocyte responses have been well recognized as being important pathways in inflammation. recently another form of inflammatory lymphocyte response has been described, the th17 pathway. th17 cells produce cytokines such as il-17a to clear extra-cellular bacteria and fungi and have been implicated in autoimmune and chronic inflammatory diseases. the th17 response in copd is unknown. methods subjects were patients with copd (ex-smokers, fev1 < 70% predicted who had not had an exacerbation for at least 1 month) and control subjects (ex-smokers and normal spirometry). serum samples were obtained for measurement of c reactive protein (crp) and il-17a, the latter measured using enzyme-linked immunosorbent assay (elisa). production of il-17a by t-cell subsets was also identified by intra-cellular cytokine staining and measured by flow cytometry. the mean fev1 of copd subjects was 42 % predicted (6.1 sem, n = 6) and mean fev1 of controls was 112 % predicted (3.0 sem, n = 4). the copd group had a higher mean level of crp 9.5 mg/l (3.9 sem) compared to the control group mean level of 4.6 mg/l (0.6 sem). the mean level of the il-17 in the copd group as measured by elisa was 22.3 pg/ml (16.9 sem, range 0-87) whilst no il-17 was measured in any of the control subjects. conclusions the findings of this pilot study suggest that il-17 may be elevated in association with crp in stable copd. airway obstruction is defined as a fev1/fvc ratio below the lower limit of normal. airway obstruction may prolong the forced expiratory time (fet). method spirometry results from 467 patients were categorized as obstructive, restrictive or normal. the mean, range and coefficient of variation were determined for fet in each diagnostic group. receiver operator characteristic (roc) curves were used to determine if fet could predict a low fev1/fvc. the number of patients with airway obstruction in five fet groups: <9; 9; 10-12; 13-14; and >14 seconds were determined. results the coefficient of variation was high for all groups. pair-wise comparisons showed a difference in mean fet between patients with normal lung function versus those with airway obstruction (p < 0.001). the best cut-point in the roc analysis of 9.895 seconds had a sensitivity of 0.66, specificity 0.77 and area under the curve of 0.743 for predicting obstruction. the technique of skeletal muscle microbiopsy has previously been validated [1] and shown to be minimally invasive and well tolerated in participants with stable copd. aim a study was undertaken to determine the feasibility and tolerability of obtaining microbiopsy muscle samples from the patient admitted for acute exacerbation of copd patient. methods written informed consent was obtained to collect the muscle, blood and sputum samples for research purposes. local anaesthetic was injected prior to the insertion of a 16 gauge bard max core disposable biopsy instrument through the associated guide needle. multiple passes (up to 6) were obtained. the patient was asked to evaluate the experience by rating it on the modified borg scale 0-10. results to date 5 patients and 3 controls have participated in this study. the gold severity ranged from 2-4 and ats exacerbation severity 2-3. the mean age 75 years (range 68-83 years), bmi mean 23.6 kg m -2 (range 17.2-27.1 kg m -2 ) and fat free mass was determined using single frequency bioimpedance. the sample mass obtained ranged from 27.2-104.1 mg, with an increasing yield occurring with increased experience of the operator. the procedure has been well tolerated, the borg scale rating ranged from 1-2/10. all patients were ambulant post procedure; no haematoma or bruising was observed in any of the subjects. conclusion the microbiopsy technique allows the collection of muscle tissue with minimal discomfort to the participant. small tissue masses such as these are sufficient to obtain measures of local markers of wasting and may prove to be a useful adjunct to the collection of sputum and blood for the measure of biomarkers in copd research. introduction older people (op) with obstructive airways disease (oad) experience multiple problems that may impact on their quality of life (qol) and disease management. these problems may relate to pathophysiology, symptoms, self management skills, psychological issues, lifestyle or other problems identified as important by the patient. aim the aim of this study was to determine the frequency of clinical problems associated with oad and to determine if a problem based assessment (pba) could adequately identify these problems. methods a multidimensional assessment tool was developed and the content compared to clinical practice guidelines. participants over 55 years with diagnosed oad underwent this assessment. results sixty-one consecutive patients, aged 59-87 years, with mean (sd) fev1 of 51.4 (17.85) % predicted were assessed. the assessment tool identified a mean (sd) of 3.03 (2.13) current and significant co morbidities with an additional 11 (3.37) clinical problems per patient. qol was increasingly impaired with an increasing number of problems (p < 0.0001). regression modelling identified that the number of identified clinical problems accounted for 55% of the qol impairment. the model demonstrated that every additional patient problem was associated with a clinically significant change in qol impairment (4.22 units) . conclusions op with oad experience multiple clinical problems and co morbidities that adversely impact their qol. a pba of op with oad identifies significant problems that may not be addressed in a diagnosis centred approach. there is a need to identify and effectively manage this array of problems in clinical practice. discussion in this diverse group of copd patients, there was a positive correlation between dlco and fev1, but not kco and fev1. the fev1/ kco plot identifies substantial numbers of patients with the potential ad and e phenotypes defined above. we intend to study inflammatory biomarkers in these groups. fat free mass index (ffmi) is a marker of morbidity and mortality in copd. measurement of ffm in the out-patient population is commonly undertaken using single frequency bioelectrical impedance analysis (bia). however the formulae to convert measured values to ffm are population dependent. schols et al (am j clin nutr, 1991) suggested that formula used for the general population may be inappropriate for patients with copd, and derived a specific formula from total body water (tbw) as measured by deuterium dilution. we compare this method of measuring ffm with 5 others, along with tbw and ffm hydration. methods tbw was measured in 31 outpatients with copd by bia and a difference method (weight-(protein+bone mineral+fat+non-bone mineral+ glycogen)) and ffm hydration was calculated. ffmi was measured by skin fold anthropometry (sfa), bia (3 separate formulae), dual energy x-ray absorptiometry (dexa) and total body potassium by g-counter (tbk). comparison between methods for tbw and ffmi was made by bland-altman analysis and between methods of calculation of ffm hydration by paired t-test. the two methods of assessment of tbw showed little difference (bias -0.04, 95% limits of agreement -5.40 to 5.31). however there was a significant difference in calculation of hydration of ffm (p = 0.0001). sfa, bia (lukaski), bia (tanita) and tbk underestimated ffmi when compared to bia (schols), with bias of -1.24, -3.87, -1.06 and -2.76 respectively. dexa however had a bias of only 0.05 and 95% loa of -3.09 to 3.21. conclusions there are differences between methods of assessment of tbw and ffmi and comparing values between methods must be done with caution. this has implications for assessment of morbidity and mortality in copd. chronic obstructive pulmonary disease (copd) has been identified as a major health problem in australia. recent studies have suggested that respiratory viral infections are the major cause of a worsening of copd; however this has not been studied in australia. aim to characterize pef changes and identify viruses during copd exacerbations. methods a pilot prospective longitudinal cohort study was done. patients had confirmed copd with fev1 <70% predicted and reversibility <10% and/or 200 ml. patients recorded daily peak expiratory flow (pef) measurements and daily chest and cold scores over a period of 2 years. sputum samples and nasal aspirates were taken at 6-month review (control visit) and whenever they had symptoms of an exacerbation (worsening of copd symptoms -seemungal et. al. am j resp crit care med, 2001). nasal aspirates and sputum samples were obtained and analysed by rt-pcr for rhinovirus (rv). result five patients have finished 2 years of study. a total of 12 exacerbations were reported based on patient symptoms. only 3 exacerbations were associated with significant reductions in pef and only one was linked to increases in nasal cold scores. all samples taken at control visits and nasal aspirates and sputum samples during exacerbations were negative for rv by rt-pcr. positive controls confirmed the accuracy of the assay. conclusion our data suggest that a symptom-based definition of copd exacerbation is not always accompanied by significant reductions in lung function parameters. these 'exacerbations' are also not associated with the commonest reported viral cause. our findings suggest that variability of copd may mimic. bronchiectasis is characterized by hypersecretion of mucus and impaired clearance that results in mucus accumulation, chronic cough, sputum production and recurrent infections. inhaled mannitol (400 mg) improves clearance of mucus by increasing the airway hydration and by reducing the viscoelastic and surface properties of mucus. however, the effect of other doses of mannitol on the clearance of mucus in patients with bronchiectasis is unknown. methods fourteen patients, age: 63.3 ϯ 5.7 yr, were studied on 5 visits. clearance of mucus was measured using 99m tc-sulphur colloid and imaging with a gamma camera at baseline and with mannitol ( weight loss and skeletal muscle atrophy are major determinants of morbidity in chronic obstructive pulmonary disease (copd), which are independent of lung function impairment. thus, we examined if a high-fat diet (hfd) protected against the wasting associated with prolonged cigarette smoke exposure (se) in mice. methods male balb/c mice were exposed to the smoke of 4 cigarettes/day, 6 days/week for 7 weeks. sham mice were handled identically without smoke exposure. mice consumed either standard laboratory chow (3.5 kcal/g, consisting of 12 % fat) or a hfd (4.3 kcal/g, 32% consisting of fat). we examined the effect of se and hfd on hind limb skeletal muscles, lung (tissue & bronchoalveolar lavage (balf)) and systemic inflammation in the 4 groups of mice (n = 8/ group). results after 7 weeks of hfd, sham and se mice were 12 and 13% heavier (respectively, p < 0.05) than chow fed animals. conversely, se significantly decreased body weight of chow and hfd fed mice by 16 and 15%, respectively, compared to sham animals (p < 0.05). the hfd did not protect against the decrease in soleus, tibialis anterior and gastrocnemius skeletal muscle weights induced by se (p < 0.05). se altered the mrna expression of a number of genes associated with the regulation of skeletal muscle mass including insulin-like growth factor-i (igf-i), atrogin-1 and interleukin (il)-6. the mrna expression of pro-inflammatory cytokines and chemokines was significantly increased by se in the lung, as were the number of inflammatory cells in balf (p < 0.05). on the other hand, although obesity has been linked to systemic inflammation, the hfd exerted little direct effect on the skeletal muscle and lung parameters measured. se and hfd had no effect on two markers of systemic inflammation, il-6 and serum amyloid a, whereas se tended to reduce circulating igf-i, an anabolic hormone. conclusions the hfd was not protective against the weight loss and skeletal muscle wasting associated with cigarette smoke exposure. supported by the nhmrc and crc for chronic inflammatory diseases. background patients with copd and bronchiectasis undertake airway clearance therapy (act) and exercise as part of physiotherapy management but it is unknown whether these treatments provoke gastro-oesophageal reflux (gor). this study aimed to determine the impact of positive expiratory pressure (pep) therapy and exercise on gastro-oesophageal function. p. aeruginosa is a significant opportunistic lung pathogen in individuals with cystic fibrosis (cf) and is associated with increased lung disease and morbidity. early intervention is beneficial for the effective clearance of p. aeruginosa and better long-term health outcomes. currently, lung flora of cf patients is monitored by regular culturing of sputum, however, children unable to expectorate are limited to annual bronchoalveolar lavages (bal), which is invasive and requires general anaesthesia. saliva is useful for clinical assays as collection is simple, non-invasive. we are developing a standardized enzymelinked immunosorbent assay (elisa) to detect respiratory infection of p. aeruginosa in cf children who cannot expectorate. methods 18 children (7-18 years) with cf and recent p. aeruginosa lung infection history and 16 non cf children (1-6 years) with no previous p. aeruginosa infection history provided saliva as positive, negative controls respectively. saliva was obtained by spitting, or absorbed using cellulose swabs and later extracted. these cell-free supernatant samples were used in an elisa anti-p. aeruginosa iga using commercial antigen. all results were standardized to account for flow using total iga expression. results median value was increased 9 fold in the recent p. aeruginosa lung infection group (mann-whitney test, n = 34, p յ 0.001). there was no significance between mucoid and non mucoid samples, and detection was independent of cfu/ml. discussion early findings support that p. aeruginosa respiratory infection can be detected through specific analysis of salivary iga expression. larger population sampling (30 positive, 90 negative) will aid selection of cut-off values for specificity and sensitivity testing in the future to objectively determine the utility of this assay as a means of monitoring for p. aeruginosa and for determining effectiveness of treatment. medical thoracoscopy is utilized widely throughout europe and northern america by thoracic physicians for the management of pleural disease, including the undiagnosed pleural effusion, malignant effusions and less commonly pneumothorax (ptx). australia has limited experience in this modality. we report the success of medical thoracoscopy in both primary and secondary ptx requiring intervention. methods data were collected from 2001 to 2007 in patients treated with medical thoracoscopy for the treatment of ptx. results 11 patients, 7 male, 4 female. average age 48 (range 19-86). 1 first episode primary spontaneous (ps) ptx, 2 third episodes of ps, 5 first secondary spontaneous (ss), 1 second ssptx, 2 third ssptx. underlying pulmonary disease in secondary ptx included: 4 chronic obstructive pulmonary disease, 1 lymphangioleiomyomatosis, 1 mesothelioma, 1 metastatic angiosarcoma and 1 was secondary to a motor vehicle accident. 7 had a history of smoking, 5 were former smokers and 2 were current smokers, with a mean 24 pack year history (range 5-45). 7 ptx were large, 4 moderate. 5 patients had an intercostal catheter (icc) inserted prior to thoracoscopy, 1 had failed pleural aspirate. there was evidence of bronchopleural fistula in 7 patients prior to the procedure. there was a median of 9 days from ptx to thoracoscopy. light sedation was used for the procedure in 10 patients, 1 required a general anaesthesia with a double lumen endotracheal tube due to anxiety. single port entry, dry talc poudrage and a 16 gauge french icc was used for all procedures. icc was removed a mean of 2 days following thoracoscopy and patients discharged on day 4. pain was the most common complication, requiring narcotic analgesia. one patient died on day 7, secondary to metastatic angiosarcoma. there has been no recurrence of ptx in any patient. conclusion medical thoracoscopy, performed by thoracic physicians is an effective procedure for the treatment of pneumothorax requiring intervention, including selected patients with evidence of bronchopleural fistula. funding nil. conflict of interest nil. nomination for young investigator award no. background lung cancer incidence and mortality are high in tasmania. australia (aihw 2003) 85/100 000 72/100 000 tasmania (cancer registry 2003) 102/100 000 89/100 000 aims and objectives (a) to determine patient demographics in southern tasmania, (b) to determine compliance to identified measures of best practice and (c) assess referral rates, clinical utility and potential delay to positron emission tomography (pet) in a regional setting. methods a prospective database collected information on local clinical practice. cases presented at a multidisciplinary lung cancer meeting over a 12 month period (march 2006 -april 2007 were analysed. data were available for n = 121/161 (75%). results are shown as mean ϯ sd. results 113 primary lung cancer cases were identified. the mean age was 71 ϯ 11 years. 58% of patients were male and 95% were current or ex-smokers. 81% were non-small cell lung cancers (nsclc). tissue diagnosis 93% time from diagnosis to surgery (27 ϯ 15 days) 82% < 42 days macroscopically complete surgical resection (9/11) 82% pet for stage iiib before radical chemoradiotherapy 75% 62% of patients presenting with early or locally advanced disease underwent further staging with pet (n = 34/55). management was changed in 50% of cases (17/34). the average time from pet referral to scan was 11 ϯ 5 days. conclusion a disproportionate number of lung cancers occurred in women. although surgery was performed within recognized timeframes, 2 of 11 patients had incomplete resections. pet influenced management decisions and was performed in a timely fashion. hp chan 1,2 , v tran 1,2 , c lewis 1,3 , p thomas exhaled breath condensate (ebc) is a simple, safe and non-invasive method of sampling breath and has the potential to investigate lung cancer and the associated neoplastic process in the lungs. increased oxidative stress has been implicated in the pathogenesis of lung cancer, and is characterized by elevated hydrogen ions, and hydrogen peroxide (h2o2), which is formed from the conversion of superoxide anions by superoxide dismutase. airway ph has already been shown to be decreased in ebc of patients with other respiratory conditions, but not in lung cancer. therefore the concentration of h2o2 and hydrogen ions in the ebc of lung cancer subjects was compared with matched controls. methods six subjects with newly diagnosed lung cancer were recruited and matched with control subjects: non-smokers, ex-smokers and smokers. ebc was collected and h2o2 was then measured by an assay method based on oxidation of 3,3',5,5'-tetramethybenzidine by horseradish peroxidase and h2o2 while ph was measured using a ph meter. results there was a significant difference (p = 0.033, anova) in h2o2 concentration between the 4 groups with the lung cancer group having elevated mean h2o2 concentration of 23.68 mm (9.15 (sem) compared to the controls: non-smokers, 17.59 mm (6.53 (sem); ex-smokers, 14.35 mm (3.79 (sem); and smokers, 5.21mm (0.69 (sem). ph did not differ significantly (p = 0.659, kruskal-wallis test) between the groups. conclusion these preliminary data suggest that there is significant difference in h2o2 concentration between the groups. the demonstration of an elevated h2o2 level in those with lung cancer indicates an increase in oxidative stress which implies that this may be part of the pathogenesis or response to neoplasia. supported by none. conflict of interest none. pro-inflammatory th1 cytokines produced by t cells and monocytes play an important role in the immune response to malignant cells. however, tumours may escape immune surveillance by inhibiting th1 response and promoting chronic inflammation at the tumour site. methods to investigate the effect of soluble factors released by lung cancer cells on t cell and monocyte pro-and anti-inflammatory cytokines, culture supernatants from several lung cancer cell lines and a normal epithelial cell line (16hbe) were cultured with whole blood for 24 hours, then for a further 16 hrs with and without stimuli. intracellular cytokine / chemokine production was determined using multiparameter flow cytometry. results in stimulated cultures, there was a significant decrease in t cell th1 pro-inflammatory cytokines ifng, tnfa and il-2 and a decrease in monocyte il-1a, il-8, il-12, tnfa, mcp-1 and mcp-3 but an increase in antiinflammatory cytokine il-10 compared with 16hbe and control media. in non-stimulated blood cultures there was an increase in all monocyte inflammatory cytokines / chemokines in the presence of lung cancer supernatants. conclusions lung cancers secrete soluble factors that inhibit the antitumour pro-inflammatory th1 response by t cells and monocytes and upregulate monocyte anti-inflammatory cytokine il-10 following "antigenic challenge". lung cancer cells may also escape immune surveillance by secreting soluble factors that cause newly recruited monocytes to release inflammatory cytokines promoting chronic inflammation at the tumour site. cytotoxic t-cells (ctl's) are important barriers against tumour cells. ctl's induce apoptosis of target cells by mechanisms that include the release of pore-forming perforin and granule associated enzymes, such as granzyme b and granulysin. proteinase inhibitor-9 (pi-9) is the only known granzyme b inhibitor and its expression has been observed in some cancers. we hypothesized that pi-9 would be differentially expressed in lung cancer cells and may inhibit granzyme b-induced apoptosis in these cells. methods we investigated pi-9, granulysin and granzyme b expression in various lung cancer cell lines (1299 ( , 1466 ( , 2009 and normal epithelial cells obtained from bronchial brushing using flow cytometry. peripheral bloodderived t-cells were then incubated with lung cancer cell line supernatants and levels of pi-9, granzyme b and t-cell reactive oxygen species (ros) were assessed. results pi-9 expression was detected in all lung cancer cell lines, (1299 (54.2%), 1466 (90.2%), 2009 (85%), sbc-1 (81%)), at much higher levels than in normal bronchial epithelial cells (8.5%). granzyme b and granulysin levels were undetectable or low in cancer cells (0-9.2%). increased expression of pi-9 and reduced levels of granzyme b were observed in cd8+ t-cells in the presence of all cancer cell supernatants tested (p < 0.05). interestingly, t-cell ros levels were significantly increased in cd8+ t-cells after incubation with cancer cell supernatants (p < 0.05). conclusions high pi-9 expression in lung cancer cells combined with a reduction in t-cell granzyme b expression and enhanced intracellular t-cell ros levels may be a mechanism of immune evasion of lung cancer cells to granzyme b-induced cytotoxicity. immunotherapy for lung malignancies such as lung cancer and mesothelioma is most likely to be successful it it can be combined with conventional tumour debulking approaches such as chemotherapy and surgery. but they scientific basis of such combinations is yet to be determined. to study this we evaluated (1) the capacity of different lung chemotherapy drugs to alter tumour antigen cross-presentation and immunogencity, (2) duration of antigen presentation and responsiveness to immunotherapy after debulking surgery with/without lymphadenectomy, and (3) the pattern of tlr agonism which best synergized with chemotherapy and surgery. we used the ab1-ha murine model of lung malignancy in balb/c mice. results (1) the antimetabolite drugs gemcitabine and pemetrexed were most immunogenic compared to the cytotoxic antibiotics doxorubicin and mitomycin c and the alkylating agent cisplatin. gemcitabine delived large amounts of tumour antigen into the cross-presentation pathway. (2) tumour antigen cross-presentation persisted for only 10 days following resection. the optimal window for immunotherapy following cancer surgery is 1 week for effector ctl stimulation and 2-4 weeks for memory ctl stimulation. (3) the viral-like tlr agonists tlr 3, 7 and 9 were the most effective adjuvant tlr molecules, with tlr 7 agonists generating the strongest systemic anti-tumour responses. conclusion these results help explain previous lung immunotherapy failures and will inform new clinical trials. background mesothelioma is a highly aggressive tumour with an increasing world wide incidence. the serum biomarker mesothelin is elevated in some individuals prior to development of clinical symptoms of the disease and may be useful for screening. we therefore studied the sensitivity and specificity of urinary versus serum levels of mesothelin for mesothelioma patients and evaluated the influence if renal function on the biomarker level. materials and methods concurrent sera and urine samples collected from patients with and control populations. mesothelin concentrations were determined by double-determinant elisa using the mesomark tm assay (fdi, pa). their estimated glomerular filtration rate (egfr) was also calculated. results mesothelin levels correlated between serum and urine samples (pearson's correlation 0.791; p < 0.0001). mesothelin levels were significantly higher in patients with mesothelioma compared to those with asbestosis and/or pleural plaques in serum (4 ϯ 0.9 versus 0.9 ϯ 0.05 nm; p < 0.0001, respectively), in urine (1.9 ϯ 0.5 versus 0.3 ϯ 0.03; p < 0.0001) and in urine following normalization using creatine levels (0.2 ϯ 0.05 versus 0.04 ϯ 0.01). age and egfr were significantly associated with mesothelin levels. conclusion the sensitivity and specificity of mesothelin in urine and in serum were comparable. urine mesothelin may prove to be a useful alternative to serum mesothelin for mass screening of asbestos-exposed individuals. patients undergoing ct coronary angiogram (cta) are often former or current smokers with a high incidence of asymptomatic lung disease. overseas reports show a rate of lung abnormalities ranging from 6.7% to 19%. there are no studies from australia and local factors such as the higher incidence of atypical mycobacteria may influence the rate of benign findings. we are therefore performing a prospective observational study to identify the prevalence and characteristics of incidental lung findings in people undergoing routine cta. methods population: 100 patients undergoing routine cta after informed consent. intervention: radiologist evaluation of lung windows on diagnostic standard workstations. comparator: uncontrolled observational study of consecutive patients. outcomes: primary: prevalence and characteristics of abnormal findings, final diagnosis (clinical judgment, biopsy or long term followup). secondary: number of downstream investigations and costs. results 25 ctas have been studied to date. in 8/25 (32%), abnormalities were noted on lung windows. in 2/25 (8%), there were lung nodules, in 2/25 (8%) there were hilar lymph node abnormalities, in 1/25 (4%), there was hemidiaphragm elevation and in 3/25 (12%) there were pleural plaques (data collection ongoing with study closure expected in february 2008). conclusions preliminary data indicate a substantial number of incidental pulmonary findings from cta; full results will be presented. further analysis is required to determine the impact (benefits, costs and harms) that may result from the concurrent examination of lung windows at routine cta. aim increased levels of nitrogen oxides (nox) and inflammatory markers have been found in bronchoalveolar fluid of lung cancer (lc) patients, but have not been investigated in exhaled breath condensate (ebc).the aim of this study was to compare nox and total protein levels in ebc of lc patients with control subjects. methods ebc was collected during tidal breathing through a glass collection device cooled to 4°c. ebc nox concentrations were measured by a fluorescent modification of the greiss method. total protein in ebc was determined employing the bicinchoninic acid (bca) assay. ebc nox data were log transformed. all data were analysed using anova and expressed as mean ϯ sem. results a total of 88 control subjects and 54 patients with primary lc were recruited. nox and protein concentrations are shown in table 1 . there was no significant difference in ebc nox levels (p > 0.05), but in total protein there was a significant difference between lung cancer patients and all control groups (p = 0.04). conclusion significantly increased ebc total protein levels were found in patients with lung cancer. these data suggest that protein mediator secretion or vascular leak may be present in those with lung cancer. future studies will focus upon the identification of these proteins. methods in this two stage case-control study 446 lung cancer cases and 484 healthy smoker controls were recruited. 180 genetic markers (snps) implicated in lung cancer were screened in our test cohort of 439 smokers and ex-smokers. 30 snps whose genotypes (co-dominant or recessive model) were associated with either the healthy smokers (protective) or lung cancer (susceptibility) phenotype were identified. after genotyping this 30 snp panel in a second cohort of 491 subjects 19 snps were chosen and assigned a simple composite genetic score that was combined with scores for age, history of copd and family history of lung cancer, weighted according to our multivariate regression analysis (n = 930 total subjects). the lung cancer risk score was linearly related to the likelihood of lung cancer with odds ratios (referenced against the lowest score quintile) ranging from 1 to 29 in the highest quintile. on receiver operator curve analyses, the auc was 0.78 and the frequency distribution showed bimodal separation between healthy smokers and lung cancer cases. utility of the score was not affected by effects of age, smoking history or lung function. we suggest that genetic data may be combined with other risk variables to define smokers or ex-smokers at risk of lung cancer for targeted interventions such as smoking cessation and early detection of lung cancer. supported by health research council, nz. conflict of interest yes. tp 144 v aiyappan 1 , a graham 2 1 department of medicine, maroondah hospital, melbourne, australia, and 2 the new disease-modifying anti-rheumatic drug (dmard) leflunomide is being used increasingly to treat inflammatory arthritis. its association with interstitial lung disease needs to be considered before combining it with methotrexate. case report a 73-year-old male who was known to have rheumatoid arthritis and was on methotrexate was admitted with progressive dyspnoea and malaise. he had been recently started on leflunomide. investigations revealed interstitial lung disease and acute renal failure. he improved on conservative treatment (stoppage of disease modifying drugs (dmard), iv fluids and steroids). review of literature an epidemiological study by suissa et al has suggested that there is increased risk of ild associated with leflunomide in patients with a history of ild or methotrexate use but they attributed this to channelling bias. there has also been a report of leflunomide associated with iga glomerulonephritis.by this presentation we aim to increase the awareness of this entity. we also suggest that any patient who is started on combination dmard (i.e. methotrexate and leflunomide) should have a baseline chest x-ray and be monitored for development of interstitial lung disease. conclusion we are reporting the first ever case of interstitial lung disease and glomerulonephritis (in the same patient), due to usage of leflunomide. this entity needs to be thought about in any patient on combination dmards. background bone morphogenic protein receptor ii (bmpr-ii) mutations are associated with pulmonary artery hypertension. failure of the growth inhibitory effects of bmp may contribute to vascular obliteration and remodelling leading to pulmonary artery hypertension (pah) [1] . pah has been observed following venous thrombembolic disease (vte), including pulmonary embolism (pe) and deep venous thrombosis (dvt) [2] . local markers of the pulmonary vascular endothelium rather than traditional markers of thromobophilia are thought to be involved [3] . methods plasma was collected from age and gender matched participants within 24 hours of diagnosis of vte and prior to commencement of warfarin therapy. plasma samples were hybridized to individual human cytokine antibody arrays, to detect protein levels of bmp2, bmp4 and bmpr-ii. results bmp2 and bmp4 levels were higher in patients with dvt than pe. no difference in the bmp level was observed between patients with pe and controls. soluble bmpr-ii receptor was lower in patients with pe than in controls or patients with dvt. conclusion in patients with pulmonary artery stress during the time of a pe the bmpr-ii receptor is reduced, which may predispose patients to vascular remodelling and obliteration. the bmp 2 and 4 levels are reduced at the same time, suggesting a possible overriding regulatory mechanism. the physiological role of bmp's and bmp receptors in patients with vte warrants further investigation. historically, cyclophosphamide has had a variable role in interstitial lung disease (ild), the rationale for its use based on the benefit seen in vasculitis and scleroderma, its rapid effect and low toxicity profile. in patients with severe progressive ild a rapidly effective, well-tolerated agent is desirable. for this reason a treatment protocol for the use of intravenous (iv) cyclophosphamide was implemented at our hospital. aim to review the indications, duration, tolerability and effect of intravenous cyclophosphamide in ild patients following the introduction of a treatment protocol. methods records of 92 patients [dlco was 40 ϯ 15% and fvc 61 ϯ 20%] completing a course of iv cyclophosphamide during 2005-6 were reviewed (excluding patients with systemic sclerosis). data covering 18 months prior to and following treatment were collected. comparative analysis of paired pulmonary function data 6 months before and after treatment was performed. 61% had underlying autoimmune disease. results primary treatment indications included progressive disease(n = 67); severe disease (n = 16); suspected vasculopathy (n = 11); bridging therapy to transplantation (n = 10); and accelerated decline (n = 5). patients received 600 mg/m 2 [mean dose 1152 ϯ 165 mg, median number of pulses 6 (1-12)]. patients with paired pulmonary function data had a difference in median change in dlco% predicted from -15.6% (-95.4 to 29.9%) before treatment to +4.25% (-17.3 to 73.9%) following treatment (p < 0.0001). this remained significant with exclusion of vasculitis, or any autoimmune disease, and independent of prior immunosuppression. therapy was well tolerated (4 withdrew from treatment, 5 deaths within 1yr, none directly related to treatment). conclusion iv cyclophosphamide is well tolerated, and associated with functional stability or improvement in the majority of patients. it remains a viable treatment alternative for consideration. pulmonary hypertension is common in interstitial lung disease (ild) and associated with a poor prognosis. as the gold-standard test, right-heart catheterization (rhc) is invasive, and resource-limited, reliable non-invasive measures of ph are needed. methods all ild patients referred for rhc during 1997-2007 were included (n = 95; 54 male; age 56.5 ϯ 12 yrs). all patients had concurrent echocardiography (tte) and pulmonary function. the relationship of rhc mean pulmonary artery pressure (mpap) to tte variables, pulmonary function, exercise capacity, as measured by six minute walk testing (6mwt, n = 58) and brain natriuretic peptide (bnp, n = 36), was examined. case a 65 year old male, non-smoker for 25 years, retired professor of anatomy (had chronic exposure to embalming fluids, formaldehyde, phenol, antifungal and other solvents, for 20 years) presented with chronic cough and phlegm production. these symptoms were worse at night (waking him several times) and early morning. his pulmonary tests were stopped due to persistent cough. a chest x-ray revealed features of longstanding interstitial lung disease. the hrct revealed widespread subpleural interlobular thickening, worse at bases, in keeping with idiopathic pulmonary fibrosis (ipf). there was minimal fibrosis and honeycombing, but no groundglass opacification, large bullae, pleural calcification or pleural plaques. however, there was associated bronchiectasis at the lung bases considered to be due to traction. the ba lavage showed 50% macrophages, 7% neutrophils, 3% lymphocytes, and 40%, eosinophils and no infection. the patient declined to have a lung biopsy. as per his past x-rays, the duration of his ipf is a little over one year. he maintains that his symptoms started only after starting irbesartan (irb). introduction transbronchial lung biopsy (tbb) has a variable and unpredictable diagnostic yield in sarcoidosis. we hypothesized that the extent and pattern of parenchymal disease on ct would predict the likelihood of a positive tbb. methods data relating to ethnicity, symptoms, pulmonary function and site and results of tbb and bronchoalveolar lavage (bal) from 70 sarcoidosis patients were recorded. all had a ct scan within 6 weeks prior to the tbb procedure. cxr stage was determined from radiology report. ct scans were scored quantitatively for patterns of parenchymal disease (nodular, reticular, consolidation, ground glass and mosaic attenuation) on a lobar basis. results 50% patients had a positive tbb (total 67% of cohort had histological confirmation). symptoms, ethnicity, treatment, lung function and cxr stage were not predictors of a positive biopsy. positive biopsy was associated with higher bal lymphocyte count (p < 0.05) and female gender (p < 0.01). a reticular pattern (p < 0.05) and higher total lung score (excluding da) (p < 0.05) on ct scan predicted a positive biopsy. in those patients with tbb from right lower lobe (53/70) the total rll score on ct was predictive of positive biopsy (p < 0.05). on multivariate analysis gender, bal lymphocytosis and total lung score were independent predictors of a positive tbb (area under roc 0.82). pulmonary arterial hypertension has two histological variants; 'arterial-only pulmonary arterial hypertension' (artpah) and 'pulmonary veno-occlusive disease' (pvod). bosentan, a dual endothelin receptor antagonist, has been found to improve haemodynamics, functional capacity and survival in artpah. however, the response to bosentan in clinically diagnosed artpah is often variable. it was hypothesized that a lack of response to bosentan therapy in clinically diagnosed artpah can be explained by misdiagnosed pvod. aims included to: (1) perform morphometric and qualitative pulmonary vessel analysis on normal controls and cases clinically diagnosed with artpah who had failed bosentan therapy; (2) ascertain if pvod is present within the case group; (3) correlate clinical variables and vessel microanatomy to identify the pathologies driving pulmonary pressure elevation. this study reviewed 14 cases of clinically diagnosed artpah (idiopathic n = 12, associated with scleroderma n = 2), who had failed bosentan therapy and had available lung tissue. controls (n = 6) were obtained from explanted lungs for other causes and a prior transthoracic echocardiogram excluded pulmonary hypertension. vessel morphometry and qualitative analysis was performed with a novel technique of smooth muscle actin immunohistochemistry counterstained with verhoeff's elastin. baseline clinical data were retrieved. we found 86% of cases had pathology confirmed pvod. only 14% of cases had artpah, the original clinical diagnosis. in pvod, significant pathology was present in all vessel types. all vessels had significant smooth muscle hypertrophy. the obstructive, collagenous, pauci-cellular intimal fibrosis of the venules (p < 0.0001) and arterioles (p < 0.0001) was considerably different to the concentric laminar proliferation of smooth muscle observed in the muscular arteries (p < 0.0001) and arterioles (p = 0.001) in artpah. artpah also had muscular artery smooth muscle hypertrophy (p = 0.007). the median time to bosentan failure was shorter in pvod than artpah (290 vs. 657 days). in conclusion, pvod is an under-diagnosed cause of pulmonary hypertension, is commonly clinically misdiagnosed as artpah and may present with a poor bosentan therapy response. finally, pvod is a vaso-occlusive, not a veno-occlusive disease, and is an independent type of pulmonary hypertension, not a subtype of pulmonary arterial hypertension. cutaneous t cell lymphomas (ctcl) are a heterogenous group of lymphoproliferative disorders. they show various clinical manifestations and diverse morphological, histological and immunological characteristics of the malignant cells. they are caused by clonally derived, skin invasive t cells. peripheral t cell lymphomas (ptcl) are generally more aggressive and have one of the lowest overall and failure-free survival rates. because of the rarity of these disorders, diagnosis and treatment remain challenging. this case report describes a 69-year-old woman presenting with progressive dyspnoea and cough, together with a distressing generalized pruritic rash. she was initially treated as left ventricular failure with the rash ascribed to a drug reaction as suggested by initial skin biopsies. the diagnosis was made on a third skin biopsy and flow cytometry of lymphocytes obtained by broncho-alveolar lavage 6 months after presentation. despite an initial response to chemotherapy she succumbed to the disease 20 months after diagnosis. clinical pathways to guide the investigation of suspected pulmonary embolism (pe) have been increasingly adopted by emergency departments (ed) worldwide. compliance with these diagnostic algorithms is critical in ensuring good patient outcomes. this study evaluated the compliance to the clinical pathway used in our ed that combines risk assessment (wells scoring system) with d-dimer test, vq scan or ctpa. the main objectives of this study were to identify those factors which contributed to compliance and to assess patient outcomes. methods a prospective observational study of 239 consecutive patients who underwent investigation for pe in our ed. patient demographics, pathway parameters and patient outcomes at 3-month follow-up were collected. case we report the case of a 37 year old woman who presented to the emergency department with a three day history of dry cough and dyspnoea. the patient was in her third pregnancy at 30 weeks gestation. she had no fever, chest pain or coryzal symptoms. the patient had presented with a right sided spontaneous pneumothorax seven months prior to the current presentation. her past medical history included placental abruption, complicating her previous two pregnancies. her second pregnancy was complicated by placental abruption at 27 weeks and the foetus had not survived. her first pregnancy was complicated by placental abruption at 36 weeks with successful delivery of the foetus. at presentation, significant findings included tachycardia, hypoxemia, tachypnoea and reduced breath sounds over the right side of the chest. chest x-ray demonstrated a large right pneumothorax. a right intercostal catheter was inserted resulting in right lung re-expansion. the catheter was removed three days later. the patient returned to hospital twenty four hours after catheter removal with a recurrent right sided pneumothorax. the patient agreed to surgical intervention involving video-assisted thoracotomy and talc pleurodesis. the patient had no further complications with the pregnancy. she delivered a healthy baby at 38 weeks gestation. discussion spontaneous pneumothorax in pregnancy is rare and there is little evidence to provide guidelines for the management of recurrent pneumothorax in high risk pregnancy. our case illustrates a successful outcome for mother and foetus with surgical intervention at 32 weeks gestation. folfox is currently the standard adjuvant treatment for locally advanced (stage iii) colon cancer and increases disease free survival. its toxicity is well tolerated with common adverse effects being paraesthesia, bone marrow suppression and gastrointestinal disturbance. pulmonary toxicity has rarely been reported. three clinical cases of acute dyspnoea following folfox therapy (2005) (2006) (2007) for stage iii colon cancer are reported. all had an anterior resection followed by 11-12 cycles of folfox. each developed rapidly progressive dyspnoea requiring hospital admission within one week of their last cycle. one patient required invasive ventilation in icu. high resolution computed tomography (hrct) showed bilateral widespread honeycomb pattern with associated ground glass opacification consistent with pulmonary fibrosis. they had reduced lung volumes and gas transfer. transbronchial biopsy and bronchoalveolar lavage in one patient showed an acute eosinophilic pneumonitis. other causes of interstitial lung disease were carefully excluded. all three patients received high dose corticosteroids with one receiving additional cyclophosphamide. the first patient showed complete recovery following an eight week course of corticosteroids, with resolution of the hrct changes and improvement in lung function. the second had symptomatic improvement of dyspnoea, but a persistent moderate reduction in gas transfer. the final patient had persisting radiographic changes and a reduced gas transfer. he remained dependant on ambulatory oxygen 6 months after his initial presentation. these patients' interstitial lung disease appears due to folfox with oxaliplatin being the most likely causative agent. the use of oxaliplatin chemotherapy has increased markedly over the last 3 years and although rare, physicians should be aware of its potential for lung toxicity. lung function testing at baseline, during and towards the end of oxaliplatin treatment should be undertaken and may allow early detection and intervention in cases of pulmonary toxicity. the forced oscillation technique (fot) with broadband signals has been employed relatively rarely in the studies on respiratory mechanics. recent work from our laboratory [1] indicated that the cheek support and the neck angle have minor influence on the impedance spectra around the first antiresonance (far,1), which makes the use of the broadband fot especially attractive in young children. methods we studied 7 healthy children (c; female: 4) and 8 children with bronchopulmonary dysplasia (bpd; female: 3), using multiple-frequency fot between 8 and 256 hz superimposed on spontaneous breathing. results groups c and bpd did not differ in age ( lung function impairment is common in children with cardiac defects associated with increases in pulmonary blood flow/pressure. to investigate the development of bronchial hyperreactivity (bhr), an aorto-caval shunt was created in a model of precapillary pulmonary hypertension. surgical shunt repair was performed to assess the reversibility of bhr. methods 26 rats were divided into 3 groups: group c (n = 10) with sham surgery, group s (n = 8) where an aorto-caval shunt was created (follow-up 4 wks), group r (n = 8) with aorto-caval shunt but surgical correction of the shunt at 4 wks (follow-up 8 wks). in all animals, respiratory input impedance (zrs) was measured at baseline and following increasing doses of methacholine (mch 2, 4, 8, 12 mcg/kg). airway resistance (raw), inertance, tissue damping (g) and elastance were estimated from the zrs spectra by model fitting. measurements were repeated in all animals at 4 wks and at 8 wks for groups r and c. results there was a significant increase in raw and g in group s and rat 4 wks at baseline and following mch ( fig.) which was reversed after surgery. to characterize the factors contributing to lung function impairment following cardiopulmonary bypass (cpb), functional residual capacity (frc), lung clearance index (lci) and respiratory mechanics were measured in children with pulmonary hypoperfusion (tetralogy of fallot, tof n = 12) and hyperperfusion (ventricular septal defect, vsd n = 12) undergoing surgical repair of congenital heart disease. methods frc and lci were measured using a sf6 washout technique and respiratory mechanics using a low frequency oscillation technique in the perioperative period. results while chest opening led to a significant improvement of lung volumes and respiratory mechanics in all patients (p < 0.001), a reduction in pulmonary blood flow during cpb decreased lung volumes and airway resistance in parallel but significantly more in children with tof compared with those with vsd. re-establishing pulmonary blood flow during cpb improved respiratory function particularly in children with tof ( figure) . conclusions sternotomy had a great impact on lung function with parallel improvement in alveolar recruitment, ventilation inhomogeneity and airway resistance. in contrast, onset of cpb led to lung function impairment with a significant drop in frc especially in children with pre-existing hypoperfused lungs. this suggest that pulmonary blood flow enhances alveolar stability through a tethering effect on the alveolar walls. children with advanced lung disease being considered for lung transplantation are likely to spend disproportionately longer periods on transplant waiting lists before appropriately sized donor organs become available. these longer waiting times reflect the lower organ donation rates seen in children; rates that are significantly lower than those reported in the adult population. we describe two children with advanced lung disease who deteriorated whilst on the waiting list for lung transplantation, and in the absence of appropriately sized donor lungs, underwent lobar lung transplantation. methods we describe the clinical course of two children, aged 9 and 13 years old, with advanced lung disease secondary to post-mycoplasma obliterative bronchiolitis and cystic fibrosis-associated bronchiectasis, respectively. results both children received a "cutdown" bilateral lobar transplant from two oversized adult brain-dead organ donors. in both cases the transplant operation involved implantation of the right middle and upper lobes, and of the left upper lobe from the donor. conclusion given the low organ donation rates in children, and in the absence of appropriately sized donor lungs, novel strategies such as lobar transplantation must be considered, particularly when children continue to clinically deteriorate whilst on the lung transplant waiting list. data from the west australian adult outcomes of extreme preterm birth study suggest that adult survivors of bronchopulmonary dysplasia (bpd) may be left with functional and structural pulmonary abnormalities, most notably emphysema. animal data suggest that the antenatal administration of corticosteroids may adversely affect lung development. we therefore sought to determine if maternal variables, including administration of corticosteroid, could predict emphysema severity in adulthood. methods bpd subjects (birthweight < 1500 g and oxygen dependence at 36 weeks post-menstrual age) born prior to 1988 were identified and recruited prospectively via the statewide neonatal follow up program as previously described. pulmonary function tests and thin selective inspiratory and expiratory computerised (ct) images were acquired and scored for emphysema severity (voxel index (%)). the obstetric history was obtained from retrospective review of case notes. results 21 adults (12 females, aged 18-34) were studied, 2 declined ct. all subjects had abnormal ct findings. fifteen (79%) had areas of emphysema. emphysema score and fev1 were not influenced by the administration of antenatal corticosteroids, indication for delivery, maternal age or presence or absence of chorioamnionitis. conclusion maternal factors, including the administration of antenatal corticosteroids, do not predict the long term respiratory outcome of bpd. the factors determining the severity of emphysema in this group remain unknown. the prevalence of childhood asthma is high in the torres strait. children have generally more severe asthma and asthma knowledge is poor. however, there is no culturally appropriate asthma education program for these children. we are conducting a randomized controlled trial to examine the additional benefits of an education intervention by indigenous health care workers (hcw) on asthma outcomes. we describe the study's objectives, design and baseline measurements. methods children with wheeze were reviewed by two paediatric respiratory physicians using a standardized protocol; children with asthma were eligible. after obtaining informed consent children were randomly allocated to: (1) three additional asthma education sessions with a hcw; or (2) no additional education from a hcw. trained hcws carried out the education sessions using culturally appropriate tools. primary outcome was the number of unscheduled hospital/doctor visits due to asthma exacerbation. all children were re-assessed at 12 months. results we enrolled 113 children aged 1 to 17 years, 81% were torres strait islanders and 12% aboriginal and torres strait islanders. the clinical spectrum of asthma was: 51% infrequent episodic asthma, 22% frequent episodic asthma and 27% chronic asthma. eighteen percent of the children knew what a written asthma action plan was; 8.5% had one. carers' assessment of knowledge of medications showed that 52% could not name any asthma medication used by their child, 40% could not explain dosage, and 67% could not explain how beta2 agonists worked. conclusions asthma knowledge and possession of asthma action plans in this cohort is poor at baseline. there is substantial room for improvement and additional asthma education by hcws potentially has significant benefits. impulse oscillometry system (ios) measures respiratory function during normal breathing by transmitting mixed frequency rectangular pressure impulses down the airways and measuring reflected pressure. computer analysis calculates respiratory impedance and its components, airways resistance and reactance, at a range of frequencies from 0.1 hz to 150 hz. no previous australian normative data exists. the ios software generates predictive normal values for each of the parameters measured including total airway resistance (r5), the proximal airway resistance (r20) as well as peripheral capacitive reactance (x5). however, they are based on german data. methods cross-sectional study of 100 community dwelling adults, with 10 males and females per 10-year cohort. inclusion criteria: age range 25-74 years, apparently good respiratory health. exclusion criteria: smokers, asthmatics and others with acute or chronic respiratory disease. both ios and spirometry were conducted on all participants. results australian predictive normal equations have been generated and compared to the current published equations. the ios parameters have been correlated with the spirometric data. results have been analysed by gender, age, height and weight and compared with the predictive normal values for each parameter provided by the german manufacturer of the ios instrument. analysis includes calculation of mean range, and lower limit of normal. conclusions a preliminary set of australian predictive equations have now been produced for the ios. these have been compared with international equations. ios has potential application in a range of respiratory disease states and in population screening for occupational health (e.g. mining, & high dust load environments). supported by phc red. rationale although clinical practice guidelines for both asthma and copd recommend spirometry for diagnosis and monitoring, beneficial effects on the management of chronic respiratory diseases in general practice have not been established. we hypothesized that spirometry would improve health outcomes compared to usual care. methods we are conducting a single masked rct with 3 arms: group a receive 3 monthly spirometry and followup, group b receive spirometry before and after the trial and group c usual care. 45 general practices were recruited though divisions of general practice in melbourne. invitations were mailed by 31 of these practices to patients who had been prescribed inhaled medications during the previous 6 months. participants returned respiratory and generic quality of life questionnaires and an asthma score card. groups a and b were tested on a micromedical turbine spirometer following ats/ers guidelines. results 351 eligible patients (275 adults, 50 children aged 8-13 and 26 youths aged 14-17 years) entered the trial. 122 were randomized to group a, 134 to group b and 95 to group c. the mean (sd) age of adult participants was 54.3 (12.7), children 10.3 (1.7) and youths 15 (1.1) years. there were 130 males and 221 females. the adults were highly symptomatic in the previous 12 months: 82% reporting wheeze, 50% chest tightness on waking, 74% shortness of breath on exertion, 61% nocturnal cough, 46% morning cough and 75% sputum. symptoms of chronic bronchitis were reported by 39% of adults and a diagnosis of copd by 19%. asthma was reported by 84%, confirmed by a doctor in 96% and 55% had experienced an attack in the last 12 months. only 35% had a written asthma action plan. 37% of adults had ever visited a hospital ed and 28% had been admitted. conclusion it is possible to recruit asthma and copd patients from general practice and to randomize them to spirometry or usual care. whether spirometry is associated with fewer symptoms, changes in medication, uptake of action plans or improvement in lung function or quality of life requires further followup. supported by nhmrc. s shah 1 , jk roydhouse 1 , b toelle 2 , s sawyer 3 , c jenkins 2 for the pace australia management committee 1 university of sydney, 2 woolcock institute of medical research, sydney, nsw 2006, and 3 royal children's hospital, melbourne, vic 3052 it is widely held that recruitment of general practitioners for research can be challenging. in this paper, we discuss the recruitment experience from a current study evaluating the impact of an educational asthma intervention on patient outcomes. our aim is to describe the two different strategies utilized to date: (1) in-house through an academic department of gp and (2) outsourced to a private gp organization. methods initial interest was generated through faxes, presentations at gp divisional meetings and newsletter advertisements. gps who expressed interest were visited by project staff to discuss the study further. a major difference was recruiting one gp per practice in the first strategy versus multiple gps per practice in the second strategy. to assess the strategies, we examined participant characteristics, number of gps recruited and number retained. results participant characteristics: under both strategies, 30% of recruits had trained in asia and 54% were women. the first strategy recruited more gps who spoke at least two languages at home (85% vs 42%) and the second strategy recruited more recently graduated gps (58% vs 50%). recruitment: the first strategy recruited 35 gps over 6 months and the second recruited 34 gps over 3 months. retention: 19 gps (54%) from the first strategy stayed in, compared to 29 (85%) from the second. conclusions whilst absolute numbers of gps recruited were similar, retention was much higher under the second strategy. recruitment in primary care is difficult and requires a range of approaches which need to be re-evaluated and adapted as necessary during the course of the study. supported by the australian government department of health and ageing. bronchiectasis is a heterogeneous condition with a large number of causative factors and range of symptoms. the classification of this condition is often confusing and hard to remember. the aim of this study was to classify non-cf bronchiectasis into different clinical phenotypes. methods 178 consecutive patients with non-cf bronchiectasis confirmed on high resolution ct scanning had a detailed clinical, spirometric and laboratory assessment performed by a respiratory physician (pk/mf/pw) and were then followed up for an average of 9 ϯ 4 years (mean and sd) for a total of over 2000 reviews. results 160 of the 178 patients (90%) could be classified as belonging to 3 phenotypic groups; 1) bronchiectasis arising in childhood, 2) bronchiectasis occurring in smokers and 3) bronchiectasis occurring in the elderly. each group had different features which are listed in the there are few data on the long term outcomes of treatment for tuberculosis (tb) by directly observed therapy (dot) in low-incidence settings. the aim of this study was to assess the incidence of recurrent tb in nsw. methods data linkage was performed within the nsw department of health tb notifications database to identify cases that had more than one tb notification between 1994 and 2006. recurrent tuberculosis was defined to include all patients with two or more culture positive episodes at least 6 months apart, where patients had received at least six months treatment for the initial episode. in cases where data contained within the notification details was not sufficient to allow us to distinguish between true cases of recurrent disease, duplication notification for the same episode or persistent disease after incomplete treatment, additional information was obtained from the area tb coordinator. results there were 5723 tb notifications between 1994 and 2006 with 3731 being culture positive. 15 cases of recurrent culture positive disease after completed treatment for the first episode were identified (recurrence rate: 0.4%). conclusions in a population with a low tb incidence, treatment of active tuberculosis with dot results in a very low rate of disease recurrence over a long period of follow-up. support nhmrc ccre in respiratory and sleep medicine. introduction rhinoviruses (rvs) are the major cause of viral-induced exacerbation of asthma. to date, the molecular mechanisms of rv pathogenesis are not understood. recent findings suggest that rv pathology may involve host cell nucleocytoplasmic trafficking, inhibiting key cell functions such as transcription and translation. the study aims to investigate the mechanism of rv 3c protease nuclear trafficking. methods hela cells were infected with rv or transfected with plasmids and cellular localization of 3c analysed at various times thereafter using immunofluorescent confocal microscopy and western blotting with specific antibodies. results 3c protease was predominantly present in nuclei of rv infected cells up to 6 hours after infection, becoming increasingly cytoplasmic thereafter. the nuclear membrane of infected cells became progressively indistinct with time. using a specific inhibitor we also found that 3c utilizes the crm-1 nuclear export pathway. 3c was predominantly in the form of 3cd in both cytoplasm and nucleus of infected cells; mature 3c protease was also detected from 6 hours after infection. deletion analysis indicats that the nuclear localization domain and a nuclear export signal are most likely to be present within the n terminal 64 amino acids. the nuclear export signal is inhibited in the full length protein, via an unknown mechanism. conclusion our data suggest that 3c and 3cd proteins localize to the nucleus in infected cells where they may play a key role in rv pathogenesis by disrupting cellular transcription and the nuclear transport machinery. chronic necrotizing pulmonary aspergillosis (cnpa) is a relatively uncommon, sub-acute, locally destructive process due to aspergillus invasion of the lung. the incidence and prognosis of cnpa are poorly described. case report we present a case of cnpa in a patient on intermittent low dose steroid therapy and recurrent refractory exacerbations of chronic obstructive pulmonary disease (copd).the patient presented with worsening shortness of breath and productive cough requiring recurrent inpatient admissions. human influenza virus is found to bind preferentially to saa2,6gal receptors found in the upper respiratory tract, while avian viruses bind to saa2,3gal receptors expressed in lower airways. this is thought to affect the ability of transmission to humans. our aim was to study the ability of avian and human influenza strains to infect bronchial epithelial cells and relate this to levels of the sialic acid receptor expression. methods calu-3 cells were used as a proximal airway cell and a549 were used as distal airway cell. human primary bronchial epithelial cells (pbecs) were obtained from healthy, asthmatic, and copd volunteers by endobronchial brushing. epithelial cells were stained with sambucus nigra lectin that binds saa2,6gal receptor, and maackia amurensis lectin ii that binds to saa2,3gal. the cells was analysed by flow cytometry. human influenza a/h3n2/wellington strain and low pathogenic avian influenza a/h11n9/sandpiper were chosen and were used at an moi of 0.005 to infect cells. the supernatants were harvested at 48 hr post infection, of which was then analysed by plaque assay for virus replication. results the calu-3 showed greater expression of saa2,6gal linkage than saa2,3gal linkage, and a549 displayed slightly higher expression of both receptors compared to pbecs. despite this human and avian influenza virus replicated to similar titre at 15,000 pfu/ml in both cell lines, but showed low replication in pbecs. background treatment of community-acquired pneumonia remains based on 'best guess' empiric algorithms because of the poor utility of current pathogen tests. furthermore our ability to stratify patients into risk groups is crude at best, relying on scores such as the pneumonia severity index or the curb-65 have major limitations. we have been slowly improving real-time pcr assays for pneumococcus as a clinical tool in patients with pneumonia. methods building on previous research we assesed two targets in the autolysin (lyta) gene and the pneumolysin (ply) gene of s.pneumoniae using the lightcycler instrument and fluorescence resonance energy transfer (fret) probes. all common s. pneumoniae serotypes were detected while other bacteria and viruses were not. the lyta target had the best sensitivity with a detection range between 21 ng to 21 fg. both assays were then applied to whole blood samples from 400 adult patients with community-acquired pneumonia, all of whom had blood cultures prior to antibiotic administration and urinary antigen testing for s.pneumoniae. the lyta pcr had the best performance characteristics with a sensitivity more than twice that of blood cultures in the clinical samples. most pcr+ve/culture -ve patients had positive urinary antigen tests. there was clinical evidence that urinary antigen +ve/ pcr -ve patients were false +ves. most significantly there was a strong correlation between quantitative bacterial count and clinical outcome. conclusions real-time quantitative pcr for pneumococcus has significant potential as both a diagnostic and therapeutic tool in patients with pneumonia. the pitjantjatjara lands are situated in the north-western corner of south australia, occupying an area of over 120 000 square kilometres with a population of approximately 3000. the population lives in small communities or homelands, and there is a high level of mobility between this region and other aboriginal communities in south australia and the northern territory. nganampa health council provides all health care services to the region. specialized support for tb control comes from both the south australia tb service based at royal adelaide hospital as well as a centre for disease control in alice springs. the prevalence of tuberculosis (tb) in this predominantly indigenous community is thought to be significantly higher than the national rate. there are considerable challenges in detecting and managing tuberculosis, relating to the community's geographical remoteness, migration of populations and access to health services. the aims of this study are to quantify the prevalence of tuberculosis in the pitjantjatjara lands, and describe the significant barriers to tb diagnosis and treatment. methods a retrospective study of all diagnoses of tuberculosis within the pitjantjatjara lands in the period 1995-2006. outcomes include measures of tuberculosis diagnosis, the rates of completed tb treatment and rates of tuberculosis drug resistance. the study will draw conclusions about the reasons for high levels of tb prevalence in this community and identify barriers to effective tuberculosis treatment. conflict of interest no. patients admitted to hospital with a diagnosis of community-acquired pneumonia (cap) are usually treated with intravenous (iv) antibiotics irrespective of pneumonia severity. available guidelines vary in recommended timing and indications for switching to oral antibiotics. aim to examine the patterns of antibiotic choice and delivery method (iv, oral and time to switch) in patients admitted with cap. methods a retrospective chart review of admissions to the respiratory unit over a 12-month period with a diagnostic-related group (drg) coding of pneumonia. 41 charts were reviewed. data collected included patient demographics, clinical features at presentation (temperature, pulse rate, respiratory rate, bp, oxygenation), initial investigations, initial antibiotic regime, time to change (iv to oral), subsequent antibiotic regime and duration, time to defervescence, length of stay and outcome. pneumonia severity was calculated using the revised british thoracic society system (curb-65), score ն 2 = severe. results 3 patients were excluded due to incorrect coding. of the 38 patients, age was 50 ϯ 21 (mean ϯ sd) yrs and 25 (66%) were male. 28 patients (74%) were febrile at presentation and the median curb-65 score was 1 (range 0-4). 37 patients (97%) received iv antibiotics. the curb-65 score was 0 or 1 (non-severe) in 25 patients and 22 of these patients received a combination of iv ceftriaxone and a macrolide. time to defervescence was 2.9 ϯ 2.3 days. time from defervescence to switching to oral therapy was 3.4 ϯ 2.8 days. in non-febrile patients, time to switch was 4.7ϯ4.3 days. length of stay was 8.7ϯ13.0 days. conclusions the time between defervescence and switch to an oral regime was relatively long, possibly contributing to an increased length of stay. many patients received ceftriaxone even with a curb-65 severity rating of 0 or 1. implementing local guideline-based treatment protocols may reduce length of stay. ultrasonic flow sensors can determine flow, volume and molar mass (mm) of the gas flow simultaneously. during tidal breathing the expired molar mass curve can be used to compute co2 over expired volume and a capnography index (cpi) can be computed. the relationship between cpi and copd classification according to gold was investigated. methods prospective, controlled trial. consecutive patients who underwent routine lung function were enrolled to participate in a tidal breathing test using an ultrasonic flow sensor. each test consisted of three tidal breathing recordings of 60 sec. flow, volume and molar mass were measured at 200 hz and data were acquired using prototype wbreath data acquisition software. mean expirograms (mm over volume) were computed and the measurements were analyzed to determine the slope of exhaled phase ii (s2), the slope of phase iii (s3) and the relationship between s2 and s3 (cpi = s3/s2). gold stages were determined from the lung function results and the ers predicted values. results 53 volunteers participated in the study with a mean age of 62 (sd 14), 23 were male, mean bmi 26 (sd 5), 17 had never smoked. the mean pack/year smoking history was 38. there was a clear relationship between gold stage and cpi: gold stage 'normal' had a mean cpi of 5.5 (sd 3.7, n = 21), stage 'severe' had a mean cpi of 13.7(sd = 3.9, n = 7). conclusion computation of cpi based on tidal breathing analysis using an ultrasonic flow and mm sensor correlates well with gold stages. it may therefore be possible to use a simple tidal breathing test to determine the severity of airways disease. background osa is common in tetraplegia and appears within weeks of injury. although cpap treatment is efficacious in able-bodied subjects, case series suggest that cpap is poorly tolerated in tetraplegia. no prospective study has examined cpap efficacy or adherence in tetraplegia. aim to determine the feasibility of cpap use to treat osa following acute tetraplegia. methods all acute admissions who consented and fulfilled the inclusion and exclusion criteria underwent full, portable polysomnography. those found to have an apnoea hypopnoea index of >10 events per hour (osa) were offered cpap, delivered via an auto-titrating device. results to date, 25 patients have been admitted (11 excluded, 3 refused consent). no significant, adverse events have been observed. two patients did not have osa. of the nine with osa, four are mid-study, two had incomplete follow-up (1 returned to uk and 1 refused 3 month assessment), two adhered with cpap and one did not due to severe, pre-existing nasal obstruction. preliminary analyses suggest that those who adhered to cpap had a marked reduction (80% compared with 10-40%) in sleepiness and a greater reduction in the functional outcomes of sleepiness compared to either those without osa or who were unable to use cpap. patient accrual, recruitment and completion rates are consistent with our initial estimates. study recruitment will be completed by end-october 2007. conclusion initial data suggest that auto-titrating cpap is a feasible treatment for osa in acute tetraplegia. these data will be used to finalize planning for a multi-national, multi-centre randomized controlled of therapy. this research was supported by the transport accident commission. visual recognition of cyanosis is an important clinical activity. cyanosis recognition is affected by lighting colour and there is anecdotal evidence that people with significant colour vision deficiencies (cvds) have particular difficulty. studies to date have centred on the colour change with oxygenation of isolated blood but it is not clear how this extrapolates to cyanotic patients in vivo. methods ten patients known to be chronically hypoxaemic and showing signs of cyanosis were recruited from the chronic respiratory program. ten normal subjects were recruited as controls. the spectral reflectances of their lips, nail beds and palm creases were measured using a topcon sr-3 telespectroradiometer. the patients were measured at rest and after exercise to lower their saturation by 5-10%. the chromaticities were calculated and plotted. results both groups showed a spread of colours but they fell into two distinct ranges. the colour difference between the groups lies very close to the colour confusions made by congenital cvds. within the cyanosed group, the colour shift was not tightly related to decreasing oxygen saturation. this is most likely due to interpersonal factors such as pigmentation and vascular perfusion that affect colour and the difficulties in measuring the colour of heterogeneous anatomical features. conclusions these results quantify the anecdotal difficulties in detecting cyanosis and suggest that observers with cvd would have problems recognizing the condition. the photographs obtained from this study will be used to compare the ability of subjects with and without cvd to detect cyanosis. supported by the nsw ambulance service. baroreflex sensitivity is depressed in osa patients during sleep but effects during wakefulness are less clear. we have now examined relationships between awake brs and severity of sleep disordered breathing (sdb). methods immediately prior to overnight polysomnography, continuous (5 min) beat-to-beat arterial blood pressure was measured via finger plethysmography (portapres) and heart rate via ecg in 20, supine, normotensive, untreated osa patients (17 males; age: 49 ϯ 15 years (mean ϯ sd); bmi: 26 ϯ 11 kg/m 2 ). spontaneous baroreflex sensitivity (brs) was calculated using the sequence technique. sdb was characterized as apnoea hyponoea index (events/hour) and arousal index (ai). data were analysed via mathematical modelling and unpaired t test. results brs fell with increasing ahi. patients with ahi > 30 events/hour (n = 9) had a significantly lower brs (8.1 ϯ 1.5 ms/mmhg) than those with ahi < 30 events/hour (19.8 ϯ 8.7 ms/mmhg, p < 0.001). brs was negatively related to both ahi and ai via fitted exponential functions (r 2 = 0.45 and 0.70, respectively). it is hypothesized that the analysis of morphology of the ecg waveform in combination with the heart rate patterns could lead to the possibility of detection of the start and duration of apnoea/hypopnoea events and consequently estimation of the apnoea-hypopnoea index (ahi). to the authors' knowledge the published ecg based algorithms for detecting sleep disordered breathing are only capable of minute by minute analysis rather than detection of individual respiratory events. methods changes to ecg parameters were investigated during respiratory events with no distinction made between apnoea and hypopnoea events. 632 isolated respiratory events and 1264 controls of identical duration were obtained from 7 polysomnographic studies, using a randomized procedure. features such as the r wave amplitude, t wave amplitude, qrs area and the r-r interval were extracted from the 2 lead ecg. a number of physiological predictors based on these features were generated. a logistic regression model was used to investigate the association between the predictors and true events, using the statistical software, stata. results univariate and multivariate analyses were performed. three multivariate models were developed; heart parameters only, ecg waveform morphology parameters only and the combinations of the two. the area under the receiver operator characteristic curves (auc) for these models were compared. the best results were obtained with the combination of morphology and heart rate parameters (auc = 0.8858 (0.0078 (sd))) compared to the morphology (auc = 0.8169 (0.0121 (sd))) and heart rate (auc = 0.7195 (0.0103 (sd))) models. the multivariate analysis has shown encouraging results indicating that an algorithm using a combination of heart rate and ecg morphological parameters could potentially be constructed that would enable the determination of individual respiratory events and subsequently an ahi. supported by the arc. introduction sacin and scond are measures of ventilation heterogeneity in acinar and conducting airways, derived from analysis of mbnw. maintaining tidal volumes of 1 l at 9-11 breaths/minute (bpm) is impossible for some. our aim was to examine the effect of different tidal volumes on sacin and scond in normals and asthmatics. methods 10 normals (23-41 yrs) and 12 asthmatics (21-63 yrs) underwent mbnw at tidal volumes of 500 ml at 20-23 bpm, 1 l at 9-11 bpm, and 2 l at 5-7 bpm. scond and sacin, were determined from the normalized phase iii slopes of breaths between turnovers (cumulative ventilation/frc) 1.5 & 6. results the mean ϯ sd %predicted fev1 was 97.3 ϯ 17% in normals and 88 ϯ 11% in asthmatics. in normals, sacin at tv of 0.5, 1 and 2 l were 0.195 ϯ 0.105 l -1 , 0.095 ϯ 0.036 l -1 and 0.058 ϯ 0.031 l -1 , respectively (p = 0.0003, anova), while scond were 0.098 ϯ 0.047 l -1 , 0.042 ϯ 0.021 l -1 and 0.029 ϯ 0.014 l -1 (p = 0.0002), respectively. in asthmatics, sacin were 0.440 ϯ 0.195 l -1 , 0.181 ϯ 0.087 l -1 and 0.100 ϯ 0.047 l -1 , respectively (p < 0.01), while scond were 0.204 ϯ 0.111 l -1 , 0.068 ϯ 0.037 l -1 and 0.031 ϯ 0.013 l -1 , respectively (p < 0.0001). conclusion increasing tidal volume while maintaining the same minute ventilation during mbnw led to large decreases in scond and sacin in both asthmatics and normals. this may be due to reduced inter-regional differences in specific ventilation with greater tv. the log-log relationship between sacin and tv allows an adjustment to be made for variations in tidal volume. funding crc for asthma and airways and nhmrc project grant #547346. dj smith 1 , k bowden 2 , t lloyd 2 , j coucher 2 , l garske 1 1 respiratory medicine, and 2 radiology, princess alexandra hospital, brisbane, australia introduction we have shown diaphragmatic flattening and decreased diaphragmatic excursion qualitatively assessed on ultrasound is strongly predictive of dyspnea severity and lower lung inflation in patients with pleural effusion. we sought to quantitatively measure diaphragm length and movement and determine how closely these are related to dyspnea severity and lung inflation. methods patients with unilateral pleural effusions had ct imaging of their diaphragm during a measured inspiratory capacity manoeuvre. maximal sagittal length was measured at tlc, and frc. patients had a baseline dyspnea index (bdi: 0-12) and respiratory function measured. results 4 patients with unilateral effusion (all right side; 3 malignant mesothelioma, 1 inflammatory) had a mean (sd) bdi of 5.5 (2.89), and tlc of 74% (3.91) predicted. the right diaphragm on the side of the effusion tended to be shorter than the left at frc (p = 0.08), and had a trend to reduced shortening with inspiration (p = 0.08). conclusions the right diaphragm is known to be longer than the left in health. the strong trend to a shorter and less mobile right diaphragm associated with effusion suggests this is a potential mechanism for dyspnea. further recruitment will enable correlation between bdi, tlc and diaphragm length and mobility. 4) ) that was slightly worse than an able bodied, control population (17.9 (3.1)), but better than an able-bodied population with untreated osa (14.5 (3.6)). the mapi predicted that 14% of the sample were likely to have osa. these data will be complimented by full sleep studies to be performed at the participants' homes in late 2007, early 2008. conclusion our interim data suggest that the rate of subjective sleep complaints are not substantially different in the population with tetraplegia compared with the able-bodied. this research was supported by the victorian neurotrauma initiative. it has long been assumed that the ventilation heterogeneity associated with lung disease could in itself affect the measurement of carbon monoxide transfer factor. the aim of this study was to investigate the potential estimation errors of carbon monoxide diffusing capacity (tlco) measurement that are specifically due to conductive ventilation heterogeneity. we induced conductive airway ventilation heterogeneity in 35 never-smoker normal subjects by histamine provocation, and related the resulting changes in ventilation heterogeneity (derived from the multiple breath washout test) to corresponding changes in diffusing capacity, alveolar volume and inspired vital capacity (derived from the single breath tlco method). average conductive ventilation heterogeneity doubled (p < 0.001), while tlco decreased by 6% (p < 0.001), with no correlation between individual data (p > 0.1). when dividing diffusing capacity by alveolar volume, the resulting transfer coefficient was not significantly different pre versus post histamine (p = 0.074). these findings can be brought in agreement with recent modelling work, where specific ventilation heterogeneity resulting from different distributions of either inspired volume or end-expiratory lung volume have been shown to affect tlco estimation errors in opposite ways. the combination of these errors appears to largely cancel out in our experimental situation of induced ventilation heterogeneity comparable to that observed in lung disease. we conclude that conductive ventilation heterogeneity per se has a negligible effect on diffusing capacity measurement. an important determinant of airway function in humans is vagal-mediated cholinergic tone in airway smooth muscle (asm). this airway tone may be altered in disease states. the use of mouse models for the study of airway diseases, including asthma, pulmonary fibrosis and copd is well established. however, it is not known whether mice actually possess basal asm tone or, if it does exist, how this tone changes in disease models. this study was undertaken to determine whether mice have detectable asm tone in vivo. methods respiratory system impedance (zrs) was measured in female adult balb/c mice using a wave-tube modification of the forced oscillation technique. zrs was measured during slow (~35 s) inflation-deflation manoeuvres between the transrespiratory pressures of 0 and 20 cmh2o. baseline lung mechanics and thoracic lung volumes (tgv) were measured before and after each mouse was allocated to one of four treatment groups: 'saline' mice received an i.p injection of saline, 'atropine' mice received i.p. atropine sulphate, 'vagotomy' mice had their left and right cervical vagus nerves isolated by blunt dissection and cut, and 'sham' mice had the area of the vagus nerves exposed but the nerves were not cut. results there were no post-treatment changes in tgv, airway resistance, tissue damping, tissue elastance, inertance or tissue hysteresivity in any of the four groups. conclusions the lack of change in lung mechanics post-atropine or postvagotomy in balb/c mice suggests that, unlike humans and many other species, the airways of mice have no baseline asm tone. supported by nhmrc grant#11488. nomination none. conflict of interest none. both male gender and increased mandibular enclosure volume predict more severe sleep disordered breathing in obstructive sleep apnoea patients. we now examine gender/body size/mandibular enclosure volume relationships for normal subjects stepwise multiple linear regression analysis was used to model body size/enclosure volume interactions. results for the whole group, mv was 261.1 ϯ 6.0 ml (mean ϯ se) while rmv was 205.1 ϯ 4.9 ml. head circumference (positive) and forehead height (negative) were both independent predictors for mv and rmv (both p < 0.02), while hip circumference was an additional positive predictive factor for rmv (p < 0.04). after adjusting for these parameters, male mv and rmv were larger than for females conclusion these findings suggest that mandibular enclosure volumes are relatively larger in males, even after adjusting for body size/cranial dimension. differing body size/mandibular enclosure volume interactions may contribute to gender influences on the severity of sleep disordered breathing. supported by nhmrc of australia nomination john read prize for sleep and physiological research tp 027 audit of ctpa in a regional hospital y raje, s vincent, g simpson department of thoracic medicine, cairns base hospital, cairns, qld 4870 since the introduction of computerized tomographic pulmonary angiograms (ctpa) at our institution the number of requests for this investigation at our institution has grown at an alarming rate. the purpose of this study was to evaluate the clinical assessment of suspected pulmonary embolism (pe). methods 50 ctpa were reviewed. results 31 female, 19 male. mean age 50 yrs (range 21-87). 26 ctpa requests came from department of medicine, 21 from emergency department, 2 from surgical teams and 1 from oncology outpatients. 36 patients presented with chest pain (pleuritic in 20 cases), 25 had dyspnea, 7 presented with collapse. 4 patients had haemoptysis. hypoxaemia was recorded in 7. none were clinically shocked and only one had a recorded tachycardia. d-dimer requested in 10 patients and was elevated in 9. arterial blood gases performed in only 10 patients (20%). 47 patients had prior chest x-ray which was normal in 24 (48%). 8 patients had consolidation on chest x-ray, 2 pleural effusions, 2 atelectasis and 1 fractured ribs. recorded risk factors included 4 patients with previous dvt or pe, 4 patients with malignancy and 6 patients were immediately post-operative. only 6 ctpas (12%) demonstrated evidence of pe. of these 2 had recent dvt and 2 were post-operative. 1 had a history of bowel cancer. there was no formal record of pre-test clinical probability of pe (eg wells' score) for any of the 50 cases. retrospective calculation of the cases of pe, 4 had a wells' score of 4.5 and 1 of 4 with the remaining patient with wells' score of under 2. only 3 patients (one with clinically probable pe) had received fractionated heparin prior to the ctpa. conclusion (1) ctpas performed at our institution have a low yield (12%).(2) pre-investigation clinical assessment was poor and there was poor adherence to published guidelines, (3) this results in many unnecessary ctpa examinations generating increased work and expense for the medical imaging department and exposes many patients to unnecessary and potentially harmful radiation exposure. the evaluation and management of hereditary hemorrhagic telangiectasia involves a multidisciplinary approach according to international guidelines. the aim of this audit was to compare the assessment process in one centre with that of the international recommendations. methods retrospective comparison was made by medical chart review of all patients with a diagnosis of hht between the years 1994 to 2006. demographic along with clinical data with diagnostic investigations, complications, treatment and genetic evaluation, including family screening was collected. the proportion of patients evaluated and managed as per the international recommendations was determined. results the audit identified 26 patients with the diagnosis of hht, with the mean age 58 years. diagnostic criteria were met in 77% of the cohort. of the known clinical features, 54% had a family history, and 81% epistaxis. cutaneous telangiectasia was present in 85% and visceral involvement in 92%. pulmonary arterio-venous malformations (pavm) were seen in 16 patients, cerebral avm in 4, gastrointestinal telangiectasia was documented in 8. one patient had a spinal (cervical) avm, and another had pulmonary hypertension in association with this condition. only 8 patients underwent diagnostic or screening investigations in accordance with the international recommendations. furthermore, one patient was referred for a genetic evaluation. conclusions this clinical audit found that 31% of patients referred to this centre were evaluated in accordance with the international recommendations. genetic assessment was lacking. the study supports the need for a coordinated, multidisciplinary approach to the evaluation and management of hht in this centre. lm young 1 , n good 1 , d milne 2 , w fergusson 1 , i zeng 1 , j kolbe 1 , ml wilsher 1 background while airflow limitation is the most common physiological impairment in sarcoidosis, there are limited data on airway hyperresponsiveness (ahr). understanding the role of ahr in sarcoidosis, if any, may help to identify individuals who might benefit from inhaled therapies. aims (1) to determine the prevalence of ahr in sarcoidosis. (2) to determine the correlation between responses to direct (using histamine) and indirect (using hypertonic saline) bronchial challenge. (3) to determine the clinical, physiological and radiological predictors of ahr. methods subjects with a diagnosis of sarcoidosis based on typical clinical presentation and compatible hrct features and/or tissue biopsy and with a baseline fev1>35% predicted were recruited. subjects underwent standard hypertonic (15% fall in fev1) and histamine (20% fall in fev1) challenge (>1 day but <7 days apart), lung function testing and high resolution computed tomography (hrct) of the chest. results the 52 subjects (48 ϯ 11 years, 35% female, 92% european, 35% stage i, 25% stage ii, 40% stage iii, 0% stage iv) had well preserved lung function overall (fev1 = 2.8l ϯ 0.7.87% predicted). ahr was detected in 5/47 (11%) to hypertonic saline and 19/43 (44%) to histamine challenge. on univariate analysis, response to histamine challenge was predicted by conglomerate fibrosis (p = 0.002) and reticular pattern (p = 0.05) on hrct. the baseline % predicted fev1 was significantly associated with ahr on univariate (p = 0.004), and multivariate analysis (p = 0.01) when adjusted by hrct patterns. conclusions there is a high prevalence of ahr using histamine challenge in this study of sarcoidosis subjects. ahr most strongly associates with baseline % predicted fev1 but also conglomerate fibrosis and reticular pattern on hrct. these findings may reflect the consequence of airway remodelling following inflammation. further studies are warranted to confirm these findings. background upper airway shunt represents a significant source of measurement artefact in the use of the forced oscillation technique (fot), with increasing importance in young children. changes in respiratory system admittance, ars (or zrs -1 ), are theoretically independent of the upper airway shunt. this study examines the possible clinical benefit of ars in preschool children by assessing any increased ability to differentiate responses to bronchial challenges in the routine clinical setting. we hypothesized the use of ars would provide improved sensitivity to clinically relevant obstruction, bronchodilator responsiveness (bdr) and airway hyper-responsiveness (ahr) in young children with respiratory disease. method previous fot measurements were re-analysed and ars calculated to derive: (1) ars reference equations in healthy young children (n = 158); (2) bdr in ars, respiratory system resistance (rrs) and reactance (xrs) in healthy children (n = 78), children with cystic fibrosis (n = 39), neonatal chronic lung disease (n = 49), asthma (n = 56) and wheeze (n = 66); (3) ahr to inhaled adenosine-5′-monosphate (amp) in 19 children. fisher's exact tests were used to assess changes in diagnostic outcomes between ars and conventional fot outcomes (rrs and xrs). results ars was no more sensitive to bronchodilator induced changes than conventional fot outcomes. amp challenges resulted in equivalent responses measured by relative changes in rrs and ars while absolute changes in ars were the least sensitive variable. conclusion this study does not support a clinical advantage in using ars in measuring responses to either inhaled bronchodilator or amp. c hollier 1,2 , c menadue 1,2 , d flunt 1,2 , aj piper 1,2 1 department of respiratory and sleep medicine, royal prince alfred hospital, nsw 2050, and 2 woolcock institute of medical research, nsw 2050 serial measurement of arterial carbon dioxide (paco2), ph and bicarbonate (hco3 -) is essential in the management of patients with hypercapnic respiratory failure (hrf). this information is usually obtained from a sample of arterial blood (abg). the procedure can be painful and distressing for patients, and is sometimes technically difficult due to obesity or contractures. our aim was to determine the validity and feasibility of arterialized venous blood (av) sampling as an alternative to abgs in measuring paco2, ph and hco3levels in patients with chronic hrf. method eighteen patients completed the study. venous blood was arterialized by heating forearm skin to a temperature of 42-45°c with an electric heating pad. an av sample was taken from a cannula positioned in a vein of the heated forearm simultaneously with an abg. in addition, the reliability of av sampling within the recommended temperature range (42-45°c) was investigated in ten healthy volunteers placed on volume cycled ventilation in order to maintain constant ventilation. av samples were taken at 0.5°c temperature intervals from 42.5-45°c results the table below summarizes results for validation of av sampling: based on the evidence that cardiovascular dynamics are altered due to obstructive sleep apnea, this study aims to identify the onset and termination of each apnea event using power spectral density (psd) and morphological features of single lead ecg signal over 5 second period. methods ecgs from 4 patients overnight sleep studies were examined for location of the pre-scored apnea events. onset (n = 1995), maximum (n = 6751) and termination (n = 1996) of each apnea event and normal events (n = 11219) were annotated on 5 second windows. features extracted were psd, amplitudes of r and t wave of 5 second ecgs. receiver operating characteristics (roc) analysis was used to gauge the event recognition ability of all features. weight loss causes an improvement in the severity of osa, however substantial weight loss is very difficult for obese patients. the very low caloric diet (vlcd) has been shown to be successful in causing significant weight loss in obese patients. this is a pilot study on the use of a formal screening protocol to identify osa patients who are potentially eligible for the supervised vlcd program offered by the endocrinology department at auckland city hospital. method 344 consecutive patients who attended the sleep laboratory at ach between june to december 2006 were screened using the protocol. patients who are eligible to be considered for the vlcd program are identified as having a combination of obesity (bmi > 30), osa (ahi > 5 on sleep study) and being residents within the auckland district healthboard region. results 243/ 344 patients screened did not fulfil the inclusion criteria: 171 lived outside the adhb region; 71 had bmi < 30; 7 patients did not have osa (ahi < 5). 101 patients fulfilled the inclusion criteria. 54/101 patients (54%) were excluded due to medical or psychiatric contraindications to vlcd.47 patients (47%) who did not have contraindications to vlcd were contacted. 33 patients were contacted successfully. 14 patients were either unavailable to phone contacts on 3 separate days or were disconnected. 12/101 patients consented to being referred (12%). 21/101 patients declined referral (21%). conclusion this pilot study is the first study using a formal comprehensive screening protocol in the recruitment of obese osa patients into a medically supervised vlcd program. only a small proportion (12%) of patients proceeded to being referred to the vlcd program. key: cord-018452-qyf2vymf authors: sica, valentina; izzo, valentina title: pathophysiologic role of autophagy in human airways date: 2016-03-07 journal: autophagy networks in inflammation doi: 10.1007/978-3-319-30079-5_16 sha: doc_id: 18452 cord_uid: qyf2vymf lung diseases are among the most common and widespread disorders worldwide. they refer to many different pathological conditions affecting the pulmonary system in acute or chronic forms, such as asthma, chronic obstructive pulmonary disease, infections, cystic fibrosis, lung cancer and many other breath complications. environmental, epigenetic and genetic co-factors are responsible for these pathologies that can lead to respiratory failure, and, even, ultimately death. increasing evidences have highlighted the implication of the autophagic pathways in the pathogenesis of lung diseases and, in some cases, the deregulated molecular mechanisms underlying autophagy may be considered as potential new therapeutic targets. this chapter summarizes recent advances in understanding the pathophysiological functions of autophagy and its possible roles in the causation and/or prevention of human lung diseases. lung diseases are some of the most common medical conditions in the world. the lung has the principal aim to mediate gas exchange [ 60 ] . for this reason, the lung can be subjected to several insults, belonging to the environment (inspiration of foreign matter, particles, smoke), reactive oxygen species (ros) production, biological origins (e.g., viruses, bacteria), changes in o 2 tension, and mechanical stresses (e.g., mechanical ventilation). it is possible to discriminate between diseases affecting: (i) the airways (asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, acute bronchitis and cystic fi brosis); (ii) the interstitium (sarcoidosis, idiopathic pulmonary fi brosis, autoimmune diseases, pneumonias and pulmonary edemas); (iii) the blood vessels (pulmonary embolism and hypertension); the pleura (pleural effusion, pneumothorax and mesothelioma); (iv) the chest wall (obesity hypoventilation syndrome and neuromuscular disorders). the development of lung diseases can be associated to both acute and chronic exposure to such insults. however, in most conditions, a favouring genetic is necessary [ 60 ] . yet, the lung has various inducible defence mechanisms to protect itself. first, constitutive and inducible stress protein and antioxidant defences; second, innate immune responses; third, pro-and anti-apoptotic mechanisms [ 84 , 85 , 103 ] . several studies have recently pinpointed the emerging role of macroautophagy (more often and hereby referred to as autophagy) in lung homeostasis and diseases. autophagy is a catabolic process that involves the sequential sequestration of cytoplasmic material within double-membraned vesicles (autophagosomes), the fusion of autophagosomes with lysosomes, and the degradation of autophagosomal cargoes (as well as of structural autophagosomal components) by lysosomal hydrolases [ 26 ] . autophagy is mediated by a genetically encoded, evolutionary conserved machinery that is connected to most, if not all, major biochemical processes of the cell, including core metabolic circuitries as well as signal transduction pathways initiated by plasma membrane receptors [ 18 ] . basically, autophagy responds to three major organismal needs: (1) it preserves cellular homeostasis in physiological conditions; (2) it plays a key role in cellular adaptation to stressful stimuli; and (3) it participates in the communication of states of the danger to the whole organism [ 21 ] . indeed, autophagy continuously operates to mediate the disposal of potentially dangerous structures that may otherwise accumulate in the cytoplasm as a consequence of normal cellular activities, like old (and damaged) organelles or protein aggregates [ 64 ] . moreover, the autophagic fl ux is highly responsive to situations in which intracellular or extracellular homeostasis is perturbed, which generally involves either an increased offer of autophagic substrates (as it occurs in the course of viral infection) or an increased need for autophagic functions or products (as it occurs in response to nutrient deprivation) [ 90 ] . in both these settings, profi cient autophagic responses are required for the optimal adaptation of cells to stress, as demonstrated in experiments involving pharmacological inhibitors of autophagy or the depletion of essential components of the autophagic machinery [ 46 ] . finally, autophagy is required for cells experiencing so-called "oncogenic stress" (i.e., the boost of cellular functions driven by activating mutations in one oncogene or loss-of-function mutation in one tumor suppressor gene) to become senescent (a cell-intrinsic oncosuppressive mechanism) while secreting immunostimulatory cytokines and expressing on their surface ligands for activatory natural killer (nk)-cell receptors (hence triggering a cell-extrinsic mechanism of tumor suppression) [ 55 ] . along similar lines, cancer cells succumbing to a peculiar form of apoptosis known as "immunogenic cell death" are able to recruit antigen-presenting cells and hence trigger an adaptive immune response only if they secrete atp as they die, a process that requires profi cient autophagic responses [ 42 , 45 ] . it should be noted that autophagy has also been causally implicated in some instances of cell death, especially in lower organisms like drosophila melanogaster [ 13 , 17 ] . however, in mammals autophagy mainly mediates robust cytoprotective functions, and -when cellular homeostasis is irremediably compromised -contributes to the maintenance of organismal homeostasis by playing a role in danger signalling. in line with this notion, defects in the autophagic machinery have been associated with a wide panel of human pathologies, including (but not limited to) malignant diseases, neurodegenerative disorders, as well as cardiovascular, renal and pulmonary conditions [ 86 ] . an accurate description of the autophagy pathway and its role in immunity and infl ammation has been provided in several previous chapters of this book; therefore, here we will focus on the impact of autophagic in the etiology and treatment of human pulmonary diseases. acute lung injury (ali) and the acute respiratory distress syndrome (ards) describe clinical syndromes of acute respiratory failure with substantial morbidity and mortality. ali is characterised by acute infl ammation that causes disruption of the lung endothelial and epithelial barriers. the ali cellular features include loss of alveolar-capillary membrane integrity, excessive transepithelial neutrophil migration, and release of pro-infl ammatory, cytotoxic mediators. the treatment of ali is predominantly based on ventilatory strategies [ 35 ] . however, prolonged exposure to high oxygen therapy (hyperoxia) can result in lung injury [ 7 ] . few studies are present in the literature concerning the role of autophagy in ali, even so these works support the hypothesis that activation of autophagy has a protective role in this disease. it has been demonstrated that prolonged hyperoxia, which causes characteristic lung injury in mice, induced the increase of lc3ii expression. moreover, in pulmonary epithelial cells, the genetic depletion of lc3 sentitizes the cells to hyperoxia-induced cell death suggesting that lc3 activation confers cytoprotection in oxygen-dependent cytotoxicity [ 93 ] . besides, the involvement of mitophagy has also been identifi ed. the ability to resist hyperoxia is proportional to pten-induced putative kinase 1 (pink1) expression. in fact, the pink1 −/− mice were more susceptible to hyperoxia when compared to wild-type mice. furthermore, genetic deletion of pink1 or pink1 silencing in the lung endothelium cells increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis and oxidant generation [ 108 ] . chronic obstructive pulmonary disease (copd) is a chronic infl ammatory lung disease that causes breathing diffi culty, cough, sputum production and dyspnoea. emphysema and chronic bronchitis can contribute to copd development. emphysema is a condition resulting from a severe damage of air sacs (the alveoli). chronic bronchitis is due to infl ammation of the lining of the bronchial tubes. the lung damage that leads to copd is caused by long-term exposure to irritating gases or particulate matter, most often from cigarette smoke (cs), air pollution or workplace exposure to dust, smoke or fumes. however, a genetic susceptibility to the disease should be considered as an important cofactor. patients with copd present increased risk of developing other pathologies, such as heart disease or lung cancer [ 53 ] . multiple molecular mechanisms, not fully understood, participate to the copd evolution and, among others, the involvement of the autophagic pathway has been pointed out [ 3 , 86 ] . in lung tissue from copd patients, an increase of autophagic vacuoles as well as several autophagy markers (lc3, atg4, atg5/12, atg7) expression has been detected [ 8 ] . these evidences are perhaps a result of defective autophagic fl ux. to corroborate this hypothesis, an increased accumulation of p62 and ubiquitinated proteins and a decreased expression levels of sirtuin 6 (sirt6) have been evaluated in lung homogenates from copd patients [ 92 ] . kuwano and colleagues hypothesize that the insuffi cient autophagic clearance is involved in the accelerated cell senescence observed in copd [ 16 , 92 ] . the cs induces mitochondrial damage, accompanied by increased ros production in vitro . the cs-induced mitophagy was inhibited by pink1 and park2 knockdown, resulting in enhanced mitochondrial ros production. moreover, a decreased expression of park2 in copd lungs compared with non-copd lungs has been detected, suggesting that insuffi cient mitophagy is a part of the pathogenic sequence and cellular senescence of copd [ 32 ] . in addition, a defective xenophagy has been observed in alveolar macrophages of smokers, suggesting that the deregulation of this selective process may contribute to recurrent infections [ 65 ] . in contrast, other fi ndings indicate that autophagy has an opposite role in copd favouring the pathological environment. it has been shown that rtp801 (also known as redd1) expression is increased in human emphysematous lungs and in lungs of mice exposed to cs, whereas rtp801 knockout mice were protected against acute cs-induced lung injury. rtp801 inhibits mammalian target of rapamycin (mtor), by stabilizing the tsc1-tsc2 inhibitory complex. the inhibition of mtor is linked to autophagy induction, but rtp801 expression enhances oxidative stress-dependent cell death, amplifying the development of cs-induced lung injury [ 105 ] . furthermore, the higher expression of autophagy proteins has been linked to lung epithelial cell death, airway dysfunction and emphysema in response to cs. genetic depletion of lc3b in vivo ( map1lc3b −/− mice) suppressed cell death and emphysematous airspace enlargement during chronic cs exposure compared to the wild type mice [ 9 ] . more recently, the same group demonstrated that mitophagy regulates necroptosis, which contributes to the copd pathogenesis. mice defi cient for pink1 were protected against mitochondrial dysfunction, airspace enlargement and mucociliary clearance (mcc) disruption during cs exposure [ 63 ] . interestingly, they identifi ed the contribution of a novel selective autophagy-dependent pathway that regulates cilia length, "ciliophagy", in the copd pathophysiological evolution. exposure to cs reduced cilia length and autophagy-impaired ( beclin 1 +/− or map1lc3b −/− ) mice resisted to the cs-induced cilia shortening via a mechanism involving histone deacetylase 6 (hdac6) [ 48 ] . accordingly, it has been shown that autophagy negatively regulate ciliogenesis by the degradation of the essential ciliary protein ift20 [ 70 ] . conversely, hedgehog (hh) signalling from primary cilia promotes autophagy [ 70 ] and autophagy promotes ciliogenesis by degrading ofd1 (oral facial digital syndrome) at centriolar satellites [ 95 ] . further studies are necessary to clarify the dual relationship between these processes [ 101 ] . in conclusion, these studies illustrate that the contribution of autophagy in copd pathophysiology is complex and show a context-specifi c role depending on the cell type and tissue as well as on the different stimuli involved. interstitial lung disease (ild) is a general category that includes all lung diseases affecting the interstitium, the tissue and space that extends throughout both lungs. among them the most common are sarcoidosis and idiopathic pulmonary fi brosis (ipf). sarcoidosis is a systemic infl ammatory disease caused by persistent reaction toward a stimulus (virus or antigens) that continues even when it is physiologically cleared from the body. lung interstitium fi brosis is the fi rst symptom in patients with sarcoidosis. conversely, ipf is characterized by specifi c fi brosis at interstitial level due to the increased extracellular matrix (ecm) protein deposition and hyper activation of myofi broblasts [ 10 ] . recently, reduced lc3ii expression and p62 accumulation has been found in lung tissue from ipf patients [ 72 ] . the reduced expression of the transcription factor foxo3a in ipf fi broblasts could be the cause for the reduction in the levels of lc3 protein as the expression of this latter is positively stimulated by foxo3a [ 30 ] . furthermore, in fi broblast of ipf patients, decreased expression in beclin-1 protein and increased expression of the anti-apoptotic protein bcl-2 have been found, confi rming a defect in the autophagy pathway at different level [ 81 ] . moreover, fi broblastic foci (ff), that are the starting point for fi brogenesis, are enriched in ubiquitinated proteins and p62, confi rming the insuffi cient autophagy at the basis of ipf pathogenesis [ 3 ] . autophagy inhibition is able to induce acceleration of epithelial cell senescence and fi broblast to myofi broblast differentiation (fmd), which have a critical role in ipf development [ 3 ] . transforming growth factor-β1 (tgf-β1) is one of the essential mediators of fi brosis since it stimulates fi broblasts to produce fi bronectin and the smooth muscle-α actin (α-sma), which is a myofi broblast marker. autophagy has been associated to fi brosis through tgf-β1. in fact, genetic deletion of lc3 or beclin 1 increases tgf-β1 activity as well as in vivo treatment with rapamycin can protect from fi brosis [ 72 ] . tgf-β1 expression seems to be dependent on il-17a, a proinfl ammatory cytokine involved in chronic infl ammation and autoimmune disease. blocking il-17a might reduce the progression of fi brosis promoting the autophagic degradation of collagen [ 61 ] . recently, lacking of matrix metalloproteinases-19 (mmp-19) has been associated with exacerbated fi brosis in the hyperplastic alveolar epithelium of ipf lungs [ 106 ] . additionally, mmp-19-defi cient mice exhibit diminished atg4c protein expression, demonstrating a direct correlation between these two pathways [ 33 ] . similar evidences from an independent group corroborate the role of autophagy in promoting fmd. in fact, atg4b-defi cient mice exhibited reduction in autophagic activity in lungs, collagen accumulation and increased protein levels of the myofibroblast biomarker α-sma [ 6 ] . pharmacological treatment with the alkaloid barberine has been proposed for ipf monitoring because of its capacity to inhibit the activation of mtor and to increase the expression of lc3 and beclin 1 in an bleomycin in vivo model of airway-fi brosis [ 11 ] . furthermore, the multiple tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of ips for its anti-fi brotic effect. it has been shown that nintedanib is able to reduce the expression of ecm proteins, fi bronectin and collagen as well as to induce a beclin 1 dependent, atg7 independent autophagy [ 76 ] . asthma is a chronic respiratory disease affecting 300 million people worldwide. asthma manifests through several symptoms including wheezing, breathlessness, and chest tightness. asthmatic airways are characterized by chronic infl ammation, eosinophil infi ltration, epithelial fi brosis, mucus hyperproduction, and goblet cell hyperplasia [ 20 ] . it is considered as chronic allergic infl ammatory disease, mostly mediated by a th2 response, but an initial th1-type immune response seems to be the trigger for the subsequent th2-type response [ 82 ] . thus, th2 hyperactivation leads to persistent airway infl ammation and the occurring of asthma phenotype [ 38 ] . emerging evidences suggest that activation of autophagy is associated with reduced lung function in asthmatic patients. in particular electron microscopy analysis of fi broblast and epithelial cells from asthmatic patients showed increased autophagic hallmarks "such as double membrane autophagosomes" compared to healthy patients [ 75 ] . unfortunately, at present, the role of autophagy in asthma is still unclear. a recent study demonstrated that two single nucleotide polymorphisms (snps), namely rs12201458 and rs510432 were associated with childhood asthma. in particular rs510432 localises at the promoter of atg5 gene and could increase its expression in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic patients [ 58 ] . another intronic snp variant (rs12212740) in atg5 gene was also shown to be associated with pre-bronchodilator forced expiratory volume in 1 second (fev1) in asthmatic patients [ 75 ] . atg5 is an essential player in the initiation of autophagy, but its role in asthma pathogenesis is controversial. on one hand atg5 could help viral elimination through the activation of xenophagy, and on the other hand it negatively regulates the antiviral properties of type i interferon (ifn) inhibiting innate anti-virus immune responses [ 36 , 90 ] . together with these fi ndings, lungs from conditional atg7 knockout mice manifest hyper-responsiveness to cholinergic stimuli, which is a common sign of asthma and chronic infl ammatory diseases [ 31 ] . asthma severity has been directly correlated with the level of autophagic response in the sputum granulocytes, peripheral blood cells and peripheral blood eosinophils of severe and non-severe asthmatic patients [ 5 ] . autophagy is also involved in the maintenance of intracellular ros homeostasis, and it has been well established that oxidative stress is associated with asthma so that exhaled levels of hydrogen peroxide (h 2 o 2 ) and nitric oxide (no) are currently used as predictors of asthma severity [ 68 ] . chronic asthma is characterized by excessive ecm deposition and proliferation of myofi broblasts, leading to fi brosis in the airway wall [ 79 ] . the accumulation of fi brotic tissue is mostly due to the production of collagen a1 and fi bronectin by the primary human airway smooth muscle through a mechanism autophagy-dependent that involves the tgfβ1. this response is reverted by the silencing of the major key autophagy-inducing gene atg5 and atg7 [ 104 ] . as already mentioned, asthma is a pathology mostly driven by th2-type cytokines. among them, il-13 is extensively produced in activated cd4 + th2 lymphocytes and is overexpressed in the airway epithelium of asthmatic patients [ 47 ] . here, il-13 is thought to be responsible for epithelial hypertrophy, mucus hypersecretion, adventitial fi brosis and goblet cell hyperplasia [ 111 ] . it directly induces hypersecretion of mucin 5ac, oligomeric mucus/gel forming (muc5ac) in airway epithelial cell and oxidant stress through a mechanism that is autophagy-dependent, as demonstrated in vitro by depletion of atg5 or atg14 in primary human trachealbronchial epithelial cells [ 15 ] . autophagy might be involved in the pathophysiology of alternaria (alt-e)associated asthma. alt-e is an outdoor allergen able to activate autophagy, which in turn stimulates epithelial cells to release il-18 [ 67 ] . this latter when produced is able to stimulate th2 differentiation from naïve cd4 + t-cells and ifn-γ production by th1 cells. il-18 level in serum of asthmatic patients might refl ect the degree of disease exacerbation [ 94 ] . cystic fibrosis (cf) is one of the most common lethal genetic diseases in caucasian population. it is an autosomal recessive disease caused by mutation in the cystic fibrosis transmembrane conductance regulator (cftr) gene. approximately 1 out of 20 caucasians are carriers for mutation in this gene. up to date over 2000 types of different mutations have been discovered and classifi ed according to the degree of functional cftr protein ( http://www.genet.sickkids.on.ca/statisticspage. html ; [ 27 ] ). among these, the most common one is the f508del-cftr. approximately 90 % of cf patients have at least one f508del-cftr allele, and about 70 % are homozygous for it. the cftr channel is located at the apical surface of epithelial cells and it is deputized to move out cl − from the cell. na + passes through the membranes passively, increasing the movement of water by osmosis. loss of functional cftr expression is thought to alter this homeostatic balance through the epithelial layer, leading to net volume depletion of mucus, increased viscosity, and ineffective bacterial clearance [ 43 , 78 ] . recurrent pulmonary infections in turn induce an increased infl ammatory response and signalling, thus starting a vicious cycle of mucus retention, infection, and infl ammation. since the cftr is localized in many organs, cf symptoms could go from malabsorption at pancreatic level and gastrointestinal obstruction to male infertility and liver disease. nevertheless, the main cause of death remains persistent and untreatable pulmonary pseudomonas aeruginosa infection. several recent studies have demonstrated an impairment of autophagy in cf. in fact, in epithelial cells, mutated/unfunctional cftr causes increased ros production with consequent increase in tissue transglutaminase type 2 (tg2) levels. tg2, in turn, leads to crosslinking of several targets including beclin 1 [ 54 , 57 ] . beclin 1 interactome displaces from the endoplasmic reticulum (er) leading to the sequestration of class iii-phosphoinositide 3-kinase (pi3k) complex, accumulation of p62 with consequent inhibition of autophagosomes formation. the resulting accumulation of aggresomes leads to proteasome overload and may promote the accumulation of mutated cftr in intracellular aggregates [ 54 ] . restoration of beclin 1 activity, depletion of p62 by genetic manipulation or treatment with autophagy-stimulatory proteostasis regulators, such as cystamine, functionally rescue the cftr mutated protein at the apical surface of epithelial cells both in vitro and in vivo [ 54 ] . heme oxygenases are enzymes involved in the catabolism of the heme ring to generate carbon monoxide, biliverdin-ixα, and ferrous iron. the inducible isoform heme oxygenase-1 (ho-1) is activated in response to stress such as oxidative stress, hypoxia, heavy metals exposure and cytokines. ho-1, together with its enzymatic products, is able to inhibit apoptosis and related cell death pathways, conferring tissue protection in case of lung or vascular injury [ 66 ] . ho-1 could represent the link between cf and impaired autophagy since its expression is increased in human bronchial cf cells. this increase has been associated either to the reduction of apoptosis/injury during p. aeruginosa challenge either to the expression of infl ammatory mediators [ 109 ] . other evidences suggesting the cytoprotective role of ho-1 in cf showed that lipopolysaccharide (lps)-challenged cf macrophages fail to compartmentalize ho-1 to the cell surface and this mechanism seems to be dependent on the reduction in caveolin-1 (cav-1) expression [ 107 ] . in fact, when ho-1 localises at the plasma membrane, is able to form a complex with cav-1, which in turn binds and detaches myd88 from its complex with tlr4 thus terminating the cell death signal [ 99 ] . autophagic clearance of bacteria (so-called xenophagy) could also be impaired in case of disease, inducing increased bacterial infection that is one of the most frequent injuries in cf patients [ 90 ] . in fact it has been demonstrated that burkholderia cenocepacia has the capacity to survive in f508del-cftr macrophages since immediately after the engulfment, the bacteria resides on lc3-positive vacuoles that appear as arrested autophagosomes [ 98 ] . this capacity is directly correlated to the levels of p62, so that its depletion leads not only to a decreased bacterial survival in macrophages but also to the release of beclin 1 from aggresomes allowing its recruitment to the b. cenocepacia vacuole and bacterial clearance via autophagy [ 2 ] . b. cenocepacia represents a serious threat for cf patients since the infection results in persistent lung infl ammation and the bacteria are resistant to most of all available antibiotics [ 1 ] . similar fi ndings showed that pharmacological or molecular inhibition of autophagy reduces the clearance of intracellular pseudomonas aeruginosa in vitro [ 37 ] . treatment of cf mice with the mtor inhibitor rapamycin decreases bacterial burden in the lungs and drastically reduces signs of lung infl ammation [ 1 ] . in a normal situation, autophagy can help not only removing polyubiquitinated protein but also controlling bacteria clearance; for these reasons novel strategies aimed at restoring autophagy are emerging as promising therapeutic approaches for cf patients [ 56 ] . aatd is a hereditary disorder characterized by a low serum level of alpha-1-antitrypsin (aat), a 52 kda serine protease inhibitor, member of the serpin family [ 29 ] . aat is essentially synthetized in the liver and secreted into the bloodstream, where it controls tissue degradation by the enzyme neutrophil elastase. the defi ciency in aat is associated with liver and lung disease due to the loss of anti-infl ammatory and antiproteolytic functions. the majority of patients with aat defi ciency are homozygotes for a missense mutation ("piz mutation": lysine replaces glutamic acid at position 342) that alters protein folding. mutant aat molecules polymerize and aggregate in the er of hepatocytes, forming large intrahepatocytic globules, the characteristic features of this disease. the proteasome is responsible for degrading the soluble form of att by means of er-associated degradation while autophagy is involved in disposal of insoluble att polymers and aggregates [ 74 ] . in fact, a signifi cant accumulation of autophagic vacuoles was found in vitro and in vivo in liver cells from aatd patients as well as in piz mouse model [ 96 , 97 ] . whereas in absence of autophagy the degradation of aat was retarded [ 39 ] . moreover, it has been demonstrated that the stimulation of autophagy by carbamazepine or rapamycin treatment or by liver-directed gene transfer of transcription factor eb (tfeb), a gene regulating lysosomal function and autophagy [ 89 ] , reduce the hepatic amount of aat as well as the hepatic fi brosis in mice expressing mutant aat [ 28 , 41 , 71 ] . although these results should be corroborated, altogether indicate that autophagy exerts a protective role in aatd and open a real possibility to treat aatd with pro-autophagic molecules. pulmonary hypertension (ph) was fi rst identifi ed in 1891 by ernst von romberg. ph is a severe and progressive disease that consists in increased blood pressure of lung vasculature and, often, can be a complication of chronic lung disease [ 88 ] . since 2008 the pathology has been classifi ed, by the world health organization (who), in fi ve groups on the basis of mechanisms underlying the pathogenesis of the multiple types of ph. the role of autophagy in pulmonary hypertension has mainly been described in correlation with pulmonary arterial hypertension (pah), who group i. little is known about the aetiology of ph, one of the most frequent genetic mutations causing idiopathic inherited form of ph is found in the gene encoding bone morphogenetic protein (bmp) receptor type-ii (bmpr2). in pah, the pulmonary artery smooth muscle cells (pasmcs) proliferate excessively and are resistant to apoptosis. chloroquine, a known inhibitor of autophagy fl ux, has been described as a drug preventing experimental pah progression. the induction of pah, by monocrotaline, in rat is associated with increased autophagy and decreased bmpr2 protein expression. the inhibition of autophagy by chloroquine ameliorates the level of bmpr2, inhibits the proliferation and stimulates apoptosis of rat pasmcs [ 52 ] . a recent publication [ 50 ] confi rms that the inhibition of autophagy, by overexpressing mtor, is a promising therapeutic strategy against pah. however, the role of autophagy in ph is still unclear and controversial, in fact, its protective role has been described in the initial phase of the pathogenesis of ph. histochemical analysis of samples obtained from human ph lungs and mouse exposed to chronic hypoxia, showed an increase in the lipidated form of lc3 and in egr-1, which regulates lc3 expression. moreover, lc3 −/− or egr-1 −/− , but not beclin 1 +/− mice are more susceptible to ph and in vitro lc3 knockdown cells showed an increase of hypoxic cell proliferation, suggesting a role for lc3 in the adaptation during vascular remodelling under hypoxia [ 49 ] . in most organs, including the lung, autophagy robustly counteracts malignant transformation, i.e. , the conversion of a healthy cell into a (pre-)neoplastic cell, and several mechanisms related to the ability of autophagy to preserve cellular or organismal homeostasis account for such a pronounced oncosuppressive activity [ 19 ] . indeed, besides being required for oncogene-induced senescence and anticancer immunosurveillance (see above) [ 112 ] , autophagy promotes the maintenance of genomic integrity by multiple mechanisms [ 25 ] . first, it mediates the degradation of damaged mitochondria, which are prone to overproduce genotoxic ros and other redox active entities of endogenous and exogenous origin [ 22 ] . second, profi cient autophagic responses appear to be required for optimal dna damage responses [ 59 ] . third, autophagy is involved in the disposal of potentially oncogenic retrotransposons and micronuclei [ 80 ] . moreover, autophagy generally mediates anti-infl ammatory effects, and chronic infl ammation is known to accelerate oncogenesis (at least in some tissues, including the lung) [ 14 ] . finally, it has been proposed that autophagy is required for the preservation of normal tissue architecture, in particular at the level of the stem-cell compartment [ 23 ] . although little is known on the deregulation of stem cells in pulmonary carcinogenesis, it cannot be excluded that autophagic defects may promote malignant transformation in the lung also via this mechanism [ 69 ] . conversely, the ability of autophagy to preserve genomic and redox homeostasis seems very relevant in the context of lung tumorigenesis, which in a signifi cant proportion of cases is associated with tobacco smoking or exposure to environmental nanoparticles like asbestos crystals [ 65 ] . indeed, the oncogenic effects of both smoking and asbestos have been linked to their ability to cause ros overgeneration along with genetic/genomic defects and chronic infl ammatory responses [ 12 ] . all these effects are limited, at least to some extent, by profi cient autophagic responses. irrespective of the precise mechanisms whereby autophagy counteracts malignant transformation in the lung, various genetic interventions aimed at specifi cally disabling autophagy in the lungs have been shown to promote malignant transformation driven by several oncogenes, including mutated b-raf proto-oncogene, serine/threonine kinase ( braf ) [ 91 ] , epidermal growth factor receptor ( egfr ) [ 100 ] , kirsten rat sarcoma viral oncogene homolog ( kras ) [ 24 , 77 ] . intriguingly enough, in one of these models, accelerated oncogenesis caused by the lung-specifi c inactivation of atg5 was linked to increased tumor-infi ltration by immunosuppressive cd4 + cd25 + foxp3 + regulatory t cells [ 77 ] . moreover, the concomitant bi-allelic inactivation of serine/threonine kinase 11 (stk11, best known as lkb1) and phosphatase and tensin homolog (pten), two tumor suppressor genes that inhibit autophagy [ 34 , 87 ] , has been shown to cause the formation of pulmonary squamous cell carcinomas that express high levels of the immunosuppressive molecule cd274 (best known as pd-l1) [ 102 ] . these latter observations strongly corroborate the notion that autophagy mediates not only cell-intrinsic, but also cell-extrinsic oncosuppression. the capacity of autophagy to preserve cellular homeostasis is benefi cial to healthy cells, but also benefi cial to transformed cells. this implies that autophagy often (but not always) promotes tumor progression, i.e., the growth and evolution of a transformed cells into an ever more malignant cancer [ 62 ] . indeed, malignant cells are often exposed to relatively adverse microenvironmental conditions, including a shortage of nutrients and oxygen (especially in poorly vascularized tumor areas), and autophagy is instrumental for these cells (as it is for their non-transformed counterparts) to cope with stress and proliferate. along similar lines, the ability of autophagy to preserve stemness is benefi cial for the host when it preserves normal tissue architecture, but detrimental when it sustains the malignant stem-cell compartment. finally, autophagy supports the survival of malignant cells in key step of tumor progression, the so-called "epithelial-to-mesenchymal transition" (emt). in this context, epithelial cancer cells "initially growing in situ " physically detach from ecm and become able to colonize surrounding tissues as well as distant organs. the emt is required for all malignancies to become locally and distantly invasive, and critically relies on profi cient autophagic responses [ 4 ] . in the presence of autophagic defects or pharmacological inhibitors of autophagy, indeed, malignant cells undergoing the emt and detaching from the ecm, succumb to a form of regulated cell death often referred to as "anoikis" [ 73 ] . corroborating these observations, the genetic and/or pharmacological inhibition of the autophagic machinery in established tumors has been shown to accelerate disease progression in various models of pulmonary oncogenesis, including (but not limited to) braf -and kras -driven tumorigenesis [ 24 , 77 , 91 ] . autophagy provides malignant cells with an increased resistance to various perturbations of homeostasis, including the lack of nutrient and oxygen that cancer cells normally experience in poorly vascularized tumor areas, as well as the presence of xenobiotics like chemotherapeutic agents and physical stress conditions like irradiation. an abundant amount of literature demonstrates indeed that chemical inhibitors of autophagy as well as genetic interventions that compromise autophagic responses accelerate (rather than inhibit) the demise of malignant cells exposed to a wide panel of chemotherapeutics or to irradiation, both in vitro and in vivo . these observations provided a strong rationale to the development of combinatorial therapeutic strategies involving chemo-or radiotherapy given in combination with an inhibitor of autophagy [ 19 ] . clinical grade highly specifi c chemical inhibitors of autophagy, however, have not yet been developed, and currently available molecules that can be used in the clinic, like chloroquine (a widely employed antimalarial agent) often operate as lysosomal inhibitors, i.e., they target several processes other than autophagy [ 83 ] . moreover, concerns have been raised that inhibiting autophagy at the whole-body level may de facto favor malignant transformation in healthy tissues, refl ecting the prominent oncosuppressive functions of autophagy in physiological conditions [ 51 ] . finally, recent data highlight the differential role of autophagy in cancer therapy in immunocompromised versus immunocompetent hosts [ 44 ] . in this setting, the response to radiotherapy of human non-small cell lung carcinoma (nsclc) or murine colorectal carcinoma (crc) cells xenografted in nude mice was significantly improved when cells were rendered autophagy-defi cient by the stable depletion of atg5 or beclin 1 [ 44 ] . however, when murine crc cells were implanted in immunocompetent syngeneic mice, the stable knockdown of atg5 compromised the therapeutic activity of irradiation, a defect that could be restored (at least in part) by the intratumoral administration of a chemical inhibitor of extracellular atpases [ 44 ] . these fi ndings demonstrate that inhibiting autophagy in immunocompetent hosts may prevent the elicitation of a therapeutically relevant immune response against dying cancer cells. in summary, although autophagy generally (but not always) promote the progression of pulmonary malignancies and increases the resistance of lung cancer cells to chemo-and radiotherapeutic regimens, additional experiments are required to understand whether combinatorial treatments involving autophagy inhibitors constitute a clinically viable approach against pulmonary neoplasms. similarly, further work is needed to clarify whether biomarkers of autophagy such as the expres-sion levels of beclin 1 or the lipidation of lc3 have a positive or negative prognostic/ predictive value in patients with lung cancer, as preliminary results are rather controversial [ 40 , 110 ] . abundant evidences indicate that autophagy actively participates in a wide range of cellular responses to both physiologic-and pathologic-related events in the diverse tissues and cell types that constitute the lung system. nevertheless, much is yet to be learnt about its biological relevance, functional targets, and role in development and disease. as described in this chapter, lungs are the fi rst line of defence against several insults and associated diseases are growing both in number and chronicisation. a clear deregulation of the autophagic machinery has been highlighted in most of the lung diseases, suggesting that this process mainly exerts a defensive role. however, in some pathological contexts, it has been reported that the activation of the autophagic process contributes to damage. as a consequence, a detailed knowledge of the molecular mechanisms at the basis of autophagy in lung pathologies is required for the development of novel diagnostic tools and promising therapeutic strategies. autophagy stimulation by rapamycin suppresses lung infl ammation and infection by burkholderia cenocepacia in a model of cystic fi brosis depletion of the ubiquitin-binding adaptor molecule sqstm1/p62 from macrophages harboring cftr δf508 mutation improves the delivery of burkholderia cenocepacia to the autophagic machinery autophagy in the pathogenesis of pulmonary disease regulation of autophagy during ecm detachment is linked to a selective inhibition of mtorc1 by perk autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target essential role for the atg4b protease and autophagy in bleomycin-induced pulmonary fi brosis oxygen toxicity and tolerance egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease autophagy protein microtubule-associated protein 1 light chain-3b (lc3b) activates extrinsic apoptosis during cigarette smoke-induced emphysema oxidative stress and pulmonary fi brosis berberine inhibits smad and non-smad signaling cascades and enhancesautophagy against pulmonary fi brosis neutralizing tumor-promoting chronic infl ammation: a magic bullet autophagy, not apoptosis, is essential for midgut cell death in drosophila autophagy in infection, infl ammation and immunity il13 activates autophagy to regulate secretion in airway epithelial cells insuffi cient autophagy promotes bronchial epithelial cell senescence in chronic obstructive pulmonary disease essential versus accessory aspects of cell death: recommendations of the nccd metabolic control of autophagy autophagy in malignant transformation and cancer progression asthma and chronic obstructive pulmonary disease to be or not to be? how selective autophagy and cell death govern cell fate mitochondria and the autophagy-infl ammationcell death axis in organismal aging the bone marrow niche, stem cells, and leukemia: impact of drugs, chemicals, and the environment autophagy suppresses progression of k-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis hallmarks of cancer: the next generation regulation mechanisms and signaling pathways of autophagy cystic fi brosis-associated liver disease an autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin z and reduces hepatic fi brosis alpha 1 anti-trypsin: one protein, many functions reduced foxo3a expression causes low autophagy in idiopathic pulmonary fi brosis fi broblasts on collagen matrices inducible disruption of autophagy in the lung causes airway hyper-responsiveness -mediated mitophagy is involved in regulation of hbec senescence in copd pathogenesis matrix metalloproteinase (mmp)-19-defi cient fi broblasts display a profi brotic phenotype lkb1 modulates lung cancer differentiation and metastasis acute lung injury: epidemiology, pathogenesis, and treatment the atg5 atg12 conjugate associates with innate antiviral immune responses autophagy enhances bacterial clearance during p. aeruginosa lung infection autophagy and role in asthma intracellular inclusions containing mutant alpha1-antitrypsin z are propagated in the absence of autophagic activity light-chain 3a autophagic activity and prognostic signifi cance in non-small cell lung carcinomas rapamycin reduces intrahepatic alpha-1-antitrypsin mutant z protein polymers and liver injury in a mouse model consensus guidelines for the detection of immunogenic cell death the molecular basis for disease variability in cystic fi brosis autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to defi cient immunogenic signalling immunogenic cell death in cancer therapy autophagy and the integrated stress response direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma histone deacetylase 6-mediated selective autophagy regulates copd-associated cilia dysfunction autophagic protein lc3b confers resistance against hypoxia-induced pulmonary hypertension mammalian target of rapamycin overexpression antagonizes chronic hypoxia-triggered pulmonary arterial hypertension via the autophagic pathway autophagic and tumour suppressor activity of a novel beclin1-binding protein uvrag chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type ii receptor degradation global burden of copd defective cftr induces aggresome formation and lung infl ammation in cystic fi brosis through ros-mediated autophagy inhibition autophagy and cellular immune responses the holy grail of cystic fi brosis research: pharmacological repair of the f508del-cftr mutation tissue transglutaminase activation modulates infl ammation in cystic fi brosis via ppargamma down-regulation functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma autophagy suppresses tumor progression by limiting chromosomal instability casarett and doull's toxicology: the basic science of poisons blocking il-17a promotes the resolution of pulmonary infl ammation and fi brosis via tgf-beta1-dependent and -independent mechanisms the ampk signalling pathway coordinates cell growth, autophagy and metabolism mitophagydependent necroptosis contributes to the pathogenesis of copd autophagy: renovation of cells and tissues identifi cation of an autophagy defect in smokers' alveolar macrophages heme oxygenase-1, a critical arbitrator of cell death pathways in lung injury and disease alternaria extract activates autophagy that induces il-18 release from airway epithelial cells hydrogen peroxide content and ph of expired breath condensate from patients with asthma and copd autophagy in cancer stem cells: a potential link between chemoresistance, recurrence, and metastasis functional interaction between autophagy and ciliogenesis gene transfer of master autophagy regulator tfeb results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin defi ciency autophagy in idiopathic pulmonary fi brosis autophagy inhibition suppresses pulmonary metastasis of hcc in mice via impairing anoikis resistance and colonization of hcc cells autophagic disposal of the aggregation-prone protein that causes liver infl ammation and carcinogenesis in α -1-antitrypsin defi ciency genetic and histologic evidence for autophagy in asthma pathogenesis novel mechanisms for the anti-fi brotic action of nintedanib a dual role for autophagy in a murine model of lung cancer cystic fi brosis airway fi brosis in asthma: mechanisms, consequences, and potential for therapeutic intervention autophagic removal of micronuclei decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fi brosisfi broblasts predominant th2-like bronchoalveolar t-lymphocyte population in atopic asthma autophagy modulation as a potential therapeutic target for diverse diseases heme oxygenase-1/carbon monoxide: from metabolism to molecular therapy autophagy in the lung autophagy in lung disease pathogenesis and therapeutics the biology and clinical relevance of the pten tumor suppressor pathway oxygen sensing, homeostasis, and disease tfeb links autophagy to lysosomal biogenesis organelle-specifi c initiation of autophagy autophagy sustains mitochondrial glutamine metabolism and growth of brafv600e-driven lung tumors autophagy induction by sirt6 through attenuation of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell senescence hyperoxiainduced lc3b interacts with the fas apoptotic pathway in epithelial cell death il-18 might refl ect disease activity in mild and moderate asthma exacerbation autophagy promotes primary ciliogenesis by removing ofd1 from centriolar satellites fasting in alpha1-antitrypsin defi cient liver: constitutive activation of autophagy retention of mutant alpha1-antitrypsin z in endoplasmic reticulum is associated with an autophagic response intracellular survival of burkholderia cepacia complex in phagocytic cells the heme oxygenase-1/carbon monoxide pathway suppresses tlr4 signaling by regulating the interaction of tlr4 with caveolin-1 egfr-mediated beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance cytoskeleton: autophagy and ciliogenesis come together loss of lkb1 and pten leads to lung squamous cell carcinoma with elevated pd-l1 expression current concepts on oxidative/carbonyl stress, infl ammation and epigenetics in pathogenesis of chronic obstructive pulmonary disease autophagy regulates tgf-beta1 induced fi brosis in human airway smooth muscle cells rtp801, a suppressor of mtor signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema matrix metalloproteinase-19 is a key regulator of lung fi brosis in mice and humans reduced caveolin-1 promotes hyperinfl ammation due to abnormal heme oxygenase-1 localization in lipopolysaccharide-challenged macrophages with dysfunctional cystic fi brosis transmembrane conductance regulator endothelial pink1 mediates the protective effects of nlrp3 defi ciency during lethal oxidant injury heme oxygenase-1 expression in human lungs with cystic fi brosis and cytoprotective effects against pseudomonas aeruginosa in vitro autophagic protein beclin 1 serves as an independent positive prognostic biomarker for non-small cell lung cancer pulmonary expression of interleukin-13 causes infl ammation, mucus hypersecretion, subepithelial fi brosis, physiologic abnormalities, and eotaxin production mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance the authors report no confl ict of interest. key: cord-342150-dadc8whz authors: lindahl, sten g. e. title: using the prone position could help to combat the development of fast hypoxia in some patients with covid‐19 date: 2020-06-17 journal: acta paediatr doi: 10.1111/apa.15382 sha: doc_id: 342150 cord_uid: dadc8whz the world is facing an explosive covid‐19 pandemic. some cases rapidly develop deteriorating lung function, which causes deep hypoxaemia and requires urgent treatment. many centres have started treating patients in the prone position, and oxygenation has improved considerably in some cases. questions have been raised regarding the mechanisms behind this. the mini review provides some insights into the role of supine and prone body positions and summarises the latest understanding of the responsible mechanisms. the scope for discussion is outside the neonatal period and entirely based on experimental and clinical experiences related to adults. the human respiratory system is a complex interplay of many different variables. therefore, this mini review has prioritised previous and ongoing research to find explanations based on three scientific areas: gravity, lung structure and fractal geometry and vascular regulation. it concludes that gravity is one of the variables responsible for ventilation/perfusion matching but in concert with lung structure and fractal geometry, ventilation and regulation of lung vascular tone. since ventilation distribution does not change between supine and prone positions, the higher expression of nitric oxide in dorsal lung vessels than in ventral vessels is likely to be the most important mechanism behind enhanced oxygenation in the prone position. hence, the prone position in infants younger than 6 months is not recommended. however, it may be discussed with regard to toddlers, pre-school and school children who suffer from acute lung insufficiency with deep hypoxaemia to such an extent that it threatens their life. but these age groups are not so well studied. the subject is highly interesting, and it will be a challenge in the future to design investigations that are able to evaluate prone and supine positions in a developing respiratory system. this paper reviews the use of supine and prone positions in adult patients with severe respiratory insufficiency. it aims to reach a conclusion about the mechanisms that explain the sometimes dramatic improvements in oxygenation when these patients are turned prone. but it has been difficult to demonstrate that the prone position used for these sick patients also improves mortality rates. such studies are difficult to perform and interpret, as the nature of the disease and the extremely complex intensive care they receive are hard to control. this makes it hard to evaluate comparable patient groups and collect reliable outcome data. this point was well made in a 2019 paper by gattinoni et al which also highlighted the impact of protective ventilator settings to avoid ventilator-induced lung injuries. 1 guérin et al examined a well-controlled and carefully selected patient population that received intensive care and assessed the mortality rates related to supine and prone positions. the authors concluded that mortality was lower in the group treated prone. 2 the world is currently experiencing an explosive pandemic caused by a new coronavirus, with a clinical presentation of a somewhat different kind. in some cases, patients experience rapidly deteriorating the function of the human respiratory system is a complex interplay of different variables, such as the rib cage, the diaphragm, abdominal distension, pleural pressure, body fluids, heart function, pulmonary and systemic circulation, lung parenchyma, the tracheobronchial tree, alveoli and central and peripheral innervation. all of these act in concert to achieve adequate gas exchange for the maintenance of life. each of them is a theoretical area in its own right, with regard to detailed mechanistic functions. it would not be possible for a review like this to provide a comprehensive overarching presentation of them all. therefore, it was necessary to prioritise key scientific areas for this mini review. that is why the finding on mechanisms and explanations will focus on three scientific areas: gravity, lung structure and fractal geometry and vascular regulation. for many years, pulmonary circulation was defined according to gravity and based on advanced investigations in upright humans. [3] [4] [5] according to these studies, pulmonary circulation was described in three zones. this was due to relationships between pulmonary artery pressure, alveolar pressure and pulmonary venous pressure from apical to basal lung regions with increasing pulmonary perfusion down the lung. in addition, a fourth zone was added for basal lung regions, where conditions allow pulmonary interstitial pressure to exceed pulmonary venous and alveolar pressures. these investigations and physiological interpretations have played an important role in developing our understanding of lung function today. this platform created new knowledge which has, step-by-step, resulted in advanced treatment of the insufficient lung. • gravity is only one variable responsible for ventilation/ perfusion matching and is executed in concert with lung structure and fractal geometry, gas distribution and regulation of lung vascular tone. • the most important mechanism is the higher expression of nitric oxide in dorsal lung vessels than in ventral vessels. chest in 1988. 10 certainly, gravity is of interest for pulmonary circulation and ventilation and influences v/q matching along the vertical axis and, more so, at the longer distances down the lung. to extrapolate the importance of gravity for pulmonary circulation, a study was performed using single photon emission tomography in volunteers subjected to hypergravity of three times normal gravity (3 g). a human centrifuge was used with the subjects in the supine position. interestingly, a paradoxical result was reached, with a shift of dominant perfusion from dependent to independent lung regions when the normal gravitational force (0 g) was changed to 3 g. 16 this paradoxical finding invited to thoughts on lung structure and to the experimental study by beck and rehder 8 using microspheres in isolated perfused dog lungs. they concluded that: the distribution of regional vascular conductances were related to the anatomic location and were not related to gravity, nor were they caused by nonuniformities in regional lung expan like all organs, the anatomy and structure of lungs are genetically determined and are important pre-requisite conditions for function. embryologic development of the lungs is characterised by dichotomous branching of both the airways and vessels. the branching follows a genetically determined geometric pattern, such as angles related to the common stem from which the dichotomous branching originates. also, the development of airways and vessels are in parallel and happens synchronously. this structural arrangement will ensure laminar transport of both gas and blood to, and from, alveoli. altered angulations of the fractal geometry often cause turbulence and increased resistance, which reduces oxygenation and gas exchange ( figure 1 ). fractal geometry of the airways and vessels has, to a large extent, been clarified by findings from the washington school of medicine in seattle. 23, 24 investigations using microspheres found that small 29 single photon emission computed tomography image of pulmonary perfusion in one supine volunteer before (a) and after (b) nos inhibition using l-nmma intravenously. r indicates the right lung the finding that vascular conductance in dogs was greater in dorsal than in ventral lung vasculature clearly indicated that there ought to be vasoactive mechanisms in the lungs that were counteracting gravity. 8 another important observation was in 1996, when hlastala et al, found that lung perfusion in standing horses was not dominantly governed by gravity. 27 this was a valuable step as it narrowed down several possibilities that might constitute the most responsible mechanism for improved v/q matching in the prone position. a next step was to explore whether no production in human lung vasculature could be responsible for improved v/q matching in the prone position. an investigation was designed to challenge the hypothesis that no plays an important role in the regulation of regional lung perfusion. nitric oxide synthase (nos) messenger ribonucleic acid (mrna) and nos activity using citrulline assay were analysed in ventral and dorsal lung tissue samples from patients subjected to lung surgery. 29 in addition, the study also assessed regional lung perfusion in volunteers by single photon emission computed tomography before and after nos inhibition. the hypothesis was supported, and it was found that mrna expression of endothelial nos was higher in dorsal than in ventral lung regions. moreover, it was found that calcium-dependent nos activity in citrulline units was higher in dorsal than ventral lung whereas calcium-independent nos activity was similar in ventral and dorsal regions (figure 2a based on this review, it is concluded that gravity is one of the variables responsible for v/q matching, but in concert with lung structure and fractal geometry, gas distribution and regulation of lung vascular tone. this conclusion is based on a long series of investigations published in leading journals and illustrates the essence of science, where brick is laid on brick in a process of continued development. in view of the unchanged ventilation distribution of prone and supine, it currently seems that the most important mechanism is different regulation of lung vascularity in dorsal and ventral lung regions, due to expression of the potent vasodilator no. this mechanism is also present in horses and in pigs making it likely that the enhanced no production in dorsal lung regions is an evolutionary trait preserved from the time when we walked on four legs. it is very likely that there is more to know, and understand, about the complex lung function which will be further elucidated in future investigations. based on the above findings, it appears that using the prone position to combat the fast development of deep hypoxia in some patients with covid-19 is a useful tool. this could even be used in spontaneously breathing patients with deep hypoxaemia prior to initiation of mechanical ventilation and extra corporeal oxygenation. the author has no conflicts of interest to declare. prone positioning in acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome distribution of blood flow in isolated lung; relation to vascular and alveolar pressures effect of lung volume on the distribution of pulmonary blood flow in man effect of extra-alveolar vessels on distribution of blood flow in the dog lung use of extreme position changes in acute respiratory failure improved oxygenation in patients with acute respiratory failure: the prone position differences in regional vascular conductances in isolated dog lungs effect of posture on inter-regional distribution of pulmonary ventilation in man the prone position in ards patients: a clinical study the prone positioning during general anesthesia minimally affects respiratory mechanics while improving functional residual capacity and increasing oxygen tension prone positioning improves pulmonary function in obese patients during general anesthesia dramatic effect on oxygenation in patients with severe acute lung insufficiency treated in the prone position pulmonary gas exchange improves in the prone position with abdominal distension pulmonary perfusion is more uniform in the prone than in the supine position: scintigraphy in healthy humans paradoxical redistribution of pulmonary blood flow in prone and supine humans exposed to hypergravity fractal properties of pulmonary blood flow: characterization of spatial heterogeneity high-resolution maps of regional ventilation utilizing inhaled fluorescent microspheres pulmonary blood flow distribution has a hilar-to-peripheral gradient in awake, prone sheep pulmonary gas-exchange analysis by using simultaneous deposition of aerosolized and injected microspheres gravity is a minor determinant of pulmonary blood flow distribution gravity is an important but secondary determinant of regional pulmonary blood flow in upright primates pulmonary blood flow remains fractal down to the level of gas exchange regional ventilation-perfusion distribution is more uniform in the prone position posture primarily affects the distribution of lung tissue with minor effect on regional blood flow and ventilation lung ventilation and perfusion in prone and supine postures with reference to anesthetized and mechanically ventilated healthy volunteers pulmonary blood flow distribution in standing horses is not dominated by gravity regional differences in endothelial function in horse lungs: possible role in blood flow distribution? regulation of regional lung perfusion by nitric oxide using the prone position could help to combat the development of fast hypoxia in some patients with covid-19 key: cord-000307-iv18eiap authors: capelozzi, vera luiza; parra, edwin roger; ximenes, manoel; bammann, ricardo helbert; barbas, carmen silvia valente; duarte, marid irmd seixas title: pathological and ultrastructural analysis of surgical lung biopsies in patients with swine‐origin influenza type a/h1n1 and acute respiratory failure date: 2010-12-17 journal: clinics (sao paulo) doi: 10.1590/s1807-59322010001200003 sha: doc_id: 307 cord_uid: iv18eiap background: cases of h1n1 and other pulmonary infections evolve to acute respiratory failure and death when co‐infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment. objective: to perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ards with confirmed h1n1. methods: lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. immunophenotyping was used to characterize macrophages, natural killer, t and b cells, and expression of cytokines and inos. results: the pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. ultrastructural analysis showed viral‐like particles in all cases. conclusions: viral‐like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by rt‐pcr on nasopharyngeal aspirates. bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair. recently, a novel swine-origin influenza a (h1n1) virus with molecular features of north american and eurasian swine, avian, and human influenza viruses [1] [2] [3] [4] has been associated with an outbreak of respiratory disease. according to the world health organization (who), between 25 april and 11 october 2009, 399,232 confirmed cases of h1n1 influenza virus and 4,735 deaths occurred throughout the world. 5 brazil reported 1,528 deaths up to 10 november 2009. 6 swine-origin influenza a (h1n1) virus infection can cause severe acute respiratory failure (arf), requiring admission to an intensive care unit (icu) in 15-30% of previously healthy young to middle-aged people. 3, 4, 7, 8 death may occur when co-infections or lung injury prevail over the immune response, resulting in a progressive worsening of lung function (low compliance and oxygenation). early diagnosis and a complete understanding of the pathological features of the h1n1 virus are important to help to improve treatment and the prognosis of this lethal disease. analysis of the lung tissue from an open lung biopsy (olb) of these severe cases can help in understanding the pathogenesis of this severe and sometimes fatal development. until now, no reports of olb findings used to guide the treatment of patients with h1n1 pneumonitis have been published, although according to many authors olb is safe and diagnostically useful in patients with arf, enabling appropriate therapy. [9] [10] [11] [12] the pathogenesis of arf associated with swine-origin influenza virus (s-oiv) infection in humans is unknown. the influenza virus triggers pulmonary inflammation owing to an infiltration of inflammatory cells and an immune response. bronchial epithelial cells are the primary target and the principal host for the virus. 13, 14 normally, influenza viruses are recognized and destroyed by innate immune mechanisms which involve macrophages, interferon (ifn) a, b and other cytokines, natural killer (nk) cells and complement. when influenza viruses escape from these early defense mechanisms, they are captured and eliminated by adaptive immune mechanisms, where t and b cells and their antigen-specific effectors (cytotoxic t lymphocytes, cytokines such as ifnc and antibodies) target the virus. additionally, antigen-specific memory cells (t and b cells) are involved in the prevention of the subsequent viral infection. 14 thus, pathological findings obtained by an olb, coupled to ultrastructural and immunologic analysis, may have an impact on decisions about changes in treatment strategies employed for these critically ill patients, and also provide a greater understanding of the pathophysiology of s-oiv infection. the objective of this study was to analyze pathologically and ultrastructurally s-oiv lung infection and the pulmonary immune response in a series of five cases with olb. we studied pathologically and ultrastructurally five patients suspected of having a pandemic s-oiv virus who developed arf requiring ventilatory support. nasal swabs for rt-pcr for h1n1 were collected from all patients. the olbs indicated by the clinicians were carried out after receiving consent from the families. these patients had a severe evolution of the virus and more information about the physiopathology of the disease was required in order to provide adequate treatment. if no improvement of the respiratory status was seen in the patients with arf after $5 days (defined as no decrease of the lung injury score) an olb was indicated. 15 lung tissue sections (4 mm thick), prepared from 10% formalin-fixed, routinely processed, paraffin-embedded blocks, were stained with hematoxylin-eosin. the following methods of histochemical staining were carried out: grocott's methenamine silver stain, brown-brenn, and ziehl-neelsen. the following pathological changes were analyzed: a) necrotizing bronchiolitis, b) alveolar collapse, c) dilatation of the airspaces, d) hyaline membrane, e) fibroplasia, f) squamous metaplasia, g) multinucleated cells, h) alveolar hemorrhage, i) acute inflammatory exudates, j) atypical pneumocytes. pathological changes were graded, using two sections, according to a five-point semiquantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1-25%, 2 = changes in 26-50%, 3 = changes in 51-75%, and 4 = changes in 76-100% of examined tissue. this semiquantitative analysis is currently routinely used in most studies of the department of pathology of the university of sã o paulo medical school. 16, 17 for immunohistochemistry, the avidin-biotin-peroxidase complex and streptavidin-biotin enzyme complex immunostaining methods were used with antibodies against: lymphocytes cd4 (clone: mo834, dilution 1:1000), cd8 (clone: m7103, dilution 120), cd20 (clone: m755, dilution 140), macrophages-histiocytes cd68 (clone: m814, dilution 130), mouse monoclonal antibodies from dako, carpinteria, ca, usa; s100 (clone: z311, dilution 11000) rabbit polyclonal antibodies from dako; cd1a (clone: mca1657, dilution 1: 200) mouse monoclonal antibodies from serotec, oxford, uk; natural killer, nk (clone: ms136p, dilution 11000) mouse monoclonal antibodies from neomarkers, fremont, ca, usa; interleukin 4 (il-4) (dilution 140), il-10 (dilution 140) goat polyclonal antibodies from r&d systems, minneapolis, mn, usa; ifnc (clone: mab285, dilution 130), mouse monoclonal antibodies from r&d systems; tumor necrosis factor alpha (tnfa) (clone: af210na, dilution 140) all mouse monoclonal antibodies from r&d systems; inducible nitric oxide synthase (inos) (dilution 1500) polyclonal rabbit from calbiochem, la jolla, ca, usa. immunohistochemical reactions were carried out in accordance with the manufacturer's instruction. diaminobenzidine was used as the color substrate, and meyer's hematoxylin was used for counterstaining. cell immunophenotypes and immune expression of cells using the different methods of immunohistochemical staining were identified and graded according to a five-point semiquantitative intensity-based scoring system as: 0 = negative, 1 = positive in 1-25%, 2 = positive in 26-50%, 3 = positive in 51-75%, and 4 = positive in 76-100% of examined tissue. 18 small blocks (1 mm 3 ) of lungs were fixed in 2% glutaraldehyde/2% paraformaldehyde in cacodylate buffer overnight, then fixed in 1% osmium tetroxide, dehydrated, and embedded in araldite. ultrathin sections obtained from selected areas were double-stained and examined in a philips tecnai 10 electron microscope at 80 kv. for each electron microscopy image (15/case), the following structural changes were analyzed: a) cytoplasmic swelling, b) degenerative changes, c) sloughing of necrotizing alveolar epithelial cell type i (aeci) and ii (aecii), d) denudation of the epithelial basement membrane, e) hyaline membranes, f) alveolar septal collapse, g) viral particles such as tubuloreticular structures (trs) and cylindrical confronting cisternae (ccc), h) multinucleated aecii. ultrastructural findings were graded according to a five-point semiquantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1-25%, 2 = changes in 26-50%, 3 = changes in 51-75%, and 4 = changes in 76-100% of examined tissue. 16, 17 five patients (two male, three female) mean age 48 years (range 35-81) were studied; only patient no 4 had preexisting medical illnesses (table 1) and chest x-ray abnormality at disease onset. all the patients presented with a 4-10 days' (median 5 days) history of shortness of breath and flu-like symptoms and rapid clinical deterioration. they were transferred to the icu for tracheal intubation and ventilation (range 8-25 days; median 17) and diagnosed as having arf. 15 all the patients received 75 mg twice a day by nasal enteral tube of olsetamivir (range 4-14 days; median 10) and intravenous steroids (range 9-20 days; median 12). after obtaining these results the dose was changed from 75 mg twice a day to 150 mg twice a day through a nasal enteral tube, in accordance with the brazilian guidelines. the presence of the h1n1 virus was confirmed in all five patients (table 1) by nasal swab or lung tissue positivity of rt-pcr according to guidelines from the centers for disease control and prevention. 19 other microbiological investigations, including the isolation of other viruses, were negative. during the evolution of disease in the patients in the icu, staphylococcus aureus was isolated from a blood culture (patients 2 and 3) and klebsiella spp were identified in tracheal aspirate specimens (patient 1). patients 1, 2 and 4 are alive, but patients 3 and 5 died of respiratory failure, with concurrent congestive heart failure, hepatic encephalopathy, and acute renal failure. table 2) . pulmonary specimens from patients 3 and 5 presented more intense changes at optical microscopy. the membranous and respiratory bronchioles were extensively compromised by epithelial necrosis, squamous metaplasia, and obliteration by fibroplasia ( figure 1a -f). the parenchyma was modified by extensive alveolar collapse, dilatation of the airspaces, alveolar hemorrhage, and sparse hyaline membrane formation ( figure 1g -i). there was interstitial thickening, with mild to moderate fibroplasia ( figure 1i ), but a disproportionately sparse infiltrate of inflammatory cells, mainly histiocytes, including multinucleated forms, lymphocytes and megakaryocytes ( figure 1j-k) . atypical bronchiolar and alveolar epithelial cells (aecs) were seen in all five patients, although the distribution was focal ( figure 1j ). these atypical forms included multinucleated giant cells with irregularly distributed nuclei ( figure 1k , l) or bronchiolar and aecs with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm ( figure 1m ). however, distinct viral inclusions were not apparent. the ultrastructural features were represented by bronchial and alveolar epithelium necrosis, a destroyed alveolar epithelium/basement membrane unity and the presence of viral-like particles (table 3) . patients 3 and 5 presented more prominent changes at submicroscopic level. cytoplasmic swelling, necrosis, and degenerative changes of the endoplasmic reticulum and other organelles were present in bronchial and aecs (figure 2a-c) . a large number of bronchiolar and aecs were detached from the basement membrane and were showing apoptosis (figure 2a, b) . lymphocytes also exhibited apoptosis. sloughing of apoptotic bronchiolar cells and aecs causing denudation of the epithelial basement membrane was followed by deposition of hyaline membranes ( figure 2d ). ultrastructural evidence of alveolar collapse was also present by the apposition of the alveolar septa ( figure 2e -g). the regenerating bronchiolar epithelium extended along the adjacent alveolar septa showing features of cells with prominent surface microvilli with decreased or absent lamellar bodies and considerable cytologic atypia ( figure 2h -l). increased myofibroblasts and collagen fibers were also present ( figure 2i ). multinucleated epithelial cells with prominent nucleoli were noted in most cases, although such cells were sparse ( figure 2k ). the proliferating bronchiolar and aecs containing trs and ccc, probably representing residual viral-like particles, were distinguished in all cases ( figure 2m -r). trs appeared as reticular aggregates of branching membranous tubules located within the cisternae of the endoplasmic reticulum ( figure 2m -o) or were compact ( figure 2q , r). ccc were identified as elongated, slightly curved cylindrical structures ( figure 2p , q), ring shaped ( figure 2r ) or fused membranous lamellae, representing cisternae of endoplasmic reticulum. (table 4) . patients 3 and 5 presented with immunologic impairment. in all patients small aggregates of macrophages, cd4+ thelper cells, cd8+ t-cytotoxic cells, cd20+ b-cells, cd1a+ dendritic cells, s100+ dendritic cells, natural killer lymphocytes were present around vessels and bronchioles. dendritic cells and tnfa were expressed sparsely in macrophages, aecs and endothelial cells, whereas ifnc was expressed in small mononucleated cells in lungs from patients with s-oiv. there was a very strong expression of il-4, il-10 and inos in small mononucleated cells. this case series documents for the first time the pathological and ultrastructural findings of lung tissue from five patients admitted to the icu with arf and s-oiv infection who were submitted to olb. s-oiv (h1n1) virus and the pulmonary syndrome is an acute respiratory illness, first identified in mexico with at present, 399,232 cases registered, 4,735 deaths, affecting more than 179 countries. 2, 5 our patients, most of them previously healthy, had an atypical influenza-like illness that progressed during a period of 5-7 days. the two patients who died showed a higher degree of pathological commitment of the disease at the olb. most of our patients were young to middle-aged and had previously been healthy. increased risk for severe s-oiv illness is found in young children, 10-19 age groups, patients older than 65 years, pregnant women, obese people and those with comorbidities. 1, 7, 20 fifteen to thirty per cent of patients with h1n1 infection required icu admission. mortality among the patients who required mechanical ventilation was around 58%. 7 in our case series the olb findings showed that the lung damage was most likely due to infection by the influenza virus. the main pathological finding revealed necrotizing bronchiolitis and dad, respiratory epithelial cells probably being the primary target of the infection. the extensive destruction of the respiratory and aecs and dysfunction in the immune and adaptative immune response led to dad. as previously reported, possible mechanisms of damage include direct injury to the respiratory and alveolar epithelium exposing the basement membrane and leading to alveolar collapse by loss of surfactant, 13,14,21 with a secondary cytokine storm. 22 this is followed by exudation of macromolecules from the circulation, which finally form hyaline membranes. activation of the cytokines is part of the immune reaction aiming to eradicate the virus. in this study, the systemic ifnc and tnfa cytokine activation probably resulted in reactive hemophagocytic syndrome in the bronchiole-associated lymphoid tissue and possibly also mediated the epithelial necrosis. 23 a mild inflammatory infiltration is most often seen in viral pneumonias; this has been explained by a cytokine-mediated blockade of lymphocytopoiesis and also by blockade of release from the bone marrow. 24 in our cases, expression in the lung of ifnc by small mononucleated cells and tnfa by macrophages and aecs was low. this finding may be supported by kim and colleagues, 25 conversely, we found a very strong expression of il-4, il-10 and inos by macrophages. the sparse inflammatory and immune reaction found in our samples, which involves targetting of the virus by nk cells, lymphocytes t and b cells, cd8+ cytotoxic t-lymphocyte cells, as well as cd1a and s100 cells, may be due to a combination of lymphoid tissue necrosis and apoptosis and exhaustion of lymphoid proliferation in response to the cytokine overdrive. in addition, the high il-10 expression associated with its anti-inflammatory action may explain the low degree of inflammation observed in our cases. taken together, our results suggest that in s-oiv infection, altered innate and adaptative immune responses may lead to incomplete virus eradication in the primary target of the infection and, consequently, imbalance between inflammation and reparation, resulting in bronchiolar obliteration and dad. dad is likely to be a consequence of bronchiolar obstruction and consequent hypoxia rather than direct invasion of the viruses. it is a severe pattern of lung injury and could be secondary to various pulmonary and extrapulmonary insults. 26 in this series of cases we found dad in which alveolar collapse was prominent, differing from classic dad found in arf or secondary to other pulmonary and extrapulmonary insults. this finding may have important implications in the ventilation strategy of the patients. 27 in addition, the presence of intra-alveolar hemorrhage may suggest virus-associated hemophagocytic syndrome. 23 the lung tissue score was obtained independently by two different investigators. the pathologic findings were graded according to a five-point semiquantitative severity-based scoring system: 0 = normal lung parenchyma; 1 = changes in 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 76-100% of the examined tissue. table 3 -semiquantitative analysis of electron microscopy. in our current series, pulmonary ultrastructural analysis was important to obtain an understanding of the pathophysiology of this new disease. first, we demonstrated apoptosis and necrosis in the bronchiolar epithelium together with viral-like particles, thus suggesting the bronchiolar epithelium as the primary target of the virus infection. second, we documented the submicroscopic pattern of a clastogenic dad in s-oiv infection. third, we found indirect evidence of virus infection in alveolar and bronchiolar epithelial cells represented by the trs and ccc. these submicroscopic structures were demonstrated ultrastructurally in the lung tissue of all the patients and their presence suggests an inactivation of the virus by oseltamivir treatment or an altered innate immune response of these patients. they appeared mainly in respiratory cells and aecs and have previously been described in a variety of cell types. 28, 29 usually, they occur in endothelial cells and lymphocytes from patients with autoimmune diseases and viral infections. 30 patients with acquired immunodeficiency syndrome present trs and ccc in these same cells. 31 the mechanism of trs and ccc production in vivo is not definitely established. nevertheless, clinical and experimental studies have shown that the presence of both structures in these diseases is directly associated with the increase of ifna and ifnb but not with ifnc. 29 one theory to explain the nature and pathogenesis of trs and ccc suggests that these structures are incomplete viral particles. 30 in our study, these viral-like particles were noted mainly in the respiratory epithelial cells, but not in the other cell types within the lung. these observations reinforce the hypothesis that the primary target cells for s-iov infection are probably the bronchiolar epithelium. the atypical morphology of the bronchiolar and alveolar epithelial cells was probably related to viral cytopathic effects or reactive changes. in fact, the presence of multinucleated epithelial cells is not exclusive to s-iov, and is seen in pneumonia caused by the family of paramyxoviridae, including parainfluenza viruses, measles, mumps, respiratory syncytial virus and, perhaps, metapneumovirus. 31 although multinucleated cells were seen in our cases, these probably reflect non-specific secondary changes. we describe a case series of five patients with influenzalike illness with pneumonia and ensuing arf who underwent olb with subsequently confirmed diagnosis by rt-pcr testing for s-iov infections. this report has some limitations. first, this study may not validate the importance of olb in this population; however, it did provide information about this new disease. second, it is difficult to compare our findings with those of others because to our knowledge no studies reporting an olb in patients with sthe lung tissue score was obtained independently by two different investigators. the pathologic findings were graded according to a five-point semiquantitative severity-based scoring system: 0 = normal lung parenchyma; 1 = changes in 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 76-100% of the examined tissue. ifn, interferon; tnfa, tumor necrosis factor a. iov have been published. although there are already many autopsy series with patients with h1n1 that can be used for comparison, the pathological findings at autopsy are modified mainly by the presence of associated co-infections and mechanical ventilation. [32] [33] [34] [35] [36] [37] [38] [39] in summary, we have presented the pulmonary pathology in a confirmed and well-defined series of cases of s-iov infection associated with arf. the pathological features, in addition to necrotizing bronchiolitis and dad, included the presence of multinucleated cells and intra-alveolar fibrin exudates (organizing pneumonia-like lesions). although each of these features is non-specific, their combined occurrence, together with positive serologic, microbiologic, and immunologic investigations and/or ultrastructural tissue examination enables the diagnosis of s-iov infection to be confirmed, and is particularly useful in clinically suspicious cases that do not fulfill the who criteria or in clinically inapparent cases. we have shown that viral-like particles can be successfully demonstrated in lung tissue by ultrastructural examination, highlighting the importance of olb, particularly in those patients without confirmation of the virus. we also showed that bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and that dad is the consequence of airways obliteration and dysfunction on innate immunity, suggesting that the treatment should be focused on epithelium repair. swine influenza 1. a (h1n1) infection in two children -southern california update: infections with a swine-origin influenza a (h1n1) virus -united states and other countries update: swine influenza a (h1n1) infections -california and texas novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans world health organization -pandemic (h1n1) 2009 -update 77 novembro de 2009. secretaria de vigilâ ncia em saú de pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico severe respiratory disease concurrent with the circulation of h1n1 influenza impact of open lung biopsy on refractory acute respiratory failure open lung biopsy in patients on mechanical ventilation with suspected diffuse lung disease a contributive result of open-lung biopsy improves survival in acute respiratory distress syndrome patients usefulness of open lung biopsy in mechanically ventilated patients with undiagnosed diffuse pulmonary infiltrates: influence of comorbidities and organ dysfunction host defense function of the airway epithelium in health and disease: clinical background defense mechanisms against influenza virus infection in the respiratory tract mucosa an expanded definition of the adult respiratory distress syndrome recruitment maneuver in pulmonary and extrapulmonary experimental acute lung injury pulmonary lesion induced by low and high positive end-expiratory pressure levels during protective ventilation in experimental acute lung injury cox-2, mmp-9, and noguchi classification provide additional prognostic information about adenocarcinoma of the lung. a study of 117 patients from brazil protocol of real time rt-pcr for swine influenza a (h1n1) human case of swine influenza a (h1n1) triple reassortant virus infection, wisconsin the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1 -a review distinctly different expression of cytokines and chemokines in the lungs of two h5n1 avian influenza patients novel swine-origin influenza a (h1n1) virus-associated hemophagocytic syndrome-a first case report hemophagocytic lymphohistiocytosis complicating influenza a infection association of tumor necrosis factor-alpha with fever and pulmonary lesion score in pigs experimentally infected with swine influenza virus subtype h1n2 diagnosis of usual interstitial pneumonia and distinction from other fibrosing interstitial lung diseases pulmonary and extrapulmonary acute respiratory distress syndrome are different significance of tubuloreticualr inclusions in the pathobiology of human diseases the acquired immunodeficiency syndrome: and ultrastructural study tubuloreticular structures and cylindrical confronting cisterae: a review lung pathology of fatal severe acute respiratory syndrome lung pathology in fatal novel human influenza a (h1n1) infection autopsy findings in eight patients with fatal h1n1 influenza pandemic influenza a (h1n1): pathology and pathogenesis of 100 fatal cases in the united states novel h1n1 influenza a viral infection complicated by alveolar hemorrhage a histopathological study on influenza a h1n1 infection in humans pathologic findings in novel influenza a (h1n1) virus (''swine flu'' ) infection: contrasting clinical manifestations and lung pathology in two fatal cases bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states pulmonary pathologic findings of fatal 2009 pandemic influenza a/ h1n1 viral infections key: cord-017856-4fccnygg authors: roden, anja c.; tazelaar, henry d. title: pathology of lung rejection: cellular and humoral mediated date: 2018-04-24 journal: lung transplantation doi: 10.1007/978-3-319-91184-7_13 sha: doc_id: 17856 cord_uid: 4fccnygg acute rejection is an important risk factor for bronchiolitis obliterans syndrome, the clinical manifestation of chronic airway rejection in lung allograft recipients. patients with acute rejection might be asymptomatic or present with symptoms that are not specific and can be also seen in other conditions. clinical tests such as pulmonary function tests and imaging studies among others usually are abnormal; however, their results are also not specific for acute rejection. histopathologic features of acute rejection in adequate samples of transbronchial lung biopsy of the lung allograft are currently the gold standard to assess for acute rejection in lung transplant recipients. acute alloreactive injury can affect both the vasculature and the airways. currently, the guidelines of the 2007 international society of heart and lung transplantation consensus conference are recommended for the histopathologic assessment of rejection. there are no specific morphologic features recognized to diagnose antibody-mediated rejection (amr) in lung allografts. therefore, the diagnosis of amr currently requires a “triple test” including clinical features, serologic evidence of donor-specific antibodies, and pathologic findings supportive of amr. complement 4d deposition is used to support a diagnosis of amr in many solid organ transplants; however, its significance for the diagnosis of amr in lung allografts is not entirely clear. this chapter discusses the currently recommended guidelines for the assessment of cellular rejection of lung allografts and summarizes our knowledge about morphologic features and immunophenotypic tests that might help in the diagnosis of amr. acute rejection is the host's response to the recognition of the graft as foreign. it can occur days, months, or even years after transplantation. rejection can be divided into cellular and humoral forms. acute cellular rejection is the predominant type of acute rejection of lung allografts. it is mediated by t lymphocytes that recognize foreign human leukocyte antigens (hla) or other antigens [1, 2] . humoral rejection is mediated by preformed or de novo recipient antibodies (therefore, also referred to as antibody-mediated rejection [amr]) against antigens of the donor organ cells. acute rejection is an important complication in patients with lung allografts. twenty-nine percent of adult patients have at least one episode of treated acute rejection between discharge from the hospital and 1 year after transplantation [3] . moreover, 3.6% and 1.8% of all deaths that occur within the first 30 days or between 30 days and 1 year following lung transplantation are due to acute rejection, respectively [3] . in addition, the frequency and severity of acute rejections are thought to represent the major risk factor for the subsequent development of bronchiolitis obliterans syndrome (bos) [1, [4] [5] [6] . hla mismatch, genetic and recipient factors, type of immunosuppression, vitamin d deficiency, and infection are risk factors of acute rejection. for instance, the recipient alloimmune response is thought to be related to the recognition of differences to donor antigens leading to acute lung allograft rejection. indeed a higher degree of hla mismatch has been shown to increase the risk of acute rejection although this effect is not consistent across all hla loci or studies [4, [7] [8] [9] [10] . mismatches at the hla-dr, hla-b [7] , and hla-a [8] loci, as well as a combination of all three loci [9] , appear specifically important. for instance, acute rejection within 2 months after transplantation has been shown to be associated with hla-dr mismatch, while acute rejection at 4 years has been found to be associated with hla-b mismatch [11] . several host genetic characteristics have been studied that may modulate acute lung rejection. for instance, a genotype leading to increased il1-production may protect against acute rejection [12] , while a multidrug-resistant genotype (mdr1 c3435t) appears to predispose to persistent acute rejection that is resistant to immunosuppressive treatment [13] . the incidence of acute rejection appears to be age-dependent, with the lowest incidence of acute rejection in infants (< age 2) [14] . however, children have a higher risk for acute rejection than adults [15] . furthermore, the registry of the international society of heart and lung transplantation (ishlt) showed that the incidence of acute rejection between discharge and 1-year follow-up was slightly higher in younger adult lung allograft recipients (age 18-34 years) (36%) [16] when compared to the entire adult population in which 29% had at least one acute rejection episode [3] . the incidence of acute rejection does not seem to change in older lung transplant recipients (age 65 and higher) [17] . regimens of immunosuppression might also play a role in acute rejection. for instance, the rate of acute rejection in the first year after transplantation was highest among recipients who were on cyclosporine-based regimens and lowest among those on tacrolimus-based regimens [18] . vitamin d deficiency might also play a role in acute rejection. a study found that 80% of lung recipients were 25(oh)d deficient around the time of transplantation and that vitamin d-deficient recipients had more episodes of acute cellular rejection and infection [19] . a similar association between vitamin d deficiency and acute rejection has been described in other solid organ recipients including the liver, kidney, and heart. although the exact mechanism for this phenomenon is not entirely clear, it is speculated that (1) vitamin d might slow down the maturation of antigen-presenting cells as in vitro studies have shown, (2) vitamin d might induce dendritic cells to acquire tolerance, and/or (3) a synergistic effect between vitamin d analogs and immunosuppressants occurs [19] . viral infections have also been thought to modulate the immune system and to increase alloreactivity. indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus, rhinovirus, coronavirus, and parainfluenza virus [20] [21] [22] . chlamydia pneumoniae infection has also been linked to the development of acute rejection in one study [23] . the significance of cmv infections and the impact of cmv prophylaxis strategies on acute rejection frequency are not clear at this time [24] . the clinical course of acute rejection can be variable. acute rejection is often identified on surveillance transbronchial biopsy in an asymptomatic patient. if symptoms occur, they might be non-specific and overlap with those seen in other complications and diseases in this patient population. these symptoms might include dyspnea, fever, leukocytosis, and a widened alveolar-arterial oxygen gradient. higher-grade rejection appears to cause more severe symptoms and can lead to acute respiratory distress [17] . in patients with rejection, pulmonary function testing may show a decrease in forced expiratory volume in 1 s (fev 1 ) and vital capacity (vc). although spirometry has a sensitivity of greater than 60% for detecting infection or rejection of grade a2 and higher, it cannot differentiate between the two [25] . furthermore, the usefulness of spirometry is diminished in single lung transplant recipients, as the dysfunction of the native lung confounds the pulmonary function test results [26] . although in approximately half of the cases of acute rejection, chest x-ray studies are normal, ill-defined perihilar and lower lobe opacities, along with septal lines and pleural effusions, may be seen. findings on ct scan might include ground-glass opacities, septal thickening, volume loss, nodules and consolidation, and pleural effusions. infiltrates observed on imaging studies during the first week after lung transplantation are usually caused by the reimplantation response, i.e., reperfusion edema and other factors. infiltrates that persist beyond the first week following transplantation suggest acute rejection or infection. however, although early, the authors of small studies have attempted to demonstrate the usefulness of chest x-rays and chest ct scans in the diagnosis of rejection, more recent data show a very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes [27] . exhaled nitric oxide (no) can also serve as a marker of lung injury; it is often increased in patients with lymphocytic bronchiolitis and acute rejection [28] [29] [30] . furthermore, in a study of inert gas single-breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection [25] . although the presentation of the patient and several ancillary studies may suggest the presence of acute allograft rejection, none of these findings are specific. therefore, tissue diagnosis is necessary for a definitive diagnosis. histopathology of adequate lung biopsy samples obtained from transbronchial biopsy is currently the gold standard to assess lung allografts for rejection and to distinguish rejection from its clinical mimickers such as aspiration, infection, drug toxicity, and recurrent disease. recently, the transbronchial cryobiopsy technique was introduced which yields larger biopsies containing more alveoli, small airways, and veins and venules while exhibiting less procedural alveolar hemorrhage and crush artifact than conventional forceps transbronchial allograft biopsies [31] [32] [33] . although cryobiopsies appear to be as safe as forceps biopsies, complications can occur which is one of the reasons that this technique has so far not been universally performed for this purpose [31] . other lung tissue specimens from lung allografts include wedge biopsies, explants for retransplant, or autopsy specimens from lung transplant recipients. wedge biopsies, although seldom obtained in clinical practice, and specimens from explants provide useful histopathologic insights into the etiology of lung allograft dysfunction in advanced stages following all possible medical interventions. cellular alloreactive injury to the donor lung affects both the vasculature and the airways [34] . perivascular mononuclear cell infiltrates are the hallmark of acute cellular rejection. these infiltrates may be accompanied by subendothelial chronic inflammation (e.g., endotheliitis or intimitis) and also by lymphocytic bronchiolitis, which is characteristic of small airway rejection. the histologic changes are divided into grades based on intensity of the cellular infiltrate and the occurrence of an accompanying acute lung injury pattern. in 1990, the ishlt sponsored the lung rejection study group (lrsg), a workshop to develop a "working formulation" for the diagnosis of lung rejection by transbronchial biopsy [35] . since then the grading scheme has been revised twice, in 1996 [36] and 2007 [34] . the grading scheme is strictly pathologic, based on morphologic features recognized in transbronchial biopsies of the allograft. clinical parameters are not considered. due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. furthermore, infection and rejection may occur together. therefore, the lrsg recommends grading rejection only after the rigorous exclusion of infection [34] . the most recent classification of lung allograft biopsies is the 2007 ishlt consensus classification of allograft rejection [34] (table 13.1 ). an attempt should be made to accurately distinguish the grade of rejection since treatment is largely dependent on the histologic grade assessed by an experienced pulmonary pathologist familiar with the histopathologic features and criteria used for grading. however, inter-and intra-observer variability in grading can impact treatment and outcome [37, 38] . two studies using the 1996 grading system found relatively good interobserver agreements for the a grades (kappa of 0.65 and 0.73) [37, 38] ; however, these results could not be replicated in another study in which the kappa was 0.47 in spite of dichotomization of the a grades to a0/a1 versus a2-4 [39] . intraobserver agreement for acute rejection has been found to be good with kappa values of 0.65 and 0.79 [37, 39] . using the revised 2007 ishlt classification, bhorade and colleagues showed an overall concordance rate of 74% for grade a and 89% for grade b specimens between a site pathologist and a central pathologist [40] . however, the weighted kappa scores in that study showed only fair to moderate agreement for a grades (kappa values varied between 0.22 and 0.48) and less than a chance agreement to moderate agreement for b grades (kappa values varied between −0.04 and 0.46). interestingly, the kappa values for a and b grades were dependent on the time that had elapsed between transplantation and biopsy. the best agreement occurred in biopsies taken within 6 weeks of transplant. slightly higher agreements (81% and 93%, for a and b grades, respectively) were shown in a study that evaluated the interobserver agreement between two transplant pathologists from the same institution using the 2007 revision grading scheme [31] . although cryobiopsies are larger and appear to be easier interpretable, interobserver reproducibility did not improve with the use of cryobiopsies in that study [31] . acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis [34] . with progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. the majority of the mononuclear cells in acute rejection are t cells, although a few studies have described increased populations of b cells or eosinophils [34, 41, 42] . the histologic features of rejection are summarized in table 13 .1. features of acute cellular rejection are lacking, although the biopsy may not be entirely normal. scattered infrequent blood vessels, particularly venules, in the alveolated lung parenchyma are surrounded by a relatively thin (ring of two to three layers) chronic mononuclear cell infiltrate ( fig. 13.1a , b). the lymphocytic rim can be loose or compact and is in general circumferential but does not spill into the adjacent interstitium. endotheliitis and eosinophils are absent. in adequately alveolated and artifact-free speci-mens, the lymphocytic infiltrates may be detected at low magnification, but often higher power study is needed to identify the infiltrates. although in mild acute rejection the perivascular infiltrate of lymphocytes is still confined to the perivascular adventitia without infiltrating the adjacent interstitium or air spaces, there are more layers of lymphocytes surrounding vessels ( fig. 13.2a, b ). in addition, the perivascular mononuclear infiltrates surrounding venules and arterioles are more frequent than in grade a1. they are typically recognizable at low magnification. these infiltrates usually consist of a mixture of small round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages, and eosinophils. the cellular infiltrates can be compact or loose. subendothelial infiltration by mononuclear cells may be noted which can be associated with hyperplastic or regenerative changes in the endothelium. concurrent lymphocytic bronchiolitis may be seen. venules and arterioles are cuffed by easily recognizable dense perivascular mononuclear cell infiltrates that are commonly associated with endotheliitis ( fig. 13 .3a-c). eosinophils and even occasional neutrophils are common. in a b moderate acute rejection, the inflammatory cell infiltrate extends into the adjacent alveolar septa where it can be associated with type ii pneumocyte hyperplasia. the inflammatory infiltrate can also extend into adjacent airspaces and be associated with collections of intra-alveolar macrophages and lymphocytes. histologic features of acute lung injury may become apparent in the form of airspace fibrin. in severe rejection, there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endotheliitis ( fig. 13 .4a-f). this may be associated with necrotic intra-alveolar epithelial cells, hemorrhage and neutrophils, and usually morphologic evidence of acute lung injury in the form of organizing pneumonia, fibrin deposition, or hyaline membranes. parenchymal necrosis, infarction, or necrotizing vasculitis may be identified; however, these features are more evident on surgical rather than transbronchial lung biopsies. it should be noted that a paradoxical diminution of perivascular infiltrates can occur as cells extend into interalveolar septa and air spaces where they are admixed with macrophages. protocol surveillance biopsies of lung allografts are performed in many institutions. even though these patients are in general asymptomatic and clinically stable, one study showed that 39% of surveillance biopsies reveal acute cellular rejection with 43% showing features of minimal rejection, 49% mild rejection, and 8% moderate rejection [43] . a more recent prospective study identified morphologic findings of acute cellular rejection only in 6% of surveillance biopsies [44] , while a retrospective study of 592 a b surveillance biopsies taken within 400 days of transplantation revealed histologic findings of either acute cellular rejection or obliterative bronchiolitis in 31% of biopsies with 36% within the first 100 days and 25% between 100 and 400 days following transplantation [45] . evidence suggests that acute cellular rejection is an important risk factor for the development of bos [24] . indeed, studies have demonstrated an increased risk of bos with single episodes, increased frequencies, and increased severity of acute cellular rejection. moreover, patients with multiple episodes of even minimal acute cellular rejection were shown to be at increased risk for bos [46] , and yet a single episode of minimal acute rejection without recurrence or subsequent progression to a higher grade has been identified as an independent significant predictor of bos [47] . because of these findings, patients who are asymptomatic but are found to have acute cellular rejection (even minimal acute cellular rejection) on a surveillance allograft biopsy might be treated accordingly. however, several centers do not utilize surveillance transbronchial lung biopsies and/or treat asymptomatic patients with no clinical evidence of allograft dysfunction. prospective well-designed clinical studies are needed to provide evidence to support surveillance transbronchial lung biopsies and therapeutic interventions. this grade applies only to small airways such as terminal or respiratory bronchioles. bronchi, if present, should be described separately. it is important to mention in the pathology report whether or not small airways are present. if no small airways are identified or the biopsy has obvious infection, the grade "bx" should be used. the r behind grades 1 and 2 denotes the revised 2007 version. the small airways appear unremarkable without evidence of bronchiolar inflammation. low-grade inflammation is characterized by lymphocytes within the submucosa of the bronchioles ( fig. 13 .5a-c). the lymphocytic infiltrates can be infrequent and scattered or form a circumferential band; however, intraepithelial lymphocytic infiltration is not present. occasional eosinophils may be seen within the submucosa. there is no evidence of epithelial damage, neutrophils, necrosis, ulceration, or significant amount of nuclear debris. in high-grade small airway inflammation, there is marked lymphocytic infiltrate of the airway epithelium and airway wall. the mononuclear cells in the submucosa appear larger, and a greater number of eosinophils and plasmacytoid cells can be seen (fig. 13.6a-c) . in addition, there is evidence of epithelial damage including necrosis, metaplasia, and marked intraepithelial lymphocytic infiltration. in its most severe form, high-grade airway inflammation is associated with epithelial ulceration, fibrinopurulent exudate, cellular debris, and neutrophils. it is important to exclude an infectious process, especially if the number of neutrophils is disproportionally high when compared to other mononuclear cells within the airway wall. small airways might not be evaluable for several reasons including lack of small airways due to sampling problems, infection, tangential cutting, artifact, etc. in patients who are known to have an infection that could cause lymphocytic bronchiolitis, the allograft biopsy should also be classified as ungradeable for small airway rejection. chronic airway rejection is restricted to submucosal and intraluminal scarring of small airways including terminal and respiratory bronchioles. when large tissue sections of the lung are examined, obliterative bronchiolitis may be recognized as a panlobar process but is usually patchy. the small airways appear similar in size to the accompanying artery with a ragged inner surface. fibrosis is not present. narrowing of the small airways due to fibrosis in the airway wall is the hallmark of chronic airway rejection. the fibrosis may be eccentric or concentric. the type of fibrosis depends on the acuteness of the process, the degree of organization, and the amount of accompanying inflammation. the fibrosis can range from loose myxoid granulation tissue with variable numbers of inflammatory cells filling or partially obstructing the airway lumen in the more acute phase (fig. 13 .7a) to dense hyalinized collagen in the wall of bronchioles that is a characteristic of the chronic phase ( fig. 13.7b) . metaplastic squamous or cuboidal epithelium may overly the bronchiolar fibrosis. sometimes, only a slit-like lumen of the airway may remain as a result of a confluent submucosal scar or intraluminal polyps of scar tissue. there may be rather prominent capillaries supplying the intraluminal fibrotic areas. ultimately, the bronchiolar lumen might be entirely occluded by dense scar tissue (fig. 13.7c, d) . in these cases, only an elastic stain highlighting residual elastic tissue, the vicinity of the scar to a pulmonary artery, and residual smooth muscle may indicate that a small airway has been replaced by fibrotic scar. in the chronic phase, inflammation may be minimal or absent. usually, the scarring process is confined exclusively to respiratory bronchioles and terminal bronchioles, although it may occasionally involve adjacent alveoli. obliterative bronchiolitis is only infrequently identified in lung allografts by transbronchial biopsy, and the sensitivity of this morphologic finding for the presence of chronic rejection is only between 15 and 28% [48] [49] [50] . in a recent study, all seven conventional transbronchial biopsies that were included from patients clinically known to have bos, the clinical equivalent to morphologic obliterative bronchiolitis, failed to reveal morphologic findings of obliterative bronchiolitis [31] . although cryobiopsies contained more small airways, all nine cryobiopsies that were also included in that study from patients with clinically proven bos did not reveal obliterative bronchiolitis in the tissue [31] . this low sensitivity is largely due to sampling and its patchy nature. therefore, bos is used and more reliable for the clinical assessment of chronic airway rejection. bos is calculated as <80% fev 1 in at least two consecutive lung function tests of the patient's maximum fev 1 posttransplantation [51] . despite the low sensitivity of transbronchial biopsies for obliterative bronchiolitis, the specificity of this morphologic finding in an allograft biopsy is high, ranging from 75 to 94% [49, 50] . therefore, an attempt to diagnose obliterative bronchiolitis should be made in lung allograft biopsies. the pulmonary arteries appear of a similar size as the accompanying airways. the intima is slender and the media not thickened. chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. wedge biopsies, explants, or autopsy material may reveal it. therefore, according to the ishlt, the d grade of rejection is not applicable to allograft transbronchial biopsies. although cryobiopsies contain a higher number of venules and small veins, in a recent small study, no difference was found in the number of cases with possible vascular rejection when compared to transbronchial biopsies [31] . vascular rejection is characterized by thickened pulmonary arteries and more often veins, due to fibrointimal connective tissue ( fig. 13.8a, b) . also, thickening is usually concentric. chronic vascular rejection may be patchy. chronic vascular rejection usually starts with intimal proliferation. subsequently, the internal elastic lamina may become fragmented and discontinuous. occasionally the underlying muscular wall becomes thinned. in approximately half of the reported cases, a concurrent endovasculitis has been observed. the process is similar in pulmonary veins, although the intimal deposits may be less cellular and more waxy, eosinophilic, and sclerotic. recanalized thrombi may mimic chronic vascular rejection. in contrast to heart allografts, chronic vascular rejection in lung transplants has not resulted in graft loss; however, some patients develop pulmonary hypertension particularly those with bos [52, 53] . infection can mimic acute cellular rejection. for instance, viral infection, particularly cmv ( fig. 13 .9a-e) but also pneumocystis jirovecii pneumonia, can be associated with perivascular mononuclear cell inflammation mimicking acute cellular rejection [54] . infection can also cause small airway inflammation imitating lymphocytic bronchiolitis. mimickers of severe acute rejection include conditions that might present with acute lung injury or diffuse alveolar damage. these conditions include infection, drug toxicity, aspiration, amr, or harvest/reperfusion injury. the presence of perivascular inflammation is helpful in establishing the diagnosis of rejection. however, perivascular inflammation is not entirely specific for acute rejection, and many other conditions may simulate or mimic alloreactive lung injury [54] . marked perivascular and/or peribronchiolar mononuclear infiltrates might also raise the possibility of posttransplantation lymphoproliferative disease (ptld), and in such cases, an appropriate workup should be performed, including doing studies for epstein-barr virus, which is ubiquitous in ptld. further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases. small airway rejection and the perivascular infiltrates of grade a rejection should be distinguished from bronchiolar-associated lymphatic tissue (balt) . balt is found in the vicinity of airways, usually contains black anthracotic pigment, and presents as a rather nodular collection of chronic inflammatory cells which does not surround a vessel (fig. 13.10 ). epithelial injury, neutrophils, or eosinophils should not be seen in balt collections [34] . originally recognized in kidney transplant patients who presented with acute allograft rejection, anti-donor antibodies, and poor prognosis [55] , amr is now well established in kidney and heart allografts. in lung transplantation, amr is still an evolving concept but likely explains acute and chronic graft dysfunction/failure in a subset of patients. evidence suggests that amr occurs due to circulating antibodies that are either (1) preformed because of pregnancy, blood transfusion, or previous organ transplantation or (2) arise de novo after transplantation due to hla mismatch. furthermore, the recent development of very sensitive and specific solid-phase flow cytometry and luminex-based methodologies has allowed for more accurate detection of antibody specificities in sensitized recipients, and it has become clear that more patients than previously expected have or develop preformed anti-hla antibodies. immune stimulation by prior infections or autoimmunity may also contribute to the development of antibodies in those patients with no identifiable risk factors. overall, these preexisting or de novo antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection), accelerated humoral rejection, and/or bos [56] . in addition, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) [57] , persistent rejection, increased bos [58] , or worse overall survival [59] in patients with anti-hla antibodies. this effect is seen both with pretransplant hla sensitization and with the development of de novo anti-hla donor-specific antibodies after transplantation [58] . about 10-15% of lung transplant recipients are pre-sensitized to hla antigens [60] . even though "unacceptable antigens" are avoided during the virtual crossmatch, patients with positive pretransplant pra are at higher risk for posttransplant complications. their posttransplant pra can stay stable or increase via generation of either donor-specific or non-donor-specific anti-hla antibodies. similarly, patients that had negative pra screening tests before transplantation can develop de novo non-donor-specific or donor-specific anti-hla antibodies after transplantation. the mechanisms by which antibodies promote lung allograft injury remain poorly understood. antibody binding to allo-hla or other endothelial or epithelial targets in the lung allograft can activate the complement cascade. complement deposits lead to endothelial cell injury, production of proinflammatory molecules, and recruitment of inflammatory cells. complement-independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation [56] . furthermore, as demonstrated by in vitro studies, anti-hla antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors [61] , potentially contributing to the generation of obliterative bronchiolitis. although the diagnosis of amr in lung allograft biopsies remains challenging, when the triple test criteria are met (graft dysfunction, positive panel reactive antibodies, and evidence of complement deposition in the graft), the disease can be life-threatening, and prognosis can be poor. although the optimal treatment of amr in the lung is currently not known due to the lack of clinical trials, treatment is typically comprised of plasmapheresis, possibly intravenous immunoglobulin (ivig), and medications such as rituximab and bortezomib, among others. as such, the associated histopathologic and clinical parameters are the subject of intense investigation. deposition of complement 4d (c4d), a complement split product, on the capillary endothelium has been suggested as a surrogate marker for amr in heart, kidney, and pancreas transplants [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] . however, the role of c4d deposition in the diagnosis of amr in lung allografts is still unclear. moreover, reproducibility of c4d deposition in allograft lung tbbx is problematic, even among pathologists who routinely evaluate c4d in lung allograft biopsies [72] . furthermore, there are currently no specific or sensitive morphologic features of amr in lung allografts, although some features that are more commonly identified in these patients have emerged in some recent studies [73] . studies have attempted to evaluate immunoglobulins (ig) and complement deposits in the subendothelial space. septal capillary deposits of igs and complement products such as c1q, c3d, c4d, and c5b-9 have been described in association with anti-hla antibod-ies [74, 75] as well as allograft dysfunction and bos [76, 77] . however, except for c4d and in some institutions c3d, these studies have in general not been implemented for the workup of lung transplant biopsies for possible amr. one of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemical as well as immunofluorescence studies. often, c4d binds to the vascular elastic lamina or shows other non-specific binding such as intracapillary serum. staining is commonly only focal, and, therefore, sensitivity and specificity have not been established. only linear, continuous luminal endothelial staining of capillaries, arterioles, and/or venules by c4d should be interpreted as positive. in addition, c4d is not specific to amr but also can be seen in infection, and harvest/reperfusion injury, or any process that is associated with complement activation. in general, the concept of specific histopathologic features associated with amr remains controversial in lung transplantation. the 2007 ishlt revised consensus classification [34] did propose histopathologic features that might be specific for amr. because of the lack of specific histologic findings of amr, a multidisciplinary approach to the diagnosis was recommended that includes the following: (1) the presence of circulating antibodies (hla antibodies, anti-endothelial and anti-epithelial antibodies), (2) focal or diffuse c4d deposition (fig. 13 .11a-c), (3) histologic features of acute lung injury or hemorrhage (diffuse alveolar damage, capillary injury associated with neutrophils and nuclear debris, i.e., capillaritis), and (4) clinical signs of graft dysfunction [78] . in 2013, the pathology council of the ishlt published findings in a summary statement with recommendations for the pathologic evaluation of amr [78] . this report included suggestions for protocol biopsies with serologic evaluation for donor-specific antibodies (dsas) at or near time of biopsy. in addition, this statement included recommendations for histopathologic patterns in amr (fig. 13. 12a-e) and indications for immunohistochemical or immunofluorescence studies to further elucidate findings in amr (box 13.1). the morphologic features were confirmed by the 2016 consensus report of the ishlt [79] . the 2016 consensus report confirmed the need for a multidisciplinary approach to establish a diagnosis of amr in the lung that "integrates the clinical presentation with available immunologic and pathologic diagnostic tools" [79] . an amr staging was also proposed (table 13. 2) [79] . recently, wallace and colleagues reported findings of the banff study of the pathology of allograft lungs with dsa [73] . nine experienced lung transplant pathologists from multiple institutions performed digital slide interpretation to study transbronchial biopsy specimens from patients with known antibody status (established within 30 days of biopsy) and negative infectious workup. the study demonstrated that biopsies from patients with dsa more commonly showed morphologic features of acute lung injury with or without diffuse alveolar damage than biopsies from patients with non-dsa or no circulating antibodies. endotheliitis was more common in patients with dsa than patients without circulating antibodies. however, there was no difference in occurrence of endotheliitis between biopsies from patients with circulating non-dsa vs dsa or non-dsa vs no circulating antibodies. specimens associated with dsa had a significant higher frequency of capillary inflammation, including neutrophilic margination, increased neutrophils, or capillaritis with karyorrhexis than patients with non-dsa or no circulating antibodies. c4d staining was positive in less than 50% of capillaries in 14% of biopsies and in more than 50% of capillaries in 7% of biopsies. while there was no difference between the groups in biopsies with <50% staining, biopsies with dsa more often had over 50% capillaries staining for c4d than biopsies without any circulating antibodies. there were no significant differences identified between hla classes of the dsa and any of the evaluated pathologic findings. taken together, this study identified capillary inflammation, acute lung injury, and endotheliitis as morphologic features in lung allograft biopsies that correlate with the presence of circulating dsa. however, none of these histopathologic features were specific to patients with dsa. morphologic findings of acute lung injury with diffuse alveolar damage had the highest odds ratio for the presence of circulating dsa. this study also cautioned the usefulness of c4d immunohistochemical stain for the diagnosis of amr in lung allografts because of its infrequent diffuse positivity. although the study shows that some morphologic features correlate with the presence of circulating dsa and, therefore, might be histopathologic markers to at least suggest the possibility of amr, the reproducibility of these morphologic features is quite problematic even among experienced lung transplant pathologists. in fact, the interobserver reproducibility kappa values ranged between 0.14 and 0.4, indicating a less than a chance to moderate agreement. the lowest agreement was noted for suspicion for aspiration (median kappa, 0.14) and the highest for acute cellular rejection, alveolar hemosiderosis, and c4d staining (median kappa, 0.4, all). although a definite diagnosis of amr seems to elude pathologic interpretation at the current time, in a fully contextualized clinical environment, the findings from the biopsy specimen may aid the clinician to make a reasonable diagnosis of amr if other relevant clinical and serologic features are present. the proposed "triple test" [78] of clinical features, serologic evidence of dsa, and pathologic findings supportive of amr including capillary inflammation, acute lung injury with or without diffuse alveolar damage, and endotheliitis may currently be the best guide to the diagnosis of amr. there is no ihslt recommendation at this time regarding the coexistence of amr and acute rejection, but it clearly does occur. hyperacute rejection is a severe form of amr mediated by preexisting antibodies to abo blood groups, hla class i or ii, or other antigens on graft vascular endothelial cells. this rejection occurs within minutes to a few hours after the transplanted organ begins to be perfused. as in any form of amr, the preexisting antibodies can result from previous pregnancies, blood transfusions, or previous transplant, and their binding to donor antigens provokes complement and cytokine activation resulting in endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. the outcome is commonly fatal. in hyperacute rejection, lungs are edematous, cyanotic, and heavy, have a firm consistency, lack crepitation, and show red hepatization [80] [81] [82] [83] . the cut surface reveals patchy poorly defined areas of hemorrhagic consolidation. anastomoses are intact and typically widely patent. histologically, alveolar hemorrhage, platelet and fibrin thrombi, neutrophilic infiltration, necrosis of vessel walls, and diffuse alveolar damage are observed [76-80, 83, 84] . c4d deposition has been described. although hyperacute rejection is a wellknown complication in kidney and heart transplantations, in lung transplantation, it appears to be rather rare with only eight cases reported. six patients died within 1 h and 13 days after transplantation [80] [81] [82] [83] [84] [85] . only two patients survived [86, 87] . one of these two patients was treated with plasmapheresis, antithymocyte globulin, and cyclophosphamide immediately after hyperacute rejection was diagnosed [86] . the other patient was highly presensitized when he underwent double lung transplantation [87] . this patient was treated with multiple plasma exchanges and intravenous immunoglobulin pre-and posttransplantation together with posttransplant rituximab and bortezomib and later with anti-c5 antibody and eculizumab. although in pretransplant, panel reactive antibodies (pras) were negative in four of the eight reported patients, crossmatch was positive in all reported cases. collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative pras may occur and pretransplantation crossmatch is not often possible [80] . at least five pieces of well-expanded alveolated parenchyma are required for adequate evaluation of a transbronchial lung allograft biopsy specimen for acute rejection by the lrsg [34] . this specimen requirement was based on the "uniform opinion of the consensus meeting." to ensure that the minimum number of required pieces of alveolated lung parenchyma is available for pathology review, it is recommended that the bronchoscopist needs to take more than five pieces. even more pieces might be necessary to provide small airways for review. interestingly, a prospective 12-month single-operator study by scott and colleagues [88] including 219 transbronchial allograft biopsies with 6 to 56 samples per procedure (mean 17.3 samples per procedure) taken from 3 lobes (or 2 lobes and the lingula of 1 lung) of 54 heart-lung transplant and 2 single lung transplant recipients revealed a sensitivity of 94% and a specificity of 90% for identification of rejection by histopathology. this study estimated that 18 samples per procedure are needed to have a 95% confidence of finding rejection. therefore, false-negative results due to patchy distribution of acute rejection are likely not uncommon. the absence of histologic and immunophenotypic features of acute rejection or antibody-mediated rejection requires clinicopathologic correlation as a negative biopsy does not necessary rule out rejection. furthermore, the bronchoscopist should be familiar with imaging studies, especially high resolution computed tomography studies if available, and aim to sample radiologically abnormal bronchopulmonary segments. if such imaging was not recently performed or the results are normal, then samples should be obtained from different lobes to try to minimize sampling error. specimens should be gently agitated in formalin to open up the alveoli. there is currently no recommendation for cryobiopsies. in a recent study using cryobiopsies to evaluate rejection in lung allografts, a median of three pieces provided twice as many alveoli and small airways than a median of ten pieces by conventional forceps biopsy [31] . the ishlt recommends a minimum of three levels from the paraffin block for hematoxylin and eosin (h&e) staining for histologic examination [34] . in addition, "connective tissue stains" such as trichrome or verhoeff-van gieson (vvg) stain are recommended to evaluate airways for the presence of submucosal fibrosis and vessels for graft vascular disease. stains for microorganisms including gomori-grocott methenamine silver stain (gms) and acid fast bacilli (afb) may be added. while silver stains are routinely performed on lung allograft biopsies in some institutions, they are currently not mandated by the lrsg because many microbiologic, serologic, and molecular techniques are available and used to identify infections in these patients [34, 89] . bal may be performed at the time of biopsy and is useful for the exclusion of infection but currently has no clinical role in the diagnosis of acute rejection. the transbronchial allograft biopsy is currently the gold standard to evaluate the graft for cellular rejection and to exclude its clinical mimickers in lung transplant patients. when reviewing transbronchial biopsy material of these patients, attention must be paid not only to features of rejection but also to its morphologic mimickers, especially infection, ptld, and abnormal drug effect. before a diagnosis of acute cellular rejection can be rendered, an infectious process should be excluded by using stains for microorganisms and/ or clinical tests including cultures of bal and/or tissue and serology. while studies to identify histopathologic and immunophenotypic features of amr are evolving, there are currently no specific morphologic findings, and clinical and serologic correlations are required for the diagnosis. prospective, well-designed long-term studies with longitudinal data of therapeutic intervention of acr on histopathology in totally asymptomatic patients with no physiological or hrct evidence of allograft dysfunction are needed to determine the clinical significance and relevance of such interventions. acute rejection and humoral sensitization in lung transplant recipients evidence for immune responses to a self-antigen in lung transplantation: role of type v collagen-specific t cells in the pathogenesis of lung allograft rejection the registry of the international society for heart and lung transplantation: thirty-second official adult lung and heart-lung transplantation report-2015; focus theme: early graft failure acute allograft rejection: cellular and humoral processes severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation bronchiolitis obliterans syndrome and restrictive allograft syndrome: do risk factors differ? mismatches at the hla-dr and hla-b loci are risk factors for acute rejection after lung transplantation does human leukocyte antigen matching influence the outcome of lung transplantation? an analysis of 3,549 lung transplantations influence of human leukocyte antigen matching on long-term outcome after lung transplantation the registry of the international society for heart and lung transplantation: twenty-seventh official adult lung and heart-lung transplant report-2010 predictors of acute rejection after lung transplantation interleukin-10 production genotype protects against acute persistent rejection after lung transplantation the impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients rejection is reduced in thoracic organ recipients when transplanted in the first year of life paediatric incidence of acute rejection and obliterative bronchiolitis: a comparison with adults the registry of the international society for heart and lung transplantation: thirtieth adult lung and heart-lung transplant report-2013; focus theme: age are symptom reports useful for differentiating between acute rejection and pulmonary infection after lung transplantation? heart lung the registry of the international society for heart and lung transplantation: 29th adult lung and heart-lung transplant report-2012 low vitamin d levels are associated with increased rejection and infections after lung transplantation clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome chlamydia pneumoniae infection after lung transplantation risk factors for bronchiolitis obliterans: a systematic review of recent publications role of pulmonary function in the detection of allograft dysfunction after heart-lung transplantation limitations of spirometry in detecting rejection after single-lung transplantation acute rejection following lung transplantation: limitations in accuracy of thin-section ct for diagnosis exhaled nitric oxide in human lung transplantation: a noninvasive marker of acute rejection serial monitoring of exhaled nitric oxide in lung transplant recipients inhaled corticosteroids and the treatment of lymphocytic bronchiolitis following lung transplantation transbronchial cryobiopsies in the evaluation of lung allografts: do the benefits outweigh the risks? cryoprobe transbronchial lung biopsy in patients after lung transplantation: a pilot safety study transbronchial cryobiopsy in lung transplantation patients: first report revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: lung rejection study group. the international society for heart transplantation revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group reliability for grading acute rejection and airway inflammation after lung transplantation analysis of the different histologic lesions observed in transbronchial biopsy for the diagnosis of acute rejection. clinicopathologic correlations during the first 6 months after lung transplantation interpretation of transbronchial lung biopsies from lung transplant recipients: inter-and intraobserver agreement interobserver variability in grading transbronchial lung biopsy specimens after lung transplantation can immunohistological analysis of transbronchial biopsy specimens predict responder status in early acute rejection of lung allografts? alemtuzumab in the treatment of refractory acute rejection and bronchiolitis obliterans syndrome after human lung transplantation the role of transbronchial lung biopsy in the treatment of lung transplant recipients: an analysis of 200 consecutive procedures prospective analysis of 1,235 transbronchial lung biopsies in lung transplant recipients yield of surveillance bronchoscopy for acute rejection and lymphocytic bronchitis/bronchiolitis after lung transplantation association of minimal rejection in lung transplant recipients with obliterative bronchiolitis the significance of a single episode of minimal acute rejection after lung transplantation the diagnosis of obliterative bronchiolitis after heart-lung and lung transplantation: low yield of transbronchial lung biopsy evaluation of transbronchial biopsy in the diagnosis of bronchiolitis obliterans after lung transplantation transbronchial biopsy in heart and lung transplantation: clinicopathologic correlations an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome pulmonary hypertension associated with lung transplantation obliterative bronchiolitis and vascular remodeling of the allograft pulmonary hypertension in patients with bronchiolitis obliterans syndrome listed for retransplantation perivascular inflammation in pulmonary infections: implications for the diagnosis of lung rejection national conference to assess antibody-mediated rejection in solid organ transplantation antibody-mediated organallograft rejection hla-specific antibodies are associated with high-grade and persistentrecurrent lung allograft acute rejection development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome pretransplant panel reactive antibody in lung transplant recipients is associated with significantly worse posttransplant survival in a multicenter study utility of peritransplant and rescue intravenous immunoglobulin and extracorporeal immunoadsorption in lung transplant recipients sensitized to hla antigens anti-hla class i antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome peritubular capillary c4d deposition and renal outcome in post-transplant iga nephropathy capillary deposition of complement c4d and c3d in pediatric renal allograft biopsies immunohistochemistry staining of c4d to diagnose antibody-mediated rejection in cardiac transplantation acute humoral rejection in kidney transplantation: ii. morphology, immunopathology, and pathologic classification chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by c4d deposits in peritubular capillaries antibodymediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation products c4d and c3d as markers c4d deposition in cardiac allografts correlates with alloantibody update on cardiac transplantation pathology pancreas allograft biopsies with positive c4d staining and anti-donor antibodies related to worse outcome for patients reproducibility of complement 4d deposition by immunofluorescence and immunohistochemistry in lung allograft biopsies banff study of pathologic changes in lung allograft biopsy specimens with donor-specific antibodies c4d deposition in lung allografts is associated with circulating anti-hla alloantibody acute humoral rejection of human lung allografts and elevation of c4d in bronchoalveolar lavage fluid c3d and the septal microvasculature as a predictor of chronic lung allograft dysfunction c3d and c4d deposition early after lung transplantation pathology of pulmonary antibody-mediated rejection: 2012 update from the pathology council of the ishlt antibody-mediated rejection of the lung: a consensus report of the international society for heart and lung transplantation hyperacute rejection after single lung transplantation: a case report fulminant hyperacute rejection after unilateral lung transplantation hyperacute rejection of a pulmonary allograft. immediate clinical and pathologic findings susceptibility of lung transplants to preformed donor-specific hla antibodies as detected by flow cytometry hyperacute rejection following lung transplantation hyperacute rejection after lung transplantation caused by undetected lowtiter anti-hla antibodies hyperacute rejection in single lung transplantation-case report of successful management by means of plasmapheresis and antithymocyte globulin treatment treatment of hyperacute antibody-mediated lung allograft rejection with eculizumab prospective study of transbronchial biopsies in the management of heartlung and single lung transplant patients practical applications in immunohistochemistry: evaluation of rejection and infection in organ transplantation key: cord-006924-1i3kf01j authors: nan title: abstracts from uscap 2020: pulmonary, mediastinum, pleura, and peritoneum pathology (1869-1980) date: 2020-03-05 journal: lab invest doi: 10.1038/s41374-020-0400-0 sha: doc_id: 6924 cord_uid: 1i3kf01j nan conclusions: a panel of immunostains including bap1, mtap, cd5, cd117 and tdt can be useful in the distinction between thymomas and thymic carcinomas with only a minority of cases being inconclusive. feasible markers of cellular senescence on paraffin but the expression of p16 protein and a low ki67 are surrogate markers of that cellular stage. sclerosing pneumocytoma (sp) is a benign tumor with a combination of patterns and a characteristic dual population of surface cells and round cells. the first express ttf1 and ck and the last only ttf1. no driver mutations have been described in this lesion. we reviewed the histological characteristics and immunohistochemical findings of 4 ba from 3 patients and compared it to 1 sp and 1 adk (from the patient with 2 ba). the antibodies used were: ttf1 (ventana, clone 8g7g3), ki67 (ventana, , p16 (cintec, e6h4) and p40 (ventana, bc28) . we also performed next-generation sequencing (ngs) (thermofisher, panel oncomine solid tumor) for all the ba and the adk. results: all 4 ba displays high expression of p16 protein in over 80% cells and a proliferative index below 3%. one of the ba contained round cells ck-and ttf1+, similar to round cells in sp. sp, sp-like cells and adk did not show a significant p16 expression. see we have identified a sp-like component that has not been described in classic ba. ba may represent another kind of benign tumor with senescent features and oncogenic alterations, similarly to nevus or pylocitic astrocytomas. these findings can be relevant in the differential diagnosis with adenocarcinomas as well as in the understanding of the malignant transformation in lung tumors. further research in a larger number of cases is needed. conclusions: in summary this study of selected images suggests that the distinction between stas and artifacts is achievable and reproducible. further work on reproducibility is needed using glass slides and frozen sections. these data suggest that these originally proposed criteria to make the distinction between artifacts and stas are readily applicable. this likely has helped the many independent investigators who have performed studies demonstrating the clinical significance of stas. design: a retrospective multi-center retrieve was performed for a ten-year period. ninety cases of mm, 50 cases of non-small cell lung adenocarcinoma (nscla) and 50 cases of breast carcinoma (bc) were retrieved from the pathology archives. all slides were reviewed and reclassified by expert pathologists. a tissue microarray (tma) was built from three sections of each case, resulting in 570 cores. immunofluorescence (if) was performed with a col (v) in-house clone (described by atayde et al, plos 2018) . images were captured through a microscope camera and the percentage of the positive area was accessed by threshold properties in the imagej software. patterns of deposition were defined as fibrillar (a linear pattern in fine bundles of intercellular deposition), surrounding (a membrane pattern, surrounding and isolating each cell) and mixed. for the mm tma, a double col (v) d2-40 stain was also performed to enhance mesothelial cell specificity. statistical analysis was performed using spss 25 by anova followed by spearman's test. a p-value of less than 0.05 was considered significant. the analysis was possible in 419 spots (74% of total):126 nscla (84%), 102 bc (68%), 229 mm (70%). the fibrillar type was expressed in 117 (93%) of nscla, 100 (98%) of bc and 9 (0.05%) of all spots of mm. the surrounding pattern was expressed in 182 (95%) of mm spots (p-value <0.05). the mixed pattern was expressed in 9 (7.7%) and 2 (1.2%) of nscla and bc spots, and when present, fibrillar dominated. average col (v) expression was: 3.58% (nscla), 12.1% (bc), and 10.65% (mm). lung expressed significantly less than the other groups (p<0.05). than that of patients with zeb1-negative tumors (781 days and 1798 days, respectively; p = 0.008). among epithelioid tumors, median survival of patients with zeb1-high tumors was significantly shorter than that of patients with zeb1-low tumors (476 days and 1798 days, respectively; p = 0.001). conclusions: as expected, biphasic morphology was associated with zeb1 staining and poorer survival. zeb1-positive epithelioid tumors had shorter median survival. furthermore, strong and diffuse positive staining in epithelioid mesotheliomas was associated with worse prognosis. use of this immunohistochemical stain may help to risk-stratify patients with epithelioid mesotheliomas, even in the absence of sarcomatoid morphology. results: using the above criteria, we identified 19 cases, of which 11 were diagnosed as lam on histology. all cases were females, ranging from 25-62 years, with an average age of 44 years (table 1 ). 1. the vegf-d levels ranged from 85 to 794 pg/dl. no case was above the present diagnostic cut-off of 800 pg/ml. all 7 cases with vegf-d levels >400pg/ml were diagnosed as lam on histology. 7 out of 9 cases with vegf-d levels <400 pg/ml did not have lam. (ppv=100%, npv=77.7%) 2. all positive cases had a typical radiological findings of multiple, diffuse, bilateral small cysts, usually <1cm. radiological findings in cases not diagnosed as lam were variable, including predilection for lower lobes, coexisting nodular lesion, varying size cysts, etc. 3. cases where the radiology was characteristic but histologically not proven to be lam, were noted to have vegf-d levels of <300pg/ml age sex vegf-d (pg/ml) radiological findings histopathology it is important to correlate radiological findings and vegf-d levels when considering the need for a surgical lung biopsy, as those cases with non-characteristic radiological findings and vegf-d <400pg/ml were unlikely to be diagnosed as lam on histology. considering that all patients with biopsy proven lam had a vegf-d levels of greater than 400 pg/ml, we speculate that this level might be used as new cut-off for triaging patients with cystic lung disease for a biopsy, in an appropriate clinical setting. lorelle brownlee 1 , robert bentham 2 , nicholas mcgranahan 3 , charles swanton 4 , david moore 2 , mariam jamal-hanjani 2 , tracerx consortium 5 results: 45 patients were identified. on fu (mean 31 months) 6 cases (13.3%) had a final diagnosis of malignancy (2 adenocarcinomas, 1 squamous cell carcinoma, 1 malt lymphoma, 1 dlbcl, 1 nsclc). of these, 3 were diagnosed on repeat cnb, 1 on wedge resection after repeat benign cnb and 2 on fu fna. other 11 cases (24.4%) with repeat biopsy and 2 cases (4.4%) with fu resection were benign (1 wedge resection and 1 lobectomy, both nodular lymphoid hyperplasia). all nodules were solid (86.7%) or part-solid on ct. most benign and all malignant cases had spiculated (24.4%) or irregular margins (55.1%). initial biopsies in malignant cases had either inflammatory (5/6) or lymphoma-like pattern (1/6). all cases with scar-like pattern (7/45, 15.5%) were benign. shorter solid lesional component on cnb correlated with final benign diagnosis (p=0.01; solid component of all malignant cases measuring ≤10 mm). cases with final malignant diagnosis also had cnb with shorter lesional tissue and higher suvmax, which approached statistical significance despite low number. conclusions: combined radiological-pathological approach may improve the diagnostic accuracy of patients with focal lung lesions and non-specific inflammation on initial cnb. good representation of the solid component of the lesions is associated with final benign diagnosis and should be considered when sampling and making management decisions. matthew cecchini 1 , tara tarmey background: many forms of interstitial lung disease (ild) are diseases of aging secondary to repetitive injury or exposures that causes an abnormal cellular senescence. a subset of ild occurs in patients with short telomeres who can also have premature greying of hair, liver fibrosis and bone marrow failure. cells with critically shortened telomeres can enter into a cellular senescence mediated in part by upregulation of the cdk inhibitor p16. despite our increasing understanding of the underlying pathogenetic mechanisms, histopathologic and radiologic features of ilds in patients with confirmed short telomeres have not been well characterized. design: cases diagnosed as positive for short telomeres (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) (2019) were identified by testing of peripheral blood granulocytes and/or lymphocytes with 10 th percentile of telomeres or less. lung explants or wedge biopsies from the patients with short telomeres were reviewed independently by 2 pathologists using defined morphologic parameters. discordant cases were reviewed by a third pathologist. high resolution ct scans were reviewed by 2 radiologists independently. immunohistochemistry for p16 (clone e6h4) was performed on a section of each case showing the most areas of active fibrosis with abundant interfaces between fibrotic and architecturally preserved lung parenchyma. all foci showing p16 positivity in both epithelial cells and associated fibroblasts were counted. cases without short telomeres were used as controls. the morphologic and radiologic features of cases with short telomeres (n=15) as compared to those without short telomeres (n=5) are outlined in table 1. among 8 cases with short telomeres that were tested for mutation, 5 (62.5%) cases had a germline mutation in a gene related to telomere maintenance (naf1, tert, rtel1, terc). the cases in the short telomere group often demonstrated features atypical for usual interstitial pneumonia (uip) on both histopathologic and radiologic examination. nine of 15 (60%) cases with short telomeres were classified as non-uip, while 3 of 5 (60%) without short telomeres were diagnosed as uip. the average number of p16-positive foci was higher in the cases with short telomeres though not statistically significant (p=0.4) ( table 1) . the majority of ilds in patients with short telomeres showed morphologic and radiologic features atypical for uip and were often diagnosed as chp or unclassifiable ild. both groups with and without short telomeres demonstrated p16-positive foci. we have recently reported on the utility of comprehensive next-generation sequencing (ngs) for distinguishing separate primary lung carcinomas (splc) from intrapulmonary metastases (ipm) in clinical practice. here, we report on detailed review of histologic challenges in determining the relationships between multifocal adenocarcinomas using ngs as a gold standard. design: a total of 70 surgically-resected adenocarcinoma pairs underwent molecular profiling using 341-468 gene hybridization-capture based ngs assay. comparative genomic profiles were used to stratify tumor pairs into clonally-unrelated (splc) and clonally-related (ipm). relationship of tumor pairs predicted by prospective histopathologic assessment was compared with ngs-based classification. histopathologic features contributing to challenges in distinguishing tumor relationships were assessed retrospectively. of 70 adenocarcinoma pairs, ngs classified 47 as splcs and 23 as ipms. prospective histologic prediction was discordant with ngs in 16 cases (23%), with significantly higher histologic misclassification rate for ngs-confirmed ipms than splcs (43% vs 13%, p=0.0003). the discordance rate was significantly higher when histologic prediction was regarded as potentially equivocal by a pathologist and confirmation by ngs was suggested (p=0.02). retrospective review highlighted several specific factors contributing to misinterpretation of ngs-defined ipms as morphologically unrelated tumors, including 1) morphologic progression leading to higher proportion of solid and micropapillary patterns in secondary tumors (n=6), and 2) presence of significant amounts of non-predominant lepidic pattern in both primary and secondary tumors (n=4). ngs-defined splcs that were initially misinterpreted as morphologically related tumors (n=6) showed closely overlapping architectural or cytologic features. comprehensive histopathologic assessment is adequate for distinguishing splcs from ipms in most cases, but has notable limitations in the recognition of a subset of cases, particularly ipms. our results support the adoption of molecular testing to supplement histologic assessment for robust discrimination of clonal relationships of multifocal adenocarcinomas in clinical practice, and we propose an algorithm incorporating specific histopathologic scenarios where molecular profiling may be most helpful. background: malignant mesothelioma (mm) is rare but lethal; some affected individuals develop mm in the setting of predisposing inherited mutations. our group found a 12% prevalence of pathogenic or likely pathogenic variants in prospectively tested mm patients. these patients showed longer survival after platinum therapy, suggesting distinct biology. germline mutated bap1 mm, the most prevalent mutated gene in mm, appears to be more indolent than wild-type. this study aims to compare the morphology of epithelioid (e-) mms with germline mutations to those without. design: eligible patients with pathologically confirmed mm underwent panel-based hereditary cancer susceptibility germline genetic testing. there were two control groups with no germline mutation matched by mm histologic subtype, age at diagnosis, and sex; 22 patients had retained bap1 and 9 had loss of bap1 on ihc. in e-mm, nuclear grade (3-tiers comprised of mitotic count and nuclear grade), presence of necrosis (yes/no), and patterns were compared using graphpad prism via fisher's exact tests. results: of 265 mms tested, 25 had germline mutations; an additional 5 cases were from other institutions (n=30). pathology was unavailable in 7. of the 23 left (table 1) , there were 22 e-mm and 1 biphasic mm with 10 gene variants. bap1 was most common (n=9). among all mms with a nuclear grade of 1 (n=79), 15 (19%) had a germline mutation. the e-mms had a lower nuclear grade than those without (p=0.01). the mitotic count was a significant contributor to lower grade (p=0.03). by site, there was a trend in lower grade for peritoneal mms (n=13) between germline mutated and non-mutated cases (p=0.06); that difference was not observed for pleural mms (n=9, p=0.24). when focusing on bap1, lower nuclear grade was also present when comparing bap1 mutated mms to sporadic bap1 ihc loss mms (p=0.05) and bap1 ihc retained mms (p=0.05). there was a trend of lower mitotic count in the bap1 germline mutated cases compared to the bap1 ihc loss and the retained cases (p=0.16, p=0.13). there was a trend of less necrosis in bap1 germline mutated mms vs mms with retained bap1 (p=0.11). solid and trabecular patterns were most frequently observed in both study cases and controls. background: lung cancer has been the most prevalent and the most deadly malignant cancer globally, which is still incurable disease in essence. immunotherapy targeting pd-1/pd-l1 represents a breakthrough in the treatment of lung cancer. nevertheless, the response rate of anti-pd-1/pd-l1 immunotherapy remains unsatisfactory, due to tumor resistance and complexity of immune microenvironment. to enable more patients to benefit from immunotherapy, a thorough understanding of the regulatory mechanisms of pd-l1 expression will be pivotal for novel combinational immunotherapies. pyruvate kinase m2 (pkm2) is a critical player of glycolysis, conducing to tumor progression and immune response. however, the correlation and clinical significance of pkm2 and pd-l1 expression in human lung adenocarcinoma (luad) remain not entirely explored. design: expression of pkm2 and pd-l1 were detected by immunohistochemistry in 74 cases of luad and the corresponding noncancerous tissues. simultaneously, multiplex immunofluorescence was used to detect pkm2, pd-l1, cd3, cd68, cd163 and pan-ck by using the opal 7-color ihc kit, combined with multi-spectral imaging system and inform software. we measured expression patterns and co-localization of these targets, evaluating their correlation with clinicopathological features and overall survival. validation of findings was conducted using mrna expression data from the cancer genome atlas (tcga) of 515 lung adenocarcinoma cases. results: co-expression of pkm2 and ck, cd3, cd68 were found. pd-l1 protein expression was detected in the tumor cells and immune cells including t cells and tumor associated macrophages (cd68+cd163+). high expression of pkm2 in tumor cells (tcs) was significantly related to lymph node metastasis and tnm stage. moreover, pkm2 expression in tcs was positively correlated with pd-l1 expression in tcs. high expression of pkm2, pd-l1, as well as both pkm2 and pd-l1 in tcs and immune cells predicted high mortality rate and worse survival, respectively. additionally, multivariate cox regression models indicated that high expression of pkm2 in tcs was an independent prognostic factor. based on tcga genomic data, high pkm2 mrna expression was significantly associated with poorer survival. background: immunotherapeutic agents have revolutionized the standard of care in patients with advanced non-small cell lung cancer (nsclc) and have emerged as novel treatment strategies as they have demonstrated promising treatment advantage over traditional chemotherapeutic agents. immunohistochemical (ihc) detection of pd-l1 expression in nsclc cases is considered to be a clinical decision-making tool to support the use of checkpoint inhibitors in nsclc patients. fda-approved assays and other antibodies are commercially available. this validation study is designed to compare a commercially available assay (28-8) with another clone (cal10), and to also determine the distribution patterns of expression of pd-l1 and pd1 in the tumor and tumor-associated environment. design: 134 consecutively diagnosed nsclcs with available whole tissue sections were evaluated for the study, which includes testing with the fda-approved pd-l1 assay 28-8 (clone 28-8; agilent/dako), pd-l1 clone cal10 (biocare medical) and pd1 clone (nat105). whole sections were chosen over tissue microarrays to adequately assess intra-tumor heterogeneity. deparaffinized tissue sections were pretreated using either online (for the two non-fda approved tests) or offline (for 28-8), followed by antibody incubation, polymer detection and dab visualization. for the 2 pd-l1 assays, the 2019-revised fda tps scoring criterion of ≥1% was employed. for the pd1 assay, immunoe cell score was employed. results: 50% of tumors (n = 67) showed tps positive expression of ≥1% using 28-8 as a gold standard assay for pd-l1. agreement between 28-8 and cal10 was 100%, with a consistently higher tps score observed with cal10 compared to 28-8. immune cell score of nat105 anti-pd1 antibody was positive in 8% of tumors (n = 12). when comparing the distribution of pd-l1 and pd1 expression on the same tumor, out of the 12 pd1 positive cases, 4 showed positive pd-l1 expression, and 8 showed a mutually exclusive expression with only pd1 positive expression. the validation study shows that pd1 and pd-l1 expression on the same tumors in nsclc is uncommon, a phenomenon that can be exploited to expand targeted therapy treatment strategies in this setting, and could possibly be expanded into other oncology settings. it also demonstrates the slightly higher sensitivity of detection achieved by clone cal10 compared to 28-8 using the tumor proportion score, which warrants an expanded evaluation. swi/snf complex, sensitizes smarca4-mutant lung tumors to chemotherapy (fillmore, nature 2015). however, the expression of ezh2 in smarca4-mutant lung tumors is unknown. we reviewed a cohort of 282 thoracic specimens sampled between 2014-19 that previously underwent next generation sequencing (ngs) of over 320 cancer-associated genes as part of routine clinical care. smarca4 frameshift, nonsense and splice site mutations were defined as loss of function (lof). ezh2 immunohistochemistry (ihc) was performed and expression was quantitatively assessed on tumor cells based on the extent of staining as follows: loss (0), weak (1+), moderate (2+) and strong (3+). a cohort of 5 lung carcinomas without smarca4-mutations were also stained as controls. we identified 13 patients (mean age 62, range 42 to 81 years, 64% male) with smarca4-mutant lung cancers (incidence 4.6%). lof mutations were present in 8 (62%) cases and included 3 nonsense, 3 frame shift and 2 splice site variants. two additional cases had smarca4 duplications. all but two cases (92%) were lung adenocarcinomas, with 1 poorly differentiated non-small cell carcinoma and 1 sarcomatoid carcinoma. all cases with lof mutations exhibited a solid growth pattern and 40% presented as distant metastatic disease. tumor tissue was available for 8 cases; ezh2 was expressed in 100% of cases with confirmed smarca4-mutations (mean 2.5 intensity). of 5 smarca4 non-mutated tumors, only 1 tumor demonstrated ezh2 expression (mean 0.2 intensity, p<0.01) conclusions: ezh2 is overexpressed in smarca4-mutant lung cancers and may serve as a potential prognostic and predictive factor for those treated with epigenetic inhibitor therapy. this is the first study to assess ezh2 expression in this subset of tumors that are known to be resistant to conventional chemotherapy. results: oncogenic rearrangements were found in 16/58 samples (28%) with contributive analyses. the most frequent event was a kif5b-ret translocation (6 patients). we also found 4 ros1 cases (cd74 and ezr), 2 nrg1 (slc3a2 and flywh1), 2 egfr (vopp1), 1 alk (eml4) and 1 ntrk1 (egfr). moreover, 4 patients had met exon14 skipping confirmed by the identification of a splice mutation. all but egfr-vopp1 fusions were targetable (18/20, 88%). ret fish was negative or equivocal in 3/6 cases, ros1 fish was negative in 1/4 positive case. alk (5a4) and ntrk1 ihc were negative in both positive cases. patients with fusions were younger than usually described and the sex ratio was close to 1. the proportion of smockers was higher in the group without oncogenic events (p = 0.045). patients without an oncogenic event were more likely to express pd-l1 (p < 0.05). five patients were treated, with partial response. the clinicoradiologic presentation of these four cases are similar to those recently reported in the literature. our small series suggest that vapi is characterized by organizing acute lung injury and should be considered in the differential diagnosis of diffuse alveolar damage of unknown etiology, particularly in younger patients. while foamy macrophages were found in 3 cases, the characteristic features of lipoid pneumonia were not evident on histologic examination. as such, the significance of lipid-laden macrophages on bal requires further investigation. background: congenital pulmonary airway malformations (cpams) are rare developmental malformations of airway branching in the lung, typically diagnosed antenatally. they are classified into 4 subtypes (type 1, type 2, type 3, and type 4) based on their histologic appearance. their origin, natural history, and genetics remain poorly understood as does their malignant potential. a subset of type 1 cpams are thought to possess malignant potential and eventually go on to develop into invasive mucinous adenocarcinoma. these lesions are characterized by areas of mucinous epithelium that can often represent a very small fraction of the total type 1 cpam. it remains to be determined if type 1 cpam lesions with mucinous epithelium are molecularly distinct from type 1 cpams that lack mucinous epithelium. design: a total of 10 type 1 cpams and 10 type 2 cpams with available slides and tissue blocks were identified. each type 1 cpam was assessed for the presence or absence of mucinous epithelium. four cases of type 1 cpam had microscopic evidence of mucinous epithelium and these areas were separately micro-dissected from areas without mucinous epithelium. genomic dna was extracted and used for molecular testing. capture-based next generation sequencing was performed at the ucsf clinical cancer genomics laboratory, using an assay (ucsf500 panel) that targets the coding regions of 480 cancer-related genes, select introns from approximately 40 genes, and the tert promoter with a total sequencing footprint of 2.8 mb results: among the four type 1 cpams that contained mucinous epithelium, a pathogenic hotspot mutation in kras codon12 was detected in all four cases. this pathogenic mutation was detected in both the mucinous and non-mucinous epithelium at roughly similar mutant allele frequency. none of the remaining 6 cases of type 1 cpams that lacked mucinous epithelium or the 10 type 2 cpams contained a kras mutation. results: biopsies with dsa had a statistically significant difference versus nabs with regards to ali (p=0.0421), presence of capillary neutrophilic inflammation (p= < 0.00001), and the presence of asw (p=0.0007). similar to earlier reports, we found a positive correlation between the capillary neutrophils, ali, and dsas. frequent asw was noted in our patients with dsas and was an easily recognized feature at low-power magnification. endothelialitis was not appreciated in any of our cases, likely due to a small sample size. complement 4d (c4d) was available in 16.6 % of specimens and showed only focal staining (<50%). c4d, acute cellular rejection, and airway inflammation did not reveal any significant statistical association with dsa status. conclusions: ali, capillary neutrophilic inflammation, and asw were morphological features found to be statistically significant in this small sample size study. these are not pathognomonic, and currently, the triple test (clinical allograft dysfunction, dsas, pathologic findings) is the best approach for the diagnosis of amr. further research investigating asw, exploring its likelihood to be included among conventional histopathologic patterns prompting clinical and serologic evaluation is needed. figure) , storiform pattern, myofibroblastic proliferation, granulation tissue, maximum nuclear length (defined in relation to lymphocyte size, right figure), nuclear atypia (grades 1-3), mitotic score of 1(0-1/2mm 2 ), 2(2-4/2 mm 2 ) or 3(≥5/2 mm 2 ) and infiltrating inflammatory cells (%). overall survival (os) was determined by kaplan-meier method and compared between groups using log-rank test. results: invasion, bland necrosis, proliferation nodules, nuclear atypia (grade >2), mitotic score (>2), nodular growth and storiform pattern showed 100% specificity (all p<0.001) but variable sensitivity (75%, 67%, 69%, 81%, 88%, 89% and 96%, respectively) for mm. nuclear length was significantly greater in mm than in cfp (median 4.0 vs 3.0x size of lymphocyte, respectively; p<0.0001). myofibroblastic proliferation and granulation tissue were more frequently observed in cfp (69% and 77%, respectively) than in mm (both 19%) (p=0.001 and p<0.001, respectively). percentage of infiltrating inflammatory cells was not significantly different (p=0.3) between mm and cfp (median 10% and 15%, respectively). there were no significant differences in gender (p=0.4), age (p=0.6), stage (p=1) and os (p=0.13) between patients with sarcomatoid mm (n=26) and dmm (n=28, defined as >50% desmoplastic component by who). conclusions: in the distinction from cfp, several features appear specific for mm, among which nuclear atypia, mitotic score, nodular growth and storiform pattern are most sensitive. myofibroblastic proliferation and granulation tissue, while not exclusive to cfp, may be a reliable indicator of benignity in absence of features specific for mm. based on this limited cohort, there appear to be no significant survival differences in patients whose tumors meet who criteria for dmm compared to those with mm showing <50% desmoplastic component. mona farahi 1 , alain borczuk 2 , kartik viswanathan 3 , michael kluger 4 , hanina hibshoosh 5 background: well-differentiated papillary mesothelioma (wdpm) is a rare indolent mesothelial tumor that is usually found incidentally in the peritoneal cavity. there is controversy about the prognosis of the disease with few reported cases of invasive and malignant behaving wdpm. thus, some consider it a tumor of uncertain malignant potential. in contrast, diffuse malignant peritoneal mesothelioma (dmpm) is an aggressive tumor associated with asbestos exposure with a poor prognosis. the distinction between these two entities relies heavily on histopathological findings. prior studies identified traf7 and cdc42 (mutually exclusive) somatic mutations in wdpm. l1 cell adhesion molecule (l1cam) an nf-kb activated gene was overexpressed in wdpm. the goal of the present study is to assess the diagnostic utility of l1cam in a larger cohort of peritoneal mesotheliomas. design: immunohistochemistry utilizing mouse monoclonal anti-l1cam antibody (clone uj127.11, sigma, l4543, 1:1000 dilution) was performed on formalin-fixed, paraffin-embedded tissue sections in 197 total cases consisting of: 41 cases of biphasic/sarcomatoid dmpm, 16 cases of wdpm, 8 cases of benign multicystic mesothelioma (bcm), and 132 cases of epithelioid dmpm. the staining was scored as either positive or negative but since 15% was the minimum percentage to show complete and intense membranous staining among the wdpm, this was chosen as the true positive cutoff. background: adenosquamous lung carcinoma (asc) is a rare yet aggressive malignancy thought to potentially arise from a bipotential undifferentiated precursor cell. a topic of controversy until coined a distinct entity in the 2015 5 th edition of the who classification of lung cancer, asc is defined as a tumor with two separate malignant populations (>10% glandular and squamous components). the coexistence of glandular and squamous differentiation within the same tumor cells has also previously been reported (pelosi, journal of thoracic oncology 2015) and termed "asc immunophenotype" due to its amphicrine phenotypic nature. the single case report showed dual ttf-1 and p40 immunohistochemical (ihc) expression, along with specific electron microscopy and molecular findings. we report here for the first time a case series with dual differentiation and further characterize the findings with molecular analysis. design: from 58 primary lung ascs diagnosed between 2017 and 2019 at our institution, we identified 7 patients (mean age 72.1 years) with dual glandular and squamous differentiation within the same population of malignant tumor cells. ihc stains for ck5/6, ck7, ttf-1, p40, and p16 were performed. the ion ampliseq tm cancer hotspot panel v2 (chp2) and variantplex myeloid kit (archerdx) next generation sequencing (ngs) assays targeting 125 oncogenes were performed on two cases. fluorescence in-situ hybridization (fish) was also performed on the two sequenced cases. ascs with dual differentiation occurred predominantly in women (85.7%, age range 60 to 85 years) with variable smoking history. all cases were poorly differentiated with a solid growth pattern and 43% (3/7) presented with distant metastasis. the following ihc stains were positive in the tumor population: ck5/6 (4/4, 100%), ck7 (3/3, 100%), ttf-1 (7/7, 100%), p40 (7/7, 100%), and p16 (7/7, 100%). ngs revealed cdkn2a and tp53 mutations in both cases; one cdkn2a variant is known to disrupt p16ink4a-and p14arf-dependent regulation of cdk4/6 and p53. loss of function atrx, bcorl1 and notch1 mutations were also identified. fish revealed cdkn2a mutations and relative loss of 9p21 in one case, and chromosome 9 monosomy in the other. we report for the first time a case series of these rare lung ascs with dual differentiation and show an association with cdkna2 mutations and corresponding p16 overexpression. background: there is considerable interobserver variability in the pathological diagnosis of uip and other ild. the variability in finding "ground truth" presents a difficult problem during the development of deep learning platforms designed to diagnose ild using ai. we describe the use of a smartphone application that allows for the collection of multiple opinions from sixteen pulmonary pathologists from nine countries as a novel method for finding "ground truth" for uip diagnoses. design: whole slide images from 14 consecutive interstitial lung disease cases were scanned by aperio cs2 at 20x objective and sliced into multiple individual 7x7mm images, resulting in 355 jpeg image patches. these individual images were randomized and shown over the internet to 16 expert pathologists from 9 countries using the novel smartphone application, bonbon system. expert lung pathologists were asked to classify each image into one of 8 categories: uip/ipf; ctd/uip; chp/uip; uip/other cause; non-uip; "not sure"; "normal"; and "exclude". images classified as "exclude" by any of the experts were deleted from analysis. all individual diagnostic classes selected for individual images were grouped by case, and the predominant class for each case, was used as "diagnosis". all diagnoses were analyzed using clustering analysis and kaplan meier statistics using jmp. interobserver agreement was calculated by fleiss kappa coefficient using r. the diagnosis of each of the 14 cases by each of the 16 pathologists showed that interobserver agreement using the 7 categories was poor (k=0. 19) . none of the cases were classified as chp-uip or uip/others by a majority of pathologists. in order to simplify the diagnoses into more clinically relevant classes, the categories uip/ipf, chp-uip, and uip/others were grouped into uip and non-uip and ctd-uip into non-uip as shown in figure 1 . clustering analysis stratified the diagnoses of each case into 3 clusters as shown in figure 2a (k= 0.77, 0.63, and 0.03, respectively), validating cluster a and b were meaningful. log rank test showed significant survival difference between uip and non-uip groups only for cases in cluster a (p=0.017) ( fig 2b) but not for cases in cluster b (fig 2 c) . eventually, 269 h&e images with high agreement of uip and non-uip group were selected as "ground truth" for ai training. conclusions: classification of image patches from wsi over app offers a useful method to standardize uip diagnosis and to develop diagnostic ai. results: precision, reproducibilty and lod were assessed using hd300 in triplicate by 2 operators on 2 instruments. the 27 ffpe and hd300 were used for accuracy. idylla™ results were in complete agreement with those obtained by ngs for egfr mutations targeted by idylla™ except for one sample. these include l858r (1), g719a (3), g719c (2), l861q (1), s768i (2), l861q (1), t790m (3), exon 19 deletions (16) and exon 20 insertion (1). the discrepant sample had an exon 19 deletion identified by ngs but was wild-type by idylla™. upon review of ngs data, an egfr synonymous snv was present downstream of the deletion which might impair the probe binding, leading to false negative results. lod of 5% vaf and tumor content of 10% were confirmed. no egfr mutations were detected by idylla tm in the samples determined by ngs as having wild-type egfr. the fully automated idylla™ system offers rapid (turnaround time of approximately 2.5 hours) and reliable testing of clinically actionable mutations in egfr directly from ffpe tissue sections. in our center, it will complement ngs testing by providing rapid egfr results within 1-2 days of diagnosis. background: identification of patients with synchronous lung adenocarcinomas has crucial implications for their staging and clinical management. these tumors are thought to represent independent primary neoplasms, but their oncogenesis is still poorly characterized. a few studies based on the analysis of limited panel of genes suggest that synchronous tumors have a high level of genetic heterogeneity. we sought to investigate the genetic mutations in a well-defined cohort of synchronous adenocarcinomas using whole-exome sequencing (wes). design: a retrospective cohort of 34 caucasian patients that underwent surgical resections for synchronous lung adenocarcinomas between 2013 and 2016 in our center was selected. all slides were reviewed by a thoracic pathologist to confirm the diagnosis based on a comprehensive histologic assessment. clinical characteristics and outcome were extracted from the electronic medical files. for each patient, three formalin-fixed paraffin embedded blocks were selected: one from two synchronous tumors and one from normal lung tissue. dna was extracted and samples were submitted to wes on an illumina hiseq platform (agilent sureselect xt). data processing was made according to gatk best practices and somatic variants (single nucleotide variants and insertion-deletions) were called using strelka and mutect. the cohort comprised 44.1% men with a mean age of 65.0 years, 91.2% had a stage i or ii disease and 94.1% were smokers or ex-smokers. mean follow-up was of 38.4 months with an 82.4% overall and 67.7% progression-free survival rates. wes showed an average of 317 called somatic variants per tumor. variants were identified in known driver genes at the following prevalences: kras 35.3%, egfr 8.8%, braf 8.8%, ros1 5.9%, alk 1.5%, met 4.4%, ret 1.5%. synchronous tumors from the same patients showed a high level of heterogeneity, as pairs shared 0 to 12 (0% to 3.3%) variants. all shared variants were likely passenger mutations, but one pair of tumors shared three variants including one in a driver gene, a kras p.g12c mutation. we showed a high level of genomic heterogeneity between two synchronous adenocarcinomas from the same patient using wes, supporting independent primary tumors. one pair of tumors had an identical kras mutation with a high level of genomic heterogeneity, emphasizing the fact that synchronous tumors can share a mutation in a frequent driver gene randomly. andréanne gagné to improve their recognition, the international association for the study of lung cancer (iaslc) recently proposed a classification based on clinicopathologic criteria. it divides msla in three categories: second primary, multiple ground glass opacities (ggo) and pneumonic type. however, the prevalence of these three subgroups remains poorly described and their clinical characteristics have been mostly described in asian cohorts. we aimed to establish the prevalence, clinicopathologic characteristics and prognosis of patients with msla in a caucasian population according to the iaslc criteria. we selected a retrospective cohort of 1012 consecutive patients, including 432 surgical patients, with a diagnosis of lung adenocarcinoma in our center between 2011 and 2014. the cohort was divided according to the iaslc classification: a group of sporadic tumors, comprising patients with one tumor and those with intrapulmonary metastasis (ipm), and a msla group further divided in second primary, multiple ggo and pneumonic type. prevalence of each group was calculated in the whole cohort. chi-square and t-tests were used to evaluate the associations between clinicopathological characteristics and the iaslc groups in surgical patients. overall survival of surgical patients was compared using a cox proportional model. : 143 patients (14.1%) with msla were identified, including 52 (5.1%) second primary, 84 (8.3%) multiple ggo and 7 (0.7%) pneumonic type. age (p=0.74), gender (p=0.38) and smoking status (p=0.77) were not associated with msla. msla patients had significantly more metachronous lung tumors (p=0.006), atypical adenomatous hyperplasia foci (p<0.001) and tumors with lepidic and acinary predominant patterns (p=0.03). there were no significant differences between the msla groups. compared with patients with a single tumor, the multiple ggo group tended to have the best prognosis (hr=0.75, p=0.41) and the second primary (hr=1.97, p=0.02) and pneumonic type (hr=2.33, p=0.41) had worse survival when using a multivariate model. to our knowledge, this is the first report to assess msla prevalence using the ialsc criteria to define patients with multiple adenocarcinomas. even though there were no differences in the clinicopathological variables between the msla groups, their survival disparity supports the iaslc classification. design: histologic slides of pulmonary lobectomies for high grade necs from the 2007-2018 period were reviewed. high grade was characterized by a mitotic count >10 per 2 mm². necrosis was subtyped as massive or punctuate. carcinoid morphology was identified according to the who morphological criteria. tumors with diffuse architecture, nuclear polymorphism and marked nucleoli were classified as "classic" lcnec. proliferation was also assessed with immunodetection of ki67. results: ten cases were available, 4 were classic lcnec, 6 had carcinoid-like features. clinical and pathological data are listed in table 1 . there were no differences between the 2 groups for clinical data. carcinoid-like subgroup had lower mitotic count (mean 28 vs 70), ki67 index (mean 54% vs 84%) and better survival (61 vs 18 months) than classic lcnec respectively. no reliable statistical analysis was allowed due to the small size of our cohort. classic lcnec (n = 4) carcinoid-like lcnec (n = 6) age mean ( conclusions: lcnecs with carcinoid features form a homogeneous subtype with precise morphological criterias. it is rare (0.3% of resected lung tumors in our center), nevertheless its real incidence is probably underestimated. indeed, for sampling reasons, we chose to report a series including only surgical specimens but we have already identified this tumor-type on biopsies from on resectable mediastinal lesions. proliferation markers are lower in carcinoid-like lcnecs than in classic lcnecs. nevertheless, results differ depending on the method of assessment. there is no overlap between the mitotic count values in both subgroups whereas ki 67 index overlaps with no upper limit value in carcinoid-like group. these results rise two questions : 1/ the choice of the method of evaluation of proliferation between mitotic count or ki67, 2/ the place of carcinoid-like lcnec within pulmonary nets classification: an intermediate group between atypical carcinoids and classic lcnecs or a high grade well differentiated net equivalent to that described in the digestive tract. however, the interests of individualizing the carcinoid-like lcnec subgroup are : diagnostic (must be distinguished from atypical carcinoid), prognostic (better than classic lcnec) and probably therapeutic (therapeutic response) jonathon gralewski background: lung cancer is a leading cause of cancer death worldwide. however, the introduction of targeted therapies in recent years has led to improved overall prognosis and survival. current guidelines for lung adenocarcinoma require epidermal growth factor receptor (egfr) exons 18-21 mutational testing. furthermore, it outlines molecular methods, such as next generation sequencing (ngs) with an acceptable turnaround time (tat) up to two weeks. this prolonged tat delays treatment decisions and increases healthcare costs. recently, a fully-automated, cartridge-based platform has been introduced with an ultra-rapid (i.e. <3 hours) sample-to-result tat that does not require traditional sample preparation, such as dna extraction, library preparation, and pcr amplification. design: twenty-one archived formalin-fixed paraffin embedded lung adenocarcinoma including 5 cytology and 16 surgical samples with previously characterized exon 18-21 egfr mutations were selected for this study. a single unstained section of the selected paraffin blocks was macrodissected, placed between filter papers, and subsequently placed into the cartridge and run for 51 egfr variant mutational analysis as per the manufacturer instructions. results were generated in <3 hours without the need for complex bioinformatics analysis and interpretation. results: of 23 previously characterized egfr mutations in 21 analyzed samples, 22 egfr variants were successfully detected by fully integrated ultra-rapid platform (96% concordance). the automated platform failed to identify an egfr g719c variant with allelic frequency of 6% that had been detected using conventional ngs platform. an additional egfr l858r variant was only seen by the cartridge based pcr platform. this variant was re-interrogated on the ngs-based platform and deemed likely a false positive result. conclusions: the cartridge-based fully integrated platform with ultra-rapid tat demonstrated high concordance with the conventional ngs-based platform. the fully-automated and integrated platform has minimized the need for significant molecular expertise and laboratory infrastructure. however, this platform does have its limitations, such as only detecting the most common egfr mutations. overall, this fully automated platform provides an ultra-rapid, and reliable cost-effective method in detecting the most common egfr variants, as outlined in the current guidelines with a detection sensitivity comparable with the conventional ngs platform. nancy greenland background: lung transplant recipients undergo bronchoalveolar lavage (bal) and biopsies to detect rejection and infection that may be antecedents of chronic lung allograft dysfunction (clad), the major limitation to long term survival. bal cytology is routinely performed but recently some centers have advocated abandoning this practice because of the low diagnostic yield. we hypothesized that inflammation observed on bal cytology would predict clad risk. we grouped diagnostic findings on bal cytology between 2012 and 2019. bronchoscopy indication, infection treatment, bal and biopsy results, and clad-free survival were abstracted from medical records. cytology associations with clinical characteristics were compared using generalized-estimating equation-adjusted logistic regression. the association between bal inflammation and clad or death were determined using time-dependent cox proportional hazards models adjusted for age, gender, diagnosis, lung allocation score, and transplant type. we evaluated 3,578 cytology reports from 491 subjects. inflammation was the most common finding (6.4%), followed by fungi (5.2% of which 0.8% were likely pathogenic). there were 5 cases of malignancy and 2 cases of cmv. inflammation on bal cytology was more common in procedures performed for symptoms (11%) versus surveillance (3%, p<0.001), associated with antimicrobial initiation (a proxy for clinically significant infection, 9% vs 5%, p<0.001), associated with acute cellular rejection (p=0.01), and linked to increased bal neutrophil and lymphocyte concentrations (p<0.001). inflammation on bal cytology was present for 32% of subjects on at least one sample, was more frequent around the time of clad onset, and was associated a 2.6-fold hazard ratio (ci 1.2-5.3) for clad or death ( figure 1 ). however, this association was not significant after adjusting for bal cell counts and acute cellular rejection (p=0.22). the presence of inflammation on bal cytology specimens is clinically significant, suggesting acute rejection or infection and increased risk of clad or death. however, other indicators of allograft inflammation can substitute for some of the information provided by bal cytology. hongxing gui background: primary pulmonary myxoid sarcoma (ppms) is an exceedingly rare low-grade lung neoplasm characterized by reticular/lacelike growth of spindle to epithelioid cells embedded in an abundant myxoid matrix. it overlaps with myxoid variant of angiomatoid fibrous histiocytoma (afh) morphologically. in terms of molecular genetics, they both have ewsr1 gene rearrangements, with ewsr1-creb1 fusion in ppms and either ewsr1-atf1 or ewsr1-creb1 fusion in afh. it is unclear whether they are distinctive entities or the same entity with a spectrum of histomorphologies. we evaluated two cases of low-grade myxoid spindle cell tumor of the lung by histomorphology, immunohistochemistry and fish studies. results: case 1 was a 49-year-old man with a 3.8 cm right lower lobe mass extending into bronchus. sections of the mass revealed proliferation of spindle cells in cords, strands and reticular patterns within an abundant myxoid stroma ( figure 1 ). the tumor cells were positive for ema and negative for desmin, ae1/3, cam5.2, ck7, ck20, ttf-1, c-kit, vimentin, cd1a, p63, and s100. case 2 was a 44year-old man with left main bronchial mass. the periphery of the mass was partially encapsulated with a lymphoid cuff. the central portion contained juxtaposed myxoid and nodular components. the former was composed of spindle cells in myxoid matrix and the latter consisted of histiocytic and spindle cells in whorled and storiform patterns ( figure 2 ). the tumor cells from both areas were diffusely positive for desmin and negative for ema, panck, ae1/3, s100, sma, cd34 and alk1. fish analysis demonstrated positive ewsr1 gene rearrangements in both cases, showing ewsr1-atf1 fusion gene in case 1 and ewsr1-creb1 fusion in case 2. we reported for the first time a case of ppms with a novel ewsr1-atf1 translocation, which is usually common in afh. the second case represented a hybrid of jaxtaposed ppms and afh components. these findings provide new evidence supporting that ppms and myxoid afh may represent a continuum with overlapping histologic, immunohistochemical and genetic features. sarika gupta 1 , sagar vishal 2 , anthony snow 3 , andrew bellizzi 1 1 university of iowa hospitals and clinics, iowa city, ia, 2 coralville, ia, 3 north liberty, ia disclosures: sarika gupta: none; sagar vishal: none; anthony snow: none; andrew bellizzi: none background: the differential diagnosis for epithelial tumors in the mediastinum includes carcinomas (ca) of the lung and thymus and thymoma. cd5 and kit are often used to distinguish thymic (+) from lung (-) ca, but these markers are rarely positive in thymoma. prior studies demonstrated frequent polyclonal pax8-positivity in thymoma (≥90%) and thymic ca (67%). we recently switched to monoclonal pax8, which has been shown to be negative in thymic tumors. given prior experience, we hypothesized that polyclonal pax8-positivity in the thymus represents cross-reactivity with another pax-family transcription factor. pax1 is normally expressed during thymic development, and a high-quality monoclonal antibody recently became commercially available. design: pax1 immunohistochemistry (ihc) (clone 5a2) was performed on tissue microarrays of 79 thymomas, 10 thymic cas, and 396 other cas with an emphasis on differential considerations and pax8-positive tumors: 175 squamous cell cas (25 lung); 41 urothelial cas; 36 lung adenocas; 28 renal cell, 25 serous, 22 endometrioid, 22 and 22 papillary and follicular thyroid, 10 breast, 7 colon, 5 esophagus, 2 poorly differentiated neuroendocrine, and 1 prostate ca. intensity (0-3+) and extent (0-100%) of expression was evaluated, and an h-score was calculated. fisher's exact and mann whitney tests were used with p<0.05 considered significant. results: pax1 was expressed by 91% of thymic tumors, including 94% of thymomas (mean/median h-score 198/210) and 70% of thymic cas (mean/median h-score 144/145). the differences in frequency (p=0.08) and h-score (p=0. 19) were not significant. pax1-positivity was noted in only 3.5% of non-thymic cas at a mean (median) h-score of 15 (5) (both p<0.0001 compared to thymic tumors); these included 6 thyroid cas (14%), 4 sccs (2%) [including 2 lung (8%)], 3 urothelial cas (7%), and 1 (10%) breast ca. two of the pax1negative thymic cas were kit-positive; all 3 were cd5-negative. detailed pax1 expression data by thymoma type are presented in the table; pax1 was very frequently, strongly expressed across types. conclusions: pax1 ihc using a novel monoclonal antibody is sensitive and specific for thymic epithelial neoplasms. occasional weak positivity in thyroid tumors may represent low-level cross-reactivity with pax8. this study exemplifies "next-generation ihc," which seeks to apply knowledge from developmental biology and molecular genetics to "intelligently design" novel ihc markers. background: tumor spread through the air space (stas) is an invasive pattern of lung cancer recently described. but there are some debates on its definition, quantification and clinical impact. in this study, we investigated the association between stas grade and clinicopathological characteristics, as well prognostic impact in resected lung cancers. design: stas has been prospectively described from 2008 and it was graded according to the distance from the edge of tumor margin as 2-tier system, i or ii from 2011. correlations between stas grade and clinicopathologic characteristics and prognostic significance were analyzed in 2000 surgically resected lung cancers. conclusions: stas was more frequently found in nets and mp-predominant adcs. it was associated with well-known aggressive features, and stas-gr ii tumors more frequently showed these features than gr i tumors in adcs. in stage ia non-mucinous adc, multivariate analysis revealed that stas grade was independent prognostic factor for rfs regardless of the extent of surgery. moreover, comparable rfs rates were observed in patients with stage ia/stas-gr ii and those with stage ib. 50% (19) 24% (10) 28% (17) tps=tumor proportion score. note that pdl1 high cases are also counted in the pdl1 positive category. *tumors with one of the following: alk fusion, nrg1 fusion, met exon 14 skipping, egfr mutation, or mutation in erbb2, braf, ret, pik3ca, or idh1/2 our data indicate that at least half of ras-driven nsclc have high pdl1 expression, an important consideration for frontline therapy selection. the majority of genomically-actionable tumors have at least low-positive expression of pdl1, highlighting the potential importance of immune checkpoint therapy in the resistant-progression setting. less than one third of genomically-negative cases have high pdl1 expression, and a significant percent are completely negative; additional innovations are needed to better tailor therapy for this patient subset. background: identification of ros1 rearrangements in advanced lung cancer carries therapeutic implications, given the available ros1targeted therapy, but can be technically challenging. immunohistochemistry (ihc) for ros1 show expression in ros1-rearranged tumors, but staining may be weak in some cases and seen in reactive pneumocytes. fluorescence in situ hybridization (fish) using break-apart probes may be difficult to interpret in cases with subtle intrachromosomal rearrangements. next-generation sequencing (ngs) can be useful but requires sufficient tissue and a turnaround time of at least 1-2 weeks. given that these current methods may have drawbacks, this study explores the utility of rna in situ hybridization (rna-ish) in detecting ros1 rearrangements in lung adenocarcinomas. results: using the ros1 rna-ish, all seven (100%) genetically-confirmed ros1-rearranged lung adenocarcinomas showed positivity; whereas none of the controls (0%) was positive. the fraction of cells in ros1-rearranged lung adenocarcinoma showing positivity ranged 60-90% (median 80%). the average number of ish signal dots was far higher in confirmed ros1-rearranged lung adenocarcinomas than controls (average: 29.6 per cell vs. 0.058 per cell). background lung showed minimal ish signal (average: 0.71 per cell). also, the positive ish signals were easily observed and could be seen using 10x objective in all cases or with a 4x objective in 6 of 7 cases. the automated ros1 rna-ish assay appears sensitive and specific in identifying ros1 rearrangements in lung adenocarcinoma, with ease of use for minimally-trained eyes. ros1 rna-ish may be a quick orthogonal tool in confirming ros1 rearrangements in clinically problematic cases. nonetheless, systematic comparison on its performance with that of ros1 ihc and fish may be warranted. on univariate analysis of all mpems, shorter os was significantly associated with year of diagnosis, asbestos exposure, poorer performance status, lymph node metastasis, higher peritoneal disease burden, absence of cytoreduction and hyperthermic intraperitoneal chemotherapy, biphasic or sarcomatoid histotype, and tumor necrosis. on univariate analysis of epithelioid mpem only, shorter os was additionally associated with nuclear pleomorphism, higher mitotic rate, higher composite nuclear grade, and non-tubulopapillary architecture. on multivariate analysis, sarcomatoid and biphasic histotypes predicted shorter os when adjusted for sex, asbestos exposure, and year of diagnosis. on multivariate analysis of epithelioid mpem only, nuclear grade and non-tubulopapillary growth were independently predictive of shorter os when adjusted for sex, year of diagnosis, and tumor necrosis. among patients with epithelioid mpem, shorter pfs after cytoreduction was associated with lymph node metastasis, tumor necrosis, nuclear pleomorphism, higher mitotic rate, higher composite nuclear grade, and solid growth. on multivariate analysis, nuclear grade and solid growth were independently predictive of shorter pfs when adjusted for year of diagnosis, sex, and necrosis. background: there is emerging evidence that vaping can result in significant lung injury, the severity of which can be variable with few cases resulting in the patient's death also been reported. there is limited data on the pathologic findings in vaping-induced lung injury. here, we report histologic and cytologic findings of four patients who developed lung disease following vaping. design: review of pathologic material and clinical information of four patients with a history of vaping (6 specimens, 3 male and 1 female patient, age 16-37 years old). we have identified 4 patients with a history of vaping who presented to the hospital with severe respiratory dysfunction. three of the patients are teenagers, while one is a younger adult and all of them required admission to the icu. the three younger patients had bronchoscopy with bronchoalveolar lavage (bal) with transbronchial biopsy in two. bal showed lipid laden macrophages in all three with acute inflammation in one. transbronchial biopsy showed intraalveolar fibrin with acute inflammation and organization in one of the biopsies. the adult patient is a known chronic alcohol and drug abuser who presented to the ed with dry cough and had subsequent wedge resection showing organizing pneumonia and chronic interstitial changes with the organizing pneumonia attributed to vaping. background: lymphangioleiyomomatosis (lam) is a rare low-grade neoplasm associated with widespread interstitial infiltration of spindle cells and subsequent cystic changes of the lesions. lam is also uniformly distributed in the lungs. target therapies such as mtor inhibitor have been to manage lam but there are no curative therapies at this juncture. lam cells are known to produce vegf-c and vegf-d with their receptor, vegfr3, which promote proliferation of lam cells also present in tumor cells. however, roles of other angiogenic factors have remained unclear. therefore, in this study, we examined the expression of angiogenic factors such as vegfr family and vasohibin (vash) and examined the correlations between these angiogenic factors and histological and clinical findings. design: 36 lam cases were obtained from 32 patients who underwent lung transplantation in tohoku university hospital from 2006 to 2018. we performed hierarchical clustering analysis to classify the cases based on the results of vegfr1, vegfr2, vegfr3, vash-1, and vash-2 immunoreactivity in lam cells. we also immunolocalized vash-1/2, cd31, and d2-40 in microvessels of the lesions. one of these clusters harbored higher vegfr1/3 and lower vegfr2 and vash-1/2, and the patients in this cluster clinically manifested symptom much older and higher p/f ratio was detected at the time of lung transplantation than those in the cluster with higher expression of all of the factors above. the cluster with higher vegfr1/2/3 expression and lower vash-1/-2 expression also demonstrated significantly higher vash-1/cd31 and vash-2/cd31 ratios in microvessels than the cluster with lower expression of all these factors above. however, there were no significant differences of lymphatic vessel strength or other histological characteristics detected in our present clustering analysis. conclusions: angiogenic factors such as vegfr2 and vash-1/-2 influenced on an early onset and progression of the clinical symptoms of lam. in addition, lam cells expressing vegfr1/2/3 promoted angiogenesis. therefore, not only vegf-c/d-vegfr3 axis but also other angiogenic factors may also enhance lam progression. background: micro-computed tomography (micro-ct), is a non-invasive method which allows 3-dimensional morphometric analysis of tissues in formalin fixed paraffin-embedded (ffpe) tissue blocks without any sectioning or loss of sample. lung adenocarcinoma has a major five tissue patterns according to the 2015 who classification (lepidic, acinar, papillary, solid and micropapillary). each tissue pattern has different prognostic indicators and outcomes. in practice, pathologists have to diagnose a predominant tissue pattern and measure each percentage of tissue pattern. the aim of this study is to analyze the structure of lung adenocarcinoma tissue patterns using micro-ct images from ffpe tissue blocks. the ffpe blocks were then sectioned, stained with hematoxylin-eosin (h&e) and scanned to create whole slide images for comparison. design: ffpe tissue blocks from five lung adenocarcinoma cases were scanned using a custom-built micro-ct scanner (nikon metrology) and digitally re-constructed for visualization and analysis using a digital image system. all h&e slides were scanned with 0.5um/pixel by nanozoomer s60 (hamamatsu photonics, japan) whole slide imaging scanner. we then investigated features of each tissue pattern and correlate between micro-ct images and whole slide images as well as histology 3d. results: micro-ct of ffpe blocks highlighted the structure of lung adenocarcinoma ( figure. 1) and normal lung tissue ( figure. 2) in 3d images. we can detect lung adenocarcinoma in a micro-ct image, and detect the tissue pattern, such as lepidic, acinar, papillary, solid, and micropapillary pattern as well as spread through air spaces (stas) (figure 1 ). we could detect the co-relation of normal lung tissue (bronchus, alveolar wall, pulmonary vessels, and visceral pleura) on h&e slides and micro-ct images ( figure 2 ). conclusions: whole block imaging by micro-ct allows for the identification of lung adenocarcinoma tissue patterns in ffpe blocks in a non-invasive and non-destructive manner. correlation between micro-ct images of ffpe blocks and h&e histology images suggests that there is potential for (1) detecting pathologic features without sectioning and staining of the tissue and (2) to measure the volume of each adenocarcinoma tissue pattern including the invasive component in a block accurately. metastasis, respectively, and more frequently associated with recurrence and death (22.6% vs. 1.9%, p<0.001 and 6.5% vs. 1.4%, p=0.05) compared with mp/s-group. survival analysis indicated that mp/s+ and mp/s <5% were associated with shorter recurrence free survival compare with mp/s-(hr=9.1, 95% ci=3.1-26.8, p<0.001; and hr=10.1, 95% ci=2.9-35.5, p<0.001, respectively). mp/s+ and mp/s<5% were more powerful predictor of recurrence than t or n stage in multivariate analyses. even very small proportion of mp/s subtype component was a significant predictive factor for recurrence in surgically resected lung adenocarcinoma. further investigation on the underlying biological mechanism of poor prognostic effect of mp/s subtype is warranted. results: patients' characteristics of 77 cases were as following; median age was 60 years old (range 33-77); 67 male and ten female; 16/16/41/4 of clinical stage i/ii/ii/vi; 21 chemotherapy, 52 chemoradiation and 4 radiotherapy; 52 adenocarcinomas, 18 squamous cell carcinomas and seven other types of histology. mpr was observed in 42 (55%), and pcr in 8(10%). the concordance rate of mpr and pcr assessment among two pathologists was high (96% and 96%). inter-observer agreement was high in mpr (kappa 0.928, p<0.001) and pcr (kappa 0.825, p<0.001). the discrepancy of mpr/pcr was due to the different judgment of tumor bed area and atypical cells whether they are benign or malignant. pathological findings of discrepancy cases had temporal and spatially heterogeneity of fibrosis, active inflammation with reactive stromal and epithelial cell changes. survival analysis will be updated at the time of presentation. our results revealed high reproducibility of mpr, and higher incidence compared to pcr, indicating mpr as a useful method for pathological therapeutic response. background: interstitial lung disease (ild) encompasses a spectrum of conditions with distinct clinical and pathologic features. a subset of ild has been linked to abnormal cellular senescence which can induce a pro-fibrotic senescence associated secretory phenotype that results in progressive pulmonary fibrosis. the senescence is mediated in part by activation of the cdk inhibitor p16 which can arrest the cell cycle and can be used to mark senescent cells. we aim to demonstrate that a distinct subset of ild associated with a senescent phenotype can be identified by expression of p16. design: 70 cases of ild diagnosed with surgical lung biopsy were identified between 2003 and 2018 at a large tertiary-care level hospital ( figure 1 ). additional p16 staining (clone e6h4, roche) was performed on representative sections with the most active fibrosis. p16 positive senescent foci were defined as a loose collection of p16-positive fibroblasts with an overlying p16-positive epithelium and scored as p16-low (0-2 foci/slide) or p16-high (≥3 foci/slide). the diagnosis was verified with the original pathology report and outcome data by time of biopsy to time of death or lung transplant. the presence of any p16-positive senescent foci was highly specific (92%) for the diagnosis of usual interstitial pneumonia (uip). in the uip group there was variable expression of p16 with a range of senescent foci between 0 and 23 per slide with 30 (67%) cases expressing some level of p16 and 20 (44%) cases expressing high levels of p16. comparing cases with high levels of p16 to cases with low to absent p16, there was a reduced survival (hr 2.26; 95% ci, 1.17 to 4.24; p = 0.016) in the p16 high group that was an independent predictor of lung transplant-free survival (figure 2) . in a sub-group analysis of only cases with a diagnosis of uip, p16 status trended towards significance (hr 2.06 ; ci, 0.99 to 4.24)( table 1) . conclusions: increased levels of p16 positive foci are highly specific for the diagnosis of uip and identify a subset of cases with a significantly worse transplant-free survival. these cases may represent an important subset of ild that may be most effectively treated with emerging classes of drugs that target senescent cells. design: 16 cases of nsclc patients who received ici were identified from departmental surgical archive and response was retrieved from the electronic medical records, including 7 cases of responders and 9 cases of non-responders. responders were defined as achieving a progression free response for more than 4 months. immunohistochemistry (ihc) studies for pd-l1, cd3, cd4, cd8, cd21, cd34, cd56, cd163, foxp3, and mmr proteins (mmrp mlh1/msh2, pms2/pms6) were performed. for cd3/4/8 ihc, the lymphocytic density (%) as well as the location (peripheral vs infiltrating) of positive t-cells were evaluated. results: all responders displayed a high pd-l1 expression (avg. of 68.8%, range 5% to 100%). the cd3/4/8 positive t-cells heavily infiltrated the tumor (avg. of 46.6%, range 30% to 80%). for non-responders, only 4 out of 9 cases displayed a lesser pd-l1 expression (avg. of 35.25%, range 1% to 70%). the cd3/4/8 positive t-cells were much less dense and infiltrative (<5%), and tended to concentrate at the periphery. the ihc results for cd56, foxp3, cd21 were negative. cd163 and cd34 had a similar pattern in both responders and non-responders. mmrp showed intact mlh1, msh2, pms2, and pms6 except for one non-responder. in addition to high pd-l1 expression, the percentage and pattern of cd3/4/8 positive t-cells appear to be more predictive of ici response in nsclc than pd-l1 alone and may easily be implemented in routine testing. the tumor microenvironment may also play an important role in facilitating immunotherapy response. however, nk-cells (cd56), tumor-associated macrophages (cd163), regulatory tcells (foxp3), and mmrp were not predictive of ici response in our pilot study. additional studies are needed to further investigate our preliminary findings. background: high-grade fetal adenocarcinoma (hfa) and enteric adenocarcinoma (ea) are both rare histopathological subtypes of lung adenocarcinoma. hfa and ea are occasionally combined with conventional-type lung adenocarcinoma, but pure types of both malignancies exist. the fact that the lungs and colon develop from the primitive striatum led us to speculate that these subtypes might share a common pathogenesis. design: among the 2253 cases of primary lung adenocarcinomas reported in our hospital, we identified 4 and 5 pure (p) hfas (0.18%) and eas (0.22%), respectively. all phfa tumors were high-grade adenocarcinomas with fetal lung morphology, necrosis, and immunopositivity for at least one of the following markers in addition to lacking morule formation: α-fetoprotein, sall-4, or glypican-3. all pea cases involved tumor cells that resembled colonic adenocarcinoma with no history of colorectal cancer. we evaluated the clinicopathological and molecular characteristics of these phfa and pea tumors. next-generation sequencing was performed using the ion-torrent personal genome machine platform and the ion ampliseq cancer hotspot panel v2. results: both phfa and pea were associated with several characteristic clinicopathological features such as smoking exposure, high incidence of lymphovascular invasion, and frequent expression of cdx2 and hnf4α. furthermore, nuclear accumulation of β-catenin was observed in 2 cases of phfa (50%) and 3 cases of pea (60%), which indicate activation of wnt signaling. the most frequently mutated gene was tp53 (3 peas), and other mutated genes that lead to the activation of wnt signaling were ctnnb1 (1 phfa) and apc (1 pea). an analysis of copy number variations revealed that smad4 deletion was the most frequently detected mutation, regardless of wnt activation (2 phfas; 2 peas). additionally, amplifications of fgfr3 were detected in 3 cases (1 ea; 2 phfas), which tend to be mutually exclusive with wnt activation. common mitogenic mutations in lung adenocarcinoma, such as egfr and kras, were not detected. conclusions: phfa and pea have similar clinicopathological features and oncogenic alterations which included frequent association with the activation of wnt signaling, amplifications of fgfr3, and smad4 deletion. recognition of these genetic subsets may help distinguish between lung adenocarcinoma with fetal and enteric morphology. background: gnas hotspot mutations have been described in indolent and slow-growing mucinous epithelial neoplasms in several organs, such as the pancreas and appendix. large genomic databases show that a subset of mucinous and non-mucinous lung adenocarcinomas harbor gnas mutations. however, the clinicopathological impact of gnas mutations on invasive mucinous adenocarcinoma of the lungs (ima) is not fully determined. we evaluated the clinicopathological and molecular characteristics of imas with gnas mutations in comparison with gnas wildtype cases. we examined egfr, kras, gnas, and tp53 mutations by pcr-direct sequencing in 80 imas. subsequently, a nanostringbased screen for 90 tyrosine kinase fusions was performed for all imas with wild type egfr and kras. next-generation sequencing using the ion-torrent personal genome machine platform and ion ampliseq cancer hotspot panel v2 or rna sequence were performed to confirm gnas mutations and tyrosine kinase fusions. mucin core proteins (muc1, muc2, muc4, muc5ac, and muc6) and differentiation transcription factors, particularly differentiating on the basis of the cellular lineage (ttf-1, cdx-2, and hnf4α) were detected by immunohistochemical staining. results: three of 80 imas (3.8%) harbored gnas mutations (2 r201h and 1 r201c). other mitogenic alterations including kras mutations (53 cases, 66.3%), tp53 mutations (11 cases, 13.8%), cd74-nrg1 fusions (2cases, 2.5%), and met exon 14 skipping (1 case, 1.25%) were detected. among 53 cases of kras mutations, g12v was the most frequent (42%), followed by g12d (34%) and g12c (13%). neither egfr mutations nor rearranged alk, ros1, ret, and ntrk were detected. all cases of gnas mutations were found in women who were never or light smokers with wild-type tp53. furthermore, gnas r201h mutations cooccurred with kras g12d mutations in two cases. in comparison with gnas wild-type cases, gnas-mutated cases were significantly associated with the female sex (p<0.05) and immunopositivity for muc4 (p<0.05). however, no significant differences were observed in other clinicopathological and immunohistochemical features, and progression-free and overall survival among both groups. conclusions: gnas-mutated imas are rare, but frequently co-occur with kras g12d mutations and immunopositivity for muc4, and are predominantly found in never-or light-smoking women; however, the prognostic impact of gnas mutations in imas is unclear. background: lung cancer is classified into small cell lung cancer (sclc) and non-small cell lung cancer (nsclc), which mainly contains adenocarcinoma (ac) and squamous cell carcinoma (sq). lung cancer subtyping plays an important role in choosing therapeutic schemes. sputum is a kind of noninvasively accessible biologic fluids containing exfoliated airway epithelial cells. although cytopathological examination of sputum is now available, its positive-rate of malignant cells is very low. it is hard to classify subtype via classic cytology on sputum specimens. we had reported that microrna panels could accurately discriminate between three subtypes of lung cancer in bronchial brushing specimens. the diagnostic value of micrornas in sputum specimens is need to be explored. in this study, 114 sputum specimens (49 ac, 39 sq, and 26 sclc) were investigated. reverse-transcriptase quantitative polymerase chain reaction (rt-qpcr) was performed to evaluate expression of 7 candidate micrornas discovered via microarrays previously. two logistic regression models constructed before was validated in the cohort of 114 sputum specimens. the area under the receiver operating characteristic curve (auc) was used to assess the diagnostic accuracy of microrna panels. the diagnostic performance was compared between microrna panels and cytology. results: panel a, consisting of mir-29a and mir-375, was built to discriminate sclc from nsclc. in the cohort of 114 sputum specimens, the auc value was 0.621 with sensitivity of 81.72% and specificity of 46.15%. similarly, panel b, consisting of mir-34a and mir-205, was used to discriminate sq from ac. in the cohort of 114 sputum specimens, the auc value was 0.678 with sensitivity of 46.94% and specificity of 82.05%. compared with cytology, microrna panels or the combination of microrna panels and cytology were of higher sensitivity and specificity in diagnosis of ac, sq and sclc. conclusions: in sputum specimens, those two microrna panels for lung cancer subtype discrimination could achieve high sensitivity and specificity. moreover, the combination of microrna panels and cytology could improve the diagnostic accuracy in further. these findings could be helpful in therapy of lung cancer. (table) . on image analysis, median total diagnostic area was 1.36 mm 2 and the median size of the largest fragment was 0.29 mm 2 (n=25). the largest fragment size was obtained for a case of diffuse large b-cell lymphoma (total area : 48.40 mm 2 , figure 1 ). there was no significant correlation between lpd diagnostic category and total fragment size (p=0.153) or largest fragment size (p=0.139). preliminary data on surgical specimens obtained by mediastinoscopy show a total diagnostic area ranging from 50.86 mm 2 to 95.30 mm 2 , with largest fragment size varying from 14.61 to 22.53 mm 2 (n=3). we report here a series of lymphoma cases diagnosed by ebus/eus, ranging across several diagnostic categories, based primarily on small size fragments. quantification of endoscopic diagnostic tissue provides insight on tissue yield, on its relationship with lymphoma subtype, and supports ebus/eus as an acceptable procedure for lymphoma diagnosis. background: light chain deposition disease (lcdd) is characterized by amorphous "glassy" deposits of immunoglobulin light chains in organs such as kidneys, heart, and liver. since these amyloid-like deposits lack the secondary structure consisting of beta-pleated sheets, they don't stain salmon-pink with congo red. additionally, a negative congo red may be attributed to suboptimal staining, especially when the deposits primarily involve organs where lcdd is rarely encountered. moreover, congo red stains are compromised when the available sections are 5 micron thick. we noticed in a case of primary lcdd of the lung that the light chain deposits stained bright red with masson trichrome and pink with sulfated alcian blue (sab) stains. in the current study, we tested whether these two stains can distinguish between amyloidosis and lcdd. we reviewed lung cases with eosinophilic deposits and immune infiltrates with negative congo red stain. we assessed trichrome and sab staining on 9 cases of lcdd and 1 control case of amyloidosis involving the lung. results: 9 cases were identified ( table 1 ). the age of the patients ranged from 44 to 75 years and 56% were females. imaging studies of 7 cases showed nodular deposits and the remaining cases showed cystic lesions and/or nodular lesions. underlying hematologic abnormalities were detected in 7 lcdd patients which included malt lymphomas (n = 6) and plasma cell neoplasm (n = 1). systemic involvement was absent in 7 cases with available information. hematoxylin and eosin stained slides of all cases showed "glassy" eosinophilic amyloid-like deposits predominantly around airways and vasculature. congo red staining was negative in all lcdd cases, while it showed salmon-pink staining of amyloid deposits in the control case. for all lcdd cases tested, trichrome stained the deposits as bright red and the sab stained the deposits as pink (figure 1 ). in contrast, amyloid deposits in the control case stained greyish blue with trichrome and bright green with sab stains, as expected. figure 1 -1926 conclusions: when congo red fails to stain "glassy" amorphous eosinophilic material salmon-pink, trichrome and sab stains might indicate the non-amyloid, light chain nature of lcdd deposits when red and pink staining is seen, respectively. these screening studies can inform downstream testing such as immunofluorescence staining for light chains, transmission electron microscopy, and typing of light chains by mass spectrometry. background: pulmonary pleomorphic carcinoma (ppc) is known for its aggressiveness and poor prognosis than other subtypes of nonsmall cell carcinoma. to better understand the molecular characteristics of ppc, we analyzed genetic alterations of ppcs and their metastatic lesions by whole exome sequencing. we included 11 ppc patients who underwent surgical resection for both primary lung cancer and metastatic lesions at seoul national university bundang hospital. carcinomatous and sarcomatous components of each primary ppc along with the metastatic lesions were microdissected. somatic mutation profiles were generated by whole exome sequencing. the majority of ppc patients were male (10 of 11, 90.9%) and smokers (10 of 11, 90.9%). carcinoma components of ppc consisted of adenocarcinoma (8 of 11, 72.7%), squamous cell carcinoma (2 of 11, 18.2%) or adenosquamous carcinoma (1 of 11, 9.1%). tp53 (70%) was the most frequently recurrent genetic alteration, followed by wdfy2 (36%), otog, pxdnl, and slitrk5 (33%). kras mutation was found in 2 cases (18.1%) and egfr mutation in one (9.1%). in addition, mutations discovered to be richer in either carcinomatous, sarcomatous or metastasis included various genes known to be associated with tumor progression and poor prognosis (flnc, nrxn1, csmd2, znf208, auts2). gene alterations associated with somatic hypermutation (pole, pold1) and epithelial-mesenchymal transition (notch1, cdh2, fn1) were also found. four cases (36.4%) had high tumor mutation burden (tmb). of the 43 altered genes investigated in depth in this study, roughly half (n=18) were shared by carcinomatous, sarcomatous and metastasis in at least one case. significant overlap of the mutation profiles between metastasis and either carcinomatous or sarcomatous component was not found. conclusions: this is the first study to analyze the molecular profile of ppc in both metastatic lesions and primary carcinomatous and sarcomatous components. sporadic genetic alterations appeared to occur among carcinomatous, sarcomatous components and metastatic lesions of ppc. various genes associated with tumor progression were altered in both primary and metastatic lesions. christin lepus 1 , julia rotow 2 , pasi janne 2 , lynette sholl 1 background: histologic transformation of egfr-mutant non-small cell lung carcinoma (nsclc) to small cell lung carcinoma (sclc) is a mechanism of acquired resistance to egfr-tyrosine kinase inhibitors (tkis) that occurs in approximately 5% of egfr-mutant nsclcs. however, the natural history/histologic progression of sclc transformation from nsclc is poorly understood. a retrospective analysis was conducted to characterize the morphologic variation during transformation of egfr-mutant nsclc to sclc. we identified 12 patients with egfr-mutant nsclc that transformed to sclc during treatment with egfr-targeted tyrosine kinase inhibitor therapy. histologic evaluation of longitudinal specimens (n=1-5 per patient) from both lung and distant metastases was performed to characterize the morphologic spectrum of lesions obtained from the initial diagnosis of nsclc to sclc transformation. results: all 12 patients had lung adenocarcinoma harboring egfr mutations at initial diagnosis (exon 19 deletion, 91.6%; l858r, 8.4%) and had been treated with at least one egfr tki prior to development of sclc. most patients (10 of 12; 83.3%) were on secondline osimertinib at the time of transformation. when transformation was first documented, four patients (33.3%) were reported to have combined adenocarcinoma and sclc or poorly differentiated carcinoma with mixed adenocarcinoma and sclc morphologic features; the remaining 8 patients (66.6%) were reported to have sclc. retrospective review of this latter group demonstrated focal nsclc-like morphology (mildly increased cytoplasm, variably prominent nucleoli, and rare gland formation) in 3 patients (37.5 %). conversely, 4 of 6 patients who had post-treatment, pre-transformation biopsies reported as adenocarcinoma showed classic adenocarcinoma architecture with superimposed small cell carcinoma-like cytomorphology, including increased nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, and finely granular chromatin. overall, a hybrid/transitional phenotype was captured in 7 of 12 patients (58.3%). among 7 patients who had tumor genotyping upon sclc transformation, all maintained the sensitizing egfr mutation and showed acquired rb loss. recognition of these transitional morphologies may facilitate earlier detection of sclc-driven resistance to egfr-targeted therapy and inform timely selection of alternate treatment regimens. yuan li background: random forest model is a recently developed machine-learning algorithm, and superior to other machine learning and regression models for its classification function and better accuracy. but it is rarely used for predicting causes of death in cancer patients. on the other hand, specific causes of death in lung cancer patients are poorly classified or predicted, largely due to its categorical nature (versus binary death/survival). we therefore tuned and employed a random forest algorithm (stata, version 15) to classify and predict specific causes of death in lung cancer patients, using the surveillance, epidemiology and end results-18 and several clinicopathological factors. the lung cancer diagnosed during 2004 were included for the completeness in their follow-up and death causes. the patients were randomly divided into training and validation sets (1:1 match). we also compared the accuracies of the final random forest and multinomial regression models. we identified and randomly selected 40,000 lung cancers for the analyses, including 20,000 cases for either set. the causes of death were, in descending ranking order, were lung cancer (72.45 %), other causes or alive (14.38%), non-lung cancer (6.87%), cardiovascular disease (5.35%), infection (0.95%), and. we found more 250 iterations and the 10 variables produced the best prediction, whose best accuracy was 69.8% (error-rate 30.2%, figure 1 ). the final random forest model with 300 iteration and 10 variables reached an accuracy higher than that of multinomial regression model (69.72% vs 64.58%). the top-5 most important factors in the random-forest model were sex, chemotherapy status, age, radiotherapy status and nodal status (figure 2 ). we optimized a random forest model of machine learning to predict the specific cause of death in lung cancer patients using a population database. the model also appears more accurate than multinomial regression model. background: primary pulmonary hematolymphoid neoplasms (phlns) are rare, and the incidence is increasing with modern diagnostic advances and treatments. flow cytometry (fc) is a proven powerful tool in the diagnosis of hematolymphoid disease, however its role in phln is not well represented. in the current study, we aim to assess the utility of fc in diagnosis of patients with phlns. we retrospectively reviewed the fc analyses of pulmonary specimens from our institutional database between 2016-2019. the specimens are comprised of pleural fluid, bronchoalveolar lavages, bronchoscopy or ct guided fnas, and vats or surgical biopsies of lung mass. phlns were detected by 10-color panel fc by identification of clonal or immunophenotypical aberrant hematolymphoid populations. primary neoplasms are classified if no extrapulmonary lesions were detected by clinical radiological work up at the time of diagnosis or within three months of diagnosis. lung involvement is also a frequent site in involvement of lymphoproliferative diseases and are referred to as secondary involvement of the lung. we retrieved 471 pulmonary specimens submitted for fc. we identified 111 cases that were positive for phlns. median age was 49 years with m:f ratio=1. 54/111 specimens aided in the diagnosis of a hematolymphoid disease. distribution are as follows: 17 pleural fluids, 33 tissue biopsies, 4 bronchoalveolar lavages. cases were classified as either primary (9/54 (17%)) or secondary (45/54 (84%)), and categorized as myeloid neoplasms, b-cell neoplasm, t-cell neoplasm, primary effusion lymphoma (pel), post-transplant lymphoproliferative disorder (ptld); and plasma cell neoplasms (table 1) . of the phlns, diffuse large b-cell lymphoma (dlbcl) was the most common diagnosis (12/54 (22%)). immunosuppression were notable in 14 patients. the remaining 57/111 cases represent followups of known lymphoproliferative diseases where fc analysis of lung specimen played a role in assessment of disease status. all fc positive cases were confirmed by tissue examination with positive predictive value (ppv) of 100%. conclusions: fc demonstrates a clear utility for immunophenotyping in the diagnosis of phln, with a perfect ppv. appreciating the spectrum of phln is helpful in disease detection. in addition, fc provides the ability to monitor residual disease and response to therapy; especially in immunosuppressed patients where expedited treatment can impact prognosis. background: piwi-interacting rnas (pirnas) are small non-coding rnas (24-35nt) that play an essential role in maintaining genome integrity trough regulation of transposable elements. their expression was tough to be limited to germinal cells and early embryogenesis but recently an implication of pirnas in cancer biology has been reported. the aim of this study was to explore the expression of pir-796 pirna, which was identified by small rnaseq, in resected nonsmall cell lung cancer (nsclc) patients, and to analyze the correlation with the clinic-pathological features. we have analyzed 191 resected nsclc samples from patients who underwent surgery in hospital clínic between 2007 and 2015. pir-796 pirna was quantified using custom taqman non-coding rna assays in tumor and normal tissue. in vitro studies using sirnas to inhibit pirna expression were performed in two lung adenocarcinoma cell lines: hcc44 and a549. results: pir-796 was overexpressed in tumor tissue compared to normal tissue (p<0.001). it also appeared to have higher expression in squamous cell carcinoma compared to adenocarcinoma histological subtype. expression of pir-796 had prognosis impact in the group of early stage (i-ii) adenocarcinoma patients. higher pir-796 levels were associated with shorter disease-free survival (p=0.045). in vitro analysis showed that the silencing of pir-796 was associated with decreased cell migration in a549 cell line (p<0.0001) and increased apoptosis in hcc44 cell line (p=0.03). in conclusion, pir-796 may have an important role in carcinogenesis in nsclc, promoting cell migration and regulating apoptosis and may be a potential new prognostic biomarker for nsclc. lucas massoth lcdd rarely involves the lungs in a nodular or diffuse pattern. we present herein a large series of pulmonary nodular lcdd. in an institutional review from >2000 patients in 2000-2019, we identified 12 specimens from 10 patients with pulmonary nodular lcdd. we reviewed clinicopathologic features and performed electron microscopy in all cases. of 10 patients (6 women, 4 men; age 42-74 [median 68] years), 7 were smokers, 4 had a history of autoimmune disease, but none had a history or evidence of a systemic lymphoproliferative/plasma cell disorder. clinical presentations were most often incidental. of 9 patients with radiology available, 3 showed a solitary nodule each; 6 showed multiple nodules (including 3 with associated cystic changes). each nodule ranged 0.6-2.1 (median 1.2) cm. by light microscopy on hematoxylin-eosin staining, each nodule appeared as amorphous eosinophilic deposits; all cases showed multinucleated giant cells engulfing the deposits and prominent plasma cell infiltrates. while the morphology was reminiscent of amyloidoma, staining for congo red was negative for amyloid in all cases. pas was positive in all 5 cases tested, and trichrome was distinctly bright red in 5 of 6 cases tested. of 8 cases with plasma cell cytoplasmic light chain expression tested by immunohistochemistry and/or in-situ hybridization, 6 were kappa-predominant, 1 lambda-predominant and 1 polytypic. immunofluorescence studies in 2 cases confirmed kappa and lambda light chain restriction in 1 case each. ultrastructurally, all cases showed extracellular electron-dense granular deposits, variably admixed with entrapped collagen fibers, often appearing perivascular, and lacking non-branching fibril formation characteristic of amyloid. of 6 patients with >2 years of follow-up (median 4.3 years), 5 were alive (the single death was unrelated to lcdd). pulmonary nodular lcdd appears clinically indolent, radiologically solitary or multifocal, and mimics amyloidoma by light microscopy. yet, special stains (negative congo red, bright red trichrome) and ultrastructural features (granular deposits with variable entrapped collagen) aid the diagnosis of lcdd and its distinction from amyloidoma. the high rate of autoimmune disease in this cohort may suggest a role of chronic immunologic stimulation in the pathogenesis of pulmonary nodular lcdd. background: molecular targeted therapies against egfr and alk have improved the quality of life of lung adenocarcinoma patients. however, targetable driver mutations are mainly found in ttf-1/nkx2-1-positive terminal respiratory unit (tru) types and rarely in non-tru types. design: to elucidate the molecular characteristics of the major subtypes of non-tru-type adenocarcinomas, we analyzed 19 lung adenocarcinoma cell lines (11 tru types and 8 non-tru types). a characteristic of non-tru-type cell lines was the strong expression of tff-1 (trefoil factor-1), a gastric mucosal protective factor. by immunohistochemistry, we examined tff-1, ttf-1/nkx2-1, hnf4-alpha, and muc5ac expressions using 238 primary lung adenocarcinomas resected at jichi medical university hospital. we also examined the correlation between tff-1 expression and clinico-pathological factors and genetic abnormalities. results: an immunohistochemical analysis of revealed that tff-1 was positive in 31 cases (13%). tff-1 expression was frequently detected in invasive mucinous (14/15(93%)), enteric (2/2(100%)), and colloid (1/1(100%)) adenocarcinomas, less frequent in acinar (5/24(21%)), papillary (7/120(6%)), and solid adenocarcinomas (2/43(5%)), and negative in micropapillary (0/1(0%)), lepidic (0/23(0%)), and microinvasive adenocarcinomas or adenocarcinoma in situ (0/9(0%)). tff-1 expression correlated with the expression of hnf4alpha and muc5ac (p<0.0001, p<0.0001, respectively) and inversely correlated with that of ttf-1/nkx2-1 (p<0.0001). these results indicate that tff-1 is characteristically expressed in non-tru-type adenocarcinomas with gastrointestinal features. tff-1-positive cases harbored kras mutations at a high frequency, but no egfr or alk mutations. tff-1 expression correlated with a poor prognosis in advanced stages. moreover, the knockdown of tff-1 inhibited cell proliferation and induced apoptosis in a tff-1-positive and kras-mutated lung adenocarcinoma cell line. these results indicate that tff-1 is not only a biomarker, but also a potential molecular target for non-tru-type lung adenocarcinomas. background: accurate and timely biomarker results are essential for the modern-day treatment of non-small cell lung cancer (nsclc). nonetheless, many institutions around the world, particularly those without in-house testing capabilities, are faced with prolonged turnaround time. this study investigates the clinical impact of implementing a rapid biomarker testing strategy. design: rapid in-house biomarker testing was implemented utilizing immunohistochemical assays for pd-l1, alk, ros, and braf v600e, as well as qpcr for egfr using the biocartis idylla technology. after a 6-month period, we performed a retrospective chart review of nsclc patients presenting pre-and post-implementation of rapid biomarker testing at our facility. results: 198 patients were included in the study (85 underwent rapid biomarker testing and 113 underwent traditional testing). the median (iqr) turnaround time for biomarker reports decreased from 61.5 (31-100) to 4 (2-7) days (p < 0.001), with turnaround time defined as the total number of days between the diagnosis and biomarker result appearing on the medical record. the mean time to initiation of systemic therapy for advanced-stage patients decreased from 52.5 days to 31.9 days (p < 0.01) the proportion of patients who had a complete biomarker report at the time of their first consult with a medical oncologist increased from 26.8% to 58.0%. similarly, the proportion of patients who had a complete biomarker report at the time of initiation of systemic therapy increased from 56.7% to 86.4%. timely biomarker results are essential for the delivery of targeted therapy and immunotherapy to nsclc patients. many centres experience delays in biomarker results for a number of reasons. in this study, we demonstrate that a small panel of rapidlyreported biomarkers can significantly reduce delays in the initiation of systemic therapy, ultimately leading to superior patient outcomes. mitra mehrad background: primary sarcomas of the thorax (heart, lung, pleura, thymus, mediastinum) are rare and little information is available on predictors of their prognosis. the national cancer institute's seer (surveillance, epidemiology, and end results) database contains abundant information on the natural history of soft tissue sarcomas. therefore, we investigated the clinical outcomes of primary thoracic sarcomas (ts) utilizing the seer database. the seer database was queried for ts entered between 1985 and 2013. ts was compared to sarcomas arising in soft tissues of the extremities and trunk. staging was performed based on the ajcc 8 th edition. multivariable cox regression was used to identify prognostic factors. kaplan-meier curves were plotted to assess cancer-specific survival (css). patients <19 years of age and those without confirmed surgical resection were excluded. results: a total of 1308 ts and 20160 soft tissue sarcomas were available for analysis. most ts arose in the lung (785; 60.0%) followed by mediastinum (224; 17.1%), heart (165; 12.6%), pleura (132; 10.1%) and thymus (2; 0.2%). the 5 most common ts were malignant solitary fibrous tumor (sft) (147; 11.2%), leiomyosarcoma (lms) (139; 10.6%), synovial sarcoma (128; 9.7%), undifferentiated pleomorphic sarcoma (ups) (126; 9.6%), and angiosarcoma (122; 9.3%), whereas in the soft tissue were ups (5477; 27.1%), lms (2357; 11.6%), myxoid liposarcoma (lps) (1544; 7.6%), well-differentiated lps (1413; 7.0%), and myxofibrosarcoma (1331; 6.6%). comparing the two groups by multivariate analysis, the ts were larger and more frequent in males (p<0.0001). they were also of higher histologic grade and stage and were less likely to receive adjuvant therapy (p<0.001). among the ts, tumors of heart origin (p<0.0001), greater than 5 cm, stage iii-iv, and histologic grade 3 had worse css (p<0.01). the more common ts tumors including malignant sft, lms, synovial sarcoma, and angiosarcoma still had worse css compared to their soft tissue counterparts even when adjusting for stage, size and treatment. conclusions: primary thoracic sarcomas show worse clinical outcome compared to soft tissue sarcomas. a thoracic specific sarcoma staging system may be more predictive of css. background: primary pulmonary mucinous adenocarcinoma (pma) may be difficult or impossible to distinguish from colorectal adenocarcinoma (crc) due to considerable morphologic and immunohistochemical (ihc) overlap. since the lung is a common site of metastasis, the need to distinguish pma from crc in the lung is a routine challenge. commonly used lineage-specific ihc markers like cdx2, ttf-1, and napsin a are helpful to distinguish non-mucinous lesions but are either insensitive or nonspecific when applied to mucinous lesions. satb2 is a relatively new ihc marker that distinguishes crc from upper gastrointestinal and pancreaticobiliary tumors. its ability to distinguish crc from pma is not yet completely elucidated. design: three tissue microarrays of lung resections containing primary pulmonary mucinous adenocarcinomas (pmas), metastatic colorectal carcinomas (crcs), and primary pulmonary non-mucinous adenocarcinomas (pnmas) were stained with ck7, ck20, satb2, cdx2, villin, ttf-1, and napsin a. two pathologists evaluated the number of positive neoplastic cells semiquantitatively, regardless of intensity: 0 (no staining), 1+ (<10%), 2+ (10-50%), 3+ (>50%). results: thirty-one pmas, 32 crcs, and 34 pnmas were assessed (table 1) . thirty (97%) of pmas and 34 (100%) of pnmas were positive (3+) for ck7, while all crcs were negative for ck7. twenty-seven (84%) of crcs and 2 (6%) of pmas were positive (3+) for satb2, and 29 (91%) of crcs and 2 (6%) of pmas were positive (3+) for cdx2. both pmas and crcs had high rates of villin positivity, with 23 (74%) and 32 (100%) positive (3+), respectively. only 4 (13%) and 7 (23%) of pmas were positive (3+) for ttf-1 and napsin a, respectively. no crcs were positive for ttf-1 or napsin a. in deciding pma vs. crc, ck7 was 97% sensitive and 100% specific for pma. satb2 was superior to cdx2 and villin but was only 96% sensitive and 94% specific for crc. conclusions: although the lineage-specific markers satb2 and cdx2 were fairly specific for crcs, a few pmas were also positive. in contrast, all crcs were negative for ck7 while almost all of the pmas were positive. lineage-specific markers ttf-1 and napsin a had a low sensitivity for pmas. villin showed a low specificity with the majority of crcs and pmas staining positively. our results suggest that a ck7-positive tumor in the lung, whether mucinous or non-mucinous, is unlikely to be of colorectal origin. lineage-specific markers such as satb2 are of questionable value when evaluating mucinous lesions in the lung. massimo milione 1 , patrick maisonneuve 2 , federica grillo 3 , alessandro mangogna 4 , giovanni centonze 5 , giovanna garzone 6 , laura cattaneo 7 , ketevani kankava 8 , adele busico 6 , paola spaggiari 9 , alessandro del gobbo 10 , luisa bercich 11 , luigi rolli 12 , elisa roca 11 , natalie prinzi 6 , giancarlo pruneri 5 , alfredo berruti 11 , ugo pastorino 12 , carlo capella 13 1 fondazione irccs istituto nazionale tumori milano, milano, italy, 2 ieo, milan, italy, 3 university of genova, genova, italy, 4 university of trieste, trieste, friuli venezia giulia, italy, 5 fondazione irccs istituto nazionale tumori milano, milan, italy, 6 irccs foundation, istituto nazionale dei tumori, milan, italy, 7 irccs foundation, istituto nazionale dei tumori, milano, italy, 8 teaching, scientific and diagnostic pathology laboratory, tbilisi state medical university, tbilisi, georgia, 9 istituti clinic humanitas, rozzano, milano, italy, 10 fondazione irccs ca granda ospedale maggiore policlinico, milan, italy, 11 university of brescia at brescia, italy, 12 irccs foundation national cancer institute, milan, italy, 13 with regards to molecular analyses, 26 cases (54,2 %) of the co-lcnec group were studied (see table 1 ). no single next-generation sequencing marker was statistically associated with os. the identification and morphologic characterization of combined features in lcnecs as well as the application of ki-67 cut off at 55% contribute in predicting clinical outcome of pure-lcnec and co-lnec patients. background: ctnnb1 encodes for β-catenin, which is a member in the wnt signal transduction pathway required for proliferation, survival and differentiation of different epithelial cells. mutation of ctnnb1 causes constitutional changes in the β-catenin protein that impedes its degradation, leading to an uncontrolled proliferation of the mutated cell. ctnnb1 exon 3 hot-spot mutations are described in various tumor types and, for instance, in endometrial cancer, are associated with high risk of disease recurrence. the role of ctnnb1, frequency and type of co-mutations has not been well characterized in non-small cell lung carcinomas (nsclc). design: between 2013-2018, lung cancer samples from 855 patients were sequenced on the ion torrent pgm with the 50 gene ampliseq cancer hotspot panel v2. our in-house sequencing database was searched to identify patients with ctnnb1 mutations; co-mutations in genes commonly altered in nsclc were also recorded. results: thirteen patients (1.5%) with ctnnb1 mutations (p.s37f (5) (one also with p.d32y), p.s37c (3), and p.s33c, p.d32h, p.g34e, p.d32v, p.d32n, in one patient each) were identified. all tumors were adenocarcinoma histology. five patients underwent lobectomy and the predominant histologic patterns were solid (2), papillary (2) and micropapillary (1) . the patients' age ranged from 54-82, at the time of diagnosis, and eight were female (61.5%). all patients were current (2) or former (11) smokers. five patients presented with stage iv disease, two with stage iii, two with stage ii, and four with stage i disease. six are deceased and seven are alive with disease. co-mutations were identified in all but one case and consisted of: braf (1 -v600e), egfr (3 -ex19 deletions), kras (4), pik3ca (3), and tp53 (3). in two patients, egfr, pik3ca and ctnnb1 mutations were co-occurring. we identified ctnnb1 mutations in 1.5% of lung adenocarcinomas, which, in our population, were associated with frequent co-mutations. additional clinicopathologic data will be aggregated from our internal patients and available databases (cbioportal) to better understand the clinical implications of ctnnb1 mutations in nsclc. co-mutation with egfr may be a mechanism of primary resistance to egfr tkis. in addition, ctnnb1 mutations make patients eligible for newer small-molecule tkis (e.g. ttki). mohammad mohammad . samples from normal tissue included pancreas (n=13), rectum/appendix/colon (n=39) and ilium/duodenum/stomach (n=13). nuclear expression for otp was interpreted as negative (<5% tumor cells stained), 1+ (<25%), 2+ (26-50%), 3+ (51-75%), and 4+ (>75%). results: six of 10 (60%) pcs were diffusely positive for otp. one of 33 (3%) pancreatic nets, 2 of 24 (8%) bladder sccs, and 1 metastatic net in the liver (negative for ttf1 and no lung mass identified) were positive for otp. all other tumors and normal tissues were negative. representative cases are shown in figure 1 . conclusions: our data demonstrated that otp expression was only rarely identified in non-pulmonary neuroendocrine tumors/carcinomas, which further validated the previous report of otp to be a highly specific marker for diagnosing pcs. the diagnostic sensitivity for pcs in this study appears to be lower than the previous report, which is probably due to the small number of cases included. caution should be taken because rare pancreatic nets and bladder sccs can be positive for otp. background: quantification of pd-l1/pd-1 expression in non-small cell lung cancer (nsclc) is not always predictive of efficacy of immune checkpoint inhibitor therapy, and response to these agents remains limited to a minority of patients. human leukocyte antigen 1 (hla-1) participates in presentation of aberrant peptide antigens, enabling cytotoxic t cells to recognize and destroy tumor cells. correspondingly, loss of hla-1 expression precludes immune recognition, and this loss is one proposed mechanism of treatment failure of immunotherapy. we hereby examine the expression of pd-l1 and hla-1 in a large cohort of nsclcs to determine the patterns and frequency of expression of these two markers. design: nsclc resection specimens from 2011-2014 classified as adenocarcinoma (ac) or squamous cell carcinoma (scc) were identified. tumor microarrays with 1.0 mm diameter cores were constructed, with 2 sections of neoplastic tissue and 1 section of uninvolved lung for each case. immunohistochemistry for pd-l1 (22c3) and hla-1 was performed. membranous tumoral pd-l1 staining was semi-quantitatively scored as <1%, 1-50%, and >50%. hla-1 was scored as intact, partial (clonal) loss, or complete loss with total absence of staining. conclusions: hla-1 loss is common in nsclc and can readily be assessed by immunohistochemical methods. in this study, there is no association between pd-l1 expression and hla-1 expression. given that hla-1 expression may be a determinant of response to immunotherapeutic agents targeting the pd-1/pd-l1 axis, it may be considered as an adjunct immunohistochemical marker prior to initiation of immune checkpoint therapy or in instances of treatment failure. background: short telomere syndromes (sts) are multisystem accelerated aging syndromes caused by inheritable gene mutations in telomere maintenance genes; frequent manifestations include bone marrow failure and interstitial lung disease (ild). insufficient information is available about histologic findings and patterns of ild in individuals with sts and whether these vary by specific inherited sts gene. our study aims to describe the morphology of lung disease in patients with sts. design: probands enrolled in the inherited hematologic disorders registry at our hospital with a personal or familial history of pulmonary fibrosis underwent genetic testing via targeted genomic capture and next-generation sequencing (ngs) of 13 sts genes. available specimens(n-8), including wedge resections(n-6) and lung explants (n-2), were analyzed for 15 histopathological features (table 1) . eight patients with known sts with ild had available histology (m=7, f=1 average age at diagnosis 56.3 years). pathognomonic findings of usual interstitial pneumonia (uip) were seen in all patients. one patient with two different mutations in the telomere-associated genes showed features of both uip and non-specific interstitial pneumonia (nsip). there was associated lymphoplasmacytic infiltration which was mild in 50% and extensive in the remainder. other histologic findings were variable, e.g. nonnecrotizing granulomas and upper lobe predominance are noted in 50% of the patients. pulmonary hypertension was seen in all of the patients except one who had a very early disease. conclusions: sts associated lung disease frequently shows uip with additional findings including inflammatory (lymphoplasmacytic infiltrate) and hypersensitivity pneumonia-like (granulomas and upper lobe predominance) features. consideration should be given to testing for sts in patients with uip with these histologic findings. it is important to identify this cohort of patients because these patients may have a more rapid progression of their ild and an increased toxicity from immunosuppressive drugs especially in post-transplant setting. if a pathogenic gene mutation is identified, first-degree family members (especially siblings) should be informed about disease monitoring options and knowing the risks of future health issues. background: genetic polymorphisms in key genes encoding enzymes involved in the bio-activation (cytochrome p450 (cyp)) or detoxification (glutathione s-transferase (gst)) of environmental carcinogens including tobacco specific nitrosamines are potential lung cancer risk factors. the frequencies of these variants and consequently their effects vary across ethnicities. the interactive effects with cyp-gst combination along with smoking have not been documented. five single nucleotide variants and two homologous deletion variants associated with cyp and gst genes and their interactive effects with smoking in non-small cell lung cancer (nsclc) were investigated design: the case control study comprised 244 cases of histologically diagnosed nsclc and 224 healthy controls. iec approval and informed consent were obtained. mean age of cases was 56.51±11.21+ 2sd, male: female ratio was 4.19, 91 were non-smokers and 153 smokers subdivided into 20 (47.7%), 20-40 (38.7%) and 40(13.6%) in pack-years. histological subtypes included 180 adenocarcinoma (adc), 57 squamous cell carcinoma (scc) and 09 adeno-squamous cases. majority were diagnosed in late stages with only 5 in stage ii, 78.2% had lymph-node metastasis and 139 cases had distant metastasis. dna was isolated from whole blood and genetic polymorphism analyses were determined by polymerase chain reaction coupled with restriction fragment length polymorphism followed by agarose and poly acrylamide gel electrophoresis for genetic variants. all the statistical analyses were performed with graph pad instat version 3.05 and spss version 16(chicago, usa). significantly high risk of nsclc and subtype adc was associated with variant cyp2a6, gstt1 and gstm1 ( conclusions: functionally relevant polymorphisms in cyp and gst genes with gene-gene and gene-environment interactions play a significant role in modifying the susceptibility to nsclc in population of indian ethnicity. design: thirty consecutive surgically resected lung-nens comprising 10 tc, 14 ac and six lcnec, all with long-term follow-up, were immunohistochemically stained for ki-67 and scanned at 40x (nanozoomer xr, hamamatsu, japan). a tailored algorithm was constructed to recognize all ki-67-stained tumor cells and the obtained patterns were described using spatial statistics, graph modeling, fractality and shannon entropy parameters. a support vector machine classifier with polynomial kernel was then trained, employing the 5 parameters that resulted most informative out of the 620 initially computed, to distinguish dead (true positive) from alive (true negative) patients. over 100 repetitions of 5-fold cross-validation, the model averaged 84.3% diagnostic accuracy in the prediction of ultimate clinical outcome (dead vs alive) in the 30 lung-nen patients under evaluation, which resulted to be independent of who classification. the corresponding values of sensitivity, specificity, ppv and npv were 73.6%, 89.6%, 78.2%, and 87.4%, respectively. the intratumor heterogeneity of ki-67 is a powerful and histology-independent resource to unravel clinical outcome of lung-nens by using machine learning algorithms. roshan raza background: cytologic diagnosis of mm is challenging since atypia in mesothelial cells is not specific for malignancy and there is no architecture to assess for invasion. loss of bap1 expression by icc and homozygous deletion of p16/cdkn2a by fish are specific but not sensitive for mm. co-deletion of mtap occurs in most mm with p16/cdkn2a deletions and can be detected by icc, which has advantages over fish. recently, 5-hmc has been reported to show 92% sensitivity and 100% specificity for distinguishing mm from benign mesothelial proliferations. to our knowledge, these 3 markers have not yet been studied in combination in cytology. herein, we determine the sensitivities and specificities of bap1, mtap and 5-hmc icc for the diagnosis of mm in cytology. design: icc with mtap, 5-hmc and bap1 was performed on all available mm cytology specimens from 2017-present and 20 benign specimens with reactive mesothelial cells on cell block. icc was scored as nuclear loss of bap1 expression, loss or marked reduction of cytoplasmic mtap expression, loss of nuclear expression of 5-hmc in at least 50% of tumor cells or non-contributory (nc) in cases without internal positive control. all available clinical next generation sequencing (ngs) data were collected. results: cytology specimens (9 fluids, 3 fna, 1 touch preparation) from 13 patients with diagnosis of mm confirmed by histology (11 epithelioid, 2 biphasic) contained adequate tumor cellularity on formalin-fixed cell blocks for study inclusion. all 20 cases with benign mesothelial cells showed retained bap1 and mtap expression and retained or <50% loss of 5-hmc expression. mm in 7, 3 and 6 patients showed bap1 loss, mtap loss and/or >50% loss of 5-hmc expression (sensitivities of 58, 23 and 46%), respectively. bap1 icc was nc in one mm specimen. while sensitivity of mtap was low, mtap loss was seen in 2 mm with retained bap1 and either retained or <50% loss of 5-hmc expression. combined sensitivity of all 3 markers was 77%. cdkn2a deletions were detected by ngs in all 3 mm with mtap loss by icc. mtap icc was retained in 2 mm with cdkn2a deletions detected by ngs. the status of the intratumoral immune microenvironment is important to guarantee the effect of immune checkpoint (ic) blockade therapy, which has been broadly used in patients with non-small cell lung carcinoma (nsclc). glucocorticoid (gc) is a hormone well-known to act strongly on the immune system. therefore, we examined the correlation between intratumorally synthesized gc through 11β hydroxysteroid dehydrogenase (hsd) 1 and the immune microenvironment in nsclc. we evaluated 125 surgical specimens from patients with nsclc (95 adenocarcinoma and 30 squamous cell carcinoma), assessing mainly the immunoreactivity for 11βhsd1 and 11βhsd2 and the levels of tumor-infiltrating lymphocytes (tils) and cd3-or cd8-positive t cells. furthermore, we examined the correlations between 11βhsd1 immunoreactivity and the therapeutic efficacy of ic blockade therapy using nine biopsy specimens from patients with nsclc. subsequently, we explored the mechanisms of gc effects on the intratumoral immune microenvironment, focusing on cytokines. results: 11βhsd1 immunoreactivity was significantly inversely correlated with the numbers of intratumoral tils, cd3-positive t cells, and cd8-positive positive t cells. additionally, we found 11βhsd1 immunoreactivity tended to be inversely correlated with the efficacy of the ic blockade therapy. according to the in vitro study, gc reduced the expression of cytokines such as il-8 and il-6, resulting in an inhibition of monocytes migration. furthermore, production of cortisol, active gc, was confirmed in the cell lines expressing 11βhsd1. conclusions: this is the first study demonstrating the significant inhibitory effects of intratumorally synthesized gc through 11βhsd1 on tissue immune microenvironment in nsclc, and the possible correlation of intratumoral 11βhsd1 status with the efficacy of ic blockade therapy. our results provided new insights into the therapeutic strategies and the efficacy prediction of ic blockade therapy. these prognostic factors may be useful to the treating physician in crafting treatment plans. this study seeks to compare these parameters between metastatic and primary sites in mm. design: paired cases of pleural mm with metastatic and primary sites were identified from the pathology archives at the participating institutions with review of h&e stained sections. histologic subtype was noted in all cases. for epithelioid mm, ng (1, 2, or 3, as previously described in the literature by kadota et. al.) and necrosis were determined. results: 60 paired cases were identified with the primary site subtype comprised of 36 epithelioid, 24 biphasic, and 0 sarcomatoid cases. 57 of 60 (95%) metastases showed epithelioid morphology; 3 (5%) metastases were biphasic. the positive predictive value of epithelioid subtype at metastatic site was 63%, sensitivity 100%, and specificity 88%. ng and the presence or absence of necrosis at metastatic sites were not correlative with primary site histologic subtype (p=0.34 and p=0.78, respectively). thirty-three pairs of metastases and primaries with epithelioid morphology were graded; 3 metastases were too small to grade. pairs were more likely to show a higher ng at primary rather than metastatic sites (p<0.01) with 6 of 8 (75%) metastatic sites with ng 1 showing ng 2 at primary site, and 3 of 16 (19%) metastatic sites with ng 2 showing ng 3 at primary site. all metastases with ng 3 had ng 3 at primary site. there were no pairs with lower ng at primary site than metastatic site. the presence or absence of necrosis at metastatic site was not predictive of the presence or absence of necrosis at primary site (p=0.39). the application of pathologic parameters to metastatic mm may not accurately predict the parameters at primary site. while sensitive, epithelioid morphology at metastatic site is not specific. metastatic sites may underestimate nuclear grade at the primary site. the presence of necrosis in a metastatic site may not be predictive of necrosis at the primary site. biopsy of the primary site may be required to more accurately classify the tumor. background: immunotherapy has dramatically changed the treatment landscape of various malignancies including lung adenocarcinomas. the 2019 nccn guidelines initially recommended single agent immunotherapy as a first line treatment option for advanced lung adenocarcinoma with pd-l1 expression levels of 50% or greater. however, given the recent data suggest that pd-l1 monotherapy is less effective in patients with egfr or alk gene alterations, the nccn panel recently deleted the recommendation for subsequent immunotherapy in these patients. we retrospectively analyzed 124 cases of lung adenocarcinoma with pd-l1 expression (>1% expression) using pd-l1 ihc 22c3 pharmdx test and correlated pd-l1 expression with the presence of egfr mutations or alk gene rearrangement in the 120 cases where all 3 tests were performed. pd-l1 expression levels were subcategorized as low expression level (1%-49% of the tumor cells express pd-l1) and high expression level (50%-100% of the tumor cells express pd-l1). results: high expression levels of pd-l1 (50%-100%) were observed in 40% (50/124) of the cases. among the 5 (4%) tumors that harbored alk gene rearrangement, 4 showed high pd-l1 expression. among the 31 (26%) tumors with detected egfr mutations, only 6 showed high pd-l1 expression. these two alterations were mutually exclusive. results are summarized in the table 1. conclusions: in our study cohort 40% (50/124) of lung adenocarcinomas with pd-l1 expression showed high (50%-100%) expression levels. 20% of these high pd-l1 expressors were positive for either egfr or alk alterations: 8% (4/50) harbored alk gene rearrangement and 12% (6/50) showed egfr mutations. we corroborated the previously reported association of alk gene rearrangement with high expression levels, and egfr mutations with low expression levels of pd-l1 in lung adenocarcinomas that express pd-l1. our study showed a substantial number (30%) of lung adenocarcinomas that express (>1%) pdl-1 have egfr mutations or alk gene rearrangement, suggesting that it is important to consider the results of these tests simultaneously in order to be able to stratify patients according to current nccn therapeutic guidelines. background: pd-l1 is a predictive marker of anti-pd-1/pd-l1 therapies for non-small cell lung cancer (nsclc). heterogeneous pd-l1 expression may cause dilemmas in anti-pd-1/pd-l1 therapies when faced with discrepant biomarker results. our aim was to comprehensively analyze the heterogeneity of pd-l1 expression defined as intratumoral area, paired samples and clones of anti-pd-l1 antibody to optimize tumor sampling and improve its accuracy. we selected 1002 nsclc surgically resected specimens, 54 cell block and 73 biopsy specimens. we analyzed the associations of pd-l1 expression with histopathological characteristics, assessed the heterogeneity between paired cell block and biopsy samples (n=54), paired biopsy and resected samples (n=19), paired two blocks of the same resected sample (n=53), paired primary and metastatic lesions (n=29), and compared the consistency of clones of pd-l1 antibody (22c3 and sp263, n=66). background: recently, we identified two young adults with idiopathic acute respiratory distress syndrome (ards) histologically characterized by diffuse alveolar injury with marked alveolar denudation, a novel pattern termed daide. both patients were exposed to trimethoprim-sulfamethoxazole (tmp-smx). here, we report four additional patients with daide following tmp-smx exposure and detail the features of all six patients. the original 2 patients were identified through routine surgical pathology, and an additional 4 patients were identified in the consultation archives. daide was identified on surgical biopsy (n=4), autopsy (n=1), or both (n=1). six patients with dad on surgical biopsy were used for comparison. clinical information was obtained from the medical record. h&e and ihc for ae1.3/cam5.2, cd68, and ck5/6 were reviewed. six otherwise healthy patients (m:f=1:5, median age: 21 years; age range: 10-37 years) initially presented with uri symptoms that rapidly progressed to ards; extensive infectious and rheumatologic workups were negative. no patients had a history of vaping. all patients were intubated and placed on ecmo for a median of 93 days (range: 1 to 190 days). surgical biopsy was performed in 5 patients (6 days before to 10 days after intubation), and autopsy was performed in 2 patients (12 and 466 days after intubation). all 5 surgical biopsies showed marked alveolar denudation with histiocytes replacing alveolar lining and nested peribronchiolar metaplasia, consistent with daide. hyaline membranes were rare when present (n=3). the 2 autopsies showed patchy alveolar-filling fibrosis and reepithelialization of most alveoli, with focal areas of alveolar denudation and histiocytic lining, suggestive of partially resolving daide. in comparison, control surgical dad cases showed prominent hyaline membranes, only focal alveolar denudation, and no significant histiocytic lining. on follow-up, daide patients underwent bilateral lung transplant (n=2); overall, 3 patients are deceased (n=3). conclusions: daide appears to be a novel variant of dad with a rapid and severe clinical course involving previously healthy patients and is characterized by extensive alveolar denudation with a lining replaced by histiocytes and a paucity of hyaline membranes. although our patients share a history of tmp-smx exposure, the role of tmp-smx in daide is currently unclear. nonetheless, patients with daide appear to have a poor prognosis. lynette sholl 1 , adrian dubuc 1 , jason hornick 2 , david chapel 1 1 brigham and women's hospital, boston, ma, 2 brigham and women's hospital, harvard medical school, boston background: the entity diffuse idiopathic neuroendocrine hyperplasia (dipnech) is a clinical syndrome defined by the who as a generalized proliferation of neuroendocrine cells either confined to the bronchial mucosa or resulting in formation of carcinoid tumorlets or tumors. when thus defined, dipnech is a diagnostic entity seen in clinically symptomatic patients presenting with histologic alterations of neuroendocrine proliferations combined with radiological features of airtrapping and multiple bilateral nodules. the significance of incidentally discovered neuroendocrine cell hyperplasia in surgically resected specimens and its correlation to radiological and clinical features is not known. this study aims to characterize the clinicopathological and radiological patterns identified in patients with histologically identified diffuse neuroendocrine cell hyperplasia (nech). we searched the pathology database starting jan, 2009 till sept, 2019. for the for the combination of the following keywords "diffuse" and "neuroendocrine cell hyperplasia (nech)'' and "lung" and identified ten cases that met the search criteria. the radiological data was reviewed by one thoracic radiologist in a blinded fashion. clinical, radiological and pathological features of these patients are depicted in table 1 . there was female predominance in our study (9/10) and all patients were above the age of 45 years. 70% (7/10) of the nech cases were diagnosed incidentally during imaging workup or postoperative surveillance for other malignancies. the remaining 3 cases were biopsied for presentation of respiratory symptoms and or radiological finding of interstitial lung disease. radiologically, bilateral involvement of lung was seen in 9 out of 10 cases with air trapping in 2 cases. the radiological diagnosis of dipnech could be made only in three cases, one of which was incidentally discovered (case 5). nine cases showed presence of carcinoid tumor or tumorlet along with nech and 4 cases showed granulomas along with nech. in conclusion, in this study, vast majority of patients with nech were incidentally discovered during surveillance and follow up imaging for an unrelated malignancy or nodules. none of these incidentally discovered patients were clinically symptomatic. the criteria for evaluation of nech in incidentally discovered asymptomatic patients is not well defined and necessitates further investigation and appropriate follow-up for an evolving interstitial lung disease. elisabeth tabb background: recent studies have implicated local microbiota in activating gamma-delta t-lymphocytes (gdtl) and subsequently inducing neutrophilic infiltration to promote oncogenesis in murine models of kras-mutant lung adenocarcinoma. this study aimed to examine the translational relevance of these preclinical findings by evaluating gdtl and tumor-associated neutrophils (tan) in human lung adenocarcinomas, including those with and without kras mutations. we quantified the numbers of gdtl and tan using immunohistochemistry for t cell receptor-gamma chain and myeloperoxidase, respectively, from 3 high-power fields (hpf; each 0.24 mm 2 ) of viable tumor areas on tissue microarray (duplicate of 2-mm core) sections of 236 human lung adenocarcinomas resected in 2010-2012. high and low gdtl or tan were defined as above or below the respective median. data were correlated with demographics, histologic features (ajcc 8 th edition), immune parameters (tumoral pd-l1 expression, cd8+ tumor-infiltrating lymphocytes [til]), molecular alterations (using a multiplex-pcr based assay), and outcome via chi-square or logrank tests as appropriate (significance: p<0.05). results: gdtl ranged 0-177 (median 3.3) per hpf (in 228 tumors with evaluable data); whereas tan ranged 0-140 (median 1.3) per hpf (in 223 tumors with evaluable data). the numbers of gdtl and tan correlated with each other (p<0.0001). both high gdtl and high tan were associated with smoking history (p<0.01; p<0.05), solid/high-grade acinar histologic patterns (p<0.0001; p<0.001), the presence of tumor necrosis (p<0.05; p<0.001), and elevated cd8+ til (p<0.001; p<0.05). high tan -but not high gdtl -was also associated with greater total tumor size (p<0.01), invasive size (p<0.05), advanced stages 3-4 (p<0.05), and worse progression-free survival (p<0.05), with a trend toward worse overall survival (p=0.05). however, gdtl and tan were not associated with tumoral pd-l1 expression or molecular alterations including kras or egfr. in human lung adenocarcinomas, we identified correlations of gdtl and tan with smoking history, aggressive histology, and elevated cd8+ til, but no associations with mutation status. tan also appeared to be associated with worse patient outcome. increased gdtl and tan may reflect aggressive tumor biology; our data also suggests that their effects may be more general and not restricted to kras-mutant tumors. background: pd-l1 expression in non-small cell lung cancer (nsclc) is used as a biomarker to treat patients with pd1 blockade therapy. pd-l1 expression may be related to tumor stromal interactions guided by underlying genotypic/phenotypic characteristics of a tumor and tumor antigenicity. increasing evidence has shown that c-met pathway activation in coordination with interferon gamma can lead to pd-l1 upregulation. in order to determine whether the underlying genomic characteristics of a tumor, including c-met status is associated with pd-l1 status, we analyzed lung nsclc that had been genomically characterized and correlated with pd-l1 status. design: immunohistochemical staining for pd-l1 was performed with clone 22c3 on a dako autostainer and scored as no expression (<1%), low expression (1-49%) or high expression (50% or greater). flourescent in situ hybridization was performed for alk, ret and ros1 (break apart probes) and c-met amplification/polysomy. mutations were detected by next generation sequencing of a 26 gene panel (cmp26, based on illumina tst26) or multiplex hotspot mutation assay (snapshot, 10 gene panel). we evaluated 103 lung nsclc (85 adenocarcinomas/favor adenocarcinoma, 7 squamous cell carcinomas, 8 not otherwise specified, 2 adenosquamous and 1 combined large cell neuroendocrine carcinoma with adenocarcinoma). no pd-l1 expression was seen in 34% (n=35) while pd-l1 expression was seen in 66% (n=68) with low expression in 25% (n=26) and high expression in 41% (n=42). among the no expression cohort there was a slight increase in egfr mutated patients as compared to pd-l1 expression cohort (23% vs. 13%) while similar frequencies in kras mutations were seen in both cohorts (no expression 40%; pd-l1 expression 38%). c-met alterations in the form of amplification, high polysomy and mutations (exon 14 skip mutations and juxtamembrane mutations) were associated with pd-l1 positive status (no pd-l1 expression 9% (n=3) vs expression 25% (n=17)), p=0.05), especially when there is high pd-l1 expression (29% (n=12); p=0.03). only in the no pd-l1 expression category was concurrent egfr mutation and c-met amplification/polysomy observed (2 of 3). higher pd-l1 expression is associated with c-met amplification, polysomy and mutations. these results indicate that patients with abnormalities in c-met may benefit from combined inhibition of pd-1 pathway and c-met pathway. background: differentiating malignant pleural mesothelioma from reactive mesothelial processes can be quite challenging. ancillary tests such as bap1 immunohistochemisry (ihc) and p16 fluorescence in situ hybridization (fish) are very helpful tools to aid in this distinction. ihc for mtap has recently been proposed as an effective surrogate marker for p16 fish, and it is an attractive alternative test due to shorter turn-around time. there is little data regarding the specificity of mtap ihc for mesothelioma, or whether it may be useful to distinguish mesothelioma from other entities in the differential diagnosis. while there are many reliable markers to distinguish epithelioid mesothelioma from adenocarcinoma, this is not true of sarcomatoid mesothelioma, which can be very difficult to distinguish from sarcomatoid carcinoma. the goal of this study was to determine if mtap loss is present in pulmonary sarcomatoid carcinoma or only in sarcomatoid mesothelioma. design: well-characterized cases of sarcomatoid carcinoma (n=35) and sarcomatoid mesothelioma (n=62) were included; diagnoses were confirmed by two thoracic pathologists with incorporation of immunophenotype, clinical and radiographic features. each case was stained for mtap (clone 2g4) and bap1 (clone c-4). successful staining was confirmed by presence of internal positive control for both stains. results: loss of mtap expression by ihc was observed in 18 of 35 pulmonary sarcomatoid carcinomas (51%); 32 of these cases also had successful bap1 staining performed, which was retained in all cases. mtap expression was lost in 38 of 62 sarcomatoid mesotheliomas (61%); bap1 was successful in all 62 cases, and showed loss in 6 (10%). in the 6 cases of sarcomatoid mesothelioma with bap1 loss, 5 also had loss of mtap, while mtap expression was retained in 1 case. conclusions: loss of mtap expression by ihc is common in pulmonary sarcomatoid carcinoma, present in half of cases. this may reflect homozygous p16 deletion, which has been described in a few cases of sarcomatoid carcinoma studied by fish analysis. this rate is similar to what is observed in sarcomatoid mesothelioma (61%). therefore, mtap loss is not specific for mesothelioma, and this stain is not useful to distinguish between these two malignancies. mtap loss is more common than bap1 loss in the setting of sarcomatoid mesothelioma (61% vs 10%, respectively). basile background: metexon 14 skipping (metex14) mutations present in 4% of lung adenocarcinoma is now becoming an important alteration to test for targeted therapy, similarly to alk. the only commercially available way to test for metex14 mutations is through next generation sequencing. there is a need for a faster and more available method to be used for the detection and validation of metex14 mutations but traditional animal base monoclonal antibody (mabs) techniques are slow and difficult to scale. a newer, faster and animal-free approach using instead b-cells cloning to generate in vitrorecombinant antibodies (rabs) is increasingly popular. here we compare a novel rabs technique to the more traditional mabs generation approach in developing a mutation-specific monoclonal immunohistochemistry (ihc) to metex14 mutation. design: using the same amino acid sequence overlapping the fusion of metexon 13 and 15, we generated a total of 72 antibody clones: 68 rabs and 4 rabbit-based mabs. the clones were validated by enzyme-linked immunosorbent assay (elisa) and ihc using a combination of synthetic peptides, metex14 mutated cell line (h596) and archival lung adenocarcinoma tissue with metex14 mutation. of the 68 rabs screened for affinity by elisa, 37 were retained for ihc validation along with all 4 mabs. using the metex14 mutated cell line, strong (3/3 staining intensity) diffuse (100% tumor cells staining) membranous staining was achieved in 4 of the rabs. seven other rabs had weak-to-intermediate (1-2/3 staining intensity) non-diffuse (5-75% tumor cell staining) membranous staining and the remaining 26 rabs showed no membranous staining. the best mabs clone only showed focal (5%) weak-to-intermediate staining with substantial background staining and the other 3 mabs were completely negative. the rabs technique was an effective approach to generate metex14 mutation-specific ihc clones. it can be scaled up more readily as opposed to the traditional animal-based hybridoma technique, and as a result, it increases its rate of success and decreases cost. this technique might allow for easier transition of mutation-based biomarkers to ihc and improve turnaround time and access for predictive tests in oncology. the top 4 rabs are currently being tested on an extended cohort of lung carcinoma tissue. (0) and weak staining (1+) were classified as negative; focal moderate staining (2+) as equivocal; patchy and diffuse moderate staining (2+) and strong staining (3+) as positive. (figure 1 ) results: among 164 cases, insm1 was positive in 16 cases (9.8%) and equivocal in 14 cases (8.5%). of 97 adenocarcinomas, insm1 was positive in 6 cases (6.2%) and equivocal in 9 cases (9.3%). the positive adc cases included 1 case with focal 3+ nuclear staining, 2 cases with diffuse 2+ staining, and 3 cases with patchy 2+ staining. of 51 squamous carcinomas, insm1 was positive in 9 cases (17.6%) and equivocal in 6 cases (11.8%). the positive sqcc cases included 1 case with patchy 3+ staining, 2 cases with diffuse 2+ staining and 6 cases with patchy 2+ staining. focal 3+ nuclear staining was seen in 1 of 16 other nsclc cases. (table 1 ) other non-small cell carcinoma conclusions: our study demonstrates that insm1 is expressed in a subset of nsclcs and suggest that caution must be exercised in interpreting insm1 staining, especially with limited sample such as biopsy and cell block sections. although insm1 is useful for the diagnosis of neuroendocrine tumors, it should not be used as a stand-alone marker in differentiating primary lung tumors. background: egfr tyrosine kinase inhibitors (tkis) therapy is a validated approach in the treatment of egfr-mutated non-small cell lung carcinoma (nsclc), but resistance universally develops and it has become a major obstacle in prolonging the survival of patients. more novel molecular biomarkers are still urgently required to elucidate the underlying mechanisms of resistance. this study aimed to investigate the role of linc00520 in the acquired resistance of nsclc to egfr-tkis. design: gene expression profiles from geo dataset were analyzed to identify the genes associated with egfr-tkis resistance. egfrmutated nsclc cell line pc9 was cultured with gefitinib for more than 6 months to acquire gefitinib-resistance, which was designated as pc9r. the expression patterns of linc00520 were characterized using reverse transcription quantitative polymerase chain reaction (rt-qpcr), and lentiviral vectors were used to infect cells to regulate the expression. cytotoxicity of egfr-tkis on infected cells was determined by cell counting kit-8 (cck-8). survival follow-up time of 948 nsclc samples from tcga dataset were enrolled in this study. in addition, statistical analysis was mainly performed by r programming language and graphpad prism 7.0 (graphpad). results: linc00520 is highly expressed in gefitinib-resistant cell line pc9r relative to pc9 (p<0.05). inhibiting linc00520 with lentivirus vectors induces apoptosis in pc9r. linc00520 could promote cell proliferation and induce resistance. statistics from tcga dataset demonstrate there is no significant difference in linc00520 expression between luad tissues (483) and normal tissues (347), but the expression level in lusc tissues (486) conclusions: linc00520 is involved in acquired resistance of egfr-tkis in nsclc. it may serve as a predictor and a potential therapeutic target for egfr-tkis resistance. ilyas yambayev background: lung cancer is the most common cause of cancer death worldwide. screening by ldct is expected to increase the frequency of early-stage nsclc of which lung adenocarcinoma (luad) is the most common subtype. although the assignment of predominant histologic subtypes is now recommended, there remains no widely accepted prognostically relevant grading system. several grading systems have been proposed however there has been no direct comparison of these grading systems in an independent cohort. here we compare several previously published architecturally based grading systems in a large cohort of stage i luad. design: h&e slides were reviewed from stage i luad resection specimens form a multi-institutional cohort of 278 patients diagnosed between 2005-2015. the staging was reassigned using ajcc 8th edition after determining the invasive size and assessing for pleural invasion. comprehensive histologic subtyping in 5% increments was performed along with mitotic figure counts and assessment for angiolymphatic invasion. this data was applied to compare recurrence-free survival rates using 4 published grading systems. the demographic, smoking status and stage characteristics are summarized in table 1 . results: figure 1 shows the kaplan meier curves for 4 published grading systems. predominant architectural pattern assignment alone was prognostically significant in stratifying patients into low ( -1968 conclusions: predominant architectural pattern assignment alone is a valuable grading system. the addition of mitotic grade and angiolymphatic invasion allows for further refinement to identify higher proportions of low-risk and a small but significant subset of very high-risk luad which might aid in the precision clinical management of early-stage luad. harbored egfr mutations. 6 lung cancers with atm p.v2424g were identified, with generally higher vafs (55-92%). 2/6 cases with atm p.v2424g also harbored the egfr p.l858r variant, while 2 cases also harbored kras codon 12 mutations. interestingly, the two kras/atm-mutated cases were from a single patient, with different kras mutations. conclusions: disease-associated brca1/2 variants are rare in lung carcinomas, and many cases were associated with kras hotspot variants, suggesting that brca1/2 mutations may be somatic in origin, likely in the setting of significant smoking history. in contrast, while atm p.v2424g is equally rare, the genomic context and vafs of the atm variants suggest possible germline events. assessment of other genes in the hr pathway is currently underway. jingping yuan 1 , huihua he 2 , lin xiong 2 , li xu 2 background: pulmonary enteric adenocarcinoma (pea) is a rare histologic type of lung adenocarcinoma. pea is composed mainly of tall columnar cells arranged in an irregular acinar or cribriform pattern with extensive central necrosis, closely resembling the appearance of intestinal epithelial and colorectal carcinomas under the microscope. immunohistochemically, pea is usually positive for ck7. however, some cases lack ck7 expression and are positive for intestinal differentiation markers, such as cdx2, villin, and ck20. for these reasons, it is difficult to distinguish between pea and pulmonary metastases of colorectal carcinoma (mcrc), so new identification methods need to be explored. satb2 expression is tissue-specific, and the only epithelial cells expressing this protein in adult tissue are the glandular cells lining the lower gastrointestinal (gi) tract. the sensitivity of satb2 in colorectal adenocarcinoma reaches 80%-97%, and their low expression in primary pulmonary tumors. therefore, this study investigated differential diagnostic values of satb2 in pea and mcrc. design: according to the who primary pea diagnostic criteria, the cases of lung adenocarcinoma were collected from patients being treated at the renmin hospital of wuhan university from 2015.1-2019.9 were screened. the specimens were independently reviewed by two pathologists, and immunohistochemical staining of lung adenocarcinoma markers (ck7, ttf-1, and napsina) and intestinal cancer markers (ck20, cdx2, and villin) was performed to aid identification. finally, after excluding possible colorectal cancer metastasis by carefully analyzing the clinical histories and imaging examinations, we recruited 51 primary pea specimens and 17 mcrc specimens for study. the sensitivity and specificity of immunomarkers satb2, ck7, ttf-1, napsina, ck20, cdx2 and villin for distinguishing pea from mcrc are evaluated. the expression rates of satb2 in pea and mcrc were 0.00% (0/51) and 100.00% (17/17), respectively. the sensitivity of satb2-, ck7+, ttf-1+, napsina+, ck20-, cdx2-and villin-for distinguishing pea from mcrc were 100.00%, 98.04%, 49.02%, 45.10%, 66.67%, 58.82%, 47.06%, respectively. the specificity of satb2-, ck7+, ttf-1+, napsina+, ck20-, cdx2-and villin-for distinguishing pea from mcrc were 100.00%, 88.24%, 88.24%, 100.00%, 82.35%, 88.24%, 88.24%, respectively. conclusions: our study shows that the sensitivity and specificity of satb2, which can all reach 100%, is much higher than those of common lung adenocarcinoma immunomarkers (ttf-1, napsina) and intestinal cancer immunomarkers (ck20, cdx2 and villin). satb2 can be viewed as the best immunomarkers for distinguishing pea from mcrc. the diagnostic value of ck7 is slightly inferior to satb2, the results of ck7 can be used as a reference for differential diagnosis of satb2. lisi yuan results: of 2296 nsclcs tested between 2017-7/2019, met ex14 variants were present in 44 (1.9%). a recurring vus not expected to impact exon 14 splicing seen in 41 cases (c.2975c>t (p.thr992ile) was excluded from analysis. in positive cases, median age was 76 (59% men; 41% women), and 46.7% were fna specimens. 32 of 44 variants were met exon 14 skipping (previously reported and/or involve the canonical recognition site), while the other 12 mutations were significant missense (3) or vus (9). of 9 vus, 5 were adjacent to the canonical splice site and likely to impact splicing, and 4 were missense variants. average allele fraction was 30.2. four cases had concomitant mutations (3=kras, 1 =egfr). of 35 cases with known clinical staging, stage 1-2=20(57%), stage 3=3 (9%), and stage 4=12(34%). of 19 resected nsclss, histological types and growth pattern included 7 lepidic predominant, 6 acinar predominant, 2 micropapillary predominant, 1 solid predominant, 1 sarcomatoid, and 2 adenosquamous. pd-l1 expression in 27 cases is shown in table 1 . stage 4 pd-l1 <50% pd-l1 > 50% pd-l1 <50% pd-l1 > 50% 12 cases (67%) 6 cases (33%) 2 cases (33%) 4 cases (67%) figure 1 -1974 conclusions: most met variants identified in our cohort (73%) are met ex14 skipping. another 11% likely result in exon 14 skipping, while the other 16% are missense variants presumably unrelated to splicing. the prevalence of met ex14 variants is lower than previously reported (1.9% vs 3%), and a large percentage of tumors has lower clinical stage and less aggressive pathologic features, both possibly reflecting sampling differences attributed to universal testing of nsclc at our institution rather than testing of only advanced disease. background: the 2015 who classification of lung tumors provided the first specialized classification for small biopsies. this article aimed to apply the newest classification to reclassify a group of small lung biopsies and analyze their status of the main driver mutations. design: 5032 cases of small lung biopsies (bronchoscopic, needle, or core biopsies) were selected, which ranged from 2015 to 2018. we applied the newest classification to reclassify them and analyzed the relationship between the diagnostic subtypes of these biopsy specimens and the mutation rates of egfr and alk. the numbers of small lung biopsies each year during 2015-2018 were respectively 1068, 1299, 1511 and 1154. there were 3280 men and 1752 women, ranging in age from 11 to 93 years (median 63 years). the most common diagnosis was primary lung cancer (3130, figure 1 -1978 figure 2 -1978 conclusions: this study gave an panoramic view on pd-l1 expression and clinicopathological profiles based on the largest chinese nsclc cohort. the discrepancy of pd-l1 expression between surgically resected specimens and biopsy specimens and metastatic lesions may result from inter/intra-tumoral heterogeneity. background: accurate assessment of pd-l1 expression is critical for selection of patients of non-small cell lung cancer (nsclc) for immunotherapy with pd-l1/pd-1 inhibitors. however, only limited reports of pd-l1expression in population of nsclc in north america are available. this study reports pd-l1 expression level in a large patient population of nsclc in canada. design: pd-l1 testing of nsclc was performed for patients from the whole province of british columbia, canada in a centralized provincial pathology laboratory at bc cancer, vancouver centre. the test used dako pd-l1 ihc 22c3 pharmdx and dako autostainer link 48 immunostainer, as it was fda approved companion diagnostic test for pembrolizumab. pd-l1 protein expression is determined by using tumor proportion score (tps), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. results: from january 2017 to march 2018, 1,716 nsclc was tested, which included 1,301(75.8%) adenocarcinoma, 284 (16.6%) squamous cell carcinoma and 131 (7.6%) nsclc, nos. pd-l1 expression level in adenocarcinoma was similar to that in squamous cell carcinoma (p>0.05), but was significantly higher than in nsclc, nos (p<0.05, table 1 ). the overall percentage of high pd-l1expression (tps³50%) was 39%. the high pd-l1 expression (tps³50%) was found in 42% distant metastases, 45% mediastinal lymph nodes, and 35% lung primary tumors. the differences in the distribution of high pd-l1 expression among distant metastatic sites, mediastinal lymph nodes metastases and primary sites had statistical significance (c2=11.8, p<0.01). conclusions: using fda approved dako pd-l1 ihc 22c3 phamadx assay, we found that adenocarcinoma had a similar pd-l1 expression level to squamous cell carcinoma but significant higher expression than nsclc, nos. the percentage of high pd-l1 expression (tps³50%) was significantly higher in mediastinal lymph nodes and distant metastatic sites than in primary sites, which suggests that pd-l1 testing of metastatic nsclc could identify more patients eligible for immunotherapy. figure 1 -1980 figure 2 -1980 conclusions: the correlations among fs, fsc, and rt are low. fs stas+ cases remain stas+ only in 60% of fsc and 48% of rt. stas shows higher correlation with grade on rt than it does on fs. these results show a lack of reliability in the assessment of stas on fs, and do not support the proposal of reporting stas in fs to make intraoperative clinical decisions, as doing so may subject patients to unnecessarily aggressive surgery. ki67% when 6 classes were used (log-rank p=0.82 and p=0.21, respectively) or when 2 classes were used with either 20% ki67% (logrank p=0.95 and p=0.53) or 40% ki67% (log-rank p=0.78 and p=0.91) as cut-point conclusions: our findings do not support the use of 40% as the minimum ki67% in lcnec as suggested by who or the use of 20% as the minimum ki67% for lcnec as described for diagnosing entero-pancreatic neuroendocrine carcinoma expression of insulinoma-associated 1 (insm1) in non-small cell lung cancers: a diagnostic pitfall for neuroendocrine tumors none background: insulinoma-associated 1 (insm1) has recently been reported as a highly sensitive and specific marker of pulmonary neuroendocrine tumors. it has also been noticed that insm1 expression can be seen, although uncommonly, in non-neuroendocrine tumors. the aim of this study was to evaluate the expression of insm1 in non-small cell cancers (nsclcs) to avoid diagnostic pitfall uv genomic signature classifies lung melanomas of unknown primary as metastases from occult cutaneous melanomas grant or research support bristol myers squibb; advisory board member, immunocore; consultant, castle biosciences; marc ladanyi: none none alterations were identified for a total of 2 -5 shared alterations per pair (mean of 3 shared alterations). the probability of chance co among the primary lung cancer, the dominant type was adenocarcinoma (1421, 28.2%), followed by nscc, favor adenocarcinoma (501, 10.0%), squamous cell carcinoma (368, 7.3%), and nscc, favor squamous cell carcinoma (360, 7.2%). the tests of the main driver mutations using arms-pcr technology demonstrated that egfr was positive in 56.1%(499/889, in adenocarcinoma and nscc, favor adenocarcinoma) 6%, 35/148) and p.s768_d770dup (20.9%, 31/148) were most frequent. 94.4% (320/339) of erbb insertions were found in adenocarcinoma, among which surgical samples were more common than small biopsies (10.1% vs 7.3%, p=0.016). among adenocarcinoma, female have higher frequencies of erbb insertions than male (11.3% vs 6.8%, p<0.001). the median age of egfr insertion carrier, erbb2 insertion carrier and non-erbb insertion carrier was 54-, 49-and 62-year old respectively (p<0.001). compared with invasive adenocarcinoma (ia) (5.6%), adenocarcinoma in situ (ais) (28.4%) and minimally invasive adenocarcinoma (mia) (20.0%) were more likely to harbor an erbb insertion (p<0.001). the pleural invasion frequency of egfr insertion carrier, erbb2 insertion carrier and non-erbb conclusions: the insertion mutations in kinase domain of egfr and erbb2 were more common in younger, female and adenocarcinoma patients. ais and mia were more frequent to harbor an erbb insertion than ia, which suggest erbb insertion may be related to the evolution of adenocarcinoma. erbb2 insertion carrier tend to have a lower pleural invasion rate while a higher lymph node metastasis rate correlation between pd-l1 expression and clinicopathological and molecular characteristics of non-small cell lung cancer: a large scale multi-centric real-world study of chinese cohort the first affiliated hospital of none background: programmed cell death ligand-1 (pd-l1) is a predictive marker of anti-pd-1/pd-l1 immune therapies for non-small cell lung cancer (nsclc).the definite relationship between pd-l1 expression and clinicopathological, molecular profiles of nsclc design: a total of 6126 nsclc specimens were enrolled from 6 centers in china. we analyzed pd-l1 (22c3) expression by immunohistochemistry on dako autostainer link 48 platform. the status of egfr was defined by rt-pcr or ngs in 2382 samples and alk was tested by ihc, fish or ngs in 1716 samples pd-l1 high expression was more frequent in egfr-wild type than in mutant type (12.9% vs. 4.7%, p < 0.001). furthermore, pd-l1 high expression was more prevalent in rare egfr mutant types than in common mutations (42.1% vs. 20.8%, p=0.031). besides, pd-l1 high expression was more frequently identified in alk fusion cases (14.6% vs. 6.5%, p= 0.001). a total of 1665 small biopsy cases included 1454 primary specimens and 211 metastatic specimens. the prevalence of pd-l1 high expression in surgical samples was much lower than in primary biopsy samples. among of them, pd-l1 high expression was also prevalent in egfr-wild type than in mutant type (32 we found that high pd-l1 expression was more prevalent in metastatic specimens than in primary biopsy specimens (30.8% vs. 21.8%). in metastatic adc specimens, the rate of high pd-l1 expression was greater than in primary adc navneet narula 4 , mari mino-kenudson 3 , andre moreira 5 1 nyu school of medicine none background: spread through air spaces (stas) has been reported to be associated with a worse prognosis in adenocarcinoma of lung. recently it has been proposed that stas be reported on frozen sections (fs) as an indication for more aggressive surgery (lobectomy vs sublobar resection). we undertook this study to evaluate the reliability of stas assessment on fs compared to fs controls (fsc) design: cases of adenocarcinoma that had fs of the tumor were identified retrospectively from two institutions. for each case, the following was recorded: presence(+)/absence(-) of stas on fs, fsc, and rt; and % of tumor patterns: lepidic(l) cross-tabulations and spearman's correlations (rs) were performed in spss (see table) of which 28/47(60%) had stas+ on fsc (rs=0.39) and 22/46(48%) had stas+ on rt (rs=0.34) (1 tumor was only present on fs/fsc). of the 40 stas+ cases on rt, 18/40(45%) did not have stas (stas-) on fs (rs=0.34). 118/165 of cases were stas-on fs; of these of the 15 g2 cases with stas+ on fs, 7 had 10% to <20% high grade pattern (m/s) we performed a retrospective review of patients with imas who had genomic analysis performed on tumors in different lobes. molecular assays included dna-based targeted next-generation sequencing (ngs) for 410-468 cancer genes combined with rna-based ngs fusion assay (archer) and non-ngs panels for a subset of cases. tumor clonal relationships were assessed by comparing somatic alterations between the separate tumor sites.results: twenty-one patients with genomically-profiled imas involving contralateral (n=19) or ipsilateral different lobes (n=2) were identified. in most patients (n=14), tumors had discrete nodular presentation. second ima presented metachronously in 11 patients with a mean latency of 4.2 years. notably, in 3 patients, contralateral spread manifested ≥8 years (up to 11 years) after initial tumor resection. genomic analysis was performed on 2 separate imas in 19 patients and 3 separate imas in 2 patients, resulting in a total of 44 genotyped tumors. comparative genomic analysis revealed that tumors in all patients shared matching driver alterations including kras (n=17), nrg1 (n=2), erbb2 (n=1) and braf (n=1). in addition, in tumor pairs profiled by ngs and archer, other shared we have encountered a group of patients with melanomas involving the lung in the absence of a clinically known primary melanoma elsewhere. a subset of patients presented with solitary large tumors. while primary pulmonary melanomas (ppm) is a category included in the thoracic who classification, given the absence of normal melanocytes in the lung its existence has been questioned. herein we investigate genomic profiles of melanomas of unknown primary origin involving the lung. in particular, we sought to determine whether uv genomic signature -a characteristic feature of most cutaneous melanomas -is present in such tumors.design: cases of melanomas involving the lung with no known primary elsewhere were identified retrospectively. the clinicopathologic characteristics of each case were annotated. all cases included in the study underwent targeted dna next-generation sequencing (ngs) interrogating up to 468 cancer genes. genomic signatures were analyzed based on a method described by alexandrov lb et al. (nature 2013; 500:415-421) .results: ten ngs-profiled melanomas involving the lung were identified. five patients had solitary lung lesions with the median size of 5.2 cm (range 2.6 to 10.1 cm). of those, 3 tumors were endo/peri-bronchial, thus meeting the suggested criteria for ppm. hilar nodes were involved in 2 patients, and 8 tumors had epithelioid morphology resembling non-small cell carcinoma. no evidence of primary melanomas was found for any patients on clinical follow-up (median 28 months; range 5 to 42 months). genomic testing revealed the following driver mutations commonly found in melanomas: braf (n=6), nras (n=1) and kit (n=1). genomic signature analysis was feasible for 8 cases harboring >20 mutations required for reliable analysis, including 4 patients with solitary masses. this revealed the presence of a dominant uv signature in all cases. in contrast, none of the primary lung carcinomas tested by the same method (n = 255) had a uv signature. the consistent presence of a uv signature provides strong support for an occult or regressed cutaneous origin of melanomas involving the lung, and argues against the concept of ppm. clinical presentation as solitary large (reaching >10 cm) masses occasionally with hilar adenopathy and epithelioid morphology may closely mimic primary lung carcinomas both clinicoradiologically and pathologically, representing a major potential diagnostic pitfall. background: pulmonary invasive mucinous adenocarcinoma (ima) commonly presents as a multifocal disease. it is widely recognized that diffuse 'pneumonic-type' ima represents aerogenous spread of a single tumor. however, imas may also present as discrete nodules in different lobes, raising the possibility of separate primary tumors. here, we explored the clonal relationship of imas involving different lobes using comparative molecular profiling.conclusions: molecular profiling supports that multifocal imas involving different lobes represent intrapulmonary spread of a single tumor rather than separate primary tumors, including tumors presenting contralaterally after a remarkably long latency (>8 years). overall, these findings reinforce the unique biology and clinical behavior of imas, and draw a sharp distinction with multifocal non-mucinous lung adenocarcinomas, which recent molecular studies confirm to represent predominantly separate primary tumors. we addressed these issues in human non-small cell lung cancer (nsclc). design: pd-l1 and b7-h3 expression in tumor cells were evaluated using immunohistochemistry. composition of tumor-infiltrating immune cells, including lymphoid cells, macrophages and dendritic cells, was analyzed using flow cytometry for fresh tissues from a prospective cohort of 71 patients with nsclc and was compared according to pd-l1 and b7-h3 expression status. ju-yoon yoon 1 , jason rosenbaum 2 we examined the local cohort of patients with the referral diagnosis of "lung cancer", sequenced by the in-house 152 gene massively parallel sequencing (mps, also known as next-generation sequencing or ngs) assay. all alterations were filtered and reviewed for disease-associated variants. for atm, our assessment was limited to p.v2424g, a variant with the highest penetrance among the atm variants, associated with increased breast cancer risks at levels comparable to disease-associated brca1/2 variants.results: 1,473 cases were successfully sequenced by the in-house solid mps assay, among which 23 patients (1.6%) were found to harbor disease-associated brca1 (14/23) or brca2 (9/23) alterations, with variant allele fractions (vafs) ranging 4-69%. these cancers were mostly adenocarcinomas (17/23, with 5 carcinoma nos and 1 scc). 10/23 harbored kras hotspot mutations, and no cases the criteria for small lung biopsies proposed by the 2015 classification of lung tumors should be applied to pathologists' daily work. it can improve the diagnostic efficiency and quality of small lung biopsies and assist oncologists in accurately understanding the pathologic diagnosis. in this way, accurate treatment and improved prognosis are more available to the patients. zhihong zhang background: to study the differential diagnosis of multi-focal lung cancer and lung cancer with pulmonary metastasis by detecting the different lesions of the same patient. to explore the differences in prognosis between mplc and im, and to explore the factors affecting the prognosis of multi-focal lung cancer and the tumor heterogeneity of multi-focal lung cancer in combination with histopathology and molecular biology.design: fifty patients with multi-focal lung cancer were screened, and the relevant clinical information was noted; the patients were diagnosed by accp standard. mutations of the lesions were detected by arms-pcr, and the detected genes included egfr, alk, ros1, met, kras, ret, her-2, braf, nras and pik3ca. the results of genetic testing were compared with those of accp standard diagnosis. we analyzed a total of 101 tumors from 50 patients. classification based on gene testing contradicted the clinicopathologic diagnosis in 10 (20%) of the comparisons, identifying independent primaries in 6 cases diagnosed as metastasis and metastases in 4 cases diagnosed as independent primaries. another 7(14%) tumor pairings were assigned an "equivocal" result based on gene testing. the results of gene testing of the remaining 33(66%) tumor pairings were consistent with the clinicopathologic diagnosis. the mutant heat map indicated that im patients have a higher rate of mutation consistency than mplc patients. the difference of prognosis between patients with mutations and those with wild-type genes patients was statistically significant (p=0.002). the difference of prognosis between patients with lymph node metastasis and those with no metastasis of lymph nodes was statistically significant (p=0.006). the difference of prognosis between patients with mplc and those with im was statistically significant (p=0.038). the difference of prognosis between patients who had different condition was statistically significant (p=0.038). multi-gene detection of multi-focal lung cancer has a certain auxiliary effect on the differential diagnosis of multiple primary lung cancer and lung cancer with pulmonary metastasis, which can complement the clinical standards, but also has some limitations. key: cord-018086-klels5e3 authors: van der kaaij, n.p.; bogers, a.j.j.c.; lachmann, b. title: ischemia-reperfusion injury of the lung: role of surfactant date: 2005 journal: yearbook of intensive care and emergency medicine 2005 doi: 10.1007/0-387-26272-5_6 sha: doc_id: 18086 cord_uid: klels5e3 nan to facilitate normal breathing with minimal effort, pulmonary surfactant lowers the surface tension at the alveolo-capillary membrane. in addition, lowering of the surface tension is important for the fluid homeostasis across the alveolo-capillary membrane. furthermore, surfactant serves as a functional barrier in the alveolus, so that the transfer of molecules across the alveolo-capillary membrane is limited. finally, surfactant protects the lung against microorganisms [10, 21, 22] . surfactant is composed of lipids (90%), of which dipalmitoyl-phosphatidylcholine (dppc) is the most surface tension lowering lipid, and surfactant associated proteins (sp) (10%). the proteins of pulmonary surfactant can be divided into two groups: the hydrophilic proteins sp-a and sp-d, and the hydrophobic proteins sp-b and sp-c. sp-b, as well as sp-c, have been demonstrated to enhance lipid insertion into the monolayer at the air/liquid interface. in this way they maintain a low surface tension, thereby protecting the surface film from being contaminated by nonsurfactant proteins, which can result in inactivation or degradation of the surfactant film. sp-a and sp-d are believed to be molecules of the innate immune system through their ability to recognize a broad spectrum of pathogens. several studies have shown that sp-a and sp-d interact with a number of viruses, bacteria and fungi, and with inhaled glycoconjugate allergens, such as pollen grains and mite allergens [10] . furthermore, sp-a has been suggested to play an important role in phospholipid secretion and recycling, formation of tubular myelin and blocking surfactant inhibition by serum proteins [21, 22] . surfactant can be divided by ultra centrifugation into two subfractions, which differ in morphological appearance and density. the heavy subtype or large aggregate subform of surfactant is highly surface active, contains a high amount of sp and is made up of tubular myelin, lamellar bodies and large vesicles. the light subtype or small aggregate subform has a poor surface lowering capacity and consists of small vesicles [22] . production and secretion of surfactant is done by the at ii cells. both at ii cells and alveolar macrophages are important for recycling of surfactant lipids, which is essential for maintaining homeostasis of the endogenous surfactant pool [22, 23] . z fluid homeostasis in the lung as discussed previously, surfactant is essential for maintaining normal fluid homeostasis in the lung and preventing pulmonary edema. fig. 2 presents a diagram of fluid balance across the lung. the normal plasma oncotic pressure of 37 cmh 2 o is opposed by the capillary hydrostatic pressure of 15 cmh 2 o, the oncotic pressure of interstitial fluid proteins of 18 cmh 2 o and by the surface tension conditioned suction pressure of 4 cmh 2 o. in general, alveolar flooding will not occur when the surfactant system is properly functioning. however, when the surface tension rises above a critical level, alveolar flooding will occur, leading to influx of proteins into the alveolar space which results in further inactivation of surfactant [10, 24] . z lung ischemia-reperfusion injury: pathophysiology lung ischemia-reperfusion injury, which occurs to a certain extent during lung transplantation, can damage the surfactant system. after the lung has been removed from the donor, the organ is hypothermically stored to reduce the rate of biochemical reactions, which results in a decreased degradation of important cellular components. nevertheless, adenosine triphosphate (atp) is depleted during ischemia, which ultimately causes inactivation of atp-dependent membrane pumps, an increase in intracellular calcium, inflammation, the formation of reactive oxygen species (ros), and cell death [10, 25] (fig. 3 ). lung ischemia-reperfusion injury: inactive atp-dependent membrane pumps and intracellular calcium accumulation under normal conditions, the action of the na + /k + -atpase pump sets up a gradient of high extracellular na + relative to intracellular levels, which in turn drives the na + /ca 2+exchanger, so that ca 2+ is pumped out of the cell. during atp depletion, the na + /k + -atpase pump becomes inactivated, leading to an increase in intracellular na + . as a result, the na + /ca 2+ pump will not function, causing ca 2+ to accumulate inside the cell. when ischemia is prolonged, the ionic balance may be so upset that the na + / ca 2+ -pump activity is reversed, resulting in import of ca 2+ in exchange for na + , thereby exacerbating calcium overload. other mechanisms contributing to high intracellular ca 2+ levels are an inactive plasmalemmal atp-dependent ca 2+ -pump, important to move ca 2+ out of the cell, liberation of stored cytoplasmic calcium due to the acidosis, and a decreased uptake by the sarcoplasmic/endoplasmic reticulum [10, 25] . cytosol elevated ca 2+ activates phospholipase a 2 , which results in the induction of arachidonic acid. arachidonic acid is normally incorporated in the cell membrane and functions as a precursor for the production of eicosanoids, consisting of thromboxanes, leukotrienes, prostacyclin and prostaglandins. the effects of the eicosanoids due to tissue injury are various [10, 25] . while thromboxanes are predominantly produced by platelets, leukotrienes are formed by leukocytes (mostly neutrophils), prostacyclin by endothelial cells and prostaglandins by smooth muscle cells. thromboxane a 2 has a potent vasoconstriction action, induces leukotriene production by neutrophils, and activates neutrophil adhesion receptors to facilitate interaction with the endothelium, which also expresses adhesion molecules due to the elevated ca 2+ . leukotrienes too cause vasoconstriction, but may as well increase vascular permeability, and enhance neutrophil accumulation, adhesion and extravasation through the endothelium. prostacyclin plays an important role in vascular function because it inhibits platelet adhe-ischemia-reperfusion injury of the lung: role of surfactant 53 fig. 3 . pathophysiology of lung ischemia reperfusion injury. see text for explanation. atp: adenosine triphosphate; amp: adenosine monophosphate; ros: reactive oxygen species sion to the vascular endothelium and is a strong vasodilator. damaged endothelial cells do not produce pgi 2 , thereby making the vessel more susceptible to thrombosis and vasospasm. prostaglandins have both a vasoconstrictor and vasodilator function. next to leukotrienes, prostaglandins can also make the vascular endothelium more`leaky' thereby promoting edema formation during inflammation [10, 25] . furthermore, increased intracellular ca 2+ causes transformation of xanthine dehydrogenase into xanthine oxidase, thereby facilitating the production of ros, as described in the next section [10, 25] . in the aerobic setting, atp is converted to urea and xanthine by the effect of xanthine dehydrogenase. however, due to the formation of xanthine oxidase in lung ischemia-reperfusion injury, hypoxanthine is broken down into ros. a second system to generate ros is by the nadph oxidase system, which is predominantly present on the membrane surfaces of monocytes, macrophages, neutrophils, and endothelial cells, and catalyzes the reduction of oxygen to superoxide and hydrogen peroxide. the superoxide anion, hydrogen peroxide and hydroxyl radical, which are all ros, are very unstable and damage cell membranes by lipid peroxidation [10, 25] . after lung ischemia-reperfusion injury, pro-inflammatory cytokines (like interleukin [il]-8, il-10, il-12, il-18, tumor necrosis factor [tnf]-a, and interferon [ifn]c) are released by macrophages due to activation during ischemia. consequently, neutrophils and lymphocytes are recruited into the lung. because of expression of adhesion molecules to both endothelium (e-selectin, p-selectin, intercellular-adhesion-molecule-1) and leukocytes (l-selectin, b-integrins), leukocytes roll (selectins), adhere (b-integrins, intercellular-adhesion-molecule-1), and extravasate into the lung tissue. macrophages and neutrophils contribute to cellular damage by the production of ros and several other mediators, such as proteolytic enzymes (gelatinases, collagenases and elastases), lysozyme, and lactoferrin [10, 25] . to summarize these different pathways: the increase in intracellular ca 2+ and na + and the formation of ros, eicosanoids, proteolytic enzymes and (phospho)lipases damage the lipid membrane of the cell, causing increased cellular permeability, the formation of cellular edema and eventually cell death. finally all these pathways lead to a disturbed surfactant system [10, 25] . to study the complex pathophysiology of lung ischemia-reperfusion injury and to investigate surfactant treatment possibilities, an animal model is often used. three major types of animal models have been reported: an isolated ex vivo, perfused lung system, a whole lung transplantation model, and an in situ warm ischemia model of the lung. the isolated, perfused lung system is a model whereby the organ is taken out of the animal, hypothermically stored for a certain period of ischemia and subsequently reperfused by the use of a langendorff system. although this model has advantages (e.g., the use of lungs of knock-out mice), the most important disadvantage clearly is the ex vivo situation, disturbing normal physiological interactions. furthermore, only short follow-up periods after reperfusion (hours) can be established [26] . animal whole lung transplantation has the advantage that it best reflects human transplantation and allows the investigation of the effect of cold ischemic storage, the use of storage solutions, and the study of allo-antigen settings. however, disadvantages are that, especially in small animals, it is a time-consuming and technically difficult procedure with often high mortality rates, limiting the study of longer follow-up periods after transplantation [3, 4, 18, 19, 27] . also, larger animals, like pigs and dogs have been used to avert the possible high mortality and the technical difficulties [6, 28±30] . nevertheless, the limitations of this model are the difficulties in activating and blocking specific pathways (due to costs and receptor specificity) and the unavailability of genetically modified animals. to overcome some of the disadvantages in transplantation models, an in situ warm ischemia model of the lung in small rodents has been developed, in which the ischemia is induced by clamping the pulmonary artery, veins and bronchus of (usually) the left lung. clamping time generally ranges from 60 to 120 minutes (van der kaaij, et al., unpublished data) [31, 32] . after declamping, reperfusion occurs. although this in vivo model is technically much easier than the aforementioned transplantation models, there are some disadvantages. firstly, warm ischemia is used, accompanied by a high metabolic rate. however, the use of short periods of warm ischemia is accepted as an accelerated model of ischemia-reperfusion injury of the lung [33] . additionally, because of often long warm ischemic periods, severe ischemia-reperfusion injury is induced resulting in still high mortality. as a result, most studies still investigate short time periods (max. 6 hours) after the start of reperfusion. only a few studies report long reperfusion times of about 1 week [27, 32] ; however, in these studies mortality was still very high after extubation of the animals. we have recently developed a model of lung ischemia-reperfusion injury in which (by adjusting the anesthetic protocol and ventilator strategy) we are able to study intervals up to months after reperfusion, with acceptable mortality (van der kaaij, et al., unpublished data). z surfactant dysfunction and lung ischemia-reperfusion injury using these experimental models, several research groups have gained valuable information on how specific parts of the surfactant system are affected by lung ischemia-reperfusion injury [2, 3, 16, 17, 34] (fig. 4) . the presence of alveolar proteins after lung ischemia-reperfusion injury has been described in many studies [2±5, 16, 19, 28, 29, 35±40] . both different warm (1±2 h [3, 36] , van der kaaij, et al., unpublished data) and cold (2±20 h [2, 4, 5, 19, 35] ) ischemic intervals have resulted in increased levels of alveolar protein between 1 and 24 hours after reperfusion. due to ros, proteolytic enzymes and phospholipases, endogenous surfactant and the endothelial and epithelial membrane are damaged. this results in leakage of proteins into the alveolus and surfactant com-ponents into the bloodstream. since surfactant is rate limiting for the transfer of proteins across the alveolo-capillary membrane and is either inactivated or lost due to the increased endothelial permeability after lung ischemia-reperfusion injury, a further influx of proteins is facilitated. because proteins, once accumulated in the alveolus, then dose-dependently inhibit surfactant, this results in a self-triggering mechanism of surfactant inactivation [24] . under normal conditions, sp-a is able to counteract the inactivating effects of serum proteins on surfactant [41] . however, after lung ischemia-reperfusion injury, a decrease in sp-a was found in human lung transplant recipients and animal models of lung ischemia-reperfusion injury [19, 42] . a decrease in sp-a was already visible after prolonged ischemic storage without reperfusion and decreased further after the start of reperfusion [2, 4, 19] . moreover, in lung transplant recipients, the level of sp-a is still low more than one year after transplantation [42] . sp-a can be degraded by ros and proteolytic enzymes. it was . edema results in dilution of the surfactant phospholipids inside the alveolus, which results in further formation of edema. a decrease in sp-a leads to [1] less inhibition of serum proteins, and [2] decreased phospholipid secretion, recycling, and formation of tubular myelin. [3] sp-b&c degradation causes less phospholipids to be inserted into the phospholipid monolayer lining the alveolar epithelium. [4] once serum proteins have infiltrated the alveolus, they compete for a place at the air-liquid interface, thereby dose-dependently inhibiting surfactant function. furthermore, once the phospholipid monolayer is damaged, the molecule transfer limiting function of surfactant is also impaired, resulting in further influx of serum proteins, so that a vicious circle has developed. [5] finally at ii cells, important in the production, recycling and secretion of surfactant phospholipids are damaged, so that less la is secreted and a smaller amount of sa is being recycled. due to these factors, a decrease in la and an increase in sa has been noticed after lung ischemia-reperfusion injury also shown that the levels of sp-a decreased with ascending severity of lung ischemia-reperfusion injury, suggesting that preservation or amelioration of sp-a is essential for improvement after lung ischemia-reperfusion injury [19] . besides the protein inhibiting function of sp-a, sp-a also plays a central role in phospholipid secretion and recycling, contributing to a decrease in surface activity of surfactant. sp-b and sp-c have also been found to be decreased after lung ischemia-reperfusion injury [28] . decreased levels or inactivation of sp-b and sp-c can result in a diminished quantity of phospholipids, but also in a changed composition of the surfactant on the surface of the alveolar epithelium, thereby impairing the surfactant lowering properties [28] . studies have reported surfactant dysfunction without a change in the overall amount of phospholipids [2, 3] . both a decrease in dppc and phosphatidyl-glycerol and an increase in sphingomyelin have been described [2, 3, 17, 28] . dppc, the saturated form of phosphatidylcholine, is the most important phospholipid known to reduce the minimum surface tension. although klepetko [29, 37±40, 44] . furthermore, it was demonstrated that when, in the case of replacement therapy, surfactant was administered just before, at or after reperfusion, it improved lung compliance and pao 2 and prevented an increase in the small aggregate/light aggregate ratio directly after lung ischemia-reperfusion injury [4, 35] . however, other studies have shown that treatment with exogenous surfactant before the onset of ischemia is more beneficial when compared to treatment at reperfusion [30, 35, 45, 46] . this can be explained by less complement activation, diminished membrane damage, and an enlarged surfactant phospholipid pool after donor treatment, thereby preventing deterioration of the entire endogenous surfactant pool [6, 46] . also, surfactant given to the donor may result in a more homogeneous distribution in the lung as compared to treatment after reperfusion, when alveolar damage has already occurred [6] . most studies investigating the effect of surfactant replacement therapy have only addressed the first hours (2±6) after reperfusion. studies on the longer term effects of lung ischemia-reperfusion injury and surfactant treatment are scarce. in this regard, erasmus and colleagues demonstrated that surfactant treatment just before reperfusion enhanced recovery from lung ischemia-reperfusion injury at one week postoperatively [27] . we confirm that lung ischemia-reperfusion injury resulted in the conversion of surfactant into less active surfactant, and impaired pao 2 and lung compliance throughout the first week after reperfusion. however, even months after reperfusion, diffuse alveolar damage and decreased lung compliance were visible. surfactant treatment before the induction of warm ischemia completely normalized these parameters from 3 to 90 days after reperfusion (van der kaaij et al., unpublished data). the rationale behind surfactant replacement therapy is to ameliorate the damage caused by ros, to decrease the inhibitory effects of serum proteins, and to preserve the levels of surfactant protein and dppc. when the quantity of surfactant is low or its composition is changed, serum proteins (like albumin, fibrin, fibrinogen, c-reactive protein [crp] , and hemoglobin) leak into the alveolus [47] . this protein leakage can be ameliorated by surfactant therapy [4, 24, 26, 48] (van der kaaij et al., unpublished data). however, some studies failed to show a decrease in leakage of serum proteins into the alveolus, which can probably be explained by the different treatment strategies [6, 35, 48] . we hypothesize that surfactant administration to the donor may be more beneficial in inhibiting serum protein leakage than treatment at the time of reperfusion. however, once alveolar proteins accumulate in the alveolus, the lung can resist against surfactant inactivation by the interference of sp-a. cockshutt et al. showed in vitro a reversed inhibition of serum proteins on the surface lowering function of surfactant when sp-a was administered [41] . with surfactant treatment the surface tension in the lung remains low, thereby maintaining the ventilation and perfusion of the lung, resulting in optimal oxygenation [6] . as mentioned earlier, an increase in the small/large aggregate ratio occurs due to lung ischemia-reperfusion injury. since most of the exogenous surfactant administered to lung is in the large aggregate subform, a larger pool of surface-active phospholipids (dppc) is created, so that the rise in small/large aggregate ratio is prevented [6, 28] . thus, the instilled exogenous surfactant protects the endogenous surfactant pool against damage. this is illustrated by the fact that the normal endogenous surfactant pool is about 10±15 mg lipid per kilogram, and that the amount of surfactant used for treatment is in the range of 50±400 mg lipid per kilogram [6] . furthermore the preservation of sp-a, sp-b, and sp-c can result in normal phospholipid recycling and secretion [28] . as already mentioned, exogenous surfactant treatment preserves the endogenous surfactant, resulting in normal endogenous sp-a, sp-b, and sp-c. moreover, it was shown that sp-a enriched surfactant was able to improve lung function after prolonged ischemia, whereas this was not possible to the same extent with sp-a deficient surfactant within one hour after reperfusion [19] . also, a decrease of the large aggregate subform was found indicating an increased recycling capacity of the sp-a enriched surfactant compared with sp-a deficient surfactant [19] . surfactant has also been shown to inhibit cytokine release from activated monocytes and macrophages [49, 50] ; the modulation of lymphocytes has also been suggested. furthermore, surfactant is known to have antioxidant capacities, resulting in reduced ros damage at the level of the alveolus [51] . surfactant treatment can thus ameliorate the accumulation and adherence of inflammatory cells, so that endothelial and at ii injury is prevented, normalizing cell permeability and surfactant recycling. semik and colleagues showed that the decreased function of at ii cells after lung ischemia-reperfusion injury is prevented by surfactant treatment [42] . z clinical studies of surfactant treatment after lung transplantation some investigators have used surfactant to treat lung transplant recipients who developed severe lung ischemia-reperfusion injury after transplantation [52±54]. in a case report by strçber and colleagues in 1995, a 26-year old woman who underwent right lung transplantation and developed severe reperfusion injury 5 hours after transplantation, was treated with intrapulmonary nebulized synthetic surfactant [53] . shortly after surfactant therapy, lung compliance, pao 2 , and tidal volume increased. moreover, 24 hours after therapy, the edematous infiltrate of the transplanted lung on chest x-ray film was resolved. another study in six lung transplant patients also suggested improvement in lung ischemia-reperfusion injury due to surfactant replacement. however, in 1 of the 6 recipients, surfactant therapy failed to improve dynamic lung compliance, which could be attributed to the application approach or the type of surfactant used (synthetic versus natural surfactant) [52] . although the use of surfactant in human lung transplantation seems promising, no prospective randomized clinical trial has so far been set up to treat severe lung ischemia-reperfusion injury. also, a clinical trial should investigate possible additional effects of donor pretreatment as compared to treatment after reperfusion. in this chapter we have discussed the effects of lung ischemia-reperfusion injury on the surfactant system. lung ischemia-reperfusion injury damages the endogenous surfactant system by the production of ros, proteolytic enzymes, and (phospho)lipases. surfactant is composed of phospholipids and associated proteins and its main function is to reduce the surface tension inside the alveolus, allowing normal breathing. impairment of the surfactant system will increase surface tension (leading to instability and collapse of alveoli), atelectasis formation, influx of serum proteins into the alveolus, pulmonary edema, decreased lung compliance, and impaired gas exchange. the use of surfactant replacement therapy (either before or after lung ischemiareperfusion injury) ameliorates lung ischemia-reperfusion injury. surfactant therapy restores the activity of the endogenous surfactant pool and reduces the inhibitory effect of serum proteins; possible other effects are that it serves as an anti-oxidant and an anti-inflammatory agent. although human data on the use of surfactant in lung transplant patients are scarce, the positive results in experimental models and a few patient reports suggest that (pre)treatment with surfactant in lung transplantation patients could improve outcome. future studies should further investigate the effect of surfactant on lung ischemia-reperfusion injury markers on both short and long term. the registry of the international society for heart and lung transplantation: seventeenth official report-2000 alterations in pulmonary surfactant composition and activity after experimental lung transplantation the function of surfactant is impaired during the reimplantation response in rat lung transplants surfactant treatment before reperfusion improves the immediate function of lung transplants in rats mitigation of injury in canine lung grafts by exogenous surfactant therapy exogenous surfactant therapy in thirty-eight hour lung graft preservation for transplantation acute pulmonary edema after lung transplantation: the pulmonary reimplantation response reperfusion injury significantly impacts clinical outcome after pulmonary transplantation predicting icu length of stay following single lung transplantation ischemia-reperfusion-induced lung injury the role of pulmonary surfactant in the pathogenesis and therapy of ards. in: vincent jl (ed) update in intensive care and emergency medicine mechanisms of repair and remodeling following acute lung injury bronchiolitis obliterans after lung transplantation: a review ischemia-reperfusion injury after lung transplantation increases risk of late bronchiolitis obliterans syndrome both alloantigen-dependent and -independent factors influence chronic allograft rejection surfactant dysfunction in lung preservation pulmonary surfactant in bronchoalveolar lavage after canine lung transplantation: effect of l-carnitine application the effect of lung preservation on alveolar surfactant sp-a-enriched surfactant for treatment of rat lung transplants with sp-a deficiency after storage and reperfusion pulmonary surfactant activity is impaired in lung transplant recipients surfactant therapy for acute lung injury/ acute respiratory distress syndrome pulmonary surfactant in health and human lung diseases: state of the art lung protective ventilation in ards: role of mediators, peep and surfactant in vivo evaluation of the inhibitory capacity of human plasma on exogenous surfactant function ischaemia-reperfusion injury intratracheal surfactant administration preserves airway compliance during lung reperfusion effects of early surfactant treatment persisting for one week after lung transplantation in rats surfactant abnormalities after single lung transplantation in dogs: impact of bronchoscopic surfactant administration surfactant function in lung transplantation after 24 hours of ischemia: advantage of retrograde flush perfusion for preservation donor lung pretreatment with surfactant in experimental transplantation preserves graft hemodynamics and alveolar morphology decreased lung ischemia-reperfusion injury in rats after preoperative administration of cyclosporine and tacrolimus effects of fr167653 on pulmonary ischemia-reperfusion injury: administration timing warm or cold ischemia in animal models of lung ischemia-reperfusion injury: is there a difference? structural and functional changes of surfactant protein a induced by ozone bronchoscopic surfactant administration preserves gas exchange and pulmonary compliance after single lung transplantation in dogs combined exogenous surfactant and inhaled nitric oxide therapy for lung ischemia-reperfusion injury in minipigs effect of hypothermic pulmonary artery flushing on capillary filtration coefficient effect of flush-perfusion with eurocollins solution on pulmonary arterial function effects of lung preservation with euro-collins and university of wisconsin solutions on endotheliumdependent relaxations low-potassium dextran solution ameliorates reperfusion injury of the lung and protects surfactant function pulmonary surfactant-associated protein a enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro ultrastructural studies of acute rejection following single lung transplantation in the rat±histological and immunohistological findings exogenous surfactant improves survival and surfactant function in ischaemia-reperfusion injury in minipigs flush perfusion with low potassium dextran solution improves early graft function in clinical lung transplantation evaluation of surfactant treatment strategies after prolonged graft storage in lung transplantation exogenous surfactant treatment before and after sixteen hours of ischemia in experimental lung transplantation increase of c-reactive protein and decrease of surfactant protein a in surfactant after lung transplantation lung preservation: the importance of endothelial and alveolar type ii cell integrity synthetic surfactant (exosurf) inhibits endotoxin-stimulated cytokine secretion by human alveolar macrophages phagocytic functions and tumor necrosis factor secretion of human monocytes exposed to natural porcine surfactant (curosurf). pediatr res characterization of antioxidant activities of pulmonary surfactant mixtures surfactant replacement in reperfusion injury after clinical lung transplantation nebulized synthetic surfactant in reperfusion injury after single lung transplantation severe reperfusion lung injury after double lung transplantation acknowledgments. the authors thank laraine visser-isles (department of anesthesiology) for english-language editing. key: cord-017021-n6rpuvwd authors: marriott, deborah j.; orla morrissey, c. title: common infections following lung transplantation date: 2018-08-31 journal: essentials in lung transplantation doi: 10.1007/978-3-319-90933-2_15 sha: doc_id: 17021 cord_uid: n6rpuvwd the lungs are the only transplanted organ in direct contact with the ‘outside world’. infection is a significant cause of morbidity and mortality in lung transplantation. early accurate diagnosis and optimal management is essential to prevent short and long term complications. bacteria, including mycobacteria and nocardia, viruses and fungi are common pathogens. organisms may be present in the recipient prior to transplantation, transmitted with the donor lungs or acquired after transplantation. the degree of immunosuppression and the routine use of antimicrobial prophylaxis alters the pattern of post-transplant infections. infection accounts for around 35% of all deaths in the first year after transplantation with bacterial pathogens responsible for approximately half of all infections [1] . the risk of infection following lung transplantation is determined by a number of factors including: • physical factors such as denervation of the allograft resulting in a reduced cough reflex and anastomotic site stenosis with distal infection • the 'net state of immunosuppression'-the result of all factors including host immune system, anti-rejection immunosuppressive therapy and concomitant viral infections such as cytomegalovirus that contribute to a patient's risk of infection • epidemiological exposure to organisms, including donor-derived infections, community acquired infections, travel related infections and healthcare associated infections • the use of prophylactic antimicrobial agents in the post-transplant period bacteria are defined by their morphology or shape and size. most pathogenic bacterial species are spherical (cocci) or rod-shaped (bacilli) and may exist as single cells (for example many of the common bacilli such as pseudomonas and stenotrophomonas) or in a variety of characteristic patterns such as s. pneumoniae (pairs of lancet shaped cocci), s. aureus (large clusters of cocci forming 'bunches of grapes') and streptococci (long chains of cocci). whilst molecular diagnostic techniques such as polymerase chain reaction (pcr) are increasingly important the basis of much microbiological diagnosis remains the characteristic appearance of the organism on a glass microscope slide when stained with dyes under a variety of conditions. common stains include the gram stain, first described by hc gram in 1884 but still in everyday use, the ziehl-neelsen or acid-fast stain for mycobacteria and the modified ziehl-neelsen stain for nocardia. the gram stain divides bacteria into gram positive or gram negative depending on the ability of the cell wall to prevent decolourisation after staining with crystal violet. it is important to remember that bacteria such as s. aureus and pseudomonas species are not stained by the ziehl-neelsen stain and conversely mycobacteria cannot be seen on a gram stain. culture techniques also differ with mycobacteria often unable to grow on conventional agar plates, requiring special growth media and prolonged culture periods. therefore if mycobacterial infection is suspected the request form for the sample must specify 'mycobacterial culture' so the appropriate investigations are performed by the laboratory. the laboratory diagnosis of important bacteria in the setting of lung transplantation is summarised in s. aureus is a common colonizer of the upper respiratory tract and skin, and is isolated with increased frequency from the sputum of patients with cystic fibrosis although the frequency decreases with age [2] . s. aureus can be acquired from the donor, the recipients own bacterial flora or the hospital environment as a healthcare associated infection, and is responsible for a wide range of health care-associated infections such as ventilator-associated pneumonia, bactereamia, and surgical site infections. isolates of s. aureus are characterised according to their susceptibility to methicillin, an anti-staphylococcal penicillin. methicillin susceptible s. aureus (mssa) is more common in community acquired infections whereas methicillin resistant s. aureus (mrsa) occurs with greater frequency in hospital acquired infections. the largest study of s. aureus following lung transplantation was a retrospective single centre study conducted over a 5 year period [3] . s aureus infection developed in 109 of 596 lung transplant (18%) recipients within 90 days of transplantation. mssa (62%) was more common than mrsa (38%) but the proportion of mrsa infections increased over time. pneumonia (48%) was the most common infection, followed by tracheo-bronchitis (26%), bacteremia (12%), intrathoracic infections (7%), and skin/soft tissue infections (7%). infected patients required longer hospital and intensive care unit stays (p < 0.0001 for both) but the 30-and 90-day mortality rates were low (7% and 12%, respectively). however infected patients had higher rates of rejection (both acute and chronic) at 1 (p = 0.048) and 3 years (p = 0.002), and higher mortality at 1 (p = 0.058) and 3 years (p = 0.009). • dicloxacillin or flucloxacillin • cefazolin or cephalothin for penicillin allergic patients (note-there is 5-10% risk of anaphylaxis in patients with documented penicillin anaphylaxis) • clindamycin is often prescribed for deep infections because it exhibits good tissue penetration. however it is a bacteriostatic antibiotic and should only be administered to patients with s. aureus bacteraemia following specialist advice • vancomycin with appropriate therapeutic drug monitoring (tdm) • teicoplanin-tdm not available in most centres. standard dosing may be inadequate, especially for bacteraemia • linezolid-superior to vancomycin for mrsa pneumonia. toxicity may occur with long-term administration unless tdm is undertaken • some isolates may be susceptible to clindamycin, cotrimoxazole and doxycycline. however these agents should not be used to treat bacteraemia infection control mssa: no specific measures required. mrsa: patients are usually placed on contact precautions (gown or apron, glove and careful hand hygiene as per 5 moments for hand hygiene) and may be isolated in a single room or cohorted with other colonised patients to prevent spread to other non-identified colonised patients. haemophilus influenzae is an important respiratory pathogen. in patients with cystic fibrosis it often causes infection early in life but is replaced by other organisms such as pseudomonas spp. over time [4] . in contrast, patients undergoing lung transplantation for other indications may be colonized with h. influenzae at any stage of life. post-transplant infection with h. influenzae is relatively uncommon. this is at least in part because of the wide spread practice of the administration of azithromycin and trimethoprim/sulphamethoxazole as prophylactic agents in the post-operative period. both these antimicrobial agents have activity against h. influenzae thereby reducing the frequency of infection. • approximately 25% of h. influenzae isolates are susceptible to ampicillin • ampicillin resistant isolates are generally susceptible to augmentin, cefuroxime and third generation cephalosporins (cefotaxime, ceftriaxone) • cephalexin is ineffective no specific infection control measures required other than standard precautions and hand hygiene. like h. influenzae s. pneumoniae is an important respiratory pathogen which is uncommon in the setting of lung transplantation, again in part because of the impact of antimicrobial prophylaxis with trimethoprim/sulphamethoxazole and azithromycin. after lung transplantation a reduction in an important component of the immune system, serum immunoglobulins, is common occurring in up to 63% of lung transplant recipients [5] . it is likely that this increases the risk and frequency of severe pneumococcal infection. • s. pneumoniae is generally susceptible to penicillin • penicillin resistant s. pneumoniae pulmonary infection can usually be successfully treated with penicillin as the concentration achieved in the lung is sufficient to exceed the threshold for efficacy • alternative treatment options for penicillin resistant s. pneumoniae causing meningitis or blood-stream include third generation cephalosporins and vancomycin no specific infection control measures required other than standard precautions and hand hygiene. pseudomonas aeruginosa is a gram negative bacillus which commonly colonises the airways of patients with cystic fibrosis but is also found in other patients proceeding to lung transplantation, for example those with chronic obstructive pulmonary disease. in many centres pseudomonas is the most common cause of post-transplantation bacterial infection. prolonged pre-transplant therapy with a variety of antibiotics frequently results in highly resistant organisms colonizing the patient at the time of transplantation. laboratory reports may refer to 'mucoid pseudomonas' isolated from a specimen. mucoid pseudomonas develops under certain environmental conditions following infection with non-mucoid species. the thick polysaccharide capsule gives the organism a 'wet' appearance when growing on an agar plate in the laboratory but more importantly renders the organism more resistant to immunological defense mechanisms such as phagocytosis and to standard anti-pseudomonas therapy. • guided by laboratory susceptibility testing, especially in patients with extensive prior antibiotic exposure • susceptibility testing of mucoid strains is less reliable than standard strains • commonly used antibiotics include aminoglycosides (gentamicin, tobramycin, amikacin), antipseudomonal beta-lactams (piperacillin-tazobactam, ceftazidime, cefepime), ciprofloxacin and meropenem. colistin may occasionally be required for extremely resistant organisms contact precautions are generally reserved for patients with multi-drug resistant pseudomonas aeruginosa. stenotrophomonas maltophilia is a gram negative bacillus which is increasingly recognized as an important pathogen of the airways in the setting of lung transplantation. the organism is widespread in the environment, found in soil, water and animal and plant material. treatment is complicated by the multi-drug resistance. • trimethoprim-sulphamethoxazole is the treatment of choice although resistance is increasingly described • ciprofloxacin is active against approximately 50% of laboratory isolates infection control • no specific infection control requirements other than standard precautions and hand hygiene. burkholderia species are gram negative bacilli closely related to pseudomonas species (in fact they were previously called pseudomonas cepacia and you will sometimes see this referred to in older literature). in the setting of cystic fibrosis and lung transplantation, the clinically important species belong to the burkholderia cepacia complex (bcc), a group of 17 genetically closely related organisms. however it has been recently recognised that not all bcc are equally pathogenic. the most important organisms include b. cenocepacia (previously named bcc genomovar iii) and burkholderia multivorans (previously bcc genomovar 2) which account for up to 97% of all burkholderia cepacia complex isolates from patients with cystic fibrosis [6] . one of the most feared organisms is burkholderia cenocepacia which can be an aggressive pathogen that is transmissible between patients and can cause epidemics. recent studies have suggested that b. cenocepacia is associated with poor outcome and is a contraindication to transplantation in many centres. therefore accurate detection and identification of burkholderia species prior to transplantation is absolutely essential: a false positive result can lead to exclusion from the transplantation waiting list whereas a false negative result can lead to poor transplantation outcome and possible cross infection between patients if appropriate infection control measures are not put in place. there is no standard treatment that can eliminate bcc. eradication of bcc is extremely difficult as many species of bcc, particularly b. cenocepacia, are intrinsically resistant via a variety of resistance mechanisms to numerous antimicrobial agents including the aminoglycosides (gentamicin, tobramycin), most antipseudomonal beta-lactam antibiotics (piperacillin-tazobactam, cefepime, ceftazidime) and colistin. rapid development of resistance may occur during therapy [6] . in a study of a large number of bcc isolates, 2621 strains of burkholderia cepacia complex isolated from 1257 cystic fibrosis patients were tested. resistance to all available antimicrobial agents was demonstrated in 18% of isolates with the most active agents, minocycline, meropenem, and ceftazidime inhibiting 38%, 26%, and 23% of strains, respectively [7] . the use of combination antimicrobial therapy to overcome these issues has not usually been successful. bcc can be spread to susceptible patients by: • person to person contact • contact with contaminated surfaces or objects • exposure to bcc in the environment contact precautions and isolation (see mrsa) may be implemented in hospital. alternately, patients colonised with bcc should not be housed next to an immunosuppressed patient. mycobacteria are bacteria forming their own genus within the phylum actinobacteria. over 190 species have been identified but not all are pathogenic (that is have the potential to cause infection in humans). mycobacteria are slender, curved rods that, unlike most bacteria, are acid fast (see preceding section). in addition, they are resistant to alkalis and dehydration meaning they can survive for long periods in the environment. the cell wall contains complex waxes and glycolipids. they multiple very slowly on special media and some clinical isolates can take 4-6 weeks to grow. based on their growth rate, catalase and niacin production and pigmentation in light or dark conditions mycobacteria are classified as mycobacterium tuberculosis complex (m. tuberculosis, m. bovis, m. africanum, m. microtii) and non-tuberculous mycobacterium (ntm). molecular techniques (e.g. pcr) can now readily differentiate between them. m. tuberculosis is transmitted from person to person. the incidence in transplant recipients is much higher than in the general population [8] . the most common cause in the transplant population is reactivation of latent infection but other causes include unrecognised transmission in the donor lungs (that is donor-derived), especially in countries where tb is endemic, and primary infection after transplantation [9] . the median time to infection from lung transplantation is 3.5 months (earlier than in renal transplant recipients) but donor-derived infections usually occur earlier, often within the first month post-lung transplant [8] . risk factors include prior residence in an endemic country, history of untreated tb, a chest x-ray which shows evidence of old healed tb, augmented immunosup-pression for rejection, use of t-cell depleting agents for immunosuppression and recipient age. the lung is the most common site of infection but in up to 33% extra-pulmonary or disseminated tb can occur with unusual presentations (e.g. skin ulcers, abscesses, tenosynovitis) [9] . fever is a very common presenting complaint as are night sweats and weight loss [9] . instead of the classical cavity that is seen on chest x-ray in immunocompetent patients, in lung transplant recipients focal infiltrates, miliary pattern, nodules or pleural effusions are more common (fig. 15 .1) [9] . the diagnosis of active tb can be challenging in lung transplant recipients with sputum samples commonly stain and culture negative. bronchoalveolar lavage (bal) with the fluid sent for acid fast bacilli (afb) staining ( fig. 15 .2) and culture is ideal. pcr testing is useful to decrease the time to diagnosis given cultures are slow to grow. biopsy of skin lesions, abscesses, soft tissue lesions or other accessible extra-pulmonary sites for afb staining, culture, histology and/or pcr can assist in the diagnosis of extra-pulmonary tb. guidelines exist for the treatment of active tb; however, there are a few specific things to note in the lung transplant setting [10] [11] [12] . • a rifamycin-based regimen (rifampicin is the most common drug used in this group) is strongly preferred because of its sterilizing capacity and ability to prevent the emergence of resistance • rifamycins interact with immunosuppressant agents. dose adjustments will be required at initiation and cessation with close monitoring of levels of immunosuppressant drugs whilst receiving a rifamycin • some centres prefer rifabutin for use in the transplant setting as it has less impact on drug metabolism than rifampicin • for localised non-severe infection and no suspicion of isoniazid resistance a fluoroquinolone could be substituted for the rifamycin with the duration extended to 12-18 months depending on the number of drugs used. otherwise a rifamycin agent should be used in the regimen. • the minimum duration is 6 months but some experts prefer a minimum of 9 months in the transplant setting. longer treatment is required for severe or disseminated infection or for infection involving the central nervous system and/or bone and joint and in pulmonary disease with ongoing afb detectable in sputum (>2 months) • streptomycin should not be used in the lung transplant setting because of the associated high-risk of nephrotoxicity. • immunosuppressive agents used to prevent rejection may only require minimal or no dose reduction. this is because immune reconstitution inflammatory syndrome (iris) can occur even when the immunosuppressant agents are not dosereduced as the anti-tb treatment can reverse some of the immunosuppressive effects of tb. screening for latent tb (prior exposure to m. tuberculosis which can reactivate and cause clinical disease) needs to be performed pre-transplant in all lung transplant candidates. two tests are available, namely, the tuberculin skin test (tst) and the interferon-gamma release assay (igra). the igra is used in most centres. screening algorithms are available [10, 13] . as the risk of reactivation and severe infection is increased in transplant recipients and the annual risk of active tb with a positive tst is 7.4%, there is a good argument for latent tb treatment. the optimal timing for latent tb treatment is pre-transplant. latent tb should be treated if: • the initial or boosted tst produces induration of ≥5 mm or a positive igra; • prior history of untreated latent tb; or • receipt of an organ from a donor known to have untreated latent tb. isoniazid (with oral pyridoxine) is the treatment of choice and has a low risk of toxicity. rifampicin for 16 weeks or isoniazid in combination with rifapentine for 12 weeks are alternative regimens, but only pre-transplantation because of drug interactions. as already stated person-to-person transmission of tb can occur. the major route is by inhalation of airborne particles. there are a number of factors that increase the risk of transmission of airborne particles including presence of untreated active pulmonary or laryngeal tb, cavitary disease, smear positivity and short time to positive m. tuberculosis culture. a number of procedures can also increase the risk of dispersal of airborne particles including intubation and bronchoscopy. patients with extrapulmonary tb are not contagious; however, concomitant pulmonary or laryngeal tb needs to be excluded firstly. immunocompromised patients with extra-pulmonary tb should be presumed to have pulmonary tb until proven otherwise. there are numerous international and national tb control guidelines on which hospitals base infection control programs for tb [14] . if tb is suspected or untreated: • the patient must be managed in airborne isolation rooms with negative pressure ventilation • masks must be worn by health-care workers when in contact with the patient and by the patient when he/she leaves the room • when tb is excluded the patient can be removed from isolation • for patients with confirmed tb isolation can be discontinued when the patient is receiving treatment, demonstrates a clinical response and has three negative afb smears from sputum • close liaison with the institutional infection control team is essential in cases of suspected or untreated tb. common ntm affecting lung transplant recipients include m. avium complex, m. kansasii and m. abscessus. these are environmental organisms so infection usually occurs via acquisition from an environmental reservoir and not person to person transmission. healthcare-associated infection from contaminated medical devices can occur and person-to-person transmission has been described with m. abscessus [15, 16] . risk factors for infection include cystic fibrosis as an underlying disease, the isolation of a ntm (particularly m. abscessus) pre-transplant and the use of rabbit anti-thymocyte globulin. median time to onset is later when compared with tb (1 year). the lungs are most commonly affected but cutaneous, soft tissue and disseminated infection can be seen, especially with m. abscessus, m. chelonae and m. kansasii [17] . with disseminated disease constitutional symptoms (e.g. sweats, tiredness, weight loss) predominate [18] . the most common radiological features seen are fibrocavitary and cavitary, nodules, bronchiectasis, tree-in-bud, and large opacities (>2 cm) [19] . diagnosis is very challenging as these are environmental organisms and it is difficult to determine whether isolation of these organisms reflects contamination/ colonization or true infection. guidelines for diagnosis exist for ntm [20] . factors such as organism burden, specific species, clinical signs and symptoms and radiological features all need to be considered when determining infection category and whether or not to treat. treatment is similar to the immunocompetent population. a multi-drug regimen is used (see table 15 .2); however, similar to tb a few specific points need to be considered in the transplant setting. • susceptibility testing should be performed to direct initial and maintenance regimens. • clarithromycin can increase serum levels of calcineurin inhibitors and rapamycin agents via the cytochrome (cyp) 3a4 pathway so with the initiation and cessation of clarithromycin the immunosuppressant agents may need dose adjustment. close monitoring of immunosuppressant concentrations is required. • the issues outlined above for rifamycin use in tb treatment also apply to the treatment of ntm. • the duration of treatment is longer than for the immunocompetent population. the minimum is usually 12 months after last positive culture; but lifelong suppressive therapy may be needed in some patients. • reduction of immunosuppression needs to be considered. • surgical resection may be required if: -large abscesses are present -there is a large burden of disease -focal disease not responding to therapy -the patient cannot tolerate therapy. m. abscessus is a particular problem in the lung transplant setting. it is increasing in incidence and can cause disseminated infection post-lung transplant which can be very difficult to eradicate. it is also resistant to many of the available antimicrobial agents and drug-related toxicity has been detected in up to 44% post-lung transplantation [21, 22] . treatment is complicated and prolonged. in some centres isolation of m. abscessus in a lung transplant candidate is considered as a strong relative contra-indication to transplantation [23] . other centres have determined that transplantation of patients with pre-transplant isolation of m. abscessus is possible with the precautions outlined in table 15 .3 [24, 25] . currently, expert opinion indicates that transplantation in those with pre-transplant isolation of m. abscessus should be decided on a case-by-case basis. as ntm are ubiquitous in the environment, transmission is usually from an environmental source. in addition, ntm are resistant to chlorine and have the ability to form bio-films. as a result infection control measures are directed at ensuring adequate disinfection of hospital equipment, rigorous and repeated surface cleaning and high-quality water supply. ongoing environmental surveillance in the hospital setting and close liaison with institutional infection control and engineering teams is critical to prevent outbreaks of ntm, particularly in the setting of construction. there are some evidence in the literature that m. abscessus has been associated with person-to-person transmission but other studies have indicated that this may not be the case [26, 27] . careful assessment of each institution's epidemiology will assist in deciding if patients with m. abscessus require airborne isolation or simply rigorous cleaning of the environment [28] . recently m. chimera contamination of heater-cooler units used in cardiac surgery has been reported resulting in cases of surgical-site and disseminated infection worldwide. new enhanced decontamination strategies have been developed and ongoing surveillance is required to ensure that these remain effective [29] . nocardia are ubiquitous, saprophytic, gram-positive bacteria that belong to the aerobic actinomycetes group. they are partially acid-fast rods that grow slowly in branching chains resembling fungal hyphae. there are more than 80 species but most infections in humans are caused by nocardia asteroides sensu stricto, n. farcinica, n. nova, and n. brasiliensis. infections with nocardia are increasing in lung transplant recipients [30] . whilst widespread throughout the world infections with nocardia have the highest frequency in dry windy climates which facilitate aerosolisation and dispersal. infections mostly occur in the first year after lung transplantation but are rare within the first month unless it is donor-derived infection. risk factors include corticosteroids (particularly in the preceding 6 months), and augmented immunosuppression (high median calcineurin inhibitor levels in the preceding 30 days) [30] . rituximab use and hypogammaglobulinaemia have also been associated with an increased risk of developing nocardia infection as has the use of alemtuzumab for treatment of allograft rejection [31] [32] [33] . inhalation is the most common route of infection therefore the lungs are most commonly affected. dissemination to other organs, particularly the skin and central nervous system (cns) has been reported in 50% of cases. the skin can also be infected by direct inoculation, especially if the lung transplant recipient is involved in outdoor activities. the most common signs and symptoms are fever, weight loss, cough, pleuritic chest pain and dyspnoea. chest imaging frequently shows irregular nodular lesions which may be cavitary (fig. 15. 3) [34] . other features include diffuse infiltrates or consolidation with associated pleural effusions. diagnosis is by microscopy, culture and histological examination of respiratory specimens (most particularly bronchoalveolar lavage fluid [bal]) or biopsy tissue (e.g. skin or brain tissue). nocardia grows on non-selective media forming characteristic white and chalky colonies. if there is a suspicion that the infection may be nocardia inform your diagnostic laboratory as the specimens require longer incubation for the growth of nocardia and in samples with mixed growth (that is multiple organisms [particularly sputum]) nocardia may be obscured. selective media can be used to improve the yield of nocardia growth (e.g. thayer-martin). nocardia has characteristic features on gram stain (see fig. 15 .4). in tissue nocardia appears as gram positive branching and beaded rods with surrounding pyogenic inflammatory reaction. it is important to determine the species and susceptibility profiles as different species have different susceptibility profiles. this information is very useful in determining the treatment regimen. antibiotics are the mainstay of treatment. the site(s) and burden of infection, the species and the potential drug-drug interactions all determine the antimicrobial regimen to be used for treatment [35] . • mild pulmonary infection-trimethoprim-sulfamethoxazole (tmp-smx) for 6-12 weeks • severe pulmonary infection (no cns involvement)-parenteral treatment with tmp-smx plus amikacin • cns infection-parenteral treatment with tmp-smx plus imipenem • multi-organ infection including the cns-intravenous (iv) amikacin added to the regimen of iv tmp-smx and imipenem. • meropenem may be used instead of imipenem as the former is less likely to precipitate seizure activity. sensitivity to meropenem must be demonstrated in the laboratory before use [36] . • linezolid has excellent in vitro activity against nocardia and has been used with success in treatment; therefore linezolid may be used as part of a multi-drug regimen [37, 38] . • if the patient has a tmp-smx allergy desensitisation should be performed if possible. parenteral treatment is continued for 3-6 week followed by oral therapy for 6-9 months. oral agents that are commonly used include tmp-smx, minocycline and/or amoxicillin-clavulanate. surgery may be required in cases of cerebral nocardiosis or large soft tissue abscesses not responding to treatment, empyema or mediastinal fluid collections and for pulmonary nocardiosis that is complicated by pericarditis. consideration should be given to reducing immunosuppression especially in cases with severe disease or those progressing on anti-microbial treatment. indefinite secondary prophylaxis is also recommended as the immunosuppression cannot be fully reversed. there are no reports of person-to-person transmission of nocardia in the literature. as nocardia are ubiquitous environmental organisms, acquisition is mostly from an environmental source. similar to ntm infection control measures in the hospital setting for nocardia are directed at disinfection of equipment and surfaces and ensuring high-quality water supply. ongoing surveillance is required to prevent outbreaks, particularly in the setting of construction. fungal infections are a significant problem in lung transplant recipients occurring in 8.6% and causing death in up 39.5% of those infected [39, 40] . the majority of infections are caused by aspergillus and candida species. cryptococcus is the third most common cause of fungal infection. the fungi that cause mucormycosis (e.g. rhizopus species), scedosporium, and fusarium are emerging and are associated with very high mortality rates (60.5%); thus, increasing emphasis is placed on early recognition, diagnosis and treatment [40] . histoplasma, coccidioides and blastomyces species are important for those who live in or have previously resided in or visited endemic areas. pneumocystis jirovecii, whilst infrequent, can cause significant morbidity and mortality. the risk factors for infection are very similar to those described above for bacterial infection. in addition, fungal infections have been implicated in triggering the development of chronic rejection (that is, chronic lung allograft dysfunction [clad]) [41] . fungi are a major problem in lung transplant recipients. the importance of thinking about fungi in any lung transplant recipient suspected of having infection cannot be over-estimated. early diagnosis and treatment is critical to optimising outcomes. prophylaxis may reduce the impact of fungal infections in lung transplant recipients but issues such as drug intolerance and drug-drug interactions and the emergence of resistance may complicate treatment and reduce overall efficacy. fungi can be a single cell or complex multicellular organisms. fungi are mainly found in soil or on dead plant matter. they can be divided up into yeasts, multicellular filamentous moulds and dimorphic fungi. yeasts are small, lemon-shaped single cells that are around the size of red blood cells. they multiply by budding a daughter cell off from the original parent cell. multicellular filamentous moulds are made up of very fine threads known as hyphae. they grow from the hyphal tips and divide repeatedly along their length creating long and branching chains. some of the hyphal branches grow into the air and spores form on these aerial branches. these spores can be carried by the wind, rain or insects to new habitats where they can germinate to start growing and producing new hyphae. the process of infection is mimicked in immunosuppressed individuals where the conidia (spores) are inhaled and with impaired immune defence mechanisms the conidia (spores) can germinate and uncontrolled hyphal growth can occur. dimorphic fungi are fungi that can exist as yeast or mould. a prime example of a dimorphic fungus is penicillium marneffei, a human pathogen that exists as a mould at room temperature but as yeast at human body temperature. aspergillus fumigatus is the most common of all aspergillus species [42] . other species that can cause infection in the lung transplant setting include a. flavus, a. terreus, a. niger and a. nidulans [42] . the importance of identifying a. terreus is that it has a different susceptibility profile to the other aspergillus species. it is resistant to amphotericin b [43] . aspergillus species commonly cause 4 types of infection in lung transplant recipients: • aspergillus colonisation • tracheobronchial aspergillosis • invasive pulmonary aspergillosis (ipa) (also known as aspergillus pneumonia) • disseminated invasive aspergillosis (ia). aspergillus colonization is defined as the detection of aspergillus in respiratory secretions by culture, pcr or by the detection of aspergillus galactomannan (a cell wall protein) in the absence of any symptoms, lesions in the airways seen on bronchoscopy or new changes seen on chest x-ray or computed tomography (ct) scan [44, 45] . aspergillus colonization has been detected pre-transplant in 8-59% of patients (most commonly in cystic fibrosis [cf] patients) and is a risk factor for post-transplant ipa and clad [3, 7] . post-transplant colonization is found in 30-40% [45] . some centres give antifungal agents to all lung transplant recipients (immediately post-transplant for 4-6 months) to minimize aspergillus colonization and its complications [45, 46] . with the use of universal prophylaxis the time to aspergillus colonisation has lengthened from 3.2 months to 6.8 months post-lung transplant [47, 48] . other centres only give antifungal treatment (for 3 months) once aspergillus is detected [45, 46] . this is known as the pre-emptive strategy. it is not known which strategy is best. tracheobronchial aspergillosis is defined as the detection of aspergillus in respiratory secretions by culture, pcr or the detection of aspergillus galactomannan with new lesions demonstrated on bronchoscopy including patches of redness (erythema), ulceration, necrosis or pseudomembranes but with no changes detected on chest x-ray or ct scan [44, 45] . the patient may be asymptomatic or may present with symptoms such as fever, cough, wheeze and/or hemoptysis [49] . it occurs in the majority of patients in the first 3 months post-lung transplant [47] . the importance of tracheobronchial aspergillosis is that the lung transplant recipient is at risk of progressing to ipa or disseminated ia [47] . • the treatment of choice is voriconazole. alternative agents include amphotericin b, posaconazole and itraconazole • combine with nebulized amphotericin b for a direct local effect [50] . • repeated bronchoscopic debridement particularly in those with large amounts of necrotic debris [51] • stenting occasionally required to maintain a patent airway the duration of treatment is dependent on the severity of the initial infection, degree of immunosuppression and response to therapy but should be given until the lesions have completely healed and potentially life-long in those with bronchial anastomotic involvement. proven ipa is defined as evidence of parenchymal (lung tissue) invasion by aspergillus hyphae or positive culture from sterile lung tissue alone or with signs/ symptoms such as fever, abnormal white cell count, new onset purulent sputum or change in the character or quantity of sputum or respiratory secretions, new onset or worsening cough, dyspnoea, tachypnoea, pleural rub, crackles or bronchial breath sounds. probable ipa is defined as signs/symptoms (as above) and new or progressive and persistent infiltrate, consolidation, cavitation or nodules and detection of aspergillus in respiratory secretions by culture, pcr or the detection of aspergillus galactomannan (single positive for bronchoalveolar lavage [bal] or 2 positives for sputum) (fig. 15 .5) [44, 45] . average time to development is 6 months [52] . in disseminated ia, respiratory disease can be associated with infection in the sinuses, orbits and central nervous system (cns). other sites where aspergillus can rarely cause infection include skin, bones, eyes (endophthalmitis), in the intra-abdominal cavity or retroperitoneum (e.g. abscess) and in the pericardium [42, 47] . • voriconazole is the treatment of choice [53] • an echinocandin (anidulafungin, caspofungin, micafungin) can be added for synergy in those with extensive disease or who are very unwell (e.g. hypoxic at presentation) [53] • treatment of disseminated disease is the same as for ipa and as for ipa treatment continues until complete resolution it is important to remember that when giving voriconazole (or other azole antifungal agents) in lung transplant recipients there are significant interactions with the immunosuppressant (e.g. tacrolimus, cyclosporine and sirolimus). dose adjustments of the immunosuppressants are required at initiation and cessation of voriconazole (or other azole) and regular monitoring of serum immunosuppressant levels is required. no specific infection control measures required. the most common infection type seen with candida species is candidaemia (infection in the bloodstream; fig. 15.6 ). this is most common during the first month post-transplant and is usually related to the recent surgery, intensive care unit stay and broad-spectrum antibiotic use peri-transplant. tissue infections can also occur and include infected pleural effusion, pleural space infection, infection of the incision sites and bronchial anastomotic site infections [50, 54] . candida species are frequently isolated from the mouth, pharynx, sputum and bal specimens but almost never spread to invade the lung tissue. universal prophylaxis targeting candida species during the first month post-transplant have been shown to be effective [55] . however, universal prophylaxis may be associated with the emergence of resistant candida strains [56] . candidaemia can manifest as fever or as severe sepsis (e.g. hypotension, tachycardia, requirement for inotrope support). invasive candidiasis is related to the site of the infection. for example, if disseminated to the skin invasive candidiasis cause skin pustules or to the eye results in endophthalmitis. blood cultures are still the gold-standard for the diagnosis of candidaemia; therefore a blood culture is required for all patients in whom candidaemia is suspected. in patients with invasive candidiasis a biopsy of the relevant tissues for staining, culture and histological examination is useful. some centres have access to beta-d-glucan testing. this non-culture based assay detects a cell wall protein of candida species and is a useful as an additional test (in addition to blood cultures and biopsy) in some patients, particularly those with intra-abdominal candidiasis. • echinocandin or liposomal amphotericin b for the treatment of candidaemia and serious candida infection [45] . • once the candida is detected and the sensitivity profile is known antifungal therapy can be altered [45] . if the isolate is sensitive to fluconazole then a change to this agent is recommended [45] • if candida is causing symptomatic infection of the urinary tract an echinocandin is not recommended as it has poor penetration into the urinary tract [45] . in this setting, fluconazole (if the isolate is sensitive) or amphotericin b and 5-flucytosine in combination (if the isolate is fluconazole-resistant) is recommended [45] . no specific infection control requirements. cryptococcus causes infection in 2% of lung transplant recipients. the most common site of cryptococcal infection is the lung (fig. 15 .7) but disseminated infection can also occur with a predilection to the central nervous system. skin involvement including cellulitis [57] and infection transmitted in the donor lungs has also been described. the median time to infection onset is 190 days. in addition to the usual diagnostic tests of culture and biopsy cryptococcal antigen assay is very useful as it is sensitive and specific and can be used to monitor disease treatment response. pre-transplant cryptococcosis has been described and is not a contra-indication to transplantation so long as disease control has been achieved with no positive cultures and cryptococcal antigen level is declining. fluconazole is continued throughout the transplant procedure and for a minimum of 6 months post-transplantation. immune reconstitution inflammatory syndrome (iris) is common with treatment of cryptococcal infection (5-14%) [58] and manifests as an apparent flare of the antifungal agents used depend on the site and burden of infection, indicating that diagnosis/exclusion of cns disease by ct scan or mri scan of head, a lumbar puncture for culture and cryptococcal antigen testing and ct of chest to determine extent of disease is critically important. • cns infection, disseminated infections or severe lung disease-liposomal amphotericin b and 5-flucytosine for a minimum of 2 weeks followed by fluconazole at high dose for 8 weeks and fluconazole at lower doses from 6 months to 1 year is recommended. • small volume pulmonary disease-fluconazole alone for 6-12 months is recommended [59] . no specific infection control requirements. the most common manifestation is pulmonary infection or infection of the cns and sinuses but gastrointestinal infection (likely through ingestion) has also been described [60] . the cumulative incidence is 0.07% and it accounts for 2% of all fungal infections. risk factors for infection include renal failure, diabetes and prior voriconazole and/or caspofungin use [61] . mucormycosis is particularly associated with tissue infarction and necrosis due to invasion of the tissue blood vessels with the growing hyphae ( fig. 15.8) . the fungi also spread rapidly along tissue planes. both these factors contribute to the high mortality rates of up to 87% seen with this infection. in view of the aggressive nature of the fungus and high mortality rate treatment requires a multi-pronged approach. • anti-fungal therapy -first-line therapy is liposomal amphotericin b. -caspofungin can be added if the infection is severe. -posaconazole or isavuconazole can be given as maintenance therapy or if the patient in intolerant of liposomal amphotericin b. • surgical debridement of all the necrotic tissue ( fig. 15.8 ), • reduction of immunosuppression • reversal of underlying factors (e.g. diabetes mellitus) no specific infection control measures required. scedosporium is an environmental organism that is recognised worldwide but has a higher incidence in specific geographical areas such as spain, the middle east and australia. it is a common fungus in floods, tsunamis and tornados resulting in a risk for transmission if the donor drowned [62] . it is commonly isolated from cf patients pre-transplant. risk factors for invasive scedosporium disease post-transplant include pre-transplant colonisation, prior receipt of amphotericin b and augmented immunosuppression. scedosporium is prone to disseminate and can be detected in blood cultures unlike other moulds such as aspergillus. in lung transplant recipients scedosporium mainly causes colonisation with invasive infection occurring in about 25%. the most common clinical manifestations of invasive disease include pneumonia, mediastinitis, fungaemia or disseminated disease [63] . progression to invasive disease is more likely in those with pre-transplant isolation; thus, if scedosporium is isolated prior to transplantation it should be treated [63] . • scedosporium is innately resistant to many of the available antifungal agents including amphotericin b. • s. apiospermum is sensitive to some of the azole antifungal agents, particularly voriconazole • a combination of voriconazole and terbinafine may be the only option against s. prolificans (now known as lomentospora prolificans) [63] . no specific infection control measures required. fusarium accounts for <1% of all invasive fungal disease in solid organ transplant patients with lung transplant recipients most commonly affected. like scedosporium whilst fusarium occurs worldwide it has a higher incidence in some countries such as in brazil, where the incidence of fusarium is second only to aspergillus. infection usually occurs within a year of transplantation and most commonly affects the lungs. outcome is poor with a 67% mortality rate. voriconazole is the most effective agent. no specific infection control measures required. histoplasma is endemic to the states bordering the ohio river valley and the lower mississippi river, usa but it has also been detected in montana and idaho. other countries and regions where it has been isolated include canada, mexico, central and south america, parts of eastern and southern europe, africa, eastern asia and australia. pulmonary and disseminated infections are the most common manifestations post-transplant. infection can range from asymptomatic to severe. the diagnosis is made by using a combination of serology (antigen and antibody), culture of respiratory secretions and biopsy with histological examination of the affected tissue [64] . routine screening pre-transplant is not recommended. serial monitoring or the administration of prophylaxis is recommended in those who had active infection prior to transplantation [65] . • mild disease-itraconazole • more severe infection-amphotericin b [66] infection control no specific infection control measures required. coccidioides are fungi that endemic to the southwest of the united states particularly the san joaquin valley, and the sonoran desert of southern california, arizona and northern mexico. in the lung transplant recipient these fungi can cause severe pneumonia or disseminated infection. disseminated infection is most commonly characterized by skin, bone and joint lesions and/or meningeal involvement. diagnosis is established by serological testing, culture or histopathology. pre-transplant assessment is required and includes a detailed past history [65] . any history of residence or travel to an endemic area requires evaluation with serological testing and chest x-ray [65] . any transplant candidate with past infection requires assessment by a specialist infectious diseases physician for clearance for transplantation [65] . in the case of active infection transplantation is deferred until the infection is quiescent (on radiology, clinically and serologically) [65] . • focal pneumonia can be treated with fluconazole • diffuse disease is treated initially with amphotericin b until clinical response followed by fluconazole or itraconazole • coccidioidal meningitis is treated with fluconazole no specific infection control measures required. blastomyces is endemic to parts of eastern north america, particularly northern ontario, south-eastern manitoba, quebec, south of the st. lawrence river, parts of the appalachian mountains and the interconnected eastern mountain chains, the west bank of lake michigan, the state of wisconsin and the entire mississippi river including the valleys of the major tributaries (e.g. ohio river). it also occurs in africa, the arabian peninsula and the indian subcontinent. similar to histoplasma and coccidioides it causes pneumonia and skin involvement (with verrucous or wartlike lesions) and is also common in the transplant recipient. diagnosis is made by culture of sputum, bal or tissue or by histopathological examination of biopsy tissue. pre-transplant assessment includes symptom assessment and chest radiography for those who live in endemic areas [65] . prophylaxis is given on a case by case basis. • liposomal amphotericin b until clinical improvement followed by oral itraconazole no specific infection control measures required. pneumocystis jirovecii was previously classified as a protozoan but with modern molecular techniques it has recently been reclassified as a fungus [67] . it was previously named p. carinii (which infects rats) but has been renamed p. jirovecii as this is the species that infects humans [68, 69] . if prophylaxis is not universally administered 5-15% of all solid organ transplant recipients develop p. jirovecii pneumonia (pjp) with the highest incidence occurring in the lung and heart-lung transplant recipients [70] . most centres administer pjp prophylaxis and as a result very few cases are now seen. the most important risk factor for pjp is corticosteroid use in combination with other immunosuppressive agents [71] . there are no good data as to a dose and duration of corticosteroids to decide when to give prophylaxis. the period of highest risk is the first 6 months post lung transplantation but most centres recommend indefinite prophylaxis [72] . previously, patients presented in respiratory failure with fever and a dry cough but as the awareness of the significance of the infection has increased and as more sensitive diagnostic tests have been developed diagnosis is made earlier when the disease in mild or indolent (that is less severe cough and dyspnoea). chest x-ray or ct scan of thorax usually demonstrates diffuse bilateral infiltrates. it is important to make a microbiological diagnosis so obtaining a respiratory specimen (induced sputum or ideally a bal) is best. a lung biopsy is rarely required. the best test is pcr although it is very sensitive so false positive results can occur. serum beta-d-glucan testing may be a useful adjunct if available [73] . like the treatment of pjp the first-line agent for prophylaxis is tmp-smp but at lower doses (1 double-strength tablet 3 times a week or a single-strength tablet daily). alternatives include dapsone, atovaquone or aerosolized pentamidine. several clusters or outbreaks of pjp have been reported, particularly in renal transplant patients. in some of these clusters or outbreaks person-to-person transmission was postulated as the cause [74] . consequently hospitalised patients with pjp should not be placed in the same room as other immunocompromised patients. otherwise standard precautions apply [75] . this review clearly illustrates that fungi are a major problem in lung transplant recipients. the importance of thinking about fungi in any lung transplant recipient suspected of having infection cannot be under-estimated. early diagnosis and treatment is critical to optimising outcomes. prophylaxis may reduce the impact of fungal infections in lung transplant recipients but issues such as drug intolerance and drug-drug interactions and the emergence of resistance may complicate and reduce its overall efficacy. further multicentre research is required to determine the optimal prophylactic strategies for lung transplant recipients. viruses are organisms that are much smaller than bacteria and are unable to be detected on routine microscopy. they are only able to survive and replicate within a living cell, using the chemical machinery of that cell to reproduce. viruses contain either deoxyribonucleic acid (dna) or ribonucleic acid (rna). important dna viruses in the setting of transplantation include the herpesvirus family whilst the rna viruses include most significant respiratory pathogens. viral infection, either primary or following reactivation of latent virus, remains an important cause of morbidity and mortality following lung transplantation. viral culture is extremely laborious and difficult and is restricted to specialist laboratories. increasingly the diagnosis of viral infection is made by pcr of peripheral blood or affected tissue with pcr available for all the members of the herpesvirus family listed below. the members of the herpesvirus family are: hsv-1 and hsv-2 cause oral and genital ulceration but occasionally cause disseminated infection, particularly in the immunocompromised host. as a rule, hsv-1 causes 80% of oral infection and 20% of genital ulceration whereas hsv-2 is responsible for 20% of oral infection and 80% of genital infection. infection may be primary, which can be severe, or reactivation from the site of latency in the neurons. the incidence of prior infection increases with age and varies according to socio-economic status, race and country of residence, with 50-96% of the general population having antibodies to hsv-1 and therefore at risk of reactivation [76, 77] . the most common manifestation of hsv-1 and hsv-2 in lung transplant recipients are mucocutaneous ulcers involving either the oral cavity or genital tract. less commonly, pneumonia, hepatitis or encephalitis may result from viral reactivation. the introduction of acyclovir in the 1980s marked the first highly effective antiviral therapy and resulted in a significant reduction in morbidity and mortality from post-transplant hsv infections. the incidence of hsv-1 and hsv-2 has fallen dramatically since ganciclovir, an anti-cmv agent with activity against hsv, has been widely used as prophylaxis against cmv infection in the transplant setting. • acyclovir/valaciclovir/famciclovir • suppressive therapy may be appropriate for frequent recurrences • the development of resistant virus is uncommon standard precautions apply to patients with active hsv lesions; however contact precautions may apply in healthcare settings if lesions are not covered and for 3 days post initiation of treatment or until crusting occurs. if hsv is disseminated contact precautions required until lesions are dried and crusted. immunocompromised staff should not care for patients. infected staff in high risk clinical areas require urgent review for leave/ redeployment. vzv primary infection results in chicken pox. the virus then lays dormant in neural tissue prior to reactivating as shingles, in particular during periods of immunosuppression. shingles may follow a single nerve pathway or dermatome, may involve multiple dermatomes or the virus may disseminate involving a variety of organs including the liver, lungs, brain and spinal cord. approximately 90% of adults in australia and the united states have antibody against vzv indicating prior infection. however, the incidence of antibody positivity varies between geographic areas with the incidence lower in tropical regions. patients who do not have antibodies to vzv should be considered for vaccination prior to transplantation. as the vaccine is a live vaccine it should not be administered after transplantation as there is insufficient safety data in immunosuppressed transplant recipients [78] . • high dose acyclovir/valaciclovir/famciclovir • ganciclovir • potential role for zoster immune globulin contact precautions for patients with active vzv lesions and for 3 days postinitiation of treatment or until crusting occurs. ebv is the causative agent of infectious mononucleosis (glandular fever), a common infection in the general population. it is also associated with the development of two cancers, nasopharyngeal carcinoma and burkitt's lymphoma. like other herpesviruses, ebv is associated with latent infection; in the case of ebv, b lymphocytes in the blood and lymphoid tissue which sets the scene for lymphoproliferative disorders. in the setting of transplantation, ebv has a clearly established role in the pathogenesis of post transplantation lymphoproliferative disorder (ptld) with up to 90% of cases associated with ebv latent infection. ptld is a spectrum of disease caused by the abnormal proliferation of lymphoid cells, with clinical manifestations varying from asymptomatic to tissue infiltration and/or focal masses in a variety of organs. figure 15 .9 is a pet-ct scan from a patient with ptld and demonstrates the widespread involvement that can occur. diagnosis is made by excisional biopsy and histological examination. high levels of ebv dna measured by pcr in peripheral blood provide supportive evidence. • reduce the level of immunosuppression • no good data to support a role for antiviral therapy (acyclovir, ganciclovir) • immunomodulatory agents such as anti cd20 (rituximab) • resection of localised lesions no specific precautions are required. cmv infection is defined as the detection of cmv replication (usually by pcr to detect cmv dna or rna in plasma or whole blood) regardless of the clinical presentation or symptoms. as with other herpesviruses, cmv infection may be the effects of cmv infection may be due to either direct tissue damage to a variety of organs (e.g. colitis) or indirect effects on the graft and the immune system (e.g. induce clad). figure 15 .10 demonstrates the 'owl's eye' appearance of cmv inclusion bodies in the bowel of a patient with cmv enteritis. there are two approaches to the prevention of cmv disease. • prophylaxis strategy-prescribing anti-cmv drugs for a defined period after transplantation (usually 6-12 months). • pre-emptive therapy-treatment with anti-cmv drugs only when the plasma or blood cmv pcr becomes positive during regular monitoring the choice of strategy varies between transplant centres and will in part be determined by the ability to rapidly and regularly perform cmv pcr on blood or plasma. in the setting of lung transplantation the prophylaxis strategy is the most frequent approach. in addition, high risk d+/r− patients are more likely to receive prolonged cmv prophylaxis. despite the various approaches to prevent cmv disease, active infection occurs in up to 30% of transplant recipients [79] . treatment options include: hhv-6 is very common in the community with approximately 95% of the general population demonstrating serological evidence of prior infection [80] . as with other herpesviruses it remains latent after primary infection and frequently reactivates after transplantation. however the significance of reactivation is uncertain as it is not reliably associated with any specific clinical syndrome. infection is most commonly asymptomatic but encephalitis, hepatitis, gastro-duodenitis and pancytopenia have been described. cmv prophylaxis does not appear to prevent hhv-6 reactivation [81] . there is limited clinical treatment data available but ganciclovir, valganciclovir and foscarnet appear to have activity against hhv-6 in laboratory testing. there may be a role for reduction of immunosuppression. no specific infection control measures required. like hhv-6, hhv-7 infection is very common in the community and reactivation can occur following transplantation. however the clinical importance of this is uncertain with no syndromes regularly associated with this virus. for this reason most laboratories do not perform pcr for hhv-7. there is minimal anecdotal data and no controlled trials for the treatment of hhv-7 although anti-cmv drugs such as ganciclovir, foscarnet and cidofovir may be effective. there are no specific infection control procedures required. along with ebv, hhv-8 is an oncogenic or cancer-causing herpesvirus. clinical manifestations include kaposi's sarcoma (ks), body cavity lymphoma and castleman's disease, a rare lymphoproliferative disorder. the prevalence of hhv-8 varies greatly, from 0 to 5% in north america and northern europe to up to 70% in regions of sub-saharan africa and the southern mediterranean where the virus is endemic [82] . previously recognized as an uncommon malignancy of elderly mediterranean men, african children, and ashkenazi jews, ks became the most common neoplasm of patients with hiv infection with an incidence >20,000 times that of the general population [83] . seropositive transplant recipients have a small risk of reactivation of latent virus and donor-derived infection has been infrequently reported. ks is the most common manifestation and body cavity lymphoma and castleman's disease are rare presentations of hhv-8. diagnosis of hhv-8 reactivation is generally by pcr whilst ks, body cavity lymphoma and castleman's disease require histological diagnosis. treatment antiviral drugs do not appear to be clinically effective. the mainstay of treatment includes: • reduction of immunosuppression or reversal of underlying immune deficiency • chemotherapy • rituximab for castleman's disease no specific infection control procedures required. respiratory viruses circulate within the community with seasonal and geographic variability. serious complications are uncommon in the non-immunocompromised host but in the setting of lung transplantation respiratory virus infections are associated with secondary bacterial infections, acute rejection and chronic graft dysfunction. increased susceptibility to respiratory viruses in lung transplant recipients is multifactorial and includes immunosuppression, impaired cough reflex, poor mucociliary clearance, altered lymphatic drainage and the direct exposure of the lung allograft to the environment. a prospective study compared 50 lung transplant recipients with respiratory virus infection with 50 uninfected recipients and demonstrated that those with a respiratory virus infection had a greater risk of acute rejection, bronchiolitis obliterans syndrome and death [84] . important respiratory viruses include: respiratory viral infections are common in lung transplantation. a recent study of 112 lung transplant recipients over a 2 year period found an infection rate of 19.3% with 61% having one or more viral infections over the study period [85] . asymptomatic carriage was uncommon (<10%) and was mainly associated with coronavirus/rhinovirus. the hospitalisation rate was 50% for influenza and parainfluenza and 16.9% for other viruses. infection control precautions for respiratory viruses include droplet precautions (single room, mask, gown and gloves for room entry) until asymptomatic and hand hygiene as per 5 moments. staff should not come to work if they have a respiratory illness and unwell visitors should not be allowed patient contact. chemoprophylaxis may be administered to patients following exposure where appropriate (see below for specific viruses). many microbiology laboratories perform a respiratory pathogen pcr diagnostic panel which includes the common respiratory viruses such as influenza a, influenza b, enterovirus, rhinovirus, coronavirus, hmpv, parainfluenza, adenovirus, rsv and non-viral organisms including bordetella pertussis, bordetella parapertussis, mycoplasma pneumoniae and pneumocystis jirovecii. testing is generally performed on nose and throat swabs (both required), a nasopharyngeal aspirate or bronchial washings. like many respiratory viruses, rsv is seasonal with a winter predominance. in healthy adults rsv is usually associated with mild, self-limited infection but in lung transplant recipients rsv can cause bronchiolitis, pneumonia and respiratory failure with a significant acute mortality up to 20% [86] and decline in lung function associated with the subsequent development and progression of bronchiolitis obliterans syndrome (bos) [87] . rsv has also been associated with acute rejection but a recent prospective study failed to confirm this finding [85] . ribavirin is a nucleoside analogue with broad range of activity against many rna viruses and, despite a lack of randomised trial data, is the cornerstone of treatment for rsv. ribavirin can be administered in 3 ways: • oral • intra-venous • aerosolised (negative pressure room and specific equipment required) advice regarding administration and dosing regimen should be sought as ribavirin has significant toxicity (primarily haematological), is teratogenic and has a very long half-life. standard and droplet precautions required. patients should be managed in a single room. influenza is a seasonal virus with the greatest incidence of infections during the winter months, although it is detectable year-round in the community. the two most frequent types are influenza a followed by influenza b. influenza viruses characteristically undergo 'antigenic drift' or minor annual changes in the surface glycoprotein that allows reinfection due to inadequate immunity. every 10 years or so 'antigenic shift' occurs secondary to the reassortment of genes between species. major outbreaks of influenza occur at this time as there is little immunity present in the community. the rate of influenza is higher after lung transplantation than other solid organ transplants [88] and may be community acquired, nosocomial or donor-derived infection. complications such as viral and bacterial pneumonia occur more frequently than in the general population. annual vaccination of transplant recipients, transplant candidates and their families is strongly recommended although the antibody response may be impaired in immunosuppressed patients [89] . treatment should be initiated in all transplant patients with suspected or proven influenza and ceased if an alternative diagnosis is made. therapeutic options include: • oseltamivir (oral, influenza a and b) • zanamivir (inhaled, influenza a and b) • amantadine (oral, influenza a only) • rimantadine (oral, influenza a only) chemoprophylaxis should be offered to patients known to be exposed to influenza virus either in the hospital or the community setting. droplet and standard precautions for duration of symptoms or until 3 days of active influenza treatment. parainfluenza viruses (piv) consist of a group of 4 serotypes, piv 1-4, which circulate year-round in the community and cause a variety of clinical presentations from the 'common cold' to bronchiolitis and pneumonia. piv 3 has been associated with large hospital outbreaks of infection due to person-to-person transmission, especially in haematology wards, with mortality rates up to 30% in outbreaks [90] . in lung transplant recipients piv infection can lead to loss of lung function and bronchiolitis obliterans syndrome. figure 15 .11 shows extensive interstitial pneumonia in a patient with severe piv infection. there are no randomised studies of antiviral therapy. however there are reports published primarily in the haematology setting suggesting ribavirin, either orally or intravenously administered, may be effective treatment. a small single centre study of rsv and piv in lung transplant recipients indicated that 33% of lung transplant patients with lower respiratory tract paramyxoviral infections who were treated with inhaled ribavirin died or did not return to baseline lung function [91] . droplet and standard precautions. hmpv was first described as recently as 2001 and has been increasingly recognised as a seasonal (predominantly late winter) respiratory pathogen causing both upper and lower respiratory tract infection. about 100% of school aged children have antibodies to this virus indicating the widespread nature of hmpv [92] . hmpv is closely related to rsv and in lung transplant recipients is thought to result in graft dysfunction. however there was mainly anecdotal data to support this until a recent review by dosanjh [93] who conducted a literature search to identify cases of both hmpv and allograft rejection within 6 months of the initial infection. 1007 lung transplantation recipients, with a total of 2883 samples, were identified. of these, 57 had hmpv without co-infection with other agents. the results of the study indicated that 35% of acute hmpv infections without co-infection were associated with acute cellular rejection within 3 months and 9.4% of the cases subsequently developed chronic allograft dysfunction/bronchiolitis obliterans syndrome suggesting that hmpv is an important pathogen in the lung transplant setting. ribavirin has been shown to have activity against hmpv in vitro [94] and in animal models of infection [95] . however no human studies in hmpv infection have been performed and the use of ribavirin remains controversial. case reports have supported ribavirin therapy with concomitant intravenous immunoglobulins (ivig) for improving symptoms. droplet and standard precautions. coronavirus and rhinovirus are the most frequent cause of the 'common cold' in the general population. however in immunocompromised patients these viruses can cause pneumonia which may be fatal, particularly in bone marrow transplant recipients [96] . persistent rhinovirus infection associated with graft dysfunction has been described in lung transplant recipients [97] . there are no specific treatment options available. decreasing immunosuppression may have a role but there is little data to support this. droplet and standard precautions. adenoviruses consist of a large group of dna viruses with over 50 types known to cause a variety of illnesses including gastroenteritis, encephalitis, hepatitis, haemorrhagic cystitis, upper and lower respiratory infections and conjunctivitis. in immunosuppressed patients adenovirus infection can develop at any time after transplantation and is associated with significant morbidity and mortality rates up to 75% [98] . adenovirus infection has been reported to be associated with organ rejection following cardiac and renal transplantation. bridges et al. reported 4 of 9 patients with adenovirus infection alone developed bronchiolitis obliterans syndrome and graft failure [99] . the registry of the international society for heart and lung transplantation: twenty-seventh official adult lung and heart-lung transplant report-2010 the changing microbial epidemiology in cystic fibrosis staphylococcus aureus infections in the early period after lung transplantation: epidemiology, risk factors, and outcomes associations between gut microbial colonization early life and respiratory outcome in cystic fibrosis what is the impact of hypogammaglobulinemia on the rate of infections and survival in solid organ transplantation? a meta-analysis burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence antimicrobial susceptibility and synergy studies of burkholderia cepacia complex isolated from patients with cystic fibrosis tuberculosis after solid-organ transplant: incidence, risk factors, and clinical characteristics in the resitra (spanish network of infection in transplantation) cohort mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management ast infectious diseases community of practice. mycobacterium tuberculosis infections in solid organ transplantation the risk of tuberculosis in transplant candidates and recipients: a tbnet consensus statement mycobacterial infections in solid organ transplant recipients tuberculosis in solid-organ transplant recipients: consensus statement of the group for the study of infection in transplant recipients (gesitra) of the spanish society of infectious diseases and clinical microbiology official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children nosocomial mycobacterium chelonae infection in laparoscopic surgery whole-genome sequencing to identify transmission of mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study the spectrum of mycobacterial infection after lung transplantation nontuberculous mycobacteria infection in solid organ transplant recipients ct findings of pulmonary non-tuberculous mycobacterial infection in non-aids immunocompromised patients: a case-controlled comparison with immunocompetent patients an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases identification of antimicrobial activity among fda-approved drugs for combating mycobacterium abscessus and mycobacterium chelonae outcomes in patients with mycobacterium abscessus pulmonary disease treated with long-term injectable drugs mycobacterium abscessus chest wall and pulmonary infection in a cystic fibrosis lung transplant recipient lung transplant outcomes in cystic fibrosis patients with pre-operative mycobacterium abscessus respiratory infections paediatric lung transplant outcomes vary with mycobacterium abscessus complex species emergence and spread of a humantransmissible multi-drug resistant nontuberculous mycobacterium mycobacterium abscessus in patients with cystic fibrosis: low impact of inter-human transmission in italy british thoracic society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (ntm-pd) microbiological problems and biofilms associated with mycobacterium chimera in heater-cooler units used for cardiopulmonary bypass risk factors, clinical characteristics, and outcome of nocardia infection in organ transplant recipients: a matched case-control study infectious complications associated with the use of rituximab for abo-incompatible and positive cross-match renal transplant recipients significant post-transplant hypogammaglobulinemia in six heart transplant recipients: an emerging clinical phenomenon? opportunistic infections in 547 organ transplant recipients receiving alemtuzumab, a humanized monoclonal cd-52 antibody ct findings of pulmonary nocardiosis ast infectious diseases community of practice. nocardia infections in solid organ transplantation clinical and laboratory features of the nocardia spp. based on current molecular taxonomy clinical experience with linezolid for the treatment of nocardia infection disseminated actinomycetoma due to nocardia wallacei invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (transnet) invasive mold infections in lung and heartlung transplant recipients: stanford university experience aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome aspergillosis in lung transplantation: incidence, risk factors and prophylactic strategies in vitro amphotericin b resistance in clinical isolates of aspergillus terreus, with a head to head comparison to voriconazole working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients international society for heart and lung transplantation guidelines for the management of fungal infections in mechanical circulatory support and cardiothoracic organ transplant recipients antifungal prophylaxis in lung transplantation aspergillus infections after lung transplantation: clinical differences in type of transplant and implications for management epidemiology of invasive fungal infections in lung transplant recipients on long-term azole antifungal prophylaxis spectrum of aspergillus infection in lung transplant recipients: case series and review of the literature candidal anastomotic infection in lung transplant recipients: successful treatment with a combination of systemic and inhaled antifungal agents anastomotic infections in lung transplant recipients incidence of invasive aspergillosis following hematopoietic stem cell and solid organ transplantation: interim results of a prospective multicentre surveillance program practice guidelines for the diagnosis and management of invasive aspergillosis. 2016 update by the infectious diseases society of america significance of blood stream infection after lung transplantation: an analysis in 176 consecutive patients a targeted peritransplant antifungal strategy for the prevention of invasive fungal disease after lung transplantation: a sequential cohort analysis epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry cellulitis caused by cryptococcus neoformans in a lung transplant recipient immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america gastrointestinal zygomycosis complicating heart and lung transplantation in a patient with eisenmenger's syndrome zygomycosis in solid organ transplant recipients: a prospective matched case-control study to assess risk for disease and outcome scedosporium apiospermum infection after neardrowning epidemiology, clinical manifestations, and outcomes of scedosporium infections among solid organ transplant recipients a multicenter evaluation of tests for diagnosis of histoplasmosis ast infectious diseases community of practice. endemic fungi in solid organ transplantation clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the infectious diseases society of america ribosomal rna sequence shows pneumocystis carinii to be a member of the fungi pneumocystis carinii: has the name really been changed? has the name really been changed? it has for most researchers opportunistic infections in patients with and patients without acquired immunodeficiency syndrome prevention of infection due to pneumocystis spp. in human immunodeficiency virus-negative immunocompromised patients risk factors for pneumocystis carinii pneumonia in kidney transplant recipients: a case-control study accuracy of β-d-glucan for the diagnosis of pneumocystis jirovecii pneumonia: a meta-analysis an outbreak of pneumocystis jiroveci pneumonia with 1 predominant genotype among renal transplant recipients: interhuman transmission or a common environmental source? guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings seroprevalence of hsv-1 and hsv-2 in the general french population the burden of infection with hsv-1 and hsv-2 in england and wales: implications for the changing epidemiology of genital herpes update on immunisations in solid organ transplant recipients: what clinicians need to know meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients a prospective survey of human herpes virus 6 infection in solid organ transplant recipients human herpesvirus-6 and -7 after lung and heart-lung transplantation principles and practice of paediatric infectious diseases kaposi's sarcoma among persons with aids: a sexually transmitted infection? clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study community respiratory viral infection in adult lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus influenza virus in solid organ transplant recipients influenza vaccine responses in lung transplant recipients molecular epidemiology of two outbreaks of parainfluenza 3 in a bone marrow transplant unit clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin pediatric human metapneumovirus infection: epidemiology, prevention and therapy respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection rhinovirus as a cause of fatal lower respiratory tract infection in adult stem cell transplant patients: a report of two cases chronic rhinoviral infection in lung transplant recipients invasive adenoviral infection in t-cell depleted hematopoietic stem cell transplantation: high mortality in the era of cidofovir adenovirus infection in the lung results in graft failure after lung transplantation there are no randomised studies of treatment. anecdotal case reports suggest cidofovir may have a role but results are mixed. droplet and standard precautions. key: cord-024183-1mrdjc39 authors: hutchison, alastair a.; leclerc, francis; nève, véronique; pillow, j. jane; robinson, paul d. title: the respiratory system date: 2013-10-08 journal: pediatric and neonatal mechanical ventilation doi: 10.1007/978-3-642-01219-8_4 sha: doc_id: 24183 cord_uid: 1mrdjc39 this chapter addresses upper airway physiology for the pediatric intensivist, focusing on functions that affect ventilation, with an emphasis on laryngeal physiology and control in breathing. effective control of breathing ensures that the airway is protected, maintains volume homeostasis, and provides ventilation. upper airway structures are effectors for all of these functions that affect the entire airway. nasal functions include air conditioning and protective reflexes that can be exaggerated and involve circulatory changes. oral cavity and pharyngeal patency enable airflow and feeding, but during sleep pharyngeal closure can result in apnea. coordination of breathing with sucking and nutritive swallowing alters during development, while nonnutritive swallowing at all ages limits aspiration. laryngeal functions in breathing include protection of the subglottic airway, active maintenance of its absolute volume, and control of tidal flow patterns. these are vital functions for normal lung growth in fetal life and during rapid adaptations to breathing challenges from birth through adulthood. active central control of breathing focuses on the coordination of laryngeal and diaphragmatic activities, which adapts according to the integration of central and peripheral inputs. for the intensivist, knowledge of upper airway physiology can be applied to improve respiratory support. in a second part the mechanical properties of the respiratory system as a critical component of the chain of events that result in translation of the output of the respiratory rhythm generator to ventilation are described. a comprehensive understanding of respiratory mechanics is essential to the delivery of optimized and individualized mechanical ventilation. the basic elements of respiratory mechanics will be described and developmental changes in the airways, lungs, and chest wall that impact on measurement of respiratory mechanics with advancing postnatal age are reviewed. this will be follwowed by two sections, the first on respiratory mechanics in various neonatal pathologies and the second in pediatric pathologies. the latter can be classified in three categories. first, restrictive diseases may be of pulmonary origin, such as chronic interstitial lung diseases or acute lung injury/acute respiratory distress syndrome, which are usually associated with reduced lung compliance. restrictive diseases may also be due to chest wall abnormalities such as obesity or scoliosis (idiopathic or secondary to neuromuscular diseases), which are associated with a reduction in chest wall compliance. second, obstructive diseases are represented by asthma and wheezing disorders, cystic fibrosis, long term sequelae of neonatal lung disease and bronchiolitis obliterans following hematopoietic stem cell transplantation. obstructive diseases are defined by a reduced fev1/vc ratio. third, neuromuscular diseases, mainly represented by dmd and sma, are associated with a decrease in vital capacity linked to respiratory muscle weakness that is better detected by pimax, pemax and snip measurements. • describe the expanded concept of breathing in terms of multiple motor functions that ensure airway protection, volume homeostasis, and ventilation. • describe the upper airway structures and how their functions affect the entire airway. • describe the nasal functions involved in air conditioning, in protection, in the maintenance of airway patency, and in the consequences of nasal bypass and obstruction. • describe the oral cavity and pharyngeal regions and control of airway patency. • describe factors involved in obstructive sleep apnea and the effects of cpap therapy. • describe the coordination of sucking, nutritive swallowing, and breathing during development and the relationship to apnea and bradycardia. • describe the importance of nonnutritive swallowing in pharyngeal clearance and how this may be influenced by cpap. • describe the three major functions of the larynx involving breathing. • describe the triple mechanism of laryngeal closure in airway protection. • describe how flow and subglottic volume are controlled during eupnea. • describe the integrative nature of the central control of laryngeal functions. • describe the roles of laryngeal muscles and their coordination with pump muscles during development and in eupnea, sighs, grunting, incremental breathing, and gasping. • describe how this information has been/ can be applied in respiratory support. • describe afferent inputs that alter laryngeal muscle functions and alter breathing and cardiovascular function. • describe the impact of central behavioral state and induced central depression upon laryngeal function and the interactions with detected changes in the gaseous environment. it is impossible for anyone to find the correct function of a part unless he is perfectly acquainted with the action of the whole instrument. galen, ad 120-200. breathing consists of multiple motor acts whose mechanical effects aim to ensure that the airway is protected, has optimal supra-and subglottic volumes, and provides vital ventilation. the upper airway extends from the nose or mouth to the larynx (seikel et al. 2005) , but its functions, in the context of ventilation, extend to the entire airway. maintenance of airway volume and ventilation is actively controlled by the centrally coordinated activities of nasal, oral, pharyngeal, and laryngeal upper airway muscles and those of the pump muscles. these act in concert with lower airway smooth muscle. effective breathing requires that the brain coordinates ventilatory functions with cardiovascular function and with other motor acts, e.g., swallowing, postural changes, and speech. adaptations in breathing must occur appropriately, rapidly, and constantly. protective and mechanical changes occur in milliseconds. changes in circulatory gas transport alter breathing within seconds. other adjustments optimize breathing in tune with changes in growth, aging, metabolism, and the environment. this chapter addresses how each upper airway structure plays critical roles in the dynamic processes of breathing. … we would do well to keep our mouths shut and reflect on the marvelous physiology of the nose. richard godfrey 1994 normal breathing occurs via the nose (fig. 4.1 ). inspired and expired air is conditioned by the nasal mucosa, whose surface area and blood supply are such that particles are filtered and temperature and humidity are modified. the nasal vascular system can achieve a 25 °c gradient between the nasal ostia and the nasopharynx (godfrey 1994) , a property that enables air breathing at very different environmental temperatures while maintaining a remarkable constancy of temperature and humidity in the lower airway, thereby optimizing cellular function and ciliary action. given its vascularity, it is not surprising that the nose is a source of nitric oxide, that trauma to the nasal passages can be induced by nasal continuous positive airway pressure (ncpap) prongs and other cannulae, and that epistaxis can require hospital admission. the nose can sniff out noxious substances and protect the airway from particles by sneezing. it can increase its resistance rapidly, switching on a watery secretion "like a tap" (godfrey 1994) , and can trigger laryngeal closure and reduced ventilatory drive (editorial 1992) . the extreme protective reflex response to nasal irritation is the dive reflex that is typified by apnea with glottic closure, bradycardia, and redistribution of blood flow to the brain, heart, and adrenals (see also sect. 47.3.1) (de burgh daly 1997; editorial 1992) . the dive reflex can be triggered by nasal water, tobacco smoke, or ice applied to the trigeminal area (angell james and de burgh daly 1969; de burgh daly 1997) . water entry into the nose is dramatic even for adults who "suffer a sense of impending suffocation and the almost impossibility of voluntarily making an inspiratory effort" (de burgh daly 1997) . exaggerated dive reflex responses can cause total upper airway closure and cardiac arrest and have been implicated in accidental and deliberately induced deaths (de burgh daly 1997). nasal breathing in the newborn and infant is the predominant but not obligatory form of breathing and is related to the high position of the epiglottis ( fig. 4 .1) (seikel et al. 2005 ). there is a nasal cycle whereby resistance alternates from one nostril to the other every 2-4 h in 80 % of humans (widdicombe 1986) . pressure in the axilla or on the lateral chest increases ipsilateral nasal resistance (widdicombe 1986) . resistance is highest in the regions adjacent to the external ostia, whose dimensions are altered by the alae nasi muscles that cause nasal flaring during exercise hyperpnea and respiratory distress, especially in newborns. newborn nasal resistance exceeds that of the adult in absolute terms but is less than that of their lower airways. nearly half of the adult's total airway resistance is nasal. thus, minor increases in nasal resistance can produce major overall effects on breathing (editorial ). in adult humans, nasal occlusion results in a switch to mouth breathing in <5 s (editorial 1992) . both this switch to oral breathing and the resumption of nasal breathing following release of the occlusion are delayed by nasal and pharyngeal anesthesia. occlusion of the nasal passages in infants results in mouth breathing in ~8 s (range <1-32 s) (editorial 1992; rodenstein 2004) . the response is quicker with advancing age but is decreased in rapid eye movement (rem) sleep, more so at 6 weeks than in the newborn period (editorial 1992) . these observations led to a proposal that the response may be related to the sudden infant death syndrome (sids) (editorial 1992) . in premature infants, the change to oral breathing results in a sixfold increase in pulmonary resistance, which over time might result in fatigue with hypoventilation (miller et al. 1987) . oral breathing places an increased burden on the lower airway to modify the temperature and humidity of inspired gas, presumably via changes in lower airway vascularity. preventing nasal breathing can decrease gas exchange and functional residual capacity (frc) (editorial 1992) . postoperative nasal packing in adults can lead to arterial hypoxemia, apnea, and sleep disturbance, with the latter also occurring in normal volunteers after local anesthesia of the nasal passages (editorial 1992) . in newborn lambs, nasal obstruction also affects gas exchange, with hypoxemia, hypercapnia, and acidosis being observed. these effects are aggravated by carotid body denervation. the impact of nasal obstruction diminishes with advancing age (editorial 1992) . blockage of the nose (choanal stenosis being an extreme example) can cause obstructive sleep apnea (osa) (marcus 2000) . human neonates whose nasal resistance is increased by birth trauma or trauma secondary to repeated suctioning breathe orally. they can have cyanotic episodes, stridor, and hypercapnia; these problems disappear with resolution of the nasal injury (miller et al. 1987) . in summary, the nose provides air conditioning, and its sensory receptors initiate reflexes that alter upper airway patency and mediate extended ventilatory changes in response to a challenge. functions in breathing …after relaxing, many sword swallowers are able to relax and breathe while swallowing a sword. from swordswallow.com 2009. the oral cavity extends from the lips to the plane of the faucial pillars, where it joins the pharynx ( fig. 4.1) . the three pharyngeal regions are the nasopharynx, from the posterior nares to the uvula; the oropharynx, from the uvula to the epiglottis; and the laryngopharynx, from the epiglottis to the esophagus. the pharynx is a conduit for air and/or food. the muscles of the tongue, especially the genioglossi, maintain patency of the oral cavity. pharyngeal patency is a function of the cavity dimensions (rodenstein 2004 ) and the balance between the opening forces exerted by pharyngeal muscle activities and the collapsing ones exerted by the tissue-intrapharyngeal pressure gradient and by pharyngeal mucosal adhesion (fig. 4.2) . patency is critically influenced by posture. narrowing occurs with neck flexion and hyperextension and can alter cavity pressures required to achieve flow. narrowing can also follow an inspiratory occlusion of the mouth/nose (a load) that augments diaphragmatic activity and thus negative pressure within the pharynx. upper and lower airway afferents induce reflex compensatory change in glossopharyngeal muscles to offset the load effect ( fig. 4 .2) (bailey and fregosi 2006) . obstructive apnea occurs during sleep in ~1-4 % of children and adults (marcus 2000) . in children, a persistent partial form of upper airway obstruction, obstructive hypoventilation, is seen (marcus 2000) . in adults, a spectrum of decreased inspiratory airflow is seen in snoring, upper airway resistance syndrome, and osa. pharyngeal osa occurs when a critical tissue pressure (p crit ) exceeds the intrapharyngeal opening pressure (fig. 4 .2) (marcus 2000; dempsey et al. 2010) and is influenced by factors that alter pharyngeal dimensions, namely, adenotonsillar hypertrophy, obesity (less common in children than adults), low subglottic airway volume, and craniofacial structural abnormalities. the major role played by the central motor output to the oral cavity and pharyngeal muscles that modify p crit is emphasized by the fact that osa occurs only in sleep, predominantly in rem sleep in children (marcus 2000) . the following factors also stress the importance of central control in osa. obstructive apnea occurring during mixed apnea in preterm newborns involves a direct central mechanism, as shown by the onset of pharyngeal and/or laryngeal closure before diaphragmatic activation (idiong et al. 1998 ) (see sect. 47.3.1) . in adults pharyngeal closure can accompany central apnea (marcus 2000) . central arousal patterns during osa differ between children and adults (marcus 2000) . other factors affecting the incidence of osa are gender, a familial tendency, and racial/ethnic factors (marcus 2000) . anti-inflammatory medications can benefit osa, suggesting a role for inflammation in its etiology (praud and dorion 2008) . therapy with ncpap improves osa by increas-ing intrapharyngeal pressure and its transverse diameter (rodenstein 2004) , by augmenting lower airway volume and probably by stimulating breathing via pharyngeal pressure sensors (angell james and de burgh daly 1969) . in children and adults with osa, cpap therapy can improve their metabolism. the abilities to suck, swallow, and breathe are developed during fetal life (harding 1986 ). at all ages, swallowing is accompanied by laryngeal closure. term newborns and infants up to 12-18 months of age can lock the larynx into the nasopharynx (figs. 4.1 and 4.3) . this separation of the nasopharyngeal-laryngeal air passage from the oropharyngeal nutritive passage enables the term infant to breathe and feed simultaneously but with decreased ventilatory drive. this ability is not developed in the preterm neonate whose sucking pattern is also immature (lau 2006) . coordination of sucking-swallowing-breathing is defined by the ability to feed "by mouth with no overt signs of aspiration, oxygen desaturation, apnea, or bradycardia" and when "a ratio of 1:1:1 or 2:2:1 suck: swallow: breathe" is attained (lau 2006) . safe swallowing occurs at end-expiration and end-inspiration (lau 2006) . in term newborns, 55 % of swallows occur at these points of the breathing cycle. in preterm infants, 55 % of swallows occur during deglutition apnea (lau 2006 nonnutritive swallowing (nns) is "essential for survival," since >2 l of oral and nasal secretions are produced daily. without nns, "the lungs rapidly fill with these secretions, producing death within a few days" (thach 2005) . when pharyngeal secretions reach a critical volume, neural receptors, in the interarytenoid notch, stimulate the laryngochemoreflex which triggers nns (thach 2005) . the critical pharyngeal volume for nns may be increased by applied positive pharyngeal pressure, providing an explanation for the decrease in nns frequency with cpap (thach 2005) . in adult humans nns occurs mainly during expiration. in infants nns occurs at any time in the respiratory cycle (thach 2005) . volitional swallowing in adults clears the pharynx and reduces the protective laryngeal adductor reflex. during sleep, the protection provided by nns and glottic closure is imperfect, and some aspiration is common in normal individuals (thach 2005) . swallow breaths are short inspirations that precede a swallow, occurring when the upper pharyngeal sphincters are closed (thach 2005) . these breaths are thought to result in pharyngeal air being inhaled as opposed to being swallowed (thach 2005) . the increased gastric air found when positive pharyngeal pressure is applied suggests that cpap can alter normal pharyngeal clearance mechanisms and infers the need to titrate the applied pressure carefully (thach 2005) . finally, swallowing may be related to sleep apnea and be part of recovery from sleep apnea (see sect. 47.3.1) (thach 2005) . in summary, patency of the oral cavity and the pharynx and the coordination with swallowing and sucking are critical for breathing. the term, but not preterm, newborn can suck and breathe simultaneously, although ventilatory drive is decreased. physiological control of breathing and swallowing involves important sensory input that limits aspiration and protects the lower airway. intensive care therapies can alter normal protective, breathing and swallowing functions. like a swiss watch, …our vocal tract depends on the precise functioning of many structures and muscles." jared diamond, 1992 (the third chimpanzee). the larynx has three major functions involving breathing. it serves as a protective structure guarding the subglottic airway; as a flow controller of subglottic absolute and tidal volumes during eupnea and complex coordinated movements; and as a vibratory modulator of flow that generates speech, song, and laughter ( fig. 4.4) . 4.1.4.1.1 laryngeal closure and airway protection protection of the subglottic airway is a centrally controlled major task of "breathing." laryngeal stimulation can trigger a clearing response, e.g., an expiration reflex or a cough, or can result in airway closure. laryngeal closure occurs at the level of the epiglottis, at the vestibular folds, and at the vocal folds. this triple mechanism has been likened to closure of a nutcracker, an analogy whose accuracy is all too evident to intensivists faced with laryngospasm (fink 1973) . and flow/volume control in adult humans the glottis opens maximally during inspiration, closes gradually to a minimum about three quarters through expiration, and then opens again in late expiration (england et al. 1982a ). glottic resistance is least at peak inspiration, increases as expiration progresses, and then decreases in late expiration to minimize inspiratory flow resistance with the onset of pump muscle activity (england et al. 1982a ). there is no sensation of expiratory glottic closure in eupnea. accurate, rapid laryngeal opening and closure maintains an optimal subglottic airway volume, ensures tidal volume changes that enable gas exchange, and enhances airway patency and surfactant secretion. the smooth tidal flow pattern, produced by laryngeal and diaphragmatic activities interacting with the mechanics of the respiratory system, avoids tissue exposure to acceleration or deceleration injuries. the same laryngeal intrinsic muscles are involved in all laryngeal functions emphasizing that the central coordinations of the motor acts of breathing are truly integrative, a fact also stressed by the impacts that laryngeal sensory inputs and the resultant vagal outputs have on the cardiovascular system. laryngeal closure is important for other motor acts, e.g., effective lifting. central control of breathing is linked to emotions. it is of note that in laughter the order of abductor-adductor activities seen in breathing is reversed (luschei et al. 2006 the intrinsic laryngeal muscles and their innervation are shown. the left panel shows from bottom to top the posterior cricoarytenoids, the sole abductors of the larynx, and the abductors: the thyroarytenoids, the lateral cricoarytenoids, the transverse and oblique arytenoids, and the aryepiglottic muscles. all of these muscles are supplied by the recurrent laryngeal nerve (x). the internal branch of the sln is sensory, while the external sln branch is motor for the cricothyroid muscles (right panel) (from o'connor, d. reproduced with permission from myer et al. (1995) . redrawn by agreement with springer-verlag) breathing and vocalizations in practices aiming to augment metabolic and mental well-being. understanding the actions of the laryngeal intrinsic muscles is a necessary first step in gaining insights into the nature of the central control of breathing. the posterior cricoarytenoids (pca) are the sole abductors of the vocal cords. there are several adductors, with the thyroarytenoids (ta) being the major ones ( fig. 4.4) . some adductors, the cricothyroid and the vocalis part of ta, enhance abduction by tensing the vocal cords. this restricts vertical movement, improving flow control in the more horizontal plane. the magnitude of abductor activity exceeds that of the adductors when glottic size is increased and vice versa, consistent with relationships between glottic size and flow resistance (england et al. 1982a; insalaco et al. 1990 ; kuna et al. 1988 ). animal studies indicate that, in general, glottic opening and closing are achieved by reciprocal laryngeal abductor and adductor activities ( fig. 4 .5) (bartlett 1989; harding 1986; hutchison et al. 1993) . concurrent actions of laryngeal abductor and adductor muscles during inspiration and expiration may occur in adult humans, but their nature is unknown kuna et al. 1988 kuna et al. , 1990 . during eupnea the inspiratory outline of the pca emg resembles the flow pattern, while the ramp shape of the diaphragm emg relates to the inspired volume trace . in animals little expiratory ta activity is seen, and trans-upper airway pressure is minimal (fig.4 .5). in eupneic expiration in adult humans, pca activity diminishes, but some ta activity is present "rounding off" the early expiratory volume the thyroarytenoid (ta) muscles, and those of the diaphragm (d), together with flow, volume, and trans-upper airway pressure changes (reproduced with permission from hutchison et al. (1993)) trace, but without the flow retardation or transupper airway resistance patterns typical of grunting (kuna et al. 1988) (fig. 4.5) . time delays exist between emg and mechanical flow changes . the timing delays differ in eupnea and grunting, reflecting different mechanoreceptor feedbacks and central outputs that affect function (fig. 4 .5) . clinically, grunting is the expiratory noise produced when air flows through a partially closed glottis (harrison et al. 1968) . total glottic closure is silent but sensed. physiologically, grunting is a breathing pattern seen throughout life. it consists of a spectrum of degrees and timings of expiratory laryngeal closure, associated with volume retention and increased subglottic pressure, followed by laryngeal opening with a rapid return to baseline end-expiratory volume (eev) (figs. 4.5 and 4.6) (see sect. 47.3.1) . in newborns with respiratory distress, harrison et al. showed that when grunting was prevented by endotracheal intubation, without positive end-expiratory pressure (peep), their oxygenation worsened (harrison et al. 1968 ). this finding established the role of airway peep and likely influenced the introduction of cpap therapy. harrison et al. also focused on venous return (harrison et al. 1968 ), stressing the importance of cardiac function in grunting. the outline of the volume-time trace in grunting (figs. 4.5 and 4.6 ) resembles that of a volume with a prolonged inspiratory time, as used in artificial ventilation to improve oxygenation. pre-surfactant, this strategy reduced the required peak inspiratory pressure and decreased the incidence of bronchopulmonary dysplasia. today, the maintenance of subglottic volume, the "open lung" approach, is a fundamental tenet of all ventilatory support. in keeping with the concept of alveolar interdependence, airway volume recruitment is achieved not by a maximal prolonged inflation but with an augmented breath (sigh) followed by an expiratory hold, mimicking the volume-time profile of grunting or of airway pressure release ventilation. spontaneous sighs can be biphasic, with inspiration being hutchison and bignall (2008)) interrupted briefly by laryngeal closure. this mechanism may reset vagal afferent feedback, avoiding a hering-breuer inflation reflex and possible loss of the acquired volume (see sect. 47.3.1) . in summary, human and animal data emphasize the importance of laryngeal control of expiratory subglottic airway volume. this central control strategy whereby airway pressure is dependent upon volume control has been shown by the study of bubble physics to produce a more stable mechanical system (hildebrandt 1974) . thus, coordinated central control of laryngeal and pump muscles is a major focus in the control of breathing. this knowledge can be applied in respiratory support (hutchison and bignall 2008) . behavioral state is a major controlling factor of breathing pattern. in fetal lambs, during the highvoltage electrocorticogram (ecog) state, apnea (no phasic laryngeal abductor or diaphragmatic activities) and tonic laryngeal adductor activity occur. laryngeal adductor activity can be enhanced by laryngeal contact with cooled lung liquid, by fetal movements, by "arousals," by swallowing, by uterine contractures, and by fetal hypoxemia. in the low-voltage ecog state, laryngeal and diaphragmatic activities are similar to those seen postnatally (harding 1986 ). hypercapnia mainly enhances fetal breathing in the low-voltage ecog state (harding 1986 ). in general, active laryngeal retardation of lung liquid egress is reported in the absence of fetal breathing movements and is related to lung growth (harding 1986 ). stimulation of the superior laryngeal nerve (sln) produces laryngeal closure (harding 1986 ) that will prevent aspiration of noxious material, e.g., meconium. the coordination of the acts of fetal swallowing (harding 1986 ) and "inspiratory" activities of the respiratory muscles stresses the developmental importance of the controller's ability to switch patterns rapidly between upper airway closure (laryngeal adduction) with lack of diaphragmatic activity (apnea) and laryngeal abductor and pump activities (see sect. 47.3.1). airway volume is high in the fetus compared to the newborn. it has been speculated that during different fetal behavioral states, the brain is setting the homeostatic limits for high and low airway volume to prepare the fetus for the major needs of subglottic volume control at birth (hutchison 2007 ) (see also sect. 47.3.1). life's greatest airway volume challenge, the establishment and maintenance of an air-filled airway at birth, is usually overcome without a hitch by breathing patterns whose complexities exceed those used during the majority of adult life. at birth, an incremental breathing pattern retains more inspired volume than is expired ( fig. 4 .6) (hutchison et al. 1994) . incremental breathing shares features with grunting with the addition of timing changes in laryngeal and pump muscle activities at end-expiration when the onset of pump muscle activity occurs before laryngeal muscles open the glottis fully. the result is a decrease in expired volume and an increase in eev (hutchison et al. 1994 ). this incremental mechanical process is aided by the airway stability accrued from the increased end-expiratory positive subglottic pressure that limits the development of a negative airway pressure during inspiration. given their tendency to chest wall distortion, it is not surprising that incremental breathing can occur in preterm neonates (eichenwald et al. 1992) . incremental breathing is also seen in gasping during acute cerebral hypoxia-ischemia (hutchison et al. 2002) . gasping is typified by brief laryngeal abductor and pump muscle activities followed by long periods of laryngeal adductor activity with apnea (hutchison et al. 2002) . subglottic pressure is increased, maintaining airway volume and likely promoting autoresuscitation (hutchison et al. 2002) . laryngeal and pump muscle activities nasal and laryngeal afferent inputs alter laryngeal abductor and adductor activities and thus glottic size. the laryngochemoreflex (lcr), whose afferent pathway is the sln, results in protective responses, including swallowing, apnea, obstructed respiratory efforts, cough, hypertension, and arousal from sleep (thach 2008 ). an obstructive apnea with bradycardia results if the lcr stimulus is persistent, especially in the immature or depressed brain. this lcr response alters with age -coughing becomes the major response. however, acquisition of a viral infection in infancy can rekindle its potency (thach 2008) . it is augmented by hypoxemia and anemia and has been implicated in apnea of prematurity and sids (thach 2008 ) (see sect. 47.3.1). other laryngeal sln afferents, including those from mechanoreceptors, drive receptors, and temperature receptors, affect laryngeal intrinsic muscle activities. although sln section does not alter the eupneic breathing pattern, these afferents are important. upper airway bypass can alter the inspiration-inhibition hering-breuer reflex. application of acid to the larynx in animals, mimicking chronic aspiration, alters the subsequent response to an applied airway load (see sect. 47.3.1). during noninvasive ventilation, nonsynchronous delivery of airflow to the upper airway in neural expiration results in laryngeal closure (rodenstein 2004; scharf et al. 1978) . thus, noninvasive ventilation and pacing of a paralyzed diaphragm should be applied synchronously with neural inspiration (rodenstein 2004; scharf et al. 1978) . in intact animals, compensatory glottic abduction follows single-breath total occlusion of inspiration or expiration at the mouth/nose. during the unimpeded expiration following a single inspiratory occlusive load, unopposed ta activity can produce laryngeal closure. thus when no air enters the lung, a compensatory reflex mechanism can prevent expiratory volume loss, maintaining absolute subglottic volume. lower airway afferent inputs affect glottic size (bailey and fregosi 2006) . stretch receptor discharges, induced by peep, decrease expiratory ta activity (harding 1986) . by contrast increased expiratory glottic adduction follows rapidly adapting receptor stimulation, e.g., with deflation or irritant stimulation secondary to a pneumothorax (bartlett 1989) , or follows c-fiber stimulation, e.g., with experimental pulmonary edema (bartlett 1989 ). direct and indirect stimulation of chest wall muscle afferents can invoke several flow patterns involving glottic closure (bartlett 1989; stecenko and hutchison 1991) . while pseudoasthma can be fabricated by partial glottic closure (rodenstein 2004) , matching laryngeal control of flow pattern to maintain optimal subglottic airway volume may explain changes reported with true increased airway resistance. subglottic inspiratory flow limitation in croup is associated with expiratory flow resistance, probably glottic in origin (argent et al. 2008) . increased lower airway resistance in adult asthmatic patients induces a breathing pattern characterized by laryngeal expiratory flow retardation (collett et al. 1983; sekizawa et al. 1987) . in adults, resistive loads applied at the mouth also decrease expiratory glottic size (brancatisano et al. 1985) . this change may occur immediately, suggesting that resistive loading, unlike total occlusive (elastic) loading, produces sudden flow changes that stimulate airway receptors to cause laryngeal adduction (brancatisano et al. 1985) . therapy with cpap reverses the expiratory laryngeal adduction in some asthmatic subjects, suggesting that airway pressure changes may alter the dynamics between stretch receptor and irritant receptor stimulations, thus changing the breathing pattern (collett et al. 1983) . in normal adults a voluntary deep breath can decrease laryngeal resistance and lower airway resistance (sekizawa et al. 1987) . by contrast, bronchoconstrictive stimulation in normal adults can result in inspiratory and expiratory glottic narrowing (higenbottam 1980 ). an increased resistance to inspiratory flow may be advantageous in that more transpulmonary pressure is applied to opening a constricted peripheral airway. if lower airway volume and resistance changes affect laryngeal function, can absence of laryngeal control affect lower airway resistance? laryngeal bypass during invasive ventilation is associated with atelectasis and with the development of increased lower airway resistance (hutchison and bignall 2008) . the latter effect may stiffen the conducting airways and thus stabilize total airway volume but demand increased effort. in summary, the data reflect the importance of laryngeal subglottic volume control as a determinant of lower airway resistance and point to interactions between lower airway afferents and coordinated laryngeal and pump muscle activities in this dynamic process (see also sect. 47.3.1). central control of coordinated laryngeal and diaphragmatic muscle activities is determined by developmental stage, behavioral states, metabolism (temperature), central inputs, and centrally acting chemicals. postnatally in lambs, increased expiratory ta activity with decreased pca activity occurs during the nrem state. by contrast, expiratory ta activity in rem is mainly absent, while pca activity is variable (harding 1986 ). in normal adult humans, expiratory ta activity is absent during stable nrem, while expiratory pca activity decreases in nrem and is variable in rem (kuna et al. 1988 . at all ages, expiratory ta activity occurs at arousal. thus behavioral state is a key factor in subglottic airway volume control that, in turn, is important in sleep apnea. increased central drive, with hyperventilation and/or hypercapnia, promotes laryngeal abduction in inspiration and expiration in adults and term newborns, in whom increased pca, diaphragmatic and intercostal muscle activities occur (bartlett 1989; insalaco et al. 1990; kuna et al. 1994; wozniak et al. 1993) . laryngeal adduction can occur during hypercapnic hyperventilation in preterm neonates, probably due to chest wall distortion (but see also sect. 47.3.1) (eichenwald et al. 1993) . this adduction also occurs in adults at the mechanical limits of airway volume ). in general, however, increased central drive decreases laryngeal resistance unless mechanical limitations are present. decreased central drive with hypocapnia diminishes expiratory glottic size and is associated with periodic breathing in adults (kuna et al. 1993; rodenstein 2004) . laryngeal closure has been noted in human newborns and infants with apnea or suspected apparent life-threatening events milner 1991, 1993) . during central apnea and periodic breathing in lambs, expiratory ta activity is noted (praud 1999) . in depressed human infants at birth, laryngeal closure can block intubation, and, in lambs, acute cerebral hypoxia-ischemia results in expiratory ta activity with laryngeal closure (hutchison et al. 2002) . centrally acting depressant drugs diminish central drive and, in lambs, produce laryngeal closure with apnea (praud 1999) . in former preterm infants, up to ~55-60 postmenstrual weeks, exposure to anesthesia and/or operative stress can result in apnea postoperatively (see sect. 47.3.1) . thus, the intensivist should avoid hypocapnia during noninvasive ventilation and be aware that central depression can result in apnea with glottic closure. hypoxia stimulates expiratory laryngeal abduction and increases ventilation in human adults and newborn lambs (england et al. 1982b; insalaco et al. 1990; praud et al. 1992) . in lambs, the increase in ventilation is dependent upon carotid body input -a sudden decrease in that input induces expiratory ta activity (praud et al. 1992 ). however, animal and human data indicate that the effects of carotid body input vary depending upon central state and the presence/absence of other inputs (de burgh daly et al. 1979 ). if animals are vagotomized and paralyzed, direct carotid body stimulation can induce expiratory ta activity, and exposure to hypoxia after airway vagal blockade or intrathoracic vagal section results in laryngeal adduction (bailey and fregosi 2006) . in adult humans, the degree of expiratory glottic adduction with hypoxia exceeds that during hypercapnia (england et al. 1982b) . thus, it is argued that the "pure" carotid body reflex response is laryngeal expiratory adduction that will retain airway volume and promote oxygenation. this pure response can be countered by the presence of ventilation which increases airway stretch receptor feedback that results in an overall expiratory laryngeal abduction response. in the author's view, a unifying explanation for the diverse findings with hypoxia is that the effect of carotid body input is to augment the central coordinated output to laryngeal and pump muscles that is selected under different conditions (see sect. 47.3.1). when central depression exists, protective inputs and/or absence of volume-related inputs produce an apneic response with glottic closure that is augmented by hypoxia. this can be seen in the "vagal" preterm infant who is sedated for a surgical procedure. at intubation laryngeal closure and apnea/bradycardia can be triggered. if hypoxemia ensues, the problems are aggravated. when tidal ventilation and airway volume feedback are restored, the impact of any ongoing hypoxemia is to augment breathing. hering and breuer found that their volumerelated reflexes were active even with hydrogen breathing. this fits the modern focus on ventilation for resuscitation from asphyxia. this chapter of upper airway physiology for the pediatric intensivist has focused on motor and specifically on laryngeal aspects relevant to the control of breathing patterns. the major message is that upper airway physiology is involved in all aspects of "breathing": protection, volume homeostasis, and ventilation; its functional impact covers the entire airway, from the nose to the alveolus. understanding upper airway physiology can guide and improve therapy, while therapy can aid a return to normal homeostasis or detract from it. current knowledge of upper airway physiology has had major implications for the application of invasive and noninvasive ventilatory support. much remains to be learned from the interactions between physics and biochemistry in the entire airway and how they affect breathing patterns. • the upper airway plays roles in all the extended functions of breathing, namely, in airway protection, in the control of supra-and subglottic volumes, and in tidal ventilation. • nasal functions include air conditioning and airway protection. stimulation can result in profuse secretions, altered patency, and the cardiorespiratory dive reflex. obstruction can affect gas exchange markedly; thus, a rapid switch to oral breathing is advantageous. • pharyngeal patency can be altered in sleep. nonnutritive swallowing is vital to minimize aspiration. therapy with cpap improves pharyngeal patency in osa but may alter pharyngeal clearing mechanisms and increase gastric air. • laryngeal closure can protect the airway rapidly. laryngospasm can be hard to treat. • controlled expiratory laryngeal closure modifies airflow and subglottic airway volume in normal breathing, at the establishment of airway volume at birth or when airway volume is threatened by mechanical, chemical, or central changes. • intrinsically or extrinsically induced changes in central state alter the laryngeal motor outputs in response to mechanical or chemical inputs. • understanding how laryngeal motor functions affect breathing patterns, airway pressures, and gas exchange is at the core of many technical and procedural advances in the provision of resuscitative measures and of invasive and noninvasive respiratory support. paul d. robinson and j. jane pillow the mechanical properties of the respiratory system are a critical component of the chain of events that result in translation of the output of the respiratory rhythm generator to ventilation. a comprehensive understanding of respiratory mechanics is therefore essential to the delivery of optimized and individualized mechanical ventilation. this chapter describes the basic elements of respiratory mechanics and reviews the developmental changes in the airways, lungs, and chest wall that impact on measurement of respiratory mechanics with advancing postnatal age. during spontaneous breathing, the forces driving inspiration are generated by the inspiratory muscles, whereas during mechanical ventilation, the forces are generated by the ventilator or by a combination of the ventilator with spontaneous inspiratory muscle contributions. the inspiratory muscle pressure needs to be sufficient to overcome three major mechanical properties of the respiratory system: elastance (e) (to overcome tissue forces required to change the lung volume (v)), resistance (r) (to overcome resistance to flow (v')), and inertance (i) (for acceleration (v'') of gas volumes). elastance is often considered in terms of its inverse, the compliance (c). the total pressure (p total ) generated is equal to the sum of the elastic (p el , proportional to volume and 1/compliance), resistive (p res , proportional to flow and resistance), and inertive (p in , proportional to acceleration and inertance) components: although pressure losses to inertia are considerable during high-frequency ventilation, the inertive component is normally a small portion of p total at normal breathing frequencies and can be effectively neglected. thus the equation driving flow during normal respiration can be simplified: nonetheless, the linearized relation of driving pressure to volume and flow does not fully characterize the pediatric respiratory system: resistance and compliance are both dependent on volume, volume history, and flow, particularly in the very small or premature neonate. further, the simplified equation does not fully incorporate the complexities of the respiratory system. almost all the tests used to measure respiratory mechanics in the infant and pediatric population have been developed in adults and then adapted for younger subjects. measurements of respiratory mechanics are most appropriately corrected to lung volume, such as functional residual capacity (frc). in the absence of a measure of frc, measurements of respiratory mechanics made in infancy are often related to body size (e.g., height/length or weight). measurements at the body surface are necessary to include the contributions of the chest wall. the chest wall contribution to respiratory mechanics can be isolated, by subtraction of the pleural measurements from those obtained at the body surface (e.g., airway opening). the respiratory system is in a passive state when there is no inspiratory muscle activity (p mus = 0). a passive condition is present in the paralyzed patient but can also occur without paralysis following hyperventilation (mortola 2004) . under passive conditions, resting lung volume is determined by to describe the principal components of respiratory mechanics of the airways, lungs, and the chest wall and the developmental and environmental considerations that impact on these measurements in the neonatal and young child two opposing forces. the outward recoil of the chest wall results from the elastic characteristics of the diaphragm/abdomen and of the rib cage. the outward recoil of the chest wall directly opposes the inward recoil of the lung. inward recoil is determined by the viscoelastic properties of the lung, including the mechanical properties of the lung tissues, and the collapsing pressure generated at the alveolar air-liquid interface. during spontaneous breathing, the passive respiratory mechanics can be measured using occlusion techniques. occlusion of the airways at lung volumes above the resting lung volume relaxes the respiratory muscles by stimulating the slowly adapting stretch receptors and the vagally mediated hering-breuer inflation reflex. passive tests assess the mechanical properties of the entire respiratory system. simultaneous measurement of transpulmonary pressure allows measurement of respiratory system compliance (c rs ) to be partitioned into lung (c l ) and chest wall (c w ) components. transpulmonary pressure (p tp ) is derived from the difference in pressure at the airway opening (p ao ) and pleural pressure (or elastic recoil pressure, p el ), measured using an esophageal balloon, liquid-filled catheter, or miniature pressure transducer. other passive measurements of respiratory mechanics include forced expiratory maneuvers to provide measures of maximal flow at frc (v′ max,frc ) (lum et al. 2006a ). partial forced expiratory flow-volume curves in infants are obtained by rapid compression of the chest through endinspiratory inflation of a jacket placed around the chest and abdomen. during tidal breathing, this rapid thoracoabdominal compression (rtc) technique provides a v′ max,frc that is similar to forced flows at low lung volumes (e.g., maximal expiratory flow mef 25 %,frc ). modification of the rtc technique by raising lung volume of the infant towards total lung capacity (so-called raised volume rtc or rvrtc) allows assessment of forced expiratory flows over a more extended range of volumes. reported measures include forced vital capacity (fvc) and forced expiratory volumes after defined time periods (e.g., 0.4, 0.5, or 1 s, termed fev 0.4 , fev 0.5 , and fev 1 , respectively) and the ratio of these values e.g., (fev t /fvc, where t is the time period chosen). in contrast, dynamic tests of respiratory mechanics allow the relative contributions of the airways and lung tissues to be partitioned, in addition to separately identifying the chest wall contribution to airway and tissue mechanical properties. dynamic mechanics derive mechanical variables from analysis of dynamic waveforms obtained either during normal (tidal) breathing or via imposed oscillatory waveforms. during tidal breathing, multilinear regression is used to determine dynamic respiratory system compliance (c dyn,rs ) and resistance (r rs ). kano and colleagues showed that consideration of volume dependence is important when using this approach to assess dynamic mechanics during mechanical ventilation. consideration of volume dependence is important because ventilatory settings may "push" the pressure-volume curve onto the flattened upper portion of the curve (kano et al. 1994 ). in the overdistended lung, fits obtained by multilinear regression are improved by the inclusion of a volume-dependent elastance term in the regression model: where p ao is the airway opening pressure, v is the tidal volume, e 1 + e 2 v is the total elastance over the cycle (e 1 and e 2 v representing the volumeindependent and volume-dependent portions of elastance, respectively), and the final term p a,ee represents the alveolar pressure at end-expiration. an important consideration during the interpretation of dynamic respiratory mechanics is the concept of frequency dependence. kano showed that increasing the ventilation rate from 30 to 80 breaths/min in a group of near-term newborn infants increased e rs and r rs by a mean of 8.3 and 17.5 %, respectively (kano et al. 1994) . frequency dependence and the mechanical properties of the respiratory system under normal tidal breathing conditions are also determined using forced oscillatory techniques (fot). these techniques are essentially noninvasive and are especially useful in infants and children as no active subject cooperation is required. the technique involves application of a pressure waveform (forcing function) to the airway opening and measurement of the resultant flow. the measured impedance reflects the complex viscoelastic resistance of the respiratory system (z rs ). simultaneous measurement of the transpulmonary pressure and hence chest wall impedance (z w ) allows determination of the lung impedance (z l ) by subtraction. the impedance (z) can be separated into the resistance (r rs ), the impedance component in phase with flow, and reactance (x rs ), representing the impedance components in phase with volume (elastance) and acceleration (inertance). the low-frequency forced oscillation technique (lfot) measures impedance over a range of frequencies (usually ~0.5-20 hz). lfot is obtained in infants by invoking the hering-breuer inflation reflex to inhibit temporarily any respiratory muscle activity, during which brief time (6-10 s) the lfot signal is applied. fitting the constant-phase model to the resultant pressure and flow data permits partitioning of respiratory mechanics into frequency-independent airway resistance (r aw ) and airway inertance (i aw ) and a constant-phase tissue component. this constant-phase tissue component is described by [(g − jh)/ωα, where g and h are coefficients for tissue damping and elastance, respectively, ω is angular frequency, and α determines the frequency dependence of the real (pressure change in phase with flow) and imaginary (pressure change in phase with volume) parts of the impedance] (hantos et al. 1992a ). the interrupter technique provides a further option to assess partitioned mechanics. the interrupter technique involves measurement of changes in airway opening pressure after a midexpiratory occlusion, which is used to calculate airway resistance (r aw ). after airway occlusion at mid-expiration, there is a biphasic change in p ao : the immediate rapid rise in p ao represents the resistive pressure drop across the conducting airways and is followed by a secondary slower increase in p ao (often referred to as p dif ) generally attributed to stress recovery in the respiratory tissues (lung and chest wall) and gas redistribution associated with ventilation inhomogeneity (bates et al. 1988) . a major limitation of this technique is that it requires mechanical ventilation and paralysis to obtain reliable data. particular challenges for lung function testing in infants and children include the marked developmental changes occurring over the first months and years of life. additional considerations include position, sleep state and sedation, and feasibility of individual tests given minimal capacity of these age groups for active cooperation and preferential nasal breathing (stocks and hislop 1996) . thus, in contrast to the adult and older child, tests in infants are performed using a mask instead of a mouthpiece and nose clips. lung development represents a period of considerable change in both lung architecture and volume and is affected by several factors including genetic, in utero and postnatal environmental exposures, somatic growth, puberty, changes in muscularity, and ongoing alveolarization. our understanding of the interactions between these factors is based on a mix of pathological and physiological data, which are predominantly cross-sectional in nature. consensus between these studies is lacking. while some of the lack of agreement is attributable to technical approaches used by different authors, a comprehensive understanding of factors influencing respiratory mechanics over time can only be answered by strong longitudinal data, which are urgently required. these tests of lung mechanics provide us with essential insight into how tissue properties change with development, and a full understanding of the changes that occur during healthy development is an important step to detect the subsequent effects of disease and response of the individual to therapeutic interventions. measurements need to be accompanied by accurate records of height (length) and weight to facilitate interpretation of longitudinal data. over the course of development, there is considerable remodelling of all of the structural components of the respiratory system, although the exact patterns of change in airways, alveoli, and blood vessels may differ with respect to each other. the term "dysanaptic growth" was originally introduced by green et al. (1974) and describes the disproportionate but physiologically normal growth of the lung relative to the airways. in infancy, airway size relative to lung volume is larger than in older children and adults. there is considerable debate in the pediatric literature about the degree, extent, and stage of development to which biological variability of airway size to lung volume occurs. the data outlined in this chapter suggest that dysanapsis is a feature of all stages of postnatal lung development. whether disproportionate growth along the length of the airway tree occurs is less clear. increased growth of the larger central airways, in relation to peripheral generations, has been described through the first year of life, after which point adult relative proportions are achieved and maintained through further growth (horsfield et al. 1987) . disproportionate central and peripheral airway growth, however, is not a consistent finding in the literature (hislop et al. 1972) . the most rapid period of tracheal growth appears to occur during the first 4 years of life (wailoo and emery 1982) . an elevated ratio of peripheral airway resistance to central airway resistance (and its contribution to total airway resistance) in young children compared to adults was originally proposed by hogg et al. (1970) , based on retrograde catheter measurements. the authors described relatively little change in the contribution of the central airways (those proximal to the 12th-15th airway generations) to conductance with age but marked increase in peripheral airway conductance from the age of 5 years of age. these changes were attributed to altered linear dimensions of the peripheral airways, based on morphometric data. the ratio of central to peripheral resistance can also be examined noninvasively by comparing measured forced expiratory flows (fef) breathing air to fef measured breathing a less-dense gas mixture of 80 % helium/20 % oxygen (heliox). fef measured breathing heliox is typically higher than during air breathing (e.g., 1.2-1.6 times at 50 % fvc). in older subjects with peripheral airway disease, this pattern is lost with ratios approaching parity (cooper et al. 1983; dosman et al. 1975) . density dependence data during infancy, in comparison to values reported in later life, appear to challenge the findings of hogg et al. (1970) . davis et al. examined the changes in density dependence through the first 2 years of life (davis et al. 1999) . fef 30-40 % higher were measured with heliox: although no relationship existed between density dependence and age, length, or fvc in the first 2 years of life (in keeping with hogg et al. (1970) ), the values obtained were similar to those reported across 16 other studies in older children ranging from school age to adulthood (summarized in table 2 of the original manuscript (davis et al. 1999) ). these equivalent cross-sectional density dependence values suggest that there is no difference in the convective accelerative and turbulent pressure loss with age, supporting the notion that the ratio of resistance of the peripheral to the central airways is similar in infants, older children, and adults. sex-specific differences in respiratory mechanics are well recognized in infants and young children and are a particularly important consideration in forced expiratory maneuvers in infants. airways are larger in females before puberty, and this difference is detectable from infancy (tepper et al. 1986a; hoo et al. 2002a) : v′ max,frc is approximately 20 % higher in girls over the first 9 months of life (hoo et al. 2002a ). similar sex-dependent increases in v′ max,frc are evident in preterm girls compared to preterm boys, suggesting such differences in lung function may be developmental rather than environmental in origin (stocks et al. 1997) . larger lungs have been reported in males between the ages of 6 weeks and 14 years (thurlbeck 1982) . presence and timing of the growth spurt is an important confounding factor, with lung growth occurring out of phase to somatic growth, especially in boys (degroodt et al. 1986; quanjer et al. 1989; schrader et al. 1984) . lung development in females is almost complete following menarche but continues throughout puberty in males (neve et al. 2002) . there is increasing evidence that the periconceptional and intrauterine exposures impact on development of the respiratory system and consequently impact on respiratory mechanics in infancy and childhood. factors known to influence lung mechanics after birth include maternal smoking, nutritional deprivation and intrauterine growth restriction, infection and/or inflammation, and maternal glucocorticoids. prenatal exposure to nicotine may cause abnormal airway branching and dimensions as well as increase airway smooth muscle and collagen deposition resulting in reduced lung function at birth that persists into early adulthood regardless of postnatal exposure (hayatbakhsh et al. 2009; svanes et al. 2004 ). lung function irregularities include airflow limitation, reduced fev 1 , and airway hyperreactivity wongtrakool et al. 2012) . chronic restriction of nutrients and/or oxygen in late pregnancy impairs development of the fetal small airways and lungs, including reduced numbers of alveoli that are also enlarged with increased septal wall thickness and basement membranes compared to nonexposed infants (pike et al. 2012) . such differences in alveolar number can influence the subsequent rate of disease progression, including an accelerated rate of decline in fev 1 with advancing age (stocks and sonnappa 2013). several cross-sectional studies have looked at the changes in airway resistance (reflecting airway size) during childhood. hall and colleagues using lfot described a quasilinear decrease in r aw with increasing length during infancy in 37 infants, including some with repeat measurements (hall et al. 2000) . lanteri and sly examined changes across the pediatric age range and also reported linear decreases in both r rs and r aw with increasing height (when plotted on a loglog plot) in 51 children (range 3 weeks-15 years) with healthy lungs (fig. 4 .7) (lanteri and sly 1993) . pooled data across the preschool age range reinforces the consistent relationship seen with increasing height (beydon et al. 2007a decreasing resistance of the respiratory system (r rs ) and airway resistance (r aw ) with increasing height. cross-sectional data taken from 51 subjects (aged 3 weeks to 15 years) (lanteri and sly 1993) . both resistance and height are plotted as natural log values but are labeled with absolute numbers for clarity examined (gerhardt et al. 1987a ). this increase occurred at a slower rate than the increase in frc, which led to a rapid drop in specific conductance ( fig. 4.8b ). this rapid decrease in specific airway resistance or conductance through early life has been described previously (doershuk et al. 1974; stocks and godfrey 1977) . tepper et al. measured maximal fef at frc, from partial expiratory flow-volume curves and corrected for changes in lung volume: they reported higher flows in the neonatal range which decreased to a steady value through the 2nd year of life, similar to those reported elsewhere in older children (tepper et al. 1986a) . differential increases in anatomic dead space volume (twofold), compared to frc (threefold), have also been shown in the pediatric population (wood et al. 1971) . rapid postnatal increases in lung volume are reported widely. frc increases by a magnitude of 40 times (from 80 ml in infants to 3,000 ml in adults) (dunnill 1982) and over ten times in total lung weight (polgar and weng 1979) . the rapid growth of the distal lung, in comparison to relatively steady changes in airway dimensions, is a major feature of respiratory system development beyond birth. after the appearance of primitive saccules at approximately 28 weeks gestation, mature alveoli with secondary septa appear from 34 weeks gestation onwards. alveolar multiplication was initially thought to cease at around 2 years of age (thurlbeck 1982) . more recent estimates indicate that increase in alveolar number continues until at least 8 years (dunnill 1982) . recent advanced imaging studies using hyperpolarized helium magnetic resonance imaging (mri) provide evidence of ongoing alveolar multiplication in adolescence (narayanan et al. 2012) . increase in alveolar size (expansion) is also evident during infancy and childhood, with recent epidemiology studies suggesting lung size increases into early adulthood, when chest wall development is complete (quanjer et al. 1989; reid 1977 ). based on cross-sectional studies, specific lung compliance falls during early infancy before remaining relatively constant from the early preschool years. tepper et al., based on measurements of compliance from the linear portion of the static pressure-volume (pv) curve in almost 50 infants, described an increase in lung compliance over the age range studied, but decreasing specific lung compliance with increasing body length in the first 2 years of life (tepper et al. 2001 ). gerhardt et al. examined similar changes in 40 infants and children over the first 5 years of life. lung compliance increased markedly (by ~20-25-fold) over the weight range examined (fig. 4 .8a) but in proportion to frc, resulting in a constant-specific compliance over the first 5 years of life. the same pattern of findings was reported in 63 children aged 2-7 years (greenough et al. 1986 ). this initial rapid period of alveolar growth is also supported by evidence from measurements using the interrupter technique and fot. lanteri and sly showed a biphasic change in p dif with age: p dif was highest in young infants, falling rapidly over the first 12 months, before increasing again after approximately 5 years of age (lanteri and sly 1993) . hall and colleagues, using lfot measures of tissue mechanics derived by fitting data to the constant-phase model, showed that both tissue parameters g and h decreased in a quasihyperbolic manner with increasing length between 7 weeks and 2 years of age (hall et al. 2000) . the difference in the pattern of change in g and h compared to the change in r aw with increasing length is further evidence of dysanapsis: tissue mechanical properties change more rapidly than airway mechanics over the first 2 years of life ( fig. 4.9 ). the chest wall of the neonate and infant, and to a lesser extent the growing child, is especially compliant compared to the compliance of the chest wall in the adult subject. a highly compliant chest wall in infancy results in less opposition to the tendency of the lung to collapse, promoting a low residual lung volume and also distortion of the chest wall, resulting in a loss of volume during inspiration. the disproportionately high compliance of the chest wall compared to the compliances of the lung means that the compliance of the respiratory system (c rs ) is approximately equal to the compliance of the lung (c l ) during childhood, especially in neonates and infancy (davis et al. 1988; gerhardt et al. 1987a) . several active (dynamic) mechanisms of respiratory mechanics serve to partially compensate for the instability in frc associated with a compliant chest wall and tendency for small airway closure during tidal breathing. infants acutely increase their end-expiratory lung volume (eelv) and maintain it above resting lung volume by modulating (abbreviating) their expiratory time (mortola and saetta 1987) ( fig. 4.10a ). in addition, they reduce expiratory flow through post-inspiratory activity of the diaphragm and inspiratory chest wall muscles (kosch and stark 1984) (fig. 4 .10b) (lopes et al. 1981) , stiffening the chest wall and by increasing laryngeal resistance (± glottic closure) briefly (kosch et al. 1988) . such dynamic modifications of respiratory mechanics occur via neural (vagal) receptor-mediated reflexes. restriction of lung function measurement to periods of quiet sleep is necessary to reduce the variability in measurements resulting from such dynamic processes (henschen and stocks 1999) . the changes in chest wall, lung, and respiratory system mechanics over the first 4 years of life were examined by papastamelos et al., in 40 subjects (papastamelos et al. 1995) . the authors used a modified mead-whittenberger technique (cook et al. 1957) : manual ventilation overrode respiratory drive and relaxed the respiratory muscles, avoiding the need for intubation or induction of a hering-breuer reflex. in this cohort, the ratio of chest wall to lung compliance fell from a mean (sd) of 2.9 (1.1) to 1.3 (0.4) (p < 0.005). chest wall to lung compliance ratios are even higher in preterm infants (gerhardt and bancalari 1980) . the stiffening of the chest wall, due to rapid rib ossification, increasing musculature, and altered chest wall configuration (bryan and wohl 1986; mead 1979; openshaw et al. 1984; leiter et al. 1986 ), is such that near-adult values, where lung and chest wall compliance are equal (mittman et al. 1965) , are reached after the first year of life. stiffening of the chest wall over the first year of life allows the infant to shift its maintenance of frc from dynamic elevation of eelv to a more passive mechanism achieved by the balance of outward recoil of the chest wall and inward recoil of the lung (colin et al. 1989 ). stiffening of the chest wall likely also explains the increase in p dif in children after age of 5 years observed by lanteri and sly (lanteri and sly 1993) . frc as a proportion of total lung capacity (tlc) increases with age through childhood, in the majority (engstrom et al. 1956; mansell et al. 1977; weng and levison 1969) , but not all (schrader et al. 1988 ), studies to date. residual volume (rv), as a proportion of tlc, also increases with age through childhood (merkus et al. 1993) , while closing capacity (measured using single-breath nitrogen washout) decreases significantly with age, as a proportion of tlc, converging towards rv (mansell et al. 1977) . this increased rv/ tlc ratio suggests that increased stiffness of the chest wall is not entirely compensated for by increase in expiratory muscle force over time (schrader et al. 1988 ). the study by merkus et al. also highlighted the need to use tlc as a measure of lung growth and not fvc, due to the fact that an increasing rv/tlc with age led to errors in maximal expired flows due to measurement at progressively higher lung volumes (merkus et al. 1993 ). the postnatal period is characterized by ongoing rapid lung development, with differing rates of growth seen in the airway, lung parenchyma, and chest wall. the dysanapsis that occurs between the components of the respiratory system has important consequences for measurements of lung mechanics performed during infancy and childhood. accounting for these developmental changes and dysanapsis is necessary to optimize disease detection and subsequent assessment of an individual's response to interventions. insights into these changes are largely derived from crosssectional studies, which have identified important sources of potential error in existing methodol. this is compared to the passive expiratory time constant (slope of the dotted line) measured after an end-inspiratory occlusion. the difference between the two slopes is generated due to active use of the respiratory muscles during expiration ("braking"). the mag-nitude of difference between the active flow-volume curvederived eelv (frc) and the passive relaxation volume (v r ) reflects the degree of active (dynamic) mechanisms employed to elevate eelv. this active use of respiratory muscles is also illustrated in (b) from data collected in a separate infant study wherein diaphragm emg activity of breaths three and four extends into expiration (both figures reproduced with permission from the publishers (mortola and saetta 1987; kosch and stark 1984) ) • the postnatal period is characterized by ongoing rapid lung development in all parts of the respiratory system (airways, lung parenchyma, and chest wall). the physiologically normal rate of change of the lung relative to the airways is disproportionate. this dysanaptic growth pattern appears to be a feature of all stages of postnatal lung development. ogy. strong longitudinal data are now urgently required to confirm these apparent patterns and answer the important questions that remain. j. jane pillow the predominant neonatal acute respiratory diseases include transient tachypnea of the newborn (ttn), respiratory distress syndrome (rds) due to surfactant deficiency and/or structural immaturity, pulmonary interstitial emphysema (pie), meconium aspiration syndrome (mas), persistent pulmonary hypertension of the newborn, pneumonia, and congenital diaphragmatic hernia (cdh). as there are no reported measurements of lung mechanics or lung function for infants with pie and pneumonia, they are not discussed further. transient tachypnea of the newborn (ttn) is characterized by delayed resorption of fetal lung fluid within the distal airspaces. the mechanical consequences of ttn include reduced lung volume (functional residual capacity, frc) and consequently also decreased lung compliance (increased elastance), tidal volume, and respiratory rate (benito zaballos et al. 1989) . although increased interstitial fluid might be expected to increase lung tissue resistance, there are no reported studies of tissue resistance in infants with ttn. • passive measures of lung mechanics provide information about the mechanics of the entire respiratory system. • dynamic measures of lung mechanics allow partitioning of the lung and chest wall components to separately identify the chest wall contribution to airway and tissue mechanical properties. • standardization of the measurement technique is essential. results generated are corrected for either lung volume (e.g., frc) or, in the absence of this, a measure of growth (e.g., weight). • a number of periconceptional, intrauterine, and postnatal factors impact on the development and mechanics of the respiratory system. important postnatal factors include maternal smoking, nutritional deprivation and intrauterine growth restriction, infection and/or inflammation, and maternal glucocorticoids. • the rate of airway growth (detected by increases in conductance or decreases in resistance) is not as rapid as the rate of frc increase with age. this greater relative increase in lung volume leads to a rapid decrease in specific resistance during early childhood. • postnatal alveolarization appears to now extend into adolescence. rapid alveolar growth occurs during infancy leading to an initial fall in specific lung compliance, which reaches a plateau during ongoing lung growth. these changes are also evidenced by the changes seen in lfot tissue parameters and interrupter technique-derived p dif . • the highly compliant chest wall of the infant leads to employment of active (dynamic) measures to maintain eelv above frc. as the ratio of chest wall to lung compliance falls and reaches the adult values of parity beyond infancy, these gradually transition to more passive mechanisms as the chest wall stiffens through early childhood. • to describe flow (f), lung volumes (v), and respiratory pressure (p) measurements together with resistance (r) and compliance (c) measurements in neonatal respiratory diseases like ttn, rds is predominantly a disease of the distal lung parenchyma. surfactant deficiency promotes atelectasis, typically characterized by low lung volumes and reduced lung compliance. lung function is also reflective of the degree of structural maturation, as surfactant deficiency is predominantly a disease of the premature infant. in the intubated infant, lung resistance measures are highly variable and not reproducible (22-32 %, icc <0.75), in contrast with less variable and more reproducible measurements of lung compliance (obtained from esophageal manometry). hence, the clinical relevance of dynamic mechanics measurements of resistance in intubated newborn infants with respiratory distress is questionable (gappa et al. 2006a) . measurements of impedance, obtained using forced oscillatory mechanics (a quasistatic lung mechanics measurement), may provide a more reliable assessment of lung mechanics. dorkin measured respiratory impedance in six paralyzed and intubated infants, three of whom also had pulmonary interstitial emphysema (dorkin et al. 1983 ). the tracheal tube contributed to almost all the inertance and approximately 50 % of the respiratory system resistance in the intubated infant. after subtraction of the impedance of the endotracheal tube, resistance ranged from 22 to 34 cm h 2 o/l/s, compliance from 0.22 to 0.68 ml/ cm h 2 o, and inertance from 0.0056 to 0.047 cm h 2 o/l/s (gappa et al. 2006a; dorkin et al. 1983) . the low-frequency forced oscillation technique (lfot) evaluates impedance simultaneously across a range of frequencies (usually ~0.5-14 hz). fitting of the resultant impedance data to the constant-phase model permits estimation of partitioned mechanical variables of the airways and the parenchymal tissues (hantos et al. 1992b ). using the lfot, impedance measurements in naïve (surfactant-deficient) newborn preterm lambs showed that respiratory system resistance (r rs ) and reactance (x rs ) are markedly frequency dependent (pillow et al. 2001a) . respiratory system resistance in the preterm infant is also dominated by the resistive properties of the tissues, contrasting sharply with the predominant airway contribution to respiratory system resistance in later life (pillow et al. 2005) . although compliance is also low (increased elastance), the tissue resistance is disproportionately greater, resulting in mechanical uncoupling of the parenchyma and increased hysteresivity (ratio of tissue resistance to tissue elastance) (pillow et al. 2001a (pillow et al. , 2005 . increased surfactant pool size (pillow et al. 2004a ) and lung volume recruitment (pillow et al. 2004b ) both enhance mechanical coupling of the tissues, evident as a lowering of the hysteresivity ratio in the lung. increased contribution of the tissues to respiratory system mechanics, and the associated increased frequency dependence, results in elevation of the resonance frequency of the lung (pillow et al. 2001a) . frequency dependence also becomes less marked with increasing postnatal age ( fig. 4 .11) (pillow et al. 2005) . variability in measurements of lung mechanics in newborn infants in part reflects lung maturation associated with advancing gestation: lung compliance increases 0.17 ml/cm h 2 o/week, to reach mean (sd) values of 2.50 (0.07) ml/cm h 2 o at term equivalent (bhutani et al. 2005) . other factors include in utero fetal exposures such as placental nutrition, inflammation, antenatal steroids, and postnatal treatments including caffeine, glucocorticoids, surfactant, and ventilation modality. early treatment with caffeine in surfactant-treated immature baboons increased c rs over the first day of life and increased ventilatory efficiency index (yoder et al. 2005) . the initial benefit of antenatal steroids on improved lung compliance at birth fades beyond 1 week after initial exposure (mcevoy et al. 2008 ). there is no evidence of that antenatal steroids have a persistent effect on lung mechanics: infants born at term after exposure to up to three courses of betamethasone at gestations between 25 and 33 weeks had no difference in gas mixing or lung volume/mechanics when compared to nonexposed term controls (hjalmarson and sandberg 2011) . like antenatal steroids, postnatal steroids also increase respiratory system compliance (durand et al. 1995) . low-dose and high-dose postnatal dexamethasone for cld achieve a similar increase in c rs suggesting equal effectiveness for improvement of lung volume (mcevoy et al. 2004) . early postnatal dexamethasone may be more effective than late dexamethasone in achieving lower r aw /r rs and higher specific conductance (sg aw ) (merz et al. 1999; vento et al. 2004) , which may indicate a degree of fixed change in the airway walls in infants receiving delayed glucocorticoid treatment. during administration, surfactant represents a fluid bolus in the airway that increases inspiratory and expiratory time constant, associated with an acute increase in respiratory system resistance. effective delivery of surfactant to the lung thus benefits from a transient increase in the inspiratory time and potentially brief increase in the delta p (peak inspiratory pressure-positive endexpiratory pressure) to overcome the increase resistance resulting from the fluid bolus. treatment with exogenous surfactant (da silva et al. 1994) or increases in positive end-expiratory pressure (peep) during recruitment from atelectasis increases lung volume: (dimitriou et al. 1999 ) lung volume increases by 61 ± 39 % within a median of 4 min after surfactant administration, with altered distribution of lung volume towards the dorsal rather than ventral compartment. the increase in lung volume after surfactant or volume recruitment maneuvers increases maximal compliance of the respiratory system, with higher tidal volumes achieved at lower pressures than required prior to treatment (miedema et al. 2011a; mcevoy et al. 2010) . importantly, the increase in compliance also impacts on the time constant (τ = r rs × c rs ) of the respiratory system. changes in the inspiratory and expiratory time constants need to be monitored as they necessitate adjustment of the inspiratory and expiratory times and influence the maximal frequency that can be used during ventilation with passive expiration. compared to treatment of rds with synchronized intermittent mandatory ventilation (simv), a small clinical trial showed that infants treated with high-frequency oscillatory ventilation (hfov) have early and sustained improvement in pulmonary mechanics and higher dynamic respiratory compliance (vento et al. 2005) . the improvement in pulmonary mechanics in patients treated with hfov is likely associated with enhanced lung volume recruitment. the neonatal lung with rds exhibits hysteresis (miedema et al. 2011b) . hence lung recruitment maneuvers that aim to recruit the lung and subsequently ventilate it on the most compliant part of the deflation pressure volume curve can achieve effective ventilation with minimum pressure and volume cost of ventilation. importantly, recruitment of the lung by increasing mean distending pressure during hfov is also associated with changes in compliance and the time constant for volume delivery during hfov (pillow 2012) . consequently, volume delivery can vary substantially over the time course of an hfov volume recruitment maneuver (miedema et al. 2012 ) and potentially result in rapid fluxes of the partial pressure of arterial carbon dioxide. the effect of compliance on volume delivery during hfov is more evident at lower frequencies (pillow 2012; pillow et al. 2001b ). the development of hfov ventilators that incorporate volume guarantee during hfov will provide protection from such rapid changes in ventilation during hfov. infants with rds often have their clinical course complicated by the persistence of a patent ductus arteriosus (pda), which presents as a leftright shunt. ligation of a pda (left-right shunt) increased dynamic compliance by 77 % in 16 newborn infants measured before and after ligation, but did not influence dynamic r rs or mean airway pressure (szymankiewicz et al. 2004a ). although meconium aspiration syndrome (mas) is often considered as a high airway resistance disease due to the presence of meconium in the airways, studies in rabbits show that the acute aspiration of meconium also causes a significant reduction in the lung-thorax compliance (sun et al. 1993 ). impaired compliance after mas is responsive to standard surfactant instillation (sun et al. 1993) . larger volume (15 ml/kg) surfactant lung lavage is an emerging treatment for mas: following surfactant lung lavage, dynamic compliance approximately doubled, and airway resistance nearly halved in a group of mas infants on mechanical ventilation (szymankiewicz et al. 2004b ). these changes in dynamic respiratory mechanics were associated with a decrease in mean (sd) airway pressure from 12.4 ± 3.6 to 5.4 ± 2.1 cm h 2 o within 48 h after surfactant lung lavage. meconium appears to have a differential effect on hyperreactivity of the airways and the lung tissue. whereas tracheal smooth muscle reactivity increases with increasing meconium concentration in response to histamine and acetylcholine, a negative correlation was observed in the lung tissue (mokry et al. 2007 ). airway hyperresponsiveness 2 weeks after birth (approximately 5 days after cessation of ecmo) is responsive to bronchodilator treatment (koumbourlis et al. 1995) as evidenced by percent change in mef 25 and the mef 25 /fvc ratio. airway hyperreactivity after mas is also reduced by budesonide (mokry et al. 2007 ). limited information is available for the longterm lung function outcomes after mas. neonates treated with ecmo for meconium aspiration have better long-term lung function outcomes than infants with diaphragmatic hernia treated with ecmo in the neonatal period (spoel et al. 2012a) , most likely reflecting differences in lung capacity and structure. at least 50 % of children who had mas in the neonatal period have evidence of trapped air on lung function during mid-late childhood. there are no published reports evaluating responsiveness to bronchodilators beyond infancy after neonatal mas. persistent pulmonary hypertension of the newborn (pphn) presents with profound hypoxia either in the absence of significant lung disease (primary pphn) or hypoxia out of proportion with the degree of respiratory disease (secondary pphn -complicating rds, meconium aspiration, pneumonia, etc.). lung function in 1-yearold infants who had pphn and who were treated with inhaled nitric oxide (ino) but not extracorporeal membrane oxygenation (ecmo) was compared with lung function outcomes of infants who were randomized to receive either ecmo or conventional management as part of the uk ecmo trial. v′ max,frc was lower than predicted in all three groups (p < 0.001). there were no statistical differences between the three groups in the z-scores for v′ max,frc (hoskote et al. 2008) . nonetheless, only 26 % of ino-treated infants had v′ max,frc z-scores below normal compared to 37 and 56 %, respectively, for ecmo and cm groups. a prospective evaluation of lung function in infants with pphn treated with/without ino was reported by dobyns and colleagues (1999) : compared to healthy control infants of the same age, there were no differences in lung volume or passive respiratory mechanics for infants with pphn, nor was there any effect of ino on later pulmonary function in the pphn group. together, these results suggest that ino treatment does not worsen outcome compared to ecmo or conventional management. infants with congenital diaphragmatic hernia have impaired airway function in the first year of life: maximal expiratory flows at frc were a mean z-score of −1.5 lower than healthy term infants (i.e., 1.5 standard deviations below the mean frc for healthy term infants) with no evidence of significant change between 6 and 12 months of age (spoel et al. 2012b) . conversely, and perhaps surprisingly, given the usual association of cdh with pulmonary hypoplasia in at least one lung, spoel noted that measured values of functional residual capacity were relatively high (47 % fell above the normal range) (spoel et al. 2012b ). as expected, mean z-score for fev 1 and fvc was negatively influenced by the presence of chronic lung disease, the duration of ventilation, and ecmo support (spoel et al. 2012a) . patients with cdh develop hyperinflation, with elevated plethysmographic frc at 1 year (hofhuis et al. 2011 ) that is still evident at 8-12 years (spoel et al. 2012a; hamutcu et al. 2004; majaesic et al. 2007 ). spoel showed that greater impairment was evident in infants with cdh who required extracorporeal membrane oxygenation (spoel et al. 2012b ). however, poorer outcome in ecmotreated cdh may reflect the initial severity of disease rather than ecmo: beardsmore showed that ecmo per se did not worsen respiratory function at 1 year of age compared to conventionally ventilated controls (beardsmore et al. 2000) . spoel and colleagues also noted deterioration in lung function over time (5-12 years) in patients with cdh who were treated with ecmo in the neonatal period (see table 4 .1) (spoel et al. 2012a ): mean (se) sds score for fev 1 after bronchodilation was higher at 5 years (−0.71 (0.40)) than at 8 years (−2.27 (0.36)) and 12 years (−2.73 (0.61)). deterioration over time may be related to maldevelopment of the alveoli and pulmonary vessels with disturbed lung growth. it is also possible that increased susceptibility to recurrent respiratory tract infections may contribute to deterioration in lung function over time. an active lifestyle and healthy eating pattern may be especially important for children with cdh to counteract the deterioration in lung function over time, given the known positive effect of participation in sports and the negative interaction of bpd is most commonly considered as the chronic respiratory condition complicating extreme prematurity and/or prolonged neonatal mechanical ventilation. factors that independently influence the tidal flow-volume-derived indices include variations in disease severity, degree of alteration to airway and lung mechanics, and also the requirement for oxygen supplementation ). there are, nonetheless, quantitative differences in tidal breathing function measured in bpd infants when compared with measurements obtained from healthy term and preterm controls. bpd infants are consistently more tachypneic than their healthy counterparts ), but differences in other lung function variables are less consistent. tidal volume (v t ) is either less than (paetow et al. 1999) or similar to (patzak et al. 1999; schmalisch et al. 2003) values obtained in healthy infants: v t relative to healthy infants may also decrease with advancing postnatal age (tepper et al. 1986b) , likely reflecting the altered maturational progression in lung structure and function. bpd infants have increased minute ventilation (mv). increased mv is primarily due to increased respiratory rate rather than increases in v, in part reflecting increased dead space ventilation. infants with bpd have an increased work of breathing. within the tidal flow-volume loop, increased work of breathing is evident from linear or concave expiratory limb morphology. bpd infants also have a less variable shape to the tidal flow-volume waveform, indicating that they are functioning near their maximum respiratory capability with minimal reserve capacity to adapt to changes in intrinsic (e.g., airway obstruction, infection, or aspiration) or extrinsic (e.g., cold stress) environment . tidal flow indices are difficult to interpret in part due to opposing effects of neurorespiratory control and lung mechanics on the morphology of the tidal flow waveform. whereas bpd infants have higher respiratory drive (quantified from the mean inspiratory flow: v t /t i where t i is inspiratory time), the ratio of the time to peak expiratory flow relative to expiratory time (t ptef :t e ) after methacholine-induced airway obstruction can either increase or decrease (clarke et al. 1994) . normal fluctuations in v t , t i , and mv seen in healthy infants in response to alternate hypoxic-normoxic breath testing are not evident in bpd infants, indicating reduced chemoreceptor sensitivity to hypoxic stimulus (calder et al. 1994) . lung and chest wall mechanics of infants with bronchopulmonary dysplasia (bpd) were extensively reviewed by gappa and colleagues (2006b) . changes are reflective of maldevelopment of the lungs and airways and remodelling of the lung parenchyma and airway walls associated with increased collagen content. in preterm infants, respiratory system compliance (c rs ) increases relative to body weight over the first 2 years of life, while an initially high respiratory system resistance (r rs ) decreases over the same period (baraldi et al. 1997a) . airway walls of preterm infants are more compliant than term infants as evidenced through measurements obtained using the high-speed interrupter technique (hit) (henschen et al. 2006) . following the nichd consensus conference in 2001 (jobe and bancalari 2001) , some investigators evaluated differences in lung function according to whether infants had mild, moderate, or severe chronic lung disease. using the single occlusion technique, hjalmarson and sandberg showed that infants with severe bronchopulmonary dysplasia (born at mean 25 weeks ga) had increased specific conductance and decreased specific compliance (sc rs ) compared to healthy preterm infants (born at mean 29 weeks gestation) (hjalmarson and sandberg 2005) . there were no differences in lung mechanics between the healthy preterm infants and those with mild or moderate bronchopulmonary dysplasia. the data published by tortorolo et al. indicate a similar trend towards lower c rs at day 7 in infants who later went on to develop mild bpd and to a greater extent in severe bpd (tortorolo et al. 2002) . their results contrast with the findings by shao and colleagues in infants of similar gestations that infants with bpd had decreased c rs (10.8 vs 16.0 ml/kpa/kg), but no difference in specific sc rs (0.69 vs 0.75 ml/kpa/kg) or r rs (6.6 vs 7.3 cm h 2 o/ml/s) compared to non-ventilated preterm infants. infants with bpd do not show an improvement in airway resistance (r aw , measured by plethysmography) over a 4-month period (shao et al. 1998) . several authors have attempted to use lung function as a predictor of bpd. the value of initial r rs for prediction of lung disease is unclear: some have found increased initial r rs is associated with respiratory outcome at 1 year (choukroun et al. 2003; lui et al. 2000; snepvangers et al. 2004) , whereas others observed that r rs is not predictive of bpd (tortorolo et al. 2002) . both lui (lui et al. 2000) and merth (merth et al. 1997) showed that early rds is not an important determinant of later lung function. serial measurements of compliance, however, show that lower c rs values after day 5 were evident in infants who later developed severe bpd. merth observed that bpd infants have reduced c rs at 1 year of age irrespective of rds (merth et al. 1997) . v′ max,frc obtained using the tidal rapid thoracic compression technique is reported consistently as being lower than healthy controls throughout the first 3 years of life, regardless of the bpd era or treatment strategies (lum et al. 2006b ). reduced expiratory flows are indicative of abnormal structural and functional development of the airways (fig. 4.12) . serial lung function measurements in infants with both bpd and "healthy" unsedated preterm infants indicate a decline over the first year of life, suggesting that factors other than bpd may contribute to abnormal airway function in preterm infants (hoo et al. 2002b) . raised volume rapid thoracic compression (rvrtc) measurements show similar mild-moderately severe airflow obstruction. however the predominance of airflow obstruction primarily at low lung volumes indicates the pathology is more likely to involve the small peripheral rather than larger central airways (lum et al. 2006b ). concurrent increased residual volume (rv), frc, and rv/ tlc (total lung capacity) ratios were more marked in infants with recurrent wheeze, indicative of a degree of hyperinflation and gas trapping. intermediate values (between healthy controls and wheezy bpd infants) were observed for rv, frc, and rv/tlc in non-wheezy preterm infants (robin et al. 2004) , likely reflective of abnormal airway growth or the effects of preterm delivery. forced deflation from frc showed that preterm infants with developing bpd have severe lower airway obstruction as early as 3-4 weeks postnatal age. increased flows and fvc following bronchodilation indicate that reopening of obstructed airways is achieved with bronchodilators (motoyama et al. 1987) . a recent systematic review and meta-analysis assessed the effect of preterm birth on later fev 1 : (kotecha et al. 2013 ) 59 studies were included in the meta-analysis, including 28 studies in children born preterm without bpd and 39 studies in children with bpd diagnosed at either 28 day of life (bpd 28 ) or 36 weeks postmenstrual age (bpd 36 ). the meta-analysis showed that just being born preterm decreased fev 1 compared to term-born controls, with further decrements in fev 1 evident for the bpd 28 and bpd 36 week groups. the mean differences (95 % ci) for % fev 1 compared with term-born controls were −7.2 % (−8.7 %, −5.6 %) for preterm group without bpd and −16.2 % (−19.9 to −12.4 %) and −18.9 % (−21.1 to −16.7 %) for the bpd 28 and bpd 36 groups, respectively. including data from preterm studies not including a control group resulted in a pooled % fev 1 estimate of 91.0 % (88.8-93.1 %) for preterm infants without bpd, 83.7 % (80.2-87.2 %) for bpd 28 , and 79.1 % (76.9-81.3 %) for bpd 36 , respectively. of interest, the authors noted that %fev 1 for bpd 28 has improved over the years. bpd survivors also have lower forced expiratory flows: 70 % of children with bpd had maximal flow at functional respiratory capacity (v max frc) below 40 % of the predicted value (baraldi et al. 1997a) . forced expiratory flow at 25 % of forced vital capacity was reduced in young adolescents born prematurely, regardless of prior bpd or level of respiratory support (anand et al. 2003) . of concern, doyle and colleagues showed that airflow limitation worsened between 8 and 18 years ). together, these data suggest that the preterm infant population is at risk of developing chronic obstructive disease and should receive long-term respiratory follow-up (baraldi and filippone 2007a; filippone et al. 2003; lum et al. 2011) . airway obstruction is accompanied by impaired diffusing capacity in children and adults born prematurely (hakulinen et al. 1996; vrijlandt et al. 2006) . similar findings in infants and toddlers with bpd in the face of normal alveolar volumes indicate parenchymal disease and impaired alveolarization (balinotti et al. 2010 ). several studies have evaluated the effect of bronchodilators on lung mechanics in infants with bpd. bronchodilators decrease r rs , without affecting c rs : ultrasonic nebulizers appear to be most effective (fok et al. 1998) . bronchial hyperresponsiveness at 2 years of age was related to mean neonatal c rs and r rs over the first 3 days in ventilated preterm neonates using breath occlusion test. c rs but not r rs was related to bronchial hyperresponsiveness at 2 years' age (snepvangers et al. 2004 ). fifty-six percent of children in the epicure study born at 25 weeks pma or less had abnormal baseline spirometry at 11 years of age, with 27 % showing a positive bronchodilator response: more marked responses were evident in the infants with prior bpd (fawke et al. 2010) . less than half of the epicure study children with abnormal lung function at 11 years were receiving treatment, suggesting more targeted respiratory follow-up is warranted. the published literature on persistence of bronchial hyperreactivity into early adulthood in the population of young adults born prior to routine antenatal glucocorticoid and postnatal surfactant treatment is conflicting: both persistence (halvorsen et al. 2004 ) and resolution (narang et al. 2008 ) of bronchial hyperresponsiveness are reported, although both studies report persistently abnormal baseline spirometry. • measurements of dynamic mechanics are most often obtained in intubated infants, but measurements of dynamic resistance are highly variable and may persistence of bronchial hyperreactivity into adulthood is unknown for the current population of premature infants. véronique nève and francis leclerc a restrictive ventilatory defect is observed when expansion of the lung is restricted because of alterations in lung parenchyma or as a consequence of extraparenchymal diseases affecting pleura, chest wall, or neuromuscular apparatus. chronic ild are characterized by derangements of the alveolar walls and loss of functional alveolar-capillary units. pediatric ild comprises a heterogeneous group of rare disorders with considerable mortality (clement 2004; fan et al. 2004; fan and langston 1993) . chronic ild in immune-competent children has been defined as the presence of respiratory symptoms and/or diffuse infiltrates on chest radiographs, abnormal pulmonary function tests (pft) with evidence of restrictive ventilatory defect and/or impaired gas exchange, and persistence of any of these findings for >3 months (clement 2004) . pft, in children over 6 years, show a reduced forced vc (fvc) and fev 1 and a normal or elevated fev 1 / fvc ratio (fan et al. 2004) (fig. 4.13) . the not be reliable. reasons for variability include the highly frequency-dependent nature of respiratory mechanics, rapidly changing nature of respiratory disease, maturation and the acute effects of antenatal exposures, and postnatal treatments and interventions. • rds is typically characterized by low lung volumes, reduced lung compliance, and elevated tissue resistance. • meconium aspiration is characterized by both increased resistance and decreased compliance. both resistance and compliance are improved by surfactant lavage. mas may be complicated by airway hyperreactivity and gas trapping beyond infancy. • infants with congenital diaphragmatic hernia have impaired airway function at 1 year of age, with evidence of continuing deterioration throughout childhood. worse lung function is likely associated with initial severity of disease and intensity/duration of mechanical ventilation. • bpd infants are typically more tachypneic than their healthy counterparts and have increased work of breathing. airway walls are more compliant and may be prone to collapse. while lung function outcomes for infants with bpd have improved over the last one to two decades, airflow limitation remains an issue for long-term respiratory function after bronchopulmonary dysplasia and may worsen with time. diffusing capacity is also impaired in children and adults born prematurely and may reflect parenchymal disease and impaired alveolarization. • although patients receiving ecmo generally have worse lung function than those treated without ecmo, this is likely due to the initial severity of lung disease and duration of mechanical ventilation rather than an effect of ecmo on long-term lung function per se. to describe flow (f), lung volumes (v), and respiratory pressure (p) measurements together with resistance (r) and compliance (c) measurements in restrictive lung diseases, obstructive lung diseases, and neuromuscular disorders (nmd) decrease in tlc, in general, is relatively less than that of vc because of normal chest wall recoil and inspiratory muscle function in most patients (martinez and flaherty 2006) . functional residual capacity (frc) and residual volume (rv) are normal or elevated resulting in increased frc/tlc and rv/tlc (gaultier et al. 1980; steinkamp et al. 1990; zapletal et al. 1985) . the latter finding suggests air trapping (fan et al. 2004) . airflow limitation has been demonstrated in some studies (fan and langston 1993) . diffusing capacity of carbon monoxide (dl co ), that evaluates the capacity of the lung to exchange gas across the alveolar capillary interface, is low in absolute term but normal when corrected for alveolar v (gaultier et al. 1980; zapletal et al. 1985) . the lung p/v curve is shifted down, and elastic recoil p at maximum inspiration is increased (fan and langston 1993; steinkamp et al. 1990; zapletal et al. 1985) . reduced c l is observed, with lower specific c l in children with fibrotic changes on transbronchial lung biopsy specimen (steinkamp et al. 1990) . the decreased c l and increased elastic recoil can increase the retractive force exerted on the walls of lung airways and reduce the airway r (r aw ) or increase fev 1 % and preserve the peak expiratory f (pef). however, once lung v becomes severely reduced, pef declines because it is then measured at a relatively small lung v (cotes et al. 2006) . pft can aid in establishing disease severity and prognosis. in nonspecific interstitial pneumonia and idiopathic fibrosis, dl co <40 % corresponds to advanced disease and predicts impaired survival. similarly, exertional desaturation <88 % at baseline testing and a decrease in fvc >10 % over the course of the short-term follow-up identify patients at particular risk of mortality (martinez and flaherty 2006) . ali/ards are characterized by an acute onset of respiratory failure, diffuse bilateral pulmonary infiltrates on the chest radiograph, the absence of clinical evidence of left atrial hypertension, and a ratio of pao 2 to fio 2 (p/f) of less than 200 mmhg (regardless of peep). acute lung injury (ali) is a subset of ards with less severe impairment in oxygenation (p/f <300) (bernard et al. 1994) . spo 2 /fio 2 (s/f) may be a good noninvasive surrogate marker for p/f in children: s/f ratios of 263 and 201 have been shown to correspond to p/f ratios of 300 and 200, respectively, the ards cutoff of 201 having 84 % sensitivity and 78 % specificity (bach and bianchi 2003) . decreased c l was associated to these items (ware and matthay 2000) . ali/ards are rare diseases in children and have mortality rates ranging from 18 to 75 % (flori et al. 2005; zimmerman et al. 2009 ). mechanical ventilation represents the main therapeutic support to maintain acceptable pulmonary gas exchange while treating the underlying disease. a "lung protective ventilation strategy" with limitation in airway p and tidal volume (vt) (the acute respiratory distress syndrome network 2000) led, in adult studies, to a reduction in mortality at day 28 and a reduction in hospital mortality (petrucci and iacovelli 2007; the acute respiratory distress syndrome network 2000) . no published data exist for children, but practice in picu is derived from adult patients (khemani and newth 2010 the most characteristic alteration in acute ards lung is marked fall in c l caused by loss of surfactant function, atelectatic lung regions, accumulation of interstitial/alveolar plasma leakage (ware and matthay 2000) , and an associated fall in frc (hammer 2001) . in adults, the decrease in c l was shown to be more important in early ards from pulmonary origin (and associated with normal chest wall c (c cw )) than in extrapulmonary ards. the latter was associated with low c cw (gattinoni et al. 1998) . a decrease in respiratory system compliance (c rs ) and a proportional decrease in tlc, fvc, and frc were also observed in children during the acute phase of ards (hammer et al. 1998; newth et al. 1997) . ali/ards may resolve completely in some patients, while in others it progresses to fibrosing alveolitis with persistent hypoxemia and a further decrease in c l (ware and matthay 2000) . fibroproliferation occurs early in ards, and its extent may be predictive of outcome (marshall et al. 2000) . the recovery phase is characterized by the gradual resolution of hypoxemia and improved c l (ware and matthay 2000) . pharmacologic interventions, such as corticosteroids, starting in the late course of ards may reduce fibrosis and may have a beneficial impact on pulmonary outcome (tang et al. 2009 ). several studies have demonstrated persistent impairment in pulmonary function of unknown long-term significance for children who required mechanical ventilation in pediatric intensive care units for respiratory failure (khemani and newth 2010) . most survivors of ards have persistent mild reductions of dl co even as long as a year after their episode. the lung v and f return to normal in most instances, although a subset of patients will have persistent impairment. both obstructive and restrictive deficits may be seen (alberts et al. 1983; elliott et al. 1981; ghio et al. 1989; hert and albert 1994; peters et al. 1989) . timing of recovery occurring in the first year after ards was described in two prospective studies of adult survivors of ards (herridge et al. 2003; mchugh et al. 1994) . after discharge from the icu, a restrictive ventilatory defect and reduced dl co were observed in almost all patients and an obstructive defect in only 5 %. improvement was observed until 6 months after discharge. a relatively static period was observed after that. by 6 months, abnormalities in fvc, tlc, and dl co were observed in 55, 45, and 26 %, respectively, of patients. patients with more severe ards, as determined by their higher ali scores (corresponding to patients ventilated for >2 weeks), were more impaired for fvc throughout the follow-up and did not return to normal pulmonary function levels (mchugh et al. 1994) . patients of herridge's study were shown to have a mild restrictive defect with mild to moderate reduction in dl co at 3 months. lung v and spirometric measurements were normal by 6 months, but dl co remained low throughout the 12-month follow-up (herridge et al. 2003) . for the pediatric age group, a few small-sized observational cohort studies have reported sequelae in 10-100 %, in mostly asymptomatic subjects (ben-abraham et al. 2002; fanconi et al. 1985; golder et al. 1998; lyrene and truog 1981; weiss et al. 1996) . obstructive (reversible and nonreversible airflow obstruction) and restrictive abnormalities have been observed after discharge. like in adults, recovery during the following months reached plateau levels at 12 months with no further improvement (golder et al. 1998) . decrease in c l was shown to persist in ≥50 % of children and adults, evaluated 1-2 months (aggarwal et al. 2000) , or 34 months after ards (klein et al. 1976) , and was associated with a restrictive defect in some patients. symptomatic upper airway obstruction as a result of laryngotracheal injury from long-term intubation has been described in minority (10 %) of adults after ards with evidence of upper airway obstruction on inspiratory and expiratory f/v curves (elliott et al. 1988 ). the prevalence of childhood obesity is increasing in developed countries. obese children have more respiratory symptoms than their normal-weighted peers, and respiratory-related disorders increase with increasing weight. the prevalence of asthma has also increased in children. both obesity and asthma have their beginnings in early childhood (litonjua and gold 2008) . wheezing, asthma, and obesity seem to be associated (guerra et al. 2002; hancox et al. 2005; schachter et al. 2001 ). however, three large studies (bibi et al. 2004; schachter et al. 2003; wickens et al. 2005) showed a clear increase in symptoms of asthma in association with obesity but not more frequent airway hyperresponsiveness in obese children (deane and thomson 2006) . c cw is reduced in obesity, because of increased adiposity in the abdomen and around the thoracic cage that restricts rib expansion and because the decreased total thoracic and pulmonary v may pull the chest wall below its resting level to a flatten portion of its p/v curve. c cw decrease correlated with co 2 retention, independent of body fat, and with shortness of breath (subramaian and strohl 2004) . c l may be decreased in some obese individuals because of a large pulmonary blood volume and intrinsic alterations of elastic characteristics of lung tissues (subramaian and strohl 2004) . as additional fat in the abdomen raises the diaphragm, the frc and expiratory reserve volume (erv) are reduced in the erected position and further reduced in the supine position (koenig 2001) . the lung bases are poorly ventilated which contributes to hypoxemia. tlc and vc may also be reduced, but rv is usually maintained (therefore rv/tlc ratio may be increased). in morbidly obese individuals, an increase in body mass correlates with reduced fev 1 (carey et al. 1999 ) and fvc (fev 1 / fvc ratio remains normal) (zerah et al. 1993) . data in children and adolescents confirm the reduced frc and static lung v (li et al. 2003) and the decrease in fvc and fev 1 with increasing proportions of body fat as a percentage of body weight (lazarus et al. 1997 ). all these effects were shown to be reversible in morbidly obese patients (with body mass index (bmi) >40) following weight loss (camargo et al. 1999; carey et al. 1999; deane and thomson 2006) . in seated patients with simple morbid obesity, inspiratory and expiratory muscle strength is normal (yap et al. 1995) . when patients are supine, maximal inspiratory p (pimax) decreases by about half as a result of overstretching of the diaphragm, causing it to operate on the descending limb of its length-tension curve (laghi and tobin 2003) . morbidly obese patients without hypoventilation (simple obesity) compensate for the respiratory load by increasing respiratory drive and diaphragmatic pressure output (pankow et al. 1998 ) and increasing rib cage contribution to tidal breathing. scoliosis is the most common abnormality of the spine with direct effect on the thoracic cage. idiopathic scoliosis (including infantile, juvenile, and adolescent) accounts for 80-85 % of scoliosis. neuromuscular scoliosis (as in duchenne muscular dystrophy (dmd) or spinal muscular atrophy (sma)) and scoliosis associated with congenital vertebral abnormalities account each for 5 % of scoliosis. infantile and congenital scoliosis, untreated, results in severe spinal deformities and is at risk of cardiopulmonary insufficiency before adulthood (canet and bureau 1990) . patients with scoliosis and a cobb angle <70° are usually asymptomatic, those with an angle between 70° and 100° often experience dyspnea on exertion, and those with the angle >100° are at risk for chronic respiratory failure (estenne et al. 1998) . scoliosis can produce severe reductions in lung v and restrictive ventilatory defect with decrease in fvc, fev 1 , and tlc as well as a normal fev 1 /fvc ratio (gagnon et al. 1989; muirhead and conner 1985; upadhyay et al. 1993; weber et al. 1975 ). among patients with moderate to severe scoliosis (35-100°), the vc decrease is related to the cobb angle. when the cobb angle exceeds 100° (severe scoliosis), vc falls to about 50 % of predicted (laghi and tobin 2003) . impaired lung growth (davies and reid 1971) , significant decrease in c l and c cw (at about 25-50 % of predicted in children and adults with scoliosis) (laghi and tobin 2003) , and/or impaired muscle strength as a consequence of inefficient coupling between the respiratory muscles and the thoracic cage account for the effect of scoliosis on lung function. in adults with scoliosis and cobb angles of 66-136°, pimax was about 50-76 % (estenne et al. 1998; laghi and tobin 2003) . decreased tlc is often associated with increased rv resulting in very high rv/tlc ratio (day et al. 1994) , probably reflecting the dysfunction of expiratory muscles, which do not allow full exhalation. significant gas trapping with elevated plethysmographic frc/helium frc ratio can occur with response to bronchodilators, indicating airway hyperresponsiveness that results from chronic airway inflammation secondary to the poor clearance of secretions (boyer et al. 1996) . the chest distortion causes airway distortion thereby contributing to a slight increase in airway r (r aw ) (canet and bureau 1990) . scoliosis, by inducing significant displacement/rotation of the intrathoracic trachea and/or main stem bronchi, can cause mechanical obstruction as shown on the f/v loop with flattening of the initial portion of the expiratory loop suggesting central airway obstruction (borowitz et al. 2001) . the decreased c rs may account for the increased work of breathing, with increased transdiaphragmatic pressure during tidal breathing; increased rib cage contribution to tidal breathing; transversus abdominis recruitment during exhalation, as observed in most patients with severe scoliosis (laghi and tobin 2003) ; and the associated increased risk of respiratory muscle fatigue and eventual respiratory failure. in contrast to idiopathic scoliosis which tends to stabilize (or progress at a lower rate) after the person reaches skeletal maturity, neuromuscular scoliosis (such as in dmd or sma) continues to worsen because of the progressive worsening of the muscle weakness. recurrent aspiration and pneumonias secondary to impaired clearance of airway secretions are potential complications. due to the severity of their primary muscle weakness, patients with neuromuscular scoliosis may not be able to maintain adequate ventilation and may develop severe atelectasis (koumbourlis 2006) . scoliosis is reported in 60-100 % of sma children and is due to the inability of the trunk muscles to support spine in the upright position. all children with type 2 sma develop scoliosis starting from around 3 years. the average curve is more than 54° at 10 years of age (mullender et al. 2008) . as the scoliosis progresses, respiratory function deteriorates, and the risk of life-threatening complications increases (rodillo et al. 1989) . spinal fusion is recommended when scoliosis progresses and reaches a cobb angle of between 40° and 60° in children ≥10 years of age (tsirikos and baker 2006) . in dmd, scoliosis incidence ranges from 75 to 95 % (muntoni et al. 2006) . dmd develop scoliosis after losing independent ambulation (in the second decade of life). once scoliosis reaches 30°, it progresses with age and growth. corticosteroids may delay the development and progression of scoliosis in dmd (muntoni et al. 2006) . optimal timing for surgical intervention is while lung function is satisfactory and before cardiomyopathy becomes severe enough to risk arrhythmia under anesthesia (cobb angle between 30° and 50°). best prognosis for recovery seems to be fvc >40 %, although others use the absolute vc of <1.9 l as an indicator of rapid progression of scoliosis and poor prognosis (finder et al. 2004) . comparison between preand postoperative pulmonary function reveals no improvement due to correction of scoliosis. long-term studies show that decline of pulmonary function in dmd patients is unchanged in operated patients compared to patients who had no surgery (muntoni et al. 2006 ). asthma is the most frequent chronic disease observed in children (prevalence ranging from 8 to 12 % (isaac 1998)). asthma is difficult to diagnose in children ≤5 years. children presenting with wheeze at an early age belong to a heterogeneous group: early transient, late onset, and persistent wheezers (martinez and helms 1998) . half of the "early" wheezing children become asymptomatic by school age. they may have diminished lung function early on, but by 6 years lung function is improved (martinez et al. 1995) . children who had wheezing that began in infancy and continued at 6 years demonstrated normal pulmonary function initially with reduced lung function by 6 years. those children with "persistent wheezing" have some ongoing chronic inflammatory process that results in airway alterations and some loss of lung function in early childhood, which extends to varying degrees in adulthood (oswald et al. 1997 ). respiratory syncytial virus (rsv) bronchiolitis in infancy is often associated with recurrent wheezing and asthma during subsequent years. however, wheezing tends to diminish by school age or adolescence. most follow-up studies of rsv bronchiolitis in infancy show that forced expiratory flow (fef) rates and fev 1 are lower at school age compared with control groups (hall et al. 1984; pullan and hey 1982) . children who had been hospitalized for rsv bronchiolitis as infants later had lung function abnormalities similar to those found in children with asthma (kattan et al. 1977; wennergren and kristjansson 2001) . increased r to airflow is the basis of the clinical manifestations of asthma, including dyspnea and wheeze. usually, airflow limitation is reversible. fixed airway obstruction may be seen in later disease stage (strachan et al. 1996) or be a component of a specific asthma phenotype (bush 2004) . airway obstruction in childhood is associated with a reduced fev 1 in adulthood (jenkins et al. 1994; roorda et al. 1994) . airflow obstruction (afo) can be assessed by body plethysmography, spirometry, forced oscillation technique (fot), and interrupter technique, but these are more difficult to perform in children <6 years. incentive spirometry can be performed in the majority of children ≥3 years (beydon et al. 2007b ) together with frc helium measurement and r measurement (using interrupter technique (r int )) (beydon et al. 2007b) , fot, or plethysmography for measurement specific r aw (sr aw ) (dab and alexander 1976) . infant pfts are mainly research tools. the fev 1 is the "gold standard" of measuring airway obstruction in children over 6 years (miller et al. 2005; pellegrino et al. 2005) . preschoolers often do not exhale for more than 1 s. therefore fev 1 may not be an accurate index of bronchial obstruction in this age group, and the utility of fev 0.5 or fev 0.75 as outcome measure in this age group has been explored (aurora et al. 2004; beydon et al. 2007b; neve et al. 2006; vilozni et al. 2005) . international guidelines (nhlbi 2002) recommend an initial evaluation and regular reeval-uation for the assessment of the severity and the control of asthma. asthma severity describes the underlying disease state as evaluated by fev 1 and daytime and nighttime symptoms, all measured before treatment. patients with moderate and severe persistent asthma are said to have values of 60-80 % and <60 % of predicted, respectively. fev 1 may not be the best measure of severity in childhood asthma because most asthmatic children have fev 1 values in the normal range independent of disease severity when clinically stable (spahn et al. 2004) . diminished fev 1 values in school-age children should identify children at risk of fixed airway obstruction at adulthood (rasmussen et al. 2002) . for children, fev1/fvc appears to be a more sensitive measure of severity in the impairment domain (nhlbi 2007) a concave shape on the f/v curve may be observed as a result of small airway obstruction (pellegrino et al. 2005) . it may be observed in asthmatic children with a normal fev 1 , together with a significant improvement following administration of a bronchodilator (basek et al. 2005; brand and roorda 2003) . a concave shape is associated with impairment in the fef between 25 and 75 % of fvc (fef 25-75 % ) that is believed to measure peripheral airway obstruction. fef 25-75 % is among the first parameters to be abnormal in pediatric asthma and often the most significantly impaired of all spirometric measures (paull et al. 2005; spahn and chipps 2006) . a fef 25-75 % ≤65 % is well correlated with bronchodilator responsiveness in asthmatic children with normal fev 1 and may suggest suboptimal asthma control (simon et al. 2010) . plethysmography may show lung distension/air trapping with larger rv and tlc in children than in adults (jenkins et al. 2003) . the rv is a sensitive parameter of airway obstruction in children, and a decrease in rv after bronchodilator administration appears to be specific for asthma diagnosis (walamies 1998) . air trapping may be observed when fvc and fev 1 values are normal in mild or moderate asthma (cooper et al. 1977) or in controlled asthma (vilozni et al. 2009 ). indeed, the principal event taking place in the asthmatic lung is the closure of small airways with increase in rv that is associated to a protective increase in tlc to preserve the functional range of fvc. once the limit of the chest wall expansion has been reached, further increase in rv will result in falls in both fvc and fev 1 (irvin and bates 2009) . frc may be elevated, due to airway closure and/or dynamically elevated with an increased expiratory time constant (stanescu 1999) . it is a compensatory mechanism that minimizes the increase in r aw and the expiratory flow limitation in obstructive airway disease. hence, at an early stage of the disease, an isolated increase in lung v may be the sole functional abnormality (landau et al. 1979; paton 2000) . in asthmatic children, frc increases with severity of asthma (greenough et al. 1987) and is more elevated in symptomatic children ). trapped air is associated with hypoxemia (wolf et al. 1983 ) but correlates poorly with f limitation and may be lacking in patients with severe asthma (basek et al. 2005; desmond et al. 1986 ). in preschool children with asthma, a 6-week inhaled corticosteroid therapy is associated with reduced hyperinflation as indicated by lower frc helium . bronchodilators were shown to decrease helium frc in 80 % of children aged 2-7 years, and the decrease was correlated to baseline frc. some children exhibited an increase in frc after bronchodilation when baseline value was low (greenough et al. 1989) . children higher baseline r int measurement was shown in young preschool asthmatics (beydon et al. 2003; nielsen and bisgaard 2001) and children with a history of wheeze (mckenzie et al. 2000) compared with healthy children. children with persistent wheeze were shown to have higher r int value than those with transient or no previous wheeze, but there was an overlap in r int values between the three groups (brussee et al. 2004 ). using fot, increased r of the respiratory system at 5 hz has been reported in asymptomatic asthmatic preschool children as compared with healthy subjects in some studies (nielsen and bisgaard 2001) . plethysmographic specific r aw (sr aw ), product of r aw , and plethysmographic thoracic gas volume (tgv) may be altered by both hyperinflation and decreased airway diameter. in preschool children, it was shown to be increased in children with a history of wheeze (lowe et al. 2002) ; it was able to identify responses to bronchodilators (nielsen and bisgaard 2001) and inhaled steroids (nielsen and bisgaard 2000) . sr aw was more strongly related to fef after 50 % of fvc has been exhaled (fef 50 % ) than to fev 1 and could be used in preschool children to predict mild airflow limitation (mahut et al. 2009 ). however, there is no established cutoff for sr aw to separate healthy subjects from asthmatics (marchal and schweitzer 2005) . in cf, the combination of mucus retention, bacterial infections, and inflammation results in obstructive lung disease primarily involving the small airways (bedrossian et al. 1976; fox et al. 1974; ratjen and grasemann 2005) . restrictive alterations develop secondary to the damage of lung parenchyma by inflammatory changes and by neutrophil degradation products. chronic airway infection, progressing to bronchiectasis, gas trapping, and hypoxemia and hypercarbia, is the hallmark of cf lung disease. pulmonary insufficiency is responsible for at least 80 % of cf-related death (cystic fibrosis foundation 2008). fev 1 is the gold standard for lung function in cf. fev 1 reflects the progression of pulmonary disease and correlates with mortality. an fev 1 of less than 30 % predicted in conjunction with other clinical indicators is used for selecting suitable candidates for lung transplantation (kerem et al. 1992) . in cf, obstruction primarily affects the small airways. an early change associated with airflow obstruction in small airways is a concave shape on the f/v curve and a reduction in the fef 25-75 % (pellegrino et al. 2005 ) such as reported in cf patients (corey et al. 1997; 1976 ). this fef often shows abnormalities in patients in whom fev 1 is in the normal range. however, the large interindividual variability of this fef must be taken into account in its interpretation. diminished fevs and fefs have also been reported in sedated infants with cf (including infants considered to be asymptomatic by their physician) using the raised volume rapid thoracoabdominal compression technique (ranganathan et al. 2002; and in preschool children using incentive spirometry (kozlowska et al. 2008; marostica et al. 2002; vilozni et al. 2007 ). other techniques detecting small airway obstruction afo in small airways is associated with lung hyperinflation as indicated by increased rv, rv/ tlc, and tlc. a small airway obstruction syndrome with decreased vc and fev 1 increased rv, but a normal fev 1 /vc ratio and tlc has also been described (stanescu 1999) . the condition is attributed to premature closing of airway leading to air trapping as demonstrated by high-resolution computed tomography (hrct) scans (cotes et al. 2006) . studies using body plethysmography have provided evidence for trapped gases in the majority of patients with cf (beier et al. 1966) . the ratio of rv to tlc that indicates hyperinflation, a key feature of cf lung disease, has been found to correlate with disease severity in numerous studies (beier et al. 1966; landau et al. 1979; landau and phelan 1973a) . percentage of children with hyperinflation and trapped gas increases with age, and children with severe hyperinflation at 6-8 years showed the most severe disease progression over time (kraemer et al. 2006) . in children <6 years, frc is the only lung volume that can be measured routinely. hyperinflation is one of the earliest features of cf (gappa et al. 2001) . elevated tgv has been found in cf infants (beardsmore et al. 1988) , and hyperinflation has been detected by the helium dilution technique in preschool children (beydon et al. 2002) . multiplebreath washout methods (mbw) may be particu-larly sensitive to changes in peripheral lung function and detect early functional abnormalities in infant and preschool children with cf (aurora et al. 2005; ranganathan et al. 2008) . frequency dependence of lung c as indicative afo in small airways has been observed in the majority of cf, but the invasive nature of this technique limits its clinical use (landau and phelan 1973a) . alterations in lung elastic recoil were found with variable frequency (cook et al. 1959; landau and phelan 1973a; mansell et al. 1974 ). as the airway disease becomes more advanced and/or more central airways become involved, fev 1 will be reduced out of proportion to the reduction in vc and an obstructive ventilatory defect be observed. severity of lung disease in cf is classified by the degree of impairment in fev 1 (with fev 1 < 50 % of predicted having severe disease) (pellegrino et al. 2005 ), (fig. 4.14) . the obstructive ventilatory defect is reversible if an improvement in fvc and/or fev 1 of at least 12 % of baseline after betaadrenergic agents is observed (pellegrino et al. 2005 ). significant reversibility can be observed in 50-60 % of cf patients, and 10-20 % show reduced lung function in response to short-acting inhaled bronchodilators (brand 2000; landau and phelan 1973b; shapiro et al. 1976 ). studies in cf have shown decreased c and increased r (cook et al. 1959 ). due to the large surface area of small airways, their contribution to r aw is relatively small. r aw measurements are therefore normal in many patients with cf until they develop disease involving the larger airways (landau and phelan 1973a) . as the disease progresses, bronchial obstruction leads to tissue destruction with bronchiectasis and the development of areas of pulmonary emphysema and fibrosis. in more advanced disease, the destruction of lung tissue leads to decreased c that parallels the decline in fev 1 (hart et al. 2002) . this increases respiratory load and favors a rapid and shallow breathing pattern that further impairs gas exchange in cf patients. the loss of lung v that develops in patients due to tissue destruction as well as the hyperinflation decreases fvc: therefore fev 1 /fvc ratio may be normal even in patients with severe disease (landau and phelan 1973a) . a small proportion of cf patients also develop a restrictive lung disease with decreased plethysmo(ries et al. 1988 ). pulmonary disease resulting from a neonatal respiratory disorder is called chronic lung disease of infancy (cld). bronchopulmonary dysplasia (bpd) (defined as the need for supplemental oxygen for at least 28 days after birth) accounts for the vast majority of cases of cld. what is now considered the "old" bpd was originally described in slightly preterm newborns who had been exposed to aggressive mechanical ventilation and high concentrations of inspired oxygen. diffuse airway damage, smooth muscle hypertrophy, neutrophilic inflammation, and parenchymal fibrosis reflected extensive disruption of relatively immature lung structures. the "new" form of bpd is interpreted as a developmental disorder: despite being delivered several weeks before alveolarization begins, these infants often have only mild respiratory distress syndrome at birth, but lung development is affected with interruption of alveolarization at a very early stage with subsequent alveolar-capillary hypoplasia (baraldi and filippone 2007b ). most of the information on long-term lung function in survivors of bpd refers to patients who had the condition before surfactant treatment was available ("old" form of bpd). in the first months of life, survivors of bpd are severely affected. infants with bpd at 28 days of age have increased total and expiratory r and severe f limitation especially at low lung v (hazinski 1990 ). neonates and young infants with cld have increased levels of frc by plethysmography and decreased frc values by nitrogen washout, suggesting the presence of trapped air (wauer et al. 1998) . specific dynamic c (c dyn ) may be reduced by more than 50 % (allen et al. 2003; gerhardt et al. 1987b ) by small airway narrowing but also by interstitial fibrosis, edema, and atelectasis (hazinski 1990 ). lung mechanics measurement that reflects the severity of neonatal disease (i.e., c rs at 10-20 days) has been shown to correlate with subsequent reduction in lung function in 2-year-old bdp (baraldi et al. 1997b; bhandari and panitch 2006) . childhood during infancy and early childhood, an improvement of airway physiology is observed, and c l improves over time (baraldi et al. 1997b; bhandari and panitch 2006; gerhardt et al. 1987b ). maximum flow rates at frc were reported to increase significantly within the first 2 years along with a reduction in r aw (farstad et al. 1995; trachsel and coates 2005) . however analysis of fef shows that substantial afo persists in numerous survivors of bdp and in preterm infants without bpd during the first 3 years of life (baraldi et al. 1997b; tepper et al. 1986b ). the degree of airflow limitation in the first years of life seems to predict later pulmonary function (at 8 years) suggesting tracking of lung function with time, negligible "catch-up" growth of the lung, and irreversible early airway-remodelling process (baraldi and filippone 2007b) . body plethysmography studies in school children and adolescents generally reveal normal tlc with hyperinflation as reflected by an increased rv and rv/tlc ratio. consistently children diagnosed with bpd are more prone to hyperinflation than prematurely born children without bpd. frc is normal or mildly increased. differences between frc determined by helium dilution technique and by plethysmography may indicate the presence of trapped air (trachsel and coates 2005) . at the age of 8 years, c dyn was reported to be 57 % of the control group, in bpd, and 74 % in prematurely born children without cld (parat et al. 1995; trachsel and coates 2005) . school-age children with a history of bpd have lower fev 1 than children born at term or those born prematurely without lung disease. they also have a lower fvc and fev 1 /fvc ratio than children born at term (allen et al. 2003; bhandari and panitch 2006) . data from 18 studies in children, adolescents, and young adults show that spirometric values are consistently lower in survivors of bpd, at any age between 6 and 20 years, than in controls born at term with fev 1 ranging from normal to severely decreased values (baraldi and filippone 2007b) . patients who were born prematurely but did not have bpd usually fare better, but they too may have airflow limitation at school age and later (baraldi and filippone 2007b) . asthma-like symptoms are often associated to spirometric evidence of airflow limitation in children who had bpd as infants. but airflow limitation is only partially reversed by β2-agonists in children suggesting a stabilized remodelling process. hrct studies have documented scattered parenchymal fibrosis and architectural distortion in many survivors of bpd, findings that are unusual in children with asthma (baraldi and filippone 2007b) . there is no evidence that children with bpd born since the introduction of surfactant-replacement therapy (i.e., "new" bpd) have better spirometric results at school age than those born in the pre-surfactant era (i.e., "old" bpd) as shown in studies evaluating cohort of infants weighting less than 1,000 g at birth or who were born before a gestational age of 29 weeks (doyle 2006; halvorsen et al. 2006) . these results suggest that prematurity itself has a very important independent influence on the long-term respiratory prognosis (baraldi and filippone 2007b) . hematopoietic stem cell transplantation (hsct) is an established therapy for many chemosensitive or radiosensitive malignancies in children. bo is the most common late noninfectious pulmonary complication (i.e., that presents after the first 100 days following transplantation) with 80 % of cases occurring between 6 and 12 months. the reported incidence range is 0-48 % (soubani and uberti 2007) . bo seems to be less common in children than in adults (cerveri et al. 2005) . the most important association with bo is chronic graft versus host disease (gvhd), especially progressive chronic gvhd which evolves without hiatus from active acute gvhd. most cases of bo are thought to be secondary to bronchial mucosal damage from gvhd with inflammation of the small airways and subsequent obliteration. the main symptoms associated with bo are dry cough, dyspnea, and wheezing. twenty percent of patients are asymptomatic. spirometry is the main tool to diagnose and follow up patients with bo following hsct. based on the experience of lung transplantation, a "bo syndrome" has been defined by pft rather than histology (estenne et al. 2002) . it was suggested that the diagnosis of bo is made when there is evidence of (1) new onset of afo with reduction in fev 1 < 75 % of predicted with a fev 1 /fvc < 0.70 not responsive to bronchodilators; (2) air trapping or small airway thickening or bronchiectasis on hrct of the chest with inspiratory and expiratory cuts with nonparenchymal involvement, rv on pft > 120 % of predicted or pathological confirmation of constrictive bronchiolitis; and (3) absence of infection in the respiratory tract documented by clinical symptoms, radiological studies, or microbiological cultures (soubani and uberti 2007) . there are some studies that suggested that a reduction in mean fef 25-75 % may precede the decline in fev 1 and is a sensitive but not specific indicator of subsequent development of bo (estenne et al. 2000; ouwens et al. 2002; patterson et al. 1996; reynaud-gaubert et al. 2000) . a pediatric study defined afo as fev 1 < 80 % and fef 25-75 % < 60% of predicted (schultz et al. 1994) . since the evolution of the disease despite treatment is particularly negative with a progressive worsening of respiratory function and a high mortality, early diagnosis is important. this can be achieved by systematic regular monitoring of the respiratory function starting from the onset of acute gvhd (cerveri et al. 2005 ). decrease in vc is closely linked to weakness of respiratory muscles and disease progression. vc below 40 % predicts nocturnal alveolar hypoventilation, and vc <25 % or <1 l, in duchenne patients, is strongly associated with respiratory failure. in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory pressures. in patients with neuromuscular disorders, respiratory failure may present either acutely as a result of pneumonia or more slowly, as a result of progressive ventilatory decompensation. dmd and sma account for the majority of patients seen in pediatric practice. the most frequently noted abnormality of lung v in patients with respiratory muscle weakness is a reduction in vc. vc can be measured in cooperative children, usually older than 6 years. vc reflects the strength of both inspiratory and expiratory muscles but is not specific, as it can be reduced by other factors than muscle weakness, such as reduction in both c l (gibson et al. 1977) and c cw in patients with chronic respiratory muscle weakness (de troyer et al. 1980) . the decreased c cw probably results from stiffening of tendons and ligaments of the rib cage and ankylosis of the costosternal and thoracovertebral joints (laghi and tobin 2003) . a decrease in c l could be related to diffuse microatelectasis in a few patients (estenne et al. 1993) . in children younger than 4 years with nmd, higher c cw normalized to body weight than in controls has been reported (papastamelos et al. 1996) . such a high c cw results in chest wall deformation during tidal breathing and excessive work of breathing. it also predisposes to atelectasis and can result in fixed deformation of the chest wall (i.e., pectus excavatum). absence of lung stretch with sigh breaths and chest wall deformities can also result in reduced lung growth for children with nmd (bach and bianchi 2003; panitch 2009 ). in patients with nmd, a decrease in vc is an early sign of respiratory impairment. decrease in vc and tlc is closely linked to a weakness of respiratory muscles (braun et al. 1983; hahn et al. 1997; ragette et al. 2002) and disease progression (inkley et al. 1974; samaha et al. 1994 ). in patients with advanced nmd, a restrictive ventilatory defect is observed with tlc reduction. in dmd, a typical evolution of lung v is observed (hahn et al. 1997) . in ambulatory dmd the lung v increases with age (ascending phase), and predicted v is almost normal (tangsrud et al. 2001) . with loss of ambulation between 7 and 12 years (gozal 2000) , measured vc remains stable (plateau phase), but % predicted values begin to decline. in the following years, vc declines by about 200 ml or 6-8 % per year (descending phase) phillips et al. 2001; rideau et al. 1981) . in sma, there is a characteristic pattern of involvement with intercostal muscle weakness and relative sparing of the diaphragm (kuzuhara and chou 1981; perez et al. 1996) . the rib cage is neither stabilized nor expanded during inspiration. over a period of time, the retraction of the rib cage has a detrimental effect on alveolar development. in type 1 sma, with disease beginning before birth, pulmonary hypoplasia has been described (cunningham and stocks 1978) . the best parameter to monitor respiratory muscle strength in children with sma over 6 years is fvc (% predicted) (manzur et al. 2003) . in sma, lung v (% predicted) decreases with age, with a greater vc decrease in type 2 than in type 3 sma. a decline in vc from 87 to 73 % was shown to occur in type 3 sma from 12 to 18 years (souchon et al. 1996) . the fvc may therefore be normal or near normal in stronger ambulant type 3 sma (samaha et al. 1994) . in type 2 sma, fvc is more severely impaired and was shown to decline from 55 % predicted to 37 %, from 10 to 16 years, with a yearly average decline by 2-5 % between 7 and 15 years of age (barois et al. 2005; herridge et al. 2003; souchon et al. 1996) . a restrictive pattern was observed in a majority (70 %) of type 2 sma children followed longitudinally . in intermediate type 1 sma, severe impairment in fvc has also been described (barois et al. 2005) with progressive decrease in fvc from 60 % predicted (at 7 years) to 16 % (at 15 years) in children without tracheotomy and from 22 % predicted (at 7 years) to 14 % (at 15 years) in children with tracheotomy (ioos et al. 2004 ). progression of scoliosis seems to contribute to vc decline in children with nmd (miller et al. 1988 ), but treatment of scoliosis with spinal stabilization did not prevent further vc decline in patients with dmd (kennedy et al. 1995; miller et al. 1988 ). in type 2 sma, beneficial effects of spinal surgery on pulmonary function remain controversial (mullender et al. 2008; wang et al. 2007 ), but the rate of pulmonary function decline may be slowed (wang et al. 2007 ). static lung v may also be affected in some nmd patients by coexistent lung or airway disease (american thoracic society, european respiratory society 2002). measurement of postural change in vc gives a simple index of weakness of the diaphragm relative to the other inspiratory muscles. a fall of 30 % or more in the supine compared with the erect posture is generally associated with severe diaphragmatic weakness (american thoracic society, european respiratory society 2002). studies in adults with generalized nmd suggest that supine vc is a simple, sensitive, and specific test for diaphragm weakness and can replace invasive diagnostic tests (fromageot et al. 2001; lechtzin et al. 2002) . thresholds of vc have been identified to predict treatable complications and outcome in patients with nmd. a vc below 60 % is a sensitive and specific predictor of the onset of sleep-disordered breathing; vc below 40 % predicts nocturnal alveolar hypoventilation, and vc < 25 % or <1 l, in dmd, is strongly associated with respiratory failure and poor survival unless patients are treated with mechanical ventilation (baydur et al. 1990; canny et al. 1989; hukins and hillman 2000; mellies et al. 2003; phillips et al. 2001 phillips et al. , 1999 ragette et al. 2002; simonds et al. 1998; wallgren-pettersson et al. 2004) . rv is usually normal or increased, the latter, particularly with marked expiratory weakness (kreitzer et al. 1978) . consequently, tlc is less markedly reduced than vc, and the rv/tlc and frc/tlc ratios are often increased without necessarily implying airway obstruction (american thoracic society, european respiratory society 2002). a hypodynamic type of ventilatory defect with increased rv, low vc, and normal tlc can be observed early in the course of nmd. such a presentation has recently been described in patients with dmd (tangsrud et al. 2001) . these children may later exhibit chest wall deformities leading to a true restrictive syndrome. r aw is normal in uncomplicated respiratory muscle weakness. the maximum expiratory and maximum inspiratory f/v curves characteristically show a reduction in those f that are most effort dependent, that is, maximum expiratory f at large lung v (including pef) and maximum inspiratory f at all lung v. with severe expiratory weakness, an abrupt fall in maximum expiratory f is seen immediately before rv is reached (vincken et al. 1987) . oscillations of maximum expiratory and/or inspiratory f, the so-called sawtooth appearance, are seen particularly when the upper airway muscles are weak and in patients with extrapyramidal disorders (american thoracic society, european respiratory society 2002; vincken and cosio 1989) . in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory p measurement. vc is normal, or only minimally reduced, if respiratory muscle strength is more than 50 % of predicted (laghi and tobin 2003) . this finding results from the sigmoid shape of the p/v relationship of the respiratory system. several invasive and noninvasive tests to assess respiratory muscle strength have been reported to be of value in testing respiratory muscle strength in patients with nmd. in children, normative data are only available for pimax, pemax, and sniff nasal inspiratory pressure (snip). pimax and pemax are tests of global inspiratory and expiratory muscle strength. they were found to be reduced in nmd patients (baydur 1991; black and hyatt 1971) . the pimax decline was related to the decline of inspiratory reserve volume and tlc, and the pemax decline was related to the decline of erv and the increase of rv in children and adults with dmd (hahn et al. 1997) . in dmd, earlier in the course of the disease, inspiratory muscle strength evaluated by pimax remained relatively well preserved (as compared with pemax) implying relative sparing of the diaphragm (hahn et al. 1997; mcdonald et al. 1995) . in patients with diaphragm weakness, a vc decline of 25 % or more following supine positioning is associated with a mean pimax decline of 18 ± 14 % (ragette et al. 2002) . sleepdisordered breathing usually develops when pimax is less than 45 cm h 2 o. diurnal hypercapnia is likely when respiratory muscle strength falls to 35 cm h 2 o (ragette et al. 2002) . the sniff is a natural maneuver which many children find much easier to perform than pimax. inspiratory muscle strength can easily be assessed by snip in children with nmd (stefanutti et al. 2000) . a recent report comparing snip and pimax in 241 patients with nmd found that the values of pimax were at least the same or even greater than the snip, particularly in patients with severe ventilatory restriction. this can be explained by the fact that patients with severe neuromuscular disorders may not be able to perform a rapid sniff maneuver owing to significant muscle atrophy (hart et al. 2003) . the reduction of pemax is the first sign of respiratory muscle dysfunction in dmd children (hahn et al. 1997; mcdonald et al. 1995) . expiratory muscles that contribute to pemax are primarily the abdominal muscles, and their strength normally exceeds that of inspiratory muscles (american thoracic society, european respiratory society 2002). therefore pemax < pimax indicates prevailing expiratory muscle weakness, a characteristic for children with type 2 and 3 sma . recurrent chest infections may therefore occur early in sma children due to a predominance of expiratory muscle weakness with insufficient cough and retention of airway secretions. in patients with muscular dystrophies, pemax >45 cm h 2 o has been found to be necessary for an effective cough (mellies and dohna-schwake 2005; mellies et al. 2001; szeinberg et al. 1988) . assessment tools to measure the different components of cough include also inspiratory vc (ivc) and the peak expiratory flow or peak cough flow (pcf). expiratory muscle weakness is often associated with a decrease in pcf and erv. the effect of coughing can be visualized on the maximum expiratory f/v curve in healthy subjects as a transient f exceeding the maximum achieved during forced expiration. the absence of such supramaximal f transients during coughing presumably results in impaired clearance of airway secretions and is associated with more severe expiratory muscle weakness (american thoracic society, european respiratory society 2002; polkey et al. 1998) . patients who could not generate peak f transients had significantly reduced pef, fvc, and pemax values ≤45 cm h 2 o (szeinberg et al. 1988) . impaired coughing leads to mucus retention, atelectasis, and recurrent pneumonia. in adults with nmd, pcf below 160-200 l/min is associated with insufficient clearance of airway secretions (bach and saporito 1996) . pcf (below 160 l/) and ivc <1.1 l seem also to be able to identify children with nmd at high risk for severe chest infections (dohna-schwake et al. 2006) . baseline pcf measurements above 160 l/ min, however, do not guarantee adequate airway clearance, because respiratory muscle function can deteriorate during respiratory infections (labanowski et al. 1996) . for this reason a peak cough expiratory flow rate of 270 l/min has been used to identify patients who would benefit from assisted cough techniques (finder et al. 2004; laroche et al. 1988) . in dmd, the likelihood of having a pcf value <270 l/min has been shown to rise significantly when fvc is < 2.1 l (gauld and boynton 2005) . the illustrations presented in this paper were captured with the jaeger program 5.20.0.52 (viasys healthcare, höchberg, germany). • a restrictive ventilatory defect of pulmonary origin is usually associated with a decrease in lung c (c l ) that can be due to an increase in the quantity of interstitial tissue in the lung, like in interstitial lung disease (ild), pulmonary fibrosis, infiltration, or edema. acute lung injury (ali) and acute respiratory distress syndrome (ards) result from pulmonary edema and inflammation. • restrictive defect that arises in the chest wall as a consequence of severe obesity or disease process affecting the ribs or the vertebral column, such as kyphoscoliosis, is associated with reduction in c cw . • in restrictive diseases, expansion of the lung is restricted because of alterations in lung parenchyma or as a consequence of extraparenchymal diseases affecting pleura, chest wall, or neuromuscular apparatus. a restrictive ventilatory defect is observed in these diseases with reduction in total lung capacity (tlc) and normal forced expiratory volume in 1 s (fev 1 )/ vital capacity (vc) ratio. • obstructive diseases of the lung are extremely common. airflow limitation is a functional consequence of asthma and chronic obstructive pulmonary disease (chronic bronchitis, emphysema), cystic fibrosis (cf), bronchiectasis, and bronchiolitis. airflow limitation is also observed in bronchiolitis obliterans (bo). an obstructive defect is defined by a reduced fev 1 / vc ratio. severity of lung function impairment is based on fev 1 % of predicted (fev 1 < 50 % corresponding to severe impairment). airflow obstruction (afo) is often reversible in asthma with improvement in fev 1 and/or fvc ≥12 % of baseline ( fig. 4.15) . afo in small airways is suspected from a concave shape on the fig. 4.15 in cystic fibrosis, as the airway disease becomes more advanced and/or more central airways become involved, fev 1 will be reduced out of proportion to the reduction in vital capacity (vc) and an obstructive ventilatory defect be observed with a reduced fev 1 /vc ratio. a fev 1 < 50 % predicted corresponds to severe impairement of lung function expiratory f/v curve. afo in small airways is associated with lung hyperinflation. in children with asthma an isolated increase in lung volume may be the sole functional abnormality. hyperinflation is one of the earliest features of cf. • decrease in vc is closely linked to weakness of respiratory muscles and disease progression. vc reflects the strength of both inspiratory and expiratory muscles, but it can be reduced by other factors than muscle weakness (as decreased c l and c cw ). a fall of 30 % or more in the supine compared with the erect posture is associated with severe diaphragmatic weakness. vc below 40 % predicts nocturnal alveolar hypoventilation, and vc <25 % or <1 l, in duchenne patients, is strongly associated with respiratory failure. in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory pressures (pimax, pemax, sniff nasal inspiratory pressure (snip)). analysis of static pulmonary mechanics helps to identify functional defects in survivors of acute respiratory distress syndrome the outlook for survivors of ards statement on the care of the child with chronic lung disease of infancy and childhood ats/ers statement on respiratory muscle testing lung function and respiratory health in adolescents of very low birth weight nasal reflexes the mechanics of breathing in children with acute severe croup quality control for spirometry in preschool children with and without lung disease multiple-breath washout as a marker of lung disease in preschool children with cystic fibrosis prevention of pectus excavatum for children with spinal muscular atrophy type 1 criteria for extubation and tracheostomy tube removal for patients with ventilatory failure. a different approach to weaning modulation of upper airway muscle activities by bronchopulmonary afferents lungfunction tests in neonates and infants with chronic lung disease: tidal breathing and respiratory control growth of lung parenchyma in infants and toddlers with chronic lung disease of infancy chronic lung disease after premature birth pulmonary function until two years of life in infants with bronchopulmonary dysplasia spinal muscular atrophy. a 4-year prospective, multicenter, longitudinal study (168 cases) respiratory functions of the larynx interrupter resistance elucidated by alveolar pressure measurement in open-chest normal dogs respiratory muscle strength and control of ventilation in patients with neuromuscular disease decline in respiratory function and experience with long-term assisted ventilation in advanced duchenne's muscular dystrophy lung function in infants with cystic fibrosis respiratory function in survivors of the united kingdom extracorporeal membrane oxygenation trial the lung in cystic fibrosis. a quantitative study including prevalence of pathologic findings among different age groups pulmonary pathophysiology in cystic fibrosis long-term assessment of pulmonary function tests in pediatric survivors of acute respiratory distress syndrome pulmonary function in newborn infants with transitory tachypnea and pneumothorax definitions, mechanisms, relevant outcomes, and clinical trial coordination pulmonary function tests in preschool children with cystic fibrosis pulmonary function tests in preschool children with asthma statement: pulmonary function testing in preschool children pulmonary outcomes in bronchopulmonary dysplasia postnatal changes in pulmonary mechanics and energetics of infants with respiratory distress syndrome following surfactant treatment the relationship between asthma and obesity in children: is it real or a case of over diagnosis? maximal static respiratory pressures in generalized neuromuscular disease relief of central airways obstruction following spinal release in a patient with idiopathic scoliosis evidence of airway obstruction in children with idiopathic scoliosis factors influencing glottic dimensions during forced expiration effect of expiratory loading on glottic dimensions in humans bronchodilators in cystic fibrosis usefulness of monitoring lung function in asthma respiratory muscle and pulmonary function in polymyositis and other proximal myopathies interrupter resistance and wheezing phenotypes at 4 years of age respiratory mechanics in children phenotype specific treatment of asthma in childhood absence of ventilatory responses to alternating breaths of mild hypoxia and air in infants who have had bronchopulmonary dysplasia: implications for the risk of sudden infant death prospective study of body mass index, weight change, and risk of adult-onset asthma in women in: chernick v (ed) kendig's disorders of the respiratory tract in children. w.b. saunders company, philadelphia canny gj, szeinberg a, koreska j, levison h (1989) hypercapnia in relation to pulmonary function in duchenne muscular dystrophy the effects of adiposity and weight change on forced expiratory volume decline in a longitudinal study of adults profiles of neuromuscular diseases. spinal muscular atrophy early respiratory system mechanics and the prediction of chronic lung disease in ventilated preterm neonates requiring surfactant treatment evaluation of a tidal expiratory flow index in healthy and diseased infants task force on chronic interstitial lung disease in immunocompetent children transition from dynamically maintained to relaxed end-expiratory volume in human infants changes in the glottic aperture during bronchial asthma studies of respiratory physiology in the newborn infant. iii. measurements of mechanics of respiration studies of respiratory physiology in children. ii. lung volumes and mechanics of respiration in 64 patients with cystic fibrosis of the pancreas occult pulmonary abnormalities in asymptomatic asthmatic children ventilation distribution and density dependence of expiratory flow in asthmatic children five-to seven-year course of pulmonary function in cystic fibrosis longitudinal analysis of pulmonary function decline in patients with cystic fibrosis lung function: physiology, measurement and application in medicine werdnig-hoffmann disease. the effects of intrauterine onset on lung growth role of positive end-expiratory pressure changes on functional residual capacity in surfactant treated preterm infants a simplified approach to the measurement of specific airway resistance effect of scoliosis on growth of alveoli and pulmonary arteries and on right ventricle direct measurement of static chest wall compliance in animal and human neonates density dependence of forced expiratory flows in healthy infants and toddlers pulmonary functions in congenital scoliosis clinical implications of chemoreceptor reflexes role of carotid-body chemoreceptors and their reflex interactions in bradycardia and cardiac arrest analysis of lung volume restriction in patients with respiratory muscle weakness obesity and the pulmonologist changing relationships between stature and lung volumes during puberty pathophysiology of sleep apnea trapped gas and airflow limitation in children with cystic fibrosis and asthma appropriate positive end expiratory pressure level in surfactant-treated preterm infants infant lung function after inhaled nitric oxide therapy for persistent pulmonary hypertension of the newborn specific airway resistance from the perinatal period into adulthood. alterations in childhood pulmonary disease predictors of severe chest infections in pediatric neuromuscular disorders respiratory system impedance from 4 to 40 hz in paralyzed intubated infants with respiratory disease the use of a helium-oxygen mixture during maximum expiratory flow to demonstrate obstruction in small airways in smokers respiratory function at age 8-9 years in extremely low birthweight/very preterm children born in victoria in 1991-1992 bronchopulmonary dysplasia in very low birth weight subjects and lung function in late adolescence the problem of lung growth effects of early dexamethasone therapy on pulmonary mechanics and chronic lung disease in very low birth weight infants: a randomized, controlled trial editorial (1992) the nose and the respiratory system developmental changes in sequential activation of laryngeal abductor muscle and diaphragm in infants hypercapnia increases expiratory braking in preterm infants pulmonary function and exercise gas exchange in survivors of adult respiratory distress syndrome upper airway obstruction following adult respiratory distress syndrome. an analysis of 30 survivors influence of human vocal cord movements on airflow and resistance during eupnea comparison of human vocal cord movements during isocapnic hypoxia and hypercapnia respiratory studies in children. i. lung volumes in healthy children, 6-14 years of age lung volume restriction in patients with chronic respiratory muscle weakness: the role of microatelectasis neck and abdominal muscle activity in patients with severe thoracic scoliosis detection of obliterative bronchiolitis after lung transplantation by indexes of ventilation distribution bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria chronic interstitial lung disease in children pediatric interstitial lung disease revisited long-term sequelae in children surviving adult respiratory distress syndrome cardiopulmonary function in premature infants with bronchopulmonary dysplasia-a 2-year follow up lung function and respiratory symptoms at 11 years in children born extremely preterm: the epicure study flow limitation in infants with bronchopulmonary dysplasia and respiratory function at school age respiratory care of the patient with duchenne muscular dystrophy: ats consensus statement the curse of adam: effort closure of the larynx pediatric acute lung injury: prospective evaluation of risk factors associated with mortality randomised crossover trial of salbutamol aerosol delivered by metered dose inhaler, jet nebuliser, and ultrasonic nebuliser in chronic lung disease helium flow-volume curves in the detection of early small airway disease supine fall in lung volumes in the assessment of diaphragmatic weakness in neuromuscular disorders pulmonary function test study and after spinal fusion in young idiopathic scoliosis lung function testing in infants with cystic fibrosis: lessons from the past and future directions lung function testing in neonates and infants with chronic lung disease: lung and chest wall mechanics acute respiratory distress syndrome caused by pulmonary and extrapulmonary disease. different syndromes? relationship between peak cough flow and spirometry in duchenne muscular dystrophy lung function in interstitial lung diseases in children chestwall compliance in full-term and premature infants pulmonary mechanics in normal infants and young children during first 5 years of life serial determination of pulmonary function in infants with chronic lung disease impairment after adult respiratory distress syndrome. an evaluation based on pulmonary mechanics in patients with respiratory muscle weakness the nose and the lower airways timing of recovery of lung function after severe hypoxemic respiratory failure in children pulmonary manifestations of neuromuscular disease with special reference to duchenne muscular dystrophy and spinal muscular atrophy variability of maximum expiratory flow-volume curves total respiratory compliance and functional residual capacity in young children abnormalities of lung mechanics in young asthmatic children effect of budesonide on pulmonary hyperinflation in young asthmatic children changes in functional residual capacity in response to bronchodilator therapy among young asthmatic children the relation of body mass index to asthma, chronic bronchitis, and emphysema clinical implications of maximal respiratory pressure determinations for individuals with duchenne muscular dystrophy diffusing capacity of the lung in school-aged children born very preterm, with and without bronchopulmonary dysplasia long-term prospective study in children after respiratory syncytial virus infection airway and respiratory tissue mechanics in normal infants pulmonary outcome in adolescents of extreme preterm birth: a regional cohort study better care of immature infants; has it influenced long-term pulmonary outcome? acute lung injury: pathophysiology, assessment and current therapy total lung capacity by n2 washout from high and low lung volumes in ventilated infants and children long-term pulmonary sequelae in children who were treated with extracorporeal membrane oxygenation for neonatal respiratory failure sex differences in the relation between body mass index and asthma and atopy in a birth cohort input impedance and peripheral inhomogeneity of dog lungs the upper respiratory tract in perinatal life the significance of grunting in hyaline membrane disease changes in pulmonary mechanics with increasing disease severity in children and young adults with cystic fibrosis limitations of sniff nasal pressure in patients with severe neuromuscular weakness maternal smoking during and after pregnancy and lung function in early adulthood: a prospective study bronchopulmonary dysplasia. in: chernick v (ed) kendig's disorders of the respiratory tract in children assessment of airway function using partial expiratory flow-volume curves: how reliable are measurements of maximal expiratory flow at frc during early infancy? new aspects of airway mechanics in pre-term infants one-year outcomes in survivors of the acute respiratory distress syndrome sequelae of the adult respiratory distress syndrome narrowing of glottis opening in humans associated with experimentally induced bronchoconstriction anatomy and physics of respiration postnatal growth and function of the pre-acinar airways lung function at term reflects severity of bronchopulmonary dysplasia effect of antenatal corticosteroid treatment on lung function in full-term newborn infants prospective longitudinal evaluation of lung function during the first year of life after extracorporeal membrane oxygenation age as a factor in the distribution of lower-airway conductance and in the pathologic anatomy of obstructive lung disease sex-specific prediction equations for vmax(frc) in infancy: a multicenter collaborative study development of airway function in infancy after preterm delivery growth of the bronchial tree in man airway function in infants treated with inhaled nitric oxide for persistent pulmonary hypertension daytime predictors of sleep hypoventilation in duchenne muscular dystrophy world federation of pediatric intensive critical care societies (wficss) non-invasive positive pressure ventilation in the preterm neonate: reducing endotrauma and the incidence of bronchopulmonary dysplasia laryngeal and diaphragmatic muscle activities and airflow patterns after birth in premature lambs respiratory muscle activities after birth in asphyxiated preterm lambs laryngeal muscle activities with cerebral hypoxia-ischemia in newborn lambs airway closure during mixed apneas in preterm infants: is respiratory effort necessary? pulmonary function in duchenne muscular dystrophy related to stage of disease thyroarytenoid muscle activity during hypoxia, hypercapnia, and voluntary hyperventilation in humans respiratory capacity course in patients with infantile spinal muscular atrophy physiologic dysfunction of the asthmatic lung factors in childhood as predictors of asthma in adult life a comparison of the clinical characteristics of children and adults with severe asthma bronchopulmonary dysplasia influence of nonlinearities on estimates of respiratory mechanics using multilinear regression analysis pulmonary function abnormalities in symptom-free children after bronchiolitis effect of spinal surgery on lung function in duchenne muscular dystrophy prediction of mortality in patients with cystic fibrosis the design of future pediatric mechanical ventilation trials for acute lung injury pulmonary function after recovery from the adult respiratory distress syndrome pulmonary complications of obesity dynamic maintenance of end-expiratory lung volume in full-term infants posterior cricoarytenoid and diaphragm activities during tidal breathing in neonates effect of preterm birth on later fev1: a systematic review and meta-analysis scoliosis and the respiratory system contribution of airway hyperresponsiveness to lower airway obstruction after extracorporeal membrane oxygenation for meconium aspiration syndrome lung function from infancy to the preschool years after clinical diagnosis of cystic fibrosis progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis respiratory muscle function in amyotrophic lateral sclerosis thyroarytenoid muscle activity during wakefulness and sleep in normal adults posterior cricoarytenoid muscle activity during wakefulness and sleep in normal adults laryngeal response to passively induced hypocapnia during nrem sleep in normal adult humans effect of hypercapnia on laryngeal airway resistance in normal adult humans preservation of the phrenic motoneurons in werdnig-hoffmann disease sleep and neuromuscular disease: frequency of sleep-disordered breathing in a neuromuscular disease clinic population disorders of the respiratory muscles the spectrum of cystic fibrosis. a study of pulmonary mechanics in 46 patients the variable effect of a bronchodilating agent on pulmonary function in cystic fibrosis small airways disease" in children: no test is best changes in respiratory mechanics with age clinical significance of severe isolated diaphragm weakness oral feeding in the preterm infant effects of body fat on ventilatory function in children and adolescents: cross-sectional findings from a random population sample of school children spirometry in the supine position improves the detection of diaphragmatic weakness in patients with amyotrophic lateral sclerosis a comparative analysis of contractile characteristics of the diaphragm and of respiratory system mechanics the effects of obesity on pulmonary function asthma and obesity: common early-life influences in the inception of disease importance of inspiratory muscle tone in maintenance of frc in the newborn specific airway resistance in 3-year-old children: a prospective cohort study early changes in respiratory compliance and resistance during the development of bronchopulmonary dysplasia in the era of surfactant therapy lung function tests in neonates and infants with chronic lung disease: forced expiratory maneuvers nature and severity of lung function abnormalities in extremely pre-term children at 11 years of age patterns of laryngeal electromyography and the activity of the respiratory system during spontaneous laughter adult respiratory distress syndrome in a pediatric intensive care unit: predisposing conditions, clinical course, and outcome relationships between specific airway resistance and forced expiratory flows in asthmatic children clinical correlations and pulmonary function at 8 years of age after severe neonatal respiratory failure lung elastic recoil in cystic fibrosis relationship of lung recoil to lung volume and maximum expiratory flow in normal children muscular dystrophy campaign sponsored workshop: recommendation for respiratory care of children with spinal muscular atrophy type ii and iii pediatric pulmonary function testing: plethysmography and gas dilution techniques pathophysiology of osas in children spirometry in 3-to 6-year-old children with cystic fibrosis fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome pulmonary function testing in idiopathic interstitial pneumonias types of asthma and wheezing asthma and wheezing in the first six years of life. the group health medical associates profiles of neuromuscular diseases. duchenne muscular dystrophy randomized, double-blinded trial of lowdose dexamethasone: ii. functional residual capacity and pulmonary outcome in very low birth weight infants at risk for bronchopulmonary dysplasia decreased respiratory compliance in infants less than or equal to 32 weeks' gestation, delivered more than 7 days after antenatal steroid therapy respiratory compliance in preterm infants after a single rescue course of antenatal steroids: a randomized controlled trial recovery of function in survivors of the acute respiratory distress syndrome airway resistance and atopy in preschool children with wheeze and cough functional significance of the area of apposition of diaphragm to rib cage pediatric pulmonary function testing: neuromuscular disorders sleep-disordered breathing and respiratory failure in acid maltase deficiency daytime predictors of sleep disordered breathing in children and adolescents with neuromuscular disorders growth of airways and air spaces in teenagers is related to sex but not to symptoms pulmonary function in infants with neonatal chronic lung disease with or without hyaline membrane disease at birth early versus late dexamethasone treatment in preterm infants at risk for chronic lung disease: a randomized pilot study changes in lung volume and ventilation during surfactant treatment in ventilated preterm infants changes in lung volume and ventilation during lung recruitment in highfrequency ventilated preterm infants with respiratory distress syndrome the effect of airway pressure and oscillation amplitude on ventilation in preterm infants oral breathing in response to nasal trauma in term infants pulmonary function and scoliosis in duchenne dystrophy standardisation of spirometry relationship between chest wall and pulmonary compliance and age effects of meconium on airway reactivity to histamine and acetylcholine in vitro mechanics of breathing measurements of respiratory mechanics in the newborn: a simple approach early onset of airway reactivity in premature infants with bronchopulmonary dysplasia the assessment of lung function in children with scoliosis a dutch guideline for the treatment of scoliosis in neuromuscular disorders muscular dystrophy campaign funded workshop on management of scoliosis in duchenne muscular dystrophy 24 the pediatric airway: an interdisciplinary approach longitudinal evaluation of airway function 21 years after preterm birth alveolarization continues during childhood and adolescence: new evidence from helium-3 magnetic resonance lung and thorax development during adolescence: relationship with pubertal status spirometry in 3-5-year-old children with asthma assessment of pulmonary function in the early phase of ards in pediatric patients global strategy for asthma management and prevention. national institute for health expert panel report 3: guidelines for the diagnosis and management of asthma the effect of inhaled budesonide on symptoms, lung function, and cold air and methacholine responsiveness in 2-to 5-year-old asthmatic children discriminative capacity of bronchodilator response measured with three different lung function techniques in asthmatic and healthy children aged 2 to 5 years changes in rib cage geometry during childhood childhood asthma and lung function in mid-adult life bronchiolar airflow impairment after lung transplantation: an early and common manifestation variability of tidal breathing flow-volume loops in healthy and sick newborns the pathophysiology of respiratory impairment in pediatric neuromuscular diseases expiratory flow limitation and intrinsic positive end-expiratory pressure in obesity developmental changes in chest wall compliance in infancy and early childhood chest wall compliance in infants and children with neuromuscular disease long-term pulmonary functional outcome of bronchopulmonary dysplasia and premature birth adult obstructive sleep apnea: pathophysiology and diagnosis a practical approach to the interpretation of lung function testing in children physiologic definitions of obliterative bronchiolitis in heart-lung and double lung transplantation: a comparison of the forced expiratory flow between 25% and 75% of the forced vital capacity and forced expiratory volume in one second rhythms and complexity of respiration during sleep in pre-term infants do nhlbi lung function criteria apply to children? a cross-sectional evaluation of childhood asthma at national jewish medical and research center interpretative strategies for lung function tests thoracoabdominal pattern of breathing in neuromuscular disorders clinical determinants of abnormalities in pulmonary functions in survivors of the adult respiratory distress syndrome lung protective ventilation strategy for the acute respiratory distress syndrome nocturnal oxygenation and prognosis in duchenne muscular dystrophy changes in spirometry over time as a prognostic marker in patients with duchenne muscular dystrophy long term respiratory consequences of intrauterine growth restriction tidal volume, recruitment and compliance in hfov: same principles, different frequency effects of gestation and antenatal steroid on airway and tissue mechanics in newborn lambs in vitro performance characteristics of high-frequency oscillatory ventilators variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry monitoring of lung volume recruitment and derecruitment using oscillatory mechanics during high-frequency oscillatory ventilation in the preterm lamb partitioning of airway and parenchymal mechanics in unsedated newborn infants the functional development of the respiratory system from the period of gestation to adulthood expiratory muscle function in amyotrophic lateral sclerosis abnormalities of functional residual capacity in symptomatic and asymptomatic young asthmatics larynx and neonatal apneas obstructive sleep disordered breathing in children: beyond adenotonsillectomy chemoreceptor and vagal influences on thyroarytenoid muscle activity in awake lambs during hypoxia wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy compilation of reference values for lung function measurements in children patterns and predictors of sleep disordered breathing in primary myopathies airway function in infants newly diagnosed with cystic fibrosis relative ability of full and partial forced expiratory maneuvers to identify diminished airway function in infants with cystic fibrosis early detection of lung disease in children with cystic fibrosis using lung function risk factors for airway remodeling in asthma manifested by a low postbronchodilator fev1/vital capacity ratio: a longitudinal population study from childhood to adulthood pediatric pulmonary function testing: cystic fibrosis the lung: its growth and remodelling in health and disease early detection of airway involvement in obliterative bronchiolitis after lung transplantation. functional and bronchoalveolar lavage cell findings respiratory function in the muscular dystrophies restricted pulmonary function in cystic fibrosis pulmonary function in bronchopulmonary dysplasia the travels of a pulmonologist through the upper airway scoliosis in spinal muscular atrophy: review of 63 cases follow-up of asthma from childhood to adulthood: influence of potential childhood risk factors on the outcome of pulmonary function and bronchial responsiveness in adulthood site of upper airway obstruction in preterm infants with problematical apnoea site of upper airway obstruction in infants following an acute lifethreatening event pulmonary function in spinal muscular atrophy fetal nicotine exposure increases airway responsiveness and alters airway wall composition in young lambs obesity is a risk for asthma and wheeze but not airway hyperresponsiveness asthma and atopy in overweight children vocal cord closure. a cause of upper airway obstruction during controlled ventilation influence of preterm onset of inspiration on tidal breathing parameters in infants with and without cld changes in the fev1-height relationship during pubertal growth respiratory muscle force and ventilatory function in adolescents obstructive lung disease in children after allogeneic bone marrow transplantation anatomy and physiology for speech, language, and hearing effect of a previous voluntary deep breath on laryngeal resistance in normal and asthmatic subjects impaired gas mixing and low lung volume in preterm infants with mild chronic lung disease the paradoxical effect of adrenergic and methylxanthine drugs in cystic fibrosis forced expiratory flow between 25% and 75% of vital capacity and fev1/forced vital capacity ratio in relation to clinical and physiological parameters in asthmatic children with normal fev1 values impact of nasal ventilation on survival in hypercapnic duchenne muscular dystrophy neonatal respiratory mechanics and development of bronchial hyperresponsiveness in preterm infants bronchiolitis obliterans following haematopoietic stem cell transplantation clinical and genetic study of chronic (types ii and iii) childhood onset spinal muscular atrophy office-based objective measures in childhood asthma is forced expiratory volume in one second the best measure of severity in childhood asthma? diagnosisrelated deterioration of lung function after extracorporeal membrane oxygenation prospective longitudinal evaluation of lung function during the first year of life after repair of congenital diaphragmatic hernia small airways obstruction syndrome phascinating physiology usefulness of sniff nasal pressure in patients with neuromuscular or skeletal disorders fibrosing alveolitis in childhood. a long-term follow-up specific airway conductance in relation to postconceptional age during infancy early life influences on the development of chronic obstructive pulmonary disease influence of ethnicity and gender on airway function in preterm infants ventilatory function in british adults after asthma or wheezing illness at ages 0-35 surfactant improves lung function and morphology in newborn rabbits with meconium aspiration parental smoking in childhood and adult obstructive lung disease: results from the european community respiratory health survey cough capacity in patients with muscular dystrophy mechanics of breathing after surgical ligation of patent ductus arteriosus in newborns with respiratory distress syndrome pulmonary function after surfactant lung lavage followed by surfactant administration in infants with severe meconium aspiration syndrome use of corticosteroids in acute lung injury and acute respiratory distress syndrome: a systematic review and meta-analysis lung function in children with duchenne's muscular dystrophy physiologic growth and development of the lung during the first year of life expiratory flow limitation in infants with bronchopulmonary dysplasia static compliance of the respiratory system in healthy infants can we breathe and swallow at the same time? some aspects of clinical relevance in the maturation of respiratory control in infants ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome the international study of asthma and allergies in childhood (isaac) steering committee. worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema postnatal human lung growth early changes of pulmonary mechanics to predict the severity of bronchopulmonary dysplasia in ventilated preterm infants pediatric pulmonary function testing: long-term sequelae of neonatal lung disease spinal muscular atrophy: classification, aetiology, and treatment of spinal deformity in children and adolescents changes in residual volume relative to vital capacity and total lung capacity after arthrodesis of the spine in patients who have adolescent idiopathic scoliosis effect of dexamethasone on tracheobronchial aspirate fluid cytology and pulmonary mechanics in preterm infants hfov in premature neonates: effects on pulmonary mechanics and epithelial lining fluid cytokines. a randomized controlled trial the role of computer games in measuring spirometry in healthy and "asthmatic" preschool children spirometry in early childhood in cystic fibrosis patients frc measurements using body plethysmography in young children flow oscillations on the flow-volume loop: clinical and physiological implications flowvolume loop changes reflecting respiratory muscle weakness in chronic neuromuscular disorders lung function and exercise capacity in young adults born prematurely normal growth and development of the trachea diagnostic role of residual volume in paediatric patients with chronic symptoms of the lower airways 117th enmc workshop: ventilatory support in congenital neuromuscular disorders -congenital myopathies, congenital muscular dystrophies, congenital myotonic dystrophy and sma (ii) consensus statement for standard of care in spinal muscular atrophy the acute respiratory distress syndrome assessment of functional residual capacity using nitrogen washout and plethysmographic techniques in infants with and without bronchopulmonary dysplasia pulmonary function in asymptomatic adolescents with idiopathic scoliosis respiratory and cardiac function in children after acute hypoxemic respiratory failure standards of pulmonary function in children relationship between respiratory syncytial virus bronchiolitis and future obstructive airway diseases obesity and asthma in 11-12 year old new zealand children in the physiology of the nose hypoxemia in attack free asthmatic children: relationship with lung volumes and lung mechanics prenatal nicotine exposure alters lung function and airway geometry through alpha7 nicotinic receptors relationship between anatomic dead space and body size in health, asthma, and cystic fibrosis laryngeal and pump muscle activities during co 2 breathing in neonates effects of posture on respiratory mechanics in obesity lung function in immature baboons with respiratory distress syndrome receiving early caffeine therapy: a pilot study lung function in children and adolescents with idiopathic interstitial pulmonary fibrosis effects of obesity on respiratory resistance incidence and outcomes of pediatric acute lung injury key: cord-003655-uo0hdrgc authors: de vries, rory d.; rennick, linda j.; duprex, w. paul; de swart, rik l. title: paramyxovirus infections in ex vivo lung slice cultures of different host species date: 2018-03-27 journal: methods protoc doi: 10.3390/mps1020012 sha: doc_id: 3655 cord_uid: uo0hdrgc in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with in vitro and ex vivo experiments. here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy. ex vivo models provide an important bridge between in vitro and in vivo experiments. the use of agarose-inflated lung slices for respiratory virus pathogenesis studies has been described previously [1] [2] [3] [4] [5] [6] . here, we describe a protocol in which agarose-inflated lung slices can be kept viable in culture for at least seven days post-necropsy of an experimental animal. the combination of these viable lung slices with recombinant viruses expressing fluorescent reporter proteins [7] [8] [9] allows for accurate, sensitive and reproducible assessment of respiratory virus infection and dissemination over time. furthermore, use of these recombinant viruses allows for real time monitoring of infection processes, using multiple methods for measurement of fluorescence (e.g., flow cytometry and confocal laser scanning microscopy). lung slices are also suitable for analysis by immunohistochemistry, thereby visualizing virus cell tropism and spatial localization of infected cells within the tissue. we have validated this technique by infecting lung slices of multiple host species (cotton rats, ferrets, dogs and macaques) with various paramyxoviruses expressing fluorescent reporter proteins (measles virus (mv), canine distemper virus (cdv), human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv)) [10] . this technique, however, is directly transferable to different host species and different viruses [11] . this protocol describes the ex vivo agarose inflation of lungs and generation of slices for virus infection. lungs should be taken from the experimental animal during necropsy, keeping the material as intact as possible and attached to the primary bronchus and trachea. using a (blunt-end) needle or flexible catheter, the fresh lungs are inflated through the trachea (or primary bronchus, if inflation of a half lung or single lobe is desired) with low-melting point agarose mixed with culture medium. after solidification on ice, slices can be prepared by hand using microtome blades and cultured in 6-or 24-well plates with culture medium, dependent on the size of the slices. these lung slices are subsequently inoculated with infectious virus (or could be used for any other experimental purpose) and can be followed in time by phase-contrast inverted light microscopy or fluorescence, when reporter viruses expressing fluorescent proteins are used (see figure 1 ). in addition, emigrant cells in the culture medium can be harvested in time and analyzed by flow cytometry. although these emigrant cells could be present due to tissue degradation, they can be detected at early time-points after refreshing the culture medium, suggesting they are mobile emigrant cells. we have detected cells from lymphoid, myeloid and epithelial origin using this method. at the end of the experiment, slices can be used as desired; potential applications include fixation followed by paraffin embedding and immunohistochemistry, or generation of single cell suspensions to be analyzed via flow cytometry. . after solidification on ice, thin slices of approximately 1-mm thick can be cut by hand (c) and are transferred to 6-or 24-well plates pre-filled with culture medium (d). slices can subsequently be inoculated with the desired virus (e) and infection of the slices is followed in time (f). in this example in panel f, a macaque lung slice infected with recombinant measles virus is shown. all animals used in these studies were housed and experiments were conducted in strict compliance with european guidelines (eu directive on animal testing 86/609/eec) and dutch legislation (experiments on animals act, 1997). protocols were approved by the independent animal experimentation ethical review committee dcc in driebergen, the netherlands. animal welfare was observed on a daily basis. critical step prepare 4% (w/v) low-melting point agarose by dissolving agarose in phosphate-buffered saline (pbs) and boil for several minutes (e.g., using a microwave oven, be careful for boiling over). as an alternative, agarose can be prepared by autoclaving. be sure to allow agarose to cool down to 42 • c (e.g., in a water bath) before inflating the lungs. the volume of low-melting point agarose should be adjusted according to the size of the lungs. resect the lungs, including the distal part of the trachea, from the experimental animal. critical step resection should be performed within 6 h after euthanasia, preferably sooner. critical step be sure not to damage the lungs while resecting, as this will interfere with the inflation process. locate the trachea (for small species) or primary bronchus (for larger species) through which you wish to inflate. for smaller mammals, inflation through the trachea works best, but for larger animals (body weight > 3-5 kg) inflation through the primary bronchus is more efficient. fill a syringe with a 42 • c pre-warmed 1:1 mixture of 4% (w/v) low-melting point agarose and lung slice medium (dmem/ham's f12 medium supplemented with 10% (v/v) fbs, penicillin, streptomycin, l-glutamine and amphotericin b). final agarose percentage is 2% (w/v). critical step avoid the formation of air bubbles in the agarose. decide on the size of the syringe dependent on the size of the trachea or bronchus through which you will inflate. either the pipette tip, blunt-end needles, catheters, cannulas the end of the syringe should be a tight fit into the trachea or bronchus. insert the end of the syringe into the trachea or primary bronchus, enforce placement inside the trachea or bronchus by clamping (or use sutures as an alternative) the trachea or bronchus around the needle. critical step be careful not to damage the trachea or bronchus while inserting the needle into the trachea or bronchus. we advise the use of pipette tips, blunt-end needles, catheters or cannulas instead of sharp needles at the end of the syringe. 6. inject the proper amount of agarose into the lungs. check inflation while injecting; keep adding agarose until the lungs are completely inflated (see video s1). critical step it is not always easy to determine the best level of inflation. in our experience, the volume used for inflation should be slightly higher than the tidal volume of the animal, which is usually around 7 ml per kg body weight. if you are inflating just one lobe of the lung, the volume should be adapted accordingly. remove needle but keep the clamp positioned on the trachea or bronchus and allow the inflated lungs to solidify on ice (5 min for smaller species and 10-30 min for larger species). transfer lungs to biosafety cabinet, remove the clamp, prepare a surface for cutting (cutting board or petri dish) and microtome blade. prepare the lungs for slicing by finding anatomically interesting locations. critical step dependent on the focus of the experiment, you have to decide which part is anatomically interesting. decide on whether you want to use for example the left or right side, and either the upper or lower parts of available lobes. to check viability, we advise to include a cut of the primary bronchus in the slice, to determine the beating of cilia indicating viable epithelial cells. start with an initial cut to ensure that a straight edge is available for cutting. now manually prepare slices of approximately 1 mm thickness. if desired, the lungs can be attached to the cutting board by needles (see video s2). as a rule of thumb, slices are thin enough for culture if you can see through the slice while cutting it and actually see the microtome blade. gently transfer cut slices into 6-, 12-, 24-, or 48-well plates (dependent on size of the slices) that were pre-filled with lung slice culture medium. infection of slices can be performed immediately. pause step slices can also be infected after 24 h of culture at 37 • c in 5% (v/v) co 2 . we have never observed discrepancies between obtained results after either direct infection or after overnight culture. transfer the lung slices into empty 6-, 12-, 24-, or 48-well plates (dependent on size of the slices). gently add virus (preferably a recombinant virus expressing a fluorescent reporter protein) in a drop wise fashion onto the lung slice. typically, we used an inoculation volume of 150 µl; always include a mock infection as negative control. we typically use 1-5 × 10 5 tissue culture infectious dose-50 (tcid 50 ) per slice; variations are possible. 3. incubate at 37 • c in 5% (v/v) co 2 for 1 h. after 1 h, add an appropriate volume of lung slice medium (inoculum can be washed away) and return plates to 37 • c in 5% (v/v) co 2 . 5. in our experience, slices can be held viable (based on beating cilia in epithelium) and followed in time for a maximum of 7 days. culture medium has to be replaced to ensure viability every other day (i.e., day 2, 4 and 6). critical step when replacing culture medium, medium should be taken off and directly replaced, not allowing the lung slices to dehydrate. beating of cilia is the best measure for viability, however when beating of cilia is no longer observed slices start disintegrating. depending on the goal of the experiment, non-viable slices should be removed or kept in culture. slices should be checked for viability on daily basis: find a bronchus with cilia under a normal light microscope. movement (see video s3) of cilia is indicative of viable epithelial cells, suggesting there are live cells in culture. if fluorescent reporter viruses were used, slices should be checked for fluorescence on a daily basis (see video s4 and figure 2 ). use a macroscopic imaging lamp as described previously [7] in combination with an emission filter to visualize fluorescence macroscopically. shine lamp directly on lung slices and find fluorescent spots. photographs can be acquired if desired. use an inverted uv microscope or confocal laser scanning microscope to identify fluorescent-positive cells. during infection and culture, effluent cells can be harvested and checked for expression of fluorescent proteins by flow cytometry if desired. harvest supernatants from slices (e.g., on day 2, 4 and 6 while replacing culture medium), transfer supernatant to tubes and centrifuge (5-10 min, 300× g). transfer pellets to 96-wells v-bottom plate, wash once in facs buffer (pbs + 0.5% (v/v) bsa + 2 mm edta). optional step to determine which subsets of cells are infected, a fluorescence activated cell sorter (facs) staining for specific cell types can be performed at this point. cells that migrate out of the lung slices are mainly lymphoid, myeloid or epithelial in origin. to study the tropism of infection you can selectively stain for example t-cells, b-cells, natural killer (nk) cells, neutrophils, granulocytes, dendritic cells, macrophages or epithelial cells, or combinations thereof. acquire cells on flow cytometer to determine number of virus-positive cells. multiple experiments can be performed at the end of culture or at desired time points. slices can be directly stained for confocal laser scanning microscopy (section 3.7.1), fixed for immunohistochemistry (section 3.7.2) or used to generate a single cell suspension for flow cytometry (section 3.7.3). critical step not all fluorophores are well-preserved by pfa fixation. sensitivity of fluorophores to fixation should be tested beforehand, and the fixative of mounting medium can be adjusted accordingly. wash slices after 10 min and permeabilize in 0.1% (v/v) triton x-100 for 30 min. wash slices and subsequently stain with fluorescent antibodies of choice. critical step to understand tissue morphology, it is important to counterstain nuclei (for example with to-pro-3 or nucblue) and add a marker of choice. we found it useful to stain cilia using an antibody against class iv β-tubulin. transfer slices to thin-bottom/glass-bottom dishes. view fluorescence by confocal laser scanning microscopy (using an inverted microscope, see figure 3 ). since slices are approximately 1-mm thick, z-stacks can be generated and from those 3d images can be rendered. in addition to using lung slices as an ex vivo culture and infection system, the method can also be used to screen the lungs of in vivo virus-infected animals for infection and determine the viral tropism (e.g., see reference [3] ). two approaches are possible: instead of inflating lungs with the 1:1 mixture of 4% (w/v) low-melting point agarose and lung slice medium, prepare a 1:1 mixture of 4% (w/v) low-melting point agarose and 4% (w/v) pfa in pbs. the rest of the protocol is identical, after preparing lung slices these can be screened by fluorescence microscopy directly, virus-positive slices can be processed further as desired (immunohistochemistry, staining for confocal laser scanning microscopy, preparation of single cell suspensions for flow cytometry). as an alternative, slices can be inflated with 1:1 mixture of 4% (w/v) low-melting point agarose and lung slice medium and kept in culture for a period of time after necropsy, to evaluate viral tropism and dissemination [4] . with the protocol described here, dependent on the size of the species used in the experiment, viable lung slices from potentially any species can be obtained for ex vivo experiments. we have observed viability of these slices for up to seven days post resection; however, this could be dependent on the species and culture conditions. infections with respiratory viruses of lung slices are normally relatively successful: figures 2 and 3 show examples of ex vivo paramyxovirus-infected lung slices. in these experiments, macaque lungs were inflated and infected with mv, which is clearly able to infect the macaque lung slices. of course, viruses corresponding to the target species should be chosen to obtain positive results. in the family of paramyxoviridae for example, viruses exclusively infecting primates or carnivores exist. measles virus, a virus of primates, successfully infected non-human primate but not dog lungs, whereas cdv, a virus of carnivores, behaved vice versa. using viruses expressing fluorescent reporter proteins allows for sensitive detection of virus-infected cells in these experiments; however, these experiments can also be performed with non-fluorescent viruses. staining should still make infected cells visible in immunohistochemistry, confocal laser scanning microscopy or flow cytometry. the model described here of course has some limitations, as a single region of the lung is directly infected, often with a relatively high inoculum. therefore, in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with proper in vitro and ex vivo experiments, indicating the potential of ex vivo experiments in these cultured lung slices. 4% agarose: prepare correct weight of hydroxyethylagarose in dpbs; • lung slice medium: dmem/ham's nutrient mixture f12 powder, glutamax, fbs, penicillin/streptomycin, amphotericin; • fluorescence activated cell sorter buffer: pbs + 0.5% (v/v) bsa + 2 mm edta (use stock of 0.5 m in distilled water); • paraformaldehyde: dissolve appropriate weight in pbs with high ph by initially supplementing with naoh. after pfa has completely dissolved, ph should be re-adjusted to 7.4. always prepare fresh or store aliquoted at −20 • c. do not use buffered formalin when directly screening fluorescence, as this will disrupt the fluorescence of most reporter proteins. supplementary materials: the following are available online at http://www.mdpi.com/2409-9279/1/2/12/s1. video s1: inflation of cotton rat lungs. syringe is filled with agarose mixed with lung slice medium and inserted into the trachea, kept in place by a suture. lungs are slowly inflated by injecting agarose and medium into the trachea; video s2: slicing of cotton rat lungs. solidified cotton rat lungs are places on petri-dish and subsequently sliced. an initial cut is made for a straight edge followed by the preparation of a single lung slice; video s3: viability screening of lung slice. a bronchus consisting of viable epithelial cells with beating cilia; video s4: fluorescence screening of live lung slice infected with a paramyxovirus expressing a fluorescent reporter protein (green). the movie initially shows a combination of normal light and fluorescent light, followed by a shutdown of the normal light, indicating the identical area but now with fluorescence light only. time for completion: 3 h 1. wash slices twice in hank's balanced salt solution (hbss) transfer slices into petri dishes and manually cut in small pieces transfer pieces into 6-well plate pre-filled with hbss + collagenase (300 units/ml) + dnase (0.15 mg/ml) incubate for 1 h on rocking platform at 37 • c in 5% (v/v) co 2 prepare single cell suspension by straining small pieces over 100 µm cell strainer centrifuge and lyse red blood cells by adding 3 ml red blood cell lysis buffer to pellet, incubate 37 • c in 5% (v/v) co 2 for three minutes centrifuge, resuspend pellet in facs buffer and perform facs staining of choice acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices responsiveness of individual airways to methacholine in adult rat lung explants early target cells of measles virus after aerosol infection of non-human primates a prominent role for dc-sign + dendritic cells in initiation and dissemination of measles virus infection in non-human primates pb1-f2 modulates early host responses but does not affect the pathogenesis of h1n1 seasonal influenza virus long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions predominant infection of cd150 + lymphocytes and dendritic cells during measles virus infection of macaques an improved plaque reduction virus neutralization assay for human metapneumovirus observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus paramyxovirus infections in ex vivo lung slice cultures of different host species asymptomatic middle east respiratory syndrome coronavirus infection in rabbits the authors wish to thank geert van amerongen for providing excellent technical assistance during the establishment of this technique. we acknowledge nih for their ongoing funding of respiratory virus pathogenesis studies, specifically grant ai099100 to w.p.d. the authors declare no conflict of interest. key: cord-001117-llb4f74a authors: ji, wen-jie; ma, yong-qiang; zhou, xin; zhang, yi-dan; lu, rui-yi; guo, zhao-zeng; sun, hai-ying; hu, dao-chuan; yang, guo-hong; li, yu-ming; wei, lu-qing title: spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments date: 2013-11-19 journal: plos one doi: 10.1371/journal.pone.0081090 sha: doc_id: 1117 cord_uid: llb4f74a background: recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. mineralocorticoid receptor (mr) has been reported as a target to regulate macrophage polarization. the present work was designed to investigate the therapeutic potential of mr antagonism in bleomycin-induced acute lung injury and fibrosis. methodology/principal findings: we first demonstrated the expression of mr in magnetic bead-purified ly6g-/cd11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (balf) from c57bl/6 mice. then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that mr antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mrna and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male c57bl/6 mice. moreover, serial flow cytometry analysis in blood, balf and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating ly6c(hi) monocyte expansion, and reduce alternative activation (f4/80+cd11c+cd206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (f4/80+cd11c-) remained unaffected by spironolactone during investigation. conclusions/significance: the present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of mr-mediated circulating monocyte and alveolar macrophage phenotype switching. idiopathic pulmonary fibrosis (ipf) is a chronic, progressive, interstitial fibrotic lung disease characterized by chronic lung inflammation, disruption of alveolar structure, interstitial fibroblast proliferation, and excessive extracellular matrix synthesis and deposition [1] [2] [3] . although evidence showed that the persistent inflammatory response is associated with progressive development of ipf, therapies currently used for ipf, namely anti-inflammatory or immunosuppressive drugs, are largely ineffective [4] . therefore, novel therapies capable of targeting inflammation without compromising body's immunity can still be a challenge in this area. macrophages in lung tissue play an important role in the clearance of pulmonary pathogens and steady-state homeostasis maintenance. emerging evidence suggests that there is a causal link between lung macrophage mediated inflammation and excessive tissue destruction elicited by variety of exogenous stimuli, i.e., silica and asbestos exposure, virus infection, etc., which will ultimately lead to a failure of inflammation resolution, a key feature that progressively promotes the development of lung fibrosis [5] [6] [7] [8] . on the other hand, macrophages are a cell population with high plasticity, and display functional diversity during different stage of inflammatory response [9, 10] . the activation state of macrophage can be generally characterized as classical activation (m1 polarization) that is associated with a th1 immune response, or alternative activation (m2 polarization) that is associated with th2 immune response [11] . in lung tissue, m1-like macrophages are the first line defense in acute lung injury and are later replaced by m2-like macrophages that contribute to tissue repair and fibrosis. it is generally believed during inflammation, myeloid ly6c hi monocytes contribute to lung macrophage replenishment [9, 12] . the results from recent basic studies indicate that manipulation of macrophage phenotype switch might be a potential target for many macrophage mediated disorders [13] [14] [15] . recently, usher and colleagues demonstrated that macrophages from mice lacking myeloid mineralocorticoid receptor (mr), exhibit a transcription profile that mimic alternatively activated macrophages, and are protected against angiotensin ii (angii) induced cardiac hypertrophy and fibrosis [16] . this work provides evidence indicating that mr in mononuclear phagocytes might be a potential target for therapeutic purpose. based on current evidence, we speculated that pharmacological inhibition of mr with clinically approved drug, may regulate lung macrophage phenotype switching, as well as their progenitors, bone marrow-derived circulating monocytes, and may confer novel therapeutic potential in a murine model of bleomycin-induced acute pulmonary injury and fibrosis. eight to ten weeks male c57bl/6 mice, weighing 16 to validate the mrna expression of mr in mouse circulating monocytes, circulating monocytes from c57bl/6 mice were purified from peripheral blood using a magnetic bead-based kit (easysep tm mouse monocyte enrichment kit, cat no. 19761, stemcell technologies, vancouver, bc, canada). the purity of enriched monocytes was confirmed by flow cytometry (see below). detailed methods for total rna isolation, reverse transcription, and real-time pcr analysis are shown below. to validate the protein expression of mr in circulating monocytes and alveolar macrophages, the purified monocytes and cells from bronchoalveolar lavage fluid (balf) were seeded on glass slides for immunohistological detection of mr. briefly, the cells were fixed with methanol, followed by permeabilization with 0.1% triton x-100. then, the cells were incubated with the primary anti-mouse mineralocorticoid receptor monoclonal antibody (1:200, ab41912, abcam, cambridge, ma, usa) at 4°c overnight. to ensure specificity, isotype control (igg2a) was prepared. for alveolar macrophages, the cells were further incubated with the primary anti-mouse f4/80 antibody (1:200, ab6640, abcam) at 37°c for 2 h. after washing with 0.01 m pbs, the cells were incubated with tetramethylrhodamine isothiocyanate (tritc)-conjugated goat anti-mouse secondary antibody [for alveolar macrophage, fluorescein isothiocyanate (fitc)-conjugated goat anti-rat secondary antibody was also added] in dark. then, cell nuclei were stained by 4,6-diamidino-2-phenylindole (dapi, sigma-aldrich, st. louis. mo, usa) with light protection. images were visualized by a fluorescence microscope (eclipse 80i, nikon, tokyo, japan). the unstained samples and samples stained with the secondary antibody without incubation with primary antibodies were used as negative controls and showed no signal during analysis. to induce pulmonary fibrosis, mice were lightly anesthetized by inhalation of ether. bleomycin a5 (2.5mg/kg body weight in 40μl saline) or saline was administered by oropharyngeal instillation as described previously [17] . animals were then randomly allocated into four treatment groups: 1) 0.9% normal saline (ns) only; 2) bleomycin (blm) only; 3) bleomycin plus 0.9% normal saline (blm+ns); 4) bleomycin plus 20mg/kg of spironolactone (blm+sp). from the day of the administration (day 0), vehicle (0.9% saline), sp (dissolved in 0.9% saline) were delivered by oral gavage once daily, and continued for 21 days. at 1, 3, 7, 14 or 21 days, animals were sacrificed by exsanguinations under sodium pentobarbital anesthesia (10 mice each time point). blood, balf and lung tissues were collected for the following assays. the balf was collected through an intratracheal cannula with three sequential 1 ml of 0.9% sterile saline and centrifuged at 300 g for 10 min at 4°c. the cell-free supernatant was stored at -80°c for analysis of cytokines. the cell pellet was resuspended in sterile 0.9% saline for total cell counts, differential cell counts, immunohistochemical staining, and flow cytometry analysis. the left lung (from which no balf was harvested) was fixed in 4% paraformaldehyde solution for 24h. after embedding in paraffin, 5 μm sections were prepared and stained with hematoxylin-eosin or masson's trichrome, and examined on a light microscope (e600pol, nikon, tokyo, japan). for detection of myofibroblasts, α smooth muscle actin (α-sma, 1:600, a2547, sigma-aldrich, st. louis. mo, usa) immunofluorescent staining was carried out as previously described [18] . for the evaluation of inflammatory response induced by bleomycin, semi-quantitative scoring criteria by szapiel and coworkers were used in a blinded fashion [19] . fibrosis and collagen was determined from 10 non-overlapping fields by using digital quantitative analysis (image pro plus software version 4.5, media cybernetics, silver spring, md, usa). the lung fibrosis index was defined as the sum of the total area of collagen in the entire visual field divided by the sum of total connective tissue area in the entire visual field. the collagen content in the whole left lung was determined by analysis of hydroxyproline as previously described [20] . in brief, lung lobes were homogenized in 1 ml of phosphate buffered saline (pbs, ph=7.4) and then hydrolyzed in 1 ml of 6 n hydrochloric acid for 16 hours at 110°c, and neutralized to ph 7.0 with naoh. chloramines t reagent (1 ml of 0.5 mol/l) was then added and the samples were left at room temperature for 20 minutes. then 20% p-dimethylaminobenzaldehyde solution (dissolved in 3.15 n perchloric acid) was added to each sample, and the mixture was incubated at 60°c for 15 minutes. absorbance was measured at 550 nm on a nanodrop 2000c spectrophotometer (thermo scientific, waltham, ma, usa). cells from blood, balf and lungs were subject flow cytometry analysis on a cytomics fc500 cytometer (beckman coulter, miami, fl, usa). all antibodies were obtained from biolegend (san diego, ca, usa). all data were analyzed with flowjo software (treestar, ashland, or, usa). for validation of the purity of magnetic bead-enriched circulating monocytes, anti-mouse cd11b-phycoerythrin (pe) (clone m1/70) and anti-mouse ly6g-percp-cy5.5 (clone 1a8) were used. for analysis of circulating monocyte subsets, ethylenediaminetetraacetic acid (edta) anti-coagulated whole blood was stained with anti-mouse cd11b-phycoerythrin (pe) (clone m1/70) and anti-mouse ly6c-fitc (clone hk1.4), incubated for 30 min at room temperature in the dark. following red cell lysis, samples were analyzed. for immunophenotypic analysis of alveolar macrophages (am), cells isolated from balf were first centrifuged (10 min at 400 g at room temperature), and the supernatant was discarded to remove dead cells. for each flow cytometry analysis, the cells were first suspended in 0.4% trypan blue in pbs, and the number of live and dead cells was measured using an automatic cell counter (counterstar tm , rui yu biotechnology co.,ltd, shanghai, china). by this method, the number of live cells in each sample is more than 95%. for subsequent flow cytometry analysis, the cells were incubated with anti-mouse f4/80-pe-cy5 (clone bm8), anti-mouse cd11c-pe-cy7 (clone n418) and anti-mouse cd206-pe (clone c068c2). following incubation, flow cytometry analysis was carried out. for immunophenotypic analysis of interstitial macrophages (ims), lung single-cell suspensions were prepared from lavaged lung (from which the balf was harvested) to reduce the contamination of am. in brief, the lower lobe of right lung were minced and incubated with 0.1 mg/ml collagenase solution (type i, sigma-aldrich) at 37°c for 60 min. after filtering through 40 μm nylon mesh, similar procedure to remove dead cells was carried out as did during sample preparation for am analysis, then the cell suspension was stained anti-mouse f4/80-fitc (clone bm8), anti-mouse cd11c-pe-cy7 (clone n418) and anti-mouse cd206-pe (clone c068c2). following incubation, samples were analyzed with flow cytometer. isotype antibodies (clone rtk2758 for f4/80; clone htk888 for cd11c; clone rtk2758 for cd206; clone rtk4530 for cd11b; clone rtk4174 for ly6c; clone rtk2758 for ly6g) were used to detect nonspecific binding. the gating strategies for analyzing am and im were according to previous report [21] . total rna from purified blood monocytes and lung tissue was isolated using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. total rna (2 μg) was reverse-transcribed into the cdna using a reverse transcription assay (promega, madison, wi, usa) in 25 μl of reaction volume according to the manufacturer's instructions. real-time pcr was performed with sybr green pcr master mix (roche diagnostics, indianapolis, in, usa) on an abi prism 7300 sequence detection system (applied biosystems, foster city, ca, usa) in triplicate and according to a two-step pcr protocol (5 min at 95°c, 40 cycles for 30 s at 95°c, 1 min at 60°c). the primer sequences are shown in table 1 . relative expression of real-time pcr products were normalized for expression of the β-actin and expressed as transcript fold change over ns mice using the 2 -△△ct method [22] . the levels of transforming growth factor β1 (tgf-β1), monocyte chemoattractant protein-1 (mcp-1)/chemokine (c-c motif) ligand 2 (ccl2), interleukin-4 (il-4), and interleukin-1β (il-1β) in the balf were measured by commercially available elisa kits (r&d systems, minneapolis, mn, usa), according to the manufacturer's instructions. all data are presented as the mean ± standard error of mean (sem). statistical analysis was performed using graphpad prism 5.0 software (graphpad, san diego, ca, usa). statistical comparison of multiple groups was performed by one-way anova with bonferroni post-hoc test or kruskal-wallis test followed by dunn's multiple comparisons (inflammation score and fibrosis index). a two-tailed p value less than 0.05 was considered statistically significant. by using magnetic bead-based monocyte enrichment method, more than 90% of the harvested cells were ly6g-cd11b+ ( figure 1a) . then we confirmed mr mrna expression in these cells by real-time pcr and pcr product electrophoresis ( figures 1b) . then, the mr protein expression of enriched monocytes was further validated by immunofluorescent staining (figure 1c) . using mouse balf, we also confirmed mr expression in alveolar f4/80+ macrophages ( figure 1d) . these results suggest that mr is expressed in mouse mononuclear phagocytes, which provides a basis for pharmacological intervention. figure 2 shows the detailed research protocol of in vivo pharmacological intervention study. figure 3 (a to h) shows the representative h.e. stained lung sections on day 7, which represents the peak magnitude of lung inflammatory response following bleomycin instillation. spironolactone treatment could significantly reduce the inflammatory response induced by bleomycin ( figure 3i) . panel j in figure 3 shows the results of differential cell counts from the balf that harvested on day 7. typically, the total fluid recovery was over 80% in all animals and the percentages of fluid recovered were not significantly different across all treatment groups. in agreement with histological findings, spironolactone treated lungs exhibited decreased total cell, macrophage, lymphocyte, neutrophil infiltration and esosinophils in alveoli. next, we measured the levels of inflammatory and profibrotic cytokines in the balf and determined related gene expression levels in lung tissue. as shown in figure 4 , compared with blm and blm+ns groups, spironolactone treatment was associated with downregulated ccl2/mcp-1, tgf-β1 and il-1β both at the mrna and the protein levels. in addition, markers for m2 polarization, such arginase-1 (arg-1) mrna level in lung tissue (figure 4g) , and il-4 protein content in balf ( figure 4f) were downregulated by spironolactone. figure 5 shows the profibrotic response using lung tissue that harvested on day 21. the histological analysis showed that mr antagonism was associated with reduced collagen deposition and α-sma positive cells (myofibroblasts). compared with ns group, the expression of type i and type iii collagen mrna in the lungs from blm and blm+ns groups were significantly upregulated, whereas spironolactone treatment could partially regress bleomycin-induced collagen expression upregulation, which was consistent with the histological findings. we next evaluated the effect of spironolactone treatment on circulating monocyte subset change. figure 6a shows the gating strategies for circulating monocyte subset analysis. as shown in figure 6b , compared with ns group, blm treated mice exhibited a significant increase of ly6c hi monocytes, starting from day 1, reaching the plateau level on day 3, then followed a gradual decrease till day 14. spironolactone treatment could significantly reduce bleomycin-induced the ly6c hi monocyte pool expansion on day 3 and thereafter. the reciprocal changes of ly6c lo monocyte subset is shown in figure 6c . using enzymatically digested lung tissue, we evaluated interstitial macrophage phenotype changes during drug intervention. as shown in figure 7b , one day after bleomycin challenge, the majority (more than 90%) of interstitial macrophages presented with a m1-like phenotype (f4/80+cd11c-cd206-), followed by a gradual decreasing trend of the proportion of m1-like macrophages, and this trend reached statistical difference on day 21. moreover, compared with blm and blm+ns groups, spironolactone has no obvious influence on interstitial macrophage phenotype switching induced by bleomycin. then we investigated the impact of spironolactone on alveolar macrophage phenotype changes. as shown in figure 8b , alveolar macrophages in ns group were mainly (more than 80%) presented with a m1-like phenotype (f4/80+cd11c +cd206-). after bleomycin challenge, there was a quick decrease of m1-like macrophage with a concomitant increase of m2-like phenotype (f4/80+cd11c+cd206+). whereas in spironolactone treated mice, this trend was partially normalized, indicating an inhibitory effect on alternative activation by mr antagonism. recent studies showed that the renin angiotensin aldosterone system (raas) plays an important role in the pathogenesis of lung injury [23] [24] [25] . in addition, the therapeutic efficacy of drug intervention targeting this system has been reported in bleomycin-induced lung injury models [26] [27] [28] [29] [30] [31] . zhao and coworker first demonstrated the therapeutical potential of spironolactone in ameliorating bleomycin-induced lung fibrosis [32] , which is also supported by a recent study [33] . a growing body of evidence suggests that manipulation of the mononuclear phagocyte phenotype switching could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis in experimental models [34] [35] [36] . it has been demonstrated that mr plays an important role in regulating myeloid cell phenotype switching in different disease conditions [16, [37] [38] [39] [40] . to our knowledge, the role of mononuclear cell mr in mediating acute lung injury induced pulmonary fibrosis has not been addressed. the present work confirmed that mr antagonism by a clinically approved drug, spironolactone, could attenuate bleomycin-induced acute lung injury and fibrosis. specifically, mr inhibition partially attenuates ly6c hi monocyte expansion in circulating compartment and normalizes disturbed balance of macrophage polarization in alveolar compartment, leading to reduced alveolitis and collagen deposition in lung tissue. these findings highlight mononuclear phagocyte mr as a promising target for ameliorating acute lung injury and profibrotic response in lungs. the raas is a hormone system which acts on multiple physiologic pathways by regulating blood pressure and fluid balance. as the terminal effector of the raas cascade, the role of aldosterone/mr signaling has been recently implicated the pathogenesis of cardiovascular diseases, insulin resistance and diabetes, and chronic inflammation associated fibrosis [41] [42] [43] . these effects are supported by the fact that in addition to the kidney, there is a wide tissue distribution of mr, such as cardiomyocytes, endothelial cells, vascular smooth muscle cells, adipocytes and macrophages [44] . here, we demonstrated that mr is expressed both in purified murine circulating ly6g-/cd11b+ monocytes and in f4/80+ alveolar macrophages, providing a basis for mr regulation of monocyte/ macrophage phenotype switching. macrophages are professional phagocytic cells with different transcriptional profiles and functional capabilities depending on their origins from various organs [45] . broadly speaking, the lung tissue contains two tissue-resident macrophage compartments, i.e., alveolar macrophages and interstitial macrophages. the traditional belief that tissue-resident macrophages are derived from circulating monocyte progenitors has been challenged by recent fate mapping studies by showing that the steady-state turnover of alveolar macrophages is extremely low: 8 to 12 months after bone marrow transplantation, 70%-60% of alveolar macrophages are host derived [35, 46] . in addition, recent studies demonstrated that lung alveolar macrophages are established prior to birth and maintains themselves subsequently during adulthood independent of replenishment from circulating monocyte input in steady state [47, 48] . on the contrary, during acute lung inflammatory response, circulating monocytes have an important impact on the lung macrophage dynamics. in general, recent studies are in agreement with the notion that following injury, there is an increased accumulation of m2-like mononuclear cells in alveoli [10, [49] [50] [51] [52] . moreover, in patients with chronic obstructive pulmonary disease, a skewing of alveolar macrophages from an m1 to m2 phenotype has been observed [50, 53] . however, with regard to the origin of these m2-like cells, some controversy existed. in an endotoxin-induced lung inflammation model, maus and coworkers showed that despite a rapid recruitment of monocytes in lung tissue, the resident alveolar macrophage pool remained static throughout the duration of inflammation and the expansion of the lung macrophage pool was mainly mediated by an influx of the circulating monocytes, followed by their differentiation into tissue macrophages [46] . in agreement with this finding, recently osterholzer et al [54] , using a gene-targeted alveolar injury model, demonstrated an increased exudate macrophages and their progenitors, ly6c hi monocytes, both exhibiting m2 polarization in alveoli. in another study [36] , gibbons and colleagues adoptively transferred ly6c hi monocytes into bleomycin-treated mice during the progressive phase of lung fibrosis, which led to an exacerbation of disease progression and an increased accumulation of m2-like macrophage in the lung. surprisingly, these alternatively activated macrophages were host derived and not from the donor ly6c hi monocytes. as a corollary, regardless of their origins, our current knowledge points to a general scheme of their relationship: initially, acute lung injury induces a rapid expansion and infiltrating ly6c hi monocytes in lung tissue, which contributes to a paralleled increase of m2like macrophages (by direct differentiation or by paracrine effects) in alveolar compartment, and the severity and persistency of m2 polarization in alveolar macrophages would ultimately influence inflammation resolution and fibrosis. the above model highlights the circulating ly6c hi monocytes as a therapeutic target. although we did not use a monocytetargeted approach to suppress ly6c hi monocytosis, it is likely that spironolactone would also exert its major pharmacological effect on circulating monocyte pool since the efficacy of orally administered drug is significantly compromised by its inability to reach alveolar space at an appropriate concentration [55] . additionally, because evidence shown that monocyte infiltration would facilitates alveolar neutrophil emigration and determines the ongoing neutrophil influx in the persistent phase of acute lung injury [56] [57] [58] , suppression of ly6c hi monocytosis by spironolactone would concomitantly lead to a decreased tissue accumulation of neutrophils, which is also observed in our study. the present work has the following limitations. first, because spironolactone has anti-androgen effect, the observed effects of this work cannot be totally ascribed to mr antagonism. indeed, there is a sex discrepancy in bleomycin-induced lung fibrosis, and estrogen may have protective effect on this model [59, 60] . in this regard, mr knockout mice are preferred to address this issue. second, we did not observed significant changes in lung interstitial macrophages by spironolactone. previous study showed this population might have a role in limiting inflammation and fibrosis [61] . thus it remains unclear whether cd206 is an appropriate m2 marker for this population as recent study showed that the change of cd206 is modest after bleomycin challenge [51] , or this population is insensitive to mr inhibition, or due to enzymatic digestion-induced surface marker loss during sample preparation, a commonly encountered technical issue. third, due to the wide distribution of mr in the body, the mechanistical explanation of global mr antagonism is fairly complex. for example, aldosterone has been implicated in the pathogenesis of pulmonary hypertension [62] , and spironolactone has been shown to attenuate experimental pulmonary hypertension via mr inhibition in pulmonary artery smooth muscle cells [63] .admittedly, bleomycin is also a frequently used tool drug to induce pulmonary hypertension [64, 65] . the downregulation of α-sma by spironolactone observed in this study, also support an antifibrotic effect of spironolactone on fibroblasts. moreover, the functional expression of mr has been demonstrated in neutrophils [66] , which may also participate in spironolactone induced amelioration of lung fibrosis, as shown by reduced neutrophil count in balf. thus, in addition to its effect on mononuclear phagocytes, the mechanisms underlying therapeutic effect of systemic use of spironolactone on bleomycin-induced lung injury is multifactorial. forth, it seems obscure to interpret the effect of mr antagonism on alveolar macrophage polarization, since macrophages lacking myeloid mr exhibit alternative activation (m2 polarization), whereas our results showed that mr inhibition could reduce alveolar m2 polarization. it should be noted the long-established binary classification of macrophage in terms of classical (m1) and alternative activation (m2) is based on in vitro studies [67] . indeed, a recent study demonstrated eplerenone, another clinically approved mr antagonist, promotes alternative activation in human monocyte-derived macrophages [68] . however, macrophages in vivo maintain their plasticity and can alter their phenotype based on the microenvironment, including cytokine milieu among other factors [10] . as pointed early, drug administration via oral route, cannot reach alveolar space at an appropriate concentration. therefore, the alterations in alveolar macrophage polarization state cannot be ascribed to mr antagonist's direct effect. it is conceivable that suppression of inflammatory (ly6c hi subset) monocyte expansion should be the direct effect by spironolactone, which ameliorates lung injury via the "ly6c hi directed pulmonary alterative activation" mechanism [36] . thus, future monocyte-targeted approaches, as well as in vitro studies are warranted to elucidate the molecular mechanism underlying suppressed ly6c hi monocytosis by mr antagonism. finally, the algorithms used in this study are relatively simple, and may inadvertently contain dendritic cells and eosinophils. more rigorous and sophisticated algorithms have been published and suggested [51, 69] . in conclusion, the present work provides the experimental evidence that mr antagonism by spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially by reducing circulating inflammatory ly6c hi role of the chemokine receptor cxcr2 in bleomycin-induced pulmonary inflammation and fibrosis incidence and prevalence of idiopathic pulmonary fibrosis: review of the literature idiopathic pulmonary fibrosis strategies for treating idiopathic pulmonary fibrosis lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s acute lung injury: how macrophages orchestrate resolution of inflammation and tissue repair macrophage-derived biomarkers of idiopathic pulmonary fibrosis the role of macrophages in healing the wounded lung macrophage biology in development, homeostasis and disease pulmonary macrophage subpopulations in the induction and resolution of acute lung injury alternative activation of macrophages: an immunologic functional perspective development of monocytes, macrophages, and dendritic cells epigenetic regulation of macrophage polarization and function suppressors of cytokine signaling 2 and 3 diametrically control macrophage polarization micrornas in immune response and macrophage polarization myeloid mineralocorticoid receptor controls macrophage polarization and cardiovascular hypertrophy and remodeling in mice aggravation of bleomycin-induced pulmonary inflammation and fibrosis in mice lacking peroxiredoxin i postinfarction healing dynamics in the mechanically unloaded rat left ventricle bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse bleomycin and il-1beta-mediated pulmonary fibrosis is il-17a dependent innate lymphoid cells mediate influenza-induced airway hyperreactivity independently of adaptive immunity analysis of relative gene expression data using real-time quantitative pcr and the 2 angiotensinconverting enzyme 2 protects from severe acute lung failure angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin ii type 1 receptor blocker inhibits pulmonary injury preventive effect of irbesartan on bleomycin-induced lung injury in mice angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury angiotensinconverting enzyme n-terminal inactivation alleviates bleomycin-induced lung injury losartan attenuates bleomycin induced lung fibrosis by increasing prostaglandin e2 synthesis angiotensin ii type 2 receptor antagonist reduces bleomycin-induced pulmonary fibrosis in mice mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models the lung macrophage: a jack of all trades fas determines differential fates of resident and recruited macrophages during resolution of acute lung injury ly6chi monocytes direct alternatively activated profibrotic macrophage regulation of lung fibrosis deletion of mineralocorticoid receptors from macrophages protects against deoxycorticosterone/salt-induced cardiac fibrosis and increased blood pressure macrophage-mediated inflammation in metabolic disease myeloid-specific deletion of the mineralocorticoid receptor reduces infarct volume and alters inflammation during cerebral ischemia nuclear receptor control of opposing macrophage phenotypes in cardiovascular disease role of the renin-angiotensin-aldosterone system in the pathogenesis of atherosclerosis mechanisms of disease: local renin-angiotensinaldosterone systems and the pathogenesis and treatment of cardiovascular disease mineralocorticoid receptors, salt-sensitive hypertension, and metabolic syndrome extrarenal effects of aldosterone geneexpression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes a vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via ccl18 alternatively activated alveolar macrophages in pulmonary fibrosismediator production and intracellular signal transduction flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung low levels of insulin-like growth factor-1 contribute to alveolar macrophage dysfunction in cystic fibrosis smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease implicating exudate macrophages and ly-6c(high) monocytes in ccr2-dependent lung fibrosis following gene-targeted alveolar injury liposome-based drug delivery to alveolar macrophages monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the ccl2-ccr2 axis in vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation monocytes control second-phase neutrophil emigration in established lipopolysaccharide-induced murine lung injury male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis what is the clinical relevance of different lung compartments? aldosterone inactivates the endothelin-b receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension mineralocorticoid receptor antagonism attenuates experimental pulmonary hypertension lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-alpha aldosterone abrogates nuclear factor kappab-mediated tumor necrosis factor alpha production in human neutrophils via the mineralocorticoid receptor transcriptional regulation of macrophage polarization: enabling diversity with identity eplerenone promotes alternative activation in human monocyte-derived macrophages identification of myeloid cell subsets in murine lungs using flow cytometry key: cord-011408-z8lw8nc6 authors: peters, matthew j. title: electronic cigarettes: tumultuous times date: 2019-11-06 journal: respirology doi: 10.1111/resp.13725 sha: doc_id: 11408 cord_uid: z8lw8nc6 see related https://onlinelibrary.wiley.com/doi/10.1111/resp.13696 'e-cigarettes will prompt young people to take up smoking. i recommend that national governments ban, or at least regulate, them' dr margaret chan-former who director-general, china daily 2015 1 in a keynote lecture at the 2017 american thoracic society conference, dr anthony s. fauci, head of the national institute of allergy and infectious diseases, described how he had been part of a briefing for president trump's transitional team in late 2016. the briefing was from a range of experts and was related to civil emergencies that trump might face during his presidency. having served under four previous presidents, dr fauci warned the team that each administration had faced at least one major infectious disease emergency-human immunodeficiency virus/acquired immune deficiency syndrome (hiv/aids) west nile virus, sars, 2009 h1n1 influenza, ebola and zika virus. less than 3 years later, it appears that donald trump's developing emergency is not an infectious disease but two phenomena related to electronic cigarettes (ec)-an extraordinary rise of ec use in youth 2 and a multistate outbreak of lung injury associated with ec product use. 3 a common link in the current outbreak, if it exists, has not been determined. diverse pathologies have been reported, as has been the case with the sporadic case reports of acute lung injury related to ec since the first use in 2012. 4 at the time of writing, there had been nearly 1604 cases reported using the centers for disease control and prevention (cdc) case definition and 34 deaths 5 -the youngest reported in the lay media being just 17. 6 respiratory failure requiring ventilatory support is common. the most common imaging findings are suggestive of bronchiolitis obliterans/cryptogenic organizing pneumonia or diffuse ground-glass opacities. 7 in the previous case reports, the course was described as favourable after cessation of ec use, but it is too early to determine the extent of residual lung injury in the current outbreak. a range of exposures are reported but, as the cdc has made clear, the investigation is ongoing. 8 many have used cannabis oil or cannabinoids, but whether that use was sporadic or regular is uncertain, and this exposure cannot explain all cases. those who have publicly claimed that contaminants must be responsible ignore the geographical dispersion of cases and the spread in time of symptom onset and seem oblivious to the reality that, from the perspective of the lung, everything in an ec is a contaminant. what is most surprising at the present time is that so many are surprised that such an outbreak might occur. the human bronchial epithelium is exposed; it is vulnerable but also resilient with innate mechanisms that are protective and also adaptive after injury. ween et al. recently reported findings of a carefully conducted study on the effects of e-liquid exposure in human bronchial epithelial cells. 9 there were three key findings. ec liquids, with a variety of constituents, induce damage that manifests as necrosis and apoptosis; macrophage efferocytosis, an adaptive mechanism that clears apoptotic cells, is compromised; and purchasers of ec liquids can have no confidence in the constituents that they are exposing their lungs to-with three versions of apple flavour having very different chemical mixes. the observations of ween et al. have even greater pertinence after the report of histopathology from 17 cases within the current outbreak. 10 open biopsy findings suggest that the dominant pathology is a form of airway-based chemical pneumonitis and not exogenous lipoid pneumonia as previously believed by some. these findings complement an nih-funded comprehensive 2017 review by chun et al. on the effects of ec on the lung, which examined a combination of in vivo and in vitro studies. 11 since that publication, we have also seen the seminal work of ghosh et al. who observed airway inflammation in a man in vivo, describing the proteomic characteristic of bronchial tissue in smokers, ec users and controls. 12 in summary, considering significant positive and negative changes, there were 292 changes seen with smoking, of which 78 were also seen with ec use. importantly, there were 113 separate proteomic changes that occurred only with ec use. this would not be unexpected by an open mind because the nature of the lung exposure is very different. youth use of ec in the united states has been described as an epidemic by the food and drug administration (fda), 13 with this assertion further supported by the latest data from the monitoring the future study. 2 ec use in the last 30 days was 25.4% in 12th grade school students, 20.2% in 10th graders and 9.0% in 8th graders, more than doubling in 2 years for each group. daily ec use as defined, not previously part of that survey, was 11.8%, 6.9% and 1.9% in the three grade groups, respectively. in an unrelated study, high or rising ec use in young people aged 16-19 years in the uk, united states and canada has not been associated with a decline in smoking, and in canada, smoking has significantly increased. 14 the always dubious proposition that ec would accurately displace smoking amongst young people without collateral damage 15 can now be filed under 'speculative fallacy'. concurrent with the lung disease outbreak and the emerging data for children, action by major regulators is being taken. in the united states, the fda has determined that juul labs adulterated its products by selling or distributing them as modified-risk tobacco products. specific concern was voiced about juul labs referring to its products as '99% safer', 'much safer', 'totally safe' and 'a safer alternative'. 16 in that warning letter, dated 9 september 2019, the fda stated that its concern is 'amplified by the epidemic rate of increase in youth use of ends products, including juul labs' products, and evidence that ec products contribute to youth use of, and addiction to, nicotine, to which youth are especially vulnerable'. on the basis of these events and data showing that fruit and candy flavours are selectively important for ec uptake by young people and never smokers, 17 a range of national and state governments have moved quickly to ban all or selected flavours and/or flavour descriptions. in the asia-pacific, india and timor leste have recently banned ec completely. in a market increasingly dominated by parties related to major tobacco companies, these decisive actions are most likely when governments can be free from influence by the tobacco industry and its acolytes. since dr chan made her statement in 2015, the world health organization (who) has been pressured to effectively dilute article 5.3 of the framework convention on tobacco control (fctc), which enshrines policy independence, by widening the pool of stakeholders. any belief that the "harm reduction" argument, or funding scientists through the foundation for a smoke-free world, could be successfully used as a pretext to create ambiguity around article 5.3, seems to have been debunked in the latest who report on the global tobacco epidemic that described tobacco industry interference as the greatest obstacle to reducing tobacco use and specified government actions that would protect policies from vested interests. 18 for those who have expressed concerns about the net benefits of ec and recommended precaution, these events bring neither a sense of vindication nor any joy-even if policy steps that we have advocated for are now being taken. young people are being harmed. far too many people of a range of ages are becoming seriously ill with lung disease, and some are not surviving their illness. it is a tumultuous time-one for concerted and continued actions. tougher controls urged for electronic cigarettes trends in adolescent vaping characteristics of a multistate outbreak of lung injury associated with e-cigarette use or vaping-united states an unexpected consequence of electronic cigarette use outbreak of lung injury associated with e-cigarette use, or vaping bronx teenager's death is the youngest vaping fatality in u.s imaging of vaping-associated lung disease outbreak of lung injury associated with e-cigarette use, or vaping effects of e-cigarette e-liquid components on bronchial epithelial cells: demonstration of dysfunctional efferocytosis pathology of vaping-associated lung injury pulmonary toxicity of e-cigarettes chronic ecigarette exposure alters the human bronchial epithelial proteome fda takes new steps to address epidemic of youth e-cigarette use, including a historic action against more than 1,300 retailers and 5 major manufacturers for their roles perpetuating youth access prevalence of vaping and smoking among adolescents in canada, england, and the united states: repeat national cross sectional surveys potential deaths averted in usa by replacing cigarettes with e-cigarettes us food & drug administration. warning letter juul labs (inc) the role of flavors in vaping initiation and satisfaction among u.s. adults world health organization. who report on the global tobacco epidemic key: cord-022136-3q24qxsr authors: maru, yoshiro title: explanation of metastasis by homeostatic inflammation date: 2016-02-02 journal: inflammation and metastasis doi: 10.1007/978-4-431-56024-1_15 sha: doc_id: 22136 cord_uid: 3q24qxsr if inflammation caused by either non-self or self molecules can disseminate throughout the body and inflammatory sites actively allow entry of circulating tumor cells and assist regrowth, then circulating tumor cells metastasize to the sites of inflammation. however, disrupted sites of homeostatic inflammation do not necessarily guarantee metastatic spread and subsequent regrowth. if inflammation caused by either non-self or self molecules can disseminate throughout the body and inflammatory sites actively allow entry of circulating tumor cells and assist their regrowth, then circulating tumor cells metastasize to the sites of inflammation. however, disrupted sites of homeostatic inflammation do not necessarily guarantee metastatic spread and subsequent regrowth. before explaining how tumor cells regrow in premetastatic organs with inflammatory properties, there is a fundamental issue of whether or not extrinsic factorinduced inflammation, systemic or localized, facilitates metastasis in the inflammatory lesion. although a rare case of metastasis of lung cancer to a minor traumatic lesion has been reported, mechanical injury seldom allows metastatic growth of tumor, otherwise surgeons lose their job. radiation to the thorax or pelvis of tumor-non-bearing normal mice increased serum tgfβ1 levels by twofold for at least 7 days [1] . mmtv/pymt transgenic model of metastatic mammary tumor showed that thoracic irradiation at 10 gy by shielding other portions of the body enhanced lung surface metastasis by threefold at 2 weeks after irradiation. this took place in a manner dependent on tgfβ1 in the tumor cells, because conditional depletion of tgfβrii using cre/lox technology reversed the effect. tgfβ1, which is induced by irradiation in a tumor-independent manner, is thought to facilitate directly the survival of the tumor cells. in this case, the lungs were not irradiated to cause inflammation but tumor burden by itself induce premetastatic conditions in the lungs and irradiation-induced serum tgfβ1 activates ctc to reach the lungs. uv-irradiated epidermal keratinocytes secrete hmgb1, which activates tlr4 to recruit neutrophils in the irradiated skin in which cd11b + ly6c + ly6g + cells dominate [2] . in transgenic mouse model of melanoma that overexpressed hgf and an oncogenic cdk4, repeatedly irradiated mice by uv for 6 weeks exhibited some melanoma cells expanding along the endothelial cells, often observed in human melanoma. this angiotropism enhanced lung metastasis, both of which were abrogated in ko mice of either tlr4 or myd88. also in this experiment, lungs were not treated by uv, but the generated melanoma by uv prepared premetastatic soil in the lungs. in both experiments, irradiation of tumor induced host cell responses, which provide favorable conditions for tumor cells to achieve metastasis, such as potentials to intravasate and survive. meta-analysis of 4049 patients who suffered from chronic liver diseases, including fatty liver, chronic hepatitis virus b or c infection, and cirrhosis, revealed significantly lower rates of liver metastasis in colorectal cancer (crc) [3] . readers should remember that the organ first encountered by circulating tumor cells from the primary tumor is liver by crc, lungs by any tumors that do not drain into the portal vein, and any organs by lung cancer. a more focused analysis of chronic viral hepatitis (cvh) b and c, liver metastasis took place in 2.86% (2 of 70 cases) compared with 16 .9% (48 of 284) in non-cvh. in c57bl/6 mice with cirrhosis, which was induced by carbon tetrachloride gastrogavage for 2 months, b16f1 melanoma cell injection via the portal vein caused seven-to ninefold increase in frequency of tumor in the liver by histological analysis in cirrhosis over the untreated group [4] . no surface metastatic nodules were found in the lungs. intravital kinetic analysis showed that the tumor cell movement was slower in the cirrhotic group. given the presence of regeneration, fiber generation and inflammatory cells in both clinical autopsy and experimental mouse settings, i assume that inflammatory nature in the liver and its matching with tumor cell property may make a difference. treatment of b16 melanoma cells with lipopolysaccharide (lps) or lipid a at 1 μg/ ml each for 48 h, which induced ccl2 expression, followed by extensive washing and subcutaneous implantation, reduced the tumor growth compared with untreated b16 cells in both wild-type and tlr4-ko mice. however, lps promotes tumor cell migration in vivo [5] . supportive evidence has been provided by experiments in which direct intratracheal administration of either escherichia coli or lps before injection of tumor cells, such as b16f10 melanoma or rm-9 cells, through the tail vein enhanced lung metastasis at 14 days after tumor challenge [6] . tumor cell injection at any time points earlier than 6 h, but not later than 3 days, of lps was effective for the enhancement. bal fluid 6 h after lps had increased amounts of il-6, g-csf, kc, sdf-1, and extracellular ubiquitin. the major chemokine receptor of neutrophils in acute lung inflammation is cxcr2, which is activated by mip-2 and kc (remember that kc is a rodent version of il-8 in humans). as with tlr4, cxcr2 is expressed in both blood and non-blood cells, both of which contribute to neutrophil accumulation and pulmonary permeability in the lungs. both ubiquitin and sfd-1 are known to bind cxcr4, and cxcr4 inhibitor amd3100 blocked the enhancement of metastasis. the reasonable but important point is that they found no metastasis in the extrapulmonary organs. in our laboratory, lps administration through a catheter only to the left bronchus with microspheres to make sure of the stimulation sites induced vascular permeability at 48 h and accumulation of intravenously injected labeled tumor cells in the left but not right lung. therefore, a local tlr4 activation of a given pulmonary segment causes a limited inflammatory space to which tumor cells are allowed to metastasize. if the lung tlr4 is activated by an endocrine manner, the whole lungs should be transformed to be premetastatic soil. mmtv-pyv mt mice display no primary mammary tumor at 9 weeks, but during the period of 18-26 weeks tumors with spontaneous metastasis appear. rheumatoid arthritis mouse model by type ii collagen injection was performed intradermally at 9 weeks of mmtv-pymt mice to induce arthritis in 11-13 weeks of tumor development [7] . the combined systems at early developmental stages of mammary tumor are assumed to see the systemic inflammatory effect at the premetastatic phase. bone metastasis took place only in the arthritis mice, and more than twofold increase in lung metastasis was observed in the same group. the arthritis induction caused leukocyte infiltration in the lungs, which was enhanced by roughly fourfold in the background of mammary tumor. elevation of factors in the lungs and circulation was detected, including il-17, il-6, and pge2. anti-il-17 antibody and cox-2 inhibitor both reduced the metastasis. the same group demonstrated that arthritis induced mast cell recruitment in the lungs in a tumorindependent manner, but tumor cell engagement enhanced the mast cell activation underpinning the enhanced metastasis [8] . anti-c-kit antibody also was effective to reduce metastasis. importantly, the detailed experiments tell us that premetastatic soil can be cultivated by inflammation of other organs. the experimental method to induce allergic inflammation in lungs has been established. intraperitoneal injections of chicken ovalbumin (ova) followed by repeated aerosol challenges can provoke mucus accumulation in respiratory tracts and airway hyperresponsiveness, serving as an asthma model [9] . the james j. lee group has demonstrated that cd4 + t-cell-dependent recruitment of eosinophils in both bronchoalveolar lavage fluid (bal) and interstitial peribronchial and perivascular areas are required for the asthmatic phenotypes by engineering eosinophil-lacking phil mice in which expression of cytocidal diphtheria toxin is congenitally driven by the eosinophil peroxidase gene promoter. gαi2-dependent ccr3 signaling promotes transendothelial migration of eosinophil [10] . when b16f10 melanoma cells were injected through the tail veil after aerosol challenges, numbers of metastatic foci on the pulmonary surface were increased by threefold [11] . t cell depletion by anti-cd4 antibody (gk1.5), corticosteroid inhalation and gαi2-ko condition, but not the phil background, abrogated the enhanced metastasis. therefore, it is not eosinophils by themselves but cd4 + tcell-dependent soil preparation to allow transendothelial migration of cells that appears to play a critical role for allergy-facilitated lung metastasis. the same group provided data of 176 breast cancer patients with lung metastasis. approximately 13% suffered from asthma but with no corticosteroid treatment, which is about twice the frequency of asthma in a random population of american females. cc10, alternatively called club cell secretory protein (ccsp) or uteroglobin (ug), is secreted by club cells in a steroid-inducible manner. cc10-ko mice are susceptible to lps inhalation with increased leukocytes in bal 24 h after lps [12] . the concentration gradient of s100a8 was detected in the lungs down to blood in cc10-ko mice [13] . cc10-ko enhanced lung metastasis after injection of b16f10 melanoma cells through the tail vein. although s100a8 was not expressed in b16f10 cells, they expressed rage whose ligand includes age and s100a8/ s100a9. an anti-rage sirna-mediated knockdown of rage in b16f10 prior to the tail veil injection suppressed lung metastasis. i assume that cc10-ko lungs are already cultivated by constant assaults of bacteria with lps for the expression of s100a8 and s100a9 before tumor cell injection. the calcineurin-nf-atc pathway has been known as located downstream of antigen-presenting cell engagement to t cells but also is activated individually by the vegf-vegfr2 and thrombin-par-1 systems [14] . both exert vascular permeability as i stated earlier. from cdna microarray experiments of huvec stimulated by vegf and thrombin, down syndrome critical region-1 (dscr-1, also called calcipressin 1, mcip-1, and adapt 78) was found dramatically induced by 22.3-fold and 17.7-fold, respectively at 1 h [15] . it turned out that dscr-1 inhibited nuclear localization of nf-atc in huvec, i.e., inhibited calcineurin activity by direct interaction. the nf-atc-mediated transcription induces expression of cytokines, such as il-6, but simultaneous induction of the inhibitory molecule dscr-1 forms a negative feedback loop to brake hyperactivation of the cytokine storm. the expression of icam-1, e-selectin, and vcam-1 were negatively affected by more than 50% in thrombin-stimulated huvec infected with dscr-1 adenovirus [16] . not only the mrna expression level but also number of genes was down-regulated by dscr-1. the same author stated that 52 of 172 thrombinactivated genes in huvec were down-regulated by dscr-1 overexpression. given that both vegfr2 and par-1 provoke inflammation, the autoinhibitory mechanism serves to avoid an overshoot and maintain the oscillating physiological levels of calcineurin-nf-atc activity. therefore, this also can be called homeostatic inflammation. adenovirus-mediated gene transfer of dscr-1 in b16 melanoma xenograft at approximately 50 mm 3 tumor size, in which angiogenesis was actively taking place, inhibited the tumor growth. however, syngrafts of murine renal carcinoma (renca) and colon cancer (mc26) in dscr-1 à ko mice displayed diminished tumor growth with significant reduction in sma + cd31 + vessels [17] . mechanistically, constitutive activation of calcineurin not only induced precocious nf-atc nuclear localization in a cyclosporine-inhibitable manner but also dephosphorylated bad to activate the apoptosis pathway in endothelial cells. cerebral hemorrhage was detected in 15% of dscr-1-ko at embryonic days 10 and 12 (e10 and e12). those experiments tell us important logics in homeostasis. there are several modes in negative feedback by molecules downstream, including catalytic inhibition of the molecule upstream by keeping the cells alive and cancellation of the biological outcome, such as growth, by eliminating the cells by apoptosis. it depends on the developmental stage of embryos and tumors. another example of endothelial homeostasis is vegf. anti-vegf antibody also resulted in side effects, such as brain hemorrhage [18] . vegf-ko mice are embryonic lethal [19] . in ectopic syngraft model with egfp-labeled b16f10 melanoma cells, premetastatic lungs were assumed by 12 days after subcutaneous implantation [20] . they found fibrinogen-fibrin deposit generated by vascular instability that is caused by expression of angiopoietin 2 (ang2), mmp3, and mmp10. they could hardly detect up-regulation of vegf in the lungs and claimed that both tgfβ and tnfα were responsible for up-regulation of those molecules, sirna-mediated knockdown of which resulted in suppressed infiltration of both myeloid cells and tumor cells. ang2 is usually expressed in vascular remodeling sites in which vessels are more plastic with loosened endothelial junctions. our experiments showed that blocking tgfβ and tnfα individually suppressed up-regulation of s100a8 and s100a9 in premetastatic lungs of tumor-bearing mice. when b16f10 melanoma cells were injected via the tail vein to dscr1-lacz mice, in which lacz expression was engineered to manifest the calcineurin-nf-atc activity, tyrosinase mrnas were not detected until day 7 after tumor cell injection. because it could be detected as early as days 3-5 before tumor cell arrival, they assumed the period as the premetastatic period. interestingly, b16f10 injection in dscr-1-ko mice significantly enhanced lung metastasis as judged by day 10. orthotopic renca tumor model and llc footpad model also aggravated lung metastasis in dscr-1-ko mice, while growth of the primary tumor was suppressed as just described. the difference between the primary sites and metastatic lungs turned out to be ang2 expression in the lungs without endothelial apoptosis. up-regulation of both vegf and activated vegfr2 was recognized in the tumor-injected dscr-1-ko mice. i have repeatedly stated the importance of vascular permeability in inflammation ( fig. 15. 1; see also chap. 14). what will happen when endothelial cells are systemically injured? it has been intensively studied from the standpoint of hypertension and vascular tone. vascular tone is how much a vessel is constricted compared with maximum dilatation with largest lumen diameters. for example, angiotensin ii (atii) (amino acids 1-8) increases vascular tone, while by atrial natriuretic peptide (anp) decreases it. an early study in 1972 showed that atii induced vascular permeability with interstitial edema in rabbit aorta and dermis in 30 min with evans blue as an indicator, which was accompanied by widening of inter-endothelial gaps by endothelial contraction [21] . atii-stimulated vessels secrete pge, which further increases permeability and induces vasodilatation. atii infusion for 14 days in mice displayed hypertension, reduced acetylcholine-mediated relaxation, increased maximum response to norepinephrine, hypertrophic remodeling with prominent macrophage infiltration, nox2 up-regulation, and nfκb activation [22] . those are attenuated in op/op mice that are deficient in m-csf showing monocytopenia and macrophage deficiency. in the same vessel injury model under the background of ldl-r-ko to induce hypercholesterolemia, development of atherosclerosis and aortic aneurysm was reduced by whole-body genetic deletion of atii type 1a receptor (at1a) [23] . the atii-induced inflammation in vessels is accompanied by leukocyte migration through activation of endothelial at1a of both endothelial cells and vascular smooth muscle cells (vsmc) and the stimulated vsmc secretes the potent permeability factor vegf and stimulated endothelial cells secrete chemokines, including ccl2, rantes, mip-1α, and ip-10 [24] . conversely, vegf-induced vascular permeability is blocked by pharmacological inhibitors against atii receptor. here the vasoconstrictor induces inflammation-mediated increase of vascular tone. in the renin-angiotensin system (ras), angiotensinogen is processed to ati (amino acids 1-10) by renin, which is further cleaved by ace to atii. both ati and atii are inactivated by ace2, a homolog of ace, to at (amino acids 1-9) and at (amino acids 1-7), respectively. ace2 negatively regulates ras. although the baseline levels of pulmonary atii were not changed in ace2-ko mice, lung injury by acid (see chap. 6) significantly augmented its levels in ace2-ko mice, which was accompanied by severe lung edema due to enhanced pulmonary vascular permeability as judged by evans blue but not to changes in pulmonary perfusion pressures. the high permeability was abrogated in at1a-ko mice or pharmacological inhibitor for at1 receptor [25, 26] . when b16f1 melanoma cells were intravenously injected, lung metastasis was reduced in at1a-ko mice [27, 28] . conversely, oral administration of atii enhanced the metastasis. the authors showed an engagement between b16f1 cells with atii-induced expression of p-selectin glycoprotein ligand-1 (psgl-1) and platelets expressing p-selectin prompted vegf release from the platelets. it is likely that atii not only stimulates pulmonary endothelial cells to induce inflammation but also b16f1 cells to induce psgl-1 expression to facilitate the engagement. the important point is that at1a-expressing tumor cells may reach the premetastatic lungs and wait for an angiogenic switch, which could be mediated by atii that plays a homeostatic role in the lung microenvironment. macrophages also express ace to generate atii [29] . tnfα can induce vascular permeability as stated before. transgenic mice of tnfα exhibited lung emphysema and severe pulmonary hypertension (ph) with vascular remodeling, such as interstitial thickening and perivascular fibrosis [30] . because nitric oxide (no) at 25 ppm failed to restore the right ventricular pressure, ph was not due to sustained vasoconstriction. in a skeletal muscle, tnfα infusion induced increased capillary permeability but only a minor effect on vascular tone [31] . the vascular permeability factor eventually increases vascular tone after sustained tnfα-induced inflammation. knockout mice of npr-a, one of the receptors for anp, displayed systemic hypertension [32] . however, lps-induced vascular permeability in the lungs could be partially attenuated by anp infusion even in npr-a-ko mice, suggesting that anp also may use other receptors, such as npr-c, for pulmonary barrier protection [33] . therefore, anp not only decreases vascular permeability but also decreases vascular tone, i.e., causes vasodilatation. lung metastasis induced by intravenous infusion of b16 melanoma cells after pretreatment with lps was reduced by 75% by anp infusion prior to the tumor cell challenge [34] . pretreatment with atii also enhanced lung metastasis, which was inhibited by candesartan, an aii receptor blocker [35] . now i state on arachidonic acid (aa) metabolites in vascular tone. in endothelial cells, aa is metabolized to (1) pgi2 by cyclooxygenase (cox), (2) 12-or 15-hete (hydroxyeicosatetraenoic acid) by lipoxygenase (lox) and 11, 12-or 14, 15-eet (epoxyeicosatrienoic acid) by cytochrome p450 epoxygenases like cyp2c8 and cyp2j2. the eets are subsequently metabolized into dhet (dihydroxyeicosatrienoic acid) with reduced biopotency by soluble epoxide hydrolase (seh) (see fig. 1 .4 in chap. 1). endothelial cells decrease their vascular tone individually by pgi2, no, and endothelium-derived hyperpolarizing factor (edhf), such as eet [36] . independence of those factors can be understood by non-no and non-pgi2-mediated relaxation even in the presence of pharmacological inhibitors against nos and cox. to complicate the circumstances, many factors participate in their release. for example, histamine induces release of pgi2 and eet. eet also is released by acetylcholine and bradykinin. eet opens ca2+-activated k-channel and hyperpolarizes the membrane. syngeneic xenograft tumor models, including b16f10 melanoma, t241 fibrosarcoma, and llc in the background of tie2 promoter-driven cyp2c8, cyp2j2 transgenic (tg) and seh-ko mice to achieve high levels of eet in endothelial cells exhibited dramatic increase in the tumor growth with enhanced tumor angiogenesis as judged by cd31-positive cells [37] . transgenic mice of tie2-seh gave opposite effects. an enhanced corneal tumor angiogenesis was observed after systemic administration of 14,15-eet. in addition, stimulation of metastasis after removal of llc primary tumor was further augmented in the background of those eet-high mice. i underline that they observed that the affected metastatic organs were multiple, including lymph nodes, liver, and lungs even without removal of the primary tumor. the eet-high tumor-bearing mice after tumor removal showed high levels of plasma vegf and the resected tumor expressed high vegf mrnas. most importantly and intriguingly, the authors performed parabiosis experiments to share circulation between, for example, a tumor-bearing tie2-seh-ko mouse as a donor with high eet and a tie2-seh-tg mouse as a recipient with low eet. this parabiosis gave enhanced tumor growth but the opposite condition, i.e., tumor in eet-low mice failed to enhance it. the shared high levels of eet were not sufficient to enhance tumor growth and endothelial eet levels were critical. it also was the case in metastasis. a tumor-bearing tie2-cyp2c8-tg mouse with high eet failed to enhance metastasis in the recipient tie2-seh-tg mouse, demonstrating that endothelial eet levels control the metastatic microenvironment. eet is produced in endothelial cells and therefore it appears to circulate and act on endothelial cells of the whole body. however, even in conditions where eet could work in an endocrine manner, local actions in each endothelial cells appear to control its bioactivity. both transgenic and ko engineering just provides artificial circumstances in which the endothelial eet simultaneously affects all endothelial cells of the whole body. benign fat tumors are functionally malignant ( fig. 15.1 ; see also chap. 14). a similarity can be recognized between cancer and adipose tissue. ccr2-and tlr4promoted myeloid cells are mobilized in the primary tumor sites, premetastatic sites, and adipose tissues in obesity. the accumulated macrophages facilitate the tumor proliferation in the primary sites and hypertrophy of the adipocytes. therefore, adipose tissues can be assumed to be benign tumors, because adipocytes do not make metastatic progression to different locations of adipose tissues nor circulating bone marrow-derived cells differentiate into adipocytes. analyses of adipose tissue of mice that underwent bmt with gfp + bone marrow revealed that there were no gfp + cells that also were positive perilipin, an adipocyte marker [38] . a situation in which tumor cells utilize host homeostatic systems was proposed by peter bannasch in 1997 [39] (fig. 15.1) . the glycogen storage system in liver is homeostatically regulated by glycogenolysis by epinephrine and glucagon and glycogenosis by insulin. as i stated in chaps. 10 and 12, glycogenolysis in liver and active glycolysis are metabolic hallmarks in cancer. however, an early preneoplastic event common to both chronic liver disease patients with a high risk for hepatocellular carcinoma and n-nitrosomorpholine-treated chemical hepatocarcinogenesis model in rats is glycogenotic activity that mimics the insulin effect [40] . this also can be recognized as a homeostatic response to a driving force for glycogenolytic activity that still is latent. a similar phenomenon in angiogenesis is observed. semaphorin3a (sema3a) is one of the endogenous angiogenesis inhibitors by signaling through cellular cytoskeleton (see chap. 16). in biopsy samples of human uterine cervical cancer patients, sema3a was highly expressed in the epithelial cells and some endothelial cells in high-grade dysplasia called cin-3, which was totally absent in cervical squamous cell carcinomas (scc) [41] . this was supported by the hpv/e2 mouse model in which cervical tumor progression could be time-dependently observed from low-grade (cin-1/2), cin-3 lesions, and scc. sema3a was highly expressed in endothelial cells in cin-3 lesions. it is likely that the up-regulation of sema3a is a homeostatic response against the angiogenic switch by the tumor before full tumor angiogenesis. the idea of lps mimicry is based on the demonstration (see below) by us and other group that md-2, the co-receptor of tlr4 (see fig. 5 .3 in chap. 5), binds not only lps but also s100a8 and saa3. in biochemical levels, we used full-length s100a8 and saa3 proteins expressed in and purified from mammalian cells, and synthetic peptides of various lengths theoretically to avoid contamination of lps. surface plasmon resonance analyses revealed that kd is 10 nm for mammalian s100a8 and 0.356 nm for mammalian saa3 when tested with baculovirus-purified tlr4/md-2 proteins [42] . the apparent kd between tlr4/md-2 and lipid a was 3 nm (see chap. 5). the synthetic peptide of 2-89 amino acids of s100a8 binds md-2 purified from baculovirus with kd 0.73 μm and stimulated cell migration in vitro in a manner dependent on tlr4, md-2, and myd88. the binding domain is localized in the c-terminal 30 amino acids s100a8 , which is supported by the results of docking simulation [43] . overlapping peptide scanning of 15 amino acids of saa3 revealed that amino acids 43-57 bind the md-2 with kd 30 μm and stimulated cell migration dependently on tlr4, md-2, and myd88. injection of the bioactive 15-mer to achieve a serum concentration around the kd value in tumor-non-bearing mice could mobilize bmdc to the lungs in an md-2-dependent manner [44] . when human peripheral mononuclear cells were stimulated by either 1 μg/ml of e. coli-derived lps or purified s100a8 with endotoxin <0.01 pg/ml for 24 h and the culture supernatants were subjected to cytokine array, both induced for example ccl2 but ip-10 and cxcl12 were induced only by lps. cytokines specifically induced by s100a8 were not found [45] . the up-regulation of s100a8 and s100a9 in premetastatic lungs is dependent on vegf and tnfα, both of which are produced by the primary tumor [46] . the lung metastasis is blocked by anti-s100a8 antibody or tlr40-ko. let us consider pneumonia. lps in the alveolar space promotes permeability in epithelial barrier. anti-s100a8 antibody can suppress transepithelial migration of leukocytes from the interstitial to alveolar space. given the bacteriocidal roles of leukocytes in the alveolar space, it is reasonable for bacteria to promote epithelial permeability for invasion and for leukocytes to counter-migrate. because anti-s100a8 antibody can block transendothelial migration of leukocytes from circulation to interstitial space, s100a8 is likely to contribute to extravasation of leukocytes in physiological and of tumor cells in pathological condition. exogenously borne microbes through the airway induce mobilization of bone marrowderived myeloid cells (bmdc) to the lungs. the triggering mechanism may involve microbial lps that activates tlr4 in club cells in terminal bronchioles. the paracrine signaling goes in the direction to the circulation side. expression of endogenous ligands, such as s100a8 and saa3, in endothelial cells in sterile premetastatic lungs is induced by primary tumor-derived growth factors, such as ccl2, from the circulation side and the paracrine signaling goes in an opposite direction from the circulation to airway side to result in amplification of saa3 in club cells. bone marrow may misrecognize those endogenous ligands as lps derived from lung infection and mobilize the myeloid cells there to battle against the phantom microbes leukocyte extravasation requires their initial attachment with endothelial cells. bone marrow leukocytes are known to up-regulate mac-1 (cd11b-cd18) expression in response to not only lps but also other inflammatory factors including tnfα, ltb4, and c5a. in neutrophils, in which roughly 40% of cytosolic protein is s100a8 and a9, native s100a8-s100a9 heterodimers purified from neutrophils failed to enhance adhesion to fibrinogen whose receptor is mac-1 at concentrations up to 2 μm, whereas e. coli-expressed s100a9 could do so by changing cd18 (¼β2 integrin) into an active form that can be recognized by monoclonal antibody mab24 [47] . if the recombinant s100a9 protein was contaminated by lps, it should be the lps activity via tlr4. if not, the activity should have been mediated by a receptor that binds s100a9 but not s100a8; the most likely one is cd147 (see below) whose activation results in expression of tnfα that may work in a paracrine fashion. s100a8 and s100a9 were claimed to be in vivo substrate of mmp9 whose knockout reduced lung metastasis [48] . the peter angel group nicely engineered hepatocyte-specific conditional double transgenic mice of s100a8 and s100a9, which displayed no disease phenotypes including inflammatory lesions [49] . both proteins were detected weakly in the liver but not increased in the serum from the baseline. interestingly, they showed roughly twofold up-regulation of serum concentration of cxcl1 with systemic enrichment of gr1 + s100a8 + monocytic cells in circulation and passively in liver. given that s100a8 was up-regulated in the serum of tumor-bearing mice in our experiments, s100a8 induction of cxcl1 is sufficient for leukocyte mobilization from the bone marrow. i am eager to see if portal injection of tlr4or rageexpressing cells can augment liver metastasis. for cxcl1, also refer to chaps. 12 and 16. plasminogen activator inhibitor-1 (pai-1) (see chaps. 3 and 12), which is induced by lps or atf3 activation, plays a defensive role in pneumonia by facilitating transepithelial migration of leukocytes into the air space. both s100a8 and saa3 can potentially activate atf3 (see below). therefore, the defensive mechanism against bacteria by mobilizing leukocytes is <hijacked> by tumor cells. just like leukocytes moving toward lps, tumor cells move toward those endogenous tlr4 ligands in the lungs resulting in metastatic dissemination. pai-1 inhibits plasminogen activator responsible for generating plasmin, which can activate mmp9 required for metastatic microenvironment. this seems paradoxical. however, s100a8 and saa3 cause vascular permeability and fibrin-fibrinogen deposition, which as a next step activates fibrinolytic events. 15.6.2.2 s100a8 and the eph-ephrin system s100a8 induces ephrin-a1 expression [50] . the original finding was that lps induces tnfα, which then promotes expression of ephrin-a1 in huvec. the prototype of the eph (the cdna was originally isolated from a nude mouse transplantable tumor named erythropoietin-producing hepatoma) family of receptor tyrosine kinases, now called epha1, was discovered by me in 1987, and currently the family is grouped into epha with ten members and ephb with six members. their ligands are either gpi-anchored ephrin-a1 through a6 or transmembrane ephrin-b1 to b3 proteins, respectively. the system forms a counter-receptor system giving both forward (ephrin to eph) and reverse (eph to ephrin) signaling. the family members play important roles in many biological settings, including ephb4ephrin-b2 in arteriovenous differentiation, ephrin-b2 in vegfr2 activity, and epha3/a5-ephrin-a2/a5 in neuronal pathfinding. systemic administration of lps mimics bacterial assaults from the most exposed organs, i.e., lungs sensing air-borne lps and liver responsible for perception of gut-derived lps via portal vein. the misrecognition by those organs was reflected in the time-dependent up-and down-regulation of both epha1/a2 and ephrin-a1 expressions, which is presumably involved in vascular permeability to allow leukocyte extravasation there [51] . a small elevation of lps from whatever tissues attacked by bacteria is homeostatically controlled by this way. we have shown that the epha1and epha2 can bind the membrane-bound ephrin-a1 in lung endothelial cells serving as adhesion molecules in a manner independent of its tyrosine kinase activity [52] . however, a soluble form of ephrin-a1, which is released from the primary tumor by adam12-mediated shedding, reaches in an endocrine fashion and disrupts the pulmonary vascular barrier disorganizing ve-cadherin. we also have shown that the ephrin-a1-fc recombinant soluble protein causes contraction of endothelial cells in a tyrosine kinasedependent manner through the sam domain binding to integrin-linked kinase and the subsequent rhoa-rock pathway [53] . in both epha1-ko and epha2-ko mice, vascular permeability in the lungs was actually increased. the information indicates the disruption of molecules that usually participate in lung homeostasis as adhesion machinery causes inflammation in the lungs, i.e., vascular permeability and cell migration. the critical mechanism is likely switching between on and off state of the epha1 tyrosine kinase activity. therefore, the mechanisms of premetastasis cannot be explained without putting into consideration of molecules directly involved in lung homeostasis. the classical danger, such as invading bacteria or tumor cells and necrotic cells, are absent in the premetastatic lungs, but both vascular permeability and cell migration actively take place as in the case of physiological conditions. however, their levels are higher than those in physiological circumstances (see below). after arrival of tumor cells, which is the true danger, danger hypothesis-based events continue as found in the primary tumor. what is the mechanism by which to explain the sustained expression of s100a8 and saa3 in the lungs while their serum levels decrease 10-14 days after implantation of tumor cells? detailed analysis of stimulation and expression pattern of s100a8, saa3, and tnfα revealed that the triggering mechanism is primary tumor-secreted ccl2 that activates ccr2 in the hyperpermeable regions in the lungs to induce s100a8 expression in the endothelial cells. s100a8 from endothelial cells is secreted into the interstitium where macrophages are located and stimulated to secrete saa3. the interstitial saa3 then stimulates tlr4 expressed in club cells in the terminal bronchiole regions, a preferential site of lung metastasis. club cells then express saa3, which stimulates their own tlr4 resulting in autoamplification of saa3 [54] (fig. 15.2) . at this stage, the paracrine cascade starting from ccl2 may be dispensable for the establishment of premetastatic milieu even if the primary tumor is removed. it is assumed that tlr4 inhibition may be one of the reasonable ways to put an end to the saa3 autoamplification. an initial lps exposure is known to induce tolerance against subsequent lps challenge. the mechanisms involve tlr4-induced activation of aryl hydrocarbon receptor (ahr) whose ligands include dioxin of environmental origin and endogenous kynurenine, a product of tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (ido1) that also is induced by tlr4 activation, as manifested by deceased serum levels of tnfα and il-6 [55] . ahr-ko mice were more sensitive to endotoxemia by impaired mitigation of the tlr4-nfκb signaling by ahr. ahr is expressed in club cells that metabolize xenobiotics and can autoamplify saa3. by utilizing ahr-expressing primary hepatocytes derived from ahr-ko mice, it was shown that a potent ahr agonist tcdd (2,3,7,8tetrachlorodibenzo-p-dioxin) repressed saa3 expression induced by il-1β and il-6 [56] . the authors showed that similar effects were observed with different ahr agonists, such as benzopyrene and naphthoflavone, and that the mechanisms involved inhibited recruitment of p65rela and c/ebpβ to the saa3 promoter. however, ongoing studies in our laboratory have shown that neither s100a8 nor saa3 lack this feedback (tsukahara and maru, unpublished results). readers should remember that ccl2 is also involved homeostatic inflammation in alveolar recruitment of monocytes (see chap. 14). we have shown that both s100a8 and ccl2 can induce vascular permeability in the lungs. i will review interesting experiments by jeffrey pollard group [57] . they defined inflammatory ly6c + (therefore gr1 + ) (see chap. 12.6.3.2) and resident ly6c -(gr1 -) monocytes sharing cd45 + cd11b + cd115 + phenotypes (cd115 is csf1 -r and almost all cd115 + cells are ly6g -), which were sorted from csf1-r-gfp transgenic mice to track down after adoptive transfer into mmtv-pymt mice. while gr1monocytes were recruited in the primary tumor, gr1monocytes were mobilized to the late-stage lung metastatic lesions and forced pulmonary metastatic nodules by intravenous injection of a pymt-induced mouse mammary tumor cell line (met-1), and intriguingly lungs 7 h after intravenous injection of met-1 cells before extravasation in the lungs, but not 7-week-old pymt mice with yet premalignant mammary tumors presumably without expression of ccl2 even if they make an entry into circulation. high levels of ccr2 expression were observed in the recruited gr1monocytes. notably, ccl2 expression levels in tumor cells were homogenous in the metastatic lung nodules but heterogenous in the primary tumor. therefore, ccl2 expression appears to confer a metastatic trait on the tumor cells. given that even ctc expressing ccl2 alone without primary tumor induced gr1 + monocytes in the lung, augmented levels of ccl2 in circulation may be misrecognized by gr1 + monocytes in the bone marrow as a danger producing ccl2 in the lungs. the recruited monocytes are destined to prepare premetastatic soil by producing vegf in contact with ccl2-secreting tumor cells and vegf works in concert with ccl2 to facilitate tumor cell extravasation into the lungs. they showed that myeloid cell-specific conditional knockout of vegf by the tamoxifen-inducible system linked to the csf1-r promoter abrogated the tumor cell seeding in the lungs. vegf by itself was not required for the recruitment of gr1monocytes since vegf-null monocytes were recruited at similar levels to the control. tlr4 is a double-edged sword but seemingly defensive. the tlr4 polymorphism d299g in the extracellular domain showed reduced lps response with a greater risk of sepsis and decreased ability for binding hmgb1, an endogenous tlr4 ligand (see part ii). the frequency of metastasis by 5 years after breast cancer surgery was 40% in d299g patients compared with 26.5% in those without polymorphism, suggesting the polymorphism is loss of defensive function [58] . autosomal recessive mutations in myd88 within the irak4-interacting domain, such as l93p result in failed interaction with irak4 leading to severe infection with pyrogenic bacteria during the early infancy [59] . compared with reactions in the initial exposures to exogenous bacteria in neonates, those in early adolescence get less and less severe. conversely, a gain of function mutation l265p in myd88, which results in constitutive myd88 signaling with production of cytokines, such as il-6, can facilitate diffuse large b-cell lymphoma progression [60] . thus, the tlr4-myd88 pathway appears to be defensive against bacteria and tumor progression, but too much defensive reaction favors tumor progression. lps reduces the expression of ccl21 through the tlr4-socs3 pathway in high endothelial venules in lymph nodes, which results in less mobilization of ccr7-expressing lymphocytes as attackers to the lymph nodes [61] . in this case, tlr4 weakens the defense via ccl21. when tumor cells express ccr7, ccl21 promotes their mobilization through afferent lymphatic ducts that are dilated by vegf-c. here, ccl21 promotes the attack. in both cases, it is favorable for attack by tumor cells. this apparent discrepancy can be easily understood by thinking of homeostatic mimicry and the hijacking idea. lps mobilizes defensive bmdc. s100a8 also mobilizes defensive bmdc [62] . mobilized bmdc participates in more s100a8 production but find no enemy or attackers to battle against in the premetastatic lungs. overexpressed s100a8 mobilizes aggressive tumor cells expressing tlr4 in postmetastatic lungs. an sirna-mediated knockdown of tlr4 in tumor cells abrogated their lung metastasis [54] . at this stage, defenders are substituted with attackers. tlr4 activation inhibited growth of breast cancer cells with wild-type p53 by increasing growth-suppressing ifnγ secretion but promotes growth of those with p53 mutations by expression progrowth cytokines, including il-6 and cxcl1 [63] . as i just stated, both plasma s100a8 and saa3 elevate in tumor-bearing mice 10 and 8 days, respectively, after subcutaneous implantation of tumor cells with recruitment cd11b + myeloid cells in the premetastatic lungs. there are some differences between intravenously injected lps and the endogenous tlr4 ligands produced in tumor burden. reciprocal bmts between wild-type and tlr4 -ko mice can theoretically generate (1) bm tlr4-ko with most of the pulmonary population of cells, such as epithelial and endothelial cells are of wild-type, and (2) lung tlr4 -ko with recruitment of a small population of wild-type cells from the bone marrow. when systemic lps was administered at 0.5 mg/kg, neutrophil recruitment in the lungs was observed within 4 h as monitored by myeloperoxidase activity and chloroacetate esterase staining in the lungs, which was abrogated in tlr4 -ko and lung tlr4 -ko, but not bm tlr4 -ko, e-and p-selectin double ko and cd18-ko [64] . lps-activated tlr4 was reflected in the p-selectin expression in the lungs in totally wild-type and bm tlr4 -ko mice but not tlr4 -ko and lung tlr4 -ko mice. collectively, lps stimulates tlr4 expression in the lung resident cells to induce neutrophil recruitment. when similar experiments were performed by tumor challenge, which increases plasma levels of both s100a8 and saa3, pulmonary recruitment of mac1 + myeloid cells was impaired in tlr4 -ko and bm tlr4 -ko mice but not lung tlr4 -ko mice, indicating that blood cell tlr4 is important, but the experiments failed to reduce the significance of lung tlr4. because club cells, which express tlr4, are derived from bm, a mixture of tlr4 + and tlr4club cells was present in lung tlr4 -ko mice. more than 20 members are known in the s100 family proteins [65] . they are homologous to each other by 25-65% in amino acid levels. information of the members with determined crystal structure revealed that they have an ef-hand motif and form antiparallel homo-or hetero-dimers. they can further associate to form high-order multimers. high-resolution nmr spectroscopy revealed that binding of ca2+ opens up the hydrophobic pocket allowing their interaction with the c-type immunoglobulin domain of rage, i.e., ca2+ molecularly switches the binding. this ca2+ sensor is engaged in a variety of biological events by functioning in intracellular and/or extracellular space. s100a8 can preferably form a hetero-dimer with s100a9. in peripheral blood cells from s100a9-ko mice, s100a8 proteins were under detection, although s100a8 mrnas were present, suggesting that s100a9 proteins are responsible for stabilization of s100a8 proteins in mice [66] . an opposite situation was reported in humans. human s100a9 proteins were unstable in the absence of s100a8 proteins [67] . both s100a8 and s100a9 proteins were initially identified in the synovial fluid of rheumatoid arthritis patients [68] . corpora amylacea in prostate glands of prostate cancer patients mainly consists of amyloid (see chap. 6) of s100a8 and s100a9 [69] . s100a8/s100a9 proteins purified from granulocytes showed a propensity to aggregate in vitro forming fibrillar structures in 8 weeks in a manner dependent on zn2+ and ca2+ as judged by atomic force microscopy and transmission electron microscopy [70] . liquid chromatography-electrospray ionization mass spectrometry of corpora amylacea revealed the presence of hemoglobin subunits and myeloperoxidase of host origin as well as bacterial dna sequences, strongly indicating the involvement of bacterial infection-induced inflammation in the formation of this crystal. s100a8 expression is up-regulated not only in pre-metastatic lungs but also in synovial fluid in rheumatoid arthritis patients, serum from sle and intestinal epithelial cells of crohn's inflammatory bowel disease and most importantly in tumors [71, 72] . in contrast to most secretory proteins exported by the er-golgi pathway, unconventional, i.e., still unknown mechanisms underline the release of il-1β, il-18, il-33, proil-1α, fgf, and s100a8, all of which lack the leader sequence [73] . it is known that il-1β secretion depends on narlp3 inflammasome and caspase-1 activation [74] . in addition to soluble factors, such as tnfα and lps, hif-1 induces expression of both s100a8 and s100a9 by binding to their hypoxia response element (hre) in the promoters in benign prostate epithelial hyperplasia bph-1 cells and prostate cancer pc-1 and du-145 cells [75] . analysis of 145 patients who were subjected to radical prostatectomy revealed similar expression patterns of s100a8 and hif-1α in immunostaining and a negative correlation between the expression levels of s100a9 and the time required for recurrence. although dexamethasone alone failed to induce s100a8 expression in 20 h but enhanced lps-stimulated expression in thioglycolate-elicited murine macrophages, it directly induced s100a8 expression in human blood monocytes. synovial knee joint biopsy of ra patients revealed that s100a8-positive cell numbers increased 24 h after intravenous administration of methylprednisolone [76] . up-regulation after steroid may imply that s100a8 is at least seemingly antiinflammatory. however, the story is not so simple. for example, increased expression of s100a8 in acute lymphoblastic leukemia with mll gene translocation confers glucocorticoid resistance on the patients. in this case, abundant intracellular s100a8 is assumed to interfere with glucocorticoid-elicited ca2+ fluxes from the er to mitochondria [77] . two receptors are known for s100a8: tlr4 and rage [78, 79] (fig. 15.3 ). more precisely, we showed that s100a8 binds the md-2 coreceptor of the tlr4-md-2 complex. only n-glycosylated rage, which constitutes only 5% of total rage, can bind s100a8 [80] . from the rage side, it can bind the authentic ligand age, s100a6, and s100a12 [81, 82] . one of the unique features of s100 proteins is that they lack signal peptides usually essential for secretory proteins, but they are actually secreted actively and passively into the extracellular space by an as yet unknown mechanism. members that participate in tumor biology include s100a2, s100a4, s100a6, s100a7, s100a8, s100a9, and s100b [83] . clinical correlation between the expression and invasive levels has been reported in s100a4 (also called metastasin) in particular. this is supported by two logical experiments, i.e., metastasis in mmtv-neu mice was enhanced when crossed with s100a4 transgenic mice, while that in mmtv-pymt with s100a4-ko was suppressed, indicating that s100a4 is necessary and sufficient for the tumor metastasis although the mechanism is still unclear [84] . both stromal cells and tumor cells are influenced in terms of expression levels of s100a4 in those tumor models. similar to s100a8, it is abundant in the synovial fluid of rheumatoid arthritis patients and in the psoriatic skin. intraperitoneal administration of anti-s100a4 antibody in the human psoriasis xenograft in scid mice was effective by reducing the epidermal thickness and ki-67+ proliferating cells [85] . while expression of s100a4 in tumor cells induces their metastatic potentials, various types of cells, including t cells, dc, macrophages, fibroblasts, and myofibroblasts, are s100a4-positive in psoriatic skin. secreted forms of s100a4 are thought to bind rage. s100a8 and s100a9 can form homo-dimers and hetero-dimers, but their partial synthetic peptides do not necessarily do so (fig. 15.3) . i mean either monomer or homo-dimer by s100a8 and heterodimer by s100a8/s100a9. in 1999, marion a. hofmann initially showed evidence of a linkage of s100 family with inflammation by s100a12 binding to rage whose authentic ligand is age (see chap. 6) pathologically essential in diabetes mellitus [82] . mice lack the s100a12 gene of which s100a8 may function in place. in a skin carcinogenesis model induced by chemicals dmba/tpa, s100a8, s100a9, mip-1α, mip-1β, and mip-2 were up-regulated [86] . those are target genes of nfκb that is activated by rage or tnfα. knockout mice of either tnfα or rage were resistant to this chemical carcinogenesis, suggesting sustained nfκb activation by a positive feed-forward loop of s100a8/s100a9 to rage to nfκb to s100a8/s100a9. reciprocal bmt experiments with wild-type and rage-ko mice revealed that rage in bone marrow cells is responsible for carcinogenesis and dermal infiltration of neutrophils and macrophages. now rage has at least five ligands, i.e., age, s100a6, s100a12 (only human), s100a8/s100a9, and hmgb1. conversely, s100a9 has at least three receptors: rage, tlr4, and emmprin. tomas leanderson group has shown that human s100a9 and dimeric s100a8-s100a9 bound human rage with kd of 38 nm and 9.4 nm, respectively [87] . it is of note that s100a12 bound rage with kd at 90 nm. they also bound the human tlr4-md-2 complex with kd of 2.1 nm and 3.8 nm, respectively. because the binding was not inhibited by simultaneous application of md-2 proteins, it is likely that s100a9 binds the tlr4 side. affinity isolation as spectrometry in search of binding proteins for s100a9 identified emmprin also called cd147 [88] . s100a9 stimulation induced expression of cxcl1, tnfα, ephrin-a1, and mmp1 in the emmprin signaling presumably via traf2. rage, another receptor for s100a9, failed to form a heterodimer with emmprin. intravenously injected emmprin-expressing melanoma cells metastasized to the skin of s100a9 but not s100a8 transgenic mice with the epidermis-specific involucrin promoter. furthermore, pull-down experiments of human monocyte-derived dendritic cells after hiv-1 infection with fc-fused cd85j, which belongs to the human leukocyte immunoglobulin-like receptor family, revealed binding of s100a9 [89] . they reported that stimulation of purified nk cells by recombinant tetrameric but not monomeric s100a9 proteins expressed and purified from e. coli in endotoxin-free conditions as they claimed increased production of tnfα and enhanced their anti-hiv-1 activity. if homeostatic, lps should have a feedback loop. see fig. 5.3 in chap. 5 for the fundamental tlr4 signaling cascade. negative regulation by a20 was stated in chap. 8. in addition, upon tlr4 activation, nfκb-mediated atf3 transcription takes place [90] . atf3 directly associates with hdac1 to inhibit tlr4-driven gene expression [90] . although lps stimulation induced expression of neutrophil chemoattractant cxcl1 in atf3-ko mice, neutrophil recruitment in the atf3-ko lungs was not observed [91] . gene expression profile analysis revealed that tiam2 was down-regulated in atf3-ko, which was responsible for the migration defect. in the ova-induced model of bronchial asthma, atf3 is up-regulated in the lungs. atf3-ko increased the hyperresponsiveness, pulmonary eosinophilia, and th2 cytokine production [92] . when you remember this model combined with lung metastasis by b16f10 melanoma cells (see above), you would expect aggravation of metastasis in the lungs. however, myeloid specific atf3-ko or general atf3-ko both reduced lung metastasis in the background of syngeneic orthotopic mmtv-pymt mammary cancer model [93] . interestingly, there was no difference in the primary tumor growth. transcription factor atf3 is activated in a variety of cells essentially by stress, such as hypoxia, il-6, and tnfα. atf3-activated macrophages, for example, switch the balance to m2 and directly activate mmp9 expression. this may be one reason to explain the metastasis-suppressing activity in the atf3-ko mice. activated tlr4 also induces pi3k-and rapl-mediated activation of cd11b, i.e., changing its conformation from an inactive to active state. as you remember that cd11b is an integrin αmβ2, this is an inside-out signal. this elicits a sequential activation of src and syk associated with itam [94] . then syk phosphorylates myd88 and trif, to which an e3 ligase cbl-b binds and ubiquitinates for proteasomal degradation. this eventually weakens the lps-induced signaling by cd11b-mediated negative feedback. calcineurin inhibitor, such as fk506, or an sirna-mediated knockdown of calcineurin in raw cells augmented the tlr4-nfκb cascade-mediated expression of tnfα [95] . conversely, a constitutively active calcineurin by deleting the autoinhibitory and calmodulin-binding regions reduced lps-stimulated activation of nfκb. given that tnfα production by fk506 was reduced in macrophages deficient in myd88, trif, tlr4, and tlr2 and that calcineurin co-immunoprecipitates with them, the mechanisms may involve interaction, direct or indirect, between calcineurin and those molecules. lps activates akt. while traf6 located downstream of tlr4 signaling can serve as a direct e3 ubiquitin ligase for akt important for its membrane translocation and activation in growth factor-activated tumor cells, akt controls regulatory micrornas [96] . for example, akt up-regulates mirna let-7e that is capable of repressing tlr4 expression within a few hours, which is restored to the baseline in 48 h. akt1-ko macrophages displayed exaggerated responses to lps. the deduced amino acid sequence portion between mouse and human saa3 are highly homologous to each other, and a single nucleotide insertion in the human saa3 exon2 results in a frameshift to generate a shorter saa3 with a unique c-terminal saa3 sequence of 12 amino acids. interestingly, this type of evolutionary genetic alteration occurs in primates, including chimpanzees ( fig. 15.4) . because no expression of human saa3 has been reported so far, it has been assumed to a pseudogene. although we could not find human saa3 transcripts in the database of the expressed pseudogenes in tumor cells [97] , elaborate qpcp analysis of human lung cancer samples in our laboratory revealed that there exists a chimeric saa2-saa3 fusion mrnas [98] . the human saa2 exon3 is connected to the human saa3 exon2 with roughly 200 kb between the 2 exons to yield the saa2-saa3 fusion protein that could be recognized by a monoclonal antibody against the unique sequence of human saa3. the corresponding gene product activates erk by binding and stimulating lox-1, an endothelial cell-specific scavenger for oxidized ldl. the binding affinity was 206 nm as judged by elisa between c-terminal synthetic 26-mer human saa3 peptide and soluble lox-1. given that neither the saa2-saa3 purified from mammalian cells nor synthetic peptide of human saa3 bound the tlr4-md-2 complex, human saa3 signaling is diverged from that of mouse. exogenous lps of initial encounter induced expression of ccr2, which in turn bound endogenous ccl2 in germfree mice, suggesting that innate stimulation by exogenous microbes induce receptors for endogenous mediators (hiratsuka and maru, unpublished data). we have observed up-regulation of individually ccl2 + and ccr2 + cells in the lungs of lps-treated but not pbs-treated germ-free neonatal mice. however, neonatal mice housed in the standard spf conditions, in which they were exposed to lps-containing bacteria, increased numbers of ccl2 + and ccr2 + cells were recognized even pbs-treated mice. in the spf neonates lps administration followed by llc injection induced the tumor cell recruitment in the lungs, which was totally abrogated in ccr2-ko mice (hiratsuka and maru, unpublished data). thus, neonatal exposure to exogenous lps activates the ccl2-ccr2 system, which leads to the endogenous tlr4 cascade of the s100a8-saa3 paracrine system. a similar situation is known in saa3 expression in adipose tissue through gut [99, 100] . levels of saa3 mrna in epididymal adipose tissue and colon of germfree swiss-webster mice were approximately ten-and sevenfold, respectively, higher than those raised conventionally. myd88-ko reduced the saa3 mrna levels by sixfold compared with the wild type. lps simulates tlr4 in the colon epithelial cells as well as macrophages to induce saa3 expression. serum levels of saa3 in conventionally raised mice were higher than those in germ-free mice and lps administration induced saa3 expression in adipose tissue. given that both authentic ligand lps and endogenous one saa3 bind tlr4 in adipose tissue macrophages to secrete saa3 by a feed-forward mechanism. thus lps-triggered and autoamplified saa3 production stimulates myeloid cell mobilization from bone marrow resulting in accumulation of macrophages in both tissues. the inflammatory response in both colon and adipose tissue is stabilized at homeostatic levels. we should pay attention to the mechanism by which lps-induced inflammation in colon can spread to other tissue(s) if lps can induce expression of endogenous tlr4 ligands, because they can function as lps in bacteria-free tissues. what are the roles in premetastatic lungs of mdsc that i stated in the chap. 12? in our initial report of premetastatic lung establishment by s100a8, we monitored mac-1 + cells. this population of cells includes cd11b + gr1 + cells, i.e., mdsc. in breast cancer ectopic xenograft model with 4t1 cells labeled by gfp, which causes spontaneous metastasis to the lungs, flow cytometric analysis and qpcr analysis of gfp of the lungs showed that mice did not have tumor cells in the lungs until 14 days after tumor implantation. coculture of sorted mdsc with normal lung cells revealed that the number of ifnγ-producing macrophages was significantly decreased [101] . histological analysis of the premetastatic lungs showed that costaining of cd11b + gr1 + and mmp9 + cells. in addition, huvec cocultured with mdsc producing mmp9 disrupted ve-cadherin of huvec. it is likely that mdsc cultivates the soil by reducing antitumor effect and by promoting pulmonary vascular permeability. mdsc accumulate not only in the premetastatic lungs but also in other parts of the body, such as the spleen, by more than fivefold in 3 weeks after tumor cell transplantation. analysis of the splenic population of mdsc revealed several interesting features: (1) mdsc failed to accumulate in tumors transplanted in s100a9-ko mice but instead cd8 + and cd4 + t cells infiltrated the tumor and 9 of 12 mice rejected tumor; (2) s100a9 overexpression induced mdsc accumulation by blocking myeloid differentiation via the nox2 complex-mediated production of ros. is s100a8-saa3 expression necessary and/or sufficient for premetastatic soil establishment? both anti-s100a8 and anti-saa3 antibodies individually blocked lung metastasis as we reported previously [42, 46] . saa3-ko mice showed no prominent phenotypes in metastasis experiments in our hands (tomita t and maru y, unpublished results). because s100a8-ko mice were embryonic lethal, we are currently trying to establish conditional s100a8-ko mice to uncover the homeostatic role of s100a8. we have discussed on inflammatory conditions, including lps administration and acute pneumonia, which promote lung metastasis. knockout mice of uteroglobin, which is an anti-inflammatory protein also called ccsp or cc10 (see chaps. 14 and 15), a specific marker of lung club cells, displayed up-regulated expression of lung s100a8 and enhanced lung metastasis when injected with b16f10 melanoma cells through the tail vein in a tumor nonbearing condition [13] . in mice with keratin 5-directed expression of iκbα to the epidermis (k5-iκbα), inflammatory hyperplasia with s100a8 + neutrophil that dominated over f4/80 + macrophages took place followed by the development of well-differentiated squamous cell carcinomas at 4 months of age. up-regulation of tnfα, s100a8, and saa3 was found in the skin lesions, which were sensitive to uvb at 500 j/m 2 to induce apoptosis [102] . the dermatitis and skin tumor formation were abrogated when k5-iκbα mice were crossed with tnfr1-ko but not ccl2-ko mice [103, 104] . murine mammary cancer met1 cells were subcutaneously implanted in k5-iκbα mice, and the tumor was resected to promote lung metastasis. in premetastatic lungs, cd11b + gr1 + cell numbers were elevated but reduced after the resection. however, the resection induced development of macroscopic metastatic nodules in the lungs but not in the skin where premetastatic factors that we proposed, such as s100a8 and tnfα, were constantly up-regulated (tomita, toftgard and maru, unpublished data). lung metastasis after resection can be explained by elimination of negative angiogenic switch on the tumor cells that reached the lungs before resection. however, the tumor cells could hardly reach the inflammatory skin, suggesting the presence of organotropic factors that control the lung metastasis. binding of receptor activator of nuclear factor kb (rank) in osteoclast precursors and rank ligand (rankl) in osteoblasts induces osteoclast differentiation in the presence of m-csf [105] (fig. 15.5) . the osteoclast precursors are derived from bone marrow hematopoietic stem cells and c-kit + m-csfr + cd11b low. this homeostasis in bone is hijacked by tumor cells to achieve bone metastasis by ectopically expressing both rankl and rank [106] . rank stimulation elicits signaling through akt and erk, inducing cell migration but not cell proliferation or cell death. rank is frequently expressed in breast and renal cell cancer [107] . rank is also expressed in normal mammary gland epithelial cells. a report of 73 clear cell carcinoma of kidney showed that rank, rankl, and the soluble form of decoy receptor for rankl called osteoprotegerin (opg) were expressed in 82%, 66%, and 71%, respectively, as judged by immunostaining. although roughly 30% of renal cell cancer patients develop bone metastasis, opg-high patients were free of bone metastasis. patients with strong expression of both rank and rankl had extramedullary metastasis to skin and liver. rank-expressing tumor cells migrate towards rankl-expressing osteoblasts in bone. on arrival, rankl in tumor cells and induced rankl in osteoblasts by tumor cell-derived pthrp stimulate rank to induce active osteoclastogenesis and subsequent osteolytic bone metastasis. given that rankl is induced by lps in osteoblasts, endogenous tlr4 ligands, such as s100a8, derived from either myeloid cells or tumor cells might play a role in osteoclastogenesis [108] . intracardiac injection of b16f10 melanoma cells that express high levels of rank resulted in metastasis in bones, ovaries, adrenal glands, and brain. opg not only inhibited rankl-dependent cell migration of tumor cells in vitro but also suppressed bone metastasis in vivo. intriguingly, however, metastasis to other organs was not affected. this information indicates that hijacking the rank-rankl system participates not only in the mobilization of tumor cells but also in organ specificity in metastasis. in addition to tumor cells, lps-activated tlr4-expressing neutrophils express rankl. this also is the case in neutrophils from the synovial fluid of exacerbated rheumatoid arthritis patients in which endogenous tlr4 ligands s100a8 and s100a9 are abundant [109] . therefore, rankl not only in tumor cells but also in neutrophils participates in osteoclastic bone resorption. cox-2 expression (see chap. 1) is observed not only in a variety of tumor cells but also stromal cells. lps induces pge2 production in macrophages. in postmetastatic bone experiments with melanoma cells, we have shown by using coculture systems and gene knockout mice that b16 melanoma cell-osteoblasts contacts resulted in osteoblastic expression of rankl in a membrane-bound (m) pges-1 (see chap. 1)-dependent manner, leading to osteoclast formation [110] . bone metastasis was abrogated in genetic background of mpges-1-ko or in the presence of an ep4 antagonist ae3-208 when melanoma cells were injected intravenously through the tail vein. thus, in this case the rank-rankl system is hijacked by melanoma cell-stimulated activation of mpges-pge2-ep4 signaling in osteoblasts. another way to hijack homeostasis is found in one of the mechanisms of brain metastasis by breast cancer and melanoma. connexin43 mediates homocellular communications in endothelial cells (see chap. 3). endothelial cell-specific knockout of connexin43 resulted in dramatic no elevation and hypotension suggesting that vascular gap junctions contributes to vascular homeostasis [111] . intraperitoneal injection of lps into mice also induced bladder connexin43 expression in bladder smooth muscle cells, which was largely blocked by an inos inhibitor. this suggests complicated mechanisms of connexin43 expression in a variety of cells in which no generation systems are involved [112] . an emt regulator twist up-regulates connexin43 expression in breast cancer cells, which mediates heterocellular communication between the cancer cells and brain endothelial cells [113] . the heterocellular connection appears to be functional as judged by the passage of calcein orange dye from the tumor cells to the brain endothelial cells, which might exchange other bioactive molecules. importantly, using 4t-1 breast cancer cells that were labeled with pkh26 membrane dye, which is retained when labeled cells are not dividing but lost after three cell divisions, the authors showed that the metastatic tumor cells that initiated heterocellular communication with brain endothelial cells may be dormant cells as exemplified by cancer stem cells. autacoids function in a paracrine fashion in physiological conditions. however, when overexpressed in pathological conditions they appear in the blood stream and affect the distant target cells in an endocrine manner. autocrine secretion of vegf in endothelial homeostasis, that of s100a8 in myeloid cells as exemplified in immune suppressive functions of mdsc and paracrine secretion of tnfα within a variety of tissues are known. plasma levels of those factors increase in tumor patients in general, which allows their action in tissues distant from the sites of original overproduction, i.e., primary tumors. for example, s100a8-a9 protein levels in plasma were elevated in gastric cancer patients [114] . although the s100a8-a9 producing primary tumor is fixed in stomach, the plasma s100a8-a9 works in an endocrine manner to stimulate bone marrow-derived mdsc, which secretes s100a8-a9 forming an autocrine loop. mdsc circulate and migrate to a tissue including the primary tumor and distant ones apart from it, in which the original functions of s100a8-a9 are transmitted. in the danger theory, matzinger proposed that the immune system is activated by damaged cells (fig. 15.6 ) [115] . this led to the molecular identification of damage signals by so-called alarmins or danger-associated molecular patterns (damps), including uric acids, hmgb1 (see chap. 6), and s100a8 (chap. 15). although matzinger described by mistake that the immune system fails to be activated by tumors, because they usually do not become necrotic to give damps [116] , current understanding is that tumors cause tissue damages and therefore premetastasis, which is devoid of tissue damages, precedes the arrival of tumor cells as a true danger. hijacking of homeostatic roles played by endogenous mediators by tumor cells gives inflammatory conditions in premetastatic tissues. tumor cells inhibition of tgf-β with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma lower incidence of hepatic metastases of colorectal cancer in patients with chronic liver diseases: meta-analysis impact of cirrhosis on the development of experimental hepatic metastases by b16f1 melanoma cells in c57bl/6 mice activation of toll-like receptor 4 on tumor cells in vitro inhibits subsequent tumor growth in vivo the ubiquitin-cxcr4 axis plays an important role in acute lung infection-enhanced lung tumor metastasis collagen induced arthritis increases secondary metastasis in mmtv-pyv mt mouse model of mammary cancer arthritis augments breast cancer metastasis: role of mast cells and scf/c-kit signaling influence of the route of allergen administration and genetic background on the murine allergic pulmonary response defining a link with asthma in mice congenitally deficient in eosinophils allergic pulmonary inflammation promotes the recruitment of circulating tumor cells to the lung clara cells attenuate the inflammatory response through regulation of macrophage behavior lack of an endogenous anti-inflammatory protein in mice enhances colonization of b16f10 melanoma cells in the lungs vascular endothelial growth factor-and thrombininduced termination factor, down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis a protein encoded within the down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling thrombin-induced autoinhibitory factor, down syndrome critical region-1, attenuates nfat-dependent vascular cell adhesion molecule-1 expression and inflammation in the endothelium targeted deletion of the calcineurin inhibitor dscr1 suppresses tumor growth bevacizumab safety in patients with central nervous system metastases heterozygous embryonic lethality induced by targeted inactivation of the vegf gene the calcineurin-nfat-angiopoietin-2 signaling axis in lung endothelium is critical for the establishment of lung metastases effects of angiotensin ii and some analogues on vascular permeability in the rabbit reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin ii-infused macrophage colonystimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury hypercholesterolemia stimulates angiotensin peptide synthesis and contributes to atherosclerosis through the at1a receptor angiotensin ii increases expression of ip-10 and the renin-angiotensin system in endothelial cells negative regulation of vegf-induced vascular leakage by blockade of angiotensin ii type 1 receptor angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin ii type 1a receptor signaling facilitates tumor metastasis formation through p-selectin-mediated interaction of tumor cells with platelets and endothelial cells the renin-angiotensin system and malignancy adipocyte-derived lipids increase angiotensin-converting enzyme (ace) expression and modulate macrophage phenotype pulmonary hypertension in tnf-alpha-overexpressing mice is associated with decreased vegf gene expression in vivo effects of tumor necrosis factor-alpha on capillary permeability and vascular tone in a skeletal muscle hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor a atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-a anp/gc-a signaling attenuates pulmonary metastasis of b16 melanoma enhanced by lipopolysaccharide or angiotensin-ii angiotensin ii type i antagonist prevents pulmonary metastasis of murine renal cancer by inhibiting tumor angiogenesis arachidonic acid metabolites as endothelium-derived hyperpolarizing factors epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice bone marrow-derived circulating progenitor cells fail to transdifferentiate into adipocytes in adult adipose tissues in mice early bioenergetic changes in hepatocarcinogenesis: preneoplastic phenotypes mimic responses to insulin and thyroid hormone enhancement and phenotypic modulation of nnitrosomorpholine-induced hepatocarcinogenesis by dehydroepiandrosterone mouse colon carcinoma cells established for high incidence of experimental hepatic metastasis exhibit accelerated and anchorageindependent growth the s100a8-serum amyloid a3-tlr4 paracrine cascade establishes a pre-metastatic phase eritoran inhibits s100a8-mediated tlr4/md-2 activation and tumor growth by changing the immune microenvironment serum amyloid a3 binds md-2 to activate p38 and nf-kappab pathways in a myd88-dependent manner s100a8 and s100a9 induce cytokine expression and regulate the nlrp3 inflammasome via ros-dependent activation of nf-kappab(1.) tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis s100a9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background hepatocyte-specific s100a8 and s100a9 transgene expression in mice causes cxcl1 induction and systemic neutrophil enrichment ephrin-a1 expression induced by s100a8 is mediated by the toll-like receptor 4 endothelial epha receptor stimulation increases lung vascular permeability adam12-cleaved ephrin-a1 contributes to lung metastasis epha1 interacts with integrin-linked kinase and regulates cell morphology and motility imbalance of clara cell-mediated homeostatic inflammation is involved in lung metastasis aryl hydrocarbon receptor control of a disease tolerance defence pathway ah receptor represses acute-phase response gene expression without binding to its cognate response element ccl2 recruits inflammatory monocytes to facilitate breasttumour metastasis toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy pyogenic bacterial infections in humans with myd88 deficiency oncogenically active myd88 mutations in human lymphoma salmonella disrupts lymph node architecture by tlr4-mediated suppression of homeostatic chemokines bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury tlr4 has a tp53-dependent dual role in regulating breast cancer cell growth endothelium-derived toll-like receptor-4 is the key molecule in lps-induced neutrophil sequestration into lungs s100 proteins in cancer loss of s100a9 (mrp14) results in reduced interleukin-8-induced cd11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro s100a9 as a pharmacological target molecule in inflammation and cancer two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis amyloid formation by the pro-inflammatory s100a8/a9 proteins in the ageing prostate biophysical characterization of s100a8 and s100a9 in the absence and presence of bivalent cations increased serum levels of s100a8/a9 and s100a12 are associated with cardiovascular disease in patients with inactive systemic lupus erythematosus phagocyte-specific s100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease active caspase-1 is a regulator of unconventional protein secretion activation of the nlrp3 inflammasome in dendritic cells induces il-1beta-dependent adaptive immunity against tumors hypoxia and hif-1 increase s100a8 and s100a9 expression in prostate cancer regulation of s100a8 by glucocorticoids elevated s100a8/s100a9 expression causes glucocorticoid resistance in mll-rearranged infant acute lymphoblastic leukemia mrp8 and mrp14 are endogenous activators of toll-like receptor 4, promoting lethal, endotoxin-induced shock rage, carboxylated glycans and s100a8/a9 play essential roles in colitis-associated carcinogenesis n(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression hexameric calgranulin c (s100a12) binds to the receptor for advanced glycated end products (rage) using symmetric hydrophobic target-binding patches rage mediates a novel proinflammatory axis: a central cell surface receptor for s100/calgranulin polypeptides s100b and s100a6 differentially modulate cell survival by interacting with distinct rage (receptor for advanced glycation end products) immunoglobulin domains s100 protein family in human cancer significance of the s100a4 protein in psoriasis rage signaling sustains inflammation and promotes tumor development identification of human s100a9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides s100a9 is a novel ligand of emmprin that promotes melanoma metastasis s100a9 protein is a novel ligand for the cd85j receptor and its interaction is implicated in the control of hiv-1 replication by nk cells systems biology approaches identify atf3 as a negative regulator of toll-like receptor 4 atf3 is a novel regulator of mouse neutrophil migration activating transcription factor 3 is a negative regulator of allergic pulmonary inflammation transcription factor atf3 links host adaptive response to breast cancer metastasis integrin cd11b negatively regulates tlr-triggered inflammatory responses by activating syk and promoting degradation of myd88 and trif via cbl-b calcineurin negatively regulates tlr-mediated activation pathways the kinase akt1 controls macrophage response to lipopolysaccharide by regulating micrornas expressed pseudogenes in the transcriptional landscape of human cancers human serum amyloid a3 (saa3) protein, expressed as a fusion protein with saa2, binds the oxidized low density lipoprotein receptor adipocyte-derived serum amyloid a3 and hyaluronan play a role in monocyte recruitment and adhesion regulation of serum amyloid a3 (saa3) in mouse colonic epithelium and adipose tissue by the intestinal microbiota gr-1+cd11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung squamous cell carcinomas and increased apoptosis in skin with inhibited rel/nuclear factor-kappab signaling tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by nf-kappab inhibition timed nf-kappab inhibition in skin reveals dual independent effects on development of hed/eda and chronic inflammation the molecular understanding of osteoclast differentiation regulation of cancer cell migration and bone metastasis by rankl increased rankl expression is related to tumour migration and metastasis of renal cell carcinomas gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via toll-like receptors surface rankl of toll-like receptor 4-stimulated human neutrophils activates osteoclastic bone resorption direct melanoma cell contact induces stromal cell autocrine prostaglandin e2-ep4 receptor signaling that drives tumor growth, angiogenesis and metastasis endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice reciprocal regulation between proinflammatory cytokine-induced inducible no synthase (inos) and connexin43 in bladder smooth muscle cells role of connexins in metastatic breast cancer and melanoma brain colonization increased myeloid-derived suppressor cells in gastric cancer correlate with cancer stage and plasma s100a8/a9 proinflammatory proteins tolerance, danger, and the extended family the danger theory: 20 years later key: cord-020764-5tq9cr7o authors: vertrees, roger a.; goodwin, thomas; jordan, jeffrey m.; zwischenberger, joseph b. title: tissue culture models date: 2010-05-21 journal: molecular pathology of lung diseases doi: 10.1007/978-0-387-72430-0_15 sha: doc_id: 20764 cord_uid: 5tq9cr7o the use of tissue cultures as a research tool to investigate the pathophysiologic bases of diseases has become essential in the current age of molecular biomedical research. although it will always be necessary to translate and validate the observations seen in vitro to the patient or animal, the ability to investigate the role(s) of individual variables free from confounders is paramount toward increasing our understanding of the physiology of the lung and the role of its cellular components in disease. additionally, it is not feasible to conduct certain research in humans because of ethical constraints, yet investigators may still be interested in the physiologic response in human tissues; in vitro characterization of human tissue is an acceptable choice. the use of tissue cultures as a research tool to investigate the pathophysiologic bases of diseases has become essential in the current age of molecular biomedical research. although it will always be necessary to translate and validate the observations seen in vitro to the patient or animal, the ability to investigate the role(s) of individual variables free from confounders is paramount toward increasing our understanding of the physiology of the lung and the role of its cellular components in disease. additionally, it is not feasible to conduct certain research in humans because of ethical constraints, yet investigators may still be interested in the physiologic response in human tissues; in vitro characterization of human tissue is an acceptable choice. tissue culture techniques have been utilized extensively to investigate questions pertaining to lung physiology and disease. the isolation and propagation of human bronchial epithelial cells has allowed investigators to begin to characterize the interactions and reactions that occur in response to various stimuli. moreover, the culture of human airway smooth muscle has allowed researchers to investigate a pathologic cascade that occurs in asthma as well as other physiologic responses in the smooth muscle of the lung. numerous lung cancer cell lines have been established to investigate their responses to chemotherapy and determine their biologic properties. overall, the use of cultured human lung tissue has provided a windfall of information on the pathogenesis of diseases that affect the lung and on the basic physiology and development of the lung in general. despite this wealth of information in the literature, this chapter is the fi rst to discuss the use of tissue culture models to examine the physiology and pathologic basis of lung diseases. in light of this, we briefl y discuss the history and principles behind the utilization of tissue culture. we then discuss the current use of tissue culture to examine many of the from henrietta lacks were cultivated into the fi rst immortal cell line-"hela." 5 hela cells are still one of the most widely used cell lines today. since the 1950s, tissue culture has become fi rmly established as a mechanism to answer many questions in biomedical research. today, tissue culture is widely used to investigate diseases that affect the lung, and through this work we have been able to increase our understanding of the pathologic cascades that occur in lung diseases, as well as the normal physiologies of the lung. tissue culture is a commonly used generic term for the in vitro cultivation of cells, attributed to the early cultures that generally consisted of heterogeneous cultures of crudely disaggregated tissues. currently, many terms are used that can be encompassed by the term: organ culture, cell culture, primary explants, and ex vivo propagation all deal with the in vitro cultivation of cells or tissues. cell culture in general can be applied either to primary cells (e.g., those with a fi nite life span) or to cell lines (e.g., hela cells). additionally, these cultures can be either a homogenous or a heterogenous group of cells. primary cell culture involves the isolation of cells from a tissue by disaggregation. single cell suspensions from tissues can be completed through either enzymatic digestion of extracellular matrix surrounding the cells-such as with ethylenediaminetetraacetic acid, trypsin, or collagenase-or mechanical disaggregation. these disaggregation procedures have the disadvantage of possibly injuring cells. if the cells of interest are adherent viable cells, they will be separated from nonviable cells when the medium is changed. alternatively, viable cells can be separated from nonviable cells prior to culture by subjecting the single cell suspension to density gradient centrifugation (e.g., hypaque). primary cells have an advantage of possessing many of the biologic properties that they possessed in vivo because they are not transformed. primary cells, unlike cell lines, are not immortal and have only a fi nite survival time in culture before becoming senescent. variant cells, however, as well as those obtained from neoplastic tissue, may proliferate infi nitely, thus becoming immortal in vitro. this will eventually allow the immortal cell to take over the culture and can be thought of as a cell line. in general, primary human cultures will survive for 30-80 passages in vitro, although this number is dependent on cell type, conditions, and possibly other unknown factors. primary cells are widely used to examine the effects of toxins, infectious agents, or other cellular interactions that would not be feasible in vivo. primary cells have a disadvantage of being a heterogeneous mixture of cells upon primary isolation, with the type of cell obtained generally a component of the disag-gregation method used. the most common contaminant seen following isolation of primary cells is cells of mesenchymal origin (e.g., fi broblasts). however, advances have been made that allow the culture of homogenous populations of cells. for instance, cell surface molecules specifi c for the cells of interest may be tagged with monoclonal antibodies. techniques such as fl uorescenceactivated cell sorting or the use of magnetic beads can be utilized to enrich the single cell suspension for the cell type of interest. additionally, some investigators have recently exploited unique characteristics of certain cells, such as the presence of p-glycoprotein or multidrug resistance-associated proteins expressed on endothelial cells, to poison other contaminating cells in culture. 6 another type of primary cell culture is "primary explants." this type of culture is not subjected to a disaggregation procedure like the primary cell technique described earlier. therefore, single cell suspensions do not occur. briefl y, tissue samples are dissected and fi nely minced. these tissue pieces are then placed onto the surface of a tissue culture plate. following plating of tissue pieces, cells have been shown to migrate out of the tissue and onto the tissue culture surface. 7 this technique is useful when cells of interest may become damaged or lost in the disaggregation technique described earlier and is often used to culture human bronchial epithelial cells. 8 cell lines are another useful source of cells to investigate questions in biomedical research. these cells have the advantage of being immortal as opposed to the fi nite life spans that primary cells possess. additionally, they are generally well studied and characterized, leaving few experimental variables to worry about. these cells however, are prone to dedifferentiation-a process by which they lose the phenotypic characteristics of the cell from which they began. many of the early cell lines were established from tumor tissue and as such possess abnormal growth characteristics. newer cell lines have been established by molecular techniques such as inserting a telomerase gene into a cell to allow it to replicate infinitely. 9 because of the phenotypic changes that allow cell lines to replicate infi nitely in culture, they are often a fi rst choice for experiments; however, they are also highly criticized in light of their nonnatural phenotype. organ culture, as the name implies, involves ex vivo culture of the whole or signifi cant portion of the organ. the main advantage to this type of culture is the retention and preservation of the original cell-cell interaction and extracellular architecture. this type of culture may be particularly important when experimental design necessitates the use of an ex vivo system, but researchers still need to retain the original organ architecture to answer questions posed. these types of cultures do not grow rapidly, however, and are therefore not suitable for experiments needing large numbers of a particular cell type. 10 advantages and limitations of tissue culture tissue culture has become the penultimate tool of the reductionist biologist. the utilization of tissue culture as a research methodology has allowed investigators to study isolated interactions in its near-normal environment. these experiments by their very nature introduce artifacts; however, they do minimize the number of confounding variables that may affect a particular experiment. for instance, tissue culture allows investigators to determine the effects of one particular treatment on a particular cell type, which would not be feasible in vivo. additionally, tissue culture models of disease allow investigators to obtain samples and make observations more readily than those done in vivo. however, it is the relative simplicity of experiments done in vitro that allows models of disease or physiology to come under frequent and warranted criticism. these models do not take into consideration the complexity of biologic systems. diminishing possible confounding variables by culturing cells in vitro brings up the constant criticism of how applicable results are because of alterations of the normal cellular environment in vivo. for example, cell-cell interactions in vitro are reduced and unnatural. moreover, the culture does not contain the normal heterogeneity and threedimensional architecture that is seen in vivo. this said, however, tissue culture biology has proved to be successful in many ways. we have briefl y discussed the advantages that experimental systems using tissue culture affords researchers studying physiology and pathogenesis. because of its ability to isolate individual variables and determine their role(s) in physiology, cell culture has become an integral tool in deciphering the pathologic cascades that occur in human disease. diseases that affect lung are no exception. many diseases that affect the lung, and humans in general, are multifactorial. this begs the question how can cell culture, because of its reductionist nature only dealing with a minimal number of variables, help to solve the unknown questions and decipher the components involved in disease? often, clinical observations, and the questions arising therein, have been the launching pad for investigation. for instance, observations of massive infl ammation in the bronchoalveolar lavage samples of patients with acute respiratory disease syndrome (ards), consistent with damage seen in histologic samples, prompted investiga-tors to determine the role(s) of infl ammation in the etiology of ards. through the use of cell culture, investigators were able to determine individual interactions that occurred in the disease process. investigators have utilized culture models employing microcapillary endothelial cells under fl ow conditions to understand the role of proinfl ammatory cytokines in the cytokinesis and emigration of neutrophils in disease. using a model of pulmonary endothelium under fl ow conditions allowed investigators to demonstrate the importance of certain proinfl ammatory cytokines in ards. 11 the role of inhaled toxicants in lung injury, and the mechanism(s) by which they cause disease, is another area of investigation that has utilized cell culture. scientists have developed diverse and unique tissue culture systems that contain air-liquid barriers of lung epithelium and subjected these cells to various gaseous toxicants to determine what occurs following inhalation of various chemicals. utilizing these types of systems, investigators are able to control the exposure time and other variables that may be diffi cult when determining inhaled toxicant effects in vivo. moreover, the use of tissue culture, as opposed to an animal model, allows investigators to observe effects kinetically, without undue changes (e.g., sacrifi ce) and expense in the experimental model. 11 a tissue culture model also permits an investigator to observe multiple changes in real time, such as cellular integrity, cell signaling and intracellular traffi cking, protein expression changes, oxidant-induced cellular damage, and more. deciphering each of these changes in an animal model would be extremely diffi cult; through employing a tissue culture model, researchers are able to tightly control the experimental system while isolating the events of interest. further examples of how tissue culture models are currently being used to elucidate questions in lung physiology and disease are discussed later in the section on lung tissue cell lines. maintaining cells in vitro was initially a very diffi cult task. many characteristics need to be fulfi lled before a successful cell culture occurs. some of these characteristics are dependent on the type of tissue being studied; others may depend on specifi c requirements of the individual cells. various chemically defi ned media are now available commercially to support the growth and differentiation of numerous cell types. the creation of defi ned media has allowed investigators to culture a multitude of cell types while controlling the local environment to answer pertinent questions. for example, glucose can be removed from a culture medium in order to study its effects on cellular metabolism, relative position in the cell cycle, and many other effects. each chemical component is known in these media. additionally, investigators can add growth factors to nourish their cell cultures. the medium chosen when culturing cells in tissue culture must fi t two main requirements: (1) it must allow cells to continue to proliferate in vitro, and (2) it must allow the preservation of the certain specialized functions of interest. 7 the most common medium formulations used currently in lung research are dulbecco's modifi ed eagle's medium, minimum essential medium, rpmi 1640, and ham's f-12. occasionally, investigators develop new medium types to attain a formulation that optimizes their own experimental conditions. fetal bovine serum is a common additive to most tissue culture media, although some investigators choose to forgo this additive for more defi ned supplementation. additionally, others may choose sera from other sources such as human serum when culturing cells of human origin. inactivation of complement by heat treating serum for 1 hr at 56°c was initially very popular in tissue culture. however, it has become clear that this treatment may in fact damage some of the proteinaceous growth factors present in the medium, rendering it less effective. currently, many experts recommend heat inactivation only if the cell type of interest is particularly sensitive to complement. 12 more specifi c examples of medium utilized in lung tissue culture models are given later in the section on lung tissue cell lines. when deciphering if the current culture conditions are suffi cient for the experimental design, the investigator must determine which cellular characteristics are important. not only are the general characteristics, such as adhesion, multiplication, and immortalization of cell types important, but so are tissue-specifi c characteristics. of importance to pulmonary research, the lung is a unique environment to simulate in vitro because of the air-liquid interface. recently, investigators have made use of culture insert wells (e.g., transwells, corning) in order to study this interaction. 6 cell adhesion nearly all normal or neoplastic human epithelial cells will attach with relative ease to tissue culture surfaces. most tissue culture models utilizing tissue of lung origin fi t this description, with the notable exception of small cell lung carcinoma cell lines. however, for culture cells that may loosely adhere, or may not adhere at all, scientists coat tissue culture surfaces with extracellular matrix proteins. incubating tissue culture surfaces with serum, as well as laminin, fi bronectin, or collagen, prior to culture has been shown to improve attachment of fi nicky cells. 8 these treatments also help in replicating the normal attachment of cells to extracellular matrix proteins in vivo. the development of continuous cell lines may be serendipitous, as was the development of early cell lines. in brief, many investigators would continue splitting primary cell cultures until one or more cell clones became immortal. unfortunately, the changes that generally occurred in culture led to cells with abnormal phenotypes that had undergone dedifferentiation. today, many investigators choose to use molecular biology techniques, exploiting our current knowledge of oncogenic viruses and enzymatic processes of cellular aging to transform primary cells in vitro to an immortal phenotype. it is known that the large t antigen present in the sv (simian virus) 40 virus is capable of transforming cells to an abnormal phenotype. 11, 13, 14 moreover, transfection of primary cells with a transposase enzyme has also been shown to induce an immortal phenotypic change while preserving most normal cellular functions and phenotypes. 11 dedifferentiation a commonly encountered problem in tissue culture is dedifferentiation. this loss of phenotype may be insignificant to the research at hand or it may be critical, and it must be dealt with on a case by case basis. when a cell culture undergoes dedifferentiation it is often unclear whether undifferentiated cells took over the culture of terminally differentiated cells or whether a primary cell of interest became immortal under the culture conditions. the functional environment in which cells are cultured is critical when correlating experimental results to those seen in vivo. we previously alluded to the importance of the environment in which cells are cultured when discussing the advantages and limitations of tissue culture. investigators have frequently strived to replicate integral in vivo environments in vitro in order to increase the signifi cance of their experimental results. the development of cell culture insert wells (e.g., transwells, corning) has allowed investigators to culture bronchial or alveolar epithelial cells at an air-liquid interface. this ability allows investigators to begin to replicate a signifi cant aspect of these cells' functional environment in vitro, thereby increasing their understanding of the effects of gaseous particles on pulmonary epithelial cells. alternatively, scientists have also cultured epithelial cells on a roller bottle apparatus. this method allows investigators to determine the amount of time the apical epithelial cell surface is in contact with the air. capillary cell cultures have also come under frequent criticism when cultured in a monolayer in a tissue culture plate. investigators have been able to utilize gel matrices in which capillary cells form tubule-like structures, more closely replicating the architecture these cells maintain in vivo. additionally, endothelial cells are constantly under fl ow conditions in vivo. addressing this condition in vitro has allowed investigators to look at the role of endothelial cells during infl ammation-helping to increase the understanding of the role endothelium plays in acute lung injury. at times, researchers may also choose to determine the effects of soluble factors (e.g., cytokines, hormones, neurotransmitters) from acute patients or animal models in a cell culture model. the milieu of soluble factors present in the serum that may play a role in a disease state is considerable. moreover, these factors may have actions alone that are different when combined with other soluble factors. reconstituting every factor presents a diffi culty in vitro and leaves the possibility that an unknown factor may be missing. to address this, investigators have harvested sera from patients or animal models and used these samples as additives in their media formulations. for instance, through the use of serum samples from an animal model of smoke/burn injury-induced acute lung injury, investigators have demonstrated that use of arteriovenous co 2 removal in acute lung injury signifi cantly reduces apoptotic cell death in epithelial cells. 15 lung tissue cell lines: establishment and signifi cance the diversity of research fi elds utilizing tissue culture models of lung diseases is extensive. in this section, we will give a brief overview of the main lung cell types that are being utilized in research today to answer pressing questions about lung physiology and the pathophysiology of pulmonary disease. included in this discussion is also an overview of cell isolation and culture. the use of normal human bronchial epithelial (hbe) cells is extensively reported in the literature. based on a method pioneered by lechner et al., 16 bronchial fragments obtained from surgery, autopsy, or biopsy specimens may be used as explants. the outgrowth of bronchial epithelial cells occurs readily from these explants when grown in medium supplemented with bovine pituitary extract and epidermal growth factor. alternatively, these cells have also been demonstrated to grow in basal keratinocyte serum-free medium without supplementation; however, they demonstrate a slower growth rate and earlier senescence. 8 cultures of hbe cells are valuable for determining the responses to toxic inhaled pollutants. in vitro exposure systems based on these methods have several advantages. first, in vitro exposure systems can be stringently controlled and reproduced much better than in animal systems; second, individual determination of the cell types' responses to pollutants allows for a better characterization of the individual involvement of the cell type to a biologic response. finally, in vitro determination of the responses to toxic agents allows investigators to observe the reactions of human cells when testing in humans is not feasible because of ethical restraints. in vitro study of the responses of bronchial cells to gaseous pollutants is not without its diffi culties. wallaert et al. 17 have described these constraints well. briefl y, because of the gaseous nature of the pollutants, culture systems should be designed that allow signifi cant exposure times to pollutants while also taking care to inhibit cells from drying out when exposed to air. to facilitate these experiments, roller bottle cultures have been developed that allow cells direct contact with the ambient air. alternatively, cells have been grown on a membrane fi lter and cultured at an air-liquid interface, which allows constant exposure to the experimental treatment. the same type of experiments that are used to determine the responses of cells to inhaled toxicants have also been used to characterize responses to inhaled pharmaceuticals. in addition to the characterization of responses to inhaled agents, epithelial cell cultures, notably alveolar epithelium obtained from fetal lung tissue, have allowed investigators to characterize the liquid transport phenotype that occurs in the developing lung. characterization of the cl − ion secretion system, which occurs in the distal lung epithelium throughout gestation, has been shown to be integral in the stimulation of growth of the developing lung by regulating liquid secretion. likewise, a phenotypic switch of na + absorptive capacity has been described toward the end of gestation, which is important for preparation of the lung to function postpartum and beyond. these culture systems have elucidated important physiologic changes that occur in the developing lung. similar experiments have demonstrated that while ion transport plays a crucial role in this process other hormones and neurotransmitters are also important. pulmonary endothelial cells represent a unique type of endothelium because of their paradoxical responses to hypoxia. this uniqueness highlights the need to utilize cell culture models of pulmonary endothelium as opposed to other endothelia when interested in investigating their role(s) in pulmonary physiology. several investigators have described the isolation and culture of pulmonary endothelial cells. persistent pulmonary hypertension of the newborn, also known as neonatal pulmonary hyper-tension, is caused by a disorder of the pulmonary vasculature from fetal to neonatal circulation, culminating in hypoxemic respiratory failure and death. the inciting events that culminate in neonatal pulmonary hypertension are multifactorial. despite this, decreased production of vasodilator molecules such as nitric oxide and prostaglandin i 2 in the pulmonary endothelium has been shown to be a critical component of disease progression. 18 primary cell cultures of human airway smooth muscle tissue can be obtained utilizing a method described by halayko et al. 19 in which they isolated and characterized airway smooth muscle cells obtained from canine tracheal tissue. briefl y, airway smooth muscle cells were obtained by fi nely mincing tissue and subjecting it to an enzymatic disaggregation solution containing collagenase, type iv elastase, and type xxvii nagarse protease. following generation of a single cell suspension, cells may be grown in dulbecco's modifi ed eagle's medium supplemented with 10% fetal bovine serum. halayko et al. 20 obtained approximately 1.3 × 10 6 smooth muscle cells per gram of tissue using this method. although halayko et al. 21 pioneered this technique using trachealis tissue, many other investigators have obtained airway smooth muscle cells from a variety of biopsy specimens. airway smooth muscle hyperreactivity and hypertrophy has been known for nearly 100 years 2 to be an important end response of asthma. the use of airway smooth muscle in vitro has been vital toward delineating the pathologic steps that occur in asthma, as well as testing of potential therapeutics that may help to decrease the morbidity and mortality of asthma. additionally, the relative paucity of in vivo models of asthma further illustrates the value of isolation and characterization of smooth muscle cells from asthmatic patients in vitro. using airway smooth muscle cell culture, investigators have characterized both the hypertrophic and hyperplastic growth of smooth muscle in individuals. investigation of the potential stimuli that lead to airway smooth muscle proliferation and hypertrophy have led researchers to implicate the mitogen-activated protein kinase family members, extracellular signal-regulated kinase-1 and -2, and the phosphoinositol-3 kinase pathways in pathogenesis. 22 additionally, mediators directing smooth muscle migration have also been observed in vitro and may play a role in the progression of asthma. platelet-derived growth factor, fi broblast growth factor-2, and transforming growth factor-β (tgf-β) have all been shown to play a role in the migratory response of smooth muscle cells seen in asthma. 22 additionally, contractile agonists such as leukotriene e 4 have been shown to potentiate the migratory responses seen with platelet-derived growth factor treatment. 22 human airway smooth muscle cell culture has also been utilized to investigate possible pharmacologic interventions for the treatment of asthma. β 2 -agonists have been shown to decrease the rate of dna synthesis and likewise decrease the hyperplasia seen in airway smooth muscle cells in response to mitogenic stimuli through an increase in cyclic adenosine monophosphate. like β 2agonists, glucocorticoids have similar antiproliferative activities. lung cancer tissue and the development of novel therapeutics culture of neoplastic cells from human tumors has allowed investigators to harvest a wealth of knowledge into the biology of lung cancers; moreover, these cultures have provided potential models to test potential therapeutics. the propagation of lung cancer cells in vitro has been covered in great depth previously. 8 in contrast to primary cell cultures, cultures of neoplastic cells are immortal, allowing their easy growth in culture with less chance of being overgrown by mesenchymal cells such as fi broblasts. the relative ease of growth in culture has led to many cell lines of lung cancer tissue. the national cancer institute, recognizing the need for a variety of lung cancer cell lines (both small cell and non-small cell), helped establish over 300 cell lines. 23 these lines are a wonderful resource for investigators given that they are extensively characterized, and many have full clinical data available. moreover, many of these cell lines are now easily available through the american type culture collection for a modest handling fee. additionally, if investigators do not wish to use currently established lung cancer cell lines, obtaining clinical samples for use in tissue culture models is relatively easy. the same methods used to obtain biopsy specimens for clinical staging can also be used to begin cell cultures. following culture and initial characterization of lung cancer cell lines, many investigators have demonstrated that lung cancer cell lines maintain a similar phenotype after establishment. specifi cally, it has been verifi ed that injection of lung cancer cell lines into nude mice exhibit similar histopathology to the original tumor, indicating minimal change occurred following establishment of the cell lines. small cell lung carcinoma (sclc) cell lines have been established from a multitude of biopsy specimens, including bone marrow, lymph nodes, and pleural effusions. 8, 24 once viable cells have been obtained from clinical samples, cells are easily maintained in a basal cell culture medium such as rpmi 1640 in a humidifi ed incubator at 37°c and 5% co 2 , although the initial isolations of sclc lines utilized hites and acl-4 media. 25 most established sclc cell lines maintain a neuroendocrine phenotype in culture; however, baillie-johnson et al. 24 noticed considerable heterogeneity in the cell lines they established, highlighting the signifi cance that establishing a cell line from the clinical sample of interest may provide investigators with a line that possesses the exact phenotypic properties of interest. small cell carcinoma poses many diffi culties to surgical treatment, owing to its early and widespread metastasis. therefore, combination chemotherapy is generally utilized in treatment. unfortunately, despite initial sensitivities, sclc tumors become resistant to further treatment. utilizing in vitro cultures of sclc cell lines, sethi et al. 26 began to describe how extracellular matrix proteins can protect sclc against apoptosis-inducing chemotherapeutics through β 1 -integrin-mediated survival signals. these data indicate that extracellular matrix proteins surrounding sclc may play a role in the local recurrence seen in patients following chemotherapy in vivo and suggest novel therapeutics aimed at blocking these survival signals. non-small cell lung carcinoma (nsclc) cell lines including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma have all been established. despite the fact that nsclc cells comprise three distinct histologic cell types, all cell types can be established relatively easily. the primary treatment protocol for patients affl icted by nsclc is generally surgical resection of the tumor; therefore, tumor cells for culture are readily available. these cell types can be grown under conditions similar to those described for sclc. infectious diseases play a unique role in lung pathology in light of their roles as either important contributors or consequences of many lung diseases. for instance, certain lung diseases may predispose patients to infection: patients affl icted with obstructive lung diseases, as well as cystic fi brosis patients, commonly suffer from severe and recurrent bacterial infections. additionally, patients may become superinfected following a viral respiratory infection. systemic infections, such as gram-negative bacterial sepsis, may lead to lung diseases such as ards. human type ii alveolar pneumocytes and acute lung injury/acute respiratory distress syndrome pulmonary alveolar type ii cells are a unique cell subset that carries out highly specialized functions that include synthesis and secretion of surfactant, a unique composition of lipoproteins that act to reduce surface tension at the alveolar air-liquid interface. 27 defi ning the molecular mechanisms leading to production of surfactant by type ii pneumocytes is important in many disease processes. the pathogenic sequence that results in ards, the most severe manifestation of alveolar lung injury, is generally thought to be initiated by a systemic infl ammatory response. 28 despite this knowledge, there still exist many questions about the initial triggers and pathologic steps that occur in ards. greater understanding of these steps may help to develop new treatment regimes. currently, treatment of ards consists of mechanical ventilation, which helps to stabilize blood gases. however, mechanical ventilation itself may provoke further infl ammation in the alveoli, thereby decreasing compliance and gas exchange in the alveoli. 29 the cell type of particular interest in ards and diffuse alveolar damage is the type ii pneumocytes. [30] [31] [32] [33] [34] until recently, studies trying to decipher the pathologic sequence in acute lung injury have had to rely on standard lung epithelial cell lines. recently, however, human type ii alveolar epithelial cells (pneumocytes) have been successfully isolated from fetal human lung tissue by collagenase digestion. 35 briefl y, fetal lung tissues were minced and incubated in a serum-free medium containing dibutyryl cyclic adenosine monophosphate for 5 days. the tissue explants were then treated with collagenase and incubated with deae-dextran to eliminate contaminating fi broblasts. cells were then plated onto tissue culture dishes treated with extracellular matrix derived from mdck cells and cultured overnight in waymouth's medium containing 10% serum. these steps resulted in relatively pure populations of human type ii pneumocytes that were then cultured at an air-liquid interface. using these methods, alcorn et al. 35 were able to maintain a primary culture that retained the morphologic and biochemical characteristics of type ii pneumocytes for up to 2 weeks. conventional bioreactors and three-dimensionality: the origins of three-dimensional culture carrel postulated that tissue development was linked to access to nutrient supply, noting that peripheral cells grew readily, and internal cells became necrotic presumably based on their distance from the nutrient source. to circumvent this issue, carrel implemented cultures on silk veils, preventing the plasma clots of the growth media from deforming or becoming spherical, thus facilitating the internal cell's ability to obtain nutrient replenishment. many attempts were made in standard culture systems (bioreactors) and other culture apparatuses to escape the constraints of two-dimensional cell culture, with the intent of yielding high-fi delity human and mammalian tissues, and thus emphasizing the need for development of three-dimensional biology. another famous researcher, leighton, improved on carrel's techniques in the 1950s and 1960s. leighton's major contribution to three-dimensional culture technology was the introduction of the idea of a sponge matrix as a substrate on which to culture tissues. 36 , 37 leighton fi rst experimented on cellulose sponges surrounded by plasma clots resident within glass tubes. he devised a system to grow 1-to 5-mm 3 tissue explants on sponges, using small amounts of chick plasma and embryo extract. after the mixture solidifi ed on the sponge leighton added the nutrient media and inserted the "histoculture" in a roller apparatus to facilitate nutrient mass transfer. he experimented with many sponge combinations, discovering that collagen-impregnated cellulose sponges were optimal for sustaining the growth of native tissue architecture. 3, 38 leighton was successful in growing many different tissue types on the sponge-matrix cultures. 3, 38 leighton also found that c3hba mouse mammary adenocarcinoma cells, when grown on sponge-matrix histoculture, aggregated "much like the original tumor, forming distinct structures within the tumors such as lumina and stromal elements, and glandular structures." an extremely important difference of this threedimensional histoculture from the standard two-dimensional culture is the apparent quiescence of the stromal component and the balanced growth of these cells with regard to the overall culture. leighton further advanced the concept of three-dimensional histoculture to histophysiologic gradient cultures. 39 these cultures are conducted in chambers that allow metabolic exchange between "the pool of medium and the culture chamber by diffusion across a membrane." histophysiologic gradient cultures mimic, to some degree, diffusion in tissues. 38 from the pioneering work of carrel and leighton, other methods of emulating three-dimensional cultures have been developed, such as embedding cells and tissues in collagenous gels of rat tail as per the techniques of nandi and colleagues. many of the advantages of threedimensional cultures seen by leighton, nandi, and others may be attributed to permitting the cells to retain their normal shape and special associations. 3 this global concept will be important as we begin to understand and recall the physical and environmental characteristics of the rotating-wall vessel systems. other methods of three-dimensional culture encompass a technique known as organ culture or culture on a fi lter, a strategy developed by strangeways 40 and fell and robinson. 41 tissue explants were grown on lens paper in a watch glass containing liquid culture medium. browning and trier 42 found "that for some tissues, it is critical to keep the cultures at the air-liquid interface," thus allowing the tissues to experience conditions similar to the in vivo environment. another strategy is the use of three-dimensional cultures known as proto-tissues, or aggregates of cells, used to form spheroids. this technique was popularized by sutherland and colleagues more than 20 years ago when they manipulated aggregates of cells into a spherical confi guration by spinning agitation of the cells in spinner fl asks. 43 this technique produced pseudo-tissue-like organoids useful for research evaluations. each of these methodologies will be of benefi t as we continue to examine strategies for achieving three-dimensional lung tissue constructs. 3, 38 finally, membrane bioreactors are capable of retaining enzymes, organelles, and microbial, animal, and plant cells behind a membrane barrier, trapped in a matrix or adherent to the membrane surface. in 1963, gallup and gerhardt 44 fi rst used the membrane bioreactor for dialysis culture of serratia marcescens. immobilized enzyme microencapsulation was pioneered by chang, 45 but butterworth et al. 46 fi rst developed the enzyme membrane reactor to successfully accomplish starch hydrolysis with α-amylase. likewise, for animal cell culturing, knazek et al. 47 fi rst cultured human choriocarcinoma cells on compacted bundles of amicon fi bers. many reviews on the particular applications of hollow fi ber and immobilized bioreactant bioreactors for enzyme catalysts, microbial cells, and animal cell culture are available. [48] [49] [50] [51] [52] [53] as presented previously, tissue-engineering applications of three-dimensional function and structure are well known in medical science research. 54 in microgravity three-dimensional aggregates form, facilitating the expression of differentiated organotypic assemblies. investigations to determine the effect of composite matrices, spiked with esterifi ed hyaluronic acid and gelatin, to augment osteochondral differentiation of cultured, bone marrow-derived mesenchymal progenitor cells and the effects of the matrix on cellular differentiation have been examined in vitro and in vivo. 54 briefl y, empty and populated matrices cultured for 28 days, with and without tgf-β 1 demonstrated the following results. cells implanted in the matrix produced a robust type ii collagen extracellular matrix in vitro. matrices placed in immunodefi cient mice yielded no differentiation in empty constructs, osteochondral differentiation in loaded implants, and an enhanced level of differentiation in preimplantation in vitro-cultured matrices containing tgf-β 1 . these results demonstrate the utility of three-dimensional matrix for presentation of bone mesenchymal progenitor cells in vivo for repair of cartilage and bone defects as well as indicate the efficacy for in vitro tissue engineering regimes. 54 these techniques lend themselves to microgravity and ground-based research tissue cultures alike. many earth-based laboratories are researching and developing hemopoietic bone marrow cultures of stem cell origin, and three-dimensional confi gurations are providing promising results as illustrated by schoeters and coworkers. 55 they report that murine bone marrow cells, cultured under long-term hemopoietic conditions, produce mineralized tissue and bone matrix proteins in vitro but only when precipitated by the presence of adherent bone stroma cells in three-dimensional collagen matrices. at a concentration of 8 × l0 6 stromal cells, mineralization occurs in 6 days. in contrast, twodimensionally oriented marrow fragments at 1 × 10 7 cells require requires more than 10 days before mineralization can similarly be detected. 55 two-dimensional long-term marrow culture facilitates and enhances expansion of the stromal component and rudimentary differentiation of osteogenic-like cells in the adherent stromal layer as verifi ed by type i collagen or cells positive for alkaline phosphatase. production of osteonectin and osteocalcin, a bone-specifi c protein, combined with calcifi cation is observed only in threedimensional cultures. these studies demonstrate the need for and benefi t of three-dimensionality and the application to the microgravity environment. 55 as we can see, this further reinforces the quest for threedimensionality and the potential of modeling the microgravity environment. investigations clearly show the need for the application of three-dimensional study techniques in lung pathophysiologic studies. interestingly, three-dimensional biology has facilitated full-scale investigations into most areas of tissue engineering, cell biology and physiology, immunology, and cancer research. anchorage-dependent cells are widely cultured on microcarriers. 56 studies show that for the purposes of improved surface-to-volume ratio and scale up, the microcarrier suspension culture provides excellent potential for high-density cell growth. 57 in addition, microcarriers serve well as structural supports for three-dimensional assembly, the composite of which is the basis for threedimensional tissue growth. 58 conventional culture systems for microcarrier cultures (i.e., bioreactors) use mechanical agitation to suspend microcarriers and thus induce impeller strikes as well as fl uid shear and turbulence at the boundary layer between the wall and the fl uid. investigators have attempted to make a complete study of the most effi cient bioreactor designs and agitation regimens. 59 they concluded that virtually all stirred-tank bioreactors operate in the turbulent regimen. it has been demonstrated that bead-to-bead bridging of cells is enhanced signifi cantly at lower agitation rates in a stirred reactor. 60 excessive agitation from either stirring or gas bubble sparging has been documented as a cause of cell damage in microcarrier cell cultures. 61, 62 to overcome the problems induced by these mechanisms, investigators developed alternative culture techniques such as porous microcarriers to entrap cells, 63 increased viscosity of culture medium, 64 bubble-free oxygenation, 65 and improved methods for quiescent inoculation. 66, 67 these steps decreased the damage attributed to turbulence and shear forces but failed to signifi cantly rectify the problems. reactor systems of substantially increased volume exhibit less agitation-related cell damage. this is presumably because of the decreased frequency of cell-microcarrier contact with the agitation devices in the systems. research-scale investigations do not afford the luxury of experimenting with large-scale production systems. therefore, if a large-volume system is indeed more quiescent, an improved bioreactor system should emulate the fl uid dynamics present in the upper regions of large-scale reactors in which cells and microcarriers reside with minimal agitation. the problem, then, is to suspend microcarriers and cells without inducing turbulence or shear while providing adequate oxygenation and nutritional replenishment. the term rotating-wall vessel comprises a family of vessels, batch fed and perfused, that embody the same fl uid dynamic operating principles. these principles are (1) solid body rotation about a horizontal axis that is characterized by (a) colocation of particles of different sedimentation rates, (b) extremely low fl uid shear stress and turbulence, and (c) three dimensional spatial freedom; and (2) oxygenation by active or passive diffusion to the exclusion of all but dissolved gasses from the reactor chamber, yielding a vessel devoid of gas bubbles and gas-fl uid interface (zero head space). 68, 69 three-dimensional models of lung disease current cell culture models have shortcomings resulting in unreliable tumor growth, uncharacteristic tumor development, nonhuman tumors, and inadequate methods of detection. cells propagated under traditional culture conditions differ widely in their expression of differentiated markers, adhesion receptors, and growth factor receptors compared with cells in situ or those grown as tissue-like structures. 70, 71 this is of concern because the phenotypic changes leading to malignant transformation often stem from alterations in the balanced and multifaceted roles of growth factors, receptors, and cytokines (reviewed by herlyn et al. 71 ). with increasing evidence of the importance of adhesive contacts, paracrine cross-talk between different cell types, and signaling cascades that link the cell with a complex substratum, there is now recognition that models must be developed that better simulate these complexities. there is still much to learn about the dynamic relationships among the different phenotypes found in the normal lung and in lung cancers. until a cell culture system is developed that allows differentiation to occur, 72 it is diffi cult to make any fi rm statement about relating effects in cell culture to clinical practice. tissue engineering is very embryonic in development and currently nearly universally focused on building replacement tissues. a new technology developed at the nasa johnson space center used to study colon cancer has been adapted to three-dimensional in vitro lung tissue culture models but has not been reported on to date. rotating-wall vessels are horizontally rotating cylindrical tissue culture vessels that provide controlled supplies of oxygen and nutrients with minimal turbulence and extremely low shear. 69 these vessels suspend cells and microcarriers homogeneously in a nutrient-rich environment, which allows the three-dimensional assembly of cells to tissue. prior to seeding rotating-wall vessels (synthecon, inc, houston, tx), cells were cultured in standard t fl asks (corning, corning, ny) in gtsf-2 medium (1993 psebm) in a humidifi ed 37°c, 5% co 2 incubator. the rotating-wall vessels were seeded with 1-2 mg/ml cultispher-gl microcarriers (hyclone laboratories, inc., logan, ut) followed by beas2-b or bzr-t33 cells (atcc, baltimore, md) at a density of 2 × 10 5 cells/ml. cultures were grown in the rotating-wall vessels for 14-21 days for formation of 3-to 5-mm diameter tumor masses. rotating-wall vessel rotation was initiated at 25 rpm and increased as aggregate size became larger. stationary control cultures were initiated under the same conditions using fep tefl on bags (american fluoroseal, columbia, md). at 24-hour intervals ph, dissolved co 2 , and dissolved o 2 were determined using a corning 238 model clinical blood gas analyzer. glucose concentration was determined using a beckman 2 model clinical glucose analyzer (beckman, fullerton, ca). cell samples were harvested every 48 hr and fi xed with omnifi x (xenetics, tustin, ca) for immunohistochemistry or fi xed with 3% glutaraldehyde/2% paraformaldehyde in 0.1 m cacodylic buffer (electron microscopy sciences, fort washington, pa) for scanning electron microscopy. cancer models already developed by nasa investigators include growth and differentiation of an ovarian tumor cell line, 72-74 growth of colon carcinoma lines, 72 and three-dimensional aggregate and microvillus formation in a human bladder carcinoma cell line. 74 in support as an appropriate model for cancer, even the most rudimentary three-dimensional cellular structures exhibit different phenotypes than cell lines cultured under twodimensional conditions. properties such as responses to tgf-β, drug resistance to cisplatin or cyclophosphamide, and resistance to apoptosis are all altered in various types of cell aggregates. 75 many investigations sustain consistent evidence that cells growing in three-dimensional arrays appear more resistant to cytotoxic chemoagents than cells in monolayer culture. 38 li et al. found that spheroids were more resistant to cytosine arabinoside by 11-fold and methotrexate by 125-fold when compared with single cell suspensions. 76 further monolayer cultures of colon carcinoma cells were sensitive to piericidin c in contrast to responses within in vivo colon tumors or three-dimensional slices of tumors grown in vitro. 77 numerous other investigations have revealed increased levels of drug resistance of spheroids compared with single cell monolayers. 3, 38 questions of poor diffusion and insuffi cient drug absorption within spheroids and a relatively frequent high proportion of resting cells have clouded differences in drug resistance, which could be the result of nutrient deprivation and hypoxia. heppner and colleagues executed precise experiments that confi rmed threedimensional structure and function as the causative agent and was responsible for drug resistance rather than simple inaccessibility to nutrients or the drug concentration. heppner embedded tumor specimens or cell aggregates in collagen gels, exposed the culture to various cytotoxic drugs, and compared the drug responses of the same cells in monolayers. these experiments revealed an increased resistance in the three-dimensional tumor arrays of a remarkable 1,000-fold greater than in monolayer cultures, and a similar result was seen in three-dimensional histocultures in collagen. the tumor cells grew in the presence of drug concentrations that rendered monolayers to a viability less than 0.1% of control cultures. amazingly, heppner observed that the cells became sensitive again when replated as monolayers and fi nally showed that even when exposed to melphalan and 5-fl uorouracil in monolayer cells transferred to collagen gels were again resistant based on three-dimensional architecture. thus, the cells were exposed to the drugs as monolayers, facilitating access to the drugs, and, once the cells were transferred after drug exposure to a threedimensional structure, high resistance to the drugs was sustained. 38, [78] [79] [80] [81] based on the caliber of data referenced above, teicher et al. 82 serially passaged through multiple (10) transfers emt-6 tumors in mice that were treated with thiotepa, cisplatin, and cyclophosphamide over a prolonged 6month period, thus producing extremely drug-resistant tumors in vivo. when these tumors were grown as monolayer cultures, they were as drug sensitive as the parental cells. kobayashi and colleagues 83 grew the same in vivo drug-resistant tumor cell lines as spheroids in threedimensional arrays, and resistance was almost 5,000 times that of the parent line with selected drugs, an example being the active form of cyclophosphamide used in vitro. similarly extreme resistance was also observed to cisplatin and thiotepa. this resistance was not seen in monolayer cultures, even when the monolayers were cultured on traditional extracellular matrix substrates. these experiments reconfi rmed that cells in a three-dimensional array are more drug resistant than monolayer cells in vitro and demonstrated that three-dimensional cellular confi gurations can and do become resistant to super pharmacologic doses of drugs by forming compact structures. 38 rotating-wall vessel tumor models several important human tumor models have been created in rotating-wall vessel cultures, specifi cally, lung, prostate, colon, and ovarian. 14, 58, 73, 84 many of these models involve cancers that are leading killers in our society. we present two such examples in this section, colon and prostate carcinoma. as previously reviewed, the literature indicates the remarkable difference between chemotherapeutic cytotoxicity in two-dimensional and three-dimensional cellular constructs, which may be predicated on a number of criteria. therefore, a threedimensional tumor model that emulates differentiated in vivo-like characteristics would provide unique insights into tumor biology. goodwin et al. 58 detail the fi rst construction of a complex three-dimensional ex vivo tumor in rotatingwall vessel culture composed of a normal mesenchymal base layer (as would be seen in vivo) and either of two established human colon adenocarcinoma cell lines, ht-29, an undifferentiated line, and ht-29km a stable, moderately differentiated subline of ht-29. each of these engineered tumor tissues produced tissue-like aggregates (tlas) with glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. necrosis was minimal throughout the tissue masses up to 60 days of culture while achieving >1.0 cm in diameter. other notable results included enhanced growth of neoplastic colonic epithelium in the presence of mixed normal human colonic mesenchyme. these results mimic the cellular differentiation seen in vivo and are similar to results obtained with other tumor types. prostate carcinoma has also been modeled in the rotating-wall vessel system by several investigators. [85] [86] [87] one of the most comprehensive descriptions of these engineered tissues is detailed by wang et al. 88 in that review, the authors describe the ability of the rotatingwall vessel system to recapitulate human prostate carcinoma (lncap) and bone stroma (mg63) to illuminate the evolution of prostate tumorigenesis to the metastatic condition. in particular, the lncap and arcap models represented in the review are known to be lethal in the human, being androgen independent and metastatic. rotating-wall vessel tla engineering also allowed indepth study of epithelial and stromal interactions, which are the facilitating elements of the continuance of lncap prostate-specifi c antigen production in vitro. when lncap was cultured in three dimensions without stroma, production of prostate-specifi c antigen ceased and metastatic markers were not observed. the authors outline the process of malignant transformation, demonstrating that these metastatic models are only possible in threedimensional tlas and are achieved by specifi c geometric relationships in three-dimensional confi guration. furthermore, they show through direct comparison with other culture systems the advantages of the rotating-wall vessel system to allow synergistic relationships to study this disease state. 88 unlike two-dimensional models, these rotating-wall vessel tumor tissues were devoid of metabolic and nutrient defi ciencies and demonstrated in vivo-like architecture. these data suggest that the rotating-wall vessel affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissues. in this section, we explore the utility of rotating-wall vessel tlas as targets for microbial infection and disease. several studies have been conducted recently that indicate that three-dimensional tissues respond to infective agents with greater fi delity and with a more in vivo-like response than traditional two-dimensional cultures. nickerson et al. 89 describe the development of a threedimensional tla engineered from int-407 cells of the human small intestine, which were used as targets for the study of salmonella typhimurium. in this study, threedimensional tlas were used to study the attachment, invasion, and infectivity of salmonella into human intestinal epithelium. immunocytochemical characterization and scanning and transmission electron microscopic analyses of the three-dimensional tlas revealed that the tlas more accurately modeled human in vivo differentiated tissues than did two-dimensional cultures. the level of differentiation in the int-407 tlas was analogous to that found in previously discussed small intestine tlas 72 and from other organ tissues reconstructed in rotatingwall vessels. analysis of the infectivity studies revealed salmonella attached and infected in a manner significantly different from that in control two-dimensional cultures. during an identical exposure period of infection with salmonella, the three-dimensional tlas displayed a minor loss of structural integrity when compared with the two-dimensional int-407 cultures. furthermore, salmonella demonstrated a greatly reduced ability to adhere, invade, and induce the apoptotic event in these int-407 three-dimensional tlas than in twodimensional cultures. this result is not unlike the in vivo human response. two-dimensional cultures were significantly damaged within several hours of contact with the bacteria; conversely, although "pot marks" could be seen on the surfaces of the three-dimensional tlas, they remained structurally sound. cytokine analysis and expression postinfection of three-dimensional tlas and two-dimensional cultures with salmonella exhibited remarkable differences in expressed levels of interleukin (il)-1α, il-1β, il-6, il-1ra, and tumor necrosis factor-α mrnas. additionally, noninfected three-dimensional tlas constitutively demonstrated elevated levels of tgf-β 1 mrna and prostaglandin e 2 compared with noninfected two-dimensional cultures of int-407. 89 as previously stated, traditional two-dimensional cell monolayers lack adequate fi delity to emulate the infection dynamics of in vivo microbial adhesion and invasion. the respiratory epithelium is of critical importance in protecting humans from disease. exposed to the environment, the respiratory epithelium acts as a barrier to invading microbes present in the air, defending the host through a multilayered complex system. 90 the three major layers of the human respiratory epithelium are pseudostratifi ed epithelial cells, a basement membrane, and underlying mesenchymal cells. ciliated, secretory, and basal epithelial cells are connected by intercellular junctions and anchored to the basement membrane through desmosomal interactions. together with tight junctions and the mucociliary layer, the basement membrane maintains the polarity of the epithelium and provides a physical barrier between the mesenchymal layer and the airway. 91, 92 infi ltrating infl ammatory and immune cells move freely between the epithelial and subepithelial compartments. airway epithelial cells play a vital role in host defense 90 by blocking paracellular permeability and modulating airway function through cellular interactions. ciliated epithelial cells block invasion of countless inhaled microorganisms by transporting them away from the airways. 93 as regulators of the innate immune response, epithelial cells induce potent immunomodulatory and infl ammatory mediators such as cytokines and chemokines that recruit phagocytic and infl ammatory cells that remove microbes and enhance protection. 90, 91, 94, 95 ideally, cell-based models should reproduce the structural organization, multicellular complexity, differentiation state, and function of the human respiratory epithelium. immortalized human epithelial cell lines, such as beas-2b, 96 primary normal human bronchial epithelial cells, 97 and air-liquid interface cultures, 98 are used to study respiratory virus infections in vitro. traditional monolayer cultures (two-dimensional) of immortalized human bronchoepithelial cells represent homogenous lineages. although growing cells in monolayers is convenient and proliferation rates are high, such models lack the morphology and cell-cell and cell-matrix interactions characteristic of human respiratory epithelia. thus, their state of differentiation and intracellular signaling pathways most likely differ from those of epithelial cells in vivo. primary cell lines of human bronchoepithelial cells provide a differentiated model similar to the structure and function of epithelial cells in vivo; however, this state is short lived in vitro. 97, 99 air-liquid interface cultures of primary human bronchoepithelial cells (or submerged cultures of human adenoid epithelial cells) are grown on collagen-coated fi lters in wells on top of a permeable fi lter. these cells receive nutrients basolaterally, and their apical side is exposed to humidifi ed air. the result is a culture of well-differentiated heterogeneous (ciliated, secretory, basal) epithelial cells essentially identical to airway epithelium in situ. 98, 100 although this model shows fi delity to the human respiratory epithelium in structure and function, maintenance of consistent cultures is not only diffi cult and time consuming but also limited to small-scale production and thus limits industrial research capability. true cellular differentiation involves sustained complex cellular interactions [101] [102] [103] in which cell membrane junctions, extracellular matrices (e.g., basement membrane and ground substances), and soluble signals (endocrine, autocrine, and paracrine) play important roles. [104] [105] [106] [107] this process is also infl uenced by the spatial relationships of cells to each other. each epithelial cell has three membrane surfaces: a free apical surface, a lateral surface that connects neighboring cells, and a basal surface that interacts with mesenchymal cells. 108 recently viral studies by goodwin et al. 109 and suderman et al. 110 were conducted with rotating-well vessel-engineered tla models of normal human lung. this model is composed of a coculture of in vitro threedimensional human bronchoepithelial tlas engineered using a rotating-wall vessel to mimic the characteristics of in vivo tissue and to provide a tool to study human respiratory viruses and host-pathogen cell interactions. the tlas were bioengineered onto collagen-coated cyclodextran beads using primary human mesenchymal bronchial-tracheal cells as the foundation matrix and an adult human bronchial epithelial immortalized cell line (beas-2b) as the overlying component. the resulting tlas share signifi cant characteristics with in vivo human respiratory epithelium, including polarization, tight junctions, desmosomes, and microvilli. the presence of tissuelike differentiation markers, including villin, keratins, and specifi c lung epithelium markers, as well as the production of tissue mucin, further confi rm these tlas differentiated into tissues functionally similar to in vivo tissues. increasing virus titers for human respiratory syncytial virus (wtrsva2) and parainfl uenza virus type 3 (wtpiv3 js) and the detection of membrane-bound glycoproteins (f and g) over time confi rm productive infections with both viruses. viral growth kinetics up to day 21 pi with wtrsva2 and wtpiv3 js were as follows: wtpiv3 js replicated more effi ciently than wtrsva2 in tlas. peak replication was on day 7 for wtpiv3 js (approximately 7 log 10 particle forming units [pfu] per milliliter) and on day 10 for wtrsva2 (approximately 6 log 10 pfu/ml). viral proliferation remained high through day 21 when the experiments were terminated. viral titers for severe acute respiratory syndrome-coronavirus were approximately 2 log 10 pfu/ml at 2 day pi. human lung tlas mimic aspects of the human respiratory epithelium well and provide a unique opportunity to study the host-pathogen interaction of respiratory viruses and their primary human target tissue independent of the host's immune system, as there can be no secondary response without the necessary immune cells. these rotating-wall vessel-engineered tissues represent a valuable tool in the quest to develop models that allow analysis and investigation of cancers and infectious disease in models engineered with human cells alone. we have explored the creation of three-dimensional tlas for normal and neoplastic studies and fi nally as targets for microbial infections. perhaps carrel and leighton would be fascinated to know that from their early experiments in three-dimensional modeling and the contributions they made has sprung the inventive spirit to discover a truly space age method for cellular recapitulation. the biochemical basis of pulmonary function the pathology of bronchial asthma three-dimensional histoculture: origins and applications in cancer research histoculture of human breast cancers studies on the propagation in vitro of poliomyelitis viruses. iv. viral multiplication in a stable strain of human malignant epithelial cells (strain hela) derived from an epidermoid carcinoma of the cervix effects of paramyxoviral infection on airway epithelial cell foxj1 expression, ciliogenesis, and mucociliary function culture of animal cells: a manual of basic technique preclinical models of lung cancer: cultured cells and organ culture human papillomavirus and the development of cervical cancer: concept of carcinogenesis relationship of alveolar epithelial injury and repair to the induction of pulmonary fi brosis generation of human pulmonary microvascular endothelial cell lines promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast lon culture and transformation of human airway epithelial cells a mechanism of hyperthermia-induced apoptosis in ras-transformed lung cells smoke/burn injury-induced respiratory failure elicits apoptosis in ovine lungs and cultured lung cells, ameliorated with arteriovenous co 2 removal clonal growth of epithelial cells from normal adult human bronchus experimental systems for mechanistic studies of toxicant induced lung infl ammation regulation of vasodilator synthesis during lung development markers of airway smooth muscle cell phenotype airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes airway wall remodelling and hyperresponsiveness: modelling remodelling in vitro and in vivo nci series of cell lines: an historical perspective establishment and characterisation of cell lines from patients with lung cancer (predominantly small cell carcinoma) cell culture methods for the establishment of the nci series of lung cancer cell lines extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo radioimmunoassay of pulmonary surface-active material in the tracheal fl uid of the fetal lamb selected anatomic burn pathology review for clinicians and pathologists overview of ventilatorinduced lung injury mechanisms surfactant protein-a levels in patients with acute respiratory distress syndrome positive end-expiratory pressure modulates local and systemic infl ammatory responses in a sepsis-induced lung injury model injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome alveolar infl ation during generation of a quasi-static pressure/volume curve in the acutely injured lung evaluation of mechanical ventilation for patients with acute respiratory distress syndrome as a result of interstitial pneumonia after renal transplantation primary cell culture of human type ii pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses collagen-coated cellulose sponge: three dimensional matrix for tissue culture of walker tumor 256 histophysiologic gradient culture of stratifi ed epithelium the three-dimensional question: can clinically relevant tumor drug resistance be measured in vitro? structural biology of epithelial tissue in histophysiologic gradient culture tissue culture in relation to growth and differentiation the growth, development and phosphatase activity of embryonic avian femora and end-buds cultivated in vitro organ culture of mucosal biopsies of human small intestine growth of nodular carcinomas in rodents compared with multicell spheroids in tissue culture dialysis fermentor systems for concentrated culture of microorganisms semipermeable microcapsules application of ultrafi ltration for enzyme retention during continuous enzymatic reaction cell culture on artifi cial capillaries: an approach to tissue growth in vitro membranes and bioreactors: a technical challenge in biotechnology membrane bioreactors: engineering aspects membrane bioreactors: present and prospects artifi cial membrane as carrier for the immobilization of biocatalysts hollow fi ber cell culture in industry hollow fi ber enzyme reactors engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges microcarrier/cultures of animal cells mammalian cell culture: engineering principles and scale-up morphologic differentiation of colon carcinoma cell lines ht-29 and ht-29km in rotating-wall vessels hydrodynamic effects on animal cells in microcarrier cultures physical mechanisms of cell damage in microcarrier cell culture bioreactors growth and death in over agitated microcarrier cell cultures cell death in the thin fi lms of bursting bubbles growth of anchoragedependent cells on macroporous microcarriers viscous reduction of turbulent damage in animal cell culture biological experiences in bubble-free aeration system critical parameters in the microcarrier culture of animal cells the large-scale cultivation of mammalian cells analysis of gravity-induced particle motion and fl uid perfusion fl ow in the nasa-designed rotating zero-head-space tissue culture vessel cell culture for threedimensional modeling in rotating-wall vessels: an application of simulated microgravity autocrine and paracrine roles for growth factors in melanoma growth-regulatory factors for normal, premalignant, and malignant human cells in vitro reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity threedimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics threedimensional modeling of t-24 human bladder carcinoma cell line: a new simulated microgravity vessel multicellular resistance: a new paradigm to explain aspects of acquired drug resistance of solid tumors comparison of cytotoxicity of agents on monolayer and spheroid systems modifi ed 2-tumour (l1210, colon 38) assay to screen for solid tumor selective agents signifi cance of three-dimensional growth patterns of mammary tissues in collagen gels assessing tumor drug sensitivity by a new in vitro assay which preserves tumor heterogeneity and subpopulation interactions factors affecting growth and drug sensitivity of mouse mammary tumor lines in collagen gel cultures origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine tumor resistance to alkylating agents conferred by mechanisms operative only in vivo acquired multicellular-mediated resistance to alkylating agents in cancer heat sterilisation to inactivate aids virus in lyophilised factor viii threedimensional growth patterns of various human tumor cell lines in simulated microgravity of a nasa bioreactor long term organ culture of human prostate tissue in a nasa-designed rotating wall bioreactor establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and psa expression three-dimensional coculture models to study prostate cancer growth, progression, and metastasis to bone threedimensional tissue assemblies: novel models for the study of salmonella enterica serovar typhimurium pathogenesis series introduction: innate host defense of the respiratory epithelium the airway epithelium: structural and functional properties in health and disease apicobasal polarization: epithelial form and function robbins infectious diseases, 6th ed. philadelphia: wb saunders epithelial regulation of innate immunity to respiratory syncytial virus epithelia cells as regulators of airway infl ammation human bronchial epithelial cells with integrated sv40 virus t antigen genes retain the ability to undergo squamous differentiation identifi cation and culture of human bronchial epithelial cells developing differentiated epithelial cell cultures: airway epithelial cells evaluation of anchorageindependent proliferation in tumorigenic cells using the redox dye alamar blue airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion disorganization of stroma alters epithelial differentiation of the glandular stomach in adult mice expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction cell replication of mesenchymal elements in adult tissues. i. the replication and migration of mesenchymal cells in the adult rabbit dermis defi ning conditions to promote the attachment of adult human colonic epithelial cells laminin expression in colorectal carcinomas varying in degree of differentiation infl uence of mammary cell differentiation on the expression of proteins encoded by endogenous balb/c mouse mammary tumor virus genes opinion: building epithelial architecture: insights from three-dimensional culture models threedimensional engineered high fi delity normal human lung tissue-like assemblies (tla) as targets for human respiratory virus infection severe acute respiratory syndrome (sars)-cov infection in a threedimensional human bronchial-tracheal (hbte) tissue-like assembly key: cord-016814-tf17dpo5 authors: enes, sara rolandsson; uriarte, juan j.; pouliot, robert a.; weiss, daniel j. title: clinical application of stem/stromal cells in copd date: 2019-08-07 journal: stem cell-based therapy for lung disease doi: 10.1007/978-3-030-29403-8_6 sha: doc_id: 16814 cord_uid: tf17dpo5 chronic obstructive pulmonary disease (copd) is a progressive life-threatening disease that is significantly increasing in prevalence and is predicted to become the third leading cause of death worldwide by 2030. at present, there are no true curative treatments that can stop the progression of the disease, and new therapeutic strategies are desperately needed. advances in cell-based therapies provide a platform for the development of new therapeutic approaches in severe lung diseases such as copd. at present, a lot of focus is on mesenchymal stem (stromal) cell (msc)-based therapies, mainly due to their immunomodulatory properties. despite increasing number of preclinical studies demonstrating that systemic msc administration can prevent or treat experimental copd and emphysema, clinical studies have not been able to reproduce the preclinical results and to date no efficacy or significantly improved lung function or quality of life has been observed in copd patients. importantly, the completed appropriately conducted clinical trials uniformly demonstrate that msc treatment in copd patients is well tolerated and no toxicities have been observed. all clinical trials performed so far, have been phase i/ii studies, underpowered for the detection of potential efficacy. there are several challenges ahead for this field such as standardized isolation and culture procedures to obtain a cell product with high quality and reproducibility, administration strategies, improvement of methods to measure outcomes, and development of potency assays. moreover, copd is a complex pathology with a diverse spectrum of clinical phenotypes, and therefore it is essential to develop methods to select the subpopulation of patients that is most likely to potentially respond to msc administration. in this chapter, we will discuss the current state of the art of msc-based cell therapy for copd and the hurdles that need to be overcome. chronic obstructive pulmonary disease (copd) is a progressive life-threatening disease that is significantly increasing in prevalence. the world health organization (who) predicts that copd will become the third leading cause of death worldwide by 2030 [1] [2] [3] . there is currently no cure for this disease, and smoking cessation remains the most prominent intervention [4] . because of the lack of effective curative pharmaceutical options and the increase in prevalence, extensive efforts have been devoted to the development of new strategies for cell replacement and tissue remodeling in copd. so far, most focus has been on mesenchymal stromal cell (msc) therapy. msc are theoretically ideal candidates for cell therapeutic approaches because of their low or absent constitutive hla class i and ii expression, allowing allogeneic administration of mscs obtained from normal healthy volunteers, and their immunosuppressive and antibacterial properties [5, 6] . in this chapter, we will examine in detail the biological rationale for use of mscs in copd, clinical trials, and the current challenges for implementing this approach as a potential therapy for copd. copd is a progressive lower respiratory condition, which has a massive impact on public health worldwide. increasing in prevalence, copd is currently responsible for over 120,000 us deaths annually and is expected to become the third leading cause of death globally in the next few years [7] . copd is most often associated with longterm smokers over the age of 40 and is thought to be driven by abnormal tissue response(s) to inhaled toxic particles over time. the life expectancy of continuous cigarette smokers is at least 10 years shorter than nonsmokers and the absolute risk of developing copd among this population has been estimated to be 15-30% [8] ; however, there is evidence for significant underdiagnosis [9, 10] . the most common symptoms of copd are chronic bronchitis (persistent cough with chronic mucus production), dyspnea (shortness-of-breath), wheezing, and chest tightness. as a progressive disease, these symptoms get worse over time. current treatments, most importantly smoking cessation, are part of a delay strategy to slow down the physiological disease progression. these physiologic changes all contribute to the impairment of efficient breathing and include: the gradual loss-of-elasticity of the lung tissue leading to collapse of airway and alveolar sacs, weakening-to-rupture of alveolar septal walls, enlargement of segmented airspace, loss of gasexchange surface area, increased mucus production, airway plugging, and airway narrowing driven by swelling and fibrosis ( fig. 6.1 ). copd is a complex pathology with a diverse spectrum of clinical phenotypes, comorbidities, and treatment profiles [11, 12] . the gold criteria have been widely utilized to help standardize the copd definitions and treatment guidelines; however, they do not fully encompass the diversity of copd phenotypes [13, 14] . the treatments available to patients diagnosed with copd are not curative and cannot completely stop disease progression; however, indicate alveolar space (side-to-side alveolar wall distance). scale bars at 4× magnification represent 500 μm and at 10× magnification 200 μm they are key to slowing disease progression and importantly to improve quality of life. the most important intervention at any stage is the cessation of smoking and/or limitation of exposure to other identified environmental risk factors. symptomatic treatment throughout disease progression often relies on bronchodilators, which are inhaled beta-agonists or muscarinic antagonists. early-stage individuals will most often be treated with short-acting bronchodilator therapies (saba/sama); however, as the disease progresses treatment will need to incorporate long-acting drugs that affect these receptors (laba/lama). unfortunately, bronchodilators can only partially resolve lung hyperinflation in emphysema [15] , becomingly increasingly less effective as the disease progresses. inhaled corticosteroids (ics), often used to treat acute respiratory exacerbations, work by interfering with the transcription pathways of key inflammation genes; however, this treatment does not always work and unfortunately can have little to no longterm benefits [16] . in addition to direct toxic effects of cigarette smoke on lung epithelial cells, there is increasing appreciation that altered or aberrant immune cell signaling significantly contributes to much of the irreparable tissue damage. smokers with undiagnosed copd normally experience lowlevel infiltration of inflammatory cells into the large airways and peripheral lung parenchyma and have what is increasingly recognized as early disease. in individuals with diagnosed copd, the inflammatory process is amplified and prolonged leading to many of the tissue-remodeling events associated with chronic bronchitis and emphysema; hallmarks of copd [17] . for example, in smoking-induced emphysema, chronically activated macrophages have been found to express upregulated levels of several proteinases and matrix metalloproteinases (mmps) in both human smokers and in mouse models of cigarette exposure [18] . macrophages also play a crucial role in triggering the initial immune response in responding to smoking induced inflammation. alveolar macrophages are usually in a quiescent state and actually work to suppress the adaptive immune system in the healthy lung; however, in chronic inflammatory situations alveolar macrophages are the main source of proinflammatory amplification and play a significant role in causing an influx of other immune cells [19] . ultimately, the disease progresses to a point where gas exchange is limited by the tissue damage and extent of hyperinflation. in many cases invasive surgical interventions are the only option; these include endobronchial valve insertion, bullectomy, lung volume reduction surgery, and lung transplantation [20] . lung volume reduction surgeries can successfully address some issues with hyperinflation in selected patients by returning some of the mechanical advantage of normal breathing. however, invasive surgeries are associated with high morbidity and operative mortality [21] [22] [23] , especially in late-stage copd patients who are often poor targets for surgical intervention. for some patients with end-stage copd, lung transplantation is the only option. however, this approach offers its own unique challenges including rejection risk, requirement for immunosuppression, and the limited supply of donor lungs. while transplanted lungs can certainly facilitate better gas exchange than severe copd lungs, the benefits are balanced by the risks, as the 5 years survival of transplant recipients is only around 50% [24] [25] [26] [27] . at present, there are no true curative treatments that can stop the progression of copd, thus new therapeutic strategies are needed. advances in cell-based therapies provide a platform for development of new therapeutic approaches in copd. at this moment, much focus has been given to msc cell-based therapies, mainly because of their immunomodulatory properties. the promising results in animal models have translated into clinical trials for treatment of copd and emphysema. searching on the clinicaltrials.gov database for trials listed through november 28 2018, using the keywords "copd" and "stromal cell"; "copd" and "mesenchymal stromal cell"; "copd" and "mesenchymal stem cell"; "emphysema" and "stromal cell"; "emphysema" and "mesenchymal stromal cell"; and "emphysema" and "mesenchymal stem cell", identified 18 studies of human clinical trials. so far, four of the studies have been completed and had their results published in the pubmed database, four are still in the process of recruiting patients, three of them are active but not recruiting patients, three have an unknown status, and four of them have been withdrawn [28] . this section will be focusing on the clinical studies that have been completed and for which results have been published (table 6 .1). in 2011, ribeiro-paes et al. conducted the first clinical investigation evaluating the safety of using bone marrow-derived mononuclear cells (bmmc) in four patients with advanced-stage copd (nct01110252). autologous bmmc were collected after 3 days of granulocyte colony stimulating factor (g-csf) stimulation, and bmmc were isolated using ficoll-hypaque premium™. the cells were further resuspended in albumin saline solution (ass) at a final concentration of 1 × 10 8 mononuclear cells/ml, and intravenously administered directly to the patients without freezing or in vitro culture procedures. the patients were evaluated by several pulmonary function tests, including forced vital capacity (fvc), forced expiratory volume in 1 s (fev1), and partial pressure of carbon dioxide (paco 2 ). [29] importantly, due to the small size of this study, lack of controls, and the lack of statistical analysis no clear conclusions can be drawn from these results. furthermore, the cells used in this study were heterogeneous mononuclear cells isolated from bone marrow aspirates, and not mscs, and therefore this study cannot be considered as the first msc study for treatment of copd patients. in 2013, weiss et al. performed a prospective, randomized, double-blind, placebo-controlled industry-sponsored trial evaluating the safety and the efficacy of intravenous allogeneic mscs (nct00683722). the study enrolled 62 patients (40-80 years of age), from six different centers, with moderate-to-severe copd (gold ii or iii). the patients were randomized into two groups, where the first group received non-hlamatched allogeneic mscs and the second group the infusions were well tolerated and no severe or fatal adverse events were observed during the msc or vehicle administration. no significant differences in fev1, fvc, and total lung capacity were seen between the groups. nor were differences in 6-min walk test or dyspnea assessment observed between the two groups. for most of the circulating inflammatory cytokines no significant differences were seen between the msctreated patients and the vehicle group. however, a decrease in the crp level in patients treated with msc compared to their baseline crp levels was observed. the most important finding in this study was that msc administration was safe in an older population of patients with moderate-tosevere copd [30] . stolk et al. performed a phase i, prospective, open-label study (nct01306513) where they aimed to assess the safety and feasibility of intravenously infused bone marrow-derived mscs for ten patients with severe emphysema that had serial lung volume reduction surgeries (lvrs). during the first lvrs bone marrow was aspirated. mscs were isolated from the bone marrow aspirates and expanded in vitro (passage 1-3) followed by cryopreservation. at three and four weeks prior to the second lvrs, mscs were intravenously administered to the patients at two different occasions. spirometry, gas transfer, lung volumes, and lung densitometry were evaluated at baseline and at the 12 months follow-up. seven patients completed the full protocol. three patients were withdrawn from the study due to problem aspirating bone marrow, no msc growth, or persistent air leak after the first lvrs. no toxicity after the msc infusions was observed and the patients did not report any symptoms that were considered related to the treatment. at 12 months follow-up, a significant increase in fev1 and body weight was observed compared to baseline levels. however, if changes in fev1 and body weight was due to msc administration or to the surgeries remain unknown, since this study protocol did not include a control group. importantly, no signs of increased pulmonary fibrosis were observed when lung tissue was evaluated by both histology and ct-derived lung density [31] . de oliveira et al. combined msc administration with one-way endobronchial valve (ebv) insertion [32] . this study was a prospective, patient-blinded, placebo (vehicle)-controlled, phase i study on ten patients with advanced heterogeneous emphysema (nct01872624). de oliveira et al. aimed to investigate the safety of combining ebv insertion with intrabronchial msc administration. the authors hypothesized that combining intrabronchial msc administration with ebv would reduce the inflammation, a common side effect of ebv placement. this study, however, was not designed to investigate msc as a treatment for copd, but rather specifically to investigate if msc treatment would enhance ebv placement by reducing the underlying inflammation. therefore, the secondary aim was to investigate if msc administration reduced the systemic inflammation. mononuclear cells (mncs) were isolated from 60 ml bone marrow aspirate collected from the iliac crest of a single healthy donor using density-gradient centrifugation. mncs were cultured at a density of 1 × 10 5 cells per cm 2 in iscove's modified dulbecco's medium supplemented with 15% fetal bovine serum, penicillin, and streptomycin at 37 °c, 5% co 2 for generation of mscs. mscs were immunophenotyped and samples were taken for microbiological and cytogenetic testing. mscs were harvested at passage three or four, diluted in saline solution, and placed in infusion bags. right before ebv insertion 10 8 mscs (in 30 ml saline) were administered to five of ten patients using a video bronchoscope with a 2.8-mm instrument channel. the patients in the vehicle group received saline. in both groups, the infusions were performed in the region where the ebvs were supposed to be placed (the segmental or subsegmental bronchus of all branches of the target lobe). immediately after the msc administration or vehicle administration and ebv insertion, a chest radiograph was performed to confirm the ebv placement. for the following 2 days, the patients were evaluated for body temperature, blood pressure, oxygen saturation, heart, and respiratory rates. arterial blood gas, complete blood count, urea, creatinine, glucose, and electrolytes were evaluated at day 0, 1, 7, 30, and 90. chest ct scans were performed at day 0, 30, and 90. circulating levels of inflammatory cytokines were assessed in serial blood samples obtained throughout the study period. efficacy was evaluated as improvement from baseline in fev1, fvc, fev1/fvc, total lung capacity, single-breath carbon monoxide diffusing capacity, the body mass index, airway obstruction, dyspnea, exercise index, and health-related quality of life (st. george's respiratory questionnaire). all ten patients completed the full protocol. the msc administration was well tolerated and all patients tolerated the ebv insertion but one, who developed pneumonia, pneumothorax, empyema, and respiratory failure. no severe adverse events were seen in the group receiving msc, but 40% in the msc group and 60% in the placebo group experienced adverse events during the study period, and importantly none of the adverse events was reported to be related to the msc administration. no difference in toxicological or lung function parameters such as fev1, fvc, and total lung capacity were observed between the groups. in accordance with data reported by weiss et al. [30] the msc treated group had significantly reduced levels in crp at day 30 and 90 post administration. patients receiving msc infusions were reported to have a significant decrease in the st. george's respiratory questionnaire scores compared to the placebo group at day 90 post administration. the authors concluded that intrabronchial msc administration in combination with ebv insertion appears to be safe in patients with severe heterogeneous emphysema. furthermore, in this study msc administration tended towards decreased circulating crp levels; however, due to the low number of recruited patients and the limited follow-up period it was not possible to evaluate if msc treatment altered the efficiency of the ebv placement or the subsequent clinical copd course. [32] . finally, armitage et al. recently published a single site, phase i study that was not listed at the nih clinicaltrials.gov database, rather only in the australian clinical trials registry (number 12614000731695), which aimed to investigate the distribution of intravenously infused mscs into copd patients. nine patients with mild-tosevere copd (gold i-iv) received infusion of low passage allogenic bone marrow-derived mscs radiolabeled with indium-111, followed by a second infusion of unlabeled mscs one week post the first administration. in similarity with the other clinical trials, all patients tolerated the msc infusions well and no infusional or short-term adverse effects were reported. following the first infusion, labeled mscs were detected in the lungs within 30 min by computed tomography (ct) scan, and remained detectible 24 h after the infusion. after 24 h, indium-111 was detected in spleen, liver, and bone marrow up to 7 days after infusion. moreover, 4 h after the first infusion the patients were assessed by single-photon emission computed tomography (spect) to evaluate msc localization within the lungs. furthermore, the amount of indium-111 positively correlated with the baseline fev1 and the diffusing capacity of the lung for carbon monoxide. in addition, this study further aimed to investigate systemic inflammation following the msc infusion. the authors were not able to detect il-1 beta, il-10, il-12p70, or il-17a; however, increased circulating levels of crp were detected at 1 h and up to 2 days after msc administration. interestingly, this study suggests that msc infusion shifted the balance towards a more anti-inflammatory profile, as the number of circulating regulatory t-cells were increased 7 days after msc administration and the proportion of dendritic cells were altered, favoring plasmacytoid dendritic cells [33] . current clinical trials that aimed to evaluate the effect of msc administration in copd patients differ in a wide range of factors such as routes of administration, number of msc administered, number of administrations, use of fresh mscs or culture-expanded mscs. furthermore, all the investigations discussed above, were phase i-ii studies that were underpowered in order to detect potential efficacy and no improved pulmonary function or respiratory quality of life was observed. although the primary end-point was safety and all studies reported that msc administration was well tolerated and no toxicity was observed, further studies, both clinical and preclinical, are needed to better understand potential therapeutic efficacy of mscs in copd. despite increasing number of preclinical studies demonstrating that msc administration could prevent or treat experimental copd and emphysema [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] , clinical studies have not been able to reproduce the preclinical results, and to date no efficacy or significantly improved pulmonary function in copd patients have been observed. in this section, we will be discussing some of the challenges in the field and the hurdles that need to be overcome in order to improve the efficacy of msc therapy in copd [51] . mscs are known to be a heterogeneous cell population [52, 53] , containing subpopulations that have been demonstrated to be functionally different from each other [54, 55] . many of the phenotypic and functional differences depend on differences in culture conditions, individual donors, the harvest site, and the tissue source [56] [57] [58] [59] . this makes it difficult to compare results between different studies, both preclinical and clinical, and importantly it hinders progression in the field. efforts should therefore be concentrated on developing standardized msc isolation methods and culture conditions. in 2006, the international society for cellular therapy published a position paper in order to address this issue. in this article, they defined minimal suggested criteria for cultured human mscs [60] . since this position paper by dominici et al. was published it has been updated once in 2012 [6] , but the msc field has advanced and today mscs are isolated from different organs and tissues and therefore these minimal criteria urgently need to be modified and updated. to date, bone marrow-derived mscs are the most widely investigated, but preclinical studies have demonstrated that mscs with immuneregulatory and regenerative properties can be isolated from other tissues such as adipose tissue, umbilical cord, and lung [58, [61] [62] [63] [64] [65] [66] . a large body of data demonstrates that mscs execute their therapeutic effects through a spectrum of paracrine activities, and interestingly preclinical data suggest that mscs isolated from different tissues have different secretome profiles [57, 67] . it is also important to realize that primary mscs change phenotype when they are isolated from their native tissue and plated on a plastic culture dish [56] . the bona-fide msc, which are thought to be small and quiescent, noncycling cells in vivo, changes phenotype into a spindle-shaped and active proliferating and secretory cell in culture [56, 68] . at early passages, mscs have a high proliferation rate but as their time in culture progresses their proliferation rate declines and they finally enter a senescence stage [69] [70] [71] . also the morphology is changed during culture expansion, mscs in early passages have a thin spindle-shaped morphology, but at higher passages mscs tend to become larger and more flattened cells with an irregular shape [56, 70] . moreover, mscs have been reported to accumulate dna damage during in vitro expansion, which could potentially lead to tumorigenesis upon implantation [72, 73] . importantly, tumor development was not observed in any of the clinical studies using msc as treatment for copd and/or emphysema patients, although longer follow-up is necessary [29] [30] [31] [32] . furthermore, the biological properties of mscs can also be strongly influenced by the cell culture medium. cell culture media are often supplemented with serum, and most often fetal bovine serum (fbs) is used. the use of fbs has several disadvantages, especially in clinical settings. first, the possibility of contamination with pathogens such as prions and viruses and the potential immune reaction to bovine proteins. second, lot-to-lot variation between different fbs batches might induce differences in msc behavior such as proliferation rates and differentiation potential, and make it difficult to standardize methods and reproducibility of results [56, 74, 75] . human platelet lysate (hpl) is as an alternative to fbs in clinical settings. hpl has the advantages of containing non-animal products and therefore no risk of xenogeneic infections and immune rejection. on the other hand, hpl is a human product and has the potential to transmit human diseases such as hepatitis b and c, and human immune deficiency virus (hiv). in similarity with fbs, hpl also brings the disadvantage of having lot-to-lot variation [75, 76] . a third option would be to use serum-free cell or synthetic culture media. these media are highly promising, but more studies are needed in order to have evaluate their utility for producing clinical grade-mscs. recently, lensch et al. demonstrated that mscs had a higher proliferation rate when growing in xeno-free medium which resulted in a greater viable cell yield compared to standard fbs containing culture medium [77] . nevertheless, further studies are needed in order to evaluate if the in vivo biological properties of msc are altered when expanded in the in vitro setting. another factor that may influence the biological function of mscs is the freezing and thawing of cells before administrated to patients. the current model for allogeneic msc use is to expand cells on plastic culture dishes following harvest and isolation from bone marrow or other source and cryopreserve the cells until usage. when it is time for administration, cells are thawed, washed, and directly administered to the patients most commonly through intravenous infusions [78] . however, a number of studies have demonstrated that mscs that have been freeze-thawed have impaired functional properties. francois et al. reported that cryopreserved mscs had impaired immunosuppressive properties [78] . in accordance with these results, moll et al. published an article where they demonstrated that freezethawed mscs had a reduced responsiveness to proinflammatory stimuli and an impaired production of anti-inflammatory mediators [79] . minor effects on gene expression of freeze-thawed mscs compared to continuously cultured mscs have been observed. however, the alterations in gene expression between different donors were larger than the effects of cryopreservation [80] . although there is a practical need for expanding and cryo-banking cells for therapeutic use [79] , most preclinical studies have been performed using log phase of growth msc. there are studies, some of them discussed above, which demonstrated that freeze-thawing procedure alters the biological properties of msc. francois et al. found that during the thawing process a heat-shock stress response was initiated that was associated with the impaired immunosuppressive properties of msc. interestingly, this response was reversible and cells were recovered after 24 h of culture [78] . these results imply that cryopreservation and banking of cells might be possible, as long as the cells are allowed to recover in culture before use. the study by cruz et al. further supports the potential of using freeze-thawed cells for clinical trials. in this study, the authors compared the therapeutic effect of continuously cultured versus freshly thawed bone marrow-derived mscs in an aspergillus hyphal extract (ahe) exposed asthma mouse model, and found no difference in therapeutic effect between the two groups [81] . utilizing plastic culture dishes are by far the most traditional way of cultivating and expanding msc; however, alternative culture systems have been developed that might mimic the in vivo situation more compared to the more traditional 2d cultivation on plastic. the use of alternative threedimensional cell culture systems can hopefully contribute to narrowing the gap between preclinical and clinical research. different groups have studied the possibility to grow mscs on plastic culture dishes coated with extracellular matrix molecules (ecm) such as collagen and fibronectin [82, 83] . ecm is a three-dimensional network composed of noncellular structures that play an important role within the lung, not only by providing structural support and adding stability but also as a bioactive environment that can influence cellular responses [84] . engler et al. demonstrated that changing the elasticity of the ecm that mscs were grown on significantly affected the msc phenotype. mscs grown on a stiffer ecm differentiated towards the osteoblast lineage, whereas mscs grown on a softer ecm differentiated towards the adipocyte lineage [85] . the msc differentiation potential could also be altered by changing the cross-linking of the collagen fibers [86] . in addition, modifications of the geometric shape, cell density, and cell size have been implicated in the differentiation potential of msc [87, 88] . interestingly, mcmurray et al. developed a nanoscale surface that maintained the phenotype and multilineage potential of longterm cultured mscs [89] . how the ecm environment affects the msc therapeutic behavior, especially in a fibrotic or emphysematous copd lung, is currently a largely untouched area that will most likely play a pivotal role in the development of successful msc-based therapies. a different approach of the three-dimensional cultures is the usage of the hanging drop model. in conformity with primary mscs, culturing mscs using the hanging drop method resulted in nondividing cells [90] , but an increased potential to differentiate towards osteoblast and adipocyte lineages was also demonstrated [91] . another strategy that has been used for msc expansion relies on culturing mscs in 3d scaffolds (decellularized lung tissue or synthetic scaffolds) [92] [93] [94] . in this system, cultivation on a plastic surface could be avoided, but a perfusion-based bioreactor system is required [56] . studies have shown that mscs cultured in lung ecm hydrogels have enhanced viability and increased expression of sox2 and oct4 compared to cells grown on plastic [95] . furthermore, changes in secretion of cytokines including il-1ra, vegf, g-csf, fgf, and hgf have been demonstrated in mscs grown in 3d culture compared to 2d [96, 97] . taken together, the traditional way of cultivating mscs as monolayer on a plastic surface may result in mscs with a different phenotype compared to mscs expanded in three-dimensional culture systems. however, whether cultivating mscs on ecm coating, in scaffolds, or in hanging drops increases the beneficial effects when used for clinical settings remains to be evaluated and further studies are needed. it is well known that oxygen levels can affect cell functions, such as differentiation, cytokine production, and proliferation [98] [99] [100] [101] . furthermore, it is also known that different adult tissues experience a wide range of oxygen levels [102] and that severe pathological inflammation can cause hypoxia, reduced ph, and oxidative stress [103, 104] . nevertheless, mscs tend to be cultured at atmospheric oxygen levels (20-21% o 2 ) which do not reflect the microenvironment they normally reside in, or the microenvironment they will encounter when administered into the diseased lung [102] . culturing mscs at oxygen levels that more closely represent their in vivo situation have a huge impact on msc behaviors. lennon et al. observed that mscs grown at lower oxygen levels had a greater number of colony-forming cells and proliferated at a higher rate compared to mscs grown at higher oxygen levels. also, lennon et al. demonstrated that mscs cultured at 5% oxygen formed more bone structures in vivo, compared to mscs grown in 20% oxygen [105] . moreover, adipose-derived mscs grown at low oxygen levels, secreted higher levels of cytokines such as vegf and fgf compared to cells cultured at 20% oxygen [102] . combining the low oxygen condition with growing the mscs in 3d cultures has been shown to increase the expression of pluripotent genes such as oct-4, sox-2, nanog, and rex-1 compared to control [99, 106] . beegle et al. reported that mscs pretreated with hypoxia before administration enhanced survival rate and cell retention compared to cell grown at 20% oxygen. taken together, these studies emphasize the importance of understanding the effects of differences in protocols, culture conditions, and oxygen levels in the context of culturing mscs for clinical trials for copd where you have gasexchange impairment, active immune response, and inflammation. despite an enormous interest in using mscs for clinical settings, the exact in vivo function is not understood, especially not within the lung. a compelling amount of data now points towards that mscs act by paracrine mechanisms rather than through engraftment [51, [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] . tracking studies of intravenous injected mscs reveal that most of the injected cells disappear after 24 h [33, 46, 110, 117] , and since mscs do not engraft it is unlikely that mscs can remodel injured tissue by differentiating into other cell types. the mechanisms by which mscs are the most likely to be involved in copd and emphysema are discussed below. immunomodulation through paracrine actions is one of the main mechanisms of actions of mscs and involves both the innate and the adaptive immune system [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] (fig. 6.2) . these effects include inhibition of t-cell [120, 121] and b-cell proliferation [127] , macrophage polarization [119] , and differentiation of t-cells towards t-regulatory cells [132] [133] [134] . the paracrine actions have been associated with several mediators such as hepatocyte growth factor (hgf), transforming growth factor beta (tgfβ), prostaglandin (pge2), il-10, ifn-gamma, tnf-stimulated gene 6 (tsg6), and indoleamine 2, 3-dioxygenase (ido) [115, 118, 119, 135, 136] . in addition to the paracrine immunomodulatory effects, mscs might activate the immune system by recognition of the immune cells. as mentioned, mscs are rapidly cleared from the lung after infusion, which was recently demonstrated to be mainly through phagocytosis by monocytes [137] . the recognition of mscs by monocytes results in a polarization of monocytes/macrophages towards an immunosuppressive phenotype that results in an immunomodulatory response [137, 138] . similar results have also been demonstrated with heatinactivated mscs, suggesting that mscs also can act in a passive immunomodulatory manner [139] . however the potency of apoptotic mscs are controversial, apoptotic mscs have been demonstrated to be completely ineffective when injected intravenously in mice [140] . msc are also known to secrete antimicrobial proteins and polypeptides that are molecules responsible for bacterial killing. mscs secrete the antimicrobial peptide, ll-37, following eschericia coli. stimulation, which was subsequently found to be responsible for the antimicrobial activity in a model of e. coli pneumonia [141] . in addition to its antimicrobial activities, ll-37 can also play an important role in inflammatory and immune modulatory actions [141, 142] . a growing body of data suggests that mscs can form links with other cells, and that they have the potential to transfer components such as mitochondria [143] [144] [145] [146] . through mitochondrial transfer msc have been demonstrated to be able to rescue epithelial cells with defective mitochondria [144] . the mitochondria transfer is thought to be via direct transfer by microtubules and tunneling nanotubes (tnt) [144, 147] . mscs can also transfer mitochondria to macrophages resulting in an increased phagocytic activity [148] . mitochondrial biogenesis is regulated by extracellular stimuli [149] and several lung diseases are associated with impaired mitochondrial biogenesis and dysfunctional mitochondria [150, 151] . however, beyond the mitochondria-derived reactive oxygen species (ros), the contribution of mitochondria in the development of copd is still under investigation [152] . in addition to mitochondria transfer through microtubules and tnt, mitochondria can also be transported via extracellular vesicles (ev) [144, [153] [154] [155] . it is also becoming increasingly clear that msc-derived evs can influence the behavior of surrounding inflammatory and structural cells. for example, evs released from mscs can stimulate bronchial epithelial cells and alveolar cells to secrete proinflammatory cytokines [156, 157] . furthermore, msc-derived evs suppress the potential of lung fibroblasts to differentiate towards myofibroblasts [158] . it is not only mitochondria that could be transferred by msc-derived evs, also other components such as microrna, proteins, lipids, dna, and mrna [159, 160] . evs are taken up by other cells, and evs derived from mscs have been demonstrated to impact immune cells. evs isolated from il-beta pretreated mscs induced macrophage polarization towards the anti-inflammatory phenotype (m2) [161] . mscderived evs have also been associated with inhibition of t-cell proliferation, inducing apoptosis of activated t-cells and promotion of regulatory t-cells [162] . msc-derived evs have been tested in experimental copd models, but further studies are needed [163] . it is now widely accepted that, following in vivo delivery, culture-derived mscs respond to the microenvironment they encounter, which in copd and emphysema could encompass everything from massive inflammatory environment to emphysematous tissue destruction. therefore, it is important to consider several important aspects of the msc preparation and administration used today. the route by which mscs are delivered into the patients most likely plays an important role in the msc potential function. despite the fact that several clinical trials has been performed using mscs for severe lung disorders [29] [30] [31] [32] [164] [165] [166] [167] [168] , the best route of administration have not been determined. in preclinical studies, two main administration routes have been evaluated: systemic administration [34, 41, 43, 45, 47, 48, 50, 169, 170] and local administration [39, 41-43, 171, 172] . copd is a systemic disease and therefore systemic administration might be better suitable for these patients. in addition, systemic administration is less invasive and has less contamination risks compared to local administration [51] . not only has the route of administration been different in the different studies conducted to date but also the number of cells administered with each injection and whether single or multiple injections were administered during the trial. according to antunes et al. a wide range of msc doses in preclinical settings have been used, from 10 4 up to 6 × 10 6 [51] . so far, bone marrow-derived mscs are the most frequently used cell source for msc-treatments, especially when used in human clinical trials. however, mscs derived from other sources such as adipose-derived, umbilical cord-derived, lungderived, and amniotic fluid-derived mscs have been evaluated for treatment of copd/emphysema models [36, 41, 50, 172] . since it is known that the environment affects msc function and viability, several preconditioning strategies have been tested. some researchers have been focusing on the effect of the inflammatory environment and the cytokines that may be encountered in the diseased lung [124, [173] [174] [175] [176] [177] [178] [179] . krampera et al. reported that mscs stimulated with ifn-gamma, increased the levels of ido produced and secreted by mscs, leading to an increased suppressive effect on t-lymphocyte proliferation. moreover, the authors were able to demonstrate that the inhibitory effects of mscs on t-lymphocyte proliferation were completely abolished when adding an ifn-gamma blocking antibody to the culture system [124] . in an ifngamma knock out mouse model, polchert et al. were able to demonstrate that endogenous ifngamma was required to initiate msc efficacy. however, after pretreatment of mscs with high doses of ifn-gamma they immediately became active [173] . also pre-stimulating mscs with a combination of inflammatory cytokines has been explored [174, 175] . another interesting approach to mimic the microenvironment is to utilize patient samples such as serum and bronchoalveolar lavage (bal) fluid from patients and pre-stimulate cells with such prior to the administration [180, 181] . moreover, attempts to improve the beneficial effects of mscs have utilized treatment with the toll-like receptor-3 ligand (poly(i:c)). the authors found that mscs pretreated with poly(i:c) had improved immunosuppressive properties, an effect that was inhibited by addition of the microrna mir-143 [182] . in addition to the inflammatory environ-ment, others have studied the effect of pretreating mscs with hypoxia and nutrient deficiency. during culture under hypoxic conditions, mscs have been shown to have decreased expression of senescence-associated beta-galactosidase and an increase in the expression of anti-apoptotic proteins such as bcl-2 and bcl-xl [183, 184] . by exposing mscs to hypoxia the hypothesis is that the cells will adapt to the ischemic environment with oxidative stress, an environment they likely will encounter in the copd lung. this might potentially enhance the time that mscs can survive and exert their therapeutic paracrine actions in the recipient lung. a different way of increasing the therapeutic effect by mscs is to genetically manipulate the cells prior to administration [185] [186] [187] . for example, jiang et al. demonstrated that after co-overexpressing the genes ang-1 and akt in mscs, an increased cell survival and improved angiomyogenesis was observed in an experimental model of acute myocardial infarction [188] . in lung, mscs overexpressing ang-1 have been demonstrated to more potently decrease lpsinduced pulmonary inflammation and proinflammatory cytokine release into the bal fluid [189] . in another study by mcginley et al., overexpression of heat shock protein 27 (hsp27) in mscs led to decreased apoptosis and improved cardiac function [190] . overexpression of manganese superoxide dismutase in adipose-derived mscs, a gene strongly upregulated during hypoxia, increased the time that the mscs were detectable in a matrigel plug implanted into a mouse model [191] . moreover, he et al. transduced mscs with angiotensin-converting enzyme 2 (ace2), an enzyme that degrades angiotensin ii and had previously been demonstrated to have a protective role against acute lung injury. the ace2transduced mscs were demonstrated to reduce pulmonary vascular permeability, normalize the expression of enos, and improve the endothelial barrier integrity, when infused into an ali-mouse model. furthermore, the ace2 overexpressing mscs also displayed an improvement in the suppression of the inflammatory response [192] . combination of different treatments could be another approach to enhance msc efficacy. this approach was used in two of the clinical trials discussed above. stolk et al. combined msc treatment with lung volume reduction surgery and de oliveira et al. with a one-way endobronchial valve insertion [31, 32] . an alternative could be to pretreat the recipient tissue with pharmacological drugs in order to make the recipient site more accessible to the infused cells [104, [193] [194] [195] . in a cardiac disease model, pharmacological pretreatment of a vasodilator drug in the recipient site of transplantation resulted in an enhanced delivery of mscs [193] . in a clinical trial using mscs for treatment of chronic heart failure, the administration site was treated with a shock wave prior to the administration of the cells. in the group receiving both the shock wave pretreatment and the msc infusion, the overall occurrence of major adverse cardiac events were significantly decreased compared to the control groups [194] .these are all important observations for potential cell-based therapies for lung diseases and should be investigated further. the lack of translating the encouraging preclinical data into clinically relevant effects in patients with copd and emphysema brings up the following question: is copd the most suitable pulmonary disease for msc-based treatment? the animal models of copd and emphysema used in the preclinical studies were optimized to detect the maximum therapeutic effects [196] , and might therefore not reflect the in vivo situation that mscs encounter when infused into patients with copd and/or emphysema. copd is characterized by tissue damage, structural changes, and inflammation, and as mentioned it is a heterogeneous disease with different degrees of fibrosis and emphysema [197] . copd patients with different phenotypes might respond differently to msc administration [198] , and choosing patients that are more likely to respond to the treatment could be one way to improve the clinical outcome. another possible way to improve the outcome could be the timing of the treatment. in animal studies, mscs are frequently administered to the animals in close proximity to the induction of the disease [42, 48, 50, 172] or even at the same time or prior to the disease induction [43, 47] . based on these preclinical findings, mscs might be more beneficial earlier in the disease than in later stages of the disease. however, copd patients tend not to seek medical attention in early stage of the disease [199] . one way to foresee which patients would most likely respond to the treatment could be to develop in vitro potency assays. even if it is widely accepted that the therapeutic effect of mscs is mainly mediated by paracrine effects, the exact mechanism of action is not determined. this makes it difficult to develop one single analytic or biological assay, and most likely, a combination of evaluating different mechanisms would be needed [200, 201] . another potential way would be to develop biomarkers to indicate which patients have an active disease and therefore might benefit more from a msc-based therapy. to date, several potential biomarkers, including circulating fragments of ecm proteins, have been shown to be increased in copd patients with an active disease e.g. in relation to acute exacerbations [202] [203] [204] . finally, broekman et al. suggested that in addition to the optimization of msc-treatment and potency assays, challenges such as improved outcome parameters needs to be addressed [196] . in parallel with the growing interest of cellbased therapies for copd and other lung diseases, an increased market for commercial stem cell therapies has developed both in the usa and globally [205] . this very unfortunate and problematic outcome might partly be due to an increased visibility to desperate patients through the internet and open social media channels [206] . these unproven and often unsafe stem cell treatments can create a situation in which desperate patients easily can be misled into participating in very expensive treatments, which are not covered by insurance. furthermore, the providers at the stem cell clinics often fail to prove safety and efficacy of their treatments failing to fulfill recognized biological and medical standards, exposing the patients to unnecessary risks and leaving the patient and their family with dashed hopes [205, 207] . these stem cell clinics have the potential to harm even more patients and their families, as well as bring the field into disrepute and hamper the progression of safe and effective msc-based therapies. therefore, organizations such as the international society for stem cell research (isscr) and the international society for cell and gene therapies (isct) have taken stances against these unethical cell-therapy clinics. also, the food and drug administration (fda) is beginning to take actions against the stem cell tourism [205, 208] . in a review by dominici et al., the authors discuss the importance of having proper communication between different players such as medical doctors, industry, patient organizations, and patients, in order to enhance credibility and patient welfare [205] . in an attempt to begin proactively addressing this issue, the american thoracic society (ats) respiratory cell and molecular biology assembly stem cell working group posted a statement online and several other related publications [207, [209] [210] [211] [212] . this statement will help to translate new scientific findings into patient education in an unbiased way and to make the public aware of the limitations and potential risks associated with such therapeutic approaches [209, 212] . however, it is not only the patients that need education, many pulmonologists are also not familiar with the stem cell field, and the ats respiratory cell and molecular biology assembly stem cell working group has developed educational resources for this audience also [207] . msc-based therapy for treatment of copd and emphysema has demonstrated promising results in animal models; however, this has not translated into clinically relevant effects in patients to date. current clinical trials have failed to demonstrate efficacy and improved lung function, but importantly they have uniformly demonstrated the msc administration to be safe. the challenges ahead for this field are to standardize the isolation and culture conditions in order to have a cell product with high quality and reproducibility, to select the proper subpopulation of patients that is most likely to respond to the cell treatment, to develop appropriate potency assays, and to improve or develop new methods to measure outcomes. furthermore, the usage of cell-free products such as evs and conditioned medium, or pretreating mscs prior to administration has demonstrated promising results. however, there is still a long way to go and many challenges are ahead before we have an optimal msc-based treatment for patients with copd and emphysema. joint statement for the diagnosis, management, and prevention of chronic obstructive pulmonary disease for gulf cooperation council countries and middle east-north africa region chronic obstructive pulmonary disease (copd) fact sheet. who projections of global mortality and burden of disease from 2002 to 2030 the 2017 global initiative for chronic obstructive lung disease report and practice implications for the respiratory therapist concise review: the bystander effect: mesenchymal stem cell-mediated lung repair mesenchymal stromal cells: new directions the future of multihospital systems. top health care financ developing copd: a 25 year follow up study of the general population copd: the dangerous underestimate of 15% determinants of underdiagnosis of copd in national and international surveys copd and its comorbidities: impact, measurement and mechanisms copd heterogeneity: implications for management the history of copd the natural history of chronic bronchitis and emphysema: an eight year study of early chronic obstructive lung disease in working men in london improvement in resting inspiratory capacity and hyperinflation with tiotropium in copd patients with increased static lung volumes how corticosteroids control inflammation: quintiles prize lecture the pathology of chronic obstructive pulmonary disease a distinctive alveolar macrophage activation state induced by cigarette smoking alveolar macrophage in the driver's seat chronic obstructive pulmonary disease 10: bullectomy, lung volume reduction surgery, and transplantation for patients with chronic obstructive pulmonary disease lung volume reduction therapies for advanced emphysema: an update complications of lung volume reduction surgery biologic lung volume reduction in advanced upper lobe emphysema: phase 2 results molecular pathogenesis of emphysema a new classification system for chronic lung allograft dysfunction bronchiolitis obliterans after human lung transplantation bronchiolitis obliterans after lung transplantation: a review unicentric study of cell therapy in chronic obstructive pulmonary disease/ pulmonary emphysema a placebo-controlled, randomized trial of mesenchymal stem cells in copd a phase i study for intravenous autologous mesenchymal stromal cell administration to patients with severe emphysema combined bone marrow-derived mesenchymal stromal cell therapy and one-way endobronchial valve placement in patients with pulmonary emphysema: a phase i clinical trial mesenchymal stromal cell infusion modulates systemic immunological responses in stable copd patients: a phase i pilot study bone marrow cells repair cigarette smokeinduced emphysema in rats mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smokeinduced damage adipose stem cell treatment in mice attenuates lung and systemic injury induced by cigarette smoking lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of elastase-injured lung autologous lung-derived mesenchymal stem cell transplantation in experimental emphysema mesenchymal stem cells alleviate airway inflammation and emphysema in copd through down-regulation of cyclooxygenase-2 via p38 and erk mapk pathways cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model intravenous and intratracheal mesenchymal stromal cell injection in a mouse model of pulmonary emphysema mesenchymal stem cell-based hsp70 promoter-driven vegfa induction by resveratrol alleviates elastase-induced emphysema in a mouse model the therapeutic effects of optimal dose of mesenchymal stem cells in a murine model of an elastase induced-emphysema tracking intravenous adipose-derived mesenchymal stem cells in a model of elastase-induced emphysema mesenchymal stem cells transplantation protects against rat pulmonary emphysema mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells lung tissue engineering technique with adipose stromal cells improves surgical outcome for pulmonary emphysema autologous transplantation of adipose tissue-derived stromal cells ameliorates pulmonary emphysema mesenchymal stromal cell therapy in copd: from bench to bedside flexible and dynamic organization of bone marrow stromal compartment bone marrow stromal stem cells: nature, biology, and potential applications isolation of functionally distinct mesenchymal stem cell subsets using antibodies against cd56, cd271, and mesenchymal stem cell antigen-1 characterization of bone marrowderived mesenchymal stromal cells (msc) based on gene expression profiling of functionally defined msc subsets a relativity concept in mesenchymal stromal cell manufacturing quantitative proteomic characterization of lung-msc and bone marrow-msc using dia-mass spectrometry msc from fetal and adult lungs possess lung-specific properties compared to bone marrow-derived msc primary mesenchymal stem cells in human transplanted lungs are cd90/ cd105 perivascularly located tissue-resident cells minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement wharton's jelly-derived cells are a primitive stromal cell population human umbilical cord mesenchymal stem cells: a new era for stem cell therapy multilineage cells from human adipose tissue: implications for cell-based therapies mesenchymal stem cells derived from human adipose tissue mesenchymal stem cells in human secondtrimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts unveiling the differences of secretome of human bone marrow mesenchymal stem cells, adipose tissue-derived stem cells, and human umbilical cord perivascular cells: a proteomic analysis low/negative expression of pdgfralpha identifies the candidate primary mesenchymal stromal cells in adult human bone marrow different facets of aging in human mesenchymal stem cells donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy from cytogenomic to epigenomic profiles: monitoring the biologic behavior of in vitro cultured human bone marrow mesenchymal stem cells a link between the accumulation of dna damage and loss of multipotency of human mesenchymal stromal cells serumfree human msc medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells comparison of synthetic media designed for expansion of adipose-derived mesenchymal stromal cells cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties? effects of freeze-thawing and intravenous infusion on mesenchymal stromal cell gene expression freshly thawed and continuously cultured human bone marrow-derived mesenchymal stromal cells comparably ameliorate allergic airways inflammation in immunocompetent mice attachment, growth, and detachment of human mesenchymal stem cells in a chemically defined medium collagen promotes higher adhesion, survival and proliferation of mesenchymal stem cells the extracellular matrixthe under-recognized element in lung disease? matrix elasticity directs stem cell lineage specification extracellular-matrix tethering regulates stem-cell fate geometric cues for directing the differentiation of mesenchymal stem cells cell shape, cytoskeletal tension, and rhoa regulate stem cell lineage commitment nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency hematopoietic stem and progenitor cell expansion in contact with mesenchymal stromal cells in a hanging drop model uncovers disadvantages of 3d culture enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine preparation of decellularized lung matrices for cell culture and protein analysis residual detergent detection method for nondestructive cytocompatibility evaluation of decellularized whole lung scaffolds enhanced human bone marrow mesenchymal stromal cell adhesion on scaffolds promotes cell survival and bone formation tunable hydrogels from pulmonary extracellular matrix for 3d cell culture changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming combination of msc spheroids wrapped within autologous composite sheet dually protects against immune rejection and enhances stem cell transplantation efficacy in situ normoxia enhances survival and proliferation rate of human adipose tissue-derived stromal cells without increasing the risk of tumourigenesis impact of low oxygen tension on stemness, proliferation and differentiation potential of human adiposederived stem cells critical effect of oxygen tension on rate of growth of animal cells in continuous suspended culture primitive human hpcs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent effect of hypoxia on human adipose-derived mesenchymal stem cells and its potential clinical applications regulation of immunity and inflammation by hypoxia in immunological niches challenges and strategies for improving the regenerative effects of mesenchymal stromal cell-based therapies cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3d constructs concise review: current status of stem cells and regenerative medicine in lung biology and diseases limited engraftment capacity of bone marrow-derived mesenchymal cells following t-cell-depleted hematopoietic stem cell transplantation plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms evolving paradigms for repair of tissues by adult stem/progenitor cells (mscs) intravenous hmscs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein tsg-6 hepatocyte growth factor mediates mesenchymal stem cell-induced recovery in multiple sclerosis models mesenchymal stromal cells: sensors and switchers of inflammation network analysis of transcriptional responses induced by mesenchymal stem cell treatment of experimental sepsis prevention of endotoxin-induced systemic response by bone marrow-derived mesenchymal stem cells in mice multipotent mesenchymal stromal cells and the innate immune system mesenchymal stromal cells and hematopoietic stem cell transplantation human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific t cells to their cognate peptide bone marrow mesenchymal stem cells induce division arrest anergy of activated t cells role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells mesenchymal stem cells inhibit human th17 cell differentiation and function and induce a t regulatory cell phenotype reciprocal interactions between human mesenchymal stem cells and gammadelta t cells or invariant natural killer t cells human mesenchymal stem cells modulate b-cell functions mesenchymal stem cell-natural killer cell interactions: evidence that activated nk cells are capable of killing mscs, whereas mscs can inhibit il-2-induced nk-cell proliferation mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle human mesenchymal stem cells inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche a new mesenchymal stem cell (msc) paradigm: polarization into a pro-inflammatory msc1 or an immunosuppressive msc2 phenotype mesenchymal-stem-cell-induced immunoregulation involves fas-ligand-/fas-mediated t cell apoptosis interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of cd4+ t-cell subsets expressing a regulatory/suppressive phenotype cell contact, prostaglandin e(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of cd4+cd25(high) forkhead box p3+ regulatory t cells bone marrow stromal cells attenuate sepsis via prostaglandin e(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production anti-inflammatory protein tsg-6 secreted by activated mscs attenuates zymosan-induced mouse peritonitis by decreasing tlr2/nf-kappab signaling in resident macrophages immunomodulation by therapeutic mesenchymal stromal cells (msc) is triggered through phagocytosis of msc by monocytic cells mesenchymal stem cells induce suppressive macrophages through phagocytosis in a mouse model of asthma inactivated mesenchymal stem cells maintain immunomodulatory capacity mesenchymal stromal cells: clinical challenges and therapeutic opportunities antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide ll-37 antimicrobial cathelicidin peptide ll-37 inhibits the lps/atp-induced pyroptosis of macrophages by dual mechanism stem cells, cell therapies, and bioengineering in lung biology and diseases. comprehensive review of the recent literature 2010-2012 mitochondrial transfer between cells can rescue aerobic respiration cell-tocell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture mechanisms of mesenchymal stem/stromal cell function characterization of intercellular communication and mitochondrial donation by mesenchymal stromal cells derived from the human lung mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ards mitochondria in mesenchymal stem cell biology and cell therapy: from cellular differentiation to mitochondrial transfer mitochondrial dysfunction increases allergic airway inflammation association between mitochondrial dysfunction and severity and outcome of septic shock mitochondria in lung diseases intra-and intercellular quality control mechanisms of mitochondria mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micrornas monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells cd18-mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles thy-1 dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation extracellular vesicle-shuttled mrna in mesenchymal stem cell communication biological properties of extracellular vesicles and their physiological functions exosomal mir-146a contributes to the enhanced therapeutic efficacy of interleukin-1beta-primed mesenchymal stem cells against sepsis microvesicles derived from mesenchymal stem cells: potent organelles for induction of tolerogenic signaling adipose stem cell-derived nanovesicles inhibit emphysema primarily via an fgf2-dependent pathway treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (start study): a randomised phase 2a safety trial a prospective, non-randomized, no placebo-controlled, phase ib clinical trial to study the safety of the adipose derived stromal cells-stromal vascular fraction in idiopathic pulmonary fibrosis a phase 1b study of placentaderived mesenchymal stromal cells in patients with idiopathic pulmonary fibrosis treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smokeinduced damage bone marrow mesenchymal stem cell transplantation for treatment of emphysemic rats mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via vegf-vegf receptors therapeutic effects of amniotic fluid-derived mesenchymal stromal cells on lung injury in rats with emphysema ifn-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression cytokine modulation of tlr expression and activation in mesenchymal stromal cells leads to a proinflammatory phenotype impaired function of bone marrow mesenchymal stem cells from immune thrombocytopenia patients in inducing regulatory dendritic cell differentiation through the notch-1/jagged-1 signaling pathway human msc suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander m2 macrophage differentiation mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell properties antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma activation of human mesenchymal stem cells impacts their therapeutic abilities in lung injury by increasing interleukin (il)-10 and il-1rn levels serum from asthmatic mice potentiates the therapeutic effects of mesenchymal stromal cells in experimental allergic asthma the toll-like receptor 3 ligand, poly(i:c), improves immunosuppressive function and therapeutic effect of mesenchymal stem cells on sepsis via inhibiting mir-143 transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan mesenchymal stem cells modified with akt prevent remodeling and restore performance of infarcted hearts hepatocyte growth factor-modified mesenchymal stem cells improve ischemia/reperfusioninduced acute lung injury in rats transfection of mesenchymal stem cells with the fgf-2 gene improves their survival under hypoxic conditions supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1 mesenchymal stem cell survival in the infarcted heart is enhanced by lentivirus vector-mediated heat shock protein 27 expression promotion of survival and engraftment of transplanted adipose tissue-derived stromal and vascular cells by overexpression of manganese superoxide dismutase mesenchymal stem cells overexpressing angiotensin-converting enzyme 2 rescue lipopolysaccharide-induced lung injury prostacyclin improves transcoronary myocardial delivery of adipose tissue-derived stromal cells effect of shock wave-facilitated intracoronary cell therapy on lvef in patients with chronic heart failure: the cellwave randomized clinical trial angiogenic pretreatment improves the efficacy of cellular cardiomyoplasty performed with fetal cardiomyocyte implantation mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease? thorax extracellular matrix remodelling in copd understanding copd: a vision on phenotypes, comorbidities and treatment approach copd: early diagnosis and treatment to slow disease progression international society for cellular therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials the challenge of defining mesenchymal stromal cell potency assays and their potential use as release criteria characterization of serological neo-epitope biomarkers reflecting collagen remodeling in clinically stable chronic obstructive pulmonary disease high levels of biomarkers of collagen remodeling are associated with increased mortality in copd -results from the eclipse study accelerated extracellular matrix turnover during exacerbations of copd positioning a scientific community on unproven cellular therapies: the 2015 international society for cellular therapy perspective science, ethics and communication remain essential for the success of cell-based therapies unproven stem cell treatments for lung disease-an emerging public health problem balancing safety and innovation for cell-based regenerative medicine statement on unproven stem cell interventions for lung diseases medical societies, patient education initiatives, public debate and marketing of unproven stem cell interventions co-opting of clinicaltrials. gov by patient-funded studies the global emergence of unregulated stem cell treatments for respiratory diseases. professional societies need to act key: cord-023303-fxus38mp authors: nan title: lung cancer/bronchology sigs: combined poster session date: 2008-03-12 journal: respirology doi: 10.1111/j.1440-1843.2008.01252_8.x sha: doc_id: 23303 cord_uid: fxus38mp nan increased airway smooth muscle (asm) in asthma may be due to hyperplasia or hypertrophy of asm cells. the contribution of extracellular matrix (ecm) within asm bundles has not previously been accounted for when estimating asm cell volume. aim to estimate the mean asm cell volume in asm bundles in asthma. methods post-mortem tissues from control subjects (c n = 9); nonfatal (nfa n = 11) and fatal (fa n = 10) cases of asthma were studied. on 30 mm transverse airway sections stained with haematoxylin, the volume density (nv) of asm cell nuclei was estimated using an optical disector (¥1000). the mean cell volume (vc = 1/nv) was calculated, correcting for the volume fraction of asm (fasm) within the asm bundle (corrected vc = 1/(nv ¥ fasm)). fasm was estimated on 0.5 mm thick sections of the same airway stained with masson's trichrome. basement membrane perimeter (pbm) was used to indicate airway size. results table shows mean ϯ sd. (one-way anova) *p < 0.05 for c v fa, nfa v fa. conclusion these data suggest that although asm area is increased in asthma, mean asm cell volume is unchanged. therefore hyperplasia, not hypertrophy, of asm cells is present in both mild and severe asthma. these results were similar for both large and small airways. asthma is characterized by airway inflammation and remodelling which contribute to airway hyperresponsiveness and episodic airflow obstruction. mast cell (mc) densities are higher on the smooth muscle (asm) in asthma so their mediators may modulate other asm functions as well as cause contraction. aim to investigate the effect of mc mediators on chemokine and extracellular matrix (ecm) production by asm cells from donors with and without asthma. methods mc were isolated from the resected lung samples of 6 patients, resuspended at 10 6 cells/ml in dmem + 10% fbs and stimulated with ige/anti-ige. supernatants (sn) were collected after 2 and 24 h and the mc lysed. sub-confluent asm cells from 6 donors with and without asthma were serum deprived for 72 h before mc sn/lysates were added in dmem + 10%fbs for 48 h. il-8 and eotaxin levels in all asm sn and mc sn/lysates were measured by elisa. fibronectin and collagen iv deposition was measured in situ by immunoassay following asm cell lysis. results in asthmatic and non-asthmatic asm cells all mc sn and lysates reduced eotaxin release by up to 47% and 58%, whereas the 0-2 h mc sn significantly increased il-8 release to 178 ϯ 35.9% (p = 0.0339) and 169 ϯ 49% (p = 0.0445) of the fbs control respectively. however, only nonasthmatic asm cell il-8 release was increased by the mc 2-24 h sn (216 ϯ 85%; p = 0.0421) and cell lysates (215 ϯ 47%; p = 0.0421). the 0-2 h mc sn also increased fibronectin deposition to 143 ϯ 16% (p = 0.008) by asthmatic asm cells only. mc sn and lysates had no effect on collagen iv deposition. conclusions activated mast cell mediators differentially modulated chemokine and ecm secretion by asm cells from donors with and without asthma. thus mast cells may modulate their own recruitment to the smooth muscle and remodelling locally in the airways in asthma. supported by nhmrc. the technique of ige passive sensitization reproduces ige-related allergic responses in vitro and studies have validated this technique for investigations modelling allergic smooth muscle responses. there are no studies investigating effects of ige sensitization on rhinovirus (rv) infection. we hypothesized that rv infection is enhanced by ige sensitization, a consequence of diminished early innate immune responses. methods beas-2b epithelial cells and primary culture airway fibroblasts were sensitized with ige 24 h-7 d prior to infection with rv16. samples of tissue culture supernatant and cell lysates were collected over a 12 h period after infection for analysis. viral replication was measured by real-time rt-qpcr and viral titration and type i interferon mrna by rt-qpcr. ige receptor mrna expression was examined using rt-pcr. results initial studies to establish the model used human serum high in ige (>1000 iu/ml), this yielded inconsistent results and it was found that purified ige (1000 iu/ml) provided more reliable responses. sensitization was established after 24 h ige incubation and was comparable with up to 7 d. rt-pcr detected mrna for the ige low affinity receptor only after sensitization. following rv16 infection, vrna was increased after 24 h in ige sensitized cells (p < 0.05), but this effect varied noticeably between and within cell lines. cellular expression of ifn-b mrna increased with viral infection but in cells sensitized with ige lower levels of expression were noted (p < 0.05). conclusions ige passive sensitization enhanced rv replication in vitro but the model is constrained by significant variability between and within cell lines. the effect of sensitization on rv replication may occur through the low affinity ige receptor. activated mast cells (mc) are present in higher numbers on the airway smooth muscle (asm) in asthma compared with other inflammatory airway diseases. matrix metallo-proteinases (mmps) cleave chemokines and alter chemokine gradients by degrading the extracellular matrix and thus may modulate mc migration to the asm. aim to determine the levels of mmp-2, mmp-9 and their inhibitors, timp-1 and timp-2, secreted by asm cells from donors with and without asthma. method confluent asm cells were washed, serum-starved for 48 h and then stimulated with th1 (il-1, tnf and ifn) or th2 (il-1, il-4 and il-13) cytokines or left unstimulated. after 4 and 24 h,the sn were collected. the relative amount of pro and active forms of mmp-2 and mmp-9 in sn were determined by gelatine zymography. timp-1 and timp-2 levels in the sn were measured by elisa. results pro-and active mmp-9 were not detected. however, pro-mmp-2 levels were high in sn of asm cells from donors with (195.6 ϯ 47.2 % positive control/10 5 cells) and without (226.5 ϯ 49.2 % positive control/10 5 cells) asthma. a trend to increased active mmp-2 production by asm cells from donors with (7.3 ϯ 2.7 % positive control/10 5 cells, n = 9) compared to without (2.9 ϯ 0.7 % positive control/10 5 cells, n = 11) asthma after 24 h was not significant (p = 0.101). timp-1 and timp-2 levels respectively were high in the sn of cells from donors with (69.4 ϯ 19.6 and 21.3 ϯ 4.7 ng/10 5 cells, n = 5) and without (57.3 ϯ 13.7 and 16.6 ϯ 3.5 ng/10 5 cells, n = 5) asthma. th1 and th2 cytokine stimulation did not affect mmp or timp release. conclusions th1 and th2 cytokines did not regulate asm cell production of mmp-2, timp-1 and timp-2. altered asm mmp-2 activity is unlikely to play a role in mc chemotaxis to asm cells from donors with asthma in vitro or their presence on the asm in asthma. there has been a marked increase in the prevalence of asthma and other allergic diseases in the last few decades. one of the explanations for this is the change in our diet. one of the characteristics of the "western diet" is a high intake of both saturated and polyunsaturated fat. this prompted us to compare the effects of high fat and low fat meals on the numbers of circulating eosinophils and other leukocytes. methods we studied 12 volunteers who had allergic rhinitis and/or asthma and a peripheral eosinophil count at baseline of ն200 ¥ 10 7 /l. this was a randomized, crossover trial with participants studied on two different days. on each occasion they arrived fasting and after bloods were drawn consumed a 3000 calorie meal. one of the meals was high in saturated fat and refined carbohydrate. the other meal was low in saturated fat and high in fruit and fibre. bloods were drawn postprandially every hour for five hours. results eosinophil counts were highest in the early morning and fell over the course of the day but the decrease was less with the high fat meal (p = 0.03). over the same period of time the increase in lymphocytes (p = 0.016) was greater with the high fat meal. the high fat meal was also associated with greater increases in triglycerides (p < 0.0001) and cholesterol (0.004). conclusions in atopic individuals a high fat meal was associated with higher circulating numbers of eosinophils and lymphocytes than an isocaloric meal that was low in fat. further studies of the effect of dietary fat on eosinophilic inflammation are warranted. supported by the university of auckland research committeee. intravenous gamma globulin therapy (ivig), which is therapeutic in a variety of immune diseases, has been reported to be effective on patients with severe steroid-dependent asthma. although fcer are known to play important roles in asthma, there are few reports about the role of fcg?receptors in asthma. fcg receptor iib (fcgriib) is unique inhibitory receptor, which suppresses immune response. in this study, we evaluated the effect of ivig in allergic airway inflammation in ova-challenged mice and the mechanism of the inhibitory effects of ivig and fcgriib. method c57bl/6 mice (wt) and fcgriib deficient mice (ko) were sensitized with ovalbumin (ova) and alum and subsequently challenged with nebulized ova. before ova challenge rabbit igg was administered intravenously. the airway inflammation and effects of igg were assessed by histology, cell counts of bal fluid and airway hyperresponsiveness. result histology showed that igg treatment ameliorated the inflammation around the airway and the vessels and hypertrophy of goblet cells induced by ova challenge. the migratory activity of dcs is modulated in inflammatory diseases such as asthma. recently, we reported that immature dcs express kinin receptors and that bradykinin (bk) significantly enhances the migration of immature dc in vitro. as kinins mediate many of the pathophysiological effects associated with asthma, we hypothesized that lys-des[arg 9 ]-bk, which is produced during inflammation and acts via the b1 receptor (b1r), would inhibit migration of mature dcs. methods day 7 cultured human monocyte-derived dcs were matured with lps, tnfa +il-1b or cd40l in the absence or presence of lys-des[arg 9 ]-bk. maturation of dc was analysed by flow cytometry (facs). b1r expression was assessed by reverse-transcriptase pcr and quantitative confocal microscopy. migration of mature dc was assessed in transwell chambers with lysdes [arg 9 ]-bk and the chemokine ccl19 used as chemoattractants. results maturation of dcs was found to result in down-regulation of b1r expression to varying degrees depending upon the maturation stimulus used. mature dcs all demonstrated an ability to migrate toward lys-des[arg 9 ]-bk and ccl19. however pre-treatment with lys-des[arg 9 ]-bk decreased the migratory ability of all mature dcs to both chemoattractants. conclusions along with chemokines, lys-des[arg 9 ]-bk is likely to play a crucial role in regulating the in vivo migration of mature dc during inflammation. the production of lys-des [arg 9 ]-bk during inflammation potentially immobilizes mature dcs thereby facilitating locally-mediated immune responses within inflamed tissues. supported by the asthma foundation of western australia. introduction alternative or aberrant splicing is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms. the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to use a new and innovative approach involving the use of the genechip (r) exon array system together with real-time quantitative pcr to study asthma candidate genes in human monocyte-derived dendritic cells. asthmatic and non-asthmatic subjects provided 20 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method, and differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data indicate splice variant expression in dendritic cells from asthmatic patients is influenced by asthma severity. conclusion exon expression array analysis has generated a number of asthma candidate genes with alternative splice variants. further studies to validate these data in a replicate data set and establish the functional significance of our findings in asthma are underway. alternative or aberrant splicing occurs in more than 70% of genes and is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms 1 . the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to study one asthma candidate gene in human stimulated and unstimulated: (1) monocytes, (2) monocytederived dendritic cells and (3) lung smooth muscle cells. methods asthmatic and non-asthmatic subjects provided 40 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method. up to 50% of the monocytes were then differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. induction experiments used 1 mg/ml lps and cells were stimulated for an optimal period of 24 hrs. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data from stimulation experiments indicate splice variant production can be regulated by the inflammatory response and that this response is influenced by asthma status. conclusion preliminary experiments have confirmed the presence of an aberrant splice variant for an asthma candidate gene in the primary cells studied. further studies to confirm these data and establish the functional significance of our findings in asthma are underway. exposure to environmental factors, such as environmental tobacco smoke (ets), plays a significant role in modulating pre-existing genetic susceptibilities to diseases including asthma. the glutathione s-transferase enzymes (gsts) play an important role in the detoxification of ets. there are several gst isoforms and gstp1 codes for the gst pi isoform, which is the primary gst isoform expressed in human lung tissue. two single nucleotide polymorphisms (snps) at positions 105 and 114 have been reported in gstp1 and associated with asthma and atopy. the aim of this study was to examine the effect of these snps in combination with ets, on asthma phenotypes in a cohort of asthmatic children. children were recruited during an acute episode requiring presentation at an emergency department. genotyping using pcr-rflp was completed on 218 children and ets exposure was determined by parental questionnaire. urinary cotinine was measured in the children and was in agreement with questionnaire responses. statistical analyses were performed using spss. there were no significant associations between the genotypes and asthma severity during acute exacerbations. significant associations were found between the snps and atopy in this population with an odds ratio of 2.77 for the 105aa genotype (p = 0.029) and or of 5.47 for the 114cc genotype (p = 0.002). however, when an interaction with ets was included, the odds ratios increased to 9.02 for 105aa (p = 0.05) and 9.17 for 114cc (p = 0.020). these results suggest that there is a significant gene/environment interaction impacting on atopy in this cohort. the rage gene encodes the receptor for advanced glycation end-products (rage), a member of the immunoglobulin superfamily. rage activation by ligands, including amphoterin and s100/calgranulins, leads to prolonged nf-kb signalling and has been associated with chronic inflammation. despite high levels of rage expression in lung tissue, little research has been undertaken into the role of rage in the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. objective determine genetic associations between functional polymorphisms in the rage promoter and severe and aspirin-sensitive asthma phenotypes. methods pcr and restriction fragment length polymorphism (rflp) were used to genotype three rage promoter polymorphisms, -429t>c, -374t>a and a 63 bp deletion from -407 to -345, in a large case-control asthma population phenotyped for asthma severity, atopy and aspirin sensitivity. results no associations were identified between any of the polymorphisms and the occurrence of asthma. however, the -374a allele was linked with both severe asthma (p = 0.013) and aspirin-sensitive asthma (p < 0.001). likewise, genotypes containing the -374a allele were strongly associated with both severe asthma (or 2.10, 95% ci 1.32-3.36) and aspirin-sensitive asthma (or 3.13, 95% ci 1.45-6.77). conclusions the -374a allele of the rage gene, previously shown to lead to a 3-fold increase in promoter activity, is associated with the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. these results suggest that increased rage expression, with a concomitant increase in nf-kb signalling, may in part contribute to the inflammatory response seen in these conditions. the global prevalence of allergic diseases is rising and australia has one of the highest prevalence rates in the world. the role of early childhood infections in the development of allergic disease remains controversial. objective to examine the association between early childhood infections and the development of allergic diseases in later childhood, in high risk children. methods data were analysed from the melbourne atopic cohort study (macs) of 620 infants with 1 or more first-degree family members with atopic disease. primary risk factors assessed were otitis media, bronchitis and gastroenteritis reported in the first two years of life. outcomes were current asthma, hay fever and eczema at 6 years of age. logistic regression was used to estimate crude and adjusted odds ratios. results asthma was the most common allergic condition (25.4%, 95% ci 21.6-29.5%), followed by eczema (24.9%, 95% ci 21.1-29.0%) and hayfever (15.6%, 95% ci 12.5-19.1%). the most commonly reported infection was otitis media (58.9%, 95% ci 54.9-62.8%), then gastroenteritis (37.7%, 95% ci 33.9-41.7%) and then bronchitis (19.4%, 95% ci 16. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] .7%). all 3 types of infection within the first 2 years of life were associated with increased risk of asthma. an increased risk of asthma at 6 years was seen with otitis media (or = 1.14, 95% ci 1.02-1.3), bronchitis (or = 1.34, 95% ci 1.0-1.8) and gastroenteritis (or = 1.23, 95% ci 0.96-1.6). when the frequency of infection was examined, those who reported at least 3 episodes of gastroenteritis had a 3-4-fold increased risk and an almost 30% absolute increased risk (rd 0.34, 95% ci 0.08-0.59). conclusion these findings appear to contradict the hygiene hypothesis. the findings for gastroenteritis are novel. further examination of these associations and possible underlying mechanisms is warranted. grant support asthma foundation of victoria, nestle. background knowledge about incident cases of asthma in australia is limited because they are not routinely reported. the ability to predict the number of new cases of asthma would be helpful in allocating resources for asthma education, management and care. data on first use of medications for asthma gives an indication of the incidence of asthma. the objective of this study was to estimate the incidence rate of asthma by investigating asthma medication use in individuals. methods pharmaceutical benefits scheme (pbs) records for all prescriptions filled for inhaled corticosteroids (alone or combined formulation), cromones and leukotriene receptor antagonists from july 2002 to june 2005 were included. using a 2-year look back window, any persons who had their first prescription for any of these drugs dispensed between july 2004 and june 2005 were assumed to be incident cases. overall and age-specific incidence rates were calculated per 100 asthma-medication-free individuals. results there were 352,082 individuals who had their first asthma medication dispensed between july 2004 and june 2005, which equates to an overall incidence rate for asthma of 1.89 per 100. the incidence was higher among children aged 0-14 years (2.07) and adults aged 65 years and over (2.45) . conclusions our estimated incidence rates were consistent with those reported by others in the literature. while the pbs database was designed for administrative purposes, it can be used to estimate incidence rates for asthma. support acam is a collaborating unit of the australian institute of health and welfare and is funded by the department of health and ageing (doha). we acknowledge the pharmaceutical pricing and estimates section of doha for provision of pbs data. keywords asthma incidence, pharmaceutical benefits scheme. rosario ampon 1 , guy marks 1 , teresa to 2 , leanne poulos 1 , anne-marie waters 1 1 australian centre for asthma monitoring (acam), sydney, australia, and 2 hospital for sick children, toronto, canada background the ability to assess individual patterns of asthma medication use would have clinical relevance in targeting effective asthma education and management for this common condition. to describe longitudinal patterns of asthma medication use, we used a population-based prescription database to follow individuals from the first time they filled an asthma prescription. asthma is more commonly listed on death certificates as an associated cause of death, in people whose deaths are attributed to other causes, than as an underlying cause of death. understanding the importance of these associations would contribute towards an overall appreciation of the impact of asthma on mortality. the objective of this analysis was to estimate the prevalence of asthma as an associated cause of death when various other diseases were attributed as the underlying cause of death. background acam currently recommend 24 indicators to measure population-level asthma health and outcomes. we examined correlations among several asthma indicators covering prevalence, morbidity and mortality to try and produce a condensed set of indicators which minimized redundancy. methods seven of the 24 indicators were included in this study: prevalence of ever having doctor diagnosed asthma, prevalence of current asthma, asthma-related general practice (gp) encounters, proportion of people with asthma with an asthma action plan (aap), hospitalizations for asthma, hospital patient days for asthma, and deaths due to asthma. a correlation matrix was created for these indicators by age groups. pearson correlation coefficients ն0.7 or յ-0.7 were considered strong. results there were strong positive correlations between prevalence of ever asthma and current asthma (r = 1.0); gp visits and aap possession (r = 0.74), hospitalization (r = 0.91) and patient days (r = 0.95); and hospitalization and patient days (r = 0.90) and aap possession (r = 0.73). recent australian reports have shown that the prevalence of asthma and respiratory symptoms has decreased over the last 10-15 years. as part of a larger study investigating child health and air quality we have collected nationwide data from schoolchildren living in act, victoria, queensland, wa and sa. methods schools were selected based on proximity to air quality monitoring stations. classes from years 3 to 6 were randomly selected and all children were invited to participate. parents self completed a questionnaire that included questions about diagnosed asthma and respiratory symptoms. results a total of 1989 children provided questionnaires for analysis. the response rate varied between states and territories and ranged from 30% to 42%. the sample comprised 51.9% girls and the mean age of children was 10.2 years. ever diagnosed asthma 27.9 current asthma ('does he/she still have asthma? ') 13.8 wheeze in the past 12 months 16.1 respiratory symptoms limiting activities 11.8 missed school due to asthma or wheezing 8.8 conclusions despite the relatively low participation rate, the prevalence estimates for current asthma are similar to those reported in the national health survey 2004-05 [1] . there is no evidence of any recent increase in the prevalence of childhood asthma. methods tahs is a longitudinal population-based respiratory study of 8583 subjects which commenced in 1968 when they were 7 years of age. since the initial study another 4 follow-ups have been conducted, including the most recent follow-up when subjects were 44 years of age. lung function of the total sample was measured at baseline and in sub-samples in 3 subsequent followups. asthma was categorized as persistent, frequent or episodic when participants reported asthma symptoms in at least 3 follow-ups, in 2 follow-ups or in 1 follow-up respectively. results by age 7 years ever asthma prevalence was 16%. at age 44, 10% of those who had not reported asthma by age 7 had asthma symptoms while 75% of those who reported asthma by age 7 had no asthma symptoms. hence over all only 25% of the asthma symptoms at age 44 were attributable to asthma developed by age 7. in contrast, 91% of the persistent and frequent asthmatics had developed their asthma by age 7. persistent and frequent asthmatics had more symptoms and poorer lung function at age 7, 14 and 44 as well as more reversibility at age 44 (p < 0.05). childhood asthmatics who also had a productive cough by age 7 were more likely to have persistent asthma than those without a cough (p < 0.05). conclusions although the majority of middle-age asthma is related to postchildhood onset asthma, most severe middle-age asthma has its origin in persistent childhood disease. having productive cough in childhood may identify high risk asthmatics who require especially rigorous management in early life. one third of women experience an improvement in asthma during pregnancy, and symptoms improve in most women in the late third trimester. we hypothesized that the exacerbation rate would be reduced and that symptoms during exacerbations would be less severe in the third trimester compared to the second trimester. methods pregnant women with asthma (n = 81) were prospectively followed from recruitment (14.8 weeks (3 sd) ) to delivery at clinic visits (18, 30, 36 weeks and during exacerbation), and fortnightly phone calls. the asthma control questionnaire (acq) was administered at each contact and exacerbations classified as severe (requiring medical intervention) or mild (selfmanaged). lung function, medication use, fractional exhaled nitric oxide (feno) and full blood counts were assessed. paracetamol is commonly used in infants as an analgesic and antipyretic. it has been hypothesized that frequent paracetamol consumption may result in reduced lung capacity to cope with oxidative stress and increase risk of respiratory disease. to date, no study has examined exposure to paracetamol during infancy, when lungs are still developing, and risk of childhood asthma. method a birth cohort of 620 infants with an atopic family history was recruited. frequency of paracetamol exposure was prospectively documented up to 2 years of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results paracetamol exposure in infancy was common (97% exposed by two years of age), with some infants receiving paracetamol on up to 77 days. it has been hypothesized that mucosal immune response requires a particular micro-flora milieu in the infant's gastro-intestinal tract, and that early life antibiotic exposure may disrupt this process and increase risk of allergic disease. method a birth cohort of 620 infants with an atopic family history was recruited. exposure to oral antibiotics was prospectively documented up to 12 months of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results by one year of age, approximately 80% of infants had received at least one course of oral antibiotics. the prevalence of current asthma in childhood was approximately 30% (148/495). frequent use of antibiotics (more than 20 days exposure during first year of life) was associated with increased risk of childhood asthma (or = 2.52, 95% ci = 1.40-4.54) when compared to infant who had not been exposed. excluding infants with a diagnosis of asthma within the first two years of life, reduced this association by about 30% (or = 1.80, 95% ci = 0.90-3.57) and adjustment for gender, parental history of asthma and number of infections in the first year of life further reduced this association (or = 1.60, 95% ci = 0.79-3.22). the increased risk of childhood asthma associated with antibiotic exposure in the first year of life is, at least in part, due to confounding with early life wheeze and infections. if real, the independent effect of antibiotic exposure on risk of childhood asthma is likely to be minimal in this high risk cohort. support dairy australia, crc for asthma and airways, vichealth, nestle. the epidemiological data on asthma suggest a gender difference that varies with age. hormonal effects have been suggested as a possible explanation for these differences. the aim of this study was to examine reproductive factors and risk of asthma among the females of the tasmanian longitudinal health study (tahs). methods the tahs is a longitudinal population-based cohort study of respiratory disease which commenced in 1968 when subjects were 7 years of age. four follow-up studies have been conducted including the current most comprehensive follow-up with subjects at 44 years of age. information has now been collected on reproductive factors such as number of pregnancies, age at pregnancies, age at menarche and contraceptive pill use as well as on asthma status. reproductive factors were examined as risk factors for asthma using multiple logistic regression to adjust for all likely confounders. results a total of 2,776 women completed the most recent postal survey. of these 355 (12.8%) had current asthma, and of these women with current asthma 73.5% (261) developed asthma after childhood. on average these women were in their mid-twenties when they developed asthma (mean ϯ sd age = 26.6 ϯ 12.5 yrs). we found with increasing age at first birth an approxi-mate~30% reduced risk of current asthma in women who developed their asthma post-childhood (trend p = 0.04). we did not observe any other associations between reproductive factors and risk of asthma. conclusions our results are consistent with the hypothesis that early pregnancy may promote asthma development by altering the immune response favouring a th2 pathway. a delay in the age of first pregnancy reduces this risk of asthma. grant support nhmrc, clifford craig foundation, victorian & tasmanian asthma foundations. introduction the association between exposure to pets in early life and subsequent development of sensitization and allergic disease remains controversial. the objective of this analysis was to examine the relationship between cat exposure before birth and development of cat sensitization over time within the melbourne atopic cohort study (macs). methods the macs is a prospective longitudinal cohort study that initially recruited 620 women antenatal in melbourne from february 1990 to november 1994. detailed information on cat exposure was collected at recruitment and frequently until two years of age. skin prick test (spt) were conducted at 6, 12, 24 months and 10 years. the data were analysed by logistic regression and using generalized estimating equations (gee) for the repeated measures design. results among 620 subjects, 169 (28.8%) had a cat before birth. at 6 months, 1.9% (n = 11) of subjects were sensitized to cat and by 10 years of age 18.8% (n = 68) were sensitized. those who did not have cat before birth belong to a higher social class, and were more likely to have a father with allergic disease than those with a cat. those who developed sensitization to cat were more likely to have a paternal family history of allergic disease and more likely to be sensitized to other allergens. we did not observe any association between exposure to cat before birth and the development of sensitization to cat at 6 months (or = 0.7, 95% ci 0. 1-3.3) , 12 months (or = 1.4, 0.5-3.9), 24 months (or = 0.76, 0.2-2.5) or 10 years (or = 0.6, 0. 2-1.4) . these crosssectional results were confirmed by the gee analysis. conclusion our results fail to show an association between cat exposure before birth and development of sensitization to cat. furthermore exposure after birth in the first 18months of life was not associated with an increased or decrease risk of sensitization to cat. our results do not support either a benefit or risk associated with cat ownership and sensitization. introduction peri-natal events influence the development of asthma and atopic diseases in childhood but the current literature is contradictory on the effect of low birth weight, small for gestational age and prematurity on asthma risk. the aim of this study was to assess the relationship between these three exposures and asthma from childhood to adulthood. aim to assess the current prevalence of dda, wheeze (<12 months), atopy and ahr in children and adults in busselton. methods an age-and sex-stratified random sample of adults, selected from the electoral roll, was invited to complete a questionnaire and attend the local study centre for assessment of atopy (allergen skin tests) and ahr (methacholine). all children from participating primary and secondary schools were also invited to attend. the prevalences of dda, wheeze, atopy, ahr and "current asthma" (wheeze + ahr) were calculated. background asthma is often associated with comorbidity, however few studies have investigated comorbidities among people with this common condition. the objective of this analysis was to describe patterns of non-respiratory comorbidity among adults hospitalized with asthma in australia. methods data on hospitalizations for people aged 15 years and over with a principal diagnosis of asthma (j45, j46) were obtained from the australian institute of health and welfare's (aihw) national hospital morbidity database for the period 2005-06. patterns of comorbidity were examined by investigating additional diagnoses for non-respiratory disease according to icd-10 diseasespecific chapters. results among people aged 15 years and over hospitalized in 2005-06 with a principal diagnosis of asthma (16,566 hospitalizations; 70% female; 47% aged 35-64 years), 33% had at least one non-respiratory comorbidity. median length of stay was higher among those with at least one comorbidity (4 days) than among those with no comorbidities (2 days). among people aged 15-64 years, the most common comorbid condition was endocrine, nutritional and metabolic diseases (19%), while among those aged 65 years and over it was diseases of the circulatory system (32%). conclusions a large proportion of asthma hospitalizations in australia are associated with non-respiratory comorbidity and a longer length of stay. further, the pattern of non-respiratory comorbidity associated with asthma hospitalizations varies by age. given our rapidly ageing population, the level of comorbidity associated with asthma has implications for coordinated health care and demand on health services. support acam is a collaborating unit of the aihw and is funded by the department of health and ageing. keywords comorbidity, hospitalization, asthma. background asthma exacerbations are often triggered by viral respiratory infections, yet the influence of respiratory infections on the morbidity of acute asthma beyond the immediate period is unknown. we examined the influence of nasopharyngeal (npa) respiratory viral, chlamydia and mycoplasma detection on asthma morbidity in children presenting to the emergency department for an acute exacerbation of asthma. methods a subset (n = 78) of the 201 children enrolled for a randomized controlled trial (rct) on the efficacy of 5 vs 3 days of oral prednisolone had an npa taken at presentation. npa were examined for chlamydia, mycoplasma and respiratory viruses (enteroviruses, coronaviruses, human metapneumovirus, adenovirus, parainfluenza, influenza, rsv, rhinoviruses) by pcr. enrolled children were aged 2-16 years with recurrent wheeze and required ն600 ?g (mdi/spacer) or ն2.5 mg (nebulized) of salbutamol to reduce tachypnoea. parents filled validated diary cards for cough and asthma severity, and completed asthma qol data at enrolment and end of weeks 1 and 2. results pcr for various viruses was positive in 42 (53.8%) children, with no significant difference in the groups the children were randomized into. rhinovirus pcr was positive in the npa of 32 children, rsv in 7, hmpv in 2, adenovirus, parainfluenza, influenza a and b in one each. specimens were negative for the other micro-organisms listed above. children with a npa viral positive state were significantly (p = 0.002) younger than those with a negative state. however, there was no difference in the any of the asthma outcomes of children whose npa was positive or negative for the micro-organisms tested. conclusions in children with an acute asthma exacerbation presenting to emergency health facilities, a respiratory virus could be identified in >50% but the presence of a respiratory virus did not influence the morbidity of the asthma exacerbation at presentation or at the end of week-1 and week-2. the university of sydney, nsw 2006, and 3 royal north shore hospital, st leonards, nsw 2065 airway wall thickness measured using hrct is reported to be increased in asthmatic compared with control subjects. however, it is unknown whether wall thickness is a fixed structural characteristic of the airways or if it responds to transient changes in bronchomotor tone or airway size. aim to determine the effects of bronchomotor tone and lung volume on airway wall area measured by hrct. methods 8 patients with doctor-diagnosed asthma had partial chest hrct scans, before and after bronchodilator (bd), at frc, tlc and a volume midway between (mid-volume). airway segments were identified between branch points and matched between consecutive lung volumes both before and after bd, and also at constant lung volume before and after bd. mean lumen areas and wall areas for each airway segment at each volume were measured using automated analysis software. paired t-tests were used to determine changes due to bd and lung inflation. results 44 airways were matched before and after bd at frc. absolute airway wall area (wa) was related to airway lumen diameter (di wood smoke air pollution is of concern with respect to respiratory health due to its complex chemical composition and potential to carry air toxics into the lower respiratory system. launceston has a long history of poor winter air quality, primarily due to use of domestic wood heaters. participants in hobart had a similar prevalence of wood heater use, but hobart does not experience the same wood smoke pollution (due to differences in regional geography , asthma control and anxiety and depression were completed at baseline, immediately following (6 wks), and 3 mths after the intervention period. results clinically and statistically (p < 0.05) significant improvements in qol were observed in the exercise group at 6 wks compared to the control group. this difference was not maintained at 3 mths. 6mwd improved at 6 wks and 3 mths in the exercise group (p < 0.01), however the difference between groups was not significant. in the exercise group there was a trend towards improved asthma control and a reduction in anxiety and depression that was not observed in the control group. *p < 0.05, change at 6 wks vs baseline; home asthma monitoring is important for measuring day-to-day variation in lung function and symptoms. this approach requires the availability of complete diaries for a comprehensive assessment. we assessed the completeness of written diaries collected as part of a nation wide study of air quality and child health. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified from a cross-sectional study. these children were asked to record symptom scores and peak expiratory flows twice daily in diaries for a five week period. the diaries and peak flow devices were explained at a face-to-face meeting with parents and children. each week diaries were mailed back and parents received a phone call to encourage completion. completeness was defined as no missing responses to symptom questions or peak flow measurements in diaries from week two to week five. results data from the first 36 children (822 day records) were available for analysis. the sample included (53%) girls, mean age 10 yrs. the overall frequencies for complete records were; morning symptoms 85%, morning peak flow 85%, evening symptoms 83% and evening peak flow 82%. there was a significant trend for more complete morning peak flow records over the four weeks (cochrane-armitage trend test p < 0.007). agreement between morning and evening symptom completeness and between morning and evening peak flow completeness was fairly poor (kappa < 0.30). conclusions the completeness of symptom and peak flow records collected in this study was very high. the comprehensive follow-up protocol implemented is likely to have had an important impact on the completeness of asthma diaries. daily peak expiratory flow (pef) monitoring has been used in epidemiological studies to assess changes in lung function over time. the value of written pef diaries has been questioned because of problems with completeness and validity. this study aimed to compare stored electronic pef data and a written diary record of those data in a panel study in children with weekly reminders to aid adherence. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified in a population study. they were given electronic pef devices with a digital readout (miniwright digital, mwd, clement clarke, uk) and written symptom and peak flow diaries and instructed in their use at a meeting with parents and children. each child was asked to complete three pef manoeuvres every morning and evening for five weeks and to record these in the written diary. background previous research suggests that comorbid anxiety is associated with lower asthma-related quality of life (aqol) in adults with asthma. however, research is scant on the role of psychological interventions in these patients. aim to evaluate the effectiveness of a four-session cognitive-behavioural therapy (cbt) intervention, in improving the aqol, in participants with anxiety and asthma. method participants identified with comorbid anxiety and asthma were randomly assigned to the cbt intervention group (n = 10) and the asthma monitoring control group (n = 8) and evaluated on aqol measures, at various intervals. results nine participants, in the cbt group, completed the study. seven participants showed a clinically significant improvement in asthma-related emotional functioning (ef) and six participants in total aqol scores, at the 5-week post-intervention assessment. additionally, six participants in the cbt group indicated clinically significant improvement in ef and five participants in total aqol scores, at the 3-month follow-up assessment. only three participants in the control group completed the study. none of these participants showed any improvement in aqol scores at the 5-week or 3-month assessment. conclusion this pilot study suggests that a higher number of participants in the cbt group showed clinically significant improvement in ef and total aqol scores with higher retention rates. further research needs to confirm these findings in a larger group, identifying the elements of a successful cbt intervention and characteristics of participants who respond to the cbt intervention. gastro-oesophageal reflux disease (gord) is a risk factor for uncontrolled asthma. we conducted an update of a systematic review to assess whether treatment of gastro-oesophageal reflux in subjects with asthma improved asthma outcomes. methods randomized controlled trials (rcts) of gord treatment in adults or children that reported asthma health outcomes and had symptomatic gord were included and assessed in accordance with the standard cochrane systematic review process. subjects received pharmacological therapies compared with conservative management. results from 261 potentially relevant studies, 19 rcts were included in the review. when compared to placebo, morning peak expiratory flow did not significantly improve (change from baseline wmd 10.43, 95% ci: -9.55 to 30.42) with proton pump inhibitor treatment (n = 7 trials involving 739 participants). asthma exacerbations were not significantly less in the intervention groups compared with the control groups (odds ratio 0.66; 0.41-1.08; n = 557). conclusions while some trials reported evidence of asthma improvement with gord therapy, overall there appears to be no statistically significant evidence of a beneficial effect. it is clear that not all persons with gord and asthma will gain improved control over their asthma with gord therapy; this may be due to the heterogeneous pathophysiology of asthma. future large-scale trials would be required to demonstrate an effect on asthma exacerbations. kel and brd were supported by a cochrane airways group scholarship. background the ats/ers task force recommend the use of metered dose inhaler (mdi) and spacer for airflow limitation reversibility testing. salbutamol given via mdi & spacer has been shown to be equivalent to a nebulizer in the clinical setting. this has not been well studied in respiratory laboratory setting. aim to compare the 2 methods of reversibility testing in a laboratory setting. methods we conducted a laboratory based crossover study in a secondary hospital. patients with asthma or copd were eligible. the patients firstly underwent spirometry and reversibility testing following a standard dose of nebulized salbutamol. they were asked to return for a second set of spirometry within the same week and at the same time of day when reversibility with an mdi and spacer was recorded. we used an incremental dose of salbutamol starting from 2 puffs and up to 8 puffs. spirometry parameters were recorded 10 minutes after each intervention. the primary outcome was the percentage change in fev1 after each intervention. side effects were monitored for. results nine patients with asthma were recruited. the mean percentage change in fev1 was higher in the nebulizer group than after only 2 puffs via mdi & spacer (15.4 ϯ 7.4 vs 6.2 ϯ 8 [mean ϯ sd], p = 0.67). however, there were no differences between the 2 arms following higher doses of bronchodilator via mdi & spacer. the mean percentage change in fev1 after 4, 6 and 8 puffs were 12.6 ϯ 11.3, 15.4 ϯ 12.3, and 17.7 ϯ 13.6 respectively (p = 0.09, 0.05 and 0.07 respectively when compared to the nebulizer group). conclusion using an mdi and spacer for bronchodilator reversibility is equivalent to that of a nebulizer and should be the standard method of testing. the dose of bronchodilator needs to be at least 4 puffs as recommended by the ats/ers; however 6 puffs correlated best with a standard nebulizer route. further increments in bronchodilator dose provided little additional bronchodilatation. the study was limited by the small number of patients. asthma guidelines recommend a stepwise approach to treatment. the role of inhaled corticosteroid (ics) and long-acting beta-agonist (laba) combination therapy in asthma written action plans is not clear. objective to assess the efficacy of adjusting ics/laba combination therapy in a written action plan compared to fixed dosing in people with asthma requiring maintenance ics. methods cochrane systematic review of randomized controlled trials comparing ics/laba combination therapy in a single inhaler that is adjusted up or down according to a written action plan (wap) to comparison 1: budesonide/ formoterol given as a fixed maintenance dose (fd) (n = 9) or comparison 2: fluticasone/salmeterol fd (n = 2). results 10 parallel randomized controlled trials describing 11 interventions met the inclusion criteria. for the trials that compared wap to fd budesonide/ formoterol there were significant reductions for the wap group in exacerbations, (rr (95%ci): 0.82 (0.70 to 0.97)), severe exacerbations (rr (95%ci): 0.61 (0.37 to 0.99)) and study medications (wmd (95%ci): -1.18 (-1.23 to -1.14)) with no difference in asthma control or adverse events. the results for the two trials reporting wap budesonide/formoterol to fd fluticasone/ salmeterol were discordant and a homogenous pooled result could not be determined. of the 318 australians who died from asthma in 2005, over two thirds were over 50 years of age. this trend resulted in the national asthma council of australia (nac) calling for better management of asthma in the elderly. we designed an educational intervention using evidence based educational strategies to improve the content and style of general practice consultations for older people with asthma. methods randomized controlled trial of a multi-faceted program consisting of a group educational session, a videotaped standardized simulated patient consultation, followed by an academic detailing session. forty-two gps were randomized into an active or a control group. gps provided the names of patients who would be happy to participate in the study and the program was evaluated by patient and gp outcomes. results gps recruited into our program reported improvements in a range of clinical areas. one hundred and ten patients were recruited, their outcomes are under analysis. conclusion gps were overwhelmingly positive about participation in this trial and our intervention successfully improved the capacity and confidence of gp's to deliver care to older people with asthma. our study also developed several tools that would enable dissemination of our findings. supported by an asthma targeted in studies where direct clinical assessment is not possible, urgent health care utilization (hcu) is often used as an indirect measure of asthma control. this study aimed to identify factors predicting urgent hcu and asthma control. methods patients in nsw with a doctor diagnosis of asthma were recruited from community pharmacies, a research volunteer database, and databases of asthma foundation nsw, to complete a questionnaire about asthma. poor asthma control was defined as asthma control questionnaire (acq) score ն1.5. urgent hcu was defined as hospitalization, ed visit, or urgent doctor visit due to asthma. multiple logistic regression was used to identify predictors of poor control and urgent hcu. results questionnaires were completed by 608 adults (61% female) with a doctor diagnosis of asthma (pharmacy 260, woolcock 299, asthma foundation 87). 87% used inhaled corticosteroid (ics) ϯ long-acting b2-agonist in the last 4 wks. median age was 56 yrs (range 12-87), and 9% were current smokers. mean acq score was 1.4 (95% ci 1.3-1.5), with 40% of participants having poor asthma control (acq ն 1.5). 28% had urgent hcu for asthma in the previous year. significant independent predictors for poor asthma control were younger age, current smoking, living in more disadvantaged areas, being retired, having only primary education, and holding a concession card. predictors for urgent hcu were younger age, being in full-time employment, having only primary education, and being of non-english speaking background. neither ics use nor possession of a written asthma action plan was associated with lower risk for either poor asthma control or hcu. conclusions poor asthma control is common in nsw even in patients using inhaled corticosteroids. although urgent hcu is often used as an indirect measure of poor asthma control, it is affected by different factors, perhaps because health care utilization represents a more complex balance between need and access. bronchial challenge tests with mannitol, to measure airway hyperresponsiveness, can take up to 30 minutes and require inhalation of up to 635 mg of mannitol. our aim was to determine if positive mannitol challenges can be detected after half the maximal dose (315 mg) using the forced oscillation technique (fot) to measure response. methods 15 non-asthmatic subjects and 52 asthmatic subjects underwent standard mannitol challenge, up to 635 mg mannitol. respiratory system conductance (grs) and reactance (xrs) was measured by fot at 6 hz during 40 sec tidal breathing immediately after each dose of mannitol. fev1 was measured after fot, within 90 sec of mannitol administration. two point dose response slope (drs), was calculated for grs (drsgrs) and xrs (drsxrs) for standard tests, up to 635 mg, and for short tests by excluding data from doses above 315 mg. ability to detect a positive test, defined as pd15fev1 < 635 mg, was determined by the area under the roc curve (auc) and repeatability by intra-class correlation coefficient (icc). results 32 asthmatic and 2 non-asthmatic subjects had positive tests, with pd15 fev1 values from 9.2 to 622 mg. auc (95%ci) did not differ between standard (std) and short tests for drsgrs (p = 0.14) or drsxrs ( combined use of inhaled steroids (ics) and long acting beta-agonists (laba) have an important role in asthma management. we used data from a 2006 population sample to examine medication use in adults and children. methods all adults (18-75 years) and children (2-17 years) from within four discrete zones in northern sydney were eligible for an interview survey, as part of a study investigating health effects associated with traffic-related air pollution. the prevalence of use of short-acting beta-agonists (saba), any ics (alone or combination) and combined formulations of ics/laba in the previous three months was estimated for the study population and those with diagnosed asthma. results there were 806 children [mean (sd) age 8.7 (4.6) years and 50% female] and 2184 adults [mean (sd) age 45.6 (14.9) years and 56% female] interviewed in 1843 households, representing an overall response rate of 33%. the prevalence of ever diagnosed asthma was 16.1% in children and 17.4% in adults. medication data were missing for 301 subjects. background asthma affects 1:9 adult australians and is a leading cause of rejection for recruitment into the australian defence force (adf). within this diagnosis there is a wide spectrum of disease activity and clinical outcomes. also asthma assessment and management has improved so that many asthmatics are now fully active without any significant disruption or risk to their lives. hypothesis: there is a subgroup of asthmatics who are at very low risk from significant adverse effects from asthma and who could be considered for recruitment to the adf. aims 1. to identify the subgroup of asthmatics who could be considered for recruitment to the adf. 2. to develop an assessment process to identify this subgroup (screening). 3. to develop a process to evaluate the outcomes of any change to the recruitment standard for asthma (evaluation). methods 1. a literature review of the natural history, assessment, management and response to treatment of mild episodic and mild persistent asthma. 2. a literature review of asthma in the military. 3. a clinical review of the outcomes of known asthmatics in the adf. 4. an expert group to review the above and to develop a screening process and an evaluation of the program. the literature review identified a subgroup of asthmatics, defined as mild episodic and mild persistent, who with appropriate management, have a low risk of significant adverse asthma outcomes. they can be identified by a combination of questionnaire, spirometry and bronchial provocation testing. a screening process has been developed which allows asthmatics to be recruited with a negative mannitol or hypertonic saline challenge on 400 mg/day or less of budesonide (or equivalent) without laba. a methodology to evaluate the impact of these changes on the recruitment standard has also been developed. alexithymia is a personality trait associated with difficulty identifying and communicating emotional and physical feelings. it has been associated with poor control of asthma and near fatal asthma. the primary objectives of this study were to: (1) identify alexithymia in a cohort of australian asthma patients; (2) investigate the relationship between alexithymia and asthma control; (3) investigate the relationship between alexithymia and asthma management. methods cross sectional study of 25 moderate to severe asthma patients recruited from royal adelaide hospital outpatients. participants were either mailed the questionnaire pack or completed it after a clinic appointment. existing validated questionnaires were used. statistical analyses were performed using spss. results 11 male (44%) and 14 female (56%) patients with moderate to severe persistent asthma (mean age 44 years, sd = 11) participated. alexithymia scores ranged from 23.0 to 76.0 (x = 48.3, sd = 13.2). 12% (n = 3) of participants could be classified high alexithymia, 32% (n = 8) borderline alexithymia and 56% (n = 14) were low alexithymia. alexithymia mean scores were not statistically different across sociodemographic variables. a positive correlation/association was found between alexithymia score and asthma control score (r = 0.57, p < 0.01), quality of life (r = -0.65, p < 0.01), and adherence (p = 0.03) but not satisfaction with communication (r = -0.27, p = 0.2) or number of hospitalizations (p = 0.25). conclusions this is the first australian study to identify alexithymia among asthma patients and investigate relationship to control as well as management and communication. associations between alexithymia and asthma control were confirmed. a larger sample size is needed to determine impact of alexithymia on self-management and provision of clinical care for asthma. port hedland is impacted by iron-containing dust particles (pm10) that may activate lung cells when inhaled. furthermore, the effects of port hedland pm10 may differ from the effects of urban pm10 impacting metropolitan areas. the aim of this study was to assess the effects of port hedland pm10 on production and release of the inflammatory cytokines, il-6 and il-8, by human airway epithelial (a549) cells, and to compare these with the effects urban pm10 from metropolitan areas. methods human airway epithelial (a549) cells were exposed to pm10 collected at port hedland and at urban locations (sydney, perth). a549 cells were exposed to a range of pm10 concentrations (20-200 mg/ml) for 24 h. lipopolysaccharide (lps) and phorbol myristate acetate (pma) were used as positive controls. supernatants from cell cultures were assayed for il-6 and il-8 using specific elisa kits. rna was extracted and reverse transcribed to cdna. il-6 and il-8 mrna expression was quantified by duplex real-time pcr using taqman primer/probes. results lps stimulated a 2.7-fold increase in il-8 release and pma stimulated a 3-fold increase in il-8 release and a 30-fold increase in il-6 release. however, neither port hedland pm10 nor urban pm10 stimulated concentration dependent release of il-6 or il-8 by a549 cells. expression of il-6 or il-8 mrna was also not altered by port hedland or urban dust. cd8+ t-cells may cause airway epithelial cell apoptosis via the granzyme pathway. we have reported increased apoptosis of airway epithelial cells and increased bal t-cell expression of granzyme b in copd, and a positive correlation between the two. we hypothesized that the increased granzyme b would also be related to smoking history (pack years -pk/y), age and severity of airflow obstruction (fev1 %pred) in patients with copd. we further hypothesized that the t-cell granzyme b expression would be higher in the airway than the peripheral blood. methods we investigated t-cell intracellular granzyme b expression in blood from copd subjects (33 current and 24 ex-smokers) and 12 never-smoker controls, and bronchoalveolar lavage (bal) and bronchial brushing (intraepithelial t-cells) from a cohort of these subjects using flow cytometry. correlations between granzyme b and pk/y, age or fev1 were performed using spearman's rank correlation. granzyme b in t-cells from blood, bal and bronchial brushings were compared. results there were significant correlations between fev1 and granzyme b expression in blood and bal (blood: r -0.444, p = 0.002; bal: r -0.368, p = 0.029). there was a significant correlation between pk/y and granzyme b expression in blood (r 0.362, p = 0.002), but not in bal. there were no significant correlations between granzyme b and age. there were no significant differences in granzyme b expression in blood, bal or intra-epithelial compartments. conclusion granzyme b is expressed at similar levels in blood, bal and intra-epithelial compartments, supporting recent opinion that copd is a systemic disease. t-cell granzyme b is related to severity of airflow obstruction and smoking history in patients with copd and may be one mechanism of apoptosis leading to lung injury and airflow obstruction in copd. jc allen 1 , t schlosser, ee ramsay 1 , q ge 2 , aj ammit 1 as development of remodelled airways is correlated with deterioration of lung function, we require therapies that reduce and reverse structural changes in remodelled airways. in asthma, corticosteroids can halt some, but not all, aspects of airway remodelling. therefore, in order to aid future design of efficacious anti-remodelling agents we need a better understanding of the molecular mechanism/s underlying the development of airway remodelling and the effectiveness of corticosteroids. hyperplasia of airway smooth muscle (asm) is a feature of the remodelled airway in asthmatics. in this study we examined the effect of corticosteroids on a key regulator of g1 progressioncyclin d1. asm cells from n = 8 non-asthmatics and n = 7 asthmatics were pretreated for 1 h with vehicle or dexamethasone (0.1 mm). the temporal kinetics of cyclin d1 mrna and protein expression were measured up to 24 h after stimulation with the mitogen platelet-derived growth factor-bb (pdgf-bb). pdgf-bb induced a significant increase in cyclin d1 mrna expression in asm from non-asthmatics (2.6 ϯ 0.3-fold) and asthmatics (2.9 ϯ 0.3-fold) after 24 h stimulation. in non-asthmatics, the corticosteroid dexamethasone significantly (p < 0.05) reduced the amount of cyclin d1 mrna expressed (to 1.6 ϯ 0.2-fold). in contrast, cyclin d1 expression in asthmatics was relatively resistant to inhibition by dexamethasone; the amount of pdgf-bb-induced cyclin d1 expression in the absence or presence of dexamethasone was not significantly different ( sphingosine 1-phosphate (s1p), a bioactive sphingolipid found elevated in the airways of asthmatics, modulates myriad airway smooth muscle (asm) functions that promote inflammation and remodelling in asthma. in this study, we uncover the molecular pathway/s underlying s1p-induced secretion of il-6, and investigate if, and how, corticosteroids inhibit il-6 secretion. using cultured asm cells from non-asthmatics, we found that s1p induces il-6 secretion from asm cells via cre, but not ap-1, c/ebp or nf-kb, transcriptional regulation of il-6 gene expression. cre-dependence was supported by s1p-induced creb phosphorylation. although the corticosteroid dexamethasone reduced s1p-induced il-6 secretion in a dose-dependant manner, this inhibition appeared to occur via a pathway independent of creb/cre, suggesting the existence of a parallel pathway. as we recently discovered that the antiinflammatory actions of corticosteroids in asm can be mediated via the induction of the endogenous mitogen-activated protein kinase (mapk) inhibitor, mapk phosphatase-1 (mkp-1), we investigated whether mapk represents the parallel pathway targeted by corticosteroids. we found that s1p can induce activation of a variety of mapk, however, only p38 mapk phosphorylation was inhibited by dexamethasone; importantly, the increase in mkp-1 after corticosteroid treatment appeared to mirror the decrease in s1p-induced p38 mapk phosphorylation. furthermore, exogenous expression of mkp-1 inhibited s1pinduced il-6 secretion. taken together, these results suggest that parallel pathways exist to induce il-6 secretion (transcriptional via creb/cre and possibly post-transcriptional via p38 mapk) and serve to underscore the importance of mkp-1 upregulation as a mechanism of action of corticocosteroids in asm. angiogenesis is a hallmark feature of asthma. angiogenic promoters, such as vegf and tgfb are reported to be increased in airways of asthmatics. tumstatin, an endogenous angiogenic inhibitor, is the non-collagenous domain-1 (nc1) of the alpha3 chain of collagen iv. decreased levels of collagen iv have been reported in the airways of asthmatics. we investigated the presence of tumstatin in the airway of asthmatics and its potential role as an angiogenic inhibitor. we detected the six a chain nc1domains of col iv and the 7s domain of the a3 chain using immunohistochemistry. the level of tumstatin in serum and bal-f was measured by dot blot. western blots were used to identify the association with the rest of the collagen iv molecule. a tube formation assay using primary pulmonary endothelial cells (ppec) was performed to evaluate the role of tumstatin in the airway. the effect of intranasal tumstatin on airway hyperresponsiveness and angiogenesis was studied in an ovalbumin mouse model. tumstatin was absent in the airways of asthmatics (n = 14) while the remaining six collagen iv a chains were present. the 7s domain of the a3 chain was present in the asthmatic airway (n = 6). tumstatin was detected in both serum and bal-f samples from asthmatic volunteers (n = 10), however the level of expression was not significantly different from that in nonasthmatics (n = 7). in asthmatic serum tumstatin was part of the whole collagen iv a3 chain. tumstatin was able to inhibit ppec tube formation in a dose related manner. tumstatin inhibited angiogenesis in the mice airways and was associated with an improvement in ahr. the fact that tumstatin is absent from asthmatic airways and inhibited airway hyperresponsiveness and angiogenesis may indicate potential for therapeutic intervention in airway remodelling. this work was supported by the crc for asthma and airways and nh&mrc. introduction epithelial egfr (epidermal growth factor receptor) expression correlates with disease severity and neutrophil infiltration in asthmatic airways. acute exacerbations of asthma and copd are also associated with steroid refractory neutrophilic inflammation, with rhinoviruses being the most common trigger. .7 mg/l and il-6: 5.8 vs. 3.6 ng/l). since il-6 stimulates the acute phase response, we correlated its levels with the other markers. only crp was strongly correlated with il-6 (spearman r = 0.58, p < 0.0001), suggesting differential regulation of saa and ip10. saa discriminated between non-pathogen (n = 10) vs. pathogen-associated (n = 41) events (saa: 9.4 vs. 44.1 mg/l p = 0.005), whereas no significant change was observed in the other markers (ip-10: 139.8 vs. 170.5 ng/l, crp: 4 vs. 10 mg/l, il-6: 4.6 vs. 7.2 ng/ l). however when aecopd marker levels were stratified on the basis of pathogen type (viral = 12, bacterial = 21, viral and bacterial = 8), none of the markers were significantly altered. conclusions ip-10 is significantly elevated during an aecopd, however only saa differentiated non-pathogen from pathogen associated events. background severe persistent asthma is characterized by structural changes in the airways-airway remodelling. airway smooth muscle (asm) cells have the potential to play a key role in these processes through the release of growth factors, cytokines and extracellular matrix (ecm) proteins. we have previously studied the effects of budesonide and formoterol individually however, the effect of their combination on these characteristics of asm cells is not known. methods asm cells from asthmatic (n = 6) and nonasthmatic (n = 6) individuals were stimulated with transforming growth factor ß (tgfß) (1 ng/ml) with or without budesonide (10 -8 m) and formoterol (10 -10 and 10 -8 m) and fibronectin levels and interleukin-6 (il-6) release were measured by elisa. bronchial rings from nonasthmatic individuals (n = 2) were incubated with tgfß with or without the drugs and ecm protein expression (fibronectin and collagen i) measured using immunohistochemistry. results in nonasthmatic cells, budesonide alone induced fibronectin deposition whether tgfß was present or not. formoterol decreased fibronectin induced by tgfß and, when combined with budesonide, reversed the increase in fibronectin. a similar pattern was observed in asthmatic cells, except that budesonide did not further increase the tgfß mediated fibronectin release. as before [1] , il-6 was induced by formoterol but inhibited by budesonide. tgfßinduced il-6 was inhibited by both drugs and their combination in both cell types. in bronchial rings the presence of either drug did not affect tgfßinduced fibronectin or collagen i. severe combined immune deficiency (scid) spontaneous mutation specifically impairs differentiation of stem cells into mature lymphocytes. nod-cb17prkd scid (known as nod-scid) lacked nk cells, hence is commonly used in cell transfer experiments for transferring tissue and haematological xenografts. the aim of this study was to establish lung inflamamtory model in nod-scid strain. methods balb/c and nod-scid balb/c mice (n = 8) were exposed to cigarette smoke for 4 days, 2 and 4 weeks (9 cigarettes/day; 5 days/week). bronchoalveolar lavage fluid (balf) and lung tissue were collected for inflammatory profiling and analysis for cytokines, chemokines and protease expression and/or activity. results nod-scid have significant accumulation of macrophages in lung after 4 days, 2 and 4 weeks smoking as compared to no smoke control (p < 0.001) that was not different to balb/c (p > 0.05). nod-scid also have increased neutrophil number after 2 and 4 weeks smoking (p < 0.001). even though myeloid cell differentiation isn't affected by scid phenotype, nod-scid have one fold less neutrophil than balb/c mice (p < 0.001) that is also reflected in the reduced expression of matrix metalloproteinase-9. consistent with the known lymphopenic phenotype, nod-scid have significant but less lymphocytes recruitment as compared to balb/c mice after 4 weeks smoking (p < 0.001) despite the enhanced expression of inteferon inducible protein 10 (lymphocytes specific chemokine) in lung. both mouse strains showed the same elevation of net gelatinase and serine protease activity in lung. nodscid mice also demonstrated comparable transcriptional induction of proinflammatory cytokines (tnfa, il-6), growth factors (gm-csf, g-csf) and chemokines (mcp-1, mip-2), indicating susceptibility to smoke-induced injury. conclusions nod-scid mice are capable to mount smoke induced inflammatory response. this model may be useful to study localization and role of immunocytes, including adoptively transfer human cells in the pathogenesis of copd. supported by the nhmrc. rhinovirus (rv) is the cause of most common colds and up to 80% of asthma attacks. in our previous studies, plasminogen activator inhibitor 2 (pai-2) was expressed at high levels and was induced in vivo and in vitro by rv infection. pai-2 may have antiviral properties suggested by antiviral activity in some models, high pai-2 expression levels and further upregulation by rv infection. methods to determine whether pai-2 has antiviral activities following rv infection, o-hela, pai-2 expression-deficient cells were first transfected with pai-2 or control genes. this was followed by infection with rv and effects on viral replication were assessed by rt-qpcr for vrna and by viral titration for virus release. ifn expression was assessed by rt-qpcr. results ifn-a and -b mrna expression were induced in response to rv infection and to pai-2 expression in cells. pai-2 expression followed by rv infection elicited a synergistic response and pai-2 over-expression reduced vrna by >5 fold and viral titre by >3 log (p < 0.05). however, this effect was not specific to pai-2, as transfection of cells with control genes/plasmids reduced viral titre to a comparableextent. one of the pathological findings in idiopathic pulmonary fibrosis (ipf) is the presence on fibroblastic foci comprising cells which exhibit mesenchymal phenotypic features such as myofibroblast-like morphology, increased asma expression and collagen deposition. currently steroid treatment in ipf has shown limited efficacy. the cellular origins of these mesenchymal cells remain unclear, but evidence from other studies suggests that epithelial cells may undergo a transition to a mesenchymal cell phenotype (emt). transforming growth factor ß has been implicated in promoting this emt. in this study we have induced a morphological change in a549 cells using tgf-ß1 and assessed the influence of glucocorticoids, and the changes to the extracellular environment of the cells, on emt. methods a549 cells were grown on uncoated plastic cultures plates or those coated with monomeric or fibrillar collagen and treated with 200-500 pm tgf-ß1. the influence of the glucocorticoid, dexamethasone (dex, 1-1000 nm), or collagen type, on emt was assessed by microscopy, rt-pcr and western blotting for markers of myofibroblast phenotype. results tgf-ß1 induced an increase in mrna expression of asma (1.5 fold), collagen (7.0 fold) and fibronectin (2.0 fold). dex (100 nm) partially inhibited the expression of collagen, but had no effect on asma levels. however, dex (100 nm) reduced asma and ctgf protein levels. dex (100 nm) also prevented the tgf-ß1-induced morphological changes, regardless of ecm matrix. conclusion glucocorticoids appear to control some of the emt phenotype changes induced by tgf-ß1. however, the inability to fully inhibit these changes may contribute to the resistance of ipf to glucocorticoids. the extracellular environment may also play a role in the development of fibroblastic foci and their pharmacological responses. defective alveolar macrophage (am) phagocytic function in the airway may perpetuate inflammation via secondary necrosis of uncleared apoptotic cells in copd. we have previously reported that low-dose azithromycin improved macrophage function in vitro, although the mechanisms for this effect were not identified. we explored the possible role of the collectin pathway in the azithromycin-mediated improvement in phagocytosis as well as possible defects in this pathway in copd subjects. methods (1) mannose binding lectin (mbl), mannose receptor (mr), surfactant protein d (sp-d) were measured in copd subjects and controls. (2) the in vitro effects of addition of rhmbl, and blocking mr with a specific antibody, on am phagocytic ability were assessed. in vitro effects of azithromycin on am expression of mr were also investigated. (3) azithromycin (250 mg orally 2¥ weekly/12 weeks) was administered to 11 copd subjects. bronchoscopies were performed prior to and 12 weeks following therapy. ex vivo assessments included am phagocytic ability, levels of mbl, sp-d and mr and apoptosis of bronchial epithelial cells. results am mr expression and levels of mbl and sp-d were significantly reduced in copd subjects vs controls. azithomycin (500 ng/ml) increased mr expression by 31% in vitro. rhmbl induced a dose-dependent increase in am phagocytic ability (up to 148%). blocking mr significantly decreased am phagocytic ability by 60%. in copd patients following azithromycin therapy, we observed improved am phagcocytic ability, increased levels of mr and reduced levels of bronchial epithelial cell apoptosis. conclusions these findings strongly implicate the mr in both the defective phagocytic function of am in copd and as a target for the azithromycinmediated improvement in phagocytic ability. obstructive sleep apnea (osa) is associated with hypoxia and increased cardiovascular morbidity. t cells and monocytes play a significant role in atherogenesis via cytokine production. there have been reports of benefits of continuous positive airway pressure (cpap) therapy in osa. the purpose of this study was to characterize leucocyte inflammatory cytokine/chemokine production by t cells and monocytes in a group of osa patients and to investigate the therapeutic effects of cpap therapy. methods a comprehensive range of intracellular t-cell and monocyte proand anti-inflammatory cytokines/chemokines was investigated in peripheral blood from 5 osa patients and 5 aged-matched control subjects (with no evidence of sleep problems) using multiparameter flow cytometry. osa patients were again studied following 7 days of cpap therapy. results in osa patients there was an increase in intracellular t-cell ifng and tnfa production but no change in il-2, il-4 or tgfb compared with control. there was an increase in intracellular monocyte il-1a, il-8, tnfa, mcp-1 and mcp-3 in osa patients but no change in il-10 or il-12. following cpap therapy, t-cell ifng and tnfa production returned to 'normal' levels. however, although intracellular monocyte cytokine/chemokine production was decreased following cpap, levels were significantly elevated compared with control. conclusions osa is associated with increased intracellular proinflammatory cytokine/chemokines, many of which are increased in atherosclerotic plaques. although one week of cpap therapy resulted in amelioration of t-cell pro-inflammatory cytokines, longer cpap use or alternative therapy may be required to reduce monocyte pro-inflammatory mediators associated with atherosclerosis in patients with osa. gp130 has been associated with the progression of fibrosis especially in patients with idiopathic pulmonary fibrosis (ipf). gp130 is the common subunit of the receptor complexes for the il-6 family of cytokines including il-11 and oncostatin m (osm), where gp130-mediated signalling leads to activation of the erk or stat pathways. we have previously demonstrated exaggerated gp130-stat signalling to be fundamental to the development of pulmonary fibrosis in a murine model of bleomycin-induced lung fibrosis. the aim of this study was to elucidate the role of the il-6 cytokine family in the development of pulmonary fibrosis by identifying which il-6 family cytokines regulate fibrosis in bleomycin treated mice, and determine the effects of these cytokines on cell function. bleomycin (0.05 u/mouse) or control saline was administered intranasally to wildtype mice (wt), genetically engineered mice containing point mutations to prevent gp130 erk signalling (gp130 757f ) or gp130 stat signalling (gp130 dstat ), and duel il-6 and il-11 a-receptor knockout mice (il-6 -/-;il-11ar -/-). the effect of bleomycin on collagen production was examined in lung tissue 30 days post treatment by hplc. there was a significant increase in collagen levels in bleomycin treated wt lungs which was further increased in gp130 757f lungs. the lungs of gp130 dstat and il-6 -/-;il-11ar -/mice were protected from fibrosis suggesting that gp130-stat signalling is important in inducing lung fibrosis which may be mediated through il-6 and/or il-11. cell proliferation was examined in lung fibroblasts isolated from wt, gp130 dstat and gp130 757f mice. il-6, il-11 and osm were significantly mitogenic for gp130 dstat cells but not for wt or gp130 757f cells, reflecting different responses to the different signalling pathways. changes in cytokine profiles are currently being examined in lung tissue and serum of control and bleomycin treated mice 0-30 days after treatment. in conclusion, il-6 and il-11 are likely to play a role in bleomycin-induced fibrosis via the gp130-stat-mediated pathway, however this may not be due to regulation of proliferation induced by these cytokines. supported by the nhmrc. mimicking viral infection by application of various toll-like receptor ligands has shown clinical promise in the treatment of persistent viral infections and more recently with malignant tumours. commercially available toll-like receptor 7 ligands (tlr7l), such as those of the imidazoquinoline family have been applied clinically for the treatment of a number of conditions including basal cell carcinoma and hpv-induced genital warts. these compounds are known to retard tumour growth indirectly by promoting activation and migration of dcs, leading to a strong th1 cellular response, and directly via release of proinflammatory cytokines and promotion of tumour cell apoptosis. malignant mesothelioma (mm), an aggressive tumour with a mean survival of 9 months, is highly resistant to chemotherapy, radiotherapy and surgery and is therefore an interesting candidate for immunotherapy in the form of tlr7 ligand treatment. whilst tlr7 is known to be selectively expressed in immune cells and its relative expression low amongst other cell and tissue types in mammals, its expression on tumour cells and the consequences of such expression on tumour growth are unknown. here we describe the presence of tlr7 (mrna and protein) directly in a range of different tumours, including several murine and human mm cell lines. reactive oxygen species (ros) produced during the innate immune response are important agents of anti-pathogen defense but may also cause oxidative lung damage. glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. methods wild-type (wt) or mice deficient in glutathione peroxidase-1 (gpx-1 -/-) were placed in a perspex chamber and exposed to cigarette (cig) smoke generated from 9 cigs per day for 4 days. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for proteomic and genomic analysis. results wt mice exposed to cig smoke for 4 days had significantly more macrophages (3.1 ϯ 0.1(sem) ¥ 10 5 ) and neutrophils (4.9 ϯ 0.4 ¥ 10 5 ) than sham-exposed mice (2.2 ϯ 0.2 ¥ 10 5 and 0, respectively) (n = 6, p < 0.05). however, gpx-1mice exposed to cig smoke had significantly greater macrophages (5.4 ϯ 0.3 ¥ 10 5 ) and neutrophils (1.2 ϯ 0.1 ¥ 10 6 ) than smokeexposed wt mice (n = 6, p < 0.001). macrophage and neutrophil numbers in sham-exposed gpx-1 -/mice (1.7 ϯ 0.3 ¥ 10 5 and 0.5 ϯ 0.4 ¥ 10 3 ) were similar to those of sham-exposed wt mice (2.2 ϯ 0.2 ¥ 10 5 and 0). in addition, we found that balf of gpx1 -/mice exposed to cig smoke had an increased proteolytic burden compared with smoke-exposed wt mice as assessed by zymography and net gelatinase activity assay. conclusions these data suggest that gpx-1 protects the lung from cigarette smoke-induced inflammation and that targeting gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role. funded by nhmrc. the becs from subjects with chronic obstructive pulmonary disease (copd) are exposed to frequent infectious and inflammatory stimuli. infection with rv is known to trigger acute exacerbations and subjects with copd are particularly susceptible. we hypothesized that exposure of copd becs to these stimuli would alter their response to rv infection. methods bec were obtained by endobronchial brushing from subjects with gold stage 3 copd (n = 4, all ex-smokers), subjects with mild persistent asthma (n = 4) and healthy controls (hc, n = 4). becs were cultured and then treated with tumour necrosis factor (tnf)a 10 ng/ml or lps 100 mg/ml for 24 hrs and then infected with rv-43, rv-1b. response was measured by release of il-8, il-6 and ip-10 mrna and by elisa. virus replication measured by cell titration assay. results infection with both rv strains led to increased release of il-8 and ip-10 in all groups. exposure of hc and asthma becs to both lps and tnf led to increased release of il-8. in these becs there was no increase in release of il-8 exposed to lps and tnf and then infected with either rv. becs from subjects with copd released significantly less il-8 in response to all conditions and rv infection compared to hcs and asthma. no differences were seen in rv replication. the aim of this study was to determine opinions and attitudes to exercise from chronic obstructive pulmonary disease (copd) subjects after completion of a 12-month maintenance exercise program. methods following completion of a 12-month exercise study, which included a supervised program (intervention, n = 18) and control group (control, n = 17), copd subjects [mean age (sd): 66 (8); mean fev1 (% predicted) = 56% (19)] were asked to complete a questionnaire. the questionnaire included closedended questions using visual analogue scales (100 mm). in copd the 6 minute walk distance (6mwd) is known to increase with test repetition (familiarization) and in response to exercise training. it is unknown whether the magnitudes of these increases are related to the degree of disability of the individual patient. methods 6mwd was measured twice before and once after an 8 week out-patient exercise program in 121 patients (82 males) aged 67ϯ8.6 yrs, fev1 37ϯ15% predicted (meanϯsd) with stable copd. the changes in 6mwd following a familiarization test and following training were compared between patients grouped according to their degree of disability (defined as the pre-training 6mwd [best of 2 tests] expressed as %predicted 6mwd). *p < 0.05 gp 3 vs gp 1. conclusions before training, 6mwd increases following a familiarization test irrespective of the level of disability. the magnitude of this increase is similar in all groups when normalized for their pre-training 6mwd. following training, the increase in 6mwd is greatest in patients with the greatest disability (lowest pre-training 6mwd). in less disabled patients, the relatively smaller increase in 6mwd following training may reflect an inability to further increase stride length, thereby reducing the responsiveness of the 6mwt in this group. supported by nhmrc. endotoxin is a stimulant of the innate immune system and is a major component of cigarette smoke. smokers have evidence of increased airway neutrophils and inflammation. we hypothesized that endotoxin levels would be higher in the bronchial lavage (bl) of subjects who were former smokers and subjects with chronic obstructive pulmonary disease (copd). methods subjects were all ex-smokers for at least 5 years (n = 10, 5 copd, 5 healthy controls) or never smokers (n = 12, 6 asthma, 6 healthy controls). bl was collected and analysed for cell count and differential, culture for microbiology. the supernatant was analysed for il-8 by elisa and endotoxin by quantitative kinetic lal assay. results median endotoxin levels were significantly higher in ex-smokers 101 compared to never smokers 6.3 u/ml (p < 0.001). there were no differences between subjects with copd and hs. subjects with copd had higher median endotoxin levels (80 u/ml), compared to asthma (5.2 u/ml) and hc (6.3 u/ml, p = 0.03). there was no correlation between endotoxin levels and bl total cell count, neutrophils (%) or fev1 % predicted. there was a strong correlation with previous packet years smoked and endotoxin levels (r = 0.72, p < 0.01). conclusions bl endotoxin levels are higher in ex-smokers, including subjects with copd. despite this there is no relationship to increased neutrophilic inflammation. copd is associated with inflammation associated with ineffective repair of the injured epithelium and loss of structural integrity. we have shown that these changes may result from dysregulated 'efferocytosis' (increased apoptosis of bronchial epithelial cells and defective clearance of these cells by alveolar macrophages (am)). we have also reported that azithromycin, at subbactericidal dose, improved am phagocytic function ex vivo. methods we administered azithromycin at low dose (250 mg/ twice weekly for 12 weeks) to 10 copd subjects (7 male, age: 62 ϯ 8 yr, 5 current/ 5 ex-smokers, fev1: 63 ϯ 9% pred, fev1/fvc: 48 ϯ 9%). the study was openlabel, uncontrolled and primarily focused on objective biological responses obtained from the bronchoscopy samples taken. phagocytic ability of am (from bal), apoptosis of bronchial epithelial cells (from bronchial brushing), markers of inflammation in blood, bal and breath condensate (crp, wcc and inflammatory cytokines), health status (st. george's respiratory questionnaire), ecg and lung function were assessed pre and post-administration of azithromycin. results azithromycin significantly improved phagocytic ability of am (by 37%) and reduced bronchial epithelial cell apoptosis (by 34%). antiinflammatory effects of azithromycin included significantly reduced blood wcc and crp. there were non-significant reductions in levels of pro-inflammatory cytokines il-8, il-6 and tnf-a in blood, bal and breath condensate, and a trend for improved health status. conclusions our findings indicate a novel approach to supplement existing therapies in copd that may improve clearance of accumulated apoptotic material and reduce the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation. background the prevalence of gastro-oesophageal reflux disease (gord) across the disease spectrum in copd and bronchiectasis is not well described. the aim of this study was to determine the prevalence of symptomatic and silent gord in copd and bronchiectasis and its effect on lung function and quality of life (qol 4] ) and 18 healthy controls were recruited. the prevalence of gord in bronchiectasis was 33%; 37% in copd; 17% in controls. in copd and bronchiectasis, total nre and ri were increased in those with distal and proximal gord compared to those without gord (all p < 0.05). there was no difference in extent or severity of bronchiectasis in patients with or without gord (all p > 0.05). in copd, the relationship between proximal gord and fev1 was small to moderate (r = 0.383). sgrq symptom scores were higher in patients with bronchiectasis with increased ri (p = 0.02). increased proximal nre was associated with reduced physical (p = 0.03) and mental health (p = 0.02) in the sf-36 in copd. conclusions gord is a co-morbidity in patients with copd and bronchiectasis. the impact of gord on disease severity requires further evaluation. funding source nhmrc, the university of melbourne, monash university, physiotherapy research foundation. chronic obstructive pulmonary disease (copd) is prevalent among older people, however little is known about the influence of ageing on airway inflammation. the aim of this study was to compare airway inflammation in older people with obstructive airway disease to groups of older and younger healthy controls. methods participants (>55 years of age) with stable airway disease and incomplete reversibility (fev1% predicted <80% and fev1/fvc < 70%; copd n = 71) and healthy controls (n = 45, 35 older >55 years and 10 younger <55 years) were recruited from the respiratory ambulatory care clinic or by advertisement. participants underwent a clinical assessment, skin allergy test, hypertonic saline challenge, sputum induction and gas diffusion studies. results participants with copd had moderate airflow obstruction (mean (sd) fev1% predicted 56 (19)) and 45 (63%) were current or ex-smokers with a median (iqr) pack year history of 36 (20-54) pack years. ageing was associated with an increase in airway neutrophils (p = 0.0001). compared to older controls, participants with copd had increased airway eosinophils and lymphopenia (p = 0.004, p = 0.003 respectively), but no difference in airway neutrophils. conclusion airway neutrophilia is a feature of ageing and is not further increased in the presence of copd. copd is associated increased numbers of airway eosinophils with reduced lymphocytes which may impact on the ability of the immune system to combat infection. supported by nhmrc, the university of newcastle. chronic obstructive pulmonary disease (copd) is third leading cause of death and fourth leading cause of disease burden in australia. mechanisms involved in emphysema severity have not been fully understood. micrornas are noncoding rnas that regulate gene expression. we hypothesize that microrna expression differs between emphysema severity in copd patients. methods mirna profiling was performed using 15k agilent human oligo mirna microarrays on total rna extracted from non-tumour lung tissue from 30 copd patients undergoing resection for lung cancer. the mirnas were quantile normalized and anova was used to find differentially expressed genes. results demographic characteristics of the copd patients (mean (sd)) were age 69 (6) years, fev1 72 (17) % predicted and fev1/fvc ratio (<70%). anova identified 31 mirnas that were differentially expressed when stratified into two classes according to kco % predicted > or <75% (t-test, p < 0.05). discussion this mirna analysis has identified mirnas that may be important in emphysema severity in copd patients. further validation will be performed using qrt-pcr and mirna assays on the training set and an independent set, and target prediction and validation. t-helper type 1 (th1) and type 2 (th2) lymphocyte responses have been well recognized as being important pathways in inflammation. recently another form of inflammatory lymphocyte response has been described, the th17 pathway. th17 cells produce cytokines such as il-17a to clear extra-cellular bacteria and fungi and have been implicated in autoimmune and chronic inflammatory diseases. the th17 response in copd is unknown. methods subjects were patients with copd (ex-smokers, fev1 < 70% predicted who had not had an exacerbation for at least 1 month) and control subjects (ex-smokers and normal spirometry). serum samples were obtained for measurement of c reactive protein (crp) and il-17a, the latter measured using enzyme-linked immunosorbent assay (elisa). production of il-17a by t-cell subsets was also identified by intra-cellular cytokine staining and measured by flow cytometry. the mean fev1 of copd subjects was 42 % predicted (6.1 sem, n = 6) and mean fev1 of controls was 112 % predicted (3.0 sem, n = 4). the copd group had a higher mean level of crp 9.5 mg/l (3.9 sem) compared to the control group mean level of 4.6 mg/l (0.6 sem). the mean level of the il-17 in the copd group as measured by elisa was 22.3 pg/ml (16.9 sem, range 0-87) whilst no il-17 was measured in any of the control subjects. conclusions the findings of this pilot study suggest that il-17 may be elevated in association with crp in stable copd. airway obstruction is defined as a fev1/fvc ratio below the lower limit of normal. airway obstruction may prolong the forced expiratory time (fet). method spirometry results from 467 patients were categorized as obstructive, restrictive or normal. the mean, range and coefficient of variation were determined for fet in each diagnostic group. receiver operator characteristic (roc) curves were used to determine if fet could predict a low fev1/fvc. the number of patients with airway obstruction in five fet groups: <9; 9; 10-12; 13-14; and >14 seconds were determined. results the coefficient of variation was high for all groups. pair-wise comparisons showed a difference in mean fet between patients with normal lung function versus those with airway obstruction (p < 0.001). the best cut-point in the roc analysis of 9.895 seconds had a sensitivity of 0.66, specificity 0.77 and area under the curve of 0.743 for predicting obstruction. the technique of skeletal muscle microbiopsy has previously been validated [1] and shown to be minimally invasive and well tolerated in participants with stable copd. aim a study was undertaken to determine the feasibility and tolerability of obtaining microbiopsy muscle samples from the patient admitted for acute exacerbation of copd patient. methods written informed consent was obtained to collect the muscle, blood and sputum samples for research purposes. local anaesthetic was injected prior to the insertion of a 16 gauge bard max core disposable biopsy instrument through the associated guide needle. multiple passes (up to 6) were obtained. the patient was asked to evaluate the experience by rating it on the modified borg scale 0-10. results to date 5 patients and 3 controls have participated in this study. the gold severity ranged from 2-4 and ats exacerbation severity 2-3. the mean age 75 years (range 68-83 years), bmi mean 23.6 kg m -2 (range 17.2-27.1 kg m -2 ) and fat free mass was determined using single frequency bioimpedance. the sample mass obtained ranged from 27.2-104.1 mg, with an increasing yield occurring with increased experience of the operator. the procedure has been well tolerated, the borg scale rating ranged from 1-2/10. all patients were ambulant post procedure; no haematoma or bruising was observed in any of the subjects. conclusion the microbiopsy technique allows the collection of muscle tissue with minimal discomfort to the participant. small tissue masses such as these are sufficient to obtain measures of local markers of wasting and may prove to be a useful adjunct to the collection of sputum and blood for the measure of biomarkers in copd research. introduction older people (op) with obstructive airways disease (oad) experience multiple problems that may impact on their quality of life (qol) and disease management. these problems may relate to pathophysiology, symptoms, self management skills, psychological issues, lifestyle or other problems identified as important by the patient. aim the aim of this study was to determine the frequency of clinical problems associated with oad and to determine if a problem based assessment (pba) could adequately identify these problems. methods a multidimensional assessment tool was developed and the content compared to clinical practice guidelines. participants over 55 years with diagnosed oad underwent this assessment. results sixty-one consecutive patients, aged 59-87 years, with mean (sd) fev1 of 51.4 (17.85) % predicted were assessed. the assessment tool identified a mean (sd) of 3.03 (2.13) current and significant co morbidities with an additional 11 (3.37) clinical problems per patient. qol was increasingly impaired with an increasing number of problems (p < 0.0001). regression modelling identified that the number of identified clinical problems accounted for 55% of the qol impairment. the model demonstrated that every additional patient problem was associated with a clinically significant change in qol impairment (4.22 units) . conclusions op with oad experience multiple clinical problems and co morbidities that adversely impact their qol. a pba of op with oad identifies significant problems that may not be addressed in a diagnosis centred approach. there is a need to identify and effectively manage this array of problems in clinical practice. discussion in this diverse group of copd patients, there was a positive correlation between dlco and fev1, but not kco and fev1. the fev1/ kco plot identifies substantial numbers of patients with the potential ad and e phenotypes defined above. we intend to study inflammatory biomarkers in these groups. fat free mass index (ffmi) is a marker of morbidity and mortality in copd. measurement of ffm in the out-patient population is commonly undertaken using single frequency bioelectrical impedance analysis (bia). however the formulae to convert measured values to ffm are population dependent. schols et al (am j clin nutr, 1991) suggested that formula used for the general population may be inappropriate for patients with copd, and derived a specific formula from total body water (tbw) as measured by deuterium dilution. we compare this method of measuring ffm with 5 others, along with tbw and ffm hydration. methods tbw was measured in 31 outpatients with copd by bia and a difference method (weight-(protein+bone mineral+fat+non-bone mineral+ glycogen)) and ffm hydration was calculated. ffmi was measured by skin fold anthropometry (sfa), bia (3 separate formulae), dual energy x-ray absorptiometry (dexa) and total body potassium by g-counter (tbk). comparison between methods for tbw and ffmi was made by bland-altman analysis and between methods of calculation of ffm hydration by paired t-test. the two methods of assessment of tbw showed little difference (bias -0.04, 95% limits of agreement -5.40 to 5.31). however there was a significant difference in calculation of hydration of ffm (p = 0.0001). sfa, bia (lukaski), bia (tanita) and tbk underestimated ffmi when compared to bia (schols), with bias of -1.24, -3.87, -1.06 and -2.76 respectively. dexa however had a bias of only 0.05 and 95% loa of -3.09 to 3.21. conclusions there are differences between methods of assessment of tbw and ffmi and comparing values between methods must be done with caution. this has implications for assessment of morbidity and mortality in copd. chronic obstructive pulmonary disease (copd) has been identified as a major health problem in australia. recent studies have suggested that respiratory viral infections are the major cause of a worsening of copd; however this has not been studied in australia. aim to characterize pef changes and identify viruses during copd exacerbations. methods a pilot prospective longitudinal cohort study was done. patients had confirmed copd with fev1 <70% predicted and reversibility <10% and/or 200 ml. patients recorded daily peak expiratory flow (pef) measurements and daily chest and cold scores over a period of 2 years. sputum samples and nasal aspirates were taken at 6-month review (control visit) and whenever they had symptoms of an exacerbation (worsening of copd symptoms -seemungal et. al. am j resp crit care med, 2001). nasal aspirates and sputum samples were obtained and analysed by rt-pcr for rhinovirus (rv). result five patients have finished 2 years of study. a total of 12 exacerbations were reported based on patient symptoms. only 3 exacerbations were associated with significant reductions in pef and only one was linked to increases in nasal cold scores. all samples taken at control visits and nasal aspirates and sputum samples during exacerbations were negative for rv by rt-pcr. positive controls confirmed the accuracy of the assay. conclusion our data suggest that a symptom-based definition of copd exacerbation is not always accompanied by significant reductions in lung function parameters. these 'exacerbations' are also not associated with the commonest reported viral cause. our findings suggest that variability of copd may mimic. bronchiectasis is characterized by hypersecretion of mucus and impaired clearance that results in mucus accumulation, chronic cough, sputum production and recurrent infections. inhaled mannitol (400 mg) improves clearance of mucus by increasing the airway hydration and by reducing the viscoelastic and surface properties of mucus. however, the effect of other doses of mannitol on the clearance of mucus in patients with bronchiectasis is unknown. methods fourteen patients, age: 63.3 ϯ 5.7 yr, were studied on 5 visits. clearance of mucus was measured using 99m tc-sulphur colloid and imaging with a gamma camera at baseline and with mannitol ( weight loss and skeletal muscle atrophy are major determinants of morbidity in chronic obstructive pulmonary disease (copd), which are independent of lung function impairment. thus, we examined if a high-fat diet (hfd) protected against the wasting associated with prolonged cigarette smoke exposure (se) in mice. methods male balb/c mice were exposed to the smoke of 4 cigarettes/day, 6 days/week for 7 weeks. sham mice were handled identically without smoke exposure. mice consumed either standard laboratory chow (3.5 kcal/g, consisting of 12 % fat) or a hfd (4.3 kcal/g, 32% consisting of fat). we examined the effect of se and hfd on hind limb skeletal muscles, lung (tissue & bronchoalveolar lavage (balf)) and systemic inflammation in the 4 groups of mice (n = 8/ group). results after 7 weeks of hfd, sham and se mice were 12 and 13% heavier (respectively, p < 0.05) than chow fed animals. conversely, se significantly decreased body weight of chow and hfd fed mice by 16 and 15%, respectively, compared to sham animals (p < 0.05). the hfd did not protect against the decrease in soleus, tibialis anterior and gastrocnemius skeletal muscle weights induced by se (p < 0.05). se altered the mrna expression of a number of genes associated with the regulation of skeletal muscle mass including insulin-like growth factor-i (igf-i), atrogin-1 and interleukin (il)-6. the mrna expression of pro-inflammatory cytokines and chemokines was significantly increased by se in the lung, as were the number of inflammatory cells in balf (p < 0.05). on the other hand, although obesity has been linked to systemic inflammation, the hfd exerted little direct effect on the skeletal muscle and lung parameters measured. se and hfd had no effect on two markers of systemic inflammation, il-6 and serum amyloid a, whereas se tended to reduce circulating igf-i, an anabolic hormone. conclusions the hfd was not protective against the weight loss and skeletal muscle wasting associated with cigarette smoke exposure. supported by the nhmrc and crc for chronic inflammatory diseases. background patients with copd and bronchiectasis undertake airway clearance therapy (act) and exercise as part of physiotherapy management but it is unknown whether these treatments provoke gastro-oesophageal reflux (gor). this study aimed to determine the impact of positive expiratory pressure (pep) therapy and exercise on gastro-oesophageal function. p. aeruginosa is a significant opportunistic lung pathogen in individuals with cystic fibrosis (cf) and is associated with increased lung disease and morbidity. early intervention is beneficial for the effective clearance of p. aeruginosa and better long-term health outcomes. currently, lung flora of cf patients is monitored by regular culturing of sputum, however, children unable to expectorate are limited to annual bronchoalveolar lavages (bal), which is invasive and requires general anaesthesia. saliva is useful for clinical assays as collection is simple, non-invasive. we are developing a standardized enzymelinked immunosorbent assay (elisa) to detect respiratory infection of p. aeruginosa in cf children who cannot expectorate. methods 18 children (7-18 years) with cf and recent p. aeruginosa lung infection history and 16 non cf children (1-6 years) with no previous p. aeruginosa infection history provided saliva as positive, negative controls respectively. saliva was obtained by spitting, or absorbed using cellulose swabs and later extracted. these cell-free supernatant samples were used in an elisa anti-p. aeruginosa iga using commercial antigen. all results were standardized to account for flow using total iga expression. results median value was increased 9 fold in the recent p. aeruginosa lung infection group (mann-whitney test, n = 34, p յ 0.001). there was no significance between mucoid and non mucoid samples, and detection was independent of cfu/ml. discussion early findings support that p. aeruginosa respiratory infection can be detected through specific analysis of salivary iga expression. larger population sampling (30 positive, 90 negative) will aid selection of cut-off values for specificity and sensitivity testing in the future to objectively determine the utility of this assay as a means of monitoring for p. aeruginosa and for determining effectiveness of treatment. medical thoracoscopy is utilized widely throughout europe and northern america by thoracic physicians for the management of pleural disease, including the undiagnosed pleural effusion, malignant effusions and less commonly pneumothorax (ptx). australia has limited experience in this modality. we report the success of medical thoracoscopy in both primary and secondary ptx requiring intervention. methods data were collected from 2001 to 2007 in patients treated with medical thoracoscopy for the treatment of ptx. results 11 patients, 7 male, 4 female. average age 48 (range 19-86). 1 first episode primary spontaneous (ps) ptx, 2 third episodes of ps, 5 first secondary spontaneous (ss), 1 second ssptx, 2 third ssptx. underlying pulmonary disease in secondary ptx included: 4 chronic obstructive pulmonary disease, 1 lymphangioleiomyomatosis, 1 mesothelioma, 1 metastatic angiosarcoma and 1 was secondary to a motor vehicle accident. 7 had a history of smoking, 5 were former smokers and 2 were current smokers, with a mean 24 pack year history (range 5-45). 7 ptx were large, 4 moderate. 5 patients had an intercostal catheter (icc) inserted prior to thoracoscopy, 1 had failed pleural aspirate. there was evidence of bronchopleural fistula in 7 patients prior to the procedure. there was a median of 9 days from ptx to thoracoscopy. light sedation was used for the procedure in 10 patients, 1 required a general anaesthesia with a double lumen endotracheal tube due to anxiety. single port entry, dry talc poudrage and a 16 gauge french icc was used for all procedures. icc was removed a mean of 2 days following thoracoscopy and patients discharged on day 4. pain was the most common complication, requiring narcotic analgesia. one patient died on day 7, secondary to metastatic angiosarcoma. there has been no recurrence of ptx in any patient. conclusion medical thoracoscopy, performed by thoracic physicians is an effective procedure for the treatment of pneumothorax requiring intervention, including selected patients with evidence of bronchopleural fistula. funding nil. conflict of interest nil. nomination for young investigator award no. background lung cancer incidence and mortality are high in tasmania. australia (aihw 2003) 85/100 000 72/100 000 tasmania (cancer registry 2003) 102/100 000 89/100 000 aims and objectives (a) to determine patient demographics in southern tasmania, (b) to determine compliance to identified measures of best practice and (c) assess referral rates, clinical utility and potential delay to positron emission tomography (pet) in a regional setting. methods a prospective database collected information on local clinical practice. cases presented at a multidisciplinary lung cancer meeting over a 12 month period (march 2006 -april 2007 were analysed. data were available for n = 121/161 (75%). results are shown as mean ϯ sd. results 113 primary lung cancer cases were identified. the mean age was 71 ϯ 11 years. 58% of patients were male and 95% were current or ex-smokers. 81% were non-small cell lung cancers (nsclc). tissue diagnosis 93% time from diagnosis to surgery (27 ϯ 15 days) 82% < 42 days macroscopically complete surgical resection (9/11) 82% pet for stage iiib before radical chemoradiotherapy 75% 62% of patients presenting with early or locally advanced disease underwent further staging with pet (n = 34/55). management was changed in 50% of cases (17/34). the average time from pet referral to scan was 11 ϯ 5 days. conclusion a disproportionate number of lung cancers occurred in women. although surgery was performed within recognized timeframes, 2 of 11 patients had incomplete resections. pet influenced management decisions and was performed in a timely fashion. hp chan 1,2 , v tran 1,2 , c lewis 1,3 , p thomas exhaled breath condensate (ebc) is a simple, safe and non-invasive method of sampling breath and has the potential to investigate lung cancer and the associated neoplastic process in the lungs. increased oxidative stress has been implicated in the pathogenesis of lung cancer, and is characterized by elevated hydrogen ions, and hydrogen peroxide (h2o2), which is formed from the conversion of superoxide anions by superoxide dismutase. airway ph has already been shown to be decreased in ebc of patients with other respiratory conditions, but not in lung cancer. therefore the concentration of h2o2 and hydrogen ions in the ebc of lung cancer subjects was compared with matched controls. methods six subjects with newly diagnosed lung cancer were recruited and matched with control subjects: non-smokers, ex-smokers and smokers. ebc was collected and h2o2 was then measured by an assay method based on oxidation of 3,3',5,5'-tetramethybenzidine by horseradish peroxidase and h2o2 while ph was measured using a ph meter. results there was a significant difference (p = 0.033, anova) in h2o2 concentration between the 4 groups with the lung cancer group having elevated mean h2o2 concentration of 23.68 mm (9.15 (sem) compared to the controls: non-smokers, 17.59 mm (6.53 (sem); ex-smokers, 14.35 mm (3.79 (sem); and smokers, 5.21mm (0.69 (sem). ph did not differ significantly (p = 0.659, kruskal-wallis test) between the groups. conclusion these preliminary data suggest that there is significant difference in h2o2 concentration between the groups. the demonstration of an elevated h2o2 level in those with lung cancer indicates an increase in oxidative stress which implies that this may be part of the pathogenesis or response to neoplasia. supported by none. conflict of interest none. pro-inflammatory th1 cytokines produced by t cells and monocytes play an important role in the immune response to malignant cells. however, tumours may escape immune surveillance by inhibiting th1 response and promoting chronic inflammation at the tumour site. methods to investigate the effect of soluble factors released by lung cancer cells on t cell and monocyte pro-and anti-inflammatory cytokines, culture supernatants from several lung cancer cell lines and a normal epithelial cell line (16hbe) were cultured with whole blood for 24 hours, then for a further 16 hrs with and without stimuli. intracellular cytokine / chemokine production was determined using multiparameter flow cytometry. results in stimulated cultures, there was a significant decrease in t cell th1 pro-inflammatory cytokines ifng, tnfa and il-2 and a decrease in monocyte il-1a, il-8, il-12, tnfa, mcp-1 and mcp-3 but an increase in antiinflammatory cytokine il-10 compared with 16hbe and control media. in non-stimulated blood cultures there was an increase in all monocyte inflammatory cytokines / chemokines in the presence of lung cancer supernatants. conclusions lung cancers secrete soluble factors that inhibit the antitumour pro-inflammatory th1 response by t cells and monocytes and upregulate monocyte anti-inflammatory cytokine il-10 following "antigenic challenge". lung cancer cells may also escape immune surveillance by secreting soluble factors that cause newly recruited monocytes to release inflammatory cytokines promoting chronic inflammation at the tumour site. cytotoxic t-cells (ctl's) are important barriers against tumour cells. ctl's induce apoptosis of target cells by mechanisms that include the release of pore-forming perforin and granule associated enzymes, such as granzyme b and granulysin. proteinase inhibitor-9 (pi-9) is the only known granzyme b inhibitor and its expression has been observed in some cancers. we hypothesized that pi-9 would be differentially expressed in lung cancer cells and may inhibit granzyme b-induced apoptosis in these cells. methods we investigated pi-9, granulysin and granzyme b expression in various lung cancer cell lines (1299 ( , 1466 ( , 2009 and normal epithelial cells obtained from bronchial brushing using flow cytometry. peripheral bloodderived t-cells were then incubated with lung cancer cell line supernatants and levels of pi-9, granzyme b and t-cell reactive oxygen species (ros) were assessed. results pi-9 expression was detected in all lung cancer cell lines, (1299 (54.2%), 1466 (90.2%), 2009 (85%), sbc-1 (81%)), at much higher levels than in normal bronchial epithelial cells (8.5%). granzyme b and granulysin levels were undetectable or low in cancer cells (0-9.2%). increased expression of pi-9 and reduced levels of granzyme b were observed in cd8+ t-cells in the presence of all cancer cell supernatants tested (p < 0.05). interestingly, t-cell ros levels were significantly increased in cd8+ t-cells after incubation with cancer cell supernatants (p < 0.05). conclusions high pi-9 expression in lung cancer cells combined with a reduction in t-cell granzyme b expression and enhanced intracellular t-cell ros levels may be a mechanism of immune evasion of lung cancer cells to granzyme b-induced cytotoxicity. immunotherapy for lung malignancies such as lung cancer and mesothelioma is most likely to be successful it it can be combined with conventional tumour debulking approaches such as chemotherapy and surgery. but they scientific basis of such combinations is yet to be determined. to study this we evaluated (1) the capacity of different lung chemotherapy drugs to alter tumour antigen cross-presentation and immunogencity, (2) duration of antigen presentation and responsiveness to immunotherapy after debulking surgery with/without lymphadenectomy, and (3) the pattern of tlr agonism which best synergized with chemotherapy and surgery. we used the ab1-ha murine model of lung malignancy in balb/c mice. results (1) the antimetabolite drugs gemcitabine and pemetrexed were most immunogenic compared to the cytotoxic antibiotics doxorubicin and mitomycin c and the alkylating agent cisplatin. gemcitabine delived large amounts of tumour antigen into the cross-presentation pathway. (2) tumour antigen cross-presentation persisted for only 10 days following resection. the optimal window for immunotherapy following cancer surgery is 1 week for effector ctl stimulation and 2-4 weeks for memory ctl stimulation. (3) the viral-like tlr agonists tlr 3, 7 and 9 were the most effective adjuvant tlr molecules, with tlr 7 agonists generating the strongest systemic anti-tumour responses. conclusion these results help explain previous lung immunotherapy failures and will inform new clinical trials. background mesothelioma is a highly aggressive tumour with an increasing world wide incidence. the serum biomarker mesothelin is elevated in some individuals prior to development of clinical symptoms of the disease and may be useful for screening. we therefore studied the sensitivity and specificity of urinary versus serum levels of mesothelin for mesothelioma patients and evaluated the influence if renal function on the biomarker level. materials and methods concurrent sera and urine samples collected from patients with and control populations. mesothelin concentrations were determined by double-determinant elisa using the mesomark tm assay (fdi, pa). their estimated glomerular filtration rate (egfr) was also calculated. results mesothelin levels correlated between serum and urine samples (pearson's correlation 0.791; p < 0.0001). mesothelin levels were significantly higher in patients with mesothelioma compared to those with asbestosis and/or pleural plaques in serum (4 ϯ 0.9 versus 0.9 ϯ 0.05 nm; p < 0.0001, respectively), in urine (1.9 ϯ 0.5 versus 0.3 ϯ 0.03; p < 0.0001) and in urine following normalization using creatine levels (0.2 ϯ 0.05 versus 0.04 ϯ 0.01). age and egfr were significantly associated with mesothelin levels. conclusion the sensitivity and specificity of mesothelin in urine and in serum were comparable. urine mesothelin may prove to be a useful alternative to serum mesothelin for mass screening of asbestos-exposed individuals. patients undergoing ct coronary angiogram (cta) are often former or current smokers with a high incidence of asymptomatic lung disease. overseas reports show a rate of lung abnormalities ranging from 6.7% to 19%. there are no studies from australia and local factors such as the higher incidence of atypical mycobacteria may influence the rate of benign findings. we are therefore performing a prospective observational study to identify the prevalence and characteristics of incidental lung findings in people undergoing routine cta. methods population: 100 patients undergoing routine cta after informed consent. intervention: radiologist evaluation of lung windows on diagnostic standard workstations. comparator: uncontrolled observational study of consecutive patients. outcomes: primary: prevalence and characteristics of abnormal findings, final diagnosis (clinical judgment, biopsy or long term followup). secondary: number of downstream investigations and costs. results 25 ctas have been studied to date. in 8/25 (32%), abnormalities were noted on lung windows. in 2/25 (8%), there were lung nodules, in 2/25 (8%) there were hilar lymph node abnormalities, in 1/25 (4%), there was hemidiaphragm elevation and in 3/25 (12%) there were pleural plaques (data collection ongoing with study closure expected in february 2008). conclusions preliminary data indicate a substantial number of incidental pulmonary findings from cta; full results will be presented. further analysis is required to determine the impact (benefits, costs and harms) that may result from the concurrent examination of lung windows at routine cta. aim increased levels of nitrogen oxides (nox) and inflammatory markers have been found in bronchoalveolar fluid of lung cancer (lc) patients, but have not been investigated in exhaled breath condensate (ebc).the aim of this study was to compare nox and total protein levels in ebc of lc patients with control subjects. methods ebc was collected during tidal breathing through a glass collection device cooled to 4°c. ebc nox concentrations were measured by a fluorescent modification of the greiss method. total protein in ebc was determined employing the bicinchoninic acid (bca) assay. ebc nox data were log transformed. all data were analysed using anova and expressed as mean ϯ sem. results a total of 88 control subjects and 54 patients with primary lc were recruited. nox and protein concentrations are shown in table 1 . there was no significant difference in ebc nox levels (p > 0.05), but in total protein there was a significant difference between lung cancer patients and all control groups (p = 0.04). conclusion significantly increased ebc total protein levels were found in patients with lung cancer. these data suggest that protein mediator secretion or vascular leak may be present in those with lung cancer. future studies will focus upon the identification of these proteins. methods in this two stage case-control study 446 lung cancer cases and 484 healthy smoker controls were recruited. 180 genetic markers (snps) implicated in lung cancer were screened in our test cohort of 439 smokers and ex-smokers. 30 snps whose genotypes (co-dominant or recessive model) were associated with either the healthy smokers (protective) or lung cancer (susceptibility) phenotype were identified. after genotyping this 30 snp panel in a second cohort of 491 subjects 19 snps were chosen and assigned a simple composite genetic score that was combined with scores for age, history of copd and family history of lung cancer, weighted according to our multivariate regression analysis (n = 930 total subjects). the lung cancer risk score was linearly related to the likelihood of lung cancer with odds ratios (referenced against the lowest score quintile) ranging from 1 to 29 in the highest quintile. on receiver operator curve analyses, the auc was 0.78 and the frequency distribution showed bimodal separation between healthy smokers and lung cancer cases. utility of the score was not affected by effects of age, smoking history or lung function. we suggest that genetic data may be combined with other risk variables to define smokers or ex-smokers at risk of lung cancer for targeted interventions such as smoking cessation and early detection of lung cancer. supported by health research council, nz. conflict of interest yes. tp 144 v aiyappan 1 , a graham 2 1 department of medicine, maroondah hospital, melbourne, australia, and 2 the new disease-modifying anti-rheumatic drug (dmard) leflunomide is being used increasingly to treat inflammatory arthritis. its association with interstitial lung disease needs to be considered before combining it with methotrexate. case report a 73-year-old male who was known to have rheumatoid arthritis and was on methotrexate was admitted with progressive dyspnoea and malaise. he had been recently started on leflunomide. investigations revealed interstitial lung disease and acute renal failure. he improved on conservative treatment (stoppage of disease modifying drugs (dmard), iv fluids and steroids). review of literature an epidemiological study by suissa et al has suggested that there is increased risk of ild associated with leflunomide in patients with a history of ild or methotrexate use but they attributed this to channelling bias. there has also been a report of leflunomide associated with iga glomerulonephritis.by this presentation we aim to increase the awareness of this entity. we also suggest that any patient who is started on combination dmard (i.e. methotrexate and leflunomide) should have a baseline chest x-ray and be monitored for development of interstitial lung disease. conclusion we are reporting the first ever case of interstitial lung disease and glomerulonephritis (in the same patient), due to usage of leflunomide. this entity needs to be thought about in any patient on combination dmards. background bone morphogenic protein receptor ii (bmpr-ii) mutations are associated with pulmonary artery hypertension. failure of the growth inhibitory effects of bmp may contribute to vascular obliteration and remodelling leading to pulmonary artery hypertension (pah) [1] . pah has been observed following venous thrombembolic disease (vte), including pulmonary embolism (pe) and deep venous thrombosis (dvt) [2] . local markers of the pulmonary vascular endothelium rather than traditional markers of thromobophilia are thought to be involved [3] . methods plasma was collected from age and gender matched participants within 24 hours of diagnosis of vte and prior to commencement of warfarin therapy. plasma samples were hybridized to individual human cytokine antibody arrays, to detect protein levels of bmp2, bmp4 and bmpr-ii. results bmp2 and bmp4 levels were higher in patients with dvt than pe. no difference in the bmp level was observed between patients with pe and controls. soluble bmpr-ii receptor was lower in patients with pe than in controls or patients with dvt. conclusion in patients with pulmonary artery stress during the time of a pe the bmpr-ii receptor is reduced, which may predispose patients to vascular remodelling and obliteration. the bmp 2 and 4 levels are reduced at the same time, suggesting a possible overriding regulatory mechanism. the physiological role of bmp's and bmp receptors in patients with vte warrants further investigation. historically, cyclophosphamide has had a variable role in interstitial lung disease (ild), the rationale for its use based on the benefit seen in vasculitis and scleroderma, its rapid effect and low toxicity profile. in patients with severe progressive ild a rapidly effective, well-tolerated agent is desirable. for this reason a treatment protocol for the use of intravenous (iv) cyclophosphamide was implemented at our hospital. aim to review the indications, duration, tolerability and effect of intravenous cyclophosphamide in ild patients following the introduction of a treatment protocol. methods records of 92 patients [dlco was 40 ϯ 15% and fvc 61 ϯ 20%] completing a course of iv cyclophosphamide during 2005-6 were reviewed (excluding patients with systemic sclerosis). data covering 18 months prior to and following treatment were collected. comparative analysis of paired pulmonary function data 6 months before and after treatment was performed. 61% had underlying autoimmune disease. results primary treatment indications included progressive disease(n = 67); severe disease (n = 16); suspected vasculopathy (n = 11); bridging therapy to transplantation (n = 10); and accelerated decline (n = 5). patients received 600 mg/m 2 [mean dose 1152 ϯ 165 mg, median number of pulses 6 (1-12)]. patients with paired pulmonary function data had a difference in median change in dlco% predicted from -15.6% (-95.4 to 29.9%) before treatment to +4.25% (-17.3 to 73.9%) following treatment (p < 0.0001). this remained significant with exclusion of vasculitis, or any autoimmune disease, and independent of prior immunosuppression. therapy was well tolerated (4 withdrew from treatment, 5 deaths within 1yr, none directly related to treatment). conclusion iv cyclophosphamide is well tolerated, and associated with functional stability or improvement in the majority of patients. it remains a viable treatment alternative for consideration. pulmonary hypertension is common in interstitial lung disease (ild) and associated with a poor prognosis. as the gold-standard test, right-heart catheterization (rhc) is invasive, and resource-limited, reliable non-invasive measures of ph are needed. methods all ild patients referred for rhc during 1997-2007 were included (n = 95; 54 male; age 56.5 ϯ 12 yrs). all patients had concurrent echocardiography (tte) and pulmonary function. the relationship of rhc mean pulmonary artery pressure (mpap) to tte variables, pulmonary function, exercise capacity, as measured by six minute walk testing (6mwt, n = 58) and brain natriuretic peptide (bnp, n = 36), was examined. case a 65 year old male, non-smoker for 25 years, retired professor of anatomy (had chronic exposure to embalming fluids, formaldehyde, phenol, antifungal and other solvents, for 20 years) presented with chronic cough and phlegm production. these symptoms were worse at night (waking him several times) and early morning. his pulmonary tests were stopped due to persistent cough. a chest x-ray revealed features of longstanding interstitial lung disease. the hrct revealed widespread subpleural interlobular thickening, worse at bases, in keeping with idiopathic pulmonary fibrosis (ipf). there was minimal fibrosis and honeycombing, but no groundglass opacification, large bullae, pleural calcification or pleural plaques. however, there was associated bronchiectasis at the lung bases considered to be due to traction. the ba lavage showed 50% macrophages, 7% neutrophils, 3% lymphocytes, and 40%, eosinophils and no infection. the patient declined to have a lung biopsy. as per his past x-rays, the duration of his ipf is a little over one year. he maintains that his symptoms started only after starting irbesartan (irb). introduction transbronchial lung biopsy (tbb) has a variable and unpredictable diagnostic yield in sarcoidosis. we hypothesized that the extent and pattern of parenchymal disease on ct would predict the likelihood of a positive tbb. methods data relating to ethnicity, symptoms, pulmonary function and site and results of tbb and bronchoalveolar lavage (bal) from 70 sarcoidosis patients were recorded. all had a ct scan within 6 weeks prior to the tbb procedure. cxr stage was determined from radiology report. ct scans were scored quantitatively for patterns of parenchymal disease (nodular, reticular, consolidation, ground glass and mosaic attenuation) on a lobar basis. results 50% patients had a positive tbb (total 67% of cohort had histological confirmation). symptoms, ethnicity, treatment, lung function and cxr stage were not predictors of a positive biopsy. positive biopsy was associated with higher bal lymphocyte count (p < 0.05) and female gender (p < 0.01). a reticular pattern (p < 0.05) and higher total lung score (excluding da) (p < 0.05) on ct scan predicted a positive biopsy. in those patients with tbb from right lower lobe (53/70) the total rll score on ct was predictive of positive biopsy (p < 0.05). on multivariate analysis gender, bal lymphocytosis and total lung score were independent predictors of a positive tbb (area under roc 0.82). pulmonary arterial hypertension has two histological variants; 'arterial-only pulmonary arterial hypertension' (artpah) and 'pulmonary veno-occlusive disease' (pvod). bosentan, a dual endothelin receptor antagonist, has been found to improve haemodynamics, functional capacity and survival in artpah. however, the response to bosentan in clinically diagnosed artpah is often variable. it was hypothesized that a lack of response to bosentan therapy in clinically diagnosed artpah can be explained by misdiagnosed pvod. aims included to: (1) perform morphometric and qualitative pulmonary vessel analysis on normal controls and cases clinically diagnosed with artpah who had failed bosentan therapy; (2) ascertain if pvod is present within the case group; (3) correlate clinical variables and vessel microanatomy to identify the pathologies driving pulmonary pressure elevation. this study reviewed 14 cases of clinically diagnosed artpah (idiopathic n = 12, associated with scleroderma n = 2), who had failed bosentan therapy and had available lung tissue. controls (n = 6) were obtained from explanted lungs for other causes and a prior transthoracic echocardiogram excluded pulmonary hypertension. vessel morphometry and qualitative analysis was performed with a novel technique of smooth muscle actin immunohistochemistry counterstained with verhoeff's elastin. baseline clinical data were retrieved. we found 86% of cases had pathology confirmed pvod. only 14% of cases had artpah, the original clinical diagnosis. in pvod, significant pathology was present in all vessel types. all vessels had significant smooth muscle hypertrophy. the obstructive, collagenous, pauci-cellular intimal fibrosis of the venules (p < 0.0001) and arterioles (p < 0.0001) was considerably different to the concentric laminar proliferation of smooth muscle observed in the muscular arteries (p < 0.0001) and arterioles (p = 0.001) in artpah. artpah also had muscular artery smooth muscle hypertrophy (p = 0.007). the median time to bosentan failure was shorter in pvod than artpah (290 vs. 657 days). in conclusion, pvod is an under-diagnosed cause of pulmonary hypertension, is commonly clinically misdiagnosed as artpah and may present with a poor bosentan therapy response. finally, pvod is a vaso-occlusive, not a veno-occlusive disease, and is an independent type of pulmonary hypertension, not a subtype of pulmonary arterial hypertension. cutaneous t cell lymphomas (ctcl) are a heterogenous group of lymphoproliferative disorders. they show various clinical manifestations and diverse morphological, histological and immunological characteristics of the malignant cells. they are caused by clonally derived, skin invasive t cells. peripheral t cell lymphomas (ptcl) are generally more aggressive and have one of the lowest overall and failure-free survival rates. because of the rarity of these disorders, diagnosis and treatment remain challenging. this case report describes a 69-year-old woman presenting with progressive dyspnoea and cough, together with a distressing generalized pruritic rash. she was initially treated as left ventricular failure with the rash ascribed to a drug reaction as suggested by initial skin biopsies. the diagnosis was made on a third skin biopsy and flow cytometry of lymphocytes obtained by broncho-alveolar lavage 6 months after presentation. despite an initial response to chemotherapy she succumbed to the disease 20 months after diagnosis. clinical pathways to guide the investigation of suspected pulmonary embolism (pe) have been increasingly adopted by emergency departments (ed) worldwide. compliance with these diagnostic algorithms is critical in ensuring good patient outcomes. this study evaluated the compliance to the clinical pathway used in our ed that combines risk assessment (wells scoring system) with d-dimer test, vq scan or ctpa. the main objectives of this study were to identify those factors which contributed to compliance and to assess patient outcomes. methods a prospective observational study of 239 consecutive patients who underwent investigation for pe in our ed. patient demographics, pathway parameters and patient outcomes at 3-month follow-up were collected. case we report the case of a 37 year old woman who presented to the emergency department with a three day history of dry cough and dyspnoea. the patient was in her third pregnancy at 30 weeks gestation. she had no fever, chest pain or coryzal symptoms. the patient had presented with a right sided spontaneous pneumothorax seven months prior to the current presentation. her past medical history included placental abruption, complicating her previous two pregnancies. her second pregnancy was complicated by placental abruption at 27 weeks and the foetus had not survived. her first pregnancy was complicated by placental abruption at 36 weeks with successful delivery of the foetus. at presentation, significant findings included tachycardia, hypoxemia, tachypnoea and reduced breath sounds over the right side of the chest. chest x-ray demonstrated a large right pneumothorax. a right intercostal catheter was inserted resulting in right lung re-expansion. the catheter was removed three days later. the patient returned to hospital twenty four hours after catheter removal with a recurrent right sided pneumothorax. the patient agreed to surgical intervention involving video-assisted thoracotomy and talc pleurodesis. the patient had no further complications with the pregnancy. she delivered a healthy baby at 38 weeks gestation. discussion spontaneous pneumothorax in pregnancy is rare and there is little evidence to provide guidelines for the management of recurrent pneumothorax in high risk pregnancy. our case illustrates a successful outcome for mother and foetus with surgical intervention at 32 weeks gestation. folfox is currently the standard adjuvant treatment for locally advanced (stage iii) colon cancer and increases disease free survival. its toxicity is well tolerated with common adverse effects being paraesthesia, bone marrow suppression and gastrointestinal disturbance. pulmonary toxicity has rarely been reported. three clinical cases of acute dyspnoea following folfox therapy (2005) (2006) (2007) for stage iii colon cancer are reported. all had an anterior resection followed by 11-12 cycles of folfox. each developed rapidly progressive dyspnoea requiring hospital admission within one week of their last cycle. one patient required invasive ventilation in icu. high resolution computed tomography (hrct) showed bilateral widespread honeycomb pattern with associated ground glass opacification consistent with pulmonary fibrosis. they had reduced lung volumes and gas transfer. transbronchial biopsy and bronchoalveolar lavage in one patient showed an acute eosinophilic pneumonitis. other causes of interstitial lung disease were carefully excluded. all three patients received high dose corticosteroids with one receiving additional cyclophosphamide. the first patient showed complete recovery following an eight week course of corticosteroids, with resolution of the hrct changes and improvement in lung function. the second had symptomatic improvement of dyspnoea, but a persistent moderate reduction in gas transfer. the final patient had persisting radiographic changes and a reduced gas transfer. he remained dependant on ambulatory oxygen 6 months after his initial presentation. these patients' interstitial lung disease appears due to folfox with oxaliplatin being the most likely causative agent. the use of oxaliplatin chemotherapy has increased markedly over the last 3 years and although rare, physicians should be aware of its potential for lung toxicity. lung function testing at baseline, during and towards the end of oxaliplatin treatment should be undertaken and may allow early detection and intervention in cases of pulmonary toxicity. the forced oscillation technique (fot) with broadband signals has been employed relatively rarely in the studies on respiratory mechanics. recent work from our laboratory [1] indicated that the cheek support and the neck angle have minor influence on the impedance spectra around the first antiresonance (far,1), which makes the use of the broadband fot especially attractive in young children. methods we studied 7 healthy children (c; female: 4) and 8 children with bronchopulmonary dysplasia (bpd; female: 3), using multiple-frequency fot between 8 and 256 hz superimposed on spontaneous breathing. results groups c and bpd did not differ in age ( lung function impairment is common in children with cardiac defects associated with increases in pulmonary blood flow/pressure. to investigate the development of bronchial hyperreactivity (bhr), an aorto-caval shunt was created in a model of precapillary pulmonary hypertension. surgical shunt repair was performed to assess the reversibility of bhr. methods 26 rats were divided into 3 groups: group c (n = 10) with sham surgery, group s (n = 8) where an aorto-caval shunt was created (follow-up 4 wks), group r (n = 8) with aorto-caval shunt but surgical correction of the shunt at 4 wks (follow-up 8 wks). in all animals, respiratory input impedance (zrs) was measured at baseline and following increasing doses of methacholine (mch 2, 4, 8, 12 mcg/kg). airway resistance (raw), inertance, tissue damping (g) and elastance were estimated from the zrs spectra by model fitting. measurements were repeated in all animals at 4 wks and at 8 wks for groups r and c. results there was a significant increase in raw and g in group s and rat 4 wks at baseline and following mch ( fig.) which was reversed after surgery. to characterize the factors contributing to lung function impairment following cardiopulmonary bypass (cpb), functional residual capacity (frc), lung clearance index (lci) and respiratory mechanics were measured in children with pulmonary hypoperfusion (tetralogy of fallot, tof n = 12) and hyperperfusion (ventricular septal defect, vsd n = 12) undergoing surgical repair of congenital heart disease. methods frc and lci were measured using a sf6 washout technique and respiratory mechanics using a low frequency oscillation technique in the perioperative period. results while chest opening led to a significant improvement of lung volumes and respiratory mechanics in all patients (p < 0.001), a reduction in pulmonary blood flow during cpb decreased lung volumes and airway resistance in parallel but significantly more in children with tof compared with those with vsd. re-establishing pulmonary blood flow during cpb improved respiratory function particularly in children with tof ( figure) . conclusions sternotomy had a great impact on lung function with parallel improvement in alveolar recruitment, ventilation inhomogeneity and airway resistance. in contrast, onset of cpb led to lung function impairment with a significant drop in frc especially in children with pre-existing hypoperfused lungs. this suggest that pulmonary blood flow enhances alveolar stability through a tethering effect on the alveolar walls. children with advanced lung disease being considered for lung transplantation are likely to spend disproportionately longer periods on transplant waiting lists before appropriately sized donor organs become available. these longer waiting times reflect the lower organ donation rates seen in children; rates that are significantly lower than those reported in the adult population. we describe two children with advanced lung disease who deteriorated whilst on the waiting list for lung transplantation, and in the absence of appropriately sized donor lungs, underwent lobar lung transplantation. methods we describe the clinical course of two children, aged 9 and 13 years old, with advanced lung disease secondary to post-mycoplasma obliterative bronchiolitis and cystic fibrosis-associated bronchiectasis, respectively. results both children received a "cutdown" bilateral lobar transplant from two oversized adult brain-dead organ donors. in both cases the transplant operation involved implantation of the right middle and upper lobes, and of the left upper lobe from the donor. conclusion given the low organ donation rates in children, and in the absence of appropriately sized donor lungs, novel strategies such as lobar transplantation must be considered, particularly when children continue to clinically deteriorate whilst on the lung transplant waiting list. data from the west australian adult outcomes of extreme preterm birth study suggest that adult survivors of bronchopulmonary dysplasia (bpd) may be left with functional and structural pulmonary abnormalities, most notably emphysema. animal data suggest that the antenatal administration of corticosteroids may adversely affect lung development. we therefore sought to determine if maternal variables, including administration of corticosteroid, could predict emphysema severity in adulthood. methods bpd subjects (birthweight < 1500 g and oxygen dependence at 36 weeks post-menstrual age) born prior to 1988 were identified and recruited prospectively via the statewide neonatal follow up program as previously described. pulmonary function tests and thin selective inspiratory and expiratory computerised (ct) images were acquired and scored for emphysema severity (voxel index (%)). the obstetric history was obtained from retrospective review of case notes. results 21 adults (12 females, aged 18-34) were studied, 2 declined ct. all subjects had abnormal ct findings. fifteen (79%) had areas of emphysema. emphysema score and fev1 were not influenced by the administration of antenatal corticosteroids, indication for delivery, maternal age or presence or absence of chorioamnionitis. conclusion maternal factors, including the administration of antenatal corticosteroids, do not predict the long term respiratory outcome of bpd. the factors determining the severity of emphysema in this group remain unknown. the prevalence of childhood asthma is high in the torres strait. children have generally more severe asthma and asthma knowledge is poor. however, there is no culturally appropriate asthma education program for these children. we are conducting a randomized controlled trial to examine the additional benefits of an education intervention by indigenous health care workers (hcw) on asthma outcomes. we describe the study's objectives, design and baseline measurements. methods children with wheeze were reviewed by two paediatric respiratory physicians using a standardized protocol; children with asthma were eligible. after obtaining informed consent children were randomly allocated to: (1) three additional asthma education sessions with a hcw; or (2) no additional education from a hcw. trained hcws carried out the education sessions using culturally appropriate tools. primary outcome was the number of unscheduled hospital/doctor visits due to asthma exacerbation. all children were re-assessed at 12 months. results we enrolled 113 children aged 1 to 17 years, 81% were torres strait islanders and 12% aboriginal and torres strait islanders. the clinical spectrum of asthma was: 51% infrequent episodic asthma, 22% frequent episodic asthma and 27% chronic asthma. eighteen percent of the children knew what a written asthma action plan was; 8.5% had one. carers' assessment of knowledge of medications showed that 52% could not name any asthma medication used by their child, 40% could not explain dosage, and 67% could not explain how beta2 agonists worked. conclusions asthma knowledge and possession of asthma action plans in this cohort is poor at baseline. there is substantial room for improvement and additional asthma education by hcws potentially has significant benefits. impulse oscillometry system (ios) measures respiratory function during normal breathing by transmitting mixed frequency rectangular pressure impulses down the airways and measuring reflected pressure. computer analysis calculates respiratory impedance and its components, airways resistance and reactance, at a range of frequencies from 0.1 hz to 150 hz. no previous australian normative data exists. the ios software generates predictive normal values for each of the parameters measured including total airway resistance (r5), the proximal airway resistance (r20) as well as peripheral capacitive reactance (x5). however, they are based on german data. methods cross-sectional study of 100 community dwelling adults, with 10 males and females per 10-year cohort. inclusion criteria: age range 25-74 years, apparently good respiratory health. exclusion criteria: smokers, asthmatics and others with acute or chronic respiratory disease. both ios and spirometry were conducted on all participants. results australian predictive normal equations have been generated and compared to the current published equations. the ios parameters have been correlated with the spirometric data. results have been analysed by gender, age, height and weight and compared with the predictive normal values for each parameter provided by the german manufacturer of the ios instrument. analysis includes calculation of mean range, and lower limit of normal. conclusions a preliminary set of australian predictive equations have now been produced for the ios. these have been compared with international equations. ios has potential application in a range of respiratory disease states and in population screening for occupational health (e.g. mining, & high dust load environments). supported by phc red. rationale although clinical practice guidelines for both asthma and copd recommend spirometry for diagnosis and monitoring, beneficial effects on the management of chronic respiratory diseases in general practice have not been established. we hypothesized that spirometry would improve health outcomes compared to usual care. methods we are conducting a single masked rct with 3 arms: group a receive 3 monthly spirometry and followup, group b receive spirometry before and after the trial and group c usual care. 45 general practices were recruited though divisions of general practice in melbourne. invitations were mailed by 31 of these practices to patients who had been prescribed inhaled medications during the previous 6 months. participants returned respiratory and generic quality of life questionnaires and an asthma score card. groups a and b were tested on a micromedical turbine spirometer following ats/ers guidelines. results 351 eligible patients (275 adults, 50 children aged 8-13 and 26 youths aged 14-17 years) entered the trial. 122 were randomized to group a, 134 to group b and 95 to group c. the mean (sd) age of adult participants was 54.3 (12.7), children 10.3 (1.7) and youths 15 (1.1) years. there were 130 males and 221 females. the adults were highly symptomatic in the previous 12 months: 82% reporting wheeze, 50% chest tightness on waking, 74% shortness of breath on exertion, 61% nocturnal cough, 46% morning cough and 75% sputum. symptoms of chronic bronchitis were reported by 39% of adults and a diagnosis of copd by 19%. asthma was reported by 84%, confirmed by a doctor in 96% and 55% had experienced an attack in the last 12 months. only 35% had a written asthma action plan. 37% of adults had ever visited a hospital ed and 28% had been admitted. conclusion it is possible to recruit asthma and copd patients from general practice and to randomize them to spirometry or usual care. whether spirometry is associated with fewer symptoms, changes in medication, uptake of action plans or improvement in lung function or quality of life requires further followup. supported by nhmrc. s shah 1 , jk roydhouse 1 , b toelle 2 , s sawyer 3 , c jenkins 2 for the pace australia management committee 1 university of sydney, 2 woolcock institute of medical research, sydney, nsw 2006, and 3 royal children's hospital, melbourne, vic 3052 it is widely held that recruitment of general practitioners for research can be challenging. in this paper, we discuss the recruitment experience from a current study evaluating the impact of an educational asthma intervention on patient outcomes. our aim is to describe the two different strategies utilized to date: (1) in-house through an academic department of gp and (2) outsourced to a private gp organization. methods initial interest was generated through faxes, presentations at gp divisional meetings and newsletter advertisements. gps who expressed interest were visited by project staff to discuss the study further. a major difference was recruiting one gp per practice in the first strategy versus multiple gps per practice in the second strategy. to assess the strategies, we examined participant characteristics, number of gps recruited and number retained. results participant characteristics: under both strategies, 30% of recruits had trained in asia and 54% were women. the first strategy recruited more gps who spoke at least two languages at home (85% vs 42%) and the second strategy recruited more recently graduated gps (58% vs 50%). recruitment: the first strategy recruited 35 gps over 6 months and the second recruited 34 gps over 3 months. retention: 19 gps (54%) from the first strategy stayed in, compared to 29 (85%) from the second. conclusions whilst absolute numbers of gps recruited were similar, retention was much higher under the second strategy. recruitment in primary care is difficult and requires a range of approaches which need to be re-evaluated and adapted as necessary during the course of the study. supported by the australian government department of health and ageing. bronchiectasis is a heterogeneous condition with a large number of causative factors and range of symptoms. the classification of this condition is often confusing and hard to remember. the aim of this study was to classify non-cf bronchiectasis into different clinical phenotypes. methods 178 consecutive patients with non-cf bronchiectasis confirmed on high resolution ct scanning had a detailed clinical, spirometric and laboratory assessment performed by a respiratory physician (pk/mf/pw) and were then followed up for an average of 9 ϯ 4 years (mean and sd) for a total of over 2000 reviews. results 160 of the 178 patients (90%) could be classified as belonging to 3 phenotypic groups; 1) bronchiectasis arising in childhood, 2) bronchiectasis occurring in smokers and 3) bronchiectasis occurring in the elderly. each group had different features which are listed in the there are few data on the long term outcomes of treatment for tuberculosis (tb) by directly observed therapy (dot) in low-incidence settings. the aim of this study was to assess the incidence of recurrent tb in nsw. methods data linkage was performed within the nsw department of health tb notifications database to identify cases that had more than one tb notification between 1994 and 2006. recurrent tuberculosis was defined to include all patients with two or more culture positive episodes at least 6 months apart, where patients had received at least six months treatment for the initial episode. in cases where data contained within the notification details was not sufficient to allow us to distinguish between true cases of recurrent disease, duplication notification for the same episode or persistent disease after incomplete treatment, additional information was obtained from the area tb coordinator. results there were 5723 tb notifications between 1994 and 2006 with 3731 being culture positive. 15 cases of recurrent culture positive disease after completed treatment for the first episode were identified (recurrence rate: 0.4%). conclusions in a population with a low tb incidence, treatment of active tuberculosis with dot results in a very low rate of disease recurrence over a long period of follow-up. support nhmrc ccre in respiratory and sleep medicine. introduction rhinoviruses (rvs) are the major cause of viral-induced exacerbation of asthma. to date, the molecular mechanisms of rv pathogenesis are not understood. recent findings suggest that rv pathology may involve host cell nucleocytoplasmic trafficking, inhibiting key cell functions such as transcription and translation. the study aims to investigate the mechanism of rv 3c protease nuclear trafficking. methods hela cells were infected with rv or transfected with plasmids and cellular localization of 3c analysed at various times thereafter using immunofluorescent confocal microscopy and western blotting with specific antibodies. results 3c protease was predominantly present in nuclei of rv infected cells up to 6 hours after infection, becoming increasingly cytoplasmic thereafter. the nuclear membrane of infected cells became progressively indistinct with time. using a specific inhibitor we also found that 3c utilizes the crm-1 nuclear export pathway. 3c was predominantly in the form of 3cd in both cytoplasm and nucleus of infected cells; mature 3c protease was also detected from 6 hours after infection. deletion analysis indicats that the nuclear localization domain and a nuclear export signal are most likely to be present within the n terminal 64 amino acids. the nuclear export signal is inhibited in the full length protein, via an unknown mechanism. conclusion our data suggest that 3c and 3cd proteins localize to the nucleus in infected cells where they may play a key role in rv pathogenesis by disrupting cellular transcription and the nuclear transport machinery. chronic necrotizing pulmonary aspergillosis (cnpa) is a relatively uncommon, sub-acute, locally destructive process due to aspergillus invasion of the lung. the incidence and prognosis of cnpa are poorly described. case report we present a case of cnpa in a patient on intermittent low dose steroid therapy and recurrent refractory exacerbations of chronic obstructive pulmonary disease (copd).the patient presented with worsening shortness of breath and productive cough requiring recurrent inpatient admissions. human influenza virus is found to bind preferentially to saa2,6gal receptors found in the upper respiratory tract, while avian viruses bind to saa2,3gal receptors expressed in lower airways. this is thought to affect the ability of transmission to humans. our aim was to study the ability of avian and human influenza strains to infect bronchial epithelial cells and relate this to levels of the sialic acid receptor expression. methods calu-3 cells were used as a proximal airway cell and a549 were used as distal airway cell. human primary bronchial epithelial cells (pbecs) were obtained from healthy, asthmatic, and copd volunteers by endobronchial brushing. epithelial cells were stained with sambucus nigra lectin that binds saa2,6gal receptor, and maackia amurensis lectin ii that binds to saa2,3gal. the cells was analysed by flow cytometry. human influenza a/h3n2/wellington strain and low pathogenic avian influenza a/h11n9/sandpiper were chosen and were used at an moi of 0.005 to infect cells. the supernatants were harvested at 48 hr post infection, of which was then analysed by plaque assay for virus replication. results the calu-3 showed greater expression of saa2,6gal linkage than saa2,3gal linkage, and a549 displayed slightly higher expression of both receptors compared to pbecs. despite this human and avian influenza virus replicated to similar titre at 15,000 pfu/ml in both cell lines, but showed low replication in pbecs. background treatment of community-acquired pneumonia remains based on 'best guess' empiric algorithms because of the poor utility of current pathogen tests. furthermore our ability to stratify patients into risk groups is crude at best, relying on scores such as the pneumonia severity index or the curb-65 have major limitations. we have been slowly improving real-time pcr assays for pneumococcus as a clinical tool in patients with pneumonia. methods building on previous research we assesed two targets in the autolysin (lyta) gene and the pneumolysin (ply) gene of s.pneumoniae using the lightcycler instrument and fluorescence resonance energy transfer (fret) probes. all common s. pneumoniae serotypes were detected while other bacteria and viruses were not. the lyta target had the best sensitivity with a detection range between 21 ng to 21 fg. both assays were then applied to whole blood samples from 400 adult patients with community-acquired pneumonia, all of whom had blood cultures prior to antibiotic administration and urinary antigen testing for s.pneumoniae. the lyta pcr had the best performance characteristics with a sensitivity more than twice that of blood cultures in the clinical samples. most pcr+ve/culture -ve patients had positive urinary antigen tests. there was clinical evidence that urinary antigen +ve/ pcr -ve patients were false +ves. most significantly there was a strong correlation between quantitative bacterial count and clinical outcome. conclusions real-time quantitative pcr for pneumococcus has significant potential as both a diagnostic and therapeutic tool in patients with pneumonia. the pitjantjatjara lands are situated in the north-western corner of south australia, occupying an area of over 120 000 square kilometres with a population of approximately 3000. the population lives in small communities or homelands, and there is a high level of mobility between this region and other aboriginal communities in south australia and the northern territory. nganampa health council provides all health care services to the region. specialized support for tb control comes from both the south australia tb service based at royal adelaide hospital as well as a centre for disease control in alice springs. the prevalence of tuberculosis (tb) in this predominantly indigenous community is thought to be significantly higher than the national rate. there are considerable challenges in detecting and managing tuberculosis, relating to the community's geographical remoteness, migration of populations and access to health services. the aims of this study are to quantify the prevalence of tuberculosis in the pitjantjatjara lands, and describe the significant barriers to tb diagnosis and treatment. methods a retrospective study of all diagnoses of tuberculosis within the pitjantjatjara lands in the period 1995-2006. outcomes include measures of tuberculosis diagnosis, the rates of completed tb treatment and rates of tuberculosis drug resistance. the study will draw conclusions about the reasons for high levels of tb prevalence in this community and identify barriers to effective tuberculosis treatment. conflict of interest no. patients admitted to hospital with a diagnosis of community-acquired pneumonia (cap) are usually treated with intravenous (iv) antibiotics irrespective of pneumonia severity. available guidelines vary in recommended timing and indications for switching to oral antibiotics. aim to examine the patterns of antibiotic choice and delivery method (iv, oral and time to switch) in patients admitted with cap. methods a retrospective chart review of admissions to the respiratory unit over a 12-month period with a diagnostic-related group (drg) coding of pneumonia. 41 charts were reviewed. data collected included patient demographics, clinical features at presentation (temperature, pulse rate, respiratory rate, bp, oxygenation), initial investigations, initial antibiotic regime, time to change (iv to oral), subsequent antibiotic regime and duration, time to defervescence, length of stay and outcome. pneumonia severity was calculated using the revised british thoracic society system (curb-65), score ն 2 = severe. results 3 patients were excluded due to incorrect coding. of the 38 patients, age was 50 ϯ 21 (mean ϯ sd) yrs and 25 (66%) were male. 28 patients (74%) were febrile at presentation and the median curb-65 score was 1 (range 0-4). 37 patients (97%) received iv antibiotics. the curb-65 score was 0 or 1 (non-severe) in 25 patients and 22 of these patients received a combination of iv ceftriaxone and a macrolide. time to defervescence was 2.9 ϯ 2.3 days. time from defervescence to switching to oral therapy was 3.4 ϯ 2.8 days. in non-febrile patients, time to switch was 4.7ϯ4.3 days. length of stay was 8.7ϯ13.0 days. conclusions the time between defervescence and switch to an oral regime was relatively long, possibly contributing to an increased length of stay. many patients received ceftriaxone even with a curb-65 severity rating of 0 or 1. implementing local guideline-based treatment protocols may reduce length of stay. ultrasonic flow sensors can determine flow, volume and molar mass (mm) of the gas flow simultaneously. during tidal breathing the expired molar mass curve can be used to compute co2 over expired volume and a capnography index (cpi) can be computed. the relationship between cpi and copd classification according to gold was investigated. methods prospective, controlled trial. consecutive patients who underwent routine lung function were enrolled to participate in a tidal breathing test using an ultrasonic flow sensor. each test consisted of three tidal breathing recordings of 60 sec. flow, volume and molar mass were measured at 200 hz and data were acquired using prototype wbreath data acquisition software. mean expirograms (mm over volume) were computed and the measurements were analyzed to determine the slope of exhaled phase ii (s2), the slope of phase iii (s3) and the relationship between s2 and s3 (cpi = s3/s2). gold stages were determined from the lung function results and the ers predicted values. results 53 volunteers participated in the study with a mean age of 62 (sd 14), 23 were male, mean bmi 26 (sd 5), 17 had never smoked. the mean pack/year smoking history was 38. there was a clear relationship between gold stage and cpi: gold stage 'normal' had a mean cpi of 5.5 (sd 3.7, n = 21), stage 'severe' had a mean cpi of 13.7(sd = 3.9, n = 7). conclusion computation of cpi based on tidal breathing analysis using an ultrasonic flow and mm sensor correlates well with gold stages. it may therefore be possible to use a simple tidal breathing test to determine the severity of airways disease. background osa is common in tetraplegia and appears within weeks of injury. although cpap treatment is efficacious in able-bodied subjects, case series suggest that cpap is poorly tolerated in tetraplegia. no prospective study has examined cpap efficacy or adherence in tetraplegia. aim to determine the feasibility of cpap use to treat osa following acute tetraplegia. methods all acute admissions who consented and fulfilled the inclusion and exclusion criteria underwent full, portable polysomnography. those found to have an apnoea hypopnoea index of >10 events per hour (osa) were offered cpap, delivered via an auto-titrating device. results to date, 25 patients have been admitted (11 excluded, 3 refused consent). no significant, adverse events have been observed. two patients did not have osa. of the nine with osa, four are mid-study, two had incomplete follow-up (1 returned to uk and 1 refused 3 month assessment), two adhered with cpap and one did not due to severe, pre-existing nasal obstruction. preliminary analyses suggest that those who adhered to cpap had a marked reduction (80% compared with 10-40%) in sleepiness and a greater reduction in the functional outcomes of sleepiness compared to either those without osa or who were unable to use cpap. patient accrual, recruitment and completion rates are consistent with our initial estimates. study recruitment will be completed by end-october 2007. conclusion initial data suggest that auto-titrating cpap is a feasible treatment for osa in acute tetraplegia. these data will be used to finalize planning for a multi-national, multi-centre randomized controlled of therapy. this research was supported by the transport accident commission. visual recognition of cyanosis is an important clinical activity. cyanosis recognition is affected by lighting colour and there is anecdotal evidence that people with significant colour vision deficiencies (cvds) have particular difficulty. studies to date have centred on the colour change with oxygenation of isolated blood but it is not clear how this extrapolates to cyanotic patients in vivo. methods ten patients known to be chronically hypoxaemic and showing signs of cyanosis were recruited from the chronic respiratory program. ten normal subjects were recruited as controls. the spectral reflectances of their lips, nail beds and palm creases were measured using a topcon sr-3 telespectroradiometer. the patients were measured at rest and after exercise to lower their saturation by 5-10%. the chromaticities were calculated and plotted. results both groups showed a spread of colours but they fell into two distinct ranges. the colour difference between the groups lies very close to the colour confusions made by congenital cvds. within the cyanosed group, the colour shift was not tightly related to decreasing oxygen saturation. this is most likely due to interpersonal factors such as pigmentation and vascular perfusion that affect colour and the difficulties in measuring the colour of heterogeneous anatomical features. conclusions these results quantify the anecdotal difficulties in detecting cyanosis and suggest that observers with cvd would have problems recognizing the condition. the photographs obtained from this study will be used to compare the ability of subjects with and without cvd to detect cyanosis. supported by the nsw ambulance service. baroreflex sensitivity is depressed in osa patients during sleep but effects during wakefulness are less clear. we have now examined relationships between awake brs and severity of sleep disordered breathing (sdb). methods immediately prior to overnight polysomnography, continuous (5 min) beat-to-beat arterial blood pressure was measured via finger plethysmography (portapres) and heart rate via ecg in 20, supine, normotensive, untreated osa patients (17 males; age: 49 ϯ 15 years (mean ϯ sd); bmi: 26 ϯ 11 kg/m 2 ). spontaneous baroreflex sensitivity (brs) was calculated using the sequence technique. sdb was characterized as apnoea hyponoea index (events/hour) and arousal index (ai). data were analysed via mathematical modelling and unpaired t test. results brs fell with increasing ahi. patients with ahi > 30 events/hour (n = 9) had a significantly lower brs (8.1 ϯ 1.5 ms/mmhg) than those with ahi < 30 events/hour (19.8 ϯ 8.7 ms/mmhg, p < 0.001). brs was negatively related to both ahi and ai via fitted exponential functions (r 2 = 0.45 and 0.70, respectively). it is hypothesized that the analysis of morphology of the ecg waveform in combination with the heart rate patterns could lead to the possibility of detection of the start and duration of apnoea/hypopnoea events and consequently estimation of the apnoea-hypopnoea index (ahi). to the authors' knowledge the published ecg based algorithms for detecting sleep disordered breathing are only capable of minute by minute analysis rather than detection of individual respiratory events. methods changes to ecg parameters were investigated during respiratory events with no distinction made between apnoea and hypopnoea events. 632 isolated respiratory events and 1264 controls of identical duration were obtained from 7 polysomnographic studies, using a randomized procedure. features such as the r wave amplitude, t wave amplitude, qrs area and the r-r interval were extracted from the 2 lead ecg. a number of physiological predictors based on these features were generated. a logistic regression model was used to investigate the association between the predictors and true events, using the statistical software, stata. results univariate and multivariate analyses were performed. three multivariate models were developed; heart parameters only, ecg waveform morphology parameters only and the combinations of the two. the area under the receiver operator characteristic curves (auc) for these models were compared. the best results were obtained with the combination of morphology and heart rate parameters (auc = 0.8858 (0.0078 (sd))) compared to the morphology (auc = 0.8169 (0.0121 (sd))) and heart rate (auc = 0.7195 (0.0103 (sd))) models. the multivariate analysis has shown encouraging results indicating that an algorithm using a combination of heart rate and ecg morphological parameters could potentially be constructed that would enable the determination of individual respiratory events and subsequently an ahi. supported by the arc. introduction sacin and scond are measures of ventilation heterogeneity in acinar and conducting airways, derived from analysis of mbnw. maintaining tidal volumes of 1 l at 9-11 breaths/minute (bpm) is impossible for some. our aim was to examine the effect of different tidal volumes on sacin and scond in normals and asthmatics. methods 10 normals (23-41 yrs) and 12 asthmatics (21-63 yrs) underwent mbnw at tidal volumes of 500 ml at 20-23 bpm, 1 l at 9-11 bpm, and 2 l at 5-7 bpm. scond and sacin, were determined from the normalized phase iii slopes of breaths between turnovers (cumulative ventilation/frc) 1.5 & 6. results the mean ϯ sd %predicted fev1 was 97.3 ϯ 17% in normals and 88 ϯ 11% in asthmatics. in normals, sacin at tv of 0.5, 1 and 2 l were 0.195 ϯ 0.105 l -1 , 0.095 ϯ 0.036 l -1 and 0.058 ϯ 0.031 l -1 , respectively (p = 0.0003, anova), while scond were 0.098 ϯ 0.047 l -1 , 0.042 ϯ 0.021 l -1 and 0.029 ϯ 0.014 l -1 (p = 0.0002), respectively. in asthmatics, sacin were 0.440 ϯ 0.195 l -1 , 0.181 ϯ 0.087 l -1 and 0.100 ϯ 0.047 l -1 , respectively (p < 0.01), while scond were 0.204 ϯ 0.111 l -1 , 0.068 ϯ 0.037 l -1 and 0.031 ϯ 0.013 l -1 , respectively (p < 0.0001). conclusion increasing tidal volume while maintaining the same minute ventilation during mbnw led to large decreases in scond and sacin in both asthmatics and normals. this may be due to reduced inter-regional differences in specific ventilation with greater tv. the log-log relationship between sacin and tv allows an adjustment to be made for variations in tidal volume. funding crc for asthma and airways and nhmrc project grant #547346. dj smith 1 , k bowden 2 , t lloyd 2 , j coucher 2 , l garske 1 1 respiratory medicine, and 2 radiology, princess alexandra hospital, brisbane, australia introduction we have shown diaphragmatic flattening and decreased diaphragmatic excursion qualitatively assessed on ultrasound is strongly predictive of dyspnea severity and lower lung inflation in patients with pleural effusion. we sought to quantitatively measure diaphragm length and movement and determine how closely these are related to dyspnea severity and lung inflation. methods patients with unilateral pleural effusions had ct imaging of their diaphragm during a measured inspiratory capacity manoeuvre. maximal sagittal length was measured at tlc, and frc. patients had a baseline dyspnea index (bdi: 0-12) and respiratory function measured. results 4 patients with unilateral effusion (all right side; 3 malignant mesothelioma, 1 inflammatory) had a mean (sd) bdi of 5.5 (2.89), and tlc of 74% (3.91) predicted. the right diaphragm on the side of the effusion tended to be shorter than the left at frc (p = 0.08), and had a trend to reduced shortening with inspiration (p = 0.08). conclusions the right diaphragm is known to be longer than the left in health. the strong trend to a shorter and less mobile right diaphragm associated with effusion suggests this is a potential mechanism for dyspnea. further recruitment will enable correlation between bdi, tlc and diaphragm length and mobility. 4) ) that was slightly worse than an able bodied, control population (17.9 (3.1)), but better than an able-bodied population with untreated osa (14.5 (3.6)). the mapi predicted that 14% of the sample were likely to have osa. these data will be complimented by full sleep studies to be performed at the participants' homes in late 2007, early 2008. conclusion our interim data suggest that the rate of subjective sleep complaints are not substantially different in the population with tetraplegia compared with the able-bodied. this research was supported by the victorian neurotrauma initiative. it has long been assumed that the ventilation heterogeneity associated with lung disease could in itself affect the measurement of carbon monoxide transfer factor. the aim of this study was to investigate the potential estimation errors of carbon monoxide diffusing capacity (tlco) measurement that are specifically due to conductive ventilation heterogeneity. we induced conductive airway ventilation heterogeneity in 35 never-smoker normal subjects by histamine provocation, and related the resulting changes in ventilation heterogeneity (derived from the multiple breath washout test) to corresponding changes in diffusing capacity, alveolar volume and inspired vital capacity (derived from the single breath tlco method). average conductive ventilation heterogeneity doubled (p < 0.001), while tlco decreased by 6% (p < 0.001), with no correlation between individual data (p > 0.1). when dividing diffusing capacity by alveolar volume, the resulting transfer coefficient was not significantly different pre versus post histamine (p = 0.074). these findings can be brought in agreement with recent modelling work, where specific ventilation heterogeneity resulting from different distributions of either inspired volume or end-expiratory lung volume have been shown to affect tlco estimation errors in opposite ways. the combination of these errors appears to largely cancel out in our experimental situation of induced ventilation heterogeneity comparable to that observed in lung disease. we conclude that conductive ventilation heterogeneity per se has a negligible effect on diffusing capacity measurement. an important determinant of airway function in humans is vagal-mediated cholinergic tone in airway smooth muscle (asm). this airway tone may be altered in disease states. the use of mouse models for the study of airway diseases, including asthma, pulmonary fibrosis and copd is well established. however, it is not known whether mice actually possess basal asm tone or, if it does exist, how this tone changes in disease models. this study was undertaken to determine whether mice have detectable asm tone in vivo. methods respiratory system impedance (zrs) was measured in female adult balb/c mice using a wave-tube modification of the forced oscillation technique. zrs was measured during slow (~35 s) inflation-deflation manoeuvres between the transrespiratory pressures of 0 and 20 cmh2o. baseline lung mechanics and thoracic lung volumes (tgv) were measured before and after each mouse was allocated to one of four treatment groups: 'saline' mice received an i.p injection of saline, 'atropine' mice received i.p. atropine sulphate, 'vagotomy' mice had their left and right cervical vagus nerves isolated by blunt dissection and cut, and 'sham' mice had the area of the vagus nerves exposed but the nerves were not cut. results there were no post-treatment changes in tgv, airway resistance, tissue damping, tissue elastance, inertance or tissue hysteresivity in any of the four groups. conclusions the lack of change in lung mechanics post-atropine or postvagotomy in balb/c mice suggests that, unlike humans and many other species, the airways of mice have no baseline asm tone. supported by nhmrc grant#11488. nomination none. conflict of interest none. both male gender and increased mandibular enclosure volume predict more severe sleep disordered breathing in obstructive sleep apnoea patients. we now examine gender/body size/mandibular enclosure volume relationships for normal subjects stepwise multiple linear regression analysis was used to model body size/enclosure volume interactions. results for the whole group, mv was 261.1 ϯ 6.0 ml (mean ϯ se) while rmv was 205.1 ϯ 4.9 ml. head circumference (positive) and forehead height (negative) were both independent predictors for mv and rmv (both p < 0.02), while hip circumference was an additional positive predictive factor for rmv (p < 0.04). after adjusting for these parameters, male mv and rmv were larger than for females conclusion these findings suggest that mandibular enclosure volumes are relatively larger in males, even after adjusting for body size/cranial dimension. differing body size/mandibular enclosure volume interactions may contribute to gender influences on the severity of sleep disordered breathing. supported by nhmrc of australia nomination john read prize for sleep and physiological research tp 027 audit of ctpa in a regional hospital y raje, s vincent, g simpson department of thoracic medicine, cairns base hospital, cairns, qld 4870 since the introduction of computerized tomographic pulmonary angiograms (ctpa) at our institution the number of requests for this investigation at our institution has grown at an alarming rate. the purpose of this study was to evaluate the clinical assessment of suspected pulmonary embolism (pe). methods 50 ctpa were reviewed. results 31 female, 19 male. mean age 50 yrs (range 21-87). 26 ctpa requests came from department of medicine, 21 from emergency department, 2 from surgical teams and 1 from oncology outpatients. 36 patients presented with chest pain (pleuritic in 20 cases), 25 had dyspnea, 7 presented with collapse. 4 patients had haemoptysis. hypoxaemia was recorded in 7. none were clinically shocked and only one had a recorded tachycardia. d-dimer requested in 10 patients and was elevated in 9. arterial blood gases performed in only 10 patients (20%). 47 patients had prior chest x-ray which was normal in 24 (48%). 8 patients had consolidation on chest x-ray, 2 pleural effusions, 2 atelectasis and 1 fractured ribs. recorded risk factors included 4 patients with previous dvt or pe, 4 patients with malignancy and 6 patients were immediately post-operative. only 6 ctpas (12%) demonstrated evidence of pe. of these 2 had recent dvt and 2 were post-operative. 1 had a history of bowel cancer. there was no formal record of pre-test clinical probability of pe (eg wells' score) for any of the 50 cases. retrospective calculation of the cases of pe, 4 had a wells' score of 4.5 and 1 of 4 with the remaining patient with wells' score of under 2. only 3 patients (one with clinically probable pe) had received fractionated heparin prior to the ctpa. conclusion (1) ctpas performed at our institution have a low yield (12%).(2) pre-investigation clinical assessment was poor and there was poor adherence to published guidelines, (3) this results in many unnecessary ctpa examinations generating increased work and expense for the medical imaging department and exposes many patients to unnecessary and potentially harmful radiation exposure. the evaluation and management of hereditary hemorrhagic telangiectasia involves a multidisciplinary approach according to international guidelines. the aim of this audit was to compare the assessment process in one centre with that of the international recommendations. methods retrospective comparison was made by medical chart review of all patients with a diagnosis of hht between the years 1994 to 2006. demographic along with clinical data with diagnostic investigations, complications, treatment and genetic evaluation, including family screening was collected. the proportion of patients evaluated and managed as per the international recommendations was determined. results the audit identified 26 patients with the diagnosis of hht, with the mean age 58 years. diagnostic criteria were met in 77% of the cohort. of the known clinical features, 54% had a family history, and 81% epistaxis. cutaneous telangiectasia was present in 85% and visceral involvement in 92%. pulmonary arterio-venous malformations (pavm) were seen in 16 patients, cerebral avm in 4, gastrointestinal telangiectasia was documented in 8. one patient had a spinal (cervical) avm, and another had pulmonary hypertension in association with this condition. only 8 patients underwent diagnostic or screening investigations in accordance with the international recommendations. furthermore, one patient was referred for a genetic evaluation. conclusions this clinical audit found that 31% of patients referred to this centre were evaluated in accordance with the international recommendations. genetic assessment was lacking. the study supports the need for a coordinated, multidisciplinary approach to the evaluation and management of hht in this centre. lm young 1 , n good 1 , d milne 2 , w fergusson 1 , i zeng 1 , j kolbe 1 , ml wilsher 1 background while airflow limitation is the most common physiological impairment in sarcoidosis, there are limited data on airway hyperresponsiveness (ahr). understanding the role of ahr in sarcoidosis, if any, may help to identify individuals who might benefit from inhaled therapies. aims (1) to determine the prevalence of ahr in sarcoidosis. (2) to determine the correlation between responses to direct (using histamine) and indirect (using hypertonic saline) bronchial challenge. (3) to determine the clinical, physiological and radiological predictors of ahr. methods subjects with a diagnosis of sarcoidosis based on typical clinical presentation and compatible hrct features and/or tissue biopsy and with a baseline fev1>35% predicted were recruited. subjects underwent standard hypertonic (15% fall in fev1) and histamine (20% fall in fev1) challenge (>1 day but <7 days apart), lung function testing and high resolution computed tomography (hrct) of the chest. results the 52 subjects (48 ϯ 11 years, 35% female, 92% european, 35% stage i, 25% stage ii, 40% stage iii, 0% stage iv) had well preserved lung function overall (fev1 = 2.8l ϯ 0.7.87% predicted). ahr was detected in 5/47 (11%) to hypertonic saline and 19/43 (44%) to histamine challenge. on univariate analysis, response to histamine challenge was predicted by conglomerate fibrosis (p = 0.002) and reticular pattern (p = 0.05) on hrct. the baseline % predicted fev1 was significantly associated with ahr on univariate (p = 0.004), and multivariate analysis (p = 0.01) when adjusted by hrct patterns. conclusions there is a high prevalence of ahr using histamine challenge in this study of sarcoidosis subjects. ahr most strongly associates with baseline % predicted fev1 but also conglomerate fibrosis and reticular pattern on hrct. these findings may reflect the consequence of airway remodelling following inflammation. further studies are warranted to confirm these findings. background upper airway shunt represents a significant source of measurement artefact in the use of the forced oscillation technique (fot), with increasing importance in young children. changes in respiratory system admittance, ars (or zrs -1 ), are theoretically independent of the upper airway shunt. this study examines the possible clinical benefit of ars in preschool children by assessing any increased ability to differentiate responses to bronchial challenges in the routine clinical setting. we hypothesized the use of ars would provide improved sensitivity to clinically relevant obstruction, bronchodilator responsiveness (bdr) and airway hyper-responsiveness (ahr) in young children with respiratory disease. method previous fot measurements were re-analysed and ars calculated to derive: (1) ars reference equations in healthy young children (n = 158); (2) bdr in ars, respiratory system resistance (rrs) and reactance (xrs) in healthy children (n = 78), children with cystic fibrosis (n = 39), neonatal chronic lung disease (n = 49), asthma (n = 56) and wheeze (n = 66); (3) ahr to inhaled adenosine-5′-monosphate (amp) in 19 children. fisher's exact tests were used to assess changes in diagnostic outcomes between ars and conventional fot outcomes (rrs and xrs). results ars was no more sensitive to bronchodilator induced changes than conventional fot outcomes. amp challenges resulted in equivalent responses measured by relative changes in rrs and ars while absolute changes in ars were the least sensitive variable. conclusion this study does not support a clinical advantage in using ars in measuring responses to either inhaled bronchodilator or amp. c hollier 1,2 , c menadue 1,2 , d flunt 1,2 , aj piper 1,2 1 department of respiratory and sleep medicine, royal prince alfred hospital, nsw 2050, and 2 woolcock institute of medical research, nsw 2050 serial measurement of arterial carbon dioxide (paco2), ph and bicarbonate (hco3 -) is essential in the management of patients with hypercapnic respiratory failure (hrf). this information is usually obtained from a sample of arterial blood (abg). the procedure can be painful and distressing for patients, and is sometimes technically difficult due to obesity or contractures. our aim was to determine the validity and feasibility of arterialized venous blood (av) sampling as an alternative to abgs in measuring paco2, ph and hco3levels in patients with chronic hrf. method eighteen patients completed the study. venous blood was arterialized by heating forearm skin to a temperature of 42-45°c with an electric heating pad. an av sample was taken from a cannula positioned in a vein of the heated forearm simultaneously with an abg. in addition, the reliability of av sampling within the recommended temperature range (42-45°c) was investigated in ten healthy volunteers placed on volume cycled ventilation in order to maintain constant ventilation. av samples were taken at 0.5°c temperature intervals from 42.5-45°c results the table below summarizes results for validation of av sampling: based on the evidence that cardiovascular dynamics are altered due to obstructive sleep apnea, this study aims to identify the onset and termination of each apnea event using power spectral density (psd) and morphological features of single lead ecg signal over 5 second period. methods ecgs from 4 patients overnight sleep studies were examined for location of the pre-scored apnea events. onset (n = 1995), maximum (n = 6751) and termination (n = 1996) of each apnea event and normal events (n = 11219) were annotated on 5 second windows. features extracted were psd, amplitudes of r and t wave of 5 second ecgs. receiver operating characteristics (roc) analysis was used to gauge the event recognition ability of all features. weight loss causes an improvement in the severity of osa, however substantial weight loss is very difficult for obese patients. the very low caloric diet (vlcd) has been shown to be successful in causing significant weight loss in obese patients. this is a pilot study on the use of a formal screening protocol to identify osa patients who are potentially eligible for the supervised vlcd program offered by the endocrinology department at auckland city hospital. method 344 consecutive patients who attended the sleep laboratory at ach between june to december 2006 were screened using the protocol. patients who are eligible to be considered for the vlcd program are identified as having a combination of obesity (bmi > 30), osa (ahi > 5 on sleep study) and being residents within the auckland district healthboard region. results 243/ 344 patients screened did not fulfil the inclusion criteria: 171 lived outside the adhb region; 71 had bmi < 30; 7 patients did not have osa (ahi < 5). 101 patients fulfilled the inclusion criteria. 54/101 patients (54%) were excluded due to medical or psychiatric contraindications to vlcd.47 patients (47%) who did not have contraindications to vlcd were contacted. 33 patients were contacted successfully. 14 patients were either unavailable to phone contacts on 3 separate days or were disconnected. 12/101 patients consented to being referred (12%). 21/101 patients declined referral (21%). conclusion this pilot study is the first study using a formal comprehensive screening protocol in the recruitment of obese osa patients into a medically supervised vlcd program. only a small proportion (12%) of patients proceeded to being referred to the vlcd program. key: cord-006289-2k8c22u8 authors: chu, shi-jye; huang, kun-lun; wu, shu-yu; ko, fu-chang; wu, geng-chin; li, rui-ying; li, min-hui title: systemic administration of fc-77 dampens ischemia–reperfusion-induced acute lung injury in rats date: 2013-06-27 journal: inflammation doi: 10.1007/s10753-013-9678-z sha: doc_id: 6289 cord_uid: 2k8c22u8 systemic administration of perfluorocarbons (pfcs) reportedly attenuates acute lung injury induced by acid aspiration and phorbol myristate acetate. however, the effects of pfcs on ischemia–reperfusion (ir)-induced lung injury have not been investigated. typical acute lung injury was induced in rats by 60 min of ischemia and 60 min of reperfusion in isolated and perfused rat lung model. rat lungs were randomly assigned to receive pbs (control), 1 % fc-77, ir only, or ir with different doses of fc-77 (0.1 %, 0.5 %, or 1 %). subsequently, bronchoalveolar lavage fluid (balf), perfusate, and lung tissues were collected to evaluate the degree of lung injury. ir caused a significant increase in the following parameters: pulmonary arterial pressure, capillary filtration coefficient, lung weight gain, lung weight/body weight ratio, wet/dry lung weight ratio, and protein concentration in balf. tnf-α and cytokine-induced neutrophil chemoattractant-1 concentrations in perfusate samples and mda concentration and mpo activities in lung tissues were also significantly increased. histopathology showed increased septal thickness and neutrophil infiltration in the lung tissues. furthermore, nf-κb activity was significantly increased in the lungs. however, pretreatment with 1 % fc-77 prior to ir significantly attenuated the increases in these parameters. in conclusion, our results suggest that systemic fc-77 administration had a protective effect on ir-induced acute lung injury. these protective mechanisms may have been mediated by the inhibition of nf-κb activation and attenuation of subsequent inflammatory response. perfluorocarbons (pfcs) are inert chemicals having high density and good oxygen-dissolving capacity; moreover, they are highly hydrophobic and lipophobic [1] . because of these advantages, liquid ventilation with pfcs was initially shown to improve acute lung injury in animal models and a few human studies. important mechanisms that attenuate lung inflammation include the distention of collapsed alveoli to improve lung compliance, improvement of oxygenation by increasing oxygen diffusion, and removal of inflammatory mediators from the alveolar regions of the lung [2] [3] [4] . however, two subsequent large clinical trials of patients with acute respiratory distress syndrome (ards) showed that liquid ventilation was not beneficial for improving survival [5, 6] . ischemia-reperfusion (ir) lung injury is a common complication of lung transplantation procedures, pulmonary embolism, shock, and trauma, and it results in increased microvascular permeability and neutrophil infiltration in the lung tissue, damaged pulmonary endothelium, and pulmonary hypertension [7, 8] . despite advances in intensive care, ards-associated morbidity and mortality remain high [7, 8] . therefore, it is important to develop new strategies to treat these potentially reversible pulmonary injuries. the results of a number of recent in vitro and in vivo studies have suggested that pfcs have anti-inflammatory effects that may directly modulate inflammatory cell function and decrease the production of inflammatory mediators [9] [10] [11] [12] . emulsified pfcs can be safely injected into the blood system of humans as a blood substitute [13] . emulsified pfcs have also been used as part of management of a variety of diseases, including polytrauma, anemia, burns, hemorrhagic shock, toxic infectious shock, and blood vessel occlusion [14] . during the early stage of acute lung injury, the origin of activated neutrophils is intravascular and not intra-alveolar; therefore, intravascularly administered pfcs will make direct contact with inflammatory cells and may be a more potent means to decrease harmful inflammatory reactions. in addition, systemic fc-77 administration has been shown to attenuate acute lung injury induced by acid aspiration and phorbol myristate acetate (pma) [15, 16] . although pfcs reportedly limit the infarction size in ir heart injury [17, 18] , the beneficial effects of fc-77 on ir-induced acute lung injury remain unclear. therefore, the present study was designed to determine whether systemic fc-77 administration ameliorates acute lung injury induced by ir. in this experiment, we used a well-established model of isolated rat lung because it enabled us to measure the pulmonary filtration coefficient (k f ), which is the most accurate indicator of pulmonary capillary permeability [19] . we prepared the isolated and perfused rat lungs in situ as previously described [20, 21] . the animals used for this study were cared for in accordance with the guidelines of the national institutes of health (national academy press, 1996) , and approval for our study protocol was obtained from the national science council and animal review committee of the national defense medical center. male sprague-dawley rats (weight 250-350 g) were anesthetized with intraperitoneal injections of pentobarbital sodium (20-25 mg/rat). tracheostomy was performed to enable ventilation with a rodent ventilator (7025; ugo basile, comerio, va, italy). the rat lungs were ventilated with 5 % co 2 in air at 65-70 breaths/minute with a tidal volume of 2 ml. after a median sternotomy was performed, heparin (1 u/g of body weight) was injected into the right ventricle, from which 10 ml of blood was collected. this blood sample was mixed with 10 ml of normal saline containing 1.5 % human serum albumin. this was subsequently used as a perfusing fluid for the isolated lungs. for constant-flow perfusion of the isolated lungs, a cannula was inserted into the pulmonary artery via a right-ventricular puncture. a tight ligature was placed around the main trunk of the pulmonary artery. a widebore cannula was inserted into the left atrium via the left ventricle to divert pulmonary venous outflow into a reservoir. the wide-bore cannula was then fixed with a ligature at the apex of the heart. another ligature was placed above the atrioventricular junction to prevent the flow of the perfusate into the ventricles. both the pulmonary arterial pressure (pap) and the pulmonary venous pressure (pvp) were recorded from side arms of the inflow and outflow cannula. a roller pump was used to provide constant perfusion flow at a rate of approximately 8-10 ml/min to stabilize pap at 15-20 cmh 2 o. pvp was set at 4-6 cmh 2 o by adjusting the height of the venous reservoir. with the isolated perfused lungs remaining in situ, the whole rat was placed on an electronic balance. the digital signals of the electronic balance were converted to analog signals with a digital-to-analog converter and were recorded on an oscillograph recorder. isolated lung preparations were used in this study only if they satisfied three criteria: (1) no leakage at the sites of cannula insertion, (2) no evidence of bleeding or edema, and (3) an isogravimetric state. the lungs were then subjected to 60 min of ischemia by stopping ventilation and perfusion. after this period of ischemia, the lungs were reperfused for 60 min and ventilated with 5 % co 2 -95 % air. k f , which is an index of microvascular permeability to water, was determined from the lung weight change caused by increased venous pressure. during ventilation and lung perfusion, pvp was rapidly elevated by 10 cmh 2 o for at least 7 min. the slow, steady phase of weight gain as a function of time (δw/δt) was plotted on a semi-logarithmic paper. the slow component was then extrapolated to zero time to obtain the initial rate of transcapillary filtration. from this plot, k f was defined as the y-intercept (g min −1 ) divided by pvp (10 cmh 2 o) and lung weight. k f was expressed in units of grams per minute of cmh 2 o −1 × 100 g [20, 21] . after experiments, the right lung was removed from the hilar region and the wet weight was determined to calculate the lw/bw ratio. a part of the right upper lung lobe was placed in an oven at 60°c for 48 h to determine the w/d lung weight ratio. balf was obtained at the end of the experimental by irrigating the left lung with saline (2 × 2.5 ml). this fluid was centrifuged at 250×g for 10 min, and the protein concentration in the supernatant was determined using a bca protein assay (pierce, rockford, il, usa). tnf-α and cinc-1 concentrations in the perfusate after the experiment were determined using an elisa kit (r&d systems inc., minneapolis, mn, usa) according to the manufacturer's instructions. lung tissue was homogenized in a 1.15 % kcl solution. an aliquot (100 μl) of the homogenate was added to a reaction mixture containing 200 μl of 8.1 % thiobarbituric acid and 700 μl of distilled water. samples were then boiled for 30 min at 100°c and centrifuged at 3,000×g for 10 min. the absorbance of the supernatant was measured spectrophotometrically at 532 nm. a part of the right lower lung lobe was freezethawed and sonicated three times. homogenates were centrifuged at 15,000×g for 10 min at 4°c. an aliquot (100 μl) of the supernatant was mixed with 900 μl of 50 mm phosphate buffer (ph 6.0) containing 0.167 mg/ ml of o-dianisidine dihydrochloride and 0.0005 % hydrogen peroxide. one unit of peroxidase activity was defined as the amount of enzyme that decomposed 1 μmol of hydrogen peroxide per minute at 25°c. hydrogen peroxide decomposition was determined from the oxidation of o-dianisidine using an absorption coefficient of 11.3 mm cm at 460 nm. nf-κb activity was assessed by the nuclear translocation and dna binding of the p65 subunit in lung tissues, using a commercially available elisa kit (transam nf-κb p65; active motif, carlsbad, ca, usa) according to the manufacturer's instructions. a part of the right lower lung lobe was stained with hematoxylin and eosin. the histopathologic assessment was performed by two pathologists blinded to the experimental condition. for each section, 10 random areas were examined at a magnification of ×400. within each field, lung injury was scored according to (1) infiltration or aggregation of neutrophils in the airspace or vessel wall, and (2) thickness of the alveolar wall. each assessment was graded on the following four-point scale: 0, 1, 2, or 3, for no, mild, moderate, or severe injury, respectively. the resulting two scores were added and presented as the lung injury score for that section [22] . an isolated lung preparation was allowed to equilibrate for 20 min. we recorded baseline pap, pvp, and weight change and determined the initial k f for 7 min. then, all parameters were allowed to return to their baseline values for 10 min. rat lungs were randomly assigned to receive pbs (control, n=6), 1 % fc-77 (drug control, n=6), ir only, or ir with different doses of fc-77 (0.1 %, 0.5 %, or 1 %; n=6/per group). fc-77 (chemical formula, c8f18; purity, 100 %; 3m company, st. paul, mn, usa) was added to the reservoir (containing 20 ml of perfusate). then the lungs were perfused and ventilated for 60 min following ir, and the k f measurement was repeated. due to the hydrophobic property of fc-77, fc-77 was premixed with 3 ml of perfusate (drawn from the reservoir) and vortexed for several seconds before adding to the reservoir. results are expressed as mean±sem. group comparisons were made by repeated measures one-way or twoway anova, followed by post hoc comparisons using a newman-keuls test. comparisons within each group for a given variable were made by paired student's t tests. a p value of <0.05 was considered statistically significant. pap was significantly increased in the ir group than in the control group (fig. 1a) . pretreatment with fc-77 at different concentrations tended to attenuate the increase in pap induced by ir; however, the attenuation was significant only in the 1 % fc-77 group when compared with that in the ir group (p<0.05). figure 1b shows the microvascular permeability changes (expressed as k f ) in the isolated rat lungs due to ir at different doses of fc-77. k f (p<0.05) significantly increased after 120 min of ir, whereas k f did not change after 120 min of perfusion in the control group. pretreatment with fc-77 at different concentrations tended to attenuate the increase in k f ; however, significant attenuation was seen only in the 1 % fc-77 group when compared with that in the ir group (p<0.05). the lung weights in the control group remained essentially constant during the 120-min experimental period (fig. 2) . in contrast, ir caused a progressive increase in lung weight. the lung weight change expressed as the lung weight gain was significantly higher in the ir group than in the control group (p<0.05). the decrease in lung weight gain was statistically significant in the 1 % fc-77 (p<0.05) group, but not in the 0.1 % or 0.5 % fc-77 groups (p>0.05), when compared with that in the ir group. the lw/bw and w/d ratios were significantly increased in the ir group compared with those in the control group (fig. 3, p<0.05) . these ratios tended to decrease in the 0.1 % and 0.5 % fc-77 groups, albeit with no statistical significance when compared with those in the ir group. however, the lw/bw and w/d ratios significantly decreased in the 1 % fc-77group compared with those in the ir group (p<0.05). protein concentration in balf was significantly higher in the ir group than in the control group (fig. 4 , p<0.05). a significant protective effect was observed in the 0.5 % and 1 % fc-77 groups (p<0.05), but not in the 0.1 % fc-77 group (p>0.05), when compared with the ir group. tnf-α and cinc-1 concentrations in perfusates significantly increased in the ir group compared with those in the control group (p<0.05). although pretreatment with 0.5 % fc-77 tended to attenuate this increase, the attenuation was not statistically significant when compared with that in the ir group. on the other hand, fig. 1 . effects of fc-77 pre-treatment on pulmonary hypertension and the pulmonary vasculature filtration coefficient (k f ). a ischemia-reperfusion (ir) caused a significant increase in pulmonary arterial pressure (δpap). pretreatment with 1 % fc-77 significantly attenuated the increase in pap after ir. b ir significantly increased k f compared with that at baseline, although k f did not change after 120 min of perfusion in the control group. the increase in k f was significantly attenuated in the 1 % fc-77 group compared with that in the ir group. *p<0.05 versus control group; # p<0.05 versus ir group. open squares, baseline; filled squares, 60 min. the attenuation was significant in the 1 % fc-77 group when compared with that in the ir group (p<0.05; fig. 5 ). as shown in fig. 6 , mda concentration and mpo activity in lung tissues significantly increased in the ir group compared with those in the control group (p< 0.05). pretreatment with 0.1 % and 0.5 % fc-77 tended to attenuate these increases, although the attenuation was not statistically significant when compared with that in the ir group. however, mda concentration and mpo activity were significantly attenuated in the 1 % fc-77 group compared with those in the ir group (p<0.05). nf-κb activity in the lungs was significantly increased after ir (fig. 7) . nf-κb activity in the lungs tended to decrease in the 0.1 % and 0.5/% fc-77 groups compared with that in the ir group, although the difference was not statistically significant. however, the attenuation was significant in the 1 % fc-77 group when compared with that in the ir group (p<0.05). the control group showed normal lung tissue architecture and no inflammation (fig. 8a) , whereas the ir group exhibited septal thickening and marked inflam-matory cell infiltration in the interstitium and alveoli (fig. 8b) . lung inflammation was significantly lower in the 1 % fc-77 group than in the ir group (fig. 8c) . lung injury scores also provided evidence that pretreatment with 1 % fc-77 significantly attenuated ir-induced acute lung injury ( fig. 8d; p<0.05 ). in this study, we demonstrated that pretreatment with fc-77 had beneficial effects on ir-induced increases in pap, k f , lung weight gain, lw/bw ratio, w/d lung ratio, protein concentration in balf, and tnf-α and cinc-1 concentrations in perfusate; and mda concentration, mpo activity, and neutrophil infiltration in lung tissues. in addition, fc-77 pretreatment also inhibited nf-κb activation in the lungs. the protective effects of fc-77 may be mediated by the inhibition of nf-κb activation and attenuation of inflammatory responses in the lungs. the major characteristic of ir-induced acute lung endothelial injury is an increase in pulmonary microvascular permeability. in this study, we demonstrated that fc-77 pretreatment could attenuate the increase in vascular permeability on the basis of evaluations of different variables, including k f , w/d lung ratio, lw/ bw ratio, and protein concentration in balf. these findings were consistent with those of previous reports where pfcs attenuated endothelial injury in acid and pma-induced acute lung injury [15, 16] . studies of ir-induced lung injury in animal models have confirmed that sequestration of neutrophils plays an important role in ir-induced lung injury [23] . activation of neutrophils and their adhesion to endothelial cells are observed during the initial inflammatory response. activated neutrophils that infiltrate the lung release reactive oxygen species, proteases, cytokines, and vasoconstricting lipids; upregulate the expression of adhesion molecules; and subsequently cause lung injury [23] . an in vitro study showed that pfcs could diffuse from the alveolar space into an adjacent pulmonary vascular endothelial layer that modulated neutrophil adhesion and decreased the number of activated neutrophils entering the injured lung [24] . however, whether pfcs had the same effects in vivo needed to be confirmed. a previous in vitro study reported that pfcs decreased the production of hydrogen peroxide and superoxide anion by rabbit and piglet alveolar macrophages after chemical stimulation [12] . steinhorn et al. also demonstrated that pfcs attenuated oleic acid-induced lung injury by reducing the production of reactive oxygen species [25] . pfcs have also been shown to inhibit neutrophil activation and chemotaxis via decreased syk phosphorylation [26, 27] . furthermore, systemic administration of pfcs can present a barrier function and prevent direct contact between neutrophils and endothelial cells in vessels [17] . in this study, these characteristics may have contributed to the capability of systemic fc-77 to attenuate mpo activity and lipid peroxidation in the lung and decrease the intensity of the inflammatory response. these results are in agreement with recent findings of decreased neutrophil accumulation, mpo activity, and oxidative damage in lung tissues by systemic pfc administration in a rat lung injury model [15] . tnf-α and il-8 are early response mediators in the pathophysiology of acute lung injury and ards [28] . previous investigations reported that anti-tnf-α or il-8 antibodies significantly attenuated ir-induced acute lung injury [29, 30] . pfcs can reduce the production of various inflammatory cytokines in vitro and in vivo, such as tnf-α and il-8 [9, 11, [31] [32] [33] [34] . in this study, the decreased pulmonary inflammation may have been caused, in part, by decreased early production of tnfα and il-8. nf-κb is a key transcription factor that is activated by a number of stimuli, including hypoxia, ischemia, inflammatory cytokines, chemokines, and oxygen radicals [35] . in addition, the promoter regions of many cytokines such as tnf-α and il-8 are controlled by nf-κb. nf-κb activation amplifies an early inflammatory response and exacerbates tissue injury [35] . previous studies demonstrated that inhibition of nf-κb activity decreased lung reperfusion injury in porcine and rabbit models [36, 37] . the inhibition was associated with the suppression of cytokine production and neutrophil infiltration [37] . pfcs have also been shown to inhibit nf-κb activation in lps-stimulated macrophages, chlamydophila pneumoniae-mediated pneumocytes, and models of respiratory syncytial virus-induced lung inflammation [15, 34, 38] . therefore, our results are compatible with those studies. although the anti-inflammatory mechanisms of pfcs are not clear, most investigators initially considered that pfcs acted as a physical barrier on cell surfaces to prevent ligand-receptor-induced signal transduction and direct injury caused by inflammatory mediators [2] [3] [4] . further investigations revealed that pfc particles were ingested early into phagocytic cells in a time-dependent manner, thus resulted in the formation of pfc-filled vacuoles [39] . pfcs were also reportedly incorporated into the cellular fig. 8 . histological appearance of lung tissue. a control group (×400). b ischemia-reperfusion (ir) (×400). c 1 % fc-77 administration prior to ir (×400). pretreatment with 1 % fc-77 significantly improved the lung pathology compared with that in the ir group. d histopathological scores for lung injury. the lung injury scores were significantly decreased in the 1 % fc-77 group compared with those in the ir group. *p<0.05 versus control group; # p<0.05 versus ir group. membranes of alveolar epithelial cells and erythrocytes; this stabilized the cellular membrane and prevented surface receptor activation [34, [40] [41] [42] . however, other important inflammatory pathways may be involved in the protective mechanisms of pfcs, and further investigations are required to clarify these pathways. under ischemic conditions, oxidative metabolism is impaired and atp stores in the lung are rapidly depleted. the high oxygen solubility of pfcs provides a higher partial pressure of oxygen in the microcirculation, thereby enhancing the oxygen flux into tissues. it has been reported that pfcs are effective for preventing ischemic heart and brain injuries by enhancing ischemic myocardial and cerebral oxygen concentration [43, 44] . however, it is not clear if pfcs could oxygenate the ischemic rat lungs in this study. additional studies are warranted to clarify this. this study had several limitations. first, fc-77 was chosen because it is widely available. however, different pfcs have various pharmacological properties, and newgeneration pfcs may be more effective in attenuating lung inflammation. second, our observation period was 1 h; therefore, the long-term effects of pfcs on ir remained unknown. studies with a longer experimental time will be necessary before extrapolating our results to the clinic. finally, although isolated lung models are widely used to investigate various physiological phenomena, the influence of extrapulmonary organs was not tested. additional studies in intact whole animals to explore these effects are necessary. in summary, we demonstrated that rats treated with fc-77 prior to the induction of ischemia had attenuated ir-induced lung injury. the observed decrease in lung damage may have been mediated by the inhibition of nf-κb activity, inflammatory reactions, and neutrophil infiltration into the lung tissues. systemic pfc administration is a new therapeutic approach. our results provide evidence for the potential of prophylactic therapy with systemic pfcs to prevent reperfusion injury in lung transplantation. understanding the fundamentals of perfluorocarbons and perfluorocarbon emulsions relevant to in vivo oxygen delivery initial experience with partial liquid ventilation in adult patients with the acute respiratory distress syndrome perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome effect of partial liquid ventilation on pulmonary vascular permeability and edema after experimental acute lung injury prospective, randomized, controlled pilot study of partial liquid ventilation in adult acute respiratory distress syndrome partial liquid ventilation in adult patients with acute respiratory distress syndrome ischemia-reperfusion-induced lung injury acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management inhibition of inflammatory responses by fc-77, a perfluorochemical, in lipopolysaccharide-treated raw 264.7 macrophages partial liquid ventilation decreases serum tumor necrosis factor-alpha concentrations in a rat acid aspiration lung injury model perfluorocarbon suppresses lipopolysaccharide-and alpha-toxininduced interleukin-8 release from alveolar epithelial cells a liquid perfluorochemical decreases the in vitro production of reactive oxygen species by alveolar macrophages engineering blood: synthetic substitutes from fluorinated compounds clinical results of perftoran application: present and future intravascular fc-77 attenuates phorbol myristate acetate-induced acute lung injury in isolated rat lungs systemic perfluorocarbons suppress the acute lung inflammation after gastric acid aspiration in rats limitation of myocardial reperfusion injury by intravenous perfluorochemicals. role of neutrophil activation dose-dependent reduction of myocardial infarct size with the perfluorochemical fluosol-da evaluation of lung injury in rats and mice fructose-1,6-diphosphate attenuates acute lung injury induced by ischemia-reperfusion in rats baicalin attenuates air embolism-induced acute lung injury in rat isolated lungs hypercapnic acidosis attenuates reperfusion injury in isolated and perfused rat lungs neutrophils and acute lung injury perflubron attenuates neutrophil adhesion to activated endothelial cells in vitro liquid ventilation attenuates pulmonary oxidative damage neutrophil activation and chemotaxis after in vitro treatment with perfluorocarbon exposure to perflubron is associated with decreased syk phosphorylation in human neutrophils cytokine-mediated inflammation in acute lung injury tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8 aerosolized perfluorocarbon suppresses early pulmonary inflammatory response in a surfactant-depleted piglet model intratracheal perfluorocarbons diminish lps-induced increase in systemic tnf-alpha perflubron decreases inflammatory cytokine production by human alveolar macrophages perfluorocarbons decrease chlamydophila pneumoniaemediated inflammatory responses of rat type ii pneumocytes in vitro nf-kappa b activation as a pathological mechanism of septic shock and inflammation pyrrolidine dithiocarbamate reduces lung reperfusion injury attenuation of lung reperfusion injury after transplantation using an inhibitor of nuclear factor-kappab perflubron reduces lung inflammation in respiratory syncytial virus infection by inhibiting chemokine expression and nuclear factor-kappa b activation leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro perfluorocarbon attenuates response of concanavalin a-stimulated mononuclear blood cells without altering ligand-receptor interaction perfluorohexane attenuates proinflammatory and procoagulatory response of activated monocytes and alveolar macrophages in vitro cellular effects of perfluorochemicals correlate with their lipid solubility effects of fluosol-da on brain edema, energy metabolites, and tissue oxygen content in acute cerebral ischemia perfluorocarbon supplementation and postischemic cardiac function this study was supported, in part, by nsc 98-2314-b-016-034-my2 from the national science council of taiwan; mab101-63 from the ministry of national defense; grants 10132, 1032, and 10137 from taoyuan armed forces general hospital; and tsgh-102-066 from tri-service general hospital, taiwan. key: cord-005774-7z6uyn6p authors: hammer, j.; newth, c. j. l. title: infant lung function testing in the intensive care unit date: 1995 journal: intensive care med doi: 10.1007/bf01704742 sha: doc_id: 5774 cord_uid: 7z6uyn6p as a result of the previous shortage of tools to assess objectively the overall physiological status of the respiratory system in infants and young children, it has been difficult to measure the degree of physiological disorder or the response to therapy in respiratory diseases such as bpd, the pediatric version of ards, bronchiolitis, pneumonia, asthma and croup in this patient population. the newborn — four-year old child is particularly difficult to study because of their lack of cooperation and size. the recent progress in computer technology made pulmonary function testing available for this age range and opened up new possibilities for monitoring changes in disease processes affecting the respiratory system. this may improve medical management of infants and children with lung and heart diseases in particular. in 1989, shannon [49] proposed in this journal that the minimum physiological information needed for the intelligent use of mechanical ventilation (particularly if lower airway and/or pulmonary parenchymal disease was apparent) required the measurement of at least 4 variables: i) arterial partial pressure of carbon dioxide; ii) arterial oxygen saturation; iii) the mechanical time constant of the lung and iv) frc. in many circumstances, arterial co(2) is approximated by alveolar (end-tidal) co(2) and the arterial oxygen saturation is obtained from pulse oximetry accurately if perfusion is adequate. the mechanical time constant and frc are easily measured by the techniques described above and together provide important information concerning appropriate ventilator settings for a given disease. the described techniques bring new insights and awareness, but also new responsibilities in the management of infants and children with respiratory compromise. not all of these techniques need to be applied to all infants in the icu. not all the assumptions upon which some of the techniques we have described are based will prove true. any such methods which do not withstand solid scientific testing must be quickly discarded and replaced with better and (hopefully) easier methods. until recently, there has been limited ability to assess objectively the deviations from normal in lung function in infants and children, either within or without the intensive care unit (icu). however, the application of the rapid data acquisition and processing abilities of the personal computer and miniaturization of equipment has led to major changes in this field, particularly for the newborn-4-year-old child age range. the modifications of old and development of new pulmonary function tests have allowed those involved in the care of such patients novel measurements and new perspectives in both the assessment and management of respiratory failure. this review will focus on techniques which are used to measure thoracoabdominal asynchrony, tidal breathing flow-volume loops, small airway function (forced expiratory maneuvers), respiratory mechanics and lung volumes in critically ill infants and children. the major goals of these techniques in an intensive care setting are to: i) understand the underlying pathophysiology, ii) aid with diagnosis, iii) provide assessment of therapeutic response, and iv) provide a guide to changes in a patient's condition which will allow timely interventions to support the patient. ideally, such devices should also provide a measure of the disease and a prediction of outcome, in addition to being inexpensive and noninvasive. however, at this point in the evolution of intensive care, our most useful monitors, diagnosticians and medical prognosticators are almost certainly highly trained and "disease-smart" physicians, nurses and respiratory therapists at the bedside. some applications of pulmonary function testing generally require that the infants are heavily sedated, and under neuromuscular blockade when mechanically assisted with ventilation. the latter situation also requires (usually) that there be no leak around the ett in order to obtain good quality studies. this can be achieved in most cases with either a cuffed ett or with an uncuffed tube with pharyngeal packing. cuffed ett are not recommended for use in children under the age of 8 years [1] , but in a recent prospective study involving 250 infants and children, using cuffed ett one-half size smaller than the calculated uncuffed ett for age, we had no greater incidence of complications either short-term (post-extubation stridor) or long-term (tracheal stenosis) [2] . we now use cuffed ett routinely in our pediatric icu in infants and young children with pulmonary disease. it is not the intent of this report to justify as "useful" or "essential" any of the techniques which are subsequently mentioned. at one end of the spectrum, demonstration of a significant reduction of mortality and morbidity associated with a change in practice provides a dra-matic justification for the use of a diagnostic or monitoring tool. unfortunately, unlike therapeutic interventions, it is rarely possible to provide evidence of such changes with routine monitoring devices in intensive care. in addition, little has been published on the efficacy or cost effectiveness of the various procedures. at the other end of the spectrum, one can argue that any intervention which provides more understanding of disease processes can be justified, providing there is little or no deleterious effect on the patient. intermittent arterial blood gas analysis is regarded as the standard diagnostic tool for respiratory failure and is fundamental for accurate assessment of pulmonary gas exchange and ventilator management [3] . however, technologic advances have made non-invasive devices available for continous monitoring of oxygen (02) and carbon dioxide (co2). these include end-tidal co2, transcutaneous co 2 and 0 2, and pulse oximetry. if the caregiver is aware of their limitations, they allow quick feedback on rapidly changing conditions and are helpful in the continuous supervision of respiratory therapy. non-invasive blood gas monitoring has the potential to reduce significantly the frequency of abg sampling. detailed descriptions of their operation and limitations are beyond the scope of this paper, but are available in recent reviews [4, 51. thoracoabdominal asynchrony (taa) and paradoxical breathing are often observed in infants and children with various forms of respiratory diseases including upper airway obstruction (uao), parenchymal processes (such as hyaline membrane disease, pneumonia and pulmonary edema), obstructive lower airways disease (asthma, bronchiolitis, bpd) and neuromuscular diseases. this phenomenon has generally been descriptive and was referred to as chest wall retractions in clinical scoring systems. however, phase angle analysis of the lissajous figure allows us to easily detect, quantify and monitor taa in a non-invasive manner [6] . in this technique, rib cage (rc) and abdominal (abd) movements are recorded by use of an uncalibrated respiratory inductance plethysmograph, the bands of which are placed at the levels of the nipples and upper abdomen. the analog output of the rc and abd movements is acquired by a computerized data acquisition system that is programmed to calculate continuously phase angles utilizing the method of agostoni and mognoni [7] . phase angle (0) is thus calculated according to the equation: m sin 0 = -s where m is the length of the midpoint of the rc excursion and s is the length depicting the abd excursion (fig. 1 ). in addition, rc and abd movements can be continuously displayed as an x-y plot giving optical information about changes in loop shape and loop direction. except during rem sleep, the rc and abd expand and decrease in synchrony in normal full-term infants and children, producing a closed or very narrow loop with a positive slope on the x-y plot (mean 0 = 8 o, range = 0 to 25 o). however, during taa the loop opens and becomes progressively wider as taa increases. paradoxical breathing also creates a closed or very narrow loop, but with a negative slope. important information can further be obtained from the loop direction. this indicates which compartment (rc or abd) precedes the other. counterclockwise loops indicate that the abd compartment (diaphragm) leads the rc as usually observed in normal quiet breathing and most forms of respiratory distress in children. clockwise loops signify the opposite which is typically associated with diaphragmatic paralysis [8] . continuous phase angle measurement is a promising non-invasive technique for the objective assessment of taa in a variety of respiratory diseases. phase angles are elevated in uao and decrease after (z-agonist therapy (fig. 2) , but demand further research and a better understanding of their predictive value for respiratory failure [6]. we have recently validated phase angle measurements by showing that they correlate with the level of the imposed respiratory load, but do not detect respiratory muscle fatigue, in uao [9]. however, it has been demonstrated that phase angle measurements correlate with transcutaneous co 2 in infants with severe laryngotracheobronchitis and provide a useful, additional way to . phase angle measurements also correlated with improvement after bronchodilator therapy in children with obstructive airway disease such as asthma and bpd [11] . the use of phase angle analysis to monitor taa in infants and children with neuromuscular disease (e.g. infant botulism, guillain-barr6 syndrome, myopathies, neuropathies, spinal cord injuries) needs to be further clarified, but offers a promising tool to monitor sprinting or weaning processes from mechanical ventilation. diaphragmatic paralysis can be easily detected at the bedside by the characteristic generation of clockwise loops, even when this may not be obvious clinically as in the case of unilateral paralysis (e.g. post cardiothoracic surgery) [8] . a problem often encountered with this technique is that at various times phase angle loops are not based on clear sinusoidal rc and abd movements and produce numerous types of non-sinusoidal patterns [12] . we have recently suggested that a sine-wave independent mathematical approach in loop analysis improves the accuracy of phase shift calculations under such circumstances [13, 141. other methods used in the assessment of respiration involve techniques measuring the "work of breathing". the classic method for "work of breathing" has been thoroughly studied in adults by collett and co-workers [15] . however, modified approaches have been used in infants. the "work of breathing" can be measured relatively non-invasively by use of an oesophageal balloon to record pleural pressure changes and subsequent calculation of the pressure-time index. this index is an estimate of the energy cost of the "work of breathing" because 02 consumption by muscle is proportional to the integral of muscle tension (or pressure) with respect to time. klein and reynolds demonstrated that when the unintegrated pleural pressure signal was used in the index, they were able to show a response to therapy with continuous inflating pressure in sleep-related uao [16] . since breathing slows and inspiratory pressure is greater with uao, the "raw" pressure-time index underestimates the true integrated pressure-time index. nonetheless, this seems a simple and effective objective measurement for upper airway obstruction. a further variation was suggested by wolfson et al. [17] in their study on helium breathing in infants with bpd. these techniques await further validation. tidal breathing flow-volume loops tidal breathing flow-volume or pressure-volume loops are increasingly used in continuous or intermittent monitoring of mechanical ventilation in both neonatal and pediatric icus. they can be measured on special "standalone" equipment, or are increasingly incorporated into modern ventilators. these allow the measurement of tidal volume (vt), tidal flows and pressures generated during mechanical and spontaneous breaths. thus, impacts of alteration in ventilator settings or lung physiology on these parameters can be readily detected [18, 19] . spontaneous tidal volume breaths and ventilator breaths can be compared (especially along the expiratory flow limb where flow-limitation may be readily seen) which enables an estimate of the ventilatory reserve provided by mechanical ventilation (fig. 3 ). the generation of inadverunless it is certain that flow is limited (effort independent) at a particular lung volume, changes in flow rates after a therapeutic maneuver (e.g. bronchodilator) may still be attributed to factors other than a simple response to therapy. we have recently shown that the fd technique is capable of producing forced expiratory flows at flow limitation in intubated animals and infants with normal and obstructed airways [22, 23] . for the test procedure, the lungs are inflated by squeezing a breathing bag filled from a continuous compressed o~ supply to +40 cmh20 inflation pressure, defined as total lung capacity (tlc). inflation pressures are held static for at least 3 s, after which a sliding valve is activated to expose the airways to a 100-l capacity, constant negative pressure source of -40 cmh20 deflation pressure. the lungs are deflated until expiratory flow ceases at residual volume (rv) or for at least 3 s. vc and mef at various subdivisions are measured by an interposed pneumotachograph. throughout the procedure the individual is usually under neuromuscular blockade and/or heavy sedation. normal values for vc and mef at the various subdivisions still need to be defined, but in our laboratory lie in the range of 50-70 ml.kg -1 for vc and 24-38 ml.kg -t 9 s -1 for mef25 and 6-15ml.kg-t.s -~ for meft0. since it has become standard of care to use inhaled bronchodilators on intubated and ventilated patients in a variety of diseases, their effectiveness with respect to bronchodilatation can easily be documented by the fd technique [24, 25] . obstructive airway and restrictive lung diseases produce very characteristic patterns (fig. 4) which are helpful in assessing the underlying pathophysiology (e.g. rsv infection causing bronchiolitis, ards or pneumonia). serial vc and mef assessments are helpful in a variety of lung diseases like bpd and ards requiring long term mechanical ventilation and document the resolution or progression of the disease process [26, 27]. compliance and resistance reflect the mechanical properties of the lungs and require the measurement of flow, volume and pressure. compliance (c) is defined as the change in volume per unit change in pressure: it must be emphasized that compliance is a function not only of the elastic properties of the respiratory system, but also of its volume. in other words, the value obtained is different at various lung volumes, dependent on the shape of the pressure-volume curve, which in turn depends on the amount of lung disease and therapeutic maneuvers such as peep or surfactant administration. sudden changes in compliance often reflect the opening and closing of individual lung units rather than changes in lung tissue and surface tension characteristics and represents the resistive properties of the airways, lung tissue and chest wall. several methods have been designed to measure compliance and resistance in ventilated infants which has led to a confusing nomenclature for the practitioner. compliance is referred to as either dynamic compliance (cdyn) when it is measured when ventilation is in motion, or as static (passive) compliance (crs) when respiratory muscles are inactive during the test procedure. the same applies to the resistance of the respiratory system, which is referred to as either dynamic (re) or total respiratory system resistance (rrs). cdyn can be simply calculated by dividing vt by the total change in pressure necessary to deliver that volume. these numbers can be easily extracted from mechanical ventilation. however, it is understood that cdyn is related to both elastic and flow resistive characteristics according to the equation of motion of the single compartment model of the respiratory system: p = vx+r f c where p = transpulmonary pressure, vx = tidal volume and f = tidal flow. thus, cdyn changes with alteration of mechanical ventilation settings including respiratory frequency, inspiratory and end-expiratory pressure [30] . the classic technique of determining cdyn is based on the measurement of oesophageal pressure as a quantification of pleural pressure [31] . this allows differentiation of cdyn into its components of lung compliance (cl) and chest wall compliance (ccw). however, ccw is usually very high in infants and its contribution to total respiratory compliance (cxox) can often be neglected [32, 33], since cl and ccw are related as follows: this technique is invasive by virtue of the need of an oesophageal catheter and the accuracy of such measurements in intubated infants and children is controversial [34, 351. newer methods measure static compliance (crs) and resistance (rrs) and are based on relaxation of both inspiratory and expiratory muscles during brief airway occlusions during exhalation. the most widely used methods are the passive deflation and the multiple occlusion techniques [36] [37] [38] . muscle relaxation is achieved either by invoking the hering-breuer inflation reflex or by use of neuromuscular blockade. we favor the use of shortterm neuromuscular blockade together with sedation for a mechanically ventilated patient in the controlled setting of an icu because it guarantees complete muscle relaxation during the whole expiratory phase. in the following we will concentrate on the discussion of the passive deflation technique (single breath occlusion) and refer the reader to recent literature for the other methods [39] . the passive deflation technique involves measuring pressure during occlusion of the airway at endinspiration and fitting a straight line to the fv-curve obtained during the subsequent passive exhalation [40, 41] . if there is no muscle activity during exhalation, the expiratory time constant (trs) or emptying time of the respiratory system will be entirely dependent on the mechanical properties of the lungs and can be described as follows: thus, both crs and rrs can be obtained from a single breath. the determination of trs gives some idea of how rapidly the lung empties following a mechanical breath. a single time constant is defined as the time required to exhale 63 % of the tidal volume. three time constants are needed to exhale 95~ of the delivered tidal volume. this permits the determination of respiratory rates allowing complete exhalation or the detection of rate settings which lead to inadvertent peep. the passive deflation technique relies on the assumption that the respiratory system can be regarded as a single compartment model. this is valid in most healthy infants especially over the tidal volume range. however, in the presence of lung disease, the respiratory system will not always behave like a single compartment model and a single time constant will not adequately describe all the respiratory mechanics [42, 43]. we have noted multiple time constants in infants with restrictive lung diseases such as acute ards or pulmonary edema, or with severe obstructive airway disease [44] . in all these circumstances crs and rrs are best measured over the longest linear fit of the passive expiratory fv-curve. however, calculation of time constants at different intercepts may give additional information and better describe the respiratory mechanics over the whole expiration phase [451. pattern recognition adds valuable information to the interpretation of results obtained by measuring respiratory mechanics. while obstructive lung disease is characterized by a concave slope of the passive expiratory fv-loop, restrictive lung disease often results in convex loop patterns. it is important to note that in the case of intubated patients, crs and rrs measurements include the physical properties of the ett. unfortunately, there is still a great lack of normal values for crs and rrs in intubated infants and children with normal lungs. according to our studies, such normal data lie in the range of 0.8-1.2ml'cmh:o -1 9 kg -1 for crs and 0.4-0.sml'cmh20-l"s (up to 1.0 with ett < 3.5 mm i.d.) for rrs [25] . pfenninger and aebi [46] have recently used the passive deflation technique for rrs and crs to compare the response to inhaled and intravenous salbutamol in ventilator-dependent infants with chronic lung disease, and concluded there was no difference. such careful objective physiological measurements should influence how weaning such infants from the ventilator is approached. although the most fundamental interest in lung volume measurements in infancy and childhood relates to the assessment of normal and abnormal lung growth [47, 48], the determination of lung volumes is an important part of the respiratory management of infants and children [49] . lung volume measurements can help in diagnosing respiratory disorders, in evaluating responses to therapy, and in finding suitable ventilator settings with respect to rate and ventilating pressures [50-52]. lung volume is also an important variable when lung mechanics are measured [53] because specific compliance and specific resistance are normalized by lung volume, i.e. the functional residual capacity (frc). currently, frc and v t are the only lung volumes that can be accurately, repeatedly, and reliably measured in infants and small children. hence, they are the only lung volumes that can be routinely determined for clinical reasons either in the icu or in the out-patient clinic. other lung volumes such as tlc, vc and rv can also be measured, but the techniques are employed mainly for research, and require an endotracheal tube. frc can be measured by three techniques: plethysmographic (infant body box), helium (he) dilution (a closed-circuit method), and nitrogen (n2) washout (in its modern form, an open-circuit method). except in the smallest of infants, sedation is required for each technique. sulphahexafluoride has recently been used in a promising washin-washout technique [54, 55] , and has now been validated [66] . the body plethysmograph technique is labor-intensive, as is the calibration of the infant body box. edberg and colleagues [56] [57] [58] have recently applied modern computing techniques to the infant body box and obtained useful data in the neonatal icu setting. however, their lung volume data was measured using the nitrogen (n2) washout method. for most workers, the use of plethysmographic methods is impractical in the icu environment, particularly for mechanically ventilated patients. the recent advent of a commercially available computerized infant box (j~tger) may modify this opinion. the closed-circuit he dilution method has been adapted to measure frc on ventilated patients by heldt et al. [59] . the patient is connected via the ett and a sliding valve to both a bag (which is situated inside a transparent plexiglas box) and to the ventilator. in normal pre-test position the patient is ventilated directly by the ventilator through the valve. the bag, which contains a known amount of gas with known he concentration (and thus a known amount of helium) is sealed and is not connected to the patient. at end-exhalation the valve is switched so that the patient is directly connected only to the he-containing bag while the ventilator ventilates the box surrounding the bag and compresses the bag accordingly. the patient is thus ventilated by the bag which is externally compressed by the ventilator cycle. after several breaths, equilibration of he concentration between the lungs and the bag is achieved and frc can be calculated in the same way as in non-ventilated subjects. helium dilution can be used in patients on very high inspired oxygen concentrations (fio2 = 0.97). however, most thermal conductivity based he analyzers are inaccurate when o2 concentrations are high. moreover, calibration depends on the oz/he mixture ratio and should be repeated each time. leak-free connections in intubated infants are more difficult to achieve in this age group, where cuffed tubes are rarely used. if the leak is minimal, it may be eliminated by gentle tracheal pressure during the recording period. although a method for correcting leaks during frci~e measurements has been described by fox et al. [60] and is currently incorporated into some automated systems. this has not been fully evaluated nor validated and may result in significant errors. the technique is based on washing out the n 2 from the lungs by giving the subject 100% o 2 to breathe. if the amount of n 2 washed out is measured and the initial alveolar n 2 concentration is known, then the lung volume from which point the washout started can be derived. in this open circuit method, the patient is switched to breathing 100% 02 and from this point the volume of n 2 exhaled is determined by integration with respect to time of the instantaneous n2 concentration flowing in the exhalation circuit multiplied by the instantaneous flow. in 1985, gerhardt and co-workers [61] devised a new open washout system to which a constant background o2 flow was delivered. the patient inhaled from and exhaled to that circuit with background flow. although the instantaneous flow rate of the washout circuit changes continuously as the subject breathes, the average flow leaving the system over time remains unchanged because the volume of gas subtracted during inspiration is added back to the system during exhalation (this is true as long as the temperature and humidity of the inhaled and exhaled gas are equal -a condition which is easy to meet by using a humidifier). because the method ignored the instantaneous change in flow and used only the average constant flow for calculation, it was essential that sampling of n 2 for concentration measurements would "see" a continuous decrease of n 2 concentration as the washout proceeds, without the effect of the respiratory phase. this was achieved by incorporating a mixing chamber in the exhalation circuit before the sampling port from which mixed expired gas was sampled for n2 analysis. the technique developed by gerhardt et al. for spontaneously breathing infants [62] is not immediately applicable to ventilated children mainly because the gas flow during calibration does not equal the flow during the test. in order to overcome this difficulty, sivan and co-workers [63] used the respiratory mass spectrometer already "in-line" for measuring the instantaneous n 2 concentration, to record the minute ventilation by the argon dilution technique [64] . at frc the patient is switched to a second ventilator delivering 100~ 02 (washout ventilator) and washout starts. this n2 washout technique (which unlike the he dilution method is limited to patients at fio 2 < 0.65) allows accurate determination of frc during mechanical ventilation and correlates well with those values produced using the douglas bag technique [52] . in patients with restrictive lung disease, including a group with ards [44], frc measured at clinically chosen levels of peep (4-10cmh20) was 45~ below predicted frc for nonintubated normal children and 60~ below that of ventilated children with normal lungs at physiological levels of peep (2-4 cmh20). the use of progressively greater levels of peep produced increases in frc towards predicted normal values [63] . however, this suggests that in ards at least, normalization of frc would require sufficient peep to contribute to barotrauma or to compromise cardiac output and systemic oxygen transport. in spontaneously breathing infants and children, frc is the same whether determined by he dilution or n2 washout methods [65] , and is in the range of 16-22 ml 9 kg-1 (mean = 20.4 ml" kg-1). the only published data on ventilated infants and children with normal lungs demonstrated frcs up to 50~ more than the nor-mal values for spontaneously breathing (i.e. not ventilated) children on peeps of 2-4 cm h20 [63] . as a result of the previous shortage of tools to assess objectively the overall physiological status of the respiratory system in infants and young children, it has been difficult to measure the degree of physiological disorder or the response to therapy in respiratory diseases such as bpd, the pediatric version of ards, bronchiolitis, pneumonia, asthma and croup in this patient population. the newborn -four-year old child is particularly difficult to study because of their lack of cooperation and size. the recent progress in computer technology made pulmonary function testing available for this age range and opened up new possibilities for monitoring changes in disease processes affecting the respiratory system. this may improve medical management of infants and children with lung and heart diseases in particular. in 1989, shannon [49] proposed in this journal that the minimum physiological information needed for the intelligent use of mechanical ventilation (particularly if lower airway and/or pulmonary parenchymal disease was apparent) required the measurement of at least 4 variables: i) arterial partial pressure of carbon dioxide; ii) arterial oxygen saturation; iii) the mechanical time constant of the lung and iv) frc. in many circumstances, arterial co2 is approximated by alveolar (end-tidal) co 2 and the arterial oxygen saturation is obtained from pulse oximetry accurately if perfusion is adequate. the mechanical time constant and frc are easily measured by the techniques described above and together provide important information concerning appropriate ventilator settings for a given disease. the described techniques bring new insights and awareness, but also new responsibilities in the management of infants and children with respiratory compromise. not all of these techniques need to be applied to all infants in the icu. not all the assumptions upon which some of the techniques we have described are based will prove true. any such methods which do not withstand solid scientific testing must be quickly discarded and replaced with better and (hopefully) easier methods. ed) textbook of pediatric intensive care. williams & wilkins recognition and management of respiratory failure noninvasive assessment of blood gases effect of positive end-expiratory pressure on respiratory compliance in children with acute respiratory failure functional residual capacity and ventilation homogeneity in mechanically ventilated small neonates measurement of functional residual capacity by sulfur hexafluoride in small-volume lungs during spontaneous breathing and mechanical ventilation immediate effects on lung function of instilled human surfactant in mechanically ventilated newborn infants with irds lung volume, gas mixing, and mechanics of breathing in mechanically ventilated very low birth weight infants with idiopathic respiratory distress syndrom ekstr6m-jodal b, hjalmarson o (1991) a plethysmographic method for assessment of lung function in mechanically ventilated very low birth weight infants a simplified method to determine functional residual capacity during mechanical ventilation effects of endotracheal tube leaks on functional residual capacity determination in intubated neonates a simple method for measuring functional residual capacity by n 2 washout in small animals and newborn infants functional residual capacity in normal neonates and children up to 5 years of age determined by a n 2 washout method functional residual capacity in ventilated infants and children the measurement of metabolic gas exchange and minute volume by mass spectrometry alone comparison of helium dilution and nitrogen washout measurements of functional residual capacity in infants and very young children measurement of functional residual capacity by sulfur hexafluoride in small-volume lungs during spontaneous breathing and mechanical ventilation key: cord-006676-a21tdgns authors: abul, h.; abul, a.; khan, i.; matthew, t.c.; ayed, a.; al-athary, e. title: levels of il-8 and myeloperoxidase in the lungs of pneumonia patients date: 2001 journal: mol cell biochem doi: 10.1023/a:1007264411006 sha: doc_id: 6676 cord_uid: a21tdgns interleukin-8 (il-8) is considered as the major polymorphonuclear neutrophils (pmns) chemoattractant cytokine in lung diseases such as asthma and adult respiratory distress syndrome (ards). however, controversial results were obtained regarding the involvement of il-8 in the pathogenesis of pneumonia. this study examines the role of il-8 in the recruitment and activation of pmns in the lung of pneumonia patients. the interesting aspect of this study is that it is a sitespecific analysis of the infected and uninfected lungs of the same patient. the level of il-8 mrna, protein and myeloperoxidase present in the cells of the bronchioalveolar lavages (bals) taken from the areas of known pneumonic consolidations on chest x-ray (infected lung) are compared with the bals obtained from areas of no obvious infiltrate (non-infected lung). the results obtained from the infected and non-infected lungs of pneumonic patients were further compared with that of a control group of non-smoking patients. the level of il-8 mrna and protein were determined by rt-pcr and elisa respectively. there was a significant increase in the level of il-8 mrna in the infected lung as compared to its level in the non-infected lung (p < 0.001). in correlation with the increase in mrna, il-8 protein concentrations in bal fluids from the infected lung were 6 fold higher than those taken from the non-infected lung (p < 0.0001). this pattern was also consistent with mpo activity in the bals (4.5 fold more mpo activity in the infected lung as compared to that of the non-infected lung), indicating that il-8 is directly implicated in neutrophil accumulation that follows acute respiratory infection. the results of the present study, therefore, indicate the involvement of il-8 in the pathogenesis of pneumonia. chemokines constitute a large family of regulatory cytokines that play a central role in immunological processes. the accumulation and appearance of polymorphonuclear neutrophils (pmns) in the tissue may be considered as an initial marker of acute inflammatory reaction [1] . neutrophils participate in the host response to a number of infectious and non-infectious diseases and in leukocyte migration [2] [3] [4] [5] [6] . they contain cytoplasmic granules that function in storage of bioactive neuromolecules (specific or secondary granules) or in fusion with phagosomes (azurophilic or primary granules). the azurophilic granules contain a variety of enzymes including myeloperoxidase, muraminidase, cathepsin a, d, e, g, 5′-nucleotidase, β-galactosidase, elastase, collagenase, azurocidin and the defensins hnp-1, hnp-2, and hnp-3, arylsulfatase, α-mannosidase, n-acetyl-β-glucosaminidase, β-glucuronidase, acid β-glycerophosphatase and cationic peptides. the specific granules on the other hand contain vitamin-b 12 -binding protein, neutral proteases, lactoferrin, alkaline phosphatase, lysozyme, and probably collagenase [7] . although the mechanisms that regulate the release of substances from both the granules are almost the same, there are certain specific stimuli such as il-8 and zymosan that induce the release of substances from secondary granules [8] . thus il-8 functions as a potent chemotacting as well as degranulating agent. recently it has been shown that depletion of neutrophils using anti-rat neutrophil antiserum reduced subsequent development of chronic delayed type hypersensitivity reactions [9] . this study clearly demonstrates the importance of neutrophil derived factors for monocyte and lymphocyte mobilization. furthermore, it is shown that neutrophils produce a number of low molecular weight factors such as leukotriene b4 (l bt4) that attract more neutrophils and monocytes to the inflammatory site [10] . t-lymphocytes have also been shown to migrate in response to il-8 both in vivo and in vitro [11, 12] . there is a clear involvement of il-8 in the pathophysiology of various respiratory diseases [13] [14] [15] [16] [17] . in asthma, airway inflammation with eosinophils, lymphocytes and neutrophils is a characteristic feature [18] [19] [20] . in correlation with this cellular migration, there is an increase in the level of il-8 in the serum, tissue and bal of asthmatics. similar to that in asthma, the involvement of il-8 has been well investigated in adult respiratory distress syndrome (ards). however, the role of interleukin in the development of pneumonia is controversial [21] . although, an increase in the level of il-8 is a good indication of the inflammatory process, this information does not contribute much to clinical diagnosis. to our knowledge, no data is available on the production of il-8 in bal fluid from the same patient (i.e. infected and non-infected lung). therefore this study is designed to measure the site-specific increase in the level of il-8 in the lung of patients with bacterial pneumonia as compared to that of the non-smoking control group. the level of il-8 mrna and protein present in the bal obtained from subsegmental bronchi of experimental and control group of patients were determined by rt-pcr assay and enzyme immunoassay respectively. in this study we also determined the level of myeloperoxidase activity in the cells collected from 1 ml of bal each from the infected and non-infected lung. myeloperoxidase, a secreted heme protein, is an attractive candidate for monitoring phagocyte mediated cellular damage [22, 23] . the study was performed on 36 patients with bacterial pneumonia who were admitted to the chest diseases hospital in kuwait. all patients underwent medical and laboratory examinations. the control group consisted of 17 non-smoking patients among which 9 patients were with chronic cough, 3 with hemoptysis and normal chest x-ray and 5 with old fibrotic shadows (table 1) . bals were obtained first from the area of known pneumonic consolidations on chest x-ray (infected lung) followed by bals from other areas with no obvious infiltrate (non-infected lung) of the same patient. bal fluids were collected from the pneumonic patient and the control group after admission to the hospital, using sterile techniques and routine respiratory care. the bronchoscope was advanced into a subsegmental bronchus. lavage was performed using 20 ml aliquots of warmed normal saline, introduced by a syringe through bronchoscopic aspiration port. a total volume of 100-120 ml saline was infused sequentially and the volume of the lavage fluid retrieval (approximately 60 ml) was pooled and transferred immediately into sterile pre-chilled polypropylene tubes. the pooled fluid was then filtered through one layer of sterile gauze and centrifuged at 1500 rpm for 15 min at 4°c. following centrifugation, 5 ml of supernatant was taken into a sterile polypropylene tube and stored at -70°c until assayed. total rna was extracted from cells contained in 1 ml of the bal obtained from the infected and non-infected lung, using the method of chomczynski and sacchi 1987 [24] . briefly, the method is as follows. cells were lysed in 0.5 ml of 4 mguanidinium isothiocyanate. the lysates were then acidified by adding 80 µl of 3 m sodium acetate at ph 4.0. subsequently, 0.5 ml of water saturated phenol and 0.1 ml chloroform were added to the cellular lysate followed by shaking at 4°c for 20 min. lysates were spun in the cold for 15 min and the supernatants were collected and extracted again with phenol-chloroform. the supernatants were finally extracted with chloroform and the aqueous layer was collected. rna was precipitated with absolute ethanol [25] . the precipitate was further centrifuged and the pellet was air-dried. the rna pellet was then dissolved in 100 µl diethylpyrocarbonate (depc) treated water. concentration of rna was determined at 260/280 nm optical absorbance. aliquots (2 µg) of total rna were annealed with 250 ng of oligo dt primer by heating at 75°c for 10 min followed by its slow cooling to 37°c. reverse transcription was carried out using 5 units of avian myeloma virus (amv) reverse transcriptase and 20 units of rna guard following the conditions described [26] . reverse transcription reaction was carried out in 50 µl total volume and an aliquot of 5-10 µl from this cdna was amplified for 35 cycles using the following pcr amplification parameters: denaturation 94°c × 30 sec, annealing 50°c × 30 sec and extension 74°c × 60 sec. the mgcl 2 was used at a concentration of 1.5 mmol/l. the pcr amplification reaction was carried out in presence of 50 pmol each of upstream (5′-gga acc att ctc act gtg tg-3′) and down stream (5′-ctc ttc aaa aac ttc tcc aca a-3′) il-8 specific primers using 1 unit of amplitaq enzyme in a thermocycler. these primers were synthesized based on human il-8 cdna sequence information [27] . pcr products were analyzed on 10% polyacrylamide gel electrophoretically [28] , stained with ethidium bromide and photographed with a gel documentation system (stratagene). all experiments were carried out under rnase free conditions and the solutions and glassware were made rnase free with depc treatment and or by autoclaving. heat sensitive solutions were made in depc treated and autoclaved water followed by their filtration through 0.45 µ size millipore filters. the concentrations of il-8 in plasma and bal fluid supernatants were assayed in duplicate, using a quantitative immunometric, 'sandwich' enzyme immunoassay technique with a detection limit of 4.7 pg/ml (amersham, uk). level of myeloperoxidase activity was estimated in the cells collected from 1 ml of bal from the infected and non-infected lung. the method was essentially the same as described earlier [29] . cells were pelleted by centrifugation at 4°c and were homogenized in 1 ml of hexadecyltrimethylammonium bromide buffer, containing 14 mm hexadecyltrimethylammonium bromide and 50 mm kpo4, ph 6.0. samples were homogenized with polytron for 1 min and were kept cold on ice. the lysates were subsequently frozen in liquid nitrogen and thawed once. the lysates were then centrifuged for 2 min in cold at 14,000 rpm and the supernatants were used to estimate the level of mpo activity. aliquots of 20 µl supernatant were mixed with 980 µl of odianisidine hcl (sigma) solution containing 16.5 mg of odianisidine hcl, 90 ml of distilled water, 10 ml of kpo4 buffer, ph 6.0, and 50 µl of 1% h 2 o 2 . absorbance was recorded at 415 nm, every 15 sec for 1 min using beckman du700 spectrophotometer. the enzyme activity was calculated (units/min/ml) by dividing the rate of the change in the absorbance by the extinction coefficient, 1.13 × 10 -2 . enzyme unit is defined as the conversion of 1 µmol of h 2 o 2 per min per ml of alveolar lavage at room temperature. under these conditions, the residual activity in the pellet was < 10%. commutations were performed using the statview 4.02 statistical package with macintosh centris 650 computer. results are expressed as means ± s.e.m. the differences between groups were analyzed by student's t-test. the differences between the groups were considered significant if p < 0.05. in equal amounts of total cellular rna, there was a significantly higher level of il-8 mrna in the infected lung as compared to that of the non-infected lung (p < 0.001; fig. 1 , lane 2). before estimating the changes in the level of il-8 mrna, we characterized the identity of il-8 pcr fragment (272 bp). for this purpose we employed hindiii restriction enzyme. this enzyme cut the il-8 pcr fragment (272 bp) of free and complex il-8 in the blood as well as the bronchial mucosa [32] , suggesting that free il-8 may have a role in the activation of eosinophils. various other convincing studies suggest that il-8 is an eosinophil and neutrophil chemoattractant [33] [34] [35] [36] . it has been shown that il-8 plays a major role in adult respiratory syndrome (ards) [37] [38] [39] . on the other hand, certain studies could not find a correlation between the percentage of pmns and the concentration of il-8 in bal fluid of patients with ards [21] , suggesting that in addition to il-8 there may be other chemoattractant agents that are involved in transendothelial migration of pmns. several investigators have demonstrated that bal fluids obtained from patients with pulmonary infection, contain potent chemotactic factors, such as the complement peptide c5a and leukotriene-b4 (ltb4) [40] . in guinea pigs, exogenous il-8 administration has been shown to recruit neutrophils in the airway lumen [41] . in addition, in vivo and in vitro studies have shown that il-8 induces the release of t-lymphocyte chemoattractants from neutrophil [35] . in vivo studies in mice with a targeted deletion of il-8 receptor homologue has shown that the total number of recruited cells to the airway lumen following a single antigen challenge was significantly low as compared to the wild type [42] . in consistent with the above studies, in the present study, high concentrations of il-8 were found in bal fluids taken from the infected lung of patients with bacterial pneumonia. these results are in agreement with other investigators [21, 43] , who reported high levels of il-8 in bal fluids of patients with different lung diseases. since alveolar macrophages are the major source of il-8 in the lung, the local production of il-8 by these cells may be responsible for the recruitment of pmns into the pulmonary interstitial or air space in a variety of lung diseases. further studies are required to determine the relationship between the severity of the concentrations of il-8 in bal fluids taken from patients with bacterial pneumonia were always high as compared to control group (p < 0.0001; fig. 2 ). furthermore, in all patients the levels of il-8 in bal obtained from the infected lung were 6 fold higher than those from the non-infected lung 130.6 ± 6 to 194.32 ± 54 pg/ml, n = 36 (p < 0.0001). this pattern of increased expression of il-8 mrna and protein was consistent with mpo activity in the lavages. in the cells from the equal amount (1 ml) of alveolar lavages there was 4.5 times more mpo activity (9.0 units/min/ml) in the right lung as compared to the level (2.0 units/min/ml) in the left lung (fig. 3 ). recent reports have considered il-8 as the most potent and major pmn chemoattractant factor in lung diseases [13] [14] [15] [16] [17] , including ards and pneumonia [21] , cystic fibrosis [30] , human immunodeficiency virus (hiv)-infected patients with pneumocystis carinii pneumonia, bacterial pneumonia, or tuberculosis [31] . several studies have convincingly shown that il-8 plays a key role in the pathobiology of asthma [18] [19] [20] . presence of il-8 has been demonstrated in the bronchioalveolar fluid (bal) of the patients with asthma [13] [14] [15] . furthermore, in asthmatics, there was an increase in the level lung diseases and the alteration of il-8 in bal fluids. nevertheless, the data presented clearly show that the concentration of il-8 in bal fluid from pneumonic patients increased in the infected lung. in the lungs, il-8 appears to be the primary chemoattractant for neutrophils [43] . in addition to various cell types [44] , il-8 is also synthesized and released by neutrophils [45] . thus, neutrophils contribute to the recruitment of additional neutrophils in an autocrine manner, by the synthesis and release of il-8. several studies suggest that il-8 also functions as a chemoattractant for eosinophils [33] [34] [35] [36] . it has been shown that major basic protein (mbp), a 13.9 kd protein located in the crystalloid core of eosinophil secondary granules, stimulates the production of il-8 through transcriptional and posttranscriptional events [46, 47] . in neutrophils, mbp stimulated il-8 production occur post-transcriptionally through stabilization of il-8 mrna [47] . in a recent study it has been noticed that type specific consequences of lung infection may be due to the type specific differences in the induction of cytokines by various infectious agents [48] . in contrast to type 5 adenovirus, type 7 adenovirus stimulated the production of il-8 in human lung alveolar epithelial cell line (a549 cells) and primary human fetal lung fibroblasts (gm5387 cells). the regulation of il-8 production, in these cells, occurred at the transcriptional level and at the level of message stability [48] . while adenovirus type 7 increased endogenous il-8 specific mrna, both serotypes (type 7 and type 5) enhanced stabilization of il-8 mrna [48] . the data presented in our study shows that there was a significant increase in the level of il-8 mrna in the infected lung as compared to its level in the non-infected lung (p < 0.001). in correlation with the increase in mrna, il-8 protein concentrations in bal fluids from the infected lung were 6 fold higher than those taken from the non-infected lung (p < 0.0001). the mechanism of il-8 specific mrna increase (transcriptional or post-transcriptional) in this study need to be elucidated. the techniques that are commonly used to quantitate inflammatory cells during the development of various diseases in the lungs include histological analysis and ex vivo radiolabeling of leucocytes and quantification of their accumulation in the lungs by counting. these techniques, however, are labor and time intensive and have practical limitations [49] [50] [51] [52] [53] . to overcome the limitations of histological and radiolabeling studies, in the present study, we have measured the myeline peroxidase (mpo) activity, in order to quantify the neutrophil accumulation. the pattern of mpo activity in the bals (4.5 fold more mpo activity in the infected lung as compared to that of the non-infected lung) was consistent with the level of il-8 mrna and protein. the results of the present study, therefore, indicate a site-specific involvement of il-8 in the pathogenesis of pneumonia. role of neutrophils and mononuclear phagocytes in host defense and inflammation mechanisms of lysosomal enzyme release from human leukocytes: microtubule assembly and membrane fusion induced by a component of complement the ability of chemotactic factors to induce lysosomal enzyme release. i. the characteristics of the enzyme release, importance of surfaces and the relation of enzyme release to chemotactic responsiveness cytochalasin b: effect of lysosomal enzyme release from human leukocytes the structure activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils some interrelations of neutrophil chemotaxis, lysosomal enzyme secretion, and phagocytosis as revealed by synthetic peptides the development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules sequential degranulation of the two types of polymorphonuclear leukocyte granules during phagocytosis of microorganisms modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, rp-3. i. inhibition by rp-3 treatment of the priming and effector phases of delayed type hypersensitivity to sheep red blood cells in rats the biologically active leukotrienes properties of the novel proinflammatory supergene 'intercrine' cytokine family the neutrophil-activating protein (nap-1) is also chemotactic for t lymphocytes inflammatory determinants of asthma severity: mediator and cellular changes in bronchoalveolar lavage fluid of patients with severe asthma interleukin 8 in bronchoalveolar lavage of asthmatic and chronic bronchitis patients production of interleukin-8, rantes and mcp-1 in intrinsic and extrinsic asthmatics il-8 is a potent eosinophil chemoattractant interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease predominant th2-like bronchoalveolar t-lymphocyte population in atopic asthma eosinophil recruitment is associated with il-5, but not with rantes, twenty-four hours after allergen challenge eosinophilic inflammation in asthma high levels of interleukin-8 in the blood and alveolar spaces of patients with pneumonia and adult respiratory distress syndrome oxygen metabolism and toxic properties of phagocytes leukocytic oxygen activation and microbicidal oxidative toxins single-step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction molecular cloning. cold spring harbor laboratory altered expression of sodium pump isoforms in the inflamed intestine of trichinella spiralis-infected rats genomic structure of the human monocyte-derived neutrophil chemotactic factor il-8 polymerase chain reaction assay of mrna using 28s rrna as internal standard measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker interleukin-8 concentrations are eluted in bronchoalveolar lavage, sputum and sera of children with cystic fibrosis interleukin-8 and granulocyte colony-stimulating factor in bronchoalveolar lavage fluid and plasma of human immunodeficiency virus infected patients with pneumocystis carinii pneumonia, bacterial pneumonia, or tuberculosis free and complexed interleukin-8 in blood and bronchial mucosa in asthma cj: interleukin-8-induced human peripheral blood b-lymphocyte chemotaxis in vitro il-8 is a potent eosinophil chemoattractant t -lymphocyte recruitment by interleukin-8. il-8-induced degranulation of neutrophils releases potent chemoattractants for human t lymphocytes both in vitro and in vivo il-8-induced tlymphocyte migration: direct as well as indirect mechanisms elevated levels of nap-1/interleukin-8 are present in the airspace of patients with adult respiratory distress syndrome and are associated with increased mortality pivotal role of interleukin-8 in the acute respiratory distress syndrome and cerebral reperfusion injury inhibition of neutrophil-mediated acute lung inflammation injury by an antibody against interleukin-8 (il-8) neutrophil chemotactic factors in bacterial pneumonia sensory neuropeptides are not directly involved in bronchial hyperresponsiveness induced by interleukin-8 in guinea-pigs in vivo interleukin-8 receptor modulates ige production and b-cell expansion and trafficking in allergen-induced pulmonary inflammation interleukin-8 (il-8): the major neutrophil chemotactic factor in the lung chemokines, inflammation and the immune system cytokine-induced neutrophil-derived interleukin-8 biology of eosinophils allergy-principles and practice post-transcriptional regulation of gro, and il-8 mrnas by il-1 type-specific induction of interleukin-8 by adenovirus il-1 is a potent inducer of eosinophil accumulation in rat skin: inhibition of response by a platelet-activating factor antagonist and an anti-human il-8 antibody the accumulation of 111 in-eosinophils induced by inflammatory mediators, in vivo increased expression of cd11b and functional changes in eosinophils after migration across endothelial cell monolayers induction of low density and up-regulation of cd11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils key: cord-006605-tsk3pakb authors: jesmin, subrina; gando, satoshi; zaedi, sohel; sakuraya, fumika title: differential expression, time course and distribution of four pars in rats with endotoxin-induced acute lung injury date: 2006-11-30 journal: inflammation doi: 10.1007/s10753-006-9017-8 sha: doc_id: 6605 cord_uid: tsk3pakb the hypothesis that the expression of protease-activated receptors (pars) protein is regulated at the level of transcription and that par isoforms, par-1, par-2, par-3, and par-4, in lung tissue show different patterns of expression in lipopolysaccharide (lps)-induced acute lung injury (ali) was tested. male wistar rats were rendered endotoxemic by intra-peritoneal injection of lps (15 mg/kg body weight). we examined the expression of protein and mrna and the immunohistochemical localization of par isoforms in lung tissues 1, 3, 6, and 10 h after lps administration. induction of ali by lps was confirmed based on histopathological changes. lps administration induced significant increases in the expression of par isoforms (protein) at the level of transcription in ali. while the time course of par-1 and -2 expressions were different, those of par-3 and -4 were almost similar. an immunohistochemical analysis showed localization of par isoforms in the vascular endothelium, alveolar epithelium, and alveolar macrophages. however, the cellular distribution patterns of par isoforms were different. we conclude that lps induces increase in protein expression of par isoforms at the level of transcription in rats with ali. the differential expression patterns (over a time course) and distribution of par isoforms suggests a distinct role for each isoform in the pathogenesis of lps-induced ali. intra-alveolar and -vascular fibrin deposition is common in acute lung injury (ali) and acute respiratory distress syndrome (ards) [1, 2] . the up-regulation of procoagulant pathways, impairment of physiological anticoagulant systems, and depression of the fibrinolytic pathway, collectively lead to florid alveolar fibrin deposition [3] . fibrin deposits, in turn, enhance the inflammatory response by increasing vascular permeability, and by activating neutrophils and endothelial cells to produce proinflammatory cytokines [2, 3] . these processes are the hallmarks of ali/ards and contribute to its pathogenesis. recent evidence suggests that progressive ali/ards is closely linked to the activation of inflammation and coagulation [3, 4] , and that protease-activated receptors (pars) are important candidates in this interaction [5] . to date, four distinct par isoforms, namely par-1, -2, -3, and -4 have been described. thrombin activates par-1, -3, and -4, whereas, trypsin and mast cell tryptase activate par-2 [5y7]. recent evidence suggests that tissue factor/factor viia (fviia) and ternary tissue factor/fviia/fxa complexes activate par-2, and par-1 and -2, respectively [8, 9] . although the distribution patterns of some of the par isoforms have been determined in several tissues, it still remains unclear in the lungs of ali/ards. we have previously demonstrated the pulmonary expression of proinflammatory cytokine tumor necrosis factor-! (tnf) and key procoagulant molecules of tissue factor, plasminogen activator inhibitor-1 (pai-1), and fibrin in rabbits with endotoxin-induced ali [10] . in addition, we were able to observe increased levels of pars protein, and to co-localize par-1 and procoagulant molecules in the alveolar epithelium and vascular endothelium. collectively, these results suggest that increase in expression of pars, together with proinflammatory cytokine and procoagulant molecules may underlie the development of ali during endotoxemia. the aims of the present study were to complement the data of a previous study by testing the hypothesis that: (1) the pulmonary expression of par isoforms (protein) is regulated at the level of transcription and that (2) the distribution pattern of each of the four par isoforms is distinct in the lung. to test this hypothesis, we performed experiments using a rat model of lipopolysaccharide (lps)-induced acute lung injury. male wistar rats (200y250 g, 8 weeks old) were used in all experiments. endotoxemia was induced by administration of bacterial lps from escherichia coli 055:b5 (15 mg/kg), dissolved in sterile saline, via i.p injection. at this dose, lps induces lung injury, as well as the expression of inflammatory cytokines. groups of animals (n=17) were killed using sodium pentobarbital (150 mg/kg bw, i.p.) at different time-points after lps administration (1, 3, 6, and 10 h). the control group received an equal volume of sterile saline (2 ml/body), without lps. at the indicated time, the blood samples were collected by cardiac puncture for blood gas analysis, and lung tissue specimens were harvested with care, frozen immediately in liquid nitrogen, and then stored at j80-c. for paraffin sections, lung tissue specimens were postfixed in 4% paraformaldehyde overnight and then embedded in paraffin. all the experimental procedures were approved by the animal care and use committee of hokkaido university graduate school of medicine animal care and use committee. in order to determine the arterial blood pressure and heart rate of rats, a microtip pressure transducer catheter (spc-320, millar instruments, houston, tx, usa) was inserted into the left carotid artery of anaesthetized animals (sodium pentobarbital (40 mg/ kg body weight, i.p.)). then, the arterial blood pressure (bp) and heart rate (hr) were monitored using a pressure transducer (model sck-590, gould, ohio, usa) and recorded (bp and hr) using a polygraph system (amplifier, ap-601g, nihon kohden, tokyo, japan; tachometer, at-601g, nihon kohden; thermalpen recorder, wt-687g, nihon kohden). lung tissues were harvested, blotted dry and weighed in order to determine the weight of the lung in the wet state and calculate the wet-to-dry weight ratio, as follows: the lung tissues were weighed; wrapped loosely in aluminum foil; placed in a drying oven overnight; and weighed again. immediately after harvest, the specimens were fixed in 4% buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into 5-mm-thick sections. after deparaffinization, tissue sections were stained using standard hematoxylin and eosin (he) staining method. morphological injury in lung was semi-quantified by two pathologists blinded to the experimental design by analyzing, from each section, about 16 randomly selected images. the average score was then determined or calculated (n=6 rat per each group). for determining the cellular distribution of proteins of interest, tissue specimens were fixed in 4% buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into 5-mm-thick sections. the sections were then deparaffinized and treated for 20 min with citrate buffer (10 mm citric acid, ph 6.0) in a microwave oven (750 w) before immunostaining. in some cases, frozen sections were fixed in acetone and air dried. endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min. one percent bovine albumin in tris was used for 30 min at room temperature to block non-specific staining caused by secondary antibodies. the sections were then incubated with primary antibodies overnight at 4-c, rinsed in phosphate buffer solution and then exposed to the fluorescence secondary antibody, rhodamine-conjugated affinipure or fluorescein-conjugated affinipure anti-sheep, anti-rabbit, anti-goat or anti-mouse igg (jackson immuno research laboratories), for 2 h according to the manufacturer_s instructions. the specificity of immunoreactivity was confirmed by negative controls where nonimmune igg was used instead of primary antibodies. the coverslips were mounted with immunon (thermo shandon). immunofluorescent images were observed using a laser scanning confocal imaging system (mrc-1024, bio-rad laboratories). immunofluorescence staining was semi-quantitated using a scale (+ to ++++) and the average score of 20 randomly selected images was calculated. two pathologists blinded to the experimental design evaluated each slide. the same set of experiments was repeated at least three times. the immunoblotting procedure used in the present study has already been described in our previous report [11] . briefly, ice-cold lung tissues were minced with scissors, homogenized, and centrifuged, followed by determination of protein concentration of the supernatant using the bicinchoninic acid protein assay (pierce biotechnology). samples were then boiled in reducing sds sample buffer for 5 min, loaded onto an sdsypage (4y15% polyarylamide) gel under reduced conditions, subjected to electrophoresis, and electrophoretically transferred to polyvinylidine difluoride filter membrane. to reduce non-specific binding, the membrane was blocked for 2 h at room temperature with 5% non-fat milk in pbs (137 mm nacl, 2.7 mm kcl, 8.1 mm na 2 hpo 4 , 1.5 mm kh 2 po 4 ) containing 0.1% tween 20, incubated overnight at 4-c with primary antibodies in pbsytween buffer, washed three times with pbsytween buffer, and then the membrane was incubated with a suitable secondary antibody coupled to horseradish peroxidase for 60 min at room temperature. the blots were washed five times in pbsytween buffer and subsequently visualized with an enhanced chemiluminescence detection system (amersham), exposed to x-ray film (fuji photo film). intensity of total protein bands per lane was evaluated by densitometry. negligible loading/transfer variation was observed between samples. in each experiment, ßactin was used as the loading control. for immunological-based detections, the following antibodies were used: anti-human par-1 rabbit polyclonal antibody, anti-human par-2 goat polyclonal antibody, anti-mouse par-3 goat polyclonal antibody and anti-mouse par-4 goat polyclonal antibody (santa cruz biotechnology); anti-rabbit fibrinogen sheep polyclonal antibody (cedarlane laboratories); anti-human fibrin mouse monoclonal antibody (chemicon international), anti-rabbit inducible nitric oxide (no) synthase (inos) mouse monoclonal antibody (affinity bioreagents, golden, co, usa), and anti-xenopus laevis ßactin mouse monoclonal antibody (abcam). in most cases, the specificity of each antibody was initially confirmed by blocking its expression using a competing peptide against which the antibody was raised. it should also be noted that no positive immunoreactivity was observed when non-immune igg was used instead of the primary antibodies. for par-1 and -2, rat liver tissue was used as positive control for both immunofluorescence staining and the immunoblot analysis, according to the manufacturer_s instructions (santa cruz biotechnology) [12] , whereas, for par-3 and -4 immunoreactivities, rat liver tissue and rat uterus were used as positive controls, respectively [12] . please note that each of the anti-par antibodies showed no cross-reactivity with other par isoforms. anti-human fibrin mouse monoclonal antibody and anti-rabbit fibrinogen sheep polyclonal antibody recognize fibrin and fibrinogen, respectively, but can also detect each other_s target peptide. the immunogen of the anti-fibrin antibody is fibrin-like ß-peptide gly-his-arg-pro-leu-asp-lys-cys. total rna samples were prepared from lung tissue specimens using the guanidinium thiocyanatephenol-chloroform single-step extraction method with isogen (nippon gene, toyama, japan), which is routinely used in our laboratory [11] . after isolation, the rna was processed as follows: treated with dnase i; quantified and then reverse transcribed to cdna by omniscript reverse transcriptase using a first-strand cdna synthesis kit (qiagen). the reverse transcription reaction was performed at 37-c for 60 min. the expression of par isoform mrnas were analyzed by real-time quantitative pcr with taqman probe using an abi prism 7700 sequence detector (perkinyelmer applied biosystems, foster, ca, usa), as previously described [13] . gene-specific primers and taqman probes were synthesized from primer express v. 1.61 software (perkinyelmer applied biosystems), according to published cdna sequences of each gene. the sequences of the oligonucleotides were as follows: tnf levels in the plasma and lung tissues were detected using an enzyme-linked immunosorbent assay (elisa) kit for screening rat tnf (pierce biotechnology, rockford, il). for inos, we used a human inos immunoassay kit (r and d systems, minneapolis, mn). the results were expressed as meantsd (n=total number of animals in each group). the means were compared by a one-way factorial analysis of variance, followed by scheffé_s test for multiple comparisons. differences were considered to be significant at a value of p<0.05. as shown in table 1 , levels of both systolic and diastolic blood pressure decreased significantly after lps administration, in comparison to control rats. the peak levels of plasma tnf and its (tnf) concentration in the lung were significantly elevated at 1 h after lps administration. similarly, levels of plasma inos and its (inos) protein and mrna expression in the lung increased after lps administration. table 2 summarizes the values for blood gases and lactate concentrations in rats before and after lps was given. arterial pao 2 was significantly reduced from control animals at all time points after lps administration. as a quantitative measure of fluid clearance in lungs, wet-to-dry weight ratios were evaluated in lungs removed from rats killed at specified times after lps administration. the effect of lps on the ratios (wet-to-dry weight ratios) occurred in a time-dependent manner, leading to a significant (p<0.05) increase from baseline (4.5t0.2) to peak value (5.9t0.2) after lps administration. the lungs from control rats (untreated) showed no detectable injury, based on the histological analysis. in contrast, 1 h after lps administration, the lungs of treated animals showed congestion (+), neutrophil infiltration (+) and thickening of alveolar septum (+). these changes were also observed at 3 and 6 h after lps administration. at 10 h following the administration of lps, the features of lung injury became more evident. the lungs showed congestion (+), infiltration of inflammatory cells in the alveoli (++) and a thickening of alveolar septum (++), which together are called glanulomatous changes. these changes are shown in fig. 1 . the relative amounts of immunodetectable fibrinogen/fibrin increased steadily following induction of sepsis with lps. relative levels of fibrinogen/fibrin vs. control (1.0) at 1, 3, 6, and 10 h after lps administration were 1.6t0.015/1.5t0.04, 2.8t0.02/2.8t0.05, 3.5t0.04/7.1t0.2, and 3.6t0.03/6.3t0.2, respectively (p<0.01). fibrin was poorly detectable in control lungs (data not shown). at 10 h after lps administration, fibrin deposition was evident in the intra-and extra-vascular spaces, in the alveoli and in the bronchial epithelium (data not shown). the par-1 specific antibody reacted with one band of õ62 kda that is consistent with the predicted molecular mass of this receptor [14, 15] . its expression increased in a time-dependent manner (fig. 2) , whereas, that of par-2 was observed at all time points after lps administration, peaking (1.9-fold) 3 h after lps administration (fig. 2) . peak expressions of par-3 and -4 proteins were seen 6 h after lps administration (fig. 2) . par-2 was detected as a band of õ55 kda, consistent with the manufacturer_s information. in addition, the molecular weights of the bands believed to be those of par-3 and -4 were comparable to those of the genbank sequences. the mrna expression of the par isoforms, as determined by real-time pcr, corresponded to levels of their respective proteins in the lung (fig. 3) . immunohistochemical staining of par-1 (fig. 4) in the control lung were essentially nil. however, 10 h after lps administration, par-1 was predominantly expressed in the endothelium of small-to micro-sized blood vessel, and vascular smooth muscle cells of medium-to large-sized blood vessel, while only a modest expression was observed in vascular smooth muscle cells of small-micro-sized blood vessels. the endothelium of medium-to large-sized blood vessels only exhibited moderate staining of par-1. the alveolar epithelium and bronchial epithelium exhibited strong positive staining of par-1, with only modest staining observed in the alveolar macrophages. similar to the pattern of par-1 immunoreactivity, par-2 (fig. 5) immunoreactivity was essentially nil in the control lungs. however, 3 h after lps administration, it (par-2) was localized in the endothelium and vascular smooth muscle cells of small to micro size blood vessels, at comparable intensities. in the vascular endothelium of medium-to large-sized blood vessels, par-2 immunoreactivity was strong, but was weakly stained in the vascular smooth muscle cells. the intensity of par-2 immunoreactivity in the alveolar and bronchial epithelia and alveolar macrophages were similar. unlike par-1 and -2, there was some slight immunostaining observed for par-3 in the control lung (fig. 6) , and by 6 h after lps administration, it (par3) was intensively expressed in both the endothelium and vascular smooth muscle cells of small-to medium-sized blood vessels. in contrast, only moderate immunoreactivity was localized in the vascular smooth muscle cells of large-sized blood vessel, but nonetheless, strong staining in the endothelium. the intensity of par-3 immunoreactivity in the alveolar and bronchial epithelia, and alveolar macrophage, was similar to par-1 and -2. immuno-expression of par-4 in control lungs was similar to par-1 and -2 (fig. 7) , and 6 h after lps administration, strong signals were localized in the vascular smooth muscle cells of small-to medium-sized blood vessels, and moderately expressed in the vascular endothelium. this was in contrast to the vascular endothelium of large-sized blood vessels, which exhibited strong par-4 immunoreactivity, with modest staining in vascular smooth muscle cells. strong positive staining of par-4 is localized in alveolar epithelium, while moderate staining is observed in alveolar macrophages and the bronchial epithelium. these results are summarized on table 3 . here, we test the hypothesis that levels of par isoforms in lungs are regulated at the level of transcription, but each (isoform) with a distinct pattern of tissue distribution. following lps administration, we observed a significant reduction in blood pressure and an increased expression of plasma inos, which occurred in parallel to inos immunoreactivity in the lung [16] . in addition, he staining demonstrated a potent infiltration of inflammatory cells and thickening of alveolar septum called glanuloma [17] . collectively, these findings support the notion that lps (via i.p.) induces sepsis and the development of ali in a rat model. the present study observed increased expression of tnf protein in both lung tissue and plasma immediately after lps administration. tnf is known to induce the expression of tissue factor, which leads to the activation of the extrinsic coagulation pathway, and increases in levels of fxa and thrombin, and ultimately, enhancement of coagulation [18] . tnf is also known to increase levels of pai-1, which, in turn, inhibit fibrinolysis [18] . following increase in tnf levels, we found a marked elevation of fibrin/fibrinogen protein levels in the lung tissues. this observation was confirmed by immunohistochemical analysis that demonstrated clear fibrin deposition in both intra-vascular and -alveolar spaces. although, in the present study, we did not measure levels of tissue factor or the pai-1, in our previous investigation these molecules were highly expressed in the lung of lps-treated rats, as revealed by western blot and immunohistochemical analyses [10] . collectively, these data suggest that enhanced inflammation, coagulation activation, and the inhibition of fibrinolysis lead to fibrin deposition in acutely injured lung, following lps administration. these results are consistent with the clinical and experimental evidence for ali and ards [ 1y 4] . here, we also found that lps induces increases in the protein expression of pars isoforms 1 to 4 in the lung of rats. the parallel changes observed in levels of both protein and mrna of par isoforms suggests that lps may exert its influence at the transcription level. the disparity in the timing of pars expression between our previous and the present studies may be due to species differences [10] . indeed, nysted et al. [19] demonstrated that stimulation with the cytokines tnf and interleukin-1 (il-1), as well as bacterial lps resulted in a five to ten fold elevation of par-2 gene expression in a dose-dependent manner using huvec. in addition, the exogenous treatment of human skeletal muscle cell and monocyte with proinflammatory cytokines increased expression of par-1 and -2 [20, 21] . lan et al. [22] demonstrated that inflammatory stimuli induced by influenza a virus expressed par-1, -2, -3, and -4 mrna in the lung of intact mice. it is interesting to note that the occurrence of peak levels for plasma and lung tnf (1 h after lps administration), coincided with the point at which significant elevations of pars-1,-2, and-3 were observed, compared to control. these results suggest that tnf, together with, or as well as, lps rapidly induce expression of pars gene expression through transcription factors in the injured rat lungs. nuclear factor-kappab (nf-kb) is a transcriptional factor that plays a critical role in sepsis by regulating gene expression of many inflammatory cellular mediators and receptors involved in immune recognition [23] . the nf-kb activators include an extensive list of grampositive and -negative bacteriae and their products such as lps; viruses and their components; protozoan parasites; cytokines (e.g., tnf, il-1); free radical; and oxidants. although determination of factors likely to regulate the transcription of pars is important, it goes beyond the aims of the present study. we believe that nf-kb may be one of the most important transcriptional factors regulating pars expression in lps-induced ali and, as such, plan study it in our future projects. although little is known about the cellular distribution of par-3 and -4 in lung tissue, pars-1 and -2 have been demonstrated in the airway epithelial and smooth muscle cells within the respiratory tract, as well as endothelial and vascular smooth muscle cells [24] . par-1 and -2 are also present in the terminal bronchial epithelium and type ii epithelial cells of the peripheral lung, and lung macrophages, mast cells, granulocytes, and lymphocytes [24] . while our previous study demonstrated the immunolocalization of par-1 in these cells and tissues in lps-treated rabbits, the present study showed strong immunoreactivities for all isoforms of pars in the endothelium, alveolar epithelium, and lung macrophages using a rat model of ali [10] . in addition to increased immunoreactivities of tissue factor and pai-1 in the endothelium and alveolar epithelium, as reported in the previous study, we also confirmed here a strong fibrin deposition in the alveolar epithelium, and intra-alveolar and -vascular spaces. both the endothelium and alveolar epithelium are known to play important roles in the pathogenesis of ali. the co-localization of the key procoagulant molecules and the four isoforms of par in these tissues imply that these molecules may also play a pivotal role in inducing permeability in endothelial and epithelial cells, as observed in lps-induced ali. in contrast to the distinct patterns in onset and course of expression, and localizations of par-1 and -2, par-3 and -4 had almost a similar pattern. it has been suggested that, in certain instances, binding of protease to one receptor can facilitate cleavage of another receptor. this appears to be the case for par-3 and -4 on mouse platelets [25] . other studies suggest that par-3 facilitates activation of par-4 in the presence of low thrombin concentration [25, 26] . some data indicate that par-3 is a cofactor for par-4 in mouse platelets: the hirudin-like site of par-3 binds and concentrates thrombin at the cell surface, and, thereby, promote cleavage of thrombin to par-4 [26] . while the pathological significance of these phenomena has not been elucidated, the co-localization and co-expression of par3 and -4, as observed in the present study, may indicate a similar cofactor relationship. a recent study provides strong evidence that neutrophil elastase-mediated apoptosis plays a pivotal role in the pathogenesis of ali, through the par-1dependent pathway [27] . vogel et al. [28] demonstrated abrogation of thrombin-induced increase in pulmonary microvascular permeability in par-1 knockout mice. furthermore, the absence of par-1 signaling appears to attenuate bleomycin-induced lung inflammation and fibrosis [29] . indeed, using par-2 knockout mouse, su et al. [30] demonstrated that par-2 activation induces lung inflammation and pulmonary edema. in addition to these studies, we found expression of par-1 and -2 and their localization in endothelium, alveolar epithelium and macrophages in the lung. taken together, these data suggest that par-1 and -2 may play a critical role in the pathogenesis of ali. however, the physiological and or pathological significance of par-3 and -4 expressions in the injured lung is currently unclear. the limitations of the present study include: (1) a narrow window of time after administration of lps was investigated to study the pulmonary expression of pars; (2) the single moderate dose of lps (and not higher dose), although able to induce endotoxemia in rat, could not induce severe endotoxemia. thus, the current investigation could not provide insights on pars expression patterns associated with severe endotoxemia. for this reason, future studies should investigate a possible correlation between the degree of severity of induced endotoxemia and the pattern of pars expression. in addition, a more extended and prolonged time course study should be undertaken that will enable examination of the pulmonary pars expression possible. in summary, we demonstrate expression of par-1, -2, -3, and -4 proteins, which occurred at the level of mrnas for up to 10 h after lps administration in a rat model of ali. the timing in expression, i.e., in onset and over the course of time, for the four isoforms of pars were distinct. they were also localized in diverse cells, including vascular endothelium, alveolar epithelium, and alveolar macrophages, a similar localization pattern as tissue factor and pai-1, observed in our previous study. in addition, we also found tnf expression, as well as intra-alveolar and -vascular fibrin deposition in the lung. collectively, these results suggest that each par isoform plays a distinct important role in the pathogenesis of lps-induced ali. coagulation, fibrinolysis, and fibrin deposition in acute lung injury coagulation abnormalities in acute lung injury and sepsis bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia coagulation and inflammation in acute lung injury thrombin signaling and protease-activated receptors proteinase-activated receptors: novel mechanisms of signaling by serine proteases proteinase-activated receptors nonhemostatic activity of coagulation factor xa: potent implications for various diseases tissue factorand factor x-dependent activation of protease-activated receptor 2 by factor viia temporal changes in pulmonary expression of key procoagulant molecules in rabbits with endotoxin-induced acute lung injury: elevated expression levels of protease-activated receptors diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type ii diabetic rat model: a possible cause for erectile dysfunction in diabetes protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue effects of exercise training on expression of endothelin-1 mrna in the aorta of aged rats thrombin and protease-activated receptor-1 agonists promote lipopolysaccharide-induced hepatocellular injury in perfused liver role of expression of thrombin receptor par-1 in muscle cells and neuromuscular junctions during the synapse elimination period in the neonatal rat immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats acute respiratory distress syndrome. a comprehensive clinical approach pathogenesis of disseminated intravascular coagulation in sepsis the proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. comparison with the thrombin receptor thrombin receptor induction by injury-related factors in human skeletal muscle cells thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation altered expression and in vivo lung function of protease-activated receptors during influenza a virus infection in mice nuclear factor-kb role of proteaseactivated receptors in airway function: a target for therapeutic intervention? par3 is a cofactor for par4 activation by thrombin protease-activated receptors in hemostasis, thrombosis and vascular biology proteaseactivated receptor-1 mediates elastase-induced apoptosis of human lung epithelial cells abrogation of thrombin-induced increase in pulmonary microvascular permeability in par-1 knockout mice absence of protease-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis protease-activated receptor-2 activation induces acute lung inflammation by neuropeptide-dependent mechanisms key: cord-005476-q6o5239w authors: griesenbach, u; geddes, d m; alton, e w f w title: gene therapy for cystic fibrosis: an example for lung gene therapy date: 2004-09-29 journal: gene ther doi: 10.1038/sj.gt.3302368 sha: doc_id: 5476 cord_uid: q6o5239w gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases. the requirement of gene transfer into the individual cell types of the complex lung structure will very much depend on the target disease. over the last decade, the gene therapy community has recognized that there is not even one vector that is good for all applications, but that the gene transfer agent has to be carefully chosen. gene therapy is particularly attractive for diseases that currently do not have satisfactory treatment options and probably easier for monogenic disorders than for complex diseases. cystic fibrosis (cf) fulfills these criteria and is therefore a good candidate for gene therapy-based treatment. this review will focus on cf as an example for lung gene therapy and discuss the progress made in this field over the last couple of years. gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases including acute respiratory distress syndrome (ards), cancer, asthma, emphysema and cystic fibrosis (cf), not least because of the comparatively easy noninvasive accessibility of the lungs through aerosols. the lung is a complex organ and can be roughly divided into two main regions: the airways, consisting of trachea, bronchi, large and small airways, which transport air to the peripheral lung, and the alveoli, where gas exchange takes place ( figure 1 ). the cell types facing the lumen vary greatly from pseudostratified, columnar ciliated and nonciliated epithelium in the larger airways, to single-layer cuboidal epithelium in the small airways and type i and ii pneumocytes in the alveolar epithelium. 1 the requirement of gene transfer into the individual cell types will very much depend on the target disease. in addition, tumour and perhaps inflammatory cells may also be important targets for gene transfer. over the last decade, the gene therapy community has recognized that there is not even one vector that is good for all applications, but that the gene transfer agent (gta) has to be carefully chosen depending on the cell type to be targeted, the number of treatments (one versus repeat administration) required, and the size and nature (secreted versus cellular product) of the gene to be delivered. gene therapy is particularly attractive for diseases that currently do not have satisfactory treatment options, and is probably easier for monogenic disorders than for complex diseases. cf fulfills these criteria and is there-fore a good candidate for gene therapy-based treatment. this review will mainly focus on cf as an example for lung gene therapy. cf is the most common lethal autosomal recessive disease in the caucasian population and affects approximately 70 000 individuals worldwide. although several organs are affected, severe lung disease is the cause of most of the morbidity and mortality in cf individuals. 2 the cf gene, the cystic fibrosis transmembrane conductance regulator (cftr), was cloned in 1989 3 and is a chloride channel located in the apical membrane of epithelial cells. mutations in the cftr gene lead to imbalanced ion and water movement across the airway epithelium, resulting in accumulation of sticky mucus, chronic bacterial infection and inflammation. proof-of-principle for cftr gene transfer was quickly established in vitro and in animal models. 4, 5 the first clinical trials in cf patients were carried out in 1993 and to date 29 trial protocols, most of which have been completed, are published (http//www.wiley.co.uk/genmed/clinica/). the initial hope was that cf gene therapy would progress rapidly, due to the ease of noninvasive access to the lungs, but delivery of the gene to the relevant cells remains a difficult task. here, we will review the considerable progress that has been made in pre-clinical and clinical gene therapy studies for cf over the last couple of years. in non-cf individuals, cftr is not expressed abundantly in the lungs, but high expression is seen in serous cells in the submucosal glands and isolated epithelial cells in the small airways. 6 it is currently unclear which of these cell types is the main target for cf gene therapy. however, given that cf, at least in the early stages, presents as a small airway disease, airway epithelial cells (aecs) are likely to be an important target. topical delivery of gta to the lung is currently the preferred method for airway gene transfer. however, before the gta can reach the surface of the epithelial cells, a number of extracellular physical and immunological barriers have to be overcome (reviewed in ferrari et al 7 and weiss 8 ). briefly, the airway epithelium in the lung is generally covered by a thin mucus layer (figure 2 ), whose main role is to trap invading foreign particles. it has been shown that mucus significantly reduces the transfection efficiency of most viral and nonviral gtas. however, transfection efficiency could be increased through pretreatment with mucolytics or the anticholinergic drug glycopyrrolate in vitro and in vivo. 9 in cf patients, particularly at later stages in the disease, the airways are also filled with sticky sputum, consisting of inflammatory cells, cell debris, mucus and dna. to avoid the confounding effect of sputum in vivo, gene transfer should ideally be carried out early in the course of lung disease, before the lungs become filled with secretions. the glycocalyx is also a barrier to gene transfer and pretreatment with neuraminidase, which removes sialic acid residues, enhances adenovirus (ad) transfection of polarized cells in vitro. 10 although not formerly shown, it is likely that cilia also lead to steric hindrance of gta to the apical surface of epithelial cells. in addition to the physical barriers, specific and nonspecific immune defences are important inhibitors of airway gene transfer. pulmonary macrophages have been shown to ingest gtas, and removal of these cells before transfection has increased reporter gene expression by 490% in animal models. 11 however, it is unlikely that removal of macrophages is clinically feasible. in addition to the cellular immune response, humoral immune responses against gta are an important problem, severely restricting the use of viral vectors for chronic diseases such as cf. despite encouraging results in nasal and pulmonary tissues of pre-clinical models 12, 13 and being well tolerated at low-to-intermediate doses in humans, 14 adenovirusmediated gene transfer in the absence of epithelial damage has been inefficient in cf patients. 15 this is mainly due to the absence of the coxsackie-adenovirus receptor (car) on the apical surface of the majority of human aecs, 16 and highlights the important differences in receptor distribution of animal models and humans. in an attempt to increase the transfection efficiency of adenoviral vectors in vivo, gregory et al 17 assessed the effects of sodium caprate (a tight junction opener) application to the luminal surface of aecs in mouse lung, with the rationale that car expression is higher on the basolateral surface of epithelial cells. gene expression in total lung homogenate was increased 25-fold, which further increased to 45-fold when adenovirus was complexed with 2-(diethylamino)ethyl ether (deae) dextran. however, it is unclear if this increase in gene expression was attributable to increased epithelial cell transfection. a controversial issue is whether such tight junction openers can be used clinically, given the heavy bacterial colonization present in the cf lung and the attendant risk of systemic invasion. in addition to problems with low transfection efficiency, the use of adenovirus for a chronic disease like cf is limited due to effective cellular and humoral immune responses against the virus. harvey et al 18 delivered three doses of ad-cftr to the lung of cf patients 3 months apart and demonstrated that after the third administration vector-specific cftr mrna was no longer detecthelper-dependent adenoviral vectors, which are depleted of all viral genes, are less immunostimulatory and have improved safety profiles compared to first-and second-generation viruses, which have only a subset of viral genes deleted. recently, it was shown that helperdependant adenovirus combined with the epithelial cellspecific cytokeratin 18 (k18) promoter leads to reduced inflammation and more prolonged expression in murine airways. 19 the use of adenoviral vectors for cf gene therapy is currently limited by low transfection efficiency and inability of repeated administration, but it remains to be seen if future virus improvements resurrect its use. aav vectors have attracted much interest due to their good safety profile, broad tissue tropism, long duration of expression, and suggestion of their superior escape from immune system surveillance compared with other viruses. several clinical trials have been carried out in the nose, sinus and single lobes of cf patients, all using the aav2-based vector tgaav-cftr (targeted genetics corp.). this vector contains the complete human cftr cdna and uses aav inverted terminal repeat (itr)based promoter elements. phase i studies aerosolizing aav2-cftr into cf patients with mild-to-moderate lung disease have been conducted. there were no safety problems and the vector was detected in the proximal airways: however, vector-specific mrna was not found. 20 a phase ii trial was also undertaken in the maxillary sinuses of cf patients. 21 although the good safety profile was confirmed, none of the primary end points, including the time to sinusitis relapse, histopathology and interleukin (il)-8 measurements, changed significantly when compared to the contralateral control sinus. most recently, results of the first repeat-administration lung trial (three doses of nebulized aav2 1 month apart) were published. the treatment was well tolerated and showed some evidence of improved lung function and reduced il-8 in induced sputum after the first administration. a follow-up trial sufficiently powered to detect pulmonary changes has recently started. 22 the small packaging capacity of aav (o5 kb) precludes the use of this vector for transfer of larger genes. although there is enough space for the cftr cdna, it is not possible to include potent promoter/ enhancer elements. thus, all clinical trials carried out with aav2-cftr have relied on the comparatively weak itr regulatory elements, which may in part explain the disappointing efficacy data described above. strategies to overcome the aav packaging problem have therefore been developed, including approaches based on transsplicing 23 and homologous recombination. 24 the basic principle of these techniques is to split the therapeutic cdna and required regulatory elements, and package them into two viruses, which when transfecting the same cell may recombine and generate a full-length therapeutic gene. one would speculate that both of these strategies would lead to reduced transfection efficiency, when compared with the administration of one intact virus to the lung. however, surprisingly, halbert et al 24 have demonstrated that aav2/6 (itr from aav2 and capsid from aav6) recombination-dependent vectors transduced lung cells in mice almost as efficiently as intact vector, with 10% of aecs being positive. several different isoforms of human aavs have been identified and further screening for new human and nonhuman primate isoforms is underway. 25 it has already been documented that a virus with aav5 or aav6 capsid protein can enter aec more efficiently than aav2 viruses, but the overall transfection efficiency is still comparatively low. 26 recently, the atomic structure of aav2 has been identified, which should enable rational engineering of vector capsids for specific cell targeting. 27 shi et al 28 have already identified specific regions within the capsid protein that can tolerate the insertion of small exogenous peptides and have made an attempt at incorporating integrin-targeting peptides into this region. it has been postulated that aav may not infect antigen-presenting dendritic cells and thereby avoids activation of the host immune system. if this is true, aavs, in contrast to other viruses, may be suitable for repeat administration. the results of repeat administration have been reported to vary greatly and may depend on the host, delivery route and aav serotype tested. 29, 30 aurichio et al 30, 31 have shown that aav2/5 can be readministered once to the mouse lung 5 months after the first delivery. most recently, fischer et al treated nonhuman primates with serial doses (three administrations) of aerosolized aav2. this study goes some way towards demonstrating that repeat administration of aav2 maybe possible, despite increasing titres of neutralizing antibodies. 32 importantly, repeat aerosolization of aav2-cftr into cf patients is safe and well tolerated 22 and phase ii efficacy trials are currently being carried out to determine if repeat administration in humans results in persistent gene expression. the murine parainfluenza virus type 1 (or sendai virus (sev)), the human respiratory syncytial virus (rsv) and the human parainfluenza virus type 3 (piv3) have all been shown to efficiently transfect aecs via the apical membrane 33,34 using sialic acid and cholesterol, which are abundantly expressed on the apical surface of aecs. these viruses have a negative-strand rna genome and replicate in the cytoplasm. they do not go through a dna intermediate and do not enter the nucleus. only sev has been assessed in animal models in vivo and is currently the most efficient virus for airway gene transfer. first-generation recombinant sev carrying cftr cdna can produce functional cftr chloride channels in vitro and after transfection of the nasal epithelium in cf knockout mice. 33 further improvements in the sev vectors have been made by deleting the f-protein from the viral backbone (df), which rendered the second-generation viruses transmission-incompetent. inoue et al 35 have further improved the df/sev vector by introducing mutations into the matrix (m) and hemagglutinin-neuraminidase (hn) proteins, which reduce the amount of virus-like particles that are produced after transfection, thereby further improving the safety profile. sev-mediated gene expression is transient (lasting for about 7 days) and currently repeated administration is inefficient. several groups, including our own, are assessing a variety of immuno-modulatory strategies to improve the use of sev for chronic lung diseases. in contrast to retroviruses, lentiviruses transfect nondividing cells and are, therefore, suitable for transfection of terminally differentiated aecs. the virus stably integrates into the genome of transfected cells and expression is therefore likely to last for the lifetime of the cell (approximately 100 days for aecs). however, when pseudotyped with the commonly used vesicular stomatitis virus g-glycoprotein (vsv-g), lentiviruses can only enter aec via the basolateral membrane, using the inorganic phosphate receptor pit2. importantly, pit2 is also expressed on the apical surface and binds amphotropic virus equally well on both membranes. 36 thus, other as yet unidentified factors contribute to the inefficient transfection of this virus via the apical membrane. vsvg-pseudotyped hiv-derived lentivirus carrying the cftr gene transiently and partially corrected the chloride defect in cf knockout mouse nose for up to 46 days. 37 however, to achieve efficient transfection in the mouse nose, pretreatment with the tight junction opener lysophosphatidylcholine was necessary. gene expression using b-galactosidase as a reporter gene was detected for up to 92 days, without a loss in transgene expression. these results may suggest hiv integration into stem/ progenitor cells in the airways. however, longer followup will be required to determine this since the duration of follow-up (92 days) overlaps closely with the projected lifetime of aecs of about 100 days. it has previously been shown that lentivirus pseudotyped with envelope glycoproteins from the filoviruses ebola or marburg transfect aecs via the apical membrane, and that folate receptor alpha (fra) is a cellular receptor for filoviruses. a recent report has shown that fra is abundantly expressed on the apical surface of primary aecs, but interestingly does not appear to be absolutely required for filovirus uptake into the cells. 38 in the presence of anti-fra-blocking antibodies, virus entry was not affected. this indicates that cellular entry of lentivirus pseudotyped with filovirus glycoproteins is likely more complex than via a single receptor. as mentioned, above members of the paramyxovirus family, such as sev and rsv, transfect aecs very efficiently. this is due to rapid interaction between the f and hn envelope glycoproteins with cholesterol and sialic acid residues on the cell surface, respectively. the f and hn proteins are therefore promising candidates for pseudotyping lenitviruses and kobayashi et al 39 have recently described successful incorporation of f and hn envelope proteins into the capsid from simian immunodeficiency virus (siv). this vector was able to transduce polarized epithelial cells from both the apical and basolateral sides and we are currently evaluating this vector for airway transduction in animal models. importantly, unless lentiviral vectors are able to hit airway stem cells efficiently, they will likely need to be re-administered and therefore will face the same immune-response problems as other viral vectors. improving the efficiency of nonviral gene transfer to aecs has been a major focus with a variety of strategies being followed. several groups are modifying polyplexes such as polylysine and polyethylenimine (pei) by adding sugars, based on the rationale that aecs express lectins, which selectively bind and internalize glycoconjugates. although glycoconjugates containing lactose have been efficient in cell culture, [40] [41] [42] their efficacy in vivo remains to be demonstrated. receptor-mediated gene delivery has been developed for aecs by targeting the serpin-enzyme complex receptor (sec-r). 43 this receptor is responsible for the uptake of serine proteases bound to their cognate inhibitors into cells. the receptor recognizes a conserved five-amino-acid-binding motif, but tolerates large variation in the attached cargo. sec-r-directed complexes are prepared by condensing plasmid dna with a covalent conjugate of a peptide receptor ligand (17 amino acids) and polylysine. ziady et al 43 have recently demonstrated partial correction of the chloride transport defect in the nasal epithelium of cf knockout mice following administration of sec-r ligand complexed to a cftr plasmid. in nondividing cells, the nuclear membrane appears to be an important barrier to gene transfer and one reason why sec-r ligand polylysine complexes transfect airway cells efficiently might be their small size. with a diameter of 18-25 nm, these nanoparticles may be able to enter the nucleus via passive diffusion through the nuclear pore complex, which has a cutoff size of about 25 nm. however, formulation and stability problems have so far prevented phase i clinical trials. peptides resembling integrin-binding domains have also been linked to plasmid dna and have been shown to transfect the airway epithelium of pigs when delivered at bronchoscopy. 44 it remains to be established if antipeptide immune responses will interfere with using peptidecarrying nonviral formulations for chronic diseases, but the risk of immune responses against the peptide can be minimized by using conserved human peptide sequences. importantly, traditionally used animal models may not be suitable to evaluate efficiency or repeat administration of human peptide formulation, if the chosen sequence is not conserved within the animal model. another nanoparticle formulation, consisting of a single plasmid molecule compacted with polyethyleneglycol (peg)-substituted polylysine (polymer of 30 lysines) has been developed. these dna nanoparticles have a rod-like structure (12-15 nm diameter, 100-150 nm length). a single-dose escalation study to evaluate the safety of nasal administration into cf patients has recently been carried out in 12 subjects. in addition to assessing safety, secondary end points included assessment of electrical correction of the ion transport defects and molecular analysis for the presence of vector-specific dna and mrna. administration of the nanoparticles was considered safe. in most patients, plasmid dna could be detected in at least one nostril. there was no evidence of vector-specific mrna in any patient, which may have been due to insufficient sensitivity of the assay. partial correction of the chloride transport defect was demonstrated in seven out of 12 patients, which persisted for up to 15 days. 45 although these initial results are encouraging, further phase ii trials will be necessary ultimately to determine the efficacy of these particles. in addition to improving nonviral dna condensing agents, several groups are improving the plasmid vectors for nonviral gene transfer. yew et al 46 have demonstrated that reduction in cpg motifs in the pdna reduces the immunostimulatory capacity of pdna after systemic administration of liposome/pdna complexes. fewer changes in blood parameters of toxicity, reduced levels of inflammatory cytokines and decreased liver damage were observed after depletion of 80% of the cpg motifs. in addition, gene expression was prolonged in immunocompetent mice. similar results were observed after topical administration of liposome/pdna complexes to the lung (rk scheule, personal communication). gill et al 47 have studied the effect of different promoters on persistence of lung gene expression by comparing the frequently used human immediate-early cytomegalovirus (cmv) promoter to the constitutive endogenous polyubiquitin c (ubc) and elongation factor 1a (ef1a) promoters. although both eukaryotic endogenous promoters lead to about 10-fold less transgene expression at day 2, duration of gene expression was significantly improved when 'naked' pdna was administered to the lung (cmv: o1 week, ubc: 416 weeks) and ubc-mediated gene expression reached cmv day 2 levels approximately 4 weeks after transfection. similar results were reporter by yew et al 48 using the ubiquitin b (ubb) promoter. promoter silencing is likely to contribute to these results and it has previously been demonstrated that the cmv promoter is silenced by tnfa and infg, which are both upregulated after gene transfer. however, it is currently unknown why the ef1a and ubc promoters are more resistant to gene silencing. despite the comparatively low transfection efficiency, nonviral gtas offer important advantages over viral gta for chronic disease. we and others are currently assessing a variety of physical delivery methods, including electroporation, magnetism, ultrasound and vibration, in an attempt to increase the transfection efficiency of nonviral formulations. electroporation has been successfully used to enhance transfection in a variety of organs including muscle. initial results for lung gene transfer are encouraging and demonstrate that the transfection efficiency of naked dna can be enhanced in the presence of electrical fields 49 (and ian pringle, personal communication). clearly, important technical questions and safety considerations have to be resolved. systemic delivery has long been postulated as a means for lung transfection and intravenous (i.v.) injection of many nonviral gtas leads to lung transfection. it is important to note that for the vast majority of gtas gene transfer is only achieved in alveolar endothelial cells and maybe pneumocytes, because the gta gets trapped in the alveolar capillaries of the pulmonary circulation, the first capillary bed encountered after i.v. administration, but are found only rarely in the conducting airways, which are the targets for cf gene therapy. to be able to transfect the conducting airway epithelium, the gta has to pass through the pulmonary circulation, reach the left side of the heart and travel from there to the bronchial circulation, which supplies the airways (figure 3) . here, the gta has to escape from the vessels and migrate through a dense layer of extracellular matrix to the basement membrane of the aecs. we have recently demonstrated that naked oligonucleotides are able to follow this route and transfect the cytoplasm of aecs efficiently. 50 koehler et al 51 have shown that the bronchial epithelium and submucosal glands can be transfected using plasmid dna complexed to the cationic liposome dodac:dope, although this was not reproducible with other lipids and appears to be a characteristic property of this particular liposome. a better understanding of liposome structure and charge interaction in the context of serum proteins will help with the rational design of nonviral gta for systemic cf gene therapy. the addition of ligands for receptor-mediated uptake may also improve the transfection efficiency of aecs after i.v. delivery and proof-of-principle for this concept has been published several years ago. ferkol et al 52 have shown that addition of ligands to the polymeric immunoglobulin receptor increases aecs transfection following systemic administration. the addition of moieties to increase organ and cell-type specific targeting will be important to minimize systemic gene transfer and toxicity. alternative non-cftr cdna nucleotide-based therapies gene repair of the endogenous cftr gene has two major advantages over traditional gene therapy. if successful, gene repair should ensure gene expression for the lifetime of the cells and appropriate control of gene expression is likely because the endogenous cftr promoter is utilized. our preliminary results indicated that the genomic cftr locus could be modified in primary rat hepatocytes, but not primary aecs, using chimeraplasts (dna/rna hybrid oligonucleotides). 53 hepatocytes have previously been shown to be easily amenable for gene repair strategies, most likely due to efficient uptake of repair molecules into the nucleus. in addition, a similar approach using small-fragment homologous recombination (sfhr) was able to reintroduce the wild-type cftr sequence into the lungs of cf knockout mice. 54 overall, the mechanisms involved in figure 3 schematic presentation of the pulmonary and bronchial circulation transporting gene transfer agents to the airway epithelium after intravenous injection (courtesy of steve smith, department of gene therapy, imperial college london). gene therapy for cystic fibrosis u griesenbach et al gene repair are not well understood and it is currently uncertain if the required 'repair' proteins are present in terminally differentiated aecs. in addition, uptake of repair oligonucleotides into the nucleus of aecs ex vivo and in vivo remains inefficient (uta griesenbach, unpublished observation) and is the first hurdle that needs to be overcome, before gene repair can be assessed. downregulation of gene expression through antisense molecules may be of therapeutical benefit in cf patients. lambert et al 55 showed that antisense inhibition of the b-cell antigen receptor-associated protein (bap) 31 increased expression of both wild-type cftr and mutant cftr and partially restored cftr chloride channel function. the exact function of bap31 is unclear, although the authors speculated that the protein may be involved in retaining mutant cftr in the er. several other chaperone proteins, mucins or the epithelial sodium channel (enac), which is hyperactive in cf, may be suitable candidates for antisense strategies. we have recently assessed rna interference-mediated gene silencing in the lungs in vivo, and although proof-ofprinciple could be demonstrated efficiency was low, 56 likely due to low transfection efficiency. splicosome-mediated trans-splicing (smart) has recently been introduced as a means to generate wild-type cftr mrna in cf xenograft models. cells were transfected with very high titres of adenovirus that produced the so-called pre-therapeutic wild-type cftr mrna molecules (ptms), which are designed to promote trans-splicing with the endogenous cftr mrna. 57 similar to gene repair, smart ensures celltype-specific expression of wild-type cftr mrna; however, the technology requires further optimization with respect to efficiency and specificity. the choice of the correct animal model is a crucial factor in developing gene therapy for cf. currently, the cf knockout mouse is the only cf animal model and although these mice do not develop the characteristic cf lung disease, they have the same ion transport defect as cf patients in their nasal epithelium. this, combined with the fact that the nasal epithelium can easily be exposed to gtas, makes the cf mouse nose an ideal organ for assessing and optimizing gene transfer. in addition, non-cf primates, 58,59 pigs 44 and most recently sheep 60 have been used to optimize airway gene transfer and allowed clinically relevant delivery methods such as nebulization to be assessed. more recently, first attempts have been made at generating cf ferret and sheep based on targeting of the cftr locus in somatic cells coupled with nuclear transfer 61 (and jim mcwhir, roslin institute, personal communication). the success of pre-clinical and clinical cf gene therapy studies stands and falls with the assays used to evaluate gene transfer. the development of new cftr-specific assays involving epithelial cell-specific detection of cftr mrna and protein, bacterial adherence to aecs, airway surface liquid height measurements and others are currently a major focus of the uk cystic fibrosis gene therapy consortium (www.cfgenetherapy.org.uk). for clinical studies, the most relevant end points are a reduction in decline of lung function over time and of episodes of infection. however, these end points are not suitable for early phase ii trials, because large patient numbers (4500) and long follow-up (412 months) would be required. 62 it is therefore crucial to identify clinical surrogate end points (such as bacterial burden, inflammatory markers and imaging) that can be assessed in smaller patient cohorts with shorter follow-up. it is unlikely that one-time administration of a short acting gta will change these clinical surrogate end points, but will more likely require repeat administration and it is therefore important to design future gene therapy trials with these surrogate end points in mind. an extensive discussion about assays is outside the scope of this review, but has recently been reviewed. 63 over the last decade, it became apparent that gene transfer to the aecs is difficult. this is perhaps unsurprising, given the lung has evolved to keep foreign particles out. the major obstacle for most viral gtas is the effective immune surveillance mechanisms in the lung, which prohibit repeat administration. many strategies to overcome this problem have already been explored, but have not yet been successful. in our view, this may be a difficult hurdle to overcome. nonviral gene transfer has traditionally been inefficient, but recently developed nanoparticles and ligand-targeting appear to be overcoming this problem. importantly, physical delivery methods to increase nonviral gene transfer are currently being assessed in the lung. although gene therapy for cf is not yet a clinical reality, the many innovative strategies currently being assessed should lead to efficient and repeatable airway gene transfer within the next few years. the cells of the pulmonary airways the molecular and metabolic basis of inherited disease identification of the cystic fibrosis gene: cloning and characterization of complementary dna non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer expression of the cystic fibrosis gene in adult human lung immunological hurdles to lung gene therapy delivery of gene transfer vectors to lung: obstacles and the role of adjunct techniques for airway administration barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract aerosol delivery of a beta-galactosidase adenoviral vector to the lungs of rodents adenovirus-mediated persistent cystic fibrosis transmembrane conductance regulator expression in mouse airway epithelium safety of local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. i. methods, safety, and clinical implications basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia enhancement of adenovirus-mediated gene transfer to the airways by deae dextran and sodium caprate in vivo airway epithelial cftr mrna expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus reduced inflammation and improved airway expression using helper-dependent adenoviral vectors with a k18 promoter a phase i study of aerosolized administration of tgaavcf to cystic fibrosis subjects with mild lung disease efficient and persistent gene transfer of aav-cftr in maxillary sinus repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial expanding aav packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison efficient mouse airway transduction following recombination between aav vectors carrying parts of a larger gene serological characterisation of human and non-human primate aavs adeno-associated virus type 5 (aav5) but not aav2 binds to the apical surfaces of airway epithelia and facilitates gene transfer the atomic structure of adeno-associated virus (aav-2), a vector for human gene therapy rgd inclusion in vp3 provides adenoassociated virus type 2 (aav2)-based vectors with a heparan sulfate-independent cell entry mechanism repeated delivery of adeno-associated virus vectors to the rabbit airway transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins successful transgene expression with serial doses of aerosolized raav2 vectors in rhesus macaques recombinant sendai virus-mediated cftr cdna transfer rsv and piv3 target human ciliated airway epithelial cells: efficient gene transfer vectors for cystic fibrosis lung disease a new sendai virus vector deficient in the matrix gene does not form virus particles and shows extensive cell-tocell spreading apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha pseudotyped lentivirus vectors derived from simian immunodeficiency virus sivagm with envelope glycoproteins from paramyxovirus sugar-mediated uptake of glycosylated polylysines and gene transfer into normal and cystic fibrosis airway epithelial cells uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells lactosylated poly-l-lysine targets a potential lactose receptor in cystic fibrosis and non-cystic fibrosis airway epithelial cells functional evidence of cftr gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of dna complexes targeted to the serpin-enzyme complex receptor evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector single dose escalation study to evaluate safety of nasal administration of cftr001 gene transfer vector to subjects with cystic fibrosis reduced inflammatory response to plasmid dna vectors by elimination and inhibition of immunostimulatory cpg motifs increased persistence of lung gene expression using plasmids containing the ubiquitin c or elongation factor 1alpha promoter high and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter electroporation-mediated transfer of the na-k-atpase b1 subunit safely increases alveolar fluid clearance in rat lungs towards nucleic acid transfer to the airway epithelium via the systemic route targeting transgene expression for cystic fibrosis gene therapy gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor conversion of wild-type cftr to the g551d mutation in primary rat hepatocytes using rna/dna oligonucleotides targeted replacement of normal and mutant cftr sequences in human airway epithelial cells using dna fragments control of cystic fibrosis transmembrane conductance regulator expression by bap31 lacz sirna and antisense dna do not decrease b-galactosidase expression in the airways of k18-lacz mice partial correction of endogenous deltaf508 cftr in human cystic fibrosis airway epithelia by spliceosome-mediated rna trans-splicing deposition and expression of aerosolized raav vectors in the lungs of rhesus macaques gene therapy for cystic fibrosis with aerosolized adenovirus-cftr: characterization of the aerosol and scintigraphic determination of lung deposition in baboons transfection efficiency and toxicity following delivery of naked plasmid dna and cationic lipid-dna complexes to ovine lung segments development of a ferret model of cystic fibrosis identifying treatments that halt progression of pulmonary disease in cystic fibrosis preclinical and clinical endpoint assays for cystic fibrosis gene therapy key: cord-009774-tqhexzdp authors: neyman, greg; irvin, charlene babcock title: a single ventilator for multiple simulated patients to meet disaster surge date: 2008-06-28 journal: acad emerg med doi: 10.1197/j.aem.2006.05.009 sha: doc_id: 9774 cord_uid: tqhexzdp objectives to determine if a ventilator available in an emergency department could quickly be modified to provide ventilation for four adults simultaneously. methods using lung simulators, readily available plastic tubing, and ventilators (840 series ventilator; puritan‐bennett), human lung simulators were added in parallel until the ventilator was ventilating the equivalent of four adults. data collected included peak pressure, positive end‐expiratory pressure, total tidal volume, and total minute ventilation. any obvious asymmetry in the delivery of gas to the lung simulators was also documented. the ventilator was run for almost 12 consecutive hours (5.5 hours of pressure control and more than six hours of volume control). results using readily available plastic tubing set up to minimize dead space volume, the four lung simulators were easily ventilated for 12 hours using one ventilator. in pressure control (set at 25 mm h(2)o), the mean tidal volume was 1,884 ml (approximately 471 ml/lung simulator) with an average minute ventilation of 30.2 l/min (or 7.5 l/min/lung simulator). in volume control (set at 2 l), the mean peak pressure was 28 cm h(2)o and the minute ventilation was 32.5 l/min total (8.1 l/min/lung simulator). conclusions a single ventilator may be quickly modified to ventilate four simulated adults for a limited time. the volumes delivered in this simulation should be able to sustain four 70‐kg individuals. while further study is necessary, this pilot study suggests significant potential for the expanded use of a single ventilator during cases of disaster surge involving multiple casualties with respiratory failure. a fter the events of september 11, 2001, and the recent hurricanes in the gulf coast, there has been a focus on anticipating the need for medical care for large numbers of victims. 1 addressing surge capacity requires a multitiered approach involving local and federal agencies as well as resource management (i.e., personnel, patient space, supplies, and special equipment). recent experiences have shown that hospitals may be rapidly extended to operate at 120%-130% capacity. 2 depending on the nature of the disaster, many otherwise plentiful hospital supplies, such as ventilators, may suddenly become insufficient to support the demand. 3 in the event of a large influx of patients in respiratory distress (e.g., a large outbreak of botulism), the number of ventilators available may not be enough to support all of the patients. while government resources would eventually be available, there may be a time when hospitals will need to provide ventilatory support to a greater number of patients than the available number of ventilators. manual ventilation (''bagging'') is possible, but it is possible that the additional personnel required would not be available. some institutions have begun to stockpile disposable automatic ventilators for use in the event of a disaster. 4 one commercially available device is vortran's automatic resuscitator, a single-patient, single-use, pressurepowered ventilator. it could be kept in stock and deployed when necessary. it runs on wall oxygen (50 psi) and is pressure cycled off the wall oxygen source, and seven can be run simultaneously off a single oxygen supply line by using the vortran e-vent case multioutlet manifold device. one advantage is that this type of automatic ventilator is gas driven (from the oxygen source) and requires no electricity (this may be an advantage in certain types of disasters), is disposable, and is modifiable to provide ventilation for up to seven individuals off one oxygen source. however, this device requires anticipatory purchase, and it lacks the computerized monitoring that standard ventilators have, necessitating more intensive staff support. 5 another option for providing increased ventilation capacity includes a proprietary device (patent pending) that is essentially a control system for splitting one ventilator to provide ventilation for two patients. 6 this does not have the advantages of the previously described disposable ventilator, because it would rely on an electrically driven ventilator. additionally, it would require the purchase of additional equipment (the proprietary control system). although larger urban hospitals may be able to justify the resources to stockpile disposable ventilators, smaller hospitals may not. in the event of a need for more ventilators than are currently available in a hospital, it may be valuable to explore methods to maximally utilize the ventilators that are already available. hospitals do have generators and would likely be able to support electrical service for a short time after the disaster. given this potential, it may be appropriate to consider a simple modification of the currently available hospital ventilators to provide more patients with ventilator support. our hypothesis was that using simple, rapidly deployable modifications, a single ventilator could be used to ventilate multiple casualties when the number of victims exceeds the number of ventilators. this was a simulator-based pilot study. this study was considered exempt after review by the institutional review board. four sets of standard ventilator tubing (hudson) were connected to a single ventilator (puritan-bennett, 840 series) via two flow splitters (one on the patient inflow limb of the circuit, and one on the patient exhaust limb). each flow splitter was constructed of three briggs t-tubes with included connection adapters (hudson) (figure 1) , with the valves removed. the briggs t-tube is utilized clinically (and generally available) for flow-by oxygen or humidity for a patient with an endotracheal or tracheos-tomy tube, or for in-line aerosol treatments of ventilated patients. the t-tubes were arranged so that the two side ports of a central t-tube were attached to the bottom ports of the two side t-tubes via adapters that come with the t-tube. the final configuration of the three t-tubes is seen in figure 2 (with a trimmed section of standard ventilation tubing at the hub for connection to the ventilator); it allowed for air flowing from the ventilator to be split evenly to four simulated patients and for the air returning from the four patients to flow back into the one exhaust port on the ventilator. the ventilator tubing was run from the inflow splitter to the outflow splitter, with four test lungs (puritan-bennett) in the center. the test lungs were used to simulate one patient each on the modified ventilator circuit. the final configuration was a simulation of four patients on a single ventilator in parallel operation (figure 3 ). to test this circuit, a time frame was arbitrarily chosen as approximately six hours. there were two reasons for this. first, this is a simple feasibility study, and we would expect someone to inspect the system at least once in six hours if ever used in a real disaster. second, we realize that beyond this feasibility study, animal studies are needed, and this could allow for observation of the function of this circuit for a longer period. finally, in many potential disaster situations, by six hours additional support may be available. pressure control operation was randomly selected (via coin toss) to precede volume control. to approximate physiologic parameters, the ventilator settings were dialed to a peak pressure of 25 cm h 2 o, 0 cm of positive end-expiratory pressure, and a respiratory rate of 16 breaths/min. the ventilator software chose an inspiratory/expiratory ratio of 1:2 automatically. after cumulative random interval inspections, total pressure control operation was 5 hours 33 minutes. volume control settings of 2,000 ml tidal volume (500 ml per test lung) and a respiratory rate of 16 breaths/min were chosen to approximate physiologic parameters. the ventilator software chose an inspiratory/expiratory ratio of 1:1 automatically. after cumulative random interval inspections, total volume control operation was 6 hours 11 minutes. during operation, the circuit was inspected at random intervals in between examinations of patients in a busy metropolitan emergency department to simulate the intervals that the circuit would be inspected in a mass casualty event. the inspections occurred approximately every 23 (ae18) minutes, and the ventilator display readouts were recorded. simultaneously, the lungs were subjectively inspected for symmetry of excursion and evidence of respiratory stacking. specifically, the examiner monitored for asymmetric inflation of individual test lungs and incomplete deflation before subsequent inflation. after the configuration was sealed, the ventilator system did not alarm. visual inspection showed roughly equivalent excursion of all lung models. no respiratory stacking was seen. averages of ventilator display readout samplings over the course of the study are presented in table 1 . a four-patient configuration operated successfully on a single ventilator for almost 12 hours. pressures did not exceed 35 cm h 2 o. airway pressures beyond 35 mm h 2 o are associated with ventilator-induced lung injury. 7 individual tidal volumes reached 471-507 ml, which approximates 7 ml/kg for a 70-kg individual. studies have shown that ventilation with 6-8 ml/kg is associated with improved outcome in injured lungs. 8 no evidence of respiratory stacking or preferential filling of individual lung simulators was observed. the chief limitation of this study is that it is a simulator study. therefore, only successful physical ventilation could be demonstrated. adequate oxygenation and the potential for ventilator-associated lung injury could not be addressed. the presumption of equal ventilation to all four lung simulators presumed equal lung physiology. a patient with asthma with greater resistance to ventilations may not receive equal ventilation with this system. further animal studies are necessary to address this concern. the inability to directly measure volumes delivered to the individual test lungs may bias the results and thus change the actual method in which this ventilator configuration would be deployed. potential infectious complications from sharing one ventilator were not investigated. again, further study in this area would be beneficial. because this was a pilot study, further research is indicated to test the efficacy and safety of the modified circuit. replication of the study in an animal model is indicated. ventilator software may allow for ventilation of more patients than was explored in this study. this may be an important consideration in usefulness of ventilators in the disaster situation. finally, development of quantitative measurement techniques of individual tidal volumes transferred would enhance further research efforts as well as clinical delivery. this pilot study suggests that the physics of a ventilator/ patient circuit could accommodate more than one american college of emergency physicians. health care system surge capacity recognition, preparedness, and response health care facility and community strategies for patient care surge capacity concept of operations for triage of mechanical ventilation in an epidemic yale new haven health automatic resuscitator (var) user's guide artificial ventilation of the lungs for emergencies principles of mechanical ventilation nadel's textbook of respiratory medicine management of post traumatic respiratory failure the authors thank the entire respiratory therapy department at st. john's medical center (detroit, mi) and dr. peter hoffmann (physics department, wayne state university, detroit, mi) for consultation regarding fundamental physics. key: cord-000492-ec5qzurk authors: devaney, james; contreras, maya; laffey, john g title: clinical review: gene-based therapies for ali/ards: where are we now? date: 2011-06-20 journal: crit care doi: 10.1186/cc10216 sha: doc_id: 492 cord_uid: ec5qzurk acute lung injury (ali) and acute respiratory distress syndrome (ards) confer substantial morbidity and mortality, and have no specific therapy. the accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ali/ards, suggest that the disease may be amenable to gene-based therapies. ongoing advances in our understanding of the pathophysiology of ali/ards have revealed multiple therapeutic targets for gene-based approaches. strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ali/ards have all demonstrated promise in preclinical models. despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ali/ards remains to be realized. multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ali/ards to the clinical setting. gene-based therapy involves the insertion of genes or smaller nucleic acid sequences into cells and tissues to replace the function of a defective gene, or to alter the production of a specifi c gene product, in order to treat a disease. gene therapy can be classifi ed into germline and somatic gene therapies. germline approaches modify the sperm or egg prior to fertilization and confer a stable heritable genetic modifi cation. somatic gene approaches use gene therapy to alter the function of mature cells. commonly used somatic gene therapy strategies include the overexpression of an existing gene and/or the insertion of smaller nucleic acid sequences into cells to alter the production of an existing gene. ali/ards may be suitable for gene-based therapies as it is an acute but relatively transient process [8] , requiring short-lived gene expression, obviating the need for repeated therapies and reducing the risk of an adverse immunological response. th e distal lung epithelium is selectively accessible via the tracheal route of administration, allowing targeting of the pulmonary epithelium [9] . th e pulmonary vasculature is also relatively accessible, as the entire cardiac output must transit this circulation. antibodies that bind antigens selectively expressed on the pulmonary endothelial surface can be complexed to gene vectors to facilitate selective targeting following intravenous administration [10] . it is also possible to use gene-based strategies to target other cells central to the pathogenesis of ali/ards, such as leuko cytes and abstract acute lung injury (ali) and acute respiratory distress syndrome (ards) confer substantial morbidity and mortality, and have no specifi c therapy. the accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ali/ ards, suggest that the disease may be amenable to gene-based therapies. ongoing advances in our understanding of the pathophysiology of ali/ards have revealed multiple therapeutic targets for genebased approaches. strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ali/ards have all demonstrated promise in preclinical models. despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ali/ ards remains to be realized. multiple barriers to eff ective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection effi ciency of nonviral delivery methods. defi cits remain in our knowledge regarding the optimal molecular targets for genebased approaches. encouragingly, recent progress in overcoming these barriers off ers hope for the successful translation of gene-based approaches for ali/ards to the clinical setting. fi bro blasts [11] . furthermore, gene-therapy-based approaches off er the potential to selectively target diff erent phases of the injury and repair process. th e potential to target specifi c aspects of the injury and repair processes such as epithelial-mesenchymal transition, fi brosis, fi brinolysis, coagulopathy and oxidative stress with these approaches is also clear. gene therapy requires the delivery of genes or smaller nucleic acid sequences into the cell nucleus using a carrier or vector. th e vector enables the gene to overcome barriers to entry into the cell, and to make its way to the nucleus to be transcribed and translated itself or to modulate transcription and/or translation of other genes. both viral and nonviral vector systems have been developed (table 1) . viral vectors are the most eff ective and effi cient way of getting larger nucleic acid sequences, particularly genes, into cells (table 1) . th e viral genome is modifi ed to remove the parts necessary for viral replication. th is segment is then replaced with the gene of interesttermed a transgene -coupled to a promoter that drives its expression. th e modifi ed genome is then encapsulated with viral proteins. following delivery to the target site, the virus binds to the host cell, enters the cytoplasm and releases its payload into the nucleus (figure 1 ). th e size of trans gene that can be used depends on the capsid size. a number of diff erent viral vectors have been used in preclinical lung injury studies to date. adenoviruses have double-stranded dna genomes, have demonstrated promise in preclinical models [12, 13] and are well tolerated at low to intermediate doses in humans [14, 15] . advantages include their ease of production, the high effi ciency at which they can infect the pulmonary epithelium [14, 16] and that they can deliver relatively large transgenes. a disadvantage of adenoviruses is their immunogenicity, particularly in repeated doses [14] . newer adenoviral vectors, in which much of the immuno genicity has been removed, hold promise [17] . while adenovirus-mediated gene transfer in the absence of epithelial damage is relatively ineffi cient [18] , this may be less of a problem in ali/ards that is characterized by widespread epithelial damage. adeno-associated viruses (aavs) are single-stranded dna parvoviruses that are replication defi cient [19] . a substantial proportion of the human population has been exposed to aavs but the clinical eff ects are unknown. aav vectors have a good safety profi le, and are less immunogenic compared with other viruses, although anti bodies do develop against aav capsid proteins that can compromise repeat administration. aav vectors can insert genes at a specifi c site on chromosome 19 . th e packaging capacity of the virus is limited to 4.7 kb, restricting the size of the transgene that can be used. aavs are less effi cient in transducing cells than adenoviral vectors. successful aav vector gene transfer has been demon strated in multiple lung cell types including lung progenitor cells, in both normal and naphthaleneinduced ali lungs [20] . aav serotypes have specifi c tissue tropisms, due to diff erent capsid proteins that bind to specifi c cell membrane receptors. aav-5 [21] and avv-6 [22] exhibit enhanced tropism for the pulmonary epi thelium [21, 22] . aavs can transduce nondividing cells and result in long-lived transgene expression. aav vectors have been used in clinical trials in cystic fi brosis patients, underlining their safety profi le [23, 24] . th ese rna viruses can transfect nondividing cells such as mature airway epithelial cells [25] . th e virus stably but randomly integrates into the genome and expression is likely to last for the lifetime of the cell (~100 days). th e transgene can be transmitted post mitosis, and there is also a risk of tumorigenesis if the transgene integrates near an oncogene. th e development of leukemias in children following gene therapy for severe combined immunodefi ciency highlights this risk [26, 27] . while lentiviral vectors may be useful to correct a gene defi ciency associated with increased risk of ali, the long-lived gene expression of lentiviral delivered genes may be more suitable for chronic diseases than for ali/ards. nonviral delivery systems, while generally less effi cient than viral vectors in transfecting the lung epithelium, are increasingly used to deliver smaller dna/rna molecules (table 1 ). strategies include the use of dna-lipid and dna-polymer complexes and naked dna/rna oligonucleotides, such as sirna [28] , decoy oligo nucleo tides [29] and plasmid dna [30] . nonviral delivery systems are less immunogenic than viral vector-based approaches, and can be generated in large amounts at relatively low cost. plasmid vectors are composed of closed circles of doublestranded dna. as naked and plasmid dna contain no proteins for attachment to cellular receptors, there is no specifi c targeting to diff erent cell types and thus it is essential that the dna is placed in close contact with the desired cell type. th ese limitations make this approach less relevant clinically. th e therapeutic dna is held within a sphere of lipids, termed a lipoplex, or within a sphere of polymers, such as polyethyleneimine, termed a polyplex. lipoplexes and polyplexes act to protect the dna, facilitate binding to the target cell membrane and also trigger endocytosis of the complex into the cell, thereby enhancing gene expression. th ese systems can be modifi ed to include a targeting peptide for a specifi c cell type, such as airway epithelial cells [31] . th ese complexes effi ciently and safely transfect airway epithelial cells [31] , and they have demonstrated promise in human studies [32] . sirnas are dsrna molecules of 20 to 25 nucleotides that can regulate the expression of specifi c genes. specifi c sirnas reduce infl ammation-associated lung injury in table 1 . viral vector-delivered gene therapy relatively easily produced immunogenic [14] adenoviral transfer of genes for a surfactant (dsdna genome) effi ciently transfect lung enzyme [49] , angiopoietin-1 [51] , hsp-70 [52] , epithelium [14, 16] apolipoprotein a-1 [53] , and na + ,k + -atpase pump can deliver larger genes [55] genes attenuate experimental ali well tolerated in lower doses [1, 3] adenoviral delivery of il-10 gene attenuates zymosan ali at low doses, but is harmful at high doses [58] adeno-associated virus good safety profi le; less limited transgene size aav vector gene transfer demonstrated in multiple vectors (ssdna genome) immunogenic diffi cult to produce in large lung cell types including progenitor cells in both inherently replication defi cient quantities normal lungs and following naphthalene-induced aav-5 and aav-6 lung epithelial ali [20] tropism [10, 11] transduce nondividing cells long-lived gene expression used in clinical trials for cf [12, 13] lentivirus vectors transduce nondividing cells [25] oncogenesis risk due to lentiviral transfer of shrna to silence cd36 gene (rna genome) integrate stably but randomly integration into genome expression suppresses silica-induced lung fi brosis into the genome [26, 27] in the rat [35] nonviral gene-based strategies plasmid transfer (closed easily produced at low cost no specifi c cell targeting electroporation-mediated gene transfer of the dsdna circles) very ineffi cient na + ,k + -atpase rescues endotoxin-induced lung injury [60] nonviral dna complexes complexes protect dna less effi cient than viral vectors cationic lipid-mediated transfer of the na + ,k + -(lipoplexes or polyplexes) complexes facilitate cellular atpase gene ameliorated high-permeability targeting [31] pulmonary edema [59] lipoplex-delivered il-10 gene decreased clp-induced ali [61] systemic cationic polyethylenimine polyplexes incorporating indoleamine-2,3-dioxygenase decreased ischemia-reperfusion ali [62] dna and rna easily produced at low cost no specifi c cell targeting specifi c sirnas reduce infl ammation-associated oligonucleotides (sirna, smaller molecules that can lung injury in humans [33] and in animal models shrna, decoy easily enter cells [28, 34] oligonucleotides) target regulation of specifi c genes shrna-based approaches have reduced lung injury in animal models [35, 36] cell-delivered gene therapy humans [33] and in animal models [28, 34] . shrna is a single strand of rna that, when introduced into the cell, is reverse transcribed and integrated into the genome, becoming heritable. during subsequent transcription, the sequence generates an oligonucleotide with a tight hairpin turn that is processed into sirna. shrnas have reduced lung injury in animal models [35, 36] . decoy oligonucleotides are double-stranded dna molecules of 20 to 28 nucleo tides, which bind to specifi c transcription factors to reduce expression of targeted genes, and have been successfully used in animal models [37, 38] . an alternative approach is to use systemically delivered cells to deliver genes to the lung. th is approach has been used to enhance the therapeutic potential of stem cellssuch as mesenchymal stem/stromal cells, which demon strate promise in preclinical ali/ards models [39] . fibroblasts have also been used to successfully deliver genes to the lung to attenuate ali [40] . preliminary data from a clinical trial in pulmonary hypertension show that endothelial progenitor cells overexpressing endothelial nitric oxide synthase (nos3) decrease pulmonary vascular resistance [41] , highlighting the potential of cell-delivered gene therapy for ali/ards. nebulization of genetic material into the lung is eff ective [42] , safe and well tolerated [32, 43, 44] . th e integrity of aav vectors [9, 43] and adenoviral virus vectors [44] are maintained post nebulization, as are cationic lipid vectors [32] and dna and rna oligonucleotides [45] . a number of gene therapy clinical trials have utilized nebulization to deliver the transgene to the lung [23, 43] , but without clear clinical benefi t to date [43, 44] . intravascular delivery approaches target the lung endothelium. th ese approaches have been successfully used in preclinical studies of cell-based gene therapies [39, 40] , and also with vectors that incorporate components such as antibodies to target antigens on the lung endothelium [10] . successful gene-based therapies require the delivery of high quantities of the gene or oligonucleotide to the pulmonary epithelial or endothelial surface, require effi cient entry into the cytoplasm of these large and insoluble nucleic acids, which then have to move from the cytoplasm into the nucleus, and activate transcription of its product. multiple barriers exist that hinder this process, not least the natural defense mechanisms of the lung, and additional diffi culties that exist in transducing the acutely injured lung (table 2 ). limitations regarding delivery technologies and defi ciencies in our knowledge regarding the optimal molecular targets also reduce the effi cacy of these approaches. th e lung has evolved eff ective barriers to prevent the uptake of any inhaled foreign particles [46] . while advantageous in minimizing the potential for uptake of external genetic material (for example, viral dna), these barriers make it more diffi cult to use gene-based therapies in the lung. barriers to entry of foreign genetic material into the lung include airway mucus and the epithelial lining fl uid, which traps and clears inhaled material. th e glycocalyceal barrier hinders contact with the cell membrane, while the tight intercellular epithelial junctions and limited luminal endocytosis further restrict entry of foreign material into the epithelial cells. transducing the acutely injured lung may be diffi cult, due to the presence of pulmonary edema, consolidated or collapsed alveoli, and additional extracellular barriers such as mucus. gene-based therapies targeted at the pulmonary epithelium may be less eff ective where there is extensive denudation of the pulmonary epithelium, as may occur in primary ards. encouragingly, there is some evidence to suggest that ali may not substantially impair viral gene transfer to the alveolar epithelium [47] . th e key limitation of nonviral vector approaches has been their lack of effi ciency in mediating gene transfer and transgene expression in the airway epithelium. viral vectors are immunogenic, due to the protein coat of the viral vector, and the immune response is related to both vector dose and number of administrations. th e potential to limit administration to a single dose in ali/ards may reduce this risk. however, the development of an infl amma tory response resulting in death following administration of a fi rst-generation adenoviral vector highlights the risks involved [48] . additional limitations of viral vectors include transgene size, which is limited by the size of the capsid that encloses the viral genes. th e therapeutic potential of gene therapy for ali/ards is underlined by a growing body of literature demon strating effi cacy in relevant preclinical models. in considering the clinical implications of these studies, it is important to acknowledge that animal models of ards do not fully replicate the complex pathophysiological changes seen in the clinical setting. th is is highlighted by the fact that many pharmacologic strategies demonstrating considerable promise in preclinical studies were later proven ineff ective in clinical trials. nevertheless, these studies provide insights into the clinical potential of these strategies. adenovirus-mediated transfer of a gene that enhances surfactant production improves lung function and confers resistance to pseudomonas aeruginosa infection ( figure 2 ) [49] . adenovirus-delivered superoxide dismutase and catalase genes protected against hyperoxic-induced, but not ischemia-reperfusion-induced, lung injury [50] . more recent studies have demonstrated the therapeutic potential of overexpression of a number of genes, including angio poietin-1 [51] , hsp-70 [52] , apolipo protein a-1 [53] , defensin î²2 [54] and the na + ,k + -atpase pump [55] . in contrast, overexpression of il-1î² can directly cause ali [56] , while overexpression of suppressor of cytokine signal ing-3 worsens immune-complex-induced ali [57] . intriguingly, intra tracheal administration of adenoviral vector incor porating il-10, prior to zymosan-induced lung injury, improved survival at a lower dose but was ineff ective and even harmful at higher doses [58] . an early murine study demonstrated that cationic lipidmediated transfer of the na + ,k + -atpase gene ameliorated high-permeability pulmonary edema [59] . electroporationassisted gene transfer of plasmids encoding for na + ,k + -atpase reverses endotoxin-induced lung injury [60] . th e lipoplex-delivered il-10 gene decreased lung and systemic organ injury induced by cecal ligation and puncture in mice [61] . systemically administered cationic polyethyleni mine polyplexes incorporating indoleamine-2,3-dioxyge nase transduced pulmonary endo thelial cells and decreased lung ischemia-reper fusion injury [62] . nf-îºb decoy oligonucleotides, incorporated into viral vectors, attenuate systemic sepsis-induced lung injury when administered intravenously (figure 3 ) [37] . in animal models, both intratracheal [34, 63] and intra venously [29, 64] administered sirna successfully silence their target genes. shrna-based approaches have been used to suppress silica-induced lung fi brosis [35] and to ameliorate lung ischemia-reperfusion-induced lung injury [36] . more recently, aerosolization of sirna that targets respiratory syncytial virus viral replication was safe and potentially eff ective in patients post lung transplant with respiratory syncytial virus infection [33] , clearly illustrating the therapeutic potential of these approaches for ali/ards. mei and colleagues enhanced the effi cacy of mesen chymal stem/stromal cells in endotoxin-induced ali by transducing them to overexpress angiopoeitin-1 (figure 4 ) [39] . mesenchymal stem/stromal cells overexpressing il-10 decreased alveolar infi ltration of cd4 and cd8 t cells following lung ischemia-reperfusion injury [65] . bone marrow stem cells expressing keratinocyte growth factor attenuate bleomycin-induced lung injury [66] . non stem cells can also be used to deliver genes to the injured lung [67] . fibroblasts overexpressing angiopoeitin-1 attenuate endotoxin-induced lung injury [40] , while fi broblasts overexpressing vascular endothelial growth factor and endothelial nitric oxide synthase can attenuate or even reverse endotoxin-induced ali [68] . advances in the identifi cation of therapeutic targets, improvements in viral and nonviral vector technologies, and regulation of gene-based therapies by temporal and spatial targeting off er the potential to translate the therapeutic promise of gene-based therapies for ali/ ards to the clinical setting (table 3) . viral vectors remain the focus of intensive research to optimize their effi ciency, to minimize their immuno genicity and to enhance their tissue specifi city [19, 31, 69, 70] . strategies to develop less immunogenic vectors have focused on modifying the naturally occurring proteins in the viral coat [71] . much research has been devoted to searching and characterizing both naturally occurring [71] and engineered capsid variants from mammalian species [72] . capsid protein modification has also been used to enhance tissue specifi city [70] . envelope protein pseudotyping involves encapsulating the modifi ed genome from one virus, such as simian immuno defi ci ency virus, with envelope proteins from another virus, such as vesicular stomatitic virus. th is encapsu lation can enhance the therapeutic potential of viral vectors, by combining the advantages of one viral genome (for example, bigger payload or site-specifi c integration) with the tissue tropism of another virus. strategies to enhance the eff ectiveness of the lipoplexes used to deliver plasmids and other dna/rna oligonucleotides involve manipulation of the lipoplex lipid content and the use of targeting peptides. th e choice of lipid infl uences expression effi ciency by enhancing release of the genetic material within the target cell [73, 74] . targeting peptides increases transfection effi ciency by directing the lipid to a particular cell membrane or cell type [31] . physical methods of plasmid delivery such as electroporation [60] and ultrasound can enhance gene transfer by bringing the plasmid dna into closer proximity with the cell membrane and/or causing temporary disruption of the cell membrane. other physical methods can also be used to increase in vivo gene transfer, including pressurized vascular delivery, laser, magnetic fi elds and gene gun delivery. th ese systems enable plasmid-based gene delivery to reach effi ciencies close to that achieved with viral vectors. successful gene therapy relies upon being able to target the injury site, and to control the duration and levels of gene expression. modifying the transgene dna to exclude nonmethylated cpg motifs, typical of bacterial dna, decreases the immune response and may increase transgene expression [75, 76] . high-effi ciency tissue-specifi c promoters may improve the effi ciency and specifi city of transgene expression. lung-specifi c promoters include surfactant promoters [77] such as the surfactant protein c promoter [78] , a ciliated cell-specifi c promoter foxj1 [79] , the cytokeratin 18 promoter [80] , and the clara cell 10-kda protein [78] . promoters can also be used to target a specifi c phase of illness, switching on when required to produce an eff ect at the optimal time point. a related approach is the development of promoters that allow for transfected genes to be turned on and off . currently, the tetracycline-dependent gene expression vector [81] is the most widely used regulated system as it has a good safety profi le. tetracycline is rapidly metabolized and cleared from the body, making it an ideal drug to control gene expression. however, the potential for an activator such as tetracycline to modulate the lung injury should be borne in mind. new-generation transactivators, with no basal activity and increased sensitivity, have now been developed [82] . in an ards context, conditional regulation of gene expression by the combined use of a lung-specifi c promoter and the tetracycline-dependent gene expression system may be a useful approach [83] . capsid protein modifi cation to reduce immunogenicity [71] capsid protein modifi cation to enhance tissue specifi city [70] envelope protein pseudotyping manipulation of lipoplex lipid content to enhance cellular uptake [73, 74] use of targeting peptides on lipoplexes and polyplexes [31] strategies to enhance gene transfer; for example, electroporation, ultrasound, gene gun delivery modifying transgene dna to eliminate bacterial motifs [75, 76] development of high-effi ciency tissue-specifi c promoters [77] [78] [79] [80] development of promoters that regulate gene expression [83] enhanced therapeutic targeting nebulization technologies [9] strategies to target the pulmonary endothelium [10] improved cellular uptake of vector surface active agents to enhance vector spread [84] reduce ubiquitination of viral capsid proteins [85] better therapeutic targets enhancement or restoration of lung epithelial and/or endothelial cell function [86] strengthening lung defense mechanisms against injury [87] speeding clearance of infl ammation and infection enhancement of the repair process following ali/ards [88] . an advantage of gene-based strategies is the ability to target specifi c cells within an organ; for example, the epithelial cells of the lung. novel nebulization technologies, which facilitate the delivery of large quantities of undamaged vector to the distal lung, demonstrate considerable promise in this regard [9] . alternative approaches to spatial targeting include targeting specifi c receptors that are plentiful on the target cell to increase transfection effi ciency. an interesting development in this regard is the targeting of systemically administered therapies to the pulmonary endothelium using antibodies to proteins expressed preferentially on these cells ( figure 5 ) [10] . in these studies, the antioxidant enzyme catalase was conjugated with antibodies to the adhesion molecule pecam, which is widely expressed on pulmonary endothelial cells, and to a nonspecifi c igg antibody. th e anti-pecam/catalase conjugate, but not the igg/catalase conjugate, bound specifi cally to the pulmonary endothelium and attenuated hydrogen peroxide injury. specifi c strategies have been developed to maximize uptake of vector into alveolar epithelial cells. it is possible to enhance lung transgene expression with the use of surface-active agents such as perfl urocarbon, which enhances the spread of vector and mixing within the epithelial lining fl uid [84] . agents that reduce ubiquitination of aav capsid proteins following endocytosis, such as tripeptide proteasome inhibitors, dramatically augment (>2,000-fold) aav vector transduction in airway epithelia [85] . ultimately, the success or failure of gene-based therapies for ali/ards is likely to rest on the identifi cation of better gene targets. ongoing advances in our understanding of the pathophysiology of ali/ards continue to reveal novel therapeutic targets for gene-based approaches. promising potential approaches include strate gies to enhance or restore lung epithelial and/or endothelial cell function [86] , to strengthen lung defense mechanisms against injury [87] , to speed clear ance of infl ammation and infection, and to enhance the repair process following ali/ards [88] . ali/ards may be a particularly suitable disease process for gene-based therapies (table 4 ). th is is supported by increasing evidence from relevant preclinical ards models for the effi cacy of gene-based therapies that enhance or restore lung epithelial and/or endothelial cell function, strengthen lung defense mecha nisms against injury, speed resolution of infl ammation and infection, and enhance the repair process following ali/ards. despite this promising preclinical evidence, the potential for gene based approaches to ali/ards in the clinical setting remains to be realized. multiple barriers exist to the successful use of gene-based therapies in the lung, which limit the effi cacy of these approaches. future research approaches should focus on overcoming these barriers, by developing more eff ective and less immunogenic vector delivery systems, developing strategies to focus gene expression on specifi c injury zones of the lung for defi ned time periods, and identifying better molecular targets that can take advantage of these potentially very powerful therapeutic approaches. abbreviations aav, adeno-associated virus; ali, acute lung injury; ards, acute respiratory distress syndrome; il, interleukin; nf, nuclear factor; shrna, small hairpin rna; sirna, small interfering rna. the authors declare that they have no competing interests. epidemiology of acute lung injury incidence and outcomes of acute lung injury one-year outcomes in survivors of the acute respiratory distress syndrome ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network pulmonary-artery versus central venous catheter to guide treatment of acute lung injury prone ventilation reduces mortality in patients with acute respiratory failure and severe hypoxemia: systematic review and meta-analysis elbourne d: effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial the acute respiratory distress syndrome optimized aerosol delivery to a mechanically ventilated rodent pecamdirected delivery of catalase to endothelium protects against pulmonary vascular oxidative stress adenoviral augmentation of elafi n protects the lung against acute injury mediated by activated neutrophils and bacterial infection aerosol delivery of a î²-galactosidase adenoviral vector to the lungs of rodents adenovirusmediated persistent cystic fi brosis transmembrane conductance regulator expression in mouse airway epithelium airway epithelial cftr mrna expression in cystic fi brosis patients after repetitive administration of a recombinant adenovirus analysis of risk factors for local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions modifi cation of nasal epithelial potential diff erences of individuals with cystic fi brosis consequent to local administration of a normal cftr cdna adenovirus gene transfer vector a phase i study of adenovirus-mediated transfer of the human cystic fi brosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fi brosis aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fi brosis. i. methods, safety, and clinical implications recent developments in adeno-associated virus vector technology analysis of adeno-associated virus progenitor cell transduction in mouse lung adeno-associated virus type 5 (aav5) but not aav2 binds to the apical surfaces of airway epithelia and facilitates gene transfer adeno-associated virus type 6 (aav6) vectors mediate effi cient transduction of airway epithelial cells in mouse lungs compared to that of aav2 vectors repeated adeno-associated virus serotype 2 aerosol-mediated cystic fi brosis transmembrane regulator gene transfer to the lungs of patients with cystic fi brosis: a multicenter, double-blind, placebo-controlled trial safety and biological effi cacy of an adeno-associated virus vector-cystic fi brosis transmembrane regulator (aav-cftr) in the cystic fi brosis maxillary sinus lentivirus vectors pseudotyped with fi loviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha gene therapy of human severe combined immunodefi ciency (scid)-x1 disease cavazzana-calvo m: insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of scid-x1 rna interference for î±-enac inhibits rat lung fl uid absorption in vivo eff ect of antisense oligonucleotides to nuclear factor-îºb on the survival of lps-induced ards in mouse electroporation-mediated transfer of plasmids to the lung results in reduced tlr9 signaling and infl ammation a receptor-targeted nanocomplex vector system optimized for respiratory gene transfer cationic lipid-mediated cftr gene transfer to the lungs and nose of patients with cystic fi brosis: a double-blind placebo-controlled trial rna interference therapy in lung transplant patients infected with respiratory syncytial virus in vivo gene silencing (with sirna) of pulmonary expression of mip-2 versus kc results in divergent eff ects on hemorrhage-induced, neutrophil-mediated septic acute lung injury silencing cd36 gene expression results in the inhibition of latent-tgf-î²1 activation and suppression of silica-induced lung fi brosis in the rat prevention of lung ischemia-reperfusion injury by short hairpin rna-mediated caspase-3 gene silencing nuclear factor-îºb decoy oligodeoxynucleotides prevent acute lung injury in mice with cecal ligation and puncture-induced sepsis eff ects of intratracheal administration of nuclear factor-îºb decoy oligodeoxynucleotides on long-term cigarette smokeinduced lung infl ammation and pathology in mice prevention of lpsinduced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1 cell-based angiopoietin-1 gene therapy for acute lung injury stem cells and cell therapies in lung biology and lung diseases calculating expected lung deposition of aerosolized administration of aav vector in human clinical studies repeated aerosolized aav-cftr for treatment of cystic fi brosis: a randomized placebo-controlled phase 2b trial aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fi brosis. ii. transfection effi ciency in airway epithelium inhibition of lung tumor growth by complex pulmonary delivery of drugs with oligonucleotides as suppressors of cellular resistance gene transfer to the lung: lessons learned from more than 2 decades of cf gene therapy acute lung injury does not impair adenoviral-mediated gene transfer to the alveolar epithelium fatal systemic infl ammatory response syndrome in a ornithine transcarbamylase defi cient patient following adenoviral gene transfer adenoviral gene transfer of a mutant surfactant enzyme ameliorates pseudomonas-induced lung injury gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cdnas protects against hyperoxia but not against ischemiareperfusion lung injury angiopoietin-1 increases survival and reduces the development of lung edema induced by endotoxin administration in a murine model of acute lung injury enhanced expression of 70-kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome human apoa-i overexpression diminishes lps-induced systemic infl ammation and multiple organ damage in mice protection against pseudomonas aeruginosa pneumonia and sepsisinduced lung injury by overexpression of î²-defensin-2 in rats overexpression of the na-k-atpase î±2-subunit improves lung liquid clearance during ventilation-induced lung injury interleukin-1î² causes acute lung injury via î±vî²5 and î±vî²6 integrin-dependent mechanisms adenoviral-mediated overexpression of socs3 enhances igg immune complex-induced acute lung injury dose-dependent improvements in outcome with adenoviral expression of interleukin-10 in a murine model of multisystem organ failure pretreatment with cationic lipid-mediated transfer of the na + k + -atpase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema electroporation-mediated gene transfer of the na + ,k + -atpase rescues endotoxin-induced lung injury interleukin-10 gene transfer: prevention of multiple organ injury in a murine cecal ligation and puncture model of sepsis nonviral gene delivery with indoleamine 2,3-dioxygenase targeting pulmonary endothelium protects against ischemia-reperfusion injury silencing of fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, infl ammation, and neutrophil infl ux after hemorrhagic shock and sepsis caveolin-1 sirna increases the pulmonary microvascular and alveolar epithelial permeability in rats interleukin-10 delivery via mesenchymal stem cells: a novel gene therapy approach to prevent lung ischemia-reperfusion injury bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fi brosis cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension microvascular regeneration in established pulmonary hypertension by angiogenic gene transfer tetracycline-inducible transgene expression mediated by a single aav vector effi cient transfection of non-proliferating human airway epithelial cells with a synthetic vector system tailoring the aav vector capsid for gene therapy artifi cial evolution with adeno-associated viral libraries analysis and optimization of the cationic lipid component of a lipid/ peptide vector formulation for enhanced transfection in vitro and in vivo stabilized integrin-targeting ternary lpd (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell cpg-free plasmids confer reduced infl ammation and sustained pulmonary gene expression toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd40 ligand to induce high amounts of il-12 targeting type ii and clara cells for adenovirus-mediated gene transfer using the surfactant protein b promoter development of lentiviral vectors with regulated respiratory epithelial expression in vivo expression of cftr from a ciliated cell-specifi c promoter is ineff ective at correcting nasal potential diff erence in cf mice a human epithelium-specifi c vector optimized in rat pneumocytes for lung gene therapy tight control of gene expression in mammalian cells by tetracycline-responsive promoters use of a new generation reverse tetracycline transactivator system for quantitative control of conditional gene expression in the murine lung construction of an rtta2(s)-m2/ tts(kid)-based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and non-human primates adenoviral vector transfection into the pulmonary epithelium after cecal ligation and puncture in rats ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins aff ects the transduction effi ciency of recombinant vectors gp130-stat3 regulates epithelial cell migration and is required for repair of the bronchiolar epithelium spatial and temporal expression of surfactant proteins in hyperoxia-induced neonatal rat lung injury intrapulmonary tnf gene therapy reverses sepsis-induced suppression of lung antibacterial host defense clinical review: gene-based therapies for ali/ards: where are we now? the present work was supported by funding from the health research board key: cord-002627-3jwu4pf2 authors: wu, nan-chun; liao, fan-ting; cheng, hao-min; sung, shih-hsien; yang, yu-chun; wang, jiun-jr title: intravenous superoxide dismutase as a protective agent to prevent impairment of lung function induced by high tidal volume ventilation date: 2017-07-26 journal: bmc pulm med doi: 10.1186/s12890-017-0448-9 sha: doc_id: 2627 cord_uid: 3jwu4pf2 background: positive-pressure mechanical ventilation is essential in assisting patients with respiratory failure in the intensive care unit and facilitating oxygenation in the operating room. however, it was also recognized as a primary factor leading to hospital-acquired pulmonary dysfunction, in which pulmonary oxidative stress and lung inflammation had been known to play important roles. cu/zn superoxide dismutase (sod) is an important antioxidant, and possesses anti-inflammatory capacity. in this study, we aimed to study the efficacy of cu/zn sod, administered intravenously during high tidal volume (htv) ventilation, to prevent impairment of lung function. methods: thirty-eight male sprague-dawley rats were divided into 3 groups: 5 h ventilation with (a) low tidal volume (ltv; 8 ml/kg; n = 10), (b) high tidal volume (htv; 18 ml/kg; n = 14), or (c) htv and intravenous treatment of cu/zn sod at a dose of 1000 u/kg/h (htv + sod; n = 14). lung function was evaluated both at baseline and after 5-h ventilation. lung injury was assessed by histological examination, lung water and protein contents in the bronchoalveolar lavage fluid (balf). pulmonary oxidative stress was examined by concentrations of methylguanidine (mg) and malondialdehyde (mda) in balf, and antioxidative activity by protein expression of glutathione peroxidase-1 (gpx-1) in the lung. severity of lung inflammation was evaluated by white blood cell and differential count in balf, and protein expression of inducible nitric oxide synthase (inos), intercellular adhesion molecule-1 (icam-1), tumor necrosis factor-α (tnf-α), matrix metalloproteinase-9 (mmp-9), and mrna expression of nuclear factor-κb (nf-κb) in the lung. we also examined protein expression of surfactant protein (sp)-a and d and we measured hourly changes in serum nitric oxide (no) level. results: five hours of ltv ventilation did not induce a major change in lung function, whereas 5 h of htv ventilation induced apparent combined restrictive and obstructive lung disorder, together with increased pulmonary oxidative stress, decreased anti-oxidative activity and increased lung inflammation (p < 0.05). htv ventilation also decreased sp-a and sp-d expression and suppressed serum no level during the time course of ventilation. cu/zn sod administered intravenously during htv ventilation effectively reversed associated pulmonary oxidative stress and lung inflammation (p < 0.05); moreover, it preserved sp-a and sp-d expressions in the lung and increased serum nitric oxide (no) level, enhancing vascular no bioavailability. conclusions: htv ventilation can induce combined restrictive and obstructive lung disorders. intravenous administration of cu/zn sod during htv ventilation can prevent lung function impairment and lung injury via reducing pulmonary oxidative stress and lung inflammation, preserving pulmonary surfactant expression, and enhancing vascular no bioavailability. methods: thirty-eight male sprague-dawley rats were divided into 3 groups: 5 h ventilation with (a) low tidal volume (ltv; 8 ml/kg; n = 10), (b) high tidal volume (htv; 18 ml/kg; n = 14), or (c) htv and intravenous treatment of cu/zn sod at a dose of 1000 u/kg/h (htv + sod; n = 14). lung function was evaluated both at baseline and after 5-h ventilation. lung injury was assessed by histological examination, lung water and protein contents in the bronchoalveolar lavage fluid (balf). pulmonary oxidative stress was examined by concentrations of methylguanidine (mg) and malondialdehyde (mda) in balf, and antioxidative activity by protein expression of glutathione peroxidase-1 (gpx-1) in the lung. severity of lung inflammation was evaluated by white blood cell and differential count in balf, and protein expression of inducible nitric oxide synthase (inos), intercellular adhesion molecule-1 (icam-1), tumor necrosis factor-α (tnf-α), matrix metalloproteinase-9 (mmp-9), and mrna expression of nuclear factor-κb (nf-κb) in the lung. we also examined protein expression of surfactant protein (sp)-a and d and we measured hourly changes in serum nitric oxide (no) level. results: five hours of ltv ventilation did not induce a major change in lung function, whereas 5 h of htv ventilation induced apparent combined restrictive and obstructive lung disorder, together with increased pulmonary oxidative stress, decreased anti-oxidative activity and increased lung inflammation (p < 0.05). htv ventilation also decreased sp-a and sp-d expression and suppressed serum no level during the time course of ventilation. cu/zn sod administered intravenously during htv ventilation effectively reversed associated pulmonary oxidative stress and lung inflammation (p < 0.05); moreover, it preserved sp-a and sp-d expressions in the lung and increased serum nitric oxide (no) level, enhancing vascular no bioavailability. (continued on next page) (continued from previous page) conclusions: htv ventilation can induce combined restrictive and obstructive lung disorders. intravenous administration of cu/zn sod during htv ventilation can prevent lung function impairment and lung injury via reducing pulmonary oxidative stress and lung inflammation, preserving pulmonary surfactant expression, and enhancing vascular no bioavailability. keywords: rats, lung function, superoxide dismutase, inflammation, oxidative stress, nitric oxide, positive pressure mechanical ventilation, surfactant protein a, surfactant protein d for more than half a century, positive-pressure mechanical ventilation has been regarded as an essential intervention assisting patients with respiratory failure in the intensive care unit and facilitating oxygenation in the operating room. on the other hand, evidence from patient's studies indicates that mechanical ventilation is one of the primary factors leading to hospital-acquired lung injury, and patients with burn injury, trauma or pre-existing respiratory diseases are particularly at high risks [40] . a recent survey of patients having cardiac surgery revealed that though most patients do not have signs of pulmonary dysfunction or lung injury prior to the surgery, some eventually develop perioperative lung injury [18] . though the exact mechanism responsible is uncertain, higher tidal volume employed commonly to reduce risks of hypoxemia and pulmonary atelectasis is thought to play a critical role [38] . pulmonary oxidative stress and lung inflammation had been implicated in the pathogenesis of ventilator-associated pulmonary dysfunction and lung injury [10, 15, 60, 68] . overinflating alveoli and repeated stretching of lung tissues can produce reactive oxygen species (ros), and diminish prostanoid synthesis that promotes redox imbalance and cyclooxygenase induction and inflammatory responses [68] . also, formation of ros disrupts the regulation of nitric oxide synthase (nos) causing nitrotyrosine accumulation, irregular tracheal mucus secretion, and increased airway resistance [63] . in addition, stretching lung tissues directly alters metabolism and secretion of pulmonary surfactant proteins (sps) that mediate alveolar surface tension and lung compliance, while surfactant protein-a (sp-a) and d (sp-d) augment pulmonary immune defense mechanisms and inhibit endogenous lipid peroxidation [4] . moreover, excessive lung stretch activates nuclear factor-κb (nf-κb) that mediates the production and release of proinflammatory cytokines and chemokines that, in turn, promote adhesion molecule expression [28] and facilitate inflammatory cell infiltration in lung [34] . for over two decades, low tidal volume ventilation has been proposed as a protective strategy; still ventilator-associated lung injury is not uncommon and, thus far, there is still no consensus regarding the optimal ventilation strategy [48] . terragni et al. [76] reported that even a well-accepted protective ventilation strategy, namely a tidal volume of 6 ml/kg and a plateau pressure ≤ 30 cm h 2 o, may not protect all patients against ventilator-induced pulmonary dysfunction. consequently, an effective pharmacological intervention may be necessary to protect ventilator-associated pulmonary dysfunction and lung injury. superoxide dismutase (sod) is an important antioxidant, active in endothelial cells, cytoplasm and mitochondrial intermembrane matrix [57] . it protects cells against superoxide damage through catalyzing the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide [50, 57] and thus inhibits peroxynitrite-mediated oxidative protein modification and cell membrane lipid peroxidation [27] . moreover, sod can facilitate vascular function, increasing nitric oxide (no) bioavailability through competing with no for superoxide anions [31] . sod was also shown to inhibit neutrophil-mediated inflammation through regulating neutrophil apoptosis [85] . despite all the benefits and no major side effects, therapeutic efficacy of sod treatment on tissue damage, in general, has been limited by its short circulatory halflife and low transcapillary permeability, due to its relatively large molecular size (molecular weight~32 kda) [84] . on the other hand, intravenous administration of cu/zn sod has been demonstrated to be effective against hyperoxia-induced lung injury [53] . though the authors did not provide explanation of why sod treatment was so effective in hyperoxic lung injury, its protective effectiveness is likely related to its substantial permeability across the pulmonary capillary. increased albumin concentration in the bronchoalveolar lavage fluid (balf) is the hallmark of hyperoxic lung injury [53] and the molecular weight (∼69 kda) of albumin is more than twice that of sod. albumin leak is also characteristic of ventilator-induced lung injury [80] and we suggest that the observed protective efficacy of intravenously administered sod may imply substantial transcapillary permeability. the aim of our study was to determine the protective efficacy of intravenously administered sod against mechanical ventilation-induced impairment of lung function. using an in vivo rat model of mechanical ventilation, we showed that 5 h of high tidal volume (htv) ventilation can induce apparent combined restrictive and obstructive lung disorder and lung injury, evident by increased lung water content and albumin content in balf. we have identified pulmonary oxidative stress and lung inflammation and reduced expression of pulmonary surfactant protein (sp)-a and d as the main contributors to the impairment of lung function and observed that intravenous administration of cu/zn sod effectively reversed mechanical ventilationassociated detrimental effects. in addition, intravenous administration of cu/zn sod steadily increased the level of serum nitric oxide (no) during the course of ventilation, suggesting that increased no bioavailability in pulmonary circulation contributes to the preservation of lung function. this study was performed on male sprague-dawley rats (biolasco co., taipei, taiwan) weighing between 250 and 300 g. rats were housed in a pathogen-free animal facility and food and water were available ad libitum. the study protocol was approved by the animal care and use committee of the fu jen catholic university and complied with the guidelines for the care and use of laboratory animals (nih guide, volume 25, number 28, 1996) . rats were anesthetized with zoletil 50 (50 mg/kg; i.p., virbac, carros cedex, france). to reduce mucous secretion, animals were given atropine (0.8 mg/kg; i.m.; sigma, mo, usa) 15 min prior to the anesthesia. the depth of anesthesia was monitored every 30 min by assessing the reflex withdrawal response to pinching of the hind paws. a tracheotomy was performed and the trachea was cannulated with a sterile polystyrene catheter (pe240), which in turn was connected to a small animal ventilator (topo small animal ventilator, kent scientific, ct, usa; constant-frequency, volumecontrolled). airway pressure was monitored continuously (deltran 6069, utah medical inc., utah, usa). the right jugular vein was cannulated with a polystyrene catheter (pe50) through which cu/zn sod or equal amounts of normal saline (0.9%) were administered. body temperature was maintained at~37°c using a heating blanket. the left femoral artery was cannulated by a polyethylene catheter (pe50), through which aortic pressure (p ao ) was monitored (deltran 6069). blood samples were collected every 60 min for assessments of blood gases (radiometer alb 5, brønshøj, denmark), blood count (fujifilm sericol retarder zv558, osaka, japan), and serum no level. hemodynamic data were recorded at 2000 hz using a 16-channel data acquisition system (model mp150, biopac systems inc., ca, us) and stored using a dual-processor laptop computer. thirty-eight rats were ventilated for 5 h, either with low tidal volume (ltv) of 8 ml/kg (n = 10) or high tidal volume (htv) of 18 ml/kg. the inspiratory/expiratory ratio for those ventilated with ltv and htv were 0.25 and 0.45, respectively. other ventilatory parameters including positive end expiratory pressure (peep = 0 cmh 2 o; positive peep has been shown to have lung protective effectiveness [1] ) fraction of inspired oxygen (0.21), respiratory rate (60 stroke/min) were identical between groups. htv-ventilated rats were administered intravenously either with cu/zn sod (htv + sod, n = 14) or with an equal amount of saline (htv; n = 14); the same amount of saline was also administered intravenously to ltv-ventilated rats. human erythrocyte cu/zn sod (s9636-15ku, sigma-aldrich co., mo, us) was administered at a dose of 1000 u/kg/h via a high-precision syringe pump (kd scientific, holliston, ma, usa) during the entire course of ventilation. the dose adopted was determined in a preliminary study, in which 4 doses (200, 500, 1000 and 2000 u/kg/h) were administered during htv ventilation, the outcome being assessed by means of lung function testing, and concentrations of methylguanidine (mg) and malondialdehyde (mda) in balf. results were dose-dependent, but no major differences were observed between rats treated with 1000 u/kg/h and those with 2000 u/kg/h. in order to maintain the blood ph within 7.30-7.45 during htv ventilation, an external dead space was added by increasing the volume of intubation tubing [61] . in another study, heat denatured cu/zn sod (1000 u/kg/h) was treated during htv ventilation; the results of lung function testing and tissue mda level were compared to those assessed in the htv group and showed no significant difference, suggesting no protective effectiveness of heat denatured cu/zn sod. at the end of study, rats were euthanized by an overdose of anesthetic (200 mg/kg i.p). the lungs and trachea were removed for balf measurements, assessments of lung water and histological analysis, and subsequent analyses of protein and mrna expression. lung function testing was conducted both at baseline and after 5 h of mechanical ventilation using a buxco forced-maneuver system (buxco research systems, wilmington, nc, usa), comprised of a plethysmograph chamber, a control panel and pressure and vacuum reservoirs. anaesthetized rats were placed inside the plethysmograph chamber with their tracheal cannulae connected to the breathing valve. the plethysmograph was connected to a differential pressure transducer and an amplifier system. after adjusting the respiratory rate of the system ventilator to match that of the animal, a series of forced maneuvers mimicking spirometric maneuvers in human subjects (i.e., inflating, occluding, and deflating the lungs at different rates) were conducted and parameters of lung function (i.e., lung volumes, airway flows and lung resistance and compliance) were calculated via the biosystem xa system (buxco) and exported to text files. pulmonary function testing at each status (baseline or post-mechanical ventilation) was repeated three times to yield averaged measurements. lungs and trachea removed at the end of experiment were weighed immediately. balf was acquired from the left lung; the right lung was used for histological examination (upper lobe), measurement of lung water (middle lobe), and analysis protein expression or mrna (lower lobe). the whole lung was inflated to 20 cmh 2 o and the right main bronchus was ligated to separate the left from the right lung. the right upper lobe bronchus was then ligated and the inflated upper right lobe was excised and immersed in 10% buffered formalin at 4°c for 24 h for tissue fixation. the middle right lobe was weighed upon dissection (wet weight) and weighed again after being dehydrated in an oven at 70°c for 7 days (dry weight). the lower lobe of right lung was sectioned and frozen immediately in liquid nitrogen and stored in a -80°c freezer for subsequent analysis of protein and mrna expression. the upper lobe of right lung was processed following dehydration (tp1020, leica biosystems, richmond, il, usa), clearing, paraffin infiltration and embedding. lung specimens were sectioned (~5 μm-thick; jung. rm2045, leica biosystems), stained with hematoxylin and eosin, and examined under a light microscope. photo images were taken by a c-mount microscope camera (whited inc., taipei, taiwan). balf was acquired by gently flushing the left lung three times through the trachea with a 1.25 ml aliquot of 0.9% saline pre-warmed to 37°c. a volume of 0.92 ±0.10 ml of lavage fluid was recovered. lavage samples were centrifuged at 800 g at 4°c for 10 min. the cell pellets were used for differential cell count and the supernatant was extracted for biomarker analyses. protein concentration (μg/ml) was measured in the supernatant of balf using spectrophotometry (multiskan fc, thermo scientific, ca, usa) with an emission wavelength at 630 nm, calibrated by a standard curve for albumin (sigma). the level of mg formation in the balf has been used as an index for pulmonary hydroxyl radical formation [74] . the lavage supernatant was diluted 1:100, and mg was measured using a spectrofluorimetric detector (jasco 821-fp fluorescence detector, tampa, fl, usa) with a fluorescent excitation maximum at 395 nm and emission maximum at 500 nm. the assay was calibrated by a standard curve of authentic mg (sigma m0377, st. louis, mo, usa), generated by various concentrations of mg (0, 50, 100, 150, and 200 mg/ml). the coefficient of variance in percentage was 3.8% with a detection limit of 1 mg/ml. the mda concentration in balf has been used as an index for pulmonary oxidative stress and lipid peroxidation [66] . mda was measured in the supernatant of balf using enzyme-linked immunosorbent assay (elisa) kits (ab46070; abcam, cambridge, ma, usa) with a detection range of 0.3-65 nmol/ml and a sensitivity of 0.208 nmol/ml. each sample was performed in duplicate and determined by an automated elisa reader at 450/540 nm wavelength. total white blood cell count and differential in balf total white blood cell (wbc) count was performed by loading 10 μl of balf onto a hemocytometer, and examined under light microscopy (400x, bx-40, olympus, tokyo, japan). differential cell count was conducted by loading 10 μl of the cell pellets on a slide, stained with liu's stain, and examined under light microscopy (1000x, olympus). one hundred cells were examined in each specimen. the level of plasma no was evaluated by the concentrations of nitrate and nitrite (metabolites of nitric oxide) using a high-performance liquid chromatography system (eno-20, eicom nox analyzer, kyoto, japan), which has a sensitivity of 30 pmol for nitrate and nitrite anion. blood samples mixed with an equal amount of methanol were centrifuged at 15,000 g at 4°c for 10 min. the top supernatant, filtered through an ultrafine membrane with a cut-off protein molecular weight of 3 kda, was injected into the hplc system. samples were separated through a strong anion-exchange column (spherisorb sax, 250 × 4.6 mm with internal diameter of 5 μm) followed by a nitrate-to-nitrite reduction process and a griess diazotization reaction. the chromophore was detected at the wavelength of 540 nm with the coefficient of variance of 3.2% and detection limit of 2 pmol. frozen lung tissues of around 200 mg were homogenized in a lysis buffer (10 mm tris · hcl, ph 7.5, 1% triton x-100, 1 mm edta, 1 mm pmsf, 10 μg/ml aprotonin, and 10 μg/ml leupeptin). the total protein concentration was determined using the bicinchoninic acid protein assay kit (sigma-aldrich, st. louis, mo, usa), measured at the absorbance wavelength of 562 nm and calibrated to a standard curve. the sample's molecular weight was determined using the precision plus protein kaleidoscope standards (10 μl, bio-rad). the protein extraction, mixed with an equal amount of sodium dodecyl sulphate (sds) buffer, was heated to 95°c for 5 min and separated on a 10% sds polyacrylamide gels in a running buffer at 80 v. these spatially separated proteins were transferred onto a nitrocellulose membrane (bio-rad) at 100 ma and at 4°c overnight. non-specific binding was blocked through placing the membrane in a blocking buffer (hycell biotechnology inc. raleigh, nc, usa) at room temperature for 1 min. blots were then incubated with the primary antibody at 4°c on an orbital shaker. in this study, the primary antibodies were glutathione peroxidase-1 (gpx-1; ab22604, abcam, cambridge, uk), tumor necrosis factor-α (tnf-α; ab1793, abcam), inos (ab15323, abcam), intercellular adhesion molecule-1 (vcam-1; mr106, novus biologicals, littletown, co, usa), matrix metalloproteinase-9 (mmp-9; ab7299, abcam), and surfactant protein a (sp-a; ab115791, abcam) and d (sp-d; ab15687, abcam). antibodies were diluted according to the instructions before use. the membranes were washed for 10 min in tris-buffered saline tween-20 (tbst) three times, followed by incubation in a diluted horseradish peroxidase-conjugated secondary antibody (emd millipore corp, billerica, ma, usa) at room temperature for 1 h. after incubation, the membrane was washed 10 min three times using tbst. blots were developed with the detection reagents (emd millipore, darmstadt, germany), and quantified using a gel documentation and image-analysis software (minichemi, sagecreation, beijing, china). the mrna of lung tissue was isolated using the rnazol reagent (molecular research center, oh, usa), followed by treatment of rnase-free dnase (qiagen kit) during column purification. reverse transcription of total mrna to cdna was carried out using the first strand cdna synthesis kit (avian myeloblastosis virus; roche applied science, penzberg, bavaria, germany) and random primers. this cdna was then used as a template for the gene-specific primer of real-time pcr. real-time pcr was conducted using platinum sybr green qpcr supermix-udg (invitrogen carlsbad, nm, usa) and an abi prism 7500 real time pcr system (applied biosystems, drive foster city, ca, usa). forward and backward primer sequences for nf-κb were ccgggcaggtctcagc and gggctgctcaatg atctcca, respectively, designed using primer express v.2.0 (applied biosystems) software. the thermal cycling conditions for real-time pcr were set at 50°c for 2 min, followed by 95°c for 10 min, and 40 cycles of melting at 95°c for 15 s, and annealing and extension at 60°c for 60 s. levels of mrna relative to an endogenous control (β-actin) were calculated by the δδc t method, using 7500 system sds software version 1.2.1.22 (applied biosystems). data were presented as mean±sem. comparisons across two sets of groups were analyzed with two-way analysis of variance, followed by tukey's post-hoc test. p < 0.05 is considered statistically significant. the hourly recorded mean aortic pressure (p mean ), peak airway pressure (p paw ) and blood gas data (ph, pco 2 and po 2 ) are summarized in table 1 . five hours of ltv ventilation did not induce significant changes in p mean , p paw or blood gas data versus those of the baseline. blood gas at baseline was not statistically different between groups. without addition of dead space, htv ventilation induces apparent respiratory alkalosis and hyperventilation. dead space addition (~3.0 ml) helped sustain blood ph, mostly within a range of 7.30 to 7.45, during htv ventilation, as well as improving respiratory alkalosis and hyperventilation. decreased pao 2 and increased paco 2 during the last hour of htv ventilation indicates impaired gas exchange, which is consistent with the interstitial edema and hyaline membrane formation observed in histological examination (see below). on the other hand, sod treatment was demonstrated to reverse the impairment of gas exchange. htv ventilation decreased p mean by 6-10 mmhg as compared with the baseline, and p mean remained relatively stable during the course of ventilation, with or without sod treatment. cu/zn sod protects against htv ventilation induced damages of lung structure five hours of ltv ventilation increased inspiratory resistance (ri) (p < 0.05), but did not alter other parameters of lung function versus the baseline. in contrast, 5 h of htv ventilation markedly increased ri (p < 0.05), while decreased maximum mid-expiratory flow (mmef)(p < 0.001), chord compliance (c chord )(p < 0.001), vital capacity (vc) (p < 0.001) and total lung capacity (tlc)(p < 0.001) versus the baseline. collectively, htv ventilation induces combined restrictive and obstructive lung disorder. in the bottom panel of fig. 2 and in fig. 3 , we showed that intravenous treatment of cu/zn sod during htv ventilation prevented impairment of lung function and lung mechanics, evident by sustained fev, expiratory flow volume relationship, ri, mmef, c chord , tlc and vc relative to the baseline. cu/zn sod reduces htv ventilation-induced increases in trans-capillary protein permeability and lung water content figure 4 shows that 5 h of htv ventilation markedly increased the content of lung water as compared with the ltv group, evident by increased wet-to-dry lung weight ratio (w/d; 5.39 ± 0.15 vs. 4.42 ± 0.11; p < 0.05) (upper panel), and lung-weight-to-bodyweight ratio (lw/bw; 0.0142 ± 0.0011 vs. 0.0120 ± 0.0006; p < 0.05) (lower panel). moreover, htv ventilation significantly increased the protein concentration in balf (pcbal; 1635±212 vs. 459±69 μg/ml; p < 0.001) (fig. 4b) . intravenous treatment of cu/zn sod notably decreased htv ventilation-induced increases in w/d (4.77 ± 0.05; p < 0.05) and lw/bw ratio (0.0124 ± 0.0007; p < 0.05), and reduced pcbal (1077.5 ± 51.6 μg/ml; p < 0.05). white blood cell (wbc) count and differential in the lung lavage has been widely used as an indicator of lung inflammation [78] . figure 5 malondialdehyde (mda) is an endogenous end-product of oxygen radical-induced and enzymatic lipid peroxidation, and has been used as a biomarker of oxidative stress [29] . the lavage level of methylguanidine (mg), an endproduction of protein catabolism, has been associated with the degree of pulmonary hydroxyl radical formation [88] . figure 6 demonstrates that 5 h of htv ventilation increased mda (3.85 ± 0.53 vs. 2.92 ± 0.34 nmol/ml; p < 0.05) and mg (158.5 ± 21.1 vs. 70.1 ± 9.6 mg/ml; p < 0.001) concentrations in balf as compared with those of the ltv group. intravenous sod treatment during htv ventilation effectively reduced mda (2.095 ± 0.35 nmol/ml; p < 0.05) and mg (77.5 ± 4.4 mg/ml; p < 0.05) in the lung lavage, indicating reduced oxidative stress and ameliorated hydroxyl radical formation and pulmonary lipid peroxidation. nitric oxide (no) plays an essential role in the modulation of pulmonary vascular, airway smooth muscle tone, non-adrenergic and non-cholinergic neurotransmission and mediation of the inflammatory response [86] . figure 7 shows hourly measured serum no in the ltv (white), htv (black) and htv + sod group (grey). we observed that though both ltv and htv ventilation decreased serum no, ltv ventilation decreased serum no primarily during the first 2 h of ventilation (p < 0.05), whereas htv ventilation decreased serum no throughout most of the 5-h ventilation (p < 0.05). notably, sod administration monotonically increased serum level of no during the course of htv ventilation. we assessed the level of pulmonary antioxidant defense via the protein expression of glutathione peroxidase-1 (gpx1) [70] , and the degree of lung inflammation by means of protein expressions of vascular cell adhesion molecule-1 (vcam-1; it mediates the adhesion of lymphocytes, monocytes, eosinophils and basophils to vascular endothelium), and inducible nitric oxide synthase (inos; it functions as a downstream inflammatory mediator of nf-κb [6] ), and matrix metalloproteinase-9 (mmp-9; it activates extracellular matrix remodeling and facilitates inflammatory cell recruitment across the epithelium [9] ), and surfactant protein a and d (sp-a and sp-d; they mediate pulmonary innate immune function [47] and the latter has been demonstrated to modulate ventilator-induced lung inflammation [67] ), and tumor necrosis factor-α (tnf-α; it functions as an essential inflammatory mediator that exerts its effector actions through activation of a transcription factor), and mrna expression of nuclear factor-κb (nf-κb; it plays b a ltv htv htv+sod though absolute macrophages (lower panel) increased after htv ventilation, the percent macrophages decreased. sod treatment during htv ventilation markedly reduced total white blood cells and the percentage of neutrophils in balf (* and # signify p < 0.05 and p < 0.001, respectively) a role in inflammation-associated cell death) [83] . figure 8 shows that htv ventilation markedly increased vcam-1, tnf-α and mmp-9 (p < 0.05), while decreased sp-a and sp-d expressions in lung (p < 0.05) as compared with those assessed in the ltv group (n = 3 each). intravenous sod administration effectively attenuated htv ventilation-associated increases in protein expressions of vcam-1, tnf-α and mmp-9, while improved suppressed protein expressions of sp-a and sp-d in lung (p < 0.05). although lung tissue expressions of gpx-1 and inos in the htv group were not significantly different from those measured in the ltv group, intravenous sod administration markedly increased the protein expression of gpx-1 while decreased protein expression of inos (p < 0.05), suggesting improved antioxidant defense and reduced inflammation in lung. as a pro-inflammatory transcription factor, nuclear factor-κb (nf-κb) plays an important role in regulating the immune and inflammatory response through upregulating inos, mmps, adhesive molecules, proinflammatory cytokines and chemokines [75] . figure 9 shows that htv ventilation upregulated nf-κb mrna expression by more than 16-fold versus that of the ltv group (p < 0.05), whereas intravenous sod administration effectively reduced htv ventilation-induced activation of nf-κb, by merely 6-fold that of the ltv group (p < 0.05), therefore limiting inflammatory response. using this in vivo rat model, we demonstrated that 5 h of ltv ventilation (8 ml/kg) increases ri, but did not significantly alter other parameters of lung function as compared to the baseline, whereas 5 h of htv ventilation (18 ml/kg) can induce apparent combined restrictive and obstructive lung disorder. in addition, htv ventilation significantly increased levels of pulmonary hydroxyl radical formation and lipid peroxidation, inflammatory cell infiltration and sequestration, and markedly increased lung inflammation. we showed that intravenous administration of sod during htv ventilation minimizes deterioration of lung function and significantly reduces associated lung inflammation and oxidative stress. to our knowledge, this is the first demonstration of apparent protective effectiveness of intravenous sod in mechanical ventilation, which may have future therapeutic significance. cu/zn sod is a homodimeric metalloenzyme with two 16.3 kda 153 amino acid subunits [50] . superoxide fig. 7 hourly measured serum no during ltv (white) and htv ventilation (black) and htv ventilation with sod administration (grey). both ltv and htv ventilation decreased serum no. ltv ventilation decreased serum no primarily during the first 2 h of ventilation (p < 0.05), whereas htv ventilation decreased serum no throughout most of the 5 h of ventilation (p < 0.05, except at 4 h). notably, sod administration monotonically increased serum no level during the course of htv ventilation is the predominant free radical produced in the biological system, and sod exerts enzymatic protection against superoxide damage by degrading superoxide radicals into oxygen and hydrogen peroxide, which in turn are converted by gpx-1 into water [84] . fukai and ushio-fukai [79] reported that sods inhibit superoxide-induced inactivation of iron-sulfur containing enzymes, such as aconitase and fumarase, and reduce the release of iron and subsequent formation for highly toxic hydroxyl radical or related iron-associated reactive species through reacting with h 2 o 2 . sod has no apparent side effects and has been shown to possess anti-inflammatory capacity against neutrophilic activity, in addition to being an antioxidant [49] . reitsma et al. [64] reported that negatively charged vascular endothelial glycocalyx tends to bind extracellular sod and facilitates its protective role. however, as a therapeutic agent, sod is limited by its relatively large molecular size and a short circulating half-life of 6 min [57] . nevertheless, exogenous treatment of sod has been demonstrated protective against hyperoxic lung injury. mikawa et al. [53] showed that intravenous administration of cu/zn sod markedly reduces hyperoxiainduced pulmonary capillary protein and water leak and decreases tissue oxidative stress and inflammation in rabbit lung. in hyperoxic mice, yen et al. [87] demonstrated that aerosol administration of cu/zn sod improves survival, reduces lung edema and parenchymal damage, and reduces systemic oxidative stress. comparable to the protective efficacy of cu/zn sod in hyperoxic lung injury, intravenous cu/zn sod treatment during htv ventilation was shown to significantly improve lung function, reduce pulmonary oxidative stress and lung inflammation, increase antioxidant defense and no bioavailability, and help sustain immune homeostasis. imbalance of sod versus gpx-1 often results in the accumulation of hydrogen peroxide and formation of hydroxyl radicals that lead to protein, lipid and even dna damage [13] . our data show that intravenous treatment of sod attenuated htv ventilation-induced pulmonary hydroxyl radical formation, lipid peroxidation, and significantly enhanced pulmonary antioxidant defense. though the exact mechanism of sod-related increasing gpx-1 is unclear, gpx-1 upregulation in response to increased cu/ zn sod has previously been observed in a murine cell line study [12] . reducing oxidative stress and increasing antioxidant activity reduce ri and improve fev. on the other hand, adding cu/zn sod may not always reduce lipid peroxidation, and that is associated with the level of iron and catalase [54] . h 2 o 2 derived by cu/zn sod, can cross cellular membrane and form hydroxyl radicals or metal-related reactive species when interacted with redoxactive transitional metal ions, such as iron or cu, via fenton reaction that leads to lipid peroxidation and cellular injury [17] . since catalase reduce h 2 o 2 into h 2 o, increased cu/zn sod in combination with catalase decreases lipid peroxidation. in this study, htv ventilation was shown to activate nf-κb and increase vcam-1, mmp-9 and tnf-α protein expressions, whilst each factor plays a role in lung function impairment, and that was effectively attenuated by intravenous treatment of cu/zn sod. vcam-1 overexpression was found in various acute and chronic lung diseases, including acute respiratory distress syndrome [15] , ventilator-induced lung injury [8] and pulmonary fibrosis [3] . cook-mills et al. showed that vcam-1 expression in lung is mediated by oxidative stress; antioxidant treatment (vitamins c and e, resveratrol, pyrrolidine dithiocarbamate and n-acetylcysteine) can inhibit vcam-1 signal transduction, thus reducing leukocyte binding to vcam-1 and decreasing vcam-1-dependent inflammation [8, 37] . recently, segui et al. [71] demonstrated that exogenous cu/zn sod downregulates endothelial vcam-1 expression that reduces leukocyte rolling and adhesion to vasculatures and leukocyte-endothelial cell interaction. mmp-9 involves wide-ranging extracellular matrix remodeling, and facilitates inflammatory cell trafficking that contribute to pathological progresses of restrictive lung disease [2] and pulmonary dysfunction in newborn babies [69] . kim et al. [33] demonstrated that inhibition of mmp-9 attenuates htv ventilation-induced lung inflammation and alveolar basement membrane damages by restricting neutrophil transmigration and tissue remodeling. treatment of cu/zn sod has been shown to suppresses mmp-9 expression by interruption of the ros-nf-κb-dependent pathway [72] and inhibition of the extracellular signal-regulated kinase (erk) pathway [23] . upregulation of tnf-α production that depletes cellular antioxidants and increases susceptibility of lung tissues to oxygen radicals [19] is associated with various lung diseases, including asthma, chronic bronchitis, copd, emphysema [45] , pulmonary fibrosis [45] , and acute respiratory distress syndrome (ards) [55] . hughes et al. [26] showed that tnf-α can potentiate histamine release at low antigen concentrations in patients with allergic asthma, causing bronchial smooth muscle contraction and increased airway resistance. also, wagner [81] showed that tnf-α induces bronchial vasoconstriction, primarily through thromboxane a2 mediated secondary release of endothelin 1. in murine, choi et al. [7] reported that blockade of tnf-α reduces late-phase airway hyper-responsiveness and airway inflammation, mainly through inactivation of phospholipase a2. in rats, guery et al. [20] presented that treatment with anti-tnf-α antibody during htv ventilation significantly reduces lung inflammation, pulmonary capillary permeability and lung edema, and damage to epithelial cells. a body of evidence suggests that activation of nf-κb plays a crucial role in ventilator-associated lung injury, while inactivation of nf-κb, by means of antibody [5] , antioxidant [11] , steroid [24] or induced pluripotent stem cells [43] , can protect against lung injury. ko et al. [35] showed that excessive stretch of lung tissue induces noninfectious lung inflammation through activation of nf-κb-interleukin(il)-6 signaling pathways. li et al. [39] reported that htv ventilation caused significant lung injury in wild-type mice, but fails to induce lung injury in protein myeloid differentiation factor 88 (myd88) deletion mice, in which nf-kb/myd88 pathway was interrupted. chiang et al. [5] showed that anti-nf-κb antibody treatment ameliorates lung injury caused by htv ventilation combined with lung ischemia and reperfusion (i/r) injury or by either insult alone, suggesting the pivotal role of nf-κb pathway in lung injury induced by htv ventilation and/or lung i/r. in autophagy-deficient mouse strains, where nf-κb was inactivated, lellouche, et al. [44] demonstrated that htv ventilation did not induce lung inflammation or lung injury. on the other hand, lin et al. [41] showed that cu/zn sod attenuates the inflammatory response in human epithelial cells through inactivation of nf-κb and activator protein-1, inhibition of jnk and p38 phosphorylation-mediated vcam expression, and decreasing tnf-α induced superoxide productions. also, yasui et al. [85] reported that exogenously added cu/zn sod regulates neutrophil apoptosis and reduces the caspase-dependent inflammatory response [56] , and that is mediated by nf-κb [36] . cu/zn sod mediates pulmonary vascular resistance and airway smooth muscle relaxation through increasing vascular nitric oxide (no) bioavailability while reducing inos expression no plays an essential role in regulating lung function in normal and disease conditions, by which no modulates pulmonary vascular smooth muscle tone [73] and airway smooth muscle relaxation [65] . no also involves fibroblast and vascular smooth muscle cell proliferation [21] , angiogenesis, and neural development [14] . no is synthesized in pulmonary endothelial cells from l-arginine by no synthases (noss) existing in three isoforms, namely neuronal (nnos or nos-1), inducible (inos or nos-2) and endothelial nos (enos or nos-3), where enos is the primary nos isoform expressed in the lung [30] . enos can be activated by agonists such as acetylcholine [14] and produces no in a continuous and lowlevel fashion [73] . as part of the immune defense, inos generates no in large quantities in response to stimuli, including tnf-α, oxidative stress, cytokines and various pro-inflammatory mediators [59] . evidence indicates that decreasing no bioavailability plays a major role in pulmonary vasoconstriction and increased airway resistance, resulting from hyperoxia [16] , pulmonary hypertension [21] or ards [77] . previous animal and patient studies revealed that large volume mechanical ventilation increases inos expression, in addition to ros production [15, 27, 42] . with the presence of ros, vascular no is consumed and no bioavailability is reduced leading to vasoconstriction [51] . moreover, no reacts with ros to form peroxynitrite, which in turn reacts with lipids, dna and proteins, causing oxidative damages to cells, resulting in cell necrosis or apoptosis [51] . previously, jung et al. [31] showed that vascular no bioavailability is strongly related to the level of extracellular sod, since sod scavenges superoxide anions that compete to bind available no. in rats, lynch et al. [46] showed that vascular cu/zn sod deficiency, by means of restricting dietary consumption of copper, can impair endothelial vasodilator function through direct inactivation of no. besides, wu et al. [82] showed that inos expression requires nadph oxidase-dependent redox signaling in microvascular endothelial cells. we showed that intravenous cu/zn sod suppresses inos expression in lung, potentially through reducing stimulation by ros, and steadily increases serum no concentration during the course of hvt ventilation, supporting increased pulmonary no bioavailability. we also conducted protein expressions of phosphorylated and unphosphorylated enos without finding significant differences among groups. mechanical ventilation has been demonstrated to affect the secretion and metabolism of pulmonary surfactants [25] . large tidal volume ventilation, though temporarily increases, eventually suppresses pulmonary surfactant secretion and decreases organized surfactant lipoproteins, and thus alters biophysical properties of surfactants that lead to decrease in lung compliance [52] and impaired pulmonary immune responses [80] . besides, the presence of pulmonary oxidative stress generated during mechanical ventilation can inactivate pulmonary antiproteases and impair surfactant function [62] . park et al. [58] showed that 80% reduction in sp-a mrna expression following ros stimulation. in addition to reducing alveolar surface tension, sp-a and sp-d play important roles in pulmonary immune responses. sp-a and sp-d, as pattern-recognition molecules of the collectin family of c-type lectins, mediate pulmonary immune defense through enhancing neutrophil uptake of bacteria [22] , and removal of microbes and their debris, pathogens, allergens, dying epithelial cells and phagocytes [80] . bridges et al. [4] showed that sp-a and sp-d can block low density lipoprotein oxidation and free-radical formation or propagation, so as to serve as potent endogenous inhibitors of lipid peroxidation and oxidative cell damage. furthermore, khubchandani and synder [32] demonstrated that sp-a offsets the inhibitory effects on surfactant function by plasma albumin leaking due to lung injury. yoshida et al. [89] showed that sp-d reduces alveolar macrophageassociated oxidant production, nf-κb activation, and mmp expression. in mice, yoshida et al. [89] reported that genetic deficiency of sp-d gene promotes alveolar macrophage infiltration, and increases mmp-2, mmp-9, and mmp-12 expressions and hydrogen peroxide formation in lung, causing progressive pulmonary emphysema. in premature newborn lambs, sato et al. [67] represented that sp-d administration inhibited ventilation-induced lung inflammation. in this study, htv ventilation was shown to reduce sp-a and sp-d expression in lung, while intravenous cu/zn sod treatment increased both sp-a and sp-d expressions that were associated with improved lung compliance, decreased mmp-9 and nf-κb expression in lung and improved lung function. as summarized in fig. 10 , 5 h of htv mechanical ventilation induces combined a restrictive and obstructive lung disorder that is associated with increased pulmonary oxidative stress, lung inflammation and inhibited expression of sp-a and sp-d (solid line: induction; dashed line: inhibition). protective efficacy of intravenous administration of cu/zn sod against htv ventilationassociated impairment of lung function is related to 4 mechanisms: (1) reductions in pulmonary oxidative stress and improvement in cellular antioxidant defense, as seen by reduced mg and mda and increased gpx-1 protein expression; (2) decreases lung inflammation, evident by reduced neutrophil and wbc count in the balf and suppressed mmp-9, vcam-1, tnf-α and inos protein expression and inhibited nf-κb mrna expression in lung; (3) increased sp-a and sp-d protein expression that improve lung compliance and antiinflammatory capacity, and (4) increased vascular no bioavailability that reduces airway resistance and increases air flow rate. acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome bronchoalveolar matrix metalloproteinase 9 relates to restrictive lung function impairment in systemic sclerosis mediators of chronic obstructive pulmonary disease pulmonary surfactant proteins a and d are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury ventilator-induced lung injury (vili) promotes ischemia/reperfusion lung injury (i/r) and nf-kappab antibody attenuates both injuries ethyl caffeate suppresses nf-kappab activation and its downstream inflammatory mediators, inos, cox-2, and pge2 in vitro or in mouse skin tnf-alpha induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase a(2) activation vascular cell adhesion molecule-1 expression and signaling during disease: regulation by reactive oxygen species and antioxidants overlapping and independent contributions of mmp2 and mmp9 to lung allergic inflammatory cell egression through decreased cc chemokines lowvolume ventilation causes peripheral airway injury and increased airway resistance in normal rabbits cyclic stretch-induced oxidative stress increases pulmonary alveolar epithelial permeability elevation in the ratio of cu/zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide cu/zn-superoxide dismutase and glutathione peroxidase during aging relative contributions of endothelial, inducible, and neuronal nos to tone in the murine pulmonary circulation alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury inhaled nitric oxide selectively reverses human hypoxic pulmonary vasoconstriction without causing systemic vasodilation superoxide dismutases: role in redox signaling, vascular function, and diseases ventilator-associated lung injury in patients without acute lung injury at the onset of mechanical ventilation human tnf-alpha in transgenic mice induces differential changes in redox status and glutathione-regulating enzymes ventilation-induced lung injury is associated with an increase in gut permeability role of nitric oxide in the pathogenesis of chronic pulmonary hypertension pulmonary surfactant proteins a and d enhance neutrophil uptake of bacteria zn-sod down-regulates mmp-9 expression via inhibition of erk ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappab and is blocked by steroids binding and uptake of surfactant protein d by freshly isolated rat alveolar type ii cells the effects of tumour necrosis factor alpha on mediator release from human lung peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase activation of nfkb and coagulation in lung injury by hyperoxia and excessive mechanical ventilation: one more reason "low and slow" is the way to go? malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury effect of adenovirus-mediated gene transfer of nitric oxide synthase on vascular reactivity of rat isolated pulmonary arteries extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecsod-deficient mice surfactant protein a (sp-a): the alveolus and beyond inhibition of matrix metalloproteinase-9 prevents neutrophilic inflammation in ventilatorinduced lung injury therapeutic effect of mahaenggamseok-tang on neutrophilic lunginflammation is associated with nf-κb suppression and nrf2 activation nf-kappab activation in myeloid cells mediates ventilator-induced lung injury the nuclear factor nf-kappab pathway in inflammation inflammatory signalings involved in airway and pulmonary diseases high tidal volumes in mechanically ventilated patients increase organ dysfunction after cardiac surgery toll-like receptor 4-myeloid differentiation factor 88 signaling contributes to ventilator-induced lung injury in mice hospital mortality, length of stay, and preventable complications among critically ill patients before and after tele-icu reengineering of critical care processes superoxide dismutase inhibits the expression of vascular cell adhesion molecule-1 and intracellular cell adhesion molecule-1 induced by tumor necrosis factor-alpha in human endothelial cells through the jnk/p38 pathways ventilatorinduced injury augments interleukin-1beta production and neutrophil sequestration in lipopolysaccharide-treated lungs suppressing nf-kappab and nkrf pathways by induced pluripotent stem cell therapy in mice with ventilator-induced lung injury impairment of autophagy decreases ventilator-induced lung injury by blockade of the nf-kappab pathway tumor necrosis factor-alpha overexpression in lung disease: a single cause behind a complex phenotype vascular superoxide dismutase deficiency impairs endothelial vasodilator function through direct inactivation of nitric oxide and increased lipid peroxidation surfactant proteins a and d protect mice against pulmonary hypersensitivity induced by aspergillus fumigatus antigens and allergens low-tidal-volume ventilation in the acute respiratory distress syndrome orgotein: a new drug for the treatment of radiation cystitis cu/zn superoxide dismutase plays important role in immune response role of free radicals in vascular dysfunction induced by high tidal volume ventilation alterations to surfactant precede physiological deterioration during high tidal volume ventilation attenuation of hyperoxic lung injury in rabbits with superoxide dismutase: effects on inflammatory mediators the role of iron in the initiation of lipid peroxidation role of tnfalpha in pulmonary pathophysiology dialysis membrane-induced neutrophil apoptosis is mediated through free radicals extracellular superoxide dismutase: a regulator of nitric oxide bioavailability mapk and jak-stat signaling pathways are involved in the oxidative stress-induced decrease in expression of surfactant protein genes inducible nitric oxide synthase contributes to ventilatorinduced lung injury activation of human macrophages by mechanical ventilation in vitro interactions of lung stretch, hyperoxia, and mip-2 production in ventilator-induced lung injury oxidative stress in pathogenesis of chronic obstructive pulmonary disease: cellular and molecular mechanisms nitric oxide inhibition of basal and neurogenic mucus secretion in ferret trachea in vitro the endothelial glycocalyx: composition, functions, and visualization peroxisome proliferator-activated receptor expression is reduced in skeletal muscle in copd the effects of eucheuma cottonii on alveolar macrophages and malondialdehyde levels in bronchoalveolar lavage fluid in chronically particulate matter 10 coal dust-exposed rats surfactant protein-d inhibits lung inflammation caused by ventilation in premature newborn lambs cyclic stretch of airway epithelium inhibits prostanoid synthesis oxidative stress and increased type-iv collagenase levels in bronchoalveolar lavage fluid from newborn babies oxidative stress and lung function down-regulation of endothelial adhesion molecules and leukocyte adhesion by treatment with superoxide dismutase is beneficial in chronic immune experimental colitis suppression of tnfalpha-induced mmp-9 expression by a cell-permeable superoxide dismutase in keratinocytes nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans nicotinamide abrogates acute lung injury caused by ischaemia/reperfusion nf-kappab: a key role in inflammatory diseases tidal hyperinflation during low tidal volume ventilation in acute respiratory distress syndrome inhaled nitric oxide in acute respiratory distress syndrome: a pilot randomized controlled study low-tidalvolume mechanical ventilation induces a toll-like receptor 4-dependent inflammatory response in healthy mice pulmonary surfactant is altered during mechanical ventilation of isolated rat lung mechanical ventilation of isolated rat lungs changes the structure and biophysical properties of surfactant tnf-alpha induced bronchial vasoconstriction inos expression requires nadph oxidasedependent redox signaling in microvascular endothelial cells methylprednisolone inhibition of tnf-alpha expression and nf-kb activation after spinal cord injury in rats therapeutic potential of superoxide dismutase (sod) for resolution of inflammation superoxide dismutase (sod) as a potential inhibitory mediator of inflammation via neutrophil apoptosis role of exhaled nitric oxide in asthma aerosolized human extracellular superoxide dismutase prevents hyperoxia-induced lung injury contribution of hydroxyl radical to the production of methylguanidine from creatinine surfactant protein d regulates nfkappa b and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways the authors would like to thank professor john v. tyberg of university of calgary, whose expertise significantly improves the manuscript. this study was supported by operating grant 104-cm-fju-07 from the chi mei foundation hospital (tainan) to drs. jiun-jr. wang and nan-chun wu. the data obtained and/or analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions ncw, ftl, chm, ycy and jjw all made substantial contributions to the conception or design of the study. ncw, ftl, ycy participated in the data acquisition. all authors participated in the data analysis. all authors participated in the interpretation of reported data. jjw prepared for the manuscript. all authors reviewed and/or critically revised the manuscript for important intellectual content and provided final approval of the version to be published. the study protocol has been approved by the animal care and use committee of the fu jen catholic university and in compliance with the guidelines for the care and use of laboratory animals (nih guide, volume 25, number 28, 1996) . not applicable. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-004092-wb150n8w authors: nieman, gary f.; gatto, louis a.; andrews, penny; satalin, joshua; camporota, luigi; daxon, benjamin; blair, sarah j.; al-khalisy, hassan; madden, maria; kollisch-singule, michaela; aiash, hani; habashi, nader m. title: prevention and treatment of acute lung injury with time-controlled adaptive ventilation: physiologically informed modification of airway pressure release ventilation date: 2020-01-06 journal: ann intensive care doi: 10.1186/s13613-019-0619-3 sha: doc_id: 4092 cord_uid: wb150n8w mortality in acute respiratory distress syndrome (ards) remains unacceptably high at approximately 39%. one of the only treatments is supportive: mechanical ventilation. however, improperly set mechanical ventilation can further increase the risk of death in patients with ards. recent studies suggest that ventilation-induced lung injury (vili) is caused by exaggerated regional lung strain, particularly in areas of alveolar instability subject to tidal recruitment/derecruitment and stress-multiplication. thus, it is reasonable to expect that if a ventilation strategy can maintain stable lung inflation and homogeneity, regional dynamic strain would be reduced and vili attenuated. a time-controlled adaptive ventilation (tcav) method was developed to minimize dynamic alveolar strain by adjusting the delivered breath according to the mechanical characteristics of the lung. the goal of this review is to describe how the tcav method impacts pathophysiology and protects lungs with, or at high risk of, acute lung injury. we present work from our group and others that identifies novel mechanisms of vili in the alveolar microenvironment and demonstrates that the tcav method can reduce vili in translational animal ards models and mortality in surgical/trauma patients. our tcav method utilizes the airway pressure release ventilation (aprv) mode and is based on opening and collapsing time constants, which reflect the viscoelastic properties of the terminal airspaces. time-controlled adaptive ventilation uses inspiratory and expiratory time to (1) gradually “nudge” alveoli and alveolar ducts open with an extended inspiratory duration and (2) prevent alveolar collapse using a brief (sub-second) expiratory duration that does not allow time for alveolar collapse. the new paradigm in tcav is configuring each breath guided by the previous one, which achieves real-time titration of ventilator settings and minimizes instability induced tissue damage. this novel methodology changes the current approach to mechanical ventilation, from arbitrary to personalized and adaptive. the outcome of this approach is an open and stable lung with reduced regional strain and greater lung protection. globally more than three million patients per year develop acute respiratory distress syndrome (ards), accounting for 10% of all intensive care unit (icu) admissions. in the united states, up to 200,000 patients a year are diagnosed with ards and 75,000 of these patients die [1] . current ards treatment is supportive: protective mechanical ventilation, typically using lower tidal volume ventilation (vt) and low-moderate positive end expiratory pressure (peep) [2] . unfortunately, current protective ventilation strategies have not lessened ards mortality rate [1, [3] [4] [5] [6] [7] [8] [9] [10] [11] . the determinant of vili is not the "mode" of ventilation, but the way parameters of the mechanical breath are set and combined. the goal of any protective mechanical breath should be maintaining functional residual capacity and increasing lung homogeneity. in this paper, we review the pathophysiology of ards in the microenvironment and identify how changes in alveolar micromechanics predispose the lung to a secondary vili. understanding how ards alters the dynamic alveolar inflation physiology enables us to adjust the mechanical breath profile (mb p -all airway pressures, volumes, flows, rates and the time at inspiration and expiration at which they are applied) necessary to minimize vili [12] . variants of the airway pressure release ventilation (aprv) mode have been used for decades with many combinations of settings (fig. 1) . in this review, we discuss the physiological impact of the time-controlled adaptive ventilation (tcav) method on ards-induced abnormal alveolar mechanics, efficacy in both translational animal models and in a retrospective clinical analysis. acute respiratory distress syndrome pathophysiology current falls into three categories: (a) normal nondependent tissue, (b) severely injured and collapsed dependent tissue, and (c) unstable tissue located between these two tissue types [13, 14] . efforts to minimize vili, block progressive acute lung injury (ali), and reduce ards mortality have resulted in two current approaches: (1) the ardsnet low vt (lvt) method is intended to protect the non-dependent normal lung tissue from overdistension (od) and reduce alveolar recruitment/ derecruitment (r/d) with positive end expiratory pressure (peep), while resting severely injured tissue by allowing it to remain collapsed throughout the ventilation cycle [2] . however, this strategy has not further reduced ards mortality [1, [3] [4] [5] [6] [7] [8] [9] [10] [11] . this suggests that our understanding of ards pathophysiology remains [12] all using the airway pressure release ventilation (aprv) mode but with different methods: a stock et al. used a cpap phase that encompassed 60% of each breath, a release phase of 1.27 s and a respiratory rate (rr) of 20/min [98] ; b davis et al. decreased the respiratory rate by prolonging both the cpap and release phase [99] ; c gama de abreau et al. adjusted their cpap and release phase to values typical of a conventional breath [100] ; d roy et al. minimized the release phase and extended cpap to occupy 90% of each breath, typical of the time-controlled adaptive ventilation (tcav) method [83] . although these studies all used the aprv mode, each differs significantly in the application methods used to set the mode incomplete, particularly in the lung microenvironment [15, 16] . indeed, the concept that the pulmonary parenchyma falls into three crudely differentiated categories according to the gravitational axis is being challenged. the current understanding is that open and collapsed tissues are not delineated into compartments, but are rather intermingled throughout the entire lung [17] [18] [19] [20] [21] . the unchanged mortality associated with the lvt method may also reflect the fact that maintaining lung tissue collapse ("resting") may not be protective [1, [3] [4] [5] [6] [7] [8] [9] [10] [11] . the atelectatic lung does not exchange gas, is susceptible to pneumonia, and may ultimately lead to collapse induration and fibrosis with the inability to re-inflate or epithelialize the airspace [22] [23] [24] . using conventional ventilation strategies, the ola has not been shown to reduce alveolar r/d-induced atelectrauma [25, 26] or improve survival [27] . in a recent rct, the ola with maximal recruitment strategy and peep set to best compliance resulted in increased mortality [27] . however, the lack of significant differences in compliance and driving pressure (∆p) between groups suggested that (1) the lungs had not been well recruited, which is essential for the ola strategy to be effective; (2) the lungs were overdistended by excessive strain following the maximal recruitment; or (3) the chosen peep was not optimal to stabilize the newly recruited lung. other research has shown [25] that ola could not be attained using peep up to 15 cmh 2 o and plateau pressure (pplat) limited to 30 cmh 2 o. while ola is theoretically lung protective, traditional recruitment maneuver (rm) + peep methods may not provide sustained recruitment, stability, and homogeneity [25, 26, [28] [29] [30] . more recent studies suggest that the lung pathology compartmentalized by gravity (i.e., normal lung tissue adjacent to acutely injured tissue) is incorrect and that regional lung strain and inflammation throughout the entire lung is the main driver of vili [16, [31] [32] [33] [34] [35] [36] . regional strain is caused with each breath by (1) alveolar and alveolar duct r/d [37] [38] [39] [40] [41] [42] [43] and (2) stress-multiplication (s-m), which cause injury to open lung areas adjacent to collapsed or edema-filled tissue [18, 19, [44] [45] [46] [47] [48] . retamal et al. used ct scans to generate volumetric strain maps revealing highly heterogeneous regional strains (caused by alveolar r/d and s-m), which suggests that there may not be a safe threshold for low vt [49] . cereda et al. hypothesized that vili is not caused by overdistension of normal lungs, but rather develops in multiple areas of excessive regional strain located throughout the lung and caused by the primary insult [17] . they showed that tissue adjacent to the primary lesion was most susceptible to secondary vili, an outcome supported by dynamic modeling of interdependent parenchyma during ali [21] . this suggests that to effectively reduce vili at the bedside, the clinician needs to know how to adjust ventilator settings (e.g., vt, pplat, peep, inspiratory and expiratory duration) to reduce r/d and s-m [50] [51] [52] . synchrotron phase-contrast imaging can measure r/d at acinar length scales over short time frames and has demonstrated that lung collapse in the microenvironment differs between normal and acutely injured lungs [53] [54] [55] [56] . scaramuzzo et al. first measured tissue collapse in the microenvironment of the normal lung with graded reductions in peep. they assessed the numerosity (asnum) and dimension (asdim) of airspaces during lung deflation and found that the primary mechanism by which the lung loses volume was reduced asnum secondary to alveolar and small airway derecruitment [53] . in a subsequent paper, scaramuzzo showed in an ards model that the mechanism of lung deflation was reduced asdim, which differs from the mechanism of normal lung deflation (asnum) [54] . broche et al. showed that "compliant collapse", which is described "as a structural collapse of the airway wall along a certain length" is the primary mechanism of airway closure in the acutely injured lung [56] . "compliant collapse" suggests that fluid movement in the microenvironment would play a role in airway collapse and reopening. thus, the function of time during inspiration and expiration, and the opening and closing pressures, would be key components in keeping the lung open and stable [57] . this work underscores the merits of an extended inspiratory duration and a brief expiratory duration to improve alveolar recruitment and stability in a rat ards model [35] , lung protection in a neonatal piglet model [58] , and reduced ards incidence and mortality in trauma patients [59] . we postulate that as the lung opens, the increase in parenchymal tethering of airways [56] and alveolar interdependence [55] reduce lung pathology as a power-law function. hamlington et al. have shown that progressive lung injury advances in power-law fashion where alveolar r/d (atelectrauma) caused the initial holes in the epithelium and that high airway pressure (volutrauma) greatly expands these holes in a power-law or rich-get-richer fashion [60] . lung protection also arguably follows a power-law function with reestablishment of parenchymal tethering, alveolar interdependence, and surfactant function all working together to accelerate recruitment and stabilization of adjacent tissue. alveoli are often misunderstood as elastic and modeled as rubber balloons with immediate size changes (volumetric distortion or strain) with application or removal of pressure (physical stress) during inspiration and expiration [15] . in reality, alveoli behave in a viscoelastic rather than an elastic manner [21, [61] [62] [63] . viscoelastic systems exhibit a time-dependent strain and can be conceptualized by the spring-and-dashpot model (fig. 2) [21] . figure 2 illustrates the strain/time curve of elastic (spring), viscous (dashpot), and viscoelastic (spring-and-dashpot) behaviors. since the lung opens and collapses as a viscoelastic system, we use the spring and dashpot to illustrate lung recruitment during inspiration and derecruitment during expiration. the initial rapid opening or collapse (strain) of lung tissue followed by a continual opening or closing over an extended period time (2-5 s) is important. viscoelastic behavior of alveolar opening and collapse begins only after the critical opening or collapse pressure for that alveolus is reached. before these critical pressures are obtained, there is no alveolar strain. however, the opening and closing pressures are not static; instead, they are dependent upon the level of surfactant deactivation and the degree of mechanical interdependence between adjacent alveolar walls and parenchymal tethering on the walls of small airways [55] . the original computational model of r/d by ma and bates was based on symmetrical bifurcations of the airway tree with each branch having an individual critical opening and collapse pressure [64] . however, this computational model no longer supported the new biological evidence on r/d at the acinar level. an alveolar interdependence component was added to the model such that the closure of a unit will impact the critical opening and collapse pressures of adjacent units [55] . fluid movement in the microenvironment during airway collapse and reopening suggests that the pressures necessary for opening and collapse are also a function of the time at which they are applied [56] . thus, a long inspiratory time with a short expiratory time would open more alveoli and prevent more alveolar collapse, as compared to the same airway pressures applied for shorter or longer amounts of time [57] . since alveoli recruit as a viscoelastic system, once critical opening and collapse pressures are reached, the longer the inspiration (fig. 2-red hold) , the more lung tissue recruited with each breath [61, [65] [66] [67] [68] . similarly, the shorter the expiratory duration (red release), the less lung tissue that will collapse. furthermore, the sustained inspiratory time causes both creep and stress relaxation, the most likely mechanism of which is redistribution of gas within the lung or opening of collapsed alveoli [69] . we postulate that this information can be used to design an mb p that will open and stabilize the acutely injured lung. the longer the inspiratory time, the more alveoli recruited. we previously quantified in vivo alveolar recruitment in real-time in a rat ards study that involved mathematical modeling. initial recruitment after the applied breath did not begin until after the first second, followed by a rapid recruitment (1-2 s). the majority of recruitment occurred in 2 s with continued gradual recruitment over the subsequent 38 s (fig. 3 ) [70] . the absence of any inflation for the first second has clinical significance since inspiratory time in most conventional ventilator settings is 0.5-1.0 s. a brief inspiratory time confines ventilation to proximal conducting/convective airways rather than allowing the time-dependent gas distribution to reach and facilitate diffusion in the distal airspace [16] . other investigators using ct scans combined with mathematical modeling also support this temporal lag in alveolar opening following an applied proximal airway pressure [71] [72] [73] . the similarities between alveolar percent recruitment/time ( fig. 3 ) coincide with the viscoelastic behavior strain/ time curves (fig. 2 , inspiration-lung recruitment). derecruitment of alveoli is also viscoelastic in nature (fig. 2 , expiration-lung derecruitment). the deflation strain/ time curve suggests that a ventilator strategy with a brief expiratory duration (red release) would minimize lung collapse, placing ventilation on the more favorable expiratory portion of the pressure-volume curve [74] . there is no mechanistic evidence that current ola protocols using a rm and titrated peep actually achieve and sustain an open lung [26, 75, 76] . the ardsnet method features a brief time at peak inspiration and an extended time at expiration (fig. 4, left) , producing an mb p that is antithetical to the tcav method (fig. 4, right) . conversely, the tcav method reconfigures time systems. an applied force (red arrows) generates a stress that results in a yield or strain once the force reaches critical opening pressure. upper left: the spring models elasticity with a rapid increase in strain leading to a plateau strain, which is distinctive of that spring. upper right: the dashpot models viscous strain, where movement of the dash progresses (dashed line) with flow of the fluid in the pot around the dash (brown arrows), which is distinctive of the viscosity of the fluid. bottom: viscoelastic behavior is modeled by the spring and dashpot, where force transfer from the spring to the dash results in a time-dependent strain with an initial rapid change in strain (1-2 s), which becomes gradual over time (2-5 s) . lung strain follows this behavior (fig. 3) . bottom left: an extended inspiratory time (hold) optimizes lung recruitment once critical opening pressure is reached. bottom right: a short expiratory time (release) minimizes lung derecruitment if it is sufficiently fast to prevent reaching the critical collapse pressure (see figure on next page.) allocation to extend inspiration using a continuous positive airway pressure phase (cpap phase) with a brief (sub-second) release for exhalation (release phase). open valve cpap is used rather than closed valve to allow the patient to spontaneously inhale or exhale with little added resistance at any time in the breathing cycle. the short expiratory time does not allow the expiratory flow to reach zero flow, and therefore, the alveolar pressure is always above the set expiratory pressure (p low ), which itself is always set at 0 cmh 2 o. the cpap phase initiates before the lung fully depressurizes (fig. 4, right) , maintaining a positive end expiratory pressure determined by the peak expiratory flow, the expiratory duration, and the compliance of the respiratory system. the gas volume released (vr) during the release phase is analogous to vt in that it equals the volume delivered during the cpap phase (we use vt in place of vr in this review for consistency). however, tcav does not aim to achieve a target vt, but rather the vt changes depending on the release time (t low ), which is adjusted by changes in respiratory system compliance (c rs ): ↓c rs = ↓vt and ↑c rs = ↑vt alveolar recruitment is not only a function of the amount of pressure applied to the lung, but also of the time during which the pressure is applied because alveoli open and collapse as a viscoelastic system (fig. 2, viscoelastic behavior) . alveolar volume change is further influenced by alveolar micro-anatomy, including parenchymal tethering and shared alveolar walls, establishing alveolar interdependence. all the above components play an important role in alveolar recruitment and derecruitment [19, 62, [77] [78] [79] . thus, the longer airway pressure is applied, the more alveoli recruited (fig. 2 , viscoelastic behavior) [70] . this time-dependent recruitment has been described by suki et al. as the "avalanche theory" of lung inflation [80] . we conducted histological measurements of terminal airspace in a rat ards model [16] and reported a redistribution of gas from alveolar ducts into alveoli with tcav, but not with a volume-controlled mode. stress relaxation occurs during the cpap phase because there is sufficient time for alveoli to be recruited. we postulate that gas is transferred from the more elastic ducts (fig. 2 , viscoelastic behavior-rapid initial strain) into the more viscous alveoli (fig. 2 , viscoelastic behavior-slow progressive strain over time) during the extended cpap phase. by comparison, the ardsnet brief inspiratory time (fig. 4 , left, duration of inspiration) method would not effectively recruit viscoelastic alveoli, allow time for tissue creep, or result in redistribution of gas from the ducts into the alveoli [16, 81] . this is supported by studies indicating that the ola, which uses occasional rms combined with a brief inspiratory duration (fig. 4, left) , has not been shown to reduce mortality. the likely reason for this lack of efficacy is that neither rms nor the brief fig. 4 the ardsnet method using the volume assist-control ventilation mode (left) has an i:e ratio of 1:3, which directs a short inspiration and a long expiration, and peep is arbitrarily set. conversely, the tcav method (right) has an i:e ratio of 12:1, which directs a long inspiration (cpap phase) and a short expiration (release phase), not allowing the lung to fully depressurize and resulting in a time-controlled peep (tc-peep, red dashed line). time controlled-peep (tc-peep) is adaptive (not arbitrary) because it is determined in real-time according to compliance, which is measured in the preceding breath by the slope of the expiratory flow curve (slope fe ) (red arrowhead on right) (fig. 6) inspiratory duration effectively opens the lung; therefore, alveolar heterogeneity and regional strain were not eliminated [26, 82, 83] . to normalize the alveolar duct to alveolar volume distribution in the acutely injured lung, it is necessary to use a combination of an extended time at inspiration (cpap phase) and short expiratory duration (release phase) (fig. 4, right) . the physiologic impact of tcav on lung recruitment over time in a brain-dead organ donor is depicted in fig. 5a , top. displayed respiratory system compliance (c rs ), driving pressure (∆p = vt/c rs ), and vt measurements are after initial transition of the brain-dead donor to tcav (tcav = 0 h) and then 12 (tcav = 12 h) and 24 (tcav = 24 h) hours on tcav. the prolonged inspiratory time (fig. 4 , right) gradually "nudges" open the lung and normalizes gas distribution within the alveoli and ducts (fig. 5a -blue collapsed lung tissue converting to open tan tissue) and the brief expiratory time prevents these newly opened alveoli from re-collapsing (fig. 4 , right) [16] . although the ∆p was slightly elevated (16.0 cmh 2 o) when tcav was first applied (t0) due to the low c rs (27 ml/cmh 2 o), it remained within the safe range due to the low vt (7.3 ml/kg). as the lung recruited over time, the vt increased (t12 = 9.2 ml/kg) without increasing ∆p, which fell into the normal lung range (9.1 cmh 2 o) due to increased c rs (59 ml/kg). continual reduction in ∆p occurred because c rs increased (t24 = 88 ml/ cmh 2 o) as the lung fully opened and ∆p fell into the normal range (6.8 cmh 2 o) (fig. 5a, top) with a vt of 10.1 ml/ kg. these data indicate how the vt can only increase if c rs increases, which personalizes the vt to the pathophysiology of the patient's lung in real-time and normalizes the tidal volume to lung volume (fig. 5a, top) . figure 5b , bottom depicts the ventilator screen and the chest radiograph (cxr) from a brain-dead donor initially on controlled mechanical ventilation (cmv) and then converted to tcav. the progressive changes in ∆p and cxr at 3 (tcav = 3 h), 29 (tcav = 29 h) and 84 (tcav = 84 h) hours on tcav are displayed. the progressive decrease in ∆p as the lung recruits is identified by the reaeration of the lung on cxr. these data suggest that an extended cpap duration for a period of hours will "nudge" alveoli open with each breath, reducing c rs and allowing ventilation at a low ∆p even with a vt higher than 6 ml/kg. the lung becomes time and pressure dependent when acutely injured, such that it will quickly collapse at atmospheric pressure [67, [84] [85] [86] . in animal ards models, the majority of lung collapse occurred in the first 4 s of exhalation with collapse as fast as 0.6 s [72] . this suggests preventing collapse of alveoli with the fastest time constants, the expiratory duration must be less than 0.6 s. markstaller et al. had similar findings in an ards porcine model with lung collapse occurring in 95% of the lung within 0.8 s [87] . lachmann was one of the first to suggest that stabilizing alveoli with heterogeneous collapse time constants could be accomplished by dramatically shortening expiratory time [88] . together, these studies suggest it is possible to stabilize alveoli with fast collapse time constants by using a brief expiratory time [72, 85, 87] . the slope of the expiratory flow curve (slope fe ) allows breath-by-breath assessment of changes in c rs (fig. 6 ) [89] . with progressive ali, edema and loss of surfactant function increases lung recoil force, causing (see figure on next page.) fig. 5 optimizing recruitment with tcav allows the lung to accommodate increased tidal volumes, without increases in driving pressure, due to a concomitant increase in compliance. a tcav-induced lung recruitment over time (0-24 h) in a brain-dead organ donor. driving pressure (δp) was calculated as tidal volume (vt) divided by respiratory system compliance (c rs ). the adaptive nature of tcav delivers low vt (7.3 ml/kg at 0 h) with lung collapse and low c rs , but adjusts vt over time (vt = 9.2 ml/kg at 12 h, vt = 10.1 ml/kg at 24 h) as the lung opens and c rs increases. notably, δp actually decreased despite increasing vt (a). b evolution of driving pressure (δp) and chest x-ray (cxr) over time: a cmv (conventional mechanical ventilation) on a brain-dead organ donor (55 kg) with baseline ventilator settings: vc-ac, vt 420, rate 24, peep 8 cmh 2 o with peak pressure 34 cmh 2 o, vt 7.9 ml/kg/predicted body weight (pbw), and δp 26 ml/cmh 2 o. chest x-ray showed severe bilateral infiltrates. tcav = 3 h: 3 h after transition to tcav with settings: cpap phase pressure = 34 cmh 2 o, release set pressure = 0 cmh 2 o, cpap time = 3.4 s, release phase duration = 0.35 s. note the lower vt of 347 ml (6.3 ml/kg/pbw), which gradually increased from a vt of 5.4 ml/kg/pbw when first transitioned to tcav (data not shown); both vts using the tcav protocol are lower than those on the conventional mode (cmv = 437 ml, 7.9 ml/kg/pbw). the cxr demonstrates radiographic clearing of densities with significant recruitment and a reduction in δp from 26 to 17 ml/cmh 2 o. tcav = 29 h: 29 h on tcav, a new chest radiograph for line placement indicated continued recruitment, and the cpap phase pressure was subsequently decreased to 29 cmh 2 o. in addition, the angle of the expiratory flow curve became less acute (fig. 6) , and the release phase duration was increased to 0.4 s. the cpap time was increased to 4.6 s because ventilation had improved. despite a lower p high , the vt continued to increase as did an improvement in c rs . the continued radiographic clearing of densities and reduction in δp fell to 14 ml/cmh 2 o despite continued vt increase. tcav = 84 h: the cpap phase pressure was further decreased to 22cmh 2 o due to continued recruitment (cxr) with a δp of 11 ml/cmh 2 o. the lungs and the heart, liver, and both kidneys from this organ donor were all successfully transplanted rapid lung collapse and decreased c rs . the collapse rate of the lung is manifested as a change in the slope of the expiratory flow curve (slope fe ), a measure of lung recoil, which is determined by c rs and both turbulent and viscous resistances [89] . brody demonstrated that (1) lung c rs could be calculated if both of these resistances are known; (2) dynamic c rs must be a constant, independent of volume; and (3) the inertia of the chest-lung system is negligible [89] . the brief release phase is passive without muscular effort or added external resistance (i.e., peep) such that the slope fe can be used as a bedside monitor to analyze the mechanical properties of the respiratory system on a breath-to-breath basis [89] . the release phase is protocolized using the tcav method for the expiratory flow to terminate (e ft ) at 75% of the expiratory flow peak (e fp ) (e fp × 75% = e ft ) (fig. 6a, b) [90] . the formula e fp × 75% = e ft was first identified empirically at the bedside to be effective at stabilizing the lung [90] and has been subsequently shown to be most effective at maintaining open and stable alveoli [35] , normalizing alveolar/alveolar duct volume distribution [16] , and resulting in homogeneously ventilated alveoli [36] . in the example presented in fig. 6b , e fp is − 50 l/min, so the expiratory flow is terminated (e ft ) at − 37.5 l/min (− 50 l/min × 75% = − 37.5 l/min). to accomplish this at the bedside, the clinician sets the ventilator to terminate the expiratory flow when it reaches 37.5 l/min (fig. 4, right) , and the cpap phase is restored (fig. 4, right) . although slope fe is not directly measured, fig. 6 personalizing the release phase using the slope of the expiratory flow curve (slope fe ). the release phase becomes briefer, directed by the slope fe with lung injury severity. a normal lung release phase is 0.5 s, with moderate ards of 0.4 s and severe ards of 0.3 s, all directed by changes in the slope fe . b the release phase duration is calculated by expiratory flow terminating (e ft ) at 75% of the expiratory flow peak (e fp ) (red arrow head). in this example, the e fp = − 50 l/min, so flow will be terminated (e ft ) at − 37.5 l/min (− 50 l/min × 75% = − 37.5 l/min). although the e ft is always at 37.5 l/min in our example, the release phase duration varies (0.3, 0.4, 0.5 s) due to changes in the slope fe (a, b) . we did not directly measure the slope of the expiratory flow curve, but by terminating expiration at 75% of the e ft , changes in the slope change the expiratory duration (a, b) . thus, the release phase is both personalized and adaptive as the patient's lungs become better or worse using the tcav method. c expiratory flow/ time graphics on a ventilator monitor from a brain-dead organ donor meeting berlin criteria for severe ards. the release phase was set using the equation: e fp × 75% = e ft . the slope fe when tcav was initially applied was 58.8°, resulting in a release phase of 0.3 s. twenty-four hours on tcav and the slope fe increased to 76.3°, resulting in a release phase of 0.5 s. the spike in the expiratory flow curve is an artifact due to compression of gas in the ventilator circuit variation in the slope causes a change in release phase duration: gradual slope = long release phase and steep slope = short release phase (fig. 6a, b, 0 .5, 0.4, 0.3 s release phase times with changes in the slope fe ). figure 6c depicts two airway flow/time curves with the slope fe circled and the angle measured on the ventilator monitor in a brain-dead donor. the top curve shows the initial application of tcav, and the bottom curve is 24 h later. with a steep slope ef , expressed as an angle (58.8°), the expiratory time is short (t low 0.3 s), and as the slope ef increases (angle goes from 58.8º to 76.3°), the expiratory duration increases (t low 0.5 s). this illustrates that the duration of the release phase changes with changing lung pathology and thus is personalized and adaptive as the patient's lung mechanics becomes better or worse (fig. 6a, b ). with cpap, the vt is directly related to c rs (fig. 5a, top) . the adaptive quality of the tcav breath allows for unique personalization of vt based on changes in lung physiology in contrast to the prevailing "one size fits all" 6 ml/kg method [83] . further, the tcav method maintains a low δp since vt decreases as c rs decreases (figs. 5a, top and 7). figure 7 presents gross lung photographs and the corresponding lung compliance (c rs ), tidal volume (vt), and driving pressure (δp) calculated from a previously published paper [82] . the animal model utilized was a clinically applicable porcine peritoneal sepsis and gut ischemia/ reperfusion (ps + i/r) ards model [83] . two groups of animals were studied: (1) ardsnet low vt (lvt) method applied after the animals desaturate and (2) the tcav method applied immediately following ps + i/r injury. the time post-ps + i/r injury that these two protocols were applied matched the time of application on patients clinically (i.e., ardsnet method is applied to patients after oxygen desaturation [2] and tcav is applied immediately upon intubation [59] ). in the ardsnet group, c rs continually decreased over the 48-h study period, whereas in the tcav group, c rs remained similar to baseline at t48 (fig. 7c) . the δp in the tcav group remained in the normal range even with elevated vt (12 ml/kg) because c rs also increased (fig. 7d) . gross photos indicate that the tcav method (fig. 7a ) maintained an open homogeneously ventilated lung without edema, whereas the ard-snet method (fig. 7b) allowed the lung to develop severe atelectasis and both intra-lobule and airway pulmonary edema. given that the inspiration:expiration (i:e) ratio for tcav is approximately 12:1, co 2 retention could reasonably be a concern. because the tcav method is such an effective lung recruitment tool, there is seldom an issue with high blood levels of co 2 once the lung is fully recruited. once recruited, there is a large surface area for co 2 diffusion and thus high concentrations of co 2 can be exhaled during the short release phase. the tcav method can be applied preemptively as soon as the patient is intubated, never giving the lung a chance to collapse and eliminating any problems with co 2 retention [50] , thus minimizing the risk of hypercapnia and eliminating the need for extracorporeal venovenous co 2 removal (ecco 2 r). in addition, if the patient is adequately hydrated, there is no negative impact on lung perfusion since lung recruitment reestablishes normal frc, which reduces pulmonary vascular resistance and right heart afterload [91, 92] . no human rcts have yet utilized the tcav method, but several recent rcts have approximated many of the settings. zhou et al. first evaluated 138 patients with a p/f less than 200 mmhg who were intubated for less than 48 h and randomized to receive either ardsnet lvt or aprv with tcav-like settings [93] . the aprv group demonstrated a significant decrease in number of days on mechanical ventilation (from 15 to 8), length of icu stay , tracheostomy requirement (29.9% to 12.7%), and a 13.4% absolute decrease in mortality (34.3% to 19.7%, p = 0.053), although the study was not sufficiently powered to show a difference in mortality. ganesan et al. conducted an rct using aprv and examined children under 12 years old with ards who had been intubated for less than 72 h and were randomized to receive either standard lvt strategy or aprv [94] . unlike the zhou trial, the aprv arm performed significantly worse, necessitating early trial termination. the investigators, however, introduced two significant and synergistically harmful changes to the tcav protocol: setting and adjusting the p high pressure of the cpap phase based on vt and improper regulation of spontaneous breathing. by limiting p high to maintain a lower vt, the investigators never opened the lung to the point necessary to eliminate regional lung strain, the same mechanism hypothesized to explain the failed art rct. their initial mean airway pressure (pmaw) difference was only 1.6 cmh 2 o despite setting p high at the pplat and then adding an additional 2 cmh 2 o. the authors even provide a table for guiding initial p high settings, which, based on the aprv arm's p/f ratio of 124 mmhg, should have resulted in an initial pmaw difference closer to 7 cmh 2 o-an almost 40% increase from what was observed. lastly, hirshberg et al. conducted an rct in adults with acute hypoxic respiratory failure and attempted to keep the vt at about 6 ml/kg. the study was stopped fig. 7 gross lung photos with corresponding driving pressure (δp), tidal volume (vt), and respiratory system compliance (c rs ) values over time [21] . two protective mechanical ventilation strategies, the tcav method (a) and the ardsnet (lvt) method (b), were tested in a clinically applicable 48-h porcine ards model of peritoneal sepsis (ps) and gut ischemia/reperfusion (i/r) injury [21] . the evolution of c rs , δp, and vt with time in each group occurred over the 48-h study period (c, d). in the ardsnet lvt method group, δp increased despite the reduction in vt because of worsening c rs . with the tcav method, δp remained low despite vt = ~ 12 ml/kg because c rs progressively increased (c, d). the personalized and adaptive vt based on lung c rs (i.e., high c rs = large vt and low c rs = small vt) was also seen in the brain-dead organ donor (fig. 5a) . gross lung photos illustrate that the tcav method (a) was lung protective, whereas the lvt method (b) resulted in severe acute lung injury. δp was calculated retrospectively and was not in the publication by roy et al. [21] early in part because the release volumes (i.e., vt) often exceeded 12 ml/kg. using the tcav protocol an increasing vt indicates that the lung is reopening and is associated with improved crs, δp, and cxr (see example, fig. 5b ). in addition, there was no evidence that the vt of 12 ml/kg caused vili since there were no significant differences in pao 2 /fio 2 (p/f) ratio, sedation, vasoactive medications, pneumothorax, or outcome between groups [95] . lastly, the t low was not set to a strict e fp × 75% = e ft . the aprv mode using different application methods has recently been shown in statistical reviews and meta-analyses of rcts to improved oxygenation, have a mortality benefit, and increase the number of ventilator-free days as compared to conventional ventilation strategies, without a higher risk of barotrauma or negative hemodynamic effects [96, 97] . neither the current lung protect and rest nor ola ventilation strategies have been effective at reducing vili and ards-related mortality below that in the arma study. for a protective ventilation strategy to be effective, it must open and stabilize the lung. dynamic physiology of alveolar volume change suggests that the use of ventilation time can solve this heretofore intractable problem. the novel use of inspiratory and expiratory times to open and stabilize the acutely injured lung may accomplish the ola goals where traditional ventilation strategies have failed. specifically, the tcav method, which uses an extended time at inspiration to open alveoli and brief expiratory time to prevent alveolar re-collapse has been shown to effectively open and stabilize the lung in animal ards models. there is a sound physiological rationale for the efficacy of the tcav method, and deviations from this method may result in a significant loss of lung protection. the combination of basic science and clinical work has given this group a paradigm changing perspective. our approach focuses on veiled mechanisms that have been largely overlooked, such as understanding the time necessary for the alveolus to open or collapse or taking advantage of biological realities, such viscoelasticity, to manage the lung. the new paradigm in tcav is configuring each breath guided by the previous one, which achieves real-time titration of ventilator settings and minimizes instability induced tissue damage. this novel methodology changes the current approach to mechanical ventilation, from arbitrary to personalized and adaptive. the outcome of this approach is an open and stable lung, which reduces regional strain and provides greater lung protection. abbreviations ards: acute respiratory distress syndrome; vili: ventilator-induced lung injury; aprv: airway pressure release ventilation; frc: functional residual capacity; tcav: time-controlled adaptive ventilation; cpap: continuous positive airway pressure; tc-peep: time controlled-positive end expiratory pressure; t low : time at low pressure; t high : time at high pressure; p high : pressure at inspiration; p low : pressure at expiration; peep: positive end expiratory pressure; e ft : expiratory flow termination; e fp : expiratory flow peak; rct : randomized controlled trial; ola: open lung approach; mb p : mechanical breath pattern; ct: computerized axial tomography. acute respiratory distress syndrome: advances in diagnosis and treatment ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries past and present ards mortality rates: a systematic review has mortality from acute respiratory distress syndrome decreased over time? a systematic review definition and epidemiology of acute respiratory distress syndrome current incidence and outcome of the acute respiratory distress syndrome lessons to learn from epidemiologic studies in ards outcome of acute respiratory distress syndrome in university and nonuniversity hospitals in germany mortality trends of acute respiratory distress syndrome in the united states from 1999 to 2013 outcomes of patients presenting with mild acute respiratory distress syndrome: insights from the lung safe study the 30-year evolution of airway pressure release ventilation (aprv) pressure-volume curve of total respiratory system in acute respiratory failure. computed tomographic scan study mechanical ventilation in adults with acute respiratory distress syndrome. summary of the experimental evidence for the clinical practice guideline looking beyond macroventilatory parameters and rethinking ventilator-induced lung injury airway pressure release ventilation reduces conducting airway micro-strain in lung injury visualizing the propagation of acute lung injury does regional lung strain correlate with regional inflammation in acute respiratory distress syndrome during nonprotective ventilation? an experimental porcine study stress distribution in lungs: a model of pulmonary elasticity alterations of mechanical properties and morphology in excised rabbit lungs rinsed with a detergent physiology in medicine: understanding dynamic alveolar physiology to minimize ventilator-induced lung injury alveolar derecruitment and collapse induration as crucial mechanisms in lung injury and fibrosis alveolitis and collapse in the pathogenesis of pulmonary fibrosis mechanical ventilation-associated lung fibrosis in acute respiratory distress syndrome: a significant contributor to poor outcome opening pressures and atelectrauma in acute respiratory distress syndrome does high peep prevent alveolar cycling? effect of lung recruitment and titrated positive endexpiratory pressure (peep) vs low peep on mortality in patients with acute respiratory distress syndrome: a randomized clinical trial alveolar instability (atelectrauma) is not identified by arterial oxygenation predisposing the development of an occult ventilatorinduced lung injury effects of respiratory rate, plateau pressure, and positive end-expiratory pressure on pao2 oscillations after saline lavage a fibre optic oxygen sensor that detects rapid po2 changes under simulated conditions of cyclical atelectasis in vitro unstable inflation causing injury: insight from prone position and paired ct scans deterioration of regional lung strain and inflammation during early lung injury tidal changes on ct and progression of ards lung inhomogeneities and time course of ventilator-induced mechanical injuries mechanical breath profile of airway pressure release ventilation: the effect on alveolar recruitment and microstrain in acute lung injury effect of airway pressure release ventilation on dynamic alveolar heterogeneity mechanisms of surface-tensioninduced epithelial cell damage in a model of pulmonary airway reopening the influence of non-equilibrium surfactant dynamics on the flow of a semi-infinite bubble in a rigid cylindrical capillary tube biomechanics of liquid-epithelium interactions in pulmonary airways tidal ventilation at low airway pressures can augment lung injury airway closure in acute respiratory distress syndrome: an underestimated and misinterpreted phenomenon alveolar volume-surface area relation in air-and saline-filled lungs fixed by vascular perfusion influence of forced inflations on the creep of lungs and thorax in the dog alveolar micromechanics in bleomycin-induced lung injury lung inhomogeneity in patients with acute respiratory distress syndrome micromechanics of alveolar edema local strain distribution in real three-dimensional alveolar geometries stress concentration around an atelectatic region: a finite element model acute respiratory distress syndrome never give the lung the opportunity to collapse preemptive mechanical ventilation based on dynamic physiology in the alveolar microenvironment: novel considerations of time-dependent properties of the respiratory system last word on viewpoint: looking beyond macroventilatory parameters and rethinking ventilator-induced lung injury regional behavior of airspaces during positive pressure reduction assessed by synchrotron radiation computed tomography the effect of positive end-expiratory pressure on lung micromechanics assessed by synchrotron radiation computed tomography in an animal model of ards dynamic mechanical interactions between neighboring airspaces determine cyclic opening and closure in injured lung individual airway closure characterized in vivo by phase-contrast ct imaging in injured rabbit lung ventilator-induced lung injury and lung mechanics limiting ventilator-associated lung injury in a preterm porcine neonatal model early application of airway pressure release ventilation may reduce mortality in high-risk trauma patients: a systematic review of observational trauma ards literature alveolar leak develops by a rich-get-richer process in ventilatorinduced lung injury lung tissue viscoelasticity: a mathematical framework and its molecular basis lung parenchymal mechanics lung parenchymal mechanics in health and disease modeling the complex dynamics of derecruitment in the lung respiratory mechanics in anesthetized paralyzed humans: effects of flow, volume, and time differential susceptibility of diaphragm muscle fibers to neuromuscular transmission failure lung mechanics. an inverse modeling approach nunn's applied respiratory physiology stress relaxation of the human lung the role of time and pressure on alveolar recruitment effect of different pressure levels on the dynamics of lung collapse and recruitment in oleic-acid-induced lung injury dynamics of lung collapse and recruitment during prolonged breathing in porcine lung injury what's new in respiratory physiology? the expanding chest wall revisited! intensive care med correlation between alveolar recruitment/derecruitment and inflection points on the pressurevolume curve ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial physiological effects of the open lung approach in patients with early, mild, diffuse acute respiratory distress syndrome: an electrical impedance tomography study airway-parenchymal interdependence. comprehensive micro-scale to meso-scale analysis of parenchymal tethering: the effect of heterogeneous alveolar pressures on the pulmonary mechanics of compliant airways a model of surfactant-induced surface tension effects on the parenchymal tethering of pulmonary airways avalanches and power-law behaviour in lung inflation viscoelastic properties of alveolar wall the effects of airway pressure release ventilation on respiratory mechanics in extrapulmonary lung injury early airway pressure release ventilation prevents ards-a novel preventive approach to lung injury predicting the response of the injured lung to the mechanical breath profile influence of inspiration to expiration ratio on cyclic recruitment and derecruitment of atelectasis in a saline lavage model of acute respiratory distress syndrome effect of tidal volume and positive end-expiratory pressure on expiratory time constants in experimental lung injury temporal dynamics of lung aeration determined by dynamic ct in a porcine model of ards open up the lung and keep the lung open mechanical compliance and resistance of the lung-thorax calculated from the flow recorded during passive expiration other approaches to open-lung ventilation: airway pressure release ventilation influence of state of inflation of the lung on pulmonary vascular resistance relation between lung volume and pulmonary vascular resistance early application of airway pressure release ventilation may reduce the duration of mechanical ventilation in acute respiratory distress syndrome airway pressure release ventilation in pediatric acute respiratory distress syndrome: a randomized controlled trial randomized feasibility trial of a low tidal volume-airway pressure release ventilation protocol compared with traditional airway pressure release ventilation and volume control ventilation protocols airway pressure release ventilation in adult patients with acute hypoxemic respiratory failure: a systematic review and metaanalysis airway pressure release ventilation during acute hypoxemic respiratory failure: a systematic review and meta-analysis of randomized controlled trials airway pressure release ventilation: a new concept in ventilatory support airway pressure release ventilation regional lung aeration and ventilation during pressure support and biphasic positive airway pressure ventilation in experimental lung injury publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. drafting of manuscript-lag, gfn, nmh, bd. critical revisions-gfn, pla, js, sjb, mm, lag, ha, mk, nmh. all authors read and approved the final manuscript. salary support for gfn, js, sjb, from nih r01 hl131143. not applicable. not applicable. not applicable. pla, gfn, mks, and nmh have presented and received honoraria and/or travel reimbursement at event(s) sponsored by dräger medical systems, inc., outside of the published work. pla, gfn, and nmh have lectured for intensive care online network, inc. (icon). nmh is the founder of icon, of which pla is an employee. nmh holds patents on a method of initiating, managing, and/or weaning airway pressure release ventilation, as well as controlling a ventilator in accordance with the same, but these patents are not commercialized, licensed, or royalty-producing. the authors maintain that industry had no role in the design and conduct of the study; the collection, management, analysis, or interpretation of the data; or the preparation, review, or approval of the manuscript. key: cord-016369-tnvlafa2 authors: lu, puxuan; zhou, boping; yuan, jing; yang, guilin title: human infected h5n1 avian influenza date: 2016-06-23 journal: radiology of influenza doi: 10.1007/978-94-024-0908-6_10 sha: doc_id: 16369 cord_uid: tnvlafa2 avian influenza is an infectious disease caused by avian influenza virus, which is also known as avian plague or european avian plague. reported, and the patients aged 1-60 years, with the same h5n1 virus strain isolated from their respiratory secretions. in december, 1997, aiv h5n1 was isolated in samples collected from a market selling alive poultry. by killing sick poultry in a large-scale, human infection was then rapidly controlled. in the year of 1997, a total of 18 cases human infected h5n1 avian infl uenza were reported, with 5 cases of death. since the fi rst occurrence of human infected h5n1 avian infl uenza in hongkong in 1997, a total of 667 cases have been defi nitively diagnosed globally, with 393 cases of death by dec. 31, 2014 . and the death rate was 58.92 %. who warned that human infected h5n1 avian infl uenza may be one of the most potentially life-threatening diseases and should be highly concerned across the world. the infected poultry by aiv h5n1 or the poultry carrying aiv h5n1, such as chicken, duck, goose, and dove, particularly chicken, are important sources of its infection. wild aquatic birds (mostly with in apparent infection) play an important role in natural spread of the disease. birds, such as swallow, chukar, bar-headed goose, crow, sparrow, and heron, may also act as the source of its infection. and migration of migrant birds also contributes to the transmission of aiv. evidence is still needed to prove whether human is also a source of its infection. and excretions. otherwise, human can also be infected after exposure to the contaminated environment by aiv. after such direct contact or exposure, the virus particles are inhaled into and attached to human upper respiratory tract for further invasion. transmission via direct or indirect contact refers to human infection of h5n1 avian infl uenza after close contact to feces of poultry/animal infected by aiv h5n1. via direct or indirect contact, the virus can be inoculated into human upper respiratory tract, conjunctival mucosa or skin wound by itself. the data from the cases with human infected h5n1 avian infl uenza indicated that human infected h5n1 avian infl uenza affects any age group and children under the age of 12 years shows a higher incidence with no gender difference. in addition, the patients from such an age group commonly experience serious conditions. in addition, human infected h5n1 avian infl uenza can also spread via other routes of transmission, including gastrointestinal tract, skin wound, conjunctiva, aerosol, and blood. it may also spread vertically or in laboratory, traffi c vehicle, and hospital. in the early stage of human infected h5n1 avian infl uenza, the patients commonly experience common infl uenza like symptoms, including fever with a body temperature persistently above 39 °c with accompanying runny nose, nasal obstruction, cough, sore throat, headache, muscular soreness and general malaise. and some patients may experience gastrointestinal symptoms including nausea, abdominal pain, and diarrhea of watery loose stools. in serious cases, the patients show persistent high fever that rapidly progresses into obvious pneumonia, acute lung injury (ali), acute respiratory distress syndrome (ards), pulmonary hemorrhage, pleural effusion, multiple organ failure, disseminated intravascular coagulation (dic), shock, and reye syndrome. secondary bacterial infection may also occur to complicate the disease that progresses into septicemia, with a mortality of up to above 50 %. due to virus infi ltration, the lung of patients with human infected h5n1 avian infl uenza is demonstrated with fl akes of opacity that is predominantly exudates by chest x-ray or chest ct scan, namely the ground glass opacity and lung consolidation. in serious cases, the lesions in lungs progress rapidly, radiologically demonstrated as large ground glass opacity and lung consolidation. in the advanced stage, both lungs are shown with diffuse consolidation. human infected h5n1 avian infl uenza is radiologically characterized by: 1. early increased density opacity and ground glass opacity in upper lung fi eld in the early stage after onset, the upper lung fi eld is demonstrated with fl akes of increased density opacity and ground glass opacity, which are the radiological signs of human infected avian infl uenza. the invasion of h5n1 avian infl uenza virus to human lung tissues causes an extensive range of lesions, with diffuse and exudative lesions in multiple lobes and multiple segments of both lungs. at the peak of their progression, most of lung fi elds in both lungs are involved, with large fl akes of cloudy dense mass like opacity or even white-lung sign. radiologically, the lesions are demonstrated with rapid changes. a lesion that is initially found in one lung fi eld may rapidly develop to involve the upper, middle and lower lung fi elds within 24-48 h, radiologically demonstrated as involvement of the whole lung lobe. 4. concurrently involved pulmonary parenchyma and interstitium due to necrosis and shedding of pulmonary alveoli, the pulmonary alveoli contain less gas but are fi lled with exudates such as various serous fl uids, fi brin, erythrocytes, and neutrophilic granulocytes. in addition, hyaline membrane obviously forms in the alveolar space. therefore, the lesions of human infected h5n1 avian infl uenza are radiologically demonstrated as alveolar exudates and lung consolidation, with the involved exudative lesions overlapping with pulmonary interstitium. after the exudative lesions are absorbed, grid like, fl akes, strips and patches of opacity is shown, which resemble to the radiological signs of viral pneumonia. the lesions are slowly absorbed. after the invasive assisting mechanical ventilation is retrieved with subsequent normal body temperature, breathing rate and wbc count, the both lungs are still radiologically demonstrated with strips, fl akes, grid like, and patches of consolidation opacity, indicating inconsistency between chest radiology and clinical symptoms due to long-term progression of the lesions. that is to say, the absorption of lesions in lung lags behind clinical manifestations. in adult patients, the pleura is involved in different degrees, with a small quantity of pleural effusion isolateral to the primary lesion as well as bilateral pleural thickening and adhesion. during the absorption stage, multiple lesions of lobular paraseptal emphysema are revealed, with no obvious signs of hilar and mediastinal lymphadenectasis. case 1 [ brief medical history ] a 31-year-old male truck driver experienced fever, cough and shortness of breath since jun. 3, 2006. after he paid a clinic visit in a local hospital, a diagnosis was made to be common cold and he received corresponding therapy. at 0:30 a.m. on jun. 10, 2006, he was hospitalized due to aggravated condition. by physical examination, the body temperature was 39.2-40 °c, heart rate 127/min, breathing rate 32/min and blood pressure 116/80 mmhg. he showed a typical development, moderate nutrition, active position and slight unconsciousness. in addition, he showed eye conjunctival congestion and edema, identically sized and round pupils and obvious cyanosis in oral lips. by percussion, the left lung fl atness; by auscultation, coarse breathing sound of both lungs that attenuated at the right lung. at d 15 prior to the onset, his wife ever went to buy an alive chicken at a market and brought it back home after having it killed on site. his wife and daughter showed no signs of pneumonia. by routine blood test, wbc 4.20 × 10 9 /l, n 0.75, l 0.24, hgb 127 g/l, rbc 4.84 × 10 12 /l, plt 132 × 10 9 /l. by blood glass analysis, ph 7.426, pao 2 63 mmhg, paco 2 34 mmhg, so 2 % 92 %. by blood biochemistry, glu 7.87 mmol/l, bun 2.8 mmol/l, cr 88 μmol/l, alt 30u/l, and ast 66u/l. t cell count in peripheral blood was 5/μl. etiologically, rt-pcr by qiagen one step rt-pcr kit was performed to examine his tracheal secretions. the examination report indicated that the target segments of h5 and n1 were amplifi ed. throat swab for rna of h5n1 (pcr) twice were positive. all the above fi ndings indicated the existence of h5n1 avian infl uenza virus. [ radiological demonstration ] see fig. 10 .1 [ diagnosis ] human infected h5n1 avian infl uenza complicated by pneumonia. [ discussion ] following the fi rst case of human infected h5n1 avian infl uenza reported in hongkong in 1997, 6 cases of death were subsequently reported. by dec. 31, 2014, a total of 40 cases was reported in china, with 27 cases of death, and the mortality was up to 67 %. the high death rate indicated strong virulence of avian infl uenza virus h5n1 and the rapid progression of the disease. chest radiological examinations are of great importance for our understandings about its radiological demonstrations, which facilitate its diagnosis, differential diagnosis and treatment. via systematic examination as well as dynamic observation and analysis, the radiological demonstrations and all the dynamic changes of the lesions were harvest from the patient. the radiological demonstrations are characterized by: 1. on d 7 after onset, the fi rst chest x-ray demonstrated large fl akes of increased density opacity in the left upper and middle lung fi elds. on d 8 after onset, the fi rst chest ct scan demonstrated large consolidation opacity with high density in the apico-posterior and anterior segments of the left lung and in the left lower lung lobe, with air bronchogram inside. the anterior segment of the right upper lung was shown with ground glass opacity. all the fi ndings indicated that ground glass opacity and large consolidation opacity are early signs of human infected avian infl uenza by chest radiology. virus h5n1 is extensive, demonstrated as diffuse and exudates lesions in both lungs with multiple segments and lobes involved. at the peak of its progression, most of the lung fi elds in both lungs is involved, demonstrated as large cloudy dense mass like opacity and even white-lung sign. 3. radiologically, the lesions change rapidly. the initial lesions in the left upper and middle lung fi elds can extend into the left lower lung fi eld within 24 h to involve the whole lung. in the following 48 h, the lesions may rapidly extend into the right upper, middle and lower lung fi elds to involve the whole lungs. 4. the lesions are absorbed slowly. in this case, on d 35 after onset, the invasive assisting mechanical ventilation was retrieved, and the patient subsequently showed normal body temperature, breathing rate and wbc count. on d 33 after onset, chest ct scan still showed strips, fl akes, grid like and patches of consolidation opacity with obvious air bronchogram inside. the follow-up ct scan was then performed each year, and the 5th chest ct scan showed interstitial fi brosis in both lungs, demonstrated as lobular septal thickening, sub-pleural arc shape linear opacity, paraseptal emphysema, ground glass opacity, bronchoectasis and small consolidation opacity. 5. the pleura is involved in different degrees. pleural effusion in a small quantity was demonstrated isolateral to the primary lesions, and bilateral pleura were shown to be thickened with adhesion. the occurrence complications following human infected avian infl uenza is the direct cause of death and should be highly concerned in clinical practice. the common complications and their prognoses are described as the following: in the most serious stage of the disease, extensive consolidation opacity in both lungs, namely the whitelung sign, indicates the occurrence of ards. in this cases, the patient showed the sign of ards on d 9 after onset. after invasive mechanical positive pressure ventilation and anti-anoxia therapy were administered, his condition was improved. and multiple organs dysfunction syndrome (mods) was controlled. common pulmonary secondary infections include pseudomonas aeruginosa infection, mycotic infection and other bacterial infections. since d 10 after onset, the patient was detected for several times with growth of pseudomonas aeruginosa in his phlegm specimens. particularly on d 20 after onset, his wbc count obviously increased and chest x-ray demonstrated patches uneven opacity in the left lower lung. after polymyxin b and other medications were administered, his pulmonary secondary infection was controlled. although his condition was stable with normal vital signs, on discharge his both lungs were shown with irregular grid like, fi brous strips and patches of opacity and accompanying local pleural thickening by thin layer ct scan. these fi ndings indicated existence of pulmonary fi brosis, whose underlying mechanism may be hyperplasia of fi brous tissues and capillaries caused by fi brosis and organization of exudates as well as formation of microthrombosis in pulmonary capillaries based on formation of alveolar hyaline membrane and fi brin exudation. however, the hypothesis needs to be proved by suffi cient evidence and the dynamic changes of pulmonary fi brosis still need to be further studied. in this case, the patient showed left pleural effusion in a small quantity on d 8 after onset by the fi rst chest ct scan. on d 33 after onset, the second chest ct scan demonstrated absorption of pleural effusion as well as local pleural thickening and adhesion. these fi ndings indicated pleura is involved in patients with human infected avian infl uenza, which has been further proved by autopsy. h5n1 avian infl uenza should be differentiated from the following diseases. the lung changes of both human infected avian infl uenza and sars are mainly parenchymal and interstitial lesions, with similar shape, development and outcome. therefore, their differential diagnosis is challenging, and sars is an acute respiratory infectious disease caused by novel corona virus. at its early stage, radiology demonstrates pulmonary interstitial changes, predominantly ground glass opacity. during its progressive stage, the lesions are demonstrated as predominantly ground glass opacity and rarely large consolidation opacity. the lung lesions of human infected h5n1 avian infl uenza show rapid changes. in some serious patients, the lesions in lungs may change within several days or even 1 day, with rapid progression from small area to large area, from upper or lower lung to the whole lung, from one lung to both lungs, and from ground glass opacity to consolidation opacity. however, in patients with sars, the lesions in lungs progress slowly, and some lesions are migratory. the lesions of human infected h5n1 avian infl uenza are in one lung or both lungs, possibly in upper and lower lung. in serious cases, the lesions are diffuse in both lungs, with more serious condition at the lower lungs. however, in the early stage of sars, the nodular lesions are commonly located in the subpleural lateral lung fi eld. based only on radiological fi ndings, pneumonia induced by h5n1, h1n1 and h7n9 avian infl uenza viruses can hardly be differentiated. and their differential diagnosis is mainly based on epidemiological history and etiological examination. pneumonia induced by h1n1, h5n1 and h7n9 avian infl uenza virus is caused by infl uenza a virus, with clinical infl uenza like manifestations. chest ct scan demonstrates the diseases with ground glass opacity and consolidation opacity of varying sizes. compared to pneumonia induced by h1n1 avian infl uenza virus, the lesions of pneumonia induced by h5n1 and h7n9 avian infl uenza virus are radiologically demonstrated with a larger range, with more rapid progression and more common air bronchogram. the lesions of pneumonia induced by h5n1 avian infl uenza virus show the more rapid progression, followed by h7n9 and then h1n1. by chest ct scan, pneumonia induced by h5n1 avian infl uenza virus is demonstrated with large ground glass opacity and consolidation opacity in both lungs that distribute extensively and progress rapidly. in some cases, the lesions are migratory, with slow absorption and obvious pulmonary interstitial fi brosis. its mortality rate is around 60 %. pneumonia induced by h7n9 avian infl uenza virus is radiologically demonstrated with initial lesions in bilateral middle and lower lungs that are predominantly ground glass opacity and consolidation opacity. these lesions show rapid progression and relatively slow absorption. and its mortality rate is about 36 %. pneumonia induced by h1n1 avian infl uenza virus is radiologically demonstrated as multiple fl akes of ground glass opacity as well as patches and large fl akes of high density consolidation, possibly with lobar or segmental atelectasis and pleural effusion. and its mortality rate is about 6 %. [ brief medical history ] a 26-year-old woman, being pregnant for more than 2 months, reported a history of fever since feb. 11, 2006, with a body temperature of above 38.5 °c and the highest body temperature of up to 40 °c. she also experienced cough with a little phlegm as well as diarrhea early after onset. in a local hospital, routine blood test showed wbc count 6.1 × 10 9 /l, gr% 74.9 % and ly% 15.6 %. chest x-ray indicated pneumonia at the right lower lung. by physical examination, breathing sound at the right lower lung was low and no other obvious positive signs. on d 7 after onset, feb. 18, 2006, she was transferred to another hospital due to persistent fever, aggravated cough with more phlegm, chest distress and diffi culty breathing. by inquiries of her epidemiological history, she reported a defi nitive history of contact to sick/dead chicken in her home 7 days prior to the onset, feb. 4, 2006. by routine blood test, wbc count 3.30 × 10 9 /l, n 0.83, l 0.15 and plt 56 × 10 9 /l. blood biochemistry showed ast 90u/l, ck 74u/l, and ldh 537u/l. by blood gas analysis, ph 7.426, pao 2 25.1 mmhg, paco 2 51 mmhg, hco 3 19.4 mmol/, and so 2 % 92 %. respiratory secretions examination on d 9 after onset (specimens collected 1 day ago), feb. 20, 2006, the virus nucleic acid positive for h5n1 avian infl uenza virus. by reexamination by national parallel laboratory, the fi nding was still positive, with h5n1 avian infl uenza virus isolation positive. the patient experienced dyspnea and rapid progression on d 7 after onset, and the condition further progressed into ards on d 8 after onset. because the simplex oxygen supplying therapy failed to maintain oxygenation, noninvasive mechanical ventilation was ordered on d 9 after onset to maintain continuous positive airway pressure (cpap) with settings of 10 cm h 2 o, fio 2 45%, and an elevated sao 2 to 90-93 %. but the noninvasive mechanical ventilation was discontinued due to shortness of breath and frequent cough of the patient. subsequently, invasive nasotracheal intubation for ventilation was performed on that day. tracheotomy was then performed for invasive ventilation 3 days after nasotracheal intubation. on d 10 after onset, her body temperature returned to normal, but on d 14 after onset, the body temperature rose again to 37.5 °c, with increased peripheral hemogram and yellowish purulent bronchial secretions. a diagnosis of secondary bacterial infection was made and the administered antibiotics were adjusted accordingly. on d 16 after onset, miscarriage occurred. and on d 18 after onset, the invasive ventilation was discontinued, which was given for 10 days. on d 21 after onset, tracheal extubation was performed. and on d 20 after onset, her hemogram returned to normal indicating controlled secondary infection and convalescent stage of the disease. on mar. 23, 2006 (d 40 after onset) , the patient was discharged according to the criteria in clinical guidelines for human infected avian infl uenza (revised version, 2005) formulated by ministry of health, p. r. china. [ radiological demonstration ] fig. 10 .2 . [ diagnosis ] human infected h5n1 avian infl uenza. [ discussion ] the patient is characterized by young woman, contact to sick/dead chicken 7 days before onset, initial symptom of fever, and normal wbc count. her condition rapidly developed into pneumonia within 1 week after onset, and further rapidly into respiratory failure and multiple organs dysfunction. chest x-ray showed early exudation opacity in the right lower lung (d 4 after onset), and exudation opacity in bilateral middle and lower lung fi elds (d 6 after onset). on d 11 after onset, chest x-ray showed the most serious conditions, with large consolidation opacity in the right lung and the left middle lung. on d 12 after onset, oxygenation was improved, but slow absorption of the lesions was revealed by both chest x-ray and chest ct scan. on discharge, chest ct scan still demonstrated extensive spots, fl akes and cords like opacity in both lungs. the case is special in the following aspects: 1. rapid progression of the condition. on d 6 after onset, chest x-ray demonstrated obvious deterioration of lesions every 3 h. clinically, the patient experienced dyspnea and blood gas analysis indicated respiratory failure. at the onset of the symptoms, the patient was pregnant for more than 2 months and miscarriage occurred on d 16 after onset, which was within the expectation of the physicians. complete curettage of uterine cavity following intraveneous anesthesia was successfully performed to remove fetus and placenta. 3. blood gas analysis and chest x-ray. on d 12 after onset, blood gas analysis, routine blood test and biochemical examination indicated improved condition of the patient. and on d 20 after onset, the above examinations indicated normal condition of the patient. however, chest x-ray and chest ct scan still demonstrated slow absorption of the consolidation opacity. on discharge, blood gas analysis indicated normal but chest ct scan demonstrated obvious pulmonary interstitial changes. this case is one of the cured cases of human infected avian infl uenza, with comparatively severe condition and only on d 14 after onset, the patient experienced re-elevated body temperature to 37.5 °c, with elevated peripheral hemogram and yellowish purulent bronchial secretions. the condition was diagnosed as secondary bacterial infection and the administered antibiotics were adjusted accordingly. on d 20 after onset, the hemogram returned to normal, indicating controlled secondary infection and convalescent stage of the disease. after her discharge, her serum collected at the convalescent stage was collaboratively administered to cure a patient with human infected h5n1 avian infl uenza in shenzhen, guangdong, china, which is known as the patient with the most serious condition among patients with h5n1 avian infl uenza. by radiology, the key points for differential diagnosis are as the following: based on early chest ct scan, the lesions are demonstrated as poorly defi ned large fl akes of increased density opacity, which are bilaterally symmetric. in combination to the clinical symptoms of high fever and cough, it should be differentiated from bacterial pneumonia of both lungs. however, the patient reported a history of contact, with normal level of peripheral wbc count. by dynamic observations via radiological examination, the lesions in both lungs progressed rapidly, with extensive involvement in bilateral lung fi elds and concurrent ground glass opacity and pulmonary consolidation opacity. all of these manifestations and fi ndings are in consistency with pneu-monia induced by human infected avian infl uenza. in addition, pneumonia induced by highly pathogenic h5n1 avian infl uenza virus should also be differentiated from sars, aids complicated by pcp, and pneumonia induced by h7n9 avian infl uenza virus. further differentiation should be comprehensively based on epidemiological history, etiological examination, clinical manifestations, and radiological fi ndings. clinical imaging diagnosis of emerging infectious diseases. beijing: people's medical publishing house radiological demonstrations of pneumonia induced by highly pathogenic h5n1 avian infl uenza virus ct demonstrations and their dynamic changes in adult patients with severe pneumonia induced by h5n1 avian infl uenza virus radiological features of lung changes caused by avian infl uenza subtype a h5n1 virus: report of two severe adult cases with regular follow-up human infected avian infl uenza key: cord-260132-lqpk3ig7 authors: quartuccio, luca; semerano, luca; benucci, maurizio; boissier, marie-christophe; de vita, salvatore title: urgent avenues in the treatment of covid-19: targeting downstream inflammation to prevent catastrophic syndrome date: 2020-04-19 journal: joint bone spine doi: 10.1016/j.jbspin.2020.03.011 sha: doc_id: 260132 cord_uid: lqpk3ig7 nan at present, healthcare systems all over the world are coping with the new coronavirus infection [1] . in particular, tremendous efforts are being made in order to support governments in the policy of infection spread containment and early detection, and researchers are working on causal treatment and the treatment of the severe and critical manifestations downstream from the viral infection [2] . as largely known, coronavirus disease 2019 (covid19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) with droplets and contact as the main way of transmission. since the first case reported in wuhan, china, in december 2019, the outbreak has gradually spread nationwide and then abroad, rapidly becoming a pandemic infection. now, coronaviruses (covs) have come back into the limelight after the severe acute respiratory syndrome coronavirus (sars-cov) in 2003 and middle east respiratory syndrome (mers-cov) outbreak in saudi arabia and south korea. the rheumatology scientific community has been involved in the management of this pandemic infection, since some treatments, usually employed in patients with inflammatory rheumatic diseases, might be useful to directly counteract the virus, as suggested for antimalarials [3] , and more recently for baricitinib, or be effective in downregulating the dangerous inflammatory pathways triggered by the virus itself [4] . a first and crucial issue to address is whether and when it is important to target the viral infection or the downstream inflammatory events as a priority for clinical purposes. our past experience in hepatitis c virus infection (hcv) and cryoglobulinemic vasculitis (i.e., an autoimmune and lymphoproliferative disease driven by a treatable infection) may be of value to this end, as it shows that, for life-threatening and severe disease manifestations, the immediate targeting of the events downstream of infection is mandatory [5] . currently, the humanized monoclonal antibody anti-interleukin-6 receptor (anti-il-6r), namely tocilizumab, appears as a promising tool to turn off the cytokine storm, which dramatically complicates the course of the infection in some patients, causing a rapidly fatal acute respiratory distress syndrome. the rationale for the use of anti-cytokine drugs is to play for time, by decreasing the dangerous inflammatory peak, while the immune system is building the adaptive response to the virus. as we write this paper, several protocols using anti-il6r treatments are starting to recruit covid-19 patients in different countries to build an evidence-based support for this treatment. tocilizumab is an effective and safe treatment for rheumatoid arthritis, for systemic and polyarticular juvenile chronic arthritis and for the most frequent systemic vasculitis in adults, i.e., giant-cell arteritis. moreover, it has been recently licensed for the treatment of cytokine storm syndrome in car-t protocols [6] . interleukin-1 (il-1) is another proinflammatory cytokine involved in the early phases of cytokine release syndrome and clinical protocols involving il-1 blockade are being adopted in several countries. a second important open and urgent question is, however, at which stage of the infection this treatment approach has to be best applied. to answer this question, it is important to carefully consider what we already know about coronaviruses from the lessons of previous epidemic infections, and to compare with covid-19 outbreak. in the 2003 sars-cov outbreak, patients complained of high fever, myalgia, dry cough, and lymphopenia as the most characteristic symptoms or signs. in about one third of the cases, patients also developed an atypical pneumonia, with acute respiratory distress as result of extensive acute lung damage [7] . these characteristics are very similar to those registered in the current outbreak [8] , where, after first systemic symptoms, dyspnoea is estimated to arise in a median of 5 days (iqr, 1-10), hospital admission occurs after 7 days, and ards after 8 days (iqr, 6-12 [8] . importantly, sars-cov patients admitted to the intensive care unit showed higher white blood cell and neutrophil counts, as well as higher levels of d-dimer, creatine kinase, and creatine, emphasizing the role of the systemic inflammation downstream the virus infection, and the transformation of the infectious disease into a systemic immunological and inflammatory disease. indeed, in nonsurvivors, all these laboratory findings were much more pronounced. as seen in the ongoing coronavirus outbreak, also in the 2003 sars-cov outbreak, several factors including advanced age, male sex, comorbidity, high levels of lactate dehydrogenase and creatine https://doi.org/10.1016/j.jbspin.2020.03.011 1297-319x/© 2020 société franç aise de rhumatologie. published by elsevier masson sas. all rights reserved. kinase, and high initial absolute neutrophil count, were significant predictive factors for intensive care unit admission and death [7] . lung pathology in 2003 sars-cov patients showed epithelial cell proliferation and desquamation, hyaline membranes formation along alveolar walls and cells infiltration (lymphocytes, neutrophils, and monocytes) during the early stage of the disease, while, of note, increased fibrosis and multinucleated epithelial giant cells formation at a later stage, highlighting the existence of a two-phase lung injury. a first acute phase with diffuse alveolar inflammation, was followed by more organized inflammation, but also permanent secondary damage [9] . furthermore, patients still manifested lung injury at a time when the viral load was falling, supporting the immune nature of the lung damage possibly independent from infection. interestingly, the lungs from actual coronavirus pneumonia patients showed alveolar exudative inflammation and interstitial inflammation, alveolar epithelium proliferation and hyaline membrane formation. most of the infiltrating lymphocytes, i.e., the major cellular component of lung inflammation, were cd4-positive t cells, attracted into the lungs from the peripheral blood, which contributed to the progressive peripheral blood lymphopenia. pulmonary interstitial fibrosis was also observed. in addition, inflammation and damage of the heart, vessels, liver, kidney and other organs were evident indicating a widespread diffusion of the virus leading to multiorgan localization of downstream pathogenetic events [10] . thus, coronavirus infection can turn into an inflammatory immune systemic disease, with clinical and subclinical simultaneous involvement of many organs. in general, the cytokine profile of sars patients showed a marked elevation of the th1 cytokine interferon (ifn)-gamma, of inflammatory cytokines interleukin (il)-1, il-6 and il-12 for at least 2 weeks after disease onset. the chemokine profile demonstrated significant elevation of neutrophil chemokine il-8, monocyte chemoattractant protein-1 (mcp-1), and th1 chemokine ifngamma-inducible protein-10 (ip-10) [11] . of note, levels of some pro-inflammatory cytokines including mcp-1, tgf-␤1, tnf-alpha, il-1beta, and il-6 in autopsy tissues from four patients who died of sars were expressed in the sars-cov-infected ace2+ cells. furthermore, sars-cov 3a protein activates the nlrp3 inflammasome in lipopolysaccharide-primed macrophages [12] . consistently, in the murine model of car t cell-induced cytokine release, the severity is not mediated by car t cell-derived cytokines, but by il-6, il-1 and nitric oxide (no) produced by recipient macrophages [13] . il-6 signalling can be targeted by tocilizumab and sarilumab (a human monoclonal antibody that targets il-6 receptor with higher affinity than tocilizumab). currently used drugs to target il-1 are anakinra (an il-1 receptor antagonist) and canakinumab (a human monoclonal antibody that binds il-1␤). importantly, they proved effective in autoinflammatory diseases, and also in macrophage activation syndrome, which rarely complicates the course of autoinflammatory diseases; there is then a strong rationale for their use in sars patients. when dealing with the antiviral response, it was already shown in 2003 that the early immunological responders (pcrpositive/antibody-positive for the virus) had a higher serum interferon-gamma profile if compared to late immunological responders (pcr-positive/antibody-negative [14] . in addition, late immunological responders tended to have higher levels of il-6 and of il-8. coronaviruses try to escape the innate immune system, the first-line of the human immunological response, by lowering the interferon response and, in this way, they buy time to replicate and undisturbedly spread in the body. in predisposed individuals (e.g., late responders) the immunological inflammatory response may acquire the characteristics of the cytokine storm syndrome, now seen also in some covid-19 patients. overall, this subset of covid-19 patients should be identified early, and higher il-6, lower lymphocyte count, and higher neutrophil count, and late seroconversion might be useful biomarkers. also, some chemokines, i.e., ip-10 (cxcl10), may be involved. it is also not surprising that older and male patients with weaker interferon-derived immune response are mostly represented in this subset. it has recently been suggested that baricitinib, a janus kinase 1 selective inhibitor, may be useful in covid-19 severe respiratory disesase [15] . as baricitinib inhibits ap2-associated protein kinase 1 (aak1) it may reduce receptor mediated endocytosis of the virus in target cells. moreover, by interfering with downstream signalling of numerous cytokines like il-6 it may also prevent subsequent cytokine release syndrome. however, jak 1 inhibition also interferes with interferon signalling, which should probably be preserved in the early phases of the disease. thus, the balance between advantageous and deleterious effects should be pondered and, the correct time frame for the use of baricitinib be clearly defined. more recently, several studies have focused on complement activation in sars-cov patients and animal models, where increased serum and lung complement cleavage products were observed. mice deficient in c3 are protected from sars-covinduced lung pathology, improve in respiratory function, and show lower levels of inflammatory cytokines/chemokines in the lung and serum [16, 17] . indeed, inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection [18] . treatments targeting complement activation with humanized anti-c5a antibody greatly reduce lung histopathologic injury and inflammation in a monkey model of virus-induced (h7n9) acute lung injury [18] . overall, there is a rationale also for targeting complement activation in covid-19 patients, and a humanized monoclonal antibody anti-c5a, namely eculizumab, currently registered for paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorder, and already effectively off-label applied in catastrophic antiphospholipid syndrome, might potentially be useful in this setting. although this does not represent the current clinical priority, it may be convenient to begin thinking of managing patients also beyond the acute phase. lung fibrosis followed sars-cov infection in some patients, and this disabling consequence should be prevented, if possible [19] . consistently, serum tgf-␤, which induced proliferation of fibroblasts, is overexpressed at the late stage of coronavirus infection [14] . it is known that tgf-␤ is an antiinflammatory cytokine that can induce fibrogenesis. more recently, it has been suggested that pulmonary fibrosis in sars-cov is caused by a hyperactive host response to lung injury mediated by epidermal growth factor receptor (egfr) signalling [20] . inhibiting egfr signalling, by the use of tyrosine kinase inhibitors like nintedanib, a drug licensed for idiopathic lung fibrosis, seems promising for interstitial lung disease in systemic sclerosis [21] , and might also be useful for covid-19. covid-19 is now a huge challenge for health systems worldwide. it has spanned the globe, now exceeding hundreds of thousands of cases and tens of thousands of deaths, and different treatment approaches are currently been investigated. the present chinese and italian experience along with the past history of sars-cov have taught us that people with weaker immune response, in particular the elderly with comorbidity, can develop an abnormal uncontrolled inflammatory response with acute and diffuse lung damage, often leading to death. we herein provide the following messages supported by the long-term clinical experience and scientific approach by rheumatologists and immunologists, and also by studies in virus-mediated autoimmune disease when the issue of targeting infection and/or downstream events was crucial. these should be of value to integrate the scientific evidence in covid-19. first, giving priority to targeting the inflammatory response to the covid19 infection appears more feasible in some patient subsets, seemingly the more severe ones, when disease has already evolved, and likely also for those in earlier stages, but at higher risk. secondly, when available, concomitant etiological, antiviral therapy remains in any case a cornerstone in all patients. immunosuppressant can make the viral clearance more difficult, and an antiviral therapy should be combined when an immunosuppressant is given, in the absence of additional data at present. thirdly, besides il-6 pathways blockers, other biologic drugs currently used by rheumatologists may be employed in selected patients. in our opinion, targeting il1 and complement activation have a strong rationale. these are assumptions and we should keep in mind also the deleterious effect of tnf-␣ blockade in septic shock. this means that we should be careful and rigorous in the evaluation of these new possibilities. finally, possible long-term sequelae, mainly lung fibrosis, should be kept in mind, monitored, and possibly treated and prevented in the near future. as countries try to buy time to save their health system from collapse through contagion containment policies, individual patients must also be helped to save time for the immune system to react effectively to the virus. global epidemiology of coronavirus disease 2019: disease incidence, daily cumulative index, mortality, and their association with country healthcare resources and economic status sars-cov-2 and covid-19: the most important research questions covid-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies a randomized controlled trial of rituximab for the treatment of severe cryoglobulinemic vasculitis fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cell-induced severe or life-threatening cytokine release syndrome a novel coronavirus associated with severe acute respiratory syndrome clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china lung pathology of fatal severe acute respiratory syndrome a pathological report of three covid-19 cases by minimally invasive autopsies plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome car t cell-induced cytokine release syndrome is mediated by macrophages and abated by il-1 blockade an interferon-gamma-related cytokine storm in sars patients baricitinib as potential treatment for 2019-ncov acute respiratory disease blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection the role of c5a in acute lung injury induced by highly pathogenic viral infections the spectrum of pathological changes in severe acute respiratory syndrome (sars) the role of epidermal growth factor receptor (egfr) signaling in sars coronavirus-induced pulmonary fibrosis inbuild trial investigators. nintedanib in patients with progressive fibrosing interstitial lung diseases-subgroup analyses by interstitial lung disease diagnosis in the inbuild trial: a randomised, double-blind, placebo-controlled, parallel-group trial key: cord-023331-jrvmgnu3 authors: nan title: asthma & allergy sig: poster session 3. physiology, environment, investigation and management date: 2008-03-12 journal: respirology doi: 10.1111/j.1440-1843.2008.01252_3.x sha: doc_id: 23331 cord_uid: jrvmgnu3 nan increased airway smooth muscle (asm) in asthma may be due to hyperplasia or hypertrophy of asm cells. the contribution of extracellular matrix (ecm) within asm bundles has not previously been accounted for when estimating asm cell volume. aim to estimate the mean asm cell volume in asm bundles in asthma. methods post-mortem tissues from control subjects (c n = 9); nonfatal (nfa n = 11) and fatal (fa n = 10) cases of asthma were studied. on 30 mm transverse airway sections stained with haematoxylin, the volume density (nv) of asm cell nuclei was estimated using an optical disector (¥1000). the mean cell volume (vc = 1/nv) was calculated, correcting for the volume fraction of asm (fasm) within the asm bundle (corrected vc = 1/(nv ¥ fasm)). fasm was estimated on 0.5 mm thick sections of the same airway stained with masson's trichrome. basement membrane perimeter (pbm) was used to indicate airway size. results table shows mean ϯ sd. (one-way anova) *p < 0.05 for c v fa, nfa v fa. conclusion these data suggest that although asm area is increased in asthma, mean asm cell volume is unchanged. therefore hyperplasia, not hypertrophy, of asm cells is present in both mild and severe asthma. these results were similar for both large and small airways. asthma is characterized by airway inflammation and remodelling which contribute to airway hyperresponsiveness and episodic airflow obstruction. mast cell (mc) densities are higher on the smooth muscle (asm) in asthma so their mediators may modulate other asm functions as well as cause contraction. aim to investigate the effect of mc mediators on chemokine and extracellular matrix (ecm) production by asm cells from donors with and without asthma. methods mc were isolated from the resected lung samples of 6 patients, resuspended at 10 6 cells/ml in dmem + 10% fbs and stimulated with ige/anti-ige. supernatants (sn) were collected after 2 and 24 h and the mc lysed. sub-confluent asm cells from 6 donors with and without asthma were serum deprived for 72 h before mc sn/lysates were added in dmem + 10%fbs for 48 h. il-8 and eotaxin levels in all asm sn and mc sn/lysates were measured by elisa. fibronectin and collagen iv deposition was measured in situ by immunoassay following asm cell lysis. results in asthmatic and non-asthmatic asm cells all mc sn and lysates reduced eotaxin release by up to 47% and 58%, whereas the 0-2 h mc sn significantly increased il-8 release to 178 ϯ 35.9% (p = 0.0339) and 169 ϯ 49% (p = 0.0445) of the fbs control respectively. however, only nonasthmatic asm cell il-8 release was increased by the mc 2-24 h sn (216 ϯ 85%; p = 0.0421) and cell lysates (215 ϯ 47%; p = 0.0421). the 0-2 h mc sn also increased fibronectin deposition to 143 ϯ 16% (p = 0.008) by asthmatic asm cells only. mc sn and lysates had no effect on collagen iv deposition. conclusions activated mast cell mediators differentially modulated chemokine and ecm secretion by asm cells from donors with and without asthma. thus mast cells may modulate their own recruitment to the smooth muscle and remodelling locally in the airways in asthma. supported by nhmrc. the technique of ige passive sensitization reproduces ige-related allergic responses in vitro and studies have validated this technique for investigations modelling allergic smooth muscle responses. there are no studies investigating effects of ige sensitization on rhinovirus (rv) infection. we hypothesized that rv infection is enhanced by ige sensitization, a consequence of diminished early innate immune responses. methods beas-2b epithelial cells and primary culture airway fibroblasts were sensitized with ige 24 h-7 d prior to infection with rv16. samples of tissue culture supernatant and cell lysates were collected over a 12 h period after infection for analysis. viral replication was measured by real-time rt-qpcr and viral titration and type i interferon mrna by rt-qpcr. ige receptor mrna expression was examined using rt-pcr. results initial studies to establish the model used human serum high in ige (>1000 iu/ml), this yielded inconsistent results and it was found that purified ige (1000 iu/ml) provided more reliable responses. sensitization was established after 24 h ige incubation and was comparable with up to 7 d. rt-pcr detected mrna for the ige low affinity receptor only after sensitization. following rv16 infection, vrna was increased after 24 h in ige sensitized cells (p < 0.05), but this effect varied noticeably between and within cell lines. cellular expression of ifn-b mrna increased with viral infection but in cells sensitized with ige lower levels of expression were noted (p < 0.05). conclusions ige passive sensitization enhanced rv replication in vitro but the model is constrained by significant variability between and within cell lines. the effect of sensitization on rv replication may occur through the low affinity ige receptor. activated mast cells (mc) are present in higher numbers on the airway smooth muscle (asm) in asthma compared with other inflammatory airway diseases. matrix metallo-proteinases (mmps) cleave chemokines and alter chemokine gradients by degrading the extracellular matrix and thus may modulate mc migration to the asm. aim to determine the levels of mmp-2, mmp-9 and their inhibitors, timp-1 and timp-2, secreted by asm cells from donors with and without asthma. method confluent asm cells were washed, serum-starved for 48 h and then stimulated with th1 (il-1, tnf and ifn) or th2 (il-1, il-4 and il-13) cytokines or left unstimulated. after 4 and 24 h,the sn were collected. the relative amount of pro and active forms of mmp-2 and mmp-9 in sn were determined by gelatine zymography. timp-1 and timp-2 levels in the sn were measured by elisa. results pro-and active mmp-9 were not detected. however, pro-mmp-2 levels were high in sn of asm cells from donors with (195.6 ϯ 47.2 % positive control/10 5 cells) and without (226.5 ϯ 49.2 % positive control/10 5 cells) asthma. a trend to increased active mmp-2 production by asm cells from donors with (7.3 ϯ 2.7 % positive control/10 5 cells, n = 9) compared to without (2.9 ϯ 0.7 % positive control/10 5 cells, n = 11) asthma after 24 h was not significant (p = 0.101). timp-1 and timp-2 levels respectively were high in the sn of cells from donors with (69.4 ϯ 19.6 and 21.3 ϯ 4.7 ng/10 5 cells, n = 5) and without (57.3 ϯ 13.7 and 16.6 ϯ 3.5 ng/10 5 cells, n = 5) asthma. th1 and th2 cytokine stimulation did not affect mmp or timp release. conclusions th1 and th2 cytokines did not regulate asm cell production of mmp-2, timp-1 and timp-2. altered asm mmp-2 activity is unlikely to play a role in mc chemotaxis to asm cells from donors with asthma in vitro or their presence on the asm in asthma. there has been a marked increase in the prevalence of asthma and other allergic diseases in the last few decades. one of the explanations for this is the change in our diet. one of the characteristics of the "western diet" is a high intake of both saturated and polyunsaturated fat. this prompted us to compare the effects of high fat and low fat meals on the numbers of circulating eosinophils and other leukocytes. methods we studied 12 volunteers who had allergic rhinitis and/or asthma and a peripheral eosinophil count at baseline of ն200 ¥ 10 7 /l. this was a randomized, crossover trial with participants studied on two different days. on each occasion they arrived fasting and after bloods were drawn consumed a 3000 calorie meal. one of the meals was high in saturated fat and refined carbohydrate. the other meal was low in saturated fat and high in fruit and fibre. bloods were drawn postprandially every hour for five hours. results eosinophil counts were highest in the early morning and fell over the course of the day but the decrease was less with the high fat meal (p = 0.03). over the same period of time the increase in lymphocytes (p = 0.016) was greater with the high fat meal. the high fat meal was also associated with greater increases in triglycerides (p < 0.0001) and cholesterol (0.004). conclusions in atopic individuals a high fat meal was associated with higher circulating numbers of eosinophils and lymphocytes than an isocaloric meal that was low in fat. further studies of the effect of dietary fat on eosinophilic inflammation are warranted. supported by the university of auckland research committeee. intravenous gamma globulin therapy (ivig), which is therapeutic in a variety of immune diseases, has been reported to be effective on patients with severe steroid-dependent asthma. although fcer are known to play important roles in asthma, there are few reports about the role of fcg?receptors in asthma. fcg receptor iib (fcgriib) is unique inhibitory receptor, which suppresses immune response. in this study, we evaluated the effect of ivig in allergic airway inflammation in ova-challenged mice and the mechanism of the inhibitory effects of ivig and fcgriib. method c57bl/6 mice (wt) and fcgriib deficient mice (ko) were sensitized with ovalbumin (ova) and alum and subsequently challenged with nebulized ova. before ova challenge rabbit igg was administered intravenously. the airway inflammation and effects of igg were assessed by histology, cell counts of bal fluid and airway hyperresponsiveness. result histology showed that igg treatment ameliorated the inflammation around the airway and the vessels and hypertrophy of goblet cells induced by ova challenge. the migratory activity of dcs is modulated in inflammatory diseases such as asthma. recently, we reported that immature dcs express kinin receptors and that bradykinin (bk) significantly enhances the migration of immature dc in vitro. as kinins mediate many of the pathophysiological effects associated with asthma, we hypothesized that lys-des[arg 9 ]-bk, which is produced during inflammation and acts via the b1 receptor (b1r), would inhibit migration of mature dcs. methods day 7 cultured human monocyte-derived dcs were matured with lps, tnfa +il-1b or cd40l in the absence or presence of lys-des[arg 9 ]-bk. maturation of dc was analysed by flow cytometry (facs). b1r expression was assessed by reverse-transcriptase pcr and quantitative confocal microscopy. migration of mature dc was assessed in transwell chambers with lysdes [arg 9 ]-bk and the chemokine ccl19 used as chemoattractants. results maturation of dcs was found to result in down-regulation of b1r expression to varying degrees depending upon the maturation stimulus used. mature dcs all demonstrated an ability to migrate toward lys-des[arg 9 ]-bk and ccl19. however pre-treatment with lys-des[arg 9 ]-bk decreased the migratory ability of all mature dcs to both chemoattractants. conclusions along with chemokines, lys-des[arg 9 ]-bk is likely to play a crucial role in regulating the in vivo migration of mature dc during inflammation. the production of lys-des [arg 9 ]-bk during inflammation potentially immobilizes mature dcs thereby facilitating locally-mediated immune responses within inflamed tissues. supported by the asthma foundation of western australia. introduction alternative or aberrant splicing is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms. the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to use a new and innovative approach involving the use of the genechip (r) exon array system together with real-time quantitative pcr to study asthma candidate genes in human monocyte-derived dendritic cells. asthmatic and non-asthmatic subjects provided 20 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method, and differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data indicate splice variant expression in dendritic cells from asthmatic patients is influenced by asthma severity. conclusion exon expression array analysis has generated a number of asthma candidate genes with alternative splice variants. further studies to validate these data in a replicate data set and establish the functional significance of our findings in asthma are underway. alternative or aberrant splicing occurs in more than 70% of genes and is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms 1 . the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to study one asthma candidate gene in human stimulated and unstimulated: (1) monocytes, (2) monocytederived dendritic cells and (3) lung smooth muscle cells. methods asthmatic and non-asthmatic subjects provided 40 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method. up to 50% of the monocytes were then differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. induction experiments used 1 mg/ml lps and cells were stimulated for an optimal period of 24 hrs. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data from stimulation experiments indicate splice variant production can be regulated by the inflammatory response and that this response is influenced by asthma status. conclusion preliminary experiments have confirmed the presence of an aberrant splice variant for an asthma candidate gene in the primary cells studied. further studies to confirm these data and establish the functional significance of our findings in asthma are underway. exposure to environmental factors, such as environmental tobacco smoke (ets), plays a significant role in modulating pre-existing genetic susceptibilities to diseases including asthma. the glutathione s-transferase enzymes (gsts) play an important role in the detoxification of ets. there are several gst isoforms and gstp1 codes for the gst pi isoform, which is the primary gst isoform expressed in human lung tissue. two single nucleotide polymorphisms (snps) at positions 105 and 114 have been reported in gstp1 and associated with asthma and atopy. the aim of this study was to examine the effect of these snps in combination with ets, on asthma phenotypes in a cohort of asthmatic children. children were recruited during an acute episode requiring presentation at an emergency department. genotyping using pcr-rflp was completed on 218 children and ets exposure was determined by parental questionnaire. urinary cotinine was measured in the children and was in agreement with questionnaire responses. statistical analyses were performed using spss. there were no significant associations between the genotypes and asthma severity during acute exacerbations. significant associations were found between the snps and atopy in this population with an odds ratio of 2.77 for the 105aa genotype (p = 0.029) and or of 5.47 for the 114cc genotype (p = 0.002). however, when an interaction with ets was included, the odds ratios increased to 9.02 for 105aa (p = 0.05) and 9.17 for 114cc (p = 0.020). these results suggest that there is a significant gene/environment interaction impacting on atopy in this cohort. the rage gene encodes the receptor for advanced glycation end-products (rage), a member of the immunoglobulin superfamily. rage activation by ligands, including amphoterin and s100/calgranulins, leads to prolonged nf-kb signalling and has been associated with chronic inflammation. despite high levels of rage expression in lung tissue, little research has been undertaken into the role of rage in the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. objective determine genetic associations between functional polymorphisms in the rage promoter and severe and aspirin-sensitive asthma phenotypes. methods pcr and restriction fragment length polymorphism (rflp) were used to genotype three rage promoter polymorphisms, -429t>c, -374t>a and a 63 bp deletion from -407 to -345, in a large case-control asthma population phenotyped for asthma severity, atopy and aspirin sensitivity. results no associations were identified between any of the polymorphisms and the occurrence of asthma. however, the -374a allele was linked with both severe asthma (p = 0.013) and aspirin-sensitive asthma (p < 0.001). likewise, genotypes containing the -374a allele were strongly associated with both severe asthma (or 2.10, 95% ci 1.32-3.36) and aspirin-sensitive asthma (or 3.13, 95% ci 1.45-6.77). conclusions the -374a allele of the rage gene, previously shown to lead to a 3-fold increase in promoter activity, is associated with the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. these results suggest that increased rage expression, with a concomitant increase in nf-kb signalling, may in part contribute to the inflammatory response seen in these conditions. the global prevalence of allergic diseases is rising and australia has one of the highest prevalence rates in the world. the role of early childhood infections in the development of allergic disease remains controversial. objective to examine the association between early childhood infections and the development of allergic diseases in later childhood, in high risk children. methods data were analysed from the melbourne atopic cohort study (macs) of 620 infants with 1 or more first-degree family members with atopic disease. primary risk factors assessed were otitis media, bronchitis and gastroenteritis reported in the first two years of life. outcomes were current asthma, hay fever and eczema at 6 years of age. logistic regression was used to estimate crude and adjusted odds ratios. results asthma was the most common allergic condition (25.4%, 95% ci 21.6-29.5%), followed by eczema (24.9%, 95% ci 21.1-29.0%) and hayfever (15.6%, 95% ci 12.5-19.1%). the most commonly reported infection was otitis media (58.9%, 95% ci 54.9-62.8%), then gastroenteritis (37.7%, 95% ci 33.9-41.7%) and then bronchitis (19.4%, 95% ci 16. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] .7%). all 3 types of infection within the first 2 years of life were associated with increased risk of asthma. an increased risk of asthma at 6 years was seen with otitis media (or = 1.14, 95% ci 1.02-1.3), bronchitis (or = 1.34, 95% ci 1.0-1.8) and gastroenteritis (or = 1.23, 95% ci 0.96-1.6). when the frequency of infection was examined, those who reported at least 3 episodes of gastroenteritis had a 3-4-fold increased risk and an almost 30% absolute increased risk (rd 0.34, 95% ci 0.08-0.59). conclusion these findings appear to contradict the hygiene hypothesis. the findings for gastroenteritis are novel. further examination of these associations and possible underlying mechanisms is warranted. grant support asthma foundation of victoria, nestle. background knowledge about incident cases of asthma in australia is limited because they are not routinely reported. the ability to predict the number of new cases of asthma would be helpful in allocating resources for asthma education, management and care. data on first use of medications for asthma gives an indication of the incidence of asthma. the objective of this study was to estimate the incidence rate of asthma by investigating asthma medication use in individuals. methods pharmaceutical benefits scheme (pbs) records for all prescriptions filled for inhaled corticosteroids (alone or combined formulation), cromones and leukotriene receptor antagonists from july 2002 to june 2005 were included. using a 2-year look back window, any persons who had their first prescription for any of these drugs dispensed between july 2004 and june 2005 were assumed to be incident cases. overall and age-specific incidence rates were calculated per 100 asthma-medication-free individuals. results there were 352,082 individuals who had their first asthma medication dispensed between july 2004 and june 2005, which equates to an overall incidence rate for asthma of 1.89 per 100. the incidence was higher among children aged 0-14 years (2.07) and adults aged 65 years and over (2.45) . conclusions our estimated incidence rates were consistent with those reported by others in the literature. while the pbs database was designed for administrative purposes, it can be used to estimate incidence rates for asthma. support acam is a collaborating unit of the australian institute of health and welfare and is funded by the department of health and ageing (doha). we acknowledge the pharmaceutical pricing and estimates section of doha for provision of pbs data. keywords asthma incidence, pharmaceutical benefits scheme. rosario ampon 1 , guy marks 1 , teresa to 2 , leanne poulos 1 , anne-marie waters 1 1 australian centre for asthma monitoring (acam), sydney, australia, and 2 hospital for sick children, toronto, canada background the ability to assess individual patterns of asthma medication use would have clinical relevance in targeting effective asthma education and management for this common condition. to describe longitudinal patterns of asthma medication use, we used a population-based prescription database to follow individuals from the first time they filled an asthma prescription. asthma is more commonly listed on death certificates as an associated cause of death, in people whose deaths are attributed to other causes, than as an underlying cause of death. understanding the importance of these associations would contribute towards an overall appreciation of the impact of asthma on mortality. the objective of this analysis was to estimate the prevalence of asthma as an associated cause of death when various other diseases were attributed as the underlying cause of death. background acam currently recommend 24 indicators to measure population-level asthma health and outcomes. we examined correlations among several asthma indicators covering prevalence, morbidity and mortality to try and produce a condensed set of indicators which minimized redundancy. methods seven of the 24 indicators were included in this study: prevalence of ever having doctor diagnosed asthma, prevalence of current asthma, asthma-related general practice (gp) encounters, proportion of people with asthma with an asthma action plan (aap), hospitalizations for asthma, hospital patient days for asthma, and deaths due to asthma. a correlation matrix was created for these indicators by age groups. pearson correlation coefficients ն0.7 or յ-0.7 were considered strong. results there were strong positive correlations between prevalence of ever asthma and current asthma (r = 1.0); gp visits and aap possession (r = 0.74), hospitalization (r = 0.91) and patient days (r = 0.95); and hospitalization and patient days (r = 0.90) and aap possession (r = 0.73). recent australian reports have shown that the prevalence of asthma and respiratory symptoms has decreased over the last 10-15 years. as part of a larger study investigating child health and air quality we have collected nationwide data from schoolchildren living in act, victoria, queensland, wa and sa. methods schools were selected based on proximity to air quality monitoring stations. classes from years 3 to 6 were randomly selected and all children were invited to participate. parents self completed a questionnaire that included questions about diagnosed asthma and respiratory symptoms. results a total of 1989 children provided questionnaires for analysis. the response rate varied between states and territories and ranged from 30% to 42%. the sample comprised 51.9% girls and the mean age of children was 10.2 years. ever diagnosed asthma 27.9 current asthma ('does he/she still have asthma? ') 13.8 wheeze in the past 12 months 16.1 respiratory symptoms limiting activities 11.8 missed school due to asthma or wheezing 8.8 conclusions despite the relatively low participation rate, the prevalence estimates for current asthma are similar to those reported in the national health survey 2004-05 [1] . there is no evidence of any recent increase in the prevalence of childhood asthma. methods tahs is a longitudinal population-based respiratory study of 8583 subjects which commenced in 1968 when they were 7 years of age. since the initial study another 4 follow-ups have been conducted, including the most recent follow-up when subjects were 44 years of age. lung function of the total sample was measured at baseline and in sub-samples in 3 subsequent followups. asthma was categorized as persistent, frequent or episodic when participants reported asthma symptoms in at least 3 follow-ups, in 2 follow-ups or in 1 follow-up respectively. results by age 7 years ever asthma prevalence was 16%. at age 44, 10% of those who had not reported asthma by age 7 had asthma symptoms while 75% of those who reported asthma by age 7 had no asthma symptoms. hence over all only 25% of the asthma symptoms at age 44 were attributable to asthma developed by age 7. in contrast, 91% of the persistent and frequent asthmatics had developed their asthma by age 7. persistent and frequent asthmatics had more symptoms and poorer lung function at age 7, 14 and 44 as well as more reversibility at age 44 (p < 0.05). childhood asthmatics who also had a productive cough by age 7 were more likely to have persistent asthma than those without a cough (p < 0.05). conclusions although the majority of middle-age asthma is related to postchildhood onset asthma, most severe middle-age asthma has its origin in persistent childhood disease. having productive cough in childhood may identify high risk asthmatics who require especially rigorous management in early life. one third of women experience an improvement in asthma during pregnancy, and symptoms improve in most women in the late third trimester. we hypothesized that the exacerbation rate would be reduced and that symptoms during exacerbations would be less severe in the third trimester compared to the second trimester. methods pregnant women with asthma (n = 81) were prospectively followed from recruitment (14.8 weeks (3 sd) ) to delivery at clinic visits (18, 30, 36 weeks and during exacerbation), and fortnightly phone calls. the asthma control questionnaire (acq) was administered at each contact and exacerbations classified as severe (requiring medical intervention) or mild (selfmanaged). lung function, medication use, fractional exhaled nitric oxide (feno) and full blood counts were assessed. paracetamol is commonly used in infants as an analgesic and antipyretic. it has been hypothesized that frequent paracetamol consumption may result in reduced lung capacity to cope with oxidative stress and increase risk of respiratory disease. to date, no study has examined exposure to paracetamol during infancy, when lungs are still developing, and risk of childhood asthma. method a birth cohort of 620 infants with an atopic family history was recruited. frequency of paracetamol exposure was prospectively documented up to 2 years of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results paracetamol exposure in infancy was common (97% exposed by two years of age), with some infants receiving paracetamol on up to 77 days. it has been hypothesized that mucosal immune response requires a particular micro-flora milieu in the infant's gastro-intestinal tract, and that early life antibiotic exposure may disrupt this process and increase risk of allergic disease. method a birth cohort of 620 infants with an atopic family history was recruited. exposure to oral antibiotics was prospectively documented up to 12 months of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results by one year of age, approximately 80% of infants had received at least one course of oral antibiotics. the prevalence of current asthma in childhood was approximately 30% (148/495). frequent use of antibiotics (more than 20 days exposure during first year of life) was associated with increased risk of childhood asthma (or = 2.52, 95% ci = 1.40-4.54) when compared to infant who had not been exposed. excluding infants with a diagnosis of asthma within the first two years of life, reduced this association by about 30% (or = 1.80, 95% ci = 0.90-3.57) and adjustment for gender, parental history of asthma and number of infections in the first year of life further reduced this association (or = 1.60, 95% ci = 0.79-3.22). the increased risk of childhood asthma associated with antibiotic exposure in the first year of life is, at least in part, due to confounding with early life wheeze and infections. if real, the independent effect of antibiotic exposure on risk of childhood asthma is likely to be minimal in this high risk cohort. support dairy australia, crc for asthma and airways, vichealth, nestle. the epidemiological data on asthma suggest a gender difference that varies with age. hormonal effects have been suggested as a possible explanation for these differences. the aim of this study was to examine reproductive factors and risk of asthma among the females of the tasmanian longitudinal health study (tahs). methods the tahs is a longitudinal population-based cohort study of respiratory disease which commenced in 1968 when subjects were 7 years of age. four follow-up studies have been conducted including the current most comprehensive follow-up with subjects at 44 years of age. information has now been collected on reproductive factors such as number of pregnancies, age at pregnancies, age at menarche and contraceptive pill use as well as on asthma status. reproductive factors were examined as risk factors for asthma using multiple logistic regression to adjust for all likely confounders. results a total of 2,776 women completed the most recent postal survey. of these 355 (12.8%) had current asthma, and of these women with current asthma 73.5% (261) developed asthma after childhood. on average these women were in their mid-twenties when they developed asthma (mean ϯ sd age = 26.6 ϯ 12.5 yrs). we found with increasing age at first birth an approxi-mate~30% reduced risk of current asthma in women who developed their asthma post-childhood (trend p = 0.04). we did not observe any other associations between reproductive factors and risk of asthma. conclusions our results are consistent with the hypothesis that early pregnancy may promote asthma development by altering the immune response favouring a th2 pathway. a delay in the age of first pregnancy reduces this risk of asthma. grant support nhmrc, clifford craig foundation, victorian & tasmanian asthma foundations. introduction the association between exposure to pets in early life and subsequent development of sensitization and allergic disease remains controversial. the objective of this analysis was to examine the relationship between cat exposure before birth and development of cat sensitization over time within the melbourne atopic cohort study (macs). methods the macs is a prospective longitudinal cohort study that initially recruited 620 women antenatal in melbourne from february 1990 to november 1994. detailed information on cat exposure was collected at recruitment and frequently until two years of age. skin prick test (spt) were conducted at 6, 12, 24 months and 10 years. the data were analysed by logistic regression and using generalized estimating equations (gee) for the repeated measures design. results among 620 subjects, 169 (28.8%) had a cat before birth. at 6 months, 1.9% (n = 11) of subjects were sensitized to cat and by 10 years of age 18.8% (n = 68) were sensitized. those who did not have cat before birth belong to a higher social class, and were more likely to have a father with allergic disease than those with a cat. those who developed sensitization to cat were more likely to have a paternal family history of allergic disease and more likely to be sensitized to other allergens. we did not observe any association between exposure to cat before birth and the development of sensitization to cat at 6 months (or = 0.7, 95% ci 0. 1-3.3) , 12 months (or = 1.4, 0.5-3.9), 24 months (or = 0.76, 0.2-2.5) or 10 years (or = 0.6, 0. 2-1.4) . these crosssectional results were confirmed by the gee analysis. conclusion our results fail to show an association between cat exposure before birth and development of sensitization to cat. furthermore exposure after birth in the first 18months of life was not associated with an increased or decrease risk of sensitization to cat. our results do not support either a benefit or risk associated with cat ownership and sensitization. introduction peri-natal events influence the development of asthma and atopic diseases in childhood but the current literature is contradictory on the effect of low birth weight, small for gestational age and prematurity on asthma risk. the aim of this study was to assess the relationship between these three exposures and asthma from childhood to adulthood. aim to assess the current prevalence of dda, wheeze (<12 months), atopy and ahr in children and adults in busselton. methods an age-and sex-stratified random sample of adults, selected from the electoral roll, was invited to complete a questionnaire and attend the local study centre for assessment of atopy (allergen skin tests) and ahr (methacholine). all children from participating primary and secondary schools were also invited to attend. the prevalences of dda, wheeze, atopy, ahr and "current asthma" (wheeze + ahr) were calculated. background asthma is often associated with comorbidity, however few studies have investigated comorbidities among people with this common condition. the objective of this analysis was to describe patterns of non-respiratory comorbidity among adults hospitalized with asthma in australia. methods data on hospitalizations for people aged 15 years and over with a principal diagnosis of asthma (j45, j46) were obtained from the australian institute of health and welfare's (aihw) national hospital morbidity database for the period 2005-06. patterns of comorbidity were examined by investigating additional diagnoses for non-respiratory disease according to icd-10 diseasespecific chapters. results among people aged 15 years and over hospitalized in 2005-06 with a principal diagnosis of asthma (16,566 hospitalizations; 70% female; 47% aged 35-64 years), 33% had at least one non-respiratory comorbidity. median length of stay was higher among those with at least one comorbidity (4 days) than among those with no comorbidities (2 days). among people aged 15-64 years, the most common comorbid condition was endocrine, nutritional and metabolic diseases (19%), while among those aged 65 years and over it was diseases of the circulatory system (32%). conclusions a large proportion of asthma hospitalizations in australia are associated with non-respiratory comorbidity and a longer length of stay. further, the pattern of non-respiratory comorbidity associated with asthma hospitalizations varies by age. given our rapidly ageing population, the level of comorbidity associated with asthma has implications for coordinated health care and demand on health services. support acam is a collaborating unit of the aihw and is funded by the department of health and ageing. keywords comorbidity, hospitalization, asthma. background asthma exacerbations are often triggered by viral respiratory infections, yet the influence of respiratory infections on the morbidity of acute asthma beyond the immediate period is unknown. we examined the influence of nasopharyngeal (npa) respiratory viral, chlamydia and mycoplasma detection on asthma morbidity in children presenting to the emergency department for an acute exacerbation of asthma. methods a subset (n = 78) of the 201 children enrolled for a randomized controlled trial (rct) on the efficacy of 5 vs 3 days of oral prednisolone had an npa taken at presentation. npa were examined for chlamydia, mycoplasma and respiratory viruses (enteroviruses, coronaviruses, human metapneumovirus, adenovirus, parainfluenza, influenza, rsv, rhinoviruses) by pcr. enrolled children were aged 2-16 years with recurrent wheeze and required ն600 ?g (mdi/spacer) or ն2.5 mg (nebulized) of salbutamol to reduce tachypnoea. parents filled validated diary cards for cough and asthma severity, and completed asthma qol data at enrolment and end of weeks 1 and 2. results pcr for various viruses was positive in 42 (53.8%) children, with no significant difference in the groups the children were randomized into. rhinovirus pcr was positive in the npa of 32 children, rsv in 7, hmpv in 2, adenovirus, parainfluenza, influenza a and b in one each. specimens were negative for the other micro-organisms listed above. children with a npa viral positive state were significantly (p = 0.002) younger than those with a negative state. however, there was no difference in the any of the asthma outcomes of children whose npa was positive or negative for the micro-organisms tested. conclusions in children with an acute asthma exacerbation presenting to emergency health facilities, a respiratory virus could be identified in >50% but the presence of a respiratory virus did not influence the morbidity of the asthma exacerbation at presentation or at the end of week-1 and week-2. the university of sydney, nsw 2006, and 3 royal north shore hospital, st leonards, nsw 2065 airway wall thickness measured using hrct is reported to be increased in asthmatic compared with control subjects. however, it is unknown whether wall thickness is a fixed structural characteristic of the airways or if it responds to transient changes in bronchomotor tone or airway size. aim to determine the effects of bronchomotor tone and lung volume on airway wall area measured by hrct. methods 8 patients with doctor-diagnosed asthma had partial chest hrct scans, before and after bronchodilator (bd), at frc, tlc and a volume midway between (mid-volume). airway segments were identified between branch points and matched between consecutive lung volumes both before and after bd, and also at constant lung volume before and after bd. mean lumen areas and wall areas for each airway segment at each volume were measured using automated analysis software. paired t-tests were used to determine changes due to bd and lung inflation. results 44 airways were matched before and after bd at frc. absolute airway wall area (wa) was related to airway lumen diameter (di wood smoke air pollution is of concern with respect to respiratory health due to its complex chemical composition and potential to carry air toxics into the lower respiratory system. launceston has a long history of poor winter air quality, primarily due to use of domestic wood heaters. participants in hobart had a similar prevalence of wood heater use, but hobart does not experience the same wood smoke pollution (due to differences in regional geography , asthma control and anxiety and depression were completed at baseline, immediately following (6 wks), and 3 mths after the intervention period. results clinically and statistically (p < 0.05) significant improvements in qol were observed in the exercise group at 6 wks compared to the control group. this difference was not maintained at 3 mths. 6mwd improved at 6 wks and 3 mths in the exercise group (p < 0.01), however the difference between groups was not significant. in the exercise group there was a trend towards improved asthma control and a reduction in anxiety and depression that was not observed in the control group. *p < 0.05, change at 6 wks vs baseline; home asthma monitoring is important for measuring day-to-day variation in lung function and symptoms. this approach requires the availability of complete diaries for a comprehensive assessment. we assessed the completeness of written diaries collected as part of a nation wide study of air quality and child health. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified from a cross-sectional study. these children were asked to record symptom scores and peak expiratory flows twice daily in diaries for a five week period. the diaries and peak flow devices were explained at a face-to-face meeting with parents and children. each week diaries were mailed back and parents received a phone call to encourage completion. completeness was defined as no missing responses to symptom questions or peak flow measurements in diaries from week two to week five. results data from the first 36 children (822 day records) were available for analysis. the sample included (53%) girls, mean age 10 yrs. the overall frequencies for complete records were; morning symptoms 85%, morning peak flow 85%, evening symptoms 83% and evening peak flow 82%. there was a significant trend for more complete morning peak flow records over the four weeks (cochrane-armitage trend test p < 0.007). agreement between morning and evening symptom completeness and between morning and evening peak flow completeness was fairly poor (kappa < 0.30). conclusions the completeness of symptom and peak flow records collected in this study was very high. the comprehensive follow-up protocol implemented is likely to have had an important impact on the completeness of asthma diaries. daily peak expiratory flow (pef) monitoring has been used in epidemiological studies to assess changes in lung function over time. the value of written pef diaries has been questioned because of problems with completeness and validity. this study aimed to compare stored electronic pef data and a written diary record of those data in a panel study in children with weekly reminders to aid adherence. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified in a population study. they were given electronic pef devices with a digital readout (miniwright digital, mwd, clement clarke, uk) and written symptom and peak flow diaries and instructed in their use at a meeting with parents and children. each child was asked to complete three pef manoeuvres every morning and evening for five weeks and to record these in the written diary. background previous research suggests that comorbid anxiety is associated with lower asthma-related quality of life (aqol) in adults with asthma. however, research is scant on the role of psychological interventions in these patients. aim to evaluate the effectiveness of a four-session cognitive-behavioural therapy (cbt) intervention, in improving the aqol, in participants with anxiety and asthma. method participants identified with comorbid anxiety and asthma were randomly assigned to the cbt intervention group (n = 10) and the asthma monitoring control group (n = 8) and evaluated on aqol measures, at various intervals. results nine participants, in the cbt group, completed the study. seven participants showed a clinically significant improvement in asthma-related emotional functioning (ef) and six participants in total aqol scores, at the 5-week post-intervention assessment. additionally, six participants in the cbt group indicated clinically significant improvement in ef and five participants in total aqol scores, at the 3-month follow-up assessment. only three participants in the control group completed the study. none of these participants showed any improvement in aqol scores at the 5-week or 3-month assessment. conclusion this pilot study suggests that a higher number of participants in the cbt group showed clinically significant improvement in ef and total aqol scores with higher retention rates. further research needs to confirm these findings in a larger group, identifying the elements of a successful cbt intervention and characteristics of participants who respond to the cbt intervention. gastro-oesophageal reflux disease (gord) is a risk factor for uncontrolled asthma. we conducted an update of a systematic review to assess whether treatment of gastro-oesophageal reflux in subjects with asthma improved asthma outcomes. methods randomized controlled trials (rcts) of gord treatment in adults or children that reported asthma health outcomes and had symptomatic gord were included and assessed in accordance with the standard cochrane systematic review process. subjects received pharmacological therapies compared with conservative management. results from 261 potentially relevant studies, 19 rcts were included in the review. when compared to placebo, morning peak expiratory flow did not significantly improve (change from baseline wmd 10.43, 95% ci: -9.55 to 30.42) with proton pump inhibitor treatment (n = 7 trials involving 739 participants). asthma exacerbations were not significantly less in the intervention groups compared with the control groups (odds ratio 0.66; 0.41-1.08; n = 557). conclusions while some trials reported evidence of asthma improvement with gord therapy, overall there appears to be no statistically significant evidence of a beneficial effect. it is clear that not all persons with gord and asthma will gain improved control over their asthma with gord therapy; this may be due to the heterogeneous pathophysiology of asthma. future large-scale trials would be required to demonstrate an effect on asthma exacerbations. kel and brd were supported by a cochrane airways group scholarship. background the ats/ers task force recommend the use of metered dose inhaler (mdi) and spacer for airflow limitation reversibility testing. salbutamol given via mdi & spacer has been shown to be equivalent to a nebulizer in the clinical setting. this has not been well studied in respiratory laboratory setting. aim to compare the 2 methods of reversibility testing in a laboratory setting. methods we conducted a laboratory based crossover study in a secondary hospital. patients with asthma or copd were eligible. the patients firstly underwent spirometry and reversibility testing following a standard dose of nebulized salbutamol. they were asked to return for a second set of spirometry within the same week and at the same time of day when reversibility with an mdi and spacer was recorded. we used an incremental dose of salbutamol starting from 2 puffs and up to 8 puffs. spirometry parameters were recorded 10 minutes after each intervention. the primary outcome was the percentage change in fev1 after each intervention. side effects were monitored for. results nine patients with asthma were recruited. the mean percentage change in fev1 was higher in the nebulizer group than after only 2 puffs via mdi & spacer (15.4 ϯ 7.4 vs 6.2 ϯ 8 [mean ϯ sd], p = 0.67). however, there were no differences between the 2 arms following higher doses of bronchodilator via mdi & spacer. the mean percentage change in fev1 after 4, 6 and 8 puffs were 12.6 ϯ 11.3, 15.4 ϯ 12.3, and 17.7 ϯ 13.6 respectively (p = 0.09, 0.05 and 0.07 respectively when compared to the nebulizer group). conclusion using an mdi and spacer for bronchodilator reversibility is equivalent to that of a nebulizer and should be the standard method of testing. the dose of bronchodilator needs to be at least 4 puffs as recommended by the ats/ers; however 6 puffs correlated best with a standard nebulizer route. further increments in bronchodilator dose provided little additional bronchodilatation. the study was limited by the small number of patients. asthma guidelines recommend a stepwise approach to treatment. the role of inhaled corticosteroid (ics) and long-acting beta-agonist (laba) combination therapy in asthma written action plans is not clear. objective to assess the efficacy of adjusting ics/laba combination therapy in a written action plan compared to fixed dosing in people with asthma requiring maintenance ics. methods cochrane systematic review of randomized controlled trials comparing ics/laba combination therapy in a single inhaler that is adjusted up or down according to a written action plan (wap) to comparison 1: budesonide/ formoterol given as a fixed maintenance dose (fd) (n = 9) or comparison 2: fluticasone/salmeterol fd (n = 2). results 10 parallel randomized controlled trials describing 11 interventions met the inclusion criteria. for the trials that compared wap to fd budesonide/ formoterol there were significant reductions for the wap group in exacerbations, (rr (95%ci): 0.82 (0.70 to 0.97)), severe exacerbations (rr (95%ci): 0.61 (0.37 to 0.99)) and study medications (wmd (95%ci): -1.18 (-1.23 to -1.14)) with no difference in asthma control or adverse events. the results for the two trials reporting wap budesonide/formoterol to fd fluticasone/ salmeterol were discordant and a homogenous pooled result could not be determined. of the 318 australians who died from asthma in 2005, over two thirds were over 50 years of age. this trend resulted in the national asthma council of australia (nac) calling for better management of asthma in the elderly. we designed an educational intervention using evidence based educational strategies to improve the content and style of general practice consultations for older people with asthma. methods randomized controlled trial of a multi-faceted program consisting of a group educational session, a videotaped standardized simulated patient consultation, followed by an academic detailing session. forty-two gps were randomized into an active or a control group. gps provided the names of patients who would be happy to participate in the study and the program was evaluated by patient and gp outcomes. results gps recruited into our program reported improvements in a range of clinical areas. one hundred and ten patients were recruited, their outcomes are under analysis. conclusion gps were overwhelmingly positive about participation in this trial and our intervention successfully improved the capacity and confidence of gp's to deliver care to older people with asthma. our study also developed several tools that would enable dissemination of our findings. supported by an asthma targeted in studies where direct clinical assessment is not possible, urgent health care utilization (hcu) is often used as an indirect measure of asthma control. this study aimed to identify factors predicting urgent hcu and asthma control. methods patients in nsw with a doctor diagnosis of asthma were recruited from community pharmacies, a research volunteer database, and databases of asthma foundation nsw, to complete a questionnaire about asthma. poor asthma control was defined as asthma control questionnaire (acq) score ն1.5. urgent hcu was defined as hospitalization, ed visit, or urgent doctor visit due to asthma. multiple logistic regression was used to identify predictors of poor control and urgent hcu. results questionnaires were completed by 608 adults (61% female) with a doctor diagnosis of asthma (pharmacy 260, woolcock 299, asthma foundation 87). 87% used inhaled corticosteroid (ics) ϯ long-acting b2-agonist in the last 4 wks. median age was 56 yrs (range 12-87), and 9% were current smokers. mean acq score was 1.4 (95% ci 1.3-1.5), with 40% of participants having poor asthma control (acq ն 1.5). 28% had urgent hcu for asthma in the previous year. significant independent predictors for poor asthma control were younger age, current smoking, living in more disadvantaged areas, being retired, having only primary education, and holding a concession card. predictors for urgent hcu were younger age, being in full-time employment, having only primary education, and being of non-english speaking background. neither ics use nor possession of a written asthma action plan was associated with lower risk for either poor asthma control or hcu. conclusions poor asthma control is common in nsw even in patients using inhaled corticosteroids. although urgent hcu is often used as an indirect measure of poor asthma control, it is affected by different factors, perhaps because health care utilization represents a more complex balance between need and access. bronchial challenge tests with mannitol, to measure airway hyperresponsiveness, can take up to 30 minutes and require inhalation of up to 635 mg of mannitol. our aim was to determine if positive mannitol challenges can be detected after half the maximal dose (315 mg) using the forced oscillation technique (fot) to measure response. methods 15 non-asthmatic subjects and 52 asthmatic subjects underwent standard mannitol challenge, up to 635 mg mannitol. respiratory system conductance (grs) and reactance (xrs) was measured by fot at 6 hz during 40 sec tidal breathing immediately after each dose of mannitol. fev1 was measured after fot, within 90 sec of mannitol administration. two point dose response slope (drs), was calculated for grs (drsgrs) and xrs (drsxrs) for standard tests, up to 635 mg, and for short tests by excluding data from doses above 315 mg. ability to detect a positive test, defined as pd15fev1 < 635 mg, was determined by the area under the roc curve (auc) and repeatability by intra-class correlation coefficient (icc). results 32 asthmatic and 2 non-asthmatic subjects had positive tests, with pd15 fev1 values from 9.2 to 622 mg. auc (95%ci) did not differ between standard (std) and short tests for drsgrs (p = 0.14) or drsxrs ( combined use of inhaled steroids (ics) and long acting beta-agonists (laba) have an important role in asthma management. we used data from a 2006 population sample to examine medication use in adults and children. methods all adults (18-75 years) and children (2-17 years) from within four discrete zones in northern sydney were eligible for an interview survey, as part of a study investigating health effects associated with traffic-related air pollution. the prevalence of use of short-acting beta-agonists (saba), any ics (alone or combination) and combined formulations of ics/laba in the previous three months was estimated for the study population and those with diagnosed asthma. results there were 806 children [mean (sd) age 8.7 (4.6) years and 50% female] and 2184 adults [mean (sd) age 45.6 (14.9) years and 56% female] interviewed in 1843 households, representing an overall response rate of 33%. the prevalence of ever diagnosed asthma was 16.1% in children and 17.4% in adults. medication data were missing for 301 subjects. background asthma affects 1:9 adult australians and is a leading cause of rejection for recruitment into the australian defence force (adf). within this diagnosis there is a wide spectrum of disease activity and clinical outcomes. also asthma assessment and management has improved so that many asthmatics are now fully active without any significant disruption or risk to their lives. hypothesis: there is a subgroup of asthmatics who are at very low risk from significant adverse effects from asthma and who could be considered for recruitment to the adf. aims 1. to identify the subgroup of asthmatics who could be considered for recruitment to the adf. 2. to develop an assessment process to identify this subgroup (screening). 3. to develop a process to evaluate the outcomes of any change to the recruitment standard for asthma (evaluation). methods 1. a literature review of the natural history, assessment, management and response to treatment of mild episodic and mild persistent asthma. 2. a literature review of asthma in the military. 3. a clinical review of the outcomes of known asthmatics in the adf. 4. an expert group to review the above and to develop a screening process and an evaluation of the program. the literature review identified a subgroup of asthmatics, defined as mild episodic and mild persistent, who with appropriate management, have a low risk of significant adverse asthma outcomes. they can be identified by a combination of questionnaire, spirometry and bronchial provocation testing. a screening process has been developed which allows asthmatics to be recruited with a negative mannitol or hypertonic saline challenge on 400 mg/day or less of budesonide (or equivalent) without laba. a methodology to evaluate the impact of these changes on the recruitment standard has also been developed. alexithymia is a personality trait associated with difficulty identifying and communicating emotional and physical feelings. it has been associated with poor control of asthma and near fatal asthma. the primary objectives of this study were to: (1) identify alexithymia in a cohort of australian asthma patients; (2) investigate the relationship between alexithymia and asthma control; (3) investigate the relationship between alexithymia and asthma management. methods cross sectional study of 25 moderate to severe asthma patients recruited from royal adelaide hospital outpatients. participants were either mailed the questionnaire pack or completed it after a clinic appointment. existing validated questionnaires were used. statistical analyses were performed using spss. results 11 male (44%) and 14 female (56%) patients with moderate to severe persistent asthma (mean age 44 years, sd = 11) participated. alexithymia scores ranged from 23.0 to 76.0 (x = 48.3, sd = 13.2). 12% (n = 3) of participants could be classified high alexithymia, 32% (n = 8) borderline alexithymia and 56% (n = 14) were low alexithymia. alexithymia mean scores were not statistically different across sociodemographic variables. a positive correlation/association was found between alexithymia score and asthma control score (r = 0.57, p < 0.01), quality of life (r = -0.65, p < 0.01), and adherence (p = 0.03) but not satisfaction with communication (r = -0.27, p = 0.2) or number of hospitalizations (p = 0.25). conclusions this is the first australian study to identify alexithymia among asthma patients and investigate relationship to control as well as management and communication. associations between alexithymia and asthma control were confirmed. a larger sample size is needed to determine impact of alexithymia on self-management and provision of clinical care for asthma. port hedland is impacted by iron-containing dust particles (pm10) that may activate lung cells when inhaled. furthermore, the effects of port hedland pm10 may differ from the effects of urban pm10 impacting metropolitan areas. the aim of this study was to assess the effects of port hedland pm10 on production and release of the inflammatory cytokines, il-6 and il-8, by human airway epithelial (a549) cells, and to compare these with the effects urban pm10 from metropolitan areas. methods human airway epithelial (a549) cells were exposed to pm10 collected at port hedland and at urban locations (sydney, perth). a549 cells were exposed to a range of pm10 concentrations (20-200 mg/ml) for 24 h. lipopolysaccharide (lps) and phorbol myristate acetate (pma) were used as positive controls. supernatants from cell cultures were assayed for il-6 and il-8 using specific elisa kits. rna was extracted and reverse transcribed to cdna. il-6 and il-8 mrna expression was quantified by duplex real-time pcr using taqman primer/probes. results lps stimulated a 2.7-fold increase in il-8 release and pma stimulated a 3-fold increase in il-8 release and a 30-fold increase in il-6 release. however, neither port hedland pm10 nor urban pm10 stimulated concentration dependent release of il-6 or il-8 by a549 cells. expression of il-6 or il-8 mrna was also not altered by port hedland or urban dust. cd8+ t-cells may cause airway epithelial cell apoptosis via the granzyme pathway. we have reported increased apoptosis of airway epithelial cells and increased bal t-cell expression of granzyme b in copd, and a positive correlation between the two. we hypothesized that the increased granzyme b would also be related to smoking history (pack years -pk/y), age and severity of airflow obstruction (fev1 %pred) in patients with copd. we further hypothesized that the t-cell granzyme b expression would be higher in the airway than the peripheral blood. methods we investigated t-cell intracellular granzyme b expression in blood from copd subjects (33 current and 24 ex-smokers) and 12 never-smoker controls, and bronchoalveolar lavage (bal) and bronchial brushing (intraepithelial t-cells) from a cohort of these subjects using flow cytometry. correlations between granzyme b and pk/y, age or fev1 were performed using spearman's rank correlation. granzyme b in t-cells from blood, bal and bronchial brushings were compared. results there were significant correlations between fev1 and granzyme b expression in blood and bal (blood: r -0.444, p = 0.002; bal: r -0.368, p = 0.029). there was a significant correlation between pk/y and granzyme b expression in blood (r 0.362, p = 0.002), but not in bal. there were no significant correlations between granzyme b and age. there were no significant differences in granzyme b expression in blood, bal or intra-epithelial compartments. conclusion granzyme b is expressed at similar levels in blood, bal and intra-epithelial compartments, supporting recent opinion that copd is a systemic disease. t-cell granzyme b is related to severity of airflow obstruction and smoking history in patients with copd and may be one mechanism of apoptosis leading to lung injury and airflow obstruction in copd. jc allen 1 , t schlosser, ee ramsay 1 , q ge 2 , aj ammit 1 as development of remodelled airways is correlated with deterioration of lung function, we require therapies that reduce and reverse structural changes in remodelled airways. in asthma, corticosteroids can halt some, but not all, aspects of airway remodelling. therefore, in order to aid future design of efficacious anti-remodelling agents we need a better understanding of the molecular mechanism/s underlying the development of airway remodelling and the effectiveness of corticosteroids. hyperplasia of airway smooth muscle (asm) is a feature of the remodelled airway in asthmatics. in this study we examined the effect of corticosteroids on a key regulator of g1 progressioncyclin d1. asm cells from n = 8 non-asthmatics and n = 7 asthmatics were pretreated for 1 h with vehicle or dexamethasone (0.1 mm). the temporal kinetics of cyclin d1 mrna and protein expression were measured up to 24 h after stimulation with the mitogen platelet-derived growth factor-bb (pdgf-bb). pdgf-bb induced a significant increase in cyclin d1 mrna expression in asm from non-asthmatics (2.6 ϯ 0.3-fold) and asthmatics (2.9 ϯ 0.3-fold) after 24 h stimulation. in non-asthmatics, the corticosteroid dexamethasone significantly (p < 0.05) reduced the amount of cyclin d1 mrna expressed (to 1.6 ϯ 0.2-fold). in contrast, cyclin d1 expression in asthmatics was relatively resistant to inhibition by dexamethasone; the amount of pdgf-bb-induced cyclin d1 expression in the absence or presence of dexamethasone was not significantly different ( sphingosine 1-phosphate (s1p), a bioactive sphingolipid found elevated in the airways of asthmatics, modulates myriad airway smooth muscle (asm) functions that promote inflammation and remodelling in asthma. in this study, we uncover the molecular pathway/s underlying s1p-induced secretion of il-6, and investigate if, and how, corticosteroids inhibit il-6 secretion. using cultured asm cells from non-asthmatics, we found that s1p induces il-6 secretion from asm cells via cre, but not ap-1, c/ebp or nf-kb, transcriptional regulation of il-6 gene expression. cre-dependence was supported by s1p-induced creb phosphorylation. although the corticosteroid dexamethasone reduced s1p-induced il-6 secretion in a dose-dependant manner, this inhibition appeared to occur via a pathway independent of creb/cre, suggesting the existence of a parallel pathway. as we recently discovered that the antiinflammatory actions of corticosteroids in asm can be mediated via the induction of the endogenous mitogen-activated protein kinase (mapk) inhibitor, mapk phosphatase-1 (mkp-1), we investigated whether mapk represents the parallel pathway targeted by corticosteroids. we found that s1p can induce activation of a variety of mapk, however, only p38 mapk phosphorylation was inhibited by dexamethasone; importantly, the increase in mkp-1 after corticosteroid treatment appeared to mirror the decrease in s1p-induced p38 mapk phosphorylation. furthermore, exogenous expression of mkp-1 inhibited s1pinduced il-6 secretion. taken together, these results suggest that parallel pathways exist to induce il-6 secretion (transcriptional via creb/cre and possibly post-transcriptional via p38 mapk) and serve to underscore the importance of mkp-1 upregulation as a mechanism of action of corticocosteroids in asm. angiogenesis is a hallmark feature of asthma. angiogenic promoters, such as vegf and tgfb are reported to be increased in airways of asthmatics. tumstatin, an endogenous angiogenic inhibitor, is the non-collagenous domain-1 (nc1) of the alpha3 chain of collagen iv. decreased levels of collagen iv have been reported in the airways of asthmatics. we investigated the presence of tumstatin in the airway of asthmatics and its potential role as an angiogenic inhibitor. we detected the six a chain nc1domains of col iv and the 7s domain of the a3 chain using immunohistochemistry. the level of tumstatin in serum and bal-f was measured by dot blot. western blots were used to identify the association with the rest of the collagen iv molecule. a tube formation assay using primary pulmonary endothelial cells (ppec) was performed to evaluate the role of tumstatin in the airway. the effect of intranasal tumstatin on airway hyperresponsiveness and angiogenesis was studied in an ovalbumin mouse model. tumstatin was absent in the airways of asthmatics (n = 14) while the remaining six collagen iv a chains were present. the 7s domain of the a3 chain was present in the asthmatic airway (n = 6). tumstatin was detected in both serum and bal-f samples from asthmatic volunteers (n = 10), however the level of expression was not significantly different from that in nonasthmatics (n = 7). in asthmatic serum tumstatin was part of the whole collagen iv a3 chain. tumstatin was able to inhibit ppec tube formation in a dose related manner. tumstatin inhibited angiogenesis in the mice airways and was associated with an improvement in ahr. the fact that tumstatin is absent from asthmatic airways and inhibited airway hyperresponsiveness and angiogenesis may indicate potential for therapeutic intervention in airway remodelling. this work was supported by the crc for asthma and airways and nh&mrc. introduction epithelial egfr (epidermal growth factor receptor) expression correlates with disease severity and neutrophil infiltration in asthmatic airways. acute exacerbations of asthma and copd are also associated with steroid refractory neutrophilic inflammation, with rhinoviruses being the most common trigger. .7 mg/l and il-6: 5.8 vs. 3.6 ng/l). since il-6 stimulates the acute phase response, we correlated its levels with the other markers. only crp was strongly correlated with il-6 (spearman r = 0.58, p < 0.0001), suggesting differential regulation of saa and ip10. saa discriminated between non-pathogen (n = 10) vs. pathogen-associated (n = 41) events (saa: 9.4 vs. 44.1 mg/l p = 0.005), whereas no significant change was observed in the other markers (ip-10: 139.8 vs. 170.5 ng/l, crp: 4 vs. 10 mg/l, il-6: 4.6 vs. 7.2 ng/ l). however when aecopd marker levels were stratified on the basis of pathogen type (viral = 12, bacterial = 21, viral and bacterial = 8), none of the markers were significantly altered. conclusions ip-10 is significantly elevated during an aecopd, however only saa differentiated non-pathogen from pathogen associated events. background severe persistent asthma is characterized by structural changes in the airways-airway remodelling. airway smooth muscle (asm) cells have the potential to play a key role in these processes through the release of growth factors, cytokines and extracellular matrix (ecm) proteins. we have previously studied the effects of budesonide and formoterol individually however, the effect of their combination on these characteristics of asm cells is not known. methods asm cells from asthmatic (n = 6) and nonasthmatic (n = 6) individuals were stimulated with transforming growth factor ß (tgfß) (1 ng/ml) with or without budesonide (10 -8 m) and formoterol (10 -10 and 10 -8 m) and fibronectin levels and interleukin-6 (il-6) release were measured by elisa. bronchial rings from nonasthmatic individuals (n = 2) were incubated with tgfß with or without the drugs and ecm protein expression (fibronectin and collagen i) measured using immunohistochemistry. results in nonasthmatic cells, budesonide alone induced fibronectin deposition whether tgfß was present or not. formoterol decreased fibronectin induced by tgfß and, when combined with budesonide, reversed the increase in fibronectin. a similar pattern was observed in asthmatic cells, except that budesonide did not further increase the tgfß mediated fibronectin release. as before [1] , il-6 was induced by formoterol but inhibited by budesonide. tgfßinduced il-6 was inhibited by both drugs and their combination in both cell types. in bronchial rings the presence of either drug did not affect tgfßinduced fibronectin or collagen i. severe combined immune deficiency (scid) spontaneous mutation specifically impairs differentiation of stem cells into mature lymphocytes. nod-cb17prkd scid (known as nod-scid) lacked nk cells, hence is commonly used in cell transfer experiments for transferring tissue and haematological xenografts. the aim of this study was to establish lung inflamamtory model in nod-scid strain. methods balb/c and nod-scid balb/c mice (n = 8) were exposed to cigarette smoke for 4 days, 2 and 4 weeks (9 cigarettes/day; 5 days/week). bronchoalveolar lavage fluid (balf) and lung tissue were collected for inflammatory profiling and analysis for cytokines, chemokines and protease expression and/or activity. results nod-scid have significant accumulation of macrophages in lung after 4 days, 2 and 4 weeks smoking as compared to no smoke control (p < 0.001) that was not different to balb/c (p > 0.05). nod-scid also have increased neutrophil number after 2 and 4 weeks smoking (p < 0.001). even though myeloid cell differentiation isn't affected by scid phenotype, nod-scid have one fold less neutrophil than balb/c mice (p < 0.001) that is also reflected in the reduced expression of matrix metalloproteinase-9. consistent with the known lymphopenic phenotype, nod-scid have significant but less lymphocytes recruitment as compared to balb/c mice after 4 weeks smoking (p < 0.001) despite the enhanced expression of inteferon inducible protein 10 (lymphocytes specific chemokine) in lung. both mouse strains showed the same elevation of net gelatinase and serine protease activity in lung. nodscid mice also demonstrated comparable transcriptional induction of proinflammatory cytokines (tnfa, il-6), growth factors (gm-csf, g-csf) and chemokines (mcp-1, mip-2), indicating susceptibility to smoke-induced injury. conclusions nod-scid mice are capable to mount smoke induced inflammatory response. this model may be useful to study localization and role of immunocytes, including adoptively transfer human cells in the pathogenesis of copd. supported by the nhmrc. rhinovirus (rv) is the cause of most common colds and up to 80% of asthma attacks. in our previous studies, plasminogen activator inhibitor 2 (pai-2) was expressed at high levels and was induced in vivo and in vitro by rv infection. pai-2 may have antiviral properties suggested by antiviral activity in some models, high pai-2 expression levels and further upregulation by rv infection. methods to determine whether pai-2 has antiviral activities following rv infection, o-hela, pai-2 expression-deficient cells were first transfected with pai-2 or control genes. this was followed by infection with rv and effects on viral replication were assessed by rt-qpcr for vrna and by viral titration for virus release. ifn expression was assessed by rt-qpcr. results ifn-a and -b mrna expression were induced in response to rv infection and to pai-2 expression in cells. pai-2 expression followed by rv infection elicited a synergistic response and pai-2 over-expression reduced vrna by >5 fold and viral titre by >3 log (p < 0.05). however, this effect was not specific to pai-2, as transfection of cells with control genes/plasmids reduced viral titre to a comparableextent. one of the pathological findings in idiopathic pulmonary fibrosis (ipf) is the presence on fibroblastic foci comprising cells which exhibit mesenchymal phenotypic features such as myofibroblast-like morphology, increased asma expression and collagen deposition. currently steroid treatment in ipf has shown limited efficacy. the cellular origins of these mesenchymal cells remain unclear, but evidence from other studies suggests that epithelial cells may undergo a transition to a mesenchymal cell phenotype (emt). transforming growth factor ß has been implicated in promoting this emt. in this study we have induced a morphological change in a549 cells using tgf-ß1 and assessed the influence of glucocorticoids, and the changes to the extracellular environment of the cells, on emt. methods a549 cells were grown on uncoated plastic cultures plates or those coated with monomeric or fibrillar collagen and treated with 200-500 pm tgf-ß1. the influence of the glucocorticoid, dexamethasone (dex, 1-1000 nm), or collagen type, on emt was assessed by microscopy, rt-pcr and western blotting for markers of myofibroblast phenotype. results tgf-ß1 induced an increase in mrna expression of asma (1.5 fold), collagen (7.0 fold) and fibronectin (2.0 fold). dex (100 nm) partially inhibited the expression of collagen, but had no effect on asma levels. however, dex (100 nm) reduced asma and ctgf protein levels. dex (100 nm) also prevented the tgf-ß1-induced morphological changes, regardless of ecm matrix. conclusion glucocorticoids appear to control some of the emt phenotype changes induced by tgf-ß1. however, the inability to fully inhibit these changes may contribute to the resistance of ipf to glucocorticoids. the extracellular environment may also play a role in the development of fibroblastic foci and their pharmacological responses. defective alveolar macrophage (am) phagocytic function in the airway may perpetuate inflammation via secondary necrosis of uncleared apoptotic cells in copd. we have previously reported that low-dose azithromycin improved macrophage function in vitro, although the mechanisms for this effect were not identified. we explored the possible role of the collectin pathway in the azithromycin-mediated improvement in phagocytosis as well as possible defects in this pathway in copd subjects. methods (1) mannose binding lectin (mbl), mannose receptor (mr), surfactant protein d (sp-d) were measured in copd subjects and controls. (2) the in vitro effects of addition of rhmbl, and blocking mr with a specific antibody, on am phagocytic ability were assessed. in vitro effects of azithromycin on am expression of mr were also investigated. (3) azithromycin (250 mg orally 2¥ weekly/12 weeks) was administered to 11 copd subjects. bronchoscopies were performed prior to and 12 weeks following therapy. ex vivo assessments included am phagocytic ability, levels of mbl, sp-d and mr and apoptosis of bronchial epithelial cells. results am mr expression and levels of mbl and sp-d were significantly reduced in copd subjects vs controls. azithomycin (500 ng/ml) increased mr expression by 31% in vitro. rhmbl induced a dose-dependent increase in am phagocytic ability (up to 148%). blocking mr significantly decreased am phagocytic ability by 60%. in copd patients following azithromycin therapy, we observed improved am phagcocytic ability, increased levels of mr and reduced levels of bronchial epithelial cell apoptosis. conclusions these findings strongly implicate the mr in both the defective phagocytic function of am in copd and as a target for the azithromycinmediated improvement in phagocytic ability. obstructive sleep apnea (osa) is associated with hypoxia and increased cardiovascular morbidity. t cells and monocytes play a significant role in atherogenesis via cytokine production. there have been reports of benefits of continuous positive airway pressure (cpap) therapy in osa. the purpose of this study was to characterize leucocyte inflammatory cytokine/chemokine production by t cells and monocytes in a group of osa patients and to investigate the therapeutic effects of cpap therapy. methods a comprehensive range of intracellular t-cell and monocyte proand anti-inflammatory cytokines/chemokines was investigated in peripheral blood from 5 osa patients and 5 aged-matched control subjects (with no evidence of sleep problems) using multiparameter flow cytometry. osa patients were again studied following 7 days of cpap therapy. results in osa patients there was an increase in intracellular t-cell ifng and tnfa production but no change in il-2, il-4 or tgfb compared with control. there was an increase in intracellular monocyte il-1a, il-8, tnfa, mcp-1 and mcp-3 in osa patients but no change in il-10 or il-12. following cpap therapy, t-cell ifng and tnfa production returned to 'normal' levels. however, although intracellular monocyte cytokine/chemokine production was decreased following cpap, levels were significantly elevated compared with control. conclusions osa is associated with increased intracellular proinflammatory cytokine/chemokines, many of which are increased in atherosclerotic plaques. although one week of cpap therapy resulted in amelioration of t-cell pro-inflammatory cytokines, longer cpap use or alternative therapy may be required to reduce monocyte pro-inflammatory mediators associated with atherosclerosis in patients with osa. gp130 has been associated with the progression of fibrosis especially in patients with idiopathic pulmonary fibrosis (ipf). gp130 is the common subunit of the receptor complexes for the il-6 family of cytokines including il-11 and oncostatin m (osm), where gp130-mediated signalling leads to activation of the erk or stat pathways. we have previously demonstrated exaggerated gp130-stat signalling to be fundamental to the development of pulmonary fibrosis in a murine model of bleomycin-induced lung fibrosis. the aim of this study was to elucidate the role of the il-6 cytokine family in the development of pulmonary fibrosis by identifying which il-6 family cytokines regulate fibrosis in bleomycin treated mice, and determine the effects of these cytokines on cell function. bleomycin (0.05 u/mouse) or control saline was administered intranasally to wildtype mice (wt), genetically engineered mice containing point mutations to prevent gp130 erk signalling (gp130 757f ) or gp130 stat signalling (gp130 dstat ), and duel il-6 and il-11 a-receptor knockout mice (il-6 -/-;il-11ar -/-). the effect of bleomycin on collagen production was examined in lung tissue 30 days post treatment by hplc. there was a significant increase in collagen levels in bleomycin treated wt lungs which was further increased in gp130 757f lungs. the lungs of gp130 dstat and il-6 -/-;il-11ar -/mice were protected from fibrosis suggesting that gp130-stat signalling is important in inducing lung fibrosis which may be mediated through il-6 and/or il-11. cell proliferation was examined in lung fibroblasts isolated from wt, gp130 dstat and gp130 757f mice. il-6, il-11 and osm were significantly mitogenic for gp130 dstat cells but not for wt or gp130 757f cells, reflecting different responses to the different signalling pathways. changes in cytokine profiles are currently being examined in lung tissue and serum of control and bleomycin treated mice 0-30 days after treatment. in conclusion, il-6 and il-11 are likely to play a role in bleomycin-induced fibrosis via the gp130-stat-mediated pathway, however this may not be due to regulation of proliferation induced by these cytokines. supported by the nhmrc. mimicking viral infection by application of various toll-like receptor ligands has shown clinical promise in the treatment of persistent viral infections and more recently with malignant tumours. commercially available toll-like receptor 7 ligands (tlr7l), such as those of the imidazoquinoline family have been applied clinically for the treatment of a number of conditions including basal cell carcinoma and hpv-induced genital warts. these compounds are known to retard tumour growth indirectly by promoting activation and migration of dcs, leading to a strong th1 cellular response, and directly via release of proinflammatory cytokines and promotion of tumour cell apoptosis. malignant mesothelioma (mm), an aggressive tumour with a mean survival of 9 months, is highly resistant to chemotherapy, radiotherapy and surgery and is therefore an interesting candidate for immunotherapy in the form of tlr7 ligand treatment. whilst tlr7 is known to be selectively expressed in immune cells and its relative expression low amongst other cell and tissue types in mammals, its expression on tumour cells and the consequences of such expression on tumour growth are unknown. here we describe the presence of tlr7 (mrna and protein) directly in a range of different tumours, including several murine and human mm cell lines. reactive oxygen species (ros) produced during the innate immune response are important agents of anti-pathogen defense but may also cause oxidative lung damage. glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. methods wild-type (wt) or mice deficient in glutathione peroxidase-1 (gpx-1 -/-) were placed in a perspex chamber and exposed to cigarette (cig) smoke generated from 9 cigs per day for 4 days. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for proteomic and genomic analysis. results wt mice exposed to cig smoke for 4 days had significantly more macrophages (3.1 ϯ 0.1(sem) ¥ 10 5 ) and neutrophils (4.9 ϯ 0.4 ¥ 10 5 ) than sham-exposed mice (2.2 ϯ 0.2 ¥ 10 5 and 0, respectively) (n = 6, p < 0.05). however, gpx-1mice exposed to cig smoke had significantly greater macrophages (5.4 ϯ 0.3 ¥ 10 5 ) and neutrophils (1.2 ϯ 0.1 ¥ 10 6 ) than smokeexposed wt mice (n = 6, p < 0.001). macrophage and neutrophil numbers in sham-exposed gpx-1 -/mice (1.7 ϯ 0.3 ¥ 10 5 and 0.5 ϯ 0.4 ¥ 10 3 ) were similar to those of sham-exposed wt mice (2.2 ϯ 0.2 ¥ 10 5 and 0). in addition, we found that balf of gpx1 -/mice exposed to cig smoke had an increased proteolytic burden compared with smoke-exposed wt mice as assessed by zymography and net gelatinase activity assay. conclusions these data suggest that gpx-1 protects the lung from cigarette smoke-induced inflammation and that targeting gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role. funded by nhmrc. the becs from subjects with chronic obstructive pulmonary disease (copd) are exposed to frequent infectious and inflammatory stimuli. infection with rv is known to trigger acute exacerbations and subjects with copd are particularly susceptible. we hypothesized that exposure of copd becs to these stimuli would alter their response to rv infection. methods bec were obtained by endobronchial brushing from subjects with gold stage 3 copd (n = 4, all ex-smokers), subjects with mild persistent asthma (n = 4) and healthy controls (hc, n = 4). becs were cultured and then treated with tumour necrosis factor (tnf)a 10 ng/ml or lps 100 mg/ml for 24 hrs and then infected with rv-43, rv-1b. response was measured by release of il-8, il-6 and ip-10 mrna and by elisa. virus replication measured by cell titration assay. results infection with both rv strains led to increased release of il-8 and ip-10 in all groups. exposure of hc and asthma becs to both lps and tnf led to increased release of il-8. in these becs there was no increase in release of il-8 exposed to lps and tnf and then infected with either rv. becs from subjects with copd released significantly less il-8 in response to all conditions and rv infection compared to hcs and asthma. no differences were seen in rv replication. the aim of this study was to determine opinions and attitudes to exercise from chronic obstructive pulmonary disease (copd) subjects after completion of a 12-month maintenance exercise program. methods following completion of a 12-month exercise study, which included a supervised program (intervention, n = 18) and control group (control, n = 17), copd subjects [mean age (sd): 66 (8); mean fev1 (% predicted) = 56% (19)] were asked to complete a questionnaire. the questionnaire included closedended questions using visual analogue scales (100 mm). in copd the 6 minute walk distance (6mwd) is known to increase with test repetition (familiarization) and in response to exercise training. it is unknown whether the magnitudes of these increases are related to the degree of disability of the individual patient. methods 6mwd was measured twice before and once after an 8 week out-patient exercise program in 121 patients (82 males) aged 67ϯ8.6 yrs, fev1 37ϯ15% predicted (meanϯsd) with stable copd. the changes in 6mwd following a familiarization test and following training were compared between patients grouped according to their degree of disability (defined as the pre-training 6mwd [best of 2 tests] expressed as %predicted 6mwd). *p < 0.05 gp 3 vs gp 1. conclusions before training, 6mwd increases following a familiarization test irrespective of the level of disability. the magnitude of this increase is similar in all groups when normalized for their pre-training 6mwd. following training, the increase in 6mwd is greatest in patients with the greatest disability (lowest pre-training 6mwd). in less disabled patients, the relatively smaller increase in 6mwd following training may reflect an inability to further increase stride length, thereby reducing the responsiveness of the 6mwt in this group. supported by nhmrc. endotoxin is a stimulant of the innate immune system and is a major component of cigarette smoke. smokers have evidence of increased airway neutrophils and inflammation. we hypothesized that endotoxin levels would be higher in the bronchial lavage (bl) of subjects who were former smokers and subjects with chronic obstructive pulmonary disease (copd). methods subjects were all ex-smokers for at least 5 years (n = 10, 5 copd, 5 healthy controls) or never smokers (n = 12, 6 asthma, 6 healthy controls). bl was collected and analysed for cell count and differential, culture for microbiology. the supernatant was analysed for il-8 by elisa and endotoxin by quantitative kinetic lal assay. results median endotoxin levels were significantly higher in ex-smokers 101 compared to never smokers 6.3 u/ml (p < 0.001). there were no differences between subjects with copd and hs. subjects with copd had higher median endotoxin levels (80 u/ml), compared to asthma (5.2 u/ml) and hc (6.3 u/ml, p = 0.03). there was no correlation between endotoxin levels and bl total cell count, neutrophils (%) or fev1 % predicted. there was a strong correlation with previous packet years smoked and endotoxin levels (r = 0.72, p < 0.01). conclusions bl endotoxin levels are higher in ex-smokers, including subjects with copd. despite this there is no relationship to increased neutrophilic inflammation. copd is associated with inflammation associated with ineffective repair of the injured epithelium and loss of structural integrity. we have shown that these changes may result from dysregulated 'efferocytosis' (increased apoptosis of bronchial epithelial cells and defective clearance of these cells by alveolar macrophages (am)). we have also reported that azithromycin, at subbactericidal dose, improved am phagocytic function ex vivo. methods we administered azithromycin at low dose (250 mg/ twice weekly for 12 weeks) to 10 copd subjects (7 male, age: 62 ϯ 8 yr, 5 current/ 5 ex-smokers, fev1: 63 ϯ 9% pred, fev1/fvc: 48 ϯ 9%). the study was openlabel, uncontrolled and primarily focused on objective biological responses obtained from the bronchoscopy samples taken. phagocytic ability of am (from bal), apoptosis of bronchial epithelial cells (from bronchial brushing), markers of inflammation in blood, bal and breath condensate (crp, wcc and inflammatory cytokines), health status (st. george's respiratory questionnaire), ecg and lung function were assessed pre and post-administration of azithromycin. results azithromycin significantly improved phagocytic ability of am (by 37%) and reduced bronchial epithelial cell apoptosis (by 34%). antiinflammatory effects of azithromycin included significantly reduced blood wcc and crp. there were non-significant reductions in levels of pro-inflammatory cytokines il-8, il-6 and tnf-a in blood, bal and breath condensate, and a trend for improved health status. conclusions our findings indicate a novel approach to supplement existing therapies in copd that may improve clearance of accumulated apoptotic material and reduce the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation. background the prevalence of gastro-oesophageal reflux disease (gord) across the disease spectrum in copd and bronchiectasis is not well described. the aim of this study was to determine the prevalence of symptomatic and silent gord in copd and bronchiectasis and its effect on lung function and quality of life (qol 4] ) and 18 healthy controls were recruited. the prevalence of gord in bronchiectasis was 33%; 37% in copd; 17% in controls. in copd and bronchiectasis, total nre and ri were increased in those with distal and proximal gord compared to those without gord (all p < 0.05). there was no difference in extent or severity of bronchiectasis in patients with or without gord (all p > 0.05). in copd, the relationship between proximal gord and fev1 was small to moderate (r = 0.383). sgrq symptom scores were higher in patients with bronchiectasis with increased ri (p = 0.02). increased proximal nre was associated with reduced physical (p = 0.03) and mental health (p = 0.02) in the sf-36 in copd. conclusions gord is a co-morbidity in patients with copd and bronchiectasis. the impact of gord on disease severity requires further evaluation. funding source nhmrc, the university of melbourne, monash university, physiotherapy research foundation. chronic obstructive pulmonary disease (copd) is prevalent among older people, however little is known about the influence of ageing on airway inflammation. the aim of this study was to compare airway inflammation in older people with obstructive airway disease to groups of older and younger healthy controls. methods participants (>55 years of age) with stable airway disease and incomplete reversibility (fev1% predicted <80% and fev1/fvc < 70%; copd n = 71) and healthy controls (n = 45, 35 older >55 years and 10 younger <55 years) were recruited from the respiratory ambulatory care clinic or by advertisement. participants underwent a clinical assessment, skin allergy test, hypertonic saline challenge, sputum induction and gas diffusion studies. results participants with copd had moderate airflow obstruction (mean (sd) fev1% predicted 56 (19)) and 45 (63%) were current or ex-smokers with a median (iqr) pack year history of 36 (20-54) pack years. ageing was associated with an increase in airway neutrophils (p = 0.0001). compared to older controls, participants with copd had increased airway eosinophils and lymphopenia (p = 0.004, p = 0.003 respectively), but no difference in airway neutrophils. conclusion airway neutrophilia is a feature of ageing and is not further increased in the presence of copd. copd is associated increased numbers of airway eosinophils with reduced lymphocytes which may impact on the ability of the immune system to combat infection. supported by nhmrc, the university of newcastle. chronic obstructive pulmonary disease (copd) is third leading cause of death and fourth leading cause of disease burden in australia. mechanisms involved in emphysema severity have not been fully understood. micrornas are noncoding rnas that regulate gene expression. we hypothesize that microrna expression differs between emphysema severity in copd patients. methods mirna profiling was performed using 15k agilent human oligo mirna microarrays on total rna extracted from non-tumour lung tissue from 30 copd patients undergoing resection for lung cancer. the mirnas were quantile normalized and anova was used to find differentially expressed genes. results demographic characteristics of the copd patients (mean (sd)) were age 69 (6) years, fev1 72 (17) % predicted and fev1/fvc ratio (<70%). anova identified 31 mirnas that were differentially expressed when stratified into two classes according to kco % predicted > or <75% (t-test, p < 0.05). discussion this mirna analysis has identified mirnas that may be important in emphysema severity in copd patients. further validation will be performed using qrt-pcr and mirna assays on the training set and an independent set, and target prediction and validation. t-helper type 1 (th1) and type 2 (th2) lymphocyte responses have been well recognized as being important pathways in inflammation. recently another form of inflammatory lymphocyte response has been described, the th17 pathway. th17 cells produce cytokines such as il-17a to clear extra-cellular bacteria and fungi and have been implicated in autoimmune and chronic inflammatory diseases. the th17 response in copd is unknown. methods subjects were patients with copd (ex-smokers, fev1 < 70% predicted who had not had an exacerbation for at least 1 month) and control subjects (ex-smokers and normal spirometry). serum samples were obtained for measurement of c reactive protein (crp) and il-17a, the latter measured using enzyme-linked immunosorbent assay (elisa). production of il-17a by t-cell subsets was also identified by intra-cellular cytokine staining and measured by flow cytometry. the mean fev1 of copd subjects was 42 % predicted (6.1 sem, n = 6) and mean fev1 of controls was 112 % predicted (3.0 sem, n = 4). the copd group had a higher mean level of crp 9.5 mg/l (3.9 sem) compared to the control group mean level of 4.6 mg/l (0.6 sem). the mean level of the il-17 in the copd group as measured by elisa was 22.3 pg/ml (16.9 sem, range 0-87) whilst no il-17 was measured in any of the control subjects. conclusions the findings of this pilot study suggest that il-17 may be elevated in association with crp in stable copd. airway obstruction is defined as a fev1/fvc ratio below the lower limit of normal. airway obstruction may prolong the forced expiratory time (fet). method spirometry results from 467 patients were categorized as obstructive, restrictive or normal. the mean, range and coefficient of variation were determined for fet in each diagnostic group. receiver operator characteristic (roc) curves were used to determine if fet could predict a low fev1/fvc. the number of patients with airway obstruction in five fet groups: <9; 9; 10-12; 13-14; and >14 seconds were determined. results the coefficient of variation was high for all groups. pair-wise comparisons showed a difference in mean fet between patients with normal lung function versus those with airway obstruction (p < 0.001). the best cut-point in the roc analysis of 9.895 seconds had a sensitivity of 0.66, specificity 0.77 and area under the curve of 0.743 for predicting obstruction. the technique of skeletal muscle microbiopsy has previously been validated [1] and shown to be minimally invasive and well tolerated in participants with stable copd. aim a study was undertaken to determine the feasibility and tolerability of obtaining microbiopsy muscle samples from the patient admitted for acute exacerbation of copd patient. methods written informed consent was obtained to collect the muscle, blood and sputum samples for research purposes. local anaesthetic was injected prior to the insertion of a 16 gauge bard max core disposable biopsy instrument through the associated guide needle. multiple passes (up to 6) were obtained. the patient was asked to evaluate the experience by rating it on the modified borg scale 0-10. results to date 5 patients and 3 controls have participated in this study. the gold severity ranged from 2-4 and ats exacerbation severity 2-3. the mean age 75 years (range 68-83 years), bmi mean 23.6 kg m -2 (range 17.2-27.1 kg m -2 ) and fat free mass was determined using single frequency bioimpedance. the sample mass obtained ranged from 27.2-104.1 mg, with an increasing yield occurring with increased experience of the operator. the procedure has been well tolerated, the borg scale rating ranged from 1-2/10. all patients were ambulant post procedure; no haematoma or bruising was observed in any of the subjects. conclusion the microbiopsy technique allows the collection of muscle tissue with minimal discomfort to the participant. small tissue masses such as these are sufficient to obtain measures of local markers of wasting and may prove to be a useful adjunct to the collection of sputum and blood for the measure of biomarkers in copd research. introduction older people (op) with obstructive airways disease (oad) experience multiple problems that may impact on their quality of life (qol) and disease management. these problems may relate to pathophysiology, symptoms, self management skills, psychological issues, lifestyle or other problems identified as important by the patient. aim the aim of this study was to determine the frequency of clinical problems associated with oad and to determine if a problem based assessment (pba) could adequately identify these problems. methods a multidimensional assessment tool was developed and the content compared to clinical practice guidelines. participants over 55 years with diagnosed oad underwent this assessment. results sixty-one consecutive patients, aged 59-87 years, with mean (sd) fev1 of 51.4 (17.85) % predicted were assessed. the assessment tool identified a mean (sd) of 3.03 (2.13) current and significant co morbidities with an additional 11 (3.37) clinical problems per patient. qol was increasingly impaired with an increasing number of problems (p < 0.0001). regression modelling identified that the number of identified clinical problems accounted for 55% of the qol impairment. the model demonstrated that every additional patient problem was associated with a clinically significant change in qol impairment (4.22 units) . conclusions op with oad experience multiple clinical problems and co morbidities that adversely impact their qol. a pba of op with oad identifies significant problems that may not be addressed in a diagnosis centred approach. there is a need to identify and effectively manage this array of problems in clinical practice. discussion in this diverse group of copd patients, there was a positive correlation between dlco and fev1, but not kco and fev1. the fev1/ kco plot identifies substantial numbers of patients with the potential ad and e phenotypes defined above. we intend to study inflammatory biomarkers in these groups. fat free mass index (ffmi) is a marker of morbidity and mortality in copd. measurement of ffm in the out-patient population is commonly undertaken using single frequency bioelectrical impedance analysis (bia). however the formulae to convert measured values to ffm are population dependent. schols et al (am j clin nutr, 1991) suggested that formula used for the general population may be inappropriate for patients with copd, and derived a specific formula from total body water (tbw) as measured by deuterium dilution. we compare this method of measuring ffm with 5 others, along with tbw and ffm hydration. methods tbw was measured in 31 outpatients with copd by bia and a difference method (weight-(protein+bone mineral+fat+non-bone mineral+ glycogen)) and ffm hydration was calculated. ffmi was measured by skin fold anthropometry (sfa), bia (3 separate formulae), dual energy x-ray absorptiometry (dexa) and total body potassium by g-counter (tbk). comparison between methods for tbw and ffmi was made by bland-altman analysis and between methods of calculation of ffm hydration by paired t-test. the two methods of assessment of tbw showed little difference (bias -0.04, 95% limits of agreement -5.40 to 5.31). however there was a significant difference in calculation of hydration of ffm (p = 0.0001). sfa, bia (lukaski), bia (tanita) and tbk underestimated ffmi when compared to bia (schols), with bias of -1.24, -3.87, -1.06 and -2.76 respectively. dexa however had a bias of only 0.05 and 95% loa of -3.09 to 3.21. conclusions there are differences between methods of assessment of tbw and ffmi and comparing values between methods must be done with caution. this has implications for assessment of morbidity and mortality in copd. chronic obstructive pulmonary disease (copd) has been identified as a major health problem in australia. recent studies have suggested that respiratory viral infections are the major cause of a worsening of copd; however this has not been studied in australia. aim to characterize pef changes and identify viruses during copd exacerbations. methods a pilot prospective longitudinal cohort study was done. patients had confirmed copd with fev1 <70% predicted and reversibility <10% and/or 200 ml. patients recorded daily peak expiratory flow (pef) measurements and daily chest and cold scores over a period of 2 years. sputum samples and nasal aspirates were taken at 6-month review (control visit) and whenever they had symptoms of an exacerbation (worsening of copd symptoms -seemungal et. al. am j resp crit care med, 2001). nasal aspirates and sputum samples were obtained and analysed by rt-pcr for rhinovirus (rv). result five patients have finished 2 years of study. a total of 12 exacerbations were reported based on patient symptoms. only 3 exacerbations were associated with significant reductions in pef and only one was linked to increases in nasal cold scores. all samples taken at control visits and nasal aspirates and sputum samples during exacerbations were negative for rv by rt-pcr. positive controls confirmed the accuracy of the assay. conclusion our data suggest that a symptom-based definition of copd exacerbation is not always accompanied by significant reductions in lung function parameters. these 'exacerbations' are also not associated with the commonest reported viral cause. our findings suggest that variability of copd may mimic. bronchiectasis is characterized by hypersecretion of mucus and impaired clearance that results in mucus accumulation, chronic cough, sputum production and recurrent infections. inhaled mannitol (400 mg) improves clearance of mucus by increasing the airway hydration and by reducing the viscoelastic and surface properties of mucus. however, the effect of other doses of mannitol on the clearance of mucus in patients with bronchiectasis is unknown. methods fourteen patients, age: 63.3 ϯ 5.7 yr, were studied on 5 visits. clearance of mucus was measured using 99m tc-sulphur colloid and imaging with a gamma camera at baseline and with mannitol ( weight loss and skeletal muscle atrophy are major determinants of morbidity in chronic obstructive pulmonary disease (copd), which are independent of lung function impairment. thus, we examined if a high-fat diet (hfd) protected against the wasting associated with prolonged cigarette smoke exposure (se) in mice. methods male balb/c mice were exposed to the smoke of 4 cigarettes/day, 6 days/week for 7 weeks. sham mice were handled identically without smoke exposure. mice consumed either standard laboratory chow (3.5 kcal/g, consisting of 12 % fat) or a hfd (4.3 kcal/g, 32% consisting of fat). we examined the effect of se and hfd on hind limb skeletal muscles, lung (tissue & bronchoalveolar lavage (balf)) and systemic inflammation in the 4 groups of mice (n = 8/ group). results after 7 weeks of hfd, sham and se mice were 12 and 13% heavier (respectively, p < 0.05) than chow fed animals. conversely, se significantly decreased body weight of chow and hfd fed mice by 16 and 15%, respectively, compared to sham animals (p < 0.05). the hfd did not protect against the decrease in soleus, tibialis anterior and gastrocnemius skeletal muscle weights induced by se (p < 0.05). se altered the mrna expression of a number of genes associated with the regulation of skeletal muscle mass including insulin-like growth factor-i (igf-i), atrogin-1 and interleukin (il)-6. the mrna expression of pro-inflammatory cytokines and chemokines was significantly increased by se in the lung, as were the number of inflammatory cells in balf (p < 0.05). on the other hand, although obesity has been linked to systemic inflammation, the hfd exerted little direct effect on the skeletal muscle and lung parameters measured. se and hfd had no effect on two markers of systemic inflammation, il-6 and serum amyloid a, whereas se tended to reduce circulating igf-i, an anabolic hormone. conclusions the hfd was not protective against the weight loss and skeletal muscle wasting associated with cigarette smoke exposure. supported by the nhmrc and crc for chronic inflammatory diseases. background patients with copd and bronchiectasis undertake airway clearance therapy (act) and exercise as part of physiotherapy management but it is unknown whether these treatments provoke gastro-oesophageal reflux (gor). this study aimed to determine the impact of positive expiratory pressure (pep) therapy and exercise on gastro-oesophageal function. p. aeruginosa is a significant opportunistic lung pathogen in individuals with cystic fibrosis (cf) and is associated with increased lung disease and morbidity. early intervention is beneficial for the effective clearance of p. aeruginosa and better long-term health outcomes. currently, lung flora of cf patients is monitored by regular culturing of sputum, however, children unable to expectorate are limited to annual bronchoalveolar lavages (bal), which is invasive and requires general anaesthesia. saliva is useful for clinical assays as collection is simple, non-invasive. we are developing a standardized enzymelinked immunosorbent assay (elisa) to detect respiratory infection of p. aeruginosa in cf children who cannot expectorate. methods 18 children (7-18 years) with cf and recent p. aeruginosa lung infection history and 16 non cf children (1-6 years) with no previous p. aeruginosa infection history provided saliva as positive, negative controls respectively. saliva was obtained by spitting, or absorbed using cellulose swabs and later extracted. these cell-free supernatant samples were used in an elisa anti-p. aeruginosa iga using commercial antigen. all results were standardized to account for flow using total iga expression. results median value was increased 9 fold in the recent p. aeruginosa lung infection group (mann-whitney test, n = 34, p յ 0.001). there was no significance between mucoid and non mucoid samples, and detection was independent of cfu/ml. discussion early findings support that p. aeruginosa respiratory infection can be detected through specific analysis of salivary iga expression. larger population sampling (30 positive, 90 negative) will aid selection of cut-off values for specificity and sensitivity testing in the future to objectively determine the utility of this assay as a means of monitoring for p. aeruginosa and for determining effectiveness of treatment. medical thoracoscopy is utilized widely throughout europe and northern america by thoracic physicians for the management of pleural disease, including the undiagnosed pleural effusion, malignant effusions and less commonly pneumothorax (ptx). australia has limited experience in this modality. we report the success of medical thoracoscopy in both primary and secondary ptx requiring intervention. methods data were collected from 2001 to 2007 in patients treated with medical thoracoscopy for the treatment of ptx. results 11 patients, 7 male, 4 female. average age 48 (range 19-86). 1 first episode primary spontaneous (ps) ptx, 2 third episodes of ps, 5 first secondary spontaneous (ss), 1 second ssptx, 2 third ssptx. underlying pulmonary disease in secondary ptx included: 4 chronic obstructive pulmonary disease, 1 lymphangioleiomyomatosis, 1 mesothelioma, 1 metastatic angiosarcoma and 1 was secondary to a motor vehicle accident. 7 had a history of smoking, 5 were former smokers and 2 were current smokers, with a mean 24 pack year history (range 5-45). 7 ptx were large, 4 moderate. 5 patients had an intercostal catheter (icc) inserted prior to thoracoscopy, 1 had failed pleural aspirate. there was evidence of bronchopleural fistula in 7 patients prior to the procedure. there was a median of 9 days from ptx to thoracoscopy. light sedation was used for the procedure in 10 patients, 1 required a general anaesthesia with a double lumen endotracheal tube due to anxiety. single port entry, dry talc poudrage and a 16 gauge french icc was used for all procedures. icc was removed a mean of 2 days following thoracoscopy and patients discharged on day 4. pain was the most common complication, requiring narcotic analgesia. one patient died on day 7, secondary to metastatic angiosarcoma. there has been no recurrence of ptx in any patient. conclusion medical thoracoscopy, performed by thoracic physicians is an effective procedure for the treatment of pneumothorax requiring intervention, including selected patients with evidence of bronchopleural fistula. funding nil. conflict of interest nil. nomination for young investigator award no. background lung cancer incidence and mortality are high in tasmania. australia (aihw 2003) 85/100 000 72/100 000 tasmania (cancer registry 2003) 102/100 000 89/100 000 aims and objectives (a) to determine patient demographics in southern tasmania, (b) to determine compliance to identified measures of best practice and (c) assess referral rates, clinical utility and potential delay to positron emission tomography (pet) in a regional setting. methods a prospective database collected information on local clinical practice. cases presented at a multidisciplinary lung cancer meeting over a 12 month period (march 2006 -april 2007 were analysed. data were available for n = 121/161 (75%). results are shown as mean ϯ sd. results 113 primary lung cancer cases were identified. the mean age was 71 ϯ 11 years. 58% of patients were male and 95% were current or ex-smokers. 81% were non-small cell lung cancers (nsclc). tissue diagnosis 93% time from diagnosis to surgery (27 ϯ 15 days) 82% < 42 days macroscopically complete surgical resection (9/11) 82% pet for stage iiib before radical chemoradiotherapy 75% 62% of patients presenting with early or locally advanced disease underwent further staging with pet (n = 34/55). management was changed in 50% of cases (17/34). the average time from pet referral to scan was 11 ϯ 5 days. conclusion a disproportionate number of lung cancers occurred in women. although surgery was performed within recognized timeframes, 2 of 11 patients had incomplete resections. pet influenced management decisions and was performed in a timely fashion. hp chan 1,2 , v tran 1,2 , c lewis 1,3 , p thomas exhaled breath condensate (ebc) is a simple, safe and non-invasive method of sampling breath and has the potential to investigate lung cancer and the associated neoplastic process in the lungs. increased oxidative stress has been implicated in the pathogenesis of lung cancer, and is characterized by elevated hydrogen ions, and hydrogen peroxide (h2o2), which is formed from the conversion of superoxide anions by superoxide dismutase. airway ph has already been shown to be decreased in ebc of patients with other respiratory conditions, but not in lung cancer. therefore the concentration of h2o2 and hydrogen ions in the ebc of lung cancer subjects was compared with matched controls. methods six subjects with newly diagnosed lung cancer were recruited and matched with control subjects: non-smokers, ex-smokers and smokers. ebc was collected and h2o2 was then measured by an assay method based on oxidation of 3,3',5,5'-tetramethybenzidine by horseradish peroxidase and h2o2 while ph was measured using a ph meter. results there was a significant difference (p = 0.033, anova) in h2o2 concentration between the 4 groups with the lung cancer group having elevated mean h2o2 concentration of 23.68 mm (9.15 (sem) compared to the controls: non-smokers, 17.59 mm (6.53 (sem); ex-smokers, 14.35 mm (3.79 (sem); and smokers, 5.21mm (0.69 (sem). ph did not differ significantly (p = 0.659, kruskal-wallis test) between the groups. conclusion these preliminary data suggest that there is significant difference in h2o2 concentration between the groups. the demonstration of an elevated h2o2 level in those with lung cancer indicates an increase in oxidative stress which implies that this may be part of the pathogenesis or response to neoplasia. supported by none. conflict of interest none. pro-inflammatory th1 cytokines produced by t cells and monocytes play an important role in the immune response to malignant cells. however, tumours may escape immune surveillance by inhibiting th1 response and promoting chronic inflammation at the tumour site. methods to investigate the effect of soluble factors released by lung cancer cells on t cell and monocyte pro-and anti-inflammatory cytokines, culture supernatants from several lung cancer cell lines and a normal epithelial cell line (16hbe) were cultured with whole blood for 24 hours, then for a further 16 hrs with and without stimuli. intracellular cytokine / chemokine production was determined using multiparameter flow cytometry. results in stimulated cultures, there was a significant decrease in t cell th1 pro-inflammatory cytokines ifng, tnfa and il-2 and a decrease in monocyte il-1a, il-8, il-12, tnfa, mcp-1 and mcp-3 but an increase in antiinflammatory cytokine il-10 compared with 16hbe and control media. in non-stimulated blood cultures there was an increase in all monocyte inflammatory cytokines / chemokines in the presence of lung cancer supernatants. conclusions lung cancers secrete soluble factors that inhibit the antitumour pro-inflammatory th1 response by t cells and monocytes and upregulate monocyte anti-inflammatory cytokine il-10 following "antigenic challenge". lung cancer cells may also escape immune surveillance by secreting soluble factors that cause newly recruited monocytes to release inflammatory cytokines promoting chronic inflammation at the tumour site. cytotoxic t-cells (ctl's) are important barriers against tumour cells. ctl's induce apoptosis of target cells by mechanisms that include the release of pore-forming perforin and granule associated enzymes, such as granzyme b and granulysin. proteinase inhibitor-9 (pi-9) is the only known granzyme b inhibitor and its expression has been observed in some cancers. we hypothesized that pi-9 would be differentially expressed in lung cancer cells and may inhibit granzyme b-induced apoptosis in these cells. methods we investigated pi-9, granulysin and granzyme b expression in various lung cancer cell lines (1299 ( , 1466 ( , 2009 and normal epithelial cells obtained from bronchial brushing using flow cytometry. peripheral bloodderived t-cells were then incubated with lung cancer cell line supernatants and levels of pi-9, granzyme b and t-cell reactive oxygen species (ros) were assessed. results pi-9 expression was detected in all lung cancer cell lines, (1299 (54.2%), 1466 (90.2%), 2009 (85%), sbc-1 (81%)), at much higher levels than in normal bronchial epithelial cells (8.5%). granzyme b and granulysin levels were undetectable or low in cancer cells (0-9.2%). increased expression of pi-9 and reduced levels of granzyme b were observed in cd8+ t-cells in the presence of all cancer cell supernatants tested (p < 0.05). interestingly, t-cell ros levels were significantly increased in cd8+ t-cells after incubation with cancer cell supernatants (p < 0.05). conclusions high pi-9 expression in lung cancer cells combined with a reduction in t-cell granzyme b expression and enhanced intracellular t-cell ros levels may be a mechanism of immune evasion of lung cancer cells to granzyme b-induced cytotoxicity. immunotherapy for lung malignancies such as lung cancer and mesothelioma is most likely to be successful it it can be combined with conventional tumour debulking approaches such as chemotherapy and surgery. but they scientific basis of such combinations is yet to be determined. to study this we evaluated (1) the capacity of different lung chemotherapy drugs to alter tumour antigen cross-presentation and immunogencity, (2) duration of antigen presentation and responsiveness to immunotherapy after debulking surgery with/without lymphadenectomy, and (3) the pattern of tlr agonism which best synergized with chemotherapy and surgery. we used the ab1-ha murine model of lung malignancy in balb/c mice. results (1) the antimetabolite drugs gemcitabine and pemetrexed were most immunogenic compared to the cytotoxic antibiotics doxorubicin and mitomycin c and the alkylating agent cisplatin. gemcitabine delived large amounts of tumour antigen into the cross-presentation pathway. (2) tumour antigen cross-presentation persisted for only 10 days following resection. the optimal window for immunotherapy following cancer surgery is 1 week for effector ctl stimulation and 2-4 weeks for memory ctl stimulation. (3) the viral-like tlr agonists tlr 3, 7 and 9 were the most effective adjuvant tlr molecules, with tlr 7 agonists generating the strongest systemic anti-tumour responses. conclusion these results help explain previous lung immunotherapy failures and will inform new clinical trials. background mesothelioma is a highly aggressive tumour with an increasing world wide incidence. the serum biomarker mesothelin is elevated in some individuals prior to development of clinical symptoms of the disease and may be useful for screening. we therefore studied the sensitivity and specificity of urinary versus serum levels of mesothelin for mesothelioma patients and evaluated the influence if renal function on the biomarker level. materials and methods concurrent sera and urine samples collected from patients with and control populations. mesothelin concentrations were determined by double-determinant elisa using the mesomark tm assay (fdi, pa). their estimated glomerular filtration rate (egfr) was also calculated. results mesothelin levels correlated between serum and urine samples (pearson's correlation 0.791; p < 0.0001). mesothelin levels were significantly higher in patients with mesothelioma compared to those with asbestosis and/or pleural plaques in serum (4 ϯ 0.9 versus 0.9 ϯ 0.05 nm; p < 0.0001, respectively), in urine (1.9 ϯ 0.5 versus 0.3 ϯ 0.03; p < 0.0001) and in urine following normalization using creatine levels (0.2 ϯ 0.05 versus 0.04 ϯ 0.01). age and egfr were significantly associated with mesothelin levels. conclusion the sensitivity and specificity of mesothelin in urine and in serum were comparable. urine mesothelin may prove to be a useful alternative to serum mesothelin for mass screening of asbestos-exposed individuals. patients undergoing ct coronary angiogram (cta) are often former or current smokers with a high incidence of asymptomatic lung disease. overseas reports show a rate of lung abnormalities ranging from 6.7% to 19%. there are no studies from australia and local factors such as the higher incidence of atypical mycobacteria may influence the rate of benign findings. we are therefore performing a prospective observational study to identify the prevalence and characteristics of incidental lung findings in people undergoing routine cta. methods population: 100 patients undergoing routine cta after informed consent. intervention: radiologist evaluation of lung windows on diagnostic standard workstations. comparator: uncontrolled observational study of consecutive patients. outcomes: primary: prevalence and characteristics of abnormal findings, final diagnosis (clinical judgment, biopsy or long term followup). secondary: number of downstream investigations and costs. results 25 ctas have been studied to date. in 8/25 (32%), abnormalities were noted on lung windows. in 2/25 (8%), there were lung nodules, in 2/25 (8%) there were hilar lymph node abnormalities, in 1/25 (4%), there was hemidiaphragm elevation and in 3/25 (12%) there were pleural plaques (data collection ongoing with study closure expected in february 2008). conclusions preliminary data indicate a substantial number of incidental pulmonary findings from cta; full results will be presented. further analysis is required to determine the impact (benefits, costs and harms) that may result from the concurrent examination of lung windows at routine cta. aim increased levels of nitrogen oxides (nox) and inflammatory markers have been found in bronchoalveolar fluid of lung cancer (lc) patients, but have not been investigated in exhaled breath condensate (ebc).the aim of this study was to compare nox and total protein levels in ebc of lc patients with control subjects. methods ebc was collected during tidal breathing through a glass collection device cooled to 4°c. ebc nox concentrations were measured by a fluorescent modification of the greiss method. total protein in ebc was determined employing the bicinchoninic acid (bca) assay. ebc nox data were log transformed. all data were analysed using anova and expressed as mean ϯ sem. results a total of 88 control subjects and 54 patients with primary lc were recruited. nox and protein concentrations are shown in table 1 . there was no significant difference in ebc nox levels (p > 0.05), but in total protein there was a significant difference between lung cancer patients and all control groups (p = 0.04). conclusion significantly increased ebc total protein levels were found in patients with lung cancer. these data suggest that protein mediator secretion or vascular leak may be present in those with lung cancer. future studies will focus upon the identification of these proteins. methods in this two stage case-control study 446 lung cancer cases and 484 healthy smoker controls were recruited. 180 genetic markers (snps) implicated in lung cancer were screened in our test cohort of 439 smokers and ex-smokers. 30 snps whose genotypes (co-dominant or recessive model) were associated with either the healthy smokers (protective) or lung cancer (susceptibility) phenotype were identified. after genotyping this 30 snp panel in a second cohort of 491 subjects 19 snps were chosen and assigned a simple composite genetic score that was combined with scores for age, history of copd and family history of lung cancer, weighted according to our multivariate regression analysis (n = 930 total subjects). the lung cancer risk score was linearly related to the likelihood of lung cancer with odds ratios (referenced against the lowest score quintile) ranging from 1 to 29 in the highest quintile. on receiver operator curve analyses, the auc was 0.78 and the frequency distribution showed bimodal separation between healthy smokers and lung cancer cases. utility of the score was not affected by effects of age, smoking history or lung function. we suggest that genetic data may be combined with other risk variables to define smokers or ex-smokers at risk of lung cancer for targeted interventions such as smoking cessation and early detection of lung cancer. supported by health research council, nz. conflict of interest yes. tp 144 v aiyappan 1 , a graham 2 1 department of medicine, maroondah hospital, melbourne, australia, and 2 the new disease-modifying anti-rheumatic drug (dmard) leflunomide is being used increasingly to treat inflammatory arthritis. its association with interstitial lung disease needs to be considered before combining it with methotrexate. case report a 73-year-old male who was known to have rheumatoid arthritis and was on methotrexate was admitted with progressive dyspnoea and malaise. he had been recently started on leflunomide. investigations revealed interstitial lung disease and acute renal failure. he improved on conservative treatment (stoppage of disease modifying drugs (dmard), iv fluids and steroids). review of literature an epidemiological study by suissa et al has suggested that there is increased risk of ild associated with leflunomide in patients with a history of ild or methotrexate use but they attributed this to channelling bias. there has also been a report of leflunomide associated with iga glomerulonephritis.by this presentation we aim to increase the awareness of this entity. we also suggest that any patient who is started on combination dmard (i.e. methotrexate and leflunomide) should have a baseline chest x-ray and be monitored for development of interstitial lung disease. conclusion we are reporting the first ever case of interstitial lung disease and glomerulonephritis (in the same patient), due to usage of leflunomide. this entity needs to be thought about in any patient on combination dmards. background bone morphogenic protein receptor ii (bmpr-ii) mutations are associated with pulmonary artery hypertension. failure of the growth inhibitory effects of bmp may contribute to vascular obliteration and remodelling leading to pulmonary artery hypertension (pah) [1] . pah has been observed following venous thrombembolic disease (vte), including pulmonary embolism (pe) and deep venous thrombosis (dvt) [2] . local markers of the pulmonary vascular endothelium rather than traditional markers of thromobophilia are thought to be involved [3] . methods plasma was collected from age and gender matched participants within 24 hours of diagnosis of vte and prior to commencement of warfarin therapy. plasma samples were hybridized to individual human cytokine antibody arrays, to detect protein levels of bmp2, bmp4 and bmpr-ii. results bmp2 and bmp4 levels were higher in patients with dvt than pe. no difference in the bmp level was observed between patients with pe and controls. soluble bmpr-ii receptor was lower in patients with pe than in controls or patients with dvt. conclusion in patients with pulmonary artery stress during the time of a pe the bmpr-ii receptor is reduced, which may predispose patients to vascular remodelling and obliteration. the bmp 2 and 4 levels are reduced at the same time, suggesting a possible overriding regulatory mechanism. the physiological role of bmp's and bmp receptors in patients with vte warrants further investigation. historically, cyclophosphamide has had a variable role in interstitial lung disease (ild), the rationale for its use based on the benefit seen in vasculitis and scleroderma, its rapid effect and low toxicity profile. in patients with severe progressive ild a rapidly effective, well-tolerated agent is desirable. for this reason a treatment protocol for the use of intravenous (iv) cyclophosphamide was implemented at our hospital. aim to review the indications, duration, tolerability and effect of intravenous cyclophosphamide in ild patients following the introduction of a treatment protocol. methods records of 92 patients [dlco was 40 ϯ 15% and fvc 61 ϯ 20%] completing a course of iv cyclophosphamide during 2005-6 were reviewed (excluding patients with systemic sclerosis). data covering 18 months prior to and following treatment were collected. comparative analysis of paired pulmonary function data 6 months before and after treatment was performed. 61% had underlying autoimmune disease. results primary treatment indications included progressive disease(n = 67); severe disease (n = 16); suspected vasculopathy (n = 11); bridging therapy to transplantation (n = 10); and accelerated decline (n = 5). patients received 600 mg/m 2 [mean dose 1152 ϯ 165 mg, median number of pulses 6 (1-12)]. patients with paired pulmonary function data had a difference in median change in dlco% predicted from -15.6% (-95.4 to 29.9%) before treatment to +4.25% (-17.3 to 73.9%) following treatment (p < 0.0001). this remained significant with exclusion of vasculitis, or any autoimmune disease, and independent of prior immunosuppression. therapy was well tolerated (4 withdrew from treatment, 5 deaths within 1yr, none directly related to treatment). conclusion iv cyclophosphamide is well tolerated, and associated with functional stability or improvement in the majority of patients. it remains a viable treatment alternative for consideration. pulmonary hypertension is common in interstitial lung disease (ild) and associated with a poor prognosis. as the gold-standard test, right-heart catheterization (rhc) is invasive, and resource-limited, reliable non-invasive measures of ph are needed. methods all ild patients referred for rhc during 1997-2007 were included (n = 95; 54 male; age 56.5 ϯ 12 yrs). all patients had concurrent echocardiography (tte) and pulmonary function. the relationship of rhc mean pulmonary artery pressure (mpap) to tte variables, pulmonary function, exercise capacity, as measured by six minute walk testing (6mwt, n = 58) and brain natriuretic peptide (bnp, n = 36), was examined. case a 65 year old male, non-smoker for 25 years, retired professor of anatomy (had chronic exposure to embalming fluids, formaldehyde, phenol, antifungal and other solvents, for 20 years) presented with chronic cough and phlegm production. these symptoms were worse at night (waking him several times) and early morning. his pulmonary tests were stopped due to persistent cough. a chest x-ray revealed features of longstanding interstitial lung disease. the hrct revealed widespread subpleural interlobular thickening, worse at bases, in keeping with idiopathic pulmonary fibrosis (ipf). there was minimal fibrosis and honeycombing, but no groundglass opacification, large bullae, pleural calcification or pleural plaques. however, there was associated bronchiectasis at the lung bases considered to be due to traction. the ba lavage showed 50% macrophages, 7% neutrophils, 3% lymphocytes, and 40%, eosinophils and no infection. the patient declined to have a lung biopsy. as per his past x-rays, the duration of his ipf is a little over one year. he maintains that his symptoms started only after starting irbesartan (irb). introduction transbronchial lung biopsy (tbb) has a variable and unpredictable diagnostic yield in sarcoidosis. we hypothesized that the extent and pattern of parenchymal disease on ct would predict the likelihood of a positive tbb. methods data relating to ethnicity, symptoms, pulmonary function and site and results of tbb and bronchoalveolar lavage (bal) from 70 sarcoidosis patients were recorded. all had a ct scan within 6 weeks prior to the tbb procedure. cxr stage was determined from radiology report. ct scans were scored quantitatively for patterns of parenchymal disease (nodular, reticular, consolidation, ground glass and mosaic attenuation) on a lobar basis. results 50% patients had a positive tbb (total 67% of cohort had histological confirmation). symptoms, ethnicity, treatment, lung function and cxr stage were not predictors of a positive biopsy. positive biopsy was associated with higher bal lymphocyte count (p < 0.05) and female gender (p < 0.01). a reticular pattern (p < 0.05) and higher total lung score (excluding da) (p < 0.05) on ct scan predicted a positive biopsy. in those patients with tbb from right lower lobe (53/70) the total rll score on ct was predictive of positive biopsy (p < 0.05). on multivariate analysis gender, bal lymphocytosis and total lung score were independent predictors of a positive tbb (area under roc 0.82). pulmonary arterial hypertension has two histological variants; 'arterial-only pulmonary arterial hypertension' (artpah) and 'pulmonary veno-occlusive disease' (pvod). bosentan, a dual endothelin receptor antagonist, has been found to improve haemodynamics, functional capacity and survival in artpah. however, the response to bosentan in clinically diagnosed artpah is often variable. it was hypothesized that a lack of response to bosentan therapy in clinically diagnosed artpah can be explained by misdiagnosed pvod. aims included to: (1) perform morphometric and qualitative pulmonary vessel analysis on normal controls and cases clinically diagnosed with artpah who had failed bosentan therapy; (2) ascertain if pvod is present within the case group; (3) correlate clinical variables and vessel microanatomy to identify the pathologies driving pulmonary pressure elevation. this study reviewed 14 cases of clinically diagnosed artpah (idiopathic n = 12, associated with scleroderma n = 2), who had failed bosentan therapy and had available lung tissue. controls (n = 6) were obtained from explanted lungs for other causes and a prior transthoracic echocardiogram excluded pulmonary hypertension. vessel morphometry and qualitative analysis was performed with a novel technique of smooth muscle actin immunohistochemistry counterstained with verhoeff's elastin. baseline clinical data were retrieved. we found 86% of cases had pathology confirmed pvod. only 14% of cases had artpah, the original clinical diagnosis. in pvod, significant pathology was present in all vessel types. all vessels had significant smooth muscle hypertrophy. the obstructive, collagenous, pauci-cellular intimal fibrosis of the venules (p < 0.0001) and arterioles (p < 0.0001) was considerably different to the concentric laminar proliferation of smooth muscle observed in the muscular arteries (p < 0.0001) and arterioles (p = 0.001) in artpah. artpah also had muscular artery smooth muscle hypertrophy (p = 0.007). the median time to bosentan failure was shorter in pvod than artpah (290 vs. 657 days). in conclusion, pvod is an under-diagnosed cause of pulmonary hypertension, is commonly clinically misdiagnosed as artpah and may present with a poor bosentan therapy response. finally, pvod is a vaso-occlusive, not a veno-occlusive disease, and is an independent type of pulmonary hypertension, not a subtype of pulmonary arterial hypertension. cutaneous t cell lymphomas (ctcl) are a heterogenous group of lymphoproliferative disorders. they show various clinical manifestations and diverse morphological, histological and immunological characteristics of the malignant cells. they are caused by clonally derived, skin invasive t cells. peripheral t cell lymphomas (ptcl) are generally more aggressive and have one of the lowest overall and failure-free survival rates. because of the rarity of these disorders, diagnosis and treatment remain challenging. this case report describes a 69-year-old woman presenting with progressive dyspnoea and cough, together with a distressing generalized pruritic rash. she was initially treated as left ventricular failure with the rash ascribed to a drug reaction as suggested by initial skin biopsies. the diagnosis was made on a third skin biopsy and flow cytometry of lymphocytes obtained by broncho-alveolar lavage 6 months after presentation. despite an initial response to chemotherapy she succumbed to the disease 20 months after diagnosis. clinical pathways to guide the investigation of suspected pulmonary embolism (pe) have been increasingly adopted by emergency departments (ed) worldwide. compliance with these diagnostic algorithms is critical in ensuring good patient outcomes. this study evaluated the compliance to the clinical pathway used in our ed that combines risk assessment (wells scoring system) with d-dimer test, vq scan or ctpa. the main objectives of this study were to identify those factors which contributed to compliance and to assess patient outcomes. methods a prospective observational study of 239 consecutive patients who underwent investigation for pe in our ed. patient demographics, pathway parameters and patient outcomes at 3-month follow-up were collected. case we report the case of a 37 year old woman who presented to the emergency department with a three day history of dry cough and dyspnoea. the patient was in her third pregnancy at 30 weeks gestation. she had no fever, chest pain or coryzal symptoms. the patient had presented with a right sided spontaneous pneumothorax seven months prior to the current presentation. her past medical history included placental abruption, complicating her previous two pregnancies. her second pregnancy was complicated by placental abruption at 27 weeks and the foetus had not survived. her first pregnancy was complicated by placental abruption at 36 weeks with successful delivery of the foetus. at presentation, significant findings included tachycardia, hypoxemia, tachypnoea and reduced breath sounds over the right side of the chest. chest x-ray demonstrated a large right pneumothorax. a right intercostal catheter was inserted resulting in right lung re-expansion. the catheter was removed three days later. the patient returned to hospital twenty four hours after catheter removal with a recurrent right sided pneumothorax. the patient agreed to surgical intervention involving video-assisted thoracotomy and talc pleurodesis. the patient had no further complications with the pregnancy. she delivered a healthy baby at 38 weeks gestation. discussion spontaneous pneumothorax in pregnancy is rare and there is little evidence to provide guidelines for the management of recurrent pneumothorax in high risk pregnancy. our case illustrates a successful outcome for mother and foetus with surgical intervention at 32 weeks gestation. folfox is currently the standard adjuvant treatment for locally advanced (stage iii) colon cancer and increases disease free survival. its toxicity is well tolerated with common adverse effects being paraesthesia, bone marrow suppression and gastrointestinal disturbance. pulmonary toxicity has rarely been reported. three clinical cases of acute dyspnoea following folfox therapy (2005) (2006) (2007) for stage iii colon cancer are reported. all had an anterior resection followed by 11-12 cycles of folfox. each developed rapidly progressive dyspnoea requiring hospital admission within one week of their last cycle. one patient required invasive ventilation in icu. high resolution computed tomography (hrct) showed bilateral widespread honeycomb pattern with associated ground glass opacification consistent with pulmonary fibrosis. they had reduced lung volumes and gas transfer. transbronchial biopsy and bronchoalveolar lavage in one patient showed an acute eosinophilic pneumonitis. other causes of interstitial lung disease were carefully excluded. all three patients received high dose corticosteroids with one receiving additional cyclophosphamide. the first patient showed complete recovery following an eight week course of corticosteroids, with resolution of the hrct changes and improvement in lung function. the second had symptomatic improvement of dyspnoea, but a persistent moderate reduction in gas transfer. the final patient had persisting radiographic changes and a reduced gas transfer. he remained dependant on ambulatory oxygen 6 months after his initial presentation. these patients' interstitial lung disease appears due to folfox with oxaliplatin being the most likely causative agent. the use of oxaliplatin chemotherapy has increased markedly over the last 3 years and although rare, physicians should be aware of its potential for lung toxicity. lung function testing at baseline, during and towards the end of oxaliplatin treatment should be undertaken and may allow early detection and intervention in cases of pulmonary toxicity. the forced oscillation technique (fot) with broadband signals has been employed relatively rarely in the studies on respiratory mechanics. recent work from our laboratory [1] indicated that the cheek support and the neck angle have minor influence on the impedance spectra around the first antiresonance (far,1), which makes the use of the broadband fot especially attractive in young children. methods we studied 7 healthy children (c; female: 4) and 8 children with bronchopulmonary dysplasia (bpd; female: 3), using multiple-frequency fot between 8 and 256 hz superimposed on spontaneous breathing. results groups c and bpd did not differ in age ( lung function impairment is common in children with cardiac defects associated with increases in pulmonary blood flow/pressure. to investigate the development of bronchial hyperreactivity (bhr), an aorto-caval shunt was created in a model of precapillary pulmonary hypertension. surgical shunt repair was performed to assess the reversibility of bhr. methods 26 rats were divided into 3 groups: group c (n = 10) with sham surgery, group s (n = 8) where an aorto-caval shunt was created (follow-up 4 wks), group r (n = 8) with aorto-caval shunt but surgical correction of the shunt at 4 wks (follow-up 8 wks). in all animals, respiratory input impedance (zrs) was measured at baseline and following increasing doses of methacholine (mch 2, 4, 8, 12 mcg/kg). airway resistance (raw), inertance, tissue damping (g) and elastance were estimated from the zrs spectra by model fitting. measurements were repeated in all animals at 4 wks and at 8 wks for groups r and c. results there was a significant increase in raw and g in group s and rat 4 wks at baseline and following mch ( fig.) which was reversed after surgery. to characterize the factors contributing to lung function impairment following cardiopulmonary bypass (cpb), functional residual capacity (frc), lung clearance index (lci) and respiratory mechanics were measured in children with pulmonary hypoperfusion (tetralogy of fallot, tof n = 12) and hyperperfusion (ventricular septal defect, vsd n = 12) undergoing surgical repair of congenital heart disease. methods frc and lci were measured using a sf6 washout technique and respiratory mechanics using a low frequency oscillation technique in the perioperative period. results while chest opening led to a significant improvement of lung volumes and respiratory mechanics in all patients (p < 0.001), a reduction in pulmonary blood flow during cpb decreased lung volumes and airway resistance in parallel but significantly more in children with tof compared with those with vsd. re-establishing pulmonary blood flow during cpb improved respiratory function particularly in children with tof ( figure) . conclusions sternotomy had a great impact on lung function with parallel improvement in alveolar recruitment, ventilation inhomogeneity and airway resistance. in contrast, onset of cpb led to lung function impairment with a significant drop in frc especially in children with pre-existing hypoperfused lungs. this suggest that pulmonary blood flow enhances alveolar stability through a tethering effect on the alveolar walls. children with advanced lung disease being considered for lung transplantation are likely to spend disproportionately longer periods on transplant waiting lists before appropriately sized donor organs become available. these longer waiting times reflect the lower organ donation rates seen in children; rates that are significantly lower than those reported in the adult population. we describe two children with advanced lung disease who deteriorated whilst on the waiting list for lung transplantation, and in the absence of appropriately sized donor lungs, underwent lobar lung transplantation. methods we describe the clinical course of two children, aged 9 and 13 years old, with advanced lung disease secondary to post-mycoplasma obliterative bronchiolitis and cystic fibrosis-associated bronchiectasis, respectively. results both children received a "cutdown" bilateral lobar transplant from two oversized adult brain-dead organ donors. in both cases the transplant operation involved implantation of the right middle and upper lobes, and of the left upper lobe from the donor. conclusion given the low organ donation rates in children, and in the absence of appropriately sized donor lungs, novel strategies such as lobar transplantation must be considered, particularly when children continue to clinically deteriorate whilst on the lung transplant waiting list. data from the west australian adult outcomes of extreme preterm birth study suggest that adult survivors of bronchopulmonary dysplasia (bpd) may be left with functional and structural pulmonary abnormalities, most notably emphysema. animal data suggest that the antenatal administration of corticosteroids may adversely affect lung development. we therefore sought to determine if maternal variables, including administration of corticosteroid, could predict emphysema severity in adulthood. methods bpd subjects (birthweight < 1500 g and oxygen dependence at 36 weeks post-menstrual age) born prior to 1988 were identified and recruited prospectively via the statewide neonatal follow up program as previously described. pulmonary function tests and thin selective inspiratory and expiratory computerised (ct) images were acquired and scored for emphysema severity (voxel index (%)). the obstetric history was obtained from retrospective review of case notes. results 21 adults (12 females, aged 18-34) were studied, 2 declined ct. all subjects had abnormal ct findings. fifteen (79%) had areas of emphysema. emphysema score and fev1 were not influenced by the administration of antenatal corticosteroids, indication for delivery, maternal age or presence or absence of chorioamnionitis. conclusion maternal factors, including the administration of antenatal corticosteroids, do not predict the long term respiratory outcome of bpd. the factors determining the severity of emphysema in this group remain unknown. the prevalence of childhood asthma is high in the torres strait. children have generally more severe asthma and asthma knowledge is poor. however, there is no culturally appropriate asthma education program for these children. we are conducting a randomized controlled trial to examine the additional benefits of an education intervention by indigenous health care workers (hcw) on asthma outcomes. we describe the study's objectives, design and baseline measurements. methods children with wheeze were reviewed by two paediatric respiratory physicians using a standardized protocol; children with asthma were eligible. after obtaining informed consent children were randomly allocated to: (1) three additional asthma education sessions with a hcw; or (2) no additional education from a hcw. trained hcws carried out the education sessions using culturally appropriate tools. primary outcome was the number of unscheduled hospital/doctor visits due to asthma exacerbation. all children were re-assessed at 12 months. results we enrolled 113 children aged 1 to 17 years, 81% were torres strait islanders and 12% aboriginal and torres strait islanders. the clinical spectrum of asthma was: 51% infrequent episodic asthma, 22% frequent episodic asthma and 27% chronic asthma. eighteen percent of the children knew what a written asthma action plan was; 8.5% had one. carers' assessment of knowledge of medications showed that 52% could not name any asthma medication used by their child, 40% could not explain dosage, and 67% could not explain how beta2 agonists worked. conclusions asthma knowledge and possession of asthma action plans in this cohort is poor at baseline. there is substantial room for improvement and additional asthma education by hcws potentially has significant benefits. impulse oscillometry system (ios) measures respiratory function during normal breathing by transmitting mixed frequency rectangular pressure impulses down the airways and measuring reflected pressure. computer analysis calculates respiratory impedance and its components, airways resistance and reactance, at a range of frequencies from 0.1 hz to 150 hz. no previous australian normative data exists. the ios software generates predictive normal values for each of the parameters measured including total airway resistance (r5), the proximal airway resistance (r20) as well as peripheral capacitive reactance (x5). however, they are based on german data. methods cross-sectional study of 100 community dwelling adults, with 10 males and females per 10-year cohort. inclusion criteria: age range 25-74 years, apparently good respiratory health. exclusion criteria: smokers, asthmatics and others with acute or chronic respiratory disease. both ios and spirometry were conducted on all participants. results australian predictive normal equations have been generated and compared to the current published equations. the ios parameters have been correlated with the spirometric data. results have been analysed by gender, age, height and weight and compared with the predictive normal values for each parameter provided by the german manufacturer of the ios instrument. analysis includes calculation of mean range, and lower limit of normal. conclusions a preliminary set of australian predictive equations have now been produced for the ios. these have been compared with international equations. ios has potential application in a range of respiratory disease states and in population screening for occupational health (e.g. mining, & high dust load environments). supported by phc red. rationale although clinical practice guidelines for both asthma and copd recommend spirometry for diagnosis and monitoring, beneficial effects on the management of chronic respiratory diseases in general practice have not been established. we hypothesized that spirometry would improve health outcomes compared to usual care. methods we are conducting a single masked rct with 3 arms: group a receive 3 monthly spirometry and followup, group b receive spirometry before and after the trial and group c usual care. 45 general practices were recruited though divisions of general practice in melbourne. invitations were mailed by 31 of these practices to patients who had been prescribed inhaled medications during the previous 6 months. participants returned respiratory and generic quality of life questionnaires and an asthma score card. groups a and b were tested on a micromedical turbine spirometer following ats/ers guidelines. results 351 eligible patients (275 adults, 50 children aged 8-13 and 26 youths aged 14-17 years) entered the trial. 122 were randomized to group a, 134 to group b and 95 to group c. the mean (sd) age of adult participants was 54.3 (12.7), children 10.3 (1.7) and youths 15 (1.1) years. there were 130 males and 221 females. the adults were highly symptomatic in the previous 12 months: 82% reporting wheeze, 50% chest tightness on waking, 74% shortness of breath on exertion, 61% nocturnal cough, 46% morning cough and 75% sputum. symptoms of chronic bronchitis were reported by 39% of adults and a diagnosis of copd by 19%. asthma was reported by 84%, confirmed by a doctor in 96% and 55% had experienced an attack in the last 12 months. only 35% had a written asthma action plan. 37% of adults had ever visited a hospital ed and 28% had been admitted. conclusion it is possible to recruit asthma and copd patients from general practice and to randomize them to spirometry or usual care. whether spirometry is associated with fewer symptoms, changes in medication, uptake of action plans or improvement in lung function or quality of life requires further followup. supported by nhmrc. s shah 1 , jk roydhouse 1 , b toelle 2 , s sawyer 3 , c jenkins 2 for the pace australia management committee 1 university of sydney, 2 woolcock institute of medical research, sydney, nsw 2006, and 3 royal children's hospital, melbourne, vic 3052 it is widely held that recruitment of general practitioners for research can be challenging. in this paper, we discuss the recruitment experience from a current study evaluating the impact of an educational asthma intervention on patient outcomes. our aim is to describe the two different strategies utilized to date: (1) in-house through an academic department of gp and (2) outsourced to a private gp organization. methods initial interest was generated through faxes, presentations at gp divisional meetings and newsletter advertisements. gps who expressed interest were visited by project staff to discuss the study further. a major difference was recruiting one gp per practice in the first strategy versus multiple gps per practice in the second strategy. to assess the strategies, we examined participant characteristics, number of gps recruited and number retained. results participant characteristics: under both strategies, 30% of recruits had trained in asia and 54% were women. the first strategy recruited more gps who spoke at least two languages at home (85% vs 42%) and the second strategy recruited more recently graduated gps (58% vs 50%). recruitment: the first strategy recruited 35 gps over 6 months and the second recruited 34 gps over 3 months. retention: 19 gps (54%) from the first strategy stayed in, compared to 29 (85%) from the second. conclusions whilst absolute numbers of gps recruited were similar, retention was much higher under the second strategy. recruitment in primary care is difficult and requires a range of approaches which need to be re-evaluated and adapted as necessary during the course of the study. supported by the australian government department of health and ageing. bronchiectasis is a heterogeneous condition with a large number of causative factors and range of symptoms. the classification of this condition is often confusing and hard to remember. the aim of this study was to classify non-cf bronchiectasis into different clinical phenotypes. methods 178 consecutive patients with non-cf bronchiectasis confirmed on high resolution ct scanning had a detailed clinical, spirometric and laboratory assessment performed by a respiratory physician (pk/mf/pw) and were then followed up for an average of 9 ϯ 4 years (mean and sd) for a total of over 2000 reviews. results 160 of the 178 patients (90%) could be classified as belonging to 3 phenotypic groups; 1) bronchiectasis arising in childhood, 2) bronchiectasis occurring in smokers and 3) bronchiectasis occurring in the elderly. each group had different features which are listed in the there are few data on the long term outcomes of treatment for tuberculosis (tb) by directly observed therapy (dot) in low-incidence settings. the aim of this study was to assess the incidence of recurrent tb in nsw. methods data linkage was performed within the nsw department of health tb notifications database to identify cases that had more than one tb notification between 1994 and 2006. recurrent tuberculosis was defined to include all patients with two or more culture positive episodes at least 6 months apart, where patients had received at least six months treatment for the initial episode. in cases where data contained within the notification details was not sufficient to allow us to distinguish between true cases of recurrent disease, duplication notification for the same episode or persistent disease after incomplete treatment, additional information was obtained from the area tb coordinator. results there were 5723 tb notifications between 1994 and 2006 with 3731 being culture positive. 15 cases of recurrent culture positive disease after completed treatment for the first episode were identified (recurrence rate: 0.4%). conclusions in a population with a low tb incidence, treatment of active tuberculosis with dot results in a very low rate of disease recurrence over a long period of follow-up. support nhmrc ccre in respiratory and sleep medicine. introduction rhinoviruses (rvs) are the major cause of viral-induced exacerbation of asthma. to date, the molecular mechanisms of rv pathogenesis are not understood. recent findings suggest that rv pathology may involve host cell nucleocytoplasmic trafficking, inhibiting key cell functions such as transcription and translation. the study aims to investigate the mechanism of rv 3c protease nuclear trafficking. methods hela cells were infected with rv or transfected with plasmids and cellular localization of 3c analysed at various times thereafter using immunofluorescent confocal microscopy and western blotting with specific antibodies. results 3c protease was predominantly present in nuclei of rv infected cells up to 6 hours after infection, becoming increasingly cytoplasmic thereafter. the nuclear membrane of infected cells became progressively indistinct with time. using a specific inhibitor we also found that 3c utilizes the crm-1 nuclear export pathway. 3c was predominantly in the form of 3cd in both cytoplasm and nucleus of infected cells; mature 3c protease was also detected from 6 hours after infection. deletion analysis indicats that the nuclear localization domain and a nuclear export signal are most likely to be present within the n terminal 64 amino acids. the nuclear export signal is inhibited in the full length protein, via an unknown mechanism. conclusion our data suggest that 3c and 3cd proteins localize to the nucleus in infected cells where they may play a key role in rv pathogenesis by disrupting cellular transcription and the nuclear transport machinery. chronic necrotizing pulmonary aspergillosis (cnpa) is a relatively uncommon, sub-acute, locally destructive process due to aspergillus invasion of the lung. the incidence and prognosis of cnpa are poorly described. case report we present a case of cnpa in a patient on intermittent low dose steroid therapy and recurrent refractory exacerbations of chronic obstructive pulmonary disease (copd).the patient presented with worsening shortness of breath and productive cough requiring recurrent inpatient admissions. human influenza virus is found to bind preferentially to saa2,6gal receptors found in the upper respiratory tract, while avian viruses bind to saa2,3gal receptors expressed in lower airways. this is thought to affect the ability of transmission to humans. our aim was to study the ability of avian and human influenza strains to infect bronchial epithelial cells and relate this to levels of the sialic acid receptor expression. methods calu-3 cells were used as a proximal airway cell and a549 were used as distal airway cell. human primary bronchial epithelial cells (pbecs) were obtained from healthy, asthmatic, and copd volunteers by endobronchial brushing. epithelial cells were stained with sambucus nigra lectin that binds saa2,6gal receptor, and maackia amurensis lectin ii that binds to saa2,3gal. the cells was analysed by flow cytometry. human influenza a/h3n2/wellington strain and low pathogenic avian influenza a/h11n9/sandpiper were chosen and were used at an moi of 0.005 to infect cells. the supernatants were harvested at 48 hr post infection, of which was then analysed by plaque assay for virus replication. results the calu-3 showed greater expression of saa2,6gal linkage than saa2,3gal linkage, and a549 displayed slightly higher expression of both receptors compared to pbecs. despite this human and avian influenza virus replicated to similar titre at 15,000 pfu/ml in both cell lines, but showed low replication in pbecs. background treatment of community-acquired pneumonia remains based on 'best guess' empiric algorithms because of the poor utility of current pathogen tests. furthermore our ability to stratify patients into risk groups is crude at best, relying on scores such as the pneumonia severity index or the curb-65 have major limitations. we have been slowly improving real-time pcr assays for pneumococcus as a clinical tool in patients with pneumonia. methods building on previous research we assesed two targets in the autolysin (lyta) gene and the pneumolysin (ply) gene of s.pneumoniae using the lightcycler instrument and fluorescence resonance energy transfer (fret) probes. all common s. pneumoniae serotypes were detected while other bacteria and viruses were not. the lyta target had the best sensitivity with a detection range between 21 ng to 21 fg. both assays were then applied to whole blood samples from 400 adult patients with community-acquired pneumonia, all of whom had blood cultures prior to antibiotic administration and urinary antigen testing for s.pneumoniae. the lyta pcr had the best performance characteristics with a sensitivity more than twice that of blood cultures in the clinical samples. most pcr+ve/culture -ve patients had positive urinary antigen tests. there was clinical evidence that urinary antigen +ve/ pcr -ve patients were false +ves. most significantly there was a strong correlation between quantitative bacterial count and clinical outcome. conclusions real-time quantitative pcr for pneumococcus has significant potential as both a diagnostic and therapeutic tool in patients with pneumonia. the pitjantjatjara lands are situated in the north-western corner of south australia, occupying an area of over 120 000 square kilometres with a population of approximately 3000. the population lives in small communities or homelands, and there is a high level of mobility between this region and other aboriginal communities in south australia and the northern territory. nganampa health council provides all health care services to the region. specialized support for tb control comes from both the south australia tb service based at royal adelaide hospital as well as a centre for disease control in alice springs. the prevalence of tuberculosis (tb) in this predominantly indigenous community is thought to be significantly higher than the national rate. there are considerable challenges in detecting and managing tuberculosis, relating to the community's geographical remoteness, migration of populations and access to health services. the aims of this study are to quantify the prevalence of tuberculosis in the pitjantjatjara lands, and describe the significant barriers to tb diagnosis and treatment. methods a retrospective study of all diagnoses of tuberculosis within the pitjantjatjara lands in the period 1995-2006. outcomes include measures of tuberculosis diagnosis, the rates of completed tb treatment and rates of tuberculosis drug resistance. the study will draw conclusions about the reasons for high levels of tb prevalence in this community and identify barriers to effective tuberculosis treatment. conflict of interest no. patients admitted to hospital with a diagnosis of community-acquired pneumonia (cap) are usually treated with intravenous (iv) antibiotics irrespective of pneumonia severity. available guidelines vary in recommended timing and indications for switching to oral antibiotics. aim to examine the patterns of antibiotic choice and delivery method (iv, oral and time to switch) in patients admitted with cap. methods a retrospective chart review of admissions to the respiratory unit over a 12-month period with a diagnostic-related group (drg) coding of pneumonia. 41 charts were reviewed. data collected included patient demographics, clinical features at presentation (temperature, pulse rate, respiratory rate, bp, oxygenation), initial investigations, initial antibiotic regime, time to change (iv to oral), subsequent antibiotic regime and duration, time to defervescence, length of stay and outcome. pneumonia severity was calculated using the revised british thoracic society system (curb-65), score ն 2 = severe. results 3 patients were excluded due to incorrect coding. of the 38 patients, age was 50 ϯ 21 (mean ϯ sd) yrs and 25 (66%) were male. 28 patients (74%) were febrile at presentation and the median curb-65 score was 1 (range 0-4). 37 patients (97%) received iv antibiotics. the curb-65 score was 0 or 1 (non-severe) in 25 patients and 22 of these patients received a combination of iv ceftriaxone and a macrolide. time to defervescence was 2.9 ϯ 2.3 days. time from defervescence to switching to oral therapy was 3.4 ϯ 2.8 days. in non-febrile patients, time to switch was 4.7ϯ4.3 days. length of stay was 8.7ϯ13.0 days. conclusions the time between defervescence and switch to an oral regime was relatively long, possibly contributing to an increased length of stay. many patients received ceftriaxone even with a curb-65 severity rating of 0 or 1. implementing local guideline-based treatment protocols may reduce length of stay. ultrasonic flow sensors can determine flow, volume and molar mass (mm) of the gas flow simultaneously. during tidal breathing the expired molar mass curve can be used to compute co2 over expired volume and a capnography index (cpi) can be computed. the relationship between cpi and copd classification according to gold was investigated. methods prospective, controlled trial. consecutive patients who underwent routine lung function were enrolled to participate in a tidal breathing test using an ultrasonic flow sensor. each test consisted of three tidal breathing recordings of 60 sec. flow, volume and molar mass were measured at 200 hz and data were acquired using prototype wbreath data acquisition software. mean expirograms (mm over volume) were computed and the measurements were analyzed to determine the slope of exhaled phase ii (s2), the slope of phase iii (s3) and the relationship between s2 and s3 (cpi = s3/s2). gold stages were determined from the lung function results and the ers predicted values. results 53 volunteers participated in the study with a mean age of 62 (sd 14), 23 were male, mean bmi 26 (sd 5), 17 had never smoked. the mean pack/year smoking history was 38. there was a clear relationship between gold stage and cpi: gold stage 'normal' had a mean cpi of 5.5 (sd 3.7, n = 21), stage 'severe' had a mean cpi of 13.7(sd = 3.9, n = 7). conclusion computation of cpi based on tidal breathing analysis using an ultrasonic flow and mm sensor correlates well with gold stages. it may therefore be possible to use a simple tidal breathing test to determine the severity of airways disease. background osa is common in tetraplegia and appears within weeks of injury. although cpap treatment is efficacious in able-bodied subjects, case series suggest that cpap is poorly tolerated in tetraplegia. no prospective study has examined cpap efficacy or adherence in tetraplegia. aim to determine the feasibility of cpap use to treat osa following acute tetraplegia. methods all acute admissions who consented and fulfilled the inclusion and exclusion criteria underwent full, portable polysomnography. those found to have an apnoea hypopnoea index of >10 events per hour (osa) were offered cpap, delivered via an auto-titrating device. results to date, 25 patients have been admitted (11 excluded, 3 refused consent). no significant, adverse events have been observed. two patients did not have osa. of the nine with osa, four are mid-study, two had incomplete follow-up (1 returned to uk and 1 refused 3 month assessment), two adhered with cpap and one did not due to severe, pre-existing nasal obstruction. preliminary analyses suggest that those who adhered to cpap had a marked reduction (80% compared with 10-40%) in sleepiness and a greater reduction in the functional outcomes of sleepiness compared to either those without osa or who were unable to use cpap. patient accrual, recruitment and completion rates are consistent with our initial estimates. study recruitment will be completed by end-october 2007. conclusion initial data suggest that auto-titrating cpap is a feasible treatment for osa in acute tetraplegia. these data will be used to finalize planning for a multi-national, multi-centre randomized controlled of therapy. this research was supported by the transport accident commission. visual recognition of cyanosis is an important clinical activity. cyanosis recognition is affected by lighting colour and there is anecdotal evidence that people with significant colour vision deficiencies (cvds) have particular difficulty. studies to date have centred on the colour change with oxygenation of isolated blood but it is not clear how this extrapolates to cyanotic patients in vivo. methods ten patients known to be chronically hypoxaemic and showing signs of cyanosis were recruited from the chronic respiratory program. ten normal subjects were recruited as controls. the spectral reflectances of their lips, nail beds and palm creases were measured using a topcon sr-3 telespectroradiometer. the patients were measured at rest and after exercise to lower their saturation by 5-10%. the chromaticities were calculated and plotted. results both groups showed a spread of colours but they fell into two distinct ranges. the colour difference between the groups lies very close to the colour confusions made by congenital cvds. within the cyanosed group, the colour shift was not tightly related to decreasing oxygen saturation. this is most likely due to interpersonal factors such as pigmentation and vascular perfusion that affect colour and the difficulties in measuring the colour of heterogeneous anatomical features. conclusions these results quantify the anecdotal difficulties in detecting cyanosis and suggest that observers with cvd would have problems recognizing the condition. the photographs obtained from this study will be used to compare the ability of subjects with and without cvd to detect cyanosis. supported by the nsw ambulance service. baroreflex sensitivity is depressed in osa patients during sleep but effects during wakefulness are less clear. we have now examined relationships between awake brs and severity of sleep disordered breathing (sdb). methods immediately prior to overnight polysomnography, continuous (5 min) beat-to-beat arterial blood pressure was measured via finger plethysmography (portapres) and heart rate via ecg in 20, supine, normotensive, untreated osa patients (17 males; age: 49 ϯ 15 years (mean ϯ sd); bmi: 26 ϯ 11 kg/m 2 ). spontaneous baroreflex sensitivity (brs) was calculated using the sequence technique. sdb was characterized as apnoea hyponoea index (events/hour) and arousal index (ai). data were analysed via mathematical modelling and unpaired t test. results brs fell with increasing ahi. patients with ahi > 30 events/hour (n = 9) had a significantly lower brs (8.1 ϯ 1.5 ms/mmhg) than those with ahi < 30 events/hour (19.8 ϯ 8.7 ms/mmhg, p < 0.001). brs was negatively related to both ahi and ai via fitted exponential functions (r 2 = 0.45 and 0.70, respectively). it is hypothesized that the analysis of morphology of the ecg waveform in combination with the heart rate patterns could lead to the possibility of detection of the start and duration of apnoea/hypopnoea events and consequently estimation of the apnoea-hypopnoea index (ahi). to the authors' knowledge the published ecg based algorithms for detecting sleep disordered breathing are only capable of minute by minute analysis rather than detection of individual respiratory events. methods changes to ecg parameters were investigated during respiratory events with no distinction made between apnoea and hypopnoea events. 632 isolated respiratory events and 1264 controls of identical duration were obtained from 7 polysomnographic studies, using a randomized procedure. features such as the r wave amplitude, t wave amplitude, qrs area and the r-r interval were extracted from the 2 lead ecg. a number of physiological predictors based on these features were generated. a logistic regression model was used to investigate the association between the predictors and true events, using the statistical software, stata. results univariate and multivariate analyses were performed. three multivariate models were developed; heart parameters only, ecg waveform morphology parameters only and the combinations of the two. the area under the receiver operator characteristic curves (auc) for these models were compared. the best results were obtained with the combination of morphology and heart rate parameters (auc = 0.8858 (0.0078 (sd))) compared to the morphology (auc = 0.8169 (0.0121 (sd))) and heart rate (auc = 0.7195 (0.0103 (sd))) models. the multivariate analysis has shown encouraging results indicating that an algorithm using a combination of heart rate and ecg morphological parameters could potentially be constructed that would enable the determination of individual respiratory events and subsequently an ahi. supported by the arc. introduction sacin and scond are measures of ventilation heterogeneity in acinar and conducting airways, derived from analysis of mbnw. maintaining tidal volumes of 1 l at 9-11 breaths/minute (bpm) is impossible for some. our aim was to examine the effect of different tidal volumes on sacin and scond in normals and asthmatics. methods 10 normals (23-41 yrs) and 12 asthmatics (21-63 yrs) underwent mbnw at tidal volumes of 500 ml at 20-23 bpm, 1 l at 9-11 bpm, and 2 l at 5-7 bpm. scond and sacin, were determined from the normalized phase iii slopes of breaths between turnovers (cumulative ventilation/frc) 1.5 & 6. results the mean ϯ sd %predicted fev1 was 97.3 ϯ 17% in normals and 88 ϯ 11% in asthmatics. in normals, sacin at tv of 0.5, 1 and 2 l were 0.195 ϯ 0.105 l -1 , 0.095 ϯ 0.036 l -1 and 0.058 ϯ 0.031 l -1 , respectively (p = 0.0003, anova), while scond were 0.098 ϯ 0.047 l -1 , 0.042 ϯ 0.021 l -1 and 0.029 ϯ 0.014 l -1 (p = 0.0002), respectively. in asthmatics, sacin were 0.440 ϯ 0.195 l -1 , 0.181 ϯ 0.087 l -1 and 0.100 ϯ 0.047 l -1 , respectively (p < 0.01), while scond were 0.204 ϯ 0.111 l -1 , 0.068 ϯ 0.037 l -1 and 0.031 ϯ 0.013 l -1 , respectively (p < 0.0001). conclusion increasing tidal volume while maintaining the same minute ventilation during mbnw led to large decreases in scond and sacin in both asthmatics and normals. this may be due to reduced inter-regional differences in specific ventilation with greater tv. the log-log relationship between sacin and tv allows an adjustment to be made for variations in tidal volume. funding crc for asthma and airways and nhmrc project grant #547346. dj smith 1 , k bowden 2 , t lloyd 2 , j coucher 2 , l garske 1 1 respiratory medicine, and 2 radiology, princess alexandra hospital, brisbane, australia introduction we have shown diaphragmatic flattening and decreased diaphragmatic excursion qualitatively assessed on ultrasound is strongly predictive of dyspnea severity and lower lung inflation in patients with pleural effusion. we sought to quantitatively measure diaphragm length and movement and determine how closely these are related to dyspnea severity and lung inflation. methods patients with unilateral pleural effusions had ct imaging of their diaphragm during a measured inspiratory capacity manoeuvre. maximal sagittal length was measured at tlc, and frc. patients had a baseline dyspnea index (bdi: 0-12) and respiratory function measured. results 4 patients with unilateral effusion (all right side; 3 malignant mesothelioma, 1 inflammatory) had a mean (sd) bdi of 5.5 (2.89), and tlc of 74% (3.91) predicted. the right diaphragm on the side of the effusion tended to be shorter than the left at frc (p = 0.08), and had a trend to reduced shortening with inspiration (p = 0.08). conclusions the right diaphragm is known to be longer than the left in health. the strong trend to a shorter and less mobile right diaphragm associated with effusion suggests this is a potential mechanism for dyspnea. further recruitment will enable correlation between bdi, tlc and diaphragm length and mobility. 4) ) that was slightly worse than an able bodied, control population (17.9 (3.1)), but better than an able-bodied population with untreated osa (14.5 (3.6)). the mapi predicted that 14% of the sample were likely to have osa. these data will be complimented by full sleep studies to be performed at the participants' homes in late 2007, early 2008. conclusion our interim data suggest that the rate of subjective sleep complaints are not substantially different in the population with tetraplegia compared with the able-bodied. this research was supported by the victorian neurotrauma initiative. it has long been assumed that the ventilation heterogeneity associated with lung disease could in itself affect the measurement of carbon monoxide transfer factor. the aim of this study was to investigate the potential estimation errors of carbon monoxide diffusing capacity (tlco) measurement that are specifically due to conductive ventilation heterogeneity. we induced conductive airway ventilation heterogeneity in 35 never-smoker normal subjects by histamine provocation, and related the resulting changes in ventilation heterogeneity (derived from the multiple breath washout test) to corresponding changes in diffusing capacity, alveolar volume and inspired vital capacity (derived from the single breath tlco method). average conductive ventilation heterogeneity doubled (p < 0.001), while tlco decreased by 6% (p < 0.001), with no correlation between individual data (p > 0.1). when dividing diffusing capacity by alveolar volume, the resulting transfer coefficient was not significantly different pre versus post histamine (p = 0.074). these findings can be brought in agreement with recent modelling work, where specific ventilation heterogeneity resulting from different distributions of either inspired volume or end-expiratory lung volume have been shown to affect tlco estimation errors in opposite ways. the combination of these errors appears to largely cancel out in our experimental situation of induced ventilation heterogeneity comparable to that observed in lung disease. we conclude that conductive ventilation heterogeneity per se has a negligible effect on diffusing capacity measurement. an important determinant of airway function in humans is vagal-mediated cholinergic tone in airway smooth muscle (asm). this airway tone may be altered in disease states. the use of mouse models for the study of airway diseases, including asthma, pulmonary fibrosis and copd is well established. however, it is not known whether mice actually possess basal asm tone or, if it does exist, how this tone changes in disease models. this study was undertaken to determine whether mice have detectable asm tone in vivo. methods respiratory system impedance (zrs) was measured in female adult balb/c mice using a wave-tube modification of the forced oscillation technique. zrs was measured during slow (~35 s) inflation-deflation manoeuvres between the transrespiratory pressures of 0 and 20 cmh2o. baseline lung mechanics and thoracic lung volumes (tgv) were measured before and after each mouse was allocated to one of four treatment groups: 'saline' mice received an i.p injection of saline, 'atropine' mice received i.p. atropine sulphate, 'vagotomy' mice had their left and right cervical vagus nerves isolated by blunt dissection and cut, and 'sham' mice had the area of the vagus nerves exposed but the nerves were not cut. results there were no post-treatment changes in tgv, airway resistance, tissue damping, tissue elastance, inertance or tissue hysteresivity in any of the four groups. conclusions the lack of change in lung mechanics post-atropine or postvagotomy in balb/c mice suggests that, unlike humans and many other species, the airways of mice have no baseline asm tone. supported by nhmrc grant#11488. nomination none. conflict of interest none. both male gender and increased mandibular enclosure volume predict more severe sleep disordered breathing in obstructive sleep apnoea patients. we now examine gender/body size/mandibular enclosure volume relationships for normal subjects stepwise multiple linear regression analysis was used to model body size/enclosure volume interactions. results for the whole group, mv was 261.1 ϯ 6.0 ml (mean ϯ se) while rmv was 205.1 ϯ 4.9 ml. head circumference (positive) and forehead height (negative) were both independent predictors for mv and rmv (both p < 0.02), while hip circumference was an additional positive predictive factor for rmv (p < 0.04). after adjusting for these parameters, male mv and rmv were larger than for females conclusion these findings suggest that mandibular enclosure volumes are relatively larger in males, even after adjusting for body size/cranial dimension. differing body size/mandibular enclosure volume interactions may contribute to gender influences on the severity of sleep disordered breathing. supported by nhmrc of australia nomination john read prize for sleep and physiological research tp 027 audit of ctpa in a regional hospital y raje, s vincent, g simpson department of thoracic medicine, cairns base hospital, cairns, qld 4870 since the introduction of computerized tomographic pulmonary angiograms (ctpa) at our institution the number of requests for this investigation at our institution has grown at an alarming rate. the purpose of this study was to evaluate the clinical assessment of suspected pulmonary embolism (pe). methods 50 ctpa were reviewed. results 31 female, 19 male. mean age 50 yrs (range 21-87). 26 ctpa requests came from department of medicine, 21 from emergency department, 2 from surgical teams and 1 from oncology outpatients. 36 patients presented with chest pain (pleuritic in 20 cases), 25 had dyspnea, 7 presented with collapse. 4 patients had haemoptysis. hypoxaemia was recorded in 7. none were clinically shocked and only one had a recorded tachycardia. d-dimer requested in 10 patients and was elevated in 9. arterial blood gases performed in only 10 patients (20%). 47 patients had prior chest x-ray which was normal in 24 (48%). 8 patients had consolidation on chest x-ray, 2 pleural effusions, 2 atelectasis and 1 fractured ribs. recorded risk factors included 4 patients with previous dvt or pe, 4 patients with malignancy and 6 patients were immediately post-operative. only 6 ctpas (12%) demonstrated evidence of pe. of these 2 had recent dvt and 2 were post-operative. 1 had a history of bowel cancer. there was no formal record of pre-test clinical probability of pe (eg wells' score) for any of the 50 cases. retrospective calculation of the cases of pe, 4 had a wells' score of 4.5 and 1 of 4 with the remaining patient with wells' score of under 2. only 3 patients (one with clinically probable pe) had received fractionated heparin prior to the ctpa. conclusion (1) ctpas performed at our institution have a low yield (12%).(2) pre-investigation clinical assessment was poor and there was poor adherence to published guidelines, (3) this results in many unnecessary ctpa examinations generating increased work and expense for the medical imaging department and exposes many patients to unnecessary and potentially harmful radiation exposure. the evaluation and management of hereditary hemorrhagic telangiectasia involves a multidisciplinary approach according to international guidelines. the aim of this audit was to compare the assessment process in one centre with that of the international recommendations. methods retrospective comparison was made by medical chart review of all patients with a diagnosis of hht between the years 1994 to 2006. demographic along with clinical data with diagnostic investigations, complications, treatment and genetic evaluation, including family screening was collected. the proportion of patients evaluated and managed as per the international recommendations was determined. results the audit identified 26 patients with the diagnosis of hht, with the mean age 58 years. diagnostic criteria were met in 77% of the cohort. of the known clinical features, 54% had a family history, and 81% epistaxis. cutaneous telangiectasia was present in 85% and visceral involvement in 92%. pulmonary arterio-venous malformations (pavm) were seen in 16 patients, cerebral avm in 4, gastrointestinal telangiectasia was documented in 8. one patient had a spinal (cervical) avm, and another had pulmonary hypertension in association with this condition. only 8 patients underwent diagnostic or screening investigations in accordance with the international recommendations. furthermore, one patient was referred for a genetic evaluation. conclusions this clinical audit found that 31% of patients referred to this centre were evaluated in accordance with the international recommendations. genetic assessment was lacking. the study supports the need for a coordinated, multidisciplinary approach to the evaluation and management of hht in this centre. lm young 1 , n good 1 , d milne 2 , w fergusson 1 , i zeng 1 , j kolbe 1 , ml wilsher 1 background while airflow limitation is the most common physiological impairment in sarcoidosis, there are limited data on airway hyperresponsiveness (ahr). understanding the role of ahr in sarcoidosis, if any, may help to identify individuals who might benefit from inhaled therapies. aims (1) to determine the prevalence of ahr in sarcoidosis. (2) to determine the correlation between responses to direct (using histamine) and indirect (using hypertonic saline) bronchial challenge. (3) to determine the clinical, physiological and radiological predictors of ahr. methods subjects with a diagnosis of sarcoidosis based on typical clinical presentation and compatible hrct features and/or tissue biopsy and with a baseline fev1>35% predicted were recruited. subjects underwent standard hypertonic (15% fall in fev1) and histamine (20% fall in fev1) challenge (>1 day but <7 days apart), lung function testing and high resolution computed tomography (hrct) of the chest. results the 52 subjects (48 ϯ 11 years, 35% female, 92% european, 35% stage i, 25% stage ii, 40% stage iii, 0% stage iv) had well preserved lung function overall (fev1 = 2.8l ϯ 0.7.87% predicted). ahr was detected in 5/47 (11%) to hypertonic saline and 19/43 (44%) to histamine challenge. on univariate analysis, response to histamine challenge was predicted by conglomerate fibrosis (p = 0.002) and reticular pattern (p = 0.05) on hrct. the baseline % predicted fev1 was significantly associated with ahr on univariate (p = 0.004), and multivariate analysis (p = 0.01) when adjusted by hrct patterns. conclusions there is a high prevalence of ahr using histamine challenge in this study of sarcoidosis subjects. ahr most strongly associates with baseline % predicted fev1 but also conglomerate fibrosis and reticular pattern on hrct. these findings may reflect the consequence of airway remodelling following inflammation. further studies are warranted to confirm these findings. background upper airway shunt represents a significant source of measurement artefact in the use of the forced oscillation technique (fot), with increasing importance in young children. changes in respiratory system admittance, ars (or zrs -1 ), are theoretically independent of the upper airway shunt. this study examines the possible clinical benefit of ars in preschool children by assessing any increased ability to differentiate responses to bronchial challenges in the routine clinical setting. we hypothesized the use of ars would provide improved sensitivity to clinically relevant obstruction, bronchodilator responsiveness (bdr) and airway hyper-responsiveness (ahr) in young children with respiratory disease. method previous fot measurements were re-analysed and ars calculated to derive: (1) ars reference equations in healthy young children (n = 158); (2) bdr in ars, respiratory system resistance (rrs) and reactance (xrs) in healthy children (n = 78), children with cystic fibrosis (n = 39), neonatal chronic lung disease (n = 49), asthma (n = 56) and wheeze (n = 66); (3) ahr to inhaled adenosine-5′-monosphate (amp) in 19 children. fisher's exact tests were used to assess changes in diagnostic outcomes between ars and conventional fot outcomes (rrs and xrs). results ars was no more sensitive to bronchodilator induced changes than conventional fot outcomes. amp challenges resulted in equivalent responses measured by relative changes in rrs and ars while absolute changes in ars were the least sensitive variable. conclusion this study does not support a clinical advantage in using ars in measuring responses to either inhaled bronchodilator or amp. c hollier 1,2 , c menadue 1,2 , d flunt 1,2 , aj piper 1,2 1 department of respiratory and sleep medicine, royal prince alfred hospital, nsw 2050, and 2 woolcock institute of medical research, nsw 2050 serial measurement of arterial carbon dioxide (paco2), ph and bicarbonate (hco3 -) is essential in the management of patients with hypercapnic respiratory failure (hrf). this information is usually obtained from a sample of arterial blood (abg). the procedure can be painful and distressing for patients, and is sometimes technically difficult due to obesity or contractures. our aim was to determine the validity and feasibility of arterialized venous blood (av) sampling as an alternative to abgs in measuring paco2, ph and hco3levels in patients with chronic hrf. method eighteen patients completed the study. venous blood was arterialized by heating forearm skin to a temperature of 42-45°c with an electric heating pad. an av sample was taken from a cannula positioned in a vein of the heated forearm simultaneously with an abg. in addition, the reliability of av sampling within the recommended temperature range (42-45°c) was investigated in ten healthy volunteers placed on volume cycled ventilation in order to maintain constant ventilation. av samples were taken at 0.5°c temperature intervals from 42.5-45°c results the table below summarizes results for validation of av sampling: based on the evidence that cardiovascular dynamics are altered due to obstructive sleep apnea, this study aims to identify the onset and termination of each apnea event using power spectral density (psd) and morphological features of single lead ecg signal over 5 second period. methods ecgs from 4 patients overnight sleep studies were examined for location of the pre-scored apnea events. onset (n = 1995), maximum (n = 6751) and termination (n = 1996) of each apnea event and normal events (n = 11219) were annotated on 5 second windows. features extracted were psd, amplitudes of r and t wave of 5 second ecgs. receiver operating characteristics (roc) analysis was used to gauge the event recognition ability of all features. weight loss causes an improvement in the severity of osa, however substantial weight loss is very difficult for obese patients. the very low caloric diet (vlcd) has been shown to be successful in causing significant weight loss in obese patients. this is a pilot study on the use of a formal screening protocol to identify osa patients who are potentially eligible for the supervised vlcd program offered by the endocrinology department at auckland city hospital. method 344 consecutive patients who attended the sleep laboratory at ach between june to december 2006 were screened using the protocol. patients who are eligible to be considered for the vlcd program are identified as having a combination of obesity (bmi > 30), osa (ahi > 5 on sleep study) and being residents within the auckland district healthboard region. results 243/ 344 patients screened did not fulfil the inclusion criteria: 171 lived outside the adhb region; 71 had bmi < 30; 7 patients did not have osa (ahi < 5). 101 patients fulfilled the inclusion criteria. 54/101 patients (54%) were excluded due to medical or psychiatric contraindications to vlcd.47 patients (47%) who did not have contraindications to vlcd were contacted. 33 patients were contacted successfully. 14 patients were either unavailable to phone contacts on 3 separate days or were disconnected. 12/101 patients consented to being referred (12%). 21/101 patients declined referral (21%). conclusion this pilot study is the first study using a formal comprehensive screening protocol in the recruitment of obese osa patients into a medically supervised vlcd program. only a small proportion (12%) of patients proceeded to being referred to the vlcd program. key: cord-022082-1dq623oe authors: greaves, peter title: respiratory tract date: 2007-09-28 journal: histopathology of preclinical toxicity studies doi: 10.1016/b978-044452771-4/50007-9 sha: doc_id: 22082 cord_uid: 1dq623oe the chapter describes different aspects of the respiratory tract. in preclinical safety studies, pathologies of the respiratory system can be a result of an intercurrent disease or can be induced by systemically administered drugs. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and technologies required to administer a drug. a complex technology is developed to support the assessment of adverse effects of inhaled substances in rodent and nonrodent species, and the extrapolation of experimental findings to humans. the nasal chambers are the structures that are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. in rodents, the relatively small size of the nose and nasal sinuses facilitates a histological examination. findings show that infectious agents cause inflammation in the nose and nasal sinuses, and this may be associated with inflammation in the conjunctiva, the middle ear, and the oral cavity. it has been observed that a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium, and to a lesser extent in respiratory and glandular epithelial cells. by far and away the most important pulmonary diseases in humans are related to the smoking of tobacco. however, occupational lung diseases caused by inhalation of industrial chemicals, particulate matter and antigens, are also important causes of morbidity and mortality. for this reason, considerable effort has been directed to the examination of airborne pollutants over recent years, including study of their effects in laboratory animals when administered by the inhalation route. extensive study has shown that a complex array of defensive mechanisms protects the lung against the adverse effects of airborne substances and pathogenic organisms. aerodynamic factors prevent access of particles larger than 10|im diameter for these are deposited on the walls of the nasal passages. particles measuring between 2 and 10 (im diameter tend to be trapped by the mucus-covered ciliated epithelium lining the bronchial tree and removed by mucociliary transport aided by the cough reflex. smaller particles may reach the alveoli where they are ingested and transported by pulmonary macrophages.^ considerations of airborne delivery to the lungs are also important in the development of therapies to be administered via the respiratory tract. whilst the inhalation route has been used for many years for volatile anaesthetic gases, the respiratory tract is increasingly being employed for delivery of therapy in not only for asthma and other lung diseases but also as a means of systemic delivery of polypeptides such as insulin. in contrast to the adverse pulmonary effects of cigarette smoke and industrial pollutants, therapeutic agents remain a relatively minor cause of pulmonary toxicity in humans although actual incidence is difficult to ascertain. however, drug-induced pulmonary disease appears to be an increasingly frequent clinical problem and the drugs associated with parenchymal pulmonary injury in humans continue to increase.^ although it is difficult to assess in the context of the underlying disease process, it has been suggested that about 10% of patients receiving wellestablished anticancer drugs develop various forms of pulmonary toxicity some novel antineoplastic therapies may have a similar liability.^ drug-induced toxicity usually occurs after exposure of lung tissue via the circulation to parent drug or metabolites, although increasingly the adverse effects of direct administration of drugs to the lungs needs consideration in preclinical studies. in patients a number of drugs have been associated with pulmonary toxicity which can occur through different mechanisms and take different forms.^"^ through their specific pharmacological action drugs can produce excessive effects on bronchial calibre or pulmonary function. drugs mediate allergic reactions in the bronchi or lungs. they may also produce a variety of obscure, diffuse pulmonary alveolar conditions including a pulmonary syndrome resembling systemic lupus er3^hematosis. as the respiratory tract is a major route by which microorganisms gain entry into the body, opportunistic pulmonary infections with bacteria, viruses, fungi or protozoa are consequences of immunosuppression or broad-spectrum antibacterial therapy. as in other organs, drugs that disturb coagulation may precipitate pulmonary thromboembolism or haemorrhage. localized lung lesions also result from accidental, diagnostic or therapeutic inhalation of xenobiotics. mucociliary clearance is also sensitive to therapeutic agents that affect the secretion of mucus and fluid, ciliary activity and transport.^ treatment with antacids or histamine h2 blockers can also increase the risk of pneumonia developing in patients in intensive care units through increasing gastric ph, which leads to an overgrowth of gram-negative bacteria in the stomach and retrograde pharyngeal colonization.^ in preclinical safety studies, pathology of the respiratory system can be the result of intercurrent disease or be induced by drugs administered systemically. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and the technologies required to administer drug. the different anatomical and physiological characteristics of the airways also influence drug toxicity, disposition and metabolism. the development of drugs to be administered by inhalation or intranasal routes is particularly difficult because of the perceived risks of high local drug concentration in respiratory tissues and their use in potentially vulnerable patients with pulmonary disease.^ a complex technology has been developed to support the assessment of the adverse effects of inhaled substances in rodent and non-rodent species and the extrapolation of the experimental findings to humans.^'^ in order to administer drugs by inhalation, it is necessary to generate aerosols (suspensions of particles in a gas) with a well-defined composition, particle size and shape. they must be delivered to the respiratory tract of laboratory animals in a way that parallels the likely human exposure. in case of therapeutic agents, this should avoid non-respiratory pathways through the skin and food. when aerosols are inhaled, various fractions of the particles are deposited at different locations in the respiratory tract. site of deposition depends primarily on particle size, but variability in the sites of deposition occurs among different laboratory animal species and humans by virtue of the differences in the size and shape of the respiratory passages as well as breathing patterns. ^^ in addition, there are different types of inhalers used in human therapy to consider in the assessment, which can deliver different materials to the lungs, for example nebulizers, propellant-driven metered dose inhalers and dry powder inhalers for asthma treatment. ^^ the subsequent fate of inhaled particles depends not only on their size but also their shape, chemical nature, and solubility in body fluids. soluble substances are absorbed into the blood stream and are removed by the pulmonary circulation. they may also undergo metabolism by enzymes present in the cell populations of the respiratory tract and reactive metabolites may cause local pulmonary damage. insoluble, inert particles are removed primarily by the mucociliary transport system of the trachea and bronchi or through phagocytosis by macrophages. overload of the lung by even relatively inert, nonfibrous particles such as titanium dioxide or carbon black may impair alveolar macrophage-mediated particle clearance. ^^ this may lead in turn to accumulation of dusts over time with eventual fibrotic and tumorigenic responses, particularly in rats.^^ measurements of respiration rate, tidal volume, airway resistance, pulmonary gas exchange and the disposition of the inhaled substances have an important place in the evaluation of chemically induced lung damage in laboratory animals.^"^'^^ however, the key component of the evaluation of the adverse effects of inhaled substances is careful morphological assessment of the fixed tissues. even though there are novel and very sensitive physiological methods for the characterization of oedema following lung injury in rodents, light and electron microscopy of lung tissue provides vital qualitative evidence of the nature of injury. ^^ the nasal chambers are the structures which are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. although these chambers are not usually examined in great detail in conventional toxicity studies in which substances are administered orally or by parenteral routes, they are carefully examined histologically when drugs are administered by inhalation. study of nasopharyngeal silicone rubber casts has shown considerable species differences in the anatomy of this part of the airway. ^^"^^ relative to total nasal length, the nasopharynx is longest in rats and shortest in humans with the dog in an intermediate position. maxilloturbinates are relatively simple structures in man and non-human primates but highly complex in dogs and rodents. as a consequence, regional nasal airflow and disposition patterns vary considerably and this influences the distribution of lesions produced by inhaled xenobiotics in the nasal cavity.^^ however, comparison of the nasal cavity of rhesus monkey and humans using magnetic resonance imaging and nasal casts have shown that many similarities in structure exist in these species. ^^ the anterior nares are lined by stratified squamous epithelium. in other zones the sinuses are covered either by respiratory or olfactory epithelium with a zone of transitional epithelium at the junction between the two types. respiratory epithelium is similar to that found elsewhere in the respiratory passages being composed of ciliated cells, serous and mucous cells, brush cells, intermediate cells and progenitor basal cells. it represents a cellular system engaged in mucociliary clearance carrying surface secretions to the nasopharynx to be cleared by swallowing. although this epithelium is similar to that lining the other large airways, key differences are the particularly rich complement of secretory cells and the complex vasculature of the nose which can modulate capillary, arterial and venous blood flow through the mucosa.^^ mucins may be particularly important. it has been postulated that they not only have a physical protective function but also possess antioxidant properties by virtue of the scavenging behaviour of their high proportion of sugar groups. ^^ the proportion of the nose lined by olfactory mucosa is variable between species, being disposed over a much larger area in dogs and rodents than in primates. ^^ however, it is structurally similar in humans and rodents. it is located in more dorsal or posterior regions of the nasal passages out of the direct line of airflow during normal respiration. olfactory mucosa is a pseudostratified columnar epithelium composed of basal cells, sustentacular cells and sensory cells with mucus-secreting bowman's glands situated in the lamina propria. basal cells are composed of two distinct types, light and dark cells. the light type represents the primitive, stem cell population. sustentacular or supporting cells are non-ciliated, columnar cells possessing microvilli that extend into the overlying layer of mucus. cell bodies of olfactory sensory neurons are situated in the middle layer of the epithelium between sustentacular and basal cells. their dendritic processes extend above the epithelial surface to end in a ciliated expansion referred to as the olfactory vesicle that is believed to be the receptor of odour perception. olfactory axons extend from the cell body, penetrate the basement membrane in bundles to become surrounded by schwann cells and eventually join with the olfactory bulb. the olfactory system is of importance in toxicology for it can be selectively damaged by xenobiotics, including therapeutic agents, presumably as a result of its high metabolizing capacity. the superficial location of neural cells in the olfactory epithelium also provides a model system for the study of the effects of xenobiotics on neural cells. submucosal mucous glands have been well characterized in the rat, hamster and dog, where they are divided into lateral nasal glands and maxillary recess glands. these are both situated in the posterior parts of the nasal cavity and composed of mucus-secreting cells.^^"^^ immunocytochemical study using antisera raised against the major isoenzymes of rat hepatic microsomal cytochromes p450 induced by (3-napthoflavone, 3-methylcholanthrene, phenobarbitone and pregnenolone-16-a-carbonitrile as well as nadph-cytochrome p450 reductase, epoxide hydrolase and glutathione s-transferases b, c and e, has shown their presence in rat nasal mucosal cells.^^ cyp2a enzymes appear to be expressed at high levels in the respiratory tract mucosa.^^"^^ this suggests that the nasal mucosa not only has a capacity for metabolizing and activating xenobiotics by oxidation, but also for hydration and inactivation of potentially toxic epoxides and conjugating electrophilic, reactive metabolites with reduced glutathione. it has been shown that the distribution of immune-reactive enzymes is different in olfactory and respiratory mucosa.^^ xenobiotics can be metabolized within both olfactory and respiratory mucosa but the olfactory regions appear to possess greatest capability for oxidative metabolism. consequently, regional differences in nasal toxicity and tumour formation from inhaled materials may not only be a response to different water solubility and deposition patterns but also differences in the formation of reactive metabolites.^'* another feature of this metabolizing activity is that it can be induced by systemically administered xenobiotics and this can alter the distribution of enzyme activity in the nasal mucosa.^^ studies of the mouse olfactory mucosa have shown that whilst typical hepatic inducers of cyp2a5 do not significantly change its expression in the mucosa, olfactory toxicants can alter the pattern of enzyme distribution.^^'^^ like many other tissues exposed to external environmental agents, the nasal mucosa possesses aggregates of lymphoid tissue in the underlying lamina propria. in rats these areas, characterized by follicles containing both t and b cell areas, are located in the ventral aspects of the lateral walls of the nasal airways at the opening of the nasopharyngeal duct.^^'^^ like the gutassociated lymphoid tissue, these nasal follicles have been shown in the rat to be covered by specialized epithelium with islands of cells with microvilli, socalled m or membranous cells. little is known of any toxicity occurring in this tissue despite its strategic position in the respiratory tract.^^ in rodents, the relatively small size of the nose and nasal sinuses facilitates histological examination. usually this area is sectioned transversely into several standardized blocks following decalcification.^^ there have been a number of detailed publications describing the histological preparation and assessment and recording of pathology of the rodent nasal cavity.^^"^^ careful standardized histological sections, careful recording of lesions with the use of diagrams of the rodent nasal cavity are useful in the assessment of lesions in the nasal cavity found in inhalation studies."*^ in larger species, particularly dogs and primates, sectioning and blocking is more complex. although dissection is required, a similar procedure following decalcification can be adopted. careful examination of haematoxylin and eosin stained sections remains paramount in the assessment of the nasal cavity, although special stains may be helpful. examination of cytokeratin expression in the respiratory mucosa has been used as a marker of epithelial differentiation in the respiratory tract>^ a test system that relates to the innervation of the nasal mucosa is that proposed by alarie.^^ the trigeminal nerve endings in the nasal mucosa of mice mediate the response to sensory irritants and this can be measured by a decrease in respiratory rate. it has been shown that a good correlation exists between the decrease in respiration rate in mice exposed to airborne chemicals and the nasal irritancy potential of the chemicals in humans.^^ this enables the detection of airborne sensory irritants and the prediction acceptable levels of exposure to the upper respiratory tract in people. microbial pathogens infectious agents cause inflammation in the nose and nasal sinuses and this may be associated with inflammation in the conjunctiva, middle ear and oral cavity. murine pathogens may cause alterations in the respiratory tract that can confound the assessment of changes induced by xenobiotics.^^ in rats, microbiological agents implicated in the development of rhinitis and sinusitis include corynebacterium kutscheri (pseudotuberculosis), streptococcus pneumonia, pasteurella pneumotropica, klebsiella pneumoniae, mycoplasma pulmonis and the sialodacryoadenitis virus or rat corona virus.^^ rats infected with the sialodacryoadenitis virus show inflammation and necrosis of the upper respiratory epithelium as well as damage to salivary and lachrymal glands. the sendai virus, a paramyxovirus, also has marked tropism for the respiratory tract, including the nasal cavity, and is associated with systemic effects that can compromise studies in laboratory rodents. occasionally, fungal infections of the airways with. aspergillus fumigatus are reported.^^ a variable that has been shown to influence the severity of the rhinitis produced by mycoplasma pulmonis is the strain of rat. following housing of lewis and fischer 344 strains together to eliminate microbial and environmental differences it was shown that the lewis strain developed a more severe rhinitis following inoculation with mycoplasma pulmonis than fischer 344 rats, although the reason for the difference was unclear."^^ rats exposed to ammonia, a common pollutant of the air in laboratory animal cages, have also been shown to develop lesions of the dorsal meatus, dorsal nasal septum and prominence of the turbinates.^^ these lesions are characterized histologically by swelling or mild degeneration of the epithelium. it appeared that ammonia exposure potentiated the acute inflammatory response of the nasal cavity to microbiological pathogens. a microorganism reported in the nasal cavity of rhesus monkeys employed in inhalation studies is the nematode of the genus anatrichosoma.^^ sections of this nematode are found in the squamous epithelium of the nasal vestibule and are associated with acanthosis and hyperkeratosis of the epithehum and a multifocal or diffuse granulomatous inflammation in the submucosa. administration of toxic or irritant substances to laboratory animals by the inhalation route produces degenerative, inflammatory and reactive changes in the nasal mucosa. the range of histological features is similar to those found in other mucosal surfaces damaged by other exogenous agents. whilst therapeutic agents administered by the inhalation route do not usually produce a severe degenerative or inflammatory responses in the nasal mucosa, at least at therapeutic doses, the simple categories proposed by hardisty and colleagues in recording of degenerative and reactive lesions following exposure to volatile chemicals are useful.'^^ categories suggested are: degeneration, regeneration, atrophy (postdegenerative), respiratory metaplasia and basal cell hyperplasia and inflammation. degeneration is usually the earliest morphological change characterized by loss of sensory and sustentacular cells resulting in a thinner mucosa. bowman's glands and nerve bundles, individual cell necrosis may be seen in more severe cases. regeneration is characterized by proliferation of basal cells associated with an epithelium that loses its regular structural features. post-degenerative atrophy usually follows severe damage and is characterized by loss of sensory and sustentacular cells. respiratory metaplasia is a process whereby the normal olfactory mucosa is replaced by pseudostratified epithelium of respiratory type often with cilia. basal cell hyperplasia represents a longer term effect where the proliferating basal cells form a distinct layer of cells below the respiratory epithelium. an example of the type and distribution of the degenerative and inflammatory conditions which can be induced by inhaled irritants is provided by the study in which swiss-webster mice were given various irritants by inhalation for periods of 6 hours per day for 5 days at concentrations that produced a 50% decrease in respiratory rate (alarie test). although the degree of histological changes varied with different agents, the changes were broadly similar in type and distribution.^^ most agents examined produced little or no alteration in the squamous mucosa lining the anterior part of the nose apart from some mild increase in thickness of the squamous layers. principal sites of damage were shown to be the anterior respiratory epithelium adjacent to the vestibule and the olfactory epithelium of the dorsal meatus. there was a distinct decrease in severity in posterior regions. histologically, the lesions in respiratory epithelium ranged from mild loss of cilia and small areas of epithelial exfoliation to frank erosion, ulceration and necrosis of the epithelium and underlying tissues including bone. variable polymorphonuclear cell infiltration was also found. in some cases, early squamous metaplasia developed on the free margins or the naso-maxillo-turbinates. changes to the olfactory epithelium varied from focal to extensive loss of sensory cells associated with damage to sustentacular cells. in severe cases, complete loss of olfactory epithelium occurred. although the degree of histological change was shown to vary with different agents, lesions induced by the more water-soluble chemicals tended to remain localized in the anterior part of the nasal cavity whereas agents with relatively low water solubility produced lung lesions in addition. it was suggested that these findings demonstrated the powerful 'scrubbing' action of the nasal cavity for water soluble, airborne xenobiotics.^^ inflammatory alterations have been induced in the nasal cavity of rodents treated with therapeutic agents at high doses by inhalation. significantly irritant substances do not make viable therapies. however, the precise relevance of such changes for human therapy by the inhalation route are sometimes questionable when the nasal damage is limited to high doses and it is not associated with alterations in other parts of the respiratory tract. in the case of tulobuterol, a 32-adrenergic receptor agonist, it was argued that the nasal inflammation induced in rats in a one month inhalation toxicity study was the result of a particularly high exposure of the nasal epithelium to drug, not representative of the likely human exposure to tulobuterol by inhalation, where little or no nasal exposure would occur.^^ rp73401 [3cyclopentyloxy)-ar-(3,5-dichloro-4-pyridy)-4-methoxybenzamide], a novel type iv phosphodiesterase inhibitor which was being developed for the treatment of asthma and rheumatoid arthritis, was also reported to produce degeneration of the olfactory epithelium in rats but neither dogs nor mice after single and repeated oral doses and by inhalation.^^ histologically, the olfactory epithelium showed necrosis of the superflcial epithelial layers including the sustentacular and sensory cells, with sparing of the basal cell layer. there was also damage to bowman's glands. the development of proliferative lesions and ultimately tumours of principally neuroectodermal origin followed chronic treatment. as rp73401 was highly metabolized and the nasal lesions could be inhibited by treatment of rats with metyrapone, a non-speciflc inhibitor of cytochromes p450, it was postulated that the changes were the result of p450mediated activation in the olfactory tissues, not linked to its pharmacological phosphodiesterase activity.^^ nasal epithelial degeneration and necrosis has also been reported in both rats and dogs treated with another candidate anti-inflammatory drug ci-959 by the intranasal route. this agent affected olfactory epithelium more than respiratory mucosa, suggesting that metabolism was important in the generation of this toxicity^^ although the nasal cavity has not been often examined histologically in great detail in toxicity studies conducted on drugs administered orally or by parenteral routes, damage to the nasal mucosa can be induced by drugs administered by these routes. one example is methimazole, a thioureylene antithyroid drug used in clinical practice where oral doses of 0.2-2mg/kg/day are employed and abnormalities of taste and smell have been described.^^ administration of methimazole at relatively high doses to long-evans rats by single oral (50mg/kg) or intraperitoneal (25mg/kg) routes was shown to produce damage to the sustentacular and sensory cells with sparing of the basal cells and basement membrane.^^ bowman's glands were also involved. methimazole is metabolized by the flavin-containing monooxygenase system and it is employed as a model substrate for this enzyme in vitro. the presence of flavin-containing monooxygenase isoforms in olfactory mucosa of long-evans rats suggested that reactive intermediates may be responsible for the nasal toxicity ^^ similar changes have been reported in mice where depletion of glutathione in the olfactory mucosa has been demonstrated also suggesting formation of local reactive metabolites.^^ histological examination has also shown that intravenous administration of a single dose of vincristine to mice damages the olfactory epithelium.^^'^^ vincristine is a vinca alkaloid derivative used in cancer therapy which has antimitotic activity and binds to tubulin. cell death was noted in olfactory cells 2-5 days after dosing with a peak of cell proliferation at 5 days and repair after about 10 days. these features resemble those that can be seen in other proliferating tissues after single doses of antimitotic drugs. the risk of damage to human olfactory cells from agents with these effects in rat nasal mucosa often remains uncertain because an understanding of relative exposure and metabolism in different species and a better understanding of the metabolic potential of human olfactory mucosa is required. a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium and to a lesser extent in respiratory and glandular epithelial cells.^^'^^ these inclusions are pas-negative and ultrastructural examination shows that they are membrane-bound, ellipsoid bodies containing homogenous electron dense matrix. their significance remains uncertain. a consensus classification for the variety of proliferative, non-neoplastic changes and atypical epithelial lesions and neoplasms found in the rat nasal cavity has been defined by schwartz and colleagues.^^ the classification of the international agency for research on cancer provides a similar perspective for rats and mice.^^'^^ proliferative lesions may be occasionally seen in untreated rodents in carcinogenicity studies but are much more commonly induced by administration of xenobiotics in inhalation carcinogenicity studies. spontaneous nasal tumours are uncommon but most often squamous in type in rats whereas in mice spontaneous squamous tumours are extremely rare and haemangiomas and respiratory adenomas predominate.^^'^^ the generally agreed categories are described below: mucous (goblet) cell hypertrophy and hyperplasia affects the nasal respiratory epithelium and are characterized by the presence of enlarged mucus-filled goblet cells, some of which form clusters suggestive on intraepithial glands. squamous cell hyperplasia is seen in the stratified squamous epithelium of the nares and is characterized by a focal increase in the number of cell layers. cells may show atypia with irregular enlarged, pleomorphic nuclei and nucleoli. squamous metaplasia occurs to respiratory epithelium under conditions of chronic damage. it is characterized histologically by the presence of three or more layers of epithelial cells with eosinophilic cytoplasm and clear cell boundaries whereas advanced lesions show typical keratinization and formation of intercellular bridges. cellular atypia may also be seen and should be characterized when found. respiratory epithelial metaplasia (of the olfactory epithelium) represents atrophy and degeneration of the olfactory epithelium with loss of sensory cells and in advanced cases loss of sustentacular cells with replacement by ciliated and non-ciliated respiratory epithelium. it may be seen as a spontaneous focal lesion in aged rats. epithelial hyperplasia with cellular atypia (atypical hyperplasia, basal cell hyperplasia, dysplasia) is a term used to embrace proliferative lesions in the respiratory and olfactory mucosa in the nasal cavity in which there is varying degrees of altered differentiation and atypia. there is perturbation of the growth pattern of the epithelium such that the changes are not those found in the normal regenerative response to transient mucosal damage. adenomas (polypoid or villous adenoma, adenomatous or villous polyp) usually develop in the anterior part of the nasal cavity and are usually exophytic lesions that develop from respiratory epithelium or nasal glands. adenomas of respiratory epithelium may be papillary in form but are, by definition, well circumscribed with minimal cellular pleomorphism and atypia. they may very occasionally occur spontaneously in aged rats.^^ adenomas of nasal glands usually show an acinar pattern. squamous cell papillomas develop in the squamous epithelium of the nares or in areas of squamous metaplasia in respiratory or olfactory epithelium. they are exophytic lesions with limited connective tissue stroma. they may develop spontaneously in aged rats.^^ carcinomas of either squamous or glandular differentiation develop in the nasal mucosa. histologically, they have similar characteristics to those in other epithelial tissues. they are rare spontaneous lesions in aged laboratory rodents but may be induced by xenobiotics administered by inhalation, orally or by the parenteral route. squamous carcinomas have been reported to develop in a small number of untreated fischer 344 rats used in carcinogenicity studies in association with point mutations in the c-h-ras and c-k-ras gene.^^ olfactory neuroblastoma (ethesioneuroblastoma, olfactory neuroepithelioma, olfactory neuroepithelial carcinoma) show olfactory differentiation and arise from olfactory epithelium. they do not seem to occur as spontaneous lesions in rats or mice and only rarely induced.^^'^^ cells are arranged in lobules or in solid sheets with scanty stroma. cells are relatively uniform with scanty cytoplasm with round or oval hyperchromatic nuclei. true rosettes with lumens or pseudorosettes are also seen. poorly differentiated tumours of this type may require ultrastructural study for diagnosis. olfactory neuroblastomas typically show the presence of electron-dense neurosecretory granules, neurofilaments or axons. as there is no detailed understanding of the biological behaviour of these neoplasms in laboratory rodents, the generic term olfactory neuroblastoma is usually preferred. they are almost always invasive tumours.^^ olfactory carcinomas forming glands, follicles and rosettes have been occasionally reported in aged syrian hamsters.^^'^^ mesenchymal neoplasms may be seen in the nasal cavity, particularly after exposure to potent carcinogens. their histological features are similar to those in the soft tissues and bone elsewhere in the body (see chapter 2). the mucosa lining the larynx and trachea becomes involved as part of an upper or lower respiratory tract infection. for instance, in rats, an acute laryngitis or tracheitis has been shown to accompany experimental infection with mycoplasma pulmonis and the sialodycroadenitis virus."^^'^^ a spontaneous degenerative condition of tracheal and laryngeal cartilage of uncertain pathogenesis associated with granulomas has been reported in fischer 344 rats.^^ the condition increases in severity and incidence with advancing age although it is seen in rats as young as 6 weeks of age. tracheal cartilage rings may also show alterations in genetically engineered animals, such as the c57bl/6j-tgn(c3-l-tag)cjeg (tag) mice that have generalized defects in cartilage development.^^ the larynx of rodents is also susceptible to the effect of inhaled substances, notably tobacco smoke but also pharmaceutical agents and propellants.^^'^^ in view of the localized nature of induced lesions in the larynx, standardized histological sectioning techniques have been proposed for rats, mice and hamsters using anatomical landmarks.^^"^^ the target site is located on the ventral floor of the larynx near the base of the epiglottis cranial to the ventral laryngeal diverticulum. lesions tend to occur in the ventrolateral region, which is covered by respiratory epithelium and the inner aspect of the arytenoid processes which is lined by squamous mucosa. the larynx responds to inhaled irritants by inflammatory, degenerative and regenerative changes in a manner similar to other regions of the respiratory tract. these include disruption of the epithelial cells, inflammatory cell exudates and infiltration, goblet cell hyperplasia and squamous metaplasia.^^ however, these changes are not specific to inhaled irritants but also occur as a response to natural respiratory tract pathogens in conventionally housed rats.'^^ the pseudostratified ciliated and non-ciliated mucosa of the trachea may also show pathological alterations in inhalations studies, although sites at the bifurcation (carina) are those often first affected. consequently, the carina should be systematically included in examination of the respiratory tract for induced lesions.^^'^^ as in the nasal passages a range of proliferative lesions including squamous hyperplasia, mucous cell hyperplasia, as well as papilloma, carcinoma and mesenchymal tumours are occasionally reported in the airways in laboratory rodents. in humans and laboratory animals, the trachea terminates at the bifurcation giving rise to two main bronchi which serve left and right lungs. depending on species, the main bronchi subdivide into further branches that enter the different lobes. various forms of branching are recognized. bronchi may arise as side-branches from a parent or stem bronchus (monopodial). the parent bronchus can divide into two equal daughter bronchus (dichotomous) or several daughter bronchi ipolychotomous)j^ study of silicone rubber casts of the respiratory tract has shown that the bronchial trees of humans and non-human primates are essentially dichotomous, in contrast to the monopodial pattern of rodents.^^ the comparatively long trachea of the dog gives rise to dichotomous upper airways but monopodial branching develops peripherally within each lobe. the size of the lungs is generally dependent on size and weight of the different species. auometric studies have shown that lung volume, alveolar surface area and diffusing capacity increase proportionally with body weight across a broad range of mammalian species, although cell size and surface area appear to be more determined by cell function rather than species size.^^ dogs have comparatively smaller body mass and higher airway dimensions compared to humans.^^ the number of lobes is species-dependent. the human lung possesses an upper and lower left lobe and an upper, middle and lower right lobe. this contrasts with the upper, middle and lower left lobes and a fourth, azygos right lobe in rhesus monkeys and baboons.^^ the dog has three lobes on both right and left sides. rats, mice and hamsters show cranial, middle, caudal and postcaval right lobes with a single, left lobe in mice and rats and a superior and inferior lobe on the left side in hamsters. cell types lining the bronchi are generally similar between species.^ the majority of cells are the ciliated cells that are accompanied by variable but relatively smaller proportions of basal cells, intermediate cells, mucous or goblet cells, serous cells, neuroendocrine and brush cells. in addition, mucous cells line the adjacent bronchial glands.^^ unlike the tracheal mucosa, which is pseudostratified, the mucosa of intra-pulmonary bronchi is non-stratified. ciliated cells are tall, columnar cells attached to basal and intermediate cells by desmosomal junctions. tight junctions exist between adjacent specialized cells at the apex. each cell possesses 200 or more cilia that are engaged in mucociliary clearance.^^ the superficial cell surface also shows a pronounced glycocalyx. the cytoplasm of ciliated cells contains scattered profiles of rough endoplasmic reticulum, a supranuclear golgi and numerous mitochondria particularly near the apex where a prominent cytoskeleton is also found. mucous or goblet cells are typical mucus-secreting cells representing about 10% of the bronchial mucosa cell population in man but less than 1% in pathogenfree rats.^ the serous cell is a cylindrical or pyramidal cell containing small, round, closely packed serous granules.^^ basal cells are compact, pyramidal cells resting on the basement membrane. they are believed to be progenitor stem cells with the intermediate cells representing an intermediate stage of cell differentiation. the mucus-secreting and ciliated cells form the cellular basis for the mucociliary clearance mechanism of the main conducting airways. the epithelium is covered by a mucous blanket that is fairly complete in humans and rabbits but patchier in rats.^ the mucous layer is segregated into an upper layer or gel phase separated from epithelial cells by a serous layer or sol phase. the complex carbohydrates of the glycocalyx and secreted mucosubstances show species-related differences in their sugar residues, which can be demonstrated histochemically by the use of labelled-lectins.^^ mucociliary clearance mechanisms are sensitive to the effects of many therapeutic agents, particularly those that alter mucins, fluid or electrolyte balance and ciliary activity. anaesthetic gases, barbiturates, narcotics and alcohol depress clearance function. by contrast, topical, oral or parenteral administration of (3-adrenergic agonists, isoprenaline and adrenalin, produce a dose-dependent stimulation of mucociliary transport by an effect on ciliary beat frequency, probably mediated by increasing levels of cyclic adenosine monophosphate in ciuated cells rather than through vascular changes. although basal mucociliary function is dependent on normal vagal tone, parasympathomimetic agents can affect mucociliary transport. acetylcholine and cholinergic agents stimulate ciliary activity whereas anticholinergic drugs, atropine and hyoscine, inhibit ciliary activity and mucociliary transport. these substances may alter deposition of inhaled particles in the lung.^ clara cells or non-ciliated bronchiolar cells located in the bronchiolar epithelium, first described by clara in 1937, are small and cylindrical in shape with highly infolded nuclei, surface microvilli, well developed golgi, abundant endoplasmic reticulum and characteristic oval, homogeneous electron-dense granules in the apical cytoplasm. in rats, rabbits and humans the granules are pas-positive, although they are usually considered pas-negative in hamster and mouse.^^ clara cells have high metabolic activity. they contain cytochrome p450-dependent enzymes and secrete a variety of proteins.^^"^^ clara cell secretory protein is the major component of their cytoplasmic granules and they have been shown to produce mucin following antigen challenge.^^ in most laboratory rodents, the conducting airways terminate abruptly at the non-cartilaginous terminal bronchiole that opens directly into an alveolar type airway, the alveolar duct which in turn communicates with the alveoli.^^ squamous epithelial or type i cells form only about 10% of all lung cells but they line over 90% of the alveolar surface, by virtue of extremely long cytoplasmic extensions. the principal gas exchange takes place across this cell. in the rat, the typical thickness of this barrier is 20 nm for a cytoplasmic extension of a type i pneumocyte, 90 nm for basal lamina and 90 nm for an endothelial cell.^^ the type i cell contains juxtanuclear mitochondria and the long smooth cytoplasmic extensions contain many ribosomes and pinocytotic vesicles. the anatomical configuration and function of type i cells render them highly vulnerable to inhaled gases and particles. the other main alveolar lining cell is the granular pneumocyte or type ii cell which constitutes about 10% of all lung cells, but which covers only about 5% of the alveolar surface.^^ this cell does not possess the long cytoplasmic processes typical of type i cells and it shows many microvilli on its luminal surface. the cell cytoplasm contains rough endoplasmic reticulum, golgi apparatus, some mitochondria and characteristic oval, osmiophilic lamellar inclusions. surfactant, a microaggregate of phospholipid and protein which modifies alveolar surface tension at low inflation volumes, is secreted by type ii alveolar cells. ultrastructural immunocytochemistry has shown the presence of surfactant apoproteins in the synthetic organelles and in the lamellar bodies of these cells, in agreement with the concept that the surfactant apoproteins are synthesized in the rough endoplasmic reticulum, glycosylated in the golgi and are stored in lamellar bodies.^^ type ii cells are more resistant to the damaging effects of xenobiotics and unlike type i cells they retain the ability to undergo mitotic division. following damage to type i cells, increased numbers of mitoses are evident in type ii cells which results in the appearance of large undifferentiated epithelial cells which ultimately differentiate into type i and type ii cells.^^ the lung also contains a dense neural network and a population of endocrinelike cells believed to be important in lung function.^^ these neurosecretory cells (kultschitsky or apud cells) are scattered sparsely in the epithelial surface of the larynx, trachea bronchi, bronchioles and alveoli. these cells are oval or cuboidal with oval nuclei, and argyrophilic cytoplasm which electron microscopic examination shows to contain dense core granules. the role of neuroendocrine cells in the lung is uncertain but immunocytochemical study has shown them to contain a number of neuroendocrine substances including neurone-specific enolase, synaptophysin, chromogranin and a variety of other peptides similar to vasoactive intestinal peptide, bombesin, calcitonin, serotonin, leu-encephalin, p endorphin and acth.^^'^^ cells lining the bronchi, bronchioles and alveolar walls are capable of metabolizing xenobiotics. immunocytochemical study has shown the presence of immune-reactive cytochromes p450, nadph cytochrome p450 reductase, epoxide hydroxylase and glutathione s-transferase in bronchial epithelial cells, ciliated bronchiolar cells, clara cells, type ii and possibly type i pneumocytes in the rat lung.^^ different cell populations contain different amounts of enzymes, clara cells containing the greatest concentrations of the phenobarbitoneinducible isoenzyme of cytochrome p450, nadph-cytochrome p450 reductase and epoxide hydrolase. studies of microsomal enzyme activities suggest that lung tissue contains fewer p450 isoenzymes than liver, principally forms cyplal cyp2b1, cyp3a2 and cyp4bl^^ whereas p450 enzyme activity is highly concentrated in specific cell types, overall microsomal enzyme activity is low compared with liver on the basis of microsomal protein weight.^^ other important cells are the pulmonary alveolar macrophages and lymphocytes. lymphocytes are found in the epithelium of the airways, in the interstitium of alveoli and as part of follicles in bronchial walls. pulmonary macrophages form part of the specific immune defence system of the lung, involving, as elsewhere in the body, antigen presentation. in the rat and mouse, distinctive populations of pulmonary macrophages have been described based on enzyme activities and reactivity to monoclonal antibodies against monocyte and macrophages surface determinates.^^'^^ bronchus associated macrophages in rat and mouse have more acid phosphatase and less non-specific esterase activity than the populations found in the pulmonary alveoli and interstitial tissues. an important aspect of the immune system is the bronchus-associated lymphoid tissue or balt, which forms part of the mucosal lymphoid system found in other epithelia. the morphology of balt is a useful guide to the nature and degree of immune stimulus in the lung. balt is organized in a way that is characteristic of other peripheral lymphoid organs. it is structurally similar in the laboratory rat, mouse, rabbit, guinea pig as well as in man but its size and prominence is species and strain-dependent as well as a function of the degree of antigenic stimulus^"^"^^. in the rat, the balt is composed of lymphoid aggregates or fouicles located mostly between a bronchus and artery with a zone of lymphocytes situated immediately under the bronchial epithelium. as in other peripheral lymphoid tissue, balt is organized into b and t cell zones but in no predetermined manner. immunocytochemical staining has shown that b and t lymphocyte zones differ in location from one aggregate to another. there are about two t lymphoc3^es for every three b cells compared with a ratio of 2:5 in rat peyer's particles.^^ the ratios may be different in other species. quantitative observations of t cell subsets using monoclonal antibodies have also shown that rat balt normally contains twice as many t-helper as t-suppressor/cytotoxic lymphocytes.^^ the t cells are confined to one or two discrete zones with a light scattering of t cells within the b cell zones and immediately under the bronchial epithelium. in common with lymph nodes, interdigitating cells are also found. the epithelium overl3dng balt shows anatomical modifications. it is composed of ciliated and non-ciliated cells covered by microvilli. in conventional, untreated laboratory rats, balt shows little activity and germinal centres are usually absent, although balt may be more prominent in some rat colonies in association with non-specific inflammatory lesions in lungs.^^'^^ in one colony of young wistar rats germinal centres were not seen in balt in untreated animals but they developed following the administration of a single intratracheal dose of lipopolysaccharide, a t cell-independent antigen.^^^ single intratracheal doses of t cell dependent antigens such as horseradish peroxidase, bovine serum albumin and bcg have been shown to produce only minor morphological changes which include expansion of the zone of lymphocytes immediately under the epithelium and infiltration of the bronchial epithelium overlying balt by lymphocytes.^^^ in addition, perivascular, peribronchial or alveolar infiltrates of small and large lymphocytes and macrophages were observed in the lungs of rats given bcg. immunoc3^ochemical study of the rat balt following intratracheal challenge with horseradish peroxidase showed that the majority of cells that infiltrated the bronchial epithelium were t helper (cd4 positive) lymphocytes. ^^^ furthermore, la antigen expression of the epithelial cells overlying the balt was shown to increase, associated with an increase in the number and size of microvilli, a more pronounced glycocalyx and a decrease in number and size of cilia. immunocytochemical study of the balt tissue in c57b1/6 mice using monoclonal antibodies to lymphoid and macrophage populations has demonstrated quite similar arrangements of cells to those in the rat with the majority of t cells belonging to the t helper (cd4 positive) class.^^ the pulmonary lymphatic system drains into mediastinal or cervical lymph nodes. although among rat strains differences in the location of lymph nodes and their drainage occur, tracer studies in the fischer 344 rat using colloidal carbon have shown that the lung lymphatics drain mainly into posterior mediastinal lymph nodes and those in the tracheal wall drains primarily to the internal jugular and posterior cervical nodes.^^^ although a variety of fixation, embedding and staining procedures are available for light and electron microscopic examination of lung tissue, there is no substitute for initial, careful visual inspection of the lungs at autopsy. uneven collapse of lungs on opening the thoracic cavity, discoloration or alteration in texture of the pleural or cut surface, congestion and presence of fluid in the larger airways may indicate structural damage. in rodent lungs, small pulmonary adenomas may be detectable by inspection in good light. fresh lung weight is also a helpful measure in lung assessment, although passive vascular engorgement can significantly affect this value. nevertheless, studies in the normal fischer 344 rat have shown that after exsanguination, wet lung weights show a close relationship to body weight and that dry weight of lungs consistently represents about 20% of the wet weights regardless of age or body weight. ^^^ an increase in wet weight over dry weight appears to be a good index of pulmonary oedema. ^^ various methods of fixation have been employed although simple immersion fixation in formalin for conventional light microscopy has the virtue of simplicity and it avoids the risk of translocation or removal of exudates from airways and alveoli. mixtures of formaldehyde, paraformaldehyde and glutaraldehyde are used in initial fixation for electron microscopy.^^ the best overall appreciation of lung architecture is achieved by instillation of fixative via the trachea under an appropriate constant pressure or by perfusion fixation of the pulmonary arteries that is less liable to dislodge intra-alveolar exudate. in a review of methods employed routinely in rodent toxicity studies, instillation of fixative via the trachea was the preferred method in most laboratories because its advantages were seen to outweigh its disadvantages.^^ the sampling procedure is an important aspect of histological examination of the bronchi and lungs, particularly those of large laboratory animals. the extent of histological sectioning in conventional toxicity studies should be modulated to take account of lesions found by macroscopic examination, the type of study and the nature of the test substance. the bronchi should be carefully sampled to allow assessment of any alterations in bronchial epithelium. morphometric analysis represents a sensitive tool of value in the evaluation of drug-induced lung changes, but it requires particularly rigorous sampling and evaluation procedures.^^^'^^^ a tiered, multiple stage or cascade sampling technique is normally considered the most appropriate for morphometric studies.^^'^ this involves dividing the lung into a series of homogeneous compartments or strata from which randomly selected samples can be examined by appropriate light or electron microscopic techniques. conventional special stains for reticulin and collagen as well as pas and alcian blue for mucins are helpful in the characterisation of lung damage and changes to the respiratory epithelium. immunocytochemistry and enzyme cytochemistry are also useful in the study of the heterogeneous cell population of the lung. xenobiotic metabolizing activity can be studied both by enzyme cytochemical methods as well as by immunocytochemical techniques using antisera specific for pulmonary monooxygenases and related enzymes.^^ important structural components, particularly collagen and laminin can be studied both at light and ultrastructural level with immunocytochemical methods.^^^ cytokeratin immunocytochemistry can be used as a method for the characterization of changes to epithelial cells.^^ clara cells can be localized by the presence of clara cell secretory protein and ciliated cells by the presence of tubulin.^^ endocrine cells are visualized by immunocytochemistry using antibodies to general neuroendocrine markers such as chromogranin and synaptophysin or regulatory peptides.^^ other useful antigens, which can be demonstrated in the lung, include surfactants, lysozyme, immunoglobulins and those of microorganisms that infect the lung.^^^ electron microscopy is particularly useful for the detailed characterization of injury to the cells of the alveolar epithelium and endothelium ( figure 6 .1). pulmonary oedema is a component of many inflammatory conditions of the lung, including those induced by infectious agents. however, the term oedema is reserved for a poorly cellular exudate characterized by the presence of pale, homogenous eosinophilic material in the alveoli, sometimes associated with a similar exudate in the lung septae and perivascular connective tissue. it occurs in a number of spontaneous conditions such as in congestive cardiac failure, metastatic pulmonary neoplasms or as an agonal change in association with pulmonary congestion and haemorrhage. drugs may induce cardiogenic pulmonary oedema as a consequence of pulmonary hypertension or impaired ventricular contractility. cardiogenic oedema is often associated with vascular congestion and red blood cells and haemoglobin may leak into airspaces. this can give rise to the presence of haemoglobin crystals within the oedema fluid in formalin-fixed tissue sections. most importantly, pulmonary oedema may be a manifestation of acute lung injury. inhalation or systemic administration of toxic chemicals may produce acute pulmonary oedema (see figure 6 .1). some substances such as phenylthiourea and a-naphlythiourea produce massive pulmonary oedema in laboratory animals when administered orally, principally as a result of damage to the endothelium of pulmonary capillaries and venules. ^^^ over 30 drugs have been reported to produce non-cardiogenic pulmonary oedema in humans, either directly or through poorly understood immunogenic mechanisms.^ another form of pulmonary oedema involves the main airways. allergic reactions in sensitized airways of asthmatic individuals is believed to result from cross-linking of ige and activation of mast cells that degranulate and release inflammatory mediators. ^^^ this has been reproduced in the main airways of rats sensitized to ovalbumin and then challenged with ovalbumin by the intratracheal route. ^^^ this treatment leads to rapid accumulation of bronchial exudate, degranulation of mast cells and the development of mucosal oedema, most marked immediately below the respiratory epithelium. congestion and haemorrhage is a frequent finding in the lungs of laboratory animals, where it is usually related to certain modes of death. it can be associated with administration of drugs and chemicals that have adverse effects on cardiac function or on the coagulation system. administration of heparin to rats produces a characteristic extravasation of blood into the air spaces.^^^ lower respiratory tract infection is generally not a major health hazard among laboratory animals but it is nevertheless an ever-present threat that can cause overt respiratory disease within a colony or develop following administration of xenobiotics. subclinical pulmonary infections and infestations can also produce histological alterations in the bronchial airways or pulmonary parenchyma which mimic changes induced by inhaled irritants or systemically administered drugs.^^'^^ furthermore, some respiratory pathogens alter immune defences and exacerbate the effects of inhaled substances. ^^^ a range of bacterial and viral pathogens may produce inflammatory lung changes.^^ typically, bacterial pathogens such as steptococcus pneumoniae produce acute bronchitis associated with a variable degree of acute inflammation of the lung parenchyma (bronchopneumonia) or a confluent lobar pneumonia. viral agents are generally associated with histological features of bronchiolitis and interstitial pneumonia, characterized by an increase in mononuclear cells in the respiratory bronchioles and alveolar septa. the histological features are variable for they depend on the particular pathogen, species and strain, immune status, presence or absence of secondary infection and the particular stage at which the infection is examined. respiratory infections are frequently mixed. changes due to secondary bacterial infection are frequently superimposed on those induced by viruses. sequential histopathological examination of the lungs of laboratory animals following inoculation with respiratory tract pathogens has been able to characterize the evolution of pathological changes produced by individual organisms. for instance, following inoculation with mycoplasma pulmonis, one of the more important respiratory pathogens among laboratory rodents both lewis and fischer 344 rats were shown to develop upper and lower respiratory tract inflammation. in the lewis strain this was characterized after 28 days by a variable acute inflammatory exudate in bronchi and bronchioles with focal bronchiectasis, inflammation and hyperplasia of the epithelium with a predominantly macrophage infiltration of the alveoli and alveolar walls.^^'^^^ these changes were associated with marked hyperplasia of the bronchusassociated lymphoid tissue (balt), which extended down the airways and blood vessels towards the periphery of the lungs. although the lymphoid hyperplasia was also found in inoculated fischer 344 rats, it was less marked and accompanied by little or no mucopurulent exudate or active inflammation of the bronchial walls. this disparity in response suggested that differences were related to the degree of lymphocyte activation in the two strains, an imbalance in regulation of lymphocyte proliferation in lewis rats, or both.^^^ other studies have been conducted in both rats and mice infected with another important respiratory pathogen of laboratory rodents, the sendai virus (parainfluenza type 1). sequential studies showed that the initial damage to bronchial and bronchiolar epithelium is associated with polymorphonuclear and lymphocytic inflammation (bronchiolitis). immunocytochemical and ultrastructural studies revealed the presence of viral antigen in the mucosa.^^^ hyperplastic and multinucleated syncytial epithelial cells develop in the hyperplastic terminal bronchiolar epithelium and the inflammatory process extended to involve peribronchial or peribronchiolar parenchyma with infiltration of alveolar walls by mononuclear cells, macrophages and neutrophils. a similar cell population accompanied by cell debris and oedema fluid develops in air spaces. pulmonary arteries show only minor involvement with inflammatory cells and focal reactive hyperplasia of the endothelium. immunocytochemistry and ultrastructural examination suggested that virus replication takes place in alveolar type i and type ii epithelial cells and macrophages but not in endothelial or interstitial cells of the alveolar septae.^^^ it was shown that when repair occurs there may be residual distortion of bronchiolar and alveolar walls by collagen and hyperplastic cuboidal epithelium may line the thickened alveolar septa. air spaces may also contain enlarged macrophages with pale vacuolated cytoplasm.^^^ strain differences in susceptibility have also been demonstrated to this virus. there is differential pulmonary interleukin 12 (il-12) gene expression between virus-susceptible brown norway rats and resistant fischer 344 rats and il-12 treatment provides protection from virus-induced chronic airway inflammation and remodelling. moreover increased tumour necrosis factor a (tnfa) expression has been shown to be an important regulatory factor in the development of sendai virus-induced bronchiolar flbrosis in infected rats.^^^ virus-inoculated brown norway rats had increased tnfa pulmonary mrna levels and increased numbers of bronchiolar macrophages and fibroblasts expressing tnfa protein compared with virus-inoculated f344 rats.^^^ the corona virus, which causes sialodacryoadenitis in many rat colonies, also produces lower respiratory tract inflammation. this is characterized by acute bronchitis and bronchiolitis with focal extension into lung parenchyma. thickened oedematous, hypercellular alveolar walls infiltrated by monocytic cells are found.^^ immunocytochemistry has shown the presence of viral antigen in bronchial and bronchiolar epithelial cells. there is also peribronchial lymphocytic infiltration and increased prominence of balt. ultimately complete resolution occurs. viruses remain a potential source of spontaneous respiratory disease in laboratory dogs. canine adenovirus type 2, parainfiuenza sv5, canine herpes virus, coronavirus and parvovirus have all been isolated from laboratory dogs developing respiratory disease.^^^ the syndrome of visceral larva migrans also incites focal inflammation, granulomas and fibrosis in the lungs of species such as dog and primate in which parasites are prevalent. the syndrome of visceral larva migrans is usually defined as that which results from the migration of nematode larvae into the viscera. it has been well described in the beagle dog lung where it results from the larvae of toxocara species or metastrongyloid nematodes.^^^'^^^ the precise identification of parasites is not always possible in tissue sections. histological appearances of infested lungs are highly variable. nematodes surrounded by granulomas and granulomatous inflammation, mostly in a subpleural location, may be visible in sections. in affected lungs there may be perivasculitis and active arteriolitis, bronchiolitis and peribronchiolitis. pleural involvement by the inflammatory process can be marked, particularly in regions overlying granulomas. scarring develops and pleural and sub-pleural fibrosis is frequently associated with epithelial hyperplasia and squamous metaplasia of the associated airways ( figure 6 .2).^^^ the lesions may sufficiently severe to resemble those induced by high doses of anticancer drugs such as bleomycin (see below). pulmonary acariasis is a common infestation of many species of non-human primates caused by various species of the mite pneumonyssus. reproduction of the mites appears to take place in the terminal bronchioles. pneumonyssus simicola is the recognized form found in rhesus monkeys.^^^ although it is most prevalent in wild caught primates, the disease is not easily eliminated during breeding in captivity. ^^^ even when eliminated by ivemectin the lesions of chronic bronchiolitis, bronchiectasis and pigmentation may persist as an incidental finding. ^^^ as the mite can produce significant destructive pulmonary pathology and render animals susceptible to secondary pulmonary bacterial infections, it can disrupt or confound the interpretation of toxicity studies performed in primates. the lesions are located most frequently in cranial lobes and are characterized by the presence of bullae distending the pleural surface, parenchymal cysts, nodules and scar tissue.^^^'^^^ histologically, there is a wide range of inflammatory activity. fully developed lesions are characterized by granulomatous bronchiolitis and peribronchiolitis with involvement of immediately adjacent alveoli. cystic lesions involving the bronchiolar walls develop around the parasites giving rise to the appearance of walled-off cysts composed of highly cellular granulation tissue, associated with neutrophils, lymphocytes, macrophages, multinucleated giant cells and various pigments (see below). in less active lesions, dilated, cystic airways with walls composed of thick bands of smooth muscle and lined by squamous or cuboidal epithelium are found. pneumocystis carinii is an important cause of pneumonia in patients with the acquired immunodeficiency syndrome (aids) as well as in other immunocompromised patients, including those treated with immunosuppressive drugs.^^"^ the natural habitat of pneumocystis carinii is pulmonary alveoli and it is widely encountered in the human population without being associated with overt disease. both clinical and experimental evidence suggests that impaired cellular immunity is much more important as a predisposing factor than impaired humoral immunity. ^^^ as in humans, laboratory animals may have latent pneumocystis infection that becomes clinically evident following immunosuppression. it has been shown in the rat that chronic administration of various regimens of adenocorticosteroids, low protein diets, cyclophosphamide and other immunosuppressive drugs with concomitant antibiotic administration to prevent other infections gives rise to typical pneumocystis pneumonia. ^^^ rodents with genetically deficient cellular immunity also develop pneumocystis pneumonia. the importance of pneumocystis pneumonia in toxicology is that it can be considered as a sentinel of chronic immune depression. in haematoxylin and eosin stained sections, pneumocystis pneumonia is characterized in both humans and rodents by the presence of alveoli filled with foamy eosinophilic material containing a few macrophages and indistinct nuclei of pneumocystis (figure 6 .2fe). ovoid or crescent-shaped structures of the organisms become clearly visible with gomori methenamine silver or toluidine blue stains. ultrastructural study of rats with pneumocystis pneumonia shows that trophozoites attach themselves most frequently to type i pneumocytes by altering their morphology to the contours of the pneumocytes rather than by a process of invasion. ^^^ systemically administered therapeutic agents may produce histological changes within the lung parenchyma that mimic components of the normal response to respiratory pathogens. however, there is no sharp separation between agents that produce pulmonary oedema with those that are associated with acute inflammatory changes and histological features overlap because an acute inflammatory process is often accompanied by exudate within airspaces. an example of drug-induced pulmonary inflammation in laboratory animals and humans is reported following the administration of interleukin 2 (il-2). il-2 is a glycoprotein lymphokine, molecular weight 15kda, which is normally produced by activated t cells and mediates immunoregulatory responses. it has been produced in large quantities by recombinant dna technology for use in tumour immunotherapy. however, high doses have been associated with a number of adverse effects, notably the 'vascular leak' syndrome, characterized clinically by pulmonary oedema, pleural effusions and ascites. ^^^ the vascular leak syndrome has been reported in laboratory animals given high doses of this agent. histological examination of the lungs of b6d2f mice developing this syndrome following administration of il-2 showed infiltration of the alveolar walls with large lymphocytes and intra-alveolar proteinaceous exudate containing large lymphocytes, macrophages and red blood cells.^^^'^^^ pulmonary venules and arterioles showed the presence of lymphocytes attached to or lying beneath the endothelium, infiltrating vessel walls or in a perivascular location where they were accompanied by oedema fluid or red blood cells. similar, but less severe changes have been demonstrated in rats given il-2.^^^ in addition, treated rats showed an infiltration of pulmonary vasculature with eosinophils probably secondary to an eosinopoietic cytokine produced by il-2 stimulated lymphocytes. immunocytochemical evaluation of the lymphoid infiltrate in mice showed that most of the cells were thy 1.2positive (cd90) lymphocytes. furthermore, co-administration of asialo gml (ganglio-n-tetrosyl-ceremide) with il-2 not only abrogated the clinical signs but also reduced the number of asialo gml-positive lymphocytes in the tissue sections. as lymphoid cells expressing lyt-2 (cds, suppressor/cytotoxic t cells) were unaffected by asialo gml treatment, it was postulated that the vascular leak syndrome (but not antitumour efficacy) in these mice was mediated by an endogeneous subset of il-2 stimulated lymphocytes or lymphokine-activated killer cells. ^^^ corresponding changes were also observed in liver and lymphoid tissue. immunocytochemical and detailed electron microscopic studies in rats have supported the concept that il-2 induces cytotoxic vascular damage that is mediated both directly by lymphokine-activated killer cells and cytotoxic t lymphocytes with secondary release of inflammatory cytokines.^^^ as in humans, severe chronic pulmonary inflammatory disease in laboratory animals may compromise pulmonary function and lead to secondary alterations in other organs. although the mechanisms were not explored in detail, a diffuse interstitial pulmonary inflammatory process with lung haemorrhage was induced in rats treated for two years with prizidilol (skandf 92657-a2), an antihypertensive agent with both vasodilator and (3 adrenoceptor blocking properties.^^^ affected animals developed dyspnoea associated with reduction in lung volume, deformity of the thoracic spinal column and marked cardiac hypertrophy. inflammation with granulomas develops in the lungs of laboratory animals under a variety of different circumstances, which have been alluded to above. a common cause in rodents is granulomatous pulmonary inflammation resulting from aspiration of stomach contents or food particles (aspiration pneumonia). this is sporadically observed in aged rodent where it is associated with general ill health, particularly resulting from pressure effects of large pituitary adenomas and subsequent disturbance of pharyngeal or laryngeal reflex mechanisms. ^^^ histologically, the lungs show peribronchial and peribronchiolar granulomatous inflammation with macrophages and foreign body cells associated with fragments of refractive vegetable matter. the associated bronchial mucosa may also show reactive changes including goblet cell hyperplasia in long-standing cases. as dogs and primates are more liable to be infested by parasites, granulomatous inflammation in response to pulmonary larvae is more common in these species. pulmonary tuberculosis represents a potential problem among non-human primate colonies in view of its insidious onset and its liability for transmission from monkeys to humans. ^^^ pathological findings are similar to those so well known in the human disease. the disease is characterized by the presence of granulomas in lung parenchyma and lymph nodes. in florid cases there may be caseation surrounded by epithelioid and multinucleated giant cells and variable numbers of lymphocytes, plasma cells and flbroblasts. diffuse granulomatous pneumonia as a result of tuberculosis is also reported in non-human primates. granulomatous pneumonitis is also produced in laboratory animals by the intravenous injection of bcg. twenty-eight days following intravenous injection of bacille calmette-guerin (bcg), the lungs of c57b1/6 mice contained numerous granulomas composed of histiocytes and round cells which were surrounded by alveoli with thickened walls and associated with mild interstitial pneumonitis. ^^^ these histological changes were associated with an increase in the number of thy 1.2-positive (cd90) cells, especially lyt-1 (cds) positive lymphocytes. the histological changes were abrogated by treatment with cyclosporin a suggesting an important role for cd5-positive lymphocytes in the development of the granulomas. discrete granulomas occur in the lungs of experimental animals in response to intra-tracheal or intravenous injection of certain relatively insoluble substances (figure 6.3) . intra-tracheal administration of insoluble polymerized dextran and latex micro-particles to mice showed that the morphology and the systemic effects of granulomas depends on the nature of the injected substances. it has been shown that large granulomas develop rapidly in the pulmonary parenchyma around dextran particles that subsequently regress quickly, whereas latex particles produce small, discrete stable granulomas. ^^^ although both forms of granulomas are of foreign body or non-immunological in type, those produced by dextran but not latex beads, are associated with anergy-like immunosuppression, probably caused by release of soluble factors from the granulomas. it has been reported that granuloma formation after instillation of sephadex beads is associated with increases in the interleukin 1-(il-1) like activity in the lung.^^^ studies comparing the effects of inhaled crystalline silica and titanium dioxide have shown a correlation between the release of the macrophage derived cytokine il-1 and granuloma formation, suggesting that il-1 might be a useful biomarker for granuloma formation. ^^^ localized, angiocentric granulomas of foreign body type, clustered around pulmonary arteries and arterioles and occasionally alveolar capillaries and venules also develop following intravenous injection of relatively insoluble polysaccharides or other polymers. ^^^ characteristic epithelioid and large, foreign body type giant cells efface the smaller vessels although overt necrosis is not usually observed (figure 6.3) . haemosiderin-laden macrophages accumulate in the alveou of laboratory animals in association with chronic pulmonary congestion and haemorrhage. similar changes occur in patients in congestive cardiac failure where the haemosiderin-laden macrophages are termed 'heart failure' cells. the lungs of non-human primates are especially liable to contain alveolar, perivascular and peribronchial aggregates of macrophages laden with various brown pigments. iron-containing pigments have been associated with the inflammatory changes produced by simian lung mites (pneumonyssus simicola) which are prevalent in many non-human primates. in addition, lungs from some primate colonies may show perivascular and peribronchial collections of brown-grey macrophages containing highly refractive spicules and plates composed of high concentrations of silica.^^^'^^^ it has been shown that in old world primates including rhesus and cynomolgus monkeys, this pigment contains fossil diatomaceous material, compatible with the concept that the animals inhale dusts containing diatoms and other silicon fragments to which they are exposed in their semi-arid, natural habitats.^^^ chronic lung injury from a variety of different causes is frequently associated with the development of pulmonary fibrosis characterized by the replacement of the normal pulmonary structure by a thickened collagenous matrix with consequent reduction in the capacity for gas exchange. regardless of the inciting agent, the fibrogenic process appears to be generally characterized by disruption of the normal alveolar-capillary structure, leakage of exudate from the vascular compartment into the airspaces, subsequent invasion by infiammatory cells and fibroblasts associated with excess matrix formation. studies in laboratory animals with different fibrogenic agents as well as in humans have suggested that central to pulmonary fibrogenesis is increased production of tnfa by macrophages.^^'^^^'^^^"^^^ this cytokine is a not only a mitogen for fibroblasts but also a potent activator and chemo-attractant for macrophages, capable of stimulating release of other cytokines and inducing expression of adhesion molecule expression on endothelial cells. moreover, it has been shown that tnfa receptor knockout mice appear protected from the fibroproliferative effects of inhaled asbestos. ^^^ pulmonary fibrosis is a common sequel of chronic lower respiratory tract inflammation. it may be associated with, or preceded by interstitial pneumonitis, characterized by infiltration of lymphocytes, plasma cells and macrophages with scattered polymorphonuclear cells. ^^^ focal pulmonary fibrosis occurs spontaneously in laboratory animals, although this is usually most prevalent in dogs and non-human primates as a response to chronic infestation by parasites, which are not easily eliminated during breeding. in humans, conditions leading to pulmonary fibrosis vary widely. they include infections, shock lung syndrome, ionizing radiation, inhalation of irritant particulate matter, exposure to antigens or excessive amounts of oxygen as well as the results of the toxicity of paraquat and a range of both cytotoxic and noncytotoxic therapeutic agents which cause pulmonary parenchymal injury.^'^^^ the principal therapeutic agents that produce pulmonary fibrosis in both humans and laboratory animals are anticancer drugs. bleomycin, a glycopeptide preparation derived from streptomyces verticillus, is the best known example but pulmonary fibrosis is also associated with the clinical use of a number of other anticancer agents, including l,3-bis-(2-chloroethyl)-l-nitrosourea (bcnu or carmustine), cyclophosphamide, busulphan, mitomycin c and methotrexate.2 '108,147-150 the precise mechanisms involved in the induction of pulmonary fibrosis by antineoplastic drugs in humans are poorly understood. the true incidence for a particular drug is difficult to estimate because of confounding factors in cancer patients, such as concomitant administration of several drugs, radiation and oxygen therapy, diffuse pulmonary cancer and opportunistic infections. it is probable that drug-induced fibrosis is accentuated by concomitant administration of several antineoplastic agents, radiation therapy, hyperoxia, preexisting pulmonary damage and age of the patient. severity is often related to total dose of drug received.^^^ novel antineoplastic drugs may also produce lung toxicity.^ bleomycin is associated with the development of interstitial pneumonia and pulmonary fibrosis in clinical use and this can be reproduced in experimental animals. the histopathological appearances of bleomycin-induced pulmonary fibrosis in patients are in many instances different from those seen in laboratory animals because the lungs of patients treated with bleomycin are modified by the primary neoplastic disease, smoking, multiple drugs, radiation therapy and secondary pulmonary infections, interstitial pneumonitis and fibrosis.^^^ it has been postulated that tnfa is an important mediator in the development of bleomycin-induced fibrosis. ^'^ in the preclinical evaluation of bleomycin beagle dogs were given cycles of drug by the intravenous route for periods of up to 26 weeks. ^^^ dogs developed anorexia, weight loss, a variety of epithelial lesions as well as focal interstitial pneumonia and fibrosis. the focal lung lesions were characterized by increased elastic fibres, reticulin, collagen and acid mucosubstances. the lesions were situated predominantly in the pleural and subpleural zones, suggestive of a potentiating effect of friction between the pleural surfaces. histologically the lesions resembled those produced by larvae migrans in the dog (see figure 6 .2a). similar histological changes have also been described in both rats and mice treated with bleomycin by both the intravenous and intratracheal route.^^^'^^^ as fibrosis is such a consistent change, bleomycin-treated rodents have been extensively employed as a model for pulmonary fibrosis. early changes include mild, diffuse increases in interstitial lymphocytes, macrophages, polymorphonuclear cells and perivascular or interstitial oedema. after about a week, interstitial infiltrates also comprise fibroblasts with early collagen deposition, associated with proliferation of macrophages and type ii pneumocytes.^^^'^^^ subsequently, the amount of interstitial collagen increases, with eventual scarring and collapse of lung tissue in proportion to the cumulative dose given. ^^^ immunohistochemical and ultrastructural study of rats and mice treated with bleomycin shows a large accumulation of immune-reactive laminin and reduplication of the basement lamina within the thickened alveolar walls. ^^^ in bleomycin-treated rats three-dimensional scanning electron microscopy shows drug-induced capillary remodelling comprising irregular alveolar and pleural capillaries with increased diameter and decreased branching. ^^^ certain strains of mice have been shown to possess greater sensitivity to bleomycin fibrogenesis. the c57bl/6 strain produces a greater fibroblastic response than dba/2 and swiss mice and the balb/c strain demonstrates a particularly poor fibroblastic response. ^^^ therapeutic use of cyclophosphamide is also occasionally associated with the development of pulmonary interstitial fibrosis.^^^'^^^ it appears to be associated with two forms of pathology: an early-onset pneumonitis and a late onset progressive pulmonary fibrosis. ^^^ similar changes have been less easy to reproduce in laboratory animals. when mice were sequentially examined for periods of up to one year after a single intravenous dose of loomg/kg of cyclophosphamide, only slight pulmonary interstitial thickening and hypercellularity was observed in association with progressive multifocal accumulation of intra-alveolar macrophages. ^^^ however, these changes were also accompanied by a progressive increase in pulmonary hydroxyproline content and a decrease in pulmonary compliance with time in treated animals compared with controls. the changes were amplified by exposure to 70% ambient oxygen. the bronchiolitis, alveolar septal infiammation and fibrosis induced by gold therapy in patients with rheumatoid arthritis is probably immune-mediated. this condition is associated with peripheral eosinophilia and drug-induced alterations to the immune system.^^^ emphysema is characterized by abnormal, permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis. three principle types, centriacinar, panacinar and distal acinar emphysema, are recognized in humans. enlargement of air spaces as a result of congenital factors or fibrous scarring are grouped separately and not regarded as emphysema. ^^^ emphysema has been reported as an age-related spontaneous change in laboratory rats.^^^ however, several experimental rodent emphysema models have been developed, using intratracheal instillation of proteolytic enzymes papain, pancreatic and neutrophil elastase. this gives rise to histological appearances resembling panacinar emphysema in humans.^^^ irritant gases, notably oxides of nitrogen, are also capable of inducing changes in the lungs of laboratory rats and hamsters following long term exposure which resemble mild human, centrilobular emphysema.^^^'^^^ a variety of different names have been applied to membrane-bound, acid phosphatase-positive cytoplasmic inclusions with a lamella or crystalloid ultrastructural matrix. these include myeloid bodies, myelinoid bodies, myelin figures or myelinosomes. these lysosomal inclusions are seen in small numbers in a variety of normal cell types but they accumulate in various organs in laboratory animals following administration of a wide variety of drugs of diverse therapeutic classes.^'^^^"^^^ the generalized accumulation of these lysosomal cytoplasmic bodies is generally called phospholipidosis, a term coined to describe the tissue accumulation of phospholipids. ^^^ at the light microscopic level, phospholipids are characterized by the increase in the number of foam cells in the airspaces. examples of drug-induced phospholipidosis include the anorectic drug chlorphentermine, tricyclic antidepressants, inhibitors of cholesterol biosynthesis such as triparanol, the antihistamine chlorcyclizine and its analogues, the selective oestrogen receptor antagonist tamoxifen, chloroquine and the cardiovascular drugs amiodarone, 4,4'diethylamino-ethoxyhexestrol and perhexiline.^^^'^^^'^^^ many tissues and organs may develop the cytoplasmic inclusions, including lymphoid cells, liver, pancreas, endocrine tissue, nervous system, muscle cells, eyes and particularly lungs. aminoglycoside antibiotics may produce laminated phospholipid inclusions in the renal tubular cell and imidazol antifungals in hepatocytes (see under liver and kidney, chapters 9 and 10). many drugs that induce phospholipidosis usually share structural features, notably a hydrophilic cationic side chain, a primary, secondary or tertiary amine and a hydrophobic region that is usually an aromatic ring or ring system. as this structural pattern renders these molecules amphiphilic, these drugs probably bind with polar lipids by means of electrostatic and hydrophobic forces.^^"^ this leads to formation of drug-lipid complexes which are poorly degraded by lysosomal enzymes and which accumulate in the cell cytoplasm to form the inclusions described above. as the binding is not covalent, its reversibility depends on the dissociation rate constant under the particular intracellular conditions and drug concentration achieved. predictions of this activity based on molecular structure have shown reasonably good correlation with the ability of compounds to produce phospholipidosis in cultured rat peritoneal macrophages. more recently other cell based systems have been proposed for screening for phospholipidosis.^^^'^^^ however these perform less well in the prediction of in vivo potency, presumably because of differences in drug disposition in blood and tissues. it should be underlined that the accumulation of foamy macrophages in the alveolar spaces may also be a spontaneous change in laboratory animals. it has long been recognized as a spontaneous alteration in ageing rats.^^^ it may also be found in lung tissue distal to bronchial lesions that impede clearance mechanisms. in contrast to drug-induced changes, the spontaneous lipidosis characterized by accumulation of alveolar foam cells occurs sporadically in older rats and is observed in both controls and treated animals. drug-induced phospholipidosis occurs within a period of several months during which lungs of control animals remain fairly free of foam cell accumulation. the lungs appear especially vulnerable to drug-induced phospholipidosis, possibly because macrophages are in very close proximity to blood-borne agents. phospholipidosis is also more clearly visible microscopically in alveoli whereas it can be easily overlooked in other organs. the continuous uptake of phospholipid-rich surfactant material from the alveoli by macrophages leads to excessive accumulation of phospholipids when their catabolism is impaired.^^^'^^^ the fact that lungs are commonly affected is a potentially useful diagnostic feature because in many organs phospholipidosis can be extremely difficult to recognize in haematoxylin and eosin stained sections. although the changes in the lungs are not specific for drug-induced phospholipidosis, an increase in the number of lipid-containing lung macrophages in treated animals compared with controls is relatively easy to detect and provides a simple way for the pathologist to screen for this effect. in severe generalized phospholipidosis in rats, the lungs show irregular pale grey or yellowish patches of discoloration of the pleura and parenchyma. this is a result of patchy or confluent aggregates of large, pale, foamy macrophages. they may be free lying or packed in alveoli and accompanied by granular, extracellular material. their abundant cytoplasm shows a vacuolated appearance in which fine eosinophilic granules are sometimes visible. the nuclei are rounded and centrally located structures of variable size (figure 6.4) . multi-laminated cells are also occasionally seen, as are vacuolated cells firmly attached to alveolar walls, probably pneumocytes. these foamy cells stain typically for phospholipids (e.g. acid haematin), although neutral lipids may also be present and stain with oil red o. semi-thin plastic-embedded sections stained with toluidine blue allow better characterization of phospholipidosis in all organs, including the lungs. the macrophages in the air spaces contain unmistakable dense, dark round cytoplasmic inclusions of variable size, some over 5 mm diameter. ^^^ plasticembedded sections also show the inclusions in other pulmonary cells including pneumocytes attached to the alveolar walls, from which they can be seen discharging into the alveolar spaces. as in other organs affected by phospholipidosis, ultrastructural examination reveals dense, multi-lamellar membranes and numerous heterogeneous dense bodies of lysosomal origin (figure 6.4) . these bodies need to be distinguished from membranous bodies that form as a result of fixation for ultrastructural study. lipids tend to leach out and become hydrated to form myelinoid membranes during glutaraldehyde fixation. these structures are subsequently fixed by osmium to give rise to electron-dense membranous figures both outside and inside cells, particularly in mitochondria where they may be mistaken for pathological lesions.^^^ the lamella patterns seen in phospholipidosis may be simple alternating dense and clear lines spaced at 4-5 nm, or more complex arrangements of clear and dense lines. the other typical crystalloid inclusions of hexagonal aggregates of tubular subunits seen in other organs are not usually found in the lungs. the significance of these various forms is uncertain but they probably represent the various phases in which phospholipids exist and are influenced by proportions of lipids present. electron microscopic examination reveals that not only are pulmonary macrophages affected by these changes but that inclusions may be present in pneumocytes types i and ii, pulmonary capillary endothelial cells, smooth muscle cells, bronchiolar epithelium and occasionally neutrophils.^^^"^^^ the changes are typically still visible several weeks after withdrawal from treatment with the offending agent. although the extent of pulmonary phospholipidosis in the lungs varies between dosage regimen and animal species, studies with chlorphentermine, 4,4'diethylaminoethoxyhexestrol and amiodarone indicate that similar cytological and ultrastructural changes occur in most laboratory animal species studied including rats, mice, hamsters, guinea pigs, rabbits and dog.^^^'^^^'^^^'^^^ safety assessment -amphiphilic drugs what are the imphcations for humans of drugs that induce phosphohpidosis in laboratory animals? novel compounds continue to be found that possess the property of producing phosphohpidosis in laboratory animals with varying degrees of severity.^^^'^^^ although not all drugs that produce phosphohpidosis in animals have been studied in humans, only very few drugs that produce phosphohpidosis in animals have been shown capable of inducing significant phosphohpidosis in human clinical practice.^ agents such as chloroquine, 4,4'diethylamindethoyhexestrol and amiodarone, which have been shown to produce phosphohpidosis in patients, can also induce cellular damage in the same organs. however the phenomenon of phosphohpidosis where phospholipids are packaged behind lysosomal membranes may not be causally related to cellular damage in humans. indeed the weight of evidence suggests that druginduced phosphohpidosis per se is an adaptive phenomenon and does not in itself have functional or deleterious consequences unless excessive. ^^^ hence, the finding of phosphohpidosis in animal studies with a novel drug requires careful assessment on a case by case basis with respect to its implications for the safety of humans. an example of this issue is the iodinated benzofuran derivative amiodarone, a potent antiarrhythmic drug effective against ventricular arrhythmia. lung toxicity continues to be a problem in patients treated for cardiac arrhythmias with this drug.^^"^ not only does phosphohpidosis occur in a wide variety of organs in laboratory animals treated with amiodarone,^^^'^^^ but also in liver, peripheral nerve cells, skin, lymphoid cells and lungs in patients at therapeutic doses.^^^'^^^'^^^ although pulmonary interstitial fibrosis occurs in association with phosphohpidosis in patients, amiodarone-induced phosphohpidosis in rodents is not associated with pulmonary fibrosis or significant functional alterations. several theories have been proposed for the pulmonary alveolitis and interstitial fibrosis in humans. the weight of evidence to date suggests that the accumulation of lipid-laden histiocytes is not causally related to the alveolitis or pulmonary fibrosis. ^^^ indeed, overall there is little evidence that the mere presence of phosphohpidosis is deleterious to the organism.^^^ cytotoxicity, possibly through the metabolite desethylamiodarone, has been proposed and an immune-mediated mechanism has been postulated, possibly favoured by the binding of drug to components of pulmonary tissue.^^^ it might also involve free radical formation or indirect influences on inflammatory mechanisms.^^^ it is also possible that pulmonary disease results from an interaction of several mechanisms and metabolic factors unique to particular patients. ^^^ despite undoubted differences in tissue and species sensitivity to development of phosphohpidosis, dose, drug disposition, metabolism and elimination and the degree of tissue exposure to drug are important considerations in safety assessment of drugs that produce phosphohpidosis in laboratory animals. although phosphohpidosis is more likely to occur at high doses employed in toxicity studies than at lower therapeutic doses used in patients, it has been suggested that this may be offset by faster ehmination of the drug, characteristic of small laboratory animals. ^^^ the potential for drugs to accumulate in critical tissues such as eye and heart is especially important when drugs are administered for long periods of time, particularly as tissue/plasma ratios of some amphiphilic drug may exceed 100, following repeated administration.^^^ consequently, although phospholipidosis may not have functional consequences, any implications for humans of drugs that induce phospholipidosis in laboratory animals can only be assessed on a case by case basis, with due consideration of mechanism, drug disposition and clinical risk-benefit analysis. it is important to underline that similar morphological changes due to the increased presence of phospholipids in lysosomes can also result from treatment with compounds that are not cationic amphiphilic structures. mechanisms include direct or indirect inhibition of lysosomal enzyme activity. this reenforces the need to understand the mechanism of any chemically induced increase of phospholipids in the lungs of laboratory animals. for example, it has been shown that when glycosaminoglycans accumulate in inherited human lysosomal disorders they inhibit other lysosomal enzymes, thereby inducing lysosomal phospholipid inclusions. ^^^ this is reflected by administration of high doses of the trypanocidal drug suramin to rats which induces intracellular storage of glycosaminoglycans associated with phospholipid inclusions in diverse organs including lungs. ^^^ although at light microscopy clear vacuoles are typically seen, electron microscopic examination shows the presence of both clear vacuoles containing glycosaminoglycans and lamellar phospholipid inclusions. a similar effect seems to have been produced in rats by elmironâ®, a semi-synthetic heparin-like macromolecular carbohydrate derivative, chemically and structurally similar to glycosaminoglycans used clinically for anticoagulant effects and interstitial nephritis.^^^ another example is the induction of lysosomal inclusions in the lungs of rat and dogs by the macrolide antibiotic erythromycin.^^^ collections of foam cells were described in the lungs and lymphatic tissues of dogs and rats treated with high oral doses. foam cells in the lung showed a pattern of small whorls in vacuoles similar to that seen with other drugs that induce phospholipidosis. in vitro studies have suggested all macrolide antibiotics have the potential to cause phospholipidosis. biochemical studies suggest that drug binds to phosphatidylinositol-containing liposomes and inhibits activity of lysosomal phospholipase in close correlation with the number of cationic groups carried by each of the drugs. ^^^ hook reviewed other agents, such as oxidant gases and insoluble particles including silica, that can also increase phospholipid levels and histological appearances of phospholipidosis in the lungs.^^^ some of these agents inhibit phospholipid catabolism in the lungs giving rise to accumulation of surfactant protein a and surfactant lipoproteins and a clinico-pathological picture similar to pulmonary alveolar proteinosis in humans. studies from humans have shown that three chnically distinct forms of this condition occur: congenital, secondary or acquired. the congenital disease is caused by a diverse range of mutations in the genes encoding surfactant proteins or the (3c chain of the receptor of granulocyte-monocyte colony stimulating factor (gm-csf). the secondary form occurs in association with conditions where there is functional impairment or reduced numbers of alveolar macrophages, such as in haematological cancers, following immune suppression or inhalation of silica or toxic fumes. acquired or idiopathic alveolar proteinosis that accounts for over 90% of all cases (0.37 per 100000 persons) has been an enigma until recently. patients are at risk from infections, particularly nocardia, and the 5 year survival rate appears to be about 75%. studies from transgenic mouse models and in humans have shown that autoantibodies against gm-csf are important in the development of the acquired form of the disease as this antibody causes a defect in macrophages function which impairs the catabolism of surfactant lipids and proteins. ^^^ in this context it is of interest to note that treatment with imatinib, a tyrosine kinase inhibitor of the bcr-abl tyrosine kinase constitutively expressed in philadelphia chromosome positive myeloid leukemia, has been associated with the accumulation of lamellar inclusions in pulmonary macrophages in a leukaemia patient, although this might have been a result of the primary disease. ^^^ studies in mice have suggested that imatinib mesylate actually inhibits the fibrogenic activity of transforming growth factor (3 and prevents fibrosis induced by bleomycin.^^^ various forms of hyperplasia are found in the airways and lungs of laboratory animals. the mucosal surface of the bronchi may show hyperplasia of the goblet cells, squamous hyperplasia or metaplasia. the cells lining the terminal bronchiole and alveolus may also show hyperplasia and squamous metaplasia. standard classifications for the characterization of these changes in histological sections have been developed for use in rodent studies.^^"^^ goblet cell hyperplasia is a well-recognized response of the mucosa of conducting airways to chronic inflammation and inhalation of irritant substances such as cigarette smoke and sulphur dioxide.^^'^^'^^^'^^^ the degree of goblet cell hyperplasia is dictated by the severity and duration of the irritation or inflammatory process. florid cases of goblet cell hyperplasia are characterized by thickening and pseudostratification of the tracheal or bronchial mucosa by a population of tall, mucus secreting cells with abundant pale cytoplasm. in addition, goblet cells extend further down the airways than in normal animals and mucus may fill or distend the airways or impact in the alveoli. in less florid cases, a simple increase in the number of goblet cells may be found without other structural change.^^ goblet cell hyperplasia of the lining epithelium may be accompanied by an increase in size of the underlying submucosal glands. this has clearly been demonstrated in patients with chronic bronchitis and in rats where submucosal glands are normally quite prominent.^^^'^^^ species differences may exist because the airways of laboratory animals are variably endowed with goblet cells and submucosal mucous glands. the normal rat has more goblet cells lining the airways than either mouse or hamster. ^^^ the factors controlling these alterations are uncertain but is has been long suggested that increased mitotic activity as well as cell conversion, probably by metaplasia of serous or clara cells to mucous cells, is involved.^^^ it has more recently been shown in mice sensitized to ovalbumin and subject to a single antigen challenge by aerosol that clara cells in the proximal airways show great plasticity and become mucin-secreting cells.^^ pharmacological agents can induce goblet or mucous cell hyperplasia. rats given six or 12 daily injections of isoprenaline, a non-selective (3 receptor agonist showed a dose-and time-dependent increase in the number and size of alcian blue-positive goblet (mucous) cells as well as serous cells in the tracheal and bronchial mucosa. this was associated with an increase in length, width and depth of submucosal glands.^^^ similar changes were produced by pilocarpine, although both alcian blue-and pas-positive cells were increased in number following this agent, suggesting that pilocarpine induced both acid and neutral glycoprotein secretion. comparison of the distribution of these changes in the rat following isoprenaline, with those of salbutamol, pilocarpine and tobacco smoke, showed that there were regional differences in the distribution of these changes in the airways.^^'* isoprenaline produced a greater increase in secretory cells in peripheral airways than tobacco smoke, which itself produces a greater increase in mitotic activity. isoprenaline and pilocarpine produced a more diffuse change than the more selective (3 agonist, salbutamol. the changes induced by these therapeutic agents are presumably the result of their pharmacological activity.^^'^ sturgess and reid showed that the changes in the rat were accompanied by hypertrophy of the pancreas, submaxillary and parotid salivary glands^^^ (see digestive system, chapter 8). unlike the rat and mouse, the hamster appears predisposed to develop minor multifocal epithelial hyperplasia of the tracheal and bronchial mucosa spontaneously with advancing age. these changes are flat or polypoid in nature and are composed of clear cells and goblet cells.^^'^^ the epithelium of the bronchi shows squamous metaplasia in response to chronic irritation or injury. it is characterized by three or more layers of epithelial cells with abundant eosinophilic cytoplasm with prominent cell boundaries. it may be associated with degenerative alterations to the mucosa or goblet cell hyperplasia. squamous metaplasia can also develop in the alveolar parenchyma as a response to prolonged damage such as produced by large burden of inhaled irritant or insoluble dusts. the metaplasia is also characterized by the presence of several layers of flattened epithelial cells showing squamous differentiation. the term pulmonary keratinizing cyst has been recommended for pulmonary cystic lesions lined by non-neoplastic squamous epithelium without excessive proliferative change.^^^ hyperplasia, bronchiolo-alveolar (type ii cell hyperplasia) hyperplasia may involve the lining epithelium of the alveoli or bronchioli. this form of hyperplasia has been termed alveolar hyperplasia, adenomatosis, alveolar bronchiolization or epithelialization. it occurs spontaneously but can be induced by infections and administration of irritant xenobiotics in rats/^'^^^"^^^ j^j^g64,209 ^^^ hamsters.^^^ histologically, the lesions consist of localized but unencapsulated foci of hyperchromatic regular, cuboidal or columnar cells investing airspaces without appreciable distortion of alveolar walls. neuroendocrine hyperplasia is well described in hamsters. although small aggregates of neuroendocrine cells (neuroepithelial bodies) are found at various levels of the bronchi and bronchioli in normal hamsters, administration of nitrosamines and 4-nitroquinoline 1-oxide produces neuroendocrine hyperplasia.^^^"^^^ hyperplastic lesions are recognizable as groups of non-ciliated cuboidal, oval or columnar cells located in the bronchial or bronchiolar epithelium. they contain argyrophilic granules that show immunoreactivity for corticotrophin (acth) and neurone-specific enolase. ultrastructural examination reveals the presence of dense-core cytoplasmic granules of apud type. proliferative changes have also been reported in other species, including rats and humans in hypoxic conditions, although it has been suggested that these changes might be a result of increased peptide content rather than cell proliferation.^^'^^^ the most frequently diagnosed neoplasm world wide is lung cancer, where it is usually caused by smoking tobacco.^^^ bronchogenic squamous carcinoma is generally the most common subtype but in north america the incidence of adenocarcinoma now exceeds that of squamous cell tumours for reasons not fully understood. some of the most aggressive subtypes are small and large cell neuroendocrine lung cancers, defined as small or large tumour cells with greater than ten mitoses per 2mm^.^^^ these seem to be seen almost exclusively in heavy cigarette smokers. in contrast to findings in people, squamous cell lung tumours are only occasionally seen arising spontaneously in laboratory animals. even laboratory animals -rats, mice, hamster, monkeys and dogs -exposed to tobacco smoke for long periods and at high doses fail to develop an increase in lung tumours.^^^'^^^ moreover, there appears to be no good experimental model for neuroendocrine lung cancer. thus, particular caution is merited if using animal models for prediction of lung tumorigenic potential of inhaled substances. by far the most common primary pulmonary neoplasms found in laboratory rats, mice and hamsters are adenomas and adenocarcinomas. these appear to develop from the bronchiolar or alveolar epithelium, although their precise histogenesis is somewhat disputed. although spontaneous squamous neoplasms are uncommon in rodents cystic keratinizing lesions can be induced in rats by high burdens of particulate material in the lungs.^^^ pleural mesotheliomas and mesenchymal neoplasms also occur in these species but are uncommon. they can be induced in rodents by mineral fibres.^^^ mesenchymal tumours have similar histological features to those in soft tissues and mesotheliomas may show either epithelial or mesenchymal differentiation or both. in most rat strains alveolar or bronchiolar neoplasms occur spontaneously in relatively small numbers, but morphologically identical neoplasia can be induced by administration of chemical carcinogens.^^^ the most common are classified as bronchiolo-alveolar adenoma (pulmonary adenoma) and bronchioloalveolar carcinoma. the national toxicology program database on control fischer 344 rats used in carcinogenicity studies indicates an overall percentage of less than 3% of animals with bronchiolo-alveolar adenomas and less than 1% with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar adenomas in different studies was between 0 and 14% in this series. histologically, bronchiolo-alveolar tumours are mostly small, discrete, rounded nodules located in the lung parenchyma and composed of fairly uniform cells with moderately hyperchromatic nuclei arranged in solid (alveolar), tubular, papillary or mixed growth patterns. they usually compress surrounding tissues without infiltration or metastatic spread (adenoma), although loss of differentiation, infiltration and spread to adjacent tissues can occur (adenocarcinoma). ultrastructural study of bronchiolar-alveolar neoplasia in fischer 344 rats has shown the presence of osmiophilic, lamellated inclusion bodies similar to those found in alveolar type ii cells. therefore it has been suggested that the neoplasms are derived from this cell type.^^^ pulmonary squamous carcinoma occurs but is a very uncommon spontaneous neoplasm in the rat.^^ the large proliferative but benign cystic lesions found in the lungs of rats following accumulation of large amounts of particulate matter have been termed pulmonary cystic keratinizing epitheliomas for they have been regarded as benign neoplasms. when these lesions show evidence of tissue invasion they are regarded as pulmonary squamous cell carcinomas. similar lesions are very occasionally reported as spontaneous lesions.^^^ analogous neoplasms are found more commonly in most strains of laboratory mice used in carcinogenicity bioassays although considerable variation in incidence is reported. they are common in strain a mice where they are observed in low frequency at 3-4 months of age and incidences reach nearly 100% by 24 months of age.^^^ fewer, but significant numbers are found in b6c3fi mice, although there is considerable inter-laboratory variation.^^^ the national toxicology program database on control b6c3fi mice used in carcinogenicity studies indicates an overall percentage of about 16% of males and 6% of females with bronchiolo-alveolar adenomas but only about 5% and 2.5% respectively with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar tumour varied considerably between studies in this series. even in the same laboratory, mice housed under similar conditions show variation in incidence in these neoplasms with time. the incidence of lung adenomas and adenocarcinomas occurring in cd-i mice used as controls in 18-month carcinogenicity bioassays in the same laboratory under similar conditions for a period of 3 years varied from between 19 and 36% in males and 6 to 16% in females.^^^ by contrast, some strains of mice such as the c5781/10j strain show a very low predisposition to the development of lung adenomas.^^^ although these mouse pulmonary adenomas and adenocarcinomas do not resemble the common lung tumours in humans, strain differences have been exploited to study genetic susceptibility and resistance to pulmonary adenomas and carcinomas.^^^'^^^ histologically, pulmonary tumours of this type in mice are generally small, sharply circumscribed nodules composed of fairly uniform, closely packed columns of cuboidal or columnar cells arranged in tubular or papillary structures with scanty fibrovascular stroma ( figure 6 .5). they may be less well differentiated, with cellular pleomorphism, and show intrabronchial growth, invade lung parenchyma and produce metastatic spread. the histogenesis of mouse pulmonary adenomas and adenocarcinomas is disputed. on the basis of sequential light and electron microscopic study of pulmonary adenomas induced in bagg-webster swiss mice by transplacental exposure to ethylnitrosourea, it has been suggested that they develop from either alveolar type ii cells or clara cells.^^^'^^^ careful, stepwise analysis using light microscopic and electron microscopic examination has suggested that adenomas can be divided into three principal groups. some are composed of solid growths of uniform cuboidal cells with expanding margins limited to alveolar septae (alveolar pattern). these cells contained concentrically arranged cytoplasmic lamellar bodies and abundant, large mitochondria similar to mitochondria found in alveolar type ii cells. tubular or papillary patterns are composed of cuboidal cells showing histological and ultrastructural features of clara cell differentiation.^^^ however, immunocytochemical studies of chemically induced and spontaneous pulmonary neoplasia in b6c3f1, balb/c or a strain mice have shown that the majority of adenocarcinomas, including those showing papillary patterns, contain surfactant apoprotein, typical of type ii antigens, suggesting that most neoplasms show alveolar type ii differentiation.^^^ however, in view of the plasticity of clara cells, this does not exclude a clara cell origin of the tumours. immunocytochemistry of specific clara cell secretory protein expression in a transgenic mouse model of lung carcinomas developing from clara cells has shown that the protein is lost during tumour cell progression.^^^ it has also been shown in strain a mice that the proportion of tumours with papillary and solid/alveolar growth patterns varies with the inducing agent.^^^ this also suggests biological differences exist between histological subtypes. very few squamous carcinomas are reported in most series of mouse studies. a chemically induced mouse model of squamous cell carcinoma has been generated by administration of n-nitroso-tris-chloroethylurea. strain differences in susceptibility to squamous cancer development have been demonstrated in this model, with nih swiss, a/j and swr/j being highly susceptible, akr/j and c57bl/6j being resistant and fvb/j and balb/cj mice showing an intermediate response to carcinogen.^^^ the high incidence and the inherent variabihty of pulmonary adenomas and adenocarcinomas in conventional mouse carcinogenicity bioassays sometimes gives rise to statistically significant differences between control and treatment groups. there is considerable risk in over-interpretation of such group differences in conventional mouse bioassays. in the analysis of group differences, consideration needs to be given to tissue sampling procedure, age-standardization, historical control incidence, effects on food intake as well as the results of mutagenicity studies and carcinogenicity bioassays in other rodent species. indeed, a considerable number of widely employed therapeutic agents of different classes have produced an increase in benign or malignant pulmonary tumours in carcinogenicity studies performed in mice without this proving of any significance to humans. davies and monro counted at least 17 drugs of this type in the 1994 physicians' desk reference of the united states.^^^ for instance, in a carcinogenicity bioassay in which cfl mice were treated for 80 weeks with the synthetic analgesic tilidine fumarate, a statistically significant difference (p < 0.01) was reported in the incidence of lung adenocarcinomas between the top dose female group (24%) and concurrent controls (10%).^^^ it was argued that group differences did not indicate tumorigenic potential of tilidine fumarate on the basis that the incidence in the high dose group was within the historical control range (27%) and that there was no tumorigenic effect in a parallel 104 week rat carcinogenicity study. a more difficult evaluation concerned metronidazole, a nitroimidazole which is an important therapeutic agent active against anaerobic organisms and trichomonas species. administration of this compound led to an increased incidence of pulmonary adenomas and carcinomas in three separate mouse carcinogenicity bioassays.^^^'^^^ the analysis of these findings was somewhat complicated by evidence that metronidazole shows mutagenic activity in bacterial assays using some strains of salmonella typhimurium. it was argued that the risk to human patients was slight because the increase in prevalence in pulmonary tumours was likely to be a result of changes in nutritional status of the mice through the effect of metronidazole on gut fiora, as similar differences could occur between ad libitum fed mice and those fed the same but restricted diet.^^^ it was also postulated that the positive findings in bacterial mutagenesis assays were an inherent part of the antibacterial activity of metronidazole as a result of nitroreduction that does not occur in normal mammalian tissues. this conclusion was supported by negative effects in hamster carcinogenicity bioassays as well as lack of excess cancer risk in women followed up for 10 years or more.^^^ the common occurrence of lung adenomas in strain a mice has been utilized in the development of a quantitative bioassay for carcinogenic activity. this followed the demonstration that administration of carcinogens such as 3-methylcholanthrene to this strain could significantly increase the incidence of pulmonary adenomas within periods of up to six months.^^^ over many years the strain a mouse pulmonary tumour assay has been used to test a large number of chemicals of different classes, including polycyclic hydrocarbons, nitrosamines, food additives, alkyl halides, metals and chemotherapeutic agents.^^^'^^^ however, as with many test systems, correlation of results in the strain a test with 2 year carcinogenicity study data and genotoxicity results have been shown to be poor so prudence is needed in the use of this test.^^^ hamsters develop lung adenomas spontaneously in small numbers with advancing age. they are composed of uniform cylindrical cells similar to those found in bronchial epithelium or goblet cells showing distinct mucus production.^^'^^'^^^ an immunohistochemical study of similar pulmonary neoplasms induced in hamsters by n-nitrosodiethylamine showed the presence of clara cell antigen in early phase of development, but as the tumours developed they became more squamous in type and showed immunoreactivity for cytokeratins.^^^ a clara cell origin was suggested for most of these neoplasms. lung defenses against infection: a clinical correlation drug-induced lung toxicity pulmonary toxicity from novel antineoplastic agents drug-induced diseases. drug-induced respiratory disease mucociliary clearance and mucus secretion in the lung risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation considerations for toxicology studies of respiratory drug products overview of inhalation toxicology inhalation exposure technology, dosimetry, and regulatory issues pulmonary deposition: determinants and measurement techniques inhalation therapy: technological milestones in asthma treatment the relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report interpretation of new techniques used in determination of pulmonary function in rodents measurement of respiratory patterns in rodents using whole-body plethysmography and a pneumotachograph evaluation of lung injury in rats and mice comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants comparative aspects of nasal airway anatomy. relevance to inhalation toxicology comparative anatomy, physiology, and function of the upper respiratory tract comparative anatomy of mammalian respiratory tracts: the nasopharyngeal region and the tracheobronchial region airflow, gas deposition, and lesion distribution in the nasal passages a comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts the upper airways. 1. nasal physiology and defence of the lungs antioxidant protection: a function of tracheobronchial and gastrointestinal mucus the histochemical and microscopical differentiation of the respiratory glands around the maxillary sinus of the rat hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin d the lateral nasal gland of the dog, its structure and secretory content sites for xenobiotic activation and detoxication within the respiratory tract: implications for chemically induced toxicity cytochrome-p450 2a of nasal epithelium -regulation and role in carcinogen metabolism purification and characterization of heterologously expressed mouse cyp2a5 and cyp2g1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes differential xenobiotic induction of cyp2a5 in mouse liver, kidney, lung, and olfactory mucosa nasal cytochrome p450 2a: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen cell-specific expression of cyp2a5 in the mouse respiratory tract: effects of olfactory toxicants biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach nasal lymphoid tissue in the rat toxicity to nasal associated lymphoid tissue histopathologic examination of the rat nasal cavity structural evaluation of the respiratory system normal histology of the nasal cavity and application of special techniques approaches to the identification and recording of nasal lesions in toxicology studies nasal diagrams -a tool for recording the distribution of nasal lesions in rats and mice histopathology of nasal olfactory mucosa front selected inhalation toxicity studies conducted with volatile chemicals cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. 1. determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung irritating properties of airborne materials to the upper respiratory tract bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man infectious diseases of the upper respiratory tract: implications for toxicology studies respiratory tract murine respiratory mycoplasmosis in lew and f344 rats: strain differences in lesion severity spontaneous proliferative lesions in the nasopharyngeal meatus of f344 rats spontaneous tumors and common diseases in three types of hamsters spontaneous tumors and common diseases in two colonies of syrian hamsters. ii respiratory tract and digestive system sialodacyoadenitis virus-associated lesions in the lower respiratory tract of rats oropharyngeal granulomas and tracheal cartilage degeneration in fischer-344 rats articular chondromatosis and chrondroid metaplasia in transgenic tag mice cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases histological sectioning of the rodent larynx for inhalation toxicity testing histologic methods and interspecies variations in the laryngeal histology of f344/n rats and b6c3f1 mice interspecies variations in the histology of toxicologically important areas in the larynges of crl:cd rats and syrian golden hamsters revised guides for organ sampling and trimming in rats and mice. part 2: a joint publication of the rita and nacad groups a comparison of the pathology of the larynx from spf, germ-free, conventional, feral and myoplasma-infected rats modelling of biological tree structures allometric relationships of cell numbers and size in the mammalian lung the ultrastructure of various cell types in the lung of the rat length and distribution of cilia in human and canine airways lectin-binding affinities of the respiratory tract. a light microscopical study of ciliated epithelium in rat, guinea pig and hamster clara cell proteins identification, cellular-locahzation, isolation, and characterization of human clara cell-specific 10-kd protein mucin is produced by clara cells in the proximal airways of antigen-challenged mice morphology of the terminal bronchiolar region of common laboratory mammals morphological changes in the lung during the life span of fischer 344 rats immunocytochemical localization of the major surfactant apoproteins in type ii cells, clara cells and alveolar macrophages of rat lung quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung what can the biology of small cell cancer of the lung teach us about the endocrine lung? the role of metabolism in chemical-induced pulmonary toxicity characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (balt) macrophages in the rat. an enzyme-cytochemical and immunocytochemical study subpopulations of lymphoid and non-lymphoid cells in bronchus-associated lymphoid tissue (balt) of the mouse bronchial lymphoid tissue. 1. morphologic characteristics the specificity of the high endothelial venule in bronchus-associated lymphoid tissue (balt) t cells and t cell subsets in rat bronchus associated lymphoid tissue (balt) in situ and in suspension have you seen this?' infiammatory lesions in the lungs of rats inflammatory lesions in the lungs of wistar rats histological changes in rat bronchus-associated lymphoid tissue after administration of five different antigens changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intracheal challenge with horseradish peroxidase patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat age-body weight relationships to lung growth in the f344 rat as indexed by lung weight measurements application of morphometric methods to study diffuse and focal injury in the lung caused by toxic agents morphometric assessment of pulmonary toxicity in the rodent lung the connective tissue of the rat lung: electron immunohistochemical studies immunohistochemical techniques and their applications in the histopathology of the respiratory system systemically applied chemicals that damage lung tissue bronchial mucosal mast cells and their implications in the pathogenesis of asthma morphological-changes in rat tracheal mucosa immediately after antigen challenge comparative studies of heparin and heparin fragments: distribution and toxicity in the rat interactions between sendai virus and bacterial pathogens in the murine lung: a review murine respiratory mycoplasmosis in f344 and lew rats -evolution of lesions and lung lymphoid-cell populations pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (sendai) virus respiratory tact lesions in weanling outbred rats infected with sendai virus increased tumor necrosis factor-alpha (tnf-alpha) gene expression in parainfluenza type 1 (sendai) virus-induced bronchiolar fibrosis il-12 reduces the severity of sendai virus-induced bronchiolar inflammation and remodeling studies of respiratory disease in random source laboratory dogs: viral infections in unconditioned dogs visceral larva migrans in the dog lesions produced by a new lung worm in beagle dogs diagnostic exercise: macaque with dyspnoea treatment of pulmonary acariasis in rhesus macaques with ivermectin a primer of primate pathology: lesions and nonlesions attachment of microbes to host cells: relevance o^ pneumocystis carinii animal model: pneumocystis carinii pneumonia in the immunosuppressed rat attachment of pneumocystis carinii to rat pneumocytes a progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin 2 or high-dose interleukin 2 alone toxicity of human recombinant interleukin-2 in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement toxicity of human recombinant interleukin-2 in the mouse is mediated by interleukin-activated lymphoc3^es. separation of efficacy and toxicity by selective lymphocyte subset depletion characterization of the pulmonary lesions induced in rats by human recombinant interleukin-2 dyspnoea and thoracic spinal deformation in rats after oral prizidilol (skandf 92657-a2) diagnostic exercise: pneumonia in a rat multidrug chemotherapy of tuberculosis in rhesus monkeys granulomatous pneumonitis induced by bacille calmette-guerin in the mouse and its treatment with cyclosporin a anergy-like immunosuppression in mice bearing pulmonary foreign body granulomatous inflammation direct evidence for granulomainducing role of interleukin-1 cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity animal model: pulmonary granulomatous vasculitis induced in rats by treatment with glucan naturally occurring diatomaceous pneumoconiosis in subhuman primates ultrastructural and micropulse analysis of simian lung mite pigments lung c3^okine production in bleomycin-induced pulmonary fibrosis treatment of human recombinant soluble tnf receptor of pulmonary fibrosis induced by bleomycin or silica in mice the physiology of transforming growth factor-a tnf-a receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos extensive laminin and basement membrane accumulation occurs at the onset of bleomycin-induced rodent pulmonary fibrosis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease methotrexate pneumonitis induced by intrathecal methotrexate therapy. a case report with pharmacokinetic data cytotoxic drug-induced pulmonary disease: update 19s0 lung toxicity associated with cyclophosphamide use. two distinct patterns interstitial pneumonitis associated with bleomycin therapy bleomycin lung toxicity: who are the patients with increased risk? preclinical toxicologic evaluation of bleomycin (nsc 125 066), a new antitumor antibiotic bleomycin-induced pulmonary toxicity in the rat intratracheal versus intravenous administration of bleomycin in mice: acute effects an investigation of possible models for the production of progressive pulmonary fibrosis in the rat. the effects of repeated intraatracheal instillation of bleomycin capillary remodeling in bleomycin-induced pulmonary fibrosis the role of strain variation in murine bleomycin-induced pulmonary fibrosis pulmonary toxicology of cyclophosphamide: a 1-year study animal models of emphysema lung scleroproteins in young and adult rats and in rats with spontaneous emphysema: comparative studies by biochemical and histochemical approach a quantitative study of stenosis in the respiratory bronchiole of the rat in n02-induced emphysema long-term sequelae of bronchiolitis induced by nitrogen dioxide in hamsters cationic amphiphilic drug-induced phospholipidosis drug-induced phospholipidoses drug-induced lipidosis and the alveolar macrophage drug-induced lysosomal disorders in laboratory animals: new substances acting on lysosomes pulmonary and generalized lyosomal storage induced by amphiphilic drugs drug-induced generalized phospholipidosis tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses fine structural alterations in the lungs of iprindole-treated rats a cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development validation of an in vitro screen for phospholipidosis using a high-content biology platform multifocal histiocytosis in the lungs of rats the induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone collecting and processing tissues for diagnostic electron microscopy chlorphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species amiodarone lung toxicity: a human and experimental study generalized phospholipidosis induced by amphiphilic cationic psychotropic drug morphological and biochemical changes in the liver of various species in experimental phospholipidosis after diethylaminoethoxyhexestrol recovery from amiodarone-induced lipidosis in laboratory animals. a toxicological study epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine d3 selective antagonist pnu-177864 myopathy related to administration of a cationic amphiphilic drug and the use of multidose drug distribution analysis to predict its occurrence drug-induced phospholipidosis: are there functional consequences? an 82-year-old man with dyspnea and pulmonary abnormalities amiodarone induced phospholipidosis. biochemical, morphological and functional changes in the lungs of rats chronically treated with amiodarone granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study amiodarone-associated pulmonary fibrosis. evidence of an immunologically mediated mechanism an evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity lipidosis induced by amphiphilic cationic drugs pathological discussion. case record of the massachusetts general hospital organomegaly and histopathology in an animal model of mucopolysaccharidosis induced by suramin lysosomal-storage disorder induced by elmiron following 90-days gavage administration in rats and mice foam cell response in the lung and lymphatic tissues during long-term high-level treatment with erythromycin interactions of macrolide antibiotics (erythromycin a, roxithromycin, erythromycylamine [dirithromycin], and azithromycin) with phospholipids: computer-aided conformational analysis and studies on acellular and cell culture models alveolar proteinosis and phospholipidosis of the lungs pulmonary alveolar proteinosis imatinib-associated pulmonary alveolar proteinosis imatinib mesylate inhibits the profibrogenic activity of tgf-(3 and prevents bleomycin-mediated lung fibrosis an experimental study of hypersecretion of mucus in the bronchial tree goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with or without the antiinflammatory agent phenylmethyloxadiazole measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis mitotic activity of airway epithelium after short exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole the effect of isoprenaline and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen experimental chronic bronchitis classification of cystic keratinising squamous lesions of the rat lung: report of a workshop pathological changes during aging in barrier-reared fischer 344 male rats neoplastic and nonneoplastic lesions in aging f344 rats histopathological profile of a wistar rat stock including a survey of the literature naturally occurring sendai disease of mice immunohistochemical demonstration of clara cell antigen in lung tumors of bronchiolar origin induced by n-nitrosodiethylamine in syrian golden hamsters sequential morphologic alterations in the bronchial epithelium of syrian golden hamsters during n-nitrosomorpholine-induced pulmonary tumorigenesis lung endocrine-like cells in hamsters treated with diethylnitrosamine: alterations in vivo and in cell culture 4-nitroquinoline 1-oxide-induced pulmonary endocrine cell hyperplasia in s3rrian golden hamsters increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine-cells of hypoxic rats the 2004 world health organization classification of lung tumors pleura, thymus and heart a review of chronic inhalation studies with mainstream cigarette smoke in rats and mice a review of chronic inhalation studies with mainstream cigarette smoke, in hamsters, dogs, and nonhuman primates animal models of mesothelioma induced by mineral fibers: implications for human risk assessment morphology of spontaneous and induced tumors in the bronchiolo-alveolar region of f344 rats spontaneous cystic keratinising epithelioma in the lung of a sprague-dawley rat strain a mouse lung tumor bioassay variability in the rates of some common naturally occurring tumors in fischer 344 rats and (c57bl/6nxc3h/hen)fl (b6c3f1) mice a carcinogenicity study in mice of a (3-adrenegic antagonist, primidolol; increased total tumour incidence without tissue specificity effect of diet on spontaneous disease in the inbred mouse strain c57b1/10j pol iota is a candidate for the mouse pulmonary adenoma resistance 2 locus, a major modifier of chemically induced lung neoplasia cancer susceptibility in the mouse: genetics, biology and implications for human cancer histogenesis of the papillary clara cell adenoma histologic and ultrastructural features of the clara cell adenoma of the mouse lung immunocytochemical localization of the surfactant apoprotein and clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice immunohistochemical analysis of clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis strain a/j mouse lung adenoma patterns vary when induced by different carcinogens a chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility marketed human pharmaceuticals reported to be tumorigenic in rodents evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate induction of lung tumors and malignant lymphomas in mice by metronidazole toxicologic evaluation of metronidazole with particular reference to carcinogenic, mutagenic, and teratogenic potential induced pulmonary tumors in mice. ii: reaction of lungs of strain a mice to carcinogenic hydrocarbons. arc/iii;es of pathology strain a mouse pulmonary tumor test results for chemicals previously tested in the national cancer institute carcinogenicity tests animal model: spontaneous carcinoma of the lung in hamsters key: cord-011781-0yswqubf authors: svanberg, emilie krite; larsson, jim; rasmussen, martin; larsson, marcus; leander, dennis; bergsten, sara; bood, joakim; greisen, gorm; fellman, vineta title: changes in pulmonary oxygen content are detectable with laser absorption spectroscopy: proof of concept in newborn piglets date: 2020-06-13 journal: pediatr res doi: 10.1038/s41390-020-0971-x sha: doc_id: 11781 cord_uid: 0yswqubf background: using an optical method based on tunable diode laser absorption spectroscopy, we previously assessed oxygen (o(2)) and water vapor (h(2)o) content in a tissue phantom of the preterm infant lung. here we applied this method on newborn piglets with induced lung complications. methods: five mechanically ventilated piglets were subjected to stepwise increased and decreased fraction of inspired oxygen (fio(2)), to atelectasis using a balloon catheter in the right bronchus, and to pneumothorax by injecting air in the pleural cavity. two diode lasers (764 nm for o(2) gas absorption and 820 nm for h(2)o absorption) were combined in a probe delivering light either externally, on the skin, or internally, through the esophagus. the detector probe was placed dermally. results: calculated o(2) concentrations increased from 20% (iqr 17−23%) when ventilated with room air to 97% (88−108%) at fio(2) 1.0. h(2)o was only detectable with the internal light source. specific light absorption and transmission patterns were identified in response to atelectasis and pneumothorax, respectively. conclusions: the optical method detected fio(2) variations and discriminated the two induced lung pathologies, providing a rationale for further development into a minimally invasive device for real-time monitoring gas changes in the lungs of sick newborn infants. impact: optical spectroscopy can detect pulmonary complications in an animal model. oxygen concentrations can be evaluated in the lungs. presents a novel minimally invasive method to detect lung oxygenation and complications. potential to be developed into a lung monitoring method in newborn infants. potential for bed-side detection of pulmonary complications in newborn infants. the pulmonary transition from intrauterine environment to normal postnatal air breathing is vulnerable and may cause a multitude of pulmonary complications that are increasingly prevalent with decreasing gestational age of the newborn infant. abnormal pulmonary aeration is common in neonatal lung diseases. despite modern synchronized respiratory support methods or minimally invasive surfactant therapy, there is still a risk of life-threatening complications such as pneumothorax, 1 and bronchopulmonary dysplasia remains a major problem and a long-term consequence of the acutely ill preterm lung. 2 thereby, a continuous measure of lung aeration may be useful for improving the precision of ventilatory support and detection of air leaks or inadequately ventilated regions of the lung. presently, most lung problems are diagnosed with pulmonary x-ray, which only gives a snapshot of the current condition and causes harmful radiation, increasing the risk of malignancy development later in life. 3, 4 research efforts have been directed towards the development of noninvasive alternatives to x-ray imaging. currently, lung ultrasound imaging 5 can be used for the detection of pneumothorax, consolidated lung parts and pleural effusions (e.g. blood). however, since nearly 100% of the ultrasound pulses are reflected by gas, the amount of air in the lungs cannot be estimated. the technique is emerging as an alternative to chest x-ray, 6 but it is user-dependent and provides only momentary information. a new optical technique called gas in scattering media absorption spectroscopy (gasmas), 7 based on tunable diode laser absorption spectroscopy (tdlas), detects gas concentration by free gas absorption in cavities surrounded by multiple light scattering media. 8 the high-resolution spectroscopic technique detects spectrally narrow gas absorption features in the surrounding wide band absorption of the tissue. we have applied this technique on phantoms of newborn lung 9-11 and on healthy newborn infants 12 and showed that pulmonary o 2 content could be detected. 13 in addition, the method allows determination of water vapor (h 2 o) absorption path length, which enables evaluation of lung volume and pulmonary o 2 concentration. 10, 13 a challenge when using optical methods in biological tissues is the strong light scattering and absorption leading to considerable attenuation of detectable light and weak signals. much of the light emitted through the skin is scattered, and only a small portion of the light reaches the detector. the approach with internal illumination would increase the amount of light reaching the detector. this approach has successfully been used in our phantom studies. 14 we hypothesized that the gasmas method with an external as well as an internal light source probe would detect changes in pulmonary o 2 gas concentration, including responses to induced local lung pathologies in newborn piglets. danish landrace newborn piglets underwent routine care at the farm together with siblings until transported to the animal facilities of copenhagen university. they were immediately taken to the surgical theater and anesthetized. the danish animal experiments inspectorate approved the study protocol (2018-15-0201-01403). all responsible researchers had current animal handling certificate and the experiments were performed according to felasa (federation of laboratory animal science associations) guidelines and national regulations. five healthy piglets, at a mean age of 14 days (range 13 −15 days) and a median weight of 3.2 kg (range 3.1-3.3 kg), were used. anesthesia was induced by an intramuscular injection of 1 ml/kg zoletil (tiletamine 25 mg/ml, zolazepam 25 mg/ml, xylazine 25 mg/ml, ketamine 25 mg/ml, methadone 4 mg/ml, buthorphanol 4 mg/ml) and was maintained by infusion of propofol (15 mg/ kg/h) and fentanyl (7 µg/kg/h). after sedation, the piglets were endotracheally intubated and connected to a pressure-controlled ventilator. at 3 h after initiation of anesthesia, 1 ml/kg zoletil was administered intravenously, and at any signs of distress or poor sedation additional dose of 0.1 ml zoletil was given. arterial co 2 was kept between 4.5 and 6.5 kpa and arterial saturation above 95%. a heat moister exchanger (humid-vent® mini, teleflex, morrisville, north carolina) was connected between the ventilator and the endotracheal tube. arterial saturation was continuously monitored by pulse oximetry on the foreleg (radical 7 ® , massimo, irvine, canada). normal rectal temperature was maintained (38.5 −39.5°c) by a heating pad. the femoral artery was cannulated for continuous invasive monitoring of mean arterial blood pressure and heart rate. both ear veins were cannulated for infusion of propofol, fentanyl and saline 0.9%. the piglet recovered for 1 h after the surgical preparation before the experiment. arterial blood gases and blood glucose were measured after the surgical preparation and during the experimental study. at the end of the study, the piglet was euthanized with 100 mg/kg pentobarbital. we conducted three interventions ( fig. 1 ) with a dermal detector probe and two different light source probes, one applied to the skin, and one internal probe located in the esophagus. the internal optical fiber probe was positioned in the esophagus at a distance of 21 cm from the snout. the localization corresponded to a position at the heart level, which was confirmed by fluoroscopy x-ray in the first two piglets (fig. 2 ). this position was kept for all the following piglets as they had similar size and weight. the detector probe was placed in the right axillary line, approximately 2 cm below the foreleg. the dermal light source probe was placed in the midclavicular line on the right side of the thorax with at least 3 cm distance to the detector. the source and detector probes were fixed with elastic black nylon straps that also prevented light dispersion from the dermal laser source. the initial procedure was a stepwise increase and decrease in inspired oxygen fraction (fio 2 ) in the ventilator settings with the light source on the skin and thereafter repeated in duplicate with the light source in the esophagus (fig. 1a) . the absorption of o 2 and h 2 o vapor was measured by the gasmas system with o 2 (fao 2 ). at each fio 2 setting, the measured o 2 and h 2 o absorption signals were averaged over 10 s and sampled ten times. after the measurement at fio 2 1.0, a pulmonary recruitment procedure was done by increasing the inspiration pressure, to reduce the risk of resorption atelectasis. 15 recruitment was deemed successful when tidal volumes were the same as before starting the experiment. the detector probe position, fixation and the stepwise changes of fio 2 were the same in the dermal and esophageal measurements (fig. 2) . we then performed a partial pulmonary atelectasis with the esophageal light probe in place (fig. 1b) . the fio 2 was set to 0.30. a 4 fr embolectomy catheter with a balloon at its tip (lemaitre® vascular, sulzbach, germany) was inserted into the trachea parallel to the endotracheal tube into the right main bronchus (fig. 1b) . in two piglets, the position was verified by fluoroscopy x-ray. partial obstruction of the distal airways was considered confirmed when the end-tidal co 2 had decreased by 50% after balloon inflation. the balloon was deflated after approximately 1.5 min or if the piglet became circulatory unstable. oxygen signals were continuously measured during the procedure, which was repeated three times. between each atelectasis, mechanical recruitment of pulmonary volume was performed. after the piglet had recovered from atelectasis, a small thoracostomy was prepared in the right axillary line, 1 cm below the dermal detector probe, and a drainage tube was placed and fixated with sutures. the piglet was ventilated with fio 2 1.0 while 100 ml of ambient air (fio 2 0.21) was injected into the pleural space through the tube (fig. 1c) . in two piglets, we confirmed the pneumothorax with fluoroscopy x-ray. oxygen signals were continuously measured during the pneumothorax phase. approximately 2 min after induction, the air was withdrawn from the pleural cavity and pulmonary recruitment followed. the procedure was repeated three times. optical experimental setup the gasmas system (gpx medical ab, sweden) measured gas absorption at two wavelengths, 764 nm for o 2 gas absorption and 820 nm for h 2 o vapor absorption. the measured gas absorption is expressed in a dimension of [%m] which is the product of the relative gas concentration [%] and the absorption path length [m]. 13 the instrument was calibrated using known concentrations and absorption path lengths. the relative humidity in the lung is approximately 100%, and together with the knowledge of tissue temperature and air pressure, the h 2 o vapor gas concentration can numerically be estimated. 16 the measured h 2 o vapor gas absorption in [%m] thus provides the absorption path length in [m] for light at 820 nm. assuming that the light at 764 nm travels the same average distance in the gas volume, the measured o 2 gas absorption in [%m] then provides the o 2 concentration in [%]. the two diode lasers providing light at 764 and 820 nm, respectively, were focused into an optical fiber (core diameter 400 µm), which provided an output power at the tissue interface of 25 and 8.1 mw, respectively. the diode lasers and the light combining optics were enclosed in a nitrogen-filled box to eliminate offsets in the absorption signal due to ambient air. the gas absorption signal was obtained by sequentially scanning the wavelength of the lasers, at a rate of 1 khz, over one of the absorption lines of the o 2 molecule at 764 nm and of h 2 o at 820 nm. the two gases were measured in sequence with a measurement time of 100 s before switching. optical fibers with two different light distributions at the distal end were employed for skin and esophageal light administration, respectively. the rationale for using internal light illumination is the fact that when light is applied on the skin and detected from the skin surface, theoretically, only 1% of the light that reaches lung tissue would reach the detector. if the light source is placed internally, e.g. through a nasogastric feeding tube or an endotracheal tube, the light travels through the air-filled lung with a shorter path through solid tissue before reaching the skin surface, thereby theoretically leading to higher light intensity at the detector with a larger portion of photons subjected to gas absorption. taking as an example if 1% of the light emitted at the surface would reach the lung tissue and then be scattered back after interacting with the gas, again only 1% of that light would reach a detector on the surface. clearly, if the light instead would only need to travel in one direction, using internal illumination, a factor of about 100 in detected intensity could typically be gained. also, by internal illumination, a larger fraction of the detected light would have passed through the gas-bearing lung tissue, laying between the light injection point and the detector. for skin light administration, a diffusing cylindrical object was used to create a homogeneous emission over an area of 1 cm 2 with an irradiance of 25 mw/cm 2 and 8.1 mw/cm 2 for 764 and 820 nm, respectively. for the internal light administration, the end of the fiber was diffusing over a longitudinal distance of 10 mm with an outer diameter of 0.9 mm, giving an irradiance of 88 and 29 mw/cm 2 for 764 and 820 nm, respectively. due to the diffuse emission and low light intensities, both fiber probe designs are eye-safe (laser class i, iec 60825-1:2014) and no significant tissue heating is expected as the irradiance is kept below 150 mw/cm 2 . 17 after propagating through the tissue, the transmitted light was detected by a photodiode (active area 10 mm × 10 mm) incorporated into a detector probe attached to the skin. the detector probe area was covered with a layer of ultrasonic gel in order to reduce offsets in the absorption signal. the detected absorption signal was intensity normalized and frequency filtered by the electronic platform, which acquires the absorption magnitude through proprietary algorithms. the oxygen concentration, x o2 , was evaluated assuming that the absorption path length of water vapor l h2o ð þ and oxygen l o2 ð þ is equal. l h2o was obtained from the absorption signal of water vapor (a h2o ) and by calculating the temperature-dependent water vapor concentration x h2o . 18 for each measurement series, i.e. a set value of inspired oxygen, the a o2 and a h2o were sequentially obtained, as described above. the oxygen concentration in the equation was determined by using the measured oxygen absorption and the median value of the water vapor absorption. every measurement series yielded between 8 and 12 data points and the fio 2 was repeated between two and five times for each piglet, with the exception for piglet number 3, where the fio 2 challenge was done only once. for statistical analyses, a one-way anova test was performed. with the internal light probe placed in the esophagus, the detected absorption signal was roughly one order of magnitude larger compared to the measurements with the dermal light source (fig. 3) . water vapor absorption was variably detected when using the skin light source (fig. 3b) . as the evaluation of o 2 concentration is dependent on the absorption of both o 2 and h 2 o, determination was not possible for all piglets. when using the internal light source, the signals from both o 2 and h 2 o were clearly detectable, and the absorption values changed with the set value of fio 2 (fig. 3c, d) . the calculated o 2 concentrations, using the internal light source, are shown in fig. 4 atelectasis was induced in four piglets while continuously monitoring the o 2 absorption and light transmission using the internal light source. during the atelectasis, the o 2 absorption signal and the light transmission were both observed to decrease over a time scale of 10−30 s (fig. 5) . when the balloon was deflated, the absorption and light transmission returned in most cases to the initial value before atelectasis. the continuously monitored o 2 absorption decreased rapidly when the pneumothorax was induced and simultaneously the light transmission increased (fig. 6a) . when the air was removed from the pleural space, the o 2 absorption and the light transmission returned to their initial values. using an optical measurement exposure time of 0.333 s in one piglet during the pneumothorax, the variation in transmission and absorption followed each breath provided by the ventilator (fig. 6b) . in this study, for the first time, we have successfully determined pulmonary o 2 concentration using an internal laser light source. we show here that the detected amount of o 2 in the lungs correlated with inhaled o 2 concentrations and that the pathophysiological changes of aeration produced in the experimental setting were detectable by the gasmas system. we were not able to estimate o 2 concentrations when using a dermal light source, due to too weak h 2 o absorption signal. using the internal light source placed in the esophagus, we detected distinct and prompt changes in absorption and transmission signals in response to stepwise changes in fio 2 , as well as to induced pneumothorax. interestingly, a breath-related change was observed both in o 2 absorption and light transmission during pneumothorax. the observed decrease in o 2 absorption during atelectasis was less robust with variations between piglets. the rapid decrease in o 2 absorption signal during the pneumothorax can be explained by the collapse of the lung enabling the light to predominately travel through a hollow, nonstructured pleural cavity. the fio 2 was 1.0, and upon lung collapse, the remaining absorption signal equivalents the o 2 content in ambient air injected into the pleural cavity. conversely, when removing the air, the incremental absorption signal can be explained by the lung gradually regaining its original gas-filled form. noteworthy, the method appeared to be sensitive enough to detect inspiration and expiration changes to each breath. the o 2 absorption decrease during atelectasis may be related to o 2 consumption and induced physical change of the lung structure resulting in a reduced absorption path length, i.e. smaller gas volume in a denser lung. this pathophysiologic difference between pneumothorax and atelectasis explains the patterns of absorption changes. thus, this experimental study provides a proof of concept that the gasmas technique has the potential to detect neonatal life-threatening pulmonary complications. the incidence of pneumothorax is up to 5−7% in infants with a birth weight less than 1500 g treated in ventilators 19 and it carries a risk of mortality. continuous monitoring of pulmonary oxygen (fig. 3c, d) . the median and the 25th and 75th quartiles are shown. changes in pulmonary oxygen content are detectable with laser absorption. . . ek svanberg et al. content could possibly reduce the time from onset of pneumothorax until diagnosis and intervention. the gasmas technique, if applied with an indwelling probe in the nasogastric tube, is not more invasive than the routinely used nava-ventilation, and according to one study no adverse effects of the nava catheter were observed. 20 we envision that the technique could be developed into a surveillance for preterm infants in need of respiration support. pulmonary pathophysiology has been subject to investigation with a range of noninvasive techniques with continuous monitoring. 21 in general, newer techniques, such as electromagnetic inductance plethysmography and electrical impedance tomography, provide clinically useful data, including the detection of pneumothoraxes. 22 the techniques, however, are restricted in the applicability due to difficulties in electrode placement on the tested subjects, especially in infants with very low birth weight. 23 optoelectronic plethysmography is a noninvasive 3d motion capture method, measuring chest wall movements, estimating lung volumes. the technique reconstructs the chest wall surface by using up to 89 surface markers (24 can be used in the infant setting) and 8 infrared cameras, mounted in the room. this is a promising research technique for lung volume assessment, but not feasible for clinical bed-side continuous monitoring, nor can it provide information on oxygen distribution throughout the lungs. 24 electrical impedance tomography 25, 26 gives information on global and regional changes in lung impedance correlating with intrathoracic changes in air content and thereby lung volume. however, this technique only gives information on a transversal "slice" of the lung and does not provide any information on o 2 content. lung ultrasound is another noninvasive technique used in intensive care units to detect e.g. atelectasis 5 ; however, it is highly user-dependent, gives no information on oxygen distribution, and does not allow for continuous surveillance. in comparison to the above mentioned, the gasmas technique enables continuous monitoring of the oxygen concentration in the lung as well as an indication of changes in gas volume with one light probe (preferably internal) and one or several detection probes. conventional pulse oximetry is routinely used for arterial oxygen saturation monitoring, and in the newborn infant information about pre-and postductal differences in oxygen saturation can be obtained. the proposed technique in the present study should be considered as a complement to pulse oximetry as it has the ability to detect lung pathologies quickly, most probably before changes in arterial oxygenation occur. a considerable signal variation in the absorption signal between the piglets during the induced atelectasis was observed. however, the decrease in transmission observed in all piglets was consistent. this is physiologically well understood, since the induced atelectasis leads to distal lung parenchyma collapse, resulting in concomitant decreased transmission of light. this needs to be further investigated, focusing on the transmission variation as a response to varying degrees of atelectasis. due to too weak h 2 o signal with dermal light administration, the pulmonary o 2 concentration could not be estimated. piglets have an elongated and narrow thorax compared to human infants and the front leg makes the apical part of the thorax less accessible. this disturbs the required balance between the need for a minimum light source-detector distance and proximity to lung tissue with volumes large enough for the light not to travel through extra-pulmonary tissue (e.g. trachea, mediastinum, and abdominal organs). light from a dermal light source will theoretically scatter more at entrance to the lungs than from an internal source. moreover, the characteristics of the skin of the piglet differ from preterm infants and thereby piglets may not be the optimal model to mimic human infants, when evaluating the dermal light source. however, using internal light administration, we obtained clear signal changes in response to the right-sided pneumothorax, suggesting robust signal conductance from source to detector. in previous studies, we have obtained strong o 2 absorption signals in healthy full-term infants with a dermal light source. 13 in the present study, when delivering the light internally, reliable pulmonary o 2 concentrations could, for the first time in an experimental animal, be determined using the h 2 o absorption signal. thus, the internal light source was superior to the dermally positioned. likewise, calculations of oxygen concentrations were possible on a 3d phantom of a newborn infant´s torso, when applying the light source internally. 14 autopsy of one piglet did not reveal any visually detected damage to the esophagus. however, we did not study the possible microscopic damages to the tissue from slight changes in pulmonary oxygen content are detectable with laser absorption. . . ek svanberg et al. temperature increases or physical damage from the optical fiber probe. the diffuse light emission was significantly lower than the maximum irradiance allowed, 150 mw/cm 2 , which when exposed on the skin would generate a maximum temperature increase of 2°c. 17 whether this local increase in temperature of the nasogastric tube could have any adverse effects on neonatal esophagus needs to be investigated. we envision that for clinical investigations to avoid contact with esophagus, the light probe would be inserted into or embedded in a nasogastric tube, and could even be used together with nava ventilation. future studies of the gasmas technique should focus on the reliability and accuracy of the measurements in sufficient number of subjects with an improved equipment developed from the prototype used in this study. further, the applicability of the technique to detect changes in lung volume parameters (e.g. tidal volume) can be studied by changing ventilator settings in the experimental model, as indicated by the possibility to follow each breath. before clinical studies in newborn infants can be designed, the safety aspects of the technique, such as heating effects, must be assessed. also, ensuring a simple and robust applicability with well-designed light probes and detectors suitable for fragile preterm infants is essential for the technique to be investigated in the neonatal intensive care unit environment. gasmas enables measurement of pulmonary o 2 concentration using an internal light probe along with distinct signal changes during pneumothorax in a neonatal piglet model. compared to electric impedance and ultrasound, gasmas has the advantage to detect oxygen content and lung volume changes of each breath. we were not able to reliably detect pulmonary o 2 content using a dermal light probe. future studies should focus on the accuracy and reliability along with safety of this method. impact of minimally invasive surfactant therapy in preterm infants at 29−32 weeks gestation ventilation in extremely preterm infants and respiratory function at 8 years cancer risks from diagnostic radiology radiation exposure from ct scans in childhood and subsequent risk of leukaemia and brain tumours: a retrospective cohort study ultrasound in neonatal lung disease. quant neonatal respiratory distress syndrome: chest x-ray or lung ultrasound? a systematic review gas in scattering media absorption spectroscopy-from basic studies to biomedical applications gas monitoring in human sinuses using tunable diode laser spectroscopy nonintrusive gas monitoring in neonatal lungs using diode laser spectroscopy: feasibility study development of a 3-dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen-laser-detector position considerations computer simulation analysis of source-detector position for percutaneously measured o2-gas signal in a three-dimensional preterm infant lung noninvasive monitoring of gas in the lungs and intestines of newborn infants using diode lasers: feasibility study diode laser spectroscopy for noninvasive monitoring of oxygen in the lungs of newborn infants comparison of dermal versus internal light administration in human lungs using the tdlas-gasmas technique-phantom studies effects of anesthesia on the respiratory system new equations for computing vapor-pressure and enhancement factor kinetics of the superficial perfusion and temperature in connection with photodynamic therapy of basal cell carcinomas using esterified and non-esterified 5-aminolaevulinic acid a simple procedure for calculating atmospheric water vapor concentration expectant management of pneumothorax in ventilated neonates electrical activity of the diaphragm (edi) values and edi catheter placement in non-ventilated preterm neonates evaluation of bedside pulmonary function in the neonate: from the past to the future electrical impedance tomography can rapidly detect small pneumothoraces in surfactant-depleted piglets lung function tests in neonates and infants with chronic lung disease: global and regional ventilation inhomogeneity optoelectronic plethysmography in clinical practice and research: a review changes in lung volume and ventilation during surfactant treatment in ventilated preterm infants assessment of lung ventilation in infants with respiratory distress syndrome using electrical impedance tomography this work was financially supported in part by the european community (eureka eurostars) neolung project (e! 9833 neo-lung), the laserlab-europe (euh2020 654148) jra project bioapp and departments of pediatrics and physics, lund university, lund, sweden. competing interests: gpx medical is a startup company responsible for the development of the gasmas prototype used in the study. d.l. and s.b. are employed by the company. e.k.s., j.l., m.r., m.l., j.b., g.g. and v.f. have nothing to disclose.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-001473-aki28lhp authors: chen, qi xing; song, sheng wen; chen, qing hua; zeng, cong li; zheng, xia; wang, jun lu; fang, xiang ming title: silencing airway epithelial cell-derived hepcidin exacerbates sepsis-induced acute lung injury date: 2014-08-06 journal: crit care doi: 10.1186/s13054-014-0470-8 sha: doc_id: 1473 cord_uid: aki28lhp introduction: the production of antimicrobial peptides by airway epithelial cells is an important component of the innate immune response to pulmonary infection and inflammation. hepcidin is a β-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. however, nothing is known about its function in lung infections and inflammatory diseases. we therefore sought to investigate the role of airway epithelial cell-derived hepcidin in sepsis-induced acute lung injury. methods: acute lung injury was induced by polymicrobial sepsis via cecal ligation and puncture (clp) surgery. adenovirus-mediated short hairpin rna specific for the mouse hepcidin gene hepc1 and control adenovirus were intratracheally injected into mice. the adenovirus-mediated knockdown of hepcidin in airway epithelial cells was evaluated in vivo. lung injury and the seven-day survival rate were assessed. the levels of hepcidin-related iron export protein ferroportin were measured, and the iron content and function of alveolar macrophages were evaluated. results: the hepcidin level in airway epithelial cells was upregulated during polymicrobial sepsis. the knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality (53.33% in ad-shhepc1-treated mice versus 12.5% in ad-shneg-treated mice, p <0.05). the knockdown of hepcidin in airway epithelial cells also led to reduced ferroportin degradation and a low intracellular iron content in alveolar macrophages. moreover, alveolar macrophages form the airway epithelial cell-derived hepcidin knockdown mice showed impaired phagocytic ability than those from the control mice. conclusions: airway epithelial cell-derived hepcidin plays an important role in clp-induced acute lung injury. the severe lung injury in the airway epithelial cell-derived hepcidin knockdown mice is at least partially related to the altered intracellular iron level and function of alveolar macrophages. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are common clinical disorders and present substantial health problems worldwide [1] [2] [3] . the incidence of ali has been reported to be 190,000 cases per year in the united states, and sepsis is one of the most commonly encountered conditions underlying the development of ali [1, 4] . through advances in supportive care, the mortality rate for patients with ali/ards has decreased over time but still remains high [5, 6] . therefore, elucidating the pathogenesis of ali/ards is necessary and may help with the development of novel therapeutic targets for ali/ards. the lung is exposed to a large number of potentially pathogenic microorganisms that are inhaled. to protect this vital organ against infection and inflammation, many defense mechanisms have been evolved in the lung to prevent the development of pulmonary infections. the secretion of endogenous cationic antimicrobial peptides by epithelial cells onto the airway surface represents an important component of immune defense in the lung [7] [8] [9] . recent studies have demonstrated the importance of these antimicrobial peptides, such as defensins and cathelicidin, in the pulmonary immune system and in pulmonary diseases [10] [11] [12] . hepcidin is a β-defensin-like antimicrobial peptide that is mainly produced by the liver. hepcidin not only shows antimicrobial activity against gram-positive bacteria, gram-negative bacteria and yeasts, but also functions as a principal iron regulatory hormone [13] [14] [15] . hepcidin binds to the iron export protein ferroportin and induces its internalization and degradation, which leads to decreased cellular iron export and increased intracellular iron retention [16, 17] . because iron is an essential nutrient for all organisms, hepcidin also restricts the iron available to invading microbes, thereby enhancing the host defense against pathogens [17] [18] [19] . furthermore, hepcidin can modulate the lipopolysaccharide (lps)-induced acute inflammatory response via the suppression of cytokine expression [20, 21] . the multiple functions of hepcidin suggest its importance in the host immune response to infection and inflammation. recent studies have reported that in addition to being produced mainly by hepatocytes, hepcidin is also expressed by other cells, such as airway epithelial cells (aecs) [22] . however, the role of aecderived hepcidin in pulmonary immune defense against infection and inflammation remains unknown. in the present study, we generated a mouse model of aec-derived hepcidin knockdown and investigated the impact of interfering with aec-derived hepcidin on the pathophysiology of sepsis-induced ali. we also explored the possible mechanisms involved. adenovirus-mediated short hairpin rna (shrna) as the mouse hepcidin gene hepc1 performs similar biological actions as human hepcidin [23, 24] , we investigated hepc1 in the present study. small interfering rna specific to the mouse hepc1 (acagaugagacagacuacadt dt) was described previously [25] , and we used this template to design the corresponding shrna (accgac agatgagacagactacattcatgagatgtagtct gtctcatctgtcttttt) with minor modifications (invitrogen, shanghai, china). an adenovirus that expresses the hepc1 shrna or a nonspecific control oligonucleotide (gcctaaggttaagtcgccctcgcgaac gaaggcgagggcgacttaaccttaggtt) was prepared. briefly, the double-strand shrna oligo was cloned into pentr/u6 vector using the block-it u6 rnai entry vector kit (life technologies, grand island, ny, usa). the expression clone was then generated by recombination of pentr/u6 entry construct and pad/block-it-dest vector using block-it adenoviral rnai expression system (life technologies) according to the manufacturer's instructions. after digested by endonuclease paci (new england biolabs, ipswich, ma, usa), the recombinant adenoviral plasmid was transfected into 293a cells. the harvested adenovirus was concentrated and purified by cscl gradient centrifugation. viral titer was determined using adeno-x rapid titer kit (clontech, mountain view, ca, usa). the recombinant virus and control virus were named ad-shhepc1 and ad-shneg, respectively. to generate a mouse model of aec-derived hepcidin knockdown, ad-shhepc1 or ad-shneg (approximately 2.2 × 10 11 viral particles) was intratracheally injected into the mice after anesthesia with chloral hydrate (400 mg/kg, intraperitoneally (i.p.)). to improve the knockdown efficiency, a second administration of the adenovirus was performed 72 hours later. twelve days after the first injection, the mice were ready used in the experiments. ali was induced by polymicrobial sepsis with cecal ligation and puncture (clp) surgery as described previously [26] . briefly, after the abdominal wall was prepared with a 10% povidone-iodine solution, a 1-cm midline abdominal incision was made. the cecum was then exposed, isolated, and ligated with a 4-0 silk ligature just distal to the ileocecal valve to avoid intestinal obstruction. the puncture was performed twice with a 22-gauge needle. then, the cecum was repositioned, and the incision was closed with a 4-0 sterile suture. sham-operated mice were underwent the same procedure but without ligation and needle perforation of the cecum. at the end of the operation, the mice were resuscitated immediately by the subcutaneous administration of saline (1 ml/mouse) and allowed free access to food and water after awakening. in the experiment to evaluate the survival rate, the mice were monitored for survival every six to twelve hours until seven days post-clp. twenty-four hours after clp, the mice were sacrificed via cervical dislocation. the chest cavity was opened via a midline incision. to perform the bronchoalveolar lavage, the lung was lavaged with 0.5 ml of chilled phosphate-buffered saline three times. in all cases, more than 90% of the total lavage volume was recovered. a 0.5-ml aliquot of balf was used for the total cell counts and bacteriologic culture. the remaining balf was centrifuged at 1000 g for five minutes, and the cell-free supernatant was stored at -80°c for further analysis. the total cell count in the balf was determined using a hemocytometer. the balf was serially diluted in phosphate-buffered saline and inoculated on luria broth agar plates. after incubation at 37°c for 16 hours, visible colonies were counted and calculated as colony-forming units/ml of balf. the total protein in the cell-free supernatant of the balf was determined by using the bca protein assay kit (pierce, rockford, il, usa). the interleukin (il)-6 level in the balf was determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (abcam, san francisco, ca, usa). in separate groups the lung was excised, weighed, and then placed in an oven at 60°c for 72 hours to achieve the dry weight. the ratio of lung wet weight to dry weight was then calculated. the lung tissues from the mice were fixed in buffered 4% paraformaldehyde solution (ph = 7.4) for 24 hours, embedded in paraffin, and sectioned at a thickness of 4 μm. the histological examination was conducted in a blinded fashion after staining with hematoxylin and eosin. for rna extraction, alveolar macrophages were isolated by adhering bronchoalveolar lavage cells to plastic for one hour at 37°c in 5% co 2 [27] . total rna of the lung and liver tissues as well as the alveolar macrophages was extracted using the trizol™ reagent. reverse transcription was performed using 1 μg of total rna with the reverse transcription system (promega, madison, wi, usa). the transcriptional level of hepcidin was quantified by real-time pcr using a standard sybr™ green pcr protocol on a cfx96 real-time pcr detection system (bio-rad laboratories inc., hercules, ca, usa). the housekeeping gene β-actin served as an internal control, and the relative expression level of hepcidin was calculated using the 2 −δδct method. immunohistochemical staining was performed following standard procedures. the formalin-fixed paraffin-embedded tissues were sliced into 4-μm-thick sections, deparaffinized, and rehydrated. for the hepcidin measurement in the alveolar macrophages, the cells were isolated by adhering bronchoalveolar lavage cells to coverslips at 37°c in 5% co 2. then, the cells were fixed with 95% ethanol for 15 minutes. antigen retrieval was performed in 10 mmol/ l citric acid buffer (ph 6.0) for 10 minutes using a 750-w microwave. endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes. after incubation with rabbit anti-mouse hepcidin antibody (1:400 dilution; abcam) overnight at 4°c, the sections were washed in phosphate-buffered saline and incubated with a polymer horseradish peroxidase-conjugated secondary antibody (zsgb-bio, beijing, china) for one hour. the sections were further incubated with dako liquid dab large-volume substrate-chromogen system (dako, glostrup, denmark) and counterstained with hematoxylin. negative controls were included in all assays by replacing the rabbit anti-mouse hepcidin antibody with nonimmune rabbit antiserum. the immunostaining was evaluated using an olympus bx-50 light microscope (olympus, tokyo, japan). the stain density was analyzed using the image pro-plus 6.0 analysis system (media cybernetics inc., silver spring, md, usa), and the level of hepcidin was measured as the integral optical density. the protein concentrations in the lung homogenate or lysate of alveolar macrophages were detected using a bca protein assay kit (pierce). the proteins (20 μg) were denatured by heating at 70°c for 10 minutes in 4 × nupage lds sample buffer (life technologies) and separated by nupage bis-tris gel electrophoresis (life technologies). then, the proteins were blotted onto polyvinylidene fluoride membrane (millipore, billerica, ma, usa). the membranes were blocked with 5% nonfat milk in tris-buffered saline with 0.05% tween-20 and incubated overnight with goat anti-ferroportin antibody (santa cruz biotechnology, dallas, tx, usa). the membranes were then washed with tris-buffered saline with 0.05% tween-20 three times for five to ten minutes each. after incubation with the related horseradish peroxidaseconjugated secondary antibody (jackson immunoresearch laboratories, inc., west grove, pa, usa), the membranes were visualized with the supersignal west pico chemiluminescent substrate (pierce). the signals were quantified using the image j software by wayne rasband (national institute of health, bethesda, maryland, md, usa). β-actin served as a protein control. the iron content in alveolar macrophages and spleen was determined by prussian blue staining using a commercially available kit according to the manufacturer's instructions (shanghai yuanye bio-technology co., shanghai, china). the stain density in the spleen was analyzed using the image pro-plus 6.0 analysis system as described above. for the iron measurement in the alveolar macrophages, the cells were isolated by adhering bronchoalveolar lavage cells to coverslips at 37°c in 5% co 2. then, the cells were fixed with 95% ethanol for 15 minutes. the cells were considered positive with the presence of bluecolored granules within intact alveolar macrophages under a microscope. at least 200 random macrophages were counted, and the results are presented in terms of the percentage of positive cells. the serum iron concentration was measured using atomic absorption spectroscopy [28] . to investigate the phagocytosis function of alveolar macrophage, alveolar macrophages were isolated as described above. the cells were then incubated with fluorescent escherichia coli (life technologies) for two hours. fluorescence from the extracellular bacteria was quenched with 0.4% trypan blue. after three washes with phosphatebuffered saline, the fluorescence was observed under a fluorescence microscope (olympus). the phagocytic index was quantified as number of fluorescent e.coli internalized by one macrophage cell counted in 10 random fields. the data are expressed as the mean ± standard deviation (sd) or median with range where applicable. the differences between the two groups were analyzed by the t test or mann-whitney u test. the survival curves were analyzed by the kaplan-meier log-rank test. the statistical analyses were performed using graphpad prism software 5.0 (graphpad software inc., la jolla, ca, usa) and spss 16.0 for windows (spss inc., chicago, il, usa). a twotailed p value of less than 0.05 was considered to be statistically significant. hepcidin expression is modulated in response to infectious and inflammatory stimuli [14, 17] . we first investigated whether the hepcidin expression level in the lung tissue changed during polymicrobial sepsis. twenty-four hours after clp surgery, the hepcidin level was significantly increased in the lung tissue, especially in the aecs (figure 1 ). this finding indicates that hepcidin derived from aecs may play an important role in sepsis-induced ali. to confirm the role of hepcidin in ali, ad-shhepc1 or ad-shneg was administered to the mice via intratracheal instillation. twelve days after administration of the adenovirus-mediated shrna, the mice were subjected to an ali model, and the hepcidin level in lung and liver were assessed at 24 hours after induction of ali. as shown in figure 2a and 2b, both the mrna and protein levels of hepcidin in aecs was significantly reduced, whereas the hepcidin expression in alveolar macrophages ( figure 2c and 2d) and hepatocytes ( figure 2e and 2f) was not affected. these results demonstrated that in the current study the intratracheal administration of ad-shhepc1 only silenced the hepcidin gene transcription in aecs, which was in accordance with previous studies that adenovirus-mediated intratracheal gene delivery specifically inhibited targeted gene expression in lung epithelial cells but not in alveolar macrophages and other organs [29, 30] . the lung injury was evaluated both in wild-type mice and adenovirus-treated mice at 24 hours after challenge with clp or a sham operation. histological characteristics of lung injury, including diffuse alveolar damage, infiltration of numerous leukocytes and interstitial edema, were observed both in the wild-type mice and ad-shnegtreated mice after clp challenge. knockdown of hepcidin in aecs led to more severe lung damage in the ad-shhepc1-treated mice after clp challenge ( figure 3a ). lung wet/dry weight ratios from the experimental mice further confirmed the histological findings ( figure 3b ). the balf analysis showed that the hepcidin knockdown mice had significantly higher cell counts and protein concentrations ( figure 3c and 3d ). of note, pulmonary bacterial colonization was much more severe in the ad-shhepc1-treated mice ( figure 3e ). however, the balf il-6 level in the hepcidin knockdown mice was not significantly different from that in the control mice ( figure 3f) . moreover, the influence of disruption of the hepcidin gene in aecs on the outcome of ali was further studied. clp challenge caused a seven-day mortality of 18.75% in the wild-type mice, comparable to a mortality of 12.5% in the ad-shneg-treated mice. however, knockdown of hepcidin in aecs significantly increased the seven-day mortality after clp surgery (53.33% versus ad-shneg-treated mice, p <0.05; figure 3g ). hepcidin regulates iron metabolism by binding to ferroportin and causing its internalization and degradation. we therefore investigated the ferroportin levels in both whole lung tissue and alveolar macrophages. as expected, the ferroportin in the control mice was almost totally degraded, whereas a higher ferroportin level was observed in the hepcidin knockdown mice ( figure 4a and 4b) . we further asked whether hepcidin gene modification in aecs had an impact on local and systemic iron metabolism. of note, the knockdown of hepcidin in aecs resulted in less iron retention in the alveolar macrophages ( figure 5a ), whereas the iron contents in the spleen macrophages and serum iron concentration between the two groups showed no significant differences ( figure 5b and 5c) . moreover, to link the intracellular iron content to the function of alveolar macrophages, we assessed the phagocytosis of alveolar macrophages, and found that the alveolar macrophages from ad-shhepc1-treated mice showed less phagocytic ability than those from the control animals ( figure 5d ). in the current study, we found that the pulmonary hepcidin level was upregulated during polymicrobial sepsis. the knockdown of hepcidin in aecs aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality. these pathophysiologic changes are at least partially related to the altered intracellular iron level and function of alveolar macrophages in the hepcidin knockdown mice. hepcidin is produced predominantly by hepatocytes. its hepatic expression can be upregulated by iron overload and inflammation and suppressed by hypoxia and anemia [14, 17] . a recent study reported that hepcidin is expressed in aecs in response to interferon-γ [22] . in the current study, an increased hepcidin level in aecs was observed during polymicrobial sepsis. because the lung is the first vital organ that is adversely affected at the onset of sepsis [31] , the elevated expression of hepcidin may protect the mice against lung injury. to support this hypothesis, knockdown of hepcidin in aecs exacerbated sepsisinduced lung injury (figure 3 ). considering that disruption of hepcidin has a deleterious effect, a low severity of clp model was used in the current study. since the underlying pathophysiology of sepsis is based on the severity grade of the clp model, the increase in lung injury parameter such as wet/dry weight ratio was minuscule but significantly different. hepcidin is a master regulator of iron metabolism via its interaction with ferroportin. hepcidin can trigger ferroportin polyubiquitination and induce ferroportin endocytosis [32, 33] . in the lung, aecs express high levels of hepcidin and are the main source of local hepcidin production. alveolar macrophages express ferroportin and are therefore target cells for hepcidin [22] . in the present study, knockdown of hepcidin in aecs impacted its interaction with ferroportin, and prevented degradation of the ferroportin protein in both alveolar macrophages and the figure 1 the expression of hepcidin in airway epithelial cells (aec) was upregulated after cecal ligation and puncture (clp) surgery. hepcidin expression was examined by immunohistochemistry. a representative image is presented (magnification: ×400) and quantified data are shown. n = 6 mice/group. *p <0.005. lung, which consequently caused intracellular iron export into the pulmonary microenvironment. the elevated iron in the lung can be used not only for invading pathogen growth and replication, resulting in more virulent and persistent infections [18] , but also to generate reactive oxygen species, leading to cell damage and lung injury [34, 35] . on the other hand, a recent study showed that iron status could impact cytoskeleton rearrangement, which is important for the phagocytic process in macrophages [36] . indeed, the alveolar macrophages from ad-shhepc1-treated mice showed less phagocytosis ability than those from the control animals. since liver is the major source of systemic hepcidin, in the current study liver hepcidin levels were not affected and circulating iron concentrations were comparable between the ad-shneg-and ad-shhepc1-treated mice. therefore, the function of circulating leukocytes should not be influenced. although the inciting injury (clp) is remote, when the bacteria circulating in the blood stream invaded the lung after clp, the decreased phagocytosis function of the alveolar macrophages from the ad-shhepc1-treated mice could result in bacterial accumulation in the lung. in addition, as hepcidin exhibits broad spectrum antimicrobial properties [37] , the knockdown of hepcidin may contribute to more severe pulmonary infections and lung injuries. using primary human aecs and alveolar macrophages, frazier et al. reported that the treatment with exogenous hepcidin did not affect ferroportin expression in the aecs and did not alter iron accumulation in both aecs and alveolar macrophages [22] . the contradictory findings between the current study and frazier's study may result from the difference between the in vivo model and ex vivo model used and the nature of hepcidin peptide. moreover, limited by the sensitivity of the prussian blue staining method, the iron content in the aecs was undetectable in this study. because the role of aecs in pulmonary iron metabolism is much less than that of alveolar macrophages [22] , the iron status in aecs may not play a major role in the pathophysiology of lung injury. the production of cytokines and other inflammatory mediators at the site of injury is a feature of the pathogenesis of ali, with il-6 being one of the hallmarks [38] . however, the balf il-6 level in the aec-specific hepcidin knockdown mice was not significantly different from that in the control mice. previous studies found that low intracellular iron in macrophages could impair the translation of specific inflammatory cytokine transcripts, such as il-6 [39, 40] . because alveolar macrophages are a major source of il-6 production during lung injury, the decreased iron content in alveolar macrophages may compromise the local inflammatory response in the aec-specific hepcidin knockdown mice. the acute respiratory distress syndrome acute respiratory distress syndrome epidemiology and outcomes of acute lung injury incidence and outcomes of acute lung injury has mortality from acute respiratory distress syndrome decreased over time? a systematic review mortality rates for patients with acute lung injury/ards have decreased over time human defensins and ll-37 in mucosal immunity hiemstra ps: defensins and cathelicidins in inflammatory lung disease: beyond antimicrobial activity expression and activity of beta-defensins and ll-37 in the developing human lung increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection high concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome protection against pseudomonas aeruginosa pneumonia and sepsis-induced lung injury by overexpression of beta-defensin-2 in rats hepcidin and iron regulation, 10 years later fine tuning of hepcidin expression by positive and negative regulators hepcidin in human iron disorders: therapeutic implications hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice hepcidin and the iron-infection axis iron in innate immunity: starve the invaders two to tango: regulation of mammalian iron metabolism hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice hepcidin expression in human airway epithelial cells is regulated by interferon-γ functional differences between hepcidin 1 and 2 in transgenic mice targeted disruption of the hepcidin 1 gene results in severe hemochromatosis antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia triggering receptor expressed on myeloid cells-2 protects against polymicrobial sepsis by enhancing bacterial clearance hussell t: a critical function for cd200 in lung immune homeostasis and the severity of influenza infection hepatic iron concentration does not predict response to standard and pegylated-ifn/ribavirin therapy in patients with chronic hepatitis c attenuation of igg immune complex-induced acute lung injury by silencing c5ar in lung epithelial cells in vivo gene silencing (with sirna) of pulmonary expression of mip-2 versus kc results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury zinc modulates the innate immune response in vivo to polymicrobial sepsis through regulation of nf-kappab hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination the iron cycle and oxidative stress in the lung efficacy and toxicity of intravenous iron in a mouse model of critical care anemia murine macrophages response to iron leap-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury selective modulation of tlr4-activated inflammatory responses by altered iron homeostasis in mice attenuated inflammatory responses in hemochromatosis reveal a role for iron in the regulation of macrophage cytokine translation submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the national science fund for distinguished young scholars (no. 30825037), a key program (no. 81130036) and program (no. 81101445) from the national natural science foundation of china, zhejiang provincial program for the cultivation of high-level innovative health talents, and zhejiang provincial natural science foundation of china (y2080253). the funding agencies did not have any role in design, collection, analysis, and interpretation of data, as well as in the writing of the manuscript and in the decision to submit it for publication. the current study explored the role of aec-derived hepcidin in polymicrobial sepsis-induced ali, which is at least partially related to the altered intracellular iron level and function of alveolar macrophages. these observations provide new insight into the pathogenesis of ali/ards and might have therapeutic implications for ali/ards. knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality. knockdown of hepcidin in airway epithelial cells led to reduced ferroportin degradation in lung and a low intracellular iron content in alveolar macrophages.these findings provide new insight into the pathogenesis of ali/ards and might have therapeutic implications for ali/ards. the authors declare that they have no competing interests.authors' contributions qxc contributed to the study design, data analysis and drafting of the manuscript. sws carried out the animal studies, analyzed the data and drafted the manuscript. qhc participated in the animal studies and performed the quantitative pcr experiment. clz carried out the immunoblot and phagocytosis assays. xz participated in the iron determination and balf analysis. jlw contributed to the study design and coordination, and helped to draft the manuscript. xmf conceived of the study and critically revised the manuscript for important intellectual content. all authors read and approved the final manuscript. key: cord-006541-ror7z8h7 authors: liu, xiaoli; zhang, hua; su, lijie; yang, peng; xin, zhiqiang; zou, junwei; ren, shuangyi; zuo, yunfei title: low expression of dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related protein in lung cancer and significant correlations with brain metastasis and natural killer cells date: 2015-07-07 journal: mol cell biochem doi: 10.1007/s11010-015-2465-4 sha: doc_id: 6541 cord_uid: ror7z8h7 dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related protein (dc-signr) is a type ii transmembrane protein which has been reported to bind a variety of pathogens as well as participate in immunoregulation. but the association between the level of dc-signr and lung cancer is unknown. to investigate the clinical diagnostic significance of dc-signr in lung cancer, we investigated serum dc-signr levels in 173 lung cancer patients and 134 healthy individuals using enzyme-linked immunosorbent assay (elisa). results showed that serum dc-signr levels in lung cancer patients were lower than that in healthy controls (p = 0.0003). a cut-off value of 3.8998 ng/l for dc-signr predicted the presence of lung cancer with 78.03 % sensitivity and 49.25 % specificity (area under the curve = 0.6212, p = 0.0003). strikingly, serum dc-signr levels were significantly higher in lung cancer patients with brain metastasis compared to those without metastasis (p = 0.0283). moreover, the serum concentrations of dc-signr in lung cancer patients also correlated significantly with serum natural killer cells percentage (p = 0.0017). in addition, immunohistochemistry assay demonstrated that the expression of dc-signr in lung tissues of 31 lung cancer patients and 13 tuberculosis patients was significantly lower than that in 18 normal lung tissues (p = 0.0418, 0.0289), and there is no significant difference between tuberculosis tissues and lung cancer tissues (p = 0.2696). these results suggest that dc-signr maybe a promising biological molecule that has the potential for clinical research of lung cancer, whereas its underlying roles are needed to be investigated in further studies. electronic supplementary material: the online version of this article (doi:10.1007/s11010-015-2465-4) contains supplementary material, which is available to authorized users. lung cancer is the leading cause of cancer death worldwide among men and the second leading cancer site in women [1] . the high mortality rate of this disease is primarily due to the difficulty of early diagnosis, the high metastatic potential, and the poor responses to chemical therapy and radiotherapy [2] . unfortunately, the unclear etiology of lung cancer presents a huge challenge for the treatment of lung cancer. many groups' results showed that smoking is the most important modifiable risk factor for lung cancer [3] [4] [5] . otherwise, lung cancer susceptibility is also determined by host genetic factors [6, 7] . infection may play a role in lung cancer, including human papilloma virus (ppv), epstein-barr virus, and human immunodeficiency virus (hiv) [8] [9] [10] . in addition, there are some biological molecules correlated with the risk of lung cancer such as p-selectin [11] and chondrolectin (chodl) [12] . other known risk factors for lung cancer include exposure to electronic supplementary material the online version of this article (doi:10.1007/s11010-015-2465-4) contains supplementary material, which is available to authorized users. several occupational and environmental carcinogens such as asbestos, arsenic, and polycyclic aromatic hydrocarbons [13] . recent studies from several groups suggest that lectins may play an important role in a variety of physiological and pathological processes, typically with tumor progression and metastasis. for example, läubli and borsig [14] reported that selectins were upregulated the pulmonary metastatic microenvironment is facilitated by p or l-selectin mediated interactions between tumor cells and blood components, which can significantly reduced metastasis. other groups results also showed that high mannose-binding lectin (mbl) levels were associated with poorer lung cancer survival [15] , and low mbl levels increased gastric cancer risk [16] . additionally, liver sinusoidal endothelial cell lectin (lsectin) can adhere to colorectal cells and plays an important role in colorectal carcinoma liver metastasis [17] . recently, our laboratory has been reported that dc-sign and dc-signr are low expressions in patients with non-hodgkin's lymphoma (nhl) and could be potentially useful in nhl clinical settings [18, 19] . dc-sign and dc-signr are novel blood-based molecular markers which can potentially be used for the diagnosis of early stage of colon cancer patients [20] . therefore, we are interested in the expression of dc-signr in lung cancer patients. dc-signr, a closest homologues to the c-type lectin superfamily members dc-sign, lsectin, and cd23, which consists of an n-terminal cytoplasmic tail, a transmembrane domain, an extracellular c-terminal neck region, and a c-type carbohydrate recognition domain (crd) [21, 22] . the crd domain of dc-signr can interact with high mannose carbohydrates, and the neck domains that support the crd may influence the ligand-binding properties of these lectins, and the intra-cellular domain located in the cytoplasmic tail containing recycling and internalization motifs [23] . dc-signr is highly expressed on liver sinusoidal endothelial cells, lymph node endothelial cells, and a significant proportion in terms of placenta capillary endothelial cells but was not expressed in peripheral bloodderived dcs [24] . furthermore, dc-signr is also expressed in the lung in both cytokeratin positive alveolar epithelia, as well as a subset of cells that co-expressed angiotensin converting enzyme-2 (ace2) but were negative for cytokeratin [25] . like dc-sign, dc-signr can recognize and bind several pathogens, including viruses, bacteria, fungi, and parasites [26] . dc-signr also recognizes endogenous ligands, such as icam molecules [27] , although their glycan epitopes have not been fully characterized. however, the role of dc-signr in lung cancer was not been elucidated. in our research, we compared the serum levels of dc-signr between lung cancer patients and healthy volunteers using an elisa. we found that lung cancer patients showed lower levels of dc-signr compared with healthy controls and that there were correlations between serum levels of dc-signr and clinical parameters of lung cancer patients. in immunohistochemistry (ihc) study, the dc-signr is lower expression in lung tissues from lung cancer patients compared to those in normal lung tissues. serum samples of 173 lung cancer patients were obtained from the first and second affiliated hospital of dalian medical university during march 2014-june 2014 and were stored at -80°c until they were analyzed. the mean age of the 173 patients was 61 years (range, 32-83 years), 102 were male and 71 were female. according to the world health organization (who) classification, the lung cancer cases consisted of small cell lung cancer (sclc) and non-small cell lung cancer (nsclc), the nsclc is further divided into three major pathologic subtypes, including adenocarcinoma (adc), squamous cell carcinoma (scc), and large cell carcinoma [28] . clinical staging of lung cancer patients was performed according to the iaslc lung cancer staging [29] . clinical data including gender, age, cancer stage, histological subtype, concentrations of tumor markers, and the percentage of t cell subsets were acquired from hospitalization records. the basic characteristics of these subjects were shown in table 1 and supplementary table 1. the control group was composed of 134 healthy blood donor volunteers. among the healthy subjects, the mean age was 56 years (range, 24-73 years). each healthy control performed routine physical examinations, and the results were within the normal range. lung cancer patients and healthy individuals with various pathogen infections and poor performance status were excluded in present study. in addition, formalin-fixed and paraffin-embedded tissues of 31 lung cancer patients, 13 lung tissues from tuberculosis patients, 18 normal lung tissues, and 9 lymph nodes from 2009 to 2015 were obtained from the second affiliated hospital of dalian medical university. lung tissues from tuberculosis patients and normal lung tissues were used as control groups, and normal lymph nodes were used as either positive or negative controls. the clinical data for these subjects are summarized in table 2 and supplementary table 2 . we used standard sandwich elisa to detect the levels of dc-signr in human serum. 96-well microplates were coated with 100 ll anti-dc-signr goat polyclonal antibody (pab, santa cruz biotechnology, inc., usa, catalog number: sc-17261) at a final concentration of 0.27 lg/ml and incubated overnight at 4°c. washed the plates 3 times with phosphate buffered saline (pbs) containing 0.05 % tween-20 (pbst) (ph 7.4), and the wells were blocked with blocking buffer (5 % bovine serum albumin) at 37°c for 1 h. the plates were washed three times with pbst. and then, 100 ll serum from lung cancer patients and healthy controls was added to the wells. as a negative control, 100 ll pbs was used. each plate was incubated at 37°c for 2 h. subsequently, pbst was used to wash the plates 3 times, and 100 ll anti-dc-signr mouse monoclonal antibody (mab, r&d systems, inc., usa, catalog number: mab16211) diluted to a concentration of 0.5 lg/ml was added to each wells. the plates were incubated at 37°c for 1.5 h. after washing, 100 ll peroxidase-conjugated goat anti-mouse (zsgb-bio) was added and the plates were incubated at 37°c for 1 h. finally, the plates were washed 3 times and incubated with 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb, tiangen bio-tech co, ltd.) at 37°c for 0.5 h, and then 2 mol/l h 2 so 4 was added to stop the reaction. the optical density (od) was measured at the 450 nm wavelength on a microplate reader. the quantitative dc-signr concentrations were determined by comparing the optical density values with the standard curve. the s100b levels in lung cancer patients with brain metastasis were detected by elisa kit (westang bio-tech, ltd). the patients who have other diseases which can affect s100b levels were excluded in this study. the concentrations of s100b were shown in supplementarytable 3. before being deparaffinized in xylene and rehydrated in a graded ethanol series, sections from paraffin-embedded blocks were incubated at 60°c for 30 min. sections were washed with pbs, and then endogenous peroxidase activity was blocked with 3 % hydrogen peroxide diluted in double-distilled water for 10 min at room temperature. antigen retrieval was performed by heating slides in citrate buffer (0.01 mol/l citric acid, ph 6.0) for 20 min in a microwave oven. after being washed with pbs, the sections were incubated with goat serum for the purpose of blocking. sections were incubated with anti-dc-signr rabbit pab (abcam, inc.) overnight at 4°c (1:250). the next day, after being washed with pbs, the sections were incubated with horseradish peroxidase-labeled anti-rabbit immunoglobulin (1:1000) (zsgb-bio) at 37°c for 1 h and were then washed again. finally, all the sections were developed with 3,3 0 -diaminobenzidine tetrahydrochloride (dab,zsgb-bio) within the cells and then counterstained with hematoxylin. photographs were recorded on an olympus bx51 microscope. the mean density of dc-signr expression was assessed using the image-pro plus version 6.0 (media cybernetics, inc. silver spring, md usa). integrated optical density (iod) sum represents the protein content of dc-signr in the area of interest (aoi), while area equals the area of aoi. (iod sum)/area stands for ''mean density''. briefly, images were captured at 2009 magnification from 3 aois/case, which were selected based on areas with maximal dc-signr staining. the mean density values are shown in supplementary table 2 . this quantitation was positively correlated with dc-signr expression in tissue. non-parametric mann-whitney u test was used to determine the statistical significance among two groups. the statistical significance among more than two groups was determined with the kruskal-wallis non-parametric test. correlation of dc-signr values with clinical parameters was tested by the non-parametric spearman's rank correlation coefficient test. in all tests, 2-sided p values below 0.05 were considered significant. all statistical analyses were performed using graphpad prism5 (graphpad software, inc., san diego, ca, usa). we detected serum dc-signr levels in 307 participants, composing of 173 with lung cancer and 134 healthy controls by elisa. the dc-signr level in the serum of patients with lung cancer (14.9434 ± 0.3152 mg/ml) was significantly lower than that in healthy controls (3.4696 ± 0.2471 mg/ml) (p = 0.0003; fig. 1a) . a roc curve is often used to assess the power of a novel serum marker for tumor prediction. in the present study, a cut-off value of 3.8998 ng/l for dc-signr predicted the presence of lung cancer with 78.03 % sensitivity and 49.25 % specificity, and the area under curve (auc) was 0.6212 (p = 0.0003) (fig. 1b) . therefore, serum dc-signr levels demonstrated low accuracy for the detection of lung cancer. serum concentrations of dc-signr correlated significantly with lung cancer patients who have brain metastasis we assessed the correlations between serum levels of dc-signr and clinical data, including gender, age, cancer stage, histologic subtype, and metastasis to different organs. strikingly, serum concentrations of dc-signr of lung cancer patients with brain metastasis were higher than that without metastasis (p = 0.0283; fig. 2b ). however, the levels of dc-signr in patients with lung cancer showed no significant difference between metastasis and non-metastasis (p = 0.3887; fig. 2a) . furthermore, the serum dc-signr levels were not significantly different between bone metastasis and non-metastasis (p = 0.3841; fig. 2b ), lung metastasis and non-metastasis (p = 0.6744; fig. 2b ), lymph node metastasis and non-metastasis (p = 0.9880; fig. 2b ). otherwise, we found that the serum levels of dc-signr correlated significantly with serum nk cells percentage with the spearman's correlation coefficient of -0.3221 (p = 0.0017; fig. 3a ). however, there was no significant correlation between serum dc-signr and cd3, with a spearman's correlation coefficient of 0.1583 (p = 0.1713; fig. 3b ). moreover, serum levels of dc-signr showed no association with cd4 with a spearman's correlation coefficient of 0.0513 (p = 0.6270; fig. 3c ). in addition, serum levels of dc-signr displayed no correlation with cd8 with a spearman's correlation coefficient of 0.0723 (p = 0.4934; fig. 3d ). no significant correlations were observed between serum levels of dc-signr and clinical parameters, such as age, gender, cancer stage, and histologic subtype contrary to above results, serum levels of dc-signr in patients with lung cancer showed no significant difference between male and female (p = 0.5723; fig. 4a ). serum levels of dc-signr also showed no significant difference between patients of b60 years old and patients [60 years old (p = 0.1213; fig. 4b ). serum levels of dc-signr expressed no association with age with a spearman's correlation coefficient of 0.0706 (p = 0.3559; fig. 4c ). furthermore, the serum dc-signr levels were not significantly different among the stage i-ii, and stage iii-iv (p = 0.9299; fig. 4d ). additionally, for different histologic subtype, no significant difference was observed between lung cancer patients with sclc and those with nsclc (p = 0.6000; fig. 4e) . similarly, the difference of serum dc-signr levels was not apparent between sclc, adc and scc (p = 0.5016; fig. 4f ). serum dc-signr levels showed no significant correlation with s100b, carcinoembryonic antigen (cea), neuron-specific enolase (nse), and cyfra21-1 in our study, the levels of dc-signr in lung cancer patients with brain metastasis is higher than that without metastasis. s100b as a marker of brain metastasis, so we compare the correlation between the two molecular. however, there is no significant correlation between dc-signr and s100b, with a spearman's correlation coefficient of 0.003847 (p = 0.9854; fig. 5a ). in present study, serum levels of dc-signr expressed no association with cea, with a spearman's correlation coefficient of 0.1982 (p = 0.1007; fig. 5b) . similarly, serum levels of dc-signr were not significantly correlated with nse, with a spearman's correlation coefficient of -0.0709 (p = 0.3742; fig. 5c ). furthermore, the data also showed no correlation between serum dc-signr and cyfra21-1 levels, with a spearman's correlation coefficient of 0.0168 (p = 0.8346; fig. 5d ). the expression of dc-signr in lung cancer patients and tuberculosis patients was significantly lower than that in normal lung tissues we determined the expression level of dc-signr in serum. moreover, it has been reported that dc-signr is fig. 2 serum dc-signr levels were significantly correlated with brain metastasis. the serum levels of dc-signr were not significantly different between metastasis and non-metastasis (p = 0.3887) (a). serum concentrations of dc-signr were higher in brain metastasis than in non-metastasis (p = 0.0283) (b). however, the serum dc-signr levels were not significantly different between bone metastasis and non-metastasis (p = 0.3841), lung metastasis and non-metastasis (p = 0.6744), lymph node metastasis and nonmetastasis (p = 0.9880) (b) mol cell biochem (2015) 407:151-160 155 expressed in the lung in both cytokeratin positive alveolar epithelia, as well as a subset of cells that co-expressed ace2 [25] . therefore we want to know whether the dc-signr expression in lung cancer tissues. we performed ihc analysis, using anti-dc-signr pab in 31 lung cancer tissues (1 sclc, 16 adc, and 14 scc) and 13 lung tissues of tuberculosis patients, and 18 normal lung tissues were used as controls. strikingly, the mean density of dc-signr expression in the lung cancer patients and tuberculosis patients was significantly lower than that in normal (figs. 6, 7) . the normal lymph nodes of tumor patients were used as positive controls and negative controls. the expression of dc-signr mainly occurs in infective diseases, such as acquired immune deficiency syndrome (aids) with hiv infection, hepatitis c infection disease, and ebola virus infection disease [26] . recently, our laboratory reported that dc-signr expression is lower in patients with nhl than in healthy individuals and higher expression in colon cancer patient serum [18, 20] . to extend the knowledge of the possible clinical applications of dc-signr, we studied the serum levels of dc-signr in patients with lung cancer, and we also studied the expression and location levels of dc-signr in lung cancer tissues. our study demonstrated that serum levels of dc-signr in lung cancer patients were significantly lower than those in healthy individuals. furthermore, we found that the serum dc-signr levels correlated significantly with brain metastasis and serum nk cells percentage in lung cancer patients. brain metastasis as one of the most dreaded complications of lung cancer has a poor prognosis [30] . up to 50 % of lung cancer patients develop brain metastasis during their course of disease [31] . the occurrence of brain metastasis is associated with poor prognosis and high morbidity in patients with advanced lung cancer, even after intensive multimodal therapy [32] . furthermore, little is known about the pathobiology and the molecular mechanisms involved in the brain metastatic cascade which limits the possibilities for focused drug development and clinical trial [33] . s100b and proapolipoprotein a1 as a serum marker of brain metastasis in lung cancer have been investigated in a recent study [34] [35] [36] . in our study, the dc-signr levels in lung cancer patients with brain metastasis are significantly higher than those without metastasis. so, we compared the correlation between serum dc-signr and s100b levels. however, there is no significant correlation between them. s100b which is a protein found in astrocytes and only released into serum when the blood-brain barrier is breached [37] . the expression of dc-signr in brain tissues is still unknown. thus, we guessed that the two molecular may have a difference mechanism to affect the brain metastasis of lung cancer and it can be investigated in further study. we also compared the serum levels of dc-signr in bone, lung, and lymph nodes metastasis with non-metastasis, respectively. but we found no significant difference between them. natural killer (nk) cells, as a major component of the innate immune system, can limit the growth and dissemination of several types of tumors [38] . unlike t cells and b cells, nk cells can directly exert cellular cytotoxicity on tumor cells without prior sensitization and secrete immunostimulatory cytokines, which control both local tumor growth and metastasis [39] . an epidemiologic survey showed that low nk cell activity is associated with increased cancer risk [40] . numerous studies have demonstrated that the infiltration of nk cells appears to have prognostic value in gastric carcinoma [41] , colorectal carcinoma [42] , and lung carcinomas [43, 44] , as a relatively higher level of nk cell infiltrate correlates with a better prognosis, thus suggesting relevant protective roles for nk cell infiltrate. in the present study, we detected a significantly negative correlation between serum levels of dc-signr and serum percentage of nk cells in lung cancer patients. the molecular mechanism for this negative correlation is not yet known. on the contrary, we found no significant correlations between serum levels of dc-signr and serum percentage of cd3, cd4, and cd8 in the present study. in order to know the dc-signr whether it has the ability to distinguish the benign and malignant diseases of the lung, we choose tuberculosis (tb) patients, which results in mycobacterium tuberculosis infections as a benign disease group. mycobacterium tuberculosis can targets dc-sign to inhibit the immuno-stimulatory function of dc through the interaction of the mycobacterial mannosylated lipoarabinomannan (manlam) to dc-sign [45] . like dc-sign, dc-signr also can capture man-lam and rapidly internalizes it to lysosomes, and may be involved in the clearance of mycobacteria [45] . tailleux l et al. [46] showed dc-sign present in the alveolar cd11b ? mus in the lungs of patients with tb. in present study, we found the dc-signr expression in lungs of patients with tb. however, there was no significantly difference between the dc-signr expression in lung cancer tissue and tuberculosis. strikingly, both of them are lower than those in normal lung tissues. in summary, we have shown that the serum level of dc-signr is a promising marker to help distinguish lung cancer patients from healthy individuals. additional studies are warranted to assess the potential value of dc-signr in vivo. these studies will most likely make dc-signr a useful biological molecule for clinical research on lung cancer mechanisms in the future. compliance with ethical standards the study was approved by the research ethics committee of dalian medical university, and fig. 6 ihc for dc-signr expression in tissues of lung cancer tissues, tuberculosis patients, and normal tissues. ihc analysis was used to determine dc-signr expression and arrows indicate positive staining. areas in the black boxes of a, c, e, g, and i were enlarged below. dc-signr expression was detected in lung tissues from tuberculosis patients (a-b). dc-signr expression was detected in the lung tissues from lung cancer patients (c-d). dc-signr expression in alveolar epithelia cells of normal lung tissues (e-f). dc-signr expression in lymphatic endothelial cells exhibited strong positive (g-h). normal lymph node tissues were used as a negative control (i-j) fig. 7 semi-quantitative image analysis of dc-signr expression in tissues. the graph displays a scatter plot of the levels of dc-signr expression in lung tissues from lung cancer patients, tuberculosis patients, and normal controls. there was a statistical significance in ihc for dc-signr expression between lung cancer tissues and normal lung tissues (p = 0.0418), tuberculosis tissues and normal controls (p = 0.0289). there was no significantly difference between lung cancer patients and tuberculosis patients (p = 0.2696). dc-signr expression in lung cancer patients was lower than in normal lung tissues informed consent was obtained from all participants, in agreement with institutional guidelines. global cancer statistics surfactant protein a suppresses lung cancer progression by regulating the polarization of tumor-associated macrophages lung cancer: epidemiology, etiology, and prevention tobacco control policy in developed countries: yesterday, today, and tomorrow mortality in relation to smoking: 50 years' observations on male british doctors genome-wide association scan of tag snps identifies a susceptibility locus for lung cancer at 15q25.1 the molecular epidemiology of lung cancer the role of human papilloma virus in lung cancer: a review of the evidence relationship between epstein-barr virus and lymphoepithelioma-like carcinoma of the lung: a clinicopathologic study of 6 cases and review of the literature hiv infection is associated with an increased risk for lung cancer, independent of smoking soluble selectin levels in patients with lung cancer chondrolectin is a novel diagnostic biomarker and a therapeutic target for lung cancer cancer of the lung selectins as mediators of lung metastasis lung cancer survival and functional polymorphisms in mbl2, an innate-immunity gene mannose-binding lectin-2 genetic variation and stomach cancer risk novel roles of liver sinusoidal endothelial cell lectin in colon carcinoma cell adhesion, migration and in vivo metastasis to the liver low expression of dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related protein in non-hodgkin lymphoma and significant correlations with lactic acid dehydrogenase and b2-microglobulin low expression of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in non-hodgkin lymphoma and a significant correlation with b2-microglobulin the clinical significance of dc-sign and dc-signr, which are novel markers expressed in human colon cancer dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr: subunit organization and binding to multivalent ligands dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection homozygous l-sign (clec4 m) plays a protective role in sars coronavirus infection dc-sign and l-sign: the signs for infection characterization of dc-sign/r interaction with human immunodeficiency virus type 1 gp120 and icam molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor the new world health organization classification of lung tumours the iaslc lung cancer staging project: proposals for the revision of the tnm stage groupings in the forthcoming (seventh) edition of the tnm classification of malignant tumours the elderly with synchronous non-small cell lung cancer and solitary brain metastasis: does palliative thoracic radiotherapy have a useful role a cancer stem cell model for studying brain metastases from primary lung cancer the molecular biology of lung cancer brain metastasis: an overview of current comprehensions and future perspectives trial design on prophylaxis and treatment of brain metastases: lessons learned from the eortc brain metastases strategic meeting s100b protein as a possible participant in the brain metastasis of nsclc s100b promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line proapolipoprotein a1: a serum marker of brain metastases in lung cancer patients serum s100b as a possible marker of blood-brain barrier disruption functions of natural killer cells natural killer cells in human cancer: from biological functions to clinical applications natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population prognostic value of intratumoral natural killer cells in gastric carcinoma the prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma prognostic significance of tumor infiltrating natural killer cells subset cd57 in patients with squamous cell lung cancer the prognostic value of natural killer cell infiltration in resected pulmonary adenocarcinoma identification of the mycobacterial carbohydrate structure that binds the c-type lectins dc-sign, l-sign and signr1 dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis acknowledgments this study was supported by grants from the chinese national natural science foundation projects (81372669,31270867, 31470800) and science and technology planning project of liaoning province, china (2012225020). the authors declare that they have no conflict of interest. key: cord-006624-or0cpc6j authors: kamler, m.; pizanis, n. title: aktueller stand der lungentransplantation: pulmonale und nichtpulmonale komplikationen date: 2013-05-31 journal: z herz thorax gefasschir doi: 10.1007/s00398-013-1005-3 sha: doc_id: 6624 cord_uid: or0cpc6j lung transplantation is an established therapeutic option for selected patients with various end stage pulmonary diseases which prolongs survival and improves quality of life. a multitude of pulmonary and non-pulmonary complications can lead to significant morbidity thus impairing short and long-term survival. early recognition and fast treatment of these complications are fundamental measures to prevent secondary destructive incidents. this article reviews the most frequent complications arising after lung transplantation. pulmonale und nichtpulmonale komplikationen beeinflussen letalität und morbidität nach einer lungentransplantation. dabei sind primäres organversagen, non-zytomegalievirus(non-cmv)-infektionen und kardiovaskuläre ereignisse im 1. jahr die häufigsten ursachen [6] . nach dem 1. jahr bleiben non-cmv-infektionen und bronchiolitis-obliterans-syndrom (bos) hauptverantwortlich. hierbei entwickelt die mehrzahl der patienten innerhalb der ersten 5 jahre eine mehr oder weniger ausgeprägte form des bos. zudem lässt die älter werdende population der transplantatempfänger erwarten, dass medizinische, insbesondere nichtpulmonale komplikationen zunehmen werden. entscheidend für die erfolgreiche therapie ist das frühe erkennen der probleme durch ein gewissenhaftes monitoring der patienten. die vorliegende übersicht behandelt die häufigsten pulmonalen und nichtpulmonalen komplikationen nach lungentransplantation. das primäre organversagen (" primary graft dysfunction", pgd) ist mit einer inzidenz zwischen 14 und 23% weiterhin eine bedeutsame komplikation in der perioperativen phase der lungentransplantation [7] . es tritt meist innerhalb von 72 h post transplantationem auf und ist nach heutiger auffassung als unabhängiger risikofaktor für ein bos zu betrachten. die schädigung des transplantats entsteht u. a. nach unterbrechung der blutversorgung bei der organentnahme, durch die kombiniert warme und kalte ischämie, durch manipulation am organ bei entnahme und implantation sowie schließlich in der reperfusionsphase. die klinischen zeichen des pgd sind hypoxämie und diffuse infiltrate im thoraxröntgenbild, begleitet von einem kapillarleck. es zeigen sich alveoläre und interstitielle ödeme, gefolgt von der entwicklung hyaliner membranen analog zum "adult respiratory distress syndrome" (ards). das erscheinungsbild der pgd ist vielfältig und graduell unterschiedlich. von milden infiltraten bis hin zu lebensbedrohlichen gasaustauschstörungen bietet sich ein breites bild. das pgd ist die führende ursache der perioperativen letalität bei der lungentransplantation, interessanterweise scheint es aber auch in mild ausgeprägten formen mit vermindertem langzeitüberleben vergesellschaftet zu sein [7] . in einer retrospektiven studie an 122 mukoviszidosepatienten nach lungentransplantation konnte gezeigt werden, dass in diesem speziellen kollektiv 2 faktoren mit pgd zusammenhängen: die ischämiezeit des 2. implantierten grafts bei doppellungentransplantation und ein erhöhter laktatspiegel im serum begleiteten ein verstärktes pgd [10] . hierbei ist die hyperlaktatämie sicherlich nicht ursäch-lich für pgd, sondern eher als sekundärer effekt bei auftretendem pgd zu sehen. pathophysiologisch ist das pgd schwer zu fassen. es erscheint klinisch ähnlich wie andere formen der akuten lungenschädigung. störungen der gerinnungskaskade, hochregulierung von adhäsionsmolekülen und endotheliale dysfunktion sind involviert. christie et al. [8] berichteten im rahmen einer großen pros pektiven studie mit über 300 patienten über die suche nach einem biomarker für pgd. sie konnten erhöhte plasmaspiegel für lösliche rezeptoren der endprodukte der glykosylierung ("soluble receptor for advanced glycation end products", srage) identifizieren. diese waren mit pgd vergesellschaftet und werden auch durch die zahl der transfundierten blutprodukte beeinflusst. "soluble receptor for advanced glycation end products" ist ein potenzieller marker der schädigung von typ-1-epithelzellen in den lungen. des weiteren zeigte sich, dass die transfusion von blutprodukten starken einfluss auf pgd hat. möglicherweise führen transfundierte "anti-human-leukocyte-antigen"(anti-hla)-antikörper und/oder die gegenwart von antikörpern in blutprodukten mit konsekutiver neutrophilenaktivierung zu einer lungenschädigung. bei der lungentransplantation kommt es zur höchsten abstoßungsrate im vergleich zu anderen transplantierten organen. zirka 55% der lungentransplantationsempfänger benötigen eine therapie für die akute organabstoßung im 1. jahr nach der transplantation [35] . junge empfänger haben dabei insgesamt das niedrigste risiko, eine akute abstoßung zu erleiden. akute abstoßungen stellen einen hauptrisikofaktor für die entwicklung einer chronischen abstoßung dar. sowohl minimale einzelepisoden als auch rezidivierende akute abstoßungsepisoden erhöhen dieses risiko. die therapie von geringgradigen abstoßungen führt zu positiven verläufen. die symptome sind unspezifisch: subfebrile temperaturen, infiltrate im röntgenbild, verschlechterung der lungenfunktion, dyspnoe, hypoxämie oder auch pleuraerguss. histologisch besteht eine lymphozytäre infiltration in den terminalen bronchioli und begleitenden gefäßen. die klassifikation wird gemäß einer standardisierten nomenklatur histologisch nach transbronchialer biopsie vorgenommen. um die diagnose besser zu definieren, werden die methoden ausgeweitet. in einer experimentellen arbeit wiesen jungraithmayr et al. [18] durch den einsatz des elektronenmikroskops aktivierte dendritische zellen sowie thrombozyten-und makrophagenakkumulationen am endothel der großen gefäße nach; dies sind wichtige neue detailinformationen zur pathophysiologie der akuten abstoßung. im rahmen einer laboranalytischen studie an 10 lungentransplantierten berichteten miyoshi et al. [28] ermutigende ergebnisse, allerdings bei kleiner fallzahl, über die evaluation eines antidonor-igm-monitorings zur frühdiagnose einer abstoßung. die anfälligkeit der lungen für verletzung, infektion und die konstante auseinandersetzung mit der umgebung über die atemluft sind höchstwahrschein-lich für hohe zahl der akuten abstoßungen verantwortlich. weitere risikofaktoren sind "hla missmatch" [30] , virale infektionen, rezidivierende abstoßungsperioden und auch genetische faktoren des empfängers [22, 38] . in einer retrospektiven analyse von 481 patienten konnten mangi et al. [25] aufzeigen, dass ein hla-missmatch am dr-locus zu vermehrten frühabstoßungen und ein hla-missmatch am b-locus zu erhöhten akuten abstoßungen im langzeitverlauf nach 4 jahren führen. bei anderen organen, so z. b. den nieren, geht selbst das vorliegen von bis zu 6 hla-missmatches nicht mit einer beeinträchtigung des graft-überlebens einher [24] ; dies allerdings unter potenter immunsuppression der modernen ära [19] . aus dem aktuellen bericht des registers der international society for heart and lung transplantation (ishlt) geht hervor, dass auch bei den lungen bis zu 6 missmatches nur in einer moderaten erhöhung der zehnjahreletalität resultieren (relatives risiko 1,06; p-wert 0,0001, [5] ). ob letztlich ein hla-missmatch wirklich zur immunologischen basis der akuten abstoßung beiträgt, ist aus den existierenden studien nicht eindeutig zu erheben. trotz der einführung neuer immunsuppressiva konnte die zahl der akuten abstoßungsepisoden nicht bedeutend reduziert werden. aktuelle regime beinhalten die gaben von kalzineurininhibitoren, kortikosteroide, mycophenolat-mofetil oder azathioprin. wenige studien untersuchten die direkte verbindung zwischen immunsuppressivaspiegel und akuter abstoßung. ahya et al. [2] fanden hohe immunsuppressivaspiegel, vergesellschaftet mit einer niedrigeren inzi[9] . im internationalen vergleich hat die verabreichung von il-2-rezeptor-(il2r)-antagonisten in der letzten dekade deutlich zugenommen, dagegen hat die applikation von atg deutlich abgenommen [6] . lange zeit war unklar, ab welchem abstoßungsgrad eine behandlung durchgeführt werden sollte. in betrachtung der problematik, dass auch leichte abstoßungen zu nachfolgenden rezidivierenden akuten abstoßungen führen könnten, sollten diese nach heutigem kenntnisstand immer behandelt werden. die initialtherapie besteht in der applikation einer steroidstoßtherapie über 3 tage. hierüber gibt es jedoch keinen klinischen therapiekonsens zwischen den zentren. für wiederholte abstoßungen wird versucht, diese z. b. mithilfe einer erneuten steroidstoßtherapie einzudämmen. andere studien berichten über den wechsel von cyclosporin a oder die applikation eines atg, il2r-antagonisten oder anderer antikörper. für therapieversager werden weitere antikörper evaluiert z. b. alemtuzumab (anti-cd52-antikörper, [20] ). versuche mit der inhalation von immunsuppressiva, z. b. cyclosporin a, erbrachten zunächst ermutigende ergebnisse. es gibt jedoch wenig konklusive daten. es entwickeln 40-50% der lungentransplantierten ein bos, die klinische manifestation der chronischen abstoßung. sie gilt weiter als eine der haupttodesursachen. die klassifikation richtet sich nach der einschränkung der einsekundenkapazität ("forciertes expiratorisches volumen in einer sekunde", fev 1 ) in bezug auf den postoperativen bestwert. histologisch finden sich entzündung und fibrose mit vermehrung des eosinophilen bindegewebes in den bronchiolen bis zur kompletten okklusion mit konsekutiver mukostase. folglich versterben diese patienten entweder an respiratorischem versagen oder an infektionen. das erscheinungsbild ist uneinheitlich sowohl bezüglich der ausprägung als auch des zeitlichen verlaufs. therapeutisch werden immunsuppressiva, fotopherese und auch bestrahlungen eingesetzt, ohne dass bisher nachweisliche erfolge erbracht wurden. gottlieb et al. [15] konnten durch den einsatz von azithromycin eine erholung der lungenfunktion erreichen, ebenso vos et al. [37] in einer kontrollierten studie. für therapieversager ist die retransplantation eine weitere therapieoption, die an ausgewählten patienten mit guten ergebnissen durchgeführt werden kann. die rolle der "gastroesophageal reflux disease" (gerd) bei der lungentransplantation ist noch nicht abschließend geklärt. in den letzten jahren gibt es eine zunehmende zahl an untersuchungen zu die primäres organversagen · bronchiolitis obliterans · gaft-abstoßung · "gastroesophageal reflux disease" · übersichtsartikel update in lung transplantation. pulmonary and non-pulmonary complications abstract lung transplantation is an established therapeutic option for selected patients with various end stage pulmonary diseases which prolongs survival and improves quality of life. a multitude of pulmonary and non-pulmonary complications can lead to significant morbidity thus impairing short and longterm survival. early recognition and fast treatment of these complications are fundamental measures to prevent secondary destructive incidents. this article reviews the most frequent complications arising after lung transplantation. primary organ failure · bronchiolitis obliterans · graft rejection · gastroesophageal reflux disease · review sem thema. insbesondere die antirefluxchirurgie ("laparoscopic antireflux surgery", lars) steht neben medikamentösen therapien im fokus. reflux bei lungentransplantationsempfängern ist mit einer erhöhten inzidenz an bos vergesellschaftet. kontrolle und therapie von gerd führen dabei wieder zu einer stabilisierung der lungenfunktion beim bos [4] . betrachtet man die aktuelle literatur, kommt es zu einem gemeinsamen auftreten von gerd und bos, aber nicht zu einer kausal begründeten vergesellschaftung. der üblicherweise durchgeführte ph-test zur diagnose von gerd ist nur ein indirekter test, um das risiko einer aspiration abzuschätzen. eine untersuchung mithilfe der ösophagealen impedanzmessung zeigte, dass bei 71% der lungentransplantationsempfänger die therapie mit protonenpumpenhemmern die absolute zahl an refluxereignissen von nichtsäurehaltigen magenbestandteilen nicht reduzieren konnte. dies konnte auch in der bronchoalveolären lavage (bal) bestätigt werden [3] . der nachweis von pepsin in der bal bei lungentransplantationsempfängern war in einer untersuchung von stovolt et al. [33] am höchsten bei patienten mit akuter abstoßung. vermutlich kommt es bei der aspiration von mageninhalten, wie pepsin und gallensäuren, zu einer beschleunigung des allograft-versagens und entwicklung von bos. hoher gallensäurespiegel in der bal ist mit schlechterer lungenfunktion und schlechterem überleben assoziiert, wie mertens et al. [26] in einer retrospektiven analyse an 37 lun-gentransplantierten belegten. in der prospektiven studie von fisichella et al. [12] an 64 lungentransplantierten patienten wurde der pepsingehalt in der bal im langzeitverlauf bestimmt. gesunde kontrollpersonen wiesen kein pepsin in der bal auf, erhöht war der gehalt dagegen bei lungentransplantationspatienten unabhängig von ihrem refluxstatus. patienten, die einem lars unterzogen wurden, hatten niedrige pepsinspiegel. bei patienten, die nachweislich aspiriert hatten, trat auch hier eine schnellere progredienz des bos auf. der vorteil der lars gegenüber der medikamentösen therapie mit protonenpumpenhemmern besteht darin, dass auch nichtsäurehaltige refluxanteile gehemmt werden, die zu einer inflammatorischen reaktion am bronchialepithel führen können [27] . zur lars werden idealerweise patienten ausgewählt, die einen positiven ösophagealen säurenachweis oder pepsin in der bal aufweisen oder bei denen die aspiration in der transbronchialen biopsie nachgewiesen wurde. trotz eines unklaren biologischen mechanismus, wie aspiration die lungen schädigen kann, steht lars im fokus der therapie von gerd bei transplantationspatienten, da höchstwahrscheinlich ein kausaler zusammenhang zwischen gerd-induzierter aspiration und der entwicklung einer lungenschädigung besteht. atemwegskomplikationen sind immer noch eine signifikante ursache der morbidität und letalität nach einer lungentransplantation. sie treten meist zwischen 2 und 9 monate nach der transplantation auf, mit einer rate von 7-35% und einer sterblichkeit von 2-4% [31] . ursächlich ist zum einen eine minderversorgung mit arteriellem blut, da lungen und bronchialsystem ohne arterielle blutversorgung über die bronchialarterien arbeiten müssen. dies trifft insbesondere für den bronchus intermedius in der perioperativen transplantationsphase zu (. abb. 1). andere faktoren, wie pgd, infektionen, abstoßungsepisoden sowie nebeneffekte der immunsuppressiva und beatmung mit positivem endexspiratorischem druck ("positive end-expiratory pressure", peep) sind zu berücksichtigen. klinisch finden sich lokale infekte, nekrosen, dehiszenz und granulationsgewebe bis hin zur stenose und bronchomalazie (. abb. 2). die therapeutischen möglichkeiten sind überwiegend interventioneller natur im sinne von medikamentöser therapie, interventioneller therapie mithilfe der laser-foto-resektion, kryotherapie oder ballondilatation von stenosen und platzierung von stents zum offenhalten einer anastomose [1] . in einer aktuellen retrospektiven analyse von fernandez-bussy et al. [11] [34] . insbesondere die postischämische stenose des bronchus intermedius kann mit der temporären stent-einlage gut therapiert werden [23] . der chirurgische ansatz ist nur bei einer ausgeprägten dehiszenz mit mediastinitis erforderlich. die interventionsmöglichkeiten sind dann begrenzt und erfordern häufig eine pneumektomie. zur prophylaxe werden verschiedene nahttechniken eingesetzt, außerdem die kürzung des spenderbronchus, soweit wie möglich, im sinne einer reduzierung des ischämischen bronchusgewebes. eine modifizierte anastomosentechnik wurde von van berkel et al. [36] vorgestellt. dabei wird der spenderbronchus bis auf die lappenkarina zurückgeschnitten, d. h. so weit wie überhaupt technisch möglich. durch diese technik konnte die atemwegskomplikationsrate eines chirurgen von 13 auf 2,1% gesenkt werden. eine exzellente serie an konsekutiven lungentransplantationen bei mukoviszidosepatienten präsentierte aktuell die gruppe aus zürich. in 100 konsekutiven patienten kam es im verlauf von bis zu 3 jahren nach transplantation zu keiner einzigen atemwegskomplikation [17] . da bei der lungentransplantation mit der durchführung der bronchialen anastomosen formal in einem unsterilen bereich gearbeitet wird, kommt es häufiger zu infektionen als bei transplantationen anderer organe. im verlauf konfrontieren ubiquitär vorhandene organismen, die per inhalationem aufgenommen werden, die transplantierte lunge. in der frühphase nach transplantation kommt es häufig zu bakteriellen pneumonien. ursächlich sind eingeschränkter hustenstoß bei postoperativem schmerz sowie die denervierung der lunge mit auch eingeschränkter lymphatischer drainage, eingeschränkte mukoziliäre funktion und im spenderorgan mitgebrachte erreger. neben pseudomonas aeruginosa, der insbesondere bei mukoviszidosepatienten auftritt, ist die prävalenz resistenter und nosokomialer keime wie staphylokokken und weiterer pseudomonas-spezies erhöht. infektionen stellen eine lebenslange gefahr bei immunsupprimierten lungentransplantierten patienten dar und repräsentieren eine der häufigsten ursachen früher und später letalität. die bestehende inflammation und die noch vorhandene immunantwort begünstigen eine akute und/oder eine chronische abstoßung [13] . hervorzuheben ist unter den viralen infektionen der cmv-befall. die cmv-infektion ist mit bis zu 33% im 1. postoperativen jahr die häufigste virale infektion bei lungentransplantierten. dabei ist der präoperative status des empfängers von großer bedeutung, da cmv-seronegative empfänger ein größeres risiko einer neuinfektion haben, wenn der spender eine seropositive konstellation aufweist. dieses missmatch (donor +, empfänger −) konnte durch das ishlt-register als risikofaktor einer erhöhten letalität identifiziert werden [5] . die diagnose sollte serologisch und durch bal erfolgen. die therapie besteht in der i.v.-gabe von ganciclovir und oraler gabe von valganciclovir. bei therapieversagen können noch virostatika wie foscarnet oder cidofovir in betracht gezogen werden [13] . im fokus bleibt aktuell die cmv-prophylaxe bzw. die empfohlene dauer der prophylaxe. mehrere studien zeigen die überlegenheit einer 12-monatigen gegenüber einer 3-monatigen prophylaxe; daten über den effekt von 6-bis 9-monatigen prophylaxen sind zurzeit noch nicht eindeutig [21] . es gilt dabei, zwischen nebenwirkungen einer langzeitprophylaxe, aber auch den kosten dieser therapie abzuwägen. da die cmv-infektion im 1. postoperativen jahr am häufigsten auftritt, ist zurzeit am ehesten eine einjährige prophylaxe zu vertreten [29] . andere ambulant erworbene respiratorische viren ("community-acquired respiratory virus", carv) umfassen adenoviren, influenzaviren sowie einige paramyxoviren ["respiratory syncytial virus" (rsv), parainfluenzavirus (piv) und "human metapneumovirus" (hmpv)]. ribavirin, inhalativ oder oral verabreicht, konnte bei rsv-und piv-infektionen erfolgreich eingesetzt werden, geht aber auch bei inhalativer gabe mit unerwünschten nebenwirkungen (bronchokonstriktion, teratogenität) einher, sodass zurzeit die orale gabe erfolgversprechender erscheint. therapiemöglichkeiten bei influenzaviren sind: amantadin, rimantadin und neuraminidaseinhibitoren (zanamivir, oseltamivir). daten über die wirksamkeit dieser therapien bei lungentransplantierten patienten sind limitiert. regelmäßige impfungen sollten nach dem 1. postoperativen jahr erfolgen, auch wenn ein protektiver titer der literatur zufolge nur bei ca. 50% der immunsupprimierten patienten erreicht werden kann. fungale infektionen treten mit einer häufigkeit von ca. 15-35% auf; in 80% der fälle sind aspergillus-oder candida-spezies vertreten [32] . sie zeigen ein bipha die wichtigsten nichtpulmonalen komplikationen und deren therapien sind in . tab. 1 zusammengefasst. pulmonale und nichtpulmonale probleme können lebensqualität und langzeitergebnisse nach lungentransplantation beeinträchtigen. neben der empfängerauswahl sind die chirurgische phase mit organexplantation, konservierung und implantation sowie die perioperative phase (bis zu 6 monate) entscheidend für das ergebnis. die erkenntnis, dass sich komplikationen gegenseitig verstärken können (wie z. b. akute abstoßungen und bos, wie virale infekte und akute abstoßungen, oder wie gerd und bos) muss zu maximalen anstrengungen der verbesserung des patienten-monitorings sowie zu frühestmöglichem therapiebeginn beim auftreten einer komplikation führen. interessenkonflikt. der korrespondierende autor gibt für sich und seinen koautor an, dass kein interessenkonflikt besteht. die sieger 2013 stehen fest in berlin wurden im rahmen einer festlichen gala der diesjährige galenus-von-pergamon-preis und der charityaward von springer medizin verliehen. überreicht wurden die begehrten medaillen vom parlamentarischen staatssekretär thomas rachel im namen der galenus-schirmherrin und bundesforschungsministerin professor johanna wanka. pulmonary complications of lung transplantation association between elevated whole blood epstein-barr virus (ebv)-encoded rna ebv polymerase chain reaction and reduced incidence of acute lung allograft rejection gastro-oesophageal reflux and gastric aspiration in lung transplant patients with or without chronic rejection early fundoplication prevents chronic allograft dysfunction in patients with gastroesophageal reflux disease the registry of the international society for heart and lung transplantation: 29th adult lung and heart-lung transplant report-2012 the registry of the international society for heart and lung transplantation: twentyeighth adult lung and heart-lung transplant report-2011 the effect of primary graft dysfunction on survival after lung transplantation plasma levels of receptor for advanced glycation end products, blood transfusion, and risk of primary graft dysfunction basiliximab as an alternative to antithymocyte globulin for early immunosuppression in lung transplantation factors associated with early graft dysfunction in cystic fibrosis patients receiving primary bilateral lung transplantation treatment of airway complications following lung transplantation the protective role of laparoscopic antireflux surgery against aspiration of pepsin after lung transplantation the lung transplant patient in the icu induction therapy with antithymocyte globulin before reperfusion long-term azithromycin for bronchiolitis obliterans syndrome after lung transplantation rabbit anti-thymocyte globulin induction therapy does not prolong survival after lung transplantation lung transplantation for cystic fibrosis: a single center experience of 100 consecutive cases ultrastructural changes in acute lung allograft rejection: novel insights from an animal study impact of human leukocyte antigen-dr mismatch status on kidney graft survival in a predominantly african-american population under the newer immunosuppressive era treatment of intractable interstitial lung injury with alemtuzumab after lung transplantation lung transplantation clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant the management of bronchus intermedius complications after lung transplantation: a retrospective study analysis of transplant outcomes after five or six human leukocyte antigen-mismatched living donor kidney transplantation predictors of acute rejection after lung transplantation bile acids aspiration reduces survival in lung transplant recipients with bos despite azithromycin gastric juice from patients "on" acid suppressive therapy can still provoke a significant inflammatory reaction by human bronchial epithelial cells elevation of antidonor immunoglobulin m levels precedes acute lung transplant rejection is prevention the best treatment? cmv after lung transplantation does human leukocyte antigen matching influence the outcome of lung transplantation? an analysis of 3,549 lung transplantations airway complications and management after lung transplantation: ischemia, dehiscence, and stenosis fungal infections after lung transplantation pepsin, a biomarker of gastric aspiration in lung allografts: a putative association with rejection airway complications after lung transplantation: longterm outcome of silicone stenting registry of the international society for heart and lung transplantation: twenty-fourth official adult lung and heart-lung transplantation report-2007 impact of anastomotic techniques on airway complications after lung transplant a randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation the impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients in der diesjährige galenus-preis für grundlagenforschung geht an das team von prof. oliver groß aus göttingen. der nephrologe hat mit arbeiten über ace-hemmer beim alport-syndrom dafür gesorgt, dass es erstmals eine therapieempfehlung für die chronisch progrediente nierenfi brose gibt. vielen der jungen patienten, die an dieser speziellen und seltenen progressiven hereditären nierenerkrankung leiden, kann mit dieser therapie womöglich die dialyse erspart bleiben. der preis ist mit 10.000 euro dotiert. key: cord-001945-ueccexxc authors: yang, ce; jiang, jianxin; yang, xuetao; wang, haiyan; du, juan title: stem/progenitor cells in endogenous repairing responses: new toolbox for the treatment of acute lung injury date: 2016-02-11 journal: j transl med doi: 10.1186/s12967-016-0804-1 sha: doc_id: 1945 cord_uid: ueccexxc the repair of organs and tissues has stepped into a prospective era of regenerative medicine. however, basic research and clinical practice in the lung regeneration remains crawling. owing to the complicated three dimensional structures and above 40 types of pulmonary cells, the regeneration of lung tissues becomes a great challenge. compelling evidence has showed that distinct populations of intrapulmonary and extrapulmonary stem/progenitor cells can regenerate epithelia as well as endothelia in various parts of the respiratory tract. recently, the discovery of human lung stem cells and their relevant studies has opened the door of hope again, which might put us on the path to repair our injured body parts, lungs on demand. herein, we emphasized the role of endogenous and exogenous stem/progenitor cells in lungs as well as artificial tissue repair for the injured lungs, which constitute a marvelous toolbox for the treatment of acute lung injury. finally, we further discussed the potential problems in the pulmonary remodeling and regeneration. with the occurrence and evolution of critical diseases (trauma, burn, infections, sepsis, hemorrhagic shock), lungs belong to the most easily injured organs. acute lung injury (ali) also constitutes the causative factor for the other organ chaos [1] . thus, it is important to prevent and cure the respiratory dysfunction for the improvement of treatment in multiple organ dysfunctions (mods) [2] . however, compelling evidence indicates that the remedy of ali and acute respiratory distress syndrome (ards) based on the ventilation function support and antiinflammatory treatment remains unsatisfied [3] [4] [5] . actually, the key point to treat the ali and ards is to realize both the structural remodeling and functional repair, and recover the normal gas exchange. presently, the potential measures to realize the repair and regeneration of injured adult lung tissues is to activate the self repairing potential through an extra-or intra-pulmonary route [6, 7] , and improve the local pulmonary microenvironment so as to promote the reconstruction of breathing function. during these complex courses, the principal biological event is that stem/progenitor cells are synergistically involved in the repair of injured lung tissues (fig. 1 ). (g-csf) is known to induce mobilization of bmscs to peripheral blood, while their increased homing to sites of injury would improve tissue healing. also, the mobilizers could induce the increase of bone marrow-derived epcs in the murine model of emphysema [10] , inducing angiogenesis in injured lungs through mobilizing epc [11] . similarly, in the patients suffered from bacteria pneumonia and ali, the number of circulating epcs is obviously increased, which is even related to their prognosis. in turn, the mobilizing capacity of bone marrowderived epcs is impaired after ards [12] , indicating the necessity of improvement of bone marrow mobilization so as to promote the pulmonary repair. meanwhile, mobilization of hspcs and colony formation capacity of peripheral blood mononuclear cells demonstrated great significance after ali [13] [14] [15] . all these findings indicate fig. 1 schematic illustration of the exogenous and endogenous stem/progenitor cells as well as the regular delivery routes in the repair and regeneration in acute lung injury that the bone marrow-derived stem/progenitor cells exhibit the mobilizing courses, and play a substantial role in the regression of excessive inflammatory responses and repair in injured lungs. in addition, recent researchers found that ali with endotoxin or no 2 does not enhance development of airway epithelium from bone marrow [16] , suggesting that the expansion and proliferation of endogenous bone marrow-derived stem/progenitor cells toward airway descendants are further required once their mobilization occurs. presently in the clinical stem cell therapy, mesenchymal stem cells (mscs) are widely used owing to the easy accessibility and low immunogenicity [17] . the allograft of bone marrow mscs are easily tolerated for the acceptors due to the low expression of major histocompatibility complex (mhc) i, ii and co-stimulator molecules in t cells. thus, these theoretically reasonable cells are further stored until use without ethical disputation. in recent years above 130 clinical trials of mscs have been registered and carried out. bone marrow mscs have been proved to efficiently alleviate the lung injury and promote the recovery courses [18] , partly due to the immunoregulatory effects [19, 20] . meanwhile, administration of mscs via the vein or trachea also reduces the lps-induced ali, alleviating the chest impact injury and hyperoxia-induced lung injury, reversing the pathological reduction of pulmonary surface area and the blunted breathing function in rodents [21, 22] . bleomycininduced inflammation, collagen deposition and fibrosis were also attenuated after the mscs injection [10] . the conditioned medium for mscs culture further showed similar therapeutic effects on pulmonary function [23] . hence, bone marrows mscs possess the prospective clinical value for the repair and regeneration in ali. concerning the protective roles of bone marrow-and peripheral blood-derived epcs in ali, recent studies showed that their peripheral infusion could lead to homing in injured lung tissues [24] , relieving the inflammatory injury [25, 26] and promote the endothelial repair and recovery of immune function dissonance [26, 27] , which may be enhanced by the treatment of simvastatin [28] . also, inhaled no contributes to the repair of injured lungs in piglets via increasing circulating endothelial progenitor cells [29] . the number of epcs is positively related to the animal survival. from the clinical point of view, the increased number of epcs in the pneumonia patients is the innocent representation of self repair in bodies. if the number of circulating epcs didn't increase in ali [12] , these patients might have distressing outcome, indicating the great necessity for supplementing the epcs in combination with immune modulatory measures. presently, the understanding of hspcs efficacy in ali remains limited. interestingly, stem/progenitor cells derived from the circulation contribute to the repair of injured lungs in surgically generated parabiotic mice [30] , indicating the potential contribution of hspc in ali. in fact, during the human prenatal development, the hspcs firstly appear in the fetal yolk sac. four months later. they transfer to the fetal livers for the further growth and differentiation. in the newborn phase, they finally locate in the bone marrow, followed with the appearance of varying stages of leukocytes, red cells and platelets. thus, the role of hspcs at least includes the immunoregulatory and repairing effects in ali. since the discovery of adscs by zuk et al. [31] , their capacity in the repair and regeneration of injured lungs has been widely investigated. adscs exhibit large reserve quantity owing to the extensively distributed adipose tissues in bodies. as compared with the bmscs, adscs were easily harvested from dumped adipose end product via the regular adipose aspiration technique. the total volume of adult bone marrow extracts is 40 ml while that of adult adipose can easily reach 500 ml. moreover, the harvest of adscs is easy to adopt as well as no potential blood-derived contamination and immunologic rejection compared with the bone marrow allograft [32] . the pickup rate of adscs is 100-500 times of that of bone marrow mscs. so, adscs can be abundantly harvested in the limited time without in vitro expansion. in a randomized, placebo-controlled pilot study, adscs showed significant protective effects on ali. optical imaging analysis further indicated that they promote the subacute airway remodeling, and ameliorates ventilator-induced lung injury in rats [33, 34] . the main protective reasons referred to enos and enos-derived no [35] . actually, adscs could secrete vascular endothelial growth factor, granulocyte colonystimulating factor (g-csf), hepatocyte growth factor (hgf), stromal derived factor-1 (sdf-1) when promoting the angiogenesis. also, they release collagen i and iii and laminin via a paracrine route [32, 36] . all these factors may play a substantial role in pulmonary structural repair and functional reconstruction in ali. placenta-derived stem/progenitor cells come from placenta, umbilical cord and amniotic fluid and their contents. among them, the placenta is structurally complicated. human placenta consists of amnion, chorion and basal deciduas. the amnion and chorion are from fetus while basal deciduas are from precursor. the placenta is the reservoir of stem and progenitor cells during the fetal development. once the fetal disengagement is finished, it is easily acquired without ethical disputation. presently, placenta-derived stem/progenitor cells are positively involved in the repair of injured lungs including fetal membrane-derived mscs, umbilical cord mscs, umbilical cord blood-derived mscs, amniotic fluid stem cells, and amnion epithelial cells, etc. [37] . previous studies have demonstrated that amniotic fluid stem cells could attenuate hyperoxia-induced ali in mice [38] , inhibiting the progression of bleomycin-induced pulmonary fibrosis via ccl2 modulation in bronchoalveolar lavage [39] . amnion epithelial cells could also act as a seed cells for the therapy of ali [40] . meanwhile, human umbilical cord mscs reduced systemic inflammation and attenuated lps-induced ali in rats [41] . human cd34+ progenitor cells from umbilical cord blood could also attenuate inflammatory lung injury following lps challenge [42] . further, intratracheal administration of umbilical cord blood-derived mscs played a pleasing role for the patient with ards [43] . therefore, placenta-derived stem/progenitor cells act as efficient candidates for the treatment of ali. human embryonic stem cell-derived progenitor cells could ameliorate sepsis-induced lung inflammatory injury via interaction with a specific population of with cd11b+ cells [44] . meanwhile, researchers could get functional airway epithelium from human embryonic stem cells through an expansive generation measure [45] . however, owing to the ethical bottleneck and law disputation, they shouldn't be recommended to be an ideal seed cells in the repair and regeneration of injured lungs. induced pluripotent stem cells (ipscs) were firstly acquired from genetically engineered fibroblasts in skin, similar to the dedifferentiation findings by fu, et al. [46] in 2001. now, it has been confirmed that adscs are easier to be transferred to ipscs than fibroblasts [47] . the embryonic stem cell-like functions of ipscs were proved occasionally since 2007. ipscs were also got from a patient with ali [48] , which could differentiate into alveolar epithelial cells in vitro for use in vivo [49] . the therapeutic capacity of ipscs might be related to the inhibition of src, nf-κb and pi3k/akt pathway as well as ip-10-dependent paracrine regulation [50] [51] [52] . in view of the initialization characteristics of ipscs from adult cells, they could pave the way to the structural remodeling and functional repair in ali without any ethical and law obstacles. meanwhile, stem/progenitor cells were widely used to act as the vectors of target genes to attenuate the inflammatory injury and promote the repair and regeneration of injured lung. recent studies have demonstrated that the genetically engineered stem cells with overexpression of cxcr4 [53] , angiotensin-converting enzyme 2 [54] , il-33 antagonist soluble il-1 receptor-like-1 [55] , keratinocyte growth factor, angiopoietin 1 [56] and dominant-negative inhibitor of ccl2 [57] could greatly facilitate treatment of ali in rodents. all these results substantially indicate that the therapeutic efficacy of genetically engineered stem/progenitor cells boosted by the stably transfected target genes in the pulmonary repair. in the adult mammalian tissues and organs, there are still some endogenous stem/progenitor cells, which distribute the predetermined microenvironment named niche. the niche supplies the repairing cells for the homeostasis and repair of tissues and organs. concerning the endogenous pulmonary stem/progenitor cells, researchers reported that they exist in the adult respiratory tissues in rodents and humans (table 1) . although there remains lack of the specific molecular markers for the lung stem/progenitor cells, their isolation and culture seems difficult, and the classification of these cells is also in controversial, they have been widely approved for the maintaining of pulmonary structural stability and functional repair. pulmonary stem/progenitor cells (trachea and bronchial stem cells, bronchiolar stem cells, bronchioloalveolar stem cells, alveolar stem cells and alveolar type ii cells, etc.) were shown to play substantial roles for the recovery of homeostasis and repair of injured tissues through molecular markers, lineage tracing and clonal analysis [58] [59] [60] [61] [62] [63] (fig. 2) . the alveolar epithelium is composed of the flat alveolar type i (at1) cells comprising 95 % of the gas-exchange surface area and less than 5 % of cuboidal alveolar type ii (at2) cells comprising the rest. once ali occurs, at1 cells showed injury, necrosis or apoptosis. then, rare, long-lived, mature at2 cells could differentiate and substitute the disabled at1 cells in the injured area [64, 65] . at this moment, some of at2 cells were found to become hypertrophy, which is easily discriminated in various injured lung tissues. meanwhile, the heterogeneity of at2 cells had been presented and classified with three subgroups including the alveolar renewal focus, alveolar repair focus and at2 replacement focus [66] . further, the subgroup of stem cells were shown to exist within the at2 cells, which were found to widely distribute in the terminal bronchiole, bronchioalveolar epithelial progenitor cell isolation anti-inflammation, tissue repair [12] hematopoietic stem/ progenitor cell isolation anti-inflammation, tissue repair [14] engineering stem/ progenitor cell ipscs isolation and genetic manipulation anti-inflammation via nf-kb and src pathway, differentiation toward alveolar epithelial cells [49] [50] [51] [52] modified mesenchymal stem cell anti-inflammation, tissue repair [53, 54, 56] duct junction (badj) and alveolus. in comparison, the contribution of at2 cells is more significant than bronchioalveolar stem cells (bascs) concerning the numerical preponderance and differentiation potential [67, 68] . some murine at2 cells can also generate bascs unexpectedly [69] . therefore, during the course of pulmonary remodeling after ali, the efficient shift of repair potential in at2 cells including the number, distribution and cellular transfer path should be undoubtedly weighted. the endogenous stem cells in the resident lung cells were further confirmed using the gfp-labeled chimera mice, which synergistically contribute to the regenerative alveolus. robust preclinical literature has showed that at2 cells could repair the injured alveolar epithelium. however, the potential of pulmonary endogenous stem cells to substituting the injured at2 cells remains unclear. to address this question, new findings suggested that the murine stem cell antigen (sca)-1 positive cell may be the endogenous lung stem cell although it could not found in rats [70] . the cell number with stem cell marker (sca-1, cd34 and c-kit) increased in the elastase-induced lung injury. the combination of hgf and elastase could synergistically increase the number of sca-1+/spc+ cells. most of the sca-1+ belong to the endogenous lung stem cells while most of the c-kit+ cells come from the bone marrow [70, 71] . therefore, the increase in the number of endogenous lung stem/ progenitor cells is in great need for the repair of injured lung tissues. in recent years, zhang and colleagues [72] found that the excitation of wnt signal pathway could significantly increase the number of bascs. the pharmacological modulators, lithium may also promote the amplification and differentiation of specific stem cell group in the lung tissues [73] , which supplies the new avenue for the endogenous repair of injured lungs on the basis of pulmonary stem cells. clara cells possess anti-inflammatory capacity and impacts the pulmonary innate immune response [74, 75] . conditional depletion of clara cells induced peribronchiolar fibrosis, and potentiated lung inflammation and alveolar dysfunction, demonstrating its role of functional repair/regeneration in ali [76] . concurrently, our studies further showed that the boosted expression of clara cells resulted in the transformation of cellular shape from sporadic cube to serried high prismatical, and the enhancement of anti-inflammatory effect of clara cell secreting protein (ccsp) after retinoic acid plus simvastatin treatment in shock-endotoxin-induced pulmonary damage [77] . ccsp, an important lung derived protective factor, may play a substantial role on the pathogenesis of ali induced by endotoxemia [78] . moreover, compelling evidence showed that the differentiation potential of clara cells towards at1 and at2 cells after severe lung injury [79, 80] . so, the improved at1 and at2 cells maybe partly derived from facultative clara cells so as to keep the integrity of alveolar walls. presently, as compared with the boosting of murine lung stem cells, the investigation of human lung stem cells (hlscs) remains superficial since their presentation by kajstura et al in 2011 [81] . the main reasons are as follows. first, there remains lack of the specific markers for hlsc. second, the acquisition of human lung tissues is limited. nonetheless, the hlscs researches proved their in vitro c-kit positive characteristics, which were further confirmed using the in vivo experimental models. also, some experimental preparations have been given for the potential clinical usage. but it remains a long way to go before the clinical engraftment for hlscs. first, how efficient is the hlscs engraftment. whether does the newborn lung tissues differentiated from hlscs possess the normal physiological function? second, how is the meanwhile, recent studies further acquired human alveolar progenitor cells (aepcs) [82] , aepcs have the endothelial phenotype with mscs character. by using the chip analysis, aepcs were found to share many genes with mscs and at2 cells, indicating the phenotype overlapping between alveolar epithelial cells and mscs. in fact, apecs possess the capacity of phenotypic conversion between the mesenchymal and epithelium, indicating their potential in the pulmonary tissue repair. the mesenchymal characteristics, especially anti-apoptotic ability may benefit the functional epithelial progenitor cells. further investigation is necessary to elucidate their detailed pathophysiological role in the repair of injured lungs. the lung mscs have the ability of self renewal and differentiate into mesenchymal cell. given the varying characteristics in the different organs, the basic criteria for lung mscs include the adhesive ability on the plastic petri dishes, and the in vitro ability of osteogenesis, adipogenesis and cartilagenesis [22] since there are no specific cellular markers on the surface of pulmonary mscs. the lung mscs can be isolated from the lung and bronchoalveolar lavage fluid. karoubi et al. [83] isolated the mscs from the human lung tissues in the surgical operation, and successfully induced their differentiation toward the at2 cells expressing aqp5 and ccsp. although the action of mscs is not completely clear in the lung regeneration, the beneficial effects of mscs on the lung injuries have been extensively investigated. mscs can secrete diverse cytokines and growth factors. co-culture of lps-stimulated lung cells and mscs could result in the reduction of pro-inflammatory cytokines, indicating that the soluble mediators may inhibit excessive inflammatory responses, or the direct interactions of lung cells and mscs could produce the anti-inflammatory effects. similar results have showed that the immunoregulatory role of mscs on immune cells other studies reported that the intra trachea administration of pulmonary stem cells with the mscs phenotype attenuated the elastase-induced emphysema [84] . the transplanted stem cells can reach the alveolar space besides some of them reserved in the alveolar wall. these results didn't support the idea of cell differentiation, but indicated their immunoregulatory effects in the injured lung tissues. in addition, new mechanisms included the mitochondria dna transmission between the mscs and other cytosolic components through intercellular bridges, which may regulate the cellular biological ability in the recipient cells [85] . hence, the protective effects of mscs are rather the anti-inflammatory effects than the differentiation towards the lung cells. the protective role of stem/progenitor cells is to release the anti-injured and pro-reparative factors mainly via the paracrine/endocrine pathways, which is primarily due to their significant apoptosis and clearance by unknown innate immune mechanisms after their transplantation. likewise, the researches concerning their microenvironment regulation demonstrated that the stem/progenitor cell-derived conditioned medium possesses the similar efficacy, suggesting that the secreting factors (il-10, il-rn, vegf, angiopoietin-1) act as the anti-inflammatory and pro-reparative mediators on the gas-blood barrier in ali [86] [87] [88] [89] . moreover, microvesicles containing anti-inflammatory mrna and mirna secreted by the stem/progenitor cells also possessed the therapeutic potential during the repairing courses [90] [91] [92] . second, bmsc could protect against oxidative stress in escherichia coli-induced ali in mice [93] , and ameliorate seawater-exposure-induced ali by inhibiting autophagy in lung tissues [94] . concurrently, they could restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury [95] . third, bmsc could reduce inflammation while enhancing bacterial clearance in bacterial pneumonia [96, 97] . fourth, the number of a population of clara cells possessing secreting capacity; named bronchioalveolar stem cells (bascs) were increased in ali [98] . the ex vivo colony formation experiments proved that the proliferation of bascs was maybe due to mscs but not growth factors. the therapeutic effect of mscs on the chronic bronchopulmonary dysplasia showed that bascs help the reconstruction of pulmonary epithelial structure. so, the repairing effects of mscs maybe realize via the stimulation of bascs proliferation, demonstrating the peculiarly promoting effects of extra pulmonary stem cells on the lung stem cells during the repair and regeneration. the previous cell therapy on ali showed that the time courses of pulmonary remodeling and functional repair varies depending on the wound agents (live bacteria, oleic acid, bleomysin, etc.) [99] , indicating the clinical therapeutic efficacy may be intrinsically related to the ali etiology. thus, it is important to make the sensible selection and stringent judgment for the initiating factors, contaminated pathogens (bacteria, virus, and fungus) and potential window phases in the stem/progenitor cell-mediated lung repair. the injured lungs may release large quantities of stressrelated neuroendocrine hormones, neuromediators and neuropeptides [100] [101] [102] , which deeply influence the biological activities of stem/progenitor cells. steadily growing evidence has been shown that glucocorticoids, epinephrine and norepinephrine may be involved in the migrating or chemotaxis activities during the mobilizing courses [103] . acetylcholine released via vagus nerve and postganglionic neurons of adrenergic nerve has also been proved to modulate these courses [104] . recently, melatonin treatment was found to improve adscs therapy for acute lung ischemia-reperfusion injury [105] . concurrently, the immune status are also related to the protective efficacy of mscs because they could reduce lung injury in immunocompromised but not immunocompetent mice [106] . thus, the consonance of neuroendocrine immune network is of great importance for the lung repair and regeneration. the cell therapy of ali suggested that the prominent advantage of exogenous stem/progenitor cells is quantity-controllable although the procedures of harvest, purification and expansion are required in most conditions. hence, the potential contamination and biosafety should be carefully considered (table 2) . once the transplantation procedure is completed, the turnover of these stem/progenitor cells should also be further controlled to lower the risk of graft rejection and teratoma formation [107] [108] [109] . in comparison, the greatest advantage of endogenous stem/progenitor cells lies in the good safety only required to use the mobilizers or activating elements for their redistribution within the bone marrow, blood and lungs. however, the constraint of cell numbers especially in the blunted mobilization responses for some patients may deteriorate the therapeutic efficacy. in addition, other important findings have reported that there exist some differences between mscs from bone marrow, placenta and umbilical cord blood in terms of their immunosuppressive properties against t cells [110] . first, the stem/progenitor cells in the same species is better than in the different species in the ali therapy [111] . likewise, the autotransplantation is preferentially selected compared with allotransplantation. second, aging stem/progenitor cells were showed to have impaired migration and anti-inflammatory responses as well as abnormal immunosuppressive properties against t cells [110, 112] , indicating the selection of young stem/ progenitor cells is in preference. third, the delivery routes for stem/progenitor cell transplantation are also comparable. the therapeutic effects via intraperitoneal route were slightly inferior to intravenous route in amiodarone induced lung injury in rats [113] . so, the reasonable transplantation route (venous, trachea, intrapleural or intraperitoneal pathway) should be carefully considered for the ideal therapeutic efficacy (fig. 1) . until now, few lungs are available for transplantation and the results have not been completely assentient. hence ways are being sought to either engraft stem/ progenitor cells or fetal lung cells that will form pulmonary structures to the severely injured lungs, or build bio-artificial lungs that completely replace the depleted. however, owing to the complicated three-dimensional tissue structure and above 40 cell types, lungs are difficult to be artificially constructed perfectly. recently, several artificial lung models have been presented. the essential researches utilized the scaffold material that supports the development of alveolar-like epithelia and endothelia from fetal lung cells. meanwhile, the decellularized lungs were also repopulated with fetal lung epithelial cells delivered via the trachea and lung endothelial or human umbilical cord vein cells through the pulmonary artery. the constructs were then cultured in a bioreactor where the cells regenerated region-specific tissue with the characteristics of normal alveolar tissue before transplantation. although these cells need to be carefully investigated before the clinical usage, the integrity of lung stroma remains to be resolved, and the gas exchange capacity is limited, the concept of bio-artificial lungs may supply a candidate replacement measure for the severe lung injury owing to its low immune rejection and controlled organ origin, which might throw sunshine on the 50 million end staged-patients with ali. to the end, the endogenous lung remodeling, repair and regeneration has become the new avenue for the refractory lung diseases. however, the following concerns remains to be added. first, most of successful ali experiments were accomplished with rodents [114] . their lungs in thoracic cavities possess the energetic proliferating capacity in the whole life period which is quite different from human lungs because the transplantation of exogenous stem/progenitor cells into matured human lung tissues seems difficult at least owing to our limited insight in the hlsc. but the initiation of endogenous stem/ progenitor (self renewal, proliferation, migration, and differentiation) is safe and maybe efficient via some modulators (kgf, hgf, retinoid acid, etc.). second, in view of the bio-safety of endogenous stem/progenitor cells and the quantity constraint of exogenous stem/progenitor cells, it may be reasonable to consider the combination of these two types of cells in the ali therapy. third, the repair and regeneration of injured lungs is complicated. we should emphasize the microenvironmental regulation via the neuroendocrine immune network [115, 116] . only in this way may the stem/progenitor cells possess the ideal biological capability (migration, mobilization, chemotaxis and homing, expansion, differentiation and proliferation). such good "soil" may benefit these "seed" cells in the remodeling and regeneration of injured lungs. fourth, regarding the complicated cell types in lungs, the integration of inflammatory modulation and pro-repair factors' increase may help inhibit their deleterious injury and promote the structural remodeling. the potential measures should consider the synergistic combination of statins plus retinoid acid, statins plus hgf, etc. fifth, concerning the stem/progenitor cell pool of the bone marrow, and the energetic reservoir of the stem/progenitor cell pool of placenta, umbilical cord and amnion during the perinatal period, the selection of compositive stem/ progenitor cell populations maybe benefit ali treatment more than a single stem/progenitor cell population, which has been partly confirmed by the previous studies of mononuclear cell populations in the bone marrow [117] . sixth, concerning their robust protective capacity via paracrine/endocrine mediators released by stem/ progenitor cells, it is valuable to develop some stem/progenitor cell-derived therapeutic fluid similar to the conditioned medium of stem/progenitor cell in combined with the microvesicles for the ali therapy. finally, from the previous experience in the research of bio-artificial lungs, it is valuable to deeply emphasize the contribution of extracellular matrix while using stem/progenitor cells, which might pave the road for the pulmonary integrity in the lung remodeling and regeneration (fig. 3 ). taken together, the remaining unknown issues include the protraction regularity of lung stem/progenitor cell lineage, the transition and turnover of extra pulmonary stem/ progenitor cell and the integration and docking between intra-and extra-pulmonary stem/progenitor cells. nonetheless, the ideal animal models, clinical samples as well as usage of intra-and extra-pulmonary stem/progenitor cells will undoubtedly contribute to the elucidation of pathophysiological mechanism of lung regeneration, and the pursuit of new measures for the refractory ali. adscs: adipose-derived stem cells; aepcs: alveolar progenitor cells; ali: acute lung injury; ards: acute respiratory distress syndrome; badj: bronchioalveolar duct junction; bascs: bronchioalveolar stem cells; bmscs: bone marrow derived mesenchymal cells; ccsp: clara cell secreting protein; epcs: epithelial progenitor cells; g-csf: granulocyte colony-stimulating factor; hgf: hepatocyte growth factor; hlscs: human lung stem cells; hspcs: hematopoietic stem/progenitor cells; ipscs: induced pluripotent stem cells; mhc: major histocompatibility complex; mods: multiple organ dysfunctions; mscs: mesenchymal stem cells; sca: stem cell antigen; sdf-1: stromal derived factor-1. authors'contributions cy, xy, hw and jd drafted the manuscript; cy and jj critically reviewed the manuscript. all authors read and approved the final manuscript. acute lung injury and the acute respiratory distress syndrome: a clinical review expressions of scavenger receptor, cd14 and protective mechanisms of carboxymethyl-beta-1, 3-glucan in posttraumatic endotoxemia in mice preventing acute lung injury and acute respiratory distress syndrome: back to square one management of acute lung injury and acute respiratory distress syndrome in children management of acute lung injury and acute respiratory distress syndrome in children: a different perspective impaired mobilization of endothelial progenitor cells in acute lung injury/acute respiratory distress syndrome: inhibition of an endogenous mechanism of lung repair clinical review: stem cell therapies for acute lung injury/acute respiratory distress syndrome-hope or hype? bone marrow-derived mesenchymal stem cells in repair of the injured lung bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema adrenomedullin regenerates alveoli and vasculature in elastase-induced pulmonary emphysema in mice hepatocyte growth factor induces angiogenesis in injured lungs through mobilizing endothelial progenitor cells mobilization of endothelial progenitor cells from bone marrow is impaired in a piglet model of acute respiratory distress syndrome acute lung injury but not sepsis is associated with increased colony formation by peripheral blood mononuclear cells acute lung injury in a healthy donor during mobilization of peripheral blood stem cells using granulocyte-colony stimulating factor alone regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways acute lung injury with endotoxin or no2 does not enhance development of airway epithelium from bone marrow stem cells and cell therapy approaches in lung biology and diseases efficacy and safety of mesenchymal stromal cells in preclinical models of acute lung injury: a systematic review protocol stem/ progenitor cells in lung development, injury repair, and regeneration role of bone marrow and mesenchymal stem cells in healing after traumatic injury mesenchymal stem cells protects hyperoxia-induced lung injury in newborn rats via inhibiting receptor for advanced glycation end-products/nuclear factor kappab signaling therapeutic effects of bone marrow-derived mesenchymal stem cells on pulmonary impact injury complicated with endotoxemia in rats stem cell conditioned medium improves acute lung injury in mice: in vivo evidence for stem cell paracrine action peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury protective effects of bone marrowderived endothelial progenitor cells and houttuynia cordata in lipopolysaccharide-induced acute lung injury in rats autologous transplantation of endothelial progenitor cells attenuates acute lung injury in rabbits autologous transplantation of peripheral blood-derived circulating endothelial progenitor cells attenuates endotoxin-induced acute lung injury in rabbits by direct endothelial repair and indirect immunomodulation repair of lipopolysaccharide-induced acute lung injury in mice by endothelial progenitor cells, alone and in combination with simvastatin inhaled no contributes to lung repair in piglets with acute respiratory distress syndrome via increasing circulating endothelial progenitor cells cells derived from the circulation contribute to the repair of lung injury multilineage cells from human adipose tissue: implications for cell-based therapies treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study optical imaging of subacute airway remodeling and adipose stem cell engraftment after airway injury autologous transplantation of adipose-derived stromal cells ameliorates ventilator-induced lung injury in rats adipose tissue-derived stem cells attenuate acute lung injury through enos and enos-derived no adipose-derived stem cells weigh in as novel therapeutics for acute lung injury pregnancy-associated progenitor cells: an under-recognized potential source of stem cells in maternal lung amniotic fluid stem cells from egfp transgenic mice attenuate hyperoxia-induced acute lung injury amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via ccl2 modulation in bronchoalveolar lavage amnion epithelial cells as a candidate therapy for acute and chronic lung injury human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate lps-induced acute lung injury in rats human cd34+ progenitor cells freshly isolated from umbilical cord blood attenuate inflammatory lung injury following lps challenge intratracheal administration of umbilical cord blood-derived mesenchymal stem cells in a patient with acute respiratory distress syndrome interaction of a specific population of human embryonic stem cell-derived progenitor cells with cd11b+ cells ameliorates sepsis-induced lung inflammatory injury expansive generation of functional airway epithelium from human embryonic stem cells dedifferentiation of epidermal cells to stem cells in vivo feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells establishment of an induced pluripotent stem cell line from a patient with acute lung injury. nan fang yi ke da xue xue bao differentiation of mouse induced pluripotent stem cells into alveolar epithelial cells in vitro for use in vivo induced pluripotent stem cell therapy ameliorates hyperoxiaaugmented ventilator-induced lung injury through suppressing the src pathway improvement of ventilator-induced lung injury by ips cell-derived conditioned medium via inhibition of pi3 k/akt pathway and ip-10-dependent paracrine regulation suppressing nf-kappab and nkrf pathways by induced pluripotent stem cell therapy in mice with ventilator-induced lung injury cxcr4 overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats mesenchymal stem cells overexpressing angiotensin-converting enzyme 2 rescue lipopolysaccharide-induced lung injury human mesenchymal stem cells overexpressing the il-33 antagonist soluble il-1 receptor-like-1 attenuate endotoxin-induced acute lung injury keratinocyte growth factor gene delivery via mesenchymal stem cells protects against lipopolysaccharide-induced acute lung injury in mice mesenchymal stem cells stably transduced with a dominant-negative inhibitor of ccl2 greatly attenuate bleomycin-induced lung damage evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung paving the road for lung stem cell biology: bronchioalveolar stem cells and other putative distal lung stem cells identification of bronchioalveolar stem cells in normal lung and lung cancer in vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations the glandular stem/progenitor cell niche in airway development and repair evidence for stem-cell niches in the tracheal epithelium airway epithelial progenitors are region specific and show differential responses to bleomycininduced lung injury progenitor cells in acute lung injury alveolar progenitor and stem cells in lung development, renewal and cancer a change in the number of ccsp(pos)/spc(pos) cells in mouse lung during development, growth, and repair foxm1 mediates the progenitor function of type ii epithelial cells in repairing alveolar injury induced by pseudomonas aeruginosa warburton d. isolation and characterization of distal lung progenitor cells isolation and characterization of murine multipotent lung stem cells intranasal hgf administration ameliorates the physiologic and morphologic changes in lung emphysema a gata6-wnt pathway required for epithelial stem cell development and airway regeneration wnt/beta-catenin signaling regulates cancer stem cells in lung cancer a549 cells clara cells impact the pulmonary innate immune response to lps airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction conditional depletion of airway progenitor cells induces peribronchiolar fibrosis retinoic acid promotes the endogenous repair of lung stem/progenitor cells in combined with simvastatin after acute lung injury: a stereological analysis polydatin up-regulates clara cell secretory protein to suppress phospholipase a2 of lung induced by lps in vivo and in vitro a cellular pathway involved in clara cell to alveolar type ii cell differentiation after severe lung injury regeneration of alveolar type i and ii cells from scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza evidence for human lung stem cells analysis of gene expression profiles in alveolar epithelial type ii-like cells differentiated from human alveolar epithelial progenitor cells identification of mesenchymal stromal cells in human lung parenchyma capable of differentiating into aquaporin 5-expressing cells pulmonary stem cells and the induction of tissue regeneration in the treatment of emphysema mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury paracrine factors from mesenchymal stem cells: a proposed therapeutic tool for acute lung injury and acute respiratory distress syndrome activation of human mesenchymal stem cells impacts their therapeutic abilities in lung injury by increasing interleukin (il)-10 and il-1rn levels mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via vegf-vegf receptors allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type ii cells by secretion of angiopoietin-1 a systematic review of preclinical studies on the therapeutic potential of mesenchymal stromal cell-derived microvesicles msc microvesicles for the treatment of lung disease: a new paradigm for cell-free therapy human mesenchymal stem cell microvesicles for treatment of escherichia coli endotoxin-induced acute lung injury in mice mesenchymal stromal cell injection protects against oxidative stress in escherichia coli-induced acute lung injury in mice bone marrow mesenchymal stem cells ameliorates seawater-exposure-induced acute lung injury by inhibiting autophagy in lung tissue conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury mesenchymal stem cells enhance survival and bacterial clearance in murine escherichia coli pneumonia mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice exogenous norepinephrine correlates with macrophage endoplasmic reticulum stress response in association with xbp-1 effects of bilateral adrenalectomy on the innate immune responses following trauma in rats corticosterone exerts immunostimulatory effects on macrophages via endoplasmic reticulum stress adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the lps-induced lung injury vagus nerve through alpha7 nachr modulates lung infection and inflammation: models, cells, and signals melatonin treatment improves adiposederived mesenchymal stem cell therapy for acute lung ischemia-reperfusion injury human mesenchymal stem cells reduce lung injury in immunocompromised mice but not in immunocompetent mice endogenous and exogenous cell-based pathways for recovery from acute respiratory distress syndrome risk of teratoma formation after transplantation of induced pluripotent stem cells effect and mechanism of acute graft versus host disease on early diffuse murine lung injury following allogeneic stem cell transplantation human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against t cells comparison of the therapeutic effects of human and mouse adiposederived stem cells in a murine model of lipopolysaccharide-induced acute lung injury aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response comparative study between intravenous and intraperitoneal stem cell therapy in amiodarone induced lung injury in rat concise review: current status of stem cells and regenerative medicine in lung biology and diseases bilateral regulatory action of corticotropin-releasing hormone on immune-mediated inflammation effects of hypothalamus destruction on the level of plasma corticosterone after blast injury and its relation to interleukin-6 in rats cell therapy demonstrates promise for acute respiratory distress syndrome-but which cell is best? the authors thank professor min zhao (university of california, davis) for his critical reading of this manuscript. we also apologize for the omission of any references due to the space constraints of this review and wish to thank members of their laboratories for helpful criticism. the authors declare that they have no competing interests. this • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-276927-rxudwp2v authors: barbas, carmen sílvia valente; matos, gustavo faissol janot; amato, marcelo britto passos; carvalho, carlos roberto ribeiro title: goal-oriented respiratory management for critically ill patients with acute respiratory distress syndrome date: 2012-08-23 journal: crit care res pract doi: 10.1155/2012/952168 sha: doc_id: 276927 cord_uid: rxudwp2v this paper, based on relevant literature articles and the authors' clinical experience, presents a goal-oriented respiratory management for critically ill patients with acute respiratory distress syndrome (ards) that can help improve clinicians' ability to care for these patients. early recognition of ards modified risk factors and avoidance of aggravating factors during hospital stay such as nonprotective mechanical ventilation, multiple blood products transfusions, positive fluid balance, ventilator-associated pneumonia, and gastric aspiration can help decrease its incidence. an early extensive clinical, laboratory, and imaging evaluation of “at risk patients” allows a correct diagnosis of ards, assessment of comorbidities, and calculation of prognostic indices, so that a careful treatment can be planned. rapid administration of antibiotics and resuscitative measures in case of sepsis and septic shock associated with protective ventilatory strategies and early short-term paralysis associated with differential ventilatory techniques (recruitment maneuvers with adequate positive end-expiratory pressure titration, prone position, and new extracorporeal membrane oxygenation techniques) in severe ards can help improve its prognosis. revaluation of ards patients on the third day of evolution (sequential organ failure assessment (sofa), biomarkers and response to infection therapy) allows changes in the initial treatment plans and can help decrease ards mortality. acute respiratory distress syndrome (ards) is due to an increase in the pulmonary alveolar-capillary membrane permeability causing lung edema rich in protein and consequently acute hypoxemic respiratory failure in genetically susceptible patients exposed to determined risk factors [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . a recent study showed that the del/del genotype (patients homozygous for the 4 base pair deletion in the promoter of nfkb1) is associated with an age-dependent increase in odds of developing ards (or 5.21, 95% ci 1. 35-20.0 ) and patients with the del/del genotype and ards also have increased hazard of 60-day mortality (hr 1.54, 95% ci 1.01-2.36) and more organ failure (p < 0.001) [15] . all age groups may be affected, although the syndrome has a higher incidence and mortality in older people [16] . the most common precipitating causes of ards are pulmonary infections, nonpulmonary sepsis, shock, gastric aspiration, thoracic trauma, fat embolism, near drowning, inhalational injury, cardiopulmonary bypass, drug overdose, acute pancreatitis, and high-risk trauma (especially traumatic brain injury) [17] . recent epidemiological studies suggested a variety of intrahospital risk factors for ards development such as multiple blood products transfusions, mechanical ventilation with high tidal volumes, excessive fluid resuscitation, and hospitalacquired pneumonia as well as high-risk surgeries (especially aortic vascular, cardiac, and acute abdomen); all risk factors are potentially preventable. chronic alcohol abuse, chronic liver disease, immunosuppression, hypoalbuminemia, and obesity are also all associated with the development of ards, whereas diabetes mellitus appears to be protective [17] . after exposure to a risk factor, there is an important activation of neutrophils and release of harmful mediators including cytokines (such as interleukins 1, 6, and 8 and soluble tumor necrosis factor-alpha receptors), proteases, reactive oxygen species, and matrix metalloproteinases leading to future damage. an overwhelming pulmonary inflammatory process is initiated leading to alveolar epithelial and vascular endothelial injury. alveolar epithelial injury of type i cells contributes to the pulmonary edema and the breakdown of this epithelial barrier exposes the underlying basement membrane, predisposing to bacteremia and sepsis. injury to type ii alveolar cells leads to an impairment of surfactant function with consequent collapse of the lungs. histopathologically there is diffuse alveolar damage with neutrophil infiltration, alveolar hemorrhage and hyaline membrane formation [18] [19] [20] [21] [22] . there are localized destruction and occlusion of the vascular bed of the lungs by intravascular thrombosis and an increment of the anatomical dead space resulting in an increase of arterial carbon dioxide associated with a poor outcome. fibrosis can be evident histologically as early as one week after the onset of ards and procollagen iii peptide, a precursor of collagen synthesis, can be elevated in bronchoalveolar lavage fluid of ards patients at the time of tracheal intubation, its increment being associated with a poor ards prognosis. vascular injury and remodeling may lead to pulmonary arterial hypertension which may compromise right ventricular function associated with a poor clinical outcome [13] . incorporation of modified risk factors such as acute increase of respiratory rate, presence of tachypnea, detection of pulse oximeter desaturation, increased necessity of oxygen supplementation, presence of low ph, acidosis, or hypoxemia in an arterial blood gas sample in clinical practice can improve the clinicians' ability to perform early diagnosis and prompt therapeutic intervention in ards [17] . the presence of these modified risk factors may alert physicians to avoid secondary hospital exposures, such as blood products transfusions, excessive fluid administration, infusion of potentially toxic drugs, high tidal volume mechanical ventilation, and gastric aspiration. implementation of ventilator associated pneumonia prevention bundles decreases the incidence of vap and can lower the incidence of ards [17] . implementation of automated ards electronic screening in usa hospitals such as "assist" (electronic alert from laboratory when the arterial blood gas analysis shows hypoxemia and the radiology department when chest x-ray shows bilateral pulmonary infiltrates) to identify intubated patients with ards in medical and surgical icus showed a sensitivity of 97.6% (95% ci, 96.8-98.4%) and specificity of 96.8% (95% ci, 96.8-98.4%) when compared to a manual screening algorithm that had a sensitivity of 57.1% (95% ci, 54.5-59.8%) and specificity of 99.7% (95% ci, 99.4-100%) in 1270 icu patients over a 21-week period during enrollment in ardsnet trials [23] . the results of this study indicated the advantages of having an in-hospital automated screening of ards over manual screening. the automated screening can increase the chances of ards diagnosis, alert the clinicians, and elicit the rapid response from the hospital team of intensivists to initiate clinical protocols and ards therapeutic interventions [24] . most hospitals and intensive care units worldwide use the standard criteria for the diagnosis of acute lung injury (ali)/ards: presence of acute hypoxemia (pao 2 /fio 2 less than 300 mmhg or 39.99 kpa for ali or less than 200 mmhg or 26.66 kpa for ards), bilateral infiltrates seen on a frontal chest radiograph that are consistent with pulmonary edema, and no clinical evidence of left atrial hypertension, or (if it is measured) a pulmonary artery wedge pressure (pawp) of less than 18 mmhg according to the 1994-1998 american-european consensus conference on ards (aecc) [25, 26] . this definition aimed to simplify and standardize the diagnosis of ards worldwide. however, in clinical practice, in order to detect and diagnose ali/ards cases, physicians must focus on patients' complaints, physical examination alterations, patients at risk of developing the disease, or patients presenting finger pulse oximeter desaturation. following the ali/ards clinical suspicion, physicians should order an arterial blood gas analysis and a chest radiograph to be able to confirm the ali/ards diagnosis. recent updates of ards definition such as the 2005 delphi consensus [8] or the berlin definition [27] were published in order to improve ards diagnosis criteria. the berlin definition reclassified ards as mild (pao 2 /fio 2 < 300 or 39.99 kpa), moderate (pao 2 /fio 2 < 200 or 26.66 kpa), and severe (pao 2 /fio 2 < 100 mmhg or 13.33 kpa) and removed the term ali and the necessity of a swan ganz catheter to access pawp. acute time frame was specified as the onset within 1 week of a known clinical insult or new or worsening respiratory symptoms chest radiography criteria were clarified and bilateral opacities consistent with pulmonary edema were maintained as the main radiological criteria of ards, but it was recognized that these findings could be demonstrated on ct scan instead of chest radiograph. the recent berlin definition of ards is a decisive step forward in refining the diagnosis of the syndrome, but pao 2 /fio 2 is influenced by ventilator settings and this fact should be considered; bilateral pulmonary infiltrates can be the result of a wide variety of acute lung diseases that should be better investigated. left and right ventricular function, pulmonary artery pressures, and volemic status could be better evaluated by bedside echocardiography and extravascular lung water can be measured using picco catheter, in order to evaluate the degree of pulmonary edema. predictors of mortality should be calculated at icu admission. with the information, the icu team can program a more careful treatment plan according to disease severity. the berlin definition shows better predictive validity for mortality compared to the aecc definition, but the absolute value of the area under the receiver operating curve is still too small (0.577), suggesting that some factors are still missing. further discussion and research are needed before we reach a comprehensive definition of ards. critical care research and practice 3 the typical findings of ards in a computer tomography reveal a heterogeneous bilateral pulmonary infiltrate predominantly in gravity-dependent regions of the lungs and more preserved lungs in nondependent lung regions. using quantitative analysis of the ct scan, the gravity-dependent pulmonary ards infiltrate is typically nonaerated lung tissue consistent with compressive atelectasis [28, 29] . lung weight assessed by ct scan is increased in ards and is correlated with the severity of the syndrome [27] . the finding of concomitant interstitial infiltrates suggests viral or mycoplasma, chlamydia or opportunistic pulmonary infections, or drug-induced lung disease. the differential diagnosis of bilateral pneumonia, alveolar hemorrhage, and acute interstitial lung disease such as acute interstitial pneumonia, hypersensitivity pneumonitis, acute eosinophilic pneumonia, and bronchiolitis obliterans with organizing pneumonia can be suggested by the characteristic ct scan findings of each specific disease [12] . the results of stepwise lung recruitment maneuvers as well as positive end-expiratory (peep) titration to keep the lungs open with minimal collapse can be assessed by computer tomography analysis [30] . this strategy is aimed at opening up the lungs and keeping the lungs open [31] as quickly and early as possible as postulated by lachmann [32] in order to have a huge improvement in lung function and avoid potential ventilator-induced lung injury. recently, our group reported the experience with maximal recruitment strategy (mrs) in 51 patients with ards. mrs consisted of 2-minute steps of tidal ventilation with pressure-controlled ventilation, fixed driving pressure of 15 cmh 2 o, respiratory rate of 10 breaths/minute, inspiratory/expiratory ratio of 1 : 1, and stepwise increments in peep levels from 10 to 45 cmh 2 o (recruitment phase). after that, peep was decreased to 25 cmh 2 o and, then, from 25 to 10 cmh 2 o (peep titration phase) in steps of 5 cmh 2 o, each one lasting 4 minutes. at each of the steps computer tomography image sequences from the carina to the diaphragm were acquired during an expiratory pause of 6-10 seconds. lung collapse was assessed online by visual inspection, for immediate clinical decision, and offline for quantitative measurements. mrs showed a statistically significant decrease in nonaerated areas of the ards lungs that was accompanied by a significant increment in oxygenation. the opening plateau pressure observed during the recruitment protocol was 59.6 (±5.9 cmh 2 o), and the mean peep titrated after mrs was 24.6 (±2.9 cmh 2 o). mean pao 2 /fio 2 ratio increased from 125 (±43) to 300 (±103; p < 0.0001) after mrs and was sustained above 300 throughout seven days. nonaerated parenchyma decreased significantly from 53.6% (interquartile range (iqr): 42.5 to 62.4) to 12.7% (iqr: 4.9 to 24.2) (p < 0.0001) after mrs. the potentially recruitable lung was estimated at 45% (iqr: 25 to 53), (figure 1 ). icu mortality was 28% and hospital mortality was 32%. the independent risk factors associated with mortality were older age and higher driving pressures (or higher delta pressure control). there were no significant clinical complications with mrs or barotrauma [33] . a better evolution of these ards patients with less necessity of oxygen supplementation in the recovery phase of the disease and a better quality of life must be tested in prospective, controlled clinical trials. a recent metaanalysis showing beneficial effects on mortality using higher peep levels compared with lower peep in ards patients corroborates the results of our clinical case series of ards patients submitted to mrs [33] . ards is a biphasic disease that progresses from an acute exudative phase, characterized by epithelial and endothelial injury, neutrophilic aggregation, formation of hyaline membranes, alveolar edema, and hemorrhage, to an organizing phase, characterized by regeneration and healing via resolution or repair with persistent intra-alveolar and interstitial fibrosis [11] . it is crucial to make the diagnosis of ards in the acute phase (preferably less than 72 hours) in order to make it possible to open up the lungs with recruitment maneuvers and keep the lungs open with sufficient peep levels to enable a more homogenous ventilation, minimizing the possible ventilator-induced lung injury (vili) triggers and allowing the recovery of the lungs [34] [35] [36] . a recent study analyzing 85 patients with ards graded into six findings according to the extent of fibroproliferation at the ct scan showed that higher ct scores were associated with statistically significant decreases in organ-failure free days as well as ventilator free days and were an independent risk factor for mortality (or = 1.2, 95% ci 1.06-1.36, p < 0.005) [37] . positron emission tomography with ( 18 f) fluorodeoxyglucose (fdg-pet) detects inflammatory cells and can assess lung inflammation in ards lungs helping in the understanding of ards physiopathology [38] [39] [40] . lung ultrasonography is a new helpful tool that can be performed at bedside without radiation exposure. thoracic ultrasound is widely used for diagnostic and therapeutic intervention in patients with pleural effusion and pneumothoraces. the assessment of lung recruitment and peep titration in ards patients at bedside using lung ultrasonography is a new promising technique [41] . currently, the two main limitations of this technique are its inability to detect lung overdistension and its operator-dependent characteristic. thoracic electrical impedance tomography (eit) is a highly promising imaging technique to apply at the bedside for peep titration in ards patients. new automated tools permit the calculation of the percentage of collapsed as well as overdistended lung tissue at decremental peep levels after lung recruitment maneuvers ( figure 3 ). the regional distribution of collapse and overdistension may provide insights about the lung pathology. this technique permits daily peep adjustments at the bedside and verification of tidal volume distribution, avoiding excessive end-expiratory collapse or tidal overdistention [42] [43] [44] [45] . one of the main sofa score during first 7 days after mrs * p < 0.005 advantages of this technique is the possibility of around the clock monitoring. further studies are needed to evaluate the clinical impact of these bedside techniques in ards patients' prognosis. randomized trials suggested that patients with acute hypoxemic respiratory failure are less likely to require endotracheal intubation when noninvasive ventilation (niv) is added to standard therapy [46] . however, most of these studies analyzed mixed causes of acute hypoxemic respiratory failure and reported the highest intubation rates for patients with ards (51 to 70%) and that the presence of ards was one factor independently associated with niv failure and higher mortalities rates (50 to 70%). recently, zhan and colleagues [47] analyzed 40 patients with ards randomly allocated to receive either noninvasive ventilation or high-concentration oxygen therapy through a venturi mask. noninvasive positive pressure ventilation decreased the respiratory rate and improved pao 2 /fio 2 with time. the proportion of patients requiring intubation and invasive mechanical ventilation was significantly lower in the noninvasive ventilation group (one of 21 versus 7 of 19; p = 0.02). therefore, noninvasive ventilation can be used as a first ventilatory support technique in selected patients with mild/moderate ards and a hemodynamic stable condition to avoid endotracheal intubation. a larger randomized trial, however, is required, with the need for intubation and mortality as the outcome of interest. a close-monitored initial trial of noninvasive ventilation should be considered in most mild/moderate ards patients, mainly the immunosuppressed ones with pulmonary infection in order to avoid intubation and invasive mechanical ventilation. however, early detection of collapse was more prominent in the right lung. after analyzing the sequence of eit images, the peep selected for this patient was 17 cmh 2 o, believed to represent the best compromise between collapse and overdistension. according to the ardsnet peep/fio 2 table, this patient had been ventilated with a peep = 24 cmh 2 o in the previous 48 hours. the patient was weaned from ventilator 3 days later. niv failure must be recognized, and a prompt intubation and mechanical ventilation must be provided in order to avoid complications. protective ards mechanical ventilation strategies with tidal volumes equal to or less than 6 ml/kg of predicted body weight have been traditionally associated with reduced mortality (when compared with 12 ml/kg of predicted body weight) [48, 49] . a recent meta-analysis, however, scrutinized the specific role of various ventilatory strategies used in randomized trials on lung protection (like plateau-pressure limitation and higher peep use) and showed that tidal volume per se is not exactly the most important parameter to prioritize. [53] demonstrated decreased lung inflammation with this protective ventilatory strategy. although these results are encouraging, the physiologic background supporting the use of p-v curves to titrate peep lacks consistency nowadays. in many different situations, investigators have reported a large dissociation between closing pressures of the lung and the calculated value for the inflection point obtained from the inspiratory p-v curve. in general, patients with high values of inflection point tend to have a more severe disease, and this may explain the relative success of this strategy. nevertheless, we will probably use better tools to titrate peep in the next few years. a more consistent use of the p-v curve has been demonstrated for the analysis of lung recruitability [54, 55] . airway pressure release ventilation is a modified form of continuous positive airway ventilation (cpap) described by stock and dows in 1987 that uses fairly high prolonged cpap levels with short and intermittent releases of the airway pressure to low cpap levels allowing ventilation and co 2 clearance. this mode of ventilatory support enhances oxygenation by augmenting alveolar recruitment and requires less sedation when used in ards patients compared to conventional mechanical ventilation [56, 57] . bipap ventilation combined with lung recruitment maneuvers can also be used in ards patients. wang and colleagues compared this modality of ventilatory support with assist/controlled volume ventilation in a prospective, randomized trial of 28 ards patients showing a better pao 2 /fio 2 ratio, pulmonary compliance, and a shorter duration of mechanical ventilation [58] . pressure support ventilation (psv) along with sufficient peep levels should be used as early as possible in ards patients to avoid respiratory muscle dystrophy and to decrease mechanical ventilation duration [32] . the reason for the improvement in oxygenation obtained with psv in ards has been challenged in the recent years [59, 60] . the apparent improvement in recruitment seems to have been overstated and there is evidence that it is related to an increased perfusion of better ventilated lung areas, but not to decreased lung collapse. growing concerns related to excessive tidal recruitment or excessive dyssynchrony during this mode of ventilation will have to be better addressed in the next years [61] . the advantages of using assist modes are to keep the respiratory muscles' activity, but sometimes it is difficult to synchronize the patients to the ventilators. recently, neurally adjust ventilation (nava) was used in ards experimental models [62] and ards patients [63] demonstrating that the ventilation cycle and the magnitude of assist breath in nava matched the patients' breath pattern better than in psv, nava improving patient-ventilator synchrony compared to psv. high frequency oscillatory ventilation (hfov) is an alternative mode of ventilatory support that can improve oxygenation by means of a higher mean airway pressure coupled with small tidal volumes generated by a piston pump oscillating at a frequency of 3-10 hz and a higher respiratory rate. however, to date there are few studies involving a small number of patients comparing hfov to conventional ventilation. a recent meta-analysis suggested a trend towards mortality benefit and more ventilator free days. however, the results of this analysis should be interpreted cautiously as the main study contributing to its results used high tidal volume in the control group rather than protective lung ventilation strategy [64] . the use of the position change (supine to prone) leads to consistent improvement in arterial oxygenation in ards patients. large randomized, controlled trials have consistently showed improvement in oxygenation without reduction in duration of mechanical ventilation or survival benefit. a recent meta-analyses suggest survival benefits in ards patients [65] or, more specifically, in a subgroup of patients with severe ards (pao 2 /fio 2 < 100 mmhg) [66] . in our experience, the prone position can be an acceptable alternative to improve oxygenation in severe ards patients with arterial pulmonary hypertension and right ventricular dysfunction, which associated with the use of inhaled nitric oxide, can minimize intrathoracic pressures to facilitate right ventricular performance. the principles of a protective ventilation with proper peep titration and minimum driving pressures should also be pursued during prone positioning protocols. clinical studies suggested that elevated pulmonary artery systolic pressure in ards patients was associated with an adverse prognosis [67] . these data have been further supported by a more recent analysis of hemodynamic data from the ardsnet fluids and catheter therapy trial (factt) [68] . the investigators assessed the transpulmonary gradient (tpg) (mean pa pressure-pulmonary capillary occlusion pressure (pcop)) and the pulmonary vascular resistance index (pvri) in a group of patients randomized to receive a pulmonary artery catheter to guide their ards management. of note, all patients received a consistent protective ventilator strategy with target tidal volume ∼6 ml/kg ideal body weight and plateau pressures maintained <30 cmh 2 o. the highest recorded daily value of tpg and pvri was used for the analysis. in the population of 475 patients randomized to receive a pulmonary artery catheter for ards management, none of the baseline measures of cardiopulmonary dysfunction, including central venous pressure, pa systolic, or diastolic pressure, pulmonary capillary occlusion pressure (paop), or cardiac index distinguished survivors from nonsurvivors. in the pulmonary artery catheter population, 73% demonstrated an elevated transpulmonary gradient (tpg > 12). patients with a tpg > 12 mmhg had a significantly greater mortality rate than patients with a tpg < 12 mmhg (30% versus 19%; p = 0.02). patients with a persistently elevated tpg through day 7 of therapy had a significantly greater mortality than patients with an elevated tpg at day 0-1 which subsequently normalized. in multivariate analysis, pulmonary vascular dysfunction as represented by an elevated tpg and pvri remained an independent predictor of an adverse outcome in the ards population. these data further support an important predictive role for pulmonary vascular disease in ards outcome [69] . in the largest published echocardiographic series of ards, 22% of patients receiving a consistent lung protective ventilation strategy (mean peep of 10 cmh 2 o and mean plateau pressure (pplat) of 23 cmh 2 o) had evidence for acute cor pulmonale. in this population, 19% demonstrated evidence of a moderate-to-large patent foramen ovale [70] . the incidence of right to left shunting increased to 34% in patients with echocardiographic evidence of acute cor pulmonale. increase of oxygenation and co 2 removal by making the ards patients' blood pass throughout a membrane oxygenator outside the body is the principle of extracorporeal membrane oxygenation that can be applied venousvenous (good for oxygenation and co 2 removal), arterialvenous (good for co 2 removal), and venous-arterial (good for cardiovascular support). early clinical trials of ecmo employed primarily an arterial-venous strategy with larger bore catheters for patients with intractable hypoxemia [71] . more modern investigations have used a safer venous-venous access approach [72, 73] . a recent uk prospective, randomized, clinical trial (cesar) showed a survival advantage in the ecmo group (63% for ecmo versus 47% for controls). nevertheless, the study was criticized as there was no standardized protocol management for the control group and some patients in the ecmo arm did not receive the proposed treatment [74] . the authors of cesar trial also recommended transferring adult patients with severe but potentially reversible respiratory failure and a ph less than 7.20 on optimal conventional management, to a center with an ecmo-based management protocol to significantly improve survival without severe disability. the authors demonstrated that this strategy is also likely to be cost effective in settings with similar services to those in the united kingdom [74] . another recent approach for application of extracorporeal carbon dioxide removal new devices (ecmo-r) in ards patients is the demonstration that in severe ards even the low tidal volume ventilation with 6 ml/kg of predicted body weight can cause tidal hyperdistension in the nondependent regions of the lungs accompanied by plateau airway pressures greater than 28 cmh 2 o and elevated plasma markers of inflammation. in this group application of ecmo-r could allow the authors to decrease the tidal volume to less than 6 ml/kg with a consequent plateau pressure less than 25 cmh 2 o that was associated with a lower radiographic index of lung injury and lower levels of lung-derived inflammatory cytokines. however, prognostic implication of this new ecmo-r devices application in clinical practice is still under investigation [75] . pumpless interventional lung assist (ila) is also used in patients with ards and is aimed at improving extracorporeal gas exchange with a membrane integrated in a passive arteriovenous shunt. ila serves as an extracorporeal assist to support mechanical ventilation by enabling low tidal volume and a reduced inspiratory plateau pressure in extremely severe ards patients. zimmermann and colleagues used ila in 51 severe ards patients and observed a decrease in paco 2 allowing the decrease in tidal volume and plateau pressure (ultraprotective ventilation) with a hospital mortality rate of 49% [76] . some authors suggest the use of combined ventilatory strategies in patients with ards. bingold and colleagues [77] successfully used superimposed high-frequency jet ventilation (shfjv) in combination with continuous positive airway pressure/assisted spontaneous breathing (cpap/asb) in five patients with h1-n1-associated ards to improve oxygenation. varpula and colleagues [78] demonstrated a significant improvement in oxygenation in 28 ards patients, when they compared apvr associated with prone ventilation to simv-pressure control/pressure support group. aprv after 24 h appears to enhance improvement in oxygenation in response to prone positioning. rival and colleagues [79] examined the effects of the prone position associated with a recruitment maneuver consisting of 45 cmh 2 o extended sigh in pressure control, in 16 ards patients. the combination of both ventilatory techniques led to the highest increase in pao 2 /fio 2 ratio without significant clinical side effects. lubnow and colleagues [80] examined the effects of 6 days of the combination of high-frequency oscillatory ventilation (hfov) and extracorporeal carbon dioxide removal with the interventional lung assist (ila) in 21 severe ards patients who failed conventional ventilation. they observed an increase in pao 2 /fio 2 ratio and ph and a decrease in paco 2 . weaning from hfov/ila was successful in 10 patients. the 30-day mortality rate was 43%, and hospital mortality rate was 57%. in conclusion, combined ventilatory strategies can be applied in severe ards patients, but the best match among all the available ventilatory techniques is still a matter of debate. pulmonary infection and sepsis are the most important triggering factors of ards. pulmonary infection has been associated with a higher risk of ards progression in comparison to nonpulmonary infection in at risk populations [81] . a wide variety of organisms can invade the respiratory tract and trigger host innate and acquired immune system initiating the inflammatory cascade of ards, sepsis, and multiple organ failure [11] . it is particularly pertinent to investigate the etiology of pulmonary infection on the first day assessing a nasal swab for a respiratory virus detection (influenza, adenovirus) lower respiratory tract secretion or a bronchoalveolar lavage fluid (balf) for bacteria (especially multiresistant species), other viruses as herpes and cytomegalovirus, coronavirus, or metapneumonic virus [82] . opportunistic agents such as pneumocystis jiroveci must be investigated in immunosuppressed patients. urinary screening for legionella species is decisive, because if positive, specific therapy must be introduced [11] . the assessment of balf on the first as well as on the third day of mechanical ventilation is of the utmost importance not only in terms of assessment of etiology of pulmonary infection but also of the assessment of proinflammatory mediators of ards (il-1, il6, il8, il 10, soluble tumor necrosis factor-alpha receptors (stnfr), and soluble intercellular adhesion molecule-1) and mediators of ventilator-induced lung injury (that can also be obtained in the plasma) such as stnfr, il6, il8, and il-10, indicators of epithelial cell injury (soluble advanced glycation end-product receptors-srage), and surfactant protein-d, components of the coagulation system (protein-c and plasminogen activator inhibitor 1) [11, 83] . elevated levels of procollagen peptide iii in lavage fluid from patients on day 3 of ards were independent risk factors for mortality [84] . procalcitonin (pct) and c-reactive protein (crp) are progressively being used in critical care setting in order to diagnose pulmonary infection and sepsis and to guide the antibiotic therapy. procalcitonin levels correlated with severe sepsis and bacteraemia [85] . a pct-based algorithm guiding initiation and duration of antibiotic therapy in critical ill patients with suspected bacterial infections was associated with a 23% relative reduction in antibiotic exposure with no significant increase in mortality [86] . the persistence of an elevated serum crp in critical ill patients with ards may alert the intensivist to a possible persistent infection or inflammatory process. at this moment, a new workup for infection and change in antibiotic therapy could help improve the patient's evolution. early and quick administration of antibiotics in sepsis and septic shock as well as early goal resuscitative measures for septic shock or early goaldirected therapy decrease mortality in this high mortality critically ill conditions [87, 88] . we also suggest that preventive measures to avoid gastric aspiration (elevated decubitus, intermittent check for residual gastric content during diet infusion) and to avoid ventilation associated pneumonia (wash hands, elevated decubitus, special endotracheal tubes) should be implemented. the resolution of pulmonary edema is central to recover from ali as it entails regression of air space inflammation and restoration of a functioning alveolar-capillary membrane. accordingly, elevated extravascular lung water measured using this technique early in the course of ali/ards, particularly if indexed to predicted body weight, was associated with a poor prognosis [89] . a study analyzing the evolution of ards patients showed that unknown-site infection (adjusted hazard ratio (hr) 3.08, 95% ci 1.37-6.90) and multiple site infection (adjusted hr 1.63, 95% ci 1.13-2.35) were associated with increased mortality [90] . in ards patients it is of considerable significance to evaluate the source of infection as well all organs and systems affected by the sepsis syndrome in order to map the organism (number of nonpulmonary organ failures), to calculate the prognostic indices (acute physiology and chronic health evaluation (apache) and simplified acute physiology score (saps)) and to plan the multiorgan system approach to treat the disease. the higher the number of multiple organ failure associated with ards, the higher the hospital mortality. trauma patients with ards are associated with lower mortality and oliguricrenal failure, while septic shock patients are associated with the highest hospital mortality rates, suggesting that during the first day of hospitalization these ards patients should be stratified and treated according to the severity of the syndrome and associated comorbidities [91] . in our case series of 51 patients with early severe ards the mean apache ii score was 20.2 ± 6.2 (predicted mortality of 40%), median sofa score (day 1) was 10 (7 to 12), median nonpulmonary organ failure was 2 (1 to 2), sepsis was present in 71% of our patients, and septic shock in 63%, vasopressors were used in 82.3% of our patients, and continuous renal replacement therapy was used in 56.8% of our patients. apache ii and day 1 sofa score were not associated with hospital mortality, but day 3 sofa score was [33] (figure 2) showing that a revaluation of the ards patients especially the ones with multiple organ failure and maintenance of sofa score higher than 8 at day 3 has to be considered in order to evaluate hidden sources of infection or to change the antibiotics according to day 1 collected cultures. in moderate-severe ards patients (pao 2 /fio 2 < 150), a phase iv randomized controlled trial comparing cisatracurium to placebo for 48 hours showed an improved critical care research and practice 9 adjusted 90-day survival rate and increased ventilator-free in the cisatracurium group without a significant increase in muscle weakness. short-term paralysis may facilitate patient-ventilator synchrony in the setting of lung protective ventilation. short-term paralysis would eliminate patient triggering and expiratory muscle activity. in combination, these effects may serve to limit regional overdistention and cyclic alveolar collapse. paralysis may also act to lower metabolism and overall ventilatory demand [92] . inhaled nitric oxide is an endogenous vasodilator that reduces v/q mismatch and improves oxygenation by pulmonary vasodilation in alveolar units that are ventilated, reducing pulmonary vascular resistance in patients with ards. a cochrane review of 14 clinical trials with 1303 patients showed only a transient improvement in oxygenation with no benefit regarding length of icu or hospital stay, ventilator-free days or survival. an increased renal impairment was observed in the inhaled nitric oxide-treated group [93] . the effects of steroids in the late-stage fibrotic phase of ards (after 7 days of onset) were tested in a phase iii study of the ards network. the study showed no mortality benefit in the treatment group, with a higher mortality in patients treated 14 days after onset [94] . recently, seam and colleagues tested the effects of methylprednisolone infusion in 55 early ards patients compared to placebo. they observed that methylprednisolone therapy was associated with greater improvement in lung injury score (p = 0.003), shorter duration of mechanical ventilation (p = 0.005), and lower intensive care unit mortality (p = 0.05) than in the control subjects. on days 3 and 7, methylprednisolone decreased interleukin-6 and increased protein-c levels (p < 0.001) compared with control subjects [95] . from the available evidence, low-dose steroids (1-2 mg/kg/methylprednisolone) may be considered in patients with severe early ards. nevertheless, it is not recommended to initiate corticosteroids beyond 14 days after the onset of ards. ketoconazole, lisofylline, sivelestat, n-acetylcysteine, and exogenous surfactant are not recommended as treatment for ards patients [12] . cumulative positive fluid balance is associated with worse clinical outcomes in patients with ards. a phase iii study conducted by the ards network (the factt study) compared liberal versus conservative fluid strategy in patients with acute lung injury. they observed an improvement in oxygenation, lung injury score (lis), and shortened duration of mechanical ventilation without any increase in other organ failure in the conservative group, despite no difference in hospital mortality [68] . beta-agonists were investigated in multicenter, prospective, randomized trials in their aerosolized presentation (the alta study) and their intravenous presentation (the balti-2 study). both studies showed no mortality benefit and betaagonists are not recommended as part of therapy for patients with ards [96] . the omega study [97] , a randomized, double-blind, placebo-controlled, multicenter trial analyzed 272 patients with early acute lung injury allocated to receive either twicedaily enteral supplementation of n-3 fatty acids, γ-linolenic acid, and antioxidants compared with an isocaloric control. enteral nutrition, directed by a protocol, was delivered separately from the study supplement. the patients that received enteral supplementation had fewer ventilator-free days (14 versus 17.2, p = 0.02), more days with diarrhea (29 versus 21%; p = 0.001), and no difference in the adjusted 60-day mortality (25.1% versus 17.6%; p = 0.11). more recently, a randomized, open-label, multicenter trial, the eden study [98] , reported 1000 patients with acute lung injury, randomized to receive either trophic or full enteral feeding for the first 6 days. initial trophic enteral feeding did not improve ventilator-free days, 60-day mortality, or infection complications but was associated with less gastrointestinal intolerance. finally, based on relevant literature articles and the authors' clinical experience, we suggest a goal-oriented management for critically ill patients with ards that can help improve clinicians' ability to care for these patients (as shown below). patients with ards. correct ards diagnosis. acute onset, increase respiratory rate, pulse oximeter desaturation and hypoxemia (pao 2 /fio 2 < 300). (i) if possible, get a computer tomography (improved diagnosis accuracy, permits differential diagnoses, and helps to set recruitment maneuvers and adequate peep levels). (ii) lung ultrasound, fdg-pet ct, electrical impedance tomography, and pressure-volume p × v curves can help assess the correct diagnosis and set protective mechanical ventilation. (iii) get nasal swab and inferior respiratory tract secretion for infection diagnosis or a bal (infection diagnosis and proinflammatory mediators and procollagen iii measurements). (iv) get hemocultures and blood for infection detection. start resuscitative measurements for septic shock and start appropriate antibiotics. critical care research and practice (v) assessment of prognostic indices (apache, saps) and sequential organ failure assessment (sofa) score. standardize initial mechanical ventilation for blood gas measurements. tidal volume: 6 ml/kg predicted body weight, peep of 5 cmh 2 o, rr = 20. classify ards severity. mild: pao 2 /fio 2 < 300, moderate: pao 2 /fio 2 < 200, and severe: pao 2 /fio 2 < 100. (i) if possible, get a doppler echocardiogram to assess left ventricular function, right ventricular function, systolic pulmonary artery pressure, and vena cava compressibility. (ii) measure extravascular lung water, if available. (a) in cases of severe ards consider recruitment maneuvers and adequate peep titration. (b) in cases of severe ards with right ventricular dysfunction and pulmonary artery hypertension consider prone position and inhaled nitric oxide. (c) in cases of excessive co 2 retention: paco 2 > 80 mmhg and ph < 7.2 consider intratracheal gas insufflation and extracorporeal co 2 removal. (i) early recognition of ards modified risk factors and avoidance of aggravating factors during hospital stay such as high tidal volume ventilation, multiple blood products transfusions, excessive fluid administration, ventilator associated pneumonia, and gastric aspiration prevention could help decrease its incidence. (ii) an early extensive clinical, laboratory, and imaging evaluation of "at risk patients" allows a correct diagnosis of ards, assessment of comorbidities, calculation of prognostic indices (apache, saps, sofa), stratification of the severity of ards, and planning a careful treatment. (iii) rapid administration of antibiotics and resuscitative measures in case of sepsis and septic shock associated with protective ventilatory strategies and early shortterm paralysis associated with differential ventilatory techniques (recruitment maneuvers with adequate peep titration, prone position, and new ecmo techniques) in severe ards can help improve its prognosis. (iv) revaluation of ards patients on the third day of evolution (sofa, biomarkers, and response to infection therapy) allows changes in the initial treatment plans and can help decrease ards mortality. (v) fibroproliferative changes on high-resolution ct in ards can predict mortality and ventilator dependency. acute respiratory distress in adults an expanded definition of the adult respiratory distress syndrome the acute respiratory distress syndrome comparison of clinical criteria for the acute respiratory distress syndrome with autopsy findings acute respiratory distress syndrome: a historical perspective acute respiratory distress syndrome: underrecognition by clinicians and diagnostic accuracy of three clinical definitions acute pulmonary edema development of a clinical definition for acute respiratory distress syndrome using the delphi technique acute lung injury and the acute respiratory distress syndrome: a clinical review acute lung injury and acute respiratory distress syndrome: diagnostic hurdles genetic variants in the angiopoietin-2 gene are associated with increased risk of ards acute respiratory distress syndrome and acute lung injury acute respiratory distress syndrome: a clinical review acute respiratory distress syndrome: pathophysiology and therapeutic options an nfkb1 promoter insertion/deletion polymorphism influences risk and outcome in acute respiratory distress syndrome among caucasians incidence and outcomes of acute lung injury early identification of patients at risk of acute lung injury: evaluation of lung injury prediction score in a multicenter cohort study diffuse alveolar damage, the pathological basis of adult respiratory distress syndrome the role of intra-alveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage the acute respiratory distress syndrome ards and diffuse alveolar damage: a pathologist's perspective clinical year in review iii: asthma, lung transplantation, cystic fibrosis, acute respiratory distress syndrome performance of an automated electronic acute lung injury screening system in intensive care unit patients introducing automated acute lung injury/acute respiratory distress syndrome electronic screening in intensive care unit practice: is it the future? the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination ventilatory, pharmacologic, supportive therapy, study design strategies, and issues related to recovery and remodeling. acute respiratory distress syndrome acute respiratory distress syndrome: the berlin definition lung structure and function in different stages of severe adult respiratory distress syndrome what has computed tomography taught us about the acute respiratory distress syndrome? reversibility of lung collapse and hypoxemia in early acute respiratory distress syndrome mechanical ventilation in acute respiratory failure: recruitment and high positive end-expiratory pressure are necessary open up the lung and keep the lung open how large is the lung recruitability in early acute respiratory distress syndrome: a prospective case series of patients monitored by computed tomography state-of-the-art mechanical ventilation understanding and avoiding ventilatorinduced lung injury: lessons from an insightful experimental study best strategy to recruit primary ards: what to look for fibroproliferative changes on high-resolution ct in the acute respiratory distress syndrome predict mortality and ventilator dependency: a prospective observational cohort study imaging in acute lung injury and acute respiratory distress syndrome assessment of lung inflammation with 18 f-fdg pet during acute lung injury lungs of patients with acute respiratory distress syndrome show diffuse inflammation in normally aerated regions: a [ 18 f]-fluoro-2-deoxy-dglucose pet/ct study bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment electrical impedance tomography bedside estimation of recruitable alveolar collapse and hyperdistension by electrical impedance tomography a unified approach for eit imaging of regional overdistension and atelectasis in acute lung injury whither lung eit: where are we, where do we want to go and what do we need to get there? does noninvasive positive pressure ventilation improve outcome in acute hypoxemic respiratory failure? a systematic review early use of noninvasive positive pressure ventilation for acute lung injury: a multicenter randomized controlled trial ventilation with lower tidal volumes as compared with traditional tidal volumes for acute 12 critical care research and practice lung injury and the acute respiratory distress syndrome pressure and volume limited ventilation for the ventilatory management of patients with acute lung injury: a systematic review and meta-analysis effects of superimposed driving-pressure on mortality in patients with acute lung injury or acute respiratory distress syndrome effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome a high positive end-expiratory pressure, low tidal volume ventilatory strategy improves outcome in persistent acute respiratory distress syndrome: a randomized, controlled trial effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial a scanographic assessment of pulmonary morphology in acute lung injury: significance of the lower inflection point detected on the lung pressure-volume curve influence of tidal volume on alveolar recruitment: respective role of peep and a recruitment maneuver airway pressure release ventilation in acute respiratory distress syndrome airway pressure release ventilation: an alternative mode of mechanical ventilation in acute respiratory distress syndrome comparison of the effects of bipap ventilation combined with lung recruitment maneuvers and low tidal volume a/c ventilation in patients with acute respiratory distress syndrome pressure support improves oxygenation and lung protection compared to pressure-controlled ventilation and is further improved by random variation of pressure support noisy pressure support ventilation: a pilot study on a new assisted ventilation mode in experimental lung injury spontaneous breathing during lung-protective ventilation in an experimental acute lung injury model: high transpulmonary pressure associated with strong spontaneous breathing effort may worsen lung injury effects of neurally adjusted ventilatory adjusted ventilatory assist on prevention of ventilator-induced diaphragmatic dysfunction in acute respiratory distress syndrome rabitts effects of neurally adjusted ventilatory assist on patientventilator synchrony in patients with acute respiratory distress syndrome high frequency oscillation in patients with acute lung injury and acute respiratory distress syndrome (ards): systematic review and meta-analysis an updated study-level meta-analysis of randomised controlled trials on proning in ards and acute lung injury prone positioning improves survival in severe ards: a pathophysiologic review and individual patient metaanalysis hemodynamic profile in severe ards: results of the european collaborative ards study comparison of two fluid-management strategies in acute lung injury pulmonary vascular dysfunction is associated with poor outcomes in patients with acute lung injury prevalence and prognosis of shunting across patent foramen ovale during acute respiratory distress syndrome extracorporeal membrane oxygenation in severe acute respiratory failure. a randomized prospective study clinical review: extracorporeal membrane oxygenation extracorporeal membrane oxygenation for ards in adults efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal carbon dioxide removal pumpless extracorporeal interventional lung assist in patients with acute respiratory distress syndrome: a prospective pilot study superimposed highfrequency jet ventilation combined with continuous positive airway pressure/assisted spontaneous breathing improves oxygenation in patients with h1n1-associated ards combined effects of prone positioning and airway pressure release ventilation on gas exchange in patients with acute lung injury prone position and recruitment manoeuvre: the combined effect improves oxygenation combination of high frequency oscillatory ventilation and interventional lung assist in severe acute respiratory distress syndrome clinical characteristics and outcomes of sepsis-related vs non-sepsis-related ards virus-induced acute respiratory distress syndrome: epidemiology, management and outcome biomarkers of acute lung injury: worth their salt? type iii procollagen peptide in the adult respiratory distress syndrome. association of increased peptide levels in bronchoalveolar lavage fluid with increased risk for death predictive value of procalcitonin decrease in patients with severe sepsis: a prospective observational study use of procalcitonin to reduce patients' exposure to antibiotics in intensive care units (prorata trial): a multicentre randomised controlled trial early goal-directed therapy in the treatment of severe sepsis and septic shock early interventions in severe sepsis and septic shock: a review of the evidence one decade later extravascular lung water indexed to predicted body weight is a novel predictor of intensive care unit mortality in patients with acute lung injury the influence of infection sites on development and mortality of ards predictors of hospital mortality in a population-based cohort of patients with acute lung injury neuromuscular blockers in early acute respiratory distress syndrome inhaled nitric oxide for acute respiratory distress syndrome (ards) and acute lung injury in children and adults efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome effects of methylprednisolone infusion on markers of inflammation, coagulation, and angiogenesis in early acute respiratory distress syndrome randomized, placebo-controlled trial of an aerosolized beta-2 adrenergic agonist (albuterol) for the treatment of acute lung injury omega-3 (n-3) fatty acid, gamma-linoleic acid (gla) and anti-oxidant supplementation in acute lung injury (omega trial) initial trophic vs full enteral feeding in patients with acute lung injury: the eden randomized trial the authors would like to thank adriana pardini for revision of the language. key: cord-016300-vw11c2wt authors: jain, kewal k. title: biomarkers of pulmonary diseases date: 2017-09-18 journal: the handbook of biomarkers doi: 10.1007/978-1-4939-7431-3_16 sha: doc_id: 16300 cord_uid: vw11c2wt lungs and airways are affected by several pathologies, the most important of which are inflammation, infection and cancer. some of the biomarkers of these pathologies are similar to those found in involvement of other organs. this chapter will briefly discuss general issues of biomarkers of pulmonary disorders listed in table 16.1. biomarkers of lung cancer are described in chapter 13. lungs and airways are affected by several pathologies, the most important of which are inflammation, infection and cancer. some of the biomarkers of these pathologies are similar to those found in involvement of other organs. this chapter will briefly discuss general issues of biomarkers of pulmonary disorders listed in table 16 .1. biomarkers of lung cancer are described in chapter 13. low lung function is associated with increased morbidity and mortality. it is therefore of interest to identify biomarkers that are associated with impaired lung function. lung function (fev1 and fvc) and a panel of 15 inflammatory biomarkers (including cytokines, chemokines, adhesion molecules, crp and wbc count) from blood samples were analysed subjects aged 70 years (kuhlmann et al. 2013) . wbc count, crp and vcam-1 were found to relate to poorer lung function. a doserelated association was found for the combination wbc count and crp towards fev1 and wbc and vcam-1 towards fvc. this indicates that combination of two biomarkers yielded more information than assessing them one by one when analysing the association between systemic inflammation and lung function. oxidative stress is the hallmark of various chronic inflammatory lung diseases. increased concentrations of ros in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. non-invasive approaches to measure oxidative stress have been investigated. the collection of exhaled breath condensate (ebc) is a noninvasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. the biomarkers of oxidative stress such as h 2 o 2 , f2-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species can be measured in ebc. oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. ebc is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. it is expected that ebc can be exploited to detect specific levels of biomarkers and monitor disease severity in response to treatment. community-acquired pneumonia (cap) is one of the most common reasons for emergency department. despite its prevalence, there are many challenges to proper diagnosis and management of pneumonia. there is no accurate and timely gold standard to differentiate bacterial from viral disease, and there are limitations in precise risk stratification of patients to ensure appropriate site-of-care decisions. clinical risk scores such as pneumonia severity index (psi) and curb-65 (confusion, urea, respiratory rate, blood pressure, age > 65 years), and blood biomarkers of different physiopathological pathways are used in predicting longterm survival in patients with cap. in a prospective study, patients admitted with cap were followed for 6 years and cox regression models as well as area under the receiver operating characteristics curve (auc) were used to investigate associations between initial risk assessment and all-cause mortality (alan et al. 2015) . initial psi and curb-65 scores both had excellent long-term prognostic accuracy, with a step-wise increase in mortality per risk class. the addition of inflammatory (pro-adrenomedullin) and cardiac (pro-atrial natriuretic peptide) blood biomarkers measured upon hospital admission further improved the prognostic capabilities of the psi. pathological changes in severe acute respiratory syndrome (sars) suggest that sars sequelae are associated with dysregulation of cytokine and chemokine production. a study from taiwan showed that cytokine or chemokine profiles in patients with sars differ markedly from those in patients with community-acquired pneumonia (cap) and control groups (chien et al. 2006) . serum levels of three cytokines were significantly elevated in sars patients versus the cap: interferon-γ-inducible protein-10 (ip-10), interleukin (il)-2, and il-6. cytokine levels began to rise before the development of chest involvement and peaked earlier than did lung injury assessed by chest x-ray. conversely, in cap patients but not sars patients or controls, levels of interferon-γ, il-10, and il-8 were elevated, and rose in tandem with radiographic changes. a further difference between groups was the ratio of il-6 to il-10, at 4.84 in sars patients versus 2.95 in cap patients. however, in both sets of patients, levels of il-6 correlated strongly with the severity of lung injury. the early induction of ip-10 and il-2, as well as the subsequent overproduction of il-6 and lack of il-10, probably contribute to the main immunopathological processes involved in sars lung injury and may be early biomarkers of lung injury. these findings differ from those observed in subjects with cap. plasma biomarkers related to inflammation − il-8 and enhanced neutrophil recruitment to the lung (icam-1) − are independently associated with increased mortality in patients with ali. higher levels of il-8 and icam-1 independently predicted death (mcclintock et al. 2008 ). in addition, lower levels of the coagulation marker protein c were independently associated with an increased risk of death. the association of lower protein c levels with non-survivors continues to support the role for disordered coagulation in ali/ards. these associations exist despite consistent use of lung protective ventilation and persist even when controlling for clinical factors that also impact upon outcomes. the two biomarkers with an independent association with mortality, il-8 and icam-1, need to be be studied further for their potential value in stratifying patients in clinical trials. acute respiratory distress syndrome (ards) is the rapid onset of respiratory failure − the inability to adequately oxygenate the blood − that often occurs in the critically ill. acute lung injury (ali) precedes ards as severe respiratory illnesses progress. both conditions can be life-threatening. in a large-scale, multicenter trial of patients with ards or ali, higher levels of nitric oxide (no) in urine were strongly associated with improved survival, more ventilator-free days, and decreased rates of organ failure (mcclintock et al. 2007 ). the authors speculated that no has a beneficial effect on ali since it scavenges oxygen free radicals that are generated during oxidative stress. since no increases microcirculation, it helps to better perfuse tissue beds in the lungs. the investigators offered an alternative hypothesis to explain their findings: no created inside the body may have a beneficial effect on organs other than the lung during ali. it might help prevent further tissue damage by improving oxygen and nutrient delivery to the tissues, while helping to decrease the amount of toxic oxygen species. the authors also speculated that no might have antibacterial effects that could be important in infectious conditions that predispose patients to ali. pulmonary surfactant, a complex of lipids and proteins, functions to keep alveoli from collapsing at expiration. surfactant proteins a (sp-a) and d (sp-d) belong to the collectin family and play pivotal roles in the innate immunity of the lung. pulmonary collectins directly bind with broad specificities to a variety of microorganism and possess antimicrobial effects. these proteins also exhibit both inflammatory and antiinflammatory functions. the collectins enhance phagocytosis of microbes by macrophages through opsonic and/or non-opsonic activities. the proteins stimulate cell surface expression of phagocytic receptors including scavenger receptor a and mannose receptor. since the expression of sp-a and sp-d is abundant and restricted within the lung, the proteins are now clinically used as biomarkers for lung diseases. the levels of sp-a and sp-d in bronchoalveolar lavage fluids, amniotic fluids, tracheal aspirates and pleural effusions reflect alterations in alveolar compartments and epithelium, and lung maturity. the determination of sp-a and sp-d in sera is a noninvasive and useful tool for understanding some pathological changes of the lung in the diseases, including pulmonary fibrosis, collagen vascular diseases complicated with interstitial lung disease, pulmonary alveolar proteinosis, acute respiratory distress syndrome and radiation pneumonitis (takahashi et al. 2006) . interstitial lung disease (ild) is defined as restrictive lung function impairment with radiographic signs of ild. kl-6, a mucinous high-molecular weight glycoprotein, is expressed on type ii pneumonocytes and is a potential biomarker of ild. a retrospective, cross-sectional analysis caucasian patients with polymyositis (pm) or dermatomyositis (dm) and ild were shown to have elevated serum levels of kl-6 compared to patients without ild (fathi et al. 2012) . at a cut-off level of 549 u/ml, the sensitivity and specificity for diagnosis of ild was 83% and 100%, respectively. the level of serum kl-6 may serve as measure of ild in patients with pm/dm, and is a promising biomarker for use in clinical practice to assess response to treatment. chronic obstructive pulmonary disease (copd) consists of two main forms − chronic bronchitis and emphysema − and sufferers usually have a combination of these conditions. there has been increasing interest in using pulmonary biomarkers to understand and monitor the inflammation in the respiratory tract of patients with copd. bronchial biopsies and bronchoalveolar lavage provide valuable information about inflammatory cells and mediators, but these procedures are invasive, so that repeated measurements are limited. sputum provides considerable information about the inflammatory process, including mediators and proteinases in copd, but samples usually represent proximal airways and may not reflect inflammatory processes in distal bronchi. analysis of exhaled breath is a noninvasive procedure so that repeated measurements are possible, but the variability is high for some assays. there is relatively little information about how any of these biomarkers relate to other clinical outcomes, such as progression of the disease, severity of disease, clinical subtypes or response to therapy. more information is also needed about the variability in these measurements. in the future pulmonary biomarkers may be useful in predicting disease progression, indicating disease instability and in predicting response to current therapies and novel therapies, many of which are now in development. the copd foundation biomarker qualification consortium (cbqc) is a unique public-private partnership established in 2010 between the copd foundation, the pharmaceutical industry, and academic copd experts with advisors from the us national heart lung & blood institute and fda (miller et al. 2016) . the initial intent of the cbqc was to integrate data collected in 2009 and submit a dossier for the qualification. this led to the fda qualification of plasma fibrinogen as a prognostic or enrichment biomarker for all-cause mortality and copd exacerbations in 2015. it is the first biomarker drug development tool qualified for use in copd under the fda's drug development tool qualification program. alpha1-antitrypsin (aat) is a plasma glycoprotein that inhibits neutrophil elastase, and individuals who inherit altered aat genes resulting in deficiency of the protein are at high risk for copd and liver cirrhosis. this deficiency can be detected by serum protein pattern studies. in the past, testing for the deficiency has been done retrospectively in patients with copd or liver disease, but the introduction of a home-administered finger-stick blood spot test for aat genotype enables affected families to construct pedigrees to enable them to identify children who are at risk for developing copd in later life and should avoid exposure to dust and smoke. extracellular matrix (ecm) remodeling of the lung tissue releases protein fragments into the blood, where they may be detected as serologic surrogate biomarkers of disease activity in copd. association of ecm turnover with severity and outcome of copd has been assessed in a prospective, observational, multicenter study, global initiative for chronic obstructive lung disease grades ii to iv, and serum samples were analyzed at stable state, during exacerbation as well as 4 weeks after exacerbation (stolz et al. 2017) . results showed that patients with the lowest levels of pro-forms of collagen type iii (pro-c3) and type vi (pro-c6) had more severe airflow limitation, hyperinflation, air trapping, and emphysema. collagen type iii (c3m) and collagen type vi (c6m) were associated with dyspnea. in conclusion, serum biomarkers of ecm turnover were significantly associated with disease severity and clinically relevant outcomes in patients with copd. lung ecm remodeling in healthy controls and copd patients was investigated in the copdgene study. the data suggest that type vi collagen turnover and elastin degradation by neutrophil elastase are associated with copd-induced inflammation (eosinophil-bronchitis) and emphysema (bihlet et al. 2017) . serological assessment of type vi collagen and elastin turnover may assist in identification of phenotypes likely to be associated with progression and amenable to precision medicine for clinical trials. lung failure, also termed "lung attack", is the most common organ failure seen in the intensive care unit. lung attacks, which effect individuals with copd are among the leading cause of visits to emergency rooms among chronic disease sufferers. other causes are neuromuscular impairment, pulmonary edema, pneumonia, and vascular diseases such as acute or chronic pulmonary embolism. when a patient is admitted into the hospital with a severe lung failure, it usually takes >3 months to get to 80% of his or her baseline health. if the patient's health is poor to start with, the new attack can be devastating or even fatal. a test that could more accurately present a patient's disease could make it easier to predict and treat copd progression to lung failure. there is need for a test that could be performed in any clinical lab and could be used far more widely than the current lung function tests, which are performed in certain centers by specially trained personnel. in 2012, canada's prevention of organ failure (proof) center of excellence in vancouver received funding from genome british columbia to develop a biomarkerbased test for determining a copd patient's risk for having a lung attack. genes and protein biomarker sets that have been discovered at proof center could have the ability to predict copd-caused lung attacks and need to be validated. circulating bnp levels were evaluated as a parameter for the presence and severity of pulmonary hypertension (ph) in patients with chronic lung disease (leuchte et al. 2006) . during a follow-up time of approximately 1 year, significant pulmonary hypertension (mean pulmonary artery pressure > 35 mm hg) was diagnosed in more than one-fourth of patients and led to decreased exercise tolerance and life expectancy. elevated bnp concentrations identified significant pulmonary hypertension with a sensitivity of 0.85 and specificity of 0.88 and predicted mortality. moreover, bnp served as a risk factor of death independent of lung functional impairment or hypoxemia. it is concluded that plasma bnp facilitates noninvasive detection of significant ph with high accuracy and can be used as a screening test for the presence of ph. in addition, bnp enables an assessment of the relevance of ph and could serve as a useful prognostic parameter in chronic lung disease. a study has revealed that serum levels of the neuroendocrine activity biomarker chromagranin a (cga) are increased in male smokers with impaired lung function, and are associated with both respiratory symptoms and the degree of airway obstruction (sorhaug et al. 2006) . the subgroup of airway epithelial cells belonging to the diffuse neuroendocrine system, termed pulmonary neuroendocrine cells, may represent a putative regulatory function of cga as a prohormone. they are considered to control growth and development of the fetal lung and regulation of ventilation and circulation, but may also have a role in the pathogenesis of smoking-induced airway disease. the findings indicate that neuroendocrine activation may be important in smoking-related airway inflammation and remodeling, and raise the possibility that cga could be of predictive value as a biomarker of prognosis in smoking-associated diseases. measurements of c-reactive protein (crp), a biomarker of inflammation, provide incremental prognostic information beyond that achieved by traditional biomarkers in patients with mild to moderate copd, and may enable more accurate detection of patients at a high risk of mortality. lung function decline is significantly related to crp levels, with an average predicted change in fev1 of −0.93% in the highest and 0.43% in the lowest quintile. however, respiratory causes of mortality are not significantly related to crp levels. genome-wide expression profiling of peripheral blood samples from subjects with significant airflow obstruction was performed to find non-invasive gene expression biomarkers for copd (bhattacharya et al. 2011) . correlation of gene expression with lung function measurements identified a set of 86 genes. a total of 16 biomarkers showed evidence of significant correlation with quantitative traits and differential expression between cases and controls. further comparison of these peripheral gene expression biomarkers with those previously identified from lung tissue of the same cohort revealed that two genes, rp9 and nape-pld, were decreased in copd cases compared to controls in both lung tissue and blood. these results contribute to our understanding of gene expression changes in the peripheral blood of patients with copd and may provide insight into potential mechanisms involved in the disease. patients with copd are often at high risk of early death and identification of prognostic biomarkers may aid in improving their survival by providing early intensive therapy for high-risk patients. a study has investigated the prognostic role of hyperuricemia at baseline on the prognosis of patients with copd by retrospective evaluation of data . hyperuricemia was found to be not associated with other baseline characteristics in patients with copd. kaplan-meier survival curve showed that patients with copd with hyperuricemia had higher risk of mortality compared with patients with normouricemia. thus, hyperuricemia is a promising biomarker of early mortality in patients with copd. decreased expression of vascular endothelial growth factor (vegf) and its receptor has been implicated in the pathogenesis of copd. levels of placenta growth factor (plgf), another angiogenic factor, are increased in the serum and bronchoalveolar lavage (bal) fluid of patients with copd and are inversely correlated with fev1 (cheng et al. 2008) . serum levels of plgf in patients with copd were more than double those in smokers and nonsmokers without copd. these findings suggest that bronchial epithelial cells can express plgf, which may contribute to the pathogenesis of copd. both plgf and vegf expression levels were increased in cultured bronchial epithelial cells exposed to pro-inflammatory cytokines such as tnfα and il-8. although the mechanisms underlying the observed detrimental effects of plgf remain to be clarified, persistent plgf expression might have adverse effects on lung parenchyma by down-regulating angiogenesis. although the aim of management of patients with asthma is to control their symptoms and prevent exacerbations and morbidity of the disease, optimal management may require assessment and monitoring of biomarkers, i.e., objective measures of lung dysfunction and inflammation. clinical observations suggest that rhinovirus infection induces a specific inflammatory response in predisposed individuals that results in worsened asthmatic symptoms and increased airway inflammation. a study has shown that ifn-γinduced protein (ip)-10 is specifically released in acute virus-induced asthma, and can be measured in the serum to predict a viral trigger of acute exacerbations (wark et al. 2007) . primary bronchial epithelial cell models of rhinovirus infection were used to identify mediators of rhinovirus infection and responded to infection with rhinovirus-16 by releasing high levels of ip-10, rantes, and il-16, as well as smaller amounts of il-8 and tnf-α. ip-10, perhaps in combination with tnf-α, might be a useful clinical marker to identify rhinovirus and other virus-induced acute asthma. additional findings suggest that ip-10 or cxcr3 (an ip-10 receptor that is highly expressed in activated t cells) might have a role in worsening of airflow obstruction and airway inflammation, and may therefore be potential therapeutic targets. international guidelines on the management of asthma support the early introduction of corticosteroids to control symptoms and to improve lung function by reducing airway inflammation. however, not all individuals respond to corticosteroids to the same extent and it would be an desirable to be able to predict the response to corticosteroid treatment. several biomarkers have been assessed following treatment with corticosteroids including measures of lung function, peripheral blood and sputum indices of inflammation, exhaled gases and breath condensates. the most widely examined measures in predicting a response to corticosteroids are airway hyperresponsiveness, exhaled no (eno) and induced sputum. of these, sputum eosinophilia has been demonstrated to be the best predictor of a short-term response to corticosteroids. more importantly, directing treatment at normalizing the sputum eosinophil count can substantially reduce severe exacerbations. the widespread utilization of sputum induction is hampered because the procedure is relatively labor intensive. the measurement of eno is simpler, but incorporating the assessment of no in an asthma management strategy has not led to a reduction in exacerbation rates. the challenge now is to either simplify the measurement of a sputum eosinophilia or to identify another inflammatory marker with a similar efficacy as the sputum eosinophil count in predicting both the short-and long-term responses to corticosteroids. airway inflammation is associated with an increased expression and release of inflammatory reactants that regulate processes of cell migration, activation and degranulation. one study was done to quantify bronchial lavage (bal) fluid and serum levels of il-8, secretory leukocyte protease inhibitor (slpi), soluble intracellular adhesion molecules-1 (sicam-1) and scd14, as surrogate markers of inflammatory and immune response in asthma and copd patients with similar disease duration time (hollander et al. 2007 ). biomarkers were measured using commercially available elisa kits. the findings show that of four measured biomarkers, only the bal il-8 was higher in copd patients when compared to asthma. severe asthma is characterized by elevated levels of proinflammatory cytokines and neutrophilic inflammation in the airways. blood cytokines, biomarkers of systemic inflammation, may be a feature of increased inflammation in severe asthma. one study found that il-8 and tnf-α levels were higher in severe asthmatics than in mild-moderate asthmatics or in controls and, in conjunction with augmented circulating neutrophils, suggest the involvement of neutrophil-derived cytokine pattern (silvestri et al. 2006) . furthermore, in patients with severe asthma, tnf-α levels were positively correlated with both exhaled nitric oxide and circulating neutrophil counts. cytokine levels were elevated even though the patients were on high-dose inhaled steroids. this finding might reflect the inability of these drugs to significantly suppress production of this cytokine by airway cellular sources including epithelial cells and inflammatory cells. in patients with severe asthma there may be an imbalance between il-8 production and the blocking capacity of il-8 autoantibodies. the findings of this study may be clinically relevant and suggest that drugs that block tnf-α release or activity might represent a new treatment option in severe asthma. airway hyperresponsiveness is the main feature of asthma and is defined as an increase in the ease and degree of airway narrowing in response to brochoconstrictor stimuli. inflammation plays a central role in the pathogenesis of asthma and much of it can be attributed to helper t cell type 2 cytokine activation, the degree of which strongly correlates to disease severity. one of the inflammatory mediators in asthma is nitric oxide (no). the exhaled no level is elevated in asthma, particularly allergic asthma during the pollen season, and can predict asthma exacerbation. it may be clinically more useful to compare exhaled no values with a subject's previous values than to compare them with a population based normal range. cough variant asthma (cva) and atopic cough both present with bronchodilator-resistant non-productive cough but may be differentiated from and other causes of chronic non-productive cough by measuring exhaled no. exhaled no levels in patients with atopic cough are significantly lower than those in patients with cva and bronchial asthma (fujimura et al. 2008 ). there are no significant difference in the exhaled no levels between patients with cva and bronchial asthma. a uk study findings show that it is feasible to measure bronchial flux no concentration ( j no) and alveolar no concentration (c alv ) in 70% of children, with c alv levels potentially reflecting alveolar inflammation in asthma (paraskakis et al. 2006) . c alv and j no were measured from the fractional exhaled no (feno 50 ) at multiple exhalation flow rates in asthmatic children. although feno 50 and jno give essentially the same information, c alv is higher in asthmatic children than in normal children. this study also highlights the relationship between poor control of asthma and c alv (a biomarker of alveolar inflammation) but further work is needed to confirm the relevance of this. a novel nanosensor can detect a possible asthma attack before it begins. the minute sensor can be fitted into a hand-held device, and when a person blows into the device, it measures the no content of their breath. use of this device would provide asthma sufferers with a simple and cost effective way to monitor their asthma inflammation. an explanation for increased levels of exhaled no is nonenzymatic generation of no from nitrite due to airway acidification in asthmatics. reduced arginine availability may also contribute to lung injury by promoting formation of cytotoxic radicals such as peroxynitrite. as arginine levels decline, nitric oxide synthase (nos) itself can begin to generate superoxide in lieu of no, thereby favoring no consumption via the generation of peroxynitrite that could induce lung injury. this reduction in bioavailability of no via formation of species such as peroxynitrite could be further amplified by the rapid loss of sod activity during the asthmatic response. plasma arginase activity declines significantly with treatment and improvement of symptoms. additional studies are needed to determine whether measurements of plasma arginase activity will provide a useful biomarker for underlying metabolic disorder and efficacy of treatment for this disease. the arginase activity present in serum probably does not accurately reflect whole body arginase activity or that compartmentalized in the lungs, since the arginases are intracellular enzymes. because arginase is induced in monocytes in response to helper t cell type 2 cytokines, it is speculated that these cells are one likely source of the elevated arginase in serum, consistent with the localization of arginase expression within macrophages in the lungs. athough exhaled no is a clinically useful biomarker of eosinophilic airway inflammation in asthma, significant validation and investigation are required before exhaled breath condensate could be utilized for making decisions in clinical practice (simpson and wark 2008) . endothelins are proinflammatory, profibrotic, broncho-and vasoconstrictive peptides, which play an important role in the development of airway inflammation and remodeling in asthma. a study has evaluated the endothelin-1 (et-1) levels in exhaled breath condensate (ebc) of asthmatics with different degree in asthma severity (zietkowski et al. 2008) . et-1 concentrations in ebc of all asthmatic patients were significantly higher than in healthy volunteers. et-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. thus, measurements of et-1 in ebc may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma. the release of et-1 from bronchial epithelium through the influence of many inflammatory cells essential in asthma and interactions with other cytokines, may play an important role in increase of airway inflammation, which is observed after postexercise bronchoconstriction in asthmatic patients. ige plays a central role in the pathophysiology of asthma. the two essential phases in this pathophysiology are sensitization to allergen and clinical expression of symptoms on reexposure to the sensitizing allergen. omalizumab (xolair, genentech) is a recombinant humanized igg1 monoclonal anti-ige antibody that binds to circulating ige, regardless of allergen specificity, forming small, biologically inert ige-anti-ige complexes without activating the complement cascade. an 89-99% reduction in free serum ige (i.e., ige not bound to omalizumab) occurs soon after the administration of omalizumab, and low levels persist throughout treatment with appropriate doses. a total serum ige level should be measured in all patients who are being considered for treatment with omalizumab, because the dose of omalizumab is determined on the basis of the ige level and body weight. the dose is based on the estimated amount of the drug that is required to reduce circulating free ige levels to less than 10 iu per milliliter. lebrikizumab (roche) is an injectable humanized mab designed to block il-13, which contributes to key features of asthma. lebrikizumab improves lung function in adult asthma patients who are unable to control their disease on inhaled corticosteroids. il-13 induces bronchial epithelial cells to secrete periostin, a matricellular protein. increased levels of periostin, a biomarker of asthma, can be measured in the blood. in the milly phase ii trial, patients with high pretreatment periostin levels had greater improvement in lung function when treated with lebrikizumab, compared to patients with low periostin levels (corren et al. 2011) .the primary endpoint of the trial showed that at week 12, lebrikizumab-treated patients had a 5.5% greater increase in lung function from the baseline compared to placebo. lebrikizumabtreated patients in the high-periostin subgroup experienced an 8.2% relative increase from baseline forced expiratory volume in 1 second (fev1), compared with placebo. in the low-periostin subgroup, those patients on the drug experienced a 1.6% relative increase in fev1, compared with placebo. these results support further investigation of lebrikizumab as a personalized medicine for patients who suffer from moderate to severe uncontrolled asthma periostin enables selection of patients who will benefit most from the drug. cystic fibrosis (cf) is the most common serious genetic disease among caucasians in the us. the disease results from a defective gene that affects multiple aspects of cellular function. its most serious symptom is a build-up of thick, sticky mucus in the airways, which can lead to fatal lung infections. the usual method for screening and diagnosis is genotyping of cystic fibrosis transmembrane conductance regulator (cftr) gene mutations. antibody microarrays have been developed as a platform for identifying a cf-specific serum proteomic signature. serum samples from cf patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to cf. the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the nfkappab signaling pathway, and in proteins that may be selectively expressed in cf-affected tissues such as lung and intestine. in several instances, the data from the antibody microarrays can be validated by quantitative analysis with reverse capture protein microarrays. in conclusion, antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from cf and control patients. saliva, because of the noninvasive collection process, shows great potential as a biological fluid for cf monitoring. extensive protein degradation and differentially expressed proteins have been identified in sputum as biomarkers of inflammation relating to pulmonary exacerbations of cf. use of fiber microarrays for measuring significant variations of the levels of six proteins in saliva supernatants -vegf, mmp-9, ip-10, il-8, il-1β and egf -as well as the correlations of these levels with clinical assessments, has demonstrated the value of saliva for cf research and monitoring (nie et al. 2015) . the prohosp study group. clinical risk scores and blood biomarkers as predictors of long-term outcome in patients with community-acquired pneumonia: a 6-year prospective follow-up study peripheral blood gene expression profiles in copd subjects biomarkers of extracellular matrix turnover are associated with emphysema and eosinophilic-bronchitis in copd increased expression of placenta growth factor in chronic obstructive pulmonary disease temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome lebrikizumab treatment in adults with asthma kl-6: a serological biomarker for interstitial lung disease in patients with polymyositis and dermatomyositis exhaled nitric oxide levels in patients with atopic cough and cough variant asthma serum and bronchial lavage fluid concentrations of il-8, slpi, scd14 and sicam-1 in patients with copd and asthma association of biomarkers of inflammation and cell adhesion with lung function in the elderly: a population-based study brain natriuretic peptide is a prognostic parameter in chronic lung disease higher urine nitric oxide is associated with improved outcomes in patients with acute lung injury biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury plasma fibrinogen qualification as a drug development tool in chronic obstructive pulmonary disease. perspective of the chronic obstructive pulmonary disease biomarker qualification consortium correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis measurement of bronchial and alveolar nitric oxide production in normal children and children with asthma parathyroid hormone as a novel biomarker for chronic obstructive pulmonary disease: korean national health and nutrition examination survey high serum levels of tumour necrosis factor-α and interleukin-8 in severe asthma: markers of systemic inflammation? the role of exhaled nitric oxide and exhaled breath condensates in evaluating airway inflammation in asthma increased serum levels of chromogranin a in male smokers with airway obstruction systemic biomarkers of collagen and elastin turnover are associated with clinically relevant outcomes in copd pulmonary surfactant proteins a and d: innate immune functions and biomarkers for lung diseases ifn-gamma-induced protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations hyperuricemia is a biomarker of early mortality in patients with chronic obstructive pulmonary disease endothelin-1 in exhaled breath condensate of stable and unstable asthma patients key: cord-009983-naht0ik6 authors: kim, yoon hee; kim, kyung won; lee, kyung eun; lee, mi‐jung; kim, sang kyum; kim, se hoon; shim, hyo sup; lee, chang young; kim, myung‐joon; sohn, myung hyun; kim, kyu‐earn title: transforming growth factor‐beta 1 in humidifier disinfectant‐associated children's interstitial lung disease date: 2015-06-25 journal: pediatr pulmonol doi: 10.1002/ppul.23226 sha: doc_id: 9983 cord_uid: naht0ik6 background: humidifier disinfectant‐associated children's interstitial lung disease has an unpredictable clinical course with a high morbidity and mortality. objectives: to evaluate the differences in clinical findings between survivors and non‐survivors of humidifier disinfectant‐associated children's interstitial lung disease. to evaluate dynamic changes in serum cytokines related to inflammation and fibrosis in lung injury, and to determine whether these changes are predictive of survival in this disease. methods: we evaluated 17 children with humidifier disinfectant‐associated children's interstitial lung disease, from whom serum samples were obtained weekly during hospitalization. the severity of chest tomographic and lung pathologic findings was scored. levels of several cytokines were measured in the serial serum samples. results: seven of the 17 children were survivors. compared to survivors, non‐survivors had greater ground‐glass attenuation on follow‐up chest tomography, higher admission neutrophil counts, and more macrophages on pathologic findings. transforming growth factor‐beta 1 persisted at an elevated level (1,000–1,500 pg/ml) in survivors, whereas it decreased abruptly in non‐survivors. at the time of this decrease, non‐survivors had clinical worsening of their respiratory failure. transforming growth factor‐beta 1 was positively correlated with pao(2)/fio(2) (r = 0.481, p < 0.0001). conclusions: non‐survivors exhibited more inflammatory clinical findings than survivors. transforming growth factor‐beta 1 remained elevated in survivors, suggesting that it affected the clinical course of humidifier disinfectant‐associated children's interstitial lung disease. the prognosis of this lung disease may depend more on controlling excessive inflammation and repairing damaged lung than on fibrosis, and transforming growth factor‐beta 1 may play a key role in this process. pediatr pulmonol. 2016;51:173–182. © 2015 wiley periodicals, inc. a severe interstitial lung disease (ild) intractable to any treatment has affected korea every spring since approximately 2006, and a similar ild led to the death of several pregnant women in 2011. [1] [2] [3] [4] [5] based on the results of an epidemiological study and an animal study, the korea centers for disease control and prevention (kcdc) disclosed that disinfectants nebulized from humidifiers might be the cause of this lethal lung injury. [5] [6] [7] this disinfectant, which was composed of [2-(2-ethoxy) ethoxyethyl] guanidium chloride (pgh), polyhexamethyleneguanidine (phmg), 5-chloro-2methylisothiazol-3(2h)-one (cmit)/ 2-methylisothiazol-3-one (mit), and didecyldimethylammonium chloride (ddac), was reported to cause fatal lung injury and has been prohibited for any use that may lead to inhalation. [6] [7] [8] [9] recently, a case-control study and a multicenter intervention study in korea provided further evidence suggesting that the cause of this fatal ild appeared to be the humidifier disinfectant. 10, 11 the american thoracic society and european respiratory society have recently updated guidelines for the diagnosis, classification, and management of ild and children's interstitial lung disease (child). [12] [13] [14] however, ild is still a very diverse disease group, with a high mortality and morbidity and an increasing prevalence. 15 there have been continuing efforts to determine reliable prognostic factors and effective treatments through increased understanding of the pathophysiology of ild. 16, 17 several possible prognostic markers have been proposed, including interleukin 8 (il-8), matrix metalloproteinase (mmp), vascular endothelial growth factor (vegf), transforming growth factor beta (tgf-b), krebs von den lungen-6 (kl-6; a representative inflammatory and fibrotic cytokine), and monocyte chemotactic protein 1 (mcp-1). however, no biomarker has been established as a standard for the management of patients with ild. [18] [19] [20] [21] [22] the current paradigm for understanding the pathophysiology of ild involves chronic inflammation by repetitive injury to lung epithelial cells, followed by aberrant repair processes leading to excessive irreversible lung fibrosis. 23 this process may progress gradually or rapidly, and it may cease when lung damage is mild or progress to severe lung dysfunction. 16 despite current medical advances, we are still unable to predict the exact progression and ultimate prognosis of this disease. existing studies, which have generally focused on investigating clinical characteristics and cytokines at a single time, have been unable to determine factors that reliably predict the progress and prognosis of ild. 21 there have been many efforts to determine the clinical prognostic factors of the humidifier disinfectant-associated children's interstitial lung disease (hd-child) because this ild progressed rapidly and was refractory to all treatment in non-survivors, whereas the clinical course in survivors tended to be highly favorable. 1, 11 disappointingly, no initial clinical characteristics, such as radiologic, pathologic, and laboratory findings, were significantly associated with survival. 1, 3, 11 the recently reported nationwide intervention study indicated that humidifier disinfectants could be the possible cause, but it mentioned the limitations caused by our lack of knowledge about the differences in individual susceptibility. 11 in this study, we compared serial changes in pulmonary inflammatory and fibrotic cytokines between survivors and non-survivors and evaluated whether patterns of change in these cytokines could be useful prognostic markers. we also examined potential correlations between changes in useful prognostic cytokines and clinical indices, such as radiologic, pathologic, and laboratory findings. a total of 17 children diagnosed with hd-child who were included in the patients reported in the previous nationwide report 11 were enrolled in this study. all children were admitted to severance children's hospital from february 2010 to may 2011, and satisfied the diagnostic criteria reported previously. 10, 11 briefly, the criteria were as follows: (1) age less than 18 years at the time of diagnosis; (2) rapid progression to acute respiratory distress; and (3) chest tomographic (ct) findings of diffuse or centrilobular ground-glass opacities and/or air leak syndrome, such as pneumothorax, pneumomediastinum, or subcutaneous emphysema. 10, 11 all children were checked to have been exposed to humidifier disinfectants before diagnosis by interviewing with their parents and confirmed with hd-child thorough the previous nationwide report. 11 serum samples were collected weekly from hospital admission until discharge or death. to determine clinical prognostic factors, we collected information regarding the patients' demographic data, laboratory findings, treatment, ct results, and pathologic findings. this study was approved by the institution review board of severance hospital (seoul, korea, irb no. 4-2013-0670). all children underwent thin-section ct scans of the chest. the ct images were reviewed and scored by two radiology specialists. details regarding the methodology used for the radiologic review and scoring are provided in an online data supplement. the pathologic diagnosis was made by lung biopsy obtained during video-assisted thoracoscopic surgery in 12 patients. these biopsies were performed within 1 week after admission. to evaluate the grade of pathologic findings, two pathologists reviewed the specimens. details of the methods used for the pathologic review and scoring are provided in an online data supplement. serum levels of the following were measured by enzyme-linked immunosorbent assay, according to the manufacturer's (r&d systems, minneapolis, mn) instructions: il-8; il-13; monocyte chemoattractant protein-1 (mcp-1); mmp-9; periostin; tgf-b1; vascular endothelial growth factor (vegf); regulated on activation, normal t cell expressed and secreted (rantes) cytokine; and granulocyte macrophage-colony stimulating factor (gm-csf). patient characteristics, laboratory data, ct results, pathologic findings, and treatments were compared between the survivor and non-survivor groups. the study protocol indicated that serum samples would be collected weekly during the hospital stay; however, in patients whose condition was extremely unstable or those who underwent concurrent blood sampling for monitoring of their treatment, not all of the serum samples for this study were collected at exact weekly intervals. since the clinical course and length of hospital stay varied widely from child to child, a substantial quantity of serum cytokine data was missing. to account for missing data and irregular intervals between serial serum cytokine collections, we used a generalized linear mixed-effect model for comparing the slope of changes in the serial serum cytokines among the survivors and non-survivors. 24 we evaluated the slope of changes as estimate values with standard errors. also, we used the slope comparison test for comparing the trend of tgf-b1 and the trends of the other cytokines according to survival. the differences of the trend of tgf-b1 and the trends of the other cytokines were significant in p < 0.05 and the more close to zero the value of estimates were, the more significant the relationship with the trend of tgf-b1 and the trends of the other cytokines were. a p-value <0.05 was considered statistically significant. statistical software (sas version 9.2, sas institute inc., cary, nc) was used for all analyses. the clinical characteristics of children in this study are shown in table 1 . the mean age of all patients was 2.6 years, and 12 children (70.6%) were male. twelve children had a weight below the 5th percentile for the same age and sex. on admission, the most common symptoms were cough, tachypnea, and fever. the ratio of arterial oxygen partial pressure to inspired oxygen fraction (pao 2 /fio 2 ) at admission of non-survivors was higher than that of survivors, although the difference between groups was not statistically significant. the admission peripheral blood leukocyte count and absolute neutrophil count of non-survivors were higher than those of survivors (p ¼ 0.025 and p ¼ 0.010, respectively). only one child had a respiratory virus detected by nasopharyngeal aspirate, and no patient had bacteria detected in sputum or blood cultures. during the hospital stay, all patients required supplemental oxygen, and all non-survivors received mechanical ventilation. no survivors received extracorporeal membrane oxygenation (ecmo). steroid therapy was used for all patients. hydroxychloroquine, cyclophosphamide, or both were administered to 14 of the 17 children. the initial and follow-up high resolution ct scores for ground-glass attenuation, septal thickening, bronchiectasis, and sum of these three findings are shown in table 2 . the change in ground-glass attenuation, septal thickening, and sum scores from initial to follow-up was greater in non-survivors than in survivors (p ¼ 0.002, p ¼ 0.002, and p ¼ 0.005, respectively). whereas air leak syndrome on the initial hrct did not differ between groups, this finding was more common on the follow-up hrct in non-survivors than survivors (p ¼ 0.021). the pathologic characteristics were bronchiolar destruction with peribronchiolar inflammation or fibrosis, accompanied by a predominantly centrilobular destructive lesion or diffuse alveolar damage, which were generally consistent with previous reports. 3, 11 additionally, type ii pneumocyte hyperplasia was found frequently in our patients. the only pathologic finding that differed significantly between survivors and non-survivors was the score for interstitial and intra-alveolar foamy macrophages. the interstitial and intra-alveolar foamy macrophages score was higher in non-survivors (p ¼ 0.026) ( table 3 ). the scores for bronchiolar and peribronchiolar destruction, type ii pneumocyte hyperplasia, intraalveolar fibrinous exudates, alveolar inflammatory cell infiltrate, and alveolar fibrosis were not different between survivors and non-survivors. the frequency of centrilobular destruction also did not differ between groups. the serial changes in cytokines (il-8, il-13, mcp-1, mmp-9, periostin, tgf-b1, vegf, rantes, and gm-csf) are shown according to survival in figure 1 and appendix 1. the tgf-b1 values of survivors were maintained at approximately 1,000-1,500 pg/ml from the admission to discharge, whereas tgf-b1 values in most non-survivors eventually decreased below 500 pg/ml. the estimate of the slope of tgf-b1 in non-survivors was à16.4469, which was significant different from the slope of à1.1223 found in survivors (p ¼ 0.0257) ( table 4) . no differences between survivors and non-survivors were noted for the trends of the serial values of other cytokines (appendix 1). the slopes for the other cytokines also did not differ significantly between groups (table 4) . comparing the trend of tgf-b1 and the trends of the other cytokines according to survival by the slope comparison analysis, the slopes of il-8, mcp-1, periostin, and vegf showed the similar trends with the slope of tgf-b1 while gm-csf showed the opposite trend with the slope of tgf-b1 in non-survival group. in survival group, only the slope of mmp-9 had the similar trend with the slope of tgf-b1 in supplementary table s1 . since tgf-b1 in most non-survivors eventually fell below 500 pg/ml, we compared the serial changes in tgf-b1 with the clinical course in each non-survivor (appendix 2). except for three children, most nonsurvivors had a point at which the tgf-b1 abruptly decreased (shown as red lines in appendix 2). among the seven non-survivors with these abrupt decreases in tgf-b1, four were transferred to the intensive care unit (icu) or managed with increasing the ventilator settings (e.g., by increasing the positive end-expiratory pressure [peep] or inspired oxygen fraction [fio 2 ]), and three other patients were initiated on ecmo at around the time that the tgf-b1 abruptly fell. in the three patients who did not demonstrate an abrupt decrease in tgf-b1 (shown as blue lines in appendix 2), the last sampling was obtained prior to worsening of their respiratory failure, as exemplified by an increase in ventilator settings or initiation of ecmo. as shown in figure 2 , the pao 2 /fio 2 ratios correlated well with tgf-b1 (pearson's correlation coefficient, r ¼ 0.481, p < 0.0001). since the values of pao 2 /fio 2 ratios and tgf-b1 were checked serially in all children, we adjusted the analysis for the serial values from each patient using a generalized linear mixed model. in the adjusted model, the pao 2 /fio 2 ratios were not correlated with tgf-b1 (adjusted r ¼ 0.375, adjusted p ¼ 0.138). since tgf-b1 has been reported to restore depleted vegf secretion after acute lung injury, 25 we reviewed the serial changes in vegf related to tgf-b1 in the seven non-survivors who showed the abrupt decrease in tgf-b1. a depicted in figure 3 , vegf decreased just after or at the same time as the fall in tgf-b1 in these nonsurvivors. this study suggested that maintaining an elevated tgf-b1 predicted eventual improvement of excessive pulmonary inflammation and a favorable outcome in hd-child. by contrast, an abrupt decrease of the elevated tgf-b1 was associated with a decrease in vegf and reflected worsening respiratory function. these findings suggest that tgf-b1 may play a key role in the repair of damaged lung and control of excessive inflammation in acute lung injury, rather functioning in promoting the progression of irreversible, fatal fibrosis. tgf-b1 is involved in lung development, inflammation, injury-related fibrosis, and repair. 26 in ild, and especially idiopathic pulmonary fibrosis (ipf), tgf-b1 has usually been considered a poor prognostic factor, inducing pulmonary fibrosis through stimulation of fibrogenic cytokines and endothelial-mesenchymal transition. it has likewise been considered a potential therapeutic target. 27 recently, tgf-b1 was reported to inhibit autophagosome activity, thus leading to pulmonary fibrosis in a lung injury model. 28 conversely, tgf-b1 is also important in the development of the immature lung of neonates, and excessive tgf-b1 may induce bronchopulmonary dysplasia (bpd), in which it may inhibit alveolarization in extreme premature lung. the specific role of tgf-b1, therefore, appears to be tightly controlled, resulting in the appropriate amount of activity at the correct time and place. 26 in an animal model of hyperoxic lung injury (a representative acute lung injury), tgf-b1 was reported to promote the repair of damaged lung and improve survival. 25 tgf-b1 also repaired damaged deoxyribonucleic acid and increased fibronectin secretion to accelerate closure of a monolayer of hyperoxic cells subjected to a scratch wound. 25 tgf-b1 has also been reported to play a key role in inhibiting excessive pulmonary inflammation. 29, 30 in the hd-child of this study, the role of tgf-b1 seemed to be very complex in relating with repairing damaged lung, controlling severe inflammation, and promoting pulmonary fibrosis. in non-survivors, the abrupt decrease in tgf-b1 correlated well with clinical deterioration in respiratory function, as exemplified by such events as increased ventilator settings (e.g., very high peep) or applying ecmo for support of impending severe respiratory failure. in addition, the pao 2 /fio 2 , which is the most widely recognized clinical index of lung injury, tended to correlate positively with tgf-b1. 31 however, this correlation was not significant statistically after adjustment for the several measurements in one subject. considering the inevitably small number of subjects and irregular sampling times and intervals because of the fatal clinical course of this disease, it does not seem reasonable to consider only the strict statistical analysis results. a correlation between changes in tgf-b1 with the clinical severity of respiratory failure and lung oxygenation index may provide additional indirect evidence for the role of tgf-b1 in repairing damaged lung and controlling excessive inflammation. vegf functions in promoting angiogenesis and increasing vascular permeability. 32 it has been identified as a reliable index in ipf, reflecting the degree of lung fibrosis on radiologic imaging and the severity of the disease. 19 paradoxically, vegf has also been suggested as a possible therapeutic agent because of its role in stimulating growth and inhibiting apoptosis of alveolar epithelial cells. 33 in premature children, vegf was reported to be elevated as a compensatory mechanism during severe respiratory distress proceeding to bpd. 34 vegf, like tgf-b1, thereby exhibits paradoxical roles in lung injury, and in our patients, it seemed to function in repair of the damaged lungs. 35 our study likewise provided evidence in humans supporting the results of an animal study in which tgf-b1 restored the depleted vegf during repair of acute lung injury. 25 il-8 is a potent pro-inflammatory cytokine that is increased in patients with ipf. 36 il-13 has been suggested as a possible therapeutic target, which was evaluated in a large-scale phase 2 study of anti il-13 in ipf. 37 the inflammatory and fibrotic cytokines mcp-1 (ccl2) and rantes were dramatically increased in a mouse model when ddac, a component of humidifier disinfectants, was injected into the trachea and increased the susceptibility to infection by host immune modulation related to toll-like receptor 4 (tlr 4). 7, 9, 22 mmp-9, periostin, and gm-csf have been reported to be important factors in pulmonary fibrosis. 21, 38, 39 these cytokines were increased in our patients with hd-child, but serial changes in these cytokines were not related to whether patients were survivors or non-survivors. the recent nationwide intervention study of hd-child in korea did not identify any useful clinical prognostic marker, 11 and clinical indices, such as radiologic, pathologic, and laboratory findings. in our current study, only small differences in clinical indices were noted between survivors and non-survivors. non-survivors were more likely to have persistent air leak syndrome and more extensive ground-glass attenuation and septal thickening on follow-up ct, which showed a potential possibility of serial ct findings as a prognostic tool; a higher neutrophil count on laboratory tests; and a greater number of macrophages on pathologic findings. the ground-glass attenuation, increased neutrophil counts, and increased macrophages reflect inflammation more than fibrosis. the prognosis of most ild is generally more closely associated with the severity of pulmonary fibrosis than the severity of inflammation because fibrosis is usually irreversible, whereas inflammation tends to be more reversible. 15, 16, 21 considering all the clinical findings of our non-survivors, the prognosis of hd-child seemed to be more closely related to excessive inflammation than fibrosis. as well, the control of excessive inflammation seemed to be a key factor in determining survival in hd-child. children are more vulnerable to lung injury following exposure to toxic materials than adults, while at the same time, repair and regeneration of damaged lung may by fig. 2 . correlation of transforming growth factor beta 1 (tgf-b1) with the ratio of arterial oxygen partial pressure and inspired oxygen fraction (pao 2 /fio 2 ). pao 2 /fio 2 ratios were well correlated with tgf-b1 (r ¼ 0.481, p < 0.0001). in the adjusted model considering the serial values in the same patient, the pao 2 /fio 2 ratios were not correlated with tgf-b1 (adjusted r ¼ 0.375, adjusted p ¼ 0.138). fig. 3 . serial changes of transforming growth factor beta 1 (tgf-b1) and vascular endothelial growth factor (vegf) during hospitalization in seven non-survivors. vegf was decreased just after or at the same time as the decrease in tgf-b1 in all non-survivors with an abrupt fall in tgf-b1. more prominent in children because alveolarization can persist up to puberty, although it is mostly complete by 18 months of age. 40, 41 since most children in this study were very young, with a mean age was 2.6 year old, it seems reasonable that excessive inflammation, followed by control of the inflammation and repair of the damaged lung, was more important than fibrosis in predicting their prognosis. also, twelve children (70.6%) were an underweight in this study and it seemed to reflect on individual functional deterioration or increasing disease susceptibility by malnutrition. 42 the strengths and limitations of this study might be considered. this study provides meaningful information about the role of tgf-b1 in controlling inflammation and repairing acute lung injury in humans, especially children. furthermore, the subjects were young children, who are usually difficult to include in research studies. our study is of particularly importance when one considers the increasing concerns in recent years about chemical terrorism. it is the only study of acute lung injury due to a toxic inhalant that provides information about the pathophysiology, related therapeutic considerations, and prognostic factors, as determined by analyzing serial changes in cytokines, instead of single data. the most important limitations are the very limited number of patients and the analyzing of the only blood samples without any lung tissue and exudates, but it was inevitable because the clinical course of hd-child was severe, aggressive, and unpredictable. the small number of subjects, plus the irregular sampling and substantial quantity of missing cytokine data, made the statistical analysis difficult, although a generalized mixed model called a spaghetti plot was used to help overcome this. also, we could only rely on interviewing with the parents whether the humidifier disinfectant was exposed to the children. a further limitation was that this study could not define a clear cause and effect relationship; this should be re-evaluated in a well-designed animal model. in conclusion, this study confirmed the possible role of tgf-b1 in the repair of damaged lung and control of vigorous inflammation, in acute lung injury associated with unexpected exposure to humidifier disinfectants in young children. it also suggested that such a repair mechanism could be more prominent in young children whose lungs are still actively growing and developing than adult. further studies involving in vitro investigations and an in vivo animal model are required to directly confirm this mechanism. epidemic acute interstitial pneumonia in children occurred during the early nationwide surveillance of acute interstitial pneumonia in korea toxic inhalational injury-associated interstitial lung disease in children two series of familial cases with unclassified interstitial pneumonia with fibrosis interim report of epidemiological investigation on lung injury with unknown cause in korea fatal misuse of humidifier disinfectants in korea: importance of screening risk assessment and implications for management of chemicals in consumer products didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice humidifier lung: possible contribution of endotoxin-induced lung injury altered pulmonary defense system in lung injury induced by didecyldimethylammonium chloride in mice inhalation toxicity of humidifier disinfectants as a risk factor of children's interstitial lung disease in korea: a case-control study humidifier disinfectantassociated children's interstitial lung disease paediatric interstitial lung disease: classification and definitions an official american thoracic society clinical practice guideline: classification, evaluation, and management of childhood interstitial lung disease in infancy an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias year in review 2012: acute lung injury, interstitial lung diseases, sleep and physiology interstitial lung disease ipf: new insight on pathogenesis and treatment peripheral blood proteins predict mortality in idiopathic pulmonary fibrosis significance of serum vascular endothelial growth factor level in patients with idiopathic pulmonary fibrosis circulating kl-6 predicts the outcome of rapidly progressive idiopathic pulmonary fibrosis re-evaluation of fibrogenic cytokines in lung fibrosis a role for mcp-1/ccr2 in interstitial lung disease in children modern concepts on the role of inflammation in pulmonary fibrosis using the general linear mixed model to analyse unbalanced repeated measures and longitudinal data tgf-beta signaling promotes survival and repair in rat alveolar epithelial type 2 cells during recovery after hyperoxic injury developmental responses to lung injury: repair or fibrosis the impact of tgf-beta on lung fibrosis: from targeting to biomarkers autophagy in idiopathic pulmonary fibrosis the integrin alpha v beta 6 binds and activates latent tgf beta 1: a mechanism for regulating pulmonary inflammation and fibrosis mechanisms of tgf-beta signaling from cell membrane to the nucleus study design strategies, and issues related to recovery and remodeling. acute respiratory distress syndrome angiogenesis in interstitial lung diseases: a pathogenetic hallmark or a bystander? vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and a549 cells pulmonary vascular endothelial growth factor and flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn novel therapeutic strategies for acute lung injury induced by lung damaging agents: the potential role of growth factors as treatment options increased expression of the interleukin-8 gene by alveolar macrophages in idiopathic pulmonary fibrosis. a potential mechanism for the recruitment and activation of neutrophils in lung fibrosis new treatment and markers of prognosis for idiopathic pulmonary fibrosis: lessons learned from translational research metalloproteinases in idiopathic pulmonary fibrosis periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis mechanisms of acute respiratory distress syndrome in children and adults: a review and suggestions for future research programming of respiratory health in childhood: influence of outdoor air pollution malnutrition and gastrointestinal and respiratory infections in children: a public health problem supporting information additional supporting information may be found in the online version of this article at the publisher's web-site the authors would like to thank all the children and their parents who were involved, for their cooperation in this study. we also thank dong wook kim, phd (medical research support section, department of biostatistics, yonsei university college of medicine) for his statistical assistance. key: cord-006452-mmdk2xom authors: chen, jing; tang, yue; liu, yun; dou, yushun title: nucleic acid-based therapeutics for pulmonary diseases date: 2018-10-18 journal: aaps pharmscitech doi: 10.1208/s12249-018-1183-0 sha: doc_id: 6452 cord_uid: mmdk2xom nucleic acid-based therapeutics present huge potential in the treatment of pulmonary diseases ranging from lung cancer to asthma and chronic pulmonary diseases, which are often fatal and widely prevalent. the susceptibility of nucleic acids to degradation and the complex structure of lungs retard the effective pulmonary delivery of nucleic acid drug. to overcome these barriers, different strategies have been exploited to increase the delivery efficiency using chemically synthesized nucleic acids, vector encapsulation, proper formulation, and administration route. however, several limitations regarding off-target effects and immune stimulation of nucleic acid drugs hamper their translation into the clinical practice. therefore, their successful clinical application will ultimately rely on well-developed carriers and methods to ensure safety and efficacy. in this review, we provide a comprehensive overview of the nucleic acid application for pulmonary diseases, covering action mechanism of the nucleic acid drugs, the novel delivery systems, and the current formulation for the administration to lungs. the latest advances of nucleic acid drugs under clinical evaluation to treat pulmonary disorders will also be detailed. due to their location and physiological function, the lungs are directly accessible to pollutants and viruses from the outside, rendering them susceptible to diseases ranging from lung cancer to chronic pulmonary diseases. among these pulmonary diseases, chronic obstructive pulmonary disease claimed 3.0 million lives in 2016, while lung cancer caused 1.7 million deaths (1) . since current treatments of these diseases have limited efficacy, many studies are being conducted to find novel effective treatments. though most lung diseases are considered to be the product of a variety of endogenous and exogenous influences, and less obviously are associated with gene replacement therapy. abnormal conditions are likely to arise from an imbalance between destructive and protective mechanisms. nucleic acids can be a new class of therapeutics to reconstitute a homeostatic balance by overexpression of protective genes or the suppression of damaging genes, which offers new strategies for the treatment of respiratory diseases (2) . the mesh-like network of blood vessels in the lungs, coupled with easy access through the pulmonary airways, enables the lungs to be targeted by both intravenous and topical routes. the latter fact makes the lung unique compared with other organs, allowing specific lung sites such as alveolar cells and bronchial epithelium to be exclusively targeted for different therapeutic applications (3) . in this review, we focus on nucleic acid-based therapies for pulmonary diseases. we discuss the hurdles nucleic acids face for translation into clinics and recent progress in the product into clinical trials. antisense oligonucleotides (asos) are single-strand dnas or rnas that selectively bind to complementary mrnas to modulate their functions. their hybridization could result in downregulation or upregulation of gene expression by diverse mechanisms. rnase h1-dependent asos could bind to target rna to form hybrid through watson-crick base pairing and downregulate translation through rnase h-induced degradation of the mrna. splice switching oligonucleotides could control the way exons skipping, modulate pre-mrna splicing, and generate novel proteins. asos can also interfere with other aspects of rna functions, such as blocking association of specific transcription factors with mrna, antagonizing microrna activities, and inhibiting rna-mediated telomerase activity (4) (5) (6) . antisense oligonucleotides are the first kind of nucleic acid drugs widely used in clinical trials. among the fdaapproved nucleic acids, aso-based drugs account for the majority as for now ( table i) . as of august 2018, only one aptamer drug and sirna drug have been approved by the fda. the first clinically approved nucleic acid drug was aso drug, vitravene (fomivirsen), indicated for cytomegalovirus retinitis in 1998. followed by kynamro (mipomersen) targeting mrna encoding apolipoprotein b for the treatment of familial hypercholesterolemia, exondys 51 (eteplirsen) designed to skip exon 51 of the dystrophin protein for the treatment of duchenne muscular dystrophy, spinraza (nusinersen) inducing the inclusion of exon 7 in the smn1 and smn2 mrna to treat spinal muscular atrophy and recently luxturna (voretigeneneparvovec-rzyl) for biallelic rpe65 mutation-associated retinal dystrophy (7, 8) . small interference rnas(sirnas) are double-strand rna molecules of 21 to 23 base pairs in length designed to silence target genes in a sequence-specific manner. after introduction into the cytoplasm, sirnas interact with multifunctional protein argonaute-2 and form the rna-induced silencing complex (risc), where one of the strands is degraded and the other strand (mostly antisense) is left as a guide to recognizing target mrna sequences. subsequently, mrnas which are perfect or nearly perfectly complementary to the sirna antisense strand are cleaved by the activated riscs (9) . the specific gene silencing effect of sirnas makes them indispensable tools for target identification and validation in drug discovery and development (10) . in 2018, onpattro (patisiran) infusion became the first fda-approved sirna drug. it is for the treatment of peripheral nerve disease caused by hereditary transthyretinmediated amyloidosis in adult patients. onpattro is designed to interfere with rna production of an abnormal form of the protein transthyretin. by preventing the production of transthyretin, the drug can help reduce the accumulation of amyloid deposits in peripheral nerves, improving symptoms, and helping patients better manage the condition. micro rnas(mirnas) are 18-24 nucleotides long, singlestranded, endogenous noncoding rna molecules that act as key regulators for a variety of cellular pathways. they can regulate gene expression by complementary binding to the core sequence in the 3′-untranslated region(3′-utr) of target mrnas (11) . either sirna or mirna could associate into the risc. unlike sirna, mirna can recognize mrna with partially complementary sequences, which means one mirna may have multiple different mrna targets (10) . hence, delivery of exogenous micrornas or microrna mimics could be particularly useful in diseases having multiple diseaserelevant targets (7) . mirnas mediate multiple biological processes, and alterations in mirna function have been associated with different diseases like cancer, metabolic disorders, and viral pathogenesis (12) . mirnas related to cancer are generally classified as tumor suppressor mirnas or tumorpromoting mirnas. tumor suppressor mirnas (e.g., let-7, mir-34 families, and mir-15/16) are responsible for suppressing oncogenes and are mostly downregulated in cancer. restoration of their normal function can be achieved by mirna replacement via administration of synthetic mirna mimics functioning similarly to the endogenous counterparts. tumor-promoting mirnas (e.g., mir-21, mir-17-92 cluster, and mir-155) are known to downregulate tumor suppressor genes and have been reported to be overexpressed in cancer (13) . asos and mirna sponges targeting tumor-promoting mirnas can be used to block aberrantly overexpressed mirnas (14) . aptamers are short oligonucleotides with unique threedimension structures that enable them to specifically recognize and bind to targeted proteins. aptamers of interest could be selected from a pool of randomized molecules by methods known as systematic evolution of ligands through exponential enrichment. therapeutic aptamers could act as inhibitors of protein function, or as targeting moieties for drug delivery (15, 16) . the use of rna-aptamers conjugates for targeted delivery of oligonucleotide molecules has been widely explored and well reviewed elsewhere (17, 18) . pegaptanib, the only aptamer that has been approved by the fda, is acting through the former way. vascular endothelial growth factor (vegf) induces angiogenesis, and increases vascular permeability and inflammation, playing a central role in the progression of age-related macular degeneration. pegaptanib could selectively bind to vegf isoform, vegf165, thereby preventing vegf165 from activating its receptors and suppressing pathological neovascularization (19) . the therapeutic and targeting properties of aptamers could be combined to construct multifunctional molecules. using an aptamer that binds to and antagonizes the receptor tyrosine kinase axl, an aptamer-mirna conjugates was developed with synergistic therapeutic effects, owing to oncosuppressive effects of the mirna and inhibitory function of the aptamer (20, 21) . barriers to nucleic acid-based therapies for pulmonary diseases the treatments for pulmonary diseases are mainly by parenteral injection and pulmonary administration through intranasal instillation, aerosol, or inhalation. hence, the first barriers that nucleic acid drugs via these two routes encounter are blood and respiratory tract (fig. 1 ). parenteral administration of unmodified nucleic acids has been set back by their very short half-life in the bloodstream, serum nuclease degradation, quick renal clearance, and poor biodistribution. the parenteral route also exposes the whole human body to nucleic acids, which may hamper the delivery efficiency to target tissues or organs (22) . to avoid enzymatic degradation and renal clearance, local drug administration routes have been proposed to directly deliver the drugs to the site of interest. pulmonary administration reveals a strong potentiality as it could transport therapeutic agents to diseased lung tissue in a non-invasive manner. while the degradation by nucleases is negligible comparing to systemic administration, delivery through the airway could be hampered by physiological barriers. the mucociliary clearance action, the surface liquid that covers the airway and macrophages along different parts of the airways, limits the transport of nucleic acids to the site of action (23) . the highly viscous mucus layer in the airways traps and prevents nucleic acids reaching the underlying epithelium and propelled them out with the impact of cillated cells (24) . thus, the development of particles that could efficiently penetrate the mucus barrier, without compromising its protective properties, is a clear challenge for improving pulmonary drug delivery (25) . even if the nucleic acids successfully penetrate through and escape from all the extracellular barriers mentioned previously, they still face the challenge to cross the cell membrane and reach the site of action in the cytoplasm or nucleus. negative charge and large molecular weight make it hard for naked nucleic acids to enter the cell. the endocytosis of nucleic acids could be improved with the help of cationic biomaterials or targeting moieties which interact with the negative proteins or receptors on the cellular surface (26) . one of the most challenging intracellular barriers for nucleic acids delivery is their tendency to remain entrapped in endosomes. intracellular nucleic acids are transported in early endosome vesicles where various nucleases exist and the ph further reduce to 4.5 in the process to late endosomes and lysosomes, and most nucleic acids degraded in the endosome before reaching the site of action (27) . the classic approach has been to use small-molecule endosomolytic agents like chloroquine to disrupt endosomes and release entrapped oligonucleotides from endosomes. two similar types of small molecules have been reported recently with these molecules substantially enhanced the pharmacological activities of oligonucleotides both in cell culture and murine model (28, 29) . although these endosomolytic agents significantly enhanced the delivery efficiency, they currently display a narrow therapeutic window for clinical use. to overcome these biological barriers, strategies like chemical modification, conjugation, vector encapsulation, and selection of administration route have been utilized to improve the delivery of nucleic acids to lungs. since naked nucleic acid is prone to degradation in the biological fluid, chemical modifications at the sugar, backbone, or the individual bases have been introduced to improve its stability and efficacy in biological systems. phosphorothioate(ps)-modified backbone is the most widely used chemistry modification to increase the nuclease resistance. based on ps backbones, nucleic acids designed with additional 2′-sugar modifications such as 2′-o-methyl (2′-ome) or 2′-o-methoxyethyl (2′-moe) can not only further enhance stability and target affinity, but also largely block the activation of toll-like receptors and reduce immune responses (30) . besides ps modification, peptide nucleic acids and phosphoramide morpholino oligomers are nucleotide analogs with strong nuclease resistance as the phosphodiester linkage is completely substituted by a polyamide backbone or a phosphorodiamidate group (31) . however, 2′-sugar modifications of asos might block the recruitment of rnaseh. therefore, bgapmers^was developed, that is asos containing a sequence of ps-modified backbone residues(bgap^) to facilitate rnase h activity and sugar-modified residues(bflanks^) on either side of the gap to increase resistance to degradation and enhance binding to target mrna (6) . beside chemical modification, conjugation strategies are often exploited to enhanced stability and delivery efficiency. representative biomolecules conjugated to nucleic acids conclude targeting ligands and membrane-active molecules, such as lipids, aptamers, peptides, carbohydrates, and polymers (32) . cholesterol attachment to nucleic acids facilitates cellular import and improves intracellular uptake via lipoproteins-mediated pathways (33) . intravenous and intraperitoneal injection of anti-mdr1 cholesterol-sirna conjugate in healthy and tumor-bearing severe combined immune deficiency mice demonstrated efficient accumulation deep in the tissue and the cytoplasm of almost all the liver and tumor cells (34) . sirnas conjugated to n-acetylgalactosamine molecule, a high-affinity ligand for the hepatocyte-specific asialoglycoprotein, are undergoing clinical trials and provided promising results (32) . antibodies or aptamers could be conjugated directly to nucleic acids to realize targeted delivery to specific tissues or cell types. because of the advantages like good reproducibility and low system toxicity, chemical modification and conjugation of nucleic acids have been paid great attention and all the four fda-approved asos are chemically modified and used without a delivery vehicle. while compared to vectorbased systems, poor delivery efficiency and limited orientation are still great concerns of nucleic acid-conjugates for their clinical translation. besides chemical modification, vectors offer important opportunities for nucleic acids to overcome delivery challenges. ideal nucleic acid delivery vectors are expected to condense and protect nucleic acids, facilitate their transport to target cells, and subcellular compartments. viruses, as naturally evolved transfection agents, could enter the cells via endocytosis and release viral genome that could replicate and transcribe into proteins for producing multiple copies. due to their higher transfection efficiency, three major classes of viral vectors, namely, adenovirus (35) , adeno-associated virus (36) , and lentivirus (37) have been extensively used in nucleic acid therapy. however, the limitation of payload, inherent immunogenicity, and the difficulty of large-scale production limited their clinical application. the advantage of non-viral vectors lies in low immunogenicity and toxicity, ease of production, and the large payload over their viral counterparts. widely investigated non-viral delivery vectors include polymers, lipids, polypeptides, and inorganic nanomaterials (such as calcium phosphate and quantum dots). most of the vectors for nucleic acids possess cationic charges that assist in loading nucleic acids through charge interactions. common non-viral delivery systems used in pulmonary diseases are listed in table ii . based on various non-viral vectors, hybrid systems made up by condensed nucleic acid/polycation complexes as the core and lipid bilayer membrane as the shell have been developed. th e use of en dogenou s pho sp holipids, su ch as dipalmitoylphosphatidylcholine, can be considered a valid approach to increase the compatibility of nanoparticles with the lung environment (46) . researchers combined a naturalderived pulmonary surfactant shell with a sirna-loaded dextran nanogel to achieve effective sirna delivery to murine alveolar macrophages, which are difficult to transfect, resulting in a substantial gene knockdown with a relatively low dose (47) (48) (49) . diverse surface modifications and conjugation of targeting agents attached to the vectors could render them desirable properties and enhance the therapeutic efficiency of nucleic acid therapy. surface modification with high molecular weight hyaluronic acid which can mediate active cd44 targeting in tumors and increase circulation time of cationic sirna lipoplexes improved the delivery efficiency and achieved supported reduction of the expression of luciferase mrna in tumor due to the sirna inhibition (52) . systemic administration of nucleic acids faces serious challenges, including rapid excretion, low bioavailability, and systemic toxicity. while local administration allows lower delivery doses and reduced side effects, making it an attractive route (53) . most of the fda-approved nucleic acid-based drugs are locally delivered: fomivirsen is delivered to the eyes by intraocular injection, spinrazais by intrathecal injection, and luxturnais by subretinal injection (7, 8) . for pulmonary disease, the target organ could be reached through systemic administration or pulmonary administration. the latter route could potentially enhance retention time of nucleic acids in the desired site of action, reduce systemic toxic effects, and provide a therapeutic solution to a range of pulmonary disorders (54) . inhalation and intranasal route represent the most common way to deliver nucleic acid into the airways due to the ease of administration and non-invasive characteristic, and are the main administration routes in clinical trials. biodistribution studies of aerosol inhalation of polyester-sirna nanoparticles to mice bearing orthotopic lung tumors showed specific accumulation in the lungs (55) . nucleic acids can be formulated into liquid aerosol generated by an inhaler or nebulizer, or dry powder aerosol for pulmonary delivery. liquid aerosol formulations were almost exclusively adopted in clinical trials involving pulmonary delivery of nucleic acids. among the three major types of inhalation devices consisting of pressurized metered dose inhalers (pmdis), nebulizers, and dry powder inhalers (dpis), pmdis and dpis are the most portable and commonly-used devices (56) . pmdis, in which the therapeutic agents are suspended in the hydrofluoroalkane (hfa) propellant, have been regarded as golden standard delivery system for asthma and chronic obstructive pulmonary disease therapies (56) . a pmdi formulation containing mannitol microparticles which encapsulated sirna polyplex nanoparticles showed good aerodynamic properties for deep lung deposition and significant gene knockdown efficiency in lung a549 cells (57) . dpis are usually thought as a better option to deliver therapeutic nucleic acids than pmdis because their dry particle form enhances the stability of nucleic acids and decreases the risk of microbial contamination (58). chow et al. first formulated naked sirna into inhalable dry powders (at 2% w/w) using spray drying technology with the incorporation of mannitol and l-leucine; the latter acted as powder dispersibility enhancer, and the integrity of sirna was well retained (59) . although systemic administration does not provide the aforementioned advantages of local delivery, for some indications like lung metastasis and pulmonary hypertension, the desired target sites might locate on the interstitium and lung alveolar and endothelial cells rather than the airway epithelium. lung metastases are expected to have an endothelial origin and therefore may be better accessible through blood vessel than through airways (60) . although intravenous injection is not direct delivery to the lung, this route is still able to achieve high levels of transgene expression in the lungs. a multifunctional lipid envelopetype nanodevice developed to target the lung endothelium was found to accumulate in the lung within 5 min after injection. this carrier did not quickly remove to other organs and remain in lungs for 6 h. based on this carrier, systemic administration of anti-cd31 sirna successfully suppressed the metastatic progression (61) . therefore, the administration route should be carefully chosen according to the therapeutic application. since the discovery of nucleic acids, their association with multiple diseases and hence the therapeutic potential have been extensively demonstrated. in the last decades, many investigations have been successfully proved the therapeutic efficiency of nucleic acids on various lung diseases ranging from cancer to pulmonary inflammatory diseases. some of the nucleic acid products have entered the clinical stage; recent clinical trials involving nucleic acid drugs for pulmonary diseases are summarized in table iii . lung cancer is the leading cause of cancer-related deaths in the usa and worldwide (62) . according to the difference in histology, 87% cases of lung cancer are classified as nonsmall cell lung cancer (nsclc) and 13% cases are small cell lung cancer (sclc). in addition to sclc and nsclc, malignant pleural mesothelioma is a rare form of lethal cancer developing in the tissue lining of the lungs (63). current treatments for lung cancers include surgical resection, chemotherapy, radiation therapy, and targeted drug therapy, but these existing therapeutics have limited efficacy, and survival rate of nsclc patients has remained low (63) . therefore, studies on target treatment of lung cancers with selective nucleic acid against oncogenic pathways have drawn intensive interest and some of them have entered clinical practice. custirsen (ogx-011) is a ps-aso inhibitor of clusterin, an anti-apoptotic chaperone protein upregulated in cancer cells in response to chemotherapy and might mediate resistance (64) . preclinical data showed that custirsen significantly decreases clusterin production, increases the sensitivity of lung cancer cells to chemotherapies, and inhibits tumor growth in lung cancer models. in the phase 2 trial of custirsen in patients who were treated with a combination of a gemcitabine/platinum doublet, serum clusterin levels were notably reduced. a larger randomized phase 3 study is needed to demonstrate the potential survival benefit of custirsen in patients with nsclc (65) . imetelstat (grn163l) is a 13 base phosphoramidate oligonucleotide conjugated to a 5-palmitoyl lipid group against the rna component of telomerase, an enzyme responsible for maintaining telomere length and crucial for the indefinite growth of tumor cells. blocking telomerase with imetelstat leads to antineoplastic effects. in a phase 2 study, imetelstat failed to improve progress-free survival rates in advanced nsclc patients with diverse telomere. but there was a trend toward survival improvement for patients with shorter telomeres. further investigations on short telomeres as predictive biomarkers are warranted for clinical development of imetelstat (66) . a lot of sirna-based therapeutics are being assessed in preclinical and clinical trials of pulmonary diseases. aln-rsv01, a sirna therapeutic directing against the mrna encoding the n protein of the respiratory syncytial virus, has completed phase ii clinical trials (67) . sirnas also hold great promise as therapeutic agents for cancer through rnai silencing oncogene expression. sirna for cancer therapies are beginning to be tested in human clinical trials, such as aln-vsp(alnylam pharmaceuticals) for the treatment of liver cancer and calaa-01(calando pharmaceuticals) as tumor inhibitor (68), and they have shown promising pharmacodynamics and tolerability. however, to extend small rna therapy to other major cancer types, including lung cancer, delivery vehicles that target nonliver tissues and specific delivery route are needed. lung cancer is an attractive cancer type for local or systemic small rna delivery treatment. various therapeutic target genes (e.g., survivin, bcl2, hdm2) for lung cancer therapy have already been identified and become targets of sirna therapy (33) . mirnas play a central and complex role in cancer development and are generally classified as tumor suppressor mirnas or tumor-promoting mirnas (oncomirnas). tumor suppressor mirnas in lung cancer include let-7 family, mir-34/449 family, mir-15/16, mir-200 family, and mir-205; oncomirnas in lung cancers include mir-17~92 cluster, mir-21, and mir-221/222 (62) . there are two approaches for mirna modulators to act as cancer therapies: exploiting antisense-based inhibitors of oncogenic mirnas or replacing downregulated tumor suppressor mirnas with synthetic mirna mimics (69) . to date, there are two tumor suppressive mirna mimics of mirna-34 (mrx34; mirna therapeutics inc.) and mirna-16 (targomirs; engeneic ltd.) that have entered clinical trials. mrx34 is a synthetic version of mir-34a encapsulated in liposomes. mir-34a is a tumor suppressor often expressed at reduced levels in a broad range of cancer types, which functions to downregulate the expression of more than 30 different oncogenes across multiple oncogenic pathways (70) . but immune-related serious side effects caused termination of the trial of mrx34. targomirs are double-strand synthetic mir-16-based microrna mimics delivered by engeneic dream vectors which are deprived from nonliving bacterial minicells with a targeting moiety (71, 72) . the mir-16 family has been implicated as tumor suppressor in a range of cancer types, and their primary targets are genes (e.g., bcl2, cdk1, and jun) involved in cancer progression. in vitro and in vivo studies showed that the restoration of expression of mir16 in malignant pleural mesothelioma induced the apoptosis of tumor cells and inhibited tumor growth. long-term survival after a short treatment period was observed in the phase 1 study. however, the safety issue and early signs of activity of targomirs still warrant further clinical trials (71) . asthma is a kind of chronic inflammatory airway disease with high prevalence, which could induce airway hyperresponsiveness, infiltration of inflammatory cells, and airway remodeling. it has been estimated that about 300 million people suffer from this disease on a global scale (73) . the current therapeutics for asthma (including inhaled β2-adrenergic receptor agonists, inhaled corticosteroids, and monoclonal antibody against ige) could effectively control the disease for most patients while there are still about 10% of the patients still out of control under the current treatments. (74) . besides, the current drugs fail to stop or reverse the airway remodeling and some of the drugs followed with concerns of long-term adverse effects, which means there are unmet needs for better drugs (75, 76) . choi et al. developed a novel therapy combining traditional drugs with novel therapeutics. in the regimen, dexamethasone (dexa) was attached to peis to act as a controller ingredient to control the airway inflammation. while sirna against vitamin d binding protein, which is a responsible molecule of allergic asthma, was delivered by dexa-pei at the same time (77) . this multi-target treatment effectively reduced the airway inflammation and secretion of inflammatory factors. asthma is a complex disease associated with the interaction between genetic, epigenetic, and environmental parameters, involved with a plethora of cells and cellular factors (59) . one direction for developing new drugs to treat asthma is to target central pathways to the pathogenesis of the disease, and nucleic acid-mediated therapies silencing the specific effector or the upstream regulator can be a potential approach. ribosomal protein s3 (rps3) was found to bind to the subunit of nf-κb complex and enhance the downstream inflammatory effect. intratracheal delivery of rps3 silencing sirna effectively alleviate airway hyperresponsiveness (ahr) and immune cell infiltration, and decreased serum total ige levels were also observed (78) . sb010, a new class of aso therapeutic sequence-specific targeting and cleaving gata3 mrna, has entered into phase 2a clinical trials. the overexpression of gata3 was found in cells involved in allergic inflammation. the results of the trial showed that inhaled sb010 significantly attenuate both the early-phase and late-phase allergen-induced asthmatic responses (79) . another aso drug tpi-asm8, developed by pharmaxis, contains two types of asos targeting the βc subunit of the il-3, il-5, gm-csf receptors (top004), and human ccr3 (top005) respectively. tpi-asm8 showed the protective effect against ige-mediated early asthmatic response and reduced eosinophilic airway inflammation (80) . chronic obstructive pulmonary disease (copd) is one of the most common chronic respiratory diseases of the airways with an increasing morbidity and mortality; it has been forecasted that copd will be ranked the fourth burden of disease worldwide by year 2030 (81, 82) . copd is characterized by progressive airflow obstruction and airway inflammatory response. current therapeutic strategies are through inhaled long-acting β2-agonists, long-acting muscarinic antagonists, and corticosteroids to dilate bronchus and suppress inflammatory, which is similar to the treatment of asthmas (81, 83) . emerging drugs in copd focus on the cellular and molecular components regulating airway inflammations (82) . phosphodiesterases (pdes) are a group of 11 different isoenzymes (pde1-11) hydrolyzing camp, increased levels of which promote airway smooth muscle relaxation and bronchodilation with anti-inflammatory responses. among the big pde family, pde4 is present in many types of cells relating to copd and thought to be a promising therapeutic target. tpi1100, a dual pde inhibitor comprising two modified asos directing against pde4b/4d and 7a, was designed to reduce the recruitment and activation of inflammatory cells in copd and shown to reduce the neutrophil influx in bronchoalveolar lavage (bal) and inflammation of smoke-exposure or lps-challenge murine models (84) . the phase i clinical trial of tpi1100 was initiated in 2009 but was withdrawn due to drug development suspension. the lungs of copd patients show that the reduction of alveolar elastic fibers and self-healing ability is impaired due to chondroitin sulfate proteoglycan versican inhibiting tropoelastin assembling into fibers. wu et al. employed a small interfering rna (sirna) against versicanin primary pulmonary fibroblasts from copd patients and enhanced the deposition of tropoelastin, which offers a new direction to lung repairment in copd therapy (85) . mirna expression has been proposed as an accessible biomarker of copd disease (86) . multiple mirnas were found altered in copd patients and murine models and could serve as potential biomarkers for the copd detection and prognosis. for example, downregulation of mir-20a, 28-3p, 34c-5p, 100, and upregulation of mir-7 and 21 have intimate association with copd development (30) . micrornas were also found to play an important role in copd muscle dysfunction and mass loss (87) . elevated mir-424-5p expression in patients with muscle wasting might contribute to the inhibition of protein synthesis and loss of muscle mass (88) . it was demonstrated that mir-422a, as a suppressor of tgf-β signaling by reducing the expression of smad4 protein, might attribute to the maintenance of muscle mass (89) . cystic fibrosis (cf) is a genetic disorder giving rise to the functional failure of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which acts as an epithelial chloride channel. the interaction of cftr and epithelial sodium channel (enac) is responsible for the homeostasis of the airways epithelial surface. the deficiency or flaw of cftr leads to hyperactivity of epithelial sodium channel. reduced chloride secretion and increased sodium absorption subsequently result in mucus dehydration, chronic infection, and airway inflammation (5, 90) . using antisense oligonucleotides that correct the basic defect at the mrna level could restore the crucial balance between enac and cftr. a recent study exploited aerosol delivery of asos in cf-like mouse models to inhibit enac activity by triggering rnase h1-dependent degradation of scnn1a mrna, which encodes the enac ɑ subunit. this strategy effectively reduced goblet cell hyperplasia and reversed cf-like symptoms, demonstrating that an enac antisense therapy may provide a potential therapy for cf (91) . the drug qr-010 is a single-stranded antisense rna-based oligonucleotide sequence designed to hybridize the sequences adjacent to the deleted f508 region in the cftr mrna to restore the full function of cftr protein in patients with the f508del mutation. preliminary studies in cell culture and mouse f508del model showed improved chloride efflux after qr-010 treatment (92) . data showed that topical administration of qr-010 to the nasal epithelium improved cftr function by measuring the nasal potential difference of f508del cf subjects (93) . a phase1b study to evaluate the safety, tolerability, and pharmacokinetics of qr-010 is ongoing in cf patients with homozygous f508del cystic fibrosis. acute respiratory distress syndrome (ards) is a type of acute diffuse lung injury with a high mortality rate, which is clinically characterized by pulmonary infiltrates, hypoxemic respiratory failure, and edema, (94) . the mild form of ards is termed as acute lung injury (ali). it is suggested that approximately 2~8 cases of ards per 100,000 population per year. ali is more common, with rates up to 25 per 100,000 per year reported (95) . the common risk factors conclude sepsis, trauma, pneumonia, and toxic inhalation (95) . current ards therapy is to improve impaired gas exchange and lung mechanics by anti-inflammatory drugs, bronchodilators, and mechanical ventilation, which show limitation in controlling the disease progression. as researchers digging into the mechanisms of ards, crucial regulatory agents participating in the initiation and progression of ards, like mirnas and cytokines, have become appealing therapeutic targets. it was found that murine ali models treated with asos against mir-155 gained the enhanced recovery of ali as evidenced by the reduction of bal protein and pro-inflammatory cytokines, and the number of bal cells (96) . nf-κb is a family of dna binding proteins involved in the expression of pro-inflammatory factors and thus the development of ards. depletion of nf-κb by specific sirna targeted nf-κb p65 in lipopolysaccharide (lps)-induced ali rat models effectively reduced levels of the pro-inflammatory cytokines and ameliorated symptoms induced by lps (97) . in vivo administration of the sis1plyase/hmgb1a/r3v6 complex reduced the s1plyase level and weakened the inflammatory response and apoptosis in an lps-induced ali model, indicating that sis1plyase and hmgb1a have a synergistic therapeutic effect for ali (98) . nucleic acid drugs hold great promises as new classes of therapeutic agents for pulmonary diseases, and some candidates have entered into clinical trials (table iii) . the unique structures of lungs enable the delivery of nucleic acid to be implemented by intravenous and pulmonary routes. inhalation and intranasal routes have been found to be ideal for effective delivery. for proper therapeutic use, researchers have modified the chemical structure of nucleic acids to increase their ability against nuclease degradation and reduce immune responses. the transition from bench to bedside of nucleic acid-based therapy also depends heavily on the availability of a safe delivery system that can facilitate trafficking into site of action. the safety issue, especially the immunogenicity of nucleic acids and their vectors, is the biggest stumbling block before nucleic acid drugs for lung diseases become available in the clinic, and further work in this area need to be thoroughly investigated. it is still necessary to identify suitable carriers with the ability to successfully reach the action site in the lung and protect the activity of nucleic acids during the delivery. with the advances and ongoing clinical trials, the future of nucleic acid drugs for pulmonary diseases remains very promising. world health organization. the top 10 causes of death gene therapy for pulmonary diseases targeted delivery of sirna to activated t cells via transferrinpolyethylenimine (tf-pei) as a potential therapy of asthma pharmacology of antisense drugs nucleic acid therapies for cystic fibrosis. nucleic acid ther [internet] rna therapeutics: beyond rna interference and antisense oligonucleotides advances in the delivery of rna therapeutics: from concept to clinical reality fda-approved oligonucleotide therapies in 2017 preclinical and clinical development of sirna-based therapeutics sirna versus mirna as therapeutics for gene silencing development of novel therapeutic agents by inhibition of oncogenic micrornas an overview of micrornas delivering the promise of mirna cancer therapeutics micrornas as cancer therapeutics: a step closer to clinical application therapeutic rna aptamers in clinical trials aptamers: molecules of great potential current progress on aptamertargeted oligonucleotide therapeutics aptamers as targeted therapeutics: current potential and challenges food and drug association on 10/12/2011 for macugen®, nda 021756 selective delivery of therapeutic single strand antimirs by aptamer-based conjugates multifunctional aptamer-mirna conjugates for targeted cancer therapy pulmonary administration of small interfering rna: the route to go? sirnabased therapies for pulmonary diseases carrier interactions with the biological barriers of the lung: advanced in vitro models and challenges for pulmonary drug delivery mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues overcoming cellular barriers for rna therapeutics breaking down the barriers: sirna delivery and endosome escape a novel family of small molecules that enhance the intracellular delivery and pharmacological effectiveness of antisense and splice switching oligonucleotides high-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides oligonucleotide therapy for obstructive and restrictive respiratory diseases nucleic-acid therapeutics: basic principles and recent applications adv drug deliv rev delivery strategies and potential targets for sirna in major cancer types. adv drug deliv rev cholesterol-containing nuclease-resistant sirna accumulates in tumors in a carrierfree mode and silences mdr1 gene a myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors impact of trem-2 gene silencing on inflammatory response of endotoxininduced acute lung injury in mice lipid nanoparticle delivery of a microrna-145 inhibitor improves experimental pulmonary hypertension delivery of therapeutic sirna to the lung endothelium via novel lipoplex formulation dacc development of spray-freeze-dried sirna/pei powder for inhalation with high aerosol performance and strong pulmonary gene silencing activity tpp-dendrimer nanocarriers for sirna delivery to the pulmonary epithelium and their dry powder and metered-dose inhaler formulations anti-inflammatory effect of anti-tnf-α sirna cationic phosphorus dendrimer nanocomplexes administered intranasally in a murine acute lung injury model dendrimer-inspired nanomaterials for the in vivo delivery of sirna to lung vasculature an inhalable β 2 -adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of sirna to lung recent advances in chitosan-based nanoparticulate pulmonary drug delivery hybrid lipid/polymer nanoparticles for pulmonary delivery of sirna: development and fate upon in vitro deposition on the human epithelial airway barrier hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of sirna to murine alveolar macrophages bio-inspired pulmonary surfactantmodified nanogels: a promising sirna delivery system surfactant protein b (sp-b) enhances the cellular sirna delivery of proteolipid coated nanogels for inhalation therapy efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers local delivery of sirna-loaded calcium phosphate nanoparticles abates pulmonary inflammation aerosol delivery of stabilized polyester-sirna nanoparticles to silence gene expression in orthotopic lung tumors inhaled gene delivery: a formulation and delivery approach gababreceptor ligand-directed trimethyl chitosan/tripolyphosphate nanoparticles and their pmdi formulation for survivin sirna pulmonary delivery dry powder formulation of plasmid dna and sirna for inhalation inhaled powder formulation of naked sirna using spray drying technology with l-leucine as dispersion enhancer delivery systems for pulmonary gene therapy t=js&page=reference&d=emed5&news=-n&an=2003346437ovidweb.cgi?t=js&page=reference&d=-emed5&news=n&an=2003346437 lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic sirna delivery to the pulmonary endothelium micrornas and lung cancers: from pathogenesis to clinical implications nanoparticle-based targeted gene therapy for lung cancer ogx-011): a second-generation antisense inhibitor of clusterin in development for the treatment of prostate cancer phase i/ii trial of custirsen (ogx-011), an inhibitor of clusterin, in combination with a gemcitabine and platinum regimen in patients with previously untreated advanced nonsmall cell lung cancer a randomized phase ii study of the telomerase inhibitor imetelstat as maintenance therapy for advanced nonsmall-cell lung cancer a randomized, double-blind, placebocontrolled study of an rnai-based therapy directed against respiratory syncytial virus therapeutic mirna and sirna: moving from bench to clinic as hyaluronic acid-conjugated lipoplexes for targeted delivery of sirna in a murine metastatic lung cancer model delivery systems and local administration routes for therapeutic sirna pulmonary delivery of therapeutic sirna micrornas in non-small cell lung cancer: current status and future therapeutic promises phase i study of mrx34, a liposomal mir-34a mimic, administered twice weekly in patients with advanced solid tumors safety and activity of microrna-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, phase 1, open-label, dose-escalation study versatile vectors for targeted drug or si/shrna cancer therapy epidemiology and economic burden of asthma new targets for drug development in asthma new therapies for asthma: is there any progress? asthma: pathogenesis and novel drugs for treatment a new combination therapy for asthma using dual-function dexamethasone-conjugated polyethylenimine and vitamin d binding protein sirna ribosomal protein s3 gene silencing protects against experimental allergic asthma allergen-induced asthmatic responses modified by a gata3-specific dnazyme antisense therapy against ccr3 and the common beta chain attenuates allergen-induced eosinophilic responses chronic obstructive pulmonary disease emerging drugs for chronic obstructive pulmonary disease emerging therapeutic strategies in copd a multi-targeted antisense oligonucleotide-based therapy directed at phosphodiesterases 4 and 7 for copd deposition of insoluble elastin by pulmonary fibroblasts from patients with copd is increased by treatment with versican sirna targeting microrna function in respiratory diseases: mini-review the role of micrornas in copd muscle dysfunction and mass loss: implications on the clinic mir-424-5p reduces ribosomal rna and protein synthesis in muscle wasting mir-422a suppresses smad4 protein expression and promotes resistance to muscle loss cystic fibrosis inhaled enac antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice strategies in early clinical development for the treatment of basic defects of cystic fibrosis 1 qr-010, an investigational rna therapeutic, improves cftr activity in cystic fibrosis subjects homozygous for the f508del mutation mechanisms and clinical consequences of acute lung injury acute lung injury antisense oligonucleotide treatment enhances the recovery of acute lung injury through il-10-secreting m2-like macrophage-induced expansion of cd4+ regulatory t cells small interfering rna targeting nf-κb attenuates lipopolysaccharideinduced acute lung injury in rats combined delivery of hmgb-1 box a peptide and s1plyase sirna in animal models of acute lung injury key: cord-005228-187d3pxz authors: wang, jian; li, fengqi; tian, zhigang title: role of microbiota on lung homeostasis and diseases date: 2017-10-09 journal: sci china life sci doi: 10.1007/s11427-017-9151-1 sha: doc_id: 5228 cord_uid: 187d3pxz the lungs, as a place of gas exchange, are continuously exposed to environmental stimuli, such as allergens, microbes, and pollutants. the development of the culture-independent technique for microbiological analysis, such as 16s rrna sequencing, has uncovered that the lungs are not sterile and, in fact, colonized by diverse communities of microbiota. the function of intestinal microbiota in modulating mucosal homeostasis and defense has been widely studied; however, the potential function of lung microbiota in regulating immunity and homeostasis has just begun. increasing evidence indicates the relevance of microbiota to lung homeostasis and disease. in this review, we describe the distribution and composition of microbiota in the respiratory system and discuss the potential function of lung microbiota in both health and acute/chronic lung disease. in addition, we also discuss the recent understanding of the gut-lung axis, because several studies have revealed that the immunological interaction among the gut, the lung, and the microbiota was involved in this issue. the microbiota represents the complex collections of microorganisms including bacteria, viruses, parasites, and fungi colonizing the body surfaces exposed to the outside world. the diversity and composition of microbiota are determined by many factors, including host genetics, environmental factors, and host immunity (rooks and garrett, 2016) . in health, the microbiota is beneficial for metabolism, development of the immune system and protection against pathogens, and in turn, the immune system will affect the composition of the microbiota (alegre et al., 2014; honda and littman, 2016) . †contributed equally to this work *corresponding authors (jian wang, email: ustcwj@mail.ustc.edu.cn; zhigang tian, email: tzg@ustc.edu.cn) in recent years, the intestinal microbiota has become the subject of extensive research, and our knowledge about the components of intestinal microbiota and their potential function is rapidly growing. the human intestinal tract harbors over 100 trillion microbial cells that contribute to physiology, metabolism, nutrition and immune functions (glenwright et al., 2017) . dysbiosis of intestinal microbiota has been linked to gastrointestinal diseases, such as inflammatory bowel disease (ibd) and obesity (round and mazmanian, 2009 ). intriguingly, intestinal microbiota is also found to influence other organs, such as brain, liver, and lung, which led to the coining of the concept of the gut-lung axis (bird, 2012; he et al., 2017; young et al., 2016) . the functions of intestinal microbiota have been widely studied and recognized. however, our understanding of the functions of microbiota at other body sites is still in its infancy. clearly, the skin, the upper respiratory tract, and the genitourinary tract also harbor diverse communities of microbiotas as well as site-specific immune networks that are involved in maintaining barrier function and local immune homeostasis (belkaid and tamoutounour, 2016; brubaker and wolfe, 2017; taylor et al., 2016) . the textbook told us that "the normal lungs are free from bacteria". however, the culture-independent techniques of microbial identification indicate the presence of a lung resident microbiota in mammals (bassis et al., 2015; morris et al., 2013) . in humans, lung microbiota have been identified in healthy donors and in patients with chronic pulmonary disease, and the composition of lung microbiota is similar as the microbiota in the upper respiratory tract, but the number is lower, likely resulting from transient entry rather than independent communities with indistinguishable structure (charlson et al., 2011) . the studies for potential functions of lung microbiota in regulating specific immunity and homeostasis have just begun. many questions need to be answered, such as what are the barrier and immune functions of lung microbiota? what is the influence of changed lung microbiota on lung health and disease? what is the possibility that using therapeutic microbiota to cure chronic pulmonary diseases? in this review, we will describe the recent understanding of lung microbiota and its relevance for pulmonary health and disease. the respiratory system is comprised by a series of organs that are responsible for taking in oxygen and expelling carbon dioxide. for the sake of convenience, the respiratory system is divided artificially into two functional parts: the upper respiratory tract and the lower respiratory tract. the upper respiratory tract consists of nostrils, nasal cavities, pharynx, epiglottis, and larynx. the lower respiratory tract includes trachea, bronchi, bronchioles, and lungs. the primary function of the lungs is to transfer oxygen from the air to the blood and to release carbon dioxide from the blood to the air. surprisingly, lefrancais et al. newly found that the lungs are a primary site of terminal platelet production and account for approximately 50% of total platelet production in mice, which means that the lungs are an organ with considerable hematopoietic potential (lefrancais et al., 2017) . the breath function of the lung determines its structure that opens to the exterior environments, which makes the lungs face the persistent challenge from foreign substances (bai et al., 2016; siu et al., 2014) . however, it is surprising that the inflammatory response seldom occurs in the lungs even though considering the large surface areas of the lungs and the huge volumes of air inspired on a daily basis (tian et al., 2016; wissinger et al., 2009) . how could the lungs ignore or tolerate harmless stimuli to prevent potentially fatal immunopathology? an immune response initiates only when a pathogen is sufficiently dangerous that it exceeds a specific threshold of immune response. the threshold of immune response is determined by many factors including environmental factors, genetics, diet, stress, age, and even preceding inflammatory events. thus, the threshold is varied between different individuals and even in different parts of the same individual (shekhar et al., 2017; snelgrove et al., 2011; wissinger et al., 2009) . the condition of the lungs demands the threshold of immune response higher to avoid overmuch and excessive inflammatory response. to achieve this purpose, the lungs execute multiple site-specific immune regulatory strategies to restrain inflammation. for example, compared with macrophages from other parts of body, alveolar macrophages, which are dominated in airway (>95%), express lower levels of mhc class ii and costimulatory molecules and display a suppressive phenotype by secreting interleukin-10 (il-10) and transforming growth factor beta (tgf-β) (thepen et al., 1994) ; airway epithelial cells secrete high levels of tgf-β, il-10 and granulocyte-macrophage colony-stimulating factor (gm-csf) to limit dc responsiveness and alveolar macrophage activation (li and flavell, 2008) ; pattern recognition receptors, especially toll-like receptors, play a critical role in activating the innate immune response and subsequent adaptive immune response. studies found that although tlr4 molecules are constitutively expressed in human alveolar and bronchial epithelial cells, they mainly express intracellularly rather than on the cell surface (guillot et al., 2004) . recently, with the development of the culture-independent technique for microbiological analysis, many studies uncovered that, except the upper respiratory tract, the lower respiratory tract including the lungs is also colonized by diverse communities of microbiota man et al., 2017) . the studies of intestinal microbiota have shown that microbiota could provide essential health benefits to the host by regulating immune homeostasis (chung and kasper, 2010) . given the beneficial function of intestinal microbiota to the intestinal tract, we cannot help but wonder if the microbiota in the lungs has the similar function as intestinal microbiota to promote the development of the lung immune system and maintain lung immune homeostasis? historically, traditional culture-based studies and classic teaching indicated that the normal lungs are free from bacteria, and this notion has persisted in contemporary medicine . however, in the past 20 years, several culture-independent techniques based on the direct amplification and analyses of 16s-rrna have been developed to study environmental microorganisms and the microbiota in human (su et al., 2012) . the application of culture-independent techniques has uncovered that the lungs are not sterile and, in fact, colonized by diverse communities of microbiotas (segal and blaser, 2014) . the microbiotas in human body primarily include six phyla: firmicutes, bacteroidetes, proteobacteria, actinobacteria, fusobacteria, and cyanobacteria (cui et al., 2014; o'dwyer et al., 2016) . the upper respiratory tract is colonized by a huge number of anaerobes and tenfold more aerobic bacteria (bassis et al., 2015; charlson et al., 2011) . the most common bacterial phyla in the lungs are bacteroidetes, firmicutes, and proteobacteria after analyzing the bacteria in bronchoalveolar lavage (bal) from a healthy adult with culture-independent techniques. the dominant genera in the lungs include prevotella, veillonella, pseudomonas, fusobacteria, and streptococcus (beck et al., 2012) . where is the lung microbiota from, and what is the relationship of lung microbiota with microbiota in other part of the body? using culture-independent methods based on rna/dna sequencing or microarrays, the investigation of lung microbiota composition does not require culture of individual microbes. however, sampling the lungs for microbiota sequencing is a technical challenge considering lung microbiota with a low biomass, and sampling lung microbiota by bronchoscopy would introduce a theoretical risk of contamination from both mouth and nose (o'dwyer et al., 2016) . charlson et al. used two bronchoscopes to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush, which could limit the risk of contamination. by analyzing the composition of the microbiota in oral wash, bronchoalveolar lavage fluid (balf), nasal swab, and gastric aspirate samples, they found that the respiratory tract harbors a homogenous microbiota that decreases in biomass from the upper respiratory tract to the lower respiratory tract, and the lung microbiota more closely resemble the oral and nose microbiota (charlson et al., 2011) , implying that the lung microbiota might originate from the upper respiratory tract by breath. in addition, the composition and diversity of the lung microbiota are mainly influenced by three factors: (i) the type and number of microbes immigrating into the lungs, (ii) the elimination race of microbes from the lungs, and (iii) the reproduction rates of the microbe itself in the lungs (bassis et al., 2015; charlson et al., 2011) . the studies on intestinal microbiota have proved that the microbiota benefits the host by improving the mucosal structure and function, shaping both the innate and adaptive immune systems, and providing the protection against harmful pathogen infection (rooks and garrett, 2016; zhang and liang, 2016) . the gut-associated lymphoid tissues including peyer's patches, isolated lymphoid follicles, and mesenteric lymph nodes are underdeveloped in germ-free mice (nakanishi et al., 2015; round and mazmanian, 2009 ). however, there are no reports to show that the lung microbiota had similar effects on the development of pulmonary mucosa-associated lymphoid tissue (gallacher and kotecha, 2016) . for maintaining the homeostasis of intestinal system, pattern recognition receptors (prrs) sense microbial compounds and induce the differentiation of regulatory t cells (treg) and th17 cells (atarashi et al., 2013; lochner et al., 2011; shaw et al., 2012; song et al., 2016) . similarly, prrs in the lungs could also sense microbial compounds from lung microbiota and shift naive t cells to th1 cells but not th2 cells. before birth, the unsound pattern of immune system is dominated by th2 cells. after birth, the polarization of naïve t cells in the lungs will switch from th2 phenotype to th1 phenotype, which will protect against neonatal asthma and allergic disease (lloyd and hessel, 2010 ) ( figure 1a ). germ-free and specific-pathogen-free mice mount immune response development toward th2 type and display susceptibility to house dust mite-induced allergic asthma (remot et al., 2017) . mucosal administration of innocuous whole bacteria or component such as lipopeptide, peptidoglycan, lps or dna can induce th1 immune response and protect mice against asthma and allergy (saeedi et al., 2015) . as discussed above, for maintaining the homeostasis of intestinal system and inhibit excessive inflammatory response, intestinal microbiota promote and maintain the differentiation of treg cells. in the lungs, the bacterial load increased during the first two weeks after birth, and the bacterial phyla shifts from gammaproteobacteria and firmicutes towards bacteroidetes. the changes of the microbiota were associated with the development of helios-negative treg cells in the lungs in a pd-l1-dependent manner. absence of microbiota or blockade of pd-l1 caused exaggerated inflammatory response to allergens through to adulthood (figure 1b) . in our previous studies, we found that the microbiota in upper respiratory tract also provided protection against lethal inflammation in the lungs caused by influenza infection in a tlr2-and alveolar macrophagedependent manner. priming spf mice with tlr2-ligand + staphylococcus aureus, which commonly colonizes the upper respiratory tract in human, promoted the differentiation of m2 macrophages with immunosuppressive function, which then significantly reduced influenza-mediated inflammatory response in the lungs (wang et al., 2013) (figure 1c ). the lung microbiota play roles in lung homeostasis maintenance. a, similar as intestinal microbiota, lung microbiota might also be recognized by pattern recognition receptors (prrs) and then promote the polarization of naïve t cells in the lungs from th2 to th1 after birth to protect against neonatal asthma and allergy. this issue needs to be determined. b, in neonate's lungs, the bacterial load increases, and the bacterial phyla shifts from gammaproteobacteria and firmicutes towards bacteroidetes. the changes of the microbiota are associated with the development of helios-negative treg cells in the lungs that subsequently inhibit the exaggerated inflammatory response to allergens through to adulthood. c, staphylococcus aureus (s. aureus), a common microbiota in upper respiratory tract and lung, promote the differentiation of m2 alveolar macrophages then provide protection against lethal inflammation in the lungs caused by influenza infection. the relevance of microbiota to intestinal health and disease has been widely demonstrated. dysbiosis of intestinal microbiota is involved in the pathogenesis of chronic bowel diseases including chronic inflammatory bowel diseases (ibd), ulcerative colitis (uc), and crohn's disease (cd) (macfarlane et al., 2009; matsuoka and kanai, 2015) . recently, the lung microbiota was also suggested to contribute to lung disease, and the changes of the lung microbiota will affect the risk of disease, the response to drugs, and the clinical outcomes (lynch, 2016) . there are many factors, such as anatomical injuries, pathological effects, physiological changes, and immune system defects, which could disrupt the lung microbiota and result in chronic lung diseases . chronic lung diseases mainly include asthma, chronic obstructive pulmonary disease (copd), cystic fibrosis (cf), and idiopathic pulmonary fibrosis (ipf). asthma is a chronic and multifactorial disease and thought to be caused by a combination of genetic and environmental factors including air pollution and allergens (ghosh et al., 2015) . asthma is more popular in developed countries, which means that the living environment has great impact on the etiology of asthma by altering the diversity and composition of the microbiota in the lungs. ege et al. have reported that children who grow up in traditional farms are exposed to a wider range of environmental microbes and show a low risk of asthma compared with the children in reference group (ege et al., 2011) . many studies on asthma patients have identified that the composition of lung microbiota in patients is different from healthy controls (hilty et al., 2010; marri et al., 2013) . there are more frequent proteobacteria and less frequent bacteroidetes in asthma patients, which might be an accurate predictor of this disease. thus, the composition of the lung microbiota and the interaction between lung microbiota and host are important for the etiology and development of asthma. copd is a type of obstructive lung disease characterized by long-term poor airflow (tan et al., 2016; tan et al., 2014) . many studies on the relationship between lung microbiota and copd have found that the lung microbiotas in patients with mild and moderate copd are similar as the lung microbiota in healthy controls (sze et al., 2014) , which is different from asthma patients who have detectably changed lung microbiota even with mild disease (marri et al., 2013) . the change of lung microbiota only can be found in patients with advanced copd . in patients with advanced copd, there are more frequent proteobacteria or firmicutes and less frequent bacteroidetes (garcia-nuñez et al., 2014; wu et al., 2014) , which is similar to the shift of the microbiota in asthma patients. however, asthma and copd are different diseases in the lungs, implying that, besides the change of the lung microbiota, other factors also contribute to the disease and play more important roles than the microbiota. cf is an inherited disorder that mainly affects the lungs. the syndrome of cf in the lungs displays a progressive development of bronchiectasis and obstructive lung disease (stenbit and flume, 2011) . the relationship between lung microbiota and the pathogenesis of cf is still controversial. specific respiratory pathogens, such as staphylococcus aureus and pseudomonas aeruginosa, can be detected increased in sputum from almost all young cf patients during both clinical stability and exacerbations (ramsey, 1996) , so the exacerbations of cf has long been considered as a result of bacterial infection. however, some studies found that antibiotic therapy has no significant influence on the process of the disease (hurley et al., 2012; smith et al., 2003) . therefore, cf exacerbation is not associated with increased bacterial density or decreased diversity, and the relationship between lung microbiota and cf pathogenesis might be more complex than we thought before. ipf is a chronic fatal remodeling lung disease characterized by a progressive decline in lung function. there are many evidences supporting that the etiology and progression of ipf are related to the change of the lung microbiota and bacterial infection (folcik et al., 2014; molyneaux et al., 2014 ). an increased bacterial burden could be detected in the balf of ipf patients compared with controls, and there were a relatively increased abundance of streptococcus, pneumococcus, or staphylococcus taxonomic groups found in human and mouse model (collard et al., 2007; han et al., 2014) . moreover, host defense and innate immunity also showed to play a role in ipf disease progression. defective tlr3 signaling leads to aberrant inflammation and promotes ipf disease progression (o'dwyer et al., 2013) . thus, it is clear that the lung microbiota is involved in the etiology or/and progression of chronic lung diseases. however, there are still several questions that remain to be answered. for example, what are the reasons that cause the change of the lung microbiota in chronic lung disease? is the change of the lung microbiota a cause or consequence of chronic lung disease? meanwhile, with the in-depth study of the relationship between lung microbiota and respiratory disease, manipulating lung microbiota might become a rational and promising way for therapies. excitingly, exposure to bacteria and their products by airway route has already been demonstrated to be useful to control allergic airway inflammation in adult mice. nembrini et al. reported that pulmonary exposure to escherichia coli results in a suppression of allergic airway inflammation through the recruitment of γδ t cells in a tlr4-dependent manner (nembrini et al., 2011) . hagner et al. found that intranasal treatment of farm-derived staphylococcus sciuri w620 could protect mice against both ovalbumin (ova)-and house dust mite extract (hdm)-induced airway hyperresponsiveness by inhibiting il-12 expression in mature dcs in a tlr2-and nod2-dependent manner (hagner et al., 2013) . these two examples show us a possible way to take advantage of lung microbiota to prevent or change disease progression in human, and there are more bacteria that could be selected as the target with increasing knowledge of lung microbiota. microbiota plays a critical role in maintaining the homeostasis of the colonized organs or tissues. however, more and more studies found that the local microbiota changes could influence the immunity at the distal tissues, especially the interaction between the intestinal tract and respiratory tract (budden et al., 2017; schleiermacher and hoffmann, 2007; trompette et al., 2014) . chronic lung disorders, such as asthma, copd, and cf, all display a component of intestinal disease manifestation and respiratory viral infections are usually accompanied by intestinal symptoms (keely et al., 2012) , which implies the existence of the immunological link between the guts and the lungs, referred to as gut-lung axis. increasing evidence shows that the complex interactions between the intestinal microbiota and host immune system are important not only for the intestinal local but also for other organs or tissues. dysbiosis of the intestinal microbiota is linkage to the pathogenesis and progression of chronic lung diseases, such as asthma. disruption of the intestinal microbiota in early life could increase the risk of the development of asthma, and restoring the changed intestinal microbiota via probiotic treatment could attenuate the risk (arrieta et al., 2015; kozakova et al., 2016; liu and marc rhoads, 2016) . in addition, the intestinal microbiota is also broadly protective against respiratory infection (tamburini and clemente, 2017) . depletion or absence of intestinal microbiota would lead to impaired immune responses following viral or bacterial respiratory infection. ichinohe et al. found that intestinal microbiota played a critical role in the generation of virus-specific cd4 + and cd8 + t cells and antibody responses after respiratory influenza virus infection (ichinohe et al., 2011) . chen et al. found that depleting intestinal microbiota by antibiotic treatment would increase the bacterial counts in blood and the lungs and the mortality of mice following respiratory escherichia coli infection (chen et al., 2011) (figure 2a) . wang, j., et al. sci china life sci december (2017) vol. 60 no. 12 evolving literature on microbiota suggested that the gut-lung axis is bi-directional, resembling a loop that can be stimulated from two sites. although little is known about the influence of lung microbiota on intestinal microbiota and intestinal immunity, some studies demonstrated that lung inflammation could affect intestinal microbiota and cause disease. in our previous study, we found that respiratory influenza infection caused immune injuries in both respiratory and intestinal mucosal tissues. further studies showed that there was no influenza virus in the intestinal tract after viral infection by an intranasal route, which ruled out the possibility that influenza virus infected and caused immune injury at intestinal local directly. finally, we found that ccl25/ccr9 axis mediated the recruitment of lung-derived ccr9 + cd4 + t cells into the intestinal tract, which then changed the composition of intestinal microbiota and caused intestinal immune injury (wang et al., 2014) . another study also found that a locally induced pulmonary allergic response could also affect the composition of the intestinal microbiota, and vice versa, the changed intestinal microbiota support inflammation in the lungs (vital et al., 2015) ( figure 2b ). more than 30 years ago, mcdermott and bienenstock found that donor-derived mesenteric lymph node b cells distributed in most mucosal tissues of recipient mice after adoptive transfer, while peripheral lymph node b cells only returned to their original site, which led to the coining of the concept of "the common mucosal immune system" (mcdermott and bienenstock, 1979) . it suggested that the mucosal immune system is a system-wide organ and immune cells interplay among different mucosal tissues (gill et al., 2010; wang and tian, 2015) . although the concept was presented over 30 years ago, some questions remain to be answered, such as how do the different mucosal sites communicate with each other? what kinds of immune cells and molecules are involved in this process? we think that the gut-lung axis is a part of the common mucosal immune system, and the studies and findings of the gut-lung axis will help us to know more about the common mucosal immune system. with the advent and development of culture-independent techniques, the microbiota was found in the lungs, which is contrary to the old notion that the lungs are a sterile organ. at the same time, many new questions arise. the studies about intestinal microbiota have shown that it plays a critical role in the development of local and even systemic immune system, but there is no evidence to support that the lung microbiota has a similar function. the dysbiosis of lung microbiota is linked to the lung chronic disease, but it is not clear whether dysbiosis is a cause or a consequence of immune dysregulation and disease initiation or progression. in the future, we may constitute a healthy lung microbiota community to further our understanding of the complexity of lung microbiota as well as their genetic and metabolic potential and even manipulate the lung microbiota as a potential therapeutic way to treat chronic lung disease. the author(s) declare that they have no conflict of interest. risk of childhood asthma treg induction by a rationally selected mixture of clostridia strains from the human microbiota respective il-17a production by γδ t and th17 cells and its implication in host defense against chlamydial lung infection analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals the microbiome of the lung the influence of skin microorganisms on cutaneous immunity gut microbiota influences liver disease the female urinary microbiota, urinary health and common urinary disorders emerging pathogenic links between microbiota and the gut-lung axis topographical continuity of bacterial populations in the healthy human respiratory tract commensal microflora contribute to host defense against escherichia coli pneumonia through toll-like receptors microbiota-stimulated immune mechanisms to maintain gut homeostasis acute exacerbations of idiopathic pulmonary fibrosis the microbiome and the lung the microbiome and the respiratory tract exposure to environmental microorganisms and childhood asthma idiopathic pulmonary fibrosis is strongly associated with productive infection by herpesvirus saimiri respiratory microbiome of newborn infants severity-related changes of bronchial microbiome in chronic obstructive pulmonary disease b lymphocytes regulate airway granulocytic inflammation and cytokine production in a murine model of fungal allergic asthma the future of mucosal immunology: studying an integrated system-wide organ structural basis for nutrient acquisition by dominant members of the human gut microbiota lung microbiota promotes tolerance to allergens in neonates via pd-l1 response of human pulmonary epithelial cells to lipopolysaccharide involves toll-like receptor 4 (tlr4)-dependent signaling pathways farm-derived gram-positive bacterium staphylococcus sciuri w620 prevents asthma phenotype in hdm-and ova-exposed mice lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the comet study gutlung axis: the microbial contributions and clinical implications disordered microbial communities in asthmatic airways the microbiota in adaptive immune homeostasis and disease results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis microbiota regulates immune defense against respiratory tract influenza a virus infection pulmonary-intestinal cross-talk in mucosal inflammatory disease colonization of germ-free mice with a mixture of three lactobacillus strains enhances the integrity of gut mucosa and ameliorates allergic sensitization the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors tgf-β: a master of all t cell trades lock"ing up allergic responses with a polish probiotic functions of t cells in asthma: more than just th2 cells restricted microbiota and absence of cognate tcr antigen leads to an unbalanced generation of th17 cells the lung microbiome and airway disease the gut microbiota in inflammatory bowel disease the microbiota of the respiratory tract: gatekeeper to respiratory health asthma-associated differences in microbial composition of induced sputum host-microorganism interactions in lung diseases the gut microbiota and inflammatory bowel disease evidence for a common mucosal immunologic system. i. migration of b immunoblasts into intestinal, respiratory, and genital tissues the role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis comparison of the respiratory microbiome in healthy nonsmokers and smokers commensal gram-positive bacteria initiates colitis by inducing monocyte/macrophage mobilization bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism the toll-like receptor 3 l412f polymorphism and disease progression in idiopathic pulmonary fibrosis the lung microbiome, immunity, and the pathogenesis of chronic lung disease management of pulmonary disease in patients with cystic fibrosis bacteria isolated from lung modulate asthma susceptibility in mice gut microbiota, metabolites and host immunity the gut microbiota shapes intestinal immune responses during health and disease the transient but not resident (tbnr) microbiome: a yin yang model for lung immune system pulmonary abnormalities in inflammatory bowel disease a brave new world: the lung microbiota in an era of change microbiotainduced il-1β, but not il-6, is critical for the development of steady-state th17 cells in the intestine cd103+ lung dendritic cells (ldcs) induce stronger th1/th17 immunity to a bacterial lung infection than cd11b hi ldcs suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain susceptibility testing of pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration airway immune homeostasis and implications for influenza-induced inflammation the roles and functional mechanisms of interleukin-17 family cytokines in mucosal immunity pulmonary exacerbations in cystic fibrosis culture-independent methods for studying environmental microorganisms: methods, application, and perspective a comparison between droplet digital and quantitative pcr in the analysis of bacterial 16s load in lung tissue samples from control and copd gold 2 gut microbiota: neonatal gut microbiota induces lung immunity against pneumonia levels of cmv-reactive antibodies correlate with the induction of cd28null t cells and systemic inflammation in chronic obstructive pulmonary disease (copd) impaired ctla-4 responses in copd are associated with systemic inflammation host-microbiome interactions in acute and chronic respiratory infections the role of alveolar macrophages in regulation of lung inflammation regional immunity in tissue homeostasis and diseases gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis alterations of the murine gut microbiome with age and allergic airway disease bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated th17 cell-dependent inflammation how lung infection leads to gut injury immune homeostasis in the respiratory tract and its impact on heterologous infection analysis of the bacterial community in chronic obstructive pulmonary disease sputum samples by denaturing gradient gel electrophoresis and real-time pcr the gut-liver-lung axis. modulation of the innate immune response and its possible role in chronic obstructive pulmonary disease innate recognition of microbial-derived signals in immunity and inflammation acknowledgements this work was supported by the national natural science foundation of china (31400783, 91542000). key: cord-004405-l5rif2lu authors: bleyer, martina; kunze, marius; gruber-dujardin, eva; mätz-rensing, kerstin title: spontaneous lung pathology in a captive common marmoset colony (callithrix jacchus) date: 2017-03-01 journal: primate biol doi: 10.5194/pb-4-17-2017 sha: doc_id: 4405 cord_uid: l5rif2lu data on spontaneous pathology are substantially scarce for common marmosets, compared to other laboratory animals, but is essential for the interpretation of histological findings in the context of toxicological and experimental studies. especially if common marmosets are used as experimental animals in respiratory research, detailed knowledge on the spectrum, occurrence, and incidence of spontaneous histopathological pulmonary lesions in this non-human primate species is required. in this study, lung tissue of 638 common marmosets from the marmoset colony of the german primate center was examined histologically. the analysis revealed a high incidence of predominantly mild and multifocal interstitial pneumonia (32.99 %) of unknown etiology in most cases. only few marmosets exhibited lobar pneumonia (1.41 %) and bronchopneumonia (0.94), which were mainly caused by bacterial pathogens such as bordetella bronchiseptica and klebsiella pneumoniae. lung immaturity and atelectasis were common histological findings in newborn marmosets. typical background lesions included anthracosis (8.15 %), hemosiderosis (1.72 %), extramedullary hematopoiesis (11.6 %), mineralization (10.97 %), and inflammatory cell foci (10.34 %). in addition, three cases of pulmonary arteriopathy (0.47 %) and 1 case of foreign-body granuloma (0.16 %) were detected in the marmoset study cohort. the high prevalence of circulatory disturbances (congestion, edema, hemorrhage) and changes in air content (secondary atelectasis, alveolar emphysema) could partly be explained by euthanasia-related artifacts or agonal changes. the present study provides a comprehensive overview of the range and incidence of spontaneous pulmonary histopathology in common marmosets, serving as valuable reference data for the interpretation of lung lesions in toxicological and experimental marmoset studies. recently, the common marmoset (callithrix jacchus) has increasingly attracted attention as a translational animal model in the field of respiratory research because of its small size, good availability, and consistent characteristics of primate lung architecture (greenough et al., 2005; lever et al., 2008; seehase et al., 2012; curths et al., 2013 curths et al., , 2014 . as a non-rodent species, the common marmoset is used in preclinical testing of drugs acting on the respiratory system and is a suitable animal model for various human pulmonary diseases, including asthma and chronic obstructive pulmonary disease (copd; seehase et al., 2012; curths et al., 2013 curths et al., , 2014 . histopathological examination of lung tissue from toxicological and experimental studies requires detailed knowledge of the spectrum of spontaneously occur-ring lung pathology of this laboratory animal species to identify possible drug-induced or disease-associated pulmonary lesions and to distinguish these from species-specific background lesions. compared to other laboratory animals, spontaneous pathology of common marmosets is less well defined. background lesions of the common marmoset in toxicological studies have previously been documented by kaspareit et al. (2006) , who also referred to pulmonary findings. david et al. (2009) also performed a retrospective study on the spontaneous pathology of common marmosets including the morphological diagnoses of pneumonia, atelectasis, pulmonary extramedullary hematopoiesis, and lymphosarcoma in the lungs. however, a detailed survey about the range and incidence of lung pathology in common marmosets does not exist in the literature so far. in order to provide reference data on spontaneous histopathological pulmonary findings in conventionally kept common marmosets, we performed a retrospective study on necropsy material of 638 common marmosets from the indoor-housed marmoset colony of the german primate center in göttingen. in this retrospective study, archived lung tissue of 638 common marmosets (317 males and 321 females) originating from the marmoset colony of the german primate center in göttingen, germany, was used. archived material included formalin-fixed or paraffin-embedded lung tissue, or histological sections of the lung, which were collected between 1997 and 2011. animals of this study were housed in small family groups in an indoor facility with a room temperature of 25 • c and relative humidity of 50-60 % on a 12 h light-dark cycle with 30 min "dawn" and "dusk" periods. care and housing conditions of the animals complied with the regulations of the european parliament and the council directive on the protection of animals used for scientific purposes (2010/63/eu), the national institutes of health guide for the care and use of laboratory animals (2010), and the applicable german animal protection law. according to necropsy records, animals underwent necropsy after spontaneous death, following euthanasia due to illness with poor prognosis, or after scheduled terminal kill in the context of experimental studies. from the latter group of marmosets only control animals or animals without treatment-related findings were considered for re-evaluation of lung histology. photographic documentation of respective macroscopic pulmonary findings and results from bacteriological culture of the lungs were available for some animals. lung tissue samples were fixed in 4 or 10 % phosphate-buffered formaldehyde, paraffin-embedded, sectioned at 3 µm, and stained with hematoxylin and eosin (he). if required for diagnostic purposes, additional stains were prepared and analyzed, including periodic acid schiff (pas) reaction, prussian blue stain, von kossa stain, masson's trichrome stain, congo red stain, grocott's methenamine silver stain, and giemsa stain. the lungs of all 638 marmosets were re-examined histologically, and findings were reported on a spreadsheet (microsoft office excel 2010) with searchable columns for morphological diagnosis/histological finding, sex, age, cause of death (if known), chronicity of lesion (in case of inflammation), and severity grade. animals were assigned to three age groups: newborn (0 to 7 days old, including supposedly timely delivered but stillborn marmosets), juvenile (7 days to 30 months old), and adult (older than 30 months). histological findings were grouped into inflammatory conditions, neoplasia, changes in air content, circulatory disturbances, pigment deposition, and miscellaneous findings. total incidences of findings were indicated in absolute numbers and percentage. in addition, absolute numbers of findings were calculated for males and females as well as different age groups. in the present study, 39 of 638 common marmosets (6.11 %) did not show any histological changes of the lungs. all spontaneous pulmonary lesions of the other animals are documented in table 1 . the most commonly observed inflammatory lung condition was constituted by interstitial pneumonia, which was observed in 206 marmosets (32.29 %). the majority of cases with interstitial pneumonia revealed a subacute course of disease with predominance of plasma cells in the inflammatory cell infiltrate. regarding severity and distributional pattern, mild multifocal or multifocal to coalescing forms predominated ( fig. 1) , while severe and diffuse cases were very rare. in two marmosets (0.31 %), interstitial pneumonia was associated with acute to subacute alveolitis. there was no histological evidence of infectious agents in all cases of interstitial pneumonia, except for one male juvenile marmoset, which showed characteristic disseminated grocott-positive blastospores and pseudohyphae in inflamed lung regions, indicating a mycotic etiology consistent with candidiasis. bacterial culture, if available, was positive in the minority of marmosets affected by interstitial pneumonia. bacterial isolates included escherichia coli, streptococcus sp., erysipelothrix rhusiopathiae, klebsiella pneumoniae, and pseudomonas aeruginosa. other forms of pneumonia were rare and included lobar pneumonia in nine marmosets (1.41 %), suppurative bronchopneumonia in six marmosets (0.94 %), and bronchointerstitial pneumonia in two marmosets (0.31 %). lobar pneumonias were further subdivided into purulent or fibrinopurulent forms according to the inflammatory exudate (fig. 2) . in two cases (0.31 %), there was fibrinopurulent pleuropneumonia. the majority of lobar pneumonias (eight of nine cases) and all suppurative bronchopneumonias were moderate to severe and acute to subacute, representing the main cause of disease or death in most cases. bacterial culture of marmoset lungs affected by purulent bronchopneumonia yielded isolates of streptococcus sp. and/or bordetella bronchiseptica in all cases. bordetella bronchiseptica was also isolated from the lungs of a juvenile female marmoset with fibrinopurulent pleuropneumonia. in three cases of lobar pneumonia, streptococcus sp., enterococcus sp., and/or klebsiella pneumoniae ssp. ozaenae could be isolated, while five cases of lobar pneumonia were negative for bacterial culture. neoplastic conditions occurred in five marmoset lungs, including lymphoma in four adult animals (0.63 %) and fibrosarcoma in one juvenile animal (0.16 %). regarding the age of animals with tumors, lymphomas affected three rather young adults (3.5 years (two cases) and 2.75 years old) and one older animal (7 years old). the fibrosarcoma occurred in a 1-year-old marmoset. in all cases, pulmonary tumors were regarded as secondary, resulting from metastatic neoplastic disease with presumptive primary tumors in the nodal or extranodal lymphatic system (lymphomas) and in the mammary gland (fibrosarcoma). immunohistochemical examinations confirmed b cell origin of at least three lymphomas (fig. 3) . one lymphoma has not been further characterized. changes in air content were commonly observed, either in otherwise healthy lungs or as an additional finding to other histological diagnoses. there was evidence of atelectasis in 215 marmosets (33.7 %), of which the majority represented subtotal secondary (acquired) atelectasis (178 of 215 cases). primary (fetal) atelectasis occurred in 37 newborn marmosets (5.8 %), of which 36 animals showed total fetal atelectasis that was regularly associated with lung immaturity. one juvenile marmoset revealed partial fetal atelectasis, also accompanied by discrete signs of lung immaturity. alveolar emphysema of variable severity and extent was present in 154 animals (24.14 %), whereas interstitial emphysema could not be observed in this study. circulatory disturbances in marmoset lungs included congestion, edema, hemorrhage, and hyaline membrane formation. acute pulmonary congestion was a common finding (41.54 %), often regarded as agonal or euthanasia-induced due to the use of barbiturates. the same might be true for pulmonary edema, which was present in 131 marmosets (20.35 %) and occurred both as an additional finding and solitarily. the majority of pulmonary edema was represented by alveolar forms (120 of 131 cases), while involvement of the interstitium was only seen in 11 cases. extravasation of erythrocytes (hemorrhage) into the interstitium or alveolar space could be seen in 39 marmosets (6.11 %) and, to some extent, was presumably also caused by euthanasia or agony. hyaline membranes were observed in the lungs of three newborn marmosets (0.47 %) with concurrent atelectasis and evidence of lung immaturity. mild to moderate deposition of coal dust in the pulmonary interstitium (anthracosis) was present in 52 mostly adult marmosets (8.15 %). in general, anthracosis was not associated with any tissue reaction (fig. 4) . hemosiderin-laden macrophages (hemosiderosis) were observed in the lungs of two juvenile and nine adult marmosets (1.72 %), which commonly showed co-existing hemosiderosis in other organs, especially in liver, spleen, and kidneys. there was no evidence of chronic heart failure in cases of pulmonary hemosiderosis. within the group of miscellaneous lung findings, extramedullary hematopoiesis, mainly characterized by megakaryocytes within alveolar septa, was observed in 74 animals (11.6 %). this predominantly affected adult marmosets, which regularly showed concurrent foci of extramedullary hematopoiesis in multiple organs (liver, spleen, etc.). the second-most-common miscellaneous finding was multifocal interstitial or subpleural mineralization, being present in 70 juvenile and adult marmosets (10.97 %), followed by disseminated inflammatory cell foci observed in 66 marmosets (10.34 %). they mainly consisted of plasma cells, macrophages, and lymphocytes and were primarily located within alveolar septa, perivascular or peribronchial/peribronchiolar (fig. 5) . cuboidal alveolar epithelium and thick fibrotic interalveolar tissue of variable extent, indicative of pulmonary immaturity, were present in 62 newborn/stillborn marmosets and in 1 juvenile animal (9.72 %). in many cases, premature lungs also showed total atelectasis and represented a common cause of death in newborn marmosets. a few animals with immature lungs also revealed accumulations of intra-alveolar amniotic fluid (fig. 6) . focal or multifocal alveolar histiocytosis, found in 26 marmosets (4.08 %), was generally associated with inflammatory lung lesions. multifocal interstitial and subpleural fibrosis was detected in juvenile and adult marmosets (3.61 %), occasionally accompanied by focal mineralization (fig. 7) . three adult marmosets (0.47 %) revealed pulmonary arteriopathy, characterized by hyperplasia and mineralization of the tunica media as well as edema and hypertrophy of the tunica intima. a focal foreign-body granuloma due to an aspirated hair fragment (fig. 8) was observed in the lung of one adult female marmoset (0.16 %). with an incidence of 32.99 %, the most common inflammatory condition in the lungs was interstitial pneumonia. however, the majority of cases were mild and were not associated with severe clinical disease or death. except for a few case reports, published data on the incidence of interstitial pneumonias in common marmoset colonies are lacking. david et al. (2009) observed pneumonias in 9 of 597 marmosets but did not provide further classification of this diagnosis. the etiopathogenesis of interstitial pneumonia generally includes aerogenous damage to the alveolar epithelium (e.g., by toxic gases or due to infection with pneumotropic viruses) or hematogenous injury to the alveolar capillary endothelium or basement membrane (e.g., in septicemia, by endotoxins from the alimentary tract, from free radicals released in acute respiratory distress syndrome, from microembolism or disseminated intravascular coagulation, in the context of hypersensitivity reactions, or due to infection with endotheliotropic viruses) (lópez, 2007) . in the common marmosets affected by interstitial pneumonia, testing for respiratory viruses was not performed routinely. therefore, a viral etiology accounting for at least a part of interstitial pneumonias cannot finally be excluded. evidence of bacterial agents was present in only a few cases, including streptococcus sp., escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and erysipelothrix rhusiopathiae. these isolates are of variable pathogenicity regarding respiratory infections but are usually not associated with interstitial pneumonia (simmons and gibson, 2012) . in some marmosets, bacterial isolates were also obtained from other organs (gall bladder, intestine) with evidence of bacteria-induced pathologic lesions suggesting septicemic distribution of the respective bacteria. mycotic interstitial pneumonia was observed in one marmoset with systemic candidiasis, which represents the most frequently occurring mycotic disease in immunocompromised non-human primates (simmons and gibson, 2012) . however, the etiology of the majority of interstitial pneumonias remains unclear. environmental influences linked to the housing conditions of the marmoset colony -e.g., room temperature, humidity, air exchange rate and air filter specifications, and aerosol formation -may represent predisposing or triggering factors for the development of interstitial lung inflammation in captive marmosets, although evidence for this assumption is lacking. in addition, fine dust pollution has to be considered as an initiating factor for interstitial lung disease, especially with regard to the atmospheric composition in the natural habitat of common marmosets, which surely differs from the artificial conditions in indoor marmoset husbandry. the presence of anthracosis in 52 marmosets (8.15 %) points to at least partial exposure of indoor-kept marmosets to the outside air. however, the association between anthracosis and interstitial pneumonia remains questionable as many animals with intrapulmonary coal dust pigment did not exhibit obvious interstitial inflammation or fibrosis, which is consistent with observations in cynomolgus monkeys (sato et al., 2012) . finally, influences like stress or immunological status of the animal, both conditions that are hard to grasp, may have an impact on the individual's disposition to develop interstitial lung inflammation (lópez, 2007) . lobar pneumonias and bronchopneumonias occurred in a small number of marmosets (1.41 and 0.94 %, respectively), were grossly evident in most cases, and were generally caused by bacterial infection resulting in severe disease and death. bordetella bronchiseptica was isolated from the lungs in all cases of bronchopneumonia and 1 case of lobar pneumonia. outbreaks of bordetellosis with characteristic pneumonic lesions have previously been described in marmoset colonies and were associated with high morbid-ity and mortality (baskerville et al., 1983; chalmers et al., 1983) . pathogenic klebsiella pneumoniae strains are known to cause purulent/fibrinopurulent pneumonias in new world monkeys (berendt et al., 1978; simmons and gibson, 2012) and could be isolated in most cases of lobar pneumonia in the common marmosets. primary pulmonary neoplasia is rare in non-human primates and is limited to a few case reports primarily referring to malignant epithelial tumors observed in different macaque species (lowenstine and osborn, 2012) . in a previous study on the incidence of pulmonary tumors in the marmoset colony of the german primate center, brack et al. (1996) reported three cases of primary lung tumors (one small-cell carcinoma, one bronchial adenoma, one squamous cell carcinoma) in 409 adult callitrichids that were examined between 1978 and 1994. however, in the present study, for which data were obtained from the time period between 1997 and 2011, pulmonary neoplasms in the marmoset colony (0.79 %) exclusively represented secondary tumors in the context of metastatic disease with primary tumors located in other organ systems (lymphatic system, mammary gland). primary lung malignancies or benign lung tumors were not observed in the present study. acquired atelectasis with patchy distribution was commonly diagnosed in the examined marmoset lungs (27.9 %). however, in most cases, obvious causative factors, e.g. compression or obstruction, could not be observed. therefore, a large portion of atelectasis probably resulted from artificial lung collapse during necropsy followed by immersion fixation with formaldehyde. congenital atelectasis mainly affected stillborn marmosets or newborn animals that died within a couple of days after birth (5.8 %). the main causes of congenital atelectasis include obstruction of airways due to aspiration of amniotic fluid and alterations in the quantity and quality of pulmonary surfactant (lópez, 2007) . in the common marmosets, atelectatic lungs regularly showed concurrent lung immaturity, suggesting surfactant deficiency. in a few cases, lung immaturity was associated with hyaline membrane formation, indicating acute respiratory distress syndrome as the likely cause of death. amniotic fluid aspiration was evident in a couple of newborn marmosets and might have caused atelectasis due to airway obstruction. alveolar emphysema is a common secondary finding in lungs affected by bronchopneumonia or lobar pneumonia and can be attributed to a valve effect elicited by exudate plugs in the intrapulmonary airways (lópez, 2007) . however, as the incidence of alveolar emphysema clearly exceeds the number of alveolar pneumonias in the marmoset study cohort, alveolar emphysema in most marmosets likely represents an agonal change or a euthanasia artifact. the same probably applies to most marmoset lungs exhibiting circulatory disturbances, including acute congestion, edema, and hemorrhage, which are frequently seen in animals euthanized with barbiturates (lópez, 2007) . hemosiderosis is a common finding in many new world monkey species, including common marmosets; mainly manifests in the liver; and is believed to be caused by highiron diets (miller et al., 1997; rensing and oerke, 2005) . as there were no signs of chronic heart failure in cases of pulmonary hemosiderosis but there was evidence of concurrent hemosiderosis in other organs, the majority of pulmonary hemosiderin deposition in the common marmosets of this study is regarded as the result of systemic iron overload due to excessive intestinal iron uptake. however, the presence of siderophages may also, to some extent, represent residua of localized pulmonary hemorrhages of undefined origin (sato et al., 2012) . the occurrence of extramedullary hematopoiesis in the lungs of adult common marmosets has previously been described by kaspareit et al. (2006) and is believed to be an incidental finding without clinical relevance (zühlke and weinbauer, 2003; chamanza et al., 2006) . subpleural mineralization macroscopically presented as subpleural plaques, which are distinctly visible at necropsy. both interstitial and subpleural mineralization was found in 70 marmosets (10.97 %) and was largely regarded to be of metastatic origin as there was co-existing mineralization of other tissues with accentuation on basal lamina structures. taking into consideration that the diet for young marmosets in the german primate center is supplemented with vitamin d to prevent rachitic lesions, soft tissue mineralization in the common marmosets was likely due to hypervitaminosis d, which is a well-known nutritional disease entity in new world monkeys (hunt, 1969; kaspareit et al., 2006; mcinnes, 2012; saravanan et al., 2015) . circumscribed areas of interstitial and subpleural fibrosis occurred in 23 juvenile and adult marmosets (3.61 %) and presumably represent residua from earlier tissue damage. mononuclear inflammatory cell foci, which were present in 66 marmoset lungs (10.34 %), are a regularly observed background finding in common marmosets and may affect different organ systems (chamanza et al., 2006; kaspareit et al., 2006) . in the lungs, it is important to distinguish between such inflammatory cell foci and interstitial pneumonia, which should be feasible regarding the extent, distribution, and severity of infiltrating inflammatory cells. the histological features of pulmonary arteriopathy observed in 3 adult marmosets (0.47 %) were indicative of pulmonary hypertension, and concurrent hypertrophic cardiomyopathy was present in at least one of the affected animals. however, the exact pathogenetic mechanisms leading to pulmonary arteriopathy remained obscure in the common marmosets. occasional occurrence of foreign-body granulomas in marmoset lungs has previously been reported by kaspareit et al. (2006) . they are usually caused by aspiration of foreign material (hair, food particles, plant fragments) (sato et al., 2012) . when small and focal as in the present case, they can be regarded as incidental microscopic findings without clinical relevance. however, aspiration of larger or sharp-edged foreign bodies may result in substantial tissue reaction and an outbreak of bordetella bronchiseptica pneumonia in a colony of common marmosets (callithrix jacchus) nonhuman primate model for the study of respiratory klebsiella pneumoniae infection tumors of the respiratory tract observed at the german primate center a survey of the pathology of marmosets (callithrix jacchus) derived from a marmoset breeding unit airway hyper-responsiveness in lipopolysaccharid-challenged common marmosets (callithrix jacchus) respiratory animal models in the common marmoset (callithrix jacchus) spontaneous pathology of the common marmoset (callithrix jacchus) and tamarins (saguinus oedipus, saguinus mystax) pneumonitis and multi-organ system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus hypervitaminosis d in new world monkeys background pathology of the common marmoset (callithrix jacchus) in toxicological studies experimental respiratory anthrax infection in the common marmoset (callithrix jacchus) respiratory system non-human primates in biomedical research: diseases background lesions in laboratory animals, a color atlas hepatic hemosiderosis in common marmosets, callithrix jacchus: effect of diet on incidence and severity husbandry and management of new world species: marmosets and tamarins, diseases, in: the laboratory primate non-human primate diseases of relevance in drug development and their impact on the interpretation of study findings, in: the non-human primate in nonclinical drug development and safety assessment histopathology of incidental findings in cynomolgus monkeys (macaca fascicularis) used in toxicity studies s: lps-induced lung inflammation in marmoset monkeys for testing of anti-inflammatory drugs bacterial and mycotic disease of non-human primates, in: non-human primates in biomedical research: diseases the common marmoset (callithrix jacchus) as a model in toxicology acknowledgements. the authors dedicate this work to franz-josef kaup, head of the pathology unit of the german primate center, who provided the essential idea and the sample material as well as financial and human resources to realize this study. the authors thank the necropsy and histology team from the pathology unit of the german primate center for skilled technical assistance.edited by: s. treue reviewed by: two anonymous referees m. bleyer et al.: lung pathology in common marmosets respiratory distress. tissue migration may lead to complications like abscess formation, pneumo-or pyothorax, or signs of penetration of other organs (lópez, 2007) .this study documents the range and incidence of spontaneous histological findings in the lungs likely to be encountered in purpose-bred common marmosets used in toxicological or experimental studies. when interpreting these findings in marmoset studies, special care should be taken to identify preexisting pulmonary disease and to distinguish speciesspecific background findings from trial-related changes. all relevant data are presented in the paper. please contact the corresponding author for further details.competing interests. the authors declare that they have no conflict of interest. key: cord-003558-7lvqpz21 authors: davies, patrick; yasin, samra; gates, simon; bird, david; silvestre, catarina title: clinical scenarios of the application of electrical impedance tomography in paediatric intensive care date: 2019-03-29 journal: sci rep doi: 10.1038/s41598-019-41774-1 sha: doc_id: 3558 cord_uid: 7lvqpz21 eit is a radiation-free functional modality that enables bedside imaging and monitoring of lung function and expansion. clinical interest in this method has been driven by the need for bedside monitoring of the dynamics of the lungs and the effects of ventilatory manoeuvres, including changes in ventilator settings, suctioning, chest drains, positioning and physiotherapy. we aimed to describe the use of electrical impedance tomography (eit) as a clinical tool in a tertiary paediatric intensive care unit. children requiring intensive care with a variety of clinical conditions had an electrode belt with 16 electrodes wrapped around the chest, which sequentially applied a small alternating current from each electrode pair. the signal gives information on both real time, regional, global, and relative data. with the correct application, and understanding of the monitor, much clinical information can be gained, with potentially significant patient benefit. we present the clinical use of eit in six conditions: asthma, ventilation weaning and expansion recoil, sequential lobar collapse, targeted physiotherapy, pleural effusion assessment, and peep optimisation. screenshots and analyses are offered displaying the pragmatic use of this technology. electrical impedance tomography is a clinically useful tool on the paediatric intensive care unit. it allows monitoring of a patient’s respiratory function in ways which are not possible through any other means. an understanding of respiratory physiology will allow use of this information to improve patient outcomes. electrical impedance tomography (eit) is a radiation-free functional modality that enables bedside imaging and monitoring of lung function and expansion. it has been evaluated in a number of pulmonary conditions in humans and animal models [1] [2] [3] [4] [5] [6] [7] [8] . it has been used in various clinical settings including acute respiratory distress syndrome (ards), establishing the best positive end expiratory pressure (peep) [8] [9] [10] [11] [12] , the response of the lungs to recruitment manoeuvres [12] [13] [14] [15] [16] and trying to minimize areas of collapse and hyperinflation 6, 17 . there are some studies that evaluate the application of high-flow nasal cannula therapy 18, 19 and quantify the extent of pulmonary oedema in acute lung injury 20 . eit however does not offer the same spatial resolution when compared with modalities such as computerised tomography (ct) scanning. it does however offer good temporal resolution and informs clinicians on real time regional ventilation distribution. the images produced can also be affected by body movement, changes in electrode contact, changes in posture 21 , as well as interference with other medical devices 22 . these need to be considered when interpreting the images. clinical interest in this method has been driven by the need for bedside monitoring of the dynamics of the lungs and the effects of ventilatory manoeuvres, including changes in ventilator settings, suctioning, chest drains, positioning and physiotherapy. our aim is to disseminate our experience of the use of electrical impedance tomography in the use of patients in paediatric intensive care, to act as a clinical primer for other clinicians and to communicate the pragmatic use of this technology. an electrode belt with 16 electrodes is wrapped around the chest, which sequentially apply a small ac current from each electrode pair. the impedance between the electrodes sending the current and the receiving electrodes will change dependant on the makeup of the matter between the respective electrodes. air has a much higher impedance than, for instance, blood. with the application of tomography, the sequential signals can be built up in to a 2 dimensional 32 × 32 pixel picture in real time with a frame rate of between 20 and 50 hz 23 . the information is visualised on a screen as a "heat map", where areas of impedance change (equivalent to ventilation) are displayed. the signal gives information on both real time, regional, global, and relative data. with the correct application, and understanding of the monitor, much clinical information can be gained, with potentially significant patient benefit. the information can be categorised in to global chest expansion, regional ventilation, and ventilatory compliance (expansion over time), with both absolute and relative figures for all 23, 24 . interpreting this data requires an understanding of ventilatory physiology and experience, however is easily learnt by interested parties. there are two main views: a live view which shows moving images of a cross section of the lung, with regional ventilation clearly seen, and an end expiratory lung impedance view, which gives breath by breath impedance of the whole lung. until now, this technology has mainly been clinically used, infrequently, in adults. we describe the clinical applications of the use of eit in children, with the physiological background and interpretation of the images. we used a draeger pulmovista 500 eit monitor, with a variety of belts which allow monitoring of children above 3.5 kg in weight. the use of the monitor was explained to all parents and verbal consent obtained. the chair of the nottingham 1 research ethics committee confirmed the united kingdom health research authority decision tool opinion, which states that this project is exempt from needing ethical approval. clinical scenarios. eit is used routinely in complex children on our picu. there was no specific case selection; we present six cases where the use of eit has been of direct clinical benefit to children being treated for a variety of conditions. we present six differing clinical scenarios; all commonly found on paediatric intensive care units, and demonstrate how eit can add important information which would not be otherwise available. we display the use of eit in: ventilatory changes were attempted with the aim of optimising his gas exchange and reducing air trapping. it was unknown whether air trapping was occurring, relative to baseline. the eit trace shows initially a change from a pip of 25 cm h 2 o to a pip of 20, with constant peep, and a change of rate from 30 to 20 ( fig. 1 , first arrow). the global impedance curve immediately begins to fall, reaching steady state around two minutes after. this signifies an overall decrease in the chest expansion (or end-expiratory lung volume [eelv] ). this reduction in eelv is a strong indicator for a significant reduction in gas trapping, which should improve chest compliance. at 6 minutes in to this recording (second arrow) the rate was increased to 25 due to a significant reduction in the minute volume. there was a small, but insignificant rise in the global expansion. the expansion remains lower than at the initial settings. tidal volumes remained static, implying no change in the compliance (and no change in overdistension) after this rate increase. this allowed increased co 2 clearance with no corresponding hyperinflation at reduced ventilatory settings. global chest impedance/end-expiratory lung impedance. a child with a diagnosis of pneumonitis whilst on volume ventilation was considered for weaning of ventilation as the ventilatory parameters had improved. however, when the peep was reduced by 2 cm h 2 o from 10 to 8, the global chest expansion displayed a sequential loss of volume over time. the peep was returned to the previous settings. on suctioning and returning to the ventilator, his oxygen saturations fell, requiring ventilation by anaesthetic bag. analysis of the eit trace showed that on returning to his ventilation, his global expansion (eelv) took a long time to return to baseline (fig. 2) . after another period of ventilation, a similar reduction in peep showed a reduction in volume (as would be expected with reduced pressures), but this was stable over time 25 . he was suctioned for secretions and had not shown instability. analysis of his eit trace showed return to near baseline within a small number of breaths. eit was thus able to illustrate a lack of resilience to initial weaning, which later improved, providing added confidence to continue weaning towards extubation. www.nature.com/scientificreports www.nature.com/scientificreports/ sequential lobar collapse: effect of postural changes. eit parameter: live regional ventilation distribution/tidal image. a 2 year old girl admitted with adenovirus and respiratory syncitial virus pneumonia with type 1 respiratory failure, ventilated on pressure control with pip 30, peep 10, rr 25-30, and fio2 0.7-0.8. she had frequent lobar collapse and she was monitored with eit which showed right sided lung collapse. she was nursed with the chest right side up, and within one hour her ventilation was balanced and equal, with expansion returning to the collapsed lung. however, after another hour the left (dependant) side lost ventilation. for the next 48 hours, continuously monitored by eit, she was repositioned as soon as one lung showed signs of loss of ventilation, usually every 3-4 hours. with this technique, the position of the patient was optimised based on lung expansion, and allowed the patient to wean ventilation and be extubated 3 days later. figure 3 shows tidal images with a relative hypoventilation of the right lung, she was repositioned with the left side of the chest up and 3 hours later, the right lung expansion had improved, at the cost of the left lung ventilation. she was then turned again. targeted physiotherapy: lobar or lung collapse: assessing progression and effect of physiotherapy. eit parameter: tidal image showing relative regional ventilation distribution. a 9 year old boy was admitted with a background of brainstem glioma when he was 2 years old, with significant sequelae including paraplegia and loss of brainstem function needing long term ventilation support 24 hours a day though a tracheostomy he was admitted with right sided total lung collapse needing picu admission and escalation of his ventilation to a picu ventilator: pressure control with pip 22, peep 10, rr 20, and fio2 80-100%.eit monitoring was instigated. he received regular airway clearance using manual techniques and mechanical insufflation-exsufflation (mi:e). observing eit during physiotherapy treatment it was clear that the exsufflation component of mi:e, despite effectively clearing secretions, was causing temporary collapse of the right lung (fig. 4) . this was not resolved by www.nature.com/scientificreports www.nature.com/scientificreports/ the 2 re-inflation breaths available on the mi:e device. the physiotherapy team were able to adjust their treatment plan to include further re-recruitment following exsufflation. this prevented deterioration in objective parameters following airway clearance. following 6 days of treatment, his right lung was clinically fully reinflated, confirmed by eit. he did not have any chest radiographs during this time. pleural effusion: quantifying respiratory compromise. eit parameter: relative regional ventilation distribution. a 10 year old with a diagnosis of chronic myeloid leukemia present ed to the hospital with disseminated varicella zoster infection with multi organ failure needing respiratory, renal. and inotropic support. she had a pleural effusion of 1 cm on the chest x-ray, which was measured at 1.8 cm on chest ultrasonography. her oxygen requirements were high, with an fio 2 of 0.8. 80%. a decision needed to be made whether insertion of a chest drain would be beneficial, with the heightened risk of an intrathoracic bleed due to the presence of disseminated intravascular coagulopathy. eit showed a very significant loss of expansion in the affected side, compared to the non affected side. on balance, a chest drain was felt to be in her best interests, and it was inserted without complication. the ventilation improved. eit pictures showed real time improvement of inflation, with aeration up to the chest wall (fig. 5) 26 . choosing the optimal peep level minimises overinflation and reduces collapse and makes physiological sense. analysis showed that the optimum peep (i.e. that which balances overdistension and collapse) was 9 cm h 2 o (fig. 6) . the patient's peep was increased to 9 cm h 2 o. www.nature.com/scientificreports www.nature.com/scientificreports/ we have presented six clinical scenarios where the use of eit has materially benefitted our paediatric intensive care patients. the information obtained by using eit is difficult or impossible to be gathered by any other non-invasive technique. conventional monitoring modalities give some feedback, but this is often delayed, inaccurate, or with a low level of accuracy 27 . the interpretation of the images is critical to the use of eit. our six scenarios can be interpreted as follows: 1. asthma: gas trapping is a difficult problem in such patients and quantifying the extent of the problem is impossible. it only becomes apparent when ventilation becomes compromised, and then it is difficult to know whether this change in ventilation is due to clinical deterioration, or the respiratory dynamics. use of www.nature.com/scientificreports www.nature.com/scientificreports/ eit gives instant, breath by breath feedback on the global chest expansion, allowing pre-emptive management of incipient gas trapping problems. 2. ventilation weaning and effects of disconnection. the patient will not display clinical signs soon after a ventilator change: it may take many hours for this to manifest. however, it can be anticipated that problems will arise if the patient is unable to maintain adequate lung expansion with the ventilatory settings which have been applied. if the global end-expiratory lung volume keeps dropping after the (expected) initial volume loss, then problems can be anticipated in the short to medium term. a patient who can hold a stable expansion after a ventilatory parameter change is likely to have a greater chance of medium term stability. current monitoring techniques are poor at predicting rapidity of atelectasis and lung collapse after weaning. knowledge of this may help to predict whether a patient is ready for extubation. the first trace shows a patient whose lungs cannot yet cope with a loss of peep, and is not ready for extubation. the second shows a patient who can rapidly restore lung volume and maintain adequate expansion making him suitable for extubation. 3. sequential lobar collapse. repositioning a patient with unilateral lobar collapse can cause respiratory instability, leading to a practice of minimal handling and reducing the frequency of unstable episodes. clinical examination is inaccurate in a small chest with many added sounds. chest radiography gives good knowledge of lobar collapse, but that may lead to worsening of gases and there is the added concern of radiation dosage. the use of eit allows for monitoring of lung inflation in real time and ensure that significant areas of dependent collapse are avoided limiting respiratory instability due to repositioning. direct visualisation identifies lung collapse rapidly making reinflation easier. 4. targeted physiotherapy. constant eit monitoring allowed us to monitor lung expansion without resorting to frequent chest radiographs. the physiotherapy team were able to quickly identify an adverse effect of treatment and adjust their plan accordingly. with standard monitoring it may not have been apparent why objective markers were deteriorating following airway clearance. 5. pleural effusion. chest x-ray, and sonography, show a depth of fluid but do not give a sense of how much this fluid affects the lung inflation. using live regional ventilation information allows precise visual quantification of this, meaning that clinical decisions can be based on more accurate information. in this case, the chest drain was high risk, but with the addition of eit data this risk was felt to be worth taking. cases of pneumothorax or foreign body would have a similar eit picture, with the ability to quantify which areas of lung are non-functional and to what degree. 6. peep optimisation. establishing the optimal peep in invasively ventilated patients is a challenge; the balance between collapse and over distension is difficult to assess and no method has been shown to prevent ventilator induced lung injury. eit can be used to evaluate the regional compliance changes at different levels of peep, measuring areas of collapse and over distention during a peep trial. both collapse and overdistension are detrimental to the patient. knowledge of the effects of the peep to the patient allows the clinician to titrate it to produce minimal detrimental effects. eit is often used for continuous monitoring. real time visualisation of chest expansion leads to knowledge of why an acute respiratory compromise has occurred. the treating team can be instantly aware of where the problem lies. we also use eit for progressing ventilation in the complex patient. no other non-invasive monitoring modality will give instant feedback over ventilatory changes. such patient's fragility means that delayed feedback from blood gas analysis or clinical change would be potentially harmful. the use of eit allows the treating team to make changes they would not have dared to otherwise make due to their fragility. by making quicker progress than anticipated we are able to minimise lung injury and maximise chances of recovery. a further clinical use on our unit is as a feedback spirometer. conscious patients of a suitable developmental age can respond well to visualisation of how to ensure that breathing is effective. there are multiple modalities of assessing a patient on mechanical ventilation (table 1 ). there is, however, no perfect modality as they all have their disadvantages. clinical examination is a single event, low resolution method of understanding lung movement. the benefits of ct scanning are offset by the radiation exposure as well as the logistical difficulties involved in taking the patient to the scanner. 28 x-rays have lower logistical www.nature.com/scientificreports www.nature.com/scientificreports/ difficulties and lower radiation, but they do not allow continuous investigation, or measurements of flow or dynamic expansion 23 . ventilator spirometry feedback allows no regional data, but does have the benefit of allowing continuous data29. lung ultrasound gives important and useful information, but is a non-continuous, low resolution modality. compared to these monitoring modalities, eit has significant benefits. it is a continuous, high resolution monitoring which allows assessment of dynamic, expansion, and flow data. with any novel monitoring technology, there is a learning curve for clinicians. although eit needs good education for treating staff, this is a very visual monitor which can give information even if the technology behind it is not well understood. our experience is that after some initial scepticism, the bedside team rapidly adopted it as the benefits were explained. there is presently little clinical evidence of the efficacy of eit in practice. good ventilatory practice, involving the avoidance of hyperinflation and/or collapse, is associated with better outcomes. the information from eit monitoring should allow better ventilatory practice, however the translation of this in to clinical outcomes is as yet unproven. suitably sized eit belts are not commercially available for paediatric patients and the use of individual electrodes is time-consuming and risks skin irritation. this severely limits the use in this population. its use in non-sedated patients is also limited due to patient anxiety and movement, causing potential image artefacts. studies have also shown a difference in skin impedance in neonates as compared to adults 30. reconstruction algorithms in current eit modalities also do not account for differences in chest shape or size for neonates5. however, as this technology becomes more widely used, and the information gained from it better understood, there will be more commercial pressure to ensure that patients of all ages and sizes are catered for. as clinical experience and knowledge increases, this may become a routine part of clinical care in complex children. with all forms of highly individualised medicine, there are concerns as to its effectiveness. population evidence allows excellent assessment of a treatment's long-term effectiveness, for a population mean. however, population evidence cannot tell the clinician whether the treatment will work for the individual patient, merely that if they treated 100 patients, it would be of overall benefit to give such a treatment. this monitor offers evidence to the clinician that the physiology has changed for the individual patient. it cannot tell the clinician whether this has long term, or outcome benefits. an excellent understanding of physiology is necessary to ensure that physiological benefits then translate to outcome benefits. electrical impedance tomography is a clinically useful tool on the paediatric intensive care unit. it allows monitoring of a patient's respiratory function in ways which are not possible through any other means. an understanding of respiratory physiology will allow use of this information to improve patient outcomes. absolute electrical impedance tomography (aeit) guided ventilation therapy in critical care patients: simulations and future trends effect of closed endotracheal suction in high-frequency ventilated premature infants measured with electrical impedance tomography assessment of lung ventilation in infants with respiratory distress syndrome using electrical impedance tomography electrical impedance tomography monitoring in acute respiratory distress syndrome patients with mechanical ventilation during prolonged positive end-expiratory pressure adjustments lung recruitment and endotracheal suction in ventilated preterm infants measured with electrical impedance tomography identification of regional overdistension, recruitment and cyclic alveolar collapse with electrical impedance tomography in an experimental ards model assessment of lung recruitment by electrical impedance tomography and oxygenation in ards patients positive end expiratory pressure titration after alveolar recruitment directed by electrical impedance tomography assessment of regional lung recruitment and derecruitment during a peep trial based on electrical impedance tomography bedside measurement of changes in lung impedance to monitor alveolar ventilation in dependent and nondependent parts by electrical impedance tomography during a positive end-expiratory pressure trial in mechanically ventilated intensive care unit patient assessment of respiratory system compliance with electrical impedance tomography using a positive endexpiratory pressure wave maneuver during pressure support ventilation: a pilot clinical study protective ventilation using electrical impedance tomography monitoring of recruitment and derecruitment by electrical impedance tomography in a model of acute lung injury end-expiratory lung impedance change enables bedside monitoring of end-expiratory lung volume change regional respiratory time constants during lung recruitment in high-frequency oscillatory ventilated preterm infants slow moderate pressure recruitment maneuver minimizes negative circulatory and lung mechanic side effects: evaluation of recruitment maneuvers using electric impedance tomography bedside selection of positive end-expiratory pressure by electrical impedance tomography in hypoxemic patients: a feasibility study oxygen delivery through high-flow nasal cannulae increase end-expiratory lung volume and reduce respiratory rate in post-cardiac surgical patients effect of high flow nasal cannula and body position on end-expiratory lung volume. a cohort study using electrical impedance tomography electrical impedance tomography (eit) for quantification of pulmonary edema in acute lung injury electrical impedance tomography: the realization of regional ventilation monitoring chest electrical impedance tomography examination, data analysis, terminology, clinical use and recommendations: consensus statement of the translational eit development study group positive end-expiratory pressure optimization using electric impedance tomography in morbidly obese patients during laparoscopic gastric bypass surgery bedside estimation of recruitable alveolar collapse and hyperdistension by electrical impedance tomography non-invasive radiation-free monitoring of regional lung ventilation in critically ill infants computed tomography-an increasing source of radiation exposure regional ventilation distribution in the first 6 months of life p.d. conceived the paper and coordinated contributions from the clinical team. p.d., c.s., s.g., s.y. and d.b. shared their eit patients and knowledge. all authors wrote and edited the paper and figures. competing interests: draeger ag paid for the article processing fee. they were only contacted after final completion of the paper. draeger had no input in to the study design or patient selection, and did not edit any drafts or proofs of the paper. there are no other competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-005573-mryrl1s1 authors: raimondi, francesco; yousef, nadya; migliaro, fiorella; capasso, letizia; de luca, daniele title: point-of-care lung ultrasound in neonatology: classification into descriptive and functional applications date: 2018-07-20 journal: pediatr res doi: 10.1038/s41390-018-0114-9 sha: doc_id: 5573 cord_uid: mryrl1s1 lung ultrasound (lus) is the latest amongst imaging techniques: it is a radiation-free, inexpensive, point-of-care tool that the clinician can use at the bedside. this review summarises the rapidly growing scientific evidence on lus in neonatology, dividing it into descriptive and functional applications. we report the description of the main ultrasound features of neonatal respiratory disorders and functional applications of lus aiming to help a clinical decision (such as surfactant administration, chest drainage etc). amongst the functional applications, we propose safe (sonographic algorithm for life threatening emergencies) as a standardised protocol for emergency functional lus in critical neonates. safe has been funded by a specific grant issued by the european society for paediatric research. future potential development of lus in neonatology might be linked to its quantitative evaluation: we also discuss available data and research directions using computer-aided diagnostic techniques. finally, tools and opportunities to teach lus and expand the research network are briefly presented. the first report on the use of lung ultrasonography (lus) in adult medicine appeared in 1995 1 and lus has been rapidly gaining popularity, also in paediatrics and in neonatology. lus is a point-ofcare, easy-to-learn, radiation-free, bedside, quick and repeatable technique. lus signs vary little by age, 2 which makes it especially suitable for use in the smallest patients and in the critical care setting. in the past 10 years, there has been a notable increment in publications on the use of lus in neonatology (fig. 1) , and even more in adult medicine. we demonstrated that launching a lus program in their neonatal intensive care unit (nicu) roughly halved the number of chest radiograms and significantly decreased the mean radiation dose/patient. 3 we present a comprehensive review on lus in neonatology with an emphasis on pathophysiology and on a classification into descriptive (qualitative) and functional (semiquantitative) applications. lus is a powerful diagnostic technique and a noninvasive research tool to describe several neonatal respiratory disorders in a qualitative manner. international lus guidelines for adult critical care include a short chapter about applications in infants, based on the little data available in 2012, and conclude that the use of descriptive lus may be of interest. 4 many studies have been published since then, and we aim to review the knowledge available today. the main lus semiology patterns are illustrated in fig. 2 and in the supplementary material s1-s5. the description of lus findings for each neonatal lung disorder is summarised in table 1 . transient tachypnoea of the neonate the main pathophysiological feature of transient tachypnoea of the neonate (ttn) is delayed lung fluid re-absorption during the foetal life transition and this creates a mainly interstitial, ab extrinseco lung oedema. lus shows a high sensitivity and specificity to detect alveolar-interstitial oedema and to estimate extravascular lung water (evlw) in adults through the evaluation of b-lines, which are vertical dynamic artefacts arising at the fluid/ air interface. 5, 6 b-lines may be sparse or confluent, creating a continuum that is generally referred to as an 'alveolar-interstitial pattern'. 5 using lus, substantial liquid retention has been demonstrated at 10 years of life in 14% of healthy neonates, while 49%, 78% and 100% of neonates had completed airway liquid clearance at 2, 4 and 24 h, respectively. 7 moreover, it seems that neonates born by an elective caesarean section have higher fluid retention early after birth than those vaginally delivered. [7] [8] [9] consistently, copetti and cattarossi showed that neonates with ttn have both interstitial oedema (represented by b-lines), and normal areas (represented by a-lines). a sharp increase in echogenicity was described in the lower lung fields of ttn neonates and the authors named this finding 'double lung' point. 10 subsequent publications showed that the double lung point is not a perfect diagnostic tool for ttn, whose ultrasound appearance may include pleural line thickness, a more diffused alveolar-interstitial pattern and the presence of normally aerated areas. 11, 12 this seems to be confirmed by the preliminary data of an ongoing multicentre, international study. 13 despite the rich semiology and the absence of a unique diagnostic sign, the distinction between ttn and respiratory distress syndrome (rds) is relatively easy (see below), but it should be kept in mind that clinical and laboratory data must always be integrated with lus to refine the diagnosis. 14 respiratory distress syndrome the typical lus appearance of respiratory distress syndrome (rds) consists of bilateral white lungs (i.e., the diffuse alveolar-interstitial pattern) with no spared areas reflecting decreased air/fluid ratio. other findings include 'sub-pleural' small consolidations and/or an irregular pleural line. 11 these results have been confirmed by multiple studies. [11] [12] [13] [15] [16] [17] since rds is a more severe and diffuse condition than ttn, the absence of spared areas (with a-lines) seems the most noticeable sign and, contrary to ttn, in the absence of any treatment, lus appearance will not improve quickly. 11 there is a high inter-observer agreement among physicians with different levels of lus expertise, which makes the differential diagnosis between rds and ttn reliable, irrespective of the operator. 18 lus is also useful in diagnosing the complications of rds, such as pulmonary haemorrhage, pneumothorax (pnx) or atelectasis. 19, 20 two pathophysiological aspects must be considered: (1) clinical and laboratory data must always be integrated with lus to refine the diagnosis of rds, especially when it may coexist with other conditions, like pneumonia, early-onset sepsis or air leaks; 15 (2) mixed-type situations may exist where fluid retention is associated with partial surfactant deficiency, as this has been recently demonstrated by lamellar body count. 21 interestingly, a semiquantitative lus score describing lung aeration (see below) correlated with lamellar body count. 22 these mixed ttn/rds cases may last longer than classical ttn and may sometimes require noninvasive respiratory support or even a surfactant. 23 unlike chest x-rays, lus appearance does not change shortly after surfactant administration, 24 and this is intrinsically due to their different properties: lus detects lung fluid content, while xrays directly detect lung aeration. surfactant replacement unavoidably implies some fluid administration even with the more concentrated surfactant preparations. animal data demonstrate an almost total evlw clearance 6 h after surfactant administration. 24 however, the same process seems more variable and heterogeneous in human neonates, as lus appearance may be influenced by respiratory support, gestational age, fluid intake, pre-existing condition (pure rds or a more complex situation with superimposed lung inflammation and surfactant catabolism, such as acute respiratory distress syndrome (ards)) and the eventual simultaneous development of broncho-pulmonary dysplasia (bpd). neonatal ards ards is an acute, life-threatening respiratory failure, characterised by extensive lung tissue inflammation, endothelial injury and both quantitative and qualitative secondary surfactant dysfunction, leading to loss of lung aeration. 25 neonatal ards shares the same biological and pathophysiological aspects of the syndrome in lung ultrasound semiology. the basic semiology patterns are illustrated: these patterns may be variably found in different respiratory disorders described in table 1 . arrows indicate the sub-pleural consolidation, the border of a consolidation, the double lung point or the lung point. the size threshold to distinguish micro-consolidations (sub-pleural) from consolidations (0.5 cm) is arbitrary. some semiology patterns are also dynamically shown in the videos in supplementary material 1 older patients: thus, signs on lung imaging are similar. lus findings in neonatal ards consist of bilateral diffuse loss of aeration, which may vary from a diffuse alveolar-interstitial to an irregular alveolar pattern with consolidations with bronchograms and/or atelectases. 26 lung imaging is one of the diagnostic criteria included in the montreux definition of neonatal ards, but this officially requires x-ray findings (diffuse, bilateral and irregular opacities or infiltrates, or complete opacification of the lungs, which are not fully explained by local effusion, atelectasis, rds, ttn or congenital lung anomalies). 25 nonetheless, lus has been used for the diagnosis of ards in adults 27 and is considered suitable in neonates if sufficient clinical expertise exists for its interpretation. 28 despite similarities with the syndrome in older patients, neonatal ards may also have different triggers, such as meconium aspiration syndrome (mas), lung haemorrhage, perinatal asphyxia or necrotising enterocolitis that are peculiar to newborn age. meconium aspiration syndrome meconium aspiration syndrome (mas) is the only ards-triggering condition for which lus findings have been formally described so far and they consist of a mix of normal lung areas, coalescent or sparse alveolar-interstitial pattern and consolidations with bronchograms. these signs are irregularly present all over the lungs and may change over time as the meconium-driven inflammation progresses; meconium plugs may also occur and create atelectases. 29 these findings were confirmed in a larger study of 117 neonates with mas and a dissociation between clinical severity and imaging findings may sometimes occur. 30 in summary, lus signs in mas include all the possible findings ranging from normally aerated zones to a complete loss of aeration, and, when the injury is sufficiently severe and diffuse, the lesions may cause an important oxygenation impairment and qualify as neonatal ards. air leak syndromes lus signs of pnx (see supplementary material s4 and s5) are the absence of lung sliding and of any other sign other than a-lines; these findings are described in detail elsewhere. 31 lus has a higher diagnostic accuracy than conventional radiology for the diagnosis of pnx in adults, as it has been demonstrated by a metaanalysis of 13 studies. 32 therefore, lus can potentially detect subclinical pnx that may go radiologically underdiagnosed and that does not require treatment. recently, a case report and two diagnostic accuracy studies suggest that lus may also be very useful in the diagnosis of neonatal pnx. [33] [34] [35] in critically ill babies, lus can be used for rapid detection of life-threatening tension pnx: an international multicentre study confirmed that lus has an optimal diagnostic accuracy and is quicker than conventional radiology. 36 lus also resulted more accurately than chest transillumination (which is also less accurate than conventional radiology). 34 a case report described the use of lus to detect and follow up neonatal interstitial emphysema. 37 no formal lus description of neonatal pneumomediastinum exists. however, pneumomediastinum has been detected in children as (1) the absence of lung sliding on parasternal scan (with normal sliding in other chest areas); (2) a parasternal 'still' lung point, since the air collection displaces the lungs laterally and reveals the border between the air-filled mediastinum and the displaced lung; this still lung point does not move with spontaneous breathing and it remains under the parasternal area; (3) impossibility to obtain a normal parasternal heart view due to air artefact, regardless of the breathing cycle. 38, 39 pneumonia lus typically shows pneumonia as the presence of consolidations with irregular borders and air bronchograms, associated with pleural line abnormalities, and alveolar-interstitial pattern in the adjacent areas if the inflammatory process is extended. the presence of these signs carries an optimal diagnostic accuracy according to a study performed on 40 neonates with symptomatic pneumonia and 40 controls. 40 similar results were obtained in a larger cohort of 3405 chinese neonates, of whom 725 were diagnosed with pneumonia by routine lus: among 81 cases without any sign of lung disease by chest radiograms, there were 32 cases with clinical and ultrasound evidence of pneumonia. 41 these data are fully consistent with those obtained in older patients. in fact, a meta-analysis of eight diagnostic studies (765 paediatric patients, including both neonates and children) yielded a sensitivity and a specificity of 96 and 93%, respectively, which is superior to the accuracy of chest radiograms and comparable to that obtained combining radiology and laboratory exams. 42 similarly, a meta-analysis of 20 studies (2513 adults) showed that lus has a high accuracy (sensitivity 85%, specificity 93%) to diagnose pneumonia defined by the combination of radiological and clinical data. 43 finally, a smaller metaanalysis of 12 studies (1515 adults) showed lus to be more accurate than conventional radiology or computerised tomography (ct) alone. 44 some points still deserve to be investigated. there are no specific data regarding pneumonia of different types (i.e. congenital, community-acquired or ventilator-associated), although lobar or haemilobar consolidations are useful to diagnose ventilatorassociated pneumonia in adults, when coupled with clinical diagnostic criteria. 45 thresholds for the size of consolidations and exact measurement methods also need to be defined. bronchiolitis and other viral low tract respiratory infections bronchiolitis, often caused by respiratory syncytial virus (rsv), is essentially an airway inflammatory disease causing obstructive respiratory failure with possible secondary involvement of the alveolar tissue. therefore, from an ultrasound point of view, bronchiolitis presents as a nonhomogeneous pleural line abnormalities (pleural line thickening and/or irregularities), small 'subpleural' and/or larger consolidations or an alveolar-interstitial pattern, in the case of parenchymal involvement. 46 for the sickest patients, consolidations may span across several intercostal spaces, 47 due to associated atelectasis, viral alveolar injury or superimposed bacterial infection. if the lung injury is severe enough, patients may qualify for rsv-induced ards and this is characterised by a shift towards a mainly restrictive and severe respiratory failure 48 with a greater loss of aeration, as described above. lus findings correlate with disease severity, with a higher proportion of hospitalised patients having positive findings compared to outpatients. 49 moreover, findings gradually resolve with clinical improvement and the lung aeration correlates with the duration of oxygen therapy both in spontaneously breathing infants 50 and in those needing noninvasive respiratory support. 47 a good concordance among operators of different expertise has been reported for the ultrasound evaluation of bronchiolitis, 49 similar to that reported for restrictive disorders. 18 bronchiolitis findings are non-specific and shared with other viral low tract respiratory infections; thus, laboratory tests are warranted to clarify the aetiology and also rule out bacterial coinfection. 51 only one study has evaluated lus during a h1n1 outbreak suggesting moderate accuracy in distinguishing viral and bacterial pneumonias, as these were showing an interstitial pattern and consolidations, respectively. 52 however, this study was performed during an outbreak and may be biased by the high disease prevalence. consistently, other case series in adults have shown nonspecific lus findings in influenza and measles. [53] [54] [55] no specific neonatal studies are available in this field. broncho-pulmonary dysplasia early prediction of worsening respiratory conditions and bronchopulmonary dysplasia (bpd) is a potentially interesting application for lus, as other techniques have failed in this regard. 56, 57 two studies published in the nineties addressed lus features of bpd using the trans-abdominal approach. the authors found that in bpd-developing babies, lus showed the persistence or the appearance of nonhomogeneous retro-diaphragmatic hyperechogenicity, which was not visible in controls. 58, 59 . since then, ultrasound technology and our understanding of bpd have greatly improved. data on transthoracic lus and bpd are currently lacking. moreover, there are no lus data describing 'developing bpd' or the new concept of chronic pulmonary insufficiency of prematurity, that may provide new interesting areas of application for lus. 60 malformations lus has been used to describe congenital pulmonary airway malformations (cpam), which have a variable appearance (a large or micro-cystic lesion or irregular consolidations) in line with the four histological types described in the most recent cpam classification. 61, 62 the gold standard to diagnose lung malformations remains the ct-scan, though lus may allow to suspect cpam in the absence of an antenatal diagnosis. 62 functional lung ultrasound lus may also be used in a more 'functional' way, to guide therapeutic interventions or to assist during invasive procedures. some examples of these applications already exist in the form of lus scores or decision-making protocols in adult critical care. 63, 64 we shall review a few neonatal data and provide specific proposals for these applications in neonatology. scores for semi-quantitative lus a basic, three-stage classification can be set as coalescent b lines (i.e. the 'white lung image'), sparse b lines and the normal, diffuse a-line pattern. using this simple system, our group monitored the postnatal lung fluid clearance and predicted nicu admission in a cohort of late preterm and term infants. 65 this information may be particularly valuable to healthcare providers in level i/ii perinatal centres. we also investigated the usefulness of lus linking a specific lus profile to a therapeutic decision: the presence of a bilateral severe alveolar-interstitial pattern reliably predicted the need for intubation in nicu-admitted preterm neonates (sensitivity 88.9%, specificity 100%). 66 similar results were found by other authors in a cohort of neonates older than 32 weeks, arbitrarily classifying the lus findings as low (normal or ttn) or high risk (rds, mas, pneumothorax or pneumonia). 67 these papers used descriptive lus to predict or guide clinical decision but did not quantify the lus findings. since lus detects the artefacts generated by the accumulation of fluid, and given that artefacts may be ranked according to the air/fluid ratio, it is possible to create scores inversely reflecting lung aeration. several semi-quantitative scores are available in adult critical care and their description is beyond our scope. nonetheless, all lus scores are based on the same semiology and, interestingly, they seem only useful in restrictive lung disorders. in fact, an obstructive condition will create air-trapping and this might not be distinguishable from a normally aerated lung at lus, as both present with a-lines. consistently, lus scores may well evaluate lung aeration, but they cannot detect over-distension, as it has been proven in ventilated adults. 68 despite these limitations, lus scores offer the advantage of allowing serial semi-quantitative evaluations of the disease severity. we described the first lus score to be used in neonates with respiratory failure modifying a score already used in adult critical care. 69 the main modifications were (1) fewer lung areas to scan given the smaller chest size; (2) use of a small linear or a microlinear 'hockey-stick' probe instead of a convex one. we were able to demonstrate that the lus score is suitable and inversely correlated to oxygenation. 70 the score is based on three chest areas for each side (upper anterior, lower anterior and lateral) and a 0-to-3 score is given for each area: more details are shown in supplementary material s6. the lus score is able to predict the need for surfactant treatment in preterm infants below 34 weeks' gestation (area under the curve: 0.93 (95% ci: 0.86-0.99; p < 0.001)), 70 and in extremely preterm neonates affected by rds (area under the curve: 0.94 (95% ci: 0.90-0.98; p < 0.001)). 71 the diagnostic accuracy was lower in late preterm and term neonates since they may be affected by various lung disorders with different appearance and severity, such as rds and ttn, but also aspiration syndromes and sepsis or ards. lus score calculation has a high inter-observer agreement regardless of the ultrasonographers' experience. 70 computer-aided image analyses provide an appealing approach for interpreting lus and different technologies are being developed. 72 for instance, ultrasound lung texture analysis has already been used to examine the foetal lung and predict the need of respiratory support. 73 we found a significant correlation between the lus score calculated by the ultrasonographer or by a supervised machine-learning approach and oxygenation indexes, while a lus score obtained with greyscale analysis, another computerised image analysis technique, did not correlate with oxygenation. 74 computer technology is progressing fast and we speculate a future when ultrasound images will be processed free of subjective interpretation. in the meantime, available data demonstrate that a visually calculated lus score is a useful and easy tool to predict surfactant need in preterm neonates with rds, to evaluate lung aeration while titrating the respiratory support or to be used as a research outcome measure. 75 lus score has its drawbacks (i.e. a semi-quantitative measure, impossibility to detect air-trapping) but is more easy and quickly available at the bedside than more complex techniques, such as electrical impedance tomography or respiratory inductance plethysmography. 76, 77 semi-quantitative lus has not only been used for respiratory failure due to a primary pulmonary disorder, but also for neonates with heart defects causing cardiogenic lung oedema. the latter is much more common in adults and, in these cases, the lus score has been calculated simply by counting b-lines, provided that there was no consolidation due to a parenchymal process. neonates with congenital heart defects predisposing to pulmonary overflow have a higher b-line count than babies without overflow 78 and the b-line score also correlates with the duration of ventilation. 79 similar applications could be proposed to evaluate pulmonary overflow in the case of haemodynamically significant patent ductus arteriosus or to guide fluid management albeit specific studies are currently lacking. standardised protocol for functional lus: the safe algorithm there is a need for specific protocols integrating lus findings into diagnostic and/or operative flow-charts. formal lus protocols for the evaluation of trauma, 80 (3) pleural effusion. the algorithm only takes a few minutes and aims to help diagnosing the most urgent treatable complications whilst awaiting expert help. a paediatric cardiologist evaluation of congenital heart defects is included in the algorithm but only when the most urgent causes have been already ruled out. safe is designed for the average neonatologist and may be applied using any probe without losing time to change it commonly used in adult critical care. thus, lus needs to be integrated into appropriate decision-making algorithms in neonatology. point-of-care ultrasound is rapidly growing and guidelines about neonatologist-performed echocardiography have been already published, 83, 84 but there are no formal algorithms for the use of lus in neonatology. a project in this direction has been specifically funded by a european society for paediatric research grant and aimed to create the 'safe protocol' (sonographic algorithm for life threatening emergencies) to standardise the use of lus in critically ill neonates. the safe protocol is designed for use in the case of unexpected and severe decompensation (bradycardia or severe desaturation requiring resuscitative manoeuvres or significantly increasing oxygen/ ventilator parameters to maintain stable oxygen saturation levels) in formerly stable infants in the nicu. it aims to help the diagnosis of the most urgent treatable complications: current knowledge on the ultrasound detection of the most critical neonatal complications was integrated into the algorithm and rapid diagnosis of unexpected and potentially fatal complications was prioritised. the safe protocol starts with an easy, subjective 'eyeball' assessment of myocardial contractility, which is comparable to other techniques which are more accurate but unsuitable during emergencies. 85, 86 then, safe uses standardised items together with a simplified and rapid rule-in/rule-out approach to detect only three main life-threatening complications. as shown in fig. 3 , the ultrasound algorithm is designed by order of urgency. hence, ruling out cardiac tamponade, which is a rare condition, is the first step in the decision tree since it may be rapidly fatal in the absence of prompt intervention followed by pneumothorax, and lastly, pleural effusion. the latter is also quite unusual, but it is associated to central venous lines often used in nicu care: current guidelines for adult critical care recommend the use of lus for the diagnosis of pleural effusion, as it outperforms chest x-rays. 4 safe is designed for the average neonatologist, it requires only minimal training and can be performed with a single ultrasound probe, as it targets basic ultrasound signs. a preliminary evaluation of safe after basic training in an academic nicu has shown that the algorithm is quick and easy to perform, even for lesserexperienced clinicians. 87 the safe protocol will need to be evaluated prospectively, as it has been done for similar algorithms (bedside lung ultrasound in emergency (blue) and fluid administration limited by lung sonography (falls)) presently used in adult critical care. [80] [81] [82] moreover, although any probe can be used, the optimal probe needs to be determined with a specific study. further work to expand the safe protocol to include other organs is ongoing. lus-guided procedures lus has been used to guide invasive procedures in order to reduce associated complications. lus guidance is recommended for chest tube placement in adults, 88 as this effectively reduces complications. 89 there are no neonatal studies about these procedures, but it is highly probable that lus may provide similar advantages, also because of the smaller patients' size. we demonstrated that the lus detection of tension pnx is extremely accurate and quicker than using conventional radiology. 36 other authors successfully performed a lus-guided drainage of a lifethreatening tension pneumomediastinum. 90 even in the absence of specific studies, the use of lus is advisable in these situations, where enough expertise exists, as lus will likely assist the operator and make the procedure easier. lus has been shown to be effective for verifying endotracheal tube (ett) position in patients of different ages. this can be achieved by assessing the normal sliding on both hemithoraces that confirms ventilation; in turn, other studies have aimed to directly visualise the ett tip position. jaeel et al. recently performed a systematic review of neonatal studies on this topic. 91 the studies report a successful visualisation of the tube tip in more than 80% of cases and this correlated with the position observed on chest radiograms in 73-100% of cases. we must acknowledge that there were variations in techniques, calculations, probes and operators' expertise across the studies: lus visualisation of ett does not seem to be straightforward and is potentially subjected to erroneous interpretation. simpler techniques (such as digital palpation of the ett tip in the suprasternal notch) have also been proposed. unless convincing evidence is published, lus cannot be recommended as a routine technique to verify ett placement, while end-tidal co 2 measurement is recognised as the gold standard. 92 lus is relatively easy to learn in vivo and we have organised successful practical courses since 2014. 93 however, some interesting bench models have been created to mimic lus semiology and teach lus-guided procedures, although they may also be useful as an educational tool for descriptive lus. models have been created with plastic phantoms or a wet sponge with or without pork ribs, but also simply using a hand with a wet foam. [94] [95] [96] [97] conclusion there is a rapid growth in the use of lus in neonatology and an increasingly large body of evidence supporting its use in neonatal respiratory care. however, the knowledge available is still far from that acquired in adult critical care. methodological stringency and multicentre studies are needed. therefore, we have founded the neolus group (neonatal lung ultrasound for the neonate and the small infant): a dedicated research network currently counting more than 150 members around the world and disposing of a dedicated page on social networks. 98 this and other initiatives will contribute to the further development of lus in neonatology. a bedside ultrasound sign ruling out pneumothorax in the critically ill. lung sliding neonatal lung ultrasound exam guidelines lung ultrasound decreased radiation exposure in preterm infants in a neonatal intensive care unit international evidence-based recommendations for point-of-care lung ultrasound the comet-tail artifact. an ultrasound sign of alveolarinterstitial syndrome lung ultrasound predicts well extravascular lung water but is of limited usefulness in the prediction of wedge pressure lung ultrasound immediately after birth to describe normal neonatal transition: an observational study lung ultrasound during the initiation of breathing in healthy term and late preterm infants immediately after birth, a prospective, observational study delayed lung liquid absorption after cesarean section at term the 'double lung point': an ultrasound sign diagnostic of transient tachypnea of the newborn lung ultrasound accuracy in respiratory distress syndrome and transient tachypnea of the newborn lung ultrasonography to diagnose transient tachypnea of the newborn is the double lung point an accurate diagnostic marker fir transient tachypnoea of the neonate? a prospective international study clinical data are essential to validate lung ultrasound diagnosis of neonatal transient tachypnea and its differentiation from respiratory distress syndrome using lung ultrasound lung ultrasound in respiratory distress syndrome: a useful tool for early diagnosis diagnostic performance of point of care ultrasonography in identifying the etiology of respiratory distress in neonates neonatal lung sonography: interobserver agreement between physician interpreters with varying levels of experience use of lung ultrasound in detection of complications of respiratory distress syndrome lung ultrasonography of pulmonary complications in preterm infants with respiratory distress syndrome. ups surfactant deficiency in transient tachypnea of the newborn a noninvasive surfactant adsorption test predicting the need for surfactant therapy in preterm infants treated with continuous positive airway pressure continuous positive airway pressure and the burden of care for transient tachypnea of the neonate: retrospective cohort study surfactant administration for neonatal respiratory distress does not improve lung interstitial fluid clearance: echographic and experimental evidence the montreux definition of neonatal ards: biological and clinical background behind the description of a new entity imaging in acute respiratory distress syndrome hospital incidence and outcomes of the acute respiratory distress syndrome using the kigali modification of the berlin definition diagnosis of neonatal ards: is montreux closer to berlin than to kigali? authors' reply lung ultrasound findings in meconium aspiration syndrome lung ultrasonography to diagnose meconium aspiration syndrome of the newborn bedside ultrasonography for diagnosis of pneumothorax pleural ultrasonography versus chest radiography for the diagnosis of pneumothorax: review of the literature and meta-analysis lung ultrasound-guided emergency pneumothorax needle aspiration in a very preterm infant lung ultrasound diagnostic accuracy in neonatal pneumothorax lung ultrasonography to diagnose pneumothorax of the newborn lung ultrasound for diagnosing pneumothorax in the critically ill neonate lung ultrasound: an useful tool for the follow-up of neonatal localized interstitial emphysema pneumomediastinum as a sonographic mimic of pneumothorax the still lung point: new sonographic evidence for pneumomediastinum lung ultrasonography for the diagnosis of severe neonatal pneumonia routine application of lung ultrasonography in the neonatal intensive care unit lung ultrasound for the diagnosis of pneumonia in children: a meta-analysis systematic review and meta-analysis for the use of ultrasound versus radiology in diagnosing of pneumonia lung ultrasound for the diagnosis of pneumonia in adults: a metaanalysis accuracy and applications of lung ultrasound to diagnose ventilator-associated pneumonia: a systematic review lung ultrasound in bronchiolitis: comparison with chest x-ray can a simple lung ultrasound score predict length of ventilation for infants with severe acute viral bronchiolitis? acute respiratory distress syndrome caused by respiratory syncytial virus point-of-care lung ultrasound in young children with respiratory tract infections and wheeze lung ultrasound: a useful tool in diagnosis and management of bronchiolitis the role of lung ultrasound in viral lower respiratory tract infections prospective application of clinicianperformed lung ultrasonography during the 2009 h1n1 influenza a pandemic: distinguishing viral from bacterial pneumonia dynamic assessment of lung injury by ultrasound in a case with h7n9 influenza early recognition of the 2009 pandemic influenza a (h1n1) pneumonia by chest ultrasound sonographic detection of radio-occult interstitial lung involvement in measles pneumonitis variables associated with the early failure of nasal cpap in very low birth weight infants reliability of cxr for the diagnosis of bronchopulmonary dysplasia sonographic prediction of chronic lung disease in the premature undergoing mechanical ventilation the value of ultrasound examination of the lungs in predicting bronchopulmonary dysplasia chronic pulmonary insufficiency of prematurity: developing optimal endpoints for drug development prenatal and postnatal management of congenital pulmonary airway malformation lung ultrasound findings in congenital pulmonary airway malformations blue-protocol and falls-protocol: two applications of lung ultrasound in the critically ill chest ultrasound in acute respiratory distress syndrome can neonatal lung ultrasound monitor fluid clearance and predict the need of respiratory support? use of neonatal chest ultrasound to predict noninvasive ventilation failure lung ultrasound as a predictor of mechanical ventilation in neonates older than 32 weeks bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment lung ultrasound in the icu: from diagnostic instrument to respiratory monitoring tool lung ultrasonography score to evaluate oxygenation and surfactant need in neonates treated with continuous positive airway pressure point-of-care lung ultrasound in neonatology: classification into lung ultrasound score predicts surfactant need in extremely preterm neonates detection of abnormalities in ultrasound lung image using multi-level rvm classification fetal lung texture team. prediction of neonatal respiratory morbidity by quantitative ultrasound lung texture analysis: a multicenter study can we assess the severity of neonatal respiratory distress by ultrasound? a comparison of three methods noninvasive high-frequency ventilation and the errors from the past: designing simple trials neglecting complex respiratory physiology chest electrical impedance tomography examination, data analysis, terminology, clinical use and recommendations: consensus statement of the translational eit development study group evaluation of bedside pulmonary function in the neonate: from the past to the future usefulness of lung ultrasound in neonatal congenital heart disease (lusnehdi): lung ultrasound to assess pulmonary overflow in neonatal congenital heart disease assessment of extravascular lung water by ultrasound after congenital cardiac surgery: lung ultrasound after congenital cardiac surgery focused assessment with sonography for trauma (fast): results from an international consensus statement relevance of lung ultrasound in the diagnosis of acute respiratory failure: the blue protocol international federation for emergency medicine consensus statement: sonography in hypotension and cardiac arrest (shoc): an international consensus on the use of point of care ultrasound for undifferentiated hypotension and during cardiac arrest targeted neonatal echocardiography in the neonatal intensive care unit: practice guidelines and recommendations for training. writing group of the american society of echocardiography (ase) in collaboration with the european association of echocardiography (eae) and the association for european pediatric cardiologists (aepc) recommendations for neonatologist performed echocardiography in europe: consensus statement endorsed by european society for paediatric research (espr) and european society for neonatology (esn) techniques for comprehensive two dimensional echocardiographic assessment of left ventricular systolic function the search for intelligent quantitation in echocardiography: "eyeball," "trackball" and beyond the safe protocol: a sonographic algorithm for life-threatening emergencies in the neonatal intensive care unit recommendations on the use of ultrasound guidance for adult thoracentesis: a position statement of the society of hospital medicine ultrasound-guided thoracentesis: is it a safer method ultrasound guided percutaneous relief of tension pneumomediastinum in a 1-day-old newborn ultrasonography for endotracheal tube position in infants and children a role of end-tidal co(2) monitoring for assessment of tracheal intubations in very low birth weight infants during neonatal resuscitation at birth creating thoracic phantoms for diagnostic and procedural ultrasound training phantom model and scoring system to assess ability in ultrasound-guided chest drain positioning randomized, noninferiority study between video versus hand ultrasound with wet foam dressing materials to simulate b-lines in lung ultrasound: a consort-compliant article economical sponge phantom for teaching, understanding, and researching a-and bline reverberation artifacts in lung ultrasound the authors are grateful to the espr pulmonology section for their support. the authors are also indebted with philippe durand (md) for the cpam image. the authors also wish to thank samsung (seoul, south korea) for the technical assistance provided. videos in the supplementary material have been taken with samsung hm70a or with general electrics ge logiq e9, using a high-frequency linear probe. the development of the safe algorithm has been supported by the espr cure & care research grant 2016 (received by ny). the online version of this article (https://doi.org/10.1038/s41390-018-0114-9) contains supplementary material, which is available to authorized users. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-011345-w0ke1tqz authors: howe, sarah l.; märz, melanie; krüger-ziolek, sabine; laufer, bernhard; pretty, chris; shaw, geoffery m.; desaive, thomas; möller, knut; chase, j. geoffrey title: measuring lung mechanics of expiratory tidal breathing with non-invasive breath occlusion date: 2020-05-14 journal: biomed eng online doi: 10.1186/s12938-020-00777-0 sha: doc_id: 11345 cord_uid: w0ke1tqz background and objective: lung mechanics measurements provide clinically useful information about disease progression and lung health. currently, there are no commonly practiced methods to non-invasively measure both resistive and elastic lung mechanics during tidal breathing, preventing the important information provided by lung mechanics from being utilised. this study presents a novel method to easily assess lung mechanics of spontaneously breathing subjects using a dynamic elastance, single-compartment lung model. methods: a spirometer with a built-in shutter was used to occlude expiration during tidal breathing, creating exponentially decaying flow when the shutter re-opened. the lung mechanics measured were respiratory system elastance and resistance, separated from the exponentially decaying flow, and interrupter resistance calculated at shutter closure. progressively increasing resistance was added to the spirometer mouthpiece to simulate upper airway obstruction. the lung mechanics of 17 healthy subjects were successfully measured through spirometry. results: n = 17 (8 female, 9 male) healthy subjects were recruited. measured decay rates ranged from 5 to 42/s, subjects with large variation of decay rates showed higher muscular breathing effort. lung elastance measurements ranged from 3.9 to 21.2 cmh[formula: see text] o/l, with no clear trend between change in elastance and added resistance. resistance calculated from decay rate and elastance ranged from 0.15 to 1.95 cmh[formula: see text] os/l. these very small resistance values are due to the airflow measured originating from low-resistance areas in the centre of airways. occlusion resistance measurements were as expected for healthy subjects, and increased as expected as resistance was added. conclusions: this test was able to identify reasonable dynamic lung elastance and occlusion resistance values, providing new insight into expiratory breathing effort. clinically, this lung function test could impact current practice. it does not require high levels of cooperation from the subject, allowing a wider cohort of patients to be assessed more easily. additionally, this test can be simply implemented in a small standalone device, or with standard lung function testing equipment. page 2 of 16 howe et al. biomed eng online (2020) 19:32 background spirometry is the most frequently performed lung function test. it is a simple and lowcost test which assesses lung health by analysing airflow and lung volume during specific breathing manoeuvres [1] [2] [3] . the results of spirometry are able to guide therapy, by indicating the type and severity of any lung condition present. however, underlying lung mechanics cannot be directly measured without further testing [4] . these mechanics are affected by lung disease, and monitoring how they change over time may provide a more accurate assessment of lung condition in response to therapy. hence, there is a need to link easily obtained spirometry data with clinically and physiologically relevant lung mechanics models. this proof-of-concept study presents a novel, model-based method of measuring lung mechanics during spirometry, or eventually with a standalone device. the model used is a dynamic elastance single-compartment lung model. the lung mechanics calculated are a constant, total system resistance, and a time-varying elastance. the dynamic elastance measured during expiration represents a combination of the lung's elastic recoil and muscular expiratory effort. lung mechanics were calculated by occluding expiratory breaths using a plethysmograph with built-in shutter, so occlusion resistance was also calculated. spirometry focuses on the results of the forced expiration manoeuvre. patients are asked to inhale as deeply as possible, then exhale as forcefully as possible until forced vital capacity (fvc) is reached. this maximum effort breath effort can be difficult to achieve for small children and other patients with limited levels of cooperation or lung function. additionally, large muscular expiratory effort can increase airway resistance as airways are constricted, and even cause gas trapping if small airways collapse. hence, the test described in this paper assesses the lung mechanics of tidal breathing. these mechanics represent lung condition at average breathing effort. average expiratory effort may prove a better marker of lung condition, and be more readily measurable on a regular basis, than peak expiratory effort. airflow measured during shuttering match simulated waveforms shown in fig. 11 . figure 1 shows typical pressure and flow waveforms measured at baseline added resistance for comparison. the average tidal flow is shown by the purple line in fig. 1 . this average flow was used to calculate flow caused by the shutter, which is shown by the dashed line in fig. 1 . the qv loop presented in fig. 2 is for this airflow attributed to the shutter. after flow attributed to the shutter was expected to have decayed away, the shutter-induced flow was still greater than zero. this non-zero flow was due to the tidal flowrate remaining elevated above the expected average tidal flow rate for the entire duration after shuttering. the response of a short linear region, typically 20-50 ml, followed by a long typically linear region with much lower or zero slope can be observed for all subjects at baseline added resistance. non-linear and non-zero regions in the qv loop represent time-varying mechanics not present in the average tidal breathing waveform. range. when collating all shuttered breaths, outliers were defined as data greater than 1.5 standard deviations above the 75th or below the 25th percentile. in addition, the decay rate was not calculated if the decaying range identified contained less than 3 datapoints. no other methods were used to exclude data from analysis. table 1 contains details of the decay rates measured for all subjects for all external resistance levels. decay rates measured range from 0.1 to 48. flow decay rate is inversely proportional to resistance. so as resistance is added to the system, measured decay rate is expected to decrease. however, this trend was only observed for 7/17 subjects (subjects 1, 3, 5, 11, 14, 16, 17) , and only for the first three resistance levels (0, 0.4, 0.8 cmh 2 os/l). figure 3 shows the trends observed for all subjects. no clear trend can be identified, with the average decay rate at each resistance level remaining unchanged. additional resistance was added to the spirometer mouthpiece via venturis specifically designed for this study. the venturi was inserted between the mouthpiece and flow sensor. this positioning added noise to the flow measurements due to turbulent airflow from the venturi. an example is shown in fig. 4 . this noise increased with increasing resistance, and was large enough for some subjects to obscure the underlying waveform, preventing any measurements of decay rate. lung elastance calculated at shutter opening is presented in table 2 , and the effect of added resistance on elastance is shown in fig. 5 . elastance values range from 3.9 to 22.1 cmh 2 o/l, and generally showed small intra-subject variation for each resistance level with a typical standard deviation less than 1.0 cmh 2 os/l. no clear trend was observed between elastance and added resistance, with elastance remaining fairly consistent between resistance levels. r rs separated from flow decay rate is presented in table 3 , and fig. 6 shows how r rs changes with added resistance. at all levels of added resistance, excluding subject 9, measured r rs was very small. the largest resistance of 2.84 cmh 2 os/l was measured for subject 12. generally, r rs was less than the value of added resistance alone. r rs does not increase proportional to added resistance, as had been expected. expiratory elastance calculated by this test is presented as positive, due to the spirometer defining positive pressure as expiration. when pressure is typically measured for calculating lung mechanics, such as during mechanical ventilation, positive pressure is defined as inspiration. as such, the negative of elastance was used to separate resistance from decay rate. table 4 shows rocc calculated for each subject, and the effect of external resistance on rocc is shown in fig. 7 . rocc ranged from 3.0 to 8.0. measured resistance is expected to increase proportional to the added resistance. on average, rocc results the exponentially decaying region was easily located for all subjects, except subject 9. the shutter duration used in this study (200 ms) was twice as long as the minimum required. longer shutter duration gives more time for subjects to react to the shutter. subject 9 appeared to react to shuttering by significantly reducing respiratory effort after approximately 100 ms of shutter closure, or an air-leak was created around the mouthpiece. this flow reduction resulted in incorrect measurements of decay rate, and, consequently, for the resistance calculated from the decay rate. an assumption used in data analysis was the airflow in response to shuttering would be superimposed on average tidal breathing. due to the reduction in airflow for subject 9, the effective airflow induced by the shutter is negative, as seen in fig. 8 . as a result, the correct region could not be identified. in general, the measured decay rates had fairly large intra-subject variation with the standard deviation often as high as 30% of the mean value. subjects 2 and 3 in particular had extremely large variation in measured decay rate. these subjects had a variety of different looking post-shutter waveforms, but no significant reduction in driving pressure. however, the changes in airflow shape indicate possible muscular reaction to shuttering. hence, even though the decaying flow is correctly identified, the decay rate calculated may be incorrect, because it is not possible to separate the effects of the shutter from unexpected muscular reaction. the degree of muscular reaction to shuttering is expected to decrease with shorter occlusion duration. hence, higher breath-to-breath consistency should be achieved by decreasing the duration closer to 100 ms. in addition, subjects in this study were fig. 8 flow measurements for subject 9 were lower than expected after shutter release. airflow measured after shuttering was less than average tidal airflow (purple line) and pressure reduced during shutter closure, indicating a muscular reflex in response to the shutter page 10 of 16 howe et al. biomed eng online (2020) 19:32 required to consciously breathe deeper than usual due to minimum flow rate limitations. if this limit were reduced or removed, breath-to-breath variation may reduce, because each breath could be driven subconsciously. the expected range of static elastance for healthy lungs is 2-10 cmh 2 os/l [5] [6] [7] . elastance measured in this study generally fit into this expected range, indicating the majority of airflow could be attributed to lung elastic recoil. there was no consistent trend between measured elastance and added resistance. however, elastance tended to be slightly higher when external resistance was added. these results suggest external resistance and the use of shuttering may not significantly affect measured elastance, but some breath-to-breath elastance change should be expected. elastance measured for subjects 11, 13, 14, and 16 was higher than expected. for healthy, tidally breathing subjects, increased elastance indicates muscular breathing effort. the additional elastance due to muscular effort cannot be separated from static elastance without further measures. hence, expiratory breathing effort during mechanics measurement could indicate restrictive disease where none is present. however, the measurement of combined elastance of tidal breathing may provide clinically relevant information. a larger than expected elastance during quiet tidal breathing indicates an elevated work-of-breathing, which could negatively impact quality of life. r rs was much smaller than expected. the typical range of airway resistance for healthy subjects is around 1.5-2.5 cmh 2 os/l [8, 9] . however, r rs was less than 1.3 cmh 2 os/l for all subjects at baseline added resistance. often, r rs calculated was less than added external resistance alone. r rs was not significantly or consistently affected by added external resistance. airflow begins in areas in the centre of airways with low skin friction effects. because airflow induced by the shutter decays quickly, typically only 20-50 ml of air is involved. the small volume does not allow enough time for air to flow from higher friction areas, resulting in a poor airway resistance estimate. as a result, monitoring decay rate of flow in response to shuttering or other pressure impulses only gives information on lung elastance. however, separate resistance measurements are possible during shuttering. rocc is an established method to measure airway resistance during tidal breathing. the combined resistance of plethysmograph and mouthpiece was 1.5 cmh 2 os/l. subtracting this resistance from rocc measured at 0 cmh 2 os/l added resistance, all subjects fell into the range 1.9-5.2 cmh 2 os/l. rocc increased as external resistance was added. however, the increase in rocc was not consistently the 0.4 cmh 2 os/l expected, showing a limitation of the single-compartment model. in general, the average tidal qv loop for all subjects showed a linear end-expiratory relationship between flow and volume, as shown in fig. 9 . this result suggests healthy lung mechanics of end-expiration are relatively constant during tidal breathing. hence, when instantaneous mechanics are measured during end-expiration, as in this study, the mechanics can be expected to represent the entire end expiratory portion. without further measures, the contribution of static lung tissue elastance cannot be separated from the dynamic elastance created by muscular breathing effort. subjects in this study showed a range of breathing effort. increased breathing effort was shown to clearly obscure passive elastance in some cases. however, subjects with respiratory illness, such as copd, may exhibit less end-expiratory breathing effort, as their capacity for breathing effort is reduced. the shutter closure duration was quite long, at 200-250 ms. reducing occlusion to 100 ms may lead to more consistent intra-and inter-subject results. in particular, because there is less time for subjects to react to shuttering, the adaption to shuttering would be limited. hence, there is a tradeoff between shutter duration, sampling rate, and sensor noise to assess mechanics in this way. although qv loops of tidal breathing suggest lung mechanics remain fairly constant at end-expiration, the shutter may cause changes in lung mechanics. the large pressure built-up in the respiratory system during occlusion may cause airways to increase in diameter, reducing their resistance to airflow. additionally, lung viscoelasticity tends to increase the pressure during shutter closure. the viscoelastic pressure build-up may increase measured elastance calculated at shutter re-opening. access to a plethysmograph, as used in this study, or a spirometer with built in shutter may be limited. to broaden access to this test, a simple, portable, hand-held device could be used to measure these lung mechanics. this proof-of-concept study describes a novel method to non-invasively measure lung mechanics of tidally breathing subjects. this test was able to identify reasonable dynamic lung elastance along with rocc. measurements of both mechanics were within the expected physiological range for healthy subjects breathing with elevated muscular effort. rocc was able to follow changes in upper airway resistance with some subjectdependent variation, as expected. clinically, this lung function test could impact current practice. it does not require high levels of cooperation from the subject, allowing a wider cohort of patients to be assessed. additionally, this test could be widely accessible as it can be implemented with either a small standalone device, or standard lung function testing equipment. the single-compartment lung model is simple and easy to understand [10] . this model has been further developed to give the dynamic elastance, single-compartment lung model, which describes the elastic properties of the lung as a combination of static and time-varying components [11] . this model has been used in the intensive care unit (icu) to estimate lung mechanics for mechanically ventilated patients spontaneously breathing on top of ventilator support [11, 12] . the model has also been extended to predict future lung condition for adult icu patients [13, 14] the model used in this study is the dynamic elastance, single-compartment lung model. respiratory muscles produce the driving pressure needed to oppose passive respiratory elastic forces, such as lung and chest recoil, and maintain airflow through respiratory airways. hence, the model is defined: where e dy is the dynamic elastance representing muscular breathing effort to create driving pressure, e rs is the sum of all passive respiratory system elastances, r rs is the combination of respiratory airway resistance and external resistances, v is volume, q is flow and t is time, direct measurement of respiratory driving pressure, e dy (t)v (t) , is not possible without highly invasive measures, such as an oesophageal balloon catheter. without direct measurement of driving pressure, lung mechanics cannot be simply identified. as an example, variations in individual lung mechanics can cause identical airflow in different subjects to be be created by vastly different driving pressures. this study makes use of two properties of the lung predicted by the single-compartment lung model. first, an exponentially decaying flow will be created in response to a large, sudden change in driving pressure [15] . this property can be shown by combining e dy and e rs into a total driving elastance term, e d . the result is a simple lung model describing the balance of elastic and resistive force in the lung: solving the resulting ode for q(t) yields: shows the decay rate of airflow in response to a change in driving pressure depends on a combination of the lung mechanics terms, e d and r rs . a second property predicted by the single-compartment lung model is that when there is no airflow, any pressure measured at the mouth must be due to elastic lung mechanics. if the airways are held open to the atmosphere, lung recoil and muscular effort will balance at atmospheric pressure. however, if breathing is occluded, the pressure measured at the mouth will be equal to the combination of driving pressure and lung recoil. a shutter built into a plethysmograph was used to induce exponentially decaying flow. the shutter occluded expiration for 200 ms, which is longer than the 100 ms minimum needed for pressure to equalise across the respiratory system [16] . when the shutter was released, pressure at the mouth dropped from driving pressure to atmospheric pressure, creating the exponentially decaying flow described by eq. 3. the lung's response to shuttering can be simulated with an electrical circuit, with the shutter modelled as a voltage-controlled switch, as shown in fig. 10 . figure 11 shows the simulated pressure and airflow measurements. the airflow measured after the shutter is re-opened is a superposition of tidal flow due to respiratory muscles and exponentially decaying flow caused by the shutter. the exponentially decaying airflow can be separated from the measured flow using adaptive filtering. the breath-to-breath variation of tidal breathing is low, with breaths typically having similar magnitude, duration, and flow profile. hence, the expected tidal airflow can be estimated by averaging all breaths before the shuttered breath. in this study, a minimum of 5 tidal breaths were recorded before each shuttered breath. the airflow created in response to the shutter re-opening is calculated by subtracting the average tidal airflow from airflow measured during shuttering. an assumption of the single-compartment lung model is that passive lung mechanics do not change during the breath. if muscular breathing effort also remains constant after the shutter is re-opened, the decay rate of flow caused by the shutter, e d /r rs , can be calculated with a linear fit to the trace of airflow vs volume (qv loop). however, the elastance and resistance cannot be separated by measuring the decay rate alone. an role of spirometry in primary care standardisation of spirometry indications for spirometry in outpatients with respiratory disease pulmonary function tests a simple method for measuring total respiratory compliance; normal values for males reference values for dynamic and static pulmonary compliance in men pulmonary compliance normal values for respiratory resistance using forced oscillation in subjects > 65 years old lung mechanics: an inverse modeling approach time-varying respiratory system elastance: a physiological model for patients who are spontaneously breathing model-based peep optimisation in mechanical ventilation development of a predictive pulmonary elastance model to describe lung mechanics throughout recruitment manoeuvres. ifac-papersonline basis function identification of lung mechanics in mechanical ventilation for predicting outcomes of therapy changes: a first virtual patient. ifac-paper-sonline expiratory model-based method to monitor ards disease state use of interrupter technique in assessment of bronchial responsiveness in normal subjects a graphical method for practical and informative identifiability analyses of physiological models: a case study of insulin kinetics and sensitivity on the separate determination of lung mechanics in in-and expiration inspiratory respiratory mechanics estimation by using expiratory data for reverse-triggered breathing cycles use of the interrupter technique in assessment of lung function not applicable. page 14 of 16 howe et al. biomed eng online (2020) 19:32 additional measure of either r rs or e d is required to separate the combined mechanics of the decay rate [17] [18] [19] .elastance is defined as the pressure in the lung per unit volume. pressure measured while airflow is occluded will approximate the respiratory driving pressure. hence, if muscular effort remains constant, the elastance after the shutter re-opens can be calculated from the pressure and volume measured momentarily before the shutter re-opens ( p 0 and v 0 , respectively):r rs was able to be separated from the decay rate after this elastance was calculated.additionally, occlusion resistance (rocc) was calculated following standard protocol [16, 20] . the gradient of pressure from 30 to 75 ms after the shutter was closed was extrapolated backwards to 15 ms before closure. the difference between this extrapolated pressure and the true pressure measurement at that time was divided by the airflow recorded at that time to produce an estimate for total airway resistance before occlusion.due to minimum flow limits built into the shuttering software employed, lung mechanics were measured while panting. for each test, the shutter was activated 5 times with a minimum of 5 normal breaths recorded before shuttering. extra resistance was added to the spirometer mouthpiece to simulate upper airway obstruction. the test was repeated twice at each resistance level. the resistances added were 0 (baseline), 0.4, 0.8, and 1.2 cmh 2 os/l, respectively. seventeen healthy subjects were enrolled in this study (8 female, 9 male, age 27±4.5, bmi 25±4, 3 smokers), where subjects were deemed healthy if they had no current respiratory disease or history of severe respiratory disease. smokers were included in this study. data were recorded using a ganshorn powercube body plethysmograph with lfx 1.8 respiratory diagnostic software. the shutter was controlled with lfx the datasets generated and analysed during the current study are not publicly available for privacy reasons but anonymised data are available from the corresponding author on reasonable request. the university of canterbury human ethics committee granted approval for this study, and the collection and use of the data analysed in this study. participants in this study gave their consent for the use of their anonymised data in this study. the authors declare that they have no competing interests. key: cord-009766-mdmqcvww authors: comerford, andrew; förster, christiane; wall, wolfgang a. title: structured tree impedance outflow boundary conditions for 3d lung simulations date: 2010-08-01 journal: j biomech eng doi: 10.1115/1.4001679 sha: doc_id: 9766 cord_uid: mdmqcvww in this paper, we develop structured tree outflow boundary conditions for modeling the airflow in patient specific human lungs. the utilized structured tree is used to represent the nonimageable vessels beyond the 3d domain. the coupling of the two different scales (1d and 3d) employs a dirichlet–neumann approach. the simulations are performed under a variety of conditions such as light breathing and constant flow ventilation (which is characterized by very rapid acceleration and deceleration). all results show that the peripheral vessels significantly impact the pressure, however, the flow is relatively unaffected, reinforcing the fact that the majority of the lung impedance is due to the lower generations rather than the peripheral vessels. furthermore, simulations of a hypothetical diseased lung (restricted flow in the superior left lobe) under mechanical ventilation show that the mean pressure at the outlets of the 3d domain is about 28% higher. this hypothetical model illustrates potential causes of volutrauma in the human lung and furthermore demonstrates how different clinical scenarios can be studied without the need to assume the unknown flow distribution into the downstream region. mechanical ventilation is an indispensable tool for the survival of critical care patients suffering from acute respiratory distress syndrome ͑ards͒ or acute lung injury ͑ali͒. however, the mortality rates of these diseases remains high, approximately 40% ͓1͔. in addition, it is widely known that the use of mechanical ventilation is itself the cause of a number of further associated complications ͓2͔, which are collectively termed ventilator induced lung injury ͑vili͒. this is despite the more recent adoption of modern protective ventilation strategies, where lower tidal volumes are administered while retaining a positive pressure at the end of expiration, which increases lung functional residual capacity and alveolar recruitment. these protocols improve the situation considerably over conventional ventilation ͓3-5͔. however, there are competing ventilator strategies each with their own merits and drawbacks, for example, the level of positive end-expiratory pressure ͑peep͒ required by the patient ͓6,7͔. biologically, these aforementioned ventilator related diseases manifest themselves at the alveolar level and are characterized by inflammation of the lung parenchyma, albeit still much controversy surrounds the precise mechanisms ͓8͔. vili can be characterized into separate but not mutually exclusive protocol dependent mechanisms: these are volutrauma ͑alveolar overdistension͒, atelectotrauma ͑recruitment-derecruitment͒, and biotrauma ͑inflammation͒ ͓9͔. predisposition to these conditions is brought about by heterogeneous lung mechanics when the patient is suffering from ards or ali ͓9͔, for example, the functional capacity of a diseased lung can be reduced resulting in alveolar overdistension ͓10͔. mortality is usually caused via indirect mechanisms resulting from vili ͓11͔ in which a cascade of events leads to sepsis or multiple organ failure. understanding the reason why the lungs still become damaged or inflamed during mechanical ventilation is a key question sought by the medical community. computational modeling of the tracheobronchial region repre-sents an approach that can provide more in depth knowledge into how the human lung functions mechanically, both during normal breathing and with the assistance of mechanical ventilation. of particular interest are what conditions within the lung mechanically differ under both diseased and healthy conditions. currently, a number of limitations exist for modeling the lower airways ͑in this paper lower airways and higher generations are used synonymously to mean peripheral vessels͒. first the resolution of ct imaging is restricted ͑maximum 0.5 mm resolution͒, hence, smaller vessels are not visible for segmentation. second, even if the entire lung was fully segmentable, currently, it is not computationally feasible to simulate the full lung tree. due to these points, alternative methods for modeling the lung must be sought. previously, there have been extensive models of nonpatient specific human lungs ͓12-14͔ based around literature data such as weibel ͓15͔ and horsfield et al. ͓16͔ . furthermore, more recent studies have utilized patient specific ct data ͓17-19͔ and ct ovine lungs ͓20͔ under a variety of boundary conditions, including zero pressure conditions, prescribed flowrate, and impedance. in addition, the simulations of refs. ͓17,21͔ included for the first time the effects of fluid-structure interaction ͑fsi͒. there has been considerable work in the area of lung impedance, specifically related to 1d transmission line models ͓22-25͔. impedance of the lung is an extremely important phenomenon as it plays a central role in the development and distribution of lung disease. more specifically, the heterogeneity of lung disease is brought about by changes in impedance conditions within the lung. these changes further affect how flow and pressure distribute through the lung. the aforementioned impedance models assume 1d fluid mechanics throughout the entire lung. however, from previous 3d simulations it is well known that the flow in the upper portion of the lower airways is not accurately modeled with 1d equations since typical bifurcating flow phenomena, such as flow recirculation, exist. for this reason, a fully coupled 1d-3d approach must be used and is hence developed in this paper. the developed impedance is based on an asymmetric structured tree methodology as introduced and used for blood in refs. ͓26-28͔. this cited work has been modified in order to represent the physiological environment in the pulmonary tree. the coupling of the 1d model to the 3d model is achieved utilizing a dirichlet to neumann approach introduced in ref. ͓29͔ for arterial blood flow. the aim of this paper is to apply this modeling approach to lung simulations and also to study the mechanics of the lung under artificial ventilation. in particular, using such an approach, the entire conducting airway can be modeled efficiently obtaining important pressure and flow information. 2 methodology 2.1 3d geometry reconstruction. the geometry used in the simulations was segmented using the commercially available segmentation software mimics ͑materialise, leuven, belgium͒. the standard ct scans had a resolution of 0.6 mm. this allowed up to seven generations of the bronchial tree to be obtained. following segmentation the outlets of the geometry were cut normal to the flow direction and the geometry was exported in stereolithography ͑stl͒ format. figure 1 shows the lung model with outlets cut normal to the flow direction. 2.2 meshing. the geometry was then meshed in the commercially available meshing software harpoon ͑sharc, manchester, uk͒. however, before meshing, the outlets here extruded within harpoon in order to satisfy the requirements of the boundary conditions ͑see sec. 2.4.2͒. in this software, a high quality mesh in the complex bronchial tree is a matter of minutes, an example mesh with extruded outlet extensions is shown in fig. 2 . the final mesh contained approximately 2 ϫ 10 6 tetrahedral elements equating to 1.6ϫ 10 6 degrees of freedom. the governing equations for airflow in the tracheobronchial region are the time dependent incompressible navier-stokes equations. where u represents the velocity vector ͑u = ͓u , v , w͔͒, p is the air pressure, is the density, and is the dynamic viscosity. the numerical simulations were performed in our in-house multiphysics research code baci. this code takes advantage of message passing interface ͑mpi͒ parallelization, hence, the solution of large scale simulations is achieved in a realistic time frame. the fluid domain was spatially discretised with stabilized finite elements. in time, the equations were discretised using a one-stepscheme. the resulting linear system was solved using the generalized minimal residual iterative solver with multilevel preconditioning, which is implemented in our code using the open-source package aztec ͓30͔. for all simulations a time step size of 0.004s was used and the newton iterations were considered converged when the residual dropped below 10 −6 . the inflow boundary conditions used in the present simulations are based around normal breathing and mechanical ventilator prescribed flowrate under inspiratory conditions. for normal breathing, a sinusoidal inspiratory profile was utilized, q͑t͒ = a sin͑t͒, where a represents the flow amplitude and the circular frequency. this was done under two conditions with a mean flow of 0.2 l/s and 0.5 l/s, respectively, ͑fig. 3͑a͒͒. this basic breathing model in the present study was only used as a benchmark since under nonmechanical ventilation conditions the upper airways ͑nasal pharynx͒ should also be included, in particular, because the glottis can produce turbulence in the trachea and the first few generations of the bronchial airways. under mechanical ventilation conditions, the endotracheal tube is inserted into the level of the trachea, hence, we have not included the upper airways in our simulations. for mechanical ventilation, a typical protective ventilator constant flow profile, see fig. 3͑b͒ , was utilized. this profile is characterized by almost constant flow, apart from a rapid acceleration and deceleration period at the beginning and end of inspiration. due to the acceleration periods and high frequency oscillations, the mechanical ventilation is expected to lead to different flow and pressure dynamics due to the considerably higher slew rates. the two aforementioned profiles ͑sinusoidal and constant flow venti-lation͒ cover the situations, which are available to the anaesthetist as control variables for ventilation. at the walls of the model all velocity components are zeroed in order to enforce the no slip condition. furthermore, since this study wants to focus on the effects of boundary conditions, the walls are assumed to be rigid. future work will address the issue of fsi as has previously been investigated by our group ͓17͔, where simpler boundary conditions were used. 2.4.1 impedance tree. the airways beyond the 3d domain are modeled using a 1d approach previously described for arterial flow ͓26,27͔. this 1d approach is modified in order to model the pulmonary tree rather than the peripheral arterial tree. in order to calculate the impedance of the pulmonary tree, the airways are modeled as 1d flexible elastic tubes. to achieve this, the linearized 1d fluid momentum equation is used and is given by where u is the velocity in the streamwise direction, t time, density, viscosity, and r the radius of the vessel. the approximation of linearized 1d momentum is reasonable for the pulmonary tree due to the high wave speed. in addition to the momentum equation, we also have the 1d continuity equation where p is the pressure, q is the flowrate, and c is the vessel compliance, which is given by the following linearized state equation ͓22͔. with r as the vessel radius, e as the elastic modulus, and h as the wall thickness. the wall thickness values are determined, based on vessel diameter, from the human horsfield lung model given in ref. ͓24͔ . equations ͑3͒ and ͑4͒ can then be formulated into an expression for impedance in the frequency domain by utilizing womersley's solution, which is an analytical solution for oscillatory flow in a circular tube ͓31͔. the derived impedance equation expresses the impedance at the root of each airway segment ͑z͑0,͒͒ as a function of the impedance in the downstream airway tree ͑z͑l , ͒͒ and is given by where g is the product of the wave speed c and the vessel compliance c, l is the vessel length, and the circular frequency. the wave speed c is given by where a 0 is the cross-sectional area, f j is a function of the zeroth and first order bessel functions as calculated from womersley's solution, and is the density and compliance. the impedance of each individual branch is then summed in series and parallel in order to obtain the total impedance of a specific tree. at bifurcation points, the impedance of the parent vessel is related to the daughter vessels using a standard bifurcation condition. where subscripts p, l, and r represent parent, left daughter, and right daughter vessels, respectively. in addition, scaling of the tree is achieved by introducing a relationship between parent and daughter vessels. r l = ␣r p and r r = ␤r p ͑9͒ where ␣ and ␤ represent parameters for tree asymmetry. for example, if ␣ = ␤ = constant, the tree would represent a weibel lung tree ͓15͔. in this paper, both symmetric and asymmetric trees were investigated. asymmetric branching was based on averaged reported values in literature ͓32,33͔ with ␣ = 0.86 and ␤ = 0.7. recursively applying eqs. ͑6͒ and ͑8͒ from the root of the tree down to a critical radius provides the impedance of the lung tree in the frequency domain. in the current model, the impedance at the root of the tree is included but in all numerical examples in this study it was considered to be zero. realistic nonzero impedance at the root of the tree will be addressed in future models, hence, taking into account peripheral compliance. this will be achieved by coupling to coupled multiscale simulations of the alveolar region ͓34,35͔. to calculate the time varying pressure at the root of a specific tree, the impedance must be first transformed into the time domain via finally, a time varying pressure is obtained at the root of the artificial tree by convoluting the impedance z͑t͒ with the history of flow at a specific outlet, i.e., inflow curves utilized in the simulations "a… light breathing "q mean =0.2 l/s… and light activity "q mean =0.5 l/s… and "b… constant flow mechanical ventilation. note the ventilation curve was chosen based on the high slew rates and oscillations, which occur in the profile to understand how these affect lung mechanics. the above method was implemented into our in-house finite element computational fluid dynamics ͑cfd͒ code as part of a outflow boundary condition, which is explained in the following section. coupling. the coupling of the impedance model to the 3d domain was achieved using a previously described dirichlet-neumann approach, see ref. ͓29͔ for full details. this method has the advantage of being mathematically well posed and has proven to be numerically stable. in addition, it also has the advantage that the solution of 1d and 3d domain is achieved simultaneously. the method was implemented both explicitly and implicitly into our finite element cfd code, however, little difference was observed between the methods and the explicit version was utilized due to the faster solution times. in brief, given the downstream domain pressure, p ‫ء‬ ͑t͒ ͑obtained from the impedance model͒, the time dependent pressure at the outflow of the 3d domain can be represented by where ⌫ s is the outflow boundary surface, w is a weighting function, ĩ is the identity tensor, is the viscous stress tensor, and n is the outward normal. the second term ͑͒ is assumed to be zero, which inherently assumes that the flow is fully developed ͑ =0͒. to satisfy this condition, the outlets of the 3d model are extruded. all outlet extrusions were deemed suitably long such that the observed profile was parabolic and streamwise independent. furthermore, the effect that these extensions have on the pressure is minimal. this was further verified using the traction free condition with and without the extensions and comparing the pressure at the equivalent location. finally, the use of these pressure based boundary conditions, in particular, how the flow distributes, was verified by looking at the mean flow division ratio as a percentage of the inlet flow. the following distribution was obtained: right lobe ͑57%͒, left lobe ͑42%͒, right superior lobe ͑19%͒, right middle lobe ͑10%͒, right inferior lobe ͑28%͒, left superior lobe ͑24%͒, and left inferior lobe ͑18%͒. these flow divisions were deemed suitable based on reported literature values ͓16͔. the major advantage of this method is that no assumption needs to be made about the flow, hence, flow distribution is completely governed by the downstream region. further validation was provided by considering the pressure drop calculated with this coupled approach, we considered the pressure drop over the whole model for the varying flowrates. this instantaneous drop was approximately 33 pa for a flowrate of 0.5 l/s; if we consider the equivalent values reported in literature ͓36͔, the value shows good agreement. the bronchial pressure drop over the acceleratory phase is given in fig. 4. the results of the simulations show well documented characteristics for flow in a bifurcating tree, in particular, centripetal acceleration due to curving vessels leads to flow phenomena such as flow recirculation and secondary flow. this all arises from the need to satisfy the continuity condition, leading to dean like vortices forming in the daughter vessels. furthermore, the nonuniform geometry causes acceleration of the fluid in the higher generations ͑for clarification the lowest generation is the trachea͒, which is not observed in more idealized geometries ͑see flow vectors in fig. 5͒ . the narrowing of the vessels in these locations leads to jetting of the flow and, hence, pressure decreases. the flow remains relatively disturbed throughout the model, reinforcing the need for 3d simulations in the lower generations of the bronchial tree. in the higher generations, the flow is relatively uniform meaning the application of the impedance boundary conditions in these loca-tions is acceptable. the most notable change between standard traction free and impedance outflow conditions is the pressure distribution, both spatially and temporally. in fig. 6 , the spatial pressure distribution is compared for the two different boundary conditions: traction free ͑up to seven generation model͒ and impedance ͑seven generation model plus up to 13 generations of the 1d tree depending on the outlet size͒. evidently, there are marked differences. for the present model, the pressure variation between different outlets did not vary considerably due to the similarity in outlet size. the time history of pressure is completely different for the two different scenarios. from a physiological prospective the above is of utmost importance because determination and understanding of correct airway pressure drop plays an important role in lung mechanics. the above immediately reinforces the need to consider the downstream domain when modeling the tracheobronchial region. to observe the difference in more detail, normalized values were plotted ͑fig. 7͒. this shows that with the impedance conditions there is a slight narrowing of the pressure-flow loop; this indicates an increase in the resistive component of impedance. overall, the two loops are very similar exhibiting nonlinear behavior with peak pressure and flow basically coinciding. this is to be expected based on the similar outlet sizes throughout the model. the normalized pressure distribution within the 3d domain is effected only marginally by the impedance of the downstream domain. the result is rather interesting, as it suggests that the opposition to flow comes mainly from the upper parts of the airway rather than the impedance of the lower tree. however, the pressure change between impedance and traction free is significant and is likely to play a central role when fluid-structure interaction of the 3d domain is considered. figure 8 shows the distribution of mean pressure along two different centerline paths: left lobe ͑gray͒ and right lobe ͑black͒. overall there is a drop in the pressure descending the vessel. however, due to disturbed flow phenomena, pressure increases are observed in the higher generations. this result is in contrast to more idealized models, which lack the pressure variation due to local curvature changes. hence the pressure in such models tends to drop in a relatively uniform manner along the vessel. the results here also indicate the strong dependence on asymmetry, which previously has been reported to be an important factor in the resulting pressure distribution. in particular the distribution between the left and right lung lobe bear no relationship. furthermore, when the results are compared with the traction free model ͑data not shown͒ greater spatial gradients in pressure are observed. finally, it is seen from the results that the largest pressure drop is observed in the higher generations. the effect of different trees on the pressure distribution in the 3d domain is significant, however, this is to be expected as the impedance is dependent on the size of the tree, i.e., the smaller the tree the greater the impedance. this is demonstrated in fig. 9 , where two different symmetric weibel trees ͑␣ = ␤ = 0.5 and 0.9͒ are attached to the end of the model. evidently the pressure in the 3d domain is considerable higher. in particular the resulting pressure in the faster diminishing tree is higher because essentially the tree can accommodate less flow ͑due to reduced cross-sectional area͒. this represents a constriction of the entire lung tree. the above suggests that the depth of the tree is the most important factor, i.e., the number of generations. this means the structure of the downstream tree is relatively insignificant to the spatial pressure distribution, especially the difference between symmetric and asymmetric trees. overall, the models utilizing an asymmetric tree are better because they are a closer representation of in vitro measured trees, however, some experimental validation based around pressure measurements is still required. in all presented models the effects on flow appear to be relatively minimal, there is a small change in the field, however, this appears to be relatively insignificant. finally, we consider how the boundary conditions can be used to simulate hypothetical disease in the human lung. the current impedance model has the advantage that no a priori assumptions need to be made about the flow since the flow in the 3d domain is governed by the peripheral vessels. this is a major advantage over previous impedance implementations. here, we consider a light constriction of the left superior lobe, i.e., a lung of reduced functional residual capacity. this could represent clinical scenarios such as alectasis or a derecruited region of the lung. this model is only indicative and is not meant to reproduce the complex nature of true lung disease since lung disease formation tends to be heterogeneous. the constriction of the airways is modeled by increasing the impedance on the outlets. here, the model responds by letting less flow into this lobe and naturally diverting flow to other regions of the lung. this shows that with our method we can simulate heterogeneous changes in the lung. furthermore, considfig. 6 comparison of pressure contours, for light activity, at maximum inspiration: "a… traction free boundary condition "up to seven generations… and "b… impedance boundary condition"up to 17 generations…. with the impedance conditions the maximum pressure is approximately 44% higher. note the lower limit of the scale is set to 5 pa for visual comparison purposes and for the traction free condition the pressure at the outlets is virtually zero, meaning the pressure in the rest of the tree would be negative. ering fig. 10 , the pressure-flow relationship is only really affected in the vicinity of the occlusion. in figs. 10͑a͒ and 10͑b͒ , it is evident that there is a widening of the loop ͑hysteresis͒ indicating an increase in the phase difference between pressure and flow. however, in other parts of the lung the relationship remains very similar ͑between disease and undiseased scenarios͒. this suggests the response is quite local, i.e., pressure and flow is increased throughout the lung, however, the dynamic between the two remains similar. under mechanical ventilation complex flow dynamics result from both geometric and temporal influences. ultimately geometry is the major factor in the resulting flow distribution, however, the rapid deceleration observed late in the inspiratory phase results in larger recirculation zones forming in the higher generations. the flowrate difference between the two different boundary conditions shows very similar profiles ͑data not shown͒. in addition, the instantaneous flowrate difference is maximal 4% higher under impedance conditions. the most nofig. 7 normalized pressure-flow relationship in the bronchial airway. evidently the pressure and flow are consistently in phase throughout the cycle. this is more noticeable for impedance conditions, where a narrowing of the loop is observed. for the impedance condition, the temporal development of pressure is more rapid. the difference in flowrate between the two conditions is marginal with the flowrate being overall reduced under impedance conditions "data not directly shown, however, at each point along the two above curves the flow is nearly identical…. table change between standard traction free and impedance outflow conditions is again the pressure distribution, both temporally and spatially. in particular the max pressure difference between the two different boundary conditions at peak inspiration is 27% in the second generation ͑traction free condition is 27% lower than impedance͒. the observed temporal gradients in pressure are interestingly high in the higher generations, however, the dynamics tended to be similar. this effect is driven by the nonuniform geometry. the actual maximum pressure level for the first four to five generations generally varies only a small amount. interestingly in the left lobe of the lung, the pressure over the cycle in the third generation is higher than in the second. this is driven by the flow route, more air is diverted into the left superior lobe, where the pressure is reduced ͑data not shown͒. this results in less air flowing into the left inferior lobe. the flow route is primarily driven by the curvature of the second generation of the left hand side of the lung, where the air is skewed toward the interior surface of the lung, hence flow continuity results in secondary currents, which lead to flow diverting back toward the anterior surface. this aforementioned phenomenon is primarily due to the nonuniformity of the geometry, meaning the cross-sectional area change plays an important role in the pressure and flow distribution. this particularly suggests that models based around circular cross sections fail to capture essential dynamics such as pressure variations due to cross-sectional changes. figure 11 compares the pressure-flow relationship between ventilation and breathing. clearly there is a change in this relationship. this is evidenced by the pressure-flow loop area increase, hence indicating that inertial effects are important ͑pressure is leading flow͒. this relationship is also seen to be much more nonlinear. this is specifically true during the acceleration phases. the small lag that does occur comes about due to transient fluid inertia, which results directly from higher slew rates observed during this ventilation protocol. ultimately the flow in the lung can be characterized as quasi-steady ͑womersley numbers ϳ1͒, which means the flow responds to the changing pressure gradient in a very rapid fashion. both boundary conditions, under mechanical ventilation, exhibit phase differences; this indicates that the upper generations contribute more dominantly to the lung impedance than the peripheral vessels, this is also backed up by the dynamics being not significantly different. however, the peripheral vessels are obviously important for correct pressure distribution/level in the lower generations, as indicated by the large difference between pressure levels. again a hypothetical diseased lung is simulated. the results are shown in fig. 12 . most interesting is the result in the superior left lobe ͑fig. 12͑b͒͒, where the increased impedance on the branch results in an in phase relationship ͑between pressure and flow͒ during acceleration and deceleration. this particularly shows a very different relationship to that of the healthy profile and highlights that the method of ventilation is important. modeling this reduced functional lung results in an overall increase in pressure in the lung, see fig. 13 . in particular, the mean pressure plotted along a centerline ͑gray centerline from fig. 8͒ is elevated throughout and at the outlet ͑located in the inferior left lobe͒ it is approximately 28% higher. the pressure is up to 38% higher in the vicinity of the occluded area. similar observations are also seen in the right lobe. these results mean that the nondiseased part of the ventilated lung is potentially at risk from volutrauma due to an increase in lobular pressure over a normal functioning lung. in this paper we have developed and used structured tree outflow boundary conditions for modeling the lower airways of the lung tree up to 17 generations. the coupling of this 1d model to the 3d domain uses a dirichlet to neumann approach. this approach is most general as it makes no assumption about the form of the downstream domain. furthermore, the method is applied in a mathematically well posed formulation, i.e., avoiding a full dirichlet problem. currently the impedance model is based purely around a structured tree, however, the present implementation alfirst the boundary conditions were compared under normal breathing conditions, demonstrating that there is a clear difference between the pressure levels. in addition, different trees were attached to the outlets, specifically, a weibel tree with different scaling coefficients. this showed that the pressure level can change significantly. the faster scaling tree coefficient ͑␣ = ␤ = 0.5͒ represents a tree of reduced volume, causing elevated pressure in the lungs. this highlights the importance of the small airways on the pressure levels in the lower generations. utilizing models in this way allows the effects of small airways to be investigated, which otherwise remains difficult ͓38͔. we further investigated the effects of constant flow ventilation on lung mechanics. the high slew rate ventilation curve resulted in larger recirculation zones in higher generations, particularly during the deceleration phase. the higher frequencies of the mechanical ventilation profile led to a different pressure-flow dynamic, in particular during the rapid acceleration/deceleration phase and where the flow became slightly negative ͑compare fig. 11͒ . the wider pressure-flow loop indicates a phase lag between pressure and flow; this is in contrast to normal breathing, where the pressure and flow remain essentially in phase. this different dynamic is due to the effects of transient fluid inertia. however, generally the flow in the lungs is quasi-steady since there is only a small lag between pressure and flow, i.e., flow responds rapidly to the changing pressure gradient. this is to be expected given the womersley number is only marginally above one for the lower generations and below one for higher generations. overall, for all models the flowrate did not change considerably. the fact that the impedance does not change the flow magnitude significantly suggests that models, which are interested in flow related phenomena only, such as particle distribution, potentially do not require such complicated impedance type boundary conditions. at the very least, such simulations can consider only resistance at the outlet terminals. however, in making such an assumption, a significant number of generations must be considered in the model in order to capture the relevant pressure-flow dynamics, i.e., the impedance is predominantly from the upstream area of the model. this is supported by previous medical observations, where it is reported that around 10% of the resistance comes from the small airways ͓39,40͔. from a flow physics per-spective, this does not mean the number of generation levels, rather it requires that the reynolds number at the outlet is sufficiently low. modeling diseased lungs is essential for understanding ventilation of diseased patients and can also provide insight into how different mechanical maneuvers affect the diseased lungs. understanding how the lungs respond under mechanical ventilation is currently an unresolved question still trying to be elucidated by the medical community ͓41͔. here, a partial obstruction of the superior left lobe was investigated in order to see the effects on the rest of the lung. this simulation also demonstrated the usefulness of the coupling method, where no assumption is made a priori about the flow into the peripheral airways, i.e., the flow is realistically governed by the impedance in this downstream region. the obstruction was not meant to model the complexity of lung disease but instead see the effects of the peripheral vessels on the lung dynamics. however, it could be indicative of a downstream derecruited region. the results demonstrated for both normal breathing and mechanical ventilation that the major changes in pressure-flow dynamics tended to be localized to the vicinity of the region, where the constricted vessels are located, i.e., the other regions of the lung showed increased pressure and flow, however, the relationship between the variables is virtually unchanged. in these other regions, the mean outlet pressure was on average approximately 28% higher, which could potentially result in overdistension ͑volutrauma͒ of the downstream alveoli due to elevated lobular pressure. the model here, although completely idealized, demonstrates that diseased lungs respond differently to healthy lungs. further work is required in this area to understand heterogeneous lung disease. this model represents a step in the direction to understand the complex lung mechanics occurring during mechanical ventilation. one of the major areas, which requires understanding is the effect of different mechanical maneuvers on lung mechanics in a diseased state. in literature, alternative approaches related to modeling the total tracheobronchial airways have been proposed ͓42,43͔. in ref. ͓42͔ , a 15 generation model of the airway system was simulated by calculating the flow in a 3d weibel triple bifurcation and then repeatedly reproducing this section in parallel, where the outflow from each section becomes the inflow for the next downstream bifurcation unit. in ref. ͓43͔ , an idealized 17 generation model based on an anatomical data set was simulated under steady state conditions, however, their mesh used in the model was very course. although these two methods provide some useful insights into flow structures in the conducting airways our approach has a number of advantages: we can model the whole conducting airway utilizing a realistic geometry for the first seven generations and then attach 1d trees as boundary conditions onto the 3d domain; the 1d domain takes into account the effects of wall movement; we are utilizing transient simulations; and we have a framework in which we have demonstrated that we can model diseased state conditions. future work will also encompass the expiration phase, which is nontrivial from a computational perspective. the simulations will also take into account fsi effects using our vast experience in fsi and the efficient implementations in our in-house code ͓21͔. this is expected, given the change observed in pressure under impedance conditions to lead to different flow and stress dynamics in the tracheobronchial region. in particular significant changes are expected to be seen in the higher generations due to the reduced wall cartilage content. currently the 1d model is attached to the outlet of the 3d model obtained from the ct data, future models will include artificial bifurcations based on morphological data to extend the 3d model a few extra generations, this is in order to limit any error associated with using a 1d model. ultimately, we are working toward a whole lung model, hence, work is currently being undertaken to couple the model presented here to models of the alveolar region ͓44,34͔, therefore we will have a model of the tracheobronchial tree coupled to the acinar region. this is expected to alter the results since this is a major part of the respiratory system. this is in the hope that further understanding of the lung, specifically the alveoli under healthy and diseased conditions can be ascertained. finally, the present models have provided detailed insight into the effects of the peripheral airways on breathing mechanics and also the use of coupling for lung simulations. incidence and outcomes of acute lung injury effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial mechanical ventilation in acute lung injury and acute respiratory distress syndrome effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome low mortality associated with volume pressure limited ventilation with permissive hypercapnia in severe adult respiratory distress syndrome higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome mechanisms of ventilator-induced lung injury: the clinician's perspective acute lung injury protective ventilation of patients with acute respiratory distress syndrome a lung computed tomographic assessment of positive end-expiratory pressure-induced lung overdistension the acute respiratory distress syndrome modelling of peak-flow wall shear stress in major airways of the lung modeling the bifurcating flow in a human lung airway airflow structures and nano-particle deposition in a human upper airway model morphometry of the human lung models of the human bronchial tree fluid structure interaction in lower airways of ct-based lung geometries an anatomically based hybrid computational model of the human lung and its application to low frequency oscillatory mechanics characteristics of the turbulent laryngeal jet and its effect on airflow in the human intra-thoracic airways characteristics of airflow in a ct-based ovine lung: a numerical study coupling strategies for biomedical fluid-structure interaction problems wave propagation, input impedance, and wall mechanics of the calf trachea from 16 to 1,600 hz airway inhomogeneities contribute to apparent lung tissue mechanics during constriction how heterogeneous bronchoconstriction affects ventilation distribution in human lungs: a morphometric model a morphometric model of lung mechanics for time-domain analysis of alveolar pressures during mechanical ventilation structured tree outflow condition for blood flow in larger systemic arteries numerical simulation and experimental validation of blood flow in arteries with structured-tree outflow conditions fractal network model for simulating abdominal and lower extremity blood flow during resting and exercise conditions outflow boundary conditions for three-dimensional finite element modeling of blood flow and pressure in arteries the physics of pulsatile flow ct-based geometry analysis and finite element models of the human and ovine bronchial tree a three-dimensional model of the human airway tree towards a comprehensive computational model for the respiratory system a nested dynamic multi-scale approach for 3d problems accounting for micro-scale multi-physics pulmonary fluid dynamics generation of an anatomically based three-dimensional model of the conducting airways the role of small airways in lung disease small airways: a time to revisit? the physiology of small airways alveolar recruitment in acute lung injury an adjustable triple-bifurcation unit model for air-particle flow simulations in human tracheobronchial airways computational model of airflow in upper 17 generations of human respiratory tract modeling the mechanical behavior of lung tissue at the micro-level we acknowledge the support of deutsche forschungsgemeinschaft ͑dfg͒ through project no. wa-1521/8. the ct images of the tracheobronchial tree have been provided by dr. med ulrich barkow and dr. med. univ. andreas strohmaier from "diagnostische radiologie," stuttgart, germany. key: cord-012884-56z95uca authors: bargagli, elena; refini, rosa metella; d’alessandro, miriana; bergantini, laura; cameli, paolo; vantaggiato, lorenza; bini, luca; landi, claudia title: metabolic dysregulation in idiopathic pulmonary fibrosis date: 2020-08-07 journal: int j mol sci doi: 10.3390/ijms21165663 sha: doc_id: 12884 cord_uid: 56z95uca idiopathic pulmonary fibrosis (ipf) is a fibroproliferative disorder limited to the lung. new findings, starting from our proteomics studies on ipf, suggest that systemic involvement with altered molecular mechanisms and metabolic disorder is an underlying cause of fibrosis. the role of metabolic dysregulation in the pathogenesis of ipf has not been extensively studied, despite a recent surge of interest. in particular, our studies on bronchoalveolar lavage fluid have shown that the renin–angiotensin–aldosterone system (raas), the hypoxia/oxidative stress response, and changes in iron and lipid metabolism are involved in onset of ipf. these processes appear to interact in an intricate manner and to be related to different fibrosing pathologies not directly linked to the lung environment. the disordered metabolism of carbohydrates, lipids, proteins and hormones has been documented in lung, liver, and kidney fibrosis. correcting these metabolic alterations may offer a new strategy for treating fibrosis. this paper focuses on the role of metabolic dysregulation in the pathogenesis of ipf and is a continuation of our previous studies, investigating metabolic dysregulation as a new target for fibrosis therapy. idiopathic pulmonary fibrosis (ipf) is a severe chronic interstitial lung disease (ild) of unknown aetiology [1] , limited to the lungs. median survival is from 3 to 5 years after diagnosis [2, 3] . the disease is characterized by a radiological and histological pattern of usual interstitial pneumonia (uip) [4] with parenchymal fibrosis and excess collagen deposition [5] . it mainly affects ex-smoker adults over 65 years, causing dyspnoea, dry cough and the progressive loss of respiratory function [6] . fibroblast/myofibroblast proliferation and the activation of alveolar epithelial cells with progressive extracellular matrix deposition in the lung parenchyma destroy lung structure [7] . antifibrotic agents (nintedanib and pirfenidone) reduce the progression of ipf [8, 9] . the pathogenesis of ipf remains unclear, despite the growing number of studies. there is an urgent need for reliable biomarkers for early diagnosis and to monitor progression. recent studies have identified potentially useful peripheral and bronchoalveolar lavage (bal) biomarkers, including chemokines, cytokines, genes and proteins [10] [11] [12] [13] [14] [15] , but the 2018 international guidelines for the clinical diagnosis of ipf do not include recommendations, due to insufficient evidence [16] . of modified extracellular lipids by foamy macrophages could be a key event in the evolution of fibrosis towards acute exacerbation [43, 44] . the dysregulation of calgranulin a8 (s100a8), a1at and albu suggests "il-12 signalling and production in macrophages" and the "production of nitric oxide and reactive oxygen species in macrophages", stressing the role of macrophages in acute exacerbation [20] . another proteomic study by landi et al. on the bal of patients with pulmonary fibrosis related to systemic sclerosis showed the dysregulation of proteins related to lipid metabolism, such as lysozyme c, fabp4, rbp4 and hp, functionally correlated with ppar-γ, underlining the idea of lipid metabolic alterations concomitant with the onset and development of fibrosis [45] . in 2013 and 2014, our proteomic studies on the bal of ipf patients, compared with the profiles of healthy smoker and non-smoker controls, reported a dysregulation in proteins involved in the raas [18] . in particular, the down-regulation of angt in the bal of ipf patients with respect to the other conditions was reported. angiotensinogen is the substrate of renin and its down-regulation in ipf strongly suggests high renin and angt consumption to produce angiotensin i. indeed, renin converts angt into angiotensin i (ang i), which is in turn cleaved into angiotensin ii (ang ii) by angiotensin-converting-enzyme (ace), also typically found in the endothelial cells of the lung capillaries [46] , where raas, essential for blood pressure control and fluid homeostasis, plays an important role in pulmonary hypertension. plasma levels of angt are reported to be modulated by hormones such as corticosteroid, oestrogen, thyroid hormones and angiotensin ii [46, 47] , suggesting that hormone imbalance could influence raas and its dysregulation. the important role of raas in metabolic regulation is also explained by its two axes: ace-2/ang 1-7/mas which has antiproliferative, antioxidant and anti-inflammatory functions with beneficial effects on hypertension, glucose intolerance and insulin resistance (ir); and the counter-regulatory ace/ang ii/at1 [48] with opposite effects. ace-2 has been shown to play a protective role in lung disease through effects mediated by the mas oncogene. in the lung of ipf patients, ace-2 is reported to be significantly depleted [49] , suggesting that raas is important in the pathogenesis of ipf. in turn, the dysregulation of raas components can trigger many metabolic functions, such as those observed in metabolic syndrome and diabetes mellitus type 2 [48] . indeed, in diabetic retinopathy, raas is an effector of vascular epithelial growth factor (vegf) that drive the leakage of retinal blood vessels [50] . vegf is also well known to be one of the major players in ipf onset and its receptor is one of the targets of nintedanib treatment [11, 51] . moreover, the production of angt and ang ii observed in white adipocyte tissue reveals the role of raas in promoting adipocyte hypertrophy and decreasing the number of small insulin-sensitive adipocytes [52] . these dysregulations, observed in metabolic disorders, also characterize aging, leading to pro-inflammatory and pro-fibrotic effects in different organs and inducing lung fibrosis, vascular dysfunction, myocardial fibrosis, nephropathy and insulin secretory defects with increased insulin resistance [53] . angiotensin ii is a versatile effector molecule with intracrine/autocrine/paracrine roles. it stimulates the adrenal cortex to secrete aldosterone [54] , a pivotal hormone that induces inflammation by the mrna expression of il-6 and tnf-α, and fibrosis by tgf-β. it promotes lipogenesis, thus increasing adipose tissue mass [46] and influencing the release of prostaglandins such as cyclooxygenase (cox) 1-derived prostaglandin e(2) [46] , found to be defective in epithelial cells and macrophages in the lungs of ipf patients [55] . cyclooxygenase is required for the production of lipid mediators such as prostanoid (e.g., prostaglandin pge2) associated with fibrotic lung disorders [56] . moreover, cox-derived prostanoids appear to play an important role in toll-like receptor response [56] . curiously, high glucose in cell cultures induces an increase in angiotensinogen, angiotensin-converting enzyme (ace) and at1r mrna levels, as well as ang ii and tgf-β1 concentrations, promoting the epithelial-mesenchymal transition and fibronectin synthesis [57] . aldosterone is also reported to impair metabolism and to stimulate the differentiation of 3t3-l1 cells and brown preadipocytes into mature adipocytes, influencing the expression of the adipokine gene [58] . alterations in raas are therefore closely related to metabolic disorders such as diabetes and dyslipidaemia, which may influence fibrosis pathways in different ways (table 1 ; figure 1 ). figure 1 ). schematic representation of the renin-angiotensin-aldosterone system (raas). agt (angt) in green represent the dysregulated protein that we found with the proteomic analysis. in red and blue, the opposite effects of raas. in green represent the dysregulated protein that we found with the proteomic analysis. in red and blue, the opposite effects of raas. our proteomic studies found dysregulated proteins such as hpt, transferrin, prdx1, prdx5, gstp1, ceru and albu in the bal of ipf patients; these proteins are related to hypoxia, oxidative stress and iron metabolism [18, 19] . the renin-angiotensin-aldosterone system that we have already described is also known to increase oxidative stress [59] , since ang ii is an inducer of reactive oxygen species [59] , which together with reactive nitrogen species are reported to be elevated in ipf [45, 97, 98] . a hypoxic stimulus, characterizing this pathology, increases the activation of hypoxia inducible factor-1α (hif-1 α), a transcription factor [60] , which together with constitutively expressed hif-1β, determines the transcription of hypoxia response elements (hres) with pivotal roles in metabolic reprogramming [61] . the transcription of hypoxia response elements triggers adaptive mechanisms, modifying glycolysis, angiogenesis, pro-survival signalling, cell proliferation and cell migration [62] . the hif-1 target genes include pyruvate dehydrogenase kinase 1, hexokinase 2 and lactate dehydrogenase, the modified expression of which gives rise to a glycolytic switch, similar to the warburg effect in cancer [62] , which also occurs in ipf, as already reported [63] . the warburg effect is accompanied by high lactic acid production which induces the epithelial-mesenchymal transition via the activation of tgf-β. moreover, the glycolytic switch to aerobic glycolysis not only produces little energy but also stabilizes hif-1α, which promotes tgf-β-induced myofibroblast differentiation [18, 60, 63, 64] . oxidative stress, the induction of hif-1α and the glycolysis switch are also related to extracellular matrix deposition, typical of fibrosis. extracellular matrix production is strongly linked to glycolysis, since the latter provides energy and building blocks for collagen production. another hypoxia response element is lysyl oxidase transcription, fundamental for collagen crosslinking, and typical of fibrotic tissue [64] . in line with the oxidative stress response and extracellular matrix deposition, stock et al. found that the sod2 mrna expression was markedly suppressed in both systemic sclerosis-ild and ipf fibroblasts, compared with non-ild controls, leading to an increasing trend in acta2 mrna expression and cell proliferation [65] . the hypoxic stimulus characterizing ipf also leads to pulmonary hypertension (ph) by progressive vasoconstriction and vascular remodelling. indeed, hypoxia induces the proliferation and differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts that ultimately, stimulate the recruitment of inflammatory cells and the release of key regulators. the proliferation and differentiation of pulmonary artery adventitial fibroblasts during chronic hypoxia are mainly dependent on the signal transduction of the p38 pathway and its downstream mediator, hif-1 [66] . studies on mesenchymal stem/stromal cells, also used in ipf therapy [67] , have shown that hif-1α expression not only influences glycolytic activity, but also mitochondrial oxidative phosphorylation under hypoxic conditions [61] . the latter normally enables the production of atp and is a major source of reactive oxygen species. in ipf, alveolar macrophages and mitochondrial reactive oxygen species are reported to be significantly more abundant and associated with a lower expression of mitochondrial-encoded oxidative phosphorylation genes [68] . another possible source of reactive oxygen species in ipf is the accumulation of iron in the lower respiratory tract, also reported to be induced by smoking. disordered iron regulation, as our findings suggested, may play a central role in the pathogenesis of oxidative stress-induced microscopic injury, triggering epithelial cell damage and fibroblast proliferation [69] . patients with ipf typically show excessive extracellular iron and macrophage hemosiderin, suggesting abnormal iron homeostasis leading to recurring microscopic injury and fibrosing damage [69] . various histological and bal ipf studies have also shown increased numbers of iron-laden macrophage clusters in the alveolar and interstitial spaces, driving macrophages to generate reactive oxygen species and leading to oxidative damage with dna breaks, lipid peroxidation and protein oxidation [70] . these findings suggest that oxidative stress and iron metabolic disorder create positive feedback, promoting the progression of fibrosis (table 1 ; figure 2 studies on mesenchymal stem/stromal cells, also used in ipf therapy [67] , have shown that hif-1α expression not only influences glycolytic activity, but also mitochondrial oxidative phosphorylation under hypoxic conditions [61] . the latter normally enables the production of atp and is a major source of reactive oxygen species. in ipf, alveolar macrophages and mitochondrial reactive oxygen species are reported to be significantly more abundant and associated with a lower expression of mitochondrial-encoded oxidative phosphorylation genes [68] . another possible source of reactive oxygen species in ipf is the accumulation of iron in the lower respiratory tract, also reported to be induced by smoking. disordered iron regulation, as our findings suggested, may play a central role in the pathogenesis of oxidative stress-induced microscopic injury, triggering epithelial cell damage and fibroblast proliferation [69] . patients with ipf typically show excessive extracellular iron and macrophage hemosiderin, suggesting abnormal iron homeostasis leading to recurring microscopic injury and fibrosing damage [69] . various histological and bal ipf studies have also shown increased numbers of iron-laden macrophage clusters in the alveolar and interstitial spaces, driving macrophages to generate reactive oxygen species and leading to oxidative damage with dna breaks, lipid peroxidation and protein oxidation [70] . these findings suggest that oxidative stress and iron metabolic disorder create positive feedback, promoting the progression of fibrosis (table 1; figure 2 ). an intriguing interactome that we observed in our work on ipf concerned fabp4, rbp4, hp, apoai and za2g, related to peroxisome proliferator-activated receptor gamma [18] . this intriguing network also emerged in our study, comparing the proteomes of the bal of patients with pulmonary fibrosis associated with systemic sclerosis and healthy controls [45] , corroborating the finding that this pleiotropic nuclear hormone receptor is involved in pathologies that progress to fibrosis. we found fabp4, also known as ap2, an adipokine primarily expressed in adipocytes and in macrophages, in bal. this adipokine plays a pivotal role in coordinating and integrating metabolic and inflammatory signalling in settings of insulin resistance, dysregulated lipid metabolism and an intriguing interactome that we observed in our work on ipf concerned fabp4, rbp4, hp, apoai and za2g, related to peroxisome proliferator-activated receptor gamma [18] . this intriguing network also emerged in our study, comparing the proteomes of the bal of patients with pulmonary fibrosis associated with systemic sclerosis and healthy controls [45] , corroborating the finding that this pleiotropic nuclear hormone receptor is involved in pathologies that progress to fibrosis. we found fabp4, also known as ap2, an adipokine primarily expressed in adipocytes and in macrophages, in bal. this adipokine plays a pivotal role in coordinating and integrating metabolic and inflammatory signalling in settings of insulin resistance, dysregulated lipid metabolism and inflammation [71] . fabp4 together with hnf4 and ppar-α are required to resist oxidative stress and regulate transcription to drive changes in lipid metabolism [72] . rbp4, another protein that we found dysregulated in bal samples, is a cytokine primarily produced by adipose tissue and connected to ppar-γ. it is the main transport protein for retinol in the blood. thus, rbp4 is known to regulate lipids, glucose metabolism and insulin resistance [73] . retinol bound to rbp4 and stra6, stimulated by retinoic acid (6), triggers the signalling cascade mediated by jak/stat (janus kinase/signal transducer and activator of transcription) that induces genes that inhibit insulin signalling and control lipid homeostasis [74] . the jak/stat pathway is crucial for transducing signals from a variety of metabolically important hormones and cytokines, including growth hormone, leptin, erythropoietin, il4, il6 and ifn-υ. this pathway is dysregulated in obesity and metabolic diseases [75] . a recent study reported the use of jak inhibitors on pathologies with lung fibrosis involvement showing that jak/stat plays a role in the development of fibrosis [76] . moreover, other studies highlight lung fibrotic stability after the administration of jak inhibitors such as baricitinib in patients with rheumatoid arthritis and lung involvement [77] . transcriptional factor ppar-γ, related to our differential proteins, is the master regulator of adipogenesis and has antifibrotic effects initiated by blocking tgf-β in organs such as the skin, lung and heart [78] . in adipogenesis, it determines an increase in mrna for lipogenic enzymes, adiponectin, insulin-dependent glucose transporter (glut4) and angt, with effects on lipid and glucose metabolism. curiously, it has been reported that the first-line antidiabetic drug metformin exerts potent antifibrotic effects by modulating metabolic pathways, activating ppar-γ signalling, inhibiting tgf-β, suppressing collagen formation and inducing lipogenic differentiation in lung fibroblasts derived from ipf patients [79] , but unfortunately it does not have clinically relevant outcomes in patients [80] . moreover, nintedanib and pirfenidone are reported to inhibit tgf-β in different ways, but do not induce lipogenesis as much as does metformin. fibroblast growth factor 1 (fgf1) is also linked to ppar-γ: mackenzie et al. reported it to be the growth factor most significantly increased in ipf patients, and that it co-localized in basal cell sheets, myofibroblast foci and surfactant protein-c positive alveolar epithelial type-ii cells, and with fgfr1, fgfr2, fgfr3, fgfr4 and myofibroblasts [81] . its relationship to energy intake has earned this growth factor the definition of metabolic regulator. different environmental signals (exposome) are reported to be sensed by ppar-γ, which drives adipose remodelling via fgf1. inadequate levels of fgf1 are also associated with insulin resistance [82] . the action of ppar-γ is likewise seen in lipofibroblast differentiation, where it prevents oxidative lung injury, promoting mesodermal differentiation and antioxidant defences. ppar-γ is also reported to indirectly stimulate lung surfactant production, so that the lung can adapt to atmospheric oxygen [83] . ppar-γ agonists promote adipocyte differentiation and adiponectin secretion [84, 85] . another recent proteomic study of ours on bal protein modulation after the nintedanib treatment of ipf patients highlighted the up-regulated apolipoprotein c3 in serum after one year of nintedanib administration, linked to ppar-γ by enrichment analysis [51] . an even greater number of studies highlight the lipid metabolic dysregulation in the onset and development of ipf, focusing on ppar-γ action and the production of adiponectins and leptins [86] . serum concentrations of adiponectin, as reported by d'alessandro et al., could be useful for predicting ipf prognosis, since they are inversely correlated with dlco percentages and body mass index. moreover, adiponectin levels in bal also demonstrated an inverse correlation with body mass index and a direct correlation with eosinophil percentages, both negative prognostic factors of ipf [86] . adiponectins are almost exclusively synthesized by adipocytes and are the most abundant adipose tissue-derived adipokine in plasma. they are a powerful hormone involved in lipid metabolism, insulin sensitization, apoptosis and inflammation, besides being a key molecule in several immunological pathways linked to carcinogenesis [87] . leptin levels are reported to be elevated in serum and correlated with the severity of lung fibrosis, since leptin significantly promotes the epithelial-mesenchymal transition in a549 cells, decreases autophagosome formation, inhibits the lipidation of lc3i to lc3ii, and up-regulates the expression of p62 by activating the pi3k/akt/mtor pathway [88] involved in the onset and development of ipf (figure 3) . interestingly, in line with our findings regarding lxr/rxr and fxr/rxr and the differentiation and activation of macrophages in ipf [20] , venosa et al. reported that treatment with fibrotic substances induces a time-related increase in large vacuolated macrophages and the accumulation of oxidized phospholipids in lung macrophages and epithelial cells, promoting the formation of foamy macrophages and their m2 activation. this treatment also increases phospholipids and cholesterol in bal fluid, with evident alteration of lipid-handling pathways under the control of the transcription factors lxr, fxr, ppar-γ and sterol regulatory element-binding protein [89] . lipid metabolism depends on proper mitochondrial function and correct interaction between mitochondria and er. correct outer mitochondrial membrane fusion is therefore essential to connect the intracellular network in order to regulate and meet cell metabolic demands. in turn, mitochondrial fusion is required for oxidative phosphorylation, mitochondrial dna biogenesis, mitophagy regulation and metabolic adaptation. chung et al. reported that mitochondrial fusion and lipid metabolism are closely linked to regulate alveolar epithelial cell ii injury and subsequent fibrotic remodelling by damaging surfactant lipid production. surfactants are a lung surface-active lipoprotein complex, containing about 90% lipids, that reduces lung surface tension, prevents the alveoli from collapsing, and plays a critical role in immune regulation [90] . mitochondria are fundamentally involved in the synthesis of fatty acids, which may be influenced by episodes of insulin-resistance, glycogenesis or the accumulation of glucose. these alterations lead to mitochondrial dysregulation that plays a role in defective autophagy, telomere attrition, altered proteostasis and cell senescence through the increased production of reactive oxygen species, decreased mitochondrial biogenesis and impaired mitochondrial macroautophagy [91, 92] . interestingly, in line with our findings regarding lxr/rxr and fxr/rxr and the differentiation and activation of macrophages in ipf [20] , venosa et al. reported that treatment with fibrotic substances induces a time-related increase in large vacuolated macrophages and the accumulation of oxidized phospholipids in lung macrophages and epithelial cells, promoting the formation of foamy macrophages and their m2 activation. this treatment also increases phospholipids and cholesterol in bal fluid, with evident alteration of lipid-handling pathways under the control of the transcription factors lxr, fxr, ppar-γ and sterol regulatory element-binding protein [89] . lipid metabolism depends on proper mitochondrial function and correct interaction between mitochondria and er. correct outer mitochondrial membrane fusion is therefore essential to connect the intracellular network in order to regulate and meet cell metabolic demands. in turn, mitochondrial fusion is required for oxidative phosphorylation, mitochondrial dna biogenesis, mitophagy regulation and metabolic adaptation. chung et al. reported that mitochondrial fusion and lipid metabolism are closely linked to regulate alveolar epithelial cell ii injury and subsequent fibrotic remodelling by damaging surfactant lipid production. surfactants are a lung surface-active lipoprotein complex, containing about 90% lipids, that reduces lung surface tension, prevents the alveoli from collapsing, and plays a critical role in immune regulation [90] . mitochondria are fundamentally involved in the synthesis of fatty acids, which may be influenced by episodes of insulin-resistance, glycogenesis or the accumulation of glucose. these alterations lead to mitochondrial dysregulation that plays a role in defective autophagy, telomere attrition, altered proteostasis and cell senescence through the increased production of reactive oxygen species, decreased mitochondrial biogenesis and impaired mitochondrial macroautophagy [91, 92] . caporarello et al. reported that restoring ppar-γ co-activator 1-alpha levels in ipf fibroblasts improved mitochondrial biogenesis and function and nad biosynthesis, and also influenced the fate of lipogenic fibroblasts through the induction of ppar-γ transcription [93] . proteomics coupled with systems biology studies, performed on bal samples from patients with ipf, healthy controls, other ilds and different phenotypes of ipf, has brought to light a number of interesting molecules that seem to be involved in the onset of fibrosis via mechanisms related to metabolic dysfunction [17] [18] [19] [20] 45] . we summarize our reports in table 1 , and the proteins indicated and discussed above have been uploaded on metacore software™ version 6.37 from clarivate analytics (clarivate analytics, boston, ma, usa; https://portal.genego.com/) in order to build the interactome reported in figure 4 . where apoa1, gstp1, a1at, angt, apoc3 (red circles) are the central functional hubs i.e., proteins with a high number of interactions with other modulators. an -omics approach to the study of ipf has made it possible to obtain a picture of an almost complete set of dysregulated proteins. bioinformatic analyses linked these proteins to precise molecular pathways, which until recently were tagged as related to specific fibrosis pathways, losing sight of their metabolic effects, and vice versa for the effect that metabolism could have on them. since recent discoveries regarding the pathogenesis of fibrosis have led to exciting therapeutic opportunities in the field of metabolic regulation, there has been a switch to research on metabolic pathways in ipf. following this suggestion, we also performed a bioinformatic analysis on metacore software by the tool "drug look up for your data" visualizing specific drug molecules ( figure 5 ) related to the differential proteins reported in table 1 , and considered a potential target of treatment. most of the reported drugs are molecules acting on metabolic and hormonal pathways such as androstanolone, linoleic acid, estradiol, nitric oxide (no), etc. based on our own as well as other findings, a fine balance between lipid metabolism and wound healing mechanisms leading to fibrosis is suggested in ipf onset and development. lipid metabolism as well as glucose anabolism and catabolism are dramatically subject to certain environmental and genetic influences, which may alter their equilibrium, leading to metabolic or fibrotic disorders. to corroborate these observations, various widespread diseases associated with the onset of fibrosis, including ipf, cirrhosis, hepatitis, non-alcoholic steatohepatitis, chronic kidney disease, myocardial infarction, heart failure, diabetes and scleroderma, share common metabolic pathways [49, 57, [94] [95] [96] . the liver and lung are reported to share many immune/inflammatory responses to damage through the lung-liver axis. metabolic dysregulation described in the liver could have repercussions at the lung level, just as the hepatitis c virus (hcv), which affects the liver, enhances the development of ipf and is considered a risk factor for ipf, and lungs and liver have resident macrophages that play key roles in mediating the immune/inflammatory response [49] . diabetes is another possible risk factor for the development of ipf [80] . glycogenesis, the accumulation of glucose and episodes of insulin-resistance also influence the synthesis of fatty acids, leading to mitochondrial dysregulation and contributing to defective autophagy, telomere attrition, altered proteostasis and cell senescence, with an increased production of reactive oxygen species, decreased mitochondrial biogenesis and impaired mitochondrial macroautophagy [91, 92] . interestingly, higher serum and bal fluid concentrations of hypomethylated cpg-rich mitochondrial dna, released by necrotic cells and viable cells in response to various stressors, have been reported in ipf patients than in healthy controls. this gives rise to the activation of macrophages and fibroblasts in certain experimental settings, inducing lung fibrosis [91] . -omics approach to the study of ipf has made it possible to obtain a picture of an almost complete set of dysregulated proteins. bioinformatic analyses linked these proteins to precise molecular pathways, which until recently were tagged as related to specific fibrosis pathways, losing sight of their metabolic effects, and vice versa for the effect that metabolism could have on them. since recent discoveries regarding the pathogenesis of fibrosis have led to exciting therapeutic opportunities in the field of metabolic regulation, there has been a switch to research on metabolic pathways in ipf. table 1 in a hub-centric network. apoa1 in hdl, gstp1, alpha 1-antitrypsin, angiotensinogen, apoc3 (red circles) are the central functional hubs i.e., the proteins with a high number of interactions with other modulators in the interactome. the red arrows indicate the inhibition, the green arrows mean inductions, and the grey arrows indicate a generic correlation. the teal highlighted lines indicate the well-known canonical molecular pathways. the relative figure legend is in supplemental material in figure s1 . following this suggestion, we also performed a bioinformatic analysis on metacore software by the tool "drug look up for your data" visualizing specific drug molecules ( figure 5 ) related to the differential proteins reported in table 1 , and considered a potential target of treatment. most of the reported drugs are molecules acting on metabolic and hormonal pathways such as androstanolone, linoleic acid, estradiol, nitric oxide (no), etc. table 1 in a hub-centric network. apoa1 in hdl, gstp1, alpha 1-antitrypsin, angiotensinogen, apoc3 (red circles) are the central functional hubs i.e., the proteins with a high number of interactions with other modulators in the interactome. the red arrows indicate the inhibition, the green arrows mean inductions, and the grey arrows indicate a generic correlation. the teal highlighted lines indicate the well-known canonical molecular pathways. the relative figure legend is in supplemental material in figure s1 . table 1 considered a potential target of treatment. based on our own as well as other findings, a fine balance between lipid metabolism and wound healing mechanisms leading to fibrosis is suggested in ipf onset and development. lipid metabolism as well as glucose anabolism and catabolism are dramatically subject to certain environmental and genetic influences, which may alter their equilibrium, leading to metabolic or fibrotic disorders. to corroborate these observations, various widespread diseases associated with the onset of fibrosis, including ipf, cirrhosis, hepatitis, non-alcoholic steatohepatitis, chronic kidney disease, myocardial infarction, heart failure, diabetes and scleroderma, share common metabolic pathways [49, 57, [94] [95] [96] . table 1 considered a potential target of treatment. mitochondrial dysregulation is linked to lipid accumulation in the lungs, influencing the lipid composition of pulmonary surfactant, which is reported to be an extracellular lipid reservoir, uniquely susceptible to direct and continuous exposure to environmental oxidants, inflammatory agents and pathogens. surfactant lipids have been recognized as bioactive molecules that maintain immune quiescence in the lung but can be remodelled by the inhaled environment into neo-lipids, which mediate key roles in inflammation, immunity and fibrosis. since surfactant composition is sensitive to circulating lipoproteins, the lipid milieu of the alveoli should be recognized as susceptible to diet and common systemic metabolic disorders [99] . idiopathic pulmonary fibrosis has long been considered to be limited to the lung compartment, although different pathways related to metabolic alterations in ipf are also typical of diseases affecting other organs. fibrosis is thought to begin in the lung as microvascular injury, leading to endothelial cell damage and alveolar epithelial injury, accompanied by the release of cytokines, chemokines and growth factors, and the induction of the coagulation cascade, triggering positive feedback that amplifies all these molecular responses [100] . hypertriglyceridemia and hyperglycaemia are widely reported to impair macro-and micro-vascular functions. the inadequate accumulation of lipid depot in the lung after particular kinds of environmental exposure could alter metabolic pathways in this organ. genetic alterations in different ipf phenotypes could promote these metabolic changes. variants in different surfactant genes could alter lipid metabolism, while telomere attrition or shortening could influence mitochondrial functions and together they could influence each other to induce a fibrotic response instead of adipose tissue accumulation. complex diseases such as ipf involve genetic and environmental risk factors and engage many cells and tissues. for this reason, they pose challenges to traditional approaches that examine individual factors in favour of multi-tissue multi -omics systems biology, to comprehensively elucidate the within-and cross-tissue molecular networks underlying gene-by-environment interactions. by combining different -omics data, bioinformatic instruments can be used to correlate the larger amount of information in a network-based systems medicine. all the metabolic pathways described are implicated in the pathogenesis of ipf through the induction of the onset and development of fibrosis. little data are available on the ipf of metabolic pathways, leading to alterations in lipid and glucose metabolism and mitochondrial dysfunctions. research is increasingly focusing on this new view of ipf as a systemic disorder. the altered metabolism of carbohydrates, lipids, proteins and hormones has been documented in the lung, liver and kidney fibrosis. correcting these metabolic alterations is becoming a new strategy for antifibrotic therapies. understanding the pathology of ipf from different but interconnected points of view, namely the exposome, genetic predisposition and dysregulated metabolic pathways, could be the key to a holistic picture of this disease. the following are available online at http://www.mdpi.com/1422-0067/21/16/5663/s1, figure s1 : legend of metacore interactome. the authors declare there was not support in the form of grant, gift, equipment, and/or drugs. clinical results of sublobar resection versus lobectomy or more extensive resection for lung cancer patients with idiopathic pulmonary fibrosis exacerbations in idiopathic pulmonary fibrosis triggered by pulmonary and nonpulmonary surgery: a case series and comprehensive review of the literature the impact of lung cancer on survival of idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis pathologic regulation of collagen i by an aberrant protein phosphatase 2a/histone deacetylase c4/microrna-29 signal axis in idiopathic pulmonary fibrosis fibroblasts the effect of cigarette smoking on bronchoalveolar lavage protein profiles from patients with different interstitial lung diseases a. mtorc1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in ipf fibroblasts efficacy and safety of nintedanib in patients with advanced idiopathic pulmonary fibrosis pirfenidone in idiopathic pulmonary fibrosis: real-life experience in the referral centre of siena bronchoalveolar lavage and serum kl-6 concentrations in chronic hypersensitivity pneumonitis: correlations with radiological and immunological features antithrombin iii as predictive indicator of survival in ipf patients treated with nintedanib: a preliminary study bal biomarkers' panel for differential diagnosis of interstitial lung diseases serum amyloid a in patients with idiopathic pulmonary fibrosis serum amyloid a: a potential biomarker of lung disorders nk and nkt-like cells in granulomatous and fibrotic lung diseases diagnosis of idiopathic pulmonary fibrosis. an official ats/ers/jrs/alat clinical practice guideline towards a functional proteomics approach to the comprehension of idiopathic pulmonary fibrosis, sarcoidosis, systemic sclerosis and pulmonary langerhans cell histiocytosis a system biology study of balf from patients affected by idiopathic pulmonary fibrosis (ipf) and healthy controls comparative proteomic analysis of bronchoalveolar lavage of familial and sporadic cases of idiopathic pulmonary fibrosis proteomic characterization of idiopathic pulmonary fibrosis patients: stable versus acute exacerbation. monaldi arch proteomic analysis in interstitial lung diseases: a review identification of the lipid biomarkers from plasma in idiopathic pulmonary fibrosis by lipidomics lipidomics applications for discovering biomarkers of diseases in clinical chemistry lipid analysis of airway epithelial cells for studying respiratory diseases lipid rafts and signal transduction the outs and the ins of sphingosine-1-phosphate in immunity mass spectrometry strategies for clinical metabolomics and lipidomics in psychiatry, neurology, and neuro-oncology top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients palmitic acid-rich high-fat diet exacerbates experimental pulmonary fibrosis by modulating endoplasmic reticulum stress protein folding and the challenges of maintaining endoplasmic reticulum proteostasis in idiopathic pulmonary fibrosis an established role? antagonistic crosstalk between nf-κb and sirt1 in the regulation of inflammation and metabolic disorders nf-κb, inflammation, and metabolic disease egln1/c-myc induced lymphoid-specific helicase inhibits ferroptosis through lipid metabolic gene expression changes resveratrol attenuates diabetes-associated cell centrosome amplification via inhibiting the pkcα-p38 to c-myc/c-jun pathway the proteome speciation of an immortalized cystic fibrosis cell line: new perspectives on the pathophysiology of the disease proteostasis network alteration in lysosomal storage disorders: insights from the mouse model of krabbe disease low-dose dihydrotestosterone drives metabolic dysfunction via cytosolic and nuclear hepatic androgen receptor mechanisms involvement of er stress, pi3k/akt activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis foxo1-mediated inhibition of stat1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease creb depletion in smooth muscle cells promotes medial thickening, adventitial fibrosis and elicits pulmonary hypertension dibutyryl-camp attenuates pulmonary fibrosis by blocking myofibroblast differentiation via pka/creb/cbp signaling in rats with silicosis foam cell formation in vivo converts macrophages to a pro-fibrotic phenotype the pro-fibrotic and anti-inflammatory foam cell macrophage paradox bronchoalveolar lavage proteomic analysis in pulmonary fibrosis associated with systemic sclerosis: s100a6 and 14-3-3ε as potential biomarkers abu-izneid, t. renin-angiotensin-aldosterone (raas): the ubiquitous system for homeostasis and pathologies angiotensinogen: hormonal regulation and relative importance in the generation of angiotensin ii antioxidant effects of oral ang-(1-7) restore insulin pathway and ras components ameliorating cardiometabolic disturbances in rats the renin angiotensin system in liver and lung: impact and therapeutic potential in organ fibrosis the renin-angiotensin-aldosterone system (raas) is one of the effectors by which vascular endothelial growth factor (vegf)/anti-vegf controls the endothelial cell barrier idiopathic pulmonary fibrosis serum proteomic analysis before and after nintedanib therapy angiotensinogen and angiotensin-converting enzyme mrna decrease and at1 receptor mrna and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of glut4 and peroxisome proliferator-activated receptor (pparγ) the ace-2 in covid-19: foe or friend? angiotensin iii stimulates aldosterone secretion from adrenal gland partially via angiotensin ii type 2 receptor but not angiotensin ii type 1 receptor reduced expression of cyclooxygenase (cox) in idiopathic pulmonary fibrosis and sarcoidosis cyclooxygenase and cytokine regulation in lung fibroblasts activated with viral versus bacterial pathogen associated molecular patterns a potential role of the renin-angiotensin-aldosterone system in epithelial-to-mesenchymal transition-induced renal abnormalities: mechanisms and therapeutic implications inflammation and fibrosis in perirenal adipose tissue of patients with aldosterone-producing adenoma moustaid-moussa, n. the renin angiotensin system, oxidative stress and mitochondrial function in obesity and insulin resistance hif-1α contributes to ang ii-induced inflammatory cytokine production in podocytes hif1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions a rassf1a-hif1α loop drives warburg effect in cancer and pulmonary hypertension involvement of the warburg effect in non-tumor diseases processes targeting metabolic dysregulation for fibrosis therapy bromodomain and extraterminal (bet) protein inhibition restores redox balance and inhibits myofibroblast activation pirfenidone inhibits hypoxic pulmonary hypertension through the nadph/ros/p38 pathway in adventitial fibroblasts in the pulmonary artery the potential of mesenchymal stem cell therapy for chronic lung disease accumulation of damaged mitochondria in alveolar macrophages with reduced oxphos related gene expression in ipf hfe gene variants and iron-induced oxygen radical generation in idiopathic pulmonary fibrosis bronchoalveolar lavage (bal) cells in idiopathic pulmonary fibrosis express a complex pro-inflammatory, pro-repair, angiogenic activation pattern, likely associated with macrophage iron accumulation high-molecular-weight adiponectin, and cardiovascular mortality among men with type 2 diabetes: a 22-year prospective study nhr-49/hnf4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting peroxisome proliferator-activated receptors-α and -γ, and camp-mediated pathways, control retinol-binding protein-4 gene expression in brown adipose tissue signaling by vitamin a and retinol-binding protein in regulation of insulin responses and lipid homeostasis combined anti-fibrotic and anti-inflammatory properties of jak-inhibitors on macrophages in vitro and in vivo: perspectives for scleroderma-associated interstitial lung disease efficacy of baricitinib in treating rheumatoid arthritis: modulatory effects on fibrotic and inflammatory biomarkers in a real-life setting adipocyte-specific repression of ppar-gamma by ncor contributes to scleroderma skin fibrosis metformin induces lipogenic differentiation in myofibroblasts to reverse lung fibrosis metformin does not affect clinically relevant outcomes in patients with idiopathic pulmonary fibrosis increased fgf1-fgfrc expression in idiopathic pulmonary fibrosis a pparγ-fgf1 axis is required for adaptive adipose remodelling and metabolic homeostasis the lung alveolar lipofibroblast: an evolutionary strategy against neonatal hyperoxic lung injury obesity, adiponectin and vascular inflammatory disease role of the adipose pparγ-adiponectin axis in susceptibility to stress and depression/anxiety-related behaviors bal and serum multiplex lipid profiling in idiopathic pulmonary fibrosis and fibrotic hypersensitivity pneumonitis molecular pathways: adiponectin and leptin signaling in cancer leptin promotes pulmonary fibrosis development by inhibiting autophagy via pi3k/akt/mtor pathway regulation of macrophage foam cell formation during nitrogen mustard (nm)-induced pulmonary fibrosis by lung lipids mitofusins regulate lipid metabolism to mediate the development of lung fibrosis mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis pgc1α repression in ipf fibroblasts drives a pathologic metabolic, secretory and fibrogenic state adipose tissue, inflammation, and cardiovascular disease angiotensin ii and its role in tubular epithelial to mesenchymal transition associated with chronic kidney disease lipid abnormalities in chronic renal failure patients undergoing hemodialysis carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis oxidative/nitrosative stress, autophagy and apoptosis as therapeutic targets of melatonin in idiopathic pulmonary fibrosis surfactant lipids at the host-environment interface. metabolic sensors, suppressors, and effectors of inflammatory lung disease recent progress in systemic sclerosis-interstitial lung disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: authors are grateful to helen hampt for careful english revision. all authors declare that they do not have any potential conflicts of interest. key: cord-017412-1avevzya authors: losada, liliana; ghedin, elodie; morris, alison; chu, hong wei; nierman, william c. title: the human lung microbiome date: 2010-10-11 journal: metagenomics of the human body doi: 10.1007/978-1-4419-7089-3_7 sha: doc_id: 17412 cord_uid: 1avevzya the human lower respiratory tract is considered sterile in normal healthy individuals (flanagan et al., 2007; speert, 2006) despite the fact that every day we breathe in multiple microorganisms present in the air and aspirate thousands of organisms from the mouth and nasopharynx. this apparent sterility is maintained by numerous interrelated components of the lung physical structures such as the mucociliary elevator and components of the innate and adaptive immune systems (discussed below) (reviewed in (diamond et al., 2000; gerritsen, 2000)). however, it is possible that the observed sterility might be a result of the laboratory practices applied to study the flora of the lungs. historically, researchers faced with a set of diseases characterized by a changing and largely cryptic lung microbiome have lacked tools to study lung ecology as a whole and have concentrated on familiar, cultivatable candidate pathogens. the human lower respiratory tract is considered sterile in normal healthy individuals (flanagan et al., 2007; speert, 2006) despite the fact that every day we breathe in multiple microorganisms present in the air and aspirate thousands of organisms from the mouth and nasopharynx. this apparent sterility is maintained by numerous interrelated components of the lung physical structures such as the mucociliary elevator and components of the innate and adaptive immune systems (discussed below) (reviewed in (diamond et al., 2000; gerritsen, 2000) ). however, it is possible that the observed sterility might be a result of the laboratory practices applied to study the flora of the lungs. historically, researchers faced with a set of diseases characterized by a changing and largely cryptic lung microbiome have lacked tools to study lung ecology as a whole and have concentrated on familiar, cultivatable candidate pathogens. with the availability of new technologies for cultivation-independent analysis of microbial populations, it is now possible to follow individuals by sampling their lung microbiome sequentially during episodes of disease and recovery in order to identify associations between the lung microbiome and health and disease. any respired contaminating particles or pathogens that evade the lung's physical and immune barriers are usually eliminated by dendritic cells and alveolar macrophages that deliver them into local draining lymph nodes. macrophages kill invading microorganisms while en route to the draining lymph nodes, and in some cases at the nodes themselves (bozza et al., 2002; kirby et al., 2009) . thus, it is not unusual to isolate viable bacteria or fungi from "normal" lung tissue (lass-florl et al., 1999) , and the term "sterile" should be applied with caution. it is perhaps more accurate to say that there is no resident flora that permanently colonizes normal lungs. even normal healthy lungs are not microbe free all the time. lower airway infections by bacteria, viruses, or fungi are among the most prevalent causes of transmissible disease in humans, with two to three million community-acquired (non-hospital-acquired) cases per year in the united states (segreti et al., 2005) . in 2006, the number of deaths attributed to pneumonia (bacterial and viral) and influenza in the united states was 60,000 (gao et al., 2008) www.cdc.gov/ nchs/fastats/deaths.htm). in 2009, nearly 9.3 million new cases of tuberculosis were reported around the world (http://www.who.int/mediacentre/factsheets/ fs104/en/index.html). with proper treatment, the lungs of individuals with these infectious diseases will revert to their normal "sterile" state. little is known about the composition of the microbial population of the upper and lower airways in health or disease. it is likely, given the multiple microorganisms already implicated in chronic lung diseases such as chronic obstructive pulmonary disease (copd) , that there are other undetected organisms and that there are complex relationships between multiple pathogens involved that are not currently understood. a few studies have examined microbial species in limited numbers of normal subjects and patients with various respiratory disorders. one study using 16s rdna clone libraries and microarrays did not detect any bacteria in the lungs of patients without respiratory disease who were briefly intubated for surgery (flanagan et al., 2007) . the same study reported that all patients intubated for longer periods had detectable 16s rdna and that the bacterial diversity present decreased during antibiotic usage. another study used 16s rdna amplification to identify bacterial species in 16 patients with ventilator-associated pneumonia (bahrani-mougeot et al., 2007) . this study identified bacterial pathogens not seen using conventional culture techniques, especially anaerobes, and found that oral bacteria could be detected in the lung. in one study, sputum samples from 25 cystic fibrosis (cf) patients were analyzed using 16s gene profiling and the authors identified an average of 7.2 species present per subject (bittar et al., 2008) . viruses have also been examined in nasal lavages in both asthmatic and normal subjects with cold symptoms using the virochip microarray (kistler et al., 2007) . the microarray technology identified more viruses than conventional culture methods and had excellent sensitivity and specificity compared with pathogen-specific polymerase chain reaction (pcr). an unexpected diversity of human coronavirus and rhinovirus strains was discovered in the subjects. so if the lung is generally sterile, why do some individuals become chronically colonized? what organisms colonize the lungs? those with physically compromised airways or immune system deficiencies are subject to chronic microbial colonization of their airways and to high-frequency episodes of viral, bacterial, or fungal lower respiratory infections. perhaps in no other body site is the direct relationship between disease and microbiome more explicit than in the lungs where there is a distinct and obvious microbial difference between normal and diseased individuals. the lower respiratory tract, composed of the trachea and lungs, is quite different in structure and function from the upper respiratory tract, which is highly colonized by microorganisms. the lungs themselves are divided into different sections according to their function and structures: the bronchi, bronchioles, and alveoli. bronchi and bronchioles are primarily conductive airways surrounded by thick cartilage that allow easy airflow into the parenchyma (or alveolar tissue) of the lungs, where gas exchange occurs. conductive airways are covered in ciliated epithelium interspersed with different types of secretory cells that release mucins, immunomodulatory proteins, surfactants, and proteases. together, the physical and chemical barriers protect against physical and biological damage by establishing a mucociliary elevator, which brings about an upward transport of a mucus stream for the lungs (fraser, 2005) . the secretory cells decrease in proportion from 20 to 30% in the trachea to less than 1% in the distal and alveolar parts of the lungs. in addition to the physicochemical protection provided by cilia and mucus, the epithelium is also protected by several immune cells, including dendritic, langerhans, t lymphocytes, and mast cells, that respond to inhaled antigens establishing a robust immunity (fraser, 2005) . it is thought that in conjunction with the physical barriers provided by the nose and upper respiratory mucosa, these defenses are enough to maintain sterility in the lower respiratory tract. the vast majority of the lung surface epithelium, however, is alveolar. it is estimated that 87% of the total volume of the lungs is alveolar, with only 6% of this being tissue and the remainder gas (stone et al., 1992) . the primary role of this tissue is to carry out gas exchange. the epithelium is mostly a continuous single layer of cells overlying a thin interstitium, which contains numerous capillaries that supply ample blood for gas exchange (fraser, 2005; stone et al., 1992) . unlike the epithelium in the conducting airways, the respiratory epithelium is not ciliated or protected by mucus. instead, it is covered by surfactant proteins that maintain the surface tension for efficient gas exchange. the lack of mucus or secretory cells is compensated by the presence of alveolar macrophages, mast cells, lymphocytes, dendritic cells, and other monocyte-like cells that protect the epithelium from potential pathogens and help maintain sterility. recent world health organisation (who) figures rank lower respiratory diseases second in an assessment of the burden of disease worldwide (http://www.who.int/ respiratory/en/). in 2006, 124, 500 people died in the us due to chronic lower respiratory disease (www.cdc.gov/nchs/fastats/deaths.htm). chronic respiratory diseases include: asthma, copd, cf, and bronchiectasis. these diseases generally lead to impaired clearance and function of the mucociliary elevator and/or the immune protection of the lung. in addition, immune deficiency such as that caused by the human immunodeficiency virus (hiv) also disrupts the typical immune homeostasis in the lungs. without the normal protective barriers, the lungs fall victim to persistent and severe colonization that can ultimately lead to death, particularly for cf patients. as discussed below, the lung microbiome in each of these diseases is very different from normal individuals. the data discussed in the following sections demonstrate a clear link between microbial colonization and severity of disease symptoms. it is unclear, however, what exact role these different microbial populations play in initiating and enhancing the progress of such chronic respiratory diseases. lastly, in some cases, the data hint that some population structures might actually be protective against further decline, but much more research needs to be conducted in this area to make a definitive claim. this chapter discusses the methods for sampling and characterizing the microbiome of the lungs. in addition, it reviews the current status of our understanding of the lung microbiome in asthma, idiopathic bronchiectasis, cf, copd, and during immune deficiency due to hiv infection. several procedures have been developed for sampling the microbial population of the human lung econiche. in order of increasing invasiveness they are sputum induction, bronchoalveolar lavage (bal), bronchial brushing, endobronchial biopsy, and transbronchial biopsy. sputum induction by inhalation of hypertonic saline is a noninvasive method to obtain samples from the lower respiratory tract for cell and microbial analysis (bickerman et al., 1958) . the quality of samples varies and can be scored on the volume of the obtained sputum plugs and the level of salivary contamination as measured by squamous cells observed by microscopy. bal is a procedure in which a bronchoscope is passed through the mouth or nose into the lungs and saline is instilled into a segment of the lung and then recollected for examination (henderson, 1994; reynolds and chretien, 1984) . bal is most commonly used to diagnose infections in both immunocompetent and immunosuppressed patients. bal is the most common procedure for sampling the lower respiratory system microbial colonization/infection status, to sample the components of the epithelial lining fluid, and to determine the protein composition of the airways. it is often used in evaluating the patient's lung immunological status by sampling cells and pathogen levels. bal is an invasive procedure and thus is less ideal for research purposes. bronchial brushing provides access to cells and microbes that are adherent to the luminal surfaces of the lower airways. in this procedure, a flexible fiber optic bronchoscope is used for brushing a targeted lesion or site (fennessy, 1967; zavala et al., 1973) , where induced sputum and bal procedures will allow sampling of cells and microbes that can be washed from the lumen surface, brushing will recover adherent cells (e.g., bronchial epithelial cells) and microbes. recently, brushing techniques have been developed to sample distal lung (i.e., small airway) epithelial cells and associated microbes (ammous et al., 2008) . this technique will enable investigators to further study the microbiome in lung diseases such as copd. endobronchial biopsy involves using the fiber optic bronchoscope to identify appropriate target sites in the lung and obtain large airway tissue samples using inserted alligator forceps, cup forceps, or curette passed through the endoscope's central channel. this procedure poses a higher risk than bal but allows sampling the invasive microbes within the airway tissue (scott et al., 1991; trulock et al., 1992) . transbronchial biopsy, the most invasive of these sampling procedures, is routinely performed for clinical care and allows clinicians and researchers to obtain distal (small) airway tissues as well as alveolar tissues. this procedure has been safely done by several research groups (balzar et al., 2005) and will likely further our understanding of the microbiome in human distal lung tissues, but carries a significant risk of complications. standard microbiological and virologic methods detect only a small proportion of the bacteria and viruses present in various body sites because the great majority of these organisms are uncharacterized or uncultivable. to understand the real diversity, culture-independent methods, such as sequencing, are thus a necessity. sequenced-based identification of microbial species is facilitated by decreased costs of sequencing, and the availability of next-generation sequencing technologies, further enhances the capacity to generate large amounts of data. for the identification of bacterial species within an environment, the amplification of 16s rrna genes (or 16s rdna) using universal primers are useful for diversity characterization because this genetic locus is present in all bacterial species (relman et al., 1991) . the nine hypervariable regions of the 16s rdna can be used for bacterial species identification (chakravorty et al., 2007; rokas et al., 2007) with some regions having better discriminatory value than others. the sequencing and phylogenetic analysis of bacterial 16s rrna derived from microbiome samples has been the primary method used to investigate bacterial diversity in the human body (bik et al., 2006; dekio et al., 2005; eckburg et al., 2005; gao et al., 2007; hugenholtz et al., 1998; hyman et al., 2005; zhang et al., 2006) . these studies have revealed a far higher level of diversity than conventional culture techniques (aly et al., 1976; bik et al., 2006; dekio et al., 2005; kazor et al., 2003; korting et al., 1988; kroes et al., 1999; paster et al., 2001) . these studies revealed that the majority of bacterial sequences correspond to uncultivated species and novel organisms. there was significant intersubject variability and variability between stool and mucosal microbial populations. for example, recent studies by blaser and colleagues at new york university have demonstrated substantial changes in the ratio of the genus streptococcus to propionibacterium in skin samples from healthy persons and in normal skin of patients with psoriasis (ratio = 0.4; n = 2, 649 clones), and from psoriatic lesion samples (ratio = 5.0; n = 1, 314 clones; p = 0.01) (gao et al., 2008) . in a lung study, bacterial diversity was analyzed in the endotrachael aspirates from seven intubated patients colonized with pseudomonas aeruginosa using both sequencing from 16s rrna clone libraries and an oligonucleotide microarray termed as the phylochip (flanagan et al., 2007) . controls were subjects briefly intubated for elective surgery. bacteria were not detected by either method in samples from the controls. sequencing from the clone libraries detected the presence of many orally, nasally, and gastrointestinal associated bacteria including known pathogens. the phylochip detected the same organisms and many additional bacterial groups present at low abundance. following antibiotic therapy, the bacterial populations' diversity decreased and was dominated by a single respiratory pathogen. in six of the seven patients, the dominant species was p. aeruginosa in spite of targeting this organism with antibiotics to which it was reportedly sensitive. the authors hypothesize that the loss of population diversity may directly contribute to pathogenicity, persistence, and development of pneumonia. similarly, amplification of regions from the 18s and internal transcribed spacer (its) regions of the rrna, a conserved fungal gene, allows discrimination of fungal species (fujita et al., 2001; makimura, 2001) . a preliminary study was undertaken to examine the efficacy of a community sequencing method to identify the fungal species in bal lung samples from 23 human subjects. the fungal its1-5.8s-its2 region was amplified and the results showed that 4 of 23 patients (17%) had fungal dna levels that could be reproducibly detected by pcr. the detected fungi included aspergillus fumigatus, candida tropicalis, and penicillium digitatum, among others (denning, unpublished) . these data agree with a culture-based study that showed that 63% of their sample population had evidence of pulmonary fungal colonization (lass-florl et al., 1999) , most commonly with a. fumigatus and other candida species, and also zygomycetes. the results also demonstrate that rrna sequencing is a viable platform for characterization of fungal communities in the human body. although there are no conserved genes that can be targeted for determination of viral diversity, whole genome shotgun of a sample enriched for viruses (such as by filtering) can lead to an effective characterization of viral communities (angly et al., 2006) . hundreds of viral genome sequences can be completed in a single sequencing reaction run using the gs-flx (454/roche) sequencing platform. using this technology and a random priming-based method, referred to as sequence independent single primer amplification (sispa), near-full-length genomes of rna or dna viruses can be sequenced. sispa can be used to sequence known and unknown viral genomes (djikeng et al., 2008) . this viral sequencing methodology can potentially be adapted for the determination of viruses within bal by enrichment using nuclease treatment and filtration followed by taking the extracted total rna and dna through the sispa process followed by sequence comparisons to known viruses. the initial studies of small 16s rrna described above hinted at great diversity within the human microbiome, yet it left important questions unanswered such as the identity of the nondominant community members and their biological roles. the applications of shotgun techniques to the study of the human microbiome (kurokawa et al., 2007; manichanh et al., 2006; zhang et al., 2006) again highlight the extent of microbial diversity associated with the human body while revealing much more of the identity and biology of nonculturable microorganisms. as a result of reduced costs and improved sequencing technologies, it is possible to perform in-depth metagenomic surveys of the human body's microbial diversity beyond the 16s rrna surveys. metagenomics, a term introduced in 1998, describes the functional and sequence-based analysis of total microbial genomes from environmental samples (handelsman et al., 1998) . metagenomics uses techniques that resemble the "whole genome shotgun" approach of single genome sequencing, but it is not limited to a single species. human metagenomics has provided insight into the complex composition of the microbiome of these several body sites, and this information has allowed us to draw tentative conclusions about the relationship between specific microbiomes and health. the human microbiome is composed of multiple "ecological niches", including the mouth (kroes et al., 1999; paster et al., 2001) , esophagus (zhang et al., 2006) , stomach (bik et al., 2006) , intestine , skin (gao et al., 2007) , and vagina (zhou et al., 2004) . our understanding of the overlap and the degree of communication between them is rudimentary at best. perhaps the most extensively studied has been the human gut microbiome where the interaction of the gut microflora, independently or through interaction with the genetic makeup of the host plays a role in obesity, crohn's disease, and ulcerative colitis (frank et al., 2007; gophna et al., 2006; turnbaugh et al., 2006) . asthma is a complex disease characterized by chronic inflammation in the lungs and reversible narrowing of the airways. symptoms include dyspnea, coughing, wheezing, airway hyper-reactivity, chronic eosinophilic atopy, and mucus hyper secretion (busse and lemanske, 2001) . about 20 million people in the us have been diagnosed with asthma; 9 million of them are children. asthma causes 4,000 deaths per year in the us and 11 million exacerbations. asthma is caused by environmental and genetic factors (martinez, 2007) , with asthma attacks resulting from immune responses to inhaled allergens. the majority of asthma exacerbations are caused by viral infections (krishnan et al., 2006) . atypical bacterial infections have also been associated with asthma exacerbations and with chronic asthma (johnston and martin, 2005; martin et al., 2001) . in susceptible individuals, the development of asthma has been associated with bacterial colonization in neonates (bisgaard et al., 2007) and viral and bacterial infections (wu and chu, 2009 ). there is abundant evidence testifying to the importance of microbes to the development and maintenance of asthma. a recent publication using a bacterial gene sequencing method suggests a disordered microbiome in asthmatic airways (hilty et al., 2010) . in the developed world there has been an increased focus on predisposing factors for asthma due to its rapidly increasing prevalence, now affecting up to a quarter of urban children (lilly, 2005) . asthma is known to be caused by environmental and genetic factors (martinez, 2007) . these factors determine asthma severity and how easily it can be treated (martinez, 2007) . many associations with asthma have been detected including exposure to cigarette smoke (thomson et al., 2004) , caesarean section birth relative to natural birth (thavagnanam et al., 2008) , early viral respiratory infections (gold and wright, 2005; harju et al., 2006) , early in life antibiotic use (marra et al., 2006) , and living in the us (gold and wright, 2005) . one theory for the cause of the increase in asthma incidence is the hygiene hypothesis (strachan, 1989) , that the rise in prevalence of asthma is a direct consequence of the success of modern hygienic practices in preventing childhood infections. this hypothesis is supported by numerous studies that have shown that children coming from a less hygienic environment have less asthma and other allergenic diseases (ball et al., 2000; celedon et al., 1999; jarvis et al., 1997) . in addition, alterations in innate immune system genes have been shown to be associated with the inception and development of asthma. these genes include the toll-like receptors and other genes such as mbl, mylk, defb1, jun, inf-î±5, and nos2a reviewed in wu and chu (2009) . asthma exacerbations have long been associated with viral infections (pattemore et al., 1992) . more recently, the use of reverse transcriptase pcr has greatly facilitated the identification of the exacerbation-associated virus. studies using this tool have suggested that 75-80% of asthma exacerbations are caused by virus infections (wark et al., 2002) . rhinovirus (rv) infections during early childhood are associated with the development of asthma, lower respiratory tract infections, and wheezing (jackson et al., 2008; lemanske et al., 2005) . they are also associated with hospitalization for asthma in adults (venarske et al., 2006) . a separate study revealed that patients with allergic asthma infected with rv had increased admissions to hospitals and that dust mite allergen was the primary allergen when these patients were skin tested with a panel of aeroallergens (green et al., 2002) . respiratory syncytial virus (rsv) in infants causes lower respiratory infection leading to pneumonia and bronchiolitis. rsv bronchiolitis is the leading cause of wheezing in infants and young children, and children infected with rsv resulting in bronchiolitis are more likely to develop wheezing and asthma later in childhood (peebles, 2004) . similarly, the human metapneumovirus (hmpv) was first isolated from children in 2001 and has been found to be associated with asthma exacerbations in both children under 5 years of age and adults (foulongne et al., 2006; williams et al., 2004) . mycoplasma pneumoniae and chlamydia pneumoniae are bacteria that attach to airway epithelial cells and cause cell damage. infections by these bacteria have been shown to be associated with asthma exacerbations (johnston and martin, 2005; lieberman et al., 2003; martin et al., 2001) . using a pcr assay, 31 of 55 patients with asthma were positive for either of these bacteria in lung tissue or bal, suggesting that some level of colonization by these bacteria may be common in asthma patients (martin et al., 2001) . studies in a mouse model suggest that preexisting allergic inflammation impairs the ability to upregulate tlr-2 and il-6 in the lungs, leading to decreased clearance of m. pneumoniae and an increase in airway inflammation (kraft et al., 2008) . evidence is accumulating that infections are associated with the induction and development of asthma. first, long-term cohort studies on the development of asthma show that most childhood asthma begins in infancy. the first episode of wheezing begins before the age of 3 and is frequently associated with lower respiratory tract viral infections, usually rsv, but also rv (gern et al., 2000; sigurs et al., 2005) . these infectious episodes and associated wheezing are strong predictors for the development of childhood asthma and atopy (devulapalli et al., 2008; kusel et al., 2007; martinez et al., 1995; singh et al., 2007) . second, many studies have associated viral infections with asthma prevalence in children (devulapalli et al., 2008; jackson et al., 2008; kusel et al., 2007; papadopoulos and kalobatsou, 2007; sigurs et al., 2005; singh et al., 2007; williams et al., 2004) . lastly, wu et al. have provided evidence to suggest that viral infections have a causal role in asthma initiation and development where they show that viral infection during the first 4 months of age is strongly correlated with the development of asthma by age 5 (wu et al., 2008) . only one-third of children with childhood wheezing and asthma, however, will develop persistent asthma symptoms in adulthood (gerritsen, 2002; taylor et al., 2005; vonk et al., 2004) . management of the symptoms with corticosteroid therapy is effective but may not alter the asthma progression (guilbert et al., 2006) . the role of infections in asthma induction and development will likely be shown to be mediated through the effect of these infections on the chronic inflammatory response in the airways of asthmatics. microbial infections can generate either a th2-or a th1-biased response that could exacerbate or attenuate asthma, respectively. in asthmatics, a pro-inflammatory th2 response persists even in the absence of allergens involving cd4+ th2 cells, eosinophils, mast cells, and the th2 cytokines il-4, il-5, il-9, and il-13 (holgate, 2008) . bacterial infections have been shown to contribute to asthma development. in a longitudinal prospective birth cohort study of 411 infants born to mothers with current or previous asthma, neonates colonized in the hypopharyngeal regions with streptococcus pneumoniae, haemophilus influenzae, or moraxella catarrhalis or a combination of these organisms were found to be at increased risk for recurrent wheezing in early childhood and asthma at age 5 (bisgaard et al., 2007) . a protective role for some bacteria has been reported . several studies have found a protective effect of mycobacterial exposure on atopy and airway inflammation (camporota et al., 2003; shirakawa et al., 1997; yang et al., 2002) . these exposures include bacillus calmette-guerin vaccination or heat-killed mycobacterium vaccae. early exposure to bacterial endotoxins may reduce future allergies or asthma (von mutius et al., 2000) , although endotoxins associated with house dust are associated with more asthma symptoms and worse lung function (dales et al., 2006; michel et al., 1996; park et al., 2001) . thus, the role of bacteria in asthma initiation and development appears to be complex. the causative interaction is likely to prove to be the interaction of bacteria and bacterial components in modulating the th1 and th2 innate immune system responses. the characterization of these interactions will be complicated by timing, dose, anatomical site, and duration of the bacterial exposure as well as the host genetic and environmental factors influencing the immune inflammatory response (holt, 1996) . it has recently been demonstrated that patients with severe asthma who are also atopic or sensitized to environmental fungi may benefit from treatment with the antifungal azole itraconazole . this observation has raised questions about the relationships among asthma severity, fungal sensitization, and fungal exposure. the issue is complicated by more than 1.5 million species of fungi that are thought to exist (hawksworth and rossman, 1997 ) and more than 80 species of fungi that have been associated with symptoms of airway allergy (horner et al., 1995) . for one species, a. fumigatus, 20 allergens are thought to participate in human airway allergies (www.allergome.org). determining the clinical relevance of fungal allergens is confounded further by extensive cross-reactivity among fungal allergens (crameri et al., 2009) . fungal allergens can induce a number of different human bronchopulmonary disorders, each with a distinct immune pathogenesis. in allergic bronchopulmonary aspergillosis (abpa), the respiratory system is chronically colonized typically with a. fumigatus. evidence now suggests that severe asthmatics without abpa are more likely to be atopic to fungi than patients with milder disease. the diagnostic label "severe asthma with fungal sensitization (safs)" has recently been applied to this group . in these patients, the fungal sensitization is most commonly a. fumigatus, candida albicans, and penicillium notatum . the association between severe asthma and fungi has been identified in numerous studies. atopy to environmental fungi has been associated with severe asthma (o'driscoll et al., 2005) . many population studies have shown an association between local fungal spore counts and medical emergencies due to asthma exacerbations (atkinson et al., 2006) . furthermore, studies have shown that fungus exposure in fungal-sensitized individuals induces asthma symptoms (malling, 1986; matheson et al., 2005; pulimood et al., 2007; salo et al., 2006; woodcock et al., 2006) . treatment of patients with safs with antifungal drugs has generally led to improvement of asthma symptoms concurrent with improvements in several markers of atopy such as reduced ige values, reduced eosinophils counts, and reductions in the level of dose of oral and systemic steroids required (pasqualotto et al., 2009) . these findings lead to the considerations of the fungal composition of the lung microbiome in asthmatic individuals and indeed in normal individuals. environmental fungi colonize the lungs of otherwise healthy people (lass-florl et al., 1999; okudaira et al., 1977) . these studies were dependent on cultivationbased methods for the detection and identification of these fungi. as a cultivationindependent method, gas chromatography/mass spectroscopy on exhaled breath has revealed the presence of fungus specific biomarkers in patients with cf with and without fungal colonization by a. fumigatus (syhre et al., 2008) . this approach was limited to analyzing for known a. fumigatus markers. the application of sequencing-based approaches for studying the lung microbiome will be essential for revealing the role of fungi in the lung microbiome and the role of the lung microbiome on asthma. cf is the most common inherited lung disease in the world. it is a severe autosomal recessive disease with an incidence of 1:2000 at birth in populations of northwestern european origin, with a mutant gene carrier frequency of 1:23 in these populations. the genetic defect occurs in the cystic fibrosis transmembrane regulator (cftr) protein, which acts to transport chloride across cell membranes. patients with cf are the archetype population with chronic bronchial colonization. symptoms include permanent bacterial colonization of the lower airways, with a formation of a biofilm, fat maldigestion, male infertility, and elevated levels of chloride in the sweat (knowles and durie, 2002) . the thick pulmonary system mucus in cf patients minimizes the effectiveness of the mucociliary elevator in clearing the lung of mucus-trapped microorganisms and other debris. as a consequence, microbes chronically colonize these patients' lungs and they suffer bouts of infection, requiring frequent hospital admission. cultures reveal a wide range of bacteria, including p. aeruginosa, mycobacteria, a. fumigatus, and sometimes viruses. the precise contributions of different microbes to patient morbidity, and the importance of inter-specific interactions remain largely unclear [reviewed in (harrison, 2007) ]. the complexity of this ecosystem is difficult to overstate. as an example of this complexity, the lungs of cf patients contain large numbers of neutrophils that migrate to this location in response to microbial colonization. these neutrophils secrete granule antimicrobial proteins called defensins that kill microbes. by analysis of cf sputum samples, the levels of extracellular defensins are sufficiently abundant that they may damage the airway epithelium (soong et al., 1997) . as another example of this inter-specific complexity, p. aeruginosa in cf lungs produces copious amounts of a tricyclic compound pyocyanin that kills competing microbes and eukaryotic cells. this compound was shown to specifically inactivate a human lung epithelial cell line vacuolar atpase (ran et al., 2003) . a study of the microbiome of the lungs was conducted to explore the hypothesis that organisms not routinely identified by culture occur in the lungs of cf patient airways and may contribute to disease. to test this hypothesis, 16s rrna sequence analysis was performed on bal samples from 42 subjects, 28 cf patients, and 14 other disease controls (harris et al., 2007) . the findings of this analysis were that, for cf subjects, a single rrna type was dominantly represented in the clone libraries prepared from lung microbiome genomic dna. this was not found in the controls. thirteen of the cf subjects' samples contained bacteria not routinely assessed by culture. candidate pathogens were identified in four cf subjects. candidate pathogens were also identified in the controls. this study documented the power of culture-independent molecular techniques to provide a broader view of the airway bacteria than standard clinical culture methods. the cf viral metagenome was explored in a recent study using five cf individuals and five individuals without disease (willner et al., 2009) . in both cohorts, the overall viral diversity was low. the cf bacteriophage communities were highly similar to each other, whereas the non-cf individual had more distinct phage communities. cf eukaryotic viral communities were dominated by a few viruses, including human herpes viruses and retroviruses. the significance of fastidious or noncultivatable organisms in the airway of cf patients is beginning to be explored. application of specific culture conditions to favor the growth of anaerobes coupled with molecular identification techniques have focused attention in cf on bacteria not routinely detected by standard culture and biochemical identification techniques (harris et al., 2007; tunney et al., 2008; worlitzsch et al., 2009) . direct, culture-independent detection techniques have identified much larger numbers of bacterial species in cf airways and have demonstrated the ability to identify likely pathogenic bacteria occurring during exacerbations when routine cultures are negative (harris et al., 2007) . these molecular identification and detection methods have identified bacteria with different antibiotic susceptibilities relative to conventional pathogens and will undoubtedly lead to novel antimicrobial intervention trials in cf (worlitzsch et al., 2009) . similar methodology to detect anaerobes or noncultivatable bacteria has not been applied systematically to patients with idiopathic bronchiectasis. bronchiectasis is characterized by chronic dilation and inflammation of the conducting airways associated with recurring infections (barker, 2002) . it is the pathologic manifestation of several genetic disorders, including cf and primary ciliary dyskinesia (pcd). however, many patients have no identifiable causes. idiopathic bronchiectasis is estimated to affect approximately 110,000 us adults (weycker et al., 2005) . symptoms include cough and chronic sputum production, recurring airway infection, dyspnea, wheezing, and chest pain (barker, 2002) . microbial infections are central to the pathogenesis and progression of disease. much of the research characterizing the composition and significance of the lower airway microbial flora has been done in cf and relatively little is known about the microbial contribution to disease pathogenesis in idiopathic bronchiectasis. however, recent observations suggest a link between the lower airway microbial flora and host disease characteristics. for example, the prevalence of idiopathic bronchiectasis associated with nontuberculous mycobacteria (ntm) appears to be increasing (billinger et al., 2009; marras et al., 2007) . both familial clustering and a characteristic "tall asthenic" phenotype (scoliosis, pectus excavatum, mitral valve abnormalities) in postmenopausal women with bronchiectasis associated with ntm infection (colombo et al., 2009; kim et al., 2008) have been reported. it is unknown whether the age, female sex, and unique body morphotype associations are seen in idiopathic bronchiectasis unassociated with ntm. correlating disease phenotype with microbial flora is dependent upon accurately categorizing the microbial status of the patients. for environmental organisms like ntm, it is important that this categorization include both accurate speciation and determination that the organism likely represents true infection rather than contamination or transient colonization. the american thoracic society and the infectious diseases society of america (ats/idsa) microbiologic diagnostic criteria for pulmonary disease based on sputum specimens call for at least two positive sputum specimens for the same species (kim et al., 2008) . concomitant recovery of filamentous fungi from airway specimens is also common in bronchiectasis, but the pathophysiologic consequences are not known. a recent study in cf patients found that a. fumigatus, like ntm, was commonly present in older patients: 75% of patients aged 16-20 years and in 60% of patients over age 20 (valenza et al., 2008) . amin and colleagues further noted that cf patients who were chronically infected with a. fumigatus (defined as two positive cultures in a given year) had significantly worse airway obstruction as evidenced by a lower forced expiratory volume in one second (fev 1 ) and significantly higher risk of pulmonary exacerbations during subsequent follow-up than patients without a. fumigatus (amin et al., 2009) . this potential negative impact on the course of bronchiectasis and a possible benefit from antifungal treatment for chronic infection in cf have prompted initiation of a multicenter clinical trial of itraconazole in cf patients in canada (amin et al., 2009; shoseyov et al., 2006) . in non-cf bronchiectasis, a recent study suggested that aspergillus is more common in patients infected with ntm than in those without ntm, and that it is commonly associated with fungal lung disease manifestations in ntm-infected patients (kunst et al., 2006) . however, outside the relatively small numbers of idiopathic bronchiectasis patients with allergic bronchopulmonary aspergillosis (abpa) or chronic necrotizing aspergillosis, the pathologic significance of these fungi has not been systematically explored in large numbers of patients and very few data are available for aspergillus species other than fumigatus or filamentous fungi other than aspergillus (kobashi et al., 2006; kunst et al., 2006; raju et al., 2008) . abpa is well described in association with bronchiectasis occurring in asthmatics and patients with cf (malde and greenberger, 2004) . the diagnostic criteria rely on an elevated total ige as well as elevated a. fumigatus-specific ige and igg in the setting of episodic bronchial obstruction, pulmonary infiltrates, and central bronchiectasis. kunst and colleagues assessed the prevalence of positive serologic markers for a. fumigatus [ige by radioallergosorbent test (rast) and precipitins] among idiopathic bronchiectasis patients and found these markers to be commonly present especially in the setting of concomitant ntm disease (kunst et al., 2006) . patients with these serologic markers more commonly had radiographic manifestations suggesting aspergillus-associated disease. while other filamentous fungi such as scedosporium species have been commonly recovered from the airways of both cf and non-cf bronchiectasis patients, the role these fungi play in disease pathogenesis remains controversial (cooley et al., 2007) . while specific ige antibody rast and precipitin assays can be prepared using allergen prepared from the isolated species and correlated with the clinical presentation of allergic bronchopulmonary mycoses, these assays have not been commonly used to characterize the clinical significance of these fungal species recovered from the lower airway (fedorova et al., 2008; lake et al., 1991) . copd is the fourth leading cause of death in the us (petty, 2000) and is expected to rank third in the world by 2020 (lopez and murray, 1998) . despite efforts aimed at smoking cessation, little impact has been made on copd incidence, and current treatments are ineffective in slowing progression of the disease. copd has been defined by the global initiative for chronic obstructive lung disease (gold) as "a disease state characterized by airflow limitation that is not fully reversible". the diagnosis of copd can also encompass those with chronic obstructive bronchiolitis and emphysema. tissue inflammation in copd is characterized by a predominant neutrophil, cd8+ lymphocyte, and macrophage infiltration (keatings et al., 1996; lacoste et al., 1993; o'shaughnessy et al., 1997; saetta et al., 1998) . it has been proposed that the mechanism of tissue damage involves the recruitment and activation of neutrophils, macrophages, and cd8+ t cells with concomitant upregulation of several cellular proteases and inflammatory cytokines. although smoking is clearly the leading risk factor for copd, not all smokers develop disease (buist and connett, 1993) . while smoking can stimulate inflammation in the lungs, smokers with copd have an increased inflammatory response than smokers without copd, and inflammation can persist despite smoking cessation (keatings et al., 1996; lacoste et al., 1993; o'shaughnessy et al., 1997; saetta et al., 1998) . these observations suggest that some other factor or factors contribute to development and perpetuation of the inflammatory response in copd. infection might be one such factor critical in triggering and perpetuating the inflammatory response in copd. the mechanism by which infections might act to promote copd progression has been termed the "vicious circle" hypothesis (sethi, 2000a; sethi and murphy, 2008) . in this scenario, smoking causes structural remodeling that renders smokers more likely to become colonized and/or less able to clear subclinical infection. defects in mucociliary clearance and surfactant abnormalities caused by smoking also contribute to the tendency to develop chronic infection (finley and ladman, 1972; honda et al., 1996; raju et al., 2008; vastag et al., 1985; verra et al., 1995) . once colonization is established, the organism or organisms recruit white blood cells to the lungs, stimulating release of inflammatory cytokines and chemokines as well as proteases. inability to clear the inciting organism perpetuates the cycle, ultimately resulting in tissue destruction, airway thickening, and clinical copd. the most commonly implicated bacteria are h. influenzae, m. catarrhalis, s. pneumoniae, and p. aeruginosa (sethi, 2004) . viruses that seem to be important in copd include adenovirus, influenzae viruses, rhinovirus, respiratory syncytial virus, and human metapneumovirus (mallia et al., 2006; martinello et al., 2006; retamales et al., 2001; seemungal et al., 2001) . these pathogens can be found in patients with copd in the stable state and during exacerbations (sethi, 2004) . the colonization seen in patients with copd is likely playing a role in disease and is not just an innocent bystander. for example, as bacterial load increases, fev 1 falls, and colonization has been associated with greater sputum purulence, increased sputum neutrophils, and increased levels of interleukin (il)-8, tumor necrosis factor (tnf)î±, and neutrophil elastase (obrian et al., 2007; patel et al., 2002; sethi, 2000b; stockley et al., 2000) . exacerbations associated with viruses are more severe and last longer than those without a viral trigger (papi et al., 2006; seemungal et al., 2001) . in addition, exacerbations associated with both bacteria and viruses may be more severe than those associated with single organisms (obrian et al., 2007) , suggesting the usefulness of metagenomic techniques in this disease. colonization with the fungus pneumocystis jirovecii (pc, formerly pneumocystis carinii f. sp. hominis) has recently been implicated in copd pathogenesis. this organism generally causes acute pneumocystis pneumonia (pcp) in patients with immunosuppression such as those infected with hiv, but colonization with the organism occurs in both hiv + and hiv â�� individuals and may be important in copd. colonization with pc is increased in hiv patients with copd and correlates with disease severity (morris et al., 2004b; probst et al., 2000) . animal models also support the role of pc colonization in copd. christensen and colleagues recently reported that in immunocompetent mice, exposure to cigarette smoke and pc colonization resulted in pulmonary function deficits and airspace enlargement characteristic of emphysema (christensen et al., 2008) . in a model of pc colonization in simian/human-immunodeficiency virus (shiv)-infected nonhuman primates (norris et al., 2006) , pc-colonized animals developed airway obstruction and radiographic emphysema while animals infected with shiv alone did not develop these changes (shipley et al., 2010) . although pulmonary infections and neoplasms associated with hiv have decreased since the availability of highly active antiretroviral therapy (haart) (palella et al., 1998) , some pulmonary conditions may actually be increasing in persons with hiv. diseases such as copd, asthma, and bronchiectasis were reported to be increased in those with hiv before the introduction of antiretroviral therapy, and a similar decrease in these conditions as seen in the opportunistic infections has not occurred after antiretroviral treatment of hiv. in fact, in a recent study of hiv + patients, almost 4% of deaths were due to obstructive airway disease in 1998, a threefold increase from the pre-haart era (louie et al., 2002) . before the haart era, hiv + subjects were noted to have an accelerated form of emphysema with significant emphysematous disease seen in subjects less than 40 years old (diaz et al., 1992 (diaz et al., , 2000 . both emphysema and airflow obstruction have been reported in hiv infection. unlike many of the acquired immunodeficiency syndrome (aids)defining opportunistic infections, hiv-associated copd may actually be more common in the current era of hiv as it is frequently reported in those without a history of aids-related pulmonary complications and the now aging hiv + population has a longer exposure to smoking and hiv. given the immunological defects seen with hiv, it is quite possible that hiv + subjects, especially those who smoke, are more prone to develop subclinical pulmonary infections, even if successfully treated with haart. the changes that occur in the lung microbiome have not been studied in hiv, but microbial colonization is a likely factor in the accelerated copd seen in this population. the vicious circle hypothesis of copd could be further worsened in hiv + patients by upregulation of hiv levels in the lung stimulated by pulmonary colonization. several studies have shown that pulmonary infections increase lung levels of hiv. koziel and colleagues reported that hiv rna was detected in 62% of patients with active lung disease compared to 16% of asymptomatic subjects, independent of clinical stage of hiv and serum hiv rna levels (koziel et al., 1999) . the lung appears to be an independent compartment for hiv replication as drug mutations found in bal differ from those in blood (white et al., 2004) . hiv in the lungs is associated with a lymphocytic alveolitis, particularly in those subjects with cd4 cell counts between 200 and 500 cells/î¼l, suggesting that the virus might act independently to stimulate pulmonary inflammation (twigg et al., 1999) . the relationship of hiv pulmonary viral levels, infections, inflammation, and copd has not been examined. pneumocystis colonization is likely important in the pathogenesis of copd in those with hiv as well as in the hivpopulation. in hiv + subjects, the prevalence of colonization is high, particularly if subjects smoke, and colonization is seen even in patients with high cd4 cell counts receiving haart (morris et al., 2004a) . anatomic emphysema is also more common in hiv + patients with pc colonization (morris, unpublished data) . it has recently been shown that pneumocystis colonization in hiv + subjects is associated with worse airway obstruction and an increased likelihood of clinical diagnosis of copd, independent of smoking history and cd4 cell count ). in addition, the shiv-infected nonhuman primates described above serve as a model for the development of copd in the setting of pc colonization and hiv-like immunodeficiency (norris et al., 2006; shipley et al., in press ). microorganisms including bacteria, fungi, and viruses play a central role in development, exacerbation, and progress of lung diseases. even though normal lungs do not have a permanent resident microbiome, diseased lungs are acutely infected and/or chronically colonized. standard laboratory practices have not properly reflected the entirety of the microbiomes, either in health or in disease, and thus newer sequence-based technologies have begun to reveal the true complexity of the lung microbiomes. much more research still needs to be conducted in order to fully understand the microbial burden of the lungs, and how this burden relates to health and disease. it is apparent that conditions that compromise the physical and immune system barriers to lung colonization by microbes result in chronic colonization and recurrent infections. these conditions include chronic inflammation as seen in hiv, asthma, and bronchiecstasis, or physical obstruction observed in cf and bronchiecstasis. the lung microbiomes in each of these conditions has not been properly explored to date, which limits our ability to make definitive conclusions on how to best manage these diseases. our understanding of the fundamental role of viruses in the initial establishment and progress of asthma underscores how little knowledge exists on the role of viral infection in other chronic respiratory diseases. in addition, the fact that some bacterial populations and/or components seem to be protective against further and severe exacerbations in asthma opens the door to questions about the role of microorganisms in protecting against other diseases. the exact nature of this protection is not clearly understood, and great benefit would come from studies that further clarify these intriguing results. furthermore, if some population structures aid in preventing disease progression, it is likely that other population structures may predispose episodes of acute acerbations and progression of the underlying disease condition. a comprehensive understanding of the dynamics of these microbiome interactions would likely result in better, more efficient therapies for these and other respiratory diseases. our developing microbiome analysis technology coupled with our increasing awareness of the potential positive and negative impact of lung population structure on respiratory system health and disease strongly supports the initiation of aggressive projects to characterize the human lung microbiome and its influence on health and disease. bacterial flora in psoriasis the effect of chronic infection with aspergillus fumigatus on lung function and hospitalization in cystic fibrosis patients variability in small airway epithelial gene expression among normal smokers the marine viromes of four oceanic regions temporal associations between daily counts of fungal spores and asthma exacerbations molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia siblings, day-care attendance, and the risk of asthma and wheezing during childhood relationship of small airway chymase-positive mast cells and lung function in severe asthma an aerosol method of producing bronchial secretions in human subjects: a clinical technic for the detection of lung cancer molecular analysis of the bacterial microbiota in the human stomach nontuberculous mycobacteria-associated lung disease in hospitalized persons childhood asthma after bacterial colonization of the airway in neonates molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients does helicobacter pylori protect against asthma and allergy dendritic cells transport conidia and hyphae of aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate th responses to the fungus the lung health study. baseline characteristics of randomized participants the effects of mycobacterium vaccae on allergeninduced airway responses in atopic asthma day-care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy a detailed analysis of 16s ribosomal rna gene segments for the diagnosis of pathogenic bacteria pneumocystis murina infection and cigarette smoke exposure interact to cause increased organism burden, development of airspace enlargement, and pulmonary inflammation in mice familial clustering of pulmonary nontuberculous mycobacterial disease infection with scedosporium apiospermum and s. prolificans cross-reactivity among fungal allergens: a clinically relevant phenomenon? airborne endotoxin is associated with respiratory illness in the first 2 years of life detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling the link between fungi and severe asthma: a summary of the evidence randomized controlled trial of oral antifungal treatment for severe asthma with fungal sensitization: the fungal asthma sensitization trial (fast) study severity of obstructive airways disease by age 2 years predicts asthma at 10 years of age the innate immune response of the respiratory epithelium emphysema-like pulmonary disease associated with human immunodeficiency virus infection increased susceptibility to pulmonary emphysema among hiv-seropositive smokers viral genome sequencing by random priming methods diversity of the human intestinal microbial flora genomic islands in the pathogenic filamentous fungus aspergillus fumigatus transbronchial biopsy of peripheral lung lesions low yield of pulmonary surfactant in cigarette smokers loss of bacterial diversity during antibiotic treatment of intubated patients colonized with pseudomonas aeruginosa human metapneumovirus infection in young children hospitalized with respiratory tract disease molecularphylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases histology and gross anatomy of the respiratory tract multiplex pcr using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains molecular analysis of human forearm superficial skin bacterial biota substantial alterations of the cutaneous bacterial biota in psoriatic lesions relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection host defence mechanisms of the respiratory system follow-up studies of asthma from childhood to adulthood metagenomic analysis of the human distal gut microbiome population disparities in asthma differences between tissue-associated intestinal microfloras of patients with crohn's disease and ulcerative colitis synergism between allergens and viruses and risk of hospital admission with asthma: case-control study long-term inhaled corticosteroids in preschool children at high risk for asthma molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products pathogenic bacteria and viruses in induced sputum or pharyngeal secretions of adults with stable asthma molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis microbial ecology of the cystic fibrosis lung where are all the undescribed fungi bronchoalveolar lavage disordered microbial communities in asthmatic airways pathogenesis of asthma infections and the development of allergy decreased contents of surfactant proteins a and d in bal fluids of healthy smokers fungal allergens impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity microbes on the human vaginal epithelium wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the association of family size with atopy and atopic disease bacterial diversity within the human subgingival crevice nontuberculous mycobacterial disease and aspergillus-related lung disease in bronchiectasis comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis, and chronic obstructive pulmonary disease allergic bronchopulmonary fungal disease caused by bipolaris and curvularia mp (1999) pulmonary aspergillus colonization in humans and its impact on management of critically ill patients rhinovirus illnesses during infancy predict subsequent childhood wheezing atypical pathogen infection in adults with acute exacerbation of bronchial asthma diversity of asthma: evolving concepts of pathophysiology and lessons from genetics the global burden of disease trends in causes of death among persons with acquired immunodeficiency syndrome in the era of highly active antiretroviral therapy species identification system for dermatophytes based on the dna sequences of nuclear ribosomal internal transcribed spacer 1. nihon ishinkin gakkai zasshi allergic bronchopulmonary aspergillosis an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study diagnosis and immunotherapy of mould allergy. iv. relation between asthma symptoms, spore counts and diagnostic tests reduced diversity of faecal microbiota in crohn's disease revealed by a metagenomic approach does antibiotic exposure during infancy lead to development of asthma?: a systematic review and metaanalysis isolation prevalence of pulmonary nontuberculous mycobacteria in ontario a link between chronic asthma and chronic infection human metapneumovirus and exacerbations of chronic obstructive pulmonary disease asthma and wheezing in the first six years of life. the group health medical associates genes, environments, development and asthma: a reappraisal changes in indoor allergen and fungal levels predict changes in asthma activity among young adults severity of asthma is related to endotoxin in house dust prevalence and clinical predictors of pneumocystis colonization among hiv-infected men association of chronic obstructive pulmonary disease severity and pneumocystis colonization airway obstruction is increased in pneumocystis-colonized human immunodeficiency virus-infected outpatients genomic sequence of the pathogenic and allergenic filamentous fungus aspergillus fumigatus pneumocystis colonization, airway inflammation, and pulmonary function decline in acquired immunodeficiency syndrome mold sensitization is common amongst patients with severe asthma requiring multiple hospital admissions comparison of skin prick tests with specific serum immunoglobulin e in the diagnosis of fungal sensitization in patients with severe asthma inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of cd8+ t lymphocytes with fev1 the effect of elevated temperature on gene transcription and aflatoxin biosynthesis studies on the fungal flora in the lung of human necropsy cases. a critical survey in connection with the pathogenesis of opportunistic fungus infections declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. hiv outpatient study investigators respiratory viruses in childhood asthma infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations house dust endotoxin and wheeze in the first year of life the effects of antifungal therapy on severe asthma with fungal sensitization and allergic bronchopulmonary aspergillosis bacterial diversity in human subgingival plaque relationship between bacterial colonisation and the frequency, character, and severity of copd exacerbations viruses as precipitants of asthma symptoms viral infections, atopy, and asthma: is there a causal relationship? scope of the copd problem in north america: early studies of prevalence and nhanesiii data: basis for early identification and intervention detection of pneumocystis carinii dna in patients with chronic lung diseases epidemic asthma and the role of the fungal mold alternaria alternata gene expression profiles of bronchoalveolar cells in pulmonary tb human targets of pseudomonas aeruginosa pyocyanin the organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients amplification of inflammation in emphysema and its association with latent adenoviral infection respiratory tract fluids: analysis of content and contemporary use in understanding lung diseases what can comparative genomics tell us about species concepts in the genus aspergillus? cd8+ t lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease exposure to alternaria alternata in us homes is associated with asthma symptoms prospective study of transbronchial biopsies in the management of heart-lung and single lung transplant patients respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease principles of antibiotic treatment of community-acquired pneumonia in the outpatient setting bacterial infection and the pathogenesis of copd infectious etiology of acute exacerbations of chronic bronchitis bacteria in exacerbations of chronic obstructive pulmonary disease: phenomenon or epiphenomenon? infection in the pathogenesis and course of chronic obstructive pulmonary disease the inverse association between tuberculin responses and atopic disorder persistent pneumocystis colonization leads to development of chronic obstructive pulmonary disease in a non-human primate model of aids aspergillus bronchitis in cystic fibrosis severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 bronchiolitis to asthma: a review and call for studies of gene-virus interactions in asthma causation purification and characterization of defensins from cystic fibrosis sputum bacterial infections of the lung in normal and immunodeficient patients bronchial inflammation: its relationship to colonizing microbial load and alpha(1)-antitrypsin deficiency distribution of lung cell numbers and volumes between alveolar and nonalveolar tissue hay fever, hygiene, and household size investigation into the production of 2-pentylfuran by aspergillus fumigatus and other respiratory pathogens in vitro and human breath samples asthma in remission: can relapse in early adulthood be predicted at 18 years of age a meta-analysis of the association between caesarean section and childhood asthma asthma and cigarette smoking the role of transbronchial lung biopsy in the treatment of lung transplant recipients. an analysis of 200 consecutive procedures detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis an obesity-associated gut microbiome with increased capacity for energy harvest lymphocytic alveolitis, bronchoalveolar lavage viral load, and outcome in human immunodeficiency virus infection prevalence and antimicrobial susceptibility of microorganisms isolated from sputa of patients with cystic fibrosis mucociliary clearance and airways obstruction in smokers, ex-smokers and normal subjects who never smoked the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers exposure to endotoxin or other bacterial components might protect against the development of atopy childhood factors associated with asthma remission after 30 year follow up neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma prevalence and economic burden of bronchiectasis different resistance mutations can be detected simultaneously in the blood and the lung of hiv-1 infected individuals on antiretroviral therapy human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals fungal contamination of bedding antibiotic-resistant obligate anaerobes during exacerbations of cystic fibrosis patients evidence of a causal role of winter virus infection during infancy in early childhood asthma role of infections in the induction and development of asthma: genetic and inflammatory drivers mycobacterial infection inhibits established allergic inflammatory responses via alteration of cytokine production and vascular cell adhesion molecule-1 expression use of the bronchofiberscope for bronchial brush biopsy. diagnostic results and comparison with other brushing techniques signature patterns revealed by microarray analyses of mice infected with influenza virus a and streptococcus pneumoniae characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods key: cord-000254-bufbjdmw authors: clement, annick; nathan, nadia; epaud, ralph; fauroux, brigitte; corvol, harriet title: interstitial lung diseases in children date: 2010-08-20 journal: orphanet j rare dis doi: 10.1186/1750-1172-5-22 sha: doc_id: 254 cord_uid: bufbjdmw interstitial lung disease (ild) in infants and children comprises a large spectrum of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. these disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. typical features of ild include dyspnea, diffuse infiltrates on chest radiographs, and abnormal pulmonary function tests with restrictive ventilatory defect and/or impaired gas exchange. many pathological situations can impair gas exchange and, therefore, may contribute to progressive lung damage and ild. consequently, diagnosis approach needs to be structured with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. several classifications for ild have been proposed but none is entirely satisfactory especially in children. the present article reviews current concepts of pathophysiological mechanisms, etiology and diagnostic approaches, as well as therapeutic strategies. the following diagnostic grouping is used to discuss the various causes of pediatric ild: 1) exposure-related ild; 2) systemic disease-associated ild; 3) alveolar structure disorder-associated ild; and 4) ild specific to infancy. therapeutic options include mainly anti-inflammatory, immunosuppressive, and/or anti-fibrotic drugs. the outcome is highly variable with a mortality rate around 15%. an overall favorable response to corticosteroid therapy is observed in around 50% of cases, often associated with sequelae such as limited exercise tolerance or the need for long-term oxygen therapy. interstitial lung disease (ild) in infants and children represents a heterogeneous group of respiratory disorders that are mostly chronic and associated with high morbidity and mortality (around 15%) [1, 2] . these disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. typical features of ild include the presence of diffuse infiltrates on chest radiograph, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange [3] . there have been many different approaches to the classification of ild, with major shifts based on clinical investigation, improvement in chest imaging, and collaboration with pathologists. in 1998, katzenstein and myers proposed four histopathologically distinct subgroups of idiopathic interstitial pneumonias: usual interstitial pneumonia (uip), desquamative interstitial pneumonia (dip) and a closely related pattern termed respiratory bronchiolitis-associated ild, acute interstitial pneumonia (formerly hamman-rich syndrome), and non specific interstitial pneumonia (nsip) [4] . in 2002, an international multidisciplinary consensus classification of idiopathic interstitial pneumonias was proposed by the american thoracic society (ats)/european respiratory society (ers) [5] . this classification defined a set of histologic pattern that provided the basis for clinico-radiologic-pathologic diagnosis, with the final pathologic diagnosis being made after careful correlation with clinical and radiologic features. however, as discussed in several reports, the classification schemes of adult ild are not satisfactory for the pediatric cases which seem to comprise a broader spectrum of disorders with a more variable clinical course [6] . in addition, pediatric histologic patterns often do not resemble pathologic features of lung tissues from adults and some forms are only observed in children younger than 2 years. among the proposed classifications for pediatric ild, one strategy frequently used is to separate the primary pulmonary disorders and the systemic disorders with pulmonary involvement. recently, an additional group has been introduced which is based on the concept that some pediatric ild are observed more frequently in infants, while others are more specific to older children. the last ers monography on ild provided a chapter on pediatric classification which is based on a clear distinction between children aged 0-2 years and children over 2 years-old [7] . indeed the stage of lung development and maturation should be taken into consideration when approaching a diagnosis of pediatric ild. in this view, a new term "diffuse lung disease" has recently been introduced that comprises a diverse spectrum of lung disorders with impaired gas exchange and diffuse infiltrates by imaging. these disorders, more prevalent in young children, include diffuse developmental disorders, lung growth abnormalities, neuroendocrine cell hyperplasia and pulmonary interstitial glycogenosis, surfactant dysfunction disorders, disorders related to systemic diseases, disorders of immunocompromised host, and disorders of normal host caused by various insults such as aspiration syndrome or infections [8] . some diseases are mostly observed in older children such as systemic diseases, idiopathic disorders as described in adults (dip, uip, nsip and lymphoid interstitial pneumonia (lip)), unclassifiable ild and also infectious disorders [9] . it is important to point out that the pathologic processes underlying the so-called diffuse lung diseases involve not only the alveolar structure but also the distal part of the small airways and the conducting zone, i.e. the terminal bronchioles. terminal bronchioles are lined with a simple cuboidal epithelium containing clara cells, basal cells and a limited number of ciliated cells. clara cells secrete nonsticky proteinaceous compounds to maintain the airway in the smallest bronchioles, which constitute the quiet zone between the conducting and the respiratory lung zones [10] . the terminal bronchioles are surrounded by a spiral of smooth muscle. each of the terminal bronchioles divides to form respiratory bronchioles which contain a small number of alveoli. consequently, the term of diffuse lung disease refers to disorders that can affect both the distal part of the conducting and the respiratory lung zones, and include ild as well as pathological processes leading to obstruction/ obliteration of small airways [8] . therefore, diffuse lung diseases encompass a broader group of diseases than ild which refers to disorders that affect the respiratory function of the lung and consequently the pulmonary structure responsible of the diffusion of gases between blood and air (i.e. the alveolar epithelium, the interstitium, and the pulmonary capillary endothelium). the present review focuses on ild in immunocompetent children, and excludes pulmonary consequences of previous lung injury in situations of chronic aspiration syndromes, resolving acute respiratory distress syndrome, and bronchopulmonary dysplasia. an estimated prevalence of 3.6 per million has been reported by dinwiddie and coworkers through a national survey of chronic ild in immunocompetent children in the united kingdom and ireland over a three year period (1995) (1996) (1997) (1998) [1] . this prevalence is certainly under-estimated due to the lack of standardized definitions and the absence of organized reporting systems. from the limited published data composed mainly of case reports and small series, it seems that pediatric ild occurs more frequently in the younger age and in boys [11] . in addition, nearly 10% of cases appear to be familial [12] . the understanding of the mechanisms underlying the development and progression of ild remains elusive [13, 14] . indeed, for a long time, chronic ild and pulmonary fibrosis were believed to result mainly from chronic inflammation following an initial injury to the alveolar epithelial lining [15, 16] . in cases of limited injury, it was thought that the reparative attempt could reverse the trend toward fibrosis. by contrast, in situations of continuing injury, the repair process driven by inflammatory molecules produced by the local cells will result in scarring and structural changes. therefore, by targeting the inflammatory response, the belief was that fibrosis could be prevented or controlled. this theory explains the large use of anti-inflammatory therapy with, however, limited clinical efficacy. based on clinical and experimental observations, a new paradigm has progressively emerged with the alveolar epithelium being viewed as a key actor in the development of ild [17] [18] [19] . following injury, alveolar epithelial cells (aec) may actively participate in the restoration of a normal alveolar architecture through a coordinated process of re-epithelialization, or in the development of fibrosis through a process known as epithelial-mesenchymal transition (emt) [20] . complex networks orchestrate emt leading to changes in cell architecture and behaviour, loss of epithelial characteristics and gain of mesenchymal properties. the reasons for epithelial cell loss and inappropriate re-epithelialisation are still debated, but ongoing apoptosis is believed to be a key component in the progression of the disorder [21] . prolonged denudation of the basement membrane contributes to altered interactions and cross-talk between aecs and mesenchymal cells, resulting in profound modifications of cell functions with imbalanced production of oxidants, proteases, and polypeptide mediators including cytokines and growth factors such as transforming growth factor (tgf)-β and endothelin (et)-1. a consequence is the perpetuation of a vicious cycle with tgf-β promoting epithelial cell apoptosis, which in turn increases the local production of tgf-β [22] . et-1 is also considered to be an important actor, based on the current knowledge of its numerous functions including fibroblast and smooth muscle cell mitogen, and stimulant of collagen synthesis [23, 24] . recent studies showed that et-1 is produced by aec, and could induce alveolar emt via stimulation of endogenous tgf-β production. ild may be caused by myriad etiologies with differing prognoses and natural history. indeed, multiple factors may injure the alveolar epithelium and initiate the development of ild [25] . the initiating injury can be introduced through the airways and the circulation, or can occur as a result of sensitization. consequently, the mechanisms underlying disease progression will be influenced by the causative event as well as by the host and the environment. these mechanisms are developed through interactions of multiple pathways, which include apoptotic pathways, developmental pathways, and endoplasmic reticulum (er) associated pathways ( figure 1 ). apotosis plays a central role in lung remodeling associated with ild [26] . an important molecule in the events associated with epithelial cell apoptosis is tgf-β, which is overexpressed in ild. downstream events linked to upregulation of tgf-β include modifications in the expression of various components of the cell cycle machinery, mainly the cyclin-dependent kinases (cdk) system that plays an essential role in ensuring proper cell cycle progression. recently, much work has been focused on the protein p21cip1, a member of the cdk inhibitor family. this protein promotes cell cycle arrest to apoptosis in cases of cellular dna damage. interestingly, upregulation of p21cip1 has been reported in the lung tissues of patients with pulmonary fibrosis, primarily in hyperplastic alveolar epithelial cells [27] the increased expression of p21cip1 can favour the process of epithelial cell apoptosis. apoptotic cells can also produce tgf-β. a consequence would be the perpetuation of a vicious cycle with tgf-β promoting epithelial cell apoptosis, which in turn increases the local production of tgf-β. recently, it has been suggested that genes associated with lung development and embryonic pathways could be involved in aberrant epithelium-mesenchymal crosstalk and epithelial plasticity, and could therefore participate in the development of chronic ild. selman and coworkers reported that lung fibrosis is characterized by enrichment for genes associated with cell adhesion, extracellular matrix, smooth muscle differentiations, and genes associated with lung development [28] [29] [30] [31] . during emt in the embryonic period, cells undergo a switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype [32] . molecular processes governing emt are induced by a cooperation of receptor tyrosine kinases or oncogenic ras (rtk/ras) pathway and tgf-β signaling [33] . recently, additional pathways and effectors have been reported to play a role in the induction of emt, such as wnt//β-catenin, notch and sonic hedgehog signalling [34] . recent reports strongly suggest that the er stress may represent an important mechanism of the altered repair process observed in the alveolar epithelium of fibrotic lung [35] . situations associated with abnormal regulation of the cascade of events leading to the formation of mature protein result in either misfolding or mistargeting of the protein. these events trigger induction of intracellular aggregate formation and er stress, which can lead to cell death through apoptosis and autophagic pathways [36, 37] . several stimuli including oxidant-antioxidant imbalance, viral proteins, inflammatory molecules, nutrient deprivation may induce er stress [38, 39] ( figure 2 ). among the cytoprotective mechanisms available are the er chaperones such as binding immunoglobulin protein (bip). interestingly, mutant bip mice have been reported to die within several hours of birth from respiratory failure due to impaired secretion of pulmonary surfactant by type 2 aec. in these animals, expression of surfactant protein (sp)-c was reduced and the lamellar bodies were malformed, indicating that bip figure 2 alveolar structure disorder-associated ild and er stress. the (endoplasmic reticulum) er and its protein maturation machinery allow the synthesis of mature secretory and membrane proteins with specific folded conformation. in situations of stress induced by genetic mutations or environmental factors, unfolded or misfolded proteins are retained in the er and induce a defence mechanism called the er stress response. the induction of er chaperones is critical to increase the er folding capacity allowing the production of correctly folded protein. when this defence mechanism is impaired, the misfolded proteins can either be degraded by the proteasome or form protein aggregates. protein aggregates are toxic and can cause conformational diseases. within the alveolar epithelium, misfolding of sp-c could trigger induction of intra-cellular aggregate formation and er stress, with consequently development of alveolar structure disorder-associated ild and conformational disease. plays a critical role in the biosynthesis of surfactant [40] . several recent reports suggest the possible implication of er stress in ild, with activation of stress response markers in fibrotic lung tissues. it is now well established that surfactant dysfunction plays an important role in the development and progression of ild. pulmonary surfactant is a multimolecular complex constituted of phospholipids and proteins secreted by type 2 aec into the alveolar space. it assures alveolar stability by reducing surface tension along the epithelial lining and this role involves mainly the lipids and the specific hydrophobic sp, sp-b and sp-c. other important players in surfactant metabolism include the atp-binding cassette, sub-family a, member 3 (abca3) and the thyroid transcription factor 1 (ttf-1). surfactant deficiency can be induced by a number of primitive and secondary mechanisms. among them are genetic defects with mutations in sp-b gene (sftpb) as well as genes coding for sp-c (sftpc), abca3, and ttf-1 [41] [42] [43] . more than 30 sftpb (located on chromosome 2) mutations have been identified among patients with a congenital deficiency in sp-b. for sftpc located on chromosome 8, at least 35 mutations have been described, localized primarily in the coohterminal brichos domain [44, 45] . a proposed function of the brichos domain is a chaperone-like activity, which could prevent misfolding and aggregation of the parent protein. alterations in the brichos domain could therefore lead to diseases through mechanisms related to abnormal protein processing and cell toxicity [46] . recently, several studies have also documented the role of abca3 deficiency in ild. abca3 functions in the transport of surfactant lipids into lamellar bodies and is required to maintain pulmonary surfactant phospholipid homeostasis. another contributor of ild is ttf-1 (nk2 homeobox 1) dysfunction. ttf-1 is a critical regulator of transcription for the surfactant protein sp-b and sp-c. it is encoded by a gene located on chromosome 14q13 and is composed of three exon and two introns [47] . it is expressed in the thyroid, brain and lung. stem cell dysfunction represents a new domain of investigation. alveolar epithelium regeneration and repair requires activation and proliferation of tissue-resident (progenitor) cells and their differentiation to replace the damaged cells [48] . however, unlike cancer cells, stem cells are not immortal and display decreasing telomere length with aging [49] . telomere shortening has been documented to be associated with reduced capacity for stem cell renewal, and decreased activity of telomerase, the polymerase responsible for telomere maintenance. the stem cells of the alveolar epithelium are the type 2 aec, and expression of telomerase has been documented in these cells [48] . experimental studies have also indicated that telomerase is expressed mainly during lung development with a peak expression before birth followed by a decrease to nearly undetectable levels in mature alveolar epithelium. interestingly, telomerase expression and activity could be reinduced in normal quiescent type 2 aec exposed to oxidative stress [50] . the current understanding is that a population of type 2 aec may have the capacity to survive injury through telomerase activation, and consequently may be responsible for repopulation of the damaged alveolar epithelium. on the basis of reports of pulmonary disorders in dyskeratosis congenita (a rare hereditary disease of poor telomere maintenance), recent and exciting findings have documented mutations in the telomerase gene in familial idiopathic pulmonary fibrosis [51] . in addition, it is likely that environmental factors such as inflammation, oxidative stress, or virus infection may modify telomerase activity and account for the development of organ-specific disease associated with telomerase dysfunction. in this view, new data in chronic respiratory diseases support the concept that alveolar stem cell dysfunction may play an important role in the rate of progression or severity in ild [52] . the question whether telomerase mutations or telomere dysfunction may be implicated in pediatric ild needs to be addressed in prospective studies, one possible tool being determination of telomere length in circulating leukocytes. the frequency of lung fibrotic disorders is much lower in children than in adults. some clinical situations have features certainly unique to children, but many of these diseases overlap their adult counterparts with the primary event being injury and damage of the alveolar epithelium [11, 13] . yet, the overall outcome and prognosis of the diseases in children are thought to be less severe than in adult patients. in addition, pediatric ild is more responsive to therapeutic strategies than adult ild [9] . these differences may be explained by the types of initial injury, which may not be similar due to changes in the host environment. another explanation is the modifications of the process of wound healing with age. comparison of the response to injury in foetal and adult skin shows clear differences [53] . skin wound healing in the foetus is characterized by complete regeneration of the skin and the absence of scar formation. progressively with age, the skin looses the capacity to regenerate the original tissue architecture with the result being scar formation that extends outside the wound bed. the process of healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors [54] . the slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. in a study on the role of aging in the development of cardiac fibrosis in the rabbit, differences in the cascade of events leading to myocardial remodeling were observed, with mainly the presence of more myofibroblasts synthesising collagen and expressing high levels of tgf-β in older animals [55] . a study of growth factors involved in skin wound healing in young and aged mice also showed age-dependent changes. expression of all the fibroblast growth factors was diminished in aged mice, even in healthy skin. in addition, the post-wound regulation of expression of these factors and of tgf-β was less pronounced and slower than in young mice. these findings are in agreement with data observed in muscle that indicated significant alterations in the tgf-β production with age [56, 57] . other potential mechanism is linked to the observation that injury in adult tissues does in certain circumstances stimulate tissue regeneration, depending on the presence of small subsets of primitive stem cells. stem cells are the self-renewing, primitive, undifferentiated, multipotent source of multiple cell lineages [49] . while such cells are critical for development and growth through childhood, residual pools of adult stem cells are hypothesized to be the source of the frequently limited tissue regeneration and repair that occurs in adults [58] . unlike embryonic stem cells, adult stem cells are not immortal, and show decreasing telomere length with increasing age. the naturally limited replacement capacity of such endogenous stem cell pools may occur via exhaustion of the stem cell pool or arise as a consequence of inherited or acquired mutations that alter proper stem cell function [59] . the limited life span of cells may result from replicative senescence in response to various stresses including dna damage, oxidants, and telomere erosion [52] . all these forms of injury have been documented in the lung from adult patients with ild. the prevalence of children ild is higher in the younger patients: more than 30% of patients are less than 2 years at diagnosis, as recorded by the recent ers task force. 7% have parental consanguinity and nearly 10% of case siblings were affected by similar diseases. there is a male predominance with a sex ratio of 1.4. the presenting clinical manifestations are often subtle and nonspecific. the onset of symptoms is, in most cases, insidious and many children may have had symptoms for years before the diagnosis of ild is confirmed. however, the majority of patients has symptoms for less than one year at the time of initial evaluation. the clinical manifestations vary from asymptomatic presentation with radiological features suggestive of ild to more characteristic presence of respiratory symptoms and signs such as cough, tachypnea and exercise intolerance [9, 60] . these varying presentations are also reflected in the report published by fan et al. who systematically evaluated the clinical symptoms and physical findings of 99 consecutive children with ild [2] . common symptoms at presentation included cough, dyspnea, tachypnea and chest wall retraction, exercise limitation and frequent respiratory infections. cough is observed in almost 75% of the patients, is normally non-productive and does not disturb sleep. tachypnea is observed in 80% of patients and is usually the earliest and most common respiratory symptom. unexplained fever is also reported in almost one third of infants. failure to thrive (37%), tiring during feeding and weight loss are also common symptoms, mainly in young patients. although a history of wheezing may be elicited in almost 50% of the patients, wheezing is documented by physical examination in only 20% of the cases. the frequent clinical findings are inspiratory crackles (44%), tachypnea and retraction. in a child with a normal birth history, these are strongly suggestive of ild. other findings associated with an advanced stage of lung disease include finger clubbing (13%) and cyanosis during exercise or at rest (28%) [9, 61] . during physical examination it is essential to check the presence of associated non-respiratory symptoms such as joint disease, cutaneous rashes, and recurrent fever suggestive of collagen-vascular disorders. details should also be obtained on precipiting factors such as feeding history, infections, or exposure to dust and drugs. in addition, information on relatives or siblings with similar lung conditions should be gathered. plain radiographs are usually performed in a child suspected of ild at first presentation, but the information provided is often limited and the key chest imaging tool for diagnosis is the high resolution computed tomography (hrct), which can visualize the parenchymal structure to the level of the secondary pulmonary lobule. hrct technique for ild diagnosis has been extensively discussed [62] [63] [64] . to optimise spatial resolution, there is a general agreement to use thin sections, the smallest field of view and a sharp resolution algorithm. the most common hrct feature of ild is widespread ground-glass attenuation. intralobular lines, irregular interlobular septal thickening and honeycombing are less common findings. large subpleural air cysts in the upper lobes adjacent to areas of ground-glass opacities have been also reported in young children with ild. these cysts are interpreted as paraseptal or irregular emphysema. hrct is useful for ild diagnosis and selection of lung area to be biopsied. it is proposed that it also may contribute to monitor disease activity and/or severity. however, evaluation is still needed to support a role of hrct as a follow up tool in pediatric patients. pulmonary function testing (pft) techniques are well established in children and adolescents. however, children aged 2-6 years represent a real challenge in pulmonary function assessment as they cannot be sedated and find it difficult to cooperate with all respiratory manoeuvres. in 2007, an ats and ers statement on pft in preschool children summarized the current knowledge on the pft techniques suitable for young children [65, 66] . although pft does not provide specific information, it represents a useful investigation for both the diagnosis and the management of ild [11] . generally, in ild, pulmonary function abnormalities reflect a restrictive ventilatory defect with reduced lung compliance and decreased lung volumes [67] [68] [69] . vital capacity (vc) is variably diminished; the decrease in total lung capacity (tlc) in general is relatively less than in vc. functional residual capacity (frc) is also reduced but relatively less than vc and tlc, and residual volume (rv) is generally preserved; thus the ratios of frc/tlc and rv/tlc are often increased. airway involvement is observed only in a minority of patients. lung diffusing capacity of carbon monoxide (dlco) or transfer factor (tlco) is often markedly reduced and may be abnormal before any radiological findings. however, dlco corrected for lung volume may also be normal in many children. hypoxemia as defined by a reduced resting arterial oxygen saturation (sao 2 ) or a reduced resting arterial oxygen tension is often present. hypercarbia occurs only late in the disease course. during exercise the above described dysfunctions become even more pronounced. thus, gas exchange during exercise might be a more consistent and sensitive indicator of early disease [3] . bronchoalveolar lavage (bal) usefully provides specimens for cytological examination, microbial cultures, and molecular analysis. besides infections, bal can be of diagnostic value in several situations. in the context of pulmonary alveolar proteinosis, bal abnormalities are characterized by milky appearance fluid, abundant proteinaceous periodic acid schiff positive material, and presence of foamy alveolar macrophages (am) [70] . bal can also be diagnostic for pulmonary alveolar haemorrhage [11] . this diagnostic is easy when the bal fluid has a bloody or pink color, but its gross appearance may be normal. microscopic analysis may then be of value by documenting the presence of red blood cells in am or haemosiderin laden am [71] . among other situations, the diagnosis of langerhans cell histiocytosis can be performed with the use of the monoclonal antibodies revealing the presence of cd1a positive cells (in more than 5% of the bal cells) [72] . lipid disorders with lung involvement represent another indication of bal. this includes congenital lipid-storage diseases (gaucher's disease and niemann-pick disease) or chronic lipid pneumonia due to chronic aspiration [73, 74] . however, in cases of aspiration syndromes, the presence of lipid laden am is sensitive but not specific [75] . in other pathological situations, bal can usefully serve to direct further investigations. accumulation of bal t-lymphocytes with prevalence of cd4+ cells is suggestive of sarcoidosis, whilst prevalence of cd8+ cells is suggestive of hypersensitivity pneumonitis [76] . also, an increase in bal eosinophils suggests pulmonary infiltrates associated with eosinophilia syndromes [77] . depending of the underlying diseases, a number of cellular and molecular investigations can be proposed including the studies of various surfactant components, phospholipids and apoproteins [78] . with increasing recognition of the different patterns of ild and their clinical significance, histological investigation has become increasingly important. depending on disorder presentation, biopsy may concern more accessible organs than the lung such as the skin or the liver in sarcoidosis. histological evaluation of lung tissue usually represents the final step in a series of diagnostic approaches. different methods may be used to obtain lung tissue. the major difference between individual methods lies mainly in balancing invasiveness against the potential for obtaining adequate and sufficient tissue for diagnosis. the techniques of choice are open lung biopsy and video assisted thoracoscopy biopsy. in children, open lung biopsy usually provides sufficient tissue with few complications related directly to the biopsy procedure [79] . video assisted thoracoscopy biopsy is an alternative to open lung biopsy, and it has been shown that the procedure can be safely performed, even in small children [80] . the place of other methods like transbronchial lung biopsy and percutaneous needle lung biopsy in appropriate diagnosis of pediatric patients with ild has to be established [81] [82] [83] . the lung histological patterns that can be observed in ild have been reviewed by the ats/ers [5] . in children, they include mainly: dip, nsip, and lip. dip is characterized by airspaces filled with am, thickened alveolar septa, scattered mixed inflammatory cells and minimal fibrosis. many alveolar spaces are lined by hyperplastic type 2 aec. recently, association with surfactant disorders has been reported [41, [84] [85] [86] . nsip encompasses a broad spectrum of abnormalities with varying degrees of alveolar wall inflammation or fibrosis. the cellular pattern of nsip is characterized by mild to moderate interstitial chronic inflammation and type 2 aec hyperplasia in inflammation areas. it has been reported in a variety of underlying conditions including connective tissues diseases and surfactant disorders. lip features include a marked diffuse infiltrate of mature lymphocytes, plasma cells and histiocytes within the pulmonary interstitium, particularly the alveolar walls. they are often associated with either connective tissues disorders or immunodeficiency states, both congenital and acquired [9] . another pattern described mainly in adults is diffuse alveolar damage (dad), which includes diffuse homogeneous thickening of alveolar interstitial walls with myofibroblast accumulation, prominent type 2 aec hyperplasia and atypia, and hyaline membranes containing surfactant proteins and cellular debris [87] . usual interstitial pneumonia (uip) is rare in children [88] . it is characterized by severe remodeling of the alveolar structure with heterogeneous appearance consisting of contiguous areas of normal lung, dense scarring, and bronchiolar abnormal proliferation. interstitial inflammation is usually mild to moderate. histologic patterns of ild unique to infancy are described below. laboratory tests are used to exclude a number of respiratory diseases in childhood that does not typically present with ild such as chronic aspiration syndromes, resolving acute respiratory distress syndrome, tuberculosis, cystic fibrosis, bronchopulmonary dysplasia and diffuse pulmonary disease such as cystic fibrosis. laboratory tests also verify the absence of immunodeficiencies [3] . when these conditions have been eliminated, the spectrum of investigations that should be performed for the diagnostic approach will be guided by the history and clinical presentation in each individual child. these investigations are discussed below for the various disorders. in addition, an increasing number of blood and bal biomarkers for evaluation of disease severity and progression is currently investigated. the studied molecules include various cytokines and chemokines, surfactant protein d, krebs von den lungen-6 antigen (kl-6), matrix metalloproteinases mmp1 and mmp7 and defensins [89] [90] [91] [92] . a large number of pathological situations can impair gas exchange and contribute to progressive lung damage and ild. consequently, diagnosis approaches need to be organized by cause, with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. indeed, in a number of pathological situations, no final diagnosis is proposed and the conclusion reported by the physician in charge of the patient is ild of unknown cause. however, information from recent studies highlights the concept that lung insults caused by substances from the environment or in the context of systemic diseases are largely under-estimated and should be more often discussed considered in the diagnostic process. based on this consideration, the following diagnostic grouping for pediatric ild can be considered 1) exposure-related ild; 2) systemic disease-associated ild; 3) alveolar structure disorder-associated ild; and 4) ild specific to infancy. accordingly, a step-by-step etiological diagnostic approach is required and is summarized in figure 3 . once the diagnosis of ild is established on clinical, radiological, and functional findings, a careful history should be obtained for potential exposure-related diseases leading to discuss the need for specific serum antibodies against offending antigens. the following step focuses on the search for systemic disease associated ild, oriented by the presence of clinical and functional extra-pulmonary manifestations. in such situations, additional investigations should include specific serum antibodies and possibly tissue biopsies in organs other than the lung. finally, elimination of these 2 groups of causes with a lung restricted expression of the disease allows discussing the potential interest of a lung biopsy. exposure-related disease refers to diseases caused by a sufficient level of exposure (dose) to components with target organ contact, and subsequent biologic changes and clinical expression. many agents have been associated with pulmonary complications of various types including ild. the adult literature has provided extensive lists of candidate molecules [93] . in children, the potential involvement of these molecules is not similar as the environmental conditions and the use of therapeutic drugs differ. it is important to point out that exposure-related diseases are certainly under-estimated in the pediatric age. one reason is linked to the fact that the diagnosis is less often discussed than in adults as pediatricians and other child health care providers do not usually have the expertise necessary to take an environmental history. in this review, the most frequent causes of exposure-related ild are discussed. hypersensitivity pneumonitis (hp) is a cell-mediated immune reaction to inhaled antigens in susceptible persons [94, 95] . in children, hp is often associated with exposure to antigens in the home environment as well as with certain hobbies. the most frequent types of hp include bird fancier's diseases, humidifier lung diseases, and chemical lung diseases. bird fancier's diseases are induced by exposure to birds with the antigens being glycoproteins in avian droppings, and on feathers. importantly, respiratory symptoms in exposed patients who have only one pet bird at home should raise the suspicion of hp [96] . humidifier lung diseases (air conditioner lung, misting fountain lung, basement lung diseases) are caused mainly by free-living amoeba and nematodes, as well as bacteria and fungi. chemical lung diseases can be induced by various inorganic antigens such as those from vaporized paints and plastics. low-molecular-weight chemicals may react with proteins in the airways, thus forming complete antigens. once exposure history is obtained, additional information is required and includes biologic tests allowing measurements of environmental contaminants and interpretation of the results by environmental medicine experts. as hp is believed to be an adult disease, children are often diagnosed at the chronic stage of the disease resulting of a long-term exposure to low levels of inhaled antigens. children can develop subtle interstitial inflammatory reactions in the lung without noticeable symptoms for months [97] . clinical features in the classic form include non productive cough, dyspnea, malaise, asthenia and occasional cyanosis [95] . lung function abnormalities are not specific and appear similar to changes observed in other ild. hrct abnormalities vary from ground glass attenuation predominantly in the mid-upper zone to nodular opacities with signs of figure 3 search for ild etiology in children. ild is defined by the presence of diffuse infiltrates on chest radiographs or chest high resolution computed tomography, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange. the search for etiology requires a systematic step-by-step diagnostic strategy for identifying: exposure-related ild; systemic disease-associated ild; alveolar structure disorder-associated ild; and ild specific to infancy. air-trapping [62, 63, 98] . laboratory tests focus mainly on the search for serum-precipitating igg antibodies against the offending antigen [95] . however, the presence of these antibodies is considered to be of questionable clinical relevance for diagnosis, as it is observed in up to 50% of serum samples of exposed but asymptomatic individuals. bal cell profile study typically shows an increase in total cell count with a remarkable elevation in the percentage of lymphocytes often over 50% with a decreased cd4/cd8 ratio [95, 97] . however, in contrast to studies in adults, the cd4/cd8 ratio could be within the normal range for children [76] . histopathologic evaluation of lung tissue is usually not necessary for the diagnosis of hp. at the present time, there is no diagnostic test that is pathognomonic for hp, and only significant predictors of hp are identified. the most significant diagnostic tool is a detailed environmental exposure history. other diagnostic features include: positive precipitating antibodies to the offending antigen; recurrent episodes of symptoms; symptoms occurring 4-8 h after exposure; occurrence of diffuse parenchymal lung disease by lung function and hrct; bal abnormalities with lymphocytic alveolitis and increased cd8+ t cells. medication, drug, radiation and tobacco exposure drugs used in inflammatory or cancer pediatric diseases can cause ild. they include anti-inflammatory agents (e.g. aspirin, etanercept), immunosuppressive and chemotherapeutic agents (e.g. azathioprine, methotrexate, cyclophosphamide), antibiotics, cardiovascular agents, and, for teenagers, illicit drugs [99, 100] . there are no distinct clinical, radiographic or pathologic patterns, and the diagnosis is usually made when a patient is exposed to medication known to result in lung disease, with a timing of exposure appropriate for disease development and elimination of other causes of ild. treatment relies on avoidance of further exposure and corticosteroids in markedly impaired patients. exposure to therapeutic radiation in the management of pediatric cancer may also results in ild. patients presenting within 6 months of therapy generally have radiographic abnormalities with ground glass patterns in both radiation-exposed and unexposed tissue [101] . the association between tobacco use and ild is less well appreciated than the relation with chronic obstructive pulmonary disease (copd). in addition, pediatric patients do not usually have a significant smoking history to develop respiratory disorders [102] . connective tissues disorders (ctd) are a heterogeneous group of immunologically mediated inflammatory diseases. their origins are multifactorial with genetic, constitutional and environmental elements contributing to their development. ctd refers to any disease that has the connective tissues of the body as a primary target of pathology. the connectives tissues are composed of two major structural proteins, elastin and collagen, with different types of collagen proteins in each tissue [103] . many ctd feature abnormal immune system activity associated with inflammation. pulmonary manifestations of ctd may include both vascular and interstitial components. from recent reports, the incidence of ild in the context of ctd appears to be higher than previously appreciated [104, 105] . importantly, ild may precede the development of clinically obvious ctd, sometimes by months or years. table 1 provides information on suggestive clinical and serological features in selected conditions. the main disorders to be considered in childhood are rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. the other include sjögren syndrome, dermatomyositis and polymyositis, ankylosing spondylitis, and mixed connective tissue disease. rheumatoid arthritis rheumatoid arthritis (ra) is an inflammatory disorder defined by its characteristic diarthroidal joint involvement. it is the most common ctd in children, but pulmonary involvement is less frequent than in adults. genetic and environmental factors seem to be important contributors of disease progression, with influence of sex (more frequent in male), presence of two copies of the hla-drb1 "shared epitope" (hla-dr se) and anticyclic citrullinated peptide antibody (anti-ccp), and possibly tobacco exposure [106, 107] .almost 50% of patients with ra have specific serologic abnormalities several years before the onset of joint symptoms, and the findings of elevated serum levels of igm rheumatoid factor or anti-ccp is associated with a high risk for the development of ra [107] . systemic sclerosis systemic sclerosis (ssc) is characterized by a progressive dermatologic abnormality [108] . its etiology remains unknown; it is believed to be a complex disease in which interactions between environmental, auto-immune, and genetic factors result in various disease phenotypes [109] . although it is a rare disease in childhood, the diagnosis is based on skin disease. cardiopulmonary complications are common and have been associated with death in young patients. almost all patients with ssc have serum antinuclear antibodies. the other autoantibody markers are listed in table 1. recently, the presence of anti-dna topoisomerase ii autoantibody has been reported to be a key factor in the development of ild, in association with class ii mhc status (hla-dr3, hla-dpbi) [110] . systemic lupus erythematosus systemic lupus erythematosus (sle) is an auto-immune disorder characterized by the involvement and dysfunction of multiple organ systems. the mechanisms of tissue injury involve autoantibody production and immunocomplex formation leading to an inflammatory process. diverse clinical phenotypes are observed, including a variety of mucocutaneous lesions, non erosive arthropathy, renal disease (glomerulonephritis and interstitial nephritis), lung disease, pericarditis, and a spectrum of neurologic disorders. laboratory abnormalities are characterized by the presence of antibodies reactive to nuclear (ana) and cytoplasmic antigens. pulmonary vasculitis are observed in vasculitic syndromes that preferentially affect small vessels (arterioles, venules, and capillaries). they include the anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis (wegener's granulomatosis, churg-strauss syndrome, and microscopic polyangitis) that share histologic similarities without immune deposits; anti-glomerular basement membrane (gbm) disease; henoch-schönlein purpura and cryoglobulinemia vasculitis. vasculitic syndromes that affect large/medium vessels (such as kawasaki's disease, polyarteritis nodosa) only occasionally affect the lung [111] . wegener's granulomatosis wegener's granulomatosis (wg) is a rare disease of uncertain cause. it seems to affect children as much as adults with an increasing reported incidence around 2.75 cases/million/year, mostly in teenagers with a reported median age of 14.2 years (4-17 years) [112, 113] . it is characterized by inflammation in a variety of tissues including blood vessels (vasculitis). wg primarily affects the upper respiratory tract, lung, and kidneys. the diagnosis is based on the combination of symptoms and a biopsy of affected tissue with necrotising granulomatous vasculitis in the absence of an infectious etiology. the diagnosis is further supported by positive blood tests for cytoplasmic-staining (c)-anca pr3 type [114] . churg-strauss syndrome churg-strauss syndrome (css) is a granulomatous small-vessel vasculitis. the cause of this allergic angiitis and granulomatosis is not known, but autoimmunity is evident with the presence of hypergammaglobulinemia, increased levels of immunoglobulin e (ige), and perinuclear-staining (p)-anca. the diagnosis relies on biopsy evidence for vasculitis and at least 4 criteria among the following: moderate to severe asthma, blood eosinophilia (at least 10%), and nonfixed pulmonary infiltrates with extravascular eosinophils on biopsy [115] . twenty-nine pediatric cases have been reported so far in the literature, with lung involvement in 72% of [116] . anti-glomerular basement membrane disease goodpasture syndrome is a rare disease that involves rapidly progressive kidney failure along with lung disease and is characterized by the deposition of anti-gbm antibodies. several cases have been reported in the pediatric literature. the autoantibodies mediate tissue injury by binding to their reactive epitopes in the basement membranes. this binding can be visualized as the linear deposition of immunoglobulin along the glomerular basement membrane. the principle component of the basement membrane is type iv collagen which can be expressed as 6 different chains, from alpha1 to alpha6. the goodpasture antigen has been localized to the carboxyl terminus of the noncollagenous domain of the alpha3 chain of type iv collagen. the anti-gbm antibody can usually be found in serum [117] . strong evidence exists that genetics play an important role. patients with goodpasture disease have an increased incidence of hla-drb1 compared to control populations [118] . granulomatous disorders are characterized by the presence of granulomas defined as a focal, compact collection of inflammatory cells in which mononuclear cells predominate. granulomas form as a result of tissue injury by a wide variety of agents including microorganisms, antigens, chemical, drugs and other irritants. in other situations including sarcoidosis, the etiologic factors remain to be determined. sarcoidosis sarcoidosis is a chronic inflammatory disease in which granulomatous lesions can develop in many organs, mainly the lung. its cause remains obscure, and most likely involves environmental and host factors [119] . the current concept is that a still unknown stimulus activates quiescent t cells and macrophages leading to recruitment and activation of mononuclear cells, with, as a consequence, granuloma formation, alveolitis, and in some cases interstitial lung fibrosis [120] . sarcoidosis is relatively uncommon among children. its diagnosis is based on a combination of suggestive clinical features, with histologically-documented noncaseating granuloma, in the absence of other known causes of granuloma formation [121] . the incidence and prevalence of sarcoidosis are reported to be influenced by age, race and geographic localization [122] . although the youngest patients reported were infants 2 and 3-months old, most of the cases in children occur in preadolescents and adolescents. from the national patient registry on patients with sarcoidosis in denmark during the period 1979-1994, 81 patients with a confirmed diagnosis were ≤16 years of age [123] . the calculated incidence was 0.29 per 100.000 person-years. in children ≤4 years of age, the incidence was 0.06; it increased gradually to 1.02 in children aged 14-15 years. marked racial differences in the incidence and prevalence of sarcoidosis have been reported by many authors [122] . various reports in the literature also indicate that race and ethnicity affect both the patterns of organ involvement and disease severity. in a follow-up study we have conducted in 21 children with pulmonary sarcoidosis, 12 children were black [124] . also the number of organs involved was higher in the black than in the caucasian children. clinical manifestations in sarcoidosis are the consequences of local tissue infiltration with sarcoid granuloma. therefore, disease expression depends on the organ or system involved and a variety of symptoms and physical findings can be observed [125] . the modes of presentation include non-specific constitutional symptoms, alone or associated with symptoms related to specific organ involvement. in the report of children with sarcoidosis in denmark, the most common non specific symptoms were asthenia, weight loss, and fever [123] . clinical findings mainly include respiratory manifestations, lymphadenopathy, skin lesions, ocular and central nervous system abnormalities. the most common radiographic findings are hilar lymph node enlargements, with or without lung changes. lung function abnormalities are frequently observed in children with restrictive pulmonary pattern and abnormal diffusing capacity [126] . other investigations such as bal documenting a lymphocytic alveolitis with increased cd4/cd8 ratio, and elevated serum angiotensin-converting enzyme may provide additional evidence of sarcoidosis [127] . other granulomatous disorders in children a number of pathological situations are associated with granulomatous disorders defined by the presence of non-caseating granuloma in biopsied tissues. infections are the main causes of other granulomatous diseases, and are in some cases related to disorders of neutrophil function such as chronic granulomatous disease (cgd) [128] . most children with cgd present with recurrent bacterial and fungal infections. the most frequently encountered pathogens are staphylococcus aureus, aspergillus, burkholderia cepacia, and enteric gram negative bacteria [129] . the most prominent pulmonary lesions include an extensive infiltration of the lung parenchyma and hilar adenopathy. in some situations, a homogeneous distribution of small granulomatous lesions can occur, with a radiological appearance of miliary tuberculosis. the other granulomatous diseases can be seen in other described diseases, such as immune disorders (including crohn's disease and histiocytosis x), hs pneumonitis, vasculitis disorders or neoplasms. lysosomal diseases gaucher's disease is an autosomal recessive disease and the most common of the lysosomal storage diseases. it is caused by a genetic deficency of the enzyme lysosomal gluco-cerebrosidase that catalyses the breakdown of glucocerebroside, a cell membrane constituent of red and white blood cells. the consequence is an accumulation of glucocerebroside in reticuloendothelial cells, leading to excessive deposition of fatty material in the spleen, liver, kidneys, lung, brain and bone marrow. pulmonary expression is mainly characterized by physiologic involvement (reduction in lung the diffusion capacity and the functional residual volume). lung imaging may show interstitial changes [130] . niemann-pick diseases are genetic diseases primarily due to deficiency of sphingomyelinase resulting in the accumulation of sphingomyelin within lysosomes in the macrophage-monocyte phagocyte system, mainly the brain, spleen, liver, lung, and bone marrow. histology demonstrates lipid laden macrophages in the marrow, as well as "sea-blue histiocytes" on pathology. the infantile form with a dominant neurologic expression is rapidly fatal. in older patients, cases of ild have been reported [131] . hermansky-pudlak syndrome is a heterogeneous group of autosomal recessive disorders associated with accumulation of a ceroid-like substance in lysosomes of a variety of tissues. it is characterized by albinism, bleeding tendency associated to poor platelet aggregation and systemic complications associated to lysosomal dysfunction. a chronic inflammatory process may explain the progressive development of ild and fibrosis [132] . familial hypercalcemia with hypocalciuria familial hypercalcemia with hypocalciuria is caused by autosomal dominant loss-of-function mutations in the gene encoding the calcium-sensing receptor (casr), a g-protein coupled membrane receptor expressed in many tissues [133] . loss-of-function mutations in casr impair the feedback inhibition of parathyroid hormone secretion in response to a rise in the blood calcium concentration. the result is hypercalcemia associated with inappropriately normal or mildly elevated levels of parathyroid hormone. in the kidneys, mutations in casr prevent the feedback inhibition of calcium reabsorption in situation of hypercalcemia, leading to relative hypocalciuria. respiratory symptoms are usually mild and associated with reduction in the lung diffusion capacity. lung histology indicates the presence of foreign body giant cells and mononuclear cells infiltrating the alveolar interstitium, without circumscribed granulomas. langerhans'-cell histiocytosis is part of the histiocytosis syndromes, which are characterized by an abnormal proliferation of langerhans' cells [134] . the langerhans cells are differentiated cells of monocyte-macrophage lineage that function as antigen-presenting cells. the origin of the expanded population of langerhans' cells is unknown; in adults, the only consistent epidemiologic association is with cigarette smoking. these cells may form tumors, which may affect various parts of the body. most cases of pediatric langerhans'-cell histiocytosis are observed in children between ages 1 and 15 years, with usually bone involvement (80%) including the skull. the tumors produce a punched-out appearance on bone x-ray, and can cause fracture without apparent traumatism. langerhans'-cell histiocytosis can also affects various organs including the lung [135] . children with pulmonary langerhans'-cell histiocytosis present in a variety of ways. they can be asymptomatic or present common symptoms such as nonproductive cough and dyspnea. hrct of the chest is a useful and sensitive tool for the diagnosis. indeed, the combination of diffuse, irregularly shaped cystic spaces with small peribronchiolar nodular opacities, predominantly in the middle and upper lobe, is highly suggestive of pulmonary langerhans'-cell histiocytosis [63] . other abnormalities include ground-glass attenuation. the presence of increased numbers of langerhans' cells in bal fluid (identified by staining with antibodies against cd1a) with a proportion greater than 5 percent is also strongly suggestive of pulmonary langerhans'-cell histiocytosis. histologically, the cellular lesions forms nodules containing a mixed population of cells with variable numbers of langerhans' cells, eosinophils, lymphocytes, plasma cells, fibroblasts, and pigmented alveolar macrophages. several forms of ild have been reported to occur with inflammatory bowel diseases (crohn's disease) and celiac disease [136] . primary biliary cirrhosis and chronic hepatitis have also been reported to be associated with parenchymal lung dysfunction [137, 138] . in addition, there are reports on ild in association with neurocutaneous disorders (tuberous sclerosis, neurofibromatosis, ataxia-telangiectasia) and amyloidosis [139] . depending on the causes, the components of the alveolar structure (the epithelium and the alveolar space, the interstitium, and the pulmonary capillary endothelium) can be involved differently and can serve as primary targets of the underlying pathological processes. based on history, clinical presentation, bal data, and, most important, on information from lung tissue studies, the disorders can be gathered in groups according to predominant structural targets (figure 4 ). the disorders affecting primarily the alveolar epithelium and the alveolar space share common histopathological description, with preserved pulmonary architecture, hyperplasia of type 2 aec, interstitial infiltrates composed of immuno/inflammatory cells and scattered myofibroblasts, and the alveolar space filled with either immuno/inflammatory cells, desquamated materials, or components derived from surfactant lipid and protein complex. in the coming years, it is likely that the list of disorders will expand rapidly with the availability of specific tissue markers. currently, the following grouping can be proposed: infections, surfactant disorders, and eosinophilic lung diseases. infections the role of infection, mainly viral, in the development and progression of ild is sustained by a number of human and experimental reports. from recent knowledge, it is strongly suggested that latent viral infections may be involved in the pathogenesis of ild, through targeting of the alveolar epithelium. the main virus implicated include adenovirus, members of human herpes virus family (epstein-barrr virus and cytomegalovirus), and respiratory syncitial virus [140] . number of other viruses can also be involved such as influenza a, hepatitis c, or even human immunodeficiency virus (hiv) in immunocompetent children [141] [142] [143] [144] . human adenovirus being predominantly respiratory pathogens, adenovirus infections can cause a variety of pulmonary symptoms and can persist for long periods of time. several studies in adult patients have indicated that the adenovirus gene product e1a could be detected figure 4 alveolar structure disorder-associated ild. depending on the causes, the alveolar structure components can be involved differently and serve as primary targets of the underlying pathological processes. based on history, clinical presentation, bal and lung tissue information, the disorders can be gathered in groups according to the predominant alveolar targets: epithelium, vascular or interstitial components. in lung tissues by in situ hybridization in up to 16% of cases of idiopathic pulmonary fibrosis. the causative role of the virus in the initiation of the disease remains uncertain, but it may be an important factor in its progression as treatment with corticosteroids may make patients more susceptible to adenovirus infection or reactivation from latency. e1a has been shown to increase the production of tgf-β and to induce lung epithelial cells to express mesenchymal markers, thereby contributing to remodeling of the alveolar structure [145] . isolation of the virus from the throat and serologic studies are diagnostic supportive, but the diagnosis is confirmed by the detection of the virus in lung tissues. epstein-barrr virus (ebv) and cytomegalovirus (cmv) are widespread pathogens that share the characteristic ability of herpesviruses to remain latent within the body over long periods. in mice, the control of herpesviruses replication have also been reported to be associated with the arrest of lung fibrosis [146] . ebv is present in all populations, infecting more than 95% of individuals within the first decades of life. infection by cmv is reported in 60% of individuals aged 6 and older and more than 90% of aged individuals have antibodies against cmv. in addition, cmv is also the virus most frequently transmitted to a developing fetus. most healthy people who are infected by ebv and cmv after birth have no symptoms, but infection is important to certain high-risk groups of infants and immunocompromised individuals. several studies in the adult literature have reported an increased incidence of ebv and cmv infection in patients with pulmonary fibrosis, associated with virus dna-positive lung tissue biopsies in several cases [147] . however, so far, no evidence of causal relationship between viruses and pulmonary fibrosis has been provided. respiratory syncytial virus (rsv) is the most common cause of viral lower respiratory tract infection. it affects people of all ages, and can cause severe disease in infants, in older immunodeficient children and the elderly. an intriguing feature of rsv infection is the susceptibility of previously infected individuals to reinfection with antigenically closely related viruses or the identical virus strain. recently, increased interest has been focused on the contribution of persistent rsv in several chronic lung diseases including chronic obstructive pulmonary disease [148] . the role of rsv in the physiopathology of theses disorders as well as and the mechanisms of its persistence remain to be elucidated [149] . interestingly, in a recent work on the histopathology of untreated human rsv infection, the presence of the virus in aec has been documented [150] . from these various data, a role of rsv in the development of ild needs to be investigated. immunostaining with rsv-specific antibodies of tissues from lung biopsy should be proposed. among the other pathogens, chlamydophila pneumoniae and mycoplasma pneumoniae are currently drawing increasing consideration. they are frequent causes of community acquired pneumonia in children. before the age of 10 years, almost 70% of children have had chlamydophila pneumoniae infection based on serological studies [151] . these pathogens are intracellular organisms that primarily infect respiratory epithelial cells and alveolar macrophages and have the propensity to persist within several cell types such as macrophages. they are well known to cause a wide variety of respiratory manifestations, with possible progression towards diffuse parenchymal diseases associated with interstitial infiltrates on chest imaging and reduction in the lung diffusion capacity [152] . regarding legionella pneumophilia infection, progression towards ild has been infrequently reported in adult patients. results from recent studies provided evidence that viruses can infect the alveolar epithelium and may be documented in lung tissues from patients using virus dna detection and immunohistochemistry. a number of specific antibodies are currently available and should prompt to investigate the presence of the above cited viruses in the lung tissues from children with ild. surfactant disorders surfactant disorders include mainly genetic surfactant protein disorders and pulmonary alveolar proteinosis the deficiency in sp-b is a rare autosomal recessive condition known to be responsible for lethal neonatal respiratory distress. rare survivals have been described in partial deficiencies [153, 154] . the sftpc mutation i73t (c.218 t > c) is the more prevalent mutation. others are described in only one family. the phenotype associated with sftpc mutations is extremely heterogeneous leading from neonatal fatal respiratory failure to children and adults chronic respiratory disease with ild [45] . recessive mutations in the abca3 gene were first attributed to fatal respiratory failure in term neonates but are increasingly being recognized as a cause of ild in older children and young adults. over 100 abca3 mutations have been identified in neonates with respiratory failure and in older children with ild [86, [155] [156] [157] [158] [159] [160] [161] . mutations in the ttf-1 gene are associated with "brainlung-thyroid syndrome" which combines congenital hypothyroidism, neurological symptoms (hypotonia, chorea), and ild of variable intensity [162] [163] [164] [165] [166] [167] [168] . so far, few mutations have been reported, mostly in exon 3 [169, 170] . pulmonary alveolar proteinosis (pap) is a rare lung disorder characterized by alveolar filling with floccular material derived from surfactant phospholipids and protein components. pap is described as primary or secondary to lung infections, hematologic malignancies, and inhalation of mineral dusts. recently, the importance of granulocyte/macrophage colony-stimulating factor (gm-csf) in the pathogenesis of pap has been documented in experimental models and in humans. gm-csf signaling is required for pulmonary alveolar macrophage catabolism of surfactant. in pap, disruption of gm-csf signaling has been shown, and is usually caused by neutralizing autoantibodies to gm-csf. therefore, the emerging concept is that pap is an autoimmune disorder resulting in macrophage and neutrophil dysfunction. in a recent report, it has been reported that gm-csf autoantibodies are normally present in healthy individuals, but at lower levels than in pap patients [171] . in addition, in vitro experiments indicated that these autoantibodies reduce gm-csp signaling similarly in healthy individuals and in pap patients. at levels above a critical threshold, gm-csf autoantibodies are associated with multiple impaired gm-csf dependent myeloid function [172] . several cases of genetic defects in the common beta chain for the gm-csf receptor have been documented [173] . eosinophilic lung diseases eosinophilic lung diseases constitute a diverse group of disorders of various origins. the diagnosis is suggested by the presence of pulmonary infiltrates on chest imaging and peripheral eosinophilia. it is confirmed by the presence of increased amounts of eosinophils in bal and/or lung tissue eosinophilia. in this section, eosinophilic vasculitis will not be discussed (see chapter 6.2.2). the search for an etiology includes a combination of clinical and laboratory investigations. eosinophilic lung diseases of known cause in children include mainly allergic bronchopulmonary aspergillosis, parasitic infections and drug reactions. eosinophilic lung diseases of unknown cause comprise loeffler syndrome (characterized by migrating pulmonary opacities), acute eosinophilic pneumonia, and chronic eosinophilic pneumonia [174, 175] . the idiopathic hyper-eosinophilic syndrome is a rare disorder observed mainly in adults; it is characterized by prolonged eosinophilia and a multiorgan system dysfunction due to eosinophil infiltration with pulmonary involvement documented in almost half of the patients [176, 177] . alveolar capillary dysplasia and pulmonary capillary hemangiomatosis the pulmonary capillaries form a dense sheet-like meshwork composed of short interconnected capillary segments. the capillary meshes are wrapped over the alveoli, with only a single sheet of capillaries between adjacent alveoli on the same alveolar duct. impaired development of this vascular network can be caused by genetic defects, prematurity or injury. aberrant angiogenesis documented in pediatric patients include mainly alveolar capillary dysplasia, and pulmonary capillary hemangiomatosis [178] . alveolar capillary dysplasia is a rare disorder, presenting with persistent pulmonary hypertension of the newborn [179] . the strongest diagnostic features are poor capillary apposition and density, allied with medial arterial hypertrophy and misalignment of pulmonary vessels [180] . pulmonary capillary hemangiomatosis is also a rare disease that is characterized by proliferation of capillary-sized vessels within the alveolar walls of the lung [181] . intimal thickening and medial hypertrophy of the small muscular pulmonary arteries are present resulting in elevated pulmonary vascular resistance. most cases appear sporadic. chest imaging shows nodular pulmonary infiltrates and septal lines. a definitive diagnosis can be made only by histologic examination. interestingly, capillary proliferation in the alveolar wall has been reported in hereditary haemorrhagic telangiectasia [182] . lymphatic disorders alveolar structure formation is characterized by refinement of the gas exchange unit and functional adaptation of endothelial cells into vessels including pulmonary lymphatics. the pulmonary lymphatic network promotes efficient gas exchange through maintaining interstitial fluid balance. lymphatic disorders can be classified as primary or secondary. congenital errors of lymphatic development can lead to primary pulmonary lymphatic disorders that include lymphangiomas and lymphangiomatosis, lymphangiectasis, and lymphatic dysplasia syndrome [183, 184] . lymphangiomas are focal proliferations of well differentiated lymphatic tissue, and lymphangiomatosis describes the presence of multiple lymphangiomas. most of these disorders are discovered in fetuses or during the early postnatal period. lymphangiectasis is characterized by pathologic dilation of lymphatics. the term "lymphatic dysplasia syndrome" includes congenital chylothorax, and the yellow nail syndrome (a triad of idiopathic pleural effusions, lymphedema, and dystrophic nails) [185] . secondary forms of lymphatic disorders result from a variety of processes such as chronic airway inflammation that impair lymph drainage and increase lymph production [186] . diffuse alveolar hemorrhage syndromes diffuse alveolar hemorrhage (dah) syndromes are caused by the disruption of alveolar-capillary basement membrane as a consequence of injury to the alveolar septal capillaries, and less commonly to the arterioles and veinules. the hallmarks are intra-alveolar accumulation of red blood cells, fibrin, and hemosiderin-laden macrophages. it is important to point out that approximately one third of patients with dah do not manifest hemoptysis, and bal can be extremely helpful if this entity is suspected by showing the presence of siderophages or red blood cells within the alveoli. dah can be observed in association with systemic findings or without evidence of associated diseases. in children, situations of dah in the context of other disorders are reported in several forms of vasculitis discussed above. other disorders that can also be accompanied by dah include pulmonary hypertension and congenital heart diseases, pulmonary veino-occlusive disease, arteriovenous malformations and hereditary haemorrhagic telangiectasia, coagulation disorders, and celiac disease [187] . in the absence of systemic findings, isolated pulmonary capillaritis should be discussed with the search for positivity of the antiglomerular basement membrane antibody with linear deposits in the lung tissue biopsy as well as suggestive serologic features such as p-anca antibodies [188] . idiopathic pulmonary hemosiderosis is a diagnosis of exclusion based on patient presentation with acute, subacute, or recurrent dah, on the results of lung biopsy showing evidence of 'bland' pulmonary hemorrhage (ie, without capillaritis or vasculitis), and after exclusion of the conditions listed above [189] . in this situation, red blood cells leak into the alveolar space without evidence of damage and/or inflammation of the alveolar capillaries. in addition, the diagnosis of idiopathic pulmonary hemosiderosis can only be considered after exclusion of diseases induced by environmental factors such as pesticide and cow's milk (heiner's syndrome) [190] . this syndrome is a hypersensitivity disease that affects primarily infants, and is caused by antibodies to cow's milk proteins. the diagnosis is supported by positive milk precipitin test and rapid improvement of symptoms and pulmonary infiltrates on chest imaging after exclusion of milk proteins. in the resolution phase of tissue injury, elimination of mesenchymal cells and recruited inflammatory cells is essential for restoration of normal cellular homeostasis. dysregulated repair process in ild is associated with accumulation and dysfunction of interstitial fibroblasts [191] . in the coming years, it is likely that progress in the understanding of the mechanisms involved in the impaired myofibroblast apoptosis as well as evasion of these cells from immune surveillance will open new areas of investigations and will provide support for the characterization of disorders that affect primarily the alveolar interstitial components in pediatric ild. indeed, recently, distinct intrinsic differences in gene expression pathways has been reported between control and lung fibrosis myofibroblasts which suggests that ild myofibroblasts are pathological cells with fundamental changes [192] . in the context of ild, pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia, and chronic pneumonitis in infancy have been reported to be exclusively observed in very young children [8] . pulmonary interstitial glycogenosis (pig) is a non lethal disease, reported in neonates with respiratory distress syndrome developed shortly after birth [193, 194] . very few cases are described so far but it seems to have a male preponderance [195] . the histological hallmark of pulmonary interstitial glycogenosis is the accumulation of monoparticulate glycogen in the interstitial cells on lung biopsy. it is thought to represent a maturation defect of interstitial cells that leads them to accumulate glycogen within their cytoplasm [8, 196] . it is discussed that pig could meet "chronic pneumonitis in infancy" as this remains a generalized term [87] . as well, pig could be considered as a premature lung disease, but more than half of published cases were in term infants [195, 197, 198] . the long term consequences in these infants need to be ascertained. neuroendocrine cell hyperplasia of infancy (nchi) is also a non lethal disease characterized by tachypnea without respiratory failure. the human airway epithelium contains highly specialized pulmonary neuroendocrine cells (pnec) system. it's function remains unknown but is hypothysed to act in modulation of fetal lung growth and in post-natal stem cell condition [199] . the pnec system permits synthesis and release of serotonin and neuropeptides such as bombesin [200] . as normal bombesin levels decrease after mid-gestation, its overexpression in nchi could be attributed to a nonregression of neuroendocrine cells [201] . clinical presentation is typically a respiratory distress in post-natal young infant (mean age 3.8 months in a large serie, but cases in older children have been reported [202] . hrct shows patchy centrally ground-glass opacifications and air trapping [203] . on lung biopsy, the histological abnormality is hyperplasia of neuroendocrine cells within bronchioles documented by bombesin immunohistochemistry. the follow-up reveals in some cases the persistence of tachypnea and oxygen requirement for several months. usually, there is a good prognosis [7, 8, 196, 202] . chronic pneumonitis in infancy was first described by katzenstein et al. [4] . the clinical and radiologic features are similar to those observed in other forms of ild. specific histologic abnormalities include diffuse thickening alveolar septa, hyperplasia of type 2 aec, and presence of primitive mesenchymal cells within the alveolar septa. in some cases, foci of pulmonary proteinosis-like material have been observed in air spaces. the prognosis has been reported to be poor with a high mortality rate. other disorders associated with pulmonary development and growth abnormalities encompass a broader spectrum of respiratory manifestations and are more adequately integrated in the classification of diffuse lung diseases [8] . management of children with ild includes administration of oxygen for chronic hypoxaemia, and maintenance of nutrition with an adequate energy intake, immunization with influenza vaccine on an annual basis is recommended along with other routine immunizations against major respiratory pathogens [11] . in addition, aggressive treatment of intercurrent infections and strict avoidance of tobacco smoke and other air pollutants are strongly recommended. a very few children do not require any treatment and recover spontaneously. in the majority of cases, treatment with immunosuppressive, anti-inflammatory, or anti-fibrotic drugs is required for weeks, months or even years [1, 9, 61] . various drugs discussed below can be used, but no guidelines for treatment of ild in children have been proposed so far. the major reason is the very limited number of pediatric patients available for a prospective clinical trial. in addition, controlled studies with a placebo arm are unacceptable because of the poor prognosis of untreated cases and the reported efficacy of anti-inflammatory therapies in a number of pediatric ild. at the present time, the main therapeutic strategy is based on the concept that suppressing inflammation may most likely prevent progression to fibrosis. among the anti-inflammatory agents used in pediatric ild, steroids are the preferred choice, administered orally and/or intravenously. this has been well illustrated by the results of the ers task force on pediatric ild [9] . oral prednisolone is most commonly administered at a dose of 1-2 mg/kg/day [1] . children with significant disease are best treated with pulsed methylprednisolone at least initially [61, 204] . this is usually given at a dose of 10-30 mg/kg/day for 3 days consecutively at monthly intervals. the minimum number of cycles recommended is 3 but treatment may need to be continued for a longer period of 6 months or more depending on response. when the disease is under control, the dosage of methylprednisolone can be reduced or the time between cycles can be spaced out. the disease may then be controlled with oral prednisolone preferably given as an alternate day regime. in few cases oral prednisolone is used from the beginning simultaneously with intravenous methylprednisolone but this is only recommended in those with very severe disease. methylprednisolone may be effective when other forms of steroids administration fail without significant side effects. an alternative to steroids is hydroxychloroquine with a recommended dose of 6-10 mg/kg/day. individual case reports have described a response to hydroxychloroquine even in the presence of steroid resistance [1, 205, 206] . some groups have proposed to base the decision as to which agent to use on the lung biopsy findings, with a preference for steroids in case of large amount of desquamation and inflammation and for hydroxychloroquine if increased amounts of collagen representing pre-fibrotic change are found. however, as documented in the ers task force on pediatric ild, the preferred choice between steroids or hydroxychoroquine in children is highly dependent on the expertise of the center in charge of the patient, and does not seem to be oriented by the histopathological pattern [9] . in case of severe disease, steroids and hydroxychloroquine may be associated. in situations of inefficiency of steroids and hydroxychloroquine, other immunosuppressive or cytotoxic agents such as azathioprine, cyclophosphamide, cyclosporine, or methotrexate may be used. these treatments have been used mainly in situation of autoimmune disorders. promising therapeutic options include macrolides. indeed, these antibiotics have been shown to display a number of anti-inflammatory and immunomodulatory actions. although the mechanisms and cellular targets specific to macrolide activity remain to be elucidated, beneficial effects in several chronic lung diseases including chronic obstructive pulmonary diseases (copd) and cystic fibrosis have been reported [207, 208] . of interest is the ability of macrolides to accumulate in host cells including epithelial cells and phagocytes. in a recent report, a favorable response to treatment with clarithromycine has been described in an adult patient with dip [209] . other new therapeutic strategies currently proposed in adult patients target fibrogenic cytokines. the th1 cytokine interferon-γ has an antifibrotic potential through suppression of th2 fibrogenic functions. antagonists to tgf-β include pirfenidone and decorin. the use of molecules directed against tnf-α such as the soluble tnf-α receptor agent etanercept is also under investigation. to date, there are no reports on the use of these novel therapies in pediatric ild. finally, in the coming years, it is likely that an expanding number of molecules aimed at favoring alveolar surface regeneration and repair through activation and proliferation of tissue-resident (progenitor) cells will come out. depending on the underlying diseases, several specific treatment strategies needs to be considered. these include whole lung lavage for pulmonary alveolar proteinosis, which has been reported to be effective by removing the material from the alveolar space [210] . gm-csf has also been shown of interest in this disease [171] . other strategies such as interferon-α for pulmonary haemangiomatosis are effective [211] . in recent years, lung transplantation has emerged as a viable option in children of all ages, even in young infants, and lung or heart-lung transplantation may be offered as an ultimate therapy for end-stage ild [11] . the outcome and survival do not seem to be different from those reported in conditions others than ild, although comparisons are difficult to establish due to the limited number of reported cases. response to treatment and outcome can be evaluated in children based on several criteria such as decrease in cough and dyspnea, increase in oxygenation at rest and sleep, and changes in pulmonary function tests [1, 11] . improvement on thoracic hrct may also be seen, but tends to occur over a much longer period of time. reports in pediatric ild have not shown a good correlation between histological findings and outcome. some children with relatively severe fibrosis on lung biopsy make good progress, whereas others with mild desquamation have a poor outcome. this is probably due to the variable severity of the disease in different parts of the lung especially in relation to the particular area biopsied, despite hrct guidance. overall a favorable response to corticosteroid therapy can be expected in 40-65% of cases, although significant sequelae such as limited exercise tolerance or the need for long-term oxygen therapy are often observed. reported mortality rates are around 15%. the outcome for infants is more variable [1, 61] . pediatric ild comprises a large spectrum of disorders, with compelling evidence that some of these disorders are observed more frequently in infants, while others are more specific to older children. ongoing basic research will provide new insights into the molecular basis of ild pathogenesis (including genetic factors causing familial disease) in children, and is expected to identify important preclinical markers of disease, pathways of disease regulation, and novel potential targets for therapeutic intervention. for the future, there is a strong need for international collaboration which will allow collecting sufficiently large cohorts of patients with specific entities in order to perform proper therapeutic trials. as a prerequisite, however, a clear and standardised classification of the histopathology of the underlying conditions is critical. such multicenter trials will help to reduce the still considerable morbidity and mortality in children with ild. abbreviations (ards): acute respiratory distress syndrome; (aec): alveolar epithelial cells ankylosing spondylitis; (ab): antibodie; (anti-ccp): anticyclic citrullinated peptide; (anti-gbm): antiglomerular basement membrane anca): anti-neutrophil cytoplasmic antibody; (ana): antinuclear antibodies u1-rnp): anti-u1-ribonucleoprotein; (sao2): arterial oxygen saturation binding immunoglobulin protein; (bal): bronchoalveolar lavage; (casr): calciumsensing receptor; (cgd): chronic granulomatous disease; (copd): chronic obstructive pulmonary disease; (css): churg-strauss syndrome; (ctd): connective tissue disorders; (cmv): cytomegalovirus; (c): cytoplasmicstaining; (dip): desquamative interstitial pneumonia; (dad): diffuse alveolar damage; (dah): diffuse alveolar hemorrhage; (dlco): diffusing capacity of the lung for carbon monoxide functional residual capacity; (sftpb): gene coding for sp-b; (sftpc): gene coding for sp-c; (gm-csf): granulocyte/ macrophage colony-stimulating factor human immunodeficiency virus; (hp): hypersensitivity pneumonitis; (ig): immunoglobulin; (ild): interstitial lung disease lymphocytic interstitial pneumonia; (mmp): metalloproteinases; (mpa): microscopic polyangiitis; (mctd): mixed connective tissue disease; (nchi): neuroendocrine cell hyperplasia of infancy; (nsip): non-specific interstitial pneumonia pulmonary function testing; (pig): pulmonary interstitial glycogenosis pulmonary neuroendocrine cells; (rv): residual volume; (rsv): respiratory syncitial virus; (ra): rheumatoid arthritis; (rnp): ribonucleoprotein; (srp): signal recognition particle systemic lupus erythematosus; (ssc): systemic sclerosis; (ttf-1): thyroid transcription factor 1; (tlc): total lung capacity; (tlco): transfer factor of the lung for carbon monoxide; (tgf): transforming growth factor; (uip): usual interstitial pneumonia idiopathic interstitial pneumonitis in children: a national survey in the united kingdom and ireland factors influencing survival in children with chronic interstitial lung disease diffuse parenchymal lung disease. cape town: karger idiopathic pulmonary fibrosis: clinical relevance of pathologic classification european respiratory society international multidisciplinary consensus. classification of the idiopathic interstitial pneumonias pediatric interstitial lung disease: children are not small adults paediatric interstitial lung disease diffuse lung disease in young children: application of a novel classification scheme task force on chronic interstitial lung disease in immunocompetent children physiology and pathophysiology of bronchial secretion interstitial lung diseases in infants and children interstitial lung disease in children -genetic background and associated phenotypes the pathogenesis of interstitial lung diseases in children lung alveolar epithelium and interstitial lung disease the role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis update in diffuse parenchymal lung disease from lung injury to fibrosis towards systems biology of human pulmonary fibrosis mechanisms of pulmonary fibrosis complex networks orchestrate epithelialmesenchymal transitions apoptosis in pulmonary fibrosis: too much or not enough? transforming growth factorbeta activation in the lung: focus on fibrosis and reactive oxygen species endothelin-1 induces alveolar epithelial-mesenchymal transition through endothelin type a receptormediated production of tgf-beta1 endothelin-1 as initiator of epithelial-mesenchymal transition: potential new role for endothelin-1 during pulmonary fibrosis idiopathic pulmonary fibrosis: multiple causes and multiple mechanisms? evolving concepts of apoptosis in idiopathic pulmonary fibrosis p21 regulates tgf-beta1-induced pulmonary responses via a tnf-alphasignaling pathway membrane type-matrix metalloproteinases in idiopathic pulmonary fibrosis matrix metalloproteases in aberrant fibrotic tissue remodeling role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs epithelial-mesenchymal interactions in the developing lung molecular mechanisms of epithelial morphogenesis the epithelial-mesenchymal transition: new insights in signaling, development, and disease epithelial endoplasmic reticulum stress and apoptosis in sporadic idiopathic pulmonary fibrosis surfactant protein c biosynthesis and its emerging role in conformational lung disease misfolded brichos sp-c mutant proteins induce apoptosis via caspase-4-and cytochrome c-related mechanisms adaptation and increased susceptibility to infection associated with constitutive expression of misfolded sp-c endoplasmic reticulum stress in alveolar epithelial cells is prominent in ipf: association with altered surfactant protein processing and herpesvirus infection aberrant quality control in the endoplasmic reticulum impairs the biosynthesis of pulmonary surfactant in mice expressing mutant bip clinical, radiological and pathological features of abca3 mutations in children aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in abca3 mutants associated with nonfatal pediatric interstitial lung disease expression of abca3, a causative gene for fatal surfactant deficiency, is up-regulated by glucocorticoids in lung alveolar type ii cells nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein c gene new surfactant protein c gene mutations associated with diffuse lung disease posttranslational processing of surfactant protein-c proprotein: targeting motifs in the nh(2)-terminal flanking domain are cleaved in late compartments structure of the human nkx2.1 gene stem/ progenitor cells in lung development, injury repair, and regeneration stem cells and their niches telomerase in alveolar epithelial development and repair mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation short telomeres are a risk factor for idiopathic pulmonary fibrosis progress and potential for regenerative medicine mammalian fetal organ regeneration role of ageing and coronary atherosclerosis in the development of cardiac fibrosis in the rabbit alterations in the tgfbeta signaling pathway in myogenic progenitors with age expression of fibroblast growth factors and their receptors during fullthickness skin wound healing in young and aged mice nuclear reprogramming: a key to stem cell function in regenerative medicine new insights into the pathogenesis and treatment of idiopathic pulmonary fibrosis: a potential role for stem cells in the lung parenchyma and implications for therapy clinical presentation of interstitial lung disease in children pediatric interstitial lung disease revisited hrct of paediatric lung disease hrct in paediatric diffuse interstitial lung disease-a review for diagnostic value of high-resolution ct in the evaluation of chronic infiltrative lung disease in children statement: pulmonary function testing in preschool children pulmonary function testing in young children lung function tests in patients with idiopathic pulmonary fibrosis. are they helpful for predicting outcome? chest lung function, breathing pattern, and gas exchange in interstitial lung disease sequential pulmonary function measurements during treatment of infantile chronic interstitial pneumonitis bronchoalveolar lavage in children. ers task force on bronchoalveolar lavage in children diagnostic value of hemosiderin-containing macrophages in bronchoalveolar lavage cd1a-positive cells in bronchoalveolar lavage samples from children with langerhans cell histiocytosis bronchoalveolar lavage cell analysis in a child with chronic lipid pneumonia value of bronchoalveolar lavage in lipidoses with pulmonary involvement lipid-laden macrophages in bronchoalveolar lavage fluid as a marker for pulmonary aspiration bronchoalveolar lavage fluid findings in children with hypersensitivity pneumonitis pulmonary infiltrates with eosinophilia syndromes in children expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease the diagnostic value of bronchoalveolar lavage in immunocompetent children with chronic diffuse pulmonary infiltrates the safety and efficacy of thoracoscopic lung biopsy for diagnosis and treatment in infants and children diagnosis of interstitial lung disease by a percutaneous lung biopsy sample combined percutaneous lung biopsy and high-resolution computed tomography in the diagnosis and management of lung disease in children a protocol for the handling of tissue obtained by operative lung biopsy: recommendations of the child pathology co-operative group abca3 mutations associated with pediatric interstitial lung disease a mutation in the surfactant protein c gene associated with familial interstitial lung disease abca3 gene mutations in newborns with fatal surfactant deficiency cellular interstitial pneumonitis in infants. a clinicopathologic study the value of classifying interstitial pneumonitis in childhood according to defined histological patterns mmp1 and mmp7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis serum kl-6 differentiates neuroendocrine cell hyperplasia of infancy from the inborn errors of surfactant metabolism serum kl-6 and surfactant proteins a and d in pediatric interstitial lung disease bronchoalveolar lavage fluid cellular characteristics, functional parameters and cytokine and chemokine levels in interstitial lung diseases pediatric environmental health. atlanta: us department of health and human services. agency for toxic substances and disease registry. division of toxicology and environmental medicine update in diffuse parenchymal lung diseases hypersensitivity pneumonitis in children bird fancier's lung: a series of 86 patients hypersensitivity pneumonitis in children: clinical features, diagnosis, and treatment computed tomography of diffuse interstitial lung disease in children interstitial lung disease associated with drug therapy interstitial lung disease induced by drugs and radiation the epidemiology of interstitial lung disease and its association with lung cancer cigarette smoking and diffuse lung disease cellular and connective tissue changes in alveolar septal walls in emphysema occult connective tissue diseases mimicking idiopathic interstitial pneumonias interstitial lung disease in connective tissue diseases non-inherited maternal human leukocyte antigen alleles in susceptibility to familial rheumatoid arthritis anticyclic citrullinated peptide antibody-negative rheumatoid arthritis: clues to disease pathogenesis current developments in pediatric systemic sclerosis current concepts in disease-modifying therapy for systemic sclerosis-associated interstitial lung disease: lessons from clinical trials mechanisms of scleroderma-induced lung disease clinical overview of vasculitic syndromes in the pediatric age classification, presentation, and initial treatment of wegener's granulomatosis in childhood incidence of wegener's granulomatosis in children wegener's granulomatosis and microscopic polyangiitis churg-strauss syndrome churg-strauss syndrome in childhood: a clinical review advances in the pathogenesis of goodpasture's disease: from epitopes to autoantibodies to effector t cells the hla-drb1*1501 allele is prevalent among japanese patients with antiglomerular basement membrane antibody-mediated disease sarcoidosis in the setting of idiopathic chronic bronchiolitis with airway colonization from p. aeruginosa: treatment with low-dose macrolides potential etiologic agents in sarcoidosis pediatric sarcoidosis childhood sarcoidosis in denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children pulmonary sarcoidosis in children: a follow-up study childhood sarcoidosis: long-term follow-up pulmonary sarcoidosis in children: serial evaluation in bronchoalveolar lavage cells during corticosteroid treatment chronic-granulomatous disease chronic granulomatous disease: the european experience pulmonary involvement in type 1 gaucher disease: functional and exercise findings in patients with and without clinical interstitial lung disease lung disease in niemann-pick disease spectrum of fibrosing diffuse parenchymal lung disease a new missense mutation in the casr gene in familial interstitial lung disease with hypocalciuric hypercalcemia and defective granulocyte function langerhans cell histiocytosis: update for the pediatrician pulmonary langerhans cell granulomatosis pulmonary manifestations in a pediatric patient with ulcerative colitis: a case report primary biliary cirrhosis complicated with interstitial lung disease: a prospective study in 178 patients hepatitis c virus enhances incidence of idiopathic pulmonary fibrosis neurofibromatosisassociated lung disease: a case series and literature review viruses as co-factors for the initiation or exacerbation of lung fibrosis chronic lung disease in human immunodeficiency virus (hiv) infected children ferri c: mixed cryoglobulinemia alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules hcv infection: pathogenesis, clinical manifestations and therapy titration of non-replicating adenovirus as a vector for transducing active tgf-beta1 gene expression causing inflammation and fibrogenesis in the lungs of c57bl/6 mice control of virus reactivation arrests pulmonary herpesvirus-induced fibrosis in ifn-gamma receptor-deficient mice herpesvirus dna is consistently detected in lungs of patients with idiopathic pulmonary fibrosis respiratory syncytial virus persistence in chronic obstructive pulmonary disease respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases the histopathology of fatal untreated human respiratory syncytial virus infection occurrence of chlamydia trachomatis and chlamydia pneumoniae in paediatric respiratory infections reduced lung diffusion capacity after mycoplasma pneumoniae pneumonia partial deficiency of surfactant protein b in an infant with chronic lung disease prolonged survival in hereditary surfactant protein b (sp-b) deficiency associated with a novel splicing mutation surfactant composition and function in patients with abca3 mutations abca3 as a lipid transporter in pulmonary surfactant biogenesis abca3 is critical for lamellar body biogenesis in vivo abca3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol genetics of pediatric interstitial lung disease usual interstitial pneumonia in an adolescent with abca3 mutations identification and characterization of a novel abca3 mutation deletion of thyroid transcription factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure choreoathetosis, hypothyroidism, and pulmonary alterations due to human nkx2-1 haploinsufficiency partial deficiency of thyroid transcription factor 1 produces predominantly neurological defects in humans and mice autosomal dominant transmission of congenital hypothyroidism, neonatal respiratory distress, and ataxia caused by a mutation of nkx2-1 brain-thyroid-lung syndrome: a patient with a severe multi-system disorder due to a de novo mutation in the thyroid transcription factor 1 gene new syndromic form of benign hereditary chorea is associated with a deletion of titf-1 and pax-9 contiguous genes five new ttf1/nkx2.1 mutations in brain-lung-thyroid syndrome: rescue by pax8 synergism in one case lethal respiratory failure and mild primary hypothyroidism in a term girl with a de novo heterozygous mutation in the titf1/nkx2.1 gene nkx2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in "brain-lung-thyroid syndrome anti-gm-csf antibodies in paediatric pulmonary alveolar proteinosis granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects analysis of the gm-csf and gm-csf/il-3/il-5 receptor common beta chain in a patient with pulmonary alveolar proteinosis chronic eosinophilic pneumonia in a 13-year-old child acute eosinophilic pneumonia. semin respir crit care med eosinophilic lung diseases: a clinical, radiologic, and pathologic overview hypereosinophilic syndromes alveolar capillary dysplasia with misalignment of pulmonary veins and anterior segment dysgenesis of the eye: a report of a new association and review of the literature alveolar capillary dysplasia: a logical approach to a fatal disease congenital misalignment of pulmonary vessels and alveolar capillary dysplasia: how to manage a neonatal irreversible lung disease? pulmonary capillary hemangiomatosis associated with primary pulmonary hypertension: report of 2 new cases and review of 35 cases from the literature pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis: a clinicopathologic study of 35 cases abnormal lymphangiogenesis in idiopathic pulmonary fibrosis with insights into cellular and molecular mechanisms therapeutic strategies for idiopathic chylothorax pulmonary lymphangiectasia british paediatric orphan lung diseases (bpold) pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation diffuse alveolar hemorrhage syndromes in children diffuse alveolar hemorrhage: diagnosing it and finding the cause pulmonary haemosiderosis in infants and children milk-induced pulmonary disease in infants (heiner syndrome) escape from the matrix: multiple mechanisms for fibroblast activation in pulmonary fibrosis evasion of myofibroblasts from immune surveillance: a mechanism for tissue fibrosis depletion of alveolar glycogen corresponds with immunohistochemical development of cd208 antigen expression in perinatal lamb lung surfactant lipid synthesis and lamellar body formation in glycogen-laden type ii cells pulmonary interstitial glycogenosis: a new variant of neonatal interstitial lung disease diffuse lung disease in infancy a proposed classification applied to 259 diagnostic biopsies pulmonary interstitial glycogenosis in identical twins neonatal pulmonary interstitial glycogen accumulation disorder a confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium pulmonary neuroendocrine cell system in pediatric lung disease-recent advances gastrin-releasing peptide gene expression in developing, hyperplastic, and neoplastic human thyroid c-cells persistent tachypnea of infancy is associated with neuroendocrine cell hyperplasia neuroendocrine cell hyperplasia of infancy (nehi) chronic interstitial lung disease in children: response to high-dose intravenous methylprednisolone pulses chloroquine treatment of interstitial lung disease in children familial interstitial lung disease in children: response to chloroquine treatment in one sibling with desquamative interstitial pneumonitis role of macrolide therapy in chronic obstructive pulmonary disease effects of prolonged use of azithromycin in patients with cystic fibrosis: a meta-analysis beneficial response to macrolide antibiotic in a patient with desquamative interstitial pneumonia refractory to corticosteroid therapy pulmonary alveolar proteinosis in children diffuse neonatal haemangiomatosis interstitial lung diseases in children this work was supported by inserm, université pierre et marie curie-paris6, paris, assistance publique-hopitaux de paris, ministère de la santé (centre de référence des maladies respiratoires rares), and comité de soutien de belleherbe. the authors would like to especially thank malika malhoul, delphine michon, alexandra blondel, aurore coulomb and hubert ducou le pointe for all of their effort towards the creation of the reference center for rare lung diseases. authors' contributions ac and nn contributed equally to this work and should be considered as joint first authors. ac, nn and hc drafted the review. re and bf have been involved in revising critically the review. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-023288-sqr33y72 authors: nan title: paediatric sig: poster session date: 2008-03-12 journal: respirology doi: 10.1111/j.1440-1843.2008.01252_11.x sha: doc_id: 23288 cord_uid: sqr33y72 nan increased airway smooth muscle (asm) in asthma may be due to hyperplasia or hypertrophy of asm cells. the contribution of extracellular matrix (ecm) within asm bundles has not previously been accounted for when estimating asm cell volume. aim to estimate the mean asm cell volume in asm bundles in asthma. methods post-mortem tissues from control subjects (c n = 9); nonfatal (nfa n = 11) and fatal (fa n = 10) cases of asthma were studied. on 30 mm transverse airway sections stained with haematoxylin, the volume density (nv) of asm cell nuclei was estimated using an optical disector (¥1000). the mean cell volume (vc = 1/nv) was calculated, correcting for the volume fraction of asm (fasm) within the asm bundle (corrected vc = 1/(nv ¥ fasm)). fasm was estimated on 0.5 mm thick sections of the same airway stained with masson's trichrome. basement membrane perimeter (pbm) was used to indicate airway size. results table shows mean ϯ sd. (one-way anova) *p < 0.05 for c v fa, nfa v fa. conclusion these data suggest that although asm area is increased in asthma, mean asm cell volume is unchanged. therefore hyperplasia, not hypertrophy, of asm cells is present in both mild and severe asthma. these results were similar for both large and small airways. asthma is characterized by airway inflammation and remodelling which contribute to airway hyperresponsiveness and episodic airflow obstruction. mast cell (mc) densities are higher on the smooth muscle (asm) in asthma so their mediators may modulate other asm functions as well as cause contraction. aim to investigate the effect of mc mediators on chemokine and extracellular matrix (ecm) production by asm cells from donors with and without asthma. methods mc were isolated from the resected lung samples of 6 patients, resuspended at 10 6 cells/ml in dmem + 10% fbs and stimulated with ige/anti-ige. supernatants (sn) were collected after 2 and 24 h and the mc lysed. sub-confluent asm cells from 6 donors with and without asthma were serum deprived for 72 h before mc sn/lysates were added in dmem + 10%fbs for 48 h. il-8 and eotaxin levels in all asm sn and mc sn/lysates were measured by elisa. fibronectin and collagen iv deposition was measured in situ by immunoassay following asm cell lysis. results in asthmatic and non-asthmatic asm cells all mc sn and lysates reduced eotaxin release by up to 47% and 58%, whereas the 0-2 h mc sn significantly increased il-8 release to 178 ϯ 35.9% (p = 0.0339) and 169 ϯ 49% (p = 0.0445) of the fbs control respectively. however, only nonasthmatic asm cell il-8 release was increased by the mc 2-24 h sn (216 ϯ 85%; p = 0.0421) and cell lysates (215 ϯ 47%; p = 0.0421). the 0-2 h mc sn also increased fibronectin deposition to 143 ϯ 16% (p = 0.008) by asthmatic asm cells only. mc sn and lysates had no effect on collagen iv deposition. conclusions activated mast cell mediators differentially modulated chemokine and ecm secretion by asm cells from donors with and without asthma. thus mast cells may modulate their own recruitment to the smooth muscle and remodelling locally in the airways in asthma. supported by nhmrc. the technique of ige passive sensitization reproduces ige-related allergic responses in vitro and studies have validated this technique for investigations modelling allergic smooth muscle responses. there are no studies investigating effects of ige sensitization on rhinovirus (rv) infection. we hypothesized that rv infection is enhanced by ige sensitization, a consequence of diminished early innate immune responses. methods beas-2b epithelial cells and primary culture airway fibroblasts were sensitized with ige 24 h-7 d prior to infection with rv16. samples of tissue culture supernatant and cell lysates were collected over a 12 h period after infection for analysis. viral replication was measured by real-time rt-qpcr and viral titration and type i interferon mrna by rt-qpcr. ige receptor mrna expression was examined using rt-pcr. results initial studies to establish the model used human serum high in ige (>1000 iu/ml), this yielded inconsistent results and it was found that purified ige (1000 iu/ml) provided more reliable responses. sensitization was established after 24 h ige incubation and was comparable with up to 7 d. rt-pcr detected mrna for the ige low affinity receptor only after sensitization. following rv16 infection, vrna was increased after 24 h in ige sensitized cells (p < 0.05), but this effect varied noticeably between and within cell lines. cellular expression of ifn-b mrna increased with viral infection but in cells sensitized with ige lower levels of expression were noted (p < 0.05). conclusions ige passive sensitization enhanced rv replication in vitro but the model is constrained by significant variability between and within cell lines. the effect of sensitization on rv replication may occur through the low affinity ige receptor. activated mast cells (mc) are present in higher numbers on the airway smooth muscle (asm) in asthma compared with other inflammatory airway diseases. matrix metallo-proteinases (mmps) cleave chemokines and alter chemokine gradients by degrading the extracellular matrix and thus may modulate mc migration to the asm. aim to determine the levels of mmp-2, mmp-9 and their inhibitors, timp-1 and timp-2, secreted by asm cells from donors with and without asthma. method confluent asm cells were washed, serum-starved for 48 h and then stimulated with th1 (il-1, tnf and ifn) or th2 (il-1, il-4 and il-13) cytokines or left unstimulated. after 4 and 24 h,the sn were collected. the relative amount of pro and active forms of mmp-2 and mmp-9 in sn were determined by gelatine zymography. timp-1 and timp-2 levels in the sn were measured by elisa. results pro-and active mmp-9 were not detected. however, pro-mmp-2 levels were high in sn of asm cells from donors with (195.6 ϯ 47.2 % positive control/10 5 cells) and without (226.5 ϯ 49.2 % positive control/10 5 cells) asthma. a trend to increased active mmp-2 production by asm cells from donors with (7.3 ϯ 2.7 % positive control/10 5 cells, n = 9) compared to without (2.9 ϯ 0.7 % positive control/10 5 cells, n = 11) asthma after 24 h was not significant (p = 0.101). timp-1 and timp-2 levels respectively were high in the sn of cells from donors with (69.4 ϯ 19.6 and 21.3 ϯ 4.7 ng/10 5 cells, n = 5) and without (57.3 ϯ 13.7 and 16.6 ϯ 3.5 ng/10 5 cells, n = 5) asthma. th1 and th2 cytokine stimulation did not affect mmp or timp release. conclusions th1 and th2 cytokines did not regulate asm cell production of mmp-2, timp-1 and timp-2. altered asm mmp-2 activity is unlikely to play a role in mc chemotaxis to asm cells from donors with asthma in vitro or their presence on the asm in asthma. there has been a marked increase in the prevalence of asthma and other allergic diseases in the last few decades. one of the explanations for this is the change in our diet. one of the characteristics of the "western diet" is a high intake of both saturated and polyunsaturated fat. this prompted us to compare the effects of high fat and low fat meals on the numbers of circulating eosinophils and other leukocytes. methods we studied 12 volunteers who had allergic rhinitis and/or asthma and a peripheral eosinophil count at baseline of ն200 ¥ 10 7 /l. this was a randomized, crossover trial with participants studied on two different days. on each occasion they arrived fasting and after bloods were drawn consumed a 3000 calorie meal. one of the meals was high in saturated fat and refined carbohydrate. the other meal was low in saturated fat and high in fruit and fibre. bloods were drawn postprandially every hour for five hours. results eosinophil counts were highest in the early morning and fell over the course of the day but the decrease was less with the high fat meal (p = 0.03). over the same period of time the increase in lymphocytes (p = 0.016) was greater with the high fat meal. the high fat meal was also associated with greater increases in triglycerides (p < 0.0001) and cholesterol (0.004). conclusions in atopic individuals a high fat meal was associated with higher circulating numbers of eosinophils and lymphocytes than an isocaloric meal that was low in fat. further studies of the effect of dietary fat on eosinophilic inflammation are warranted. supported by the university of auckland research committeee. intravenous gamma globulin therapy (ivig), which is therapeutic in a variety of immune diseases, has been reported to be effective on patients with severe steroid-dependent asthma. although fcer are known to play important roles in asthma, there are few reports about the role of fcg?receptors in asthma. fcg receptor iib (fcgriib) is unique inhibitory receptor, which suppresses immune response. in this study, we evaluated the effect of ivig in allergic airway inflammation in ova-challenged mice and the mechanism of the inhibitory effects of ivig and fcgriib. method c57bl/6 mice (wt) and fcgriib deficient mice (ko) were sensitized with ovalbumin (ova) and alum and subsequently challenged with nebulized ova. before ova challenge rabbit igg was administered intravenously. the airway inflammation and effects of igg were assessed by histology, cell counts of bal fluid and airway hyperresponsiveness. result histology showed that igg treatment ameliorated the inflammation around the airway and the vessels and hypertrophy of goblet cells induced by ova challenge. the migratory activity of dcs is modulated in inflammatory diseases such as asthma. recently, we reported that immature dcs express kinin receptors and that bradykinin (bk) significantly enhances the migration of immature dc in vitro. as kinins mediate many of the pathophysiological effects associated with asthma, we hypothesized that lys-des[arg 9 ]-bk, which is produced during inflammation and acts via the b1 receptor (b1r), would inhibit migration of mature dcs. methods day 7 cultured human monocyte-derived dcs were matured with lps, tnfa +il-1b or cd40l in the absence or presence of lys-des[arg 9 ]-bk. maturation of dc was analysed by flow cytometry (facs). b1r expression was assessed by reverse-transcriptase pcr and quantitative confocal microscopy. migration of mature dc was assessed in transwell chambers with lysdes [arg 9 ]-bk and the chemokine ccl19 used as chemoattractants. results maturation of dcs was found to result in down-regulation of b1r expression to varying degrees depending upon the maturation stimulus used. mature dcs all demonstrated an ability to migrate toward lys-des[arg 9 ]-bk and ccl19. however pre-treatment with lys-des[arg 9 ]-bk decreased the migratory ability of all mature dcs to both chemoattractants. conclusions along with chemokines, lys-des[arg 9 ]-bk is likely to play a crucial role in regulating the in vivo migration of mature dc during inflammation. the production of lys-des [arg 9 ]-bk during inflammation potentially immobilizes mature dcs thereby facilitating locally-mediated immune responses within inflamed tissues. supported by the asthma foundation of western australia. introduction alternative or aberrant splicing is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms. the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to use a new and innovative approach involving the use of the genechip (r) exon array system together with real-time quantitative pcr to study asthma candidate genes in human monocyte-derived dendritic cells. asthmatic and non-asthmatic subjects provided 20 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method, and differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data indicate splice variant expression in dendritic cells from asthmatic patients is influenced by asthma severity. conclusion exon expression array analysis has generated a number of asthma candidate genes with alternative splice variants. further studies to validate these data in a replicate data set and establish the functional significance of our findings in asthma are underway. alternative or aberrant splicing occurs in more than 70% of genes and is a major contributor to protein diversity, in which a single gene can generate structurally and functionally distinct protein isoforms 1 . the role of alternative splicing in asthma pathogenesis has not been previously investigated. we hypothesized that specific alternatively spliced asthma candidate genes contribute to the development of asthma. we chose to study one asthma candidate gene in human stimulated and unstimulated: (1) monocytes, (2) monocytederived dendritic cells and (3) lung smooth muscle cells. methods asthmatic and non-asthmatic subjects provided 40 ml of blood from which peripheral blood mononuclear cells (pbmc) were isolated by ficoll-paque gradient centrifugation. monocytes were separated from other leukocytes by adherence method. up to 50% of the monocytes were then differentiated into dendritic cells following incubation with defined concentrations of gm-csf and il-4. induction experiments used 1 mg/ml lps and cells were stimulated for an optimal period of 24 hrs. rna was isolated and reverse transcribed for real-time semi-quantitative pcr and densitometry. chi squared test was used to assess associations between alternative splicing and asthma. results data from stimulation experiments indicate splice variant production can be regulated by the inflammatory response and that this response is influenced by asthma status. conclusion preliminary experiments have confirmed the presence of an aberrant splice variant for an asthma candidate gene in the primary cells studied. further studies to confirm these data and establish the functional significance of our findings in asthma are underway. exposure to environmental factors, such as environmental tobacco smoke (ets), plays a significant role in modulating pre-existing genetic susceptibilities to diseases including asthma. the glutathione s-transferase enzymes (gsts) play an important role in the detoxification of ets. there are several gst isoforms and gstp1 codes for the gst pi isoform, which is the primary gst isoform expressed in human lung tissue. two single nucleotide polymorphisms (snps) at positions 105 and 114 have been reported in gstp1 and associated with asthma and atopy. the aim of this study was to examine the effect of these snps in combination with ets, on asthma phenotypes in a cohort of asthmatic children. children were recruited during an acute episode requiring presentation at an emergency department. genotyping using pcr-rflp was completed on 218 children and ets exposure was determined by parental questionnaire. urinary cotinine was measured in the children and was in agreement with questionnaire responses. statistical analyses were performed using spss. there were no significant associations between the genotypes and asthma severity during acute exacerbations. significant associations were found between the snps and atopy in this population with an odds ratio of 2.77 for the 105aa genotype (p = 0.029) and or of 5.47 for the 114cc genotype (p = 0.002). however, when an interaction with ets was included, the odds ratios increased to 9.02 for 105aa (p = 0.05) and 9.17 for 114cc (p = 0.020). these results suggest that there is a significant gene/environment interaction impacting on atopy in this cohort. the rage gene encodes the receptor for advanced glycation end-products (rage), a member of the immunoglobulin superfamily. rage activation by ligands, including amphoterin and s100/calgranulins, leads to prolonged nf-kb signalling and has been associated with chronic inflammation. despite high levels of rage expression in lung tissue, little research has been undertaken into the role of rage in the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. objective determine genetic associations between functional polymorphisms in the rage promoter and severe and aspirin-sensitive asthma phenotypes. methods pcr and restriction fragment length polymorphism (rflp) were used to genotype three rage promoter polymorphisms, -429t>c, -374t>a and a 63 bp deletion from -407 to -345, in a large case-control asthma population phenotyped for asthma severity, atopy and aspirin sensitivity. results no associations were identified between any of the polymorphisms and the occurrence of asthma. however, the -374a allele was linked with both severe asthma (p = 0.013) and aspirin-sensitive asthma (p < 0.001). likewise, genotypes containing the -374a allele were strongly associated with both severe asthma (or 2.10, 95% ci 1.32-3.36) and aspirin-sensitive asthma (or 3.13, 95% ci 1.45-6.77). conclusions the -374a allele of the rage gene, previously shown to lead to a 3-fold increase in promoter activity, is associated with the chronic inflammatory asthma phenotypes of severe and aspirin-sensitive asthma. these results suggest that increased rage expression, with a concomitant increase in nf-kb signalling, may in part contribute to the inflammatory response seen in these conditions. the global prevalence of allergic diseases is rising and australia has one of the highest prevalence rates in the world. the role of early childhood infections in the development of allergic disease remains controversial. objective to examine the association between early childhood infections and the development of allergic diseases in later childhood, in high risk children. methods data were analysed from the melbourne atopic cohort study (macs) of 620 infants with 1 or more first-degree family members with atopic disease. primary risk factors assessed were otitis media, bronchitis and gastroenteritis reported in the first two years of life. outcomes were current asthma, hay fever and eczema at 6 years of age. logistic regression was used to estimate crude and adjusted odds ratios. results asthma was the most common allergic condition (25.4%, 95% ci 21.6-29.5%), followed by eczema (24.9%, 95% ci 21.1-29.0%) and hayfever (15.6%, 95% ci 12.5-19.1%). the most commonly reported infection was otitis media (58.9%, 95% ci 54.9-62.8%), then gastroenteritis (37.7%, 95% ci 33.9-41.7%) and then bronchitis (19.4%, 95% ci 16. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] .7%). all 3 types of infection within the first 2 years of life were associated with increased risk of asthma. an increased risk of asthma at 6 years was seen with otitis media (or = 1.14, 95% ci 1.02-1.3), bronchitis (or = 1.34, 95% ci 1.0-1.8) and gastroenteritis (or = 1.23, 95% ci 0.96-1.6). when the frequency of infection was examined, those who reported at least 3 episodes of gastroenteritis had a 3-4-fold increased risk and an almost 30% absolute increased risk (rd 0.34, 95% ci 0.08-0.59). conclusion these findings appear to contradict the hygiene hypothesis. the findings for gastroenteritis are novel. further examination of these associations and possible underlying mechanisms is warranted. grant support asthma foundation of victoria, nestle. background knowledge about incident cases of asthma in australia is limited because they are not routinely reported. the ability to predict the number of new cases of asthma would be helpful in allocating resources for asthma education, management and care. data on first use of medications for asthma gives an indication of the incidence of asthma. the objective of this study was to estimate the incidence rate of asthma by investigating asthma medication use in individuals. methods pharmaceutical benefits scheme (pbs) records for all prescriptions filled for inhaled corticosteroids (alone or combined formulation), cromones and leukotriene receptor antagonists from july 2002 to june 2005 were included. using a 2-year look back window, any persons who had their first prescription for any of these drugs dispensed between july 2004 and june 2005 were assumed to be incident cases. overall and age-specific incidence rates were calculated per 100 asthma-medication-free individuals. results there were 352,082 individuals who had their first asthma medication dispensed between july 2004 and june 2005, which equates to an overall incidence rate for asthma of 1.89 per 100. the incidence was higher among children aged 0-14 years (2.07) and adults aged 65 years and over (2.45) . conclusions our estimated incidence rates were consistent with those reported by others in the literature. while the pbs database was designed for administrative purposes, it can be used to estimate incidence rates for asthma. support acam is a collaborating unit of the australian institute of health and welfare and is funded by the department of health and ageing (doha). we acknowledge the pharmaceutical pricing and estimates section of doha for provision of pbs data. keywords asthma incidence, pharmaceutical benefits scheme. rosario ampon 1 , guy marks 1 , teresa to 2 , leanne poulos 1 , anne-marie waters 1 1 australian centre for asthma monitoring (acam), sydney, australia, and 2 hospital for sick children, toronto, canada background the ability to assess individual patterns of asthma medication use would have clinical relevance in targeting effective asthma education and management for this common condition. to describe longitudinal patterns of asthma medication use, we used a population-based prescription database to follow individuals from the first time they filled an asthma prescription. asthma is more commonly listed on death certificates as an associated cause of death, in people whose deaths are attributed to other causes, than as an underlying cause of death. understanding the importance of these associations would contribute towards an overall appreciation of the impact of asthma on mortality. the objective of this analysis was to estimate the prevalence of asthma as an associated cause of death when various other diseases were attributed as the underlying cause of death. background acam currently recommend 24 indicators to measure population-level asthma health and outcomes. we examined correlations among several asthma indicators covering prevalence, morbidity and mortality to try and produce a condensed set of indicators which minimized redundancy. methods seven of the 24 indicators were included in this study: prevalence of ever having doctor diagnosed asthma, prevalence of current asthma, asthma-related general practice (gp) encounters, proportion of people with asthma with an asthma action plan (aap), hospitalizations for asthma, hospital patient days for asthma, and deaths due to asthma. a correlation matrix was created for these indicators by age groups. pearson correlation coefficients ն0.7 or յ-0.7 were considered strong. results there were strong positive correlations between prevalence of ever asthma and current asthma (r = 1.0); gp visits and aap possession (r = 0.74), hospitalization (r = 0.91) and patient days (r = 0.95); and hospitalization and patient days (r = 0.90) and aap possession (r = 0.73). recent australian reports have shown that the prevalence of asthma and respiratory symptoms has decreased over the last 10-15 years. as part of a larger study investigating child health and air quality we have collected nationwide data from schoolchildren living in act, victoria, queensland, wa and sa. methods schools were selected based on proximity to air quality monitoring stations. classes from years 3 to 6 were randomly selected and all children were invited to participate. parents self completed a questionnaire that included questions about diagnosed asthma and respiratory symptoms. results a total of 1989 children provided questionnaires for analysis. the response rate varied between states and territories and ranged from 30% to 42%. the sample comprised 51.9% girls and the mean age of children was 10.2 years. ever diagnosed asthma 27.9 current asthma ('does he/she still have asthma? ') 13.8 wheeze in the past 12 months 16.1 respiratory symptoms limiting activities 11.8 missed school due to asthma or wheezing 8.8 conclusions despite the relatively low participation rate, the prevalence estimates for current asthma are similar to those reported in the national health survey 2004-05 [1] . there is no evidence of any recent increase in the prevalence of childhood asthma. methods tahs is a longitudinal population-based respiratory study of 8583 subjects which commenced in 1968 when they were 7 years of age. since the initial study another 4 follow-ups have been conducted, including the most recent follow-up when subjects were 44 years of age. lung function of the total sample was measured at baseline and in sub-samples in 3 subsequent followups. asthma was categorized as persistent, frequent or episodic when participants reported asthma symptoms in at least 3 follow-ups, in 2 follow-ups or in 1 follow-up respectively. results by age 7 years ever asthma prevalence was 16%. at age 44, 10% of those who had not reported asthma by age 7 had asthma symptoms while 75% of those who reported asthma by age 7 had no asthma symptoms. hence over all only 25% of the asthma symptoms at age 44 were attributable to asthma developed by age 7. in contrast, 91% of the persistent and frequent asthmatics had developed their asthma by age 7. persistent and frequent asthmatics had more symptoms and poorer lung function at age 7, 14 and 44 as well as more reversibility at age 44 (p < 0.05). childhood asthmatics who also had a productive cough by age 7 were more likely to have persistent asthma than those without a cough (p < 0.05). conclusions although the majority of middle-age asthma is related to postchildhood onset asthma, most severe middle-age asthma has its origin in persistent childhood disease. having productive cough in childhood may identify high risk asthmatics who require especially rigorous management in early life. one third of women experience an improvement in asthma during pregnancy, and symptoms improve in most women in the late third trimester. we hypothesized that the exacerbation rate would be reduced and that symptoms during exacerbations would be less severe in the third trimester compared to the second trimester. methods pregnant women with asthma (n = 81) were prospectively followed from recruitment (14.8 weeks (3 sd) ) to delivery at clinic visits (18, 30, 36 weeks and during exacerbation), and fortnightly phone calls. the asthma control questionnaire (acq) was administered at each contact and exacerbations classified as severe (requiring medical intervention) or mild (selfmanaged). lung function, medication use, fractional exhaled nitric oxide (feno) and full blood counts were assessed. paracetamol is commonly used in infants as an analgesic and antipyretic. it has been hypothesized that frequent paracetamol consumption may result in reduced lung capacity to cope with oxidative stress and increase risk of respiratory disease. to date, no study has examined exposure to paracetamol during infancy, when lungs are still developing, and risk of childhood asthma. method a birth cohort of 620 infants with an atopic family history was recruited. frequency of paracetamol exposure was prospectively documented up to 2 years of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results paracetamol exposure in infancy was common (97% exposed by two years of age), with some infants receiving paracetamol on up to 77 days. it has been hypothesized that mucosal immune response requires a particular micro-flora milieu in the infant's gastro-intestinal tract, and that early life antibiotic exposure may disrupt this process and increase risk of allergic disease. method a birth cohort of 620 infants with an atopic family history was recruited. exposure to oral antibiotics was prospectively documented up to 12 months of age. interviews were conducted at 6 and 7 years to ascertain asthma in the previous 12 months. results by one year of age, approximately 80% of infants had received at least one course of oral antibiotics. the prevalence of current asthma in childhood was approximately 30% (148/495). frequent use of antibiotics (more than 20 days exposure during first year of life) was associated with increased risk of childhood asthma (or = 2.52, 95% ci = 1.40-4.54) when compared to infant who had not been exposed. excluding infants with a diagnosis of asthma within the first two years of life, reduced this association by about 30% (or = 1.80, 95% ci = 0.90-3.57) and adjustment for gender, parental history of asthma and number of infections in the first year of life further reduced this association (or = 1.60, 95% ci = 0.79-3.22). the increased risk of childhood asthma associated with antibiotic exposure in the first year of life is, at least in part, due to confounding with early life wheeze and infections. if real, the independent effect of antibiotic exposure on risk of childhood asthma is likely to be minimal in this high risk cohort. support dairy australia, crc for asthma and airways, vichealth, nestle. the epidemiological data on asthma suggest a gender difference that varies with age. hormonal effects have been suggested as a possible explanation for these differences. the aim of this study was to examine reproductive factors and risk of asthma among the females of the tasmanian longitudinal health study (tahs). methods the tahs is a longitudinal population-based cohort study of respiratory disease which commenced in 1968 when subjects were 7 years of age. four follow-up studies have been conducted including the current most comprehensive follow-up with subjects at 44 years of age. information has now been collected on reproductive factors such as number of pregnancies, age at pregnancies, age at menarche and contraceptive pill use as well as on asthma status. reproductive factors were examined as risk factors for asthma using multiple logistic regression to adjust for all likely confounders. results a total of 2,776 women completed the most recent postal survey. of these 355 (12.8%) had current asthma, and of these women with current asthma 73.5% (261) developed asthma after childhood. on average these women were in their mid-twenties when they developed asthma (mean ϯ sd age = 26.6 ϯ 12.5 yrs). we found with increasing age at first birth an approxi-mate~30% reduced risk of current asthma in women who developed their asthma post-childhood (trend p = 0.04). we did not observe any other associations between reproductive factors and risk of asthma. conclusions our results are consistent with the hypothesis that early pregnancy may promote asthma development by altering the immune response favouring a th2 pathway. a delay in the age of first pregnancy reduces this risk of asthma. grant support nhmrc, clifford craig foundation, victorian & tasmanian asthma foundations. introduction the association between exposure to pets in early life and subsequent development of sensitization and allergic disease remains controversial. the objective of this analysis was to examine the relationship between cat exposure before birth and development of cat sensitization over time within the melbourne atopic cohort study (macs). methods the macs is a prospective longitudinal cohort study that initially recruited 620 women antenatal in melbourne from february 1990 to november 1994. detailed information on cat exposure was collected at recruitment and frequently until two years of age. skin prick test (spt) were conducted at 6, 12, 24 months and 10 years. the data were analysed by logistic regression and using generalized estimating equations (gee) for the repeated measures design. results among 620 subjects, 169 (28.8%) had a cat before birth. at 6 months, 1.9% (n = 11) of subjects were sensitized to cat and by 10 years of age 18.8% (n = 68) were sensitized. those who did not have cat before birth belong to a higher social class, and were more likely to have a father with allergic disease than those with a cat. those who developed sensitization to cat were more likely to have a paternal family history of allergic disease and more likely to be sensitized to other allergens. we did not observe any association between exposure to cat before birth and the development of sensitization to cat at 6 months (or = 0.7, 95% ci 0. 1-3.3) , 12 months (or = 1.4, 0.5-3.9), 24 months (or = 0.76, 0.2-2.5) or 10 years (or = 0.6, 0. 2-1.4) . these crosssectional results were confirmed by the gee analysis. conclusion our results fail to show an association between cat exposure before birth and development of sensitization to cat. furthermore exposure after birth in the first 18months of life was not associated with an increased or decrease risk of sensitization to cat. our results do not support either a benefit or risk associated with cat ownership and sensitization. introduction peri-natal events influence the development of asthma and atopic diseases in childhood but the current literature is contradictory on the effect of low birth weight, small for gestational age and prematurity on asthma risk. the aim of this study was to assess the relationship between these three exposures and asthma from childhood to adulthood. aim to assess the current prevalence of dda, wheeze (<12 months), atopy and ahr in children and adults in busselton. methods an age-and sex-stratified random sample of adults, selected from the electoral roll, was invited to complete a questionnaire and attend the local study centre for assessment of atopy (allergen skin tests) and ahr (methacholine). all children from participating primary and secondary schools were also invited to attend. the prevalences of dda, wheeze, atopy, ahr and "current asthma" (wheeze + ahr) were calculated. background asthma is often associated with comorbidity, however few studies have investigated comorbidities among people with this common condition. the objective of this analysis was to describe patterns of non-respiratory comorbidity among adults hospitalized with asthma in australia. methods data on hospitalizations for people aged 15 years and over with a principal diagnosis of asthma (j45, j46) were obtained from the australian institute of health and welfare's (aihw) national hospital morbidity database for the period 2005-06. patterns of comorbidity were examined by investigating additional diagnoses for non-respiratory disease according to icd-10 diseasespecific chapters. results among people aged 15 years and over hospitalized in 2005-06 with a principal diagnosis of asthma (16,566 hospitalizations; 70% female; 47% aged 35-64 years), 33% had at least one non-respiratory comorbidity. median length of stay was higher among those with at least one comorbidity (4 days) than among those with no comorbidities (2 days). among people aged 15-64 years, the most common comorbid condition was endocrine, nutritional and metabolic diseases (19%), while among those aged 65 years and over it was diseases of the circulatory system (32%). conclusions a large proportion of asthma hospitalizations in australia are associated with non-respiratory comorbidity and a longer length of stay. further, the pattern of non-respiratory comorbidity associated with asthma hospitalizations varies by age. given our rapidly ageing population, the level of comorbidity associated with asthma has implications for coordinated health care and demand on health services. support acam is a collaborating unit of the aihw and is funded by the department of health and ageing. keywords comorbidity, hospitalization, asthma. background asthma exacerbations are often triggered by viral respiratory infections, yet the influence of respiratory infections on the morbidity of acute asthma beyond the immediate period is unknown. we examined the influence of nasopharyngeal (npa) respiratory viral, chlamydia and mycoplasma detection on asthma morbidity in children presenting to the emergency department for an acute exacerbation of asthma. methods a subset (n = 78) of the 201 children enrolled for a randomized controlled trial (rct) on the efficacy of 5 vs 3 days of oral prednisolone had an npa taken at presentation. npa were examined for chlamydia, mycoplasma and respiratory viruses (enteroviruses, coronaviruses, human metapneumovirus, adenovirus, parainfluenza, influenza, rsv, rhinoviruses) by pcr. enrolled children were aged 2-16 years with recurrent wheeze and required ն600 ?g (mdi/spacer) or ն2.5 mg (nebulized) of salbutamol to reduce tachypnoea. parents filled validated diary cards for cough and asthma severity, and completed asthma qol data at enrolment and end of weeks 1 and 2. results pcr for various viruses was positive in 42 (53.8%) children, with no significant difference in the groups the children were randomized into. rhinovirus pcr was positive in the npa of 32 children, rsv in 7, hmpv in 2, adenovirus, parainfluenza, influenza a and b in one each. specimens were negative for the other micro-organisms listed above. children with a npa viral positive state were significantly (p = 0.002) younger than those with a negative state. however, there was no difference in the any of the asthma outcomes of children whose npa was positive or negative for the micro-organisms tested. conclusions in children with an acute asthma exacerbation presenting to emergency health facilities, a respiratory virus could be identified in >50% but the presence of a respiratory virus did not influence the morbidity of the asthma exacerbation at presentation or at the end of week-1 and week-2. the university of sydney, nsw 2006, and 3 royal north shore hospital, st leonards, nsw 2065 airway wall thickness measured using hrct is reported to be increased in asthmatic compared with control subjects. however, it is unknown whether wall thickness is a fixed structural characteristic of the airways or if it responds to transient changes in bronchomotor tone or airway size. aim to determine the effects of bronchomotor tone and lung volume on airway wall area measured by hrct. methods 8 patients with doctor-diagnosed asthma had partial chest hrct scans, before and after bronchodilator (bd), at frc, tlc and a volume midway between (mid-volume). airway segments were identified between branch points and matched between consecutive lung volumes both before and after bd, and also at constant lung volume before and after bd. mean lumen areas and wall areas for each airway segment at each volume were measured using automated analysis software. paired t-tests were used to determine changes due to bd and lung inflation. results 44 airways were matched before and after bd at frc. absolute airway wall area (wa) was related to airway lumen diameter (di wood smoke air pollution is of concern with respect to respiratory health due to its complex chemical composition and potential to carry air toxics into the lower respiratory system. launceston has a long history of poor winter air quality, primarily due to use of domestic wood heaters. participants in hobart had a similar prevalence of wood heater use, but hobart does not experience the same wood smoke pollution (due to differences in regional geography , asthma control and anxiety and depression were completed at baseline, immediately following (6 wks), and 3 mths after the intervention period. results clinically and statistically (p < 0.05) significant improvements in qol were observed in the exercise group at 6 wks compared to the control group. this difference was not maintained at 3 mths. 6mwd improved at 6 wks and 3 mths in the exercise group (p < 0.01), however the difference between groups was not significant. in the exercise group there was a trend towards improved asthma control and a reduction in anxiety and depression that was not observed in the control group. *p < 0.05, change at 6 wks vs baseline; home asthma monitoring is important for measuring day-to-day variation in lung function and symptoms. this approach requires the availability of complete diaries for a comprehensive assessment. we assessed the completeness of written diaries collected as part of a nation wide study of air quality and child health. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified from a cross-sectional study. these children were asked to record symptom scores and peak expiratory flows twice daily in diaries for a five week period. the diaries and peak flow devices were explained at a face-to-face meeting with parents and children. each week diaries were mailed back and parents received a phone call to encourage completion. completeness was defined as no missing responses to symptom questions or peak flow measurements in diaries from week two to week five. results data from the first 36 children (822 day records) were available for analysis. the sample included (53%) girls, mean age 10 yrs. the overall frequencies for complete records were; morning symptoms 85%, morning peak flow 85%, evening symptoms 83% and evening peak flow 82%. there was a significant trend for more complete morning peak flow records over the four weeks (cochrane-armitage trend test p < 0.007). agreement between morning and evening symptom completeness and between morning and evening peak flow completeness was fairly poor (kappa < 0.30). conclusions the completeness of symptom and peak flow records collected in this study was very high. the comprehensive follow-up protocol implemented is likely to have had an important impact on the completeness of asthma diaries. daily peak expiratory flow (pef) monitoring has been used in epidemiological studies to assess changes in lung function over time. the value of written pef diaries has been questioned because of problems with completeness and validity. this study aimed to compare stored electronic pef data and a written diary record of those data in a panel study in children with weekly reminders to aid adherence. methods children who had ever been diagnosed with asthma and had respiratory symptoms in the last year were identified in a population study. they were given electronic pef devices with a digital readout (miniwright digital, mwd, clement clarke, uk) and written symptom and peak flow diaries and instructed in their use at a meeting with parents and children. each child was asked to complete three pef manoeuvres every morning and evening for five weeks and to record these in the written diary. background previous research suggests that comorbid anxiety is associated with lower asthma-related quality of life (aqol) in adults with asthma. however, research is scant on the role of psychological interventions in these patients. aim to evaluate the effectiveness of a four-session cognitive-behavioural therapy (cbt) intervention, in improving the aqol, in participants with anxiety and asthma. method participants identified with comorbid anxiety and asthma were randomly assigned to the cbt intervention group (n = 10) and the asthma monitoring control group (n = 8) and evaluated on aqol measures, at various intervals. results nine participants, in the cbt group, completed the study. seven participants showed a clinically significant improvement in asthma-related emotional functioning (ef) and six participants in total aqol scores, at the 5-week post-intervention assessment. additionally, six participants in the cbt group indicated clinically significant improvement in ef and five participants in total aqol scores, at the 3-month follow-up assessment. only three participants in the control group completed the study. none of these participants showed any improvement in aqol scores at the 5-week or 3-month assessment. conclusion this pilot study suggests that a higher number of participants in the cbt group showed clinically significant improvement in ef and total aqol scores with higher retention rates. further research needs to confirm these findings in a larger group, identifying the elements of a successful cbt intervention and characteristics of participants who respond to the cbt intervention. gastro-oesophageal reflux disease (gord) is a risk factor for uncontrolled asthma. we conducted an update of a systematic review to assess whether treatment of gastro-oesophageal reflux in subjects with asthma improved asthma outcomes. methods randomized controlled trials (rcts) of gord treatment in adults or children that reported asthma health outcomes and had symptomatic gord were included and assessed in accordance with the standard cochrane systematic review process. subjects received pharmacological therapies compared with conservative management. results from 261 potentially relevant studies, 19 rcts were included in the review. when compared to placebo, morning peak expiratory flow did not significantly improve (change from baseline wmd 10.43, 95% ci: -9.55 to 30.42) with proton pump inhibitor treatment (n = 7 trials involving 739 participants). asthma exacerbations were not significantly less in the intervention groups compared with the control groups (odds ratio 0.66; 0.41-1.08; n = 557). conclusions while some trials reported evidence of asthma improvement with gord therapy, overall there appears to be no statistically significant evidence of a beneficial effect. it is clear that not all persons with gord and asthma will gain improved control over their asthma with gord therapy; this may be due to the heterogeneous pathophysiology of asthma. future large-scale trials would be required to demonstrate an effect on asthma exacerbations. kel and brd were supported by a cochrane airways group scholarship. background the ats/ers task force recommend the use of metered dose inhaler (mdi) and spacer for airflow limitation reversibility testing. salbutamol given via mdi & spacer has been shown to be equivalent to a nebulizer in the clinical setting. this has not been well studied in respiratory laboratory setting. aim to compare the 2 methods of reversibility testing in a laboratory setting. methods we conducted a laboratory based crossover study in a secondary hospital. patients with asthma or copd were eligible. the patients firstly underwent spirometry and reversibility testing following a standard dose of nebulized salbutamol. they were asked to return for a second set of spirometry within the same week and at the same time of day when reversibility with an mdi and spacer was recorded. we used an incremental dose of salbutamol starting from 2 puffs and up to 8 puffs. spirometry parameters were recorded 10 minutes after each intervention. the primary outcome was the percentage change in fev1 after each intervention. side effects were monitored for. results nine patients with asthma were recruited. the mean percentage change in fev1 was higher in the nebulizer group than after only 2 puffs via mdi & spacer (15.4 ϯ 7.4 vs 6.2 ϯ 8 [mean ϯ sd], p = 0.67). however, there were no differences between the 2 arms following higher doses of bronchodilator via mdi & spacer. the mean percentage change in fev1 after 4, 6 and 8 puffs were 12.6 ϯ 11.3, 15.4 ϯ 12.3, and 17.7 ϯ 13.6 respectively (p = 0.09, 0.05 and 0.07 respectively when compared to the nebulizer group). conclusion using an mdi and spacer for bronchodilator reversibility is equivalent to that of a nebulizer and should be the standard method of testing. the dose of bronchodilator needs to be at least 4 puffs as recommended by the ats/ers; however 6 puffs correlated best with a standard nebulizer route. further increments in bronchodilator dose provided little additional bronchodilatation. the study was limited by the small number of patients. asthma guidelines recommend a stepwise approach to treatment. the role of inhaled corticosteroid (ics) and long-acting beta-agonist (laba) combination therapy in asthma written action plans is not clear. objective to assess the efficacy of adjusting ics/laba combination therapy in a written action plan compared to fixed dosing in people with asthma requiring maintenance ics. methods cochrane systematic review of randomized controlled trials comparing ics/laba combination therapy in a single inhaler that is adjusted up or down according to a written action plan (wap) to comparison 1: budesonide/ formoterol given as a fixed maintenance dose (fd) (n = 9) or comparison 2: fluticasone/salmeterol fd (n = 2). results 10 parallel randomized controlled trials describing 11 interventions met the inclusion criteria. for the trials that compared wap to fd budesonide/ formoterol there were significant reductions for the wap group in exacerbations, (rr (95%ci): 0.82 (0.70 to 0.97)), severe exacerbations (rr (95%ci): 0.61 (0.37 to 0.99)) and study medications (wmd (95%ci): -1.18 (-1.23 to -1.14)) with no difference in asthma control or adverse events. the results for the two trials reporting wap budesonide/formoterol to fd fluticasone/ salmeterol were discordant and a homogenous pooled result could not be determined. of the 318 australians who died from asthma in 2005, over two thirds were over 50 years of age. this trend resulted in the national asthma council of australia (nac) calling for better management of asthma in the elderly. we designed an educational intervention using evidence based educational strategies to improve the content and style of general practice consultations for older people with asthma. methods randomized controlled trial of a multi-faceted program consisting of a group educational session, a videotaped standardized simulated patient consultation, followed by an academic detailing session. forty-two gps were randomized into an active or a control group. gps provided the names of patients who would be happy to participate in the study and the program was evaluated by patient and gp outcomes. results gps recruited into our program reported improvements in a range of clinical areas. one hundred and ten patients were recruited, their outcomes are under analysis. conclusion gps were overwhelmingly positive about participation in this trial and our intervention successfully improved the capacity and confidence of gp's to deliver care to older people with asthma. our study also developed several tools that would enable dissemination of our findings. supported by an asthma targeted in studies where direct clinical assessment is not possible, urgent health care utilization (hcu) is often used as an indirect measure of asthma control. this study aimed to identify factors predicting urgent hcu and asthma control. methods patients in nsw with a doctor diagnosis of asthma were recruited from community pharmacies, a research volunteer database, and databases of asthma foundation nsw, to complete a questionnaire about asthma. poor asthma control was defined as asthma control questionnaire (acq) score ն1.5. urgent hcu was defined as hospitalization, ed visit, or urgent doctor visit due to asthma. multiple logistic regression was used to identify predictors of poor control and urgent hcu. results questionnaires were completed by 608 adults (61% female) with a doctor diagnosis of asthma (pharmacy 260, woolcock 299, asthma foundation 87). 87% used inhaled corticosteroid (ics) ϯ long-acting b2-agonist in the last 4 wks. median age was 56 yrs (range 12-87), and 9% were current smokers. mean acq score was 1.4 (95% ci 1.3-1.5), with 40% of participants having poor asthma control (acq ն 1.5). 28% had urgent hcu for asthma in the previous year. significant independent predictors for poor asthma control were younger age, current smoking, living in more disadvantaged areas, being retired, having only primary education, and holding a concession card. predictors for urgent hcu were younger age, being in full-time employment, having only primary education, and being of non-english speaking background. neither ics use nor possession of a written asthma action plan was associated with lower risk for either poor asthma control or hcu. conclusions poor asthma control is common in nsw even in patients using inhaled corticosteroids. although urgent hcu is often used as an indirect measure of poor asthma control, it is affected by different factors, perhaps because health care utilization represents a more complex balance between need and access. bronchial challenge tests with mannitol, to measure airway hyperresponsiveness, can take up to 30 minutes and require inhalation of up to 635 mg of mannitol. our aim was to determine if positive mannitol challenges can be detected after half the maximal dose (315 mg) using the forced oscillation technique (fot) to measure response. methods 15 non-asthmatic subjects and 52 asthmatic subjects underwent standard mannitol challenge, up to 635 mg mannitol. respiratory system conductance (grs) and reactance (xrs) was measured by fot at 6 hz during 40 sec tidal breathing immediately after each dose of mannitol. fev1 was measured after fot, within 90 sec of mannitol administration. two point dose response slope (drs), was calculated for grs (drsgrs) and xrs (drsxrs) for standard tests, up to 635 mg, and for short tests by excluding data from doses above 315 mg. ability to detect a positive test, defined as pd15fev1 < 635 mg, was determined by the area under the roc curve (auc) and repeatability by intra-class correlation coefficient (icc). results 32 asthmatic and 2 non-asthmatic subjects had positive tests, with pd15 fev1 values from 9.2 to 622 mg. auc (95%ci) did not differ between standard (std) and short tests for drsgrs (p = 0.14) or drsxrs ( combined use of inhaled steroids (ics) and long acting beta-agonists (laba) have an important role in asthma management. we used data from a 2006 population sample to examine medication use in adults and children. methods all adults (18-75 years) and children (2-17 years) from within four discrete zones in northern sydney were eligible for an interview survey, as part of a study investigating health effects associated with traffic-related air pollution. the prevalence of use of short-acting beta-agonists (saba), any ics (alone or combination) and combined formulations of ics/laba in the previous three months was estimated for the study population and those with diagnosed asthma. results there were 806 children [mean (sd) age 8.7 (4.6) years and 50% female] and 2184 adults [mean (sd) age 45.6 (14.9) years and 56% female] interviewed in 1843 households, representing an overall response rate of 33%. the prevalence of ever diagnosed asthma was 16.1% in children and 17.4% in adults. medication data were missing for 301 subjects. background asthma affects 1:9 adult australians and is a leading cause of rejection for recruitment into the australian defence force (adf). within this diagnosis there is a wide spectrum of disease activity and clinical outcomes. also asthma assessment and management has improved so that many asthmatics are now fully active without any significant disruption or risk to their lives. hypothesis: there is a subgroup of asthmatics who are at very low risk from significant adverse effects from asthma and who could be considered for recruitment to the adf. aims 1. to identify the subgroup of asthmatics who could be considered for recruitment to the adf. 2. to develop an assessment process to identify this subgroup (screening). 3. to develop a process to evaluate the outcomes of any change to the recruitment standard for asthma (evaluation). methods 1. a literature review of the natural history, assessment, management and response to treatment of mild episodic and mild persistent asthma. 2. a literature review of asthma in the military. 3. a clinical review of the outcomes of known asthmatics in the adf. 4. an expert group to review the above and to develop a screening process and an evaluation of the program. the literature review identified a subgroup of asthmatics, defined as mild episodic and mild persistent, who with appropriate management, have a low risk of significant adverse asthma outcomes. they can be identified by a combination of questionnaire, spirometry and bronchial provocation testing. a screening process has been developed which allows asthmatics to be recruited with a negative mannitol or hypertonic saline challenge on 400 mg/day or less of budesonide (or equivalent) without laba. a methodology to evaluate the impact of these changes on the recruitment standard has also been developed. alexithymia is a personality trait associated with difficulty identifying and communicating emotional and physical feelings. it has been associated with poor control of asthma and near fatal asthma. the primary objectives of this study were to: (1) identify alexithymia in a cohort of australian asthma patients; (2) investigate the relationship between alexithymia and asthma control; (3) investigate the relationship between alexithymia and asthma management. methods cross sectional study of 25 moderate to severe asthma patients recruited from royal adelaide hospital outpatients. participants were either mailed the questionnaire pack or completed it after a clinic appointment. existing validated questionnaires were used. statistical analyses were performed using spss. results 11 male (44%) and 14 female (56%) patients with moderate to severe persistent asthma (mean age 44 years, sd = 11) participated. alexithymia scores ranged from 23.0 to 76.0 (x = 48.3, sd = 13.2). 12% (n = 3) of participants could be classified high alexithymia, 32% (n = 8) borderline alexithymia and 56% (n = 14) were low alexithymia. alexithymia mean scores were not statistically different across sociodemographic variables. a positive correlation/association was found between alexithymia score and asthma control score (r = 0.57, p < 0.01), quality of life (r = -0.65, p < 0.01), and adherence (p = 0.03) but not satisfaction with communication (r = -0.27, p = 0.2) or number of hospitalizations (p = 0.25). conclusions this is the first australian study to identify alexithymia among asthma patients and investigate relationship to control as well as management and communication. associations between alexithymia and asthma control were confirmed. a larger sample size is needed to determine impact of alexithymia on self-management and provision of clinical care for asthma. port hedland is impacted by iron-containing dust particles (pm10) that may activate lung cells when inhaled. furthermore, the effects of port hedland pm10 may differ from the effects of urban pm10 impacting metropolitan areas. the aim of this study was to assess the effects of port hedland pm10 on production and release of the inflammatory cytokines, il-6 and il-8, by human airway epithelial (a549) cells, and to compare these with the effects urban pm10 from metropolitan areas. methods human airway epithelial (a549) cells were exposed to pm10 collected at port hedland and at urban locations (sydney, perth). a549 cells were exposed to a range of pm10 concentrations (20-200 mg/ml) for 24 h. lipopolysaccharide (lps) and phorbol myristate acetate (pma) were used as positive controls. supernatants from cell cultures were assayed for il-6 and il-8 using specific elisa kits. rna was extracted and reverse transcribed to cdna. il-6 and il-8 mrna expression was quantified by duplex real-time pcr using taqman primer/probes. results lps stimulated a 2.7-fold increase in il-8 release and pma stimulated a 3-fold increase in il-8 release and a 30-fold increase in il-6 release. however, neither port hedland pm10 nor urban pm10 stimulated concentration dependent release of il-6 or il-8 by a549 cells. expression of il-6 or il-8 mrna was also not altered by port hedland or urban dust. cd8+ t-cells may cause airway epithelial cell apoptosis via the granzyme pathway. we have reported increased apoptosis of airway epithelial cells and increased bal t-cell expression of granzyme b in copd, and a positive correlation between the two. we hypothesized that the increased granzyme b would also be related to smoking history (pack years -pk/y), age and severity of airflow obstruction (fev1 %pred) in patients with copd. we further hypothesized that the t-cell granzyme b expression would be higher in the airway than the peripheral blood. methods we investigated t-cell intracellular granzyme b expression in blood from copd subjects (33 current and 24 ex-smokers) and 12 never-smoker controls, and bronchoalveolar lavage (bal) and bronchial brushing (intraepithelial t-cells) from a cohort of these subjects using flow cytometry. correlations between granzyme b and pk/y, age or fev1 were performed using spearman's rank correlation. granzyme b in t-cells from blood, bal and bronchial brushings were compared. results there were significant correlations between fev1 and granzyme b expression in blood and bal (blood: r -0.444, p = 0.002; bal: r -0.368, p = 0.029). there was a significant correlation between pk/y and granzyme b expression in blood (r 0.362, p = 0.002), but not in bal. there were no significant correlations between granzyme b and age. there were no significant differences in granzyme b expression in blood, bal or intra-epithelial compartments. conclusion granzyme b is expressed at similar levels in blood, bal and intra-epithelial compartments, supporting recent opinion that copd is a systemic disease. t-cell granzyme b is related to severity of airflow obstruction and smoking history in patients with copd and may be one mechanism of apoptosis leading to lung injury and airflow obstruction in copd. jc allen 1 , t schlosser, ee ramsay 1 , q ge 2 , aj ammit 1 as development of remodelled airways is correlated with deterioration of lung function, we require therapies that reduce and reverse structural changes in remodelled airways. in asthma, corticosteroids can halt some, but not all, aspects of airway remodelling. therefore, in order to aid future design of efficacious anti-remodelling agents we need a better understanding of the molecular mechanism/s underlying the development of airway remodelling and the effectiveness of corticosteroids. hyperplasia of airway smooth muscle (asm) is a feature of the remodelled airway in asthmatics. in this study we examined the effect of corticosteroids on a key regulator of g1 progressioncyclin d1. asm cells from n = 8 non-asthmatics and n = 7 asthmatics were pretreated for 1 h with vehicle or dexamethasone (0.1 mm). the temporal kinetics of cyclin d1 mrna and protein expression were measured up to 24 h after stimulation with the mitogen platelet-derived growth factor-bb (pdgf-bb). pdgf-bb induced a significant increase in cyclin d1 mrna expression in asm from non-asthmatics (2.6 ϯ 0.3-fold) and asthmatics (2.9 ϯ 0.3-fold) after 24 h stimulation. in non-asthmatics, the corticosteroid dexamethasone significantly (p < 0.05) reduced the amount of cyclin d1 mrna expressed (to 1.6 ϯ 0.2-fold). in contrast, cyclin d1 expression in asthmatics was relatively resistant to inhibition by dexamethasone; the amount of pdgf-bb-induced cyclin d1 expression in the absence or presence of dexamethasone was not significantly different ( sphingosine 1-phosphate (s1p), a bioactive sphingolipid found elevated in the airways of asthmatics, modulates myriad airway smooth muscle (asm) functions that promote inflammation and remodelling in asthma. in this study, we uncover the molecular pathway/s underlying s1p-induced secretion of il-6, and investigate if, and how, corticosteroids inhibit il-6 secretion. using cultured asm cells from non-asthmatics, we found that s1p induces il-6 secretion from asm cells via cre, but not ap-1, c/ebp or nf-kb, transcriptional regulation of il-6 gene expression. cre-dependence was supported by s1p-induced creb phosphorylation. although the corticosteroid dexamethasone reduced s1p-induced il-6 secretion in a dose-dependant manner, this inhibition appeared to occur via a pathway independent of creb/cre, suggesting the existence of a parallel pathway. as we recently discovered that the antiinflammatory actions of corticosteroids in asm can be mediated via the induction of the endogenous mitogen-activated protein kinase (mapk) inhibitor, mapk phosphatase-1 (mkp-1), we investigated whether mapk represents the parallel pathway targeted by corticosteroids. we found that s1p can induce activation of a variety of mapk, however, only p38 mapk phosphorylation was inhibited by dexamethasone; importantly, the increase in mkp-1 after corticosteroid treatment appeared to mirror the decrease in s1p-induced p38 mapk phosphorylation. furthermore, exogenous expression of mkp-1 inhibited s1pinduced il-6 secretion. taken together, these results suggest that parallel pathways exist to induce il-6 secretion (transcriptional via creb/cre and possibly post-transcriptional via p38 mapk) and serve to underscore the importance of mkp-1 upregulation as a mechanism of action of corticocosteroids in asm. angiogenesis is a hallmark feature of asthma. angiogenic promoters, such as vegf and tgfb are reported to be increased in airways of asthmatics. tumstatin, an endogenous angiogenic inhibitor, is the non-collagenous domain-1 (nc1) of the alpha3 chain of collagen iv. decreased levels of collagen iv have been reported in the airways of asthmatics. we investigated the presence of tumstatin in the airway of asthmatics and its potential role as an angiogenic inhibitor. we detected the six a chain nc1domains of col iv and the 7s domain of the a3 chain using immunohistochemistry. the level of tumstatin in serum and bal-f was measured by dot blot. western blots were used to identify the association with the rest of the collagen iv molecule. a tube formation assay using primary pulmonary endothelial cells (ppec) was performed to evaluate the role of tumstatin in the airway. the effect of intranasal tumstatin on airway hyperresponsiveness and angiogenesis was studied in an ovalbumin mouse model. tumstatin was absent in the airways of asthmatics (n = 14) while the remaining six collagen iv a chains were present. the 7s domain of the a3 chain was present in the asthmatic airway (n = 6). tumstatin was detected in both serum and bal-f samples from asthmatic volunteers (n = 10), however the level of expression was not significantly different from that in nonasthmatics (n = 7). in asthmatic serum tumstatin was part of the whole collagen iv a3 chain. tumstatin was able to inhibit ppec tube formation in a dose related manner. tumstatin inhibited angiogenesis in the mice airways and was associated with an improvement in ahr. the fact that tumstatin is absent from asthmatic airways and inhibited airway hyperresponsiveness and angiogenesis may indicate potential for therapeutic intervention in airway remodelling. this work was supported by the crc for asthma and airways and nh&mrc. introduction epithelial egfr (epidermal growth factor receptor) expression correlates with disease severity and neutrophil infiltration in asthmatic airways. acute exacerbations of asthma and copd are also associated with steroid refractory neutrophilic inflammation, with rhinoviruses being the most common trigger. .7 mg/l and il-6: 5.8 vs. 3.6 ng/l). since il-6 stimulates the acute phase response, we correlated its levels with the other markers. only crp was strongly correlated with il-6 (spearman r = 0.58, p < 0.0001), suggesting differential regulation of saa and ip10. saa discriminated between non-pathogen (n = 10) vs. pathogen-associated (n = 41) events (saa: 9.4 vs. 44.1 mg/l p = 0.005), whereas no significant change was observed in the other markers (ip-10: 139.8 vs. 170.5 ng/l, crp: 4 vs. 10 mg/l, il-6: 4.6 vs. 7.2 ng/ l). however when aecopd marker levels were stratified on the basis of pathogen type (viral = 12, bacterial = 21, viral and bacterial = 8), none of the markers were significantly altered. conclusions ip-10 is significantly elevated during an aecopd, however only saa differentiated non-pathogen from pathogen associated events. background severe persistent asthma is characterized by structural changes in the airways-airway remodelling. airway smooth muscle (asm) cells have the potential to play a key role in these processes through the release of growth factors, cytokines and extracellular matrix (ecm) proteins. we have previously studied the effects of budesonide and formoterol individually however, the effect of their combination on these characteristics of asm cells is not known. methods asm cells from asthmatic (n = 6) and nonasthmatic (n = 6) individuals were stimulated with transforming growth factor ß (tgfß) (1 ng/ml) with or without budesonide (10 -8 m) and formoterol (10 -10 and 10 -8 m) and fibronectin levels and interleukin-6 (il-6) release were measured by elisa. bronchial rings from nonasthmatic individuals (n = 2) were incubated with tgfß with or without the drugs and ecm protein expression (fibronectin and collagen i) measured using immunohistochemistry. results in nonasthmatic cells, budesonide alone induced fibronectin deposition whether tgfß was present or not. formoterol decreased fibronectin induced by tgfß and, when combined with budesonide, reversed the increase in fibronectin. a similar pattern was observed in asthmatic cells, except that budesonide did not further increase the tgfß mediated fibronectin release. as before [1] , il-6 was induced by formoterol but inhibited by budesonide. tgfßinduced il-6 was inhibited by both drugs and their combination in both cell types. in bronchial rings the presence of either drug did not affect tgfßinduced fibronectin or collagen i. severe combined immune deficiency (scid) spontaneous mutation specifically impairs differentiation of stem cells into mature lymphocytes. nod-cb17prkd scid (known as nod-scid) lacked nk cells, hence is commonly used in cell transfer experiments for transferring tissue and haematological xenografts. the aim of this study was to establish lung inflamamtory model in nod-scid strain. methods balb/c and nod-scid balb/c mice (n = 8) were exposed to cigarette smoke for 4 days, 2 and 4 weeks (9 cigarettes/day; 5 days/week). bronchoalveolar lavage fluid (balf) and lung tissue were collected for inflammatory profiling and analysis for cytokines, chemokines and protease expression and/or activity. results nod-scid have significant accumulation of macrophages in lung after 4 days, 2 and 4 weeks smoking as compared to no smoke control (p < 0.001) that was not different to balb/c (p > 0.05). nod-scid also have increased neutrophil number after 2 and 4 weeks smoking (p < 0.001). even though myeloid cell differentiation isn't affected by scid phenotype, nod-scid have one fold less neutrophil than balb/c mice (p < 0.001) that is also reflected in the reduced expression of matrix metalloproteinase-9. consistent with the known lymphopenic phenotype, nod-scid have significant but less lymphocytes recruitment as compared to balb/c mice after 4 weeks smoking (p < 0.001) despite the enhanced expression of inteferon inducible protein 10 (lymphocytes specific chemokine) in lung. both mouse strains showed the same elevation of net gelatinase and serine protease activity in lung. nodscid mice also demonstrated comparable transcriptional induction of proinflammatory cytokines (tnfa, il-6), growth factors (gm-csf, g-csf) and chemokines (mcp-1, mip-2), indicating susceptibility to smoke-induced injury. conclusions nod-scid mice are capable to mount smoke induced inflammatory response. this model may be useful to study localization and role of immunocytes, including adoptively transfer human cells in the pathogenesis of copd. supported by the nhmrc. rhinovirus (rv) is the cause of most common colds and up to 80% of asthma attacks. in our previous studies, plasminogen activator inhibitor 2 (pai-2) was expressed at high levels and was induced in vivo and in vitro by rv infection. pai-2 may have antiviral properties suggested by antiviral activity in some models, high pai-2 expression levels and further upregulation by rv infection. methods to determine whether pai-2 has antiviral activities following rv infection, o-hela, pai-2 expression-deficient cells were first transfected with pai-2 or control genes. this was followed by infection with rv and effects on viral replication were assessed by rt-qpcr for vrna and by viral titration for virus release. ifn expression was assessed by rt-qpcr. results ifn-a and -b mrna expression were induced in response to rv infection and to pai-2 expression in cells. pai-2 expression followed by rv infection elicited a synergistic response and pai-2 over-expression reduced vrna by >5 fold and viral titre by >3 log (p < 0.05). however, this effect was not specific to pai-2, as transfection of cells with control genes/plasmids reduced viral titre to a comparableextent. one of the pathological findings in idiopathic pulmonary fibrosis (ipf) is the presence on fibroblastic foci comprising cells which exhibit mesenchymal phenotypic features such as myofibroblast-like morphology, increased asma expression and collagen deposition. currently steroid treatment in ipf has shown limited efficacy. the cellular origins of these mesenchymal cells remain unclear, but evidence from other studies suggests that epithelial cells may undergo a transition to a mesenchymal cell phenotype (emt). transforming growth factor ß has been implicated in promoting this emt. in this study we have induced a morphological change in a549 cells using tgf-ß1 and assessed the influence of glucocorticoids, and the changes to the extracellular environment of the cells, on emt. methods a549 cells were grown on uncoated plastic cultures plates or those coated with monomeric or fibrillar collagen and treated with 200-500 pm tgf-ß1. the influence of the glucocorticoid, dexamethasone (dex, 1-1000 nm), or collagen type, on emt was assessed by microscopy, rt-pcr and western blotting for markers of myofibroblast phenotype. results tgf-ß1 induced an increase in mrna expression of asma (1.5 fold), collagen (7.0 fold) and fibronectin (2.0 fold). dex (100 nm) partially inhibited the expression of collagen, but had no effect on asma levels. however, dex (100 nm) reduced asma and ctgf protein levels. dex (100 nm) also prevented the tgf-ß1-induced morphological changes, regardless of ecm matrix. conclusion glucocorticoids appear to control some of the emt phenotype changes induced by tgf-ß1. however, the inability to fully inhibit these changes may contribute to the resistance of ipf to glucocorticoids. the extracellular environment may also play a role in the development of fibroblastic foci and their pharmacological responses. defective alveolar macrophage (am) phagocytic function in the airway may perpetuate inflammation via secondary necrosis of uncleared apoptotic cells in copd. we have previously reported that low-dose azithromycin improved macrophage function in vitro, although the mechanisms for this effect were not identified. we explored the possible role of the collectin pathway in the azithromycin-mediated improvement in phagocytosis as well as possible defects in this pathway in copd subjects. methods (1) mannose binding lectin (mbl), mannose receptor (mr), surfactant protein d (sp-d) were measured in copd subjects and controls. (2) the in vitro effects of addition of rhmbl, and blocking mr with a specific antibody, on am phagocytic ability were assessed. in vitro effects of azithromycin on am expression of mr were also investigated. (3) azithromycin (250 mg orally 2¥ weekly/12 weeks) was administered to 11 copd subjects. bronchoscopies were performed prior to and 12 weeks following therapy. ex vivo assessments included am phagocytic ability, levels of mbl, sp-d and mr and apoptosis of bronchial epithelial cells. results am mr expression and levels of mbl and sp-d were significantly reduced in copd subjects vs controls. azithomycin (500 ng/ml) increased mr expression by 31% in vitro. rhmbl induced a dose-dependent increase in am phagocytic ability (up to 148%). blocking mr significantly decreased am phagocytic ability by 60%. in copd patients following azithromycin therapy, we observed improved am phagcocytic ability, increased levels of mr and reduced levels of bronchial epithelial cell apoptosis. conclusions these findings strongly implicate the mr in both the defective phagocytic function of am in copd and as a target for the azithromycinmediated improvement in phagocytic ability. obstructive sleep apnea (osa) is associated with hypoxia and increased cardiovascular morbidity. t cells and monocytes play a significant role in atherogenesis via cytokine production. there have been reports of benefits of continuous positive airway pressure (cpap) therapy in osa. the purpose of this study was to characterize leucocyte inflammatory cytokine/chemokine production by t cells and monocytes in a group of osa patients and to investigate the therapeutic effects of cpap therapy. methods a comprehensive range of intracellular t-cell and monocyte proand anti-inflammatory cytokines/chemokines was investigated in peripheral blood from 5 osa patients and 5 aged-matched control subjects (with no evidence of sleep problems) using multiparameter flow cytometry. osa patients were again studied following 7 days of cpap therapy. results in osa patients there was an increase in intracellular t-cell ifng and tnfa production but no change in il-2, il-4 or tgfb compared with control. there was an increase in intracellular monocyte il-1a, il-8, tnfa, mcp-1 and mcp-3 in osa patients but no change in il-10 or il-12. following cpap therapy, t-cell ifng and tnfa production returned to 'normal' levels. however, although intracellular monocyte cytokine/chemokine production was decreased following cpap, levels were significantly elevated compared with control. conclusions osa is associated with increased intracellular proinflammatory cytokine/chemokines, many of which are increased in atherosclerotic plaques. although one week of cpap therapy resulted in amelioration of t-cell pro-inflammatory cytokines, longer cpap use or alternative therapy may be required to reduce monocyte pro-inflammatory mediators associated with atherosclerosis in patients with osa. gp130 has been associated with the progression of fibrosis especially in patients with idiopathic pulmonary fibrosis (ipf). gp130 is the common subunit of the receptor complexes for the il-6 family of cytokines including il-11 and oncostatin m (osm), where gp130-mediated signalling leads to activation of the erk or stat pathways. we have previously demonstrated exaggerated gp130-stat signalling to be fundamental to the development of pulmonary fibrosis in a murine model of bleomycin-induced lung fibrosis. the aim of this study was to elucidate the role of the il-6 cytokine family in the development of pulmonary fibrosis by identifying which il-6 family cytokines regulate fibrosis in bleomycin treated mice, and determine the effects of these cytokines on cell function. bleomycin (0.05 u/mouse) or control saline was administered intranasally to wildtype mice (wt), genetically engineered mice containing point mutations to prevent gp130 erk signalling (gp130 757f ) or gp130 stat signalling (gp130 dstat ), and duel il-6 and il-11 a-receptor knockout mice (il-6 -/-;il-11ar -/-). the effect of bleomycin on collagen production was examined in lung tissue 30 days post treatment by hplc. there was a significant increase in collagen levels in bleomycin treated wt lungs which was further increased in gp130 757f lungs. the lungs of gp130 dstat and il-6 -/-;il-11ar -/mice were protected from fibrosis suggesting that gp130-stat signalling is important in inducing lung fibrosis which may be mediated through il-6 and/or il-11. cell proliferation was examined in lung fibroblasts isolated from wt, gp130 dstat and gp130 757f mice. il-6, il-11 and osm were significantly mitogenic for gp130 dstat cells but not for wt or gp130 757f cells, reflecting different responses to the different signalling pathways. changes in cytokine profiles are currently being examined in lung tissue and serum of control and bleomycin treated mice 0-30 days after treatment. in conclusion, il-6 and il-11 are likely to play a role in bleomycin-induced fibrosis via the gp130-stat-mediated pathway, however this may not be due to regulation of proliferation induced by these cytokines. supported by the nhmrc. mimicking viral infection by application of various toll-like receptor ligands has shown clinical promise in the treatment of persistent viral infections and more recently with malignant tumours. commercially available toll-like receptor 7 ligands (tlr7l), such as those of the imidazoquinoline family have been applied clinically for the treatment of a number of conditions including basal cell carcinoma and hpv-induced genital warts. these compounds are known to retard tumour growth indirectly by promoting activation and migration of dcs, leading to a strong th1 cellular response, and directly via release of proinflammatory cytokines and promotion of tumour cell apoptosis. malignant mesothelioma (mm), an aggressive tumour with a mean survival of 9 months, is highly resistant to chemotherapy, radiotherapy and surgery and is therefore an interesting candidate for immunotherapy in the form of tlr7 ligand treatment. whilst tlr7 is known to be selectively expressed in immune cells and its relative expression low amongst other cell and tissue types in mammals, its expression on tumour cells and the consequences of such expression on tumour growth are unknown. here we describe the presence of tlr7 (mrna and protein) directly in a range of different tumours, including several murine and human mm cell lines. reactive oxygen species (ros) produced during the innate immune response are important agents of anti-pathogen defense but may also cause oxidative lung damage. glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. methods wild-type (wt) or mice deficient in glutathione peroxidase-1 (gpx-1 -/-) were placed in a perspex chamber and exposed to cigarette (cig) smoke generated from 9 cigs per day for 4 days. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for proteomic and genomic analysis. results wt mice exposed to cig smoke for 4 days had significantly more macrophages (3.1 ϯ 0.1(sem) ¥ 10 5 ) and neutrophils (4.9 ϯ 0.4 ¥ 10 5 ) than sham-exposed mice (2.2 ϯ 0.2 ¥ 10 5 and 0, respectively) (n = 6, p < 0.05). however, gpx-1mice exposed to cig smoke had significantly greater macrophages (5.4 ϯ 0.3 ¥ 10 5 ) and neutrophils (1.2 ϯ 0.1 ¥ 10 6 ) than smokeexposed wt mice (n = 6, p < 0.001). macrophage and neutrophil numbers in sham-exposed gpx-1 -/mice (1.7 ϯ 0.3 ¥ 10 5 and 0.5 ϯ 0.4 ¥ 10 3 ) were similar to those of sham-exposed wt mice (2.2 ϯ 0.2 ¥ 10 5 and 0). in addition, we found that balf of gpx1 -/mice exposed to cig smoke had an increased proteolytic burden compared with smoke-exposed wt mice as assessed by zymography and net gelatinase activity assay. conclusions these data suggest that gpx-1 protects the lung from cigarette smoke-induced inflammation and that targeting gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role. funded by nhmrc. the becs from subjects with chronic obstructive pulmonary disease (copd) are exposed to frequent infectious and inflammatory stimuli. infection with rv is known to trigger acute exacerbations and subjects with copd are particularly susceptible. we hypothesized that exposure of copd becs to these stimuli would alter their response to rv infection. methods bec were obtained by endobronchial brushing from subjects with gold stage 3 copd (n = 4, all ex-smokers), subjects with mild persistent asthma (n = 4) and healthy controls (hc, n = 4). becs were cultured and then treated with tumour necrosis factor (tnf)a 10 ng/ml or lps 100 mg/ml for 24 hrs and then infected with rv-43, rv-1b. response was measured by release of il-8, il-6 and ip-10 mrna and by elisa. virus replication measured by cell titration assay. results infection with both rv strains led to increased release of il-8 and ip-10 in all groups. exposure of hc and asthma becs to both lps and tnf led to increased release of il-8. in these becs there was no increase in release of il-8 exposed to lps and tnf and then infected with either rv. becs from subjects with copd released significantly less il-8 in response to all conditions and rv infection compared to hcs and asthma. no differences were seen in rv replication. the aim of this study was to determine opinions and attitudes to exercise from chronic obstructive pulmonary disease (copd) subjects after completion of a 12-month maintenance exercise program. methods following completion of a 12-month exercise study, which included a supervised program (intervention, n = 18) and control group (control, n = 17), copd subjects [mean age (sd): 66 (8); mean fev1 (% predicted) = 56% (19)] were asked to complete a questionnaire. the questionnaire included closedended questions using visual analogue scales (100 mm). in copd the 6 minute walk distance (6mwd) is known to increase with test repetition (familiarization) and in response to exercise training. it is unknown whether the magnitudes of these increases are related to the degree of disability of the individual patient. methods 6mwd was measured twice before and once after an 8 week out-patient exercise program in 121 patients (82 males) aged 67ϯ8.6 yrs, fev1 37ϯ15% predicted (meanϯsd) with stable copd. the changes in 6mwd following a familiarization test and following training were compared between patients grouped according to their degree of disability (defined as the pre-training 6mwd [best of 2 tests] expressed as %predicted 6mwd). *p < 0.05 gp 3 vs gp 1. conclusions before training, 6mwd increases following a familiarization test irrespective of the level of disability. the magnitude of this increase is similar in all groups when normalized for their pre-training 6mwd. following training, the increase in 6mwd is greatest in patients with the greatest disability (lowest pre-training 6mwd). in less disabled patients, the relatively smaller increase in 6mwd following training may reflect an inability to further increase stride length, thereby reducing the responsiveness of the 6mwt in this group. supported by nhmrc. endotoxin is a stimulant of the innate immune system and is a major component of cigarette smoke. smokers have evidence of increased airway neutrophils and inflammation. we hypothesized that endotoxin levels would be higher in the bronchial lavage (bl) of subjects who were former smokers and subjects with chronic obstructive pulmonary disease (copd). methods subjects were all ex-smokers for at least 5 years (n = 10, 5 copd, 5 healthy controls) or never smokers (n = 12, 6 asthma, 6 healthy controls). bl was collected and analysed for cell count and differential, culture for microbiology. the supernatant was analysed for il-8 by elisa and endotoxin by quantitative kinetic lal assay. results median endotoxin levels were significantly higher in ex-smokers 101 compared to never smokers 6.3 u/ml (p < 0.001). there were no differences between subjects with copd and hs. subjects with copd had higher median endotoxin levels (80 u/ml), compared to asthma (5.2 u/ml) and hc (6.3 u/ml, p = 0.03). there was no correlation between endotoxin levels and bl total cell count, neutrophils (%) or fev1 % predicted. there was a strong correlation with previous packet years smoked and endotoxin levels (r = 0.72, p < 0.01). conclusions bl endotoxin levels are higher in ex-smokers, including subjects with copd. despite this there is no relationship to increased neutrophilic inflammation. copd is associated with inflammation associated with ineffective repair of the injured epithelium and loss of structural integrity. we have shown that these changes may result from dysregulated 'efferocytosis' (increased apoptosis of bronchial epithelial cells and defective clearance of these cells by alveolar macrophages (am)). we have also reported that azithromycin, at subbactericidal dose, improved am phagocytic function ex vivo. methods we administered azithromycin at low dose (250 mg/ twice weekly for 12 weeks) to 10 copd subjects (7 male, age: 62 ϯ 8 yr, 5 current/ 5 ex-smokers, fev1: 63 ϯ 9% pred, fev1/fvc: 48 ϯ 9%). the study was openlabel, uncontrolled and primarily focused on objective biological responses obtained from the bronchoscopy samples taken. phagocytic ability of am (from bal), apoptosis of bronchial epithelial cells (from bronchial brushing), markers of inflammation in blood, bal and breath condensate (crp, wcc and inflammatory cytokines), health status (st. george's respiratory questionnaire), ecg and lung function were assessed pre and post-administration of azithromycin. results azithromycin significantly improved phagocytic ability of am (by 37%) and reduced bronchial epithelial cell apoptosis (by 34%). antiinflammatory effects of azithromycin included significantly reduced blood wcc and crp. there were non-significant reductions in levels of pro-inflammatory cytokines il-8, il-6 and tnf-a in blood, bal and breath condensate, and a trend for improved health status. conclusions our findings indicate a novel approach to supplement existing therapies in copd that may improve clearance of accumulated apoptotic material and reduce the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation. background the prevalence of gastro-oesophageal reflux disease (gord) across the disease spectrum in copd and bronchiectasis is not well described. the aim of this study was to determine the prevalence of symptomatic and silent gord in copd and bronchiectasis and its effect on lung function and quality of life (qol 4] ) and 18 healthy controls were recruited. the prevalence of gord in bronchiectasis was 33%; 37% in copd; 17% in controls. in copd and bronchiectasis, total nre and ri were increased in those with distal and proximal gord compared to those without gord (all p < 0.05). there was no difference in extent or severity of bronchiectasis in patients with or without gord (all p > 0.05). in copd, the relationship between proximal gord and fev1 was small to moderate (r = 0.383). sgrq symptom scores were higher in patients with bronchiectasis with increased ri (p = 0.02). increased proximal nre was associated with reduced physical (p = 0.03) and mental health (p = 0.02) in the sf-36 in copd. conclusions gord is a co-morbidity in patients with copd and bronchiectasis. the impact of gord on disease severity requires further evaluation. funding source nhmrc, the university of melbourne, monash university, physiotherapy research foundation. chronic obstructive pulmonary disease (copd) is prevalent among older people, however little is known about the influence of ageing on airway inflammation. the aim of this study was to compare airway inflammation in older people with obstructive airway disease to groups of older and younger healthy controls. methods participants (>55 years of age) with stable airway disease and incomplete reversibility (fev1% predicted <80% and fev1/fvc < 70%; copd n = 71) and healthy controls (n = 45, 35 older >55 years and 10 younger <55 years) were recruited from the respiratory ambulatory care clinic or by advertisement. participants underwent a clinical assessment, skin allergy test, hypertonic saline challenge, sputum induction and gas diffusion studies. results participants with copd had moderate airflow obstruction (mean (sd) fev1% predicted 56 (19)) and 45 (63%) were current or ex-smokers with a median (iqr) pack year history of 36 (20-54) pack years. ageing was associated with an increase in airway neutrophils (p = 0.0001). compared to older controls, participants with copd had increased airway eosinophils and lymphopenia (p = 0.004, p = 0.003 respectively), but no difference in airway neutrophils. conclusion airway neutrophilia is a feature of ageing and is not further increased in the presence of copd. copd is associated increased numbers of airway eosinophils with reduced lymphocytes which may impact on the ability of the immune system to combat infection. supported by nhmrc, the university of newcastle. chronic obstructive pulmonary disease (copd) is third leading cause of death and fourth leading cause of disease burden in australia. mechanisms involved in emphysema severity have not been fully understood. micrornas are noncoding rnas that regulate gene expression. we hypothesize that microrna expression differs between emphysema severity in copd patients. methods mirna profiling was performed using 15k agilent human oligo mirna microarrays on total rna extracted from non-tumour lung tissue from 30 copd patients undergoing resection for lung cancer. the mirnas were quantile normalized and anova was used to find differentially expressed genes. results demographic characteristics of the copd patients (mean (sd)) were age 69 (6) years, fev1 72 (17) % predicted and fev1/fvc ratio (<70%). anova identified 31 mirnas that were differentially expressed when stratified into two classes according to kco % predicted > or <75% (t-test, p < 0.05). discussion this mirna analysis has identified mirnas that may be important in emphysema severity in copd patients. further validation will be performed using qrt-pcr and mirna assays on the training set and an independent set, and target prediction and validation. t-helper type 1 (th1) and type 2 (th2) lymphocyte responses have been well recognized as being important pathways in inflammation. recently another form of inflammatory lymphocyte response has been described, the th17 pathway. th17 cells produce cytokines such as il-17a to clear extra-cellular bacteria and fungi and have been implicated in autoimmune and chronic inflammatory diseases. the th17 response in copd is unknown. methods subjects were patients with copd (ex-smokers, fev1 < 70% predicted who had not had an exacerbation for at least 1 month) and control subjects (ex-smokers and normal spirometry). serum samples were obtained for measurement of c reactive protein (crp) and il-17a, the latter measured using enzyme-linked immunosorbent assay (elisa). production of il-17a by t-cell subsets was also identified by intra-cellular cytokine staining and measured by flow cytometry. the mean fev1 of copd subjects was 42 % predicted (6.1 sem, n = 6) and mean fev1 of controls was 112 % predicted (3.0 sem, n = 4). the copd group had a higher mean level of crp 9.5 mg/l (3.9 sem) compared to the control group mean level of 4.6 mg/l (0.6 sem). the mean level of the il-17 in the copd group as measured by elisa was 22.3 pg/ml (16.9 sem, range 0-87) whilst no il-17 was measured in any of the control subjects. conclusions the findings of this pilot study suggest that il-17 may be elevated in association with crp in stable copd. airway obstruction is defined as a fev1/fvc ratio below the lower limit of normal. airway obstruction may prolong the forced expiratory time (fet). method spirometry results from 467 patients were categorized as obstructive, restrictive or normal. the mean, range and coefficient of variation were determined for fet in each diagnostic group. receiver operator characteristic (roc) curves were used to determine if fet could predict a low fev1/fvc. the number of patients with airway obstruction in five fet groups: <9; 9; 10-12; 13-14; and >14 seconds were determined. results the coefficient of variation was high for all groups. pair-wise comparisons showed a difference in mean fet between patients with normal lung function versus those with airway obstruction (p < 0.001). the best cut-point in the roc analysis of 9.895 seconds had a sensitivity of 0.66, specificity 0.77 and area under the curve of 0.743 for predicting obstruction. the technique of skeletal muscle microbiopsy has previously been validated [1] and shown to be minimally invasive and well tolerated in participants with stable copd. aim a study was undertaken to determine the feasibility and tolerability of obtaining microbiopsy muscle samples from the patient admitted for acute exacerbation of copd patient. methods written informed consent was obtained to collect the muscle, blood and sputum samples for research purposes. local anaesthetic was injected prior to the insertion of a 16 gauge bard max core disposable biopsy instrument through the associated guide needle. multiple passes (up to 6) were obtained. the patient was asked to evaluate the experience by rating it on the modified borg scale 0-10. results to date 5 patients and 3 controls have participated in this study. the gold severity ranged from 2-4 and ats exacerbation severity 2-3. the mean age 75 years (range 68-83 years), bmi mean 23.6 kg m -2 (range 17.2-27.1 kg m -2 ) and fat free mass was determined using single frequency bioimpedance. the sample mass obtained ranged from 27.2-104.1 mg, with an increasing yield occurring with increased experience of the operator. the procedure has been well tolerated, the borg scale rating ranged from 1-2/10. all patients were ambulant post procedure; no haematoma or bruising was observed in any of the subjects. conclusion the microbiopsy technique allows the collection of muscle tissue with minimal discomfort to the participant. small tissue masses such as these are sufficient to obtain measures of local markers of wasting and may prove to be a useful adjunct to the collection of sputum and blood for the measure of biomarkers in copd research. introduction older people (op) with obstructive airways disease (oad) experience multiple problems that may impact on their quality of life (qol) and disease management. these problems may relate to pathophysiology, symptoms, self management skills, psychological issues, lifestyle or other problems identified as important by the patient. aim the aim of this study was to determine the frequency of clinical problems associated with oad and to determine if a problem based assessment (pba) could adequately identify these problems. methods a multidimensional assessment tool was developed and the content compared to clinical practice guidelines. participants over 55 years with diagnosed oad underwent this assessment. results sixty-one consecutive patients, aged 59-87 years, with mean (sd) fev1 of 51.4 (17.85) % predicted were assessed. the assessment tool identified a mean (sd) of 3.03 (2.13) current and significant co morbidities with an additional 11 (3.37) clinical problems per patient. qol was increasingly impaired with an increasing number of problems (p < 0.0001). regression modelling identified that the number of identified clinical problems accounted for 55% of the qol impairment. the model demonstrated that every additional patient problem was associated with a clinically significant change in qol impairment (4.22 units) . conclusions op with oad experience multiple clinical problems and co morbidities that adversely impact their qol. a pba of op with oad identifies significant problems that may not be addressed in a diagnosis centred approach. there is a need to identify and effectively manage this array of problems in clinical practice. discussion in this diverse group of copd patients, there was a positive correlation between dlco and fev1, but not kco and fev1. the fev1/ kco plot identifies substantial numbers of patients with the potential ad and e phenotypes defined above. we intend to study inflammatory biomarkers in these groups. fat free mass index (ffmi) is a marker of morbidity and mortality in copd. measurement of ffm in the out-patient population is commonly undertaken using single frequency bioelectrical impedance analysis (bia). however the formulae to convert measured values to ffm are population dependent. schols et al (am j clin nutr, 1991) suggested that formula used for the general population may be inappropriate for patients with copd, and derived a specific formula from total body water (tbw) as measured by deuterium dilution. we compare this method of measuring ffm with 5 others, along with tbw and ffm hydration. methods tbw was measured in 31 outpatients with copd by bia and a difference method (weight-(protein+bone mineral+fat+non-bone mineral+ glycogen)) and ffm hydration was calculated. ffmi was measured by skin fold anthropometry (sfa), bia (3 separate formulae), dual energy x-ray absorptiometry (dexa) and total body potassium by g-counter (tbk). comparison between methods for tbw and ffmi was made by bland-altman analysis and between methods of calculation of ffm hydration by paired t-test. the two methods of assessment of tbw showed little difference (bias -0.04, 95% limits of agreement -5.40 to 5.31). however there was a significant difference in calculation of hydration of ffm (p = 0.0001). sfa, bia (lukaski), bia (tanita) and tbk underestimated ffmi when compared to bia (schols), with bias of -1.24, -3.87, -1.06 and -2.76 respectively. dexa however had a bias of only 0.05 and 95% loa of -3.09 to 3.21. conclusions there are differences between methods of assessment of tbw and ffmi and comparing values between methods must be done with caution. this has implications for assessment of morbidity and mortality in copd. chronic obstructive pulmonary disease (copd) has been identified as a major health problem in australia. recent studies have suggested that respiratory viral infections are the major cause of a worsening of copd; however this has not been studied in australia. aim to characterize pef changes and identify viruses during copd exacerbations. methods a pilot prospective longitudinal cohort study was done. patients had confirmed copd with fev1 <70% predicted and reversibility <10% and/or 200 ml. patients recorded daily peak expiratory flow (pef) measurements and daily chest and cold scores over a period of 2 years. sputum samples and nasal aspirates were taken at 6-month review (control visit) and whenever they had symptoms of an exacerbation (worsening of copd symptoms -seemungal et. al. am j resp crit care med, 2001). nasal aspirates and sputum samples were obtained and analysed by rt-pcr for rhinovirus (rv). result five patients have finished 2 years of study. a total of 12 exacerbations were reported based on patient symptoms. only 3 exacerbations were associated with significant reductions in pef and only one was linked to increases in nasal cold scores. all samples taken at control visits and nasal aspirates and sputum samples during exacerbations were negative for rv by rt-pcr. positive controls confirmed the accuracy of the assay. conclusion our data suggest that a symptom-based definition of copd exacerbation is not always accompanied by significant reductions in lung function parameters. these 'exacerbations' are also not associated with the commonest reported viral cause. our findings suggest that variability of copd may mimic. bronchiectasis is characterized by hypersecretion of mucus and impaired clearance that results in mucus accumulation, chronic cough, sputum production and recurrent infections. inhaled mannitol (400 mg) improves clearance of mucus by increasing the airway hydration and by reducing the viscoelastic and surface properties of mucus. however, the effect of other doses of mannitol on the clearance of mucus in patients with bronchiectasis is unknown. methods fourteen patients, age: 63.3 ϯ 5.7 yr, were studied on 5 visits. clearance of mucus was measured using 99m tc-sulphur colloid and imaging with a gamma camera at baseline and with mannitol ( weight loss and skeletal muscle atrophy are major determinants of morbidity in chronic obstructive pulmonary disease (copd), which are independent of lung function impairment. thus, we examined if a high-fat diet (hfd) protected against the wasting associated with prolonged cigarette smoke exposure (se) in mice. methods male balb/c mice were exposed to the smoke of 4 cigarettes/day, 6 days/week for 7 weeks. sham mice were handled identically without smoke exposure. mice consumed either standard laboratory chow (3.5 kcal/g, consisting of 12 % fat) or a hfd (4.3 kcal/g, 32% consisting of fat). we examined the effect of se and hfd on hind limb skeletal muscles, lung (tissue & bronchoalveolar lavage (balf)) and systemic inflammation in the 4 groups of mice (n = 8/ group). results after 7 weeks of hfd, sham and se mice were 12 and 13% heavier (respectively, p < 0.05) than chow fed animals. conversely, se significantly decreased body weight of chow and hfd fed mice by 16 and 15%, respectively, compared to sham animals (p < 0.05). the hfd did not protect against the decrease in soleus, tibialis anterior and gastrocnemius skeletal muscle weights induced by se (p < 0.05). se altered the mrna expression of a number of genes associated with the regulation of skeletal muscle mass including insulin-like growth factor-i (igf-i), atrogin-1 and interleukin (il)-6. the mrna expression of pro-inflammatory cytokines and chemokines was significantly increased by se in the lung, as were the number of inflammatory cells in balf (p < 0.05). on the other hand, although obesity has been linked to systemic inflammation, the hfd exerted little direct effect on the skeletal muscle and lung parameters measured. se and hfd had no effect on two markers of systemic inflammation, il-6 and serum amyloid a, whereas se tended to reduce circulating igf-i, an anabolic hormone. conclusions the hfd was not protective against the weight loss and skeletal muscle wasting associated with cigarette smoke exposure. supported by the nhmrc and crc for chronic inflammatory diseases. background patients with copd and bronchiectasis undertake airway clearance therapy (act) and exercise as part of physiotherapy management but it is unknown whether these treatments provoke gastro-oesophageal reflux (gor). this study aimed to determine the impact of positive expiratory pressure (pep) therapy and exercise on gastro-oesophageal function. p. aeruginosa is a significant opportunistic lung pathogen in individuals with cystic fibrosis (cf) and is associated with increased lung disease and morbidity. early intervention is beneficial for the effective clearance of p. aeruginosa and better long-term health outcomes. currently, lung flora of cf patients is monitored by regular culturing of sputum, however, children unable to expectorate are limited to annual bronchoalveolar lavages (bal), which is invasive and requires general anaesthesia. saliva is useful for clinical assays as collection is simple, non-invasive. we are developing a standardized enzymelinked immunosorbent assay (elisa) to detect respiratory infection of p. aeruginosa in cf children who cannot expectorate. methods 18 children (7-18 years) with cf and recent p. aeruginosa lung infection history and 16 non cf children (1-6 years) with no previous p. aeruginosa infection history provided saliva as positive, negative controls respectively. saliva was obtained by spitting, or absorbed using cellulose swabs and later extracted. these cell-free supernatant samples were used in an elisa anti-p. aeruginosa iga using commercial antigen. all results were standardized to account for flow using total iga expression. results median value was increased 9 fold in the recent p. aeruginosa lung infection group (mann-whitney test, n = 34, p յ 0.001). there was no significance between mucoid and non mucoid samples, and detection was independent of cfu/ml. discussion early findings support that p. aeruginosa respiratory infection can be detected through specific analysis of salivary iga expression. larger population sampling (30 positive, 90 negative) will aid selection of cut-off values for specificity and sensitivity testing in the future to objectively determine the utility of this assay as a means of monitoring for p. aeruginosa and for determining effectiveness of treatment. medical thoracoscopy is utilized widely throughout europe and northern america by thoracic physicians for the management of pleural disease, including the undiagnosed pleural effusion, malignant effusions and less commonly pneumothorax (ptx). australia has limited experience in this modality. we report the success of medical thoracoscopy in both primary and secondary ptx requiring intervention. methods data were collected from 2001 to 2007 in patients treated with medical thoracoscopy for the treatment of ptx. results 11 patients, 7 male, 4 female. average age 48 (range 19-86). 1 first episode primary spontaneous (ps) ptx, 2 third episodes of ps, 5 first secondary spontaneous (ss), 1 second ssptx, 2 third ssptx. underlying pulmonary disease in secondary ptx included: 4 chronic obstructive pulmonary disease, 1 lymphangioleiomyomatosis, 1 mesothelioma, 1 metastatic angiosarcoma and 1 was secondary to a motor vehicle accident. 7 had a history of smoking, 5 were former smokers and 2 were current smokers, with a mean 24 pack year history (range 5-45). 7 ptx were large, 4 moderate. 5 patients had an intercostal catheter (icc) inserted prior to thoracoscopy, 1 had failed pleural aspirate. there was evidence of bronchopleural fistula in 7 patients prior to the procedure. there was a median of 9 days from ptx to thoracoscopy. light sedation was used for the procedure in 10 patients, 1 required a general anaesthesia with a double lumen endotracheal tube due to anxiety. single port entry, dry talc poudrage and a 16 gauge french icc was used for all procedures. icc was removed a mean of 2 days following thoracoscopy and patients discharged on day 4. pain was the most common complication, requiring narcotic analgesia. one patient died on day 7, secondary to metastatic angiosarcoma. there has been no recurrence of ptx in any patient. conclusion medical thoracoscopy, performed by thoracic physicians is an effective procedure for the treatment of pneumothorax requiring intervention, including selected patients with evidence of bronchopleural fistula. funding nil. conflict of interest nil. nomination for young investigator award no. background lung cancer incidence and mortality are high in tasmania. australia (aihw 2003) 85/100 000 72/100 000 tasmania (cancer registry 2003) 102/100 000 89/100 000 aims and objectives (a) to determine patient demographics in southern tasmania, (b) to determine compliance to identified measures of best practice and (c) assess referral rates, clinical utility and potential delay to positron emission tomography (pet) in a regional setting. methods a prospective database collected information on local clinical practice. cases presented at a multidisciplinary lung cancer meeting over a 12 month period (march 2006 -april 2007 were analysed. data were available for n = 121/161 (75%). results are shown as mean ϯ sd. results 113 primary lung cancer cases were identified. the mean age was 71 ϯ 11 years. 58% of patients were male and 95% were current or ex-smokers. 81% were non-small cell lung cancers (nsclc). tissue diagnosis 93% time from diagnosis to surgery (27 ϯ 15 days) 82% < 42 days macroscopically complete surgical resection (9/11) 82% pet for stage iiib before radical chemoradiotherapy 75% 62% of patients presenting with early or locally advanced disease underwent further staging with pet (n = 34/55). management was changed in 50% of cases (17/34). the average time from pet referral to scan was 11 ϯ 5 days. conclusion a disproportionate number of lung cancers occurred in women. although surgery was performed within recognized timeframes, 2 of 11 patients had incomplete resections. pet influenced management decisions and was performed in a timely fashion. hp chan 1,2 , v tran 1,2 , c lewis 1,3 , p thomas exhaled breath condensate (ebc) is a simple, safe and non-invasive method of sampling breath and has the potential to investigate lung cancer and the associated neoplastic process in the lungs. increased oxidative stress has been implicated in the pathogenesis of lung cancer, and is characterized by elevated hydrogen ions, and hydrogen peroxide (h2o2), which is formed from the conversion of superoxide anions by superoxide dismutase. airway ph has already been shown to be decreased in ebc of patients with other respiratory conditions, but not in lung cancer. therefore the concentration of h2o2 and hydrogen ions in the ebc of lung cancer subjects was compared with matched controls. methods six subjects with newly diagnosed lung cancer were recruited and matched with control subjects: non-smokers, ex-smokers and smokers. ebc was collected and h2o2 was then measured by an assay method based on oxidation of 3,3',5,5'-tetramethybenzidine by horseradish peroxidase and h2o2 while ph was measured using a ph meter. results there was a significant difference (p = 0.033, anova) in h2o2 concentration between the 4 groups with the lung cancer group having elevated mean h2o2 concentration of 23.68 mm (9.15 (sem) compared to the controls: non-smokers, 17.59 mm (6.53 (sem); ex-smokers, 14.35 mm (3.79 (sem); and smokers, 5.21mm (0.69 (sem). ph did not differ significantly (p = 0.659, kruskal-wallis test) between the groups. conclusion these preliminary data suggest that there is significant difference in h2o2 concentration between the groups. the demonstration of an elevated h2o2 level in those with lung cancer indicates an increase in oxidative stress which implies that this may be part of the pathogenesis or response to neoplasia. supported by none. conflict of interest none. pro-inflammatory th1 cytokines produced by t cells and monocytes play an important role in the immune response to malignant cells. however, tumours may escape immune surveillance by inhibiting th1 response and promoting chronic inflammation at the tumour site. methods to investigate the effect of soluble factors released by lung cancer cells on t cell and monocyte pro-and anti-inflammatory cytokines, culture supernatants from several lung cancer cell lines and a normal epithelial cell line (16hbe) were cultured with whole blood for 24 hours, then for a further 16 hrs with and without stimuli. intracellular cytokine / chemokine production was determined using multiparameter flow cytometry. results in stimulated cultures, there was a significant decrease in t cell th1 pro-inflammatory cytokines ifng, tnfa and il-2 and a decrease in monocyte il-1a, il-8, il-12, tnfa, mcp-1 and mcp-3 but an increase in antiinflammatory cytokine il-10 compared with 16hbe and control media. in non-stimulated blood cultures there was an increase in all monocyte inflammatory cytokines / chemokines in the presence of lung cancer supernatants. conclusions lung cancers secrete soluble factors that inhibit the antitumour pro-inflammatory th1 response by t cells and monocytes and upregulate monocyte anti-inflammatory cytokine il-10 following "antigenic challenge". lung cancer cells may also escape immune surveillance by secreting soluble factors that cause newly recruited monocytes to release inflammatory cytokines promoting chronic inflammation at the tumour site. cytotoxic t-cells (ctl's) are important barriers against tumour cells. ctl's induce apoptosis of target cells by mechanisms that include the release of pore-forming perforin and granule associated enzymes, such as granzyme b and granulysin. proteinase inhibitor-9 (pi-9) is the only known granzyme b inhibitor and its expression has been observed in some cancers. we hypothesized that pi-9 would be differentially expressed in lung cancer cells and may inhibit granzyme b-induced apoptosis in these cells. methods we investigated pi-9, granulysin and granzyme b expression in various lung cancer cell lines (1299 ( , 1466 ( , 2009 and normal epithelial cells obtained from bronchial brushing using flow cytometry. peripheral bloodderived t-cells were then incubated with lung cancer cell line supernatants and levels of pi-9, granzyme b and t-cell reactive oxygen species (ros) were assessed. results pi-9 expression was detected in all lung cancer cell lines, (1299 (54.2%), 1466 (90.2%), 2009 (85%), sbc-1 (81%)), at much higher levels than in normal bronchial epithelial cells (8.5%). granzyme b and granulysin levels were undetectable or low in cancer cells (0-9.2%). increased expression of pi-9 and reduced levels of granzyme b were observed in cd8+ t-cells in the presence of all cancer cell supernatants tested (p < 0.05). interestingly, t-cell ros levels were significantly increased in cd8+ t-cells after incubation with cancer cell supernatants (p < 0.05). conclusions high pi-9 expression in lung cancer cells combined with a reduction in t-cell granzyme b expression and enhanced intracellular t-cell ros levels may be a mechanism of immune evasion of lung cancer cells to granzyme b-induced cytotoxicity. immunotherapy for lung malignancies such as lung cancer and mesothelioma is most likely to be successful it it can be combined with conventional tumour debulking approaches such as chemotherapy and surgery. but they scientific basis of such combinations is yet to be determined. to study this we evaluated (1) the capacity of different lung chemotherapy drugs to alter tumour antigen cross-presentation and immunogencity, (2) duration of antigen presentation and responsiveness to immunotherapy after debulking surgery with/without lymphadenectomy, and (3) the pattern of tlr agonism which best synergized with chemotherapy and surgery. we used the ab1-ha murine model of lung malignancy in balb/c mice. results (1) the antimetabolite drugs gemcitabine and pemetrexed were most immunogenic compared to the cytotoxic antibiotics doxorubicin and mitomycin c and the alkylating agent cisplatin. gemcitabine delived large amounts of tumour antigen into the cross-presentation pathway. (2) tumour antigen cross-presentation persisted for only 10 days following resection. the optimal window for immunotherapy following cancer surgery is 1 week for effector ctl stimulation and 2-4 weeks for memory ctl stimulation. (3) the viral-like tlr agonists tlr 3, 7 and 9 were the most effective adjuvant tlr molecules, with tlr 7 agonists generating the strongest systemic anti-tumour responses. conclusion these results help explain previous lung immunotherapy failures and will inform new clinical trials. background mesothelioma is a highly aggressive tumour with an increasing world wide incidence. the serum biomarker mesothelin is elevated in some individuals prior to development of clinical symptoms of the disease and may be useful for screening. we therefore studied the sensitivity and specificity of urinary versus serum levels of mesothelin for mesothelioma patients and evaluated the influence if renal function on the biomarker level. materials and methods concurrent sera and urine samples collected from patients with and control populations. mesothelin concentrations were determined by double-determinant elisa using the mesomark tm assay (fdi, pa). their estimated glomerular filtration rate (egfr) was also calculated. results mesothelin levels correlated between serum and urine samples (pearson's correlation 0.791; p < 0.0001). mesothelin levels were significantly higher in patients with mesothelioma compared to those with asbestosis and/or pleural plaques in serum (4 ϯ 0.9 versus 0.9 ϯ 0.05 nm; p < 0.0001, respectively), in urine (1.9 ϯ 0.5 versus 0.3 ϯ 0.03; p < 0.0001) and in urine following normalization using creatine levels (0.2 ϯ 0.05 versus 0.04 ϯ 0.01). age and egfr were significantly associated with mesothelin levels. conclusion the sensitivity and specificity of mesothelin in urine and in serum were comparable. urine mesothelin may prove to be a useful alternative to serum mesothelin for mass screening of asbestos-exposed individuals. patients undergoing ct coronary angiogram (cta) are often former or current smokers with a high incidence of asymptomatic lung disease. overseas reports show a rate of lung abnormalities ranging from 6.7% to 19%. there are no studies from australia and local factors such as the higher incidence of atypical mycobacteria may influence the rate of benign findings. we are therefore performing a prospective observational study to identify the prevalence and characteristics of incidental lung findings in people undergoing routine cta. methods population: 100 patients undergoing routine cta after informed consent. intervention: radiologist evaluation of lung windows on diagnostic standard workstations. comparator: uncontrolled observational study of consecutive patients. outcomes: primary: prevalence and characteristics of abnormal findings, final diagnosis (clinical judgment, biopsy or long term followup). secondary: number of downstream investigations and costs. results 25 ctas have been studied to date. in 8/25 (32%), abnormalities were noted on lung windows. in 2/25 (8%), there were lung nodules, in 2/25 (8%) there were hilar lymph node abnormalities, in 1/25 (4%), there was hemidiaphragm elevation and in 3/25 (12%) there were pleural plaques (data collection ongoing with study closure expected in february 2008). conclusions preliminary data indicate a substantial number of incidental pulmonary findings from cta; full results will be presented. further analysis is required to determine the impact (benefits, costs and harms) that may result from the concurrent examination of lung windows at routine cta. aim increased levels of nitrogen oxides (nox) and inflammatory markers have been found in bronchoalveolar fluid of lung cancer (lc) patients, but have not been investigated in exhaled breath condensate (ebc).the aim of this study was to compare nox and total protein levels in ebc of lc patients with control subjects. methods ebc was collected during tidal breathing through a glass collection device cooled to 4°c. ebc nox concentrations were measured by a fluorescent modification of the greiss method. total protein in ebc was determined employing the bicinchoninic acid (bca) assay. ebc nox data were log transformed. all data were analysed using anova and expressed as mean ϯ sem. results a total of 88 control subjects and 54 patients with primary lc were recruited. nox and protein concentrations are shown in table 1 . there was no significant difference in ebc nox levels (p > 0.05), but in total protein there was a significant difference between lung cancer patients and all control groups (p = 0.04). conclusion significantly increased ebc total protein levels were found in patients with lung cancer. these data suggest that protein mediator secretion or vascular leak may be present in those with lung cancer. future studies will focus upon the identification of these proteins. methods in this two stage case-control study 446 lung cancer cases and 484 healthy smoker controls were recruited. 180 genetic markers (snps) implicated in lung cancer were screened in our test cohort of 439 smokers and ex-smokers. 30 snps whose genotypes (co-dominant or recessive model) were associated with either the healthy smokers (protective) or lung cancer (susceptibility) phenotype were identified. after genotyping this 30 snp panel in a second cohort of 491 subjects 19 snps were chosen and assigned a simple composite genetic score that was combined with scores for age, history of copd and family history of lung cancer, weighted according to our multivariate regression analysis (n = 930 total subjects). the lung cancer risk score was linearly related to the likelihood of lung cancer with odds ratios (referenced against the lowest score quintile) ranging from 1 to 29 in the highest quintile. on receiver operator curve analyses, the auc was 0.78 and the frequency distribution showed bimodal separation between healthy smokers and lung cancer cases. utility of the score was not affected by effects of age, smoking history or lung function. we suggest that genetic data may be combined with other risk variables to define smokers or ex-smokers at risk of lung cancer for targeted interventions such as smoking cessation and early detection of lung cancer. supported by health research council, nz. conflict of interest yes. tp 144 v aiyappan 1 , a graham 2 1 department of medicine, maroondah hospital, melbourne, australia, and 2 the new disease-modifying anti-rheumatic drug (dmard) leflunomide is being used increasingly to treat inflammatory arthritis. its association with interstitial lung disease needs to be considered before combining it with methotrexate. case report a 73-year-old male who was known to have rheumatoid arthritis and was on methotrexate was admitted with progressive dyspnoea and malaise. he had been recently started on leflunomide. investigations revealed interstitial lung disease and acute renal failure. he improved on conservative treatment (stoppage of disease modifying drugs (dmard), iv fluids and steroids). review of literature an epidemiological study by suissa et al has suggested that there is increased risk of ild associated with leflunomide in patients with a history of ild or methotrexate use but they attributed this to channelling bias. there has also been a report of leflunomide associated with iga glomerulonephritis.by this presentation we aim to increase the awareness of this entity. we also suggest that any patient who is started on combination dmard (i.e. methotrexate and leflunomide) should have a baseline chest x-ray and be monitored for development of interstitial lung disease. conclusion we are reporting the first ever case of interstitial lung disease and glomerulonephritis (in the same patient), due to usage of leflunomide. this entity needs to be thought about in any patient on combination dmards. background bone morphogenic protein receptor ii (bmpr-ii) mutations are associated with pulmonary artery hypertension. failure of the growth inhibitory effects of bmp may contribute to vascular obliteration and remodelling leading to pulmonary artery hypertension (pah) [1] . pah has been observed following venous thrombembolic disease (vte), including pulmonary embolism (pe) and deep venous thrombosis (dvt) [2] . local markers of the pulmonary vascular endothelium rather than traditional markers of thromobophilia are thought to be involved [3] . methods plasma was collected from age and gender matched participants within 24 hours of diagnosis of vte and prior to commencement of warfarin therapy. plasma samples were hybridized to individual human cytokine antibody arrays, to detect protein levels of bmp2, bmp4 and bmpr-ii. results bmp2 and bmp4 levels were higher in patients with dvt than pe. no difference in the bmp level was observed between patients with pe and controls. soluble bmpr-ii receptor was lower in patients with pe than in controls or patients with dvt. conclusion in patients with pulmonary artery stress during the time of a pe the bmpr-ii receptor is reduced, which may predispose patients to vascular remodelling and obliteration. the bmp 2 and 4 levels are reduced at the same time, suggesting a possible overriding regulatory mechanism. the physiological role of bmp's and bmp receptors in patients with vte warrants further investigation. historically, cyclophosphamide has had a variable role in interstitial lung disease (ild), the rationale for its use based on the benefit seen in vasculitis and scleroderma, its rapid effect and low toxicity profile. in patients with severe progressive ild a rapidly effective, well-tolerated agent is desirable. for this reason a treatment protocol for the use of intravenous (iv) cyclophosphamide was implemented at our hospital. aim to review the indications, duration, tolerability and effect of intravenous cyclophosphamide in ild patients following the introduction of a treatment protocol. methods records of 92 patients [dlco was 40 ϯ 15% and fvc 61 ϯ 20%] completing a course of iv cyclophosphamide during 2005-6 were reviewed (excluding patients with systemic sclerosis). data covering 18 months prior to and following treatment were collected. comparative analysis of paired pulmonary function data 6 months before and after treatment was performed. 61% had underlying autoimmune disease. results primary treatment indications included progressive disease(n = 67); severe disease (n = 16); suspected vasculopathy (n = 11); bridging therapy to transplantation (n = 10); and accelerated decline (n = 5). patients received 600 mg/m 2 [mean dose 1152 ϯ 165 mg, median number of pulses 6 (1-12)]. patients with paired pulmonary function data had a difference in median change in dlco% predicted from -15.6% (-95.4 to 29.9%) before treatment to +4.25% (-17.3 to 73.9%) following treatment (p < 0.0001). this remained significant with exclusion of vasculitis, or any autoimmune disease, and independent of prior immunosuppression. therapy was well tolerated (4 withdrew from treatment, 5 deaths within 1yr, none directly related to treatment). conclusion iv cyclophosphamide is well tolerated, and associated with functional stability or improvement in the majority of patients. it remains a viable treatment alternative for consideration. pulmonary hypertension is common in interstitial lung disease (ild) and associated with a poor prognosis. as the gold-standard test, right-heart catheterization (rhc) is invasive, and resource-limited, reliable non-invasive measures of ph are needed. methods all ild patients referred for rhc during 1997-2007 were included (n = 95; 54 male; age 56.5 ϯ 12 yrs). all patients had concurrent echocardiography (tte) and pulmonary function. the relationship of rhc mean pulmonary artery pressure (mpap) to tte variables, pulmonary function, exercise capacity, as measured by six minute walk testing (6mwt, n = 58) and brain natriuretic peptide (bnp, n = 36), was examined. case a 65 year old male, non-smoker for 25 years, retired professor of anatomy (had chronic exposure to embalming fluids, formaldehyde, phenol, antifungal and other solvents, for 20 years) presented with chronic cough and phlegm production. these symptoms were worse at night (waking him several times) and early morning. his pulmonary tests were stopped due to persistent cough. a chest x-ray revealed features of longstanding interstitial lung disease. the hrct revealed widespread subpleural interlobular thickening, worse at bases, in keeping with idiopathic pulmonary fibrosis (ipf). there was minimal fibrosis and honeycombing, but no groundglass opacification, large bullae, pleural calcification or pleural plaques. however, there was associated bronchiectasis at the lung bases considered to be due to traction. the ba lavage showed 50% macrophages, 7% neutrophils, 3% lymphocytes, and 40%, eosinophils and no infection. the patient declined to have a lung biopsy. as per his past x-rays, the duration of his ipf is a little over one year. he maintains that his symptoms started only after starting irbesartan (irb). introduction transbronchial lung biopsy (tbb) has a variable and unpredictable diagnostic yield in sarcoidosis. we hypothesized that the extent and pattern of parenchymal disease on ct would predict the likelihood of a positive tbb. methods data relating to ethnicity, symptoms, pulmonary function and site and results of tbb and bronchoalveolar lavage (bal) from 70 sarcoidosis patients were recorded. all had a ct scan within 6 weeks prior to the tbb procedure. cxr stage was determined from radiology report. ct scans were scored quantitatively for patterns of parenchymal disease (nodular, reticular, consolidation, ground glass and mosaic attenuation) on a lobar basis. results 50% patients had a positive tbb (total 67% of cohort had histological confirmation). symptoms, ethnicity, treatment, lung function and cxr stage were not predictors of a positive biopsy. positive biopsy was associated with higher bal lymphocyte count (p < 0.05) and female gender (p < 0.01). a reticular pattern (p < 0.05) and higher total lung score (excluding da) (p < 0.05) on ct scan predicted a positive biopsy. in those patients with tbb from right lower lobe (53/70) the total rll score on ct was predictive of positive biopsy (p < 0.05). on multivariate analysis gender, bal lymphocytosis and total lung score were independent predictors of a positive tbb (area under roc 0.82). pulmonary arterial hypertension has two histological variants; 'arterial-only pulmonary arterial hypertension' (artpah) and 'pulmonary veno-occlusive disease' (pvod). bosentan, a dual endothelin receptor antagonist, has been found to improve haemodynamics, functional capacity and survival in artpah. however, the response to bosentan in clinically diagnosed artpah is often variable. it was hypothesized that a lack of response to bosentan therapy in clinically diagnosed artpah can be explained by misdiagnosed pvod. aims included to: (1) perform morphometric and qualitative pulmonary vessel analysis on normal controls and cases clinically diagnosed with artpah who had failed bosentan therapy; (2) ascertain if pvod is present within the case group; (3) correlate clinical variables and vessel microanatomy to identify the pathologies driving pulmonary pressure elevation. this study reviewed 14 cases of clinically diagnosed artpah (idiopathic n = 12, associated with scleroderma n = 2), who had failed bosentan therapy and had available lung tissue. controls (n = 6) were obtained from explanted lungs for other causes and a prior transthoracic echocardiogram excluded pulmonary hypertension. vessel morphometry and qualitative analysis was performed with a novel technique of smooth muscle actin immunohistochemistry counterstained with verhoeff's elastin. baseline clinical data were retrieved. we found 86% of cases had pathology confirmed pvod. only 14% of cases had artpah, the original clinical diagnosis. in pvod, significant pathology was present in all vessel types. all vessels had significant smooth muscle hypertrophy. the obstructive, collagenous, pauci-cellular intimal fibrosis of the venules (p < 0.0001) and arterioles (p < 0.0001) was considerably different to the concentric laminar proliferation of smooth muscle observed in the muscular arteries (p < 0.0001) and arterioles (p = 0.001) in artpah. artpah also had muscular artery smooth muscle hypertrophy (p = 0.007). the median time to bosentan failure was shorter in pvod than artpah (290 vs. 657 days). in conclusion, pvod is an under-diagnosed cause of pulmonary hypertension, is commonly clinically misdiagnosed as artpah and may present with a poor bosentan therapy response. finally, pvod is a vaso-occlusive, not a veno-occlusive disease, and is an independent type of pulmonary hypertension, not a subtype of pulmonary arterial hypertension. cutaneous t cell lymphomas (ctcl) are a heterogenous group of lymphoproliferative disorders. they show various clinical manifestations and diverse morphological, histological and immunological characteristics of the malignant cells. they are caused by clonally derived, skin invasive t cells. peripheral t cell lymphomas (ptcl) are generally more aggressive and have one of the lowest overall and failure-free survival rates. because of the rarity of these disorders, diagnosis and treatment remain challenging. this case report describes a 69-year-old woman presenting with progressive dyspnoea and cough, together with a distressing generalized pruritic rash. she was initially treated as left ventricular failure with the rash ascribed to a drug reaction as suggested by initial skin biopsies. the diagnosis was made on a third skin biopsy and flow cytometry of lymphocytes obtained by broncho-alveolar lavage 6 months after presentation. despite an initial response to chemotherapy she succumbed to the disease 20 months after diagnosis. clinical pathways to guide the investigation of suspected pulmonary embolism (pe) have been increasingly adopted by emergency departments (ed) worldwide. compliance with these diagnostic algorithms is critical in ensuring good patient outcomes. this study evaluated the compliance to the clinical pathway used in our ed that combines risk assessment (wells scoring system) with d-dimer test, vq scan or ctpa. the main objectives of this study were to identify those factors which contributed to compliance and to assess patient outcomes. methods a prospective observational study of 239 consecutive patients who underwent investigation for pe in our ed. patient demographics, pathway parameters and patient outcomes at 3-month follow-up were collected. case we report the case of a 37 year old woman who presented to the emergency department with a three day history of dry cough and dyspnoea. the patient was in her third pregnancy at 30 weeks gestation. she had no fever, chest pain or coryzal symptoms. the patient had presented with a right sided spontaneous pneumothorax seven months prior to the current presentation. her past medical history included placental abruption, complicating her previous two pregnancies. her second pregnancy was complicated by placental abruption at 27 weeks and the foetus had not survived. her first pregnancy was complicated by placental abruption at 36 weeks with successful delivery of the foetus. at presentation, significant findings included tachycardia, hypoxemia, tachypnoea and reduced breath sounds over the right side of the chest. chest x-ray demonstrated a large right pneumothorax. a right intercostal catheter was inserted resulting in right lung re-expansion. the catheter was removed three days later. the patient returned to hospital twenty four hours after catheter removal with a recurrent right sided pneumothorax. the patient agreed to surgical intervention involving video-assisted thoracotomy and talc pleurodesis. the patient had no further complications with the pregnancy. she delivered a healthy baby at 38 weeks gestation. discussion spontaneous pneumothorax in pregnancy is rare and there is little evidence to provide guidelines for the management of recurrent pneumothorax in high risk pregnancy. our case illustrates a successful outcome for mother and foetus with surgical intervention at 32 weeks gestation. folfox is currently the standard adjuvant treatment for locally advanced (stage iii) colon cancer and increases disease free survival. its toxicity is well tolerated with common adverse effects being paraesthesia, bone marrow suppression and gastrointestinal disturbance. pulmonary toxicity has rarely been reported. three clinical cases of acute dyspnoea following folfox therapy (2005) (2006) (2007) for stage iii colon cancer are reported. all had an anterior resection followed by 11-12 cycles of folfox. each developed rapidly progressive dyspnoea requiring hospital admission within one week of their last cycle. one patient required invasive ventilation in icu. high resolution computed tomography (hrct) showed bilateral widespread honeycomb pattern with associated ground glass opacification consistent with pulmonary fibrosis. they had reduced lung volumes and gas transfer. transbronchial biopsy and bronchoalveolar lavage in one patient showed an acute eosinophilic pneumonitis. other causes of interstitial lung disease were carefully excluded. all three patients received high dose corticosteroids with one receiving additional cyclophosphamide. the first patient showed complete recovery following an eight week course of corticosteroids, with resolution of the hrct changes and improvement in lung function. the second had symptomatic improvement of dyspnoea, but a persistent moderate reduction in gas transfer. the final patient had persisting radiographic changes and a reduced gas transfer. he remained dependant on ambulatory oxygen 6 months after his initial presentation. these patients' interstitial lung disease appears due to folfox with oxaliplatin being the most likely causative agent. the use of oxaliplatin chemotherapy has increased markedly over the last 3 years and although rare, physicians should be aware of its potential for lung toxicity. lung function testing at baseline, during and towards the end of oxaliplatin treatment should be undertaken and may allow early detection and intervention in cases of pulmonary toxicity. the forced oscillation technique (fot) with broadband signals has been employed relatively rarely in the studies on respiratory mechanics. recent work from our laboratory [1] indicated that the cheek support and the neck angle have minor influence on the impedance spectra around the first antiresonance (far,1), which makes the use of the broadband fot especially attractive in young children. methods we studied 7 healthy children (c; female: 4) and 8 children with bronchopulmonary dysplasia (bpd; female: 3), using multiple-frequency fot between 8 and 256 hz superimposed on spontaneous breathing. results groups c and bpd did not differ in age ( lung function impairment is common in children with cardiac defects associated with increases in pulmonary blood flow/pressure. to investigate the development of bronchial hyperreactivity (bhr), an aorto-caval shunt was created in a model of precapillary pulmonary hypertension. surgical shunt repair was performed to assess the reversibility of bhr. methods 26 rats were divided into 3 groups: group c (n = 10) with sham surgery, group s (n = 8) where an aorto-caval shunt was created (follow-up 4 wks), group r (n = 8) with aorto-caval shunt but surgical correction of the shunt at 4 wks (follow-up 8 wks). in all animals, respiratory input impedance (zrs) was measured at baseline and following increasing doses of methacholine (mch 2, 4, 8, 12 mcg/kg). airway resistance (raw), inertance, tissue damping (g) and elastance were estimated from the zrs spectra by model fitting. measurements were repeated in all animals at 4 wks and at 8 wks for groups r and c. results there was a significant increase in raw and g in group s and rat 4 wks at baseline and following mch ( fig.) which was reversed after surgery. to characterize the factors contributing to lung function impairment following cardiopulmonary bypass (cpb), functional residual capacity (frc), lung clearance index (lci) and respiratory mechanics were measured in children with pulmonary hypoperfusion (tetralogy of fallot, tof n = 12) and hyperperfusion (ventricular septal defect, vsd n = 12) undergoing surgical repair of congenital heart disease. methods frc and lci were measured using a sf6 washout technique and respiratory mechanics using a low frequency oscillation technique in the perioperative period. results while chest opening led to a significant improvement of lung volumes and respiratory mechanics in all patients (p < 0.001), a reduction in pulmonary blood flow during cpb decreased lung volumes and airway resistance in parallel but significantly more in children with tof compared with those with vsd. re-establishing pulmonary blood flow during cpb improved respiratory function particularly in children with tof ( figure) . conclusions sternotomy had a great impact on lung function with parallel improvement in alveolar recruitment, ventilation inhomogeneity and airway resistance. in contrast, onset of cpb led to lung function impairment with a significant drop in frc especially in children with pre-existing hypoperfused lungs. this suggest that pulmonary blood flow enhances alveolar stability through a tethering effect on the alveolar walls. children with advanced lung disease being considered for lung transplantation are likely to spend disproportionately longer periods on transplant waiting lists before appropriately sized donor organs become available. these longer waiting times reflect the lower organ donation rates seen in children; rates that are significantly lower than those reported in the adult population. we describe two children with advanced lung disease who deteriorated whilst on the waiting list for lung transplantation, and in the absence of appropriately sized donor lungs, underwent lobar lung transplantation. methods we describe the clinical course of two children, aged 9 and 13 years old, with advanced lung disease secondary to post-mycoplasma obliterative bronchiolitis and cystic fibrosis-associated bronchiectasis, respectively. results both children received a "cutdown" bilateral lobar transplant from two oversized adult brain-dead organ donors. in both cases the transplant operation involved implantation of the right middle and upper lobes, and of the left upper lobe from the donor. conclusion given the low organ donation rates in children, and in the absence of appropriately sized donor lungs, novel strategies such as lobar transplantation must be considered, particularly when children continue to clinically deteriorate whilst on the lung transplant waiting list. data from the west australian adult outcomes of extreme preterm birth study suggest that adult survivors of bronchopulmonary dysplasia (bpd) may be left with functional and structural pulmonary abnormalities, most notably emphysema. animal data suggest that the antenatal administration of corticosteroids may adversely affect lung development. we therefore sought to determine if maternal variables, including administration of corticosteroid, could predict emphysema severity in adulthood. methods bpd subjects (birthweight < 1500 g and oxygen dependence at 36 weeks post-menstrual age) born prior to 1988 were identified and recruited prospectively via the statewide neonatal follow up program as previously described. pulmonary function tests and thin selective inspiratory and expiratory computerised (ct) images were acquired and scored for emphysema severity (voxel index (%)). the obstetric history was obtained from retrospective review of case notes. results 21 adults (12 females, aged 18-34) were studied, 2 declined ct. all subjects had abnormal ct findings. fifteen (79%) had areas of emphysema. emphysema score and fev1 were not influenced by the administration of antenatal corticosteroids, indication for delivery, maternal age or presence or absence of chorioamnionitis. conclusion maternal factors, including the administration of antenatal corticosteroids, do not predict the long term respiratory outcome of bpd. the factors determining the severity of emphysema in this group remain unknown. the prevalence of childhood asthma is high in the torres strait. children have generally more severe asthma and asthma knowledge is poor. however, there is no culturally appropriate asthma education program for these children. we are conducting a randomized controlled trial to examine the additional benefits of an education intervention by indigenous health care workers (hcw) on asthma outcomes. we describe the study's objectives, design and baseline measurements. methods children with wheeze were reviewed by two paediatric respiratory physicians using a standardized protocol; children with asthma were eligible. after obtaining informed consent children were randomly allocated to: (1) three additional asthma education sessions with a hcw; or (2) no additional education from a hcw. trained hcws carried out the education sessions using culturally appropriate tools. primary outcome was the number of unscheduled hospital/doctor visits due to asthma exacerbation. all children were re-assessed at 12 months. results we enrolled 113 children aged 1 to 17 years, 81% were torres strait islanders and 12% aboriginal and torres strait islanders. the clinical spectrum of asthma was: 51% infrequent episodic asthma, 22% frequent episodic asthma and 27% chronic asthma. eighteen percent of the children knew what a written asthma action plan was; 8.5% had one. carers' assessment of knowledge of medications showed that 52% could not name any asthma medication used by their child, 40% could not explain dosage, and 67% could not explain how beta2 agonists worked. conclusions asthma knowledge and possession of asthma action plans in this cohort is poor at baseline. there is substantial room for improvement and additional asthma education by hcws potentially has significant benefits. impulse oscillometry system (ios) measures respiratory function during normal breathing by transmitting mixed frequency rectangular pressure impulses down the airways and measuring reflected pressure. computer analysis calculates respiratory impedance and its components, airways resistance and reactance, at a range of frequencies from 0.1 hz to 150 hz. no previous australian normative data exists. the ios software generates predictive normal values for each of the parameters measured including total airway resistance (r5), the proximal airway resistance (r20) as well as peripheral capacitive reactance (x5). however, they are based on german data. methods cross-sectional study of 100 community dwelling adults, with 10 males and females per 10-year cohort. inclusion criteria: age range 25-74 years, apparently good respiratory health. exclusion criteria: smokers, asthmatics and others with acute or chronic respiratory disease. both ios and spirometry were conducted on all participants. results australian predictive normal equations have been generated and compared to the current published equations. the ios parameters have been correlated with the spirometric data. results have been analysed by gender, age, height and weight and compared with the predictive normal values for each parameter provided by the german manufacturer of the ios instrument. analysis includes calculation of mean range, and lower limit of normal. conclusions a preliminary set of australian predictive equations have now been produced for the ios. these have been compared with international equations. ios has potential application in a range of respiratory disease states and in population screening for occupational health (e.g. mining, & high dust load environments). supported by phc red. rationale although clinical practice guidelines for both asthma and copd recommend spirometry for diagnosis and monitoring, beneficial effects on the management of chronic respiratory diseases in general practice have not been established. we hypothesized that spirometry would improve health outcomes compared to usual care. methods we are conducting a single masked rct with 3 arms: group a receive 3 monthly spirometry and followup, group b receive spirometry before and after the trial and group c usual care. 45 general practices were recruited though divisions of general practice in melbourne. invitations were mailed by 31 of these practices to patients who had been prescribed inhaled medications during the previous 6 months. participants returned respiratory and generic quality of life questionnaires and an asthma score card. groups a and b were tested on a micromedical turbine spirometer following ats/ers guidelines. results 351 eligible patients (275 adults, 50 children aged 8-13 and 26 youths aged 14-17 years) entered the trial. 122 were randomized to group a, 134 to group b and 95 to group c. the mean (sd) age of adult participants was 54.3 (12.7), children 10.3 (1.7) and youths 15 (1.1) years. there were 130 males and 221 females. the adults were highly symptomatic in the previous 12 months: 82% reporting wheeze, 50% chest tightness on waking, 74% shortness of breath on exertion, 61% nocturnal cough, 46% morning cough and 75% sputum. symptoms of chronic bronchitis were reported by 39% of adults and a diagnosis of copd by 19%. asthma was reported by 84%, confirmed by a doctor in 96% and 55% had experienced an attack in the last 12 months. only 35% had a written asthma action plan. 37% of adults had ever visited a hospital ed and 28% had been admitted. conclusion it is possible to recruit asthma and copd patients from general practice and to randomize them to spirometry or usual care. whether spirometry is associated with fewer symptoms, changes in medication, uptake of action plans or improvement in lung function or quality of life requires further followup. supported by nhmrc. s shah 1 , jk roydhouse 1 , b toelle 2 , s sawyer 3 , c jenkins 2 for the pace australia management committee 1 university of sydney, 2 woolcock institute of medical research, sydney, nsw 2006, and 3 royal children's hospital, melbourne, vic 3052 it is widely held that recruitment of general practitioners for research can be challenging. in this paper, we discuss the recruitment experience from a current study evaluating the impact of an educational asthma intervention on patient outcomes. our aim is to describe the two different strategies utilized to date: (1) in-house through an academic department of gp and (2) outsourced to a private gp organization. methods initial interest was generated through faxes, presentations at gp divisional meetings and newsletter advertisements. gps who expressed interest were visited by project staff to discuss the study further. a major difference was recruiting one gp per practice in the first strategy versus multiple gps per practice in the second strategy. to assess the strategies, we examined participant characteristics, number of gps recruited and number retained. results participant characteristics: under both strategies, 30% of recruits had trained in asia and 54% were women. the first strategy recruited more gps who spoke at least two languages at home (85% vs 42%) and the second strategy recruited more recently graduated gps (58% vs 50%). recruitment: the first strategy recruited 35 gps over 6 months and the second recruited 34 gps over 3 months. retention: 19 gps (54%) from the first strategy stayed in, compared to 29 (85%) from the second. conclusions whilst absolute numbers of gps recruited were similar, retention was much higher under the second strategy. recruitment in primary care is difficult and requires a range of approaches which need to be re-evaluated and adapted as necessary during the course of the study. supported by the australian government department of health and ageing. bronchiectasis is a heterogeneous condition with a large number of causative factors and range of symptoms. the classification of this condition is often confusing and hard to remember. the aim of this study was to classify non-cf bronchiectasis into different clinical phenotypes. methods 178 consecutive patients with non-cf bronchiectasis confirmed on high resolution ct scanning had a detailed clinical, spirometric and laboratory assessment performed by a respiratory physician (pk/mf/pw) and were then followed up for an average of 9 ϯ 4 years (mean and sd) for a total of over 2000 reviews. results 160 of the 178 patients (90%) could be classified as belonging to 3 phenotypic groups; 1) bronchiectasis arising in childhood, 2) bronchiectasis occurring in smokers and 3) bronchiectasis occurring in the elderly. each group had different features which are listed in the there are few data on the long term outcomes of treatment for tuberculosis (tb) by directly observed therapy (dot) in low-incidence settings. the aim of this study was to assess the incidence of recurrent tb in nsw. methods data linkage was performed within the nsw department of health tb notifications database to identify cases that had more than one tb notification between 1994 and 2006. recurrent tuberculosis was defined to include all patients with two or more culture positive episodes at least 6 months apart, where patients had received at least six months treatment for the initial episode. in cases where data contained within the notification details was not sufficient to allow us to distinguish between true cases of recurrent disease, duplication notification for the same episode or persistent disease after incomplete treatment, additional information was obtained from the area tb coordinator. results there were 5723 tb notifications between 1994 and 2006 with 3731 being culture positive. 15 cases of recurrent culture positive disease after completed treatment for the first episode were identified (recurrence rate: 0.4%). conclusions in a population with a low tb incidence, treatment of active tuberculosis with dot results in a very low rate of disease recurrence over a long period of follow-up. support nhmrc ccre in respiratory and sleep medicine. introduction rhinoviruses (rvs) are the major cause of viral-induced exacerbation of asthma. to date, the molecular mechanisms of rv pathogenesis are not understood. recent findings suggest that rv pathology may involve host cell nucleocytoplasmic trafficking, inhibiting key cell functions such as transcription and translation. the study aims to investigate the mechanism of rv 3c protease nuclear trafficking. methods hela cells were infected with rv or transfected with plasmids and cellular localization of 3c analysed at various times thereafter using immunofluorescent confocal microscopy and western blotting with specific antibodies. results 3c protease was predominantly present in nuclei of rv infected cells up to 6 hours after infection, becoming increasingly cytoplasmic thereafter. the nuclear membrane of infected cells became progressively indistinct with time. using a specific inhibitor we also found that 3c utilizes the crm-1 nuclear export pathway. 3c was predominantly in the form of 3cd in both cytoplasm and nucleus of infected cells; mature 3c protease was also detected from 6 hours after infection. deletion analysis indicats that the nuclear localization domain and a nuclear export signal are most likely to be present within the n terminal 64 amino acids. the nuclear export signal is inhibited in the full length protein, via an unknown mechanism. conclusion our data suggest that 3c and 3cd proteins localize to the nucleus in infected cells where they may play a key role in rv pathogenesis by disrupting cellular transcription and the nuclear transport machinery. chronic necrotizing pulmonary aspergillosis (cnpa) is a relatively uncommon, sub-acute, locally destructive process due to aspergillus invasion of the lung. the incidence and prognosis of cnpa are poorly described. case report we present a case of cnpa in a patient on intermittent low dose steroid therapy and recurrent refractory exacerbations of chronic obstructive pulmonary disease (copd).the patient presented with worsening shortness of breath and productive cough requiring recurrent inpatient admissions. human influenza virus is found to bind preferentially to saa2,6gal receptors found in the upper respiratory tract, while avian viruses bind to saa2,3gal receptors expressed in lower airways. this is thought to affect the ability of transmission to humans. our aim was to study the ability of avian and human influenza strains to infect bronchial epithelial cells and relate this to levels of the sialic acid receptor expression. methods calu-3 cells were used as a proximal airway cell and a549 were used as distal airway cell. human primary bronchial epithelial cells (pbecs) were obtained from healthy, asthmatic, and copd volunteers by endobronchial brushing. epithelial cells were stained with sambucus nigra lectin that binds saa2,6gal receptor, and maackia amurensis lectin ii that binds to saa2,3gal. the cells was analysed by flow cytometry. human influenza a/h3n2/wellington strain and low pathogenic avian influenza a/h11n9/sandpiper were chosen and were used at an moi of 0.005 to infect cells. the supernatants were harvested at 48 hr post infection, of which was then analysed by plaque assay for virus replication. results the calu-3 showed greater expression of saa2,6gal linkage than saa2,3gal linkage, and a549 displayed slightly higher expression of both receptors compared to pbecs. despite this human and avian influenza virus replicated to similar titre at 15,000 pfu/ml in both cell lines, but showed low replication in pbecs. background treatment of community-acquired pneumonia remains based on 'best guess' empiric algorithms because of the poor utility of current pathogen tests. furthermore our ability to stratify patients into risk groups is crude at best, relying on scores such as the pneumonia severity index or the curb-65 have major limitations. we have been slowly improving real-time pcr assays for pneumococcus as a clinical tool in patients with pneumonia. methods building on previous research we assesed two targets in the autolysin (lyta) gene and the pneumolysin (ply) gene of s.pneumoniae using the lightcycler instrument and fluorescence resonance energy transfer (fret) probes. all common s. pneumoniae serotypes were detected while other bacteria and viruses were not. the lyta target had the best sensitivity with a detection range between 21 ng to 21 fg. both assays were then applied to whole blood samples from 400 adult patients with community-acquired pneumonia, all of whom had blood cultures prior to antibiotic administration and urinary antigen testing for s.pneumoniae. the lyta pcr had the best performance characteristics with a sensitivity more than twice that of blood cultures in the clinical samples. most pcr+ve/culture -ve patients had positive urinary antigen tests. there was clinical evidence that urinary antigen +ve/ pcr -ve patients were false +ves. most significantly there was a strong correlation between quantitative bacterial count and clinical outcome. conclusions real-time quantitative pcr for pneumococcus has significant potential as both a diagnostic and therapeutic tool in patients with pneumonia. the pitjantjatjara lands are situated in the north-western corner of south australia, occupying an area of over 120 000 square kilometres with a population of approximately 3000. the population lives in small communities or homelands, and there is a high level of mobility between this region and other aboriginal communities in south australia and the northern territory. nganampa health council provides all health care services to the region. specialized support for tb control comes from both the south australia tb service based at royal adelaide hospital as well as a centre for disease control in alice springs. the prevalence of tuberculosis (tb) in this predominantly indigenous community is thought to be significantly higher than the national rate. there are considerable challenges in detecting and managing tuberculosis, relating to the community's geographical remoteness, migration of populations and access to health services. the aims of this study are to quantify the prevalence of tuberculosis in the pitjantjatjara lands, and describe the significant barriers to tb diagnosis and treatment. methods a retrospective study of all diagnoses of tuberculosis within the pitjantjatjara lands in the period 1995-2006. outcomes include measures of tuberculosis diagnosis, the rates of completed tb treatment and rates of tuberculosis drug resistance. the study will draw conclusions about the reasons for high levels of tb prevalence in this community and identify barriers to effective tuberculosis treatment. conflict of interest no. patients admitted to hospital with a diagnosis of community-acquired pneumonia (cap) are usually treated with intravenous (iv) antibiotics irrespective of pneumonia severity. available guidelines vary in recommended timing and indications for switching to oral antibiotics. aim to examine the patterns of antibiotic choice and delivery method (iv, oral and time to switch) in patients admitted with cap. methods a retrospective chart review of admissions to the respiratory unit over a 12-month period with a diagnostic-related group (drg) coding of pneumonia. 41 charts were reviewed. data collected included patient demographics, clinical features at presentation (temperature, pulse rate, respiratory rate, bp, oxygenation), initial investigations, initial antibiotic regime, time to change (iv to oral), subsequent antibiotic regime and duration, time to defervescence, length of stay and outcome. pneumonia severity was calculated using the revised british thoracic society system (curb-65), score ն 2 = severe. results 3 patients were excluded due to incorrect coding. of the 38 patients, age was 50 ϯ 21 (mean ϯ sd) yrs and 25 (66%) were male. 28 patients (74%) were febrile at presentation and the median curb-65 score was 1 (range 0-4). 37 patients (97%) received iv antibiotics. the curb-65 score was 0 or 1 (non-severe) in 25 patients and 22 of these patients received a combination of iv ceftriaxone and a macrolide. time to defervescence was 2.9 ϯ 2.3 days. time from defervescence to switching to oral therapy was 3.4 ϯ 2.8 days. in non-febrile patients, time to switch was 4.7ϯ4.3 days. length of stay was 8.7ϯ13.0 days. conclusions the time between defervescence and switch to an oral regime was relatively long, possibly contributing to an increased length of stay. many patients received ceftriaxone even with a curb-65 severity rating of 0 or 1. implementing local guideline-based treatment protocols may reduce length of stay. ultrasonic flow sensors can determine flow, volume and molar mass (mm) of the gas flow simultaneously. during tidal breathing the expired molar mass curve can be used to compute co2 over expired volume and a capnography index (cpi) can be computed. the relationship between cpi and copd classification according to gold was investigated. methods prospective, controlled trial. consecutive patients who underwent routine lung function were enrolled to participate in a tidal breathing test using an ultrasonic flow sensor. each test consisted of three tidal breathing recordings of 60 sec. flow, volume and molar mass were measured at 200 hz and data were acquired using prototype wbreath data acquisition software. mean expirograms (mm over volume) were computed and the measurements were analyzed to determine the slope of exhaled phase ii (s2), the slope of phase iii (s3) and the relationship between s2 and s3 (cpi = s3/s2). gold stages were determined from the lung function results and the ers predicted values. results 53 volunteers participated in the study with a mean age of 62 (sd 14), 23 were male, mean bmi 26 (sd 5), 17 had never smoked. the mean pack/year smoking history was 38. there was a clear relationship between gold stage and cpi: gold stage 'normal' had a mean cpi of 5.5 (sd 3.7, n = 21), stage 'severe' had a mean cpi of 13.7(sd = 3.9, n = 7). conclusion computation of cpi based on tidal breathing analysis using an ultrasonic flow and mm sensor correlates well with gold stages. it may therefore be possible to use a simple tidal breathing test to determine the severity of airways disease. background osa is common in tetraplegia and appears within weeks of injury. although cpap treatment is efficacious in able-bodied subjects, case series suggest that cpap is poorly tolerated in tetraplegia. no prospective study has examined cpap efficacy or adherence in tetraplegia. aim to determine the feasibility of cpap use to treat osa following acute tetraplegia. methods all acute admissions who consented and fulfilled the inclusion and exclusion criteria underwent full, portable polysomnography. those found to have an apnoea hypopnoea index of >10 events per hour (osa) were offered cpap, delivered via an auto-titrating device. results to date, 25 patients have been admitted (11 excluded, 3 refused consent). no significant, adverse events have been observed. two patients did not have osa. of the nine with osa, four are mid-study, two had incomplete follow-up (1 returned to uk and 1 refused 3 month assessment), two adhered with cpap and one did not due to severe, pre-existing nasal obstruction. preliminary analyses suggest that those who adhered to cpap had a marked reduction (80% compared with 10-40%) in sleepiness and a greater reduction in the functional outcomes of sleepiness compared to either those without osa or who were unable to use cpap. patient accrual, recruitment and completion rates are consistent with our initial estimates. study recruitment will be completed by end-october 2007. conclusion initial data suggest that auto-titrating cpap is a feasible treatment for osa in acute tetraplegia. these data will be used to finalize planning for a multi-national, multi-centre randomized controlled of therapy. this research was supported by the transport accident commission. visual recognition of cyanosis is an important clinical activity. cyanosis recognition is affected by lighting colour and there is anecdotal evidence that people with significant colour vision deficiencies (cvds) have particular difficulty. studies to date have centred on the colour change with oxygenation of isolated blood but it is not clear how this extrapolates to cyanotic patients in vivo. methods ten patients known to be chronically hypoxaemic and showing signs of cyanosis were recruited from the chronic respiratory program. ten normal subjects were recruited as controls. the spectral reflectances of their lips, nail beds and palm creases were measured using a topcon sr-3 telespectroradiometer. the patients were measured at rest and after exercise to lower their saturation by 5-10%. the chromaticities were calculated and plotted. results both groups showed a spread of colours but they fell into two distinct ranges. the colour difference between the groups lies very close to the colour confusions made by congenital cvds. within the cyanosed group, the colour shift was not tightly related to decreasing oxygen saturation. this is most likely due to interpersonal factors such as pigmentation and vascular perfusion that affect colour and the difficulties in measuring the colour of heterogeneous anatomical features. conclusions these results quantify the anecdotal difficulties in detecting cyanosis and suggest that observers with cvd would have problems recognizing the condition. the photographs obtained from this study will be used to compare the ability of subjects with and without cvd to detect cyanosis. supported by the nsw ambulance service. baroreflex sensitivity is depressed in osa patients during sleep but effects during wakefulness are less clear. we have now examined relationships between awake brs and severity of sleep disordered breathing (sdb). methods immediately prior to overnight polysomnography, continuous (5 min) beat-to-beat arterial blood pressure was measured via finger plethysmography (portapres) and heart rate via ecg in 20, supine, normotensive, untreated osa patients (17 males; age: 49 ϯ 15 years (mean ϯ sd); bmi: 26 ϯ 11 kg/m 2 ). spontaneous baroreflex sensitivity (brs) was calculated using the sequence technique. sdb was characterized as apnoea hyponoea index (events/hour) and arousal index (ai). data were analysed via mathematical modelling and unpaired t test. results brs fell with increasing ahi. patients with ahi > 30 events/hour (n = 9) had a significantly lower brs (8.1 ϯ 1.5 ms/mmhg) than those with ahi < 30 events/hour (19.8 ϯ 8.7 ms/mmhg, p < 0.001). brs was negatively related to both ahi and ai via fitted exponential functions (r 2 = 0.45 and 0.70, respectively). it is hypothesized that the analysis of morphology of the ecg waveform in combination with the heart rate patterns could lead to the possibility of detection of the start and duration of apnoea/hypopnoea events and consequently estimation of the apnoea-hypopnoea index (ahi). to the authors' knowledge the published ecg based algorithms for detecting sleep disordered breathing are only capable of minute by minute analysis rather than detection of individual respiratory events. methods changes to ecg parameters were investigated during respiratory events with no distinction made between apnoea and hypopnoea events. 632 isolated respiratory events and 1264 controls of identical duration were obtained from 7 polysomnographic studies, using a randomized procedure. features such as the r wave amplitude, t wave amplitude, qrs area and the r-r interval were extracted from the 2 lead ecg. a number of physiological predictors based on these features were generated. a logistic regression model was used to investigate the association between the predictors and true events, using the statistical software, stata. results univariate and multivariate analyses were performed. three multivariate models were developed; heart parameters only, ecg waveform morphology parameters only and the combinations of the two. the area under the receiver operator characteristic curves (auc) for these models were compared. the best results were obtained with the combination of morphology and heart rate parameters (auc = 0.8858 (0.0078 (sd))) compared to the morphology (auc = 0.8169 (0.0121 (sd))) and heart rate (auc = 0.7195 (0.0103 (sd))) models. the multivariate analysis has shown encouraging results indicating that an algorithm using a combination of heart rate and ecg morphological parameters could potentially be constructed that would enable the determination of individual respiratory events and subsequently an ahi. supported by the arc. introduction sacin and scond are measures of ventilation heterogeneity in acinar and conducting airways, derived from analysis of mbnw. maintaining tidal volumes of 1 l at 9-11 breaths/minute (bpm) is impossible for some. our aim was to examine the effect of different tidal volumes on sacin and scond in normals and asthmatics. methods 10 normals (23-41 yrs) and 12 asthmatics (21-63 yrs) underwent mbnw at tidal volumes of 500 ml at 20-23 bpm, 1 l at 9-11 bpm, and 2 l at 5-7 bpm. scond and sacin, were determined from the normalized phase iii slopes of breaths between turnovers (cumulative ventilation/frc) 1.5 & 6. results the mean ϯ sd %predicted fev1 was 97.3 ϯ 17% in normals and 88 ϯ 11% in asthmatics. in normals, sacin at tv of 0.5, 1 and 2 l were 0.195 ϯ 0.105 l -1 , 0.095 ϯ 0.036 l -1 and 0.058 ϯ 0.031 l -1 , respectively (p = 0.0003, anova), while scond were 0.098 ϯ 0.047 l -1 , 0.042 ϯ 0.021 l -1 and 0.029 ϯ 0.014 l -1 (p = 0.0002), respectively. in asthmatics, sacin were 0.440 ϯ 0.195 l -1 , 0.181 ϯ 0.087 l -1 and 0.100 ϯ 0.047 l -1 , respectively (p < 0.01), while scond were 0.204 ϯ 0.111 l -1 , 0.068 ϯ 0.037 l -1 and 0.031 ϯ 0.013 l -1 , respectively (p < 0.0001). conclusion increasing tidal volume while maintaining the same minute ventilation during mbnw led to large decreases in scond and sacin in both asthmatics and normals. this may be due to reduced inter-regional differences in specific ventilation with greater tv. the log-log relationship between sacin and tv allows an adjustment to be made for variations in tidal volume. funding crc for asthma and airways and nhmrc project grant #547346. dj smith 1 , k bowden 2 , t lloyd 2 , j coucher 2 , l garske 1 1 respiratory medicine, and 2 radiology, princess alexandra hospital, brisbane, australia introduction we have shown diaphragmatic flattening and decreased diaphragmatic excursion qualitatively assessed on ultrasound is strongly predictive of dyspnea severity and lower lung inflation in patients with pleural effusion. we sought to quantitatively measure diaphragm length and movement and determine how closely these are related to dyspnea severity and lung inflation. methods patients with unilateral pleural effusions had ct imaging of their diaphragm during a measured inspiratory capacity manoeuvre. maximal sagittal length was measured at tlc, and frc. patients had a baseline dyspnea index (bdi: 0-12) and respiratory function measured. results 4 patients with unilateral effusion (all right side; 3 malignant mesothelioma, 1 inflammatory) had a mean (sd) bdi of 5.5 (2.89), and tlc of 74% (3.91) predicted. the right diaphragm on the side of the effusion tended to be shorter than the left at frc (p = 0.08), and had a trend to reduced shortening with inspiration (p = 0.08). conclusions the right diaphragm is known to be longer than the left in health. the strong trend to a shorter and less mobile right diaphragm associated with effusion suggests this is a potential mechanism for dyspnea. further recruitment will enable correlation between bdi, tlc and diaphragm length and mobility. 4) ) that was slightly worse than an able bodied, control population (17.9 (3.1)), but better than an able-bodied population with untreated osa (14.5 (3.6)). the mapi predicted that 14% of the sample were likely to have osa. these data will be complimented by full sleep studies to be performed at the participants' homes in late 2007, early 2008. conclusion our interim data suggest that the rate of subjective sleep complaints are not substantially different in the population with tetraplegia compared with the able-bodied. this research was supported by the victorian neurotrauma initiative. it has long been assumed that the ventilation heterogeneity associated with lung disease could in itself affect the measurement of carbon monoxide transfer factor. the aim of this study was to investigate the potential estimation errors of carbon monoxide diffusing capacity (tlco) measurement that are specifically due to conductive ventilation heterogeneity. we induced conductive airway ventilation heterogeneity in 35 never-smoker normal subjects by histamine provocation, and related the resulting changes in ventilation heterogeneity (derived from the multiple breath washout test) to corresponding changes in diffusing capacity, alveolar volume and inspired vital capacity (derived from the single breath tlco method). average conductive ventilation heterogeneity doubled (p < 0.001), while tlco decreased by 6% (p < 0.001), with no correlation between individual data (p > 0.1). when dividing diffusing capacity by alveolar volume, the resulting transfer coefficient was not significantly different pre versus post histamine (p = 0.074). these findings can be brought in agreement with recent modelling work, where specific ventilation heterogeneity resulting from different distributions of either inspired volume or end-expiratory lung volume have been shown to affect tlco estimation errors in opposite ways. the combination of these errors appears to largely cancel out in our experimental situation of induced ventilation heterogeneity comparable to that observed in lung disease. we conclude that conductive ventilation heterogeneity per se has a negligible effect on diffusing capacity measurement. an important determinant of airway function in humans is vagal-mediated cholinergic tone in airway smooth muscle (asm). this airway tone may be altered in disease states. the use of mouse models for the study of airway diseases, including asthma, pulmonary fibrosis and copd is well established. however, it is not known whether mice actually possess basal asm tone or, if it does exist, how this tone changes in disease models. this study was undertaken to determine whether mice have detectable asm tone in vivo. methods respiratory system impedance (zrs) was measured in female adult balb/c mice using a wave-tube modification of the forced oscillation technique. zrs was measured during slow (~35 s) inflation-deflation manoeuvres between the transrespiratory pressures of 0 and 20 cmh2o. baseline lung mechanics and thoracic lung volumes (tgv) were measured before and after each mouse was allocated to one of four treatment groups: 'saline' mice received an i.p injection of saline, 'atropine' mice received i.p. atropine sulphate, 'vagotomy' mice had their left and right cervical vagus nerves isolated by blunt dissection and cut, and 'sham' mice had the area of the vagus nerves exposed but the nerves were not cut. results there were no post-treatment changes in tgv, airway resistance, tissue damping, tissue elastance, inertance or tissue hysteresivity in any of the four groups. conclusions the lack of change in lung mechanics post-atropine or postvagotomy in balb/c mice suggests that, unlike humans and many other species, the airways of mice have no baseline asm tone. supported by nhmrc grant#11488. nomination none. conflict of interest none. both male gender and increased mandibular enclosure volume predict more severe sleep disordered breathing in obstructive sleep apnoea patients. we now examine gender/body size/mandibular enclosure volume relationships for normal subjects stepwise multiple linear regression analysis was used to model body size/enclosure volume interactions. results for the whole group, mv was 261.1 ϯ 6.0 ml (mean ϯ se) while rmv was 205.1 ϯ 4.9 ml. head circumference (positive) and forehead height (negative) were both independent predictors for mv and rmv (both p < 0.02), while hip circumference was an additional positive predictive factor for rmv (p < 0.04). after adjusting for these parameters, male mv and rmv were larger than for females conclusion these findings suggest that mandibular enclosure volumes are relatively larger in males, even after adjusting for body size/cranial dimension. differing body size/mandibular enclosure volume interactions may contribute to gender influences on the severity of sleep disordered breathing. supported by nhmrc of australia nomination john read prize for sleep and physiological research tp 027 audit of ctpa in a regional hospital y raje, s vincent, g simpson department of thoracic medicine, cairns base hospital, cairns, qld 4870 since the introduction of computerized tomographic pulmonary angiograms (ctpa) at our institution the number of requests for this investigation at our institution has grown at an alarming rate. the purpose of this study was to evaluate the clinical assessment of suspected pulmonary embolism (pe). methods 50 ctpa were reviewed. results 31 female, 19 male. mean age 50 yrs (range 21-87). 26 ctpa requests came from department of medicine, 21 from emergency department, 2 from surgical teams and 1 from oncology outpatients. 36 patients presented with chest pain (pleuritic in 20 cases), 25 had dyspnea, 7 presented with collapse. 4 patients had haemoptysis. hypoxaemia was recorded in 7. none were clinically shocked and only one had a recorded tachycardia. d-dimer requested in 10 patients and was elevated in 9. arterial blood gases performed in only 10 patients (20%). 47 patients had prior chest x-ray which was normal in 24 (48%). 8 patients had consolidation on chest x-ray, 2 pleural effusions, 2 atelectasis and 1 fractured ribs. recorded risk factors included 4 patients with previous dvt or pe, 4 patients with malignancy and 6 patients were immediately post-operative. only 6 ctpas (12%) demonstrated evidence of pe. of these 2 had recent dvt and 2 were post-operative. 1 had a history of bowel cancer. there was no formal record of pre-test clinical probability of pe (eg wells' score) for any of the 50 cases. retrospective calculation of the cases of pe, 4 had a wells' score of 4.5 and 1 of 4 with the remaining patient with wells' score of under 2. only 3 patients (one with clinically probable pe) had received fractionated heparin prior to the ctpa. conclusion (1) ctpas performed at our institution have a low yield (12%).(2) pre-investigation clinical assessment was poor and there was poor adherence to published guidelines, (3) this results in many unnecessary ctpa examinations generating increased work and expense for the medical imaging department and exposes many patients to unnecessary and potentially harmful radiation exposure. the evaluation and management of hereditary hemorrhagic telangiectasia involves a multidisciplinary approach according to international guidelines. the aim of this audit was to compare the assessment process in one centre with that of the international recommendations. methods retrospective comparison was made by medical chart review of all patients with a diagnosis of hht between the years 1994 to 2006. demographic along with clinical data with diagnostic investigations, complications, treatment and genetic evaluation, including family screening was collected. the proportion of patients evaluated and managed as per the international recommendations was determined. results the audit identified 26 patients with the diagnosis of hht, with the mean age 58 years. diagnostic criteria were met in 77% of the cohort. of the known clinical features, 54% had a family history, and 81% epistaxis. cutaneous telangiectasia was present in 85% and visceral involvement in 92%. pulmonary arterio-venous malformations (pavm) were seen in 16 patients, cerebral avm in 4, gastrointestinal telangiectasia was documented in 8. one patient had a spinal (cervical) avm, and another had pulmonary hypertension in association with this condition. only 8 patients underwent diagnostic or screening investigations in accordance with the international recommendations. furthermore, one patient was referred for a genetic evaluation. conclusions this clinical audit found that 31% of patients referred to this centre were evaluated in accordance with the international recommendations. genetic assessment was lacking. the study supports the need for a coordinated, multidisciplinary approach to the evaluation and management of hht in this centre. lm young 1 , n good 1 , d milne 2 , w fergusson 1 , i zeng 1 , j kolbe 1 , ml wilsher 1 background while airflow limitation is the most common physiological impairment in sarcoidosis, there are limited data on airway hyperresponsiveness (ahr). understanding the role of ahr in sarcoidosis, if any, may help to identify individuals who might benefit from inhaled therapies. aims (1) to determine the prevalence of ahr in sarcoidosis. (2) to determine the correlation between responses to direct (using histamine) and indirect (using hypertonic saline) bronchial challenge. (3) to determine the clinical, physiological and radiological predictors of ahr. methods subjects with a diagnosis of sarcoidosis based on typical clinical presentation and compatible hrct features and/or tissue biopsy and with a baseline fev1>35% predicted were recruited. subjects underwent standard hypertonic (15% fall in fev1) and histamine (20% fall in fev1) challenge (>1 day but <7 days apart), lung function testing and high resolution computed tomography (hrct) of the chest. results the 52 subjects (48 ϯ 11 years, 35% female, 92% european, 35% stage i, 25% stage ii, 40% stage iii, 0% stage iv) had well preserved lung function overall (fev1 = 2.8l ϯ 0.7.87% predicted). ahr was detected in 5/47 (11%) to hypertonic saline and 19/43 (44%) to histamine challenge. on univariate analysis, response to histamine challenge was predicted by conglomerate fibrosis (p = 0.002) and reticular pattern (p = 0.05) on hrct. the baseline % predicted fev1 was significantly associated with ahr on univariate (p = 0.004), and multivariate analysis (p = 0.01) when adjusted by hrct patterns. conclusions there is a high prevalence of ahr using histamine challenge in this study of sarcoidosis subjects. ahr most strongly associates with baseline % predicted fev1 but also conglomerate fibrosis and reticular pattern on hrct. these findings may reflect the consequence of airway remodelling following inflammation. further studies are warranted to confirm these findings. background upper airway shunt represents a significant source of measurement artefact in the use of the forced oscillation technique (fot), with increasing importance in young children. changes in respiratory system admittance, ars (or zrs -1 ), are theoretically independent of the upper airway shunt. this study examines the possible clinical benefit of ars in preschool children by assessing any increased ability to differentiate responses to bronchial challenges in the routine clinical setting. we hypothesized the use of ars would provide improved sensitivity to clinically relevant obstruction, bronchodilator responsiveness (bdr) and airway hyper-responsiveness (ahr) in young children with respiratory disease. method previous fot measurements were re-analysed and ars calculated to derive: (1) ars reference equations in healthy young children (n = 158); (2) bdr in ars, respiratory system resistance (rrs) and reactance (xrs) in healthy children (n = 78), children with cystic fibrosis (n = 39), neonatal chronic lung disease (n = 49), asthma (n = 56) and wheeze (n = 66); (3) ahr to inhaled adenosine-5′-monosphate (amp) in 19 children. fisher's exact tests were used to assess changes in diagnostic outcomes between ars and conventional fot outcomes (rrs and xrs). results ars was no more sensitive to bronchodilator induced changes than conventional fot outcomes. amp challenges resulted in equivalent responses measured by relative changes in rrs and ars while absolute changes in ars were the least sensitive variable. conclusion this study does not support a clinical advantage in using ars in measuring responses to either inhaled bronchodilator or amp. c hollier 1,2 , c menadue 1,2 , d flunt 1,2 , aj piper 1,2 1 department of respiratory and sleep medicine, royal prince alfred hospital, nsw 2050, and 2 woolcock institute of medical research, nsw 2050 serial measurement of arterial carbon dioxide (paco2), ph and bicarbonate (hco3 -) is essential in the management of patients with hypercapnic respiratory failure (hrf). this information is usually obtained from a sample of arterial blood (abg). the procedure can be painful and distressing for patients, and is sometimes technically difficult due to obesity or contractures. our aim was to determine the validity and feasibility of arterialized venous blood (av) sampling as an alternative to abgs in measuring paco2, ph and hco3levels in patients with chronic hrf. method eighteen patients completed the study. venous blood was arterialized by heating forearm skin to a temperature of 42-45°c with an electric heating pad. an av sample was taken from a cannula positioned in a vein of the heated forearm simultaneously with an abg. in addition, the reliability of av sampling within the recommended temperature range (42-45°c) was investigated in ten healthy volunteers placed on volume cycled ventilation in order to maintain constant ventilation. av samples were taken at 0.5°c temperature intervals from 42.5-45°c results the table below summarizes results for validation of av sampling: based on the evidence that cardiovascular dynamics are altered due to obstructive sleep apnea, this study aims to identify the onset and termination of each apnea event using power spectral density (psd) and morphological features of single lead ecg signal over 5 second period. methods ecgs from 4 patients overnight sleep studies were examined for location of the pre-scored apnea events. onset (n = 1995), maximum (n = 6751) and termination (n = 1996) of each apnea event and normal events (n = 11219) were annotated on 5 second windows. features extracted were psd, amplitudes of r and t wave of 5 second ecgs. receiver operating characteristics (roc) analysis was used to gauge the event recognition ability of all features. weight loss causes an improvement in the severity of osa, however substantial weight loss is very difficult for obese patients. the very low caloric diet (vlcd) has been shown to be successful in causing significant weight loss in obese patients. this is a pilot study on the use of a formal screening protocol to identify osa patients who are potentially eligible for the supervised vlcd program offered by the endocrinology department at auckland city hospital. method 344 consecutive patients who attended the sleep laboratory at ach between june to december 2006 were screened using the protocol. patients who are eligible to be considered for the vlcd program are identified as having a combination of obesity (bmi > 30), osa (ahi > 5 on sleep study) and being residents within the auckland district healthboard region. results 243/ 344 patients screened did not fulfil the inclusion criteria: 171 lived outside the adhb region; 71 had bmi < 30; 7 patients did not have osa (ahi < 5). 101 patients fulfilled the inclusion criteria. 54/101 patients (54%) were excluded due to medical or psychiatric contraindications to vlcd.47 patients (47%) who did not have contraindications to vlcd were contacted. 33 patients were contacted successfully. 14 patients were either unavailable to phone contacts on 3 separate days or were disconnected. 12/101 patients consented to being referred (12%). 21/101 patients declined referral (21%). conclusion this pilot study is the first study using a formal comprehensive screening protocol in the recruitment of obese osa patients into a medically supervised vlcd program. only a small proportion (12%) of patients proceeded to being referred to the vlcd program. key: cord-016690-3gsq724l authors: li, hongjun title: hiv/aids related respiratory diseases date: 2013-09-30 journal: radiology of hiv/aids doi: 10.1007/978-94-007-7823-8_17 sha: doc_id: 16690 cord_uid: 3gsq724l lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging findings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging fi ndings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. pneumocystis has been believed to be a kind of protozoon. recently, based on its ultrastructure and ribosomal rna phylogenetic analysis, pneumocystis is now believed to be a kind of fungus, with high affi nity to the lung tissues. due to the compromised immunity, 95 % aids patients sustain different types of pulmonary infections, of which pcp is the most common life-threatening opportunistic infection with an incidence rate of about 60-85 %. about 90-95 % patients suffering from aids complicated by pcp are adolescents and adults with their cd4 t cell counts being less than 200/ μl. clinical manifestations of typical pcp are fever, cough (dry cough without phlegm), dyspnea, chest distress and shortness of breath. dyspnea is shown as progressive difficulty in breathing, which initially occurs after physical activities and develops into diffi culty breathing even in resting state. pcp is commonly accompanied by weight loss, fatigue, anemia, general upset and lymphadenectasis. all these symptoms are non-specifi c, but patients often report subjective feelings of severe symptoms while physical signs are mild. by auscultation, the lungs are normal or with slightly dry, moist rales. these are the clinical fi ndings characteristic to aids complicated by pcp. in most patients with pcp, the serum ldh level increases but it is non-specifi c. in cases of aids complicated by pcp, the blood po2 reduces, commonly being lower than 70 mmhg in patients in the middle and advanced stages. the diagnostic imaging for pcp includes chest x-ray and ct scanning. due to the low resolution of chest x-ray, its demonstrations are negative for pcp patients in the early stage or only include thickened pulmonary markings and decreased pulmonary transparency. however, ct scanning demonstrates tiny lesions or more detailed changes in lungs. especially with the application of hrct, the detection rate of pcp lesions has been greatly improved. it has been internationally reported that nearly 10 % of pcp patients show negative fi ndings by the chest x-ray but with abnormal fi ndings by hrct. due to the rapid progression of pcp as well as its complex pathological changes, ct scanning demonstrations are diverse with specifi city. according to different pulmonary ct scanning demonstrations in different stages of the illness, pcp is divided into early stage (exudative and infi ltrative stage), middle stage (fusion and parenchymal stage) and advanced stage (absorption or fi brosis stage). the early typical manifestations include intrapulmonary multiple miliary nodules, mainly distributed in both middle and lower lung fi elds. it may be accompanied by enlarged hilar shadow, which should be differentiated from acute miliary tuberculosis. the middle stage is a period of infi ltration. as the disease progresses, miliary and patchy shadows fuse and expand into a dense infi ltrative shadow with even density, showing a diffuse ground glass liked change. the typical manifestations include bilaterally symmetric foci with the hilus as the center. the foci infi ltrates from the hilus to bilateral pulmonary interstitium, progressing from the both middle lungs to both lower lungs. hrct can more clearly demonstrate the foci, showing a map liked or gravel road liked appearance, with clearly demonstration of gas containing bronchus penetrating the foci. the pulmonary apex is involved later. the exterior stripe of the lung fi eld has increased transparency, showing typical willow leaf sign or moon bow sign which is the manifestation of compensatory pulmonary emphysema. during the late compensatory repair period, the intrapulmonary lesions are mainly parenchymal changes and fi brosis, with large fl aky high density shadows as well as cords liked and reticular changes. pneumothorax, mediastinal emphysema, pneumatocele, pleural effusion and other complications may occur, with an incidence of pneumatocele in about 10-20 % patients. the autopsy grossly demonstrates swelling of the lung tissue, and the alveoli are fi lled with large quantity foamy liquid. the pathological changes mainly manifested as interstitial pneumonia, with early manifestations of increased permeability of the capillary wall basement membrane in the alveolar walls, which leads to fl uid exudation. the pneumocystis carinii proliferate in large quantity and adhere to cause degeneration of the type i alveolar epithelial cells and shedding of the basement membrane. vascular congestion, edema as well as infi ltration of lymphocytes, plasma cells and mononuclear cells can be found in the pulmonary interstitium. due to the extensive existence of aspergillus in natural world, sputum smear positive often fails to defi ne its invasive infection. in the cases with aspergillus infection, hyphae can be found in the sputum. fungal infections often occur in patients with cd4 t cell count below 100/μl, of which the most common pulmonary infection is aspergillus infection, followed by penicillium marneffei infection. pulmonary infections caused by candida albicans and histoplasma are rarely found. the incidence of cryptococcal pulmonary infection is still in a disagreement, which is increasing recently. there are also some common endemic fungal infections, such as the most commonly found fungal infections of aids complicated by penicillium infections in guangxi zhuang autonomous region and hong kong, china. aspergillus has an extensive existence in the natural world. aids complicated by aspergillus infection is related to the application of corticosteroid hormone or broad-spectrum antibiotics, which occurs commonly in the advanced/critical stage of aids. the cases of pulmonary fungal infections, with fi ndings of hyphae (aspergillus or candida) or yeast in cytoplasm (histoplasma capsulatum) in tissue sections and simultaneous fi ndings of histiocytic reactions including the infi ltration of neutrophilic granulocyte and the necrosis of histocytes, can be diagnosed as having invasive fungal infection. candida albicans is yeast liked fungus, which is widespread in the natural world. it can parasitize in the mocous of skin, oral cavity, intestinal tract and vagina of the human being. candida albicans cannot cause disease in immunocompetent people but is pathogenic in immunocompromised population. after its invasion into the tissues, it turns into mycelia and multiplies in large quantity with great toxicity. it also has the ability to fi ght against phagocytosis. clinically, its infection is characterized by a chronic onset and clinical symptoms of low and moderate grade fever but rarely high fever, cough, shortness of breath, cyanosis, irritation or dysphoria. the pulmonary signs include weakened breathing sounds by auscultation and obvious moist rales of lungs. the serious cases may have symptoms of systemic poisoning. the illness is prolonged and repeated during its whole progression. by diagnostic imaging demonstrations, it can be divided into the following types: (1) bronchitis type, with chest x-ray demonstrations of increased pulmonary markings in lower fi elds of both lungs; (2) pneumonia type, commonly with accompanying extrapulmonary lesions. the lesions are mainly located in the middle and lower lung fi elds and lesions in the lower lung fi eld are more common. the apex is generally not involved. the lesions are recurring one after another. a small number of patients may sustain complications of exudative pleurisy. (3) disseminated type, with miliary shadows, diffuse nodular shadows or multiple small abscesses. the lesions often involve the middle and lower lungs. chest x-ray demonstrates thickened pulmonary markings and accompanying spots, small fl akes and large fl akes of parenchyma shadows, in manifestations of bronchial pneumonia. in some serious cases, the foci may fuse together and enlarge to involve the entire lobe. ct scanning demonstrates pulmonary nodules and few have ground glass liked changes of the lungs. pathological changes include acute infl ammatory lesions in the lungs, alveolar exudation and infi ltration of monocytes, lymphocytes and neutrophils. acute disseminated lesions often cause multiple small abscesses, central caseous necrosis, spores and hyphae in and around the lesions. histoplasma capsulatum belongs to moniliales family, deuteromycetes class and fungal kingdom, whose growth requires organic nitrogen. it is often isolated from the soil with abundant contents of birds or bats faeces and spreads along with chickens, birds, dogs, cats, and mice. when the conidia and mycelial fragments of histoplasmosis are inhaled, most can be expelled by the defense mechanism of the human body. granulomas may form, but in immunocompromised patients, it may cause disseminated histoplasmosis. when the cd4 t cell count in aids patients is less than 150/μl, histoplasma capsulatum infection of lungs may occur. histoplasma capsulatum pneumonia has a higher incidence in south america, africa and india. in the slight cases, the clinical manifestations are similar to symptoms of the cold, with low-grade fever, cough, and general upset. in the serious cases, there are symptoms of infl uenza, including chills, high fever, cough, chest pain, dyspnea, fatigue and poor appetite. in the cases of acute cavity, thin-walled cavity may form within a month. complications may be pericarditis, arthritis, skin nodules, rash fi brous mediastinitis and mediastinal granuloma. diagnostic imaging demonstrations are non-specifi c, with scattering pulmonary acinus exudation, multiple nodules in a diameter of about 3 mm with accompanying thickened septa, and formation of granulomas with accompanying calcifi cation. it should be differentiated from bacterial pneumonia, tuberculosis and other pulmonary fungal infections by laboratory tests to defi ne the diagnosis. the specifi city of the glycogen antigen detection of histoplasma capsulatum is up to 98 %. mucor spreads through the respiratory tract. it commonly invades the blood vessels, especially arteries. it reproduces locally or causes thrombosis and embolism. clinical manifestations are high fever, cough, sputum, shortness of breath, chest distress, chest pain and hemoptysis (pulmonary artery involvement). the diagnostic imaging demonstrates fl akes infl ammatory foci, with manifestations of pulmonary cavity and pulmonary infarction. the pathological changes are hemorrhagic infarction of local tissue, pneumonia and exudation of neutrophils. hemorrhagic infarction of local tissue may be related to hyphae induced minor arteries lesions. it is estimated that one third of the world population was/is infected with tubercle bacillus and 9 % of them are aids patients. the who reported that there are 88,000 newly infected patients of tb each year and 8.4 % of them are caused by aids. it is estimated that each year in 1,000 hiv infected patients, 35-162 sustain active tb, and there is a great risk of active tb progressed from the latent tuberculosis infection. hiv infected patients with tuberculosis are commonly young and middle aged adults, with more male patients than female patients. tuberculosis can occur at any stage of aids and at any level of cd4 t cell counts. it has been internationally reported that hiv infection complicated by tb has no specifi c imaging demonstrations. it has an acute onset, with an incidence of acute onset 2.5 times as high as that in non-hiv infected patients. the lesions are morphologically diverse, which are different from non-hiv infected patients with tb. hiv infection complicated by tb has commonly an acute onset, while tb in non-hiv infected patients is commonly secondary to other lesions, with cavities, fi brosis, pleural thickening and calcifi cation. a study conducted in china has demonstrated that for aids complicated by tb, the acute cases mainly have military and exudative lesions, with an incidence of 33 and 49 % respectively; while the incidence of chronic cases including cavity, fi brosis and calcifi cation is declining, being 11, 11 and 2 respectively. a later occurrence of tuberculosis in hiv infected patients indicates a more seriously immunocompromised immunity, with less typical clinical manifestations and imaging demonstrations. when the cd4 t cell count level is above 350-400/μl, the systemic symptoms are fever, chills, night sweating, fatigue, poor appetite and weight loss. respiratory symptoms are cough, expectoration, hemoptysis, chest pain and dyspnea. it manifests as primary tuberculosis, with its foci distributing in the middle and lower lungs, involving multiple lobes and segments. when the cd4 t cell count decrease, the impact of tb increase including the occurrence of extrapulmonary tuberculosis and disseminated disease. when the cd4 t cell count drops below 200/μl, pulmonary tuberculosis manifests as acute onset (such as miliary tuberculosis) or extrapulmonary tuberculosis (such as ileocecal tuberculosis) and peripheral lymph nodes tuberculosis. its difference from the clinical manifestations of non-hiv infected patients is as the following: (1) more common pulmonary infi ltration with multiple involvements and rare cavities; (2) higher incidence of dissemination (87-96 %) commonly along with blood fl ow and higher incidence of extrapulmonary tuberculosis (60-70 %); (3) more common lymph node tuberculosis, such as hilar, mediastinal and extrapleural lymphadenectasis; (4) lower positive rate of tuberculin test (ppd); (5) more patients with no expectoration, with sputum smear for acid-fast bacilli staining is negative; (6) higher incidence of resistant strains, high recurrence rate, and higher mortality (table 17 .1 ). foci in the cases with aids complicated by pulmonary tuberculosis are change quickly. after anti-tb treatment, the lesions are absorbed quickly. those receiving no anti-tb therapy, the foci tend to fuse together to form a mass or diffusely distribute. bacterial septicemia often occurs in aids patients. many opportunistic pathogens can cause respiratory infections, including bacterial bronchitis, pneumonia and pleuritis. the incidence rate of bacterial pneumonia (bp) is 3-5 %. bp has a larger range of impact on hiv infected patients than on non-hiv infected groups. repeated episode of bp is considered to be the fi rst manifestations of latent hiv infection. therefore, for those individuals who have recurrent pneumonia without other risk factors, they should be alert to hiv infection. the common pathogenic bacteria include streptococcus pneumoniae, staphylococcus aureus, rhodococcus equi, haemophilus and pseudomonas aeruginosa. as non-hiv infected patients, the most common pathogens of pneumonia are streptococcus pneumoniae and haemophilus infl uenzae. legionella and klebsiella are also common. many factors, such as the reduced t lymphocytes in hiv infected patients, manufacturing disorders of neutrophils, mononuclear cells and cytokines, and dysfunctional b lymphocytes, provide chances for opportunistic bacterial infections. in addition, the application of broad-spectrum antibiotics also increases the chance of opportunistic infections. bp can occur in any stage of hiv and at any level of cd4 t cell count. when the cd4 t cell count decreases, the incidence of bp also increases. the clinical manifestations of hiv infected patients are the same as non-hiv infected patients, with acute onset (3-5 days) , high fever (39-40 °c) , chills, chest pain, dyspnea, cough, purulent sputum (bloody or rusty). being different from non-hiv infection, pulmonary infection in hiv-infected patients is often recurrent. the imaging demonstrations of hiv/aids related bacterial pneumonia are similar to those in non-hiv infected patients. most cases of streptococcal pneumonia and haemophilus pneumonia have unilateral, confi ned and partially fused foci with accompanying pleural effusion. the imaging demonstrations include thickened and deranged pulmonary markings, alveolar fi lling of infl ammatory exudates with the progression of the illness, large fl aks infl ammatory infi ltration shadows or parenchymal shadows, bronchial gas fi lling phase in the parenchymal shadows. the lesions distribute along the pulmonary segments or lobes, rarely with accompanying pleural effusion. during the absorption period, the density of the parenchymal shadows gradually reduces and the scope narrows down. there may be cavities in some individual cases. but in most cases it is completely absent after poor effi cacy and more side effects favorable effi cacy and less side effects 3-4 weeks. lesions absorption are slow in elderly patients and recurrent patients, which is diffi cult to be completely absorbed and often develop into organic pneumonia. rhodococcus equi was initially discovered in 1923 and nominated as corynebacterium equi. after structure analysis of the cell wall, it was found to be different from corynebacterium, and therefore it is classifi ed into rhodococcus. rhodococcus equi is generally considered as the pathogens of horses, pigs and cattle. human rhodococcus equi infection is rare. but in recent years, due to an increase in patients with immunodefi ciency syndrome, reports of rhodococcus equi induced human respiratory infection and sepsis are increasing. rhodococcus equi is an intracellular facultative parasitic bacterium. its optimum temperature for growth is 30 °c, and suitable temperature for its growth is 10-40 °c. the acid-fast staining for rhodococcus equi shows uncertain results. due to its various morphology, it is commonly mistaken as diphtheroids bacilli, bacillus or micrococcus. on sheep blood agar plate, the bacterium can have synergistic hemolysis with staphylococcus aureus, mononuclear listeria and corynebacterium pseudotuberculosis. toxicity mechanisms of rhodococcus equi has been recently discovered the existence of toxic plasmid, which provides a new idea for the full understanding of its pathogenesis. clinical symptoms are usually cough, orange red sputum, high fever and other symptoms. e marchiori et al. in 2005 studied fi ve cases of aids complicated by rhodococcus equi pulmonary infection. all the patients had a case history of cough and fever history for 1-2 months with accompanying shortness of breath and chest pain. li et al. in 2009 [ 106 ] studied a group of 13 cases. all patients had fever, with a body temperature being 38-40 °c, cough, orange red sputum. the typical clinical manifestations of the disease are fever, dyspnea and chest pain. other symptoms such as weight loss, diarrhea and joint pain are not representative. based on the course of the disease, the diagnostic imaging demonstrations of rhodococcus equi pulmonary infection can be divided into early stage, showing round liked fl aky blurry shadows surrounding unilateral hilum that has blurry boundary; middle stage (parenchymal change), showing central sphere liked high density shadow surrounding unilateral hilum, in parenchymal changes and with clear boundary; advanced stage (necrosis) showing secondary cavity of the pulmonary mass, possibly with hydropneumothorax and pleurisy. the imaging demonstrations are characteristic, but lack of specifi city. and it should be differentiated from pulmonary tumors. the pathological changes include the most commonly chronic suppurative bronchopneumonia and extensive pulmonary abscesses. the histopathology demonstrates massive bleeding in alveolar space, large quantity erythrocytes, intact cellular wall and large quantity epithelial cells. the predominant pathological changes may also be fi broblasts, with parenchymal changes of lung tissue and thickened alveolar septa. accumulating piles of strip liked purple rhodococcus equi can be found by pas staining. common pathogenic viruses of the opportunistic pulmonary infections in hiv infected patients are cytomegalovirus (cmv) and infl uenza virus. cmv is the most common pathogen of hiv/aids related pulmonary infection. by autopsy, 49-82 % patients with hiv/aids have cmv infection, only second to pneumocystis carinii pneumonia. moskowitz et al. [ 32 ] reported that among the direct causes of death in aids patients, 19 % is due to pulmonary cytomegalovirus infection. because of the lack of typical clinical manifestations and sensitive examinations for its early diagnosis, the defi nitive diagnosis rate of cytomegalovirus pneumonia is only 13-24 % before autopsy. the clinical manifestations of cmv infection are non-specifi c. the systemic symptoms are fever, soreness of joints and muscles. respiratory symptoms are paroxysmal dry cough, progressive shortness of breath, diffi culty in breathing during activities. pulmonary cmv infection may develop secondary fungal infection or be complicated by bacterial, fungal, and pneumocystis carinii infections. the cytomegalovirus can widely spread in the organs and tissues of the infected patients, and the infections can directly lead to the damage of infected host cells. in addition, the virus can also cause pathogenic effects via immune pathological mechanism. some scholars classifi ed cmv pneumonia into diffuse, miliary necrosis and cytomegalic. diffuse and cytomegalic cmv pneumonia are often accompanied by diffuse alveolar damage (dad), which is more common in the diffuse type of cmv pneumonia but less common in cytomegalic type of cmv pneumonia. the pathological basis of diffuse small nodules in lungs is hemorrhagic necrosis. sometimes cmv infection is concurrent with other infections in the lungs, and even co-infects one cell. pulmonary parenchymal changes indicate bacterial and fungal infections, such as fi ndings of inclusion bodies in the cells, commonly known as eagle's eye sign. the imaging demonstrations of cytomegalovirus pneumonia are diverse. some studies summarize that the lungs commonly have manifestations of diffuse interstitial infi ltration or alveolar infi ltration, with ground glass liked changes, pulmonary parenchymal changes, grid liked changes, thickend bronchial wall, bronchiectasis, pulmonary nodules or masses. the principal changes include the early lesions of ground glass liked changes and advanced lesions of pulmonary masses. lymphoid interstitial pneumonia is the abnormal hyperplasia of the pulmonary lymphoid tissue. its occurrence is related to autoimmune diseases, and is believed to be a direct response of the lungs to hiv. the clinical manifestations are recurrent infections, poor appetite, hepatomegaly and splenomegaly, and arrested development. the diagnostic imaging demonstrates no characteristic changes by ct scanning, with thickened bronchial wall, diffuse central lobular nodules or bronchiectasis, grid liked and cords liked shadows in uneven thickness. the pathological changes include accumulating lymphocytes and plasma cells that are mixed to infi ltrate the pulmonary interstitium and expand to surrounding areas of the bronchi. toxoplasma pneumonia is caused by the infection of the intracellular parasite, toxoplasma gondii. ludlam et al. in 1963 fi rstly proposed the concept of pulmonary toxoplasmosis, which was believed to cause atypical pneumonia [ 107 ] . the clinical manifestations are cough and expectoration. in the serious cases, dyspnea and cyanosis can occur. in the chronic cases, there are long term low grade fever, cough, weight loss and enlarged lymph nodes. the diagnostic imaging demonstrates bronchopneumonia, interstitial pneumonia and pleurisy. (1) bronchial pneumonia is also known as lobular pneumonia, with scattered patchy and blurry density shadows. (2) interstitial pneumonia has typical manifestations of reticular and nodular shadows. (3) pleurisy is rare, showing pleural effusion, limited diaphragmatic activity. the imaging demonstrations are non-specifi c, which can be defi ned in combination with the etiologic examinations. the pathological changes are congestion and edema of the surrounding connective tissue of the alveolar wall and bronchial walls, widened pulmonary interstitium, small quantity serous fi brin exudation from alveoli and pulmonary interstitium, and infi ltration of macrophages and lymphocytes. toxoplasma cysts and tachyzoites may be found in pulmonary interstitium and macrophages as well as alveolar epithelium. kaposi's sarcoma, a vascular tumor, was discovered in 1872, and is also known as multiple hemorrhagic sarcomas, multiple vascular sarcomas, or multiple pigmented sarcomas. kaposi's sarcoma is believed to be the defi ning tumor of aids. outbreak of ks occurred in male homosexuals in europe and the united states. data show that in about 30 % caucasian homosexuals, kaposi's sarcoma is a major complication of in hiv/aids patients. it has been confi rmed that, though kaposi's sarcoma has strong invasion, the disease itself has little impact on the mortality of aids. the cause of death in majority of the patients is still opportunistic infections. the clinical manifestations include face and neck lesions in dark red to purple red plaques. the plaques do not fade away when pressed, with surrounding brown ecchymosis. it commonly involves multiple organs including lungs, spleen, oral cavity, lymph nodes, gastrointestinal tract and liver. the lungs are the major target of invasion. the diagnostic imaging demonstrates hilar lymphadenectasis and its surrounding nodular infi ltration, bilateral interstitial changes, and pleural effusion that are its typical x-ray demonstration. early pathological changes are similar to those of common angioma; with gathering of capillaries into groups containing histocytes engulfed hemosiderin and orderly arranged vascular endothelial cells. it further progression see active proliferation of endothelial cells and fi broblasts, increased nuclear mitosis with anaplasia, and scattered lymphocytes and histocytes between blood vessels. in the advanced stage, occlusion and necrosis of the vascular lumen can be found. irregular lumen and fi ssures of the new capillaries can be commonly found in the tumor, fi lled with blood and common hemorrhage. in china, ks is relatively rare. its defi nitive diagnosis can be made by pathological examination. other hiv/aids related malignancies include burkitt's lymphoma, non-hodgkin's lymphoma, hodgkin's lymphoma and lung cancer. in summary, hiv/aids related pulmonary infections are important diseases in the disease spectrum of hiv/aids imaging. the diagnostic imaging is irreplaceable examinations for pulmonary infections. early prevention and correct diagnosis are the keys to improve the quality of life and prolong the lives of patients. the complexity and multiplicity of hiv/aids related pulmonary diseases present challenges for the clinicians. firstly, hiv/aids related diseases should be optimally classifi ed. each type should has a disease spectrum, which can be used for exclusion in combination with immunological indices to make the diagnosis and differential diagnosis. the diagnosis of hiv/ aids related pulmonary infections should be made in combination with case history and laboratory tests for targeting individualized diagnosis to serve clinical practice. carnii pneumonia (pcp) the pathogen is the trophozoites and cysts produced by pneumocystis carinii, principally living in the lungs. pneumocystis carinii was used to being categorized as as protozoon, but recently, it is believed to be belonged to fungus according to its ultrastructure and pneumocystis ribosomal rna phylogenetic analysis. the main infection route of pcp is airborne transmission and reactivation of in vivo latent pneumocystis carinii. infl ammatory and immune responses of the host include phagocytosis of pneumocystis carinii by the alveolar macrophages, infi ltration of lymphocytes in peribronchial and vascular area, proliferation of type ii alveolar cells, local and systemic increase of antibody. by naked eyes observation, there are extensive and diffuse invasion of lungs, which is soft like waterlogged sponge and in milky white with black spots. the fi lled foamy substance in the alveoli and bronchioles is a mixture of necrotic fungus and immunoglobulin. the alveolar septum has infi ltration of plasma cells and lymphocytes, resulting in thickened alveolar septa up to 5-20 times as the normal thickness that occupy 3/4 of the entire lung volume. the cysts are fi rstly located in the macrophage cytoplasm of the alveolar septa. subsequently, the alveolar cells containing cysts sheds off into the alveolar space. after the rupture of the cystic wall, sporozoite is discharged to turn into free trophozoites, which gains its access into the alveolar space. the alveolar exudates include plasma cells, lymphocytes and histocytes ( fig. 17 .1a-c ). the clinical symptoms include dry cough, shortness of breath and an indoor hypoxia. about 95 % aids patients have multi-pathogens induced pulmonary infections. the most signifi cant laboratory abnormality in most pcp patients is hypoxemia. based on correlation between pcp and arterial oxygen partial difference, hypoxemia is divided into three degrees. the slight cases at indoor conditions have their pao 2 being above 70 mmhg, or alveolar-arterial oxygen pressure difference being less than 35 mmhg, or both. the moderate and severe cases have their pao 2 being usually less than 70 mmhg, or alveolar-arterial oxygen pressure difference being above 35 mmhg, or both. the most common manifestations of aids complicated by pcp are progressive subacute onset of dyspnea, fever, dry cough and chest distress, the symptoms aggravating in a few days or weeks. pulmonary examination is usually negative in slight cases. as the disease aggravates, the cases show shortness of breath, cyanosis, tachycardia, and diffuse dry rales. pneumocystis carinii infection accounts for 60-85 % of aids patients, which is one of the major causes of death in aids patients. the diagnostic imaging examinations include chest x-ray, ct scanning and nuclear medicine examination. (1) chest x-ray is the conventional examination for screening. early lesions tend to be missed for the diagnosis due to the limited resolution or atypical lung lesions. (2) ct scanning with high resolution is superior to chest x-ray. (3) nuclear medicine examination demonstrates increased uptake of the isotope-labeled monoclonal antibodies in the lung tissues of the pcp patients. (1) by tracheal suction or lung tissue biopsy, the detection rate of pneumocystis carinii is up to 90 %. by tissue section staining, abundant protozoa can be found in intra-alveolar foamy eosinophil substance mass (by methenamine silver nitrate staining, the dark brown round or oval shaped cysts can be found in a diameter of 6-8 μm out of the cells). (2) by elisa, pneumocystis igg antibody can be detected and by latex particle agglutination test, the protozoa antigen can be detected. (3) molecular biology techniques, such as pcr can be applied for early diagnosis. the following examinations are non-specifi c, but can be used to assess the severity of pcp and its progression. (1) by arterial blood gas analysis, the patients may show reduced blood oxygen saturation and respiratory alkalosis. (2) by serum enzyme spectrum analysis, the patients may show increased ldh. (3) it can be detected to have increased alveolar-arterial oxygen partial pressure difference. in the early stage (exudation period), alveolar fl uids exudate, with diffuse granular shadows in the bilateral lung fi elds extend from the hilum to the surrounding. in the middle stage (infi ltration and fusion period), the intrapulmonary lesions fuse, with ground glass liked or cloudy shadows that are bilaterally symmetric like butterfl y wings. in the middle and advanced stages (parenchymal changes period), the lung tissues show parenchymal changes, with high density shadows and accompanying air bronchogram. the lung periphery shows stripes of transparent shadows. in the advanced stage (pulmonary fi brosis period), the pulmonary interstitium is thickened in dense cords liked appearance, with interval irregular patchy shadows. the pulmonary ventilation improves and the lung periphery shows dense parenchymal shadows with emphysema, pneumomediastinum and pneumothorax. in the early stage (exudation period), the lesions radiatus develop from the hilum to lung fi eld. in the early stage, the diffuse exudative lesions distribute as pulmonary acinus, with changes similar to pulmonary interstitial changes. it was believed to be interstitial pneumonia. however, acute pcp is actually exudation of alveoli and spaces containing a male patient aged 31 years was confi rmatively diagnosed as having aids by the cdc. he complained of dyspnea, cyanosis and wheezing for 3 weeks, with obviously decreased oxygen saturation. his cd4 t cell count was 45/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of dyspnea, cyanosis and wheezing for 8 weeks, with obviously decreased oxygen saturation. her cd4 t cell count was 45/ μl. patients with acquired immunodefi ciency. the early symptoms include fever, dry cough and shortness of breath. the advanced symptoms are serious dyspnea, cyanosis, progressive hypoxemia and respiratory failure. by pulmonary examinations, scattered dry and moist rales can be heard. trophozoites of pneumocystis cysts can be found by liquid giemsa staining. slight and moderate interstitial infl ammation responses mainly involve lymphocytes and alveolar macrophages. the detection of cysts containing sporozoites is the basis to defi ne the diagnosis. chest x-ray chest x-ray demonstrations of pcp can be classifi ed into four types. (1) early pulmonary interstitial infi ltration and diffuse miliary alveolar exudation; (2) in the middle stage, there are alveolar exudates, with fusion and parenchymal changes; (3) in the middle-advanced stage, diffuse parenchymal changes; (4) pulmonary interstitial fi brosis and lung cavity or lung bulla, as well as pneumothorax and emphysema. for the cases with negative or atypical fi ndings by chest x-ray, ct scanning with high resolution should be performed. ct scanning demonstrates early lesions of multiple symmetric diffuse miliary nodal shadows, which have clear boundaries. in the middle stage, there are thin cloudy shadows or ground glass liked density shadows. in the middleadvanced stage, the lung tissues show parenchymal shadows, with trachea-bronchial sign. in the outer strip of the lung, a transparent area in shape of willow leaf can be demonstrated. in the advanced stage, fi brous cords liked shadows are demonstrated some lung tissues with compensatory emphysema and even pulmonary pseudocysts. the intake of the isotope-labeled monoclonal antibody by lung tissues of pcp patients increases. hiv/aids related pcp should be differentiated from bacterial pneumonia, pulmonary tuberculosis, viral pneumonia, fungal pneumonia, ards, and lymphocytic interstitial pneumonia (lip). bacterial pneumonia has more focal lesions but less diffuse lesions. pulmonary mycobacterium tuberculosis infection has manifestations of military pulmonary tuberculosis by chest x-ray, which is diffi cult to be differentiated from early pcp. hiv/ aids related pcp shows miliary nodules, which further fuse into cloudy or ground glass liked shadows or parenchymal changes. the lesions are commonly symmetrical, with the hilus as the center. the clinical manifestations include fever, dry cough or accompanying diffi culty breathing, and even cyanosis. but in the cases of pulmonary mycobacterium tuberculosis infections, most show miliary nodules, which further fuse into large nodules or mass. after about 2 weeks treatment in the early stage, the military nodules in both lungs can be absent, with common clinical symptom of high fever. correlation studies of miliary tuberculosis and peripheral blood cd4 t cell count have demonstrated that the general incidence of miliary tuberculosis is low, only 6-9 %, but it is the main manifestation of hiv/aids related pulmonary miliary tuberculosis. generally, when cd4 t cell count is below 200/μl, the incidence of cavity lesions is 29 %, noncavity lesions 58 %, complicated by pleural effusion 11 % and lymphadenectasis 20 %. when cd4 t cell count is between 200 and 390/μl, the incidence of cavity lesions and non-cavity lesions each accounts for 44 %, complicated pleural effusion 11 % and lymphadenectasis 14 %. when cd4 t cell count above 400/μl, the manifestation is commonly pneumonia type, in fl aky shadows or parenchymal shadows in just one pulmonary segment. the incidence of cavity lesions is 63 %, non-cavity lesions 33 %, complicated by pleural effusion 3 % and no lymphadenectasis. chest x-ray demonstrates cytomegalovirus pneumonia negative in 1/3 patients. the foci are commonly bilateral, with reticular particles in 33 % patients, alveolar foci in 22 % patients, nodular foci in 11 % patients, complicated by cavity in 11 % patients, cysts in 6 % patients, pleural effusion in 33 % patients and lymphadenectasis in 11 % patients. the incidence of diffuse foci in the cases of cryptococcus neoformans pneumonia is 76 %, interstitial foci or mixed foci 76 %, alveolar foci 19 %, nodular foci 5 %, lymphadenectasis 11 % and pleural effusion 5 %. hiv/aids related pcp is more likely to occur in children with aids, which presents diffi culty for its differentiation form lymphoid interstitial pneumonia. however, lymphoid interstitial pneumonia commonly has a chronic onset, with commonly manifestations of cough and dry rales. systemic lymphadenectasis and enlargement of salivary glands can also be found. by lung tissues biopsy, ebv-dna1 can be detected, which provides basis for their differentiation. pneumocystis, a unicellular organism, is the pathogen of pneumocystis carinii pneumonia. pneumocystis carinii pneumonia is one of common opportunistic infections in aids patients, which is also the leading cause of death in aids patients. in the initial episode of pcp, most patients have a cd4 t cell count of less than 100/μl. diagnostic imaging demonstrates bilaterally symmetrical ground glass liked shadows, which can be diffusely distributed and tend to mainly involve the periphery of the hilus or the middle and lower lung fi elds. hrct scanning is commonly applied to assess early pcp that is demonstrated negative by chest x-ray. hrct scanning demonstrates bilaterally symmetric patchy or fused ground glass liked shadows. the pathological basis of ground glass liked shadows and parenchymal areas refl ect that the acinus is fi lled by the foamy exudates, which are composed of surface active substances, cellulose and cell debris. all of the ground glass liked shadows, overlapping septa and the intralobular linear shadows are in gravel road liked manifestation. the septa and intralobular linear shadows demonstrate pulmonary interstitial edema or cellular infi ltration. in the middle-advanced stage of pcp, there are manifestations of small pulmonary nodules, pulmonary parenchymal changes, thickened interlobular septa, intralobular linear shadows, mass like lesions, pleural effusion, and lymphadenectasis. the cysts tend to mainly involve the upper lobes, which can be unilateral or bilateral pulmonary cysts, pneumothorax, mild or severe interstitial fi brosis and traction bronchiectasis. hrct scanning demonstrations of pcp are non-specifi c. its diagnosis should be in combination with hivph13 and etiological examinations. bacterial infections mycobacterium tuberculosis is still an important pathogen for pulmonary infection in hiv positive patients. since the mid-1980s, the main cause of the increasing incidence of tuberculosis is the prevalence of hiv infection. the incidence of tuberculosis in aids patients is 200-500 times higher than the general population. hiv infection is the most dangerous factor for progression of latent tuberculosis into active tuberculosis. tubercle bacillus belongs to mycobacterium family of mycobacterium genus, which is divided into types of human, bovine and murine. the main cause of human tuberculosis is human mycobacterium tuberculosis, which is known as acid-fast bacilli. tubercle bacillus wall is the complex containing high molecular weight fatty acids, lipids, proteins and polysaccharides, which are related to its pathogenicity and immune responses. lipid can cause the infi ltration of human monocytes, epithelial cells and lymphocytes to form tuberculous nodules. its protein contents can cause allergic reactions, and infi ltration of neutrophils and mononuclear cells. polysaccharides participate in certain immune responses (such as agglutination). these pathogenic factors lay the foundation for the occurrence of tuberculosis in aids patients. human immunity, allergic responses as well as the number and pathogenicity of tubercle bacilli are closely related to the quality, range, spreading rate and the progression of tuberculosis. its pathological changes are characterized by exudation, infi ltration, proliferation and hyperplasia, degenerative necrosis (caseous necrosis) and cavity formation. the manifestations include congestion, edema and infi ltration of leukocytes. the exudative lesions occur in early stage of tuberculosis infl ammation or when the lesions deteriorate. it can also be found in the serosa tuberculosis. there is neutrophilic granulocytes in the exudative lesions, which are gradually substituted by monocytes (phagocytes). the engulfed tubercle bacilli can be found in the large mononuclear cells. the exudative lesions are absorbed and dissipated through the phagocytosis of the mononuclear-phagocyte system, even with no scar. when large mononuclear cells engulf and digest tubercle bacilli, the phospholipid of the bacteria render the large mononuclear cells to enlarge and be fl at, similar to epithelial cells, which is known as epithelioid cells. these epithelioid cells gather into groups, with central langhans giant cells that pass the messages of the bacteria antigens to lymphocytes. surrounding the langhans giant cells, there are often many lymphocytes to form typical tuberculous nodules, which are characteristic lesions of tuberculosis. this is why it is called tuberculosis. in the tuberculous nodules, tubercule bacilli are usually undetectable. proliferation based lesions often occur in the cases with less bacteria invasion and when human cells mediated immunity is predominant. degeneration often occurs on the basis of the exudative or proliferative lesions. tubercle bacilli overcome macrophages and then continually proliferate in large quantity. after the cells become cloudy and swelling, the foci show fatty degeneration, dissolved into fragments, until the occurrence of necrosis. after the death of infl ammatory cells, proteolytic enzymes are released to dissolve the tissues that results in necrosis, which is coagulative necrosis. by naked eyes observation, they are yellowish gray, with loose and brittle quality like caseous. therefore it is known as caseous necrosis. microscopic examination demonstrates an area of solid and eosin staining red necrotic tissues with no tuberculosis. tubercle bacilli in the foci of caseous necrosis proliferate in large quantity to cause liquefaction, which is related to infi ltration of neutrophile granulocytes and large monocytes. part of liquefi ed caseous necrotic substances can be absorbed and part can be discharged by the bronchus to form cavities. otherwise, it may cause intrapulmonary spreading along with bronchi. the small caseous necrosis or proliferative lesions can be shrunk and absorbed after treatment, with only residues of slight fi brous scars. due to the compromised immunity in aids patients, the lesions rarely show fi ber tissues proliferation, but form cords liked scar. calcifi cation rarely occurs. if the necrotic lesions erode the blood vessels, tubercle bacilli can cause systemic miliary tuberculosis along with blood fl ow, including brain, bones and kidneys. large quantity sputum containing tubercle bacilli gains its access into the gastrointestinal tract. it can also cause intestinal tuberculosis and peritoneal tuberculosis. pulmonary tuberculosis can cause tuberculosis pleurisy via direct spreading to the pleura ( fig. 17 .19a-c ). clinically, it is a chronic progression, with rare acute onset. the clinical symptoms are commonly systemic, with fever and fatigue. the respiratory symptoms include cough and hemoptysis. pulmonary tb can be divided into primary and secondary, with the initial episode commonly being primary (type i). the residual bacteria after primary infection can cause secondary infection (type ii-iv) when the immunity is compromised via spreading along blood fl ow or direct spreading. it is common in hiv positive children. most cases are asymptomatic, sometimes with symptoms of low grade fever, mild cough, sweating, rapid heartbeat, and poor appetite. hiv/aids related miliary tuberculosis is one of the major manifestations of pulmonary tuberculosis, which is more common. the onset of acute miliary tuberculosis is rapid, with symptoms of chills and high fever with a body temperature up to 40 °c, mostly remittent fever or continuous fever. there may be decreased leukocytes count and accelerated sedimentation rate. the progression of subacute and chronic hematogenous disseminated pulmonary tuberculosis is relatively slow. infi ltrative pulmonary tuberculosis in aids patients commonly occurs in both middle and lower lung fi elds, with fl aky and fl occulent foci or parenchymal changes in lobes or segments. caseous lesions are rare. the early stage of infi ltrative pulmonary tuberculosis is commonly asymptomatic, with later occurrence of fever, cough, night sweating, chest pain, weight loss, expectoration and hemoptysis. this type of pulmonary tb rarely occurs in aids patients. in non-aids patients, chest x-ray demonstrates three major changes, namely cavity, fi brosis, and bronchial dissemination. in the aids patients, the pulmonary manifestations include single or multiple nodular shadows with clear boundaries. tuberculous pleuritis is an exudative infl ammation caused by the direct invasion of tubercle bacillus from the primary lesion near the pleura into the pleura, or hematogenous dissemination via the lymphatic vessels to the pleura. the routes for occurrence of tuberculous pleurisy include: (1) the bacteria in the hilar lymph tuberculosis counterfl ow to the pleura along lymph vessels. (2) tb lesions adjacent to pleura rupture to cause direct access of the tubercle bacilli or products of tuberculosis infection into the pleural cavity. (3) acute or subacute hematogenous disseminated tuberculosis causes pleuritis. (4) due to the increased allergic responses, the pleura highly respond to tuberculosis toxins to cause exudation. (5) thoracic tuberculosis and rib tuberculosis rupture into the pleural cavity. clinically, pleuritis can be divided into three types, dry pleuritis, exudative pleuritis and tuberculous empyema (rare). the common clinical manifestations are fever, cough with accompanying chest pain of the affected side and shortness of breath. (1) sputum smear examination is simple to manipulate, with high accuracy rate. the fi ndings of the tubercle bacilli can defi ne the diagnosis. it still is the golden criteria for the diagnosis of pulmonary tuberculosis. (2) sputum tubercle bacilli culture has high reliability. tubercle bacilli drug sensitivity test can be performed but requires 6-8 weeks to obtain the results. therefore, its application is limited. (1) tuberculin purifi ed protein derivative (ppd) test is commonly used. its positive result is one of the evidence confi rming a past history of tb infection. (2) bactec test can be performed to detect the metabolites of mycobacterium tuberculosis. generally, mycobacterium can be detected in 2 weeks. the quantity of mycobacteria can affect the period required for test results. (3) pcr has poor specifi city but high sensitivity of up to 98-100 %. both can be applied to observe the enlarged lymph nodes in the chest and mediastinum. in addition, they can be applied to obtain specimens for biopsy, which facilitates the diagnosis and differential diagnosis. diagnostic imaging examinations include chest x-ray and ct scanning. chest x-ray can demonstrate the location, quality and range of the lesions. it can also help to assess the therapeutic effi cacy. ct scanning can demonstrate small or hidden lesions, with a high resolution. primary pulmonary tuberculosis, also known as primary syndrome, is rare in adult aids patients. chest x-ray demonstrates intrapulmonary patchy or large fl aky parenchymal changes, hilar and mediastinal lymphadenectasis in connection to irregular cords liked shadows (located between intrapulmonary lesion and the hilum). lymph node tuberculosis is demonstrated to have mediastinal lymphadenectasis that sometimes fuse into mass. in aids patients, simple mediastinal lymph node tuberculosis is more common than primary syndrome. tuberculosis (1) the acute cases are demonstrated to have diffused miliary nodules in both lungs with even distribution, even size and even density. (2) the subacute and chronic cases are demonstrated to have nodules in both lungs, with uneven distribution, uneven size and uneven density. sometimes calcifi cation occurs in the nodules, with fi brous cords and thickened pleura. infi ltrative pulmonary tuberculosis are demonstrated to have patchy parenchymal changes in the middle and lower lung fi elds as well as parenchymal changes, cavities and fi brous cords liked foci in the segments and lobes. it can also occur in the upper lung fi elds, commonly with accompanying mediastinal and hilar lymph node tuberculosis. it commonly occurs in the advanced stage of aids,, with manifestations of pulmonary interstitial fi brosis and formation of cavities. this type of pulmonary tuberculosis is less common. it rarely occurs, mostly in the early stage of aids. it is rare in the middle and advanced stages of aids. dry pleuritis has manifestations of blunt costophrenic angle and limited diaphragm mobility. exudative pleuritis is manifested as small quantity pleural effusion and thickened pleura, commonly with encapsulated effusion of the lateral pleura. calcifi cation is rare. a male patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. he complained of dull chest pain, dyspnea, fever, night sweating, fatigue and anorexia. his cd4 t cell count was 65/μl. a female patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. she complained of dull chest pain, dyspnea, fever, night sweating and fatigue. her cd4 t cell count was 85/μl. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he was infected hiv via blood transfusion, with complaints of cervical lymph node tuberculosis, ascites and abdominal infection, fungal stomatitis, biliary stones with infection and severe anemia. his cd4 t cell count was 33/μl. a female patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and night sweating. her cd4 t cell count was 73/μl. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she was hospitalized due to complaints of chest distress, cough and expectoration for 2 months, with after noon low grade fever and weight loss. on admission, she was confi rmed hiv positive and a cd4 t cell count of 120/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 71/μl. a female patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 891/μl. a female patient aged 34 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 51/μl. cough expectoration, chest pain, dyspnea, fever, night sweating, fatigue, anorexia, lymphadenectasis and rapid progression of the conditions. ppd skin test with a resulted diameter of more than 5 mm should be considered as tuberculosis infection. but its positive rate remains low. the culture of sputum and bronchoalveolar lavage fl uid can detect the pathogens. nucleic acid analysis or dna probe technique, pcr and chromatography can be applied to detect tubercle bacilli. the commonly used diagnostic imaging examinations include chest x-ray and ct scanning. the main demonstrations include (1) intrapulmonary and extrapulmonary lymphadenectasis; (2) miliary tuberculosis manifestations; (3) infi ltrative (pneumonia type) pulmonary tuberculosis; (4) pulmonary interstitial fi brosis, cavity, pulmonary emphysema, nodules, emphysema, and bronchiectasis with accompanying infections. in the cases with their cd4 t cell count being above 400/μl, the imaging demonstrations are similar to those of non-hiv/aids patients with pulmonary tuberculosis. in the cases with their cd4 t cell count being above 400/μl, the manifestations are intrapulmonary large fl aky parenchymal changes, surrounding satellite lesions as well as mediastinal and hilar lymphadenectasis. sometimes the manifestations may be only mediastinal lymphadenectasis and their fusion into mass. in the cases with cd4 t cell count being above 200/μl, there may be accompanying extrapulmonary tuberculosis, such as tuberculous peritonitis, bone tuberculosis, brain tuberculosis and splenic tuberculosis. in the cases with their cd4 t cell count being lower than 100/μl, the manifestations are mostly miliary tuberculosis. hiv/aids related tuberculosis should be principally differentiated from pneumocystis carinii pneumonia, fungal infections, other pneumonia and lung cancer. hiv/aids related tuberculosis should be differentiated from pcp. pcp is mainly manifested as multiple lesions with hilum as the center to extending symmetrically to outside of the lungs. in the advanced stage, pcp has main lesions of pulmonary interstitial fi brosis, with less accompanying mediastinal and hilar lymphadenectasis. the laboratory tests can facilitate to defi ne the diagnose. hiv/aids related tuberculosis should be differentiated from fungal infections. fungal infections are relatively less common than tuberculosis. the imaging fi ndings of hiv/aids related pulmonary fungal infections are diverse, with manifestations of miliary, fl aky fl occulent liked, parenchymal, mass, and interstitial changes. in general, the diffuse lesions are mainly interstitial changes, while confi rmed lesions, compared to tb lesions, are more likely to have thick walled cavities. satellite lesions of fungal infections are less than those of tuberculosis, with less accompanying mediastinal and hilar lymphadenectasis. sometimes laboratory tests are necessary to defi ne the diagnosis. hiv/aids related tuberculosis should be differentiated from non-tuberculosis mycobacteria pneumonia. their imaging fi ndings are similar to each other, which presents challenges for their differential diagnosis. molecular biology examinations play an important role in the differentiation. hiv/aids related tuberculosis should be differentiated from other pneumonia. non-bacterial pneumonia (mycoplasma, viral and allergic) often shows patchy shadows, which are similar to the manifestations of early infi ltrative pulmonary tuberculosis. when bacterial pneumonia shows lobar lesions, it may be confused with tuberculous caseous pneumonia, which should also be differentiated for the diagnosis. symptoms of mycoplasma pneumonia are mild, with imaging fi ndings being always inconsistency with the clinical symptoms, which usually subside within 2-3 weeks. in the cases of allergic pneumonia, eosinophils in the blood increase, with intrapulmonary mobile shadows, which are the basis for their differentiation. bacterial pneumonia can have acute onset, with chills, high fever, rust colored sputum, and streptococcus pneumoniae positive. recovery is rapid after antibiotic treatment and all these symptoms can subside within 1 month. hiv/aids related tuberculosis should be differentiated from pulmonary abscesses. in the cases of infi ltrative pulmonary tuberculosis with cavities, it should be differentiated from pulmonary abscess. especially, tuberculosis with cavities in the apical segment of inferior lobe should be differentiated from acute pulmonary abscess. chronic fi brous cavity tuberculosis should be differentiated from chronic pulmonary abscess. the key points for the differentiation are tubercle bacilli positive by sputum culture in the cases of tb, while tubercle bacilli negative by sputum culture in the cases of abscesses. pulmonary abscess has an acute onset, with increased leukocytes and neutrophils as well as favorable therapeutic effi cacy of antibiotics. but sometimes tuberculosis with cavity may develop into bacterial infection, with undetectable tubercle bacillus by sputum culture. hiv/aids related tuberculosis should be differentiated from lung cancer. the central type of lung cancer has nodular shadow in the hilum or hilar and mediastinal lymph node metastasis, which should be differentiated from lymphatic tuberculosis. the peripheral type of lung cancer has small fl aky infi ltration and nodules in the periphery of the lungs, which should be differentiated from tuberculoma or tuberculosis infi ltrative lesions. lung cancers occur commonly in people aged above 40 years. the central type mainly is squamous carcinoma and the cases often have a history of long term smoking, with symptoms of no fever but diffi culty breathing or chest distress as well as gradually increasing chest pain. there are also symptoms of irritated cough with blood phlegm and progressive weight loss. the cases with supraclavicular metastasis have palpable harden lymph nodes. the intrapulmonary nodules can lobulated with fi ne spikes, no satellite lesions, generally no calcifi cation and possible vacuole sign. the peripheral type of lung cancer shows pleura invagination sign. tuberculin test often shows negative in the cases of lung, positive or weakly positive in the cases with tb, and negative or weak positive in aids patients. hiv infection is known to be the main factor for the development of latent tuberculosis into active tuberculosis. immunosuppression are similar to those in the cases with primary tuberculosis, with characteristic abnormal manifestations of hilar and/or mediastinal lymphadenectasis and parenchymal changes of the air chambers. ct scanning demonstrates enlarged nodules with low density. enhanced scanning demonstrates marginal enhancement of the lymph nodes. the incidence of military tuberculosis in aids patients is increasing due to reduced thymic t lymphocytes in aids patients and the defects of delayed allergic responses, which result in the formation of granulomas and impaired functions to kill bacilli and confi ne the lesions. nontuberculous mycobacteria (ntm) refer to the mycobacteria except for mycobacterium tuberculosis complex (human, cattle, african and vole) and mycobacterium leprae. the most commonly known nomination is nontuberculous mycobacteria (ntm). more than 100 kinds of ntm have been found so far. according to berger manual of systematic bacteriology, ntm is divided into two categories, rapid growth type and slow growth type. ntm are widely spread in nature, such as soil, dust, fl owing water and raw milk. under a microscope, ntm is morphologically similar to tubercle bacilli, with red stained fi ndings by acid-fast staining. according to the growth of ntm in solid medium, the runyon classifi cation divides ntm into the following four groups, light chromogenic bacteria; dark chromogenic bacteria that can cause cervical lymphnoditis in children, intrapulmonary or extrapulmonary infections and abrasive abscess; non-chromogenic bacteria including mycobacterium avium complex, intracellular mycobacteria that can cause pulmonary infections, lymphnoditis, arthritis and meningitis; rapid growth bacteria including mycobacterium fortuitum, mycobacterium, mycobacterium abscessus that can cause pulmonary diseases and skin infections. immunocompromised populations, such as hiv infected patients, patients with neoplasms, patients with long-term use adrenocortical hormone or immunosuppressive agents, are more susceptible to disseminated ntm infection. immunocompetent people may have mycobacterium kansasii and mycobacterium avium infections. it was reported in the united states that the occurrence of mycobacterium avium complex infection in hiv positive patients is up to more than 95 %. the pathological changes of ntm infections are similar to those of tuberculosis. ntm lymphnoditis is pathologically characterized by granulomatous infl ammation. tuberculous nodules formed by epithelioid cells and langhans giant cells are rare, with no accompanying central caseous necrosis. due to the weak pathogenicity of ntm, the pathological changes are slight, but there is difference in the pathological changes of ntm infections in terms of location, type and host. cavities are common in the cases with pulmonary ntm infection, commonly being multiple or multilocular thin wall cavities. the pleuron is rarely involved, with non-specifi c pathological changes of infl ammation but with large quantity pathogens of ntm. patients often have a history of chronic obstructive pulmonary disease, tuberculosis, silicosis, pulmonary abscess, bronchiectasis, cystic fi brosis, diabetes, ulcer as well as use of hormone or immunosuppressive agents. its occurrence is more common in males than in females. the symptoms include cough, expectoration, hemoptysis, chest pain, diffi culty breathing, low grade fever, weight loss and fatigue. the symptoms are nonspecifi c and the conditions progress slowly. for patients with suspected diagnosis of pulmonary ntm infection, sputum smear for acid-fast staining, sputum culture and bronchial lavage specimen culture can be performed. the positive fi ndings should be identifi ed with two to three times repeated culture. the same fi nding of ntm can defi ne the diagnosis. pathological biopsy can be performed for the diagnosis of ntm lymphnoditis. using 16s-23 srdna gene spacer sequence (igs) of ntm for pcr-restriction fragment length polymorphism analysis (pcr-rflp), ntm species can be identifi ed, which is more accurate, faster and simpler than the conventional morphological and biochemical examinations. mycobacterium tuberculosis and ntm have common antigen. ppd skin test produces cross-reaction, but there are still differences between mycobacterium tuberculosis and ntm. ppd-t of the mycobacterium tuberculosis and ppd-ntm of ntm are simultaneously obtained for mantoux skin tests. the induration diameter of ppd-t in ntm patients is generally within 15 mm. for the cases with the induration diameter of ppd-ntm skin test being 5 mm larger or over 25 % larger than that of ppd-t skin test, ntm infection can be confi rmed. both are the most commonly used imaging examinations. a female patient aged 26 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress for half a month, fever for 10 days, 1 day after cesarean section and fi nding of hiv positive for 1 day. her cd4 t cell count was 54/μl, with treponema pallidum antibody negative and ppd test negative. imaging demonstrations include various lesions such as infi ltration, cavity, nodules, fi brous caseation and extensive fi ber contraction in unilateral or bilateral lungs. the incidence of cavity is up to 80 %, being singular or multiple. cavities caused by intracellular mycobacterium are mostly found in the pleura, with thin wall and less surrounding exudates. the incidence of non-tuberculous mycobacterial infections in aids patients is high. mac infection is usually caused by the initial exposure rather than the reactivation of latent pathogens. in patients with complications of mac related lung diseases, most of the imaging fi ndings are normal. the most common manifestation is mediastinal or hilar lymphadenectasis. and the pulmonary symptoms are similar to those of tuberculosis. in the cases with multiple patchy parenchymal changes, cavities can be found, as well as nodules with blurry boundaries, pleural effusion and rarely found miliary nodules. sputum or bronchoalveolar lavage fl uid culture positive, clinical symptoms, imaging fi ndings, and response to treatment can defi ne the diagnosis. staphylococcus aureus is a gram positive coccus and is coagulase positive staphylococcus. the pathogenic substances of staphylococcus aureus mainly are toxins and enzymes, such as hemolytic toxins, leukocidin and enterotoxin, which play a role in hemolysis, necrosis, killing leukocytes and vascular spasm. the staphylococcus aureus coagulase is the main reason for suppurative infection. pneumonia caused by inhaled staphylococcus aureus through the respiratory tract often shows lesions in the large lobes or extensive fusion of bronchopneumonia lesions. bronchial and alveolar rupture allows gas to enter the pulmonary interstitium, which is communicated with the bronchi. in the cases of bronchiolar blockage by necrotic tissues or pus, the one-way valve effect is formed to cause tension pulmonary emphysema. in the cases with superfi cial pulmonary emphysema with excessively high tension, it ruptures to form pneumothorax or pyopneumothorax, as well as bronchooleural fi stula ( fig. 17.45a, b ) . the symptoms include chills, persistent high fever, cough, expectoration, chest pain and other symptoms. there is no sign in the early period. symptoms are scattered moist rales in both lungs, being in consistency to severe toxic symptoms and respiratory symptoms. yellow purulent sputum is the typical characteristics of staphylococcus aureus pneumonia. in the cases with larger lesions or fusion of lesions, signs of parenchymal changes, pneumothorax or pyopneumothorax can be found. in the sputum or pleural fl uid smears examinations, the bacteria with a concentration being no less than 107 cfa/ml is the pathogen, the bacteria with a concentration being 105-107 cfa/ml is the suspected pathogen, and the bacteria with a concentration being less than 105 cfa/ml is the contaminated bacteria. there are increased wbc count and neutrophils, leftward migration of the nucleus and possibly no increase of wbc count in aids patients. immunofl uorescence, enzyme-linked immunosorbent assay and counter immunoelectrophoresis can be performed to detect serum antigen or antibody of the pathogenic bacteria, which can defi ne the diagnosis. polymerase chain reaction has certain signifi cance in pathogen detection. the protected bronchoscopic specimen (pbs) and bronchoalveolar lavage (bal) can be applied to collect the specimen, which has reduced chances of specimens contamination by oral bacteria. biphasic tv monitors guided pulmonary puncture and suction for pulmonary tissues examinations can be performed to detect the real pathogenic bacteria. both are the most commonly used imaging examinations. the diagnostic imaging demonstrates staphylococcus aureus pneumonia as lesions in the inner zone of both middle lower lungs. there are singular or multiple parenchymal changes in patchy or lobar distribution that may fuse into large fl akes. it may be complicated by cavity and pulmonary emphysema, with surrounding compensatory emphysema. a male patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever with a body temperature of about 39 °c and chest pain for 2 months. his cd4 t cell count was 31/μl. chills, fever, cough, expectoration, chest pain and other symptoms commonly; hemoptysis and dyspnea rarely the patients may be found with fever appearance, rarely shortness of breath and cyanosis. in the serious cases, the body temperature can be as high as 39-40 °c and blood pressure decreases, with signs of shock. by chest examinations, decreased ipsilateral respiratory motion; increased or decreased fremitus, dull sound in percussion; bronchial breathing sounds or moist rales by auscultation; rarely pleural friction and weakened breathing sounds. increased wbc count and neutrophils, possible the nucleus left shift; no increase or even decrease of wbc count in aids patients; staphylococcus aureus positive by blood culture. sputum or pleural fl uid smears examinations for pathogenic bacteria culture is positive, and antibiotic sensitivity test is positive. by chest x-ray and ct scanning, the most common demonstrations are lesions of bronchial pneumonia. the fi ndings of pulmonary emphysema and cavities can facilitate the diagnose. the lesions should be differentiated from infi ltrative parenchymal bronchioloalveolar carcinoma. the smaller lesions should be differentiated from pulmonary infarction. the large lesions should be differentiated from obstructive pneumonia. it is diffi cult to identify the types of common bacterial pneumonia simply by chest x-ray and ct scanning. in combination to the laboratory tests, the diagnosis can be defi ned. the incidence of pyogenic bacterial infection is increasing in aids patients, which is caused by their weakened cellular and humoral immunity. the manifestations of these most common bacterial infections are similar to those of non-hiv infected patients by chest x-ray. bacterial pneumonia and purulent bronchitis are the most common causes of pulmonary infections in aids patients. particularly, they are frequently found in patients with a history of intravenous drug abuse and smokers. they are histologically characterized by infl ammations of the bronchi and bronchioles as well as infl ammatory exudates and mucus in the airway lumens. ct scans facilitates the diagnosis of bronchiolitis and early bronchial pneumonia. the demonstrations are characterized by (1) small centrilobular nodular shadows, which is the cross sectional demonstration of bronchioles fi lled with infl ammatory substances and its surrounding infl ammations; rhodococcus equi infection is one of the zoonotic diseases, which commonly occurs in the grazing areas. patients with t lymphocyte immunodefi ciency caused by aids and other factors are especially susceptible to the infection. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi infection in human is rare. but in recent years, due to an increase of patients with immunodefi ciency syndrome, reports on rhodococcus equi caused human respiratory infections and sepsis are increasing. in the past, the toxicity mechanism of rhodococcus equi was mostly speculated. until recently, the damage process of toxic plasmid to human tissue is discovered, which presents a new way for the study of the pathogenesis of rhodococcus equi infections. rhodococcus equi is one of the facultative parasites in the cells and its optimum growth temperature is 30 °c, with a suitable growth temperature of 10-40 °c. acid-fast staining of rhodococcus equi shows uncertain results. due to its morphological diversity, it is often mistaken as diphtheroid bacillus, bacillus or micrococcus. in sheep blood agar, rhodococcus equi can have synergistic hemolysis with staphylococcus aureus, listeria monocytogenes and corynebacterium pseudotuberculosis, which is a characteristic manifestation of rhodococcus equi. the most common pathological changes in rhodococcus equi infection are chronic purulent bronchitis and extensive lung abscess. imaging often demonstrates subacute pneumonia, commonly with cavities. the clinical manifestations are poor appetite, drowsiness, fever and shortness of breath. studies by e marchiori et al. [ 30 ] in 2005 revealed that all the 5 cases of aids complicated by rhodococcus equi pulmonary infection have cough and fever lasting for 1-2 months, with accompanying shortness of breath and chest pain. all the 13 cases, studied by li et al. in 2011 [ 106 ] , have fever with a body temperature up to 38-40 °c and cough. in addition, there are also expectoration with orange red sputum in 10 cases, hemoptysis in 4 cases, dyspnea in 11 cases, moist rales of lungs in 13 cases, emaciation in 6 cases, poor appetite in 6 cases, diarrhea in 2 cases, joint pain in 1 case, oral candidiasis infections in 13 cases, oral herpes in 4 cases, chest pain in 4 cases, no obvious symptoms in 1 case and hepatitis b in 3 cases. typical clinical manifestations of this disease are fever, cough, dyspnea and chest pain, while others such as emaciation, diarrhea and joint pain are not representative symptoms. identifi cation of the bacteria various specimens were inoculated on blood plates at a temperature of 35 °c for 18-24 culture, with bacteria growth of 18 strains. they are biologically characterized by a diameter of about 0.5 mm, non-transparent and slight yellowish colonies. after 48-72 h, the colonies expand to 1-2 mm, which can be emulsifi ed in mucous fl uid liked state. most of the colonies produce orange and orange red pigments, which can be cultured in ordinary agar. histopathological fi ndings are typical for rhodococcus equi infection. h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells, possibly predominant fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining demonstrates scattered or clustered rhodococcus equi in pink or purplish red. both are the most commonly used imaging examinations. biphasic tv monitor guided lung puncture can be performed to suck lung tissues for biopsy, based on which the real pathogenic bacteria can be detected. the typical demonstrations include central hilar sphere liked shadow with increased density in unilateral lung, accounted for 70 %. there are also manifestations of exudative infi ltration and large fl aky or spherical mass shadows in the right or left hilar area. the lesions are in patchy or fl aky appearance, radiating from the hilum to the lung fi eld with blurry boundaries. a male patient aged 30 years was confi rmatively diagnosed as having aids by the cdc. he complained of recurrent fever, cough and chest pain for 13 days; and was found hiv positive for 8 days. he was also a carrier of hepatitis c virus, with symptoms of fever with no known causes, cough, chest distress and weak limbs. by examinations, he was found to have complexion of chronic conditions; many moist and dry rales by cardiopulmonary auscultation. he had a past history of hiv positive for 8 years, with drug abuse and extramarital affairs. the history of present illness includes fever, paroxysmal cough with a little whitish yellow thick sputum since may 8, 2008 . he also had subjective paroxysmal dull pain in the left chest, and hemoptysis once which was bright red with blood clot in a volume of about 80 ml. his cd4 t cell count was 10/μl. by sputum culture, rhodococcus equi was found positive. after receiving antibiotic treatment, his conditions improved and he was discharged from the hospital. catalase test of the strain is positive, which is confi rmed as rhodococcus equi by api coryne system. examinations h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells or mainly fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining and masson staining demonstrate scattered or clustered distribution of rod rhodococcus equi in pink or purplish red. aids complicated by pulmonary rhodococcus equi infection shows subacute infl ammation. firstly, there are exudates in the surrounding area of unilateral hilum as well as sphere shaped mass shadows which is centrally dense and peripherally blurry, with its apex pointing to the hilum. the lesions can be complicated by cavities and fl uid level, with thick wall of the cavities. as the disease progresses, the abscess cavities have increased tension, with gradually thinner abscesss cavity walls and uneven wall thickness, even showing pleural effusion. these are its characteristic imaging fi ndings. it often needs to be differentiated from pneumocystis carinii pneumonia, tuberculosis, staphylococcus aureus pneumonia, central type lung cancer and other diseases. imaging fi ndings of pneumocystis carinii pneumonia usually are ground glass liked changes in the lung fi eld, with parenchymal changes and centrilobular nodules. tuberculosis often shows miliary tuberculosis, with lymphadenectasis, large tubercles and parenchymal changes. these characteristic pathological and imaging fi ndings of staphylococcus aureus pneumonia are similar to those of bronchial pneumonia (lobular pneumonia). the lesions are nodules with blurry boundaries in a diameter of 4-10 mm. the disease progresses rapidly, while pulmonary rhodococcus equi infection has a chronic progression. hrct scanning demonstrates staphylococcus aureus pneumonia as centrilobular nodules and branch linear shadows (tree buds sign), which can be found in 40 % patients, with 15-30 % will develop into commonly singular pulmonary abscess. chest ct scanning demonstrates round liked abscess cavity with thick wall and liquid level in it. the inner wall of the abscess cavity is often irregular, with various changes within short period. the central type of lung cancer is commonly demonstrated as round liked shadow of unilateral hilum with rough boundary. lobulation or bronchial stenosis sometimes occurs. however, aids complicated by rhodococcus equi pneumonia is demonstrated as sphere liked mass in the hilum; mostly sphere liked increased density shadow with hilum as the center. the shadow is centrally dense and peripherally blurry, with no bronchial stenosis. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi is generally believed to be the pathogen for horses, pigs and cattle. [ 106 ] showed a ct4 t cell count of lower than 49/μl. all the results are in consistency. in conclusion, aids complicated by pulmonary rhodococcus equi infection is mainly subacute infl ammation. there are exudation around the unilateral hilum as well as centrally dense and peripherally blurry sphere shaped mass shadows, with secondary cavities and parenchyma changes and even pleural effusion. all of these are characteristic imaging demonstrations. most cases of hiv positive complicated by respiratory or pulmonary diseases are caused by aspergillus fumigatus. aspergillus fumigatus belongs to filamentous fungi, which is a common opportunistic fungus and has a wide distribution in the nature. as conditional pathogenic bacteria, it can parasitize in the human skin and upper respiratory tract. human has certain resistance to aspergillus so it commonly fails to cause diseases. in immunocompromised aids patients, the pathogenic bacteria can pass through the defects in the skin and mucous membrane into the blood flow to infect the tissues and organs. aspergillus commonly violates bronchus and lung, with involvements of rhinal sinuses, external auditory canal, eye and skin. otherwise, it disseminates to organs of the body along with blood fl ow. the early lesions are diffuse infi ltrative and exudative changes. and advanced lesions are necrosis, pyogenesis or granuloma. large quantity hyphae can be found in the lesions. the hyphae penetrate the blood vessels to cause vasculitis, perivascular infl ammation and thrombosis. and thrombosis can cause ischemia and necrosis of the tissue. according to the pathological changes and imaging fi ndings, it can be divided into three major types: vascular invasion type, bronchopneumonia type and allergic bronchopulmonary aspergillosis type. (1) the vascular invasion type is the result caused by toxins released in the process of aspergillus spreading extensively from the primary focus to the lungs. vascular infi ltration of the pulmonary parenchyma and coagulative necrosis are believed to be the cause of vascular occlusion and pulmonary infarction. (2) bronchopneumonia type is acute bronchitis caused by inhalation of aspergillus spores. in the cases of hyphae invasion into the lung tissues, extensive infi ltrative pneumonia or focal granuloma are resulted in. it can also cause necrosis, pyogenesis and multiple small abscesses. spherical pulmonary aspergillosis is often secondary to bronchiectasis, tuberculosis, carcinous cavity and other lung diseases. mycelia multiply and gather in the cavities of the lungs to form a spherical mass with fi brin and mucosal cells, which are called aspergillar glomera, which do not invade the lung tissue. (3) allergic bronchopulmonary aspergillosis type is the proliferation and germination of inhaled aspergillus spore in the airway, often showing obvious related mucosal lesions and eventually resulting in bronchiectasis ( fig. 17.64a-c ) . the cases with acute onset have symptoms of high fever or irregular fever, cough, shortness of breath and green purulent sputum. the cases with a chronic onset have symptoms of repeated cough and hemoptysis, which are similar to those of tuberculosis. the pulmonary signs are not obvious, with occasional fi ndings of moist rales. most cases are asymptomatic and sometimes there are fever, cough, shortness of breath, and mucous purulent sputum. the main symptoms are persistent fever, cough and chest pain. in the serious cases, there is dyspnea. by microscopic examination of sputum, aspergillus hyphae can be found. the culture for aspergillus fumigatus is positive. serum ige is commonly above 2,500 μg/l. skin test for aspergillus antigen is positive. serum anti-aspergillus antigen igg antibody precipitin is positive. puncture of lungs and pleura for biopsy facilitates the diagnosis of pulmonary fungal infections. both are the most commonly used imaging examinations. hiv/aids related aspergillus bronchopneumonia is commonly demonstrated to have increased pulmonary markings, diffuse patchy blurry shadows and mass shadows in both lungs. spherical pulmonary aspergillosis is commonly demonstrated to have sphere liked aspergillar glomera suspending in the cavities to form a crescent shaped transparent area, in characteristic meniscus sign, rolling ball sign and fi ngertip sign. meniscus sign is nominated due to a meniscus liked space between the aspergillar glomera growing in the cavity and the cavity wall. rolling ball sign means that the aspergillar glomera moves along with the changes of posture. fingertip sign indicates that the substance formed by aspergillar glomera in dilated bronchi is in a fi nger shape, sometimes in v shape sign and y shape sign. invasive lesions refer to lesions invading or destroying lung structures, such as pneumonia, parenchymal changes and necrosis. a female patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress, with increased eosinophilic granulocytes. her cd4 t cell count was 45/μl. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 96/μl. a male patient aged 38 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 76/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized due to complaints of fever for 1 week, chest distress and shortness of breath for 1 day. on admission, he was confi rmed as hiv positive, with a cd4 t cell count of 9/μl. by physical examinations, he was in poor physical conditions, respiratory rate 27/min, lips cyanotic, coarse breathing sounds of both lungs with small quantity dry rales. his conditions progressed rapidly and death occurred due to respiratory failure after 3 days. lung and pleura puncture for biopsy can detect the growth of aspergillus hyphae. sputum culture can detect aspergillus hyphae, with fi ndings of aspergillus fumigatus positive. in the cases with allergic bronchopulmonary aspergillosis, the serum ige is above 2,500 μg/l. skin test of aspergillus antigen is positive. the serum anti-aspergillus antigen igg antibody precipitin is positive. characteristic ct scanning demonstrations of hiv/aids related parasitic aspergillar glomera include pulmonary cavities or cavity lesions with spherical contents, smooth boundaries of the spherical contents with even density, lunate shaped or ring shaped transparent shadows between cavity or cavity walls and the contents, migration of the contents with the body postures. according to the pathological and imaging demonstrations, it can be divided into three major types: in the early stage, ct scanning demonstrates soft tissue density nodules or light ground glass liked halo sign around the mass, which is the evidence for the diagnosis of the invasion type pulmonary aspergillosis. air cresent sign refers to round pulmonary infi ltration with accompanying central necrosis and surrounding lunate or ring shaped cavity. other noncharacteristic ct scanning demonstrations include multiple lobular parenchyma lesion shadows or lobular fusion shadows, parenchyma lesion shadows in the lobes, segments and subsegments, nodular or mass shadows and thin/thick wall cavities or low density areas in the mass shadows. it is demonstrated to have parenchymal lesions around the airway or/and central small nodules in the lower lobes. the parenchymal lesions prove the occurrence of mycotic bronchopneumonia. the most common imaging fi nding is the thickened bronchial wall. central bronchiectasis is its characteristic demonstration. in the cases of dilated bronchi containing sputum bolt or mucus, it shows fi ngertip shaped or toothpaste shaped shadow, which should be considered as its characteristic demonstration. hiv/aids related pulmonary aspergillosis should be differentiated from congenital bronchial atresia. most cases of the congenital bronchial atresia are atresia at the proximal pulmonary segment of the bronchi, often with a clearly defi ned mass. in the typical cases, there are bronchial branches and more branches in fi ngertip shape, pointing to the pulmonary hilum. confi ned pulmonary air retention in the pulmonary lobe and segment of the atresic bronchi is the important evidence for the diagnosis of congenital bronchial atresia. hiv/aids related pulmonary aspergillosis should be differentiated from allergic bronchial-pulmonary aspergillosis. in the cases of allergic bronchial-pulmonary aspergillosis have no clearly defi ned mass, with demonstrations of v shaped, y shaped, grapes shaped or fi ngertip shaped shadows with clearly defi ned boundaries, which are characteristic in those patients with bronchial asthma or a case history of exposure to dusts containing fungi. there is also increased proportion of eosinophilic granulocytes in the peripheral blood. detection of aspergillus in phlegm can defi ne the diagnosis. hiv/aids related pulmonary aspergillosis should be differentiated from central lung cancer. central lung cancer also can cause mucus impaction of the distal bronchi, with manifestations of bronchial arctia and/or truncation, and the surrounding soft tissue mass shadows. hiv/aids related pulmonary aspergillosis should be differentiated from pulmonary cavities and abscesses induced by dissolved tuberculoma, secondary pulmonary tb, chronic lung abscess and peripheral lung cancer as well as cystic bronchiectasis. except aspergilloma, spheric morphology caused by other causes is commonly irregular. the cavity contents cannot migrate with body postures, which is the key point for the differential diagnosis. hiv/aids related pulmonary aspergillosis can be caused by many pathogenic bacteria and aspergillus fumigatus is the most common one. the infection is often caused by inhaled aspergillus fumigatus in the environment. vascular invasion type of pulmonary aspergillosis usually has multiple lesions and nodular changes. generally in pathology, the center of nodule presents typical pale color; commonly with fi brous ring surrounding the nodules resulted from hemorrhage and/or lung parenchymal changes. histologically, they are characterized by coagulative necrosis of the lung tissues, infi ltration of large quantity hyphae in the necrotic tissue, pulmonary vascular infi ltration, but usually without responses of vasculitis and thrombosis. the enzymes released by neutrophile granulocytes can cause the separation of necrotic tissue from its adjacent lung tissues to form necrotic mass in the cavities. airway invasion type of pulmonary aspergillosis, also called aspergillus bronchopneumonia, accounts for 15-30 % of invasive aspergillosis. the most common imaging fi ndings are unilateral/bilateral fl aky parenchymal changes, centrilobular small nodules and branches liked linear shadows (tree buds sign). histologically, it is characterized by necrosis and infi ltration of neutrophil granulocytes. the lesions surround the bronchiole and the bronchiole. the invasion of the pulmonary artery can cause bleeding of the adjacent pulmonary parenchyma. allergic bronchopulmonary aspergillosis rarely has lesions, with no unknown pathogenesis, which is generally believed to be related to type i and type ii allergic reactions. it usually shows obvious asthma related mucosal lesions. hyphae generated by aspergillus fumigatus can induce the production of mucus and additional mucosal lesions, eventually leading to bronchiectasis. dilatate bronchial lumen is fi lled with mucus or with absence of epithelium, which is replaced by a granulomatous infl ammatory infi ltration. the most common imaging manifestations are migratory fl occulent, branched y shaped and v shaped (fi ngertip sign) shadows in the pulmonary parenchyma, which are related to the infi ltration of eosinophils. pathologically, bronchial cystic dilatation in the pulmonary segment and sub-segment occurs, with large quantity eosinophils in the bronchial mucus and scattered broken aspergillus hyphae. in combination with the case history, the diagnosis can be defi ned. compromised immunity is an important cause of cryptococcosis, especially in patients with aids or abnormal lymphoproliferative diseases. cryptococcus neoformans, a single phase mould, exists widely in the natural world. the cryptococcus has a diameter of less than 10 μm, which can be inhaled into the human body via respiratory tract. under the impact of a high concentration carbon dioxide, it forms a clearly defi ned protective layer composed of polysaccharide capsule to antagonize the defense mechanisms of the host. thus, lung infection occurs after its inhalation in immunocompetent people, which is commonly asymptomatic. of cryptococcus, inhalation of cryptococcus by aids patients can lead to hilar lymphadenopathy, as well as singular or multiple subpleural small nodules, being similar to those in the cases of mycobacterium tuberculosis infection. in the early stage of cryptococcal infection, only a mild infl ammatory reaction or diffuse infi ltrative exudative changes occur. but in the advanced stage, necrosis, suppuration or granuloma is formed. large quantity hyphae can be found in the focus. in the cases with hyphae penetrating the blood vessels, vasculitis, perivascular infl ammation and thrombosis occur. and thrombosis leads to ischemia and necrosis of the tissue (fig. 17.77 ). pulmonary cryptococcus infection in aids patients often is extensively disseminating, with symptoms of fever, cough, diffi culty breathing, expectoration, chest pain caused by pleuritis, and even acute respiratory distress syndrome (ards). hiv/aids related pulmonary cryptococcus infection has no characteristic imaging demonstrations. chest x-ray and ct scanning show multiple morphology of the lesions. in the slight cases, there are thickened pulmonary markings in both lower lungs or isolated nodular shadows, and occasionally cavities. in the cases of acute interstitial infl ammation, there are diffuse infi ltrative or miliary foci, with infi ltration, nodules or exudation in any lobe which is more common in bilateral middle and lower lungs, in unilateral lung or confi ned to one lobe. the foci may be isolated huge spherical or multiple nodular, without obvious surrounding infl ammatory responses, similar to those of tubercles or tumors. otherwise, they are diffuse miliary shadows or fl aky infi ltrative shadows. a male patient aged 60 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized on 2009-2-19 due to headache and vomiting for more than 10 days. in the csf, cryptococcus was found. by blood and sputum culture, cryptoccocus was detected. the diagnosis was cryptococcal meningitis, cryptococcal pneumonia and cryptococcal sepsis. after receiving amphotericin b antifungal treatment and dehydration, headache and vomitting were relieved. but chest ct scanning reexamination demonstrated increased pulmonary lesions, which was considered as tuberculosis. on 2009-4-1, he was given herv anti-tuberculosis treatment. twenty days ago he sustained weakened lower limbs, which gradually aggravated and completely paralyzed in the recent 1 week, his cd4 t cell count was 34/μl. hiv/aids related pulmonary cryptococcus infections should be differentiated from tuberculosis, primary or metastatic lung cancer. tuberculosis mostly is secondary tuberculosis, which is caused by repeated infections of tubercule bacillus. lesions show fl aky or fl occulent shadows in the two upper lungs, with blurry boundaries. the wrapped necrotic foci by fi bers develop into nodules. it can also show miliary shadows, mostly with mediastinal lymphadenectasis. it should also be differentiated from primary or metastatic lung cancer. cryptococcus is a relatively common pathogen of pulmonary fungal infection, and mostly develops in aids patients. usually, it is a disseminated disease, with common involvement of the central nervous system and the lungs. in immunocompetent patients, the nodular granuloma caused by the pathogen is similar to those of other pulmonary fungal infections. in patients with serious immunosuppression, wide tissue infi ltration of the pathogens may occur in the lungs. liked shadows, parenchymal changes of air cavity and miliary nodules. the pathogenic fungi can be found mainly in the pulmonary interstitium. imaging fi ndings include singular or multiple nodules or masses, parenchymal changes of lung lobes and lung segments with clear or unclear boundaries in size of 1-10 cm. there may be also miliary lesions, lymphadenectasis and cavity shadows. candida is an opportunistic pathogen, which widely exists in nature. candida albicans parasitize in the oral cavity, laryngopharynx, upper respiratory tracts, vaginal and intestinal mucosa of human being. pulmonary and bronchial moniliasis is commonly caused by candida albicans which has the strongest pathogenicity. after its invasion into the tissues, candida transforms into hyphae and multiplies in a large quantity, with strong toxicity and ability to fi ght against phagocytosis. aids patients may have disseminated pathological changes. only when the immunity is compromised, the pathogen invades into the bronchus or lungs to cause diseases. therefore, pulmonary candida infection is commonly secondary. candidosis can cause acute infl ammation in bronchus and lungs, mainly exudation of neutrophils, which can be divided into two types: bronchitis type and pneumonia type. the pathological changes in the early stage are acute suppurative infl ammation, accompanied with the formation of abscesses. by the naked eyes observation, they are large fl aky parenchymal changes, with central grayish white coagulative necrosis. under a microscope, the lesions are large fl aky caseous necrosis, accompanied with the formation of abscesses, and surrounding infi ltration of hyphae and phagocytes. in the advanced, there are caseous necrosis, formation of cavities, fi brosis and granuloma. symptoms in aids patients are mild, with frequent cough, with a small amount of white mucous phlegm or thick phlegm, no fever or low grade fever; scattered spots of white membranes in the mucosa of oral cavity, throat and bronchus. dry rales can be heard occasionally in both lungs. in aids patients, the manifestations are mostly acute pneumonia or sepsis, with chills, fever, cough, expectoration of white mucous jelly liked phlegm or thick phlegm often with blood or necrotic tissue. the thick sputum, candidal hyphae and shedding cell debris can be condensed into small colloid clumps, with yeast smell. other symptoms include even haemoptysis and diffi culty breathing. dry and moist rales can be heard in lungs. symptoms may be diffi culty breathing, rhinocnesmus, runny nose and sneezing. wheezing rales can be heard in both lungs. chest x-ray chest x-ray demonstrates nodular shadows and fl aky parenchyma changes in unilateral or bilateral lungs. sometimes there is miliary infection. ct scanning demonstrates most lesions in the middle and lower lung fi elds, with rare involvement of the apex. there are thickened lung markings or diffuse small fl aky/patchy shadows, some of which can fuse into large fl aky dense shadows, with blurry boundaries. nodules, due to bleeding around it, may be surrounded by ground glass liked shadows, which is necrotic bronchopneumonia, usually accompanied with a large quantity neutrophils. cough expectoration with white mucous phlegm or thick phlegm, hemoptysis and shortness of breath. examinations of the oral cavity and the throat demonstrate spots liked white membrane covering the surface, and dry and moist rales in the lungs. successive cultures of phlegm, lung tissue, pleural fl uid or cerebrospinal fl uid repeatedly demonstrate the same strain of candida, or direct microscopic fi ndings of large quantity pseudohyphae or hyphae and groups of spores can defi ne the diagnosis. it is demonstrated to have thickened and deranged lung markings in double lung, diffuse small fl aky/patchy shadows, fusion of some small shadows into large fl aky dense shadows, with blurry boundaries, enlarged hilum and blurry structures. the conditions progress rapidly, with repeated lesions occurrence. hiv/aids related pulmonary candida infection should be differentiated from bacterial pneumonia. bacterial pneumonia often has symptoms such as high fever, cough, expectoration, chest pain and shortness of breath. ct scanning demonstrates fl occulent infi ltrative shadows or parenchyma changes and cavities. the pathogen can be detected in the sputum or chest liquid. hiv/aids related pulmonary candida infection should be differentiated from virus pneumonia. viral pneumonia fi rstly causes upper respiratory tract infection, which spread downward to cause pulmonary infl ammation. the demonstrations a male patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever, cough, expectoration, chest pain and shortness of breath, with pulmonary parenchymal changes sign and moist rales. his cd4 t cell count was 18/μl. include ground glass liked changes in the lung fi elds or mass shadows. the defi nitive diagnosis should be based on throat swabs, virus isolation from the sputum and serum specifi c antibodies test. hiv/aids related pulmonary candida infection should be differentiated from pulmonary tuberculosis. in the early stage, the symptoms and signs include irritative dry cough, expectoration, hemoptysis and cavities in lungs. (detailed manifestations of tuberculosis see the section about tuberculosis in this chapter) its diagnosis mainly should be based on chest x-ray and fi ndings of tubercule bacillus in sputum or other specimens, or tuberculosis specifi c pathological changes. hiv/aids related pulmonary candida albicans is a widespread dimorphism bacteria. the oval shaped budding yeasts and hyphae both can be found in the tissues. candidiasis is a common disease in aids patients. chest x-ray demonstrates unilateral or bilateral patchy parenchymal changes of the air cavity and nodules with unclear boundaries. miliary lesions are common. hrct demonstrates multiple nodular shadows in both lungs, often accompanied with parenchymal changes. its defi nitive diagnosis should be based on the fi ndings of candida albicans in the tissues. penicillium marneffei (pm) is a newly found penicillium in 1956, which is a special strain with a distribution in south east asia and southern china. rhizomys is its natural host. in 1973, disalvo et al. [ 12 ] reported the fi rst case of natural human pm infection. in 1984, the fi rst case of human pm infection in china was reported in guangxi zhuang autonomous zone. pm is an opportunistic pathogen and immunocompromised people are susceptible to its infection. its spreading is along with soil contaminated by rhizomys feces to invade human body via the respiratory tract, the digestive tract and skin defects. pm infection is believed to be one of the most common opportunistic infections in aids patients in southeast asia, which has an increasing incidence. pm is the only dimorphic fungus in hyphomycetes penicillium, which is a special strain of penicillium. at different culture temperatures, it shows conversion of biphasic forms: fungal phase at 25 °c and yeast phase at 37 °c. the fungal phase is the hyphae of many cells, with certain biological morphology, such as penicillus, conidiophore, chain liked conidiospore and chains between spores. the yeast phase shows unicellular or bicellular form. in the growth process of pm, large quantity bright rosy or dark rosy pigments are produced, which is characteristically pm. the pigment of yeast phase is secondary metabolites of cells with strong hydrophobicity, which can promote the adhesion of conidiums in fungal phase and cells in yeast phase to the alveolar macrophages and other cells surface in the human body. the pigment monoclonal antibody (mab) can interrupt the pathological process of adhesion. in addition, this pigment can determine the expression of cluster-encoding genes mbr through diffusion and penetration of drugs to the cell membrane, thus preventing the penetration of hydrophilic antifungal drugs, such as fl uconazole. that is to say, it improves the natural antifungal resistance level of pm. the soluble components of the pigment in fungal phase can trigger the generation of anti-conidium antibody (only igg) in animals to prevent its spreading in the body. the phenomenon proves that, in terms of tissue invasion, fungal phase is less powerful than yeast phase. conidium in fungal phase is the carrier of pathogen while the cells in yeast phase are the real pathogenic factors. when pm spreads to the target organ along with the blood fl ow, it is engulfed by mononuclear phagocytes. in the cases of replication itself and further spreading, reactive proliferation of phagocytes is caused. mononuclear phagocyte system has strong defense ability. in the cytoplasm of proliferated mononuclear phagocytes, various amounts of pm can be found. pm mainly invades into the body via the respiratory tract, digestive tract and skin defects. in immunocompetent people, local abscesses form in the invasion site, which is characterized by the thick mucus fl uid, with mainly necrosis and liquefaction. vascular reactions and exudation of neutrophil leukocytes and body fl uids is less than abscesses induced by common purulent bacteria. the clinical manifestation is confi ned suppurative infl ammation. when the immunity is compromised, due to the insuffi ciency of immunologic factors, it is diffi cult for the immune cells to restrict and digest the engulfed pathogens, which leads to confi ned suppurative reaction. therefore, it often presents with diffuse lesions. the pulmonary lesions are principally interstitial exudative infl ammation. the typical penicillium marneffei disease has acute or subacute onset, along with fever, chills and shivers, cough and expectoration, hemoptysis, shortness of breath, abdominal pain, diarrhea, bloody stool, fatigue, central necrotic papula mainly in the head and face and scattering in the trunk and extremities as well as hepatosplenomegaly. bone marrow smear and pas staining can be performed to detect the pathogen. blood, bone marrow, pleuroperitoneal fl uid, phlegm and skin defect tissue are collected for the culture at double temperatures with sabouraud'broth medium. at the temperature of 25 °c, the colony is in dark red with villous surface, with surrounding red wine liked pigments to gradually spreading into the medium. biopsy of lymph nodes and skin defects with pas staining and wright & gimsa staining can be performed. wbc count, hemochrome, platelets, ast and cd4 t cell count. ct scanning and routine chest x-ray are the diagnostic imaging examinations of choice, which can facilitate to understand the size, morphology, location, quantity and density of the lesions. it demonstrates multiple small nodular shadows in the lungs, multiple honeycomb liked cavities in both lungs and mediastinal lymphadenectasis. abdominal scanning demonstrates different degrees of hepatic, splenic and retroperitoneal lymphadenectasis, which can fuse into a huge mass. routine chest x-ray it demonstrates thickened, deranged and blurry pulmonary markings, small cavities, military nodular shadows, mass liked shadows, spots and patchy shadows, ground glass liked changes, pleuritis and pleural effusion. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. her husband had a history of drug abuse and she complained of abdominal pain and fever for more than 2 months, with accompanying face rash and diarrhea. her cd4 t cell count was 5/μl. by examinations, she sustained skin palpula, abdominal tenderness, central concave skin rashes on the face, neck, and upper limbs. there was a palpable mass in the upper left abdomen, hard and tenderness, in a size of 12 × 12 cm. more than 2 months before her admission, she had persistent dull abdominal pain that is commonly in the upper left abdomen, with accompanying fever and face skin rashes that is gradually increasing and spreads to the neck and upper limbs. she also had hepatosplenomegaly, abdominal aortic lymphadenectasis and ascites. a female patient aged 51 years was confi rmatively diagnosed as having aids by the cdc. she had an unhealthy sexual life, with complaints of fever and cough for more than 1 month. her cd4 t cell count was 7/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he had been found to be hiv positive for 5 years, with complaints of irregular fever, cough, fatigue and dizziness for 10 days to be hospitalized. by examinations, his cd4 t cell count was 40/μl, hiv positive, subcultivation of strains demonstrated typical biphasic penicillium. by fungus culture, typical penicillus was found. by bone marrow smear, the round corpuscles mainly located in the macrophages. a male patient aged 32 years was confi rmatively diagnosed as having aids by the cdc. he had a history of extramarital affair, with complaints of fever, cough, chest distress, shortness of breath, fatigue, poor appetite, poor sleep, weight loss and shortness of breath after activities for more than 3 months. his cd4 t cell count was 23/μl. chest x-ray and ct scanning both demonstrate multiple nodules in different sizes and cavity shadows. the cavities cluster into honeycomb liked changes with uneven thickness of the walls, clear boundaries and surrounding infl ammatory exudates. some of the nodules infuse into mass dense shadows. lesions in both lungs have a symmetrical or asymmetric distribution, with no characteristic leions. mediastinal lymph nodes are obviously enlarged. sputum smear for direct microscopy demonstrates candida. pms are found by fi brobronchoscopy lavage smear and biopsy. (1) candida skin test shows positive. (2) fluorescent antibody test for pms is performed in procedures of direct smear, fungal colony culture and histopathological examination of the tissue sections. metabolites test of pm and pcr can be performed to determine the gene sequences of pm for the early diagnosis. hiv/aids related penicillium marneffei pneumonia should be differentiated from bronchiectasis and blood borne staphylococcus aureus pulmonary abscess. although the cases of bronchiectasis are demonstrated to have clustering round shadows on the cross sections, the wall is thinner and even, accompanying to the spots liked vascular shadows. some lesions have typical railway liked bronchial dilation signs. it is demonstrated to have multiple small cavity lesions in both lungs in line with the evenly distributing blood borne lesions. the lesions are rarely clustering. the wall of the cavities is thick and even with surrounding marginal exudates and blurry boundaries. with the steady increasing of hiv infections, reports on the complication of pm disease have also been increasing recently. the disease can be localized but mostly disseminated, with the involvement of lungs, liver, skin, lymph nodes and other tissues and organs. therefore, it is known as penicilliosis marneffei or disseminated penicillium marneffei infection in literature reports. due to the insuffi cient knowledge about the disease, diagnosis is delayed or missed. in thailand, penicilliosis marneffei has been the indicator disease of aids. about 20 % aids patients are infected by pm and 70 % have necrotic papula, which is characteristically disseminated penicillium marneffei infection. ct scanning demonstrates hiv/aids complicated by disseminated penicillium marneffei infection as fl aky parenchyma shadows in the lungs, clustering of cavities and nodular shadows, mediastinal lymphadenectasis and pleural infl ammation responses, which can facilitate its clinical diagnosis. mucormycosis is a rare kind of conditional fungal disease, with its pathogen, mucor, distributing widely in the natural world. it generally fails to cause diseases, but can cause systematic infections in immunocompromised people. mucor often invades into the human body via the nose to cause paranasal sinuses and orbital infections, which can further invade into the brain to cause meningitis and frontal abscesses. pulmonary mucormycosis is only second to the nasal-cerebral infection. it can spread via the respiratory tract to cause pulmonary infections. in addition, there are also skin and gastrointestinal mucormycosis as well as disseminated mucormycosis. the pathological changes of hiv/aids related pulmonary mucormycosis are mainly hemorrhagic necrotic infl ammation. the defense mechanism of immunocompetent people is to kill the fungal spores with the phagocytosis of macrophages and oxidation killing mechanism. in immunocompromised or immunodefi cient patients, the macrophages are often too weak to restrain the engulfed spores from germinating. therefore, the disease occurs. vascular vessels are susceptible to the invasion of mucor, especially the arteries. mucor can locally multiply to cause the formation of blood clots and embolization, and disseminate to other organs along with the blood fl ow. the main lesions are hemorrhage and necrosis of local tissues and exudation of neutrophils. the lesions of hemorrhage and necrosis are possibly related to the arteriole lesions caused by hyphae. the clinical manifestation of hiv/aids related pulmonary mucormycosis is a nonspecifi c pneumonia. the most common symptoms reported in literatures are persistent high fever, cough, hemoptysis, chest pain and diffi culty breathing. it has a rapid progression, with a high mortality rate of 65-96 %. lung lesions are hemorrhagic infarction or pneumonia, which can cause high fever, cough, expectoration, shortness of breath, chest distress, chest pain, hemoptysis (pulmonary artery involved) and other symptoms. moist rales can be heard in both lungs and pleural rubs can be heard in the cases of pleura involvement. 1. assisted by bronchofi broscopy, lung biopsy can be performed. 2. histological examinations include bronchial lavage fl uid examination, exploratory thoracotomy and puncture of lung tissues for biopsy. 3. chest x-ray and ct scanning are conventional effective examinations. the lesions are frequently found in the dorsal and medial segments of both lungs. early exudation shows miliary shadows, cavity shadows with no wall or with thin wall and small bronchiectasis shadows. their further progression may cause fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion, with accompanying mediastinal lymphadenectasis. 1. the fi ndings of mucor or their hyphae by sputum and bronchofi broscopic biopsy. 2. the diagnostic imaging demonstrates lesions commonly in the dorsal and medial segments of both lungs. there are diffuse scattering miliary shadows, cavity shadows with no wall or with thin walls and small bronchiectasis shadows. they further progress into fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion. 3. often with accompanying mediastinal lymphadenectasis. chest x-ray of pulmonary mucormycosis demonstrates progressive infi ltrated parenchymal changes, or masses, nodules, cavities and pleural effusion. it needs to be differentiated from miliary tuberculosis. hiv/aids related miliary tuberculosis are commonly demonstrated as chronic blood borne disseminated tuberculosis, with lesions distributing symmetrically in both lungs. its long term progression causes fusion into masses. otherwise, it can be cured by anti-tb therapies. the early lesions are no-wall cavities or thin wall cavities and small bronchioectasis shadows, based on which the differential diagnosis can be made. almost all cases of hiv/aids related pulmonary mucormycosis are found in immunocompromised patients. chest x-ray demonstrates parenchyma changes and isolated or multiple nodules or masses. the parenchyma changes are patchy or fuse, with unilateral or bilateral distribution. about 20 % patients have pleural effusion and less than 10 % patients have hilar or mediastinal lymphadenosis. ct scanning demonstrates singular or multiple nodules or masses, commonly with clustering or honeycomb liked cavities. cytomegalovirus pneumonia has extensive pathological changes in the lungs. pathologically, it shows interstitial pneumonia, with the lesions randomly blood borne distributing in the lungs. the distribution can be diffuse, panlobular or focal. the target cells of pathological changes include alveolar cells and macrophages. diffused pulmonary interstitial edema and fi brosis as well as alveolar swelling, focal necrosis, bleeding and hyperplasia occur after cmv infections to cause hypoxemia. gross observation of fresh specimens demonstrates pulmonary surface edema and fl aky blooding spots. fixed specimens demonstrate brown hard lung tissues. under a microscope, pulmonary interstitial congestion as well as infi ltration of lymphocytes and mononuclear cells can be found, with the involved epithelial cells enlarged. in the pulmonary interstitium and alveoli, there are intranuclear inclusions, cytoplasmic inclusions and fl uid containing abundant proteins. the classical intranuclear inclusions can be found in the cells, purplish red or purplish blue, round or oval, with surrounding halos in eagle eyes sign. atypical cytomegalic inclusions in cells are slender, long and round liked with abundant cytoplasm and accentric nucleolus, which are blurry, unclear and atypical ( fig. 17 .97a-e ). immunohistochemitry demonstrates hiv p24 antigen positive. the systemic symptoms of cmv infection include fever, joint and muscle soreness and pain, abdominal distension and orthostatic hypotension. the respiratory symptoms include paroxysmal dry cough, diffi culty breathing, cyanosis and three depressions sign. according to the imaging fi ndings of cmvp, cmv pneumonia can be classifi ed as diffuse, miliary and mass types, among which the diffuse type is the most common. cytomegalovirus can be separated from respiratory secretions culture and urine culture by using human embryonic fi broblasts. by urine sediment smear, giant cell with inclusions can be found. by using immunofl uorescence, indirect hemagglutination inhibition and complement fi xing test, the antibody titer can be found increased. indirect immunofl uorescence test and immunoenzymic staining test can be applied to detect the anti-cmv-igg and igm antibody. in addition, enzyme-linked immuno sorbent assay (elisa) can also be performed to detect the anti-cmv-igg and igm antibody. cmv-igm antibody positive indicates a recent infection, which has diagnostic value. a single serum cmv-igg antibody positive indicates a previous infection. and during the acute and recovery phases, double serum cmv-igg antibody titer being no less than four times increase has diagnostic value, indicating a recent infection. pcr can be applied to quantitatively determine the viral load in the whole blood, blood plasma, leukocytes, urine, bronchoalveolar lavage fl uid (balf), cerebrospinal fl uid and the tissue specimens, which is believed to be the best way for the diagnosis of invasive cmv infection. chest x-ray is the most commonly used examination. chest ct scanning is superior to chest x-ray in terms of resolution and detection rate of the lesions. pulmonary demonstrations by cmvp include diffuse interstitial infi ltration and alveolar infi ltration to form reticular shadows, nodules and parenchymal changes. a baby boy aged 6 months was confi rmatively diagnosed as having aids by the cdc. he was infected via vertical transmission from mother to child, with recurrent cough after being born and the most recent cough for 4 days as well as wheezing cough in throat for 1 day before he was hospitalized. he had a past history of premature birth, with primary apnea and bronchial pneumonia and was hospitalized for treatment. later, he was admitted for three times due to cough, which was diagnosed as interstitial pneumonia. by examinations, wbc 16.3 × 10 9 /l, lym lymphocyte count 11.7 × 10 9 /l, cmv-ab weak positive, blood sedimentation 11 mm/h, and tuberculosis antibody negative. after treated by broad-spectrum antibiotic therapy, the therapeutic effi cacy is unfavorable. cytomegalovirus (cmv) infection is an important cause of pneumonia in patients with compromised immunity. imaging fi ndings include nodular shadows with blurry boundaries and bilateral fl aky parenchymal changes of the lungs. the nodules tend to be bilaterally symmetric or asymmetric, with centrilobular distribution. histopathological manifestations are nodular alveolar hemorrhage, necrosis and infl ammatory lesions, and diffused alveolar lesions. the nodules tend to have a centrilobular distribution, indicating occurrence of bronchiolitis. in aids patients, if the diameter of nodules is under 10 cm, it is most likely to be viral infection. the size of the nodules can facilitate the differential diagnosis of pulmonary infections. herpes simplex viral pneumonia (hsvp) often occurs in the upper respiratory tract, and rarely in the lower respiratory tract. human herpes simplex virus can be divided into two types, namely herpes simplex virus type i (hsv-i) and herpes simplex virus type ii (hsv-ii). herpes simplex viral pneumonia mostly occurs in patients with immunodefi ciency. herpes simplex viral pneumonia can be caused by hsv-i and hsv-ii, both of which have a nucleocapsid with 20 surfaces. the thickness of the nucleocapsid is about 100 nm, which is composed of 162 capsomeres. the nucleocapsid contains the core of the virus dna. the virion gains the phospholipid rich viral envelope when it passes through the nuclear envelope. the nucleocapsid gemmates after it passes through the nuclear membrane and is released to the cell surface. the nucleocapsid can also be released outside the cells or gains its access into the neighbour cells for further reproduction. herpes simplex virus replicates itself in the cell nucleus to produce histopathologic changes of herpes virus replication, with visible cowdry a type intranuclear inclusions. the pathogenesis process of herpes simplex virus infection in the body can be divided into fi ve stages: initial skin mucosa infection, acute ganglia infection, latent infections, re-activation, and recurrent infections in susceptible hosts. patients infected by herpes simplex virus can produce igm, igg and iga antibodies to fi ght directly against virus protein, which may play a role in changing the severity of the infection. interferon also participates in the control of herpes simplex infection by inhibiting the virus or regulating the defense mechanism. genetic factors may be also related to the herpes virus infection. cellular immunity can confi ne the infection. herpes virus cannot reproduce in the alveolar macrophages of human body, which is also the reason why herpes virus is less than cytomegalovirus in lungs. currently, it is believed that herpes simplex virus is an important pathogen of respiratory infections, especially in immunocompromised patients. localized herpes simplex viral pneumonia occurs due to the direct spreading of virus in the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is caused by the virus spread from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia caused by hsv-i or hsv-ii has been reported, and both are related to diffused infections. but in patients without herpes simplex viral infection in skin mucosa, herpes simplex viral pneumonia can also occur. herpes simplex viral pneumonia is caused by the direct spreading of the virus from the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is cause by the spreading of the virus from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia cause by either hsv-i and hsv-ii have been reported, both of which are related to diffuse infections. in such cases, the lung tissues may have infl ammatory infi ltration, lung parenchyma necrosis, bleeding, cellular swelling and round, diffuse interstitial pneumonia. and in most cases, there are accompanying cellular changes of herpes virus infection such as the intranuclear eosinophilic inclusions, necrotic herpes simplex viral trachitis. herpes simplex viral bronchitis has demonstrations of mucosa erythema, edema, exudation and ulcer, with coverage of the surface by fi brous purulent membranous secretions. the common initial clinical symptoms of herpes simplex viral pneumonia are shortness of breath, cough, and fever with a body temperature being higher than 38.5 °c, decreased wbc count, hypoxemia, respiratory dysfunctioning and azotemia. hsv pneumonia may be accompanied by mucocutaneous lesions by hsv, which show earlier than those of pneumonia. there may be concurrent fungus, cytomegalovirus or bacteria infection. herpes simplex viral tracheobronchitis may show tracheal or bronchial spasm or stenosis. etiological examinations hsv can be detected in tracheobronchial secretions, bronchoalveolar lavage fl uid and lung tissues. early sampling should be performed under the guidance of a bronchofi broscope. tissue culture is the most sensitive and specifi c method for the diagnosis, which can also be used for the classifi cation of the virus. papanicolaou (pap) or tzank test is a fast and cheap method for cellular diagnosis. elisa can be used to detect herpes simplex virus, with a sensitivity of up to 95 % and a high specifi city. chest x-ray demonstrations are less valuable for the differential diagnosis. pulmonary ct scanning can be applied for the differential diagnosis. herpes simplex viral pneumonia includes three types, namely local, multiple or diffuse interstitial infi ltration. in the early stage, typical hilar or diffuse interstitial shadows with increased density can be found, with thickened bronchial wall. as the disease progresses, cloudy or patchy alveolar tamponade and fusion can be found. chest x-ray may demonstrate negative for herpes simplex viral trachitis and bronchitis. herpes simplex viral pneumonia should be differentiated from bacterial pneumonia, cmv pneumonia, and infl uenza pneumonia. hiv/aids related herpes simplex viral pneumonia is mostly demonstrated by multiple signs, including small nodules, ground glass liked shadows and patchy parenchymal changes. the nodules are in centrilobular distribution, mostly with accompanying branches liked shadows (tree buds sign). chest x-ray demonstrates diffuse lung parenchymal changes. imaging fi nding are parenchymal change areas with bilaterally blurry boundaries. generally, the nodules have a diameter of 2-10 mm. ct scanning with high resolution demonstrates nodules with surrouding ground glass liked density lesions. lymphoid/lymphocytic interstitial pneumonia (lip) is more common in children with aids. the cdc in the united states has defi ned lip in children under the age of 13 years as the diagnostic indicator of aids. the predictive diagnostic criteria include chest x-ray demonstration reticular nodular changes in pulmonary interstitium of both lungs for no less than 2 months, undetectable pathogens and no responses to the antibiotic therapy. currently, hiv/aids related lymphocytic interstitial pneumonia is considered to be related to hiv and epstein-barr virus, human t cell leukemia-lymphoma type i virus (htlv-i) and hiv-i. the infection of the above viruses causes pulmonary lymphatic hyperplasia and other systemic diseases. about 22-75 % children with hiv infection sustain lip, and 3 % in adults. most cases of non-hiv infected patients with lymphoid interstitial pneumonia are women, at average age of 56 years old, more commonly in the age group of 40-50 years old and above 70 years old. the pathological manifestations are infi ltration of small and mature lymphocytes as well as plasma cells in alveolar septum and the alveolus, extensive interstitial fi brosis and non-caseous granuloma. it is characterized by diffuse infi ltration of lymphocytes, plasmocytes and histocytes in the pulmonary interstitium. the lymph follicle with germinal center is more common. hyperplasia occurs in type ii alveolar epithelium, and the macrophage increases in the alveolar cavity. there are rare or mild intraalveoli organization and macrophage aggregation. staining of the immune globulin light chain demonstrates poly-clone b cells. the clinical symptoms are in progressive development, with cough and suffocation, rare hemoptysis and sjogren syndrome commonly in mouth and eyes. by examinations, the signs have slight difference between adults and children. in children, there are lymphadenectasis, hepatosplenomegaly, enlargement of parotid gland, clubbing fi ngers and wheezing sound. in adults, there are lymphadenectasis, slight fi ne bubbling rales, as well as hepatosplenomegaly and enlargement of parotid gland in 1/3 patients. 1. peripheral hemogram demonstrates increased lymphocytes and eosinophilic granulocytes. 2. myelogram demonstrates increased lymphocytes, plasmocytes and eosinophils. 3. blood biochemical examination demonstrates increased immune globulin, predominantly lgm. 4. blood gas analysis demonstrates hyoxemia. 5. pathogenic examinations by bronchofi broscopy, bronchial alveolar lavage and biopsy can defi ne the diagnosis. 6. pulmonary function examinations demonstrate restrictive ventilatory disorder, lower lung compliance and impaired diffusion function. impaired diffusion function is a more sensitive indicator in monitoring the progress of the disease. 7. chest x-ray is the most commonly used imaging examination, while chest ct scanning is commonly applied for the differential diagnosis. chest x-ray demonstrates hiv/aids related lymphoid interstitial pneumonia as reticular or reticular nodular shadows of lung markings in both lungs. hrct demonstrates bilateral diffuse ground glass liked density shadows. perivascular thin-walled pneumatocele is common. pneumatocele induced by lip is commonly found in the middle lung fi eld, which probably is due to the valve effects caused by infi ltration of cells around bronchioles. manifestations of pneumatocele, together with ground glass liked density shadows, highly indicate lip. centrilobular and subpleural small nodules and thickened intralobular septa can be occasionally found. dysfunctional diffusion is a more sensitive indicator in monitoring the progress of the disease. hiv/aids related lymphoid interstitial pneumonia should be differentiated from allergic pneumonia, carcinomatous lymphangitis and pneumocystis carinii cysts. ct scanning with high resolution demonstrates characteristic lesions of hiv/aids related lymphoid interstitial pneumonia, including intralobular linear shadows and honeycomb liked changes, with common involvement of the subpleural area and the basal lung. its characteristic manifestations are clustering gas containing thin-walled cyst in a diameter of 2-10 mm, with clear cyst wall. shared wall between cysts is its characteristic demonstration. the surrounding ground glass liked density indicates infl ammation. intralobular linear shadows indicate interstitial fi brosis. pulmonary toxoplasmosis (pt) is caused by the toxoplasma parasitizing in cells. ludlam et al. fi rstly proposed the concept of pulmonary toxoplasmosis in 1963 [ 107 ] , arguing that toxoplasma can cause atypical pneumonia. later, there are some pathological reports about pulmonary toxoplasmosis or disseminated toxoplasmosis with lung involvement. in recent years, due to the global prevalence of aids, the incidence of pulmonary toxoplasmosis is increasing, with most cases being disseminated toxoplasmosis with lung involvement. pt has been one of the important opportunistic infections in patients with immunosuppression, especially aids patients. hiv/aids related pulmonary toxoplasmosis is a zoonotic disease, with cats as its main transmission source, followed by pigs and sheep. people are infected by intake the water or food contaminated by cats' feces or without cooked meat. immunocompromised aids patients are susceptible to this disease, and its occurrence is rare in immunocompetent people. after its invasion into the human body, the sporozoite in the cystozygote and intracystic cystozoite overfl ows to penetrate the intestinal wall mucosa and spread to the whole body tissues along with blood or lymph fl ow. the brain, heart, lymph nodes, and lung are the most vulnerable tissues and organs for the infection. any abnormality in the process of defense mechanism can cause impaired immune functions to eliminate the toxoplasma, which ultimately causes systemic and pulmonary infections. pulmonary toxoplasma infection may also be caused by the blood borne spreading of reactivated toxoplasma infection in other body parts, with no exclusion of reactivated pulmonary infection or primary pulmonary infection. ludlam et al. generally nominated toxoplasmosis as atypical pneumonia [ 107 ] . catterall et al. divided toxoplasmosis into three types: necrosis, infl ammatory infi ltration and toxoplasma invasion [ 109 ] . it can also be classifi ed as type a: subclinical or occult infection; type b: interstitial and atypical pneumonia; type c: necrotic pneumonia; type d: lobar pneumonia; and type e: granulomas pneumonia (toxoplasmoma). by naked eyes observation, the involved lungs are solid, with congestion and red brown section. the pleura have bleeding spots, with moderate peribronchial lymphadenectasis. under a light microscope, there is exudation of serous fl uids in alveolar cavities, occasional formation of transparent membrane or fi brin purulent exudation, infi ltration of small quantity neutrophils, proliferation and shedding of alveolar wall cells, and trophozoite and/or cysts of toxoplasma in epithelial cells and macrophages. the pulmonary interstitium may have infi ltration of lymphocytes and plasmocytes as well as visible fi broblasts and macrophages. the granuloma changes are also found in the lung tissues, with central stripes or localized necrosis and surrounding lymphocytes and small quantity multinucleated giant cells. it is diffi cult to fi nd toxoplasma in granuloma, but it can be found in the normal tissues around or near the granuloma. almost all cases of aids complicated by pulmonary toxoplasmosis are caused by disseminated toxoplasmosis with pulmonary involvement. it is commonly diffuse pulmonary infl ammation with serious symptoms, including high fever, cough, cyanosis, breathing diffi culty, possible occurrence of skin rashes, lymphadenectasis and meningitis. the chronic cases may have long-term low grade fever, cough, and weight loss. direct light microscopy of the specimen smear and enprint such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissue or other living tissues can be performed for pathogen examinations. sabin proposed that the staining test have high sensitivity and specifi city according to the fi ndings that mixture of fresh toxoplasma with normal serum can be stained deep by alkaline methylene blue staining, while its mixture with immune serum can be stained light or blank by the same staining. other assays including indirect fl uorescent antibody, indirect blood coagulation, and complement fi xation test can provide valuable reference for the diagnosis. toxoplasm is tested in pathological eaminations that can provide valuable reference for the diagnosis. chest x-ray is the most commonly used diagnostic imaging examination. and chest ct scanning can be applied for the differential diagnosis. imaging fi ndings of hiv/aids related pulmonary toxoplasmosis can be divided into four types: bronchial pneumonia, interstitial pneumonia, pleuritis and complication of cardiovascular disease. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings that distribute along with the bronchi in the middle and lower lung fi elds, scattered patchy shadows with uneven density and blurry boundaries, fusion of some shadows into large fl aky shadow and widened hilar shadow. the type of interstitial pneumonia is demonstrated as reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with stripes and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of complication of cardiovascular disease is demonstrated as heart failure (acute pulmonary edema), with signs of pericardial effusion. a male patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough and fever. his cd4 t cell count was 29/μl. by direct light microscopy, toxoplasma tachyzoites can be found in the specimens such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissues or other living tissues. the fl uorescent antibody and complement fi xation test are positive. the biopsy tissue culture and inoculation test are positive. in the lesions and their surrounding tissues of interstitial e c d fig. 17 . 105 (continued) pneumonia, necrotic bronchitis or granuloma, toxoplasma can be found. the diagnostic imaging demonstrates any one type of pulmonary toxoplasmosis, including bronchial pneumonia, interstitial pneumonia, pleuritis and cardiovascular disease, can be used as the evidence for the diagnosis of pulmonary toxoplasmosis. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings with a distribution in both middle and lower lung fi elds along with the bronchi, scattered patchy shadows with uneven density and blurry boundaries, fusion of some patchy shadows into large fl aky shadow and widened hilum. the type of interstitial pneumonia has typical demonstrations of reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with strip and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of cardiovascular disease may have signs of heart failure (acute pulmonary edema), and signs of pericardial effusion. hiv/aids related pulmonary toxoplasmosis should be clinically differentiated from infectious mononucleosis and mycoplasma pneumonia. with primary pulmonary lesions. pulmonary low malignant b cell lymphoma is the most common primary pulmonary lymphoma which is derived from mucosa related lymphoid tissue. the manifestations include slowly decreased alveolar transparency. pulmonary high malignant b cell lymphoma is extremely rare, which often occurs with singular lesion and primary disease such as immunodefi ciency. hiv/aids related malignant lymphoma is mostly caused by compromised immunity. hiv/aids related hodgkin's lymphoma is relatively rare. there are also reports about hiv/aids related t cell lymphoma with pulmonary involvement. hiv/aids related malignant lymphomas are mostly highly malignant large cells lymphoma. cerebral lymphoma is one of the defi ning diseases of aids. it has been reported that the clinical incidence of pulmonary infi ltration by malignant lymphoma is 10-20 %, but 29-50 % by autopsy. in the early stage, it is commonly asymptomatic. with its progression, symptoms of dry cough, suffocation, and small quantity clear phlegm occur. mediastinal lymphadenosis includes lymphadenectasis to compress the trachea by, blood vessels and nerves and lead to breathing diffi culty, superior vena caval obstruction syndrome, and hoarse voice. the pulmonary parenchyma lesions include reticular structure in the lungs. the clinical symptoms are cough, expectoration, suffocation and breathing diffi culty. a male patient aged 43 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and cough for more than 1 month. his cd4 t cell count was 56/μl. in patients with hiv/aids related kaposi's sarcoma, its serologic positive rate is 100 %. kaposi's sarcoma cells can produce il-6, during which il-6 plays a role as an autocrine factor to maintain the cell growth, paracrine cytokines, stim-ulate proliferation of other interstitial cells and induct the vascular growth. therefore, kaposi's sarcoma is a kind of tumor with abundant blood vessels. before the application of harrt, the incidence of kaposi's sarcoma in male homosexuals is 21 %. after the clinical application of harrt treatment, the incidence is decreasing. in addition to hhv-8, some studies indicated that most patients with kaposi's sarcoma have hia-dr5 alleles, suggesting a possible relationship between kaposi's sarcoma and the heredity. there is no obvious difference between hiv/aids related kaposi's sarcoma and classic kaposi's sarcoma in pathological changes. early pathological manifestations are chronic infl ammation or granulomatous infl ammation, with formation of new vascular and lymphatic vessels and accompanying edema and bleeding. the fi ndings of large and protruding endothelial cells in granuloma tissue with accompanying erythrocytic exudation and hemosiderin particles have great signifi cance for the early diagnosis. the pathological changes in the advanced stage are signifi cant proliferation of the endothelial cells, and proliferation of fi broblasts around capillaries. in the advanced stage, the lesions are often accompanied by extensive connective tissue hyperplasia, which presents diffi culty for its differentiation from common sarcoma. when it is diffi cult to defi ne the diagnosis by light microscopy, immunohistochemical examinations can be used to defi ne the diagnosis. the pathological changes are characterized by lesions confi ned to the epithelial lamina propria, gathering of spindle cells with mild heteromorphism around many lacuna vasorum with irregular lumen, erythrocytic exudation and hemosiderin sedimentation. the atypical lacuna vasorum can be compressed by proliferative spindle cells to be absent. vascular endothelial cells and peripheral spindle cells may have mitotic phase in the advanced stage, with increased heteromorphism cells. the infl ammatory cells are mainly plasma cells, with acidophilic corpuscles and pas staining positive, which can assist the pathological diagnosis. pulmonary kaposi's sarcoma in aids patients rarely has symptoms. it is commonly concurrent with pulmonary opportunistic infections, with symptoms of cough, diffi culty breathing and fever. other symptoms are related with the location of the tumors. the involvement of trachea or bronchi can cause luminal stenosis. the mediastinal tumor can compress and obstruct lymph vessels to cause pulmonary edema or a large quantity pleural fl uids, which result in respiratory diffi culty, and even respiratory failure. (1) sampling by bronchoscopy or endoscopy to prepare pathological section. (2) chest x-ray demonstrates its typical manifestation of pleural effusion. dr demonstrates enlarged and deranged hilum in both lungs in bird nest liked appearance. there is light density fl aky shadows in the both lower lungs. ct scanning demonstrates multiple rounds liked nodular shadows in the middle and lower lung fi elds of both lungs with clear boundaries. there are also mediastinal and hilar lymphadenectasis, with common involvement of the pleura and bilateral pleural effusion in a small quantity. a male patient aged 33 years was confi rmatively diagnosed as having aids by the cdc. he had been detected as hiv positive for 5 months, with complaints of recurrent cough and nausea for 10 days and was hospitalized on jan. 7, 2004. the transmission route was unknown because he denied histories of intraveneous drug abuse, paid blood donation, blood transfusion and unhealthy sexual behaviors. five months ago, he was diagnosed as aids in the stage of aids in our hospital, and hospitalized to treat pcp, with a cd4 t cell count of 17/μl. his symptoms were quickly relieved after pcp treatment and he continued the antiviral therapy for almost 5 months after being discharged. by physical examinations, he was in poor spiritual condition, a light blue nodule in size of 0.5 × 0.5 cm in the left upper chest wall with medium hardness, palpable lymph nodes in size of 1.0 × 2.0 in the opisthotic area and inguen, no tenderness and being movable. by the digital rectal examination, a palpable prominent nodule with wide base at 4 cm 7 points away from the anus, with fl exible texture and smooth surface. by the auxiliary examinations, wbc 3.9 × 10 9 /l, neμt 48.3 %, lym 34.9 %, mon 7.4 %, eos 9.0 %, rbc 3.27 × 10 12 /l, hgb 126 g/l, plt 210 × 10 9 /l, routine urine test normal, blood sedimentation 16 mm/h. by hepatitis b examinations, hbsag, anti-hbe and anti-hbe positive. his cd4 t cell count was 91/μl. by abdominal b ultrasound, multiple low echo nodules in the abdominal cavity, the largest in size of 1.2 × 1.0 cm, which are suspected to be enlarged lymph nodes. on jan. 14, he received inguinal lymph node biopsy, with pathological report of kaposi's sarcoma. during the treatment and following up, the involvement of lungs, digestive tract, lymph nodes and skin is suspected, with the diagnosis of phase ii kaposi's sarcoma and chemotherapy was recommended. reexamination by chest x-ray demonstrated normal cardiopulmonary phrenic. abdominal b ultrasound failed to fi nd enlarged lymph nodes. ct scanning demonstrated shrinkage of lesions in both lungs and mediastinal lymph nodes, with only palpable soybean sized submandibular lymph node. by examinations after chemotherapy, cd4 t cell count 67/μl, viral load 63,000 copies/ml. the patients had multimorphological erythema drug eruptions, which was suspected as drug allergies of chemotherapy, which were absent after symptomatic treatment. the following ups so far show no recurrence of kaposi's sarcoma, with his cd4 t cell count fl uctuating around 400/μl. he can work as usual. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he had a history of homosexual behaviors, with complaints of fever and cough for 2 months as well as chest distress for more than 20 days. since, july 2010, fever with a body temperature of about 37.5-37.8 °c occurs, with cough, yellowish bloody sputum, and dark purplish patchy skin rashes. by examinations, his anti-treponema pallidum antibody positive, multiple dark purplish patchy skin rashes on the face, eyelid, lower jaw, hairline, chest and abdomen with skin surface desquamation, palpable bilateral cervical lymphadenectasis and the largest in size of 10 × 19 mm. by laboratory tests, wbc 5.98 × 10 9 /l, n 78.74 %, rbc 2.22 × 10 12 /l, hgb 71 g/l, plt 204 × 10 9 /l, cd4 12/μl. a male patient aged 27 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough for more than 2 months, chest distress for more than 1 month, and bloody sputum for half a month and was hospitalized. he had a history of homosexual behaviors. by examinations on admission, multiple round purplish blue skin rashes nodules on the limbs. his cd4 t cell count was 9/μl. demonstrations can also be fl aky fl occulent areas with blurry density or parenchymal density areas along with bronchi. 3. lung puncture for histopathological biopsy demonstrates irregular vascular lumen in the dermis, proliferation of endothelial cell with accompanying heteromorphism. in some cases, there are tumor masses composed of spindle cells and epithelial cells. other sarcoma and vascular tumor hiv/aids related kaposi's sarcoma should be differentiated from other sarcoma and vascular tumor. ks invasion of the digestive mucosa can cause bleeding and upper gastrointestinal symptoms. the pathological lesions can be diagnosed by upper gastrointestinal endoscopy or biopsy. in the cases of no fever and exclusion of infections, the typical imaging demonstrations and bronchoscopy fi ndings can defi ne the diagnosis of pulmonary ks. hiv/aids related kaposi's sarcoma should be differentiated from pneumocystis carinii pneumonia. the lesions of pcp are mostly symmetric ground glass liked density shadows extending outwards from the hilum in both lungs. in the middle and advanced stages, nodules, fi brosis and cavities occur, rarely with pleural effusion. lung cancer commonly refers to the cancer in lung parenchyma, usually does not include those mesodermal tumors originating from other pleura, or other malignancies like carcinoid, malignant lymphoma, or metastatic malignancies for other body parts. therefore, the following lung cancer we are discussing about refers to the malignancies originating from bronchial, or bronchiolar epithelial cells, accounting for 90-95 % of the lung parenchyma malignancies. the cause of lung cancer is still not completely known. data have indicated that the risk factors of lung cancer include smoking (including second-hand smoke), asbestos, radon, arsenic, ionizing radiation, halogen alkenes, polycyclic aromatic compounds and nickel. long-term smoking can cause proliferation of the bronchial mucosal epithelial cells and proliferation of squamous epithelium to induce squamous epithelium carcinoma or undifferentiated small cell carcinoma. non-smokers can also develop lung cancer, but adenocarcinoma is more common among them. long-term exposure to radioactive substances, like uranium and radium, and its derivatives; carcinogenic hydrocarbons, like arsenic, chromium, nickel, copper, tin, ferri, coal tar, bitumen oil, petroleum, asbestos and mustard gas, all can induce lung cancer, which is commonly squamous carcinoma and undifferentiated small cell carcinoma. some chronic pulmonary diseases, such as tuberculosis, silicosis and pneumoconiosis, can concurrent with lung cancer. in the cases with these chronic pulmonary diseases, the incidence of cancer is higher than the general population. in addition,bronchopulmonary chronic infl ammation and pulmonary fi brous scar lesions may cause metaplasia or hyperplasia of squamous epithelium during their healing processes, based on which some cases can develop into cancer. the internal factors of the human body include family heredity, compromised immunity, metabolism and endocrine dysfunction. the lung cancers distribute more in the right lung than in the left lung, more in the upper lobe than in the lower lobe. its locations range from the major bronchus to the bronchioles. the central type of lung cancer has its origination from the major bronchial lobes and locates adjacent to the pulmonary hilum. the peripheral type of lung cancer has its origination from the lower parts of pulmonary segment bronchi and locates in the peripheral areas in the lungs. in the growth process of the lung cancer, it causes the extension and dilation of the bronchial walls, and penetrates the bronchial walls to invade the adjacent lung tissues and form masses. meanwhile it intrudes into the bronchi to cause luminal stenosis or obstruction. with its further progression and dissemination, it spreads from the lungs and directly extends into the chest walls, mediastinum, heart, major vessels and other adjacent organs and tissues. lung cancer can also transfer to other parts of the body along with blood and lymph fl ows or disseminates to other pulmonary lobes via the respiratory tract. the growth rate and transferring paths of lung cancer depend on its histological types, differentiation degree and other biological characteristics. lung cancer has no special symptoms in the early period, only has the common symptoms with common respiratory diseases, including cough, bloody sputum, low grade fever, chest pain and chest distress. therefore, it is often misdiagnosed. (1) face and neck edema; (2) hoarse voice is the most common symptom; (3) shortness of breath. lung cancer tends to occur distant metastases in the early stage. in the cases with metastatic lesions to the brain, the patients sustain persistent headache and blurry vision. in the cases with metastatic lesions to the bone, bone destruction may occur to cause fracture. (1) restrictive wheezing sound, mostly occurring in the inspiratory phase and recurring after cough; (2) hoarse voice, caused by lymph nodes transferring to compress and invade the recurrent laryngeal nerve; (3) superior vena cava syndrome, caused by the compresses or invasion to the superior vena cava by the mass and venous obstruction, with edema in the head, face, neck, and upper limbs, varicose veins and edema in the upper chest, and accompanying dizziness, chest distress, shortness of breath and other symptoms; (4) horner's syndrome, with enophthalmos of the affected side, blepharoptosis, shrinkage of the pupils, eye fi ssure stenosis, increased skin temperature in the upper chest of the affected side and no sweating due to compression or invasion of the apical cancer to the cervical sympathetic ganglia; (5) should and arm pain, which is radial burning pain in the shoulder and upper limbs of the affected side due to compression or invasion of apical cancer to the brachial plexus nerve; (6) phrenic nerve paralysis, with symptoms of shortness of breath and chest distress due to invasion to the phrenic nerve; (7) dysphagia, caused by compressed esophagus by mediastinal lymphadenectasis; and diffi culty breathing caused by compressed trachea by mediastinal lymphadenectasis; (8) pericardial effusion, shortness of breath, arrhythmia and heart dysfunctions due to pericardial invasion; (9) pleural metastasis, with chest pain and cancerous pleural effusion; (10) lung cancer metastasis, spreading of lung cancer along with blood fl ow to the bone, liver, brain, kidney, adrenal gland and subcutaneous tissues. intrapulmonary metastasis is also common. metastasis to different locations shows different symptoms and signs. (11) extrapulmonary signs, commonly including joint pain or joint hypertrophy, clubbing fi ngers and mental disorders. the diagnostic imaging is the most commonly used and an important examination for the diagnosis of lung cancer. it can facilitate to fi nd some specifi c manifestations in the lesions, which provide clues for the diagnosis of lung cancer. it is also the main basis for the staging of lung cancer, but fails to defi ne the qualitative diagnosis. chest x-ray is the main examination for the diagnosis of lung cancer. anteroposterior and lateral chest x-ray are used for preliminary screening. chest ct is the diagnostic imaging examination of the choice for the diagnosis of lung cancer. for the central type of lung cancer in the early stage, there are direct signs to defi ne the diagnosis. in the early stage, thin layer scanning with a layer thickness of 1.5-4 mm can be performed to observe the bronchial changes. mr imaging can demonstrate intraluminal nodules, luminal thickness and luminal stenosis of the bronchi from the transverse, coronal, and sagittal perspectives. mr imaging demonstrates favorably cancer in the lesions of obstructive pneumonia, and masses covered by the hilum. pet/ct scanning can be used for the screening of lung cancer metastasis and assessing the therapeutic effi cacy after treatment. dsa is used for infusion chemotherapy of bronchial artery in the cases of primary lung cancer. bronchoscopy is an important examination for the diagnosis of lung cancer. the pathological changes of the endothelium and the lumen of bronchi can be directly observed by using bronchoscopy. for the cases with caner or cancerous infi ltration by bronchoscopy, sampling of the tissues under the guidance of bronchoscopy for biopsy can be performed. otherwise, bronchial secretions can be suctioned under the guidance of bronchoscopy for cytological examinations to defi ne the diagnosis and the histological classifi cation. in most cases of primary lung cancer, the shed cancer cells can be found in the sputum, which can also facilitate to defi ne its histological classifi cation. after several examinations and short-term exploratory therapies, the qualitative diagnosis cannot be defi ned and the possibility of lung cancer cannot be excluded. therefore, exploratory thoracotomy can be performed if the patient's physical conditions permit. chest x-ray early lesions are confi ned within the bronchi, causing valve ventilatory disorder and changes of obstructive emphysema. the manifestations include restrictive pulmonary gas increase and sparse lung markings. in the cases with certain degree of bronchostenosis due to unfavorable discharge of the secretions, obstructive pneumonia occurs, showing patchy blurry shadows. in the cases with complete blockage of the bronchi, obstructive atelectasis occurs, showing decreased pulmonary volume, increased density and migration of the mediastinum to the affected side. obstructive pulmonary bronchiectasis has demonstrations of intrapulmonary cords liked shadows. lung a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of repeated cough for more than 1 month and reported to have a history of unhealthy sexual behaviors. his cd4 t cell count was 65/ μl. cancer in the middle and advanced stages are mainly manifested as hilar mass and atelectasis. the mass has a high density with clear boundary. however, the cancer cannot be observed due to its common immersion in the large fl aky obstructive pneumonia lesion or large quantity pleural effusion. atelectasis is commonly manifested as shrinkage of pulmonary segments or shrinkage of unilateral lung, with high density. the shadow of atelectasis widens at the hilum to show prominent mass. in the cases of central type lung cancer in the right upper lobe, a transverse s shape is at the hilum (commonly known as pancoast cancer). early diagnosis of central type lung cancer by plain chest x-ray only shows some indirect pulmonary manifestations caused by bronchial obstruction. and these indirect signs are not characteristically lung cancer. in the cases of local obstructive emphysema, these indirect signs can be caused by foreign substances in the bronchi or early infl ammation. obstructive pneumonia is diffi cult to be differentiated from common pneumonia. obstructive atelectasis needs to be differentiated from many other conditions. (1) pathological changes in the bronchial lumen including polypoid, nodular or fl at papula masses. benign tumor has smooth boundary and malignant tumor has unsmooth boundary, commonly with wider base and thickened lumen wall. even the slight bronchial changes caused by the central type of lung cancer can be demonstrated by thin layer ct scanning, including slightly thickened bronchial wall, intraluminal small nodules and lumen stenosis or obstruction. in the middle and advanced stages, the direct signs of the central type lung cancer include thickened bronchial wall, irregular or unsmooth lining of the bronchial lumen. bronchial obstruction is suddenly truncation or gradual thinning of the lumen to obstruction. (2) hilar mass locates adjacent or around the bronchi, with smooth or arch shaped boundary. the indirect signs of the central type lung cancer in the middle and advanced stages include secondary changes to bronchial stenosis. obstructive pneumonia is manifested as patchy blurry shadows or parenchymal changes of the pulmonary segments/lobes, and decreased lung volume. mr imaging demonstrates the tumor as long t1 and long t2 signals. in the cases of central type lung cancer with secondary obstructive atelectasis and obstructive pneumonia, enhanced t1 demonstrates the tumor in the lesion of pulmonary atelectasis and obstructive pneumonia. the signal of atelectasis is higher than that of the tumor. pet/ct scanning can demonstrates increased and thick stained metabolites of metastatic lesions or residual lesions, which has a diagnostic sensitivity of above 90 %, and a reported specifi city of 80-90 %. in addition, it can be applied for the clinical consideration of it hilar, mediastinal lymph node metastasis and extrathoracic distant metastasis, which is an important method to decide clinical stages before lung cancer therapy. but pet has false negative diagnosis in the diagnosis of lung cancer with decreased metabolites, especially the alveolar cell carcinoma. for the diagnosis of pneumonia and pulmonary tuberculosis, it also has false positive results. dsa can demonstrate the blood supply of the tumors. miliary tuberculosis is more common in young adults, with obvious symptoms of systemic toxicity. anti-tuberculosis drug therapy can relieve the symptoms, with gradually absorbed lesions. (3) chest x-ray demonstrates hilar lymph node tuberculosis as mass shadows in the hilum of lung, which may be misdiagnosed as the central type lung cancer. hilar lymph node tuberculosis is more common in teenagers, commonly with symptoms of tuberculosis infection but rarely hemoptysis. lung cancer can be concurrent with pulmonary tuberculosis. (1) bronchial pneumonia; obstructive pneumonia induced by early lung cancer can be misdiagnosed as bronchial pneumonia. bronchial pneumonia has an acute onset with more obvious symptoms of infection. chest x-ray demonstrates patchy or spots shadows, with blurry boundaries and uneven density. the lesions are not confi ned within one segment or one lobe. (2) pulmonary abscesses; central necrosis and liquefaction of the lung cancer results in cancerous cavities. by chest x-ray, the central type lung cancer can be misdiagnosed as pulmonary abscesses. in the acute period, a pulmonary abscess has obvious symptoms of infection, with large quantity purulent sputum. chest x-ray demonstrates thin cavity wall, smooth inner wall, liquid level and infl ammatory changes in the surrounding lung tissues or pleura. pulmonary benign tumors including hamartomas, fi broma and chondroma have slow growth. chest x-ray demonstrates round liked mass shadow, with homogeneous density without lobation. joint united nations programme on hiv/aids (unaids) surveillance for aidsdefi ning opportunistic illnesses, 1992-1997 imaging and pathology of hiv related cytomegalovirus pneumonia roentgenographic patterns of pneumocystis carinii pneumonia in 104 patients with radiology distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids patients atlas of differential diagnosis in hiv/aids. beijing: pmph the national institutes of health (nih) the centers for disease control and prevention (cdc), and the hiv medicine association of the infectious diseases society of america (hivma/idsa) guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents acute respiratory failure associated with cryptococcosis in patients with aids: analysis of predictive factors pulmonary cryptococcosis: localized and disseminated infections in 27 patients with aids penicillium marneffei agent d'une mycose du system reticuloendothelial infection caused by penicillium marneffei description of fi rst natural infection in man cd4+ t cell-mediated fatal hyperinfl ammatory reactions in mice infected with penicillium marneffei infections caused by penicillium marneffei in china and southeast asia: review of eighteen published case and report of our mo re chinese cases disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis and treatment, and review of the literature imaging of thoracic pathology in patients with aids clinical and radiographic features of hiv-related pulmonary tuberculosis according to the level of immunosuppression global tuberculosis incidence and mortality during 1990-2000. bull world health organ tuberculosis in patients with human immunodefi ciency virus infection pulmonary tuberculosis in kigali, rwanda. impact of human immunodefi ciency virus infection on clinical and radiographic presentation relationship of the manifestations of tuberculosis to cd4 cell counts in patients with human immunodefi ciency virus infection radiology of pulmonary tuberculosis and human immunodefi ciency virus infection gulu, uganda bacterial pneumonia in persons infected with the human immunodefi ciency virus. pulmonary complications of hiv infection study group microbiology of community-acquired bacterial pneumonia in persons with and at risk for human immunodefi ciency virus type 1 infection. implications for rational empiric antibiotic therapy the intracellular bacterium rhodococcus equi requires mac21 to mammalian cells virulence of rhodococcus equi isolates from patients with and without aids association between large plasmid and 15 to 17 kilo dalton antigens in virulent rhodococcus equi rhodococcus equi plasmids:isolation and partial characterization rhodococcus equi pneumonia in aids: high-resolution ct fi ndings in fi ve patients cytomegalovirus pneumonitis in patients with aids: fi ndings in an autopsy series immediate causes of death in acquired immunodefi ciency syndrome the causes of death in patients with humanimmunodefi ciency virus infection: aclinical and pathologic study with emphasis on the role of pμlmonary diseases cytomegalovirus pneumonitis: spectrum of parenchymal ct fi ndings with pathologic correlation in 21 aids patients rhodococcus equi -an increasingly recognized opportunistic pathogen a highly representative two hybrid genomic library for the yeast yarrowia lipolvtica rhodococcus equi infection in hiv infected patients rhodococcus equi infection in patients with and without human immunodefi ciency virus infection radiological fi ndings in nine aids patients with rhodococcus equi pneumonia comparison of nucleic acid amplifi cation, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals cytokine modulation alters pulmonary clearance of rhodococcus equi and development of granulomatous pneumonia diseases of the lung: radiologic and pathologic correlations pneumonia: high-resolution ct fi ndings in 114 patients radiologic distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids patients high-resolution ct in the evaluation of clinically suspected pneumocystis carinii pneumonia in aids patients with normal, equivocal, or nonspecifi c radiographic fi ndings pneumocystis carinii pneumonia: spectrum of parenchymal ct fi ndings pulmonia in patient with aids discontinuation of prophylaxis for pneumo-cystis carinii pneumonia in hiv-infected patients treated with highly active antiretroviral therapy carcinoma of the lung in hiv-positive patients: fi ndings on chest radiographs and ct scans pulmonary complications of hiv-associated malignancies pneumocystis carinii infection and aids comparison of histologic stains in the diagnosis of pnemocystis carinii tuberculosis in patients with human immunodefi ciency virus infection comparative histopathological study of pulmonary tuberculosis in human immunodeficiency virus-infected and non-infected patients pulmonary tuberculosis in aids patients: transient chest radiographic worsening after initiation of antiretroviral therapy active pulmonary tuberculosis in patients with aids: spectrum of radiographic fi ndings (including a normal appearance) effect of hiv status on chest radiographic and ct fi ndings in patients with tuberculosis pulmonary mycobacterium avium complex disease without dissemination in hiv-infected patients mycobacterium avium complex infection in the acquired immunodefi ciency syndrome pulmonary disease due to infection by mycobacterium avium complex in patients with aids cd4 t lymphocyte count and the radiographic presentation of pulmonary tuberculosis. a study of the relationship between these factors in patients with human immunodefi ciency virus infection tuberculosis in the aids era mycobacterial pseudotumors of lymph node mycobacterial spindle cell pseudotumor of lymph node managenent of the hiv-infected patient tuberculosis in the aids era cytomegalovirus pneumonia in aids patients atypical cytomegalic cells are diagnostic for cytomegaloviral infection in aids cytomegalovirus as a primary pulmonary pathogen in aids infections with cryptococcus neoformans in the acquired immunodefi ciency syndrome endemic fungal pneumonia in immunocompromised patients disseminated histoplasmosis in adis: fi ndings on chest radiographs opportunistic fungal pneumonia pulmonary aspergillosis in the acquired immunodeficiency syndrome coccidioidomycosis during human immunodefi ciency virus infection. a review of 77 patients cryptococcal pulmonary infection in patients with aids: radiographic appearance pulmonary aspergillosis in patients with aids. clinical and radiographic correlations cryptococcal pneumonia in aids: is cryptococcal meningitis preceded by clinically recognizable pneumonia pulmonary manifestations of disseminated cryptococcosis in patients with clinical and bacteriological aspects of nocardiasis lung abscess in patients with aids focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease rhodococcus equi endobronchial mass with lung abscess in a patient with primary pulmonary aids-related lymphoma: radiographic and ct fi ndings the expanding challenge of hiv-associated malignancies the pulmonary manifestations of aids-related non-hodgkin's lymphoma varied appearance of aids-related lymphoma in the chest lymphoma versus aids intrathoracic kaposi's sarcoma in women with intrathoracic kaposi's sarcoma. ct fi ndings distribution of human herpesvirus 8 dna in tumorous and nontumorous tissue of patients with acquired immunodefi ciency syndrome with and without kaposi's sarcoma kaposi's sarcoma in lymphnodes concurrent with hodgkin's disease the natural history of kaposi's sarcoma in the acquired immunodefi ciency syndrome pulmonary toxoplasmosis in patients in-fected with human immunodefi ciency virus: a french national survey diagnostic imaging, preautopsy imaging and autopsy fi ndings of 8 aids cases analysis on the imaging features of aids with pulmonary fungal infection liver injury in hiv-1-infected patients receiving non-nucleosides reverse transcriptase inhibitors-based antiretroviral therapy mri demonstrations of aids complicated by toxoplasma gondii infection in cervical spinal cord: with 3 cases reports diagnostic imaging in aids in china: current status and clinical application imaging and pathological fi ndings of aids complicated by pulmonary rhodococcus equi infection magnetic resonance spectroscopy in the diagnosis of cognitive impairment in aids patients ct image demonstrations of hiv-seropositive tuberculosis and their relationship with cd4+ t-lymphocyte count positron emission tomography of 18fdg uptake in localized pulmonary infl ammation penicilliosisde rhizomys sinensis accumulation of nuclear mitochondrial dna in the frontal cortex cells of patients with hivassociated neurocognitive disorders imaging fi ndings of aids complicated with pulmonary rhodococcus equi infection and correlated with pathology pulmonary toxoplasmosis? pathogenesis and prevention of rhodococcus equi infection pulmonary toxoplasmosis a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough, fever and no sputum. her cd4 t cell count was 45/ μl. filariform larvae of strongyloides stercoralis invade into skin or mucosa and reach the lungs through lymphatic vessels or venous system and the right heart. they develop into schistosomula in 3-30 days. a few schistosomula develop mature in the lungs or bronchi. most schistosomula penetrate the pulmonary capillaries into alveoli to cause a series of respiratory symptoms. in the cases of serious infection and in patients with compromised immunity, disseminated lesions occur in lungs and other organs. hiv/aids related pulmonary strongyloidiasis can have manifestations of local small bleeding spots, pimples, migratory linear or strips urticaria on skin, as well as manifestations of allergic bronchitis, lobular pneumonia or asthma. aids patient may have severe diffuse infection and systemic infection, with symptoms of fever, severe cough, expectoration, hemoptysis, shortness of breath, breathing diffi culty, and asthma. 1. laboratory tests 2. the pathological examinations include biopsy tissue culture and inoculation test. 3. chest x-ray is the most commonly used examination. there are demonstrations of small spots and fl aky shadows, thickened hilar shadow and thickened pulmonary markings. mitchell reported in 1992 that interstitial or alveolar infi ltration in both lungs accounts for 62 %, nodular shadows in both lungs 15 %, hilar or mediastinal lymphadenectasis 26 %, pleural fl uids 42 %, septal line 25 %, mediastinal lymphadenectasis and ascites are the important clues for the diagnosis. 1. the clinical manifestations of hiv/aids related pulmonary strongyloidiasis are non-specifi c. its diagnosis depends on the etiological examinations. the fi ndings of strongyloides sterocralis in the patient's sputum and feces can defi ne the diagnosis. increases to 20 × 10 9 ⁄l; eosinophils granulocytes 25-30 %, or even as high as 70-80 %; the serum total lge level increases by 50 %; 90 % cases with blood serum lgg and lge of fi lariform larvae antigen positive. in the cases with femal strongyloides stercoralis parasitizing in the bronchial epithelium, rhabditiform larva, fi lariform larva, schistosomula, adult strongyloides stercoralis and the eggs can be found in fresh phlegm, which can defi ne the diagnosis. 3. pathological examinations including biopsy tissue culture and inoculation test are positive. 4. by chest x-ray, the lungs have small spots and fl akes of shadows, thickened hilar shadow and thickened pulmonary markings. hiv/aids related pulmonary strongyloidiasis should be differentiated from hiv/aids related pulmonary toxoplasmosis, infectious mononucleosis and mycoplasma pneumonia. pulmonary infi ltration of hiv/aids related malignant lymphoma commonly has three types: primary pulmonary lymphoma, which is rare and accounts for 0. 3. the diagnostic imaging examinations are the most commonly used examinations for the diagnosis. the imaging demonstrations of hiv/aids related malignant lymphomas include: 1. mediastinal lymphadenectasis is the most commonly found pulmonary manifestations of malignant lymphoma. the lesions are mainly located in anterior and middle mediastinum, in asymmetric wave liked or lobulated mass. it occurs unilaterally or bilaterally, isolated or fusion. 2. the incidence of pulmonary parenchymal lesions is 20-30 %. chest x-ray demonstrates mediastinal lymph nodes extending directly into the lungs, which is susceptible to confusion with pneumonia. they are demonstrated as round shadows in the lung fi elds or distribute in the whole lung fi elds. chest x-ray demonstrates the lymphatic spread of lesions as military nodules in different sizes or isolated intrapulmonary nodules or cavities, commonly accompanying with mediastinal hilar lymphadenectasis. in the cases with its occurrence secondary to endobronchial membrance, obstructive pneumonia or atelectasis can be caused. some patients may have diffuse pulmonary interstitial changes. pulmonary infi ltration by non-hodgkin's lymphoma can also be divided into four types: (1) nodular type; (2) pneumoniaalveolar type; (3) bronchial-vascular-lymphatic type, which can be further divided into the central bronchialvascular type, and diffuse lymphatic type; (4) diffuse lymphatic type can have lesions of reticular or reticular nodular infi ltration and its progression into patchy changes. 3. miliary-blood borne spreading type is rare. 4. the pleural lesions is mainly pleural effusion, with bloody or serous pleural fl uid. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and chest pain for more than 2 months. his cd4 t cell count was 36/μl. (1) tuberculoma is diffi cult to be differentiated from the peripheral type of lung cancer. tuberculoma is more common in young adults, with a long term course of illness. the lesions are commonly found in the apical posterior segment of the upper lobe or dorsal segment of the lower lobe. chest x-ray demonstrates the lesions with uneven density and satellite lesions. (2) miliary tuberculosis is diffi cult to be differentiated from diffuse bronchioloalveolar carcinoma. key: cord-264308-y6xuxj16 authors: liu, rui; an, liwei; liu, ge; li, xiaoyu; tang, wei; chen, xulin title: mouse lung slices: an ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses date: 2015-05-26 journal: antiviral res doi: 10.1016/j.antiviral.2015.05.008 sha: doc_id: 264308 cord_uid: y6xuxj16 the influenza a virus is notoriously known for its ability to cause recurrent epidemics and global pandemics. antiviral therapy is effective when treatment is initiated within 48 h of symptom onset, and delaying treatment beyond this time frame is associated with decreased efficacy. research on anti-inflammatory therapy to ameliorate influenza-induced inflammation is currently underway and seems important to the impact on the clinical outcome. both antiviral and anti-inflammatory drugs with novel mechanisms of action are urgently needed. current methods for evaluating the efficacy of anti-influenza drugs rely mostly on transformed cells and animals. transformed cell models are distantly related to physiological and pathological conditions. although animals are the best choices for preclinical drug testing, they are not timeor cost-efficient. in this study, we established an ex vivo model using mouse lung slices to evaluate both antiviral and anti-inflammatory agents against influenza virus infection. both influenza virus pr8 (h1n1) and a/human/hubei/3/2005 (h3n2) can replicate efficiently in mouse lung slices and trigger significant cytokine and chemokine responses. the induction of selected cytokines and chemokines were found to have a positive correlation between ex vivo and in vivo experiments, suggesting that the ex vivo cultured lung slices may closely resemble the lung functionally in an in vivo configuration when challenged by influenza virus. furthermore, a set of agents with known antiviral and/or anti-inflammatory activities were tested to validate the ex vivo model. our results suggested that mouse lung slices provide a robust, convenient and cost-efficient model for the assessment of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. this ex vivo model may predict the efficacy of drug candidates’ antiviral and anti-inflammatory activities in vivo. influenza a virus (iav) is still a threat to human health and poses a global concern due to its unpredictable, pandemic potential and pathogenesis. as high evolutionary rates of the influenza virus makes vaccination strategies difficult, anti-influenza drugs are crucial for the control of influenza pandemics. antiviral therapy is generally licensed for use within 48 h of influenza illness onset, and delaying treatment is associated with decreased drug efficacy and increased morbidity and mortality (kandun et al., 2008) . although influenza-induced pathology is still unclear, the uncontrolled immune response may be the major contributor to influenza virus-induced mortality (de jong et al., 2006; iwasaki and medzhitov, 2011; kobasa et al., 2007) . thus, studies on the modulation of the host immune response are currently underway. several animal studies have shown that anti-inflammatory agents http://dx.doi.org/10.1016/j.antiviral.2015.05.008 0166-3542/ó 2015 elsevier b.v. all rights reserved. abbreviations: tnf-a, tumour necrosis factor alpha; il-6, interleukin 6; rantes, regulated on activation, normal t cell expressed and secreted; mip-3a, macrophage inflammatory protein 3 alpha; ip-10, interferon-gamma induced protein 10; il-10, interleukin 10; il-1b, interleukin 1 beta; ifn-c, interferon gamma; ppar-c, peroxisome proliferator-activated receptor gamma; egcg, epigallocatechin gallate; cxcr3, chemokine (c-x-c motif) receptor 3; tnfr1, tumour necrosis factor alpha receptor 1; il-1r, interleukin 1 receptor; mip-1, macrophage inflammatory protein 1; ccr2, c-c chemokine receptor type 2; gm-csf, granulocyte-macrophage colony-stimulating factor; i.p., intraperitoneal; balf, bronchoalveolar lavage fluid; rig-i, retinoic acid-inducible gene i; tlr 7/8, toll-like receptor 7/8; tlr 3, toll-like receptor 3; munana, 2 0 -(4-methylumbelliferyl)-a-d-acetylneuraminic acid. can protect mice from death against influenza infection. 15-deoxy-d 12,14 -prostaglandin j 2 (15d-pgj 2 ) has been shown to protect 79% of mice from death against lethal influenza infection through manipulation of the ppar-c pathway, whereas it does not inhibit virus replication (cloutier et al., 2012) . the p38 inhibitor significantly protects mice from lethal influenza infection without affecting virus replication (borgeling et al., 2014) . statins not only reduce the levels of ldl-cholesterol, but they also counteract the inflammatory changes associated with acute coronary syndrome and improve survival in patients with influenza. similarly, in patients hospitalised with laboratory-confirmed seasonal influenza, statin treatment is associated with a 41% reduction in 30-day mortality (fedson, 2013) . therefore, strategies targeting aberrant host immune responses may be good complements for existing antiviral drugs. the routine strategies for anti-influenza drug development rely primarily on cell-based assays in primary screening followed by animal studies. however, the cost of in vivo studies is very high due to the use of animals and a large quantity of investigational compounds. lung slices, which provide a bridge between single cells and whole animals, are broadly used in physiology and toxicity studies (morin et al., 2013; sanderson, 2011) . different from single cell lines, lung slices possess multiple cell types and preserve the physiological and functional cellular relationships within the body. cell-cell and cell-matrix interactions result in lung slices closely resembling the morphology and functionality of the lung. whereas the evaluation of the efficacy of a drug based on animal studies is expensive and time-consuming, the lung slice model may serve as a valuable tool for efficacy tests of compounds for the treatment of influenza. with the development of tissue slicers, which produce slices rapidly and reproducibly, an increasing number of studies have employed lung slices to explore the interaction of hosts and pathogens (chakrabarty et al., 2007; londt et al., 2013; punyadarsaniya et al., 2011; seehase et al., 2012; van poucke et al., 2010; wu et al., 2010) . recently, several studies have shown that pig lung slices can support influenza virus replication (londt et al., 2013; van poucke et al., 2010) . another study demonstrated that influenza virus infection can induce robust cytokine and chemokine responses in human lung slices (wu et al., 2010) . theoretically, the three-dimensional lung slice culture system can be used to evaluate the potency of both antiviral and anti-inflammatory agents against influenza virus infection. in the current study, we showed that influenza viruses can efficiently replicate in mouse lung slices and induce significantly elevated levels of cytokines and chemokines. a panel of antiviral and anti-inflammatory agents were tested for their antiviral activities and/or anti-inflammatory effects in the mouse lung slices. the results from the lung slice model are consistent with those from mouse studies. our results showed that the lung slice model provides a robust, convenient and cost-economical method for the screening and evaluation of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. 6-8-week old balb/c mice were purchased from changsha laboratory animal center (hunan province, china) and were housed under specific-pathogen-free condition. all experiments were conducted according to the protocol approved by the animal care and use committee of wuhan institute of virology, chinese academy of sciences (wiva08201201). mouse adapted a/puertorico/8/34 (h1n1) and mouse adapted a/human/hubei/3/2005 (h3n2) were propagated in the allantoic cavity of 10-day-old specific-pathogen-free embryonated chicken eggs for 48 h. the allantoic fluids were collected and filtered with 0.22 lm filter and stored at à80°c. the virus strains were provided by the virus collection at wuhan institute of virology, chinese academy of sciences, china. 15-deoxy-d 12,14 -prostaglandin j 2 (15d-pgj 2 ), ribavirin and the neuraminidase substrate 2 0 -(4-methylumbelliferyl)-a-d-acetylneuraminic acid (munana) were purchased from sigma-aldrich. egcg was purchased from sichuang weikeqi biological technology co. ltd. (china). glycyrrhizin was purchased from shanghai hanxiang biological technology co. ltd. (china). the p38 pathway inhibitor sb203580, the erk pathway inhibitor u0126 and the sapk/jnk pathway inhibitor sp600125 were purchased from beyotime institute of biotechnology (china). oseltamivir carboxylate (gs 4071) was obtained from toronto research chemicals (canada). all compounds were initially dissolved in dimethyl sulfoxide (dmso, sigma-aldrich). mouse lung slices were prepared using a modification of a protocol that has been previously reported (bauer et al., 2010) . after anesthetisation by intraperitoneal injection of sodium pentobarbital (75 mg/kg), the mouse was bled through the abdominal aorta. then, the trachea was exposed, dissected from surrounding tissues and was cannulated with an 18-gauge needle. through the cannula, the lung was inflated with 1.3 ml of 2% low-melting agarose (bio-rad) dissolved in hank's buffered saline solution (hbss) solution. the whole animal was cooled with ice for 10 min to solidify the agarose and, thereby, the lung. then, the lung was taken out en bloc from the thoracic cavity and placed in the slice culture medium (dulbecco's modified eagle medium: nutrient mixture f-12, dmem/f-12, gibco) at 4°c for an additional 15 min to completely solidify the agarose. the culture medium was supplemented with 100 units/ml of penicillin, 100 lg/ml streptomycin and 250 ng/ml of amphotericin to avoid contamination. the lung lobe was afterwards dissected and cut to create a flat surface at the end of the primary bronchus. another flat surface was cut approximately 0.8 cm from the first surface. the cube was maintained in the pre-chilled slice culture medium prior to or during the slicing. the cube was cut into slices of desired thickness using a vibratome slicer (leica, vt1200s). each mouse lung cube generated at least 24 250-lm slices. the slices were then transferred into a 48-well cell culture plate and covered with 250 ll of slice culture medium in each well. the medium was changed every hour at least three times before virus infection to remove cell debris. the lung slice viability was assessed by bronchoconstriction and live/dead staining. the bronchoconstriction was monitored under a microscope when adding or removing 10 à4 m acetyl-ß-methylcholine chloride (sigma-aldrich). the photos were taken using a nikon inverted research microscope ti eclipse. for live/dead staining experiments, the slices were incubated with calcein am (1 lm) and propidium iodide (pi, 1 lg/ml) for 20 min at room temperature. a nikon multiphoton confocal microscope a1 mp + was used to record the images. the lung slices were infected with 200 ll of 10 5 pfu/ml influenza viruses for 2 h. virus diluent was used as a negative control. after the incubation, the viruses were discarded, and the slices were washed twice with phosphate buffered saline (pbs); then, fresh medium was added. at the indicated time points, the supernatants were harvested, and the virus titres and expression levels of cytokines and chemokines were detected. the slices were stored at à80°c until rna extraction was performed. for the drug addition assay, 250-lm-thick lung slices were prepared as described in section 2.3. after the last wash, the slices were infected with 200 ll of 10 5 pfu/ml pr8 virus. after incubation for 2 h, the virus was discarded, and the slice was washed with pbs. for the measurement of the z 0 factor, each 48-well plate (one lung slice per well) was processed as follows: one column was uninfected, one column was infected and mock treated, the rest of columns were infected and treated with 200 ll of six concentrations of 3-fold dilutions of ribavirin starting from 200 lm. in total, four plates were processed. for the lung slice model validation assay, each 48-well plate contained two agents, and each data point had three lung slices. na activity and ip-10 level were determined 48 h post infection. the virus titres were determined by a tcid 50 assay. briefly, madin-darby canine kidney (mdck) cells were maintained in dmem supplemented with 10% foetal bovine serum and 100 units/ml of penicillin/streptomycin. mdck cells were seeded into 96-well cell culture plates. twenty-four hours later, 10-fold serial dilutions of the supernatant were inoculated on an mdck monolayer at 37°c for 72 h, and the cytopathic effects were examined. the virus titres were calculated by the reed-muench method (reed and muench, 1938) . 2 0 -(4-methylumbelliferyl)-a-d-acetylneuraminic acid (munana) is a fluorometric substrate of the neuraminidase of the influenza virus. the virus containing sample was mixed with 20 lm munana, which is dissolved in a mes solution (33 mm 2-[n-morpholino] ethanesulfonic acid and 4 mm cacl 2 , ph = 6.5), and incubated at 37°c for 2 h in a 96-well black optiplate (perkinelmer). the reactions were stopped by adding 0.14 m naoh in 83% ethanol, and the fluorescence signal was recorded at 355 nm (excitation) and 485 nm (emission) using the wallac envision multilabel reader (perkinelmer). the protein levels of tnf-a, il-6, il-10, il-1b, ifn-c, mip-3a, ip-10, and rantes were measured using commercial elisa kits (boster biotechnology company, wuhan, china). for the anti-inflammatory assay using ip-10 as readout, the protein levels of ip-10 were measured using duoset elisa development kits (r&d). drug toxicity was assessed by mtt assay. the slices were incubated with 200 ll serially diluted concentrations of drugs for 48 h. then the drugs were removed and the slices were washed once with pbs and incubated with mtt (0.5 mg/ml) for 40 min at 37°c. after the formazan was completely dissolved in 200 ll of dmso, 100 ll of dmso solution was transferred to a 96-well plate. the optical density (od) of the dissolved formazane was measured at 540 nm. the 150 lm thick lung slices were cut and exposed to 200 ll of 10 6 pfu/ml pr8 (h1n1) or hubei (h3n2) viruses. after incubation for 2 h, the viruses were removed and fresh medium were added. 24 h later, the slices were fixed with 4% paraformaldehyde for 2 h at room temperature. the slices were then incubated in blocking buffer (pbs containing 3% bsa, 1% triton x-100 and 10% normal goat serum) for another 3 h. afterwards, the slices were immunoprobed with antibody against nucleoprotein of influenza a virus (1:100, santa cruz sc-80481) and in binding buffer (pbs containing 3% bsa and 1% triton x-100) overnight at 4°c. after washing three times with pbs, the slices were incubated with dapi (200 lg/ml, sigma-aldrich) and tritc-conjugated goat anti-mouse igg (1:100) for 1 h. fluorescent images were obtained using a nikon multiphoton confocal microscope a1 mp + (nikon). total rna was extracted from lung slices with the e.z.n.a™ microelute total rna kit according to the animal tissue protocol (omega). reverse transcription was conducted using a random hexamer primer. real-time quantitative pcr was performed using the itaq™ universal sybr ò green supermix (bio-rad). the pcr conditions were 94°c for 30 s and 40 cycles of 94°c for 15 s and 60°c 30 s. the primers for cytokines and chemokines were synthesised according to previously published data (giulietti et al., 2001) . mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg). twenty microlitres of 10 5 pfu/ml pr8 virus was inoculated nasally. the mock group was inoculated with virus diluent. on day 3 post infection, the mock mice and infected mice were sacrificed the tracheas and lungs were removed and washed three times with an injection of 2 ml of pbs containing 0.1% bsa. after centrifugation at 3000 rpm, the bronchoalveolar lavage fluids (balf) were stored at à80°c. statistical analysis was performed by the unpaired t-test. correlation of cytokines/chemokines between ex vivo and in vivo was evaluated using a linear regression analysis using graphpad prism 5.0 software. cc 50 and ec 50 values were determined using non-linear regression using graphpad prism 5.0. statistical significance was determined as follows: *, 0.01 < p < 0.05; **, 0.001 < p < 0.01. the data in the figures are represented as the means ± sem. the z 0 factor was calculated according to the method described previously (zhang et al., 1999) . to establish the ex vivo lung slice model for influenza virus infection, the viability of lung slices must be maintained. we first tested how long the mouse lung slices can remain viable under the ex vivo culture condition. mouse lung slice viability was assessed based on the bronchoconstriction assay and live/dead viability assay. as shown in fig. 1 , a strong bronchoconstriction (compare a with b) could be induced on days 1, 3 (data not shown) and 5 after treatment with 0.1 lm acetyl-ß-methylcholine chloride. the removal of the drug resulted in relaxation of the airway (fig. 1c) . to further assess the viability of lung slices, the live/dead staining method was used to evaluate whether the alveolar architecture remained intact and alive after preparation and ex vivo culture. as shown in fig. 1 , the slices were almost 100% green (viable cells) directly after preparation (fig. 1d ), whereas treatment of the slices with 1% triton-100 resulted in only red nuclei staining (dead cells, fig. 1e ). in addition, the mouse lung slices stayed alive and the alveolar architecture in lung slices remained intact up to 5 days after preparation ( fig. 1f-h) . collectively, these results suggest that the mouse lung slices can be kept alive for at least 5 days after preparation. because the lung slices can survive under ex vivo conditions, we assessed whether influenza viruses can infect and replicate in the mouse lung slices. first, lung slices with different thicknesses were generated to determine the optimal thickness of lung slice suitable for the infection of influenza viruses. as expected, 250-lm thick slices produced more infectious virions 48 h post-infection than 125-lm thick slices. however, the virus yields of 500 and 1000-lm thick slices were similar to those of 250-lm thick slices ( fig. 2a) . these results suggest that 250-lm thick lung slices are the best for infection with influenza virus, balancing cost and efficiency. next, the growth curves of influenza viruses in mouse lung slices were obtained. as shown in fig. 2d and e, both pr8 (h1n1) and hubei (h3n2) viruses demonstrated time-dependent replication and reached their maximum titre 48 h post infection, though the virus titres ex vivo are much lower than those in cell culture. neuraminidase (na) activity results ( fig. 2b and c) showed that both virus strains have similar kinetics in virus replication and infectivity ( fig. 2d and e) , suggesting that na activity can precisely represent the virus replication and infectivity in lung slices. to further correlate na activity and virus titre, ribavirin and u0126 were tested for dose dependent inhibition. as shown in fig. s1 , both compounds exhibit dose dependent inhibition of na activity or virus infectivity, indicating that na activity can be used to the 250-lm thick lung slices were prepared, and the bronchoconstriction was observed 5 days after preparation. the untreated slices (a) were incubated with 0.1 lm acetyl-ß-methylcholine chloride at 37°c for 5 min (b) in a 24-well cell culture plate. then, the drug was removed, lung slices were washed twice with pbs and incubated with fresh culture medium at 37°c for 10 min (c). the images were captured using a nikon's eclipse ti inverted microscope. red, blue and black arrows show the lung bronchus, pulmonary artery and pulmonary vein, respectively. scale bar = 200 lm. (d-h) live/dead staining assay. lung slices were stained with calcein-am (1 lm) and propidium iodide (pi, 1 lg/ml) for 20 min at room temperature on days 1, 3 and 5 after preparation (lower panels, f-h). the middle panel shows a viable slice directly after preparation (d), and a slice with complete loss of activity (treated by 1% triton-100) (e) for comparison. calcein-am and pi were used to simultaneously determine the live and dead cells. after being washed twice with pbs, images were taken using a nikon multiphoton confocal microscope a1 mp + with excitation at 800 nm, and an emission filter of 500/50 nm for calcein-am and 625/50 nm for pi. scale bar = 50 lm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) represent virus replication in the primary screening. to further confirm the infection of the lung slices by influenza virus, an immunofluorescence assay (ifa) was used to stain the nucleoprotein (np) of influenza virus on the lung slices. as shown in fig. 2f , the lung slice was strongly infected by influenza virus. overall, these results demonstrated that lung slices support influenza virus replication. one aim of our study was to evaluate the anti-inflammatory effects of potential agents in lung slices. many cytokines and chemokines are elevated in influenza-infected mice and seem important in influenza-induced inflammation. despite complications in the cytokine storm induced by influenza virus infection, we chose eight important cytokines and chemokines (belisle et al., 2010; brandes et al., 2013; lauder et al., 2013; szretter et al., 2007; wang et al., 2013; weiss et al., 2010) to study the inflammatory response following influenza virus infection. first, we examined the mrna levels of cytokines and chemokines in influenza virus-infected lung slices by real time rt-pcr. as shown in fig. 3a and b, exposure to 10 5 pfu/ml doses of pr8 and hubei viruses resulted in elevated mrna levels of most cytokine and chemokine genes tested. at 12 h post infection, the gene transcriptions were elevated and were highly activated at 48 h post infection. the cytokines and chemokines with the highest levels of induction in transcription by both viruses were rantes and ip-10. because the cytokine and chemokine responses mediated by the two viruses at transcription level are similar, the pr8 influenza virus was used for the steady protein level analysis. we then further determined the protein levels of selected cytokines and chemokines in response to influenza virus infection at 24 h and 48 h post infection. as shown in fig. 3c , after exposure to pr8 virus for 24 h, the protein levels of all tested cytokines and chemokines were significantly elevated except for il-1b and il-10. ifn-c was undetectable in the supernatant of the lung slices. the fold increase over mock for tnf-a, mip-3a, il-6, rantes and ip-10 were 2.1, 3.3, 2.4, 1.7 and 8.9, respectively. at 48 h post infection, higher levels of cytokines and chemokines were induced (fig. 3d) , and the fold increases were 4, 4.4, 3, 4 and 19, respectively. ip-10 was the most highly stimulated chemokine at both 24 h and 48 h post infection, which is consistent with the results of its mrna response to virus infection. taken together, our results suggest that a strong the supernatants were collected at indicated time points to perform the na activity assay (b and c) and tcid 50 assay (d and e). (f) infection of lung slices was assessed by the ifa assay. slices with a thickness of 150 lm were exposed to 200 ll of 10 6 pfu/ml pr8 (h1n1) or hubei (h3n2) viruses for 24 h. the slices were fixed with 4% paraformaldehyde and analysed by ifa. the images were captured using a nikon multiphoton confocal microscope a1 mp + . the virus diluent was used as a negative control (nc) in the infection. scale bar = 1000 lm. the right panels show enlarged regions of interest from the left panels. inflammatory response represented by the highly elevated mrna and protein levels of cytokines and chemokines are triggered by the infection of influenza viruses in the ex vivo mouse lung slice. our results demonstrated that the ex vivo model supports influenza virus infection (section 3.2) and exhibits an inflammatory response following influenza virus infection (section 3.3). to meet the goal of this study in the establishment of an ex vivo mouse slice model for the screening and evaluation of both antiviral and anti-inflammatory drugs against influenza infection in one assay, ensuring that the ex vivo model has similar patterns in influenza-induced cytokine and chemokine responses is critical. as shown in fig. s2 , the inflammatory responses in vivo on day 3 post-infection appeared to be most robust. the cytokine/chemokines levels in vivo on day 3 were chosen to compare with that ex vivo. the results from mouse studies showed that ifn-c, il-1b, tnf-a, and il-10 exhibited lower levels of activation (181 pg/ml, 115 pg/ml, 198 pg/ml and 278 pg/ml, respectively), whereas mip-3a, il-6, rantes and ip-10 exhibited higher levels of activation in bronchoalveolar lavage fluid (balf, 625 pg/ml, 982 pg/ml, 1216 pg/ml and 5000 pg/ml, respectively). the elevated expression of most of the tested cytokines and chemokines in the supernatant of lung slices demonstrated the same expression pattern compared with that in the mouse model (figs. 3c, d and 4a) . to evaluate the correlation between ex vivo and in vivo levels of cytokines and chemokines, a linear regression analysis model was employed. the levels of cytokines and chemokines in the supernatants of the lung slices 24 h post infection (fig. 3c ) were compared to that in the balf of mice 3 day post infection. the results show that there is a positive correlation between ex vivo and in vivo expression of selected cytokines and chemokines with a coefficient of correlation of 0.75 (fig. 4b ). more specifically, ip-10, rantes and mip-3a are perfectly correlated. the coefficient of correlation between cytokines and chemokines in the supernatant of lung slices 48 h post infection (fig. 3d) and that in the balf of mice 3 day post infection was 0.61 (fig. s3 ). our results here demonstrated that the ex vivo mouse lung slice model resembles the in vivo mouse model in response to influenza virus infection. in addition, ip-10, rantes and mip-3a may serve as readouts in the screening and evaluation of anti-inflammatory agents against influenza infection. to assess the quality of the mouse lung slice model, we used ribavirin, an antiviral drug, as a reference in a pilot experiment to test the z 0 factors for the measurements of antiviral and anti-inflammatory activities. the z 0 factor, a screening window coefficient, can be used to evaluate the quality of assays (zhang et al., 1999) . neuraminidase was chosen as a readout for antiviral activity, which was confirmed to correlate well with the production of infectious virions (fig. 2b-e) . ip-10 was chosen as a readout f nc pr8 hubei fig. 2 (continued) to represent the influenza virus-induced inflammation for three reasons: (a) it exhibits good correlation between in vivo and ex vivo models at the protein level, (b) it is the most highly induced chemokine in the mouse model and in the lung slice model, and (c) ip-10 à/à mice or mice treated with anti-ip-10 antibody demonstrate global down-regulated cytokines and chemokines and can survive after challenged by a lethal dose of influenza virus (wang et al., 2013) . therefore, ribavirin was used to determine its na and ip-10 inhibition effects to test the performance of the lung slice model. four replicate plates were processed to determine the z 0 factor. as shown in table 1 , a z 0 factor ranging from 0.53 to 0.71 for na inhibition and 0.58 to 0.73 for ip-10 inhibition demonstrated that the quality of the lung slice model for the evaluation of both antiviral and anti-inflammatory effects is satisfactory. to further validate our mouse lung slice model, a panel of agents with known antiviral or anti-inflammatory activities in vivo was tested for antiviral and anti-inflammatory effects in the lung slice model. among these agents, ribavirin, oseltamivir and gemacrone are anti-influenza drugs. egcg and u0126 have both antiviral and anti-inflammatory effects in vivo. 15d-pgj 2 and sb203580 have anti-inflammatory activities in vivo. all of these to show the induced changes, cytokines and chemokines were placed in separate panels according to scales of the protein levels. each data point was from three lung slices, and virus diluent was used as a negative control. comparisons between the infected group and the negative control were performed by the unpaired ttest (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01). the data are expressed as the means ± sem. nd, not detected (for real-time pcr, no amplification was detected during the 40 cycles. for elisa, the signal was below the detection limit of the commercial kit). agents have been reported to protect mice from death against lethal influenza infection. as shown in table 2 , all of the antiviral agents (ribavirin, oseltamivir, and gemacrone) efficiently inhibited virus replication in the lung slices, and the ip-10 levels were decreased as a result of the inhibition of viral replication (fig. s4) . unexpectedly, oseltamivir inhibited only virus replication in the lung slices without interfering with ip-10 expression (fig. s4) . in addition to their antiviral effects, u0126 and egcg possess anti-inflammatory effects (droebner et al., 2011; ling et al., 2012; pinto et al., 2011; ranjith-kumar et al., 2010) . we found that both u0126 and egcg can dose dependently decrease na levels and ip-10 levels in the lung slice model. 15d-pgj 2 and sb203580 were chosen because they protect mice from influenza infection by anti-inflammatory effects only (borgeling et al., 2014; cloutier et al., 2012) . as expected, both 15d-pgj 2 and sb203580 dose dependently decreased ip-10 levels, whereas neither inhibited virus replication in lung slices, suggesting they improved mouse survival through their anti-inflammatory mechanisms (fig. s4) . to further validate the model, we also tested six agents (dicyclomine, clotrimazole, fenofibrate, proadifen, benzydamine and nafronyl oxalate) with known antiviral activity in mdck cells ) that failed to protect mice from lethal doses of influenza virus infection (fig. s5) . results showed that dicyclomine, clotrimazole, fenofibrate, proadifen and benzydamine inhibited neither viral replication represented by na activity nor inflammatory response represented by ip-10 levels in mouse lung slices (fig. s4) . however, nafronyl oxalate, although not showing any antiviral activity, inhibited the inflammatory response (with ip-10-ec 50 of 22.1 lm, fig. s4 ), although it did not improve mouse survival rate (fig. s5) . taken together, our results suggest that with the benefits of avoiding the overuse of animals and expensive compounds, the mouse lung slice model is robust and cost-efficient for screening and evaluating both antiviral and anti-inflammatory drugs against the influenza infection in one assay. overall, the mouse lung slice can serve as a predictive model of the efficacy of drug candidates on the mouse model. current anti-influenza drug screening is based primarily on transformed cells in the form of cell-based assays. though the transformed cells are convenient for high throughput screening and the in vitro study of potency and mechanisms, they are distantly related to physiological and pathological conditions. as a consequence, results based on transformed cells may suffer from simplification. current treatments for influenza rely on the use of antiviral drugs; however, the window for antiviral therapy is narrow. several reports have shown that anti-inflammatory agents can be effective in reducing the death rate against the infection of lethal influenza (borgeling et al., 2014; cloutier et al., 2012) . comparisons between infected mice and mock mice were performed by the unpaired t-test (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01). the data are expressed as the means ± sem. nd, not detected (the signal was below the detection limit of the commercial kit). (b) comparison of the cytokine and chemokine levels in response to influenza infection between the ex vivo and in vivo models. after subtraction of the corresponding levels of the mock group, the correlation of the virus-induced il-1b, ifn-c, tnf-a, il-10, mip-3a, il-6, rantes and ip-10 in balf of day 3 post infection ( fig. 4a ) and lung slice supernatants (fig. 3c) were analysed by a linear regression analysis model. table 1 the pilot experiment of lung slices model using ribavirin. 250 lm thick lung slices were prepared as described in section 2.3. the lung slices were infected with 200 ll of 10 5 pfu/ ml pr8 virus. after 2 h incubation on 37°c, the viruses were discarded and the lung slices were washed twice with pbs. then six concentrations of ribavirin starting from 200 lm and with 3-fold serial dilution were added. na activities and ip-10 levels in culture supernatants were determined 48 h post infection as described in sections 2.7 and 2.8. s/b-na a s/b-ip-10 b z 0 -na c z 0 -ip-10 d na-ec 50 (lm) e ip-10-ec 50 (lm) f moreover, current antiviral drug screening is performed primarily using cell-based assays. the cell-based assays available for the development of anti-inflammatory agents against influenza virus infection are largely targeting single host factors or pathways that play a role in the influenza-induced inflammatory response. such strategies could be very risky for the development of anti-inflammatory drugs and lead to failure in animal tests or in clinical trials. whereas animal models, e.g., mice and ferrets, are good for efficacy studies in of both antiviral and anti-inflammatory drugs, they are not time-or cost-efficient. to overcome this problem, at least in part, we have established the mouse lung slice model to test drugs for antiviral and anti-inflammatory activities in one ex vivo assay with the convenience of the in vitro cell-based assay. the goals for this model are as follows: (i) the mouse slices can remain alive long enough for efficient influenza virus infection and inflammatory response; (ii) the inflammatory response is similar to that in the lung of mouse; (iii) to choose the best readouts for the antiviral and anti-inflammatory activities in the model; and (iv) to validate the model using antiviral and anti-inflammatory agents with confirmed efficacy in vitro and in vivo. the tissue viability of the lung slices was the first concern in establishing the lung slice model. mouse lung slices can survive and maintain their native structure for at least three days after preparation (sanderson, 2011; siminski et al., 1992) . the methods to monitor the viability of lung slices include the measuring of the ldh release, ciliary activity, bronchoconstriction induced by stimuli, and live/dead staining (sanderson, 2011) . in our research, results from bronchoconstriction experiments and live/dead staining experiments demonstrated that lung slices remained alive for at least 5 days. moreover, the infection of lung slices by influenza viruses and the kinetics of virus growth are better indications that the mouse lung slices stay viable for at least the first 2 days after preparation. our second goal was to establish an ex vivo model for the infection of mouse lung slices by influenza viruses and the induction of inflammatory responses similar to that in the mouse model. our results demonstrated that influenza viruses replicate efficiently in the mouse lung slices. these results are consistent with previous reports showing that the influenza virus can replicate in pig lung slices (van poucke et al., 2010). by ifa, mouse lung slices were clearly shown to be infected by influenza virus. to analyse the inflammatory response of lung slices following the infection of the influenza virus, we measured the levels of cytokine and chemokine and compared them with those in the balf of mice. our results showed that a positive correlation exists between the ex vivo and in vivo models in the expression of selected cytokines and chemokines with a coefficient of correlation of 0.75. ifn-c and il-1b were significantly induced in vivo but not ex vivo. this may be due to an influx of multiple types of immune cells in the lung in vivo but not in the lung slice. the lower levels of il-1b in lung slices may be due to the observation that the epithelial cells and macrophages secreted less il-1b than neutrophils in responds to influenza infection (brandes et al., 2013) . the observation that ifn-c secretion was not detected in lung slices can be explained by the fact that the primary source of ifn-c are nk cells, cd4 + th1 cells and cd8 + t cells (schroder et al., 2004) , which are absent in the lung slice. despite the discrepancy in some of the cytokine and chemokine responses, ip-10, rantes and mip-3a are perfectly correlated between the ex vivo and in vivo models. therefore, ip-10, rantes and mip-3a may represent the inflammatory response following influenza virus infection in the ex vivo model. the choice of readout for antiviral effects is straight forward and was determined previously in our lab. the fluorometric substrate of the neuraminidase of the influenza virus, munana, is used for detection of virus neuraminidase activity, which represents the levels of virus replication . the virus replication level determined by na activity is proportional to that of the tcid 50 as shown in fig. 2 (compare 2b and 2d, 2c and 2e). however, the choice of readout for anti-inflammatory activity is much more complicated because no known cytokine or chemokine is widely accepted to represent the inflammatory response to influenza infection. based on the comparison between ex vivo and in vivo responses of the selected cytokines and chemokines following influenza virus infection, ip-10, rantes and mip-3a may serve as readouts for influenza-induced inflammation. in the recent literature, to elucidate which cytokine and chemokine is more important in the influenza-induced innate immune response, cytokines or their receptor gene were knocked out in mice. tnfr1 à/à , il-6 à/à , mcp-1 à/à , il-1r à/à , mip-1 à/à , ccr2 à/à , and gm-csf à/à mice did not survive influenza infection; however, ip-10 à/à mice did survive influenza infection, suggesting that ip-10 may play an table 2 the evaluation of a panel of antiviral and anti-inflammatory agents in mouse lung slice model. the 250 lm thick lung slices were prepared as described in section 2.3. the slices were infected with 200 ll of 10 5 pfu/ml pr8 virus. after 2 h incubation on 37°c, the viruses were removed and the lung slices were washed twice with pbs. then drugs with serially diluted concentrations were added and maintained for 48 h, and the na activities and ip-10 levels were determined as described in sections 2.7 and 2.8. important role in the aberrant cytokine responses during influenza infection (darwish et al., 2011; wang et al., 2013) . ip-10 has also been proposed to be a poor prognostic indicator for patients with severe acute respiratory syndrome (sars), which is clinically similar to severe influenza infection (nelli et al., 2012) . although we did not compare the proteome changes induced by influenza, the rationale to choose a single factor to prove the concept has been previously reported (cameron et al., 2008) . taking these results into consideration, we chose ip-10 as a readout for the evaluation of anti-inflammatory drugs, and ip-10 appeared to be valid for representing the inflammatory response in the validation test of a panel of antiviral and anti-inflammatory agents. of course, if a critical study of the efficacy of a candidate compound is required, multiple readouts determined by the levels of multiple cytokines and chemokines can be used to assess the inhibitory effects against the inflammatory response following influenza virus infection. very often, compounds showing antiviral or anti-inflammatory activity in the single cell system fail in the animal assay. to validate this precision cut mouse lung slice model in screening and evaluation of both antiviral and anti-inflammatory drugs against influenza virus infection in one assay using na and ip-10 as readouts, four categories of known antiviral and anti-inflammatory drugs were tested for their antiviral and anti-inflammatory activities. the results were compared with that from mouse studies conducted by others and us. the four categories of agents are: (i) agents with only antiviral effects in vitro and in vivo; (ii) agents with both antiviral and anti-inflammatory effects in vitro and in vivo; (iii) agents with only anti-inflammatory effects in vitro and in vivo; (iv) agents with antiviral effects in vitro which failed in the mouse model. the agents of the first three categories showed antiviral or anti-inflammatory effects in the lung slice model. among the agents from the category iv, all failed to show any antiviral or anti-inflammatory activity on the mouse lung slice model, except that nafronyl oxalate was shown to inhibit ip-10 release. overall, this validation testing of a group of agents with known antiviral or anti-inflammatory activities demonstrated that the mouse lung slice model is valid, convenient and cost-efficient for screening and evaluation of both antiviral and anti-inflammatory drugs in one ex vivo assay. it appears that only drugs showing either antiviral or anti-inflammatory activity in the mouse lung slice model may protect mice from infection of lethal influenza, though exceptions may occur. in summary, we established and validated a mouse lung slice model to evaluate compounds for antiviral and anti-inflammatory effects in one assay. importantly, employing the lung slice model is economical in term of experiment costs, in compound synthesis and animal use. for the development of antiviral drugs, the mouse lung slice model is more similar to the mouse model. for the development of anti-inflammatory drugs, the mouse lung slice model is suitable for drugs targeting multiple targets and pathways involved in the inflammatory response following influenza infection and more similar to the mouse model. this model can be useful in the secondary screening for selection of drug candidates with potent antiviral or (and) anti-inflammatory activities before testing in an animal model. notably, the mouse lung slice model should be used with caution in testing anti-inflammatory drugs against influenza viruses other than h1n1 in two aspects. firstly, to make sure that the virus being tested can replicate efficiently on mouse lung slices and induce cytokine/chemokine responses similar to that in vivo. secondly, the readout(s) for the inflammatory response can be different and should be determined experimentally ex vivo and in vivo. considering that the infection of many respiratory viruses causes acute inflammation in the human lungs and induce similar inflammatory responses, it is likely that the anti-inflammatory drugs developed using the influenza virus infected mouse lung slice model may also be effective in the treatment of infection of other respiratory viruses. taken together, our research demonstrates that it is beneficial to use the mouse lung slice model to develop new drugs with either antiviral or anti-inflammatory activities in the treatment of influenza virus infection. screening and identification of inhibitors against influenza a virus from a us drug collection of 1280 drugs treating viral exacerbations of chronic obstructive pulmonary disease: insights from a mouse model of cigarette smoke and h1n1 influenza infection genomic profiling of tumor necrosis factor alpha (tnf-alpha) receptor and interleukin-1 receptor knockout mice reveals a link between tnf-alpha signaling and increased severity of 1918 pandemic influenza virus infection inhibition of p38 mitogen-activated protein kinase impairs influenza virusinduced primary and secondary host gene responses and protects mice from lethal h5n1 infection a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection gene expression analysis of host innate immune responses during lethal h5n1 infection in ferrets human lung innate immune response to bacillus anthracis spore infection the prostanoid 15-deoxy-delta12,14-prostaglandin-j2 reduces lung inflammation and protects mice against lethal influenza infection immunomodulatory therapy for severe influenza fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia antiviral activity of the mekinhibitor u0126 against pandemic h1n1v and highly pathogenic avian influenza virus in vitro and in vivo treating influenza with statins and other immunomodulatory agents an overview of real-time quantitative pcr: applications to quantify cytokine gene expression oseltamivir-ribavirin combination therapy for highly pathogenic h5n1 influenza virus infection in mice a new shield for a cytokine storm factors associated with case fatality of human h5n1 virus infections in indonesia: a case series aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus interleukin-6 limits influenza-induced inflammation and protects against fatal lung pathology germacrone inhibits early stages of influenza virus infection amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea enhanced infectivity of h5n1 highly pathogenic avian influenza (hpai) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage precision cut lung slices as an efficient tool for in vitro lung physiopharmacotoxicology studies mammalian innate resistance to highly pathogenic avian influenza h5n1 virus infection is mediated through reduced proinflammation and infectious virus release inhibition of influenza virus-induced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses green tea catechin, epigallocatechin gallate, suppresses signaling by the dsrna innate immune receptor rig-i a simple method of estimating fifty per cent endpoints exploring lung physiology in health and disease with lung slices interferon-gamma: an overview of signals, mechanisms and functions lps-induced lung inflammation in marmoset monkeys -an acute model for anti-inflammatory drug testing long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution monoclonal antibody against cxcl-10/ip-10 ameliorates influenza a (h1n1) virus induced acute lung injury ifn-gamma treatment at early stages of influenza virus infection protects mice from death in a nk cell-dependent manner innate immune response to h3n2 and h1n1 influenza virus infection in a human lung organ culture model comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted a/california/04/2009 (h1n1) a simple statistical parameter for use in evaluation and validation of high throughput screening assays supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral.2015.05. 008. key: cord-009764-m9flptcv authors: bossé, ynuk title: the strain on airway smooth muscle during a deep inspiration to total lung capacity date: 2019-01-18 journal: j eng sci med diagn ther doi: 10.1115/1.4042309 sha: doc_id: 9764 cord_uid: m9flptcv the deep inspiration (di) maneuver entices a great deal of interest because of its ability to temporarily ease the flow of air into the lungs. this salutary effect of a di is proposed to be mediated, at least partially, by momentarily increasing the operating length of airway smooth muscle (asm). concerningly, this premise is largely derived from a growing body of in vitro studies investigating the effect of stretching asm by different magnitudes on its contractility. the relevance of these in vitro findings remains uncertain, as the real range of strains asm undergoes in vivo during a di is somewhat elusive. in order to understand the regulation of asm contractility by a di and to infer on its putative contribution to the bronchodilator effect of a di, it is imperative that in vitro studies incorporate levels of strains that are physiologically relevant. this review summarizes the methods that may be used in vivo in humans to estimate the strain experienced by asm during a di from functional residual capacity (frc) to total lung capacity (tlc). the strengths and limitations of each method, as well as the potential confounders, are also discussed. a rough estimated range of asm strains is provided for the purpose of guiding future in vitro studies that aim at quantifying the regulatory effect of di on asm contractility. however, it is emphasized that, owing to the many limitations and confounders, more studies will be needed to reach conclusive statements. the beneficial effect of a deep inspiration (di) on respiratory mechanics has long been an important topic of discussion among lung physiologists [1] [2] [3] [4] [5] [6] [7] [8] . however, the mechanism accountable for the respiratory relief that is normally afforded by a di is still not clear [7, 9] . several mechanisms are likely involved and each mechanism may contribute to various extents in different individuals. however, one mechanism that arguably received the most attention is the transient change of length (i.e., strain or stretch) that airway smooth muscle (asm) undergoes during the di . indeed, the expansion of lung volume during a di modifies the caliber of the airways (table 1) ; the word "caliber" herein refers to the cross-sectional area of the airway lumen. the increase in caliber during a di results from increasing radial stress that stems from the tethering force of the lung parenchyma. since asm is embedded within the airway wall in an orientation that is nearly orthogonal to the long axis of the airways [97, 98] , an increase in airway caliber necessarily implies that asm is transiently elongated. the question of whether, and to what extent, strain affects the contractility of asm is easier to investigate in vitro. this is because in vitro experiments can specifically control or monitor the length of asm. several types of asm preparations derived from resected or postmortem lung specimens are used to study asm mechanics. they include rings, spirals and segments of bronchi; strips and segments of tracheas; precision cut lung slices; and strips of lung parenchyma. our understanding of asm mechanics evolves quickly due to the use of these in vitro preparations. they can truly address questions that would otherwise be impossible to investigate in vivo. nowadays, the tools to study asm mechanics in vitro can recreate the dynamic lung movements that occur during breathing maneuvers . they thus emulate the in vivo environment within which asm normally resides and operates. this was a steppingstone in the field of asm mechanics. it has provided instrumental insights regarding the response of asm to stress and strain. these in vitro studies demonstrated that perturbing the length of asm greatly affects its contractile capacity. this effect is certainly relevant to the bronchodilator effect of a di. however, the reported in vitro results are also plagued with a lack of consistency. the main concern relates to the size of the effect, i.e., the magnitude by which strain decreases the contractile capacity of asm. however, the discrepancies go as far as being contradictory, as some studies reported that strain increases both airway responsiveness in vivo [99] [100] [101] and the contractile capacity of asm in vitro [102] . the controversy also extends to studies performed with isolated asm cells, where it was shown that strain can both decrease and increase surrogates for asm contractility [31, [103] [104] [105] [106] [107] . all together, these findings are conducive to alternative conclusions regarding the potential contribution of asm strain to the bronchodilator effect of di. the purpose of the present review is not to discuss the variety of factors that may be responsible for the inconsistencies reported in in vitro studies, but rather to focus on one possible source of error, namely the magnitude by which asm is strained during a di. i attempted, based on published studies on human subjects in vivo, to determine the physiological range of strains that asm undergoes in vivo during a di from functional residual capacity (frc) to total lung capacity (tlc). the first section describes the methods that can be used to estimate asm strain. the second section discusses the limitations associated with those methods and to which extent they may affect the accuracy of the reported strain. finally, the third section describes the confounders that affect asm strain and that inevitably contribute to its variability. [49] v d (using co 2 as the tracer gas) 5 healthy baseline exercise, co 2 inhalation (3-6%) or both all 13.9% [50] v d (using co 2 as the tracer gas) 1 healthy baseline always from tlc, expired to a given lung volume, and then inhaled different allocated tidal volume to subsequently measure v d on the subsequent expiration. all 20.8% [51] whole-body plethysmography 24 the complexity of the question in hand stems from the difficulty to assess asm strain in vivo. although refining technologies are currently emerging, no direct measurement of asm strain during a di has yet been performed in vivo. however, many technologies provide reliable measures of luminal volume, airway caliber and resistance. these measurements at different lung volumes then allow approximation of the extent to which asm is strained during a di from frc to tlc. this section describes the methods that may be used to assess airway wall strain in vivo in humans. the major strengths of each method are also briefly stated. 2.1 dead volume. the measurement of dead volume (v d ) is the more archaic [108] but still useful way to estimate the luminal volume of the airway tree at any given lung volume. the change in v d from one lung volume to the other then allows estimation of the strain undergone by the airway tree during such an excursion of lung volume ( table 1 ). the measurement relies on the fact that the last part of the inspired air in a breath does not reach the zone of gas-exchanging units. that last part is rather contained in the v d (also called the dead space), which includes the upper airways down to the terminal bronchi. since air in the v d does not mix with the alveolar gas, the composition of gas at the mouth during an expiration suddenly changes when it transitions from the air that was contained in the v d to the air that was contained in the alveolar space. the volume of air that comes out of the lungs before this transition of air composition is v d . because diffusion inevitably occurs at the boundary between v d and alveolar volume, and that the amount of diffusion is time-dependent, appropriate timing during the respiratory maneuver is essential to obtain an accurate measure of v d . fowler was the first to use the single-breath nitrogen (n 2 ) washout to measure v d [49] . this technique has been adopted and is still used [109] [110] [111] . alternatively, some have used co 2 as the tracer gas to estimate v d [57] . johns and coworkers have demonstrated that a breath-by-breath analysis of v d is possible using co 2 as the tracer [67] . by progressively changing the lung volume, one can then obtain a slope of the relationship between v d and end-inspiratory lung volume. this slope has been abbreviated dv d and has been used to assess airway distensibility (i.e., the ease by which the airways dilate in response to increasing lung volume) [67] . dv d is advantageous because it can be measured quickly in a noninvasive manner. . although x-rays in humans may provide enough resolution to detect abnormal size of the trachea [112] , it is not sufficient to quantitate the differences that may occur during an excursion of lung volume from frc to tlc. the resolution can be improved by contrasting agents. for example, dionosil oily inserted through the cricothyroid ligament under local anesthesia, has been used successfully to measure the changes in diameter of large and relatively smaller airways during a full inspiration in man [55] . fluoroscopy is somewhat better, as it allows motion recording. it has been used without contrast agents to monitor changes in airway caliber during breathing maneuvers. it was to assess the degree of collapsibility of the trachea during coughing in controls and in cases of acquired tracheomalacia [60] . fluoroscopy may be appropriate to diagnose and assign a degree of tracheomalacia, as it was done in that study. however, it does not seem to provide note: aoct-anatomical optical coherence tomography, copd-chronic obstructive pulmonary disease, di-deep inspiration, fev 1 -forced expiratory volume in 1 s, fot-forced oscillation technique, frc-functional residual capacity, hrct-high-resolution computed tomography, laba-long-acting b2agonist, mch-methacholine, pc 20 -the provocative concentration of methacholine causing a 20% decline in fev 1 , raw-airway resistance, rv-residual volume, saba-short-acting b2-agonist, sbnw-single breath nitogen washout, tlc-total lung capacity, vc-vital capacity, and v d -dead volume. a it was assumed that asm is arranged orthogonally in relation to the long axis of the airways. b it was assumed that the volume of the airways expands isotropically during an increase in lung volume from frc to tlc. c it was assumed that the changes in resistance are inversely proportional to the changes in airway luminal radius at the fourth power. the resolution to quantitatively evaluate the changes in airway luminal area during a di. whole-body plethysmography. the measurement of airway resistance (raw) by whole-body plethysmography (raw (pleth) ) is routinely performed in laboratories of lung physiology. raw (pleth) truly represents the resistance attributed to the viscous flow of air passing through the airways. the practical and theoretical principles involved were previously expatiated [113] . briefly, the subject is enclosed in a hermetic box of known internal volume. the subject is then instructed to pant inside the box into a tube connected to an open shutter and a pneumotachograph. the shutter is then suddenly closed while the subject tries to keep panting. the airflow at the mouth and the pressures at the mouth and inside the box are monitored continuously. the variations of pressure inside the box (dpbox) when the shutter is open reflect corresponding but opposite changes in alveolar pressure (dpalv). using the law of boyle-mariotte (v 1 p 1 ¼ p 2 v 2 ), the dpbox are converted into changes in volume (dvbox: often called the shift volume). the dvbox happens to be the mirror image of the changes in thoracic gas volume (tgv) caused by rarefaction and compression of air during the panting, which are required to generate the driving pressure (dpalv) that draws air in-and-out of the lungs. when the shutter is closed, the dvbox can be related to the changes in alveolar pressure (dpalv), since dpalv are then the same as the changes in mouth pressure. once the relationships between both airflow/dvbox (when the shutter is open) and dpalv/dvbox (when the shutter is closed) are known, the dpalv required to accommodate any chosen airflow (usually 1 l/s) can be determined. the ratio of dpalv/airflow is raw (pleth) . conveniently, the same panting maneuver and boyle-mariotte's law also allow the tgv from which the subject is panting to be measured. the panting can be performed at different lung volumes to measure the relationship between raw (pleth) and lung volume. from this relationship, the airway wall strain from frc to tlc can be roughly estimated ( table 1 ). the limitations are discussed below. the greatest advantages of the whole-body plethysmography are that it is noninvasive and nonionizing. deflation. the interrupted deflation can be used to measure pulmonary resistance (r l ) and to estimate airway resistance (or conductance). the subject is instructed to take a di to tlc and then to expire slowly (sometimes at a controlled rate) while the flow is being interrupted at the mouth for a short period at specified intervals. the flow at the mouth and p l need to be measured simultaneously. p l is measured conventionally using an oesophageal balloon to estimate pleural pressure and to subtract it from airway opening pressure. during the measurement, the change in lung volume is being tracked by integrating flow. this is required to relate punctual changes in p l caused by the interruptions to the lung volumes at which they were measured. the increase in p l caused by the interruption is the driving pressure required to accommodate the airflow (and the lung tissue flow) just preceding the interruption. since the resistance attributed to tissue flow (sometimes called tissue viscance) is small [114] , especially during deflation, the rise in p l caused by the interruption of flow is mainly ascribed to the frictional resistance to airflow within the airway tree. by dividing the increase in p l to the flow at the mouth, airway resistance can then be calculated at every lung volume at which the interruption took place. the change in resistance between frc and tlc can then be converted to estimate asm strain the same way it is done for raw (pleth) (discussed below) ( table 1 ). the advantage of the interrupted deflation in comparison to the measurement of raw (pleth) is that the breathing maneuver performed by the subject is much simpler. technique. the acoustic reflection technique is an especially good tool to diagnose tracheal stenosis and malacia [115] . the use of this technique to assess large airway caliber has also been validated in glass tube models and in humans [116, 117] . changes in large airway caliber caused by changing lung volumes were also estimated ( table 1 ). the acoustic reflection technique consists of a sound wave emitted by a loudspeaker that is traveling along the airways where it is reflected and perceived by a recording microphone located on the external device near the subject's mouth. the time gap between the emitted sound and the reflections is a measure of the distance between the microphone and the points within the airway where those reflections come from. the amplitude of the reflections is a proxy of the change in airway cross-sectional areas at those specified airway locations. the greatest advantages of the acoustic reflection technique are that it is noninvasive, nonionizing, and quick. it is also easy for the subject, as no complicated breathing maneuvers are required. 2.6 high-resolution computed tomography. high-resolution computed tomography (hrct) has first been used by wilson and coworkers in 1983 to assess airway caliber at different lung volumes [64] . since then, hrct was used extensively to assess the bronchodilator effect of di ( table 1 ). the breathing protocol used varied widely, but a scan is normally taken at frc and then at tlc and the differences in airway caliber at corresponding airway locations are measured. hrct is still considered by some the gold standard for imaging the airways. this is because it assesses directly the geometry of individual airways. it is also noninvasive and does not require sedation. the tools to analyze ct scan have also evolved to provide an accurate measure of airway wall thickness. another important asset is the fact that many airways of different sizes can be assessed simultaneously. magnetic resonance imaging (mri) is a technology that is currently emerging to image the geometry of airways in vivo in humans. the technology has evolved to offer a good temporal resolution (scanning time of 0.2 s), providing images without blurring, even during cough [65] . although mri is primarily used in the field of pulmonary medicine to image ventilation defects [118] [119] [120] [121] [122] , it can now be used to obtain descent three-dimensional reconstruction of the tracheobronchial tree [123] . however, the resolution does not equate hrct [124] . despite other limitations that will be discussed in sec. 3, mri still represents an appealing noninvasive and nonradiating alternative strategy to image airway caliber. further developments are awaited before mri can accurately assess the changes in airway caliber that occur during a breathing maneuver from frc to tlc (table 1 ). probe. the pitot static probe is a device used to measure the velocity of a moving fluid. it has several applications in modern world. a miniature pitot static probe (few centimeters long and 3 mm external diameter) can be inserted into the airway tree to assess airway caliber on the basis of physiological measurements. the probe is positioned at desired heights down the airway tree, which is determined prior the measurements using a bronchoscope. the probe has an opening at the distal end leading to a first catheter coming out at the mouth, which is used to measure impaction pressure (also called total pressure or ptot). the probe also has side holes that merge into a distinct catheter to measure lateral pressure (plat). the difference between impaction pressure and lateral pressure is the drop of pressure due to convective acceleration (i.e., the kinetic energy of the gas passing the cross-sectional area). assuming incompressible air flowing in a tube with a blunt velocity profile, bernoulli equation can then be used to estimate air velocity (ptot ¼ plat þ qv 2 /2, where q is air density and v is velocity). the flow at the pitot static probe can also be calculated from the mouth flow and correcting for difference in air pressure at that location (barometric pressure þ plat) using boyle's law. with this local flow and velocity, one can than calculate the cross-sectional area at the location of the pitot static probe ( is flow in l/s and v is velocity in m/s). calculating the cumulative expelled volume by integrating the flow at the mouth allows for changes in airway caliber to be related to changes in lung volume. theoretically, one maneuver would allow the measurement of airway caliber at every lung volume. the greatest advantage of the pitot static probe is thus to follow the kinetics of change in airway caliber. the insertion of an esophageal balloon as a proxy of pleural pressure (ppl) can also provide additional insights. for example, by relating airway caliber to transmural pressure (ptm, which is plat-ppl), airway compliance (caw) can be obtained. the pitot static probe is not often used to assess airway caliber at different lung volumes. in fact, only one study documented the utility of the pitot static probe for that purpose. in that study, the measurement was made during a maximal expiratory flow-volume maneuver (mefv), i.e., the subject was instructed to inhale to tlc and then provide a maximal expiratory effort to rv. several limitations are associated with this method. of special concern is the instability of the probe during the maneuver. the plugging of the end and side holes by mucus and secretions is also common. maximal reproducible efforts are also required, which can be demanding for the subject. these limitations, together with the other limitations discussed in sec. 3, may have dissuaded many investigators from using this method. technique. this technique consists of forcing the movement of air in or out the respiratory track using different devices and to then calculate impedance from the resulting flow and pressure that are measured at different locations in relation to the subject (reviewed in ref. [125] ). the most common devices force air directly into the subject's mouth and the resulting flow and pressure within the mouth are used to calculate impedance (input impedance). the motion of air imposed by the device can also take different shapes, but sinusoidal forcing at one or several simultaneous frequencies are often used. during the measurement, the subject is usually instructed to keep breathing normally at tidal volume. the forced oscillation is thus simply superimposed on top of the natural motion of air resulting from breathing. the forced oscillation technique (fot) has gained tremendous momentum in human respiratory research. this is because it provides valuable readouts pertaining to the mechanics of the respiratory system, including respiratory system resistance (rrs) and reactance (xrs) at every tested frequencies. these measurements allow one to infer on phenomena as complex as recruitment-derecruitment and airway caliber heterogeneity. one of the greatest advantages of the fot is its fine time resolution. because of this fine time resolution, it is possible to measure the kinetics of events happening over a short time-scale, e.g., such as the extent and dynamics of airway dilatation during a di. its usefulness is also ascribed to its noninvasiveness, the ease with which it is operated and the very low level of cooperation that is required from the tested subjects. forced oscillation technique can be used to estimate asm strain during a di from frc to tlc because rrs and its inverse, respiratory system conductance (abbreviated grs), represent proper proxies of airway caliber when measured near the resonant frequency (6-10 hz) [70] . this is especially true at zero flow, when the resistance is not affected by frictional airway resistance to airflow. many have measured rrs (or grs) to assess the changes in airway caliber during a di (see table 1 ). 2.10 anatomic optical coherence tomography. anatomic optical coherence tomography (aoct) is an in vivo imaging technology that broadcasts live the interior of the airways [126] . aoct uses a fiber-optic probe that passes through a bronchoscope and emits a beam of near-infrared light toward the airway wall. the resulting reflections are detected and analyzed using lowcoherence interferometry, which allows the measurement of the position of the airway wall in relation to the probe. by rotating the probe, one can obtained two-dimensional cross-sectional images of the lumen, and by moving the probe forward or backward, these serial two-dimensional images can provide a full 3d representation of the airway lumen. although not used extensively so far, aoct is likely to provide insightful information regarding the excursion of airway caliber during a di. i am aware of only one published study that has used aoct for that purpose [85] (table 1) . a new cousin of oct has also recently been developed, called orientation-resolved oct (or-oct) [127] . or-oct uses the birefringent property of asm to capture its exact localization within the wall of large airways. asm in humans in vivo can be visualized with a sensational level of resolution by this method [127] . or-oct should ultimately enable the direct measurement of asm strain during a di from frc to tlc. none of the aforementioned methods directly measure asm strain. they rather reported a change in the cross-sectional area of the lumen in individual airways, a change in luminal volume of the entire airway tree or a change in resistance to airflow in the airways or to the whole respiratory system. this obliges the transformation of the original data into radial airway wall strain. for the sake of this review, it was then assumed that the airway wall strain can be used as an appropriate proxy of asm longitudinal strain. this section describes the limitations associated with several methods, as well as the limitations associated with the transformations and assumptions that were made to convert the original data into asm strain. muscle. the strain on the airway wall reported by most methods during the di refers to the change in perimeter at the apical surface of the epithelium (i.e., the size of the lumen). this is true for imaging methods such as bronchography, hrct, mri, and aoct, but also for the methods relying of physiological measurements, such as v d and resistance (plethysmography, interrupted deflation, acoustic reflection technique, pitot static probe, and fot). it is important to understand that the change in luminal geometry does not represent precisely the change in perimeter occurring at the middle of asm. due to the combined thickness of the epithelium, the lamina propria, and the asm, the extent by which asm is stretched during a di is slightly overestimated when the luminal geometry (radius, diameter, perimeter, and cross-sectional area) is used to calculate the change in asm length (fig. 1) . the size of this effect is more prominent in smaller airways. in fact, the size of this effect increases as the ratio of the perimeter measured at the middle of asm to the perimeter of the lumen increases. the thickening of the airway wall observed in some respiratory disorders also amplifies the size of this effect (fig. 1 ). smooth muscle alignment. the reported strain (the one estimated in table 1 ) represents more accurately the radial strain on the airway wall than the longitudinal strain on the asm. this is because the orientation of asm within the airway wall varies. here, i assumed that asm is arranged in an orthogonal fashion in relation to the long axis of the airways. this assumption is correct for the trachea and the main stem bronchi. however, asm is oriented differently in smaller airways. the angle of orientation of asm relative to the long axis of the airways is on average 75 deg [98] . however, this angle is obviously another parameter that is variable from one airway generation to another, as well as from one asm bundle to another. the implication here is that the strain reported is somewhat overestimated compared to the real strain on the long axis of the asm bundle. for example, when one assumes a radial airway wall strain (viz., an increase of airway perimeter) of 25% with no longitudinal airway strain (viz., no lengthening of the airway) and an asm bundle oriented 75 deg off the long axis of the airway, the strain on the long axis of the asm bundle is 23.5% (fig. 2) . the magnitude by which asm strain is overestimated depends on how far off the angle of orientation deviates from 90 deg. the reality, however, is that the airway is also strained on its longitudinal axis (this is discussed below). if both the radial strain and the longitudinal strain of the airway increase by 25%, the asm are then also strained by 25% regardless of the orientation of the asm bundle in relation to the long axis of the airway (fig. 2 ). containing airway smooth muscle. although the bundles of asm completely encircle the small airways, it is not the case for the trachea and the main stem bronchi. in fact, asm occupies approximately 1/3 and 2/3 of the total airway wall circumference in the trachea and the main stem bronchi, respectively. the remaining circumference is made up of cartilage. since the cartilage is stiffer than the asm, the strain is not distributed homogeneously along the entire circumference of the airway. in reality, near 100% of the change in circumference is taken up by 1/3 to 2/3 of the airway wall; the portion occupied by asm and free of cartilage. asm strain during a di in the trachea and the main stem bronchi may thus be well beyond the one estimated based on the change in airway caliber. dimensions. the estimation of asm strain during a di from frc to tlc is sometimes accomplished by measuring a change in volume. this is the case for the methods measuring and comparing v d at different lung volumes. to convert a change in volume into radial airway wall strain (or $asm longitudinal strain), an additional step is required. this is because not only the caliber but also the length of the airways fluctuates during breathing maneuvers [128] . the relative extent by which the length of the airways elongate and shorten during breathing maneuvers compared to the extent by which they dilate and narrow have been poorly studied. one can assume that the percentage change is equal in every directions; so that the luminal airway volume expands or shrinks isotropically without changing shape during both inflation and deflation. some experimental data support this assumption [58, [129] [130] [131] . studies showing that dv d is linearly related to dlung volume also support this assumption [64, 67] . however, other evidences suggest that it is not always the case [132] . in fact, the accuracy of this assumption seems greatly affected by the starting lung volume and the magnitude of the lung volume excursion [80, 84, 130] . the strain in one direction relative to the other directions is also likely to vary between airways from different individuals and between airways within the same individual. regardless, the degree by which the deformation of the airways deviates from isotropy obviously affects the precision with which asm strain can be estimated by these methods. fig. 1 the changes in luminal geometry overestimate the stretch the asm undergoes during a deep inspiration (di). the schematic illustrates a normal (left) and an asthmatic (right) airways at frc and after a di to tlc. the dimensions are zoomed but at scale to an average airway of the ten generation. the springs represent lung recoil. they are stretched at tlc relative to frc. the arrow at approximately 2 o'clock is the radius (in mm) of the airway lumen (r l ). the arrow at approximately 1 o'clock is the radius up to the middle of the airway smooth muscle layer (r m ). the material composing the airway wall was considered inextensible. notice the thinning of the airway wall when the lungs are inflating to tlc. it this schematic it was assumed that the luminal geometry at frc was equal between normal and asthmatic. it was further assumed that the di was increasing luminal radius by 25% in both normal and asthmatic, so that the luminal geometry at tlc was also equal between normal and asthmatic. the schematic demonstrates that a 25% increase of luminal perimeter (p l ) only causes a 21.2% increase of the perimeter at the middle of the asm layer (p m ). this effect is further amplified in asthmatic because of a thicker airway wall (25 versus 18.2%). the mathematics is developed in the middle. other abbreviations: a aw -area of the airway wall from the lumen to the middle of the asm layer, a l -luminal area, a m -area internal to the middle of the asm layer, and %d-change in percentage. due to the elongation of the airway tree during a di, the relative distance of a specific point within an airway to a recording device at the mouth increases. this is an issue for several methods. this vertical displacement is important to consider in order to allocate a change in caliber from frc to tlc at specific airway locations. otherwise, erroneous changes are caused by comparing a upper (and larger) part of the airway to a lower part due to lung and airway elongation at tlc. for the acoustic reflection technique, coronal radiographs can be acquired. the radiograph allows the identification of the carina and to subsequently measure its distance from the microphone. the other measured areas can then be allocated to specific airway locations. since the carina is moving at different locations during a breathing maneuver, several radiographs for monitoring its location at different lung volumes is ideally required. however, this is not always done [62] . it is thus likely that a small variability arises due to a vertical displacement of the airways relative to the recording microphone. to limit this problem, the cross-sectional area is usually averaged over a few centimeter-long segment. the vertical displacement is also an issue for the pitot static probe. indeed, the distance of the probe from the mouth is always the same, as the catheter is fixed at the mouth. this means that the probe is actually moving in relation to the airway tree during the forced expiratory maneuver. this is not taken into account with the pitot static probe. the issue of vertical displacement is more easily overcome by hrct. this is because an image of the entire (or a major portion of the) lungs is obtained. landmarks can thus be identified to assess the vertical displacement of the lungs during the di. the strain can be applied in the three dimensions, as well as in two directions (stretch or compress). the focus of this review is on the expansion of airway caliber during a di causing a stretch to asm. however, the elongation of the airways during a di also implies that asm is strained in at least another dimension, namely on its transversal axis. the effect of transversal strain on asm contractility has not been studied sufficiently. one study demonstrated that cyclical lengthening of isolated airways mainly increases their contractile capacity [133] . the third dimension (thinner versus thicker airway wall) is also important to consider. one may think that an increase in p l should thicken the airway wall, for the same reasons as it increases the length and the perimeter of the airways. however, the materials constituting the airway wall hardly change in volume during a di compared to the air-filled compartments (i.e., the lumen of the airways and the alveoli). increasing the length of an airway and its caliber during a di to tlc should thus exert a tensile stress on the airway wall, which tends to make it thinner. many ct studies actually reported that the airway wall is thinner at tlc, supporting that the airway wall is compressed in this dimension during a di [92, 94] . this was also shown in cat lobes frozen at different volumes [131] , as well as in isolated airways stretched to the letter a represents the airway perimeter and, in this example, is set to 10 mm. the letter b is the airway longitudinal distance covered by the asm bundle going around the full circumference of the airway. finally, the letter c represents the length of the asm bundle and, in this example, is set at an angle 75 deg off the long axis of the airway. using trigonometry, b and c can be determined. now imagine that this airway is stretched radially to increase its perimeter by 25%. on the flattened airway shown in (a), this would increase the height of the rectangle by 25% without changing its length (b). compared to (a), the length of a would increase by 25%, the length of b would remain unchanged and the length of c would increase by 23.5%. the angle of c would also change to 77.9 deg. therefore, a radial stretch to the airway increasing its perimeter by 25% is expected to strain the asm bundle by only 23.5%. now imagine that the airway length is also strained by 25%. on the flattened airway shown in (b), this would increase the length of the rectangle by 25% (c). compared to (a), the length of a, b, and c would all increase by 25%. in contrast to (b), the angle of c would remain unchanged. therefore, when both radial and longitudinal strains are applied simultaneously at the same magnitude on an airway, the strain on the asm is also of this magnitude. elongated lengths [133] . overall, these suggest that the airway wall is stretched in two dimensions and compressed in one dimension during a di to tlc. the asm should thus be longer and wider but thinner at tlc. this is different from stretching an asm bundle in vitro, where it becomes longer but consequently less wide and less thick. thus, in contrast to in vivo, the asm in vitro is stretched in only one dimension and compressed in two directions. the effects of varying the orientation and the direction of strain on asm contractility have been largely overlooked. only few studies conducted with isolated cells have examined the effect of strain orientation on asm contractility [31, [103] [104] [105] [106] [107] . the findings suggest that strain orientation has a major impact on the outcome. to the best of our knowledge, no study has yet investigated the impact of strain orientation and direction, as well as their different combinations, on the contractility of asm tissues. these studies are clearly warranted. airways. the effect of the upper airways (mouth, larynx, and pharynx) cannot always be discounted. whether the upper airways can be considered a mere extension of the lower airways can be justifiably questioned. some evidence suggests that the cross-sectional area of upper airways is also dependent on lung volume [134] . however, the upper airways also have an intricate geometry. the concepts of fluid mechanics that apply to a single rigid cylindrical tube in which the flow regime is laminar might not be relevant. for example, the flow path during an expiration bends when it reaches the larger space of the mouth. the flow path also encounters many protruding structures and diverticula that can alter its flow regime such as the vocal cords, the uvula, the epiglottis, the tongue, and the alimentary and nasal canals. all these structure are likely to foster turbulent flow. the measurements of resistance by the fot, the interrupted deflation, and the whole-body plethysmography are affected by upper airways. in the case of raw (pleth) , attempts were made to subtract upper airway resistance from raw in order to obtain lower raw [56] . in these experiments, the pressure difference between two lateral taps, one at the mouth and one inserted midstream in the tracheal lumen via a puncture 2-3 cm below the cricoid cartilage, was measured. this difference in pressure over the mouth flow was then used to estimate upper raw and to subtract it from raw (pleth) [56] . the acoustic reflection technique is one convenient method to assess upper airways. this is because the time at which the signal is collected can be allocated to a specific distance from the mouth. the intensity of the signal is thus dictated only by the structural features that are in place at that distance. however, it does not provide information regarding the structural features altering the signal. therefore, a notch, a dent or a circular decrease in caliber are equally detected. capturing the excursion of small airway caliber during a di is a challenge. indeed, many methods described in sec. 2 are restricted to the assessment of large airways. this is probably the main limitation of the acoustic reflection technique. according to the results obtained with rigid casts of the airway tree, the areas determined acoustically agreed well with the real areas for airway segments extending up to 6 cm past the carina [135] . therefore, the airways further down the main bronchi cannot be assessed. this obviously restrains the use of the acoustic reflection technique for the assessment of airway wall strain during di. also, only one measurement per distance can be obtained. so beyond the branching point, the signal emanates from the cumulative effect of the two main bronchi and their respective contribution cannot be distinguished. the pitot static probe is also limited to the assessment of the large airways. the lowest position measured in brackel and coworkers' study was at the entrance of the middle lobe [66] . in this case, the penetration depth of the device is limited by its size in relation to airway caliber. the same constraints apply to aoct. high-resolution computed tomography is probably the best method to assess the small airways. yet, adequate resolutions are restricted to airway size over $1 mm of internal diameter. the contribution of the small airways is certainly embodied in measurements such as v d , raw (pleth) , and fot. however, the extent by which the small airways contribute to the overall signal cannot be quantified. fot was initially suggested by some to distinguish the small from the large airways. the rationale lies in the traveling distances of the oscillations at different frequencies. while oscillations at low frequencies travel far down into the lungs, the traveling distance of high frequencies are shorter. therefore, while the low frequencies should probe the entire airway tree, the high frequencies should only probe the large airways. by looking at the frequency-dependence of rrs, or by using a simpler readout such a r 5-19 (which is the subtraction of rrs at 19 hz from the rrs at 5 hz), it was suggested that the degree of obstruction of the small airways can be deduced. although this line of reasoning is logical, this is no longer the prevailing belief. first, the penetration depth of the forced oscillations at different frequencies cannot be ascertained. it is thus not sure from how far deep into the lungs the signals used to calculate rrs at different frequencies emanate. second, and most importantly, computational models have clearly demonstrated that the frequencydependence of resistance is particularly sensitive to the pattern of constriction in peripheral airways [136] [137] [138] . on one hand, a homogeneous pattern of small airway constriction increases rrs equally throughout the frequency range. on the other hand, a heterogeneous pattern of small airway constriction increases rrs way more at low than at high frequencies. therefore, the frequencydependence of rrs (or r [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ) is now considered a readout that is more useful to quantify the degree of airway narrowing heterogeneity in the periphery than to assess the degree of small airway obstruction. near total lung capacity. the measurements made at tlc are sometimes difficult and sometimes just not possible. raw (pleth) , for example, cannot be measured at tlc but only near tlc. the measurement requires a certain amount of air to flow in-and-out of the lungs to calculate raw (pleth) . this panting maneuver near tlc is also difficult for the subject. the measurement of r l with the interrupted deflation also required a certain amount of air to evacuate the lungs in order to measure the change of p l caused by the interruption. therefore, the value at tlc cannot be determined. holding the breath at tlc during hrct can also be difficult, especially for older and/or sicker patients. concerning the pitot probe, the airway caliber at different positions were grouped in volume range (e.g., the caliber between 100% and 80% of fvc). the total strain experienced by asm may thus have been underestimated because many measurements were not at tlc. notwithstanding this possibility, the error associated with not assessing airway caliber at tlc compared with near-tlc would be small since the airways are stiffer in that range of lung volume. the variation in transmural pressure may disproportionately exceed the strain on asm. in fact, submaximal volumes and/or p l are generally sufficient to achieve maximal dilation [130, 139] . other issues are also more common at or near tlc than other lung volumes and can affect the accuracy of the measurement. for example, glottic closure commonly occurs at extreme volumes (rv and tlc). therefore, the changes in airway caliber sometimes need to be assessed within these volumes (i.e., a little above rv and a little below tlc), which may slightly underestimate the full range of airway wall excursion that occurs during a di from frc to tlc. 3.10 bronchial tree geometry. the estimation of asm strain during a di from frc to tlc is often accomplished by measuring the changes in resistance at different lung volumes. this is the case for the whole-body plethysmography, the interrupted deflation, and the fot. to convert resistance into asm strain, some arithmetics are needed. the calculation is based on poiseuille's equation. this equation states that within a tube with a perfect cylindrical geometry in which flow is running in a laminar fashion, airway resistance is inversely proportional to the luminal radius at the fourth power. a change in airway resistance at different lung volumes can thus be converted into asm strain, as the percent change in radius is the same as the percent change in airway perimeter (viz., $asm length). as aforementioned, the airways also elongate during a di. according to poiseuille's equation, resistance to airflow is proportional to the length of the airway. so while the increase in caliber at tlc decreases resistance, the elongation increases resistance. as resistance is related to the radius at the fourth power, the effect of changing caliber largely predominates over the effect of changing the length of the airway. however, the estimation of asm strain based on measurements of resistance might be slightly underestimated because the increase in airway length is usually not taken into account. it is understood that the airway tree is way more complicated than a single cylindrical tube with identical size on both ends. it is also understood that the flow is not always laminar. the airway tree branches in different directions, contains numerous bifurcations, and many bronchi are bending and exhibit a tapered end. all these elements obviously disturb flow and increase the degree of turbulence. notwithstanding this complexity, the most accurate computational model of the human lungs is the singlecompartment model [140] . this model is merely constituted of a single airway with a perfectly cylindrical geometry that runs into a single elastic compartment. the resistance that stems from the viscous flow of air within the airways in a real airway tree, as well as its response to different interventions, thus seem to be predicted quite precisely just by altering the caliber of a single conducting airway in the modeled lungs. it seems that, sometimes, the gestalt behaves as a simple element. there is thus no reason to treat it differently as such in order to extract information that would otherwise be impossible to infer. the presence of chokepoints (sometimes called stenosis) is also important to consider. the excursion of asm length at these chokepoints during breathing maneuvers can be greatly altered [141] . chokepoints also obstruct the dynamics of fluid. consequently, they severely increase resistance without necessarily affecting the caliber along the entire airway. the presence of chokepoints is thus likely to insert inaccuracies in the estimation of asm strain in methods using measures of resistance. in addition, chokepoints affect the regional transmural pressures, especially during forced maneuvers. while the transmural pressure downstream of the chokepoint increases during expiration (as the luminal pressure increases in the airways and alveoli subtended by the choked airway), it decreases upstream of the chokepoint. the opposite occurs during inspiration. either way, the transmural pressures are altered. the strain asm undergoes is thus attended to be affected in these regions. also, these chokepoints are usually not present at every lung volume. the chokepoint may thus affect resistance at low lung volumes but not at high lung volumes, and thus affect the change measured between two lung volumes. more generally, the entire geometry of the airway tree (not only chokepoints) is likely to change at different lung volumes, which inevitably affects differently the resistance to airflow. 3.11 chest wall. the chest wall can also affect some measurements. this is the case for the fot. the estimated change in airway caliber calculated from the change in rrs from frc to tlc needs to be taken with caution. this is because rrs at volumes surrounding frc is compounded by the resistance of the chest wall (rcw), which is not the case at tlc [71] . consequently, the delta of rrs from frc to tlc overestimates asm strain. this is consistent with brown and coworkers' study, in which the estimated asm strain by fot using rrs was greater than the one predicted based on delta v d assessed by the single-breath nitrogen washout [73] . r l estimated with the esophageal balloon can remedy this overestimation by discarding the effect of the chest wall. 3.12 ionizing radiation. the exposure to ionizing radiation is obviously a major concern. it is probably the most important downside of hrct. repeated measures should absolutely be avoided. unfortunately, the investigators are left with a few snapshots at a few chosen lung volumes to quantify the excursion of airway caliber, which is far from ideal. radiation is also a concern with bronchography, although the doses used are lower. many factors influence the estimated strain asm undergoes during a di from frc to tlc and should thereby be considered as confounders. the confounders can be related to methodological procedures (an intervention or the breathing maneuvers during and preceding the measurement), which can either be controlled or not. alternatively, some confounders are inherent biological factors that cannot be controlled. this section describes the many procedural and biological confounders that are known to affect airway wall strain during a di. the magnitude by which the airways are strained during a di is dictated in great part by the swing in transmural pressure (i.e., the pressure across the airway wall). the swing in transmural pressure during a di depends on the extent by which the lung parenchyma is pulling the airway open, which, in turn, depends on the transpulmonary pressure (p l ). a greater swing in p l and/or lung volume during a di inflicts a greater excursion of airway caliber and, thereby, a greater stretch to asm. p l and the lung volume attained at tlc are variable between individuals. these biological factors thus affect asm strain during a di and should thus be considered as important confounders. as discussed above, many methods that are used to estimate asm strain from frc to tlc rely on tricky breathing maneuvers near tlc (e.g., raw (pleth) ) or a holding maneuver at tlc (e.g., hrct). the level of inspiration that is achieved/maintained by the subjects during such maneuvers relative to their maximum also contributes to variability [141] . it is also important to mention that considerable regional differences in p l exist within the lungs. during the same breathing maneuvers, the airways in lower lobes are more strained than airways of the same generation in the apical lobes [87, 88, 92, 96] . the estimated strain asm undergoes is thus highly influenced by the region of the lungs in which the airways are embedded. pressure and volume. the pressure-volume curve of the lungs is not linear, so as the curve defining the relationship between transmural pressure and airway radius. by changing the starting (frc) transpulmonary pressure or lung volume not only the swing in pressure and/or volume from frc to tlc may be affected [86] but it also implies that the airways are now operating on a different part of the transmural pressure-radius curve. this is important as it indicates that the same swing in pressure now translates to different changes in radius and thus to different degrees of asm strain. this was predicted by computational modeling [142] and clearly shown in vitro [29, 39] . an increase in frc (i.e., hyperinflation) also impairs the ability to generate high p l [59] . consequently, it reduces the pressure distending the airways during a di. in fact, hyperinflation is a strong predictor of the bronchodilator effect of di [86] . more severe is the hyperinflation, smaller is the excursion of airway caliber from frc to tlc and less efficient is the bronchodilator effect of a di [86] . lung volume and p l at frc also vary between individuals. these biological factors are thus important confounders that affect the strain asm undergoes during a di. all the methods used to estimate asm strain during a di rely on measurements made at frc. they are thus influenced by the starting lung pressure and volume. 4.3 hysteresis. the pressure-volume curve of the lungs during inflation and deflation do not overlap. in fact, when the two curves are combined they form a loop that rotates clockwise, meaning that the volume at any given pressure is greater during deflation. this is called hysteresis and is due to the resistive component of the lungs, which is chiefly perceived during inflation but dissipated and barely not perceived during deflation. this hysteretic behavior also applies independently to both the airways and the lung parenchyma. therefore, when tracking the transpulmonary pressure and the cross-sectional area of an airway during breathing, a loop is also formed. however, the direction of rotation depends of the relative hysteresis between the airway and the surrounding parenchyma. when the airway is more hysteretic than the parenchyma the loop rotates clockwise. inversely, when the parenchyma is more hysteretic than the airway the loop rotates anticlockwise. in healthy individuals, airway hysteresis dominates and is largely due to asm tone [57, 58] . this was also shown in isolated airways [132, 143] . hysteresis has important implications. it indicates that the caliber of an airway at a particular lung volume varies according to whether it follows a deflation from a higher lung volume or a inflation from a lower lung volume [58] . after either a deflation or an inflation, the caliber can be larger or smaller depending on whether airway or parenchymal hysteresis predominates. for example, when airway hysteresis predominates, the caliber of the airways at frc after a di to tlc is greater than the caliber at frc prior to the di. in essence, this is the definition of the bronchodilator effect of a di. hysteresis also implies that the estimated strain asm undergoes during a di is not the same when the measurements are made from frc to tlc than when they are made from tlc to frc. an important procedural confounder is thus related to whether the change in airway caliber is measured during inflationary maneuvers versus deflationary maneuvers. the methods used to estimate asm strain described in sec. 2 involve either types of maneuvers. for example, the measurements of r l at different lung volumes with the interrupted deflation are performed during deflation. the pitot static probe also takes the measurement during a forced deflationary maneuver. contrastingly, the change in crosssectional area of the airway lumen with the mri is measured during inflation. this is because the image has to be captured during the flow of hyperpolarized gas ( 3 he or 129 xe) into the conducting airways in order to achieve a better contrast between the airways and the parenchyma. this also implies that the image is not obtained at fixed volumes but rather during a dynamic change in lung volume. the caliber at which an airway settles after a perturbation, such as a di, also takes time due to hysteresis. the timing at which the measurements are made can thus affect the results and should thereby be considered as a confounder. likewise, some methods used to estimate asm strain require interferences with the normal pattern of breathing. hrct, for example, requires breath-holding to assess airway caliber at different lung volumes. the breathhold usually decreases the bronchodilator effect of a di [3, 144] . however, whether the breath-hold overestimates or underestimates airway wall strain depends: 1-on the relative hysteresis between the airways and the parenchyma; 2-at which lung volume it is measured; and 3-whether the lungs were inflating or deflating before the breath-hold. regardless, the longer time that is allowed for the airways to settle at a new lung volume is likely to affect its caliber. consequently, the estimated strain excursion undergone by asm during such a change in lung volume will also be different compared to the one caused by the same change in volume with no breath-hold. all the other methods using measurements made at static (or quasi-static) lung volumes instead of dynamic lung volumes are likely to be affected by this procedural confounder. other procedures that clearly affect the estimation of asm strain include forced breathing maneuvers, such as forced expiratory volume in 1 s (fev 1 ) and coughing. the transitory peak of negative airway transmural pressure achieved during these maneuvers can transiently narrow the airway lumen to a level beyond that observed at the end of the maneuver [65, 74, 78, 79] . this phenomenon is also known for causing dynamic collapses and expiratory flow limitation. one method used to estimate asm strain that can certainly be affected by forced maneuvers is the pitot static probe. the flow rate (or speed of inspiration) during the di is also important to consider. a di at a high flow rate is more effective to bronchodilate the airways than a di at slow flow rate [3, 4, 144] . this was also demonstrated with isolated airways [30, 145] and asm strips stretched at different rates [16] . an increase in transmural pressure was proposed to account for the greater bronchodilator effect observed at high flow rate [144] . with regards to the estimation of asm strain, both inspiratory and expiratory forced maneuvers should be considered as procedural confounders. beyond the different breathing maneuvers used during the measurements (inflation versus deflation, static versus dynamic, slow versus forced maneuvers), the sequence of events preceding the measurements should be considered. the most prominent example is the bronchoprotective effect of a di [146] [147] [148] [149] [150] [151] . so a di, or a series of dis, prior to a bronchoconstriction elicited by inhaled methacholine, facilitates bronchodilatation induced by a subsequent di postmethacholine [101] . the time elapsed between the di premethacholine and the subsequent di under a bronchoconstricted state also affects its bronchodilator effect [148] . the time spent without di under bronchoconstriction also affects the excursion of airway caliber during a di because it increases narrowing [149, 152] . the bronchodilator effect of a di is also not the same after a sequence of dis than after a single di [149] . all together, these results indicate that the strain asm undergoes during a di is affected by lung history. the geometry of the airways at frc can also be affected if the preceding measurements were performed at rv compared to if they were performed at tlc [57] . the caliber of the airways at a given lung volume is greater when preceded by measurements done at a higher lung volume [114] . as explained above, part of it is due to hysteresis. notably, history can affect the measurements in both directions, as it can either increase or decrease the estimated strain asm undergoes during a di. lung history thus represents a serious procedural confounder and should be controlled whenever possible. 4.6 posture. the lungs undergo important deformation during changes in posture. this is because it changes the direction of the gravitational forces, which then affects the local p l and the gradient of p l [153] . a computational model predicted that a change of $10 cmh 2 o can occur at specific locations in the lungs during a change in posture from supine to prone [153] . in turn, this can be expected to change lung volume by approximately twofold, depending on which part of the pressure-volume curve this change in pressure occurs. this is then predicted to strain asm by $26%, assuming that the perimeter of the airways changes proportionally to the changes in the cube root of lung volume. a change in posture also affects the starting (frc) lung volume, engendering the consequences stated above. for example, frc is decreased in supine posture [154] . the methods used to estimate asm strain involved measurements at different postures. the measurements of airway resistance by fot and plethysmography are usually performed seated upright. contrastingly, hrct and mri are usually performed supine. the changes in gravitational forces due to these different postures are likely to affect asm strain differently in different locations of the lungs. the posture should thus be considered a procedural confounder. all the elements that constitute the airway wall can be organized differently, in addition to vary in absolute amount and relative proportion [155] . these elements include the cellular and the extracellular components of the airway wall, as well as other structures such as the cartilages. any elements constituting the airway wall should thus be considered as biological confounders. this is because any change in the structural composition of an airway is likely to modify its mechanical properties and thereby affects the strain asm undergoes during a di from frc to tlc. many of these elements vary in healthy subjects according to airway size. small airways, for example, has a structural composition that makes them more compliant [66, 76, 84, 85, 91, 141] . their percentage of dilation during a di is thus greater compared to larger airways [55, 141] . interestingly, it was shown in mice that the specific compliance of the small airways are greater than the specific compliance of the lungs [156] . the p l at which the airway dilation plateaus during an inflation maneuver also varies with airway size. in healthy individuals, the p l at which this plateau occurs increases progressively with increasing airway size [80, 84] . this indicates that maximal airway dilation occurs rapidly in small airways during inflation, while the large airways continue to dilate until later on in the maneuver. this is concerning for the methods that use the change in the entire volume of the airway tree in order to estimate the longitudinal strain on asm. this is because these methods assume that all the airways expand to the same extent. as stated above, airways of different size have different compliance. their compliance also varies according to the pressure range across which they are studied [80, 84] . in airway generations 0 (trachea) through 5, the curve that describes the relationship between luminal area and p l in every generation is sigmoidal. in fact, most of the change in luminal area occurs within a narrowed range of p l of about 15 cmh 2 o [85] . therefore, a limited window exists where changes in p l actually cause changes in airway caliber. this implies that no change in airway caliber occurs at either low and high p l . contrastingly, in the steeper part of the curve, large changes in caliber occur across a very small range of p l . similar to the mouse study stated above [156] , the specific compliance of the small airways in dogs and sheep are greater than the specific compliance of the lungs in that steeper part of the curve [130, 157] . collectively, these last observations suggest that the starting p l , the maximal p l attained during the di, and the curve describing the relationship between p l and the caliber of the airways under scrutiny need to be known in order to allocate an exact amount of strain on asm during a di. as mentioned above, the swing in transmural pressure during a di is a major determinant of asm strain. apart from the transpulmonary pressure, the transmural pressure across the wall of any airway is influenced by the stiffness of the surrounding lung parenchyma and the degree of interdependence between the airway and the parenchyma [158] . a more compliant parenchyma may dampen the swing in distending pressure generated by lung inflation and thus limit dilation. a loss of connectivity between the airway and the parenchyma, due to alveolar detachments [159] or geometric decoupling due to thicker adventitia [160] , also limits the transmission of pressure across the airway wall. the stiffness of the surrounding parenchyma and the level of interdependence between the airways and the parenchyma are thus biological confounders that are important to consider. 4.9 age. aging influences the mechanical properties of the lungs and its constituents [161] . age should thus be considered a biological confounder. for example, the extensibility (i.e., the ease by which it is elongated) of extraparenchymal airways, such as the trachea, decreases with age [162] . in contrast, intraparenchymal airways and the parenchyma seem to be more distensible [141] . indeed, loss of lung elastic recoil was reported with aging [163] . the larger percentage change in caliber of small versus large airways during a change in lung volume also seems to vanish with age, probably due to an increase in lung compliance [141] . age can thus affect asm strain during a di from frc to tlc due to various reasons. these phenomena may account for the attenuated bronchodilator effect of di with aging [164] and the lack of bronchoprotective effect of di in infants [151] . 4.10 health status. respiratory disorders can influence to various extent and by several ways the strain asm undergoes during a di. the driving pressure, the mechanical properties of the airways and the lung parenchyma, as well as the forces of interdependence can all be affected by diseases. in fact, changes in the structural composition of the airways, a process called remodeling, are common in many respiratory disorders and can have a major impact on the mechanical properties of the airway wall [165] . airway distensibility is decreased in asthma [59, 64, 66, 67, 69, 70, 86] , especially at high [75] but also at low lung volumes [166] . this seems to be mainly caused by airway remodeling [69, 75] but also inflammation [166, 167] . asthmatic patients also tend to exhibit airways of smaller caliber [68, 70, 85, 168] . the sigmoidal curve that describes the relationship between p l and airway caliber is also shifted to the left in asthmatic patients, at least when a bronchodilator (salbutamol) is administered prior to measurements [85] . this suggests that the changes in airway caliber due to breathing maneuvers occur at lower p l in asthma [85] . this may explain why fluctuation of rrs during tidal breathing is increased in asthma [72] . interestingly, this increased tidal fluctuation of rrs also negatively correlates with the bronchodilator effect of di [72] . this is somewhat logical. by operating at a steeper part of the sigmoidal curve during tidal breathing, less of the total possible strain is available during a di to tlc. in contrast to asthmatic patients, the lungs of nonasthmatic individuals seem to breath in a range of p l below the one where airway caliber is greatly affected by small changes in p l (based on small tidal fluctuation of rrs [72] ). however, for the same reason, the total possible strain the airways can undergo is still available and may occur during a di to tlc. the benefit is a greater stretch to asm and, thus, a commensurately greater bronchodilator effect of di. finally, a decline in lung elastic recoil was also reported in longstanding asthma [169] [170] [171] . all these phenomena may account for the attenuated bronchodilator effect of di in asthma [70, 72, 167, [172] [173] [174] [175] [176] [177] [178] [179] , as well as its attenuated bronchoprotector effect [150, 172, 174] . airway distensibility also seems to be attenuated in copd patients [76, 180] , especially in emphysema-predominant compared to airway-predominant subtypes of copd [88] . in this case, the lower apparent distensibility seems mainly attributed to a loss of recoil due to parenchymal destruction [59, 76] , as well as hyperinflation, which limits inspiratory capacity and the driving p l during the di [76] . these phenomena may account for the attenuated bronchodilator effect of di in copd [76, 167, 180] . in contradistinction, the distensibility of the airways is increased in patients with cystic fibrosis [63] . lung volume dependence of tracheal cross-sectional area in these patients was calculated to 0.22 cm 2 /l, compared to almost nil in healthy subjects. cystic fibrosis patients also demonstrate increased lung compliance [181] , which may limit the swing in p l . therefore, the reported increase in tracheal distensibility may be attributable to flaccidity, as previously reported in older patients with cystic fibrosis, as well as postmortem on isolated tracheas from patients with cystic fibrosis [112] . bronchiectatic bronchi, which are common in cystic fibrosis patients, are also prone to bronchomalacia. it is important to mention though that the incidence of bronchomalacia is high (70%) in patients with bronchiectasis with or without coexistent lung disorders [182] . even nonrespiratory disorders are sometimes important to consider. allogeneic haematopoietic stem-cell transplantation, for example, increases the changes in airway caliber during a lung excursion from frc to tlc [90] . this seems to be entirely related to an increase in lung parenchymal stiffness rather than an increase in airway distensibility. obesity is another example that can significantly impact asm strain during a di, mainly by changing lung volumes [183] . this phenomenon may account for the attenuated bronchodilator effect of di in obesity [184] [185] [186] . most respiratory disorders also affect the airways nonuniformly [187, 188] . the amount of strain the asm experiences during a di is thus expected to vary according to this patchy pattern of affection. overall, diseases can exert a huge and variable impact on the strain asm undergoes during a di. they should thus be considered as biological confounders. among all the elements that constitute the airway wall, asm is one that is very special. this tissue is endowed with the ability to quickly adjust the mechanical properties of the airway wall. greater is its level of activation, stiffer are the airways. in turn, stiffer airways are intuitively expected to expand less in response to a di. however, this is not always the case. the change in airway caliber caused by a simulated di is sometimes greater when asm is activated by methacholine than when it is relaxed by atropine [130] . this is because increasing the level of asm activation may also cause airway constriction, which affects asm strain during a di for at least two reasons. first, it modifies the length of asm before the di, which impacts its contractile capacity. more precisely, shortening the length of asm decreases its contractile capacity [47, 189] . second, airway constriction also changes the positions over which the airways are operating on the pressure-radius curve [142] . following airway narrowing, the di thus takes place on a more compliant part of the pressure-radius curve and thereby increases the strain for any given stress [29] . therefore, while the maximal distention attained by the airway during a di is certainly limited by the level of asm activation, the strain excursion may not. these have important implications for in vitro studies that attempt to emulate in vivo situations. for example, it may be appropriate to impose an equivalent level of strain on asm during a simulated di with or without spasmogeninduced contraction. however, this simulation only recreates adequately the in vivo situation if the asm is also adjusted to a shorter starting length in the presence of a spasmogen. the intrinsic level of asm activation is tuned by many spasmogens and bronchodilators that are produced endogenously [190] . the intrinsic level of asm activation should thus be considered a biological confounder. however, some interventions also affect the level of asm activation, which consequently changes the stiffness of the airway wall and the strain caused by a di. on one hand, many methods used to estimate asm strain during a di intentionally manipulate the level of asm activation with bronchoactive substances (bronchoconstrictor such as methacholine or bronchodilator such as salbutamol). some studies are actually designed to dissociate the active contribution of asm in determining airway wall stiffness from the passive structural components of the airway wall [58, 75, 166, 191] . these studies investigated the response to dis before and after the activation or the inhibition of asm. on the other hand, many methods used to estimate asm strain during a di unintentionally manipulate the level of asm activation by utilizing topical or systemic anesthesia. this is the case for aoct [85] . for these reasons, the level of asm activation should sometimes be considered a procedural confounder. some patients also take medications with either short or long duration of action. those are likely to affect the stress-strain relationship of the airway wall and thus the distension of the airways during a di. they should thus be considered as confounders. the duration of withholding from these medications prior to measurements is also an important confounder to take into account. apart from medications, some interventional procedures also have the potential to affect asm strain during a di. this is the case for bronchial thermoplasty, which is an alternative treatment for severe refractory asthma. it was demonstrated in dogs that bronchial thermoplasty increases airway caliber at any airway pressure from 0 to 30 cmh 2 o [192] . the change in airway caliber though from 0 to 30 cmh 2 o was identical pre-versus postbronchial thermoplasty. based on these observations, geometrical considerations need to be taken into account to understand the impact of bronchial thermoplasty on asm strain. airway caliber changes proportionately to the square of airway perimeter (viz., $asm length). the same change in caliber induced by rising the pressure from 0 to 30 cmh 2 o in an airway starting at a larger caliber implies that asm strain was attenuated postbronchial thermoplasty. therefore, the initial (frc) length of asm may be greater after bronchial thermoplasty, but the strain asm undergoes during the di may be attenuated. the goal of this review was to estimate the strain asm undergoes in vivo in humans during a di from frc to tlc. this transient stretch is important to quantify as it was proposed to contribute to the beneficial effect of di by decreasing the contractile capacity of asm. however, it remains unclear to what extent asm is strained during a di. despite this gap in knowledge, the effect of strain on asm contractility has been tested extensively in vitro. a variable range of strains at different frequencies and for different durations in a variety of asm preparations have been tested. we came to learn that the dynamic environment in which asm operates in vivo has the potential to greatly affect its contractile capacity. in general, the in vitro results indicate that the decline in contractile capacity is largely dictated by the amount of strain [11] [12] [13] [14] [15] 17, 23, [28] [29] [30] , which is consistent with in vivo results in rabbits [24] and humans [3, 5, 86, 139, 193, 194] , as well as with isolated cells [107] . together, these studies support the hypothesis that the beneficial effect of di on respiratory mechanics is related to the decrease in the contractility of asm elicited by the stretch. however, the in vitro results are also variable, sometimes contradictory, and it is still uncertain if the strain required to significantly decrease the contractile capacity of asm is physiologically attainable [29, 33] . incorporating an appropriate range of strains in these in vitro studies is obviously essential in order to understand the integrated role of asm in respiratory mechanics. as seen in sec. 2 of this review, many former methods and more recent technological advents can be used to estimate asm strain in humans in vivo during a di. on one hand, some methods assess the distensibility of the entire airway tree. these methods have the advantage to assess the effect of all the airways combined, but provide little insights on the localized response in any given airway and miss completely in reporting the spatial heterogeneity of the response. they are also unable to distinguish between radial versus longitudinal airway strain. among those methods, the measurement of v d , rrs near the resonance frequency with the fot and raw by whole-body plethysmography are the most relevant. the fot is especially appealing and increasingly used in clinical settings. combined with imaging technique and computational modeling, fot may eventually be able to document the changes in airway caliber during breathing maneuvers with a tremendous temporal and spatial resolution [195, 196] . on the other hand, other methods assess individual airway distensibility. these methods have the advantage to measure directly the localized response, but usually fail to assess the small peripheral airways. the better being hrct, which can provide adequate resolution for airways down to $1 mm of internal diameter. the other methods include bronchoscopy, fluoroscopy, acoustic reflection technique, mri, pitot static probe, and aoct. these methods were all very useful as they have clearly demonstrated that asm strain during a di differs according to airway size and location. as seen in sec. 3 of this review, several limitations need to be considered as none of these methods assess asm strain directly. each of these limitations can putatively add inaccuracies that are sometimes not negligible. a few assumptions also need to be made in order to transform the original data into asm strain. the veracity of these assumptions can certainly be argued. owing to these limitations and assumptions, it is understood that the strains reported in this review are nothing more than estimations. as seen in sec. 4 of this review, a single value of asm strain cannot be attributed to a di. this is because many procedural and biological confounders contribute to the variability between individuals and between airways within the same individual, as well as to the temporal variability of any given airway of a given individual [96] . at this point, it seems more appropriate to provide a rough estimated range of strains that is physiologically relevant. despite the different setbacks of each method and the many confounders, the level of concordance between the methods for predicting the level of asm strain during a di is decent (table 1) . this suggests that, although a greater level of temporal and spatial resolution can now be achieved, the new and refined technologies have simply confirmed the estimations made previously with antecedent methods. set apart the stiffer trachea and main stem bronchi, the longitudinal strain asm undergoes during a di from frc to tlc is estimated to reside within the range of 15-30%. these values should assist investigators who attempt to impose a physiologically relevant level of strain to different asm tissues in in vitro settings in order to mimic a di. it is also important to realize that the value of strain that i was chasing in this review is the maximal attainable strain, i.e., the one achieves at tlc. involuntary dis, or should i call them sighs, which occur spontaneously every few minutes [194, 197, 198] , do not reach tlc. provided that an in vitro study is interested to investigate the regulatory effect of these sighs on asm contractility, the range of acceptable strains is quite large. this is because beyond all the factors discussed in this review, the size of the breath during a sigh is obviously variable. the strain asm undergoes during sighs is thus commensurately variable. any intermediate values of strain occurring between frc to another higher lung volume anywhere from end-tidal inspiration to tlc can justifiably be used to simulate these spontaneous respiratory motions of the lungs. i think the one advice to take from this review is to stay within the reported range of strains and, more importantly, to never exceed the limit of what is physiologically attainable. to simulate more accurately particular in vivo situations, more data will be needed. indeed, owing to the number of biological confounders affecting the variability, more studies using different populations of patients and controlling for as many confounders as possible are required. these studies will allow us to adjust the strain in our in vitro settings according to the particular real-life situations that we are trying to model. in turn, applying a correct range of strains in our in vitro settings should: 1-ameliorate the validity of these in vitro simulations; 2-justify the incorporation of those in vitro results in computational models that attempt to predict the consequences of those findings at higher biological length scales; 3-leap forward our understanding of the regulatory role of lung movements on the contractile capacity of asm; and 4-allow to correctly infer on the potential contribution of asm strain to the bronchodilator effect of a di. effect of a previous deep inspiration on airway resistance in man bronchodilation and bronchoprotection by deep inspiration and their relationship to bronchial hyperresponsiveness bronchodilatation induced by deep breaths in relation to transpulmonary pressure and lung volume effect of inspiratory flow rate on bronchomotor tone in normal and asthmatic subjects attenuation of induced bronchoconstriction in healthy subjects: effects of breathing depth the influence of lung distension upon the response of the bronchioles to epinephrine and to histamine the dynamic face of respiratory research: understanding the effect of airway disease on a lung in constant motion airway narrowing in asthma: does speed kill? airway smooth muscle stretch and airway hyperresponsiveness in asthma: have we chased the wrong horse? mechanical properties of asthmatic airway smooth muscle decrease of airway smooth muscle contractility induced by simulated breathing maneuvers is not simply proportional to strain dilatation of the constricted human airway by tidal expansion of lung parenchyma airway smooth muscle, tidal stretches, and dynamically determined contractile states effects of length oscillation on the subsequent force development in swine tracheal smooth muscle relaxation of activated airway smooth muscle: relative potency of isoproterenol vs. tidal stretch contractile force of canine airway smooth muscle during cyclical length changes dynamic equilibration of airway smooth muscle contraction during physiological loading dynamically determined contractile states of airway smooth muscle airway hyperresponsiveness, remodeling, and smooth muscle mass: right answer, wrong reason? disrupting actin-myosin-actin connectivity in airway smooth muscle as a treatment for asthma? selected contribution: effect of chronic passive length change on airway smooth muscle length-tension relationship responsiveness of the human airway in vitro during deep inspiration and tidal oscillation responsiveness of the isolated airway during simulated deep inspirations: effect of airway smooth muscle stiffness and strain effect of tidal volume and frequency on airway responsiveness in mechanically ventilated rabbits latrunculin b increases force fluctuation-induced relengthening of ach-contracted, isotonically shortened canine tracheal smooth muscle mek modulates force-fluctuation-induced relengthening of canine tracheal smooth muscle steroids augment relengthening of contracted airway smooth muscle: potential additional mechanism of benefit in asthma airway narrowing and bronchodilation to deep inspiration in bronchial segments from subjects with and without reported asthma can tidal breathing with deep inspirations of intact airways create sustained bronchoprotection or bronchodilation? bronchodilatory response to deep inspiration in bronchial segments: the effects of stress vs. strain airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain human trachealis and main bronchi smooth muscle are normoresponsive in asthma tidal stretches do not modulate responsiveness of intact airways in vitro potent bronchodilation and reduced stiffness by relaxant stimuli under dynamic conditions maintenance of airway caliber in isolated airways by deep inspiration and tidal strains tnf and il-1b exposure increases airway narrowing but does not alter the bronchodilatory response to deep inspiration in airway segments pharmacological bronchodilation is partially mediated by reduced airway wall stiffness effects of simulated tidal and deep breathing on immature airway contraction to acetylcholine and nerve stimulation hyperinflation of bronchi in vitro impairs bronchodilation to simulated breathing and increases sensitivity to contractile activation can breathing-like pressure oscillations reverse or prevent narrowing of small intact airways? human airway smooth muscle is structurally and mechanically similar to that of other species effect of inflation on trachealis muscle tone in canine tracheal segments in vitro pressure-volume and length-stress relationships in canine bronchi in vitro mechanical modulation of pressure-volume characteristics of contracted canine airways in vitro force fluctuation induced relengthening of acetylcholine-contracted airway smooth muscle an in vitro study examining the duration between deep inspirations on the rate of renarrowing smooth muscle in human bronchi is disposed to resist airway distension length oscillation induces force potentiation in infant guinea pig airway smooth muscle lung function studies; the respiratory dead space alveolar and total ventilation and the dead space problem factors affecting the pulmonary dead space as determined by single breath analysis the relationship between airway resistance, airway conductance and lung volume in subjects of different age and body size respiratory dead space measurements in a model lung and healthy human subjects according to the single breath method physiological factors affecting airway resistance in normal subjects and in patients with obstructive respiratory disease changes in calibre of the smaller airways in man measurement of upper and lower airway resistance and conductance in man relative hysteresis of the dead space and lung in vivo factors influencing pulmonary resistance pulmonary conductance and elastic recoil relationships in asthma and emphysema acquired tracheomalacia relationship of tracheal size to maximal expiratory airflow and density dependence in vivo estimation of tracheal distensibility and hysteresis in normal adults tracheal size and distensibility in patients with cystic fibrosis the lack of distensibility of asthmatic airways evaluation of tracheal collapsibility in patients with tracheomalacia using dynamic mr imaging during coughing central airways behave more stiffly during forced expiration in patients with asthma airway distensibility in healthy and asthmatic subjects: effect of lung volume history high-resolution computed tomographic evaluation of airway distensibility and the effects of lung inflation on airway caliber in healthy subjects and individuals with asthma reduced airway distensibility, fixed airflow limitation, and airway wall remodeling in asthma selected contribution: airway caliber in healthy and asthmatic subjects: effects of bronchial challenge and deep inspirations tracking variations in airway caliber by using total respiratory vs. airway resistance in healthy and asthmatic subjects airway re-narrowing following deep inspiration in asthmatic and nonasthmatic subjects a comparison of two methods for measuring airway distensibility: nitrogen washout and the forced oscillation technique tracheobronchomalacia: comparison between end-expiratory and dynamic expiratory ct for evaluation of central airway collapse airway distensibility in adults with asthma and healthy adults, measured by forced oscillation technique the airway response to deep inspirations decreases with copd severity and is associated with airway distensibility assessed by computed tomography airway dimensions at inspiratory and expiratory multisection ct in chronic obstructive pulmonary disease: correlation with airflow limitation multi-detector ct evaluation in patients suspected of tracheobronchomalacia: comparison of end-expiratory with dynamic expiratory volumetric acquisitions tracheomalacia in adults with cystic fibrosis: determination of prevalence and severity with dynamic cine ct a method to determine in vivo, specific airway compliance, in humans airway distensibility and volume recruitment with lung inflation in copd the effect of airway remodelling on airway hyper-responsiveness in asthma airway resistance at maximum inhalation as a marker of asthma and airway hyperresponsiveness the bronchodilator response of in vivo specific airway compliance in adults with asthma elastic properties of the central airways in obstructive lung diseases measured using anatomical optical coherence tomography bronchodilation response to deep inspirations in asthma is dependent on airway distensibility and air trapping influence of inspiration level on bronchial lumen measurements with computed tomography association between airway caliber changes with lung inflation and emphysema assessed by volumetric ct scan in subjects with copd evaluation of the trachea and intrathoracic airways by the acoustic reflection method in children with cystic fibrosis airway distensibility with lung inflation after allogeneic haematopoietic stem-cell transplantation airway distensibility by hrct in asthmatics and copd with comparable airway obstruction effect of inspiration on airway dimensions measured in maximal inspiration ct images of subjects without airflow limitation acute administration of ivacaftor to people with cystic fibrosis and a g551d-cftr mutation reveals smooth muscle abnormalities measurement of intraindividual airway tone heterogeneity and its importance in asthma assessment of airway distensibility by the forced oscillation technique: reproducible and potentially simplifiable the effect of disease and respiration on airway shape in patients with moderate persistent asthma three-dimensional mapping of smooth muscle in the distal conducting airways of mouse, rabbit, and monkey directional preference of airway smooth muscle mass increase in human asthmatic airways is a myogenic response involved in deep inspiration-induced bronchoconstriction in asthmatics? the mechanism of deep inspiration-induced bronchoprotection: evidence from a mouse model deep breaths, methacholine, and airway narrowing in healthy and mild asthmatic subjects intraluminal pressure oscillation enhances subsequent airway contraction in isolated bronchial segments universal physical responses to stretch in the living cell selected contribution: mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells mechanical strain increases velocity and extent of shortening in cultured airway smooth muscle cells airway smooth muscle tone modulates mechanically induced cytoskeletal stiffening and remodeling reinforcement versus fluidization in cytoskeletal mechanoresponsiveness the volume of the dead space in breathing and the mixing of gases in the lungs of man measurements of the dead space volume functional measurements of the peripheral airways in copd single-breath washout tests to assess small airway disease in copd radiologic and pathologic abnormalities of the trachea in older patients with cystic fibrosis and working group for body plethysmography of the german society for pneumology and respiratory care effects of lung volume, volume history, and methacholine on lung tissue viscance tracheal stenosis measured by the acoustic reflection technique airway area by acoustic reflections measured at the mouth reproducibility and accuracy of airway area by acoustic reflection changes in regional airflow obstruction over time in the lungs of patients with asthma: evaluation with 3he mr imaging the variability of regional airflow obstruction within the lungs of patients with asthma: assessment with hyperpolarized helium-3 magnetic resonance imaging the difference in ventilation heterogeneity between asthmatic and healthy subjects quantified using hyperpolarized 3he mri imaging the lungs in asthmatic patients by using hyperpolarized helium-3 magnetic resonance: assessment of response to methacholine and exercise challenge probing airway conditions governing ventilation defects in asthma via hyperpolarized mri image functional modeling quantification of airway diameters and 3d airway tree rendering from dynamic hyperpolarized 3he magnetic resonance imaging investigation of hyperpolarized 3he magnetic resonance imaging utility in examining human airway diameter behavior in asthma through comparison with high-resolution computed tomography oscillation mechanics of the respiratory system phenotyping airway disease with optical coherence tomography birefringence microscopy platform for assessing airway smooth muscle structure and function in vivo x-ray studies on bronchial movements effect of lung inflation on bronchial length and diameter in excised lungs effect of lung inflation and airway muscle tone on airway diameter in vivo terminal bronchiole diameter changes with volume in isolated, air-filled lobes of cat lung hysteresis of contracted airway smooth muscle cyclical elongation regulates contractile responses of isolated airways lung volume dependence of pharyngeal cross-sectional area in patients with obstructive sleep apnea tracheal cross-sectional areas from acoustic reflections in dogs relationship between heterogeneous changes in airway morphometry and lung resistance and elastance airway inhomogeneities contribute to apparent lung tissue mechanics during constriction airway constriction pattern is a central component of asthma severity: the role of deep inspirations deep inspiration volume and the impaired reversal of bronchoconstriction in asthma the linear single-compartement model elastic properties of airways in human lungs post mortem nonlinear compliance modulates dynamic bronchoconstriction in a multiscale airway model rigidity of tracheae and bronchi during muscular constriction factors influencing the bronchodilator effect of a deep inspiration in asthmatic patients with provoked bronchoconstriction mechanical properties of contracted canine bronchial segments in vitro influence of the previous deep inspiration on the spirometric measurement of provoked bronchoconstriction in asthma deep inspiration-induced bronchoprotection is stronger than bronchodilation importance of the time interval between fev 1 measurements in a methacholine provocation test time course of increased airway narrowing caused by inhibition of deep inspiration during methacholine challenge potent bronchoprotective effect of deep inspiration and its absence in asthma volume history and effect on airway reactivity in infants and adults airway narrowing associated with inhibition of deep inspiration during methacholine inhalation in asthmatics supine and prone differences in regional lung density and pleural pressure gradients in the human lung with constant shape influence of sleep on lung volume in asthmatic patients and normal subjects airway hyperresponsiveness in asthma: geometry is not everything! airway distension during lung inflation in healthy and allergic-sensitised mice in vivo effect of lung inflation in vivo on airways with smooth muscle tone or edema airway-parenchymal interdependence abnormal alveolar attachments with decreased elastic fiber content in distal lung in fatal asthma the consequences of chronic allergic inflammation the senile lung. comparison with normal and emphysematous lungs-1: structural aspects tracheal extension in respiration physiological changes in respiratory function associated with ageing the bronchodilatory effect of deep inspiration diminishes with aging mechanisms of airway remodeling effect of airway smooth muscle tone on airway distensibility measured by the forced oscillation technique in adults with asthma bronchial inflammation and airway responses to deep inspiration in asthma and chronic obstructive pulmonary disease bronchial measurements in patients with asthma: comparison of quantitative thin-section ct findings with those in healthy subjects and correlation with pathologic findings association between peripheral airway function and neutrophilic inflammation in asthma unsuspected loss of lung elastic recoil in chronic persistent asthma unsuspected pseudophysiologic emphysema in chronic persistent asthma correlation between airway inflammation and loss of deep-inhalation bronchoprotection in asthma regulation of bronchomotor tone by lung inflation in asthmatic and nonasthmatic subjects the lack of the bronchoprotective and not the bronchodilatory ability of deep inspiration is associated with airway hyperresponsiveness airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration effects of deep inhalation in asthma: relative airway and parenchymal hysteresis effects of volume history during spontaneous and acutely induced air-flow obstruction in asthma relating maximum airway dilation and subsequent reconstriction to reactivity in human lungs reversibility of induced bronchoconstriction by deep inspiration in asthmatic and normal subjects bronchodilatory effect of deep inspiration is absent in subjects with mild copd lung elastic recoil in cystic fibrosis excessive collapsibility of bronchi in bronchiectasis: evaluation on volumetric expiratory high-resolution ct the effects of body mass index on lung volumes a deep breath bronchoconstricts obese asthmatics impaired response to deep inspiration in obesity deep inspiration avoidance and airway response to methacholine: influence of body mass index intrasubject variability in airway inflammation in biopsies in mild to moderate stable asthma heterogeneity of airway wall dimensions in humans: a critical determinant of lung function in asthmatics and nonasthmatics does the length dependency of airway smooth muscle force contribute to airway hyperresponsiveness? asthmatic airway hyperresponsiveness: the ants in the tree relationship between improved airflow limitation and changes in airway calibre induced by inhaled anticholinergic agents in copd effect of bronchial thermoplasty on airway distensibility bronchodilator effects of exercise hyperpnea and albuterol in mild-to-moderate asthma bronchomotor effect of bronchoconstriction-induced deep inspirations in asthmatics respiratory impedance measurements for assessment of lung mechanics: focus on asthma oscillometry and pulmonary mri measurements of ventilation heterogeneity in obstructive lung disease: relationship to quality of life and disease control spontaneous sigh rates during sedentary activity: watching television vs reading pattern of ventilation in young adults key: cord-026005-f2khcjdy authors: lópez, alfonso; martinson, shannon a. title: respiratory system, mediastinum, and pleurae date: 2017-02-17 journal: pathologic basis of veterinary disease doi: 10.1016/b978-0-323-35775-3.00009-6 sha: doc_id: 26005 cord_uid: f2khcjdy nan diseases of the respiratory system (respiratory apparatus) are some of the leading causes of morbidity and mortality in animals and a major source of economic losses. thus veterinarians are routinely called to diagnose, treat, and implement health management practices to reduce the impact of these diseases. in companion animals, diseases of the respiratory tract are also common and, although of little economic significance, are important to the health of the animals and thus to clinicians and pet owners. in the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections. to facilitate the understanding of the structure and function, it is convenient to arbitrarily divide the respiratory system into conducting, transitional, and gas exchange systems ( fig. 9-1 ). the conducting system includes nostrils, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and extrapulmonary and intrapulmonary bronchi, all of which are largely lined by pseudostratified, ciliated columnar cells, plus a variable proportion of secretory goblet (mucous) and serous cells (figs. 9-2 and 9-3 and efig. 9-1 ). the transitional system of the respiratory tract is composed of bronchioles, which are microscopic structures that serve as a transition zone between the conducting system (ciliated) and the gas exchange (alveolar) system (see fig. 9 -1). the disappearance of cilia in the transitional system is not abrupt; the ciliated cells in the proximal bronchiolar region become scarce and progressively attenuated, until the point where distal bronchioles no longer have ciliated cells. normal bronchioles also lack goblet cells but instead have other types of secretory cells, notably club cells (formerly clara cells) and neuroendocrine cells. club cells, also referred to as secretory bronchiolar cells, contain numerous biosynthetic organelles that play an active role in detoxification of xenobiotics (foreign substances), similar to the role of hepatocytes ( fig. 9-4) . club cells are also critical stem cells in the repair and remodeling of not only the bronchioles but also of most of the respiratory tract. in addition, club cells contribute to the innate immunity of the lung by secreting protective proteins (collectins) and pulmonary surfactant (see b) . in carnivores and monkeys, and to a much lesser extent in horses and human beings, the terminal portions of bronchioles are lined not only by cuboidal epithelium but also by segments of alveolar capillaries. these unique bronchioloalveolar structures are known as respiratory bronchioles ; also see fig. 9 -1). the gas exchange system of the respiratory tract in all mammals is formed by alveolar ducts and millions of alveoli ( fig. 9-6 ; also see fig. 9 -1). the surface of the alveoli is lined by two distinct types of epithelial cells known as type i (membranous) pneumonocytes and type ii (granular) pneumonocytes ( fig. 9-7) . all three-the conducting, transitional, and exchange systems of the respiratory system-are vulnerable to injury because of constant exposure to a myriad of microbes, particles and fibers, and toxic gases and vapors present in the air. vulnerability of the respiratory system to aerogenous (airborne) injury is primarily because of (1) the extensive area of the alveoli, which are the interface between the blood in alveolar capillaries and inspired air; (2) the large volume of air passing continuously into the lungs; and (3) the high concentration of noxious elements that can be present in the air (table 9 -1). for human beings, it has been estimated that the surface of the pulmonary alveoli is approximately 200 m 2 , roughly the area animal and cultured for microbes, yeasts, and fungi, many species of bacteria are recovered, such as mannheimia (pasteurella) haemolytica in cattle; pasteurella multocida in cats, cattle, and pigs; and bordetella bronchiseptica in dogs and pigs. the organisms that constitute the normal flora of the respiratory tract are restricted to the most proximal (rostral) region of the conducting system (nasal cavity, pharynx, and larynx). the thoracic portions of the trachea, bronchi, and lungs are considered to be essentially sterile. the types of bacteria present in the nasal flora vary considerably among animal species and in different geographic regions of the world. some present in the nasal flora are pathogens that can cause important respiratory infections under some circumstances. for instance, mannheimia (pasteurella) haemolytica is part of the bovine nasal flora, yet this bacterium causes a devastating disease in cattle-pneumonic mannheimiosis (shipping fever). experimental studies have established that microorganisms from the nasal flora are continuously carried into the lungs via tracheal air. despite this constant bacterial bombardment from the nasal flora and from contaminated air, normal lungs remain sterile because of their remarkably effective defense mechanisms. the conducting portion of the respiratory system is lined by pseudostratified columnar ciliated epithelium (most of the nasal cavity, paranasal sinuses, part of the larynx, and all of the trachea and bronchi), olfactory epithelium (part of the nasal cavity, particularly ethmoidal conchae), and squamous epithelium (nasal vestibulum and parts of the larynx). the pattern of injury, inflammation, and host response (wound healing) are characteristic for each of these three types of epithelium independent of its anatomic location. pseudostratified ciliated epithelium, which lines most of the nasal cavity and nasopharynx, part of the larynx, and all of the trachea and bronchi, is exquisitely sensitive to injury. when these cells are irreversibly injured, whether caused by viral infection, trauma, or inhalation of toxic gases, the ciliated cells swell, typically lose their attachment to underlying basement membrane, and rapidly exfoliate ( fig. 9-8) . a transient and mild exudate of fluid, plasma proteins, and neutrophils covers the ulcer. in the absence of of a tennis court. the alveolar surface of the equine lung is estimated to be approximately 2000 m 2 . it has also been estimated that the volume of air reaching the human lung every day is approximately 9000 l. lungs are also susceptible to blood-borne (hematogenous) microbes, toxins, and emboli. this fact is not surprising because the entire cardiac output of the right ventricle goes into the lungs, and approximately 9% of the total blood volume is within the pulmonary vasculature. the pulmonary capillary bed is the largest in the body, with a surface area of 70 m 2 in the adult human; this area is equivalent to a length of 2400 km of capillaries, with 1 ml of blood occupying up to 16 km of capillary bed. the respiratory system has its own normal flora (microbiota), as does any other body system in contact with the external environment. if a sterile swab is passed deep into the nasal cavity of any healthy (rhinoviruses), infectious bovine rhinotracheitis (bovine herpesvirus 1), feline rhinotracheitis (felid herpesvirus 1), and viruses of the canine infectious respiratory disease (cird) group such as canine adenovirus 2 (cav-2) and canine parainfluenza virus (cpiv) . if damage to the mucociliary blanket becomes chronic, goblet cell hyperplasia takes place, leading to excessive mucus production (hypersecretion) and reduced mucociliary clearance, and when there is loss of basement membrane, repair is by fibrosis and granulation tissue (scarring). in the most severe cases, prolonged injury causes squamous metaplasia, which together with scarring causes airway obstruction and an impediment to mucociliary clearance. in laboratory rodents, hyperplastic and metaplastic changes, such as those seen in nasal polyps and squamous metaplasia, are considered a prelude to neoplasia. the second type of epithelium lining the conducting system is the sensory olfactory epithelium, present in parts of the nasal mucosa, notably in the ethmoidal conchae. the patterns of degeneration, exfoliation, and inflammation in the olfactory epithelium are similar to those of the ciliated epithelium, except that olfactory complications or secondary bacterial infections, a specific type of progenitor cells known as basal cells or nonciliated secretory cells (preciliated cells) , which are normally present in the mucosa, migrate to cover the denuded basement membrane and undergoes mitosis, eventually differentiating into new ciliated epithelial cells (see fig. 9 -8). cellular migration, proliferation, and attachment are regulated by locally released interleukins (il-1β, il-2, il-4, and il-13), growth factors, integrins and extracellular matrix (ecm) proteins such as collagen, and fibronectin. the capacity of ciliated epithelium to repair itself is remarkably effective. for example, epithelial healing in an uncomplicated ulcer of the tracheal mucosa can be completed in only 10 days. this sequence of cell degeneration, exfoliation, ulceration, mitosis, and repair is typically present in many viral infections in which viruses replicate in nasal, tracheal, and bronchial epithelium, causing extensive mucosal ulceration. examples of transient infections of this type include human colds epithelium. neurons in the olfactory mucosa have the unique ability to regenerate, a fact that is being explored as a potential source of new neurons in the treatment of spinal cord injury. squamous epithelium, located in the vestibular region of the nose (mucocutaneous junction), is the third type of epithelium present in the nasal passages. compared with ciliated and olfactory epithelia, nasal squamous epithelium is quite resistant to all forms of injury. the pharyngeal mucosa, composed of squamous epithelium, has similar patterns of necrosis and inflammation as the oral mucosa (see chapter 7). the patterns of necrosis, inflammation, and repair in intrapulmonary bronchi are similar to those previously described for the nasal and tracheal epithelium. in brief, injury to ciliated bronchial epithelium may result in degeneration, detachment, and exfoliation of necrotic cells. under normal circumstances, cellular exfoliation is promptly followed by inflammation, mitosis, cell proliferation, cell differentiation, and finally by repair ( fig. 9-9 and see . depending on the type of exudate, bronchitis can be fibrinous, catarrhal, purulent, fibrinonecrotic (diphtheritic), and sometimes granulomatous. when epithelial injury becomes chronic, production of mucus is increased via goblet cell hyperplasia (chronic catarrhal inflammation). this form of chronic bronchitis is well illustrated in habitual smokers who continually need to cough out excessive mucus secretions (sputum). unfortunately, in some cases, excessive mucus cannot be effectively cleared from airways, which of the bronchial wall or cylindrical when destruction involves a large segment of a bronchus. grossly, bronchiectasis is manifested by prominent lumps in the lungs (bosselated appearance or having rounded eminences) resulting from distention of bronchi with exudate, which results in a concurrent obstructive atelectasis of surrounding parenchyma ( fig. 9-11 ). the cut surfaces of dilated bronchi are filled with purulent exudates; for this reason, bronchiectasis is often mistaken for pulmonary abscesses. careful inspection, usually requiring microscopic examination, confirms that exudate is contained and surrounded by remnants of a bronchial wall lined by squamous epithelium and not by a pyogenic membrane (connective tissue) as it is in the case of a pulmonary abscess. the squamous metaplasia further interferes with the normal function of the mucociliary escalator. the epithelial lining of the bronchiolar region (transitional zone) is exquisitely susceptible to injury, particularly to that caused by some respiratory viruses (bovine parainfluenza virus 3, bovine respiratory syncytial virus, adenovirus, or canine distemper virus), oxidant gases (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], or ozone [o 3 ]), and toxic substances (3-methylindole or paraquat). the precise explanation as to why bronchiolar epithelium is so prone to injury is still not clear, but it is presumably due in part to (1) its high vulnerability to oxidants and free radicals; (2) the presence of leads to chronic obstructive bronchitis and emphysema (see . chronic bronchial irritation causes squamous metaplasia of highly functional but vulnerable ciliated epithelium to nonfunctional, but more resistant, squamous epithelium. squamous metaplasia has a calamitous effect on pulmonary clearance because it causes a structural loss and functional breakdown of portions of the mucociliary escalator. hyperplasia of bronchial glands occurs frequently in chronic bronchitis, which translates to an increase of the reid index (bronchial-gland to bronchial-wall ratio) (efig. 9 -2). this index is less than 30% in the healthy human lung and in the lungs of most domestic species, except for cats, which generally have an index higher than 40%. the term airway remodeling encompasses all the structural changes that accompany chronic bronchitis such as hypertrophy and hyperplasia of smooth muscle, submucosal glands, and goblet cells; fibrosis; and increased bronchial vascularity. bronchiectasis is one of the most devastating sequelae to chronic remodeling of the bronchi. it consists of a pathologic and permanent dilation of a bronchus with rupture of the bronchial wall as a result of obstruction or chronic inflammation. destruction of walls occurs in part when proteolytic enzymes and oxygen radicals released from phagocytic cells during chronic inflammation degrade and weaken the smooth muscle and cartilage (chondromalacia) that help to maintain normal bronchial diameter ( fig. 9-10 ). bronchiectasis may be saccular when destruction affects only a small localized portion . this same type of lesion is seen in viral or mechanical injury to the mucosa of the conducting system. two days after exposure, the basement membrane is lined by rapidly dividing preciliated cells, some of which exhibit mitotic activity (inset). ten days after injury, the nasal epithelium is completely repaired. h&e stain. b, schematic representation of the events of injury and repair in the respiratory mucosa of the conducting system. blue cell, ciliated mucosal epithelial cell; pink cell, goblet cell; red cell, neutrophil. (a from lópez a, prior m, yong s, et al: am j vet res 49:1107 -1111 , 1988 into well-organized, microscopic polyps inside the bronchiolar lumen. the external surface of the exudate eventually becomes covered by ciliated cells. this lesion is referred to as bronchiolitis obliterans, and the polyps may become so large as to cause airflow impairment ( fig. 9 -12 and see fig. 9 -9). in mild but persistent bronchiolar injury, goblet cells normally absent from bronchioles proliferate from basal cells, resulting in goblet cell metaplasia and causing a profound alteration in the physicochemical properties of bronchiolar secretions ( fig. 9-13 ). the normally serous bronchiolar fluid released by club (clara) cells becomes a tenacious material when mucus produced by goblet cells is added. as a result of increased viscoelasticity of the mucus, bronchiolar secretions cannot be removed effectively by ciliary action, leading to plugging and obstruction of distal airways. under such conditions, often grouped as chronic obstructive pulmonary disease, coughing is required to clear mucus from obstructed bronchioles. pulmonary emphysema and atelectasis are further sequelae to bronchiolar metaplasia and mucous hypersecretion blocking or partially blocking the lumens of these bronchioles. these two inflation abnormalities are characteristically present in chronic obstructive pulmonary disease (copd), which is called "recurrent airway obstruction (rao or "heaves") in horses (see recurrent airway obstruction, under disorders of horses). peribronchiolar club (clara) cells rich in mixed function oxidases, which locally generate toxic metabolites (see fig. 9 -4); and (3) the tendency for pulmonary alveolar macrophages and leukocytes to accumulate in this region of the lungs. depending on the types of injury and inflammatory response, bronchiolitis is classified as necrotizing, suppurative, catarrhal (mucous metaplasia), or granulomatous. once injury to bronchiolar ciliated cells becomes irreversible, the cells degenerate and exfoliate into the bronchiolar lumen, leaving a denuded basement membrane. repair in the bronchiolar region is similar to, but less effective than, that in the tracheal or nasal mucosa. under normal circumstances, recruited phagocytic cells remove exudate and cell debris from the lumina of affected bronchioles, thus preparing the basement membrane to be repopulated with new, undifferentiated cells originating from a rapidly dividing pool of club (clara) cells. after several days, these proliferating cells fully differentiate into normal bronchiolar cells. in severe acute injury, such as that caused by aspiration pneumonia or by highly pathogenic microorganisms, exudate attaches and cannot be removed from the basement membrane of bronchioles. the exudate becomes infiltrated by fibroblasts, which form small nodular masses of fibrovascular tissue that develop postviral bronchiolitis is associated with increased expression of tlrs and unusual susceptibility to inhaled endotoxin. hyperreactive animals typically have an increased number of mast cells, eosinophils, and t lymphocytes in the airway mucosa. clinically, airway hyperresponsiveness is characterized by an exaggerated bronchoconstriction after natural exposure to mild stimuli, such as cold air, or after animals are experimentally exposed to aerosols of histamine or methacholine. because of their extremely delicate structure, alveoli are quite vulnerable to injury once the local defense mechanisms have been overwhelmed. the alveolar wall is a thin membrane formed by a core of interstitium supporting an extensive network of alveolar capillaries. fibroblasts (septal cells), myofibroblasts, collagen, elastic fibers, and few interstitial macrophages and mast cells constitute the alveolar interstitium. the wall of the alveolar capillaries facing the airspace is remarkably thin and has three layers composed of vascular endothelium, basal lamina, and alveolar epithelium. these three layers of the alveolar capillaries constitute what is customarily referred to as the blood-air barrier (see fig. 9 -7). the epithelial side of the alveolus is primarily lined by rather thin type i proliferation of lymphocytes (balt hyperplasia) is also a common microscopic lesion seen in chronic bronchiolitis. airway hyperresponsiveness, or hyperreactive airway disease, is another sequela of bronchiolar injury arising from gene-environment interactions. it develops in human beings and animals (experimentally) after a transient and often innocuous viral infection of the lower respiratory tract or from exposure to certain allergens. experimental work has shown that airway hyperreactivity in club (clara) cell edema. alveolar repair is possible as long as the basement membrane remains intact and lesions are not complicated by further injury or infection. within 3 days, cuboidal type ii (granular) pneumonocytes, which are the precursor cells and more resistant to injury, undergo mitosis and provide a large pool of new undifferentiated cells . these new cells repave the denuded alveolar basement membrane and finally differentiate into type i pneumonocytes. when alveolar injury is diffuse, proliferation pneumonocytes, which are arranged as a very delicate continuous membrane extending along the alveolar surface (see fig. 9 -7). type i pneumonocytes are particularly susceptible to noxious agents that reach the alveolar region either aerogenously or hematogenously. injury to type i pneumonocytes rapidly causes swelling and vacuolation of these cells . when cellular damage has become irreversible, type i cells detach, resulting in denudation of the basement membrane, increased alveolar permeability, and alveolar figure 9-15 hyperplasia of type ii pneumonocytes. a, acute alveolar injury, crude oil aspiration, cow. note proliferation of cuboidal epithelial cells (type ii pneumonocytes) (arrows) along the luminal surface of the alveolar wall. during alveolar repair, type ii pneumonocytes are the precursor cell for necrotic and lost type i pneumonocytes. h&e stain. b, chronic alveolar injury, interstitial pneumonia, horse. note entire alveolar membrane lined with cuboidal type ii pneumonocytes (arrowheads). the alveolar interstitium is expanded with inflammatory cells, and the alveolar lumens contain cell debris mixed with leukocytes. h&e stain. ( (2). necrosis of these cells leads to transient alveolar edema (area that is pink) (3), which is followed by hyperplasia of type ii pneumonocytes (4), stem cells that differentiate (5) into type i pneumonocytes as part of alveolar repair and healing (6). (courtesy dr. a. lópez, atlantic veterinary college.) more information on postmortem examination of the lung can be found at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. microbes, toxins, and pneumotoxicants can gain access into the respiratory system by the following routes (table 9 -2; also see table 9 -1): aerogenous, hematogenous, direct extension, and by local production of free radicals and toxic metabolites. pathogens, such as bacteria, mycoplasmas, and viruses, along with toxic gases and foreign particles, including food, can gain access to the respiratory system via inspired air. this is the most common route in the transmission of most respiratory infections in domestic animals. some viruses, bacteria, parasites, and toxins can enter the respiratory system via the circulating blood. this portal of entry is commonly seen in septicemias, bacteremias, and with protozoa and viruses that target endothelial cells. also, circulating leukocytes may release infectious organisms such as retroviruses and listeria monocytogenes while traveling through the lungs. of type ii pneumonocytes becomes so spectacular that the microscopic appearance of the alveolus resembles that of a gland or fetal lung; this lesion has been termed epithelialization or fetalization. although it is part of the normal alveolar repair, hyperplasia of type ii pneumonocytes can interfere in gas exchange and cause hypoxemia. in uncomplicated cases, type ii pneumonocytes eventually differentiate into type i pneumonocytes, thus completing the last stage of alveolar repair (see fig. 9 -14). in some forms of chronic interstitial lung injury, the surface of the alveolar basement membrane could become populated with migrating bronchiolar cells, a process known as alveolar bronchiolization or lambertosis. in severe cases, lambertosis, a metaplastic change, can be mistaken microscopically with alveolar adenomas. type i pneumonocytes are one of the three structural components of the blood-air barrier, so when these epithelial cells are damaged, there is an increase in alveolar capillary permeability and transient leakage of plasma fluid, proteins, and fibrin into the alveolar lumen (see fig. 9-14) . under normal circumstances, these fluids are rapidly cleared from the alveolus by alveolar and lymphatic absorption, and necrotic pneumonocytes (type i) and fibrin strands are phagocytosed and removed by pulmonary alveolar macrophages. when there is persistent and severe injury, fibroblasts and myofibroblasts may proliferate in the alveolar walls (alveolar interstitium), causing alveolar septal fibrosis, whereas in other forms of severe injury, fibroblasts and myofibroblasts actively migrate from the interstitium into the alveolar spaces, causing intraalveolar fibrosis. these two types of alveolar fibrosis are most commonly seen in toxic and allergic pulmonary diseases and have a devastating effect on lung function. endothelial cells are also major players in the normal and abnormal physiology of the alveolus (see . these cells trap and share circulating antigens with intravascular and interstitial macrophages. the junction between alveolar endothelial cells is not as tight as that of the type i pneumonocytes, allowing some movement of fluid and small-size molecular weight proteins into the alveolar interstitium. endothelial cells maintain an intimate cell contact with erythrocytes and leukocytes passing through the lung, since the lumen of alveolar capillaries is slightly smaller (5.0 µm) than the diameter of red and white blood cells. erythrocytes are easily deformable, so their transit time through the alveolar capillaries is shorter than that of leukocytes, which are less deformable cells. this longer transit time of leukocytes and their close cellular contact with alveolar endothelial cells have major impacts in lung inflammation and acute respiratory distress syndrome (ards). on a minute-to-minute basis, the pulmonary defense mechanisms deal effectively with noxious stimuli and mild tissue injury without the need for an inflammatory response. however, if normal defense mechanisms are ineffective or insufficient (overwhelmed), the inflammatory process is rapidly turned on as a second line of defense. postmortem examination of the respiratory tract should always be conducted in a thorough and systematic manner and include the conducting system (trachea, bronchi, and bronchioles), the lungs, and the thoracic cavity and pleura. detailed record keeping and photographic documentation are essential elements of a thorough examination. normal lungs typically have a homogeneous pink color and are slightly deflated from loss of negative intrathoracic pressure. the e-sections that follow describe a systematic approach to this process. 480.e1 chapter 9 respiratory system, mediastinum, and pleurae the respiratory tract should always be examined in a systematic manner. to determine whether negative pressure is present in the thoracic cavity, the diaphragm is punctured through the abdominal cavity before the thoracic cavity has been opened. when the diaphragm is punctured in a fresh carcass, the loss of negative pressure in the thorax causes the diaphragmatic cupola to drop back caudally toward the abdominal cavity, and at the same time, there is an audible sound caused by the inrush of air into the thorax. lack of this movement may be an indication of advanced pneumothorax, pleural effusion, or the presence of uncollapsed lungs caused by pulmonary edema, pneumonia, fibrosis, or emphysema. in carcasses that have been dead for a long time, pulmonary air and gas produced by saprophytic bacteria leak into the pleural cavity, reducing the negative thoracic pressure and collapsing the lung. the rib cage must be removed by cutting along the costosternal joints and along the neck of the ribs (close to the costovertebral joints) in such a way that pleural adhesions and abnormal thoracic contents can be observed and grossly quantified (e.g., 200 ml of clear, yellow fluid). the tongue, pharynx, esophagus, larynx, trachea, and thoracic viscera (lungs, heart, and thymus) should be removed as a unit (often called the pluck) and placed on the necropsy table. the pharynx and esophagus are opened starting at the pharynx by a single cut with scissors along the dorsal midline and are inspected for ulcers, foreign bodies, and neoplasms. the larynx and trachea must be examined by opening both along the dorsal midline from cranial to caudal ends and then extending the incision into the large bronchi of the caudal lung lobes. normal tracheobronchial mucosa has a smooth and glistening pearl-colored surface with empty lumina in airways. the presence of foamy fluid in airways indicates pulmonary edema. feed particles may suggest aspiration; however, careful examination of the mucosa is required because aspiration of ingesta from stomach or rumen into the lungs commonly takes place agonally or can be displaced into these areas when the carcass is moved. the lungs should be examined before incision. normal lungs typically have a homogeneous pink color (see fig. 9 -16). external changes include the presence of rib imprints on the pleural surface when lungs fail to collapse. in addition, the lungs should be inspected for changes in color and texture and distribution of lesions. color changes can be various shades of red, indicating hypostatic congestion, hyperemia (acute pneumonia), and hemorrhage; dark blue collapsed lobules or areas are indicative of atelectasis; pale pink to white lungs indicate notable anemia, fibrosis, or emphysema; and uniformly or patchy yellow-brown lungs indicate chronic passive congestion and pulmonary fibrosis likely secondary to chronic heart failure. lungs from exsanguinated animals are generally paler than the normal pink color because of reduced blood in the pulmonary tissue. lungs with postmortem autolysis show green discoloration, a change that is also seen in other organs (efig. 9 -3). a covering of yellowish material on the pleural surface indicates accumulation of fibrin. because it is impossible to describe the texture of normal lungs, experience in palpation is required to appreciate the actual texture of a normal lung. texture is determined by gently palpating the surface and parenchyma of the lungs. normal texture can change to firm, hard, elastic (rubbery), or crepitus (with a crackling sound or feeling). for a detailed description of lung texture, see the section on classification of pneumonias in domestic animals. palpation of the lungs, which should be gentle, also permits detection of nonvisible nodules or abscesses in the parenchyma. knowing the distribution of a lesion in the lungs also facilitates diagnosis because particular etiologic agents cause lesions with specific distribution. distribution of lesions is generally described as focal, multifocal, locally extensive, or diffuse. according to their topography, pulmo-nary lesions can also be classified as cranioventral, dorsocaudal, and so on. necropsy reports must also contain an estimate of the extent of the pulmonary lesions, preferably expressed as a percentage of the volume of the lungs affected. for instance, a report may read "cranioventral consolidation involving 40% of the lungs." if the lungs have focal lesions, a rough estimate of the number should also be included in the report. for instance, "numerous (approximately 25), small (1 to 2 cm in diameter), hard nodules were randomly distributed in all lung lobes." two methods are used to examine the nasal structures. the first is making a midsagittal cut through the head and removing the nasal septum; the second is making several transverse sections of the nose at the level of the second premolar teeth. this latter method is preferred when examining pigs suspected of having atrophic rhinitis or animals suspected of having nasal neoplasms. microscopic examination of pulmonary tissue is routinely done in diagnostic laboratories. samples of normal and abnormal lungs, along with other appropriate tissue, should always be submitted in 10% buffered-neutral formalin for histopathologic evaluation. a minimum of four lung samples (left cranial, left caudal, right cranial, and right caudal) should be taken for histopathologic examination in animals with a history of respiratory signs. to improve fixation, a paper towel can be placed over the samples of lung floating in fixative. when detailed evaluation of the alveolar walls is required, lungs can be fixed by a gentle intratracheal injection of fixative; however, this technique displaces transudates and exudates and can artificially cause distention of the perivascular and peribronchial spaces. lung biopsy specimens are taken only sporadically because complications often outweigh the diagnostic value. however, the use of new techniques, such as endoscopic-directed biopsies, has notably reduced some of these complications. biopsies of the lungs are recommended in cases of chronic persistent pulmonary disease unresponsive to treatment or intrathoracic masses of undetermined origin. endoscopic-directed biopsies of the nasal and bronchial mucosa are routinely used in clinical practice and generally have a much better diagnostic value. two valuable diagnostic tools in human medicine, bronchoalveolar lavage (bal) and transtracheal wash (ttw), have in recent years become more widely used in veterinary clinical diagnosis of respiratory ailments, particularly in horses, dogs, and cats. the basis of bal and ttw is sampling the lung or trachea of a living animal by infusing sterile fluid into the trachea or deep lung (respectively) and retrieving it to determine the cellular and biochemical composition of this fluid. in other words, the composition of the fluid reflects what is present in the bronchioloalveolar spaces and trachea. these procedures are performed by inserting a tube directly through the larynx into the trachea or bronchus, or transtracheally by inserting a tube through a needle percutaneously into the cervical trachea. microscopic examination of properly collected, stored, and processed samples may reveal many erythrocytes and siderophages in pulmonary hemorrhage or left-sided heart failure; inclusion bodies or syncytial cells in viral pneumonias; increased number of leukocytes in pulmonary inflammation; abundant mucus in asthma or equine recurrent airway obstruction (rao); the presence of pulmonary pathogens, such as parasites, fungi, and bacteria; or tumor cells in cases of pulmonary neoplasia. in the healthy animal, 80% to 95% of the bal cells are pulmonary alveolar macrophages (see fig. 9-19, a) . clearance, or a combination of both is the underlying pathogenetic mechanism in many pulmonary diseases ( fig. 9-17) . the anatomic configuration of the nasal cavity and bronchi plays a unique role in preventing or reducing the penetration of noxious material into the lungs, especially into the alveoli, which is the most vulnerable portion of the respiratory system. the narrow nasal meatuses and the coiled arrangement of the nasal conchae generate enormous turbulence of airflow, and as a result, physical forces are created that forcefully impact particles larger than 10 µm onto the surface of the nasal mucosa ( fig. 9-18 ). although particles smaller than 10 µm could escape trapping in the nasal cavity, these mediumsized particles meet a second barrier at the tracheal and bronchial in some instances, pathogenic organisms can also reach the pleura and lungs through penetrating injuries, such as gunshot wounds, migrating awns, or bites, or by direct extension from a ruptured esophagus or perforated diaphragm. the lungs, particularly the bronchioles and alveoli, are vulnerable to endogenous injury caused by the local generation of free radicals during inflammation or by toxic metabolites generated by club (clara) cells (see fig. 9 -4, b). inflammatory processes in the respiratory system, particularly those caused by infectious organisms, can spread to contiguous or distant tissues. for instance, rhinitis may spread into the sinuses causing rhinosinusitis. similarly, laryngeal inflammation may spread into the lungs when exudate in the larynx is aspirated. lung disease can have profound systemic effects when cytokines, produced locally during necrosis or inflammation, are released into circulation. as a result of the enormous vascular bed present in the lung, sepsis and septic shock often develop when proinflammatory molecules overwhelm the antiinflammatory response during the so-called "cytokine storm." it is axiomatic that a particle, microbe, or toxic gas must first gain entry to a vulnerable region of the respiratory system before it can induce an adaptive immune response or have a pathologic effect. the characteristics of size, shape, dispersal, and deposition of particles present in inspired air are studied in aerobiology. it is important to recognize the difference between deposition, clearance, and retention of inhaled particles. deposition is the process by which particles of various sizes and shapes are trapped within specific regions of the respiratory tract. clearance is the process by which deposited particles are destroyed, neutralized, or removed from the mucosal surfaces. the difference between what is deposited and what is cleared from the respiratory tract is referred to as retention. the main mechanisms involved in clearance are sneezing, coughing, mucociliary transport, and phagocytosis (table 9 -3). abnormal retention of particles resulting from increased deposition, decreased same rate in all levels of a conducting system, a "bottleneck" effect would be created in major airways as the minor but more numerous airways enter the bronchi. for this reason, the mucociliary transport in proximal (rostral) airways is physiologically faster than that of the distal (caudal) ones. ciliary activity and mucus transport increase notably in response to stimuli such as in respiratory infections. the mucociliary blanket of the nasal cavity, trachea, and bronchi also plays an important role in preventing injury from toxic gases. if a soluble gas contacts the mucociliary blanket, it mixes with the mucus, thus reducing the concentration of gas reaching deep into the alveoli. in other words, mucus acts as a "scavenger system," whereby gases are solubilized and subsequently cleared from the respiratory tract via mucociliary transport. if ciliary transport is reduced (loss of cilia) or mucus production is excessive, coughing becomes an important mechanism for clearing the airways. in addition to the mechanical barrier and physical transport provided by the mucociliary escalator, other cells closely associated with ciliated epithelium contribute to the defense mechanism of the conducting and transitional systems. among the most notable are the microfold (m) cells, which are modified epithelial cells covering the bronchial-associated lymphoid tissue (balt), both of which are strategically situated at the corner of the bifurcation of bronchi and bronchioles, where inhaled particles often collide with the mucosa because of inertial forces. from here, inhaled particles and soluble antigens are phagocytosed and transported by macrophages, dendritic cells, and other professional antigen-presenting cells (apcs) into the balt, thus providing a unique opportunity for b and t lymphocytes to enter into close contact with inhaled pathogenic substances. pulmonary lymphocytes are not quiescent in the balt but are in continual traffic to other organs and contribute to both cellular (cytotoxic, helper, and suppressor t lymphocytes) and humoral immune responses. immunoglobulin a (iga), produced by mucosal plasma cells, and, to a lesser extent, immunoglobulin g (igg) and m (igm) play important roles in the local immunity of the conducting and transitional systems, especially with regard to preventing attachment of pathogens to the cilia. chronic airway diseases, especially those caused by infectious agents such as mycoplasmas or retroviruses, are often accompanied by severe hyperplasia of the balt. the mucociliary clearance terminates at the pharynx, where mucus, propelled caudally from the nasal cavity and cranially from the tracheobronchial tree, is eventually swallowed and thus eliminated from the conducting system of the respiratory tract. some respiratory pathogens, such as rhodococcus equi, can infect the intestines after having been removed and swallowed from the respiratory tract into the alimentary system. alveoli lack ciliated and mucus-producing cells; thus the defense mechanism against inhaled particles in the alveolar region cannot be provided by mucociliary clearance. instead, the main defense mechanisms of alveoli (exchange system) are phagocytosis provided by the pulmonary alveolar macrophages and antimicrobial molecules of the alveolar lining fluid ( fig. 9-19 ). pulmonary alveolar macrophages are highly phagocytic cells, which are not to be confused with pulmonary intravascular macrophages, and are derived largely from blood monocytes and, to a much lesser extent, from a slowly dividing population of interstitial macrophages. after a temporary adaptive stage within alveolar interstitium, blood monocytes reduce their glycolytic metabolism and increase their oxidative metabolism to function in an aerobic rather than an anaerobic environment. pulmonary alveolar macrophages contribute to the bifurcations. abrupt changes in the direction of air (inertia), which occurs at the branching of major airways, cause particles in the 2-to 10-µm size range to collide with the surface of bronchial mucosa (see fig. 9 -1). because the velocity of inspired air at the level of the small bronchi and bronchioles has become rather slow, inertial and centrifugal forces no longer play a significant role in the trapping of inhaled particles. here, in the transitional (bronchiolar) and exchange (alveolar) regions, particles 2 µm or smaller may come into contact with the mucosa by means of sedimentation because of gravitation or by diffusion as a result of brownian movement. infective aerosols containing bacteria and viruses are within the size range (0.01 to 2 µm) that can gain access to the bronchiolar and alveolar regions. in addition to size, other factors, such as shape, length, electrical charge, and humidity, play an important role in mucosal deposition, retention, and pathogenicity of inhaled particles. for example, particles longer than 200 µm may also reach the lower respiratory tract provided their mean aerodynamic diameter is less than 1 µm. asbestos is a good example of a large but slender fiber that can bypass the filtrating mechanisms by traveling parallel to the airstream. once in the terminal bronchioles and alveoli, asbestos fibers cause asbestosis, a serious pulmonary disease in human beings. in summary, the anatomic features of the nasal cavity and airways provide an effective barrier, preventing the penetration of most large particles into the lungs. once larger particles are trapped in the mucosa of conducting airways and small particles are deposited on the surface of the nasal, tracheal, or bronchoalveolar mucosa, it is crucial that these exogenous materials be promptly removed to prevent or minimize injury to the respiratory system. for these purposes, the respiratory system is equipped with several defense mechanisms, all of which are provided by specialized cells operating in a remarkably well-coordinated manner. conducting system (nose, trachea, and bronchi) mucociliary clearance is the physical unidirectional movement and removal of deposited particles and gases dissolved in the mucus from the respiratory tract. mucociliary clearance, also referred to as the waste disposal system, is provided by the mucociliary blanket (mucociliary escalator) and is the main defense mechanism of the conducting system (nasal cavity, trachea, and bronchi) (see figs. 9-2 and 9-3). mucus acts primarily as a barrier and a vehicle, and it is a complex mixture of water, glycoproteins, immunoglobulins, lipids, and electrolytes. these substances are produced by goblet (mucous) cells, serous cells, submucosal glands, and fluid from transepithelial ion and water transport. once serous fluid and mucus are secreted onto the surface of the respiratory mucosa, a thin, double-layer film of mucus is formed on top of the cells. the outer layer of this film is in a viscous gel phase, whereas the inner layer, which is in a fluid or sol phase, is directly in contact with cilia (see fig. 9 -3 and see efig. 9 -1). the respiratory system of a healthy human produces approximately 100 ml of mucus per day. each ciliated cell in the conducting system has approximately 100 to 200 motile and chemosensory cilia (6 µm long), beating metachronously (forming a wave) at a ciliary beat frequency of approximately 1000 strokes per minute, and in a horse, for example, mucus moves longitudinally at a rate of up to 20 mm per minute. rapid and powerful movement of cilia creates a series of waves that, in a continuous and synchronized manner, propel the mucus, exfoliated cells, and entrapped particles out of the respiratory tract to the pharynx. the mucus is finally swallowed or, when present in large amounts, is coughed up out of the conducting system. if mucus flow were to move at the activated alveolar macrophages. similarly, inhaled particles, such as dust, pollen, spores, carbon, or erythrocytes from intraalveolar hemorrhage, are all phagocytosed and eventually removed from alveoli by pulmonary alveolar macrophages. most alveolar macrophages leave the alveoli by migrating toward the bronchiolar (transitional) region until the mucociliary blanket is reached. once there, pulmonary macrophages are removed in the same way as any other particle: along the mucociliary flow to the pharynx and swallowed. in the cat, as many as 1 million macrophages per hour move out from the alveoli into the conducting system and pharynx. destruction and removal of inhaled microbes and particles by alveolar macrophages is a well-orchestrated mechanism that engages many cells, receptors (i.e., toll-like receptors [tlrs]), and pulmonary secretions in the lung. the cell-to-cell interactions are complex and involve pulmonary alveolar macrophages, pneumonocytes, endothelial cells, lymphocytes, plasma cells, natural killer (nk) cells, and dendritic cells. antibodies are also important in the protection (acquired immune response) of the respiratory tract against inhaled pathogens. iga is the most abundant antibody in the nasal and tracheal secretions and prevents the attachment and absorption of antigens (immune exclusion). igg and, to a lesser extent, ige and igm promote the uptake and destruction of inhaled pathogens by phagocytic cells (immune elimination). igg is the most abundant antibody in the alveolar surface and acts primarily as an opsonizing antibody for alveolar macrophages and neutrophils. in addition to antibodies, there are several secretory molecules locally released into the alveoli that constitute the alveolar lining material and contribute to the pulmonary defense mechanisms. the most important of these antimicrobial products are transferrin, anionic peptides, and pulmonary surfactant (table 9 -4). to facilitate phagocytosis and discriminate between "self" and "foreign" antigens, pulmonary alveolar macrophages are furnished with a wide variety of specific receptors on their cell surfaces. among the most important ones are fc receptors for antibodies; complement receptors (for c3b, c3a, and c5a); tumor necrosis factor (tnf) receptor; and cd40 receptors, which facilitate phagocytosis and destruction of opsonized particles. toll-like receptors (tlrs) recognize microbial components, and apoptosis stimulating fragment (fas) receptors are involved in apoptosis and in the phagocytosis of apoptotic cells in the lung. "scavenger receptors," which are responsible for the recognition and uptake of foreign particulates, such as dust and fibers, are also present on pulmonary alveolar macrophages. lungs are also susceptible to hematogenously borne microbes, toxins, or emboli. the hepatic (kupffer cells) and splenic macrophages are the primary phagocytic cells responsible for removing circulating bacteria and other particles from the blood of dogs, some rodents, and human beings. in contrast, the cell responsible for the removal of circulating particles, bacteria, and endotoxin from the blood of ruminants, cats, pigs, and horses is mainly the pulmonary intravascular macrophage, a distinct population of phagocytes normally residing within the pulmonary capillaries (see fig. 9 -7). in pigs, 16% of the pulmonary capillary surface is lined by pulmonary intravascular macrophages. in ruminants, 95% of intravenously injected tracer particles or bacteria are rapidly phagocytosed by these intravascular macrophages. studies have shown that an abnormally reduced number of kupffer cells in diseased liver results in a compensatory increase in pulmonary intravascular macrophages, even in animal species in which these phagocytic cells are normally absent from the lung. in some abnormal conditions, such as sepsis, pulmonary innate and adaptive immune response by rapidly attaching and phagocytosing bacteria and any other particles reaching the alveolar lumens. the number of free macrophages in the alveolar space is closely related to the number of inhaled particles reaching the lungs. this ability to increase, within hours, the number of available phagocytic cells is vital in protecting the distal lungs against foreign material, particularly when the inhaled particle load is high. unlike that of tissue macrophages, the life span of alveolar macrophages in the alveoli is notably short, only a few days, and thus they are continuously being replaced by newly migrated blood monocytes. alveolar phagocytosis plays a prominent role in the innate defense mechanism against inhaled bacteria without the need of an inflammatory reaction. bacteria reaching the alveoli are rapidly phagocytosed, and bactericidal enzymes present in lysosomes are discharged into the phagosome containing the bacteria (see b) . except for some facultative pathogens that are resistant to intracellular killing (e.g., mycobacterium tuberculosis, listeria monocytogenes, brucella abortus, rhodococcus equi, and some salmonella spp.), most bacteria reaching the lungs are rapidly destroyed by (ros) not only induce extensive pulmonary injury but also impair the defense and repair mechanisms in the lung. oxygen and free radical scavengers, such as catalase, superoxide dismutase, ubiquinone, and vitamins e and c, are largely responsible for protecting pulmonary cells against peroxidation. these scavengers are present in alveolar and bronchiolar epithelial cells and in the extracellular spaces of the pulmonary interstitium. in summary, the defense mechanisms are so effective in trapping, destroying, and removing bacteria that, under normal conditions, animals can be exposed to aerosols containing massive numbers of bacteria without any ill effects. if defense mechanisms are impaired, inhaled bacteria colonize and multiply in bronchi, bronchioles, and alveoli, and they produce infection, which can result in fatal pneumonia. similarly, when blood-borne pathogens, inhaled toxicants, or free radicals overwhelm the protective defense mechanisms, cells of the respiratory system are likely to be injured, often causing serious respiratory diseases. for many years, factors such as viral infections, toxic gases, stress, and pulmonary edema have been implicated in predisposing human beings and animals to secondary bacterial pneumonia. there are many pathways by which the defense mechanisms can be impaired; only those relevant to veterinary species are discussed. viral agents are notorious in predisposing human beings and animals to secondary bacterial pneumonias by what is known as viral-bacterial synergism. a good example of the synergistic effect of combined virus-bacterial infections is documented from epidemics of human beings with influenza virus in which the mortality rate has been significantly increased from secondary bacterial pneumonia. the most common viruses incriminated in predisposing animals to secondary bacterial pneumonia include influenza virus in pigs and horses; bovine herpesvirus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv) in cattle; canine distemper virus (cdv) in dogs; and felid herpesvirus 1 (fehv-1) and feline calicivirus (fcv) in cats. the mechanism of the synergistic effect of viral-bacterial infections was previously believed to be the destruction of the mucociliary blanket and a concurrent reduction of mucociliary clearance, but in experimental studies, viral infections did not significantly reduce the physical removal of particles or bacteria out of the lungs. now, it is known that 5 to 7 days after a viral infection, the phagocytic function of pulmonary alveolar macrophages and, to a lesser extent, the mucociliary clearance are notably impaired (see fig. 9 -8). other mechanisms by which viruses impair defense mechanisms are multiple and remain poorly understood (box 9-1). immunization against viral infections in many cases prevents or reduces the synergistic effect of viruses and thus the incidence of secondary bacterial pneumonia. certain gases also impair respiratory defense mechanisms, rendering animals more susceptible to secondary bacterial infections. for instance, hydrogen sulfide and ammonia, frequently encountered on farms, especially in buildings with poor ventilation, can impair pulmonary defense mechanisms and increase susceptibility to bacterial pneumonia. the effects of environmental pollutants on the defense mechanisms of human beings and animals living in crowded and polluted cities remain to be determined. excessive release of cytokines by pulmonary intravascular macrophages may result in acute lung injury. existing in an oxygen-rich environment and being the site of numerous metabolic reactions, the lungs also require an efficient defense mechanism against oxidant-induced cellular damage (oxidative stress). this form of damage is caused by inhaled oxidant gases (e.g., nitrogen dioxide, ozone, sulfur dioxide, or tobacco smoke), by xenobiotic toxic metabolites produced locally, by toxins reaching the lungs via the bloodstream (e.g., 3-methylindole and paraquat), or by free radicals (reactive oxygen species) released by phagocytic cells during inflammation. free radicals and reactive oxygen species anomalies 2 localized congenital anomalies of the nasal cavity are rare in domestic animals and are often merely part of a more extensive craniofacial deformity (e.g., cyclops) or a component of generalized malformation (e.g., chondrodysplasia). congenital anomalies involving the nasal cavity and sinuses, such as choanal atresia (lack of communication between the nasal cavity and pharynx), some types of chondrodysplasia, and osteopetrosis, are incompatible with life. examples of nonfatal congenital anomalies include cystic nasal conchae, deviation of the nasal septum, cleft upper lip (harelip and cheiloschisis), hypoplastic turbinates, and cleft palate (palatoschisis) (see fig. 7 -32). bronchoaspiration and aspiration pneumonia are common sequelae to cleft palate. nasal and paranasal sinus cysts are slowly growing and expansive lesions that mimic neoplasia and cause severe cranial deformation in horses. as in other organs or systems, it is extremely difficult to determine the actual cause (genetic vs. congenital) of anomalies based on pathologic evaluation. metabolic disturbances affecting the nasal cavity and sinuses are rare in domestic animals. immunodeficiency disorders, whether acquired or congenital, are often associated with increased susceptibility to viral, bacterial, and protozoal pneumonias. for example, human beings with acquired immunodeficiency syndrome (aids) are notably susceptible to pneumonia caused by proliferation of pneumocystis (carinii) jirovecii. a similar ubiquitous organism, which under normal circumstances is not pathogenic, is also found in the pneumonic lungs of immunosuppressed pigs, foals, dogs, and rodents. pigs infected with the porcine reproductive and respiratory syndrome (prrs) virus frequently develop pneumocystis carinii infection ( fig. 9-20) . arabian foals born with combined immunodeficiency disease easily succumb to infectious diseases, particularly adenoviral pneumonia. combined infections with two respiratory viruses, such as canine distemper virus (cdv) and canine adenovirus 2 (cav-2), are sporadically reported in immunosuppressed puppies. also, large doses of chemotherapeutic agents, such as steroids and alkylating agents, cause immunosuppression in dogs, cats, and other animals, increasing susceptibility to secondary viral and bacterial infections. stress, uremia, endotoxemia, dehydration, starvation, hypoxia, acidosis, pulmonary edema, anesthesia, and ciliary dyskinesia are only some of the many conditions that have been implicated in impairing respiratory defense mechanisms and consequently predisposing animals to develop secondary bacterial pneumonia. the mechanisms by which each of these factors suppresses pulmonary defenses are diverse and sometimes not well understood. for example, hypoxia and pulmonary edema decrease phagocytic function of pulmonary alveolar macrophages and alter the production of surfactant (abnormal head tilt and abnormal gait), which in severe cases may lead to emaciation. based on the nature of exudate, rhinitis can be classified as serous, fibrinous, catarrhal, purulent, or granulomatous. these types of inflammatory reactions can progress from one to another in the course of the disease (i.e., serous to catarrhal to purulent), or in some instances exudates can be mixed, such as those seen in mucopurulent, fibrinohemorrhagic, or pyogranulomatous rhinitis. microscopic examination of impression smears or nasal biopsy, and bacterial or fungal cultures are generally required in establishing the cause of inflammation. common sequelae of rhinitis are hemorrhage, ulcers, and, in some cases, nasopharyngeal polyps (hyperplasia) arising from inflamed mucosa. rhinitis also can be classified according to the age of the lesions as acute, subacute, or chronic; to the severity of the insult as mild, moderate, or severe; and to the etiologic agent as viral, allergic, bacterial, mycotic, parasitic, traumatic, or toxic. serous rhinitis. serous rhinitis is the mildest form of inflammation and is characterized by hyperemia and increased production of a clear fluid locally manufactured by serous glands present in the nasal submucosa. serous rhinitis is of clinical interest only. it is caused by mild irritants or cold air, and it occurs during the early stages of viral infections, such as the common cold in human beings, upper respiratory tract infections in animals, or in mild allergic reactions. catarrhal rhinitis. catarrhal rhinitis is a slightly more severe process and has, in addition to serous secretions, a substantial increase in mucus production by hypersecretion of goblet cells and mucous glands. a mucous exudate is a thick, translucent, or slightly turbid viscous fluid, sometimes containing a few exfoliated cells, leukocytes, and cellular debris. in chronic cases, catarrhal rhinitis is characterized microscopically by notable hyperplasia of goblet cells. as the inflammation becomes more severe, the mucus is infiltrated with neutrophils, giving the exudate a cloudy appearance. this exudate is referred to as mucopurulent. purulent (suppurative) rhinitis. purulent (suppurative) rhinitis is characterized by a neutrophilic exudate, which occurs when the nasal mucosa suffers a more severe injury that generally is accompanied by mucosal necrosis and secondary bacterial infection. cytokines, leukotrienes, complement activation, and bacterial products cause exudation of leukocytes, especially neutrophils, which mix with nasal secretions, including mucus. grossly, the exudate in suppurative rhinitis is thick and opaque, but it can vary from white to green to brown, depending on the types of bacteria and type of leukocytes (neutrophils or eosinophils) present in the exudate . in severe cases, the nasal passages are completely blocked by the exudate. microscopically, neutrophils can be seen in the submucosa and mucosa and form plaques of exudate on the mucosal surface. neutrophils are commonly found marginated in vessels, in the lamina propria, and in between epithelial cells in their migration to the surface of the mucosa. fibrinous rhinitis. fibrinous rhinitis is a reaction that occurs when nasal injury causes a severe increase in vascular permeability, resulting in abundant exudation of plasma fibrinogen, which coagulates into fibrin. grossly, fibrin appears as a yellow, tan, or gray rubbery mat on nasal mucosa. fibrin accumulates on the surface and forms a distinct film of exudate sometimes referred to as pseudomembrane ( fig. 9-22 ). if this fibrinous exudate can be removed, leaving an intact underlying mucosa, it is termed a croupous or pseudodiphtheritic rhinitis. conversely, if the pseudomembrane is difficult to remove and leaves an ulcerated mucosa, it is referred to as diphtheritic or fibrinonecrotic rhinitis. the term diphtheritic was derived from human diphtheria, which causes a severe and destructive inflammatory process of the nasal, tonsillar, pharyngeal, and laryngeal mucosa. nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses and human beings (see nasal amyloidosis, in disorders of horses). congestion and hyperemia. the nasal mucosa is well vascularized and is capable of rather dramatic variation in blood flow, whether passively as a result of interference with venous return (congestion) or actively because of vasodilation (hyperemia). congestion of the mucosal vessels is a nonspecific lesion commonly found at necropsy and presumably associated with the circulatory failure preceding death (e.g., heart failure, bloat in ruminants in which the increased intraabdominal pressure causes increased intrathoracic pressure impeding the venous return from the head and neck). hyperemia of the nasal mucosa is seen in early stages of inflammation, whether caused by irritation (e.g., ammonia and regurgitated feed), viral infections, secondary bacterial infections, toxemia, allergy, or trauma. hemorrhage. epistaxis is the clinical term used to denote blood flow from the nose (nosebleed) regardless of whether the blood originates from the nasal mucosa or from deep in the lungs, such as in horses with "exercise-induced pulmonary hemorrhage." unlike blood in the digestive tract, where the approximate anatomic location of the bleeding can be estimated by the color the blood imparts to fecal material, blood in the respiratory tract is always red. this fact is due to the rapid transport of blood out of the respiratory tract by the mucociliary blanket and during breathing. hemorrhages into the nasal cavity can be the result of local trauma, can originate from erosions of submucosal vessels by inflammation (e.g., guttural pouch mycosis), or can be caused by neoplasms. hemoptysis refers to the presence of blood in sputum or saliva (coughing or spitting blood) and is most commonly the result of pneumonia, lung abscesses, ulcerative bronchitis, pulmonary thromboembolisms or hemorrhage, and pulmonary neoplasia. inflammation of the nasal mucosa is called rhinitis, and inflammation of the sinuses is called sinusitis. these conditions usually occur together, although mild sinusitis can be undetected. clinically, rhinosinusitis is characterized by nasal discharge. rhinitis. the occurrence of infectious rhinitis presupposes an upset in the balance of the normal microbial flora of the nasal cavity. innocuous bacteria present normally protect the host through a process called competitive exclusion, whereby potential pathogens are kept at a harmless level. disruption of this protective mechanism can be caused by respiratory viruses, pathogenic bacteria, fungi, irritant gases, environmental changes, immunosuppression, local trauma, stress, or prolonged antibacterial therapy. inflammatory processes in the nasal cavity are not life-threatening and usually resolve completely. however, some adverse sequelae in cases of infectious rhinitis include bronchoaspiration of exudate leading to bronchopneumonia. chronic rhinitis often leads to destruction of the nasal conchae (turbinates), deviation of the septum, and, eventually, craniofacial deformation. also, nasal inflammation may extend into the sinuses causing sinusitis; into facial bones causing osteomyelitis; through the cribriform plate causing meningitis; into the eustachian tubes causing otitis media or guttural pouch empyema (eustachitis) in horses; and even into the inner ear causing otitis interna and vestibular syndrome the nasal septum has been removed to expose nasal conchae. the nasal mucosa is hyperemic and covered by yellow-white purulent exudate (arrows). inset, histological section showing submucosal congestion and edema and also large aggregates of neutrophils on the superficial mucosa (asterisk). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) microscopically, the lesions include a perivascular edema with fibrin, a few neutrophils infiltrating the mucosa, and superficial plaques of exudate consisting of fibrin strands mixed with leukocytes and cellular debris covering a necrotic and ulcerated epithelium. fungal infections, such as aspergillosis, can cause a severe fibrinonecrotizing rhinitis. granulomatous rhinitis. granulomatous rhinitis is a reaction in the nasal mucosa and submucosa that is characterized by infiltration of numerous activated macrophages mixed with a few lymphocytes and plasma cells (figs. 9-23 and 9-24). in some cases, chronic inflammation leads to the formation of polypoid nodules that in severe cases are large enough to cause obstruction of the nasal passages ( fig. 9-25 ). granulomatous rhinitis is generally associated with chronic allergic inflammation or infection with specific organisms, such as fungi (see fig. 9 -24), tuberculosis, systemic mycosis (see section on granulomatous pneumonia), and rhinosporidiosis ; also see fig. 9 -25). in some cases, the cause of granulomatous rhinitis cannot be determined. sinusitis. sinusitis occurs sporadically in domestic animals and is frequently combined with rhinitis (rhinosinusitis), or it occurs as extend into the adjacent bone (osteomyelitis) or through the ethmoidal conchae into the meninges and brain (meningitis and encephalitis). nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses. unlike amyloidoses in other organs of domestic animals where amyloid is generally of the reactive type (amyloid aa), equine nasal amyloidosis appears to be of the immunocytic type (amyloid al). affected horses with large amyloid masses have difficulty breathing because of nasal obstruction and may exhibit epistaxis and reduced athletic performance; on clinical examination, large, firm nodules resembling neoplasms (amyloidoma) can be observed in the alar folds, rostral nasal septum, and floor of nasal cavity. microscopic lesions are similar to those seen in other organs and consist of a deposition of hyaline amyloid material in nasal mucosa that is confirmed by a histochemical stain, such as congo red. progressive ethmoidal hematoma. progressive ethmoidal hematoma (peh) is important in older horses and is characterized clinically by chronic, progressive, often unilateral nasal bleeding. grossly or endoscopically, an ethmoidal hematoma appears as a single, soft, tumor-like, pedunculated, expansive, dark red mass arising from the mucosa of the ethmoidal conchae ( fig. 9-28) . microscopic examination reveals a capsule lined by epithelium and hemorrhagic stromal tissue infiltrated with abundant macrophages, most of which are siderophages. viral infections. viruses, such as equine viral rhinopneumonitis virus, influenza virus, adenovirus, and equine picornavirus, cause mild and generally transient respiratory infections in horses. the route of infection for these respiratory viruses is typically aerogenous. all of these infections are indistinguishable clinically; signs consist mainly of malaise, fever, coughing, conjunctivitis, and nasal a sequela to penetrating or septic wounds of the nasal, frontal, maxillary, or palatine bones; improper dehorning in young cattle, which exposes the frontal sinus; or maxillary tooth infection in horses and dogs (maxillary sinus). based on the type of exudate, sinusitis is classified as serous, catarrhal, fibrinous (rare), purulent, or granulomatous. paranasal sinuses have poor drainage; therefore exudate tends to accumulate, causing mucocele (accumulation of mucus) or empyema (accumulation of pus) ( fig. 9-27 ). chronic sinusitis may promised horses, particularly in arabian foals with inherited combined immunodeficiency disease. bacterial infections. strangles, glanders, and melioidosis of horses are all systemic bacterial diseases that cause purulent rhinitis and suppuration in various organs. these diseases are grouped as upper respiratory diseases because nasal discharge is often the most notable clinical sign. strangles. strangles is an infectious and highly contagious disease of equidae that is caused by streptococcus equi ssp. equi (streptococcus equi) . it is characterized by suppurative rhinitis and lymphadenitis (mandibular and retropharyngeal lymph nodes) with occasional hematogenous dissemination to internal organs. unlike streptococcus equi ssp. zooepidemicus (streptococcus zooepidemicus) and streptococcus dysgalactiae ssp. equisimilis (streptococcus equisimilis), streptococcus equi is not part of the normal nasal flora. infection occurs when susceptible horses come into contact with feed, exudate, or air droplets containing the bacterium. after penetrating through the nasopharyngeal mucosa, streptococcus equi drains to the regional lymph nodes-mandibular and retropharyngeal lymph nodes-via lymphatic vessels. the gross lesions in horses with strangles (mucopurulent rhinitis) correlate with clinical findings and consist of copious amounts of mucopurulent exudate in the nasal passages with notable hyperemia of the nasal mucosa. affected lymph nodes are enlarged and may contain abscesses filled with thick purulent exudate (purulent lymphadenitis). the term bastard strangles is used in cases in which hematogenous dissemination of streptococcus equi results in metastatic abscesses in such organs as the lungs, liver, spleen, kidneys, or brain or in the joints. this form of strangles is often fatal. common sequelae to strangles include bronchopneumonia caused by aspiration of nasopharyngeal exudate; laryngeal hemiplegia ("roaring"), resulting from compression of the recurrent laryngeal nerves by enlarged retropharyngeal lymph nodes; facial paralysis and horner syndrome caused by compression of sympathetic nerves that run dorsal to the medial retropharyngeal lymph node; and purpura hemorrhagica as a result of vasculitis caused by deposition of streptococcus equi antigen-antibody complexes in arterioles, venules, and capillaries of the skin and mucosal membranes. in severe cases, nasal infection extends directly into the paranasal sinuses or to the guttural pouches via the eustachian tubes, causing inflammation and accumulation of pus (guttural pouch empyema). rupture of abscesses in the mandibular and retropharyngeal lymph nodes leads to suppurative inflammation of adjacent subcutaneous tissue (cellulitis), and in severe cases the exudate escapes through cutaneous fistulas. strangles can affect horses of all ages, but it is most commonly seen in foals and young horses. it is clinically characterized by cough, nasal discharge, conjunctivitis, and painful swelling of regional lymph nodes. some horses become carriers and a source of infection to other horses. glanders. glanders is an infectious world organization for animal health (oie)-notifiable disease of equidae caused by burkholderia mallei (pseudomonas mallei) that can be transmitted to carnivores by consumption of infected horsemeat. human beings are also susceptible, and untreated infection is often fatal. this gramnegative bacterium has been listed as a potential agent for biologic warfare and bioterrorism. in the past, burkholderia mallei was found throughout the world, but today, glanders has been eradicated from most countries, except for some areas in north africa, asia, and eastern europe. there also have been sporadic outbreaks reported in brazil. the pathogenesis of glanders is not fully understood. results from experimental infections suggest that infection occurs discharge varying from serous to purulent. viral respiratory infections are common medical problems in adult horses. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr) is caused by two ubiquitous equine herpesviruses (ehv-1 and ehv-4) and may be manifested as a mild respiratory disease in weanling foals and young racehorses, as a neurologic disease (myeloencephalopathy), or as abortion in mares. the portal of entry for the respiratory form is typically aerogenous, and the disease is generally transient; thus the primary viral-induced lesions in the nasal mucosa and lungs are rarely seen at necropsy unless complicated by secondary bacterial rhinitis, pharyngitis, or bronchopneumonia. studies with polymerase chain reaction (pcr) techniques have demonstrated that, like other herpesviruses, ehv-1 and ehv-4 persist in the trigeminal ganglia for long periods of time (latency). reactivation because of stress or immunosuppression and subsequent shedding of the virus are the typical source of infection for susceptible animals on the farm. equine influenza. equine influenza is a common, highly contagious, and self-limiting upper respiratory infection of horses caused by aerogenous exposure to type a strains of influenza virus (h7n7 [a/equi-1] and h3n8 [a/equi-2]). equine influenza has high morbidity (outbreaks) but low mortality, and it is clinically characterized by fever, conjunctivitis, and serous nasal discharge. it occurs mainly in 2-to 3-year-old horses at the racetrack. as with human influenza, equine influenza is usually a mild disease, but occasionally it can cause severe bronchointerstitial pneumonia with pulmonary edema. in some horses, impaired defense mechanisms caused by the viral infection are complicated by a secondary bacterial bronchopneumonia caused by opportunistic organisms (streptococcus zooepidemicus, staphylococcus aureus, or bacteroides sp.) found in the normal flora of the upper respiratory tract. uncomplicated cases of equine influenza are rarely seen in the postmortem room. equine influenza virus (h3n8) recently did an equine to canine "host-jump" causing extensive outbreaks of respiratory disease in dogs (see pneumonias of dogs). other equine respiratory viruses. equine picornavirus, adenovirus, and parainfluenza virus produce mild and transient upper respiratory infections (nasopharynx and trachea) in horses, unless complicated by secondary pathogens. in addition to reduced athletic performance, infected horses may have a temporary suppression of cell-mediated immunity leading to opportunistic infections such as pneumocystis carinii pneumonia. fatal adenoviral infections with severe pneumonia or enteritis occur commonly in immunocomdisorders of ruminants (cattle, sheep, and goats) infectious bovine rhinotracheitis. infectious bovine rhinotracheitis (ibr), or "rednose," occurs worldwide and is a disease of great importance to the cattle industry because of the synergism of the ibr virus with mannheimia haemolytica in producing pneumonia. the causative agent, bovine herpesvirus 1 (bohv-1), has probably existed as a mild venereal disease in cattle in europe since at least the mid-1800s, but the respiratory form was not reported until intensive management feedlot systems were first introduced in north america around the 1950s. typically, the disease is manifested as a transient, acute, febrile illness, which results in inspiratory dyspnea caused by obstruction of the airways by exudate only in very severe cases. other forms of bohv-1 infection include ulcerative rumenitis; enteritis; multifocal hepatitis in neonatal calves; nonsuppurative meningoencephalitis; infertility; and in experimental infections, mastitis, mammillitis, and ovarian necrosis. except for the encephalitic form, the type of disease caused by bohv-1 depends more on the site of entry than the viral strain. like other herpesviruses, bohv-1 also can remain latent in nerve ganglia, with recrudescence after stress or immunosuppression. this virus also causes bovine abortion, systemic infections of calves, and genital infections such as infectious pustular vulvovaginitis (ipv) and infectious balanoposthitis (ibp). the respiratory form of ibr is characterized by severe hyperemia and multifocal necrosis of nasal, pharyngeal, laryngeal, tracheal, and sometimes bronchial mucosa ( fig. 9 -29 and see fig. 9 -22). as in other respiratory viral infections, ibr lesions are microscopically characterized by necrosis and exfoliation of ciliated cells followed by repair. secondary bacterial infections of these areas of necrosis result in the formation of a thick layer of fibrinonecrotic material (diphtheritic) in the nasal, tracheal, and bronchial mucosa (see fig. 9 -22). intranuclear inclusion bodies, commonly seen in herpesvirus infections, are rarely seen in field cases because inclusion bodies occur only during the early stages of the disease. the most important sequela to ibr is bronchopneumonia, which is caused either by direct aspiration of exudate from airways or as a result of an impairment in pulmonary defense mechanisms, thus predisposing the animal to secondary bacterial infection, most frequently mannheimia haemolytica (see pneumonic mannheimiosis discussion). postmortem diagnosis of ibr is confirmed by isolation of the virus or its identification by immunohistochemistry or pcr in affected tissues. other causes of rhinitis. nasal granulomas occur in cattle presumably as a result of repeated exposure to an unidentified inhaled antigen. nasal granulomas (atopic rhinitis) are reported mainly in cattle in australia, south africa, and the united kingdom, where affected cattle develop multiple, small, pink or red, polypoid nodules, starting in the nasal vestibule that in time extend into the caudal aspect of the nasal septum (see fig. 9 -23). these nodules are composed of fibrovascular tissue mixed with lymphocytes (granulation tissue) superficially lined by hyperplastic epithelium with abundant mast cells and eosinophils in the lamina propria (nasal eosinophilia). the microscopic features suggest that hypersensitivity type i (immediate), type iii (immune complex), and type iv (delayed) may be involved in nasal granulomas of cattle. bovine (idiopathic) nasal granuloma must be differentiated from nasal mycetomas, nasal rhinosporidiosis, and nasal schistosomiasis, which also cause the formation of nodules in the nasal mucosa of cattle. an eosinophilic material consistent with the splendore-hoeppli phenomenon is occasionally observed in bovine mycotic granulomas. this phenomenon seen in some mycotic or bacterial infections is microscopically via the ingestion of contaminated feed and water and, very rarely, via inhalation of infectious droplets. the portals of entry are presumed to be the oropharynx or intestine, in which bacteria penetrate the mucosa and spread via lymph vessels to regional lymph nodes, then to the bloodstream, and thus hematogenously to the internal organs, particularly the lungs. lesions in the nasal cavity start as pyogranulomatous nodules in the submucosa; these lesions subsequently ulcerate, releasing copious amounts of burkholderia mallei-containing exudate into the nasal cavity (see fig. 4-25, a) . finally, ulcerative lesions in conchal mucosa heal and are replaced by typical stellate (star-shaped), fibrous scars. in some cases, the lungs also contain numerous gray, hard, small (2 to 10 mm), miliary nodules (resembling millet seeds) randomly distributed in one or more pulmonary lobes because of the hematogenous route. microscopically, these nodules are typical chronic granulomas composed of a necrotic center, with or without calcification, surrounded by a layer of macrophages enclosed by a thick band of connective tissue infiltrated with macrophages, fewer giant cells, lymphocytes, and plasma cells. cutaneous lesions, often referred to as equine farcy, are the result of severe suppurative lymphangitis characterized by nodular thickening of extended segments of lymph vessels in the subcutaneous tissue of the legs and ventral abdomen (see fig. 4 -25, c). eventually, affected lymph vessels rupture and release large amounts of purulent exudate through sinuses to the surface of the skin. melioidosis (pseudoglanders). melioidosis (pseudoglanders) is an important, life-threatening disease of human beings, horses, cattle, sheep, goats, pigs, dogs, cats, and rodents caused by burkholderia pseudomallei (pseudomonas pseudomallei). this disease in horses is clinically and pathologically similar to glanders, hence the name pseudoglanders. in human beings, this infection can cause severe sepsis and septic shock and has also been considered to have potential for biologic welfare. melioidosis is currently present in southeast asia and, to a much lesser extent, in northern australia and some european countries where the causative organism is frequently found in rodents, feces, soil, and water. ingestion of contaminated feed and water appears to be the main route of infection; direct transmission between infected animals and insect bites has also been postulated as a possible mechanism of infection. after gaining entrance to the animal, burkholderia pseudomallei is disseminated by the bloodstream and causes suppuration and abscesses in most internal organs, such as nasal mucosa, joints, brain and spinal cord, lungs, liver, kidneys, spleen, and lymph nodes. the exudate is creamy or caseous and yellow to green. the pulmonary lesions in melioidosis are those of an embolic bacterial infection with the formation of pulmonary abscesses, which can become confluent. focal adhesive pleuritis develops where abscesses rupture through the pleura and heal. rhinosporidiosis. the protistan parasite, rhinosporidium seeberi, causes nasal infection in human beings, horses, mules, cattle, dogs, and cats. gross lesions vary from barely visible granulomas to large expansive polypoid nodules that may be mistaken as tumors. these granulomatous nodules are detected by direct observation when present in the nasal mucosa close to the nares or by rhinoscopy when located in the deep nasal cavity. the offending organism, rhinosporidium seeberi, is readily visible in histologic preparations and in impression smears, appearing as a large (400 µm), oval sporangium containing thousands of endospores (see fig. 9 -26). rhinosporidium seeberi was once considered a mycotic agent, but recent phylogenetic investigations suggest that it is an aquatic protistan parasite of the class mesomycetozoea. giant, basophilic, intranuclear inclusion bodies in the nasal epithelium, particularly in the nasal glands ( fig. 9-32 ). immunosuppressed piglets can develop a systemic cytomegalovirus infection characterized by necrosis of the liver, lungs, adrenal glands, and brain with intralesional inclusion bodies. inclusion body rhinitis is clinically a characterized by a deeply eosinophilic homogeneous material surrounded by bacteria or mycelia. it is thought to result from a localized antigen-antibody response in tissue. oestrus ovis. oestrus ovis (diptera: oestridae; nasal bot) is a brownish fly about the size of a honeybee that deposits its first-stage larvae in the nostrils of sheep in most areas of the world. microscopic larvae mature into large bots (maggots), which spend most of their larval stages in nasal passages and sinuses, causing irritation, inflammation, and obstruction of airways. mature larvae drop to the ground and pupate into flies. this type of parasitism in which living tissues are invaded by larvae of flies is known as myiasis ( fig. 9-30 ). although oestrus ovis is a nasal myiasis primarily of sheep, it sporadically affects goats, dogs, and sometimes human beings (shepherds). the presence of the larvae in nasal passages and sinuses causes chronic irritation and erosive mucopurulent rhinitis and sinusitis; bots of oestrus ovis can be found easily if the head is cut to expose the nasal passages and paranasal sinuses. rarely, larvae of oestrus ovis penetrate the cranial vault through the ethmoidal plate, causing direct or secondary bacterial meningitis. other causes of rhinitis. infectious rhinitis is only sporadically reported in goats, and most of these cases are caused by pasteurella multocida or mannheimia haemolytica. the lesions range from a mild serous to catarrhal or mucopurulent inflammation. foreign body rhinitis caused by plant material is sporadically seen cattle, sheep, and goats ( fig. 9-31 ). inclusion body rhinitis. inclusion body rhinitis is a disease of young pigs with high morbidity and low mortality caused by a porcine cytomegalovirus (suid herpesvirus-2) and characterized by a mild rhinitis. this virus commonly infects the nasal epithelium of piglets younger than 5 weeks and causes a transient viremia. because this disease is seldom fatal, lesions are seen only incidentally or in euthanized animals. in uncomplicated cases, the gross lesion is hyperemia of the nasal mucosa, but with secondary bacterial infections, mucopurulent exudate can be abundant. microscopic lesions are typical and consist of a necrotizing, nonsuppurative rhinitis with toxigenic strains of pasteurella multocida. the only lesion associated with infection with bordetella bronchiseptica alone is a mild to moderate turbinate atrophy (nonprogressive atrophic rhinitis), but this bacterium actively promotes the colonization of the nasal cavity by pasteurella multocida. the toxigenic strains of pasteurella multocida produce potent cytotoxins that inhibit osteoblastic activity and promote osteoclastic reabsorption in nasal bones, particularly in the ventral nasal conchae, where abnormal bone remodeling results in progressive atrophy of conchae. the degree of conchal atrophy in pigs with atrophic rhinitis varies considerably, and in most pigs, the severity of the lesions does not correspond to the severity of the clinical signs. the best diagnostic method of evaluating this disease at necropsy is to make a transverse section of the snout between the first and second premolar teeth. in normal pigs, conchae are symmetric and fill most of the cavity, leaving only narrow airspaces (meatuses) between coiled conchae. the normal nasal septum is straight and divides the cavity into two mirror-image cavities. in contrast, the septum in pigs with atrophic rhinitis is generally deviated and the conchae appear smaller and asymmetric ( fig. 9-33 ). conchal atrophy causes dorsal and ventral meatuses to appear rather enlarged, and in the most advanced cases, the entire nasal conchae may be missing, leaving a large, empty space. it may seem logical to assume that after loss of conchae in an obligate nasal breather, such as the pig, the filtration defense mechanism of the nasal cavity would be impaired, thus enhancing the chances of aerogenous infections in the lung. however, the relationship between atrophic rhinitis, pneumonia, and growth rates in pigs is still controversial. osteoclastic hyperplasia and osteopenia of the conchae are the key microscopic lesions in atrophic rhinitis. depending on the stage of the disease, mucopurulent exudate may be found on the surface of the conchae. hyperplastic or metaplastic changes can occur in the nasal epithelium and glands, and infiltrates of lymphoplasmacytic cells can be present in the lamina propria. in summary, atrophic rhinitis is an important disease in pigs worldwide; morphologic characterized by a mild and transient rhinitis, causing sneezing, nasal discharge, and excessive lacrimation. atrophic rhinitis. a common worldwide disease of pigs, atrophic rhinitis (progressive atrophic rhinitis) is characterized by inflammation and atrophy of nasal conchae (turbinates). in severe cases, atrophy of the conchae may cause a striking facial deformity in growing pigs because of deviation of the nasal septum and nasal bones. the etiopathogenesis of atrophic rhinitis is complex and has been a matter of controversy for many years. pathogens historically associated with atrophic rhinitis include bordetella bronchiseptica, pasteurella multocida, haemophilus parasuis, and viral infections such as porcine cytomegalovirus (inclusion body rhinitis). in addition, predisposing factors have included genetic makeup, environment, and nutritional deficiencies. the cause of atrophic rhinitis is currently believed to be a combined infection by specific strains of bordetella bronchiseptica producing dermonecrotic toxin and linguatula serrata. linguatula serrata is a rare but highly specialized pentastomid parasite that shares some morphologic features with arthropods and annelids and causes infection when dogs consume uncooked ruminant meat containing infective larvae. it occurs primarily in carnivores, although sheep and goats may become aberrant hosts. human beings can also acquire the infection by ingesting raw ovine or caprine meat. the adult parasite is found throughout the nasal passages and sometimes can reach the sinuses and middle ear by moving through the exudate in the eustachian tubes. in common with other nasal parasites, linguatula serrata acts as an irritant, causing sneezing, catarrhal inflammation, and epistaxis. the eggs of this parasite leave the host in the exudate, which is coughed up or swallowed and eliminated in the feces. the nasal cavity and paranasal sinuses of dogs can occasionally be infested with other parasites, including mites (pneumonyssus caninum) and rhinosporidium seeberi (see figs. 9-25 and 9-26). allergic rhinitis. allergic rhinitis (hay fever; nasolacrimal urticaria), which is so common in human beings sensitized and reexposed to inhaled pollens or allergens, has been reported only sporadically in dogs and cats. hay fever in human beings and animals is a type i hypersensitivity reaction in which an ige-mediated degranulation of mast cells results in an acute rhinitis and conjunctivitis. microscopically, the nasal mucosa is edematous and infiltrated with numerous eosinophils, neutrophils, and some macrophages. clinically, allergic rhinitis is characterized by profuse serous nasal discharge and lacrimation. other causes of rhinitis. a nonspecific (idiopathic) chronic lymphoplasmacytic rhinitis is occasionally seen in dogs. immotile cilia syndrome (ciliary dyskinesia), a congenital disease, reduces mucociliary clearance and is an important factor in recurrent canine rhinosinusitis, bronchitis, bronchiectasis, and pneumonia. feline viral rhinotracheitis. feline viral rhinotracheitis (fvr) is a common, worldwide respiratory disease of cats caused by felid herpesvirus 1 (fehv-1). the disease causes an impairment of pulmonary defense mechanisms predisposing cats to secondary bacterial pneumonia or to a coinfection with feline calicivirus. the virus also can remain latent in ganglia. the vast majority of cats that recover from fvr become carriers and shed fehv-1, either spontaneously or following stress. susceptible animals, particularly kittens with low maternal immunity, become infected after exposure to a diseased or carrier cat. replication of fehv-1 in the nasal, conjunctival, pharyngeal, and, to a lesser extent, tracheal epithelium causes degeneration and exfoliation of cells. lesions caused by fehv-1 are fully reversible, but secondary infections with bacteria, such as pasteurella multocida, bordetella bronchiseptica, streptococcus spp., and mycoplasma felis, can cause a chronic, severe suppurative rhinitis and also conjunctivitis. intranuclear inclusion bodies are rarely seen in cats with fvr because inclusions are only present during the early stages of infection and have already disappeared by the time the cat is presented for diagnosis. respiratory sequelae to fvr can include chronic bacterial rhinitis and sinusitis with persistent purulent discharge; lysis of nasal bones, which can lead to conchal atrophy; permanent damage to the olfactory epithelium; and secondary bacterial pneumonia. in addition to rhinitis and interstitial pneumonia, fvr also causes ulcerative keratitis, hepatic necrosis, emaciation, abortion, and diagnosis is simple, but additional understanding of the pathogenesis will be necessary before effective preventive measures can be established. atrophic rhinitis is clinically characterized by sneezing, coughing, and nasal discharge. obstruction of the nasolacrimal duct is common and results in accumulation of dust and dried lacrimal secretions on the skin inferior to the medial canthus of the eye. viral infections. dogs have no specific viral infections affecting exclusively the nasal cavity or sinuses. acute rhinitis and sinusitis occurs as part of the canine infectious respiratory disease (cird) group caused by several distinct viruses, such as canine distemper virus, cav-1 and -2, canine parainfluenza virus, reovirus, and canine herpesvirus. the viral lesions in the respiratory tract are generally transient, but the effect of the virus on other tissues and cells can be fatal, as in distemper encephalitis in dogs. bacterial infections. as in other species, secondary bacterial rhinitis, sinusitis, and pneumonia are possible sequelae of respiratory viral infections; bordetella bronchiseptica, escherichia coli, and pasteurella multocida are the most common isolates in dogs with bacterial rhinitis. mycotic infections. aspergillus spp. and penicillium spp. cause mycotic rhinitis and sinusitis in dogs (canine nasal aspergillosis) ( fig. 9-34 ). nasal biopsies reveal extensive necrosis of the nasal epithelium and thick plaques of fibrinopurulent exudate mixed with many fungal hyphae. cryptococcus neoformans and blastomyces dermatitides infections of the nasal cavity occur sporadically in dogs ( fig. 9-35 ). lesions are characterized by mucosal granulomas containing periodic acid-schiff (pas)-positive fungal organisms, and the infection is clinically characterized by mucopurulent nasal discharge. fvr; these two viral infections account for 80% of all cases of feline respiratory diseases. a febrile systemic hemorrhagic syndrome with high mortality (up to 50%) has been reported in cats infected with virulent strains of fcv. feline chlamydiosis. feline chlamydiosis is a persistent respiratory infection of cats caused by chlamydophila felis. infection results in a conjunctivitis (similar to the conjunctivitis seen in human trachoma caused by chlamydia trachomatis) and serous or mucopurulent rhinitis. in the past, chlamydophila felis was incriminated as the agent responsible for "feline pneumonitis," but its role in causing bronchointerstitial pneumonia in cats has been seriously challenged in recent years (see pneumonias of cats). mycotic infections. the most common mycotic infection in the feline nasal cavity is caused by cryptococcus neoformans and cryptococcus gatti, but not all animals exposed to these fungi necessarily develop cryptococcosis unless they are immunosuppressed. stillbirths. clinical signs of fvr infection are characterized by lethargy, oculonasal discharge, severe rhinitis, and conjunctivitis. feline calicivirus. feline rhinitis can be caused by different strains of feline calicivirus (fcv). it is an important infection of the respiratory tract of cats, and depending on the virulence of the strain, lesions vary from a mild oculonasal discharge to severe rhinitis, mucopurulent conjunctivitis, and ulcerative gingivitis and stomatitis. the lesions, in addition to rhinitis and conjunctivitis, include acute, diffuse interstitial pneumonia with necrotizing bronchiolitis (see pneumonias of cats) and in some cases prominent ulcers of the tongue and hard palate. primary viral lesions are generally transient, but secondary bacterial infections (bordetella bronchiseptica, pasteurella multocida, or escherichia coli) are a common complication. some kittens develop lameness after infection or vaccination with calicivirus because of an acute and self-limiting arthritis ("limping kitten syndrome"). carrier state and virus shedding from oronasal secretions and feces are natural sequelae after recovery from the acute phase of the disease. clinical and pathologic features of fcv disease are strikingly similar but not identical to those of hemorrhage, increased lacrimation as a result of obstruction of nasolacrimal ducts, and sneezing. in some instances, it is not possible to clinically or grossly differentiate neoplasms from hyperplastic nodules or granulomatous rhinitis. some neoplasms may infiltrate adjacent bone structures and produce notable facial deformities, loss of teeth, exophthalmus, and nervous signs. large neoplasms also project into the meatuses, narrow the lumen, and interfere with airflow, causing stertorous breathing (see . biopsies, as well as brush and imprint cytology, have proven effective in the antemortem diagnosis of nasal neoplasms, particularly in those of epithelial lineage. a unique group of nasal carcinomas (enzootic nasal tumors, enzootic intranasal tumors, and enzootic nasal carcinoma) of sheep and goats arise from the surface epithelium and glands of the ethmoidal conchae. these types of carcinomas are caused by betaretroviruses in sheep (entv-1) and goats (entv-2). the enzootic nasal tumor has been successfully transmitted to susceptible animals by the lesions vary from discrete nasal granulomas to large confluent masses of mucopurulent exudate filling the entire nasal cavity and paranasal sinuses. microscopic examination of the exudate reveals the typical thick-walled pas-positive organisms (see fig. 9 -35). mycoplasma felis can also cause mucopurulent conjunctivitis and a mild upper respiratory infection, with clinical signs and lesions overlapping those seen with chlamydiosis, fvr, and fcr infections. respiratory infections and bronchopneumonia in cats may also be associated with the immunosuppressive effects of feline retroviruses such as feline leukemia virus (felv) and feline immunodeficiency virus (fiv). nasal aspergillosis and allergic rhinosinusitis are sporadically reported in cats (see disorders of the conducting system: species-specific diseases of the nasal cavity and paranasal sinuses: disorders of dogs: mycotic infections). neoplasms of the nasal cavity and paranasal sinuses may arise from any of the tissues forming these structures, including bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), connective tissue (fibroma or fibrosarcoma, myxoma or myxosarcoma), and blood vessels (hemangioma or hemangiosarcoma), and from all the different types of cells of glands and lining epithelium (adenoma, carcinoma, or adenocarcinoma). nasal tumors originating from stromal tissues, such as bone, cartilage, and connective tissue, are morphologically indistinguishable from those seen in other sites. in general, nasal neoplasms are rare in domestic animals, except for enzootic ethmoidal tumor (retroviral) in sheep and goats, which can occur in several animals in a herd (see the next section). in companion animals, nasal neoplasms are most common in dogs, particularly in medium to large breed dogs such as the collie, airedale terrier, basset hound, and german shepherd. the cat and the horse are less frequently affected. the main sites in order of frequency are the nasal passages and sinuses for dogs, the tip of the nose and nasal passages for cats, and the maxillary sinus and nasal passages for horses. the majority of neoplasms in the nasal cavity are malignant. benign nasal neoplasms (papilloma and adenoma) are rare and generally are either solitary or multiple, well-delineated nodules. in contrast, nasal carcinomas and nasal sarcomas are generally larger but vary in size and are often pale and multilobulated masses composed of fleshy to friable tissue (figs. 9-36 and 9-37). malignant neoplasms are locally invasive and tend to infiltrate sinuses, meninges, frontal brain, olfactory nerves, and vessels resulting in epistaxis. carcinomas vary from anaplastic (poorly differentiated) to well differentiated, in which cell and tissue morphology retains some glandular (adenocarcinoma) or squamous cell patterns. because nasal tumors in dogs and cats are usually large and invasive at the time of diagnosis, prognosis is usually poor and survival times are short. sarcomas originating in the nasal cavity and paranasal sinuses are less common than carcinomas. mesenchymal tumors can arise from bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), blood vessels (hemangioma or hemangiosarcoma), and connective tissue (fibroma or fibrosarcoma). overall, benign epithelial and mesenchymal tumors are less common than their malignant counterparts. secondary tumors in the nasal cavity are rare, with lymphoma being the most common secondary tumor in the nasal cavity of domestic animals ( fig. 9-38 ). nasal neoplasms become secondarily infected by bacteria, and clinical signs often overlap with those of infectious rhinitis and include catarrhal or mucopurulent nasal discharge, periodic anomalies 3 congenital anomalies of the pharynx, guttural pouches, larynx, and trachea are rare in all species. depending on their location and severity, they may be inconsistent with postnatal life, pose little or no problem, interfere with quality of life, or manifest themselves in later life. if clinical signs of respiratory distress, such as stridor, coughing, dyspnea, or gagging, do occur, they are usually exacerbated by excitement, heat, stress, or exercise. brachycephalic airway syndrome. see disorders of the conducting system: species-specific diseases of the pharynx, guttural inoculation of cell-free tumor filtrates. enzootic nasal tumors are typically invasive but do not metastasize ( fig. 9 -39). in some regions of the world, ethmoid tumors have been reported in horses and pigs, particularly in those farms where the endemic nasal tumors of ruminants are known to occur. nonneoplastic exophytic masses that resemble neoplasms are commonly found in horses, cats, and, to a lesser extent, other species. in horses, polyps tend to form in the ethmoidal region, whereas in cats, polyps are most frequently found in the nasopharynx and eustachian tubes. the pathogenesis of these benign growths is uncertain, although in many cases they follow chronic rhinitis or sinusitis. most recently, lymphatic obstruction secondary to inflammation has been postulated as the main culprit. grossly, polyps appear as firm, pedunculated nodules of various sizes protruding from the nasal mucosa into the nasal passages or nasopharynx ( fig. 9 -40); the surface may be smooth, ulcerated, secondarily infected, and hemorrhagic. microscopically, polyps are characterized by a core of wellvascularized stromal tissue that contains inflammatory cells and are covered by pseudostratified or squamous epithelium (see fig. 9 -40). nasal and paranasal sinus cysts are common idiopathic lesions in horses and are medically important because they clinically mimic table 1 -1 for potential, suspected, or known genetic disorders. tracheal collapse and tracheal stenosis. tracheal collapse with reduction in tracheal patency occurs in toy, miniature, and brachycephalic breeds of dogs, in which the condition is also called tracheobronchial collapse or central airway collapse. the defect also occurs in horses, cattle, and goats. by radiographic, endoscopic, or gross examination, there is dorsoventral flattening of the trachea with concomitant widening of the dorsal tracheal membrane, which may then prolapse ventrally into the lumen ( fig. 9-41 ). most commonly, the defect extends the entire length of the trachea and only rarely affects the cervical portion alone. affected segments with a reduced lumen contain froth and even are covered by a diphtheritic membrane. in horses, the so-called scabbard trachea is characterized allergic reactions. grossly, the mucosa of the epiglottis and vocal cords is thickened and swollen, often protrudes dorsally onto the epiglottic orifice, and has a gelatinous appearance ( fig. 9-43 ). laryngeal and tracheal hemorrhage. hemorrhages in these sites occur as mucosal petechiae and are most commonly seen in coagulopathies; inflammation; septicemia and sepsis, particularly in pigs with classical swine fever (hog cholera); african swine fever or salmonellosis; and horses with equine infectious anemia. severe dyspnea and asphyxia before death can cause congestion, ecchymosis, and petechiae in the laryngeal and tracheal mucosa; this lesion must be differentiated from postmortem imbibition of hemoglobin in autolyzed carcasses (see chapter 1). by lateral flattening so that the tracheal lumen is reduced to a narrow vertical slit. segmental tracheal collapse causing stenosis has been associated with congenital and acquired abnormalities. in severe cases, abnormal cartilaginous glycoproteins and loss of elasticity of tracheal rings causes the trachea to collapse. in some other cases, it is an acquired tracheal lesion that follows trauma, compression caused by extraluminal masses, peritracheal inflammation, and flawed tracheotomy or transtracheal aspirate techniques. other tracheal anomalies include tracheoesophageal fistula, which is most commonly found in human beings and sporadically in dogs and cattle. congenital fistulas can occur at any site of the cervical or thoracic segments of the trachea. acquired tracheoesophageal fistula can be a complication of improper intubation, tracheotomy, or esophageal foreign body. laryngeal hemiplegia. laryngeal hemiplegia (paralysis), sometimes called roaring in horses, is a common but obscure disease characterized by atrophy of the dorsal and lateral cricoarytenoid muscles (abductor and adductor of the arytenoid cartilage), particularly on the left side. muscular atrophy is most commonly caused by a primary denervation (recurrent laryngeal neuropathy) of unknown cause (idiopathic axonopathy) and, to a much lesser extent, secondary nerve damage (see the section on denervation atrophy in chapters 14 and 15). idiopathic laryngeal hemiplegia is an incurable axonal disease (axonopathy) of the cranial laryngeal nerve that affects mostly larger horses. secondary laryngeal hemiplegia is rare and occurs after nerve damage caused by other pathologic processes such as compression or inflammation of the left recurrent laryngeal nerve. the medial retropharyngeal lymph nodes are located immediately ventral to the floor of the guttural pouches. as a result of this close anatomic relationship, swelling or inflammation of the guttural pouches or retropharyngeal lymph nodes often results in secondary damage to the laryngeal nerve. common causes of secondary nerve damage (wallerian degeneration) include guttural pouch mycosis, retropharyngeal abscesses, inflammation because of iatrogenic injection into the nerves, neck injury, and metastatic neoplasms involving the retropharyngeal lymph nodes (e.g., lymphosarcoma). grossly, the affected laryngeal muscle in a horse with laryngeal hemiplegia is pale and smaller than normal (muscle atrophy) . microscopically, muscle fibers have lesions of denervation atrophy (see chapters 14 and 15). atrophy of laryngeal muscles also occurs in dogs as an inherited condition (siberian husky and bouvier des flanders), as a degenerative neuropathy in older dogs, secondary to laryngeal trauma in all species (e.g., choke chain damage), or secondary to hepatic encephalopathy in horses. the abnormal inspiratory sounds (roaring) during exercise in horses with laryngeal hemiplegia are caused by paralysis of the left dorsal and lateral cricoarytenoid muscles, which cause incomplete dilation of the larynx, obstruction of airflow, and vibration of vocal cords. laryngeal edema. laryngeal edema is a common feature of acute inflammation, but it is particularly important because swelling of the epiglottis and vocal cords can obstruct the laryngeal orifice, resulting in asphyxiation. laryngeal edema occurs in pigs with edema disease; in horses with purpura hemorrhagica; in cattle with acute interstitial pneumonia; in cats with systemic anaphylaxis; and in all species as a result of trauma, improper endotracheal tubing, inhalation of irritant gases (e.g., smoke), local inflammation, and animal species is classified as fibrinous, catarrhal, purulent, or granulomatous (figs. 9-44 and 9-45). chronic polypoid tracheitis occurs in dogs and cats, probably secondary to chronic infection. the most common causes of tracheitis are viral infections, such as those causing infectious bovine rhinotracheitis (see fig. 9 -29), equine viral rhinopneumonitis, canine distemper, and feline rhinotracheitis. viral lesions are generally mild and transient but often become complicated with secondary bacterial infections. at the early stages, the mucosa is notably hyperemic and can show white foci of necrosis. in the most severe cases, the affected mucosa detaches from the underlying basement membrane, causing extensive tracheal ulceration. chemical tracheitis is also commonly seen after aspiration (see fig. 9 -45). also, inhalation of fumes during barn fires can cause extensive injury and necrosis of the tracheal mucosa. in forensic cases, the presence of carbon pigment in the mucosal surface of trachea, bronchi, and bronchioles indicates that the burned animal was alive during the fire. parasitic infections of the larynx and trachea can cause obstruction with dramatic consequences, but burdens sufficient to cause such effects are not commonly seen in veterinary practice. besnoitiosis (besnoitia spp.) . besnoitiosis (besnoitia spp.) is caused by several species of this apicomplexan coccidian parasite, whose life cycle is still unknown. this parasite can cause pedunculated lesions on the skin, sclera, mucosa of the nasal cavity, and larynx of horses and donkeys, cattle, goats, and wild animals. besnoitiosis has been reported from africa, central and south america, north america, and europe. grossly, pale, round, exophytic nodules up to 2 cm in diameter can be observed protruding from mucosal surfaces. microscopically, these nodules consist of finger-like projections covered by hyperplastic and sometimes ulcerated epithelium containing numerous thick-walled parasitic cysts with little inflammatory response. cattle. see disorders of cattle. inflammation of the pharynx, larynx, and trachea are important because of their potential to obstruct airflow and to lead to aspiration pneumonia. the pharynx is commonly affected by infectious diseases of the upper respiratory and upper digestive tracts, and the trachea can be involved by extension from both the lungs and larynx. pharyngeal obstruction and perforation. intraluminal foreign bodies in the pharynx, such as medicament boluses, apples, or potatoes, can move down and obstruct the larynx and trachea. also, pharyngeal obstruction can be caused by masses in the surrounding tissue, such as neoplasms of the thyroid gland, thymus, and parathyroid glands. a number of nonspecific insults can cause lesions and clinical signs. trauma may take the form of penetrating wounds in any species: perforation of the caudodorsal wall of the pharynx from the improper use of drenching or balling guns in sheep, cattle, and pigs; choking injury because of the use of collars in dogs and cats; and the shearing forces of bite wounds. the results of the trauma may be minimal (local edema and inflammation) or as serious as complete luminal obstruction by exudate. foreign bodies may be lodged anywhere in the pharyngeal region; the location and size determine the occurrence of dysphagia, regurgitation, dyspnea, or asphyxiation. pigs have a unique structure known as the pharyngeal diverticulum (4 cm long in adult pigs), which is located in the pharyngeal wall rostral and dorsal to the esophageal entrance. it is important because barley awns may lodge in the diverticulum, causing an inflammatory swelling that affects swallowing. the diverticular wall may be perforated by awns or drenching syringes, which results in an exudate that can extend down the tissue planes between muscles of the neck and even into the mediastinum. the pharynx of the dog may also be damaged by trauma from chicken bones, sticks, and needles, resulting in the formation of a pharyngeal abscess. equine inflammation of the trachea (tracheitis). the types of injury and host inflammatory responses in the trachea are essentially the same as those described for the nasal mucosa. although tracheal mucosa is prone to aerogenous injury and necrosis, it has a remarkable capacity for repair. according to the exudate, tracheitis in all palate, are important causes of respiratory problems and reduced athletic performance in horses. an undersized epiglottis is prone to being entrapped below the arytenoepiglottic fold, causing an equine syndrome known as epiglottic entrapment. this syndrome also occurs in horses with lateral deviation and deformity of epiglottis, epiglottic cysts, or necrosis of the tip of the epiglottis. hypoplastic epiglottis also occurs in pigs. dorsal displacement of the soft palate, particularly during exercise, narrows the lumen of the nasopharynx and creates abnormal air turbulence in the conducting system of horses. epiglottic entrapment is clinically characterized by airway obstruction, exercise intolerance, respiratory noise, and cough. subepiglottic and pharyngeal cysts. anomalous lesions, such as subepiglottic and pharyngeal cysts, are occasionally seen in horses, particularly in standardbred and thoroughbred racehorses. these cysts vary in size (1 to 9 cm) and occur most commonly in the subepiglottic area and to a lesser extent in the dorsal pharynx, larynx, and soft palate. cysts are lined by squamous or pseudostratified epithelium and contain thick mucus. large cysts cause airway obstruction, reduced exercise tolerance, or dysphagia and predispose to bronchoaspiration of food. equine pharyngeal lymphoid hyperplasia. equine pharyngeal lymphoid hyperplasia, or pharyngitis with lymphoid follicular hyperplasia, is a common cause of partial upper airway obstruction in horses, particularly in 2-and 3-year-old racehorses. lymphoid hyperplasia is also seen in healthy horses as part of a response to mild chronic pharyngitis, which in many instances tends to regress with age in older animals. the cause is undetermined, but chronic bacterial infection combined with environmental factors may cause excessive antigenic stimulation and lymphoid hyperplasia. the gross lesions, visible endoscopically or at necropsy, consist of variably sized (1 to 5 mm) white foci located on the dorsolateral walls of the pharynx and extending into the openings of the guttural pouches and onto the soft palate. in severe cases, lesions may appear as pharyngeal polyps. microscopically, the lesions consist of large aggregates of lymphocytes and plasma cells in the pharyngeal mucosa. clinical signs consist of stertorous inspiration, expiration, or both. inflammation of guttural pouches. the guttural pouches of horses are large diverticula (300 to 500 ml) of the ventral portion of the auditory (eustachian) tubes. these diverticula are therefore exposed to the same pathogens as the pharynx and have drainage problems similar to the sinuses. although it is probable that various pathogens, including viruses, can infect them, the most common pathogens are fungi, which cause guttural pouch mycosis and guttural pouch empyema in the horse. eustachitis is the term used for inflammatory processes involving the eustachian (pharyngotympanic) tube. because of the close anatomic proximity of guttural pouches to the internal carotid arteries, cranial nerves (vii, ix, x, xi, and xii), and atlantooccipital joint, disease of these diverticula may involve these structures and cause a variety of clinical signs in horses. guttural pouch mycosis occurs primarily in stabled horses and is caused by aspergillus fumigatus and other aspergillus spp. infection is usually unilateral and presumably starts with the inhalation of spores from moldy hay. grossly, the mucosal surfaces of the dorsal and lateral walls of the guttural pouch mucosa are first covered by focal, rounded, raised plaques of diphtheritic (fibrinonecrotic) exudate, which with time can become confluent and grow into a large fibrinonecrotic mass ( fig. 9-46) . microscopically, the lesions are severe necrotic inflammation of the mucosa and submucosa with widespread vasculitis and intralesional fungal hyphae. necrosis of the mammomonogamus (syngamus) spp. mammomonogamus (syngamus) laryngeus is a nematode that is seen attached to the laryngeal mucosa of cattle in tropical asia and south america, and cats (gapeworm: mammomonogamus ierei) in the caribbean and southern united states. occasionally, human beings with a persistent cough or asthma-like symptoms have the parasite in the larynx or bronchi. oslerus (filaroides) osleri. see disorders of dogs. b a c empyema of guttural pouches is a sequela to suppurative inflammation of the nasal cavities, most commonly from streptococcus equi infection (strangles). in severe cases, the entire guttural pouch can be filled with purulent exudate ( fig. 9 -47). the sequelae are similar to those of guttural pouch mycosis except that there is no erosion of the internal carotid artery. it is clinically characterized by nasal discharge, enlarged retropharyngeal lymph nodes, painful swelling of the parotid region, dysphagia, and respiratory distress. guttural pouch tympany develops sporadically in young horses when excessive air accumulates in the pouch from the one-way valve effect caused by inflammation or malformation of the eustachian tube. arabian and german warm-blooded horses are particularly susceptible to develop guttural pouch tympany. it is generally unilateral and characterized by nonpainful swelling of the parotid region. cattle. tracheal edema and hemorrhage syndrome of feeder cattle, also known as the honker syndrome or tracheal stenosis of feedlot cattle, is a poorly documented acute disease of unknown cause, most often seen during the summer months. severe edema and a few hemorrhages are present in the mucosa and submucosa of the dorsal surface of the trachea, extending caudally from the midcervical area as far as the tracheal bifurcation. on section, the tracheal mucosa is diffusely thickened and gelatinous. clinical signs include inspiratory wall of the guttural pouches can extend into the wall of the adjacent internal carotid artery causing hemorrhage into the lumen of the guttural pouch and recurrent epistaxis. invasion of the internal carotid artery causes arteritis, which can also lead to formation of an aneurysm and fatal bleeding into the guttural pouches. in other cases, the fungi may be angioinvasive, leading to the release of mycotic emboli into the internal carotid artery, generally resulting in multiple cerebral infarcts. dysphagia, another clinical sign seen in guttural pouch mycosis, is associated with damage to the pharyngeal branches of the vagus and glossopharyngeal nerves, which lie on the ventral aspect of the pouches. horner's syndrome results from damage to the cranial cervical ganglion and sympathetic fibers located in the caudodorsal aspect of the pouches. finally, equine laryngeal paralysis (hemiplegia) can result from damage to the laryngeal nerves as previously described in the section on laryngeal hemiplegia. pekingese, and others. the defects are a result of a mismatch of the ratio of soft tissue to cranial bone and the obstruction of airflow by excessive length of the palatine soft tissue. secondary changes, such as nasal and laryngeal edema caused by forceful inspiration, eventually lead to severe upper airway obstruction, respiratory distress, and exercise intolerance. tracheal hypoplasia. tracheal hypoplasia occurs most often in english bulldogs and boston terriers; the tracheal lumen is decreased in diameter throughout its length. canine infectious respiratory disease. canine infectious respiratory disease (cird), formerly called canine tracheobronchitis or kennel cough, is a highly contagious group of infectious diseases characterized clinically by an acute onset of coughing notably exacerbated by exercise. the term is nonspecific, much like the dyspnea that can progress to oral breathing, recumbency, and death by asphyxiation in less than 24 hours. necrotic laryngitis. necrotic laryngitis (calf diphtheria, laryngeal necrobacillosis) is a common disease of feedlot cattle and cattle affected with other diseases, with nutritional deficiencies, or housed under unsanitary conditions. it also occurs sporadically in sheep and pigs. necrotic laryngitis, caused by fusobacterium necrophorum, is part of the syndrome termed necrotic stomatitis or laryngeal necrobacillosis, which can include lesions of the tongue, cheeks, palate, and pharynx. an opportunistic pathogen, fusobacterium necrophorum produces potent exotoxins and endotoxins after gaining entry either through lesions of viral infections, such as ibr and vesicular stomatitis in cattle, or after traumatic injury produced by feed or careless use of specula or balling guns. the gross lesions, regardless of location in the mouth or larynx (most common in the mucosa overlying the laryngeal cartilages), consist of well-demarcated, dry, yellow-gray, thick-crusted, and fibrinonecrotic exudate ( fig. 9 -48) that in the early stages is bounded by a zone of active hyperemia. deep ulceration develops, and if the lesion does not result in death, healing is by granulation tissue formation. microscopically, the necrotic foci are first surrounded by congested borders, then by a band of leukocytes, and finally the ulcers heal by granulation tissue and collagen (fibrosis). the lesions can extend deep into the submucosal tissue. numerous bacteria are evident at the advancing edge. there are numerous and important sequelae to calf diphtheria; the most serious is death from severe toxemia or overwhelming fusobacteremia. sometimes, the exudate may be copious enough to cause laryngeal obstruction and asphyxiation or be aspirated and cause bronchopneumonia. the clinical signs of necrotic laryngitis are fever, anorexia, depression, halitosis, moist painful cough, dysphagia, and inspiratory dyspnea and ventilatory failure because of fatigue of the respiratory muscles (diaphragmatic and intercostal). laryngeal contact ulcers. ulcerative lesions in the larynx are commonly found in feedlot cattle. grossly, the laryngeal mucosa reveals circular ulcers (up to 1 cm in diameter), which may be unilateral or bilateral and sometimes deep enough to expose the underlying arytenoid cartilages. the cause has not been established, but causal agents, such as viral, bacterial, and traumatic, have been proposed, along with increased frequency and rate of closure of the larynx (excessive swallowing and vocalization) when cattle are exposed to market and feedlot stresses such as dust, pathogens, and interruption of feeding. contact ulcers predispose a calf to diphtheria (fusobacterium necrophorum) and laryngeal papillomas. ulceration of the mucosa and necrosis of the laryngeal cartilages have also been described in calves, sheep, and horses under the term laryngeal chondritis. laryngeal abscesses involving the mucosa and underlying cartilage occur as a herd or flock problem in calves and sheep, presumably caused by a secondary infection with trueperella (arcanobacterium) pyogenes. anomalies 5 brachycephalic airway syndrome. brachycephalic airway syndrome is a clinical term that refers to increased airflow resistance caused by stenotic nostrils and nasal meatuses and an excessively long soft palate. these abnormalities are present in brachycephalic canine breeds such as bulldogs, boxers, boston terriers, pugs, a "common cold" in human beings or bovine respiratory disease complex (brdc) in cattle. the infection occurs commonly as a result of mixing dogs from different origins such as occurs at commercial kennels, animal shelters, and veterinary clinics. between bouts of coughing, most animals appear normal, although some have rhinitis, pharyngitis, tonsillitis, or conjunctivitis; some with secondary pneumonia become quite ill. the pathogenesis of cird is complex, and many pathogens and environmental factors have been incriminated. bordetella bronchiseptica, canine adenovirus-2 (cav-2), and canine parainfluenza virus-2 (cpiv-2) are most commonly implicated. the severity of the disease is increased when more than one agent is involved or if there are extreme environmental conditions (e.g., poor ventilation). for example, dogs asymptomatically infected with bordetella bronchiseptica are more severely affected by superinfection with cav-2 than those not carrying the bacterium. other agents are sometimes isolated but of lesser significance and include canine adenovirus-1 (cav-1: infectious canine hepatitis virus), reovirus type 1, canid herpesvirus-1 (cahv-1), canine respiratory coronavirus (crcov), and mycoplasma species. depending on the agents involved, gross and microscopic lesions are completely absent or they vary from catarrhal to mucopurulent tracheobronchitis, with enlargement of the tonsils and retropharyngeal and tracheobronchial lymph nodes. in dogs with bordetella bronchiseptica infection, the lesions are suppurative or mucopurulent rhinitis and tracheobronchitis, and suppurative bronchiolitis. in contrast, when lesions are purely viral, microscopic changes are focal necrosis of the tracheobronchial epithelium. sequelae can include spread either proximally or distally in the respiratory tract, the latter sometimes inducing chronic bronchitis and bronchopneumonia. oslerus (filaroides) osleri. oslerus (filaroides) osleri is a nematode parasite of dogs and other canidae that causes characteristic protruding nodules into the lumen at the tracheal bifurcation. they are readily seen on endoscopic examination or at necropsy. in severe cases, these nodules can extend 5 cm cranially or caudally from the tracheal bifurcation and even into primary and secondary bronchi. the disease occurs worldwide, and oslerus osleri is considered the most common respiratory nematode of dogs. the gross lesions are variably sized, up to 1 cm, submucosal nodules that extend up to 1 cm into the tracheal lumen ( fig. 9 -49, a). microscopically, nodules contain adult parasites with a mild mononuclear cell reaction; with the death of the parasite, an intense foreign body reaction develops with neutrophils and giant cells b) . clinically, it can be asymptomatic, although it most often causes a chronic cough that can be exacerbated by exercise or excitement. severe infestations can result in dyspnea, exercise intolerance, cyanosis, emaciation, and even death in young dogs. neoplasms of the guttural pouches occur rarely in horses and are usually squamous cell carcinomas. laryngeal neoplasms are rare in dogs and extremely so in other species, although they have been reported in cats and horses. the most common laryngeal neoplasms in dogs are papillomas and squamous cell carcinomas. other less common tumors are laryngeal rhabdomyoma, previously referred to as laryngeal oncocytoma, and chondromas and osteochondromas. lymphoma involving the laryngeal tissue is sporadically seen in cats. when large enough to be obstructive, neoplasms may cause a change or loss of voice, cough, or respiratory distress with cyanosis, collapse, and syncope. other signs include dysphagia, anorexia, and exercise intolerance. the neoplasm is sometimes visible from the oral cavity and causes swelling of the neck. the prognosis is poor because most lesions recur after excision. tracheal neoplasms are even more uncommon than those of the larynx. the tracheal cartilage or mucosa can be the site of an osteochondroma, leiomyoma, osteosarcoma, mast cell tumor, and carcinoma. lymphoma in cats can extend from the mediastinum to involve the trachea. each lung is subdivided into various numbers of pulmonary lobes (see fig. 9 -16). in the past, these were defined by anatomic fissures. however, in current anatomy, lobes are defined by the ramification of the bronchial tree. following this criterion, the left lung of all domestic species is composed of cranial and caudal lobes, whereas the right lung, depending on species, is composed of cranial, middle (absent in horse), caudal, and accessory lobes. each pulmonary lobe is further subdivided by connective tissue into pulmonary lobules, which in some species (cattle and pigs) are rather prominent and in others are much less conspicuous. from a practical standpoint, identification of the lungs among different species could be achieved by carefully observing the degree of lobation (external fissures) and the degree of lobulation (connective tissue between lobules). cattle and pigs have well-lobated and well-lobulated lungs; sheep and goats have well-lobated but poorly lobulated lungs; horses have both poorly lobated and poorly lobulated lungs and resemble human lungs; finally, dogs and cats have well-lobated but not well-lobulated lungs. the degree of lobulation determines the degree of air movement between the lobules. in pigs and cattle, movement of air between lobules is practically absent because of the thick connective tissue of the interlobular septa separating individual lobules. this movement of air between lobules and between adjacent alveoli (via the pores of kohn) constitutes what is referred to as collateral ventilation. this collateral ventilation is poor in cattle and pigs and good in dogs. the functional implications of collateral ventilation are discussed in the section on pulmonary emphysema. the lungs have an interconnecting network of interstitial stromal tissue supporting the blood and lymphatic vessels, nerves, bronchi, bronchioles, and alveoli. for purposes of simplicity, the pulmonary interstitium can be anatomically divided into three contiguous compartments: (1) bronchovascular interstitium, where main bronchi and pulmonary vessels are situated; (2) interlobular interstitium separating pulmonary lobules and supporting small blood and lymph vessels; and (3) alveolar interstitium supporting the alveolar walls that contain pulmonary capillaries and alveolar epithelial cells (no lymphatic vessels here) (see discussion on the blood-air barrier in the section on alveoli). pulmonary changes, such as edema, emphysema, and inflammation, may affect one or more of these interstitial compartments. anomalies 6 congenital anomalies of the lungs are rare in all species but are most commonly reported in cattle and sheep. compatibility with life largely depends on the type of structures involved and the proportion of functional tissue present at birth. accessory lungs are one of the most common anomalies and consist of distinctively lobulated masses of incompletely differentiated pulmonary tissue present in the thorax, abdominal cavity, or subcutaneous tissue virtually anywhere in the trunk. large accessory lungs can cause dystocia. ciliary dyskinesia (immotile cilia syndrome, kartagener's syndrome) is characterized by defective ciliary movement, which results in reduced mucociliary clearance because of a defect in the microtubules of all ciliated cells and, most important, in the ciliated respiratory epithelium and spermatozoa. primary ciliary dyskinesia often associated with situs inversus has been reported in dogs, which as a result usually have chronic recurrent rhinosinusitis, pneumonia, and infertility. pulmonary agenesis, pulmonary hypoplasia, abnormal lobulation, congenital emphysema, lung hamartoma, and congenital bronchiectasis are occasionally seen in domestic animals. congenital melanosis is a common incidental finding in pigs and ruminants and is usually seen at slaughter (fig. 9-50 ). it is characterized by black spots, often a few centimeters in diameter, in various organs, mainly the lungs, meninges, intima of the aorta, and caruncles of the uterus. melanosis has no clinical significance, and the texture of pigmented lungs remains unchanged. congenital emphysema is sporadically seen in dogs (efig. 9 -4). pulmonary calcification ("calcinosis"). calcification of the lungs occurs in some hypercalcemic states, generally secondary to hypervitaminosis d or from ingestion of toxic (hypercalcemic) plants, such as solanum malacoxylon (manchester wasting disease), that contain vitamin d analogs. it is also a common sequela to uremia and hyperadrenocorticism in dogs and to pulmonary necrosis (dystrophic calcification) in most species. calcified lungs may fail to collapse when the thoracic cavity is opened and have a characteristic "gritty" texture ( fig. 9-51) . microscopically, lesions vary from calcification of the alveolar basement membranes (see by pathologists. recent investigations suggest that excessive lipid originates from the breakdown products of neoplastic cells. bronchial and bronchiolar obstructions such as those caused by lungworms can also cause alveolar lipidosis. the pathogenesis relates to the inability of alveolar macrophages that normally remove part of the surfactant lipids to exit the lung via the mucociliary escalator. exogenous lipid pneumonia. another form of lipid pneumonia occurs accidentally in cats or horses given mineral oil by their owners in an attempt to remove hairballs or treat colic (aspiration pneumonia). to achieve gaseous exchange, a balanced ratio of the volumes of air to capillary blood must be present in the lungs (ventilation/perfusion ratio), and the air and capillary blood must be in close proximity across the alveolar wall. a ventilation-perfusion mismatch occurs if pulmonary tissue is either collapsed (atelectasis) or overinflated (hyperinflation and emphysema). the term atelectasis means incomplete distention of alveoli and is used to describe lungs that have failed to expand with air at the time of birth (congenital or neonatal atelectasis) or lungs that have collapsed after inflation has taken place (acquired atelectasis or alveolar collapse) (figs. 9-52 and 9-53). during fetal life, lungs are not fully distended, contain no air, and are partially filled with a locally produced fluid known as fetal lung fluid. not surprisingly, lungs of aborted and stillborn fetuses sink when placed in water, whereas those from animals that have breathed float. at the time of birth, fetal lung fluid is rapidly reabsorbed and replaced by inspired air, leading to the normal distention of alveoli. congenital atelectasis occurs in newborns that fail to inflate their lungs after taking their first few breaths of air; it is caused by obstruction of airways, often as a result of aspiration of amniotic fluid and meconium (described in the section on meconium aspiration syndrome) (see fig. 9 -52). congenital atelectasis also develops when alveoli cannot remain distended after initial aeration because of an alteration in quality and quantity of pulmonary surfactant produced by type ii pneumonocytes and club (clara) cells. this infant form of congenital atelectasis is referred to in human neonatology as infant respiratory distress syndrome (irds) or as hyaline membrane disease because of the clinical and microscopic features of the disease. it commonly occurs in babies who are premature or born to diabetic or alcoholic mothers and is occasionally found in animals, particularly foals and piglets. the pathetic, gasping attempts of affected foals and pigs to breathe have prompted the use of the name "barkers"; foals that survive may have brain damage from cerebral hypoxia (see chapter 14) and are referred to as "wanderers" due to their aimless behavior and lack of a normal sense of fear. acquired atelectasis is much more common and occurs in two main forms: compressive and obstructive (see fig. 9 -53). compressive atelectasis has two main causes: space-occupying masses in the pleural cavity, such as abscesses and tumors, or transferred pressures, such as that caused by bloat, hydrothorax, hemothorax, chylothorax, and empyema ( fig. 9-54 ). another form of compressive atelectasis occurs when the negative pressure in the thoracic cavity is lost because of pneumothorax. this form generally has massive atelectasis and thus is also referred to as lung collapse. obstructive (absorption) atelectasis occurs when there is a reduction in the diameter of the airways caused by mucosal edema and inflammation, or when the lumen of the airway is blocked by fig. 9 -51) to heterotopic ossification of the lungs (efig. 9 -5). in most cases, pulmonary calcification in itself has little clinical significance, although its cause (e.g., uremia or vitamin d toxicosis) may be very important. alveolar filling disorders are a heterogeneous group of lung diseases characterized by accumulation of various chemical compounds in the alveolar lumens. the most common are alveolar proteinosis, in which the alveoli are filled with finely granular eosinophilic material; pulmonary lipidosis, in which alveoli are filled with macrophages containing endogenous or exogenous lipid; and alveolar microlithiasis, in which the alveoli contain numerous concentric calcified "microliths" or "calcospherites." a similar but distinct concretion is known as corpora amylacea, which is an accumulation of laminated bodies composed of cellular debris, lipids, proteins, and possibly amyloid. for most alveolar filling disorders, there is little host response, and in many cases, it is an incidental finding. most of the alveolar filling disorders originate from inherited metabolic defects in which alveolar cells (epithelial or macrophages) cannot properly metabolize or remove lipids or proteins, whereas others result from an excessive synthesis of these substances in the lung. endogenous lipid (lipoid) pneumonia. endogenous lipid pneumonia is an obscure, subclinical pulmonary disease of cats and occasionally of dogs, which is unrelated to aspiration of foreign material. although the pathogenesis is not understood, it is presumed that lipids from pulmonary surfactant and from degenerated cells accumulate within alveolar macrophages. accumulation of surfactant lipids can occur in metabolic abnormalities of alveolar macrophages or in bronchial obstruction where surfactant-laden macrophages cannot exit the lungs via the mucociliary escalator. the gross lesions are multifocal, white, firm nodules scattered throughout the lungs (efig. 9-6) . microscopically, the alveoli are filled with foamy lipid-laden macrophages accompanied by interstitial infiltration of lymphocytes and plasma cells, fibrosis, alveolar epithelialization, and, in some cases, cholesterol clefts and lipid granulomas. lipid (lipoid) pneumonia occurs frequently in the vicinity of cancerous lung lesions in human beings, cats, and dogs. the reason for this association remains unknown and frequently unrecognized appearance of atelectasis is more common in species with poor collateral ventilation, such as cattle and pigs. the extent and location of obstructive atelectasis depends largely on the size of the affected airway (large vs. small) and on the degree of obstruction (partial vs. complete). atelectasis also occurs when large animals are kept recumbent for prolonged periods, such as during anesthesia (hypostatic atelectasis). the factors contributing to hypostatic atelectasis are a combination of blood-air imbalance, shallow breathing, airway obstruction because of mucus and fluid that has not been drained from bronchioles and alveoli, and from inadequate local production of surfactant. atelectasis can also be a sequel to paralysis of respiratory muscles and prolonged use of mechanical ventilation or general anesthesia in intensive care. in general, the lungs with atelectasis appear depressed below the surface of the normally inflated lung. the color is generally dark blue, and the texture is flabby or firm; they are firm if there is concurrent edema or other processes, such as can occur in ards or "shock" lungs (see the section on pulmonary edema). distribution and extent vary with the process, being patchy (multifocal) in congenital atelectasis, lobular in the obstructive type, and of various degrees in between in the compressive type. microscopically, the alveoli are collapsed or slitlike and the alveolar walls appear parallel and close together, giving prominence to the interstitial tissue even without any superimposed inflammation. pulmonary emphysema. pulmonary emphysema, often simply referred to as emphysema, is an extremely important primary disease in human beings, whereas in animals, it is always a secondary condition resulting from a variety of pulmonary lesions. in human medicine, emphysema is strictly defined as an abnormal permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of alveolar walls (alveolar emphysema). this definition separates it from simple airspace enlargement or hyperinflation, in which there is no destruction of alveolar walls and which can occur congenitally (down syndrome) or be acquired with age (aging lung, sometimes misnamed "senile emphysema"). the pathogenesis of emphysema in human beings is still controversial, but current thinking overwhelmingly suggests that destruction of mucus plugs, exudate, aspirated foreign material, or lungworms (see fig. 9 -53). when the obstruction is complete, trapped air in the lung eventually becomes reabsorbed. unlike the compression type, obstructive atelectasis often has a lobular pattern as a result of blockage of the airway supplying that lobule. this lobular lungs are extremely well-vascularized organs with a dual circulation provided by pulmonary and bronchial arteries. disturbances in pulmonary circulation have a notable effect on gaseous exchange, which may result in life-threatening hypoxemia and acidosis. in addition, circulatory disturbances in the lungs can have an impact on other organs, such as the heart and liver. for example, impeded blood flow in the lungs because of chronic pulmonary disease results in cor pulmonale, which is caused by unremitting pulmonary hypertension followed by cardiac dilation, right heart failure, chronic passive congestion of the liver (nutmeg liver), and generalized edema (anasarca). hyperemia is an active process that is part of acute inflammation, whereas congestion is the passive process resulting from decreased outflow of venous blood, as occurs in congestive heart failure ( fig. 9-56 ). in the early acute stages of pneumonia, the lungs appear notably red, and microscopically, blood vessels and alveolar capillaries are engorged with blood from alveolar walls is largely the result of an imbalance between proteases released by phagocytes and antiproteases produced in the lung as a defense mechanism (the protease-antiprotease theory). the destructive process in human beings is markedly accelerated by defects in the synthesis of antiproteases or any factor, such as cigarette smoking or pollution, that increases the recruitment of macrophages and leukocytes in the lungs. this theory originated when it was found that human beings with homozygous α 1 -antitrypsin deficiency were remarkably susceptible to emphysema and that proteases (elastase) inoculated intratracheally into the lungs of laboratory animals produced lesions similar to those found in the disease. more than 90% of the problem relates to cigarette smoking, and airway obstruction is no longer considered to play a major role in the pathogenesis of emphysema in human beings. primary emphysema does not occur in animals, and thus no animal disease should be called simply emphysema. in animals, this lesion is always secondary to obstruction of outflow of air or is agonal at slaughter. secondary pulmonary emphysema occurs frequently in animals with bronchopneumonia, in which exudate plugging bronchi and bronchioles causes an airflow imbalance where the volume of air entering exceeds the volume leaving the lung. this airflow imbalance is often promoted by the so-called one-way valve effect caused by the exudate, which allows air into the lung during inspiration but prevents movement of air out of the lung during expiration. depending on the localization in the lung, emphysema can be classified as alveolar or interstitial. alveolar emphysema characterized by distention and rupture of the alveolar walls, forming variably sized air bubbles in pulmonary parenchyma, occurs in all species. interstitial emphysema occurs mainly in cattle, presumably because of their wide interlobular septa, and lack of collateral ventilation in these species does not permit air to move freely into adjacent pulmonary lobules. as a result, accumulated air penetrates the alveolar and bronchiolar walls and forces its way into the interlobular connective tissue, causing notable distention of the interlobular septa. it is also suspected that forced respiratory movements predispose to interstitial emphysema when air at high pressure breaks into the loose connective tissue of the interlobular septa ( fig. 9-55 ). sometimes these bubbles of trapped air in alveolar or interstitial emphysema become confluent, forming large (several centimeters in diameter) pockets of air that are referred to as bullae (singular: bulla) (see efig. 9 -4); the lesion is then called bullous emphysema. this lesion is not a specific type of emphysema and does not indicate a different disease process but, rather, is a larger accumulation of air at one focus. in the most severe cases, air moves from the interlobular septa into the connective tissue surrounding the main stem bronchi and major vessels (bronchovascular bundles), and from here it leaks into the mediastinum, causing pneumomediastinum first, and eventually exits via the thoracic inlet into the cervical and thoracic subcutaneous tissue causing subcutaneous emphysema. note that mild and even moderate alveolar emphysema is difficult to judge at necropsy and by light microscopy unless special techniques are used to prevent collapse of the lung when the thorax is opened. these techniques include plugging of the trachea or intratracheal perfusion of fixative (10% neutral-buffered formalin) before the thorax is opened to prevent collapse of the lungs. important diseases that cause secondary pulmonary emphysema in animals include small airway obstruction (e.g., heaves) in horses and pulmonary edema and emphysema (fog fever) in cattle (see fig. 9 -55) and exudates in bronchopneumonia. congenital emphysema occurring secondary to bronchial cartilage hypoplasia with subsequent bronchial collapse is occasionally reported in dogs. severe and persistent cases of heart failure, the lungs fail to collapse because of edema and pulmonary fibrosis. terminal pulmonary congestion (acute) is frequently seen in animals euthanized with barbiturates and should not be mistaken for an antemortem lesion. hypostatic congestion is another form of pulmonary congestion that results from the effects of gravity and poor circulation on a highly vascularized tissue, such as the lung. this type of gravitational congestion is characterized by the increase of blood in the lower side of the lung, particularly the lower lung of animals in lateral recumbency, and is most notable in horses and cattle. the affected portions of the lung appear dark red and can have a firmer texture. in animals and human beings who have been prostrated for extended periods of time, hypostatic congestion may be followed by hypostatic edema, and hypostatic pneumonia as edema interferes locally with the bacterial defense mechanisms. pulmonary hemorrhage. pulmonary hemorrhages can occur as a result of trauma, coagulopathies, and disseminated intravascular coagulation (dic), vasculitis, sepsis, and pulmonary thromboembolism from jugular thrombosis or from embolism of exudate from a hepatic abscess that has eroded the wall and ruptured into the caudal vena cava (cattle). a gross finding often confused with intravital pulmonary hemorrhage is the result of severing both the trachea and the carotid arteries simultaneously at slaughter. blood is aspirated from the transected trachea into the lungs, forming a random pattern of irregular red foci (1 to 10 mm) in one or more lobes. these red foci are readily visible on both the pleural and the cut surfaces of the lung, and free blood is visible in the lumens of bronchi and bronchioles. rupture of a major pulmonary vessel with resulting massive hemorrhage occurs occasionally in cattle when a growing abscess in a lung invades and disrupts the wall of a major pulmonary artery or vein ( fig. 9-58 ). in most cases, animals die rapidly, often with spectacular hemoptysis, and on postmortem examination, bronchi are filled with blood (see fig. 9 -58). pulmonary edema. in normal lungs, fluid from the vascular space slowly but continuously passes into the interstitial tissue, where it is rapidly drained by the pulmonary and pleural lymphatic vessels. clearance of alveolar fluid across the alveolar epithelium is also a major mechanism of fluid removal from the lung. edema develops when the rate of fluid transudation from pulmonary vessels into the interstitium or alveoli exceeds that of lymphatic and alveolar removal ( fig. 9-59 ). pulmonary edema can be physiologically classified as cardiogenic (hydrostatic; hemodynamic) and noncardiogenic (permeability) types. hydrostatic (cardiogenic) pulmonary edema develops when there is an elevated rate of fluid transudation because of increased hydrostatic pressure in the vascular compartment or decreased osmotic pressure in the blood. once the lymph drainage has been overwhelmed, fluid accumulates in the perivascular spaces, causing distention of the bronchovascular bundles and alveolar interstitium, and eventually leaks into the alveolar spaces. causes of hemodynamic pulmonary edema include congestive heart failure (increased hydrostatic pressure); iatrogenic fluid overload; and disorders in which blood osmotic pressure is reduced, such as with hypoalbuminemia seen in some hepatic diseases, nephrotic syndrome, and protein-losing enteropathy. hemodynamic pulmonary edema also occurs when lymph drainage is impaired, generally secondary to neoplastic invasion of lymphatic vessels. permeability edema (inflammatory) occurs when there is excessive opening of endothelial gaps or damage to the cells that constitute the blood-air barrier (endothelial cells or type i pneumonocytes). hyperemia. pulmonary congestion is most frequently caused by heart failure, which results in stagnation of blood in pulmonary vessels, leading to edema and egression of erythrocytes into the alveolar spaces. as with any other foreign particle, erythrocytes in alveolar spaces are rapidly phagocytosed (erythrophagocytosis) by pulmonary alveolar macrophages. when extravasation of erythrocytes is severe, large numbers of macrophages with brown cytoplasm may accumulate in the bronchoalveolar spaces. the brown cytoplasm is the result of accumulation of considerable amounts of hemosiderin; these macrophages filled with iron pigment (siderophages) are generally referred to as heart failure cells ( fig. 9-57 ). the lungs of animals with chronic heart failure usually have a patchy red appearance with foci of brown discoloration because of accumulated hemosiderin. in this type of edema is an integral and early part of the inflammatory response, primarily because of the effect of inflammatory mediators, such as leukotrienes, platelet-activating factor (paf), cytokines, and vasoactive amines released by neutrophils, macrophages, mast cells, lymphocytes, endothelial cells, and type ii pneumonocytes. these inflammatory mediators increase the permeability of the blood-air barrier. in other cases, permeability edema results from direct damage to the endothelium or type i pneumonocytes, allowing plasma fluids to move freely from the vascular space into the alveolar lumen ( fig. 9 -60 and see fig. 9 -14). because type i pneumonocytes are highly vulnerable to some pneumotropic viruses (influenza and brsv), toxicants (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], hydrogen sulfide [h 2 s], and 3-methylindole), and particularly to free radicals, it is not surprising that permeability edema commonly accompanies many viral or toxic pulmonary diseases. a permeability edema also occurs when endothelial cells in the lung are injured by bacterial toxins, sepsis, ards, dic, anaphylactic shock, milk allergy, paraquat toxicity, adverse drug reactions, and smoke inhalation (efig. 9-7) . the concentration of protein in edematous fluid is greater in permeability edema (exudate) than in hemodynamic edema (transudate); this difference has been used clinically in human medicine to differentiate one type of pulmonary edema from another. microscopically, because of the higher concentration of protein, edema fluid in lungs with inflammation or damage to the blood-air barrier tends to stain more intensely eosinophilic than that of the hydrostatic edema from heart failure. grossly, the edematous lungs-independent of the cause-are wet and heavy. the color varies, depending on the degree of congestion or hemorrhage, and fluid may be present in the pleural cavity. if edema is severe, the bronchi and trachea contain considerable amounts of foamy fluid, which originates from the mixing of edema fluid and air ( fig. 9-61 ). on cut surfaces, the lung parenchyma oozes fluid like a wet sponge. in cattle and pigs that have distinct lobules, the lobular pattern becomes rather accentuated because of edematous distention of lymphatic vessels in the interlobular septa and the edematous interlobular septum itself ( fig. 9-62 ). severe pulmonary edema may be impossible to differentiate from peracute pneumonia; (h&e)-stained sections (see fig. 9 -60), particularly if a fixative such as zenker's solution, which precipitates protein, is used. acute respiratory distress syndrome. acute (adult) respiratory distress syndrome (ards; shock lung) is an important condition in human beings and animals characterized by pulmonary hypertension, intravascular aggregation of neutrophils in the lungs, acute lung injury, diffuse alveolar damage, permeability edema, and formation of hyaline membranes ( fig. 9-63) . these membranes are a mixture of plasma proteins, fibrin, surfactant, and cellular debris from necrotic pneumonocytes (see fig. 9-55, b) . the pathogenesis of ards is complex and multifactorial but in general terms can be defined as diffuse alveolar damage that results from lesions in distant organs, from generalized systemic diseases, or from direct injury to the lung. sepsis, major trauma, aspiration of gastric contents, extensive burns, and pancreatitis are some of the disease entities known to trigger ards. all these conditions provoke "hyperreactive macrophages" to directly or indirectly generate overwhelming amounts of cytokines causing what is known as a "cytokine storm." the main cytokines that trigger ards are tnf-α, interleukin (il)-1, il-6, and il-8, which prime neutrophils previously recruited in the lung capillaries and alveoli to release cytotoxic enzymes and free radicals. these substances cause severe and diffuse endothelial and alveolar damage that culminates in a fulminating pulmonary edema (see fig. 9 -63). ards occurs in domestic animals and explains why pulmonary edema is one of the most common lesions found in many animals dying of sepsis, toxemia, aspiration of gastric contents, and pancreatitis, for example. a familial form of ards has been reported in dalmatians. the pulmonary lesions in this syndrome are further discussed in the sections on interstitial pneumonia and aspiration pneumonia in dogs. neurogenic pulmonary edema is another distinctive but poorly understood form of life-threatening lung edema in human beings that follows cns injury and increased intracranial pressure (i.e., head injury, brain edema, brain tumors, or cerebral hemorrhage). this type of pulmonary edema can be experimentally reproduced in laboratory animals by injecting fibrin into the fourth ventricle. it involves both hemodynamic and permeability pathways presumably from massive sympathetic stimulation and overwhelming release of catecholamines. neurogenic pulmonary edema has sporadically been reported in animals with brain injury or severe seizures or after severe stress and excitement. pulmonary embolism. with its vast vascular bed and position in the circulation, the lung acts as a safety net to catch emboli before they reach the brain and other tissues. however, this positioning is often to its own detriment. the most common pulmonary emboli in domestic animals are thromboemboli, septic (bacterial) emboli, fat emboli, and tumor cell emboli. pulmonary thromboembolism (pte) refers to both local thrombus formation and translocation of a thrombus present elsewhere in the venous circulation ( fig. 9-64 ). fragments released inevitably reach the lungs and become trapped in the pulmonary vasculature ( fig. 9-65 and see fig. 9 -64). small sterile thromboemboli are generally of little clinical or pathologic significance because they can be rapidly degraded and disposed of by the fibrinolytic system. larger thromboemboli may cause small airway constriction, reduced surfactant production, pulmonary edema, and atelectasis resulting in hypoxemia, hyperventilation, and dyspnea. parasites (e.g., dirofilaria immitis and angiostrongylus vasorum), endocrinopathies (e.g., hyperadrenocorticism and hypothyroidism), glomerulopathies, and hypercoagulable states can be responsible for pulmonary arterial thrombosis and pulmonary thromboembolism in dogs (efig. 9-8) . pieces of this fact is not surprising because pulmonary edema occurs in the very early stages of inflammation (see efig. 9-7) . careful observation of the lungs at the time of necropsy is critical because diagnosis of pulmonary edema cannot be reliably performed microscopically. this is due in part to the loss of the edema fluid from the lungs during fixation with 10% neutral-buffered formalin and in part to the fact that the fluid itself stains very poorly or not at all with eosin because of its low protein content (hemodynamic edema). a protein-rich (permeability) edema is easier to visualize microscopically because it is deeply eosinophilic in hematoxylin and eosin fig. 9-104) . 1, normal alveolar capillary externally covered by type i and type ii pneumonocytes and internally by vascular endothelium (see fig. 9 -14 for more detail). 2, at the early stages of sepsis, proinflammatory cytokines (interleukin 1 [il-1] and tumor necrosis factor [tnf]) cause circulating neutrophils to adhere to the endothelial surface. following a "cytokine storm," the marginated neutrophils further activated by inflammatory mediators suddenly release their cytoplasmic granules (proteolytic enzymes and elastases myeloperoxidase) into the surrounding milieu (arrows). 3, enzymes released by these neutrophils cause injury to type i pneumonocytes (arrows) and endothelial cells (arrowheads), disrupting the blood-air barrier and causing permeability edema (curved arrows), alveolar hemorrhage (double-headed arrow), and exocytosis of neutrophils into the alveolar space (double-headed arrow). 4, extravasated plasma proteins admixed with surfactant and cell debris form thick hyaline membranes along the alveolar wall. 5, in the unlikely event that the animal survives, the healing process starts with alveolar macrophages removing cellular debris, reabsorption of edema, and hyperplasia of type ii pneumonocytes (double-headed curved arrow) that subsequently differentiate into type i pneumonocytes (see fig. 9 b a recognized in the bovine lung after strong pneumatic stunning at slaughter (captive bolt) ( fig. 9-66, a) . although obviously not important as an antemortem pulmonary lesion, brain emboli are intriguing as a potential risk for public health control of bovine spongiform encephalopathy (bse). fragments of hair can also embolize to the lung following intravenous injections (see fig. 9-66 , b). hepatic emboli formed by circulating pieces of fragmented liver occasionally become trapped in the pulmonary vasculature after severe abdominal trauma and hepatic rupture (see fig. 9-66, c) . megakaryocytes trapped in alveolar capillaries are a common but incidental microscopic finding in the lungs of all species, particularly dogs (see fig. 9-66, d) . tumor emboli (e.g., osteosarcoma and hemangiosarcoma in dogs and uterine carcinoma in cattle) can be numerous and striking and the ultimate cause of death in malignant neoplasia. in experimental studies, cytokines released during pulmonary inflammation are chemotactic for tumor cells and promote pulmonary metastasis. pulmonary infarcts. because of a dual arterial supply to the lung, pulmonary infarction is rare and generally asymptomatic. however, pulmonary infarcts can be readily caused when pulmonary thrombosis and embolism are superimposed on an already compromised pulmonary circulation such as occurs in congestive heart c d thrombi breaking free from a jugular, femoral, or uterine vein can cause pulmonary thromboembolism. pulmonary thromboembolisms occur in heavy horses after prolonged anesthesia (deep vein thrombosis), recumbent cows ("downer cow syndrome"), or in any animal undergoing long-term intravenous catheterization in which thrombi build up in the catheter and then break off (see fig. 9 -65). septic emboli, pieces of thrombi contaminated with bacteria or fungi and broken free from infected mural or valvular thrombi in the heart and vessels, eventually become entrapped in the pulmonary circulation. pulmonary emboli originate most commonly from bacterial endocarditis (right side) and jugular thrombophlebitis in all species, hepatic abscesses that have eroded and discharged their contents into the caudal vena cava in cattle, and septic arthritis and omphalitis in farm animals (see fig. 9 -64). when present in large numbers, septic emboli may cause unexpected death because of massive pulmonary edema; survivors generally develop pulmonary arteritis and thrombosis and embolic (suppurative) pneumonia, which may lead to pulmonary abscesses. bone marrow and bone emboli can form after bone fractures or surgical interventions of bone. these are not as significant a problem in domestic animals as they are in human beings. brain emboli (i.e., pieces of brain tissue) in the pulmonary vasculature reported in severe cases of head injury in human beings have recently been pulmonary macrophages (alveolar, intravascular, and interstitial), which have an immense biologic armamentarium, are the single most important effector cell and source of cytokines for all stages of pulmonary inflammation. these all-purpose phagocytic cells modulate the recruitment and trafficking of blood-borne leukocytes in the lung through the secretion of chemokines (see etable 9 -1). before reviewing how inflammatory cells are recruited in the lungs, three significant features in pulmonary injury must be remembered: (1) leukocytes can exit the vascular system through the alveolar capillaries, unlike in other tissues, where postcapillary venules are the sites of leukocytic diapedesis (extravasation); (2) the intact lung contains within alveolar capillaries a large pool of resident leukocytes (marginated pool); and (3) additional neutrophils are sequestered within alveolar capillaries within minutes of a local or systemic inflammatory response. these three pulmonary idiosyncrasies, along with the enormous length of the capillary network in the lung, explain why recruitment and migration of leukocytes into alveolar spaces develops so rapidly. experimental studies with aerosols of endotoxin or gram-negative bacteria have shown that within minutes of exposure, there is a significant increase in capillary leukocytes, and by 4 hours the alveolar lumen is filled with neutrophils. not surprisingly, the bal fluid collected from patients with acute pneumonia contains large amounts of inflammatory mediators such as tnf-α, il-1, and il-8. also, the capillary endothelium of patients with acute pneumonia has increased "expression" of adhesion molecules, which facilitate the migration of leukocytes from capillaries into the alveolar interstitium and from there into the alveolar lumen. in allergic pulmonary diseases, eotaxin and il-5 are primarily responsible for recruitment and trafficking of eosinophils in the lung. movement of plasma proteins into the pulmonary interstitium and alveolar lumen is a common but poorly understood phenomenon in pulmonary inflammation. leakage of fibrinogen and plasma proteins into the alveolar space occurs when there is structural damage to the blood-air barrier. this leakage is also promoted by some types of cytokines that enhance procoagulant activity, whereas others reduce fibrinolytic activity. excessive exudation of fibrin into the alveoli is particularly common in ruminants and pigs. the fibrinolytic system plays a major role in the resolution of pulmonary inflammatory diseases. in some cases, excessive plasma proteins leaked into alveoli mix with necrotic type i pneumonocytes and pulmonary surfactant, forming microscopic eosinophilic bands (membranes) along the lining of alveolar septa. these membranes, known as hyaline membranes, are found in specific types of pulmonary diseases, particularly in ards, and in cattle with acute interstitial pneumonias such as bovine pulmonary edema and emphysema and extrinsic allergic alveolitis (see pneumonias of cattle). in the past few years, nitric oxide has been identified as a major regulatory molecule of inflammation in a variety of tissues, including the lung. produced locally by macrophages, pulmonary endothelium, and pneumonocytes, nitric oxide regulates the vascular and bronchial tone, modulates the production of cytokines, controls the recruitment and trafficking of neutrophils in the lung, and switches on/off genes involved in inflammation and immunity. experimental work has also shown that pulmonary surfactant upregulates the production of nitric oxide in the lung, supporting the current view that pneumonocytes are also pivotal in amplifying and downregulating the inflammatory and immune responses in the lung (see etable 9 -1). as the inflammatory process becomes chronic, the types of cells making up cellular infiltrates in the lung change from mainly neutrophils to largely mononuclear cells. this shift in cellular composition is accompanied by an increase in specific cytokines, such as failure. it also occurs in dogs with torsion of a lung lobe (fig. 9-67) . the gross features of infarcts vary considerably, depending on the stage, and they can be red to black, swollen, firm, and cone or wedge shaped, particularly at the lung margins. in the early acute stage, microscopic lesions are severely hemorrhagic, and this is followed by necrosis. in 1 or 2 days, a border of inflammatory cells develops, and a few days later, a large number of siderophages are present in the necrotic lung. if sterile, pulmonary infarcts heal as fibrotic scars; if septic, an abscess may form surrounded by a thick fibrous capsule. in the past three decades, an information explosion has increased the overall understanding of pulmonary inflammation, with so many proinflammatory and antiinflammatory mediators described to date that it would be impossible to review them all here (see chapters 3 and 5). pulmonary inflammation is a highly regulated process that involves a complex interaction between cells imported from the blood (platelets, neutrophils, eosinophils, mast cells, and lymphocytes) and pulmonary cells (type i and ii pneumonocytes; endothelial and club [clara] cells; alveolar and intravascular macrophages; and stromal interstitial cells, such as mast cells, interstitial macrophages, fibroblasts, and myofibroblasts). blood-borne leukocytes, platelets, and plasma proteins are brought into the areas of inflammation by an elaborate network of chemical signals emitted by pulmonary cells and resident leukocytes. long-distance communication between pulmonary cells and blood cells is largely done by soluble cytokines; once in the lung, imported leukocytes communicate with pulmonary and vascular cells through adhesion and other inflammatory molecules. the best known inflammatory mediators are the complement system (c3a, c3b, and c5a), coagulation factors (factors v and vii), arachidonic acid metabolites (leukotrienes and prostaglandins), cytokines (interleukins, monokines, and chemokines), adhesion molecules (icam and vcam), neuropeptides (substance p, tachykinins, and neurokinins), enzymes and enzyme inhibitors (elastase and antitrypsin), oxygen metabolites (o 2 •, oh•, and h 2 o 2 ), antioxidants (glutathione), and nitric oxide (e -table 9 -1). acting in concert, these and many other molecules send positive or negative signals to initiate, maintain, and, it is hoped, resolve the inflammatory process without causing injury to the lung. chapter 9 respiratory system, mediastinum, and pleurae ewith several episodes of hemorrhage are characterized by large areas of dark brown discoloration, largely in the caudal lung lobes. microscopically, lesions are alveolar hemorrhages, abundant alveolar macrophages containing hemosiderin (siderophages), mild alveolar fibrosis, and occlusive remodeling of pulmonary veins. recurrent airway obstruction. recurrent airway obstruction (rao) of horses, also referred to as copd, heaves, chronic bronchiolitis-emphysema complex, chronic small airway disease, alveolar emphysema, and "broken wind," is a common clinically asthma-like syndrome of horses and ponies. rao is characterized by recurrent respiratory distress, chronic cough, poor athletic performance, airway neutrophilia, bronchoconstriction, mucus hypersecretion, and airway obstruction. the pathogenesis is still obscure, but genetic predisposition, t h 2 (allergic) immune response, and the exceptional sensitivity of airways to environmental allergens (hyperreactive airway disease) have been postulated as the basic underlying mechanisms. what makes small airways hyperreactive to allergens is still a matter of controversy. epidemiologic and experimental studies suggest that it could be the result of preceding bronchiolar damage caused by viral infections; ingestion of pneumotoxicants (3-methylindole); or prolonged exposure to organic dust, endotoxin, and environmental allergens (molds). it has been postulated that sustained inhalation of dust particles, whether antigenic or not, upregulates the production of cytokines (tnf-α, il-8, and monokine-inducible protein ) and neuropeptides (neurokinin a [nka], neurokinin b [nkb], and substance p), attracting neutrophils into the bronchioloalveolar region and promoting leukocyte-induced bronchiolar injury. summer pasture-associated obstructive pulmonary disease (spaopd) is a seasonal airway disease also reported in horses with similar clinical and pathologic findings. more recently, the term inflammatory airway disease (iad) has been introduced in equine medicine to describe rao-like syndrome in young horses 2 to 4 years old. the lungs of horses with heaves are grossly unremarkable, except for extreme cases in which alveolar emphysema may be present. microscopically, the lesions are often remarkable and include goblet cell metaplasia in bronchioles; plugging of bronchioles with mucus mixed with few eosinophils and neutrophils (see fig. 9 -13); peribronchiolar infiltration with lymphocytes, plasma cells, and variable numbers of eosinophils; and hypertrophy of smooth muscle in bronchi and bronchioles. in severe cases, accumulation of mucus leads to the complete obstruction of bronchioles and alveoli and resultant alveolar emphysema characterized by enlarged "alveoli" from the destruction of alveolar walls. feline asthma syndrome. feline asthma syndrome, also known as feline allergic bronchitis, is a clinical syndrome in cats of any age characterized by recurrent episodes of bronchoconstriction, cough, or dyspnea. the pathogenesis is not well understood but is presumed to originate, as in human asthma, as a type i hypersensitivity (igemast cell reaction) to inhaled allergens. dust, cigarette smoke, plant and household materials, and parasitic proteins have been incriminated as possible allergens. this self-limited allergic disease responds well to steroid therapy; thus it is rarely implicated as a primary cause of death except when suppressed defense mechanisms allow a secondary bacterial pneumonia. bronchial biopsies from affected cats at the early stages reveal mild to moderate inflammation characterized by mucosal edema and infiltration of leukocytes, particularly eosinophils. increased numbers of circulating eosinophils (blood eosinophilia) are present in some but not all cats with feline asthma. il-4, interferon-γ (ifn-γ), and interferon-inducible protein (ip-10), which are chemotactic for lymphocytes and macrophages. under appropriate conditions, these cytokines activate t lymphocytes, regulate granulomatous inflammation, and induce the formation of multinucleated giant cells such as in mycobacterial infections. inflammatory mediators locally released from inflamed lungs also have a biologic effect in other tissue. for example, pulmonary hypertension and right-sided heart failure (cor pulmonale) often follows chronic alveolar inflammation, not only as a result of increased pulmonary blood pressure but also from the effect of inflammatory mediators on the contractibility of smooth muscle of the pulmonary and systemic vasculature. cytokines, particularly tnf-α, that are released during inflammation are associated, both as cause and as effect, with the systemic inflammatory response syndrome (sirs), sepsis, severe sepsis with multiple organ dysfunction, and septic shock (cardiopulmonary collapse). as it occurs in any other sentinel system where many biologic promoters and inhibitors are involved (coagulation, the complement and immune systems), the inflammatory cascade could go into an "out-of-control" state, causing severe damage to the lungs. acute lung injury (ali), extrinsic allergic alveolitis, ards, pulmonary fibrosis, and asthma are archetypical diseases that ensue from an uncontrolled production and release of cytokines (cytokine storm). as long as acute alveolar injury is transient and there is no interference with the normal host response, the entire process of injury, degeneration, necrosis, inflammation, and repair can occur in less than 10 days. on the other hand, when acute alveolar injury becomes persistent or when the capacity of the host for repair is impaired, lesions can progress to an irreversible stage in which restoration of alveolar structure is no longer possible. in diseases, such as extrinsic allergic alveolitis, the constant release of proteolytic enzymes and free radicals by phagocytic cells perpetuates alveolar damage in a vicious circle. in other cases, such as in paraquat toxicity, the magnitude of alveolar injury can be so severe that type ii pneumonocytes, basement membranes, and alveolar interstitium are so disrupted that the capacity for alveolar repair is lost. fibronectins and transforming growth factors (tgfs) released from macrophages and other mononuclear cells at the site of chronic inflammation regulate the recruitment, attachment, and proliferation of fibroblasts. in turn, these cells synthesize and release considerable amounts of ecm (collagen, elastic fibers, or proteoglycans), eventually leading to fibrosis and total obliteration of normal alveolar architecture. in summary, in diseases in which there is chronic and irreversible alveolar damage, lesions invariably progress to a stage of terminal alveolar and interstitial fibrosis. for pneumonia, see section species-specific pneumonia of domestic animals. exercise-induced pulmonary hemorrhage. exercise-induced pulmonary hemorrhage (eiph) is a specific form of pulmonary hemorrhage in racehorses that occurs after exercise and clinically is characterized by epistaxis. because only a small percentage of horses with bronchoscopic evidence of hemorrhage have clinical epistaxis, it is likely that eiph goes undetected in many cases. the pathogenesis is still controversial, but current literature suggests laryngeal paralysis, bronchiolitis, and extremely high pulmonary vascular and alveolar pressures during exercise, alveolar hypoxia, and preexisting pulmonary injury as possible causes. eiph is seldom fatal; postmortem lesions in the lungs of horses that have been affected disease may be known by different names. in pigs, for instance, enzootic pneumonia and mycoplasma pneumonia refer to the same disease caused by mycoplasma hyopneumoniae. the word pneumonitis has been used by some as a synonym for pneumonia; however, others have restricted this term to chronic proliferative inflammation generally involving the alveolar interstitium and with little or no evidence of exudate. in this chapter, the word pneumonia is used for any inflammatory lesion in the lungs, regardless of whether it is exudative or proliferative, alveolar, or interstitial. on the basis of texture, distribution, appearance, and exudation, pneumonias can be grossly diagnosed into four morphologically distinct types: bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia. by using this classification, it is possible at the time of a necropsy to predict with some degree of certainty the likely cause (virus, bacteria, fungi, or parasites), routes of entry (aerogenous vs. hematogenous), and possible sequelae. these four morphologic types allow the clinician or pathologists to predict the most likely etiology and therefore facilitate the decision as to what samples need to be taken and which tests should be requested to the diagnostic laboratory (i.e., histopathology, bacteriology, virology, or toxicology). however, overlapping of these four types of pneumonias is possible, and sometimes two morphologic types may be present in the same lung. the criteria used to classify pneumonias grossly into bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia are based on morphologic changes, including distribution, texture, color, and general appearance of the affected lungs (table 9 -5). distribution of the inflammatory lesions in the lungs can be (1) cranioventral, as in most bronchopneumonias; (2) multifocal, as in embolic pneumonias; (3) diffuse, as in interstitial pneumonias; or (4) locally extensive, as in granulomatous in the most advanced cases, chronic bronchoconstriction and excess mucus production may result in smooth muscle hyperplasia and obstruction of the bronchi and bronchioles and infiltration of the airway mucosa by eosinophils. a syndrome known as canine asthma has been reported in dogs but is not as well characterized as the feline counterpart. few subjects in veterinary pathology have caused so much debate as the classification of pneumonias. historically, pneumonias in animals have been classified or named based on the following: 1. presumed cause, with names such as viral pneumonia, pasteurella pneumonia, distemper pneumonia, verminous pneumonia, chemical pneumonia, and hypersensitivity pneumonitis 2. type of exudation, with names such as suppurative pneumonia, fibrinous pneumonia, and pyogranulomatous pneumonia 3. morphologic features, with names such as gangrenous pneumonia, proliferative pneumonia, and embolic pneumonia 4. distribution of lesions, with names such as focal pneumonia, cranioventral pneumonia, diffuse pneumonia, and lobar pneumonia 5. epidemiologic attributes, with names such as enzootic pneumonia, contagious bovine pleuropneumonia, and "shipping fever" 6. geographic regions, with names such as montana progressive pneumonia 7. miscellaneous attributes, with names such as atypical pneumonia, cuffing pneumonia, progressive pneumonia, aspiration pneumonia, pneumonitis, farmer's lung, and extrinsic allergic alveolitis until a universal and systematic nomenclature for animal pneumonias is established, veterinarians should be acquainted with this heterogeneous list of names and should be well aware that one the parts of the face with the tip of your finger has been advocated by some pathologists. the texture of a normal lung is comparable to the texture of the center of the cheek. firm consolidation is comparable to the texture of the tip of the nose, and hard consolidation is comparable to the texture of the forehead. the term consolidation is frequently used to describe a firm or hard lung filled with exudate. pneumonias ( fig. 9-68) . texture of pneumonic lungs can be firmer or harder (bronchopneumonias), more elastic (rubbery) than normal lungs (interstitial pneumonias), or have a nodular feeling (granulomatous pneumonias). describing in words the palpable difference between the texture of a normal lung compared with the firm or hard texture of a consolidated lung can be a difficult undertaking. an analogy illustrating this difference based on touching changes in the gross appearance of pneumonic lungs include abnormal color, the presence of nodules or exudate, fibrinous or fibrous adhesions, and the presence of rib imprints on serosal surfaces (see fig. 9 -68). on cut surfaces, pneumonic lungs may have exudate, hemorrhage, edema, necrosis, abscesses, bronchiectasis, granulomas or pyogranulomas, and fibrosis, depending on the stage. palpation and careful observation of the lungs are essential in the diagnosis of pneumonia. (for details, see the section on examination of the respiratory tract.) bronchopneumonia refers to a particular morphologic type of pneumonia in which injury and the inflammatory process take place primarily in the bronchial, bronchiolar, and alveolar lumens. bronchopneumonia is undoubtedly the most common type of pneumonia seen in domestic animals and is with few exceptions characterized grossly by cranioventral consolidation of the lungs (fig. 9-69 and see fig. 9 -68). the reason why bronchopneumonias in animals are almost always restricted to the cranioventral portions of the lungs is not well understood. possible factors contributing to this topographic selectivity within the lungs include (1) gravitational sedimentation of the exudate, (2) greater deposition of infectious organisms, (3) inadequate defense mechanisms, (4) reduced vascular perfusion, (5) shortness and abrupt branching of airways, and (6) regional differences in ventilation. the term cranioventral in veterinary anatomy is the equivalent of "anterosuperior" in human anatomy. the latter is defined as "in front (ventral) and above (cranial)." thus, applied to the lung of animals, "cranioventral" means the ventral portion of the cranial lobe. however, by common usage in veterinary pathology, the term cranioventral used to describe the location of lesions in pneumonias has come to mean "cranial and ventral." thus it includes pneumonias affecting not only the ventral portion of the cranial lobe (true cranioventral) but also those cases in which the pneumonia has involved the ventral portions of adjacent lung lobes-initially the middle and then caudal on the right and the caudal lobe on the left side. bronchopneumonias are generally caused by bacteria and mycoplasmas, by bronchoaspiration of feed or gastric contents, or by improper tubing. as a rule, the pathogens causing bronchopneumonias arrive in the lungs via inspired air (aerogenous), either from infected aerosols or from the nasal flora. before establishing infection, pathogens must overwhelm or evade the pulmonary defense mechanisms. the initial injury in bronchopneumonias is centered on the mucosa of bronchioles; from there, the inflammatory process can spread downward to distal portions of the alveoli and upward to the bronchi. typically, for bronchopneumonias, the inflammatory exudates collect in the bronchial, bronchiolar, and alveolar lumina leaving the alveolar interstitium relatively unchanged, except for hyperemia and possibly edema. through the pores of kohn, the exudate can spread to adjacent alveoli until most or all of the alveoli in an individual lobule are involved. if the inflammatory process cannot control the inciting cause of injury, the lesions spread rapidly from lobule to lobule through alveolar pores and destroyed alveolar walls until an entire lobe or large portion of a lung is involved. the lesion tends to spread centrifugally, with the older lesions in the center, and exudate can be coughed up and then aspirated into other lobules, where the inflammatory process starts again. at the early stages of bronchopneumonia, the pulmonary vessels are engorged with blood (active hyperemia), and the bronchi, bronchioles, and alveoli contain some fluid (permeability edema). in cases in which pulmonary injury is mild to moderate, cytokines locally released in the lung cause rapid recruitment of neutrophils and alveolar macrophages into bronchioles and alveoli ( fig. 9-70 and see fig. 9 -69). when pulmonary injury is much more severe, proinflammatory cytokines induce more pronounced vascular changes by further opening endothelial gaps, thus increasing vascular permeability resulting in leakage of plasma fibrinogen (fibrinous exudates) and sometimes hemorrhage in the alveoli. alterations in permeability can be further exacerbated by structural damage to pulmonary capillaries and vessels directly caused by microbial toxins. filling of alveoli, bronchioles, and small bronchi with inflammatory exudate progressively obliterates airspaces, and as a consequence of this process, portions of severely affected (consolidated) lungs sink to the bottom of the container when placed in fixative. the replacement of air by exudate also changes the texture of the lungs, and depending on the severity of bronchopneumonia, the texture varies from firmer to harder than normal. the term consolidation is used at gross examination when the texture of pneumonic lung becomes firmer or harder than normal as a result of loss of airspaces because of exudation and atelectasis. (for details, see the discussion of lung texture in the section on classification of pneumonias in domestic animals). inflammatory consolidation of the lungs has been referred to in the past as hepatization because the affected lung had the appearance and texture of liver. the process was referred to as red hepatization in acute cases in which (1) congestion, (2) red hepatization (liver texture), (3) gray hepatization, and (4) resolution. because of the use of effective antibiotics and prevention, pneumococcal pneumonia and its four classic stages are rarely seen; thus this terminology has been largely abandoned. currently, the term bronchopneumonia is widely used for both suppurative and fibrinous consolidation of the lungs because both forms of inflammation have essentially the same pathogenesis in which the pathogens reach the lung by the aerogenous route, injury occurs initially in the bronchial and bronchiolar regions, and the inflammatory process extends centrifugally deep into the alveoli. it must be emphasized that it is the severity of pulmonary injury that largely determines whether bronchopneumonia becomes suppurative or fibrinous. in some instances, however, it is difficult to discriminate between suppurative and fibrinous bronchopneumonia because both types can coexist (fibrinosuppurative bronchopneumonia), and one type can progress to the other. suppurative bronchopneumonia. suppurative bronchopneumonia is characterized by cranioventral consolidation of lungs (see figs. 9-68 and 9-69), with typically purulent or mucopurulent exudate present in the airways. this exudate can be best demonstrated by expressing intrapulmonary bronchi, thus forcing exudate out of the bronchi (see fig. 9 -69). the inflammatory process in suppurative bronchopneumonia is generally confined to individual lobules, and as a result of this distribution, the lobular pattern of the lung becomes notably emphasized. this pattern is particularly obvious in cattle and pigs because these species have prominent lobulation of the lungs. the gross appearance often resembles an irregular checkerboard because of an admixture of normal and abnormal (consolidated) lobules (see fig. 9 -69). because of this typical lobular distribution, suppurative bronchopneumonias are also referred to as lobular pneumonias. different inflammatory phases occur in suppurative bronchopneumonia where the color and appearance of consolidated lungs varies considerably, depending on the virulence of offending organisms and chronicity of the lesion. the typical phases of suppurative bronchopneumonia can be summarized as follows: 1. during the first 12 hours when bacteria are rapidly multiplying, the lungs become hyperemic and edematous. 2. soon after, neutrophils start filling the airways, and by 48 hours the parenchyma starts to consolidate and becomes firm in texture. 3. three to 5 days later, hyperemic changes are less obvious, but the bronchial, bronchiolar, and alveolar spaces continue to fill with neutrophils and macrophages, and the affected lung sinks when placed in formalin. at this stage, the affected lung has a gray-pink color, and on cut surface, purulent exudate can be expressed from bronchi. 4. in favorable conditions where the infection is under control of the host defense mechanisms, the inflammatory processes begin to regress, a phase known as resolution. complete resolution in favorable conditions could take 1 to 2 weeks. 5. in animals in which the lung infection cannot be rapidly contained, inflammatory lesions can progress into a chronic phase. approximately 7 to 10 days after infection, the lungs become pale gray and take a "fish flesh" appearance. this appearance is the result of purulent and catarrhal inflammation, obstructive atelectasis, mononuclear cell infiltration, peribronchial and peribronchiolar lymphoid hyperplasia, and early alveolar fibrosis. complete resolution is unusual in chronic bronchopneumonia, and lung scars, such as pleural and pulmonary fibrosis; bronchiectasis as a consequence of chronic destructive bronchitis (see bronchiectasis [dysfunction/responses to injury and patterns of injury]); atelectasis; pleural adhesions; and lung abscesses may remain unresolved there was notable active hyperemia with little exudation of neutrophils; conversely, the process was referred to as gray hepatization in those chronic cases in which hyperemia was no longer present, but there was abundant exudation of neutrophils and macrophages. this terminology, although used for and applicable to human pneumonias, is rarely used in veterinary medicine primarily because the evolution of pneumonic processes in animals does not necessarily follow the red-to-gray hepatization pattern. bronchopneumonia can be subdivided into suppurative bronchopneumonia if the exudates are predominantly composed of neutrophils and fibrinous bronchopneumonia if fibrin is the predominant component of the exudates (see table 9 -5). it is important to note that some veterinarians use the term fibrinous pneumonia or lobar pneumonia as a synonym for fibrinous bronchopneumonia, and bronchopneumonia or lobular pneumonia as a synonym for suppurative bronchopneumonia. human pneumonias for many years have been classified based on their etiology and morphology, which explains why pneumococcal pneumonia (streptococcus pneumoniae) has been synonymous with lobar pneumonia. in the old literature, four distinct stages of pneumococcal pneumonia were described as lumen of bronchiole capillary pulmonary defense mechanisms to allow them to colonize the lungs and establish an infection. suppurative bronchopneumonia can also result from aspiration of bland material (e.g., milk). pulmonary gangrene may ensue when the bronchopneumonic lung is invaded by saprophytic bacteria (aspiration pneumonia). fibrinous bronchopneumonia. fibrinous bronchopneumonia is similar to suppurative bronchopneumonia except that the predominant exudate is fibrinous rather than neutrophilic. with only a few exceptions, fibrinous bronchopneumonias also have a cranioventral distribution (fig. 9-72 and see fig. 9-68) . however, exudation is not restricted to the boundaries of individual pulmonary lobules, as is the case in suppurative bronchopneumonias. instead, the inflammatory process in fibrinous pneumonias involves numerous contiguous lobules and the exudate moves quickly through pulmonary tissue until the entire pulmonary lobe is rapidly affected. because of the involvement of the entire lobe and pleural surface, fibrinous bronchopneumonias are also referred to as lobar pneumonias or pleuropneumonias. in general terms, fibrinous bronchopneumonias are the result of more severe pulmonary injury and thus cause death earlier in the sequence of the inflammatory process than suppurative bronchopneumonias. even in cases in which fibrinous bronchopneumonia involves 30% or less of the total area, clinical signs and death can occur as a result of severe toxemia and sepsis. the gross appearance of fibrinous bronchopneumonia depends on the age and severity of the lesion and on whether the pleural surface or the cut surface of the lung is viewed. externally, early stages of fibrinous bronchopneumonias are characterized by severe congestion and hemorrhage, giving the affected lungs a characteristically intense red discoloration. a few hours later, fibrin starts to permeate and accumulate on the pleural surface, giving the pleura a ground glass appearance and eventually forming plaques of fibrinous exudate over a red, dark lung (see fig. 9 -72). at this stage, a yellow fluid starts to accumulate in the thoracic cavity. the color of fibrin deposited over the pleural surface is also variable. it can be bright yellow when the exudate is formed primarily by fibrin, tan when fibrin is mixed with blood, and gray when a large number of leukocytes and fibroblasts are part of the fibrinous plaque in more chronic cases. because of the tendency of fibrin to deposit on the pleural surface, some pathologists use the term pleuropneumonia as a synonym for fibrinous bronchopneumonia. on the cut surface, early stages of fibrinous bronchopneumonia appear as simple red consolidation. in more advanced cases (24 hours), fibrinous bronchopneumonia is generally accompanied by notable dilation and thrombosis of lymph vessels and edema of interlobular septa (see fig. 9-72, b) . this distention of the interlobular septa gives affected lungs a typical marbled appearance. distinct focal areas of coagulative necrosis in the pulmonary parenchyma are also common in fibrinous bronchopneumonia such as in shipping fever pneumonia and contagious bovine pleuropneumonia. in animals that survive the early stage of fibrinous bronchopneumonia, pulmonary necrosis often develops into pulmonary "sequestra," which are isolated pieces of necrotic lung encapsulated by connective tissue. pulmonary sequestra result from extensive necrosis of lung tissue either from severe ischemia (infarct) caused by thrombosis of a major pulmonary vessel such as in contagious bovine pleuropneumonia or from the effect of necrotizing toxins released by pathogenic bacteria such as mannheimia haemolytica. sequestra in veterinary pathology should not be confused with "bronchopulmonary sequestration," a term used in human pathology to describe a congenital malformation in which whole lobes or parts of the lung develop without normal connections to the airway or vascular systems. for a long time. "enzootic pneumonias" of ruminants and pigs are typical examples of chronic suppurative bronchopneumonias. microscopically, acute suppurative bronchopneumonias are characterized by hyperemia, abundant neutrophils, macrophages, and cellular debris within the lumen of bronchi, bronchioles, and alveoli (see fig. 9 -70). recruitment of leukocytes is promoted by cytokines, complement, and other chemotactic factors that are released in response to alveolar injury or by the chemotactic effect of bacterial toxins, particularly endotoxin. in most severe cases, purulent or mucopurulent exudates completely obliterate the entire lumen of bronchi, bronchioles, and alveoli. if suppurative bronchopneumonia is merely the response to a transient pulmonary injury or a mild infection, lesions resolve uneventfully. within 7 to 10 days, cellular exudate can be removed from the lungs via the mucociliary escalator, and complete resolution may take place within 4 weeks. in other cases, if injury or infection is persistent, suppurative bronchopneumonia can become chronic with goblet cell hyperplasia, an important component of the inflammatory process. depending on the proportion of pus and mucus, the exudate in chronic suppurative bronchopneumonia varies from mucopurulent to mucoid. a mucoid exudate is found in the more chronic stages when the consolidated lung has a "fish flesh" appearance. hyperplasia of balt is another change commonly seen in chronic suppurative bronchopneumonias; it appears grossly as conspicuous white nodules (cuffs) around bronchial walls (cuffing pneumonia). this hyperplastic change merely indicates a normal reaction of lymphoid tissue to infection. further sequelae of chronic suppurative bronchopneumonia include bronchiectasis (see figs. 9-10 and 9-11), pulmonary abscesses, pleural adhesions (from pleuritis) ( fig. 9-71) , and atelectasis and emphysema from completely or partially obstructed bronchi or bronchioles (e.g., bronchiectasis). clinically, suppurative bronchopneumonias can be acute and fulminating but are often chronic, depending on the etiologic agent, stressors affecting the host, and immune status. the most common pathogens causing suppurative bronchopneumonia in domestic animals include pasteurella multocida, bordetella bronchiseptica, trueperella (arcanobacterium) pyogenes, streptococcus spp., escherichia coli, and several species of mycoplasmas. most of these organisms are secondary pathogens requiring a preceding impairment of the fulminating hemorrhagic bronchopneumonia can be caused by highly pathogenic bacteria such as bacillus anthracis. although the lesions in anthrax are primarily related to a severe septicemia and sepsis, anthrax should always be suspected in animals with sudden death and exhibiting severe acute fibrinohemorrhagic pneumonia, splenomegaly, and multisystemic hemorrhages. animals are considered good sentinels for anthrax in cases of bioterrorism. interstitial pneumonia refers to that type of pneumonia in which injury and the inflammatory process take place primarily in any microscopically, in the initial stage of fibrinous bronchopneumonia, there is massive exudation of plasma proteins into the bronchioles and alveoli, and as a result, most of the airspaces become obliterated by fluid and fibrin. leakage of fibrin and fluid into alveolar lumina is due to extensive disruption of the integrity and increased permeability of the blood-air barrier. fibrinous exudates can move from alveolus to alveolus through the pores of kohn. because fibrin is chemotactic for neutrophils, these types of leukocytes are always present a few hours after the onset of fibrinous inflammation. as inflammation progresses (3 to 5 days), fluid exudate is gradually replaced by fibrinocellular exudates composed of fibrin, neutrophils, macrophages, and necrotic debris ( fig. 9-73 ). in chronic cases (after 7 days), there is notable fibrosis of the interlobular septa and pleura. in contrast to suppurative bronchopneumonia, fibrinous bronchopneumonia rarely resolves completely, thus leaving noticeable scars in the form of pulmonary fibrosis and pleural adhesions. the most common sequelae found in animals surviving an acute episode of fibrinous bronchopneumonia include alveolar fibrosis and bronchiolitis obliterans, in which organized exudate becomes attached to the bronchiolar lumen (see fig. 9-12) . these changes are collectively referred to as bronchiolitis obliterans organizing pneumonia (boop), a common microscopic finding in animals with unresolved bronchopneumonia. other important sequelae include pulmonary gangrene, when saprophytic bacteria colonize necrotic lung; pulmonary sequestra; pulmonary fibrosis; abscesses; and chronic pleuritis with pleural adhesions. in some cases, pleuritis can be so extensive that fibrous adhesions extend onto the pericardial sac. pathogens causing fibrinous bronchopneumonias in domestic animals include mannheimia (pasteurella) haemolytica (pneumonic mannheimiosis), histophilus somni (formerly haemophilus somnus), actinobacillus pleuropneumoniae (porcine pleuropneumonia), mycoplasma bovis, and mycoplasma mycoides ssp. mycoides small colony type (contagious bovine pleuropneumonia). fibrinous bronchoa b n alveolar epithelium. inhaled antigens, such as fungal spores, combine with circulating antibodies and form deposits of antigen-antibody complexes (type iii hypersensitivity) in the alveolar wall, which initiate a cascade of inflammatory responses and injury (allergic alveolitis). hematogenous injury to the vascular endothelium occurs in septicemias, sepsis, dic, larva migrans (ascaris suum), toxins absorbed in the alimentary tract (endotoxin) or toxic metabolites locally generated in the lungs (3-methylindole and paraquat), release of free radicals in alveolar capillaries (ards), and infections with endotheliotropic viruses (canine adenovirus and classical swine fever [hog cholera]). interstitial pneumonias in domestic animals and human beings are subdivided based on morphologic features into acute and chronic. it should be kept in mind, however, that not all acute interstitial pneumonias are fatal and that they do not necessarily progress to the chronic form. acute interstitial pneumonias. acute interstitial pneumonias begin with injury to either type i pneumonocytes or alveolar capillary endothelium, which provokes a disruption of the blood-air barrier and a subsequent exudation of plasma proteins into the alveolar space (see fig. 9-14) . this leakage of proteinaceous fluid into the alveolar lumen constitutes the exudative phase of acute interstitial pneumonia. in some cases of diffuse alveolar damage, exuded plasma proteins mix with lipids and other components of pulmonary surfactant and form elongated membranes that become partially attached to the alveolar basement membrane and bronchiolar walls. these membranes are referred to as hyaline membranes because of their hyaline appearance (eosinophilic, homogeneous, and amorphous) microscopically (see figs. 9-55 and 9-63). in addition to intraalveolar exudation of fluid, inflammatory edema and neutrophils accumulate in the alveolar interstitium and cause thickening of the alveolar walls. this acute exudative phase is generally followed a few days later by the proliferative phase of acute interstitial pneumonia, which is characterized by hyperplasia of type ii pneumonocytes to replace the lost type i pneumonocytes (see fig. 9 -15). type ii pneumonocytes are in fact progenitor cells that differentiate and replace necrotic type i pneumonocytes (see fig. 9-14) . as a consequence, the alveolar walls become increasingly thickened. this process is in part the reason why lungs become rubbery on palpation, what prevents their normal collapse after the thorax is opened, and why the cut surface of the lung has a "meaty" appearance (see fig. 9 -74). of the three layers of the alveolar walls (endothelium, basement membrane, and alveolar epithelium) and the contiguous bronchiolar interstitium (see fig. 9-7) . this morphologic type of pneumonia is the most difficult to diagnose at necropsy and requires microscopic confirmation because it is easily mistaken in the lung showing congestion, edema, hyperinflation, or emphysema. in contrast to bronchopneumonias, in which distribution of lesions is generally cranioventral, in interstitial pneumonias, lesions are more diffusely distributed and generally involve all pulmonary lobes, or in some cases, they appear to be more pronounced in the dorsocaudal aspects of the lungs (see fig. 9 -68). three important gross features of interstitial pneumonia are (1) the failure of lungs to collapse when the thoracic cavity is opened, (2) the occasional presence of rib impressions on the pleural surface of the lung indicating poor deflation, and (3) the lack of visible exudates in airways unless complicated with secondary bacterial pneumonia. the color of affected lungs varies from diffusely red in acute cases to diffusely pale gray to a mottled red, pale appearance in chronic ones. pale lungs are caused by severe obliteration of alveolar capillaries (reduced blood-tissue ratio), especially evident when there is fibrosis of the alveolar walls. the texture of lungs with uncomplicated interstitial pneumonia is typically elastic or rubbery, but definitive diagnosis based on texture alone is difficult and requires histopathologic examination. on a cut surface, the lungs may appear and feel more "meaty" (having the texture of raw meat) and have no evidence of exudate in the bronchi or pleura (fig. 9-74 ). in acute interstitial pneumonias, particularly in cattle, there is frequently pulmonary edema (exudative phase) and interstitial emphysema secondary to partial obstruction of bronchioles by edema fluid and strenuous air gasping before death. because edema tends to gravitate into the cranioventral portions of the lungs, and emphysema is often more obvious in the dorsocaudal aspects, acute interstitial pneumonias in cattle occasionally have a gross cranioventral-like pattern that may resemble bronchopneumonia, although the texture is different. lungs are notably heavy because of the edema and the infiltrative and proliferative changes. the pathogenesis of interstitial pneumonia is complex and can result from aerogenous injury to the alveolar epithelium (type i and ii pneumonocytes) or from hematogenous injury to the alveolar capillary endothelium or alveolar basement membrane. aerogenous inhalation of toxic gases (i.e., ozone and no 2 ) or toxic fumes (smoke inhalation) and infection with pneumotropic viruses (influenza, herpesviruses, or canine distemper virus) can damage the figure 9 -74 interstitial pneumonia, lung, feeder pig. a, the lung is heavy, pale, and rubbery in texture. it also has prominent costal (rib) imprints (arrows), a result of hypercellularity of the interstitium and the failure of the lungs to collapse when the thorax was opened. b, transverse section. the pulmonary parenchyma has a "meaty" appearance and some edema, but no exudate is present in airways or on the pleural surface. this type of lung change in pigs is highly suggestive of a viral pneumonia. (courtesy dr. a. lópez, atlantic veterinary college.) pneumonia are centered in the alveolar wall and its interstitium, a mixture of desquamated epithelial cells, macrophages, and mononuclear cells are usually present in the lumens of bronchioles and alveoli. ovine progressive pneumonia, hypersensitivity pneumonitis in cattle and dogs, and silicosis in horses are good veterinary examples of chronic interstitial pneumonia. pneumoconioses (silicosis and asbestosis), paraquat toxicity, pneumotoxic antineoplastic drugs (bleomycin), and extrinsic allergic alveolitis (farmer's lung) are well-known examples of diseases that lead to chronic interstitial pneumonias in human beings. massive pulmonary migration of ascaris larvae in pigs also causes interstitial pneumonia ( fig. 9-77 ). there is an insidious and poorly understood group of chronic idiopathic interstitial diseases, both in human beings and in animals, that eventually progress to terminal interstitial fibrosis. these were originally thought to be the result of repeated cycles of alveolar injury, inflammation, and fibroblastic/myoblastic response to an unknown agent. however, aggressive antiinflammatory therapy generally fails to prevent or reduce the severity of fibrosis. now, it is acute interstitial pneumonias are often mild and transient, especially those caused by some respiratory viruses, such as those responsible for equine and porcine influenza. these mild forms of pneumonia are rarely seen in the postmortem room because they are not fatal and do not leave significant sequelae (see the section on defense mechanisms/barrier systems). in severe cases of acute interstitial pneumonias, animals may die of respiratory failure, usually as a result of diffuse alveolar damage, a profuse exudative phase (leakage of proteinaceous fluid) leading to a fatal pulmonary edema. examples of this type of fatal acute interstitial pneumonia are bovine pulmonary edema and emphysema, and ards in all species. chronic interstitial pneumonia. when the source of alveolar injury persists, the exudative and proliferative lesions of acute interstitial pneumonia can progress into a morphologic stage referred to as chronic interstitial pneumonia. the hallmark of chronic interstitial pneumonia is fibrosis of the alveolar walls (with or without intraalveolar fibrosis) and the presence of lymphocytes, macrophages, fibroblasts, and myofibroblasts in the alveolar interstitium (figs. 9-75 and 9-76). in other cases, these chronic changes are accompanied by hyperplasia and persistence of type ii pneumonocytes, squamous metaplasia of the alveolar epithelium, microscopic granulomas, and hyperplasia of smooth muscle in bronchioles and pulmonary arterioles. it should be emphasized that although the lesions in interstitial the term bronchointerstitial pneumonia is used in veterinary pathology to describe cases in which microscopic lesions share some histologic features of both bronchopneumonia and interstitial pneumonia (efig. 9-9 ). this combined type of pneumonia is in fact frequently seen in many viral infections in which viruses replicate and cause necrosis in bronchial, bronchiolar, and alveolar cells. damage to the bronchial and bronchiolar epithelium causes an influx of neutrophils similar to that in bronchopneumonias, and damage to alveolar walls causes proliferation of type ii pneumonocytes, similar to that which takes place in the proliferative phase of acute interstitial pneumonias. it is important to emphasize that bronchointerstitial pneumonia is a microscopic not a gross diagnosis. examples include uncomplicated cases of respiratory syncytial virus infections in cattle and lambs, canine distemper, and influenza in pigs and horses. embolic pneumonia refers to a particular type of pneumonia in which gross and microscopic lesions are multifocally distributed in all pulmonary lobes. by definition, lung injury is hematogenous, and the inflammatory response is typically centered in pulmonary arterioles and alveolar capillaries. lungs act as a biologic filter for circulating particulate matter. sterile thromboemboli, unless extremely large, are rapidly dissolved and removed from the pulmonary vasculature by fibrinolysis, causing little, if any, ill effects. experimental studies have confirmed that most types of bacteria when injected intravenously (bacteremia) are phagocytosed by pulmonary intravascular macrophages, or they bypass the lungs and are finally trapped by macrophages in the liver, spleen, joints, or other organs. to cause pulmonary infection, circulating bacteria must first attach to the pulmonary endothelium with specific binding proteins or simply attach to intravascular fibrin and then evade phagocytosis by intravascular macrophages or leukocytes. septic thrombi facilitate entrapment of bacteria in the pulmonary vessels and provide a favorable environment to escape phagocytosis. once trapped in the pulmonary vasculature, usually in small arterioles or alveolar capillaries, offending bacteria disrupt endothelium and basement membranes, spread from the vessels to the interstitium and then to the surrounding lung, finally forming a new nidus of infection. embolic pneumonia is characterized by multifocal lesions randomly distributed in all pulmonary lobes (see fig. 9 -68 and e-figs. 9-10 and 9-11). early lesions in embolic pneumonia are characterized grossly by the presence of very small (1 to 10 mm), white foci surrounded by discrete, red, hemorrhagic halos ( fig. 9-78 ). unless emboli arrive in massive numbers, causing fatal pulmonary edema, embolic pneumonia is seldom fatal; therefore these acute lesions are rarely seen at postmortem examination. in most instances, if unresolved, acute lesions rapidly progress to pulmonary abscesses. these are randomly distributed in all pulmonary lobes and are not restricted to the cranioventral aspects of the lungs, as is the case of abscesses developing from suppurative bronchopneumonia. the early microscopic lesions in embolic pneumonias are always focal or multifocal ( fig. 9-79) ; thus they differ from those of endotoxemia or septicemia, in which endothelial damage and interstitial reactions (interstitial pneumonia) are diffusely distributed in the lungs. when embolic pneumonia or its sequela (abscesses) is diagnosed at necropsy, an attempt should be made to locate the source of septic emboli. the most common sources are hepatic abscesses that have ruptured into the caudal vena cava in cattle, omphalophlebitis in farm animals, chronic bacterial skin or hoof infections, and a contaminated catheter in all species (see fig. 9-64) . valvular or mural endocarditis in the right heart is a common source of septic emboli and embolic pneumonia in all species. most frequently, bacterial proposed that a genetic mutation alters the cell-cell communication between epithelial and mesenchymal cells in the lung. this aberrant cellular communication leads to an overexpression of inflammatory and repair molecules (i.e., il-4, il-13, tgf-β1, and caveolin), leading to increased apoptosis and interstitial deposition of extracellular matrix (ecm). the chronic interstitial (restrictive) diseases in human medicine include "idiopathic pulmonary fibrosis," "nonspecific interstitial pneumonia," "unusual interstitial pneumonia," and "cryptogenic organizing pneumonia," also referred to as idiopathic bronchiolitis obliterans organizing pneumonia (idiopathic boop). feline idiopathic pulmonary fibrosis is an example of this type of progressive interstitial disease in veterinary medicine. it has been reported that in rare cases, chronic alveolar remodeling and interstitial fibrosis can progress to lung cancer. the lung has numerous circular areas of hemorrhage distributed randomly throughout all lung lobes (embolic pattern [see fig. 9 -68]). these foci arise from injury to the microvasculature in alveolar septa and the visceral pleura secondary to lodgment of bacterial or fungal emboli (septic emboli) from valvular or mural endocarditis in the right heart or from other bacterial or fungal diseases where the bacterium or fungus gains access to the circulatory system as occurs in many bacterial and fungal enteritides or pneumonias caused by salmonella spp., e. coli, or aspergillus spp. the pathogenesis of granulomatous pneumonia shares some similarities with that of interstitial and embolic pneumonias. not surprisingly, some pathologists group granulomatous pneumonias within one of these types of pneumonias (e.g., granulomatous interstitial pneumonia). what makes granulomatous pneumonia a distinctive type is not so much the portal of entry or site of initial injury in the lungs but, rather, the unique type of inflammatory response that results in the formation of granulomas, which can be easily recognized at gross and microscopic examination. as a rule, agents causing granulomatous pneumonias are resistant to intracellular killing by phagocytic cells and to the acute inflammatory response, allowing prolonged persistence of these agents in tissues. the most common causes of granulomatous pneumonia in animals include systemic fungal diseases, such as cryptococcosis (cryptococcus neoformans and cryptococcus gatti), coccidioidomycosis (coccidioides immitis), histoplasmosis (histoplasma capsulatum), and blastomycosis (blastomyces dermatitidis) (see fig. 9 -35). in most of these fungal diseases, the port of entry is aerogenous, and from the lungs the fungi disseminate systemically to other organs, particularly the lymph nodes, liver, and spleen. filamentous fungi such as aspergillus spp. or mucor spp. can also reach the lung by the hematogenous route. granulomatous pneumonia is also seen in some bacterial diseases, such as tuberculosis (mycobacterium bovis) in all species and rhodococcus equi in horses. sporadically, aberrant parasites such as fasciola hepatica in cattle and aspiration of foreign bodies can also cause granulomatous pneumonia (efig. 9-12 granulomatous pneumonia is characterized by the presence of variable numbers of caseous or noncaseous granulomas randomly distributed in the lungs (see fig. 9 -68). on palpation, lungs have a typical nodular character given by well-circumscribed, variably sized nodules that generally have a firm texture, especially if calcification has occurred ( fig. 9-80) . during postmortem examination, granulomas in the lungs occasionally can be mistaken for neoplasms. microscopically, pulmonary granulomas are composed of a center of necrotic tissue, surrounded by a rim of macrophages (epithelioid cells) and giant cells and an outer delineated layer of connective tissue commonly infiltrated by lymphocytes and plasma cells ( fig. 9-81 ). unlike other types of pneumonias, the causative agent in granulomatous pneumonia can, in many cases, be identified isolates from septic pulmonary emboli in domestic animals are trueperella (arcanobacterium) pyogenes (cattle), fusobacterium necrophorum (cattle, pigs, and human beings), erysipelothrix rhusiopathiae (pigs, cattle, dogs, and human beings), streptococcus suis (pigs), staphylococcus aureus (dogs and human beings), and streptococcus equi (horses). granulomatous pneumonia refers to a particular type of pneumonia in which aerogenous or hematogenous injury is caused by organisms or particles that cannot normally be eliminated by phagocytosis and that evoke a local inflammatory reaction with numerous alveolar and interstitial macrophages, lymphocytes, a few neutrophils, and sometimes giant cells. the term granulomatous is used here to describe an anatomic pattern of pneumonia typically characterized by the presence of granulomas. g g but yet unproven that viral infections may also predispose horses to airway hyperresponsiveness and recurrent airway obstruction (rao). equine influenza. equine influenza is an important and highly contagious flulike respiratory disease of horses characterized by high morbidity and low mortality and explosive outbreaks in susceptible populations. it is an oie-notifiable disease. two antigenically unrelated subtypes of equine influenza virus have been identified (h7n7 [a/equi-1] and h3n8 [a/equi-2]). the course of the disease is generally mild and transient, and its importance is primarily because of its economic impact on horse racing. the types of injury and host response in the conducting system are described in the section on disorders of the nasal cavity and paranasal sinuses of horses. uncomplicated lesions in the lungs are mild and self-limiting bronchointerstitial pneumonia. in fatal cases, the lungs are hyperinflated with coalescing areas of dark red discoloration. microscopically, there is a bronchointerstitial pneumonia characterized by necrotizing bronchiolitis that is followed by hyperplastic bronchiolitis, hyperplasia of type ii pneumonocytes, hyaline membranes in alveoli, and sporadic multinucleated giant cells. the microscopic changes are ards in severe and fatal cases. the influenza virus antigen can be readily demonstrated in ciliated cells and alveolar macrophages. clinical signs are characterized by fever, cough, abnormal lung sounds (crackles and wheezes), anorexia, and depression. secondary bacterial infections (streptococcus equi, streptococcus zooepidemicus, staphylococcus aureus, and escherichia coli) commonly complicate equine influenza. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr), or equine herpesvirus infection, is a respiratory disease of young horses that is particularly important in weanlings between 4 and 8 months of age and to a much lesser extent in young foals and adult horses. the causative agents are ubiquitous equine herpesviruses (ehv-1 and ehv-4) that in addition to respiratory disease can cause abortion in pregnant mares and neurologic disease (equine herpes myeloencephalopathy) (see the section on disorders of the nasal cavity and paranasal sinuses of horses). the respiratory form of evr is a mild and a transient bronchointerstitial pneumonia seen only by pathologists when complications with secondary bacterial infections cause a fatal bronchopneumonia microscopically in sections by pas reaction or grocott-gomori's methenamine silver (gms) stain for fungi or by an acid-fast stain for mycobacteria. viral infections of the respiratory tract, particularly equine viral rhinopneumonitis and equine influenza, are important diseases of horses throughout the world. the effects of these and other respiratory viruses on the horse can be manifested in three distinct ways. first, as pure viral infections, their severity may range from mild to severe, making them a frequent interfering factor in training and athletic performance. second, superimposed infections by opportunistic bacteria, such as streptococcus spp., escherichia coli, klebsiella pneumoniae, rhodococcus equi, and various anaerobes, can cause fibrinous or suppurative bronchopneumonias. third, it is possible equine henipavirus (hendra virus). fatal cases of a novel respiratory disease in horses and human beings suddenly appeared in approximately 1994 in hendra, a suburb of brisbane, australia. this outbreak was attributed to a newly recognized zoonotic virus that was tentatively named equine morbillivirus. now called hendra virus (hev), this emerging viral pathogen is currently classified as a member of the genus henipavirus (includes hendra virus and nipah virus), in the family paramyxoviridae. fruit bats (flying foxes) act as natural reservoirs and are involved in the transmission by poorly understood mechanisms. the lungs of affected horses are severely edematous with gelatinous distention of pleura and subpleural lymph vessels. microscopically, the lungs have diffuse alveolar edema associated with vasculitis, thrombosis, and the presence of multinucleated syncytial cells in the endothelium of small pulmonary blood vessels and alveolar capillaries. the lymphatic vessels are notably distended with fluid. the characteristic inclusion bodies seen in other paramyxovirus infections are not seen in horses; however, the virus can be easily detected by immunohistochemistry in pulmonary endothelial cells and alveolar epithelial cells (pneumonocytes). clinical signs are nonspecific and include fever, anorexia, respiratory distress, and nasal discharge. equine multinodular pulmonary fibrosis. equine multinodular pulmonary fibrosis is a lung disease characterized by well-demarcated fibrotic nodular lesions in the lung (efig. 9-13 ). until recently, the pathogenesis was unclear, but recent studies proposed equine herpesvirus 5 (ehv-5) as the putative etiology. grossly, the lungs show multifocal to coalescing, firm tan nodules scattered in all pulmonary lobes, which resemble pulmonary neoplasia. microscopically, alveolar walls are thickened due to collagen deposition, infiltration of lymphocytes and macrophages, and cuboidal cells lining the alveolar walls. the alveolar lumens contain neutrophils and macrophages, some of which may contain a large eosinophilic intranuclear inclusion body. typical clinical signs include weight loss, low-grade fever, and progressive exercise intolerance. this condition has a poor prognosis. rhodococcus equi. rhodococcus equi is an important cause of morbidity and mortality in foals throughout the world. this facultative intracellular gram-positive bacterium causes two major forms of disease: the first involves the intestine, causing ulcerative enterocolitis, and the second severe and often fatal bronchopneumonia. although half of foals with pneumonia have ulcerative enterocolitis, it is rare to find animals with intestinal lesions alone. occasionally, infection disseminates to lymph nodes, joints, bones, the genital tract, and other organs. because rhodococcus equi is present in soil and feces of herbivores (particularly foals), it is common for the disease to become enzootic on farms ("hot spots") where the organism has been shed earlier by infected foals. serologic evidence of infection in horses is widespread, yet clinical disease is sporadic and largely restricted to young foals or to adult horses with severe immunosuppression. virulence factors encoded by plasmids (virulenceassociated protein a [vapa gene]) are responsible for the survival and replication of rhodococcus equi in macrophages, thus determining the evolution of the disease. this bacterium has also been sporadically incriminated with infections in cattle, goats, pigs, dogs, and cats, and quite often in immunocompromised human beings, for example, those infected with the aids virus, after organ transplantation, or undergoing chemotherapy. it is still debatable whether natural infection starts as a bronchopneumonia (aerogenous route) from which rhodococcus equi reaches the intestine via swallowed sputum or whether infection starts as an enteritis (oral route) with a subsequent bacteremia into the lungs. (streptococcus equi, streptococcus zooepidemicus, or staphylococcus aureus) . uncomplicated lesions in evr are seen only in aborted fetuses or in foals that die within the first few days of life. they consist of focal areas of necrosis (0.5 to 2 mm) in various organs, including liver, adrenal glands, and lungs. in some cases, intranuclear inclusion bodies are microscopically observed in these organs. outbreaks of interstitial pneumonia in donkeys have been attributed to multiple strains of asinine herpesviruses (ahv-4 and -5). clinically, horses and donkeys affected with the respiratory form of evr exhibit fever, anorexia, conjunctivitis, cough, and nasal discharge. equine viral arteritis. equine viral arteritis (eva), a pansystemic disease of horses, donkeys, and mules caused by an arterivirus (equine arteritis virus [eav]), occurs sporadically throughout the world, sometimes as an outbreak. this virus infects and causes severe injury to macrophages and endothelial cells. gross lesions are hemorrhage and edema in many sites, including lungs, intestine, scrotum, and periorbital tissues and voluminous hydrothorax and hydroperitoneum. the basic lesion is fibrinoid necrosis and inflammation of the vessel walls (vasculitis), particularly the small muscular arteries (lymphocytic arteritis), which is responsible for the edema and hemorrhage that explain most of the clinical features. pulmonary lesions are those of interstitial pneumonia with hyperplasia of type ii pneumonocytes and vasculitis with abundant edema in the bronchoalveolar spaces and distended pulmonary lymphatic vessels. viral antigen can be detected by immunoperoxidase techniques in the walls and endothelial cells of affected pulmonary vessels and in alveolar macrophages. clinical signs are respiratory distress, fever, abortion, diarrhea, colic, and edema of the limbs and ventral abdomen. respiratory signs are frequent and consist of serous or mucopurulent rhinitis and conjunctivitis with palpebral edema. like most viral respiratory infections, eva can predispose horses to opportunistic bacterial pneumonias. african horse sickness. african horse sickness (ahs) is an arthropod-borne, oie-notifiable disease of horses, mules, donkeys, and zebras that is caused by an orbivirus (family reoviridae) and characterized by respiratory distress or cardiovascular failure. ahs has a high mortality rate-up to 95% in the native population of horses in africa, the middle east, india, pakistan, and, most recently, spain and portugal. although the ahs virus is transmitted primarily by insects (culicoides) to horses, other animals, such as dogs, can be infected by eating infected equine flesh. the pathogenesis of african horse sickness remains unclear, but this equine orbivirus has an obvious tropism for pulmonary and cardiac endothelial cells and, to a lesser extent, mononuclear cells. based on clinical signs (not pathogenesis), african horse sickness is arbitrarily divided into four different forms: pulmonary, cardiac, mixed, and mild. the pulmonary form is characterized by severe respiratory distress and rapid death because of massive pulmonary edema, presumably from viral injury to the pulmonary endothelial cells. grossly, large amounts of froth are present in the airways, lungs fail to collapse, subpleural lymph vessels are distended, and the ventral parts of the lungs are notably edematous (see fig. 4-40) . in the cardiac form, recurrent fever is detected, and heart failure results in subcutaneous and interfascial edema, most notably in the neck and supraorbital region. the mixed form is a combination of the respiratory and cardiac forms. finally, the mild form, rarely seen in postmortem rooms, is characterized by fever and clinical signs resembling those of equine influenza; it is in most cases transient and followed by a complete recovery. this mild form is most frequently seen in donkeys, mules, and zebras and in horses with some degree of immunity. detection of viral antigen for diagnostic purposes can be done by immunohistochemistry in paraffin-embedded tissues. chapter 9 respiratory system, mediastinum, and pleurae clinically, rhodococcus equi infection can be acute, with rapid death caused by severe bronchopneumonia, or chronic, with depression, cough, weight loss, and respiratory distress. in either form, there may be diarrhea, arthritis, osteomyelitis, or subcutaneous abscess formation. parascaris equorum. parascaris equorum is a large nematode (roundworm) of the small intestine of horses; the larval stages migrate through the lungs as ascarid larvae do in pigs. it is still unclear whether migration of parascaris equorum larvae can cause significant pulmonary lesions under natural conditions. experimentally, migration of larvae results in coughing, anorexia, weight loss, and small necrotic foci and petechial hemorrhages in the liver, hepatic and tracheobronchial lymph nodes, and lungs. microscopically, eosinophils are prominent in the interstitium and airway mucosa during the parasitic migration and in focal granulomas caused by dead larvae in the lung. dictyocaulus arnfieldi. dictyocaulus arnfieldi is not a very pathogenic nematode, but it should be considered if there are signs of coughing in horses that are pastured together with donkeys. donkeys are considered the natural hosts and can tolerate large numbers of parasites without ill effects. dictyocaulus arnfieldi does not usually become patent in horses, so examination of fecal samples is not useful; bal is only occasionally diagnostic because eosinophils (but not parasites) are typically found in the lavage fluid. mature parasites (up to 8 cm in length) cause obstructive bronchitis, edema, and atelectasis, particularly along the dorsocaudal lung. the microscopic lesion is an eosinophilic bronchitis similar to the less acute infestations seen in cattle and sheep with their dictyocaulus species. the results of experimental studies suggest that natural infection likely starts from inhalation of infected dust or aerosols. once in the lung, rhodococcus equi is rapidly phagocytosed by alveolar macrophages, but because of defective phagosome-lysosome fusion and premature lysosomal degranulation, bacteria survive and multiply intracellularly, eventually leading to the destruction of the macrophage. interestingly, rhodococcus equi appears to be easily killed by neutrophils but not macrophages. released cytokines and lysosomal enzymes and bacterial toxins are responsible for extensive caseous necrosis of the lungs and the recruitment of large numbers of neutrophils, macrophages, and giant cells containing intracellular gram-positive organisms in their cytoplasm. depending on the stage of infection and the immune status and age of affected horses, pulmonary lesions induced by rhodococcus equi can vary from pyogranulomatous to granulomatous pneumonia. in young foals, the infection starts as a suppurative cranioventral bronchopneumonia, which progresses within a few days into small variable-size pulmonary abscesses. these abscesses rapidly transform into pyogranulomatous nodules, some of which become confluent and form large masses of caseous exudate ( fig. 9-82 ). microscopically, the early lesion starts with neutrophilic infiltration, followed by an intense influx of alveolar macrophages into the bronchoalveolar spaces. this type of histiocytic inflammation persists for a long period of time because rhodococcus equi is a facultative intracellular organism that survives the bactericidal effects of equine alveolar macrophages. in the most chronic cases, the pulmonary lesions culminate with the formation of large caseonecrotic masses with extensive fibrosis of the surrounding pulmonary parenchyma. pcr analysis of tracheobronchial aspirates has successfully been used as an alternative to bacteriologic culture in the diagnosis of rhodococcus equi infection in live foals. b a rhinotracheitis (ibr)/bovine herpes virus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv); and noninfectious interstitial pneumonias, such as bovine pulmonary edema and emphysema, reinfection syndrome, and many others. bovine enzootic pneumonia. enzootic pneumonia, sometimes simply referred to as calf pneumonia, is a multifactorial disease caused by a variety of etiologic agents that produces an assortment of lung lesions in young, intensively housed calves. the hostmicrobial-environmental triad is central in the pathogenesis of this disease. morbidity is often high (up to 90%), but fatalities are uncommon (>5%) unless management is poor or unless new, virulent pathogens are introduced by additions to the herd. enzootic pneumonia is also called viral pneumonia because it often begins with an acute respiratory infection with bpiv-3, brsv, or possibly with one or more of several other viruses (adenovirus, bohv-1, reovirus, bovine coronavirus [bcov] , and bovine rhinitis virus). mycoplasmas, notably mycoplasma dispar, mycoplasma bovis, ureaplasma, and possibly chlamydophila, may also be primary agents. following infection with any of these agents, opportunistic bacteria, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, histophilus somni, mannheimia haemolytica, and escherichia coli, can cause a secondary suppurative bronchopneumonia, the most serious stage of enzootic pneumonia. the pathogenesis of the primary invasion and how it predisposes the host to invasion by the opportunists are poorly understood, but it is likely that there is impairment of pulmonary defense mechanisms. environmental factors, including air quality (poor ventilation), high relative humidity, and animal crowding, have been strongly incriminated. the immune status of the calf also plays an important role in the development and severity of enzootic pneumonia. calves with bovine leukocyte adhesion deficiency (blad), which prevents the migration of neutrophils from the capillaries, are highly susceptible to bronchopneumonia. lesions are variable and depend largely on the agents involved and on the duration of the inflammatory process. in the acute phases, lesions caused by viruses are those of bronchointerstitial pneumonia, which are generally mild and transient, and therefore are seen only sporadically at necropsy. microscopically, the lesions are necrotizing bronchiolitis, necrosis of type i pneumonocytes with hyperplasia of type ii pneumonocytes, and mild interstitial and alveolar edema. in the case of bpiv-3 and brsv infection, intracytoplasmic inclusion bodies and the formation of large multinucleated syncytia, resulting from the fusion of infected bronchiolar and alveolar epithelial cells, can also be observed in the lungs (fig. 9-83) . airway hyperreactivity has been described in calves after brsv infection; however, the significance of this syndrome in relation to enzootic pneumonia of calves is still under investigation. the mycoplasmas also can cause bronchiolitis, bronchiolar and alveolar necrosis, and an interstitial reaction, but in contrast to viral-induced pneumonias, mycoplasmal lesions tend to progress to a chronic stage characterized by striking peribronchiolar lymphoid hyperplasia (cuffing pneumonia). when complicated by secondary bacterial infections (e.g., pasteurella multocida and trueperella pyogenes), viral or mycoplasmal lesions change from a pure bronchointerstitial to a suppurative bronchopneumonia (fig. 9-84) . in late stages of bronchopneumonia, the lungs contain a creamy-mucoid exudate in the airways and later often have pulmonary abscesses and bronchiectasis (see fig. 9-11) . note that the same viruses and mycoplasmas involved in the enzootic pneumonia complex can also predispose cattle to other diseases, such as pneumonic mannheimiosis (mannheimia aspiration pneumonia. aspiration pneumonia is often a devastating sequela to improper gastric tubing of horses, particularly exogenous lipid pneumonia from mineral oil delivered into the trachea in treatment of colic. gross and microscopic lesions are described in detail in the section on aspiration pneumonias of cattle. opportunistic infections. chlamydophila (chlamydia) spp., obligatory intracellular zoonotic pathogens, can cause systemic infection in many mammalian and avian species; in horses, they can also cause keratoconjunctivitis, rhinitis, pneumonia, abortion, polyarthritis, enteritis, hepatitis, and encephalitis. serologic studies suggest that infection without apparent disease is common in horses. horses experimentally infected with chlamydophila psittaci develop mild and transient bronchointerstitial pneumonia. there are unconfirmed reports suggesting a possible association between these organisms and recurrent airway obstruction in horses. detection of chlamydial organisms in affected tissue is not easy and requires special laboratory techniques such as pcr, immunohistochemistry, and fluorescent antibody tests. horses are only sporadically affected with mycobacteriosis (mycobacterium avium complex, mycobacterium tuberculosis, and mycobacterium bovis). the intestinal tract and associated lymph nodes are generally affected, suggesting an oral route of infection with subsequent hematogenous dissemination to the lungs. the tubercles (granulomas) differ from those in ruminants and pigs, being smooth, gray, solid, sarcoma-like nodules without grossly visible caseous necrosis or calcification (efig. 9-14) . microscopically, the tubercles are composed of macrophages, epithelioid cells, and multinucleated giant cells. fibrosis increases with time, accounting in part for the sarcomatous appearance. adenovirus infections occur commonly in arabian foals with combined immunodeficiency (cid), a hereditary lack of b and t lymphocytes. in cases of adenoviral infection, large basophilic or amphophilic inclusions are present in the nuclei of tracheal, bronchial, bronchiolar, alveolar, renal, and intestinal epithelial cells. as it occurs in other species, infection with a unique fungal pathogen known as pneumocystis carinii typically occurs in immunosuppressed or immunoincompetent individuals such as arabian foals with cid (see fig. 9 -20). diagnosis of pneumocystis carinii requires microscopic examination of lungs and special stains. idiopathic interstitial pneumonia. interstitial and bronchointerstitial pneumonias of undetermined cause that can progress to severe pulmonary fibrosis have been reported in foals and young horses. the gross and microscopic lesions are reminiscent of those of bovine pulmonary edema and emphysema or ards. the lungs are notably congested and edematous and microscopically are characterized by necrosis of the bronchiolar epithelium, alveolar edema, hyperplasia of type ii pneumonocytes, and hyaline membranes. the cause of this form of equine interstitial pneumonia is not known, but toxic and particularly viral causes have been proposed. bovine respiratory disease complex (brdc) and acute undifferentiated respiratory disease are general terms often used by clinicians to describe acute and severe bovine respiratory illness of clinically undetermined cause. these terms do not imply any particular type of pneumonia and therefore should not be used in pathology reports. clinically, the brd complex includes bovine enzootic pneumonia (multifactorial etiology); pneumonic mannheimiosis (mannheimia haemolytica); respiratory histophilosis (histophilus somni), previously known as respiratory hemophilosis (haemophilus somnus); mycoplasma bovis; respiratory viral infections, such as infectious bovine 528.e1 chapter 9 respiratory system, mediastinum, and pleurae pneumonic mannheimiosis (shipping fever) is the most important respiratory disease of cattle in north america, particularly in feedlot animals that have been through the stressful marketing and assembly processes. mannheimia haemolytica biotype a, serotype 1 is the etiologic agent most commonly responsible for the severe pulmonary lesions. a few investigators still consider that pasteurella multocida and other serotypes of mannheimia haemolytica are also causes of this disease. even after many years of intense investigation, from the gross lesions to the molecular aspects of the disease, the pathogenesis of pneumonic mannheimiosis remains incompletely understood. experiments have established that mannheimia haemolytica a1 alone is usually incapable of causing disease because it is rapidly cleared by pulmonary defense mechanisms. these findings may explain why mannheimia haemolytica, despite being present in the nasal cavity of healthy animals, only sporadically causes disease. for mannheimia haemolytica to be established as a pulmonary infection, it is first required that stressors impair the defense mechanisms and allow the bacteria to colonize the lung (see section on impairment of defense haemolytica). clinically, enzootic pneumonia is usually mild, but fatal cases are occasionally seen even in farms with optimal health management. pneumonic mannheimiosis (shipping fever). shipping fever (transit fever) is a vague clinical term used to denote acute respiratory diseases that occur in cattle several days or weeks after shipment. the disease is characterized by a severe fibrinous bronchopneumonia, reflecting the fact that death generally occurs early or at an acute stage. because mannheimia haemolytica (formerly pasteurella haemolytica) is most frequently isolated from affected lungs, the names pneumonic mannheimiosis and pneumonic pasteurellosis have been used synonymously. it is known that pneumonic mannheimiosis can occur in animals that have not been shipped and that organisms other than mannheimia haemolytica can cause similar lesions. therefore the term shipping fever should be relinquished in favor of more specific names, such as pneumonic mannheimiosis or respiratory histophilosis. irregular areas of coagulative necrosis are typically bordered by a rim of elongated cells often referred to as oat-shaped cells or oat cells that are degenerating neutrophils mixed with a few alveolar macrophages (see fig. 9 -86). in the early stages of necrosis, there is no evidence of vascular thrombosis, suggesting that necrosis is primarily caused by the cytotoxin of mannheimia haemolytica and is not the result of an ischemic change. the interlobular septa become distended with protein-rich edematous fluid, and the lymphatic vessels contain fibrin thrombi. the trachea and bronchi can have considerable amounts of blood and exudate, which are transported by the mucociliary escalator or coughed up from deep within the lungs, but the walls of the trachea and major bronchi may or may not be involved. because of the necrotizing process, sequelae to pneumonic mannheimiosis can be serious and can include abscesses, encapsulated sequestra (isolated pieces of necrotic lung), chronic pleuritis, fibrous pleural adhesions, and bronchiectasis. clinically, pneumonic mannheimiosis is characterized by a severe toxemia that can kill animals even when considerable parts of the lungs remain functionally and structurally normal. cattle usually become depressed, febrile (104° to 106° f [40° to 41° c]), and anorexic and have a productive cough, encrusted nose, mucopurulent nasal exudate, shallow respiration, or an expiratory grunt. hemorrhagic septicemia. pneumonic mannheimiosis should not be confused with hemorrhagic septicemia (septicemic pasteurellosis) of cattle and water buffalo (bubalus bubalis) caused by inhalation or ingestion of serotypes 6:b and 6:e of pasteurella multocida. this oie-notifiable disease does not occur in north america and currently is reported only from some countries in asia, africa, and recently in germany. in contrast to pneumonic mannheimiosis, in which lesions are always confined to the lower respiratory tract, the bacteria of hemorrhagic septicemia always disseminates hematogenously to other organs. at necropsy, typically, generalized petechiae are present on the serosal surfaces of the intestine, heart, and lungs and in skeletal muscles. superficial and visceral lymph nodes are swollen and hemorrhagic. variable lesions include edematous and hemorrhagic lungs with or without consolidation; hemorrhagic enteritis; blood-tinged fluid in the thorax and abdomen; and subcutaneous edema of the head, neck, and ventral abdomen. bacteria can be cultured from blood, and animals have high fever and die rapidly (100% case fatality). respiratory histophilosis (haemophilosis). respiratory histophilosis is part of the histophilus somni (haemophilus somnus) disease complex, which has at least eight different clinicopathologic forms, each one involving different organs. this complex includes septicemia, encephalitis (known as thrombotic meningoencephalitis [tme]), pneumonia (respiratory histophilosis), pleuritis, myocarditis, arthritis, ophthalmitis, conjunctivitis, otitis, and abortion. the portals of entry for the different forms of histophilosis have not been properly established. the respiratory form of bovine histophilosis is the result of the capacity of the bacterium to induce both suppurative and fibrinous bronchopneumonia (efig. 9-15 ). the latter is in some cases indistinguishable from that of pneumonic mannheimiosis. the pathogenesis of respiratory histophilosis is still poorly understood, and the disease cannot be reproduced consistently by administration of histophilus somni alone. like mannheimia haemolytica, it requires predisposing factors such as stress or a preceding viral infection. histophilus somni is often isolated from the lungs of calves with enzootic pneumonia. the capacity of histophilus somni to cause septicemia and localized infections in the lungs, brain, eyes, ear, heart, mammary gland, male and female genital organs, or placenta is perhaps attributable to specific virulence factors, such as immunoglobulin-binding proteins (igbps) and lipooligosaccharide (los). also, histophilus mechanisms). these stressors include weaning, transport, fatigue, crowding, mixing of cattle from various sources, inclement weather, temporary starvation, and viral infections. horizontal transmission of viruses and mannheimia haemolytica occurs during crowding and transportation of cattle. viruses that most commonly predispose cattle to pneumonic mannheimiosis include bohv-1, bpiv-3, and brsv. once established in the lungs, mannheimia haemolytica causes lesions by means of different virulence factors, which include endotoxin, lipopolysaccharide, adhesins, and outer membrane proteins; however, the most important is probably the production of a leukotoxin (exotoxin), which binds and kills bovine macrophages and neutrophils. the fact that this toxin exclusively affects ruminant leukocytes probably explains why mannheimia haemolytica is a respiratory pathogen in cattle and sheep but not in other species. during mannheimia haemolytica infection, alveolar macrophages, neutrophils, and mast cells release maximum amounts of proinflammatory cytokines, particularly tnf-α, il-1, il-8, adhesion molecules, histamine, and leukotrienes. by locally releasing enzymes and free radicals, leukocytes further contribute to the injury and necrosis of bronchiolar and alveolar cells. the gross lesions of acute and subacute pneumonic mannheimiosis are the prototypic fibrinous bronchopneumonia, with prominent fibrinous pleuritis ( fig. 9-85 and see fig. 9 -72) and pleural effusion. lesions are always cranioventral and usually ventral to a horizontal line through the tracheal bifurcation. the interlobular septa are distended by yellow, gelatinous edema and fibrin. the "marbling" of lobules is the result of intermixing areas of coagulation necrosis, interlobular interstitial edema, and congestion ( fig. 9-86) . microscopically, lung lesions are evident 4 hours after experimental infection in which neutrophils fill the bronchial, bronchiolar, and alveolar spaces. within 24 to 48 hours, the cytotoxic effect of mannheimia haemolytica is manifested by necrosis of individual alveolar cells and fibrin begins to exude into the alveoli from increased permeability of the air-blood barrier. these changes are exacerbated by endothelial swelling, altered platelet function, increased procoagulant activity, and diminished profibrinolytic activity in the lungs. by 72 hours, alveolar macrophages start to appear in the bronchoalveolar space. at this time, large and the pulmonary defense mechanisms. lung lesions are typically those of a chronic bronchopneumonia with numerous well-delineated caseonecrotic nodules (fig. 9-87 and e-fig. 9-16) . microscopically, lesions are quite characteristic and consist of distinct areas of pulmonary necrosis centered on bronchi or bronchioles. the lesion is formed by a core of fine eosinophilic granular debris surrounded by a rim of neutrophils, macrophages, and fibroblasts (see fig. 9-87) . although the origin of the caseonecrotic lesions is under investigation, recent studies incriminate reactive oxygen species (ros) and reactive nitrogen species (rns) as the major contributors for cell injury in the lung. the diagnosis is confirmed by isolation or somni has the ability to undergo structural and antigenic variation, evade phagocytosis by promoting leukocytic apoptosis, inhibit intracellular killing, reduce transferrin concentrations, and induce endothelial apoptosis in the lungs of affected calves. mixed pulmonary infections of histophilus somni, mannheimia haemolytica, pasteurella multocida, trueperella pyogenes, and mycoplasmas are fairly common in calves. mycoplasma bovis pneumonia. mycoplasma bovis is the most common mycoplasma sp. isolated from pneumonic lungs of cattle in europe and north america. pulmonary infection is exacerbated by stress or any other adverse factor (e.g., viral infection) that depresses n control programs for infectious disease. it was eradicated from north america in 1892 and from australia in the 1970s, but it is still enzootic in large areas of africa, asia, and eastern europe. the etiologic agent, mycoplasma mycoides ssp. mycoides small colony type, was the first mycoplasma isolated and is one of the most pathogenic of those that infect domestic animals. natural infection occurs in cattle and asian buffalo. the portal of entry is aerogenous, and infections occur when a susceptible animal inhales infected droplets. the pathogenic mechanisms are still inadequately understood but are suspected to involve toxin and galactan production, unregulated production of tnf-α, ciliary dysfunction, immunosuppression, and immune-mediated vasculitis. vasculitis and thrombosis of pulmonary arteries, arterioles, veins, and lymphatic vessels lead to lobular infarction. the name of the disease is a good indication of the gross lesions. it is a severe, fibrinous bronchopneumonia (pleuropneumonia) similar to that of pneumonic mannheimiosis (see figs. 9-72 and 9-85) but having a more pronounced "marbling" of the lobules because of extensive interlobular edema and lymphatic thrombosis. typically, 60% to 79% of lesions are in the caudal lobes (not cranioventrally), and pulmonary sequestra (necrotic lung encapsulated by connective tissue) are more frequent and larger than pneumonic mannheimiosis. unilateral lesions are common in this disease. microscopically, the appearance again is like that of pneumonic mannheimiosis, except that vasculitis and thrombosis of pulmonary arteries, arterioles, and capillaries are much more obvious and are clearly the major cause of the infarction and thrombosis of lymphatic vessels in interlobular septa. mycoplasma mycoides ssp. mycoides small colony type remains viable in the sequestra for many years, and under stress (e.g., starvation), the fibrous capsule may break down releasing mycoplasma into the airways, thus becoming a source of infection for other animals. clinical signs are those of severe sepsis, including fever, depression, and anorexia followed by severe respiratory signs such as opened-mouth breathing, dyspnea and coughing, and crepitation and pleural friction on thoracic auscultation. vaccination is highly effective in preventing the disease. bovine tuberculosis. tuberculosis is an ancient, communicable, worldwide, chronic disease of human beings and domestic animals. it continues to be a major problem in human beings in underdeveloped countries, and it is on the rise in some industrialized nations, largely because of the immunosuppressive effects of aids, immigration, and movement of infected animals across borders. the world health organization (who) estimates that more than 1 million people die of tuberculosis and 8 million new cases appear each year, mostly in developing countries. mycobacterium tuberculosis is transmitted between human beings, but where unpasteurized milk is consumed, mycobacterium bovis from the milk of cattle with mammary tuberculosis is also an important cause of human tuberculosis. mycobacterium bovis infections have also been reported in a number of domestic and wild mammalian species; in some countries, wildlife reservoirs exist and may act as a source of infection for cattle. bovine tuberculosis is primarily caused by mycobacterium bovis, but infection with mycobacterium tuberculosis, the pathogen of human tuberculosis, and mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) can occur sporadically. tuberculosis can be acquired by several routes, but infection of the lungs by inhalation of mycobacterium bovis is the most common in adult cattle, whereas ingestion of infected milk is more predominant in young animals. organisms belonging to the mycobacterium avium complex can also infect cattle, but for infection caused by these organisms, the term atypical mycobacteriosis (not tuberculosis) is currently preferred. immunohistochemical labeling of tissue sections for mycoplasma antigens. mycoplasma bovis is also incriminated in arthritis, otitis, mastitis, abortion, and keratoconjunctivitis. contagious bovine pleuropneumonia. contagious bovine pleuropneumonia is an oie-notifiable disease of historic interest in veterinary medicine because it was the object of early national a b c than 90% of bovine cases, a chronic, moist cough can progress to dyspnea. enlarged tracheobronchial lymph nodes can contribute to the dyspnea by impinging on airways, and the enlargement of caudal mediastinal nodes can compress the caudal thoracic esophagus and cause bloating. interstitial pneumonias. atypical interstitial pneumonia (aip) is a vague clinical term well entrenched in veterinary literature but one that has led to enormous confusion among veterinarians. it was first used to describe acute or chronic forms of bovine pneumonia that did not fit in any of the "classic" forms because of the lack of exudate and lack of productive cough. microscopically, the criteria for diagnosis of aip in cattle were based on the absence of obvious exudate and the presence of edema, interstitial emphysema (see the section on pulmonary emphysema), hyaline membranes, hyperplasia of type ii pneumonocytes, and alveolar fibrosis with interstitial cellular infiltrates. at that time, any pulmonary disease or pulmonary syndrome that had a few of the previously mentioned lesions was traditionally diagnosed as aip, and grouping all these different syndromes together was inconsequential because their etiopathogenesis were then unknown. field and laboratory investigations have demonstrated that most of the bovine syndromes previously grouped under aip have rather different causes and pathogeneses ( fig. 9-88) . furthermore, what was "atypical" in the past has become so common that it is fairly routine nowadays to find "typical cases" of aip. for all these reasons, investigators, largely from britain, proposed that all these syndromes previously clustered into aip should be named according to their specific cause or pathogenesis. the most common bovine syndromes characterized by edema, emphysema, hyaline membranes, and hyperplasia of type ii pneumonocytes include bovine pulmonary edema and emphysema (fog fever), "extrinsic allergic alveolitis" (hypersensitivity pneumonitis), "reinfection syndromes" (hypersensitivity to dictyocaulus sp. or brsv), milk allergy, ingestion of moldy potatoes, paraquat toxicity, toxic silo gases, mycotoxins, and others. acute bovine pulmonary edema and emphysema (fog fever). acute bovine pulmonary edema and emphysema (abpee), known in britain as fog fever (no association with atmospheric conditions), occurs in cattle usually grazing "fog" pastures (i.e., aftermath or foggage, regrowth after a hay or silage has been cut). epidemiologically, abpee usually occurs in adult beef cattle in the fall when there is a change in pasture from a short, dry grass to a lush, green grass. it is generally accepted that l-tryptophan present in the pasture is metabolized in the rumen to 3-methylindole, which in turn is absorbed into the bloodstream and carried to the lungs. mixed function oxidases present in the nonciliated bronchiolar epithelial (club) cells metabolize 3-methylindole into a highly pneumotoxic compound that causes extensive and selective necrosis of bronchiolar cells and type i pneumonocytes (fig. 9-89 and see fig. 9 -88) and increases alveolar permeability, leading to edema, thickening of the alveolar interstitium, and alveolar and interstitial emphysema. 3-methylindole also interferes with the lipid metabolism of type ii pneumonocytes. the gross lesions are those of a diffuse interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema (see fig. 9-55, a) . the lungs are expanded, pale, and rubbery in texture, and the lesions are most notable in the caudal lobes. microscopically, the lesions are alveolar and interstitial edema and emphysema, formation of characteristic hyaline membranes within alveoli (see fig. 9-55, b) , and in those animals that survive for several days, hyperplasia of type ii pneumonocytes and alveolar interstitial fibrosis. respiratory infection usually starts when inhaled bacilli reach the alveoli and are phagocytosed by pulmonary alveolar macrophages. if these cells are successful in destroying the bacteria, infection is averted. however, mycobacterium bovis, being a facultative pathogen of the monocytic-macrophage system, may multiply intracellularly, kill the macrophage, and initiate infection. from this first nidus of infection, bacilli spread aerogenously via airways within the lungs and eventually via the lymph vessels to tracheobronchial and mediastinal lymph nodes. the initial focus of infection at the portal of entry (lungs) plus the involvement of regional lymph nodes is termed the primary (ghon) complex of tuberculosis. if the infection is not contained within this primary complex, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. hematogenous dissemination occurs sporadically when a granuloma containing mycobacteria erodes the wall of a blood vessel, causes vasculitis, and allows the granuloma to discharge mycobacteria into the alveolar circulation. if dissemination is sudden and massive, mycobacteria are widely disseminated and numerous small foci of infection develop in many tissues and organs and the process is referred to as miliary tuberculosis (like millet seeds). the host becomes hypersensitive to the mycobacterium, which enhances the cell-mediated immune defenses in early or mild infections but can result in host-tissue destruction in the form of caseous necrosis. the evolution and dissemination of the pulmonary infection are closely regulated by cytokines and tnf-α production by alveolar macrophages. unlike abscesses that tend to grow rather fast, granulomas evolve slowly at the site of infection. the lesion starts with few macrophages and neutrophils ingesting the offending organism, but because mycobacterium organisms are resistant to phagocytosis, infected macrophages eventually die, releasing viable bacteria, lipids, and cell debris. cell debris accumulates in the center of the lesion, whereas viable bacteria and bacterial lipids attract additional macrophages and a few lymphocytes at the periphery of the lesion. some of these newly recruited macrophages are activated by local lymphocytes and become large phagocytic cells with abundant cytoplasm resembling epithelial cells, thus the term epithelioid macrophages. multinucleated giant cells (also macrophages) appear at the edges of the lesion, and finally the entire focus of inflammatory process becomes surrounded by fibroblasts and connective tissue (see fig. 9 -81). it may take weeks or months for a granuloma to be grossly visible. bovine tuberculosis, the prototype for granulomatous pneumonia, is characterized by the presence of a few or many caseated granulomas (see fig. 9 -80). the early gross changes are small foci (tubercles) most frequently seen in the dorsocaudal, subpleural areas. with progression, the lesions enlarge and become confluent with the formation of large areas of caseous necrosis. calcification of the granulomas is a typical finding in bovine tuberculosis. single nodules or clusters occur on the pleura and peritoneum, and this presentation has been termed pearl disease. microscopically, the tubercle is composed of mononuclear cells of various types. in young tubercles, which are noncaseous, epithelioid and langhans' giant cells are at the center, surrounded by lymphocytes, plasma cells, and macrophages. later, caseous necrosis develops at the center, secondary to the effects of cell-mediated hypersensitivity and enclosed by fibrosis at the periphery. acid-fast organisms may be numerous but more often are difficult to find in histologic section or smears. clinically, the signs of tuberculosis relate to the dysfunction of a particular organ system or to general debilitation, reduced milk production, and emaciation. in the pulmonary form, which is more grossly, the postmortem lesions vary from subtle, gray, subpleural foci (granulomatous inflammation) to severe lesions, in which the lungs are firm and heavy and have a "meaty appearance" because of interstitial pneumonia (efig. 9 -17) with type ii pneumonocyte hyperplasia, lymphocytic infiltration, and interstitial fibrosis. characteristically, discrete noncaseous granulomas formed in response to the deposition of antigen-antibody complexes are scattered throughout the lungs. chronic cases of extrinsic allergic alveolitis can eventually progress to diffuse fibrosing alveolitis. clinically, it can be acute or chronic; the latter has a cyclical pattern of exacerbation during winter months. weight loss, coughing, and poor exercise tolerance are clinical features. full recovery can occur if the disease is recognized and treated early. reinfection syndrome. hypersensitivity to reinfection with larvae of dictyocaulus viviparus is another allergic syndrome manifested in the lungs that causes signs and lesions indistinguishable from abpee, with the exception of eosinophils and possibly larvae in the alveolar exudate. the hypersensitivity reaction in the lung causes diffuse alveolar damage and edema, necrosis of type i pneumonocytes, and hyperplasia of type ii pneumonocytes. in the later stages of the disease, there is formation of small granulomas with interstitial infiltrates of mononuclear cells. it has been suggested but not confirmed that emphysema with diffuse proliferative alveolitis and formation of hyaline membranes can also occur sporadically in the late stages of brsv infection in cattle. presumably, this disease shares many similarities with "atypical" infections occasionally seen in children with respiratory syncytial virus (rsv human strain), in which a hypersensitivity to the virus or virus-induced augmentation of the immune response results in hypersensitivity pneumonitis (see fig. 9 -88). brsv infection is also known to enhance hypersensitivity to environmental allergens in cattle. other forms of bovine interstitial pneumonia. inhalation of manure ("pit") gases, such as nitrogen dioxide (no 2 ), hydrogen interstitial cell infiltrates, fibrosis, emphysema acute proliferation phase hyperplasia of type ii pneumonocytes clinically, severe respiratory distress develops within 10 days of the abrupt pasture change, and cattle develop expiratory dyspnea, oral breathing, and evidence of emphysema within the lungs and even subcutaneously along the back. experimentally, reducing ruminal conversion of l-tryptophan to 3-methylindole prevents the development of abpee. a number of other agents cause virtually the same clinical and pathologic syndrome as is seen in abpee. the pathogenesis is assumed to be similar, although presumably other toxic factors are specific for each syndrome. one of these pneumotoxic factors is 4-ipomeanol, which is found in moldy sweet potatoes contaminated with the fungus fusarium solani. mixed function oxidases in the lungs activate 4-ipomeanol into a potent pneumotoxicant capable of producing irreversible oxidative injury to type i pneumonocytes and bronchiolar epithelial cells, presumably through lipoperoxidation of cell membranes. similarly, purple mint (perilla frutescens), stinkwood (zieria arborescens), and rapeseed and kale (brassica species) also cause pulmonary edema, emphysema, and interstitial pneumonia. extrinsic allergic alveolitis. extrinsic allergic alveolitis (hypersensitivity pneumonitis), one of the most common allergic diseases in cattle, is seen mainly in housed adult dairy cows in the winter. this disease shares many similarities with its human counterpart known as farmer's lung, which results from a type iii hypersensitivity reaction to inhaled organic antigens, most commonly microbial spores, mainly of the thermophilic actinomycete, saccharopolyspora rectivirgula (micropolyspora faeni), commonly found in moldy hay. this is followed by an antibody response to inhaled spores and local deposition of antigen-antibody complexes (arthus reaction) in the lungs (see fig. 9 -88). because it affects only a few animals of the herd or the sporadic person working in a farm, it is presumed that intrinsic host factors, such as dysregulation of dendritic cells, t lymphocytes, igg, interleukins, ifn-γ, and surfactant, are involved in the pathogenesis of the disease. chapter 9 respiratory system, mediastinum, and pleurae efigure 9 -17 interstitial pneumonia, adult cow. note meaty appearance of the pulmonary parenchyma and mild edematous distention of the interlobular septa. inset, thick hyaline membranes (arrows) lining hypercellular alveolar walls. hypersensitivity pneumonia was suspected. (courtesy dr. a. lópez, atlantic veterinary college.) gases, inhalation of no 2 (silo gas) also causes bronchiolitis, edema, and interstitial pneumonia and, in survivors, bronchiolitis obliterans ("silo filler's disease"). smoke inhalation resulting from barn or house fires is sporadically seen by veterinarians and pathologists. in addition to skin burns, animals involved in fire accidents suffer extensive thermal injury produced by the heat on the nasal and laryngeal mucosa, and severe chemical irritation caused by inhalation of combustion gases and particles in the lung. animals that survive or are rescued from fires frequently develop nasal, laryngeal, and tracheal edema, and pulmonary hemorrhage and alveolar edema, which are caused by chemical injury to the blood-air barrier or by ards caused by the excessive production of free radicals during the pulmonary inflammatory response (see efig. 9-7) . microscopic examination of the lungs often reveals carbon particles (soot) on mucosal surfaces of the conducting system. verminous pneumonia (dictyocaulus viviparus). pulmonary lesions in parasitic pneumonias vary from interstitial pneumonia caused by migrating larvae to chronic bronchitis from intrabronchial adult parasites, to granulomatous pneumonia, which is caused by dead larvae, aberrant parasites, or eggs of parasites. in many cases, an "eosinophilic syndrome" in the lungs is characterized by infiltrates of eosinophils in the pulmonary interstitium and bronchoalveolar spaces and by blood eosinophilia. atelectasis and emphysema secondary to the obstruction of airways by parasites and mucous secretions are also common findings in parasitic pneumonias. the severity of these lesions relates to the numbers and size of the parasites and the nature of the host reaction, which sometimes includes hypersensitivity reactions (see section on reinfection syndrome). a common general term for all of these diseases is verminous pneumonia, and the adult nematodes are often visible grossly in the airways ( fig. 9-90) . dictyocaulus viviparus is an important pulmonary nematode (lungworm) responsible for a disease in cattle referred to as verminous pneumonia or verminous bronchitis. adult parasites live in the bronchi of cattle, mainly in the caudal lobes, and cause severe bronchial irritation, bronchitis, and pulmonary edema, which in turn are responsible for lobular atelectasis and interstitial emphysema. atelectasis is confined to the lobules of the lungs ventilated by the obstructed bronchi (dorsocaudal). interstitial emphysema (interlobular) is caused by forced expiratory movements against a partially obstructed single bronchus. in addition to the inflammation of bronchial mucosa, bronchoaspiration of larvae and eggs also causes an influx of leukocytes into the bronchoalveolar space (alveolitis). verminous pneumonia is most commonly seen in calves during their first summer grazing pastures that are repeatedly used from year to year, particularly in regions of europe that have a moist cool climate. the parasite can overwinter in pastures, even in climates as cold as canada's, and older animals may be carriers for a considerable length of time. at necropsy, lesions appear as dark or gray, depressed, wedgeshaped areas of atelectasis involving few or many lobules usually along the dorsocaudal aspect of the lungs. on cut surface, edematous foam and mucus mixed with white, slender (up to 80-mm long) nematodes are visible in the bronchi (see fig. 9 -90). in the most severe cases, massive numbers of nematodes fill the bronchial tree. microscopically, the bronchial lumens are filled with parasites admixed with mucus because of goblet cell hyperplasia, and there is squamous metaplasia of the bronchial and bronchiolar epithelium because of chronic irritation. there are also inflammatory infiltrates in the bronchial mucosa; alveolar edema; hyperplasia of balt sulfide (h 2 s), and ammonia (nh 3 ), from silos or sewage can be a serious hazard to animals and human beings. at toxic concentrations, these gases cause necrosis of bronchiolar cells and type i pneumonocytes and fulminating pulmonary edema that causes asphyxiation and rapid death (see fig. 9-60) . like other oxidant secretory granules released by club cells contain several proteins, such as surfactantlike protein, antiinflammatory protein (cc10), and bronchiolar lining proteins. b, ros produced by club cells are also absorbed into capillaries within the lamina propria and are transferred by the circulatory system to pulmonary capillaries where they disrupt the air-blood barrier, causing degeneration and necrosis of type i pneumonocytes. this process leads to leakage of plasma fluid (alveolar edema [pink color]) and extravasation of erythrocytes (alveolar hemorrhage) and neutrophils (inflammation). ingested pneumotoxicants can be metabolized by the liver, leading to release of ros into the circulatory system that then disrupts the air-blood barrier in a similar manner. fig. 9 -77). microscopically, there are focal intraalveolar hemorrhages caused by larvae migrating through the alveolar walls. some larvae admixed with edematous fluid and cellular exudate (including eosinophils) may be visible in bronchioles and alveoli. the alveolar walls are thickened because of edema and a few inflammatory cells. clinical signs include cough and expiratory dyspnea to the point of oral breathing. hydatid cysts, the intermediate stage of echinococcus granulosus, can be found in the lungs and liver and other viscera of sheep and to a lesser extent in cattle, pigs, goats, horses, and human beings. the adult stage is a tapeworm that parasitizes the intestine of canidae. hydatidosis is still an important zoonosis in some countries, and perpetuation of the parasite life cycle results from animals being fed uncooked offal from infected sheep and consumption of uninspected meat. hydatid cysts are generally 5 to 15 cm in diameter, and numerous cysts can be found in the viscera of affected animals ( fig. 9-91 ). each parasitic cyst is filled with clear fluid; numerous daughter cysts attach to the wall, each containing several "brood capsules" with protoscolices inside. hydatid cysts have little clinical significance in animals but are economically important because of carcass condemnation. aspiration pneumonias. the inhalation of regurgitated ruminal contents or iatrogenic deposition of medicines or milk into the trachea can cause severe and often fatal aspiration pneumonia. bland substances, such as mineral oil, may incite only a mild suppurative or histiocytic bronchopneumonia, whereas some "home remedies" or ruminal contents are highly irritating and cause a fibrinous, necrotizing bronchopneumonia. the right cranial lung lobe tends to be more severely affected because the right cranial bronchus is the most cranial branch and enters the ventrolateral aspect of the trachea. however, the distribution may vary when animals aspirate while in lateral recumbency. in some severe cases, pulmonary necrosis can be complicated by infection with saprophytic organisms present in ruminal contents, causing fatal gangrenous pneumonia. aspiration pneumonia should always be considered in animals whose swallowing has been compromised-for example, those with cleft palate or hypocalcemia (milk fever). on the other hand, neurological diseases such as encephalitis (e.g., rabies) or encephalopathy (e.g., lead poisoning) should be investigated in animals in which the cause of aspiration pneumonia could not be caused by persistent immunologic stimuli; hypertrophy and hyperplasia of bronchiolar smooth muscle because of increased contraction and decreased muscle relaxation; and a few eosinophilic granulomas around the eggs and dead larvae. these granulomas, grossly, are gray, noncaseated nodules (2 to 4 mm in diameter) and may be confused with those seen at the early stages of tuberculosis. the clinical signs (coughing) vary with the severity of infection, and severe cases can be confused clinically with interstitial pneumonias. expiratory dyspnea and death can occur with heavy parasitic infestations when there is massive obstruction of airways. a different form of bovine pneumonia, an acute allergic reaction known as reinfection syndrome, occurs when previously sensitized adult cattle are exposed to large numbers of larvae (dictyocaulus viviparus). lesions in this syndrome are those of a hypersensitivity pneumonia as previously described. other lung parasites. ascaris suum is the common intestinal roundworm of pigs; larvae cannot complete their life cycle in calves, but the larvae can migrate through the lungs and cause severe pneumonia and death of calves within 2 weeks of infection. infection is usually acquired from the soil on which infested pigs were previously kept. the gross lesions are a diffuse interstitial pneumonia with hemorrhagic foci, atelectasis, and interlobular edema and it also occurs in canada, europe, australia, and probably elsewhere. this disease has two major clinicopathologic forms: one involves the central nervous system of goat kids and young goats and is characterized by a nonsuppurative leukoencephalomyelitis; the other form involves the joints of adult goats and is characterized by a chronic, nonsuppurative arthritis-synovitis. in addition, infection with cae virus can cause chronic lymphocytic interstitial pneumonia. the lentivirus of cae, caprine arthritis and encephalitis virus (caev), is closely related to visna/maedi virus and, in fact, cross infection with cae virus in sheep has been achieved experimentally. similar to maedi, cae infection presumably occurs during the first weeks of life when the doe transmits the virus to her offspring through infected colostrum or milk. horizontal transmission between infected and susceptible goats via the respiratory route has also been described. after coming into contact with mucosal cells at the portal of entry, the virus is phagocytized by macrophages, which migrate to the regional lymph nodes. infected macrophages are disseminated hematogenously to the central nervous system, joints, lungs, and mammary glands. like maedi, there is some evidence that the recruitment of lymphocytic cells results from dysregulation of cytokine production by infected macrophages and lymphocytes in affected tissues. it can take several months before serum antibodies can be detected in infected goats. grossly, the interstitial pneumonia is diffuse and tends to be most severe in the caudal lobes. the lungs are gray-pink and firm in texture with numerous, 1-to 2-mm, gray-white foci on the cut surface. the tracheobronchial lymph nodes are consistently enlarged. microscopically, the alveolar walls are thickened by lymphocytes and conspicuous hyperplasia of type ii pneumonocytes ( fig. 9-93 ). one important difference between the pneumonias of cae and maedi is that in cae the alveoli are filled with proteinaceous eosinophilic material (alveolar proteinosis), which in electron micrographs has structural features of pulmonary surfactant. the pulmonary form of cae can be mistaken for parasitic pneumonia (muellerius capillaris) because these two diseases have lymphocytic interstitial pneumonia and can coexist in the same goat. explained otherwise. depending on the nature of the aspirated material, histopathologic evaluation generally reveals foreign particles such as vegetable cells, milk droplets, and large numbers of bacteria in bronchi, bronchioles, and alveoli (efig. 9-18 ). vegetable cells and milk typically induce an early neutrophilic response followed by a histiocytic reaction with "foreign body" multinucleated giant cells (see efig. 9-12 ). special stains are used for the microscopic confirmation of aspirated particles in the lung (e.g., pas for vegetable cells and oil red-o for oil or milk droplets). maedi (visna/maedi). maedi is an important, lifelong, and persistent viral disease of sheep and occurs in most countries, except australia and new zealand. maedi means "shortness of breath" in the icelandic language, and it is known as graaff-reinet disease in south africa, zwoegerziekte in the netherlands, la bouhite in france, and ovine progressive pneumonia (opp) in the united states. more recently, the disease has also been referred to as ovine lentivirusinduced lymphoid interstitial pneumonia or simply lymphoid interstitial pneumonia (lip). maedi is caused by visna/maedi virus (vmv), a nononcogenic small ruminant lentivirus (srlv) of the family retroviridae that is antigenically related to the lentivirus causing caprine arthritisencephalitis (cae). seroepidemiologic studies indicate that infection is widespread in the sheep population, yet the clinical disease seems to be rare. the pathogenesis is incompletely understood, but it is known that transmission occurs largely vertically, through ingestion of infected colostrum, and horizontally, via inhalation of infected respiratory secretions. once in the body, the ovine lentivirus causes lifelong infections within monocytes and macrophages, including alveolar and pulmonary intravascular macrophages; clinical signs do not develop until after a long incubation period of 2 years or more. pulmonary lesions at the time of death are severe interstitial pneumonia and failure of the lungs to collapse when the thorax is opened. notable rib imprints, indicators of uncollapsed lungs, are often present on the pleural surface ( fig. 9-92 ). the lungs are pale, mottled, and typically heavy (two or three times normal weight), and the tracheobronchial lymph nodes are enlarged. microscopically, the interstitial pneumonia is characterized by balt hyperplasia and thickening of alveolar walls and peribronchial interstitial tissue by heavy infiltration of lymphocytes, largely t lymphocytes (see fig. 9 -75). recruitment of mononuclear cells into the pulmonary interstitium is presumably the result of sustainable production of cytokines by retrovirus-infected pulmonary macrophages and lymphocytes. hyperplasia of type ii pneumonocytes is not a prominent feature of maedi, likely because in this disease there is no injury to type i pneumonocytes, but there is some alveolar fibrosis and smooth muscle hypertrophy in bronchioles. secondary bacterial infections often cause concomitant bronchopneumonia. enlargement of regional lymph nodes (tracheobronchial) is due to severe lymphoid hyperplasia, primarily of b lymphocytes. the virus can also infect many other tissues, causing nonsuppurative encephalitis (visna), lymphocytic arthritis, lymphofollicular mastitis, and vasculitis. maedi is clinically characterized by dyspnea and an insidious, slowly progressive emaciation despite good appetite. death is inevitable once clinical signs are present, but it may take many months. caprine arthritis-encephalitis. caprine arthritis-encephalitis (cae) is a retroviral disease of goats (small ruminant lentivirus) that has a pathogenesis remarkably similar to that of visna/maedi in sheep. it was first described in the united states in the 1970s, but such as pasteurella multocida, pneumonia may progress to fibrinous or suppurative bronchopneumonia. one might expect some specific evidence pointing to the infectious agents (e.g., large intranuclear inclusion bodies in epithelial cells with adenoviral infection), but this is often not the case, either because examination is seldom done at the acute stage when the lesions are still present or because secondary bacterial infections mask the primary lesions. in the late stages, chronic enzootic pneumonia is characterized by hyperplastic bronchitis, atelectasis, alveolar and peribronchiolar fibrosis, and marked peribronchial lymphoid hyperplasia (cuffing pneumonia). ovine pneumonic mannheimiosis. ovine pneumonic mannheimiosis is one of the most common and economically significant diseases in most areas where sheep are raised. it is caused by mannheimia haemolytica and has a pathogenesis and lesions similar to those of pneumonic mannheimiosis of cattle. colonization and infection of lungs are facilitated by stressors such as changes in weather; handling; deworming; dipping; viral infections such as parainfluenza virus 3 (piv3), respiratory syncytial virus (rsv), and adenovirus; and probably chlamydiae and bordetella parapertussis infections. lesions are characterized by a severe fibrinous bronchopneumonia (cranioventral) with pleuritis ( fig. 9-94 and e-fig. 9-19 ). subacute to chronic cases progress to purulent bronchopneumonia, and sequelae include abscesses and fibrous pleural adhesions. a similar form of pneumonic mannheimiosis has been reported with increased frequency in bighorn sheep. septicemic pasteurellosis. septicemic pasteurellosis, a common ovine disease, is caused by bibersteinia trehalosi (formerly pasteurella trehalosi or mannheimia haemolytica biotype t) in lambs 5 months of age or older or by mannheimia haemolytica (biotype a) in lambs younger than 2 months of age. both organisms are carried in the tonsils and oropharynx of clinically healthy sheep, and under abnormal circumstances (particularly under stress from dietary or environmental changes) bacteria can invade adjacent tissues, enter the bloodstream, and cause septicemia. gross lesions include a distinctive necrotizing pharyngitis and tonsillitis; ulcerative esophagitis (efig. 9-20) ; severe congestion and edema of the lungs; focal hepatic necrosis; and petechiae in the mucosa of the tongue, esophagus, and intestine and particularly in the lungs and pleura. clinically, goats are active and afebrile but progressively lose weight despite normal appetite. the encephalitic or arthritic signs tend to obscure the respiratory signs, which are only evident on exertion. secondary bacterial bronchopneumonia is common in affected animals. bacterial pneumonias. in the past, pasteurella haemolytica was incriminated in four major ovine diseases known as (1) acute ovine pneumonic pasteurellosis (shipping fever), (2) enzootic pneumonia (nonprogressive chronic pneumonia), (3) fulminating septicemia, and (4) mastitis. under the new nomenclature, mannheimia haemolytica is responsible for ovine pneumonia resembling shipping fever in cattle (ovine pneumonic mannheimiosis), septicemia in young lambs (younger than 3 months of age), and ovine enzootic pneumonia and sporadic severe gangrenous mastitis in ewes. bibersteinia (pasteurella) trehalosi (formerly pasteurella haemolytica biotype t) is the agent incriminated in septicemia in lambs 5 to 12 months old. chronic enzootic pneumonia. in sheep, this entity is a multifactorial disease complex that, in contrast to ovine pneumonic mannheimiosis, causes only a mild to moderate pneumonia and it is rarely fatal. it generally affects animals younger than 1 year of age. significant costs associated with chronic enzootic pneumonia include reduction of weight gain, labor costs, veterinary fees, and slaughterhouse waste. the modifier "chronic" is used here to avoid any confusion with pneumonic mannheimiosis ("acute enzootic pneumonia"). it is also sometimes called atypical pneumonia, chronic nonprogressive pneumonia, proliferative pneumonia, or other names. chronic enzootic pneumonia is a clinical epidemiologic term and does not imply a single causal agent but is the result of a combination of infectious, environmental, and managerial factors. the list of infectious agents involved in ovine enzootic pneumonia includes mannheimia haemolytica, pasteurella multocida, parainfluenza virus 3 (pi-3), adenovirus, reovirus, respiratory syncytial virus (rsv), chlamydiae, and mycoplasmas (mycoplasma ovipneumoniae). in the early stages of enzootic pneumonia, a cranioventral bronchointerstitial pneumonia is characterized by moderate thickening of alveolar walls because of hyperplasia of type ii pneumonocytes. in some cases, when lungs are infected with secondary pathogens, viviparus of cattle. as seen in cattle with dictyocaulus viviparus, areas of atelectasis secondary to bronchiolar obstruction are present, particularly along the dorsal caudal aspects of the caudal lung lobes. microscopically, affected lungs are characterized by a catarrhal, eosinophilic bronchitis, with peribronchial lymphoid hyperplasia and smooth muscle hyperplasia of bronchi and bronchioles. bronchioles and alveoli can contain edematous fluid, eosinophils, and parasitic larvae and eggs. microscopic granulomas caused by aspirated eggs can be observed in the distal lung. the clinical signs (cough, moderate dyspnea, and loss of condition) and lesions relate mainly to obstruction of the small bronchi by adult worms and filaria. anemia of undetermined pathogenesis and secondary bacterial pneumonia are common in small ruminants with this parasitic disease. muellerius capillaris. muellerius capillaris, also called the nodular lungworm, occurs in sheep and goats in most areas of the world and is the most common lung parasite of sheep in europe and northern africa. it requires slugs or snails as intermediate hosts. the lesions in sheep are typically multifocal, subpleural nodules that tend to be most numerous in the dorsal areas of the caudal lung lobes ( fig. 9-95, a) . these nodules are soft and hemorrhagic in the early stages but later become gray-green and hard or even calcified. microscopically, a focal, eosinophilic, and granulomatous reaction occurs in the microscopically, the hallmark lesion is a disseminated intravascular thrombosis often with bacterial colonies in the capillaries of affected tissues. the alveolar capillaries contain bacteria and microthrombi, and the alveolar lumens have fibrin and red blood cells. mannheimia haemolytica and bibersteinia trehalosi are readily isolated from many organs. affected animals usually die within a few hours of infection, and these animals only rarely have clinical signs such as dullness, recumbency, and dyspnea. contagious caprine pleuropneumonia. a number of mycoplasma spp., often referred to as the "mycoides cluster," can produce respiratory tract infections in goats; however, only mycoplasma capricolum ssp. capripneumoniae is considered to cause contagious caprine pleuropneumonia. this disease is the goat counterpart of contagious bovine pleuropneumonia in cattle; sheep do not have a corresponding disease. this oie-notifiable disease is important in africa, the middle east, and areas of asia, but it is also seen elsewhere. the gross lesions caused by mycoplasma capricolum ssp. capripneumoniae are similar to those of the bovine disease and consist of a severe, often unilateral fibrinous bronchopneumonia and pleuritis; however, distention of the interlobular septa (which are normally not as well developed in goats as in cattle) and formation of pulmonary sequestra are less obvious than in the bovine disease. clinically, contagious caprine pleuropneumonia is similar to contagious bovine pleuropneumonia, with high morbidity and mortality, fever, cough, dyspnea, and increasing distress and weakness. other small ruminant mycoplasmas. pneumonia, fibrinous polyarthritis, septicemia, meningitis, mastitis, peritonitis, and abortion are possible manifestations of disease caused by mycoplasma mycoides ssp. mycoides large colony type and mycoplasma mycoides ssp. capri. the pathogenicity of other mycoplasmas, such as mycoplasma ovipneumoniae, mycoplasma arginini, and mycoplasma capricolum ssp. capricolum, in sheep and goats is still being defined and specific description of the lesions would be premature. these organisms probably cause disease only in circumstances similar to those for enzootic pneumonia, where host, infectious, and environmental factors create a complex interaction in the pathogenesis of the disease. it has been suggested that igg antibodies directed against ovine mycoplasmal antigens cross-react with ciliary proteins, causing inflammation and ciliary dysfunction, a condition in lambs referred to as coughing syndrome. tuberculosis. although tuberculosis has generally been considered uncommon in sheep and goats, caprine tuberculosis has become a significant disease in areas of spain and europe. mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) is the most common cause, but infection with mycobacterium bovis or with the mycobacterium avium complex does occur when the disease is prevalent in other species in the locality. the pulmonary form, similar to that seen in cattle, is characterized by a granulomatous pneumonia with multiple, large, caseous, calcified, and well-encapsulated granulomas scattered throughout the lungs. intralesional acid-fast organisms within macrophages are not as abundant as in bovine tuberculosis. staphylococcus aureus. young sheep (2 to 12 weeks old) are susceptible to staphylococcus aureus septicemia (tick pyemia). this bacterium causes disseminated inflammation and abscesses in the joints, heart, liver, kidneys, and cns, and in the lung it can also produce bronchopneumonia and pulmonary abscesses (efig. 9-21 ). dictyocaulus filaria. dictyocaulus filaria, also called the large lungworm, is a serious, worldwide, parasitic disease of the lungs, most commonly of lambs and goat kids but occurring in adults as well. the life cycle and lesions are similar to those of dictyocaulus epithelial cells spreads rapidly throughout the nasal, tracheal, and bronchial mucosa, with the more severe outbreaks reflecting more involvement of intrapulmonary airways and secondary infection with pasteurella multocida, trueperella (arcanobacterium) pyogenes, or haemophilus spp. although uncommon, human beings infected with swine influenza (h1n1) can transmit the virus to pigs; therefore it is important that veterinarians or workers with influenza-like illness stay away from pig farms. natural transmission of h1n1 and h5n1 from human beings to ferrets (mustela putorius furo) and from human beings to cats and dogs has also been reported. pulmonary lesions caused by influenza virus alone are rarely seen in the postmortem room because this disease has a very low mortality rate unless complicated with secondary bacterial infections. grossly, a copious catarrhal to mucopurulent inflammation extends from the nasal passages to the bronchioles, with the volume of mucus being sufficient to plug small airways and cause a lobular or multilobular atelectasis in the cranioventral regions of the lungs. the appearance can be similar grossly, although not microscopically, to that of mycoplasma hyopneumoniae. fatal cases have severe alveolar and interstitial pulmonary edema. microscopically, the lesions in uncomplicated cases are typical of a virus-induced, necrotizing bronchitis-bronchiolitis, which in severe cases extends into the alveoli as bronchointerstitial pneumonia. it is characterized by necrosis of the bronchial/bronchiolar epithelium, thickening and infiltration of the alveolar wall with mononuclear cells and aggregates of macrophages, neutrophils, mucus, and some necrotic cells within the alveolar lumen. if these changes are extensive enough, the lumen of bronchioles can be occluded by exudate, causing lobular atelectasis. viral antigen can be demonstrated in infected epithelial cells by immunoperoxidase techniques. in the later stages of alveolar inflammation, neutrophils are progressively replaced by intraalveolar macrophages, unless the pneumonia is complicated by secondary bacterial infections. recent serologic surveys indicate that infection is also prevalent in wild pigs. clinically, a sudden onset of fever, nasal discharge, stiffness, labored breathing, weakness or even prostration, followed by painful and often paroxysmal coughing, is seen in animals of all age groups and may affect most of the herd. the outbreak subsides virtually without mortality within 1 or 2 weeks; the clinical appearance is much more alarming than the pathologic changes, unless the pigs have secondary infection with bacteria. infection can be confirmed using pcr in secretions collected with nasal swabs. the most important effect of most outbreaks of influenza is severe weight loss, but pregnant sows may abort or give birth to weak piglets. porcine reproductive and respiratory syndrome. a disease originally named mystery swine disease was first recognized in the united states in 1987. in 1990, it was seen in europe, and the disease now occurs worldwide in most major pig-raising countries. in 1991, dutch investigators isolated a virus as the etiologic agent; porcine reproductive and respiratory syndrome virus (prrsv) is currently classified in the genus arterivirus of the family arteriviridae. as its name implies, prrs is characterized by late-term abortions and stillbirths and respiratory problems. the respiratory form is generally seen in nursery and grow/finish pigs. the pathogenesis has not been completely elucidated, but it is presumed that there is a mucosal portal of entry with virus replication in macrophages of the lymphoid tissue, followed by viremia and finally dissemination of infected macrophages to the lungs and other organs, such as the thymus, liver (kupffer cells), spleen, lymph nodes, and intestine. the pulmonary alveolar and intravascular macrophages are the major targets for prrs virus, which induces apoptosis of these cells. the virus also downregulates the innate immune response by subpleural alveoli where the adults, eggs, and coiled larvae reside ( fig. 9-95, b) . clinical signs are usually not apparent. goats differ from sheep by having diffuse interstitial rather than focal lesions, and the reaction to the parasites seen microscopically varies from almost no lesions to a severe interstitial pneumonia with heavy infiltrates of mononuclear cells in alveolar walls resembling cae or mycoplasmal infections. secondary effects of muellerius capillaris infection in sheep and goats include decreased weight gain and possibly secondary bacterial infections. protostrongylus rufescens. protostrongylus rufescens is a worldwide parasite of sheep, goats, and wild ruminants. it requires an intermediate snail as a host. infection is usually subclinical, but protostrongylus rufescens can be pathogenic for lambs and goat kids and can cause anorexia, diarrhea, weight loss, and mucopurulent nasal discharge. the adult parasite lives in bronchioles as dictyocaulus spp., but it causes pulmonary nodules similar to those of muellerius capillaris. porcine pneumonias are unequivocally a major obstacle for the contemporary swine industry. the incidence, prevalence, and mortality rates of pneumonias in pigs depend on a series of complex, multifactorial interactions. among the most commonly recognized elements linked to porcine pneumonias are the following: • host (age, genetic makeup, immune status) • infectious agents (viruses, bacteria) • environmental determinants (humidity, temperature, ammonia concentrations) • management practices (crowding, mixing of animals, air quality, nutrition, stress) because of the nature of these multifactorial interactions, it will become obvious in the following paragraphs that more often than not a specific type of pneumonia frequently progresses to or coexists with another. the term porcine respiratory disease complex (prdc) has been introduced in clinical practice to describe pigs with signs of respiratory infection involving combined bacterial and viral infections. commonly implicated microbes include porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), porcine circovirus 2 (pcv2), porcine respiratory coronavirus (prcov), mycoplasma hyopneumoniae, and pasteurella multocida. swine influenza (swine flu). swine influenza is a highly contagious acute respiratory viral disease of swine that is caused by swine influenza virus (siv), a type a influenza virus of the family orthomyxoviridae. it is generally accepted that swine influenza resulted from adaptation of the type a influenza virus that caused the human influenza pandemic during world war i. the most common subtypes of siv currently circulating in pigs are h1n1, h1n2, and h3n2. swine influenza is enzootic worldwide and is known to infect human beings who are in close contact with sick pigs. in 2009, an outbreak of swine-human influenza (h1n1), presumably transmitted from pigs to human beings, emerged in mexico and rapidly spread to many countries throughout the world. this new "pandemic" was attributed to a triple-reassortant of influenza a virus containing gene segments of swine, eurasian avian, and human strains. human infection with this novel strain affected mainly children and young adults, as well as individuals of any age with an underlying debilitating condition. transmission between influenza-infected and susceptible pigs occurs mainly by aerosol or oral route. siv attaches to and replicates within epithelial cells of the upper respiratory tract; the infection of similar inclusions are occasionally seen in bronchial glandular and renal epithelial cells. the lungs show thickening of the alveolar walls because of hyperplasia of type ii pneumonocytes and interstitial infiltrates of mononuclear cells, peribronchiolar fibrous hyperplasia, and necrotizing bronchitis/bronchiolitis. circovirus can be confirmed in affected tissue by immunohistochemical or pcr techniques. dual infections with pcv2 and prrsv frequently occur in pigs, and secondary infections with pneumocystis carinii are commonly seen in pigs with this coinfection. characteristically, alveoli are filled with a distinctive foamy exudate that contains the organism, which is not visible in h&e-stained sections but is easily demonstrated with gomori's methenamine silver stain (see fig. 9-20) . in human beings, pneumocystis (carinii) jirovecii pneumonia (pneumocystosis) is one of the most common and often fatal complications in aids patients. as in aids patients, abnormal populations of cd4 + and cd8 + t lymphocytes have been incriminated as the underlying mechanism leading to pneumocystosis in foals and pigs. nipah virus. nipah virus belongs to the paramyxoviridae family and shares a genus (henipavirus) with the closely related hendra virus (see section on pneumonias of horses). another emerging zoonotic disease, nipah virus caused a major epidemic with significant human mortality in southeast asia in 1998 and 1999. people handling pigs were primarily affected. similar to hendra virus, fruit bats (flying foxes) act as natural reservoir and are involved in the transmission to pigs by poorly understood mechanisms. in pigs, this virus infects the respiratory system resulting in pneumonia with syncytial cells occurring in the vascular endothelium and in the respiratory epithelium at all levels of the lung. disease is spread to human beings via the respiratory route. human-to-human transmission of this virus has been reported in more recent outbreaks. other viral pneumonias of pigs. porcine respiratory coronavirus (prcov) is sporadically incriminated in pneumonia in pigs. this viral pneumonia is generally mild, and most pigs fully recover if the pneumonia is not complicated with other infections. lesions in the lung are those of bronchointerstitial pneumonia with necrotizing bronchiolitis. interestingly, infections with porcine and other respiratory coronaviruses have been used to investigate the pathogenesis of severe acute respiratory syndrome (sars), an emerging and highly contagious condition in human beings that is attributed to a novel human coronavirus (sars-cov). the relationship between sars-cov and animal coronavirus is still under investigation. other viruses rarely incriminated in porcine respiratory disease complex (prdc) include paramyxovirus, encephalomyocarditis virus, hemagglutinating encephalomyocarditis virus, and adenovirus. petechial hemorrhages in the lung and pulmonary edema may be seen with african swine fever, classical swine fever, and pseudorabies virus infections. porcine enzootic pneumonia. porcine enzootic pneumonia, a highly contagious disease of pigs caused by mycoplasma hyopneumoniae, is grossly characterized by suppurative or catarrhal bronchopneumonia ( fig. 9-96 and efig. 9-23 ). when its worldwide prevalence and deleterious effect on feed conversion are taken into account, this disease is probably the most economically significant respiratory disease of pigs. although an infectious disease, it is very much influenced by immune status and management factors, such as crowding (airspace and floor space), ventilation (air exchange rate), concentrations of noxious gases in the air (ammonia and hydrogen sulfide), relative humidity, temperature fluctuations, and mixing of stock from various sources. it has been demonstrated with inhibiting interferons and deregulates the adaptive immune response, thus interfering with the normal defense mechanisms predisposing pigs to septicemia and bacterial pneumonia. the most common opportunistic organisms are streptococcus suis, salmonella choleraesuis, mycoplasma hyopneumoniae, haemophilus parasuis, bordetella bronchiseptica, pasteurella multocida, and pneumocystis carinii. dual viral infections with prrsv and porcine circovirus 2 (pcv2), siv, and porcine respiratory coronavirus (prcov) are commonly found in pigs, and such coinfections increase the severity of disease. on postmortem examination, pulmonary lesions vary from very mild changes characterized by failure of the lung to collapse when the thorax is opened and the presence of rib imprints (see fig. 9 -74) to severe changes manifested by consolidation of the lung in cases that have been complicated with bacterial pneumonia. tracheobronchial and mediastinal lymph nodes are typically enlarged. microscopically, pulmonary changes are those of interstitial pneumonia characterized by thickening of alveolar walls by infiltrating macrophages and lymphocytes and mild hyperplasia of type ii pneumonocytes. necrotic cells are scattered in the alveolar lumens. unlike some other viral infections, bronchiolar epithelium does not appear to be affected. diagnosis of prrs in tissue collected at necropsy can be confirmed by immunohistochemistry and pcr techniques. infected pigs may become carriers and transmit the infection through body fluids and semen. clinically, prrs in nursery and young growing animals is characterized by sneezing, fever, anorexia, dyspnea, cough, and occasional death. some piglets develop severe cyanosis of the abdomen and ears, which explains why this syndrome was named blue ear disease when first described in europe. porcine circovirus-associated disease. another emerging porcine syndrome, characterized clinically by progressive emaciation in weaned pigs, was originally described in the 1990s in canada, the united states, and europe. since then, it has disseminated to many countries, causing economic devastation in pig farms worldwide. because of the clinical signs and lesions in many organs, this syndrome was named postweaning multisystemic wasting syndrome (pmws). porcine circovirus 2 (pcv2) has been incriminated as the etiologic agent and is a member of the circoviridae family. pcv2 has been associated with a number of syndromes in pigs, including systemic pcv2 infection (the preferred term for pmws because it may also affect mature pigs), pcv2-associated pneumonia, pcv2-associated enteritis, porcine dermatitis and nephropathy syndrome (pdns), pcv2-associated reproductive failure, and, most recently, pcv2-associated cerebellar vasculitis. the diseases caused by pcv2 are now collectively known as porcine circovirus-associated disease (pcvad); the most common manifestations are systemic pcv2 infection (pmws) and pcv2-associated pneumonia as part of the porcine respiratory disease complex. all of these manifestations affect more than one organ, and there is substantial overlap between the syndromes. at necropsy, pigs with systemic pcv2 infection (pmws) and pcv2-associated pneumonia are often in poor body condition, and the most remarkable changes, not considering other possible secondary infections, are enlargement of the superficial and visceral lymph nodes and a mild interstitial pneumonia characterized by failure of the lungs to collapse when the thorax is opened. jaundice is occasionally observed. microscopically, the lymphoid tissues show lymphoid depletion, histiocytic replacement of follicles, and notable proliferation of parafollicular histiocytes, some of which fuse and form syncytial cells (granulomatous lymphadenitis); necrosis of the lymphoid follicles is seen less often. in some cases, large basophilic inclusion bodies are present singly or as grapelike clusters (botryoid inclusions) within the cytoplasm of macrophages, particularly in peyer's patches, spleen, and lymph nodes (efig. 9-22 ). chapter 9 respiratory system, mediastinum, and pleurae peribronchial, bronchiolar, and alveolar interstitium. additional virulence factors include the ability of mycoplasma hyopneumoniae to cause immunosuppression, reduce the phagocytic activity of neutrophils in the lung, and change the chemical composition of mucus. all of these functional alterations can predispose the lung to secondary bacterial infections. the lesions caused by mycoplasma hyopneumoniae start as a bronchointerstitial pneumonia and progress to a suppurative or mucopurulent bronchopneumonia once secondary pathogens are involved (commonly seen at necropsy). in most pigs, gross lesions affect only portions of the cranial lobes, but in more severely affected pigs, lesions involve 50% or more of the cranioventral portions of the lungs (see fig. 9 -96). the affected lungs are dark red in the early stages but have a homogeneous pale-gray ("fish flesh") appearance in the more chronic stages of the disease. on cut surface, exudate can easily be expressed from airways, and depending on the stage of the lesions and secondary infections, the exudate varies from purulent to mucopurulent to mucoid. microscopic lesions are characterized by an influx of macrophages and neutrophils into the bronchi, bronchioles, and alveoli, and with time there is also notable balt hyperplasia (see fig. 9-96, b) . in some cases, accumulation of exudate can be severe enough to cause occlusion of bronchioles and atelectasis of the corresponding lobules. the suppurative bronchopneumonia may be accompanied by a mild fibrinous pleuritis, which is often more severe if other organisms, such as mycoplasma hyorhinis, pasteurella multocida, or actinobacillus pleuropneumoniae, are also involved. abscesses and fibrous pleural adhesions are sequelae of chronic complicated infections. clinically, enzootic pneumonia occurs as a herd problem in two disease forms. a newly acquired infection of a previously clean herd causes disease in all age groups, resulting in acute respiratory distress and low mortality. in a chronically infected herd, the mature animals are immune and clinical signs are usually apparent only in growing pigs at times of particular stress such as at weaning. in such herds, coughing and reduced rate of weight gain are the most notable signs. porcine pasteurellosis. porcine pasteurellosis is an infectious disease complex with unclear pathogenesis that includes primary infections by pasteurella multocida alone (primary pasteurellosis) or, more frequently, after the defense mechanisms are impaired and a secondary bacterium colonizes the lung (porcine pneumonic pasteurellosis). in rare cases, pasteurella multocida causes acutely fatal septicemias in pigs (primary septicemic pasteurellosis). it is important to remember that pasteurella multocida serotypes a and d are both part of the normal nasal flora and are also causative agents of bronchopneumonia, pleuritis, and atrophic rhinitis in pigs. pasteurella multocida is one of the most common secondary pathogens isolated from the lungs of pigs with swine influenza virus (siv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus 2 (pcv2), pseudorabies (suhv-1), classical swine fever (hog cholera), enzootic pneumonia, and porcine pleuropneumonia. secondary infections with pasteurella multocida notably change the early and mild bronchointerstitial reaction of enzootic and viral pneumonias into a severe suppurative bronchopneumonia with multiple abscesses and sometimes pleuritis. the other important role of pasteurella multocida in porcine pneumonias is as a cause of a fulminating, cranioventral, fibrinous bronchopneumonia (pleuropneumonia) after influenza virus infection or stress from inadequate ventilation resulting in high levels of ammonia in the air. the nature of the lesion and the predisposing factors of poor management or coexisting viral infections suggest that fulminating porcine pasteurellosis has a pathogenesis similar to that of pneumonic mannheimiosis of cattle. pharyngitis with subcutaneous cervical edema, fibrinohemorrhagic polyarthritis, and focal lymphocytic pcr that mycoplasma hyopneumoniae is present in the air of infected farms. the causative agent, mycoplasma hyopneumoniae, is a fastidious organism and very difficult to grow; thus the final diagnosis is frequently based on interpretation of lesions alone or supported by ancillary tests to detect this mycoplasma in affected lungs by immunohistochemistry, immunofluorescence, or pcr. the bronchopneumonic lesions of porcine enzootic pneumonia are in most cases mild to moderate, and thus mortality is low unless complicated with secondary pathogens, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, bordetella bronchiseptica, haemophilus spp., mycoplasma hyorhinis, and other mycoplasmas and ureaplasmas. although the pathogenesis of porcine enzootic pneumonia is not completely elucidated, it is known that mycoplasma hyopneumoniae first adheres to the cilia of the bronchi by means of a unique adhesive protein, produces ciliostasis, and finally colonizes the respiratory system by firmly attaching to the ciliated epithelial cells of the trachea and the bronchi of the cranioventral regions of the lungs. once attached to the respiratory epithelium, it provokes an influx of neutrophils into the tracheobronchial mucosa; causes extensive loss of cilia (deciliation); stimulates an intense hyperplasia of lymphocytes in the balt; and attracts mononuclear cells into the factors have been identified. these factors allow actinobacillus pleuropneumoniae to attach to cells; produce pores in cell membranes; damage capillaries and alveolar walls, resulting in vascular leakage and thrombosis; impair phagocytic function; and elicit failure of clearance mechanisms. the gross lesions in the acute form consist of a fibrinous bronchopneumonia characterized by severe consolidation and a fibrinous exudate on the pleural surface. although all lobes can be affected, a common site is the dorsal area of the caudal lobes. in fact, a large area of fibrinous pleuropneumonia involving the caudal lobe of a pig's lung is considered almost diagnostic for this disease (fig. 9-97) . on cut surface, consolidated lungs have notably dilated interlobular septa and irregular but well-circumscribed areas of necrosis caused by potent cytotoxins produced by actinobacillus pleuropneumoniae. except for the distribution, pulmonary lesions of porcine pleuropneumonia are identical to those of pneumonic mannheimiosis of cattle. the microscopic lesions are also very similar and include areas of coagulative necrosis surrounded by a thick cluster of "streaming (oat-shaped/oat cell) leukocytes" and notable distention of the interlobular septa because of severe edema and lymphatic thrombosis. bronchioles and alveoli are filled with edematous fluid, fibrin, neutrophils, and few macrophages (see fig. 9 -97). pigs with the chronic form have multiple pulmonary abscesses and large (2 to 10 cm) pieces of necrotic lung encapsulated by connective tissue (sequestra)-changes frequently seen in slaughterhouses. interstitial nephritis are also present in porcine pneumonic pasteurellosis. sequelae of porcine pneumonic pasteurellosis include fibrous pleuritis and pericarditis, pulmonary abscesses, so-called sequestra, and usually death. in contrast to ruminants, mannheimia haemolytica is not a respiratory pathogen for pigs, but in some instances, it can cause abortion in sows. porcine pleuropneumonia. porcine pleuropneumonia is a highly contagious, worldwide disease of pigs caused by actinobacillus (haemophilus) pleuropneumoniae (app), which is characterized by a severe, often fatal, fibrinous bronchopneumonia with extensive pleuritis (pleuropneumonia). survivors generally develop notable residual lesions and become carriers of the organisms. porcine pleuropneumonia is an increasingly important cause of acute and chronic pneumonias, particularly in intensively raised pigs (2 to 5 months old). transmission of actinobacillus pleuropneumoniae occurs by the respiratory route, and the disease can be reproduced experimentally by intranasal inoculation of the bacterium. considered a primary pathogen, actinobacillus pleuropneumoniae can sporadically produce septicemia in young pigs and otitis media and otitis interna with vestibular syndrome in weaned pigs. two biovars and 15 serotypes of the organism have been identified; all serotypes can cause the disease, but differences in virulence exist. the pathogenesis is not yet well understood, but specific virulence factors, such as rtx toxins (hemolytic/cytolytic toxins apx i to apx iv), capsular factors, fimbriae and adhesins, lipopolysaccharide, and permeability tuberculosis. tuberculosis is an important disease in domestic and wild pigs that has a much greater prevalence in pigs than in cattle or other domestic mammals in many countries. porcine tuberculosis is attributed to infection with mycobacterium bovis and porcine mycobacteriosis to infection with mycobacterium avium complex. a common scenario in small mixed-farming operations is the diagnosis of avian tuberculosis at the time that pigs are slaughtered, and the source is ingestion of tuberculous chickens or contaminated litter. as would be expected, granulomas are found in the mesenteric, mandibular, and retropharyngeal lymph nodes; to a lesser extent in the intestine, liver, and spleen; and only in rare cases in the lung. the route of infection in pulmonary tuberculosis and mycobacteriosis of pigs is most often hematogenous after oral exposure and intestinal infection. lung lesions are those of a granulomatous pneumonia. the microscopic lesions are basically those of tubercles (granulomas), but the degree of encapsulation, caseation, and calcification varies with the type of mycobacterium, age of the lesion, and host immune response. other bacterial pneumonias of pigs. septicemias in pigs often cause petechial hemorrhages in the lung and pulmonary edema. salmonellae, escherichia coli, and listeria monocytogenes can cause severe interstitial pneumonia in very young animals. salmonella choleraesuis causes a necrotizing fibrinous pneumonia similar to porcine pleuropneumonia, and salmonella typhisuis causes a chronic suppurative bronchopneumonia. in high health herds, actinobacillus suis may cause fibrinohemorrhagic pleuropneumonia and is easily confused with porcine pleuropneumonia. metastrongylosis. metastrongylus apri (elongatus), metastrongylus salmi, and metastrongylus pudendotectus (lungworms) of domestic and feral pigs occur throughout most of the world and require earthworms as intermediate hosts for transmission. the incidence of disease has therefore decreased with development of confinement housing. the importance of pig lungworms is mainly because infection results in growth retardation of the host. clinical signs include coughing because of parasitic bronchitis. the gross lesions, when noticeable, consist of small gray nodules, particularly along the ventral borders of the caudal lobes. the adult worms are grossly visible in bronchi, and microscopically, the parasites cause a catarrhal bronchitis with infiltration of eosinophils and lobular atelectasis ( fig. 9-99) . ascaris suum. the larvae of ascaris suum can cause edema, focal subpleural hemorrhages, and interstitial inflammation (see fig. 9 -77). along their larval migration tracts, hemorrhages also occur in the liver and, after fibrosis, become the large white "milk spots" seen so frequently as incidental findings at necropsy. it has been reported that ascaris suum may cause immunosuppression in severely affected pigs. pigs can be killed if exposed to an overwhelming larval migration. other causes of pneumonia. foreign body granulomatous pneumonia occurs frequently in pigs after inhalation of vegetable material (starch pneumonia), presumably from dusty (nonpelleted) feed. lesions are clinically silent but are often mistaken for other pneumonic processes during inspection at slaughterhouses. microscopically, pulmonary changes are typical of foreign body granulomatous inflammation in which variably sized feed particles are surrounded by macrophages and neutrophils, and often have been phagocytosed by multinucleated giant cells. feed (vegetable) particles appear as thick-walled polygonal cells that stain positive with pas because of their rich carbohydrate (starch) content (see efig. 9-12) . clinically, porcine pleuropneumonia can vary from an acute form with unexpected death and blood-stained froth at the nostrils and mouth to a subacute form characterized by coughing and dyspnea accompanied by clinical signs of sepsis such as high fever, hypoxemia, anorexia, and lethargy (efig. 9-24) . a chronic form is characterized by decreased growth rate and persistent cough. animals that survive often carry the organism in the tonsils, shed the organism, and infect susceptible pigs. haemophilus pneumonia. in addition to glasser's disease characterized by polyserositis (pericarditis, pleuritis, peritonitis, polyarthritis, and meningitis) (efig. 9-25) , some serotypes of haemophilus parasuis (originally haemophilus influenzae suis) can also cause suppurative bronchopneumonia that in severe cases can be fatal. the causal organism, haemophilus parasuis, is usually carried in the nasopharynx of normal pigs and requires abnormal circumstances such as those following stress (weaning and cold weather) or viral infections (swine influenza or pcv2). specific pathogen-free (spf) pigs seem to be particularly susceptible to glasser's disease (arthritis and serositis) but not to pulmonary infection (bronchopneumonia). streptococcal pneumonia. streptococcus suis is a common cause of porcine disease worldwide and a serious zoonosis capable of causing death by septic shock or meningitis and residual deafness in butchers, veterinarians, and pig farmers. typically, streptococcus suis gains entrance to the susceptible young pig through the oropharyngeal mucosa and is carried in the tonsils, nasal mucosa, and mandibular lymph nodes of healthy animals, particularly in survivors of an outbreak. infected sows can abort or vertically transmit the infection to their offspring. some serotypes of streptococcus suis cause neonatal septicemia, and this can result in suppurative meningitis, otitis, arthritis, polyserositis, myocarditis, valvular endocarditis, and embolic pneumonia ( fig. 9-98 ). other serotypes of streptococcus suis can reach the lung by the aerogenous route and cause a suppurative bronchopneumonia, in combination with pasteurella multocida, escherichia coli, or mycoplasma hyopneumoniae, or in combination with actinobacillus pleuropneumoniae, which causes a fibrinous bronchopneumonia. coinfections of streptococcus suis with pcv2 and prrsv are also frequently seen in some farms. gross lesions in the acute stages include serous to catarrhal to mucopurulent nasopharyngitis and conjunctivitis. the lungs are edematous and have a diffuse interstitial pneumonia ( fig. 9 -100) microscopically characterized by necrotizing bronchiolitis, necrosis and exfoliation of pneumonocytes, mild alveolar edema, and, several hours later, thickening of the alveolar walls because of interstitial mononuclear cell infiltrates and hyperplasia of type ii pneumonocytes. secondary infections with bordetella bronchiseptica and mycoplasmas are common and induce life-threatening suppurative bronchopneumonia. the thymus may be small relative to the age of the animal because of viral-induced lymphocytolysis. microscopically, eosinophilic inclusions are present in the epithelial cells of many tissues, in the nuclei or cytoplasm, or in both (see fig. 9 -100). they appear early in the bronchiolar epithelium but are most prominent in the epithelium of the lung, stomach, renal pelvis, and urinary bladder, making these tissues good choices for diagnostic examination. viral inclusions are rarely seen in the later stages of this disease. the suppurative secondary bronchopneumonias often hinder the detection of viral lesions in the lung, particularly because bronchiolar cells containing inclusion bodies exfoliate and mix with the neutrophils recruited by the bacterial infection. distemper virus antigens can be readily demonstrated in infected cells by the immunoperoxidase technique (see fig. 9 -100), which can also be used in skin biopsies for the antemortem diagnosis of canine distemper. distemper virus also has a tendency to affect developing tooth buds and ameloblasts, causing enamel hypoplasia in dogs that recover from infection. of all distemper lesions, demyelinating encephalomyelitis, which develops late, is the most devastating (see chapter 14). sequelae to distemper include the nervous and pneumonic complications mentioned previously and various systemic infections, such as toxoplasmosis and sarcocystosis, because of depressed immunity. persistent viral infection occurs in some dogs that survive the disease, and they may become carriers and the source of infection for other susceptible animals. clinical signs consist of biphasic fever, diarrhea, vomiting, weight loss, mucopurulent oculonasal discharge, coughing, respiratory distress, and possible loss of vision. weeks later, hyperkeratosis of the foot pads ("hard pad") and the nose are observed, along with nervous signs, including ataxia, paralysis, convulsions, or residual myoclonus (muscle twitches, tremors, and "tics"). in general, inflammatory diseases of the lungs are less of a problem in dogs than in food-producing species and can be subdivided in two major groups, infectious and noninfectious pneumonias. "canine infectious respiratory disease" (cird) is the term currently used by clinicians to describe a heterogeneous group of respiratory infections in dogs; these diseases were previously clustered under the name of infectious tracheobronchitis or "kennel cough." cird is the canine counterpart of brd and prd complexes in cattle and pigs, respectively. the most common viruses in cird include canine parainfluenza virus (cpiv), canid herpesvirus 1 (cahv-1), canine adenovirus-2 (cav-2), canine respiratory coronavirus (crcov), canine distemper virus (cdv), and canine influenza virus (civ). bordetella bronchiseptica, streptococcus equi ssp. zooepidemicus, and mycoplasma spp. are the most frequent bacterial isolates in cird. it has been recently recognized that animal shelters are an important source of viral and bacterial infections for dogs and cats. uremia and paraquat toxicity are perhaps the two most notable noninfectious causes of canine respiratory disease. canine distemper. canine distemper is an important and ubiquitous infectious disease of dogs, other canidae, wild felidae, mustelidae, and marine mammals throughout the world. it is caused by a morbillivirus that is antigenically related to the human measles, rinderpest (officially eradicated in 2011), "peste de petit ruminants," and phocine distemper viruses. canine distemper virus (cdv) is transmitted to susceptible puppies through infected body fluids. the virus invades through the upper respiratory tract and conjunctiva, proliferates in regional lymph nodes, becomes viremic, and in dogs with an inadequate antibody response, infects nearly all body tissues (pantropic), particularly the epithelial cells. distemper virus hampers the immune response, downregulates cytokine production, and persists for a long time in some tissues. cdv can target the lungs either directly as a viral pneumonia or indirectly by its immunosuppressive effects rendering the lungs susceptible to secondary bacterial and protozoal infections, or as a coinfection with other viruses such as canine adenovirus-2 and canid herpesvirus 1. 15:292-294, 2003.) inclusion bodies occur within epithelial cells in early lesions. cahv-1 has also been identified as a cause of ulcerative keratoconjunctivitis in older dogs. canine influenza (canine flu). canine influenza is an emerging contagious respiratory infection of dogs that was first described in the united states and subsequently in other countries. it has a high morbidity (close to 100%), but the mortality, as with most other influenza infections, is relatively low (less than 8%). this disease, first diagnosed in greyhounds, is caused by a novel influenza-a virus (canine influenza virus or civ), a mutation from a previously recognized h3n8 strain of equine influenza virus. dog-to-dog transmission does occur and therefore this infection must be distinguished from other viruses of the canine infectious respiratory disease (cird) group. pulmonary lesions are generally mild and transient, but infected dogs are susceptible to secondary bacterial bronchopneumonia. the most relevant lesions in dogs dying unexpectedly from canine influenza are pleural and pulmonary hemorrhages. microscopically, there is necrotizing tracheitis, bronchitis, and bronchiolitis with exudation of neutrophils and macrophages. in severe cases, hemorrhagic interstitial or bronchointerstitial pneumonia may be accompanied by vasculitis and thrombosis. influenza antigen can be demonstrated by immunohistochemistry in airway epithelium and alveolar macrophages. clinically, dogs with canine influenza are lethargic, inappetent, and hyperthermic and frequently cough and show nasal discharge. these signs resemble those seen in dogs with kennel cough or secondary bacterial pneumonia. in addition, there are confirmed cases of canine influenza caused by the porcine h1n1 presumably transmitted from infected pet owners. bacterial pneumonias. dogs generally develop bacterial pneumonias when the pulmonary defense mechanisms have been impaired. pasteurella multocida, streptococcus spp., escherichia coli, klebsiella pneumoniae, and bordetella bronchiseptica can be involved in pneumonia secondary to distemper or after aspiration of gastric contents ( fig. 9-102 and efig. 9-27 ). streptococcus zooepidemicus can cause acute and fatal hemorrhagic pleuropneumonia with canine adenovirus type 2 infection. cav-2 infection is a common but transient contagious disease of the respiratory tract of dogs, causing mild fever, oculonasal discharge, coughing, and poor weight gain. the portal of entry is generally by inhalation of infected aerosols followed by viral replication in the surface cells of the upper respiratory tract, mucous cells of the trachea and bronchi, nonciliated bronchiolar epithelial cells, and type ii pneumonocytes. pulmonary lesions are initially those of bronchointerstitial pneumonia, with necrosis and exfoliation of bronchiolar and alveolar epithelium, edema, and, a few days later, proliferation of type ii pneumonocytes, mild infiltration of neutrophils and lymphocytes in the alveolar interstitium, and hyperplastic bronchitis and bronchiolitis. large basophilic intranuclear viral inclusions are typically seen in bronchiolar and alveolar cells ( fig. 9-101) . infection with cav-2 is clinically mild unless complicated with a secondary bacterial infection or coinfections with other viruses such as distemper virus. experimental work suggests cav-2 reinfection may lead to hyperreactive airways, a nonspecific condition in which the bronchial mucosa becomes highly "responsive" to irritation such as that caused by cold air, gases, or cigarette smoke. however, it is not clear if this outcome is true in natural infections. canid herpesvirus 1. canid herpesvirus 1 (cahv-1) can cause fatal systemic disease in newborn puppies and is probably a contributing factor in "fading puppy syndrome." hypothermia has been suggested as a pivotal component in the pathogenesis of fatal infections in puppies. many dogs are seropositive, suggesting that transient or subclinical infections are more common than realized; the virus remains latent in the trigeminal and other ganglia and can be reactivated after stress, resulting in asymptomatic transmission of cahv-1 virus to offspring via the placenta, thus resulting in abortion or stillbirths. in puppies, cahv-1 causes ulcerative tracheitis, interstitial pneumonia (efig. 9-26) , and focal necrosis and inflammation in the kidneys, liver, and brain. eosinophilic intranuclear aspiration pneumonia starts as an acute necrotizing bronchitis and bronchiolitis caused by aspiration of irritant materials such as gastric acid or a caustic material administered by mouth. the aspirate also contains potentially pathogenic bacteria, and because the mucociliary apparatus is damaged and these bacteria are not removed, they settle into the ventral portions of the lung (from gravity) and provoke a fibrinosuppurative and necrotizing bronchopneumonia. b, bronchoalveolar spaces are filled with neutrophils, macrophages, and bacteria (arrows). h&e stain. inset, large colonies of bacteria (arrows). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) a b infection; thus it most frequently affects outdoor and hunting dogs. from the lung, infection is disseminated hematogenously to other organs, mainly bone, skin, brain, and eyes. pulmonary lesions are characterized by multifocal to coalescing pyogranulomatous pneumonia, generally with firm nodules scattered throughout the lungs (fig. 9-103) . microscopically, nodules are pyogranulomas with numerous macrophages (epithelioid cells), some neutrophils, multinucleated giant cells, and thick-walled yeasts (see fig. 9 -35, c). yeasts are 5 to 25 µm in diameter and are much better visualized when they are stained with pas reaction or gomori's methenamine silver stain. nodules can also be present in other tissues, chiefly lymph nodes, skin, spleen, liver, kidneys, bones, testes, prostate, and eyes. this fungus can be easily identified in properly prepared and stained transtracheal washes or lymph node aspirates. clinical signs can reflect involvement of virtually any body tissue; pulmonary effects include cough, decreased exercise tolerance, and terminal respiratory distress. coccidioidomycosis. coccidioidomycosis (san joaquin valley fever), caused by the dimorphic fungus coccidioides immitis, occurs mainly in animals living in arid regions of the southwestern united states, mexico, and central and south america. it is a primary respiratory tract (aerogenous) infection commonly seen at slaughterhouses in clinically normal feedlot cattle. in dogs, coccidioidomycosis also has an aerogenous portal of entry and then a b hemorrhagic pleural effusion in dogs. death is generally a consequence of severe sepsis and septic shock or from β-hemolytic streptococcal bacteremia causing emboli in the lungs, liver, brain, and lymph nodes. the primary source of the infection cannot be determined in most cases. dental disease in dogs may be a source of systemic and pulmonary infection, a concept wellrecognized in human medicine for many years. the role of mycoplasmas in canine pneumonia is still uncertain because these organisms are frequently isolated from normal nasopharyngeal flora. tuberculosis is uncommon in dogs because these animals appear to be quite resistant to infection; most cases occur in immunocompromised dogs or in dogs living with infected human beings. dogs are susceptible to the infection with mycobacterium tuberculosis, mycobacterium bovis, and mycobacterium avium complex, and therefore canine infection presupposes contact with human or animal tuberculosis. the clinicopathologic manifestation is pulmonary after inhalation or alimentary after oral exposure, but in most cases infection is disseminated to lymph nodes and visceral organs. the gross lesions are multifocal, firm nodules with necrotic centers, most often seen in the lungs, lymph nodes, kidneys, and liver. diffuse granulomatous pleuritis and pericarditis with copious serofibrinous or sanguineous effusion are common. microscopically, granulomas are formed by closely packed macrophages but with very little connective tissue. mycotic pneumonias. mycotic pneumonias are serious diseases seen commonly in animals in some regions. there are two main types: those caused by opportunistic fungi and those caused by a group of fungi associated with systemic "deep" mycoses. all of these fungi affect human beings and most domestic animals but are probably not transmitted between species. aspergillosis. opportunistic fungi, such as aspergillus spp. (particularly aspergillus fumigatus), are important in birds, but in domestic animals, they mainly affect immunosuppressed individuals or those on prolonged antibiotic therapy. the pulmonary lesion is a multifocal, nodular, pyogranulomatous, or granulomatous pneumonia. microscopically, there is necrosis and infiltrates of neutrophils, macrophages, and lymphocytes, with proliferation of fibroblasts eventually leading to encapsulation of the granuloma. fungal hyphae are generally visible in the core of the lesion and in the walls of blood vessels. systemic mycoses (dimorphic fungal infections) . systemic (deep) mycoses are caused by blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans/ cryptococcus gatti (see fig. 9 -35). blastomycosis mainly affects dogs and is discussed here, whereas cryptococcosis is discussed in the section on pneumonias of cats. in contrast to other fungi, such as aspergillus spp., organisms of the systemic mycosis group are all primary pathogens of human beings and animals and thus do not necessarily require a preceding immunosuppression to cause disease. these fungi have virulence factors that favor hematogenous dissemination and evasion of immune and phagocytic responses. systemic dissemination is often exacerbated by the administration of immunosuppressant drugs such as corticosteroids. these fungi are usually detected by cytological evaluation of affected tissues. blastomycosis. blastomycosis occurs in many countries of the north american continent, africa, the middle east, and occasionally in europe. in the united states, it is most prevalent in the atlantic, st. lawrence, and ohio-mississippi river valley states, compared with the mountain-pacific region. blastomyces dermatitidis is a dimorphic fungus (mycelia-yeast) seen mainly in young dogs and occasionally in cats and horses. this fungus is present in the soil, and inhalation of spores is considered the principal route of from the alveolar interstitium associated with larvae or dead worms because little reaction develops to the live adults. crenosoma vulpis. crenosoma vulpis is a lungworm seen commonly in foxes and sporadically in dogs with access to the intermediate hosts-slugs and snails. the adult lungworms live in small bronchi and bronchioles in the caudal lobes, causing eosinophilic and catarrhal bronchitis manifested grossly as gray areas of inflammation and atelectasis. in some animals, crenosoma vulpis causes bronchiolar goblet cell metaplasia and mucous obstruction, resulting in lobular atelectasis due to the valve effect of the mucous plug. eucoleus aerophilus. eucoleus aerophilus (capillaria aerophila) is a nematode parasite typically found in the trachea and bronchi of wild and domestic carnivores. in some cases, this parasite may also involve the nasal passages and sinuses. although generally asymptomatic, some dogs cough because of the local irritation caused by the parasites on the tracheal or bronchial mucosa. paragonimus spp. paragonimus kellicotti in north america and paragonimus westermani in asia are generally asymptomatic fluke infections in fish-eating species. the life cycle involves two intermediate hosts, the first a freshwater snail and the second a freshwater crab or crayfish; in north america, cats and dogs acquire infection by eating crayfish. gross lesions include pleural hemorrhages where the metacercariae migrate into the lungs. later, multifocal eosinophilic pleuritis, and subpleural cysts up to 7 mm long containing pairs of adult flukes, are found along with eosinophilic granulomas around clusters of eggs. like many other parasitic pneumonias, lesions and scars are more frequent in the caudal lobes. pneumothorax can occur if a cyst that communicates with an airway ruptures to the pleural surface. other parasitic infections. angiostrongylus vasorum and dirofilaria immitis are parasites of the pulmonary arteries and right ventricle and, depending on the stage, can produce different forms of pulmonary lesions. adult parasites can cause chronic arteritis that leads to pulmonary hypertension, pulmonary arterial thrombosis, interstitial (eosinophilic) granulomatous pneumonia, pulmonary interstitial fibrosis, congestive right-sided cardiac failure, and eventually caudal vena caval syndrome. other lesions include pleural petechial hemorrhages and, in later stages, diffuse pulmonary hemosiderosis and multifocal pulmonary infarcts. larvae and eggs also cause alveolar injury, thickening of the alveolar walls with eosinophils and lymphocytes (interstitial pneumonia), and multifocal or coalescing granulomas with giant cells (parasitic granulomas). pneumocystis carinii has been reported as a sporadic cause of chronic interstitial pneumonia in dogs with a compromised immune system (see pneumonias of horses; also see fig. 9 -20). aspiration pneumonia. aspiration pneumonia is an important form of pneumonia that occurs in dogs when vomit or regurgitated materials are aspirated into the lungs, or when drugs or radiographic contrast media are accidentally introduced into the airways (efig. 9-28) . as in other animal species, aspiration pneumonia may be unilateral or may more often affect the right cranial lobe ( fig. 9-104 ). the severity of lesions depends very much on the chemical and microbiologic composition of the aspirated material. in general, aspiration in monogastric animals, particularly in dogs and cats, is more severe because of the low ph of the gastric contents (chemical pneumonitis). in severe cases, dogs and cats die rapidly from septic shock and ards (see fig. 9 -63), which is microscopically characterized by diffuse alveolar damage, protein-rich pulmonary edema, neutrophilic alveolitis, and formation of typical hyaline membranes along the alveolar walls (see fig. 9-104 ). in animals that survive the acute stages of aspiration, pulmonary lesions progress to bronchopneumonia. aspiration pneumonia is a common sequela to disseminates systemically to other organs. clinical signs relate to the location of lesions, so there can be respiratory distress, lameness, generalized lymphadenopathy, or cutaneous lesions, among others. the lesions caused by coccidioides immitis consist of focal granulomas or pyogranulomas that can have suppurative or caseated centers. the fungal organisms are readily seen in histologic or cytologic preparation as large (10 to 80 µm in diameter), double-walled, and highly refractile spherules containing numerous endospores (see fig. 9-35, d) . histoplasmosis. histoplasmosis is a systemic infection that results from inhalation and, in dogs, possibly ingestion of another dimorphic fungus, histoplasma capsulatum. histoplasmosis occurs sporadically in dogs and human beings and, to a lesser extent, in cats and horses. bats often eliminate histoplasma capsulatum in the feces, and droppings from bats and birds, particularly pigeons, heavily promote the growth and survival of this fungus in the soil of enzootic areas. pulmonary lesions are grossly characterized by variably sized, firm, poorly encapsulated granulomas and, sometimes, more diffuse involvement of the lungs. microscopically, granulomatous lesions typically have many macrophages filled with small (1 to 3 µm), punctiform, intracytoplasmic, dark oval bodies (yeasts) (see fig. 9 -35, a) that are best demonstrated with pas reaction or gomori's methenamine silver stain. similar nodules or diffuse involvement can be present in other tissues, chiefly lymph nodes, spleen, intestine, and liver. toxoplasmosis. toxoplasmosis is a worldwide disease caused by the obligate intracellular, protozoal parasite toxoplasma gondii. cats and other felidae are the definitive hosts in which the mature parasite divides sexually in the intestinal mucosa. human beings, dogs, cats, and many wild mammals can become intermediate hosts after accidental ingestion of fertile oocysts shed in cat feces or ingestion of undercooked or raw meat containing tissue cysts, and fetuses can be infected transplacentally from an infected dam. in most instances, the parasite infects many cells of different tissues and induces an antibody response (seropositive animals) but does not cause clinical disease. toxoplasmosis is often triggered by immunosuppression, such as that caused by canine distemper virus. toxoplasmosis is characterized by focal necrosis around the protozoan. pulmonary lesions are severe, multifocal necrotizing interstitial pneumonia with notable proliferation of type ii pneumonocytes and infiltrates of macrophages and neutrophils. other lesions in disseminated toxoplasmosis include multifocal necrotizing hepatitis, myocarditis, splenitis, myositis, encephalitis, and ophthalmitis. the parasites appear microscopically as small (3 to 6 µm) basophilic cysts that can be found free in affected tissues or within the cytoplasm of many epithelial cells and macrophages (see efig. 8-8) . similar findings can be seen sporadically in dogs infected with neospora caninum and sarcocystis canis, and immunohistochemistry would be required to differentiate those protozoal organisms from toxoplasma gondii. filaroides hirthi. filaroides hirthi, a lungworm of the alveoli and bronchioles of dogs, has long been known as a cause of mild subclinical infection in large colonies of beagle dogs in the united states. however, it can on occasion cause severe and even fatal disease in individual pets, presumably as a result of immunosuppression. clinical signs may include coughing and terminal respiratory distress. grossly, the lesions are multifocal subpleural nodules, often with a green hue because of eosinophils, scattered throughout the lungs. microscopically, these nodules are eosinophilic granulomas arising 548.e1 chapter 9 respiratory system, mediastinum, and pleurae other pneumonias. idiopathic pulmonary fibrosis is a rare condition of uncertain etiology reported in the west highland white terrier breed that shares similarities with human and feline idiopathic pulmonary fibrosis. microscopically, there is diffuse interstitial pneumonia and progressive alveolar fibrosis with capillary obliteration, hyperplasia of type ii cells, some of which exhibit cellular atypia, and finally hypertrophy and hyperplasia of smooth muscle. the interstitial fibrosis eventually spills over alveolar spaces causing conspicuous intraalveolar fibrosis. although upper respiratory tract infections are common and important in cats, pneumonias are uncommon except when there is immunosuppression or aspiration of gastric contents. viral infections such as feline rhinotracheitis and calicivirus may cause lesions in the lungs, but unless there is secondary invasion by bacteria, they do not usually cause a fatal pneumonia. feline rhinotracheitis. feline rhinotracheitis is an important viral disease of cats caused by the ubiquitous felid herpesvirus 1 (fehv-1). this infection affects primarily young or debilitated cats causing inflammation in the nasal, ocular, and tracheal mucosa and, to a much lesser extent, the lung (see species-specific diseases of the nasal cavity and paranasal sinuses). when lungs are affected, fehv-1 causes bronchointerstitial pneumonia with necrosis of bronchiolar and alveolar epithelium, thickening of the alveolar walls, and extensive permeability edema. eosinophilic intranuclear inclusion bodies may be seen in infected epithelial cells early in infection. feline calicivirus. feline calicivirus (fcv) causes upper respiratory disease, stomatitis, conjunctivitis, and, to a lesser extent, interstitial pneumonia. microscopically, affected lungs exhibit the typical pattern of bronchointerstitial pneumonia with necrotizing bronchiolitis, thickening of alveolar walls, occasionally hyaline membranes, hyperplasia of type ii pneumonocytes, and macrophages admixed with cellular debris in the alveolar lumens. because pulmonary lesions are similar to those caused by fehv-1, isolation or in situ detection is required for final diagnosis. feline infectious peritonitis. feline infectious peritonitis (fip) is caused by fip virus (fipv), a mutated form of feline enteric cleft palate, and in dogs with megaesophagus secondary to either myasthenia gravis or persistent right aortic arch. it is also an important complication of general anesthesia or neurologic diseases affecting laryngeal function. paraquat. paraquat, a broad-spectrum herbicide widely used in gardening and agriculture, can cause severe and often fatal toxic interstitial pneumonia (pneumonitis) in dogs, cats, human beings, and other species. after ingestion or inhalation, this herbicide selectively accumulates in the lung where paraquat toxic metabolites are produced by club (clara) cells. these metabolites promote local release of free radicals in the lung, which causes extensive injury to club cells and to the blood-air barrier, presumably through lipid peroxidation of type i and ii pneumonocytes and alveolar endothelial cells (see fig. 9 -89). paraquat toxicity has been used experimentally as a model of oxidant-induced alveolar injury and pulmonary fibrosis. soon after poisoning, the lungs are heavy, edematous, and hemorrhagic because of extensive necrosis of epithelial and endothelial cells in the alveolar walls. the lungs of animals that survive acute paraquat toxicosis are pale, fail to collapse when the thorax is opened, and have interstitial emphysema, bullous emphysema, and occasionally pneumomediastinum. microscopic findings in the acute and subacute phases include necrosis of type i pneumonocytes, interstitial and alveolar edema, intraalveolar hemorrhages, and proliferation of type ii pneumonocytes. in the chronic stages (4 to 8 weeks later), the lesions are typically characterized by severe interstitial and intraalveolar fibrosis. uremic pneumopathy. uremic pneumonopathy (pneumonitis) is one of the many extrarenal lesions seen in dogs with chronic uremia. lesions are characterized by a combination of pulmonary edema and calcification of vascular smooth muscle and alveolar basement membranes. in severe cases, alveolar calcification prevents lung collapse when the thorax is opened. in the more advanced cases, the lungs appear diffusely distended, pale red or brown in color, and show a rough pleural surface with rib imprints (see fig. 9 -51). on palpation, the pulmonary parenchyma has a typical "gritty" texture because of mineralization of the alveolar and vascular walls, which are best visualized microscopically by using special stains such as von kossa (see fig. 9 -51). because this is not primarily an inflammatory lesion, the term pneumonitis should not be used. fig. 9-63) . a, note that the lungs did not collapse when the thorax was opened (loss of negative pressure) and as a result fill almost the entire thoracic cavity. the cranioventral aspects of the lung are consolidated with hemorrhage. b, alveolar capillary congestion, thick hyaline membranes along the alveolar septa (arrows), and intraalveolar hemorrhage. these microscopic changes are typical of the diffuse alveolar damage seen in lungs with ards. h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) feline calicivirus has removed chlamydophila felis from its previously overstated importance as a lung pathogen. tuberculosis. cats are susceptible to three types of mycobacterial infections: classic tuberculosis, feline leprosy, and atypical mycobacteriosis. classic tuberculosis in cats is rare and generally caused by mycobacterium bovis and mycobacterium microti but also, to a lesser extent, by mycobacterium tuberculosis. nosocomial tuberculosis (mycobacterium bovis) in cats has been reported with increased frequency. the usual route of infection for feline tuberculosis is oral, through infected rodents/meat or unpasteurized milk, so the granulomatous lesions are mainly in the intestine and mesenteric lymph nodes where they may disseminate through infected phagocytes to other organs. the solid and noncaseated appearance of tuberculous nodules is grossly similar to that of neoplasms, so they must be differentiated from pulmonary neoplasms (e.g., lymphoma). classic tuberculosis with dermal lesions in cats should be differentiated from feline leprosy (localized skin granulomas) caused by mycobacterium lepraemurium and other nonculturable species of acid-fast bacilli. atypical mycobacteriosis is caused by contamination of a skin wound with saprophytic and nonsaprophytic mycobacteria such as those of the mycobacterium avium complex. advances in pcr techniques have notably reduced the time required for etiologic diagnosis of mycobacteriosis in veterinary diagnostic laboratories. cryptococcosis. cryptococcosis (pulmonary cryptococcus neoformans or cryptococcus gatti) is the most frequent systemic mycosis in cats, and lesions are akin to those discussed in the section on mycotic pneumonias of dogs. it occurs worldwide in all species but is diagnosed most frequently in cats, horses, dogs, and human beings. some healthy dogs and cats harbor cryptococcus in the nasal cavity and become asymptomatic carriers. clinical infection may occur in immunocompetent cats and in cats that are immunologically compromised, such as by felv, fiv, malnutrition, or corticosteroid treatment. lesions can occur in nearly any tissue, resulting in a wide c coronavirus (fecv), and is one of a few viral infections of domestic animals that result in pyogranulomatous pneumonia. this disease is microscopically characterized by a vasculitis affecting many tissues and organs ( fig. 9-105) . other viral pneumonias. other viruses sporadically incriminated in feline interstitial pneumonia are cowpox virus (cpxv) and influenza a h1n1. pasteurellae. bacteria from the nasal flora such as pasteurella multocida and pasteurella-like organisms are occasionally associated with secondary bronchopneumonia in cats ( fig. 9-106) . pasteurella multocida also causes otitis media and meningitis, but its role as a respiratory pathogen is mainly associated with pyothorax. interestingly, there are reports of pasteurella multocida pneumonia in older or immunosuppressed human beings acquired through contact with domestic cats. mycoplasmas. mycoplasmas are often isolated from the lungs of cats with pulmonary lesions but are not definitively established as primary pathogens in feline pneumonias. feline pneumonitis. the term feline pneumonitis is a misnomer because the major lesions caused by chlamydophila felis (formerly chlamydia psittaci) are severe conjunctivitis and rhinitis (see species-specific diseases of the nasal cavity and paranasal sinuses). the elucidation of the importance of feline viral rhinotracheitis and organism infects erythrocytes in the erythrocytic stage of disease and multiplies in intravascular macrophages/monocytes, including those in the alveolar capillaries (efig. 9-29) , during the leukocytic stage of disease. aspiration pneumonia. aspiration pneumonias are common in cats as a result of vomiting, regurgitation, dysphagia, or anesthetic complication or after accidental administration of food, oral medicaments, or contrast media into the trachea (iatrogenic). pulmonary lesions are similar to those described for dogs, and the type of lung lesion depends on the chemical and bacterial composition of the aspirated material (see the section on aspiration pneumonia of dogs). feline idiopathic pulmonary fibrosis. feline idiopathic pulmonary fibrosis is a rare, progressive, and fatal disease of cats of uncertain etiology characterized by multifocal fibrotic nodules subpleurally and randomly in the lung making the pleural surface resemble nodular cirrhosis of the liver ( fig. 9-108) . microscopically, the affected alveolar and peribronchiolar interstitium is thickened by excessive fibrosis, abundant deposition of extracellular matrix, and hypertrophy of smooth muscle. some investigators suggest an intrinsic cellular defect in type ii pneumonocytes as the underlying cause. the alveolar walls are diffusely lined by cuboidal hyperplastic type ii pneumonocytes, and the alveolar lumens often contain exfoliated cells and necrotic debris. this feline condition has morphologic features similar to "equine multinodular pulmonary fibrosis" and "cryptogenic pulmonary fibrosis" in human beings. fetal pneumonias. pneumonia is one of the most frequent lesions found in fetuses submitted for postmortem examination, particularly in foals and food-producing animals. because of autolysis, lack of inflation, and the lungs being at various stages of development, fetal lesions are often missed or misdiagnosed. in the nonaerated fetal lung, the bronchoalveolar spaces are filled with a viscous, locally produced fluid known as lung fluid or lung liquid. it has been estimated that an ovine fetus produces approximately 2.5 ml of "lung fluid" per kilogram of body weight per hour. in the variety of clinical signs. however, granulomatous rhinitis, sinusitis, otitis media and interna, pneumonia, ulcerative dermatitis, and meningoencephalitis are most common. the pulmonary lesion in cryptococcosis is a multifocal granulomatous pneumonia and, like those occurring in other internal organs, they are small, gelatinous, white foci. the gelatinous appearance is due to the broad mucous capsule around the yeast (see fig. 9-35, b) . microscopically, lesions contain great numbers of fungal organisms (4 to 10 µm in diameter without the capsule) and only a few macrophages, lymphocytes, and multinucleated giant cells. this thick polysaccharide capsule does not stain well with h&e, and thus there is a large empty space or halo around the yeast. feline lungworm. aelurostrongylus abstrusus, known as feline lungworm, is a parasite that occurs in cats wherever the necessary slug and snail intermediate hosts are found. it can cause chronic respiratory disease with coughing and weight loss and, sometimes, severe dyspnea and death, particularly if there are secondary bacterial infections. the gross lesions are multifocal, amber, and subpleural granulomatous nodules up to 1 cm in diameter throughout the lungs. on incision, these nodules may contain viscous exudate. microscopically, the adult parasites, eggs, and coiled larvae are in the bronchioles and alveoli, where they cause catarrhal bronchiolitis, hyperplasia of submucosal glands, and, later, granulomatous alveolitis, alveolar fibrosis, and fibromuscular hyperplasia ( fig. 9-107) . during routine examination of feline lungs, it is quite common to find fibromuscular hyperplasia in bronchioles and arterioles in otherwise healthy cats. it was alleged in the past that this fibromuscular hyperplasia was a long-term sequela of subclinical infection with aelurostrongylus abstrusus. however, this view has been challenged; thus the pathogenesis and significance of pulmonary fibromuscular hyperplasia in healthy cats remains uncertain. in severe cases, fibromuscular hyperplasia is grossly visible in the lungs as white subpleural nodules. other parasitic pneumonias. toxoplasma gondii, paragonimus kellicotti, and dirofilaria immitis can also affect cats (see the section on parasitic pneumonias of dogs). cytauxzoon felis is an apicomplexan hemoparasite that affects domestic and wild felidae. the diseases that cause fetal pneumonia in farm animals. gross lesions in the lungs are generally undetected, but microscopic lesions include focal necrotizing interstitial pneumonia and focal necrosis in the liver, spleen, or brain. fetal bronchointerstitial pneumonia also occurs in some viral abortions, such as those caused by infectious bovine rhinotracheitis (ibr) virus and bovine parainfluenza virus 3 (bpiv-3) in cattle and equine viral rhinopneumonitis (evr) in horses. fetal pneumonias in dogs and cats are infrequently described, perhaps because aborted puppies and kittens are rarely submitted for postmortem examination. with advancements in molecular biology techniques, the etiologic diagnosis of abortions and their association with pulmonary fetal lesions is rapidly improving. neonatal pneumonias and septicemias. these entities are rather common in newborn animals lacking passive immunity because of the lack of either ingestion or absorption of maternal colostrum (failure of passive transfer or hypogammaglobulinemia). in addition to septicemias causing interstitial pneumonia, farm animals with hypogammaglobulinemia can develop bronchopneumonia by inhalation of bacterial pathogens. these include histophilus somni and pasteurella multocida in calves; streptococcus spp. in foals; and escherichia coli, listeria monocytogenes, and streptococcus suis in pigs. meconium aspiration syndrome. meconium aspiration syndrome (mas) is an important but preventable condition in human babies that originates when amniotic fluid contaminated with meconium is aspirated during labor or immediately after birth. the pathogenesis of mas is basically the same as in those of fetal bronchopneumonia (see fig. 9-109) . fetal hypoxia, a common event during dystocia or prolonged parturition, causes the fetus to relax the anal sphincter and release meconium into the amniotic fluid. aspiration of meconium can occur directly from aspirating contaminated amniotic fluid before delivery (respiratory movements with an open glottis) or immediately after delivery when the meconium lodged in the nasopharynx is carried into the lung with the first breath of air. this latter form of aspiration is prevented in delivery rooms by routine suction of the nasopharynx in meconium-stained babies. mas is well known in human babies, but the occurrence and significance in animals remains largely unknown. mas has been reported in calves, foals, piglets, and puppies. although pulmonary lesions are generally mild and transient, aspiration of meconium can be life-threatening for newborn babies and animals because it typically occurs in compromised neonates already suffering from intrauterine hypoxia and acidosis. neonatal acidosis is known to impair colostrum absorption in calves. common mas sequelae are lobular atelectasis, pulmonary hypertension, and possibly airway hyperreactivity. in the most severe cases of mas, focal (patchy) atelectasis can be observed grossly in the lung, indicating failure of the lungs to be fully aerated because of the mechanical obstruction and the chemical effect of meconium on pulmonary surfactant (see fig. 9 -52). microscopically, meconium and keratin exfoliated from skin of the fetus into the amniotic fluid are present in bronchi, bronchioles, and alveoli and accompanied by mild alveolitis characterized by infiltration of leukocytes followed by alveolar macrophages and occasional giant cells (efig. 9-30) . lung cancer in animals is rare, unlike in human beings, in which the incidence is alarming and continues to be the number one cause of death due to cancer in canada, the united states, and europe. interestingly, prostatic and breast cancers, so much feared by men fetus, this fluid normally moves along the tracheobronchial tree, reaching the oropharynx, where a fraction is swallowed into the gastrointestinal tract, and a small portion is released into the amniotic fluid. at the time of birth, the lung fluid is rapidly reabsorbed from the lungs by alveolar absorption and lymphatic drainage. aspiration of amniotic fluid contaminated with meconium and bacteria from placentitis is the most common route by which microbial pathogens reach the fetal lungs. this form of pneumonia is secondary to fetal hypoxia and acidosis ("fetal distress"), which cause the fetus to relax the anal sphincter, release meconium into the amniotic fluid, and, in the terminal stages, inspire deeply with open glottis, resulting in the aspiration of contaminated fluid ( fig. 9-109 ). gross lesions are only occasionally recognized, but microscopic changes are similar to those of a bronchopneumonia. microscopically, bronchoalveolar spaces contain variable numbers of neutrophils, macrophages, epidermal squames, and pieces of meconium that appear as bright yellow material because of its bile content. in contrast to postnatal bronchopneumonia, lesions in fetuses are not restricted to the cranioventral aspects of the lungs but typically involve all pulmonary lobes. in cattle, brucella abortus and trueperella (arcanobacterium) pyogenes are two of the most common bacteria isolated from the lungs of aborted fetuses. these bacteria are usually present in large numbers in the amniotic fluid of cows with bacterial placentitis. inflammation of the placenta interferes with oxygen exchange between fetal and maternal tissue, and the resultant fetal hypoxia induces the fetus to "breathe" with an open glottis and aspirate the amniotic fluid. aspergillus spp. (mycotic abortion) and ureaplasma diversum cause sporadic cases of placentitis, which results in fetal pneumonia and abortion. in addition to the respiratory route (aspiration), pathogens, such as bacteria and viruses, can also reach the lungs via fetal blood and cause interstitial pneumonia. listeriosis (listeria monocytogenes), salmonellosis (salmonella spp.), and chlamydiosis (chlamydophila abortus [c. psittaci]) are the best known examples of blood-borne primary benign neoplasms of the lungs, such as pulmonary adenomas, are highly unusual in domestic animals. most primary neoplasms are malignant and appear as solitary masses of variable size that, with time, can metastasize to other areas of the lungs and to distant organs. it is sometimes difficult on gross and microscopic examination to differentiate primary lung cancer from pulmonary metastasis resulting from malignant neoplasms elsewhere in the body. it is often difficult to determine the precise topographic origin of a neoplasm within the lungs-for example, whether it originates in the conducting system (bronchogenic carcinoma), transitional system (bronchiolar carcinoma), exchange system (alveolar carcinoma), or bronchial glands (bronchial gland carcinoma). according to the literature, pulmonary carcinomas in animals arise generally from club (clara) cells or type ii pneumonocytes of the bronchioloalveolar region, in contrast to those in human beings, which are mostly bronchogenic. tumors located at the hilus generally arise from major bronchi and tend to be a solitary large mass with occasional small metastasis to the periphery of the lung. in contrast, tumors arising from the bronchioloalveolar region are often multicentric with numerous peripheral metastases in the lung parenchyma. because of histologic architecture and irrespective of the site of origin, many malignant epithelial neoplasms are classified by the all-encompassing term of pulmonary adenocarcinomas. dogs and cats are the species most frequently affected with primary pulmonary neoplasms, largely carcinomas, generally in older animals. the mean age for primary lung tumors is 11 years for dogs and 12 years for cats. pulmonary carcinomas in other domestic animals, except for retrovirus-induced pulmonary carcinoma in sheep, are less common, possibly because fewer farm animals are allowed to reach their natural life span. these neoplasms can be invasive or expansive, vary in color (white, tan, or gray) and texture (soft or firm), and often have areas of necrosis and hemorrhage, which result in a "craterous" or "umbilicate" appearance. this umbilicate appearance is frequently seen in rapidly growing carcinomas in which the center of the tumoral mass undergoes necrosis as a result of ischemia. some lung neoplasms resemble pulmonary consolidation or large granulomas. cats with moderately differentiated neoplasms had significantly longer survival time (median, 698 days) than cats with poorly differentiated neoplasms (median, 75 days). dogs with primary lung neoplasms, grades i, ii, and iii, had survival times of 790, 251, and 5 days, respectively. ovine pulmonary adenocarcinoma (ovine pulmonary carcinoma). ovine pulmonary adenocarcinoma, also known as pulmonary adenomatosis and jaagsiekte (from the south african afrikaans word for "driving sickness"), is a transmissible, retrovirus-induced neoplasia of ovine lungs caused by jaagsiekte sheep retrovirus (jsrv). it occurs in sheep throughout the world, with the notable exception of australia and new zealand; its incidence is high in scotland, south africa, and peru and unknown but probably low in north america. this pulmonary carcinoma behaves very much like a chronic pneumonia, and jsrv shares many epidemiologic similarities with the ovine lentivirus responsible for maedi and the retrovirus responsible for enzootic nasal carcinoma in small ruminants. pulmonary adenomatosis has been transmitted to goats experimentally but is not known to be a spontaneous disease in that species. ovine pulmonary adenocarcinoma affects mainly mature sheep but can occasionally affect young stock. intensive husbandry probably facilitates horizontal transmission by the copious nasal discharge and explains why the disease occurs as devastating epizootics with 5% to 80% mortality when first introduced into a flock. differential diagnosis between maedi and pulmonary adenomatosis can prove difficult because both diseases often coexist in the same flock and women, are a distant second. to say that cigarette smoking is responsible for this epidemic of lung cancer is unnecessary. although dogs have been proposed as valuable "sentinels" for environmental hazards, such as exposure to passive smoking, asbestos, dyes, and insecticides, it is not known if the prevalence of canine lung tumors has increased in geographical areas with high contamination. alterations in genes (oncogenes) and chromosomes and changes in biologically active molecules have been linked to lung cancer in recent years. as with many other forms of cancer, epidemiologic studies indicate that the incidence of pulmonary neoplasms increases with age, but there are still insufficient data to confirm that particular canine or feline breeds have a higher predisposition to spontaneous lung neoplasms. a standard nomenclature of pulmonary neoplasms in domestic animals is lacking, and as a consequence, multiplicity of names and synonyms occur in the veterinary literature. some classifications are based on the primary site, whereas others emphasize more the histomorphologic type. the most common types of benign and malignant pulmonary neoplasms in domestic mammals are listed in box 9-2. clinically, the signs of pulmonary neoplasia vary with the degree of invasiveness, the amount of parenchyma involved, and locations of metastases. signs may be vague, such as cough, lethargy, anorexia, weight loss, and perhaps dyspnea. in addition, paraneoplastic syndromes, such as hypercalcemia, endocrinopathies, and pulmonary hypertrophic osteoarthropathy, have been associated with pulmonary neoplasms. primary neoplasms of the lungs. primary neoplasms of the lungs arise from cells normally present in the pulmonary tissue and can be epithelial or mesenchymal, although the latter are rare. any malignant tumor metastatic from another body location (e.g., osteosarcoma in dogs, uterine carcinoma in cows, and malignant melanoma in horses) box 9-2 classification of pulmonary neoplasms 553.e1 chapter 9 respiratory system, mediastinum, and pleurae abundant cytoplasm containing numerous acidophilic granules, which are positive for pas and for s-100 protein using immunohistochemistry. although this tumor can cause bronchial obstruction and respiratory signs, in most cases, it is an incidental finding in older horses submitted for postmortem examination. lymphomatoid granulomatosis. lymphomatoid granulomatosis is a rare but interesting pulmonary disease of human beings, dogs, cats, and possibly horses and donkeys characterized by nodules or large solid masses in one or more lung lobes. these frequently metastasize to lymph nodes, kidneys, and liver. microscopically, tumors are formed by large pleomorphic mononuclear (lymphomatoid) cells with a high mitotic rate and frequent formation of binucleated or multinucleated cells. tumor cells have a distinct tendency to grow around blood vessels and invade and destroy the vascular walls. lymphomatoid granulomatosis has some resemblance to lymphoma and is therefore also referred to as angiocentric lymphoma; phenotypic marking confirms that neoplastic cells are a mixed population of plasma cells, b and t lymphocytes, and histiocytes. cerebral and cutaneous forms of lymphomatoid granulomatosis have also reported in human beings, dogs, and cats. secondary neoplasms of the lungs. secondary neoplasms of the lungs are all malignant by definition because they are the result of metastasis to the lungs from malignant neoplasms elsewhere. because the pulmonary capillaries are the first filter met by tumor emboli released into the vena cava or pulmonary arteries, secondary neoplasms in the lung are relatively common in comparison to primary ones. also, secondary tumors can be epithelial or mesenchymal in origin. common metastatic tumors of epithelial origin are mammary, thyroid ( fig. 9-111) , and uterine carcinomas. tumors of mesenchymal origin are osteosarcoma ( fig. 9-112, a) ; hemangiosarcoma ( fig. 9-112, b) ; malignant melanoma in dogs; lymphoma in cows, pigs, dogs, and cats ( fig. 9-113) ; and vaccineassociated sarcoma in cats. usually, secondary pulmonary neoplasms are multiple; scattered throughout all pulmonary lobes (hematogenous dissemination); of variable size; and, according to the growth pattern, can be nodular, diffuse, or radiating (efig. 9-31) . the appearance of metastatic neoplasms differs according to the type of neoplasm. for example, dark red cystic nodules containing blood indicate hemangiosarcoma, dark black solid nodules indicate melanoma, and hard solid nodules (white, yellow, or tan color) with bone spicules indicate osteosarcoma. the gross appearances of or in the same animal. death is inevitable after several months of the initial onset of respiratory signs, and a specific humoral immune response to jsrv is undetectable in affected sheep. during the early stages of ovine pulmonary carcinoma, the lungs are enlarged, heavy, and wet and have several firm, gray, variably sized nodules that in some cases can be located in the cranioventral lobes mimicking a bronchopneumonic lesion ( fig. 9-110, a) . in the later stages, the nodules become confluent, and large segments of both lungs are diffusely, but not symmetrically, infiltrated by neoplastic cells. on cross section, edematous fluid and a copious mucoid secretion are present in the trachea and bronchi ( fig. 9-110, b) . microscopically, the nodules consist of cuboidal or columnar epithelial cells lining airways and alveoli and forming papillary or acinar (glandlike) structures (see fig. 9-110, a) . because the cells have been identified ultrastructurally as originating from both type ii alveolar epithelial cells and club (clara) cells, the neoplasm is considered a "bronchioloalveolar" carcinoma. sequelae often include secondary bronchopneumonia, abscesses, and fibrous pleural adhesions. metastases occur to tracheobronchial and mediastinal lymph nodes and, to a lesser extent, to other tissues such as pleura, muscle, liver, and kidneys. neoplastic cells stain strongly positive for jsrv using immunohistochemistry. clinically, ovine pulmonary adenocarcinoma is characterized by a gradual loss of condition, coughing, and respiratory distress, especially after exercise (e.g., herding or "driving"). appetite and temperature are normal, unless there are secondary bacterial infections. an important differentiating feature from maedi (interstitial pneumonia) can be observed if animals with pulmonary adenomatosis are raised by their hind limbs; copious, thin, mucoid fluid, produced by neoplastic cells in the lungs, pours from the nostrils of some animals. carcinoid (neuroendocrine) tumor of the lungs. carcinoid tumor of the lungs is a neoplasm presumably arising from neuroendocrine cells and is sporadically seen in dogs as multiple, large, firm pulmonary masses close to the mainstem bronchi. it has also been reported in the nasal cavity of horses. tumor cells are generally polygonal with finely granular, pale, or slightly eosinophilic cytoplasm. nuclei are small, and mitotic figures are absent or rare. granular cell tumor. granular cell tumor is a rare and locally invasive tumor that has been reported mainly in human beings and older horses. the cell origin of this tumor was thought to be the myoblast, but it is currently presumed to be schwann cells, which are normally present in the bronchovascular bundles of the lung. microscopically, neoplastic cells are large, polyhedron-shaped with metastatic carcinomas are generally similar to the primary neoplasm and sometimes have umbilicated centers. proper diagnoses of pulmonary neoplasms in live animals require history, clinical signs, radiographs, cytologic analysis of bal fluid, and, when necessary, a lung biopsy. identification of a specific lineage of neoplastic cells in biopsy or postmortem specimens is often difficult and requires electron microscopy or immunohistochemical techniques. electron microscopy allows identification of distinctive cellular components such as osmiophilic lamellar phospholipid nephritic bodies in alveolar type ii epithelial cells or melanosomes in melanomas. immunohistochemical staining is also helpful in identifying tumor cells. the thoracic wall, diaphragm, and mediastinum are lined by the parietal pleura, which reflects onto the lungs at the hilum and continues as the visceral pleura, covering the entire surface of the lungs, except at the hilus where the bronchi and blood vessels enter. the space between the parietal and visceral pleura (pleural space) is only minimal and under normal conditions contains only traces of clear fluid, which is a lubricant, and a few exfoliated cells. samples of this fluid are obtained by thoracocentesis, a simple procedure in which a needle is passed into the pleural cavity. volumetric, biochemical, and cytologic changes in this fluid are routinely used in veterinary diagnostics. anomalies 7 congenital defects are rare and generally of little clinical significance. cysts within the mediastinum of dogs and, less often, cats in severe cases, the amount of fluid present in the thoracic cavity can be considerable. for instance, a medium-size dog can have 2 l of fluid, and a cow may accumulate 25 l or more. excessive fluid in the thorax causes compressive atelectasis resulting in respiratory distress (see fig. 9 -54). hydrothorax is most commonly seen in cattle with right-sided heart failure or cor pulmonale (hydrostatic) (efig. 9 -32); dogs with congestive heart failure (hydrostatic), chronic hepatic disease (hepatic hydrothorax) ( fig. 9-114) , or nephrotic syndrome (hypoproteinemia); pigs with mulberry heart disease (increased vascular permeability); and horses with african horse sickness (increased vascular permeability). hemothorax. blood in the thoracic cavity is called hemothorax, but the term has been used for exudate with a sanguineous component. causes include rupture of a major blood vessel as a result of severe thoracic trauma (e.g., hit by car); erosion of a vascular wall by malignant cells or inflammation (e.g., aortitis caused by spirocerca lupi); ruptured aortic aneurysms; clotting defects, including coagulopathies; warfarin toxicity; disseminated intravascular coagulation (consumption coagulopathy); and thrombocytopenia. hemothorax is generally acute and fatal. on gross examination, the thoracic cavity can be filled with blood, and the lungs are partially or completely atelectatic ( fig. 9-115 ). chylothorax. the accumulation of chyle (lymph rich in triglycerides) in the thoracic cavity ( fig. 9-116 ) is a result of the rupture of major lymph vessels, usually the thoracic duct or the right lymphatic duct. the clinical and pathologic effects of chylothorax are similar to those of the other pleural effusions. causes include thoracic neoplasia (the most common cause in human beings but a distant second to idiopathic cases in dogs), trauma, congenital lymph vessel anomalies, lymphangitis, dirofilariasis, and iatrogenic rupture of the thoracic duct during surgery. the source of the leakage of chyle is rarely found at necropsy. when the leakage of chyle occurs in the abdominal cavity, the condition is referred to as chyloabdomen. cytologic and biochemical examination of fluid collected by thoracocentesis typically reveals large numbers of lymphocytes, lipid droplets, few neutrophils in chronic cases, and high triglyceride content. can be large enough to compromise pulmonary function or mimic neoplasia in thoracic radiographs. these cysts may arise from the thymus (thymic branchial cysts), bronchi (bronchogenic cysts), ectopic thyroid tissue (thyroglossal duct cysts), or from remnants of the branchial pouches, and they are generally lined by epithelium and surrounded by a capsule of stromal tissue. anomalies of the thoracic duct cause some cases of chylothorax. pleural calcification. pleural calcification is commonly found in dogs and less often in cats with chronic uremia. lesions appear as linear white streaks in parietal pleura, mainly over the intercostal muscles of the cranial part of the thoracic cavity. the lesions are not functionally significant but indicate a severe underlying renal problem. vitamin d toxicity (hypervitaminosis d) and ingestion of hypercalcemic substances, such as vitamin d analogs, can also cause calcification of the pleura and other organs. pneumothorax. pneumothorax is the presence of air in the thoracic cavity where there should normally be negative pressure to facilitate inspiration. human beings have a complete and strong mediastinum so that pneumothorax is generally unilateral and thus not a serious problem. in dogs, the barrier varies, but in general it is not complete, so often some communication exists between left and right sides. there are two main forms of pneumothorax. in spontaneous (idiopathic) pneumothorax, air leaking into the pleural cavity from the lungs occurs without any known underlying disease or trauma. in secondary pneumothorax, movement of air into the pleural cavity results from underlying pulmonary or thoracic wall disease. the most common causes of secondary pneumothorax in veterinary medicine are penetrating wounds to the thoracic wall, perforated esophagus, iatrogenic trauma to the thorax and lung during a transthoracic lung biopsy or thoracoscopy, tracheal rupture from improper intubation, and rupture of emphysematous bullae or parasitic pulmonary cysts (paragonimus spp.) that communicate with the thoracic cavity. pneumothorax and pneumomediastinum caused by high air pressure (barotrauma) are also well documented in cats after equipment failure during anesthesia. clinical signs of pneumothorax include respiratory distress, and the lesion is simply a collapsed, atelectatic lung. the air is readily reabsorbed from the cavity if the site of entry is sealed. pleural effusion. pleural effusion is a general term used to describe accumulation of any fluid (transudate, modified transudate, exudate, blood, lymph, or chyle) in the thoracic cavity. cytologic and biochemical evaluations of pleural effusions taken by thoracocentesis are helpful in determining the type of effusion and possible pathogenesis. based on protein concentration and total numbers of nucleated cells, pleural effusions are cytologically divided into transudates, modified transudates, and exudates. hydrothorax. when the fluid is serous, clear, and odorless and fails to coagulate when exposed to air, the condition is referred to as hydrothorax (transudate). causes of hydrothorax are the same as those involved in edema formation in other organs: increased hydrostatic pressure (heart failure), decreased oncotic pressure (hypoproteinemia, as in liver disease), alterations in vascular permeability (inflammation), or obstruction of lymph drainage (neoplasia). in cases in which the leakage is corrected, if the fluid is a transudate, it is rapidly reabsorbed. if the fluid persists, it irritates the pleura and causes mesothelial hyperplasia and fibrosis, which thickens the pleura. from a perforated esophagus. chronic injury typically results in serosal fibrosis and tight adhesions between visceral and parietal pleurae (see fig. 9 -71). when extensive, these adhesions can obliterate the pleural space. pleuritis or pleurisy. inflammation of the visceral or parietal pleurae is called pleuritis, and according to the type of exudate, it can be fibrinous, suppurative, granulomatous, hemorrhagic, or a combination of exudates. acute fibrinous pleuritis can progress with time to pleural fibrosis ( fig. 9-117 ). when suppurative pleuritis results in accumulation of purulent exudate in the cavity, the lesion is called pyothorax or thoracic empyema ( fig. 9-118) . clinically, pleuritis causes considerable pain, and in addition, empyema can result in severe toxemia. pleural fibrous adhesions (between parietal and visceral pleura) and fibrosis are the most common sequelae of chronic pleuritis and can significantly interfere with inflation of the lungs. pleuritis can occur as an extension of pneumonia, particularly in fibrinous bronchopneumonias (pleuropneumonia), or it can occur alone, without pulmonary involvement ( fig. 9-119 ). bovine and ovine pneumonic mannheimiosis and porcine and bovine pleuropneumonia are good examples of pleuritis associated with fibrinous bronchopneumonias. polyserositis in pigs and pleural empyema, particularly in cats and horses, are examples of pleural inflammation in pleural tissue is readily susceptible to injury caused by direct implantation of an organism through a penetrating thoracic or diaphragmatic wound; by hematogenous dissemination of infectious organisms in septicemias; or by direct extension from an adjacent inflammatory process, such as in fibrinous bronchopneumonia or in contrast to those with the effusive ("wet") form, in which thoracic involvement is primarily that of a pleural effusion. cytologic evaluation of the effusion typically shows a low to moderate cellularity with degenerated leukocytes, lymphocytes, macrophages, and mesothelial cells, and a pink granular background as a result of the high protein content. pleuritis is also an important problem in horses. nocardia spp. can cause fibrinopurulent pneumonia and pyothorax with characteristic sulfur granules. although mycoplasma felis can be isolated from the respiratory tract of normal horses, it is also isolated from horses with pleuritis and pleural effusion, particularly during the early stages of infection. the portal of entry of this infection is presumably aerogenous, first to the lung and subsequently to the pleura. the pleural surface of the lung is often involved in neoplasms that have metastasized from other organs to the pulmonary parenchyma and ruptured the visceral pleura to seed the pleural cavity. mesothelioma is the only primary neoplasm of the pleura. which involvement of the lungs may not accompany the pleuritis. pleural inflammation is most frequently caused by bacteria, which cause polyserositis reaching the pleura hematogenously. these bacteria include haemophilus parasuis (glasser's disease) (see , streptococcus suis, and some strains of pasteurella multocida in pigs; streptococcus equi ssp. equi and streptococcus equi ssp. zooepidemicus in horses; escherichia coli in calves; and mycoplasma spp. and haemophilus spp. in sheep and goats. contamination of pleural surfaces can be the result of extension of a septic process (e.g., puncture wounds of the thoracic wall and, in cattle, traumatic reticulopericarditis) and ruptured pulmonary abscesses (e.g., trueperella pyogenes). in dogs and cats, bacteria (e.g., nocardia, actinomyces, and bacteroides) can cause pyogranulomatous pleuritis, characterized by accumulation of blood-stained pus ("tomato soup") in the thoracic cavity. this exudate usually contains yellowish flecks called sulfur granules ( fig. 9-120 ), although these are less common in nocardial empyema in cats. many species of bacteria, such as escherichia coli, trueperella pyogenes, pasteurella multocida, and fusobacterium necrophorum, can be present in pyothorax of dogs and cats. these bacteria occur alone or in mixed infections. the pathogenesis of pleural empyema in cats is still debatable, but bite wounds or penetration of foreign material (migrating grass awns) are likely. pyogranulomatous pleuritis with empyema occurs occasionally in dogs, presumably associated with inhaled small plant material and penetrating (migrating) grass awns. because of their physical shape (barbed) and assisted by the respiratory movement, aspirated grass awns can penetrate airways, move through the pulmonary parenchyma, and eventually perforate the visceral pleura causing pyogranulomatous pleuritis. cats with the noneffusive ("dry") form of feline infectious peritonitis (fip) frequently have focal pyogranulomatous pleuritis, mesothelioma is a rare neoplasm of the thoracic, pericardial, and peritoneal mesothelium of human beings that is seen most commonly in calves, in which it can be congenital. in human beings, it has long been associated with inhalation of certain types of asbestos fibers (asbestos mining and ship building) alone or with cigarette smoking as a probable cocarcinogen; no convincing association between the incidence of mesothelioma and exposure to asbestos has been made in domestic animals. in animals, there may be pleural effusion with resulting respiratory distress, cough, and weight loss. mesothelioma initially causes a thoracic effusion, but cytologic diagnosis can be difficult because of the morphologic resemblance of malignant and reactive mesothelial cells. during inflammation, mesothelial cells become reactive and not only increase in number but also become pleomorphic and form multinucleated cells that may be cytologically mistaken for those of a carcinoma. grossly, mesothelioma appears as multiple, discrete nodules or arborescent, spreading growths on the pleural surface ( fig. 9-121) . microscopically, either the mesothelial covering cells or the supporting tissue can be the predominant malignant component, so the neoplasm can microscopically resemble a carcinoma or a sarcoma. figure 9 -120 nocardiosis. a, chronic pleuritis (nocardia asteroides), pleural cavity, cat. the pleural cavity is covered with abundant red-brown ("tomato soup") exudate" (syringe). once considered to be pathognomonic of nocardia spp. infection, it is no longer regarded as being diagnostic of nocardiosis. the fluid contains abundant protein, erythrocytes, granulomatous inflammatory cells, and sulfur granules. b, chronic pleuritis (nocardia asteroides), visceral pleura, dog. the thickened pleura has a granular pink-gray appearance because of granulomatous inflammation and the proliferation of fibrovascular tissue of the pleura. c, chronic pleuritis (nocardia asteroides), dog. the pleura has been thrown up into villous-like projections composed of abundant fibrovascular tissue and granulomatous inflammation. leakage from the neocapillaries of the fibrovascular tissue is responsible for the hemorrhagic appearance of the pleural exudate. h&e stain. d, chronic pleuritis (nocardia asteroides), thoracic cage, parietal pleura, cat. large pieces of exudate, which contain yellow sulfur granules, are present on the thickened pleura. although considered malignant, mesotheliomas rarely metastasize to distant organs. secondary neoplasms of the pleura. secondary tumors may also spread into the visceral and parietal pleura. thymomas are rare neoplasms that grow in the cranial mediastinum of adult or aged dogs, cats, pigs, cattle, and sheep. thymomas are composed of thymic epithelium and lymphocytes (see chapter 13). old age, both in human beings and in animals, is known to be a risk factor for pulmonary infections, but the precise mechanisms involved in this increased susceptibility are still under investigation. some studies have shown that in aged individuals the antibacterial properties provided by surfactant proteins, proinflammatory cytokines, and complement are altered. pulmonary hyperinflation (often referred to as senile emphysema) has been reported as an age-related change in human and canine lungs. other age-related changes described in canine lungs include mineralization of bronchial cartilage, pleural and alveolar fibrosis, and heterotopic bone formation (so-called "pulmonary osteomas"). we thank all pathologists at the atlantic veterinary college, university of prince edward island for providing case material. suggested readings are available at www.expertconsult.com. lung section showing a distended and partially occluded blood vessel (center of figure) containing large granular cells. these large cells are macrophages, and their cytoplasm is filled with myriad merozoites isolation of porcine circoviruslike viruses from pigs with a wasting disease in the usa and europe exercise-induced pulmonary hemorrhage effect of mucociliary transport relies on efficient regulation of ciliary beating epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats canine h3n8 influenza virus infection in dogs and mice failure of respiratory defenses in the pathogenesis of bacterial pneumonia in cattle the respiratory system advances in diagnosis of respiratory diseases of small ruminants canine nasal disease transmission of equine influenza virus to dogs dear jd: bacterial pneumonia in dogs and cats acute respiratory distress syndrome in dogs and cats: a review of clinical findings and pathophysiology inflammatory response to infectious pulmonary injury laryngeal paralysis: a study of 375 cases in a mixed-breed population of horses stem cells of the respiratory tract exudative pleural disease in small animals bovine respiratory disease research pulmonary thromboembolism coccidioidomycosis in dogs and cats: a review cousens c: pathology and pathogenesis of ovine pulmonary adenocarcinoma prognosis factors for survival in cats after removal of a primary lung tumor: 21 cases (1979-1994) the acute respiratory distress syndrome: from mechanism to translation endogenous lipid pneumonia in cats: 24 cases (1985-1998) retroviral infections in sheep and goats: small ruminant lentiviruses and host interaction canine and feline nasal neoplasia the acute respiratory distress syndrome equine respiratory medicine and surgery canine pleural and mediastinal effusions: a retrospective study of 81 cases a review of histiocytic diseases of dogs and cats estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle polymicrobial respiratory disease in pigs current state of knowledge on porcine circovirus type 2-associated lesions common and emerging infectious diseases in the animal shelter chronic rhinitis in the cat advances in the understanding of pathogenesis, and diagnosis and therapeutics of feline allergic asthma mannheimia haemolytica and bovine respiratory disease detection of respiratory viruses and bordetella bronchiseptica in dogs with acute respiratory tract infections current perspectives on the diagnosis and epidemiology of mycoplasma hyopneumoniae infection mannheimia haemolytica: bacterialhost interactions in bovine pneumonia acute lung injury review rhodococcus equi: the many facets of a pathogenic actinomycete tumors of the respiratory system key: cord-024141-9sdbhw2g authors: liu, haiyan; zhang, jun; lin, fan title: lung and mediastinum date: 2017-09-02 journal: handbook of practical fine needle aspiration and small tissue biopsies doi: 10.1007/978-3-319-57386-1_6 sha: doc_id: 24141 cord_uid: 9sdbhw2g fine needle aspiration and small tissue biopsies have become a primary modality to achieve a definitive diagnosis of a mass-like lesion of the lung and mediastinum. this chapter delineated cytologic and histologic features of common and rare neoplastic and nonneoplastic mass-like lesions of the lung and mediastinum. the utilities and pitfalls of commonly used diagnostic immunohistochemical (ihc) stains, such as ttf1, napsin a, p40 and ck5/6, and small diagnostic ihc panels, were described. multiple challenging and yet practical cases at the end of the chapter were used to reemphasize important points illustrated throughout the chapter. • the concept of personalized medicine and the remarkable advances in lung cancer genetics and therapy in the past decade have changed lung pathology practice dramatically. • the 2015 world health organization (who) classification of tumors of the lung specifies that immunohistochemistry is required for lung cancer diagnosis, not only for small biopsies and fine needle aspiration (fna) specimens but also for certain resected specimens such as solid adenocarcinoma (adc), nonkeratinizing squamous cell carcinoma, large cell carcinoma, neuroendocrine tumors, and sarcomatoid carcinomas. • new criteria and terminology for the diagnosis of lung cancer based on small biopsies and cytology are proposed in the 2015 who classification and are summarized in tables 6.1 and 6.2. • the role of cytopathologists has expanded to not only making a specific diagnosis, including histopathological subtyping of tumors, but also to thoughtfully utilizing the limited material for necessary genetic studies to help personalize treatment strategies for advanced lung cancer patients. • thyroid transcription factor 1 (ttf1) and napsin a are accepted markers for adc differentiation; p40 is reported to be the most specific and sensitive marker for squamous cell differentiation. a reasonable recommendation is that, when immunohistochemistry is deemed necessary, at least one antibody each for squamous and glandular differentiation, but no more than two antibodies, should be used for an initial workup (e.g., ttf1 and p40 or p63). thus a limited panel of ttf1 and p40 (or p63) is suggested for subtyping the tumor to preserve tissue for molecular testing. • molecular testing for epithelial growth factor receptor (egfr) mutation and anaplastic lymphoma kinase (alk) rearrangement is recommended in tumors classified as adc and in cases where an adc component cannot be excluded. • when evaluating a computed tomography (ct)guided fna of a lung lesion in the periphery, mesothelial cells are frequently seen on smears; reactive mesothelial cells may mimic carcinoma, and caution should be taken. • diagnosis of adc with a lepidic growth pattern can be challenging in fna specimens. both quality (degree of atypia) and quantity (groups of atypical cells) need to be considered. in a typical case of adc with a lepidic growth pattern, the smears tend to be very cellular, containing many groups of atypical epithelial cells. in contrast, in a reactive condition, such as pulmonary infarction, atypical reactive pneumocytes are usually less numerous. • in mucinous adc with a lepidic growth pattern, the neoplastic epithelial cells may mimic pulmonary macrophages. features such as a mucinous background, cytoplasmic mucin/vacuoles, eccentrically located nuclei, and more abundant cytoplasm are helpful in reaching a correct diagnosis. • basaloid squamous cell carcinoma may mimic small cell carcinoma; therefore, cellblock preparation in conjunction with immunostains will be helpful. • infection such as aspergillosis may result in squamous cells with significant cytological atypia; therefore, infectious etiologies should be excluded before a diagnosis of well-differentiated squamous cell carcinoma is rendered. • a lesion from the mediastinum should be considered if cytological features do not fit the description of a typical "lung lesion." • thymoma is rare in young patients and children. in lymphocyte-dominant thymoma, the main component of the aspirate may contain cortical thymocytes with expression of terminal deoxynucleotidyl transferase (tdt) and cluster of differentiation (cd)99; therefore, caution should be taken to avoid misdiagnosing it as a lymphoblastic lymphoma. • thymic neuroendocrine neoplasm may mimic an epithelial-dominant thymoma; therefore, immunohistochemistry should be performed in cases with equivocal features. tables 6.1 and 6.2 summarize new criteria and terminology for the diagnosis of lung cancer based on small biopsies and cytology from the 2015 who classification. upper specific viral infections, such as herpes simplex ( fig. 6 .6) and cytomegalovirus ( fig. 6.7) , can cause significant cytosevere acute respiratory syndrome (sars) caused by a novel coronavirus (sars-cov) became a worldwide outbreak in 2003, affecting more than 8000 patients, with a fatality rate of 9.2%. sars-cov belongs to a family of large, positive, single-stranded ribonucleic acid (rna) viruses. the key pathologic finding is diffuse alveolar damage (dad). depending on different phases in the disease progression, the composition of inflammatory cells may vary; however, macrophages (including multinucleated forms) and lymphocytes usually predominate. other pathologic findings, such as fibrosis, prominent vascular injury, hemophagocytosis, squamous metaplasia, apoptosis, and atypical pneumocytes, including multinucleated giant pneumocytes with irregularly distributed nuclei or pneumocytes with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm, were reported. ancillary tests, such as in situ hybridization, immunohistochemistry, viral isolation, or reverse transcription polymerase chain reaction (rt-pcr), are necessary to confirm the diagnosis. representative images are shown in fig. 6 .8a, b. fna is a useful means of diagnosing pulmonary fungal infection, which should be suspected whenever there is granulomatous inflammation. silver or periodic acid-schiff (pas) stains are used on cellblock sections. the common fungal infections include (1) candida species; (2) aspergillus species, table 6 .3. pulmonary strongyloidiasis affects immunocompetent and, more commonly, immunosuppressed persons presenting with pneumonitis with hemoptysis. the etiological agent is the nematode. strongyloides stercoralis in sputum is shown in fig. 6 .15. infection by mycobacterium tuberculosis is often a granulomatous inflammation containing clusters of epithelioid histiocytes, lymphocytes, and langhans giant cells, with or without necrosis. in immunocompromised patients there may be abundant acid-fast organisms without obvious granulomatous inflammation. there are pulmonary macrophages and neutrophil-predominant mixed inflammatory cells in the background acquired immunodeficiency syndrome (aids) patients are especially susceptible to mycobacterium aviumintracellulare, an acid-fast organism producing negative images on romanowsky stain. special stains on cellblock section are particularly helpful. nocardia, a weakly acid-fast filamentous organism, often infects immunocompromised patients, producing cavitary nodules on radiographs, which may mimic a neoplastic process. • sarcoidosis is characterized by non-caseating granulomas in many organs, most commonly the lung. • chest x-ray: bilateral hilar adenopathy is a classic finding; variable lung parenchyma changes, from normal, diffuse reticular, or ground glass opacities, nodular consolidation or cystic scarring. • ct and positron emission tomography (pet) scan can also be used. • aggregates of epithelioid histiocytes, with or without schaumann and asteroid bodies • multinucleated giant cells and lymphocytes an essential part of the diagnosis of sarcoidosis is the exclusion of alternative possibilities: • granulomas caused by infectious agents, such as mycobacterial infection and fungal infection • drug-induced, hypersensitivity pneumonitis, or foreign body granulomatosis • wegener granulomatosis is characterized by necrotizing vasculitis and may present as a lung mass with or without involvement of nasal passages and kidneys. • multiple pulmonary nodules on imaging • neutrophils, giant cells, necrotic collagen, and epithelioid histiocytes • the findings are nonspecific; therefore, serologic studies are necessary. (c) gms stain reveals small oval or cup-shaped yeast forms, cellblock the 2015 who classification of tumors of the lung, pleura, thymus and heart has been published recently. there are numerous important changes in the classification of lung tumors, as summarized in table 6 .4. fig. 6 .16a, b. • round to multilobulated, well-circumscribed tumor nodules composed of mesenchymal tissues, including chondroid or chondromyxoid tissue, fat, connective tissue, smooth muscle, and bone in various proportions, intermixed with clefts of respiratory epithelial cells • chondromyxoid tissue usually predominates. • representative images are shown in fig. 6 .16c, d. • immunohistochemistry is usually not necessary for diagnosis. • pulmonary hamartomas have a high frequency of translocation t(3;12)(q27-28;q14-15) leading to a gene fusion of the high mobility group protein gene at-hook 2 (hmga2) and the lipoma preferred partner (lpp) gene. • most often young patients, under the age of 40 • male = female • the most common endobronchial mesenchymal lesion in childhood • chest x-ray: a peripheral, discrete, solitary nodule. if endobronchial location, post-obstructive pneumonia and atelectasis may be evident. fig. 6 .17a, b. • positive for vimentin, smooth muscle actin (sma), and rarely to desmin • negative for myogenin, myoglobin, cd117, and s100 protein • focal cytokeratin (ck) positivity was reported in 1/3 of cases, likely due to alveolar entrapment. • anaplastic lymphoma kinase 1 (alk1) expression was noted in 40% of cases. • p53 is negative; however, positivity is associated with recurrence and malignant transformation. • representative images are illustrated in fig. 6 .17c, d. squamous cell carcinomas are malignant tumors that either morphologically show squamous cell differentiation (keratinization and/or intercellular bridges) or are morphologically undifferentiated non-small cell carcinomas but show squamous cell differentiation immunohistochemically. the 2015 who classifications of tumors of the lung reclassified squamous cell carcinomas into keratinizing, nonkeratinizing, and basaloid subtypes. the nonkeratinizing tumors require immunohistochemical proof of squamous differentiation. • about 20% of all pulmonary malignancies • clinical presentation is similar to other non-small cell carcinomas. • usually a central mass with cavitation and post-obstructive pneumonia keratinizing squamous cell carcinoma: • abundant dyshesive cells with dense cytoplasm, may be orangeophilic • polygonal, rounded, or elongated cells • tadpole or fiber-like cells • pleomorphic, pyknotic nuclei with obscured nucleoli and chromatin detail • anucleated cells and twisted keratin strands (herxheimer spirals) • representative images are shown in fig. 6 .18a-d. nonkeratinizing or basaloid squamous cell carcinoma: • cohesive groups of cells with larger nuclei and coarsely granular chromatin • cyanophilic cytoplasm on pap stain • rare or no keratinization • representative images are shown in fig. 6 .19a-f. who classification of tumors of the lung: keratinizing, nonkeratinizing, and basaloid subtypes: 1. keratinizing squamous cell carcinomas exhibit recognizable keratinization, keratin pearls, and/or intercellular bridges, as illustrated in fig. 6 .20a, b. 2. nonkeratinizing squamous cell carcinomas are without recognizable keratinization, keratin pearls, or intercellular bridges, as illustrated in fig. 6 .20c. 3. basaloid squamous cell carcinomas are tumors with a basaloid component in greater than 50% of the tumor, regardless of the presence of any keratinization, as illustrated in fig. 6 .20d. • immunohistochemistry is required for the diagnosis of nonkeratinizing squamous cell carcinomas. • squamous cell carcinomas express p40, p63, and ck5/6. • ttf1 is usually negative in keratinizing squamous cell carcinomas, may be weakly focally positive in nonkeratinizing squamous cell carcinomas. • representative images are illustrated in fig. 6 .21a-d. • squamous cell carcinomas of lung are characterized by complex genomic alterations, frequently involving the following pathways: deletion of chromosome 9p (cdkn2a) was observed in 72% of cases. • the most common primary pulmonary malignancy, accounting for about 40% of cases. • mortality and incidence rates have generally been highest in high-income countries but are now declining, especially in younger males and females. • has been more common in men than in women, but has begun to converge. histologic subtype other than a lepidic pattern; tumor cells infiltrating myofibroblastic stroma. 5. mia diagnosis is excluded if the tumor invades lymphatics, blood vessels, air spaces, or pleura, contains tumor necrosis, and spreads through air spaces. 6. the cell type is mostly nonmucinous, but rarely may be mucinous. • the 2015 who classification of tumors of lung classifies adc as lepidic, acinar, papillary, micropapillary, or solid according to the predominant pattern after a comprehensive histologic subtyping to identify all of the different histologic patterns in 5% increments. • other variants of adcs include invasive mucinous adc, colloid adc, fetal adc, and enteric adc. • representative images are shown in fig. 6 .23a-d. • positive for ttf1 and napsin a: commonly used markers for pneumocytes, with comparable sensitivity, about 75%; much lower ttf1 expression in solid adcs and mucinous adcs • positive for ck7 • negative for squamous cell markers; however, p63 positivity was reported in up to 30% of lung adcs. • egfr, kirsten rat sarcoma viral oncogene (kras), and alk mutations are specific for lung adcs. the adcs with egfr or alk alteration are usually located in the periphery; in contrast, the adcs with kras mutation are frequently located in hilar region. the prevalence for kras and egfr mutations is 10-30%; the transforming fusion gene echinoderm microtubule-associated proteinlike 4 (eml4)-alk is found in 5% of lung adcs. the characteristics of patients with egfr mutation are asians, never smokers, with nonmucinous tumors; for alk mutation, patients are younger age, male gender, and never or light smokers; for kras mutation patients are non-asians, smokers, with invasive mucinous adcs usually negative for ttf1, and positive for mucin (muc) 2,5,6 immunophenotypes. this group of tumor represents a morphologic and biologic spectrum of tumors that is classified by the 2015 who classification of tumors of the lung into four types: preinvasive lesion (including diffuse idiopathic pulmonary neuroendocrine cell hyperplasia), carcinoid tumors (including typical and atypical carcinoid tumors), large cell neuroendocrine carcinoma (lcnec), including combined lcnec, and small cell carcinoma (including combined small cell carcinoma). carcinoid tumors are neuroendocrine epithelial malignancies, including typical carcinoids, defined as those with <2 mitoses/per 2 mm 2 , lacking necrosis, ≥0.5 cm in size, and atypical carcinoids, which are those with 2-10 mitoses/per 2 mm 2 and/or foci of necrosis. • account for <1% of all lung cancers. • majority (70-90%) are typical carcinoids. • more common in female, white, and <60 years old. • clinical syndromes are uncommon, including carcinoid syndrome, cushing syndrome, and acromegaly. • chest x-ray: usually a centrally located, lobulated mass with a prominent endobronchial component; one third of tumors in periphery. • ct using intravenous contrast medium shows considerable enhancement; calcification, especially in centrally located tumors; atelectasis; bronchiectasis; and hyperlucency for tumors with bronchial involvement. • loosely cohesive groups and single uniform cells with granular nuclei and ample eosinophilic cytoplasm and naked nuclei. • round, columnar, or plasmacytoid cells, forming acinar or rosette-like structures. • branching capillaries. • clean background. • usually uniform polygonal or spindled cells arranged in organoid or trabecular patterns. • other growth patterns also seen: rosette formation, papillary, pseudoglandular, or follicular patterns. • significant pleomorphism and prominent nucleoli may be seen in typical carcinoids. • the differential features for atypical carcinoids are the presence of 2-10 mitoses per 2 mm 2 and/or necrosis. these changes may be focal. • representative images are shown in fig. 6 .24c-f. • immunohistochemistry is recommended for the diagnosis of neuroendocrine tumors, not only in small biopsy or cytology cases but also in resected specimens. • the typical phenotype expresses neuroendocrine markers (cd56, synaptophysin, chromogranin); most are positive for panck and negative for high molecular weight cytokeratins (hmwcks) and ttf1. however, ttf-1 expression was reported in 43-53% of cases. • the proliferative index (ki-67) is valuable in distinguishing carcinoids from high-grade neuroendocrine carcinomas, especially in small biopsies or cytology specimens with significant crush artifact. small cell carcinomas have high proliferative index (>50%) in contrast to <10-20% in carcinoids. • ki-67 is not recommended for the distinction of typical from atypical carcinoids due to the lack of cutoff value. • representative images are shown in fig. 6 .25a-d. • heavy smokers in >90% of cases. • paraneoplastic syndrome is uncommon. • usually a periphery mass with irregular margin, with or without intratumoral calcification • the tumors are usually large, showing central inhomogeneous enhancement on ct with contrast. • cavitation uncommon • overlapping with other neuroendocrine tumors and adcs • loosely cohesive or single, monotonous tumor cells with a hyperchromatic nuclear chromatin pattern but easily appreciated nucleoli, nuclear membrane irregularity, and preserved moderate to abundant, delicate cytoplasm • necrosis or apoptotic debris may be seen. • representative images are shown in fig. 6.26a • the proliferative index (ki-67) is usually in the range of 40-80%. • necrosis is common. • representative images are shown in fig. 6 .26c, d. • immunohistochemical markers such as synaptophysin, chromogranin, and cd56 are required to confirm neuroendocrine differentiation of the tumor. • cd56 was reported in 92-100% of cases, chromogranin in 80-85%, and synaptophysin in 50-60%. • about 50% of cases are positive for ttf1. • panck, low molecular weight cytokeratin (lmwck), and ck7 are expressed in either dot-like or diffuse cytoplasmic patterns. • cd117 positivity was reported in >70% of cases. • thirteen percent of all newly diagnosed lung cancers are small cell carcinomas. • virtually all are heavy smokers, male predominant. • usually a centrally located lobulated mass, with occasional endobrochial involvement; 5% peripherally located • a large hilar mass with bulky mediastinal lymph nodes is characteristic; invasion of hilar vessels and vena cava is common; cavitation is rare. • small cells with hyperchromatic nuclei, powdery chromatin texture, indistinct nucleoli, nuclear molding, and scant cytoplasm • marked mitosis and single cell necrosis • nuclear debris and crush artifact in the background • representative images are shown in fig. 6 .27a-d. • reserve cell hyperplasia • carcinoids • small blue cell tumors, such as lymphoma, ewing sarcoma, and rhabdomyosarcoma • non-small cell carcinoma • pulmonary blastoma • merkel cell carcinoma • the tumor usually presents in a sheetlike pattern. • the tumor cells are small, containing round, oval, or spindled nuclei, with a fine granular chromatin pattern, inconspicuous nucleoli, and scant cytoplasm, without defined cell borders. • nuclear molding is frequent. • high mitotic rate is seen, at least 10 mitoses/per 2 mm 2 , with an average of 60 mitoses/per 2 mm 2 . the proliferative index (ki-67) is >50%, with an average of >80%. • may show extensive necrosis • representative image is shown in fig. 6 .27e. • immunohistochemical studies may be required to confirm neuroendocrine and epithelial differentiation of the tumor. • ck ae1/3, cam5.2, and mnf116 are positive in nearly 100% of cases, with either dot-like, paranuclear, or diffuse cytoplasmic staining patterns. • neuroendocrine markers (cd56, synaptophysin, and chromogranin) are reactive in the majority of cases; cd56 and synaptophysin are usually diffuse and strong, but chromogranin is often focal and weak. • <10% of small cell carcinomas may lack or have only very focal expression of neuroendocrine markers. • ttf1 is positive in up to 90-95% of cases. • cd117 positivity was reported in 60% of cases. • representative images are shown in fig. 6 .27f-j. the features of differential diagnosis for neuroendocrine tumors are summarized in table 6 .5. • about 2.3% of all pulmonary malignancies. • usually in the sixth decade, male predominant. • most patients are smokers. • chest x-ray: usually a peripheral mass, rarely may be cavitated • single cells or loose clusters • vesicular, pleomorphic nuclei with irregular nuclear membrane, multiple nucleoli and high nuclear-tocytoplasmic ratio • ill-defined, feathery cytoplasm types. therefore, molecular testing is recommended for large cell carcinomas. sarcomatoid carcinoma is a general term that includes pleomorphic carcinoma, carcinosarcoma, and pulmonary blastoma, which are individual entities in the 2015 who classification. this group of tumors accounts for 2-3% of all cancer cases in a surgical series and <1% of all lung cancers. definitive diagnosis of this group of tumors is very difficult or impossible in small biopsies and cytology specimens. the characteristic features of individual tumors in this group are summarized in table 6 .6. representative images are illustrated in fig. 6 .28a-h. this is a new entity in the 2015 who classification of tumors of lung. the group of carcinomas associated with • affects people of all ages, although, it was originally reported in children and younger adults • male equals female • fewer than 100 cases reported • usually presents at an advanced stage, with pleural effusion, chest pain, weight loss, and respiratory symptoms • chest x-ray: extremely rapid-growing tumor, with complete opacification of the thorax within 2-8 weeks • ct: a hypoattenuating, heterogeneously enhancing, often extensively necrotic mass with poorly defined, infiltrative borders. high fluorodeoxyglucose (fdg) uptake is characteristic. • usually cellular smears • discohesive clusters and single of small to intermediate size, monomorphic cells with irregular nuclear membrane, granular to coarse chromatin pattern, and discrete nucleoli • mitoses, necrotic debris, and crush artifact are common. • representative image is shown in fig. 6 fig. 6 .29b. • speckled nuclear positivity in more than 50% of tumor cells with nut antibody is a constant finding and is diagnostic. • broad-spectrum cytokeratins are positive in majority of cases. • other epithelial markers, such as epithelial membrane antigen (ema), epithelial cell adhesion molecule (berep4), and carcinoembryonic (cea) antigen showed variable results. • most of cases are positive for p63/p40 and cd34. • occasional reactivity to synaptophysin, chromogranin, and even ttf1 was observed. • nut carcinomas are defined by the presence of nut gene rearrangement, which is a chromosomal translocation between the nut gene (nutm1) on chromosome 15q14 and other genes: bromodomain containing 4 (brd4) on chromosome 19p13.1 (70%), brd3 on chromosome 9q34.2 (6%), or an unknown partner gene (24%). the most common metastatic tumors are carcinomas, especially from the gastrointestinal tract, gynecological tract, breast, urothelial, head and neck, prostate, and other sites, followed by sarcomas, melanomas, and germ cell tumors. sex and age distribution depend on tumor types, such as colorectal cancers in elderly patients of both sexes, breast cancer and melanoma in younger adults, and germ cell tumors and sarcomas in young adults or children. metastases can be single or multiple, usually involving the lung parenchyma and the pleura. however, bilateral, multiple, peripherally located round, variable-size nodules (hematogenous metastases), or diffuse thickening of the interstitium (lymphangitic carcinomatosis) are typical. cytological features, histological findings, and immunoprofile are the same as the primary tumors. the differential immunophenotypes for the most common metastatic carcinomas are summarized in table 6 .7. the who classifications of tumors of the pleura, 2015, are summarized in table 6 .8. • usually occurs in older adults, ≥60 years • more often in men • a strong association with asbestos exposure • pleural effusion is very common. • variety of presentations, classically exhibiting a diffuse circumferential ring of nodular pleura associated with ipsilateral effusion • less commonly presents with pleural effusion without obvious pleural nodularity • pleural plaques, especially associated with calcifications, are suggestive of asbestos exposure. • hypercellular specimen with mixed epithelial and spindle cells. • can be epithelial or spindle cell dominate. • resemble ordinary mesothelial cells. • tumor giant cells, granulomas, and psammoma can be seen. • representative images are shown in fig. 6 .30a-c. • pulmonary adc (table 6 .9) • metastatic carcinoma • reactive mesothelial cells (table 6 .10) • account for 60-80% of malignant mesotheliomas • variety of histological patterns. most common patterns: solid, tubulopapillary, and trabecular; less common patterns: micropapillary, adenomatoid (microcystic), clear cell, transitional, deciduoid, and small cell showing varying degree of cellularity from hyalinized acellular to highly cellular stroma. myxoid changes may be seen in 5-10% of cases, with nests of bland-looking, vacuolated epithelioid cells floating in the matrix. • representative images are shown in fig. 6 .30d-f. • characterized by a proliferation of spindle cells arranged in fascicles or with a haphazard pattern and involves the adipose tissue of the parietal pleura or the adjacent lung parenchyma. • the tumor cells show variety of morphology, from plump to thin, elongated cells with scant cytoplasm. • the degree of nuclear atypia, mitotic activity, and necrosis vary from minimal to moderate to marked. • characterized by areas of atypical spindle cells arranged in a so-called patternless pattern within a dense, hyalinized, fibrous stroma constituting at least 50% of the tumor • invasion of adipose tissue is the most reliable criterion to distinguish desmoplastic mesothelioma from organizing pleuritis. • show any combination of the patterns described above constituting at least 10% of the tumor • positive for ck ae1/3, calretinin, ck5/6, wt1, and d2-40. • negative for berep4, moc-31, b72.3, and cea. • see tables 6.8 and 6.9 for differential diagnosis. • multiple chromosomal alterations, more common with chromosomal losses, especially on chromosomal arms 1p, 3p, 4q, 6q, 9p, 13q, 14q, and 22q; alterations in several tumor suppressor genes, including neurofibromin 2 (nf2), cdkn2a (p16ink4a), cdkn2b (p15ink4b), and brca1-associated protein 1 (bap1). • representative images are shown in fig. 6 .31a-f. • most common in sixth to seventh decades • no sex predisposition • accounting for <5% of primary pleural tumors • unknown etiology • slow-growing, relatively benign tumors, up to 10% malignant • usually asymptomatic, incidental finding • usually solitary, well-circumscribed mass arising from visceral pleura • can be multiple and distributed throughout the pleural cavity • cellular smear with bland-appearing, small, oval to spindled cells • no mitosis or necrosis • representative images are shown in fig. 6.32a fig. 6 .32c, d. • positive for signal transducer and activator of transcription 6 (stat6): > 95% of cases, specific. • positive for cd34, b-cell cll/lymphoma 2 (bcl2), and cd99: nonspecific. • occasionally may be positive for sma, epithelial membrane antigen (ema), keratin, s100, or desmin. • representative images are shown in fig. 6 .32e, f. • the tumor harbors characteristic gene fusion ngfi-a binding protein 2 (nab2)-stat6. the mediastinum can be divided into four hypothetical compartments: superior, anterior, middle, and posterior. the distribution of organs and tumors of the mediastinum is summarized in table 6 .11. in adults, the distribution of tumors is as follows: 46% thymic epithelial tumors, 23% lymphomas, 16% endocrine tumors, and 15% germ cell tumors. in children, the frequency of tumors is 47% neurogenic neoplasms, 19% germ cell tumors, 12% lymphomas, 10% thymic epithelial tumors, 6% cysts, and 6% mesenchymal tumors. the 2015 who classification of tumors of the thymus is summarized in table 6 .12. selected entities will be discussed in the section below. • bronchogenic cyst -any part of the mediastinum • enteric cyst -posterior mediastinum • thymic cyst -anterior mediastinum • pericardial cyst -middle mediastinum • hypocellular smear • dependent upon the type of the cyst thymoma • rare malignancy overall, but the most common mediastinal tumors in adults • median age of 50 years, rare in children • may present as a mass lesion or an incidental finding • may be associated with myasthenia gravis, pure red cell aplasia, hypogammaglobulinemia (good syndrome), and/ or other autoimmune disorders • usually a mass lesion, well-circumscribed or invasive borders, depending on the subtypes of thymoma • usually moderate to hypercellular smear • two populations of relatively bland neoplastic epithelial cells and lymphocytes • the proportion of epithelial cells and lymphocytes is dependent upon subtype. • in epithelial subtype, epithelial cells are dominant; in lymphocyte-predominant subtype, small mature lymphocytes are dominant; and in spindle cell type, spindle epithelial cells are predominant. • neoplastic epithelial cells tend to be cohesive and have a delicate nuclear membrane, fine nuclear chromatin, and small nucleoli. • few or no mitoses are present. • representative images are shown in fig. 6 .33a-f. • type a thymoma: 1. complete or incomplete fibrous capsule may display coarse lobulation with thick fibrous bands. 2. microcystic pattern is most common, more prominent in subcapsular areas. 3. other patterns: rosettes (with or without a central lumen), glandular or glomeruloid structures, masson's hemangioma-like papillary projections in cystic spaces, meningioma-like whorls, fascicular growth, and storiform growth. 4. hassall corpuscles are absent. 5. the tumor cells are spindled and/or oval-shaped with bland nuclei, finely dispersed powdery chromatin, and inconspicuous nucleoli. 6. low mitotic activity, usually <4 mitoses/per 2 mm 2 . 7. no or very few immature lymphocytes. 8. when hypercellularity, increased mitotic counts, and focal necrosis are present, designate as atypical type a thymoma variant. 9. representative images are shown in fig. 6.34a (a) predominance of polygonal epithelial cells forming solid sheets, resulting in a pink appearance on h&e; (b) paucity of admixed nonneoplastic immature t cells. • epithelial cells: usually positive for ae1/3, ck7, ck19, p63, pax8, forkhead box n1 (foxn1), cd57, and cd205; negative for ema, cd117, and cd5. • small lymphocytes (thymocytes) are mature t-cells, but positive for cd3, cd5, tdt, cd99, with a high mib1 (ki-67) proliferative index. • representative images are shown in fig. 6 .34e-h. thymic carcinomas include of a variety of histological types of tumors, accounting for approximately 22% of all thymic epithelial neoplasms. the squamous cell carcinomas are the most common type, accounting for approximately 70% of all thymic carcinoma cases. lymphoepithelioma-like carcinomas are rare, accounting for 6-32% of all thymic carcinomas; sarcomatoid carcinomas account for 5-10%; and basaloid carcinomas account for <5%. other types, such as mucoepidermoid carcinomas, clear cell carcinomas, adcs, nut carcinomas, and undifferentiated carcinomas, are very few. the characteristic features of the three most common types are summarized in table 6 .13. thymic neuroendocrine tumors are categorized into two major groups: (1) low-grade typical carcinoids and intermediategrade atypical carcinoids, which always show characteristic morphological and immunohistochemical neuroendocrine features, and (2) high-grade lcnecs and small cell carcinomas, which may lack some neuroendocrine features. • no established association with smoking. • more aggressive clinical course: propensity for recurrence, lymph node or distant metastases, and tumorassociated death. • atypical carcinoids account for the majority of the tumors. • only carcinoids reported in setting of multiple endocrine neoplasia type 1 (men1). • average age at presentation: 49 years. • carcinoids show a strong male predominance. • typically a lobulated, heterogeneous mass in the anterior mediastinum • same as pulmonary neuroendocrine tumors; please refer that section in this chapter. • representative images are shown in fig. 6 .36a-i. • pulmonary neuroendocrine tumors: careful clinical and radiological correlation is the primary way to distinguish pulmonary from thymic neuroendocrine tumors. • thymoma, especially type a, to distinguish from carcinoids. • thymic carcinoma usually shows focal and/or weak expression of neuroendocrine markers. the same as pulmonary neuroendocrine tumors, respectively. same as the pulmonary neuroendocrine tumors, respectively. 1. mature teratoma: cytology diagnosis is difficult; may be paucicellular with few anucleated squamous cells and macrophages in a proteinaceous background; ciliated bronchial cells, smooth muscle, and cartilage are present; a mucoid background with bland-looking signet ring cell-like mucus cells may be seen. 2. immature teratoma: may be cellular, composed of aggregates or individual small, round, hyperchromatic cells with high nuclear-to-cytoplasmic ratio, and inconspicuous nucleoli; rare rosettes with neuropils, rhabdomyoblasts, immature cartilage, and blastemalike stromal cells may be identified. 2. immature teratoma: the immature teratomatous elements are mostly cellular spindle mesenchymal components, but immature neural and epithelial elements can be seen; frequent mitoses; embryonic rhabdomyoblastic tissue, blastomatous tissue resembling embryonic kidney or lung, primitive neuroectodermal tumor (pnet), and other epithelial or mesenchymal malignant transformations can be seen. the immunophenotypes of mediastinal germ cell tumors are summarized in table 6 .14. sex determining region y box 10 (sox10) and cytoplasmic staining for vimentin and human melanoma black 45 (hmb45), focally for melanoma antigen recognized by t cells (mart1) and s100 (both nuclear and cytoplasmic), while ck ae1/3 and neuroendocrine markers (synaptophysin, chromogranin and cd56) were nonreactive. representative images are illustrated in fig. 6 .39e-h. the overall findings are those of a metastatic spindle cell melanoma. the diagnosis of metastatic spindle cell melanoma can be challenging on fna specimens. the spindled tumor cells can display a wide range of morphologies, from deceptively bland-appearing reactive fibroblastlike cells to highly pleomorphic, high-grade sarcomatous spindle cells. the diagnostic cytological features of conventional melanoma, such as a dispersed single cell pattern, eccentric nuclei, cytoplasmic melanin pigments, intranuclear pseudoinclusions, prominent macronucleoli, and bi-or multinucleations, are often subtle or even lacking. for the index case, the presence of melanin pigments is a valuable clue to raise the suspicion for melanoma which, in conjunction with the immunophenotype, justified the diagnosis of spindle cell melanoma. the presence of cytoplasmic melanin (continued) immunohistochemistry plays a critical role in the diagnosis of spindle cell melanoma, especially in the clinical scenario of metastasis. metastatic spindle cell melanomas tend to lose expression of some melanoma markers. piao and colleagues reported that spindle cell melanomas express s100, hmb45, and mart1 in 67%, 50%, and 18% of cases, respectively. although the sensitivity of s100 for melanoma in general is very high (~93-100%), its specificity is low. the expression of s100 can be seen in a variety of tumors. hmb45 is expressed in melanocytic tumors, also in clear cell sarcomas, perivascular epithelial cell tumors (pecomas), melanocytic schwannomas, meningeal melanocytomas, some ovarian steroid tumors, and renal cell carcinomas with the t(6;11)(p29;q12) translocation. the sensitivity of hmb45 for melanoma in general is in the range of 70-90%, but only 0-30% for desmoplastic melanomas. mart1 has a sensitivity of ~85-97% for primary and ~57-92% for metastatic melanomas, but only 0-33% for desmoplastic melanomas; its specificity for melanoma is 95-100%. mart1 expression is also seen in pecomas and clear cell sarcomas. sox10 is a transcription factor that is essential for the survival of neural crest-derived cells and for the maintenance of the multipotency of neural crest cells. the cells derived from neural crest multipotential cells include neurons and glial cells in the peripheral nervous system, melanocytes of the skin, c cells of the thyroid, catecholaminergic cells of the adrenal gland, and cartilage and bone of the face. studies have shown that, among tumors, sox10 is commonly expressed in melanomas, including desmoplastic melanomas, tumors with schwann cell differentiation, myoepithelial cell tumors of the soft tissue and some salivary gland neoplasms, particularly those with myoepithelial differentiation, and acinic cell carcinomas. when compared with other melanocytic-associated markers, sox10 is highly sensitive and specific and can assist in the differential diagnosis of melanomas. even in desmoplastic melanomas, sox10 expression was reported in 100% of cases. studies of sox10 expression in tumors of the nervous system reported that sox10 is commonly expressed in schwannomas (100%), neurofibromas (98-100%), and, less frequently, mpnsts (~50-55%). the differential diagnosis of this case is summarized in table 6 .15. learning objectives 1. to describe the cytological features of this entity 2. to become aware of the subtle differences in cytology and to raise the question of a metastasis 3. to become familiar with the potential utility of immunohistochemical markers in the diagnosis of this tumor a 72-year-old female with a past medical history of noninvasive low-grade papillary urothelial carcinoma a year ago presented with coughing and chest pain for month. ct imaging of the chest revealed multiple lung nodules and lymphadenopathy. an ultrasound-guided fna and biopsy of the hilar lymph node were performed. the smears were highly cellular, composed of loosely cohesive clusters and single spindle to plump cells with hyperchromatic nuclei, coarse chromatin, occasional discernible to prominent nucleoli, and abundant delicate cytoplasm without defined borders. some of the cells appeared spindled, columnar or racket-shaped, with eccentrically located nuclei and cytoplasmic tails, also called cercariform cells. abundant necrosis was noted in the background. representative images of the fna smears and biopsy are shown in fig. 6 .40a-f. the cytological features raise a differential diagnosis that includes a non-small cell carcinoma of lung, especially squamous cell carcinoma, a sarcomatoid carcinoma, and metastatic carcinoma, especially metastatic urothelial carcinoma. a panel of immunoassays was performed, revealing that the tumor cells were positive for ck7, ck20 (focal), ck5/6, uroplakin ii, p40, and placental s100 (s100p) while negative for ttf1, napsin a, synaptophysin, and chromogranin. representative images are illustrated in fig. 6 .40g-j. the final diagnosis is metastatic carcinoma with urothelial differentiation there are limited reports in literature regarding the cytology of metastatic urothelial carcinomas. the following features were reported: (1) loosely cohesive cells occurring singly and in syncytial clusters; (2) large hyperchromatic nuclei with irregularly distrib-uted granular chromatin and abundant granular or fibrillar cytoplasm; (3) distinct cell borders; (4) multilayered papillary fragments; (5) cells with eccentric nuclei, multiple nucleoli, and intracytoplasmic vacuoles; and (6) cercariform cells, "bipolar" cells, and spindled-shaped cells. among those, multilayered papillary fragments and cercariform cells are the most helpful features in the distinction of urothelial carcinoma from others. the cercariform cells, first described in 1993 by johnson and kini and further defined in 1995 by powers and elbadawi, are fusiform, pyramidal, or racket-like cells with eccentric nuclei that form non-tapering, flattened, bulbous, or fishtail-like cytoplasmic extensions in varying lengths. the presence of a small vacuole in the bulbous tail was also a helpful criterion. these cells, which are encountered in 57-100% of metastatic urothelial carcinomas, are interpreted in favor of urothelial carcinoma, particularly when they are observed in large numbers. however, they are not specific and must be considered alongside other clinical and morphological characteristics and immunohistochemical phenotypes. the cytological features of metastatic urothelial carcinomas are very difficult to differentiate from mesenchymal tumors and squamous cell carcinomas. immunohistochemical analyses play a crucial role. urothelial cell carcinomas express both ck7 and ck20. in addition, they express hmwcks (ck5/6, ck903), p63, gata binding protein 3 (gata3), uroplakin ii, and s100p. gata3 is a zinc finger transcription factor with a diverse range of biologic roles. gata3 is a newer generation of urothelial specific marker. gata3 expression was reported in approximately 80% of urothelial carcinomas, over 90% of breast carcinomas, 100% of parathyroid gland tissue or tumors, salivary gland tumors, especially the salivary ductal carcinomas (~90%), and transitional proliferations of the gynecological tract. in addition, 0-12% of pulmonary squamous cell carcinomas and approximately 10% of pancreatic carcinomas were also reported to be positive for gata3, although weakly and focally in the majority of positive cases. the immunohistochemical differential diagnosis of metastatic urothelial carcinomas is summarized in table 6 .16. met uca metastatic urothelial carcinoma, sqcc squamous cell carcinoma, ca carcinoma, ck cytokeratin, ttf1 thyroid transcription factor 1, gata3 gata binding protein 3, s100p placental s100 a gata3 expression in squamous cell carcinoma of lung was reported in 0-12% of cases; when positive, it is usually weak and focal. comprehensive cytopathology cytology: diagnostic principles and clinical correlates the art & science of cytopathology fine needle aspiration cytology of metastatic urothelial carcinoma: study of seven cases with review of literature transthoracic fine-needle aspiration cytology of non-invasive, low-grade urothelial carcinoma with lung metastasis: a case report with review of the literature comprehensive genomic characterization of squamous cell lung cancers lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore nut midline carcinomas in the thymic region pathology and pathogenesis of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome diagnostic histopathology of tumors who classification of tumours of the lung, pleura, thymus and heart. lyon: iarc who panel. the 2015 world health organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 international association for the study of lung cancer diagnosis of lung adenocarcinoma in resected specimens: implications of the 2011 international association for the study of lung cancer pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) ancillary tests utility of gata3 immunohistochemistry in differentiating urothelial carcinoma from prostate adenocarcinoma and squamous cell carcinomas of the uterine cervix, anus, and lung neural crest progenitors and stem cells expression of the urothelial differentiation markers gata3 and placental s100 (s100p) in female genital tract transitional cell proliferations temporally regulated neural crest transcription factors distinguish neuroectodermal tumors of varying malignancy and differentiation selective immunohistochemical markers to distinguish between metastatic high-grade urothelial carcinoma and primary poorly differentiated invasive squamous cell carcinoma of the lung placental s100 (s100p) and gata3: markers for transitional epithelium and urothelial carcinoma discovered by complementary dna microarray induction of the neural crest and the opportunities of life on the edge diagnostic utility of sox10 to distinguish malignant peripheral nerve sheath tumor from synovial sarcoma, including intraneural synovial sarcoma sorting out sox10 functions in neural crest development unknown primary/undifferentiated neoplasm immunohistochemical evaluation of gata-3 expression in er-negative breast carcinomas immunohistochemical evaluation of gata3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas sox10-a marker for not only schwannian and melanocytic neoplasms but also myoepithelial cell tumors of soft tissue: a systematic analysis of 5134 tumors sox10: a pan-schwannian and melanocytic marker value of gata3 immunostaining in tumor diagnosis: a review value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update value of sox10 immunostaining in tumor diagnosis survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor sox10 and extrinsic combinatorial signaling urinary bladder and urachus the application of immunohistochemical biomarkers in urologic surgical pathology rabbit polyclonal anti-sox10 is a reliable ihc marker for melanoma and its mimics handbook of practical immunohistochemistry: frequently asked questions lymphoepithelioma-like carcinoma sarcomatoid carcinoma clinical features 1. 70% of all cases 2. most common in sixth decade 3. men slightly more than women 4. <5% associated with myasthenia gravis cartilage, spicules of bone, patches of pigmented tissue, or brain tissue may be discernible; an admixture of ectoderm, endoderm, and mesoderm is seen, assembled in either a disorganized or organized pattern. learning objectives 1. to describe the cytological features of this entity 2. to become aware of its cytologic atypia, a potential mimic of malignancy 3. to become familiar with the utility of lab tests in the diagnosis of this entity case a 14-year-old female with a medical history of rett syndrome presented with respiratory illness, fatigue, and pneumonia for 2-3 months. respiratory secretion for culture and rt-pcr and bronchial washing for cytology and differential cell count were received. the bronchial washing specimen revealed scattered atypical squamoid cells with large nuclei, high nuclearto-cytoplasmic ratio, and hyperchromatic, smudged chromatin with a suggestion of nuclear inclusions in background of intense acute inflammation, reactive respiratory epithelial cells, and pulmonary macrophages. the cellblock material showed similar findings. immunohistochemically, the scattered atypical cells were decorated by ck7, but nonreactive to p40; the profile suggested that those cells are pneumocytes. representative images are shown in fig. 6 .38a-d.sars, a new human emergent infectious disease mainly involving the lower respiratory tract, became a worldwide outbreak in 2003, affecting more than 8000 patients, with a fatality rate of 9.2%. a novel coronavirus (sars-cov), a member of a family of large, positive, single-stranded rna viruses, was identified as the etiological agent for this disease. the key pulmonary pathology is that of dad, featured by pronounced pulmonary edema and hyaline membrane formation. there are interstitial thickening, fibrosis, and intraalveolar exudates with granulation tissue formation, as well as sparsely inflammatory cell infiltrates, including macrophages (often multinucleated forms) and lymphocytes. depending on different phases in the disease progression, the degree of fibrosis and the composition of inflammatory cells may vary. other pathologic findings, such as prominent vascular injury, hemophagocytosis, squamous metaplasia, apoptosis, and atypical pneumocytes, including multinucleated giant pneumocytes with irregularly distributed nuclei or pneumocytes with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm, were reported. however, distinct viral inclusions were not apparent. ancillary tests, such as in situ hybridization, immunohistochemistry, viral isolation, or rt-pcr are necessary to confirm the viral infection.for the index case, serology testing and culture of the respiratory secretion were positive for coronavirus oc43. electron microscopy (em) revealed viral-like particles in pneumocytes, but not in macrophages or other cell types of lung. the findings confirmed the viral infection.the atypical pneumocytes, metaplastic squamous cells, and apoptosis may mimic malignancy. caution should be exercised in the interpretation of specimens from patients with a history of sars and coronavirus infection. the awareness of clinical history and the limited amount of atypical cells should alert the pathologists to avoid overdiagnosis in this clinical setting. learning objectives 1. to describe the cytological features of this entity 2. to become aware of the common differential diagnoses of this tumor 3. to become familiar with the potential utility of the immunohistochemical markers in the diagnosis of this tumor an 81-year-old gentleman with no documented past history of malignancy presented with dizziness and difficulty of breathing. ct imaging of the chest revealed multiple lung nodules and lymphadenopathy. in addition, a possible brain lesion was noted on imaging. an ultrasound-guided fna of the hilar lymph node was performed. the smears were highly cellular, composed of clusters, aggregated cell groups, and abundant single spindled to plump cells with hyperchromatic nuclei without prominent nucleoli. some rosette-like structures were seen, as well as scattered pigment-laden macrophages. the pigments were dusky and brown granules on pap stain and blue-black on diff-quik stain. abundant necrosis was noted. representative images of the fna smears and cellblock material were shown in fig. 6 .39a-d.the cytological features raise a differential diagnosis that includes neuroendocrine tumor, spindle cell tumors, including sarcomatoid carcinoma and metastatic melanoma of the spindle cell type. a limited panel of immunoassays was performed, revealing tumor cells with strong and diffuse nuclear staining for (continued) key: cord-005941-e4fvj54l authors: hamm, h.; fabel, h.; bartsch, w. title: the surfactant system of the adult lung: physiology and clinical perspectives date: 1992 journal: clin investig doi: 10.1007/bf00180279 sha: doc_id: 5941 cord_uid: e4fvj54l pulmonary surfactant is synthesized and secreted by alveolar type ii cells and constitutes an important component of the alveolar lining fluid. it comprises a unique mixture of phospholipids and surfactant-specific proteins. more than 30 years after its first biochemical characterization, knowledge of the composition and functions of the surfactant complex has grown considerably. its classically known role is to decrease surface tension in alveolar air spaces to a degree that facilitates adequate ventilation of the peripheral lung. more recently, other important surfactant functions have come into view. probably most notable among these, surfactant has been demonstrated to enhance local pulmonary defense mechanisms and to modulate immune responses in the alveolar milieu. these findings have prompted interest in the role and the possible alterations of the surfactant system in a variety of lung diseases and in environmental impacts on the lung. however, only a limited number of studies investigating surfactant changes in human lung disease have hitherto been published. preliminary results suggest that surfactant analyses, e.g., from bronchoalveolar lavage fluids, may reveal quantitative and qualitative abnormalities of the surfactant system in human lung disorders. it is hypothesized that in the future, surfactant studies may become one of our clinical tools to evaluate the activity and severity of peripheral lung diseases. in certain disorders they may also gain diagnostic significance. further clinical studies will be necessary to investigate the potential therapeutic benefits of surfactant substitution and the usefulness of pharmacologic manipulation of the secretory activity of alveolar type ii cells in pulmonary medicine. summary. pulmonary surfactant is synthesized and secreted by alveolar type ii cells and constitutes an important component of the alveolar lining fluid. it comprises a unique mixture of phospholipids and surfactant-specific proteins. more than 30 years after its first biochemical characterization, knowledge of the composition and functions of the surfactant complex has grown considerably. its classically known role is to decrease surface tension in alveolar air spaces to a degree that facilitates adequate ventilation of the peripheral lung. more recently, other important surfactant functions have come into view. probably most notable among these, surfactant has been demonstrated to enhance local pulmonary defense mechanisms and to modulate immune responses in the alveolar milieu. these findings have prompted interest in the role and the possible alterations of the surfactant system in a variety of lung diseases and in environmental impacts on the lung. however, only a limited number of studies investigating surfactant changes in human lung disease have hitherto been published. preliminary results suggest that surfactant analyses, e.g., from bronchoalveolar lavage fluids, may reveal quantitative and qualitative abnormalities of the surfactant system in human lung disorders. it is hypothesized that in the future, surfactant studies may become one of our clinical tools to evaluate the activity and severity of peripheral lung diseases. in certain disorders they may also gain diagnostic significance. further clinical studies will be necessary to investigate the potential therapeutic benefits of surfactant substitution and the usefulness of pharmacologic manipulation of surfactant (=surface active agent) is a material capable of lowering surface tension. the existence of a pulmonary surface active substance was first postulated by van neergard in 1929 [181] . he found the calculated surface tension of the alveolar air-liquid interface to be too high to prevent endexpiratory alveolar collapse and atelectasis. therefore, he predicted the presence of an agent able to exert and maintain a low alveolar surface tension as a prerequisite for the adequate ventilation of the peripheral airways and for normal lung function. it was almost another 30 years until pattle [135] and clements [20] found a substance in lung edema fluid and in lung extracts that indeed lowered the surface tension dramatically. the material was found to be composed of a phospholipid and a protein fraction. in 1959, avery and mead [4] drew attention to the role of a surfactant deficit in hyaline membrane disease (irds) of premature infants. thus, clinical relevance and a first therapeutic perspective became apparent in surfactant research. more than another 30 years later, the understanding of the pulmonary surfactant system has grown tremendously. the precise composition of the surfactant is known down to the genetic codes of surfactent-specific proteins, making the industrial production of different surfactants a realistic prospect. much has been learnt about surfactant synthesis in the alveolar type ii cell and its regulation and metabolism. intratracheal surfactant re-placement is on the verge of becoming a routine life-saving therapy in irds. accumulating evidence suggests that in adult respiratory distress syndrome (ards) a similar disturbance of the surfactant system is involved which may possibly be ameliorated by substitution therapy. in recent years, surfactant functions other than the maintenance of normal lung function have come into view. perhaps most important among these findings is that surfactant plays a major role in pulmonary defense mechanisms and local immunomodulation. therefore, the role of surfactant in different lung diseases and in the defense against various environmental impacts on the respiratory tract is attracting growing attention. the purpose of this article is to present a review of the current knowledge on the pulmonary surfactant system with emphasis on possible clinical implications and future perspectives for adult pulmonary medicine. surfactant is a complex mixture of lipids and proteins ( fig. 1) . additionally, carbohydrate components are found in both the lipid [165] and the protein fractions [189] , but their precise functions remain to be established. most of the present data on surfactant composition is based on analyses of lung lavages [65, 174] , which are thought to reflect adequately the situation in the alveolar lining fluid. however, it has to be kept in mind that lavage specimens may to some degree be contaminated with lipids of nonsurfactant origin, e.g., lipids stemming from cell membranes or lipids secreted by airway epithelial cells [93] . fewer data exist on the intracellular surfactant composition, e.g., in the lamellar bodies of alveolar type ii cells. however, the surfactant composition of the intra-and extracellular compartments seems to be similar [55, 65] . the pool size of extracellular surfactant has been investigated in animals and ranges from about 10-15 mg/kg body weight in adults. mature newborns have 5-to 10-fold higher values [83] . assuming similar values in man, a 70 kg person would thus have an estimated alveolar surfactant pool of approximately 0.7-1.0 g. however, there are no available data on the normal surfactant pool size in man, and there may possibly be considerable interindividual variations. lipids are the major surfactant component by weight (fig. 1) . they make up about 85%-90% of whole isolated surfactant [65] . approximately 90% of this lipid fraction consists of a mixture of phospholipids. the remaining 10% are composed of other lipids, mainly cholesterol, which seems to be blood-derived and is of uncertain functional significance [66] . phospholipids combine hydrophobic and hydrophilic properties and are therefore involved in the coating of boundary areas and surfaces. they possess the ability to achieve low surface tensions at air-liquid interfaces and support, for example, the formation of micelles and lamellae. however, phospholipids not only have structural functions but may in many ways be involved in different dynamic biological processes [64] . the phospholipid composition of human lung surfactant is shown in fig. 1 . in other mammals, this distribution is very much the same. none of these phospholipids is unique to surfactant, but in contrast to the phospholipid profile in other organs, the relative concentrations of phospatidylcholine and phosphatidylglycerol are higher. surfactants of amphibians and birds lack phosphatidylglycerol, suggesting that this phospholipid was introduced late in evolution [65] . in human fetal lung development, phosphatidylglycerol becomes detectable only late in pregnancy and may serve as an indicator of fetal pulmonary maturity [59] , although it does not seem to contribute to the reduction of alveolar surface tension [15] . phosphatidylcholine accounts for approximately 80% of total surfactant phospholipids and for about two-thirds of whole surfactant (fig. 1 ). approximately 70% of its fatty acids are saturated under normal conditions [65] , the most common saturated acid being palmitic acid. dipalmitoylphosphatidylcholine (dppc) is the surfactant component which is predominantly responsible for the reduction of alveolar surface tension [9] . its hydrophilic (choline) residue associates with the alveolar liquid phase while the hydrophobic (palmitic acid) residue reaches into the air phase [197] . pattle [135] first noted that a protein component in surfactant material seemed necessary for proper surfactant function. in 1973, king et al. [94] could, for the first time, demonstrate the existence of specific surfactant proteins. by weight, protein accounts for approximately 10% of whole isolated surfactant. about 80% of this protein portion consists of contaminating serum proteins while only 20% are made up by the surfactant-specific proteins (fig. 1) . four surfactant specific proteins (sp) have so far been identified (table 1) . a simplified nomenclature of these proteins has recently been proposed [145] and is increasingly being accepted, despite certain difficulties and disadvantages [33] . the first three proteins are simply termed surfactant protein a, b, and c in descending rank of their molecular masses. more recently, a fourth protein called sp-d has been described. the primary structures of surfactant proteins a, b, and c have been identified, and their commercial production by modern techniques of molecular biology is possible [67, 158] . sp-a is the major surfactant protein in regard to relative abundance as well as size. in vivo, sp-a is found as a group of isoforms with a molecular weight ranging from approximately 28 to 36 kda, depending on the extent of posttranslational modifications [67] . it has structural homologies with clq, a protein of the classical complement path-way [171, 182] , and contains a collagen-like domain [28] which is the probable association site of sp-a monomeres. after alveolar secretion, sp-a is predominantly :found as a multimeric molecule resembling a flower bouquet [182] . recent evidence suggests that in man, there are at least two different sp-a subtypes encoded on two separate genes [44, 188] . this may have structural implications for the arrangement of the naturally occurring sp-a multimeres [183] , but the functional significance of these findings awaits further clarification. sp-a seems to play an important role in the formation of a preliminary alveolar surfactant layer called tubular myelin which is found immediately after alveolar secretion [145, 181, 191] . in concert with sp-b and sp-c, sp-a probably enhances the surface activity of the surfactant monolayer [43] . however, the importance of the presence of sp-a regarding this aspect of surfactant function is still debated [146] . sp-a seems to be unique among the surfactant specific proteins as it apparently has additional functions in the surfactant complex apart from influencing surface activity. the structural homologies to the complement protein clq stimulated investigations of possible common biological functions of these two proteins. indeed, it was found that the presence of sp-a enhances the phagocytosis of opsonized sheep erythrocytes by macrophages and monocytes in a concentration-dependent manner [171] . furthermore, sp-a is able to increase the phagocytosis of staphyloccocus aureus [178] , herpes simplex virus type 1 [178] , and colloidal gold particles [43] . thus, sp-a seems to play an important role in the local host defense mechanisms of the lung. another probable function of sp-a is its ability to regulate the alveolar surfactant concentration. in vitro, sp-a inhibits the secretion of phosphatidylcholine from cultured alveolar type ii cells [34] and enhances the uptake of surfactant lipids [198] . possibly, these sp-a effects are mediated by an alveolar type ii cell receptor [157] . sp-b is a small protein of a molecular weight of approximately 8 kda under reducing conditions [186] . although it is very hydrophobic, it remains soluble in aqueous solutions to some extent. sp-b forms thiol-dependent oligomers of different sizes with the dimer probably being the most common form in vivo [84, 185] . it has no known immunomodulatory or regulatory function but seems to be a key protein in the formation of a functionally optimal and stable surfactant monolayer on the alveolar surface [5, 23, 169] . also, sp-b seems to play a role in the formation of tubular myelin in cooperation with sp-a [191] . its amino acid sequence contains high amounts of cysteine, suggesting that disulfide bridges may be important to the role of this protein in the surfactant complex [67] . indeed, intramolecular disulfide bridges seem to contribute to the structural properties of the sp-b polypeptide chain, and an intermolecular disulfide link may explain the frequent natural occurrence of sp-b dimers [84] . furthermore, sp-b has a strong positive net charge (at physiological ph) which seems to be important to the interaction between sp-b and the anionic phospholipids [23, 84] . however, the structural interaction between sp-b and other surfactant components still has to be more clearly defined. sp-c is a very small protein with a molecular weight of approximately 5 kda. it is extremely hydrophobic, which is in part due to a high content of the hydrophobic amino acid valine [145] . it is therefore only soluble in organic solvents. small size, hydrophobicity, and low immunogenicity make the investigation of this protein a difficult task. as far as its functional role in the surfactant complex is presently understood, it contributes to the formation and stabilization of the alveolar surfactant monolayer in cooperation with sp-b [169] . probably, sp-c has no role in tubular myelin formation [191] . the molecular structure and most of the properties of sp-c are substantially different from sp-b, suggesting that both proteins have separate roles in the surfactant complex. indeed, in vitro studies indicate that sp-c may be more important to the adsorption of phospholipids, while sp-b supports the reduction of surface tension more effectively [199] . there seem to be no similarities of sp-c to other proteins of known functions that would suggest an additional role of sp-c [185] . sp-d is a collagenous glycoprotein synthesized by alveolar type ii cells which has only recently been described [137, 138] . the question still remains whether this protein is a true surfactant protein or a protein that is synthesized independently of the surfactent pathway and is only functionally associated with the surfactant complex. in rat bronchoalveolar lavage (bal) fluids, the total sp-d content was found to be approximately 12% of that of sp-a [99] . it has a molecular size of approximately 43 kda (reduced) and appears to build polymeric complexes comprised of the 43-kda subunits. sp-d has certain structural similarities with sp-a and probably is readily soluble in the alveolar milieu. like sp-a, sp-d does not contribute to the surface activity of the surfactant complex [137] . its function is still hypothetical. structural analogies with proteins like mannosebinding protein, conglutinin, and sp-a suggest that it may have a role in local host defense [184] , perhaps by functioning like an opsonin. a recent study indicates that sp-d may also have regulatory functions by counteracting the inhibitory effects of sp-a on phospholipid secretion by alveolar type ii cells [98] . the site of alveolar surfactant synthesis and secretion is the cuboidal alveolar type ii cell which covers less than 5% of the alveolar surface. there is evidence to suggest that surfactant synthesis and secretion in the lung are not exclusively restricted to the alveolar type ii cell but that they may also take place in higher parts of the airways, for instance in clara cells [3, 170] and possibly even in the tracheal epithelium [8] . this may contribute to normal mucociliary function [190] . however, the significance of these findings awaits further investigation. the alveolar surfactant components are synthesized and assembled in the endoplasmic reticulum of alveolar type ii cells and then transferred to the golgi apparatus prior to forming socalled lamellar bodies in the cytoplasm (fig. 2) . this process has been followed by autoradiography and by immunocytochemistry for phospholipids and sp-a [33] . as shown by transmission electron microscopy [122], lamellar bodies undergo a process of maturation while travelling through the cytoplasm and are eventually transported into the alveolar space by merocrine secretion after fusion with the cell membrane. here, the lamellar bodies rapidly transform into tubular myelin, an intermediate surfactant material that is composed of a lattice of highly ordered tubules. sp-a is thought to play a role in the formation of tubular myelin and has recently been located at the corners of the tubular framework by immune electron microscopic techniques [181] . another recent in vitro study [191] suggests that in tubular myelin formation the presence of sp-b but not of sp-c is necessary in addition to sp-a. finally, this material is spread to reach its definitive form, the surfactant monolayer (figs. 3, 4) . pulmonary surfactant is not a static accessory of the alveolus but undergoes a constant dynamic process of turnover and metabolism. this review will present only a short summary of the present knowledge on these processes. for more detailed information, the interested reader is referred to a number of recently published reviews which emphasize these aspects [17, 18, 56, 65, 174-176, 185, 196, 197] . basically, all phospholipid components of surfactant seem to be synthesized and incorporated into the lamellar bodies within the alveolar type ii cell. this is supported by findings that the phospholipid composition of isolated lamellar bodies is virtually identical to that of bal [197] . dppc is the best studied phospholipid regarding intracellular synthesis pathways. it is de novo synthesized from blood-derived phospholipid precursors and can probably also be remodelled from unsaturated or recycled phosphatidylcholine. less evidence exists on the synthesis and precise pathways of secretion of the surfactant-specific proteins. alveolar sp-a gene expression is restricted to the alveolar type ii cell as shown by in situ hybridization [141] . it is synthesized as a preprotein of approximately 29 kda and a second variant of approximately 31 kda. different posttranslational modifications of this protein like sialylation, acetylation, and sulfation have been described [145] . single sp-a monomeres are oligomerized to hexameric bundles resembling flower bouquets [182] . probably, surfactant proteins and the phospholipids are all assembled and introduced into the lamellar bodies within the type ii cell before secretion [185] . however, many details of this process remain to be investigated. for instance, it is not yet quite certain whether all of the individual proteins are introduced into the lamellar bodies or if some of them join the surfactant complex after secretion into the alveolar space [56] . fur-thermore, no evidence exists on the possible association of sp-d with intracellular lamellar bodies and their secretion. surfactant synthesis has been found to be influenced by a number of different stimuli [7] (table 2). glucocorticoids, camp, oestrogens, and thyroid hormones, among others, have been described as enhancing surfactant synthesis. however, the in vivo role and importance of these factors is not clearly determined. some of these stimuli, e.g., glucocorticoids, may vary in their effects depending on dose and time [105] , and there may be different pathways for the regulation of surfactant phospholipid and protein synthesis [56] . a recent in vivo study [42] has investigated the influence of exogenously administered glucocorticoids and of adrenalectomy on the regulation of surfactant proteinsl glucocorticoid administration resulted in the accumulation of mrnas of surfactant proteins sp-a, b, and c, with the highest response being sp-b mrna. however, adrenalectomy did not change the mrna levels but decreased the total pulmonary sp-a levels. this study demonstrates that exogenous glucocorticoids enhance surfactant protein synthesis and suggests that adrenal hormones may have a role in the pulmonary response to stress. on the other hand, endogenous steroids under normal conditions do not seem to be important to baseline surfactant protein synthesis at the mrna level but may to a minor degree contribute to translational or posttranslational processing. the inhibition of surfactant production is possibly controlled by a feedback mechanism involving a surfactant protein [172] . surfactant secretion into the alveolar space is accomplished by exocytosis of the lamellar bodies. experimental data suggest that various stimuli like high volume lung inflation and increased ventilation rate, adrenergic agents, estrogens, and thyroid hormones may enhance surfactant secretion, while beta-blockade and an sp-a-dependent feedback mechanism have inhibitory effects [18, 33, 65] (table 2 ). sp-d seems to counteract the inhibitory effect of sp-a [98] . again, the in vivo significance of these experimental data remains under discussion. turnover studies with different labeled surfacrant phospholipids after secretion have demonstrated half-lives of between 15 and 30 h [60, 155] . the fate of secreted surfactant material seems to be determined by five mechanisms: -intraalveolar catabolism -phagocytosis and degradation by alveolar macrophages [110, 118] -removal by the mucociliary escalator -recycling into the alveolar type ii cell -redistribution into other surrounding tissue clearance studies in rabbits [140] have shown that approximately 7% of radiolabeled phosphatidylcholine is removed via the upper airways in 24 h, suggesting that this pathway is only of minor importance. further work by the same group [139] supports evidence that most surfactant material is probably redistributed into the surrounding tissue or is recycled into alveolar type ii cells. many aspects of the regulation of these processes remain to be clarified. sp-a has been shown to enhance the uptake of liposomes into the alveolar type ii cell [198] . this process is probably mediated by an sp-a receptor on the epithelial surface of type ii cells, which also controls the reuptake of sp-a [157] . s u r f a c t a n t a n d l u n g f u n c t i o n this review will only give a short introduction into the role of surfactant in alveolar stability and in the work of breathing. the interested reader is referred to a number of articles [22, 69, 70, 136, 194] which discuss these aspects in detail. the lowering of surface tension is the best known function of surfactant material and led to its discovery. however, this classical surfactant function probably was not the initial reason for the development of this material in evolution since animals with less complex lung architectures and thus without a need for surface-tension lowering agents already possess a pulmonary surfactant system [52] . surfactant material has been shown to reduce the surface tension at the alveolar air-liquid interface down to levels that are required for normal ventilation of the peripheral lung. it reduces the respiratory work load throughout the respiratory cycle and improves lung compliance. the most important surfactant component in this regard is saturated phosphatidylcholine. other surfactant components like sp-a and more importantly, sp-b and sp-c, have been described to enhance the surface activity of this phospholipid. the hydrophobic saturated fatty acids of saturated phosphatidylcholine are aligned in parallel and rise out of the liquid phase into the alveolar air. the hydrophilic choline residues are packed in the aqueous phase of the alveolus. this arrangement remains stable through ventilatory compression and extension of the alveolus and reduces the strong alveolar cohesive forces close to zero. thus, alveolar surfactant material successfully prevents alveolar collapse and atelectasis as observed in surfactant-deficient lungs, e.g., in irds. surfactant material may contribute to pulmonary defense mechanisms and local immunomodulation in four different ways: -support of nonspecific defense mechanisms -direct bactericidal properties of surfactant components -immunomodulatory action on lymphocytes -augmentation of macrophage activities in the alveolar milieu surfactant is part of the alveolar and bronchial epithelial lining fluid which is thought to act as a nonspecific barrier against adhesion and invasion of microorganisms. also, surfactant has antioxidant activities [115] which may contribute to the protection of the alveolar epithelium by scavenging toxic (reduced) oxygen species. several reports have addressed the possible antibacterial properties of surfactant material. studies of rat alveolar lining material identified long-chain free fatty acids as bactericidal surfactant components and demonstrated their antibiotic action against pneumococci in vitro [24] . however, studies of human alveolar lining material obtained by bal could not demonstrate antibacterial effects against pneumococci or haemophilus influenzae [86] . the in vivo significance of these findings is still uncertain, and the antibiotic effect of surfacrant remains controversial. lung surfactant has been shown to influence the activities of lymphocytes and macrophages. these influences are probably of significant in vivo importance for the maintenance of a balance between excessive immune responses and favorable cellular defense mechanisms. surfactant suppresses the activation and the proliferative response of lymphocytes to various stimuli in a dose-dependent manner [17, 164, 192] . this suppressor activity is contained in the lipid fraction of surfactant [17] . the major surfactant phospholipids phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were shown to be responsible for this immunoregulatory effect. the mechanism of this effect has not yet been clarified but may be related to changes in cell membrane dynamics [193] . surfactant exerts its effects only on the resting lymphocyte or on the early stage of lymphocyte activation. activated lymphocytes are not affected. the suppression seems to be largely irreversible, even after the removal of surfactant material from the medium. the inhibitory effects of surfactant have been shown for a variety of lymphocyte activities such as proliferation, differentiation, immunoglobulin production, and natural killer cell activity [10, 17, 164, 192, 193] . nearly all studies on the influence of surfactant on alveolar macrophage activity report an enhancement of macrophage functions. in detail, it has been shown that surfactant material supports phagocytosis [131, 177] and intracellular killing [88, 131] of staphylococcus aureus and the phagocytosis of herpes simplex virus type 1 [178] . it may also enhance the migration of alveolar macrophages [159] and their cytotoxicity against tumor cells [13] . it has to be stressed that these studies report the results of in vitro investigations mostly with animal material. thus, the significance of these findings for normal human lung defense mechanisms is not yet definitely established. in general, alveolar macrophages are thought to be less active than blood monocytes or macrophages residing in other tissues [75, 150] . recent studies [171, 177, 178] have shown that sp-a is probably responsible for the enhancement of alveolar macrophage functions as isolated sp-a had the same stimulant effect on macrophages as whole surfactant, while surfactant lipids had no effect [177] . probably, this macrophage stimulation is mediated by a macrophage receptor which binds sp-a. the specific binding and uptake of sp-a by macrophages has been demonstrated by electron microscopy [110] . a recent report [109] suggests that the sp-a receptor may be identical with the leukocyte clq receptor, which is a tempting hypothesis since sp-a has structural homologies with the complement protein clq. surfactant material, as part of the alveolar epithelial lining fluid, is thought to represent a first defense line against inhaled particles and gases reaching the alveolar space. apart from building a "mechanical" barrier, it probably plays an active role in the elimination of foreign particles, e.g., by enhancing macrophage activities and by exerting antioxidant effects [115] against a variety of oxidant gases. on the other hand, the surfactant system itself may be damaged by inhaled particles and gases. a number of studies have been published addressing the impact of air pollutants and other toxicants on the pulmonary surfactant system. varying study designs such as the use of different animal or in vitro models and different doses and exposure times have led to divergent and sometimes conflicting results. furthermore, some of the studies focussing only on phospholipid alterations leave some doubt as to whether these changes are truly related to surfactant abnormalities or rather reflect other mechanisms like unspecific cell membrane damage. it certainly has to be kept in mind that the surfactant system is only one of the potential targets of pollutants and toxicants reaching the lung periphery, and hazardous effects on the surfactant system may be of a direct or indirect or as yet unknown nature. this review will only give a short overview of the known or proposed effects of some pollutants and toxicants on the surfactant system. for further information, the reader is referred to a number of reviews and articles focussing on this subject [40, 55, 119, 120, ozone is a major component of photochemical smog. it acts as a highly aggressive oxidant and leads to the transudation of blood proteins and to edema in the alveolar space even at comparatively low concentrations. furthermore, chronic low-dose ozone exposure is known to increase the susceptibility to pulmonary infections. it is believed that the pulmonary toxicity of ozone is at least in part due to impairment of the surfactant system [55] . several reports support this hypothesis. in rats exposed to 0.3 ppm of ozone for 16 days, giant lamellar bodies were observed in the alveolar type ii cells after day 11. this could suggest that ozone may impair surfactant secretion [163] . short-term exposure (2.5 h) of isolated rat alveolar type ii cells to variable amounts of ozone resulted in impaired intracellular synthesis of phospholipids [57] . exposure of bonnet monkeys to variable low-dose concentrations of ozone for 21-90 days led to changes of fatty acid compositions and a marked increase in phosphatidylcholine levels in lung lavage fluids [149] . short-term (1-8 h), high dose (3 ppm) ozone exposure of rats resulted in ultrastructural alterations of intracellular lamellar bodies and inhibited proper unfolding of secreted lamellar body membranes in the alveolar space [6] . in vitro ozone exposure of sp-a led to impairments of important physiologic sp-a functions like self-association and sp-a-mediated lipid aggregation [132] . these studies sugest that ozone even at low levels leads to changes in surfactant metabolism and secretion and to alterations of composition and properties of the secreted surfactant material. thus, it seems likely that the pulmonary toxicity of ozone is in part due to impairment of the surfacrant system. one of the many remaining questions is whether ozone-induced surfactant abnormalities are also involved in the increased susceptibility to pulmonary infections of chronically exposed individuals. the majority of atmospheric nitrogen oxides is derived from natural sources. however, in urban areas, nitrogen oxides from energy utilization largely determine air pollution levels with these gases [123] . the pulmonary toxicity of nitrogen dioxide is similar to that of ozone, inducing free radical reactions and lipid autoxidation [55] . probably, both these air pollutants have synergistic toxic effects on the lung. short-term exposure (5 h) of rats to high levels (40 ppm) of nitrogen dioxide resulted in phosphatidylcholine and phosphatidylglycerol accumulation in lung tissue with a peak at 48 h postexposure. incorporation studies suggested that this increase was due to enhanced phospholipid synthesis [14] . long-term exposure (9 months) to low levels (2.9 ppm), by contrast, led 645 to a significant decrease in the lung lipid content and changes in the phospholipid fatty acid composition [2] . these studies may indicate that the acute effect of nitrogen dioxide on alveolar type ii cells is enhanced surfactant lipid synthesis, while chronic low-dose exposure leads to a decrease in surfactant synthesis capacity. however, the evidence is still scarce and not all observed phospholipid changes are necessarily related to the surfactant system. further studies are necessary to define more precisely the possible impact of nitrogen dioxide on alveolar type ii cells and surfactant material. also, in view of a more realistic approach to urban air pollution, it seems important to learn more about the co-toxicity of ozone and nitrogen dioxide. the toxicity of hyperbaric oxygen or oxygen at high concentrations is well-known and represents one of the problems of mechanical high oxygen ventilation, e.g., in intensive care units. the toxic effect is due to aggressive oxygen-derived free radicals which attack various cell constituents and probably also the surfactant system. in detail, it has been found that rabbits exposed to 100% oxygen for 64 h exhibited a marked decrease in phosphatidylcholine synthesis and cell lipid content followed by a recovery to normal patterns and subsequently supranormal levels beginning 3 days postexposure [74] . the same group [114] showed that intratracheal surfactant substitution significantly diminished the progression of hyperoxic injury. in rats exposed to 85% oxygen for 72 h, increased levels of phosphatidylcholine and sp-a were found in lung lavages [1261. it was concluded that hyperoxic lung injury is not due to intraalveolar decreases of these two major surfactant components. however, in another animal study dppc was decreased and the pg:pi ratio was markedly lower than baseline values after 4-5 days exposure to 100% oxygen. longer periods of exposure resulted in a further drop of dppc values [95] . pulmonary oxygen toxicity does not seem to be consistently related to changes of surface tension measured in lung lavage fluids of exposed animals [1] . in conclusion, hyperoxic lung injury may be associated with alveolar type ii cell changes in surfactant biosynthesis. however, different studies have found partly conflicting results, and the way in which hyperoxic lung injury contributes to quantitative and functional changes of alveolar surfactant is still poorly understood. it should also be remembered that the oxidant attack of oxygen is not limited to type ii cells or surfactant, and thus, some of the described phospholipid changes may not exclusively reflect surfactant abnormalities. cigarette smoke is a complex mixture of particles and gases. a reduced yield of phospholipids from bal fluids of smokers compared with nonsmokers has been described [41] . this difference was interpreted to be partly due to lower lavage fluid recovery from smokers related to their known tendency to bronchoconstriction. additionally, it was thought to reflect the enhanced phagocytosis activity of alveolar macrophages [21] . another group found no such quantitative differences between smokers and nonsmokers but described a decreased phospholipid/protein ratio in smokers [108] . in rats exposed to cigarette smoke, a decrease of surfactant material in lung lavages was found. additionally, a progressive injury of alveolar type ii cells was observed over time as determined by electron microscopy, indicating that type ii cells and therefore possibly the surfactant system may be one of the targets of cigarette smoke in the peripheral lung [102] . in vitro studies showed that smoke particles but not the gas phase of cigarette smoke interacted with a surface film of surfactant and altered its surface active properties in such a way that the maximum surface area was reduced, but the minimum surface tension was increased [68] . this may possibly contribute to altered mechanical properties of the lungs of smokers. in conclusion, only a few studies have so far investigated the possible impacts of cigarette smoke on the surfactant system. thus, our knowledge about the effects of this important pulmonary toxicant is still very fragmentary and awaits further investigations. smoke generated from the burning of polyurethane foam has been shown to increase significantly the total phospholipid content of lung lavages from rats after short-term exposure [134] . diesel exhausts were shown to induce pulmonary phospholipidosis in rats [37] . in another study, short-term exposure of rats to 6 mg/m 3 diesel exhaust resulted in an increased labeling index in type ii cells and enhancement of whole lung d n a synthesis [195] . additionally, lavage phospholipid values were increased, and there was evidence of reversible alterations of fatty acid and phospholipid metabolism. hydrogen sulfide is an irritant gas with toxic effects on the respiratory tract. an animal study suggests that higher doses impair the ability of sur-factant to lower surface tension. however, this does not seem to be due to a direct effect of hydrogen sulfide on surfactant material but due to surfactant inhibitors in the pulmonary edema fluid induced by hydrogen sulfide [53] . dusts, especially those with a high fibrogenic potential, seem to stimulate the production of surfactant [55] . silica (usually quartz dust) inhalation leads to a striking increase in the alveolar surfactant phospholipid and sp-a content [31, 90] . recently, the accumulation of sp-d has also been reported [29] . morphologically, these observations are accompanied by type ii cell hypertrophy and hyperplasia [90, 119] . the lungs of silica-exposed animals share common features with alveolar proteinosis in man so that they may be used as animal models of this disease. asbestos inhalation provokes a very similar accumulation of surfactant material in the alveolar space [38, 55] . the heavy metal cadmium is a known pulmonary toxicant. the main sources of human exposure are cigarette smoke, automobile emissions, and metal-processing plants. in rats, the inhalation of cadmium chloride led to an early decrease of phospholipids in lung lavage, accompanied by an increase in tissue phospholipids. after 4 days, levels of lavage phospholipids then markedly increased above normal values [16] . in vitro studies with alveolar type ii cell cultures exposed to cadmium chloride demonstrated inhibition of surfactant secretion, while cadmium alone had no such effect [40] . paraquat, a commercially important herbicide, has marked toxic effects on the lung, particularly on the alveolar type ii cell. in vivo and in vitro studies have shown that the synthesis of surfactant phospholipids decreases after exposure. however, it is not yet clear whether this effect on surfactant production is the primary cause for paraquat-associated respiratory failure. a major problem in the clinical treatment of paraquat poisoning is the synergistic toxicity of high oxygen mechanical ventilation [55] . the number of available studies on surfactant changes in human lung diseases is still limited. however, with further improvement of investigative tools and increasing interest in possible clinical implications, it should be expected that such studies will prosper in the near future. the main material for surfactant studies in man is bal because it is available at a low risk to the patient and gives access to all alveolar surfactant components. nev-647 ertheless, this method has its limitations. bal does not give direct insight into cellular changes of alveolar type ii cells and methods of quantification of the obtained epithelial lining fluid are limited by the complex nature of fluid dynamics during the procedure [92, 111] . also, not all phospholipids in bal fluid must necessarily be surfactant phospholipids. they may in part stem from airway epithelial cell secretions [93] or from cell membranes (alveolar cells, but also leucocytes, macrophages, etc.), which seems particularly important to consider in inflammatory lung diseases (e.g., [148] ). nonetheless, the first clinical studies have shown that quantitative changes of surfactant components in different lung diseases can be found in comparison with healthy controls. normal values for sp-a in human bal are found in the range of about 0.5-3 ~tg/ml lavage fluid ( [62, 116, 142] and own unpublished data). normal total phospholipid levels seem to vary considerably among individuals and also among laboratories [61, 76, 77, 81, 116, 143, 154] . additionally, chromatographic determination of the distribution of individual bal phospholipids (fig. 1) is often used to describe surfactant abnormalities. recently, simpler enzymatic methods were recommended for phospholipid analysis in bal fluids [48] . however, at present, only phosphatidylcholine and phosphatidylglycerol may be quantified by this method. another frequent approach is to investigate the biophysical properties (ability to lower surface tension) of the obtained surfactant material. various conditions such as severe trauma, major surgical procedures, burns, sepsis, acute pancreatitis, and aspiration pneumonia are capable of inducing this form of acute lung injury. it may rapidly progress to respiratory failure and continues to have a high mortality of around 50%-65% [36, 45] with hardly a change over the years in spite of many improvements in modern intensive care medicine. ards may be triggered or aggravated by high oxygen mechanical ventilation which is necessary in many of these patients. surfactant changes are thought to play an important role in the pathogenesis of this condition. however, it should be emphasized that ards is a severe, multifactorial disease in which surfactant is only one piece of the puzzle. serious disturbances of surfactant phospholipid composition and surfactant function as well as a reduced sp-a content in the bal have been described in animal models [104, 168] and in man [11, 61, 143, 144] , while the total phospholipids have not consistently been found to be reduced. the pathogenesis of these changes seems complex and is still only partly understood. again, some of the reported phospholipid changes may not be directly related to surfactant abnormalities but may be caused by other mechanisms like breakdown of cell membranes. one of the major mechanisms leading to surfactant disturbances in ards is probably connected to a massive fluid and protein accumulation in the alveolar compartment. especially in the early stages of ards, the increased permeability of the alveolocapillary barrier leads to noncardiogenic pulmonary edema with high concentrations of plasma-derived proteins. edema fluid and coagulated proteins block the alveolar air spaces and impair normal gas exchange [36, 161] . several lines of evidence suggest that many of these plasmaderived proteins also have a strong potential for inactivation of surfactant material [71, 97, 161] . in detail, this has been experimentally demonstrated for fibrinogen [46] , fibrin monomers [160] albumin [73] , and even hemoglobin [72] . these results suggest that in ards a major problem is probably not only the postulated deficit of alveolar type ii cell function but also a relative deficit of functionally intact surfactant material due to massive protein inactivation. it remains to be investigated whether or not the protein inactivation of alveolar surfactant is, to a minor degree, also relevant to other pulmonary diseases. another possible mechanism of surfactant inactivation in ards is that surfactant phospholipids may be degraded by phospholipase a2, an enzyme which is probably involved in ards caused by pancreatitis or sepsis [35, 82] . furthermore, it has been shown that e. coli endotoxin reduces surfactant synthesis in vitro [103] and in vivo [130] . many authors have called for clinical studies to investigate the benefit of surfactant substitution therapy in ards [36, 100, 112, 125] , but there are still numerous problems to be solved like dosage, timing, and delivery method [71] which dampen the enthusiasm for patient trials. much of the optimism is certainly due to the fact that ards shares common features with irds in which surfactant replacement is on the verge of becoming a standard therapy. additionally, animal studies (e.g., [101] ) and human case reports [85, 100, 127, 152] support the hope for a beneficial effect of surfactant replacement therapy in ards. the first controlled clinical trials are presently under way. even if beneficial effects on survival can be demon-strated, a significant mortality will probably remain, since the cause of death in ards is not invariably related to respiratory failure [121] . idiopathic pulmonary fibrosis (ipf) is a progressive fibrosing lung disease of unknown origin which involves alveolar epithelial injury and alveolar type ii cell proliferation [91] . total phospholipids in bal were found to be reduced, with decreases of pg and dppc and an increase in pi [76] . another study [154] reported similar findings in 15 untreated patients. total phospholipids in bal were less than half that of controls, with raised percentages of pi and lowered pg. the severity of these changes correlated with more advanced histopathologic fibrosis. in 28 patients (including the 15 patients of the former study), the sp-a content of bal was reported to be significantly lower than in normal controls [116] . in 32 patients with untreated ipf, the pg level was lowered, and its increase after the commencement of steroid therapy seemed to indicate clinical improvement [81] . these studies suggest that surfactant studies may be of clinical value to assess the prognosis and proper management in ipf [107] . sarcoidosis is a generalized granulomatous disease of unknown origin which frequently involves the lung. in 3 patients, total phospholipids in bal were not significantly decreased, and changes in phospholipid composition were not found [76] . in partial agreement with these observations, no significant changes in total bal phospholipids were found in 12 untreated patients with sarcoidosis [154] , but there was an increase in the pg:pi ratio. if confirmed, these findings could be of interest as a clinical tool to separate sarcoidosis from ipf in the differential diagnosis of fibrosing lung diseases. another study of 13 untreated patients described a decrease of dppc in bal [12] . in 8 untreated patients with active sarcoidosis, our group found raised sp-a levels in bal in comparison with healthy controls [63] . further studies are necessary to confirm surfactant changes in pulmonary sarcoidosis and to evaluate their role in this disease and their potential in the differential diagnosis of fibrosing lung disorders. the known immunoregulatory role of surfactant makes this pulmonary disease an interesting object of surfactant studies. however, only a few reports have so far addressed the role of surfactant in hypersensitivity pneumonitis (hp). in a recent study, 3 untreated patients with hp are mentioned whose sp-a values in bal were lower than in normal subjects [116] . by contrast, our own preliminary data from bal fluids of 8 patients with untreated, active hp show higher sp-a values than controls [63] . also, the sp-a content of alveolar macrophages (obtained by bal) as assessed by immunocytochemistry was elevated in untreated hp patients in comparison with healthy controls [54] . another recent study demonstrated that acute immune lung injury in guinea pigs is augmented in animals with partial surfactant depletion while surfactant replacement ameliorated the parameters of lung injury [153] . this prompted a somewhat optimistic comment that surfactant replacement might be useful in the therapy of cell-mediated immune diseases of the lung [151] . it is an attractive hypothesis that surfactant abnormalities may play a role in the pathogenesis of pneumonia and/or that surfactant changes occur as a consequence of alveolar infection. as an example, viral infection could damage alveolar surfacrant, facilitating the secondary invasion of bacteria. as yet, only a few studies have investigated these questions, so that our knowledge of the role of surfactant in pneumonia is still rather incomplete. in patients with bacterial pneumonia, changes in the fatty acid composition of bal phospholipids have been described [11] . in an animal model of pneumocystis carinii pneumonia, increased amounts of total phospholipids and decreases in the percentage of pc were observed [162] . it was hypothesized that these findings contribute to the altered lung mechanics and respiratory distress in this disease. however, it should be stressed once more that phospholipid changes may not necessarily reflect true surfactant abnormalities. in 22 patients with acquired immunodeficiency syndrome (aids)-related pneumonia (mostly p. carinii pneumonia), a marked increase of sp-a in bal was reported in comparison with 21 healthy controls [142] . phospholipid analysis was not done, and it remained unclear whether the observed changes were primarily related to human immunodeficiency virus (hiv) infection or to pneumonia. further studies of the reactions and the potential role of the surfactant system in bacterial or viral invasion of the alveolar space are certainly necessary and may be awaited with interest. this is a rare disease in which for unknown reasons the alveolar type ii cell synthesizes and secretes excessive amounts of abnormal surfactant material. lungs of silica-exposed animals share common features with human alveolar proteinosis (see above), but there is no evidence that dust exposure has a role in the pathogenesis of this disease in man. a typical finding is the accumulation of tubular myelin-like multilamellated structures in the alveoli [78] . the bal fluid is characterized by increased content of total phospholipids with a relative decrease in pg and an increase in pi [77] . diagnosis is usually made histologically but may also be made by the demonstration of excessively high sp-a levels in bal or simply in sputum [113] . further studies of surfactant material and alveolar type ii cells of these patients may possibly help to identify the cause of this condition, which is presumably related to a disturbance of the normal type ii cell regulation. radiation pneumonitis and subsequent fibrosis are known problems after radiotherapy of thoracic organs. animal studies have shown that the number of lamellar bodies in type ii cells drops dramatically immediately after radiation and that this is accompanied by an increase in lavage surfactant content [40] . in vitro studies by the same group demonstrated that this is a direct effect of radiation on type ii cells and that these cells exhibit changes which may indicate a switch of phospholipid synthesis to cell membrane repair after radiation damage. these experimental findings indicate that radiation may lead to massive surfactant secretion from type ii cells early after exposure followed by a sharp drop in further surfactant synthesis. in 4 patients with pleural mesothelioma, hemithorax irradiation caused protracted accumulation of proteins in the alveolar epithelial lining fluid which may inhibit the surface activity of surfactant. no significant changes in total phospholipid content were found, but pg, pi, pc, and sp-a levels were decrased, while the sphingomyelin concentrations were markedly increased [62] . however, the raised sphingomyelin levels in this study probably originate from other sources than alveolar surfactant. the changes were most evident 4 months after the completion of radiotherapy. unfortunately, immediate or early effects of radiation were not investigated. further work will be 649 necessary to determine the role of surfactant abnormalities in the pathogenesis of radiation pneumonitis. drug-induced pulmonary disease (dipd) is often accompanied by histological changes of alveolar type ii cells like dysplasia and proliferation [25, 26] . therefore, it is reasonable to expect changes of the type ii cell surfactant production in druginduced lung injury. however, only a few drugs which are potentially able to induce dipd have so far been investigated in this respect. polychemotherapy has in one report been described as inducing decreased phosphatidylcholine levels and increased phosphatidylglycerol levels in bal of patients with bronchial carcinoma [156] . bleomycin is an antineoplastic drug which has a known capability to induce fibrosing lung disease. in animal studies, bleomycin lung injury is frequently used as a model of pulmonary fibrosis [25] . bleomycin induces proliferation of type ii cells and giant intracellular lamellar bodies in mice. in rats with bleomycin lung disease, the bal after days 14 and 30 revealed increased amounts of total phospholipids, with increased percentages of pc and pi, while that of pg was decreased. these changes coincided with an altered lung compliance [173] . another study described an initial decrease of total phospholipids after 4 days and a subsequent 2.5-fold increase over control animals on days 21 and 28 [80] . the percentage of pg was reduced, and that of pi was increased. sp-a levels did not change throughout the experiment. from these results, a rather general conclusion was drawn that sp-a is insensitive to lung injury and repair. decreased bal phospholipids were also found in the early phase of fibrosis in hamsters. the surface-active properties of surfactant were inhibited and lung pressure-volume curves deteriorated [133] . in conclusion, bleomycin apparently leads to a decrease of total phospholipid values within the first days of lung injury, followed by an increase above normal values with a decreased pg: p! ratio. it remains to be confirmed that these observations adequately and specifically reflect the injury of alveolar type ii cells. it seems surprising that sp-a, a more specific secretory product of alveolar type ii cells than phospholipids, did not change in the one study cited above. the conclusion that sp-a is insensitive to lung injury is not convincing, since changes in sp-a levels have been reported in idio-pathic pulmonary fibrosis and other lung disorders. it has been hypothesized that an increase of alveolar surfactant material may contribute to the pathogenesis of pulmonary fibrosis by activating alveolar macrophages which in turn stimulate fibroblasts [39] . however, there is no experimental support to this idea, and from a clinical point of view, this hypothesis appears doubtful since most patients with alveolar proteinosis do not tend to develop pulmonary fibrosis. amphiphilic drugs like amiodarone, propranolol, chloramphenicol, and chlorpromazine may interact with pulmonary phospholipids and thus surfactant phospholipids, causing pulmonary phospholipidosis. a proposed mechanism is that normal phospholipid degradation is impaired by binding to the drugs. inhibition of phospholipases may also be involved [87] . it seems reasonable to suspect surfactant abnormalities in many other drug-induced lung disorders because dipd is often associated with morphological alterations of type ii cells. as an example, we recently observed morphological changes of alveolar type ii cells in a case of acute mesalazine alveolitis [187] . subsequent analysis of the bal fluid of this patient revealed an increase of sp-a content approximately 10-fold above healthy controls. byssinosis is a lung disease observed in cotton workers. clinically, patients present with fever, flulike symptoms, and bronchoconstriction. lipopolysaccharides from gram-negative bacteria found in respirable cotton dusts are thought to be responsible for this disease. a recently published in vitro study suggests that cotton extracts cause biophysical alterations of the lung surfactant [30] . it is hypothesized that these effects play a part in the pathogenesis of byssinosis. bal from lung transplants of 11 dogs were recently investigated with the principle aim of finding surfactant phospholipid changes specific to infection or rejection [89] . this differential diagnosis represents one of the major problems in the treatment of lung recipients. however, the data obtained in this study were essentially inconclusive. the optimism that surfactant abnormalities specific to rejection or infection will be found in the future seems somewhat questionable because both are inflammatory processes with presumably similar responses of alveolar type ii cells. another recent study [96] determined dppc levels in bal of excised dog lungs during hypothermic storage (4 â° c for 24 h) and after left lung transplantation (6 dogs, 24-h postoperative observation period). a decrease of dppc levels was found in both situations. however, in a second group of 6 transplanted dogs receiving l-carnitine infusions preand postoperatively, dppc levels and oxygen tension were higher postoperatively than in the group not treated with carnitine. it was concluded that the drop in dppc levels reflected ischemic damage to alveolar type ii cells and that carnitine (a cofactor for fatty acid transport into mitochondria) improved surfactant synthesis and therefore pulmonary gas exchange in the transplants. however, analysis of other bal phospholipids and surfactant-specific proteins is lacking in this study, and it remains to be confirmed that carnitine infusions really have such in vivo effects. a current review of this subject [128] outlines some of the possible perspectives of surfactant analysis and treatment in lung transplantation in more detail. certainly, much more work has to be done to assess the usefulness of surfactant studies or even surfactant replacement therapies in lung recipients. as outlined before, surfactant therapy may prove to have beneficial effects on the course of ards and is now being investigated in clinical trials. presently, there is no convincing evidence to suggest that such a treatment may also be of use in other adult lung diseases. a number of surfactant preparations are now in use, and some of them are already marketed for the treatment of irds. their composition and therefore their properties vary considerably, and it is not yet clear which preparation will be the best considering efficacy, safety, availability, and price. it seems possible that different surfactants will in the future prove optimal for different indications, thus perhaps leading to a variety of specifically designed preparations. bovine surfactant preparations (e.g., survanta, surfactant-ta, alveofact) are organic solvent extracts of minced cow lungs and contain phospholipids in a natural composition plus sp-b and sp-c but no sp-a. in a randomized controlled trial of a bovine surfactant preparation (single dose) for the prevention of irds [51] , it could be demon-651 strated that the survival rate without bronchopulmonary dysplasia was significantly improved. furthermore, there was a tendency to a better overall survival rate and a reduction in total time of mechanical ventilation. a single-dose regimen of survanta reduced the severity of respiratory distress and the frequency of pneumothorax but not the mortality in another randomized controlled study of irds [79] . survanta has also been reported to improve lung recoil but not arterial blood gases in a rabbit lung model of ards [101] . surfactant-ta in a randomized controlled trial has been shown to diminish the amount of respiratory support necessary in premature infants with irds [49] . another similar study demonstrated the reduction of intracranial hemorrhage and bronchopulmonary dysplasia in infants surviving irds [47] . in an anecdotal report of 2 cases of ards, surfactant-ta was also reported to have beneficial effects, although the dose was unusually low [127] . porcine surfactants (e.g., curosurf) are organic solvent extracts from minced porcine lungs with a composition comparable to bovine surfactants. beneficial effects of curosurf have been described in 3 patients with ards [152] and in a series of children with severe irds [166] . also, natural surfactants from amnion fluid or bal have been used. a serious drawback of all natural surfactants is their limited availability and their high prices. alec is a simple preparation of only two phospholipids, dppc and pg (in a weight proportion of 7:3). exosurf (or exosurf neonatal) is a mixture of dppc, hexadecanol, and tyloxapol and also does not contain surfactant-specific proteins. in a recently published, large, multicenter trial in infants with irds [106] , exosurf in a two-dose regimen was shown to reduce mortality and perinatal morbidity. however, in a sheep model of ards, aerosolized exosurf failed to demonstrate a beneficial effect [200] . presently, great efforts are being made to produce synthetic surfactants which resemble natural surfactants more closely. the genes of sp-a, b, and c have been cloned so that these proteins can be produced by methods of recombinant dna technology [158] . surfactant phospholipids can easily be produced by chemical synthesis. thus, different surfactant preparations can now be designed and studied in vitro and in vivo. these syn-thetic surfactants will have the advantage of high quality and nearly unlimited availability, which is an important prerequisite for pharmacological trials and subsequent clinical use on a larger scale. however, several remaining issues will have to be solved, e.g., the optimal composition of such "designer surfactants." probably, only the phospholipids dppc and pg will be necessary in conjunction with surfactant proteins to guarantee full surfactant efficacy [158] . another issue which is presently debated is whether or not sp-a is a necessary component of synthetic surfactant preparations. bovine and porcine surfactant preparations without sp-a have already been shown to be effective, and it is feared that the addition of sp-a may increase the immunogenicity and impair the stability of the preparation. however, the addition of sp-a enhances the biophysical activity and increases the resistance of surfactants against inhibitory proteins in vitro, which seems an important aspect, especially when treating ards [179] . most data on surfactant dosage in adults are derived from animal studies or clinical trials of irds treatment (e.g., [27, 47, 58, 79, 166] ). usually, a single dose or a two-dose regimen is preferred over repeated surfactant instillations. an adequate single dose for the treatment of ards is thought to be in the range of 50-300 mg phospholipids/kg body weight [36, 71, 83, 85, 100] . alternatively, for example, a cumulative total dose of 4 g has been used [152] . surfactant preparations containing proteins should be expected to have a potential for sensitization of a patient to foreign proteins. data from children with irds treated with exogenous surfactant indicate that circulating surfactant-anti-surfactant immune complexes frequently occur [117] . however, many irds patients without substitution therapy also seem to have such circulating immune complexes [167] , and negative effects have not yet been observed. a recently published study [19] demonstrated igm antibodies to surfactant specific proteins in patients with severe irds and showed that the antibody occurrence decreased after surfactant treatment. it was concluded that irds can lead to a leak of surfactant-specific proteins into the circulation and that surfactant treat-652 ment may reduce this leak by reducing the lung damage. another issue is that exogenous surfactant could interfere with endogenous surfactant synthesis and secretion. indeed, in vitro evidence on surfactant regulation (see above) would suggest that surfactant substitution could have such unwanted effects, e.g., by feedback inhibition of type ii cells. however, a recent in vivo study in rabbits [129] has shown that this was not the case. on the contrary, administration of different surfactant preparations tended to stimulate endogenous surfactant synthesis and secretion. in conclusion, several studies and existing clinical experience suggest that surfactant substitution therapy is not associated with serious risks. however, possible long-term effects are not yet known, and further studies should continue to monitor patients for potential side effects of surfactant treatment. pharmacologic improvement of surfactant abnormalities or deficits in human lung diseases, especially in ards, would be of considerable clinical value. despite some encouraging in vitro and animal studies, no clinical studies have yet convincingly demonstrated the usefulness of theoretically promising pharmacologic agents. one of the problems in ards is probably that successful pharmacological substances would require very strong stimulatory effects on alveolar type ii cells to overcome not only the alveolar surfactant deficit but also the inhibitory effects of exudated proteins in the alveoli. steroids are known to interfere with many of the mechanisms thought to be involved in ards. their actions include beneficial effects on surfactant synthesis. however, a number of large clinical trials have not been able to prove a clinical benefit of steroid therapy in ards (for review see [50] ). this is not necessarily due to a failure of steroids to enhance surfactant synthesis but may simply reflect the multiple disturbances associated with ards. steroids are frequently and with some success used in mothers at risk of premature delivery to prevent irds (for review see [124] ). beta-agonists are able to enhance surfactant synthesis and secretion from alveolar type ii cells in vitro. these agents are also used to suppress premature labor in mothers and possibly accelerate fetal lung maturation as a beneficial side effect [124, 125] . however, no clear evidence has so far been presented to support such in vivo effects of beta-agonists. ambroxol, a drug which is primarily marketed as a mucolytic agent, seems to enhance surfactant production and secretion [32, 147] and has been reported to be useful for the prevention and treatment of irds (for review see [125]). we were not able to find studies investigating a potential use of ambroxol in ards. this review has attempted to summarize briefly the present knowledge on the pulmonary surfactant system and has tried to outline some of the available information which may in the future become relevant to clinical pulmonary medicine. after more than 60 years of research, the surfactant system of the human lung has not yet become part of routine diagnostic or therapeutic considerations in adult pulmonary medicine. however, with growing knowledge from basic research, surfactant studies are beginning to give us some new insights into the mechanisms involved in various lung diseases and in the degree of involvement of alveolar type ii cells. thus, a variety of possible perspectives have now arisen, ranging from diagnostic to therapeutic implications and preventional aspects. it does not seem likely that surfactant analyses will in the future be used primarily to arrive at a specific diagnosis of a disease since there are probably not many conditions which feature characteristic surfactant abnormalities. however, present evidence fosters speculations that surfactant studies may prove useful in giving some information about the activity, intensity, and perhaps the duration of some pulmonary diseases or pollutant exposure, and they may be found helpful in the differential diagnosis of fibrosing lung disease. furthermore, it can be speculated that surfactant studies may help to monitor the effects of therapies and to assess the prognosis of various lung diseases. however, much more work has to be done to investigate these hypotheses, and possible useful results will have to be weighed against the established clinical tools. therapeutic perspectives are at present mainly focussed on ards. here, the first results of clinical trials will soon be available and are awaited with interest. other indications of surfactant therapy are not yet clearly visible and remain highly speculative. however, the known role of the surfactant system in pulmonary host defense mechanisms and local immunomodulation will continue to stimulate clinical interest in its role in inflammatory and immunologic disorders of the lung. surfactant in pulmonary oxygen toxicity long-term nitrogen dioxide exposure surfactant apoprotein a (sp-a) is synthesized in airway cells surface properties in relation to atelectasis and hyaline membrane disease surfactant protein sp-b induces ordering at the surface of model membrane bilayers ozone stress initiates acute perturbations of secreted surfactant membranes hormonal regulation of pulmonary surfactant chemical structure of phospholipids in the lungs and airways of sheep surface tension induced by dipalmitoyl lecithin in vitro under physiological conditions lung derived surface active material (sam) inhibits natural killer cell tumor cytotoxicity changes in fatty acids in phospholipids of the bronchoalveolar fluid in bacterial pneumonia and in adult respiratory distress syndrome decreased phosphatidylcholine in the lung fluid of patients with sarcoidosis enhancement of macrophage and monocyte cytotoxicity by the surface active material of lung lining fluid sequential changes in phospholipid composition and synthesis in lungs exposed to nitrogen dioxide structure and function of phosphatidylglycerol-deficient lung surfactant toxicity of inhaled cadmium chloride: early responses of the antioxidant and surfactant systems in rat lungs immunomodulation by pulmonary surfactant regulation of lung surfactant secretion surfacrant proteins and anti-surfactant antibodies in sera from infants with respiratory distress syndrome with and without snrfactant treatment surface tension of lung extracts smoking and pulmonary surfactant pulmonary surface tension and alveolar stability pulmonary surfactant protein b (sp-b): structure-function relationships role of surfactant free fatty acids in antimicrobial defenses drug-induced pulmonary disease. part 1: cytotoxic drugs drug-induced pulmonary disease. part 2: noncytotoxic drugs decreased mortality rate among small premature infants treated at birth with a single dose of synthetic surfactant: a multicenter controlled trial lung surfactant associated proteins and type iv collagen share common epitopes surfactant protein d. increased accumulation in silica-induced pulmonary lipoproteinosis biophysical alteration of lung surfactant by extracts of cotton dust effects of silica on the composition of the pulmonary extracellular lining the pharmacology of ambroxol review and new results pulmonary surfactant pulraonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type ii cells acute tung injury induced by phospholipase a 2 surfactant replacement in adult respiratory distress syndrome pulmonary phospholipidosis in rats respiring air containing diesel particulates pathogenesis of pulmonary fibrosis induced by chrysotile asbestos. virchows arch experimental bleomycin lung in mice physiologic and toxicologic responses of alveolar type ii cells low yield of pulmonary surfactant in cigarette smokers in vivo regulation of surfactant proteins by glucocorticoids sixty years of surfactant research isolation and characterization of cdna clones for the 35-kda pulmonary surfactant-associated protein adult respiratory distress syndrome -prognosis after onset artificial pulmonary surfactant inhibited by proteins surfactant replacement therapy with a single postventilatory dose of a reconstituted bovine surfactant in preterm neonates with respiratory distress syndrome: final analysis of a multicenter, doubleblind, randomized trial and comparison with similar trials lipid content of alveolar lining material collected by bronchoalveolar lavage randomized controlled trial of exogenous surfactant for the treatment of hyaline membrane disease pharmacological treatment of the adult respiratory distress syndrome a multicenter randomized controlled clinical trial of bovine surfactant for prevention of respiratory distress syndrome pulmonary defense mechanisms in boa constrictor effects of hydrogen sulfide exposure on surface properties of lung surfactant increased surfactant protein a (sp-a) content in human alveolar macrophages in hypersensitivity pneumonitis lung surfactant and pulmonary toxicology synthesis and assembly of lung surfactant effects of ozone on phospholipid synthesis by alveolar type ii cells isolated from adult rat lung clinical experience with exogenous natural surfactant absence of phosphatidylglycerol (pg) in respiratory distress syndrome in the newborn analysis of labeling and clearance of lung surfactant phospholipids in rabbit evidence of lung surfactant abnormality in respiratory failure changes in surfactant in bronchoalveolar lavage fluid after hemithorax irradiation in patients with mesothelioma surfactant protein a in bronchoalveolar lavage of sarcoidosis and hypersensitivity pneumonitis phospholipids as dynamic participants in biological processes lung surfactant surfactant cholesterol metabolism of the isolated perfused rat lung pulmonary surfactant apoproteins: a review of protein and genomic structure lung lipids and disease. respiration 55 pulmonary surface film stability and composition the role of lung surfactant role of pulmonary surfactant in the development and treatment of adult respiratory distress syndrome effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity surface property changes from interactions of albumin with natural surfactant and extracted lung lipids type ii pneumocyte changes during hyperoxic lung injury and recovery down-regulation of immune responses in the lower respiratory tract: the role of alveolar macrophages changes in phospholipids in bronchoalveolar lavage fluid of patients with interstitial lung diseases alterations of acidic phospholipids in bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis dissolution and reassembly of tubular myelin-like multilamellated structures from the lungs of patients with pulmonary alveolar proteinosis a multicenter, randomized, placebo-controlled trial of surfactant therapy for respiratory distress syndrome surface and tissue forces, surfactant protein a, and the phospholipid components of pulmonary surfactant in bleomycin-induced pulmonary fibrosis in the rat changes in phosphatidylglycerol in bronchoalveolar lavage fluids from patients with cryptogenic fibrosing alveolitis changes of alveolar stability and phospholipids in pulmonary surfactant in acute pancreatitis surfactant for the treatment of respiratory distress syndrome surfactant protein b : disulfide bridges, structural properties, and kringle similarities human alveolar lining material and antibacterial defenses types of interaction of amphiphilic drugs with phospholipid vesicles enhancement of bactericidal capacity of alveolar macrophages by human alveolar lining material analysis of bronchoalveolar lavage and pulmonary alveolar surfactant for diagnosis of rejection and infection in heart-lung transplantation alveolar type ii cells, surfactant protein a (sp-a), and the phospholipid components of surfactant in acute silicosis in the rat structure of alveolar epithelial cells in patients with fibrotic lung disorders fluid dynamics during bronchoalveolar lavage secretions from primary hamster tracheal surface epithelial cells in culture: mucin-like glycoproteins, proteoglycans, and lipids isolation of apoproteins from canine surface active material -and pneumonia-induced lung injury. ii. properties of pulmonary surfactant pulmonary surfactant in bronchoalveolar lavage after canine lung transplantation inactivation of exogenous surfactant by pulmonary edema fluid surfactant protein d (sp-d) counteracts the inhibitory effect of surfactant protein a (sp-a) on phospholipid secretion by alveolar type ii cells characterization of pulmonary surfactant protein d: its copurification with lipids animal models and clinical pilot studies of surfactant replacement in adult respiratory distress syndrome surfactant replacement improves lung recoil in rabbit lungs after acid aspiration injury to type ii pneumocytes in rats exposed to cigarette smoke endotoxin suppresses surfactant synthesis in cultured rat lung cells functional abnormalities of lung surfactant in experimental acute alveolar injury in the dog glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein a in fetal human lung a controlled clinical trial of synthetic surfactant in infants weighing 1250 g or more with respiratory distress syndrome bronchoalveolar lavage lipids in idiopathic pulmonary fibrosis biochemical analyses of bronchoalveolar lavage fluids of healthy human volunteer smokers and nonsmokers human leucocyte clq receptor binds other soluble proteins with collagen domains specific interaction of lung surfactant protein a (sp-a) with rat alveolar macrophages limitations of using urea to quantify epithelial lining fluid recovered by bronchalveolar lavage surfactant in adult respiratory distress syndrome surfactant apoprotein.-a concentration in sputum for diagnosis of pulmonary alveolar proteinosis sublethal hyperoxic injury to the alveolar epithelium and the pulmonary surfactant system characterization of antioxidant activities of pulmonary surfactant mixtures idiopathic pulmonary fibrosis. abnormalities in the bronchoalveotar lavage content of surfacrant protein a immunologic consequences of exogenous surfactint administration degradation of pulmonary surfactant disaturated phosphatidylcholines by alveolar macrophages hypertrophy and hyperplasia of alveolar type ii cells in response to silica and other pulmonary toxicants adverse effects of toxins and drugs on the surfactant systems causes of mortality in patients with the adult respiratory distress syndrome toxicological data on nox: an overview pulmonary surfactant: physiology, pharmacology and clinical uses exogenous surfactant treatment for the adult respiratory distress syndrome? a historical perspective effects of pulmonary oxygen injury on airway content of surfactant-associated protein a surfactant for adults with respiratory failure surfactant analysis and replacement therapy: a future tool of the lung transplant surgeon surfactant treatments alter endogenous surfactant metabolism in rabbit lungs bacteremia-induced suppression of alveolar surfactant production rat lung lavage surfactant enhances bacterial phagocytosis and intracellular killing by alveolar macrophages structural and functional changes of surfactant protein a induced by ozone changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis effects of smoke inhalation on surfactant phospholipids and phospholipase a2 activity in the mouse lung properties, function and origin of the alveolar lining layer the relation between surface tension and area in the alveolar lining film purification and biochemical characterization of cp4 (sp-d), a collagenous surfactant-associated protein surfactant protein d is a divalent cation-dependent carbohydrate-binding protein clearance of surfactant phosphatidylcholine from adult rabbit lungs clearance of surfactant phosphatidylcholine via the upper airways in rabbits localization of surfactant protein synthesis in human lung by in situ hybridization increased recovery of surfactant protein a in aids-related pneumonia surfactant abnormalities in patients with respiratory failure after multiple trauma altered pulmonary surfactant in uncomplicated and septicemia-complicated courses of acute respiratory failure a proposed nomenclature for pulmonary surfactant-associated proteins the role of surfactant-associated proteins biochemical modification of pulmonary surfactant after bromhexine derivatc injection rheological and transport properties of airway secretions in cystic fibrosis -relationships with the degree of infection and severity of the disease changes in lipids of lung lavage in monkeys after chronic exposure to ambient levels of ozone immunologic system in the respiratory tract pulmonary surfactant as a physiologic immunosuppressive agent curstedt t (1989) the adult respiratory distress syndrome: first trials with surfactant replacement pulmonary surfactant suppresses the immune lung injury response to inhaled antigen in guinea pigs idiopathic pulmonary fibrosis -abnormalities in bronchoalveolar lavage fluid the surfactant system of the lung changes in pulmonary surfactant composition following macc chemotherapy for lung carcinoma binding and uptake of pulmonary surfactant protein (sp-a) by pulmonary type ii epithelial cells lung surfactant: a biotechnological challenge alveolar macrophage migration -influence of lung lining material alteration of surfactant function due to protein leakage: special interaction with fibrin monomer surfactant abnormalities and adult respiratory failure surfactant phospholipids and lavage phospholipase a2 in experimental pneumocystis carinii pneumonia giant lamellar bodies in alveolar type ii cells of rats exposed to a low concentration of ozone the effect of pulmonary surface-active material on the generation and expression of murine b-and t-lymphocyte effector functions in vitro isolation and characterization of a sulfated glyceroglucolipid from alveolar lavage of rabbit early versus late surfactant replacement therapy in severe respiratory distress syndrome surfactant replacement: immunological considerations surfactant abnormality after endotoxin-induced lung injury in guinea pigs structure-function relationships of bovine pulmonary surfactant proteins sp-b and sp-c the proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry human pulmonary surfactant protein (sp-a), a protein structurally homologous to clq, can enhance fcr-and crl-mediated phagocytosis altered lipid synthesis in type ii pneumocytes exposed to lung surfactant correlation of changes in pulmonary surfactant phospholipids with compliance in bleomycin-induced pulmonary fibrosis in the rat synthesis of surfactant lipids in the adult and fetal lung: pathways and regulatory aspects metabolism of phospholipids in the lung aspects of metabolism and storage of pulmonary surfactant: experiments with isolated type ii pneumocytes and lamellar bodies pulmonary surfactant protein a enhances the host-defence mechanism of rat alveolar macrophages surfactant protein a is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein a neue auffassungen fiber einen grundbegriff der atemmechanik surfactant protein a is localized at the corners of the pulmonary tubular myelin lattice macromolecular organization of natural and recombinant lung surfactant protein sp 28-36 structural comparison of recombinant pulmonary surfactant protein sp-a derived from two human coding sequences: implications for the chain composition of natural human sp-a surfactant proteins and sp-d function and regulation of expression of pulmonary surfactant-associated proteins identification of surfactant proteolipid sp-b in human surfactant and fetal lung mesalazine alveolitis isolation and characterization of the human pulmonary surfactant apoprotein gene glycosylation and secretion of surfactant-associated glycoprotein a endobronchial surface active phospholipids: clinical conclusions changes in lipid structure produced by surfactant proteins sp-a, sp-b, and sp-c immunoregulatory properties of pulmonary surfactant: effect of lung lining fluid on proliferation of human blood lymphocytes immunosuppression by pulmonary surfactant: mechanisms of action relations among recoil pressure, surface area, and surface tension in the lung effects of short-term exposure to diesel exhaust on lung cell proliferation and phospholipid metabolism clearance and recycling of pulmonary surfactant metabolism and turnover of lung surfactant surfactant apoprotein mr=26.000-36.000 enhances uptake of liposomes by type ii ceils role of bovine pulmonaryassociated proteins in the surface active property of phospholipid mixtures effects of aerosolized artificial surfactant on repeated oleic acid injury in sheep acknowledgements. we would like to thank prof. k. morgenroth, ruhr-universitfit bochum, for the excellent electron mi-croscopic photographs (figs. 2-4) and prof. schfifer (byk-gulden co., konstanz, germany), prof. akino and prof. kuroki (sapporo medical college, japan) for aiding our laboratory with sp-a antigen and antibodies. key: cord-016790-by7cxz1g authors: ahuja, jitesh; kapnadak, siddhartha g.; pipavath, sudhakar title: imaging of lung transplantation date: 2018-04-24 journal: lung transplantation doi: 10.1007/978-3-319-91184-7_19 sha: doc_id: 16790 cord_uid: by7cxz1g lung transplantation has become a viable treatment option for end-stage lung disease. common indications for lung transplantation are chronic obstructive pulmonary disease (copd), idiopathic pulmonary fibrosis, cystic fibrosis, alpha-1 antitrypsin deficiency, and pulmonary arterial hypertension. either single or bilateral lung transplantation can be performed, but bilateral lung recipients appear to have a better median survival than single lung recipients. complications after lung transplantation are common and may have nonspecific clinical and radiologic manifestations. the time point at which these complications occur relative to the date of transplant is crucial in formulating a differential diagnosis and recognizing them accurately. significant advances in imaging techniques and recognition of air trapping in exhalation images and other patterns /distribution of parenchymal abnormalities have led to routine use of hrct for diagnostic evaluation in patients manifesting respiratory decline in the lung transplant recipient. lung transplantation is a viable treatment option for end-stage lung disease. common indications for lung transplantation are chronic obstructive pulmonary disease (copd), idiopathic pulmonary fibrosis, cystic fibrosis, alpha-1 antitrypsin deficiency, and pulmonary arterial hypertension (pah). other less common indications include end-stage sarcoidosis, lymphangioleiomyomatosis (lam), pulmonary langerhans cell histiocytosis/granulomatosis (plch/g), and other interstitial lung diseases. either single or bilateral lung transplantation can be performed. bilateral transplantation is required for patients with suppurative lung diseases including cystic fibrosis and most patients with pah. while suitable outcomes may be achieved with single lung transplantation for other diagnoses, there is a trend in recent years for use of bilateral transplantation, largely related to a suggestion of better long-term survival seen in registry data [1] . specifically, according to the 2014 international society for heart and lung transplantation (ishlt) registry report, the median survival for all adult recipients is 5.7 years, but bilateral lung recipients appear to have a better median survival than single lung recipients (7 versus 4.5 years, respectively) [1] . overall survival after lung transplantation has improved due to refinement in surgical technique, improvement in organ procurement and preserving techniques, and advances in medical management and immunosuppression. the most common cause of mortality in first 30 days after lung transplantation is primary graft dysfunction. infection is the most common cause of mortality in the first 6 months and chronic rejection thereafter [1] . complications after lung transplantation are common and may have nonspecific clinical and radiologic manifestations. in addition, frequently, several of these complications may coexist and complicate their presentation. in order to reduce mortality and morbidity among lung transplant recipients, it is important to recognize these complications accurately and in time. the time point at which these complications occur relative to the date of transplant is crucial in formulating a differential diagnosis and recognizing them accurately. in this chapter, we will describe the radiologic manifestations of various pulmonary complications occurring after lung transplantation. complications are classified chronologically into groups based on the time from transplant: immediate (<24 h), early (24 h to 1 week), intermediate (8 days to 2 months), primary late (2-4 months), and secondary late (≥4 months) (table 19 .1). size mismatch between the donor lung and recipient thoracic cavity may result in mechanical complications. most centers accept a size difference ranging from 10 to 25% [2] . if the donor lung is too large for recipient thoracic cavity, atelectasis and impaired ventilation may result. atelectasis may be evident on immediate postoperative radiograph. on the other hand, when a small allograft lung is used in single lung transplantation for emphysema, the hyperexpanded native lung may compress the allograft, resulting in restrictive pulmonary function ( fig. 19.1a, b) . native lung volume reduction surgery performed at the time of, or after, transplantation may help prevent this complication [3] . lung torsion is a rare but serious complication that can occur due to discrepancy in size between the recipient's enlarged thoracic cavity and donor's small lung. imaging will show torsion of the hilar structures including vasculature and airway. it can lead to collapse of a lobe or entire lung due to airway compromise or expansible consolidated lobe due to hemorrhagic infarction. once lung torsion is suspected, immediate surgery is indicated to avoid death from parenchymal necrosis. hyperacute rejection is caused by the presence of preformed antibodies to donor organ-specific human leukocyte antigen (hla) or abo antigen. abo matching and improved antibody detection and crossmatch techniques have made hyperacute rejection very rare after lung transplanta primary graft dysfunction is a form of non-cardiogenic pulmonary edema that usually occurs by postoperative day 1, peaks in severity by day 4, and generally improves by day 7. it is diagnosed after excluding left heart failure, fluid overload, infection, and acute transplant rejection. the pathogenesis of primary graft dysfunction is multifactorial including interruption of lymphatic and bronchial circulation, donor organ ischemia, surgical trauma, denervation of donor lung, and decreased surfactant production [5] . chest radiography and ct manifestations are nonspecific and include perihilar and lower lobe predominant ground-glass opacity, and/or consolidation, interlobular septal thickening, and peribronchial air-space opacities (figs. 19.2 and 19.3). pleural complications-including pleural effusion, hemothorax, pneumothorax, and empyema-occur in 22-34% of patients after transplantation [6, 7] . existing pleural abnormalities before lung transplantation can predispose to pleural complications. for instance, pleural infection or inflammation can result from endstage lung diseases for which lung transplantation is required such as cystic fibrosis. invasive procedures required to diagnose (wedge biopsy) or treat (pleurodesis or pleural drain for pneumothorax or pleural effusion) end-stage lung disease may result in pleural abnormalities. pneumothorax is the most common pleural complication [7] airway anastomotic complications should be suspected if pneumothorax persists or enlarges after the early postoperative period (>7 days after transplantation). pleural effusion is also a common complication secondary to increased capillary permeability and impaired lymphatic drainage of the allograft lung during the early postoperative period ( fig. 19 .5a, b). pleural fluid in the immediate postoperative period is often hemorrhagic and becomes progressively less hemorrhagic by 7 days. pleural effusion usually resolves by 2 weeks. persistent or enlarging pleural effusion should raise suspicion for empyema or left heart failure. hemothorax usually results from surgical complications ( fig. 19.6a, b) . persistent air leak, empyema, and hemothorax are associated with increased mortality and should be promptly recognized and managed [7, 8] . acute rejection usually occurs after the second postoperative week and is classically caused by cell-mediated immune response. approximately 80% of acute rejections occur in first 3 months, and 50% of patients have at least one episode of acute rejection in the first postoperative year [9, 10] . repeated episodes of acute rejection are considered a risk factor for the development of chronic rejection [9, 11] . when present, radiographic manifestations are nonspecific and similar to primary graft dysfunction including ground-glass opacities, consolidation, interlobular septal thickening, and airway anastomotic complications include bronchial dehiscence, bronchial stenosis, or bronchomalacia. reported incidence of airway complications is approximately 15%, but there has been a decrease in the airway complication rate due to the refinement in surgical technique, improvement in immunosuppression, and donor organ preservation technique [12] . the most important factor predisposing to airway complication is donor bronchus ischemia, due to disruption of the native bronchial circulation. recurrent bronchial dehiscence usually occurs in the first 2-4 weeks after transplantation affecting 2-3% patients [9] . ct usually shows extralumi-nal foci of air in perianastomotic region, and occasionally bronchial defects can be visualized (figs. 19.10a-c, 19.11a, b, and 19.12a, b) . indirect signs include persistent or enlarging air leak, pneumothorax, or pneumomediastinum. bronchial dehiscence may resolve spontaneously or progress to bronchial stenosis. bronchial stenosis and bronchomalacia are late complications usually occurring at least 2-4 months after transplantation. bronchial stenosis occurs in 10% of subjects and is usually seen at the anastomotic site or distally [13] . ct shows bronchial irregularity and focal stenosis (figs. 19.13a-d and 19.14a, b) . management includes resection of the granulation tissue and balloon dilation with or without stenting. bronchomalacia or transient airway narrowing can be diagnosed with bronchoscopy or ct. inspiratory and expiratory ct images are invaluable in the diagnosis of bronchomalacia ( fig. 19.15a, b) . a diagnosis is suggested when there is more than 50 (liberal) to 70% (conserva-tive) dynamic narrowing of the airway or lunate shape of the airway on expiration. vascular anastomotic complications are uncommon. pulmonary artery stenosis is more common than pulmonary vein stenosis. it can occur in the early or late postoperative period [14] . contrastenhanced ct angiogram is the best available noninvasive modality to assess narrowing or occlusion of the anastomotic site. this may cause pulmonary infarction more readily due to the absence of bronchial artery circulation in the early postoperative period. treatment options include angioplasty and stenting. a b fig. 19.12 (a, b) infection is the most common complication and a leading cause of morbidity and mortality after lung transplantation. patients are at increased risk of infection because of immunosuppression, impaired mucociliary clearance, absence of cough reflex, disruption of lymphatic drainage, and direct communication between the allograft and the atmosphere. infectious risks vary based on factors which include the time elapsed since transplantation. bacterial infections are the most common cause of pneumonia with incidence highest in the first 4 weeks after lung transplantation [15] . gramnegative organisms such as klebsiella species and pseudomonas aeruginosa are common causative agents. infection by gram-positive organisms including staphylococcus aureus is also observed. during the first postoperative month, 35-70% recipients develop bacterial pneumonia [16] . imaging features are similar to the non-transplant population and include lobar or multifocal consolidation, ground-glass opacity, nodules, and pleural effusion [13, 15, 17] ( fig. 19.16a, b) . fungal infections are caused by aspergillus and less commonly by the candida species. pneumocystis pneumonia is uncommon these days because of routine chemoprophylaxis. fungal infections can occur at any time but usually peak between 10 and 60 days after transplantation. these are less common but carry higher mortality compared to bacterial or viral infections. patterns of fungal infection include angioinvasive pulmonary infection, ulcerative tracheobronchitis, and bronchial anastomosis infection [16] . ct in angioinvasive fungal infection shows ill-defined nodules or mass-like consolidation with surrounding ground-glass opacity (halo sign) indicating hemorrhage [13, 15] ( fig. 19.17a, b) . on ct tracheobronchitis shows diffuse central airway wall thickening and surrounding mediastinal fat stranding. bronchial a b fig. 19.14 (a, b) anastomosis infection can cause bronchial dehiscence and show small foci of extra-luminal air adjacent to the airways. cytomegalovirus (cmv) is the most common opportunistic infection and the second most common cause of pneumonia in lung transplantation patients. cmv pneumonia usually occurs between 1 and 6 months from transplantation [13] . cmv-seronegative recipients who receive cmv-seropositive donor lungs are at increased risk for developing primary infection. ct shows varying patterns including ground-glass opacity, centrilobular nodules with tree-in-bud pattern, consolidation, and interlobular septal thickening [13, 17] (fig. 19.18a, b) . in addition to cmv, other community acquired viruses such as adenovirus, respiratory syncytial virus (rsv), and influenza and parainfluenza viruses may infect lung transplant recipients. viral infections can predispose lung transplant recipients to a b fig. 19.16 (a, b) obliterative bronchiolitis [18, 19] , a manifestation of chronic rejection. chronic rejection is a clinicopathologic syndrome most often characterized by obliterative bronchiolitis, affecting 50% of lung transplant recipients at 5 years [20] , and the term bronchiolitis obliterans syndrome ( bos) is used to describe the clinical situation. it is a major factor limiting the long-term survival in lung transplant recipients. it usually occurs 6 months after transplantation, and risk factors include prior episodes of recurrent acute rejection or infections, particularly cmv pneumonia. obliterative bronchiolitis is the most common histologic pattern seen in bos and manifests as eosinophilic hyaline fibrosis in respiratory bronchiolar walls with luminal occlusion [20] . due to patchy distribution of the disease, transbronchial biopsy may be negative. in such cases diagnosis is made clinically on the basis of otherwise unexplained, persistent decrease in fev1 indicative of small airway disease. pulmonary function tests classically show an obstructive pattern, but combined obstruction and restriction may be seen [21] . high-resolution ct shows bronchial wall thickening, bronchiectasis, and heterogeneous areas of hyperlucent (dark) interspersed with adjacent normal lung parenchyma (mosaic attenuation) that is accentuated in exhalation images. thus, it is very important to obtain paired inspiratory and expiratory hrct images when chronic rejection is suspected clinically. on expiration hrct, normal lung parenchyma becomes greyer, and hyperlucent (pathologic) lung remains dark indicating air trapping (figs. 19.19a, b and 19.20a, b) . thus, the differentiation between normal and abnormal lung becomes more pronounced on expiration ct [13, 22] . organizing pneumonia pattern similar to patients with cryptogenic organizing pneumonia occurs in 10-28% of patients after lung transplantation and may be associated with both acute and chronic rejection [15, 23] or may represent infection. it is characterized by the presence of inflama b fig. 19.19 (a, b) a 33-year-old woman with bolt in 2007 for cystic fibrosis. ct during inspiration (a) and expiration (b) shows patchy areas of air trapping (asterisks in b), consistent with bos a b fig. 19.20 (a, b) a 72-year-old woman with bolt in 2013 for ipf. ct shows bronchiectasis (arrows in a and b) and mosaic attenuation, consistent with obliterative bronchiolitis indicating chronic lung allograft dysfunction of the bos variety matory granulation tissues within the alveoli, alveolar sacs, and ducts. it responds rapidly to high-dose corticosteroids. bronchoscopy is often done to exclude infection before starting on highdose corticosteroids. ct shows peripheral, peribronchovascular, or perilobular consolidation (fig. 19.21) . frequently, peripheral arc-like consolidation with central ground-glass opacity (reverse halo/atoll sign) is seen [15, 24] . post-transplant lymphoproliferative disorder is a spectrum of disease varying from benign lymphoid hyperplasia to high-grade lymphoma. it typically manifests within 1 year after transplantation but can be seen from 1 month to several years after transplantation. ptld risk is increased significantly in epstein-barr virus (ebv) seronegative recipients receiving lungs from seropositive donors. the other major risk factor is augmented immunosuppression. incidence varies between 2.8 and 6% at 1 year after transplantation [25, 26] . it is more common with lung transplantation than with other solid organ transplantation. the majority of cases of ptld is of b cell origin and associated with ebv primary infection. a much smaller proportion of ptlds are of t cell origin, hodgkin's type, or, rarely, plasma cell type such as multiple myeloma. these categories are more likely to be ebv-negative. ebv-associated ptld presents early (within 1 year), usually has a benign course and is intrathoracic, whereas ebv-negative ptld presents late (median onset 50-60 months posttransplant), generally more aggressive and extrathoracic. early disease responds to antiviral therapy and reduction in immunosuppression. late disease generally requires chemotherapy and radiation therapy [25] . ct shows solitary or multiple nodules with peripheral and basal predominant distribution (figs. 19.22a, b and 19.23a, b) . mass-like consolidation and mediastinal and hilar lymphadenopathy are also commonly seen. rarely, interlobular septal thickening can be seen. pleural and chest wall masses with or without pleural effusion and/or pericardial effusion have been described [27, 28] . biopsy is often needed to differentiate ptld from infection [29, 30] . upper lobe fibrosis is uncommon and occurs 1-4 years after transplantation. the exact cause for this is unknown but is described as a component of chronic allograft dysfunction (clad) demonstrating restrictive physiology hypothesized to be a variant of chronic rejection [21, 31] . clinically, patients present with restrictive allograft syndrome (ras), which is marked by restrictive pulmonary function testing and often demonstrates a more aggressive and treatment-refractory course compared to obliterative bronchiolitis. ct shows interlobular septal thickening, traction bronchiectasis, volume loss, and architectural distortion [31] (fig. 19.24a, b) . recurrence of primary disease in transplanted lungs is uncommon with reported incidence of 1% [32] . sarcoidosis is the most common disease recurring after transplantation reported in approximately 35% of patients [32, 33] . other diseases that can recur include lymphangioleiomyomatosis (lam), pulmonary langerhans cell histiocytosis (plch), pulmonary alveolar proteinosis (pap), and multifocal adenocarcinoma (previously bronchioloalveolar carcinoma). recurrence of primary disease has been reported as early as 2 weeks and as late as 2 years after transplantation. the radiologic manifestations are similar to primary disease [32] . bronchoscopy and transbronchial biopsy are frequently performed after lung transplantation to aide in the diagnosis of the various aforementioned complications [34] . transbronchial biopsy itself can lead to various complications including pneumothorax, hemothorax, and pulmonary hemorrhage. solid and cavitary nodules with a b fig. 19.23 (a, b) surrounding ground-glass opacity may be an incidental finding in the allograft up to 1 month as a result of transbronchial biopsy in the corresponding location, and thus such a finding must be interpreted with caution and not confused with a fungal infection [35] . the temporal relationship to biopsy and location of nodules at the known biopsy sites helps distinguish these nodules from infection. a wide spectrum of complications with overlapping radiologic findings may occur after lung transplantation. familiarity with radiologic appearance of these complications in correlation with clinical manifestations and time course since transplantation is very helpful in the management of these patients. the threshold for performing ct should be low to detect infections in this immunocompromised population at an early stage. ct protocols should be tailored to suspected complications such as performing inspiration and expiration ct when chronic allograft dysfunction is suspected and performing ct angiogram when vascular complications are suspected. accurate tailoring of ct protocols increases the yield of imaging and is useful in appropriate retrieval of specimens/samples from abnormal airways and pulmonary parenchyma in identifying the specific pathology. follow-up imaging is essential and is guided by patient's clinical course and pulmonary function tests. the registry of the international society for heart and lung transplantation: thirty-first adult lung and heart-lung transplant report--2014; focus theme: retransplantation donor criteria and evaluation single lung transplantation followed by contralateral bullectomy for bullous emphysema hyperacute rejection following lung transplantation reperfusion edema after lung transplantation: radiographic manifestations pleural complications in lung transplant recipients acute and chronic pleural complications in lung transplantation pleural space complications associated with lung transplantation acute pulmonary allograft rejection. mechanisms, diagnosis, and management the registry of the international society for heart and lung transplantation: twentysixth official adult lung and heart-lung transplantation report-2009 acute and chronic rejection after lung transplantation airway complications after lung transplantation: a review of 151 anastomoses imaging of lung transplantation: review vascular complications of lung transplantation postoperative complications of lung transplantation: radiologic findings along a time continuum infections after lung transplantation ct findings of pneumonia after lung transplantation cytomegalovirus replication within the lung allograft is associated with bronchiolitis obliterans syndrome imaging of the chest after lung transplantation bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria a new classification system for chronic lung allograft dysfunction early bronchiolitis obliterans following lung transplantation: accuracy of expiratory thin-section ct for diagnosis bronchiolitis obliterans organizing pneumonia (boop) in lung transplant recipients bronchiolitis obliterans with organizing pneumonia versus chronic eosinophilic pneumonia: high-resolution ct findings in 81 patients posttransplant lymphoproliferative disorder: incidence, presentation, and response to treatment in lung transplant recipients lymphoproliferative disease after lung transplantation: comparison of presentation and outcome of early and late cases intrathoracic lymphoproliferative disorders in the immunocompromised patient: ct findings post-transplant lymphoproliferative disorder: intrathoracic manifestations post-transplantation lymphoproliferative disorder: the spectrum of imaging appearances lymphoproliferative disorders after lung transplantation: imaging features fibrosis of the upper lobes: a newly identified late-onset complication after lung transplantation? frequency and ct findings of recurrent disease after lung transplantation sarcoidosis: recurrence of primary disease in transplanted lungs prospective study of the value of transbronchial lung biopsy after lung transplantation transplanted lungs: nodules following transbronchial biopsy key: cord-023509-tvqpv6fp authors: corrin, bryan; nicholson, andrew g. title: occupational, environmental and iatrogenic lung disease date: 2011-03-02 journal: pathology of the lungs doi: 10.1016/b978-0-7020-3369-8.00007-0 sha: doc_id: 23509 cord_uid: tvqpv6fp nan in practice the term is confined to the effects of mineral dust on the lungs. diseases caused by organic dusts are not included among the pneumoconioses and, in medicolegal practice at least, the presence of dust alone is insufficient to indicate pneumoconiosis: for compensation to be considered, the mineral dust must alter the structure of the lung and cause disability. the british industrial injuries advisory council defined pneumoconiosis as 'permanent alteration of lung structure due to the inhalation of mineral dust and the tissue reactions of the lung to its presence, excluding bronchitis and emphysema' . 4 parkes recommends that cancer and asthma caused by mineral dust should also be excluded from the definition, an opinion with which we concur. 5 to reach the lung, dust particles have to be very small. particle density and shape also affect the aerodynamic properties of dust. host factors such as airflow characteristics, airway branching patterns and airway disease also affect dust deposition. three deposition mechanisms are recognised ( fig. 7.1.1 ): 1. inertial impaction: when air streams change direction or velocity, the inertia of the entrained particles causes them to maintain their original direction for a distance that depends upon their density and the square of their diameter. the same rules govern a car approaching a bend too fast: the car crashes into the outside of the bend. 2. sedimentation (gravitational settlement): under the influence of gravity, particles settle with a speed that is proportional to their density and the square of their diameter. 3. diffusion: very small airborne particles acquire a random motion as a result of bombardment by the surrounding gas molecules. inhaled dust particles are liable to sediment out in the alveoli if they have a diameter in the range of 1-5 µm, are roughly spherical in shape, and in density approximate to that of water. larger or denser particles impact or precipitate on the walls of the conductive airways and are rapidly removed by ciliary action. smaller particles may reach the alveoli but do not sediment so readily and many are therefore exhaled. very small particles are deposited on the walls of alveoli by diffusion but because they are so small the total amount of dust deposited in this way is insignificant compared with that deposited by sedimentation ( fig. 7.1. 2). direct measurement shows that most lung dust (96%) has a particle diameter less than 2.5 µm. 6 fibrous dust particles behave differently. fibres over 100 µm in length may reach the alveoli if they are very thin and remain aligned with the air stream. fibre penetration is inversely related to path length and the number of bifurcations. 7 tall people have longer conductive airways and experience more deposition in these sites than short people who have greater alveolar deposition for the same level of exposure. 8 slightly more dust is deposited in the right lung than the left, probably because the right main bronchus is more in line with the trachea, and is broader and shorter than the left, and carries 55% of the inhaled air. 9, 10 dust clearance from the lung inhaled dust that settles in the conductive airways is removed within a day or two by ciliary action. only dust that reaches the alveoli is liable to cause pneumoconiosis and much of this is also removed, but the clearance rate here is much slower: many coalminers continue to expectorate mine dust years after retirement. alveolar clearance is gravity largely effected by macrophages, principally via the airways to the pharynx but also via lymphatics to the regional lymph nodes. the airway and interstitial routes interconnect at the bronchiolar level 11 where some dust-laden macrophages leave the interstitium for the air space. 12 this interconnection is probably the route utilised by circulating macrophages clearing other parts of the body of endogenous or exogenous particulate matter via the lung. 13 long asbestos fibres present a particular problem to macrophage clearance. some minerals, notably chrysotile asbestos, undergo slow physicochemical dissolution in the lungs. only a small fraction of the inhaled dust gains access to the interstitium, a necessary step if it is to cause pneumoconiosis. some free dust enters through the bronchus-associated lymphoid tissue 11, 12 and some is taken up by, or pierces, the alveolar epithelium ( fig. 1.34 , p. 21). [14] [15] [16] some of this is transported within hours to the hilar lymph nodes. 17 so rapid is this translocation that it is thought not to involve most, the lesions are more numerous and better developed in the upper lobes than the bases but the reverse is true of asbestosis. the reasons for this are complex but undoubtedly involve the dust deposition:clearance ratio for the effect of the dust will depend upon both its amount and the duration of its stay in the lungs. there are well-recognised regional differences in the distribution and clearance of inhaled material, which in turn are dependent upon man's upright posture, the consequent gravitational forces being maximal at the apices. 35 when standing at rest, the apices of the lungs are hardly perfused, so that lymph formation and clearance are much better at the bases. [36] [37] [38] similarly, the apices are relatively less well aerated; alveoli in the lower lobes receive more air than those in the upper lobes. 37, 39 the greater respiratory excursions at the bases are thought to promote macrophage mobility there. it is to be expected therefore that the bases would both receive and clear more dust than the apices, rendering it difficult to predict on theoretical grounds which parts of the lungs carry the heaviest dust burden. in fact, more dust of all types is found in the upper lobes, the part most severely affected by every type of pneumoconiosis except asbestosis. 40, 41 the predilection of asbestos to affect the periphery of the lower lobes is attributed to the dangerous long asbestos fibres preponderating there. 41, 42 pulmonary reactions to mineral dust the main tissue reaction to mineral dust is fibrosis. silica is highly fibrogenic and is therefore very likely to cause pneumoconiosis. carbon is non-fibrogenic and therefore, unless there are complications, coal pneumoconiosis causes little disability. tin too is harmless, and stannosis therefore unimportant, although the chest radiograph is highly abnormal because tin is very radiopaque. stannosis is one of several terms that specify pneumoconiosis due to a particular mineral, the best known being silicosis, asbestosis and anthracosis. the blackness of carbon and red-brown colour of iron give ample evidence, both naked-eye and microscopically, of the type and amount of these dusts when they are present in the lung ( fig. 7.1.3 ), but other inorganic dusts may be more difficult to identify. however, a flick-out substage condenser and polaroid filters to test for refractility and birefringence respectively are useful adjuncts that are too often neglected by the histopathologist. crystalline silica is traditionally regarded as being only weakly birefringent, in contrast to silicates which generally show up brightly with simple crossed polaroid filters. 43 however, with modern microscope lamps, if the light source is set at high intensity when using polaroid filters, both silica and silicates are birefringent. 44 mineralogists use polarising microscopy for analysis, but only by studying large polished crystals with controlled orientation of the light. the small dust particles found in tissue sections are too small to permit analysis by this technique but it is nevertheless very useful for detecting their presence ( fig. 7.1.4) . particle shape gives a useful indication of mineral type but appearances are sometimes deceptive: the plate-like crystals of talc are seldom observed as such, usually being viewed edge-on, when they appear to be needle-shaped. occasionally, stains can be used to identify minerals, e.g. a modified perls' reaction for inhaled iron, and irwin's aluminon stain for aluminium, but these too have largely been replaced by modern analytical techniques. ultrafine dust particles are particularly liable to be transported across the alveolar epithelium. 11 the integrity of the alveolar epithelium is very important to dust translocation from the air spaces to the interstitium. much more dust reaches the interstitium if the epithelium is damaged. 18, 19 it is widely thought that macrophages that have left the interstitium for the alveolar space never return, 17, 20 but this is probably untrue. 21 heavily laden macrophages accumulate in alveoli bordering the terminal and respiratory bronchioles, eventually filling them completely. erosion of the alveolar epithelium permits re-entry of these macrophages into the interstitium, 22 very close to foci of bronchial mucosaassociated lymphoid tissue (malt), which are found near the terminal bronchioles. 23 these aggregates guard the mouths of lymphatics, which commence at this point; alveoli are devoid of lymphatics. dustladen interstitial macrophages accumulate in and around the bronchial malt, which macklin therefore referred to as dust sumps. 24 most pneumoconiotic lesions are found in the region of the dust sumps and are therefore focal. asbestosis is diffuse rather than focal because the long asbestos fibres are not readily mobilised and cannot be concentrated in the centriacinar dust sumps. this is also seen on occasion with platy non-fibrous dusts such as talc, mica, kaolinite and feldspar. [25] [26] [27] [28] [29] [30] [31] [32] [33] within the dust sumps the dust particles are not static. they are constantly being freed and reingested by interstitial macrophages and, because these cells are mobile, successively inhaled dusts soon become intimately mixed. 34 macrophages play an important role in pneumoconiosis and if the dust is fibrogenic the repeated phagocytosis of indestructible mineral particles results in constant fibroblast stimulation. the zonal distribution of pneumoconiosis pneumoconiosis affects both lungs but seldom evenly and some pneumoconioses show characteristic patterns of lung involvement. in microincineration combined with dark-field microscopy can also be used to demonstrate small particles. incombustible mineral particles that cannot be seen with bright-field or polarising microscopy are rendered visible by this technique and their position on the slide can be compared with tissue reactions evident in a serial section that has not been incinerated. microincineration has, however, also been largely replaced by modern analytical techniques that will now be considered. analytical electron microscopy is very helpful in identifying minerals, whether applied to lung digests or tissue sections. [45] [46] [47] [48] scanning electron microscopy permits the examination of thicker sections than transmission electron microscopy but does not detect very small particles. however, scanning electron microscopy allows more tissue to be examined and avoids the difficulty of cutting mineral particles with an ultramicrotome. mineral particles in a 5-µm thick deparaffinised section can be recognised in a scanning electron microscope set to collect the back-scattered electrons. 46 the instrument can then be focused on points of potential interest and switched to x-ray diffraction, which provides information on crystal structure (fig. 7.1.5) . alternatively, elemental analysis may be undertaken with either energy-dispersive or wavelength-dispersive x-ray spectroscopy. with energy-dispersive x-ray spectroscopy, all elements of atomic number above 11 are identified, whilst with wavelength-dispersive x-ray spectroscopy the section can be scanned for one particular element. with the former technique different elements are shown graphically as individual peaks, the heights of which are proportional to the amounts of the different elements within the particle studied, thereby giving information on probable molecular formula ( fig. 7.1.6) . thus, different silicates can be distinguished from each other and also from silica, which registers as pure silicon, oxygen (atomic number 8) not being detected. the fact that the elements of low atomic number that constitute organic chemicals are not detected means that any minerals present (except beryllium, atomic number 4) can be recognised easily in tissue sections. only particles can be analysed however: elements present in only molecular amounts cannot be detected by x-ray analysis. the detection of trace amounts of substances such as beryllium requires bulk chemical analysis or techniques that are not widely available such as atomic absorption spectrometry, neutron activation analysis and microprobe mass spectrometry. 49, 50 the last of these techniques can also provide molecular (as opposed to elemental) analysis of organic as well as inorganic particles. 51 another analytical technique of interest is microscopic infrared spectroscopy which provides data on the compound nature of microscopic particles in tissue sections ( fig. 7.1.7) . micro-raman spectroscopy is also useful in this respect. some metals cause hypersensitivity, which can be identified by exposing the patient's lymphocytes to metals and measuring their reaction in vitro. 52 radiological grading of pneumoconiosis a scheme for grading pneumoconiosis radiologically by comparison with standard radiographs has been adopted by the international labour organisation (ilo) and is widely used. 53 small opacities (up to 1 cm diameter) are graded by their profusion, 1, 2 and 3 indicating increasing numbers, and by their size, increasing through p, q and r if rounded and s, t and u if irregular. type p opacities are described as punctiform and measure up to 1.5 mm in diameter; larger lesions up to 3 mm in diameter (type q) are described as micronodular or miliary; and those over 3 mm and up to 1 cm in diameter (type r) are described as nodular. irregular opacities cannot be sized so accurately, s, t and u indicating fine, medium and coarse respectively. large opacities (over 1 cm diameter) are graded by their combined size, increasing through a, an opacity measuring between 1 and 5 cm in diameter; b, one or more opacities whose combined area does not exceed the equivalent of one-third of the area of the right lung field (when they are regrouped in the mind's eye or measured with a transparent ruler); and c, one or more opacities whose combined area exceeds one-third of the area of the right lung field (when similarly regrouped). in coalworkers, small opacities (up to 1 cm diameter) correspond to simple coalworker's pneumoconiosis and large opacities (over 1 cm diameter) to complicated coalworker's pneumoconiosis, which is also known as progressive massive fibrosis. silicotic lesions have been identified in the lungs of egyptian mummies, and the injurious effects on the lungs of inhaling mine dust have been recognised for more than 400 years. as long ago as the sixteenth century in joachimsthal, bohemia (now jachymov, czech republic), diseases of miners' lungs were attributed to the dust the miners breathed. silicosis, tuberculosis and lung cancer are all now known to have been prevalent among the miners in this region, the cancer being largely attributable to the high level of radioactivity in the mines. silicosis was recognised in the uk soon after the discovery in 1720 that the addition of calcined flint to the clay from which china is made produced a finer, whiter and tougher ware. the preparation and use of this flint powder were highly dangerous, causing the condition known as potter's rot, one of the first of the many trade names by which silicosis has since been known. aluminium oxide (alumina) now provides a safe, effective substitute for flint in this industry. in 1830 it was noted that sheffield fork grinders who used a dry grindstone died early, and amongst other preventive measures it was recommended that the occupation should be confined to criminals: fortunately for them, the substitution of carborundum (silicon carbide) for sandstone was effective enough. however, silicosis still occurs in some miners, tunnellers, quarrymen, stone dressers and metal workers. silica in one form or another is used in many trades -in the manufacture of glass and pottery, in the moulds used in iron foundries, as an abrasive in grinding and sandblasting, and as a furnace lining that is refractory to high temperatures. rocks such as granite and sandstone are siliceous and their dusts are encountered in many mining and quarrying operations. in coal mining in the uk the highest incidence of the disease was in pits where the thinness of the coal seams required the removal of a large amount of siliceous rock, a process known as 'hard heading' . in south africa, silicosis causes a high mortality among the gold miners on the witwatersrand, where the metallic ore is embedded in quartz. slate is a metamorphic rock that contains both silica and silicates, and slateworkers develop both silicosis and mixed-dust pneumoconiosis. 55, 56 nor are rural industries immune from the disease, particularly if ventilation is inadequate, as it is in certain african huts where stone implements are used to pound meal and the occupants develop mixed-dust pneumoconiosis. 57 silicosis and mixed-dust pneumoconiosis have also been reported in dental technicians. 58 desert sand is practically pure silica but the particles are generally too large to reach the lungs. however, silicosis has been reported in inhabitants of the sahara, libyan and negev deserts and those living in windy valleys high in the himalayan mountains, [59] [60] [61] [62] [63] [64] [65] whilst in california the inhalation of dust raised from earth has led to silicate pneumoconiosis in farm workers, 66 horses 67 and a variety of zoo animals. 68 the silica in rocks such as granite, slate and sandstone is largely in the form of quartz and this is therefore the type of silica encountered in most of the industries considered above. cristobalite and tridymite, which are possibly even more fibrogenic than quartz, are more likely to be encountered in the ceramic, refractory and diatomaceous earth industries where processing involves high temperatures. many workers with silicosis are asymptomatic. as a general rule, exposure to silica dust extends over many years, often 20 or more, before the symptoms of silicosis first appear: by the time the disease becomes overt clinically, much irreparable damage has been inflicted on the lungs. the initial symptoms are cough and breathlessness. from then onwards, respiratory disability progresses, even if the patient is no longer exposed to silica dust. ultimately, there may be distressing dyspnoea with even the slightest exercise. silicosis sometimes develops more rapidly, perhaps within a year or so of first exposure. such 'acute silicosis' was observed in the scouring powder industry in the 1930s when these cleansing agents consisted of ground sandstone mixed with a little soap and washing soda. 69, 70 the additives were considered to have rendered the silica in the sandstone more dangerous but it is possible that the rapidity of onset of the disease merely reflected the intensity of the dust cloud to which the packers were exposed. confusingly, the term 'acute silicosis' has since been applied to a further effect of heavy dust exposure in tunnellers, sand blasters and silica flour workers, namely pulmonary alveolar lipoproteinosis (see below), 71, 72 whilst the terms 'accelerated silicosis' or 'cellular phase silicosis' have been substituted for 'acute silicosis' in referring to the rapid development of early cellular lesions. 43, 73 the time from first exposure to the development of symptoms (the latency period) is inversely proportional to the exposure level. however, it is evident that a certain amount of silica can be tolerated in the lungs without fibrosis developing, indicating either a time factor in the pathogenetic process or a threshold dust load that has to be reached before fibrosis develops. silica particles that are roughly spherical in shape and of a diameter in the range of 1-5 µm sediment out in the alveoli and are concentrated within macrophages at macklin's dust sumps, as explained previously (see p. 27). early lesions, as seen in so-called accelerated or cellular phase silicosis, consist of collections of macrophages separated by only an occasional wisp of collagen. the early lesions have been likened to granulomas and on occasion have been mistaken for langerhans cell histiocytosis or a storage disorder, but langerhans cells are scanty and the histiocytes contain dust particles rather than accumulated lipid or polysaccharide. the macrophages of the early lesion are gradually replaced by fibroblasts and collagen is laid down in a characteristic pattern. the mature silicotic nodule is largely acellular and consists of hyaline collagen arranged in a whorled pattern, the whole lesion being well demarcated ( fig. 7.1.8 ) and sometimes calcified. small numbers of birefringent crystals are generally evident within the nodules when polarising filters are used, but these mainly represent silicates such as mica and talc, inhaled with the silica. silica particles are generally considered to be only weakly birefringent, 43 but fairly strong birefringence is evident in strong light (see above). 44 silicotic nodules develop first in the hilar lymph nodes and are generally better developed there than in the lungs. [74] [75] [76] indeed, silicotic nodules are occasionally found in the hilar lymph nodes of persons who have no occupational history of exposure to silica and whose lungs are free of such lesions, the silica in the nodes being presumed to represent inhaled particles derived from quartz-rich soil. 77 severely affected lymph nodes often calcify peripherally, giving a characteristic eggshell-like radiographic pattern. this is sometimes the only radiological abnormality. 76 such enlarged lymph nodes may occasionally press upon and obstruct adjacent large bronchi 78 or result in a left recurrent laryngeal nerve palsy, 79 so simulating malignancy. 80 sometimes the nodules develop within the walls of major bronchi, occasionally causing a middle-lobe syndrome (see p. 92). 81 silicotic nodules are also found along the lines of the pleural lymphatics 75, 82 where they have been likened to drops of candle wax on the visceral pleura. very rarely, silica-induced fibrosis is more pronounced in the pleura than in the lungs. 83 lung tissue between the nodules is often quite normal and not until the process is very advanced is there any disability ( fig. 7. 1.9b). in severe cases large masses of fibrous tissue are formed, which may undergo central necrosis and cavitation ( fig. 7.1.10 ). 84 on close inspection it is evident that these consist of conglomerations of many silicotic nodules closely packed together. in such severe cases cor pulmonale develops. occasionally, silicotic nodules develop in the abdominal as well as the thoracic lymph nodes, and in the liver, spleen, peritoneum and bone marrow. [85] [86] [87] [88] [89] in about 10% of cases, the typical pulmonary nodules that predominantly affect the upper lobes are accompanied by diffuse fibrosis that is maximal in the lower lobes. 27, 33, [90] [91] [92] the latter may show 'honeycombing' and closely resemble idiopathic pulmonary fibrosis. the association is too common to be explained by chance and the diffuse fibrosis is therefore regarded as a further manifestation of the pneumoconiosis, possibly due to an interaction between the dust and the immunological factors discussed below. the pathogenesis of silicosis has excited much interest and many different theories have been advanced over the years. an early theory held that the hardness of the silica was responsible, but this was discounted by the observation that silicon carbide (carborundum) is harder than silica but is non-fibrogenic. theories based on the piezoelectric property and on the solubility of silica were successively abandoned although the latter had a long period of popularity. it gained support from kettle's experiments which showed that fibrosis developed about chambers placed in an animal's peritoneal cavity if the chambers contained silica powder sealed in by a collodion membrane through which solutes such as silicic acid could pass. however, it was later shown that the pores in a collodion membrane are quite irregular in size and when the experiments were repeated using chambers guarded by millipore membranes, no fibrosis developed, despite solutes being able to diffuse out. 93 the solubility theory also fails to take account of the differing fibrogenicity of the various forms of silica despite them being of similar solubility. 54 furthermore, if the outer, more soluble layer of the particles is removed by etching, fibrogenicity is increased although solubility is decreased. in line with this, freshly fractured crystalline silica is more pathogenic in every respect than its aged equivalent, 94 which may partly explain the severity of silicosis in trades such as sandblasting. these observations suggest that the fibrogenicity of silica is connected with its surface configuration. it is now known that uptake of the silica by macrophages is necessary for silicosis to develop. if silica and macrophages are enclosed together in peritoneal millipore chambers, a soluble product of the macrophages diffuses out and causes fibrosis. this observation led to the realisation that the fibrogenicity of the various crystalline forms of silica correlated well with their toxicity to macrophages and for a time macrophage death was thought to be necessary. 95 it is now considered that before the macrophages are killed by the ingested silica, they are stimulated to secrete factors that both damage other con stituents of the lung and promote fibrosis. [96] [97] [98] [99] [100] [101] [102] transforming growth factor-β is one fibrogenic factor that has been implicated in the pathogenesis of silicosis. [103] [104] [105] toxic damage to macrophages is due to silica particles injuring the phagolysosomal membranes, so releasing acid hydrolases into the cytoplasm. 95 it is important in the pathogenesis of the disease indirectly because when the macrophage crumbles, the silica particles are taken up by fresh macrophages and the fibrogenic process continues. it has been suggested that early involvement of the hilar lymph nodes in the fibrogenic process promotes the development of the disease in the lung by delaying dust clearance. 74 immunological factors have been implicated in the pathogenesis of silicosis because many patients with silicosis have polyclonal hypergammaglobulinaemia, rheumatoid factor or antinuclear antibodies, and because there is a well-recognised association between autoimmune diseases such as systemic sclerosis and rheumatoid disease and exposure to silica. 43, [106] [107] [108] [109] the relation of immunity to dust exposure appears to be a reciprocal one: on the one hand, the presence of dust results in rheumatoid lesions in the lungs being more florid (see caplan's syndrome, p. 341), whilst on the other, non-specific immunisation of rabbits with horse serum results in experimental silicotic lesions being larger and more collagenous. 110 it is doubtful whether pneumoconiosis and autoimmune disease play a causative role in each other but one seems to aggravate the other and may lead to its earlier development. one of the commonest and most feared complications of silicosis is chronic respiratory tuberculosis. 109 once this infection has been added to the silicosis, the prognosis rapidly worsens. it is thought that in the presence of silica, the tubercle bacilli proliferate more rapidly because the ingested silica particles damage phagolysosomal membranes and thereby interfere with the defensive activity of the macrophages. the synergistic action of silica dust has long been held responsible for the inordinately high incidence of respiratory tuberculosis in mining communities. many former south african gold miners now have acquired immunodeficiency syndrome (aids) as well as silicosis and tuberculosis has consequently reached almost epidemic proportions amongst these men. phagocyte damage by ingested dust particles may also cause some cases of chronic necrotising aspergillosis complicating pneumoconiosis. 111 a series of studies suggesting that there might be a link between silica inhalation and lung cancer was reviewed by the international agency for research on cancer in 1987, leading to the conclusion that the evidence for carcinogenicity of crystalline silica in experimental animals was sufficient, while in humans it was limited. 112 subsequent epidemiological publications were reviewed in 1996, when it was concluded that the epidemiological evidence linking exposure to silica to the risk of lung cancer had become somewhat stronger 113 but that in the absence of lung fibrosis remained scanty. 113 the pathological evidence in humans is also weak in that premalignant changes around silicotic nodules are seldom evident. 114 nevertheless, on this rather insubstantial evidence, lung cancer in the presence of silicosis (but not coal or mixed-dust pneumoconiosis) has been accepted as a prescribed industrial disease in the uk since 1992. 115 some subsequent studies have provided support for this decision. 116 in contrast to the sparse data on classic silicosis, the evidence linking carcinoma of the lung to the rare diffuse pattern of fibrosis attributed to silica and mixed dusts is much stronger and appears incontrovertible. 33, 92 alveolar lipoproteinosis in response to heavy dust exposure a further complication of exposure to silica is the development of alveolar lipoproteinosis (see p. 317). 71, 72, 117, 118 very heavy experimental exposure to silica, and indeed other dusts, stimulates hypersecretion of alveolar surfactant to such an extent that the normal clearance mechanism is overwhelmed. [119] [120] [121] [122] [123] [124] [125] alveolar macrophages are enlarged by numerous phagolysosomes distended by lamellar bodies that represent ingested surfactant. the alveoli are filled by such cells and, having a foamy cytoplasm, they produce the appearances of endogenous lipid pneumonia, similar to that more usually encountered as part of an obstructive pneumonitis distal to a bronchial tumour. the macrophages gradually disintegrate and the free denatured surfactant slowly becomes compacted, during which time its staining with both eosin and the periodic acid-schiff reagents intensifies until the appearances are finally those of alveolar lipoproteinosis. this process prevents the aggregation and concentration of the dust in the usual foci and thereby hinders the development of silicosis. lipoproteinosis and silicosis may be seen in conjunction but, more often, different areas of the lung show one or the other. the lipoproteinosis has its own severe impact on lung function, but, unlike silicosis, is potentially reversible (by massive alveolar lavage). occasional patients exposed to silica develop renal disease. [126] [127] [128] [129] two mechanisms appear to operate. first, translocation of silica particles from the lungs leads to their deposition in the renal interstitium with resultant nephrotoxity. second, silica stimulates an autoimmune response characterised by the formation of various antibodies, notably rheumatoid factor and antinuclear antibodies, which leads to the development of immune complex-mediated glomerulonephritis. 128, 129 amorphous silica manmade submicron forms of silica, variously known as amorphous, vitreous, colloidal, synthetic or precipitated silica, are widely used in industry. they consist of pure non-crystalline silicon dioxide. particle size ranges from 5 to 200 nm but aggregates of the particles measure from 1 to 10 µm. industrial surveys suggest that inhalation of such dust is harmless, observations that are in accord with the results of animal experiments. 54 an amorphous silica is the principal component of the fossilised remains of diatoms that constitute the sedimentary rock, diatomite ( fig. 7.1.11 ). this is generally obtained by open-cast mining, following which the rock is crushed and calcined. the calcined product is used in filters, insulation material and as a filler. being amorphous, the silica in diatomite is harmless, but calcining (>1000°c) results in its conversion to crystalline forms of silica. diatomaceous earth pneumoconiosis is unusual and its risk appears to be related to the amount of cristobalite and tridymite (two forms of crystalline silica) produced in the calcining process. 130 the silicates are complex compounds in which silicon and oxygen form an anion combined with cations such as aluminium and magnesium: talc, for example, is a hydrated magnesium silicate with the formula mg 3 si 4 o 10 (oh) 2 . silicates include fibrous forms (asbestos and the zeolites), plate-like forms (talc and mica) and clays (kaolinite and fuller's earth). in histological sections, the platy talc and mica particles are generally cut tangentially and therefore appear needleshaped (see fig. 7 .1.4). they are strongly birefringent whereas the clays are only weakly so. talc particles in the lung exceeding 5 µm in length should arouse suspicion of intravenous drug abuse. 131 of the fibrous silicates, zeolite is used as a building material in certain communities, notably in central turkey. pneumoconiosis is not a problem but zeolites are of medical interest because, like asbestos, they present a mesothelioma risk. asbestos is dealt with separately (see below). pneumoconiosis has been described with various non-fibrous silicates, notably in the rubber industry, which uses talc and, less commonly, mica as lubricants. other occupations posing a risk include the extraction of kaolinite from china clay (kaolin), 27, 132, 133 and in the open-cast and underground mining of fuller's earth (montmorillonite, bentonite and attapulgite clays, which were originally used in 'fulling' (degreasing) wool). 134, 135 however, all these substances are commonly contaminated with silica, asbestos or both, and it has been questioned whether in pure state they are at all fibrogenic. the modifying effect of inert substances such as iron on that of silica is well known (see mixed-dust pneumoconiosis, below) and it has been suggested that talc, mica and fuller's earth act in a similar way in regard to their more fibrogenic contaminants, the pneumoconioses attributed to them in reality representing mixed-dust pneumoconiosis or asbestosis. contrary evidence comes from reports of pulmonary fibrosis in persons heavily exposed to pure talc, mica or kaolin. 32 all these silicates are evident in the tissues as plate-like birefringent crystals which often provoke a foreign-body giant cell reaction (see fig. 7 .1.3) and may result in fibrotic nodules. large focal lesions resembling the progressive massive fibrosis of coalworkers may be produced, and also a diffuse 'asbestosis-like' form of pneumoconiosis, the latter attributed to poor macrophage mobilisation of the plate-like particles. [27] [28] [29] [30] [31] [32] [132] [133] [134] [135] [136] [137] [138] it would appear therefore that silicates are indeed fibrogenic if enough is inhaled; they appear to vary in fibrogenicity but in all cases they are less fibrogenic than silica. inert dusts are non-fibrogenic and therefore of little clinical consequence, although elements of high atomic number can give rise to a striking chest radiograph. 139 it should be noted however that inert or lowly fibrogenic materials may be associated with substances of medical importance, for example, kaolin, bentonite and barytes (barite) may all be contaminated with silica 27, 134, 140 and talc may be contaminated with asbestos. the best known of the inhaled inert mineral dusts is carbon while, of the remainder, iron is the most widespread. others include tin and barium. with all these dusts, particles retained in the lung are gathered at macklin's dust sumps by heavily laden macrophages which are lightly bound together there by a few reticulin fibres. collagen is not formed and the worker suffers no ill-effects. the lungs take on the colour of the dust and in siderosis assume a deep brick-red hue. carbon deposition is commonly found in the lungs, particularly those of city dwellers and tobacco smokers. it is also the principal constituent of coal, which is dealt with separately below, and large amounts of pure carbon may be inhaled by workers involved in the manufacture of carbon black, carbon electrodes and charcoal. [141] [142] [143] [144] although carbon is regarded as being non-fibrogenic, the very heavy lung burdens encountered in industries such as these may lead to the complicated form of pneumonconiosis known as progressive massive fibrosis that is more commonly encountered in coal workers (see p. 340). heavy pure carbon deposition may also be acquired domestically when wood is burnt in buildings devoid of a chimney, so-called 'hut lung', 145 a term that is also applied to the domestic acquisition of carbon mixed with silica or silicates, resulting in forms of mixeddust pneumoconiosis. 57, 64, 65 anthracofibrosis is a term introduced by chinese bronchoscopists for bronchial stenosis or obliteration associated with carbon pigmentation of the mucosa. 146 although the original description incriminated tuberculosis, mixtures of various mineral dusts acquired at work or domestically are a more likely cause. [147] [148] [149] [150] iron dust in the lungs was first described by zenker in 1867, when he also introduced the terms siderosis and pneumonokoniosis. 3 zenker was describing a woman who coloured paper with iron oxide powder ('rouge'), a substance which is still encountered by some workers engaged in polishing silver, glass, stone and cutlery. siderosis is also found in welders, iron foundry fettlers, steel workers, boiler scalers and haematite miners and crushers. iron dust particles are reddish-brown but in the lung may be masked by carbon 151 : when evident, or revealed by microincineration, they resemble haemosiderin and generally give a positive perls' reaction, but particularly with haematite, heat (60-80 o c) and concentrated (12n) hydrochloric acid may be necessary. 152 haematite miners in both the uk (cumbria) and france (lorraine) have an increased risk of bronchial carcinoma, but radon gas rather than haematite is the suspected carcinogen. radon is a decay product of uranium. minute amounts are present in all rocks but local concentrations occur and these are liable to build up in mines if ventilation is limited. silver, as well as iron, is found in the lungs of silver polishers, where it stains elastin in alveolar walls and pulmonary vessels grey. such argyrosiderosis is as harmless as siderosis. tin miners are subject to silicosis but not stannosis because the ore, which is found in association with siliceous rocks, contains only low concentrations of the metal. tin smelters, on the other hand, and factory workers exposed to high concentrations of tin dust or fume, are liable to inhale large amounts of this inert metal and develop the striking chest radiograph of stannosis. they remain in good health however for tin is completely non-fibrogenic. tin particles in the lung resemble carbon but are strongly birefringent and remain after microincineration: microprobe analysis provides positive identification. other inert dusts include barium, which also has a high atomic number and is therefore radiopaque, 139 and minerals of low radiodensity such as limestone, marble and cement (all chiefly composed of calcium carbonate) and gypsum (hydrated calcium sulphate). however, the extraction of barium ore (almost entirely in the form of barium sulphate, which is known as barytes in europe and barite in the usa) may entail exposure to silica and silicates. pure baritosis resembles stannosis and siderosis. the term 'mixed-dust pneumoconiosis' refers to the changes brought about by inhaling a mixture of silica and some other less fibrogenic substance such as iron, carbon, kaolin or mica. 33, 151, [153] [154] [155] the proportion of silica is usually less than 10%. typical occupations include foundry work and welding and the mining of coal, haematite, slate, shale and china clay. the action of the silica is modified and, although fibrotic nodules are formed, they lack the well-demarcated outline and concentric pattern of classic silicosis. the lesions are found in a centriacinar position and are stellate in outline with adjacent scar emphysema. they are firm and generally measure no more than 5 mm in diameter. they closely resemble the fibrotic nodules of simple coal pneumoconiosis (see below). confluent lesions also occur on occasions. these resemble the progressive massive fibrosis of coalworkers and appear to represent a single large lesion rather than a conglomeration of individual nodules, as in advanced silicosis. abundant dust is generally evident in lesions of all sizes; this consists of black carbon or brown iron mixed with crystals of varying degrees of birefringence, silicates generally being strongly birefringent and silica weakly so. calcification is unusual. mixed-dust pneumoconiosis carries an increased risk of pulmonary tuberculosis, but not to the same degree as silicosis. in some cases the stellate nodules are accompanied by diffuse fibrosis, as in silicosis and again possibly involving interactions between the dust and immunological factors. involvement of the bronchi with consequent stenosis (so-called anthracofibrosis) is described above. the term 'anthracosis' was initially applied to changes observed in a coalminer's lung 157 but is now often extended to include the common carbon pigmentation of city dwellers' lungs, and the term 'coal pneumoconiosis' is more appropriate to a special form of pneumoconiosis to which coalworkers are subject, particularly those who work underground. the principal constituent of coal, carbon, is non-fibrogenic, so suspicion has naturally fallen on the ash content of mine dust, some of which derives from the coal, some from adjacent rock strata and some from stone dust laid in the roadways to minimise the risk of coal dust explosions. coal itself appears to be the responsible agent because coal-trimmers, working in the docks and not exposed to rock dust, also develop the disease. 158 coalminers encountering siliceous rock are, of course, also liable to develop silicosis like other underground workers. coal consists largely of elemental carbon, oxygen and hydrogen with traces of iron ore and clays such kaolinite, muscovite and illite, but no silica. the mineral content varies with the type and rank (calorific value) of the coal. all coal derives from peat, the youngest type being lignite and the oldest anthracite, with bituminous (house) coal in between. as it ages, the oxygen and mineral constituents diminish and the coal hardens. lignite is soft and said to be of low rank, anthracite hard and of high rank, with bituminous coal intermediate. although high-rank coal is of low mineral content, its dust is more toxic to macrophages in vitro and is cleared more slowly in vivo. this observation may explain why, in the uk, high-rank coal is associated with a higher prevalence of coal pneumoconiosis. the low mineral content of high-rank coal is reflected in the mineral content of the lungs of those who hew such coal in the uk, but in the ruhr, in germany, and in pennsylvania, in the usa, anthracite miners' lungs contain more silica than those who hew bituminous coal, the silica presumably deriving from other sources. not surprisingly, the presence of silica is reflected in the tissue reaction to the inhaled dust, resulting in a more fibrotic reaction very analogous to mixed-dust pneumoconiosis. a spectrum of changes is therefore encountered in coalminers' lungs, ranging from coal pneumoconiosis through mixeddust pneumoconiosis to silicosis; the findings in any individual depend upon the nature of the coal being mined and the type of work undertaken. in high-rank british collieries the development of coal pneumoconiosis appears to depend on the total mass of dust inhaled, whereas in low-rank british collieries the mineral content of the lung dust appears to be more important. 159 this may explain apparently contrary data drawn from different coalfields -data based on coals of different composition that are not strictly comparable. some workers have stressed the importance of silica in the dust whereas others, particularly in the high-rank coalfields of south wales, have been unable to detect any association between silica and the level of pneumoconiosis. both findings may be correct, but only for the particular group of miners examined in each case. 160 the lesions of coal pneumoconiosis are generally focal and fall into one or other of two major types, simple and complicated, depending upon whether the lesions measure up to or over 1 cm; simple corresponds to categories 1-3 of the ilo grading system (see p. 331) and complicated, which is also known as progressive massive fibrosis, to ilo categories a-c. more diffuse interstitial fibrosis has been reported in about 16% of welsh and west virginian coalminers, usually involving those carrying a particularly heavy dust burden; it runs a more benign course than non-occupational interstitial fibrosis (idiopathic pulmonary fibrosis). 161 similar findings have been reported from france. 162 simple coal pneumoconiosis consists of focal dust pigmentation of the lungs, which may be associated with a little fibrosis and varying degrees of emphysema. its clinical effects are relatively minor. some degree of black pigmentation (anthracosis) of the lungs is common in the general urban population, especially in industrial areas, but much denser pigmentation is seen in coalminers, whose lungs at necropsy are black or slate-grey. black pigment is evident in the visceral pleura along the lines of the lymphatics and on the cut surface where it outlines the interlobular septa and is concentrated in macklin's centriacinar dust sumps ( fig. 7.1.12 ). the dust is generally more plentiful in the upper parts of the lungs and in the hilar lymph nodes, possibly due to poorer perfusion and consequently poorer lymphatic drainage there (see p. 21). 162 two forms of coal dust foci are recognised, macules and nodules, the former being soft and impalpable and the latter hard due to substantial amounts of collagen. both lesions are typically stellate but the more fibrotic the nodules, the more rounded they become, until it is difficult to distinguish them macroscopically from those of silicosis. in these circumstances reliance has to be placed on the whorled pattern of the collagen that is evident microscopically in silicosis. the stellate nodules are analogous to those seen in mixed-dust pneumoconiosis caused by mixtures of silica and inert dusts other than carbon (see above). with polarising filters, small numbers of birefringent crystals may be seen in both macules and nodules, usually representing mica or kaolinite derived from rock that bordered the coal. macules consist of closely packed dust particles, free or within heavily laden macrophages, so that the lesion appears black throughout ( fig. 7.1.13 ). appropriate stains show that the dust-laden macrophages and free dust are lightly bound by reticulin. very little collagen is evident. although striking in their appearance, dust macules are thought to have little effect on lung function. nodules contain substantial amounts of collagen and are thought to have an adverse, but limited, effect on respiration. they vary from a heavily pigmented, stellate lesion, which apart from its collagen content resembles the dust macule ( fig. 7. 1.14), to one that is less pigmented and more circumscribed. the stellate, heavily pigmented type of nodule is seen in lungs that have a relatively low ash content whilst the more rounded and less pigmented nodule is seen in lungs with relatively high ash loads. 163 radiologically (see p. 321), p-type opacities correspond to macules, q-type opacities to the stellate nodules that resemble those of mixeddust pneumoconiosis and r-type opacities to the rounded nodules that resemble those of silicosis. 53, 164 thus, the radiological changes of simple coalworker's pneumoconiosis are due to the dust and the small amount of collagen present and do not reflect any emphysema that may also be present. however, pulmonary dust foci are often associated with emphysema ( fig. 7.1.15 ) and the severity of the emphysema appears to correlate with the dust load. the prevalence of chronic bronchitis and emphysema is high in the coal industry and it has long been debated whether occupation or cigarette smoking is the major factor contributing to emphysema in coalminers. [165] [166] [167] [168] as well as mineral dust, nitrous fumes from shot-firing form another occupational hazard of coal mining. heppleston made a special study of the emphysema found in coalminers, claiming that it differs from centriacinar emphysema, as seen in smokers in the general population, and attributing it to the dust. 169 he introduced the term 'focal emphysema of coalworkers' to describe this special process. others find it very difficult to identify any convincing difference between the emphysema of coalworkers and that encountered outside the industry but heppleston based his claims on the study of serial sections. by this means he showed that, although both forms affect respiratory bronchioles, the focal emphysema of coalworkers affects more proximal orders of these airways and is not associated with the bronchiolitis seen with centriacinar emphysema. furthermore, focal emphysema is a dilatation lesion whereas coniosis, also known as progressive massive fibrosis, can have very serious consequences. particularly when the lesions are large, it is associated with productive cough, breathlessness, significant impairment of lung function and premature death. the major factor accounting for the development of progressive massive fibrosis appears to be the sheer bulk of coal dust in the lung, rather than coal rank or the silica content of the mine dust. 179 progressive massive fibrosis has occasionally been recorded in dockers loading silica-free coal into the holds of ships 158 and in workers exposed to pure carbon in the manufacture of carbon black and carbon electrodes. [141] [142] [143] progressive massive fibrosis is characterised by large (over 1 cm) black masses, situated anywhere in the lungs but most common in the upper lobes. the lesions may be solitary or multiple and very large, occupying most of the lobe and even crossing an interlobar fissure to involve an adjacent lobe (figs 7.1.3b, 7.1.16). they cut fairly easily, often with the release from a central cavity of black fluid flecked by cholesterol crystals. for many years it was believed that the condition was the result of synergism between mycobacterial infection and dust but the failure of the attack rate to decrease as tuberculosis declined negated this view. 180 today, more emphasis is placed on total dust load for the lesions tend to affect lungs that carry an unduly heavy dust burden. if the remainder of the lung shows little evidence of dust accumulation, the possibility of the masses representing caplantype lesions (see below) should be considered. centriacinar emphysema involves destruction of adjacent alveolar walls. by definition, therefore, focal emphysema is not a true emphysema at all (see p. 102). however, it has been shown that mineral dusts cause elastin and collagen breakdown in the rat lung. 170 focal emphysema may progress to the destructive centriacinar form and this has strengthened claims that mine dust plays a causal role in centriacinar emphysema. 1, [171] [172] [173] [174] [175] [176] in the uk, these claims have been accepted and chronic bronchitis and emphysema in coalminers and metal production workers have been accepted as prescribed industrial diseases since 1992. 177 in germany too, chronic obstructive pulmonary disease is now compensatable as an occupational disease. the conditions for compensation in the uk were initially: • underground coal mining for a minimum of 20 years in aggregate • forced expiratory volume in 1 second at least 1 litre below that expected or less than 1 litre in total • radiological category of at least 1/1. however the last of these criteria has now been dropped. the inclusion of a time element and the omission of some estimate of dust load (such as radiological category) have been criticised, with some justification. 178 as with lung cancer caused by chromates benefit is paid irrespective of smoking habits. whereas simple coal pneumoconiosis, particularly the macular variety, has little effect on lung function, complicated coal pneumomicroscopically, the lesions consist of dust and connective tissue intermixed in a random fashion. central necrosis and cavitation commonly occur. the necrosis is thought to be ischaemic. 181 it is amorphous or finely granular, and eosinophilic apart from abundant dust particles and cholesterol crystal clefts. the fibrotic component in a complicated pneumoconiotic lesion is rich in fibronectin, with collagen only more abundant at the periphery. 182 two types of progressive massive fibrosis are recognisable, corresponding to the two types of nodule described in simple coal pneumoconiosis. 163 the first appears to have arisen by enlargement of a single nodule, whereas the second is a conglomeration of individual lesions, each of which corresponds to the more circumscribed type of nodule seen in simple coal pneumoconiosis. the ash content of the lungs bearing these two types of progressive massive fibrosis varies in the same way as with the two types of simple pneumoconiotic nodules, the enlarged single lesion being found in lungs with a relatively low ash content, and the conglomerate lesion in lungs with a relatively high ash content. the second type resembles the conglomerate nodules of large silicotic lesions but lacks the characteristic whorled pattern of the latter. the diffuse interstitial fibrosis found in a minority of coalworkers is associated with heavy dust deposition. it may progress to honeycombing but, as with the focal forms and unlike idiopathic interstitial fibrosis, it is better developed in the upper zones, the reasons for which are discussed above (see the zonal distribution of pneumoconiosis, p. 329). the pathogenesis of coal pneumoconiosis has much in common with that of silicosis, and indeed many other pneumoconioses. it involves the promotion of fibrogenic factor synthesis and release by cells phagocytosing the inhaled dust. several such factors have now been identified, the degree of fibrosis produced varying with the amount of dust inhaled and the ability of its constituents to promote the production of the responsible cytokines. these include plateletderived growth factor, insulin-like growth factors 1 and 6, transforming growth factor-β and tumour necrosis factor-α. 101, 183, 184 as with other minerals, the indestructability of the dust perpetuates the process. as in silicosis, immunological factors appear to be involved, for there is an increased prevalence of rheumatoid arthritis 185 and of circulating autoantibodies [186] [187] [188] in miners with coal pneumoconiosis. rheumatoid factor has also been demonstrated within the lung lesions. 189 these abnormalities are generally more pronounced in miners with complicated pneumoconiosis but are also found in those with the simple variety. it is also possibly pertinent to the immunological basis of coal pneumoconiosis that some of the pulmonary manifestations of rheumatoid disease are more pronounced in coalminers. this was first pointed out by caplan and will be considered next. caplan described distinctive radiographic opacities in the lungs of coalminers with rheumatoid disease, 190 and it is now recognised that similar lesions may develop in rheumatoid patients exposed to siliceous dusts. the development of such rheumatoid pneumoconiosis does not correlate with the extrapulmonary or serological activity of the rheumatoid process. nor is there a strong relation to dust burden: caplan lesions are characteristically seen in chest radiographs that show little evidence of simple coal pneumoconiosis. pathologists recognise the lesions as particularly large necrobiotic nodules similar to those seen in rheumatoid patients who are not exposed to dust (fig. 7.1.17) . however, because of their large size (up to 5 cm diameter) they may be confused with progressive massive fibrosis undergoing central ischaemic necrosis (see above) or silicosis complicated by caseating tuberculosis. such errors will be less likely if the radiological evolution of the lesions is considered for they tend to cavitate and undergo rapid remission, only to be succeeded by others. they are also well demarcated radiologically. pathologically, they resemble rheumatoid nodules in showing peripheral palisading but differ in their large size and the presence of dust. 191 the dust accumulates in circumferential bands or arcs within the necrotic centres of the lesion (fig. 7.1.18 ), an arrangement that suggests periodic episodes of inflammatory activity. caplan lesions differ from tuberculosis in lacking satellite lesions and tubercle bacilli, and from progressive massive fibrosis in showing characteristic bands of dust pigmentation (table 7. asbestosis is defined as diffuse interstitial fibrosis of the lung caused by exposure to asbestos dust. 192, 193 it does not cover asbestos-induced carcinoma of the lung or asbestos-induced pleural disease. the development of asbestosis depends on the presence of fairly large dust burdens: this is in contrast to mesothelioma and other forms of asbestos-induced pleural disease, which, although also dose-related, occur following the inhalation of far smaller amounts of asbestos dust. asbestos is a generic term for more than 30 naturally occurring fibrous silicates, fibre being defined as an elongated particle with a length-tobreadth (aspect) ratio of at least 3. asbestos fibres have a high aspect ratio, generally over 8. based on their physical configuration they can be divided into two major groups, serpentine and amphibole. the physical dimensions and configuration of asbestos fibres are strongly linked to their pathogenicity. chrysotile (white asbestos) is the only important serpentine form. it accounts for most of the world production of asbestos of all types ( 194 being a serpentine mineral, chrysotile consists of long, curly fibres that can be carded, spun and woven like cotton ( fig. 7.1.19 ). the curly chrysotile fibres are carried into the lungs less readily than the straight amphibole asbestos fibres, and once there undergo physicochemical dissolution and are cleared more readily. they readily fragment into short particles that are easily ingested by macrophages and in the acidic environment of the macrophage phagolysosome they are particularly unstable. the half-life of chrysotile in the lungs is estimated to be in the order of only a few months. 195, 196 not surprisingly therefore chrysotile is the least harmful type of asbestos in respect of all forms of asbestos-induced pleuropulmonary disease. [197] [198] [199] it may nevertheless cause pulmonary fibrosis if sufficient is inhaled. 200, 201 in contrast to chrysotile, amphibole forms of asbestos consist of straight rigid fibres that are stable within the lung. they do not fragment, they are insensitive to chemical attack and their clearance halflives are in the order of decades rather than months. 196 the main amphibole forms of asbestos of commercial importance are crocidolite (blue asbestos) and amosite (brown asbestos). crocidolite, reputedly the most dangerous in regard to all forms of asbestos-related disease, was formerly mined in western australia (wittenoom) and south africa (cape province and the transvaal); it was the principal amphibole used in the uk. amosite, the name of which derives from the acronym for the former asbestos mines of south africa company in the transvaal, was the principal amphibole used in north america. amphiboles are no longer imported by the developed countries but much remains in old lagging and presents a considerable dust hazard when this is removed. tremolite, a further amphibole asbestos, contaminates quebec chrysotile deposits, montana vermiculite and many forms of commercial (non-cosmetic) talc and is responsible for much of the asbestos-related disease in chrysotile miners and millers. 202 another amphibole asbestos, anthophyllite, was formerly mined in finland. it causes pleural plaques (see p. 716) but not lung disease, possibly because its fibres are relatively thick ( fig. 7.1.20) . 203 erionite is a zeolite rather than a type of asbestos but is comparable in form to amphibole asbestos and is also biopersistent. it is found . these coated structures are termed 'asbestos bodies' . because other fibres may gain a similar coat, the non-specific term 'ferruginous body' has been advocated. however, coated carbon fibres (so-called coal bodies) are easily recognised as such by their black core. 206 in practice, ferruginous bodies with the appearance of asbestos bodies almost always prove to have an asbestos core. 207, 208 long fibres are more likely to be coated than short ones, which are cleared more quickly: in one study few fibres less than 5 µm in length were coated and few fibres over 40 µm in length were uncoated. 209 amphiboles form bodies more readily than chrysotile. a comparison of light and electron microscopic fibre counts found that 0.14% of chrysotile, 5% of crocidolite and 26.5% of amosite formed bodies. 210 nevertheless, sufficient chrysotile fibres are coated to permit recognition of asbestosis by standard histological criteria (diffuse fibrosis and asbestos bodies), even if chrysotile is the only asbestos present. 211 despite the biodegradability of chrysotile, asbestos body numbers do not materially diminish with time. 212 very occasionally however a patient with diffuse pulmonary fibrosis and a history of asbestos exposure has no evident asbestos bodies but analysis shows a fibre burden within the range found in asbestosis, justifying fibre analysis in such cases. 212a there is evidence that alveolar macrophages are involved in the coating of asbestos fibres to form asbestos bodies and that the bodies are less harmful to the macrophages than uncoated fibres. 213 asbestos bodies give a prussian blue reaction for iron when stained by perls' method and their yellow-brown colour makes them easily recognisable in unstained films of sputum or in unstained histological sections. sections may be cut 30 µm thick to increase the yield and help identify bodies that lie at an angle to the microtome blade. there is a good correlation between the numbers of asbestos bodies seen in lung sections and those in tissue digests. 214, 215 the bodies may be found singly or in irregular clumps or stellate clusters. they are unevenly distributed but in well-established asbestosis they are easily found. if they are not evident, asbestos burden may be assessed quantitatively in tissue digests (see below). their presence in lung tissue, sputum or bronchoalveolar lavage fluid merely confirms exposure, not the presence of disease. however, the number of asbestos bodies in lavage fluid correlates well with lung asbestos burden 216, 217 and the number in sputum correlates with the duration and intensity of exposure. [216] [217] [218] fibre counts 193, 208, [219] [220] [221] [222] [223] quantitation is desirable in certain circumstances (box 7.1.1), in which case it is best effected on 2-cm 3 blocks of fixed or fresh lung tissue obtained from three different sites, avoiding tumour and thickened pleura. the tissue blocks are digested with caustic soda or bleach, following which the fibres may be collected on a millipore membrane or viewed in suspension in a red blood cell-counting chamber. if phase contrast optics are used both coated and uncoated fibres can be assessed. 219 alternatively, dark ground illumination can be used to demonstrate uncoated fibres. 221 however, electron microscopy is to be preferred as it detects far more fibres than are visible by light microscopy and can also provide information on fibre type. it is important that the laboratory is well practised in fibre analysis and has established its own control range for the general population as well as asbestosis as most lungs contain some asbestos. ambient fibres are generally shorter than 5 µm and some workers therefore confine their counts to fibres that are at least as long as this. 224 justification for this comes from animal experiments demonstrating that long fibres cause more inflammation, chromosomal damage, fibrosis, lung tumours and mesotheliomas than short fibres, [225] [226] [227] [228] and from studies in humans suggesting that long fibres in parts of central turkey where it causes both mesothelioma and a pattern of interstitial pulmonary fibrosis that is comparable to asbestosis. 204, 205 asbestos use and exposure exposure to asbestos occurs in countries where it is extracted ( asbestos is used particularly for fireproofing, in heat and sound insulation and for strengthening plastics and cement. thus, unless adequate precautions are taken, exposure is experienced by dockers unloading asbestos in the close confines of a ship's hold, by thermal insulation workers (laggers and strippers) in shipyards, power stations, train maintenance depots, factories and other large buildings, by construction workers such as carpenters cutting asbestos building panels, and by workers making asbestos products such as fireproof textiles, brake and clutch linings, and specialised cement. as well as such direct exposure, exposure may also be: • indirect, as experienced by the families of asbestos workers • paraoccupational, as experienced by those working alongside an asbestos worker • neighbourhood, as experienced by those living downwind of an asbestos works or mine • ambient, as experienced by those living or working in a building containing asbestos. exposure to asbestos incorporated in the structure of a building carries a negligible health risk if the asbestos material is well maintained to prevent shedding of dust. stripping asbestos out is more dangerous than maintaining it in situ, but maintenance is sometimes neglected. the near indestructibility of asbestos accentuates the health problems that its ubiquity poses. because of their aerodynamic properties, fibres of 100 µm or more in length may reach the finer bronchioles and alveoli. once impacted, the sharp asbestos fibres become coated with a film of protein that is rich in iron. the coating is thickest at the ends of the fibres, giving a 229 other human studies have shown that, although asbestos load is maximal in the upper lobes, more long fibres are found at the bases, where fibrosis is most marked. 41,230 a further reason for limiting attention to the longer fibres is that the shorter ones are cleared more easily and their number therefore varies with the time lapsed since last exposure. for these reasons asbestos regulations in many countries now limit attention to fibres that are over 5 µm in length and have a length-to-diameter (aspect) ratio greater than 3: such fibres have become known as regulatory or world health organization (who) fibres. values are best expressed as fibres/g dry lung. by light microscopy, normal values range up to 50 000: over 20 000 is seen with mesotheliomas, and over 1 000 000 in asbestosis (table 7 .1.5). 216, 224, 231, 232 however, compared with electron microscopy, light microscopy is relatively insensitive, showing only 26.5% of the amosite, 5% of the crocidolite and 0.14% of the chrysotile. 210 light microscopic counts correlate poorly with severity of asbestosis 219 and electron microscopy non-asbestos fibres commonly found in the lung include mullite, which derives from fly ash. this may constitute up to 70% of the total fibre burden (see table 7 .1.6) and is thought to be harmless. there is no firm evidence that manmade fibres present a health hazard 237 but in certain localities natural non-asbestos mineral fibres, zeolites for example, are important causes of mesothelioma (see p. 718) and also cause interstitial pulmonary fibrosis. 204 in contrast to the first half of the twentieth century, much of the asbestosis encountered today is asymptomatic, identified radiologically or histologically in lungs resected for carcinoma or removed at autopsy. symptomatic cases are characterised by an insidious onset of breathlessness, a dry cough and crackles over the lower lung fields. finger clubbing is a variable feature. lung function tests show a restrictive respiratory defect. radiology initially shows small irregular basal opacities that gradually coalesce to become linear, coarsen and eventually progress to a honeycomb pattern of small cysts. the principal differential diagnosis, both clinically and pathologically, is from idiopathic pulmonary fibrosis. this is aided by the slow progression of asbestosis, which often extends over 20 years, as opposed to an average course of 2-3 years from presentation to death for the idiopathic condition. most cases of asbestosis are diagnosed solely on the occupational history and these clinicoradiological features. recourse to histology is unusual but biopsy (preferably as a wedge of lung) may be undertaken if the clinical features are atypical. histology also arises when the pathologist samples lung parenchyma remote from a resected carcinoma (the universal importance of which cannot be overemphasised). asbestosis (established) over 1 000 000 over 100 000 000 the light microscopic counts include total fibres (coated and uncoated). the electron microscopic counts include only amphibole asbestos. results from different laboratories vary and these figures, derived from several sources, 216, 231, 232 provide only a general guide. reliable results depend upon counts being made regularly and the normal range from that laboratory being ascertained. ratios of counts obtained by electron and light microscopy vary greatly but approximate to 100. is better in this respect. [197] [198] [199] by transmission electron microscopy, values may range up to 5 000 000 in controls, with asbestosis generally above 100 000 000 and mesotheliomas found at any level down to 1 000 000, all these figures representing amphibole fibres/g dried lung (see table 7 .1.5). 216, 231, 232 it should be noted that counts from different parts of the same lung may vary widely; 42,231-235 caution should therefore be exercised in interpreting a count obtained on a single sample. there is also wide discrepancy between laboratories, even when analysing the same sample. 234 results obtained in an individual case therefore have to be evaluated against a standard set of values unique to that laboratory. electron microscopy also provides valuable information on the type of fibre. chrysotile differs physically from the amphiboles in two respects: its fibres are both curved and hollow (figs 7.1.20 and 7.1.22). with an electron microscope equipped for microprobe analysis, the various forms of asbestos may also be distinguished from other fibres and from each other (box 7.1.2), 230, 236 an important point as the amphibole forms of asbestos are far more dangerous than chrysotile (table 7 .1.6). [197] [198] [199] coroners require autopsy verification of the diagnosis in all suspected cases and this also necessitates hystology. when the lungs from a patient with asbestosis are seen at autopsy, pleural fibrosis is often found, and although this may also be attributable to asbestos exposure it is to be regarded as an independent process and not part of the asbestosis: it is dealt with separately on page 715. slicing the lung affected by asbestosis shows a fine subpleural fibrosis, especially of the lower lobes ( fig. 7 .1.23). in severe cases the fibrosis often extends upwards to involve the middle lobe and lingula, and sometimes the upper lobes also. microcystic change associated with the fibrosis develops in advanced cases and in severe disease there may be cysts over 1 cm in diameter. however, these classic changes are seldom seen in developed countries today. following decades of dust suppression in asbestos factories, current patients have mild to moderate asbestosis and are dying of related cancer or of non-pulmonary disease. 238 in some of these cases the asbestosis is only detectable microscopically. fixation of the lungs through the bronchi and the use of heard's barium sulphate impregnation technique facilitate demonstration of the fibrosis (see p. 757 and fig. 7.1.23 ). the mild degree of asbestosis currently encountered is of little functional significance but is often critical in determining whether an associated carcinoma of the lung should be attributed to asbestos exposure (see below). the histological diagnosis of asbestosis requires an appropriate pattern of interstitial fibrosis associated with the presence of asbestos bodies. both components must be present. the fibrosis is paucicellular, lacking any significant degree of inflammation and being collagenous rather than fibroblastic. it is generally considered that asbestosis begins about the respiratory bronchioles and alveolar ducts where most of the asbestos fibres impact. 15 alveolar walls attached to these bronchioles show fine interstitial fibrosis. however, this early lesion has to be interpreted with caution because it is not specific to asbestos, being found with other inhaled mineral dusts 239, 240 and even in many cigarette smokers who have not been so exposed. 241 it more likely represents a non-specific reaction to a variety of inhaled particles. it may cause mild airflow obstruction but is not associated with the radiographic, clinical or restrictive changes of classic asbestosis. as the disease progresses, the focal changes join up so that the basal subpleural regions show widespread interstitial fibrosis and eventually complete destruction of the alveolar architecture. in severe cases there may be honeycombing and metaplastic changes in the alveolar and bronchiolar epithelium. apart from the presence of asbestos bodies the changes resemble those of non-specific interstitial pneumonia, or more rarely usual interstitial pneumonia. fibroblastic foci may be found but they are uncommon. there is often an increase in alveolar macrophages but the desquamative interstitial pneumonia that has been reported in association with asbestos 242, 243 is not to be regarded as a variant of asbestosis 192 ; concomitant smoking is a more likely cause. 241 a variety of other non-specific inflammatory processes such as organising pneumonia have been reported in asbestos workers and if localised some have been suspected of representing malignancy until biopsied. 244 several schemes have been proposed for grading the extent and severity of asbestosis. these are of value in epidemiological studies but should only be applied to cases meeting the histopathological criteria for a diagnosis of asbestosis. one such scheme is shown in box 7.1. 3. 193,245,246 in well-established asbestosis asbestos bodies are numerous and easy to find, aggregates of them sometimes forming clumps ( fig. 7.1.24) . in earlier lesions a detailed search may be necessary, in which fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli 2 b extension of fibrosis to involve alveolar ducts and/or two or more tiers of alveoli adjacent to the respiratory bronchiole, with sparing of at least some alveoli between adjacent bronchioles 3 fibrotic thickening of the walls of all alveoli between at least two adjacent respiratory bronchioles 4 honeycomb change a an average score is obtained for an individual case by adding the scores for each slide (0-4), then dividing by the number of slides examined b grade 1 and, to a lesser extent, grade 2 need to be distinguished from smoking-induced peribronchiolar fibrosis and mixed-dust pneumoconiosis. case the examination of unstained or perls-stained sections facilitates their identification. minimum criteria for the diagnosis of asbestosis require the identification of diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or other mass lesion and the presence of either two or more asbestos bodies in tissue with a section area of 1 cm 2 or a count of uncoated asbestos fibres that falls in the range recorded for asbestosis by the same laboratory. 192, 193 there are marked variations in the concentration of asbestos fibres between samples from the same lung 216, 235 and it is therefore recommended that at least three areas be sampled, the apices of the upper and lower lobes and the base of the lower lobe. 223 the equivalent of mallory's alcoholic hyalin of the liver has been described in the lungs in asbestosis, 193, 247 and subsequently in other [248] [249] [250] [251] it is seen as small eosinophilic cytoplasmic inclusions within hyperplastic type ii alveolar epithelial cells (fig. 7.1.25a ). electron microscopy shows that the inclusions consist of a tangle of tonofilaments ( fig. 7.1.25b ) and by immunocytochemistry a positive reaction is obtained with antibodies to cytokeratin, both these features being typical of mallory's hyalin in the liver. the inclusions also react for ubiquitin, the accumulation of which is indicative of cellular damage, in particular faulty proteinolysis. 251 the differential diagnosis of asbestosis includes pulmonary fibrosis due to many other causes, any of which may of course affect an asbes-tos worker as much as members of the general population. the proportion of diffuse pulmonary fibrosis in asbestos workers that is not attributable to asbestos has been estimated to be as high as 5% and likely to rise as the risk of asbestosis diminishes with better industrial hygiene. 252 the principal differential diagnosis of asbestosis is from idiopathic pulmonary fibrosis. both diseases affect the bases and periphery of the lungs predominantly. in the late stages, cystic change is more evident in idiopathic pulmonary fibrosis but this criterion is not totally reliable. nor is the presence of pleural fibrosis, although it is usually present in asbestosis and is seldom found in idiopathic pulmonary fibrosis. asbestosis seldom progresses or does so very slowly after exposure ceases 253, 254 whereas idiopathic pulmonary fibrosis typically proves fatal within 3-4 years from onset. the fibrosis of asbestosis is generally paucicellular: inflammation is not a feature and the fibroblastic foci that characterise the usual interstitial pneumonia pattern of fibrosing alveolitis are seldom observed in asbestosis. very often the distinction from idiopathic pulmonary fibrosis has to be based on the amount of asbestos in the lung and, if asbestos bodies are not readily identifiable, this has to depend on fibre counts. errors are made both by overlooking substantial numbers of asbestos bodies completely and by ascribing undue importance to scanty bodies. if considering the possibility of minimal asbestosis (to justify attributing carcinoma of the lung to asbestos, for example) it should be remembered that a little peribronchiolar fibrosis is also characteristic of smokers' lungs, centriacinar emphysema and early mixeddust pneumoconiosis. [239] [240] [241] 255 as described above, at least two asbestos bodies/cm 2 in the presence of interstitial fibrosis distant from any lung cancer or other mass lesion is required for a diagnosis of asbestosis. 192 although the causes of asbestosis and idiopathic pulmonary fibrosis are very different, they resemble each other in several ways, suggesting that similar pathogenetic mechanisms may operate. 105, [256] [257] [258] in both these diseases there is degeneration of the alveolar epithelium and capillary endothelium, with patchy loss of the former, 256 and bronchoalveolar lavage shows an increase in macrophages that might perpetuate the damage by releasing lysosomal enzymes, nitric oxide and hydroxyl radicals. 257, [259] [260] [261] both diseases are also characterised by an increased prevalence of circulating non-organ-specific autoantibodies. 262 experimentally, asbestos exposure leads to the activation of a variety of fibrogenic cytokines at sites of lung injury. 105, [263] [264] [265] [266] [267] [268] inhaled asbestos activates a complement-dependent chemoattractant for macrophages 269 and macrophage stimulation involves the secretion of fibroblast stimulating factors, [270] [271] [272] asbestos being intermediate between haematite and silica in regard to macrophage-mediated fibrogenicity. 273 the epithelial damage could be mediated directly by the needle-like asbestos fibres or indirectly through enhanced phagocyte generation of free radicals (which is much greater with amphibole asbestos than with either chrysotile or silica). 268, 274 fibrogenic cytokines released by activated pulmonary phagocytes and regenerating alveolar epithelial cells in asbestosis include tumour necrosis factor-α and transforming growth factor-β, 268 as in idiopathic pulmonary fibrosis. as a result of better industrial hygiene, asbestosis is less severe today than in earlier years when it followed much heavier exposure, with the consequence that death from respiratory failure and cor pulmonale is less common and sufferers are surviving longer. there is therefore now a greater risk of asbestos-related cancer eventually developing. asbestos exposure predisposes to two varieties of malignant neoplasm, carcinoma of the lung and mesothelioma of the pleura and peritoneum. the risk of malignancy increases with dose but the relative risk of carcinoma is much smaller than that of mesothelioma. for example, with heavy exposure, as in lagging, the risk of mesothelioma is increased 1000-fold whereas it is increased only fivefold for lung cancer. hence, with light exposure there is a substantial risk of mesothelioma but only a small risk of lung cancer. asbestosis requires heavy exposure and in one group of patients with asbestosis, 39% died of pulmonary carcinoma, 10% of mesothelioma and 19% of other respiratory diseases. 238 although there were many earlier reports, the link with carcinoma of the lung may be considered to have been firmly established by 1955, 275 that between crocidolite asbestos and mesothelioma by 1960, 276 and that between amosite asbestos and mesothelioma by 1972. 277 mesothelioma is considered on page 717. in regard to carcinoma of the lung, asbestos is not such a potent pulmonary carcinogen as cigarette smoke but together their effects are multiplicative rather than additive (table 7 .1.7). 278, 279 however, the risk attributable to asbestos is the same regardless of smoking history, being increased fivefold in both smokers and non-smokers. there is usually a latent period in excess of 20 years between first exposure to asbestos and the development of lung cancer and the risk increases the greater the cumulative exposure. the increased risk involves carcinomas of all the histological types encountered in the lung, although adenocarcinoma has been disproportionately overrepresented. 245, [280] [281] [282] [283] [284] [285] it is uncertain 114 whether the increased risk of carcinoma is caused by the asbestos 192, [286] [287] [288] [289] [290] [291] or the asbestosis. [292] [293] [294] [295] [296] [297] [298] [299] the latter view envisages the carcinoma arising in the foci of alveolar epithelial hyperplasia and dysplasia that commonly accompany any interstitial fibrosis (see carcinoma complicating idiopathic pulmonary fibrosis, p. 275). however, most carcinomas complicating asbestosis arise in the bronchi rather than the alveolar tissue. on the other hand, more arise in the sites worst affected by asbestosis, the lower lobes and the periphery of the lung, than in the general population ( fig. 7.1.26) . 238, 245, [281] [282] [283] [284] [285] the majority view has been that asbestosis is a necessary precursor of the carcinoma but evidence to the contrary is finding increasing support (table 7 .1.8). 192 in the uk, industrial compensation was formerly only awarded to an asbestos worker for carcinoma of the lung if there was also asbestosis or diffuse pleural fibrosis but new rules were introduced in 2006. asbestosis remains a sufficient criterion but diffuse pleural thickening is not and asbestosis is no longer a necessary criterion: asbestos is deemed to have been responsible if the patient worked in asbestos textile manufacture, spraying, lagging or gas mask manufacture for at least 5 years before 1975 or 10 years after 1975. the basis for these changes is the premise that heavy asbestos exposure is sufficient in itself to account for carcinoma of the lung. 278 together, these factors have a multiplicative rather than additive effect mortality ratio non-smoking controls 1 non-smoking asbestos workers 5 cigarette-smoking controls 11 cigarette-smoking asbestos workers 53 asbestosis associated with carcinoma of the lung. the asbestosis has been highlighted by barium sulphate impregnation and is seen as a grey subpleural band to the right of the picture. although the carcinoma has arisen in the same lobe as the asbestosis it has not obviously arisen in an area affected by asbestosis. 1. asbestosis diagnosed clinically, radiologically or histologically or a minimum count of 5000 asbestos bodies per gram dry lung tissue (/g dry), or an uncoated asbestos fibre burden of 2 million amphibole fibres more than 5 µm in length/g dry, or 5 million amphibole fibres more than 1 µm in length/g dry or estimated cumulative exposure to asbestos of at least 25 fibres/ ml-years or an occupational history of 1 year of heavy exposure to asbestos (e.g. manufacture of asbestos products, asbestos spraying) or 5-10 years of moderate exposure (e.g. construction or ship-building) and 2. a minimum lag time of 10 years lung fibre counts in the asbestosis range (see table 7 .1.5) provide valuable evidence of such exposure. compensation standards for asbestos-associated lung cancer in different countries are shown in box 7. 1.4. 192,289 asbestos-induced airway disease although asbestosis causes a restrictive respiratory defect, airflow limitation is also seen in this disease. much of the airflow limitation is attributable to cigarette smoking but it is also seen in non-smoking asbestos workers and is worse in those with asbestosis. 300 the pathological basis of this appears to be small-airways disease (see p. 123). 301 it is possibly a non-specific reaction to inhaled dust or cigarette smoke. 302 because it is not established that this lesion progresses to interstitial alveolar fibrosis (asbestosis) the term 'asbestos airways disease' is suggested. 302 fibrosis limited to the bronchioles is specifically excluded from the definition of asbestosis in the latest guidelines (although these retain grade 0 for fibrosis limited to the bronchiolar walls). 193 it should also be noted that, although emphysema is considered to be a destructive rather than fibrotic condition, a little focal the presence of asbestos-related bilateral pleural plaques or asbestos-related bilateral pleural thickening and occupational exposure and a lag time of at least 12 years the presence of asbestosis or pleural plaques or diffuse pleural thickening or fibre-years of exposure only fibre-years of exposure are taken into account exposure, at least 10 years' latency and asbestos-related pleural or parenchymal changes asbestosis is not required but smoking is taken into consideration attempts are made to quantify separately the attributability to asbestos, smoking and other factors (e.g. radon) fibrosis is generally evident in this common condition 255 and does not necessarily indicate early asbestosis. aluminium has been implicated in the development of respiratory disease during the refining of its principal ore, bauxite, to yield various aluminium oxides (aluminas), in the preparation of the metal by smelting alumina, in the production of corundum abrasive and in the production of special aluminium powders used in explosives. bauxite is a mixture of various aluminium oxides, hydroxides and silicates, iron oxide and titanium dioxide. the oxides of aluminium are obtained by differential heating of the ore and the respiratory effects of this work appear to be no more than mild airway irritation. it is generally accepted that aluminium oxide is inert. aluminium is prepared by the electrolytic reduction of its oxide dissolved in sodium aluminium fluoride (cryolite), a process releasing a considerable amount of fluoride-rich effluent. exposed workers have complained of what is termed pot-room asthma. the pathology of this condition is not well described but the pathogenesis is thought to involve irritation rather than allergy. 303 the abrasive corundum is formed from bauxite mixed with coke and iron heated in an electric arc furnace, a process in which workers may be exposed to the fumes of alumina and free silica. in the past some of these workers developed diffuse pulmonary fibrosis (shaver's disease) 369 and, although this was initially attributed to the aluminium, it is now agreed that the free silica was the responsible agent. the exposure to free silica has been reduced and the disease is now regarded as historic. aluminium powder holds a paradoxical position in regard to lung disease. in certain industries it has caused very severe pulmonary fibrosis, yet in others it has proved harmless. indeed, at one time canadian miners breathed aluminium dust before work, in the belief that this would reduce the danger of silica in the mine dust 304 and more recently silicosis has been treated by such means in france. 305 it is questionable whether this practice is effective but it at least appears to cause no harm. the explanation for these contradictory observations probably lies in differing methods of manufacture of aluminium powder. aluminium metal appears to be an inert substance but this is only because it has a high affinity for oxygen and the surface layer of aluminium oxide so formed is firmly bound to the underlying metal, unlike ferric oxide which permits further rusting of iron. granular aluminium powders, produced in a ball mill or from a jet of molten aluminium, therefore acquire a protective coat of surface oxide and are inert. with stamped aluminium powders, however, surface oxidation is prevented by lubricants added to aid the separation of these flake-like particles. the usual lubricant (stearin) contains stearic acid and this polar compound combines with the underlying metal, which is thereby protected from both atmospheric oxidation and the action of body fluids when such dust is inhaled. in certain circumstances, however, non-polar lubricants in the form of mineral oils have been substituted for stearin. this happened in germany during the second world war when munition production was stepped up but stearin was difficult to obtain, 306, 307 and in the uk in the 1950s to make the powder darker for purely commercial reasons. 308 in vitro, oil-coated stamped aluminium powder reacts with water to produce aluminium hydroxide, which affords the underlying metal no protection against further attack, so that aluminium hydroxide continues to be formed. 309 this substance is a protein denaturant, once used in the tanning industry, and it is believed that this property underlies the very ex ceptional cases of severe pulmonary fibrosis that have occurred in connection with stamped aluminium powder produced with mineral oil rather than stearin. 309, 310 the fibrosis has a very characteristic pattern, affecting the upper lobes and progressing rapidly, the interval from onset of symptoms to death being as short as 2 years. 308 there is marked shrinkage of the lungs with gross elevation of the diaphragm and buckling of the trachea (fig. 7.1.27 ). the lungs are grey ( fig. 7.1.28) and microscopically, numerous small black jagged particles are seen. these can be shown to contain aluminium with irwin's aluminon stain or by microprobe analysis. 311 what appears to be a different pathological effect of aluminium dust on the lungs is the rare development of granulomatous disease resembling sarcoidosis and berylliosis. 312, 313 this represents hypersensitivity to the metal, amenable to confirmation with a lymphocyte transformation test similar to that used to diagnosis berylliosis (see below). rare cases of desquamative interstitial pneumonia and pulmonary fibrosis have been reported in aluminium welders. 311, 314 elements with atomic numbers from 57 (lanthanum) to 71 (lutetium) are known as the lanthanides or rare earth metals. they are used in many manufacturing processes, including the production of hightemperature ceramics and the grinding of optical lenses. carbon arc lamps used in reproduction photography emit appreciable quantities of oxidised lanthanides, particularly cerium oxide, and there are reports of pneumoconiosis in exposed individuals. 315 the pathological changes reported have varied from granulomatous nodules to diffuse interstitial fibrosis indistinguishable from the idiopathic variety except for the presence of rare earth elements (usually cerium) detected by polarising light microscopy and electron microprobe analysis. 315 hard metal is a tungsten alloy containing small amounts of cobalt, titanium, molybdenum and nickel. it is exceptionally tough and once formed can only be worked with diamond. it is used in the tips of drill bits, on abrasive wheels and discs, and in armaments. interstitial lung disease is liable to arise in its manufacture or in those using hard metal as an abrasive. 316 experimental work suggests that cobalt is the dangerous constituent 317 but this element is soluble and, unless industrial contact has been recent, analysis of lung tissue usually shows tungsten and titanium but no cobalt. the role of cobalt is also indicated by the development of similar interstitial lung disease in diamond polishers using high-speed polishing discs made with a diamond-cobalt surface that lacked tungsten carbide and the other constituents of hard metal. 318, 319 hard-metal lung disease and cobalt lung take two forms, an industrial asthma and interstitial fibrosis. the latter has a diffuse lower zonal distribution and the appearances mimic idiopathic pulmonary fibrosis. however, an unusual feature is the presence of moderate, or perhaps only small numbers, of giant cells (fig. 7.1.29a, b) . 316, 320 not only are there multinucleate alveolar macrophages but syncytial cell forms develop in the alveolar epithelium. electron microscopy confirms that these are multinucleate type ii pneumocytes (fig. 7.1.29c ). 316 such epithelial changes are well known in measles pneumonia but the viral inclusion bodies that characterise this infection are not found in hard-metal pneumoconiosis. the changes are those initially described as a particular pattern of idiopathic interstitial pneumonia termed giant cell interstitial pneumonia or gip (see p. 264). elemental analysis shows that many, but not all, cases of gip represent hardmetal disease. the exceptions seldom give a history of cobalt exposure and must be presumed to represent true idiopathic cases. conversely, epithelial giant cells are not always found in hard-metal pneumoconiosis and so their presence, although highly characteristic, is neither totally specific nor totally sensitive. beryllium is the lightest of metals. it has an atomic weight of 4 and special properties that make it especially useful in many applications. it is more rigid than steel, has a high melting point and is an excellent conductor of heat and elecricity. unfortunately, the inhalation of beryllium dust or fume is exceedingly dangerous. 321, 322 those who worked with beryllium compounds before precautionary measures were taken suffered a high morbidity and mortality. sometimes, the escape of dangerous fumes from the factories was on such a scale that people living in nearby houses, downwind from the places in which these materials were being worked, contracted and occasionally died from berylliosis ('neighbourhood cases'). 323 alternatively, contamination of a beryllium worker's clothes might lead to berylliosis in a temperatures. the alloys of beryllium are also now widely used, especially those with copper, on which it confers elasticity and resistance to fatigue. alloy manufacture and the machining of beryllium alloys are therefore further activities that entail a risk of berylliosis, as is the recovery of the metal in the recycling of scrapped electronic and computer parts. seemingly innocuous occupations such as dental laboratory technician are not without risk of chronic berylliosis. 329 there are good grounds for regarding chronic berylliosis as being an allergic condition. many of those affected react strongly to skin tests with dilute solutions of beryllium salts, although these must be undertaken with care: occasionally in a highly sensitised person even so small an exposure may evoke a systemic reaction. the skin reaction is of the delayed type, occurs in only 5% of exposed individuals, is not associated with a clear-cut dose-response curve and represents a granulomatous response. further evidence for the disease having an allergic basis derives from bronchoalveolar lavage, which demonstrates an excess of t-helper lymphocytes that proliferate in vitro on exposure to beryllium salts. 330 a positive transformation test given by these lymphocytes is a more reliable indicator of disease than in vitro blood lymphocyte transformation testing, 52 which is safe but not wholly reliable and indicates only sensitization, rather than berylliosis. susceptibility to berylliosis varies widely from person to person and it is notable that chronic pulmonary disease is strongly associated with the hla antigen dpβ1 and the glu69 gene. 331, 332 the importance of genetic factors is supported by a report of the disease in identical twins. 333 chronic berylliosis is thought to be initiated by the metal binding to tissue proteins and acting as a hapten to initiate a delayed hypersensitivity response characterised by a proliferation of t-helper lymphocytes. these sensitised cells in turn secrete a variety of cytokines (e.g. interleukin-2, tumour necrosis factor-α and interferon-γ) that recruit and activate macrophages, which mature into epithelioid cells. the resultant epithelioid cell granulomas destroy the lung tissue and lead to pulmonary fibrosis. if beryllium enters the subcutaneous tissues through a cut or abrasion, as often happened in the earlier days of fluorescent lamp manufacture, a sarcoid-like granuloma soon appears at the site; in time, the overlying epidermis may break down to form an ulcer. even more serious are the lesions produced by the inhalation of beryllium. chronic pulmonary berylliosis takes the form of a widespread granulomatous pneumonia with a histological picture identical to that of sarcoidosis (fig. 7.1.30a ). both berylliosis and sarcoidosis affect the upper lobes more than the lower (fig. 7.1.30b ) and in both diseases the granulomas are preferentially distributed along lymphatics and may involve adjacent blood vessels. in neither condition is there widespread necrosis but in both diseases the granulomas occasionally display a little central necrosis or hyalinisation. as in sarcoidosis, the hilar lymph nodes may be involved but, unlike sarcoidosis, not in isolation. over a period of many years, the sarcoid-like granulomas gradually undergo progressive fibrosis, with consequent impairment of pulmonary function. in the later stages, when the disease has become chronic, dispersal of beryllium from its site of initial absorption may lead to generalisation of the disease and to the appearance of similar granulomas elsewhere, particularly in the liver, kidneys, spleen and skin, but this is unusual. relative. 324 beryllium compounds may also cause contact dermatitis and conjunctivitis. 325 beryllium is also classified as a probable pulmonary carcinogen, 326 but this is controversial. two forms of berylliosis are recognised, acute and chronic. acute berylliosis was first reported in germany in 1933 327 and is now largely of historical interest, being only encountered as a result of rare accidental or unexpected exposure. it follows the inhalation of a soluble beryllium salt and represents chemical injury, the pathology being that of diffuse alveolar damage (see p. 136). further consideration will be confined to chronic berylliosis, which is allergic in nature. chronic berylliosis was first reported in 1946 in the fluorescent lamp industry. 328 beryllium has now been replaced in this application but it has since proved to be of great value in the nuclear, electronic, computer and aerospace industries and the production of refractory materials and crucibles that are to be subjected to particularly high and there is a lifelong risk of disease. progression often entails alternating exacerbations and remissions, long after exposure has ceased. in keeping with the view that berylliosis is a hypersensitivity reaction, very little beryllium is necessary to cause the disease. particulate beryllium is so scanty in the affected tissues and the atomic number of beryllium so low that electron microprobe analysis is generally unsuitable for its detection. furthermore conventional detectors are protected by a beryllium window. however, the substitution of a polymeric window has enabled beryllium to be detected by electron microprobe analysis, presumably in a patient with fairly heavy exposure. 334 ion or laser microprobe mass spectroscopy can also detect very small amounts of beryllium in tissue sections but these techniques are not widely available. the differential diagnosis of chronic berylliosis is from sarcoidosis, to which it is identical morphologically. [335] [336] [337] however, as noted above, it is unusual for berylliosis to cause significant hilar lymphadenopathy in the absence of pulmonary disease, which is a common feature of sarcoidosis. extrathoracic granulomas, erythema nodosum and uveitis, which are all common in sarcoidosis, are unusual in berylliosis. however, one group found that 6% of patients initially diagnosed as having sarcoidosis actually had chronic berylliosis. 338 similar findings have been reported by others. 324, 339 any patient thought to have sarcoidosis who has worked with or near metals should be offered a beryllium lymphocyte transformation test. a list of laboratories performing this test can be found at www.dimensional. com/~mhj/medical_testing.html. although polyvinyl is not a mineral and the reaction of the lungs to its presence is therefore not a true pneumoconiosis, it is generally so termed and is dealt with here for convenience. workers are exposed to polyvinyl chloride dust in the milling and bagging of this plastic and micronodular opacities may be detected in their lungs radiologically. however, the material is non-fibrogenic and histology merely shows a foreign-body reaction to the dust particles. 340 the radiological opacities may abate when exposure ceases. 341 nevertheless, one polyvinyl chloride worker developed systemic sclerosis, 342 which is a recognised complication of silicosis (see p. 335). polyvinyl chloride is produced from vinyl chloride monomer, which has a causal association with angiosarcoma of the liver and probably other forms of cancer, including carcinoma of the lung (see p. 534). in the late 1990s a characteristic lung disease was identified in workers at several factories producing plush material by spraying nylon flock on to an adhesive backing material. [343] [344] [345] [346] the flock fibres are too large to be inspired but may be mixed with smaller nylon shards of respirable size. the workers complained of cough and breathlessness and were found to have a restrictive ventilatory defect with interstitial markings on radiography. their symptoms improved on removal from the workplace but relapsed on return to work. pathologically, there was lymphocytic bronchiolitis and peribronchiolitis with widespread lymphoid hyperplasia represented by lymphoid aggregates. chronic berylliosis is characterised by the gradual onset of cough, shortness of breath, chest pain, night sweats and fatigue. these symptoms may develop within a few weeks of exposure or many years later. once the worker is exposed, the beryllium is retained in the tissues granulomas were not identified. the histological appearances suggest a severe immunological reaction and raise possibilities such as rheumatoid disease and sjögren's syndrome but consideration of the clinical and serological setting and the occupation should permit recognition of the cause. the industrial production of popcorn and other foodstuffs appears to carry a risk of obstructive airway disease. [347] [348] [349] [350] biopsy of affected workers has shown peribronchiolar fibrosis and granulomas and air sampling has identified many volatile organic compounds, of which the flavouring agent diacetyl (2,3-butanedione) is suspected of being responsible for the bronchiolitis. it is difficult to continue paint spraying (air brushing, aerographics) without adequate respiratory protection but in the early 1990s several small aerographic factories operated in the neighbourhood of alicante, southeastern spain without any concern for the workers' health. the workers were required to paint patterns on textiles using a hand-held spray gun. the atmospheric pollution was intense but complaints of respiratory difficulties were met with reassurances and the workers urged to continue. this they did because of the otherwise poor economy, often returning to work when disabling breathlessness had settled down. a change of paint (to acramin f) may have contributed because the worst-affected workers were employed at two plants that had made this switch. their illness has been described as the 'ardystil syndrome' after the name of one of these factories. some workers were left with permanent respiratory disability. one required a lung transplant and 6 others died. [351] [352] [353] [354] transbronchial biopsy showed organising pneumonia, which in the fatal cases had progressed to irreversible interstitial fibrosis. a similar outbreak of respiratory disease was subsequently reported in algerian textile factories where acromin f was applied by the same technique. 355, 356 acromin f is marketed as a paste and used as such without ill-effect. its use in heavy spray form appears to be responsible for the 'ardystil syndrome' . workers in engineering workshops may be exposed to the prolonged inhalation of fine sprays or mists of the longer-chain hydrocarbons that constitute many mineral oils. this may result in exogenous lipid pneumonia, 357 which is described on page 314, or extrinsic allergic alveolitis. [358] [359] [360] the vapour of shorter-chain hydrocarbons such as paraffin oil (kerosene: c 10-16 ) and petrol (gasoline: c 4-12 ) and gaseous hydrocarbons such as propane may act as acute asphyxiants or central nervous system depressants but have negligible pulmonary toxicity. however, if they are ingested or aspirated in their liquid form they are acutely toxic to the lungs, producing a chemical pneumonitis with the features of diffuse alveolar damage. ingestion may be accidental or deliberate (see fig. 4 .19, p. 142) whereas aspiration is generally inadvertent, occurring in siphoning accidents, such as those experienced by fairground operatives who 'breath or eat fire' ('fire-eater's lung'). 361, 362 animal experiments involving the intratracheal injection of kerosene resulted in acute pulmonary exudates, which cleared except for residual bronchiolitis. 363 welder's pneumoconiosis, first recognised in 1936, 364 essentially represents the fairly harmless deposition of iron in the lungs (siderosis -see p. 337). however, welders may suffer various ill-effects from the inhalation of substances other than iron (table 7.1.9). some of these are para-occupational risks, that is, encountered by welders because they work near another process and are inadvertently exposed: thus, shipyard welders may be exposed to asbestos, 365 and those in foundries to silica. welders may therefore develop a mixed-dust pneumoconiosis (see p. 337), rather than just siderosis. however, one analytical investigation identified excess amounts of iron alone in association with pulmonary fibrosis; the silicon content did not differ from that in controls. 366 more directly, welders may be exposed to asbestos insulation that they themselves use, while welders of special steel alloys run the risk of metal-induced asthma, metal fume fever, polymer fume fever and the consequences of toxic metal fume inhalation, 367 all of which are described separately in this chapter, as is lung disease in aluminium welders. chronic bronchitis has been attributed to the inhalation of low concentrations of irritants such as ozone and nitrogen dioxide by welders but this risk is unproven and the subject of much controversy. welders may also inhale carcinogenic hexavalent chromium compounds in the course of their work and therefore develop lung cancer. the term 'welder's lung' is often applied indiscriminately to any of these diseases and, as it has no specific meaning, is best avoided. dust, fume and gas are some of the terms used to describe different physical forms of respirable agents. they are defined in table 7 .2.1 on the finely divided fume of several metals is highly toxic to the lungs and capable of producing severe acute and chronic damage to both the conductive airways and the alveoli, resulting in acute tracheobronchitis and bronchiolitis, diffuse alveolar damage, obliterative bronchiolitis and pulmonary fibrosis. important metal fumes in this respect include aluminium, which is released together with silica fume in bauxite smelting (see shaver's disease, 369 above), cadmium from welding or cutting special steels, chromium from cutting its alloys or in the manufacture of chromates, cobalt released in the production and use of its alloys (see hard-metal disease, above), mercury released in various industries and in the home, 370 nickel carbonyl released during the purification of metallic nickel or the manufacture of nickel alloys 371 and beryllium (see above). many irritant gases cause severe acute and chronic damage to both the conductive airways and alveoli. the changes are non-specific and similar to those wrought by toxic metal fumes (see above) and viruses amongst other agents. they consist of acute tracheobronchitis and bronchiolitis, obliterative bronchiolitis, diffuse alveolar damage and pulmonary fibrosis. the gases liable to produce such damage include oxides of nitrogen, sulphur dioxide, ozone, phosgene, chlorine, ammonia and various constituents of smoke, notably acrolein. some of these are also touched upon in chapter 7.2 because they are of general as well as occupational importance, although there is no rigid difference between general and occupational pollution. ozone, sulphur dioxide and nitrogen dioxide are oxidising gases that may be found together as industrial atmospheric pollutants. each is capable of producing diffuse alveolar damage by means of its oxidising properties and the release of free active radicals. in addition, they cause damage to distal airways, particularly terminal and respiratory bronchioles, with resulting bronchiolitis. oxides of nitrogen may be encountered with fatal consequences by farmhands seeking to free a blockage in a silo when they encounter pockets of this gas that have accumulated on top of the fermenting silage: the term 'silo-filler's disease' is generally applied to the initial haemorrhagic oedema or the obliterative bronchiolitis that develops in those who survive the initial chemical injury. [372] [373] [374] [375] asphyxia due to the farmhand encountering pockets of carbon dioxide is a further hazard within agricultural silos. other farmhands have suffered from the inhalation of toxic gases or bacteria when handling liquid manure. [376] [377] [378] [379] welding, which is considered below, may also involve exposure to toxic gases such as oxides of nitrogen. ozone, the principal oxidant gas of photochemical smog, produces pulmonary changes at relatively low levels and may be encountered at higher concentrations in various industries. potentially dangerous levels of ozone are produced from atmospheric oxygen by ultraviolet radiation given off in welding while ozone is used in industry to sterilise water, bleach paper, flour and oils, and mask the odour of organic effluents. the damage wrought by ozone is predominantly centriacinar in distribution, affecting terminal and respiratory bronchiolar epithelium and proximal alveolar epithelium. [380] [381] [382] there is loss of cilia and necrosis of centriacinar alveolar type i epithelial cells. the changes are dose-dependent and, in one study, the youngest animals were most sensitive. 383 in long-term experiments, hyperplastic bronchiolar clara and ciliated cells extended peripherally to line alveolar ducts. 384 the role of granulocytes is stressed in some experimental studies 385 and it is notable that neutrophil migration is prominent when the human lungs are damaged by ozone. 386 aldehydes such as acetaldehyde, formaldehyde and acrylic aldehyde (acrolein) are widely used in the plastics and chemical industries. the first is a liquid and the others are water-soluble gases. pathologists are of course familiar with formaldehyde solution from its use as a disinfectant and histological fixative. all these aldehydes are intensely irritant and their acute effects generally prevent prolonged exposure to high concentrations. chronic effects include skin sensitivity and asthma, and in rats nasal carcinoma. however, the doses to which these experimental animals were exposed far exceed any that are likely to be encountered by humans, in whom there is no convincing evidence of aldehyde-induced cancer. 387 ammonia gas is extensively used in industry as a raw material, notably in the manufacture of nitrogenous products such as fertilisers and plastics. it is highly soluble and its acute irritative effects are mainly felt in the eyes, nose and throat, but high levels affect the major airways, possibly leading to them being blocked by exudates. survival usually brings full recovery but bronchiectasis and obliterative bronchiolitis have been described. chlorine gas is widely used in the chemical industry. it is transported and stored under pressure in liquid form. heavy exposure through its accidental release or use as a war gas has proved fatal through its acute toxicity causing exudative airway occlusion and pulmonary oedema. survivors usually recover completely but, as with nitrogen dioxide and ammonia, there is a risk of obliterative bronchiolitis. phosgene (carbonyl chloride, cocl 2 ) is a poisonous, colourless gas that was responsible for thousands of deaths during world war i, when it was used in chemical warfare. it is used industrially in the preparation of some organic chemical compounds and is formed, perhaps inadvertently, 388 by the combustion of methylene chloride in products such as paint strippers. phosgene causes injury to terminal bronchioles and alveoli, with resulting oedema and hyaline membrane formation. the mechanism of cell damage is uncertain but it may depend on inactivation of intracellular enzymes by the gas. longterm problems are rare but chronic bronchitis and emphysema have been described in survivors. mustard gas (bichloroethyl sulphide, c 4 h 8 cl 2 s) is a further agent that has been used in chemical warfare. it is primarily a skin vesicant but when inhaled it results in widespread epithelial destruction and pulmonary oedema. survivors may be left with irritant-induced asthma (reactive airways dysfunction), chronic bronchitis, tracheobronchomalacia, bronchiectasis and bronchiolitis obliterans. [389] [390] [391] thionyl chloride is used in the manufacture of lithium batteries where it is liable to result in the release of sulphur dioxide and hydrochloric acid fumes. workers in such factories have developed lung injury varying from mild, reversible interstitial disease to severe obliterative bronchiolitis. 392 hydrogen sulphide is the principal chemical hazard of natural gas production. high levels of the gas also buid up in sheds housing large numbers of pigs, the source here being the pig manure. once inhaled the gas is rapidly absorbed into the blood stream. the effects are therefore widespread but include the usual respiratory effects of irritant gases, varying from sneezing to pulmonary oedema and acute respiratory distress, depending upon the exposure. in alberta 221 cases were identified over a 5-year period. the overall mortality was 6%; 5% of victims were dead on arrival at hospital. most required admission to hospital but the survivors experienced no long-term adverse effects. 392a the danger of asphyxia from the inhalation of gases devoid of oxygen is fairly widespread in industry. 393 it generally arises from the use of inert gases, which, being non-toxic, give a false sense of security. pockets of these gases tend to form in confined spaces. anoxic death from the accumulation of methane is well known in mines and has also occurred in slurry pits and sewers. anoxic asphyxia in diving (and anaesthesia) has resulted from the incorrect connection of gas cylinders or failure to notice that a mixed gas contains insufficient oxygen. deaths have occurred in welding when argon or carbon dioxide has been used to shield the weld and prevent oxidation of the metals at the high temperatures employed. deaths have also resulted from inadvertent entry to discharged oil tanks filled with nitrogen to reduce the risk of explosions, or from the formation of pockets of nitrogen gas applied in liquid form to freeze the contents of damaged pipes so that they can be repaired without the necessity to drain down. the respiration of a gas devoid of oxygen causes loss of consciousness within seconds because it not only fails to provide oxygen but removes that present in the pulmonary arterial blood. the changes at autopsy are those common to cellular hypoxia. they include cerebral and serosal petechiae and pulmonary congestion and haemorrhage but these features are not specific and are not always present. the cause of death can generally only be surmised from the circumstances surrounding the death. occupational asthma is the commonest cause of work-related respiratory disease in many western countries (table 7 .1.10). [394] [395] [396] the reported incidence ranges from 13 per million workers in south africa to 174 per million workers in finland. 395, 396 it occurs in many industries (table 7 .1.11) 397 and occupational factors can be identified as contributing to asthma in about 15% of adult cases. 398 over 250 aetio399 in the uk a third are organic, a third chemical, 6% metallic and the rest miscellaneous. the commonest, in descending order, are isocyanates, flour and grain, laboratory animals, glutaraldehyde, solder or colophony and hardening agents. 400 atopy appears to predispose to occupational asthma when the allergen is of high molecular weight but not when it is of low molecular weight. for example, atopic individuals are particularly prone to develop asthma if employed in the manufacture of biological detergents, whereas atopy does not increase the risk of asthma from sensitisation to toluene di-isocyanate, which is a serious health problem in the manufacture of polyurethane. similarly, platinum salts are such potent sensitising agents that nearly all those exposed to them develop asthma. asthma-provoking metals other than platinum include chromium, cobalt, nickel and vanadium, all of which are used in steel alloys, and possibly aluminium (see pot-room asthma, p. 357). other asthma-inducing factors encountered in industry include grain and flour dust, certain wood dusts, soldering fluxes containing colophony (pine resin), epoxy resin hardeners such as phthallic anhydride, isocyanate-containing foams and paints, formaldehyde and the excreta of laboratory animals. contaminated humidifiers may cause occupational asthma as well as humidifier fever and extrinsic allergic alveolitis. 401 pathologically, occupational asthma is identical to nonoccupational asthma (see p. 109). byssinosis is a further form of occupational asthma, 402 one encountered in the cotton industry. the sensitising agent is a component of the cotton bract, which is the part of the cotton harvest other than the cotton fibre. bract consists of dried leaf, other plant debris and soil particles and contains a variety of fungal and bacterial residues, including lipopolysaccharide endotoxin, but the exact nature of the sensitising agent remains unknown. 403 the endotoxin is unlikely to be responsible for byssinosis but may be the cause of so-called mill fever, a self-limiting illness characterised by malaise, fever and leukocytosis that is experienced by many people on first visiting a cotton mill. dust levels and the risk of byssinosis are particularly high in the carding rooms where the raw cotton is teased out before it is spun. affected workers are worse when they return to work after the weekend break, a feature attributed to antibody levels having built up during this brief respite from the cotton dust. there is no link with atopy and the fluctuating antibodies are precipitins of the immunoglobulin g class. complement activation by both arms of the complement cascade has been reported. 404, 405 when the lancashire economy was largely cotton-based, necropsies on workers suffering from byssinosis generally showed gross emphysema, and this came to be accepted as evidence of byssinosis. however, it is now realised that in this heavily industrialised part of the uk, emphysema is as common in the general population as in cotton workers and it can no longer be considered a component of byssinosis. other findings in byssinosis are more commensurate with asthma, namely an increase in bronchial muscle and mucous cells. 406 no granulomas or other evidence of extrinsic allergic alveolitis are found. fever may be the predominant feature in a variety of occupational illnesses and the unifying term 'inhalation fever' has been proposed. 407 however, the individual occupations are of interest and these conditions will therefore be considered separately. mill fever has been mentioned above under byssinosis. humidifier fever is an acute illness characterised by malaise, fever, myalgia, cough, tightness in the chest and breathlessness, all of which are worse on monday mornings if the humidifier responsible is at work rather than home. the chest complaints, and their aggravation on return to work after the weekend, are features shared with byssinosis (see above) but the general complaints fit better with extrinsic allergic alveolitis (see p. 279). humidifier fever develops in circum-stances that also lead to the development of a form of extrinsic allergic alveolitis, and not surprisingly the same name has been extended to this latter condition, with inevitable confusion. both diseases are caused by microbiological contamination of humidifiers or air conditioners so that a fine spray of microorganisms is emitted into the office, factory or home. investigations have generally shown the baffle plates of the air conditioner to be covered with a slime of bacteria, fungi or protozoa (mainly amoeba and ciliates), and extracts of this have been used to identify precipitins in the patient's sera, as in extrinsic allergic alveolitis. however, unlike extrinsic allergic alveolitis, humidifier fever resolves within a day and leaves no permanent injury. for this reason there is seldom the opportunity to study the tissue changes, and partly for this reason it remains unclear whether the disease is mediated by immune complexes, as in extrinsic allergic alveolitis, or by endotoxins derived from the contaminants. a febrile illness occurring in precipitin-negative farm-workers after heavy exposure to fungi in their silos was attributed to inhaled fungal toxins and named pulmonary mycotoxicosis. 409 it is also known as precipitin test-negative farmer's lung and organic dust toxic syndrome. 410 the condition is generally self-limiting and is seldom biopsied but desquamative interstitial pneumonia and diffuse alveolar damage have been reported. 411, 412 metal fume fever this is a self-limiting acute illness characterised by fever, sweating, myalgia, chest pain, headache and nausea, that comes on monday mornings when occupational exposure is experienced after a weekend's respite, as with bysinnosis and humidifier fever; during the week tolerance develops. 368, 413 the disease involves the release of cytokines such as tumour necrosis factor and is presumed to have an allergic basis. 414 the metals involved are chiefly zinc, copper and magnesium, and, to a lesser extent, aluminium, antimony, iron, manganese and nickel. occupations at risk include any that generate such metal fumes, but particularly welding. it is most commonly associated with welding zinc-coated surfaces. if the symptoms persist, alternative diagnoses, such as acute cadmium poisoning and other specific toxic metal fume diseases, should be suspected: these are not self-limiting and may cause severe bronchiolitis or diffuse alveolar damage (see above). this illness resembles metal fume fever except that it occurs without regard to previous exposure: no tolerance develops and there is therefore no particular susceptibility on mondays. the polymers concerned are quite inert, except when heated to produce fume: polytetrafluorethylene (ptfe, teflon, fluon, halon) is a notable example. as with other self-limiting diseases, little is known of the tissue changes. 54 environmental irradiation chiefly affects the skin but in some parts of the world rocks near the surface release significant amounts of radon gas. this carcinogen is liable to accumulate in buildings and be inhaled, so subjecting the occupants to an increased risk of lung cancer. the installation of underfloor ventilation is therefore advocated in such areas. this subject is explored more fully on page 533. the body is vulnerable to both increases and decreases in pressure and it is the lungs that often bear the brunt of the damage. increased pressure may result in blast injury or crushing of the chest while decreased pressure may result in the lungs literally bursting or dissolved gases being released within the blood (caisson disease), or the vascular alterations that underlie mountain sickness developing. some of these pressure changes entail a risk of pneumothorax and it is essential that this is properly investigated postmortem by the chest being opened under a water seal. loud music has been incriminated as a specific form of air pressure change causing pneumothorax and metereologists have shown that 'spontaneous' pneumothoraces tend to occur in clusters associated with natural drops in atmospheric pressure. 1,2 explosions may cause injury by the body being violently thrown against a less moveable object, by objects being thrown against the body or by the blast wave hitting the body. these mechanisms often act together but sometimes there is only blast injury, to which the lungs are particularly vulnerable. for a time it was considered that the damage was direct, the blast wave travelling down the airways to injure the lungs. however, at the start of the second world war, experiments conducted in the uk showed that the lungs were injured indirectly, the blast wave being transmitted to them through the chest wall: pulmonary blast injury is worst on the side of the body towards the explosion, and can be reduced by protective clothing. 3 underwater explosions are particularly dangerous because water is incompressible. there may be severe internal injury but no external evidence of damage other than a trickle of blood from the mouth or nose. this is because the injury is rate-dependent. quite small thoracic deform-ation may produce severe pulmonary damage if peak compression is attained very quickly, typically in less than 5 ms. conversely, severe chest wall distortion may produce only minor pulmonary contusion if this time is extended beyond 20 ms. 4 at necropsy, the lungs are contused, with blood evident in the airways and parenchyma. depending on the force of the blast, the haemorrhage may be pinpoint, patchy or confluent. it tends to follow the lines of the ribs and may be accompanied by pleuropulmonary lacerations having the same distribution. in this case there will also be haemothorax, pneumothorax and possibly air embolism. patchy pulmonary haemorrhages cuff the blood vessels. 5, 6 in patients who survive for a few days, the lungs resemble the liver macroscopically and histologically show chronic interstitial inflammation and fibrosis as well as haemorrhage. 7 other injuries are often present and fat embolism, aspiration pneumonia, fluid overload and infection may all be added to the effects of the blast wave. 'chest squeeze' is another form of barotrauma caused by high pressure but here the body is compressed rather than subject to a sudden wave of pressure as in blast injury. it is experienced by divers who descend very deeply, thereby subjecting their bodies to such high pressure that their chest walls are literally crushed, so that their ribs break and their lungs are severely compressed. more common mishaps experienced by divers include drowning and decompression sickness, both of which are dealt with below, and neurological syndromes such as nitrogen narcosis, which will not be considered further. 8 'burst lung' is the most acute form of decompression sickness. 9 it is experienced by divers and submariners making rapid ascents from depth and by aviators who ascend too rapidly in unpressurised aeroplanes, experience failure of a plane's pressure system or have to eject at high altitudes. injury to the lung is caused by trapped alveolar gas expanding so rapidly that it exceeds total lung capacity before it can escape through the trachea. the lungs literally burst: the alveolar walls rupture and blood mixes directly with alveolar air. the victim experiences chest pain and there may be blood-stained froth at the mouth or frank haemoptysis. air may enter the alveolar walls to cause interstitial emphysema or air embolism. asthmatics may be at particular risk because of regional air-trapping. 10 . diving mammals such as porpoises and whales are protected from such dangers of peripheral air-trapping by cartilage extending far out into the finest conductive airways so that these passages never close, even at the end of full expiration (fig. 7.2.1) . 11, 12 patients requiring positive-pressure artificial respiration are also at risk of burst lung, but the complications of the resultant interstitial emphysema differ from those experienced by divers. in divers, the chest wall is buttressed by the surrounding water and air in the interstitium is liable to track towards the hilum of the lungs and enter pulmonary veins, with resultant cerebral and coronary air embolism, either of which may prove fatal. 13 iatrogenic burst lung, on the other hand, takes place in patients whose chest wall is not so buttressed, and then outward rupture of the interstitial air is more likely, resulting in pneumothorax. extension of the interstitial emphysema to the mediastinum, neck and chest wall is also more likely in such patients, resulting in surgical emphysema at these sites. however, there are exceptional cases marked by both cerebral embolism and extensive air tracking. 14 the same circumstances that lead to burst lung may also cause decompression sickness, which is also known as caisson disease. 9 in this condition there is a sudden release of nitrogen gas that has gone into solution in the lipids of adipose tissue and of myelinated nervous tissue at the higher pressure: the released nitrogen gains access to the blood stream in which it forms bubbles. 15 doppler ultrasound techniques show that this is quite customary when divers ascend from depth, 16 but the lungs generally provide an effective filter so that there are no untoward systemic effects, although there may be sudden chest pain on deep inspiration ('the chokes'). gradual decompression permits the nitrogen to diffuse across the alveolar membranes and be exhaled. if, however, substantial amounts of nitrogen are released from solution, sufficient pulmonary arteries may be blocked to cause pulmonary hypertension, with resultant opening of arteriovenous communications or a patent foraman ovale, so permitting the gas to enter the systemic circulation. this is often followed by limb pains ('the bends') and perhaps cerebral symptoms ('the staggers'). fatal cases are characterised by gas bubbles within blood vessels throughout the body and froth in the heart chambers. delayed effects include ischaemic necrosis of bones and other tissues. 17 deep-diving mammals are protected by the same mechanism that prevents them suffering from burst lung. they exhale before diving and during the dive the chest is compressed to the extent that virtually all the gas in the lungs passes into the cartilage-buttressed nonrespiratory airways (see fig. 7.2.1) , resulting in very little to be absorbed by the blood. the pulmonary collapse also serves to reduce buoyancy. the distribution of the little gas that is absorbed is minimised by bradycardia. 18 many viscera experience anaerobic respiration but hypoxia is minimised in the heart and musculature by high levels of haemoglobin and myoglobin. the brain is further protected by the supplying arteries drawing on oxygen stored in an unusual spongelike cervical organ known as the rete mirabilis. mountain sickness is due to reduced atmospheric pressure brought about more slowly than that responsible for decompression sickness .19,20 it may be acute or chronic. acute mountain sickness is likely to be experienced by anyone who ascends above 3000-4000 m without a period of acclimatisation at intermediate levels. symptoms are as liable to occur in people born at high altitude who return after a few weeks spent at sea level as in those who go to the mountains for the first time: acclimatisation is obviously short-lived and is therefore necessary whenever an ascent is to be made. the ill-effects are commonly precipitated by exercise. in the susceptible, acute mountain sickness commonly appears within 3 days of ascent. the basis of acute mountain sickness is tissue hypoxia. it results in deteriorating intellectual and psychological function, headache, nausea, vomiting, and more rarely pulmonary and cerebral oedema. high-altitude pulmonary oedema is characterised by increasing dyspnoea, cyanosis and a dry cough, and later the production of copious, frothy sputum, which sometimes becomes blood-stained. 21 the pulmonary artery pressure is markedly raised but wedge pressures are normal, indicating that the left side of the heart is unaffected and that pulmonary venous constriction is unlikely to be an important contributory factor. the pulmonary oedema fluid has a high protein content 22 and the condition has been characterised as a non-cardiogenic high-permeability oedema associated with excessive pulmonary hypertension. 23, 24 hypoxia is a well-known cause of pulmonary arteriolar constriction but in acute mountain sickness the vascular response appears to be exaggerated for the pulmonary artery pressure is considerably higher than is usual for the altitude. an association with certain hla complexes (hla-dr6 and hla-dq4) suggests that this has a genetic basis. 25 although arteriolar constriction only tends to protect the pulmonary capillaries, it could explain the oedema if the process was patchy -as is the resultant oedema -for patchy arteriolar constriction would subject the rest of the lung to abnormally high pressures and lead to capillary stress failure in these areas (see pp. 402 and 448). 26, 27 measurements of capillary pressure suggest that this is indeed the case. 28 furthermore, vasodilators such as calcium channel blocking agents and inhaled nitric oxide gas 23, 29, 30 have been used with success to counter acute mountain sickness, supporting the idea that hypoxic vasoconstriction plays a central role. autopsy shows the lungs to be heavy and firm. the cut surface weeps oedema fluid, which is often blood-stained, but a striking feature is the patchy distribution of the changes. areas of haemorrhagic oedema alternate with others that contain clear oedema fluid and others that are normal apart from overinflation. pulmonary arterial thrombi are commonly found. microscopy confirms the presence of haemorrhagic oedema and may show neutrophils and hyaline membranes in the alveoli. the alveolar capillaries are congested and may contain thrombi. there may also be an increase in mast cells and rarely pulmonary infarction. the right ventricle is commonly dilated whereas the left ventricle is normal. highlanders generally show right ventricular hypertrophy and increased muscle in their pulmonary arteriesm, changes that are not apparent in lowlanders. 31, 32 chronic mountain sickness prolonged residence at high altitude leads to hypoxic pulmonary hypertension (see p. 424), an increase in red cell mass and cor pulmonale. livestock taken from lowland plains to high-altitude pastures suffer similarly but the natural stock of the himalayas and ethiopian highlands are apparently immune. so too are other species long established at high altitude such as the llama and yak. these species are said to have adapted to their climate, that is, the forces of natural selection have bred out the pulmonary vasoconstrictive response to hypoxia. cattle of european origin and humans acclimatise to high altitude by processes such as increasing their red cell mass but generally they are not adapted like native species and suffer hypoxic pulmonary hypertension at altitudes in excess of 3000 m. certain himalayan highlanders may be an exception to this in that their small pulmonary arteries are reported not to show the muscularisation that characterises hypoxic pulmonary hypertension. 33 in cattle of european origin, the dependent oedema of right-sided cardiac failure caused by hypoxic pulmonary hypertension affects the breast (brisket) particularly and in the rocky mountains of north america such cattle are said to have 'brisket disease' . 34 a human counterpart of this has been described in children of chinese ancestry who have been taken to reside in tibet and who have developed a fatal form of subacute infantile mountain sickness. 35 a small minority of permanent residents in the andes develop the changes of chronic mountain sickness to a marked degree and are said to suffer from monge's disease. 36 the basis of this is alveolar hypoventilation, which leads to a progressive fall in systemic arterial oxygen saturation and elevation of haemoglobin concentration to an unusually severe degree. the latter averages about 25 g/dl, which exceeds even the 20 g/dl found in healthy high-altitude residents. patients with monge's disease are so deeply cyanosed that their lips are virtually black. their pulmonary artery resistance is also markedly raised. the cause of the alveolar hypoventilation is uncertain but the only cases of monge's disease that have come to necropsy had conditions such as kyphoscoliosis that predispose to alveolar hypoxia. drowning is defined as suffocation by submersion, and usually occurs in water. it is the commonest cause of accidental death among divers but 96% of drowning accidents do not involve deep descents. falling into quite shallow water is a particularly common cause of drowning in young children. in adults, men outnumber women by 4 to 1. more die in fresh water than the sea, not because it is more hazardous to the lungs than sea water, but because unguarded inland waters and swimming pools are visited more frequently. alcohol consumption contributes to many deaths by drowning. drowning is not simply a matter of being unable to keep one's head above water. this may be merely a secondary event. for example, the entry dive may result in underwater head injury, or the exertion of swimming may precipitate a heart attack. furthermore, the struggling swimmer going down for the third time ('drowning not waving') is the exception: most drowning is characterised by the swimmer failing to surface or quietly dropping beneath the surface without anyone noticing. swimming underwater can be extremely hazardous if it is preceded by hyperventilation, a danger that needs to be more widely appreciated. hyperventilation results in undue loss of carbon dioxide so that instead of hypercapnia forcing the swimmer to surface to breathe, progress under water may be continued until hypoxia causes sudden loss of consciousness. panic contributes to many swimming accidents and is often precipitated by the inadvertent aspiration of just a little water. most people are naturally buoyant, but only slightly so. with the lungs fully expanded the average adult has a positive buoyancy of about 2.5 kg, which is sufficient to keep the head out of the water if the rest of the body is submerged. if an arm (weight about 3 kg) is raised to wave for help, the head will go down. if the swimmer shouts, exhalation reduces buoyancy to neutral at normal end-expiration and to negative at residual volume. buoyancy cannot be regained when the head is submerged and unless able to swim to the surface, the person will continue to sink. autopsy generally shows that the lungs are full of water, but some victims die of 'dry drowning' due to laryngospasm. events may also be modified by the temperature of the water. sudden immersion in cold water may result in tachycardia, hypertension and hyperventilation, making it difficult for the victim to keep the airways free of water. it may also result in sudden death due to ventricular fibrillation. even a good swimmer loses consciousness within an hour of immersion in very cold water. drowning is then inevitable unless a correctly fitted life jacket is worn, in which case there is a danger of death from hypothermia. however, as in open heart surgery, cold prolongs the interval before there is irreversible brain damage. if the person is rescued, water in the lungs is quickly absorbed, even if it is saline, and therefore hyperosmolar:aspirated sea water is quickly equilibrated by pure water joining it from the blood but the alveolar epithelial barrier remains impermeable to protein and once osmotic equilibrium is reached, all is quickly reabsorbed. [37] [38] [39] fresh water is absorbed even more quickly. it is unnecessary to tip the patient to hasten this process. any water recovered in this way comes from the stomach and time that should be devoted to mouth-to-mouth breathing and cardiac massage is lost. these resuscitative efforts may need to be prolonged as fresh water in particular inactivates alveolar surfactant, leading to alveolar collapse which persists until the surfactant is replenished. very few victims who are resuscitated on site fail to survive, and very few who cannot be resuscitated on site recover later. interchange of fluid between the blood and air spaces may cause major fluctuations in plasma volume with consequent changes in ionic concentrations and haemolysis. hypervolaemia may cause circulatory problems but hyperkalaemia consequent upon the haemolysis is not thought to be as important as was formerly believed: ventricular fibrillation following submersion is more likely to be a complication of hypothermia than of electrolyte imbalance. circulatory collapse may ensue shortly after rescue. this is due to loss of the circulatory support provided by the pressure the water exerts on the body, which results in a considerable increase in cardiac output while the body is immersed. on leaving the water the loss of this support results in a tendency to venous pooling. although this is countered by baroreceptor responses, these are reduced by prolonged immersion in cold water. circulatory collapse is believed to be the cause of death in many persons who perish within minutes of rescue. to counter this effect, patients should be lifted out of the water in the prone position. it can be seen that, in fatal cases, the pathologist is faced with several possibilities. thus, death may have been due to: • natural causes before the body entered the water • unnatural causes before entry, the body merely being disposed of in the water • natural causes in the water • injuries received in the water from impact with rocks, a boat or a ship's propeller, or in tropical waters from predators such as a crocodile or a shark (any of which may also be incurred after death, as may disfigurement by fish and rats) • 'dry drowning' • true drowning • hypothermia • circulatory failure after rescue. true drowning is indicated by froth in the airways and heavy waterfilled lungs. both fresh and salt water contain numerous microscopic algae known as diatoms and those representative of the water in which the drowning occurred are found in the lungs. unless death occurred before submersion, diatoms are also found in other viscera because these tiny life forms easily enter the circulation. thus, the presence of diatoms in digests of organs such as the kidneys, liver, brain and bone marrow suggests that death was due to drowning. because they have a siliceous capsule, diatoms are resistant to putrefaction as well as digestion and can be identified in the body long after death. however, a positive test is not always accepted as proof of drowning and a negative test does not exclude drowning. the various physical forms in which respirable environmental agents may be encountered are defined in table 7 .2.1. some effects of inhalant lung injury are recognised as distinct disease entities and are dealt with elsewhere: for example, the pneumoconioses on page 327, extrinsic allergic alveolitis on page 279, chronic bronchitis on page 98 and lung cancer on page 532. other respirable agents, such as lead fume and carbon monoxide gas, exert their harmful effects elsewhere in the body and will not be considered further. this section is concerned with toxic substances that may be inhaled by the general public. those that are more likely to be encountered in the workplace or in war zones are considered on page 355. the lungs have a rather stereotyped pattern of response to inhaled toxins, displaying degenerative changes and inflammation of varying degree, the former sometimes amounting to necrosis. in general, the site of maximal absorption or injury is related to solubility (for gases and vapours) and particle size (for aerosols such as dusts, fog, fumes, mists, smog and smoke): the less water-soluble and the smaller the particle size, the further down the respiratory tract the agent will penetrate ( fig. 7 [40] [41] [42] thus, ammonia produces intense congestion of the upper respiratory passages and laryngeal oedema whereas phosgene has little effect on these sites but causes pulmonary oedema. 40 air pollution [43] [44] [45] [46] [47] the toxic (as opposed to allergenic) air pollutants thought to pose the greatest threat to the lungs comprise smoke particles, sulphur dioxide, oxides of nitrogen, various aldehydes and ozone. smoke and sulphur dioxide derive particularly from the combustion of fossil fuels in domestic fires and power stations, nitrogen dioxide is an important car exhaust and domestic gas appliance pollutant and ozone is the principal photochemical product of smog. aldehydes such as formaldehyde and acrylic aldehyde (acrolein) also contribute to general air pollution because they are released in the combustion of diesel oil and petrol. collectively, these pollutants have been incriminated in the exacerbation (rather than causation) of asthma. they also predispose to respiratory infection and result in airway inflammation and hypersecretion. 48, 49 their effect on children is of particular concern because development of the lungs is known to continue well into childhood and damage to the lungs before their growth is complete is likely to be irreparable. at the other extreme of life episodes of severe air pollution are known to hasten the deaths of many patients with chronic airway disease. particularly high concentrations of the agents responsible for air pollution may be encountered in industry and their effects are therefore also considered in chapter 7.1, on occupational diseases of the lung. many of the polycyclic hydrocarbons found in polluted air are carcinogenic (see p. 532) and it is therefore not surprising that urban air pollution has been found to be associated with excess mortality from lung cancer. 50 domestic air pollution is rife in many of the poorer parts of the world due to the burning of biomass (wood, dried cow dung, bagasse, straw) in unventilated living rooms for heating and cooking. the women are particularly at risk of developing chronic bronchitis while their children have an increased incidence of acute respiratory infections. 51,52,52a volcanic ash (tephra) irritates the eyes, skin and respiratory tract and in some eruptions may contain much free silica (e.g. montserrat in 1995 and mount st helens, washington state, usa in 1980) or be associated with the release of radon gas (e.g. the azores in 1957). 53 the destruction of the world trade center in 2001 caused massive air pollution of new york city that had lasting respiratory effects on survivors, rescue workers and local residents. [54] [55] [56] at the time of the disaster there was much smoke from combustion of aeroplane fuel and flammable materials in the building while the collapse of the twin towers released dust from cement and dry-wall partitions that was highly alkaline. [57] [58] [59] this caused considerable irritation of the eyes and the conductive airways. a year later many victims were still suffering from bronchial hyperreactivity and poor ventilatory function, in a so-called reactive airways dysfunction syndrome 54, 55 and there was continuing spirometric decline 5 years later. 60 the respirable portion of the dust formed only a small fraction of the whole but given the level of exposure its future effects cannot be discounted, particularly as it contained substances such as asbestos. unusual effects attributed to the disaster include acute eosinophilic pneumonia and granulomatous pneumonitis. 61, 62 allergenic air pollutants are dealt with in detail in the sections on asthma (see p. 109) and extrinsic allergic alveolitis (see p. 279). allergenic air pollution is generally occupational or domestic but periodic widespread air pollution was responsible for the epidemics of asthma seen in barcelona in the 1980s, which were eventually traced to ships discharging cargoes of soya flour (see p. 114). smoking-related diseases figure large throughout this book and in this section they are merely summarised collectively. of the greatest importance, both in the number of patients they affect and in their clinical effects on the individual, are the various forms of chronic obstructive lung disease and lung cancer, but there are many other respiratory diseases associated with smoking, and a few that are less common in smokers (box 7.2.1). 63 not surprisingly, these diseases are often encountered in combination and sometimes one may obscure another. for example, a cigarette smoker may have emphysema in the upper lobes and idiopathic pulmonary fibrosis in the lower lobes. 64, 65 alternatively, langerhans cell histiocytosis and desquamative interstitial pneumonia may affect the same parts of the lungs, in which case the focal lesions of the former may be masked by the latter condition. 63 the term 'smoking-related interstitial lung disease' has been introduced to cover a spectrum of interstitial diseases related to smoking 63, 66, 67 as well as being used in a more restricted sense to describe a combination of air space enlargement and interstitial fibrosis predominantly affecting the lower lobes. 68,69,69a quite advanced interstitial fibrosis has been reported in smokers with no clinical evidence of interstitial lung disease. 69b early changes detectable in smokers include chronic bronchiolitis, fibrosis of the bronchiolar wall and mild peribronchiolar interstitial fibrosis. 70, 71 even earlier changes are detectable at the molecular level: as many as 152 smoking-responsive genes that are significantly up-regulated or down-regulated have been identified in normal cigarette smokers. 72 there is marked individual variation, which may explain why many lifelong heavy smokers experience no respiratory problems. histological evidence that a patient smokes is provided by an increase in the number of alveolar macrophages and a characteristic brown discoloration of cytoplasm due to the phagocytosis of tar and other particulate matter derived from tobacco smoke (fig. 7.2.3) . cigarette smokers are at greater risk of lung disease than cigar and pipe smokers, probably because they inhale more deeply. they do this because cigarette smoke is more acid than cigar and pipe smoke and its nicotine content is therefore absorbed more easily through the lungs than the buccal mucosa. smokers obviously put their own health at greatest risk but the lesser hazards of passive smoking are now well recognized (see p. 532). passive smoking involves both the smoke exhaled by others and that coming from smouldering tobacco between puffs, the latter being known as sidestream smoke. the harmful effects of maternal smoking on the unborn child also come in this category. they include increased airway responsiveness and reduced lung function during the neonatal period and an increased risk of sudden infant death syndrome. reduced numbers of alveolar attachments to the bronchioles have been demonstrated in such infants. 73 smoking is also associated with disease of other organs (e.g. carcinoma of the oesophagus and bladder) but these are outwith the remit of this text. tobacco smoking by waterpipe (shisha, hubble-bubble) is enjoying a rise in popularity, both in its heartland, the middle east, and western countries, and wherever it is practised it is widely perceived as being less dangerous than smoking cigarettes. 74 this is probably a mis conception. what evidence there is suggests that waterpipe tobacco smoking is just as harmful as cigarette smoking, if not more so. 75 the lungs may be injured in burned patients in many ways (box 7.2.2) 76,77 but an important consideration when a body is recovered from a fire is whether death was due to the fire or took place beforehand, the latter raising the possibility of foul play. a vital reaction to the skin burns and the presence of soot in the lower airways provide evidence that death occurred in the fire but an absence of soot from the airways may be due to death occurring rapidly, from asphyxia or poisoning by gases released in the conflagration. soot is cleared rapidly and if the patient survives a few days an absence of soot from the airways is to be expected. 76 lung injury may result directly from heat and smoke inhalation or indirectly from the release of mediators associated with blast injury or shock. although air temperature in a fire may reach very high temperatures thermal injury seldom extends beyond the carina but more extensive injury from heat alone was seen in men exposed to steam escaping from a fractured boiler pipe. 78 those dying immediately showed coagulative necrosis of the respiratory mucosa down to the level of the alveolar ducts and alveolar congestion and oedema, while those surviving a little longer exhibited diffuse alveolar damage. the diffuse alveolar damage probably represented a manifestation of shock from their extensive cutaneous scalding whereas the mucosal necrosis is directly attributable to heat. diffuse alveolar damage is extrinsic allergic alveolitis sarcoidosis blast injury asphyxia poisoning by combustion products (e.g. carbon monoxide, cyanide) direct thermal injury (largely limited to the trachea) irritant smoke, fume and gas (e.g. oxides of nitrogen, ammonia, acrolein, sulphur dioxide) hypovolaemic shock secondary to skin loss septicaemic shock from: infected skin burns infected central lines secondary viral and bacterial pneumonia fluid overload tracheostomy complications, including tracheobronchitis, pneumonia and barotrauma oxygen toxicity absorption of toxic topical disinfectants thromboembolism uraemia usually part of systemic multiorgan failure in these patients, and is the leading cause of death in burns. 79 the ubiquity of plastics today means that smoke contains numerous irritants, including isocyanates, aldehydes and fluorinated organic chemicals. irritant smoke products have two principal effects. firstly, they cause an immediate painful stimulation of the eyes and respiratory tract which at low concentrations may prevent escape and at high concentrations may cause laryngeal spasm and death. secondly, they cause bronchopulmonary injury some hours after exposure. burned patients dying within 4-12 days often show tracheobronchial necrosis and diffuse alveolar damage with prominent hyaline membranes. 76, 77, 80 secondary herpesvirus infection is often present. 81, 82 the respiratory changes caused by heat and smoke are non-specific and careful consideration of the many causes of lung injury in burned patients listed in box 7.2.2 and of the clinical circumstances and management is generally required. often it will be concluded that the cause of the lung injury is multifactorial. long-term consequences of smoke inhalation include bronchiectasis and obliterative bronchiolitis. 83 methyl isocyanate, the chemical released at bhopal the bhopal catastrophe of 1984 was caused by the accidental release of 30 tons of methyl isocyanate gas (ch 3 -n=c=o) from a pesticide plant. 84 over 200 000 people were exposed, of whom 2500 died, mostly within hours of exposure, and 60 000 were seriously injured. the victims complained of intense ocular and respiratory irritation. some survivors were left with persistent respiratory impairment, which was thought to be due to obliterative bronchiolitis. 85, 86 methyl isocyanate is an extremely potent respiratory irritant, destroying the epithelium throughout the conducting airways, with comparatively less parenchymal injury. in survivors, epithelial regeneration, often involving squamous metaplasia, quickly commences, but not before endobronchial granulation tissue projections have developed, resulting in obliterative bronchiolitis. tear gases are chemical irritants delivered as an aerosol for the purpose of riot control. they react with mucocutaneous sensory nerve receptors causing intense irritation of the eyes, mucous membranes and skin. the respiratory effects are mainly concentrated on the upper tract so that there is violent sneezing, severe rhinorrhoea and cough but there may also be tracheobronchitis and rarely pulmonary oedema. 87 patients with pre-existent asthma or chronic obstructive pulmonary disease are most severely affected while others may be left with reactive airways dysfunction. toxins reaching the lungs via the blood stream may be drugs, food contaminants, metabolites produced elsewhere in the body, or chemicals ingested intentionally or accidentally, either in the home or the workplace. the lungs are selectively damaged by certain blood-borne toxins for a variety of reasons. for example, the herbicide paraquat is preferentially taken up by the lungs because of its molecular homology with certain endogenous substances. as detailed below, the type i alveolar epithelial cells are the cells that bear the brunt of the damage in paraquat poisoning. on the other hand, the alveolar capillary endothelium has its own selective uptake mechanisms (see metabolic functions of the pulmonary endothelium, p. 23) which may be responsible for it being selectively damaged by other chemicals. the bronchiolar clara cells are selectively injured by some ingested chemicals because they are equipped to deal with inhaled xenobiotics, but occasionally this activity results in metabolites that are extremely toxic. an example of this from veterinary medicine is provided by the furan-derivative 4-ipomeanol, which is found in mouldy sweet potatoes and results in acute pulmonary oedema in cattle fed such a diet. when this chemical is injected into mice, the bronchioles are denuded of clara cells whereas the intervening ciliated cells are completely unaffected. the selective damage to the bronchiolar clara cells appears to stem from the oxidative efficiency of their p-450 cytochromes, 88 which is much higher than those of the liver. chemicals having a similarly selective effect on bronchiolar clara cells include 3-methylfuran, carbon tetrachloride, naphthalene and 1,1-dichloroethylene, the last of which is a volatile compound that is widely used in the plastics industry. procarcinogens may be activated in the airways by similar mechanisms. paraquat is a dipyridylium compound that is widely used in agriculture as a herbicide. it kills all green plants but is inactivated on contact with the soil. it is applied as a spray and if the manufacturer's instructions are followed there is no danger to health. most fatal cases of paraquat poisoning, both accidental and suicidal, have been due to ingestion of the 20% aqueous solution gramoxone. the less concentrated granular form weedol is unlikely to be ingested accidentally but may be taken suicidally. 89 paraquat is not absorbed by the intact skin but repeated or prolonged application damages the epidermis so that absorption into the blood stream with consequent systemic effects is possible, but rare. 90 although paraquat has toxic effects on the liver, kidneys and myocardium, these are transient and attention has centred on the pulmonary changes, which are usually fatal. following suicidal ingestion of large amounts of paraquat, death from multiorgan failure and pulmonary haemorrhage occurs within a few days, whereas most victims of accidental paraquat poisoning die from progressive pulmonary fibrosis between 10 and 14 days after ingestion. in those who survive longer, a honeycomb pattern of pulmonary fibrosis may be apparent. 91 paraquat is a powerful oxidant and owes its toxicity to the production of active oxygen radicals. the lungs are particularly susceptible because paraquat is concentrated there by an active uptake mechanism in the alveolar epithelium. the inadvertent uptake of paraquat probably stems from a similarity between the molecular arrangement of its quaternary nitrogen atoms and the amine groups of endogenous oligoamines such as putrescine, spermidine and spermine, which are concerned in alveolar epithelial cell division and differentiation (fig. 7.2.4) . 92 this results in paraquat levels being 6-10 times higher in the lung than in the plasma. once taken up by the lung, paraquat is not metabolised but participates in redox cycling so that superoxide radicals are constantly produced. epithelial injury is proportional to the concentration of paraquat, while it is lessened by hypoxia and antioxidants such as superoxide dismutase, and potentiated by increased concentrations of oxygen. [93] [94] [95] [96] the high concentration of oxygen in the alveoli is a further reason why the lungs are particularly vulnerable to paraquat. knowledge of the toxic effects of paraquat comes from observations on autopsy series 89, 97, 98 and from experimental studies that have enabled the sequence of pulmonary changes to be observed. [99] [100] [101] [102] in accordance with paraquat being taken up by the alveolar epithelium, electron microscopy shows that these cells suffer more profound damage than the endothelium. 99 type i epithelial cells swell and undergo necrosis (fig. 7. 2.5), 103 whilst type ii cells, although remaining capable of proliferation, show ultrastructural evidence of damage with derangement of cell organelles. 99, 100 histological changes in the lungs follow the pattern of diffuse alveolar damage, with a characteristic feature of the early exudative phase being intense vascular congestion and alveolar haemorrhage. 89, 97, 104 hyaline membranes are most clearly seen by about 5 days (fig. 7.2.6 ) and epithelial proliferation and fibrosis are conspicuous by about 14 days. the pattern of pulmonary fibrosis in paraquat poisoning has been disputed. some authors have stressed its interstitial position, whereas others have clearly demonstrated that it is intra-alveolar. 98, 102, [104] [105] [106] [107] however, as described on page 148, it generally assumes an obliterative pattern of intra-alveolar fibrosis in which the lumina of several adjacent alveoli are totally effaced, rendering them completely airless (see fig. 4 .24, p. 148). a new multisystem disease appeared abruptly in the environs of madrid in 1981. [108] [109] [110] over 20 000 people were affected and about 1 in 60 died. the disease was initially thought to be mycoplasma pneumonia but was soon found to be associated with the use of adulterated oil sold illicitly by door-to-door salesmen. although it was sold for culinary purposes the oil had been produced for industrial use in steel manufacture. it consisted of rapeseed and olive oil mixed with liquified animal fat, aniline and other organic chemicals. it has not been possible to identify the exact chemical responsible for the disease or to reproduce the changes in other species but the later induction of similar pathological changes by another substance contaminated with an aniline derivative is possibly relevant (see l-tryptophan-induced eosinophilia-myalgia syndrome, p. 389). 111 some clinical and pathological features of the disease suggest that immune mechanisms may also be involved. the initial clinical features included fever, respiratory distress, cough, haemoptysis, skin eruptions and marked eosinophilia. radiographs suggested pulmonary oedema and sometimes showed pleural effusion. about 5% of patients died at this stage but most recovered quickly. however, within a few weeks many were readmitted to hospital with nausea, vomiting, diarrhoea and abdominal pain. about a quarter then proceeded to develop weakness, myalgia, weight loss, scleroderma-like skin signs and pulmonary hypertension. 112, 113 many of these patients died after a long, wasting illness or are permanently disabled with neurological and hepatic disorders. in the early phase the lungs showed the most severe changes, which consisted of a combination of diffuse alveolar damage, eosinophilic infiltrates and arterial luminal narrowing by endothelial swelling and vacuolation, intimal foam cell infiltration and a non-necrotising vasculitis. 109, 112, 114 there was also capillary thrombosis, which later extended into arteries and veins, culminating in fibrosing obliteration of these blood vessels. in some patients dying of haemoptysis, dilated thin-walled blood vessels were identified in the mucosa of major blood-filled airways. late features in the lungs included plexogenic arteriopathy (see p. 420), possibly secondary to changes in the liver. similar inflammatory and vascular changes were seen in many other tissues. notable extrapulmonary features included fasciitis, vasculitis, neuronal degeneration, perineuritis, hepatic injury and tissue eosinophilia. sauropus androgynus is a vegetable that is widely cultivated for the table in many south-eastern asian countries. it is apparently harmless when cooked but recently there has been a vogue in taiwan for consuming large amounts of its unprocessed juice, blended with that of guavas or pineapple, because of its supposed efficacy as a slimming aid and in blood pressure control. coincident with this fad there has been an upsurge in patients with symptoms of obstructive lung disease. within a 4-month period more than 60 such patients were seen at one hospital. [115] [116] [117] they had four features in common: recent consumption of uncooked s. androgynus juice, fixed ventilatory obstruction, radiological evidence of bilateral bronchiectasis and an absence of any previous chronic respiratory disease. four patients agreed to undergo open-lung biopsy. this showed chronic bronchiolitis or obliterative bronchiolitis of constrictive pattern. the lymphocytes were mainly t cells but immunofluorescent and electron microscopy showed no evidence of an immune process. four patients underwent single-lung transplantation. the excised lungs showed sclerotic obliteration of bronchial arteries in the walls of bronchi 4-5 mm in diameter with segmental necrosis of bronchi 2-4 mm in diameter. the changes were considered to fit best with segmental ischaemic necrosis of bronchi at the watershed zone of the bronchial and pulmonary vasculature. 118 further patients have required lung transplantation but public education of the dangers of this herbal medicine now appears to have been successful. 119 alcohol and nicotine outstrip all other recreational drugs in popularity and their effects are of course well known. those of tobacco smoking are summarised above and dealt with in detail in the chapters on obstructive lung disease (chapter 3) and carcinoma of the lung (chapter 12.1). less well known is the lung disease that results from smoking blackfat tobacco, a practice popular with guyanese indians. blackfat is the trade name of a type of tobacco that is flavoured with mineral oil, some of which vaporises and is inhaled when the tobacco is smoked, to cause exogenous lipid pneumonia (see p. 314 [ch8] ). 120 in recent years the smoking of two other substances, marijuana and cocaine, has gained in popularity. it would not be surprising if the long-term effects of smoking these substances were similar to those of cigarette smoking but as yet it is too early to judge. however, the short-term effects are similar to those of tobacco smoking and this bodes badly for their ultimate effects. marijuana consists of the dried leaves of the cannabis plant, also known as hemp, as opposed to hashish, which is the plant's resin, and a further extract known as 'weed oil' . all these substances are smoked because they contain cannabis alkaloids which have psychoactive effects. however, this habit also exposes the lungs to many of the same respiratory irritants that are found in tobacco smoke. initial exposure to marijuana smoke often results in coughing while habitual smokers produce black sputum. bronchial biopsy shows inflammation and squamous metaplasia and bronchoalveolar lavage demonstrates increased numbers of cells, which are predominantly macrophages but also include neutrophils. [121] [122] [123] [124] [125] these changes are virtually identical to the short-term effects of tobacco smoke and are therefore likely to be similarly followed by the development of chronic obstructive lung disease and lung cancer. indeed, the dangers of smoking marijuana are probably greater than those of smoking tobacco as compared with tobacco smoking it is associated with a fivefold greater increase in blood carboxyhaemoglobin and a threefold increase in the amount of tar inhaled. 126 it is estimated that three cannabis cigarettes result in the same degree of bronchial damage as 20 tobacco cigarettes. 127 there is also evidence that the effects of smoking marijuana and tobacco are additive. 128 not surprisingly therefore, epidemiological studies report a doserelated impairment of large-airway function in marijuana smokers. 129 there are also several reports attributing pneumothorax to marijuana smoking ( fig. 7.2.7) . 130, 131 the pneumothorax may be spontaneous or develop during the deep, sustained inspiratory effort involved in smoking marijuana (or cocaine), which may be enhanced by a partner applying positive ventilatory pressure by mouth-to-mouth contact. thoracoscopy in such cases has shown predominantly apical, irregular bullous emphysema, while lung biopsy has demonstrated widespread alveolar filling by heavily pigmented macrophages. 131, 132 evidence is also beginning to accumulate that long-term cannabis use increases the risk of lung cancer. 133 smoking cannabis in the form of weed oil is also reported to result in exogenous lipid pneumonia. 134 cocaine cocaine hydrochloride is a fine white powder derived from the leaves of the plant erythroxolon coca by a complex chemical process. it is heat-labile and therefore cannot be smoked. users inject it intravenously or inhale it unheated through the nose, the latter practice being known as 'snorting' . however, a heat-stable free-base form that can be smoked is easily prepared from the hydrochloride with baking powder and a solvent such as ether. this process results in a crystalline deposit that is known as 'rock' because of its appearance or 'crack' because of the crackling sound it emits when heated. when smoked, the cocaine is readily absorbed and an intense surge of euphoria is experienced within 8 seconds. the intravenous route takes twice as long and 'snorting' several minutes. the hard addict therefore prefers to smoke 'crack' . a variety of pulmonary complications of smoking free-base cocaine has been reported. 128, [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] acute effects include cough, shortness of breath, chest pain and haemoptysis. asthma may be aggravated, black sputum is produced, and pneumothorax and interstitial emphysema have resulted from valsalva manoeuvres undertaken in the belief that they promote even more rapid absorption. biopsy has shown pulmonary congestion and oedema, organising pneumonia, haemorrhage, haemosiderosis, diffuse alveolar damage and interstitial pneumonia or fibrosis. less common effects include eosinophilic pneumonia, extrapulmonary eosinophilic angiitis, medial thickening of pulmonary arteries and the barotrauma described above (see fig. 7.2.7) . severe burning of the airways has also been seen due to 'crack' being smoked before all the ether used in its preparation has evaporated. 'snorting' unheated cocaine has its own complications: substances such as cellulose or talc with which the drug is 'cut' (mixed as a diluent) are liable to provoke a foreign-body giant cell reaction in the lungs (fig. 7.2.8 ). 145 however, particles of foreign material larger than those in the usual respirable range (allowing for the fibrous shape of substances such as cellulose) should suggest intravenous use (see 'filler embolism', below). heroin is usually injected, but it may be smoked, when, as with marijuana, it is liable to lead to a very pronounced macrophage response. intravenous heroin abuse sometimes causes the sudden onset of a potentially fatal high-permeability pulmonary oedema (fig. 7.2.9 ). intravenous abuse of heroin and other drugs is also liable to cause 'filler embolism', which will now be considered. 'filler embolism' is the result of illicit drug usage in which compounds designed for oral use are injected intravenously to heighten their effects. oral preparations consist largely of fillers such as talc or starch and this insoluble particulate matter accumulates in the pulmonary capillaries. it provokes a foreign-body giant cell reaction, thrombosis and fibrosis and may cause pulmonary hypertension ( fig. 7 this 'designer' drug, taken for its central stimulant activity (street names 'ice' or 'u-4-e-uh', pronounced euphoria), is related to the appetite suppressor aminorex, discussed on page 424, and has similarly been associated with pulmonary hypertension. assessing whether a particular clinical manifestation represents an adverse drug reaction considers previous experience with the drug, alternative aetiological agents, the timing of events, drug levels, and the effect of withdrawing the drug and rechallenge with the drug. 7 it is worth bearing in mind that: • one drug may cause several patterns of disease. • one pattern of disease may be produced by a variety of drugs. • a drug reaction may develop long after the drug has been withdrawn. • a drug reaction may develop suddenly even though the dose of the drug has not been altered. • drug effects may be augmented by factors such as age, previous radiotherapy and elevated oxygen levels. • drug reactions may be localised. • many drugs cross the placenta to affect the fetus. an alternative classification of adverse drug reactions, which is more appropriate to pathology practice and which will be followed here, is one based on the pattern of disease. some pathological patterns of drug-induced lung disease are shown in table 7 central depression of respiration occurs as a side-effect of barbiturates, morphine and its derivatives, and even mild sedatives, and may be particularly troublesome in patients suffering from chronic obstructive lung disease. ventilation in such patients may be largely dependent on hypoxic respiratory drive and treatment with oxygen may therefore also have an adverse effect on respiration by lowering the degree of hypoxia and so diminishing the stimulation of the respiratory centre. peripheral impairment of the respiratory drive may be brought about by aminosides and other antibiotics, while corticosteroids may result in a myopathy affecting the respiratory muscles. other iatrogenic hazards affecting the peripheral nerves controlling respiration include nerve root disease complicating immunisation and surgical damage to the spinal and phrenic nerves. asthmatic patients are particularly susceptible to exacerbations of their disease by drugs (box 7.3.2). this effect may occur either as a predictable pharmacological side-effect of the drug or as an idiosyncratic response. examples of the former include β-adrenergicic antagonists and cholinergic agents while examples of the latter include sensitivity to the colouring agent tartrazine, for which reason many manufacturers have eliminated tartrazine from their red, orange and yellow tablets. allergic bronchoconstriction also forms part of generalised anaphylactic reactions induced by vaccines and antisera and occurs as a localised response to penicillin, iodine-containing contrast media, iron dextran and other medicaments. bronchospasm may also be initiated by the non-specific irritant effect of inhaling nebulised drugs if they are prepared as a hypotonic solution, a side-effect that is prevented by using isotonic solutions. aspirin and other non-steroidal anti-inflammatory agents aspirin-induced asthma has been recognised for many years and more recently several of the newer anti-inflammatory drugs have been found to exacerbate asthma in certain sensitive individuals. the basis for this is uncertain but the likelihood of an individual antiinflammatory drug provoking an asthmatic response is related to its potency as an inhibitor of prostaglandin cyclooxygenase pathway, resulting in the production of leukotrienes. [8] [9] [10] as well as asthma being exacerbated by drugs, the disease has been caused by occupational exposure in the pharmaceutical industry to certain drugs which can be inhaled during manufacture, notably penicillin, cephalosporin, methyldopa, cimetidine and piperazine. obliterative bronchiolitis of the constrictive type has been reported with penicillamine 11,12 and gold 13, 14 but in many cases it is possibly the underlying condition rather than the drug that is res ponsible (see p. 123). this is often rheumatoid disease, which is sometimes complicated by bronchiolitis obliterans whether the patient is under treatment or not. 15 organising pneumonia extending into peripheral bronchioles (see p. 120) may be seen with a variety of drugs but results in a restrictive rather than obstructive lung defect and is to be regarded as a cytotoxic effect of the drug acting primarily at the alveolar level (see below). raw sancropus androgyns taken as a slimming aid causes severe obliterative bronchiolitis (see p. 376). the cytotoxic effects of drugs may be acute or chronic, leading to changes as varied as pulmonary oedema, diffuse alveolar damage, pulmonary haemorrhage and haemosiderosis, organising pneumonia, interstitial pneumonitis and interstitial fibrosis. 16, 17 some of the most severe acute effects are seen with the chemotherapeutic agents used in malignant disease 18 but they are also recorded with drugs that are not traditionally thought to be cytotoxic, e.g. desferrioxamine administered as a prolonged intravenous infusion in acute iron poisoning. 19 pulmonary toxicity due to busulphan was first described in 1961, 20 and has been the subject of several subsequent studies. [21] [22] [23] [24] it remains the mainstay of treatment for chronic myeloid leukaemia. like other alkylating agents, it acts by cross-linking dna strands. clinical estimates of the incidence of pulmonary toxicity vary around 4% but subclinical damage is thought to be much more common. although not strictly dose-dependent, toxicity is rarely seen with a total cumulative dose of less than 500 mg. synergy with radiation and other cytotoxic drugs occurs. 25 similar effects have been reported for most cytotoxic agents, particularly bleomycin. 26 pulmonary toxicity is seen less commonly with other alkylating agents, such as cyclophosphamide and melphalan. [27] [28] [29] [30] bleomycin is a cytotoxic antibiotic derived from streptomyces species. it is widely used in the treatment of neoplasms such as lymphomas and germ cell tumours, and is thought to produce its therapeutic and toxic effects by altering the normal balance between oxidants (active oxygen radicals) and antioxidant systems. 26 bleomycin produces superoxide radicals when incubated with oxygen and iron in vitro. oxygen enhances its effects, 31 a fact well known to anaesthetists who accordingly take care to limit concentrations of inspired oxygen to 30% in patients on bleomycin who are undergoing surgery. [32] [33] [34] radiotherapy and cytotoxic agents such as bleomycin are also synergistic. bleomycin is preferentially concentrated in the lungs and pulmonary fibrosis can be produced in animals when it is administered intravenously, intraperitoneally or by intratracheal instillation. electron microscopy shows that the early changes consist of swelling and vesiculation of endothelial cells, interstitial oedema and type i epithelial cell necrosis. 35, 36 the reported incidence of bleomycin toxicity varies from 2 to 40% depending on the type of patient being treated and on dosage. in general, toxic effects increase with age and cumulative dose: above a total dose of about 500 units they rise significantly. the acute morphological changes attributable to drugs include pulmonary oedema and diffuse alveolar damage. acute pulmonary oedema is seen in heroin addicts who die while injecting themselves intravenously but it is also seen in patients administered a variety of drugs therapeutically, for example hydrochlorothiazide, salicylate, opiates, vinorelbine,and desferrioxamine. the oedema is of the high permeability type (see p. 402), rich in protein, and is occasionally haemorrhagic or accompanied by the hyaline membranes of diffuse alveolar damage. diffuse alveolar damage has alveolar epithelial necrosis as its basis (figs 7.3.1 and 7.3.2). however, the continuing action of many cytotoxic drugs affects the regeneration process so that atypical type ii epithelial cells develop, a characteristic feature that was first described with busulphan and subsequently with bleomycin. 21, 37 these two drugs differ chemically but both act (by different mechanisms) on dna. the atypical cells have abundant deeply eosinophilic or amphophilic cytoplasm and large nuclei, which may be multiple but are usually single. the nuclei measure up to 12 µm and are densely stained throughout or contain either large homogeneous deeply eosinophilic inclusions or clear vacuoles (fig. 7.3.3) . electron microscopy distinguishes the inclusions from nucleoli and shows them to consist of tubular aggregates derived from the internal nuclear membrane. 37 airway epithelium shows similar nuclear changes and often undergoes squamous metaplasia. the presence of such cells in sputum specimens submitted for cytology can lead to a misdiagnosis of malignancy. fibrosis may follow diffuse alveolar damage or develop insidiously, perhaps many years after drug therapy ceased (fig. 7.3.4) . 38 it may be both interstitial and intra-alveolar. the interstitial component is often accompanied by a non-specific chronic inflammatory infiltrate. the proportions of inflammation, which is potentially reversible, and fibrosis, which when collagenous is irreversible, obviously bear on the prognosis. however, most case reports antedate the recent classification of interstitial pneumonia described in chapter 6 and it is uncertain how their pathological appearances would now be classified. the majority lack the classic features of usual interstitial pneumonia and fibrotic non-specific interstitial pneumonia. many show overlapping patterns of intersitital pneumonia and this alone should arouse suspicion that a drug may have been responsible. however, some cytotoxic drugs result in pulmonary changes by more than one mechanism: for example, methotrexate may produce hypersensitivity reactions with granuloma formation [41] [42] [43] [44] or pulmonary eosinophilia 45 as well as diffuse alveolar damage. pulmonary toxicity is also occasionally seen in patients undergoing treatment with gold salts for rheumatoid disease: in addition to diffuse alveolar damage, there may be eosinophilia and dermatitis in these cases, again indicating possible hypersensitivity. 46 nitrofurantoin is another example of a drug resulting in a variety of patterns of alveolar injury: diffuse alveolar damage, desquamative interstitial pneumonia, giant cell interstitial pneumonia, organising pneumonia and eosinophilic pneumonia have all been recorded in association with this drug. [47] [48] [49] it should also be noted that in patients with neoplastic disease, clinical features suggestive of a pulmonary drug reaction may be due to factors other than drugs. in leukaemic patients, for example, these include direct infiltration of the lungs by leukaemic cells, opportunist infection and, if bone marrow transplantation has been undertaken, the effects of irradiation and possibly graft-versus-host disease. phospholipidosis is encountered with drugs such as the antidysrhythmic agent amiodarone, 50 which block lysosomal enzymes involved in the breakdown of complex lipids. this leads to their accumulation throughout the body but the effect is most marked in tissues that take up the drug and contain cells rich in lysosomes. the lung fulfils both these requirements through its rich complement of alveolar macrophages. these cells accumulate the enzyme substrate (phospholipid) in their cytoplasm with the result that large foam cells fill the alveoli (fig. 7.3.6 ). the appearances are those of endogenous lipid pneumonia, similar to that seen in obstructive pneumonitis. however, with amiodarone cytoplasmic vacuolation is also seen in epithelial and interstitial cells. the phospholipid inclusions contained within the vacuoles are particularly well seen in unstained frozen sections viewed by polarised light. 51 identical changes to those induced by amiodarone were seen in the lungs of rats exposed to very high levels of the antidepressant drug iprindole 52 and the anorectic drug chlorphentermine. 53 these three compounds, iprindole, chlorphentermine and amiodarone, all belong to the amphiphilic group of drugs which block lysosomal phospholipase and sphingomyelinase. although their pharmacological actions are very different, a molecular homology is apparent (fig. 7.3.7) . it is likely that all patients receiving substantial amounts of amiodarone develop phospholipidosis throughout the body, but this is generally well tolerated. only a minority experience respiratory impairment and in these there is also evidence of pulmonary inflammation and fibrosis, which is possibly mediated immunologically. 54 these patients generally have a restrictive lung deficit, the onset of which may be acute or chronic. bronchoalveolar lavage shows foamy macrophages but these cells indicate exposure to the drug rather than drug toxicity; nor are they specific to amiodarone, being observed on occasion with other drugs. lymphocytes of suppressor type may also be detected on lavage. 54 histologically, amiodarone toxicity is diagnosed on a combination of phospholipidosis and interstitial pneumonia and fibrosis. occasionally the hyaline membranes of diffuse alveolar damage are superimposed on the interstitial changes (see fig. 7.3.2) . [55] [56] [57] in some patients the fibrosis is intraalveolar rather than interstitial and the appearances are those of organising pneumonia. 58 the process may be localised and mimic a neoplasm radiologically. 59, 60 amiodarone toxicity is probably dose-dependent but there is considerable individual variation in the amount required, 61 ,62 which appears to be under genetic control. 63 amiodarone toxicity is uncommon in patients taking daily doses of 200 mg or less whereas the there are drugs that undoubtedly cause a usual interstitial pneumonia pattern, for example the chemotherapeutic agents and nitrofurantoin (fig. 7. 3.5), while others, for example the statins, are recorded as having induced a non-specific interstitial pneumonia pattern. 39 a drug history is therefore imperative when assessing any patient with diffuse parenchymal lung disease. organising pneumonia similar to the cryptogenic condition described on page 308, and probably similarly reversible with steroids, has been encountered with a variety of drugs, including amiodarone, sulphasalazine and pencillamine. 40 penicillamine has also been incriminated in the development of both diffuse alveolitis and bronchiolitis obliterans, but both these changes could well be due to the underlying rheumatoid disease for which the pencillamine is administered. 15 in busulphan lung there may be an organising intraalveolar fibrinous exudate, 21 which at its most extreme results in irreversible effacement of the alveolar architecture by sheets of loose connective tissue (see p. 148). with continued experimental administration of the drug iprindole mentioned above, the phospholipidosis it produced gradually evolved into alveolar proteinosis (more properly called lipoproteinosis; see p. 316), 65 but this has not been reported as a drug effect in humans. alveolar proteinosis has however been recognised in a number of patients receiving chemotherapy for conditions such as leukaemia. the mechanism here is probably based on the cytotoxic action of the drug and the material filling the alveoli may represent the detritus of degenerate alveolar cells rather than excess pulmonary surfactant, as in the primary auto-immune form of alveolar proteinosis. eosinophilic pneumonia, the pathology of which is described on page 461, may be caused by several drugs, including nitrofurantoin, para-aminosalicylic acid, sulphasalazine, phenylbutazone, gold compounds, aspirin and penicillin (see box 9.3, p. 460). 66, 67 it may also follow radiation to the chest. 68 the tissue eosinophilia is generally accompanied by a rise in the number of eosinophils in the blood. the clinical picture varies from transient asymptomatic opacities on a chest radiograph to a life-threatening illness with severe respiratory distress and hypoxaemia, so-called acute eosinophilic pneumonia (see p. 462). the reaction is often associated with a florid rash. withdrawal of the drug may be all that is required to effect resolution but corticosteroids are usually given as they produce a marked improvement. this syndrome of necrotising granulomatosis, vasculitis and eosinophilia in asthmatic patients, which is described more fully on page 465, has been reported when leukotriene receptor antagonists have been used to treat asthma. however, it is likely that the syndrome has been merely unmasked by the antileukotriene permitting a reduction in corticosteroid dose rather than representing a direct effect of the antileukotriene. 69, 70 mesalazine has also been implicated in inducing a vasculitis during treatment for inflammatory bowel disease. 71 the eosinophilia-myalgia syndrome was identified in the usa in 1989 and quickly identified as being due to the ingestion of ltryptophan from one particular japanese supplier. withdrawal of this substance led to the virtual elimination of the disease, but not before 2000 patients had been affected, 1 in 60 fatally. [72] [73] [74] [75] [76] cases were subsequently described in europe where there were further fatalities. l-tryptophan is an essential amino acid that is freely available to the public: its purchase does not require a medical prescription. it has been promoted as a dietary supplement and as an agent against insomnia and premenstrual tension. women in the reproductive years preponderated in the patients affected by the resultant eosinophiliamyalgia. the clinicopathological features of the syndrome are similar to those of the spanish toxic oil syndrome (see p. 375) and differ more in degree than type. the discovery of an aniline-derived contaminant in the tryptophan-induced condition is a further link connecting these two syndromes. 77 an immune basis is suggested by the identification of t lymphocytes activated against fibroblasts in the eosinophilia-myalgia syndrome. 78 the illness is a multisystem disorder and besides blood eosinophilia and myalgia there may be arthralgia, fever, rash and involvement of the lungs, liver and central nervous system. as in the toxic oil syndrome, there is fasciitis, wasting and muscle pain associated with blood and tissue eosinophilia. the lungs are affected in 60% of cases. pulmonary symptoms have included cough, dyspnoea and chest pain. radiographs have shown diffuse bilateral infiltrates and pulmonary hypertension has been documented in a few cases. 79 histology of the lungs shows an oedematous myxoid intimal thickening affecting small pulmonary blood vessels and a diffuse interstitial lymphocytic and eosinophilic infiltrate. 72, 73, 75, 76, 80 these cells may also be seen within the walls of the thickened blood vessels (fig. 7.3.8) . 72, 76 massive ingestion of l-tryptophan has resulted in the appearances of an organising pneumonia. 81 as an adverse drug reaction, granulomatous alveolitis is best exemplified by the extrinsic allergic alveolitis of pituitary snuff-takers, but it is also encountered on rare occasions with cytotoxic and other drugs, including methotrexate, bacille calmette-guérin (bcg) immunisation, interferons, ciprofloxacin, antiviral therapy and tumour necrosis factor antagonists. [42] [43] [44] 48, [82] [83] [84] [85] [86] [87] [88] [89] [90] the histological appearances may suggest extrinsic allergic alveolitis or sarcoidosis but the centri-acinar or lymphangitic concentration of these conditions is usually lacking. however, unless an infective agent can be demonstrated the diagnosis generally requires consideration of the clinical and environmental details, including any drug regimen. exogenous lipid pneumonia may result from the unintentional aspiration of various fat-based medicaments such as liquid paraffin, oily nose drops and petroleum jelly or of fat-rich dietary supplements in the form of ghee. [91] [92] [93] [94] [95] [96] [97] the consumption of liquid paraffin as an aperient is common in some countries and may be taking place without the knowledge of the patient's medical practitioner. regurgitation and aspiration of ingested oil are especially likely to happen during sleep in the presence of a hiatus hernia or when the oesophagus fails to empty completely into the stomach because of achalasia of the cardia. the aspiration of vegetable oil occurred in the past from the use of menthol in olive oil for the treatment of tuberculous laryngitis, and occasionally from the use of iodinated vegetable oils for bronchography. [98] [99] [100] [101] more recently exogenous lipid pneumonia has developed from the constant sucking of lollipops formulated for the administration of the analgesic fentanyl but also containing a stearate component. 102 the treatment of epistaxis by nasal packing with paraffin gauze has also led to exogenous lipid pneumonia. the pathology of exogenous lipid pneumonia is described on page 314. other medicines may also be aspirated unwittingly, for example a ferrous sulphate tablet may cause brown iron staining and necrosis of the bronchus at the point of impact, progressing to bronchial stenosis. [103] [104] [105] distal infection is then likely, as with any foreign body. barium sulphate aspiration may complicate gastrointestinal radiography. 106 large amounts may impair ventilation but being inert there is no permanent injury to the lungs, although the striking changes are evident on the chest radiograph. an outbreak of pulmonary hypertension affecting many swiss, austrian and german patients in the period 1966-68 was probably due to the anorectic drug aminorex, 107 which was accordingly withdrawn with regression in the number of new cases. the pathology in these patients was identical to that of primary pulmonary hypertension (see p. 420) and it proved impossible to reproduce the condition in laboratory animals but the epidemiological evidence that aminorex was to blame is very strong. fenfluramine and phentermine, further anorectic drugs that are chemically similar to aminorex, have also been associated with such plexogenic pulmonary hypertension, [108] [109] [110] [111] [112] and with fibroproliferative plaque on the tricuspid valve and pulmonary arteries. 113 pulmonary hypertension due to pulmonary veno-occlusive disease has sometimes complicated the use of cytotoxic chemotherapeutic agents 114 or followed bone marrow transplantation. 115 non-steroidal anti-inflammatory agents such as indomethacin and diclofenac cross the placenta and, if given in late pregnancy, may cause premature closure of the ductus arteriosus, resulting in severe neonatal pulmonary hypertension. 116, 117 pulmonary hypertension is a well-recognised association of human immunodeficiency virus (hiv) infection but until recently has been unexplained. now, however, evidence is emerging that the highly active antiretroviral therapy administered to hiv-positive patients might be responsible for the pulmonary hypertension. 118 the older high-oestrogen contraceptive drugs carried a slight risk of thromboembolism but this is not seen with the newer preparations. pulmonary thromboembolism has also occurred with a drug-induced lupus syndrome associated with anticardiolipin antibodies. chemotherapeutic drugs such as mitomycin may cause widespread smallvessel thrombosis resulting in the haemolytic-uraemic (thrombotic microangiopathic) syndrome. there is prominent involvement of pulmonary vessels and patients often suffer from respiratory as well as renal insufficiency, and pulmonary hypertension. the syndrome can develop during treatment or up to several months after the drug has been withdrawn. pulmonary thromboembolism is also recorded as a complication of immunoglobulin infusion. 119 non-traumatic fat embolism has resulted from the agglutination or 'creaming' of fat emulsions administered intravenously as a source of calories to debilitated patients. [120] [121] [122] [123] [124] [125] the agglutinated liposomes occlude fine blood vessels throughout the body, causing effects such as priapism, osteonecrosis and pancreatitis. they may be demonstrated in the pulmonary capillaries but the lungs have considerable vascular reserve and it is uncertain what effect the vascular occlusion has on pulmonary function. agglutination of these fat emulsions is particularly common in severely ill patients and this has been attributed to the elevated blood levels of acute-phase proteins, especially c-reactive protein, that are found in the very ill. the agglutination is also induced by calcium and may be brought about by administering calcium and other mineral supplements through the same venous line as the fat. once agglutinated, the fat is less soluble and may be demonstrated in paraffin sections. sudan black is especially useful for this purpose (fig. 7.3.9 ). microvascular crystal embolism is a further risk of parenteral nutrition, the crystals representing various calcium salts that may precipitate in the circulation. 126 transient diffusion abnormalities attributed to oil embolism are very common in patients undergoing lymphangiography but serious respiratory impairment is limited to those patients with pre-existing lung disease or in whom substantial amounts of contrast medium are injected rapidly. [127] [128] [129] [130] other emboli of an iatrogenic nature described in pulmonary arteries include the broken-off ends of intravenous catheters and cannulas, particles from dialysis tubing, 131 prosthetic implants of substances such as teflon and silicone 88, [132] [133] [134] [135] and various materials injected to occlude abnormal blood vessels. 136, 137 diffuse pulmonary haemorrhage diffuse pulmonary haemorrhage may result from interference with the clotting mechanism by anticoagulants 138 or from widespread pulmonary capillaritis, the latter reported in leukaemic patients treated with retinoic acid. 139 pulmonary haemorrhage has also been reported as an idiosyncratic reaction to lymphangiography media 140 and as a complication of immunoglobulin infusion, 141 while the development of anti-basement membrane antibodies resulting in goodpasture's syndrome has been attributed to penicillamine. 141a infection is a common pulmonary hazard in any patient receiving corticosteroids, chemotherapy or any other immunosuppressant drug. viral, bacterial, fungal and protozoal infections, often in combination, may all develop in the lungs of such patients and tissue reactions may be atypical. pneumocystis jiroveci, for example, may elicit a granulomatous reaction or cause diffuse alveolar damage rather than the usual foamy alveolar exudate (see p. 226). metastatic calcification, described on page 489, may result from any drug causing hypercalcaemia, e.g. high doses of vitamin d, calcium and inorganic phosphate or excessive alkali intake in the treatment of peptic ulceration. carcinoma of the lung may be promoted by drugs. arsenicals cause squamous metaplasia of the bronchi and occasionally squamous carcinoma, while peripheral scar cancers, usually adenocarcinomas, have developed in lungs showing fibrosis due to drugs such as busulphan. drugs may result in a variety of pleural diseases. 142 common examples include effusions, chronic inflammation and fibrosis. these are usually encountered in isolation but may be associated with chronic interstitial pneumonia or fibrosis. sometimes there is also serological evidence of systemic lupus erythematosus: many drugs, including hydantoin, practolol, procainamide, hydralazine and sulphonamides, are associated with the development of a syndrome resembling systemic lupus erythematosus that includes pleural disease. whether the drugs are directly responsible for the syndrome or merely promote the development of latent natural disease is uncertain. ergotamine derivatives such as methysergide and bromocriptine are notable for the production of pleural fibrosis, which is sometimes associated with mediastinal and retroperitoneal fibrosis large amounts or prolonged treatment are generally required to produce this effect. [143] [144] [145] in patients given practolol, pleural thickening has become evident several years after the drug was discontinued. this shows the need for a careful drug history in any patient with unexplained pleural fibrosis. reports of radiation-induced lung damage began to appear soon after ionising radiation became widely used in the treatment of malignant disease. [146] [147] [148] despite refinements in radiotherapy techniques it is often impossible to avoid irradiating small areas of lung when treating cancer of the lung, breast, spine, thymus and oesophagus. parts of the lungs are also included in 'mantle' irradiation of mediastinal lymph nodes affected by lymphoma. occasionally, the whole of both lungs is irradiated, as in the treatment of widespread pulmonary metastases or as part of whole-body irradiation prior to marrow transplantation for the treatment of leukaemia. radiation pneumonitis, usually localised, is estimated to affect about 8% of patients. 149 therapeutic irradiation is given as divided doses over several weeks in order to minimise damage to adjacent tissue. the effects of such fractionated treatment are cumulative. in the lungs an early exudative phase soon passes and progressive damage becomes apparent only after months or even years. 150, 151 the changes are generally confined to the area of lung that is irradiated but are widespread when the whole body is irradiated prior to bone marrow transplantation or there is accidental whole-body irradiation. however, localised irradiation of the lung has been followed by abnormalities in non-irradiated areas. these include bilateral alveolar exudates, 152 migratory organising pneumonia affecting both lungs 153, 154 and fulminant bilateral interstitial pneumonia. 155 the likelihood of lung injury is increased by the simultaneous use of cytotoxic drugs and oxygen therapy. 156 furthermore, chemotherapy following irradiation may result in exacerbation of the injury in areas previously irradiated, a phenomenon termed 'recall pneumonitis' . 157, 158 in the long term, irradiation also results in an increased incidence of lung carcinoma. this was seen in patients given therapeutic irradiation to the spine for ankylosing spondylitis 159 and is still encountered on occasion following irra diation for breast cancer. 160 the pathogenesis of radiation injury is described on page 146. radiation damage to the lung is traditionally separated into fulminant acute injury coming on within days, subacute pneumonitis developing within several weeks (typically 2-3 months) and interstitial fibrosis slowly evolving from the subacute stage or making itself apparent years later. the migratory organising pneumonia referred to above is an unusual further effect, as is chronic eosinophilic pneumonia. 68 in the pleura, radiation causes fibrinous effusions and adhesions. pleural effusion and pulmonary oedema may be augmented by the long-term effects of radiation on the heart. fulminant acute injury is an unusual and unexpected effect of therapeutic radiation but one that is likely to come to the attention of the pathologist as an autopsy is often requested. the clinical features are those of acute lung injury and the pathological changes are those of diffuse alveolar damage. the cause is likely to be accidental overdosage, augmentation of the radiation damage by accompanying oxygen therapy or treatment with cytotoxic drugs. occasionally however these factors can be excluded, in which case the damage has to be ascribed to 'hypersensitivity' . subacute radiation pneumonitis is encountered more commonly. after an interval of about 2-3 months the patient complains of shortness of breath and a non-productive cough. the chest radiograph shows hazy opacification proceeding to more dense consolidation. lung biopsy shows alveolar and interstitial oedema, possibly with residual hyaline membranes, proliferation of atypical alveolar epithelial cells and interstitial fibroblasts and organising thrombosis. later, as the process advances, there is widespread fibrosis comparable to that illustrated in figure 4 .24 on page 148 and ultimately dense scarring (fig. 7.3.10) . tracheal and aortic injury may complicate radiation treatment of tracheal lesions, sometimes resulting in an aortotracheal fistula. 161 patients requiring mechanical ventilation are liable to suffer lung injury in a number of ways. in addition to effects of barotrauma such as pneumothorax and surgical emphysema, they often develop diffuse alveolar damage. the high oxygen tension that is often combined with mechanical ventilation is a major factor 162-164 but mechanical forces other than the high pressures responsible for barotrauma can also contribute to this form of lung injury, notably by resulting in excessive end-expiratory stretch and repeated collapse/recruitment of the alveolar walls. 165, 166 low tidal volume ventilation is therefore a fundamental part of the management of diffuse alveolar damage. although oxygen is necessary to life, it is cytotoxic in high concentrations. severe hyperoxia damages dna, inhibits cellular proliferation and ultimately kills cells. its toxicity is thought to be due to the intracellular production of active oxygen radicals, some of which derive from activated neutrophils attracted to the site of injury. [167] [168] [169] [170] under normal conditions most of the oxygen is reduced to water by cytochrome oxidase, and any active radicals produced are eliminated by superoxide dismutase, catalase and other antioxidants. however, these defence mechanisms may prove inadequate when active radicals are produced in excess. 171 problems are likely to arise in clinical practice when lung disease necessitates the concentration of oxygen in the inspired air being raised in order to maintain normal blood levels of oxygen and prevent cerebral hypoxia. [172] [173] [174] a 'safe' level for oxygen administration is not firmly established and, because of species differences in susceptibility to oxygen, caution is needed in extrapolating from animal studies. however, animal experiments have shown that previous damage to the lungs renders them unduly sensitive to oxygen 175,176 and conversely that prior exposure to high levels of oxygen confers some resistance to subsequent oxygen exposure. 177 clinical studies suggest that less than 50% oxygen (at atmospheric pressure) can be tolerated for long periods without ill effect. little, if any, serious lung damage results from administration of 100% oxygen for up to 48 hours but concentrations between 50% and 100% carry a risk of damage if this period is exceeded. 171, 178 extracorporeal oxygenation of the blood circumvents the problem but if it is to be prolonged it becomes a major undertaking that poses its own hazards; it is therefore generally reserved for patients who remain hypoxaemic despite other measures. 179 intravenous blood oxygenators are employed to minimise the supplementation of inspired oxygen and partial liquid ventilation utilising perfluorocarbon has also been used. 180 experimentally, disruption of cd40 binding to reduce the release of proinflammatory cytokines has shown promising results in blunting oxygen-induced lung injury. 181 none of the morphological changes attributable to oxygen toxicity is specific. 174 the earliest ultrastructural change in experimental oxygen poisoning is swelling of endothelial cells, the cytoplasm of which becomes grossly oedematous and vacuolated. swelling and fragmentation of type i epithelial cells follow and these cells become separated from their basement membrane, which is then coated by thin strands of protein. 178 this coating is replaced by proliferating type ii cells by the 12th day. with recovery in room air the lungs practically return to normal. 182 the full clinical picture of oxygen poisoning is the acute respiratory distress syndrome and the corresponding pathological changes are those of diffuse alveolar damage, 174 as described on page 136. patients with hypovolaemic shock or undergoing major surgery often require massive blood transfusions and this provides another possible cause of pulmonary damage. although hypervolaemia is the commonest cause of pulmonary oedema after blood transfusion, transfusion-related acute lung injury is more often fatal. platelet and white cell aggregates are known to develop in stored blood, but a relationship between the number of microaggregates transfused and the degree of respiratory impairment has not been convincingly demonstrated. leukocyte antibodies are a more likely cause of lung injury in these patients. such antibodies are often found in multiparous female donors as a result of sensitisation by fetal white cells during pregnancy. alternatively, the recipient may have developed them during pregnancy or as a result of previous blood transfusions. the implicated antibodies are thought to initiate alveolar capillary damage within hours of transfusion by stimulating granulocyte aggregation. 183, 184 electron microscopy has shown capillary endothelial damage with activated granulocytes in contact with alveolar basement membranes. 185 cardiopulmonary bypass entails oxygenation and circulation of the blood by extracorporeal devices, so permitting major heart surgery. in the early days of such surgery it was not unusual for patients to develop fatal respiratory insufficiency in the postoperative period. this led to the term 'postperfusion lung' . electron microscopic studies showed alveolar damage with degranulation of neutrophils in pulmonary capillaries. 186, 187 the syndrome is now less common but infants remain susceptible. 188 the most likely explanation is that the synthetic materials with which blood comes into contact during the bypass procedure are able to activate complement. this is mediated by hageman factor (factor xii) and the alternative pathway. aggregation of neutrophils leads to their sequestration in the lungs and damage results from their release of lysosomal enzymes and active radicals. [188] [189] [190] the process is delayed by hypothermia. 189 a postcardiac injury syndrome develops after a variety of myocardial or pericardial injuries: it has been described after cardiac surgery (postpericardiotomy syndrome), myocardial infarction (dressler's syndrome), blunt trauma to the chest, percutaneous puncture of the heart and implantation of a pacemaker. 191 there is a delay of anything between a few days and a few months between the cardiac injury and the onset of symptoms, which comprise chest pain, breathlessness, dyspnoea and fever. examination usually reveals haemorrhagic pleural or pericardial effusions and pulmonary infiltrates. the syndrome usually resolves spontaneously and few pathological studies have therefore been conducted. however, the changes of diffuse alveolar damage have been reported, principally hyaline membrane formation and type ii pneumocyte hyperplasia. 192 the pathogenesis is obscure. antibodies reacting with myocardial antigens often develop after cardiac surgery but there is no relationship between these and the development of the syndrome. [192] [193] [194] this minimally invasive technique is used to destroy lesions as varied as pulmonary metastases and the connection between the left atrium and ectopic foci in the muscular sleeves that surround the terminations of the pulmonary veins (see p. 76). the former may be complicated by pneumothorax and the latter by pulmonary vein stenosis. 195, 196 central venous cannulation (synonym: catheterisation) is widely used in treating seriously ill patients and may give rise to serious complications. the commonest early complications related to the respiratory tract are caused by local trauma: they include pneumothorax, subcutaneous emphysema, haemothorax and air embolism. infection occurs later, causing endocarditis, septic emboli and lung abscesses. 197 thrombosis is another common late complication: one autopsy study of patients with central venous lines showed that 15% had major pulmonary emboli and 65% had microscopic emboli in their pulmonary arteries. 198 pulmonary artery cannulation, for example with a swan-ganz catheter, may result in pulmonary infarction or any of the traumatic complications of central venous catheterisation mentioned above. 199, 200 tracheotomy entails a small immediate risk of haemorrhage from damaged subthyroidal arteries, while an endotracheal tube predisposes to infection, as with all foreign bodies. infection is also promoted by the filtering action of the upper respiratory air passages being bypassed. the latter factor also necessitates humidification of the inspired air and on occasion the humidifier or ventilator has become contaminated so that an aerosol of bacteria is introduced directly into the lower respiratory tract. 201 high-pressure ventilation may also lead to interstitial emphysema, pneumothorax and surgical emphysema. asphyxia may follow an endotracheal tube becoming blocked by secretions or through it being badly positioned. secretions need to be constantly removed yet repeated suctioning to achieve this has led to cardiac dysrrythmia and even cardiac arrest. 202 if the balloon on the endotracheal tube is too near the tracheostomy it may act as a fulcrum, causing the tip of the tube to press into the tracheal wall. pressure necrosis and perforation may follow, leading to mediastinitis, tracheo-oesophageal fistula or erosion of a large blood vessel. these are also complications of tracheobronchial laser therapy. pressure from the balloon may lead to a tracheal diverticulum and after the tube is withdrawn the trachea may become narrowed at either the site of the incision or further down where the balloon on the tracheal tube causes pressure. small, shallow ulcers generally heal quickly but deeper ulcers cause necrosis of the tracheal cartilage, and healing is then often accompanied by fibrous stenosis (fig. 7.3 .11) or web formation. this results in wheezing and dyspnoea but not before the trachea has narrowed to 30% of its original size, which may take months. earlier narrowing may be caused by oedema or a fibrinous pseudomembrane. 203, 204 sometimes the stenosis takes the form of a large mass of granulation tissue at the tracheostomy site, a so-called granuloma ball. in children especially, intubation may lead to tracheomalacia so that after the tube is removed the airway collapses. 205 necrotising sialometaplasia is a further complication of prolonged intubation. 206 the incidence of such posttracheostomy complications can be minimised by careful placement of the stoma and tube, avoidance of large apertures and high cuff pressures, elimination of heavy connecting equipment and meticulous care of the tracheostomy. nasogastric feeding tubes may of course lead to aspiration lesions in the lungs and even fatal asphyxia if they are inadvertently allowed to enter the trachea rather than the oesophagus. bronchoscopy is generally a safe, almost routine procedure. a review of 23 862 patients who underwent bronchoscopy identified severe complications in 152 (0.637%), of whom three died. 207 the fatal cases comprised a 78-year-old with coronary heart disease who developed cardiac arrest and two patients who had had tracheal transplantation for oesophageal cancer and required bronchoscopic laser treatment but died of airway obstruction. the pleural cavity is intubated in the treatment of pneumothorax and pleural effusions the tube being placed anteriorly to drain air and posteriorly to drain fluid. complications include laceration of an intercostal artery or vein, the lung, the diaphragm and the heart. pneumonectomy has been practised since the 1930s, since when the mortality associated with this operation has dropped from over 50% to near zero in the best hospitals. risk factors include underlying lung disease, other medical conditions and more extensive procedures such as pleuropneumonectomy and pneumonectomy combined with chest wall resection. the anatomical changes that take place soon after pneumonectomy have been extensively studied by radiologists who describe the air-filled postpneumonectomy space gradually filling with fluid and contracting as the mediastinum shifts and the ipsilateral dome of the diaphragm rises. 209 much of the space is filled by fluid within 2 weeks but complete opacification may take up to 6 months. rapid filling in the immediate postoperative period suggests haemorrhage or chylothorax. however, fluid accumulation is normally rapid after pleuro-pneumonectomy and may compromise the function of the other lung. pathologists conducting autopsies long after the operation may find complete fibrous obliteration of the postpneumonectomy space, coupled with mediastinal shift and elevation of the hemidiaphragm, but often there is persistent brown fluid, which may be clear, cloudy or occasionally purulent. 210 the remaining lung is generally enlarged, with its volume greater than predicted. animal studies have shown that if one lung is excised early in life the enlargement is partly due to enhanced growth but later it represents only dilatation of existing air spaces. hepatocyte growth factor is thought to be involved in the proliferation of residual lung cells following pneumonectomy. 211 pulmonary complications include those typically seen after other thoracic procedures, such as haemorrhage and infection, and those unique to the postpneumonectomy state, namely anastomotic dehiscence and postpneumonectomy pulmonary oedema. the latter presents as the acute respiratory distress syndrome and represents the early stages of diffuse alveolar damage. it follows severe shift of the heart and mediastinum, which is commoner in children and young adults, in whom the tissues are more compliant. [212] [213] [214] [215] the condition complicates up to 4% of lung resections 216, 217 and is commoner following excision of the right lung when severe herniation of the left lung into the postpneumonectomy space stretches the trachea and left main bronchus and the latter is compressed between the left pulmonary artery in front and the arch of the aorta behind. in the long term the compression can result in bronchomalacia and postobstructive bronchiectasis. if postpneumonectomy oedema develops the immediate postoperative mortality is high -50% following pneumonectomy, 42% following lobectomy and 22% following sublobar resections. 217, 218 the pathogenesis is probably multifactorial but apart from factors such as fluid overload and high inspired oxygen concentrations there is probably an element of alveolar wall injury, induced by oxidant generation secondary to lung stretching and general surgical trauma. 219, 220 occupational dust exposure and chronic obstructive pulmonary disease -a systematic overview of the evidence occupational exposures and the risk of copd: dusty trades revisited the origin of the term 'pneumonokoniosis' industrial injuries advisory council. pneumoconiosis and byssinosis. london: hmso; 1973 aerosols: their deposition and clearance human lung parenchyma retains pm2.5 airway branching patterns influence asbestos fiber location and the extent of tissue injury in the pulmonary parenchyma and lymph nodes in infants' lungs pulmonary sumps, dust accumulations, alveolar fluid and lymph vessels candida lung abscesses complicating parenteral nutrition a diffuse form of pulmonary silicosis in foundry workers inhalation of china stone and china clay dusts: relationship between mineralogy of dust retained in the lungs and pathological changes talc pneumoconiosis. a report of six patients with postmortem findings diffuse 'asbestosis-like' interstitial fibrosis of the lung pulmonary pathology in workers exposed to nonasbestiform talc talc pneumoconiosis -a pathologic and mineralogic study diffuse interstitial fibrosis in nonasbestos pneumoconiosis -a pathological study the disposal of coal and haematite dusts inhaled successively vertical gradient of alveolar size in lungs of dogs frozen intact apical localization of phthisis distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive co 2 apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and progressive massive fibrosis of the lung effect of inspiratory flow rate on the regional distribution of inspired gas apical scars and their relationship to siliceous dust accumulation in non-silicotic lungs topographic distribution of asbestos fibres lung structure as a risk factor in adverse pulmonary responses to asbestos exposure. a case-referent study in quebec chrysotile miners and millers particle deposition in casts of the human upper tracheobronchial tree differences in particle deposition between the two lungs pulmonary retention of ultrafine and fine particles in rats experimental study of the dust-clearance mechanism of the lung the lung: an excretory route for macromolecules and particles phagocytic potential of pulmonary alveolar epithelium with particular reference to surfactant metabolism chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages the uptake of mineral particles by pulmonary epithelial cells translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships patterns of particle deposition and retention after instillation to mouse lung during acute injury and fibrotic repair silica deposition in the lung during epithelial injury potentiates fibrosis and increases particle translocation to lymph nodes the permeability of lung parenchyma to particulate matter the migration of bronchoalveolar macrophages into hilar lymph nodes etude au microscope electronique du granulome pulmonaire silicotique experimental the postnatal development of lymphoreticular aggregates in human lung in relation to occupational and non-occupational exposure the distribution of amosite asbestos in the periphery of the normal human lung diseases associated with exposure to silica and nonfibrous silicate minerals detection of silica particles in lung tissue by polarizing light microscopy a practical method for the identification of particulate and crystalline material in paraffin-embedded tissue specimens particles in paraffin sections demonstrated in the backscattered electron image quantitative analysis of inorganic particulate burden in situ in tissue sections microanalysis in histopathology ion microprobe mass analysis of beryllium in situ in human lung: preliminary results laser probe mass spectrometry (lamms) analysis of beryllium, sarcoidosis and other granulomatous diseases molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by fourier transform laser microprobe mass spectrometry value of beryllium lymphocyte transformation test in chronic beryllium disease and in potentially exposed workers radiographic and pathologic correlation of coal workers' pneumoconiosis slateworker's pneumoconiosis transkei silicosis three cases of dental technician's pneumoconiosis related to cobaltchromium-molybdenum dust exposure: diagnosis and follow-up deposition of silicosis dust in the lungs of the inhabitants of the sahara regions sulla possibilita e sulla frequenza della silicosi pulmonare tra gli abitanti del deserto libico simple siliceous pneumoconiosis of bedouin females in the negev desert simple siliceous pneumoconiosis in negev bedouins silicosis in a pakistani farmer silicosis in a himalayan village population -role of environmental dust non-occupational pneumoconiosis at high altitude villages in central ladakh silicate pneumoconiosis of farm workers silicate pneumoconiosis and pulmonary fibrosis in horses from the monterey-carmel peninsula comparative pathology of silicate pneumoconiosis two cases of acute silicosis -with a suggested theory of causation acute silicosis silicose aigue fatale par inhalation volontaire de poudre à recurer acute silicosis in tombstone sandblasters accelerated silicosis in scottish stonemasons the relation between fibrosis of hilar lymph glands and the development of parenchymal silicosis distribution of silicotic collagenization in relation to smoking habits silicosis presenting as bilateral hilar lymphadenopathy silicotic lymph node lesions in nonoccupationally exposed lung carcinoma patients obliterative central bronchitis due to mineral dust in patients with pneumoconiosis left recurrent laryngeal nerve palsy associated with silicosis anthracotic and anthracosilicotic spindle cell pseudotumors of mediastinal lymph nodes: report of five cases of a reactive lesion that simulates malignancy right middle lobe atelectasis associated with endobronchial silicotic lesions pleural pearls following silicosis: a histological and electronmicroscopic study silicainduced pleural disease: an unusual case mimicking malignant mesothelioma factors associated with massive fibrosis in silicosis silicosis of systemic distribution hepatosplenic silicosis hepatic silicosis silicotic lesions of the bone marrow: histopathology and microanalysis peritoneal silicosis pulmonary fibrosis in non-ferrous foundry workers irregular opacities in coalworkers' pneumoconiosis -correlation with pulmonary function and pathology pneumoconiosis-related lung cancers -preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis the effects of dusts on peritoneal cells within diffusion chambers freshly fractured quartz inhalation leads to enhanced lung injury and inflammation: potential role of free radicals an examination of the cytotoxic effects of silica on macrophages activity of macrophage factor in collagen formation by silica fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages effect of sio 2 -liberated macrophage factor on protein synthesis in connective tissue in vitro the regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis the ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro cytokines and cytokine network in silicosis and coal workers' pneumoconiosis activation of murine macrophages by silica particles in vitro is a process independent of silica-induced cell death localization of type i procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis transforming growth factor-beta (tgfbeta) in silicosis mechanisms in the pathogenesis of asbestosis and silicosis changes in the composition of lung lipids and the 'turnover' of dipalmitoyl lecithin in experimental alveolar lipo-proteinosis induced by inhaled quartz case report: pulmonary alveolar proteinosis and aluminum dust exposure induction of surfactant protein (sp-a) biosynthesis and sp-a mrna in activated type ii cells during acute silicosis in rats silicon nephropathy: a possible occupational hazard rapidly progressive silicon nephropathy silica and glomerulonephritis: case report and review of the literature rapidly progressive crescenteric glomerulonephritis in a sandblaster with silicosis diatomaceous earth pneumoconiosis particle size for differentiation between inhalation and injection pulmonary talcosis kaolin pneumoconiosis. a case report kaolin pneumoconiosis. radiologic, pathologic and mineralogic findings silicosis in wyoming bentonite workers pneumoconiosis due to fuller's earth pulmonary disease caused by the inhalation of cosmetic talcum powder talc dust pneumoconiosis mica-associated pulmonary interstitial fibrosis baritosis: a benign pneumoconiosis silicosis in barium miners scleroderma in gold-miners on the witwatersrand with particular reference to pulmonary manifestations immunological factors in the pathogenesis of the hyaline tissue of silicosis mini-review -immunologic aspects of pneumoconiosis adverse effects of crystalline silica exposure chronic necrotizing pulmonary aspergillosis in pneumoconiosis -clinical and radiologic findings in 10 patients silica and some silicates. monographs on the evaluation of the carcinogenic risk of chemicals to humans occupational lung disease: 1. exposure to crystalline silica and risk of lung cancer: the epidemiological evidence do silica and asbestos cause lung cancer? report by the industrial injuries advisory council in accordance with section 171 of the social security administration act 1992 on the question whether lung cancer in relation to occupational exposure to silica should be prescribed. london: hmso; 1992 crystalline silica exposure, radiological silicosis, and lung cancer mortality in diatomaceous earth industry workers acute silicoproteinosis acute silicolipoproteinosis atypical reaction to inhaled silica alveolar proteinosis. its experimental production in rodents experimental endogenous lipid pneumonia and silicosis pathogenesis of experimental pulmonary alveolar proteinosis pneumoconiosis in carbon electrode makers graphite pneumoconiosis of electrotypers case report: a case of wood-smoke-related pulmonary disease hut lung. a domestically acquired particulate lung disease bronchial stenosis due to anthracofibrosis anthracofibrosis attributed to mixed mineral dust exposure: report of three cases anthracofibrosis, bronchial stenosis with overlying anthracotic mucosa: possibly a new occupational lung disorder occupational anthracofibrosis pneumoconiosis and other causes of death in iron and steel foundry workers histochemical study of certain iron ore dusts mixed-dust pneumoconiosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation chronic interstitial pneumonia in silicosis and mix-dust pneumoconiosis: its prevalence and comparison of ct findings with idiopathic pulmonary fibrosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation coal pneumoconiosis coal workers' pneumoconiosis pneumoconiosis in coal trimmers dust exposure, dust recovered from the lung, and associated pathology in a group of british coalminers the relationship between the mass and composition of coal mine dust and the development of pneumoconiosis interstitial fibrosis in coal miners -experience in wales and west virginia coal worker's pneumoconiosis: ct assessment in exposed workers and correlation with radiographic findings variations in the histological patterns of the lesions of coal workers' pneumoconiosis in britain and their relationship to lung dust content comparison of radiographic appearances with associated pathology and lung dust content in a group of coalworkers small airways disease and mineral dust exposure. prevalence, structure and function coal mining, emphysema, and compensation revisited coal mining, emphysema, and compensation revisited -reply coal mining and chronic obstructive pulmonary disease: a review of the evidence the pathogenesis of simple pneumokoniosis in coal workers mineral dusts cause elastin and collagen breakdown in the rat lung: a potential mechanism of dust-induced emphysema emphysema in coal workers' pneumoconiosis pulmonary disability in coal workers' pneumoconiosis quantified pathology of emphysema, pneumoconiosis, and chronic bronchitis in coal workers emphysema and dust exposure in a group of coal workers coal worker's lung: not only black, but also full of holes contributions of dust exposure and cigarette smoking to emphysema severity in coal miners in the united states report by the industrial injuries advisory council in accordance with section 171 of the social security administration act 1992 on the question whether bronchitis and emphysema in coal miners and metal production workers should be prescribed. london: hmso; 1992 the new prescription: industrial injuries benefits for smokers? exposure to respirable coalmine dust and incidence of progressive massive fibrosis the attack rate of progressive massive fibrosis ischemic necrosis in anthracosilicosis the composition of massive lesions in coal miners mechanisms of fibrosis in coal workers' pneumoconiosis: increased production of platelet-derived growth factor, insulin-like growth factor type i, and transforming growth factor beta and relationship to disease severity secretion and mrna expression of tnf alpha and il-6 in the lungs of pneumoconiosis patients a broader concept of caplan's syndrome related to rheumatoid factors differential agglutination test in rheumatoid arthritis complicated by pneumoconiosis circulating antinuclear antibody and rheumatoid factor in coal pneumoconiosis serologic changes in pneumoconiosis and progressive massive fibrosis of coal workers immunological investigations of coalworkers' disease certain unusual radiological appearances in the chest of coal-miners suffering from rheumatoid arthritis pathological studies of modified pneumoconiosis in coal-miners with rheumatoid arthritis (caplan's syndrome) asbestos, asbestosis, and cancer: the helsinki criteria for diagnosis and attribution report of the asbestosis committee of the college of american pathologists and pulmonary pathology society the asbestos cancer epidemic rapid short-term clearance of chrysotile compared with amosite asbestos in the guinea pig persistence of natural mineral fibers in human lungs: an overview a pathological and mineralogical study of asbestos-related deaths in the united kingdom in 1977 correlation between fibre content of the lungs and disease in naval dockyard workers correlation between fibre content of the lung and disease in east london asbestos factory workers the effects of the inhalation of asbestos in rats mass and number of fibres in the pathogenesis of asbestos related lung disease in rats fiber burden and patterns of asbestos-related disease in chrysotile miners and millers carcinogenesis and mineral fibres lung diseases due to environmental exposures to erionite and asbestos in turkey prospective study of mesothelioma mortality in turkish villages with exposure to fibrous zeolite analysis of ferruginous bodies in bronchoalveolar lavage from foundry workers analysis of the cores of ferruginous (asbestos) bodies from the general population fiber counting and analysis in the diagnosis of asbestos-related disease concentrations and dimensions of coated and uncoated asbestos fibres in the human lung comparison of the results of asbestos fibre dust counts in lung tissue obtained by analytical electron microscopy and light microscopy asbestos bodies and the diagnosis of asbestosis in chrysotile workers asbestos fiber content of lungs with diffuse interstitial fibrosis: an analytical scanning electron microscopic analysis of 249 cases comparison of phagocytosis of uncoated versus coated asbestos fibers by cultured human pulmonary alveolar macrophages numbers of asbestos bodies on iron-stained tissue sections in relation to asbestos body counts in lung tissue digests asbestos content of lung tissue in asbestos associated diseases: a study of 110 cases analysis and interpretation of inorganic mineral particles in 'lung' tissues asbestos bodies in bronchoalveolar lavage in relation to asbestos bodies and asbestos fibres in lung parenchyma asbestos bodies in the sputum of asbestos workers: correlation with occupational exposure the optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to human pathology reaction assessment of mineral fibres from human lung tissue detection of asbestos fibres by dark ground microscopy analysis of lung asbestos content guidelines for mineral fibre analyses in biological samples: report of the ers working group relationship between occupations and asbestos-fibre content of the lungs in patients with pleural mesothelioma, lung cancer, and other diseases inflammation-generating potential of long and short-fibre amosite asbestos samples asbestos-stimulated tumour necrosis factor release from alveolar macrophages depends on fibre length and opsonization the pathogenicity of long versus short fibre amosite asbestos administered to rats by inhalation and intra-peritoneal injection cytogenetic and pathogenic effects of long and short amosite asbestos exposure and mineralogical correlates of pulmonary fibrosis in chrysotile asbestos workers quantitative assessment of inorganic fibrous particulates in dust samples with an analytical transmission electron microscope fibre distribution in the lungs and pleura of subjects with asbestos related diffuse pleural fibrosis malignant mesothelioma in women observations on the distribution of asbestos fibres in human lungs analysis of asbestos fibres and asbestos bodies in tissue samples from human lung: an international interlaboratory trial distribution and characteristics of amphibole asbestos fibres in the left lung of an insulation worker measured with the light microscope the identification of asbestos dust with an electron microprobe analyser symposium on man-made mineral fibres mortality in cases of asbestosis diagnosed by a pneumoconiosis medical panel small-airway lesions in patients exposed to nonasbestos mineral dusts small airways disease and mineral dust exposure relation of smoking and age to findings in lung parenchyma: a microscopic study electron microscopic studies in desquamative interstitial pneumonia associated with asbestos desquamative interstitial pneumonia associated with chrysotile asbestos fibres localized inflammatory pulmonary disease in subjects occupationally exposed to asbestos criteria for the diagnosis and grading of asbestosis report of the pneumoconiosis committee of the college of american pathologists and the national institute for occupational safety and health cytoplasmic hyalin in asbestosis further observations on cytoplasmic hyaline in the lung lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore lung pathology of fatal severe acute respiratory syndrome immunohistochemical detection of ubiquitin-positive intracytoplasmic eosinophilic inclusion bodies in diffuse alveolar damage idiopathic pulmonary fibrosis in asbestosexposed workers mortality of workers certified by pneumoconiosis medical panels as having asbestosis radiographic evidence of asbestos effects in american marine engineers chronic airflow obstruction in lung disease. philadelphia: saunders fine structural changes in cryptogenic fibrosing alveolitis and asbestosis pulmonary asbestosis and idiopathic pulmonary fibrosis: pathogenetic parallels crocidoliteinduced pulmonary fibrosis in mice hydroxyl radicals are formed in the rat lung after asbestos instillation invivo asbestos fibers and interferon-gamma up-regulate nitric oxide production in rat alveolar macrophages oxidant sand antioxidant mechanisms of lung disease caused by asbestos fibres antibodies in some chronic fibrosing lung diseases asbestos causes translocation of p65 protein and increases nf-kappa b dna binding activity in rat lung epithelial and pleural mesothelial cells rapid activation of pdgf-a and -b expression at sites of lung injury in asbestos-exposed rats upregulation of the pdgf-alpha receptor precedes asbestos-induced lung fibrosis in rats tnf-alpha receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses the molecular basis of asbestos induced lung injury inhaled asbestos activates a complementdependent chemoattractant for macrophages alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis cellular and molecular basis of the asbestos-related diseases up-regulated expression of transforming growth factor-α in the bronchiolar-alveolar duct regions of asbestos-exposed rats a study of macrophage-mediated initiation of fibrosis by asbestos and silica using a diffusion chamber technique enhanced generation of free radicals from phagocytes induced by mineral dusts mortality from lung cancer in asbestos workers diffuse pleural mesotheliomas and asbestos exposure in northwestern cape province carcinogenicity of amosite asbestos asbestos exposure, cigarette smoking and death rates smoking, exposure to crocidolite, and the incidence of lung cancer and asbestosis pathology of carcinoma of the lung associated with asbestos exposure a study of the histological cell types of lung cancer in workers suffering from asbestosis in the united kingdom lung cancer cell type and asbestos exposure histological type of lung carcinoma in asbestos cement workers and matched controls incidence of lung cancer by histological type among asbestos cement workers in denmark lung cancer in asbestos cement workers in denmark asbestos and lung cancer in glasgow and the west of scotland exposure to crocidolite and the incidence of different histological types of lung cancer asbestos and lung cancer asbestos and lung cancer: is it attributable to asbestosis or to asbestos fibre burden? in: corrin b, editor. pathology of lung tumours asbestos fibreyears and lung cancer: a two phase case-control study with expert exposure assessment the quantitative risks of mesothelioma and lung cancer in relation to asbestos exposure is asbestos or asbestosis the cause of the increased risk of lung cancer in asbestos workers? relation between asbestosis and bronchial cancer in amphibole asbestos miners asbestosis as a precursor of asbestos related lung cancer -results of a prospective mortality study asbestos exposure, asbestosis, and asbestosattributable lung cancer asbestos and lung cancer -reply criteria for attributing lung cancer to asbestos exposure asbestosis: a marker for the increased risk of lung cancer among workers exposed to asbestos asbestos, asbestosis, and lung cancer: a critical assessment of the epidemiological evidence airway function in lifetime-nonsmoking older asbestos workers severe diffuse small airways abnormalities in long term chrysotile asbestos miners morphology of small-airway lesions in patients with asbestos exposure does aluminum smelting cause lung disease? the treatment of silicosis by aluminum powder silicose aigue. caractéristiques cliniques, radiologiques, fonctionnelles et cytologiques du liquide bronchoalveolaire. a propos de 6 observations pathologie and pathogenesis der aluminiumlunge die aluminiumlunge -eine neue gewerbeerkrankung. z f d ges pulmonary fibrosis in workers exposed to finely powdered aluminium aluminium pneumoconiosis i. in vitro comparison of stamped aluminium powders containing different lubricating agents and a granular aluminium powder effect on the rat lung of intratracheal injections of stamped aluminium powders containing different lubricating agents and of a granular aluminium powder desquamative interstitial pneumonia in an aluminum welder sarcoidlike lung granulomatosis induced by aluminum dusts aluminum induced pulmonary granulomatosis aluminum welding fume-induced pneumoconiosis rare earth (cerium) pneumoconiosis rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review hard metal disease interstitial lung disease and asthma in hard-metal workers: bronchoalveolar lavage, ultrastructural, and analytical findings and results of bronchial provocation tests the biological action of tungsten carbide and cobalt: studies on experimental pulmonary histopathology cobalt lung in diamond polishers rapidly fatal progression of cobalt lung in a diamond polisher hard metal pneumoconiosis and the association of tumor necrosis factor-alpha the pulmonary toxicity of beryllium beryllium and lung disease recent chronic beryllium disease in residents surrounding a beryllium facility nonoccupational beryllium disease masquerading as sarcoidosis -identification by blood lymphocyte proliferative response to beryllium beryllium disease international agency for research on cancer. an evaluation of carcinogenic risk to humans. overall evaluation of carcinogenicity: an updating of iarc monographs vols 1-42 uber eine apparatur zur erzeugung niedriger staubkonzentrationen von grosser konstanz und eine methode zur mikrogravinctrischen staubbestemmung. anwendung bei der untersuchung von stauben aus der berylliumsgewinnung delayed chenical pneumonitis occurring in workers exposed to beryllium compounds chronic beryllium disease in a dental laboratory technician maintenance of alveolitis in patients with chronic beryllium disease by berylliumspecific helper t cells hla-dpb1 glutamate 69: a genetic marker of beryllium disease immunogenetic basis of environmental lung disease: lessons from the berylliosis model beryllium disease in identical twins beryllium detection in human lung tissue using electron probe x-ray microanalysis diagnostic criteria for chronic beryllium disease (cbd) based on the uk registry 1945-1991 united kingdom beryllium registry: mortality and autopsy study lung disease and the lightest of metals misdiagnosis of sarcoidosis in patients with chronic beryllium disease diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients the pathology of interstitial lung disease in nylon flock workers flock worker's lung: broadening the spectrum of clinicopathology, narrowing the spectrum of suspected etiologies polyethylene flock-associated interstitial lung disease in a spanish female popcorn workers' lung clinical bronchiolitis obliterans in workers at a microwave-popcorn plant clinical bronchiolitis obliterans in workers at a microwave-popcorn plant bronchiolitis obliterans syndrome in popcorn production plant workers popcorn worker's lung' in britain in a man making potato crisp flavouring toxicity in textile airbrushing in spain outbreak of organising pneumonia in textile printing sprayers epidemic outbreak of interstitial lung disease in aerographics textile workersthe 'ardystil syndrome': a first year follow up organizing pneumonia in textile printing workers: a clinical description outbreak of pulmonary disease in textile dye sprayers in algeria five-year follow-up of algerian victims of the ''ardystil syndrome mineral oils and petroleum 357. skorodin ms, chandrasekhar aj. an occupational cause of exogenous lipoid pneumonia machine operator's lung. a hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols metalworking fluid-associated hypersensitivity pneumonitis: a workshop summary an outbreak of hypersensitivity pneumonitis at a metalworking plant: a longitudinal assessment of intervention effectiveness stenius-aarniala b. fire-eater's lung fire-eater's lung: seventeen cases and a review of the literature pulmonary mechanics in dogs given different doses of kerosene intratracheally x-ray appearance of the lungs of electric arc welders the health of welders in naval dockyards: the risk of asbestosrelated diseases occurring in welders welders' pneumoconiosis: tissue elemental microanalysis by energy dispersive x ray analysis the respiratory health of welders toxic fumes and gases 368. nemery b. metal toxicity and the respiratory tract lung changes associated with the manufacture of alumina abrasives acute inorganic mercury vapor inhalation poisoning nickel poisoning ii. studies on patients suffering from acute exposure to vapors of nickel carbonyl silo-filler's disease -a syndrome caused by nitrogen dioxide silo-filler's disease: nitrogen dioxide-induced lung injury silo filler's disease silo-filler's disease in new york state dung lung: a report of toxic exposure to liquid manure death caused by fermenting manure acute toxic exposure to gases from liquid manure slurry lung': a report of three cases pulmonary lesions in rats exposed to ozone pathology of adult respiratory distress syndrome response of macaque bronchiolar epithelium to ambient concentrations of ozone ultrastructural alterations of alveolar tissue of mice differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months ozone-induced acute tracheobronchial epithelial injury -relationship to granulocyte emigration in the lung ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy a cohort study of workers exposed to formaldehyde in the british chemical industry -an update pulmonary toxicity following exposure to methylene chloride and its combustion product, phosgene the diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases tracheobronchomalacia and air trapping after mustard gas exposure an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients thionyl-chloride-induced lung injury and bronchiolitis obliterans hydrogen sulfide poisoning: review of 5 years' experience anoxic asphyxia anoxic asphyxia -a cause of industrial fatalities: a review sword '94: surveillance of work-related and occupational respiratory disease in the uk occupational asthma as identified by the surveillance of work-related and occupational respiratory diseases programme in south africa incidence of occupational asthma by occupation and industry in finland sword '93: surveillance of work-related and occupational respiratory disease in the uk american thoracic society statement: occupational contribution to the burden of airway disease aetiological agents in occupational asthma reported incidence of occupational asthma in the united kingdom, 1989-97 occupational asthma in a factory with a contaminated humidifier the pathology of byssinosis byssinosis: a review in vitro alternative and classical activation of complement by extracts of cotton mill dust: a possible mechanism in the pathogenesis of byssinosis antibody independent complement activation by card-room cotton dust occupational fevers 407. raskandersen a, pratt ds. inhalation fever -a proposed unifying term for febrile reactions to inhalation of noxious substances humidifier fever pulmonary mycotoxicosis organic dust toxicity (pulmonary mycotoxicosis) associated with silo unloading desquamative interstitial pneumonitis and diffuse alveolar damage in textile workers: potential role of mycotoxins pulmonary mycotoxicosis: a clinicopathologic study of three cases pulmonary involvement in zinc fume fever cytokines in metal fume fever music: a new cause of primary spontaneous pneumothorax atmospheric pressure influences the risk of pneumothorax: beware of the storm experimental study of blast injuries to the lungs biophysics of impact injury to the chest and abdomen histologic, immunohistochemical, and ultrastructural findings in human blast lung injury histologic, immunohistochemical, and ultrastructural findings in human blast lung injury the ultrastructure of rat lung following acute primary blast injury disorders associated with diving decompression disorders in divers intrapulmonary air trapping in submarine escape training casualties airway structure and alveolar emptying in the lungs of sea lions and dogs the structure and function of the small airways in pinniped and sea otter lungs mechanisms of death in shallow-water scuba diving pulmonary barotrauma in submarine escape training the pathophysiology of decompression sickness hemodynamic changes induced by recreational scuba diving dysbaric osteonecrosis: aseptic necrosis of bone deep diving mammals: dive behavior and circulatory adjustments contribute to bends avoidance current concepts: high-altitude illness illnesses at high altitude high altitude pulmonary edema high altitude pulmonary oedema; characteristics of lung lavage fluid prevention of high-altitude pulmonary edema by nifedipine pulmonary circulation at high altitude association of high-altitude pulmonary edema with the major histocompatibility complex pathogenesis of high-altitude pulmonary oedema: direct evidence of stress failure of pulmonary capillaries high-altitude pulmonary edema: current concepts high-altitude pulmonary edema is initially caused by an increase in capillary pressure when lungs on mountains leak -studying pulmonary edema at high altitudes inhaled nitric oxide for high-altitude pulmonary edema lung pathology in high-altitude pulmonary edema pathological features of the lung in fatal high altitude pulmonary edema occurring at moderate altitude in japan lack of smooth muscle in the small pulmonary arteries of the native ladakhi -is the himalayan highlander adapted? aspects of hypoxia pulmonary blood vessels and endocrine cells in subacute infantile mountain sickness the human pulmonary circulation: its form and function in health and disease pulmonary edema associated with salt water near-drowning -new insights the effect of salt water on alveolar epithelial barrier function near-drowning: clinical course of lung injury in adults haggard hw. action of irritant gases upon the respiratory tract regional respiratory tract absorption of inhaled reactive gases respiratory irritants encountered at work health effects of outdoor air pollution committee of the environmental and occupational health assembly of the committee of the environmental and occupational health assembly of the fine particulate air pollution and mortality in 20 us cities, 1987-1994 health effects of diesel exhaust emissions respiratory changes due to long-term exposure to urban levels of air pollution: a histopathologic study in humans chronic glandular bronchitis in young individuals residing in a metropolitan area an association between air pollution and mortality in 6 united states cities chronic bronchitis in women using solid biomass fuel in rural peshawar indoor air pollution: a poverty-related cause of mortality among the children of the world risk of copd from exposure to biomass smoke human health impacts of volcanic activity persistent hyperreactivity and reactive airway dysfunction in firefighters at the world trade center the world trade center residents' respiratory health study: new-onset respiratory symptoms and pulmonary function pulmonary function after exposure to the world trade center collapse in the new york city fire department characterization of the dust/smoke aerosol that settled east of the world trade center (wtc) in lower manhattan after the collapse of the wtc 11 chemical analysis of world trade center fine particulate matter for use in toxicologic assessment longitudinal assessment of spirometry in the world trade center medical monitoring program acute eosinophilic pneumonia in a new york city firefighter exposed to world trade center dust granulomatous pneumonitis following exposure to the world trade center collapse smoking-related interstitial lung diseases: a concise review concomitant upper-lobe bullous emphysema, lower-lobe interstitial fibrosis and pulmonary hypertension in heavy smokers: report of eight cases and review of the literature heterogeneity in combined pulmonary fibrosis and emphysema smoking-related interstitial lung diseases challenges in pulmonary fibrosis 4: smoking-induced diffuse interstitial lung diseases combined pulmonary fibrosis and emphysema: a distinct underrecognised entity smoking-related changes in the background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course clinical assembly contribution to the celebration of 20 years of the ers. smoking-related lung diseases: a clinical perspective clinically occult interstitial fibrosis in smokers: classification and significance of a surprisingly common finding in lobectomy specimens bronchiolar inflammation and fibrosis associated with smoking -a morphologic cross-sectional population analysis relation of smoking and age to findings in lung parenchyma: a microscopic study variability in small airway epithelial gene expression among normal smokers airway alveolar attachment points and exposure to cigarette smoke in utero patterns of global tobacco use in young people and implications for future chronic disease burden in adults tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic pathology of the lung in fatally burned patients respiratory tract pathology in patients with severe burns heat injuries to the respiratory system death in the burn unit: sterile multiple organ failure pulmonary interstitial edema and hyaline membranes in adult burn patients herpesvirus infections in burn patients pulmonary herpes simplex in burns patients long-term course of bronchiectasis and bronchiolitis obliterans as late complication of smoke inhalation disaster at bhopal: the accident, early findings and respiratory health outlook in those injured chronic lung inflammation in victims of toxic gas leak at bhopal respiratory morbidity 10 years after the union carbide gas leak at bhopal: a cross sectional survey management of the effects of exposure to tear gas ingested toxic agents 88. boyd mr. evidence for the clara cell as a site of cytochrome p450-dependent mixed-function oxidase activity in lung pulmonary histological appearances in fatal paraquat poisoning pulmonary damage due to paraquat poisoning through skin absorption paraquat induced pulmonary fibrosis in three survivors kinetics and cellular localisation of putrescine uptake in human lung tissue enhancement of oxygen toxicity by the herbicide paraquat hypoxic protection in paraquat poisoning the effects of variable o 2 tension and of exogenous superoxide dismutase on type ii pneumocytes exposed to paraquat paraquat-induced injury of type ii alveolar cells the changing pattern of paraquat poisoning in man remodeling of the alveolar structure in the paraquat lung of humans: a morphometric study experimental paraquat poisoning the pathogenesis and structure of paraquatinduced pulmonary fibrosis in rats pulmonary ultrastructure after oral and intravenous dosage of paraquat to rats patterns of pulmonary structural remodeling after experimental paraquat toxicity experimental models of interstitial pneumonia: paraquat, iprindole fine structure of the lung lesion in a case of paraquat poisoning fatal pulmonary intra-alveolar fibrosis after paraquat ingestion paraquat lung: a reappraisal an immunohistochemical study of the fibrosing process in paraquat lung injury toxic-allergic syndrome caused by ingestion of rapeseed oil denatured with aniline pathology of a new toxic syndrome caused by ingestion of adulterated oil in spain clinical epidemiology of toxic oil syndrome 3-(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome pulmonary hypertension due to toxic oil syndrome. a clinicopathologic study pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome pulmonary vascular lesions in the toxic oil syndrome in spain outbreak of bronchiolitis obliterans associated with consumption of sauropus androgynus in taiwan histopathological study of sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans sauropus androgynus-constrictive obliterative bronchitis/bronchiolitishistopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression segmental necrosis of small bronchi after prolonged intakes of sauropus androgynus in taiwan sauropus bronchiolitis -reply the lipoid pneumonia of blackfat tobacco smokers in guyana a morphometric analysis of the male and female tracheal epithelium after experimental exposure to marijuana smoke differential examination of bronchoalveolar lavage cells in tobacco cigarette and marijuana smokers tracheobronchial changes in habitual, heavy smokers of marijuana with and without tobacco airway inflammation in young marijuana and tobacco smokers an unusual cause of patchy ground-glass opacity pulmonary hazards of smoking marijuana as compared with tobacco british lung foundation. a smoking gun? tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco effects of cannabis on pulmonary structure, function and symptoms large lung bullae in marijuana smokers the pulmonary pathology of illicit drug and substance abuse bong lung: regular smokers of cannabis show relatively distinctive histologic changes that predispose to pneumothorax cannabis use and risk of lung cancer: a case-control study exogenous lipid pneumonia related to smoking weed oil following cadaveric renal transplantation a review of the respiratory effects of smoking cocaine medical complications of cocaine abuse blackened bronchoalveolar lavage fluid in crack smokers -a preliminary study pulmonary histopathology in cocaine abusers pulmonary complications of crack cocaine: a comprehensive review cocaine-induced churg-strauss vasculitis pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization crack lung: an acute pulmonary syndrome with a spectrum of clinical and histopathologic findings effects of 'crack' cocaine on pulmonary alveolar permeability pulmonary hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature cellulose granulomas in the lungs of a cocaine sniffer intravenous injection of talc-containing drugs intended for oral use. a cause of pulmonary granulomatosis and pulmonary hypertension autopsy findings in drug addicts pulmonary 'mainline' granulomatosis: talcosis of intravenous methadone abuse self induced pulmonary granulomatosis. a consequence of intravenous injection of drugs intended for oral use the pulmonary vascular lesions of intravenous drug abuse microcrystalline cellulose pulmonary embolism and granulomatosis talc granulomatosis: laboratory findings similar to sarcoidosis postmortem findings of pulmonary lesions of older datum in intravenous drug addicts. a forensic-pathologic study recreational use of aminorex and pulmonary hypertension fatal drug reactions among medical inpatients drug-related hospital admissions: a review of australian studies published 1988-1996 incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies (see comments) the quality in australian health care study adverse reactions to drugs drug-induced pulmonary disease -an update an algorithm for the operational assessment of adverse drug reactions. ii. demonstration of reproducibility and validity aspirin idiosyncrasy and tolerance aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks; diagnostic and physiopathological implications mechanism of aspirin sensitivity bronchiolitis and bronchitis in connective tissue disease. a possible relationship to the use of penicillamine bronchiolitis obliterans in a patient with localized scleroderma treated with d-penicillamine constrictive bronchiolitis obliterans following gold therapy for psoriatic arthritis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease progressive airway obliteration in adults and its association with rheumatoid disease drug-induced infiltrative lung disease drug-induced and iatrogenic infiltrative lung disease interstitial lung disease associated with drug therapy pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning interstitial pulmonary fibrosis following busulfan therapy busulphan lung report of a case and review of the literature busulfan lung: report of two cases and review of the literature a 60-year-old man with pulmonary infiltrates after a bone marrow transplantation -busulfan pneumonitis drug-induced pulmonary disease lung parenchymal injury induced by bleomycin pathology of high dose intermittent cyclophosphamide therapy cyclophosphamide pneumonitis pulmonary histopathologic changes associated with melphalan therapy melphalan-induced pulmonary interstitial fibrosis potentiation of bleomycin-induced lung injury by exposure to 70% oxygen factors influencing postoperative morbidity and mortality in patients treated with bleomycin bleomycin therapy and anaesthesia risk factors of anesthesia and surgery in bleomycin-treated patients the pathogenesis of bleomycin-induced pulmonary damage in mice experimentally induced bleomycin sulfate toxicity origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity late bcnu lung -a light and ultrastructural study on the delayed effect of bcnu on the lung parenchyma statin-induced fibrotic nonspecific interstitial pneumonia bronchiolitis obliterans organizing pneumonia during treatment with acebutolol and amiodarone methotrexate-induced pneumonitis (baltimore) bronchoalveolar lavage findings suggest an immunologic disorder pneumonitis complicating low-dose methotrexate therapy for rheumatoid arthritis -discrepancies between lung biopsy and bronchoalveolar lavage findings methotrexate pneumonitis: review of the literature and histopathological findings in nine patients the pulmonary toxicity of antineoplastic agents relationship of gold and penicillamine therapy to diffuse interstitial lung disease desquamative interstitial pneumonia following long-term nitrofurantoin therapy two unusual pathological reactions to nitrofurantoin: case reports bronchiolitis obliterans organising pneumonia associated with the use of nitrofurantoin amiodarone lung toxicity: a human and experimental study effect of amiodarone on the lung shown by polarized light microscopy fine structural alterations in the lungs of iprindole-treated rats effects of chlorphentermine on the rat lung amiodarone-induced hypersensitivity pneumonitis. evidence of an immunological cell-mediated mechanism amiodarone pneumonitis: three further cases with a review of published reports amiodarone-associated pulmonary toxicity. a clinical and pathologic study of eleven cases amiodarone lung: pathologic findings in clinically toxic patients amiodarone-induced bronchiolitis obliterans organizing pneumonia (boop) amiodarone-related pulmonary mass and unique membranous glomerulonephritis in a patient with valvular heart disease: diagnostic pitfall and new findings nodular amiodarone lung disease amiodarone pneumonitis: no safe dose amiodarone pneumonitis: no safe dose susceptibility to amiodarone-induced pulmonary toxicity: relationship to the uptake of amiodarone by isolated lung cells acute amiodarone-induced pulmonary toxicity following lung resection pulmonary alveolar proteinosis developing from desquamative interstitial pneumonia in long term toxicity studies of iprindole in the rat eosinophilic lung diseases mesalazine-induced eosinophilic pneumonia chronic eosinophilic pneumonia after radiation therapy for breast cancer churg-strauss syndrome in patients receiving montelukast as treatment for asthma inhaled corticosteroids and churg-strauss syndrome: a report of five cases pulmonary migratory infiltrates and pachypleuritis in a patient with crohn's disease immunodeficiency virus-infected patients receiving antiretroviral therapy ciprofloxacin-induced acute interstitial pneumonitis pulmonary granulomas after tumour necrosis factor alpha antagonist therapy pulmonary sarcoidosis developing during infliximab therapy sarcoid-like granulomatous disease following etanercept treatment for ra pneumonia due to liquid paraffin: with chemical analysis a case of chronic paraffin pneumonitis liquid paraffin pneumonia -with chemical analysis and electron microscopy paraffinoma confirmed by infrared spectrophotometry foreign body granulomata of the lungs due to liquid paraffin exogenous lipoid pneumonia due to nasal application of petroleum jelly reaction of human lungs to aspirated animal fat (ghee): a clinicopathological study clinical reactions following bronchography the reaction of pulmonary tissue to lipiodol experimental study of bronchographic media on lung a method for the identification of lipiodol in tissue sections of lungs, lipids, and lollipops bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration pulmonary disease associated with l-tryptophan-induced eosinophilic myalgia syndrome. clinical and pathologic features a case of the eosinophilia-myalgia syndrome associated with use of an l-tryptophan product an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use l-tryptophan and the eosinophiliamyalgia syndrome: pathologic findings in eight patients histopathologic features of the l-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome 3-(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome immunemediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by l-tryptophan pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome tryptophan-induced lung disease -an immunophenotypic, immunofluorescent, and electron microscopic study bronchiolitis obliterans organizing pneumonia associated with massive l-tryptophan ingestion pulmonary disease complicating intermittent therapy with methotrexate case records of the massachusetts general hospital. a 28-year-old man with increasing dyspnea, dry cough, and fever after chemotherapy for lymphoma interferon-alpha therapy associated with the development of sarcoidosis proliferation and differentiation in mammalian airway epithelium sarcoid-like pulmonary disorder in human review of the literature severe barium sulfate aspiration into the lung: clinical presentation, prognosis and therapy aminorex and the pulmonary circulation pulmonary hypertension and fenfluramine irreversible pulmonary hypertension after treatment with fenfluramine dietary pulmonary hypertension appetite-suppressant drugs and the risk of primary pulmonary hypertension fatal pulmonary hypertension associated with short-term use of fenfluramine and phentermine autopsy findings of heart and lungs in a patient with primary pulmonary hypertension associated with use of fenfluramine and phentermine pulmonary veno-occlusive disease following therapy for malignant neoplasms pulmonary veno-occlusive disease in an adult following bone marrow transplantation: case report and review of the literature indomethacin in the treatment of premature labor. effects on the fetal ductus arteriosus prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application roles and mechanisms of human immunodeficiency virus protease inhibitor ritonavir and other anti-human immunodeficiency virus drugs in endothelial dysfunction of porcine pulmonary arteries and human pulmonary artery endothelial cells pulmonary embolism after intravenous immunoglobulin fat embolism in infancy after intravenous fat infusions pulmonary fat accumulation after intralipid infusion in the preterm infant intralipid microemboli the pathogenesis of fat embolism pulmonary lipid emboli in association with long-term hyperalimentation the impact of intravenous fat emulsion administration in acute lung injury microvascular pulmonary emboli secondary to precipitated crystals in a patient receiving total parenteral nutrition -a case report and description of the high-resolution ct findings pulmonary complications following lymphangiography with a note on technique changes in pulmonary function due to lymphangiography pulmonary complications of lymphangiography respiratory distress syndrome from lymphangiography contrast medium spallation and migration of silicone from blood-pump tubing in patients on hemodialysis acute pneumonitis after subcutaneous injections of silicone in transsexual men pulmonary granulomas secondary to embolic prosthetic valve material pulmonary teflon granulomas following periurethral teflon injection for urinary incontinence acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis isobutyl-2-cyanoacrylate pulmonary emboli associated with occlusive embolotherapy of cerebral arteriovenous malformations hemorragie alveolaire diffuse secondaire a l'utilisation d'anticoagulants oraux diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome intrapulmonary hemorrhage with anemia after lymphangiography alveolar hemorrhage as a complication of treatment with abciximab d-penicillamine induced goodpasture's syndrome in wilson's disease drugs and the pleura pleuropulmonary changes induced by ergoline drugs pleuropulmonary disease as a side-effect of treatment with bromcriptine pleuropulmonary disease due to pergolide use for restless legs syndrome fibrosis of the lung following roentgen-ray treatments for tumor radiation reaction in the lung radiation pneumonitis: experimental and pathologic observations radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors the pathogenesis of radiationinduced lung damage radiation pneumonitis: a review adult respiratory distress syndrome after limited thoracic radiotherapy migratory bronchiolitis obliterans organizing pneumonia after unilateral radiation therapy for breast carcinoma migratory organizing pneumonitis 'primed' by radiation therapy hamman-rich syndrome 'primed' by radiation? recall' pneumonitis: adriamycin potentiation of radiation pneumonitis in two children recall lung pneumonitis due to carmustine after radiotherapy pulmonary radiation injury mortality from cancer and other causes after radiotherapy for ankylosing spondylitis increased risk of lung cancer after breast cancer radiation therapy in cigarette smokers aortotracheal fistula secondary to bacterial aortitis respirator lung -a misnomer pathology of adult respiratory distress syndrome pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project lung injury caused by mechanical ventilation ventilator-induced lung injury oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes in vitro damage of rat lungs by oxygen metabolites intercellular adhesion molecule-1 contributes to pulmonary oxygen toxicity in mice -role of leukocytes revised intercellular adhesion molecule-1 expression on the alveolar epithelium and its modification by hyperoxia normobaric oxygen toxicity of the lung oxygen pneumonitis in man pathology of pulmonary oxygen toxicity diffuse alveolar damage -the role of oxygen, shock and related factors diffuse interstitial pulmonary fibrosis. pulmonary fibrosis in mice induced by treatment with butylated hydroxytoluene and oxygen potentiation of diffuse lung damage by oxygen: determining values resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung ultrastructural observations on the development of the alveolar lesions extracorporeal membrane oxygenation for adult respiratory failure pulmonary pathology of patients treated with partial liquid ventilation disruption of the cd40-cd40 ligand system prevents an oxygen-induced respiratory distress syndrome pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. ii ultrastructural and morphometric studies diffuse alveolar damage, respiratory failure and blood transfusion pulmonary injury -secondary to extracorporeal circulation fine structural changes in the lungs following cardiopulmonary bypass complement and the damaging effects of cardiopulmonary bypass acute lung injury during cardiopulmonary bypass: are the neutrophils responsible? inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies the pleuropulmonary manifestations of the postcardiac injury syndrome the postmyocardial infarction syndrome. the nonspecificity of the pulmonary manifestations the postcardiac injury syndromes antiheart antibodies following open heart surgery: incidence and correlation with postpericardiotomy syndrome analysis of the factors associated with radiofrequency ablation-induced pneumothorax irreversible intrapulmonary vascular changes after pulmonary vein stenosis complicating catheter ablation for atrial fibrillation pulmonary venous stenosis after treatment for atrial fibrillation right sided infective endocarditis as a consequence of flow directed pulmonary artery catheterisation complications and consequences of endotracheal intubation and tracheotomy. a prospective study of 150 critically ill adult patients pathologic changes of the trachea after percutaneous dilatational tracheotomy pseudomonas aeruginosa respiratory tract infections in patients receiving mechanical ventilation cardiac arrhythmias resulting from tracheal suctioning obstructive fibrinous tracheal pseudomembrane -a potentially fatal complication of tracheal intubation 12-h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial tracheobronchomalacia in children necrotizing sialometaplasia (adenometaplasia) of the trachea severe complications of bronchoscopy the postpneumonectomy state evaluation of post-pneumonectomy space by computed tomography the postpneumonectomy space: factors influencing its obliteration hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice severe airway obstruction caused by mediastinal displacement after right pneumonectomy in a child. a case report postpneumonectomy syndrome: diagnosis, management, and results treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis postpneumonectomy syndrome: another twist acute lung injury and acute respiratory distress syndrome after pulmonary resection the mortality from acute respiratory distress syndrome after pulmonary resection is reducing: a 10-year single institutional experience prevalence and mortality of acute lung injury and ards after lung resection lung injury following pulmonary resection in the isolated, blood-perfused rat lung the pathogenesis of lung injury following pulmonary resection it is estimated that 5% of all hospital admissions are due to effects of therapeutic drugs, that 10-18% of inpatients experience a drug reaction and that 3% of deaths in hospital may be related to drug therapy. [1] [2] [3] [4] the lungs are often involved in these adverse reactions. the mechanism of an adverse drug reaction may be based on:• overdosage: toxicity linked to excess dose, or impaired excretion, one classification of adverse drug reactions is that based upon the type of drug (box 7.3.1). 6 this is not adopted here but in passing it is worth noting that pharmacists are generally very helpful in supplying details of adverse reactions to specific drugs. alternatively, information on the long list of potentially pneumotoxic drugs may be obtained at http://www.pneumotox.com. a useful scheme for chapter key: cord-018134-k4vdqlgs authors: eisenberg, ronald l. title: pneumonia date: 2019-11-01 journal: what radiology residents need to know: chest radiology doi: 10.1007/978-3-030-16826-1_6 sha: doc_id: 18134 cord_uid: k4vdqlgs this chapter describes the imaging patterns of pneumonia (lobar, lobular, interstitial, round) and its complications (abscess, empyema, pneumatocele); bacterial, fungal, and viral infections; and the many manifestations of pulmonary tuberculosis. electronic supplementary material : the online version of this chapter (10.1007/978-3-030-16826-1_6) contains supplementary material, which is available to authorized users. • any pneumonia developing in a patient at least 48 hours after being hospitalized • second most common nosocomial infection (after urinary tract infections) • unlike community-acquired pneumonia, hap is usually caused by a bacterial infection, rather than a virus • high morbidity and mortality rates and the primary cause of death in intensive care units • any pneumonia developing in a patient at least 48-72 hours after endotracheal tube intubation • typically affecting critically ill patients in an intensive care unit, vap develops in up to 30% of ventilated individuals and is associated with a mortality rate of up to 80% lobar pneumonia • homogeneous consolidation of all or a substantial percentage of a single lobe • sharply marginated by one or more fissures ( [1] • silhouette sign when the consolidation is adjacent to the heart, aorta, or hemidiaphragm ( fig. 6 • foreign material entering into the tracheobronchial tree secondary to gastroesophageal reflux, altered mental status (drug overdose, anesthesia), or neurologic disorder (stroke, traumatic brain injury) • often develops in intubated patients, despite the presence of an inflatable cuff • usually occurs in the most dependent portions of the lung ○ supine -superior and posterior basal segments of the lower lobes or posterior segment of the upper lobes ( fig. 6 .8) ○ upright -lower lobes (typically on the right, because the right main bronchus runs more vertically and is wider) • rapid appearance of air-space consolidation, especially in bedridden patients. • noninfectious aspiration -usually clearance in less than 1 week ○ mendelson syndrome -large-volume aspiration of gastric acid, which produces a chemical pneumonitis and acute lung injury (even ards) and a diffuse radiographic pattern simulating pulmonary edema ( • radiographic clearance of pneumonia usually lags well behind clinical improvement • follow-up chest radiographs are recommended in approximately 4-6 weeks to ensure complete resolution of the consolidation and to assess persistent abnormality of the lung parenchyma (scarring, bronchiectasis) • failure of a pneumonia to resolve by 8 weeks suggests an inaccurate diagnosis or an endobronchial obstruction as a cause of postobstructive pneumonia ( fig. 6 .11) • especially in patients with smoking history or over age 40, ct should be considered to exclude an underlying bronchial lesion (see fig. e6 .13) • irregular infectious cavity containing necrotic debris or fluid. • often an air-fluid level, which usually has the same extent on both frontal and lateral views thicker, often irregular wall disparity in length of air-fluid levels air-fluid levels of relatively equal lengths split pleura sign on ct empyema necessitans (fig. 6.16) • chronic empyema draining via a sinus tract into the subcutaneous tissues of the chest wall, most commonly related to tuberculosis or fungal infection (actinomycosis, aspergillosis, blastomycosis, mucormycosis) • loculated pleural fluid collection or mass with associated rib destruction and often bubbles of loculated gas in soft tissues • gram-negative bacterial pneumonia that is most common in debilitated middle-aged and older men with alcoholism (about two-thirds of cases); high mortality rate • tends to form a voluminous exudate that produces a homogeneous parenchymal consolidation containing an air bronchogram • lobar enlargement (especially the right upper) with the characteristic bulging fissure sign (fig. 6 .17) ○ bulging fissure sign also in haemophilus influenzae pneumonia (predominantly in compromised hosts, such as chronic pulmonary disease, immune deficiency, alcoholism, diabetes) (see fig. e6 .22) • most frequently result from infectious particles reaching the lung from an infected heart valve (especially the tricuspid), intravenous catheter, or injected debris • persons at risk include drug abusers, immunocompromised patients, individuals with septal defects, and those with indwelling venous catheters, pacemakers, or prosthetic heart valves • initially, multiple ill-defined round or wedge-shaped opacities with a swirling pattern that are usually peripheral and tend to involve the lower lobes (starry night sign -mimicking the brush strokes in van gogh's painting of that name) • cavitary pulmonary nodules tend to develop rapidly (1-2 days) • transient, rapidly changing, migrating, nonsegmental areas of parenchymal consolidation, which are associated with blood eosinophilia and minimal (or no) pulmonary symptoms • bilateral patchy consolidations with ill-defined margins that are predominantly located in the periphery of the lung • may produce single or multiple air-space nodules with surrounding ground-glass opacities (fig. 6 .20) • unlike chronic eosinophilic pneumonia (see below), the transient air-space abnormalities resolve in some areas and reappear in others over days • loeffler's syndrome (also known as simple pulmonary eosinophilia) is applied to idiopathic cases; a similar imaging pattern can occur in response to parasitic infection or be drug-induced chronic eosinophilic pneumonia (fig. 6.21; see fig. e6 .26) • classic appearance of multifocal areas of consolidation in both lungs, especially the upper lobes, reflecting inflammatory eosinophils filling alveoli and infiltrating the interstitium • characteristic peripheral predominance ("reverse pulmonary edema pattern") • rapid response to steroid therapy (clinical improvement within hours, radiographic clearing within a few days) fig. 6 .20 loeffler's syndrome. peripheral air-space nodule with surrounding ground-glass opacity in the right lower lobe (arrow). follow-up study showed that the nodule had disappeared [7] fungal pneumonia aspergillosis • common fungus found in soil, on plants, and in decaying matter, as well as in household dust and building materials, which does not harm persons with normal immune systems and no allergic hypersensitivity • invasive aspergillosis (most aggressive form) is essentially limited to debilitated patients, diabetics, and neutropenic individuals with severely compromised immune systems (organ or bone marrow transplants, high-dose steroids or chemotherapy, lymphoma, leukemia) • central mass within a cavity in invasive aspergillosis is almost always necrotic lung (fig. 6.22 ; see figs. e6.27-e6.29) • ct halo sign -early finding of a zone of ground-glass opacity (usually related to hemorrhage) surrounding a nodule or mass is strongly suggestive of invasive aspergillosis in an immunocompromised patient (fig. 6. 23; see fig. e6 .30) • aspergilloma -solid homogeneous, rounded, mobile mycetoma that develops in a pre-existing cyst or cavity (primarily upper lobe) in a patient with underlying lung disease and is separated from its wall by a crescentic air space (air crescent sign) (see fig. e6 .31) • histoplasmosis -central united states; nodules often calcify ( fig. 6 .24) • coccidioidomycosis -southwestern united states (also northern mexico and central and south america) • actinomycosis, nocardia -pleural effusion and extension to the chest wall are common (may develop empyema) (see fig. e6 .32) • candidiasis, aspergillosis, sporotrichosis, and mucormycosisessentially limited to debilitated patients and those with underlying diseases (diabetes mellitus, lymphoma, leukemia) (see fig. e6 .33) • caused by a yeast-like fungus and almost exclusively seen in immunosuppressed patients (especially aids, lymphoproliferative diseases, or renal transplants) • initially, bilateral diffuse interstitial opacities spreading outward from the hila • if untreated, this soon progresses to a homogeneous diffuse alveolar consolidation that may simulate pulmonary edema • thin-walled, air-filled lung cysts (especially apical and subpleural) occur in about 40% of patients and may cause a pneumothorax • thick-walled cavities usually indicate superinfection • hilar adenopathy and significant pleural effusions are rare (their presence should raise the possibility of an alternate diagnosis) • although a clinical diagnosis, may appear on chest radiographs as bilateral hilar enlargement due to lymphadenopathy (see fig. e6 .37) • important to look for medial displacement of gas within the stomach and splenic flexure caused by splenomegaly varicella (chickenpox) pneumonia (fig. 6.27; see fig. e6.38) • diffuse distribution of small (1-10 mm), poorly defined nodules, which may coalesce to produce extensive bilateral fluffy infiltrates that tend to develop near the hilum and lung bases • healed varicella pneumonia classically appears as tiny military calcifications scattered widely throughout both lungs (develops several years after the pulmonary infection) • no calcification of hilar lymph nodes (unlike histoplasmosis or tuberculosis, the two other major causes of diffuse pulmonary calcifications) • cytomegalovirus, which typically occurs in immunosuppressed individuals (especially after transplantation) (fig. 6 .28) • respiratory syncytial virus in infants and young children (see fig. e6 .39) • although traditionally considered a disease of children and young adults, with the dramatic decrease in the prevalence of tuberculosis (especially in children and young adults), primary pulmonary disease can develop at any age • primary tuberculosis may affect any lobe, so that the diagnosis cannot be excluded because the infection is not in the upper lobe fig. 6 .28 tree-in-bud pattern (cytomegalovirus). centrilobular groundglass opacities in addition to nodules and "tree-in-bud" opacities in a patient with chronic myelogenous leukemia who underwent bone marrow transplantation [1] • "latent" tb refers to someone who has a positive tst with no history of tuberculous infection or imaging evidence of active or old disease (most often detected when undergoing routine screening for employment or school) • "inactive" tb refers to someone with imaging evidence of prior tuberculosis but no sign of active disease imaging • one or more foci of lobar or segmental air-space consolidation that is usually homogeneous, dense, and well defined (fig. 6 .29) • if multiple, randomly distributed throughout the lungs • cavitation is infrequent. • characteristic apical pleural thickening and fibronodular appearance in one or both upper lungs (fig. 6 .31; see fig. e6 .44) • ct -enlarged lymph nodes typically have a low-density center (due to caseous necrosis) with rim enhancement (reflecting granulomatous tissue); may demonstrate subtle cavitation that is not visible on chest radiographs (see fig. e6 and is almost diagnostic of postprimary tuberculosis • because an apical lesion may be obscured by overlying clavicle or ribs, an apical lordotic view is often of value • cavitation is common (about 50%) and characteristic of postprimary disease (figs. 6.33 and 6.34; see fig. e6 .47) • the presence of cavitation indicates that the disease is highly contagious, and this finding alone warrants putting the patient in respiratory isolation • air-fluid levels in cavities are uncommon and usually a manifestation of superinfection (see fig. e6 .48) • tuberculous cavities may result in endobronchial spread and the classic "tree-in-bud" pattern of centrilobular bronchial dilatation and filling by mucus, pus, or fluid, associated with a linear a b note the thickening of bronchial walls (white arrow) [1] branching pattern that resembles a budding tree and is generally more pronounced in the lung periphery (see figs. e6.49-e6.51) • other complications of cavitation include rupture into the pleural space (leading to empyema or bronchopleural fistula) and the development of a pseudoaneurysm of the pulmonary artery (rasmussen aneurysm) • pleural effusion and lymph node enlargement are rare in postprimary tuberculous disease (though common and sometimes the only finding in primary disease) • as the disease heals, fibrotic changes develop in the surrounding lung, which cause volume loss in the affected segment with displacement of the fissures and hilar structures (see fig. e6 .52) • ct -because the diagnosis of typical postprimary tuberculosis is generally evident on chest radiographs, ct is primarily used to assess the extent and nature of the disease (more sensitive for demonstrating cavitation and such complications as vascular erosion, rupture into the pleural space, and miliary and endobronchial spread) • hematogenous dissemination that usually occurs in patients with altered host resistance to the primary infection • almost invariably leads to a dramatic febrile response with night sweats and chills • there may be minimal symptoms in severely debilitated patients, especially elderly persons and those receiving steroids imaging • diffuse pattern of innumerable tiny (1-2 mm), discrete, relatively well-defined pulmonary nodules distributed uniformly throughout both lungs (fig. 6.35 ; see figs. e6.53 and e6.54) • ct -may detect the presence of diffuse lung involvement when corresponding chest radiographs are normal or show only minimal or limited disease clinical imaging: an atlas of differential diagnosis lower lobe-predominant diseases of the lung mosaic attenuation multiple cystlike lung lesions in the adult pulmonary tuberculosis: role of radiology in diagnosis and management eosinophilic lung diseases: a clinical, radiologic, and pathologic overview upper lobe-predominant diseases of the lung tree-in-bud pattern key: cord-006700-df8ard9o authors: müller-redetzky, holger c.; suttorp, norbert; witzenrath, martin title: dynamics of pulmonary endothelial barrier function in acute inflammation: mechanisms and therapeutic perspectives date: 2014-03-06 journal: cell tissue res doi: 10.1007/s00441-014-1821-0 sha: doc_id: 6700 cord_uid: df8ard9o the lungs provide a large inner surface to guarantee respiration. in lung alveoli, a delicate membrane formed by endoand epithelial cells with their fused basal lamina ensures rapid and effective gas exchange between alveolar and vascular compartments while concurrently forming a robust barrier against inhaled particles and microbes. however, upon infectious or sterile inflammatory stimulation, tightly regulated endothelial barrier leakiness is required for leukocyte transmigration. further, endothelial barrier disruption may result in uncontrolled extravasation of protein-rich fluids. this brief review summarizes some important mechanisms of pulmonary endothelial barrier regulation and disruption, focusing on the role of specific cell populations, coagulation and complement cascades and mediators including angiopoietins, specific sphingolipids, adrenomedullin and reactive oxygen and nitrogen species for the regulation of pulmonary endothelial barrier function. further, current therapeutic perspectives against development of lung injury are discussed. the inner walls of blood vessels are covered with a continuous endothelial cell monolayer, constituting a semi-permeable barrier between blood and interstitium. neighbouring endothelial cells (ecs) are closely connected to each other by interendothelial junctions. under physiologic conditions, the endothelial monolayer actively controls paracellular and transcellular extravasation of proteins, solutes and fluids, thereby adjusting interstitial fluid homeostasis (komarova and malik 2010) . in lung alveoli, endo-and epithelial cells and their merged basal laminas build a delicate membrane of less than 1 μm thickness, which ensures rapid and effective gas exchange between alveolar and vascular lumens while at the same time forming a robust barrier against inhaled particles and microbes. moreover, this sophisticated structure importantly contributes to central metabolic and immunologic functions of the lung. however, upon infectious or sterile inflammatory stimulation via either the alveolar (e.g., in pneumonia and mechanical ventilation) or the vascular lumen (e.g., in bacteremia and sepsis), pulmonary endothelial barrier homeostasis may be disturbed, resulting in increased permeability, protein-rich fluid extravasation, lung oedema and finally acute respiratory distress syndrome (ards) with mortality rates ranging from 27 to 45 % depending on severity (ranieri, et al. 2012) (fig. 1) . pneumonia is the most prevalent infectious disease worldwide and the third most frequent cause of death (world health organisation 2013) . pneumonia is also the most frequent cause of sepsis, a systemic inflammatory response of the organism, which may originate from infections at any other site of the body (abdominal, blood stream, urogenital, etc.) . in both pneumonia and sepsis, the initial innate immune response to invading bacteria, viruses or fungi is insufficient to avert the infection. despite subsequent antibiotic treatment, the interaction of pathogens and host defence culminates in complex inflammatory responses. liberation of inflammatory mediators, recruitment and activation of leukocytes to the lungs and activation of complement and coagulation cascades, are initiated, contributing to pulmonary endothelial hyperpermeability and ards development. for patients with ards, mechanical ventilatory support is an inevitable and life-saving treatment but may also perpetuate the inflammatory response and further enhance pulmonary endothelial barrier dysfunction (verbrugge et al. 2007) . specific pharmacologic therapies aiming at improvement of endothelial barrier function in patients with pneumonia, sepsis and/or ards are lacking. however, recent experimental studies enhanced our understanding of endothelial pathomechanisms contributing to the development of ards, potentially providing the basis for novel therapeutic strategies. therefore, we try here to give an overview on recent insights into the mechanisms of pulmonary endothelial barrier dysfunction in acute inflammation. inside endothelial cells, filaments of polymerised actin molecules together with polymerised tubulin molecules (microtubules) build the cytoskeleton, which is connected to glycocalyx (yoneda and couchman 2003) , focal adhesions and junctional proteins. whereas two types of endothelial junctions, adherens junctions (aj) and tight junctions (tj), are known, the current concept is that a sealing belt of tight junctions is present in ec of the blood-brain barrier but play a minor, if any, role in the barrier function of pulmonary endothelium. consisting in vascular endothelial (ve) cadherin and catenin, ajs maintain the tight connection of adjacent ecs and regulate the paracellular passage of fluids and solutes smaller than 3 nm in radius across the endothelial monolayer (komarova and malik 2010) . in parallel, larger molecules including hormones, drugs, albumin and albumin-bound substances are transported across the endothelial barrier by transcellular trafficking. caveolar vesicles formed at the luminal side of ecs take up the molecules to be transported, cross the ecs and release the molecules at the abluminal surface by exocytosis (predescu et al. 2007 ). these two different transport mechanisms actively regulate endothelial permeability and thereby tissue fluid homeostasis. pathogens entering the alveolar compartment by inhalation or via the bloodstream are recognized by pathogen recognition receptors (prrs). the heterogeneous group of prrs comprises toll-like receptors (tlrs), cytosolic nod-like receptors (nlrs), rig-i-like receptors (rlrs) and dna sensors (opitz et al. 2010) . in alveoli, these receptors are expressed in epithelial cells, macrophages, dendritic cells, ecs and in subsequently recruited immune cells. prrs sense highly conserved microbial molecules called pathogen-associated molecular patterns (pamps) and specific endogenous molecules liberated by cell injury called danger-associated molecular patterns (damps). prr activation evokes cellular production of inflammatory cytokines, interferons and chemokines on transcriptional and post-translational levels (opitz et al 2010) , resulting in the activation of locally distributed cells and the recruitment of neutrophils and macrophages. thus, upon microbial infection and "sterile" tissue damage by various insults, prrs are central contributors to the inflammatory response. when being controlled, these inflammatory mechanisms are a prerequisite for pathogen clearance and thus for survival. however, control is frequently lost and once the inflammatory cascade is on track even effective antibiotic treatment is unable to stop it, which can (partly) be explained by ongoing pamp and damp release from dying bacteria and injured cells, respectively. inappropriate inflammation induces further unchecked synthesis of cytokines, chemokines and lipid mediators, accumulation and activation of leukocytes, uncontrolled activation of complement and coagulation cascades and last but not least endothelial barrier dysfunction. fig. 1 a airspace-derived activation of the endothelium by mediators, bacterial toxins or physical stress due to mechanical ventilation starts a complex interplay of various inflammatory cascades resulting in vascular permeability. monocytes (m) are recruited to the endothelium (ec) and facilitate its further activation by secretion of tnfα, thereby augmenting the recruitment of neutrophils (pmn). activated platelets stimulate pmn. endothelium-pmn contact leads to permeability (1). upon stimulation pmn undergo netosis, liberating neutrophil extracellular traps (nets) consisting in dna and histones that cause endothelial toxicity and barrier breakdown (2). specific soluble mediators also increase permeability (3). neutrophil-platelet complexes activate blood coagulation. central effector proteases like thrombin directly mediate vascular permeability. further, thrombin activates complement factor c5 to c5a-a permeability increasing anaphylatoxin (4). tnf tumor necrosis factor; il-1β interleukin-1β; ros/rns reactive oxygen and nitrogen species. b intracellular signalling regulates endothelial permeability. endothelial contraction results from actin myosin interaction after mlc-phosphorylation, which is regulated by myosin light chain kinase (mlck) and myosin light chain phosphatase (mlcp). mlcp is inhibited by rhoa-rock signalling while mlck is activated by c-src, rhoa and ca 2+ /calmodulin. ca 2+ enters the cytosol from endoplasmatic reticulum (er) or extracellular space. downstream of platelet activating factor (paf) and paf receptor (pafr), phospholipase c (plc) hydrolyses posphatidyl inositol bisphosphate (pip) into inositol 1,4,5-triphosphate (ip3) and diacylglycerol (dag). ip3 mediates ca 2+ liberation from the er while dag opens transient receptor potential canonical (trpc) channels in the cellular membrane. the resulting increase of intracellular ca 2+ leads to the activation of protein kinase c (pkc) α, to further rhoa activation and to ca 2+ /calmodulin complexes, altogether finally leading to mlck activation. actin polymerisation forms stress fibres associated with endothelial contraction. various stimuli like il-1β or mechanical force activate mitogen-activated protein kinase (mapk) p38 (p38), which activates mapk activated protein kinase 2 (mk2), which phosphorylates heat shock protein 25 (hsp25) leading to actin polymerisation. adherence junctions (aj) are mandatory for the sealing of intercellular contacts. ve-cadherin is anchored in peripheral cortical actin to the cytoskeleton. ve-cadherin phosphorylation leads to ve-cadherin internalisation and thereby to increased endothelial permeability. rhoa and c-src phosphorylate vecadherin. rac-1 and p190rhoagap (p190) functionally antagonise rhoa activity. p190rhoagap is recruited to the aj by p120-catenin (p120), which itself inhibits ve-cadherin internalisation. rock inhibits p190rhoagap and pkcα inactivates p120-catenin thereby augmenting destabilisation of aj. iqgap1 recruits and stabilises rac-1, protecting against ve-cadherin internalisation not only direct prr ligation by the pathogen but also liberated pathogenic factors may activate prr-dependent inflammatory cascades. for example, cell wall peptidoglycan of streptococcus pneumoniae activates tlr-2 (schroder et al. 2003) , while the pneumococcal exotoxin pneumolysin is recognized by tlr-4 and nlrp-3 (malley et al. 2003; witzenrath et al. 2011) . bacterial toxins rapidly compromise endothelial cell function (rubins et al. 1992; suttorp et al. 1988 suttorp et al. , 1990 suttorp et al. , 1991 . pneumolysin, for example, may rapidly induce (1) ca 2+ influx and (2) liberation of platelet activating factor (paf) followed by thromboxane release (lucas et al. 2012; witzenrath et al. 2007 ). ca 2+ increase and thromboxane receptor ligation both activate myosin light chain kinase (mlck) via pkcα and rho-kinase dependent signaling (hippenstiel et al. 1997; lucas et al. 2012; witzenrath et al. 2007 ). mlck phosphorylates mlc and subsequent actinmyosin-dependent cytoskeletal contraction evokes disruption of ajs, interendothelial gap formation and paracellular permeability (shen et al. 2010 ). in addition, pneumolysin is a cholesterol-dependent cytolysin that kills ecs by pore formation (tilley et al. 2005) . thus, pathogens may induce endothelial injury via host-dependent inflammatory and via direct mechanisms. upon acute inflammation, neutrophils and distinct monocyte subsets among other recruited leukocytes are involved in the pathophysiology of pulmonary vascular barrier failure. platelets also contribute to vascular injury by activating neutrophils and liberating soluble factors that directly interact with vascular barrier integrity. neutrophils are rapidly recruited to the lung upon different insults (grommes and soehnlein 2011; yoshida et al. 2006 ). in the lungs, the capillary compartment is the place of neutrophil transmigration, in contrast to other vascular beds where neutrophils pass the endothelial barrier in the venules. upon stimulation by various inflammatory agents, the cytoskeleton of the neutrophils changes by forming peripheral actin rims, which leads to neutrophil stiffening and trapping in the capillary bed (yoshida et al. 2006) . although the initial trapping is independent from expression of integrins and selectins on the cell surface (yoshida et al. 2006) , further recruitment may indeed depend on selectins and integrins in distinct scenarios (reviewed in grommes and soehnlein 2011) . however, endothelial leukocyte adhesion and alveolar recruitment of neutrophils does not induce significant vascular permeability per se (martin et al. 1989; rosengren et al. 1991) . although not yet shown for the pulmonary endothelium, studies performed in huvecs or cremaster vessel preparations reveal that during the process of neutrophil transmigration, endothelial disruption seems to be controlled by the formation of "endothelial domes / transmigratory cups" (carman and springer 2004; phillipson et al. 2008 ) that encapsulate the neutrophil and further by ring-like structures of neutrophil lfa-1 and endothelial icam-1 around the invading leukocyte, thereby potentially sealing the barrier through diapedesis (shaw et al. 2004) . however, activated neutrophils contribute to vascular permeability by (1) secretion of soluble factors causing endothelial contraction, (2) contact mediated mechanisms and (3) generation of reactive oxygen species. amongst others, soluble factors of neutrophils include tnf-α, which binds to tnf-α receptor 1 and 2 and is known to induce vascular permeability. notably, although tnf-α leads to mlck and rho kinase (rock)-dependent actin stress fibre generation in endothelial cells, this is probably not the main mechanism of tnf-α induced endothelial permeability, as blocking rock or mlck did not ameliorate transcellular electric resistance (petrache et al. 2001 ). however, tnf-α also induced p38 mapk-dependent disarrangement of the microtubule system and thereby loss of intercellular ve-cadherin resulting in barrier disruption. blocking microtubule breakdown strongly protected against barrier failure induced by tnf-α (petrache et al. 2003) . further soluble factors include: (1) thromboxane a2, which is processed by endothelial cyclooxygenase-2 (cox2) from neutrophil-derived arachidonic acid, binds to the thromboxane receptor and may induce permeability in endothelial cells (kim et al. 2010 ); (2) leukotriene a4, which is processed by endothelial ltc4 synthetase and binds to the endothelial cysteinyl lt receptor subtype 2 (cyslt2r); and (3) cxcl1, -2, -3, -8 which bind to cxcr2 and are involved in endothelial barrier disruption (extensively reviewed in (distasi and ley 2009)). further, neutrophil -endothelial contact via icam-1 and lfa-1/mac-1 leads to (1) rapid increase of intracellular ca 2+ , which mediates actin polymerisation and endothelial contraction as well as disassembly of adherence junctions due to phosphorylation of ve-cadherin and (2) to the secretion of heparin-binding protein, which is also secreted by neutrophils upon binding of ltb4 to the blt1 receptor, finally resulting in barrier failure by endothelial contraction (for detailed review of underlying mechanisms, refer to distasi and ley 2009). activation of neutrophils in the pulmonary microvasculature leads to endothelial hyperpermeability by generation of reactive oxygen species. gao et al. (2007) observed that ros generation upon tnf-α stimulation depends on class 1a phosphoinositide 3 kinase and cd11b/cd18, resulting in nadph oxidase activation and finally generation of ros, causing pulmonary hyperpermeability (see below). platelets secrete various mediators upon activation, including thromboxane, thereby decreasing endothelial barrier integrity as observed in human umbilical vein endothelial cells (huvec) and in vivo (kim et al. 2010) . moreover, platelets mediate vascular permeability in infection and inflammation indirectly via activation of neutrophils (he et al. 2006; looney et al. 2009; zarbock et al. 2006 ). clark and colleagues have shown that platelets are activated by stimulation of tlr4 on their surface in murine sepsis (clark et al. 2007 ). upon activation, platelets secrete thromboxane, which is mandatory for the formation of permeability-mediating platelet-neutrophil complexes. in contrast, neutrophils solely attached to the endothelium after activation by tnf-α do not increase vascular permeability (he et al. 2006) . in mouse models of transfusion-related acute lung injury (trali) platelets are crucial for the development of permeability and pulmonary neutrophil sequestration (looney et al. 2009 ). further, platelets are involved in the generation of neutrophil extracellular traps (nets). neutrophils can undergo a process termed netosis in which the neutrophil expels its condensed dna, to which histones, antimicrobial peptides and enzymes like myeloperoxidase are bound. nets can bind and kill bacteria and thus contribute to the innate immune response against invading pathogens (brinkmann et al. 2004 ). on the other side, nets can be harmful. nets are involved in thrombus generation and cause endothelial permeability and sepsis related organ failure (caudrillier et al. 2012; clark et al. 2007; saffarzadeh et al. 2012) . in trali, platelets are mandatory for net formation in the lung and inhibition of platelet aggregation ameliorated net generation and consecutively pulmonary permeability (caudrillier et al. 2012 ). among peripheral blood monocytes a population of gr-1 high /ccr2 + /cxccr1 low monocytes can be defined, which are delivered from the bone marrow to sites of inflammation. this population rapidly homes in the pulmonary microvasculature upon lipopolysaccharide (lps) infusion or the onset of injurious mechanical ventilation and primes the lung for the development of pulmonary oedema formation when a second hit like lps, zymosan or ventilator-induced lung injury (vili) occurs wilson et al. 2009 ). the mechanism by which this damage is mediated is not fully clarified but the recruited monocytes secrete tnf-α and activate endothelial cells in a paracrine fashion, thereby directly or indirectly contributing to endothelial barrier dysfunction (o'dea et al. 2005) . further, they are involved in the process of neutrophil recruitment in ali (dhaliwal et al. 2012) . although the underlying mechanisms of leukocyte mediated barrier failure are of highest scientific interest, therapeutic interference to ameliorate acute lung injury by depletion or blocking of cell recruitment should raise concerns as neutrophils and monocytes are key players of pulmonary and systemic innate immune responses and therapeutic intervention at this level might leave the patient functionally immunosuppressed. elevated fibrin turnover is a hallmark of acute lung injury regardless of its genesis and may correlate with the severity of the diseases (glas et al. 2013; prabhakaran et al. 2003) . intrapulmonary fibrin deposition results from tissue factorfactor vii pathway activation, reduced pulmonary fibrinolytic capacity due to elevation of plasminogen activator inhibitor 1 (pai-1) concentrations, diminished absolute and relative protein c activity due to reduced protein c production and shedding of thrombomodulin, an important activator of protein c on the cell surface, as well as reduced antithrombin iii levels (hofstra et al. 2011; prabhakaran et al. 2003; ware et al. 2003) . pulmonary coagulopathy occurs after alveolar flooding with protein-rich fluid due to high permeability oedema, resulting in alveolar fibrin deposition but coagulopathy also contributes to inflammation and vascular permeability itself, thereby aggravating the disease. thrombin, the central protease of the coagulation pathway activating fibrinogen, mediates proinflammatory effects by binding to protease activated receptors (par), thereby causing secretion of cytokines or leading to liberation of vascular endothelial growth factor (vegf), which contributes to vascular permeability (hippenstiel et al. 1998 ). furthermore, thrombin can directly cause endothelial cell contraction and processing of complement factor c5a from c5, a potent anaphylatoxin causing inflammation and vascular permeability (cirino et al. 1996; glas et al. 2013; huber-lang et al. 2006; khan et al. 2013; liu et al. 2010) . the complement system is part of the innate immune system and is also involved in functions of the adaptive immune response (mastellos et al. 2003) . the complement cascade can be activated by the classical, the lectin and the alternative pathways (markiewski and lambris 2007) . antigen-antibody complexes activate the classical pathway by binding c1q, thereby processing c1s, while in the lectin pathway mannose binding lectins (mbl) bind to pathogen associated molecular patterns on bacteria, assembling with mannose binding lectin proteases (mblp) 1 and 2 thereafter. c1a and mbl/ mblp1+2 subsequently interact with c2 and c4, processing the c3 convertase c4b2a. the alternative pathway is activated after contact with, e.g., bacterial surfaces by spontaneous hydrolysis of c3, which forms together with factor bb the alternative c3 convertase c3bbb. both c3 convertases process c3 to c3aan anaphylatoxinand c3b, which is part of the c5 convertase. the c5 convertase cleaves c5 into c5aa second anaphylatoxinand c5b, the latter one being part of the membrane attack complex that leads to cell lysis, while c3a and c5a contribute to inflammation and vascular permeability. both c3a and c5a induce stress fibre generation in endothelial cells and thereby endothelial contraction. notably, the response was only of short duration after c3a stimulation, while being prolonged after c5a exposition (schraufstatter et al. 2002) . c5a-induced permeability was more severe and phosphaditiyinositol-3 kinase-, src kinaseand epidermal growth factor (egf) receptor-dependent, while c3a mediated its effects via rho kinase-controlled pathways (schraufstatteret al. 2002) . neutralising c5a in rodent models of acute lung injury and systemic inflammatory responses reduced permeability in various organs including the lung (liu et al. 2010) . however, in c3-deficient mice, immune complex-mediated lung injury including vascular permeability was not attenuated, while c5a deficiency proved to be protective (huber-lang et al. 2006 ). this observation led to the understanding that c5a can be alternatively processed by the protease thrombin defining another alternative pathway for complement activation downstream of c3a. thus, targeting c5a rather than c3a to ameliorate vascular permeability seems to be reasonable. a study by kahn and colleagues also even observed aggravated microvascular injury in c3-deficient mice suffering from acute rejection after trachea transplantation, while antagonisation of c5a was highly protective. again, thrombin-mediated c5a activation accounted for this observation (khan et al. 2013 ). toll-like receptor 4 (tlr4) dependent signaling tlr4 is central for recognition of exogenous (e.g., lps) and endogenous (e.g., high mobility group box-1, oxidised phospholipids) pro-inflammatory stimuli (imai et al. 2008; park et al. 2004) . systemic lps levels have been linked to severity of sepsis and related organ failure (marshall et al. 2004) . lps induced vascular permeability (mehta and malik 2006) and mice deficient for tlr4 were protected against lung injury due to different stimuli including lps, oleic acid, cecal ligation and puncture and gut or lung ischemia/reperfusion injury (ben et al. 2012; hilberath et al. 2011; imai et al. 2008; tauseef et al. 2012; zanotti et al. 2009 ). various signalling cascades have been linked to tlr4-mediated pulmonary permeability. oxidised phospholipids induced tlr4dependent activation of trif (tir domain-containing adapter-inducing interferon-β) and traf6 (tnf receptorassociated factor 6) leading to nf-κb-dependent il-6 liberation, which contributed to lung oedema (imai et al. 2008) . after binding to the tlr4/md2 receptor complex, lps induced nf-κb activation via myd88, irak (interleukin-1 receptorassociated kinase)1, irak2 and irak4 (kawagoe et al. 2008; medvedev et al. 2002) . further, recognition of lps by tlr4 increased intracellular diacylglycerol (dag) levels, activating transient receptor potential canonical (trpc) 6 channels and leading to calcium influx, thereby activating mlck, which facilitates myosin light chain (mlc) phosphorylation inducing endothelial cell contraction. mlck activation further augmented lps-induced nf-κb-related inflammatory responses that contribute to vascular leakage (mehta and malik 2006; tauseef et al. 2012) . further, tlr4 activation evoked phosphorylation of src-kinase and consecutively of ve-cadherin and p120, ultimately resulting in destabilisation of adherence junctions (gong et al. 2008) . tlr-4 is involved in the proinflammatory response to hmgb-1 in monocytes, which again was found to be myd88-, irak1,2,4-and nf-κb-dependent (park et al. 2004 ). moreover, hmgb-1 was linked to lung oedema formation in ventilator-induced lung injury (ogawa et al. 2006 ). however, hmgb-1 also induced endothelial permeability via the receptor for advanced glycation end products (rage) (wolfson et al. 2011) . in summary, tlr4 is often critically involved in the regulation of vascular barrier function during lung inflammation. thus, enthusiasm was aroused by the development of eritoran, an inhibitor of lps-binding to the tlr-4 adaptor molecule md-2. eritoran reduced pulmonary inflammation in different animal models (mullarkey et al. 2003) as well as in humans exposed to lps bolus infusion (lynn et al. 2003) . in a phase ii clinical trial, patients with severe sepsis treated with eritoran tended to have reduced mortality as compared to placebotreated patients (tidswell et al. 2010 ). however, a recent multicentre phase iii study found no impact of eritoran on mortality or relevant secondary outcome parameters in sepsis (opal et al. 2013) , questioning the rationale of tlr4 inhibition for the treatment of sepsis and related organ failure including ards. although not proven by current data, it is tempting to speculate that targeting a single prr was unsuccessful because of the pleiotropic immune activation by various pamps and damps involving different prrs and downstream signaling pathways in sepsis. angiopoietin-1 (ang-1) to ang-4 are ligands of the receptor tyrosine kinase tie2. ang-1 and -2 are well-known regulators of angiogenesis, inflammation and vascular leakage (reviewed in david et al. 2013; eklund and saharinen 2013) , whereas the role of ang-4 and its murine orthologue ang-3 has not been extensively investigated. tie2 is abundantly expressed in endothelium and also in pmns and a subpopulation of monocytes (lemieux et al. 2005; wong et al. 2000) . ang-1 is constitutively expressed in different cell types, including pericytes surrounding the vasculature, vascular smooth muscle cells, fibroblasts, thrombocytes and megakaryocytes (eklund and saharinen 2013) . steady tie2 activation by ang-1 importantly contributes to endothelial quiescence and barrier integrity. in contrast, ang-2 is expressed in endothelial cells, stored in weibel-palade bodies (fiedler et al. 2004 ) and rapidly released upon activation by inflammatory stimuli including tnf-α and thrombin (fiedler et al. 2004 (fiedler et al. , 2006 . ang-2 acts as an antagonist of ang-1 at the tie2 receptor, thus confirming endothelial quiescence and perpetuating proinflammatory, barrier-disintegrating mechanisms (fiedler et al. 2006; scharpfenecker et al. 2005) ang-2 mrna expression is increased upon stimulation by tnf-α, thrombin, hyperoxia, vegf, pdgf and many other factors (augustin et al. 2009 ). in 2006, parikh and colleagues reported that ang-2 serum levels were generally increased in patients with sepsis, being even more increased when sepsis was accompanied by ards (parikh et al. 2006 ). in subjects with acute lung injury, plasma ang-2 had a prognostic value for mortality in non-infection-related but not in infection-related, acute lung injury (calfee et al. 2012) . in two experimental models of sepsis, ang-2 heterozygous mice had reduced ang-2 levels and were protected against lung injury, indicating that ang-2 plays a pathogenetic role besides being a marker of disease severity (david et al. 2012) . the perception of ang-2 being of central pathophysiologic importance in sepsis is being supported by the recent observation that ang-2 antibody treatment attenuated acute pericyte loss, permeability, hypotension and mortality in mice subsequent to intravenous lps injection (ziegler et al. 2013) . in vitro, ang-2 increased and ang-1 suppressed, endothelial adhesion molecule expression and pmn adhesion (fiedler et al. 2006; gamble et al. 2000) . ang-2 may also be able to directly recruit inflammatory cells, because the 20 % monocytes expressing tie-2 have been shown to display chemotaxis towards ang-2 in vitro (murdoch et al. 2007 ). moreover, mice genetically overexpressing ang-1 or being treated with ang-1 showed reduced pulmonary cytokine and adhesion molecule expression, pmn infiltration and vascular leakage in endotoxin-or hydrogen peroxide-induced lung injury (mammoto et al. 2007; mccarter et al. 2007; witzenbichler et al. 2005; xu et al. 2008 ). ang-1 reduced pro-inflammatory gene expression and mediator production probably via interaction of the phosphorylated tie-2 receptor with currently unidentified inhibitors of nf-κb (hughes et al. 2003) . in addition to regulating inflammation, ang-1 and -2 directly alter endothelial integrity. in mice, ang-1-induced tie-2 receptor phosphorylation stimulated the p190rhogtpaseactivating protein (p190rhogap) via pi3-kinase and rac1 to inactivate rhoa, resulting in reduced f-actin stress fibre formation and diminished endothelial permeability (mammoto et al. 2007 ). for rac-1 activation by ang-1, iq domain gtpase-activating protein-1 (iqgap-1) is required . in line, the ability of ang-1 to reduce endotoxemia-induced pulmonary vascular leakage was abolished by downregulation of p190rhogap in mice (mammoto et al. 2007 ). further, ang-1 (1) interfered with the inositol triphosphate (ip3) receptor, thereby blocking trpc1-dependent ca 2+ influx and reducing endothelial hyperpermeability in vitro (ahmmed et al. 2004; jho et al. 2005 ); (2) increased the presence of junctional ve-cadherin protein via extracellular signal-regulated kinase (erk) 1/2dependent activation of sphingosine kinase 1, thereby strengthening the tethering forces between adjacent endothelial cells ; and (3) decreased basal and vegfinduced phosphorylation and subsequent internalisation of ve-cadherin (gavard et al. 2008) . adenoviral ang-1 gene transfer as well as administration of mesenchymal stem cells transfected with ang-1 almost completely abolished pulmonary hyperpermeability induced by subsequent lipopolysacharide injection witzenbichler et al. 2005 ). however, both approaches for ang-1 delivery were far from translation into effective clinical therapies. in this respect, the development of vasculotide, a pegylated 7mer peptide that activates tie-2 (tournaire et al. 2004 ) and the demonstration of vasculotide´s therapeutic potential in established abdominal sepsis in mice (kumpers et al. 2011 ) may represent important milestones on the long way from understanding the importance of tie-2 for endothelial barrier function to the clinical application of tie-2 activation. sphingolipids, a class of lipids containing sphingoid bases as a backbone, form a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. some sphingolipids regulate biological processes, including sphingomyelin, ceramide, sphingosine and sphingosine-1phosphate. the current understanding of the role of these four and other sphingoid bases in acute lung injury has been recently reviewed in detail (natarajan et al. 2013; uhlig and yang 2013) . ceramide is derived from palmitoyl-coa and serine in a multi-step process or from sphingomyelin by sphingomyelinase. ceramide is deacylated to sphingosine (sph) through the action of ceramidases (canals et al. 2011) and sph is rapidly phosphorylated by sphingosine kinase (sphk)-1 and -2 to sphingosine-1-phoshate (s1p). s1p is either cleaved by s1p lyase (s1pl) to ethanol-amine phosphate and trans-2-hexadecenal, or dephosphorylated to sphingosine by s1p phosphatases 1 and 2 (s1ppase) or by lipid phosphate phosphatases (lpp). ceramide deteriorates and s1p improves, barrier integrity. of note, the gram-negative endotoxin lps and the pneumococcal exotoxin pneumolysin disrupt the pulmonary endothelial barrier in a platelet-activating factor (paf)-dependent manner (uhlig and yang 2013; witzenrath et al. 2007) , with paf increasing vascular permeability by an acid sphingomyelinase (asmase)-dependent process (goggel et al. 2004 ). in brief, asmase-produced ceramide recruits caveolin-1, enos and trpc-6 channels into caveolae. no usually blocks trpc6 channels but caveolin-1 inhibits no production by enos, resulting in trpc6 activation followed by an increase of [ca 2+ ] i , mlck activation, mlc phosphorylation and finally ec contraction and paracellular permeability (uhlig and yang 2013) . s1p is produced by platelets, erythrocytes, hematopoietic and vascular endothelial cells (hanel et al. 2007; tani et al. 2005; venkataraman et al. 2008; yatomi et al. 1995) . coordinated biosynthesis and degradation maintain s1p concentrations in plasma and tissues in the range required for most favourable physiologic functions, which include regulation of cell proliferation, differentiation, survival, migration, morphogenesis and barrier function (natarajan et al. 2013) . using mice that selectively lack s1p in the plasma, camerer and colleagues noted that basal plasma levels of s1p maintain endothelial barrier function. as compared to wild-type littermates, mice with a lack of plasma s1p had increased pulmonary vascular leak and demonstrated enhanced susceptibility to paf stimulation, a phenotype reversed by s1p transfusion (camerer et al. 2009) . s1p acts as an intracellular messenger (le stunff et al. 2004) or as an extracellular ligand of five cell surface receptors (s1p1-5), which are differentially expressed and coupled to various g proteins (uhlig and yang 2013) . vascular endothelial cells primarily express s1p1, s1p2 and s1p3. physiologic s1p plasma concentrations (0.5-1 μm) maintain microvascular barrier integrity via ligation of the g i -coupled s1p1 and exogenous addition of s1p to lung ecs increased monolayer integrity rapidly and dose-dependently through s1p1. s1p binding to s1p1 induces rac activation, peripheral mlc phosphorylation, adherens junction assembly and cortactin translocation, which protects endothelium from barrier-disruptive effects of thrombin . moreover, teijaro and colleagues recently observed that endothelial s1p1 critically regulates innate immune responses in influenza pneumonia. activation of endothelial s1p1 attenuated cytokine storm, immune cell recruitment and mortality during infection with human pathogenic influenza virus , suggesting that in this case endothelial cells are conducting the innate immunity orchestra (iwasaki and medzhitov 2011) . in addition to extracellular receptor-dependent effects of s1p, intracellular s1p enhanced barrier integrity independently from s1p receptors requiring rac-1 and sphk1 -/mice were more susceptible to lps-induced lung injury compared with wild-type mice (wadgaonkar et al. 2009 ). along the same line, lps evoked increased expression and activity of the s1p catabolising s1pl, thereby reducing s1p levels. constitutive reduction of s1pl expression in vivo (s1pl +/mice) or in ecs (by sirna) reduced lung injury and inflammation upon lps stimulation (zhao et al. 2011) . most importantly, infusion of s1p reduced lung microvascular leakage and also cytokine release, leukocyte infiltration and histologic tissue changes in numerous different in vivo models of lung injury, including ischemia/reperfusion, pancreatitis and endotoxin challenge in mice and dogs mcverry et al. 2004; okazaki et al. 2007; peng et al. 2004 ). however, s1p at supraphysiologic local concentrations (>5 μm) mediates rhoa-dependent barrier disruption through ligation of s1p2 and s1p3, which couple to gi, gq and g12/13 (sammani et al. 2010; siehler and manning 2002; wang and dudek 2009 ). moreover, s1p stimulates contraction of human bronchial smooth muscle cells (rosenfeldt et al. 2003) , enhances murine airway hyperresponsiveness (roviezzo et al. 2007 ) and evokes bradycardia through s1p3 (forrest et al. 2004 ). the latter findings suggest a rather small therapeutic window for s1p, which may limit the therapeutic potential of s1p and drugs that increase s1p production or reduce s1p catabolism. therefore, s1p receptor agonists have gained considerable interest. for example, intratracheal as well as intravenous delivery of the s1p1 agonist sew-2871 reduced lung permeability after endotoxin injection (sammani et al. 2010 ) and the s1p receptor 1 and 3-5 ligand aal-r reduced lung permeability and mortality after influenza infection in mice ). closer to clinical application is a derivative of the fungal metabolite myriocin, fingolimod (fty720), which holds structural similarities with s1p and has been approved as an immunosuppressive agent for the treatment of multiple sclerosis (brinkmann et al. 2010 ). in addition to its immunosuppressive effects, fty720 enhanced endothelial barrier function in vitro (sanchez et al. 2003) and in vivo (dudek et al. 2007 ) and ameliorated lps-evoked lung injury in mice natarajan et al. 2013 ). however, we recently observed that, although lower concentrations of fty720 enhanced barrier integrity in endothelial cell monolayers (0.01-1 μm fty720) and in mechanically ventilated mice (0.1 mg/kg fty720), higher concentrations (10-100 μm fty720) evoked apoptosis and barrier dysfunction in vitro and in mechanically ventilated mice (2 mg/kg) but not in spontaneously breathing mice (müller et al. 2011 ). if these experimental findings are translatable into the clinical setting, they suggest that, in fingolimod-treated ventilated patients with multiple organ dysfunction syndrome, in whom hepatic metabolism of fty720 is hampered, increased fty720 plasma concentrations could harm lungs that are sensitised by mechanical ventilation towards barrier-destabilising effects of the drug. despite recent studies providing valuable insights into possible mechanisms of barrier regulation by fty720, the mode(s) of action remain unclear. fty720 is partly phosphorylated by sphk2, thereby increasing its affinity to s1p1 and s1p3 (billich et al. 2003) . nevertheless, reduction of vegfinduced permeability by fty720 was independent from s1p1 expression (sanchez et al. 2003 ) and endocytosis and degradation of s1p1 by fty720 has been proposed (cyster 2005) . several further concepts may possibly explain fty720-induced barrier enhancement and have recently been reviewed (natarajan et al. 2013) . notably, fty720, like s1p, induces bradycardia and dyspnea along with fev1 (forced expiratory volume in 1 s) reductions (kappos et al. 2006 ). in conclusion, caution is warranted when considering fty720 for therapeutic lung barrier enhancement in critically ill patients. reactive oxygen species (ros) and reactive nitrogen species (rns) are crucial regulators of cellular function. ros and rns are tightly counterbalanced by antioxidant systems as superoxide dismutase or glutathione. however, excessive ros/rns production or critical reduction of their antioxidative counterparts leads to oxidative stress, which is involved in the pathogenesis of lung injury and particularly vascular permeability. among other molecules displaying oxidative properties, superoxide anions (o 2 -), hydroxyl radical ( oh), hydrogen peroxide (h 2 o 2 ) and hypochloric acid (hocl) are summarised as ros, while metabolites of the nitric oxide ( no) metabolism like nitrite (no 2 -) or peroxynitirite (onoo -) with oxidative power are termed rns. both ros and rns are physiological mediators of functional cell regulation. ros derived from mitochondrial oxidative phosphorylation can modulate the specific cellular pattern by reacting with redox-reactive cysteine residues, thereby altering enzyme activities and controlling cellular signalling (ray et al. 2012) . under inflammatory conditions, endothelial nadph oxidases, xanthine oxidase, cyclooxygenase and enos are involved in increased ros/rns production. neutrophils deliver even higher amounts of ros due to nadph oxydase activity, which are in part further processed to hocl by myeloperoxidase activity. in addition, neutrophils produce rns by inos . ros and rns contribute to acute lung injury upon different insults. perfusion of isolated rabbit lungs with h 2 o 2 evoked lung oedema (hippenstiel et al. 2002; seeger et al. 1995) . h 2 o 2 exposure resulted in a rapid and substantial decrease in endothelial camp content and the effects of h 2 o 2 on endothelial permeability were inhibited by adenylate cyclase activation (suttorp et al. 1993b) . vili increased xanthine oxydoreductase (xor) activity and blocking xorprotected mice from pulmonary hyperpermeability (abdulnour et al. 2006) . ros signalling leads to mapk activation, which is involved in permeability generation in mice subjected to vili (dolinay et al. 2008; park et al. 2012) . underlying mechanisms are proinflammatory functions of this pathway and phosphorylation of heat shock protein 25 (hsp25), which mediates stress fibre generation and endothelial contraction (abdulnour et al. 2006; damarla et al. 2009; dolinay et al. 2008) . further, mice deficient for the transcription factor nrf2 exhibited increased lung injury and permeability in vili due to significantly reduced antioxidative capacity and could be rescued from exacerbation of lung injury by supplementing the antioxidant n-acetyl-cysteine (papaiahgari et al. 2007) . no, the most prominent rns, is a highly diffusible and reactive free radical gas, produced from l-arginine in the lung by constitutively expressed endothelial no synthase (enos) in endothelial cells and by inducible nos (inos) in macrophages. expression of enos usually stays constant while enos activity can be rapidly increased, whereas inos expression is inducible but the activity is usually more or less constant. numerous inflammatory incidents induce no production and release, including endothelial stimulation by bacterial pore-forming toxins (suttorp et al. 1993a ). the plethora of no´s biologic effects includes control of vascular tone and permeability, regulation of mitochondrial respiration and adhesion of platelets and leukocytes. no supports protection of cells against oxidant injury and microbial threats but can also have detrimental properties, e.g., activation of inflammatory processes, enzyme inhibition and dna damage. most probably, these cellular responses are differentially regulated by specific no concentrations (thomas et al. 2008) . the majority of no effects are mediated by (1) nitrolysation of cysteine residues, (2) reaction with transition metals like ion, zinc and copper and (3) formation of onoothrough reaction with o 2 -, which leads to nitration of proteins involved in the regulation of cellular function (korhonen et al. 2005) . inhaled nitric oxide (ino) is used as rescue therapy in individual cases of hypoxic respiratory failure in adults, children and newborns along with respiratory support and other appropriate treatments. the inhaled vasodilator reduces pulmonary arterial pressure without causing systemic vasodilation and selectively redistributes pulmonary blood flow towards ventilated lung regions, thereby reducing shunt flow and improving oxygenation (raoof et al. 2010) . nevertheless, although improvement of blood gases has been regularly noted during the first 24 h of treatment, ino does not increase ventilator-free days or survival of ards patients (afshari et al. 2011) . in addition to its vasodilatory properties, no has endothelial barrier-regulating effects in the lungs but the published experimental studies paint a dichotomous picture. inhaled no was shown to protect against pulmonary barrier dysfunction in isolated perfused and ventilated rabbit lungs upon oxidative stress or ischemia/reperfusion (kavanagh et al. 1994; poss et al. 1995; schutte et al. 2001b) . moreover, ino reduced pulmonary transvascular albumin flux in patients with acute lung injury (benzing et al. 1995 ). the precise mechanisms accounting for the stabilising effect of no remain to be elucidated but may involve increase of cyclic guanosine monophosphate (cgmp) through activation of guanylate cyclase (gc). no-induced barrier protection in rabbit lung ischemia/reperfusion was associated with increased cgmp production and could be further enhanced by inhibition of the cgmp-specific phosphodiesterase (pde) 5 (schutte et al. 2000) . also, increase of cgmp by no (donors) and/or inhibition of cgmp-specific pde 2 strengthened the endothelial barrier in pulmonary ecs upon h 2 o 2 treatment (seeger et al. 1995; suttorp et al. 1996) , in ecs and perfused mouse lungs stimulated with thrombin (seybold et al. 2005) and in mice with severe streptococcus pneumoniae pneumonia (witzenrath et al. 2009 ). the barrier-stabilising effects of no and cgmp may be partly explained by negative regulation of specific endothelial trp channels (yin et al. 2008) , some of which are central for [ca 2+ ] i increase, pulmonary endothelial cell contraction and lung hyperpermeability in response to various stimuli (alvarez et al. 2006; boueiz and hassoun 2009; hamanaka et al. 2007; jian et al. 2008; kuebler et al. 2010; tiruppathi et al. 2002; yin et al. 2008) . on the other hand, endogenous no synthesis contributed to lung injury in hypoxic ischemia/reperfusion of isolated rabbit lungs (schutte et al. 2001a) . moreover, inos expression was upregulated in response to mechanical ventilation in mice and ventilated inos -/mice as well as inos inhibitortreated mice had reduced lung inflammation and permeability compared with control wt mice . in line, pharmacologic inhibition of nos prevented the development of pulmonary hyperpermeability in rats subjected to vili (choi et al. 2003) . gain and loss of function studies have provided evidence for a contribution of soluble gc activation to ventilator-induced lung injury in mice (schmidt et al. 2008) . further, ino significantly increased endothelial permeability in rats with pseudomonas aeruginosa pneumonia independently from the inflammatory response (ader et al. 2007 ). thus, the individual effects of no on pulmonary vascular barrier function seem to depend on local no concentrations and the precise pathologic conditions. imatinib has been suggested for the treatment of increased vascular permeability. the tyrosine kinase inhibitor imatinib targets c-abl kinase, platelet-derived growth factor-derived receptors, c-kit, arg kinase and discoid domain receptors 1 and 2 and has been implemented into treatment of chronic myelogenous leukaemia. recently, imatinib was found to protect against endothelial barrier dysfunction evoked by thrombin in isolated endothelial cells, by vegf in a murine skin model and in the context of polymicrobial sepsis in mice. as the underlying mechanism, inhibition of arg kinase followed by augmented rac1 signalling and stabilised intercellular junctions and cell matrix adhesion has been identified (aman et al. 2012; chislock and pendergast 2013) . case reports have been published describing reduction of pulmonary oedema in the context of pulmonary venooclusive disease and resolution of bleomycin-induced pneumonitis (carnevale-schianca et al. 2011; overbeek et al. 2008) . with respect to clinical development, additional preclinical evidence for imatinib efficacy in ards is required. further, possible relevant undesirable effects have to be considered including cerebral haemorrhage particularly in patients with compromised coagulation, as malfunction of coagulation is also a major issue in sepsis patients (hoeper et al. 2013) . adrenomedullin (am) is an endogenous peptide with potent barrier protective properties that is expressed in various cells of the vascular system including endothelial and vascular smooth muscle cells and also in cardiomyocytes, epithelial cells and leukocytes. the am gene encodes for a preproadrenomedullin, which is processed to pro-am, from which am and proam n-terminal 20 peptide (pamp) are generated. a m i d a t i o n b y p e p t i d o g l y c i n e a l p h a a m i d a t i n g monooxygenase (pam) is crucial for biologic function of the active am peptide (temmesfeld-wollbruck et al. 2007b) . am binds to the calcitonin receptor like receptor (crlr), which assembles with receptor activity-modulating proteins (ramp) 2 and 3. in endothelial cells, binding of am to the receptor results in intracellular accumulation of the second messenger camp and in activation of various kinases including protein kinase a (pka), pkc, map kinases and others (hippenstiel et al. 2002; temmesfeld-wollbruck et al. 2007b) . mice deficient for am, crlr, pam or ramp2 die prematurely of hydrops fetalis, which highlights the role of am for vascular barrier integrity (bonder et al. 2009; caron and smithies 2001; cyster 2005; czyzyk et al. 2005; ichikawa-shindo et al. 2008) . am is up-regulated under inflammatory conditions like sepsis or experimental lung injury (agorreta et al. 2005; cheung et al. 2004; matheson et al. 2003 ) and mice heterozygous for am exhibit an aggravated inflammatory response and organ damage following lps challenge (dackor and caron 2007) . treatment with exogenous am protected against pulmonary hyperpermeability induced by various stimuli like staphylococcus aureus alpha toxin, hydrogen peroxide, lipopolysaccaride (lps) or hyperoxia and ventilator-induced lung injury (hippenstiel et al. 2002; itoh et al. 2007; müller et al. 2010; temmesfeld-wollbruck et al. 2007a) . am also protected against barrier breakdown in the gut after challenge with staphylococcus aureus alpha toxin and in ischemia reperfusion injury and stabilised the blood-brain barrier (brell et al. 2005a, b; higuchi et al. 2008; honda et al. 2006; kis et al. 2003; temmesfeld-wollbruck et al 2007a ). at least two major mechanisms may contribute to the impressive function of am. first, am leads to the relaxation of the contractile apparatus of the endothelial cell by avoiding the generation of actin stress fibres and actin myosin interaction (temmesfeld-wollbruck et al. 2007b) . we and others have observed a rise of intracellular camp upon am stimulation of endothelial cells, leading to the inhibition of mlc phosphorylation, thereby blocking actin-myosin interactionmediated cell contraction induced by thrombin or hydrogen peroxide in vitro, or evoked by mechanical ventilation in vivo (brell et al. 2005b; hocke et al. 2006; müller et al. 2010 ). however, equally potent barrier protective effects of am are observed in gut epithelial cells that were not dependent on intracellular camp increase (temmesfeld-wollbruck et al. 2009 ). second, besides reducing cell contraction am increases intercellular adherence, thereby mediating barrier stabilisation. in rat intestine, staphylococcus alpha toxin infusion induced vascular hyperpermeability accompanied by loss of ve-cadherin in submucosal blood vessels, which was avoided by am treatment (hocke et al. 2006) . in endothelial cells, am protected against the loss of ve-cadherin and occludin derangement due to thrombin or staphylococcus alpha toxin stimulation and am enhanced the expression of claudin-5 in brain microvascular endothelial cells (hocke et al. 2006; honda et al. 2006) . immunomodulating effects of am have been described (gonzalez-rey et al. 2006 ); however, we observed that the strong barrier protection of am is not coupled to anti-inflammatory properties (müller et al. 2010) . although the underlying and obviously cellspecific mechanisms of am-mediated barrier protection partly remain elusive, the powerful properties observed in complex models regardless of the stimulus and independent from immunosuppressive effects indicate a high translational potential for am. acute inflammatory diseases including pneumonia and sepsis may result in ards, which is still associated with unacceptably high mortality. research has been successfully uncovering basic disease mechanisms, leading to improvements in therapy including ventilation and resuscitation strategies. nevertheless, although the pulmonary endothelium has long been noted to be central in the pathogenesis of ards and scientists have been elucidating innumerable important mechanisms of permeability increase, most therapeutic strategies to improve ards outcome based on the understanding of lung endothelial barrier dysfunction have so far been frustrating. these drawbacks should be understood as important sources of perception and it might be worth considering some general aspects when moving forward in this field. first, to regain endothelial barrier function once the endothelium is severely injured may be a barely achievable objective. interestingly, the only strategies so far decreasing mortality in ards, reduction of tidal volume and probably early prone positioning, short-term use of neuromuscular blockers and oesophageal pressure-guided positive endexspiratory pressure adjustment (guerin et al. 2013; network ards 2000; papazian et al. 2010; talmor et al. 2008) , are aimed at alleviation of further inflammatory stress by mechanical ventilation, thus being of a rather preventive nature. it may be promising to focus on strategies that decelerate the progress of "uncomplicated" pneumonia or sepsis to ards instead of trying to reverse severe parenchymal inflammation and injury. therefore, clinical and biological predictors of progress towards ards need to be identified and future therapies should be started before full-blown ards has developed. however, this notion should not encourage the performing of experimental studies in which the treatment of interest is commenced before onset of the initial disease (pneumonia or sepsis in this case), because such a preventive strategy can rarely be translated into clinics. second, the "real life aspect" needs to be respected. icu patients are frequently prone to ards due to multiple simultaneous incidents, unlike, e.g., lps-treated mice, which means that numerous redundant pathways may be differentially involved and should probably be addressed therapeutically at the same time. further, important inter-individual differences need to be considered. third, complexity is an important issue. as our understanding of central contributors to lung injury is growing, we are becoming aware of the differential effects one and the same pathomechanistic system may have. for example, s1p seems to differentially affect endothelial integrity, depending on s1p concentration, receptor expression and the exact local cellular setting, which implements a further dimension into the picture of barrier destructing mechanisms. probably, systems biology combined with mathematical multi-scale models that integrate knowledge from experimental studies (in vitro, in vivo and in silico), clinical trials and clinical and biological predictors of the individual patient will facilitate development of successful novel therapies and improvement of ards prevention. since the first description of ards in 1967, researchers have made great efforts to unravel the mechanisms contributing to endothelial dysfunction in the lung in order to develop novel therapies. walking all the way to where we are standing today has sometimes been frustrating and possibly not even half of the whole distance has been accomplished. nevertheless, considering the high morbidity and mortality of ards, it is worth trying hard to proceed. mechanical stress activates xanthine oxidoreductase through map kinase-dependent pathways inhaled nitric oxide increases endothelial permeability in pseudomonas aeruginosa pneumonia inhaled nitric oxide for acute respiratory distress syndrome and acute lung injury in adults and children: a systematic review with meta-analysis and trial sequential analysis adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and lps protein kinase calpha phosphorylates the trpc1 channel and regulates store-operated ca2+ entry in endothelial cells transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury effective treatment of edema and endothelial barrier dysfunction with imatinib control of vascular morphogenesis and homeostasis through the angiopoietin-tie system tlr4 mediates lung injury and inflammation in intestinal ischemiareperfusion inhaled nitric oxide reduces pulmonary transvascular albumin flux in patients with acute lung injury phosphorylation of the immunomodulatory drug fty720 by sphingosine kinases sphingosine kinase regulates the rate of endothelial progenitor cell differentiation regulation of endothelial barrier function by reactive oxygen and nitrogen species adrenomedullin treatment abolishes ileal mucosal hypoperfusion induced by staphylococcus aureus alpha-toxin-an intravital microscopic study on an isolated rat ileum adrenomedullin reduces staphylococcus aureus alpha-toxin-induced rat ileum microcirculatory damage neutrophil extracellular traps kill bacteria fingolimod (fty720): discovery and development of an oral drug to treat multiple sclerosis plasma angiopoietin-2 in clinical acute lung injury: prognostic and pathogenetic significance sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases a transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them complete resolution of life-threatening bleomycin-induced pneumonitis after treatment with imatinib mesylate in a patient with hodgkin's lymphoma: hope for severe chemotherapy-induced toxicity? extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene platelets induce neutrophil extracellular traps in transfusion-related acute lung injury increased adrenomedullin expression in lungs in endotoxaemia abl family kinases regulate endothelial barrier function in vitro and in mice systemic microvascular leak in an in vivo rat model of ventilator-induced lung injury thrombin functions as an inflammatory mediator through activation of its receptor platelet tlr4 activates neutrophil extracellular traps to ensnare bacteria in septic blood chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury angiopoietin-1 requires iq domain gtpase-activating protein 1 to activate rac1 and promote endothelial barrier defense angiopoietin-2 may contribute to multiple organ dysfunction and death in sepsis* mending leaky blood vessels: the angiopoietin-tie2 pathway in sepsis monocytes control second-phase neutrophil emigration in established lipopolysaccharide-induced murine lung injury opening the flood-gates: how neutrophilendothelial interactions regulate permeability mitogenactivated protein kinases regulate susceptibility to ventilatorinduced lung injury pulmonary endothelial cell barrier enhancement by fty720 does not require the s1p1 receptor angiopoietin signaling in the vasculature the tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell weibel-palade bodies angiopoietin-2 sensitizes endothelial cells to tnfalpha and has a crucial role in the induction of inflammation immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions blockade of class ia phosphoinositide 3-kinase in neutrophils prevents nadph oxidase activation-and adhesion-dependent inflammation sphingosine 1-phosphate promotes endothelial cell barrier integrity by edg-dependent cytoskeletal rearrangement angiopoietin-1 prevents vegfinduced endothelial permeability by sequestering src through mdia bronchoalveolar hemostasis in lung injury and acute respiratory distress syndrome paf-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide tlr4 signaling is coupled to src family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia urocortin and adrenomedullin prevent lethal endotoxemia by downregulating the inflammatory response contribution of neutrophils to acute lung injury prone positioning in severe acute respiratory distress syndrome trpv4 initiates the acute calcium-dependent permeability increase during ventilatorinduced lung injury in isolated mouse lungs erythrocytes store and release sphingosine 1-phosphate in blood leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells gut hyperpermiability after ischemia and reperfusion: attenuation with adrenomedullin and its binding protein treatment resolution of toll-like receptor 4-mediated acute lung injury is linked to eicosanoids and suppressor of cytokine signaling 3 glucosylation of small gtp-binding rho proteins disrupts endothelial barrier function vegf induces hyperpermeability by a direct action on endothelial cells adrenomedullin reduces endothelial hyperpermeability perturbation of endothelial junction proteins by staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin imatinib mesylate as add-on therapy for pulmonary arterial hypertension: results of the randomized impres study pulmonary activation of coagulation and inhibition of fibrinolysis after burn injuries and inhalation trauma adrenomedullin improves the blood-brain barrier function through the expression of claudin-5 generation of c5a in the absence of c3: a new complement activation pathway the antiinflammatory endothelial tyrosine kinase tie2 interacts with a novel nuclear factor-kappab inhibitor abin-2 the gpcr modulator protein ramp2 is essential for angiogenesis and vascular integrity identification of oxidative stress and tolllike receptor 4 signaling as a key pathway of acute lung injury adrenomedullin ameliorates lipopolysaccharideinduced acute lung injury in rats a new shield for a cytokine storm angiopoietin-1 opposes vegf-induced increase in endothelial permeability by inhibiting trpc1-dependent ca2 influx high vascular pressure-induced lung injury requires p450 epoxygenasedependent activation of trpv4 oral fingolimod (fty720) for relapsing multiple sclerosis effects of inhaled no and inhibition of endogenous no synthesis in oxidantinduced acute lung injury sequential control of tolllike receptor-dependent responses by irak1 and irak2 targeting complement component 5a promotes vascular integrity and limits airway remodeling thromboxane a(2) increases endothelial permeability through upregulation of interleukin-8 chronic adrenomedullin treatment improves blood-brain barrier function but has no effects on expression of tight junction proteins regulation of endothelial permeability via paracellular and transcellular transport pathways nitric oxide production and signaling in inflammation vascular barrier regulation by paf, ceramide, caveolae, and no -an intricate signaling network with discrepant effects in the pulmonary and systemic vasculature the synthetic tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis generation and metabolism of bioactive sphingosine-1-phosphate angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1 sphingosine-1-phosphate and its analogue fty720 diminish acute pulmonary injury in rats with acute necrotizing pancreatitis silencing of c5a receptor gene with sirna for protection from gram-negative bacterial lipopolysaccharide-induced vascular permeability platelet depletion and aspirin treatment protect mice in a twoevent model of transfusion-related acute lung injury protein kinase c-alpha and arginase i mediate pneumolysin-induced pulmonary endothelial hyperpermeability blocking of responses to endotoxin by e5564 in healthy volunteers with experimental endotoxemia recognition of pneumolysin by toll-like receptor 4 confers resistance to pneumococcal infection angiopoietin-1 requires p190 rhogap to protect against vascular leakage in vivo the role of complement in inflammatory diseases from behind the scenes into the spotlight diagnostic and prognostic implications of endotoxemia in critical illness: results of the medic study effects of leukotriene b4 in the human lung recruitment of neutrophils into the alveolar spaces without a change in protein permeability complement: structure, functions, evolution, and viral molecular mimicry adrenomedullin is increased in the portal circulation during chronic sepsis in rats the acute respiratory distress syndrome cell-based angiopoietin-1 gene therapy for acute lung injury sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury dysregulation of lps-induced toll-like receptor 4-myd88 complex formation and il-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells signaling mechanisms regulating endothelial permeability prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1 inhibition of endotoxin response by e5564, a novel toll-like receptor 4-directed endotoxin antagonist adrenomedullin attenuates ventilator-induced lung injury in mice the sphingosine-1 phosphate receptor agonist fty720 dose dependently affected endothelial integrity in vitro and aggravated ventilator-induced lung injury in mice expression of tie-2 by human monocytes and their responses to angiopoietin-2 sphingosine-1-phosphate, fty720, and sphingosine-1-phosphate receptors in the pathobiology of acute lung injury ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia mobilization and margination of bone marrow gr-1high monocytes during subclinical endotoxemia predisposes the lungs toward acute injury contribution of high-mobility group box-1 to the development of ventilator-induced lung injury sphingosine 1-phosphate inhibits ischemia reperfusion injury following experimental lung transplantation innate immune recognition in infectious and noninfectious diseases of the lung possible role of imatinib in clinical pulmonary veno-occlusive disease genetic and pharmacologic evidence links oxidative stress to ventilatorinduced lung injury in mice neuromuscular blockers in early acute respiratory distress syndrome excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein mitogen-activated protein kinase phosphatase-1 modulates regional effects of injurious mechanical ventilation in rodent lungs protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury differential effect of mlc kinase in tnf-alpha-induced endothelial cell apoptosis and barrier dysfunction the role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability endothelial domes encapsulate adherent neutrophils and minimize increases in vascular permeability in paracellular and transcellular emigration inhaled nitric oxide prevents the increase in pulmonary vascular permeability caused by hydrogen peroxide elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury molecular determinants of endothelial transcytosis and their role in endothelial permeability acute respiratory distress syndrome: the berlin definition severe hypoxemic respiratory failure: part 2-nonventilatory strategies reactive oxygen species (ros) homeostasis and redox regulation in cellular signaling sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells leukotriene b4-induced neutrophil-mediated endothelial leakage in vitro and in vivo sphingosine-1-phosphate/sphingosine kinase pathway is involved in mouse airway hyperresponsiveness toxicity of pneumolysin to pulmonary endothelial cells in vitro neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones differential effects of sphingosine 1-phosphate receptors on airway and vascular barrier function in the murine lung phosphorylation and action of the immunomodulator fty720 inhibits vascular endothelial cell growth factor-induced vascular permeability the tie-2 ligand angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice complement c3a and c5a induce different signal transduction cascades in endothelial cells lipoteichoic acid (lta) of streptococcus pneumoniae and staphylococcus aureus activates immune cells via toll-like receptor (tlr)-2, lipopolysaccharide-binding protein (lbp), and cd14, whereas tlr-4 and md-2 are not involved the pde inhibitor zaprinast enhances no-mediated protection against vascular leakage in reperfused lungs endogenous nitric oxide synthesis and vascular leakage in ischemic-reperfused rabbit lungs short-term "preconditioning" with inhaled nitric oxide protects rabbit lungs against ischemia-reperfusion injury hydrogen peroxide-induced increase in lung endothelial and epithelial permeability-effect of adenylate cyclase stimulation and phosphodiesterase inhibition tumor necrosis factor-alpha-dependent expression of phosphodiesterase 2: role in endothelial hyperpermeability coordinated redistribution of leukocyte lfa-1 and endothelial cell icam-1 accompany neutrophil transmigration myosin light chain kinase in microvascular endothelial barrier function pathways of transduction engaged by sphingosine 1-phosphate through g protein-coupled receptors bacterial exotoxins and endothelial permeability for water and albumin in vitro effects of escherichia coli hemolysin on endothelial cell function adenosine diphosphate-ribosylation of g-actin by botulinum c2 toxin increases endothelial permeability in vitro pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells role of phosphodiesterases in the regulation of endothelial permeability in vitro role of nitric oxide and phosphodiesterase isoenzyme ii for reduction of endothelial hyperpermeability mechanical ventilation guided by esophageal pressure in acute lung injury mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets tlr4 activation of trpc6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation endothelial cells are central orchestrators of cytokine amplification during influenza virus infection adrenomedullin reduces vascular hyperpermeability and improves survival in rat septic shock adrenomedullin and endothelial barrier function adrenomedullin reduces intestinal epithelial permeability in vivo and in vitro the chemical biology of nitric oxide: implications in cellular signaling phase 2 trial of eritoran tetrasodium (e5564), a toll-like receptor 4 antagonist, in patients with severe sepsis structural basis of pore formation by the bacterial toxin pneumolysin impairment of store-operated ca2+ entry in trpc4(-/-) mice interferes with increase in lung microvascular permeability a short synthetic peptide inhibits signal transduction, migration and angiogenesis mediated by tie2 receptor sphingolipids in acute lung injury vascular endothelium as a contributor of plasma sphingosine 1-phosphate lung protective ventilatory strategies in acute lung injury and acute respiratory distress syndrome: from experimental findings to clinical application differential regulation of sphingosine kinases 1 and 2 in lung injury suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus regulation of vascular permeability by sphingosine 1-phosphate protein c and thrombomodulin in human acute lung injury role of lung-marginated monocytes in an in vivo mouse model of ventilator-induced lung injury protective role of angiopoietin-1 in endotoxic shock role of platelet-activating factor in pneumolysin-induced acute lung injury phosphodiesterase 2 inhibition diminished acute lung injury in murine pneumococcal pneumonia the nlrp3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia hmgb1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption the angiopoietins, tie2 and vascular endothelial growth factor are differentially expressed in the transformation of normal lung to non-small cell lung carcinomas the 10 leading causes of death in the world mesenchymal stem cell-based angiopoietin-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice sphingosine-1-phosphate: a platelet-activating sphingolipid released from agoniststimulated human platelets negative-feedback loop attenuates hydrostatic lung edema via a cgmp-dependent regulation of transient receptor potential vanilloid 4 regulation of cytoskeletal organization by syndecan transmembrane proteoglycans neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats novel critical role of toll-like receptor 4 in lung ischemiareperfusion injury and edema complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation protection of lps-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis acknowledgement this work was funded by the deutsche forschungsgemeinschaft (sfb-tr84 b2, z2 to n.s. and c3, c6 to m.w.) key: cord-285270-amh99u0j authors: husain, shahid; mooney, martha l.; danziger-isakov, lara; mattner, frauke; singh, nina; avery, robin; ison, michael; humar, atul; padera, robert f.; lawler, leo p.; fisher, andy; drew, richard j.; gould, kate f.; sole, amparo; studer, sean; munoz, patricia; singer, lianne g.; hannan, margaret title: a 2010 working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients date: 2011-03-17 journal: j heart lung transplant doi: 10.1016/j.healun.2011.01.701 sha: doc_id: 285270 cord_uid: amh99u0j nan in the absence of standardized diagnosis and the presence of unique clinical syndromes, it is not surprising that considerable differences exist in the number of reported incidences of disease and the outcomes of various infections in cardiothoracic transplant (cttx) recipients. publications to date have employed variable and heterogeneous definitions of cttx-related infections, thereby limiting the comparison between the types and incidence of infections and the generalizability of these data across transplant centers. currently, there are no standard international definitions for infections uniquely related to cttx, with the exception of chagas disease and toxoplasmosis. 1 the purpose of the present working formulation is to provide consensus-derived expert opinion of definitions for infections in cttx for epidemiologic, research and registry data use. standard definitions of infections specifically related to cttx will allow for meaningful comparison of the type and incidence of these infections between different types of cttxs, different regimens of immunosuppression and between different transplant centers, thereby improving the reporting of infection-related morbidity and mortality after cardiothoracic transplantation. the definitions proposed herein are suitable for epidemiologic investigations and are intended to facilitate clinical decision-making. the definitions described in what follows have been reviewed and approved by a multidisciplinary working group of the international society for heart and lung transplantation (ishlt). these definitions were adapted from surveillance definitions of healthcare-associated sinusitis, tracheobronchitis and pneumonia, used in reporting to the national healthcare safety network (nhsn) and the centers for disease control and prevention's (cdc) surveillance system for patient and healthcare personnel safety. 2 definitions of invasive fungal infection (ifi) were based on those proposed by the european organization for research and treatment of cancer and the mycoses study group of the national institutes of health (eortc/msg), 3 whereas definitions from the american society of transplantation and other source documents represented the foundation for defining viral infections. 1, 4 bacterial infection overview bacterial infections are a major contributor of complications in the early post-transplant period in heart-and lung-transplanted patients. 5, 6 some bacterial infections (e.g., pre-transplant colonization or donor-derived infections) have unique issues and implications in cttx recipients 5, [7] [8] [9] [10] [11] [12] ; therefore, the definitions for these infections for epidemiologic, research or ishlt registry purposes are specifically addressed herein. many other bacterial infections (e.g., methicillin-resistant staphylococcus aureus or vancomysin-resistant enterococcus) are present similarly across hospitalized patients and solid-organ transplant (sot) recipients and are therefore not directly addressed in what follows. the existing literature in cttx has largely classified bacterial infections as "early" (e.g., post-operative) or "late" onset after transplantation, allowing transplant clinicians to determine the source of these infections and focus prevention strategy and early empirical antibiotic treatment regimens on the temporal onset of these infections. a further timeline is used to classify all infections diagnosed in the hospital setting as nosocomial, with onset 48 hours after the patient is admitted to the hospital, and communityacquired infection, with onset at the time of admission or within 48 hours of admission. the latter definitions of infection may be artificial in the setting of cttx as some infections, although related to healthcare and immunosuppression, may not occur within the established time-line of nosocomial infections. to fully appreciate the impact of the potential source of infection, we propose using the categories of nosocomial (after 48 hours of hospitalization) and community-acquired (prior to 48 hours of hospitalization) with the added category of community-acquired "transplant-related" infections. this category would include infections by pathogens acquired by the cttx patient prior to time of transplantation and that are clearly related to the immunocompromised state of the cttx patient after transplantation that may increase the risk for specific bacterial pathogens that are not common in the community. these pathogens may be related to the donor or the recipient via pre-transplant colonization of the respiratory or gastrointestinal (gi) tract and can be therefore regarded as "transplant-related" in this setting. 12, 13 it is also to be noted that community-acquired pneumonia may be transplant-related if caused by organisms that are typically associated with transplants (e.g., fungal, multidrug-resistant or atypical bacteria). respiratory bacterial infections occur frequently in lung transplant recipients. in one study, 72 episodes per 100 lung transplants per year were reported. 14 the definitions of bacterial pneumonia present significant challenges in cttx. frequent use of empirical anti-bacterial agents prior to specimen collection and the possibility of concurrent allograft rejection make the use of standard guidelines, as presented by the centers for disease control and prevention (cdc) for healthcareassociated infections (hcais), difficult to apply. 2 in addition, some microbiologic diagnostic procedures may not be routinely practiced at many transplant centers and this may limit the employment of diagnostic criteria for infections that require quantification of bacterial colonyforming units per milliliter in the bronchoalveolar lavage (bal) samples. this methodology has not been validated in the immunocompromised host and is not standardized across institutions. further, the thresholds proposed may underestimate the episodes of bacterial pneumonia in the cttx population, 20 where early empirical intervention with anti-microbial agents prior to obtaining the samples with suspected pneumonia is common practice. presence of endobronchial stents in lung transplant recipients further complicates the picture in defining various clinical syndromes. for these reasons, a specific classification of bacterial pneumoniae in cttx recipients is proposed based on radiographic findings, clinical symptoms, microbiology and histopathology (including consideration of acute rejection in lung transplant patients). in lung transplant recipients, the use of differential cytology in bal may be helpful. [21] [22] [23] the predominance of neutrophils with intracellular bacteria (hematoxylin-eosin and gram stain) is more suggestive of the presence of a bacterial pneumonia than a high proportion of lymphocytes or eosinophils in bal. on the other hand, a lymphocytic or eosinophilic bal could indicate an acute graft reaction, although cytomegalovirus (cmv), other viruses and atypical pathogens would need to be ruled out. acute rejection (ar) of the graft in lung transplant recipients presents a significant consideration in the diagnosis of all pneumonias, including those due to bacteria. there are frequent clinical scenarios where distinction between rejection and infection is critically dependent upon histopathologic findings. in many cases, evidence for infection and rejection coexist. therefore, in the setting of lung transplantation, the diagnosis of bacterial (or any) pneumonia is more precisely defined by the confirmation or exclusion of ar when microbiologic criteria are not met. 24 the determination of ar requires histologic examination. if an ar is documented and clinical and laboratory criteria for bacterial pneumonia are also fulfilled, the diagnosis of ar and concomitant pneumonia is possible. 24 accordingly, pneumonia should be indicated as pneumonia combined with an ar. • direct examination by light microscopy [gram stain, modified acid-fast bacilli (afb) stain for nocardia spp, afb stains for mycobacteria]. • culture (including rapid culture methods for legionella spp, mycobacteria spp and prolonged culture periods for detection of bacteria-causing infective endocarditis). • bal cell analysis: rule-out contamination with ͻ1% epithelial cells, 20 definitions of bacterial pneumonia and colonization in cttx are given in table 1a , whereas the definitions of tracheobronchitis are given in table 1b . 1. ventilator-associated pneumonia should be designated when reporting data. a distinction should be made between non-invasive and invasive ventilation. 2. aspiration pneumonia should be considered if the criteria are fulfilled for pneumonia (table 1a ). the cause of this type of pneumonia should be noted. 3. multiple or concurrent episodes of post-transplant pneumonia may occur. to determine a new episode in a single patient, resolution of the initial infection must be determined by clinical, laboratory or histologic evidence. the isolation of a new pathogen alone is not indicative of a new episode of pneumonia. in contrast, a second pneumonia may develop in a patient after single lung transplantation. here, the contralateral lung may develop an "independent" pneumonia by another organism. 4. sputum samples are frequently contaminated with airway colonizers (e.g., coagulase-negative staphylococcus and enterococcus spp), and therefore must be interpreted cautiously. 5. the interferon-gamma release assay (igra) serum test is not recommended for diagnosis of acute tracheobronchitis disease, although a positive result is an indication of latent disease or recent infection and a useful screening test if baseline igra testing is performed prior to transplantation. 25 6. episodes of airway colonization are not recorded as infections. 7. histologic representation of chronic graft rejection may not impact the diagnosis of bacterial pneumonia. therefore, it is not included as criteria for pneumonia definition. 26 8. the definition of "possible pneumonia" category allows recording of pneumonia after lung transplantation even if required diagnostic procedures were missed, which may occur with prior anti-microbial treatment or delay in diagnostic testing, etc. 9. in lung transplant recipients, it is desirable to always give additional information if evidence of acute graft rejection exists either by clinical or by histopathologic diagnosis. 10. it is possible to have concurrent infections-pneumonia with sinusitis or anastomotic tracheobronchitis. 11. quantification of organisms in bal is not considered essential for the diagnosis of ventilator-associated pneumonia (vap). 27 however, invasive diagnostics may help withdraw anti-bacterial therapy, which may prevent further emergence of multi-resistant organisms in future. 28,29 12. the category of bacterial tracheobronchitis is classified into probable and proven categories. they can only be diagnosed in the presence of bronchoscopic findings. we have refrained from using the term microbiogically negative tracheobrochitis as it requires more evidence. 13. endobronchial stent-associated tracheobronchitis or bronchial anastomotic infections, both fungal and bacterial, are categorized as probable (table 2 ). 14. no attempt is made to redefine atypical mycobacterial infections or pulmonary tracheobronchitis in lung transplant recipients and the use of existing definitions from european and north american societies are encouraged until further data emerge. 30 -33 15 . it is preferable to document the use of antibiotics in patients with pneumonia at the time of culture data collection. cardiothoracic transplant recipients are at an increased risk for viral infections with severe clinical sequelae. some viral infections have unique considerations and implications in cttx recipients. the definitions for these viral infections are specifically addressed herein and may be used for epidemiologic, research or registry purposes in cttx recipients. many other non-respiratory viral infections present similarly across sot recipients. diagnosis and management of these viral infections have been addressed adequately in other guidelines 1 and therefore will not be addressed herein. respiratory viral infections, including newly emerging viruses, occur frequently in lung transplant recipients. 34 some epidemiologic studies have suggested an association between respiratory viral infection and the development of bronchiolitis obliterans syndrome (bos). [35] [36] [37] [38] [39] [40] these studies yielded mixed results and the association between respiratory virus infection and bos remains unclear. 41, 42 the recent availability of molecular diagnostics, including pcr and multiplex gene techniques for the recovery of many viruses simultaneously from a single specimen, increased the recovery of pathogens in respiratory infections that previously were considered to be of undetermined etiology. 42 other viruses have been identified and are of uncer definitions for cmv infection and disease, especially for use in research, have been reported in the literature and used in other studies. 1, 4 the methodology for cmv recovery has shifted at many centers over the past decade from conventional viral culture methods and antigenemia toward quantitative molecular diagnostics, including pcr and hybrid capture assays. [45] [46] [47] however, the issues related to the recovery of cmv in bal fluid in the absence of histopathologic evidence of cmv remain unresolved, 48 and investigations are ongoing to resolve this issue. asymptomatic viral shedding in the upper oropharynx by cmv is distinguished from active cmv disease in these definitions and will assist in further assessing the role of cmv in cttx. in early studies, the recovery of cmv by viral culture in the absence of tissue diagnosis was considered diagnostic of cmv pneumonitis. 49, 50 however, further studies did not suggest that cmv recovery from bal was predictive of subsequent cmv pneumonitis. [51] [52] [53] [54] [55] with the advent of sophisticated molecular diagnostics, the recovery of cmv from bal became more specific and reproducible compared with conventional or shell-vial culture, 53 and additional studies suggested that cmv viral load in bal fluid may be correlated with invasive cmv pneumonia. 2, 56, 57 however, the utility of cmv viral load in bal in predicting cmv pneumonitis remains uncertain in studies to date. • molecular diagnostics (from whole blood, plasma, bal or tissue). -quantitative dna pcr or hybrid capture assays. -qualitative pcr. -genotypic resistance testing. • antigen pp65. • viral culture (conventional or shell-vial centrifugation). • in situ immunohistochemistry. • serology: not recommended for diagnosis. definitions for respiratory viral infections are given in table 3a and 3b. cttx recipients in general and lung transplant recipients in particular have the highest risk of mold infection. 58 recently published data suggest the cumulative incidence rate at 1 year to be 8%. 58, 59 among mold infections, the overwhelming majority of infections are due to aspergillus spp, followed by scedopsorium spp and zygomycetes. 60 despite the advancement in anti-fungal therapy, mortality remains at approximately 29% in aspergillus infections. 60 candida species was noted to be a major pathogen during the early cttx period, although it is rarely seen in lung transplant recipients in the current era. 61 although the incidence of invasive candidiasis has remained low in lung transplant recipients, this was the most common fungal infection noted in heart transplant recipients. 60 fungal infections in lung transplant recipients have certain characteristics that make them unique compared with other sot recipients as well as other immunocompromised hosts. this includes the presence of certain risk factors, such as airway ischemia and native lung or unique clinical syndromes, including tracheobronchitis, bronchial anastomotic infection and colonization-syndromes observed only in lung transplant recipients ( figure 2 ). 62 rejection syndromes in lung transplant recipients further complicate the clinical presentation. diagnosis of fungal infection based on histology alone may not be as accurate due to the concomitant presence of acute or chronic rejection in these individuals. 24 similarly, it is not only the unique clinical syndromes of fungal infection that set them apart from the other immunocompromised hosts, but the diagnostic utility of non-invasive testing also is different. serum galactomannan has markedly lower sensitivity (30%) in lung transplant recipients as compared with other immunocompromised hosts. 63, 64 similarly, the sensitivity of other serologic markers such as serum cryptococcal or coccidiodal antigen, histoplasma urine antigen may be variable. [65] [66] [67] [68] the use of bal for gm has resulted in sensitivities of ͼ66% when a 0.58 or 0.66 optical density (od) index was used as a cutoff. 69,70 a higher cutoff (1.5 od) yielded better results in one study. 71 we suggest the use of bal gm in the diagnosis of invasive aspergillosis (ia) in lung transplant recipi-ents. similarly, fungal pcrs, especially from bal specimens, are more likely to be less sensitive for the diagnosis of disease than bal specimens from other immunocompromised hosts owing to colonization of the airways. cell wall components of fungi have also been used in the diagnosis of fungal infections. currently available ␤-glucan is non-specific and is negative in cases of cryptococcosis and zygomycosis. 72 in a recent study of lung transplant recipients, serum ␤-d-glucan sensitivity was reported to be 93%, whereas specificity was merely 71%. 73 the mycoses study group (msg) and the european organization for research and treatment (eortc) recently updated the definitions of fungal infections in immunocompromised hosts. 3 these definitions represent an excellent attempt to standardize the reporting of fungal infections in studies. however, they fail to address the unique nature of clinical syndromes in lung transplant recipients, particularly colonization, tracheobronchitis and bronchial anastomotic infections. 8, 74, 75 also, the radiologic presentation of invasive mycoses in cardiothoracic organ transplant recipients may not conform to the classical "halo sign" presentation in neutropenic or stem a in the absence of biopsy categorize as probable: in the presence of histologic findings of both acute rejection and fungal invasion it should be classified as acute rejection with proven fungal infection. b the presence of mosaic appearance and ground-glass opacity may represent development of bronchiolitis obliterans syndrome or obliterative bronchiolitis. c isolation of non-pathogenic molds in culture (e.g., cladosporium spp, phialemonium, chaetomium, cunninghamella, syncephalastrum, curvularia, dactylaria, graphium or phialophora) or other non-pathogenic fungi [e.g., penicillium (non-marnefii), paecilomyces or basidiomyctes] do not qualify for the "probable" category. they should only be considered in the "proven" category. cell transplant recipients ( figure 2 ). 76 moreover, the definitions do not account for the differences in the sensitivity of serologic tests, particularly galactomannan in lung transplant recipients. 69, 70, 77, 78 in addition, the category of possible fungal infection might not be applicable in lung transplant recipients owing to a multitude of possible diagnoses in these patients. the american society of transplantation (ast) also put forward a set of definitions to be used in the study of these infections in sot recipients. 1 the ast definitions do take into account some unique clinical syndromes in lung transplant recipients but lack the detailed description of clinical syndromes. reported studies of fungal infections in lung transplant recipients used diverse definitions. 14,79 -83 the following sets of definitions are proposed to standardize the reporting of fungal infections, particularly mold and yeast (endemic mycoses, candida spp and cryptococcus spp) infections in cttx recipients, especially among general and lung transplant recipients. the isolation of non-pathogenic molds or other non-pathogenic fungi in bal or sputum is not believed to satisfy the microbiologic criteria for the diagnosis of probable invasive fungal infections in these patients without histologic confirma-tion (tables 4a and 4b) . however, these definitions of fungal infections do not address pneumocystis jiroveci infection, which has previously been adequately defined for use in cttx. 1 • direct examination by light microscopy (gram, giemsa and calcofluor stains). • culture. • histopathology: routine stains (hemotoxylin-eosin), special (gomori methenamine silver, mucicarmine, periodic acid-schiff), direct immunofluorescence and in situ hybridization). histopathologic diagnosis is useful in establishing the diagnosis of endemic fungi because of their distinctive morphology. 3 however, confusion may occur when attempting to differentiate the hyaline molds that commonly cause invasive disease. 84 fusarium spp and scedosporium spp cannot be distinguished from aspergillus spp in tissue sections and even the zygomycetes, which are morphologically quite distinct from fusarium spp, the presence of mosaic appearance and ground-glass opacity may represent development of bronchiolitis obliterans syndrome or obliterative bronchiolitis. a in the absence of biopsy categorized as probable: in the presence of histologic findings of both acute rejection and fungal invasion it should be classified as acute rejection with proven fungal infection. b isolation of non-pathogenic molds in culture (e.g., cladosporium spp, phialemonium, chaetomium, cunninghamella, syncephalastrum, curvularia, dactylaria, graphium or phialophora) or other non-pathogenic fungi [e.g., penicillium (non-marnefii), paecilomyces or basidiomyctes] do not qualify for the "probable" category. they should only be considered in the "proven" category. definitions of fungal pneumonia, tracheobronchitis, bronchial anastomotic infection and colonization in cttx are given in tables 4a, 4b, 4c , and 4d, respectively. non-cttx-specific infections, such as urinary tract infection (uti), surgical site infection (ssi), bloodstream infection (bsi), infective endocarditis (ie), clostridium difficile infection (cdi) and skin and soft tissue infections (sstis), are not included herein. 2,86 -94 the consensus opinion of the ishlt id council encourages the use of previously published international definitions for these infections, which have been well established outside of the cttx population. the use of these standard definitions will allow for intercenter comparisons of rates and types of infections that should not be significantly impacted by the transplant. american society of transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation cdc/nhsn surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group definitions of cytomegalovirus infection and disease in transplant recipients post-operative nosocomial infections after lung and heart transplantation infectious complications in pulmonary allograft recipients infection in the transplanted and native lung after single lung transplantation ulcerative tracheobronchitis after lung transplantation. a new form of invasive aspergillosis infectious complications of lung transplantation. impact of cystic fibrosis single lung transplantation: a temporal look at rejection, infection, and survival impact of graft colonization with gram-negative bacteria after lung transplantation on the development of bronchiolitis obliterans syndrome in recipients with cystic fibrosis impact of bacterial and fungal donor organ contamination in lung, heart-lung, heart and liver transplantation effectiveness of a hospitalwide programme to improve compliance with hand hygiene pneumonia after lung transplantation in the resitra cohort: a multicenter prospective study hand hygiene compliance by physicians: marked heterogeneity due to local culture? attitudes toward practice guidelines among intensive care unit personnel: a cross-sectional anonymous survey why healthcare workers don't wash their hands: a behavioral explanation hand hygiene among physicians: performance, beliefs, and perceptions thoracic organ transplantation may not increase the risk of bacterial transmission in intensive care units bronchoalveolar lavage to diagnose respiratory infections comparison of induced sputum and bronchoalveolar lavage in lung transplant recipients inflammatory cells and activation markers in bal during acute rejection and infection in lung transplant recipients: a prospective, longitudinal study bronchoalveolar lavage and transbronchial lung biopsy during acute rejection and infection in heart-lung transplant patients. studies of cell counts, lymphocyte phenotypes, and expression of hla-dr and interleukin-2 receptor revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection comparison of quantiferon-tb gold with tuberculin skin test for detecting latent tuberculosis infection prior to liver transplantation revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group a randomized trial of diagnostic techniques for ventilator-associated pneumonia diagnostic techniques for ventilator-associated pneumonia: conflicting results from two trials invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia. a randomized trial non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation an official ats/ idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases tuberculosis after solid-organ transplant: incidence, risk factors, and clinical characteristics in the resitra (spanish network of infection in transplantation) cohort tuberculosis in solidorgan transplant recipients: consensus statement of the group for the study of infection in transplant recipients (gesitra) of the spanish society of infectious diseases and clinical microbiology human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death detection of epstein-barr virus dna in peripheral blood is associated with the development of bronchiolitis obliterans syndrome after lung transplantation comparison of pcr, antigenemia assay, and rapid blood culture for detection and prevention of cytomegalovirus disease after lung transplantation a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients metapneumovirus and acute wheezing in children human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus cytomegalovirus infection in isolated lung transplantations quantitative analysis of cytomegalovirus viremia in lung transplant recipients cytomegalovirus infection and pneumonitis. impact after isolated lung transplantation international consensus guidelines on the management of cytomegalovirus in solid organ transplantation cytomegalovirus viremia in lung transplant recipients receiving ganciclovir and immune globulin bronchioloalveolar lavage in the diagnosis of cmv pneumonitis in lung transplant recipients: an immunocytochemical study quantification of cytomegalovirus dna in bal fluid: a longitudinal study in lung transplant recipients predictive value of cytomegalovirus dna detection by polymerase chain reaction in blood and bronchoalveolar lavage in lung transplant patients clinical utility of cytomegalovirus viral load in bronchoalveolar lavage in lung transplant recipients correlation between viral loads of cytomegalovirus in blood and bronchoalveolar lavage specimens from lung transplant recipients determined by histology and immunohistochemistry evaluation of interleukin-6 and interleukin-10 in lung transplant patients with human cytomegalovirus infection human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients detection of cmv pneumonitis after lung transplantation using pcr of dna from bronchoalveolar lavage cells fungal infection in lung transplantation invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (transnet) epidemiology and outcome of invasive fungal infections in solid organ transplant recipients anastomotic infections in lung transplant recipients unique characteristics of fungal infections in lung transplant recipients prospective assessment of platelia aspergillus galactomannan antigen for the diagnosis of invasive aspergillosis in lung transplant recipients diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis pulmonary cryptococcosis in solid organ transplant recipients: clinical relevance of serum cryptococcal antigen histoplasmosis in solid organ transplant recipients histoplasmosis in solid organ transplant recipients: 10 years of experience at a large transplant center in an endemic area disseminated coccidioidomycosis in a liver transplant recipient with negative serology: use of polymerase chain reaction performance characteristics of the platelia aspergillus enzyme immunoassay for detection of aspergillus galactomannan antigen in bronchoalveolar lavage fluid aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients diagnosis of invasive aspergillosis in lung transplant recipients by detection of galactomannan in the bronchoalveolar lavage fluid evaluation of a (1¡3)-beta-d-glucan assay for diagnosis of invasive fungal infections the (1,3)␤-d-glucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation pulmonary aspergillosis in cystic fibrosis lung transplant recipients saprophytic fungal infections and complications involving the bronchial anastomosis following human lung transplantation aspergillus infections after lung transplantation: clinical differences in type of transplant and implications for management bronchoalveolar lavage galactomannan in diagnosis of invasive pulmonary aspergillosis among solid-organ transplant recipients prospective assessment of platelia aspergillus galactomannan antigen for the diagnosis of invasive aspergillosis in lung transplant recipients voriconazole prophylaxis in lung transplant recipients aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome antifungal prophylaxis with voriconazole or itraconazole in lung transplant recipients: hepatotoxicity and effectiveness aspergillus infections in lung transplant recipients: risk factors and outcome fungal infections after lung transplantation the impact of invasive fungal diseases on survival after lung transplantation the use of the mucicarmine stain for a rapid presumptive identification of cryptococcus from culture definition, clinical profile, microbiological spectrum, and prognostic factors of early-onset prosthetic valve endocarditis the international sepsis forum consensus conference on definitions of infection in the intensive care unit broadened definition for hospital-acquired infective endocarditis new diagnostic criteria for infective endocarditis. a study of sensitivity and specificity new criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings recommendations for surveillance of clostridium difficile-associated disease clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america practice guidelines for the diagnosis and management of skin and soft-tissue infections key: cord-016617-qadf0xut authors: lagstein, amir; myers, jeffrey title: airway pathology in lung transplants date: 2013-06-14 journal: bronchiolitis obliterans syndrome in lung transplantation doi: 10.1007/978-1-4614-7636-8_2 sha: doc_id: 16617 cord_uid: qadf0xut the histologic diagnosis of lung transplant rejection is based on the assessment of perivascular mononuclear cell inflammation, airway inflammation and fibrosis, and vasculopathic changes. this chapter describes the pathologic features of acute and chronic rejection of the small airways (i.e., lymphocytic and obliterative bronchiolitis). as transbronchial lung biopsy is the mainstay for the assessment of rejection, a brief discussion of some of the limitations of this technique is provided from the pathologist’s perspective. several important and common entities that can mimic airway rejection are described with practical guidance for distinguishing these potential confounders on transbronchial biopsy. the non-rejection findings that are discussed include the normal biopsy, nonspecific forms of chronic bronchiolitis, cytomegalovirus and pneumocystis pneumonia, bronchiolitis obliterans-organizing pneumonia, and aspiration pneumonia. lesions of the small airways are an important manifestation of both acute and chronic rejection of the pulmonary allograft, and two major forms are recognized. the fi rst, lymphocytic bronchiolitis (lb), describes chronic mononuclear cell infl ammation of the epithelium and submucosa of the distal small airways (i.e., at the level of and distal to the membranous bronchioles). the second, obliterative (or constrictive) bronchiolitis (ob), refers to partial or complete fi brous scarring of the distal airways and it is often, but not always, pauci-infl ammatory. the term "obliterative bronchiolitis" is preferred over the term "bronchiolitis obliterans," so as not to confuse the former with the much more common bronchiolitis obliterans-organizing pneumonia (boop), also termed simply organizing pneumonia (op), an unrelated disease process with different clinical, radiologic, and pathologic features. ob is the histologic correlate of the clinically defi ned bronchiolitis obliterans syndrome (bos) and remains the gold standard for its defi nitive diagnosis [ 1 ] . while ob is perhaps best known as the central feature of chronic lung rejection, it may also occur in a number of non-transplant settings. patients with connective tissue diseases, especially rheumatoid arthritis, are perhaps the most commonly affected with ob outside of the transplant setting [ 2 ] . in addition, ob is an uncommon complication of various viral infections of the respiratory tract, particularly in children [ 3 , 4 ] , and is also a rare manifestation of medication toxicity (e.g., d -penicillamine ) [ 5 ] and inhalational injury from various toxins such as ammonia [ 6 ] , smoke [ 7 ] , and cocaine [ 8 ] . recently, ob has been described as an occupational lung disorder of microwave popcorn workers (possibly related to diacetyl exposure, a butter-fl avoring agent) [ 9 , 10 ] . ob is also a manifestation of graft-versus-host-disease in allogeneic bone marrow transplant recipients [ 11 ] . interestingly, lesions histologically identical to lb have been observed in some of these conditions [ 11 -13 ] , providing a putative link between lb and ob. indeed, lb is now widely accepted as not only a bona fi de manifestation of acute rejection, but as an important risk factor for developing chronic airway rejection. the occurrence of ob outside of the transplant setting has contributed to our understanding of the etiology and pathogenesis of this still enigmatic disorder [ 14 -16 ] . however, post-transplant-related cases remain the most common and increasingly, the best understood examples of ob. as a consequence of the great success of modern immunosuppressive drugs, surgical techniques, and management of infections, with the attendant increase in allograft longevity, ob has emerged as the major long-term obstacle to both graft and patient survival in lung transplantation [ 17 ] . this challenge has led to greater emphasis on its early recognition, with the corresponding hope that early treatment can delay or prevent its development [ 18 ] . lymphocytic and obliterative bronchiolitis are part of the formal histologic classifi cation system of lung rejection, developed by the lung rejection study group of the international society of heart and lung transplantation (ishlt). the classifi cation system is published as an ongoing series of working papers in order to maintain up-to-date and standardized nomenclature, and it has undergone two major revisions since the original working formulation was published in 1990 [ 19 -21 ] . these changes refl ect a combination of advances in the fi eld of transplant rejection, experience with the application of the earlier grading schemes, and consensus expert opinion. the current ishlt working formulation [ 21 ] recommends histologic assessment of rejection along four lines: grade a for perivascular infl ammation (acute cellular rejection [acr]), grade b for airway infl ammation (lymphocytic bronchiolitis), grade c for airway fi brosis (obliterative bronchiolitis), and grade d for chronic vascular rejection (graft atherosclerosis). acr is graded on the severity and density of perivascular and interstitial mononuclear cell infl ammation in the following manner: a0, no perivascular infi ltrates; a1, minimal acute rejection; a2, mild acute rejection; a3, moderate acute rejection; and a4, severe acute rejection. lymphocytic bronchiolitis is graded as: b0, no airway infl ammation; b1r, low-grade airway infl ammation; and b2r, high-grade airway infl ammation. constrictive bronchiolitis and chronic vascular rejection are not graded but are designated as being either absent (c0 and d0) or present (c1 and d1). because bronchoscopy with transbronchial biopsy (tbbx) is the mainstay for assessment of lung rejection, the classifi cation system includes an "ungradeable" score for each parameter, designated by an x after the letter (e.g., ax), if it cannot be assessed in the sample. this refl ects the limitations arising from the necessarily limited amount of tissue obtainable with tbbx, the patchiness of the histologic fi ndings in graft rejection, and potential confounding factors, particularly concomitant infection. this classifi cation system can also be applied to larger specimens, such as surgical lung biopsies, explanted allografts, and autopsy material, with the recognition that some fi ndings, especially chronic vascular rejection (grade d), are relatively uncommon and virtually never identifi ed on tbbx. acr and graft atherosclerosis will not be further discussed as they are beyond the scope of this chapter. the interested reader will fi nd a good discussion of these topics elsewhere [ 21 -23 ] . the focus of this chapter is the pathology of the small airways in acute and chronic rejection, with only brief discussion of the potential signifi cance of large airway infl ammation (bronchitis). in addition to rejection, the lung transplant patient is at greater risk for a variety of insults that manifest predominantly, both clinically and pathologically, as airway or airway-based abnormalities. most importantly, this includes opportunistic infections. boop and aspiration pneumonia also occur more commonly in transplanted patients and are sometimes overlooked as potential causes of allograft dysfunction. we will also briefl y review two entities that may be mistaken for rejection by the pathologist-the normal biopsy and nonspecifi c forms of chronic bronchiolitis. first however, given the central role that tbbx plays in the management of rejection, we will briefl y review the limitations of tbbx, from the perspective of the pathologist. there continues to be a spirited debate within the transplant community regarding the utility of the surveillance tbbx (i.e., one that is performed in the asymptomatic patient according to a predetermined schedule) as compared to the clinically indicated tbbx (i.e., one that is performed after the development of signs or symptoms) [ 24 , 25 ] . like any procedure tbbx has intrinsic benefi ts and costs; it is not our intention to enter into the debate regarding the most appropriate role for tbbx. our focus is instead on the histopathologic fi ndings that facilitate accurate and timely diagnosis in lung transplant patients. accurate interpretation of tbbx performed for rejection can be challenging for two main reasons. the fi rst, which is common to currently available techniques for retrieving lung tissue with tbbx, stems from the small size and necessarily limited amount of tissue obtainable via the fl exible bronchoscope. the second, which is unique to lung transplant patients, is the difficulty in separating bona fi de rejection from other processes with a similar appearance. these potential confounders are discussed in greater detail in the latter sections of this chapter. there is an inherent challenge in interpreting small pieces of tissue that may be crushed or torn. in addition, small pieces of tissue are more diffi cult to interpret due to problems stemming from oblique (or tangential) sectioning. this is unavoidable in tbbx. however, the problem of limited tissue is ameliorated, to a degree, by the goal for "adequacy" in rejection tbbx. the ishlt recommends that adequate biopsy tissue sampling consists of at least fi ve pieces of alveolated parenchyma, recognizing that the bronchoscopist may need to submit more pieces than this in order to increase the chances of including histologically assessable bronchioles within the submitted sample. moreover, the bronchoscopist may need to submit more than fi ve pieces as some, and sometimes all, of the submitted pieces are invariably comprised only of bronchial wall, exfoliated epithelium, and/or blood (see fig. 2 .1 ). as mentioned above, rejection is a histologically patchy phenomenon. while higher grades of acute rejection are, by defi nition, more diffuse processes, lower grades of rejection, including grade a2 (the traditional clinical threshold for pulse therapy), can still be very patchy. moreover, increasing evidence suggests that episodes of even minimal acute rejection or low-grade lb are associated with higher subsequent rates of chronic rejection/bos [ 26 ] . therefore, obtaining adequate biopsies may be expected to increase the diagnostic yield of tbbx and allow clinicians to appropriately treat an acute rejection episode in patients who would have otherwise gone untreated. the sensitivity of tbbx for the detection of rejection is less than 100 % even when technically adequate. the yield of tbbx should be distinguished from its sensitivity . the diagnostic yield of tbbx is the percentage of biopsies performed that are "positive" for rejection. comparing experiences between centers is challenging, in part, because positivity is not uniformly defi ned. some have defi ned as "positive" any biopsy with at least grade a1 rejection, while others defi ne as comparing an adequate ( a ) and a suboptimal ( b ) transbronchial biopsy (tbbx) at low magnifi cation (hematoxylin and eosin, original magnifi cation ×20). the tbbx in ( a ) is very generous and contains about eight substantial fragments of alveolated parenchyma. both large and small airways are well represented (although diffi cult to discern at this magnifi cation). by comparison, the tbbx in ( b ) is unsuitable for assessment of rejection. while it consists of about fi ve fragments of tissue, only three are adequately alveolated. however, even these are small, torn, and show signifi cant crush by the forceps. the other fragments are crushed bronchial wall, blood, and exfoliated epithelium, which are not useful for a meaningful histological assessment. the biopsy in (a) obviously provides much more information and is also easier to interpret "positive" biopsies having at least grade a2 rejection, any form or grade of rejection (whether types a, b, or c), and even infection. diagnostic yield also will vary depending on whether a tbbx is performed for clinical indications or surveillance. given this variability, diagnostic yield is best understood as an institution-specifi c parameter and is perhaps most useful as a measure of quality control and improvement. by contrast, sensitivity of rejection tbbx is defi ned in a standard fashion, which is the fraction of patients with a disease (transplant rejection) who have the disease (rejection) on testing (tbbx). the numerator (i.e., number of patients with rejection on tbbx) is the same whether measuring diagnostic yield or sensitivity, but the denominator is different ( yield = number of patients tested; sensitivity = number of patients with rejection). the sensitivity of tbbx varies with the number of pieces obtained. earlier studies utilizing transplanted animals that were sacrifi ced [ 27 ] showed that fi ve pieces of lung tissue were required to achieve a sensitivity of 92 % for the detection of at least mild rejection. in contrast, a study of 219 tbbx from 54 heart-lung transplant recipients by scott et al. [ 28 ] showed that in the clinical setting 18 samples per procedure may be necessary to have a 95 % confi dence of fi nding rejection. tbbx is a relatively insensitive method for detecting ob, possessing a sensitivity ranging from 15 % to nearly 40 % [ 29 , 30 ] . the low sensitivity of tbbx for ob likely stems from three factors: the diffi culty in sampling small airways on tbbx, the notoriously patchy nature of ob, and a presumed diffi culty of the biopsy forceps in acquiring fi brotic tissue. the specifi city of tbbx for rejection is also less than 100 %, due to the technical challenges of tbbx interpretation and the presence of confounding variables, especially infection. in selected situations, when a tbbx is inadequate or inconclusive and the clinical situation demands a defi nitive diagnosis, wedge lung biopsy may be a useful option. in a study of 48 open lung biopsies performed on 42 lung transplant patients from an institution performing surveillance tbbx [ 31 ] , a clinically unsuspected diagnosis was made in 14 (29 %) of the 48 biopsies, all of which resulted in changes to patient treatment. however, this study does not explicitly state the rate of discordance between prior tbbx and wedge biopsy. nonetheless, it does suggest that wedge lung biopsy can be useful in clinically deteriorating transplant patients for whom tbbx is non-diagnostic. lymphocytic bronchiolitis describes chronic mononuclear cell infi ltrates involving the small airways. the current ishlt working formulation subdivides lb into low-grade (b1r) and high-grade (b2r) forms. the "r" in the category designation stands for "revised," as it refl ects a modifi cation of the 1996 working formulation in collapsing the previous four tier grading system (minimal, mild, moderate, severe) into two (low grade and high grade). in addition, there is a category for no airway infl ammation (b0) and an ungradeable category (bx) for those cases in which small airways are not present, are not assessable (due to tangential sectioning for example), or for which the infl ammation cannot be confi dently ascribed to rejection. indeed, potential confounders that mimic lb have delayed acceptance of utilizing lb as an independent or sole marker for establishing or grading acute rejection. these potential confounders are discussed in detail in the second portion of this chapter (see section on "non-rejection findings"). low-grade lb is characterized by relatively sparse peribronchiolar lymphocytic infl ammation, often in a circumferential or partially circumferential distribution. the lymphocytic infi ltrates are localized to the submucosa, which is not expanded, and there should be no evidence of associated epithelial injury. although the ishlt defi nition of low-grade lb restricts the mononuclear infi ltrates to the submucosa, scattered intra-epithelial lymphocytes can be observed in otherwise histologically straightforward cases of low-grade lb. for that reason a diagnosis of low-grade lb is appropriate when the infi ltrates are not overly dense and localized predominantly to the submucosa. high-grade lb, by contrast, is characterized by more frequent and increasingly dense peribronchiolar lymphoplasmacytic infi ltrates. the lymphocytes may be larger low-grade lymphocytic bronchiolitis, tbbx (hematoxylin and eosin, original magnifi cation ×200). mildly to moderately dense lymphocytic infl ammation localized predominantly to the bronchiolar submucosa. lymphocyte crush artifact is prominent, which is common in forceps biopsies. while there are scattered intra-epithelial lymphocytes ( arrows ), there is no evidence of epithelial cell injury and possess an activated or plasmacytoid appearance. plasma cells may be present in either low-grade or high-grade lb, but are more numerous in high-grade lb. in contrast to low-grade lb, the denser collections of mononuclear cells (lymphocytes and plasma cells) in high-grade lb tend to infi ltrate the basement membrane and epithelium of the bronchioles. in such cases, there will usually be evidence of associated epithelial injury, ranging from epithelial cell apoptosis and necrosis to frank mucosal ulceration. squamous metaplasia of bronchiolar epithelium may also be present and is testimony to attempts at epithelial regeneration. in addition to mononuclear cells, polymorphonuclear leukocytes, including eosinophils, may be seen with high-grade lb. neutrophils are usually seen in cases with attendant epithelial necrosis or ulceration. if the luminal infi ltrates are frankly purulent, or if there is evidence of distal airspace involvement with neutrophilic infl ammation, then acute infection (bronchopneumonia) becomes a more likely explanation for the fi ndings. ob is the histologic fi nding of complete or partial bronchiolar fi brosis, whereas bos is clinically defi ned as a persistent decline in forced expiratory volume (fev1) compared to an established post-transplant baseline. while ob is the presumed histologic correlate of bos, the terms are not interchangeable because some transplant patients develop airfl ow limitation from other causes [ 1 ] . high-grade lymphocytic bronchiolitis, tbbx (hematoxylin and eosin, original magnification ×100). very dense lymphoplasmacytic infl ammation involving the epithelium and submucosa of a bronchiole ( center ). the epithelium lining the adjacent larger airway ( top ) shows no mucosal infl ammation. in other levels, the bronchiolar epithelium was necrotic and denuded a great deal of basic science, animal model, and clinical research has begun to clarify the pathogenesis of ob and the risk factors predisposing to it [ 15 , 16 ] . both acr and lb are well-established risk factors. non-immunologic risk factors, including cytomegalovirus (cmv) and non-cmv infection, boop, donor age, and graft ischemia time (among others), have also been implicated as risk factors for developing ob/bos. recently, there has been increasing attention on the existence and possible role for antibody-mediated rejection (amr) as a risk factor for ob/bos [ 32 ] . this stems from the occurrence of ob in patients with no evidence of antecedent acr [ 33 ] , evidence of septal capillary injury in cases of otherwise unexplained graft dysfunction [ 34 ] , complement deposition in capillary endothelium [ 35 ] , and the uncommon but well-documented occurrence of graft dysfunction in patients who developed donor-specifi c anti-human leukocyte antigen (anti-hla) antibodies and capillaritis on biopsy [ 36 ] . despite tantalizing evidence of a possible link between amr and lung allograft dysfunction there are persistent unresolved questions regarding its diagnosis and signifi cance. capillaritis has been proposed as a histologic marker of amr in tbbx but distinguishing capillaritis from simple neutrophil margination (diapedesis) in small specimens is challenging at best. bronchopneumonia must be rigorously excluded in this setting since neutrophil margination is common in acute infection. in the nontransplant setting necrotizing capillaritis is virtually always associated with clinical and histologic evidence of diffuse alveolar hemorrhage. immunohistochemical stains for c4d are of limited value given that interpretation is plagued by nonspecifi c background staining of endothelial cells and elastic tissue. furthermore, there is a poor correlation between linear c4d staining, the presence of necrotizing capillaritis, and the development of donor-specifi c hla alloantibodies [ 37 ] . thus, substantial diffi culties remain before amr can be embraced as a distinct clinicopathologic form of lung rejection. collagen fibrosis involving and expanding the bronchiolar submucosa is the histologic hallmark of ob. the fibrosis may be eccentric or concentric and in more advanced lesions results in complete obliteration of the airspace lumen. ob may be more difficult to recognize in the late fibrotic stage as the airways are completely scarred and therefore difficult to recognize. key to identifying these focal scars as former airways includes the presence of an associated similar-caliber artery or the presence of residual fascicles of smooth muscle within the fibrosis. in most cases, the fibrosis is not accompanied by inflammation but persistence of mononuclear cell inflammation of the sort and character of lb may be noted in some cases. indeed, lb and ob may coexist. mucostasis and/or accumulation of foamy, lipid-laden histiocytes within peribronchiolar air spaces may be present as nonspecific markers of small airways dysfunction of any cause. occasionally these finding are present in the absence of diagnostic small airways changes and are suggestivebut not diagnostic of-bronchiolar pathology. large airway infl ammation/lymphocytic bronchitis (fig. 2.6 ) lymphocytic bronchiolitis, as the name implies, affects the small airways-that is, the distal-most portions of the conducting bronchioles and the respiratory bronchioles of the allografted lung. the signifi cance of lb with respect to lung rejection is now well established. occasionally, similar appearing infl ammation of the large conducting cartilaginous airways may also occur, with or without associated lb [ 38 ] . unlike lb, much less is known about the signifi cance of isolated large airway infl ammation visà-vis rejection. early studies found increased numbers of specialized leu-7 (cd57)positive t lymphocytes in the mucosa of donor bronchi with morphologic evidence of airway injury and observed an association between lymphocytic bronchitis and subsequent ob [ 39 -41 ] . in another study, yousem and colleagues also found that "chronic infl ammation of the bronchi" was associated with subsequent development of ob, with a sensitivity and specifi city of 83 % and 100 %, respectively, although the number of cases was very small [ 42 ] . large airway bronchial fi brosis has also been observed in some lung allografts with coexisting ob [ 43 ] . these studies suggested that bronchial mucosa may be a target for rejection prompting the use of the combined term "lymphocytic bronchitis/bronchiolitis" (lbb) to refer to the mononuclear cell infi ltrates jointly affecting the bronchi and the bronchioles. as is true in small airways, infl ammation in large airways is not specifi c for rejection and is commonly present with clinical (or subclinical) infection, aspiration, chronic obstructive pulmonary disease, and other inhalational injuries. indeed, the ishlt working formulation recognizes that large airway infl ammation is most commonly associated with infection and aspiration and does not currently identify or grade "lymphocytic bronchitis" as such [ 21 ] . bronchiectasis has also been described in lung transplant patients with bos, although it is not known if this is a consequence of infection, rejection, ischemic injury, or some combination of these factors [ 44 ] . dense mononuclear cell infl ammation involving a bronchus. both the wall and epithelium is involved; the latter shows evidence of injury in the form of sloughing and regenerative atypia. there is evidence of small airway involvement as well (lymphocytic bronchiolitis); a tangential portion of an affected bronchiole is seen in the section ( arrow ). other fragments (not shown) also showed lymphocytic bronchiolitis. cultures for microorganisms and other microbiological assays were negative for infection the signifi cance of lymphocytic bronchitis in a tbbx depends upon not only the morphologic features but also the clinical context. to help distinguish lymphocytic bronchitis from nonspecifi c forms of chronic bronchitis the term should be limited to cases in which dense collections of lymphocytes are confi ned to the bronchial submucosa and submucosal glands, often infi ltrating into the bronchial epithelium. with more intense degrees of infl ammation, greater numbers of transformed lymphocytes, immunoblasts, and even eosinophils are present, and there may be evidence of epithelial injury including apoptosis, squamous metaplasia, or ulceration. neutrophils should not be abundant and there should not be evidence of viral cytopathic change or aspiration, features which would point to another etiology. when narrowly defi ned in this way lymphocytic bronchitis is not a common fi nding and, when present, is often seen in combination with other fi ndings typical of acute rejection, usually lb. such cases should be graded conventionally as per ishlt guidelines, with or without mention of the presence of lymphocytic bronchitis, since in any event clinical decision making will be based on the formal "a-b-c" rejection grade. lymphocytic bronchitis is uncommon as a truly isolated fi nding, and when present without other corroborative histologic support for a diagnosis of rejection its signifi cance is uncertain. normal (bronchus-associated lymphoid tissue) (fig. 2.7 ) the airways, as in other non-sterile mucosal sites with a more-or-less constant exposure to the external environment, possess a mucosa-associated lymphoid tissue (malt tissue) specifi cally referred to as bronchus-associated lymphoid tissue (balt). in the large airways, these comprise circumscribed submucosal aggregates (primary follicles) of lymphocytes. they are usually not very prominent, unless there has been antigenic stimulation, in which case there may be balt hyperplasia which may be associated with germinal center formation (secondary follicles). the circumscription and submucosal localization of balt follicular aggregates is not likely to be confused with lymphocytic bronchitis and bronchiolitis. however, in the intermediate and small airways, the lymphocytes may extend into the overlying epithelium ("lymphoepithelium"), which is focally attenuated as it is in other malt sites. it is important not to confuse this normal fi nding with lb (or with any other pathology). the key features distinguishing balt and lymphoepithelium from lb are that the lymphoid infi ltrates of the latter are denser, are not circumscribed, and do not form primary or secondary follicles; establishing this may require assessment of multiple consecutive tissue levels. furthermore, lb may be associated with epithelial damage including epithelial cell necrosis, mucosal ulceration, and squamous metaplasia, particularly when high grade (b2r). lastly, lb is often associated with the perivascular lymphoid infi ltrates of acr. chronic bronchiolitis is a histopathologic term referring to chronic infl ammation involving bronchiolar and peribronchiolar interstitium with or without fi brosis [ 45 ] . chronic bronchiolitis is a nonspecifi c fi nding; its signifi cance is defi ned by the histopathologic and clinical context [ 46 ] . for example, chronic bronchiolitis is a common fi nding in other primary pathologic processes, such as hypersensitivity pneumonia. in hypersensitivity pneumonia, chronic bronchiolitis is only one component of a unique combination of equally nonspecifi c fi ndings that is characteristic only when present collectively. chronic bronchiolitis is uncommon as an isolated primary pathologic process and occurs in surprisingly heterogeneous clinical contexts. in smokers with evidence of obstructive airways disease, chronic bronchiolitis corresponds to the small airways disease thought to account for airfl ow limitation in patients with emphysema and chronic bronchitis [ 47 ] . occasional unexplained chronic bronchiolitis occurs in nonsmokers with airfl ow limitation who lack other features of emphysema, chronic bronchitis, or asthma (i.e., idiopathic small airways disease). chronic bronchiolitis does not by itself predict for physiologically significant obstructive airways disease, however, and in some patients may actually be affi liated with evidence of restrictive lung disease. bronchus-associated lymphoid tissue, wedge biopsy (hematoxylin and eosin, original magnifi cation ×200). balt, a normal fi nding, comprises mucosal lymphoid aggregates associated with large and/or small airways. the aggregates are comprised of well-circumscribed subepithelial primary or secondary lymphoid follicles. in the small airways, as seen here, the lymphocytes may focally percolate among the epithelial cells ("lymphoepithelium," arrow ). it may sometimes be diffi cult to distinguish balt from bronchiolitis (of any cause) on tbbx, particularly when the sample is very small, fragmented, or crushed given the nonspecifi c nature of chronic bronchiolitis and the wide range of potential causes and associations, attributing bronchiolitis to rejection in transplant patients requires careful integration of not only histopathologic but also clinical, physiologic, and radiologic data. infectious complications are a major obstacle to both short-term and long-term survival in lung transplantation. non-cmv infections are the leading cause of morbidity and mortality in the fi rst year status post-transplantation, and remain the second leading cause of mortality thereafter, preceded only by bos [ 17 ] . pneumonia, particularly bacterial pneumonia, is the most common infection affecting lung transplant patients, especially in the early post-transplant period, although mycobacterial, viral, and fungal pneumonia all occur at an increased frequency in lung transplant patients [ 48 ] . for the pathologist, the diagnosis of acute bronchopneumonia due to pyogenic bacteria or granulomatous infection is generally straightforward and not likely to be confused with acute rejection; the former entities are characterized by suppurative or granulomatous infl ammation involving the airspaces, while acute rejection is typifi ed by mononuclear/lymphocytic infl ammation in the perivascular and peribronchiolar interstitium. certain infectious agents produce a cellular interstitial pneumonia that is more likely to be confused with acute rejection. in particular, two important opportunistic pathogens, cmv and pneumocystis jirovecii , cause an infectious pneumonia that may show prominent chronic interstitial infl ammation (i.e., chronic interstitial pneumonia) that closely resembles acute rejection [ 49 , 50 ] . in a study of cmv and pneumocystis pneumonia diagnosed by open lung biopsy and tbbx, tazelaar [ 50 ] noted perivascular lymphocytic infi ltrates similar to those seen in acute rejection in 42 % of cmv cases and 21 % of pneumocystis cases. such results reiterate the need for the pathologist to at least consider the possibility of infection in every transplant tbbx and to rigorously exclude-or include-infection with ancillary special stains in selected cases. a tbbx diagnosis of infection that includes perivascular lymphoid infi ltrates does not preclude the possibility of concomitant rejection, however, and should be regarded as indeterminate ("axbx") in this regard. if clinically warranted, a subsequent tbbx following appropriate antimicrobial treatment may be more helpful in evaluating for rejection without the confounding effects of infection. this serves as a reminder that the ultimate diagnosis in any individual patient should be the result of integration with all available clinical data, including those from microbiologic and serologic studies. among non-alloimmune risk factors for the development of ob/bos, pulmonary infection due to cmv has been the most extensively studied, with relatively fewer reports analyzing non-cmv viruses, bacteria, and fungi including pneumocystis [ 14 , 16 ] . bacterial and pneumocystis pneumonia have not been clearly shown to be signifi cant risk factors for ob/bos, while studies assessing the signifi cance of cmv pneumonia on the development of ob/bos have shown inconsistent results [ 14 , 16 ] . at this time, pulmonary infections, in general, and viral respiratory pathogens, in particular, are considered to be possible risk factors for ob/bos, perhaps by potentiating the effects of acute rejection. cytomegalovirus pneumonia (fig. 2.8 ) the key to the diagnosis of cmv pneumonia is the recognition of characteristic viral cytopathic changes caused by cmv infection, of which there are three-cytomegaly, nuclear inclusions, and cytoplasmic inclusions. cellular and nuclear enlargement (cytomegaly) is perhaps the most easily recognizable alteration. the intranuclear inclusions consist of centrally placed amorphous basophilic inclusions, usually with a clear halo separating them from the nuclear membrane. the cytoplasmic inclusions, which are not seen in every infected cell, are also basophilic and coarsely granular. the latter often stain positively with the gomori methenamine silver (gms) method. these viral cytopathic changes can affect virtually any cell, including pneumocytes, interstitial cells, and endothelial cells. while some cases may show numerous cmv virocytes, other cases may show only a few or rare infected cells, particularly in the limited samples that tbbx provides. an immunohistochemical stain for cmv is widely available and can be very helpful in confi rming the diagnosis, especially in subtle cases. in addition to the characteristic altered cells, cmv pneumonia typically elicits a predominantly chronic infl ammatory cell reaction involving the interstitium and the airways that may be nearly indistinguishable from acr and lb. in more severe cases, it may also cause diffuse alveolar damage (dad) and/or fi brinous airspace exudate. as stated above, unless the viral cytopathic changes are recognized, the case is likely to be misdiagnosed as acute rejection. the viral changes caused by cmv must be distinguished from those due to herpes simplex virus (hsv). hsv infection does not result in signifi cant cytomegaly, nor does it cause intracytoplasmic inclusions. in addition, hsv infection produces ground glass intranuclear inclusions that are usually prominently eosinophilic and with a margin of peripherally condensed chromatin. pneumocystis jirovecii pneumonia (fig. 2.9 ) there are a number of histologic changes that can be seen in pneumocystis pneumonia. the classic change is the presence of an eosinophilic "frothy" alveolar exudate on hematoxylin and eosin (h&e) staining. on higher power, this exudate possesses a honeycomb-like or microcystic appearance, representing numerous organism cysts and it is pathognomonic for the disease. this frothy exudate may be associated with features of dad including hyaline membranes. granulomatous infl ammation-necrotizing, non-necrotizing, or both-is an uncommon manifestation of pneumocystis pneumonia that is often associated with lymphocytic infl ammation and clusters of histiocytes. other less common changes include areas of necrosis, calcifi cation, and a pulmonary alveolar proteinosis-like reaction. tbbx is a sensitive technique for the detection of pneumocystis pneumonia. if the characteristic frothy eosinophilic alveolar exudates are present, then the diagnosis is straightforward and can be made even in the absence of special stains. ( a ) on low power, there is a dense predominantly chronic infl ammatory infi ltrate involving the bronchus and subjacent alveolar tissue(hematoxylin and eosin, original magnifi cation ×100). such an appearance is reminiscent of high-grade acr with lymphocytic bronchitis/bronchiolitis. ( b ) on higher magnifi cation, an endothelial cell with cmv viral cytopathic change is seen ( arrow ) (hematoxylin and eosin, original magnifi cation ×400). this comprises nucleomegaly and cytomegaly and basophilic ground glass nuclear inclusions. there may also be basophilic intracytoplasmic granules, although these are somewhat diffi cult to discern even at this magnifi cation. an immunohistochemical stain for cmv was also positive (not shown). note the marked lymphohistiocytic infl ammation ( c ) silver stains, such as the gomori methenamine silver (gms) stain, demonstrate the yeast forms, which are 4-6 μm in diameter and helmet-shaped, crescentic, or spherical (gms, original magnifi cation ×600). note the internal dot-like enhancement inside the cysts ( arrow ), a feature which helps distinguish pneumocystis from histoplasma spp. yeast forms occasionally, however, only hyaline membranes, a chronic interstitial pneumonia, or granulomas are present. for that reason, it is important to maintain a low threshold for performing special stains, especially stains such as a gms stain that highlight pneumocystis organisms. bronchiolitis obliterans-organizing pneumonia (fig. 2. bronchiolitis obliterans-organizing pneumonia (boop), also termed organizing pneumonia (op), is a nonspecifi c manifestation of acute lung injury. as such, it can be caused by or associated with a wide variety of insults and conditions, including infectious pneumonia, medications, aspiration of gastric contents, radiation, or connective tissue disease [ 51 ] . the etiology is usually not apparent on the basis of the histologic fi ndings alone. boop may also be seen as a nonspecifi c secondary change in other primary processes. idiopathic boop, also termed cryptogenic op or cop, refers to a distinct syndrome of unknown cause with characteristic clinical and radiographic features and boop as an isolated fi nding on lung biopsy [ 52 ] . spontaneous remission may occur, and in those patient requiring treatment it tends to be a steroid-responsive disease, although relapses are common. these features are in contrast to ob, which is typically insidious and progressive and not marked by relapses or remissions. boop is a fairly common fi nding in rejection biopsies [ 53 ] , reemphasizing the importance of its distinction from ob by the reviewing pathologist. indeed, in an earlier review of organizing pneumonia-like reactions in allograft biopsies, yousem and colleagues described boop as most commonly occurring in the setting of acute rejection [ 53 ] . several groups have also found boop to be a risk factor for ob/bos [ 54 , 55 ] . as such, boop has been proposed to be included in the histologic classifi cation of lung rejection [ 56 ] , although this has not been adopted. boop is characterized by fusiform proliferations of spindled fi broblastic and myofi broblastic cells set within a pale-staining myxoid matrix containing abundant mucopolysaccharides (ground substance), a combination of fi ndings sometimes described as fi bromyxoid plugs of "young" fi brosis. a key feature defi ning boop is the localization of these fi bromyxoid plugs to the lumens of the distal bronchioles ("bronchiolitis obliterans") and alveolar airspaces and ducts ("organizing pneumonia"). this distribution accounts for its typical whorled and serpentine appearance. involvement of the bronchiolar lumens causes small airway dysfunction, which in turn results in a variably prominent accumulation of foamy macrophages, sometimes referred to as endogenous lipoid pneumonia. boop may be accompanied by abundant airspace fi brin, lending an eosinophilic appearance to the process. associated infl ammation can be highly variable, from negligible to dense infi ltrates, and is usually comprised of chronic infl ammatory cells, mostly lymphocytes and plasma cells. the infl ammatory cells can be found within the fi bromyxoid tissue or alveolar septal walls or both. however, if alveolar septal and perivascular mononuclear infi ltrates are prominent, then high-grade acr should be strongly considered as the underlying etiology. neutrophils and histiocytes may also be found, but if prominent, an infectious etiology should be suspected and the use of special stains for microorganisms may be helpful in further evaluating for that possibility. the organizing phase of dad may be indistinguishable from boop in small biopsies. boop can usually be distinguished by the intraluminal localization of the fi broblastic plugs and the absence of hyaline membranes but these helpful clues are not always easily discerned in tbbx. dad typically occurs in the setting of the notice the presence of an associated cellular chronic interstitial pneumonia, a common associated fi nding in boop (of any cause), and one that should be distinguished from the perivascular mononuclear cell infi ltrates of acr adult respiratory distress syndrome (ards) and for that reason can usually be separated from boop by correlating with the patient's clinical status in histologically challenging cases. boop is usually easily distinguishable from ob, even in small biopsies, as both the location (airspaces in the former, submucosa in the latter) and constitutive elements (fi bromyxoid tissue in the former, collagen fi brosis in the latter) are distinctly different. aspiration pneumonia (fig. 2.11 ) patients who have undergone lung transplantation are at a signifi cantly increased risk for gastroesophageal refl ux and aspiration [ 57 -59 ] , possibly due to impaired cough refl ex and mucociliary clearance mechanisms. while massive acute aspiration is not a clinically occult condition, chronic, low-level episodes of repeated aspiration pose a more diffi cult diagnostic challenge; in fact, chronic aspiration is often clinically unsuspected [ 60 , 61 ] . chronic gastroesophageal refl ux and aspiration have been implicated as non-alloimmune risk factors for the development of ob/bos [ 62 ] , and anti-refl ux therapy utilizing medical (macrolide antibiotics) and surgical (gastric fundoplication) modalities has resulted in improved lung function in several studies [ 63 -66 ] . thus, aspiration is a treatable cause of pulmonary allograft dysfunction and it is a diagnosis the pathologist is often in a unique position to make. the morphologic features of particulate aspiration are suffi ciently unique that the diagnosis can often be made on tbbx. aspiration pneumonia is characterized by airway-centered granulomatous infl ammation that is often necrotizing. the granulomas typically elicit an associated boop response, which is often quite prominent and is sometimes the dominant fi nding. acute and chronic bronchitis and bronchiolitis are a nearly constant fi nding and thus the pathologist must take care before ascribing bronchiolitis to rejection or to infection. the defi ning feature of aspiration pneumonia is the presence of exogenous aspirated foreign material, either in an extracellular location or within giant cells or both. the aspirated material is of two major kinds-foodstuffs and inorganic crystalline "fi llers" derived from oral medications; the presence of either substance in the appropriate histologic context is diagnostic. the foodstuffs have a varying appearance depending on the age of the process. they include recognizable skeletal muscle and plant cell walls derived from consumed meats and vegetables, respectively; the latter may be refractile and either weakly or strongly birefringent on polarized microscopy. older organic material tends to have a pale, amorphous eosinophilic appearance, and is more diffi cult to recognize. the most common inorganic fi llers include microcrystalline cellulose, which is strongly birefringent, and crospovidone, which has an amorphous densely basophilic appearance. these exogenous compounds must not be confused with various endogenous materials that can be found within giant cells, including blue bodies, asteroid bodies, and birefringent calcium salts. as mentioned above, the granulomas in aspiration sometimes show central suppurative necrosis, wherein the giant cells surround pockets of neutrophils. the latter feature, while nonspecifi c (as it can be seen with certain infections, wegener granulomatosis, and rheumatoid . multinucleated giant cells are engulfi ng aspirated exogenous substances. the amorphous pale-staining material within the upper giant cell ( arrow ) is partially digested foodstuff while the birefringent, cracked, crystalline material in the lower giant cell ( asterisk ) is microcrystalline cellulose, a common inorganic fi ller utilized in oral medications. ( b ) polarized light microscopy can be helpful in identifying and/or confi rming polarizable substances in suspected cases of aspiration (hematoxylin and eosin, original magnifi cation ×600). certain crystalline fi llers, such as microcrystalline cellulose (as seen here) are strongly polarizable. plant cell walls from aspirated foods vary greatly in their strength of polarization nodules) is not common and is therefore a potential clue to the diagnosis. if suppurative granulomas are present in a tbbx, this should prompt the pathologist to search carefully for any associated exogenous aspirated substances. occasionally, no aspirated material can be found, a problem more common in small biopsies, and a confi dent diagnosis of aspiration pneumonia may not be possible. in immunocompromised patients the differential diagnosis for otherwise unexplained granulomatous infl ammation includes mainly opportunistic infections and should prompt appropriate special stains and microbiological assays. organisms that may cause suppurative granulomatous infl ammation resembling that seen in aspiration include, most commonly, nocardia , actinomyces , and blastomyces species. bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria pulmonary manifestations of rheumatoid arthritis. semin respir crit care med epidemiology and etiology of acute bronchiolitis in hong-kong infants postinfectious bronchiolitis obliterans in children: clinical and pulmonary function fi ndings fatal ammonia inhalation. a case report with autopsy fi ndings long-term course of bronchiectasis and bronchiolitis obliterans as late complication of smoke inhalation a review of the respiratory effects of smoking cocaine clinical bronchiolitis obliterans in workers at a microwave-popcorn plant diacetyl-induced lung disease the histological spectrum of pulmonary graft-versus-host disease in bonemarrow transplant recipients interstitial pneumonitis and lymphocytic bronchiolitis/bronchitis as a direct result of acute lethal graft-versus-host disease duplicate the histopathology of lung allograft rejection lymphocytic airway infi ltration as a precursor to fi brous obliteration in a rat model of bronchiolitis obliterans risk factors for bronchiolitis obliterans: a systematic review of recent publications obliterative bronchiolitis or chronic lung allograft rejection: a basic science review bronchiolitis obliterans syndrome-risk factors and therapeutic strategies the registry of the international society for heart and lung transplantation: twenty-eighth adult lung and heart-lung transplant report-2011 early detection of chronic pulmonary allograft dysfunction by exhaled biomarkers a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection-lung rejection study-group revision of the 1990 working formulation for the classifi cation of pulmonary allograft rejection: lung rejection study group revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection allograft vascular disease: comparison of heart and other grafted organs pulmonary arteriosclerosis in long-term human heart-lung transplant recipients bronchoscopic monitoring after lung transplantation the controversial role of surveillance bronchoscopy after lung transplantation association of minimal rejection in lung transplant recipients with obliterative bronchiolitis the sensitivity of transbronchial biopsy for the diagnosis of acute lung rejection prospective study of transbronchial biopsies in the management of heart-lung and single lung-transplant patients the diagnosis of obliterative bronchiolitis after heart-lung and lung transplantation: low yield of transbronchial lung biopsy diagnosis of chronic lung transplant rejection by transbronchial biopsy the utility of open lung biopsy following lung transplantation antibody-mediated rejection in lung transplantation: myth or reality? c3d and the septal microvasculature as a predictor of chronic lung allograft dysfunction pulmonary capillaritis: a possible histologic form of acute pulmonary allograft rejection use of c4d as a diagnostic adjunct in lung allograft biopsies acute antibody-mediated rejection after lung transplantation immunoperoxidase staining for c4d on paraffi n-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofl uorescence lymphocytic bronchitis bronchiolitis in lung allograft recipients lymphocytic bronchitis associated with graft-versus-host disease in recipients of bone-marrow transplants diagnosis of rejection of lung allografts diagnosis of lung allograft rejection by bronchial intraepithelial leu-7 positive t lymphocytes large airway infl ammation in heart-lung transplant recipients-its signifi cance and prognostic implications obliterative bronchiolitis in lung allografts removed at retransplant for intractable airway problems bronchiolitis obliterans after lung transplantationa review bronchiolitis: the pathologist's perspective bronchiolar disorders the nature of smallairway obstruction in chronic obstructive pulmonary disease infections relevant to lung transplantation lung-transplant pathology-a comparativestudy of pulmonary acute rejection and cytomegaloviral infection perivascular infl ammation in pulmonary infections: implications for the diagnosis of lung rejection katzenstein and askin's surgical pathology of non-neoplastic lung disease organizing pneumonia. features and prognosis of cryptogenic, secondary, and focal variants griffi th bp. interstitial and airspace granulation-tissue reactions in lung-transplant recipients organizing pneumonia following pulmonary transplantation and the development of obliterative bronchiolitis risk factors for the development of obliterative bronchiolitis after lung transplantation cryptogenic organizing pneumonia is an important cause of graft dysfunction and should be included in the classifi cation of pulmonary allograft rejection lung transplantation exacerbates gastroesophageal refl ux disease gastro-oesophageal refl ux and gastric aspiration in lung transplant patients with or without chronic rejection gastroesophageal refl ux and lung transplantation pulmonary disease due to aspiration of food and other particulate matter: a clinicopathologic study of 59 cases diagnosed on biopsy or resection specimens diffuse bronchiolar disease due to chronic occult aspiration gastroesophageal refl ux as cause of obliterative bronchiolitis-a case-report early fundoplication prevents chronic allograft dysfunction in patients with gastroesophageal refl ux disease improved lung allograft function after fundoplication in patients with gastroesophageal refl ux disease undergoing lung transplantation azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation azithromycin reverses airfl ow obstruction in established bronchiolitis obliterans syndrome key: cord-102958-q8jamg07 authors: hahka, taija m.; xia, zhiqiu; hong, juan; kitzerow, oliver; nahama, alexis; zucker, irving h.; wang, hanjun title: resiniferatoxin (rtx) ameliorates acute respiratory distress syndrome (ards) in a rodent model of lung injury date: 2020-09-14 journal: biorxiv doi: 10.1101/2020.09.14.296731 sha: doc_id: 102958 cord_uid: q8jamg07 acute lung injury (ali) is associated with cytokine release, pulmonary edema and in the longer term, fibrosis. a severe cytokine storm and pulmonary pathology can cause respiratory failure due to acute respiratory distress syndrome (ards), which is one of the major causes of mortality associated with ali. in this study, we aimed to determine a novel neural component through cardiopulmonary spinal afferents that mediates lung pathology during ali/ards. we ablated cardiopulmonary spinal afferents through either epidural t1-t4 dorsal root ganglia (drg) application or intra-stellate ganglia delivery of a selective afferent neurotoxin, resiniferatoxin (rtx) in rats 3 days post bleomycin-induced lung injury. our data showed that both epidural and intra-stellate ganglia injection of rtx significantly reduced plasma extravasation and reduced the level of lung pro-inflammatory cytokines providing proof of principle that cardiopulmonary spinal afferents are involved in lung pathology post ali. considering the translational potential of stellate ganglia delivery of rtx, we further examined the effects of stellate rtx on blood gas exchange and lung edema in the ali rat model. our data suggest that intra-stellate ganglia injection of rtx improved po2 and blood acidosis 7 days post ali. it also reduced wet lung weight in bleomycin treated rats, indicating a reduction in lung edema. taken together, this study suggests that cardiopulmonary spinal afferents play a critical role in lung inflammation and edema post ali. this study shows the translational potential for ganglionic administration of rtx in ards. respiratory failure due to acute respiratory distress syndrome (ards) is one of the major causes of mortality associated with acute lung injury (ali) including covid-19. [1] [2] [3] [4] [5] most forms of ali/ards are also associated with acute cytokine release, pulmonary edema and in the longer term, fibrosis. 5, 6 however, the mechanisms underlying these pathological changes in the lungs during ali/ards are not fully understood. in particular, a neural component that mediates lung pathology during ali/ards has been less considered. sensory neurons innervating the heart and lung enter the central nervous system by one of two routes; through the vagus nerve into the brain stem (medulla) with cell bodies residing in the nodose ganglia and directly into the spinal cord where cell bodies reside in the dorsal root ganglia (drg). afferents are composed of elements that respond to a variety of sensory modalities including mechanical deformation, heat, cold, ph, and inflammatory mediators, just to name a few. 7 the reflex effects following stimulation of these afferents depends on the type of stimulus and the neural pathway involved. activation of vagal afferent pathways tends to be sympatho-inhibitory and antiinflammatory. 8, 9 on the other hand, activation of spinal afferents tends to be sympathoexcitatory and pro-inflammatory. [10] [11] [12] [13] [14] [15] it is well known that small diameter spinal transient receptor vanilloid 1 (trpv1)-positive afferent c-fibers contain neuropeptides such as substance p (sp) and calcitonin gene related peptide (cgrp). 16 these peptides tend to dilate adjacent vasculature and increase microvascular permeability. 16 in the lung, this can cause pulmonary edema resulting in reduced oxygen diffusion and 4 promote immune cell infiltration resulting in neural inflammation. therefore, in the current study we hypothesized that ablation of lung afferent innervation (thoracic spinal) by application of an ultrapotent, selective afferent neurotoxin, resiniferatoxin (rtx) will modify the course of the pathology including lung edema and local pulmonary inflammation associated with progressive ali. committee of the university of nebraska medical center and performed in accordance with the national institutes of health's guide for use and care of laboratory animals and with arrive guidelines. 17, 18 experiments were performed on adult, male, 200-250g sprague-dawley rats purchased from the charles river laboratories. animals were housed on-site and given a one-week acclimation period prior to experimentation. food and water were supplied ad libitum, and rats were on 12-hour light/dark cycles. rats were randomized into three groups and evaluated at 1-week post-instillation as follows: sham rats, bleomycin (bleo)-exposed rats with saline (epidural or intrastellate injection), and bleo-exposed rats with rtx (epidural or intra-stellate injection). bleo (2.5 mg/kg, ~0.15 ml) was instilled intra-tracheally to the lungs under 3% isoflurane anesthesia. sham control rats underwent intra-tracheal instillation of saline. animals were treated with rtx or vehicle (phosphate buffered saline) by either the epidural t1-t4 drgs route (6 µg/ml, 10µl/per ganglia) or intra-stellate ganglia administration (50 µg/ml, 5 µl/per side) 3 days following bleomycin delivery ( figure 1 ). in a pilot experiment, the upper thoracic spinal afferents were ablated by epidural application of rtx as previously described. 11 briefly, rats were anesthetized using 2%-3% isoflurane:oxygen mixture. rats were placed in the prone position and a small midline incision was made in the region of the t13-l1 thoracic vertebrae. following dissection of the superficial muscles, two small holes (approximately 2 mm x 2 mm) were made in the left and right sides of t13 vertebrae. a polyethylene catheter (pe-10) was inserted into the subarachnoid space via one hole and gently advanced about 4cm approximating the t1 level. the upper thoracic sympathetic afferent ganglia were ablated by injecting resiniferatoxin (rtx; sigma aldrich), an ultra-potent agonist of the trpv1 receptor into the aubarachnoid space via the catheter. rtx (1 mg; sigma aldrich) was dissolved in a 1:1:8 mixutre of ethanol, tween 80 (sigma-aldrich), and isotonic saline. the first injection of rtx (6 µg/ml, 10ul) was made at a very slow speed (~ 1 minute) to minimize the diffusion of the drug. the catheter was then pulled back to t2, t3 and t4, respectively to perform serial injections (10 ul/each) at each segment. the catheter was withdrawn and the same injections were repeated on the other side. silicone gel was used to seal the hole in the t13 vertebra. the skin overlying the muscle were closed with a 3-0 polypropylene simple interrupted suture, and betadine was applied to the wound. for post-procedure pain management, buprenorphine (0.05 mg/kg) was subcutaneously injected immediately after surgery and twice daily for 2 days. rats were anesthetized using 2%-3% isoflurane:oxygen mixture. after the trachea was cannulated mechanical ventilation was started (model 683, harvard apparatus, south natick, ma). the skin from the rostral end of the sternum to the level of third rib was incised. portions of the superficial and deep pectoral muscles and the first intercostal muscles were cut and dissected. to localize the left or right stellate ganglion, the left or right precava vein were separated with a hooked glass or steel rod laterally away from the brachiocephalic artery to expose the internal thoracic artery and the costocervical 7 artery, which are descending branches of the right subclavian artery. stellate ganglia and ansa subclavia are located medially to the origins of the internal thoracic and costocervical arteries. then, rtx (5µl, 50 mg/ml) was injected into the ganglia with a 5µl hamilton syringe (microliter #95, hamilton, reno, nv, usa.) over 30 s bilaterally. an image of this procedure is shown in figure 2 . following these maneuvers, the thorax between the 1 st and 2nd intercostal spaces was closed with continuous 4-0 dexon ii coated braided absorbable polyglycolic acid suture and the skin was closed with 3-0 polypropylene suture and the chest evacuated. betadine was applied to the wound and the rats were allowed to recover from the anesthesia. for post-procedure pain management, buprenorphine (0.05 mg/kg) was injected subcutaneously immediately after surgery and twice daily for 2 days. the artery on the ventral aspect of the rat tail was used for the collection of small amounts of blood (~0.1 ml) for analyzing arterial blood gas at day 7 post bleomycin. the animal was restrained with a commercial restrainer so that its tail was accessible. the tail was prepared aseptically by alternating alcohol prep pads and iodine prep pads three times and the artery was punctured using a 24 g needle. a small volume of blood (~0.1 ml) was gently aspirated into the syringe for blood gas analysis (istat, abbott, chicago, il, usa). after sample collection, the needle was removed, and a gauze swab was pressed firmly on the puncture site to stop bleeding. rats were anaesthetized with pentobarbitone (40 mg/kg). evans blue, 20 mg/kg (10 mg/ml, dissolved in saline + 100 ie per ml heparin) was administered intravenously. right panel: at day 0, bleomycin or saline was given intra-tracheally; at day 3, resiniferatoxin or vehicle was given into epidural space or into stellate ganglia; at say 7, the rats were sacrificed. the procedure by which the stellate ganglia were exposed and injected in rats under anesthesia is shown in figure 2 . plasma extravasation (evans blue) was used to assess vascular permeability after acute lung injury. as shown in figure 3 , bleomycin-treated lungs exhibited a wide distribution of evans blue areas in both sides. the highest intensity of evans blue was shown at the medial aspect of each lung. the evans blue areas were largely reduced following epidural rtx treatment at the 7-day time point after bleomycin administration (figure 3) . three pro-inflammatory tissue cytokines were prevalent in the lung following bleo treatment are shown in figure 4 . il-6, il-1ß and ifný were markedly elevated following bleo treatment. these cytokine levels were normalized in epidural rtx treated rats. plasma extravasation in response to bleo was also reduced after stellate injection of rtx ( figure 5) . as can be seen, there was a marked reduction in evans blue dye in the lung following stellate injection of rtx. arterial blood gas data were evaluated in rats treated with vehicle vs rtx intra-stellate. compared to sham rats, wet lung weight (wlw) as well as the ratio of wlw to bw was significantly higher in the bleo+veh rats, which was significantly reduced by intrastellate injection of rtx. these data suggest that intra-stellate injection of rtx reduces lung edema post bleo. the data from this study provides proof of principle and is highly suggestive of an important role for trpv1-positive spinal afferent-mediated neuroinflammation in acute lung injury and progression to acute respiratory distress. the evidence provided demonstrates that ablation of trpv1 sensory afferents in the presence of acute lung injury using rtx delivered by either of two routes that target cardiopulmonary afferents leads to a rapid reduction in lung microvascular permeability and a reduction in tissue and plasma inflammatory markers. while pulmonary function per se was not directly measured in this series of experiments, arterial blood gas data strongly suggest an improvement in gas exchange. the improved body weight and reduced lung weight in rats with lung injury after receiving stellate ganglia administration of rtx suggest potential clinical benefits from reduced lung edema, and protective effects for nonpulmonary organs that would otherwise be impacted by the pulmonary triggered systemic inflammatory process. the lung is innervated by a dual sensory system including vagal and spinal afferents. both vagal and spinal afferent fibers are composed of a (high conduction velocity) and c-fiber (low conduction velocity) axons. these fibers and their sensory endings express a variety of membrane receptors that mediate ion channel function including traditional na + , k + and ca 2+ channels (both voltage and ligand gated). importantly, non-specific cation channels that are highly permeable to calcium are expressed mostly in small diameter c-fibers. 19, 20 these include at least 30 members of the transient receptor potential family including transient receptor potential a (trpa) and trpv receptors. trpv1 receptors transduce sensations of heat and neuropathic pain in the periphery. 1 7 estimates are that approximately 60 percent of thoracic drg neurons are positive for trpv1. 21 upon activation, trpv1 channels are highly permeable to calcium. 19, 20 high levels of intracellular calcium are toxic and thus damage or kill these specific afferent neurons. thus, a unique strategy has been developed to modulate the pathological effects of trpv1 afferent neurons. the ultrapotent neurotoxin, rtx binds avidly to the trpv1 receptor. after initial stimulation, high intracellular levels of calcium mediate inhibition of neuronal function. site-specific delivery of rtx can be used to intervene in various conditions to alleviate pain, inflammation, fibrosis and plasma extravasation. it has been shown that rtx-induced trpv1 sensory afferent deletion can block the afferent-contained neuropeptide release and reduce inflammatory pain. 22 cardiopulmonary spinal afferents can also be targeted with rtx by either application into the epidural space at thoracic levels t1-t4 11 (with some spread to higher and lower segments) or by injection into the stellate ganglia. while drgs are considered exclusively sensory in nature, the stellates contain soma for sympathetic efferent fibers and fibers of passage for thoracic afferents as they course through drgs and enter the spinal cord. it should be noted that in humans the stellate ganglia can be easily identified, and that this type of transcutaneous procedure can be performed with fluoroscopic or ultrasound guidance (intra-ganglionic or nerve 'block' approach). compared to epidural delivery that requires relatively larger injection volume (~80 µl for bilateral injection) to sufficiently cover the t1-t4 drgs, intra-stellate injection requires a much smaller volume (10 µl for bilateral injection), which reduces the risk of systemic absorption of rtx and allows a higher dose of rtx to be used for local injection. 1 8 the current data clearly show that intra-stellate ganglia injection of rtx markedly attenuated lung extravasation post ali, suggesting that a large proportion of thoracic afferents passing through the stellates innervate the lungs. taken together, we believe that intra-stellate ganglia delivery of rtx should be a clinically feasible intervention to treat acute lung injury compared to the epidural approach. the preliminary data presented here for epidural administration of rtx provides proof of principle that it reduces plasma extravasation in the lung. the main focus of this study was on the therapeutic effect of the stellate ganglia approach on lung pathology in our ali rat model. prior work from this laboratory has demonstrated that ablation of cardiac trpv1positive afferents reduces sympathetic nerve activity and cardiac remodeling in a post myocardial infarction model of chronic heart failure. 23 trpv1-expressing cardiopulmonary afferents participate in a sympatho-excitatory reflex that has been termed the cardiac afferent sympathetic reflex (csar) 24 and the pulmonary afferent sympathetic reflex (psar) 25 . the csar is augmented in heart failure along with cardiac afferent discharge in response to bradykinin or capsaicin. 23 epicardial administration of rtx reduces sympathetic outflow to the heart and kidneys and improves cardiac diastolic function while reducing fibrosis and cytokine content in the heart. 23 furthermore, cardiac application of local anesthetic lowers sympathetic nerve activity in anesthetized vagotomized animals suggesting tonic input from these spinal afferents in heart failure. 15, 26 on the other hand, it has been widely reported that activation of trpv1expressing afferents causes secretion of neuropeptides such as substance p (sp) and calcitonin gene-related peptide (cgrp). [27] [28] [29] [30] [31] released sp, but not cgrp, in sensory 1 9 endings binds neurokinin (nk) 1 receptors on blood vessels and causes vasodilation and increased vascular permeability that allows loss of proteins and fluid (plasma extravasation) thus promoting the regional accumulation of monocytes and leukocytes contributing to inflammation. [32] [33] [34] in the lung, this process not only impairs alveolar gas exchange but may initiate and exacerbate a fulminant cytokine storm from adjacent cells and from circulating macrophages. 6 the current study supports the idea that selective ablation of trpv1 afferents mitigates neuroinflammation in the lung by inhibiting trpv1 afferent-mediated plasma extravasation, at least in the bleomycin model of ali. importantly, although we did not directly measure respiratory parameters such as minute ventilation and respiratory rate in vehicle and rtx treated bleomycin rats, we observed significantly reduced lung weight and improved blood gas parameters including blood ph, po 2 and so 2 in bleomycin rats treated with intra-stellate ganglia injection of rtx, suggesting an improvement in lung function. combined with the results shown in the current model of ali, the potential of rtx to rescue lung function and protect multiple organs from collateral damage due to lung injury triggered inflammation is very encouraging and warrants additional studies as a rescue therapy for patients with lung injuries or infections resulting in inflammatory -mediated pneumonia. clinical assessments including potential acceleration for return to normal function or long-term protective effects on lung fibrosis should also be explored in upcoming studies. in the current study, we chose the bleomycin-induced lung injury model to study the role of pulmonary spinal afferent ablation in lung pathology after acute lung injury. intratracheal injection of bleomycin has been widely used to evoke pulmonary interstitial lesions in animal models. 35, 36 bleomycin-induced lung injury is primarily mediated by 2 0 alveolar epithelial damage resulting in the release of large number of inflammatory cells and cytokines. following pulmonary insult with bleomycin at day 0, inflammation progresses to peak levels around day three. pulmonary edema, respiratory distress, body and organ weight loss associated with systemic inflammation is observed up to day 10. 35 the model was chosen as it closely reproduces important aspects of ards including local inflammation, cytokine storm, progression to respiratory distress, and multi-organ impact. the timing of therapeutic intervention (day 3) was also carefully selected to coincide with high levels of inflammatory mediators and lung damage that would be found in infectious disorders including in covid-19 patients progressing to ventilatory support. we acknowledge a limitation that since the clinical etiology of lung injury is variable (e.g. viral/bacterial infection, chemical and surgical), the bleomycin model does not completely mimic all pathological characteristics of lung injury in humans. while we considered the lipopolysaccharide (lps)-induced lung injury model, lps has been shown to have a direct effect on sympathetic and parasympathetic afferent and efferent neurons in addition to its effect on the lung itself, 35, 37-40 and thus was less suitable for use in this study. as far as a viral infection model is concerned, we have not attempted to directly apply our findings to relevant diseases such as covid 19 , although there may be some phenotypic overlap between the bleomycin lung injury and viral infection models. more work needs to be done to validate the efficacy of rtx in other lung injury models. 2 1 our data suggest that pulmonary spinal afferent ablation by intra-stellate injection of rtx reduces plasma extravasation and local pulmonary inflammation post bleomycininduced lung injury which results in improved blood gas exchange. these findings suggests that local stellate application of rtx could be used as a potential clinical intervention to mitigate lung pathology after ali. this study was supported by sorrento therapeutics inc. dr. hanjun wang is also supported by margaret r. larson professorship in anesthesiology. dr. irving h. zucker is supported in part by the theodore f. hubbard professorship for cardiovascular research. the lung/rtx project is currently sponsored by sorrento therapeutics inc. lb: pathogenesis of acute respiratory distress syndrome clinical features of patients infected with 2019 novel coronavirus in uk: covid-19: consider cytokine storm syndromes and immunosuppression sa: respiratory support for patients with covid-19 infection clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china cs: acute respiratory distress syndrome the trp superfamily of cation channels. science&#039;s stke d: vagal afferent activation suppresses systemic inflammation via the splanchnic antiinflammatory pathway anti-inflammatory properties of the vagus nerve: potential therapeutic implications of vagus nerve stimulation hj: trpv1 (transient receptor potential vanilloid 1) cardiac spinal afferents contribute to hypertension in spontaneous hypertensive rat hj: sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats the paradoxical role of the transient receptor potential vanilloid 1 receptor in inflammation p: the insulin receptor is colocalized with the trpv1 nociceptive ion channel and neuropeptides in pancreatic spinal and vagal primary sensory neurons the innervation of the kidney in renal injury and inflammation: a cause and consequence of deranged cardiovascular control ih: cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states chapter 14 -nociceptive physiology dg: improving bioscience research reporting: the arrive guidelines for reporting animal research nr: guide for the care and use of laboratory animals role of calcium ions in the positive interaction between trpa1 and trpv1 channels in bronchopulmonary sensory neurons intrathecal resiniferatoxin in a dog model: efficacy in bone cancer pain hj: identification of cardiac expression pattern of transient receptor potential vanilloid type 1 (trpv1) receptor using a transgenic reporter mouse model mj: deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control ih: cardiac sympathetic afferent denervation attenuates cardiac remodeling and improves cardiovascular dysfunction in rats with heart failure hl: cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats hj: sympathoexcitation in response to cardiac and pulmonary afferent stimulation of trpa1 channels is attenuated in rats with chronic heart failure w: interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the nts at1 receptors in heart failure tc: impact of neuropeptide substance p an inflammatory compound on arachidonic acid compound generation hydrogen sulfide and substance p in inflammation sensory-nerve-derived neuropeptides: possible therapeutic targets the role of neuromodulators (substance p and calcitonin gene-related peptide) in the development of neurogenic inflammation in the oral mucosa 3h]resiniferatoxin autoradiography in the cns of wild-type and trpv1 null mice defines trpv1 (vr-1) protein distribution kl: nk-1 receptor mediation of neurogenic plasma extravasation in rat skin nw: desensitization of the neurokinin-1 receptor (nk1-r) in neurons: effects of substance p on the distribution of nk1-r, galphaq/11, g-protein receptor kinase-2/3, and betaarrestin-1/2 je: pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin: pathology and stereology w: lipopolysaccharide-mediated inflammatory priming potentiates painful post-traumatic trigeminal neuropathy cg: lipopolysaccharides and trophic factors regulate the lps receptor complex in nodose and trigeminal neurons novel pathway for lps-induced afferent vagus nerve activation: possible role of nodose ganglion gm: lipopolysaccharide induces substance p in sympathetic ganglia via ganglionic interleukin-1 production key: cord-018659-rxzy6k3b authors: danziger-isakov, lara; munoz, flor m.; estabrook, michele title: posttransplant complications and comorbidities date: 2018-01-08 journal: solid organ transplantation in infants and children doi: 10.1007/978-3-319-07284-5_71 sha: doc_id: 18659 cord_uid: rxzy6k3b infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. bacteria · cytomegalovirus · infectious complication · nontubercular mycobacteria · pediatric lung transplantation · respiratory virus bacteria account for about 50% of infections post lung transplant with pneumonia being most frequent. other sites include nosocomial central lineassociated bacteremia, urinary tract, and surgical site infections. the greatest risk is within the first year after transplantation, particularly in the first 3-6 months. donor infection and recipient airway colonization are also risk factors (speich and van der bij 2001; aguilar-guisado et al. 2007; parada et al. 2010; burguete et al. 2013; yun et al. 2015) . one of the largest studies of primarily adults found that 75% of infections occurred within the first year posttransplant and 42% occurred within the first 3 months. the majority, 48%, was bacterial (parada et al. 2010; burguete et al. 2013) . another study showed similar results but found that bacteremia, both primary and catheter associated, was the most common infection in the first month after transplant with pneumonia becoming most frequent after 2 months. multidrug-resistant bacteria including methicillin-resistant staphylococci, vancomycin-resistant enterococci, and carbapenem-resistant or extended-spectrum betalactamase producing gram-negative bacilli were involved in 66% of infections. bacterial infections were significantly more common in those colonized with multidrug-resistant gram-negative bacilli than those who were not (yun et al. 2015) . a pediatric study of 42 children and 49 lung transplants found that half of the infections were bacterial with 42% occurring within 3 months after transplant and 80% in the first year. the lung was the most common site (72%) and pseudomonas aeruginosa was the most common organism. bacterial infections were felt to contribute to pulmonary dysfunction (bronchiolitis obliterans) but were not the primary cause of death (metras et al. 1999) . recent data from the registry of the international society for heart and lung transplantation reported that non-cytomegalovirus infection was the cause of death in 24% of lung transplant recipients in the first year after transplant (benden et al. 2013) . one of the largest studies of pneumonia in 236 lung transplant recipients (aguilar-guisado et al. 2007) found that the most common etiology was bacterial in 83%. gram-negative bacilli accounted for 60% with pseudomonas aeruginosa being the most frequent isolate in 25%, followed by acinetobacter baumannii in 14%. staphylococcus aureus was the etiology in 14%. the probability of 1-year survival was significantly higher in those recipients who did not have an episode of pneumonia (aguilar-guisado et al. 2007 ). late-onset communityacquired pneumonia with streptococcus pneumoniae also occurs (de bruyn et al. 2004 ). survival after lung transplantation is limited by the high incidence of chronic lung allograft dysfunction (clad) that has two forms: bronchiolitis obliterans syndrome (bos) and restrictive allograft syndrome (ras). the role of infection in the development of clad has recently been reviewed (martin-gandul et al. 2015; gregson 2016) . while acute infection with community-acquired respiratory viruses is recognized as a risk, pseudomonas aeruginosa and staphylococcus aureus are increasingly recognized as well. one study of lung transplant recipients with cystic fibrosis found that loss of colonization with pseudomonas was protective against the development of bos (gottlieb et al. 2009 ). two further studies found that infections due to gram-positive bacteria, primarily staphylococcus aureus, increased the hazard risk for bos (gupta et al. 2009; valentine et al. 2009 ). the underlying allograft inflammatory state in the setting of infection also appears to be important in determining the development of clad (gregson 2016). cystic fibrosis (cf) is a common, underlying diagnosis in children who undergo lung transplantation. the registry of the international society for heart and lung transplantation reported that half of children <10 years of age and 70% of children aged 11 through 17 years had cf (benden et al. 2013) . cf-specific bacterial pathogens including multidrug-resistant (mdr) or pan-resistant bacteria persist in the paranasal sinuses and upper airways and can be a cause of posttransplant pneumonia. pseudomonas aeruginosa is most common, but other organisms include stenotrophomonas maltophilia, achromobacter xylosoxidans, and burkholderia cepacia complex (shoham and shah 2013) . in lung transplant recipients, there is increasing resistance in gram-negative bacilli including extended-spectrum beta-lactamase, ampc betalactamase, and carbapenemase (shoham and shah 2013; van duin and van delden 2013) . pseudomonas aeruginosa infection occurs in up to 80% of patients with cf and bronchiectatic lung disease, and pretransplant colonization is a significant risk factor for infection after transplant. mdr p. aeruginosa has a prevalence rate from 10% to 45% in patients with cf (shoham and shah 2013) . survival posttransplant in patients colonized with pan-resistant p. aeruginosa before transplant was similar to those with sensitive organisms at 1 year (88% vs. 96%) but lower at 3 years (63 vs. 91%) (hadjiliadis et al. 2007; shoham and shah 2013) . however, the 2006 update of the international guidelines for the selection of lung transplant candidates stated that colonization with multidrug or pan-resistant p. aeruginosa was not a contraindication because it has not been shown to significantly affect shortterm survival (orens et al. 2006) . a recent study in lung transplant recipients with cf reported that infection with pan-resistant achromobacter xylosoxidans and stenothrophomonas maltophila also did not reduce survival after lung transplantation (lobo et al. 2015) . burkholderia cepacia complex (bcc) is comprised of several different species that colonize the respiratory tract in 15-22% of patients with cf. most infections are caused by b. cenocepacia (genomovar iii) and b. multivorans (genomovar ii). pretransplant colonization with b. cenocepacia has been associated with increased posttransplant mortality (relative risk 8.4) with one study reporting 1-year survival of 29% compared to 92% in those uninfected and is considered by many centers as a contraindication to transplant (shoham and shah 2013) . recipients colonized with b. multivorans did not have decreased survival while b. gladioli had an increased mortality risk but not as high as b. cenocepacia. bcc has an 80% prevalence of multidrug resistance (shoham and shah 2013) . methicillin-resistant staphylococcus aureus (mrsa) has been increasingly recognized as a significant bacterial pathogen post lung transplantation. a study of lung transplant recipients found that 18% had s. aureus infection in the first 90 days with 62% being methicillin sensitive (mssa) and 35% being mrsa. the site of infection was pneumonia 48%, tracheobronchitis 26%, and bacteremia 12%. colonization before transplant with mrsa was a risk factor for mrsa infection posttransplant but was not associated with increased mortality at 30 and 90 days post onset of infection (shields et al. 2012) . a second study had a calculated incidence rate of mrsa of 76 cases per 1000 transplant years with the median onset of 3 months posttransplant. the most common site was the lower respiratory tract and 31% of mrsa infections were associated with bacteremia. the direct mortality after mrsa infection was 17.6% (manuel et al. 2009 ). nontuberculous mycobacteria (ntm) are ubiquitous bacteria found in environmental sources including water, soil, plants, and animals. exposure is felt to be from the environment but more recently patient-to-patient transmission has been proposed for m. abscessus complex (bryant et al. 2013) . pretransplant infection is confined primarily to the lungs, with abnormal parenchyma such as cystic fibrosis or bronchiectasis being a risk factor. posttransplant infection can involve asymptomatic colonization, invasive lung disease, skin and soft tissue infection, and central lineassociated bacteremia (griffith et al. 2007; keating and daly 2013; smibert et al. 2016) . ntm isolation from respiratory cultures in lung transplant candidates is common particularly in those with cystic fibrosis. one study (chalermskulrat et al. 2006 ) reported a 20% colonization rate with 45% of isolates being m. avium complex (mac) and 41% m. abscessus. isolation after transplant is also common from 13 to 22% with mac accounting for about 70%. invasive disease after transplant is much less common, however, occurring in fewer than 5% of lung transplant recipients (chalermskulrat et al. 2006; chernenko et al. 2006; huang et al. 2011; knoll et al. 2012) . pretransplant colonization has been associated with an increased risk of posttransplant ntm infection as well as invasive disease but only for m. abscessus (chalermskulrat et al. 2006) . while ntm isolation and disease particularly with m. abscessus is associated with increased complications post lung transplant, it has not been associated with increased mortality and is not considered an absolute contraindication to transplantation (chalermskulrat et al. 2006; knoll et al. 2012; qvist et al. 2013) . case reports of two adolescents with cystic fibrosis and pretransplant m. abscessus infection showed that when antibiotic therapy led to afb stain negativity at the time of transplant, the outcome was favorable even in the face of positive cultures (zaidi et al. 2009 ). diagnosis of ntm disease is based on criteria published by the american thoracic society/ infectious diseases society of america that include clinical and microbiological criteria (griffith et al. 2007) . compatible symptoms and radiological changes consistent with ntm infection with other etiologies excluded must be accompanied by: positive culture from at least two sputum samples, positive culture from one bronchial lavage or wash, or lung biopsy with consistent pathology and positive culture. treatment depends on accurate identification and susceptibility testing of the organism. guidelines are available and consultation with an infectious disease expert is recommended (griffith et al. 2007; keating and daly 2013) . obtaining cultures of respiratory, blood, urine, and wound samples with accurate identification and determination of drug sensitivity is critical in the treatment of bacterial infection post lung transplant. consultation with a transplant infectious diseases physician and pharmacist is recommended when designing antibiotic therapy for multi-and pan drug-resistant organisms to maximize effectiveness and minimize toxicity. removal of sources of infection such as central venous lines and drainage of focal fluid collections should be undertaken when feasible. there are no well-conducted studies that have addressed the optimal choice of agent, duration, and efficacy of antibiotic prophylaxis for lung transplantation. in the absence of positive cultures from the donor or recipient, prophylactic regimens of 48-72 h and no longer than 7 days are recommended (bratzler et al. 2013 ). in recipients with cf, broad-spectrum antibiotics are administered at the time of transplant and are selected to cover the pretransplant bacterial pathogens and associated resistance patterns (hirche et al. 2014) . most centers treat recipients with a history of p. aeruginosa infection with two-drug antipseudomonal therapy for 2-3 weeks postoperatively to reduce the risk of pneumonia and colonization of the allograft (shoham and shah 2013). large multicenter prospective studies of adult sot recipients reported that the most common fungal organisms in lung recipients were aspergillus (63%), candida (23%), and other molds (10%) while zygomycosis, cryptococcosis, and endemic fungi were uncommon (neofytos et al. 2010 ). more recent data suggest an emergence of non-aspergillus mold infections (neofytos et al. 2013; chong et al. 2015) . pediatric studies have reported an incidence of pulmonary fungal infection from 10.5 to 20%, with aspergillus being the most common organism in two studies (danziger-isakov et al. 2008; liu et al. 2009b) . a single center study of 55 lung transplant recipients (liu et al. 2009b) found a higher incidence of posttransplant fungal colonization (60%) compared to adult patients (30-40%). however, posttransplant colonization was not associated with invasive pulmonary disease, and pulmonary fungal infections were not associated with chronic allograft rejection or death (liu et al. 2009b) . a larger retrospective multicenter study with patients transplanted in 1988-2006 found tacrolimusbased immunosuppression, cytomegalovirus sero-mismatch, age over 15 years, and prior episode of rejection greater than a2 were risks for pulmonary fungal infection, but the study did not investigate colonization as a risk factor (danziger-isakov et al. 2008) . additionally, pulmonary fungal infection was independently associated with decreased 12-month survival. mortality for proven and probable infection was 38 and 11%, respectively, similar to what has been reported for adults (danziger-isakov et al. 2008) . bronchial airway anastomotic complications occurred in 14% of 214 pediatric lung transplant recipients in a single center cohort, and this complication was associated with prior episodes of posttransplant fungal infection (choong et al. 2006) . these studies indicate that fungal infection in pediatrics significantly impact posttransplant morbidity and potentially mortality. while candida species isolated from respiratory secretions may represent normal commensal flora, invasive infections due to candida species have been reported in pediatric lung transplant recipients. in addition to bronchial anastomotic infection, pleural infection, pulmonary fungal infections, and bloodstream infections appear in the pediatric literature (danziger-isakov et al. 2005; danziger-isakov et al. 2008; liu et al. 2009b ). non-albicans species including c. krusei, c. glabrata, c. parapsilosis, and c. dubliniensis can all cause disease but may have differing antibiotic susceptibilities. identification to the species level is important to facilitate optimum treatment especially as non-albicans species have been associated with increased mortality (andes et al. 2016). aspergillus species cause posttransplant infections including tracheobronchitis with anastomosis infection, invasive pulmonary disease, and disseminated disease (hosseini-moghaddam and husain 2010). risk factors for invasive disease include ischemia at the anastomosis site, single lung transplant, hypogammaglobulinemia, placement of bronchial stent, cmv infection, and colonization (robertson et al. 2009; hosseini-moghaddam and husain 2010; chong et al. 2015) . as with candida species, speciation of aspergillus is important. while a. fumigatus causes the majority of disease, other species including a. niger, a. terreus, a. flavus, and a. ustus appear to be increasing in prevalence especially with the use of inhaled amphotericin as prophylaxis (hosseini-moghaddam and husain 2010; peghin et al. 2016) . prompt diagnosis of invasive aspergillus infection is imperative to improve outcome; however, newer diagnostics have not been specifically evaluated in pediatric lung transplantation. in pediatric cancer patients, the sensitivity and specificity of galactomannan (gm), beta-dglucan, and pcr-based assays were highly variable (lehrnbecher et al. 2016) . in adult lung transplant recipients, the serum gm assay has excellent specificity but poor sensitivity while bronchoalveolar lavage gm appears more promising for diagnosis with a sensitivity of 88-100% and specificity of 89-90% depending on the cutoff used for diagnosing invasive pulmonary aspergillosis (husain et al. 2004; pasqualotto et al. 2010; luong et al. 2011 ). further, a pan-aspergillus real-time pcr assay also performed well with a sensitivity and specificity of 100% and 93%, respectively (luong et al. 2011). as newer diagnostics emerge, their utility in pediatric lung transplantation will need assessment. histoplasmosis, blastomycosis, and coccidioidomycosis are endemic fungi with restricted geographical distribution. they are found in the environment as molds and the route of infection is inhalation of spores. posttransplant disease with these organisms is rare in adults and has not been reported in the pediatric lung transplant literature to date (neofytos et al. 2010; assi et al. 2013 ). treatment of invasive fungal infection in pediatric lung transplant recipients should include input from an infectious diseases specialist particularly regarding drug choice and dosage. several national and international guidelines present treatment recommendations for invasive fungal infections (pappas et al. 2016; patterson et al. 2016) . new antifungal agents have emerged in the past decade including second-generation azole medications and echinocandins (lewis 2011). while these agents are improving outcomes related to fungal infections, clinicians must pay careful attention to therapeutic drug monitoring, interactions with immunosuppressive agents (both calcineurin inhibitors and mtors), and medication side effects to reduce potential complications. despite the significant morbidity and mortality associated with fungal infections following lung transplantation, there are not established guidelines for prophylaxis. in pediatrics, the impact of prophylaxis to prevent colonization and progression to infection is uncertain. several small, nonrandomized clinical trials in adult recipients have demonstrated efficacy ranging from 80 to 100% (hosseini-moghaddam and husain 2010; brizendine et al. 2011). three main approaches have been used in lung transplant recipients: universal prophylaxis, targeted prophylaxis, and pre-emptive therapy. universal prophylaxis is given to all recipients immediately post transplantation while targeted prophylaxis is given to patients with known risk factors (neoh et al. 2011) . further, response to positive cultures on routine posttransplant bronchoscopy may prompt initiation of pre-emptive therapy, but the optimal response to positive bal cultures is unclear (avery 2011). while inhaled amphotericin has recently been linked to a decrease in posttransplant aspergillus, amphotericin-resistant strains have emerged indicating that intervention is not benign (peghin et al. 2016) . a recent world-wide survey of antifungal prophylaxis (neoh et al. 2011 ) showed a highly variable approach with the majority (58%) using universal prophylaxis that primarily focused on preventing aspergillus infections. a survey of centers performing pediatric lung transplantation (50% exclusively pediatric) revealed an equally variable approach. universal prophylaxis is provided in 28% of centers, while 48% use targeted prophylaxis primarily to patients with cystic fibrosis or pretransplant fungal colonization (mead et al. 2014) . the focus of prophylaxis includes both aspergillus and candida species with most centers reporting the use of either voriconazole or inhaled amphotericin. additionally, the duration of prophylaxis is widely distributed from 3 to 6 months to more than 12 months. the optimal approach for fungal prophylaxis in pediatric lung transplant recipients is undefined and there are sparse data for this population to guide recommendations. the introduction of preventative antiviral regimens has improved the natural history of cytomegalovirus (cmv) after adult lung transplantation (patel and paya 1997; zamora et al. 2004; chmiel et al. 2008) ; however, cmv remains associated with increased morbidity and mortality after transplantation (husni et al. 1998; monforte et al. 2001; ruttmann et al. 2006; chmiel et al. 2008) . to improve the clarity of cmv reporting in the literature, specific definitions have been suggested and updated with diagnostic evolution (humar and michaels 2006; husain et al. 2011; ljungman et al. 2017) . cmv infection refers to the presence of active replicating virus by any method without associated symptoms. cmv syndrome includes the presence of virus plus one or more associated symptoms including fever, fatigue/malaise, leukopenia, atypical lymphocytes, thrombocytopenia, or transaminitis. those with evidence of tissue invasion are defined as end-organ cmv disease. newer definitions take into account the availability of quantitative cmv pcr testing, but a specific viral load in bal to determine cmv pneumonitis has not yet been established (ljungman et al. 2017 ). cytomegalovirus (cmv) occurs in approximately 30% of pediatric lung transplant recipients (danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ) and is associated with decreased survival in this population (metras et al. 1999; danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ). the largest multicenter study from the international pediatric lung transplant collaborative identified cmv donor seropositivity, a2 rejection, and transplant in the earliest era of transplantation (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) as increased risks for cmv. progression from cmv infection to disease occurred in 22% (danziger-isakov et al. 2009 ). interestingly, cmv developed in 7% of cmv dà/rà recipients and induction therapy increased the risk for cmv in this group (danziger-isakov et al. 2009 ). the optimal preventative regimen against cmv remains uncertain in pediatric lung transplant recipients. controversies include choices around the use of universal prophylaxis, risk-based prophylaxis, or pre-emptive therapy and duration of prevention strategy (danziger-isakov et al. 2003a) . as the merits and potential disadvantages in the limited population of pediatric transplant recipients are difficult to discern, extrapolation from the adult lung transplant population directs current practice (kotton et al. 2013) . data from adult lung transplantation indicates that prolonged prophylaxis (9-12 months) with valganciclovir has both short-and long-term benefits preventing cmv events and decreasing risk for bronchiolitis obliterans syndrome (finlen et al. 2011; mitsani et al. 2010; palmer et al. 2010 ). pre-emptive therapy is not currently recommended for high-risk cmv d+/rà lung transplant recipients (kotton et al. 2013; . antiviral complications including nephrotoxicity, bone marrow suppression, gastrointestinal manifestations, and the development of viral resistance mutations must be considered when developing prevention strategies (mitsani et al. 2010; danziger-isakov and mark baillie 2009) . a study in pediatric transplantation showed safety and efficacy of an oral valganciclovir dosing algorithm, but no pediatric lung transplant recipients were enrolled (vaudry et al. 2009 ). pediatric studies have assessed long-term intravenous ganciclovir and the adjunctive use of cmv hyperimmune globulin (cmvig) (spivey et al. 2007; ranganathan et al. 2009 ). in a study of nine pediatric lung transplant recipients, 12 weeks of intravenous ganciclovir was feasible, safe, and effective prevention, although cases of catheter-related bloodstream infections did occur when the catheters remained in place beyond the 12-week ganciclovir administration period (spivey et al. 2007 ). cmvig administration as part of a prevention regimen was associated with a threefold decrease in cmv infection but did not impact the incidence of cmv disease or other posttransplant morbidities and mortality in a multinational retrospective study (ranganathan et al. 2009 ). each institution should assure that a consistent prevention strategy and adequate monitoring are in place (kotton et al. 2013 ). cmv monitoring is an integral part of any prevention strategy and potentially allows identification of cmv infection prior to the development of cmv symptoms or end-organ disease. viral culture or a pp65 antigenemia assay has been replaced by more sensitive polymerase chain reaction (pcr) (weinberg et al. 2000) . as interassay and intercenter variability has been reported for pcr testing even in controlled research settings; utilization of a consistent assay is crucial so that results can be compared for an individual subject over time (pang et al. 2009; rychert et al. 2014 ). based on multicenter retrospective evaluation of pediatric lung transplant recipients (danziger-isakov et al. 2009 ), the highest risk period for cmv infection occurs during the first 6 weeks after discontinuation of prophylaxis; thus, appropriate monitoring should occur during this high-risk period. additionally, evaluation for cmv should occur with new onset symptoms suspicious for cmv infection including fever, fatigue, and lymphadenopathy even in cmv dà/rà patients. increased frequency of cmv surveillance is suggested during periods of increased immunosuppression, but not limited to cytolytic therapy for refractory rejection, plasmapheresis, or prolonged lymphopenia (kotton et al. 2013) . additional monitoring for cmv-specific immunity continues to develop (westall et al. 2008; kumar et al. 2009; snyder et al. 2011; manuel et al. 2013b ) and may be employed in the future to personalize cmv prevention strategies. treatment of cmv infection and disease relies primarily on antiviral therapy and, if possible, decreasing immunosuppression. a multicenter randomized clinical trial of predominantly adult kidney transplant recipients reported noninferiority of oral valganciclovir compared to intravenous ganciclovir for nonlife-threatening cmv disease; however, no pediatric patients were enrolled (asberg et al. 2007 ). current recommendations from the transplant society consensus statement include the use of intravenous ganciclovir for pediatric-aged patients as first-line therapy with acknowledgement that some experts would use oral valganciclovir for cmv infection (kotton et al. 2013) . oral ganciclovir, acyclovir, famciclovir, or valacyclovir should not be used to treat cmv. adjunctive therapy with immunoglobulins for severe pneumonitis (either intravenous immunoglobulin or cmvig) can be considered (kotton et al. 2013) . resistant cmv is rarely reported in pediatric transplant recipients (martin et al. 2010; kim et al. 2012 ), but concern may prompt consideration for alternative antiviral therapy including high-dose ganciclovir, foscarnet, and cidofovir (kotton et al. 2013) . newer antiviral agents are under investigation as options for either prevention or treatment including brincidofovir, letermovir, and maribavir. emerging data on the adoptive transfer of cmv-specific t-cells and the use of small-molecule drugs such as sirolimus, leflunomide, and artesunate may alter the future of treatment, but currently no data related to these interventions exist for pediatric lung transplant recipients. human herpes virus 6 and 7 epidemiology and risk human herpes virus (hhv) 6 and 7 are ubiquitous, common viruses that cause mild infections in young children so frequently that by 5 years of age, practically all children have been infected. there are two types of hhv-6, and although the epidemiology of hhv-6a is not clearly defined, hhv-6b is the most common cause of pediatric infections. young infants, especially those under 2 years of age, are at risk for community-acquired or nosocomial hhv-6 infection after solid organ transplantation, while infection may also be acquired through the allograft or as a reactivation of a prior infection in older children. overall, symptomatic or significant infection with hhv-6 after lung transplantation is uncommon, and the overall incidence in solid organ transplant recipients has been reported to be less than 1% . hhv-7 infection seems to be common, but its clinical manifestations are less well characterized. the most typical disease manifestation of hhv-6 infection is roseola infantum (also known as exanthem subitum or sixth disease), a classic febrile illness in young children where the resolution of a high fever of short (3-5 days) duration is followed by the appearance of a characteristic erythematous rash. while young children may present with roseola after lung transplantation, the most likely clinical manifestation in these patients may be a nonspecific febrile illness that may or may not be associated with an erythematous diffuse rash, hepatitis, pneumonia, encephalitis, and leukopenia. hhv-7 coinfection with hhv-6 is reported frequently, and hhv-7 infection alone appears to be asymptomatic or associated with milder clinical manifestations. diagnosis hhv-6/7 infection is confirmed by detection of the virus in otherwise sterile samples (blood, csf, tissue) by nucleic acid identification (pcr) or consistent histopathologic changes. quantification of viral load might be helpful to assess the progression of viremia. however, there is no known clinically relevant viral load threshold to predict progression or severity of disease. immunohistochemical staining is available in some laboratories and might be helpful to determine the presence of infection in specific organs. however, hhv-6/7 latency in human cells may result in the identification of these viruses in samples without correlation with infection. the first step in the treatment of suspected or confirmed hhv-6/7 infection in immunocompromised solid organ transplant patients is decreasing immunosuppression to allow the host's immune system to control the virus. there are no specific antivirals recommended for treatment of hhv-6/7. however, antiviral activity has been described with ganciclovir and its oral form valganciclovir, foscarnet, and cidofovir. consultation with an infectious diseases expert for the antiviral management of these infections is recommended. there are no vaccines available for the prevention of hhv-6/7 infections. suppression may be observed with antivirals used after transplantation for cmv, such as ganciclovir and valganciclovir; however, specific antiviral prophylaxis for hhv-6/7 is not recommended. hand hygiene is the most effective method to prevent transmission. hhv-8, known as the cause of kaposi's sarcoma, is an oncogenic virus associated with a variety of malignancies (primary effusion lymphoma and castleman disease) and other disease syndromes such as febrile illness, bone marrow suppression, hemophagocytosis, and multiorgan failure in highly immunocompromised patients, including transplant recipients . however, the incidence of hhv-8 infection and disease in children is very rare in the united states. residence in hhv-8 endemic areas is a risk factor, as is receipt of an organ from a donor coming from an endemic area. hhv-8 serology is not routinely obtained in solid organ recipients or donors. as with other human herpesviruses, latency can be established. decreasing immunosuppression is recommended, while treatment of associated malignant disease may also include surgical debulking, cytotoxic chemotherapies, and antivirals (for which the efficacy has not been established). adenoviruses commonly cause self-limited respiratory and gastrointestinal infections in immunocompetent children, but infections can be severe in lung and other solid organ transplant recipients. adenovirus infections are more common in pediatric than in adult transplant recipients. primary adenovirus infections may be acquired after transplantation in young children, while reactivation of latent infection or infection from the transplanted organ may occur in older children and adolescents (florescu et al. 2013) . lung transplant patients are at particularly high risk for complicated respiratory tract infection though inhalation of infected aerosol particles or direct contact transmission from infected individuals. gastrointestinal infection may occur via fecal-oral transmission. most infections occur in the first few months after transplantation, or during periods of enhanced immunosuppression. nosocomial and community exposures may be the source of infection. clinical manifestations of adenovirus depend on the organ affected. adenovirus infection can result in severe respiratory disease in lung transplant recipients, including rapidly progressive, necrotizing and potentially fatal pneumonia, as well as development of chronic lung disease with fibrosis and bronchiectasis (liu et al. 2010) . adenovirus may also cause gastroenteritis, hepatitis, meningoencephalitis, and disseminated disease with multiorgan involvement (florescu et al. 2013) . asymptomatic infection, defined as the identification of adenovirus in clinical specimens by nucleic acid detection (pcr) or culture, has been reported more commonly in adults. in children, persistent and rising viremia should be considered a concerning sign of end organ infection and risk for disseminated disease. graft failure may result from acute adenovirus infection after lung transplantation (doan et al. 2007 ). the diagnosis of adenovirus infection requires the presence of consistent clinical symptoms and the identification of adenovirus by viral culture, molecular methods, direct antigen detection, or characteristic histopathology. most adenovirus serotypes (with the exception of adenovirus 40 and 41 which cause gastroenteritis) can be isolated in cell culture; however, diagnosis by pcr is more commonly used and available. the sensitivity of rapid antigen detection tests is variable and not reliable in immunocompromised hosts. while adenovirus can be identified in respiratory secretions, stool, and urine, these are places where prolonged shedding after infection may occur. therefore, the diagnosis of acute infection is more reliable when viral identification is associated with consistent clinical symptoms, or when adenovirus is found in otherwise sterile specimens such as blood and cerebrospinal fluid or in tissues. rising viremia and detection of virus in two or more sites is considered consistent with invasive adenovirus disease. a viral load cutoff or threshold does not exist to predict the progression of disease or its outcome. however, higher and/or persistent viral loads are concerning for progressive or disseminated disease and typically indicate the need to intervene. decreasing the level of immunosuppression to allow for the host's immune response to handle the virus is the most important treatment strategy to manage adenovirus infections in young solid organ transplant patients. in certain cases, antiviral treatment may be useful, if instituted with the guidance and follow-up of a pediatric infectious diseases specialist. while there are no approved adenovirus-specific antivirals, some agents such as cidofovir have activity against adenovirus and have been used empirically for treatment. however, use of this agent is limited by its propensity to cause nephrotoxicity and bone marrow suppression. close follow up and monitoring for these side effects is recommended. the standard dose of cidofovir in children is 5 mg/kg once weekly. however, more frequent, lower dosage (1 mg/kg three times per week) and pre-and post-dose hydration have been used in an attempt to reduce the risk of renal toxicity (doan et al. 2007) . treatment is usually continued until resolution of viremia and/or symptoms, with close monitoring for side effects. other antivirals have been evaluated for treatment of adenovirus, including a lipid conjugate of cidofovir (cmx001, chimerix inc.), which is administered orally and has a lower risk for nephrotoxicity; however, its use remains experimental at this time. lung transplant patients with severe infection may have hypogammaglobulinemia, and in these cases, administration of intravenous immunoglobulin (ivig) for replacement has been used, although its benefit has not been proven (mawhorter and yamani 2008 ). an effective novel treatment strategy has been developed with the use of antigen-specific cytotoxic t lymphocytes (ctl) directed against adenovirus in hematopoietic stem cell transplant recipients; ctls have not been evaluated in lung or other pediatric solid organ transplant recipients (leen et al. 2009 ). prolonged shedding after resolution of the acute infection may occur; therefore, strict hand hygiene and disease prevention strategies need to be implemented. there are no licensed vaccines for the prevention of adenoviruses. epidemiology and risk pediatric solid organ transplant recipients and particularly lung transplant recipients are at increased risk of medical complications and mortality when acquiring common respiratory viral infections (manuel et al. 2013a) . common respiratory viruses that circulate with well-described seasonality in the united states include influenza virus, respiratory syncytial virus (rsv), human metapneumovirus, human rhinovirus, parainfluenza viruses, coronaviruses, and other respiratory viruses that are being more frequently described, such as bocaviruses. lung transplant recipients are at risk for community and nosocomial exposures during the typical time of circulation of these viruses. infection with respiratory viruses may also increase the risk for secondary bacterial pneumonia and other bacterial or fungal infections, particularly in the first few months after transplant (liu et al. 2009a) . after an acute lower respiratory virus infection, the risk for graft rejection or chronic allograft dysfunction may increase as shown in adult lung transplant recipients; however, this is controversial and has not been shown in pediatric lung transplant recipients to date (liu et al. 2010; liu et al. 2009a; vu et al. 2011) . although upper respiratory infections may present similarly in solid organ transplant recipients as in immunocompetent hosts, progression to lower respiratory tract disease manifestations with tachypnea, cough, abnormal breath sounds, hypoxemia, and respiratory failure is a concern in lung transplant recipients. clinical deterioration due to respiratory viruses is more frequently reported in the period of highest immune suppression shortly after transplant. prompt diagnosis with viral detection using nucleic acid amplification methods (pcr) is recommended in immunocompromised hosts. viral cultures could be obtained but are not as useful given that results are delayed in comparison with pcr. pcr platforms that test for multiple viruses at the same time are most helpful in lung transplant recipients. rapid antigen detection tests are no longer recommended for influenza due to their variable sensitivity; but they could still be useful for the diagnosis of rsv. respiratory secretions including nasal wash or swabs, naropharyngeal aspirates, and tracheal or broncheoalveolar lavage can be used. these viruses do not tend to be associated with viremia. supportive measures must be instituted promptly in lung transplant recipients with progression to lower respiratory tract disease. the need for invasive mechanical ventilation or other higher level of supporting measures such as extracorporeal membrane oxygenation (ecmo) is not uncommon in patients with severe disease. specific antiviral treatment is available for influenza a and b infection. immediate initiation of neuraminidase inhibitor (oseltamivir and zanamivir) therapy in lung transplant patients with fever and/or other respiratory symptoms during the period of influenza circulation may decrease the risk of complications and death associated with influenza. although influenza antivirals are usually preferred within 48 h of the onset of clinical symptoms, lung and other solid organ transplant patients have improved outcomes even with later treatment initiation (kumar et al. 2010 ). in some cases, a more prolonged duration of antiviral therapy has been used given these patient's immune-suppressed status and prolonged viral shedding. intravenous peramivir (also a neuraminidase inhibitor) is now licensed for adults, with clinical studies underway in children and adolescents. intravenous administration might be preferred in patients who have inadequate enteral absorption and who are severely ill with influenza. ribavirin, an aerosolized antiviral with in vitro activity against rsv, parainfluenza, human metapneumovirus, and other viruses, is fda approved but not routinely recommended for treatment of these infections due to lack of definitive efficacy. however, ribavirin has been used early in the course of rsv and other respiratory virus infections, as well as in more severe cases of respiratory disease, in lung transplant patients due to its antiviral effects. no randomized controlled trials have been performed although data from adult lung transplantation has indicated a potential response to aerosolized, intravenous, and oral ribavirin (glanville et al. 2005; pelaez et al. 2009; li et al. 2012 ). an inhaled small-interfering rna that targets rna (aln-rsv-001) has also been investigated as a therapy for rsv in adult lung transplantation showing potential reduction in bronchiolitis obliterans syndrome after rsv infection (zamora et al. 2011; gottlieb et al. 2016) . utilization of an rsv antibody preparation (monoclonal antibody) along with antiviral treatment in severe cases has been reported to reduce rsv-associated mortality in some cases (chavez-bueno et al. 2007 ). similar to the management of other viral infections, decreasing immune suppression is advisable when respiratory viral infections are identified. influenza immunization prior to and/or after transplantation for the recipient and all close contacts and family members is recommended to prevent infection and severe disease. inactivated influenza vaccine should be administered ideally prior to the start of the season, to ensure optimal protection. however, after transplant, and in some patients prior to transplant depending on their underlying diagnosis or need for chronic steroid or other medication use, the immune responses to vaccination might be suboptimal in lung and other solid organ transplant recipients. therefore, vaccination of close contacts and avoidance of contact with sick individuals become important measures for prevention of infection (avery et al. 2013) . prophylactic antivirals may also help decrease the risk of infection and complications in exposed unvaccinated or unprotected transplant recipients. there are no other vaccines available for the prevention of respiratory infection in most pediatric lung transplant recipients. however, palivizumab, a monoclonal antibody against rsv, can be used during the rsv season in young children less than 2 years of age who are lung transplant recipients, immunosuppressed, or who have underlying chronic lung or hemodynamically unstable heart disease (american academy of pediatrics committee on infectious diseases and american academy of pediatrics bronchiolitis guidelines committee 2014). all lung and solid organ transplant patients with suspected or known respiratory viral infections need to be isolated from other patients using standard contact and droplet precautions. posttransplant, infections remain a significant factor causing both morbidity and mortality in pediatric lung transplant recipients. pathogens are diverse including bacteria, fungi, and viruses with timing of events dependent on time from transplant. all events can have both immediate and long-term consequences in this at-risk population. prevention, identification, and early intervention for infectious events can improve outcomes after pediatric lung transplantation. lung infections in pediatric lung transplantation: experience in 49 cases cytomegalovirus disease among donor-positive/recipient-negative lung transplant recipients in the era of valganciclovir prophylaxis nebulized amphotericin b prophylaxis for aspergillus infection in lung transplantation: study of risk factors epidemiology and outcome of invasive fungal infections in solid organ transplant recipients epidemiology, risk factors, and outcomes of clostridium difficile infection in kidney transplant recipients antifungal prophylaxis in lung transplantation-a world-wide survey international guidelines for the selection of lung transplant candidates: 2006 update -a consensus report from the pulmonary scientific council of the international society for heart and lung lransplantation extended valganciclovir prophylaxis to prevent cytomegalovirus after lung transplantation: a randomized, controlled trial interlaboratory comparison of cytomegalovirus viral load assays clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america early and late infections in lung transplantation patients diagnosis of invasive aspergillosis in lung transplant recipients by detection of galactomannan in the bronchoalveolar lavage fluid infections in solid-organ transplant recipients practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america 10 years of prophylaxis with nebulized liposomal amphotericin b and the changing epidemiology of aspergillus spp. infection in lung transplantation efficacy of oral ribavirin in lung transplant patients with respiratory syncytial virus lower respiratory tract infection nontuberculous mycobacterial disease is not a contraindication to lung transplantation in patients with cystic fibrosis: a retrospective analysis in a danish patient population cytomegalovirus immunoglobulin decreases the risk of cytomegalovirus infection but not disease after pediatric lung transplantation human herpesviruses 6, 7 and 8 in solid organ transplant recipients cytomegalovirus in solid organ transplantation hypogammaglobulinemia: incidence, risk factors, and outcomes following pediatric lung transplantation combined cmv prophylaxis improves outcome and reduces the risk for bronchiolitis obliterans syndrome (bos) after lung transplantation multicenter comparison of laboratory performance in cytomegalovirus and epstein-barr virus viral load testing using international standards staphylococcus aureus infections in the early period after lung transplantation: epidemiology, risk factors, and outcomes impact of multidrug-resistant organisms on patients considered for lung transplantation mycobacterium abscessus complexa particular challenge in the setting of lung transplantation polyfunctional cytomegalovirusspecific immunity in lung transplant recipients receiving valganciclovir prophylaxis epidemiology and management of infections after lung transplantation safety and efficacy of prolonged cytomegalovirus prophylaxis with intravenous ganciclovir in pediatric and young adult lung transplant recipients effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome multidrug-resistant gram-negative bacteria infections in solid organ transplantation valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients (2013) (6) key: cord-010078-8lkkez3n authors: nan title: invited speakers date: 2010-11-24 journal: respirology doi: 10.1111/j.1440-1843.2010.01863.x sha: doc_id: 10078 cord_uid: 8lkkez3n nan the physiology of respiration reaches its extreme limits when man is exposed to the effects of high altitude, which is basically a decrease in ambient pressure and temperature. the process of acclimatization of humans to high altitude occurs within minutes of ascent, starting with increases in cardiac output, and ventilation. these result in acute changes in pao 2 and paco 2 . slower changes of acclimatization include increase in hemoglobin concentration (polycythemia), and muscle capillarity, but decrease in mitochondrial volume density and cellular aerobic capacity. through acclimatization, animals and human inhabitants of high altitude areas have developed adaptations that allow them to function as near normal as those living at sea level. humans who undergo acute ascent to high altitude could acclimatize, but some fail to do so. if there is failure to acclimatize, oxygen diffusion impairment results, due to decreased partial pressure, and lower affi nity of hb for oxygen. in addition, there is signifi cant increase in ventilation/perfusion heterogeneity. the resulting hypoxia leads to hypoxic pulmonary vasoconstiction (hpv) which results from multiple components: changes in epithelial cell wall that lead to intracellular calcium increase and/or calcium sensitization. subsequently, pulmonary hypertension develops, with actual breaks in the capillary endothelium leading to an infl ammatory process (seen more during exercise), and decrease in alveolar fl uid clearance. these mechanisms contribute to the development of alveolar edema, high altitude pulnary edema (hape), which is one of the two major diseases due to acute mountain sickness (ams). both hape and the other ams, high altitude cerebral edema (hace) could be potentially fatal, and must be recognized and treated early. knowledge of the pathophysiology of the ams would allow more rational approaches to their prevention and treatment. high altitude illnesses can develop among healthy individuals who sojourn for recreation and work: cerebral form called acute mountain sickness (ams) and potentially fatal pulmonary form called high altitude pulmonary edema (hape). ams is generally self-limited. high-altitude cerebral edema (hace) is likely a continuum of ams and the end-stage of ams. ams is not a precondition for the development of hape. hape develops in non-acclimatized mountaineers after rapid ascent to altitudes above 2500 m. hape may develop even in the absence of ams. severe ams may be a risk factor for hape. the altitude, the rate of ascent to new altitude, (>300 m/day to an altitude above 4000 m), and individual susceptibility are major determinants of ams and hape. on ascent to high altitudes all people have swelling of the brain. patient with ams often experience "hangover headache." other symptoms occur within the fi rst 6 to 36 h. these include malaise, anorexia, nausea and vomiting, and insomnia. ams patient must be evaluated for signs of global encephalopathy rather than focal fi ndings, although retinal hemorrhage is commonly seen. when these are present, the subject has hace until proven otherwise. patient may die if not treated promptly. brain herniation is the usual cause of death. the hallmark of hape is an excessively elevated pap which precedes the development of pulmonary edema. symptoms are incapacitating fatigue, chest tightness, dyspnea with effort, orthopnea, cough, and pink frothy sputum in advanced stage of disease. prevention of all altitude complications requires ascending at an increment rate to allow acclimatization. at >3000 m, one should not spend subsequent nights 300 m higher than the previous night. trekker must take a rest day every 2 to 3 days. anyone with ams should not ascend until symptoms are resolved. acetazolamide and dexamethazone are effective in prevention and treatment of ams from proceeding to hape or hace. at the fi rst sign of hace, patients should descend to a lower altitude while supplementary oxygen is given. increasing oxygenation is the highest priority in the treatment of and prevention of hape. if supplemental oxygen is unavailable, then descent, use portable hyperbaric chamber, or both become lifesaving. nifedipine is necessary only when supplemental oxygen is unavailable or descent is impossible. because of its pulmonary vasodilatory effects, phosphodiesterase inhibitors can be used for prevention and treatment of hape. rudolf virchow in 1856 described that the 3 determinant risk factors for venous blood clot formation are stasis, endothelial injury and hypercoagulable state. dr simpson et al 1940, a british surgeon observed that during the london blitz, the world war ii, britons who were forced to remain on sitting in cramped position and deck chairs for hours during the air raids developed fatal pulmonary embolism. it was suggested in 1954 by homans that "prolonged dependency stasis" or immobilization is one factor that predisposes patients to develop thrombosis in the deep veins of the legs. several risk factors have been identifi ed for developing venous thromboembolism (vte). travel is one of the transient risk factors. it is not confi ned to one mode of travel such as air travel. it is also incriminated to other modes of land travel such as car, bus and train. the term "economy class syndrome" was proposed by symington and stock (1966) and by the group of cruickshank (1988) for venous thromboembolism occurring in patients during air fl ight travel. it is usually seen in patients sitting in limited or cramped circumstances in the economy coach or tourist class seats, however it is also found out that patients who were also seated in the business class also develop this syndrome. factors implicated were the long duration of travel, immobilization or inactivity in sitting position, and the low cabin pressure, low humidity and dehydration during air fl ights. in several studies performed on large airports in europe, the presence of genetic factors such as factor v leiden and environmental factors such as the use of oral contraceptives predispose patients several fold to develop venous thromboembolism. signs and symptoms pertaining to vte develop not only during and after the fl ight but also several weeks after the travel. nowadays, airlines as well as bus companies have advisories and measures impose to prevent development of vte and deaths due to vte during the travel period. with the steadily increasing use of air travel, more and more patients with pulmonary disease are fl ying long distances, at high altitude in partially pressurised aircraft. this is associated with long periods of reduced mobility and exposure to reduced inspired pressures of oxygen and reduced barometric pressure. some individuals therefore may be at risk of barotrauma, hypoxia or venous thrombo-embolism (vte). therefore it is important to identify these individuals and adequately assess the real risk entailed by fl ying. the effect of reduced atmospheric pressure is a potential risk for patients with recent or pre-existing pneumothorax but otherwise is unlikely to be associated with risk other than that due to the associated reduced inspired oxygen fraction (fio 2 ), typically 0.15 (15%) in a commercial aircraft at cruising altitude. the reduced (fio 2 ) may be problematic for patients with hypoxic lung disease or in patients with other co-morbidities that may exacerbated by hypoxia. medical history, lung function and resting oxygen saturation will help identify patients at risk although it is diffi cult to predict the clinical effects of altitude from tests (even hypoxic challenge tests) conducted at sea level. there is currently a lack of good data defi ning the clinical outcomes due to hypoxia during fl ight in patients with lung or other diseases. the risk of vte increases with duration of fl ight above four hours, presumably related to the duration of immobility, although the role of prolonged hypoxia remains to be determined. preventive measures are now currently invoked on most airlines and guidelines for the use of antithrombotic agents are available, stratifi ed by risk. it is anticipated that guidelines will continue to be updated as new data are made available. cardio-pulmonary exercise testing is now well accepted as an appropriate test for the investigation of shortness of breath on exertion. in addition the test has been found to be useful for the assessment of pulmonary vascular dysfunction and the assessment of fi tness for major thoracic surgery. even though there are well described and internationally accepted protocols to perform the test, the interpretation of a cardio-pulmonary exercise test often leaves the interpreting physician confused. importantly with the multiple facets of the test (respiratory, cardiac, peripheral vascular) that need to be interpreted it is easy for the interpreting physician to look at a certain aspect of the test relating to their specialty and to give the other facets relatively little attention. in this presentation we review the process of interpreting cardio-pulmonary exercise tests. in addition we will interpret a number of tests based on our previous discussion on how to interpret these tests. finally we will review the literature regarding new interpretive strategies for exercise induced pulmonary vascular disease. bronchoscopy as an image-guided intervention has benefi tted from advances in optical and non-optical imaging technologies. some current bronchoscopy advances incorporate higher resolution ccd (charged-couple device) digital-"chip" technology and magnifi cation lenses to enhance the image resolution. the hope is that improved visualization combined with analysis of concomitant tissue biopsies may realize so-called "in-vivo endoscopic diagnosis" without the need for tissue biopsies, however studies of highmagnifi cation endo-cytoscopy, co-focal micro-endoscopy and optical coherence tomography (oct) remain investigational. further limiting these near-histologic resolution imaging modalities is the need for an initial screening of "highly suspicious" mucosa to focus attention upon. to facilitate identifi cation of abnormal airway mucosa, there are advances in the bronchoscopic detection of dysplastic and malignant mucosa. newer generations of autofl uorescent (af) bronchoscopes combine video ccd technology with af signaling to enhance the visual resolution of the images. non-af technologies being evaluated for the same purpose include fi ltered-light narrow band imaging (nbi) and post image-capture processing by a number of other spectral estimation technologies (set). bronchoscopic image guided interventions (bigi) also benefi tted from advances in non-traditional bronchoscopy technologies. foremost has been endobronchial ultrasound (ebus), initially designed as radial probes modeled after intravascular us and modifi ed for the airways. while useful in advancing our understanding of endobronchial mucosal structure, predicting tumor invasion depth and responsiveness to endobronchial interventions, radial ebus did not permit real-time guidance. dedicated linear-array ebus bronchoscope has changed this dramatically as the 7.5 mhz needle-puncture ebus bronchoscope has increased diagnostic accuracy of peri-bronchial lymph nodes/masses from a previous average of <50% to >90% for even small targets (<1 cm) in experienced hands. simultaneously miniaturization of radial ebus probes (thin 1.4 mm) and incorporation of guide-sheaths have increased the utility of ebus in evaluating parenchymal lung pathology. concomitant work in image processing of radiology imaging data (dicom data of chest ct images) has made available a number of "virtual bronchoscopic navigation" programs to assist the bronchoscopists in navigating towards smaller peripheral focal targets, and to improve the historic diagnostic yield of smaller (<2 cm) peripheral nodules from 20-30% up to 65-80%. these systems include passive endobronchial "road-maps" view (similar to "mapquest"/"google earth") and more technology enhanced electromagnetic navigation bronchoscopy (enb) (similar to gps guidance). all these ancillary technologies have spurred improvements in the basic bronchoscope, as thinner bronchoscopes capable of reaching peripheral segments (2.8 mm and 3.5 mm with 1.2 mm working channel; 4.0 mm with 2.0 mm working channel) are coupled with new biopsy instruments. the eventual development of steerable single fi ber scanning endoscopes with multi-wavelength imaging may change our current concepts of the bronchoscopes and how we can use them. journal compilation © 2010 asian pacifi c society of respirology pg 03-02 diagnostic tests pleural effusions are common and often present diagnostic challenges. the new british thoracic society guidelines 2010 on investigation of pleural effusions detailed some of the new approaches to undiagnosed pleural effusions. traditional teaching recommends measurement of blood and pleural fl uid protein and ldh levels as the fi rst step of investigation to categorize the effusion into a 'transudate' and 'exudate' using light's criteria. the need to apply this to all effusions is questionable in 2010. current efforts focus on the development of disease-specifi c diagnostic tools incorporating clinical, radiologic and biochemical parameters. • elevated ntpro-bnp levels in pleural fl uids are useful in confi rming cardiac failure as the etiology of a pleural effusion, especially in patients whose fl uid may be falsely elevated into the 'exudative' range by concurrent diuretic therapy. • pleural ntpro-bnp levels are elevated in cardiac failure effusions, but not in other transudative effusions (eg hepatic hydrothorax). • pleural fl uid ntpro-bnp appears a better marker than pleural fl uid bnp. variations in accuracy may also in part depend on the commercial kits used. adenosine deaminase: • ada measurements in pleural fl uids are useful in the diagnosis of tb pleural effusions with a sensitivity and specifi city of 92 and 90% respectively. limiting the test to lymphocytic pleural effusions will further improve the diagnostic accuracy. • ada is cheap and fast to perform and is now widely used in endemic countries. false positives can occur with bacterial infections, rheumatologic effusions, and occasionally malignant effusions. false negatives are uncommon, and therefore present a valuable 'rule-out' test in regions of low tb rates. • ada is at least as diagnostically useful as pleural fl uid total interferon-gamma levels. • igras have been tested in pleural fl uid and blood of patients with tb pleural effusions in several studies. the diagnostic sensitivity and specifi city are poor and igras are not recommended for the investigation of tb pleuritis. • serum mesothelin is a fda-approved test for the diagnosis and monitoring of mesothelioma. • pleural fl uid mesothelin adds information to pleural fl uid cytology in the diagnosis of mesothelioma, providing a diagnostic sensitivity of 71% (specifi city 90%). elevated pleural fl uid mesothelin levels suggest epithelioid or biphasic mesothelioma or occasionally metastatic carcinomas. procalcitonin: • early evidence suggest that serum level of procalcitonin may aid differentiation of pleural infection from pleural effusions of non-infective etiologies. the value of pleural fl uid procalcitonin level is limited. management strategies imaging guidance for pleural procedures: • pleural procedural complications are often under-estimated and underreported. studies have now shown that mandatory imaging guidance (especially bedside pleural ultrasound), and restricting procedural privilege to certifi ed trained clinicians can signifi cantly reduce complication rates from pleural procedures. this practice is now incorporated into many national and professional society guidelines. intrapleural therapy for pleural infection/empyema: • recent clinical trials on intrapleural delivery of fi brinolytics alone have failed to improve important clinical outcomes of pleural infection. however, the combination of tissue plasminogen activator and dnase has shown promising results. • recent studies have revealed increasing concerns of complications of talc pleurodesis, and randomized studies have shown a much lower success rate than previous non-randomized literature, even in selected patients. the concept of drainage without needing to create pleurodesis has growing appeal and the use of indwelling pleural catheters is now regarded as fi rst-line therapy in increasing number of centers. chronic respiratory disease (crd) is non-communicable respiratory disease including asthma, chronic obstructive pulmonary disease (copd), allergic rhinitis, idiopathic pulmonary hypertension, hypersensitivity pneumonitis, occupational respiratory disease. among these crd asthma and copd are important for regional health. facts of asthma 300 million people suffer from asthma. 255,000 people died of asthma in 2005. prevalence of asthma has increased or is increasing. asthma is the most common disease among children over 80% of asthma death occurs in low and lower-middle income countries. asthma is underdiagnosed and under-treated (who, 2010). facts of copd copd is a life-threatening lung disease that interferes with normal breathing. it is more than a "smoker's cough". an estimated 210 million people have copd worldwide. more than 3 million people died of copd in 2005, which is equal to 5% of all deaths globally that year. almost 90% of copd deaths occur in low-and middle-income countries. the primary cause of copd is tobacco smoke (through tobacco use or second-hand smoke). the disease now affects men and women almost equally, due in part to increased tobacco use among women in high-income countries. copd is not curable, but treatment can slow the progress of the disease. total deaths from copd are projected to increase by more than 30% in the next 10 years without interventions to cut risks, particularly tobacco smoking (who, 2010). prevention and control of crd in asia pacifi c were held by dokkyo medical university group, later designated as who collaborating centre for prevention and control of crd (du-wcc). seven countries and a district in asia pacifi c joined the meeting. prevalence of asthma in adults was reported from 0.99 to 7.2% with a median of 4.0% based on 13 reports. prevalence of childhood asthma (13-14 y/o) was from 0.8 to 13.2% with median of 7.7% based on 13 reports. prevalence of copd was 3.5 to13.1% with a median of 7.1% based on 9 nation-wide surveys. in spirometry-based survey reported, prevalence of copd was 8.5% in adults 40 years and over in japan, 8% in adults 45 years and over, and 8.2% in adults 40 years and over in china. management in most of the countries gina and gold were adopted for their national guidelines. major risk factors for crd, especially for copd were smoking and indoor air pollution for cooking/heating. pharmacological early interventions have been reported to improve the prognosis of asthma and copd. occupational respiratory diseases are disorders which are induced by occupational and industrial conditions. providing information of the risks of industrial activities would reduce this disorder. strategic direction for the prevention and control of crd most of crd are treatable and at least partially preventable. development of user-friendly guides for prevention and control of crd for offi cials in health care, fi rst-line health-care givers and patients and their family and its implementation would decrease the burden of these crd. the scientifi c foundation of asthma diagnosis and management has grown in leaps and bounds. evidence-based strategies to control asthma and treat its exacerbation are published yearly in the gina guidelines. the 10-year finland study showed that these strategies work. while cases treated did increase (through better detection), the hospital days and cost per case markedly decreased. the study also showed that widespread adaption and effective implementation of these strategies is best done through a national program. cmes for medical practitioners are important but are of limited reach. all stakeholders must be enlisted to buy-in. for asthma, the target stakeholders are the health care personnel, nurses and village health volunteers included; the patients and their families; the government and its public health offi cials; the asthma advocacy groups; the community-at-large; and the pharmaceutical industry. the idea is to present the problem to them, include their inputs in the formulation of the plan, collegially decide on target indicators of success and engage them to work for the implementation of the program in the context of what each one can do best. duplicating the finnish experience is a big challenge in the asia pacifi c region. while most countries have their own adaptation of the gina guidelines, few have working national asthma programs. in developing economies, the health infrastructure is not that well developed yet to absorb all guideline recommendations. spirometry may not be widely available nor affordable. government spending for health is commonly below the 5% of gdp level that who recommends. in the philippines, signifi cant out-of-pocket health expense is borne by the patient. furthermore, programs like tb control, dengue treatment and malaria eradication, which are no longer concerns in developed countries, compete for the meager public health funds. for low income countries, the international recommendations may have to be rewritten to emphasize on simple algorithm for separating non-infectious from infectious respiratory illnesses; practical objective measurements for diagnosis and management such as peak fl ow; available, affordable, and low-risk medications recommended for asthma control; and a simple regimen for recognizing severe asthma (gina). to be viable, the national asthma program will have to piggy back to the existing national health delivery infrastructure which must ensure, among others, access to free or cheap medication. lung cancer and copd commonly coexist in smokers, and the presence of copd increases the risk of developing lung cancer. in addition to smoking cessation and preventing smoking initiation, understanding shared mechanisms in these smoking-related lung diseases is critical, to develop new methods of prevention, diagnosis and treatment of lung cancer and copd. common mechanisms may involve infl ammation, abnormal repair, oxidative stress, epithelial-mesenchymal transition, altered nicotine receptor biology and epigenetic alterations. strategies to study genomics and epigenomics, in addition to gene-environment interaction, will yield greater insight into the shared pathogenesis of lung cancer and copd. copd clinical guidelines are important to guide diagnosis and management of people with copd. the australian 'copdx' guidelines are evidence-based guidelines that are prepared by the australian lung foundation and thoracic society of australia and new zealand. relevant literature is searched regularly and evaluated by a clinical committee. updates are then produced regularly during the year. challenges regarding critical appraisal, resources and dissemination to clinicians will be discussed. national copd guidelines in this region are different from country to country, but basically are adapted from gold. copd prevalence in 12 asia pacifi c countries and region estimated by regional copd working group was 6.3%. vietnam has the highest prevalence: 6.7%. the copd management and guideline implementation problems in the asia pacifi c region are: smoking, biomass using are common; continuous medical education (cme) for health workers are not compulsory; lack of device and personnel for performing proper spirometric tests; over burden for health workers; low access to medical care and low affordability for copd medications. all of these problems result in that copd diagnosis are mostly in late stage, high rate of emergency room visit, icu admission and hospitalization. the consensus is expected to cover following resolutions: reducing the smoking and biomass smoke exposure, screening for copd in large scale using questionnaires and confi rming by spirometry, advocacy for compulsory cme on copd, establishing asthma and copd outpatient care unit (acocu) in different levels of health care settles and introducing copd medications into insurance medication list. infections caused by environmental mycobacteria are more common than tuberculosis in many parts of the world. the more than 120 species of mycobacteria have similarities, but generally the diseases and hosts fi t in specifi c patterns. disease due to environmental mycobacteria can be diffi cult to diagnose and treat and can confuse workup for tuberculosis. mycobacteria have low virulence and even lower invasiveness. they form biofi lms that protect them and allow long term persistence. the treatment is often frustrating for the patients and physicians. learning their metabolic mechanisms and attacking them should be the strategy for combating the disease caused by these organisms. lung cancer and copd commonly coexist in smokers, and the presence of copd increases the risk of developing lung cancer. in addition to smoking cessation and preventing smoking initiation, understanding shared mechanisms in these smoking-related lung diseases is critical, to develop new methods of prevention, diagnosis and treatment of lung cancer and copd. common mechanisms may involve infl ammation, abnormal repair, oxidative stress, epithelial-mesenchymal transition, altered nicotine receptor biology and epigenetic alterations. strategies to study genomics and epigenomics, in addition to gene-environment interaction, will yield greater insight into the shared pathogenesis of lung cancer and copd. airway epithelial cells, which are the fi rst line of cells to contact with inhaled substances such as microorganisms, play an important role in the host defense by two major mechanisms. first, they actively contribute to the innate immune system by recognition of the pathogen and production of antimicrobial substances and cytokines. second, they provide a passive barrier function that prevents invading microorganisms, air pollutants and airborne allergens into the internal milieu. on the other hands, airway epithelial cells are involved in the production of airway infl ammation in asthma and copd by excessively and un-regulatory expressing pro-infl ammatory and pro-allergic cytokines, executing apoptosis and losing barrier function. there is very close relationship between epithelial barrier function and innate immune response of epithelial cells. for instance, losing barrier function results in not only allowing foreign substance and pathogens to invade into the internal milieu but also enhancing innate immune responses. asthma and copd are different diseases, but they may have the same mechanism in the pathogenesis of exacerbation of these diseases in term of losing epithelial barrier functions. in this symposium, we will present the latest information on and the regulatory mechanism of airway barrier function and discuss in the context with asthma and copd pathogenesis and exacerbations. key words; airway epithelial cells, barrier function, asthma, copd patients with severe and diffi cult-to-treat asthma ("refractory asthma", approximately 5% of total asthma) have impaired health status refl ected by persistent symptoms, severe airfl ow limitation and frequent asthma exacerbations despite taking maximally recommended doses of inhaled corticosteroids and long-acting β 2 -agonists. a better understanding is thus needed regarding factors associated with such troublesome condition and, in our cross-sectional observational study, clinical and demographic characteristics of patients fulfi lling the american thoracic society workshop criteria for refractory asthma (ajrccm, 2000, group a) were compared with those of patients with severe persistent asthma defi ned on the basis of the gina guideline (group b). there were no signifi cant differences between the two groups with respect to age, gender, smoking status, disease duration, pulmonary function (fev 1 , pef, dlco), or markers of airway infl ammation in the induced sputum (eosinophils, neutrophils, ecp, tryptase). however, in contrast to group b, all patients in group a were adult-onset, and 93% of the patients already had severe symptoms at the time of disease onset. prevalence of atopy, postbronchodilator fev 1 /fvc ratio and fev 1 reversibility were signifi cantly less in group a than in group b. patients in group a complained of copious amounts of phlegm associated with chronic sinusitis and/or chronic bronchitis, and showed high concentrations of mucin (muc5ac + muc5b) in the sputum. in addition, nasal clearance time assessed by saccharine test was signifi cantly longer in the group a than in the group b patients, indicating impairment of airway mucociliary clearance. these fi ndings and other pathophysiological and clinical data suggest that "refractory asthma" may be a different form of asthma (phenotype) rather than a progression of asthma severity during follow-up of natural history of the disease. furthermore, it is likely that irreversible airway narrowing possibly due to airway remodeling and airway mucus hypersecretion are important factors contributing to the pathogenesis of severe and diffi cult-to-treat (refractory) asthma. the results prompt for further longitudinal studies and interventions to defi ne the mechanisms of this unique phenotype of asthma. journal compilation © 2010 asian pacifi c society of respirology drug development is a long and expensive process. on average it takes at least 10 years and more than a billion dollars to develop a compound from basic science discovery through clinical trials and fi nal approval by regulatory authorities of a new therapeutic. one of the main obstacles to development of new compounds is the diffi culty in obtaining good pre-clinical proof of effi cacy for a new drug. most of this is currently obtained from experiments using animal models of disease or cell lines, neither of which refl ect well human disease nor predict whether responsiveness in these models predicts responsiveness in human disease. recent studies have focused on developing methods that employ human cells or tissues taken from the relevant organ and from relevant patient populations. of these models, the explant model, which uses whole tissue samples, is the closest to the in vivo situation because it maintains the complex cell-to-cell interactions. in asthma, studies have shown that this model can sometime be even better than in vivo study. thus, for example, the explant model vivo offers several advantages over in vivo allergen challenge of asthmatic volunteers. first, repeat bronchoscopy to sample the airways after initial allergen challenge is not required. second, tissue responses of more severe asthmatics, who for safety reasons cannot be challenged with allergen in vivo, can be studied. third, problems of dilution of secreted mediators during bal are avoided and released mediators are not consumed by in-coming infl ammatory cells, thus increasing the sensitivity of the model. finally, and most importantly when seeking pre-clinical proof of concept of drug effi cacy, the model allows testing of novel compounds at an early stage before its full safety profi le is established, a process that is both expensive and time-consuming. we have previously shown that the asthmatic airways generate increased t cell chemotactic activity compared to healthy controls. using a highly selective ccr4 antagonist we have recently shown that the ccr4-chemokine axis plays a key role at least in the traffi cking of t cells into the asthmatic airways. having established this, we then showed that predominantly the ccr4+ t cells are recruited in response to allergen stimulation. we have further shown that the selective removal of these ccr4+ t cell from blood signifi cantly reduced allergic infl ammation as shown by a marked reduction in the production of the th2 cytokines il-4, il-5 and il-13 but with no consequences for th1 responses. taken together, our studies have strongly suggested that inhibiting the migration of t cell to the asthmatic airway by targeting ccr4 is likely to abrogate the allergic infl ammation in the airways without affecting immune responses that serve to protect against infection. these studies have also shown the value of using such ex vivo models of asthma to provide proof of concept for new drugs, giving the pharmaceutical industry the necessary pre-clinical proof to proceed with confi dence into further clinical development. sleep-disordered breathing (sdb) or obstructive sleep apnea (osa) is a prevalent but largely undiagnosed sleep disorder. apnea-hypopnea index (ahi: the number of apneas and hypopneas per hour of sleep) is used to classify sdb severity. in icsd-2 (international classifi cation of sleep disorders ver. 2), "osa syndrome" was defi ned as ahi ≥ 5 with hypersomnolence/ daytime symptoms or as ahi ≥ 15 regardless of the symptoms. epidemiological studies clarifi ed that sdb is associated with increased likelihood of hypertension, cardiovascular disease, stroke, motor vehicle accidents, depression, diminished quality of life, and even mortality. clinical guidelines for hypertension put weights on sdb as a cause of hypertension. international diabetes federation (idf) made a consensus statement on sleep apnea and type 2 diabetes. "overlap syndrome" (coexist of copd and sdb) was reported to have much higher mortality than sdb alone. sleepiness was thought to be a major symptom for osa syndrome. it is true that there is a signifi cant trend that the severe the sdb is the more the subjects had sleepiness. however, the majority of sdb subjects (even the majority of subjects with ahi ≥ 15) do not have sleepiness (ess: epworth sleepiness scale >10). the berlin sleep questionnaire was used to screen high or low risk subjects for sdb. four-item screening tool was also developed (gender, bmi, blood pressure, snoring frequency). these tools may be useful, when certifi ed with sleep monitoring in each population. prevalence of ahi ≥ 15, estimated from two-stage sampling, was 9-14% in male and 2-7% in female. two-stage sampling is oversampling the subjects with sleepiness or snoring to perform sleep monitoring, and weighting of results to the survey sample. when all the participants underwent sleep monitoring, the prevalence of ahi ≥ 15 was 22-25% in male and 10-12% in female. there is a strong need for better recognition, screening and treatment of sdb. more studies are needed, especially for long-term outcomes of asymptomatic sdb. genome-wide association studies may be useful to elucidate causes or underlying mechanisms of sdb. continuous positive airway pressure (cpap) is a standard treatment for patients with obstructive sleep apnea (osa), especially for moderate to severe osa. the mechanism of action is to provide a pneumatic splint to preserve upper airway. the pressure level required to maintain airway patency is determined by manual pressure titration by a sleep technologist during attended laboratory polysomnography (psg) to eliminate obstructive respiratory-related events (e.g., apneas, hypopneas, respiratory effort-related arousals [rera], and snoring). despite wide acceptance as a standard therapy for treatment of osa patients, very few pap titration protocols have been published so far, and there are inconsistency and variations in cpap titration protocol among clinical sleep laboratories. for this reason, the pap titration task force developed evidence-and consensus-based standardized pap titration protocol and published its guideline entitled "clinical guidelines for the manual titration of positive airway pressure in patients with obstructive sleep apnea" in journal of clinical sleep medicine 2008. in this lecture, i will explain about manual cpap titration guidelines as below based on publications which are recommended by positive airway pressure titration task force of the american academy of sleep medicine (aasm): (1) important considerations prior to cpap titrations. (2) criteria for cpap pressure to be increased. (2) minimum and maximum starting cpap pressure. (3) an interval and minimum pressure to eliminate obstructive respiratory events. (4) different algorithms for cpap pressure to be increased to eliminate obstructive respiratory events observed for patients ≥12 years and <12 years. interventional bronchoscopy has typically been associated with obstructive tumor removal to regain central airways patency; such interventions, whether with rigid or fl exible instruments were also limited to the navigable fourth or fi fth generation airways. tissue destructive techniques included ablative heat techniques (laser, electrocautery and argon plasma coagulation), cold techniques (cryotherapy) and mechanical coring with the rigid bronchoscope. a current new crop of tissue debridement devices include modifi cations of established technologies: fl exible co2 laser fi ber usable beyond the trachea, cryotherapy using non-contact surface cryospray, rotational microdebrider devices adopted from otolaryngology, pulsating balloon resectors. the latter three devices do not involve heat that may cause post-treatment infl ammation and cartilage destruction and subsequent airway fi brosis or malacia. previous direct intra-lesional injection, with mitomycin or steroid was directed towards non-malignant fi brotic lesions, conversely on-going studies with cytotoxic agents (5fu) and compounds thought to have immune adjuvant effects (pts) are demonstrating potential utility in endobronchial tumors. photo-dynamic therapy (pdt) compounds with shorter half-lives require fewer bronchoscopies and have shortened photo-toxicity side effects. therapeutic bronchoscopic image guided interventions (bigi) have benefi tted from advances in "virtual bronchoscopic navigation" software available to improve reaching small peripheral lesions. for radiation therapy for focal lung lesions not resectable because of patient co-morbidities or preference, the accurate placement of fi ducial markers (gold) are used to direct high-dose rate external-beam intentiy modulated radiation therapy (imrt) including cyberknife machine. trials also demonstrate feasibility and effi cacy of treating peripheral lesions by high-dose rate (hdr) brachytherapy through catheters placed with image guidance. one area of new focus is bronchoscopy in the management of chronic obstructive lung diseases (old) including emphysema and severe asthma. based on lung volume reduction surgery for severe emphysema with heterogeneous distribution and air-trapping, non-surgical bronchoscopic lung volume reduction (blvr) has taken on a number of innovative approaches including exclusion by spigots (watanabe), valves (emphysys, spiration), metallic coil retraction (pneumrx), airway bypass to relieve trapped gas (broncus), atelectasis by bio-glue (aeris) or by heat steaming (uptake). although none of the clinical devices in trials have shown unqualifi ed success, some devices are now being marketed (europe), or are available on a compassionate basis for management of broncho-pleural fistulas (bpf). airway radio-frequency ablation (rfa) of airway smooth muscle is usa-fda approved for management of severe asthma. future innovations in interventional bronchoscopy will likely incorporate advances in diagnostic bronchoscopy such as video-autofl uorescence and "in-vivo biopsy" techniques to guide local endobronchial therapies for in-situ cancers; image processing software to design custom stents for compromised airways; and drug-eluting stents to maintain airway integrity in a variety of malignant and benign airway diseases. pleuroscopy describes a minimally invasive procedure that provides the physician a window into the pleural space. it refers to a procedure that is performed in an endoscopy suite or operating room with the patient under conscious sedation and local anesthesia. increasingly these procedures are being performed by nonsurgeon pulmonologists to diagnose pleural pathology such as pleural effusions or pleural carcinomatosis; talc pleurodesis and chest tube placement under direct visual guidance. pleuroscopy was fi rst conceived in a report dated 1866, which documented the fi rst endoscopic examination of the pleural space by richard cruise in a 11 year old girl with empyema. it did not gain widespread application until 1910 when hans christian jacobaeus published his technique also known as the jacobaeus operation. in this procedure he created a pneumothorax by severing adhesions using galvanocautery that collapsed the underlying lung, and allowed safe entry as well as unobstructed examination of the pleural space. since then, pleuroscopy has been applied both as a diagnostic and therapeutic tool. for a hundred years, rigid endoscopic instruments such as stainless steel trocars and telescopes have been pivotal in the technique. smaller telescopes and instruments have been applied with excellent views of the pleural space and comparable diagnostic yield. a signifi cant advance is the creation of fl exrigid pleuroscope that is fashioned like the fl exible bronchoscope. the fl ex-rigid pleuroscope consists of a handle, and a shaft that measures 7 mm in outer diameter, 22-cm proximal rigid portion and 5-cm fl exible distal end. the fl exible tip is movable by a lever on the handle, which allows 2-way angulation 160 degrees up and 130 degrees down. it has a 2.8 mm-working channel that accommodates biopsy forceps, needles and other accessories, and is compatible with various electrosurgical and laser procedures. the fl ex-rigid pleuroscope allows autoclaving. a notable advantage is its easy interface with existing processors and light sources made by the manufacturer for fl exible bronchoscopy or gi endoscopy at no additional costs. although certain endoscopic characteristics such as nodules, polypoid masses and "candle wax drops" are suggestive of malignancy, early stage mesothelioma can resemble pleural infl ammation. autofl uorescence and narrow band imaging have been incorporated to white light pleuroscopy to enhance diagnostic accuracy. both modes of imaging discriminate early malignant lesions from non-specifi c infl ammation, aid in selecting appropriate sites for biopsy and better delineate tumor margins for more precise staging, but are of little value at present in clinical practice since most patients with malignant pleural effusions have extensive pleural involvement that is easy to diagnose with white light pleuroscopy for pleuroscopic guided pleural biopsies, specimens obtained with the rigid forceps are larger than those with the fl ex-rigid pleuroscope since they are limited by size of the fl exible forceps, which in turn depends on the diameter of the working channel. the fl exible forceps also lacks mechanical strength in obtaining pleural specimens of suffi cient depth, which can be overcome by the use of insulated tip (it) diathermic knife. full thickness parietal pleural biopsies are obtained with it knife, and the electrocautery knife is particularly useful when smooth thickened lesions are encountered, of which nearly half are due to mesothelioma. to improve analgesia before talc poudrage, lidocaine can be administered to the parietal pleura via spray catheter inserted through the working channel of the pleuroscope. similarly talc poudrage can be administered under visualization using the spray catheter. with the introduction of the fl ex-rigid pleuroscope, similar in design and handling to the fl exible bronchoscope, and compatible with standard light source and video processor available in most bronchoscopy suites, pleuroscopy will enjoy an expanded interest as more practitioners acquire the skill. the fl exrigid pleuroscope is a signifi cant invention in the history of minimally invasive pleural procedures and will revolutionize the practice of pulmonary medicine by replacing conventional biopsy methods in future. the common known causes of interstitial lung disease (ild) are drug toxicities, environmental exposures and collagen vascular disease (cvd). among these causes, drug or environmental exposures can be excluded by medical history. however, cvd-related ild may often be confused with idiopathic interstitial pneumonia (iip) because the radiological and histological characteristics of cvd-related ild are often indistinguishable from those of their idiopathic counterparts and occasionally, systemic manifestations of the underlying cvd develop several months or years after the diagnosis of ild. early diagnosis of occult cvd is very important in patients presenting with ild, because there are signifi cant differences in prognosis between the iip and cvd-ild groups. patients with cvd-ild survive longer than those with iip. additionally, different treatment regimens and evaluation for additional systemic involvement or malignancy may be needed in patients with cvd-ild. although, cvd-ild and iip is often considered indistinguishable, there are some clues that can help clinicians detect occult cvd in patients presenting with ild. first, a thorough medical history and physical examination can detect occult cvd. its importance cannot be overemphasized. the cvds frequently associated with ild are scleroderma, rheumatoid arthritis (ra), polymyositis/dermatomyositis (pm/dm), sjögren's syndrome, mixed connective tissue disease (mctd), undifferentiated connective tissue disease (uctd) and systemic lupus erythematosus (sle). therefore, symptoms and signs that occur frequently in these cvds should be searched for. these symptoms and signs include raynaud's phenomenon, gastro-esophageal refl ux disease, telangiectasis, dry eyes, dry mouth, arthritis, the characteristic skin lesions of dm (heliotrope rash, gottron's papule, mechanic's hand) and various serositis etc. second, certain fi ndings on hrct can help in the diagnosis of cvd. although the parenchymal abnormalities are similar to their idiopathic counterparts, the presence of airway-related abnormalities -mosaic attenuation, bronchial wall thickening, and nodules -are more common in cvd-ild. the presence of extrapulmonary abnormalities may also provide important clues to the underlying diagnosis. patients with cvd more frequently have pleural and pericardial effusions, pericardial thickening, enlarged pulmonary artery and esophageal dilatation. hrct can also show joint abnormalities or soft tissue calcifi cations. third, there are some serologic tests that can help in the diagnosis of cvd even in patients with obscure symptoms. high titers of antinuclear antibody and rheumatoid factor are often found in patients with cvd. other more disease specifi c tests currently available are anti-ssa/ssb antibody for primary sjögren's syndrome, anti-scl-70 antibody for systemic sclerosis, anti-jo-1 antibody for pm/dm, anti-u1 ribonucleoprotein (rnp) antibody for mctd, antibody to cyclic citrullinated peptides (ccp) for rheumatoid arthritis and so on. fourth, the frequent pathologic patterns of ild associated with cvd are nsip, uip, op, lip and dad. among them, nsip is the most frequent pathologic pattern in cvd-ild. therefore, pathologic pattern consistent with nsip should raise suspicions about the possibility of cvd. other pathologic fi ndings that may be suggestive of an underlying cvd include follicular bronchiolitis and lymphoid follicles. however, it is still impossible to diagnosis all occult cvds at the outset of ild because the initial clinical presentations can be essentially indistinguishable from those of iip. therefore, close follow up for a developing cvd is very important especially in patients with nsip. the role of pathological diagnosis for non-neoplastic lung disease is important and critical. however, agreement of pathological diagnosis in iips may not be that high. despite the expectations after publication of 2002 ats/ers classifi cation of idiopathic interstitial pneumonias (iips), interobserver variability in the pathological diagnosis of iips is still problematic. there are several major reasons for the poor agreement in pathological diagnosis of iips in which the biggest reason is a lack of specifi c and diagnostic fi nding to any type of iips. in the session, i would fi rst share the virtual steps of making diagnosis on surgical lung biopsy with audience, indicate recent data of inter-observer agreement in iips cases, and then, introduce factors behind the poor agreements followed by several possible solutions to this important issue. children's interstitial lung disease (child) differs from adult interstitial lung disease in that certain classic idiopathic pneumonias described in adults are not seen in children and unique forms of interstitial lung disease are found in infants and young children but not in adults. the most common form of idiopathic interstitial pneumonia in adults is idiopathic pulmonary fi brosis (ipf), also known as cryptogenic fi brosing alveolitis (cfa), a progressive and fatal disorder, defi ned pathologically as usual interstitial pneumonia (uip). uip is characterized by a heterogeneous mixture of normal lung, mild infl ammation, and fi brosis and the presence of fi broblastic foci, felt to be the leading edge of fi brosis. previously, although many infants and children were given the diagnosis of ipf, cfa, or uip, they did not have the characteristic fi broblastic foci. thus although the uip pattern is occasionally seen in the context of another primary disorder, such as abca3 mutations, true ipf/uip does not exist in children. the tendency to use the term ipf in children merely serves to obscure the real diagnosis and creates anxiety in families whose affected children may not actually have a fatal disorder. unique conditions have been described mainly in infants and young children that do not occur in adults. these include growth abnormalities, inborn errors of surfactant metabolism, neuroendocrine cell hyperplasia of infancy (nehi), and pulmonary interstitial glycogenosis (pig). growth abnormalities occur as a consequence of an early insult to the lung that results in retarded or arrested lung development and alveolar simplifi cation. risk factors associated with growth abnormalities include prematurity, congenital heart disease, and chromosomal defects, most commonly down syndrome. the major advance in child has been the discovery of genetic mutations that lead to surfactant dysfunction. these include mutations in the sp-b, sp-c, abca3, ttf-1, and gm-csfra genes. clinical presentation can vary from severe respiratory failure at birth leading to death (sp-b, abca3 mutations) to more insidious onset with chronic lung disease (sp-c, abca3, ttf-1, gm-csfra mutations). nehi is a chronic benign form of child presenting in the fi rst year of life with tachypnea, crackles, hypoxemia, characteristic features of symmetric ground glass densities in the right middle lobe and lingula and central lung regions on hrct, and a mixed restrictive/obstructive pattern on infant lung function testing. lung biopsy shows increased numbers of neuroendocrine cells and neuroepithelial bodies in the distal airways with otherwise normal lung architecture. pig is another benign form of child seen in infants and characterized by interstitial widening with glycogen-rich interstitial cells. pig is seen as a primary disorder ("pure" pig) and as a patchy disorder seen in the background of some other primary disorder, such as a growth abnormality ("patchy" pig). in conclusion, it is important to recognize the differences between pediatric and adult interstitial lung disease so that the proper diagnosis and prognosis can be given and the appropriate treatment applied. journal compilation © 2010 asian pacifi c society of respirology not to treat acute bronchitis with initial antibiotics, with the following exceptions. those at high risk of serious complications because of preexisting co-morbidity, patients over 65 years of age with acute cough and two or more of the following, or patients over 80 years of age with one or more of the following; (1) admission to hospital in the previous year (2) type 1 or type 2 diabetes (3) history of congestive heart failure (4) current use of oral glucocorticoids. clinicians need to address patients' concerns, perspectives, and expectations about the treatment and explain to patients that antibiotics are not necessary for a self-limiting respiratory tract infection. physicians should tell patients that antibiotic use increases the risk of an antibiotic resistant infection. and physicians also need to spend time answering questions and offer a contingency plan if symptoms worsen, and advise patients to return for a consultation if symptoms are not starting to settle in accordance with the expected course of the illness or if symptoms worsen signifi cantly. some physicians are certain that patients will benefi t from antibiotics and prescribe for expectation of fast relief. they are mostly comfortable with their prescribing decisions by their clinical experiences. taiwan's study demonstrates substantial variations among physician groups in the practice of prescribing antibiotics for viral respiratory infections. older physicians and those practicing in clinics rather than medical centers were signifi cantly more likely to prescribe antibiotics, and dispensing doctors in contrast to those without dispensing privileges or on-site pharmacists were signifi cantly highly prescribing antibiotics. statistical data from nhic in korea showed that general physicians in clinics prescribe antibiotics in 68% of acute bronchitis patients, while doctors at tertiary hospitals showed less but still fairly high rate of 51%. efforts and interventions to reduce the potentially inappropriate prescription of antibiotics should target modifi able factors. quality improvement (qi) strategies like using active clinician education, delayed prescriptions and targeting management, may yield reductions in antibiotic use. anti-tussives are occasionally useful and can be offered for short-term symptomatic relief of coughing. a meta-analysis and systematic review found that beta-2-agonists were not effective for the treatment of acute bronchitis or cough of <4 weeks duration in children or in adults unless airfl ow obstruction was present. summary acute bronchitis is one of most commonly diagnosed and treated diseases in daily clinical practice. however, since it is mostly a self limiting disease, the standardization of diagnosis and treatment has long been neglected leaving various controversies in the management, particularly the use of antibiotics. the inappropriate prescription of antibiotics for acute bronchitis will surely lead to the emergence of resistant organisms in the community let alone the increase of socio-economic burden. further attention and research is needed for the reasonable approach to the treatment of acute bronchitis in order to prevent overuse of antibiotics and improve health-economy. prevalence acute bronchitis is one of the most common conditions encountered in clinical practice, accounted for approximately 10 million visits to korean physicians in 2008, and consistently ranks among the top 10 reasons for ambulatory visits in the united states. defi nition acute bronchitis refers to a clinical syndrome distinguished by a relatively brief, self-limited infl ammatory process of large and midsized airways that is manifested predominantly by cough with or without phlegm production which lasts for up to 3 weeks and absence of fi ndings suggestive of pneumonia. acute bronchitis should be distinguished from acute exacerbations of chronic bronchitis and acute infl ammation of the small airways -asthma or bronchiolitis. those with underlying lung disease, congestive heart failure, or a compromised immune system are considered to be at high risk for complications of acute bronchitis. etiology acute bronchitis is one of the most common causes of antibiotic abuse. in healthy communities, there is little evidence of bacterial infection in people with bronchitis, but there are few practical studies to distinguish between bacterial and viral bronchitis. within this context, the use of antibiotics to treat acute bronchitis is controversial but common in real practice. viruses are usually considered the most common cause of acute bronchitis but have been isolated in a minority of patients. those isolated in acute bronchitis include, in order of frequency of occurrence, are infl uenza, parainfl uenza, respiratory syncitial virus (rsv), coronavirus, adenovirus, and rhinovirus. the yield of specifi c pathogens varies according to several factors, including the presence or absence of an epidemic, the season of the year, and the infl uenza vaccination status of the population. bacterial pathogens are thought to play a very minimal role in acute bronchitis. the bacteria that have been causally linked to acute bronchitis in otherwise healthy individuals include only mycoplasma pneumoniae, chlamydophila pneumoniae and bordetella pertussis. antibiotic treatment of patients with pertussis is indicated to limit transmission, but there are no compelling data to support the prospect that cough will be less severe or less prolonged with antibiotic therapy. clinical manifestations acute bronchitis cannot be distinguished from upper respiratory infections in the fi rst few days. acute bronchitis is suggested by the persistence of cough for more than fi ve days, and most often lasts from 10 to 20 days. approximately 50% of patients with acute bronchitis report the production of purulent sputum. it usually represents sloughing of cells from the tracheobronchial epithelium, along with infl ammatory cells, and does not signify bacterial infection. pulmonary function test fi ndings consistent with bronchial hyperresponsiveness are common. fev1 less than 80% at the initial visit was present in 40% of adults from the mid western united states with no history of underlying lung disease. pft abnormalities are usually transient, typically resolving after 2 to 3 weeks, although they may last as long as 2 months. fever is a relatively unusual sign in acute bronchitis and, when accompanying cough, suggests either infl uenza or pneumonia. diagnosis acute bronchitis is established in a patient who has the sudden onset of cough, with or without sputum expectoration, and without evidence of pneumonia, the common cold, acute asthma, or an acute exacerbation of chronic bronchitis. the absence of the following fi ndings reduces the likelihood of pneumonia suffi ciently to eliminate the need for a chest radiograph: (1) heart rate >100 beats/min; (2) respiratory rate >24 breaths/min; (3) oral body temperature of >38°c; and (4) chest examination fi ndings of focal consolidation, egophony, or fremitus. chest radiography should be reserved for use in patients with any of these fi ndings or cough lasting >3 weeks. an exception, however, is a cough in elderly patients; pneumonia in elderly patients is often characterized by an absence of distinctive signs and symptoms. rapid diagnostic tests exist for several pathogens currently linked to acute bronchitis. patients with severe paroxysmal cough, with or without post-tussive vomiting should be evaluated for pertussis regardless of the immunization history. rapid tests should be used primarily when the suspected organism is treatable, the infection is known to be circulating in the community, and the patient has suggestive symptoms or signs. treatment statistical data from korea national health insurance corporation (nhic) shows that approximately 50-60% of patients with acute bronchitis receive antibiotics despite the evidence that, with few exceptions, they are ineffective. meta-analyses of randomized, controlled trials all concluded that routine antibiotic treatment is not justifi ed. the decision not to use an antibiotic should be addressed individually and explanations should be offered because many patients expect to receive an antibiotic based on previous experiences and public expectations. main challenges for appropriate antibiotic use in acute bronchitis are the diagnosis is based on clinical fi ndings, without standardized diagnostic methods and sensitive or specifi c confi rmatory laboratory tests. how to identify accurately the few patients who are seriously ill or whose symptoms could be meaningfully ameliorated by prompt antibiotic treatment is not standardized. recent studies have suggested that the annual decline in fev1 is greater in gold stage ii than in later stages of the disease. (1) if the decline in pulmonary function predominantly occurs early in the course of the disease, then it is logical that diagnosis and intervention aimed at reducing the progression of the disease should mainly occur in the early stages of the disease. severity of copd at diagnosis differs enormously on where it is done: population based studies, screening or hospital patients. epidemiologic studies: prevalence, underdiagnosis and severity there are increasingly more data on the prevalence and distribution of copd from around the world. the prevalence of chronic obstructive pulmonary disease (copd) varies from country to country, mainly due to the effects of cumulative exposure to smoking and the increased life span of the population. (2) (3) (4) (5) systematic review of epidemiological studies concluded that the prevalence of copd, in adults aged 40 years and more, worldwide ranges around 9-10%. (6) these differences may be related, at least in part, to differences in genetic background, smoking habits and exposure to other environmental risk factors, and are accompanied by differences in diagnostic rates and in management of the disease around the world. there is a large underdiagnosis of copd with about only one out of three or four of all subjects fulfi lling diagnostic criteria of copd identifi ed by the health care system. (2) (7) (8) copd in general population is diagnosed at earlier stages. data from latinamerica (platino), similar from those from spain, showed severity was distributed as follows: stage i, 59 % and stage ii, 33.8%. screening for copd screening, combined with smoking cessation advice, help motivated smokers to attempt quitting smoking. (10) in general practice, when individuals were preselected on the basis of smoking age and respiratory symptoms chronic cough was a better predictor of airfl ow obstruction than other symptoms, such as wheeze and dyspnoea. age was also a good predictor of obstruction; smokers over 60 with cough had a 48% chance of having an obstruction. (11) diagnosis at the hospital the decrease in lung function is gradual. the disease is usually diagnosed late because patients may adapt to the condition or doctors may not notice the symptoms. by then, the patient is diagnosed at the hospital, when lung function is often poor, sometimes less than 50% of normal. the relationship between lung disease and increased body weight can take two forms: the effects of increased body weight on the normal respiratory system and the association of increased body weight with diseases of the respiratory system. obesity (body mass index, bmi, greater than 30 kg/m 2 ) can reduce normal static lung volumes (principally functional residual capacity, but also total lung capacity and residual volume at very high bmi), ventilation (particularly during sleep and exercise) and gas exchange (increased gas transfer). the epidemic of overweight and obesity has been associated with the increased prevalence of asthma through proposed mechanisms such as dietary effects, reduced lung volumes and lung recoil affecting airway responses to breathing, a general infl ammatory effect and through associated sedentary lifestyle. however more recent epidemiological data suggests the association between asthma and obesity is not as close as once thought. overweight and obesity clearly are associated with the high prevalence of obstructive sleep apnoea with increased body weight increasing the likelihood of upper airway collapse on a background of reduced upper airway dimensions and snoring and reducing minute ventilation in patients with obstructive sleep apnoea, predisposing to hypoventilation and chronic hypercapnoeic respiratory failure. the increased load to breathing also affects breathing, especially during sleep, in patients with respiratory muscle weakness or abnormal chest wall mechanics due to kyphoscoliosis or previous chest wall surgery such as thoracoplasty. increased body weight will also exacerbate the effects on symptoms of existing chronic respiratory diseases including asthma, chronic obstructive pulmonary disease and interstitial lung diseases. avoidance of weight gain and continued efforts at weight loss remain an important goal in patients with respiratory disease. this presentation will be a review of recent fi ndings and implications from imaging studies in asthma. the use of 3-dimensional imaging in asthma has provided useful insights into the understanding of pathophysiology of disease, which may have implications on how asthma is treated. small airways disease plays a major role in asthma and has traditionally been diffi cult to probe. however, the combined use of new imaging methods combined with complex lung function has provided useful insights into pathophysiology. findings and implications from hrct, pet, spect and mri will be discussed. the assessment of pulmonary function has changed very little over the past 30 to 40 years. all techniques performed in the laboratory still view the lung as a very simple single compartment unit. measures of volume and fl ow only assess disease in the medium to large airways whilst the small airways receive relatively little attention. however there are a number of emerging techniques on the horizon that have the potential to give great insight in to the periphery of the lung where most respiratory disease emanates. the measurement of mechanics using the forced oscillation technique and gas mixing using the multiple breath nitrogen washout test are now emerging as very powerful tools for assessment of peripheral lung function. in this presentation we will be discussing the state of the art in terms of assessing pulmonary function and what may we expect to see in the future. copd is a major public health problem in asia. copd prevalence was 8.2% (12.4% in males and 5.1% in females) in china (>40 yrs), 9.5% in japan (>40 yrs). in most of the developing countries in asia, copd is always underdiagnosed by physicians owning to lack of routine spirometry test and only based on symptomatic diagnosis. smoking is the most important risk factor contributing to development of copd. however, copd prevalence was 4.2% in chinese non-smokers (>40 yrs) and 8.8% in korea (>45 yrs), similar to those in mexico city (6.2%) and caracas (6.6%). in china, non-smokers accounted for 38.6% of copd patients compared with 24.9% in usa and 22.9% in the uk. exposure to environmental tobacco smoke (passive smoking) and indoor air pollution (particularly the coal and biomass combination) may contribute to the higher prevalence of copd in developing countries. to reduce the risk factors (smoking, coal or biomass fuel for cooking, indoor and outdoor air pollution) are the priority for reducing the prevalence in the asian developing countries. government in some countries had made some effect in tobacco control and reducing air pollution. unlike hypertension or diabetes, the management of copd is only based on symptomatic treatment, owing to lack of specifi c biomarkers. annual lung function test with spirometer is the only parameter in detecting early stage of copd. data showed that more reversibility of fev1 was demonstrated in stages i-ii copd patients with ics/laba or tiotropine administration, as compared with those in stages iii-iv. an intervention study at the community level has shown that early intervention (improvement of indoor ventilation, bronchodilators) was able to reverse rapid annual fev, decline in copd patients. more affordable medication should be developed in the low income countries. data showed that oral administration of carbocysteine (thio compounds) reduced exacerbation rate by 24.5%, which was consistent with inhaled administration of fluticasone/formoterol (seretide) or tiotropine. in addition, orally administered low dose teophylline (100 mg, bid) improved pre-bronchodilator fev, and reduced exacerbation rate. there was a synergistic effi cacy in fev1, with the combination of teophylline and inhaled corticosteroids. chronic obstructive pulmonary disease (copd) is characterized by the presence of airfl ow limitation due to loss of lung elasticity and/or airway narrowing. the pathological hallmark of loss of lung elasticity is emphysema and airway wall remodeling contributes to the airway narrowing. using computed tomography (ct) these lesions can be assessed by measuring low attenuation areas (laa) and airway wall thickness/luminal area, respectively. recently, copd has been widely recognized as a systemic infl ammatory disease, and body weight loss is one of its clinical features. traditionally, the patients who had copd with predominant emphysema and a low body mass index (bmi) were called "pink-puffers". however, the relationship between body weight loss and emphysema had not been assessed. based on these back ground, we have evaluated the body composition, emphysema and airway dimensions in 201 copd patients using ct images. bmi was signifi cantly lower in the emphysema dominant phenotype compared to the airway dominant phenotype. furthermore, bmi correlated with laa% (ρ = −0.557, p < 0.0001) but not with wa%. chest subcutaneous fat mass was also correlated with laa% (ρ = −0.307, p < 0.0001). these data indicated that the "pink-puffer hypothesis" is correct in some aspects. next, we postulated that reduced leptin and leptin receptor signaling could contribute the development of emphysema. serum leptin was correlated with bmi which was correlated with dl co /v a . the expression of leptin and leptin receptor was evaluated pcr in 52 human lung tissues. both genes were detected in the lung tissues, but the expression of leptin gene was low. the leptin receptor gene expression was signifi cantly lower in copd patients and it was signifi cantly correlated with dl co /v a . the leptin receptor gene expression in the lung did not correlated with body weight. these data suggested that the patients' physique can be associated with the relative contribution of emphysematous lesions in copd and leptin and leptin receptor system might affect the mechanism of developing emphysema. nutritional support has been one of possible clinical approaches as a copd therapy these days. however, its effect is still controversial. to clarify the relationship between low bmi and emphysema and to classify the phenotypes of copd based on the patients' physique may help to fi ne the defi nite targets for nutritional support even in the early stage of copd. journal compilation © 2010 asian pacifi c society of respirology sy 07-03 at present copd is often only treated in gold stage iii or iv 1 . it is without question possible to diagnose copd earlier. if spirometry would be more readily performed in general practice, copd could be diagnosed in gold stage i and ii, as is evident from the practice of "spirometry days" organized by the belgian thoracic society in which the majority of the patients were diagnosed in gold stage ii 1 . whether gold stage i and ii truly represent the "early" stages of the disease may be debated 2 , but this defi nition of early copd could certainly be used as an operational defi nition. it is clear now that spirometry is required for early diagnosis of copd 3 . although at present there is no irrefutable evidence that early treatment of copd is warranted, there is accumulating suggestive evidence that early treatment of copd may result in better outcomes. this evidence is primarily related to secondary analyses of the torch 4 and uplift 5,6 studies. first, it was demonstrated in a secondary analysis of the torch study, that inhaled corticosteroids, long-acting betaagonists and their fi xed combination reduced the rate of decline of fev 1 by 13, 13 and 16 ml, respectively 7 . if treatment indeed reduces the progression of the disease, then an easy case for early treatment is made. in addition, a subgroup analysis demonstrated that except for the effect on the sgrq (st. george's respiratory questionnaire) all other treatment effects were numerically larger in gold stage ii than in gold stage iii and iv 8 . a subgroup analysis of the uplift study demonstrated numerically greater treatment effects in gold stage ii as well. in addition, tiotropium reduced the rate of decline of fev 1 in these patients (albeit only by 6 ml/year), which was not the case in the later gold stages 9 . finally, in patients not taking any medication at the onset of the study (maintenance naïve patients) tiotropium substantially reduced the rate of decline of fev 1 and the rate of deterioration of the sgrqscore 10 . taken together these data demonstrated that: 1) large treatment effects were obtained in early disease; 2) indications of disease modifi cation were present in early disease. hence, they support early treatment of copd. pulmonary rehabilitation is now the standard of care for patients with chronic obstructive pulmonary disease (copd) who remain symptomatic despite bronchodilator therapies. combining the best of interprofessional, personalized and evidence-informed care, pulmonary rehabilitation allows clinicians and their patients to realize signifi cant benefi ts in a variety of important patientcentered copd outcomes. the fundamental elements required for an effective pulmonary rehabilitation program will be discussed, and the scientifi c evidence supporting their effectiveness will be summarized. issues relating to optimal site of delivery, components of effective rehabilitation programming, duration of rehabilitation, timing of rehabilitation and target populations will be reviewed. recent developments in this rapidly expanding area will also be highlighted. lastly, methods to establish or enhance an existing pulmonary rehabilitation program will be discussed, with the goal of fully realizing the many substantive benefi ts of pulmonary rehabilitation in copd. however, only very low gene transfer seen after a second dose with either 14 day and 7 day spacing. we attribute this to rapid upregulation of neutralizing antibodies against adenovirus. anti-tumor humoral immune responses were seen almost all patients with reactions seen against known meso tumor antigens (sv40 large t antigen and mesothelin) and against unknown proteins in cell lysates. given the caveats of phase 1 trials (small numbers, different doses, heavily pretreated patients), we still saw clinical responses (defi ned as prolonged stable disease, prolonged survival, partial or complete responses by modifi ed resist criteria, decreased metabolic tumor activity by pet scanning, or "mixed" responses) in about 1/3 of the patients. we are currently administering two doses spaced only three days apart. this appears to be well tolerated. based on strong preclinical data supporting the combination of gene therapy and chemotherapy, we have started a trial using ad.inf instillation into the fi rst treatment cycle of fi rst line (cisplatin/pemetrexed) or second line chemotherapy (gemcitabine). our groups is also generating "designer chimeric t cells" in which a single chain antibody fragment is linked to the transmembrane and cytoplasmic regions of the t-cell receptor. this artifi cial t-cell receptor is then transduced into t-cells that are then reinfused. the t-cells are then activated by cells expressing mesothelin. preclinical data show striking activity against mesothelin-expressing tumors in mice. mesotheliomas make large amounts of the immunoinhibitory cytokine, transforming growth factor-beta (tgf-β). preclinical studies using tgf-β blockers have shown activity in mouse models of mesothelioma and a clinical trial using an anti-tgf-β antibody is now underway at the university of pennsylvania. in summary, gene-based and immunotherapies are being actively studied in mesothelioma and have shown some promising results. future trials are focusing on combining these approaches with chemotherapy and surgery. given the relatively mild and non-overlapping toxicities, we believe these, or other gene therapy and/or immunologic approaches will soon become part of the standard therapeutic armamentarium. the burden of asbestos-related diseases (ards), in particular mesothelioma, has been shouldered mostly by the developed countries of the west. this is a consequence of the heavy dependence on asbestos use up to around the 1970s by those countries. in contrast, the majority of asian countries started to depend on asbestos since then and has not yet reached the suffi cient latency time for the related diseases to manifest. japan is an exception, because it heavily used asbestos during the period to catch-up with the west. japan has now become one of the global epicenters of ards. it is a tragic consequence of experiencing the burden of ards that many developed countries decided to move towards banning asbestos or a de facto non-dependence. in the asia-pacifi c, this group comprises australia, new zealand, japan, korea and singapore (the "forerunner" group in terms of ards). in contrast, the many other countries in asia still use asbestos at substantial levels, turning asia into the world's center of asbestos consumption today. however, as these countries start to use up the latency time, and manage to improve medical recognition, reporting and recording, ards will soon emerge as a major public health issue in the region. early indications of this forecast do exist. not only should lessons be learnt from the experiences incurred by the "forerunner" group of countries, but more importantly, they should crystallize in international collaboration involving national administrators, academia, ngos and international organizations, for the effective recognition and countermeasures of ards. i will also refer to the progress made and hurdles encountered by the asian asbestos initiative and the who global plan of action on the elimination of asbestos-related diseases. at the international level, ards present a domino-effect that needs to be coped with. sy 08-03 interstitial pneumonia (ip) can be classifi ed into two groups in terms of known causes. pneumoconiosis, drug-induced pneumonitis, radiation-induced pneumonitis, and hypersensitivity pneunonitis (hp) are categorized as ip with known causes. on the other hand, idiopathic interstitial pneumonias (iips), which include idiopathic pulmonary fi brosis (ipf), nonspecifi c interstitial pneumonia (nsip), cryptogenic organizing pneumonia (cop), and desquamative interstitial pneumonia (dip), have no known causes. most physicians tend to diagnose of ip patients as iips without intensive examinations. however, some environmental and occupational lung diseases, especially asbestosis and chronic hp, should carefully be differentiated from iips. asbestosis is one type of pneumoconiosis, which is induced by asbestos exposure with a latent period of usually more than 10 years. bilateral fi ne crackles can be frequently auscultated and pulmonary function test shows restrictive and diffusing impairments. chest hrct shows subpleural dot, subpleural curvilinear shadow, ground glass opacity, interlobular septal thickening, traction bronchiectasis, and honeycombing. in our case series of asbestosis (n = 51) in yokosuka, a town of shipyard for more than 100 years, honeycombing was seen in 6 cases (12%). chronic hp is an allergic disease induced by long-term exposure to antigens. major causative antigens are avian dropping and feather, mold, and bacteria. chest hrct tends to show traction bronchiectasis and honeycombing in advanced stages, which are similar to ipf. in surgical lung biopsy, most cases are classifi ed as nsip-like and uip-like patterns. to clarify the importance of unrecognized exposure to avian antigen, we precisely reviewed 56 patients with bird-related chronic hp. in the 56 patients, 24 patients were bird-breeders with direct exposure to avian antigen by contacting their own birds, whereas 32 patients seemed to be exposed to wild birds, neighbor's birds, feather duvets, stuffed bird, and fertilizer with chicken droppings without recognition of avian contact. number of patients is very limited both in asbestosis and chronic hp, which suggests that there is a small group of subjects who are susceptible to develop pulmonary fi brosis after exposure to asbestos or antigens. however, genetic background of susceptibility to pulmonary fi brosis has not elucidated. in our case series of chronic hp (n = 184), 22 cases (12%) had the fi rst-degree family members with ip, who might have common environmental and/or genetic factors. further studies are needed to determine host susceptibility to pulmonary fi brosis, which might contribute to clarify the pathogenesis of ip. long acting beta-2 agonists has been in the doctors' armory of asthma medications for about 20 years (available in 1990 in uk and 1994 in usa). there is little doubt that laba when combined with inhaled corticosteroids can improve asthma symptoms for a subsection of people with asthma, and is generally superior to add-on leukotriene receptor antagonist. 1 indeed addition of laba to inhaled corticosteroids is in all major asthma guidelines as a step-up therapy. however, after the salmeterol multicentre asthma research trial was prematurely halted, a focus on effi cacy and safety of the wide use of laba severe pulmonary arterial hypertension (pah) is a fatal condition associated with complex pathobiology. vasoconstriction, thrombosis, and remodeling of the pulmonary vessel wall contribute to increased pulmonary vascular resistance in pah. the pathology of pulmonary hypertension can be classifi ed into endothelial, smooth muscle, and/or adventitial lesions, although not all compartments of the pulmonary artery wall are involved in each case of severe pah. the classic lesion of severe pah is the plexiform lesion, an abnormal proliferation of predominantly endothelial cells. smooth muscle thickening can be seen in all forms of the disease but is not a constant feature in the idiopathic pulmonary arterial hypertension. the adventitia is often markedly remodeled in patients with certain forms of collagen vascular diseases associated with severe pah, most notably scleroderma. the obligatory lining of pulmonary arteries with a monolayer of endothelial cells is disrupted in severe pah. the three-dimensional vascular pattern is rather suggestive of an intravascular tumor-like proliferation (tumorlet), instead of a retracted scar if this lesion would represent an abnormal healing to an injury to the vascular wall. the evidence of a tumorlike endothelial cell proliferation was provided by the demonstration that plexiform lesions in patients with idiopathic pah were preferentially monoclonal, whereas similar lesions in lung of patients with secondary pah due to congenital heart malformations were polyclonal. monoclonality was also observed in plexiform lesions of patients from anorexigen-induded pah. vasoconstriction has been related to abnormal potassium channels and to endothelial dysfunction. endothelial dysfunction leads to impaired production of vasodilators such as nitric oxide and prostacyclin along with overexpression of vasoconstrictors such as endothelin (et)-1. many of these abnormalities not only elevate vascular tone and promote vascular remodeling but also represent logical pharmacological targets. recent genetic and pathophysiologic studies have emphasized the relevance of several mediators in this condition, including nitric oxide, prostacyclin, et-1, serotonin, angiopoietin-1, and members of the transforming-growth-factor-beta superfamily. disordered proteolysis of the extracellular matrix is also evident in pah. the unraveling of the pathobiology of severe pah may lead us to novel therapies and approaches to better treat the disease. unfractionated heparin (ufh) and low-molecular-weight-heparin (lmwh), acted by enhancing the ability of antithrombin (at) to inhibit coagulation proteases, have been used as initial anticoagulant therapy for vte. they are delivered intravenously or by subcutaneous injection. subcutaneous fondaparinux, a synthetic pentasaccharide with specifi c anti-factor xa activity, is also recommended as the initial treatment for vte according to recent guideline. oral vitamin k antagonists, acting by reducing the activity of several coagulation proteases, are used for long-term anticoagulation. the major disadvantage of vitamin k antagonists is the need for frequent coagulation monitoring to maintain a therapeutic level. new anticoagulants, including oral direct thrombin inhibitors, such as dabigatran, and oral anti-factor xa inhibitors, such as rivaroxaban and apixaban, are emerging in recent clinical trials.11 these new drugs may replace heparins and vitamin k antagonists, which are expected to have a huge impact on the treatment of vte in the near future. thrombolytic therapy should immediately be used in patients with massive (high risk) pte. the effect of thrombolysis in patients with submissive (intermediate risk) pte remains controversial. further stratifi cation of these patients is necessary. patients with multiple poor prognostic indicators such as right heart dysfunction, thrombolytic therapy should be considered. several countries have started to support activities raising public awareness of vte, with the goal to decrease mortality and morbidity. in us, national institutes of health (nih) and centres for disease control (cdc) have fostered thrombosis activities to improve the prevention of vte and its long-term complications. in the united kingdom, a thrombosis group has been formed to promote awareness among parliamentarians about the risk and management of vte; to increase knowledge of its causes, effects, and treatments; and to monitor the implementation of government initiatives and other researches being and this program has corrected the wrong perception that pte is a rare disease in china pulmonary hypertension (ph) is a common complication of chronic respiratory diseases, such as chronic obstructive pulmonary disease (copd) or interstitial lung diseases (ild). ph associated with respiratory diseases is classifi ed as diagnostic group iii according to the current clinical classifi cation of dana point 2008. it is suggested that the pulmonary vascular abnormalities originate at an early stage of the diseases. the functional (hypoxic vasoconstriction) and morphological factors (vascular remodeling, destruction of the pulmonary parenchyma) explain the elevation of pulmonary vascular resistance that leads to ph. the ph is mild to moderate in copd with mean pulmonary artery pressure (mpap) usually ranging between 20 and 25 mmhg, however, worsening during exercise and exacerbations. a small proportion of copd patients may exhibit severe ph defi ned by a resting mpap >35 to 40 mmhg, whose prognosis is particularly poor. ph is relatively frequent in advanced ild, particularly in idiopathic pulmonary fi brosis, which predicts a poor prognosis. the diagnosis of ph is suggested by doppler echocardiography, but the confi rmation still requires right heart catheterization. the potential treatments of ph associated with respiratory diseases are as follows: 1) treat underlying lung disease; 2) provide supplemental oxygen therapy when appropriate; 3) rehabilitation; 4) treat right heart failure; 5) consider vasomodulator therapy; and 6) consider lung transplantation when indicated. journal compilation © 2010 asian pacifi c society of respirology sy 11-01 tobacco use is the most common preventable cause of death. about half of the people who don't quit smoking will die of smoking-related problems. the epidemic varies among countries and is increasingly affecting developing countries, where most of the world's smokers (82%) live. close to half of all men in low income countries smoke daily and this has been increasing. legislation the framework convention on tobacco control (fctc) was adopted by who member countries in may 2003 to commit all countries to protect nonsmokers from tobacco smoke in public places, to eliminate all tobacco advertising, promotion and sponsorship, to require warning labels of cigarette packs and to prohibit misleading tobacco product descriptors such as "light" and "mild". even though many countries have passed legislation mandating smoke-free environments and the total global population covered by comprehensive smoke-free laws increased from 3.1% to 5.4% in one year, the overwhelming majority of countries still have no smoke-free laws, very limited laws, or ineffective enforcement. compliance with smoke-free laws is low. treatment to aid smoking cessation support for smoking cessation or "treatment of tobacco dependence" refers to a range of techniques including motivation, advice and guidance, counselling and appropriate pharmaceutical aids, all of which aim to encourage and help tobacco users to stop using tobacco and to avoid subsequent relapse. the success of these interventions depends on their synergistic use in the context of a comprehensive country tobacco-control strategy. in many countries, provision for treatment, training of health-care providers, education and information on the wide use of cessation is scarce. therapies, as well as fi nancial resources are limited and rarely incorporated into standard health care. also, smoking cessation is not seen as a public health priority and is not necessarily approached as a key tobacco-control strategy. smoking cessation services are most effective when they are part of a coordinated tobacco control programme. brief cessation counselling is relatively inexpensive when integrated into existing primary health-care services, is usually well received by patients, and is most effective when it includes clear, strong and personalized advice to quit. communication technologies -such as telephone quitlines, text messaging, interactive telephony, and online counselling -as well as psychological and behavioural modifi cation therapies, offer important support. cessation prescription medicines, available in many countries like nicotine replacement therapies, bupropion and varenicline can double the likelihood that someone will successfully quit. bronchiectasis is an old disease that we all treat, but surprisingly little about this disease has been well studied. the defi nition, diagnosis, natural history, pathogenesis and treatment are all uncertain. this talk examines these points in the light of new information about biofi lms and "normal" bacteria. it asks more questions than it answers, but the questions raise thoughts on whether our usual approaches are the best. the mediastinum is generally split into three compartments strictly for the purpose of classifi cation of the most likely abnormality in the individual compartment. there are no anatomical boundaries or fascial planes that divide one compartment from the other. classically, the compartments have been classifi ed as anterior, middle, and posterior. a recent change in classifi cation has used the categories of anterior, middle-posterior, and paravertebral compartments. approximately 50% of mediastinal tumors are located in the anterior compartment, 25% in the middle compartment, and 25% in the posterior compartment. asymptomatic masses are more likely to be benign than malignant, and symptomatic masses are more likely to be malignant than benign; however, there is a large variation the most common tumors in the anterior mediastinum consist of thymoma, lymphoma, germ cell tumors, and thyroid tumors. thymoma is by far the most common anterior mediastinal tumor and approximately 2/3 are encapsulated and non-invasive while 1/3 are invasive. the most common paraneoplastic syndromes associated with thymoma include myasthenia gravis, hypogammaglobulinemia, and pure red blood cell aplasia. benign teratomas occur in both male and females, while malignant germ cell tumors of the mediastinum are almost exclusively in males. lymphoma can occur most commonly in the anterior or middle mediastinum and may be associated with systemic symptoms and occasionally superior vena cava syndrome. the most common abnormalities in the middle mediastinum include lymphoma, granulomatous disease, and developmental cysts. bronchogenic cysts are almost always benign, although they can cause symptoms such as obstructive pneumonia and are generally treated with surgical resection. recent mediastinal compartment classifi cation has switched from posterior mediastinum to naming this the paravertebral compartment. it is located posterior to an imaginary line drawn to connect the anterior aspects of the vertebral bodies on the lateral chest radiograph. the most common tumors in this location are neurogenic tumors, meningoceles, or thoracic spine lesions. the most common neurogenic tumors in adults are neurilemomas, and they are almost always benign. neurofi bromas also occur in this compartment. they are frequently benign, but may be malignant, especially in patients with neurofi bromatosis. in these individuals, malignant tumors of the nerve sheath origin are more common. ganglioneuroma, ganglioneuroblastoma, and neuroblastoma are more common neurogenic tumors in children or adolescents.. cough is an important lung defense. in a refl ex manner, noxious agents are expelled from the airways as these are sensed by the receptors in the airway epithelium. in addition to appreciating its cleansing function, understanding cough refl ex is important in the diagnosis and treatment of common clinical conditions. most diseases associated with cough are transient. a problem arises when cough persists and becomes chronic. an in-depth search for the underlying pathology is warranted since treatment directed to the cause of the cough is curative in over 90% of cases. an anatomic-diagnostic protocol ensures a systematic search for cough etiology but unfortunately, this approach can be lengthy and in many settings, impractical. thus an empiric syndromic approach is now recommended. this paradigm shift is borne by the following premises: (1) the current awareness of the relative frequency of the disorders (alone or in combination) that can cause cough; (2) the sensitivity and specifi city of many (but not all) diagnostic tests in predicting the cause of cough has been established; (3) a sequential evaluation and treatment for the common causes of cough using a combination of selected diagnostic tests and empiric therapy has been proven effective; and (4) a sequential and additive therapy is often crucial because more than one cause of cough is frequently present. a recent study by dr. aileen wang on "the management of chronic cough in a tertiary center: an asian perspective" showed that even in a filipino immunocompetent population, the most common causes of chronic cough is asthma, post-nasal drip syndrome (pnds) and gastroesophageal refl ux disease (gerd). the study concluded that: (1) the accp recommendations are generally applicable to an asian setting. research of viruses, their structure, pathogenicity and host-interactions has burgeoned. we now understand how viruses infect cells, how they replicate, how they interfere with host defences and how they interact with other tissue events and pathologies. rhinovirus infection causing the common cold is the most frequent and 'common' infection in humans. it is also implicated in most exacerbations of asthma and copd. the patho-biology of rhinovirus will be used to illustrate virus pathogenicity, virus-host-interactions and to highlight potential future therapeutic options. sy 12-02 much hope has been placed in the discovery of biomarkers to help understand the mechanisms of diseases such as copd, help stratify the disease better and guide treatment. it is also hoped that some of these could speed up drug discovery by serving as surrogate markers that respond to novel drugs within a shorter timescale than is the case with current drug trials in which the main outcome is the spirometric measurement of forced expiratory volume in one second (fev1). although studies in patients with copd have identifi ed several biomarkers, most of these need to be validated and their prognostic value is unclear. many of the markers have been identifi ed in blood and although it is recognised that there are non-pulmonary consequences of copd, some of which can be viewed as systemic biomarkers measured and/or generated in the lungs are likely to be most informative. there are several methods to identify and quantify biomarkers of copd and different biological samples (blood, bal, sputum and lung tissue) can be used to provide material for such analyses. whilst most studies to date used commercial immunoassays (elisa), there has been a keen interest in applying unbiased approaches such as proteomics. we have recently completed a large programme of work which applied 2-dimenshional electrophoresis and mass spectrometric analysis to identify biomarkers of copd. induced sputum was obtained from copd patients with a spectrum of disease severity and control subjects. two-dimensional gel electrophoresis and mass spectrometric identifi cation of differentially expressed proteins was fi rst applied to induced sputum from gold stage 2 copd patients and healthy smoker control subjects. initial results thus obtained were validated by a combination of immunoassays (western blotting and elisa) applied to a large subject cohort. the biomarkers were localised to bronchial mucosa by immunohistochemistry. of 1325 individual protein spots identifi ed, 37 were quantitatively and 3 qualitatively different between the two groups. 40 protein spots were subjected to tandem mass spectrometry, which identifi ed 15 separate protein species. seven of these were further quantifi ed in induced sputum from 98 individuals. using this sequential approach, two of these potential biomarkers (apolipoprotein a1 and lipocalin-1) were found to be signifi cantly reduced in copd patients when compared to healthy smokers. their levels correlated with fev1/fvc, indicating their relationship to disease severity. in summary, a potential role for apolipoprotein a1 and lipocalin-1 in innate defence has been postulated previously; our discovery of their reduction in copd indicates a defi cient innate defence system in airways disease that could explain increased susceptibility to infectious exacerbations. persistent chronic infl ammation, repetitive tissue injury, and dysregulated epithelial repair leading to tissue remodeling and fi brosis are the hallmarks of chronic lung diseases, such as chronic obstructive pulmonary disease (copd), chronic asthma, pneumoconiosis, pulmonary fi brosis and sarcoidosis. the innate immunity system with its pattern recognition receptors are recently identifi ed to involve in the pathogenesis of these chronic lung diseases. the neutrophilic infi ltration of the airway mediated through t h 17 and il-17 family may play an important role in steroid-resistant asthma and status asthmaticus. in addition, the epigenetic modifi cation of gene expressions and cellular senescence may modulate the progression of chronic asthma and copd. histone deacetylase 2 (hdac2), which can be inactivated by oxidative stress or pi3k-akt pathway, may regulate corticosteroid-related anti-infl ammatory response. manipulation of hdac2 activity is a new treatment direction in steroid-resistant asthma and copd. sirt1, a nad + -dependent deacetylase, is an important signaling pathway related to cell survival, dna repair, and apoptosis. the sirt1 is decreased in alveolar macrophages of smokers and copd patients, and associated with pro-infl ammatory response through the activation of nf-κb. understanding the complexity of infl ammatory and epigenetic regulations in chronic lung diseases may potentiate the development of novel therapies. fibrosis is a common fi nding in chronic lung diseases, with tgf-β-related pathways play important roles. myofi broblasts are the principal effective cells in the fi brogenic process. they can be evolved from the activation of resident pulmonary fi broblasts, marrow-derived fi brocytes, or the trans-differentiation of pulmonary epithelial cells through epithelial mesenchymal transition (emt). further understandings on the emt process in lung tissue repair may help to elucidate the mechanism of lung remodeling and fi brosis. in addition, lung stem/progenitor cell study appears to be a new and potential fi eld in lung injury and repair. it is anticipated that more clear mechanisms behind lung remodeling and fi brosis can be identifi ed and new treatment modalities be developed accordingly. sy 12-04 cancers are genetic diseases with constitutional genomic variations that are present in normal cells contributing to an individual's risk of developing cancer such as lung cancer. furthermore, cancer cells acquire genetic mutations and genome wide changes in their dna as well as their epigenome compared to their normal cellular counterparts. some of these mutations have turned out to be important driver mutations and are associated with exquisite sensitivity to targetted cancer therapies. some of these genetic changes include egfr and eml4-alk fusion gene mutations. epigenetic changes include methylation abnormalities as well as histone modifi cations, and possess the characteristic of potential reversibility which is attractive for the development of new therapies. the human genome project and rapid technological advances including deep dna sequencing have contributed signifi cantly to the ability to detect cancer specifi c genetic, genomic and epigenomic aberrations. these genetic and genomic abnormalities have promise across the translational spectrum from identifying individual susceptibility to cancers, early diagnosis markers, molecular pathology and tailoring of treatment (predictive biomarkers) as well as informing on outcome (prognostic biomarkers). much work remains to be done to validate clinical utility and translate these potential useful biomarkers into useful clinical tools. this session will review some of the developments in the genomics and epigenomics of lung cancer and mesothelioma. asthma is a disease that is diagnosed largely on a history of variable symptoms and the demonstration of variable airway narrowing. it is associated with airway infl ammation (cd4 t-lymphocytes, b-cell lymphoid aggregates, macrophages, eosinophils and, in adults, neutrophils) and airway wall remodelling (airway wall thickening, principally increased airway smooth muscle and deposition of collagen below the basement membrane, with minor encroachment on the airway lumen). it is not associated with a prominent effect on the lung parenchyma. the cause(s) of asthma remains unknown and the severity of disease remains largely constant. copd is diagnosed with a spirometer and is defi ned as a fi xed reduction in fev1, relative to fvc. when caused by cigarette smoke it is associated with airway infl ammation (cd8 t-lymphocytes, b-cell lymphoid aggregates, macrophages and neutrophils) and airway remodelling (mild increase in airway wall thickness including airway smooth muscle with encroachment on the airway lumen) and tissue destruction (emphysema and loss of small conducting airways). the severity of copd increases with age and continued exposure to irritants. copd has many causes including smoking cigarettes, burning of biomass fuels, asthma and early life respiratory illness and exposures (viral infections, bronchopulmonary dysplasia). as a group, patients with asthma have reduced lung function, in relation to disease severity, and may have a slightly increased rate of decline in lung function. patients with asthma who smoke have reduced lung function and an accelerated decline in lung function. apart from stopping smoking there is no current treatment that can improve the rate of decline in lung function in patients with asthma or copd. therefore prevention, early intervention and uncovering the unknown environmental and genetic contributions to airway diseases remain critical. sy 13-02 situation the isaac and ariap studies have showed the different prevalence but heavy burden caused by asthma in asia pacifi c countries is common. goals with inhaled corticosteroids and other medications, the goals in managing asthma in developing countries should attain those stated in gina: control of symptoms, maintaining normal activity levels, normal pulmonary functions, preventing asthma exacerbations, avoiding adverse affects and preventing asthma mortality. in developing countries, avoiding the abuse of short-acting beta 2 agonists, systemic corticosteroids and antibiotics is also a major problem. accessing medical care and medications is crucial. steps a core group, preferably at an university medical center, is the basic step. the asthma and copd outpatient care unit (acocu) run by this core group will help in getting experience and capacity building. the following steps are gina dissemination and implementation, patient club formation, increasing awareness, training doctors and nurses, advocacy, researches, efforts for the availability of affordable asthma drugs, and multiplying acocu in the whole country. the sustainability of acocu is assured by successful implementation of gina. a network of acocu will encourage and improve the activities of acocus. successful provincial acocu will encourage the building of district and even commune acocus. diffi culties the continuous medical education for all doctors on gina, the medication affordability, spirometers and mechanism to maintain an acocu. despite the advances in asthma diagnosis and treatment, slta cases continue to present in the er, sometimes leading to unnecessary mortalities. these are highly preventable situations with inhaled steroid based chronic therapy. with expert care, most patients get through er/icu urgent phase with good outcomes. these strategies are published and updated regularly in the international asthma guidelines. the finnish experience confi rms that a national program that pushes for implementation of guideline recommendations is able to reduce asthma hospitalizations and cost of care. not all countries have such a program but in most developed economies, the cost of asthma care including medications is covered provided the guidelines are followed. unfortunately, even in these affl uent countries, a subset on patients still lands in the er with sltas. while there may have been a failure of health care delivery, the cross-country existence of this problem raises the prospect that some patients are prone to life-threatening exacerbation. sltas may have its own specifi c risk factors that are distinct from the risk factors for simple hospital admission for acute severe asthma. by defi ning risk factors for slta within the population of those admitted to hospital with acute asthma, we may be able to develop specifi c interventional strategies to reduce its occurrence. reported slta risk factors include advancing age, chronic severe asthma, increased infl ammation markers, asthma exacerbated by pneumonia and low nutritional status. other risk factors include recent hospital admission, prior intubation, steroid dependence, non-adherence to inhaled corticosteroids, psychological or psychosocial problems, lack of access to medical care, lower fev1, and current cigarette-smoking. a specifi c phenotype of severe brittle asthma has been reported. the backbone of er management for slta includes quick assessment of severity, oxygen therapy, early use of systemic steroids and repetitive saba bronchodilator administrations. enhancements include the use of saba with high intrinsic effi cacy, the addition of ipratropium in refractory cases and the concurrent administration of inhaled corticosteroid for its non-genomic, airway edema-reducing effect. mgso4 can also be considered for refractory cases. when ventilator support is needed, niv may be attempted in some patient. for intubated cases, the ventilatory strategy includes low tv and rr, high i : e ratio, and monitoring for the development of dynamic hyperinfl ation. the burden of the slta problem can be mitigated by identifying its phenotype a priori, providing preventive therapy before actual exacerbation occurs and putting in the er/icu the appropriate treatment protocols. journal compilation © 2010 asian pacifi c society of respirology available treatment of asthma using inhaled corticosteroids and long-acting inhaled β2-agonists (labas) is highly effective and safe. importantly, it is also relatively inexpensive. however, many patients remain poorly controlled despite the use of optimal treatment. most advances in asthma therapy have been achieved by improving these drugs and more recently several promising once-a-day labas have been developed. new corticosteroids are also being developed with differential effect on trans-activation and trans-repression of pro-infl ammatory transcription factors, thus giving them a better therapeutic index. the big challenge in asthma is posed by corticosteroid unresponsiveness which is relative and therefore requires high doses to achieve symptom control which inevitably leads to side-effects. one option being pursued is to develop activators of the nuclear enzyme histone-deactylase (hdac)2 which is recruited to the gene initiation site of pro-infl ammatory mediators. there is an increasing appreciation that asthma is not a single disease and is increasingly seen as a syndrome consisting of several phenotypes. so far, two relatively clear subsets have been identifi ed: eosinophilic and neutrophilic forms of asthma. with this notion in mind, attempts are being made to develop more-specifi c inhibitors for a range of mediators with the hope that sub-phenotypes of asthma will be identifi ed that respond well to either single mediator inhibitors or a combination of these. a number of cytokine modulators have been tested in clinical trials, the most notable example being anti-tnf inhibitors which is felt to be more relevant to neutrophilic asthma. unfortunately, large clinical trials with tnf inhibitors have not found them to be very effective. treatment with blocking antibody for the eosinophils growth factor, il-5, has been slightly more effective, with early clinical trials showing that the treatment reduces the frequency of exacerbations in patients who have eosinophilia. whilst the exact mechanisms leading to the development of these two subphenotypes is not fully understood, it is thought that eosinophilia represents a risk factor for exacerbations which has led to eosinophils counts in sputum being used as a guide to treatment; this has been benefi cial in reducing exacerbations. neutrophilic forms of asthma represent a special challenge because patients with neutrophilia tend not to respond well to corticosteroids, making them reliant on bronchodilators. such patients' asthma may be driven by mechanisms that involve il-17 which induces the production of neutrophil chemoattractants by the epithelium, which makes il-17 and its chemo-attractant axis a target for novel therapies. the major unmet need in asthma is the treatment of infections. there are early indicators of antibiotic treatment (macrolides) being effective in the treatment of severe asthma. but the real hope comes from novel strategies aimed at the effects of viruses which are the cause of most acute exacerbations, both in milder and more severe forms of disease. recent studies have identifi ed a defi ciency in type i interferons (ifn), the production of which by the bronchial epithelium -the prime target of virus infection -has been shown to be reduced when epithelial cells from asthmatic are grown in culture and infected ex vivo. in the acute care setting, niv must usually start without delay to avoid further deterioration and an increased likelihood of failure. thus, the decision to start must be made quickly based on a bedside assessment. i recommend a simple two step process, the fi rst of which is to assess the patient's need for ventilatory assistance. if the patient has increased dyspnea (moderate to severe) and evidence of increased work of breathing including tachypnea (>24/min in obstructive diseases and >30/min in hypoxemic respiratory failure), increased accessory muscle use or abdominal paradox, the patient needs ventilatory assistance. arterial blood gases are helpful in making this assessment, but i discourage awaiting blood gas results before starting if the need is obvious, because the window of opportunity may close if initiation is too delayed. i do recommend obtaining baseline blood gases, however, and using them for comparison with later measurements to make certain that the patient is responding. the second simple step is to make sure patients have no contraindications to niv. these include patients with a need for immediate intubation by virtue of a respiratory arrest, hypotensive shock, or uncontrolled arrhythmias or upper gastrointestinal bleeding. the inability to fi t a mask because of a facial deformity, recent facial surgery or burns is also a contraindication. relative contraindications include agitation that prevents the patient from tolerating the mask, increased secretions or diminished ability to protect the airway. patients with these contraindications are at increased risk of failure if placed on niv and should be promptly intubated. patients with multiple risk factors for niv failure should be started only by experienced personnel under very close monitoring. in patients with hypercapneic respiratory failure, these include higher acute physiology scores, marked tachypnea, greater acidemia at baseline and a worse neurological score. in hypoxemic respiratory failure, risk factors for niv failure include the diagnosis of ards or pneumonia, greater age, hypotension and the failure to improve oxygenation substantially within the fi rst hour. although patients at high risk of niv failure can still be given a trial if the clinicians judge it to be indicated, but they must be watched very closely in an icu, with plans to intubate if there is no improvement within the fi rst hour or two. just as the decision to endotracheally intubate a patient in respiratory failure is a clinical judgment that requires the consideration of multiple factors, so is the decision to implement noninvasive ventilation. in the largest rct to date, cpap and nppv performed similarly, both improving dyspnea scores and ph more rapidly than with oxygen alone, but neither lowered intubation nor mortality rate (the major outcome variable) compared to oxygen-treated controls. however, this study enrolled patients whose intubation rate was slightly below 3% in all of groups, including controls, suggesting that they were too mildly ill to manifest a signifi cant mortality benefi t. meta-analyses of the rcts on cpap or nppv compared with o 2 therapy alone have confi rmed the benefi ts described above, even showing a signifi cant mortality benefi t with cpap. meta-analyses comparing the 2 modalities show equivalency of nppv and cpap with regard to reduction of intubation, lengths of stay and mortality, and with similar myocardial infarction rates. therefore, by virtue of its greater simplicity and potentially lower cost, cpap alone is generally regarded as the initial noninvasive modality of choice for cardiogenic edema patients. but because some studies have found that nppv reduces dyspnea and improves gas exchange more rapidly than cpap alone, nppv is preferred by some initially and can be substituted for cpap if patients treated initially with cpap remain dyspneic or hypercapnic. the success of noninvasive positive pressure to treat cardiogenic pulmonary edema has encouraged its extension into the pre-hospital setting. an emerging trend is to provide cpap devices on ambulances for initial therapy of cardiogenic pulmonary edema, a practice that has been associated with decreased need for intubations in the fi eld. patients with advanced chronic obstructive pulmonary disease (copd) experience poor quality of life and very high levels of symptom burden, including intractable shortness of breath, activity limitation, fatigue, social isolation, anxiety and depression. many of the these burdens are shared with caregivers, and resources in the community to support individuals and their families with chronic illness in the community are often lacking. with the recognition that patients with advanced copd and their caregivers have so many unmet needs, there is a growing acceptance for the need to improve the care and quality of life for patients with advanced copd. while signifi cant gaps in our knowledge and understanding of this area remain, factors contributing to these adverse experiences will be discussed. the importance of prevention, relief, reduction, and soothing of symptoms, without affecting a cure, will be emphasized as an integral component of the care provided for these patients. techniques and tools to optimize the care of patients with advanced copd, including optimizing pharmacologic therapies, inter-professional team care, anticipating and appropriately initiating end-of-life planning, patient and caregiver advocacy, as well as timely and effective communication will be reviewed. withdrawal or withholding life support in medically futile cases has been recognized as an ethical and a legal procedure. it is based on the inherent right of a person to autonomy in making health care decisions. the western model however may not apply to the asian setting being widely varied in terms of cultures, religions and economic progress. more than an individual decision, it may actually be a communal decision with a heavy reliance on input of relations, especially the elders. life support withdrawal often entails complete discontinuation of all measures. efforts to avoid feelings of guilt or abandonment may make families opt for partial withdrawals even when they are not shown to be any more benefi cial. studies have shown that distrust with the medical system does play a major role. active discussions may be diffi cult with reticent cultures or when there are gender differences between patients or their families and the physicians. in this era of globalization and migrations, an understanding of these differences may minimize potential confl icts that arise out of these discussions. awareness that the western approach may not fi t the asian medical model allows the health care providers to be sensitive to the needs and wants of their patients and their families. it is hoped that the data reviewed spurs the development of asia pacifi c guidelines that try to fi nd some uniformity in the diversity of the region. screening for lung cancer is not currently recommended by any major medical organization. multiple phase ii non-randomized trials of computed tomography (ct) screening have yielded enticing results. they have demonstrated that ct screening detects smaller size lung cancer of 12-15 mm in diameter. it has been documented that the chest radiographs miss 70-80% of the cancers detected by screening ct. in prevalence studies, 60-80% of detected cancers are stage i. when ct screening results were compared to a validated control group, ct has been shown to detect 3 times more lung cancer than would be expected and results in ten times more thoracic operation than would be expected. additionally, no decrease in advanced stage cancers or decrease in lung cancer deaths were observed. to date, multiple small randomized controlled screening trials (rct) have been reported, but they have been too small to assess if ct screening reduces mortality. a meta-analysis of baseline fi ndings from six small randomized controlled trials observed more stage i and more total lung cancers in the ct screened group. for every 1000 individuals screened with low dose ct, 9 stage i nsclc and 235 false positive nodules were detected and 4 thoracic operations were performed for benign nodules. the two large rct of ct screening that may defi natively answer the question of ct screening and its ability to decrease lung cancer mortality are the national cancer screening trial (nlst) and the nederlands-leuvens longkanker screenings onderzoek (nelson) trial. mortality results from those two trials are anticipated in 2011 and 2015 respectively. a recent report from the nelson trial validated the use of ct volumetric assessment of nodules to assess malignancy and determine which nodules should be treated surgically. currently, there is considerable effort to identify susceptibility genes for lung cancer with particular interest in 15q 24-25 which is strongly associated. this region contains several genes of interest, including three genes that encode nicotinic acetylcholine receptor subunits. however, these genes may just be associated with nicotine dependence. a recent report utilizing gwas (genome wide association scan) methodology identifi ed 2 snps at 13q31.3 associated with lung cancer susceptibility in never smokers. an enormous research effort is underway related to biomarkers in airway epithelial cells, blood, sputum, breath, and urine for early diagnosis or prediction of high risk. intense efforts are devoted to develop models of risk for determining which individuals should be offered screening. journal compilation © 2010 asian pacifi c society of respirology sy 16-02 lung cancer is the leading death-related cancer worldwide. molecular targeted therapy appears to be an alternative approach for patients with non-small cell lung cancer (nsclc). the epidermal growth factor receptor (egfr) is one of these targets, responsible for the cell growth, proliferation, apoptosis and metastasis of the tumors. egfr-tyrocine kinase inhibitor (tki) has been applied to target egfr and suppress the development of tumors. some egfr-tkis, including gefi tinib and erlotinib, have been approved, while the others are still under development or in clinical trials. several studies demonstrated that egfr somatic mutations might predict the high response rate and greater survival benefi t of egfr-tki. in addition, egfr amplication, k-ras mutation, met amplication or the egfr t790m mutation might predict the clinical effect of these drugs. both erlotinib and gefi tinib have been undergoing several clinical trials for nsclc treatment as a single drug or in combination with chemotherapy. br.21 trial showed that erlotinib improved survival with 731 previously treated nsclc patients in a randomized multicenter during phase iii study. thus, erlotinib was approved to be the second or third-line treatment of advanced nsclc patients. however, isel failed to demonstrate a statistically important benefi t of gefi tinib in overall survival as compared with placebo. different study population, dosing and drugs of br.21 and isel might explain the different results. in ipass trial, 1217 clinical selected nsclc patients with asian origin and characterised by adenocarcinoma histotype were treated with gefi tinib or paclitaxel/carboplatin doublets as the fi rst line therapy. the results showed that gefi tinib had the superiority in terms of progression free survival (pfs) in patients with egfr mutation. in eortc 08021 and perol trials, gefi tinib and erlotinib maintenance therapy showed the trend of improved pfs, but not overall survival in advanced nsclc patients. the toxicity of gefi tinib and erlotinib includes diarrhea, rash, etc., which can be well-tolerated. novel egfr-tkis include vandetanib, sorafenib, sunitinib, and cediranib, of which some are under evaluation in phase iii trials as monotherapy or in combination with standard chemotherapy. vandetanib targets both egfr and vegfr and was tested in the second phase trial, suggesting the addition of vandetanib to the single chemotherapy might improve response rates and survival. sorafenib has been applied to different carcinoma histology and in combination with different chemotherapy. when combined with paclitaxel and carboplatin to treat patients with squamous cell cancer, no survival benefi t was seen. however, another clinical trial was launched to investigate the effect of sorafenib, in which squamous cell cancer were not eligible. novel egfr-tkis are under development with hope of overcoming resistance to egfr-tki gefi tinib and erlotinib. there is a great need of further clinical trials. egfr-tki is one of the important alternatives in treatment of nsclc and has shown promising potential in the future. more promising results may come out if the combination and sequence of egfr-tki with traditional therapies, like chemotherapy, radiotherapy and surgery, can be optimized. there is also a need of disease-specifi c biomarkers to predict the effect of the drugs and identify the patients most likely to benefi t from the drugs. lung cancer is the number one cause of cancer death. most cases are found after distant metastasis, and outcome of drug therapy for these patients used to be disappointing. however, we have faced a new paradigm shift, i.e., the molecular targeted therapy and the individualized therapy. many promising data has reported from not only western countries but also asian countries such as the effi cacy of egfr tki to tumors with mutated egfr, that of alk inhibitor to tumor with eml4-alk fusion protein, and that of pemetrexed to non-small non-squamous cell lung cancers. new questions have emerged from these new evidences derived from some important clinical trials. among them, questions regarding with ethnic difference would be one of the most important issues. is survival data same between asian and caucasian? (data from japan lung cancer registry study as well as some global trials have shown survival of asian patients with lung cancer appears to be obviously better than that of caucasian.) why egfr mutation is frequent in asian patients? is only egfr gene status related with prognosis of lung cancer? what would be the cause of alk abnormality? is the criteria of pathological diagnosis for lung cancer same between asian countries and western countries? here, newest evidences for treatment of lung cancer will be presented, and importance of ethnic difference and asian trials will be discussed. the burden of chronic obstructive pulmonary disease (copd) is growing. despite these growing numbers, many patients with patients with copd remain undiagnosed, the greatest number being those with milder disease. delays in the diagnosis of copd are common. evidence suggests that patients with mild copd experience increased symptoms, reduced activity levels and exercise capacity, and impaired health-related quality of life. this growing body of evidence has made it clear that mild copd is not 'normal'. with recognition of this reality and efforts to appropriately recognize copd at an earlier stage, clinicians must be aware of the various therapeutic options for their patients. the defi nition of mild copd will be discussed, as well as effective strategies for the targeted early diagnosis of copd. the numerous and varied disease manifestations and consequences for patients with milder copd will be reviewed. in addition, practical and effective management options available to clinicians caring for patients with mild copd will also be examined. clinicians have been long aware that neither the traditional distinctions of "emphysema" versus "chronic bronchitis" nor the traditional clinical phenotypes of "blue bloater" and "pink puffer" are suffi cient to categorize patents that suffer from chronic obstructive pulmonary disease (copd). recently, the global initiative for chronic obstructive lung disease (gold) workshop has used quantitative measures (fev1 and fev1/fvc ratio) to defi ne copd, but this defi nition fails to take into account the full heterogeneity of copd. with an increased understanding of pathophysiologic variation, copd now clearly represents a spectrum of overlapping diseases with important extrapulmonary consequences. a "phenotype" describes the outward physical manifestations of a particular disease, and comprises anything that is part of the observable structure, function or behavior of an individual. such phenotypic distinctions in copd include: frequent exacerbator, pulmonary cachectic, rapid decliner, airways hyperresponsiveness, impaired exercise tolerance, and emphysema versus airways disease. these variable manifestations, each with unique prognostic, clinical and physiologic implications, represent distinct phenotypes within copd. while all of these phenotypes have smoking as a common risk factor, the other risk factors that determine these phenotypes remain poorly understood. an individual smoker has variable expression of each phenotype and there is mounting evidence that copd phenotypes have different clinical outcomes. these phenotypes can be broadly classifi ed into one of three groups: clinical, physiologic and radiographic. thus, the paradigm that copd is one disease may be incorrect, and suggests that copd should be considered as a spectrum of smoking-related diseases. failure to consider copd phenotypes is likely to limit the power of therapeutic trials since not all copd patients are likely to benefi t from each therapy. the challenge to future copd researchers is to better characterize these phenotypes and identify their risk factors. measurement of fractional exhaled nitric oxide (feno) is an attractive biomarker of diseases where airway eosinophilia dominates. indeed even before any randomized controlled trials were published some were advocating treatment tailored in accordance to feno data. commercially available bench-top and portable feno analyzers are now readily available and in some countries, feno measurements attract a payment. however, despite the ease of measuring feno, it has its drawbacks in biological and measurement issues. biologically feno is signifi cantly infl uenced by atopy, intake of caffeine, exercise, ethnicity, etc on the measurement front, variabilites include: feno measured by different analyzers may provide different values and on-line vs off-line measurements. the cut-off for determining 'abnormally high results' is yet unknown. not surprisingly, there is discordance on the effi cacy of tailoring asthma medications in accordance to sputum eosinophils [1] and feno [2] in people with asthma, although both are eosinophilia infl ammatory markers. tailoring of medications in accordance to sputum eosinophilia (compared to standard practice) significantly reduced exacerbations in adults with asthma (odds ratio 0.47, 95%ci 0.28, 0.87). in contrast, the benefi t of tailoring of medications in accordance to feno was, at best, modest. the utility and limitations of using feno levels in the clinical setting will be discussed in this talk. shortness of breath and activity limitation are cardinal symptoms experienced by patients suffering from respiratory illness or disease. cardiopulmonary exercise testing (cpet) allows for the objective evaluation of these symptoms, recognizing that exercise involves the effective integration of respiratory, cardiovascular, neuromuscular and metabolic functions. the organs involved in these varied and important roles have a sizeable reserve, with the consequence that clinical manifestations of a disease state or abnormality may not become readily apparent until the functional capacity of the organ(s) is markedly impaired. objective assessment of various parameters during exercise, which places an increased physiologic demand on the functional reserve capacity of these organs, can provide a sensitive method for the early detection of abnormal function and responses(s). the results from exercise testing also parallel functional capacity and quality of life more closely than measurements obtained only at rest, and have been shown to accurately predict important outcomes, such as mortality, in a variety of patients and clinical circumstances. a brief overview of normal exercise physiology and characteristic responses demonstrated by patients with various disorders frequently assessed by the pulmonologist will be offered. in addition, a summary of the indications, conduct, and practical interpretation of cpet will be presented in this session. effect of tiotropium on outcomes in patients with moderate chronic obstructive pulmonary disease (uplift): a prespecifi ed subgroup analysis of a randomised controlled trial obstructive lung disease and low lung function in adults in the united states: data from the national health and nutrition examination survey international variation in the prevalence of copd (the bold study): a population-based prevalence study chronic obstructive pulmonary disease in fi ve latin american cities (the platino study): a prevalence study prevalence of copd in spain: impact of undiagnosed copd on quality of life and daily life activities global burden of copd: systematic review and metaanalysis prevalence of chronic obstructive pulmonary disease in china. a large, population-based survey diagnostic labeling of chronic obstructive pulmonary disease in fi ve latin american cities copd prevalence in a random population survey: a matter of defi nition treatment of copd: the sooner the better clinical copd phenotypes: a novel approach using principal component and cluster analyses offi ce spirometry signifi cantly improves early detection of copd in general practice: the didasco study salmeterol and fl uticasone propionate and survival in chronic obstructive pulmonary disease clinical trial design considerations in assessing long-term functional impacts of tiotropium in copd: the uplift trial a 4-year trial of tiotropium in chronic obstructive pulmonary disease mortality in the 4-year trial of tiotropium (uplift) in patients with chronic obstructive pulmonary disease effi cacy of salmeterol/fl uticasone propionate by gold stage of chronic obstructive pulmonary disease: analysis from the randomised, placebo-controlled torch study effects of tiotropium on outcomes in patients with moderate chronic obstructive pulmonary disease (uplift): a prespecifi ed subgroup analysis of a randomised controlled trial tiotropium as a first maintenance drug in copd: secondary analysis of the uplift trial treating tobacco use and dependence: 2008 update. clinical practice guideline tobacco atlas the mpower package. geneva, world health organization implementing smoke-free environments. geneva, world health organization curbing the epidemic: governments and the economics of tobacco control clinical and public health signifi cance of treatments to aid smoking cessation we report that pulmonary emphysematous lesions appear to be a dynamic phenomenon that involves not only the gradual loss of alveolar structure, but apoptosis, cellular proliferation, and cellular senescence as well. cellular proliferation compensates for increased alveolar cell apoptosis in chronic obstructive pulmonary disease (copd) patients. however, smoking, age, and the increased cell cycle turnover that compensates for apoptosis accelerate alveolar cell senescence, thereby halting cellular proliferation and tipping the balance toward apoptosis, which, in turn, promotes the formation of emphysematous lesions. as a result, alveolar cells disappear and the emphysematous lesions progress. at the same time, cellular senescence is thought to induce infl ammation. more specifi cally, senescent alveolar cells induce infl ammation by producing various infl ammatory cytokines in tissue. lymphocytes and clara cells may also age more rapidly in the lungs of copd patients. lymphocyte senescence may induce an autoimmune reaction and increase susceptibility to infection, and clara cell senescence may impair airway regeneration as well as sustain airway infl ammation. thus, cellular senescence may be involved in arrested tissue repair, chronic infl ammation, and increased susceptibility to infection, which are the typical features of copd. there is increasing recognition that copd is an increasing global burden. new drug treatments continue to emerge suggesting that copd is more responsive to treatments than previously thought. however, there is still much that is unknown about copd that will contribute to further advances in treatment and management. pulmonary imaging can contribute by providing information on how structure and function relate to relevant clinical parameters, such as disease progression, treatment responses and exacerbations. in other words, imaging can help characterise copd in terms of clinical outcomes or phenotypes. there have been many advances in imaging methodology, including ct, mri, spect and pet. recent fi ndings from research studies using innovative methods of studying structure and particularly function, in copd will be reviewed. their clinical implications will be discussed. the spectrum of children's interstitial lung disease (child) encompasses a large, heterogeneous group of pediatric diffuse lung disorders that are diffi cult to diagnose and treat. as the differential diagnosis is large, a systematic approach is needed for accurate diagnosis. the classic fi rst step of obtaining a detailed history and performing a careful physical examination remains essential for providing diagnostic clues as well as assessing severity of illness. as examples, a history of hemoptysis and fatigue would suggest an alveolar hemorrhage syndrome; exposure to avian antigens, hypersensitivity pneumonitis; and a history of adenovirus pneumonia, bronchiolitis obliterans. the presence of growth failure, crackles, loud p2, and clubbing on physical examination would point to a severe and progressive lung process with cor pulmonale. recent advances in diagnostic modalities have greatly improved the ability of clinicians to identify these disorders. in infants and children with diffuse lung disease, genetic testing can be diagnostic for surfactant dysfunction mutations (sp-b, sp-c, abca3, ttf-1, gm-csfra receptor). infant lung function testing has proven useful for assisting in the diagnosis of certain disorders, such as neuroendocrine cell hyperplasia of infancy (nehi) and distinguishing nehi from surfactant mutations. hrct may detect extent and severity of disease, but can also be useful in diagnosing specifi c disorders, such as nehi (symmetric ground glass densities in the right middle lobe and lingula and the central lung regions), bronchiolitis obliterans (mosaic perfusion, vascular attenuation, and central bronchiectasis), hypersensitivity pneumonitis (ill-defi ned centrolobular micronodules), and pulmonary alveolar proteinosis (crazy-paving). bronchoalveolar lavage can aid in the diagnosis of specifi c conditions, such as alveolar hemorrhage syndromes (hemosiderin-laden macrophages), aspiration (lipid-laden macrophages), hypersensitivity pneumonitis and sarcoidosis (lymphocytosis), eosinophilic pneumonias (eosinophilia), and histiocytosis (cd 1a + cells). lung biopsy performed by video-assisted thoracoscopic surgery (vats) has largely supplanted conventional open lung biopsy as the procedure of choice as it is equally accurate, but associated with less morbidity. although lung biopsy remains the gold standard for diagnosis of child, it must be interpreted in the context of the clinical and radiologic fi ndings. it should be emphasized that although lung biopsy can be diagnostic in some disorders, such as bronchiolitis obliterans, it may not be necessary because less invasive studies such as hrct may be suffi cient for diagnosis. finally, some pulmonary vascular disorders, such as pulmonary vein stenosis or atresia, may mimic child. for these disorders, echocardiography, mra, or cardiac catheterization may be required for diagnosis. with a systematic approach and improved diagnostic capabilities, it is reasonable to expect that a specifi c diagnosis can now be made in the vast majority of child cases. key: cord-008510-mnpu27kl authors: lipscomb, mary f.; bice, david e.; lyons, c. richard; schuyler, mark r.; wilkes, david title: the regulation of pulmonary immunity date: 2008-04-10 journal: adv immunol doi: 10.1016/s0065-2776(08)60634-3 sha: doc_id: 8510 cord_uid: mnpu27kl thechapter describes the cells and structures of the lung that participate in pulmonary immunity and how the lung responds to challenges fromforeign antigens, with particular emphasis on animal models that have been developed to explore these issues. some ligands-receptor interactions are specific while others are not, and it is the particular pattern of surface molecules and secreted factors expressed by interacting immune cells that determines the type of immune response that develops during central processing. the cells that are the major initiators and regulators of immunity in the lung include macrophages, dendritic cells (dcs), and lymphocytes, each expressing surface molecules and secretory products that depend on perturbations in the environments. immune cells and structures of the lung and lung immunity to noninfectious particulate and soluble antigens are discussed. several models for regulation of pulmonary immunity such as models for immunity in lung infections, models for hypersensitivity lung disease, models for lung transplantation, and graft versus host are also presented. demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immune-suppressed hosts, and to suppress manifestations of immunologically mediated lung disease. the human lung is exposed daily to over 10,000 liters of inspired, ambient air and the continuous aspiration of small amounts of nasopharyngeal secretions during sleep (kikuchi et al., 1994) . depending on the quality of air in the environment or the resident flora in the nasopharynx, the respiratory tree faces the enormous task of oxygenating blood across a moist, thin alveolar-capillary wall (approximately 1 pm) and yet resisting infection. mechanical mechanisms and other innate host defenses are important in preventing infection, but acquired immunity is essential to prevent recurrent and chronic infection as made strikingly evident by the increased numbers and severity of pulmonary infections in immunocompromised hosts (newhouse et al., 1976; mason and nelson, 1992) . the lung, as is true of other epithelial surfaces that interface with the environment, has developed several strategies to avoid infection. as an important component ofthese strategies, the host must be able to downregulate both nonspecific and immune-mediated inflammation. failure to regulate local immunity results in diseases such as asthma, hypersensitivity pneumonitis, and perhaps sarcoidosis and idiopathic interstitial pneumonitis (holt, 1993; djukanovic et al., 1990; o'connor and fitzgerald, 1992) . thus, the desire to enhance protective pulmonary immune responses by vaccination and to prevent or control unwanted responses underlie the need to study basic immune mechanisms in the lung. furthermore, understanding critical immunoregulatory mechanisms may lead to strategies for preventing and controlling lung transplant rejection and immune-mediated lung damage in bone marrow transplant patients. a number of recent reviews have discussed important issues in the development of pulmonary immune responses (agostini et al., 1993; hance, 1993; lipscomb et al., 1993a; gyetko and toews, 1993; holt, 1993; bice, 1993) . the goal of this chapter is to describe the cells and structures of the lung that participate in pulmonary immunity and to summarize studies that help explain how the lung responds to challenges with foreign antigens, with particular emphasis on animal models that have been developed to explore these issues. features of the immune apparatus that are unique to the lung will be highlighted, and important questions currently under investigation will be indicated. an important challenge is to understand how the host protects itself from infection yet regulates immunity to prevent tissue damage. tissue culture has been a powerful tool for understanding how immunologically relevant cells interact. the study of how various extracellular signals influence gene expression in cultured cells has given important insight into how the milieu could influence cell behavior at various anatomic sites. however, because it is not yet possible to know what all of the influences within a tissue are, hypotheses generated from cells in culture must be tested in uiuo. for example, alveolar macrophages (am) exist attached to epithelial cells and migrate within a layer of surfactant rather than attached to plastic in a layer of medium. a recent review in this series discussed the importance of placing lymphocytes in their spatial context within the host to properly understand their function (kroemer et al., 1993) . this consideration is especially important in the lung. in considering the development of pulmonary immunity in a spatial context, it is useful to divide the evolution of an immune response in the lung into three distinct but overlapping phases (see fig. 1 ; lipscomb, 1993a) : (1) in the afferent phase, antigen reaches the lung, is fig. 1. a model for afferent, central processing, and efferent phases of a pulmonary immune response. in the afferent phase (aff), antigen that reaches bronchoalveolar spaces can directly enter lymphatics (l) or be processed by intraepithelial or interstitial dendritic cells (dc) that enter lymphatics and migrate to lung-associated lymph nodes (laln). in the central processing phase (cp) antigen on lung dcs (or, perhaps, antigen processed by resident lymph node dcs) present antigen to t lymphocytes (t) to initiate the expansion of t cell clones that, in turn, may help b cells expand when b cell immunoglobulin receptors recognize "native" antigenic determinants. in the efferent phase (eff), recently activated t cells and b cells (not shown) leave lalns and circulate; they are subsequently recruited to the lung at sites of inflammation. exposure of t cells to relevant antigen results in the release of cytokines that amplify an inflammatory response by recruiting nonspecific effector cells, such as monocytes (m), to the site and activating them to kill and/or growth-inhibit microorganisms. take up by antigen-presenting cells (apcs), and presented to naive t cells expressing the relevant t cell receptors (tcrs); (2) in the central processing phase, specific lymphocyte clones are expanded and differentiate; and (3) in the effector phase, effector t cells and b lymphoblasts find their way to pulmonary sites requiring expression of a specific immune response. at each phase, events must be tightly regulated to allow an effective immune response yet avoid excess, potentially destructive inflammation. the location in the respiratory tract where each of these phases occurs is somewhat controversial, but the bulk of evidence indicates that in the normal host after reaching the lungs, antigen is carried on apcs, in phagocytes, or free in lym-phatic fluid to draining lung-associated lymph nodes (lalns) (lauweryns and baert, 1976-1977; lehnert, 1992) where central processing occurs. effector cells are released into the efferent lymph and reach the blood stream where they are recruited from the vasculature into the lung (berman et al., 1990) . many investigations have considered the lung in the broader context of mucosal immunity. this conceptual framework is useful, but the respiratory tract has several distinctive features that require that initiation and expression of lung immunity be considered separately from immune responses at other mucosal sites. the respiratory tract shares features of other organs, i.e., skin, gut, and urogenital tract in which an epithelial layer interacts with the environment. for internal organs, the concept of a common mucosal system was developed based on evidence that immune lymphocytes generated at one surface migrated to both homologous and distant mucosal sites (mcdermott and bienenstock, 1979; mcghee et al., 1992) . we will return to this concept shortly, but in addition to this concept, the lung must be understood immunologically from the point of view that both an upper and lower respiratory tract system exist (kaltreider, 1976; kazmierowski et al., 1977) , and each system exhibits distinctive as well as common immune mechanisms. the upper respiratory tract starts at the nares and extends to the level of the terminal bronchioles. a pseudo stratified to single layered columnar epithelium covers vascularized connective tissue, the lamina propria, which, depending on the level of the airway, also contains variable numbers of mucous glands, smooth muscle, and, in the larger airways, is bounded by cartilage. by contrast, in the lower respiratory tract (which by definition includes the alveolar ducts and alveoli), the epithelium is markedly attenuated and frequently separated from the pulmonary capillary endothelium by only a fused basement membrane. the mechanisms of antigen handling and the types, location, and numbers of immunologically relevant cells differ even within areas of the upper respiratory tract, but are most strikingly different between the upper versus lower respiratory tracts. for example, mucociliary clearance is the major mechanism for clearance of particulates in the upper tract. in contrast, phagocytosis by resident am which subsequently attain the level of the upper tract to be removed by the mucociliary elevator characterizes particulate clearance in the lower tract (lauweryns and baert, 1976-1977; lehnert, 1992) . another distinctive feature of the upper versus lower tracts relates to the organization of lymphoid tissue. in the upper tract, lymphocytes reside in both aggregates and diffusely distributed along the mucosa of the upper tract, and in some animal species may infiltrate the epithelium (bienenstock et al., 1973a,b; sminia et al., 1989) . in the lower tract, lymphocytes are present in variable numbers both within alveoli and in the interstitium, but, in the normal host, organized aggregates do not occur (sminia et al., 1989; pabst, 1992) . other important features of the upper versus lower tract involve the relative importance of iga as the protective antibody. thus, igasecreting b cells occur in the mucosa of the upper tract, and iga is the major immunoglobulin in secretions of the upper respiratory tract; although iga is present in the lower tract, igg and igm predominate in bronchoalveolar lavage (bal) fluids (kaltreider, 1976) . the development of immunity in the lung, as elsewhere, requires that relevant cells display appropriate surface molecules for contact and secrete appropriate factors. some ligand-receptor interactions are specific while others are not, and it is the particular pattern of surface molecules and secreted factors expressed by interacting immune cells that determines the type of immune response that develops during central processing. furthermore, at sites of pulmonary inflammation, other patterns of adhesion molecules and cytokine/chemokine expression by immune and parenchymal cells direct the tempo and magnitude of accumulation of recruited cells. many recent studies emphasize the critical importance of cytokines and chemokines and the expression of adhesion molecules in regulating pulmonary inflammation and immunity (kunkel et al., 1989; stein-streilein and phipps, 1993; redington et al., 1993; standiford et al., 1993; jordana et al., 1993; lukacs et al., 1994a) . the cells that are the major initiators and regulators of immunity in the lung include macrophages, dendritic cells (dcs), and lymphocytes, each expressing surface molecules and secretory products that depend on perturbations in the environments. however, other cells in the milieu, e.g., epithelial cells, fibroblasts, mast cells, and various recruited blood leukocytes, also play important regulatory roles which will only be briefly examined in subsequent sections. first, however, the special feature of lung macrophages, dcs, and lymphocytes are discussed. originally proposed as important apcs in the lung (lipscomb, 1988) , this diverse group of cells is now best understood in the context of lung immunity as phagocytes and as regulators of both immunity and nonspecific inflammation (holt, 1986; lipscomb et al., 1993a) . indeed, the bulk of evidence indicates lung macrophages are unlikely apcs in the initiation ofprimary immune responses. these cells reside within the airways at all levels of the respiratory tract, in the lamina propria, the interstitium, the alveolar regions and pleura, and within pleural spaces; in ruminant species, lung macrophages are found within the pulmonary capillaries (lehnert, 1992; brain, 1992) . all lung macrophages originate from the bone marrow (van oud alblas et al., 1983; springmeyer et al., 1982; godleski and brain, 1972) , but maintenance of at least some of these resident populations partly derives from self-replicating pools (pinkett et al., 1966; bowden and adamson, 1980; sorokin et al., 1984; tarling et al., 1987; shellito et al., 1987) . pulmonary macrophages are both phenotypically and functionally diverse, even within a single compartment. because of ready access to bronchoalveolar macrophages using bal, the diversity of these cells has been most frequently studied. macrophages obtained by lavage include resident am as well as macrophages that reside within the lumen of the bronchi and bronchioles (intraluminal macrophages); a distinction between these two cell populations cannot be readily made. however, the vast majority of cells obtained during a human lavage are from the alveoli, since the technique is performed with a wedged flexible fiberoptic bronchoscope and the large alveolar surface area relative to the bronchial surface area is sampled (reynolds, 1987; american thoracic society, 1990 ). in small rodents, e.g., mice and rats, a catheter is typically placed in the trachea and a larger proportion of the recovered cells are from the bronchi and bronchioles, but the majority of cells are still from alveoli if the procedure is performed correctly. functional attributes of subpopulations of resident am have been studied. most studies exploit the differences in density (murphy and herscowitz, 1984; shellito and kaltreider, 1985; oghiso, 1987) , predominantly a function of cell size. another fractionation technique depends on differences in the cells' capacity to be readily lavaged, a function of their adherence to epithelium and/or their residence in the bronchial lumen versus in alveoli . within subpopulations, am differ in expression of class i1 major histocompatibility antigens (mhc), fc and complement receptors, phagocytic capacity, responses to chemotactic stimuli, cytotoxicity, cytokine production, and capacity to suppress in vitro immune responses murphy and herscowitz, 1984; shellito et al., 1983; shellito and kaltreider, 1985; oghiso, 1987) . furthermore, the size, function, and phenotype of am shifts during an inflammatory response (van oud alblas et al., 1983) . although alternate explanations for these changes have been offered, the most likely one is that am recently arrived from the peripheral blood are similar in the size and phenotype to circulating monocytes, the precursors of am. thus, in the study of am and their subpopulations and, indeed, in any study of lung immunity, it has become a dictum that investigators must be careful to avoid low levels of chronic inflammation by housing experimental animals in specific pathogen-free environments. because respiratory infections are so common in nearly all animal species and can markedly affect experimental results, this consideration cannot be overstated. interstitial macrophages (im) when compared with am also exhibit size, functional, and phenotypic differences. these differences may reflect the stage of differentiation from blood precursors, but more likely reflect the environment and physiological roles of phagocytes in these two distinct locations. important membrane molecules that determine the function of macrophages include complement, fc, mannose, and scavenger receptors, as well as class i and i1 mhc, adhesion, and other signaling molecules. important differences in many of these exist depending on the location of lung macrophages. for example, in mice, im express c3 receptors, whereas most resident am do not (van oud alblas and van furth, 1979) . few studies exist on the role of interstitial, pleural, or intravascular lung macrophages in immune responses. however, numerous studies have examined the role of am in stimulating mitogen, alloantigen, and antigen stimulated responses in vitro. not surprisingly, these studies have often reached conflicting conclusions, a result of the differing animal species used, the level of superimposed am suppressive activity, and the assay procedure that was used (holt, 1986; lipscomb, 1988) . most studies support the concept that am are poor apc for priming t cells even when they express high levels of class i1 mhc, as human am do (holt, 1979; ansfield et al., 1979; toews et al., 1984a; lipscomb et al., 1986) . bronchoalveolar cells, which contain up to 90% am, fail to act as effective apc in vitro because they are either actively suppressive or because they fail to express some other poorly understood accessory function which may manifest as poor lymphocyte-accessory cell binding (shellito et al., 1983; lyons et al., 1986; kradin et al., 1987) . most current evidence indicates that dcs are the most efficient apc in stimulating naive t cells, especially cd4 t cells, although class i1 mhc-positive cells of many cell types are capable of stimulating recently primed t cells (steinman, 1991; croft, 1994) . variable contamination of cells with lung dcs (pollard and lipscomb, 1990) and the presence of recently activated t cells in responder populations may well explain reports that am can function as effective apc (rich et al., 1987) . mechanisms utilized by populations of bal cells to suppress immune responses are likely to depend on variables of the assay systems and are particularly dependent on animal species. in dogs, pgez production (demenkoff et d., 1980) and the synergistic activity of pgez and oxygen radicals (kaltreider et al., 1986) were shown to be important. in humans, am/lymphocyte contact leading to inhibition of receptor-induced intracellular calcium increases in responder t cells has been described (yarbrough et al., 1991) . resident murine macrophages, particularly at high numbers relative to numbers of stimulator lung dc, suppress a mixed lymphocyte reaction (mlr) by secreting tgfp (lipscomb et al., 1993b) . furthermore, nitric oxide (no) made by murine am may inhibit the development of potent apc cell function of lung dc . evidence that am suppression plays a role in vivo has been indirectly shown by depleting am with an intratracheal dose of liposomes containing a macrophage cytotoxic drug (dichloromethylenediphosphonate) followed by immunizing via the respiratory tract. the animals demonstrated increased numbers of antibody-forming cells (afcs) in lalns compared to immunized controls pretreated with only liposomes (thepen et al., 1989) . igg, iga, and ige afcs were all increased. am-suppressive activity is important to prevent the development ofhypersensitivity reactions, but in circumstances in which lungimmunity is important for protection, am-suppressive activity could be counterproductive. however, suppressive activity of murine macrophages can be inhibited by exposure to gm-csf and to a lesser extent by other selected cytokines (bilyk and holt, 1993) . the major role of ams seems to be to phagocytose and remove potentially dangerous particulates and soluble antigens from the alveoli and to inhibit local lung immune responses. however, immunity may develop in the presence of otherwise suppressive ams by the recruitment of leukocytes to the alveolus with opposing activity or by strong environmental influences that result in cytokine secretion that diminishes am suppressor function. immunologists have long recognized that adherent cells are required for optimal in vitro immune responses whether one measures t cell lymphoproliferative responses, t cell cytokine production, or tdependent b cell responses. they have also developed an increasing appreciation of the role of dcs in priming naive t cells since they were first identified in the spleen (steinman and cohn, 1973) . since then, the role of dcs in immune responses initiated at the epithelial surface is being clarified. little doubt exists that dcs play a pivotal role in initiating immune responses in the skin (reviewed by steinman, 1991) . furthermore, substantial support has developed for the concept that intraepithelial dcs are functionally different from those that have migrated into regional lymph nodes. thus, freshly isolated langerhans cells are capable of processing antigen and stimulating t cell clones, but they require "maturation" in vitro before they are fully effective in stimulating naive t cells in an mlr; at this time they have a markedly reduced capacity to process antigen (romani et al., 1989) . data indicate that lung dcs share a number of characteristics of skin dcs and likely also play a critical role in initiating lung immunity (holt, 1993) . apcs must not only process antigen and express it in the context of class i1 mhc, but also express appropriate accessory molecules to enhance the interaction of the apcs with t cells. recent studies emphasize the importance of the interaction of â�¬37 on the apc cell surface with cd28 and/or ctla4 expressed on responder t cells to deliver a second signal (reviewed by june et al., 1994) . failure to trigger this second signal may cause t cells interacting with apcs via only the tcr-peptide/mhc i1 interface to become anergic (harding et al., 1992; boussiotis et al., 1993; chen and nabavi, 1994) and produce a state of tolerance in oivo (van goo1 et al., 1994) . b7 is a receptor family of at least two molecules in the immunoglobulin supergene family. b7-1, (cd80), and b7-2 (cd86) are both expressed constituitively on dcs in contrast to macrophages and b cells that must be activated to express these molecules (vandenberghe et al., 1993) . other important accessory molecules, usually identified phenotypically by standard immunocytochemistry and functionally by the ability of specific antibodies to block an immune function, include cd40, cd54 (icam-l), and cd58 (lfa-3) (steinman, 1991) . present on many lung dcs, but data are incomplete on the expression of cd40, cd80, and cd86 family. the high levels of constitutive class i1 mhc and the dendritic shape of dcs has been exploited to examine their location in tissue sections of lung by a number of investigators. dcs form an interdigitating network in the airway epithelium of all species in whom they have been investigated (sertl et al., 1986; holt, 1993) similar to the network described for skin langerhans cells. intraepithelial dcs are particularly dense in the trachea and gradually diminish in concentration as the airways branch, but are increased at sites of chronic inflammation . dcs also exist in the connective tissue surrounding bronchi and bronchioles, in perivascular connective tissue, in alveolar septa, in the pleura, and in very small numbers in alveolar spaces (sertl et al., 1986; holt and schon-hegrad, 1987; kradin et al., 1991; havenith et al., 1992; van haarst et al., 1994) . in rats, intraperitoneal injections of ifny increased the numbers of intraepithelial and septa1 dcs without increasing their accessory cell function , and inoculation of bacillus calmette-guerin (bcg) increased the numbers of dc that could be lavaged from the alveolus (havenith et d., 1992) . thus, the numbers of lung dcs in various anatomic sites depend on signals delivered that are coincident with inflammation. lung dcs have been isolated with a variable degree of success utilizing adherence, density, and class i1 mhc expression properties; their function and other phenotypic features have been studied. lung macrophages are the most difficult cells to separate from dcs in single cell suspensions of lung cells. effective procedures for isolating fairly pure dc populations require exploiting the phagocytic and autofluorescent properties of lung macrophages (nicod et al., ,1989a pollard and lipscomb, 1990; havenith et al., 1993a) . using these techniques, the function of dc-enriched lung cells has been assessed. functional assays have included stimulation of periodate-treated lymphocytes, mlrs, and antigen-induced stimulation of either memory t cells or lymphoblasts. all have shown that lung dcs function as well or better than dcs from other sources rochester et al., 1988; pollard and lipscomb, 1990) . dcs isolated from whole lung preparations are phenotypically heterogeneous, and cell markers differ somewhat among animal species and even within individuals of a species. an example of this latter variation relates to cdla (okt6), expressed by human langerhans cells in the skin, which has been variously described as being present on from less than 1% (sertl et al., 1986; nicod et al., 1987) to 30% (van haarst et al., 1994) of lung dcs in man. this antigen may be important in stimulating y6 t cells, a potentially important interaction for host defenses in the lung in view of observations that y6 t cells may recognize heat-shock proteins of mycobacterium tuberculosis (mtb) (born et al., 1991, kaufmann and kabelitz, 1991) . variation in expression of cytoplasmic and surface markers also occurs within populations of lung dcs. for example, in the mouse, the interdigitating cell antigen (nldc-145) and cr3 are present on about half of lung dcs, while the majority of lung dcs express cd25 and the heat-stable antigen (as defined by j11d; pollard and lipscomb, 1990) . the latter two markers are also uniformly expressed on murine thymus and skin dcs, but are absent from the majority of splenic dcs , indicating the likelihood of a closer relationship of lung dcs to tissue dcs rather than to dcs that primarily home from bone marrow to lymphoid tissue. additional evidence that lung dcs are distinct from the majority of splenic dcs is that lung dcs fail to express the splenic dc marker recognized by the monoclonal antibody 33d1 (pollard and lipscomb, 1990) . in both rat and mouse, another heterogeneous marker is fcrii, positive on about half of lung dcs (pollard and lipscomb, 1990; xia et al., 1991) . interestingly, langerhans cells freshly isolated from the skin express fcrii which is downregulated as the cells mature in culture. thus, it is possible that the fcr expression in the lung denotes a population similar to freshly isolated skin dcs. in the rat, dcs in the epithelium lining the airways are more likely to be fcr+ , whereas parenchymal dcs are nearly uniformly fcr. murine dc in the epithelium of the trachea also express fcrii (sertl et al., 1986) . this suggests the possibility that intraepithelial dcs are poised to take up and process antigens at the epithelial/environment interface and enter the interstitium to traffic to laln. thus, at least some interstitial dcs which fail to express fcr may be in transit. on the other hand, the presence of fcr-dcs in lung parenchyma, including alveolar septa, may indicate distinct immune functions for this subset of lung dcs. no difference was found in mice in the capacity of the fcr+ or fcr-subsets to stimulate an mlr (pollard and lipscomb, 1990) . however, in the rat, the fcr-population was a more potent stimulator of naive t cells in an mlr and in responses to lectins, but fcrand fcr+ were equally capable of presenting soluble and particulate antigens to antigen-sensitized t cells (kradin et al., 1993) . of interest in these later studies was that 70% of fcr-and only 39% of fcr+ cells expressed the adhesion molecule cd54 (kradin et al., 1993) , an observation that might partly explain the increased ability of the fcrcells to stimulate an mlr. in elegant studies in which lung intraepithelial dcs were isolated from the rat, these cells were shown to present antigen to primed t cells more efficiently than did parenchymal dcs. in man, further enrichment for fcr+ decreased the ability of lung cells enriched in dc to stimulate an mlr , although these studies are complicated by the large numbers of contaminating fcr+ macrophages in the dc populations. nevertheless, fcr positivity has been a useful marker for identifying subsets of lung dcs and the majority of data are consistent with the concept derived from langerhans cells that a population of fcr+ intraepithelial dcs may take up antigen from the bronchial lumen and differentiate into cells that can stimulate naive t cells upon migration into draining lymph nodes. dcs in all tissue sites originate from the bone marrow (steinman, 1991) . they can be cultured from precursors in bone marrow and peripheral blood using gm-csf and, for adult human dcs, from peripheral blood with il4 (inaba et al., 1992 (inaba et al., ,1993 thomas et al., 1993; sallusto and lanzavecchia, 1994) . in one study, ia+ lung dcs were first recognized in rat lung parenchyma in fetal life at day 15 of gestation . intraepithelial dcs were present by day 17 and continued to increase during postnatal development . comparison of fetal lung dcs demonstrated that they were not as efficient as adult dcs in stimulating an immune response, but were fully functional at birth. in contrast, another study failed to detect ia+ dcs in rat lung epithelium or parenchyma until birth, and the numbers increased rapidly until 3 weeks of age when they approximated those found in adults . ia+ dcs were first observed in the nasal turbinates and intensity of ia staining increased in time, first in the trachea and later in lung parenchyma, compatible with environmental exposure effecting the change. using another marker for dc, ox62 (which also detects y6 t cells), these same authors found cd3-negative, 0x62-positive dcs at all levels of the lung in fetal rat lung and speculated that environmental influences upregulated ia expression and subsequent function of these cells. consistent with this speculation was that ifny increased the numbers of la+ cells in airway epithelium while steroid inhalation decreased the numbers relative to control rats. the conflicting findings of the two groups may be based on the differences in the housing environments for the two groups of rats. thus, nelson and collaborators (1994) used dust-free bedding in contrast to mccarthy et al. (1992) . it was quite likely that in utero influences affected the numbers and function of lung dcs. numerous studies have demonstrated that dcs from spleen, lymph node, and skin have extraordinarily potent ability to immunize recipient animals following inoculation either iv or subcutaneously (knight et al., 1983; mckinney and streilein, 1989; sornasse et al., 1992) . lung dcs have not been used to immunize experimental animals to date, but several lines of evidence suggest that they have an important role in uiuo. as discussed, their location within the epithelium places them in an optimal position to take up and process antigens that breach the epithelial barrier; perivascular and septa1 dcs should be poised to process antigens that reach the lung via the vasculature. studies have shown that splenic dcs instilled into the lung may reach laln (havenith et ul., 1993b) and if pulsed with antigen may induce an immune response (havenith et al., 1993~) . in this latter study, ams pulsed with antigen were also capable of initiating an immune response, but in contrast to dcs heat-killed ams were also able to immunize suggesting that ams stimulated responses by having the antigen reprocessed by the host's own apcs. additionally, explanted lung dcs had gained the ability to stimulate primed t cells following the intratracheal delivery of the relevant antigens . last, consistent with the dynamic activity of epithelial dcs in carrying environmental antigens into laln, irradiation of rats resulted in a loss of 85% of resident dcs by 72 hr and reconstitution of tracheal dcs from bone marrow precursors by 10 days . lung dcs most surely play a major role in regulating lung immunity and, as discussed previously, are likely themselves regulated by multiple environmental factors. it remains to be determine whether antigen presented by lung dcs is more likely to result in th1 versus th2 responses in the lungs and whether bypass of dcs in immunization of the host is more likely to deliver a tolerogenic signal, or whether other factors are more important in determining these outcomes. compartments (holt and schon-hegrad, 1987; stein-streilein, 1988; been described either by evaluating markers in situ or by obtaining cells from bal or collagenase digestion of lung tissue. as with lung macrophages and dcs, lung lymphocytes are a dynamic population with the capacity to enter and leave the lung depending on influences in the milieu. in several species including rat, rabbit, and chicken, large numbers of lymphocytes are located in organized bronchus-associated lymphoid tissue (balt), which is discussed below. however, balt is not constitutive in all species and not regularly seen in hamsters, mice, and humans. in these latter species, the majority of lung lymphocytes are present in the interstitiurn, diffusely scattered in the mucosa, alveolar septa, or pleura. also, depending on the species, there may be an intraepithelial and/or an intravascular pool of lymphocytes (pabst, 1990) . a population of lung lymphocytes exists in the bronchoalveolar spaces and is recovered by bal. in animals kept in specific pathogenfree environments, lymphocytes range from 5 to 10% of cells recovered by bal (agostini et al., 1993; pabst, 1990) . in normal, nonsmoking humans the figures vary from less than 8 to 20% (daniele et al., 1975; davidson et al., 1985; becker et al., 1990) . the proportion of t cells to b cells and nk cells varies to some degree with the compartment in which the lymphocytes are present. the composition of lymphocytes in bal fluids is generally representative of the proportion of t cells, including cd4 and cd8 t cells, and b cells found in peripheral blood. interestingly, in humans, differences in the phenotype and function of nk cells occur between the peripheral blood and the alveolus. thus, nk cells in human alveoli fail to express cytolytic activity and are largely negative for cd16, although cd16-expressng nk cells with cytolytic activity exist in the interstitium . relatively high numbers of cytolytically active nk cells also exist in the interstitium of mice. fifteen to 20% of cells fractionated on nylon wool columns following isolation from enzyme-digested lungs express the allotypic nk1.1 marker, detectable in c57bu6 mice . when leukocytes are isolated from the lung parenchyma of guinea pigs, mice, rats, and humans, the relative percentage of lymphocytes varies from 15 to over 50%. the variance likely depends on the strain of animal, whether they are kept specifically pathogen-free, and the rigor by which small monocytes are excluded (much of the data are derived from examining wright-giemsa-stained cytospin preparations) (lipscomb et al., 1982; stein-streilein et al., 1983; holt and schon-hegrad, 1987; nicod et al., 1989a,b) . flow cytometric analysis of t cell and b cell populations of lung lymphocytes indicates that the relative proportion of b cells is either increased or the same, and the cd4/cd8 ratios are either decreased or similar compared to peripheral blood abraham et al., 1990; marathias et az., 1991; huffnagle et al., 1994) . intraepithelial lymphocytes are common in the gut mucosa and exist in lung epithelium of some animal species, but are relatively less common than those in the gut (holt and schon-hegrad, 1987; fournier et al., 1989) . in humans, no b cells were found in airway epithelium, and cd8 outnumbered cd4 t cells (fournier et al., 1989) . in an examination of epithelium of the upper respiratory tract (in the nose and covering the tonsil and adenoids), both b cells and t cells were found. notably, y6 t cells occurred in aggregates where they comprised up to 30% of the total t cells, although in general 80-90% of t cells expressed the cup tcr (graeme-cook et al., 1993) . in studies examining cells isolated from human lung parenchyma, y6 t cells made up less than 5% of t cells and were cd3+, cd4-, and cd8(abraham et al., 1990; marathias et al., 1991) . in a study of mice, on the other hand, 8-20% of resident lung lymphocytes were cd3+ and ap tcr-, and were presumably y6 t cells. y6 t cells increased following aerosol delivery of m . tuberculosis (augustin et al., 1989) . the regulation of movement of lymphocytes into and out of the lung is still incompletely understood. nevertheless, with increased information about the role of adhesion molecules in regulating the traffic of lymphocytes (springer, 1994) , investigators are beginning to unravel what regulates steady-state movement of cells into the lung as well as possible preemptive signals that occur with inflammation. research on emigration of cells into the lung has centered on the accumulation of neutrophils within pulmomary vasculature and their immigration into the air spaces of the lung because these cells are likely to play a critical role in the development of the acute respiratory distress syndrome. recent information indicates that the adhesion molecules, p-and e-selectin, the integrins, and icam-1 and icam-2 play important roles in the accumulation of neutrophils in the lung (pilewski and albelda, 1993) , the relevance of each adhesion molecule likely depends on the inflammatory stimulus that provokes the recruitment (hellewell et al., 1994; doerschuk et az., 1990; doerschuk, 1992; mulligan et al., 1993a-e) . the regulation of lymphocytes into the lungs is less well understood even though, as mentioned above, evidence exists that lymphocytes isolated from laln or collected from the efferent lymphatics of laln have a predisposition to return to the lungs (mcdermott and bienenstock, 1979; spencer and hall, 1984; joel and chanana, 1987) . several studies have demonstrated that t lymphocytes isolated from lung lavages of normal humans predominantly express a memory phenotype, e.g., they are cd45ro+, cd45ra-cells (saltini et al., 1990; becker et al., 1990) . other important markers for naive versus memory t cells are the hyaluronic acid receptor (cd44) and l-selectin. naive t cells express low levels of cd44 and high levels of l-selectin, memory t cells express high levels of cd44 and low levels of l-selectin (reviewed in sprent, 1994) . thus, with the recognition that memory t cells accumulate preferentially in the lung, the issue becomes what is the stimulus for their entry and why do naive cells fail to accumulate. the role of naive t cells with their multitude of diverse receptors is to continuously recirculate through secondary lymphoid organs, i.e., spleen and lymph nodes, so that antigen-bearing apcs can interact with appropriate t cells at that site. this apc-t cell interaction results in clonal expansion and subsequently provides challenged tissues with a population of cells able to specifically react and protect the host. thus, naive t cells have receptors that allow them to migrate to secondary lymphoid organs (butcher et al., 1990; jutila 1994) . in contrast, lymphocytes that must enter challenged or inflamed tissues might be expected to express a different set of homing receptors. indeed, this is the case. although accumulation in markedly inflamed sites seems to be nonspecific, when only low levels of inflammation exist, where immune cells accumulate may be determined in the draining lymph nodes from which they derive (picker, 1994) . teleologically, this would be a more efficient way for the immune system to selectively redirect cells to sites where they are needed. indeed, evidence suggests memory lymphocytes bearing the cutaneous lymphocyteassociated antigen seem to specifically home to the skin (reviewed in picker, 1994) . studies also suggest that memory cells in the lung may have a unique set of homing receptors compared to memory cells in the skin or at mucosal sites . thus, cd3+, cd45ro high/cd45ra low t cells in the skin were e-selectin+ and cla+, but were a4p7-and aefi7-, while lung memory t cells were e-selectin-, cla-, and a4p7-, but 50% of the cells were aep7-. the lung phenotype was different from the overall memory t cell phenotype in blood, suggesting that there might be an unidentified receptor on lung t cells that specifically selected them for emigration into the lung. an important issue is whether any naive lymphocytes traffic through the lungs. naive t cells migrate into peripheral lymph nodes via high endothelial venules (hev) using an l-selectin/peripheral lymph node addressin interaction. some animal species have balt in which hev are present. thus, in these animals, naive cells could enter the lungs and be available for primary immune responses to develop at these sites. however, evidence to support this possibility does not exist. despite scanty information about the migration of naive and long-term memory t and b cells into the lungs, several studies have noted that lymphoblasts (or recently divided lymphocytes) have a tendency to migrate into both inflamed and uninflamed lung (daniele et az., 1977; berman et al., 1990) . the location and mechanisms of lymphocyte transmigration are under investigation. e. organized balt macrophages, dcs, and lymphocytes are diffusely distributed throughout all of the compartments of the lung. in addition, in most animal species examined, at least some organized lymphoid tissue may be found in variable amounts lining bronchi and bronchioles. these structures have been recognized for many years, but were first carefully described in 1973 (bienenstock et al., 1973a,b) . more recently, some controversy about these structures has been raised because of the inability to readily detect them in all species. thus, they are relatively rare in normal humans, cats, and young pigs (pabst, 1990 (pabst, ,1992 . when first described they were compared to intestinal peyers patches; they had the appearance of a follicle without a capsule. furthermore, lymphocytes infiltrates the overlying bronchial epithelium which demonstrated alterations in morphology compared to adjacent epithelial cells. they were originally described in the bronchial mucosa of all species examined, e.g., rabbits, guinea pigs, rats, mice, dogs, pigs, chickens, and man (bienenstock et al., 1973a) . these early studies revealed that neonatal thymectomy failed to affect the normal development of balt in rats and chicks, and that tritiated thymidine labeling and autoradiography indicated that there was rapid cell proliferation of cells constituting balt (bienenstock et al., 1973b) . transplantation of fetal lungs into extrapulmonary sites did not interrupt the development of the balt, although it was not as cellular, suggesting that antigenic stimulation was required for full development. since these early studies, two comprehensive reviews have summarized the morphology and function of balt (mcdermott et al., sminia et al., 1989 ). an individual aggregate of balt or bronchusassociated lymphoid unit (balu), a term defined by sminia and col-leagues, consists of a focal area of t and b cells admixed with fibroblasts, reticulum cells, macrophages, interdigitating cells which are comparable to ia-positive dcs, and follicular dendritic cells. balus have no capsule, subcapsular sinuses, nor afferent lymphatics. nevertheless, they have peripheral sinus-like lymphatics which subsequently drain into lymph nodes (lauweryns and baert, 1976-1977) . furthermore, there are arterioles, capillaries, and venules which include high endothelical venules (hev) (otsuki et al., 1989) . the structure of a balu with the demonstration of hev indicates the likelihood that naive t cells might migrate to these structures and initiate a primary immune response against an antigen translocated from the bronchial lumen, comparable to the role for peyers patches in the gut. although early studies failed to demonstrate antigen could cross the overlying epithelium, more recent studies have suggested that soluble antigens might be translocated from the bronchial lumen across the epithelium overlying a balu (fournier et al., 1977; myrvik and ockers, 1982; . in balus lymphocytes are partitioned into b and t cell areas with central b cells surrounded by t cells. the dome over these areas and underneath the epithelium is a mixture of b and t cells (sminia et al., 1989) . in balus, igm-and igg-bearing cells are present in significant numbers together with iga-positive cells which contrasts with peyers patches in which iga cells predominate (sminia et al., 1989) . in rats, balt has been examined with monoclonal antibodies to determine the type of t cells present; cdcpositive cells outnumber cd8positive cells, but in the b cells aggregates nearly all of the t cells are cdcpositive cells (sminia et al., 1989) . important studies relating to the traffic of t and b cells to balus have been published (van der brugge-gamelkoorn and kraal, 1985) demonstrating in vitro binding of equal numbers of t and b lymphocytes to both rat and guinea pig balus. this finding is clearly different from peyers patch binding of t and b cells in which the numbers of b cells that bind are much greater than t cells that bind at a ratio of five b cells for every t cell . this finding corresponds to the increase in b cells in peyers patches relative to the numbers of b cells in balus (crawford and miller, 1984) . taken together, these studies indicate that the specificity of hev in balus is different from that in peyers patches and more closely resembles the specificity of the hev in mesenteric lymph nodes (van der brugge-gamelkoorn and kraal, 1985) . it is important to address the issue of a similarity of balt with gutassociated lymphoid tissue. balt is prominent in certain species including chicken, rabbit, and rat, but is clearly much less prominent in other species, including man. in some members of these species, balt may be completely absent (pabst, 1990) . nevertheless, even in man, other studies have shown that after birth, there is a gradual increase in loose aggregates of lymphoid cells that collect beneath the epithelium, particularly at points of bifurcation of bronchi. in man, mouse, and hamsters, these collections of lymphoid cells do not involve the epithelium nor modify the epithelium over the aggregate. in view of the relative lack of prominence of these structures in certain species, it suggests that their role in protective immune responses in the lung is not essential. since an important function in the gut is for these structures to initiate iga responses, it is possible that the lack of well-organized balt in some species predicts that local initiation of iga responses is not required for health. it also suggests that the common mucosal system in which iga b cell precursors are developed in the gut and migrate to the lung may function to successfully protect the host from lung infections. since the late 1960s, investigators have been systematically exploring mechanisms in the development of immune responses to particulate and soluble protein antigens in the lung (pepys, 1969) . a major impetus was to understand what caused hypersensitivity lung diseases, such as asthma and hypersensitivity pneumonitis, to develop in some individuals, but not in others, although antigenic exposures were the same. models to examine immune responses to various respiratory antigens were developed in many animal strains, including mice, rats, hamsters, guinea pigs, ferrets, dogs, monkeys, horses, and cattle; antigens were delivered via aerosol, intranasal, intratracheal, or intrabronchial instillation. the end point for immunity in experimental animals was generally measured by assessing the development of hypersensitivity disease clinically and morphologically, measuring serum and/ or bronchoalveolar antibody, or characterizing some aspect of cellmediated immunity (cmi) such as migration inhibition, delayed-type hypersensitivity (dth) via skin test, or lymphoproliferation (richerson, 1972; newhouse et al., 1976; kaltreider, 1976 ; kazmierowski et al., 1977 ; ganguly and waldman, 1977) . among the important findings of these early studies were that soluble antigens instilled into the lungs, in contrast to particulate antigens, often failed to produce immunologic lung damage (schatz et al., 1977; fink, 1988) , and that soluble antigen repeatedly instilled into the lung could lead to local tolerance (ratzjczak et al., 1980; holt and leivers, 1982) . interpretation of early studies did not benefit from the current perspective that the type of immune response that develops, i.e., cmi versus antibody (including the predominant isotype of the antibody), is regulated by cytokines secreted by t cells and other cells present at sites of antigen deposition (mosmann and coffman, 1989) . more recent studies have expanded the important concepts derived from earlier studies by focusing on the regulatory mechanisms in the development of lung immunity. in order to understand how various forms of antigen might reach the immune apparatus and, therefore, antigen clearance is discussed. then, while t and b cell immunity are clearly interdependent, studies that have focused on measuring specific t cell responses versus immunoglobulin synthesis in response to noninfectious lung antigens are summarized. models that specifically examine the lung's response to infectious agents and alloantigens or lead to hypersensitivity disease are covered later. many studies have shown that the lalns are responsible for primary immune responses after lung immunization (bice et al., 1980b; kaltreider et al., 1983; stein-streilein and hart, 1980; lipscomb et al., 1982) . lalns function as effective filters to remove particulate materials cleared from the lower respiratory tract via the lymphatics (brain et az., 1978; green et al., 1977 ; morrow, 1972) . although lalns are largely responsible for the induction of immunity after primary immunization, the mechanisms responsible for the clearance of antigen from the lung to lalns are not completely understood. most antigen deposited in the lung is cleared by phagocytosis by ams and neutrophils that transport foreign material up the mucociliary escalator and out of the lung, although some antigen is transported to lalns where an immune response is produced. at least some of this latter antigen is carried free in lymphatic fluid and apparently occurs in circumstances of limited inflammation (lauweryns and baert, 1976-1977) . the induction of pulmonary inflammation by antigen exposure appears particularly important in the translocation of immunogens from the lung to lalns. exposure of the lung to noninflammatory doses of antigen often fails to induce immune responses (yoshizawa et al., 1982; bice et al., 1991) . it is possible that an immune response to airway antigens requires a dose that overwhelms normal phagocytic and clearance mechanisms (bice and muggenburg, 1988 ; bice et al., 1991) . the observation that elevated immune responses are produced in lalns if antigen is deposited in the lungs of animals that have inhaled inflammagens further supports the importance of pulmonary inflammation in the translocation of antigen from the lung to the lalns (bice et al., 1985,198713) . it is possible that inflammation may also alter the relative proportion of antigen reaching lalns in cells as opposed to antigen free in lymphatic fluid. in dogs inoculated via the airways with sheep red cells, a large number of neutrophils enter the lung from the vasculature with a peak response about 1 day after instillation of antigen . furthermore, neutrophils can phagocytize particles in the alveoli and migrate to the lalns carrying the particles (harmsen et al., 1987) . ams can also phagocytize particles in the lung and transport them to lalns (corry et al., 1984; harmsen et al., 1985) . the relative contribution of neutrophils and am in the translocation of antigen from the lung to lalns is not known. however, neutrophils with phagocytized particles reach lalns earlier than ams and, may be more important for antigen transport to lalns than ams. antigen transported to the lalns may be released from both neutrophils and ams and reprocessed by resident apcs to initiate pulmonary immunity. in addition to ams and neutrophils, as previously discussed, lung dcs also likely carry antigen to the lalns, and recent studies in which pulsed splenic dcs inoculated into the trachea were capable of immunizing the hosts supports this concept (havenith et al., 1993~) . whether antigen or lung dc initiates a different type of t helper subset response than antigen arriving in phagocytes or free in lymph has not been determined. nevertheless, if lung inflammation enhances the transport of antigens from the lung to the lalns, it is possible that inhalation of materials that induce pulmonary inflammation might lead to increased recognition of airborne antigens. thus, pulmonary inflammation caused by inhaled pollutants (osebold et al., 1980) and passive cigarette smoke (murray and morrison, 1988; ehrlich et al., 1992) might increase the immune recognition of allergens and be responsible for increasing rates of asthma (evans et al., 1987; platts-mills et al., 1991) . in addition, inflammation induced by pulmonary viral infections may also be important in the induction of immunity to low levels of environmental antigens, e.g., allergens responsible for asthma (castleman et al., 1990; duff et al., 1993) . b. t cell-mediated lung immunity several important questions relate to the development of cmi in the lung and include (1) do antigens instilled into the lung cause local and/or systemic cmi? (2) does the form of antigen, i.e., soluble, particulate, expressed by viable microorganisms influence the out-come? (3) does iv or subcutaneous inoculation of similar antigens generate similar degrees of cmi in the lung? (4) what redirects the immune t cells back into the lungs? inoculation of antigens into the lung can result in both local and systemic cmi (kaltreider, 1976) . as discussed previously, both soluble and particulate antigens can induce immunity, but soluble antigens induce a less easily detectable cmi response than a similar antigen delivered in particulate form as shown by experiments in which either soluble or aggregated human serum albumin were used as the immunogen (burrell and hill, 1975; hill and burrell, 1979) . infectious organisms which replicate in situ are even more capable of producing cmi both locally and systemically (waldman et al., 1972; spencer et al., 1974; ganguly and waldman, 1972; lipscomb et al., 1982) . haptens, such as trinitrobenzene (stein-streilein, 1983) , or metals (parker and turk, 1978) , such as beryllium oxide , may also induce cmi in the lung following direct instillation into appropriate animal models. haptens and metals are agents that lead to sensitization of humans exposed to these agents in the workplace and are associated with hypersensitivity reactions. a number of early studies addressed the issue of whether subcutaneous, iv, or direct lung instillation of antigens resulted in differences in the expression of cmi. in three separate studies, guinea pigs injected via the lung with human y-globulin (hgg), dnp-hgg, or heatkilled influenza virus accumulated specific t cells among lymphocytes recovered from the lungs as measured by migration inhibition factor release or by antigen-induced lymphoproliferation assays. however, subcutaneous or iv injection of these antigens failed to result in measurable specific t cell accumulation in the lungs, although systemic cmi could be measured in lymphocytes from nodes draining the subcutaneous inoculation site or from spleen (waldman and henney, 1971; nash and holle, 1973; lipscomb et al., 1982) . however, the initiation of a mild inflammatory response in the lungs resulted in the accumulation of immune t cells in the lungs of the animals immunized via the extrapulmonary route (waldman et al., 1972; nash and holle, 1973; lipscomb et al., 1982) . using a live, attenuated rubella vaccine or another live strain of rubella virus, the kinetics of a local cmi response was studied following the inoculation of guinea pigs either subcutaneously or by an intranasal inoculation (morag et al., 1974) . in these studies, migration inhibition factor activity was the parameter for measuring cmi responses and was initially detected in the lungs 2 weeks after immunization, peaked at 4 weeks, but was no longer detectable by 6 weeks. when primary immune responses are generated in the laln, what stimulus recruits cells back to the lungs? lymphoblasts rapidly exit lymph nodes during developing immune responses (joel and chanana, 1987) . these cells enter the circulation and, under the control of adhesion molecules and locally generated chemotactic and other adhesion molecule-stimulating cytokines, are recruited into inflamed lungs (berman et al., 1990) . as previously discussed, although markedly inflamed lungs nonspecifically recruit both t cells and b cells, memory t cells generated in lalns may also have homing molecules that specifically direct their return to the lung ). an important factor in retaining recruited immune-specific cells is the continued presence of specific antigens within tissue and at least two groups have shown that this can occur (lipscomb et al., 1982 lyons and lipscomb, 1983; emeson et al., 1982) . thus, t cell blasts enriched for two different antigens and labeled with two distinguishable radioisotopes were shown to be retained in lung lobes nonspecifically, but with an additional selectivity in lung lobes containing the relevant antigen (lipscomb et al., 1982) . selective retention was induced by antigen carried by apc deposited in the lungs and was class i1 mhc restricted, suggesting that the t cells were retained in the lung lobes by binding to the apc in vivo . these studies added validity to a concept that four general mechanisms relate to recruitment and retention of immune cells in the lungs: (1) recently activated t and b cells, i.e., lymphoblasts, from any lymphoid tissue are nonspecifically recruited into inflamed lungs; (2) cells recently activated in lalns express adhesion molecules that are uniquely designed to target their binding to lung endothelium; (3) matrix and lung parenchymal cell adhesion molecules expressed under the control of local environmental perturbations facilitate the emigration of immune cells; and (4) antigen expressed on the appropriate mhc in the lung leads to preferential retention and further expansion in the lung. specificity of recruitment for b cells has not been shown (see below), but in the presence of retained antigen and specific t helper cell recruitment, specific b cells could divide and differentiate. c. b cell-dependent lung immunity antigen-specific antibody produced in lalns after a primary lung immunization is released into blood (bice et az., 1980a; shopp and bice, 1987) . in addition, large numbers of antigen-specific igg, iga, and igm afcs produced in lalns also enter the blood after lung immunization of sevral species, e.g., dogs, cynomolgus monkeys, chim-panzees, humans (bice et al., 1980aj982b; kaltreider et al., 1981; mason et al., 1985; weissman et al., 1994) . in studies of larger animals, afcs in blood are recruited into a lung lobe exposed to antigen, but significantly fewer afcs are found in the other lung lobes of the same animal that are exposed to saline or nothing. there appear to be two factors that control the entry of afcs into the lung. first, afcs must have been recently produced in an immune response . the lymphoid tissue in which they are produced exerts no control on their entry into the lung because afcs produced in the popliteal lymph nodes enter the lung at the same rate as afcs produced in lalns (hillam et al., 1985) . second, afcs enter sites of inflammation produced by instillation of antigen into the lung (bice et al., 1982a; hillam et al., 1985) . however, the recruitment of afcs into inflammatory sites in the lung is not antigen specific because they also enter lung lobes inflamed by instillation of particles or other inflammatory agents. plasma cells are found in the alveoli and interstitial lung tissues of immunized lung lobes suggestig that afcs that enter the lung mature to plasma cells (bice et al., 1987a) . most antigen-specific igm, igg, and iga antibody in the lung after a primary exposure to antigen is produced locally by these cells (hill et al., 1983) . the few afcs in control lung lobes exposed to saline also actively produce antigenspecific antibody (bice et al., 1980a71989) . the results of several studies show that afcs in the lung after a primary immunization are recruited into the lung and are not produced locally (mason et al., 1985; bice et al., 1989) . in addition, cell numbers are not amplified by interaction with antigen that might have been retained in the lung after primary immunization (bice et al., 1982a) . in addition to afcs, large numbers of other lymphocytes enter the lung with a peak response occurring between 7 to 14 days after immunization with a mean of 25% of the total lavage cells being lymphocytes . species differences exist in the release of afcs into the blood from lalns and in the recruitment of afcs into the lung. dogs (bice et al., 1982b) , nonhuman primates (bice et al., 1982a , mason et al., 1985 , and humans (stevens et al., 1979; lue et al., 1988; weissman et al., 1994) all have large numbers of afcs in their blood after immunization, and blood afcs enter the lung. in contrast, data from a single immunization of the lungs of rats, guinea pigs, rabbits, and mice suggest that few or no afcs are released into blood, and that relatively few afcs appear subsequently in the lung (bice and shopp, 1988) . however, the use of adjuvants and large doses of antigen appears to increase the number of afcs in the lung of guinea pigs and mice (shopp and bice, 1987; curtis and kaltreider, 1989) . it is possible that the strain of species being evaluated may also be important, although no data are available that compare pulmonary b cell responses in different strains of laboratory animals. although primary immune responses are not produced in the lung independently of secondary lymphoid tissues, data suggest that memory responses may be detected in the lung to antigen challenges that are independent of lalns (mason et al., 1985; ada, 1986, 1987; bice et al., 1991) . the most logical explanation for the production of afcs and antibody in the lung after an antigen challenge is that immune memory cells are recruited into and/or develop in the lung after a primary immunization. unlike a primary immune response, most specific igg and iga antibody produced in an immunized lung after a rechallenge with antigen appears to come from local immune memory b cells and localized production of afcs, rather than by afcs recruited into the lung from blood. only minimal specific igm is produced in the lung after antigen rechallenge (bice et al., 1991) . specific antibody continues to be produced in the lung for several years after the last exposure to antigen (bice et al., 1991) . lavage fluid from immunized and challenged lung lobes contained significantly more specified igg several years after the last exposure to antigen than was present in lavage fluid from control lung lobes. thus, once an intense, localized antibody response was established in the lung, immune mechanisms supported continued localized antibody production for several years after the last exposure to antigen, but only at the site of antigen exposure. although afcs were identified in lavage fluid from exposed lung lobes several years after antigen challenge, it was possible that cells in interstitial lung tissue, as well as in lalns or distant lymphoid tissues, were all important in long-term antibody production. however, the evaluation of antibody production in lung and various extrapulmonary tissues showed that most long-term antibody production occurred in interstitial tissue in the immunized lung lobe . cells from control lung lobe tissue, from lalns that received lymphatic drainage from the immunized lung lobes, from spleen, gutassociated lymph nodes, or popliteal lymph nodes did not produce significant levels of antibody 2 years after the last antigen challenge. therefore, immune cells retained in lung tissue previously exposed to antigens may be an important source of antibody to protect the lung. in addition, the absence of antibody production in lalns or in other distant lymphoid tissue suggests that antibody produced in lung tissue exposed to antigen could possibly enter the bloodstream and provide immune protection for unexposed lung lobes and extrapulmonary tissues. two possible mechanisms could be responsible for long-term antibody production in lung lobes previously exposed to antigen. first, antigen retained in the lung, possibly on follicular dendritic cells, could stimulate antibody production by antigen-specific memory b lymphocytes that migrate through the lung. alternatively, b lymphocytes recruited into or produced in the lung in response to the initial antigen challenge might live for several years and continuously secrete antibody. because continuous antibody production occurs only in lung lobes exposed to antigen, an antigen depot may be essential. data have been published that support both possibilities (tew et al., 1990; peeters and carter, 1981) . in summary, studies suggest that pulmonary humoral immunity can be maintained both by continued long-term spontaneous antibody production and by antigen challenge restimulating local pulmonary memory b cells to secrete antibody. despite the ability of the lung to express both natural and acquired immunity, respiratory tract infections are the most common type of infections experienced by humans. certainly the common cold alone wins this competition hands down! vaccination has been a powerful intervention to protect against many respiratory tract infections including the bacteria bordetella pertussis, corynebacteria diphtheriae, hemophilus influenzae, streptococcus pneumoniae, and m . tuberculosis and viruses, including one virus that primarily infects the respiratory tract, i.e., influenza, and several which initially infect via the respiratory tract, rubeola, rubella, and mumps. a major impetus for current research in infectious diseases is to learn more about natural host defenses in infections and how the immune system amplifies these defenses (mason and nelson, 1992) . by better understanding these strategies, the hope is to optimize vaccination of immunocompetent hosts and perhaps even hosts who are immunosuppressed, yet retain some capacity to respond immunologically. alternatively, in immunosuppressed individuals, once we are better able to understand how immunologically derived cytokines function in the normal host during infections, recombinant forms might be administered as replacement. the use of animal models has considerably enhanced our understanding of lung infections and the role of immunity in controlling them. in most pneumonias caused by extracellular bacteria, recruited phagocytes and opsonins, especially antibody and complement, are required to effectively control infections, even when antibiotics are used. thus, the goal for vaccination in these pneumonias is to raise the level of local antibody. for chronic pneumonias and pneumonias caused by intracellular microorganisms, the goal of vaccination is less clear, although protection against the viruses listed previously correlates with serum antibody levels. the best evidence supports the probability that enhancing cmi would be protective for immunization against many obligate and facultative intracellular bacteria, fungi, and parasites (lipscomb, 1989; campbell, 1993) . in this section, examples of animal models of infectious disease that address how pulmonary immunity develops to various etiologic agents are discussed, and the type(s) of immunity that afford protection are indicated. while investigators have used experimental models to study nearly all of the infectious agents that produce respiratory infections, space dictates that only a few representative studies be included here. the administration of s. pneumoniae into the lung leads to a rapid accumulation of neutrophils in the alveolar space. the development of this local inflammatory response during s. pneumoniae pulmonary infections was noted in early histopathological studies (loosli, 1940; wood, 1941; loosli, 1942) . subsequent studies confirmed the requirement for intact granulocyte function in the host to eradicate the pulmonary infection (wood et al., 1946; heidbrink et al., 1980) . studies have examined the mechanisms responsible for neutrophil recruitment during the early stages of s. pneumoniae pulmonary infection bruyn et al., 1992) . several groups demonstrated that animals systemically decomplemented with cobra venom factor had an impaired ability to recruit neutrophils and to clear the organisms from the bronchoalveolar space. a role for c5 in the recruitment of neutrophils in response to intratracheally delivered s. pneumoniae was assessed by using congenic c5-sufficient (c5+) and c5-deficient (c5-) mice . the results indicated that c5 was important in producing optimal, early neutrophil recruitment and bacte-rial clearance in response to s . pneumoniae, but other chemotaxins must be involved, because chemotactic activity and neutrophil recruitment was found in both c5+ and c5-mice. once phagocytic effector cells were recruited into the lung, neutrophils and macrophages required the opsonins, immunoglobulin and complement, for efficient phagocytosis of the s . pneumoniae (guckian et al., 1980; coonrood and yoneda, 1981) . the presence of c3b on the surface of the pneumococci is vital for phagocytosis. c3b can be deposited on s . pneumoniae by either the classical complement pathway through interaction with antibodies or through the alternative complement pathway (winkelstein, 1981; joiner et al., 1980) . streptococcus pneumoniae that are not killed by the initial pulmonary inflammatory reaction drain to lalns and eventually enter the systemic circulation resulting in a bacteremic phase (austrian, 1981) . during this phase, type-specific antibody to capsular polysaccharide is produced. it has been demonstrated through passive immunization studies that the presence of type-specific antibody (igg and igm) in the serum is protective against severe pneumococcal infection (musher et al., 1990) . nontype-specific antibodies to s . pneumoniae are made during infection, including antibodies to cell wall components (brown et al., 1983) , surface protein a (szu et al., 1983) , and the f polysaccharide (au and eisenstein, 1981) . in general, most animal studies indicate that these latter antibodies play a minimal role in providing effective protection against infection by s . pneumoniae (szu et al., 1986; brown et al., 1983) . the role of secretory iga in the prevention of pneumococcal disease is unclear, although one mouse model demonstrated that s . pneumoniae-specific iga could "arm" lung lymphocytes which subsequently demonstrated antibacterial action against s . pneumoniae (sestini et al., 1988) . iga has been reported to fix complement and act as an opsonin (hiemstra et al., 1988; gorter et al., 1989) and thus could play a role preventing s. pneumoniae infection. finally, humoral factors other than antibody have been implicated in protection against s . pneumoniae, particularly c-reactive protein which can activate complement and act as an opsonin when bound to the capsule. although a role for c-reative protein has been demonstrated in clearing s. pneumoniae from the bloodstream (volanakis and kaplan, 1971; horowitz, et al., 1987) , a role for c-reactive protein in the pulmonary stages of infections has not been demonstrated. models for acute pulmonary infection with h . influenza have been developed in the rat (wallace et al., 1989) and mouse (esposito and pennington, 1984; toews et al., 1984b) . the latter method delivers a reproducible bolus of organisms to the lower respiratory tract via an endobronchial catheter. using this technique it was determined that the clearance of both typable and nontypable h . influenza from lungs occurred at a very similar rate. the clearance of the organisms appeared to occur in two phases. during the initial 6 hr postinoculation, the organisms increased in numbers three-to fivefold, while during the next 18 hr the organisms were rapidly cleared (toews et al., 198413) . studies indicated that the rapid clearance phase corresponded to the influx of neutrophils into the lung and that the presence of these leukocytes was vital for effective clearance (toews et al., 1985) . the effect of specific antibody to the h . in.uenza on the rate of pulmonary clearance was examined using both active immunization and passive administration of immune sera. the results indicated that the presence of specific antibody in the serum and the bal fluid of immunized mice correlated with an increased rate of clearance from the lung. that systemic igc could provide protection in the lower respiratory tract of animals was also demonstrated by experiments showing enhanced clearance of h. influenza from the lungs of mice that had received immune sera. taken together, these results indicated that in the presence of elevated titers of serum igg, protective antibodies could enter the airways of infected lungs to provide protection against pulmonary pathogens. in contrast to the organisms discussed previously, previous immunization with s. aureus does not appear to enhance clearance or provide protective antibody in pulmonary infections ( jakab, 1976) . recent studies suggest that the pulmonary clearance of this organism may be dependent on locally produced opsonins that enhance phagocytosis by am. surfactant protein a, produced by type i1 pneumocytes, can bind to s. aureus and increase phagocytosis, while this protein does not enhance uptake of s. pneumoniae by am (mcneely and coonrood, 1993) . lung infections with two microorganisms, mtb and cryptococcus neoformans (cne) , are discussed in this section as examples of infections requiring intact cmi for resolution. animal models of chronic lung infections with several other important pathogenic organisms have been studied, including pneumocystis carinii (walzer, 1984; shel-lit0 et al., 1990; harmsen and stankiewicz, 1990; boylan and current, 1992) , histoplasma capsdatum (baughman et al., 1986; defaveri and graybill, 1991; fojtasek et al., 1993; allendoerfer et al., 1993) , blastomyces dermitiditis (morozumi et al., 1982; moser et al., 1988; frey et al., 1989; williams et al., 1994) , paracoccidiodes braziliensis (brummer et al., 1984; defaveri et al., 1989) , coccidiodes immitis (cox et al., 1988) , chlamydia trachomatis and psittaci , rhodococcus equi (kanaly et al., 1993) , and mycobacterium auiumintracellulare (takashima and collins, 1988) . although infection with legionella pneumophila can cause an acute pneumonia in susceptible hosts, it is a facultative intracellular bacterium; cmi is thought to be necessary for resolution of the infection. an animal model to study this infection has also been developed (skerrett and martin, 1991) . a central role for cmi (in which t cells recruit and activate macrophages) in controlling intracellular bacterial infections was first proposed by george mackaness using a listeria monocytogenes murine infection model (mackaness, 1964) . after finding an important role for cmi in controlling an aerogenous listeria infection in mice, mackanew extended his studies to propose that cell-mediated hypersensitivity might be an important cause of lung disease (mackaness, 1971 ). however, he and his collaborators observed that, in contrast to protection afforded by active immunization, adoptive transfer of listeria immune splenocytes seemed to afford only minor protection against an aerosolized infection (truitt and mackaness, 1971 ). in retrospect, adoptive immunity might have been transferred more successfully if lalns or lung lymphocytes from aerosol-infected mice had been used instead of spleen cells from systemically immunized mice; these latter cells likely homed inefficiently to the infected lung (huffnagle et al., 1991b) . nevertheless, in experiments with virulent mtb, when organisms were given iv, the lung developed effective resistance, although less effectively than spleens and livers (mackaness, 1971) . these early studies suggested that cmi in the lung might be more rigidly downregulated, perhaps to prevent excessive damage to delicate structures. however, mackaness offered an additional explanation, e.g., lung infections with mtb may be more difficult to control locally because organisms are sequestered in am, macrophages that, in contrast to recruited monocytes, might resist activation signals delivered by t cells. the role of pulmonary immunity during mtb infection has been analyzed in a variety of animal models (smith and wiegehaus, 1989) including rabbits (lurie, 1964) , mice (orme and collins, 1984; north and izzo, 1993) , and guinea pigs . initial experiments involved intranasal or intratracheal inoculation and the development of the middlebrook chamber (middlebrook, 1952) provided a means of aerosolizing mtb into animals. early studies examined the number of mtb required for a reproducible infection in animals. mice exposed to a mist of virulent mtb developed discreet lesions that were progressively fatal over a 10-21 week period (schwabacher and wilson, 1937) . these initial studies were extended by comparing aerosol versus intranasal delivery of mtb and it was found that both routes produced similar pathology. it was observed that a deposited inoculum of about 100 organisms was required for reproducible infection, while a dose of approximately 12,000 organisms resulted in death (glover, 1944) . resident am undoubtedly play a role during an mtb infection. mtb deposited into the lung are rapidly taken up by am. evidence for a role for am in defense against mtb partly comes from epidemiologic studies examining mtb infections in individuals with silicosis (snyder, 1978) . silica exposure results in the uptake of silica particles by am. these silica particles remain in the phagolysosomes of am throughout the life of the individual (allison and d'accy hart, 1968 ) and likely affect their function. essentially all epidemiological studies examining the incidence of mtb infections in a silica-exposed population have concluded that the incidence of mtb infections in this group is significantly higher than the incidence of mtb infection in a non-silicaexposed population (snyder, 1978) . several studies have shown that both human and/or mouse am are stimulated to produce chemotactic factors and cytokines in response to mtb or components of the mtb cell wall (barnes et al., 1992; chatterjee et al., 1992) . these released products may represent an early native defense system against mtb. thus, chemotactic factors can act to recruit neutrophils and monocytes from the circulation, while amreleased cytokines, such as tnfa, can activate both local am and newly recruited cells. indeed, some studies indicate that cytokineactivated am and/or monocytes can inhibit the growth of or kill mtb (crowle, 1990; rastogi, 1990; denis, 1991a) . more recent data suggest that macrophage cytokines, including il12, may enhance the development of the t h l subset leading to protective immunity (hsieh et al., 1993) . data indicating that avirulent mtb can elicit a greater cytokine response from macrophages than virulent mtb have lead to the hypothesis that the observed differences in mtb virulence may be due to an intrinsic ability of virulent mtb to prevent or decrease the release of factors by am (barnes et al., 1992; chatterjee et al., 1992; roach et al., 1993) . ethnic differences observed in susceptibility to mtb (coultas et al., 1993) might be due to a genetic disposition for a poor initial response by am to mtb. a role for cmi was demonstrated for protection against mtb infection (suter, 1961; leveton et al., 1989) . in a guinea pig model, it was demonstrated that in animals given a low dose of mtb, the organisms replicated in a log phase until days 19 or 20, after which exponential growth ceased . the decrease in growth coincided with the onset of tuberculin skin test sensitivity and the development of detectable bacillemia. bacteriostasis ensued over the next 40-50 days after which the numbers of mtb in lung were gradually reduced. although these data were consistent with a role for the development of an acquired cmi response for resolution of the mtb infection, it was not until orme and collins (1984) , by examining the immune response in a mouse model, that direct evidence was provided for a role of t cells in pulmonary immunity against mtb. in a series of adoptive transfer experiments, they removed splenic t cells from a mouse that had received an iv inoculation of mycobactel-ium bovis or bcg. after injecting these bcg-immune t cells into thymectomized, sublethally irradiated nonimmune mice, the mice were challenged with an aerosol dose of mtb that deposited lo4 organisms into the lungs. two important findings in these studies were (1) adoptively transferred immune t cells enhanced clearance of mtb from the lung; and (2) by differentially removing subsets of t cells with specific antisera, the skin test tuberculin sensitivity was dissociated from protective antituberculous immunity which indicated that separate populations of t cells may be responsible for the two events. in a followup study (orme, 1987) , only mtb-immune cd8 t cells adoptively transferred protection to mice challenged with a lethal aerosol inoculum (1.5 x lo5 organisms) of mtb, while either cd4 or cd8 cells could transfer protection to mice exposed to a low dose (500 organisms) of mtb. in comparison to the pulmonary inoculation studies, models using intraperitoneal or iv routes of inoculation have produced different results. in an intraperitoneal model, an mtb-reactive cd4 t cell clone provided both a dth response and protection as measured by the growth of mtb in the peritoneum (pedrazzini and louis, 1986 ). similarly, an mtb-immune cd4 t cell clone provided protection, as measured by reduced splenic cfu, following an iv mtb infection. another study using in vivo depletion of t cell subsets demonstrated that depletion of cd4 t cells decreased resistance to iv infection, while depletion of cd8 t cells did not have a significant effect (pedrazzini et al., 1987) . in contrast, in a similar model, transgenic mice incapable of producing cd8 t cells were shown to have a decreased resistance to mtb compared to normal mice (flynn et al., 1992) . a role for y6 t cells in pulmonary defenses against mtb is unresolved (o'brien et al., 1989; kaufmann and kabelitz, 1991) . because y6 t cells release ifny, it is tempting to speculate that these t cells represent an initial defense mechanism in the lung to provide activating cytokines to enhance local effector mechanisms to help control the infection until the development of protective immunity by ap t cells. initial studies demonstrated an increase in the number of lung y6 t cells after an intratracheal dose of ppd (janis et al., 1989) . other studies suggested that many y6 t cells responded to the heat-shock protein of mtb (born et al., 1991; kaufmann and kabelitz, 1991) . other studies suggested they may play a role in granuloma formation (modlin et al., 1989) . however, in humans with active mtb infections, there was no increase in y6 t cells in the granuloma as determined by immunohistochemical staining (tazi et al., 1991) . more work in animal models and human natural infections is required to define the role of y6 t cells in mucosal immunity, particularly regarding their role in mtb infections. complex interactions exist in the development of protective immunity by t cells and the type of cytokines produced during an infection. similar to the studies that show an important protective role for t h l cells that preferentially secrete ifny in leishmania infections (locksley et al., 1991) , it is likely that mechanisms for production of appropriate cytokines are critical in the development of protective immunity against mtb (flesch, 1990; denis, 1991b; kawamura et al., 1992; barnes et al., 1993; orme et al., 1993) as well as in the maintenance of a resistant state during the chronic infection stage. an important role for ifny in mtb resistance was recently demonstrated in both an aerosol and an iv mtb infection model. comparing infected normal and ifny knockout mice (cooper et al., 1993; flynn et al., 1993) , it was demonstrated that a lack of ifny resulted in a significant increase in mtb susceptibility. however, since ifny was absent throughout the course of infection, it was unclear at what stage in the immune response ifny was required (flynn et al., 1993) . indeed, ifny may be important for all aspects of the response to mtb including t cell development, cell recruitment, and activation of effector mechanisms. other studies in mice examined the granulomatous response to ivinjected bcg in animals that had received neutralizing antibody to tnfa (kindler et al., 1989) . these and other studies (amiri et al., 1992) indicate that tnfa also plays a critical role in protection against m tb, particularly in the development and maintenance of granulomas. attempts to vaccinate animals with avirulent or killed mtb have provided important data regarding potential vaccines. in general, studies suggest that to enhance the immune response against virulent mtb, viable organisms must be used (larson and wicht, 1962) . the route of immunization with viable organisms can be either iv or by aerosol. further, vaccination does not prevent infection, but rather limits tissue destruction and the degree of hematogenous dissemination . immunization with nonviable cellular elements does not afford protection. in summary, the development of protective immunity to a pulmonary infection with mtb requires the coordinated activity of multiple cell types, particularly macrophages and t cells. the continued study ofthe mtb pulmonary infection should aid in understanding the mechanisms for developing effective cmi in the lung and suggest strategies to enhance pulmonary defenses. cne is an encapsulated yeast found in desiccated form in soil, particularly in areas contaminated by pigeon feces. cne usually causes only an asymptomatic infection in humans following inhalation. normal individuals typically clear the organisms, but in those who are susceptible, particularly those with defects in cmi, the organism may disseminate via the bloodstream and produce an extrapulmonary infection, usually meningitis. mice have been used as experimental models for studying the host defenses against this microorganism, which because of the capsule resists endocytosis and thus typically replicates in tissues in an extracellular location. in murine models the organism was frequently inoculated iv or ip, although it had been established many years ago that mice housed on contaminated bedding (smith et al., 1964) , exposed to aerosols (karaoui et al., 1977) , or that received intranasal inoculations of the organism (ritter and larsh, 1963 ) developed infection. these early studies validated the concept that the organism was acquired by the respiratory tract. murphy and her colleagues have contributed substantially to the understanding of immune and natural defense mechanisms in protection against this yeast and demonstrated that animals inoculated intranasally developed pulmonary infection that disseminated, but following the development of dth gradually cleared the infection (lim et al., 1980a) . furthermore, these investigators demonstrated that transfer of t cell-enriched splenocytes from mice immunized by an intranasal infection was capable of protecting mice against an iv challenge (lim et al., 1980b) . of importance was that in these studies, passive transfer of serum failed to protect mice. we and others developed an intratracheal inoculation infection model with cne to study pulmonary immune mechanisms in mice (hill and harmsen, 1991; huffnagle et al., 1991a; huffnagle and lipscomb, 1992 ). in our model, the organism is inoculated in small amounts directly into the trachea and yeasts in the lungs are quantitated by homogenizing the organ and measuring colony-forming units (cfu). over an initial 7 days, the organisms grow rapidly followed by a gradual decrease in cfu in appropriate mouse strains, a process referred to as "lung clearance" (huffnagle and lipscomb, 1992 ). an important aspect of this model, as is true of many other lung infection models, is that both the strain ofmicroorganism and the strain of mouse determine whether the infection will be cleared from the lung and at what rate (huffnagle et al., 1991a) . in studies using a relatively low virulence encapsulated yeast, athymic nude mice, mice with severe combined immunodeficiency (scid), or mice depleted of cd4 and cd8 t cells were incapable of pulmonary clearance (huffnagle et al., 1991a,b; huffnagle and lipscomb, 1992; hill and harmsen, 1991) . furthermore, protection of the lung was adoptively transferred to scid mice by splenic lymphocytes, but adoptive immunity was more effective if lymphocytes isolated from the lungs and lalns of animals that had been immunized during a lung infection were used (huffnagle et al., 1991b) . interestingly, using a more virulent organism, cd4 t cells were responsible for increased resistance to the highly virulent organism following extrapulmonary spread, but did not demonstrate an effect in controlling the infection within the lung (mody et al., 1990) . these latter studies demonstrated, as did the earlier studies of mackaness, a dichotomy between the ability of immunized animals to demonstrate effective immunity in the lung compared to extrapulmonary organs. what is the role of cd4 and cd8 t cells in immune protection? the absence of either reduced the numbers of inflammatory cells, including macrophages, but they were even more profoundly decreased when both were absent (huffnagle et al., 1994) . a role for cd8 t cells was repeatedly shown in strains of mice that demonstrated acquired resistance to low-virulence cne, i.e., balb/c, c.b-17 (congenic to balb/c, but with the igh locus of c57bl/6 mice), and cba mice (hill and harmsen, 1991; huffnagle et al., 1991a; mody et al., 1993) . a role for cd8 t cells in this infection was particularly curious, although cd8 cells clearly participate in the development of immunity to intracellular organisms. mechanisms proposed include secretion of ifny or lysis of infected targets following recognition by cd8 t cells of peptides in the context of class i mhc (kaufmann, 1988) . however, it is not known how cd8 cells function in murine cne disease in which the organism is primarily extracellular. one important mechanism may be related to their capacity to enhance either the clonal expansion or the recruitment of cd4 cells to the lung (huffnagle et al., 1994) . thus, cd8 t cell depletion of cne-infected mice reduced the numbers of cd4 t cells in infected lungs. a second role may relate to the finding that lung cells isolated from cd4 t cell-depleted animals were capable of secreting ifny in mitogen-stimulated cultures, suggesting cd8 cells in this setting could also contribute to ifny production and play a role in macrophage activation. it was also demonstrated that cd8 cells played a critical role in the development of dth to cne in cneinfected mice and could adoptively transfer dth . this is an important observation because it proves cd8 t cells can recognize antigens of extracellular organisms in the context of class i mhc. thus, it is possible that cd8 t cells might lyse am that phagocytose the organism, but cannot kill it, so that activated macrophages or other effector cells may play a role. c.b-17 mice were particularly adept in the development of a t h l response characterized by clearing cne from their lungs, and this ability was related to enhanced secretion of il-2 and ifny by laln cells early in infection . the heightened resistance in c.b-17 mice correlated with expression of the inducible nitric oxide synthase (inos) gene in the lungs, was accompanied by secretion of no by lung cells during the early clearance phase, and was completely abrogated by both anti-ifny treatment and feeding animals an inhibitor of no production (lovchik et al., 1995) . thus, in c.b-17 mice, clearance in the lung was related to the capacity of the animals to make ifny and no. if t cells are necessary to protect lungs from cne infections, is the effector mechanism mediated mainly by activation of macrophages? the answer to this question is still uncertain, but rat am activated by ifny were able to inhibit the growth of cne (mody et al., 1991) . furthermore, prolonged incubation with gm-csf also activated am for cne growth inhibition . murine macrophages from the peritoneum of bcg-immunized mice inhibited the growth of cne in vitro by an arginine-dependent mechanism and were related to no production (granger et al., 1988; alspaugh and granger, 1991) consistent with the in vivo data of lovchik (1995) . an important aspect of growth inhibition by this no-dependent mechanism was that it did not require endocytosis, although endocytosis enhanced the growth inhibition (granger et al., 1986) . others have demonstrated that ifnyactivated mouse macrophages kill cne, but determined that a secreted protein was important (flesch et al., 1989) . activated human macro-phages make little if any no unlike rat and mouse macrophages leaving open the question of what effector mechanism human macrophage may use to growth inhibit cne. human neutrophils and macrophages not only inhibited growth, but killed, cne in cultures that include fresh complement (miller and mitchell, 1991) . the organism fixes complement by the alternate pathway resulting in c3bi binding to the yeast capsule and allowing phagocytosis by cr3-positive neutrophils and macrophages (kozel and pfrommer 1986; kozel et al., 1988) . this mechanism was shown to play an important role in clearing cne from the pulmonary vasculature during fungemic states in mice (lovchik and lipscomb, 1993) . however, cne in tissues tend not to provoke brisk inflammation, and bronchoalveolar spaces (and cerebrospinal fluid) do not contain significant complement. furthermore, am may not express cr3. thus, t cells must amplify effector systems by recruiting and/or activating nonspecific effectors or by themselves becoming direct effectors. recent studies indicated that human nk cells and t cells had direct activity in vitro against cne (levitz et al., 1994; murphy et al., 1993) , although there was conflicting evidence that human nk cells had no growth-inhibiting activity unless antibody against the organism was present (miller et al., 1990) . we demonstrated that murine nk cells had a minor effect against the organism following iv inoculation, but failed to play a role in early lung clearance if the organism was inoculated via the trachea . recently a t cellindependent, partially protective host defense mechanism was found in lung clearance in scid mice and balb/c mice depleted of cd4 andcd8tcells. athyl+,cd4-,cd8-,asialogml-cell was responsible (hill and dunn, 1993) . further studies are clearly indicated to examine the role of this cell in animal models and to identify its origin, particularly in view of the possible importance of these cells in human cne infections. histologic examination of murine lungs during the clearance phase demonstrated that yeasts were surrounded by macrophages with an activated appearance (hill, 1992; huffnagle and lipscomb, 1992) . this appearance was similar to that seen in the lungs of humans with cryptococcomas who are known to be able to resolve their infections without antibiotic therapy. when the lung becomes inflamed and complement is available, neutrophils may play some role in killing cne in the lung, although the relative importance of neutrophils over t cells and activated macrophages remains to be clarified. continued studies using a murine cne lung infection model should help elucidate the mechanisms that lead to the development of a thl-like response in lalns early during infection and subsequent recruitment of lymphocytes and macrophages into the lung. a closer examination of what effector mechanisms are at work in the lungs of animals that clear a cne infection, particularly in resistant mouse strains that may not utilize no from activated macrophages, may help elucidate host defense mechanisms in man. c. viral pneumonias viruses are intracellular organisms that usurp host cellular machinery to replicate. viral entry into cells can be blocked by antibodies. however, once inside the cell, the virus is resistant to both antibody and t cell recognition until viral peptides are presented in the context of class i mhc antigens on the cell surface and allow specific cytotoxic t cells to lyse the infected cell (zinkernagel, 1993) . as viral replication ensues and particles are released from the cell surface, antibody again has an opportunity to block the further spread of the virus. in general, cytotoxic t cells play an important role in controlling local viral replication, while antibody can prevent initial infection and extracellular spread within the host. in some viral infections, there seems to be a relatively minor role for cd4 t cells (zinkernagel, 1993) . however, in a number of viral infections in mice, depletion of cd4 t cells increased mortality and reduced the rate of clearance. the role for cd4 cells may relate to providing help for cytolytic t lymphocyte (ctl) development (reiss and burakoff, 1981) and for b cell production of high-affinity igg and iga antibodies. the role of y6 t cells, nk cells, and macrophages in acute viral infections is still not clear. furthermore, whether memory ctls play an important role in preventing recurrent infection is also uncertain (zinkernagel, 1993) . it is also uncertain whether persistence of long-term memory t or b cells against viruses requires the continued presence of virus or viral particles (zinkernagel, 1993; sprent, 1994) . a large number of viruses infect the respiratory tract, including rhinoviruses, coronaviruses, adenoviruses, influenza, and parainfluenza viruses, respiratory syncytial virus (rsv), measles, mumps, and rubella viruses. good models in mice exist for both rsv and influenza a infections and are discussed to highlight experimental models that have provided insight into immune defenses against viral respiratory tract infections. immunization against influenza a with killed or fractionated viral antigens protects against influenza, but immunization against rsv has been problematic (salk and salk, 1977; wright et al., 1982; graham et al., 1993; alwan et al., 1994) . protection against rsv, which produces a bronchiolitis in infants and is the most common cause for hospitalizing infants in western countries, was not afforded by immunization with formalin-inactivated virus. subsequent infection after such immunization sometimes resulted in unusually severe infections and even death (kapikian et al., 1969; kim et al., 1969) . the mechanism is unknown, but various theories include immune complex disease, a cd4 t cellmediated dth reaction, or a ctl-mediated pneumonitis (graham et al., 1993) . recent efforts using rsv infections in mice have sought to understand what the mechanisms for protection might be, and why immunization might lead to enhanced pathogenicity with a subsequent challenge. depleting mice of either cd4 or cd8 cells reduced the disease in the lung following an initial rsv infection, but also enhanced virus replication (graham et al., 1991) . thus, control of viral replication during even a primary infection resulted in lung pathology. at least two groups attempted to determine whether various viral subunits might initiate protective immunity, yet cause minimal pathology. mice vaccinated either parenterally or by intranasal inoculation, followed by nasal rsv challenge, lead to the expression of cytokine mrna in the lungs (graham et al., 1993) . the specific cytokine mrna detected was dependent on whether live, heat-killed, or subunit vaccines were given. inactivated virus or subunit fusion (f) protein induced cytokine expression that suggested a th2-like lymphocyte response with increased il-4 mrna relative to ifny expression. in contrast, when mice were primed with parenteral or nasal live virus, t h l responses were prominent. formalin-fixed virus and the f protein component were somewhat protective. however, the most effective protection was induced by immunizing intranasally with the live virus. furthermore, this immunization protocol resulted in the least lung pathology after rechallenge. experiments were designed to determine which t cell types caused pathology and whether specific rsv subunits evoked specific pathology-producing immune t cells. cell lines were developed from immune lymphocytes of mice immunized against the f protein, the major surface glycoprotein (g), and a 22-kda matrix protein expressed by recombinant vaccinia virus (alwan et al., 1994) . f protein lead to the development of both ctl and cd4 t cells with a t h l phenotype. g protein facilitated the development of cd4 cells with a th2 phenotype. immune cells from the 22-kda protein-immunized mice resulted in predominantly cd8 ctl. representative cell lines from each of these groups transferred both protection and pathogenic effects to rsv-infected mice, but the th2 cells seemed to be the most damaging. furthermore, combinations of lines afforded the greatest protection. thus, protection is often synonymous with pathology and it may be difficult to dissoeiate the two. l, it has been clear for some time that serum antibody correlates with protection against influenza viruses. influenza viruses exhibit antigenic drift and shift that requires individuals be immunized yearly for protection against the prevalent virus strain (zinkernagel, 1993; salk and salk, 1977) . while immunization against influenza has been successful, it is possible that new immunization protocols might be developed that would be broadly protective. in contrast to b cell epitopes, t cell epitopes may be cross-reactive; cytotoxic t cells seem to play an important role in controlling influenza infections (zinkernagel and althage, 1977) . early studies established that recovery of mice from infections with influenza a required the development of a ctl response to the virus, and protection was afforded by the adoptive transfer of immune cells into naive-infected hosts (yap et al., 1978; lukacher et al., 1984) . further studies indicated that both class i and i1 mhc-restricted t cell clones could promote recovery from a lethal pulmonary infection (mcdermott et al., 1987) . these t cell clones were preferentially retained in lungs of influenza-infected mice, independent of any viral antigenic specificity, and migrated from the pulmonary vessels into the bronchiolar lumens. thus, the immune cells accumulated at a site appropriate to provide protection against a viral challenge. mice die within 6 days of lethal influenza infections. an array of cytokines could be detected in bal fluids in these mice, but none that were unequivocally indicative of a t cell response (hennet et al., 1992) . thus, while il-la, il-lp, il-6, tnfp, gm-csf, ifny, and leukotriene b4 were identified in lavages, il-2, il-3, and il-4 were not. on the other hand, in sublethal infections in which the influenza infection was resolved, examination of cells from lavages as well as from lalns demonstrated t cell cytokine production (carding et al., 1993; sarawar et al., 1993) . in a primary infection, the kinetics of cytokine mrna was compatible with an initial response occurring in the regional lymph nodes with the effector t cells appearing later in the lungs. among the ap t cells, transcripts for ifny and tnfp were predominantly found in cd8 cells, but there was a tendency for il-4 and il-10 to appear in cd4 cells. interestingly, y8 t cells were identified and expressed il-2, il-4, and ifny. during a secondary response, t cell cytokine mrna was found almost simultaneously in lalns and in the lung (carding et al., 1993) . in related studies, mrna was detected by in situ hybridization and by cytokine production identified in individual cells by elispot. the majority of cells in lavages produced il-2, il-4, and ifny with relatively little tnf and il-10. depletion of cd4 and cd8 cells caused a significant reduction in il-2-and il-4-producing cells, but ifny-producing cells remained and were likely cd4-, cd8ap, or y6 t cells; both populations were present during the infection. these studies are typical of recent studies examining cytokine patterns in various lung infections in the lung attempting to learn what determines the type of immune response that develops to airway antigens. b2-microglobulin-deficient mice were used to determine the effect of ifny on influenza clearance. mice did not develop cd8 ctl because of the absense of class i mhc, but residual cd4 cells were capable of mediating clearance, possibly related to the development of antibody of the igg2a subclass (sarawar et al., 1994) . treatment of the mice with anti-ifny antibody delayed clearance for at least 3 days, whereas antibody to il-4 had no effect. however, all mice survived and eventually cleared the virus. notably, neither antibody to ifny nor il-4 altered the cytokine profiles detected in freshly isolated lung lymphocytes. the conclusion was that although ifny played an important role in viral clearance, its role was not to drive cd4 cells to become t h l t cells. y6 t cells developed as a prominent component of the late inflammatory process during murine influenza infections . these cells expressed all known y genes, although some predominated at times. a suggested role for these cells was that they recognized heat shock proteins on inflammatory macrophages and decreased their numbers. however, close examination of the data failed to show an inverse relationship of the numbers ofy6 t cells and lung macrophages . in another study, y6 t cells were found to be noncytolytic, but expressed mrna for ifny, gm-csf, and tnfp (eichelberger et al., 1991) . the hypothesis was presented that these cells, through their capacity to make cytokines, provided nonspecific protection against secondary infections. thus, although 76 t cells are a component of the host response to viral lung infections, their role remains unknown. blue ribbon panel on vaccine research was convened in 1993 by anthony fauci, director of the niaid, to assess the long-term goals for vaccine research and to recommend immediate priorities for the institute. among the panel's recommended priorities was the development of vaccines for respiratory infections of children, and to improve the current vaccines for pertussis and measles. an additional priority was to develop vaccines for reemerging infectious diseases, including influenza because of its inherent problems of antigenic drift and shift. the jordan report, a publication of the division of microbiology and infectious diseases of the niaid, has reviewed on a yearly basis the progress in vaccine research. in the 1993 report, respiratory tract infectious diseases for which vaccines were either being developed or improved were listed and discussed. they included the bacteria groups a and b streptococci, h. influenzae type b, nontypable h. influenzae, neisseria meningitidis, s . pneumoniae, b . pertussis, pseudomonas aeruginosa, and m . tuberculosis; the viruses rubeola, rubella, adenoviruses, influenza, and parainfluenza viruses, and respiratory syncytial virus; mycoplasma pneumoniae; and the fungi, h. capsulatum, c. immitis, and c . neoformans. thus, an enormous scientific effort is being directed at designing vaccines to protect the host from respiratory pathogens. and yet the report also highlighted the gaps in our knowledge about normal host defenses at mucosal surfaces, what immune defenses we should attempt to enhance with vaccinations, and the best methods for immunization. important issues for vaccine research have been discussed in recent publications (lambert, 1993) including reviews on the possibilities of immunization against tuberculosis (kaufmann and young, 1992 ) and on novel approaches to vaccination such as inoculating polynucleotides encoding antigens directly into muscle (donnelly et al., 1994) . important general goals for any vaccine are that it be efficacious, easy to store, easy to give, and be free of side effects. specific goals for vaccines for respiratory infections are (1) the immune response generated must be protective (a corollary is that the immune response to a microbial challenge should not cause lung pathology.); (2) the immune response must be quickly available in the respiratory tract and at the site within the respiratory tract where the microorganism is likely to seek entrance and/or produce disease; and (3) immune memory should be long term. the study of the type of protective immune responses that develop during natural infections in man or induced infections in experimental animals has provided important clues to what responses should be enhanced by immunization. this has been a productive approach for viral infections and those caused by intracellular pathogens. however, host responses that can prevent a second infection may differ from the immune responses that bring an acute infection under control. for example, in many viral infections, although cytotoxic t cells may bring a primary infection under control, antibody may prevent reinfection. another example of this principle is that although cmi controls cne infections in mice, in special circumstances, antibodies to cne can be protective (mukheqjee et al., 1992) . the isotype of the predominating antibody in preventing infection may also be extremely important, e.g., upper tract infections may be more dependent on iga responses, while in the alveolar spaces, igg may be more effective. as already discussed, an important consideration for viral vaccines is whether subunit vaccines are as effective as live attenuated organisms in initiating the desired type of immune response. an innovative strategy for generating cmi responses against nonviable antigens is to inoculate a regulatory cytokine at the time of antigen delivery (afonso et al., 1994) . the vaccine could even consist of a fusion protein of the antigen and the cytokine (tao and levy, 1993) . the immune response at the time of challenge must develop in the correct site. if the organism infects the upper respiratory tract, local iga is important. if infection is initiated in the lower respiratory tract, it may be sufficient that protective cells and antibody are available in circulation if recruitment can occur immediately following challenge. however, if significant inflammation is required before recruitment of immune cells occurs, some clinical manifestations of infection must necessarily develop, before the protective response neutralizes the infection. feasible strategies for generating iga in the upper respiratory tract are to aerosolize the antigen, to deliver the relevant antigens on the surface of a nonpathogenic microorganism, such as streptococcus gordonii, that have the potential to colonize the nasopharynx (pozzi et al., 1992) , or to immunize with oral vaccines with antigens either chemically bound to cholera toxin b-subunit (mcghee et al., 1992) , enclosed in a liposome or biodegradable microsphere (mestecky and eldrigde, 1991) , or encoded in a plasmid carried by live attenuated salmonella spp. (ckdenas and clements, 1992) . ample evidence has documented that iga precursors generated in the gut home to bronchial mucosa (weisz-carrington et al., 1987; chen et al., 1987; rued1 et al., 1994) , although the converse does not appear to occur to an appreciable extent (mcdermott and bienenstock, 1979; joel and chanana, 1987; vancott et d . , 1994) . however, a cautionary note in relationship to immunizations that enhance iga responses is that they could also stimulate ige responses and lead to allergic responses in the lungs. oral immunization with protein antigen and cholera toxin resulted in anaphylaxis in mice following an intraperitoneal antigen challenge (snider et al., 1994 ). an important issue for any vaccine is whether long-term memory is possible. based on the data derived from immunization with subunit vaccines or killed microorganisms, it seems unlikely that sufficient t cell memory could be induced for long-term cmi protection against respiratory tract infections. however, in the presence of continuous antigen, such as would occur with low levels of replicating attenuated viruses or retained intracellular microorganisms in ia-positive apc, memory t cells should persist. furthermore, follicular dendritic cells are present in balt, and long-term b cell memory could also persist due to the retention of nonviable antigens in the form of antigen-antibody complexes on these cells. evidence that long-term b cell memory occurs in the lungs of dogs has been obtained as discussed previously. however, it is important to remember that this observation was made in animals in which the antigen was directly instilled into the lung. therefore, it is not obvious that extrapulmonary immunization would induce retention of long-term local b cell memory, and suggests that lung immunity might be more effective if primary immune responses to pulmonary pathogens were boosted by intranasal or aerosol antigen delivery. animal models of human lung disease have been used to test hypotheses under well controlled conditions and to dissect mechanisms of injury, inflammation, and repair. these models have been particularly useful in distinguishing direct lung toxicity from injuries that result from immune mechanisms. to prove that a lung injury is immune mediated requires previous exposure to an appropriate agent, evidence of a specific immune response, and evidence that the injury involves recognized immunologic mechanisms. early work concentrated on dissecting isolated aspects of the immune response, i.e., the role of antibody, antibody plus complement, or t cells, emphasizing in vivo analogs of in vitro events. current models of immune-mediated injury emphasize the interrelationships among these aspects of the immune response. in particular, the importance of t cells (especially cd4 t cells) in regulating the type of the immune response is better appreciated. early studies of immune-mediated lung disease in animals were performed by administering different antigens via different routes and with different adjuvants to guinea pigs (richerson, 1972) . immune responses varied depending on the type of antigen, method of immunization, and the presumed predominant response. animals immunized with ovalbumin (ova) in complete freund's adjuvant (cfa) developed specific complement-activating antibody and hemorrhagic pneumonitis with a predominant neutrophil response after aerosol antigen challenge. in contrast, animals immunized with aba-n-acetyltyrosine in cfa exhibited dth without demonstrable serum antibody. later, ova aerosol challenge in sensitized animals produced scattered focal areas of alveolitis with thickening and increased cellularity of alveolar septa and alveolar filling with mononuclear cells. other work (brentjens et al., 1974) confirmed the hemorrhagic nature of immune complex-mediated lung disease. hemorrhagic neutrophilic pneumonitis could be transferred with serum and suppressed by administration of cobra venom factor, which depletes c' in vivo (roska et al., 1977) . richerson and colleagues described the results of aerosol ova exposure of rabbits systemically sensitized to ova in cfa. acute exposure lead to transient foci of acute pulmonary inflammation (richerson et al., 1971) , whereas chronic exposure caused decreasing inflammation (richerson et al., 1978) . t cells were prominent in both the acute and the chronic lesions (upadrashta et al., 1988) . inhalation of muramyl dipeptide could substitute for systemic immunization with ova in freund's adjuvant . the pulmonary inflammatory response could be decreased by administration of cyclosporin a at the time of aerosol challenge (kopp et al., 1985) , implicating dth in the etiology of pulmonary inflammation in the rabbit ova model. diminution in the pathologic response in the lungs despite continuing challenge could be produced by either repeated iv or aerosol exposure of rabbits to ova and was not associated with decreased antigen-specific lymphocyte proliferation nor decreased blood or bal antibody response (richerson et al., 1981; butler et al., 1982) . a similar decrease in pulmonary inflammation has been observed in rabbits, guinea pigs, and mice subjected to repeated exposures to micropolyspora faeni, the agent that causes farmer's lung disease in humans (schuyler et al., 1983 (schuyler et al., ,1987 (schuyler et al., ,1992 , and thermoactinomycetes vulgaris, which causes humidifier lung (takizawa et al., 1989) . the decrease in pulmonary inflammation during continued challenge has been attributed to desensitization, defined as suppression of preexisting dth by administration of homologous antigen. however, desensitization is clearly not present in the above models of lung disease, because lymphocyte proliferation and antibody responses were not depressed. since desensitization in other systems can be achieved by administration of an antigen by an unusual route, such as orally (weigle, 1973) , it is rather surprising that immunologic desensitization was not evident in models of repetitive pulmonary instillation of antigen. two other possible mechanisms, increased degradation of inhaled antigen or increased suppression of lymphocyte proliferation by am, were not present in these models (schuyler and schmitt, 1985; kopp et al., 1988) . additional possibilities, including decreased exposure of the lung to antigen due to changes of clearance mechanisms or change of t lymphocyte subtypes, were not investigated. b. immune complex-mediated lung injury ward and colleagues extended these studies on immune complexmediated lung injury using intratracheal instillation of antibody to bovine serum albumin (bsa), followed by iv administration of bsa. lung injury was measured using morphology, leakage of labeled intravascular protein and red blood cells into the lung, and quantitation of the neutrophil enzyme, myeloperoxidase, in the lung (johnson and ward, 1974) . marked differences were shown in responses to instilled igg versus iga immune complexes. igg immune complex-mediated damage was characterized by neutrophil infiltration into the lung and evidence of increased pulmonary vascular permeability. it was neutrophil dependent (warren et al., 1991) and required il-lp and platelet-activating factor (warren, 1992) , which were likely produced by tnfa-stimulated macrophages (warren et d., 1990) . expression of vla-4 and cd18 (mulligan et d., 1993b,c) , functioning cr1 receptors (mulligan et al., 1992a) , and cdlla (but not cdllb), and icam-1 expression, were also involved (mulligan et al., 1993~) . there was upregulation by elam-1 on pulmonary venules and capillary endothelium, perhaps mediated through a rat analog to il-8 (mulligan et al., 1991 (mulligan et al., ,1993a , and upregulation of icam-1 expression modulated through tnfa (mulligan et al., 1993a) . in contrast, iga immune complex lung injury was characterized by accumulation of mononuclear cells (warren et al., 1991) , perhaps mediated through monocyte chemoattractant protein 1 (mcp-1) ( jones et al., 1992) . the injury was neutrophil and tnfa independent and was not modulated by increases of elam-1 expression (mulligan et al., 1992c) or tnfa secretion, despite a tnfa-induced increase of elam-1 expression. iga-mediated injury was similar to igg immune complex injury in that it was vla-4, cd18, and icam-1 dependent, but was dissimilar in that it is more dependent on c d l l b than c d l l a expression (mulligan et az., 1993d) . iga immune complex lung injury was apparently mediated through nitric oxide or its derivatives . il-4 and il-10 protected against the pulmonary response to igg immune complexes, whereas only il-10 protected against iga immune complex-mediated injury (mulligan et al., 1993e) . c. asthma models there are multiple animal (e.g., primate, sheep, guinea pig, dog, rabbit, rat) models of asthma which have been used to explore pathophysiologic aspects of asthma (wegner et al., 1991; abraham, w; murray et al., 1991; lukacs et al., 1994b; yamaya et al., 1990; du et al., 1992; waserman et al., 1992; coyle et al., 1990) and as a method to test the effectiveness of various therapeutic agents. although all have some resemblance to human asthma, there are substantial differences, especially in regard to the physiologic response to airway challenge and in methods to induce immune hyperreactivity. typical protocols use intraperitoneal administration of antigen with aluminum hydroxide and/or bordetella adjuvants. guinea pigs immunized intraperitoneally with ova in aluminum hydroxide adjuvant, often with the addition of pertussis (handley et al., 1992; mauser et al., 1993) , can be induced to form ige and igg, antibodies, exhibit airway and blood eosinophilia, and display early and late-phase bronchoconstriction (cerasoli et al., 1991) when reexposed to ova. these animals also demonstrate increased bronchial reactivity to histamine or acetylcholine administered iv. using this method of sensitization, guinea pigs exhibit strain differences in blood eosoniphilia and bronchial hyperreactivity (winthereik et al., 1992) . increased responsiveness to acetylcholine after antigen challenge and late-phase bronchoconstriction correlates with bal neutrophilia (asano et al., 1994) . this method of immunization is very different from that which occurs in humans, and the pulmonary physiologic response of guinea pigs is dissimilar to human asthma. guinea pigs exposed to various parasite antigens also exhibit airway hyperreactivity (yamaya et al., 1990) . nonhuman primates exposed to ascaris suum antigen via the airway exhibit immediate skin test reactivity and either early or both early and late increases in airway resistance (patterson and harris, 1992) . dual responses are associated with more bal eosinophils and a greater increase of bal neutrophils (gundel et al., 1992) . hirshman and colleagues found that ascaris-sensitized basenji greyhound dogs exhibited a number of changes similar to those in humans with asthma including increased specific and nonspecific airway reactivity and increased numbers of bal mast cells (hirshman et al., 1980 (hirshman et al., , 1986 . spontaneous and induced histamine release from bal mast cells was increased compared to control animals (hirshman et al., 1988) . tracheal muscle from these animals exhibited impairment of the usual increase in cyclic amp in response to isoproterenol (emala et al., 1993) , and coincident with the measurement of collateral airway resistance, high resolution ct scanning detected airway narrowing (herold et al., 1991; corddry et al., 1991) . chronic treatment with methylprednisolone decreased nonspecific airway reactivity and bal eosinophil number (darowski et al., 1989) . others have induced an asthma-like syndrome with increased bronchial reactivity and reaginic antibody in dogs immunized intraperitoneally as puppies with hapten-carrier complexes in aluminum hydroxide adjuvant (kepon et al., 1977) . later work extended this model by using ragweed antigen (baldwin and becker, 1993; becker et al., 1989) . immunized dogs exhibited immediate-and late-phase skin test reactivity (becker et al., 1988) , increased antigen-specific and nonspecific bronchial reactivity, and increased bal mast cells, eosinophils, and histamine (becker et al., 1989) . t cells appear to be important in animal models of asthma. frew and colleagues demonstrated a substantial influx of non-cd8 (presumably cd4+) t cells into bronchial wall mucosa and adventitia of aerosol antigen challenged guinea pigs undergoing late-phase bronchoconstriction (frew et al., 1990) . using picryl chloride epicutaneous sensitization, mice challenged with intranasal hapten exhibited peribronchiolar cellular infiltration and increased pulmonary resistance in vivo (garssen et al., 1991) . hypersensitivity to carbacol was present in tracheas from such animals and could be transferred with t cells from sensitized animals. this phenomenon could not be produced in athymic mice (garssen et al., 1991) . cyclosporin a and fk506 administration prevented the development of both the late asthmatic response and bronchial hyperresponsiveness after antigen challenge (fukuda et al., 1991) . il-4-deficient and class i1 mhc-deficient mice which lack mature cd4+ t cells could not express peribronchiolar inflammation or bal lymphocytosis and eosinophilia when exposed to ova (brusselle et al., 1994) . exposure of mice to certain parasites, e.g., schistosomes, also caused the appearance of intrapulmonary and bal eosinophilia via an il-cdependent mechanism (lukacs et al., 1994b) , perhaps by altering the balance of th2 versus t h l t cell numbers in the lungs. antibody to il-5 can ablate the eosinophilic airway response to ova exposure in sensitized guinea pigs and can even block the ovainduced increased sensitivity to substance p (chand et al., 1992; mauser et al., 1993) . eosinophil infiltration into the tracheas of sensitized mice after aerosol antigen challenge is dependent on cd4 cells and il-5 (nakajima et al., 1992) and can be blocked by inoculations of ifny (iwamoto et al., 1993) . gelfand and colleagues developed a model of asthma in balb/c mice (typically high ige responders) induced by repetitive inhalation of ova. these animals exhibited increased specific ige production, increased sensitivity to iv methacholine, and evidence of sensitized cells in lalns and spleen capable of producing specific ige and iggl. isolated trachea from sensitized animals were hyperresponsive to electrical field stimulation. specific ige antibody and increased airway reactivity could be induced in naive recipients by transfer of sensitized cells from lalns, but not spleen cells, followed by a single aerosol ova exposure. low-ige responding sjl/ l mice failed to develop either ige antibodies or increased bronchial responsiveness, although they developed specific igg antibodies (larsen and wicht, 1962) . local airway challenge, as well as systemic sensitization, was required for the development of airway hyperreactivity . this suggested that local factors in addition to systemic sensitization were required for bronchial hyperreactivity. in this model, ifny administration during ova sensitization both decreased specific ige production and ablated increased airway reactivity. the effect of ifny was dependent on the route of administration. systemic administration decreased serum-specific ige, but not lalnspecific ige production. perhaps most importantly, the ova-induced increase of airway reactivity was ablated by airway, but not systemic, ifny administration (lack et al., 1994) . these results were compatible with an ifny-induced shift from a predominant th2 to a t h l t cell response and also gave evidence for compartmentalization of both systemic and airway immune responses. thus, airway hyperresponsiveness correlated with laln, but not systemic sensitization. d. t cell-mediated hypersensitivity t cell-mediated inflammation in the lung can result in pulmonary fibrosis or hypersensitivity pneumonitis (hp). exposure of mice to a hapten instilled into the lungs caused systemic sensitization as measured by ear swelling after reexposure to the sensitizing hapten (stein-streilein, 1983) . furthermore, following epicutaneous sensitization with lipophilic trinitrophenylchlorobenzene and an intratracheal challenge with the water-soluble hapten, trinitrophenyl, pulmonary fibrosis developed. intratracheal rechallenge with an unrelated hapten (dinitrophenol) did not produce pulmonary fibrosis (stein-streilein et al., 1987) . this model was similar to previous models of contact sensitivity using the skin for both sensitization and challenge and was consistent with a t cell-mediated dth process (polack, 1980) . different inbred mouse strains of animals exhibited coincidence of skin reactivity and the ability to develop pulmonary fibrosis (kimura et al., 1992) . induction of tolerance by injection of hapten-coupled splenocytes before sensitization depressed both the skin and the pulmonary responses following tracheal challenge (kimura et al., 1993) . the inflammatory and fibrotic responses to intratracheal hapten challenge were transferred with immune lymphocytes, but not with immune serum. in uivo administration of anti-cd4 and anti-cd8 antibodies to sensitized mice prevented or ameliorated the inflammatory and fibrotic responses to tracheal challenge. the development of pulmonary fibrosis is associated with bal il-2 activity and lymphotoxin mrna in bal nonadherent cells (garcia et al., 1992 ). an increased ratio of procollagen type 1:iii mrna in the fibroblasts from immunized, challenged animals developed, indicating that qualitative as well as quantitative collagen differences occurred (stein-streilein et al., 1992). bleomycin is a mixture of glycoproteins from streptomyces uerticullus used clinically for its antineoplastic properties, but which predictably causes pulmonary fibrosis, in a hamster model, intratracheal administration of a single dose of bleomycin to experimental animals caused pulmonary fibrosis which resembled clinical pulmonary interstitial fibrosis, albeit with some differences in the pattern of fibrosis from usual interstitial fibrosis (snider et al., 1978) . evidence has accumulated that cytokines are important mediators in animal fibrosis models. increased tgfp (khalil et al., 1989) , tnfa (piguet et al., 1989a) , il-1 and il-6 (jordana et al., 1988) , mcp-1 (brieland et al., 1993) , and macrophage-derived growth factor for fibroblasts (denholm and phan, 1989) have been detected in lungs or pulmonary cells derived from animals exposed to bleomycin. administration of anti-tnfa can prevent fibrosis (piguet et al., 1989a) . although many of the cytokine studies concentrated on the role of am, recent reports indicate that pulmonary endothelial cells constitutively produced il-6 which is increased by exposure to bleomycin (karmiol et al., 1993) . mcp-1 is produced by fibroblasts (rolfe et al., 1992) and tgfp is produced by pulmonary artery endothelial cells (phan et al., 1991 or lung fibroblasts (breen et al., 1992) . therefore, the source of cytokines in bleomycin-induced pulmonary fibrosis could include nonmacrophage pulmonary cells as well as macrophages. despite the evidence of toxicity of bleomycin-induced macrophagederived cytokines, several studies have suggested that t cells may also be important. different strains of mice responded differently to intratracheal bleomycin (schrier et al., 1983a) . pulmonary fibrosis did not occur in athymic nude mice (schrier et al., 198313) and depletion of both cd4 and cd8 t cells prevented fibrosis (jordana et al., 1988) . cormier and colleagues described a model of hp associated with alveolitis and fibrosis, and which was caused by repeated pulmonary instillation of m. fueni. it was associated with increased bal il-la, il-6, and tnfa (denis et al., 1991) . cyclosporin a administration blocked pulmonary fibrosis, but not alveolitis, and bal il-la and tnfa, but not il-6 (denis et ul., 1992a), which suggested a role for t cells in producing fibrosis. pulmonary fibrosis was also prevented by anti-tnfa antibody (denis et al., 1991) . evidence was also found for a role for am-secreted tgfp in promoting fibrosis (denis and ghadirian, 1992a) . secretion of tnfa, which probably originated from pulmonary macrophages, was fostered by csf-1 and gm-csf secreted by lymphocytes exposed to m. faeni . in contrast to an adoptive transfer model of hp to be described, in uivo depletion of t cells did not substantially affect the pulmonary histologic response to m. faeni . this might be related to repetitive challenges with an agent which has adjuvant effects (bice et al., 1974) , so that inflammation in this model was macrophage, rather than lymphocyte, driven. pulmonary fibrosis induced by repeated challenges with m. fueni, but not an increase of bal inflammatory cells, was reduced by administration of anti-cd1 la, implicating integrins in the processes that lead to fibrosis in this model (denis and bisson, 1994) . lymphocytes can be implicated in other models of hp. cyclosporin a administration ameliorated pulmonary lesions in animals subjected to airway challenges with t. uulgaris (takizawa et al., 1988) . nude mice did not exhibit pulmonary lesions of hp after exposure which was able to produce lesions in t cell-sufficient littermates. the ability to express pulmonary lesions could be transferred with t cells from sensitized mice (takizawa et al., 1992) . schuyler and colleagues have developed an adoptive transfer murine model of experimental hp using m. faeni. cells for adoptive transfer were obtained from spleen, peripheral lymph nodes, lalns, and peritoneal exudate from immunized animals. the cells were restimulated in culture with relevant antigen and could then transfer to naive recipients a susceptibility for increased lung inflammation following an intratracheal rechallenge with antigen (schuyler et al., 1991) . ifnr and il-2 were present in substantial quantities in cultures (fei et al., 1993) , and cd4 cells were required at the beginning of culture to generate effective cells for the adoptive transfer (schuyler et al., 1994) . the transferred cells were a mixture of naive and memory cd4 t cells, as defined by cd44, cd45rb, and l-selectin expression (schuyler et al., 1994a (schuyler et al., ,1992 . successful transfer was also dependent on the presence of cd4 t cells in the recipient (schuyler et al., 1994b) , suggesting the necessity of an important interaction between host and recipient cd4 t cells. despite different methods to develop models for lung allografting or gvhd, the immunologic responses in the lung show similar histologic patterns. several studies have documented these similarities (atkinson et al., 1971; emeson et al., 1982; piguet et al., 1989b; randhawa and yousen, 1992; stein-streilein et al., 1981; yousem et al., 1990) . the histoincompatible allografted lung is recognized as "foreign" by the recipient and, therefore, is subject to rejection (randhawa and yousem, 1992) . in gvhd, donor immunocompetent allogenic cells recognize the recipient as foreign (farrara and deeg, 1991) . in both of these disorders, pulmonary manifestations of lung allograft rejection and gvhd may be separated into acute and chronic changes. in acute lung allograft rejection, the initial pathologic lesions of perivascular mononuclear cell infiltrates is termed "minimal rejection" or grade 1 (yousem eta,?., 1990) . a more severe perivascular mononuclear cell infiltrate consisting of activated lymphocytes, plasma cells, and macrophages is called "mild acute rejection" or grade 2 (yousem et al., 1990) . in some instances rare eosinophils are present (yousem et al., 1990) . vascular changes may include degeneration of the endothelium (endothelialitis) (yousem et al., 1990; randhawa and yousem, 1992) , and lymphocytic infiltration of the bronchioles may be present (yousem et al., 1990) . in grade 3 acute lung cancer allograft rejection, also known as moderate acute rejection, infiltrates progress and become more apparent around pulmonary veins, arterioles, and peribron-(gvhd) in the lung: a pathologic comparison chiolar areas (yousem et al., 1990 ). in grade 4 or "severe acute rejection" mononuclear cell infiltrates extend into air spaces and involve vessels and bronchioles. necrotizing vasculitis and parenchymal necrosis may also be visible (randhawa and yousem, 1992) . in contrast to acute lung allograft rejection, the pulmonary pathology of acute gvhd has not been assigned histologic grades. beschorner et al. (1978) first described the acute changes of gvhd in the lung in recipients of bone marrow transplants. in these patients, the pathology was limited to that of lymphocytic bronchitis (beschorner et al., 1978) . more recently, atkinson et al. (1991) reported an acute pulmonary syndrome after bone marrow transplantation that resembled acute gvhd of the lung. the lesions included lymphocytic peribronchial infiltrates, bronchial epithelial degeneration, and lymphocytic perivascular infiltrates (atkinson et al., 1991) . relative to the histology of acute lung allograft rejection, the pulmonary changes observed in acute gvhd of the lung are analogous to a grade 2 rejection response. in animal models, investigators have reported acute "gvhd-like" changes in the lung (piguet et al., 1989b; stein-streilein et al., 1981; wilkes et al., 1994a) . in these studies the histologic lesions included alveolitis, lymphocytic bronchitis, and vasculitis. the histologic changes of acute gvhd were analogous to grade 4 or severe acute rejection in a lung allograft. in both allograft rejection and gvhd of the lung, the pathologic lesions of the airway began at the level of the bronchioles and extended into alveolar spaces. the chronic stage of lung allograft rejection and gvhd is associated with the development of bronchiolitis obliterans (bo) (farrara and deeg, 1991; rhandhawa and yousem, 1992; yousem et al., 1990 ). bronchiolitis obliterans is not a lesion specific to allograft rejection or gvhd and, in fact, has been associated with a variety of conditions (epler, 1988) including toxic fume inhalation, rheumatoid arthritis, penicillamine use, postinfectious etiologies, as well as idiopathic causes. the histology of bo shows granulation tissue plugs within the lumens of the small airways, epithelial cell damage, mononuclear cell infiltrates, and, at times, complete obstruction of the airways (epler, 1988 ; randhawa and yousem, 1992) . bronchiolitis obliterans observed in chronic lung allograft rejection and gvhd involve the membranous and respiratory bronchioles, and possibly involve more proximal airways (randhawa and yousem, 1992) . in contrast to the pathology observed in the acute disease, lymphocytic perivascular infiltrates are present in only 40% of bo cases associated with chronic lung allograft rejection (randhawa and yousem, 1992) . the degree of vascular involvement in bo associated with chronic gvhd is unknown but is likely less than that of acute gvhd. both canine and rat models have been utilized to study lung transplantation and the immunopathogenesis of rejection (benfield, 1976; prop et al., 1985a,b) . however, the availability of more immunological reagents has allowed the rat model developed by marck, prop, and wildevuur to be more extensively studied (marck et al., 1983) . orthotopic transplantation of the brown norway rat lung allografts (rt") into lewis (rt') rats resulted in histological and immunological changes analogous to that of human lung transplantation (marck et al., 1983; prop et al., 1985a,b) . the rejection process occurred in four phases (prop et al., 1985a,b) : (1) the latent phase which occurred immediately after transplantation in which no immunological activity was described in the graft (day 1 after transplantation); (2) the vascular phase, characterized by infiltration of balt and perivascular tissue by lymphocytes (days 2 or 3 after transplantation); (3) the alveolar phase, with mononuclear cell infiltration of the alveolar walls (days 4 or 5 after transplantation); and (4) the destruction phase, characterized by intraalveolar edema and destruction of airways and vessels by infiltrating mononuclear cells (day 6 after post-transplantation). significantly, these four phases resemble somewhat the four grades associated with acute rejection in humans (yousem et al., 1990) . while these studies described the histological changes associated with acute rejection, only two reports currently exist in the literature describing animal models of chronic rejection, known as bo (hertz et al., 1993; uyama et al., 1992) . utilizing the previously described rat lung allograft model, rats made tolerant to their allografts by cyclosporin developed the typical changes of bo around 6 months after transplantation. the airway lesions were associated with upregulated class i1 mhc expression on the epithelium in the large airways, aggregates of dcs in the submucosa, and ulcerated epithelium (uyama et al., 1992) . in a murine model utilizing hetertopically transplanted airways, the characteristic lesions of bo developed in the allograft after 21 days (hertz et al., 1993) . a significant difference between the rat models and that of clinical transplantation is that only one or two doses of the immunosuppressant drug, cyclosporin, results in indefinite acceptance of the donor rat lung (uyama et al., 1992) . human lung allograft recipients usually require life-long therapy to prevent rejection (trulock, 1993) . however, without cyclosporin, the rat lung allograft undergoes a rapid rejection process which usually results in the destruction of the allograft in 7 or 8 days posttransplantation (prop et al., 1985a,b) . although lung transplantation has become an increasingly utilized modality for the treatment of many endstage lung diseases (trulock, 1993) , the lung allograft, in both animal models and humans, is more prone to rejection than other solid organs (prop et al., 1985a,b; trulock, 1993) . the presence 6f many immunocompetent cells present in the donor lung that can stimulate a rejection response may be the explanation (prop et al., 1985a,b; trulock, 1993) . notably, despite the large numbers of t lymphocytes present in the lung and thus carried into the recipient, the clinical syndrome of systemic gvhd has not been reported in human lung allografted individuals. however, gvhd in lung transplantation was reported in an animal model in which the recipient was rendered severely immunoincompetent by total body irradiation (prop et al., 1989) . acute lung allograft rejection is believed to be initiated by donor lung apc, i.e., dcs and perhaps macrophages, interacting with recipient lymphocytes (winter et al., 1989) . although there is no direct evidence that these accessory cells mediate allograft rejection, several studies suggest their role in the rejection responses. acute rejection episodes commonly occur at a time when there is an abundance of donor dcs and lung macrophages, i.e., the first 8 to 12 weeks after transplantation, and diminish when these cells are replaced by those of the recipient (paradis et al., 1985; uyama et al., 1993) . utilizing a murine model of renal transplantation in which dcs had been depleted, lechler demonstrated that repletion of dcs resulted in the rejection of the allograft (lechler and batchelor, 1982) . similarly, blocking antibodies to dcs resulted in the prolongation of survival of murine pancreatic islet allografts (faustman et al., 1984) . as discussed previously, dcs exist within the epithelium and subepithelial areas of the bronchi/bronchioles, areas that are involved in both acute and chronic rejection. additionally, ifny, a cytokine crucial to the rejection process (o'connell et al., 1993) , was shown to upregulate the number of dcs in the interstitium surrounding pulmonary capillaries, within the alveolar interstitium, and in the bronchial epithelium . finally, dcs accumulate in areas of bo during the course of chronic allograft rejection (uyama et al., 1992) . collectively, these studies suggest a central role for dcs in the pathogenesis of lung allograft rejection. lung macrophages, although suppressive of many immune cell functions, may also be involved in the initiation of the rejection response. lung accessory cell-lymphocyte interactions occur through cytokines and intercellular signals and result in upregulated cellular and humoral immunity (wilkes and weissler, 1994) . cellular immunity is crucial in lung allograft rejection (prop et al., 1985a,b) and may result from the differential stimulation of t h l versus th2 cells. t h l lymphocytes play a significant role in the pathogenesis of solid organ allograft rejection (jordan et al., 1991; o'connell et al., 1983) . for example, in a murine example of pancreatic islet rejection, allograft infiltrating lymphocytes preferentially expressed mrna for il-2 and ifny, and not il-4 (o'connell et al., 1983) . similarly, in rat lung allografts, ifny mrna was expressed during the rejection episodes (jordan et al., 1991) . the clinical importance of t h l lymphocytes in allograft rejection is exemplified by the fact that the primary immunosuppressive agent used in recipients of human lung allografts is cyclosporin, which preferentially inhibits il-2 and ifny production from lymphocytes (cockfield et al., 1993) . in contrast, th2 lymphocyte activity, i.e., production of il-4 and il-10, which downregulates t h l activity, has been strongly associated with prevention of allograft rejection (gorczynski and wojcik, 1994) . both allogeneic am and parenchymal lung dcs were potent inducers of ifny, but not il-4, from lymphocytes (wilkes and weissler, 1994) . collectively, these data suggest that allograft rejection is in part mediated by lung macrophages and dcs stimulating t h l lymphocytes. cytokines from t h l and th2 lymphocytes can both result in specific immunoglobulin production (kitani and strober, 1993) . therefore, the upregulated t h l lymphocyte activity observed in allograft rejection might be responsible for the enhanced local immunoglobulin production observed during the rejection process (wilkes et al., 1994b) . ifny, a t h l cytokine, can stimulate igg2a from murine b lymphocytes (kitani and strober, 1993) . furthermore, ifny production, induced by human lung macrophages, selectively stimulated igg2 production from allogeneic peripheral blood mononuclear cells (wilkes and weissler, 1994) . in recipients of lung allografts undergoing rejection, wilkes (1995) demonstrated that local production of igg2 was selectively upregulated and, thus, served as a marker for the rejection response (wilkes et al., 1994b) . few studies have demonstrated a role for alloantibodies in mediating the process of lung allograft rejection. coronary atherosclerosis secondary to murine cardiac allograft rejection was in part mediated by antibodies directed against the donor coronary epithelium (russell et az., 1994) . similarly, igg2, but not iggl, igg3, or igg4, produced locally during human lung allograft rejection, preferentially bound to perivascular and peribronchial extracellular connective tissue matrices which are the anatomic locations involved in the rejection process (wilkes, manuscript in preparation) . taken together, these studies suggest a role for a thl-dependent humoral responses in the pathogenesis of lung allograft rejection. while lung accessory cell-t lymphocyte interactions initiate organ rejection (winter et al., 1989) , the production of proinflammatory cytokines, il-6 and tnfa, has been identified as a mediator ofthe rejection process saito et al., 1993) . evidence that tnfa was involved in rejection was demonstrated by demeester who reported that tnfa mrna and protein were upregulated in lung tissue during acute rejection of rat lung allografts . significantly, anti-tnfa antibodies reduced the vasculitis and hemorrhagic lesions in rejecting lung allografts (saito et al., 1993) . similarly, il-6 was upregulated in the lung during rejection and was postulated to be clinically important in following the activity of the rejection process . c. models of graft versus host disease in the lung gvhd is a systemic process, and relatively few have described pulmonary involvement in animal models of gvhd (piguet et al., 198913; stein-streilein et al., 1981) . stein-streilein et al. (1981) , utilizing a murine model, reported gvhd "reactions" in the lung. in these studies, suspensions of parental (mha) lymph node cells were instilled into the trachea of f1 hybrid (mha x cb) recipient hamsters. the histology observed in the recipient lungs showed mononuclear cell infiltration in the interstitium, alveolar, peribronchiolar, and perivascular areas. some of the animals developed thymic atrophy and splenomegaly which suggested a systemic component to the disease process (stein-streilein et al., 1981) . interestingly, when the cells were given iv or intracutaneously, the animals developed systemic gvhd without any distinctive pulmonary pathology (stein-streilein et al., 1981) . piguet also studied the pulmonary disease associated with gvhd. in these studies, irradiated f1 hybrid (cba x b10) mice were injected with either parental t lymphocyte-depleted bone marrow cells or with parental bone marrow cells together with suspensions of lymph node cells as a source of t lymphocytes. in addition to the induction of systemic gvhd, the histology of the lung was similar to that reported by stein-streilein. additionally, these investigators demonstrated the central role of t lymphocytes in the lung pathology of gvhd in that injection of t lymphocyte-depleted bone marrow cells did not induce pulmonary pathology (piguet et al., 1989b) . wilkes et al. (1994a) recently reported that allogeneic (c57bl/6) bal accessory cells (>go% macrophages), when instilled intratracheally into the lungs of normal balb/c mice weekly for 4 weeks, induced a lymphocytic alveolitis, bronchitis, and vasculitis analogous to gvhd of the lung or acute lung allograft rejection. unlike other animal models of gvhd (piguet et al., 1989b; stein-streilein et al., 1981) , these recipient mice had no evidence of systemic disease. additionally, if no further allogeneic challenges were performed, the pulmonary lesions eventually healed. as previously stated, the clinical manifestations of chronic gvhd in the lung are associated with the development of bo (farrara and deeg, 1991; randhawa and yousem, 1992; yousem et al., 1990) . in contrast, no animal models of chronic gvhd have reported an association with this type of pulmonary disease. the immunopathogenesis in animal models of gvhd has been well described (antin and farrara, 1992; piguet et al., 198913) . similar to acute lung allograft rejection, acute gvhd has been associated with the production of several proinflammatory cytokines including tl-1, tnfa, and il-6 ( antin and farrara, 1992; piguet et al., 1989b) . in fact, il-1 receptor antagonist was shown to significantly inhibit gvhd . relative to the lung, anti-tnfa antibodies partially prevented the pulmonary pathology of gvhd (piguet et al., 1989b) . while proinflammatory cytokines are involved in the pathogenesis of gvhd, t lymphocytes initiate the process (antin and farrara, 1992) . t h l lymphocytes are crucial in acute gvhd. in amurine model of gvhd, ifny and il-2 were preferentially produced in the course of acute gvhd (allen et al., 1993) with similar findings to those reported by other investigators (antin and farrara, 1992) . therefore, the immune response to alloantigens in both lung allograft rejection and gvhd seems to be associated with upregulated t h l lymphocyte activity. a role of humoral immunity in gvhd has not been well defined. however, wilkes et al. reported (1994a) that allogeneic bal cells instilled into murine lungs resulted in the predominant local production of igg2a. additionally, only igg2a was shown to be deposited in the perivascular and peribronchiolar extracellular connective tissues, the same anatomic locations involved in lung allograft rejection and gvhd of the lung. these data suggest that locally produced immunoglobulins recognize component(s) of the extracellular connective tissue matrix and may be involved in the pathogenesis of gvhd of the lung. no evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. what is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. perhaps even the common cold might be conquered. considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade. purification and characterization of intraparenchymal lung lymphocytes. 1. lmmunol the potential role of bradykinin antagonists in the treatment of asthma the adjuvant effect of interleukin-12 in a vaccine against leishmania major pulmonary immune cells in health and disease: lymphocytes. (eur. respir differential cytokine expression in acute and chronic murine graft-versus-host-disease transfer of protective immunity in murine histoplasmosis by a cd4+ t-cell clone potentiation by silica ofthe growth ofmycobacterium tuberculosis in macrophage cultures inhibition of cyptococcus neofomans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis distinct types of lung disease caused by functional subsets of antiviral t cells clinical role of bronchoalveolar lavage in adults with pulmonary disease tumour necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected scid mice hyporesponsiveness of canine bronchoalveolar lymphocytes to mitogens: inhibition of lymphocyte proliferation by alveolar macrophages cytokine dysregulation and acute graft-versushost disease possible mechanisms of airway hyperresponsiveness after late asthmatic response in guinea pigs an acute pulmonary syndrome possibly representing acute graft-versus-host disease involving the lung interstitium evaluation of the role of the pneumococcal forsman antigen (f-polysaccharide) in the cross-serotype protection induced by pneumococcal subcellular preparations resident pulmonary lymphocytes expressing the y / 6 t-cell receptor some observations on the pneumococcus and on the current status of pneumococcal disease and its prevention bronchoalveolar eosinophilic cells in a canine model of asthma: two distinctive populations cytokine production induced by mycobacterium tuberculosis lipoarabinomannan. relationship to chemical structure patterns of cytokine production by mycobacterium-reactive human t-cell clones the pathogenesis ofexperimental pulmonary histoplasmosis. correlative studies of histopathology, bronchoalveolar lavage, and respiratory function cutaneous allergic response in atopic dogs: relationship of cellular and histamine responses development of chronic airway hyperresponsiveness in ragweed-sensitized dogs characterization of normal human 541-547. lung lymphocytes and interleukin-2-induced lung t cell lines immunologic and infectious reactions in the lung lymphocyte recruitment to the lung lymphocytic bronchitis associated with graft-versus-host disease in recipients ofbone marrow transplants adjuvant properties of micropolyspora faeni immune responses after localized lung immunization in the dog regional immunologic responses following localized deposition of antigen in the lung recruitment of antibody-forming cells in the lung after local immunization is nonspecific the evaluation of lung immunity in chimpanzees effects of inhaled diesel exhaust on immune responses after lung immunization identification of plasma cells in lung alveoli and interstitial tissues after localized lung immunization comparative lung immunotoxicity of inhaled quartz and coal combustion fly ash inflammation and recruitment of immune cells into the lung long-term maintenance of localized antibody responses in the lung long-term antibody production after lung immunization and challenge: role of lung and lymphoid tissues pulmonary responses to antigen localized immune memory in the lung antibody responses after lung imrrlunization 693-698. studies on macrophage populations in the airways and the lung wall of spf mice in the steady-state and during respiratory virus infection inhibition ofthe immunosuppressive activity ofresident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor the role of gamma delta t lymphocytes in infection b7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance role of monocytes and interstitial cells in the generation of alveolar macrophages. i. kinetic studies of normal mice improved rat model of pneumocystis carinni pneumonia: induced laboratory infections in pneumocytis-free animals biologic potential of pulmonary macrophages mechanisms, measurement, and significance of lung macrophage function bleomycin regulation of transforming growth factor-p mrna in rat lung fibroblasts experimental immune complex disease of the lung expression of monocyte chemoattractant protein-1 (mcp-1) by rat alveolar macrophages during chronic lung injury localization of complement component 3 on streptococcus pneumoniae: anti-capsular antibody causes complement deposition on the pneumococcal capsule murine model ofparacoccidioidomycosis. production of fatal acute pulmonary or chronic pulmonary and disseminated disease: immunological and pathological observations attenuation of allergic airway inflammation in il-4 deficient mice mechanisms of host defense against infection with streptococcus pneumoniae the effect ofrespiratory immunization on cell-mediated immune effector cells of the lung organ specificity of lymphocyte migration: mediation by highly selective lymphocyte interaction with organspecific determinants on high endothelial venules the local and systemic iga and igg antibody responses of rabbits to a soluble inhaled antigen. measurement of responses in a model of acute hypersensitivity pneumonitis t cell involvement in resistance to facultative intracellular pathogens of the lung oral immunization using live attenuated salmonella spp. as carriers of foreign antigens late dominance of the inflammatory process in murine influenza by y/6+ t cells activation of cytokine genes in t cells during primary and secondary murine influenza penumonia viral bronchiolitis, during early life induces increased numbers of bronchiolar mast cells and airway hyperresponsiveness airway eosinophils from actively sensitized guinea pigs exhibit enhanced superoxide anion release in response to antigen challenge anti-il-5 monoclonal antibody inhibits allergic late phase bronchial eosinophilia in guinea pigs: a therapeutic approach structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor in uitro induction of t cell anergy by blocking b7 and early t cell costimulatory molecule etc-i/b7-2 effect of granulocyte-macrophage colony-stimulating factor on rat alveolar macrophages anticryptococcal activity in uitro active synthesis of hemagglutinin-specific immunoglobulin a by lung cells of mice that were immunized intragastrically with inactivated influenza virus vaccine regulation of ifn-y and tumor necrosis factor-a expression in uiuo complement and opsonins in alveolar secretions and serum of rats with pneumonia due to streptococcus pneumoniae disseminated tuberculosis in interferon y gene-disrupted mice use of collateral airways to assess airway reactivity the migration of bronchoalveolar macrophages into hilar lymph nodes respiratory diseases in minorities of the united states induction and expression of cell-mediated immune responses in inbred mice infected with coccidioides immitis the requirement 211 for platelets in allergen-induced late asthmatic airway obstruction. eosinophil infiltration and heightened airway responsiveness in allergic rabbits activation of naive, memory and effector t cells intracellular killing of mycobacteria the cell surface of mouse dendritic cells: facs analyses of dendritic cells from different tissues including thymus characterization of bronchoalveolar lymphocytes during a specific antibody-forming cell response in the lungs of mice immunocompetent cells from the lower respiratory tract of normal human lungs the bronchoalveolar lymphocyte. studies on the life history and lymphocyte traffic from blood to the lung corticosteroids decrease airway hyperresponsiveness in the basenji-greyhound dog model of asthma differentiation and activation phenotypes of lung t lymphocytes differ from those of circulating t lymphocytes hypersensitivity pneumonitis to paracoccidioides brasiliensis antigens in mice immunohistopathology of murine pulmonary histoplasmosis during normal and hypersensitive conditions the bimodal expression of tumor necrosis factor-a in association with rat lung reimplantation and allograft rejection alveolar macrophage suppression of canine bronchoalveolar lymphocytes: the role of prostaglandin ez in the inhibition of mitogen-responses the effects of bleomycin on alveolar macrophage growth factor secretion killing of mycobacterium tuberculosis within human monocytes: activation by cytokines and calcitriol involvement of cytokines in determining resistance and acquired immunity in murine tuberculosis tumor necrosis factor plays an essential role in determining hypersensitivity pneumonitis in a mouse model murine hypersensitivity pneumonitis: a study of cellular infiltrates and cytokine production and its modulation by cyclosporin a. a m peyer's patches t cells in hypersensitivity pneumonitis: effects of in oiuo depletion in a mouse model transforming growth factor+ is generated in the course of hypersensitivity pneumonitis: contribution to collagen synthesis murine hypersensitivity pneumonitis: production and importance of colony-stimulating factors in the course of a lung inflammatory reaction blockade of leukocyte function-associated antigen (lfa-1) in a murine model of lung inflammation cd18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits the role of cdl8-mediated adhesion in neutrophil sequestration induced by infusion of activated plasma in rabbits immunization with polynucleotides. a novel approach to vaccination morphometric changes during the early airway response to allergen challenge in the rat risk factors for acute wheezing in infants and children: viruses, passive smoke, and ige antibodies to inhalant allergens childhood asthma and passive smoking activation status ofthe cd4-8-y8-t cells recovered from mice with influenza pneumonia impaired betaadrenergic receptor activation of adenylyl cyclase in airway smooth muscle in the basenji-greyhound dog model of airway hyperresponsiveness antigen-induced recruitment of circulatinglymphocytes to the lungs and hilar lymph nodes ofmice challenged intratracheally with alloantigens bronchiolitis obliterans and airways obstruction associated with graft-versus-host disease experimental pneumonia due to haemophilus influenzae: observations on pathogenesis and treatment national trends in the morbidity and 481-496. 7, 140-148. 728-734. mortality of asthma in the us. prevalence, hospitalization and death from asthma over two decades graft-versus-host disease prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody t cell growth factors in spleen cell cultures in experimental hypersensitivity pneumonitis hypersensitivity pneumonitis fungicidal activity of ifn-y-activated macrophages. extracellular killing of cryptococcus neoformans activation oftuberculostatic macrophage functions by gamma interferon, interleukin-4, and tumor necrosis factor major histocompatibility complex class i-restricted t cells are required for resistance to mycobacterium tuberculosis infection an essential role for interferon y in resistance to mycobacterium tuberculosis local immunity in lung-associated lymph nodes in a murine model of pulmonary histoplasmosis bronchial lymphoepithelial nodules in the rat: morphologic features and uptake and transport of exogenous proteins intraepithelial t-lymphocyte subsets in the airways of normal subjects and of patients with chronic bronchitis lymphocytes and eosinophils in allergeninduced late-phase asthmatic reactions in the guinea pig divergent patterns of pulmonary blastomycosis induced by conidia and yeasts in athymic and euthymic mice inhibition of antigeninduced late asthmatic response and bronchial hyperresponsiveness by cyclosporin and fk 506 respiratory tract cell-mediated immunity persistent interleukin-2 activity and molecular evidence for expression of lymphotoxin in the hapten-immune model for pulmonary interstitial fibrosis t-cell-mediated induction of airway hyperreactivity in mice infection of mice with mycobacterium tuberculosis (bovis) by the respiratory route the origin of alveolar macrophages in mouse radiation chimeras intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection a role for nonspecific (cyclosporin a) or specific (monoclonal antibodies to icam-1, lfa-1, and il-10) immunomodulation in the prolongation of skin allografts after antigen-specific pretransplant immunization or transfusion complement mediated enhancement of iga induced hzoz release by human polymorphonuclear leukocytes immunohistochemical characterization of intraepithelial and subepithelial mononuclear cells of the upper airways role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice priming immunization determines t helper cytokine mrna expression patterns in lungs of mice challenged with respiratory syncytial virus macrophage-mediated fungistasis in oitro: requirements for intracellular and extracellular cytotoxicity specific amino acid (l-arginine) requirement for the microbiostatic activity of murine macrophages defense mechanisms of the respiratory membrane the role of opsonins in recovery from experimental pneumococcal pneumonia antigen-induced acute and late-phase responses in primates immunology of the aging lung a canine model of berylliuminduced granulomatous lung disease pulmonary immune cells in health and disease: dendritic cells and langerhans' cells induction by aerosol allergen of sustained and nonspecific ige-mediated airway hyperreactivity in the guinea pig cd28-mediated signalling co-stimulates murine t cells and prevents induction of anergy in t-cell clones host-parasite relationships in experimental airborne tuberculosis. vi. influence of vaccination with bacille calmette-guerin on the onset and/or extent of hematogenous dissemination of virulent mycobacterium tuberculosis to the lungs the role of macrophages in particle translocation from lungs to lymph nodes migration of neutrophils from lung to tracheobronchial lymph node requirement for cd4+ cells in resistance to pneumocystis carinii pneumonia in mice effect of bacillus calmette-gubrin inocuation on numbers of dendritic cells in bronchoalveolar lavages of rats separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization migration of dendritic cells into the draining lymph nodes of the lung and intratracheal instillation t cell priming in situ by intratracheally instilled antigen-pulsed dendritic cells granulocyte participation in early lung clearance of pneumococci from murine lung disparate role of the &integrin cd18 in the local accumulation of neutrophils in pulmonary and cutaneous inflammation in the rabbit a kinetic study of immune mediators in the lungs of mice infected with influenza a virus assessment of pulmonary airway reactivity with high-resolution computed tomography reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways activation of complement by human serum iga, secretory iga and igal fragments cell-mediated immunity to soluble and particulate inhaled antigens a t cell-independent protective host response against cryptococcus neoformans expressed at the primary site of infection in the lung intrapulmonary growth and dissemination of an avirulent strain of cryptococcus neoformans in mice depleted of cd4+ or cd8+ t cells cd4+ t cells cause multinucleated giant cells to form around crypto-coccus neofonans and confine the yeast within the primary site of infection in the respiratory tract evaluaiton of the pulmonary immune response by analysis of bronchoalveolar fluids obtained by serial lung lavage lung localization of antibodyforming cells stimulated in distant peripheral lymph nodes the bensenji-greyhound dog model of asthma: reactivity to ascaris suum, citic acid and methacholine increased metachromatic cells and lymphocytes in bronchoalveolar lavage fluid of dogs with airway hyperreactivity enhanced bronchoalveolar lavage mast cells histmine releasability in allergic dogs with and without airway hyperresponsiveness role ofcytokines in the clearance of cryptococcus neofonans pulmonary infections in mice alveolar macrophages. 11. inhibition of lymphocyte proliferation by purified macrophages from rat lung down-regulation of immune responses in the lower respiratory tract: the role of alveolar macrophages regulation of antigen-presenting cell function(s) in lung and airway tissues tolerance induction via antigen inhalation: isotype specificity, stability, and involvement of suppressor t cells localization of t cells, macrophages and dendritic cells in rat respiratory tract tissue: implications for immune function studies alveolar macophages: functional heterogeneity within macrophage populations from rat lung preparation of interstitial lung cells by enzymatic digestion of tissue slices: preliminary characterization by morphology and performance in functional assays extraction of immune and inflammatory cells from human lung parenchyma: evaluation of an enzymatic digestion procedure mhc class i1 antigen-bearing dendritic cells in pulmonary tissues of the rat. regulation of antigen presentation activity by endogenous macrophages populations immunoregulation of asthma: control of t-lymphocyte activation in the respiratory tract downregulation of the antigen-presenting cell function(s) ofpulmonary dendritic cells in vivo by resident alveolar macrophages origin and steady-state turnover of class i1 mhc-bearing dendritic cells in the epithelium of the conducting airways blood clearance of streptococcus pneumoniae by c-reactive protein development of thl cd4 + t cells through il-12 produced by listeriainduced macrophages hapten-immune pulmonary interstitial fibrosis (hipif) in mice requires both cd4+ and cd8+ t lymphocytes animal model of human disease. pulmonary cryptococcosis immunity to a pulmonary cryptococcus neoformans infection requires both cd4+ and cd8+ t cells t cell-mediated immunity in the lung: a cryptococcus neoformans pulmonary infection model using scid and athymic nude mice the role of cd4+ and cd8+ t cells in the protective inflammatory response to a pulmonary cryptococcal infection identification of proliferating dendritic cell precursors in mouse b1ood granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class 11-negative progenitor in mouse bone marrow interferon y regulates antigen-induced eosinophil infiltration into the mouse airways by inhibiting the inflitration of cd4+ t cells factors influencing the immune enhancement of intrapulmonary bactericidal mechanisms activation of gamma delta t cells in the primary immune response to mycobacterium tuberculosis distribution of lung-associated lymphocytes from the caudal mediastinal lymph node: effect of antigen acute immunologic pulmonary alveolitis complement and bacteria: chemistry and biology in host defense potential role of monocyte chemoattractant protein llje in monocyte/macrophage-dependent iga immune complex alveolitis in the rat influenza virus-specific antibody-secreting cells in the murine lung during primary influenza virus infection persistence of influenza virus-specific antibodysecreting cells and b-cell memory after primary murine influenza virus infection cytokine gene activation in rat lung allografts: analysis by northern blotting spontaneous in uitro release of alveolar-macrophage cytokines after the intratracheal instillation of bleomycin in rats cytokines in lung and airways fibrosis the b7 and cd28 receptor families function and regulation of leukocyte homing receptors expression of immune mechanisms in the lung the effect of splenectomy on the appearance of specific antibody-forming cells in the lungs of dogs after intravenous immunization with sheep erythrocytes the mechanism of appearance of specific antibody-forming cells in lungs of inbred mice after intratracheal immunization with sheep erythrocytes the capacity of normal murine alveolar macrophages to function as antigen-presenting cells for the initiation of primary antibody-forming cell responses to sheep erythrocytes in uitro failure of pulmonary clearance of rhodococcus equi infection in cd4+ t-lymphocyte-deficient transgenic mice an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine role of macrophages in immunity and pathogenesis of experimental cryptococcosis induced by the airborne route-part i: pathogenesis and acquired immunity of cryptococcus neoformans regulation of rat pulmonary endothelial cell interleukin-6 production by bleomycin: effects of cellular fatty acid composition cd8+ t lymphocytes in intracellular microbial infections gamma/delta t lymphocytes and heat shock proteins vaccination against tuberculosis and leprosy ifn-gamma-producing ability as a possible marker for the protective t cells against mycobacterium bouis bcg in mice pulmonary host defense: coordinated interaction of mechanical, cellular and humoral immune systems of the lung a canine model for reagenic hypersensitivity and allergic broncho constriction macrophage production of tranforming growth factor b and fibroblast collagen synthesis in chronic pulmonary inflammation high incidence of silent aspiration in elderly patients with community-acquired pneumonia respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine delayed type hypersensitivity responses regulate collagen deposition in the lung tolerance to hapten prevents specific delayed type hypersensitivity and pulmonary interstitial fibrosis in the mouse model the inducing role of tumor necrosis factor in the development of bactericidal granulomas during bcg development regulation of cy subclass germ-line transcripts in human peripheral blood b cells induction of immune responses in uiuo with small number of veiled (dendritic) cells cyclosporine immunodulation in a rabbit model of chronic hypersensitivity pneumonitis alveolar macrophage immunosuppression is maintained in rabbit models of hypersensitivity pneumonitis activation of the complement system by cryptococcus neofonnans leads to binding of ic3b to the yeast role of the capsule in phagocytosis of cryptococcus neoformans the poor accessory cell function of macrophages in the rat may reflect their inability to form clusters with t cells accessory cells of the lung. i. interferon-gamma increases ia+ dendritic cells in the lung without augmenting their accessory activities fcr' subsets of ia+ pulmonary dendritic cells in the rat display differences in their abilities to provide 64-68 210-218. accessory co-stimulation for naive (ox-22+) and sensitized (ox-22-) t cells. am compartmentalization of the peripheral immune system cellular and molecular aspects of granulomatous inflammation nebulized but not parenteral ifn-y decreases ige production and normalizes airways function in a murine model of allergic sensitization new vaccines for the world-needs and prospects airway response to electrical field stimulation in sensitized inbred mice: passive transfer of increased responsiveness with peribronchial lymph nodes studies of resistance to experimental tuberculosis in mice vaccinated with living attenuated tubercle bacilli and challenged with virulent organisms alveolar clearance and the role of the pulmonary lymphatics. i n "lung disease state of the art restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells pulmonary and thoracic macrophage subpopulations and clearance of particles from the lung t-cell-mediated protection of mice against virulent mycobacterium tuberculosis direct activity of human t lymphocytes and natural killer cells against cryptococcus neoformans transfer of immunity to cryptococcosis by tenriched splenic lymphocytes from cryptococcus neoformans sensitized mice the antigen-induced selective recruitment of specific t lymphocytes to the lung persistence of influenza as an immunogen in pulmonary antigen-presenting cells human alveolar macrophages: hla-dr-positive macrophages that are poor stimulators of a primary mixed leukocyte reaction role of natural killer cells in resistance to cyptococcus neoformans infections in mice the role of t lymphocytes in pulmonary microbial defense mechanisms a role for tgf-@ in the suppression by murine bronchoalveolar cells of lung dendritic cell initiated immune responses the alveolar macrophage: role in antigen presentation and accessory cell function induction of thl and th2 cd4+, subsets during murine leishmania major infection pathogenesis and pathology of lobar pneumonia the histogenesis of cells in experimental pneumonia in the dog role for c5 and neutrophils in the pulmonary intravascular clearance of circulating cyptococcus neoformans a role for gamma interferum induced nitric oxide in pulmonary clearance of crypt0 coccus neotormans systemic immunization with pneumococcal polysaccharide vaccine induces a predominant igaz response of peripheral blood lymphocytes and increases of both serum and secretory anti-pneumococcal antibodies in oiuo effector function of influenza virus-specific cytotoxic t lymphocyte clones is highly specific inflammatory granuloma formation is mediated by tnf-a-inducible intercellular adhesion molecule-1 interleukin4dependent pulmonary eosinophil infiltration in a murine model of asthma resistance to tuberculosis: experimental studies in native and acquired defense mechanisms alveolar macrophages in pulmonary immune responses. i. role in the initiation of primary immune responses and in the selective recruitment of t lymphocytes to the lung inability of human alveolar macrophages to stimulate resting t cells correlates with decreased antigen-specific t cell macrophage binding the immunological basis of acquired cellular resistance the j. bums amberson lecture-the induction and expression of cell-mediated hypersensitivity in the lung most human pulmonary infiltrating lymphocytes display the surface immune phenotype and functional responses of sensitized t cells lung transplantation in the rat normal host defenses and impairments associated with the delayed resolution of pneumonia local pulmonary immune responsiveness h e r multiple antigenic exposures in the cynomolgus monkey inhibitory effect of the trfk-5 anti-il-5 antibody in a guinea pig model of asthma inhibition of interleukin-1 by an interleukin-1 receptor antagonist prevent grant-versus-host disease ontogeny of ia+ accessory cells in fetal and newborn rat lung evidence for a common mucosal immunologic system. i. migration of b immunoblasts into intestinal, respiratory, and genital tissues the structural basis for immunity in the respiratory tract characterization and in oiuo distribution of influenza-virus-specific tlymphocytes in the murine respiratory tract the mucosal immune system: from fundamental concepts to vaccine development on the extraordinary capacity of allogeneic epidermal langerhans cells to prime cytotoxic t cells in oioo comparison of the opsonic activity of human surfactant protein a for staphylococcus aureus and streptococcus pneumoniae with rabbit and human macrophages targeting and controlled release of antigens for the effective induction of secretory antibody responses an apparatus for airborne infection of mice human natural killer cells do not inhibit growth of cryptococcus neofomans in the absence of antibody killing of cryptococcus neofomans strains by human neutrophils and monocytes lymphocytes bearing antigen-specific gamma delta t-cell receptors accumulate in human infectious disease lesions depletion of 1915-1921 cd4+ (l3t4') lymphocytes in uiuo impairs murine host defense to cryptococcus neoformans interferony activates rat alveolar macrophages for anticryptococcal activity depletion of murine cd8+ t cells in uiuo decreases pulmonary clearance of a moderately virulent strain of cryptococcus neoformans cd8 cells play a critical role in delayed type hypersensitivity to intact cryptococcus neoformans development and characteristics of in-uitro correlates of cellular immunity to rubella virus in the systemic and mucosal sites in guinea pigs protection against pulmonary blastomycosis: correlation with cellular and humoral immunity in mice after subcutaneous nonlethal infection lymphatic drainage of the lung in dust clearance fungal-strain-dependent alterations in the time course and mortality of chronic murine pulmonary blastomycosis th1 and th2 cells: different patterns of lymphokine secretion lead to different functional properties protective murine monoclonal antibodies to cryptococcus neoformans role of endothelial-leukocyte adhesion molecule 1 (elam-1) in neutrophilmediated lung injury in rats neutrophil-dependent acute lung injury. requirement for p-selectin (gmp-140) protective effects of soluble cr1 in complement-and neutrophil-mediated tissue injury lung injury after deposition of iga immune complexes. requirements for cd18 and l-arginine tumor necrosis factor alpha regulates in uiuo intrapulmonary expression of icam-1 inhibition of lung inflammatory reactions in rats by an anti-human il-8 antibody role of leukocyte adhesion molecules in complementinduced lung injury role ofbeta 1, beta 2 integrins sci icam-1 in lung injury after deposition of igg and iga immune complexes protective effects of il-4 and il-10 against immune complex-induced lung injury heterogeneity among alveolar macrophages in humoral and cell-mediated immune responses: separation of functional subpopulations by density gradient centrifugation on percoll direct interactions of human lymphocytes with the yeast-like organisms cryptococcus neofonnans passive smoking and the seasonal difference of severity of asthma in children potential use ofselective phosphodiesterase inhibitors in the treatment of asthma quantitative relationship between anticapsular antibody measured by enzyme-linked immunosorbent assay or radioimmunoassay and protection of mice against challenge with streptococcus pneumonia serotype 4 transport of horseradish peroxidase from the vascular compartment to bronchial-associated lymphoid tissue cd4+ t-lymphocytes and interleukin-5 mediate antigen-induced eosinophil infiltration into the mouse trachea local and systemic cellular immune responses in guinea-pigs given antigen parenterally or directly into the lower respiratory tract development of the airway intraepithlial dendritic cell network in the rat from ciass i1 major histocompatibility (1a)-negative precursors: differential regulation of ia expression at different levels of the respiratory tract lung defense mechanisms mononuclear cells in human lung parenchyma. characterization of a potent accessory cell not obtained by bronchoalveolar lavage separation of potent and poorly functional human lung accessory cells based on autofluorescence mononuclear cells from human lung parenchyma support antigen-induced t lymphocyte proliferation adhesion molecules on human lung dendritic cells and their role for t-cell activation granuloma formation in severe combined immunodeficient (scid) mice in response to progressive bcg infection. tendency not to form 91 granulomas in the lung is associated with faster bacterial growth in this organ. am stimulation of a major subset of lymphocytes expressing t cell receptors y6 by an antigen derived from mycobacterium tuberculosis unmodified pancreatic islet allografl rejection results in the preferential expression of certain t cell activation transcripts morphologic and functional heterogeneity among rat alveolar macrophage fractions isolated by centrifugation on density gradients adoptive protection of the mycobacterium tuberculosis-infected lung. dissociation between cells that passively transfer protective immunity and those that transfer delayed-type hypersensitivity to tuberculin the kinetics of emergence and loss of mediator t lymphocytes acquired in response to infection with mycobacterium tuberculosis cytokine secretion by cd4 t lymphocytes acquired in response to mycobacterium tuberculosis infection studies on the enhancement of allergic lung sensitization by inhalation of ozone and sulfuric acid aeroso1 lymphocyte subpopulations in high endothelial venules and lymphatic capillaries of bronchus-associated lymphoid tissue (balt) in the rat compartmentalization and kinetics of lymphoid cells in the lung is balt a major component of the human lung immune system? hla phenotype of lung lavage cells following heart lung transplantation delay in the development of the allergic response to metals following intratracheal instillation ige-mediated rhesus monkey asthma: natural history and individual animal variation functional analysis in uitro and in uiuo of mycobacterium bouis strain bcg-specific t cell c1ones importance of l3t4+ and lyt-2+ cells in the immunologic control of infection with mycobacterium bods strain bacillus-calmette-guerin in mice: assessment by elimination of t cell subsets in uiu0 regulation of the ige antibody response in mice. 11. radioresistance of established ige antibody production hypersensitivity diseases of the lungs due to fungi and organic dusts stimulation of rat endothelial cell transforming growth factor+ production by bleomycin regulation of rat pulmonary artery endothelial cell transforming growth factor+ production by il-1 / 3 and tumor necrosis factor-a control of lymphocyte homing differential expression of lymphocyte homing receptors by human memoryleffector t cells in pulmonary versus cutaneous immune effector sites tumor necrosis factor/cachetin plays a key role in bleomycin-induced pneumopathy pneumopathies of the graft-versus-host reaction adhesion molecules in the lung mixed hematopoietic and pulmonary origin of "alveolar macrophages" as demonstrated by chromosome markers epidemiologist of the relationship between exposure to indoor allergens and asthma immunologic aspects of contact sensitivity: an experimental study characterization of murine lung dendritic cells: similarities to langerhans cells and thymic dendritic cells delivery and expression of a heterologous antigen on the surface of streptococci lung allograft rejection in the rat. accelerated rejection caused by graft lymphocytes lung allograft rejection in the rat. corresponding morphological rejection phases in various rat strain combinations acute graft-versus-host disease after lung transplantation the pathology of lung transplantation killing intracellular mycobacteria in in uitro macrophage systems: what may be the role of known host microbicidal mechanisms? the role of cytokines in the pathogenesis of allergic asthma specificity of the helper t cells for the cytolytic t lymphocyte response to influenza viruses aerosolized antigen exposure without adjuvant causes increased ige production and increased airway responsiveness in the mouse bronchoalveolar lavage spectrum ofimmunoregulatory functions and properties of human alveolar macrophages acute experimental hypersensitivity pneumonitis in rabbits chronic experimental hypersensitivity pneumonitis in rabbits antigenspecific desensitization in a rabbit model of acute hypersensitivity pneumonitis chronic hypersensitivity pneumonitis produced in the rabbit by the adjuvant effect of inhaled muramyl dipeptide (mdp). a m acute experimental hypersensitivity pneumonitis in the guinea pig the infection ofwhite mice following an intranasal instillation of cryptococcus neoformans macrophage activation: lipoarabinomannan from avirulent and virulent strains of mycobacterium tuberculosis differentially induces the early genes c-fos, kc, je, and tumor necrosis factor-alpha. 1. lmmunol dendritic cells from rat lung are potent accessory cells expression and regulation of human fibroblast-derived monocyte chemtactic peptide-1 expression of interleukin-6 in association with rat lung reimplantation and allograft rejection presentation of exogenous protein antigens by dendritic cells to t cell clones immune-complex disease in guinea pig lungs immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens the participation oftumor necrosis factor in the pathogenesis of lung allograft rejection in the rat control of influenza and poliomyelitis with killed virus vaccines efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colonystimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a increased airways responsiveness in mice depends on local challenge with antigen biased accumulation of t lymphocytes with "memory"-type cd45 leukocyte common antigen gene expression on the epithelial surface of the human lung cytokine profiles of bronchoalveolar lavage cells from mice with influenza pneumonia: consequences of cd4+ and cd8+ t cell depletion administration of anti-ifn-y antibody to pz-microglobulin-deficient mice delays influenza virus clearance but does not switch the response to a t helper cell 2 phen0type immunopathogenesis of hypersensitivity pneumonitis studies on the density, distribution and surface phenotype ofintraepithelial class i1 mhc antigen (1a)-bearing dendritic cells (dc) in the conducting airways the role of strain variation in murine bleomycin-induced pulmonary fibrosis the effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis pulmonary response to repeated exposure to micropolysporu faeni prolonged exposure to m . fueni in strain i1 guinea pigs experimental murine hypersensitivity pneumonitis adoptive transfer of experimental hypersensitivity pneumonitis: cd4 cells are memory and naive cells experimental hypersensitivity pneumonitis: effect of cd4 cell depletion the inoculation ofminimal doses oftubercle 1123-1140 dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura iga-driven antibacterial activity against streptococcus pneumoniae by mouse lung lymphocytes heterogeneity of immunologic function among subfractions of normal rat alveolar macrophages. 11. activation as a determinant of functional activity immune functions of murine alveolar macrophages: binding of lymphocytes and support of lymphocyte proliferation maintenance ofthe normal rat alveolar macrophage cell population. the roles of monocyte influx and alveolar macrophages proliferation in situ a new model of pneumocystis carinii infection in mice selectively depleted of helper t lymphocytes the pulmonary antibody-forming cell response in the guinea pig after intratracheal immunization alveolar macrophage activation in experimental legionellosis structure and function of bronchus-associated lymphoid tissue (balt) animal model: experimental airborne tuberculosis in the guinea pig what animal models can teach us about the pathogenesis oftuberculosis in humans production of ige antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein ag and cholera toxin chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin the relationship between tuberculosis and silicosis antigen-pulsed dendriticcells can efficiently induce an antibody response in uioo development of macrophages in the lungs of fetal rabbits, rats, and hamsters studies on the lymphocytes of sheep. iv. migration patterns of lung-associated lymphocytes efferent from the caudal mediastinal lymph node local and systemic cell-mediated immunity after immunization of guinea pigs with live or killed m . tuberculosis by various routes t and b memory cells traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm alveolar macrophage kinetics and function after interruption of canine marrow function interleukin-8: a major mediator of acute pulmonary inflammation identification of a novel cell type in peripheral lymphoid organs of mice. i. morphology, quantitation, tissue distribution the dendritic cell system and its role in immunogenicity comparison of intratracheal and intravenous inoculation of sheep erythrocytes in the induction of local and systemic immune responses craft-versushost reaction in the lung natural killer cells in mouse lung: surface phenotype, target preference, and response to local influenza virus infection pulmonary interstitial fibrosis induced in hapten-immune hamsters persistent pulmonary interstitial fibrosis, induced by the immune response to tnp, is associated with altered mrna procollagen type 1:iii ratio allergic contact dermatitis induced by intratracheal administration of hapten immunobiology of lymphocytes in the lung effect of route of immunization on development of antibody-forming cells in hilar lymph nodes characterization of acirculating subpopulation ofspontaneous antitetanus toxoid antibody producing b cells following in viuo booster immunization differences in the migration of b and t lymphocytes: organ-selective localization in vioo and the role of lymphocyteendothelial cell recognition passive transfer of acquired resistance to infection with mycobacteria by means of cells protection against pneumococcal infection conferred by phosphocholine-binding antibodies: specificity of the phosphocholine binding and relation to several types rabbit antibodies to the cell wall polysaccharide of streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci t-cell-mediated immunity in persistent mycobacterium intracellulare infections in mice experimental hypersensitivity pneumonitis in the mouse. histologic and immunologic features and their modulation with cyclosporin a spontaneous regression in murine hypersensitivity pneumonitis: lack of immunologic tolerance hypersensitivity pneumonitis in athymic nude mice idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for b-cell lymphoma self-renewal of pulmonary alveolar macrophages: evidence from radiation chimera studies gamma/ delta t-lymphocytes are not increased in number in granulomatous lesions of patients with tuberculosis or sarcoidosis the maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigenbinding dendritic cells as accessory cells alveolar macrophage elimination in oioo is associated with an increase in pulmonary immune response in mice. 1. e x p comparative accessory cell function of human peripheral blood dendritic cells and monocytes the accessory cell function of human alveolar macrophages in specific pulmonary clearance of encapsulated and unencapsulated haemophilus injluenzae strains effect of systemic immunization on pulmonary clearance of haemophilus inpuenzae type b the role of c5 in polymorphonuclear leukocyte recruitment in response to streptococcus pneumoniae cell-mediated resistance to aerogenic infection of the lung management of lung transplant rejection t cell localization in rabbit models of acute and chronic hypersensitivity pneumonitis late airway changes caused by chronic rejection in rat lung allografts. 7'ransplantation replacement of dendritic cells in the airways of rat lung allografts contribution of antibodysecreting cells induced in mucosal lymphoid tissues ofpigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge in situ expression of b7/bb1 on antigen presenting cells and activated b cells; an immunohistochemical study the combination of anti-b7 monoclonal antibody and cyclosporin a induces alloantigen-specific anergy during a primary mixed lymphocyte reaction distribution and immunophenotype of mononuclear phagocytes and dendritic cells in the human lung origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intra-alveolar administration of aerosolized heat-killed bcc early pulmonary granulocyte recruitment in response to streptococcus pneumoniae cell-mediated immunity and antibody responses in the respiratory tract after local and systemic immunizati0ns respiratory and systemic cellularand humoral immune responses to influenza virus vaccine administeredparenterally or by nose drops an animal model demonstration of enhanced clearance of nontypable haemophilus inflenzae from the respiratory tract after antigen stimulation of gut-associated lymphoid tissue experimental models of pneumocytis carinii infections intrapulmonary tumor necrosis factor triggers local platelet-activating factor production in rat immune complex alveolitis contrasting roles for tumor necrosis factor in the pathogenesis of iga and igg immune complex lung injury relationship between interleukin-1 /3 and platelet-activating factor in the pathogenesis of acute immune complex alveolitis in the rat the relationship 3746-3750. 128,276-281. asthmatic responses and antigen-specific immunoglobulin adhesion molecules in the pathogenesis of asthma immunological unresponsiveness natural killer cell function in human lung is compartmentalized intrapulmonary antigen deposition in the human lung: local responses gut-associated lymphoid tissue as source of an iga immune response in respiratory tissues after oral immunization and intrabronchial challenge alloantigen-induced immunoglobulin production in human lung: differential effects of accessory cell populations on igg synthesis allogeneic alveolar macrophages stimulate selective production of igg2a in recipient murine lung increased bronchoalveolar igc2/igcl ratio is a marker for human lung allograft rejection role in oiuo for gamma interferon in control of pneumonia caused by chlamydia trachomatis in mice development of pulmonary infection in mice inoculated with blastomyces hrmatitidis conidia the role of complement in the host's defense against streptococcus pneumonia proteins of the respiratory tract after heart-lung transplantation genetic control of eosinophilia in guinea pig strains inbred for high or low bronchial allergic reactivity studies on the mechanism ofrecovery in pneumococcal pneumonia. i. the action of type specific antibody upon the pulmonary lesion of experimental pneumonia studies on the mechanism of recovery in pneumococcal pneumonia. iv. the mechanism of phagocytosis in the absence of antibody administration of a highly attenuated, live respiratory syncytial virus vaccine to adults and children accessory cells of the lung. 11. ia+ pulmonary dendritic cells display cell surface antigen heterogeneity mechanisms of decrease in cytoplasmic motility of alveolar macrophages during immediate asthmatic response in dogs human alveolar macrophages inhibit receptor-mediated increases in intracellular calcium concentration in lymphocytes enhancement of systemic immune response by immunization into chronically inflamed lungs a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: lung rejection study group antiviral protection by virus-immune cytotoxic t cells: infected target cells are lysed before infectious virus progeny is assembled immunity to viruses. i n "fundamental immunology the authors thank their secretarial staff for their excellent help in preparing the manuscript, specifically patricia sanchez, becky snyder, rebecca flores, gerri chavez, and christine cave. key: cord-023239-06a03o14 authors: nan title: ii. topic sessions date: 2016-06-10 journal: pediatr pulmonol doi: 10.1002/ppul.23455 sha: doc_id: 23239 cord_uid: 06a03o14 nan although the majority of children with asthma achieve symptom control on low or moderate doses of maintenance inhaled steroids, there is a small proportion that remain uncontrolled despite high doses of prescribed maintenance therapy. these children are prescribed treatments equivalent to stage 4/5 of the british thoracic society (bts) guidelines for asthma management, and either need at least this amount of therapy to achieve control, or have persistent symptoms and frequent exacerbations despite maximal treatment. children with poor control despite maximal prescribed therapy have problematic severe asthma 1 . however, the reasons for poor control may be very varied and can broadly be divided into two sub-categories. the first, "difficult asthma" is the term used to describe patients whose asthma is difficult to control because of a failure to address the basics of asthma management, an incorrect diagnosis has been made, or there has been a failure to address associated comorbidities. underlying reversible and modifiable factors that can result in poor control include poor adherence, unfavourable environmental exposures such as tobacco smoke and aero-allergens to which the patient is sensitised, poor inhaler technique and psychosocial issues 2 . if modifiable factors are successfully identified and addressed, then control can be achieved in children with difficult asthma without the need for escalating therapy or additional invasive investigations. a multidisciplinary team (mdt) is critical to enable modifiable factors to be identified and addressed in children with difficult asthma. the team must include specialist respiratory nurses, a psychologist, pharmacist, physiotherapist and medical staff. significant resources are therefore required to manage paediatric difficult asthma optimally and only specialist centres should be tasked with the assessment of these patients. although this may have an impact on healthcare resources, long term benefits for lung health are significant. the second sub-category of children that have poor asthma control despite maximal therapy are those with true severe asthma. these patients remain with persistent symptoms, or can only be controlled on maximal doses of maintenance therapy, often including oral steroids, after underlying reversible or modifiable factors have been identified and addressed 3 . importantly, more than half of all children with problematic severe asthma have difficult asthma because of underlying modifiable or reversible factors preventing asthma control 4 . therefore, the overall approach to managing a child with problematic severe asthma includes an initial step to identify and treat difficult asthma, and if symptoms persist after this, true severe asthma can be confirmed, which requires additional investigation and management 5 . very clear criteria and definitions that allow distinctions between difficult and severe asthma have been specified for both adults and children aged six years and above by the european respiratory society and american thoracic society 3 . an important point to consider when faced with a child that has poor asthma control despite maximal doses of prescribed maintenance therapy is that once above a threshold of treatment (>800mcg/day or equivalent of budesonide), the child should be referred to a specialist for further management. the national review of asthma deaths in the uk identified 20% of asthma deaths occurred in patients who should have been referred to a specialist for management of problematic asthma 6 . the modifiable factors that result in a child having difficult asthma may be identified extremely efficiently if the mdt approach described is adopted. however, what remains equally important is the continuing assessment and follow-up of patients with difficult asthma in order to ensure: 1. maintenance therapy is reduced to the minimal amount needed to achieve control 2. symptoms do improve after all modifiable factors have been addressed, and there is no progression to true severe asthma à either after short term follow-up or in the longer term 3. the basics of inhaler technique / device / adherence / allergen exposure are all being maintained a retrospective analysis of follow-up of children with difficult asthma for up to six years revealed that those in whom underlying modifiable factors were identified and addressed had an improvement in lung function and reduction in exacerbations over time, while being able to reduce maintenance dose of inhaled steroids such that the majority fell below the threshold for problematic severe asthma 4 . however, there was a large drop out in the number of patients that could be traced for the full six years, highlighting the need for better prospective longitudinal data of outcomes for children with difficult asthma. these missing data are essential in light of recent cohort studies that have followed children with severe asthma to adulthood and shown the irreversible reduction in lung function and prevalence of copd 7 . asthma is one of the most common chronic diseases in children, with a high prevalence in many developed and developing countries. worldwide prevalence of asthma in children varies from 1.6-36.8% according to the international study of asthma and allergies in childhood (issac) study. (1) despite its high prevalence, information about the prevalence of severe asthma in children is unknown, particularly in countries in transition. some estimates come from different studies that have shown that the prevalence of severe asthma in a general population is approximately 0.5-5% among children with asthma, however its true prevalence in a low-income country is unknown. (2) (3) (4) according to issac phase iii, the centers with the highest prevalence of severe asthma symptoms were mostly from english language countries, latin america, africa, the indian subcontinent and the eastern mediterranean. (5) lack of control of the disease has been attributed to various factors such as low accessibility to basic medications, weak healthcare services, poor compliance with prescribed therapy, lack of asthma education, and social and cultural factors. in general, asthma in both children and adults represents a significant problem in public health given the reduced quality of life, school or work absenteeism and increased healthcare costs, especially in countries in transition. in addition, asthma severity and control in childhood are of particular importance as they have been shown to translate into asthma morbidity in adulthood. (6) practical guidelines addressing the management of severe asthma in children have pointed out various aspects important in the development of this condition: medication issues, the environment, asthma education, comorbidity, and psychological problems. worldwide, but particularly in countries in transition, both intrinsic (race, ethnicity, weight) and extrinsic (exposure to allergens, indoor or outdoor pollutants) factors may overlap in a single child to enhance or diminish asthma control and severity. different to many developing countries from other continents, asthma is highly prevalent in latin america. moreover, issac phase iii showed that asthma prevalence in this region is still on the rise. furthermore, evidence suggests that poorly controlled asthma in some areas of latin america leads to significant economic costs attributed to emergency and unscheduled visits, and high mortality rates from asthma. (7) similar to other regions, asthma control is not obtained in most patients, despite available management guidelines and evidence of ics as controllers. several surveys have shown that close to 2.4% of all patients met all the gina criteria for total asthma control, proposing under-recognition of uncontrolled asthma, underuse of appropriate controller treatment, inadequate patient education, and patient denial as possible explanations. (8) also, several risk factors such as poverty, environmental factors, diet, genetics, vitamin d deficiency and tobacco smoking have detrimental effects on asthma control. cross-sectional data from 616 children with asthma in costa rica suggested that low serum vitamin d detected in children with mild to moderate asthma is associated with asthma severity. (9) since the development of worldwide guidelines on the diagnosis and management of asthma, special attention on achieving and maintaining asthma control as the key goal in asthma treatment has been a priority. in clinical studies of children with asthma, satisfactory asthma control can be achieved and maintained in most patients by regular treatment with ics. nevertheless, large population-based surveys consistently show that poor asthma control is common in many children with asthma, despite ics treatment. (10) other several studies have shown a reduction in the number of hospitalizations caused by asthma in various countries in transition when effective preventive and controller measures are implemented, (11) mainly avoidance of risk factors, importance on the use of basic medications and patient education. patients should be educated about the cause of asthma, what triggers the condition, how it should be monitored and managed and, importantly, the outcomes that can be expected and when to recognize lack of asthma control. moreover, health professionals should also be educated regarding under-recognition and under-treatment of asthma, as patients or parents tend to deny the severity of symptoms. in a recent study performed in costa rica (12) , we aimed to examine trends in hospitalization and mortality due to asthma over a 15-year period (1997-2011) , in particular following a national asthma plan (nap). this nap consisted of education meetings at all major public health care centers, emphasizing early diagnosis, early treatment using ics as first-line therapy for asthma control, early use of reliever medication to treat exacerbations, appropriate referral to specialists for asthma care, and avoidance of common allergen sources (e.g. dust-mite and cockroaches) or tobacco smoke. concurrent with this program, general practitioners, pediatricians and internists were first allowed to prescribe ics for asthma (only pulmonologists or allergists could prescribe ics before 2003). as a result of the implementation of the nap, the total number of asthma hospitalizations in costa rica in both children and adults decreased by approximately 53% over this period. in children younger than 10 years, hospitalizations for asthma were reduced by 57% in boys and 54% in girls between 1997 and 2011. in addition, the number of deaths due to asthma decreased by 80% over the 12-year period, with a more marked reduction occurring after implementation of the nap. in parallel with the decrement in asthma hospitalization and mortality, the number of prescriptions for ics (beclomethasone) increased by 129%. in summary, asthma prevalence in deprived regions is high and shows increased severity. reasons for inadequate asthma control in poor populations include low accessibility to effective controller medications, weak infrastructure of health services for the management of chronic disease, poor adherence to therapy, lack of educational approaches, and social, cultural and language barriers. however, recent studies have shown several alternatives to control its burden and improve outcomes. there is urgent need for more research into severe asthma, in particular in children in countries in transition. it has now become a much used adage that asthma is not a single disease but rather multiple diseases which present with common symptoms [1, 2] . this paradigm has been fundamental in shaping the way we think about asthma and possible approaches to treatment and management strategies. if one is not treating a single disease when we talk about what is commonly known as the syndrome of asthma, then we need a more personalised medication strategy to treat these different syndromes. alongside this acknowledgment of moving medicine towards more personalised treatment and management strategies, statistics and machine learning have been instrumental in helping us to shape the face of medicine by fostering engagement between clinicians, basic scientists, statisticians and mathematical modellers in order to attain a more unbiased approach to classifying different subgroups of patients using probabilistic models. the proliferation of genetic, molecular, clinical and biological data has made it necessary to use a cross-disciplinary approach to understanding the underlying mechanisms which precipitate distinct profiles of asthma and allergic disease during childhood. statistical analysis to understand subtypes of childhood wheezing the seminal paper by martinez et al. [3] was the first to propose the existence of different subgroups of childhood wheezing. based on visual assessment of patterns of wheeze during childhood using data from the tuscan children's respiratory study, they identified four groups of wheezers: "no wheeze", "transient early wheeze", "late-onset wheeze" and "persistent wheeze". this classification has been used as a classical basis for subsequent definitions of distinct subgroups of wheeze and has provided the building block for statistical pattern recognition-based methods to identify heterogeneous groups of children based on probabilistic modelling of the longitudinal profiles of asthma and wheeze over time. one such statistical technique is latent class analysis. latent class analysis assumes that the longitudinal fluctuation observed in data is measured with uncertainty. some of this uncertainty is due to random error, but another element of this uncertainty may be due to the existence of a subgroup or latent class which explains some of the heterogeneity in clinical measures which is not directly observed. henderson et al. were the first to apply such models using a data-driven approach based on wheeze observations from the avon longitudinal study of parents and children [4] . using latent class analysis based on parental reporting of wheeze, this group identified two additional phenotypes to those identified by martinez et al.: "prolonged early" and "intermediateonset" wheeze. this classification has been replicated in other studies. [5] one of the caveats of basing these modelling strategies on parental reporting of wheeze is that parents may not be able to correctly ascertain a clinical diagnosis of wheeze [6] . in light of this, belgrave et al. extended these methods by jointly modelling data from both parental questionnaires and general practitioner records which provided complementary data to give a more accurate measure of wheeze [7] . this model identified two classes of persistent wheeze: a "persistent controlled wheeze" group and a "persistent troublesome wheeze" group who had poorer lung function and more reactive airways compared to the other wheeze groups, including the "persistent controlled wheeze" group. where machine learning begins and statistical modelling ends identifying consistently defined and optimal numbers of subgroups of wheeze across different cohorts is challenging. within the era of "big data" rather than focusing on traditional statistical methodology, the medical field is looking towards data science as a means to extract knowledge and meaning from the vast quantity of information provided by clinical data. to achieve this, both traditional statistical inference methods based on robust assumptions and machine learning models which are more amenable to data complexity, breadth and depth. although there is overlap between the functionality of machine learning and statistics, the flexibility of machine learning is driven towards learning from data and integrating new information in order to update models and create more accurate models with better model performance. the programmatic focus of machine learning which incorporates vast amounts of computational power provides an excellent framework where tools traditionally used for statistical modelling would be unable to accommodate large, multi-scale datasets. in the near future, the capability of machine learning to be able to learn from data interactively may facilitate computer-assisted reasoning in identifying subgroups of patients. identifying such subgroups may be crucial in proposing effective personalised treatment strategies. such an approach will also allow us to capitalise on the existent data. as data-transparency and data-sharing become more widespread in the global community, we will have a better understanding of the evolution of asthma and allergic diseases. research into identifying heterogeneous subgroups of asthma and allergic disease has reached crucial milestones. we have moved from a subjective approach to classifying subgroups of wheezers, whereby the clinician gives a clinical assessment or diagnosis of the most likely subgroup based on observed clinical history, and we are moving towards computer-assisted reasoning, whereby we can use new information to predict the most likely class assignment based on models derived from prospective data. such reasoning would also allow us to model the evolution of asthma and allergic diseases in the future. populations of microbes (such as bacteria and yeasts) inhabit the skin and all mucosal surfaces. healthy individuals host thousands of different types of bacteria and different body sites have their own distinctive communities, with estimates suggesting that 50%-90% of all the cells in the human body are microbes. the highest density and greatest diversity of bacteria is found within the gastrointestinal tract. research suggests that the relationship between the microbiome and humans is not only commensal (a non-harmful coexistence), but is a mutualistic, symbiotic relationship with benefits for both (1) . even though we live in such a "dirty" bacterial world, infections due to bacteria are relatively very rare in individuals with a competent immune system. the microorganisms that make up a microbiome perform a wide range of useful functions, such as fermenting unused energy substrates, educating the immune system, preventing growth of pathogens, regulating the development of organs such as the gut, producing vitamins for the host and producing hormones to influence host metabolism such as directing the host to store fats. in particular, specific microbe-host interactions are thought to be critical for inducing mucosal tolerance and immune regulatory cells such as tregs. why do we develop "tolerance" to the microbes living in us and on us? perhaps we should consider tolerance as an alternative defense strategy. the continuous effort involved in destroying the microbes that surround us would impair organ function and require vast amounts of energy, which is not compatible with life. for this reason, it makes much more sense to have robust tolerance mechanisms that work in tune with potent effector responses, to ensure optimal host fitness. an intriguing question is that posed by the concept of the hygiene hypothesis in that altered exposure to microbes may influence the induction of tolerogenic immune responses, thereby making individuals more susceptible to react aggressively to nondangerous encounters with antigens such as allergens. the balance between immune tolerance and inflammation is regulated through the crosstalk between epithelial and immune cells with the microbiome involving many signaling pathways and molecules. direct contact with bacterial-associated structures can activate receptors (e.g. tlrs) on host cells, which induce signaling cascades resulting in both innate and adaptive polarized immune responses. the microbiome is also metabolically active and microbial metabolites have been shown to exert significant effects on host immune signaling networks (e.g. scfas and biogenic amines). the biogenic amine histamine can promote either pro-or anti-inflammatory effects depending on which of its four receptors are activated (2) . some, but not all, commensal bacteria express histidine decarboxylase (the enzyme needed to convert histidine to histamine). lactobacillus saerimneri 30a produces high levels of biologically active histamine and feeding this strain to mice resulted in a deterioration in health, particularly in histamine receptor 2 knock-out mice (3). significant efforts are underway to determine the positive and negative health effects associated with production of histamine by the microbiota (4). abnormalities in microbiome composition and/or metabolic activity have been shown in a wide range of disease states including type-2 diabetes, obesity, inflammatory bowel disease, colorectal cancer and allergies. efforts to use microbiome-associated therapeutics (e.g. probiotics) have clearly shown beneficial effects in animal models, with inconsistent findings in humans probably due to differences in the bacterial strains used. one probiotic bacterium that has shown consistent immunoregulatory effects in murine models and humans is b. longum subsp. longum 35624. murine models have demonstrated that oral consumption of this strain results in the induction of treg cells and these treg cells dampen nfkb activation, preventing excessive inflammation induced by salmonella infection (5, 6) . similarly, in humans, oral consumption induces treg cells, which is associated with increased secretion of il-10 by peripheral blood cells (7) . interestingly, this strain reduces systemic pro-inflammatory biomarkers in patients with psoriasis, ibs patients with chronic fatigue syndrome and patients with ulcerative colitis (8) . the mechanism involved includes the recognition of this bacterium via tlr-2/6 and dc-sign by myeloid dendritic cells and tlr-9 by plasmacytoid dendritic cells, resulting in changes in dendritic cell cytokine secretion and the production of metabolites such as retinoic acid (9) . however, these effects and mechanisms are not seen even with closely related bacterial strains, suggesting that the careful selection of microbes is essential for the future clinical development of immunotherapeutic microbes for allergy and asthma. overall, it can be concluded that the vast majority of microbes, which interact continuously with the host, are not bad. certain specific microbes can positively influence the host, while there is a minority that can have negative effects on the host. gwas findings are based upon association p-values below 5 ã 10 à8 , socalled "genome-wide significance", as well as replication in independent populations. this generally requires very large sample sizes and in order to obtain these, a 'team science' approach has been used where several studies have combined their data in meta-gwas. the largest gwas on asthma to date combined data from 23 different studies involving more than 26,000 individuals from the gabriel consortium and identified 6 genome-wide significant asthma loci.(1) similar meta-gwas have been conducted, for example by the eagle consortium, revealing a number of susceptibility loci for asthma-related traits, including feno,(2) eczema,(3) and allergic sensitization.(4) it could be expected that the large heterogeneity in disease phenotypes introduced by combining many different studies in meta-gwas would preclude valid discoveries. nevertheless, gwas on asthma and the related traits have resulted in identification of relatively few, but robust, loci with more consistency between studies compared to previous candidate gene studies. one example of this is the first large-scale gwas on allergic sensitization.(4) by meta-analysis of data from 16 different studies, it included a discovery phase of approximately 5,800 cases and 10,000 controls and a similar-sized replication phase. allergic sensitization was assessed objectively and defined by elevated levels of allergen-specific ige and/or a positive skin prick test. this study identified 10 loci associated with allergic sensitization at the genome-wide significant level and with robust replication. simultaneously, another large gwas was performed on allergic symptoms including approximately 54,000 individuals. (5) in spite of the large phenotype differences between the two studies, there was a high agreement in results with all of the 10 genome-wide significant loci from the sensitization study also showing strong association in the study on allergic symptoms, and previous gwas findings were confirmed. there has been some disappointment with the results from gwas. the identified loci only explain a minor part of the heritability, and the susceptibility variants identified in gwas are mainly common variants with relatively small effect sizes (often with odds ratios around 1.1 per risk allele) with no clinical relevance on the individual level. (1, 4) on the other hand, gwas have identified novel and robust susceptibility loci, with the potential to provide important understanding of disease mechanisms. also, comparison of results from gwas on different diseases and traits have increased the understanding of the mechanistic relationship between these, for example the relationship between allergic sensitization and asthma,(4) between allergen-specific ige and total ige levels, (4) and between atopy and autoimmunity. (3) (4) (5) larger, consortium-based studies on asthma and the related phenotypes are ongoing and are expected to identify many novel susceptibility loci. novel loci discovered from these larger studies are likely to have even smaller effect sizes than the ones previously found but, from the perspective of understanding disease pathology, each novel locus may potentially pinpoint a novel mechanism and a potential treatment target. furthermore, the era of genome-wide nucleotide sequencing applied on gene expression-and epigenome-profiling has brought new possibilities of combining gwas data with data from large public 'omics repositories. these data will increase the usefulness of gwas data by providing understanding of functional effects related to susceptibility loci, and future gwas on asthma and related diseases will be a part of integrated approaches to discover how different molecular layers modulate the genetic effect on disease, and will thereby be a central component in the attempt to tailor and improve medical treatment. asthma is a highly heterogeneous disease probably consisting of several subtypes of disease associated with different functional mechanisms. genetic loci may be involved in specific disease mechanisms and thereby help understanding this heterogeneity. for example, the strongest asthma locus identified in gwas, the 17q12-21 locus, seems strongly associated with an asthma phenotype characterized by onset in early childhood (1) and recurrent, severe exacerbations (6) and was stronger associated with asthma than allergic rhinitis. (5) in contrast, another locus at chromosome 11q13 has been associated with multiple allergy-related phenotypes, including allergic sensitization,(4) allergic symptoms,(5) eczema,(3) and asthma, suggesting a different, allergy-related, disease mechanism. the heterogeneous nature of asthma suggests that an alternative to increasing sample size in genetic studies is to focus on more specific phenotypes. such phenotypes are likely closer associated to specific mechanisms and the genetic substrate and might therefore increase study power. this was demonstrated by a gwas focusing on a specific asthma phenotype characterized by onset in early childhood and recurrent, severe exacerbations. (6) in spite of the relatively small sample size, this study resulted in association results of the same magnitude as previous much larger gwas (1) and with much larger effect sizes, particularly for the children with the highest number of exacerbations. one novel asthma gene, cdhr3, was identified, and it was confirmed, in a collaborative effort involving several birth cohort studies, that the cdhr3 locus was strongly associated with asthma exacerbations in the first years of life, both in individuals of european and non-european ancestry. these results highlight the potential of future genetic studies focusing on more homogenous phenotypes. one important future step is the translation of genetic associations to disease mechanisms. a major limitation of gwas is that they often merely identify a susceptibility locus without any clear relationship to a specific gene or biological function. two examples of this are the 17q12-21 and 11q13 loci mentioned above, where the underlying mechanisms are still poorly understood several years after their discovery, even though these loci are strong and probably central to the pathogenesis of asthma and allergy. one example of a gwas discovery where the functional mechanism might have been identified is cdhr3. in the discovery study (6) , it was suggested that the association to asthma was caused by a specific functional variant affecting surface expression of cdhr3. a later study reported that cdhr3 functions as a rhinovirus c receptor and showed that the functional variant associated with asthma exacerbations increases rhinovirus c binding and replication. (7) this potentially explains the underlying mechanism of this locus and identifies a target for future asthma and virology research. another major future challenge is to understand how genetic susceptibility interacts with environmental factors. gene-environment interactions are not accounted for in normal gwas and that might be one reason for the large heritability not explained by gwas findings. one important environmental risk factor for childhood asthma and other wheezing disorders is viral infections, and focusing on this environmental factor might be a tool to understanding mechanisms of asthma genes. (8) as an example, children with 17q12-21 risk variants seem more susceptible to rhinovirus infections, (9) and the finding that cdhr3 seems to be a rhinovirus c receptor (7) indicates that children carrying cdhr3 risk variants will have a specific susceptibility to rhinovirus c infections, a hypothesis that is currently being tested. only a few genome-wide gene-environment interaction studies have been performed, and the results of these have generally been disappointing without convincing findings. there are many inherent challenges in such studies. first, they might require even larger sample sizes than normal gwas, and exact information on environmental exposures is difficult to obtain in such large-scale studies. furthermore, the effect of a specific environmental exposure can be difficult to disentangle from that of other related environmental factors. an alternative approach is to perform cell or animal models where specific exposures can be controlled.(8) a recent study investigated the potentially protective effect of endotoxin and farm dust exposure in a mouse model of house dust mite-sensitized asthma.(10) it was found that a20 was an important mediator of the protective effects of endotoxin exposure, and this was validated in human bronchial epithelial cells. furthermore, a potential modifying effect of a20 was supported by 'look up' of snps located near the human tnfaip3 gene using data from an earlier genome-wide interaction study. this potential gene-environment interaction needs to be replicated, but this study exemplifies how mechanistic studies targeting specific environmental exposures and the use of experimental models can facilitate identification of genes involved in gene-environment interactions. in conclusion, improved understanding of the genetic architecture of asthma and other childhood wheezing disorders will require a combination of gwas focusing on more homogeneous subtypes of disease, geneenvironment interaction studies in birth cohorts and in cell models, and integration with other types of omics data. this challenge can only be overcome by a 'team science' approach bringing together many studies to provide sufficient statistical power and bringing together researchers from many disciplines to translate clinical associations to mechanistic understanding. such studies present great challenges but also the opportunity to understand asthma pathogenesis and heterogeneity, and ultimately to improve prevention and treatment of disease. recently, who definitions have changed to classify children with lower chest indrawing as having pneumonia rather than severe pneumonia and recommending treatment with oral antibiotics as ambulatory cases. [1] however, a recent meta-analysis reported that no single clinical feature is sufficient to accurately diagnose radiological pneumonia and that the who recommended diagnostic signs alone lack sufficient sensitivity or specificity, particularly for identifying children who need antibiotics. [2] radiological diagnosis of pneumonia has relied largely on changes on chest x-ray, principally consolidation or interstitial infiltrates. [3] however, chest x-rays are subject to variable interpretation, expose a child to ionizing radiation and require infrastructure and skill to do. recently, chest ultrasound has been suggested as a feasible imaging modality for diagnosis of childhood pneumonia. ultrasound has several advantages including that it can be used as a point-of-care test, can be taught to non-radiologists, is quick to perform and does not involve exposure to radiation. initial studies suggest that it has high sensitivity and specificity for pneumonia compared to chest x-rays. [4, 5] diagnosis of the etiology of pneumonia remains challenging as bacteremia is rare, distinguishing colonizing from pathogenic organisms may not be possible on respiratory specimens and co-infections are common. improvements in specimen collection and improved molecular techniques for detection of organisms have enabled more accurate detection of organisms, however ascribing etiology may be difficult unless the organism is invariably pathogenic. advances in specimen collection include the use of induced sputum in infants and young children, which provides a better specimen for detection of specific pathogens such as b. pertussis or m. tuberculosis. [6] urine antigen detection has not proven to be useful for pneumococcal pneumonia or for pulmonary tuberculosis in children. [7, 8] for induced sputum, testing of sequential, repeat specimens provides a higher yield for pathogens such as m. tuberculosis. [9] careful attention to specimen collection methods and use of different specimens may maximize the yield especially in the context of new sensitive molecular detection techniques. [10] with the availability of improved tools for etiological diagnosis, and with better vaccine coverage for conjugate vaccines, including pneumococcal conjugate vaccine, viral pathogens especially rsv and other bacteria, such as s. aureus or pertussis, are emerging as prominent causes of childhood pneumonia. [11] [12] [13] in areas of high tb prevalence, m. tuberculosis has been reported to be associated with acute pneumonia in children, with culture confirmed disease occurring in approximately 8% of cases. [14] however, better tools for detection of potential pathogens have also provided data on the complexity of etiology, with several potentially pathogenic organisms frequently identified in a single pneumonia episode. further delineation of the interactions between different organisms and pneumonia pathogenesis is needed. asthma affects as many as 334 million people of all ages in all parts of the world and is the commonest long-term respiratory condition affecting children in developed countries, the prevalence and morbidity varying by ethnic group 1 . accurate diagnosis and effective management of respiratory diseases such as asthma requires objective measures of lung function, but reliable use of such measures is only possible if appropriate normative ranges are available to distinguish the effects of disease and treatment from those of growth and development. evidence for ethnic differences in lung function ethnic differences in lung function have been well documented 2 . in the past, attempts to interpret observed ethnic differences in lung function were often confounded by selection bias related to use of small population samples that were not necessarily representative or generalizable, use of different methods, equipment and quality control (qc) criteria, failure to adjust for other important determinants of lung function, including socio-economic circumstances and/or inappropriate statistical analyses. in recent years, many of these problems have been addressed by applying standard methodology, inclusion criteria and qc to large, ethnically homogenous groups. current research shows that after adjusting for age, sex and standing height, forced expired volume in 1 sec (fev 1 : a measure of airway calibre) and forced vital capacity (fvc: a measure of lung size) are both reduced by approximately 14% in individuals of african ancestry (black) across the entire life span when compared with those of european ancestry (white) [3] [4] [5] [6] . similar though smaller reductions have been observed among south asian (from indian subcontinent) [7] [8] [9] and south-east asian (e.g. china, thailand, malaysia, etc) 6 subjects. since these "ethnic" reductions in fev 1 and fvc are generally proportional, the fev 1 /fvc ratio, which is the most commonly used outcome to assess airways obstruction, is usually independent of ethnic background [5] [6] [7] , suggesting that there are no structural or functional ethnic differences in lung design. thus the observed ethnic differences in lung function appear to be primarily limited to lung size rather than airway or dynamic respiratory characteristics. however, the same adjustment factor cannot be used for all lung volume outcomes. for example, there is evidence that the lower fvc found among black children can be attributed at least in part to a relatively high residual volume, suggesting that factors such as anatomic differences in diaphragmatic position or respiratory muscle strength might contribute to some of the observed differences 5 . furthermore, lung function indices that are internally adjusted for the size of the individual's resting lung volume, such as the lung clearance index (lci: a measure of gas mixing efficiency) 10, 11 or specific airways resistance (sraw: a measure of airway calibre adjusted for lung volume) 11 , do not appear to be influenced by ethnic background. nevertheless, since larger sample sizes will be required to confirm these findings, data interpretation of lci and sraw from non-white subjects should currently be undertaken with caution. recently, the global lung function initiative (gli) collated results from >74,000 healthy non-smokers aged 3-95 years to create the first allage, multi-ethnic reference equations for spirometry with appropriate age dependent lower limits of normal 6 . prediction equations were derived using the lms method, which allows simultaneous modelling of the mean (mu), the coefficient of variation (sigma) and skewness (lambda) of the distribution, and reference equations were derived for caucasians (white); african-americans (black), north-and south-east asians. these equations enable assessments to be evaluated over the entire age range using a single reference data set, thereby avoiding the errors that can occur when switching between equations, particularly during the transition between paediatric and adult care 12 . defining ethnicity ethnicity is extremely difficult to define. self-assigned ethnicity may differ from observer-assigned ethnicity and in certain countries it is against the law to record ethnic origin for any purpose. furthermore, in recent censuses in both the uk (2011) and us (2010), mixed-race populations have been shown to be the fastest-growing ethnic group. thus, classifying ethnicity may become an increasingly complex task! could differences in body proportions explain the ethnic differences in lung function? standing height is a major determinant of lung volumes, reflecting the fact that lung size is adapted to our metabolic needs. however this is not ideal since the size of the lungs is more closely related to thoracic size than leg length and differences in body proportions may underpin much of the observed ethnic variation in lung function. the size and lung function in children (slic) study was designed to improve normative reference ranges for lung function by taking differences in body physique into account to facilitate early diagnosis and treatment of lung disease in all children, irrespective of ethnic background 7 . however, of the numerous additional anthropometric measurements undertaken to quantify body physique, only sitting height and chest width significantly contributed to the prediction of spirometric lung function. chest dimensions and lean mass also significantly predicted fev 1 and fvc within each ethnic group, but did not affect differences between groups. the persistence of ethnic differences after adjustment for sitting height, chest dimensions, body composition and socio-economic factors may reflect the fact that some factors affecting chest size such as diaphragmatic position or muscle strength cannot be assessed by anthropometry, and emphasises the importance of taking ethnicity into account when interpreting spirometry data 7 . while some studies have shown an association between socio-economic conditions (sec) 13, 14 and lung function and suggested that this is a key factor in explaining ethnic differences in lung function 15 , there is increasing evidence that the contribution of sec to variability of lung function is very small except under the most adverse of conditions 7, 14, 16 . a recent study in india 8 , using identical equipment and techniques as those used in the slic study found that while average fev 1 and fvc in urban indian children were similar to those in indian children residing in the uk, they were significantly higher than in semiurban and rural indian children (by $6% and 11% respectively). these results probably reflect the marked differences in the degree of social deprivation between the uk and india 7,8 and suggest that there may be a threshold effect of poverty on lung function. adjusting for sitting height has been shown to reduce the contribution of sec to ethnic variability 7, 14 . the use of inappropriate reference equations and misinterpretation can lead to serious errors with respect to both under-and over-diagnosis. in the past, attempts to correct for ethnic differences, if made at all, tended to apply the same fixed adjustment factor across all ages 2 , all ethnic groups, both sexes and all spirometric outcome measures, an approach now shown to be oversimplistic 14, 17 . in addition to errors relating to ethnic differences in lung function, misdiagnosis may also occur when fixed cut-offs, such as 80% predicted fev 1 or 0.7 fev 1 /fvc are used; particularly in young children and elderly adults. while %predicted has historically been used to interpret lung function results, z-scores are more appropriate as they take into account the between-subject variability of measurements for any given outcome at any given age, as well as the predicted value 17 . similarly, use of <0.7 as a fixed threshold for abnormal fev 1 /fvc can lead to gross under-diagnosis of airway obstruction in the young and over-estimation in the elderly 17 . with exception of extreme deprivation, ethnic differences in lung function cannot be explained by socio-economic factors. after adjusting for confounders, genetic factors do contribute to ethnic differences in body physique and lung function. given the marked ethnic differences in lung function, the magnitude of which are similar across the entire life span, it is essential that lung function results in children are interpreted using ethnic specific equations whether in clinical practice or epidemiological research. although gli-2012 do not and never will cover all ethnic groups, appropriate use of age, height and sex adjusted values of fev 1 /fvc ratio derived from these equations (which is consistent across all ethnic groups) will facilitate better identification of airway obstruction in children irrespective of ethnic background. failure to adjust lung function for ethnic differences will result in overestimation of both the severity of airway obstruction and the severity and prevalence of restrictive lung disease. it is now recognised that asthma is a complex, heterogeneous disease. therefore, we need to move away from offering a single approach to management for all children and consider the identification of individual phenotypes for each child to enable optimal treatment and control. the specific facets of the disease that need to be considered and defined in each child include: i. an accurate description of symptom pattern (exacerbations alone, or persistent symptoms with and without exacerbations), ii. the nature of airway inflammation (eosinophilic, neutrophilic or non-inflamed), iii. the type and degree of structural airway changes (remodelling). although asthma control can be achieved in most children with low-moderate doses of inhaled steroids, we remain unclear about the choice of optimal maintenance therapy for each child. how should a decision between regular inhaled steroids, or leukotriene receptor antagonists be made? when additional therapies are required to achieve control, a scientific rationale for add-on therapies also is unavailable. the majority of decisions about therapies are made using a "trial of treatment" approach 1 . if one approach is not successful, then another is adopted without a clear thought process dictating choice of treatments. it is apparent that we need to change our current one size fits all approach to the management of asthma in children. although perhaps less important for children with mild or moderate disease, this becomes extremely important when we consider those with more severe disease. personalised medicine for severe asthma although atopy, airway hyperresponsiveness, eosinophilic inflammation and remodelling are the cardinal pathophysiological features of paediatric asthma, we now know that each of these features can be present to very different degrees in the individual child 2-4 . pathology has been most studied in children with severe disease, and although features such as eosinophilic inflammation and increased airway smooth muscle represent the patients as a group, there is huge overlap between children with and without asthma, and a huge spread of severity of these features within the group of children with severe asthma 2 . this within-group variability means assessments need to be made in the individual before deciding on the most appropriate add-on therapy. a proposed approach to identifying the "individual phenotype" in children with severe asthma is to split response to steroids into different domains (bossley c et al. j allergy clin immunol 2016, in press). not all children with asthma have abnormal lung function, not all have inflammation or remodelling, the response to a trial of systemic steroids can therefore be split into the following: i. lung function response, ii. inflammation response (exhaled nitric oxide and sputum eosinophils) and iii. symptom response. we have analysed this approach in 54 patients with severe therapy resistant asthma and shown a similar proportion of children (approx. 40%) responded to systemic corticosteroids in each domain, but there were no reliable predictors of a response pattern. furthermore, only 13% were complete responders (response in all domains), 15% were nonresponders (no response in any domain) and the majority (72%) were partial responders (response in >1 domain). these data highlight that childhood severe asthma is heterogeneous and a complete response in symptoms, inflammatory and physiological parameters is rare (bossley c et al. j allergy clin immunol 2016, in press). individual response patterns to systemic steroids need to be applied in the future to guide the choice of addon therapies in each child as a step towards achieving personalised medicine. subsequently, this multi-domain approach was applied clinically to identify characteristics of responders to the add-on therapy omalizumab. it became apparent that only those with a positive response in the inflammation domain (a significant reduction in exhaled nitric oxide after a trail of systemic steroids) had a beneficial response from omalizumab 5 . as increasing numbers of add-on therapies become available for use, specifically in the context of severe asthma, we need to better define pathophysiological phenotypes in individual patients and we need to understand the mechanisms mediating disease in children. in addition, we now need to incorporate individual genotypes into our definition of phentoypes to more accurately define treatment responses 6 , as has been successfully done for response to montelukast in preschool wheeze 7 . not only will this individualised approach allow us to discover novel molecular targets that will be effective specifically in the paediatric population, but it will also enable us to objectively choose the best therapy tailored to the individual child. europe consistently report that 20-40% of children with a recognized asthma diagnosis require acute medical care yearly. this is a reflection of the inadequacy of the available treatment options for prevention and treatment of exacerbations, suggesting that asthma with severe exacerbations may represent a distinct subtype of disease and demonstrating a need for improved understanding of its pathogenesis. asthma heritability is estimated at 50-80%. a number of genes have been verified in genome-wide association studies (gwas), but still the genetic background of asthma remains poorly understood. larger gwas may reveal new susceptibility loci with smaller effects, but due to the large heterogeneity of asthma (1), an alternative strategy may be to increase phenotype specificity. a specific phenotype is likely to be closer related to a specific pathogenetic mechanism and may therefore markedly increase the power of genetic studies. this was the background for a gwas focusing on a particular asthma phenotype defined by repeated, severe exacerbations in early childhood. (2) a sufficient number of cases were obtained by identification of children with recurrent acute hospitalizations for asthma between 2 and 6 years of age in the danish national patient register, and extraction of dna from dried blood spots from the danish newborn screening biobank. the case phenotype was rare with only 1/1000 of children born in denmark between 1982 and 1995 fulfilling the inclusion criteria. the final study comprised 1,173 children with repeated hospitalization and 2,511 healthy controls. five loci were identified with genome-wide significant association (pvalue < 5 ã 10-8): gsdmb, il33, rad50, il1rl1 and cdhr3. even though the sample size of this gwas was less than one fifth of the largest published gwas on asthma from the gabriel consortium,(3), it identified a similar number of genome-wide significant loci with similar statistical significance. the effect estimates were remarkably high with odds ratios between 1.4 and 2.3 per risk allele, compared to the odds ratios around 1.1-1.2 usually found in gwas on complex traits. further increasing phenotype specificity by stratified analysis in the 358 children with the highest number of exacerbations resulted in a further increase in effect estimates, with odds ratios between 1.6 and 2.7 per risk allele, and strong statistical significance. these strong results demonstrate the value of focusing on a more specific phenotype in asthma genetics. furthermore, it indicates that studies on this severe and early-onset phenotype is a "cost effective" approach whereby methodologies requiring large resources and/or strong statistical power can be applied in a limited number of individuals and still provide powerful results. the top-locus in this study, at chromosome 17q12-21 near gsdmb/ ormdl3, has consistently been associated with childhood onset asthma. (3) (4) (5) the effect size in the present study was remarkable with an or of 2.3 (p-value ¼ 1.3 ã 10 à48 ) and increasing to 2.7 for the children with highest number of exacerbations. this suggests an important role for this locus in severe exacerbations in early childhood in line with a previous report from the copsac 2000 birth cohort study.(5) cdhr3 had not previously been associated with asthma or any other disease. the association with asthma was replicated in the publically available gabriel results (3) protein structure modeling showed that the risk-associated variant is located at the interface between two domains where it could be involved in disulfide rearrangement and interfere with inter-domain stabilization, overall protein stability or conformation, in agreement with the observation in experimental studies of altered cell surface expression. (2) the biological function of cdhr3 is unknown but it seems to be a highly plausible asthma gene. it belongs to the cadherin gene family of transmembrane proteins involved in several cellular processes including epithelial polarity, cell-cell interaction, and differentiation (6) and is highly expressed in the lungs. also, other members of the cadherin family have been associated with asthma and related traits, including e-cadherin. (7) recently, it was reported that cdhr3 functions as a receptor for rhinovirus c. (8) cdhr3 was differentially expressed in epithelial cells susceptible to rhinovirus c infection compared to unsusceptible cells, and its expression on epithelial cells enabled rhinovirus c binding and replication. importantly, introduction of the risk variant at rs6967330 by transfection resulted in 10-fold increased rv-c binding and progeny yield compared to the non-risk variant. these data provide strong evidence that cdhr3 is a rhinovirus c receptor and that the association signal in the cdhr3 gene might result from increased susceptibility to rv-c infections. this finding is in line with the exacerbationrelated phenotype from the discovery gwas, since rhinovirus c has been reported to be the most common viral trigger of severe asthma exacerbations in children and associated with more severe disease and higher rates of hospital readmissions compared to other respiratory viral infections.(9,10) if correct, this would indicate that children with the cdhr3 risk variants are specifically susceptible to rhinovirus c infections compared to illnesses triggered by other viruses, a hypothesis that is currently being tested. in conclusion, the strong results found in this gwas on childhood asthma with severe exacerbations demonstrate the value of specific phenotyping in the search for asthma genes. focusing on this extreme subtype of disease might reveal mechanisms that would not be revealed in studies of milder disease, but might also increase the understanding of general asthma mechanisms. identification of cdhr3 as a risk gene might be one of the first examples where the underlying mechanism of an asthma gwas finding is understood. future studies of this gene may improve understanding and treatment of asthma exacerbations in childhood. the timing of bacterial colonization early in life is thought to be important for appropriate immune education and the transmission from mother to the fetus during pregnancy and birth is being better described. cultures of meconium have shown diverse groups of gram-positive and gram-negative bacteria, possibly not all derived post-delivery. the development of the gut microbiome is a dynamic process and early colonization with bacteroides and bifidobacterium species might play a crucial role in the development of immune regulation (1) . factors that can influence early life colonization include antibiotic treatment, method of delivery, maternal and infant diet and biodiversity in the home, surrounding environment and in family members. the gut microbiome increases in diversity during the first years of life. germ-free mice, which are not exposed to live bacteria, display exaggerated th2 and ige responses, associated with diminished polarization of treg cells. monocolonization of the mice with specific microbes, but not all microbes, suppresses the ige response and promotes treg differentiation (2) . however, certain immunological changes, such as increased inkt numbers in the mucosa, cannot be reversed following colonization of mice later in life (3). interestingly, more severe allergic responses and anaphylaxis were observed in mice who received a microbiome transplant from allergic animals, suggesting that certain microbial species can actually promote allergic responses (4). the immune system at birth is dominated by th2 cells. however, the human fetus has a functional immune system at a relative early status of development comprising cd4þ and cd8þ t cells but also foxp3þ treg cells. one concept gaining support is that the developing fetus may become educated by whole bacteria or their genetic material that is provided via maternal serum. dna from bifidobacteria and lactobacilli, two genera typically used as probiotics, are found in human placenta. in contrast, in utero exposure to potentially pathogenic bacteria such as ureaplasma species leads to immune dysregulation commonly ending in fatal complications. maternal consumption of probiotic-containing food components may reduce the risk for childhood allergic diseases and mouse models demonstrate a reduced risk of inflammatory bowel diseases. epigenetic mechanisms may be critical since application of acinetobacter lwoffii to pregnant mice reduced the airway hypersensitivity response of the offspring. the promoter region of ifn-g in cd4þ t cells of the offspring had high levels of histone-4 acetylation, associated with enhanced transcription, while the il-4 promoter region had lower levels of histone-4 acetylation (5). moreover, exposure of pregnant mothers to the farm environment, which have high levels of acinetobacter lwoffii, was associated with dna demethylation of the foxp3 locus and methylation of the th2-associated genes rad50 and il-13. since gut microbiota composition during the first months of life seems to be important for development of appropriate immune regulatory networks and thereby influence later life disease risk, intervention with probiotics, prebiotics or synbiotics might be most effective at this age or even during pregnancy. probiotics can be defined as live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host. notably, the definition of a probiotic does not differentiate between the wide range of potential health benefits and it is clear that not all probiotics will influence the immune system in the same way. findings observed with one probiotic strain cannot be extrapolated to other probiotic strains. current evidence does not indicate that the probiotics clinically tested to date reduce the risk of children developing allergy but there are significant differences between studies such as the use of different probiotic strains, different age groups and different endpoints (6) . despite very poor quality of evidence, it has been suggested that there may be benefits in specific high risk groups, such as pregnant women at high risk of having an allergic child, in women who breastfeed infants at high risk of allergy and in infants at high risk of developing allergy. in general, probiotic-supplemented formula was found to be well tolerated and safe for infants. in conclusion, a better description of the bacterial strains and metabolites, which influence immune function, is required in order to allow for the improved design and selection of future probiotic strains for prevention and treatment of allergic disorders (7). people with cystic fibrosis (cf) are living longer lives than ever in the past. the median predicted survival in developed countries is now above 40 years of age and adults with cf are outnumbering pediatric patients in several regions. various reasons may explain such improvement in life expectancy, including the establishment of cf-dedicated and multidisciplinary centers; greater attention to nutritional issues and use of pancreatic enzymes replacement therapy; airway clearance techniques tailored to individual needs and attitudes; infection control measures; use of antibiotics both chronically by inhalation and aggressively to treat pulmonary exacerbations; mucolytic and airway hydration therapies; and liver and lung transplantation (1, 2). on account of the overwhelming evidence that organ impairment begins very early, even in asymptomatic cf infants, there is now general consensus that at least some of these strategies of care should be implemented as soon as possible in order to prevent or delay irreversible structural lung damage. indeed, this has possibly been the main argument in favor of cf nbs (3). the strength of such argument has been tested by several studies and considering different approaches. randomized studies à only two randomized trials on newborn screening for cf have been completed (5, 6, 7). these evaluations need many years of follow-up and, given the high degree of evidence in favor of cf newborn screening presently available, further implementation of similar studies seems improbable and possibly non ethical. observational studies à although most of these studies confirm clinical benefits from early diagnosis of cf, their results are hampered by several biases inherent to the methodological approach. the constant improvement in treatment and the consequent longer survival has an influence on the comparison of screened individuals and unscreened historical controls. on the other hand, examining the clinical evolution of screened infants and unscreened controls from different geographical areas but born in the same years may be affected by different care practices. finally, ascertainment biases may also have an impact on the assessment of outcomes, as patients presenting clinically are likely to have more severe cf than those identified through screening or unscreened patients with very critical disease may have died before being diagnosed. health economics studies à these studies use surrogate end-points, such as the quantity of treatment needed to remain healthy, and are based on the assumption that the optimal management offered to cf patients makes it harder to detect evidence of better clinical outcome in those diagnosed by screening. late-diagnosed patients may show clinical pictures similar to those diagnosed early, but at the expense of a considerably heavier burden of care (8) . most of these studies have focused on respiratory and nutritional outcomes and on hta assessments. their overall results clearly point in the direction of a positive effect on height and weight, of longer survival and of health service savings in populations screened at birth for cf. positive effects may also be obtained in several other domains, namely: -the prevention of salt loss syndrome thanks to early beginning of salt supplementation the opportunity of surveying from birth the natural history of cf -a better understanding of the early stages of cf. the possibility of testing presymptomatic therapeutic strategies, both conventional and patient targeted. cystic fibrosis (cf) is associated with the presence of two cf-causing mutations, one in each parental cftr gene, resulting in the absence or abnormality of the cftr protein and defect in electrolyte transport across epithelial membranes, the most well known being sweat chloride >60 mmol/l. even in 2016, cf remains by essence a clinical diagnosis. the wide range and severity of symptoms/ organs involved between and within individuals makes it a clinical decision as to whether or not a person should be managed as a cf patient. this is especially the case in a small number of ambiguous or atypical cases. in 1998, a first diagnosis consensus listed criteria for cf diagnosis: (i) one or more of the phenotypic features of the disease or (ii) cf in a sibling or (iii) a positive immunoreactive trypsin (irt), in association with at least one other feature, including a positive sweat test result on two occasions, a cf-causing mutation in each cftr gene or an abnormal nasal potential difference (npd) (1). this consensus statement of the us cystic fibrosis foundation was later modified in europe based on the concept of cftr dysfunction included in the diagnosis algorithm (2) . most atypical cf patients are diagnosed based on sweat tests and/or genetic analysis. these "mild cf" individuals usually present later in their lives with pancreatic sufficiency and milder respiratory disease. they frequently carry wide clinical spectrum mutations. the difficulty occurs when patients present with clinical symptoms suggestive of cf and a sweat chloride value in the intermediate range (30-59 mmol/l). among these subjects, those with abnormalities in npd measurement or 2 identified cftr mutations have, on average, more severe lung disease than the remaining subjects, although their disease symptoms are milder than those in subjects with a sweat chloride concentration above 60 mmol/l. therefore, from a physician's and also from a patient's perspective, these individuals must be differentiated from subjects with the classical life-shortening form of cf. the remaining cases, termed "possible" or "borderline", are difficult to classify because there is poor agreement between sweat test results and prognosis on the one hand and the frequent presence of at least one cftr mutation of uncertain clinical relevance on the other. the term "cftr-related disorders" (cftr-rds) designates these varied conditions, which include multi-system disease and monosymptomatic disorders associated with cftr dysfunction but which do not fulfill the diagnostic criteria for cf (3). this encompasses 3 main clinical entities with cftr dysfunction: cbavd (congenital bilateral absence of the vas deferens), acute recurrent or chronic pancreatitis and disseminated bronchiectasis. diagnosis of cbavd is based on impalpable vas deferens on scrotal examination. even if in a proportion of men scrotal palpable vas deferens are present, surgical exploration reveals a fibrous cord or a non-permeable duct. cbavd males have either a severe and a mild/variable (88%) or two mild/ variable (12%) cftr mutations (4) . approximately 34% of men with cbavd have a cftr mutation in one gene and the splicing variant ivs8-5t on the other allele, often in association with a longer polymorphic dinucleotide repeat, a combination that does not result in cf, but reduces levels of functional cftr protein in wolffian tissues, which constitutively produce less full-length cftr mrnas than other tissues (5) . about 30% of patients with idiopathic chronic pancreatitis or recurrent acute pancreatitis are found to carry cftr mutations. no specific cftr mutations have been reported, but rare class 4 or class 5 mutations are often found (6) . an increased incidence of cftr gene mutations has been found in bronchiectasis. according to the studies, at least 1 cftr mutation is found in 10-50%, and 2 mutations in 5-20% of cases. mutations found are mostly uncommon and likely to result in residual cftr function (7) . no specific cftr mutation is associated directly with bronchiectasis. (2) . these patients must be monitored carefully for development of any complications and appropriate therapy implementation. it should be pointed out, however, that labeling patients with mild or unclear manifestations with a cf diagnosis may have negative implications such as psychological, reproductive, social, employment, and insurance issues. therefore the explanation of the diagnostic challenge, including also prognosis, must be fully and honestly explained to the patient and or his family. department of pediatrics, cf and pcd center, hadassah hebrew-university medical center, mount scopus, jerusalem, <eitank@hadassah.org.il> effective mucociliary clearance (mcc) in the respiratory system requires proper mucus production and functioning airway surface fluid layer as well as competent and coordinated ciliary beating. the vital role of these systems is best demonstrated in patients with genetic defects such as primary ciliary dyskinesia (pcd) and cystic fibrosis (cf), both of which are characterized by impaired mcc leading to acute and chronic sino-pulmonary infections. pcd is caused by defects in genes that encode the structure or regulate the movement or function of the respiratory cilia. cf is caused by mutations in the cftr gene causing abnormality in the airway surface fluid layer, with production of thickened and viscous mucus leading to impaired mcc. in both diseases, recurrent and chronic respiratory infections and persistent inflammation cause progressive lung damage. most patients with cf suffer from pancreatic insufficiency (cf-pi); however, approximately 15% have sufficient pancreatic enzyme production to maintain normal fat absorption (cf-ps). patients with pcd are similar to patients with cf-ps in that they have normal pancreatic function, and are usually without the nutritional deficiencies that are typically associated with more severe pulmonary disease in cf. in addition, pcd and cf-ps are often diagnosed at a later age and have better survival compared to cf-pi (1,2). therefore when comparing cf and pcd, one should differentiate between patients with cf-pi and cf-ps. santamaria et al. compared chest hrct scan scores for patients with pcd and a group of age-and gender-matched cf patients and showed that patients with pcd had significantly less structural damage than cf patients (3) . a recent study comparing between pcd and cf-ps and cf-pi revealed that patients with pcd had disease severity in terms of pulmonary function and structural abnormality similar to patients with cf-ps, which was significantly less severe when compared to patients with cf-pi (4). furthermore, when comparing structural abnormalities by hrct, there was a significant disparity in the distribution of the structural changes in the lungs between the three groups of patients: in pcd, the upper lung zones were relatively preserved and most changes were localized to the middle and lower lobes, whereas in cf-pi, the upper lobes were remarkably involved. in cf-ps, there was no characteristic distribution of the structural damage (4) . other studies showed that in pcd, contrary to cf groups, there was no correlation between fev 1 and ct score and between fev 1 and age (3) (4) (5) (6) (7) (8) , which provides further support to the understanding that, in pcd, lung function is not a strong indicator of severity of lung disease and therefore, follow-up by low radiation chest hrct scans should be considered. it is important to note that, in general, patients with pcd receive less intensive therapy (9) . they are not always followed regularly in specialized centers, and many are not adherent to routine treatments. the most common bacterial infection in pcd patients is h. influenzae, which is significantly less common in older cf patients (4, 10) . in cf, chronic infection with p. aeruginosa is associated with a more severe lung disease (11) . however, among patients with pcd, there was no correlation between p. aeruginosa infection and pulmonary function or hrct severity score, suggesting a different role for this microorganism in the pathogenesis of pulmonary disease in pcd (4). bush et al. compared the mucous properties in both diseases and demonstrated that inflammation, measured by il-8 concentration, was greater in pcd sputa, and that there were no significant differences in biophysical or transport properties of sputum between the two groups; however, survival in patients with pcd was generally better (12). ratjen et al. (13) assessed the inflammatory response in the airways of cf and pcd patients during pulmonary exacerbation. in stable pcd patients, no significant differences were found in sputum inflammatory markers between individuals colonized with different bacterial pathogens. however, higher bacterial density for s. aureus and h. influenzae was found in patients with cf versus pcd, and the absolute neutrophil counts were higher in pcd patients. while sputum elastase activity was similar in pcd and cf at the time of exacerbation, it decreased with antibiotic therapy in pcd but not cf patients. thus, pcd patients differ from those with cf in their responses to treatment of pulmonary exacerbations, with higher neutrophil elastase activity persisting in the cf airways at the end of treatment. joenesen et al. (14) measured the difference in breath profiles of patients with pcd and cf, with and without distinct chronic lung infections, using an electronic nose. no significant difference was found between the breath profiles of pcd patients with a chronic pa infection and pcd patients without a chronic infection. however, there was a significant difference between the breath profiles of cf patients with a chronic pa infection and cf patients without a chronic pa infection, suggesting a different response to infection between pcd and cf. in conclusion, although pcd and cf are both characterized by impaired mcc and respiratory infections, patients with pcd have a different lung disease expression compared to patients with cf-ps and with cf-pi, as assessed by fev 1 , hrct, nutritional status and bacterial infection on sputum cultures. in pcd, normal fev 1 can be maintained over time in spite of severe structural damage. this suggests a greater involvement of the large airways in pcd and the small airways in cf. furthermore, p. aeruginosa infection is less common in pcd than in cf. bronchopulmonary dysplasia (bpd) is the most important complication following mechanical ventilation in preterm infants and no definite therapy can eliminate this complication. although the mechanism is not completely clear, pulmonary inflammation is believed to play a central role in the pathogenesis. glucocorticoid is one of the most effective therapies to treat or prevent bpd. however, systemic glucocorticoid therapy is not generally recommended because of long-term adverse events (1,2). our previous pilot study in neonates and studies in animals indicated that surfactant can be used as a vehicle to deliver a topical glucocorticoid, budesonide, to the lung periphery and effectively suppress lung inflammation and lung injury (3.4.5). the mechanism for the effective delivery of budesonide using surfactant as vehicle is based on a physical phenomenon, the "marangoni effect": in the interface between high and low surface tension, a convection force is generated and this force can be used as a vehicle to facilitate the delivery of medication (6) . this is an important delivery method because inhaled glucocorticoid is technically difficult and the effect has been shown to be limited (7, 8 there was no significant difference between the groups during the study in serum electrolytes, glucose, bun and in blood pressure, and in physical growth. there was no significant difference between the groups in neuromotor function, and in mdi, pdi and in neurodevelopmental impairment (ndi) score when examined at 2-3 years of corrected age. we concluded that in very low birth weight infants with severe respiratory distress syndrome, intra-tracheal administration of surfactant/budesonide compared with surfactant alone significantly decreased the incidence of bpd or death without apparent short term or long term adverse effect. further large-sample, double-blind trials are warranted. measuring lung function in "non-collaborating" children has always been one of most difficult tasks for pediatric pulmonologists. this is because young children are not able to perform the voluntary forced expiratory maneuvers generally used in adults and schoolchildren. in infants and children up to 2 years, this problem has been generally overcome by the use of sedation, although this contributes to make lung function measurements less suitable for routine clinical use in this age group. preschool children (2-5 years) are too old to be sedated and yet too young to properly perform the forced expiratory maneuvers required for spirometry. for this age group, several techniques that just require tidal breathing have been implemented during the past decades. the american thoracic society/european respiratory society (ats/ers) working group on lung function in young children has published technical recommendations for most infant (1,2) and preschool techniques (3) and their clinical applications have also been recently summarized (4) . this lecture will focus on the most used pulmonary function tests (pfts) in infants and preschool children. chloral hydrate (80-100 mg/kg, maximum 1 g) is commonly used to sedate infants and young children up to 2 years for performing lung function testing. however, chloral hydrate is no longer available in the u.s.a. and the use of other sedatives might lead to different results (4). the most commonly used pfts in infants are the raised volume rapid thoracoabdominal compression and infant plethysmography. other pfts that are performed during tidal breathing (e.g.: tidal breathing measurements, multiple breath washout, forced oscillation technique) are more suitable to be used without sedation, especially in younger infants. the raised volume rapid thoracoabdominal compression (rvrtc) allows for the measurement of forced expiratory flow and volume in sedated infants (2) . repeated inflations using a pressure of 30 cmh 2 o are applied through a facemask and an inflatable jacket is then activated to rapidly compress the infant's chest and abdomen to obtain forced vital capacity (fvc), forced expiratory volume in 0.5 seconds (fev 0.5 ) and forced expiratory flow (fef) at defined proportions of fvc. to ensure that flow limitation has been reached, the inflation pressure of the jacket is increased at each maneuver until no further increase in flow is noticed. recently published reference equations using a current commercially available device (5) will improve the interpretation of the results. rvrtc has been successfully used in children with all kinds of respiratory diseases, including children with cystic fibrosis (cf), children born prematurely, and those with recurrent wheezing (4), showing its capability to distinguish disease populations from healthy control subjects and to detect lung function changes in clinical intervention trials. however, its long-term clinical utility still remains to be established. moreover, the need for sedation along with the time and resource intensity required are other important limitations for its use in routine clinical practice (4) . infant plethysmography is used to measure functional residual capacity (frcpleth) in sedated infants (1) . specific airway resistance (sraw) can also be measured, provided that a proper electronic thermal compensation is applied to the system to account for thermal artifacts. this technique is based on the same principle (boyle's law) as plethysmography for older subjects and uses an infant whole body plethysmograph where the infant lies supine breathing through a facemask sealed with silicon putty (1). infant plethysmography has been successfully applied to children with lung disease, especially cf and bronchopulmonary dysplasia (bpd) (4). however, as for rvrtc, its long-term clinical utility remains to be ascertained and its role in routine clinical practice is hence very limited. preschool children (2-5 years) are too old to be sedated, but also too young to properly perform the forced expiratory maneuvers required for spirometry. for this age group, several techniques that just require tidal breathing have been implemented during the past decades, allowing for lung function to be measured in awake children (3) . also, modified acceptability criteria for spirometry have been proposed for the use in preschool children (3) . it is important to highlight that the feasibility of any lung function technique in preschool children strongly depends on the capability of the operator of keeping the child quiet and focused (3). spirometry has been proposed for preschool children using modified acceptability criteria (3). since the forced expiratory volume in 1 second (fev 1 ) often cannot be obtained in preschoolers due to their different lung physiology, the use of fev in 0.5 (fev 0.5 ) or 0.75 seconds (fev 0.75 ) is recommended in this age group. also, fvc should not be reported if flow stops at more than 10% of peak flow (early termination), but fev may still be reported. less stringent repeatability criteria have also been proposed in preschool children: at least two acceptable maneuvers should be obtained with the two fvc and fev within 100 ml or 10%, but in case of a single acceptable maneuver, this should be recorded nevertheless (3) . spirometry is reported to be feasible in 55-85% of 4-5 year old children, but its feasibility tends to be much lower in younger children (4) . global multiethnic reference equations including preschool children have recently be published (6) . spirometry has been reported to discriminate healthy controls from preschool children with cf and with recurrent wheezing, although substantial overlap between groups may occur and bronchodilator response appears to be more sensitive than baseline values (4). however, a careful and rigorous approach to the use of spirometry must be taken in preschool children and several gaps in our knowledge still limit the application of this technique to clinical practice in this age group (4). the interrupter technique is based on the principle that a sudden flow interruption at the mouth during tidal breathing would make alveolar pressure rapidly equilibrate with mouth pressure, thus allowing an estimation of alveolar pressure by measuring mouth pressure. the interrupter resistance (r int ) is then calculated dividing the change in mouth pressure by the flow measured immediately before the interruption ("classical" technique) or immediately after the interruption ("opening" technique). measuring r int has been proved to be particularly suitable for preschool children, its feasibility being generally higher than 80% in this age group (4). proper reference values have been published (7) and cut-off values for the bronchodilator response have also been reported. r int is able to detect changes in the airway caliber and has been successfully used in preschool children with recurrent wheezing (4). however, its utility in clinical care remains to be established, especially by longitudinal studies (4) . the forced oscillation technique (fot) is used to measure the impedance of the respiratory system (z rs ) during tidal breathing by applying, through a mouthpiece and a filter, low-frequency pressure oscillations generated by a loudspeaker (usually 4-48 hz) (3) . changes in flow and pressure measured at the mouth are used to calculate z rs and its two components, resistance (r rs , reflecting frictional losses) and reactance (x rs , reflecting elastic properties at low frequencies and inertial forces at higher frequencies). forcing signals based on sinusoidal waves or impulses have been used, both as single-frequency or composite signals. frequencies between 5 and 10 hz are considered to reflect the mechanical properties of the total airways. fot has a good feasibility in preschool children (>80%) and several reference equations have been published (8) . fot has been used in many studies on children with recurrent wheezing, showing a good capability in discriminating health from disease, especially when bronchodilator response is used (4). however, for this technique as well, longitudinal studies on its clinical utility in young children are still needed (4). the multiple breath washout (mbw) is based on the washout of an inert gas (typically n 2 washout using 100% o 2 ) to measure ventilation inhomogeneity and frc during tidal breathing (3). non-resident inert gases have also been used. the lung clearance index (lci, the number of lung volumes expressed as frcs required to washout the inert gas) is the most commonly used mbw index. the general standard operating procedure for this technique has been recently reported (9) . lci has a good feasibility in preschool children (nearly 80%). lci has been successfully used in preschool children with cf (4), proving to be more sensitive than spirometry and plethysmography in detecting abnormal lung function. however, longitudinal studies on the clinical utility of mbw in preschool children are lacking (4) and more data are needed before lci or other mbw indices can be recommended in the routine clinical management of patients with cf (10). specific airway resistance (sraw) can be measured at tidal breathing in preschool children using a whole body plethysmograph. since sraw is the product of airway resistance by the thoracic gas volume, it can be calculated without the need to breathe against a closed valve (11), provided that a proper electronic thermal compensation is applied to obviate the need for the panting maneuver. the measurement of sraw has a good feasibility in young children and reference values are also available (11) . however, the lack of consensus on measurement methods and outcome measures makes it difficult to compare results among centers and methodological techniques are urgently needed for this technique. an accurate assessment of pulmonary function is now possible in infants and preschool children using a number of techniques. although these techniques have proven to be powerful research tools, further studies are needed to ascertain their utility in the clinical care of infants and young children with lung disease. . past studies have shown that persistent echocardiographic evidence of ph beyond the first few months of life is associated with up to 40% mortality in infants with bpd. the association of ph with poor survival in bpd has continued into the recent era of the "new bpd," especially in infants with severe disease who require prolonged support with mechanical ventilation. thus, developing insights into the pathogenesis and pathobiology of ph and related pulmonary vascular disease (pvd) in bpd continue as an important challenge and may help to improve early and late cardiopulmonary outcomes after preterm birth. mechanisms that coordinate normal vascular growth and alveolarization during development or cause abnormal lung growth in bpd are poorly understood. disruption of key signals between airway epithelium and endothelial cells can alter vascular and alveolar growth, resulting in decreased arterial and airspace structure. for example, hyperoxic lung injury in newborn animals decreases expression of the critical proangiogenic and endothelial cell survival factor, vascular endothelial growth factor (vegf). early impairment of vegf production inhibits vascular growth and impairs endothelial function, which leads to ph. in addition, disruption of angiogenesis due to adverse antenatal factors, such as chorioamnionitis, preeclampsia or maternal smoking, and postnatal events after premature birth, can cause vascular injury that not only lead to ph but can also impair distal lung growth. ongoing laboratory studies suggest that the developing endothelial cell plays a key role in the regulation and coordination of epithelial growth and distal airspace structure through the production of critical "angiocrines," such as nitric oxide (no), hepatocyte growth factor, vitamin a, insulin growth factor-1 and others. thus, since angiogenesis is necessary for normal alveolarization, it has been suggested that protecting the developing pulmonary vasculature from early injury may not only lower pvr and improve gas exchange, but may enhance distal lung growth and improve long term outcomes. abnormalities of the pulmonary circulation in severe bpd include altered tone and reactivity, structure and growth, which can cause right heart failure, impaired gas exchange, pulmonary edema, decreased exercise capacity and other clinical problems. physiologic abnormalities of the pulmonary circulation in bpd include elevated pulmonary vascular resistance (pvr) and abnormal vasoreactivity, as evidenced by the marked vasoconstrictor response to acute hypoxia and by impaired gas exchange due to abnormal distribution of lung blood flow. abnormal pulmonary vascular structure also contributes to high pvr due to increased smooth muscle cell hyperplasia and altered vascular compliance caused by increased production of an abnormal extracellular matrix. growth of the distal lung circulation is abnormal in infants with severe bpd, and decreased arterial growth (angiogenesis) reduces vascular surface area that further impairs gas exchange and increases the risk for the development of ph and impaired exercise capacity in older children. prominent bronchial or other systemic-to-pulmonary collateral vessels were noted in early morphometric studies of infants with bpd, and can be readily identified in many infants during cardiac catheterization. although these collateral vessels are generally small, large collaterals may contribute to significant shunting of blood flow to the lung, causing edema and need for higher fio 2 . in addition, recent autopsy studies suggest the presence of striking intrapulmonary anastomotic, or "shunt," vessels that link the distal pulmonary and bronchial vessels, and may contribute to poor oxygenation. past clinical studies have further shown that metabolic function of the pulmonary vasculature is impaired in bpd, as reflected by the lack of pulmonary clearance of circulating norepinephrine during passage through the lung, which may contribute to left ventricular dysfunction and systemic hypertension. clinical studies have recently shown that early echocardiographic findings of pvd after preterm birth are strongly associated with the development and severity of bpd and ph at 36 weeks corrected age. interestingly, these findings were not only associated with a worse respiratory course during the initial hospitalization, but also late respiratory outcomes, including respiratory exacerbations, hospitalizations and the need for asthma medications. ongoing studies are exploring the impact of ph-specific drug therapies, such as sildenafil and other agents, on ph and related complications. thus, pvd in preterm infants with bpd is characterized by altered lung vascular development, growth, structure, and function, which precede the onset of measureable ph. pvd due to disruption of normal pulmonary vascular development in association with preterm birth is an important determinant of the pathobiology of bpd and contributes significantly to morbidity and mortality. exposure to adverse stimuli during the antenatal and/ or early postnatal periods impairs normal pulmonary vascular development and creates an imbalance between risk and resiliency factors. recent studies have revealed the magnitude of ph in preterm infants, but many aspects of pvd remain understudied, and ongoing investigations continue to explore risk factors, mechanisms of disease, and long-term outcomes. prospective studies are needed to definitively establish standardized clinical criteria for pvd and ph in bpd, and to determine the best methods for early diagnosis, risk stratification and disease monitoring. larger collaborative studies and improved clinical infrastructure to conduct these important investigations will provide answers to these critical questions. recent evidence suggests that cftr does not act as a pure ion channel but as a platform for multiple cellular signaling pathways. importantly, the protein interactomes of wt-and f508del-cftr are rather different, and there is growing consensus that indirect measures that avoid the enhanced degradation of f508del-cftr may restore its function. recently, we discovered that cftr orchestrates a proteostatic network that influences multiple cellular functions by acting as a hub protein. this hub-dysfunction model proposes that the proteostasis network is widely deranged, both in transgenic cf mice and in primary nasal epithelial cells freshly collected from cf patients bearing f508del-cftr either in homozygous or compound heterozygous form, at two levels. firstly, autophagy, the major mechanism determining cytoplasmic protein turnover, is blocked due to tissue transglutaminase (tg2)-mediated depletion of the essential autophagy-related protein beclin 1 (becn1), leading to secondary accumulation of the autophagic substrate sqstm1/p62. secondly, peptide fragments released from proteolytically-cleaved f508del-cftr provoke an over-activation of a pleiotropic protein kinase (protein kinase ck2), which in turn contributes to f508del-cftr degradation. combined inhibition of tg2 by cysteamine, which is fda-approved for the treatment of cystinosis, and over-active ck2 by the over-the-counter greentea flavonoid epigallocatechin-gallate (egcg) respectively rescue and stabilize a functional f508del-cftr protein at the pm, both in mice and in primary nasal cells from cf patients bearing f508del-cftr or other class ii-cftr mutations. pre-clinical evidence on transgenic mice has provided the mechanistic proof-of-concept for using this combination of proteostasis regulators as an alternative cftr-repairing therapy. moreover the combination treatment reduces lung inflammation and this beneficial effect persists up to 2 weeks following cysteamine withdrawal provided that egcg was administered during washout. this prompted an open-label phase-2 trial to assess the individual response to the synergistic combination of cysteamine and egcg in cf patients bearing different cftr mutations. the combination treatment was well tolerated and decreased sweat chloride from baseline while increasing the abundance and function of cftr protein and restored autophagy in nasal cells. notably, the treatment decreased cxcl8 and tnf-a in the sputum and improved respiratory function. these positive effects were particularly strong in patients carrying f508del-cftr (or other class ii) mutations in homozygosity or heterozygosity, whereas patients with class i cftr mutation failed to respond to therapy. altogether, these results suggest that the combination treatment acts "on target", according to the hypothesis underpinning our drug design. discordance in therapeutic response rate complicates mutation-specific approaches, thus entailing the need of patient-centered (personalized) approaches to assess drug efficacy. testing the putative individual responsiveness to treatment by appropriate biomarkers before in vivo therapy should support the decision to treat. we show that restoring cftr function in vitro in nasal cells in response to cysteamine plus egcg, is highly predictive of whether the combination treatment will restore cftr function in vivo. hence, this in-vitro assay may constitute a tool to guide the clinical development of cf treatments, allowing to select patients for new therapeutic options. general frame for care infants with cf must receive care in an accredited cf care center. they must be reviewed in clinic frequently after diagnosis, for example once a month during the first 6 months of age, every 2 months until 1 year of age, and every 3 months thereafter (3). after initial diagnosis, the cf center should contact the primary care professionals for regular ambulatory follow-up to implement therapeutic strategy. parents of infants with cf should be offered access to genetic advice and counseling. the standard childhood immunization schedule must be applied in accordance with national guidelines. anti-influenza vaccination is recommended for the infant from the 6 th month of life and for all household members and healthcare providers. according to french guidelines, vaccination against chicken pox could be recommended. growth targets should reflect genetic potential, sibling height and local population demographics (1) . french guidelines recommend to catch-up birth weight percentile at 6 months (3). at 2 years, weight-for-height should be at the 50 th percentile and height at the target height percentile (target height: average of the height of the 2 parents plus 6.5 cm for boys and minus 6.5 cm for girls) (3). energy intake evaluation should be performed by a dietician on a regular basis and adapted to achieve the objectives of weight-for-height growth. energy intake could be as much as 150% of the daily recommended calorie intake for the same age in the general population (4). breast feeding is encouraged, all the more that recent data acknowledge its protective effect against pseudomonas aeruginosa infection (4, 5). formula with hydrolyzed cow's milk protein is recommended in infants with risks of malabsorption, or severe undernourishment. sodium chloride supplementation is systematic, particularly in the case of breast feeding and should be adapted to natriuresis (6) . it should be increased during periods of hot weather and all other causes of high salt loss (diarrhea, fever, ileostomy, etc.). at initial diagnosis, infants must have pancreatic function assessed by stool fecal elastase. if elastase is normal, repeat assessment is recommended. pancreatic enzyme replacement therapy should be started at diagnosis in case of clinical symptoms of exocrine pancreatic insufficiency even before obtaining the results of the elastase assay. the starting dose could be 2.000 iu lipase per 100 ml of milk. in case of persistence of symptoms of pancreatic insufficiency despite a maximum dose of 10.000 ui/kg/day of lipase, it may be necessary to evaluate the patient's compliance and the methods of conservation and administration of the pancreatic extracts. in case of poor weight-for-height growth despite an adapted substitutive pancreatic opotherapy, an evaluation is necessary including a dietetic review, a search for sodium insufficiency and other etiologies of malabsorption. in case of persistence of symptoms of exocrine pancreatic insufficiency despite a maximum dose of 10.000 ui/kg/day of lipase and in the absence of other etiologies, the administration of gastric secretion inhibitors may be envisaged. bacterial cultures of bronchial flora should be performed at each session of physiotherapy or, in case of abnormal clinical status, ideally on bronchial secretions expectorated or obtained by sputum induction (7). a chest x-ray should be performed at baseline and annual assessment, and, in case of clinical abnormality. high resolution computed tomography should complete the assessment in case of clinical or radiological abnormality and/or at initial assessment according to local practice to detect early bronchiectasis (8) . systematic respiratory physiotherapy is recommended from the time of diagnosis. the frequency of sessions of physiotherapy depends on the clinical status of the infant. regular therapy might be recommended even in the asymptomatic infant (3). any evidence of respiratory infection justifies performing a respiratory culture and adapted antibiotic treatment of the isolated pathogens. infection by staphylococcus aureus sensitive to meticillin should be treated by adapted antibiotherapy. in case of isolation of s. aureus resistant to meticillin, a treatment aiming eradication is recommended. evidence of p. aeruginosa justifies systematic antibiotic treatment, even in the asymptomatic infant. although there is still no consensus, treatment might begin with an inhaled antibiotic, eventually associated with oral ciprofloxacin. in case of persistence of p. aeruginosa after initial therapy, or if the infant presents with severe clinical signs, intravenous antibiotics should be considered (1, 2, 3) . for other pathogens, there is less clear agreement and treatment should be guided by local policies. in the absence of clinical improvement despite an adapted antibiotherapy, bronchial sampling by bronchoalveolar lavage should be considered and non-infectious causes should be searched for, including gastroesophageal reflux, asthma and an ent cause. respiratory syncytial virus (rsv) may have adverse effects on respiratory status in patients with cf (9) . there is insufficient evidence to support systematic recommendation of palivizumab in the cf infant even if some small studies suggest that there could be benefit from the use of rsv prophylaxis in infants with cf (10). us and french guidelines state that palivizumab could be discussed, namely for the infant of less than 6 months of age during an epidemic period (2,3). finally, dornase alfa, 7% hypertonic saline might be used in symptomatic infants (2) . with increasing numbers of infants with cf being diagnosed by newborn screening across most of europe and in north america, we will have the opportunity for large cohort follow-up and randomized controlled trials. this will help to establish still lacking best available evidence to harmonize therapeutic strategy in infants newly diagnosed with the final aim of improving clinical status at later ages. department of pediatrics, cf and pcd center, hadassah hebrew-university medical center, mount scopus, jerusalem, israel <eitank@hadassah.org.il> bronchiectasis is the distraction of the normal anatomy of conducting airways that results in impaired mucociliary clearance leading to chronic cough, sputum production, and recurrent infections and inflammation that cause further damage to the bronchial and bronchiolar walls leading to a vicious cycle of airway injury. the prevalence of non-cf bronchiectasis (ncfb) in children differs between developed and poor countries. in the developed world, the most common cause of bronchiectasis in children is cystic fibrosis (cf), followed by primary ciliary dyskinesia and immune deficiencies. however, up to half of cases remain without a known etiology. in developing countries, a systematic review of 989 children (1) demonstrated that an etiology was identified in 63% of children, with a previous severe pneumonia of bacterial or viral etiology and b-cell defects as the most common identified disorders. bronchiectasis should be suspected in patients who present with chronic productive cough of mucopurulent sputum. physical findings in bronchiectasis patients are nonspecific but may include crackles and wheezes on lung examination and clubbing of the digits. pulmonary function testing results generally show airflow obstruction. the diagnosis of bronchiectasis is confirmed by hrct scan which is now the gold standard for diagnosis. these include bronchial dilatation (an internal bronchial diameter greater than the diameter of the accompanying bronchial artery [i.e., the "signet ring" formation]) and a lack of bronchial tapering on sequential slices (2) . patients in whom bronchiectasis has been diagnosed should be evaluated for potential underlying causes. they need to undergo chest ct scan to define the extent of their disease. patients with focal disease require bronchoscopy to evaluate for a localized airway obstruction as the cause of the bronchiectasis. patients with diffuse bronchiectasis should be assessed for underlying systemic abnormalities including congenital disorders, chronic aspiration, impaired mucociliary clearance and systemic or local innate immune dysfunction. all patients with bronchiectasis should have a regular routine microbiological examination of their sputum for routine bacterial and ntm organisms. pulmonary exacerbations of ncfb are known to be associated with poor outcomes, and infections are common causes. gram-negative bacteria are isolated more frequently in patients with ncfb, with h. influenzae and p. aeruginosa representing the majority of identified species. however, up to 40% of sputum samples fail to grow any pathogenic bacteria (3). patients with sputum samples dominated by p. aeruginosa (pa) had a higher frequency of exacerbation and poorer lung function compared to patients whose samples were dominated by other organisms (4). nontuberculous mycobacteria (ntm) are opportunistic pathogens that afflict patients with preexisting lung disease; in particular those with ncfb, shown in a meta-analysis by chu et al. to be prevalent in nearly 10% of the patients (5). respiratory viruses were found in nearly 50% of exacerbations. the goals of bronchiectasis treatment are to reduce the number of exacerbations and to improve quality of life. if an underlying systemic etiology such as immune deficiency is identified, it should be addressed. pharmacologic agents and the mechanical mobilization of secretions have been evaluated to a limited degree in patients with non-cf bronchiectasis. short-acting or long-acting bronchodilator adrenergic and anticholinergic agents are commonly prescribed, but there have been no randomized controlled trials to support their use. pulmozyme had adverse effects when studied in patients with non-cf bronchiectasis. inhaled mannitol showed improved time to first exacerbation and quality of life. nebulized hypertonic saline solution (7%) have shown promise in the treatment of patients with both cf and non-cf bronchiectasis, but long-term prospective trials are needed. the role of the use of maintenance antibiotic therapy is uncertain in patients with non-cf bronchiectasis. rotating oral antibiotic strategies have been commonly used. for exacerbations, antibiotic therapy should be tailored to their sputum microbiology results. severe exacerbations, particularly in patients who are infected with organisms that are resistant to therapy with oral quinolones, require iv antibiotic therapy. azithromycin has been shown to attenuate muc5ac and muc2 gene expression, thereby suppressing the synthesis of mucin on human airway epithelial cells. clinically, this was demonstrated in a study that found that mean 24-hour sputum volume and qol were significantly lower in patients with bronchiectasis after 12 weeks of azithromycin compared with control subjects (6) . a recent randomized, double-blind, placebo-controlled trial in adults assigning patients to receive 500 mg azithromycin or placebo three times a week for 6 months, showed that azithromycin significantly reduced the exacerbation rate with no significant effect on fev1 (7) . based on the above and other studies, it is recommended that all patients with ncfb be treated with azithromycin. long-term inhaled antibiotics are used for patients with uncontrolled ncfb, but until more recently, data on their efficacy have been lacking. the use of mechanical aids, including chest physical therapy with postural drainage, active cycle of breathing, oscillatory positive expiratory pressure devices, and high frequency assisted airway clearance, also constitute potential adjunct therapies for patients with bronchiectasis. though these modalities are considered to be standard therapy for patients with cf bronchiectasis, their utility is less well proven in patients with non-cf bronchiectasis. it was shown that comprehensive medical care in children with ncfb was associated with a decrease in exacerbation rates (8) . these findings further exemplify the importance not only of identifying ncfb in pediatric patients, but also of ensuring that they receive close surveillance. treatment burden with lack of immediate apparent outcomes cause patients to avoid daily therapy and seek therapy only for exacerbations. resectional surgery and lung transplantation are rarely required. surgical treatment has classically been an option for patients who have localized bronchiectasis with persistent symptoms despite maximal therapy, or recurrent infections with resistant pathogens (9). the prognosis for patients with bronchiectasis is variable given the heterogeneous nature of the disease. because there are so few randomized controlled trials of therapies for non-cf bronchiectasis, patients must be evaluated and treated on an individual basis in a tailored, patient-focused approach in a specialized center to optimally evaluate and treat individuals with bronchiectasis. in humans, the dominant innervation to the airways is provided by the parasympathetic vagus nerve, whose activation induces the release of acetylcholine [1] . acetylcholine, the primary parasympathetic neurotransmitter in the airways, interacts predominantly with nicotinic receptors and the five muscarinic receptor subtypes. in addition to its well-known functions, i.e. bronchoconstriction and mucus secretion regulation, there is evidence that acetylcholine might also modulate inflammatory cell chemotaxis and activation and participate in signaling events that lead to airway wall remodeling [2] . these findings can have significant implications for anticholinergic therapy of diseases characterized by airway inflammation, bronchial obstruction and mucus hypersecretion, since a variety of data indicate that the function of muscarinic receptors is altered in these patients. nicotinic acetylcholine receptors (nachrs) are ligand-gated ion channels, formed by five homologous or identical subunits, arranged to form a central ion channel [3, 4] . depending on the subunit composition, nachrs show different kinetics and pharmacological properties. in lung tissues, the "muscle" nachrs are localized at the neuromuscular junctions of the smooth muscle cells, whilst the "neuronal" nachrs are expressed by autonomic ganglia, but also by almost every cell type, including bronchial and alveolar epithelial cells, endothelial cells, pulmonary neuroendocrine cells, submucosal glands, airway and vascular smooth muscles, fibroblasts and alveolar macrophages [4] . although nachrs are classically linked to the depolarization of the plasma membrane required for neurotransmission, non-neuronal nachrs in the lung act most frequently as calcium channels and have been linked to regulatory proteins controlling cell proliferation [2] [3] [4] . the functional role of nachrs is particularly complex and depends on subunit composition, dose response, and duration of ligand stimulation. although nachr activation often leads to a positive feedback loop that induces receptor expression, chronic stimulation of nachrs can produce channel desensitization and decreased activity. the majority of studies of nachr function in the lung are related to the effects of nicotine, i.e. to tobacco-induced mutagenesis and lung carcinogenesis, whilst little is known on the physiological functions in regulating lung growth and repair, airway epithelial cell proliferation and differentiation and electrolyte transport [3] . muscarinic receptors belong to the large family of g protein-coupled receptors, characterized by seven transmembrane domains. out of the five subtypes identified, only m1, m2 and m3 receptors have been detected in the airway and lung tissues of most mammals, including humans. almost all airway and lung cell types express muscarinic receptors. m1 receptors are present mainly in the peripheral lung tissue and in the alveolar walls: they are expressed by airway epithelial cells, where they modulate electrolyte and water secretion, by goblet cells, where they regulate mucus production, and by the ganglia, where they facilitate parasympathetic neurotransmission. m2 and m3 receptors represent the major populations in the large airways. m2 receptors are expressed by neurons, where they function as autoreceptors inhibiting the release of acetylcholine from both preganglionic nerves and from parasympathetic nerve terminals. in airway smooth muscles, they modulate different ion channels involved in cell contraction, effects that require concomitant m3 receptor-mediated release of calcium from intracellular stores. in fibroblasts and smooth muscles, m2 receptors stimulate cell proliferation and modulate cellular responses associated with airway remodeling [2, 4] . m3 receptors are the dominant receptor subtype in the regulation of airway smooth muscle contraction and of mucus secretion from submucosal glands and goblet cells [3] . m3 receptors can also favor airway smooth muscle proliferation, increasing the responses to epidermal growth factor and platelet-derived growth factor [3] . acetylcholine, in addition to the parasympathetic nerve, is also synthesized and released by a large number of non-neuronal cells, including neuroendocrine, ciliated, basal and secretory epithelial cells where it can act as an autocrine or paracrine signaling molecule. secretory and ciliated cells release acetylcholine into the luminal periciliary fluid, whereas endocrine and basal cells secrete acetylcholine basally [3] . current knowledge suggests that the local auto/paracrine production of acetylcholine by epithelial cells may play a role in regulating various aspects on the innate mucosal defense mechanisms, including mucociliary clearance. acetylcholine is known to increase ciliary beat frequency in the airways and to modulate the release of inflammatory mediators by these cells through m3 receptors and to affect inflammatory cells involved in the pathogenesis of obstructive airway diseases [3] . expression of muscarinic receptors has also been shown by most inflammatory cells, including macrophages (m1-m3) , t-and b-lymphocytes (m1-m5), mast cells (m1), neutrophils (m1-m3) and eosinophils (m1). in these cells, muscarinic receptors appear to be involved in cell proliferation and release of pro-inflammatory mediators [2, 3] . arteries, veins and bronchopulmonary anastomoses also express muscarinic receptors (m3) and dilate in response to acetylcholine released by vagal nerve stimulation. the postganglionic nerve fibers do not form defined synapses to their target cells but a terminal meshwork called 'autonomic plexus' with numerous varicosities, called sites of transmitter release, in variable and only rarely close contact to cells, such as airway smooth muscle [4] . release of acetylcholine from the parasympathetic nerve terminals in the airways appears to be under complex prejunctional regulatory mechanisms. the available data indicate that acetylcholine release can be enhanced by a variety of pro-inflammatory mediators (histamine, bradykinin, neuropeptides) and by b 2 -adrenergic agents, whist it is under the inhibitory control of muscarinic autoreceptors and downregulated by eicosanoids, such as pge 2 , opioids, nitric oxide and a 2 -adrenergic agents [5] . the activity of m3 receptors in smooth muscle appears to be spared or even increased in asthmatics, possibly because of a greater affinity of the acetylcholine binding site. there is also no evidence that muscarinic receptors are overexpressed or upregulated in airway smooth muscle in disorders characterized by bronchial obstruction or hyperresponsiveness although an acquired loss or impairment of neuronal m2 receptor function may be involved in their pathogenesis [15] . these functional changes occur after exposure to allergens, infectious agents (viruses) or pollutants (ozone) and result in increased acetylcholine release from parasympathetic nerves [6] . m2 autoreceptors dysfunction in allergic asthma is caused by the eosinophil basic protein released by activated eosinophils that, upon binding to m2 autoreceptor sialic acids, acts as an allosteric antagonist [3] . with the same mechanism, an early recruitment and activation of eosinophils is thought to cause the airway hyperreactivity that follows environmental ozone exposure [3] . in contrast, viral respiratory infections are purported to induce bronchial hyperresponsiveness through different mechanisms, including: a) the inhibition of m2 receptor synthesis, mediated by the release of interferon-g by activated cd8þ t-lymphocytes; b) the production of neuraminidase, that determines functional impairment of m2 receptor activity by cleaving their sialic acid; c) m2 receptor dysfunction, caused by the activation of the substance p (nk1) receptor overexpressed by influenza, parainfluenza and respiratory syncytial virus [3] . interestingly, increased substance p production has been reported in patients with asthma and gastroesophageal reflux, a disorder that recognizes vagus-mediated oesophageal-tracheobronchial reflexes in its pathogenesis. experiments performed in humans have corroborated the relevance of pathogenesis of m2 autoreceptors in generating airflow limitation showing that m2 receptor selective agonists inhibit cholinergic-induced bronchoconstriction in normal individuals but not in asthmatic patients [3] . defects in m2 autoreceptor activity may also explain bronchoconstriction induced by b-blockers in asthma. these drugs can increase cholinergic tone downregulating the action of endogenous catecholamines on b 2 -adrenoceptors present on cholinergic nerves [3] . thus, in extreme synthesis, the three muscarinic receptor subtypes expressed in the airways have different, somehow conflicting functions: m1 and m3 receptors facilitate cholinergic-induced events, including bronchoconstriction and mucus glands secretory activities, whilst m2 receptors have a feedback inhibitory function, regulating the release of acetylcholine from cholinergic nerve endings. this information is of great importance to understand the activity of the three anti-cholinergic agents that can be used to treat patients with reversible airway obstruction. two of these, ipratropium and oxitropium bromide, are short-acting and non-selective muscarinic antagonists. because of the lack in selectivity, they also block m2 receptors, increasing acetylcholine release, and therefore reducing the degree of their "useful" action on m1 and m3 receptors [3] . in contrast, the more recent longacting anticholinergic drug tiotropium bromide is characterized by a kinetic selectivity for m1 and m3 receptors over m2 receptors: it dissociates rapidly from m2 receptors and very slowly from m1 and m3 receptors [3, 7] . to date, the anti-cholinergic agents most commonly used to treat respiratory disorder in childhood is the "non-selective" ipratropium bromide which, alone or associated with inhaled b 2 -adrenoceptors agonists, has been demonstrated to significantly improve pulmonary function and clinical outcomes in acute asthma, in preschool wheezing, although no long-term assessments have been included [3, 8] . interestingly, preliminary data show that inhaled tiotropium bromide, once daily, is well tolerated and also improves lung function in pediatric patients with cystic fibrosis [9] and in asthmatic adolescents, symptomatic despite inhaled corticosteroids [10] . evidence from experimental models also suggest that tiotropium bromide may also modulate the acetylcholine-induced inflammatory and remodeling changes induced in the airways by a variety of stimuli, leading to hopes of having favorable clinical responses in other respiratory disorders. a relevant role in the pathogenesis of obstructive airway disorders is thought to be played by an increased acetylcholine release, at least in part due to m2 receptor dysfunction. the most commonly prescribed short-acting anticholinergic drug, ipratropium bromide, is not selective for muscarinic receptor subtypes. despite some efficacy in the most common pediatric airway diseases such as asthma and pre-school wheeze and cystic fibrosis, ipratropium bromide is not commonly prescribed as a standalone medication. the more recently introduced anticholinergic drug, tiotropium bromide, has advanced pharmacologic properties such as long duration of action and a functional selectivity for m1 and m3 receptors over m2 receptors, and has shown a good efficacy and safety profile in adult respiratory disorders, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. ongoing studies are now under way to define its therapeutic role for pediatric airway diseases. inhalation of smoke from datura strammonium, a member of the deadly nightshade family, was recommended for the treatment of asthma in 17th century ayuverdic literature. general gent, himself an asthmatic, on return from india in the early 19th century, was reported to have brought this therapy to england. strammonium and belladonna cigarettes were widely used to treat respiratory disease until the middle of the 20 th century. however there were frequent side effects, including tachycardia, hallucinations, and even addiction. with the introduction of synthetic atropine derivatives with fewer side effects, there has been a renewed interest in anticholinergic therapy for asthma. bronchial smooth muscle tone is predominantly set by cholinergic activation. patients with asthma have increased bronchial smooth muscle tone and mucus hypersecretion, likely as a result of cholinergic activity. anticholinergic medications can relax smooth muscle in children with acute asthma, these drugs also appear to have anti-inflammatory properties, and may reduce goblet cell hyperplasia driven by neutrophil elastase à a feature of severe asthma known to be resistant to steroid therapy. the short-acting anticholinergic agents, ipratropium bromide and oxitropium bromide, have been used in asthma for many years, primarily for acute asthma in the emergency department. paradoxically, although the addition of an anticholinergic medication to a beta agonist can decrease acute asthma severity and hospital admission, studies suggest that continuing the anticholinergic while the patient is in hospital does not hasten recovery or decrease length of hospital stay. however these studies have been small and potentially underpowered. until the past decade, these results have dampened enthusiasm for studying anticholinergic medications as maintenance asthma therapy. this has changed with long-acting anticholinergic (lama) bronchodilators under investigation or are available for treating lung disease: these include tiotropium, aclidinium, glycopyrronium, glycopyrrolate and umeclidinium. the once-daily lama, tiotropium bromide, is demonstrated to improve lung function and decrease the risk of exacerbation in adolescents and adults with moderate to severe asthma, despite the use of inhaled corticosteroids (ics) and long-acting b 2 -agonists (labas). in september 2015, the fda in the united states approved tiotropium for the long-term, maintenance treatment of asthma in patients 12 years of age and older. tiotropium by respimat soft-mist inhaler is now included in the global initiative for asthma report (gina) 2015 global strategy for asthma management and prevention. in phase 3 studies, tiotropium improved asthma symptoms in 68% of enrolled subjects and decreased exacerbations by 21% whilst having a safety profile similar to that of placebo. studies also show that tiotropium was effective in improving pulmonary function (fev1) and decreasing asthma attacks in children age 6-11 with poor asthma control despite use of a medium dose of ics with or without a leukotriene modifier. there was no difference in effectiveness when comparing the fda-approved dose or 2.5 mcg (2 â 1.25 mcg) once daily tiotropium to a higher dose of 5 mcg. initial studies in children younger than 6 years do not appear to show benefit. with increasing knowledge about the diverse actions of the cholinergic system in asthma and the role of muscarinic receptors in the airway, we are gaining an increased appreciation of how anticholinergic medication can play an important role in treating children and adults with chronic and poorly-controlled asthma. the 2008 ers task force opted to not use the term asthma to describe preschool wheezing illness since there was insufficient evidence showing that the pathophysiology of preschool wheezing illness is similar to that of asthma in older ages. the task force referred to pre school wheezing and described episodic (viral wheeze) for children who wheeze intermittently and are well between episodes versus multiple-trigger wheeze for children who wheeze both during and outside discrete episodes.(2) we will therefore in our current discussion refer to this young age morbidity as an entity that should be discussed separately from asthma, acknowledging that much has yet to be learned on the nature of this entity. amongst the many mechanism of virus-induced airway hyperreactivity; a common phenomenon in pediatric practice related to this young age group, studies have shown that cholinergic overactivity such as through the modulation of substance p may mediate virus-induced airway hyperreactivity. virus-specific cd8þ t lymphocytes may induce cholinergic activation through m2 receptor dysfunction.(3) hence anticholinergic medications may have a role in viral-induced wheeze with compounds that display selectivity for m1 and m3 muscarinic receptors over m2 receptors having advantages over nonselective compounds. a number of small studies addressed the role of anticholinergics in acute bronchiolitis but failed to show a role for this acute intervention. a study on 69 infants who were randomly assigned to receive nebulized salbutamol, ipratropium bromide or placebo resulted in faster improved clinical scores and oxygen saturation levels in the bronchodilator groups than in the placebo, but no effect to change the natural course of the disease. (4) in studies on this topic from 1981, inhaled ipratropium bromide administered to wheezy children (3 -32 months of age) improved lung function when measured by total body plethysmography and forced oscillation technique. (5) the authors were unable to differentiate between responders and nonresponders by clinical or by physiological parameters, but submitted that the differential distribution of obstruction between small and large airways may underlie response or lack thereof; and that subjects with a predominance of large airways obstruction were the responders to inhaled ipratropium. a logical if unproven additional speculation was that anticholinergics decrease airway secretions and with it reduce large airway resistance. a cochrane review examining the effect of adding ipratropium bromide to b 2 -agonists in wheezy infants (6) suggested that the combined therapy improved symptom scores after 24 hours compared to the use of jb 2 -agonist alone. the ers task force cited above(2) offered evidence-based recommendations on the definition, assessment and treatment of wheezing disorders in preschool children. addition of ipratropium bromide to short acting b 2 -agonists was suggested for patients with severe wheeze. in the 2014 review of the task force recommendations no reference was made to the use of anticholinergic medications. (7) tracheobronchomalacia it is widely believed amongst pediatric pulmonologists that administration of b 2 -agonists in infants with airway structural instability, predominantly tracheobronchomalacia is detrimental, while the use of anticholinergics for bronchodilatation is safe. this notion derives from a study of only 3 infants with intrathoracic tracheomalacia, using infant pulmonary function testing and demonstrating that flows improved significantly after administration of metacholine but worsened after administration of albuterol.(8) these results suggest that in patients with abnormally collapsible tracheas or large bronchi, stimulation of the smooth muscle can improve airway stability, thereby increasing forced expiratory flows, while relaxation of airway smooth muscle by bronchodilators can exacerbate obstruction. the sole support for this observation comes from a review of a series of patients with tracheobronchomalacia from chile, in whom beta-agonist medications were discontinued while the anticholinergics were not. (9) the effect of anticholinergic medication has not been assessed directly in any study, and thus whether this class of medications may have a different effect compared to beta 2 -agonists in such pathology has not been established. further studies on the effect of the various bronchodilators for such pathologies using newer technologies to assess airway resistance (e.g., forced oscillation) should be undertaken. while more invasive and challenging, a technique of direct quantitative assessment of tracheal collapsibility in infants with tracheomalacia has been described, and may be the most adequate technique to answer this important clinical question. (10) tiotropium bromide in pediatric use -asthma and the asthma-copd overlap syndrome ipratropium bromide has a limited role in childhood asthma, largely due to lack of selectivity. the more recently introduced long-acting muscarinic antagonists/anticholinergic (lama), tiotropium bromide, presents advanced pharmacologic properties such as selectivity for m3 muscarinic receptors over m2 receptors and long duration of action. a high safety profile and increasing evidence of efficacy have rendered it a mainstay medication for copd with an emerging role in adult asthma. few studies have emerged on its role in the treatment of childhood asthma and defining its therapeutic niche for pediatric airway diseases. in a recent 1-year randomized controlled trial, tiotropium add-on therapy in adolescents with moderate asthma, (11) significantly improved lung function and was safe and well tolerated when added to at least ics maintenance therapy. a study of 71 pediatric patients with asthma and chronic cough from an asthma center (12) concluded that tiotropium can be beneficial in 3 distinct patient populations: add-on therapy to asthmatics on maximal maintenance medication, an alternative to highdose inhaled steroids in patients who are experiencing significant side effects, and patients with bronchorrhea as their predominant symptom manifested by a chronic productive cough, the latter population is most likely explained by its drying effect on airway secretions. a recent editorial (13) states "approximately 1 in 12 people worldwide are affected by asthma or chronic obstructive pulmonary disease (copd); once regarded as two distinct disease entities, these two conditions are now recognized as heterogeneous and often overlapping conditions. the term "asthma-copd overlap syndrome" (acos) has been applied to the condition in which a person has clinical features of both asthma and copd". in recent years multiple reports describing this interface between asthma and copd have been published recognizing that the demarcation line between these two entities is difficult to define. while the precise definition in various populations is still being worked out, and it is obvious that the majority of such patients are adults, there is early recognition that some pediatric populations, who are viewed as asthmatic, yet have no airway reversibility, may constitute an early presentation of the overlap syndrome. the mainstay therapies for copd are long-acting inhaled bronchodilators, including longacting b2-agonists (labas) and lamas, with its characteristic member being tiotropium bromide. in patients with copd they are recognized as being equally effective because they reduce air trapping by relaxing airway smooth muscle as a result of reducing the effects of intrinsic cholinergic tone. it is therefore intriguing to speculate that once a better definition of the overlap syndrome emerges in pediatrics, an important role for tiotropium is likely to emerge particularly as a potential steroid sparing medication. peter d sly, ao mbbs, md, dsc, fracp, fers, f thor soc, fapsr, fahms p.sly@uq.edu.au the measurement of lung function is of major importance in clinical practice or respiratory medicine and in respiratory research. much has been learned about the risk factors underlying respiratory disease by measuring lung function in patients and comparing it with that in healthy controls. however, for managing an individual patient or assessing risk of disease onset or progression, it is necessary to know whether an individual's lung function is "normal" or "abnormal". over the years, a number of sets of normative equations have been produced by individual research groups in different parts of the world. these have been incorporated into commercially-available spirometers and used in populations other than those in which the data were collected. this situation was far from ideal, especially as some of the normative equations were many decades old. what is the gli? data were obtained from 73 centers in 33 countries (n ¼ 160,330) ; however not all could be used due to lack of data on ethnicity (which is illegal in france!), small numbers, missing data, lack of quality control and other factors. data were also pooled by region with data from europe, israel, while fev 1 and fvc varied between ethnic groups, they did so proportionally, meaning that fev 1 /fvc was independent of ethnicity. the lower limit of normality for fev 1 and fvc showed age dependence that differed between males and females, reaching 80% by mid-childhood and falling progressively below 80% from approximately 40 years of age. the rate of fall in the lower limit of normal for fev 1 and fvc was identical for women but fvc declined more slowly in males. a ratio of fev 1 / fvc >0.7 is taken to indicate pathological airflow limitation; however, the proportion of the healthy non-smoking population with fev 1 /fvc >0.7 rises steadily to 20-25% at 80 years of age. how well do the gli reference equations predict lung function in people in individual countries? given that the gli reference equations were compiled by pooling data from a variety of sources, one might expect that the equations would provide good estimates of lung function for populations that were well represented in the pooled data whereas they may not for populations either not included or underrepresented in the pooled data. indeed this appears to be the case, with the gli equations adequately representing lung function in australasian caucasians 2 , but not performing as well for adults in brazil 3 , north africa 4 , madagasca 5 , and children in poland 6 and peri-urban and rural india 7 . further study is required to ascertain how widely the gli reference equations can and should be applied. what constitutes "normal" data? an important consideration when creating reference equations is what characterizes a "normal" population and who should be excluded? the dataset used to construct the gli reference equations excluded ever smokers, but is this reasonable? if 20-30% of an adult population smoke, should they be excluded from equations designed to the lung function of that population? maternal smoking during pregnancy results in long-term reduction in lung function 8 but is not generally taken into consideration when defining a healthy population. "healthy" children are often defined as those with no prior asthma or hospitalization for respiratory problems, born full term with birth weight ! 2.5 kg and asymptomatic at the time of testing 9 . however, lum et al. 9 recently demonstrated that with the exception of clear-cut factors such as current and chronic respiratory disease, including children born prematurely or with low birth weight, prior asthma and mildly symptomatic made little difference to the reference equations but increased the generalizability to the target population. this debate continues! the implications of switching to the gli equations will depend on how well the gli equations represent lung function of the local population. in poland, a switch from the 1998 polish reference values to the 2012 gli would see an increase in diagnosis of obstructive lung disease from 17.5% to 20.3% and an increase in diagnosis of restrictive lung disease from 3.8% to 7.6%. whether this represents an over-diagnosis with gli or an under-diagnosis with the old equations is a matter of clinical judgment. the impact on parents and children with cystic fibrosis is likely to be substantial as families tend to focus on lung function, especially fev 1 expressed as a percent of predicted as evidence of the state of the child's lung disease. a change in number for a technical reason must be balanced against the likelihood of creating anxiety in the clinic population. respiratory symptoms are very frequent in infants and young children. special emphasis has been put on symptoms signaling bronchial obstruction and bronchial hyperresponsiveness as these may be associated with early onset of asthma. since the early 1980 0 s, several research groups have been focusing on early events in the development of asthma, especially seeking potential risk factors for predicting persistent symptoms. structural changes in the bronchial mucosa and lung function impairment in children with early obstructive symptoms have also been studied. it was documented that eosinophilic inflammation and remodeling (particularly epithelial basement membrane thickening and increased airway smooth muscle mass) are consistently present in patients with persistent asthma. interestingly, some markers of inflammation and even those of initial remodeling have already been described in children before the clinical diagnosis of asthma could be confirmed 1 . this finding supports the hypothesis of remodeling not being a late consequence of a long lasting eosinophilic inflammation but that it may run in parallel with the development of asthma, if not even precede or initiate inflammation in the bronchial mucosa. this hypothesis was later supported by further research based on bronchial biopsies in infants. eosinophilic inflammation and some markers of remodeling have been documented in the bronchial mucosa of symptomatic children as early as in the second year of life 2 . in a recent study, we were able to show that basement membrane thickening could be found even in young children at risk of developing asthma even without a history of recurrent wheeze 3 . however, the significance of these findings in terms of long term prognosis still remains less documented. it is known that airway hyperresponsiveness in infancy is associated with persistent symptoms later in childhood 4 . also, reduced airway patency at birth was shown to be linked to an increased risk of developing asthma and severe bronchial hyperresponsiveness by the age of 10 years 5 . long-term follow up of children investigated in infancy and reassessed in later childhood have so far showed that reduced baseline lung function in symptomatic infants was significantly associated with subsequent respiratory morbidity as well as with the need of anti-asthma medication at the age of 3 years. in addition, the usage of inhaled corticosteroids at the age of 3 years also seems to be in positive correlation with basement membrane thickening and increased number of mast cells in bronchial mucosa in bioptic samples taken earlier in infancy 6 . this study has thus suggested that early morphological changes in the airway wall might indeed play a role in determining subsequent respiratory morbidity. on the other hand, at the next follow-up of these children at the age of eight years, the positive correlation between current respiratory symptoms and markers of inflammation and remodeling described in infancy was no longer found 7 . this finding is consistent with the results of the follow-up of our group of children where we did not find a significant correlation between lung function (both fev 1 and fvc) measured in preschool age and basement membrane thickness measured earlier in infancy and toddler's age both in the risk group and control group of children (unpublished data). more recently, airway smooth muscle mass has come into the center of interest of many researchers in respiratory medicine. smooth muscle hyperplasia and hypertrophy in the bronchial wall of patients with asthma are considered to be a consistent feature of bronchial remodeling. it is notably a possible dysfunction of newly formed smooth muscle bundles that deserves attention and more studies in this area are urgently required. the first works in children have shown that the increase in the airway smooth muscle mass in the bronchial wall might be associated with school age asthma 8 . lately, another study has described a negative correlation between the airway responsiveness at the age of 8 years and airway smooth muscle mass in infancy 7 . however, this area of airway remodeling still remains poorly understood, especially with regard to its role in childhood asthma. based on currently available data, reduced lung function at birth or in early childhood is apparently associated with the persistence of symptoms and the decrease in lung function in later life. however, it still has not been reliably confirmed whether this low lung function has any correlation with early signs of airway remodeling. more long-term follow-up studies are needed in pediatric patients comprising both tissue biopsies taken in early age followed by longitudinal long-term lung function monitoring. nasal and sinus disease is universal in cystic fibrosis (cf). because nasal and sinus disease usually coexist, we will refer to this as "sinonasal disease". since the mucosa of the sinuses and upper respiratory tract and the mucosa of the lower respiratory tract are similar, disease may be similar in both locations and sinonasal disease could influence the severity of pulmonary disease. this view of the "universal airway" has been demonstrated in patients with pulmonary conditions, such as asthma and copd. in these diseases, an improvement in sinus health is reflected by an improvement in the lower airway disease. this has not been well studied in cf but the implications of this relationship combined with increasing life span makes an understanding of sinonasal disease important to the care of these patients. mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene in cf carriers appear to be independently associated with a higher prevalence of sinonasal disease; 36% of carriers reported chronic rhinosinusitis compared to the 13-14% in the general population. the bacterial flora of the sinuses changes with patient age, can include anaerobes and fungi, and often mirrors the organisms present in the lower respiratory tract. a link between sinus infection and lower respiratory tract infection may contribute to morbidity following lung transplantation and immunosuppression. somewhat surprisingly, the prevalence of otitis media in cf appears to be no greater than in an age-matched general population. endoscopy and computerized tomography have broadened our understanding of how cf affects the sinuses. endoscopic sinus exams are almost always abnormal and give a better indication of the presence of nasal polyposis than physical examination of the nose alone. nasal polyps become more common with age and may represent a proliferative airway repair mechanism. sinus ct has demonstrated several anomalies characteristic of sinonasal disease in cf such as bulging or displacement of the lateral nasal wall, demineralization of the uncinate process, and hypoplasia or aplasia of the paranasal sinuses. serious complications of sinonasal disease in cf are rare and include mucoceles and periorbital abscesses. these usually require surgery. there are few randomized, controlled trials evaluating medical or surgical treatments of cf sinus disease. sinus surgery may provide some benefit, though there are no established selection criteria for appropriate candidates. the trend today in neonatal intensive care units (nicus) is to be as gentle and less invasive as possible in the care of neonates. this attitude takes place in every field of neonatology, and will discuss its implementation specifically in the respiratory care administered to premature infants with respiratory distress syndrome (rds). 1, 2 prenatal corticosteroid therapy is recommended in all pregnancies with threatened preterm labor below 34 weeks' gestation. recently, it was shown that such therapy could also be beneficial in late preterm infants as it significantly reduced the rate of a neonatal composite of respiratory treatments in the first 72 hours or stillbirth or neonatal death within 72 hours after delivery. 3 at delivery, the term stabilization and not resuscitation is preferred for the vast majority of very preterm infants. only a minority of babies should require delivery room intubation. neopuff can be helpful in the delivery room and the transport to the nicu, and enables the administration of continuous positive airway pressure (cpap) and intermittent positive pressure ventilation under controlled conditions. recent large trials that reflect current practice (including greater utilization of maternal steroids and routine post delivery stabilization on ncpap) demonstrated less risk of bronchopulmonary dysplasia (bpd) or death when using early stabilization on ncpap with selective surfactant administration to infants requiring intubation. the comprehensive strategy to prevent bpd in the nicu is based on ventilatory and non-ventilatory measures. 4 the ventilatory route allows an individualized endotracheal intubation approach. recent studies concluded that early nasal cpap (ncpap) is a safe alternative to immediate intubation even in extremely low birth weight (elbw) infants. 1, 2, 4 endotracheal intubation and ventilation can result in significant damage to premature lungs and are independently associated with cerebral palsy. furthermore, despite new modes of ventilation and surfactant, bpd remains a significant morbidity and its incidence was correlated with the use and length of endotracheal mechanical ventilation. bpd in itself is associated with adverse neurodevelopmental outcome. thus, we need to avoid endotracheal ventilation, if possible. when the infant requires nasal respiratory support (nrs), we should aim for adequate oxygenation (spo 2 of 90-95%), 1 permissive hypercapnia (paco 2 of 45-55 mm hg, ph >7.22) and gentle ventilation, similarly as in endotracheal ventilation. 1, 4 ncpap is recommended as the early primary treatment of active respiratory distress syndrome (rds) (to avoid intubation or as part of the insure [intubation surfactant extubation] approach), or later, post extubation at rds resolution, in order to allow shortening of the duration of endotracheal ventilation and to treat apnea of prematurity. 1, 2 recent studies 1, 4 report comparable rates of bpd in elbw infants treated initially with ncpap as compared to endotracheal ventilation with surfactant administration. can we enhance ncpap and get better outcome for nrs by using nasal intermittent positive pressure ventilation (nippv)? nippv was defined as a method of augmenting ncpap by delivering ventilator breaths via nasal prongs. the rationale behind the use of nippv is the administration of "sigh" to the infant, thus opening microatelectasis and recruiting more ventilation units. it was shown that synchronized nippv (snippv) compared with ncpap may improve the patency of the upper airway, could activate the respiratory drive, improves thoraco-abdominal synchrony, stabilizes the chest wall, improves lung mechanics and decreases the work of breathing in premature infants. when nippv was compared to ncpap for the different indications of nrs, it was shown to enhance the potential of nrs. 4 a recent meta-analysis demonstrated a relative risk reduction for intubation in the first 72 hours in the nippv group compared with ncpap (rr 0.60, 95% ci 0.43, 0.83). 5 the nippv trial 6 was a large international multicenter randomized trial powered to study the important outcome of bpd, recruiting 1,009 extremely low birth weight babies, and it showed no difference between babies randomized to nippv compared with cpap. snippv vs. ncpap for later use, post extubation at rds resolution, as a "bridge" to spontaneous unsupported breathing, was shown to be more effective than ncpap. a pooled meta-analysis showed that snippv was more effective than ncpap in preventing failure of extubation [rr 0.21 (0.10, 0.45)] and the number needed to treat was only 3 infants to prevent one extubation failure. 7 snippv vs. ncpap, post extubation, also tended to decrease the rate of bpd. snippv may also be more effective than ncpap for apnea of prematurity. 4 a meta-analysis regarding apnea of prematurity suggests that snippv is more efficacious with apnea that is frequent or severe. however, the studies performed addressed short-term outcomes and as such could not properly address the incidence of requirement for reintubation. thus, more studies are needed before recommending snippv as standard of care for apnea of prematurity. while non-invasive ventilation is probably safe, its success depends on gestational age. the data indicate that surfactant may still have a significant role in the treatment of rds, especially in elbw infants. recent studies reported on an intubation rate of $50% in their ncpap group in elbw infants. 1, 2, 4 this leads us to the insure approach. this approach may allow the infant to benefit from both surfactant and nrs. a cochrane review 8 concluded that the insure approach with ncpap compared with later selective surfactant administration, continued mechanical ventilation, and extubation from low respiratory support was associated with less need for mechanical ventilation, lower incidence of bpd and fewer air leak syndromes. another option for surfactant application to the trachea without endotracheal intubation was described by using a thin catheter in spontaneously breathing preterm infants receiving ncpap. this technique was reported to reduce the need for mechanical ventilation. 9 there are ongoing trials with inhaled surfactant. to summarize, ncpap is still the most common mode of non invasive respiratory support worldwide. 1, 2 the available evidence supports the preference of early or later use of nippv/snippv compared to ncpap because of minimizing the use and the length of endotracheal ventilation. 4 there are data to suggest that this approach may also reduce the rate of bpd, however this has yet to be shown. 4 the results of a large international rct comparing both primary and post-extubation use of nippv with ncpap, with a composite primary outcome of death or bpd at 36 weeks' corrected age, indicate no additional benefit, or risk, conferred by nippv in comparison to ncpap. 6 whether nippv/snippv is more beneficial than ncpap within the insure approach needs to be shown. recently, heated, humidified high-flow nasal cannula (hhhfnc) is frequently used as a mode of nrs. high flows result in washout of anatomical and physiological dead space and contribute to improved fractions of alveolar gases with respect to carbon dioxide as well as oxygen and decrease the work of breathing and the energy cost of gas conditioning. hhhfnc probably creates positive end expiratory pressure (peep) that may contribute to its beneficial effect. however, the peep that is not monitored had raised concerns regarding the safety of hhhfnc in terms of air leak. recent prospective studies support the notion that hhhfnc is as effective as ncpap for early stages of rds, post extubation 10 and for apnea of prematurity. yet, more studies, especially in the initial treatment of rds and in elbw infants, are needed before adopting hhhfnc as an alternative mode of nrs in these conditions. new modes of nrs such as neurally adjusted ventilator assist (nava), and nasal high frequency ventilation, need to be further studied before concluding on benefits for the short and long term outcomes in premature infants. non-ventilatory measures in the treatment of rds, such as caffeine, nutrition, fluid and pda management and postnatal steroids in certain conditions should be included in the care of premature infants with rds in order to minimize the rate of bpd. 1, 4 the noninvasive ventilator strategy needs to be confirmed by large prospective randomized controlled trials (with long-term follow up) in order to assure it is applicable to most elbw infants. furthermore, the strategy needs to be tailored to individualized infants according to the infant's maturation; antenatal steroid treatment and severity of rds; general condition; and to certain practical nicu conditions such as experience, personnel and timing during the day. for many years, it has been generally accepted that the pathophysiology of rsv bronchiolitis is driven by the inflammatory response evoked by horizontal (i.e., interpersonal) transmission of the virus in the first few months after birth (1). however, a recently published study has brought to the forefront a striking new idea: rsv may be transmitted vertically from the respiratory tract of the mother to the lungs of the fetus (2) . until now, we believed that when a pregnant woman got a cold, the developing fetus was protected by the placenta from rsv and other respiratory viruses. in this study, pregnant rats were inoculated with a recombinant rsv strain that could be tracked through expression of a red fluorescent protein (rrrsv). the same virus was subsequently found in 30 percent of fetuses exposed in utero, as well as in the lungs of 40 percent of newborn rats and 25 percent of rats born to inoculated mothers when tested in adulthood. these data provide proof of concept for the transplacental transmission of rsv from mother to offspring and the persistence of vertically transmitted virus in lungs after birth. notably, the intrauterine rsv infection changed expression and function of critical neurotrophic pathways that control the development of cholinergic nerves in the budding airways and lung tissues (3). these changes in cholinergic innervation of the fetal respiratory tract resulted in the development of postnatal airway hyperreactivity upon reinfection with the same virus (2) . the airway smooth muscle tone was normal in the absence of stimulation and its contraction was normal in the absence of either maternal or neonatal infection. but in pups reinfected with rsv after prenatal exposure to the virus, markedly potentiated contractile responses were measured after either electrical nerve stimulation or methacholine inhalation, suggesting the involvement of both pre-and postjunctional mechanisms. these findings are consistent and provide a plausible mechanism to the epidemiologic evidence that early-life rsv infection -or possibly reinfection -predisposes a subpopulation of children to recurrent wheezing and asthma that typically spans through the first decade of life even in the absence of atopic phenotype (4). to our knowledge this is the first report of vertical transmission of rsv, or for that matter any common respiratory virus. a number of infectious agents, including herpesviruses and retroviruses, have been shown to cross the placenta and establish persistent infection in offspring. the new evidence extends this possibility to other infections, such as rsv, once regarded as temporary and localized and that instead may be longer lasting and more pervasive than we thought. also, as shown for other viral pathogens, if rsv seeds the fetus before full t-cell maturation, this could lead to induction of prenatal tolerance and justify the limited synthesis of interferon and other inflammatory cytokines that have been noted when newborns develop severe infections (5) . vertical rsv and asthma -the general concept that we have been working under for decades is that nothing bad happens in the lungs until the baby is born -even with serious conditions such as cystic fibrosis -and that the lungs are "clean" of pathogens at birth. but if human studies replicate the findings from animal models outlined above, our understanding of the pathogenesis of rsv infections would be completely changed. it would turn back the clock of respiratory developmental diseases by months and mean that we would need to start thinking about lung development and pathology during pregnancy rather than at birth. this could create a paradigm shift by extending our focus on prevention from the first few years after birth to also include the last few months before birth. this new paradigm is in line with the emerging evidence that many (or most) chronic inflammatory, degenerative, and even neoplastic diseases plaguing adults have their origins from often-subtle events occurring during fetal life. the "foetal programing hypothesis" was originally formulated by dr. david barker more than two decades ago to explain the extensively reproduced and confirmed epidemiologic evidence that low birth weight predisposes to cardiovascular disease in late adulthood (6) . dr. barker died aged 75 in september 2013, leaving the legacy of this initially controversial, but now widely accepted, idea that common chronic illnesses such as cancer, cardiovascular disease and diabetes result not always from bad genes and an unhealthy adult lifestyle, but from poor intrauterine and early postnatal health. in one of his last public speeches, he argued: "the next generation does not have to suffer from heart disease or osteoporosis. these diseases are not mandated by the human genome. they barely existed 100 years ago. they are unnecessary diseases. we could prevent them had we the will to do so." we believe the same concepts can be extended to chronic obstructive airway diseases like asthma and copd. asthma is the final product of complex interactions between genetic and environmental variables. prenatal events like the intrauterine exposure to viruses with specific tropism for the developing respiratory epithelium (7) or imbalanced maternal diet (8) will cause a shift in the trajectory of structural and functional airway development towards a hyperreactive phenotype. the same intrauterine exposures can affect gene expression via epigenetic modifications like dna methylation, histone acetylation, and by altering the relative expression of regulatory micro-rnas (9) . the resulting neonatal phenotype will predispose the child to aberrant responses to common respiratory infections and airborne irritants, thereby increasing the risk of obstructive lung disease later in life. postnatal events, such as exposure to indoor and outdoor pollutants and allergens, can further shift the equilibrium of the adult phenotype by exacerbating airway inflammation and hyperreactivity (10) . the continuous range of possible developmental trajectories and multiple sequential events acting during development will define the severity and duration of disease. the incidence, severity and mortality from childhood pneumonia has declined substantially in the last decade due to improved socioeconomic conditions, better access to care, wider implementation of effective management and preventative strategies and development and availability of improved vaccines, particularly the pneumococcal (pcv) and h influenzae type b (hib) conjugate vaccines. [1] however, pneumonia remains the leading cause of childhood mortality globally outside the neonatal period and a major cause of morbidity and hospitalization despite good immunization coverage. [2, 3] further, early childhood pneumonia has increasingly been associated with the development of chronic noncommunicable respiratory diseases into childhood and adulthood, such as asthma or chronic obstructive airways disease (copd). [4, 5] with improved global coverage of the newer conjugate vaccines, it is likely that viral causes of pneumonia may be responsible for an increasing proportion of pneumonia cases. [6] however, defining the etiology of pneumonia may be challenging as it can be difficult to distinguish colonizing from pathogenic organisms in respiratory specimens, blood culture rarely is positive and pneumonia, especially severe disease, may frequently be due to multiple co-pathogens. the development of better methods for specimen collection and of molecular diagnostics have provided more sensitive techniques to define potential etiologic agents but further compound the difficulty of ascribing pathogenicity. [7, 8] despite these limitations, studies in the post-pcv era have reported an increasing predominance of viruses in childhood pneumonia cases, with a virus identified in 70-90% of cases. [9, 10] in children vaccinated with 13valent pcv (pcv13), rsv has been reported to be the predominant pathogen in case control analyses from both high income countries and lowmiddle country settings. however, there is frequent co-occurrence of other potential pathogens with rsv, including bacteria and other viruses. [9] children under 6 months of age are at highest risk of rsv disease. [11] to adequately interpret data on viruses in the context of childhood pneumonia, the prevalence of these in healthy control children must be considered. using case control designs, viruses identified in association with pneumonia have been rsv, influenza virus and human metapneumovirus (hmpv); adenovirus, parainfluenza virus and coronavirus have been variably associated with pneumonia while the prevalence of rhinovirus has consistently been similar in cases and controls. [9, 10, 12, 13] the use of quantitative measurements of viral load has not shown to be useful in distinguishing cases from controls except for rsv and for hmpv, but the presence of these alone is sufficient to ascribe etiology. these studies indicate that rsv is a major cause of pneumonia in the era of conjugate vaccines for bacterial pathogens, particularly in young infants. however they also highlight the limitations of current diagnostic strategies, particularly the poor sensitivity of current tests for bacterial etiology and the potential for incorrectly assigning etiology based on molecular diagnostics. they also provide further data on the complexity of ascribing pneumonia etiology, showing interactions between multiple potential pathogens. despite these limitations, the emerging data indicate that a key strategy for reducing the burden of childhood pneumonia lies in prevention of rsv disease in young children. identifying the etiology of pneumonia is key for initiating appropriate management strategies particularly use of antibiotics and to guide development of new vaccines. the reduction in bacterial pneumonia through conjugate vaccines underscores the need to reconsider the empiric treatment of pneumonia in settings where there are strong immunization programs. case management with antibiotics for pneumonia or severe pneumonia in the world health organization integrated management of childhood illness (imci) program has been a highly effective strategy for reducing mortality prior to widespread conjugate vaccine availability [14] , but defining the residual burden and identifying clinical or laboratory features that distinguish bacterial from viral pathogens will be important before any change in pneumonia strategy can be recommended globally. late preterm (lp) newborns (born at 34-0/7 to 36-6/7 weeks gestational age) comprise the fastest growing subset of neonates, accounting for approximately 74% of all preterm births and about 8-9% of total births in the us [1] . "late preterm" infants are born near term, but are "immature". the late premature birth interrupts normal in utero fetal development during the last 6 weeks of gestation that are probably a "critical period" of growth and development of the fetal lungs [2] . three factors play a role in the respiratory vulnerability of lp infants [2] : 1. prematurity with its developmental and consequently physiologic components; 2. heightened rate of respiratory morbidity in the neonatal period; 3. short-term pulmonary outcome respiratory complications are the prime morbidities of lp infants [2] . a large retrospective study [3] found that the odds of respiratory morbidity (respiratory distress syndrome [rds] , transient tachypnea of the newborn [ttn], pneumonia, respiratory failure, surfactant administration, and mechanical ventilation) decreased significantly with each advancing week of gestation up to 38 weeks compared with 39 to 40 weeks. despite a relatively low absolute risk for rds or ttn at 34 weeks compared with more premature infants, this rate poses an increased risk for lp infants when compared with term infants [2] . acknowledgement of these morbidities led to studies aiming to decrease this burden. a recent large randomized controlled study [4] showed that administration of betamethasone to women at risk for late preterm delivery significantly reduced the rate of a neonatal composite of respiratory treatments in the first 72 hours (the use of continuous positive airway pressure or high-flow nasal cannula for at least 2 hoursr, supplemental oxygen with a fraction of inspired oxygen of at least 0.30 for at least 4 hours, extracorporeal membrane oxygenation, or mechanical ventilation) or stillbirth or neonatal death within 72 hours after delivery. of note, neonatal hypoglycemia was more common in the betamethasone group than in the placebo group. late prematurity may affect the respiratory system in the long term [2] . several studies reported an association of preterm birth (30-36 weeks' ga) without clinical lung disease with altered lung development and function [2] . friedrich et al. [5] in a longitudinal study found that despite normal lung volume, healthy preterm infants had persistently reduced airflow through the age of 16 months and concluded that preterm birth in itself was associated with altered lung development. a single study [6] showed a potential improvement, especially for large airway function, with advancing age. a recent large prospective cohort study showed that the number of hospitalizations caused by respiratory problems during the first year of life was doubled in moderately/late preterm (32-36 weeks' ga) compared with term infants [7] . at preschool age, moderately preterm infants revealed more nocturnal cough or wheeze during or without a cold and increased use of inhaled steroids. at the age of 5 years, rates of respiratory symptoms between moderate and early preterm born (<32 weeks' ga) children were similar; both were higher than in term born children. whether lp birth is associated with airway disease such as asthma in early childhood remains controversial [2] . different findings in published studies could result from the different methods of asthma diagnosis, age groups at diagnosis, and from the difficulties in diagnosing asthma in early childhood. a recent study [8] found that late preterm birth history is not independently associated with childhood asthma until 7 years of age. lp infants are more vulnerable to viral respiratory infections, particularly rsv, which are more severe in these infants vs. term infants. the pernicious combination of rsv bronchiolitis affecting an a priori compromised lung/ airways of lp infants may have a lasting effect on respiratory function and consequent long-term morbidity [2] . long-term persistence of an early decrease in pulmonary function tests (pft) was demonstrated by a longitudinal follow-up into early adulthood for an unselected random population in the tucson children's respiratory study [9] . these observations suggest that the notion of a "critical developmental period" for the respiratory system does exist. deficits in lung function during early life, especially if associated with lower respiratory illnesses (especially rsv), increase the risk for chronic obstructive pulmonary disease later in adult life [10] . summary lp infants are born during a "critical developmental time period" for the lungs. this may result in short and long-term pulmonary consequences. in addition, to screen the population at high-risk for disease. therefore, the effectiveness of early case finding should be a priority, but it depends on several factors such as health care system, contact tracing, and laboratory diagnosis. the diagnosis of tb in children is a common clinical challenge, and relies on a careful assessment of history of exposure, clinical examination, and relevant investigations. the most recommended approach to the diagnosis of tb in both children and adults is based on the who guidelines recommendations from 2010 and 2014 (table 1) . (2, 3) important factors to consider in all children with suspected tb is the endemic setting as well as the age and immune status of the child. in countries with a low incidence of tb, a positive contact with a case in combination with suggestive symptoms makes diagnosis more straightforward. in high tb endemic areas, a history of tb contact remains important, but is much less sensitive, given that transmission often occurs through unknown source cases. (5) laboratory tests for the diagnosis of infections can be grouped into two groups: detection of microbes (or components) and detection of components of the immune response to the microbe. the sensitivity of the first group will depend on the quality of the specimen and the concentration of microorganisms. this group includes microscopy, culture, elisa, and nucleic acid detection (pcr). the second group measures the activity of the immune system against microbe-specific antigens in the possibly infected host. this category includes antibody detection and activated t cells. the gold standard for the diagnosis of tb is bacillary detection by smear or culture. in adults, microscopy can detect up to 60% à 70% of culture-positive samples. in children, this does not work as well due to limited access to appropriate body specimens, and also because children usually have paucibacillary disease, since cavitating disease is rare in children. studies have shown that under best circumstances, acid-fast bacilli sputum smear is positive in only about 10-15% of children with tb while culture gives a better yield of 30-40%. until recently, the diagnosis of ltbi has been based exclusively on the tst, which has relatively poor sensitivity and specificity. despite these limitations, it remains the standard of care for diagnosis of ltbi worldwide, particularly in low-income countries. interferon gamma release assays (igras) measure the in vitro response to specific m. tuberculosis antigens. although they offer several advantages over tst such as better specificity, single visit, little inter-observer variability, and no booting effect; they have not been found better than tst, and are not able to predict the risk of infected individuals developing active tb disease. given their increased cost, replacing tst by igras as a public health intervention in resourceconstrained setting is not recommended. novel approaches to confirmation of tb have been developed. these include methods based on rapid culture techniques and genotypic techniques that improve detection of m. tuberculosis. an example is the xpert mtb/rif assay, which is a fully automated realtime dna based test that can detect both tb and rifampicin resistance in less than two hours. (3, 6, 7) as expected, it should be used rather than conventional microscopy and culture in children suspected of having mdr-tb. the clinical diagnosis of primary tb in children remains challenging because of non-specific signs and symptoms and difficulty with acquiring diagnostic specimens. because of this, the diagnosis of primary tb in practice, relies on a combination of clinical features and chest x-ray (cxr) findings. the detection of lymphadenopathy in the hilar and para-tracheal regions on the frontal cxr, supported by identification of subcarinal lymphadenopathy on the lateral cxr, represent a useful surrogate marker of tb at relatively low cost. however, sensitivity and specificity for identifying lymphadenopathy on cxr in children is relatively poor with significant inter-observer variation in the interpretation of radiographs, complicated further by poor quality of radiographs. affecting both accuracy and observer agreement is the lack of standardized imaging criteria and lymph nodes sizecriteria for a positive diagnosis of primary tb. attempts are therefore being made to establish 'objective' chest radiograph signs backed up by a standard set of images as a guide. ultrasound is an especially attractive imaging alternative to cxr as it does not involve radiation or require sedation and because it is relatively cheap and mobile. ultrasound of the mediastinum has been used to detect mediastinal lymphadenopathy and can also be used to detect extrapulmonary tb through abdominal imaging, at the same sitting. it is particularly useful in rural settings where no other imaging is available. the ability to store digital ultrasound images and cine-loops also enables teleradiology support by expert interpretation and opinion, from a distance. computed tomography (ct) and magnetic resonance imaging (mri) are obvious diagnostic imaging considerations that will improve diagnostic accuracy of primary tb, but the radiation dose in ct, the need for anesthesia in mri, the limited availability and high cost are real barriers to their clinical utility. mri is preferred to ct because it does not involve ionizing radiation. however, the disadvantages of mri for lung imaging (poor signal generated from the air in the lungs and movement artefacts from breathing), the cost and the requirement for the child to keep still for a prolonged period (requiring anesthesia) have slowed its use in thoracic infections. yet, whole body mri, including thoracic imaging is mainstream for detecting lymphadenopathy in childhood lymphoma. the preferred imaging technique varies with the suspected pathology and available equipment. dynamic imaging techniques such as inspiratory/ expiratory cxr, fluoroscopy, and inspiratory/expiratory or cine ct permit the lungs and airways to be imaged at different phases of the respiratory cycle. inspiratory/expiratory cxr and inspiratory/expiratory chest ct have long been the preferred initial imaging methods for detecting foreign body aspiration or bronchiolitis obliterans, respectively, on the basis of air trapping rather than direct visualization of the airway obstruction. fluoroscopy has historically been the preferred noninvasive method for diagnosing tracheobronchomalacia due to its ease of performance, even in uncooperative patients, and its high specificity, but it is limited by its subjective interpretation, low sensitivity, poor depiction of the paratracheal structures, and inability to simultaneously display the anteroposterior and lateral walls of the airway and quantify luminal cross-sectional area 1 . in infants and children too young to comply with breath-hold instructions, inspiratory/expiratory phases can be simulated by imaging during right/left lateral decubitus or prone/supine positioning. controlled-ventilation ct under sedation or anesthesia also permits inspiratory/expiratory imaging of the lungs and airways in uncooperative patients. dynamic cine ct technique allows the airways to be imaged sequentially during successive phases of the respiratory cycle, but coverage was initially limited to short (4 cm or less) segments of the airway, resulting in sampling misregistration and preventing synchronous evaluation of the true extent and severity of airway collapse during the same phase of the respiratory cycle 1 . made possible by recent technologic advances including more rapid gantry rotation and wider detector arrays (up to 16 cm craniocaudal coverage), dynamic volumetric cine ct now allows all or nearly all of the lungs and central airways to be imaged rapidly and sequentially throughout the respiratory cycle without the need for sedation or intubation. this technique is capable of providing multiplanar, 3d and 4d information about the airways during normal tidal breathing or forced expiratory maneuvers, as well as depicting the relationship of the airways to the adjacent vasculature if intravenous contrast is administered 2 . with dynamic volumetric cine ct, intrinsic and extrinsic causes of airway narrowing can be distinguished and fixed airway stenosis can be differentiated from expiratory central airway collapse due to tracheobronchomalacia (softening of tracheobronchial cartilage) or excessive dynamic airway collapse (inward bulging of the posterior membrane) 3 . tracheobronchomalacia is primary (congenital) in approximately 1/2100 children and often resolves in isolated mild to moderate cases by 2 years of age as the cartilage geometry and composition matures and posterior membrane tone develops. tracheobronchomalacia is often accompanied by gastroesophageal reflux disease and is associated with other foregut anomalies, especially esophageal atresia and tracheoesophageal fistula. tracheobronchomalacia can be secondary to extrinsic compression, chronic airway inflammation, intubation, or positive pressure ventilation and is identified in about onefourth of children with chronic respiratory symptoms or signs such as wheezing, barking cough, recurrent respiratory tract infection, apnea, cyanotic spells, or difficulty weaning from respiratory support 4 . tracheobronchomalacia was originally defined as >50% reduction in airway cross-sectional diameter during coughing, but false positives are very common with this definition, especially for the bronchi in which physiologic expiratory airway narrowing is more pronounced than for the trachea. the shape and cross-sectional area of the airway lumen can be precisely determined by ct, but there is no current consensus on the optimal threshold degree of expiratory airway collapse for a diagnosis of tracheobronchomalacia among children of varying ages with or without coexisting lung disease during either tidal breathing or forced expiration. expiratory collapse of normal airways can occur in the setting of obstructive lung disease such as asthma or bronchopulmonary dysplasia due to increased pleural pressure and increased peripheral airways resistance that reduces airway transmural pressure 4 . dynamic volumetric cine ct provides objective information to classify expiratory central airway collapse according to the femos (functional status, extent, morphology, origin, severity) system 5 , but it should be noted that the degree of luminal narrowing is only one factor in airflow limitation. evidence of airway compression or expiratory collapse on imaging does not necessarily indicate a condition requiring therapeutic intervention, and correlation with the clinical symptoms, signs, risk factors, and pulmonary function tests is necessary to determine the functional significance 1, 4 . in addition to the noninvasive nature, the advantages of dynamic volumetric cine ct over bronchoscopy include the ability to directly evaluate for vascular structures or soft tissue masses that impinge on the airway, depict the airways distal to a narrowing impassable by bronchoscope, and assess the lung parenchyma for conditions such as air trapping that may be associated with dynamic central airway collapse 2 . a disadvantage of ct is the exposure to ionizing radiation. for perspective, dynamic airway ct incurs a radiation dose similar or less to than that from a year of natural background radiation exposure 6 . dynamic cine magnetic resonance imaging (mri) avoids exposure to ionizing radiation and is capable of imaging the central airways and vasculature 7 , but is limited by a longer scan time, more frequent need for sedation/anesthesia and less detailed depiction of the lung parenchyma compared to ct. additional studies in children are needed to determine how the anatomic and functional information provided by dynamic ct is best applied to the diagnosis, treatment planning, and post-therapeutic monitoring of pediatric airway disorders. the main driving force to develop sophisticated mri sequences for pediatric chest imaging is that mri is a radiation-free technique. this is especially important for children who are more sensitive to ionizing radiation than adults [8] . this justifies the use of chest mri for short-and long-term follow-up of chronic lung diseases such as cystic fibrosis (cf), so as to reduce the lifelong cumulative radiation dose [1] . chest mri is challenging because of the magnetic heterogeneous environment in the chest region [2] . lung parenchyma is a low proton density structure and hence has a reduced signal-to-noise ratio [3] . in addition, the numerous airtissue interfaces within a voxel induce strong localized microscopic magnetic field gradients, which produce extensive mri signal dephasing leading to extremely short t2 star (t2 ã ) and geometric distortions. these effects become stronger at higher magnetic field strengths (i.e. 3 t), which are increasingly used in clinical settings for enhanced signal-to-noise ratio [1] . however, signalto-noise ratio in cases of lung pathology, such as pneumonia, edema, tumors and atelectasis, is increased by higher fluid content and amount of tissue. these conditions result in higher proton density and improved visualization [4] . moreover, mri has the advantage of integrating anatomical and functional information in a single examination, a possibility not as readily available with other imaging modalities. mri can provide functional information regarding lung perfusion using gadolinium contrast [5] , lung mechanics using dynamic acquisitions [6] , and ventilation using inhaled hyperpolarized gases [7] , oxygen enhancement or dynamic motion-based methods [8] . moreover, dwi is able to give new insight in the management of pneumonia, especially in cf patients [9] . chest mri has reached the point where it can be used in routine clinical practice. although mri cannot yet be compared to ct for anatomical detail, new sequences allow acquisition of lung images with high diagnostic quality in less than 15 s, which makes mri feasible in a clinical setting. mri can be considered an alternative to ct for the diagnosis of lung diseases and for monitoring response to treatment in pediatric lung disease. moreover, in some diseases that require long-term follow-up, such as cystic fibrosis, mri can play an important role in reducing lifelong radiation exposure related to repeated ct scans. furthermore, mri has the ability to offer functional information: information regarding lung mechanics, perfusion and ventilation can provide new insight in different pediatric lung diseases. this functional information can not only improve our understanding with regard to the pathophysiology of pediatric lung diseases, it can also open new diagnostic and therapeutic options. obstructive sleep apnea syndrome (osas) is characterized by prolonged partial airway obstruction and/or intermittent complete obstruction (obstructive apnea) during sleep, affecting about 2% to 3% of children [1] . osas is a complex syndrome with multiple etiologic factors: the main causative factor is adenotonsillar hypertrophy while other conditions, such as craniofacial dysmorphism, obesity, hypotonic neuromuscular diseases, despite inducing reduction of the caliber of the upper airways, are commonly mistreated [2] . adenotonsillectomy has been considered for many years the only treatment in children with osas although its efficacy remains uncertain, depending on the severity and on the presence of other co-morbidities, [3] . since a residual osa is reported in a large proportion of children after adenotonsillectomy [3] , and children with osa display a complex phenotype (mild or major craniofacial anomalies, and/or comorbid obesity, and/or adenotonsillar enlargement), a multi-therapeutic approach to pediatric osas and a defined timing of therapy are required [3, 4] . a narrow upper airway accompanied by maxillary constriction and mandibular retrusion is commonly reported in children with osas [5] . the skeletal conformation showing hyperdivergent skeletal growth pattern associated with posterior displacement of the tongue base, increases the upper airway narrowing and craniomandibular, intermaxillary, goniac and mandibular angles leading to a high-arched (ogival) palate [6] . rapid maxillary expansion (rme) is the most common dento-facial orthopedic procedure used in young patients to treat maxillary transverse deficiencies, starting up to 4 years of age. recently, it has been demonstrated to be efficacious to treat osas in children with a narrow palate and malocclusion: a significant reduction in the apnea-hypopnea index and in diurnal symptoms after six months of therapy with rme [7] , and positive long-term effects in children with osa and malocclusions treated with rme have been reported [8] . similar results were obtained after one year of treatment with rme in 16 preschool and school-aged non obese children with osas and dental malocclusions with a significant drop in clinical symptoms as well as apnea-hypopnea index [8] . this study also demonstrated that starting treatment early when the bone is still extremely plastic and its growth rate is maximum increases the percentage of success of rme treatment. a two-year follow up after the end of the rme application was performed in the same population of children confirming a stable decrease in apnea-hypopnea index, an increase of mean overnight oxygen saturation and a persistent improvement in clinical symptoms [9] . finally, a recent randomized study showed preliminary results regarding the effect of rme applied before adenotonsillectomy compared to the effect of rme applied after surgery, in children with osa. no significant differences between the two different approaches were described [10] . in conclusion, orthodontic treatment is a valid treatment for osa, improving clinical symptoms, respiratory parameters measured during psg with long lasting effect. the widening of the maxilla, the corrections of dental malocclusions and the correct relationships between maxillary and mandibular arches with respect to the anterior cranial base, are the main craniofacial changes induced by rme that may explain the efficacy of orthodontic therapy. orthodontic therapy should be encouraged in pediatric osas, and an early approach may permanently modify nasal breathing and respiration, thereby preventing obstruction of the upper airway. towards the turn of the century, david gozal's group published a series of papers that raised important questions. in a sample of 297 1 st grade pupils whose school performance was in the lowest decile of their class ranking, they found that 18% had sleep-associated intermittent hypoxia and/or hypercapnia; school performance improved in those whose parents had opted for adenotonsillectomy (1) . they then showed that 13% of 14-15 year olds with poor school performance had parent-reported snoring at age 4-5, compared to only 5% among those with good school performance (2). finally, a group of first graders with snoring, but no obstructive sleep apnea, i.e. an obstructive apnea index <1, performed worse on measures related to attention, social problems and visuospatial function than non-snorers, suggesting that simple snoring may not be as benign as hitherto widely believed (3) . against this background, we set out to perform the hannover study on sleep apnea in childhood (hassac), a community-based cross-sectional study on several aspects of sleep-disordered breathing (sdb) in 1144 primary school children incorporating a two-phase sequential screening procedure: participants were screened for symptoms and signs of sdb using an sdbquestionnaire and home pulse oximetry (hpo), those with outlying results on either screening method subsequently underwent an abbreviated home polysomnography (hpsg) for a final diagnosis of obstructive sleep apnea syndrome (osas). overall, participants were representative of the underlying population of third-graders in the study region. we found that 10.1% of this cohort were habitual snorers, while the population prevalence for osas was 2.8% (4, 5) . we then wanted to know how these symptoms affected behavior and academic achievements. for this, we used parental questionnaires and collected teachers' ratings, and defined poor school performance as grade 4 or worse in the last school report form, or requirement for special assistance, with this classification roughly corresponding to the lowest quintile of a class. we found that children with habitual snoring, compared to those who never snored, had 3-10 times the odds for daytime symptoms such as hyperactivity, difficulty concentrating, falling asleep while watching tv or at school or having peer problems, and 2-3 times the odds for poor school performance in mathematics, science and spelling (4, 6) . there was a clear dose-effect gradient, i.e. the proportion of children with poor school performance increased with increasing frequency of parent-reported snoring. considering its high prevalence, and assuming a causal link to disturbed behavior, habitual snoring appeared to be a substantial public health problem in primary school children. given this association, we wanted to know how this is mediated, i.e. whether this is mainly through detrimental effects of intermittent hypoxemia or more likely due to recurrent arousal. contrary to our hypothesis, the increased odds for poor school performance or daytime symptoms associated with habitual snoring stayed the same once children exhibiting intermittent desaturation in their overnight pulse oximetry recording had been excluded, suggesting that even so-called benign snoring, i.e. snoring without hypoxemia, may in fact not be benign. if not via intermittent desaturation, could the relationship with poor school performance be mediated via frequent arousals elicited by recurrent obstructive apnea? to address this question, we took advantage of the fact that children with an abnormal questionnaire score in our hassac study also underwent hpsg. thus, we re-analyzed our data on the relationship of snoring with daytime symptoms and poor school performance after excluding all children with a mixed-obstructive apnea/hypopnea index (maohi) !0.5, but again, the risk for poor school performance was not reduced among snorers after excluding those with recurrent apneas. given that simple snoring has such a strong association with daytime symptoms à are these reversible? in our hassac study, we could collect 1year follow-up data in 82 snorers and 80 controls. among these, 42 snorers (51%) had stopped snoring. while their scores for emotional problems, hyperactivity and problems with peers improved, their school performance did not (6) . this is in line with other data suggesting that reduced scores in executive functioning and iq seen in children prior to adenotonsillectomy may not improve following this operation (7) . similarly, in the avon longitudinal study on parents and children (alspac), even those whose sdb symptoms peaked at age 30 months and abated thereafter still had almost twice the odds for hyperactivity and 60% higher odds for behavioral problems at age 7 years (8). taken together, there is now a growing body of evidence that frequent snoring in children may not be as benign as previously thought, but may instead be associated with impaired behavior and poor academic achievements. these problems may even persist after snoring ceases, which à if these statistical associations were confirmed as causal à would argue for their early recognition and treatment. here, it is encouraging to see that in another longitudinal study on snoring and daytime symptoms, the proportion of children who did not snore at age 2 and 3 years was 42% in those who were breastfed for less than 1 month, but 83% in those who were breastfed for 12 months or longer (9) , suggesting that breastfeeding may reduce the risk of snoring during early childhood. in addition, given the limited availability of sleep labs, we urgently need better and easier-toperform screening methods to identify those who may need treatment for their snoring, e.g. in whom poor school performance can be predicted from a screening test (10) . also, interventions such as nasal steroids, montelukast or orthodontic treatment may deserve further study. diagnosis of osas using home respiratory polygraphy (hrp). alonso-alvarez et al. 3 prospectively assessed the diagnostic reliability of hrp in children aged 2 to 14 years with a clinical suspicion of osas. they found a sensitivity of 91% and a specificity of 94% and concluded that hrp emerges as a potentially useful and reliable approach for the diagnosis of moderate/ severe osas in children. drug-induced sedation endoscopy (dise) aims to reproduce upper airway obstruction during sleep and is gaining increasing popularity, with the hope of guiding efficient surgery and cure osdb children. in a meta-analysis, galluzi et al. 4 concluded that dise may benefit a minority of children with osas, and should only be used in children with unremarkable clinical evaluation or upon persistent osas after at. obstructive sleep-disordered breathing and obesity pathogenesis of osas in obese adolescents. literature on the pathogenesis of osas in adolescents is very limited. schwab et al. 5 prospectively compared upper airway magnetic resonance imaging in 137 adolescents aged 12 to 16 years. results indicated that lymphoid tissue, rather than other soft tissue components (tongue, lateral pharyngeal walls, parapharyngeal fat pads), are the primary upper airway anatomical risk factors for osas. while the pathogenesis of osas is clearly multifactorial (e.g., decreased upper airway reflexes in osas obese adolescents) and often require additional treatment, the results are clinically important since they suggest that at should still be considered as the first-line treatment in adolescents with osas. osdb and metabolic syndrome. in a systematic assessment of the literature on the interactions between sleep, osdb, obesity and disruptions of metabolic homeostasis in children and adolescents, hakim et al. concluded that obesity and osdb appear to contribute to the initiation and progression of each other, and that both are linked to the metabolic phenotype 6 . one intriguing mechanism postulates that osdb/ disrupted sleep as well as other factors favoring obesity, such as high-fat/ fructose diet, disrupt the gut microbiome and lead to increased systemic levels of lipopolysaccharides, in turn promoting inflammation and metabolic dysfunction. treatment of sleep-disordered breathing in children watchful waiting. chervin et al. 7 followed 192 children aged 5 to 9 years with mild/moderate osas after seven months of watchful waiting only. they found resolution of osas in 42% of the children. independent predictors of resolution were lower ahi and normal waist circumference. the authors concluded that, in practice, a baseline low ahi and normal waist circumference, or low pediatric sleep questionnaire and snoring score, may help identify an opportunity to avoid at. myofunctional therapy (mt). camacho et al. 8 performed a meta-analysis of the use of mt as a treatment for osas in adults and children. although the total number of patients (especially children, n ¼ 25) was low, the effects were highly significant. overall, mt decreased ahi by 50-60% in pediatric and adult patients. in children, a positive effect was reported when used as the only treatment in mild osas as well as to consolidate osas cure after at þ rapid maxillary expansion. the authors concluded that mt could be an adjunct to other osas treatments in patients of all ages. evolution of obstructive sleep-disordered breathing in children evolution in preschool children with osdb. walter et al. 9 investigated the long-term evolution of osdb in preschool-aged children with normal weight. half of the preschoolers with osdb were treated, most often by adenoidectomy and/or tonsillectomy. overall, osdb resolved in half of the children, either spontaneously (35%) or with treatment (57%). however, 40% still had osas, similarly to observations in school-aged children. intriguingly, complete resolution of osdb at three years post-treatment was more likely in preschoolers with moderate/severe osas compared to those with mild osas or primary snoring. long-term evolution of osas. spilsbury et al. 10 reported results on both remission and incidence of osas in 490 participants who underwent psg at 8-11 and 16-19 years of age. the authors first observed that osas in middle childhood usually remitted by adolescence. secondly, while habitual snoring and obesity predicted osas at each time point, distinct additional risk factors for osas were found in middle childhood vs. adolescence. hence, prematurity, a disadvantaged neighborhood or african-american origin also predicted osas in middle childhood, while risk factors in adolescents included male sex and previous at. finally, obesity, but not habitual snoring, in middle childhood predicted adolescent osas. these results confirm that prevention and treatment of obesity appears of utmost importance in the fight against pediatric osas. evolution of osas after treatment. lee et al. 11 performed a meta-analysis of psg findings after at for osas in 3413 obese and non-obese children. the overall success rate was 51% for postoperative ahi < 1 event /h (34% for obese vs. 49% for non-obese). postoperative ahi was positively correlated with ahi and body mass index before surgery. the authors concluded that residual osas after at persists in about 50% of children, especially in children with severe osas and obesity. a plea to abandon asthma as a disease concept asthma endotypes: a new approach to classification of disease entities within the asthma syndrome interpretative strategies for lung function tests applicability of the global lung function spirometry equations in contemporary multiethnic children impact of ethnicity and extreme prematurity on infant pulmonary function interpretation of pediatric lung function: impact of ethnicity multi-ethnic reference values for spirometry for the 3-95-yr age range: the global lung function 2012 equations lung function in children in relation to ethnicity, physique and socioeconomic factors disparities in pulmonary function in healthy children across the indian urban-rural continuum etiology of ethnic differences in childhood spirometry age and height dependence of lung clearance index and functional residual capacity ethnic differences in fraction of exhaled nitric oxide and lung function in healthy young children stitching and switching: the impact of discontinuous lung function reference equations influence of socioeconomic status on lung function and prediction equations in indian children ethnic differences in adolescent lung function: anthropometric, socioeconomic, and psychosocial factors the use of ethnically specific norms for ventilatory function in african-american and white populations lung function in the children of immigrant and uk-born south-asian mothers the global lung function initiative: dispelling some myths of lung function test interpretation addition to inhaled corticosteroids of longacting beta2-agonists versus anti-leukotrienes for chronic asthma. the cochrane database of systematic reviews pediatric severe asthma is characterized by eosinophilia and remodeling without t(h)2 cytokines novel concepts in airway inflammation and remodelling in asthma long-term effectiveness of a staged assessment for paediatric problematic severe asthma the utility of a multidomain assessment of steroid response for predicting clinical response to omalizumab asthma genetics and personalised medicine. the lancet. respiratory medicine intermittent montelukast in children aged 10 months to 5 years with wheeze (wait trial): a multicentre, randomised, placebo-controlled trial endotyping asthma: new insights into key pathogenic mechanisms in a complex, heterogeneous disease a genome-wide association study identifies cdhr3 as a susceptibility locus for early childhood asthma with severe exacerbations a large-scale, consortium-based genomewide association study of asthma effect of 17q21 variants and smoking exposure in early-onset asthma chromosome 17q21 gene variants are associated with asthma and exacerbations but not atopy in early childhood molecular evolution of the cadherin superfamily altered expression of epithelial junctional proteins in atopic asthma: possible role in inflammation cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication human rhinovirus species c infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions bacterial strain-specific induction of foxp3þ t regulatory cells is protective in murine allergy models microbial exposure during early life has persistent effects on natural killer t cell function a microbiota signature associated with experimental food allergy promotes allergic sensitization and anaphylaxis epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes sheikh a; eaaci food allergy and anaphylaxis guidelines group. eaaci food allergy and anaphylaxis guidelines. primary prevention of food allergy european cystic fibrosis society standards of care: best practice guidelines european cystic fibrosis society standards of care: framework for the cystic fibrosis centre european best practice guidelines for cystic fibrosis neonatal screening evidence for newborn screening for cystic fibrosis neonatal screening for cystic fibrosis in wales and the west midlands: clinical assessment after five years of screening nutritional benefits of neonatal screening for cystic fibrosis. wisconsin cystic fibrosis neonatal screening study group bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis economic implications of newborn screening for cystic fibrosis: a cost of illness retrospective cohort study the diagnosis of cystic fibrosis: a consensus statement h cuppens, and m sinaasappel on behalf of the diagnostic working group. cystic fibrosis: terminology and diagnostic algorithms recommendations for the classification of diseases as cftr-related disorders spectrum of cftr mutations in cystic fibrosis and in congenital absence of the vas deferens in france variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign mutations of the cystic fibrosis gene in patients with chronic pancreatitis cftr gene mutations in adults with disseminated bronchiectasis consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice cftr cftr biomarkers: time for promotion to surrogate endpoint? genotype-phenotype correlations in cystic fibrosis primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children structural and functional lung disease in primary ciliary dyskinesia differences in disease expression between primary ciliary dyskinesia and cystic fibrosis with and without pancreatic insufficiency progression of lung disease in primary ciliary dyskinesia: is spirometry less accurate than ct? longitudinal lung function and structural changes in children with primary ciliary dyskinesia evaluation of pulmonary disease using static lung volumes in primary ciliary dyskinesia lung function in patients with primary ciliary dyskinesia: a cross-sectional and 3-decade longitudinal study management of primary ciliary dyskinesia in european children: recommendations and clinical practice primary ciliary dyskinesia: diagnostic and phenotypic features on behalf of the ecfs patient registry steering group. factors associated with fev1 decline in cystic fibrosis: analysis of the data of the ecfs patient registry mucus properties in children with primary ciliary dyskinesia: comparison with cystic fibrosis changes in airway inflammation during pulmonary exacerbations in patients with cystic fibrosis and primary ciliary dyskinesia exhaled breath analysis using electronic nose in cystic fibrosis and primary ciliary dyskinesia patients with chronic pulmonary infections committee on fetus and newborn. postnatal corticosteroids to treat or prevent chronic lung disease in preterm infants committee on fetus and newborn. policy statement-postnatal corticosteroids to prevent or treat bronchopulmonary dysplasia early intratracheal instillation of budesonide using surfactant as a vehicle to prevent chronic lung disease in preterm infants: a pilot study intratracheal budesonide supplementation in addition to surfactant improves pulmonary outcome in surfactant-depleted newborn piglets surfactant and corticosteroid effects on lung function in a rat model of acute lung injury introduction to marangoni convection inhaled versus systemic corticosteroids for preventing chronic lung disease in ventilated very low birth weight preterm neonates early inhaled glucocorticoid therapy to prevent bronchopulmonary dysplasia intratracheal administration of budesonide/surfactant to prevent bronchopulmonary dysplasia plethysmographic measurements of lung volume and airway resistance ers/ats task force on standards for infant respiratory function testing raised volume forced expirations in infants: guidelines for current practice statement: pulmonary function testing in preschool children an official american thoracic society workshop report: optimal lung function tests for monitoring cystic fibrosis, bronchopulmonary dysplasia, and recurrent wheezing in children less than 6 years of age new reference ranges for interpreting forced expiratory manoeuvres in infants and implications for clinical interpretation: a multicentre collaboration multi-ethnic reference values for spirometry for the 3-95-yr age range: the global lung function 2012 equations reference ranges for interrupter resistance technique: the asthma uk initiative respiratory impedance and bronchodilator responsiveness in healthy children aged 2 to 13 years consensus statement for inert gas washout measurement using multiple-and single-breath tests multiple-breath washout as a lung function test in cystic fibrosis. a cystic fibrosis foundation workshop report plethysmographic measurements of specific airway resistance in young children chronic lung disease after premature birth value of echocardiography in assessing the outcome of bpd pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era pulmonary vascular response to to oxygen in infants with severe bpd pulmonary vascular extraction of circulating norepinephrine in infants with bpd histologic evidence of intrapulmonary arteriovenous shunt vessels in premature infants with severe bpd pulmonary vascular disease in bpd: physiology, diagnosis and treatment bronchopulmonary dysplasia: nhlbi workshop on the primary prevention of chronic lung diseases guidelines on the early management of infants diagnosed with cystic fibrosis following newborn screening cystic fibrosis foundation evidence à based guidelines for management of infants with cystic fibrosis recommandations nationales pour la prise en charge du nourrisson d epist e atteint de mucoviscidose. consensus de la f ed eration des centres de ressources et de comp etences de la mucoviscidose growth and pulmonary outcomes during the first 2 years of life of breastfed and formula-fed infants diagnosed with cystic fibrosis through the wisconsin routine screening program do infants with cystic fibrosis need a protein hydrolysate formula? a prospective, randomized, comparative study evaluation of salt supplementation in cystic fibrosis infants induced sputum compared to bronchoalveolar lavage in young, non-expectorating cystic fibrosis children risk factors for bronchiectasis in children with cystic fibrosis palivizumab for prophylaxis against respiratory syncytial virus infection in children with cystic fibrosis palivizumab immunoprophylaxis effectiveness in children with cystic fibrosis the etiologies of non-cfbronchiectasis in childhood: a systematic review of 989 subjects ct of airways disease and bronchiectasis an investigation into causative factors in patients with bronchiectasis rapid identification of bacterial species associated with bronchiectasis via metagenomic approach a novel microbiota stratification system predicts future exacerbations in bronchiectasis prevalence of nontuberculous mycobacteria in patients with bronchiectasis: a meta-analysis efficacy of azithromycin in the treatment of bronchiectasis azithromycin for prevention of exacerbations in non-cystic fibrosis bronchiectasis (em-brace): a randomised, double-blind, placebo-controlled trial respiratory exacerbations in indigenous children from two countries with noncystic fibrosis chronic suppurative lung disease/bronchiectasis localised pulmonary resection for bronchiectasis in hypogammaglobulinaemic patients autonomic regulation of the airways regulation of airway inflammation and remodeling by muscarinic receptors: perspectives on anticholinergic therapy in asthma and copd treatment of disorders characterized by reversible airway obstruction in childhood: are anticholinergic agents the answer? the airway cholinergic system: physiology and pharmacology tiotropium bromide (ba 679 br), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease pulmonary neuronal m2 muscarinic receptor function in asthma and animal models of hyperreactivity anticholinergics in the treatment of children and adults with acute asthma: a systematic review with metaanalysis the development of anticholinergics in the management of copd tiotropium in asthmatic adolescents symptomatic despite inhaled corticosteroids: a randomised dose-ranging study treatment of disorders characterized by reversible airway obstruction in childhood: are anti-cholinergic agents the answer? definition, assessment and treatment of wheezing disorders in preschool children: an evidence-based approach mechanisms of virus induced exacerbations of asthma efficacy of salbutamol and ipratropium bromide in the management of acute bronchiolitis--a clinical trial bronchodilator effect of inhaled ipratropium bromide in wheezy toddlers anticholinergic drugs for wheeze in children under the age of two years classification and pharmacological treatment of preschool wheezing: changes since 2008 effect of altering smooth muscle tone on maximal expiratory flows in patients with tracheomalacia tracheobronchomalacia in pediatric patients: clinical experience quantitative assessment of tracheal collapsibility in infants with tracheomalacia tiotropium add-on therapy in adolescents with moderate asthma: a 1-year randomized controlled trial tiotropium use in pediatric patients with asthma or chronic cough: a case series the asthma-copd overlap syndrome multi-ethnic reference values for spirometry for the 3-95 year age range: the global lung function 2012 equations the global lung initiative 2012 reference values reflect contemporary australasian spirometry comparison between reference values for fvc, fev 1 , and fev 1 /fvc ratio in white adults in brazil and those suggested by the global lung function initiative 2012 the recent multi-ethnic global lung initiative 2012 (gli 2012 ) reference values don't reflect contemporary adult's north african spirometry spirometry reference values for malagasy adults aged 18-73 years the influence of the reference values on the interpretation of lung function in children: comparison of the global lung initiative 2012 and polish 1998 reference values disparities in pulmonary function in healthy children across the indian urban-rural continuum persistent effects of maternal smoking during pregnancy on lung function and asthma in adolescents how "healthy" should children be when selecting reference samples for spirometry? markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma airway remodeling and inflammation in symptomatic infants with reversible airflow obstruction structural changes in the bronchial mucosa of young children at risk of developing asthma childhood asthma and increased airway responsiveness: a relationship that begins in infancy reduced lung function at birth and the risk of asthma at 10 years of age lung function, airway remodelling and inflammation in symptomatic infants: outcome at 3 years lung function, airway remodeling, and inflammation in infants: outcome at 8 years increased airway smooth muscle in preschool wheezers who have asthma at school age european association of perinatal medicine. european consensus guidelines on the management of neonatal respiratory distress syndrome in preterm infants-2013 update surfactant and noninvasive ventilation nichd maternal-fetal medicine units network. antenatal betamethasone for women at risk for late preterm delivery a comprehensive approach to the prevention of bronchopulmonary dysplasia nasal intermittent positive-pressure ventilation vs. nasal continuous positive airway pressure for preterm infants with respiratory distress syndrome: a systematic review and meta-analysis a trial comparing noninvasive ventilation strategies in preterm infants nasal continuous positive airway pressure versus nasal intermittent positive ventilation for preterm neonates: a systematic review and meta-analysis early surfactant administration with brief ventilation vs. selective surfactant and continued mechanical ventilation for preterm infants with or at risk for respiratory distress syndrome avoidance of mechanical ventilation by surfactant treatment of spontaneously breathing preterm infants (amv): an open-label, randomised, controlled trial high-flow nasal cannulae in very preterm infants after extubation respiratory syncytial virus infection and bronchiolitis vertical transmission of respiratory syncytial virus modulates pre-and postnatal innervation and reactivity of rat airways the role of neurotrophins in inflammation and allergy rsv and asthma: speed-dating or long-term relationship? vertical transmission of respiratory syncytial virus to fetuses in utero conveys elevated nerve growth factor expression and airway hyperreactivity upon repetitive post-natal rsv re-challenge infections fetal nutrition and cardiovascular disease in adult life alternative mechanisms for respiratory syncytial virus (rsv) infection and persistence: could rsv be transmitted through the placenta and persist into developing fetal lungs? maternal high-fat diet in pregnancy results in metabolic and respiratory abnormalities in offspring microrna-221 modulates rsv replication in human bronchial epithelial cells by targeting ngf expression less air pollution leads to rapid reduction of airway inflammation and improved airway function in asthmatic children child pneumonia at a time of epidemiological transition incidence and severity of childhood pneumonia in the first year of life in a south african birth cohort: the drakenstein child health study global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis early life origins of chronic obstructive pulmonary disease wheezing rhinovirus illnesses in early life predict asthma development in high-risk children childhood pneumonia: the role of viruses specimen collection for the diagnosis of pediatric pneumonia laboratory methods for determining pneumonia etiology in children community-acquired pneumonia among u. s. children respiratory viruses associated with communityacquired pneumonia in children: matched case-control study global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis a preliminary study of pneumonia etiology among hospitalized children in kenya the effect of case management on childhood pneumonia mortality in developing countries highly prevalent clinical issues infant mortality statistics from the 2006 period linked birth/infant death data set late preterm infants: near term but still in a critical developmental time period consortium on safe labor. respiratory morbidity in late preterm births nichd maternal-fetal medicine units network. antenatal betamethasone for women at risk for late preterm delivery growth rate of lung function in healthy preterm infants effect of late preterm birth on longitudinal lung spirometry in school age children and adolescents moderately preterm children have more respiratory problems during their first 5 years of life than children born full term risk of asthma in late preterm infants: a propensity score approach poor airway function in early infancy and lung function by age 22 years: a non-selective longitudinal cohort study overview of issues in the longitudinal analysis of respiratory data assistant professor, department of pediatrics, school of medicine, university of costa rica. <quiquesoto@gmail.com> bibliography 1. global tuberculosis report 2013. geneva. world health organization. 2013. 2. world health organization who world health organization who. guidance for national tuberculosis programmes on the management of tuberculosis in children a refined symptom-based approach to diagnose pulmonary tuberculosis in children proportion of tuberculosis transmission that takes place in households in a high-incidence area assessment of the xpert mtb/rif assay for diagnosis of tuberculosis with gastric lavage aspirates in children in sub-saharan africa: a prospective descriptive study evaluation of the xpert mtb/rif assay at a tertiary care referral hospital in a setting where tuberculosis and hiv infection are highly endemic treatment of paediatric tb: revised who guidelines immune-based diagnostics for tb in children: what is the evidence? paediatric respiratory reviews interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review systematic review: t-cell-based assays for the diagnosis of latent tuberculosis infection: an update tracheobronchomalacia in children: review of diagnosis and definition dynamic volume cta of the airway and vasculature in children: technical report tracheobronchomalacia and excessive dynamic airway collapse paediatric tracheomalacia description of a multidimensional classification system for patients with expiratory central airway collapse comparison of standard-dose and reduced-dose expiratory mdct techniques for assessment of tracheomalacia in children real-time, cine magnetic resonance imaging for evaluation of the pediatric airway general hospital ca' foncello, treviso (italy) à <gmorana61@gmail.com> 2 department of radiology, sophia children's hospital mri of the lung (2/3). why ... when ... how? mr imaging of pulmonary parenchyma impact of lung volume on mr signal intensity changes of the lung parenchyma /3): methods contrast-enhanced 3d mri of lung perfusion in children with cystic fibrosis-initial results spirometer-controlled cine magnetic resonance imaging used to diagnose tracheobronchomalacia in paediatric patients imaging of lung function using hyperpolarized helium-3 magnetic resonance imag-ing: review of current and emerging translational methods and ap-plications epidemiology of pediatric obstructive sleep apnea upper airway collapsibility in children with obstructive sleep apnea syndrome adenotonsillectomy outcomes in treatment of obstructive sleep apnea in children: a multicenter retrospective study pediatric obstructive sleep apnea: complications, management, and long-term outcomes craniofacial morphology in preschool children with sleep-related breathing disorder and hypertrophy of tonsils craniofacial modifications in children with habitual snoring and obstructive sleep apnoea: a case-control study randomized controlled study of an oral jaw-positioning appliance for the treatment of obstructive sleep apnea in children with malocclusion rapid maxillary expansion in children with obstructive sleep apnea syndrome: 12 months follow-up efficacy of rapid maxillary expansion in children with obstructive sleep apnea syndrome: 36 months of follow-up adeno-tonsillectomy and rapid maxillary distraction in pre-pubertal children, a pilot study germany <christian-f.poets@med.uni-tuebingen.de> references 1. gozal d. sleep-disordered breathing and school performance in children snoring during early childhood and academic performance at ages thriteen to fourteen years neurobehavioral implications of habitual snoring in children snoring, intermittent hypoxia and academic performance in primary school children population prevalence of obstructive sleep apnoea in a community of german third graders. the european respiratory journal: official journal of the european society for habitual snoring, intermittent hypoxia, and impaired behavior in primary school children adenotonsillectomy and neurocognitive deficits in children with sleep disordered breathing sleep-disordered breathing in a population-based cohort: behavioral outcomes at 4 and 7 years persistent snoring in preschool children: predictors and behavioral and developmental correlates predicting poor school performance in children suspected for sleep-disordered breathing utility of symptoms to predict treatment outcomes in obstructive sleep apnea syndrome pediatric osas: oximetry can provide answers when polysomnography is not available reliability of home respiratory polygraphy for the diagnosis of sleep apnea in children drug induced sleep endoscopy in the decision-making process of children with obstructive sleep apnea understanding the anatomic basis for obstructive sleep apnea syndrome in adolescents obesity and altered sleep: a pathway to metabolic derangements in children? childhood adenotonsillectomy trial. prognosis for spontaneous resolution of osa in children myofunctional therapy to treat obstructive sleep apnea: a systematic review and meta-analysis long-term improvements in sleep and respiratory parameters in preschool children following treatment of sleep disordered breathing remission and incidence of obstructive sleep apnea from middle childhood to late adolescence polysomnographic findings after adenotonsillectomy for obstructive sleep apnea in obese and non-obese children: a systemic review and meta-analysis this last year has seen a number of significant advances in the field of pediatric sleep-disordered breathing. the following is a personal selection of a few publications. overnight polysomnography (psg) is considered necessary to diagnose children suspected of sleep-disordered breathing (sdb). in practice, however, most children do not have access to overnight psg, due to the lack of sleep laboratories worldwide. the quest for a simpler means to diagnose sdb, or at least to prioritize children for referral to a sleep laboratory, remains a high priority. questionnaire. in a prospective study in children aged 5 to 9 years with obstructive sleep apnea syndrome (osas), rosen et al. 1 found that, conversely to psg, the pediatric sleep questionnaire results reflect osasrelated impairment in behavior, quality of life and sleepiness as well as predict their improvement post-adenotonsillectomy (at). the authors concluded that while psg is needed to diagnose osas, results from a careful clinical assessment provide important adjunctive information on comorbidities and their improvement after surgery. overnight oximetry in osdb children. kaditis et al. 2 performed a systematic analysis of the literature on the use of nocturnal oximetry in children with obstructive sdb (osdb ¼ from primary snoring to osas). their conclusion confirmed that overnight oximetry (spo 2 ) is useful for diagnosing osdb and for predicting post-at complications in a child with a history suggestive of osdb. overall, a desaturation index (!4%) higher than 2 episodes/hour can predict both mild and moderate-to-severe osdb, while criteria based on clusters of desaturation such as the mcgill oximetry score can predict moderate-to-severe osdb. key: cord-006862-5va1yyit authors: nan title: its asm 2012 date: 2012-11-04 journal: ir j med sci doi: 10.1007/s11845-012-0856-z sha: doc_id: 6862 cord_uid: 5va1yyit nan it is my great pleasure to welcome you to limerick, host city to the 2012 irish thoracic society annual scientific meeting. we look forward to a very exciting program, offering the best of original research and state of the art guest lectures against the backdrop of warmth and conviviality for which the meeting is known. thank you to all those who submitted abstracts and case studies this year-we received over 180 for presentation, reflecting the high quality and innovative work taking place in research centres throughout the island and further afield. i would also like to thank the abstract review committee and judges for their time and expertise in what is never an easy task due to the increasingly high standard of submissions received. special features of the meeting include guest lectures on 'asthma genomics and the respiratory biome', 'use of niv in acute respiratory failure' and 'the role of nasal electrophysiology in the difficult cf diagnosis' as well as a symposium on 'chronic respiratory failure'. i am delighted to welcome distinguished guest speakers from the uk and usa who will share their expertise on these topics. welcome also to the patient and professional organisations represented. networking and sharing information on the wealth of activities taking place across the respiratory healthcare community has become an integral part of the meeting. i would like to extend a particular welcome to the exhibitors and sponsors of this year's meeting. we are very grateful for their continued support, without which the meeting would not be possible. we present the case of an 18-year-old gentleman who had an inflammatory neuropathy secondary to a thymoma. he underwent a vats thymectomy, the first case in ireland. the surgery involved a 3 cm transverse incision above the supra-sternal notch, partial mobilization of the thymus, and 2-port placement with full thymic mobilization and excision. the advantages of a vats approach include lower blood loss (125 vs. 186 mls), lower post-operative ventilation rates (4.2 vs. 16.2 %), shorter lengths of hospitalisation (5.6 vs. 8.1 days), and improved cosmesis. the patient made an excellent recovery, discharged to home on post-operative day 4. we report a case of pleural giant b cell lymphoma in a 69 year old man induced by immunosuppressive therapy for rheumatoid arthritis (ra). despite adequate control of his ra with anti-tnf treatment, this was substituted with leflunomide and prednisolone after a myocardial infarction. he subsequently developed recurrent bloody exudative pleural effusions containing a lymphomatous cell population. prolonged remission was obtained with the anti cd20 antibody therapy, rituximab. although the attribution of lymphoma with anti-tnf therapy and immunosuppressive treatment remains controversial [1] [2] , the association is more than spurious [3] . despite a low risk of lymphoma from biological therapies, continued vigilance is warranted. pericardial atresia is a rare cardiothoracic malformation. it is mostly diagnosed incidentally, on surgery, or autopsy. it usually has benign symptoms, but herniation of the heart through a partial defect can be fatal. we describe an unusual case of 54 years old zimbabwean lady who presented with severe headache, syncope, bradycardia and orthostatic hypotension. she was also noted to have elevated d-dimers and was hypoxic on room air. a chest x-ray reported cardiomegaly. a ct pulmonary angiogram demonstrated no embolism, but did show features consistent with absence of the pericardium. further cardiac mri confirmed this finding in addition to left ward rotation of heart into left thoracic cavity and four chamber dilatation. a review of the available literature on clinical presentation, diagnostic assessment and therapeutic options is presented. and soft tissue, including primitive neuroectodermal tumors (pnets). it responds relatively well to a combination of surgical resection, chemotherapy and radiation therapy. we describe a 25 years old polish girl who presented with 01 year history of right scapular, paraspinal and anterior chest wall pain. imaging confirmed 5.4 9 4.3 cm mixed solid and cystic mass in right lung apex invading into supraclavicular muscles and neural foramen. the biopsy and immunotyping revealed small round ''blue cells'' soft tissue tumour equivalent of ees/pnet. she had neoadjuvant chemotherapy followed by surgical resection of the tumour with a good result and further planned to continue chemotherapy and evaluate treatment response. a 42 year old man with known kartagener's associated bronchiectasis presented with increased cough and sputum production associated with pyrexia. radiologically, he had a right lower lobe infiltrate but sputum culture and urinary legionella antigen were negative. however, he failed to improve on a penicillin. bronchoscopy was performed and lavage specimens grew legionella pneumophila serogroup 3. he recovered following treatment with levofloxacin. there was no history of foreign travel but he did sleep in a camper van. there are 15 known subgroups of legionella pneumophila and only serogroup 1 is detected by urinary antigen. culture remains the gold standard. a 41 year old lady was referred to the haematology service with a relapse of follicular lymphoma grade 3a. following salvage chemotherapy and pre transplant conditioning with alemtuzumab, she had a non myeloablative allogenic stem cell transplant. as part of the investigation for recurrent lower respiratory tract infections a bronchoscopy revealed a large exophytic tumour occluding the anterior orifice of the left upper lobe. biopsy and culture confirmed mycobacterium kansasii. the patient had an excellent clinical, microbiological and radiological response to treatment with resolution of the tumour at bronchoscopy. our case demonstrates an unusual presentation of mycobacterium kansasii. atypical mycobacterial infections although rare must be considered in this patient group. a 35-year-old woman presented in symptomatic hypercapnic respiratory failure. proximal muscle and neck flexor weakness was noted on examination. pulmonary function testing revealed restrictive physiology. creatinine kinase and serological testing were normal and acid-maltase deficiency was excluded. deltoid muscle biopsy was consistent with myofibrillar myopathy. myofibrillar myopathies are rare, encompassing a heterogenous group of sporadic and familial neuromuscular disorders characterised by slowly progressive muscular weakness. cardiac and respiratory muscles are involved in only a small subset of people. our patient has improved with non-invasive ventilation. further molecular genetic analysis is pending. a 74-year-old lady presented with dysphagia. she had a right lower lobe pneumonectomy for a t1nomo non-small cell bronchial carcinoma 3 years previously. a computed tomography scan of the thorax showed oesophageal dilatation, accounting for the presenting complaint, and an incidental thrombus in the pulmonary artery stump (pas). the formation of a pas thrombus is a common radiological finding following pneumonectomy [1] . there is no strong evidence to suggest anticoagulation is beneficial in this group of patients. as no respiratory symptoms were present, anticoagulation could not be justified. a 3-month interval scan showed no thrombus propagation and the patient remained asymptomatic. department of respiratory medicine, beaumont hospital, dublin 9 , ireland a 44 year old gentleman presented to the respiratory clinic with a 4/12 history of progressive dyspnoea on exertion associated with cough. he reported longstanding lower limb weakness, progressing to involve his upper limbs. arterial blood gas demonstrated partially compensated hypercapnoeic respiratory failure whilst pfts showed a classic restrictive pattern. electromyography was abnormal and a muscle biopsy confirmed a diagnosis of nemaline myopathy. he was commenced on nocturnal non-invasive ventilation and his respiratory function improved significantly. nemaline rod myopathy is a rare congenital myopathy that typically presents in childhood. presentation in adulthood with respiratory difficulties is highly unusual. jo-1 positive polymyositis is an inflammatory myopathy and commonly presents with progressive muscle weakness and basal predominant interstitial lung disease. this is a case report of two women with different clinical presentations of jo-1 positive polymyositis who had bibasal interstitial lung disease on high resolution ct thorax. they both had worsening pulmonary function studies and one of them required icu admission and intubation for management of severe respiratory distress. they both responded to different regimens of iv cyclophosphamide and long term oral immunosuppressants. these cases illustrate the respiratory manifestations of jo-1 positive polymyositis and its treatment. 65 year old male with a recent history of travelling abroad presented with 6 week history of dry cough and 5 day history of progressive sob on exertion and one episode of haemoptysis. cxr showed rul consolidation and rll effusion, ctpa was negative for pe. subsequently he developed recurrent spikes of temperature and septic screen was negative. he had three different antibiotics regime with no improvement. thereafter multiple blood cultures, vasculitic, typical and atypical microorganism, hiv, tb screen and bronchial washings came back as negative. repeat ctpa and serial cxr confirmed persistent bilateral consolidation. on day 5 of admission he developed new systolic murmur, a-fib and type 2 respiratory failure. tte and subsequent toe confirmed severe mr with mitral valve vegetation and prolapse treated with mitral valve replacement. dublin 9 , ireland 43 years old male admitted 1 year ago with mesenteric ischaemia diagnosed& nstemi, had emergency small bowel resection with right hemicolectomy. admission has been complicated over 1 year by wound infection, multiple pneumonias, multiple line infections, multiple admissions to itu, later diagnosed with short bowel syndrome, and c difficile colitis; and thrombosis of superficial venous system. due to difficult iv access and low albumin. recently admitted to itu deteriorated due to acute electrolyte disturbance. had transhepatic line (tipss) inserted by ir due to difficult iv access and low albumin. day 4 became acutely sob, cxr complete opacification of right lung. chest drain inserted had drained 9 litres of milky fluid, tubogram confirmed leak through diaphragm into pleura. a. mohamed, r. smyth, j.j. gilmartin university college hospital galway, co galway, ireland a 69 year old man presented with a 1 year history of progressive dyspnea. physical examination revealed a right pleural effusion. a chest radiograph and ct scan confirmed the presence of an effusion with thoracic lymphadenopathy and large mesenteric and para-aortic masses. a pleural aspiration was performed and 1 litre of milky white fluid was removed. pleural fluid analysis noted a protein concentration of 44 g/l and a triglyceride level of 1869 mg/dl. cytology revealed a wbc count of 2240/ml with 100 % of these being mononuclear. subsequent lymph node and bone marrow biopsy confirmed the diagnosis of chylothorax with small lymphocytic lymphoma. a 60-year-old gentleman presented with sub-acute onset of dyspnea associated with diffuse bilateral infiltrates on chest radiograph. his respiratory failure worsened despite broad spectrum antibiotics and he required intubation. he had recent type 2 diabetes mellitus that was well controlled on oral hypoglycaemics. bronchial washings were positive for pcp on silver stain. despite extensive testing a cause for immunological deficiency could not be identified. he developed ards related fibrosis and died. pcp is the most common opportunistic infection in aids patients [1] , it has been rarely reported in previously well, immunocompetent patients. an 83 year old non-smoking female presented to the er with a 3 week history of progressive dyspnoea and cough. the patient also recounted difficulty reclining at night to the point where she slept in a sitting position. examination revealed mild expiratory wheeze and hypoxemia on room air. initial clinical impression was of an infective exacerbation of late onset asthmatic bronchitis. cxr demonstrated a large hiatus hernia. pulmonary function studies showed a mixed obstructive and restrictive pattern. ct thorax revealed large diaphragmatic hernia with evidence of compression of the main bronchi. bronchoscopy showed dynamic airway collapse with complete obstruction of the left and right main stem bronchi and distal trachea on coughing. this represents an unusual cause of dynamic airway collapse and the imaging and literature are reviewed. a 48 year old lifelong non-smoker with asthma presented to er with a 3 days history of worsening breathlessness. on examination he was unable to complete sentences and reduced air entry through out chest.cxr showed right middle lobe consolidation. he had been treated as infective exacerbation of asthma. but unfortunately he deteriorated later on that day progressing to respiratory and circulatory failure and subsequently to disseminated intravascular coagulation. although having best of icu care he passed away the following day. all of his investigations including blood culture, atypical pneumonia screen, h1n1 serology were came back negative. his post mortem examination hasn't revealed any cause other than right sided pneumonia contributing to his death. this represents a shocking but mysterious case of pneumonia as the cause of death. a 54 year old gentleman was referred to the rapid access lung cancer service with a 3 week history of progressive sob and new pleural effusion. of note, he had recently presented to his general practitioner with two soft tissue lesions on the right flank. on examination, axillary lymphadenopathy was palpable with a noted interval increase in the size of the cutaneous lesions. trucut biopsy of the skin lesion and pleural aspiration revealed a histological diagnosis of mantle cell lymphoma. subsequent radiology showed extensive systemic disease and chemotherapy was offered. our case illustrates the extranodal involvement that can occur in mantle cell lymphoma, however, patients rarely present with initial cutaneous involvement as in our case. a 66 year old gentleman was referred to our lung cancer clinic with abnormal chest x-ray and palpable supraclavicular node. a staging ct thorax suggested lung primary with mediastinal and supraclavicular nodes. a biopsy of the palpable node showed hodgkin's disease (hd). a ct-guided biopsy of the lung lesion favoured lung primary and mediastinal nodal sampling revealed hd. he proceeded to lobectomy for excision of the primary lung lesion and the pathology returned a non hodgkins lymphoma. this case illustrates a presentation of dual pathologies: both hodgkins and non hodgkins lymphoma in the same patient mimicking locally advanced lung cancer. a 54-year-old man presented with a 3-month history of back pain and cough. he had a normal chest radiograph and was reassured. subsequent haemoptysis in this patient prompted ct imaging which showed a large left lower lobe mass that was concealed by the cardiac shadow. further mri imaging revealed extensive spinal metastases and thus stage iv disease. we wish to present a case series of five patients with benignappearing single plane chest radiography. further imaging revealed advanced stage lung cancer in these patients. these cases illustrate limitations of single plane radiography and identify anatomical areas where tumours may be missed such as behind the heart. they emphasise the importance of obtaining lateral films and ct imaging in patients with a reassuring single plane chest radiograph where clinical suspicion persists. a 40 year old latvian security guard with a background of relapsing polychondritis and recurrent escherichia coli lrti's, represented to hospital with recurrent haemoptysis and pyrexia of unknown origin. hrct thorax revealed innumerable randomly distributed pulmonary micronodularity. bronchoscopy and bal were unremarkable. fresh sputum cytology confirmed the diagnosis of pulmonary nematodes consistent with strongyloides stercoralis. the patient was treated with high dose ivermectin, resulting in complete resolution of the pulmonary micronodularity. this case report discusses the diagnostic criteria for relapsing polychondritis, the international issues of pulmonary nematodes and the importance of understanding the three radiological subtypes of pulmonary micronodularity (random, centrilobular and peri-lymphatic). a 21 year old gentleman with severe autism spectrum disorder (asd) presented acutely to the emergency department with behavioural disturbance. his prior history was notable for a right lower lobe infiltrate on chest radiography identified 1 year prior to admission. his behavioural disturbance manifested by self harm, and had previously precluded in-hospital investigation. ct thorax confirmed right lower lobe consolidation and flexible bronchoscopy identified a 5.5 cm tree branch within a segmental bronchus of the right lower lobe. this case highlights the importance of flexible bronchoscopy and the difficulties of access to appropriate care for those patients with asd. a 63 year old male was admitted with pleuritic chest pain after knee replacement. routine bloods, including liver function tests were normal. a chest radiograph showed right upper quadrant calcification. ct pulmonary angiography was normal but a ct abdomen showed an area of calcification in segment 7 of the liver with low attenuation centrally consistent with a hydatid cyst. hydatid disease is caused by ingestion of the dog tapeworm echinococcus granulosus. it is uncommon within ireland. treatment includes monitoring of chronic cysts, medical therapy with antibiotics in combination with either surgery of percutaneous drainage. a 61 year old female smoker was admitted with right sided pleuritic chest pain. ct thorax showed extensive right lung consolidation and bronchiectasis. bronchoscopy showed unexpected thickening and nodularity of the upper trachea. biopsies confirmed tracheal amyloidosis and immunohistochemical staining of the deposits was negative for serum amyloid a protein, transthyretin, and kappa and lambda immunoglobulin light chains, indicating amyloid of non-aa type. there was no evidence of amyloid at any other location. patients with tracheobronchial amyloidosis may be asymptomatic or present with dyspnoea, cough, haemoptysis or recurrent pneumonia. department of respiratory medicine, beaumont hospital, dublin 9 , ireland a 17-year old male presented to beaumont hospital with dyspnoea, cough and haemoptysis due to alveolar haemorrhage, requiring icu admission for high frequency oscillatory ventilation and iv cyclophosphamide. originally diagnosed with a pauci-immune vasculitis, a lung biopsy confirmed a diagnosis of pulmonary capillaritis requiring escalating treatment with pulsed iv steroids and immunosuppression. while initially clinically stable, he has had multiple icu admissions, and during an exacerbation presents with symptoms of dyspnoea, a dropping haemoglobin and bilateral infiltrates on chest radiograph. he is now managed by an adult respiratory service with specialist advice, with iv steroids, iv ig and rituximab. we discuss a minimally invasive approach with video assisted thoracoscopic surgery (vats) in performing thymectomy procedures for cases of myasthenia gravis. a 21 year old female was referred with a background history of myasthenia gravis. despite optimal medical therapy m.n. still experiences persistent symptoms and occasional hospitilization for iv immunoglobulins. a ct thorax showed an enlarged thymus. using a three port vats technique the thymectomy was completed and she was discharged 3 days post operation. this case illustrates the possible benefits of minimally invasive approach to thymectomy and avoidance of sternotomy for cases of myasthenia gravis. we report the case of a 20 y.o man who presented with classical pancoast syndrome symptoms caused by a large apical ewing's sarcoma of the chest wall. neoadjuvant chemotherapy localized the tumour to the first rib. complete enbloc surgical resection of the 62 mm residual mass (entire first rib, partial second rib and sublobar lung) was accomplished by a combined anterior hemiclamshell and posterior approach. ypt0. the c8 nerve root was spared and this led to a complete resolution of symptoms. we report the case of an 81 y.o. who underwent curative resection by right sided lobectomy for a pt2an0 adenocarcinoma. she developed this uncommon syndrome of intracardiac shunting of blood immediately post extubation. this syndrome causes profound dyspnoea as a result of arterial hypoxia which is accentuated by the upright position and relieved by recumbency. echocardiography based on clinical suspicion was diagnostic. this was successfully treated by the placement of an occluder device the patent foramen ovale. the case of a 54 year old male smoker with a 6 week history of haemoptysis, night sweats and dyspnoea on exertion is presented. chest radiography confirmed a right upper lobe thick-walled cavity with adjacent nodularity. both transbronchial and percutaneous biopsy sampling was non-diagnostic and microbiological testing was negative. following an episode of large volume haemoptysis he underwent emergency lobectomy. final histopathologic examination confirmed an anaplastic lymphoma kinase (alk)-negative pulmonary anaplastic lymphoma, an extremely rare cause of pulmonary cavitation. we report a g551d homozygote: fev 1 24 % predicted, oxygendependent with recurrent exacerbations (5 in 6 months) awaiting transplant assessment, started on ivacaftor through a named patient program. within 8 weeks transplant assessment was deferred, fev 1 had increased to 35 % predicted, continuous oxygen was discontinued, sweat chloride had fallen from 92 to 46 mmol/l and exacerbation rate decreased to 0 in 6 months prospectively. a 52 year old lady presented with headache and cough. neuroimaging raised the possibilitiy of cns mycobacterial infection. pansensitive mycobacterium tuberculosis was cultured from bronchoalveolar lavage. the patient experienced a type 1 hypersensitivity reaction following the first dose of intravenous rifampicin. the patient was commenced therefore on iv amikacin, oral moxifloxacin and ethambutol. intravenous desensitisation to rifampicin was carried out using a 1 day protocol. this was successful allowing the patient to return to oral therapy for the duration of her treatment. ipf and panca-positivity predating vasculitis is known but not widely appreciated. we describe two cases of ipf, initially anca-negative who became anca-positive with associated vasculitis. a 70-year-old, house-wife diagnosed with ipf in 2007 was anca-negative. in 2010 she was more breathless with borderlinepositive panca (repeat negative). in 2012, she developed acute mononeuritis multiplex, with a highly positive panca, responsive to immunosuppression. ipf remained stable throughout. a 67-year-old male, with established ipf (anca-negative), presented acutely with alveolar haemorrhage, renal failure and now panca-positive, responsive to plasma exchange, haemodialysis and immunosuppression. these cases support the rationale for serial anca measurements in ipf. bronchoscopy in a 49 year old female 14 months after radical chemoradiotherapy and oesophageal stent insertion demonstrated stent erosion into the proximal trachea with recurrent oesophageal scc obstructing the carina. a 20 9 40 mm covered ultraflex tracheal stent was deployed, with an oesophageal stent telescoped proximally into the displaced oesophageal stent. imaging out ruled a leak facilitating oral intake. six weeks later, staged cryotherapy and stenting of the carinal obstruction was successfully performed. cork university hospital, wilton, cork, ireland, 2 mercy university hospital, cork a 62-year-old male was reviewed with increasing shortness of breath, hoarseness and stridor. his past medical history was remarkable for supraglottic amyloidosis. these lesions were thermally ablated in 2003. he was followed up routinely. a ct thorax and subsequent bronchoscopy and biopsy was undertaken. at bronchoscopy he was found to have two large nodular protrusions that were biopsied. pulmonary amyloidosis is rare and manifestations include tracheobronchial infiltration, parenchymal infiltration (amyloidomas) persistent pleural effusions and pulmonary hypertension. symptoms of tracheobronchial amyloidosis can include hoarseness, stridor, dyspnoea and overt airway obstruction [1] . treatment involves invasive bronchoscopic therapies such argon photocoagulation (apc) and occasionally surgery [2] . we present two mushroom workers with bird fancier's lung. workers presented with progressive dyspnoea, cough and sweats, with features of hypersensitivity pneumonitis on hrct, pfts and bal/tbbx. serological studies to aspergillus fumigatus, saccharopolyspora rectivirgula, thermophilic actinomyces were negative but positive for avian proteins. workplace process analysis revealed chicken litter as fundamental in mushroom compost production. both workers received corticosteroids with symptomatic and radiological improvement. workplace relocation resulted in complete resolution of symptoms in one worker. the second worker remains exposed, wearing appropriate ppe with ongoing medical surveillance. detailed workplace analysis may be required in proper diagnosis of work-related respiratory diseases. wheeze is a continuous musical sound that lasts longer than 250 ms [1] . upper airway obstruction is commonly misdiagnosed as asthma. we describe four cases presenting with upper airway obstruction of different aetiologies. a 15-year-old female was referred with 'poorly controlled asthma'. inspiratory stridor was noted on physical exam and spirometric flow volume loops showed variable extrathoracic airway obstruction. laryngobronchoscopy confirmed paradoxical vocal cord movement. ir j med sci (2012) 181 (suppl 10):s369-s437 a 58-year-old female was referred with 'poorly controlled asthma'. physical exam revealed inspiratory stridor and spirometric flow volume loops showed fixed upper airway obstruction. larynogbronchoscopy revealed subglottic stenosis. a 75-year-old male with a 20-pack-year history of smoking was referred with worsening wheeze. physical exam and investigations revealed subtotal tracheal compression secondary to a retrosternal goitre which was successfully resected with resolution of symptoms. a 56-year-old male was referred with wheeze, fatigue and intermittent apnoea while sleeping on his left side. physical exam revealed inspiratory stridor and spirometric flow volume loops showed fixed upper airway obstruction. ct thorax and bronchoscopy revealed a congenital double aortic arch, splitting to come around the trachea (see fig. 1 ) causing tracheal compression. the four cases show the importance of considering misdiagnosed upper airway obstruction in the assessment of wheeze. a 62 year male smoker with gastro oesophageal adenocarcinoma developed dyspnoea on treatment with epirubicin, oxaliplatin, and capecitabine, raynaud's phenomenon was noted and hrct showed bilateral interstitial infiltrates. scl 70 antibodies were positive and thoracoscopic lung biopsy confirmed scleroderma-associated interstitial lung disease. patients receiving pneumotoxic agents should be assessed thoroughly for other causes before a diagnosis of drug-induced interstitial lung disease is considered. mid-western regional hospital limerick, limerick, ireland a 38 year old woman presented with recurrent lower respiratory tract infections and weight loss of over a stone over the last 6 months. she was a smoker of 20 pack year. ct showed diffusely abnormalities in both lungs. there was ill-defined pulmonary nodules with small cavitating lung lesions. specimens taken from vats showed numerous airway-centred and airway-destructive nodules composed of eosinophils and large histiocytes with vesicular chromatin that are cd1a positive. this is consistent with langerhans cell histiocytosis. she was advised on smoking cessation which she adhered to. follow-up cxr showed complete resolution of the multi-focal interstitial infiltrates several studies reported relationship between anca-associated vasculitides and malignancies (pre-existing or developed during patients follow up), bringing to discussion the putative role of tumor antigen in driving the auto-immune response. we describe the case of 45 years old male with e-cadherin genetic mutation, mandibular cementoma, horse shoe kidney, alopecia and nails dystrophy who presented with haemoptysis and bilateral pulmonary infiltrates 2 years after his first asthma diagnosis. bronchial washings demonstrated pigment laden cells consistent with alveolar haemorrhages. serological tests showed positive c-anca. there was no renal involvement. he was successfully treated with high dose of corticosteroids. we present an interesting case of endobronchial tb in patient who lacked normal host immunity. a gentleman (59 years) presented with haemoptysis on a background of ulcerative colitis managed with mesalanine and azathioprine. bronchoscopy demonstrated a mass partially occluding the left main bronchus. pan-sensitive mtb was isolated and he was treated for 9 months. four months following presentation an endobronchial resection was performed. however, repeat bronchoscopy following nine months of treatment demonstrated a persistent lesion in the left main bronchus and a new lesion in the trachea. the recurring lesion was characterised by the persistent afb, that were stainable, but did not grow. conclusion: the immune derangement, characteristic of inflammatory bowel disease patients, contributed to his aberrant persisting host response to dead organism. [1] . in these two cases dipnech was diagnosed along side carcinoid tumour via lobectomy and mediastinal lymph node sampling. one patient was symptomatic for at least 18 months prior to diagnosis complaining of episodic wheeze, flushing and diarrhoea. this patient was treated with a somatostatin analogue post operatively. dipnech has rarely been described and its prognosis and management have varied from several different case reports [2] . these two cases highlight, that although a rare entity, it needs to be considered in cases of symptomatic cough and wheeze with radiological findings suggestive of discrete pulmonary nodules. in march 2011, the first case of a subsequent outbreak of pansensitive mycobacterium tuberculosis, beijing strain, was diagnosed in a man from an irish prison. last year, the first 11 patients in this sequence were presented. herein we discuss eight further cases connected to this outbreak. all cases, but one, are males of european origin aged between 20 and 50 years. clinical presentations ranged from classical symptoms of night sweats and weight loss to acute abdomen. four of the pulmonary tb cases had, on initial presentation, acid fast bacilli positive sputum on microscopy and culture. the radiological disease was primarily consolidation or cavitating lesion in the upper to mid zones. more than a year later, this case series continues to highlight the ongoing and mounting difficulties of managing this irish prison outbreak. a 36 year-old irish male presented with a 1 month history of dyspnoea, unsteady gait, numbness over both flanks and weight loss. he attended 8 months earlier at another centre with swollen painful right testes. cxr demonstrated bilateral hilar lymphadenopathy, and rightsided pleural effusion. ct tap revealed marked mediastinal lymphadenopathy with right sided pleural effusion, mild splenomegaly, ill-defined right testes, and bilateral inguinal lymphadenopathy. serum ldh and ace were elevated. thoracentesis revealed serosanguinous exudative fluid. transbronchial and inguinal lymph node biopsy revealed evidence of non-caseating granulomas. mri spine revealed abnormal cystic areas throughout thoracic and lumbar spine. a diagnosis of sarcoidosis with multisystem involvement was made and high dose steroids were commenced with good clinical response. pleural and spinal cord sarcoidosis is a rare and interesting presentation. histology showed chronic inflammatory change and ulceration of the bronchi with no evidence of vasculitis or granuloma. no malignancy was evident. pulmonary changes similar to this have been described in patients with uc. alpha-1 antitrypsin deficiency (aatd) is an autosomal co-dominant genetic disorder associated with a substantially increased risk for the development of chronic obstructive pulmonary disease (copd) and liver disease. ats/ers guidelines recommend testing of all individuals with copd and poorly controlled asthma. the objective of the study was to investigate the diagnostic experiences of zz aatd individuals in ireland. a total of 74 zz aatd individuals completed a questionnaire at an alpha-1 clinic in relation to their diagnostic experiences and clinical presentation. the mean age of symptom onset was 37.8 â± 1.6 years (range 0.03-80); mean age of diagnosis was 44.1 â± 1.6 years (range 0.03-80). the interval between onset of symptoms and aatd diagnosis was 7 years. the smoking history analysis revealed 67 % were past smokers, 32 % never smokers and 1 % were currently smoking. for the past smokers cohort 36 % stopped smoking within the first 12 months of a diagnosis of aatd; 24 % stopped smoking after the first 12 months of a diagnosis of aatd and 40 % had stopped smoking prior to a diagnosis of aatd. our results further underline the need for increased awareness and early detection of symptomatic aatd individuals in the irish population, especially among the copd population. aat deficiency (aatd) results from mutations in the serpina1 gene, classically presenting with early-onset emphysema and/or liver disease. the most common mutation causing aatd is the z mutation, with the s mutation weakly associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. ats/ers guidelines advocate screening all copd, poorlycontrolled asthma, and cryptogenic liver disease patients, as well as first degree relatives of known aatd patients. over 8,500 individuals have been screened to date following ats/ ers guidelines in the national targeted detection programme. sequencing of the serpina1 gene was performed to identify rare mutations. we have identified 117 zz, 123 sz, 42 ss, 1249 mz, 876 ms, and over 30 individuals with clinically significant rare phenotypes (e.g. iz, fz, is, null, mmalton). this yields gene frequencies of 0.064 and 0.095 for s and z, respectively, in this targeted population. a number of rare and novel serpina1 mutations have also been identified. our results underline the need for increased awareness and early detection of aatd. all copd patients should be tested for aatd as per ats/ers guidelines. our data demonstrates that aatd in ireland is not a rare disease but a disease that is rarely diagnosed. rationale: alpha-1 antitrypsin (aat) deficiency (aatd) is genetic disease that results in low levels of aat and predisposes individuals to developing chronic obstructive pulmonary disease (copd). the z-allele is responsible for [95 % cases of aatd. key studies have demonstrated that excessive infiltration of neutrophils into the lung and neutrophil derived proteins play a pathological role in aatd lung disease. in particular a key cytokine associated with copd disease progression is tnf-alpha which is also the cause of many problems associated with autoimmune diseases. the aim of this study was to determine if there is a novel autoimmune element driving inflammation in aatd and examine aat impact on this. methods: plasma and neutrophils were isolated from mm controls, asymptomatic zz aatd and aatd patients receiving augmentation therapy. tnf-alpha was quantified by a sandwich elisa. evaluation of neutrophil degranulation was carried out by western blot analysis of neutrophil supernatants for markers of tertiary (mmp-9) and secondary granules (lactoferrin) and via flow cytometry. autoantibodies against neutrophil granule proteins were quantified in plasma by elisa. results: our results demonstrate that there are high levels of tnfalpha in aatd plasma compared to controls (p = 0.01). in vitro, tnf-alpha caused an increase in the rate of degranulation of tertiary and secondary granules from zz aatd neutrophils compared to mm cells (p \ 0.05). analysis of autoantibodies against major neutrophil granule proteins revealed a high titer of anti-lactoferrin igg autoantibodies present in patients with zz aatd. treatment of aatd patients with aat augmentation therapy resulted in a decrease in the plasma levels of neutrophil granule proteins while also reducing the titer of anti-lactoferrin igg autoantibodies (p \ 0.05). conclusion: this study has uncovered that tnf-alpha inflammatory signaling pathway can result in the development of an autoimmune element in aatd. furthermore it highlights aat therapy can impact on neutrophil degranulation and thereby reduce development of novel anti-lactoferrin autoantibodies. chronic hypoxia (ch) exposure induces diaphragmatic remodelling similar to chronic obstructive pulmonary disease (copd). elucidating the underlying mechanisms may inform therapeutic strategies to combat muscle dysfunction in copd. this study investigates the effects of ch on redox homeostasis in mouse diaphragm muscle. c57bl6 j mice were exposed to one and 6 weeks of ch (10 % f i o 2 ) or normoxia. following treatment, excised muscles were homogenised and incubated with carbonyl-or thiol-reactive fluorophores before gel electrophoresis and fluorescence scanning. optical density (od) of fluorescence was normalised to total protein, determined by colloidal coomassie staining. a nine-fold increase in free thiol groups was observed after 1 week of ch (281.7 â± 85.2 vs. 30.8 â± 5.3; mean od â± sem, ch v normoxia, n = 7 per group; p = 0.0006, student's unpaired t test), while there was a significant decrease after 6 weeks. a significant increase in carbonylation was observed after 6 weeks of ch (526.7 â± 72.4 vs. 70.7 â± 11.7; mean od â± sem, ch vs. normoxia, n = 8 per group; p \ 0.0001). we have demonstrated that despite a reduction in oxygen tension and a large, initial increase in free thiol groups, 6 weeks of ch significantly increases oxidative stress in mouse diaphragm muscle. changes in redox homeostasis are likely to affect redox-malleable proteins that are central to muscle performance. a-1 antitrypsin (aat) is a 52-kda glycosylated-protein synthesised in the liver which functions as a serine protease inhibitor. the pizz variant is associated with early onset emphysema. we hypothesize that a difference in the number of isoforms and the n-glycosylation pattern of pimm and pizz aat protein exists. the aim of this study was to compare the isoform composition of aat from pimm controls with that of pizz individuals. aat from pimm and pizz individuals was extracted and purified from plasma using alpha-1-antitrypsin select-affinity chromatography medium. isoelectric-focussing of purified aat was performed followed by 2d-page. gels were stained with coomassie brilliant blue and were immuno-blotted for aat. eight and six isoforms of pimm and pizz-aat were identified by 2d-page, respectively. densitometric analysis demonstrated higher protein expression of pimm-aat compared to pizz-aat. the pi range of pimm-aat was 4.70-5.18 and the range for pizz-aat was 4. 78-5.28 . the pizz-aat demonstrated a 0.08-0.13 pi cathodal shift of all bands, supporting the hypothesis that the pizz-aat protein is differentially n-glycosylated. this study confirms the presence of multiple isoforms of pimm-aat and demonstrates at least 6 different isoforms of pizz-aat. this requires further investigation to establish differences in n-glycan groups and possible functional consequences. understand the therapeutic potential of alpha-1 antitrypsin alpha-1 antitrypsin (aat) is a glycosylated protease inhibitor found in human plasma. aat deficiency predisposes individuals to early onset emphysema and treatment currently consists of weekly intravenous infusions of purified plasma aat. although aat has previously been shown to exert anti-inflammatory properties by binding interleukin-8 and apolipoprotein b-100, the latter being implicating in atherogenesis, hypothesized that aat may have multiple binding partners and that these complexes are involved in additional regulatory and anti-inflammatory pathways. the aim of this study is to identify all proteins that interact with aat as it circulates throughout the body in order to fully understand its therapeutic potential. to examine aat's interaction with potential linker proteins, permeation chromatography of plasma through su-perose75 10/300 gl was performed. protein profiles were visualized by coommassie blue staining of sds-page gels and western blotting. immuno-bands were quantified by densitometry. aat eluted with molecular masses of approximately 600 and 350kda indicating multiple binding partners, with the remainder eluting at the predicted molecular mass of 50kda. protein identification by mass spectrometry (lc ms/ms) is required to identity novel binding partners. identification of all proteins that bind to aat will progress our understanding of the molecular mechanisms by which aat regulates inflammation. ultimately identification of new functions of aat may be utilised to develop novel treatment options for chronic inflammatory diseases including cystic fibrosis, copd and severe asthma. no potential conflict of interest is reported. transthoracic echocardiography, neurocognitive/psychological assessments were also performed. on follow-up, all patients had normal renal function. two of seven had reduced respiratory and cardiac functions. 2 had slightly reduced neurocognitive functions and 3 had decreased feeling of well-being, and 0 from depression. in this small follow up study of a cohort of severe h1n1 patients, there was good recovery. given the initial severity of their respiratory decline i.e., requiring ventilation, this group did not seem to suffer severe chronic respiratory functional limitation. cryptogenic organising pneumonina (cop) is a disease of unknown cause, which can occur in the context of connective tissue disease. a more aggressive variant termed rapidly-progressive cop follows a fulminant course, leading to respiratory failure, and has high mortality [1] . a 16-year-old gentleman with a history of dermatomyositis presented with dry cough and dyspnoea on exertion of 2-months duration. computed tomography (ct) of the chest revealed subpleural patchy ground glass opacities involving both lungs. bronchoalveolar lavage (bal) showed benign bronchial epithelial cells with 50 % macrophages, 30 % neutrophils, 10 % lymphocytes and 10 % eosinophils. staphylococcus and hemophilus were isolated on culture. broad-spectrum antibiotics and steroids were commenced. there was evident improvement clinically, and on pulmonary function tests, however, no significant change on ct chest neccesitated vats biopsy.histopathologic findings were consistent with cop. 24 h after vats biopsy patient developed acute onset dyspnoea with respiratory failure requiring mechanical ventilation and intensivecare-unit admission. ct chest (see fig. 1 ) showed diffuse worsening of ground glass appearance and left lung consolidation. despite best efforts to rescuscitate the patient he passed away 11 h later. this represent a fatal case of rapidly progressive cop resistant to steroids. rapid deterioration resulted in poor prognosis with no lea way of trial of immunosupressive therapy. over a 4 week period thirty seven patients were admitted by nchds to cork university hospital with clinical diagnosis of community acquired pneumonia. an audit was performed on the choice of antibiotic by nchd in comparison to the local antimicrobial guidelines based on a patients curb-65 score. this audit also compared the admitting nchd's interpretation of a chest x-ray and this was compared to the formal report given by the radiology department. the audit highlighted that antimicrobial prescribing in adherence with local guidelines weakened with an increasing curb-65 score. in total 56.7 % of patients were prescribed antibiotics according to the local guidelines. this fell to 13.6 % for a curb-65 score of 3/5. approximately 30 % of nchds incorrectly diagnosed an infiltrate on chest x-ray which was later refuted by the formal radiology report. antibiotic resistance is a growing global concern and adherence to selected local and regional guidelines for prescription of antibiotics is paramount in reducing the spread of resistance pathogenic bacteria [1] . reference: rapid administration of guidelines-compliant empiric antibiotic therapy in emergency department (ed) can reduce mortality in patients admitted with community-acquired pneumonia (cap) [1] . a 5-week prospective audit was conducted in january 2012 to assess adherence to local guidelines and use of curb-65 score in treatment of such patients. all patients admitted via ed during acute medical take with symptoms and signs of chest infection and new localising radiological shadowing were included. there were 32 relevant admissions (18 males, age 42-92 years, mean 72.7). curb-65 was calculated in 13 (40.6 %) patients. seven patients (21.9 %) received antibiotics according to guidelines. in patients without curb-65 estimation adherence to guidelines was 15.7 % vs 29.4 % in those with curb-65. the results of the audit were presented at hospital grand rounds and new copies of guidelines were circulated to each ward and ed. a laminated copy was placed in a prominent position in ed. a 5 week prospective re-audit was conducted in april 2012 (29 admissions, 15 males, age 25-97, mean 74.7 years) and demonstrated an increase in appropriate antimicrobial prescribing to 34.6 % vs 21.9 % in the original audit. appropriate antibiotics prescribing was again higher when curb-65 was calculated. medicine is an evolving field with increased pressure to produce an optimally functioning health care system within budget constraints. there is a current vogue of specialisation, with medical practitioners moving further from general medicine, to condition specific delivery of services. our key concern was whether the case mix breakdown warranted further stratification of the general respiratory outpatient clinic based on conditions. data of 1932 patients attending a respiratory clinic was collected for 1 year. the primary diagnosis was coded using international classification of diseases (10th revision) coding scheme and analysed using a statistical analysis package (spss). asthma (n = 444, 22.98 %), copd (n = 269, 13.92 %) and sarcoidosis (n = 162, 8.38 %) accounted for 45.28 % of patients and the remainder of the top fifteen conditions were all respiratory in nature. 10 .45 % (n = 202) of attendances were for non-respiratory diseases as the clinic also provides follow-up for general medical patients post hospital admission. the gender mix was male (n = 906) 47.1 %; female (n = 1019) 52.9 %. the mean age was 55.5 years (sd = 27.306). the difference in mean age for asthma (48.41 sd 19.08) and copd (74.36 sd 39.51) was significant at 17.079 (p = 0.000). the above analysis reveals a strong case for the creation of three specialist outpatient clinics, for asthma, copd and sarcoidosis. the single breath method to measure diffusion capacity requires a subject to inspire a gas mixture followed by a 10 s (s) breath hold. we sought to determine if breath hold time reduction had a significant effect on measured lung diffusion for carbon monoxide (dlco) values. forced spirometry and co diffusion by the single breath method (dlcosb) were performed in duplicate with breath hold for 10 s, 8 s and 6 s in 30 controls (fev 1 107 â± 12.04 % predicted), 30 severe copd patients (fev 1 37.2 â± 7.92 % predicted), and 30 patients with interstitial lung disease (ild) (fev 1 69.5 â± 17.61 % predicted). there was no significant difference between dlcosb and dlco(va) measured at 10, 8 and 6 s in the control (p = 0.4431) and interstitial lung disease (ild) groups (p = 0.5915). however, there was a significant difference between dlcosb (p = 0.0003) and dlco(va) (p = 0.0183) measured at 10, 8 and 6 s in the chronic obstructive pulmonary disease (copd) group. in the presence of severe airway obstruction the dlco decreases with breath hold time reduction. however, in healthy controls and patients with ild, there was no significant change in the dlco when breath hold time is reduced from 10 to 6 s. this could allow for a reduction in breath hold time when measuring the dlco in patients with advanced ild who are unable to breath hold for 10 s. current guidelines recommend mechanical insufflation-exsufflation (mi-e) for airway clearance [1] . the aim of this study was to determine the current use of mi-e in neurological conditions by physiotherapists in the uk. a questionnaire was sent to relevant members of the exercise has been shown to improve quality of life in respiratory patients. through exercise, pulmonary rehabilitation operated on the concept of encouraging people with chronic obstructive pulmonary disease (copd) to improve their exercise capacity and subsequently reducing the incidence of copd exacerbation and admissions to hospital. methods: this cross sectional study was conducted between january and february 2011 at the mid-western regional hospital, limerick, ireland. all patients attending our respiratory clinic over a period of 4 weeks were invited to complete a questionnaire on arrival at the clinic. findings: the total number of participants was seventy-eight. asthma was the most frequently listed respiratory illness (n = 21).fifty-two patients said they exercised (66.7 % of the study population). higher levels of exercise participation were seen in the younger age groups (p = 0.585 introduction: respiratory diseases, largely represented by copd, are the third most common cause of acute hospital admission.our aim was to audit the prescribing habits of inhaled, nebulised medication and oxygen by doctors in a general hospital. methods: all adult patients admitted medically with chronic respiratory diseases that were on inhaled or nebulised medication were included prospectively (jan to june 2012) in this study. a proforma was used to collect data from the patients. results: there were 30 patients (67 % male). the mean age was 70. 20 patients (67 %) had a diagnosis of copd and the others had asthma and pulmonary fibrosis. seven patients (23 %) were currently smoking. four patients (13 %) were on long term oxygen treatment. in only 50 % of patients the correct dose of inhaler was prescribed. 6 patients (20 %) had the correct inhaler device charted. none of the patients had their inhaler technique checked on admission. in only 27 % the dose of nebulised medication was charted. 21 patients were given oxygen of which only 4 was prescribed (19 %). conclusion: this audit proves that our prescribing habits of inhaled, nebulised drugs and oxygen are not good. we intend to present this data to our colleagues and reaudit again. adherence to inhaled medications is difficult to assess. a prospective, observational study on patient inhaler usage while in hospital was carried out. the hypothesis was as inhalers are left at the bedside and not administered directly, doses are being missed. a device was designed that makes an acoustic record each time an inhaler was used. the drug prescription sheets on medical wards were screened to identify patients who were prescribed fluticasone/salmeterol via diskus. patients were then approached and asked to participate. the devices were analysed by two independent investigators. doses were classified as early if \6 h and late if [18 h were between doses. errors were classified as not priming the device correctly, blowing into the device, insufficient inhalation, inadequate breath hold. among 41 patients, taking 326 doses, 51(16 %) doses were taken too early, while separately 70 (21 %) doses were missed. in addition, patients blew into the inhaler 75 (15 %) times and inadequately inhaled or failed to hold breath 81 (25 %). overall, 204 (43 %) had an error either in timing or technique. none of these irregularities were documented in the drug prescription sheet. in conclusion, administration of inhalers should be directly supervised by staff, documented and an action plan for patients that are unable to use inhalers be drawn up. results: 20 patients participated in the audit. 80 % knew the names of their respiratory medication, and 55 % knew the general indication for their respiratory medication. 30 % understood the specific indication for their preventer inhaler, 40 % for their reliever inhaler, and 0 % for the combination inhaler. 80 % of patients knew the correct frequency of use of their inhaler, with 55 % of patients demonstrating adequate inhaler technique. 70 % of participants had previously being reviewed and educated by a respiratory nurse. conclusion: a significant percentage of respiratory patients lack adequate knowledge of their respiratory medication; this is despite a majority having previously been educated on this medication. recommendations: ongoing education and regular assessment of respiratory patients needs to occur. ir j med sci (2012) hcws are a cohort at risk of mycobacterium tb infection. qft-g, a lab based assay, is free of the bias and errors of tst placement or reading with the need for 48-72 h follow-up for interpretation eliminated. 34.8 % of our cohort had a false positive tst, which calls the sensitivity of this test into question. qft-g has been proven to approach 98 % sensitivity [1] and 89 % specificity [1] and is therefore a suitable replacement for tst in hcw screening. while there has been an increased recognition of non-tuberculous mycobacteria (ntm) as a clinical problem, much of this experience has come from specialised population such as cystic fibrosis patients. we evaluated our experience in a general respiratory service. positive non tuberculous mycobacterial culture results in st. vincent's university hospital from january 2007 to july 2012 were reviewed. patients with a known diagnosis of cystic fibrosis were excluded. fifty-six patients were identified with positive cultures for nontuberculous mycobacteria. thirty-eight isolates were from the respiratory tract, of which fourteen samples were sputum samples, thirteen samples were from both bronchial lavage and sputum and nine samples were bronchial lavage. the medical records of thirteen patients (seven female, six male) with probable disease were reviewed. the most frequent isolated ntm in our institution are mycobacterium avium (n = 9). two patients had mycobacterium szulgai. mean age was 61.6 â± 11.5 years; all except one patient had underlying respiratory disease. five of the thirteen patients received some treatment for ntm but only three completed a full course due to intolerability to the medications and also side-effects. our studies confirm that ntm primarily affects patients with chronic lung disease and that the treatment for this disorder is poorly tolerated. lymphopenia in active mycobacterium tuberculosis (mtb) infection is a common and well-documented finding. rather than being an epiphenomenon, this effect likely contributes to pathogen persistence in the host and the lack of a meaningful response during chronic mtb infection. our study was designed to determine the baseline and post-treatment values of total lymphocyte count and its subsets in hiv-negative patients diagnosed with active pulmonary mtb. we prospectively recruited hiv-negative patients diagnosed with pulmonary mtb infection over a 6-month period. pre and post treatment analysis of total lymphocyte count and its subsets were performed at baseline and after 6 months of tb chemotherapy. a control group comprising of patients with community acquired pneumonia also had pre-treatment lymphocyte counts performed. ten patients with active mtb infection and seven comparable controls were recruited over a 6-month period. baseline total lymphocyte count was lower in the study group (1483.2 â± 629.6) compared to control (1770.1 â± 579.6). treatment was associated with significant improvements in total lymphocyte, b-cells, cd4, cd8 (p \ 0.001) and nk cell (p \ 0.05) counts. recovery of total lymphocyte count in the control group was not significant (p = 0.17). our study demonstrates treatment of active mtb in hiv-negative patients is associated with significant improvements in total lymphocyte count and its major subsets. pleural tuberculosis is a diagnostic challenge. ada is a biomarker that has been proposed to diagnose tuberculous pleurisy but not routinely used [1] . we aim to evaluate the sensitivity and specificity of ada in the diagnosis of tuberculous effusions and to improve current practice. we prospectively examined ada levels from 45 patients with pleural effusions and followed the clinical course to establish the final diagnosis via culture, histology and clinical diagnosis. data were analysed using mann-whitney u test. there were 3 cases of tuberculous effusions with mean ada levels of 43 â± 14.7 iu/l (ci 6.4-80 iu/l) while the mean ada of non-tuberculous effusions were 22.9 â± 34 iu/l (ci 12.3-33.5 iu/l, p value = 0.041*). true positive rate was 2/45 and true negative rate was 38/45. false positive rate was 4/45 while false negative rate 1/45. if 47 iu/l is taken as cut value, the specificity is 97 % and the sensitivity is 33 %. we describe four cases of tb occurring during pregnancy and post partum. in all cases the women were non-nationals with a mean age of 35 years. in two cases the tb presented as vertebral osteomyelitis, in one case tb lymphadenitis and in one case as miliary pulmonary tb. the diagnosis and treatment of tb in pregnancy presents many challenges. the cases discussed highlight some of the complexities which we encountered. these included dealing with language and cultural barriers, multi-disciplinary management of tb osteomyelitis in a pregnant woman; involving close collaboration with the obstetric and orthopaedic teams and managing adverse effects of anti-tuberculous medications in the pregnant patient. most samples tested showed no evidence of mycobacteria, indicating need for improved case selection prior to testing. median time to zn testing is as recommended by guidelines, though there is room for improvement. in an unselected population, likelihood of negative results is high after 2 weeks of negative cultures. however, clinical suspicion should remain high until culture negativity is declared at 7 weeks, particularly when there is a high clinical probability of mycobacterial disease. background: a number of patients with bronchiectasis have a middle lobe/lingula predominant radiological pattern. other than an association with non-tuberculous mycobacteria there is a paucity of published data on this subgroup of patients. methods: we retrospectively analysed data from all patients with non-cf bronchiectasis who underwent bronchoscopy with bal in a university hospital over a 12 month period. radiological features, demographic data and microbiology were reviewed. results: 71 patients with bronchiectasis were assessed. 22 (30 %) had predominant middle lobe/lingular bronchiectasis. in this group 18 (82 %) patients were females compared to 31 (63 %) (p = 0.11) of other patients and mean age was 57 â± 12 compared to 62 â± 12 years (p = 0.73). bal microbiology in the middle lobe/lingular group revealed no growth in 11 (50 %), h influenzae in 6 (27 %), s aureus in 3 (14 %), mac in 3 (14 %) and other 2 (9 %). conclusion: middle lobe/lingula predominant bronchiectasis is a common radiological pattern particularly in females and only a minority have ntm infection. cystic fibrosis (cf) is characterised by neutrophil-dominated airway inflammation, in part attributable to the potent chemotactic agent leukotriene b 4 (ltb 4 ). the aim of this study was to investigate the ability of exogenous alpha-1 antitrypsin (aat) to inhibit ltb 4 signaling. the biological consequence of the described aat induced inhibition was investigated at the level of neutrophil released proteolytic enzymes. circulating neutrophils isolated from healthy control volunteers (n = 10) were stimulated with ltb 4 (25-200 nm/2 9 10 7 ) in the presence and absence of aat (1.8-27.5 lm) for increasing increments of time (0, 5, 10 and 20 min). the level of degranulated proteins in surrounding supernatants was determined by western blot analysis. the ability of aat to bind ltb 4 was assessed specrophometrically with uv spectra recorded on a jenway 6405 spectrophotometer at 25â°c. our in vitro data has shown that levels of degranulated mpo, ll-37 and mmp-9 (markers of primary, secondary and tertiary granule release, respectively) were significantly decreased in the presence of aat (p \ 0.05). the mechanism of inhibition involved direct binding of aat to ltb 4 as reduced vibrational fine structure of the ltb 4 / aat uv absorbance spectrum indicated complexation of the two molecules. the results of this study indicate that aat can inhibit ltb 4 signaling thereby reducing the proteolytic activity of neutrophils and propose aat aerosolized augmentation therapy as an effective treatment for ltb 4 associated pulmonary diseases including cystic fibrosis and severe asthma. the modified-shuttle-walk-test (mswt) is increasingly used in cystic fibrosis (cf) patients. however, few studies have correlated mswt with severity of disease or assessed the prognostic value of these tests. the aim of this study was to see if a correlation existed between mswt and forced expiratory volume in 1-s (fev1) and/or cf-able score. a total of 33 mswt assessments were analysed. correlations (spearman) among fev1, cf-able-score, percentage predicted distance travelled and percentage predicted distance travelled to desaturation were calculated. nine out of 33 mswt showed desaturation. the mean distance travelled was 1,073 m; 73.5 % of predicted, and mean distance to desaturation was 655.5 m; 44.9 % predicted. there was a significant correlation between distance travelled and fev1 (r = 0.755/p \ 0.001) ir j med sci (2012) 181 (suppl 10):s369-s437 and inverse correlation with cf-able-score (r = -0.739/p \ 0.001). there was a significant but poor correlation between distance to desaturation with fev1 (r = 0.736/p \ 0.001) and cf-able-score (r = -0.502/p = 0.003). however, the presence of desaturation during testing did not correlate with fev1 (r = -0.388/p = 0.028) or correlate significantly with cf-able-score (r = 0.072/p = 0.695). in conclusion there is significant correlation between total distance walked and both fev1 and cf-able-score, however, the absence of a correlation with the presence of desaturation during testing highlights the usefulness of mswt as a possible independent predictive measure, with further study needed. prolonged antibiotic therapy for cystic fibrosis (cf) exacerbations leads to increased picc (peripheral inserted central catheter) use. consequently incidence of venous thromboembolism (vte) has risen. rates of picc induced thrombosis in adults are 8.2 % [1] . we aimed to ascertain prevalence of picc induced thrombosis in adult cf patients. a retrospective review of radiology was conducted on patients who had picc insertion for antibiotics for cf exacerbations from january 2010 to december 2011. we analyzed patients with confirmed vte on doppler ultrasound and recorded patient demographics, size of picc and site of insertion. 308 piccs were inserted, 21 (6.8 %) had vte. 10 (47.6 %) were female and 11 (52.4 %) were male with symptomatic vte, presenting with arm swelling and pain. further complications were 1 (5.8 %) with superior vena-cava syndrome, 2 (11.8 %) with pulmonary embolism. of these, 4 were treated with 6 months anticoagulation therapy, and 13 were anticoagulated for 3 months once repeat doppler ultrasound confirmed no thrombosis. our rate of vte was 6.8 %, lower than in existing studies. piccinduced thrombosis depends on the population studied, as well as acquired thrombophilia secondary to inflammation, or deficiencies of anticoagulant proteins (protein c and s) due to vitamin k deficiency and/or liver dysfunction. studies have documented very poor real-life adherence to nebulised antibiotic therapies [1] . no data exists on real-life experience with inhaled antibiotics. consecutive adult cf patients commencing inhaled antibiotic therapy (tip) were recruited over a 10-month period. a questionnaire recording safety, efficacy, lung function and adherence at time of recruitment, assessed traditional nebulised treatment (tis) versus new inhaled therapy (tip) at 3, 6 and 9 months. wilcoxons rank test and paired sample t-tests were employed for statistical analysis. 69 patients have been enrolled to date. 1 patient died (unrelated to the drug). 1 patient received a lung transplant. 6/67 (9 %) discontinued tip; 5 due to cough/bronchospasm and 1 due to refractory oral candidiasis. 7/69 (10 %) were intolerant of tis prior to enrolment, with 5/7 (71 %) subsequently tolerating tip. there was a significant increase in mean adherence score from 2.1 in the tis group to 2.9 in the tip group (p value 0.001). there was no significant difference in cough, lung function, or adverse events between the groups. in a real-life clinical setting with new inhaled antibiotic therapy (tip) we demonstrate, improved tolerability, adherence, lower discontinuation rates and stable clinical phenotype. also subgroup analysis supports a trial of this in those who failed traditional nebulised treatment. aim to assess the clinical utility of a hand-held nno analyser to differentiate between pcd, respiratory disease and healthy subjects. clinically stable patients were recruited over a 6-month period. each subject completed compatible pcd phenotype proforma, nno analysis (niox mino ã� ), and one nasal brushing for electron microscopy (em) analysis. nno was measured using passive sampling at a flow rate of 5 ml/s during tidal breathing. em images will be reviewed internally and externally at an international centre of excellence (unc chapel hill). independent t-tests were used to compare mean nno values between groups. 26 subjects were recruited (n = 4 pcd, n = 6 cf, n = 7 non-cf/ non-pcd bronchiectasis, n = 4 copd, n = 5 healthy subjects). mean nno levels (ppb â± sd) were 24 â± 13.6 (pcd), 40 â± 39 (cf), 251 â± 273 (non-cf/non-pcd bronchiectasis), 263 â± 148 (copd) and 419 â± 76 (healthy control). although nno levels were reduced in pcd when compared to copd (p = 0.018) and healthy subjects (p \ 0.0001), there was no statistically significant difference between nno levels in pcd and cf (p value 0.49). results of em analysis are pending. in this study, the hand-held niox mino ã� nno analyser distinguished patients with pcd and cf from patients with copd and healthy subjects but not cf from pcd. ir j med sci (2012) studies suggest that incorrect usage of inhalers impacts negatively on asthma control. the aim of this study was to evaluate inhaler technique and symptom control in patients with severe asthma. patients referred to a newly established clinic in cork university hospital were consecutively recruited over a 6 month period. inhaler technique was assessed using a validated scoring system and instruction on correct usage given if scores were suboptimal. patients completed a validated asthma control questionnaire (acq) and asthma quality of life questionnaire (aqlq). at a follow-up clinic 3 months later technique was reassessed and acq repeated. results at baseline and follow-up were compared using standard statistical methods. 46 patients were recruited (female = 74 %), and 40/46 were followed up. mean[sd] fev1 % predicted at baseline = 76.5 % (21.5). 63 % of patients were classified as incorrect inhaler users initially, decreasing to 20 % at follow up, indicating a significant improvement in inhaler usage post-training (p = 0.003). acq scores improved significantly from median (range) 2.5 (0.14-4.60) to 2.0 (0-4.60), p = 0.002. the aqlq results indicated that patients' qol is moderately affected by asthma; median (range) score of 4.75 (2.2-6.75). this study demonstrates the importance of formally assessing inhaler technique in patients with severe, long-standing asthma as part of their clinical review. asthma is a chronic airway disease characterized by airway inflammation, bronchial hyperresponsiveness and airflow obstruction. patients with persistent symptoms despite maximum treatment as per gina guidelines are considered to have severe persistent asthma. omalizumab is a recombinant humanized monoclonal antibody licensed as an add-on therapy in these patients. the aim of this study is to assess the clinical benefit amongst responders to omalizumab therapy at a tertiary referral centre. this was a retrospective audit assessing the effects on asthma control, frequency of exacerbation and hospitalisation rates over 6 months before and after therapy. the study included 30 responders (14 females). there was a reduction in exacerbation and hospitalization rates following initiation of omalizumab, 73 and 91 %, respectively (p value .0001). the number of exacerbations decreased from 3.48 â± 2.20 to 0.93 â± 0.83 and the mean number of admissions from 1.07 â± 1.1 to 0.1 â± 0.40 over the study duration (p \ 0.001). there was 73 % reduction in the weekly need for rescue salbutamol with mean of 30.33 â± 6.49 puffs to 8.23 â± 1.51 puffs after omalizumab (p \ 0.0001). seventy-nine percent of patients were able to reduce their maintenance oral corticosteroid. in summary, responders to omalizumab therapy are less likely to experience an asthma exacerbation and hospitalisation. they were also more likely to reduce maintenance corticosteroid therapy as well as the need for rescue reliever therapy. these data suggest that omalizumab has proven effective in improving health outcomes for a cohort of carefully selected patients with severe allergic asthma in ireland. bronchial thermoplasty (bt) is a bronchoscopic procedure aimed at reducing the mass of airway smooth muscle and attenuating bronchoconstriction in severe asthmatic patients failing medical therapy. we report our experience with the first four patients treated with bt. between december 2011 and august 2012, four patients with severe asthma per gina guidelines, underwent three sessions of bronchial thermoplasty, 4 weeks apart. stringent entry criteria were required, including ongoing symptoms despite optimal medical management with the use of ics and laba's. two patients had a limited response to omalizumab. thus far, four patients met study entry criteria. three females and 1 male. the mean age was 61 years (sd 10.6). the frequency of severe asthma exacerbations was 4-6 per year. the mean fev1 and fev1/fvc prior to procedure was 63.6 and 56.4 %, respectively. the mean fev1 and fev1/fvc after the procedure was 62.1 and 60.3 %, respectively. patients reported a subjective strong improvement in quality of life post bt with more symptom-free days and less use of rescue inhalers. this emerging data relating to bt in an irish population is consistent with international data sets. bronchial thermoplasty is an additional treatment option for patients with severe asthma. approximately 40-60 % of patients with asthma have gastrooesophageal reflux (gor) and it has been postulated that this may worsen asthma severity. this study was undertaken to examine the incidence of gor in an irish steroid-dependent severe asthma cohort. 16 patients with severe asthma were recruited into this descriptive study from the severe asthma clinic in cork university hospital. our cohorts mean age was 52 years. the mean (sd) fev1 was 2.00 (0.58) l (73 % predicted). the mean time from asthma diagnosis was 28.2 (13.3) years with the patients being steroid dependent on oral steroid therapy for mean 7.3 (7.9) years with a mean dose of 8.75 mg prednisolone. 11 (69 %) reported symptoms of gor; with 14 being concomitantly treated with proton pump inhibitor. ten patients had undergone a barium swallow with five demonstrating gor radiological evidence. a further patient previously had undergone fundoplicative surgery. there was no association between gor and cumulative systemic steroid dose or fev1 in a subgroup analysis. in our study of steroid dependent asthmatics, 50 % of those formally assessed were found to have evidence of gor on barium swallow, which is consistent with reported research [1] . the incidence of gor did not depend on cumulative steroid exposure or fev1. introduction: incorrect inhaler usage is a significant problem in asthma management, resulting in poor control of asthma symptoms. the ability of patients to correctly use their inhaler might be directly linked to inhaler technique education. education may result in better inhalation technique, improved compliance and asthma control. the economic burden of asthma is very substantial and is one of the highest among chronic diseases. in the united states of america, approximately 5-7 billion dollars is wasted because of inhaler misuse per year (fink, 2005) . research question: ''what is the impact of a nurse-led education programme in promoting compliance with inhaler use in patients with asthma''. methodology: this is a quantitative study engaging a quasi-experimental pre-test and post-test design. a cohort of 21 patients who met the inclusion criteria were recruited from the out-patient department over a period of six months. during each stage, the patient was asked to demonstrate how they take their inhaler. any errors in technique were identified and rectified. their demonstration was measured through observation and the use of an inhaler proficiency schedule (ips). the participant was also asked a series of specific questions in relation to their condition, confidence level with self-administration of their inhaler, and adherence to prescribed frequency of use. the findings in this study show that inhaler education improves technique, promotes compliance and increases participant confidence levels in taking an inhaler, and as a result asthma symptoms improve. it also emerged that participants believed they were taking their inhaler correctly and so assumed that education drives were not targeted at them. 84 % (437) felt that the service provider had assisted in their success with therapy. overall, respondents use their devices as prescribed, keep in contact with their hospital department and service provider, and most feel their quality of life has improved as a result of starting treatment with cpap. obstructive sleep apnoea syndrome (osas) is characterized by repetitive upper airway (ua) obstruction during sleep. alcohol consumption increases osas severity by diminishing ua muscle tone, aggravating snoring and osas-related daytime symptoms. we hypothesized that behavioural adaptation could lead to reduced alcohol consumption in subjects with more severe osas. the influence of anthropometric, social and demographic variables, along with osas severity on alcohol consumption among subjects undergoing inpatient sleep studies was examined. regression analyses were utilised to identify independent predictors of alcohol consumption, and generate odds ratios (aor) for excessive alcohol consumption by osas severity. 926 subjects were assessed; 30.5 % were female, 64.7 % in paid employment, and 57.7 % married. 32.1 % had no osas [apnoeahypopnoea index (ahi) \5] and 25.2 % severe osas (ahi [30). alcohol consumption was 8.34 (â±12.0) u/week, with 10.7 % exceeding recommended limits. stepwise regression revealed male gender and employment status, but not ahi, as independent predictors of increased alcohol use. no difference in adjusted mean alcohol intake by osas severity class was observed. severe osas patients tended towards increased odds of excess alcohol consumption compared to those without (aor 1.76; 95 % ci 0.98-3.16; p = 0.059). increasing osas severity is not associated with lower alcohol consumption; rather, the reverse may be more likely. in our sleep clinic, ess is a poor predictor of ahi, sacs did not perform as expected, and a combined measure is of limited utility. following polysomnography diagnosis, untreated osas cases were assessed. cpap compliant subjects were re-assessed *12 weeks later. body composition was assessed by bio-electrical impedence analysis. sensewear armband ã� (swa) measured free-living ee. swa data was included if average weartime was [90 %. 15 subjects (10 male) (mean age, 54.7 years) were included. restrained-eating score was inversely associated with osas severity (14.9, 12.9, 12.7 in mild, moderate and severe, respectively). conversely, both uncontrolled eating score (17.7, 18.6, 19.6) and emotional eating score (5.5, 6.5, 7.1) were positively associated with osas severity. bmi (p = 0.006) and fat % (p = 0.003) were significantly higher in severe versus mild osas. cognitive-restraint was inversely associated with bmi and fat %, whereas both uncontrolledand emotional-eating were positively associated with these parameters. among this sample, more severe disease was associated with adverse eating behaviors. nutritional counseling targeting specific eating behaviors may be beneficial in osas. significant osa (ahi [5) was present in 80 % of the clinical group and 74 % of the screened group. no correlation of clinical significance was proven between ahi and ess in either group. ess provides useful information on subjective sleepiness but this study might suggest that it is not a reliable predictor of the presence of osa or its severity. decreased energy expenditure (ee) contributes to overweight. we investigated free-living ee and body composition in obstructive sleep apnoea syndrome (osas). following polysomnography (psg) diagnosis, untreated osas cases wore the sensewear armband ã� (swa) for *7 days, including 2 weekend days. swa quantifies free-living ee and physical activity (pa). data was included if weartime was [90 %. body composition was assessed with bio-electrical impedance analysis. upper airway muscle dysfunction is implicated in the pathophysiology of obstructive sleep apnoea syndrome (osas). pharyngeal dilator muscle inotropes may serve as adjunct therapies. we hypothesized that tempol, a superoxide scavenger, would increase sternohyoid muscle power under conditions of oxidative stress (hypoxia). excised sternohyoid muscles from adult male wistar rats, were connected to a dual-mode force transducer, between stimulating electrodes, in a bath of krebs solution at 35â°c, in either high oxygen (control) or low oxygen (hypoxia) â±10 mm tempol. stress and shortening were measured in muscles contracting from zero up to isometric load under tetanic conditions. peak power was determined. sternohyoid peak power was 2.4 â± 0.4 and 1.1 â± 0.2 w/cm 2 in control and hypoxic conditions (drug-free), respectively, and 4.2 â± 0.4 and 1.8 â± 0.5 w/cm 2 in control and hypoxic conditions (+tempol), respectively. two-way anova revealed that hypoxia (p \ 0.0001) and tempol (p = 0.003) were significant factors without drug-gas interaction. tempol increased the power-load relationship over the early (0-30 %) portion of the load step test and this was significant under hypoxic conditions. we conclude that tempol increases sternohyoid muscle power under control and hypoxic conditions. our results suggest that antioxidant therapy may be useful in the treatment of osas and other muscle weakness disorders. r. lee obstructive sleep apnoea syndrome (osas) is reported as common among ipf patients [1] . we determined the prevalence of the disorder in a cohort of ipf patients not on long term oxygen therapy and medically stable, excluding patients with active coronary disease and diabetes mellitus. 20 patients with ipf patients attending a specialized clinic underwent overnight polysomnography following a night of acclimatization. a quality of life questionnaire (sf-36) and epworth sleepiness score (ess) were also completed. statistical analysis was by student-t and man-whitney u-testing. 14 of the 20 patients were male and mean age was 67.9 â± 12.3 (sd). 75 % were current or ex-smoker. only 35 % of the patients received steroids at some time in their treatment. 9 patients had significant sleep-disordered breathing (sdb) based on the standard definition of ahi c5/h but only 2 were sleepy (ess c10), thus having osas. bmi correlated positively with ahi (r = 0.59, p = 0.006). bmi was 28.5 â± 4.6 kg/m 2 but higher in the osas/sdb group (p = 0.05). no difference in quality of life was evident between those with or without sdb or osas. we conclude that sdb and osas are as prevalent in ipf as a similar general population and bmi is the principal predictor of ahi in these patients. studies to date reveal wide variability (16-56 %) in the prevalence of fungus in the cf airway using culture-based methods. this study profiles the fungal microbiota of the cf airway using high-throughput-sequencing, and correlates this with standard culture-based methods and clinical phenotype. 55 clinically stable adult cf patients were prospectively recruited, donating one or more sputum samples. culture-based methods were employed at time of sampling. high-throughput bar-coded sequencing targeting the internal transcribed spacer (its) and small sub-unit (ssu) regions was used to profile the fungal microbiota, with subsequent sequencing on a 454 genome sequencer flx platform. baseline fev 1 % predicted, genotype, gender, bmi and pseudomonas status, were recorded by retrospective review of medical notes. in a total of 83 samples, culture-based methods detected fungus (aspergillus spp. and candida spp. only) in 13 patients. highthroughput-sequencing identified rich fungal communities in greater than 90 % of the patient sputum samples, with over 82 % of the species found not detected by culture. fungi detected included c. albicans, c. dubliniensis saccharomyces cerevisiae, malassezia spp., fuscoporia ferrea, fusarium culmorum, acremonium strictum, thanatephorus cucumeris and cladosporium spp. a comparison of patient status with diversity and species richness of fungal microbiota identified that lower fungal diversity associates with decreased lung function. aim: to identify common anaerobes and their proteases and assess their ability to cleave natural host innate human antiproteases such as alpha one antitrypsin (aat). method: we prospectively recruited 130 patients at our site in beaumont hospital. we obtained both sputum and bronchoalveolar lavage fluid (balf) at both stable and pre and post exacerbation timepoints. all samples were processed using both anaerobic bacteriologic techniques and 16s r rna sequencing methods. supernatants from p. melaninogenica were cultured in luria-bertani broth (lb) broth, (sigma l7275-100tab) and basal anaerobic media (bam) broth under strict anaerobic conditions in an anaerobic cabinet (davidson & hardy). protease production was measured using sensolyte red protease assay (anaspec). this assay measures matrix metalloproteinase (mmp) activity in broth. the days with highest protease production were recorded. native aat was incubated for selected time points with supernatant and cleavage products visualised by sds-page electrophoresis and western blotting analysis using specific antibodies raised against the antiproteases. results: using sputum and broncheoalvelar lavage from patients with cf, prevotella species accounts for 45 % of anaerobic samples identified from our group and p melaninogenica is the most common anaerobe grown from this group. the sensolyte red protease assay showed p. melaninogenica cultures produced the highest levels of active proteases on day 4, and 5. the western blot analysis demonstrated that when day 4 and 5 supernatants were incubated with aat, this antiprotease was degraded to give a distinct cleavage pattern. conclusion: this study is examining for the first time the pathogenicity of anaerobic bacteria found in cf lung and shows that the proteases produced by anaerobic bacteria are destroying host defense mechanisms and that this may impact other natural host innate antiproteases in the cf lung and play a role in inflammation. cf is a genetic disease with a high prevalence in ireland. in cf lungs chronic bacterial infection contributes to progressive respiratory failure. in particular, pseudomonas aeruginosa (pa), forms biofilms in the lungs which significantly contributes to antibiotic resistance. we have previously published on the importance of mif as a key inflammatory mediator in cf [1, 2] . building on this work, we hypothesised that mif enhances biofilm formation in the cf lung contributing to enhanced antibiotic resistance. using in vitro biofilm formation methods and qpcr we examined the effects of mif (100 ng/ml) on the growth, antibiotic resistance and gene expression of pa (strain pao1). our results to date have shown that mif significantly enhances biofilm formation of the pao1 strain of pa (p \ 0.05). in addition we have found a significant earlier induction of specific quorum sensing genes in response to mif. mif in pa cultures is associated with significantly less bacterial killing following antibiotic treatment. this raises the possibility of mif as an adjunct therapy with antibiotics by significantly this supports our hypothesis of mif inhibitors as an adjunct therapy improving the antibacterial effectiveness of antibiotics. macrophage migration inhibitory factor (mif) was one of the first cytokines to be discovered. mif is produced by a wide variety of tumours and is thought to play an important role in tumour progression. mif possesses a unique enzymatic activity linked to this role in cancer. to investigate this further we designed and evaluated a panel of small molecular weight inhibitors of mif and looked at their ability to block mif activity in vitro and in vivo. the small molecules were found to specifically inhibit the enzyme activity of mif when co-incubated with recombinant mif and its substrate. the inhibitors also significantly reduced cellular proliferation induced by treatment with recombinant mif (proliferation reduced by [45 %, p \ 0.0001) and significantly inhibited lpsinduced tnf-a production (tnf-a reduced by [48 %, p \ 0.001). in vivo, the inhibitors were found to reduce tumour growth in a subcutaneous model of lewis cell carcinoma (tumour volume reduced by [70 %, p \ 0.05). here we present data describing a number of novel small molecular weight inhibitors of mif found to be effective in vitro and in vivo. these inhibitors have the potential to be developed for therapeutic use in a cancer setting. idiopathic pulmonary fibrosis (ipf) is a progressive disease characterized by fibrosis. il-13 is a proinflammatory cytokine that has been shown to play a role in many fibrotic diseases including ipf. il-13 also induces the expression of, and binds to, one of its receptors, il-13ra2, which has been thought to function as a non-signaling decoy receptor. the cxc chemokine receptor 3 (cxcr3) and its ligands-cxcl9, cxcl10, and cxcl11-have been implicated in vascular remodeling and fibroblast motility during the development of the disease. in this study, cultured pulmonary fibroblasts from wild type and cxcr3-deficient mice were treated with various cytokines, and the expression levels of il-13ra2 and cxcr3 were measured. we demonstrate for the first time the expression of cxcr3 in cultured pulmonary fibroblasts from mice. also, il-13 was shown to downregulate basal and ligand-induced cxcr3 expression in fibroblasts. using wild-type and cxcr3-deficient animals, cxcr3 was found to be necessary for the il-13 mediated upregulation of il-13ra2, and blocking cxcr3 significantly reduced the basal expression of il-13ra2. manipulation of the cxcr3-mediated regulation of il-13ra2 or the il-13 mediated downregulation of cxcr3 may represent novel therapeutic modalities in cases of acute lung injury or chronic inflammation that may progress to fibrosis. epithelial cell to mesenchymal transition (emt), whereby epithelial cells undergo transition to a mesenchymal phenotype, giving rise to fibroblasts and myofibroblasts has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (ipf). alveolar epithelial cells (aec) are recognised to undergo emt in response to various stimuli including transforming growth factor-b1 (tgf-b1). comparison of gene expression, migration and chemokine secretion in normal and transitioned aec with primary pulmonary fibroblasts derived from normal and ipf patients was performed. a549 cells underwent 24 h (h) serum starvation followed by 48 h treatment with tgf-b1 10 ng/ml. total rna was extracted from a549 cells and primary human pulmonary normal and ipf fibroblasts. changes in expression of a panel of tgf-b target genes was determined by real time polymerase chain reaction array with subsequent validation. migration studies of the various cell types in response to serum and enzyme-linked immunosorbent assay (elisa) of cxcl5, cxcl8 and il-6 levels in cell supernatants were performed. transitioned aec assumed a mesenchymal phenotype, exhibiting a marked reduction in differential gene expression when compared to fibroblasts. migration in response to serum by transitioned aec was increased significantly compared to normal or ipf fibroblasts as was production of cxcl5 and cxcl8. background: aatd disease is a hereditary disorder leading to the development of emphysema. our group has published first evidence of upr activation within the endoplasmic reticulum (er) of zz monocytes. here we study mirna regulation of upr in healthy and emphysematous zz monocytes. method: monocytes mirnas were profiled using nanostring technologies. mirdip portal and kegg database identified mirna targets and gene networks. transfections with 60 nm anti-mir were performed using siport-neofx. mrna, mirna and protein were measured by qrt-pcr, taqman mirna assay and western blotting. results: sixty mirnas were differentially expressed in zz versus mm monocytes. mir-199a-5p is overexpressed by [40-fold and predicted to target multiple genes which are enriched for pathways in the er stress response. emphysematous versus healthy zz patients have decreased mir-199a-5p expression. mir-199a-5p inhibition increased expression of two arms of the upr; grp78 and atf6. conclusion: mirnas are differentially expressed in zz monocytes and may play a role in the upr. mir-199a-5p, predicted to target upr genes, is overexpressed in healthy zz monocytes, and negatively regulates the upr. emphysematous zz patients may have lost this protective mirna regulation leading to increased er stress in monocytes, contributing to the inflammatory milieu of aatd lung disease. 6.10 alpha-1 antitrypsin augmentation therapy is associated with decreased neutrophil adam-17 activity, plasma tnf-a levels and normalisation of neutrophil apoptosis alpha-1 antitrypsin deficiency (aatd) is characterised by neutrophil driven lung destruction and early emphysema in a low alpha-1 antitrypsin (aat) and high neutrophil elastase (ne) environment in the lungs of affected individuals. timely and effective neutrophil programmed cell death is essential for the resolution of inflammation and we have previously shown that neutrophils apoptosis is accelerated in aatd individuals. endoplasmic reticulum (er) stress is associated with the release of the pro-apoptotic cytokine tnf-a and, on the cell surface the activity of the sheddase adam-17 leads to the release of tnf-a from its membrane bound to its soluble form. the aim of our study was to determine if aat augmentation therapy can normalise the accelerated neutrophil apoptosis seen in aatd through inhibition of adam-17 activity and resultant tnf-a release. neutrophils were isolated from aatd individuals receiving aat augmentation therapy pre and post treatment. the kinetics of apoptosis were measured by caspase-3 cleavage utilising western blotting and cd16b expression by facs analysis. adam-17 activity measured using a fluorogenic peptide substrate. plasma tnf-a levels were measured by elisa. er stress was determined using the er stress marker grp-78. adam-17 activity was increased in individuals with aatd. in addition, adam-17 activity, plasma tnf-a levels and caspase-3 cleavage were reduced after augmentation therapy (p \ 0.05). cd16b expression was increased after therapy indicating normalisation of apoptosis. grp-78 expression was unchanged. from our data we have demonstrated that aat augmentation therapy can normalise neutrophil apoptosis by ameliorating adam-17 activity and resultant tnf-a release. the observed normalisation of neutrophil apoptosis may lead to reduced inflammation and a reduction in recurrent infections which characterises patients with aatd. to identify lung-selective mirnas, extracted rna was probed to mirna microarrays (mra-1001; 1,719 human mirnas), and results confirmed by taqman analysis. in silico analysis using targetscan and microrna.org identified genes targeted by identified mirnas. using a subtractive mirna strategy, 238 lung-selective hypoxic responsive mirnas were identified (anova p \ 0.05); including mir-125a-5p and mir-424. in silico analysis predicted that mir-125a-5p targets erythropoietin, which has a well-documented role to play in endothelial repair and angiogenesis. furthermore, mir-424 targets cullin 2, which has previously been shown to stabilize hypoxia-inducible factor-a and promote angiogenesis. we conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-selective alterations in mirna expression. mir-125a-5p and mir-424 may play important roles in pulmonary vascular remodelling and angiogenesis. further studies of these mirnas may uncover novel treatment strategies for hypoxic lung disease. this research project is funded by science foundation ireland. to assess whether mal plays a role in killing of intracellular mtb, we infected murine wild-type and mal knockout macrophages with virulent (h37rv) mtb. we found that mal deficient cells were unable to kill mycobacteria. human macrophage cell lines transfected with sirna against mal showed the same deficiency in killing mycobacteria. we then proceeded to evaluate key macrophage mechanisms of killing mycobacteria. we found that phagolysosomal maturation and autophagy were mal-dependent in murine and human macrophages. pro-inflammatory cytokine production was also mal dependent. we then sought to determine the effect of the common mal s180l polymorphism on macrophage responses to mtb. primary bone marrow derived macrophages from mice with the sl and ll polymorphisms displayed impaired mycobactericidal activity and phagolysosomal maturation. mal plays a critical role in determining macrophage responses to mtb through a pathway culminating in phagolysosomal maturation and killing of intracellular bacteria. asthma has been linked to the vitamin d deficient (vdd) state. we investigated whether vdd was associated with impaired lung function and inflammation. patients with respiratory symptoms (asthmatic and non-asthmatic) underwent spirometry and had serum analyzed for total immunoglobulin e (ige), high sensitive c-reactive protein (hscrp), eosinophil cationic protein (ecp), and 25-hydroxyvitamin d we examined 93 caucasians (mean age 52 years; mean bmi 26 kg/m 2 , mean fev 1 = 91.4 % predicted). mean 25(oh)d was 37.7 nmol/l. 76 % of recruits were vdd, 15 % were insufficient, while only 9 % were vitamin d sufficient. vitamin d levels were positively associated with fev 1 (r 2 = 0.01, p = 0.017). 92 % of patients with airway obstruction (fev1\80 % predicted) were vdd. ecp, hscrp and ige were non-significantly elevated in the vdd state compared to sufficiency. however, all patients with ige doctors' education about inhaled respiratory medication is extremely important in management of copd and asthma. while exercise-induced oxygen desaturation is a widely used clinical measure in ipf, data on sleep-related desaturation are lacking. we compared gas exchange during sleep and exercise in a cohort of ipf patients attending a specialized clinic not on long term oxygen therapy and medically stable, excluding patients with active coronary disease and diabetes mellitus. 20 ipf patients underwent overnight polysomnography, including transcutaneous carbon dioxide (p tc co 2 ) measurement, after a night of acclimatization. cardiopulmonary exercise testing was performed by incremental cycle ergometer. pulmonary function and awake arterial blood gases were also measured. statistical analysis included student-t and man-whitney u-testing. 9 patients had significant sleep-disordered breathing (sdb) based on an apnoea-hypopnoea index (ahi) [5. fev1 was 84.0 â± 18.7 % (sd) and diffusion (dlco) 51.1 â± 15.0 % predicted. pao 2 was 11.0 â± 1.4kpa and paco 2 5.3 â± 0.47 kpa. p tc co 2 rose by 1.13 â± 0.83 kpa during sleep (p \ 0.001) consistent with hypoventilation. the minimum oxygen saturation during sleep was lower than exercise (83 â± 7.41 vs. 92.6 â± 4 %), p = 0.007 and the fall in oxygen saturation was also greater during sleep (10.7 â± 7.0 vs. 4.8 â± 3.6, p \ 0.001). we conclude that ipf patients desaturate more during sleep than exercise and suggest that nocturnal oxymetry be considered part of the clinical assessment of such patients. diabetes mellitus (t2dm) causes increased risk of cardiovascular death, while glycosylated hemoglobin (hba1c) level predicts longterm cardiovascular mortality in non-diabetics. while obstructive sleep apnoea syndrome (osas) is associated with adverse cardiometabolic outcomes, it remains unclear if this effect is independent of obesity and other confounders. we examined the relationship of osas severity with t2dm prevalence and hba1c levels in a large european population. subjects attending university-affiliated sleep laboratories across 19 countries were prospectively assessed. all underwent overnight sleep studies, with bloods drawn to assess glycaemic health. the relationship of osas severity with t2dm prevalence, and hba1c levels in non-diabetics was examined with regression models adjusting for confounding factors, including obesity. 9,666 subjects were assessed, 72.2 % male, 50.8 % obese, and 80 % with an apnoea-hypopnoea index (ahi)[5 events/h. following adjustment for confounding factors, moderate and severe osas remained significant predictors of t2dm (adjusted odds ratio 1.51; 95 %ci 1.15-1.98; p = 0.003). in non-diabetics ahi (standardized b 0.101; p \ 0.0001), and mean spo 2 (standardized b -0.119; p \ 0.0001) were significant independent predictors of elevated hba1c levels. osas severity and nocturnal hypoxaemia predict both prevalent t2dm and hba1c levels even after rigorous adjustment for confounding variables including obesity, which may contribute to excess mortality in osas populations. background: portable devices that determine tst may act as an adjunct to level 3 diagnostic tests for osa. the swa is such and measures tst using a proprietary algorithm. calculation of tst could improve the accuracy of a level 3 diagnostic device. aim: correlation of tst by swa and npsg, in a population with and without sleep apnoea. 89 consecutive patients undergoing npsg because of a suspicion of osa wore an swa on the same night. patients were stratified by the presence and severity of osa. correlation coefficient for tst were determined between swa and npsg for all subjects and in the osa subgroups. results: the prevalence of a normal psg, mild moderate and severe osa was 22 (24.7 %), 31 (34.8 %), 12 (13.4 %) and 24 (26.9 %) of 89 subjects. and the respective correlation coefficients were r = 0.68, 0.74, 0.85 and 0.25. clinically important differences are presented with bland-altman plots (figs. 1, 2) . correlation of tst between the two methods was weakest in those with severe osa. conclusion: the determination of tst by swa in a population with severe osa is likely to be unreliable. npsg remains the gold standard for determination of tst. the relationship between lung cancer and pulmonary fibrosis remains poorly understood. the aim of this study was to conduct a descriptive analysis of clinical data collected from a cuh cohort of patients with both ild and lung cancer. a database of 637 patients with a histological diagnosis of lung cancer between august 2008 and december 2011 was reviewed. 35 patients with established ild on ct scan were identified. data from clinical notes and radiology patterns were reviewed and analysed. the male to female ratio was 1.7:1. all were smokers. 86 % of carcinomas in these patients were non-small cell lung cancer (nsclc). 49 % of patients had usual interstitial pneumonia pattern, 46 % had non-specific fibrosis, and 5 % had asbestosis. the overall median survival was 6 months (sem 0.725; 95 % ci 4.58 to 7.42). median survival for patients with early stage disease who underwent surgery (n = 13) was 7 months, followed by those who received chemo-radiotherapy (6 months), those who received no intervention (5 months), and those who received radiotherapy alone (2 months). survival for patients with lung cancer and ild was lower than published figures for patients with lung cancer alone. surgical candidates had the best survival though the survival benefit was very modest, while patients who received no intervention or radiotherapy alone faired very poorly. pleural ultrasound has a number of advantages over traditional imaging modalities with regard to visualisation of pleural pathology, in particular, pleural effusions. these include portability, the absence of radiation, dynamic imaging as well as increased sensitivity versus computed tomography scans in terms of differentiating between pleural fluid, thickening and masses [1, 2] . we present a case series of patients who underwent pleural ultrasound under the care of respiratory physicians trained in this technique in our hospital from january to august 2012. in total, 78 ultrasound scans were carried out in the 8 month period on a total of 65 patients using a portable ultrasound machine. the average patient age was 67.3 years (range 29-90 years) and 68 % were male. based on ultrasound findings, the physician proceeded directly to aspiration on 52 occasions (66 %) and a total of 13 chest drains were inserted (17 %). of those that were aspirated, the vast majority were exudative in nature (n = 45, 86.5 %). 13 (25 %) of the aspirates were due to malignant effusions. no procedure-related complications occurred. this case series highlights that imaging at the bedside is a feasible and, with the proper training, very safe method for managing pleural effusions. this audit shows that the vast majority of patients with mesothelioma are male with a poor prognosis regardless of therapeutic approach. approximately 60 % die in the hospital/hospice setting. finally, more patients with mesothelioma should be considered for clinical trials. aat deficiency (aatd) results from mutations in the serpina1 gene, classically presenting with copd and liver disease. the most common mutation causing aatd is the z mutation, with the s mutation weakly associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. ats/ers guidelines advocate screening all copd, poorly-controlled asthma, and cryptogenic liver disease patients, as well as relatives of known aatd individuals. over 8,500 individuals have been screened following ats/ers guidelines as part of the national aatd targeted detection programme. rare and novel mutations were identified by dna sequencing of the serpina1 gene. a number of rare serpina1 mutations including i, f, x christchurch , z bristol , and m malton were identified. the i mutation (arg39cys) was present at a relatively high frequency (0.0043) with over 70 cases identified. the f mutation (arg223cys) was found in 20 cases. in addition, 2 novel null mutations were identified, q0dublin and q0cork. current testing of suspected aatd cases is often limited and can miss rare and novel clinically significant serpina1 mutations. our findings underline the need for a comprehensive diagnostic work up of all patients with low aat levels including phenotyping, genotyping and if necessary, dna sequencing of the serpina1 gene. we previously observed that weaning-failure patients experience increased intensity of dyspnea (ajrccm 2009;179:a3808). we also observed that patients reported different qualitative sensations suggesting that more than one mechanism may contribute to dyspnea. the purpose of this study is to determine whether dyspnea experienced in weaning-failure patients is related to changes in pco 2 or increase in respiratory effort or both. methods: tracheostomized patients who were being weaned from prolonged mechanical ventilation at a specialized facility were enrolled. dyspnea, transdiaphragmatic pressure-time product (ptpdi), minute ventilation, and transcutaneous pco2 (ptcco 2 ) were measured during a 1-h trial of spontaneous breathing. patients who developed respiratory distress during the trial were considered weaning failures. patients who tolerated the trial and continued to breathe unassisted for at least 24 h after the trial without signs of distress were considered weaning successes. results: 28 patients were studied; 11 were women; age, 65 + 3 (se) years; duration of ventilation before the study, 28 + 2 days. fourteen patients were weaning successes; 14 patients were weaning failures. in the failures, dyspnea score increased from 4.5 + 0.8 at the start to 6.9 + 0.9 at the end of the trial (p \ 0.01). the increase in dyspnea in the failures was accompanied by increases in minute ventilation (p \ 0.01) and ptcco2 (p \ 0.01); ptpdi, an index of patient effort, remained unchanged during the trial. in the successes, dyspnea, minute ventilation and ptcco 2 did not increase during the trial; ptpdi, however, decreased from the start to the end of the trial (p = 0.04). these findings suggest that an increase in pco 2 , a major driver of minute ventilation, contributes to an increase in dyspnea during weaning failure. that the increase in dyspnea in the failures was not accompanied by an increase in ptpdi together with the successes exhibiting a decrease in ptpdi without any change in dyspnea suggests that effort is not a major determinant of dyspnea during weaning failure. conclusion: dyspnea increases in weaning-failure patients but not in weaning-success patients and the increase in dyspnea is accompanied by increase in pco 2 and minute ventilation but not by an increase in respiratory effort. background: respiratory disease constitutes one of ireland's greatest public health challenges. patients with respiratory disease are often undiagnosed despite symptoms and risk factors for lung disease. the purpose of this study was to determine the prevalence of respiratory symptoms and disease in a targeted population screening program. study design: subjects were asked to complete a questionnaire on respiratory symptoms and risk factors as part of well-publicised free spirometry testing on world spirometry day. multiple linear regression analysis was performed to identify factors contributing to variation in population fev 1 . logistic regression was used to identify predictors of airflow obstruction (fev 1 /fvc \70 %) followed by predictive model development and roc curve analysis to determine model diagnostic accuracy. results: ten centers throughout ireland participated in the study. analysis was limited to an initial discovery cohort of 555 patients (57 % female; age = 60 years (range 9-85); fev 1 = 100 % predicted(range 37-155 %). factors associated with reduced population ir j med sci (2012) 181 (suppl 10):s369-s437 fev 1 (% predicted) were smoking history, male gender, lower educational status and history of existing lung disease. in those with no history of lung disease (n = 372), 22 % had abnormal spirometry with 15 % demonstrating airflow obstruction. predictors of airflow obstruction were age, presence of cough and number of pack-years of smoking. presence of cough, age [60 years and exposure [15 pack years were associated with highest sensitivity and specificity for identifying airflow obstruction although predictive ability was only fair (roc auc = 0.70). conclusions: demographic and socioeconomic factors influence lung health in ireland. undiagnosed respiratory disease is common, particularly airflow obstruction and targeted screening is justified to identify patients with respiratory disease early. bronchiolitis affects one-third of babies in their first year of life. half of those hospitalised will have persistent cough and wheeze. to map this epidemic, we investigated all bronchiolitis admissions to tallaght hospital in the last 5 years. this will aid future planning of the service and provide an insight into the epidemic in the irish population. from 2007 until 2012, 1,408 children were admitted to tallaght hospital due to bronchiolitis. we analysed these on the basis of time of year of admission, length of stay, gender and age and compared them to national and international data. the busiest month was december, with 24.2 % of admissions. however, there was a significant increase in the incidence of bronchiolitis in the early spring of 2011 and 2012 (more than doubled) compared to previous years. the average length of stay is 2.92 days, male sex had 61 % dominance and average age was 30.29 weeks, in keeping with international data. there has been in a significant shift in the timing and incidence of bronchiolitis in tallaght hospital in the last 2 years. we explored the reasons for this, with special attention to rsv incidence, possible climate causes, vaccine programs and exposure risk. we identified 14 children, 8 males and 6 females. mean weight was 5.9 kg (range 2.53-13 kg). mean age was 8.1 months (range 9 days-35 months). stridor was the commonest presenting symptom 71 %. diagnosis was confirmed by micro-laryngobroncoscopy and supplemented by ct in 64 %. 83 % had complete tracheal ring stenosis and 85 % had concurrent cardiac anomalies. two patients had bronchus suis. extracorporeal life support (ecls) was utilized in one patient preoperatively. cardio pulmonary bypass (cpb) or ecls was utilised for the repair. laryngeal release was required in 3/14 patients. 8 patients underwent end-end anastomosis, 5 slide and 1 double slide tracheoplasty. a polydiaxanone suture was used for all anastomosis. mean (cpb) time was 93.7 min (range 25-255 min). mean cross clamp time was 31.5 min (range 24-39 min). mean length of ventilation was 5 days (range 0.5-16 days). mean icu length of stay was 12.5 days (range 2-60 days). there were two hospital mortalities. one patient only required re-intervention with balloon dilation. 92 % were symptom free on a mean follow up of 16.9 months (range 2 weeks-7 years). distal tracheal stenosis can be managed effectively utilizing cpb that also allows concurrent correction of congenital heart anomalies. mayo general hospital, 2 midlands regional hospital background and aim: attempting to reduce unnecessary attendances of well patients at outpatient clinics is prudent. this study evaluated the asthma control test (act)t and respiratory proforma, with feedback through mobile texts, in children with asthma, to determine attendance at clinic or not. methods: patients between 4 and 11 years with a diagnosis of asthma were eligible for inclusion. the parent was surveyed, by post, 2 weeks prior to the clinic date and asked to complete the asthma control test (act) and a respiratory proforma which assessed uacs symptoms, medication usage inclusive of intensification episodes and medical concerns. mobile telephone numbers were requested. parents mailed their responses in a supplied stamped envelope supplied. respondents were divided into two categories (a) act score greater than 19 and a non concerning respiratory proforma, who were texted not to attend the clinic but supplied with another outpatient appointment and (b) the remainder were texted to attend the clinic. results: over 13 clinics the parents of 199 eligible children were surveyed. one hundred and forty-one (71 %) replied of whom 103 (73 %) were well and did not attend the clinic but rebooked. of 38 who attended, 8 had new symptoms of uacs and 5 had pneumonia. of 58 who did not reply, 10 forgot to reply, 10 came to clinic with completed questionnaires, 6 had good control. thirty-five did not attend the clinic of whom 21 were discharged to the family doctor. conclusion: asthma care through postal survey with mobile text feedback is an option in the outpatient setting. background and aims: asthma is common in paediatrics with the most difficult to manage being those less than 5 years. this study evaluated the impact of a nurse delivered education program developed for parents of children under 5 years in terms of knowledge gained and parental empowerment. methods: twenty parents of children age 4-5 years were invited to attend five 1 h educational sessions which related to asthma pathophysiology, signs and symptoms, clinical assessment and medication use. a specific educational program was developed. prior to enrollment each parent was administered two surveys; (1) a previously tested asthma questionnaire containing 83 statements, and (2) a survey of parental concerns related to asthma. one month after the program was completed parents asthma knowledge and perceptions of empowerment were reassessed. results: while 20 parents were enrolled data sets for 19 were available for analysis. the pre-intervention mean knowledge level was 43.1 (52 %) (range 27-56) and post knowledge level was mean 65.5 (79 %) (range 61-72, paired t test �). the parental survey identified asthma recognition and poor coping skills as major themes which the educational program addressed. conclusion: a targeted asthma educational program improves parental knowledge and enhances parental empowerment. written action plans (w.a.p.) are recommended in international guidelines for the management of asthma [1] . despite this, uptake remains poor [2] . a qualitative prospective study of parents of children attending the paediatric asthma out-patient clinic at cork university hospital was performed to examine if; (1) written action plans are valued by parents. (2) they assist in recognition of symptoms. (3) parents commence appropriate treatment at home and identify when to seek medical advice as a result of w.a.p. (4) parents feel assured by possession of w.a.p. thirty parents of children aged 2-16 years were interviewed by the paediatric asthma nurse specialist to assess level of asthma control, knowledge of treatment and level of concern. parents were provided with a colour coded w.a.p. and all aspects of treatment were discussed. a follow up telephone interview was performed 6 months later. in the pre intervention group only 6/30 felt they had enough information to manage their child's asthma; this increased to 30/30 post intervention (p \ 0.05). 29/30 knew the location of their w.a.p.s. there were no incorrect responses regarding dose/frequency of medication. 29/30 subjects had dropped a level of concern regarding their child's asthma (p \ 0.05). with sufficient written information and education, the anxiety and concern that many parents undergo while managing a child with asthma, can be reduced. eight patients (5 male) underwent respiratory (ecls). there were 3-preterm, 3-term neonates, 1-infant and 1-child. indications included, congenital diaphragmatic hernia-3, bronchiolitis-2, primary pulmonary hypertension-1, pertussis-1 and complete tracheal ring stenosis-1. 75 % of patients were transferred to sweden or uk. eight children (6 males) underwent 9 (ecpr) runs, with a mean age of 2.1 years (range 5 weeks-8 years). 7/8 had underlying congenital heart disease, of which 2 had univentricular pathology. mean conventional (cpr) time before initiation of (ecpr) was 55 min (range 35-90 min). in the respiratory (ecls) cohort mortality was 87.5 %. the only survivor was treated in ireland. in the (ecpr) cohort our survival rate of 50 % exceeded the international extracorporeal life support organization published results of 40 %. all ecpr patients were treated in ireland. currently there is no funding for pediatric respiratory (ecls) in ireland. patients are being treated abroad at significant expense, family inconvenience and mortality. these results would suggest a change in health policy is overdue! recent evidence has confirmed a high prevalence of bronchiectasis and impaired lung function in school aged children with cf despite little in the way of symptoms of lung disease in this group in preschool years. if we are to significantly improve long term outcomes in cf we must gain a greater understanding of lung disease in the preschool years and intervene earlier with disease modifying treatments before irreversible lung disease occurs. the key to understanding early lung disease in greater detail lies in the design of robust, comprehensive, well powered longitudinal studies. shield cf was established in 2010 with these requirements in mind and is a framework through which we can start to answer some important questions. shield cf is centred around the annual cf bal surveillance programmes in our institutions. currently the shield cf programme includes: â�¢ bal-immediately processed, aliquoted and biobanked â�¢ whole blood-immediately processed, aliquoted and biobanked â�¢ oropharyngeal swab-processed for rna extraction â�¢ clinical information-baseline and ongoing information related to lung health entered onto online database from individual centres. in the future we plan to include: â�¢ lung function measured by lung clearance index (lci) â�¢ lung structure determined by chest ct to date 68 patients have been recruited with a total of 100 samples (n = cf, n = control). shield cf has contributed samples to 4 different international multicentre studies. table 1 below summarises key baseline findings within shield cf to date. within the next year shield cf will incorporate 100 preschool children between the three centres. aim of our study was to audit hospitalization for copd exacerbations with respect to patient characteristics, diagnosis and standards of care. all patients admitted to roscommon hospital with a diagnosis of aecopd from july 1st to december 31st 2011 were included. medical notes were reviewed for data collection. 51 patients were included in the study. there was a frequent failure to objectively confirm the diagnosis of copd by spirometry. only 11 (21.5 %) patients had spirometry performed at any stage. 13 patients were current smokers. inhaler technique was assessed in only 13 patients. 24 (47 %) patients received chest physiotherapy. 2 out of 13 current smokers had documented smoking cessation advice, and received smoking cessation pharmacotherapy. 47 (92 %) patients were treated with antibiotics. management of aecopd in our hospital is frequently suboptimal, and may be managed better with respiratory physician involvement. there should be more frequent spirometric confirmation of diagnosis, more conservative use of antibiotics, better screening for ltot and improvement in smoking cessation service. many copd patients return for review at the respiratory outpatient department when clinically stable. they are often reviewed by less experienced nchds who may lack the knowledge and confidence to discharge them. furthermore many beneficial clinical and lifestyle interventions are not commenced. through a series of pdsas, a checklist was developed and implemented to ensure that copd patients received appropriate interventions and highlight which patients could be safely discharged to the care of their general practitioner. this combined a checklist of criteria for optimisation of copd patient management devised in the respiratory department as well as both the validated copd assessment test (cat) and modified medical research council dyspnoea scale. discharged patients had a cat score of b15 and no significant outstanding treatment modifications. the checklist was completed in 54 copd patients of whom 12 (22 %) were suitable for discharge. interventions such as optimised pharmacological therapy, assessment of inhaler technique and vaccination education were not instituted in 15, 23 and 86 % of patients, respectively. we conclude that an opd discharge checklist is an appropriate intervention to improve quality of care for patients with copd and facilitate the discharge of stable patients from a respiratory opd. pulmonary rehabilitation (pr) is a multidisciplinary approach to improving the exercise capacity and symptoms of people with copd and ild. however, compliance is often suboptimal. this study investigated whether the education and literacy level of patients may affect attendance and completion of pr. patients were divided into two groups based on diagnosis; copd or ild. nine factors were studied: sex, age, baseline activity level, education, literacy, social isolation, transport to programme, oxygen requirements and anxiety and depression scores. completion of pr was defined as attending [65 % of the classes. our findings demonstrated that 15 % of copd patients failed the literacy test compared to 0 % of ild patients. 29 % of copd patients had only primary level education in contrast to 0 % of ild patients. 29 % of copd patients completed third level education compared to 57 % of ild patients. 22 % of copd patients travelled to pr in their own car in contrast to 86 % of ild patients. 43 % of copd patients got public transport to pr compared to 0 % of ild patients. 86 % of copd patients and 71 % of ild patients completed pr. although there were significant differences in educational achievements between groups, this did not affect their compliance in completing pr. osteoporosis has not been fully evaluated in copd patients. the development of osteoporosis among copd patients is multifactorial. the objectives of this study were: (1) to explore the prevalence of osteoporosis among copd patients, (2) to observe any correlations between t-scores and different disease related variables. 60 copd patients attending respiratory clinics were randomly assigned for dexa scanning. 28 pts were excluded because of the coexistence asthma, age [80 years and early menopause. the mean age of the studied patients was 67 (range 52-78) years, and 69 % were female. mean fev1 of these patients was 55 %. seventy-two percent had osteoporosis, and 22 % had osteopenia. mean t-score was -2.4 (male -3.2, female -2.1). t-score was noted to have positive correlation with age (r = 0.484, p = 0.005), but no correlation with bmi (r = 0.173, p = 0.314) and fev1 (r = -0.12, p = 0.513) was noted. statistical difference in t-score was observed between patients with normal/reduced mobility vs. poor mobility (p = 0.045) but no difference was observed among patients with steroids inhalers alone (over the last 2 years) or in combinations with oral steroids (p = 0.825). very high prevalence of osteoporosis was noted among our cohort of copd patients. surprisingly, there was no association of bmd with fev1, bmi and corticosteroid exposure. the aim of this study was to determine the benefits of standardised reassessment of long term oxygen therapy (ltot) patients. long-term oxygen therapy (ltot) is the treatment proven to improve survival in chronic obstructive pulmonary disease (copd) patients with chronic respiratory failure. this study was prompted by an absence of any formal or regular assessment of ltot after initial prescription. the patient's oxygen requirements were assessed carrying out abg analysis after the patient had been taken off oxygen for thirty minutes. the patient then participated in a six minute walk test (6mwt) to determine the need for ambulatory oxygen. 15 of the 30 patients contacted attended for ltot reassessment. 20 % patients no longer met the criteria for ltot i.e. pao 2 [7.3 kpa. 33 % required a decrease in their static oxygen requirements. 46.67 % required an increase in their ambulatory oxygen requirements. 13.33 % needed a prescription for ambulatory oxygen. 33 % had a sub therapeutic oxygen prescription. in total 100 % of participants required a change to their oxygen prescription. current procedures for the assessment of ltot result in a large proportion of recipients not having appropriate prescription. there is a need for the initiation of standardised regular reassessment of all ltot patients. with increased ambulatory treatment of copd exacerbation, there is a need for opd assessment of blood gases and ph. we looked at the correlation between arterial blood gas (abg) and capillary blood gas (cbg) in a stable population of opd patients with copd. patients attending for oxygen assessment had a capillary blood sample taken from the fingertip pulp. this was analysed using the epoc ã� point of care analysis system. an arterial blood sample was obtained from the radial artery and analysed using a radiometer blood analyser. 15 patients attended for oxygen assessment. we used pearson's correlation to compare the results of abg and cbg. it showed a moderately high correlation of pco 2 at 76 % (p = 0.001). a correlation of 80 % with po 2 (p = 0.001). the hco 3 and base excess correlations were extremely high at 96 % (p = 0.000) and 97 % (p = 0.000), respectively. independent sample t tests were used to look at the agreement between abg and cbg values. it showed that cbg could be useful in predicting ph, hco 3 and base excess but not very useful clinically in predicting pco 2 and po 2 . cbg could be used in the opd setting to monitor blood ph and may be useful in copd outreach assessments. identifying lung disease early is very important and can be done with spirometry testing. early detection of these diseases can greatly improve outcomes and quality of life for patients. members of the public were given the opportunity to have their lung function tested for free, as part of the world spirometry day 2012 campaign. spirometry was performed by 3 respiratory scientists. access to smoking cessation advice, inhaler technique and the benefits of exercise to maintain healthy lungs were also provided. 142 patients were tested. 38 % (n = 55) were male and 61 % (n = 87) were female. 16 % (n = 24) were under 44 years. 83 % (n = 118) were [44 years. 15 % (n = 22) reported that they had a previous lung function test. 80 % (n = 115) reported they never had a lung function test performed. 2 % (n = 3) did not answer. 36 % (n = 8) of those previously tested reported a history of known lung disease. 7 % (n = 9) who were never tested reported a history of lung disease. 45 % (n = 65) were non-smokers, 17 % (n = 25) were current smokers, 34 % (n = 49) were ex-smokers and 2 % (n = 3) unknown smoking status. 64 % (n = 92) achieved normal spirometry. 29 % (n = 42) results were abnormal. 5 % (n = 8) results were unreliable. of the 42 abnormal results; 90 % (n = 38) demonstrated obstructive lung disease, 2 % (n = 1) restrictive lung disease & 7 % (n = 3) mixed lung disease. implementing and evaluating palliative care responses for patients with advanced respiratory disease. this multi-site action research project used mixed methods data collection strategies including qualitative interviews with patients and families, expert focus groups and quantitative methods such as education surveys, evaluation and chart audits. study findings demonstrate that palliative care interventions can be implemented within respiratory services for those patients with advanced disease. the study methodology of action research ensured that all key stakeholders in the service delivery across several sites contributed to sustainable organisational change which delivered measurable care improvements for patients throughout the research process. interventions that have developed include; shared training and education across sites, multi-disciplinary team meetings, pulmonary rehabilitation session on coping, death reviews and the development of a respiratory palliative care pathway for patients. palliative care interventions are feasible within care delivery models for patients with advanced respiratory disease. referral links and a care pathway between the hospital and hospice settings are at the cornerstone of this care delivery model. half of all patients admitted with copd in ireland are either dead (8 %) or re-admitted to hospital (41 %) within 90 days [1] . variation in the management of copd patients may contribute to this. implementation of care pathways has been suggested to improve outcomes such as mortality, admission rates and length of stay. the acmb was introduced as a sticker in the healthcare record in feb 2012. a cross-sectional audit reviewed staff practice prebundle implementation, post-bundle and following an educational drive. 8 % of patients in the pre-bundle group received nebulised bronchodilators within 30 min of presentation, in comparison to[50 % in the post-education group. prescription of oral corticosteroids improved with a corresponding decrease in patients receiving none. there is ongoing use of intravenous corticosteroids ([40 %). the use of intravenous antibiotics was unchanged at approximately 40 % although over 80 % of patients received an antibiotic recommended in the acmb. 58 % of pre-bundle patients received an abg, 22 % within 30 min; following the educational drive this increased to 100 % with 30 % within the timeframe. further education may be required to decrease the frequency of intravenous medications along with examination of barriers to managing patients within the acmb timeframe. the recent ers audit of irish patients with exacerbations of copd was the first nationwide assessment of survival and readmission rates for this patient cohort. it showed an average length of stay of 7 days, a 90 day readmission rate of 41 % and mortality rate of 8.3 %. our copd outreach provides supported or pre-emptive discharge as an alternative to hospital treatment for copd exacerbations. we undertook an audit of patients treated by the outreach team over 10 years to compare rates of these values with those from the ers audit. among 234 patients who died in this period 20 (11 %) died in the 90 day period from discharge and 356 (39 %) were readmitted. of those that died, 100 % were readmitted within 90 days and 95 % died in hospital within a short period of readmission. of overall readmissions (186) 20 % were from early discharge programme, 132 (14 %) assisted and 39 (4 %) prevent readmissions. of note 0 % of prevent readmission patients died within 90 days. the median length of stay was 2 days. these data indicate that survival and readmission rates are the same whether patients are treated in hospital or by outreach teams, however, with shorter lengths of stay the later is associated with substantively lower costs. future research will need to focus on identifying the factors that contribute to the high readmission rates. the central purpose of pulmonary rehabilitation is to reduce morbidity by improving functional capacity through exercise. it is still unknown if improvements in functional capacity are maintained in the long-term and lead to increased physical activity levels. the hypothesis of this study was that pulmonary rehabilitation would lead to a sustained increase in standard outcome measures and in daily physical activity. a prospective study of 47 subjects with copd was performed, registered at clinicaltrials.gov (clinical trial number nct 0112943). the primary outcome was a maintained improvement in standard outcome measures with a secondary aim of an increase in daily physical activity. a convenient sample of the cohort (n = 17) was re-evaluated at a third time point at 1 year. a 7 weeks hospital based outpatient pulmonary rehabilitation programme led to a significant reduction in total energy expenditure (p \ 0.044) and breathlessness (borg, p \ 0.011) and improved exerthese findings show that while pulmonary rehabilitation increased exercise capacity this was not transmitted into increased daily physical activity. alternative methods to alter/affect behavioural change may need to be addressed. the national clinical programme for chronic obstructive pulmonary disease (copd) aims to improve quality, access and cost of care for patients with copd. pulmonary rehabilitation (pr) has been proven to meet these aims. the purpose of this study was to audit pr throughout ireland. hospitals and all local health areas (lha) were surveyed about prp in their catchment areas -ongoing programmes, length of and numbers on waiting lists, and enrolments in prp in the first 6 months of 2012. those without access to prp were asked about barriers to setting up such programmes. descriptive statistics were used. all hospitals (n = 39) and lha (n = 32) responded re access. 84 % of hospitals (n = 33) and 47 % of lha (n = 15) have access to pr. hospital settings have mean waiting lists 12.6 months(1,18), mean numbers waiting 75(6,160) and mean number enrolled 37(5,49), (respondents = 14). respondents (n = 2) from the community showed waiting lists of 6 and 10 months, numbers waiting 40 and 95 and numbers enrolled 27 and 31. 'black spots' with no access were identified. barriers that may be amenable to intervention include increased support for primary care teams, facilitation of appropriate referrals and the development of spirometry services in the community. patients were selected as per gold guidelines. the duration of the study was 12 weeks. four primary care centres were selected, one each in longford, athlone, tullamore and mountmellick. one spirometry clinic was held every week on a rotational basis with the intention of accommodating 10 patients per clinic. it was free service and intended for patients who had not previously undergone spirometry testing. the gp practise booked patients directly into the spirometry clinic. a total of 104 patients were booked across the 4 spirometry clinics. of the 104 patients booked, 9 % did not attend and 7 % cancelled their appointment. 3 out of the 4 clinics utilized all available slots with one clinic utilizing only 50 % of available slots. a total of 82 patients were tested. out of these, 35 had normal spirometry, 11 had gold stage 1 mild copd, 22 had stage 2 moderate copd, 5 had stage 3 severe copd and 3 had stage 4 very severe copd, 2 had possible restrictive lung disease and 2 unreliable data. success and efficiency of this type of service is heavily dependent on spirometry clinics being gp driven. a number of advantages, disadvantages and recommendations arose out of this study. 9 .15 an audit of steroid and antibiotic therapy for acute exacerbations of copd in a cork hospital sought to determine compliance with these guidelines in the mercy university hospital, cork. a retrospective review was conducted on patients admitted with exacerbations of copd from 1st june 2012 to 1st august 2012. forty-six patients attended the ed with exacerbations of copd. thirty-three charts were available for review. of the 33 patients, 18 (54.5 %) were prescribed iv hydrocortisone, despite being able to take oral prednisolone, 9 (27.3 %) were prescribed oral prednisolone and 6 (18.2 %) were not prescribed steroids. eight patients were prescribed co-amoxiclav alone, 1 patient was prescribed clarithromycin alone and no patients were prescribed doxycycline alone. four were treated with piperacillin/tazobactam, 1 patient with piperacillin/tazobactam and linezolid, 7 patients with co-amoxiclav and clarithromycin, one patient with moxifloxacin, 2 patients with ciproxin and for 9 patients, antibiotics were not prescribed. of the 7 patients prescribed co-amoxiclav and clarithromycin, 4 had infiltrates on cxr. the prescription of antibiotics and steroids in the muh in patients with acute exacerbations of copd did not meet guidelines as per the national copd acute management bundle. though niv significantly improves outcomes in acute copd patient care, there is no compulsory niv training programme for nchds. we assessed nchd's knowledge of the use of niv in copd using a questionnaire, based on bts guidelines, with reliability assessment in a subset of nchds. experience level, knowledge of niv contraindications and settings were examined. 45 questionnaires were completed. inter-rater agreement was good (kappa 0.77, se 0.07, 95 % ci 0.62-0.92). 58 % had previously worked in respiratory medicine. 80 % had commenced patients on niv and/or titrated settings on call. 19 % had received training in the use of niv. 49 % adjusted niv settings incorrectly for hypercapneic acidemia. 80 % used epap incorrectly. those with respiratory work experience had greater niv titration experience (p = 0.0002) and superior knowledge of niv settings (p = 0.009). though 55 % of interns had previously titrated settings, their confidence tended to be lower (p = 0.09) and their knowledge of niv settings was significantly inferior to more experienced doctors (p = 0.001). though niv is widely used by nchds of all grades, the present study shows training and knowledge deficiencies, especially among interns and those not in a respiratory post. there is a need for structured training in this key skillset among nchds. table 1 . conclusions: approximately one-third of the patients in 2011 who presented to the ralc were classified as asymptomatic. approximately 2/3 of asymptomatic and only 1/3 of symptomatic patients had early stage cancer (1 and 2) which was a statistically significant difference. there was no difference in smoking history gender, tissue type and ecog status at presentation between symptomatic and asymptomatic patients. lung cancer is the most common cause of cancer death in ireland. a recent large screening trial demonstrated a 20 % survival advantage with low-dose ct over chest x-ray [1] . however, the false positive rate of 96.4 % for screening detected nodules is a major drawback. a lung cancer biomarker would lead to improved specificity, reduced costs and a reduction in unnecessary procedures for patients with ct-detected pulmonary nodules. biomarkers would also be useful for monitoring response to therapy or disease progression and may reveal the molecular mechanisms underlying cancer development. proteomics represents an important tool for identifying novel cancer biomarkers. recent advances in mass spectrometry allow rapid and accurate analysis of several thousand proteins in a single study [2] . we prospectively recruited 185 patients presenting to beaumont hospital rapid access lung clinic between april 2011 and july 2012. paired bronchoalveolar lavage (bal) and serum samples were obtained. patients were subsequently grouped after clinical and mdt follow-up into (1) benign, (2) possible (for surveillance), and (3) confirmed lung cancer. samples were then analysed by orbitrap mass spectrometry and candidate lung cancer biomarkers identified based on the clinical and histological characterisation. principal components analysis (pca) of the 5215 peptides found to be differentially expressed between control bal and cancer bal was performed to determine any outliers in the data and also to identify group clustering as per diagnosis. leave-one-out cross validation (loocv) of a 5 protein plasma combination demonstrated an accuracy of *78.7 % (auc: 0.813) for distinguishing benign conditions of the lung from cancer (sclc/ nsclc) (fig. 2) . further analysis and validation is underway. trapped lung after malignant pleural fluid drainage is a contra-indication to talc pleurodesis. vats identifying patients amenable to talc may be prohibited by co-morbidities. initial experience with a miniinvasive alternative is described in three patients, two transferred with debilitating dyspnoea and previous multiple admissions for drainage procedures. ultrasound was performed and pleurx ã� insertion under la (single in 1 patient, bilateral in 2), with palliation of symptoms facilitating discharge in all three. a rapid access lung cancer clinic (rac) with radiological support was recently introduced aiming to both decrease the time to diagnosis of patients, whether negative or positive, and also to decide the most appropriate investigation for each patient with on-site radiologist support. in this observational study, we used a historical cohort of the last fifty red flag patients assessed at the respiratory clinic before the introduction of the rac, and compared it with the first thirty-five patients seen at the rac. time to diagnosis and the number of invasive investigations [bronchoscopy/fine needle aspiration (fna)] were compared as outcome measures. continuous variables were compared using mann-whitney u test and categorical variables using fischer's exact test. patients were similar in terms of demographics. there was a statistically significant reduction in the time to diagnosis (p \ 0.0001, mann-whitney u), whether negative or positive, following the introduction of the rac (fig. 1) . there was also a statistically significant reduction in the number of bronchoscopies (p \ 0.006, fischer's exact test) carried out after the introduction of the rac. these results demonstrate that the rac with radiological support decreases both the time to diagnosis and the number of invasive investigations that may not have yielded a diagnosis. tbna has been widely available for sampling mediastinal lymph nodes (ln) for over two decades. unfortunately, blind tbna has low sensitivity and limited access to only certain ln stations. ebus-tbna has revolutionised ln sampling demonstrated in many prospective multicentre trails. over a 13-month period, 34 patients with evidence of lymphadenopathy on chest ct, underwent ebus-tbna. 36 procedures were performed on 34 patients (23 males, mean age of 60.9 years). diagnostic yield, and sensitivity were calculated by reviewing clinical notes, radiological imaging, cytology, transbronchial/endobronchial biopsy, bal, and mediastinoscopy reports, and redo ebus-tbna reports from another centre. in 75 % of procedures, ln were sampled. 33.3 % of ebus-tbna samples were diagnostic (3 cases for sarcoid, and 8 for lung cancer). diagnostic yield was compared in 1st 6.5-month period versus 2nd and returned 25 versus 41.2 %. sensitivities for sarcoidosis and lung cancer were calculated at 75 and 57.1 %, respectively. review of current data shows diagnostic yield and sensitivities varies significantly, however, our results were below current published standards. radiologically guided lung biopsy is a relatively safe procedure to reach a histological diagnosis for suspicious lung lesions. we audited the performance of university hospital galway against the bts guidelines. this was a cross-sectional study. all patients who had radiologically guided percutaneous lung biopsies between january 2009 and october 2011 were included. primary outcome was the ability to reach a histological diagnosis. secondary outcome was development of pneumothorax or other complications. 94 biopsies were performed. 51 were males and 43 were females. mean age was 69.7 years. mean lesion size was 3.8 â± 2.2 cm. the procedure was done under ct-guidance in 84 patients (89.4 %), fluoroscopy in 7 patients (7.4 %) and ultrasound in 3 patients (3.2 %). the overall diagnostic rate for benign and malignant causes was 88.3 %. malignancy was diagnosed on 78 biopsies (83 %). sensitivity for detection of malignancy for lesions [2 cm in size was (94.3 %). the procedure was complicated by pneumothorax in 26 patients (27.7 %). only 5 patients (5.3 %) required chest tube insertion. we achieved a higher diagnostic rate than the level set by the bts but pneumothorax rate was slightly higher. this could be because the majority of our samples were taken using large bore cutting needles. sarcoidosis is a systemic granulomatous disease of unknown aetiology that primarily affects the lung. several reports have suggested that analysis of cd4+/cd8+ lymphocytes can be used to differentiate sarcoidosis from other causes of interstitial lung disease. the aim of this study was to evaluate the diagnostic utility of bal fluid cd4/cd8 ratio in diagnosing pulmonary sarcoidosis. this was a retrospective cohort study. study population included all patients who had bal fluid obtained during fibre-optic bronchoscopy in university hospital galway between november 2008 and june 2010. outcome variable was cd4+/cd8+ ratio as calculated on flow-cytometry performed on bal fluid. 104 patients got bal fluid analysed. 75 (72.1 %) were found suitable for analysis by flow-cytometry. 71 had a transbronchial biopsy performed. out of 37 patients with histological evidence of sarcoidosis, only 31 patients had bal fluid suitable for analysis. 19 (61.3 %) of them had a cd4/cd8 ratio higher than 3.5. sensitivity of cd4/cd8 ratio [3.5 in diagnosing pulmonary sarcoidosis was 61 % with a specificity of 67 %. positive predictive value was 73 % and a negative predictive value was 53.8 %. the distribution of cd4/cd8 ratios in patients with biopsy-proven sarcoidosis suggests that substitution of bronchoalveolar lavage cellular analysis for transbronchial biopsy is not advisable. we previously reported that smoking cessation (sc) services are available but lacked uniformity or consistency countrywide [1] . we found that (45 %) of service providers (sp) were collecting and analysing data on pregnancy status but 30 % did not analyse it and 11 % did not collect any data on pregnancy. 1 we aimed to look at outcomes of sc in pregnancy, provide evidence based data to improve services for this vulnerable population and aid their evaluation and effectiveness. using a census of all known smoking cessation programmes throughout ireland [1] , sps were asked to pilot a treatment database for a 3 month period. many different data were entered including pregnancy status and treatment outcomes. the data were returned to tfri for analysis using a statistical package spss. a database was piloted over a 3 month period by 39 sps. a convenience sample of 1,490 patients was recruited while attending sc service throughout ireland and their data were entered into the database. a total of 118 pregnant women attended the smoking cessation services during this period which represents 15.6 % of the females treated. they achieved a quit rate of 16.5 % at 4 weeks and 16.5 % at 3 months compared with 35.2 and 20.3 % of the rest of the female population treated in the same time period. the poor outcomes may be a result of the paucity of services in ireland for this population group. the findings will facilitate planning and delivery of effective sc programmes to pregnant women ensuring equity in service provision. maternal smoking in pregnancy is an important risk factor for low birth weight (lbw) (\2,500 g) and prematurity (\37 weeks gestation) [1] . the purpose of this study was to examine smoking rates and prevalence of associated adverse birth outcomes in the coombe women and infants university hospital, dublin over a 10 year period 2000-2009. a cross-sectional observational study was conducted using routinely collected data from the euroking k2 maternity system from january 2000-december 2009 (n = 77,533). smoking prevalence declined significantly from 29.6 to 17.4 % over the period. rates in teenage mothers remained very high (44.3 % in 2009). smoking prevalence was almost twice as high in mothers of lbw compared to normal birthweight babies, one and a half times higher in mothers of preterm babies compared to full term and more than twice as high in mothers of small for gestational age (sga) babies compared to non sga. a statistically significant decline was seen in the prevalence of sga babies in the period. no statistically significant change was seen in the prevalence of lbw or preterm babies. prevalence rates in pregnancy are high in ireland compared with other developed countries. increased focussed efforts are needed to reduce smoking rates. smoking-related diseases account for over 7,000 admissions per year in ireland. 50 % of all smokers will die of smoking-related disease, and smokers on average die 10 years younger than non-smokers. the aim of this project was to assess attitudes and beliefs in smokers and ex-smokers. patients who were smokers or ex-smokers were interviewed using a standardised questionnaire. candidates were recruited from respiratory clinics and general-inpatient wards in beaumont hospital. data regarding underlying medical history was acquired from medical records. 104 patients were included, 54 ex-smokers and 50 smokers. there were no differences in gender or medical history. ex-smokers had a mean age of 66.5 years compared to 56.9 years in smokers (p = 0.0017). smokers had longer smoking history; 39.14 years compared to 32.9 years (p = 0.047). smokers reported lower expectations regarding the benefits of smoking-cessation than the exsmokers; only 60 % believed there would be short-term health benefit and only 74 % believed quitting was worthwhile compared to 88.9 and 94.4 %, respectively, in the ex-smoking group (p = 0.0025/p = 0.01) there is significant variation amongst smokers and ex-smokers regarding attitudes to smoking-cessation; this is despite receiving the same smoking-cessation advice and having low fagerstrom-scores. of note current-smokers were younger but had longer smoking histories and smoked from an earlier age. results: a total of 248 patients have been referred, 63.7% (n = 271) male and 36.7% (n = 157) females. the mean age at referral was 63.9years. 44.6% of patients admitted to being current smokers, of these 61.3% were male and 38.7% were female. 34.1% had a previous history of smoking 43.9% of patients were referred from their gp, 39.7 % were referred from another consultant based with the mwrh and 12.6% from another centre. a total of 134 patients referred to the clinic subsequently had a diagnosis of cancer. 113 patients had a diagnosis of lung cancer (adenocarcinoma = 36, scc = 48, other nsclc = 14 and small cell = 15). 21 patients were diagnosed with a malignancy other then lung cancer, the most common being lymphoma, or metastatic lung disease. male patients accounted for 69.9 % (n = 93) and female 30.1% (n = 40) of those diagnosed with cancer, with a mean age across both genders of 68.27years. conclusions: the results of the audit would suggest that the ralc clinic is in line with national trends as regards smoking prevalence and age. the referral pathway needs to be the focus for the future facilitated by early referral from gp's. this will enable the ralc clinic to prioritise prompt investigations and treatments to optimise survival. sarcoidosis. we aimed to assess the diagnostic accuracy of ebus[1] . twenty ipf patients attending a specialized clinic were recruited. their blood samples were collected. full pulmonary function and awake arterial blood gas, high resolution computed tomography of chest (hrct) were performed. ccl 18 was quantified using a enzyme-linked immunosorbent assay. ccl 18 levels are presented as median â± interquartile range. correlations were analysed using pearson and simple regression. median concentration of ccl18 was higher in ipf patients [64,149.5 pg/ml (43,673.9-94,731.23)] compared to healthy controls [28,600 pg/ml (15,800-35,700), p = 0.047] [2] . ccl 18 levels were inversely correlated with diffusing capacity for carbon monoxide (dlco) (r = -0.55, p = 0.019)* and awake carbon dioxide (co 2 ) tension (r = -0.59, p = 0.008*). positive correlations were observed between a-a gradient and ccl 18 concentration (r = 0.646, p = 0.007*) and co 2 rise during sleep (r = 0.73, p = 0.011*) but no relationship between ccl 18 and fibrosis score, mean reticulation score or mean ground glass score on hrct. ccl 18 levels correlate with physiological marker of fibrosis. we found significant correlation between ccl 18 and markers of daytime hyperventilation and nocturnal hypoventilation. ccl 18 may be a useful marker of disease activity in ipf. 2012 were reviewed. 198 patients with normal cxr who had ctpa performed were included in study. in patients who had no pe but incidental findings (n = 120), further review was done to see if these findings would account for presentation. in patients with normal cxr and negative ctpa, an alternative diagnosis directed by incidental findings was able to account for presentation in 55.8 % (n = 67) of cases. of these, respiratory causes accounted for 91 % (n = 61), with pneumonia being the commonest overall cause at 59.7 %. other causes included atelectasis 22 %, emphysema 19 %, effusion 27 %, collapse 7 %, lesion 4 % and pneumothorax 3 %. ctpa is able to offer an alternative diagnosis in patients who had initial normal cxr and negative ct pa. our study is suggestive of high incidence of pneumonia in patients scanned for suspected pe. ctpa is the investigation of choice for suspected pulmonary embolism (pe). determining pretest probability for risk of pe using risk assessment scores and d-dimer is an important step in diagnosis. to establish positive predictive value of clinical and lab-based risk assessment for diagnosis of pe and to determine whether or not there was an overuse of ctpa in our service. a retrospective audit identified patients (n = 198) who had ctpa performed from january 2010-march 2012. patients were classified as being low, intermediate or high risk for pe, based on wells criteria [1] . cases were reviewed to establish if pretest assessments were used correctly in predicting probability of pe and need for ctpa. in high risk group (wells score [6) (n = 8), there were 5 diagnosed pes (ppv 62.5 %). in intermediate risk group (wells score c2 b 6) (n = 61), there were 14 pes (ppv 22.95 %). in low risk group (wells score b2) (n = 129) there were 5 pes (npv 97.2 %). in our sample, wells scores had reasonable predictive value for diagnosis of pe in high risk patients. positive predictive value in intermediate group was significantly lower and ctpa may be overused in this group. negative predictive value for low risk group was very high. therapeutic thoracentesis: the role of ultrasound and pleural manometry ultrasound-guided thoracentesis the utilization of endobronchial ultrasound for sampling of primary lung we performed a retrospective audit of mesothelioma patients diagnosed in the whsct between 2000 and 2012 and compared our practice against guidelines. 27 patients (23 male, 4 female) were diagnosed-mean age 64.7 years, range 37-85 years. asbestos exposure was documented in 21 patients (77.8 %). 16 patients (59 %) had right-sided chest disease with 11 (41 %) having left-sided disease /hospice. 4 patients are still alive. all patients were known to a lung cancer specialist nurse pilcher alfred hospital intensive care, melbourne, australia we examined the use of bronchoscopy in a 45 bed icu/hdu over a 12 month period. our aim was to establish the number of procedures done, the indications and complications. we also performed an audit of procedure documentation and set-up. 616 procedures were identified over the period in question. 68 % patients were male and ages ranged from 19 to 94 with a mean of 48 years. various indications were documented with infection (19 %), tracheostomy insertion (14 %) and difficulties with oxygenation (12 %) being the most common interstitial lung disease and pulmonary vascular disease chairs d. o'callaghan, mater misericordiae university hospital we now characterize potential mechanisms associated this b cell phenotype in sarcoidosis. serum was collected from 25 treatment-naive, sarcoidosis patients and 14 control patients. b cell activating factor (baff; r&d systems) and soluble (s) cd27 (ebioscience) levels were measured by elisa. expression of b cells in sarcoidosis granulomas were assessed using standard immunohistochemistry. all subjects signed an informed consent prior to participation. b cells were detected in sarcoid granulomas, but using serial sections no cd27 positive b cells were identified with respect to reduced cd27 b cells in sarcoidosis, we found no evidence that these cells are sequestered in sarcoid granulomas and that the key critical b cell cytokine, baff is appropriately elevated. scd27 is increased raising the possibility that the phenotype in this population reflects shedding of cd27 from the surface of memory b cells the diagnosis of sarcoidosis required the presence of non-caseating granulomata with negative ziehl-neelson stain and fungal stain plus negative culture for mycobacterium tuberculosis (tb), other bacteria and fungi. results: 30 patients were tested. 17/31 (56.6 %) had positive tblb. 13/31 had negative tblb (43.3 %). of the negative tblb group, 3/30 (10 %) had positive ebb. tb cultures on all tblb biopsy samples were negative and bal was negative for bacterial and fungal growth in all cases. no patient suffered complications secondary to the procedure. conclusion: tblb remains a useful diagnostic test in the diagnosis of sarcoidosis serum fibroblastic growth factor-23 in acute sarcoidosis p other serum biochemical markers measured included calcium, phosphate, 24 h urinary calcium, parathyroid hormone (ipth) and 25-hydoroxy vitamin d 3 . results: 26 subjects were male and 17 were female. mean (sd) ipth was 25.35 (12) ng/l, serum calcium 2.32 (0.16) mmol/l, serum phosphate 1.07 (0.21) mmol/l, serum 25-hydroxy vitamin d 3 38 (18.5) nmol/l, and 24 h urinary calcium excretion was 4.8 (2.8, 7.5) mmol/l. fgf-23 was detectible only in those patients who also had hypercalciuria and was elevated in all those with hypercalcaemia. after adjusting for covariates using stepwise multivariate linear regression fgf-23 was independently associated with serum calcium conclusions: this study describes the distribution and determinants of serum fgf-23 in acute sarcoidosis for the first time. evidence is accumulating that fgf-23 may have a pathogenic role in adverse cardiovascular outcome, whether this applies to patients with sarcoidosis and normal kidney function merits further investigation convex probe endoscopic and endobronchial ultrasound (eus/ebus) for the diagnosis of sarcoidosis ebus and eus guided lymph node aspiration is a minimally invasive procedure widely used for diagnosis and staging of lung cancer. there has been increasing interest in utilising these modalities for the diagnosis of benign conditions including pirfenidone is an emerging therapy for limited ipf mcnicholas department of respiratory medicine, st. vincent's university hospital, dublin idiopathic pulmonary fibrosis (ipf) patients have reduced exercise capacity [1], which correlates with parameters of pulmonary function and arterial oxygen tension resting pulmonary function testing and arterial blood gases were recorded. statistical analyses included student-t and mann-whitney u testing. maximal work load was reduced at 53.1 â± 4.6 % predicted corrected for sex, age and height. vo 2max was moderately reduced at 70.9 â± 3.8 % predicted. mean pre and post exercise borg score was 0 and 3. a positive correlation was observed between vo 2max and fvc (r = 0 exercise testing in the evaluation of diffuse interstitial lung disease pathophysiology of activity limitation in patients with interstitial lung disease diagnostic serum biomarkers to distinguish idiopathic pulmonary fibrosis from scleroderma-associated lung disease and healthy controls b. kennedy 1 , p. branagan cork idiopathic pulmonary fibrosis (ipf) may be difficult to differentiate from scleroderma-associated interstitial lung disease 20 potential biomarkers were measured by elisa or biochip immunoassay. differences between groups were calculated by unpaired t-tests. data are presented as mean â± sem. mean serum levels of kl-6 (1037 â± 234 vs. 207.4 â± 48 ng/ml elevated serum sp-d may help differentiate ipf from ssc-ild and healthy controls. the elevated serum sp-d in ipf compared to ssc-ild may reflect increased leakage from pulmonary capillaries arising from more extensive alveolar injury bone marrow and peripheral blood derived cd14 + cells as agents of tissue remodelling in idiopathic pulmonary fibrosis b cd14 + cells originate in bone marrow, circulate and then migrate to tissue where they may differentiate into macrophages. we investigated extrapulmonary cd14 + cells as a source of mediators of tissue remodelling in ipf. peripheral blood (pb) and bone marrow (bm) were obtained from 9 1000/ml, p = 0.3) cd14 + cells. ipfs and controls did not differ significantly in ccl2, ccl18, tgf-b1, ccl3, mmp7, mmp9 or timp1expression in either pb or bm cd14 + cells. ipfs and controls do not demonstrate any quantitative differences in the expression of mediators of tissue remodelling in pb or bm cd14 + cells. nevertheless ccl18 as an indicator of pulmonary fibrotic activity in idiopathic interstitial pneumonias and systemic sclerosis circulating levels of the chemokine ccl18 but not cxcl16 are elevated and correlate with disease activity in rheumatoid arthritis diffusing capacity of carbon monoxide (dlco) are the physiologic characteristics mostly correlated with hrct findings. high fibrosis score and extent of the reticulation and honeycombing on ct are associated with increased the risk of death [1]. we aim to investigate the correlations between radiological and physiological features of ipf. 18 patients with ipf were recruited from our database. full pulmonary function testing and awake arterial blood gas were performed. survival data were collected after a period of follow up. hrct was scored by a core radiologist on-site. data was analysed using linear regression and spearman correlation high-resolution computed tomography in idiopathic pulmonary fibrosis: diagnosis and prognosis ct features of lung disease in patients with systemic sclerosis: comparison with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia incidental abnormalities found on negative ct pulmonary angiograms performed on patients for suspected acute pulmonary embolism and with normal chest x-ray primary chest wall tumours and lung cancer invasion of the chest wall are the usual indications for chest wall resection and reconstruction. we evaluated all patients who underwent chest wall resection â± reconstruction in our unit from february 2001 to july 2012. 96 patients were identified. reconstruction was completed using either a composite of marlex mesh and methyl-metacrylate or a goretex sheet. reconstruction of the chest wall prevents post operative complications and restores the respiratory dynamics by avoiding paradoxical or harmful movements. in the majority of cases, it was possible to approximate the soft tissue over the reconstruction. in selected cases, our plastic surgical colleagues used a variety of flaps to approximate the defect.we looked at mean length of stay, histological types, morbidity and survival. there were no intra-operative mortalities. 30 day mortality was 5.2 %.chest wall resection is not a common procedure but may be performed in high volume centres with low morbidity and mortality. in those with primary chest wall tumours, a wide resection margin is associated with low recurrence rates. in patients with nsclc (t3) involving the chest wall, en bloc lung and chest wall resection has a proven curative benefit with excellent 5 year survival. fibreoptic bronchoscopy is considered a safe diagnostic tool [1] . it is suggested, however, that post-bronchoscopy complication rate increases with age [2] . we decided to study the complication rate and the outcomes of bronchoscopy in patients over 80 years in our institution. a retrospective review of case notes of patients over 80 years who underwent bronchoscopy between september 2009 and november 2011 was performed. data on complications experienced during and after bronchoscopy and the influence of the results on subsequent management were collated and analysed.ninety-six patients were included. mean age was 82.8 years (sd 2.98). thirty subjects (31.25 %) had a documented lung disease. fifty-nine patients (61.45 %) were current or ex-smokers. indications for bronchoscopy were; to evaluate for malignancy (93.8 %) and to evaluate for tb (6.2 %). post bronchoscopy complications were noted in eight (8.2 %) cases including hypoxia (3.1 %), infection (2.1 %), tachycardia (1 %) haemoptysis (1 %) and pneumothorax (1 %). six patients required post bronchoscopy treatment for complications. malignancy was diagnosed in twenty cases (20.8 %) and infection was detected in six (6.2 %). as a result of bronchoscopy, management was altered in fifty-one patients.in conclusion, bronchoscopy is relatively safe and has good diagnostic utility in patients aged more than eighty years. the introduction of an ebus-tbna program more than halved the numbers of endobronchial brushings without a statistically significant change in the number of combined bals and washings or biopsies. we plan to investigate the impact of ebus-tbna on the diagnostic yield of brushings biopsies bal and washings. the use of fibreoptic bronchoscopy in an intensive care unit tbna and eus-fna in patients with suspected sarcoidosis after its implementation at a university hospital. methods: we retrospectively analysed data on all patients with suspected sarcoidosis who underwent ebus/eus since the start of the service. sensitivity and diagnostic yield were calculated based on cytology results and further invasive diagnostics for negative samples. resuts: over 10 months 27 patients (18 males 9 females) with a mean age of 42 â± 10 were assessed. 63 % had stage i disease. 24 underwent ebus and 3 eus. 61 lymph nodes (lns) were sampled in total with a mean of 2.3/patient. diagnostic yield was 85.2 % with 23 patients diagnosed with sarcoidosis and 1 patient with tuberculosis. there were 3 false negatives yielding a sensitivity for detecting sarcoidosis of 88 %. tumour necrosis factor alpha (tnf-a) inhibitors have had a huge impact on the treatment of inflammatory arthritis, inflammatory bowel disease and psoriasis. there is also much interest in the use of tnf-a inhibitors in the treatment of refractory sarcoidosis [1] . paradoxically sarcoidosis in response to tnf-a inhibitors is increasingly recognised with over 37 cases reported to date [2] . we report a series of three cases of sarcoidosis that developed on tnf-a inhibitors.a 41 year old lady with a history of severe crohn's disease developed a right upper lobe consolidation and respiratory failure following two doses of adalimumab. investigations for tuberculosis were negative. a ct guided biopsy of a persistent right upper lobe infiltrate confirmed non caseating granulomas consistent with sarcoidosis.a 41 year old gentleman on etanercept for ankylosing spondylitis presented with dyspnoea and a dry cough. cxr showed diffuse fibrotic change. ct findings and a transbronchial biopsy showing multinucleated giant cells led to a diagnosis of sarcoidosis.a 40 year old man on adalimumab for rheumatoid arthritis was admitted with cough and purulent sputum. cxr and ct thorax confirmed mediastinal and hilar adenopathy. investigations were negative for tuberculosis. endobronchial ultrasound guided needle aspiration showed non caseating granulomas consistent with sarcoidosis.sarcoidosis in response to tnf-a inhibitors is a rare but increasingly recognised condition. following exclusion of tuberculosis a high index of clinical suspicion is needed to prevent a delay in diagnosis. the distribution of cd4/cd8 ratios in patients with biopsy-proven sarcoidosis suggests that substitution of bronchoalveolar lavage cellular analysis for transbronchial biopsy is not advisable. pirfenidone has been approved for the treatment of mild to moderate ipf in europe. pirfenidone regulates the activity of tgf-b and tnfa. we evaluated a single centre experience with pirfenidone.a retrospective cohort design was used to study ipf patients prescribed pirfenidone. a titrating dose of pirfenidone was commenced on patients with an fvc[50 % predicted and dlco[35 %. primary outcome was change in percentage predicted forced vital capacity (fvc). secondary outcome was change in percentage predicted transfer factor (dlco).26 symptomatic patients were prescribed pirfendione. the mean age was 67.8 years. 1 patient died due to an exacerbation of ipf, 6 others discontinued pirfenidone secondary to adverse-events. 12 patients reached target dose. 7 subjects continued pirfenidone at a reduced dose. 13 participants reported side effects likely related to pirfenidone. the most commonly reported side effects were gastrointestinal disturbance and photosensitivity. no significant decline in fvc or dlco was noted in patients who continued pirfenidone in 48.5 weeks follow up. key: cord-023216-avn8f2w3 authors: nan title: symposium summaries date: 2004-10-18 journal: pediatr pulmonol doi: 10.1002/ppul.20142 sha: doc_id: 23216 cord_uid: avn8f2w3 nan the creation of mouse models of cf has provided new opportunities to elucidate disease pathogenesis, correlate genotype with phenotype, and evaluate the safety and efficacy of novel cf therapies. epithelial tissues from cf mice exhibit the loss of camp-stimulated clsecretion characteristic of the human disease, and cf mouse models display a range of abnormal intestinal and pulmonary phenotypes dependent on the cftr genotype, independently segregating modifier genes, and environmental factors (diet, pathogens) (1) . studies in cf mice may also be helpful in answering the question: how much cftr is enough? quantifying the level of gene and protein expression necessary for restoring lung and intestinal function is of paramount importance for the design of somatic gene therapy or protein therapy protocols. one strategy is to reduce the expression levels or activity of cftr in wild type mice by genetic or pharmacological approaches (e.g. insertional mutagenesis; sirna; specific cftr inhibitors) and determine the threshold level at which the disease symptoms become manifest. a typical example is the creation of the exon 10 insertional cf mouse ( cftr tm1hgu ) . unlike the cf mice generated by gene replacement (knockout null mice), the original mutant mouse with a mixed background showed residual low levels of normal cftr mrna (<10% in the lungs) as a result of exon skipping and aberrant splicing (2) , associated with a 30-50% residual intestinal and nasal camp-mediated chloride secretion and only mild intestinal obstruction (95% preand post-weaning survival on solid diet); outbred mf1/129 cftr tm1hgu mice raised outside the isolator displayed signs of inflammation, reduced mucociliary clearance , and impaired airway clearance of aerosolized, cf associated bacteria (1) , suggesting that a low residual expression of normal cftr may ameliorate intestinal obstruction but not cf lung pathophysiology. recently, the phenotypic evaluation of two cftr tm1hgu inbred mutant strains (named cf/1-cftr tgh(neoim)hgu and cf/3-cftr tgh(neoim)hgu ) generated in hannover showed similarly low cftr transcript level in the intestine, a low residual amount (10-20%) of mature cftr protein in the apical membrane of the enterocytes, but close to normal (60-70%) chloride secretory responses to camp in the ussing chamber (3) . these findings suggest that ~20% of wild-type cftr protein is sufficient to normalize transepithelial intestinal chloride secretion, survival and weight gain. in contrast to intestine, the level of cftr mrna, immunostained cftr protein, and camp-stimulated chloride secretion in the nasal epithelum of these mice was almost normal, but the amiloride-sensitive sodium absorption, previously found to be 8 fold increased in cftr-null mice (4), remained ~2-fold above normal values. this finding suggests that a complete correction of the hyperabsorption of sodium in the airways, a known hallmark of cf, is difficult to reach even at virtually normal cftr protein levels. as yet the consequence of this persistent abnormality in sodium transport for lung pathophysiology remains to be explored. an alternative strategy is to express human cftr (hcftr) in cftr-/-mice and to examine rescue of the gut defect by the transgene. for example, a transgenic mouse expressing hcftr cdna under control of the intestinal fatty-acid binding protein (fabp) promoter showed normal survival and functional correction of ileal goblet cell and crypt hyperplasia, and a ~25% rescue of transepithelial chloride current (5) ; assuming that 100% chloride secretion corresponds with ~20% mature cftr (cf. cf/1 mice), it follows that ~5% cftr protein is sufficient to eliminate intestinal pathology. the notion that only partial correction of cftr channel activity may have a significant clinical impact was confirmed by replicating the g480c trafficking mutant in the murine cftr gene (6): 8% of mature cftr protein corresponded with ~40% residual camp-stimulated jejunal chloride secretion and the complete absence of intestinal pathology. insight into the question: how much cftr is enough, is also relevant for attempts to ameriolate cf disease pharmacologically, e.g. by promoting the correct processing and trafficking of f508del cftr by small molecules emerging from high-throughput screens, by altering chaperone function (7) or by suppressing premature stop mutations (8) . one approach is to treat cf mutant mice in vivo, or isolated tissues ex vivo with the rescue compounds/ approaches and compare the gain in cftr protein expression with the extent of functional rescue and the amelioration of disease symptoms. for example, ex vivo incubation of ileal mucosa from congenic fvb cftr tm1eur mice homozygous for the f508del mutation at low temperature (12h, 26 0 c) resulted in a gain in mature cftr protein (band c on a western blot) from a starting value of ~4% to a maximal level of 25% of the amount in cftr +/+ ileum, concomittant with the full restoration of camp/genistein-stimulated transepithelial chloride current (9) . assuming that the potentiator genistein fully corrects the ~3-fold lower open probability of f508del cftr channels, this outcome confirms that ~20% of mutant cftr protein, in the presence of a potentiator, is sufficient to fully restore intestinal chloride transport. complete functional rescue could also be reached by exposing the ileal mucosa for 6 h at 37 0 c to the proteasome/calpain inhibitor alln, likewise resulting in a gain in steady state levels of apical cftr protein up tõ 20% of normal levels (9) . importantly, even untreated rotterdam fvb cftr tm1eur mice maintained on solid food (hope farms srm-a) did not show signs of intestinal or lung pathology despite a low level of mature f508del cftr protein (~4%) in the intestine and a reduced level in the nasal epithelium (~30%). however a shift to a different solid diet (rm3, special diet services) caused 100% mortality by intestinal obstruction after 3-4 days, whereas changing the background from fvb to c57bl/6 provoked clear symptoms of lung pathology, without altering the nasal bioelectrics. these findings not only define optimal conditions for testing the efficacy of f508del cftr rescue approaches in these mice, but stress the importance of diet composition and genetic modifiers in cf pathology irrespective of the level of transepithelial chloride and sodium transport. what is termed a "genotype-driven" design. the second approach is to select patients with a similar level of disease severity and then determine their genotypes in a "phenotype-driven" study. both approaches have been utilized extensively in cf research, and each approach has yielded a number of important insights: 1) a small amount of wild-type cftr rna transcript ameliorates disease severity. "leaky" splice-site mutations that permit production of some normally-spliced cftr transcript can moderate disease severity in the pancreas, lungs and vas deferens. correlating estimates of the amount of wild-type transcript associated with "leaky" splice-site mutations demonstrates that approximately 3-4% wild-type transcript is associated with a less severe pancreatic disease, and approximately 8-10% allows individual to escape from cf lung disease (1;2). 2) partially functional cftr can reduce disease severity. phenotype-driven studies searching for mutations occurring in patients with mild forms of cf revealed a disproportionate number of amino acid substitutions (3;4) . analysis of cftr bearing these substitutions in cell-based systems has revealed that many retain chloride channel properties of the wild-type protein (5) . in general, the degree of residual chloride channel function of cftr correlates with the severity of the cf phenotype, in particular pancreatic status and degree of sweat chloride abnormality (6;7). correlation of chloride channel function and the severity of lung disease is less clear since there is a paucity of mutations that are convincingly associated with an improved pulmonary outcome. one exception is the a455e mutation that confers a milder lung phenotype (8) , cftr bearing this mutation has altered processing and chloride channel properties (9) . 3) increasing the amount of a partially functioning protein can avoid the life-limiting pulmonary complications of cystic fibrosis. the best example of this phenomenon is the r117h mutation. this mutation occurred at least twice in human evolution in different cftr genes, one bearing an efficient splice site (7t) in intron 8, and the other bearing an inefficient splice site (5t) in intron 8 (10) . cftr bearing r117h is properly folded and targeted to apical membranes however conduction of chloride via the cftr channel is reduced from wild-type cftr (5) . genes with r117h and 5t have reduced levels of full-length transcript leading to reduced production of partially functional cftr bearing r117h. individuals bearing r117h-5t and a severe cf mutation present with cystic fibrosis manifesting as progressive lung disease, pancreatic sufficiency, absence of the vas deferens in males and elevated sweat chloride concentration. on the other hand, r117h with the efficient 7t variant results in normal levels of full-length transcript bearing the r117h mutation leading to normal levels of cftr protein bearing this mutation. males with r117h-7t and a severe cf mutation generally have congenital absence of the vas deferens, a form of male infertility seen in cf males, and females generally have no clinical phenotype. importantly, lung disease is usually absent in these individuals. thus, increasing the amount of a partially functional cftr mutant can alleviate the lifelimiting complication of cf. 4) tissues affected in cf require different levels of cftr function to operate normally. an early revelation of genotype/phenotype studies was that the presence of one or two "mild" mutations was associated with a milder form of pancreatic disease but not milder lung disease or sweat gland abnormalities (11) . from a large number of studies, it appears that the sinuses are most sensitive to reduction in cftr function, followed by the vas deferens, lungs, sweat gland, and pancreas. thus, the level of correction needed to reverse cf pathology will depend upon the target organ. studies of genotype/phenotype conducted in cf strongly suggest that correction of most features of the disease will occur when 10% of normal cftr function is achieved. however, individuals bearing genotypes that are predicted to generate this level of cftr function have minimal differences in biochemical measurements such as sweat chloride levels and nasal potential difference measurements. improvement in measurable cftr function in vivo may require 25% function based on what is observed in cbavd males. males who have mild mutations in one cftr gene and a severe mutation in the other cftr gene can present with normal or near normal lung function, borderline to elevated sweat chloride levels and nasal potential difference measurements that are abnormal, but can be distinguished from those who have life limiting forms of cf (12) or are normal (13) . the cf respiratory disease phenotype includes thick mucus secretion and colonization of the lung with bacteria, such as pseudomonas aeruginosa (psa). presumably, repeated bacterial infections compounded by an inability to clear the infecting organism results in a profound neutrophil migration into the airways and the secretion of a variety of inflammatory mediators, including neutrophil elastase, reactive oxygen species, and cytokines, such as the chemokine il-8. the existence of an underlying defect in the airway immune response of cf patients remains controversial; however, increasing evidence suggests that the cf lung exhibits an exaggerated immune response even in the absence of bacterial infection (1;2). moreover, increasing evidence suggests that the cystic fibrosis transmembrane conductance regulator (cftr) may directly or indirectly regulate cfassociated airway inflammation and bacterial clearance. in the cf community, the advance of gene therapy has precipitated the question of how much cftr expression and function is required for the correction of cftr-related functions, including the resolution of inflammation and bacterial clearance within the cf lung. studies examining the effect of cftr on these responses have been performed utilizing both in vitro and in vivo model systems. specifically, in vitro studies have focused upon the effect of cftr expression and / or function in airway epithelial cell models on inflammatory mediator expression, nf-b activation, and bacterial adherence. in these studies, a variety of non-cf, cf, and cftr corrected-cf primary and immortalized airway epithelial cell lines have been utilized; however, little consensus has been reached on the role that cftr plays in regulating inflammatory and bacterialrelated responses. for example, several reports suggest that mutant cftr expression and / or function correlates directly with enhanced il-8 and il-6 (3;4) protein secretion, reduced rantes (5), il-10 (6), and inos (7) production, exaggerated nf-b activation (8) , and altered bacterial adherence (9;10). in contrast, additional studies indicate that little or no correlation exists between mutant cftr expression and il-8 (5;11-13) or il6 (11;14) and il-6 (14) production and nf-b activation (14;15) . with regard to in vivo studies examining the role of cftr in the pathophysiology of the cf lung, there is a general consensus that, when challenged with psaembedded agarose beads, mice lacking functional cftr exhibit heightened pulmonary inflammatory responses as compared with wild-type controls (16;17) ; such responses include increased production of tnf␣ and il-1␤. there is less agreement, however, between in vivo studies that examine the role of cftr in the bacterial clearance of an infected cf lung. a collection of recent studies indicate that correction of mutant cftr in the lungs of murine cf models enhances the clearance of burkolderia cepacia (18) , but not of psa (16;17) . interestingly, some studies have proposed that nutritional deficits, which result from cf-related intestinal defects, contribute to the exaggerated immune response in both cf patients and cf mouse models (19) . in contrast, a recent report demonstrates that cftr knock-out (ko) and gut-corrected cftr ko mice displayed similar indices of inflammation when challenged with psa (20) . this study also showed that cftr ko mice and wildtype control littermates exhibited similar lung inflammatory responses in the presence of a psa infection (20) . such discrepancies between these collective in vitro and in vivo studies may be due to differences in the airway epithelial cell and mouse models utilized, tissue culture conditions, exogenous stimuli, bacterial strain, and type of bacterial infection. importantly, these discrepancies highlight the on-going controversies regarding the role of cftr in regulating inflammatory responses within the cf lung and, therefore, make it difficult to project the amount of cftr required to attenuate such responses. in conclusion, the relationship between the level of cftr expression and excessive inflammatory responses warrants further study. the earliest concept of cf pathophysiology was that disease arises because affected organs are blocked by abnormally thick secretions 1, 2 . thick mucus was initially attributed to a defect in mucin molecules, but the discovery of electrolyte disturbances in sweat gradually led to a radically different concept: the basic defect is in ion transport, which leads to thickened secretions because of deficient water within mucus. it was long known that the first step in water secretion is often mediated by anion secretion. with the discovery that the defective cf gene codes for cftr, a channel for chloride and bicarbonate, it was immediately apparent that defective secretion of electrolytes and water could account for observed pathology in many cf-affected organs such as the vas deferens, pancreas, and intestines. an exception is the sweat duct, where cftr anion channels play an important role in electrolyte absorption, leaving water behind 3 . what about airways? people with cf typically die from unremitting lung infections. both ciliated surface epithelia and submucosal gland serous cells express cftr, and decreased anion-driven fluid secretion from both of these cell types contributes to mucus stasis. in addition, cf airways have an additional defect: lack of cftr disinhibits enac, leading to increased sodium-dri-ven fluid absorption: if extreme enough, hyper-absorption alone can produce mucus stasis and a cf-like disease 4 . we propose still a third defect: gland mucus contains a rich complement of proteins that act as anti-microbials, anti-oxidants, and inhibitors of serine proteases. we hypothesise that the bio-availability of these compounds is reduced in cf because of the altered mucus properties(a precedent for this concept has been established in cf mouse intestines 5 . furthermore, the ability of mucus to inhibit bacterial growth wanes with time, so static mucus eventually becomes a bacterial growth medium 6 . our laboratory has recently focused our efforts on gland secretion and serous cell models. glands are the major secretory organs of healthy lungs and gland serous cells express cftr . when provoked, glands produce copious amounts of mucus that traps pathogens and inhibits their growth while they are swept from the airways 7 . mucus clearance normally keeps the airways sterile, and it has been proposed that altered gland function in cf airways impairs the mucus shield e.g. 8, 9 . early studies of airway glands established many of their properties, but were carried out before it was understood that cftr is expressed in serous cells. since then, studies by ballard and colleagues, and more recently by our lab and the verkman lab, have expanded our understanding of glands and have established that cf glands have defective secretion. we developed an optical method for rapid and accurate assessment of single gland secretion rates 10 and are using it to study detailed features of single gland secretion in sheep, pigs and humans. glands secrete to both vip and ach. gland secretions are hypotonic 11 , and secretions to both mediators are slightly acidic relative to bath 12 . we found that in humans with cf, the glands are completely refractory to vip or forskolin 13 , and produce about half normal amounts of fluid in response to acetylcholine. secretions to ach have increased viscosity 11 . to look more closely at gland secretion, we have developed methods to isolate single glands or portions of glands, so that responses of single tubules and single cells can be monitored with nomarski differential interference contrast microscopy. these studies reveal that mucus is a highly heterogeneous substance, and provide evidence for exocrine, apocrine and holocrine secretion within a single gland. surprisingly, vip, which produces zero mucus outflow from cf glands, produces clear evidence of secretion when individual cf gland tubules are monitored, suggesting that proteins are being released, but without sufficient water to exit from the gland duct. these findings accord well with earlier predictions made by ballard and colleagues using pharmacological block of fluid secretion 14 . a complementary approach is to use cell culture models of serous cells. to help bridge the gap between secretion studies of glands and electrophysiological studies of cell sheets, we designed a 'virtual gland' that allows us to establish secretion rates (jv) and collect secreted fluid from secretory cells such as calu-3 cells 15 . with that link established, we can more easily interpret electrophysiological studies of calu-3 cells now being carried out in many laboratories. recent work from our laboratory indicates that calu-3 cells express kir 4.2 and possess an apical k + channel with properties similar to kir 4.2. they also possess an apical h-k atpase, resulting in the co-ordinate secretion of h + (via the h-k atpase) and hco 3 -(via cftr). this system provides one method whereby an initially isotonic secretion can be rendered hypotonic by the subsequent conversion of h 2 co 3 into co 2 and h 2 o. the hallmark of cystic fibrosis (cf) lung disease is chronic colonization/infection of the airways by a variety of bacteria, including h. influenzae, s. aureus, and p. aeruginosa. in cf, bacteria grow in regions of the lung that are normally sterile. the clinical course of cf lung disease correlates with the acquisition of bacterial infection and its progression. while the pathogenic mechanisms linking cftr mutations to cf lung disease continue to be a subject of research and debate, it is accepted that the normal host defense barriers in the lung are fundamentally altered. whatever the cause of the underlying propensity for infection, it is lung specific. the result is a characteristic susceptibility to bacterial infection. these features indicate that cf impairs the innate defenses of the lung. prominent components of innate immunity include the peptides and proteins secreted into airway surface liquid (asl). the airway epithelium directly interacts with the environment in a dynamic fashion. because of this interplay between the host and environment, systems evolved to clear or inactivate the pathogens encountered (see fig) . these defense systems include components of both innate and adaptive immunity. innate immunity complements adaptive immunity by its "ever ready" nature, as many of its components are continuously present and require no previous encounter or memory for their activity (4) . in the airways, the main cellular components of innate immunity are the epithelium and its products, and macrophages, dendritic cells, nk cells, cytotoxic t cells, and neutrophils. innate responses may be triggered via pathogens interacting with pattern recognition receptors such as the toll-like receptor (tlr) family (26, 41). the airway epithelium senses and responds to microbial challenges and can vectorially secrete its specialized products in the apical or basolateral direction to mount the appropriate response. in addition to serving as an important physical barrier, two major functions of the epithelia lining the conducting airways and submucosal glands are the production and modification of asl and the secretion of factors that contribute to host defenses. these agents act by a variety of mechanisms including disruption of microbial cell walls, sequestration of nutrients, and acting as decoys of microbial attachment. presumably, asl composition provides an optimal microenvironment for the function of the microbicidal compounds. asl contains several products with bacteriostatic or bactericidal activities, forming an important line of defense (8, 31, 40) . the specialized cells of submucosal glands (serous cells) also contribute to the secretion of airway surface fluid and are a major source of the production of macromolecules including antimicrobials and mucins (25, 43). the antimicrobial peptides and proteins produced by surface and submucosal gland epithelia act in a broad-spectrum fashion, in some cases exerting antimicrobial effects against bacteria, fungi and viruses. major asl protein components in terms of concentration include lysozyme, lactoferrin, and secretory leukocyte proteinase inhibitor (slpi) (8, 9, 20, 39, 42) . additional components are surfactant proteins a and d (sp-a, sp-d), collectin family members that are opsonins for bacteria and viruses (11, 22, 23) , the alpha and betadefensins (32), cathelicidins (2) , ngal (lipocalin 2) (10), and members of the lipid transfer/lipopolysaccharide binding protein family (3) . while the activities of many asl components have been defined, it is clearly a complex milieu, containing >1000 proteins (24), and the identity and function of many constituents are unknown. the pulmonary host defense defect in cf is complex. it is likely that the nature of the defect changes as the disease progresses. early lung disease is characterized by intermittent bacterial infection and the onset of inflammation. hallmarks of established lung disease include established bacterial infection with organisms living in biofilms, chronic neutrophilic inflammation, and progressive bronchiectasis. in the ~15 yrs since the cftr gene was discovered, many ideas emerged to explain the link between mutations and lung disease. one hypothesis is that cftr mutations alter the processing of cell surface asialoglycoproteins such that the cf epithelia are more suitable for bacterial attachment (17) . a second is that cftr at the cell surface functions as a bacterial receptor for clearance by epithelial phagocytosis; mutant cftr is unable to perform this function (28). a third hypothesis is that cftr mutations cause increased nacl and liquid absorption across epithelia, diminishing the asl volume and impairing normal mucociliary clearance. as a secondary effect of impaired clearance, bacterial adherence and colonization of the airways begins (5, 6) . a fourth theory is that mutations in cftr impair the normal ability of epithelia to modify the electrolyte composition of airway surface liquid (asl) by reabsorbing nacl (36). as a result, asl ionic strength increases and this secondarily impairs the function of many endogenous antimicrobials (35, 36, 40) . alterations in the composition of secretions from submucosal glands may contribute to dehydration of the asl layer and increased viscosity of cf secretions (18, 19) . furthermore, the trapped mucus may create an environment that is well suited for bacteria such as pseudomonas to live as biofilms (33, 34) . in addition to primary or secondary cftr-associated changes that lead to pulmonary disease manifestations, polymorphisms in other genetic loci may influence the cf phenotype (12, 29, 30) . several candidate modifier genes have been proposed for cf including mannose binding protein (14) , hbd-1 and hbd-2 (30), alpha 1-antitrypsin enhancer (16) , and hla class ii (1) . these disease pathogenesis hypotheses are not necessarily mutually exclusive, and underscore controversies in our current understanding of cf lung disease. a theme common to all these hypotheses is that altered innate defenses predispose to an increased susceptibility to colonization with bacteria. the chronic infection and inflammation associated with cf causes epithelial injury and repair responses (21) . this may also cause the epithelium to be impaired. for example, the production of the collectins sp-a and d progressively declines with disease progression (27). proteolytic enzymes, arising from cf airway secretions or resident bacteria, also cleave and inactivate many proteins and receptors involved in innate and adaptive immune responses, further impairing host defense. for example, pseudomonas proteases cleave igg and destroy its opsonic activity (13) . there is also evidence for host or pathogen derived enzymatic cleavage of transferrin, lactoferrin, and slpi (7, 38) . recently, taggart et al demonstrated that the cysteine protease cathepsins b, l, and s, in concentrations that are present in cf secretions, degrade and inactivate hbd-2 (37). in addition, the protease rich environment of the cf airways has been recently shown to cleave tlrs-2 and -4 that may further impair innate immune signaling (15) . the exuberant production of mucus and dna released from host cells and bacteria creates a complex anionic matrix that may also bind cationic antimicrobial proteins and further impair their activity. in summary, the single gene defect in cf causes complex primary and secondary host defense defects that initiate and then perpetuate progressive chronic lung disease. understanding these defects may lead to improved treatment for cf lung disease. emerging evidence from in vitro and in vivo studies suggests that airway surface liquid (asl) volume depletion initiates the cascade of events that results in cf lung disease. although regional differences may exist in the relative contributions of the superficial epithelium and glands to asl volume depletion, a common sequence follows that is manifest by periciliary liquid volume depletion, concentration of mucins in the mucus layer, adhesion of the mucus layer to airway surfaces, delayed ciliary and cough-dependent clearance, inflammation perhaps without overt bacterial infection, and ultimately acquisition of bacterial infection and promotion of bacterial biofilms. four topics that pertain to this scenario will be reviewed. • mechanisms of autoregulation of asl volume by normal and cf airway epithelia: the focus will be on the signals that sense airway surface liquid volume and the mechanisms by which these signals coordinate the deactivation/activation of epithelial ion channels (enac, cftr, cacc) to control asl volume and hence surface mucus clearance. • regional differences in airway surface liquid volume regulation in normal and cf: data will be reviewed on the expression in the normal airway of cftr in the proximal versus distal regions of the lung and the superficial versus gland epithelia. in addition, studies on the volume regulatory capacity of the normal and cf bronchiolar region will be reviewed. • functional consequences of depleted asl volume: the focus will be on mucus adhesion as being an initiating lesion in the progression of the obstructive and infectious components of cf lung disease. data demonstrating adhesion both in vitro and in vivo will be reviewed, as well as inferences as to the nature of the adhesive interactions. • methods of restoring asl volume therapeutically: the actions of hypertonic saline in restoring asl volume in normal and cf airway epithelia in vitro will be reviewed, and correlates to recent in vivo studies of the effects of hypertonic saline on mucus clearance and lung function made. in addition, the actions of selected novel small molecule pharmaceutical compounds will be reviewed. in sum, it appears that studies focused on the hypothesis that asl volume depletion is the initiating event in cf lung disease have been fruitful in regard to generating follow-on studies investigating the details of the pathogenetic sequence of cf disease, including the importance of mucus adhesion to airway surfaces and the importance of concentrated mucins in promoting not only the acquisition of infection but the biofilm mode of bacterial growth that characterizes cf. the charge now is to assign in different lung regions the relative roles of superficial epithelia versus glands in order to design effective therapies for early and, it is hoped, established cf lung disease. mucociliary clearance (mc) represents the first line of innate defense against the environmental contaminants and diverse exogenous sources of injuries to which the airways are exposed (1) . the ability of the airways to clear the mucus is depending on several factors including the number of ciliated cells and ciliary beat frequency (cbf), the volume of the airway surface liquid, the degree of hydration and the rheological properties of the periciliary (sol) and gel airway mucus. the airway secretory glandular cells may also markedly contribute to the hydration of the sol and gel mucus and therefore may directly influence the airway mucus transport (2) . there is now increasing evidence that the airway surface liquid (asl) covering the surface of the normal airways is an isotonic liquid, characterized by an optimal height (near the length of the cilia), and ideal viscosity and surface properties. the asl volume must be adjusted continuously in relation to the environment of the airway epithelium. any mechanical stimulation such as excess mucus load or external injury will first stimulate cbf, activate surface effectors and regulate the volume of asl (3) . different studies using freshly excised tissues and primary cell cultures have shown that the steady-state periciliary layer volume is maintained by a balance between na + absorption and clsecretion (1). tarran (3) has demonstrated that normal airway epithelium senses and autoregulates asl height by adjusting the rates of na + absorption and clsecretion to maintain mucus transport. it is well accepted that excess liquid at the periciliary layer level may be removed by a transepithelial enac transport. on the opposite, enac inhibition will induce clsecretion controlled by cftr activity. in these conditions, it can be easily speculated that in cf airways, the upregulation of na + and downregulation of clrelated to cftr defect will lead to a depleted asl associated with abnormal mucus transport. in cf patients with mild disease, compared with age-matched normal subjects, a reduced mucociliary clearance has been reported (4). a progressive decrease in mc is generally associated with increasing disease severity (5) . in patients with advanced cf disease, inflammation, infection and remodeling of the airway may be directly responsible of the decreased mc. whether mc is delayed early in cf infants before any infection is still unknown. most of the mc studies in cf mice have reported a large variability of the data (6) . in the normal murine lower airways according to the mouse strain, the mucociliary clearance varies from 0 to 4.5 mm/min, the lowest values being related to the low number of ciliated cells rather than to a decreased ciliary activity. in cf mice, the cbf is not decreased. although we and others (7) have reported a decreased rate of mc in cf mice, other investigators failed to demonstrate a difference (6) . interestingly, overexperession of enac in a recently published mouse model showed that the decreased volume of asl in the lower airways was associated with a large decrease (~ 60 %) in mc (8) . although the specific contribution of abnormal biochemical and rheological properties to the impairment of mucus transport at an advanced stage of the disease is accepted, the impairment of mc in early cf disease is still not clearly defined. secretory proteins, mucins and lipids are major components of airway mucus that contribute to the antibacterial defense of the airway epithelium but also contribute to the physical (adhesion), rheological (visco-elasticity) and transport capacity of the airway mucus. secreted mucins such as muc 5ac are reported to be enriched in the sol phase of cf mucus and mucins sulphation and sialylation are increased in cf secretions (9) . we have also earlier shown in secretions from adult cf patients that the phospholipid content moves towards a profile of poorly lubricant and therefore more adhesive mucus characterized by a decreased content in surface-active molecules such as phosphatidylglycerol and phosphatidylcholine (10) . whether these biochemical and rheological changes are constitutive or related to a possible defective intracellular acidification due to cftr mutation is unknown. in human fetal tracheal xenografts in the scid mice (11), we could not demonstrate any dehydration nor increased viscosity of the airway liquid collected in the lumen of the cf xenografts despite the fact that we observed an increased number of amiloride-sensitive na + channel and an inefficient camp-dependent clchannel. in fetal airway mucosa, volume activated clchannels may also contribute to the fluid secretion as previously shown in the late stages of lung development and therefore could compensate the defective mucus fluid hydration related to cftr mutation. one important cause of impaired mucus transport in cf may be related to an abnormal glandular secretion. when the secretion response to glandular agonists is inhibited in pig airways, the mucus liquid becomes less hydrated and more viscous (12, 13) and mc is markedly decreased, likely due to an uncoupling between mucin and liquid secretion. abnormal secretion by cf glands is expected on the basis that cftr is expressed in airway glandular serous cells not only at the apical membrane edith puchelle, sonia baconnais, jean-marie zahm inserm umr-s 514, reims, france surface but also at the level of membrane secretory granules (14) . moreover, fluid secretions are reduced and viscosity is increased in cf gland cells and in pig tissue treated by cftr inhibitors (15) . the implication of abnormal airway submucosal gland function in cf has been suggested. wine and joo (2) hypothesized that exocytosed mucus may remain as condensed packets for a long time after secretion and that, as in cf intestinal crypts, the granule contents may be trapped as undispersed granules within the lumen. using phase contrast dynamic videomicroscopy, we could confirm in cf glandular cells that under basal conditions, mucus exocytosis and expansion of secretory granules is markedly delayed. in parallel, we could demonstrate by quantitative dark field imaging and x-ray microanalysis on freeze dried airway glandular cell cryosections, that the mucus hydration of the cf secretory granules is significantly decreased and the ion content (na + , mg 2+ , p, s and cl -) is significantly higher compared with the non-cf secretory granule mucus content (baconnais et al., unpublished data). our data provide strong evidence that in cf cells, the alterations in ion composition and water content within the serous granules may partly explain the formation of thick mucus plugs lying over a dehydrated periciliary layer on airway surfaces. as proposed by j. wine (2) "too little water, to late" could explain that the secretory granules of glandular cells cannot correctly expand and may remain anchored to the surface of the cells. in such conditions, the glandular mucus cannot correctly disperse in the airway surface liquid. taken altogether, these results suggest that the abnormal exocytosis associated with abnormal ion and water content of secretory granules inside the glandular cells may accentuate the dehydration of the periciliary layer and contribute to the deficient mucus transport in cf airways. other alternative pathways may partly compensate this glandular mucus and asl dehydration and more or less prevent a major early failure of mc. nevertheless, the inadequate hydration of epithelial fluid at the apical surface and in intracellular secretory granules level favour the hypothesis that in cf, decreased mucus transport is a reality. lung disease in patients with cystic fibrosis (cf) is associated with neutrophil-dominant lower airway inflammation and increased lower airway concentrations of pro-inflammatory cytokines and neutrophil elastase even among infants and young children with cf (1, 2) . several studies report a robust inflammatory response in the airways of both bacterial culture-positive and culturenegative children; some studies show a greater inflammatory response in those patients with at least 5x10 4 colony forming units/milliliter of bacteria in their bronchoalveolar lavage (bal) fluid (1) . lung disease in cf can have regional heterogeneity that complicates understanding the relationship between initial airway infection and the inflammatory response (1). there is emerging computed tomography evidence for destructive regional inflammation/infection, i.e. bronchiectasis, in stable young children with cf (4) (5) (6) . such data emphasize the need for more research on the pathogenesis and treatment of early lung disease in cf, and the further development of outcome measures to assess global and regional progression of early lung disease. currently, lower airway inflammation and infection in non-expectorating patients can only be assayed by bal. there is a growing interest in proteomic analysis of blood and exhaled breath condensate to identify non-invasive markers of lower airway inflammation and infection. patients with cf acquire a unique set of respiratory tract pathogens in an age-dependent sequence. early airway infections are most frequently caused by s. aureus and non-typeable h. influenzae (1) . there is controversy on the role of s. aureus and h. influenzae in the progression of early lung disease. pseudomonas aeruginosa (pa) is a ubiquitous environmental organism and the most significant pathogen in cystic fibrosis (cf) lung disease. pa prevalence increases with age, with positive respiratory tract cultures in 15-30% of infants, 30-40% of children 2-10 y.o.,~60% of adolescents, and ~80% of adults with cf (2). reported risk factors for early pa infection include prior s. aureus respiratory tract infection, chronic anti-staphylococcal antibiotic treatment, female gender, delta f508 homozygous genotype, and attendance at cf clinics without cohorting (2) . there is currently no optimal method to assess lower airway pa infection in young children. sources of respiratory tract secretions for culture include expectorated or induced sputum, oropharyngeal (op) swabs, endolaryngeal suction, and bal fluid. sputum expectoration is the least invasive and most widely used method in older children, but children < 6 years of age can rarely expectorate. sputum induction with hypertonic saline is generally not successful in children < 8 years of age. op cultures are widely used as a surrogate for lower airway cultures in non-expectorating cf patients, but have poor sensitivity and positive predictive value for lower airway pa infection; op pa isolates can have different genotypes than bal isolates from the same patient (2) . despite the limitations, the current u.s. standard of care is to collect quarterly op cultures. the host immune response to pa antigens appears to be the earliest marker of pa infection; this is controversial and likely depends on the assays performed (2, 3) . recent u.s. studies found serologic responses to pa exotoxin a and whole-cell membrane proteins occurred prior to cultureevidence of pa infection in young children (2) . the danish cf center uses precipitins against pa antigens as a marker of chronic infection (3); they have reported reduced serologic responses to pa with intensive anti-pa treatment. limitations of serological markers include lack of commercially available standardized assays, and lack of specificity to the site of pa infection (i.e. upper or lower airway). generally, patients with cf are initially infected with unique environmental isolates of pa (1,2). regardless of treatment, patients commonly have a period of intermittent isolation of pa from the respiratory tract prior to becoming chronically infected (1) . the duration of intermittent pa + cultures can vary widely, and this complicates the decision regarding aggressive early intervention. early pa isolates are generally non-mucoid in phenotype, highly antibiotic sensitive, and present at relatively low density (2). these features suggest a "window of opportunity" for early intervention with antibiotics to possibly delay or prevent chronic pa infection and lung disease progression. early age at pa acquisition adversely affects lung disease and survival; yet, acquisition of pa does not appear to cause an immediate and rapid decline in lung function (2) . the early clinical features of pa infection are subtle and variable, including slightly reduced lung function, and poorer chest radiograph and clinical scores (2). by school age, the impact of early pa isolation is established. among patients from whom pa was isolated during the first 5 years of life, there was a 2.6 times higher risk of death, on average a 14% predicted lower fev 1 , and 7% lower weight percentile at 8 year follow-up compared to patients without pa isolated early in life (2) . danish investigators were the first advocates for aggressive treatment of early pa infection in cf (3). subsequently, most published studies have shown a microbiologic effect of antibiotics for early pa infection based on transient eradication of pa from upper and lower respiratory cultures (2) . there has been no randomized, placebo-controlled trial to evaluate clinical efficacy and safety, including drug toxicities and emergence of resistant pa or new pathogens. most studies suggest that recurrent pa infection is inevitable regardless of the initial treatment regimen (2) . the potential sources for recurrent or persistent infection include the sinuses, undetected residual lower airway infection, or re-infection with an environmental isolate. current options for early anti-pa treatment include oral quinolones, inhaled tobramycin and colistin, and iv antibiotics (1-3). macrolides have not been investigated for this indication. attractive features of oral quinolones include ease of administration, evidence for efficacy in older patients, and accumulating safety data in pediatrics. quinolone monotherapy may result in rapid emergence of resistance. limited safety data exist for inhaled tobramycin in young children, but it has a well-established safety profile in patients > 6 years of age (1) . inhaled colistin at the time of pa acquisition is used extensively in some european centers, primarily in combination with oral ciprofloxacin (3) . iv antibiotics are established treatment for pulmonary exacerbation in chronic pa infection (1); their role in the therapy of initial pa infection deserves further investigation. an ultimate goal for treatment of early pa infection is to pre-scribe the least invasive and safest treatment for the shortest duration necessary to achieve both microbiologic and clinical benefit. the pathogenesis of cf lung disease is due to dysfunction of cftr in the airway epithelium and results in impaired chloride secretion and increased sodium absorption, leading to dehydrated hyperviscous mucus secretions and impaired mucus clearance. the hallmarks of cf lung disease are chronic bacterial infection, mostly notably pseudomonas aeruginosa, and exaggerated neutrophil-mediated inflammation, eventually leading to airway obstruction and death. although the role of bacteria in cf lung pathology is well established the consequences of respiratory viral infection in cf infants are less well understood. although viral infection of the lung can result in serious illness, viral infection of the cf lung may have more severe consequences. there is also the likelihood that viral infection can establish an environment that is favorable for subsequent bacterial infection, a process termed "bacterial superinfection". the upper respiratory tracts of healthy individuals often contain commensal bacteria such as haemophilus influenzae and streptococcus pneumoniae but are asymptomatic for infection. however, these pathogens can become a threat when concurrent with a respiratory viral infection. of the common viruses infecting the respiratory tract of humans, influenza, respiratory syncytial virus (rsv) and parainfluenza virus (piv) result in the highest morbidity and mortality especially amongst children. in particular, rsv is the most common respiratory virus associated with the development of otitis media in children and is often associated with the respiratory bacterial pathogen nontypable haemophilus influenzae (nthi). nthi is responsible for a significant proportion of bacterial conjunctivitis, otitis media, sinusitis, bronchitis and pneumonia all of which may also be associated with concurrent viral infection. many mechanisms have been proposed to result in bacterial superinfection including disruption of innate immune mechanisms, e.g., impaired cough reflex; reduced efficiency of mucociliary clearance; the accumulation of excess or altered airway secretions; and, reduced macrophage-mediated phagocytosis. substantial evidence also exists for viral-induced up-regulation of bacterial adherence receptors although whether these changes occur at the lumenal surface of the respiratory epithelium or as altered secretory products is unknown. although viral-induced pathology and bacterial superinfection can occur in otherwise normal individuals, patients with cf or other underlying lung diseases are considered 'high-risk' for developing a greater severity of disease. more rapid pulmonary deterioration in cf patients has been associated with respiratory viral infections (1) and rsv infection produced more severe and prolonged disease in cf infants compared to infection of non-cf infants (2) . however, respiratory viral infections were found to just as likely occur in cf infants as in non-cf infants but more likely to result in lower respiratory tract infection, impaired pulmonary function, and hospitalization for cf infants (3) . in combination with these clinical data there also exists ample anecdotal evidence that cf patient lung disease gets much worse after viral infection. in terms of specific interactions with respiratory pathogens there is good clinical and experimental evidence that h. influenzae superinfection is associated with a concurrent airway viral infection and h. flu is recognized as an early bacterial pathogen in cf airways. however, data for an association of p. aeruginosa superinfection with airway viral infection is lacking. in the context of cf lung disease and the consequences of viral infection in cf individuals a number of important questions remain to be addressed: another rarely encountered species, b. ubonensis, should also be considered a member of the bcc based on phylogenetic assessment. all of these 'new' species have been identified among banked cf sputum isolates previously identified merely as 'b. cepacia,' indicating that all have caused infection in cf patients for many years. however, the distribution of these species in cf is quite disproportionate. studies from several countries indicate that b. cenocepacia (formerly 'b. cepacia genomovar iii') is the most prevalent bcc species recovered from cf patients. b. multivorans is the next most common species, with the remaining bcc species generally being recovered from relatively small numbers of patients. careful assessment of cf sputum isolates has also identified strains that, although clearly members of the bcc, can not be placed definitively into one of the ten currently described species; these 'species indeterminate' strains may represent still additional bcc species that await definition. genotyping (dna fingerprinting) analyses have demonstrated that in some cf care centers multiple patients are infected with the same bcc strain. some strains are even more broadly distributed. among these are strain et12, prevalent in eastern canada and the uk, and strain phdc, which has been recovered from cf patients in 24 us states and, most recently, in europe (1). et12 and phdc, as well as several other common strains reported to date, reside in the species b. cenocepacia. shared strains belonging to other bcc species, including b. cepacia, b. multivorans, and b. dolosa, have been less frequently identified (2) . the extent to which such common strains are inherently transmissible between patients is unclear and must be assessed in the context of the infection control measures employed to prevent their spread. further, available data suggest that bcc species differ with respect to their preferred niche(s) in the natural environment. a better understanding of the ecology of bcc species (and specific strains) is needed to assess the risk posed by natural reservoirs and to develop strategies to prevent acquisition from the environment. limited clinical outcomes data, including studies in lung transplant recipients, have shown an association between infection with b. cenocepacia and greater rates of morbidity and mortality (3, 4) . these observations have led some to conclude that b. cenocepacia is the "bad cepacia," implying that all strains in this species are easily transmissible and cause particularly severe infection, while other bcc species present little risk. however, most studies have involved small numbers of patients infected with a limited set of common strain types, with results being extrapolated broadly to the entire species. in fact, many, if not most, cf patients infected with b. cenocepacia harbor seemingly 'unique' strains (i.e., not shared by other patients), and many patients have remained chronically infected with this species for prolonged periods with little apparent impact on progression of lung disease. furthermore, infection with bcc species other than b. cenocepacia has been associated with sepsis and death (so-called cepacia syndrome). rather than ascribing degrees of relative virulence to entire bcc species, it seems more likely that virulence and transmissibility vary among specific bcc strains. of course, as yet undefined human host factors almost certainly contribute to clinical outcome, and so broad conclusions regarding relative bcc virulence will be difficult. a number of recent investigations employing improved animal and in vitro models of infection have yielded important insight into potential bcc virulence factors and pathogenic mechanisms. the molecular events underlying the ability of bcc to adhere to, invade and elicit a robust pro-inflammatory response from human airway epithelia have been studied by using immortalized cell lines as well as primary well differentiated human cell cultures (5, 6) . similar to p. aeruginosa, bcc are capable of expressing homoserine lactones likely involved in quorum sensing and biofilm formation (7) . a number of genes required for bacterial survival in vivo have been recently identified (8) , and provide an exciting opportunity to define potentially completely novel virulence factors. the availability of the complete genome sequence of b. cenocepacia strain j2315 (a representative of the et12 lineage) provides a critical asset to investigations of bcc pathogenesis. the sequencing of several other bcc strains and the development of a burkholderia dna microarray are underway and will surely further enhance ongoing research efforts. in summary, during the past several years, a great deal has been learned regarding bcc taxonomy, clinical microbiology and epidemiology. clearly, there is now a need for a more complete understanding of bcc ecology and for large scale outcomes studies to better assess the relative risk of infection with specific bcc strains. ultimately, better definition of the bacterial virulence factors and human host factors that determine diseases progression is necessary to devise novel therapeutic and preventative strategies. increasingly, the emergence of cf respiratory pathogens, including bacteria with intrinsic or acquired antimicrobial resistance, non-tuberculous mycobacterium (ntm) species and fungi, challenge clinicians to better characterize their prevalence and epidemiology, pathogenecity in cf lung disease, optimize treatment of infection, and prevent acquisition in both healthcare and non-healthcare settings. staphylococcus aureus binds avidly to respiratory mucin and is often is the first pathogen to colonize the respiratory tract, with a prevelance of ~45% by one year of age. s. aureus may cause chronic inflammation of the cf respiratory tract by up-regulation of proinflammatory cytokines exotoxin-mediated immune dysregulation and tissue damage. (ref) currently, 9.2% of cf patient s. aureus strains are resistant to methicillin (mrsa). mrsa strains may be transmitted via direct contact in and outside of healthcare settings, may cause transient or chronic respiratory colonization, and have been associated with severity of underlying disease and adverse outcomes in both cf and non-cf patients (cosgrove, others) although the impact among cf patients is limited to date, there is growing concern about genetically distinct community-acquired mrsa (ca-mrsa) strains that posses a unique resistance cassette and cause virulent soft tissue infections and necrotizing pneumonia mediated by the cytotoxin panton-valentine leukocidin (ref). stenotrophomonas maltophilia and achromobacter xylosoxidans are intrinsically multidrug-resistant gramnegative pathogens whose acquisition is promoted by use of broad-spectrum antimicrobials. they can cause healthcare-associated infections, including pulmonary exacerbations among cf patients (refs). among cf patients, current prevalence rates of 9.4% for s. maltophilia and 5.2% for a. xylosoxidans likely are underestimates because of the failure to use appropriate selective media and misidentification. although many patients have only transient colonization with these organisms and their pathogenecity remains poorly defined, longterm colonization s. maltophilia was associated with decreased lung function 2 to 7 years after acquisition in one study (ref) . studies have suggested that acquisition may occur both from environmental sources or from person to person. saphrophytic fungi, including aspergillus spp. and scedosporium apiospermum, are ubiquitous and may be found in soil and in the hospital environment associated with construction activities or water intrusion. aspergillus spp., predominantly aspergillus fumigatus, and can colonize the lungs of cf patients and can cause allergic bronchopulmonary aspergillosis (apba) in up to 7.8% of patients and rarely aspergilloma and invasive aspergillosis (285). registry data likely underestimates the prevalence of aspergillus. in the aerolized tobramycin trial, up to 25% of cf patients were colonized with aspergillus spp ( ) person-to-person transmission remains a rare but potential risk among patients with wound or tracheal infection (ref) . further studies are needed to assess the impact of use of selective media and infection control precautions on the prevalence of these emerging pathogens and to better define risk factors for acquisition in and outside of healthcare settings, the clinical impact and treatment strategies. it is pertinent to look at our evolving understanding of the clinical manifestations of cf in the context of key milestones that have occurred since the recognition of cf as a distinct clinical entity in 1938 (1) . from initial recognition/description through 1953, most patients with cf died in infancy or early childhood of pneumonia and severe malnutrition. the diagnosis was suggested by clinical features along with evidence of exocrine pancreatic deficiency on duodenal fluid analysis, and then confirmed at autopsy. recognition of the sweat gland defect in 1953 (2) and development of the quantitative pilocarpine iontophoresis sweat test in 1959 (3) led to the inclusion of a positive sweat test result as an essential criterion for the diagnosis of cf. recognition of physiologic abnormalities of sweat and serous-secreting glands led to a change in terminology from mucoviscidosis to cystic fibrosis. from 1960 to 1989, the cf phenotype was better defined and it became clear that cf was characterized by significant phenotypic heterogeneity (4). during this time it was recognized that 1) there is an important subset of patients with chronic sinopulmonary disease typical of cf, pancreatic sufficiency and borderline or normal sweat electrolyte concentrations (variant, atypical or non-classic cf) (5) and 2) almost all males with cf are infertile secondary to bilateral absence of the vas deferens (6) . there were two highly significant laboratory observations during this period; raised immunoreactive trypsin levels in dried blood spots could be used for newborn screening and a raised bioelectric potential difference across cf respiratory epithelia could be used as a diagnostic tool. the identification of the cftr gene in 1989 led to significant advances in our understanding of genotypephenotype correlations and also introduced the possibility of using mutation analysis for diagnostic (including prenatal) testing and carrier screening. it became recognized that there is a subset of "mild" mutations which are associated with pancreatic sufficiency and, in some instances (missense mutation a455e) with a less severe pulmonary phenotype. also, it was recognized that there are a number of disorders (cbavd, idiopathic chronic pancreatitis, chronic rhinosinusitis, abpa, diffuse panbronchiolitis) in which cftr mutations (often associated with residual cftr function) are frequently identified, but in which a diagnosis of cf may not be appropriate. this led to the introduction of diagnostic terms such as cftr or cftr-related disease or cf-like disease. further complicating the issue of "what is a cf diagnosis?" were the observations that cf mutations cannot be identified in a subset of patients who have clinical features typical of cf (7) , and that some individuals who carry two known cf mutations may remain asymptomatic over extended periods of time. also, it has become clear that differences in endorgan sensitivity to cftr protein deficiency and the genetic context in which a mutation exists can influence the cf phenotype. intron 8 variants (8) and modifier genes (9) may play an important role in determining the severity of the pulmonary and gastrointestinal manifestations seen in patients with cf. in 2004, there is still controversy regarding the diagnosis of cf. the overwhelming majority of patients fall into the category of "classic cf" in which there are typical clinical features in association with elevated sweat chloride concentrations and, in the majority of cases, two identified cf mutations. however, there remains a small but increasing subset of patients, often adults, who have atypical clinical features, a normal or borderline sweat test result and identification of one or no cf mutations, and a subset of asymptomatic newborns identified by newborn screening (two cf mutations and/or a positive sweat test result) but who lack clinical criteria to support a cf diagnosis. this has led to the somewhat controversial proposal (10) that it might be appropriate to have a diagnostic category of "genetic pre-cf" for some individuals and raises the issue of how a disease such as cf is defined in the genomic era in which gene alterations are likely to be found in the absence of clinical features (11) . it is clear that a genetic abnormality by itself does not equal a disease and that both clinical criteria and genetic abnormalities will need to be used to define a disease state. peadar g noone, m.d. the diagnosis of cystic fibrosis is straightforward in the majority of patients, particularly pancreatic insufficient (pi) patients. diagnostic criteria (set forth by a consensus statement, drawn up by a panel of experts in 1998) include clinical / phenotypic data, as well as data on cftr genetics and function; these can be applied in most clinical situations (1). the criteria were designed to encompass as many of the potential clinical scenarios as possible; the potential pitfalls that may occur with the laboratory tests of cftr were discussed. an emphasis was placed that the guidelines are unlikely to cover every possible scenario (practicing clinicians will quickly agree). difficulties may be encountered in patients with atypical disease that do not conform to traditional clinical definitions, in conjunction with cftr data that may be difficult to interpret -establishing the line between proven cf and "cf-associated" diseases thus may not be easy for the clinician. as new knowledge is gained through clinical research and clinical practice, and with the potential for increasing presentation of atypical patients the guidelines will need to be refined and updated. the traditional laboratory investigation of cftr function is the sweat test. it is strongly recommended that this test be carried out in a center experienced in its performance, and that the only acceptable test is the pilocarpine iontophoresis sweat test. furthermore, clconcentrations rather than na + concentrations are regarded as more accurate for discriminating between cf and non-cf, though sweat na + may still be used in some laboratories. the literature in regard to sweat clconcentrations has varied somewhat. the older literature recommends a clconcentration of 70 mmol/l as diagnostic of cf; these data are based on reference values obtained from pi cf patients during the 1960's, and do not include pancreatic sufficient (ps) patients, or any recently described phenotypes (2) . in view of the increasing awareness of atypical and "variant" cf, a level > 60 mmol/l is now regarded as diagnostic for cf (1) . what is more difficult to interpret is a level less than 60 mmol/l, although in the context of a compatible phenotype, the higher the clconcentration, the more suspicious of cf the clinician is likely to be. somewhat arbitrarily, a level of between 40 and 60 mmol/l is regarded as a gray zone (previously 50-60 mmol/l), often including ps / atypical cf patients. complementation with nasal potential difference (npd) measures can be useful. npd is now more widely available, has been standardized, and can be performed safely in all age groups, including neonates (3). npd requires significant technical experience and validation for each center / operator, and across groups of sufficient patients and controls. the best evidence of cf is a raised basal pd consistent with increased na + absorption across airway epithelia. across 8 different sites in the us, standaert et al found that patients with cf (n = 31) had a mean basal pd (mv) of -45.3+11.4 (vs -18.2+8.3 for non-cf). similarly, a reduced or absent cftr-mediated clconductance (mv) was present in patients with cf (+3.2+3.5 as compared with -23.7+10.2 for non-cf). despite these advances in npd measurement techniques and standardization, difficulties arise in atypical cf patients who may have normal or high normal na + transport (up to -30 mv), although usually such patients have reductions in cftr-mediated clconductance, intermediate between that of cf and non-cf (4). as a technical note, inflammation of the nasal mucosa renders the test very difficult to perform and interpret (it may cause a falsely low basal pd and apparently absent clconductance because of loss of epithelial integrity). finally, molecular testing demonstrating two "cf-causing" mutations (i.e. associated with altered cftr synthesis from a premature termination signal, alternations in intron splice sites, or novel amino acid sequences) is strong evidence of cf (1) . difficulties arise when faced with one cf-causing mutation, after completion of currently available commercial tests. then the issue arises as to how aggressive to be in searching for another cf-causing mutation on the opposite allele. exactly defining a cf-causing mutation can be a difficult process, even after consultation with a cf molecular geneticist or other experts in the field of "atypical cf" is necessary an example of clinical phenotypes described since the consensus statement include patients with chronic pancreatitis of unknown etiology, some of whom have what is now termed cftr-related pancreatitis (4) . initial cftr genetic tests showed that some of these patients were carriers of a severe cf mutation, and only after extensive tests or gene sequencing did a subset turn out to have a mild or "variable" mutation in cftr on the opposite allele. sweat chloride values were less than 60 mmol/l in some, with a mean value for all cftr compound heterozygotes of 54 mmol/l (n = 6). nasal pd tests showed values intermediate between that of normal and "classic" cf, supporting the concept of a subset of cftr mutations resulting in some residual cftr function and intermediate cftr physiology (5) . the flip side of the coin are patients with a phenotype strongly resembling cf but with normal tests of cftr structure / function (6) . such patients have some phenotypic features of cf, for example sino pulmonary disease, congenital absence of the vas deferens, gastrointestinal disease, or elevated sweat chlorides (>40 mmol/l). proportions of patients referred with these criteria (n = 74) had two (n = 29), one (n = 15), or no (n = 30) mutations in cftr. as with the patients with idiopathic pancreatitis, those patients referred already known to have one cf mutation were most likely to have another mutation (associated with reduced cftr function) on the opposite allele. the hypothesis was raised that a non-classic cf like phenotype may result from causes outside the cftr gene. finally, it used to be thought that isolation of mucoid pseudomonas aeruginosa in the airway secretions of someone with bronchiectasis was strongly suspicious of cf. however, other diseases such as primary ciliary dyskinesia may also be associated with such respiratory tract microbiology. thus, interpretation of the "clinical phenotype" portion of the current definition of a cf diagnosis is difficult when one considers these groups of patients. studies are underway looking at the issue of defining more precisely the diagnostic criteria of sweat / npd / molecular tests in the context of different phenotypes ranging from classic to mild cf to heterozygote and non-cf individuals. defects in the cftr gene cause a wide spectrum of manifestations, from classic cystic fibrosis (cf) diagnosed in childhood with multi-system involvement including sino-pulmonary disease and pancreatic insufficiency, to monosymptomatic conditions not diagnosed until much later in life (e.g. congenital absence of vas deferens (cbavd), recurrent pancreatitis). there is now confusion between what is considered cystic fibrosis and how to label other disorders associated with cftr mutations. there is a growing population of patients in whom a diagnosis of cf can neither be made nor ruled out. the lack of a diagnosis can be stressful for the individual and health care professionals currently have difficulty providing prognostic guidelines. the discovery of the cf gene has not been as helpful as hoped in these situations. the common cf-causing mutations detected on standard screening panels are usually seen in patients with classic disease, in whom the diagnosis can be made by a positive sweat test. patients who present with atypical manifestations are more likely to have less common mutations; however extensive genetic testing of the remaining 1,000 mutations or variants in cftr is not financially feasible or practical. the standard diagnostic sweat test has not been validated across this new clinical spectrum and so its role in assessing patients presenting with unusual manifestations is unclear. we know that patients presenting with atypical features may have negative or "borderline" sweat test results. newer investigations examining the function of cftr, such as measurement of nasal transepithelial potential difference, have not been standardized or validated at all. in an effort to refine the diagnostic criteria for cf, we have initiated the systematic recruitment of subjects in well-defined cohorts defined as follows: 1. typical cf 2. atypical cf (i.e. pancreatic sufficiency) 3. other conditions related to cftr(e.g. cbavd, idiopathic recurrent pancreatitis) 4. obligate cf heterogyzgotes 5. unaffected controls the cf patients have been clinically diagnosed and identified from the toronto cf clinics (pediatric and adult), with 550 current patients who have been extensively genotyped, classified as pancreatic sufficient or insufficient and who are followed regularly with clinical information prospectively collected in an extensive database. patients with unusual manifestations or disorders associated with cftr mutations are recruited from three sources: a cohort of men with cbavd from a male infertility clinic; patients with idiopathic, recurrent pancreatitis after extensive investigations to rule out other etiologies of pancreatitis; and patients referred to the toronto cf clinics with bronchiectasis of unknown etiology. identification of subjects in the latter category has been enhanced by mailings to pulmonologists and general practitioners in the province and cf clinics in canada. presentations to the medical personnel in the toronto and regional cf clinics have been made to alert them to the opportunity to refer patients in whom cf has been queried but not established or excluded. obligate heterozygotes are the siblings or parents of patients with classic cf. control subjects included siblings of known cf patients who do not carry cf gene mutations, as well as unaffected volunteers. all subjects are fully investigated with clinical assessment; complete gene scanning of cftr with multiplex heteroduplex shift analysis and characterization of dna fragments by direct sequencing analysis; sweat chloride testing, using the gibson and cooke method, and nasal transepithelial potential difference measurement performed (using method of knowles) on the same day as the sweat test. these measurements are prospectively performed in these well-defined patient and control populations, and will form the basis for classification and regression tree analysis as described later in this symposium. this novel, multi-variable approach to defining the reference parameters of these diagnostic tests will help us to refine the diagnostic process. the ultimate goal is a redefinition of the diagnostic criteria for cystic fibrosis, and a more precise definition of related syndromes. according to the 1998 cf consensus statement [1] , a diagnosis of cf is established when a cftr-associated phenotype is present in conjunction with either two abnormal sweat chloride measurements, or two disease causing cftr mutations, or an abnormal nasal potential difference (pd) result. although the sensitivity and specificity of these guidelines are high when applied to pancreatic insufficient patients and healthy controls, the growing number of patients identified with pancreatic sufficiency as well as cftr-associated clinical phenotypes has resulted in numerous diagnostic dilemmas for which current guidelines are frequently inadequate. the reference range for sweat chloride, established in 1972, was based on assessments of primarily severe cf patients and healthy controls (who were not genotyped). similarly, the cf-causing mutations included in the consensus statement do not reflect current knowledge. furthermore, objectively defined guidelines and reference values for nasal pd as a diagnostic test have not been established. our group is currently evaluating a cohort of patients presenting with diverse phenotypes associated with mutations of the cftr gene to redefine the laboratory parameters that confirm or exclude a cf diagnosis. study subjects are recruited from among conventionally diagnosed cf patients, healthy controls, obligate heterozygotes, and query cf patients presenting with infertility, pancreatitis, or sino-pulmonary disease. patient evaluation includes comprehensive medical history, sweat chloride, nasal pd measurements on the same day, pulmonary function testing, serum trypsinogen, and extensive cftr mutation testing by multiplex hereroduplex analysis and dna sequencing. methods for characterizing this increasingly heterogeneous population are necessary to refine diagnostic criteria used for clinical and research purposes. in order to identify structure and "natural" grouping within the spectrum of cf disease observed in our data, cluster analysis [2] , an exploratory technique, is used. the most commonly used hierarchical procedures, agglomerative methods, successively fuse similar individuals or groups together in clusters of increasing size and heterogeneity, ending with a single group containing all subjects. the resulting sequence of clusters is represented graphically using a dendogram, a tree diagram where individuals correspond to the 'leaves' whose 'stems' are grouped together at 'nodes'. the distances between clusters, a measure of the difference between the groups, is represented by the lengths of the stems. the dendogram thus provides a useful visual representation of the clustering algorithm that can assist in making decisions about the number of clusters present in the data. to gain further insight into the underlying factors defining the individual clusters, a figure is derived that borrows from the concept of microarray data analysis. the variables used in the cluster analysis are depicted in separate columns across the x-axis, with individual subjects each represented as a single row across the figure. shades of gray, ranging from white for values in the normal range to black for values representing greatest disease severity, are used to represent each data value. using this figure, researchers can simultaneously visualize all the variables included in the cluster analysis, revealing the underlying structure of the data and unusual observations within the clusters. classification analysis is then used to define a rule that can be used to assign new subjects to one of the clusters. unlike cluster analysis where no assumptions are made about the number of groups or their structure, in classification analysis the groups are known and are used as the basis for developing a diagnostic rule. the classification and regression tree (cart) method of breiman et al. [3] is ideally suited for developing diagnostic rules ( figure 1 ). this method uses recursive partitioning to segregate patients into one of two or more known groups. the decision tree representation incorporates complex interactions in a transparent and intuitive rule that can easily be applied to new cases. moreover, the method does not rely on normal distribution assumptions and is particularly effective for developing rules in the presence of a large number of variables. prior distributions and cost of misclassification can be incorporated into the model to optimize the diagnostic rule for use in a clinical or screening situation. cutoff values selected by cart can be further adjusted to optimize sensitivity and specificity evaluated by normal distribution assumptions, where appropriate. statistical analysis of the cystic fibrosis diagnosis cohort will identify homogeneous subgroups within the spectrum of cftr disease, providing alternative labels for subjects with an uncertain diagnosis of cf. longitudinal studies of these patient groups will refine models for prognosis and survival, assisting in frequency of follow-up and treatment decisions and providing patients and their families with more precise information. supported by nih dk9096-10 and ccff. figure 1 : 60 controls, 35 obligate heterozygotes, and 89 patients with cf were used as a training sample for cart analysis to establish a diagnostic rule based on sweat chloride and change in nasal pd from infusion to the final value after superfusion with isoproterenol (⌬am+clfr+iso). the first split based on a value of ⌬am+clfr+iso of 6.5mv has perfect sensitivity and specificity for discriminating cf from non-cf. further splits based on sweat chloride at 25.5 and 82.5 mmol/l segregate the majority of controls and cfpi patients at the extremes, with overlapping groups in the intermediate range. finally, a split based on a ⌬am+clfr+iso of -6.5mv further discriminates between controls and an intermediate group of controls and heterozygotes. by basing the initial split on ⌬am+clfr+iso rather than sweat chloride, the cart rule correctly classifies cf-ps patients with sweat chloride values in the normal range. jane l. burns, m.d. antibiotic therapy is a mainstay of treatment for cf patients hospitalized with a pulmonary exacerbation. in order to use antimicrobial agents effectively, it is important to understand the individual patient's microbiology, the patterns of antibiotic resistance within your cf center, the activity of the drugs in cf, and the optimum mode of administration. using each patient's culture and susceptibility data as a guide, it is usually possible to identify a combination of two or three agents from different antimicrobial classes (e.g., β-lactams, aminoglycosides, quinolones) that demonstrates in vitro activity against the predominant pathogens. the use of agents from different classes with different mechanisms of action offers the potential for synergistic activity. it also decreases the likelihood of development of antibiotic resistance. even if susceptibility data are unavailable, knowing the organisms most recently cultured from a respiratory sample may help direct therapy. while pseudomonas aeruginosa is often susceptible to a combination of a β-lactam and aminoglycoside, burkholderia cepacia complex is uniformly resistant to the aminoglycosides and stenotrophomonas maltophilia is never susceptible to the carbapenem antibiotics. the patterns of antibiotic resistance within a given cf center may also influence the choice of antimicrobial therapy. for example, methicillin-resistant staphylococcus aureus is being seen increasingly at some cf centers (1) and may be susceptible to clindamycin and trimethoprim/sulfamethoxazole, or perhaps only to vancomycin. knowledge of the local resistance patterns can help determine the best choice of agents. the pharmacokinetics of antimicrobial agents in cf patients may be distinct from those of other individuals. because many cf pathogens are relatively antibioticresistant, optimizing drug levels at the site of infection in the lung is critical. for drugs where serum monitoring is performed, such as the aminoglycosides, the use of a previously determined dose is optimum, unless renal function has changed. for other agents, where monitoring is not available, the use of cf-specific dosing is recommended (2) . the route of administration is the final decision that must be made in identifying antibiotics for inpatient treatment. questions that often arise include the necessity for parenteral rather than oral administration of quinolones and the advisability of combined inhaled and parenteral tobramycin. these decisions may need to be individualized for each patient. once therapy has been initiated, clinical response is often adequate to determine hospital course and the advisability of continuing therapy as an outpatient. if a patient is not improving as expected, additional culture and susceptibility testing may be indicated. physiotherapy is one of the cornerstones of treatment in cf and is part of the multi-disciplinary treatment approach. p. aeruginosa may survive longer in airways abundantly filled with sputum, and by removing infected lung secretions the rate of proteolytic tissue damage can be decreased 1 . combining intravenous anti-biotic therapy with intensified airway clearance physiotherapy has been shown to improve lung function more than iv anti-biotic treatment alone 2 . higher sputum volumes are often associated with increased inflammation, lung obstruction and destruction. inflammatory processes increase metabolism and in turn minute ventilation and work of breathing 3 . there are many reasons why patients with cf may be admitted to hospital. some of the most common reasons include acute flare up of cf lung disease, intestinal obstruction, preparation for elective surgery and for management of cf complications such and hemoptysis and pneumothorax. the objectives of hospital admissions vary depending on the reason for the admission. in the case of acute exacerbation, the main objective will be to restore optimal physical function as quickly as possible. broadly speaking, this is achieved through a multi-disciplinary approach including inhalation therapy, clearance of sputum and mucus plugs and pharmacotherapy to reduce infection and inflammation which in turn may decrease the work of breathing. further, a graduated exercise program to assist in sputum mobilization 4 and increase physical working capacity and musculo-skeletal well-being (including attention to posture, muscle strength and mobility) should commence as soon as appropriate. nutritional and psychosocial support are also part of the care package. ongoing age appropriate education about effective and efficient airway clearance, inhalation therapy and the need to adhere to treatment regimens should be incorporated in treatment sessions. each treatment session should be seized as an opportunity for education. establishing good rapport, offering age appropriate treatment and gaining the patient and family's trust and confidence are crucial in the delivery of effective treatment. an inpatient admission should include: • relevant past history • recently recommended home physiotherapy program including inhalation therapy (agents, order and timing), airway clearance therapy (act) and physical exercise program and adherence • the possibility of gastroesophageal reflux 5 in relation to physiotherapy • clinical status including subjective and objective measures of the following -amount, color, consistency and ease of expectoration of sputum -oximetry/pulmonary function tests/peak expiratory flow rate -breath sounds on auscultation, respiratory rate and pattern of breathing -exercise tolerance (current activity & incidental exercise/ exercise tolerance tests) -musculo-skeletal problems (posture, pain, muscle tightness/weakness, oedema) -urinary incontinence during coughing and forced expirations establishment of a treatment program incorporated in the multi-disciplinary care plan: 1. inhalation therapy: 6-8 broncho-dilator, mucolytic (pulmozyme, isotonic or hypertonic saline etc) inhaled anti-microbial and anti-inflammatory agents. the timing and order of these treatments in relation to act and exercise are important and need to be established. 2. choice of airway clearance therapy: [9] [10] [11] [12] [13] positioning manual techniques effective forced expirations the active cycle (huffing) of breathing positive expiratory pressure autogenic therapy drainage oscillating positive expiratory non-invasive pressure therapy ventilation 3. establishment of an exercise program to: -increase aerobic capacity, improve muscle strength and joint mobility -o 2 therapy if needed -promote normal postural alignment and physical function -motivate increased exercise participation in the short and long term 4. discharge planning occurs once the objectives of the admission have been achieved. a home treatment program needs to be negotiated with each patient individually. an early review of progress is usually organized in the cf outpatient department. the dosage and number of treatments per day need to be individualized to each patient's needs. acutely ill patients may need shorter treatments spread over the 24 hour period which should be available 7 days/week 14 . treatments should be timed before meals or at least 2 hours after to allow gastric emptying (which is delayed in some patients) 15 in order to minimize gastroesophageal reflux during act. appropriate information needs to be recorded in each patient's unit record to meet communication and medico-legal requirements. the quality of treatment and adherence are crucial in achieving optimal outcomes short and longterm. noreen roth henig, m. d. the majority of icu admissions for cf occur in the setting of respiratory complications of cf including pulmonary exacerbations from chronic infection leading to respiratory failure, hemoptysis and pneumothoraces. admission to an intensive care unit (icu) for progressive respiratory failure has different implications than admission for hemoptysis or a pneumothorax, an extrapulmonary manifestation of cf, or for post-surgical care. critical care for patients with cystic fibrosis encompasses a large number and wide range of important issues. the critically ill cf patient has needs specific to the underlying disease that distinguishes them from other icu patients. the cf team is well positioned to guide the intensivists in these issues as well as in endof-life discussions. factors such as local cf center practices, icu norms, patient preferences, and the availability of lung transplantation predictably influence admissions and outcomes in the icu. in the largest published american series of adult cf patients in the icu, 74% of the 135 admissions were for a cf specific respiratory event. [1] extrapulmonary manifestations of cf account for additional icu admissions, although the exact number is not known. two such extrapulmonary indications for icu admission are heat stroke and hematemesis from esophageal varices secondary to cirrhosis. renal failure due to drug toxicity, narcotic overdoses, and anaphylaxis to antibiotics are unfortunate complications of usual cf care that may result in icu admissions. in the largest european experience published, post-surgical admissions make up the majority of icu admissions (18 of 31 admissions). [2] in that series, 16 of the 18 admissions were for pleurodesis. in other centers, abdominal and pelvic surgeries are more common indications for icu admission. although published experience in this area is limited, it appears that cf patients admitted to an icu for any indication other than progressive respiratory failure are likely to survive to discharge from the hospital. although most physicians feel comfortable utilizing icu resources for potentially reversible problems in cf patients, progressive respiratory failure presents a unique challenge. an early multicenter study of patients who received endotracheal intubation with mechanical ventilation for respiratory failure showed abysmal outcomes: 85% of patients died within 6 weeks of intubation, either on or off the ventilator. heavy sedation and immobilization of the patient often accompany endotracheal intubation and mechanical ventilation and compromise airway clearance. suctioning of secretions through an endotracheal tube, either blindly or with a bronchoscope, is less effective than a cough and expectoration in an awake patient. in addition, patients who cough while intubated are perceived as "bucking" the vent, uncomfortable, and at risk of self-extubation, and are commonly treated with heavier sedation and/or neuromuscular blockers. the combination of poor airway clearance and the potential for the development of myopathies from immobilization and drugs may actually contribute to poor outcomes in cf patients. non-invasive mechanical ventilation (niv) provides ventilatory support to patients with respiratory failure without the limitations of sedation and immobility imposed by endotracheal intubation. the advantage is that patients can cough and expectorate while using niv. it is increasingly used in patients with cf for a variety of indications and in both inpatient and outpatient settings. the uses of niv are beyond the scope of this discussion, but its increasing use and acceptance has changed how end of life care in cystic fibrosis has undergone a revolution in the past decade. prior to the 1990s, end of life care for patients with advanced cf lung disease was based on the assumption that aggressive interventions were inappropriate; the advent of lung transplantation unraveled this consensus about the use of aggressive care at the end of life. there is anecdotal evidence that patients with end-stage cf lung disease may now see their choices as a stark dichotomy: either aggressive technologic intervention in the hopes of life extension, or acceptance of limited lifespan in order to receive high quality palliative care. this "either transplantation or palliation" model fails to recognize that the goals of pursuing restorative therapy and palliation are not by their nature in conflict, and that a mixed model of end of life care, incorporating the new realities of lung transplantation, is possible. although patients and physicians may not explicitly acknowledge it, opting for lung transplantation is certain to alter care in the final months of life. patients on the transplant list are more likely to opt for aggressive measures to sustain life in order to increase the chance of receiving a suitable organ. as a result, transplantation requires a reordering of priorities for patients and families, and a re-envisioning of the good death. it is unrealistic to assume that patients and their families have given no thought to the matter; autobiographical accounts of life with cf contain frank discussions about the circumstances of dying, both before and after transplantation. it is clear that the topic, while difficult, is important to patients, and it is clear that patients and families can both consider transplantation and make end of life plans. consideration of transplantation requires that the patient, family, and physician directly confront the lethal and where cf patients are treated. for instance, in the american study of cf patients in the icu, almost a third with infective exacerbations were treated with niv. in some hospitals, niv is used on the general medical ward and patients who fail niv do not opt for endotracheal intubation. all patients with cf in the icu require skilled attention to cf specific care, including attention to nutritional needs, enzyme supplementation, bowel care, drug clearance, hydration, and diabetes management. the nutritional needs of cf patients include overall high caloric needs, the decision between parenteral and enteral feeding, and the use of powdered enzyme supplements through feeding tubes vs. elemental formulas. distal intestinal obstructive syndrome is a potentially devastating complication of icu care and results from immobilization, dehydration, narcotic analgesics, and the difficulty in determining appropriate enzyme doses in enterally fed patients. hydration is a difficult management issue in cf patients who are prone to dehydration but who may have pulmonary edema as a contributor to the respiratory failure. drug metabolism, especially of aminoglycosides, presents unique challenges and often results in the need for dose alterations. a final issue is diabetes management. critical illness may worsen glucose control in cf, even in patients previous not recognized to have impaired glucose tolerance or cf-related diabetes. critically ill cf patients likely benefit from the same aggressive glucose control as other critically ill patients. [3] inevitably, discussions of end-of-life occur in the icu. ideally, the cf center team is aware of a patient's preferences regarding end-of-life care including aggressive interventions such as icu admission, mechanical ventilation and lung transplantation, although when faced with imminent death, a patient's perspective on end-of-life issues may change. additionally, the availability of transplantation, both lung and liver, may change the decision making paradigm, and even the local practices of the transplant center may influence end-of-life care. ultimately, the guiding principle that there is no "one-sizefits-all" approach to cf and critical care allows flexibility and individualized care at a most challenging time. nature of cystic fibrosis. physicians, eager to provide hope for recovery, may downplay the struggle that transplantation can be for patients and families, yet this does patients and families a great disservice. discussion of transplantation needs to be initiated at a point that allows adequate time for the family and patient to come to terms with the threat of approaching death and the uncertain prospect of transplantation. since administration of the usual therapies for cf only rarely causes distressing symptoms for patients, caregivers may not face difficult decisions about discontinuing therapies. at the end of life, intravenous or inhaled antibiotics, pancreatic supplements, oxygen, and the rest of the usual daily therapies for cf can be continued in most cases without a great burden to patients both on and off the transplant waiting list. there is one notable example: if bipap has been instituted as a measure of symptom relief, its removal may be both psychologically and symbolically difficult for the patient, family and clinician; the decision to remove assisted ventilation may be a visible sentinel of the approaching end of life or of the "failure" of transplant listing. pain control. initiation of pain control therapies such as opiates may have also have powerful symbolic importance for the patient family and clinician. yet skilled pain control is simply the duty of compassionate care, and concerns over the symbolic nature of opiate use should be so great that the result is a patient in avoidable pain. the common fear of respiratory suppression following use of opiates in chronic lung disease is unfounded, and concerns over addiction in end of life care have been dispelled by years of experience with their use in a wide variety of palliative situations. retrospective studies suggest that the frequency of headache and chest pain in cf patients increases steeply in the final year of life, and opiates should be used in sufficient amounts to relieve both acute and chronic pain. hemoptysis. patients with a history of marked hemoptysis may have a major bleeding episode as the final event in life. this possibility should be discussed and plans made as to what actions will be taken. simple measures such as a supply of dark towels and plans for rapid control of the symptoms of dyspnea and choking can lessen the suffering of both the patient and the family in such an event. respiratory failure. for most patients, death will follow a gradual decline in ventilation, with the accompanying "narcosis" of elevated co2 levels. yet for many, there can be waxing and waning consciousness, as well as changes in work of breathing, and pharmacologic therapies for control of dyspnea will be needed. although there is no prospective research on the use of opiates to control the dyspnea of cf respiratory failure, the use of opiates in other deaths due to copd strongly suggests that moderate doses of opiate can provide excellent control of end-stage dyspnea. there may well be other family members with cf, or a previous death from cf in the family. these deaths may have formed a "script" for the family as an example of either a good or bad death, and an open exploration of the family's view of the previous death can be invaluable in uncovering unspoken expectations. in addition, there is often a tight circle of friendship among patients with cf, and the death of one member of the circle will have powerful and long lasting effects on the other patients, particularly if death occurs suddenly. clinicians too may have a "script" of how they expect a patient's dying to proceed, and should be aware that their expectations may conflict with those of the patient or family. clinicians should openly discuss their approaches to end of life care and transplantation with one another. consultation with palliative care teams within the hospital can bring a valuable perspective. until recently, most cf clinicians have operated on the assumption that the care they provided was uniformly excellent. however, it has become increasingly clear that there are differences in outcomes among different cf clinics, suggesting that there are more and less effective approaches for providing cf care. thus, while we await the expected advances in care that will be provided in the long run by breakthroughs in biomedical research, we have the opportunity to take better advantage of existing knowledge to rapidly improve the outcomes for people with cf. there are several key strategies that healthcare providers can use in order to make positive changes in the care they deliver. these include: 1. appreciate that changes must be made to the system of healthcare delivery. the first step, and one that is often the most difficult for physicians, is to understand that simply working harder within a non-supportive system will not yield the results desired. the model of the individual physician who by force of intellect and will establishes the correct diagnosis and prescribes the appropriate therapy to cure a patient is anachronistic and does not apply to the care of patients with cf. in caring for a chronic disease like cf, multiple caregivers must communicate and integrate a complex set of data and then prescribe therapy based on the appropriate use of that data. while it is incumbent on the system to ensure that providers are knowledgeable regarding best, or ideal practices, it further needs to support consistent application of those interventions that the provider knows to be optimal. variation in outcomes (when adjusted for variation in risk) is then due to variation in the system's ability to provide this support in a consistent manner. in general, cf care providers have successfully adopted a longitudinal approach to dealing with this chronic disease. in addition, it is useful to conceptualize and work towards instituting an idealized system of chronic care delivery that consists of various interdependent components inside and outside the local clinic setting. wagner's chronic illness care model provides a useful framework for consideration and may be summarized as follows (see also http://www.improvingchroniccare.org): a. community resources -medical center-based subspecialists should form partnerships with community organizations and primary care providers to support and develop interventions and fill gaps in needed services. furthermore, providers should publicly advocate for policies that improve patient care. b. overall health delivery system -organizations should create a culture that promotes safe, high quality care. all levels of the organization should visibly support improvement and provide incentives based on quality of care. there should be open and systematic handling of errors and quality problems and care should be coordinated within and across organizations. c. patient and family self-management -individual resources should be exploited by training and empowering patients and their families in self-assessment, goal-setting, action planning, problem-solving and follow-up. furthermore, patients' input should be sought in planning all aspects of care as well as delivery system design. d. delivery system design includes the structure and function of the clinic, from the telephone to the reception area to the examination room. it is essential to define roles and distribute tasks among team members, use planned interactions, ensure regular followup, and give care that patients understand. e. decision support comprises the promotion of evidence-based care at the provider-patient interface. this is accomplished via the use of clinical tools, guidelines and algorithms to ensure that intended care is actually prescribed, and reliance on rote memory is minimized. f. clinical information systems -at the individual patient level, the system might provide timely reminders for providers and patients, facilitate individual patient care planning, and allow data to be easily shared in order to coordinate care. at the clinicwide population level, the system can help to identify relevant subpopulations for proactive care, and allow providers to monitor performance of the practice team and care system. it is the lack of the latter data that keeps many providers in the dark regarding the true effectiveness of their care. an effective organizational change strategy is an essential component of improvement work. without a disciplined approach, practitioners who are newly aware of the extent of their system's deficiencies will attempt immediate, large dramatic changes which fail either in their planning stage because they get bogged down in endless preparatory meetings or self-destruct in their implementation phase because of the number of unanticipated problems encountered. one effective approach, is the langley et al's plan/do/study/act (pdsa) cycle. to implement the process, the first step is to plan the details of a small test of change [plan] . the planned change is then carried out [do] . once the change is attempted on a small scale, data on its effectiveness is gathered [study]. following discussion of what was learned by the initial endeavor, the change strategy is then modified and reattempted [act] . through repeated use of this cycle, small hunches, theories, and ideas gradually coalesce to into significant changes that consummate in significant improvement. the essential key to success is the use of small changes that are easily accomplished, followed by the analysis of data to evaluate the impact of the intervention. the use of data on performance is essential to recognize where opportunities for improvement exist, and also in order to garner feedback on what changes truly result in improved outcome. once an organization determines specific actions that must take place in order to improve outcomes, it needs to track the consistency with which those actions are taken. improved performance on these "process" measures can be measured as a preliminary step to improvement in the outcome measures that are the true goal of the work. feedback must be received promptly and on a regular basis, and data should be reported visually in a way that can be understood and used by members of the care team as well as interested outsiders. the synergy that derives from collaboration among workers investigating the same problem is well known to scientific researchers, the most successful of whom are typically embedded in networks of cooperating laboratories within and outside of their home institutions. this strategy is equally effective for the development and spread of innovations for improvement in the delivery of health services. the most commonly used cooperative model is one that recognizes "best practices" and attempts to copy and spread approaches used by providers whose outcomes are the best within their field. however, novel and effective ideas for how to accomplish certain specific goals exist even at centers whose overall performance is average, especially if they are actively striving to improve their outcomes. thus, collaboration among various centers and healthcare workers who are trying to accomplish the same or similar goals is an important and effective strategy to accelerate change. supported in part by cff schech01c0qi. the steady increase in median survival for those with cystic fibrosis (cf) is, in large part attributed to cf center-based care and the introduction of new therapies. however, variation in practice patterns and utilization of existing therapies, as well as patient outcomes, suggests less than optimal application of existing knowledge in caring for those with cf. epidemiologic data identifies "average" cf care patterns and outcomes, but does not inform cf care providers on strategies for benchmarking above average, or "going beyond good enough". improving cf center care using existing therapies and scientific knowledge requires thoughtful examination of current care patterns to identify potential areas for improvement. the national initiative for children's healthcare quality (nichq) recruited 16 cf care centers of varying demographics across the united states to create a collaborative group. this group focused on two goals to improve cf center care: improving nutrition and reducing environmental tobacco smoke (ets) exposure. this collaborative group of centers spent eighteen months working towards these goals utilizing the care model for child health. the experience of one pediatric cf center within the collaborative is presented demonstrating successful strategies for improving outcomes in nutritional status and reducing ets exposure, through appropriate process changes, partnership with families, consistent aggressive nutritional interventions, and application of existing knowledge. practical application of elements within the care model for child health incorporated within the collaborative are shared including changes in clinical information systems, decision support, delivery system design, family education, and a prepared, proactive cf care team. the results of the collaborative efforts resulted in a steady increase in median weight percentile for all children, and an even greater increase in those children in nutritional failure at the first recorded encounter. key factors in the achievement of the collaborative goals included: nutrition focus and process changes, and engaging families and patients to teach about enteral supplementation options. 4. preview of patients prior to clinic, in addition to postclinic review. 5. improving self-management by providing specific nutrition education (monthly newsletters) and written self-management plan at each visit. targeted efforts to reduce environmental tobacco smoke (ets) exposure resulted in a 75% documented "no smoking policy" for all environments for children within the center, and a 25% "quit-rate" among parental smokers. a simple three-pronged strategy of: a) systematic assessment of ets, b) clinician education in tobacco cessation counseling, and c) identification and provision of appropriate support materials to families, allowed for significant success in the overall reduction of ets exposure. the experience of this center demonstrates the potential to improve the care and outcomes of children with cf through systematic application of existing knowledge and attention to basic tenants of the cf center-based interdisciplinary care model. identification of variation in practice allows for focused interventions in areas where changes can, and will drive cf care beyond "good enough". it is the goal of all health care practitioners to render optimal care to those who seek their assistance. to this end, the learning leadership collaborative of the cystic fibrosis foundation has begun the promotion of the education of our community in the processes of quality improvement. as the project for the mini-atp course at intermountain health care we developed a standardized approach to relate to our patients the importance of their nutritional status with the goal of having no patients in need of urgent nutritional rehabilitation by june of 2004. noting the great variance in the rate of nutritional compromise among the many cf centers, and accepting the concept that a calorie will function similarly regardless of geography, it was our hypothesis that raising awareness of nutritional status in our patient population, when coupled with specific recommendations for nutritional intervention, would result in a significant improvement in the nutritional state of our patient population. in september of 2003 we stratified our pediatric and adult population into three categories (green, yellow, and red) based on weight/height percentile for patients less than 2 years of age, bmi percentile for pediatric patients greater than 2 years of age, and bmi in adult patients equal to or greater than 20 years of age. the three categories of green, yellow, and red rendered visual reminders to institute category and age determined protocols for nutritional maintenance or rehabilitation. a flowchart was created to assist in accurately stratifying each patient at each visit. in october of 2003, we began universal distribution of growth or bmi charts at all regular visits. we also began institution of a "calorie contest" designed to raise the awareness of patients and their families regarding the caloric content of varied food combinations. those found in need of nutritional rehabilitation were treated in accordance with established cf guidelines. we tracked our adherence to our own program by recording all of the above interventions in a flow sheet placed at the front of each of our patient's paper charts. our team rapidly adapted to our new formalized process and we were able to achieve greater than 80% adherence to all of our interventions by 12/1/03. to monitor the effect of our interventions we created graphical displays of the monthly total of patients within each category as % of total monthly attendees. these data were graphed within control limits calculated from retrospective analysis of monthly data for 6 months prior to our interventions. upon reviewing our statistical process charts of our outcomes up until june 2004, we have not achieved the lofty goal of remitting our entire patient population from nutritional compromise. however, there has been notable improvement in both the pediatric and adult program outcome data as manifested by more visits per month with patients classified as "green zone". the data indicate a more prominent effect in the adult population, which has prompted us to readdress our process to further augment our positive outcomes. this process has served to enhance the nutritional status of our cf patient population and has provided a mechanism by which we may continue to improve many aspects of patient care. in our lives, sickness comes to loved ones, accidents leave their cruel marks of remembrance, and tiny legs that once ran are imprisoned in a wheelchair. mothers and fathers who anxiously await the arrival of a precious child sometimes learn that all is not well with this tiny infant. a missing limb, sightless eyes, a damaged brain, or the term "cystic fibrosis" greets the parents, leaving them baffled and filled with sorrow. sixteen years ago i had visions of what parenting a child would be like. my visions were based on hopes and dreams, which didn't include a life threatening illness. i remember waiting in a tiny room with staunch white walls that seemed to enclose us with its smells of alcohol. my husband and i anxiously waited the arrival of the pulmonary doctor as we nervously passed our two month old back and forth between us. i remember the doctor's red, white and blue tie, his graying hair and the subtle way he cleared his throat as he pronounced, "your son has cystic fibrosis." i felt isolated, overwhelmed and numb as we left the hospital corridor. there followed the inevitable blaming, the condemnation, and the perennial questions: "why such a tragedy in our family?", "how did this happen?", "where was god?". why, where, how-those recurring words-did not bring back our lost dreams, our son's perfect body, and the plans we had as parents. i have learned that self-pity, personal withdrawal, or deep despair will not bring peace, assurance, or help, which i needed to courageously, continue on my parenting journey. rather, i had to find a way to go forward, look upward, and move onward. sixteen years later the cystic fibrosis parent-to-parent support network is proving to be just this type of help by offering comfort, hope and encouragement from parents who are reliable allies that immediately understand the situation because they have been through it themselves. imagine the atmosphere of a room filled with ninetyeight mothers of children with cystic fibrosis. each of these mothers was experiencing a different place on the cystic fibrosis journey; some were newly diagnosed, and others were more experienced dealing with various struggles of cf. each mother was given the opportunity to ask a question or give advice structured around the challenge of cystic fibrosis; "how do i get my threeyear-old to sit still while taking a breathing treatment?", "i feel like all i do is give breathing treatments, chest physical therapy and medicine all day. what can i do to manage my time better?", "how can i get my teenager to take his enzymes when he goes out with friends?". how did they get support that day? a connection was made between mothers; the loneliness and despair were replaced with friendship and companionship. the robert wood johnson pursuing perfection grant supporting healthcare quality improvement at cincinnati children's hospital encouraged focus on the viewpoint of patients and parents and perspective of their needs in an effort to improve care. a qualitative study of parent to parent self-help conducted by parents and researchers and published in the journal of pediatric psychology (1) determined that an overwhelming majority of parents were positively helped by parent to parent support. this type of support was shown to increase parents' acceptance of their situations and their sense of coping. parents were also able to define their roles in the healthcare process, perfect their self-management skills and in turn feel more confident in caring for their children. with support from the cincinnati children's hospital and the pursuing perfection grant, and knowledge and experience from studies (2,3), a core group pf patients, parents and cf center staff was formed. the core group identified a void in support that the parents could fill. the next step was to promote a parent-to-parent support network with the goals of providing strategies for improved patient care, tips from other parents, coping skills, and hope and comfort. i don't want to make it sound like things just fell into place, because in actuality, it took a lot of hard work. as with any change, there were concerns and conflicts. we experienced challenges but we were able to work through them. the legal department wondered whether parents would be compliant with hippa. there were concerns with training and how well the staff and parents would work together. any of these concerns could have been used as an excuse to quit. but, as john f. kennedy said, "the problems of the world cannot possibly be solved by skeptics or cynics whose horizons are limited by the obvious realities. we need…to dream of things that never were." we have a great team of people who, despite the difficulties, are committed to succeeding and reaching a common goal. in fact look at what we have been able to accomplish so far. one-to-one support is a personalized type of support that is made available to parents particularly during periods of increased stress, such as at the time of diagnosis. parents are carefully matched with a mentoring parent who understands their situation. our team consists of tracey blackwelder cincinnati children's hospital, cincinnati, ohio twelve diverse mentoring parents who were selected by the healthcare team. these selected parents were trained in communication and listening skills. quarterly newsletter is a quarterly parent-to-parent support newsletter that is mailed to all families of patients of the cincinnati cystic fibrosis clinic. this newsletter is coordinated and edited by parents, and is written with the parent's perspective. it is an effective way to stay in touch with parents and it provides continuous uplifting stories, updates, self-management skills, and influential hints and tips that help parents feel motivated and successful. social functions offer an opportunity to share experiences, strengths and hope. these functions enable parents to connect with others and find their way out of isolation, frustration and hopelessness and turn their challenges into positive forces. socializing also provides a venue for information regarding successful care strategies. promoting awareness is an important component of parent-to-parent support to reach families. stewart henderson britt said, "doing business without advertising is like winking at a girl in the dark. you know what you're doing, but nobody else does." we connect with parents through brochures, voice mail, list-serve web service and e-mail. in summary, the parents and staff are working together as a team. just like two oxen yoked together can accomplish more than they can separately when working for a common goal, our combined team of parents and staff are accomplishing amazing things as we work together to "change the outcome" in our pursuit of perfect care. ainbinder,j., blanchard, l., singer, g.h.s., sullivan the us cystic fibrosis foundation patient registry has been collecting annual data from over 20,000 patients cared for at accredited cf care centers since 1982. this data has been used to document the steady improvement in the survival of people with cf, and improvements in lung function and in nutritional status. how can we now use it to identify opportunities for change and to monitor progress in the care of people with cf? for the past 5 years we have examined the variation in treatments and outcomes between care centers and given feedback reports from the registry to the centers on their processes and outcomes. the amount of variation at the center level is completely invisible to providers who care for patients one at a time in a busy clinic, so these reports are an important first step towards understanding each center's care and outcomes in the context of cf care in the us. clinicians want to know how they are doing; eventually patients and families are going to demand to know as well. there has been a steady improvement in those measures that have been highlighted in the reports. in the past five years, the rate of screening for cf related diabetes nationally has gone from 60% to 82%, and in 2002 six centers reported screening 100% of appropriate patients. the proportion of patients adhering annually to standards for numbers of pulmonary function tests, sputum cultures, and outpatient visits continues to rise. however, the center reports are limited to working within the framework of the clinical practice guidelines for cystic fibrosis. the guidelines summarize the best evidence for different aspects of cf management, but they also define those areas of clinical care that experts consider important. there is center level variation in every aspect of patient care: antibiotics are used chronically in over 60% of patients at some centers, zero percent at others (see figure on next page); tobi is used in 23 to 100% of pseudomonas aeruginosa positive patients; pulmozyme is used in 16 to 100% of patients; annual hospitalization rates vary from 0 to 80% of adults, with median lengths of stay from 2 to 15 days; and home iv antibiotic use rates range from 0 to 71%, with median courses between 5.5 and 29 days. nutritional processes and outcomes are similarly variable. does all this variation matter? we have started the process of answering the question. "are some centers the 'best' and what does that mean for patient care?" by examining lung function and nutrition outcomes for children and adults annually from 1998 to 2001, ranking centers and selecting those that were reliably in the top 10 in several categories we identified 7 centers with consistently good outcomes across patient groups. since 1997 the median age of death is 28.8 years for the "best" centers compared to a mean of 23.2 years for the rest of the country, and median survival is almost 7 years longer (p<0.0001). this analysis is the basis for the cf foundation's vision statement: "we believe that the life expectancy of cf patients can be extended by 5 to10 years through the consistent implementation of existing evidence-based care." what are these centers doing to be so successful? the "best" centers are diverse; small and large, academic, for profit, rural and urban. the registry can suggest areas in which to focus further study but it does not collect the level of information that is required to completely describe exemplary cf care. how could a clinically useful data instrument describe a center where there are fail safe systems in place, where every patient gets everything they need at every visit, where handoffs between care givers are smooth, where culture results are never lost, where patients and families are well informed, activated, responsible, and supported? the registry allows us to report what is currently achievable by some clinical centers and sets the performance possibilities for the present. the highest performing centers don't necessarily use more healthcare resources than other centers. benchmarking visits by clinic teams may help us to understand the fine detail of care that produces the greatest success. what can be done right now to accelerate the improvement in cf care that we have seen over the past decades? historically, the registry has been used for epidemiologic purposes; the conversion to a patient management and quality improvement tool requires that the clinical practice guidelines be written in such a way that adherence is quantifiable. we must keep the guidelines up to date for capturing and measuring exemplary cf care. already portcf helps track individual patient results, and has some important patient management and reminder tools. portcf is being enhanced so that center level reports will be available much more quickly, and eventually, the reports will be available as the data is collected. but data that is not examined is like a tree falling in the woods. the patient registry gives a broad view of cf care in the us and allows us to track progress and focus our attention on achievable goals. variation is a treasure, but it does not yield its secrets easily and to learn what creates success is going to require the concerted efforts of all the players -clinicians, patients and families, basic scientists, and the foundation. cystic fibrosis foundation patient registry 2002 annual report to the center directors, bethesda, maryland. the year 2003 was a one of dramatic change for the cf foundation patient registry. first, the implementation of new regulations pertaining to patient privacy necessitated that all care centers gain approval from their local institutional review boards (irbs) for submission of data to the registry. a formal consenting/ assenting process was required at all but 4 care centers and 3 affiliate centers. despite these logistical challenges, 2003 registry data was submitted from 195 sites including 118 care centers, 24 adult programs with distinct registry center numbers, and 53 affiliate centers. patient retention in the registry was excellent. we observed a modest (approximately 6%) drop in patient numbers as compared to 2002, but fully expect the numbers to rebound in 2004. the care center teams deserve accolades for their work on behalf of the patient registry. the transformation of the patient registry from the collection of year-end summary data to the web-enabled, encounter-based port cf application was the other major change in 2003. the web-based application has opened up new challenges and opportunities. a number of resources can now be placed at the fingertips of our care teams including consensus documents and patient education materials. ongoing data entry throughout the year provides the potential for real time feedback of information to the care team. for example, the current iteration of port cf includes reporting features such as the patient roster, patients due for a visit, and the patient summary reports. the summary report displays complications, respiratory microbiology, and graphical trends of pulmonary function and nutritional status. the query tool provides the capability to identify subsets of patients, e.g., those with cf-related diabetes, which require special monitoring. our intent with these reports and query capabilities is to provide access to patient and center level information that will facilitate health care delivery. the future for port cf holds even greater promise. our quality improvement activities are focused on three core care areas in which suboptimal outcomes are associated with morbidity and mortality: 1) growth and nutrition, 2) pulmonary, maintenance and exacerbations, and 3) cfrelated diabetes mellitus. the significant variability in these outcomes throughout the care center network suggests that they are high leverage areas for accelerating the rate of improvement in cf care. we are currently collecting data in port cf on the pertinent medical outcomes in these areas, but many of the associated practice patterns are not being captured. collecting this data throughout the year and making individual and aggregate information about these practices available to the care centers will be instrumental in supporting further improvement activities. decision support in the form of reminders and algorithms can also be incorporated for key care decisions. further enhancements to port cf will occur in concert with an updating of our clinical practice guidelines. our goal is to create a dynamic process with better integration of port cf, the practice guidelines and clinical care (see figure) . standing committees will be formed in the three core care areas. the growth and nutrition committee has recently been constituted and will be the first area for enhancements to port cf. one of the first tasks for the committees will be to develop a list of strong recommendations for care based on the scientific evidence and clinical experience. work done in our quality improvement collaboratives will also inform the process. the strong recommendations of the guidelines committees will serve as a basis for new questions and reports derived from the new data for port cf. while striving to incorporate value-added data fields into port cf, an important consideration will be the impact of data collection and data entry on care centers. in summary, port cf is a critical tool for supporting improvement activities and for tracking the outcomes of those efforts. further evolution of port cf will occur in concert with ongoing quality improvement activities and an updating of our clinical practice guidelines. the continued success of the registry is dependent upon the commitment and hard work of the care center teams and the willingness of patients and families to share their data. the response to recurrent injury, in liver and in other organs is one of wound healing. many different types of injury (i.e., chronic hepatitis, ethanol, biliary tract disease, cystic fibrosis, iron or copper overload, etc.) lead to injury and ultimately to hepatic fibrosis and cirrhosis. cirrhosis in turn leads to hepatocellular dysfunction and portal hypertension, each of which have grave clinical sequelae. cirrhosis in cystic fibrosis results in the same clinical complications as is found in patients with other forms of liver disease. extensive investigation over the past 20 years has established that the effector in the liver wound healing process is the hepatic stellate cell (also known as a lipocyte or ito cell). a central feature of the wounding response to liver injury is the transformation of resident stellate cells from a "quiescent" (normal) to an "activated" (injured liver) state ( figure 1 ). characteristics of this transition include morphologic and functional changes. morphologic changes include loss of vitamin a, acquisition of stress bundles, and development of prominent rough endoplasmic reticulum (1) (2) (3) (4) . one of the earliest described and most prominent effects of stellate cell activation is production of increased quantities of extracellular matrix, including types i, iii and iv collagens, fibronectin, laminin and proteoglycans, some of which are increased by greater than 50-fold (3). further, the available evidence now indicates that the overall increase in extracellular matrix protein deposition typical of cirrhosis can largely be ascribed to excess production by stellate cells (5) . thus, a great deal of emphasis has been placed on elucidating mechanisms underlying stimulation of fibrogenesis by stellate cells. a number of events, typically acting in concert, play a role in stimulating stellate cell fibrogenesis (6) (7) (8) (9) (10) (11) (12) (13) . for example, stellate cells produce chemotactic factors (14) , appear to be important in matrix remodeling (15) , and produce a number of "vasoactive peptides" (8, 9) , including the potent vasoconstrictor, endothelin-1 (et-1) (9, 16). indeed, et-1 and vasoactive peptides have emerged as important components of the fibrogenic response. an important emerging theme is that current methods for precise diagnosis and quantitation of the effects of therapy are required. liver biopsy remains the gold standard for assessment of liver fibrosis. however, it is not without potential problems. therefore, a number of methods have been used to non-invasively assess fibrosis. these include imaging techniques, non-specific or indirect markers of liver fibrosis, and tests that more directly assess the fibrogenic biology present in the liver. such non-invasive tests are likely to emerge as critical components of the clinical armamentarium in management of hepatic fibrosis. a number of therapies (i.e., colchicines, ursodeoxycholic acid, ptu, vitamin e, etc…) have been tested in patients with fibrosing liver disease. for the most part, these approaches have proved to be ineffective. however, newer preclinical studies have highlighted a number of putative therapies to abrogate fibrogenesis. such therapies are targeted at reducing or removing the primary injury, modulation of stellate cell activation, stimulation of matrix degradation or stimulation of stellate cell death. for example, interferon gamma, a cytokine with potent immunomodulatory activity, has profound effects on multiple aspects of stellate cell activation. these data have led to clinical trials in humans in which interferon gamma is being examined in patients with fibrosis. other specific, mechanism based, "anti-fibrotic" therapies are rapidly emerging. liver disease is a major complication in many patients with cystic fibrosis (cf). the precise prevalence of cfassociated liver disease (cfld) is difficult to determine as no reliable method of diagnosis currently exists. traditional measures of liver function do not correlate with the severity of liver disease and there is no specific cf genetic association. thus the diagnosis is not usually made until cirrhosis or complications such as portal hypertension are established. while clinical and imaging studies may detect these late changes, and invasive liver biopsy, which is not always reliable, due to the focal nature of fibrotic changes might aid diagnosis, the challenge exists to develop a sensitive, reliable non-invasive assay to identify cf patients who are at risk of developing severe liver disease, which might also serve as a guide to monitoring progression or response to therapy. although the basic mechanisms remain unclear, abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cfld. we have documented a definitive role for hepatic stellate cells in the fibrogenesis associated with cfld, and suggested a potential mechanism for the induction of hsc collagen gene expression, through the production of the cytokine tgf-beta(1) by bile duct epithelial cells (1) . accumulated bile acids have been proposed as potential mediators of cytokine production, and , indeed, we recently found a correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression (2) . a potential monitoring role for serum bile acids and serum measures of components of hepatic matrix remodelling, which may serve as markers of early fibrosis is suggested by these studies. we thus have evaluated, by preliminary cross-sectional study, the utility of selected serum markers ie bile acids, hyaluronic acid (ha), collagen type iv (cl-iv), prolyl hydroxylase (ph), matrix metalloproteinases (mmps), and tissue inhibitors of mmps (timps), by their correlation with the degree of hepatic fibrosis in patients with cfld. we compared with serum levels from patients with cf and no liver disease (cfnold), and age matched control data. methods: bile acid composition was determined by gas-liquid chromatography/mass spectrometry in bile, urine, and serum samples from 30 children with cfld, 15 children with cf but without liver disease (cfnold), and 43 controls. sera from 36 cfld, 37 cfnold and 38 control subjects were assessed for timp-1, cliv, mmp-2, ha and ph by enzyme immunoassay. serum markers were correlated with blinded hepatic fibrosis score on liver biopsies from 30 cfld subjects.results: there were significant correlations between serum cholic acid levels and the ratio of serum cholic acid/chenodeoxycholic acid with hepatic fibrosis score, the degree of inflammation, and limiting plate disruption. timp-1, ph, and cl-iv in cfld, there was a negative correlation between fibrosis score and both timp-1 (r = -0.39, p = 0.06) and ph (r = -0.48, p = 0.008). there were no differences in timp-1, cl-iv, ha or ph between cfnold and controls, nor in mmp-2 between the three groups. the correlation between both cholic acid and cholic acid/ chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in cfld. elevated levels of serum timp-1, cl-iv and ph in cfld suggests that these may be indicators of fibrogenesis in cf. higher levels of timp-1 and ph were associated with mild fibrosis, and these, in concert with serum bile acid measurements may be useful markers for the early detection of cfld. we suggest that these serum markers be subject to longitudinal study in cf as markers of progression of cfld, and/or response to future antifibrogenic therapies. clinical and biochemical parameters have been shown to be very insensitive in the identification of cystic fibrosis associated liver disease (cfld) (1). thus, many centers have begun to investigate the use of radiologic screening for cfld. this is challenging in that the histologic findings with cfld can range from steatosis through early fibrosis to biliary cirrhosis (2) . the primary therapy that is currently available, ursodeoxycholic acid, has been suggested to be most effective in early disease (3, 4) , but may improve advanced cfld (5) . thus, the challenge for treatment remains the identification of "early" cfld for potential treatment and the identification of "silent" advanced cfld to institute appropriate anticipatory management. to that end, radiologic studies offer noninvasive imaging to assess the liver. this summary will discuss ultrasonography, ct scanning, mr scanning and radioisotope excretion studies. ultrasonography (us) utilizes short pulses of high frequency sound that are reflected back at the interfaces of tissues of different acoustic properties. us is low in cost, portable and involves no radiation exposure. it is limited by the inability to penetrate bone or air which can prevent a complete examination of abdominal organs and occasionally by the depth of penetration required. us is the imaging modality of choice to screen for biliary disease in cf. cholelithiasis and biliary dilation can easily be determined. us has also been suggested as a tool to detect early changes in liver architecture in cfld (6) (7) (8) . us has an excellent sensitivity and specificity for end stage cirrhosis with portal hypertension (9) as the findings of a nodular liver, splenomegaly and varices are generally due to cirrhosis. however, the goal of screening for cfld would be to identify early liver disease prior to the progression to cirrhosis. two ultrasound classifications for cfld have been proposed (6, 8) : in montreal, they have demonstrated minimal interobserver variability in the scoring and interpretation of us (8, 9) . abnormal us findings were more common in patients with abnormal ast, alt or ggt. however, only about 50% of patients with abnormal ast, alt or ggt had an abnormal us and 10-20% with of patients normal values had an abnormal us (9) . no study has investigated inter-center variability using either scoring system or the correlation between early us findings and liver biopsy evidence of fibrosis in cf. us can be used to follow patients for the development of us abnormalities. in a longitudinal us study by leanerts, serial us was performed for an average of 6 us per patient (8) . of the 106 normal patients at the start of the study, 18 (17%) had any abnormalities identified during longitudinal screening. of these, 5 developed and continued with a heterogeneous liver, 6 developed a nodular liver and 8 progressed to portal hypertension. kaplan meier risk analysis predicted that approximately 15% of subjects would develop a nodular liver on us by 18 years of age. in this study, thirty subjects received ursodeoxycholic acid during the course of the study, clearly not all due to us abnormalities as 10-30% had biochemical abnormalities despite a normal us. it is difficult to make a firm recommendation about the use of us in screening for cfld. the sensitivity and specificity of early us findings for fibrotic cfld have never been studied, making interpretation of the meaning of these findings difficult at present. it seems likely that us will increase the identification of "silent" cf associated liver imaging abnormalities, but the timing, frequency and cost effectiveness of the routine use of us in screening for cfld remains to be determined. a prospective study of the sensitivity of this modality would be helpful in our interpretation of these studies. computed tomography (ct) detects small differences in the attenuation of x-rays to identify structures not visible on standard radiographs (10) . the addition of oral and iv contrast improves the identification of tissues and blood vessels and can detect differences in perfusion in organs such as the liver. much like us, ct is very effective at identifying end stage cirrhosis with portal hypertension. the ct findings in end stage cirrhosis from a variety of causes have been published (11) . however, accurate ct imaging of the liver requires iv contrast and radiation exposure. in biliary cirrhosis, the primary end stage lesion of cf, nodularity of the liver is common and easily seen on ct. however, there are no studies of ct findings in early cfld. similar to us, there have been no studies correlating ct and early hepatic fibrosis in cf or other liver diseases. at present, ct cannot be recommended for screening for cfld given the need for contrast and the radiation exposure compared to us. however, ct is likely the most sensitive imaging modality for screening for hepatic cancer in patients with cirrhotic cfld. magnetic resonance imaging (mri) detects the density of protons in tissue water and lipids and their relaxation times (10) . in mri, the magnetic environments of protons in fat, intracellular water and extracellular water have different relaxation properties leading to sharp contrast differentiation between tissues with differing contents of fat or water. mri is very valuable for non-invasive imaging the biliary and pancreatic tree (magnetic resonance cholangiopancreatography). mri findings in cf have been presented with excellent demonstration of biliary and pancreatic abnormalities (12) . it is the imaging modality of choice for hemagiomas. the value of mri in diffuse parenchymal liver disease is still investigational (13) . similar to us and ct, mri can detect the gross abnormalities of cirrhosis and portal hypertension. at present, the use of mri for screening for early cfld would require further investigation. hepatobiliary scintigraphy. after iv injection, several radioisotopes are extracted by the liver and excreted into the bile and eventually into the intestine. these are detected by a scintillation camera. in cf, abnormalities detected have included dilated intrahepatic and extrahepatic bile ducts and delayed excretion and intestinal appearance of the radioisotope (14) (15) (16) . delayed intestinal appearance seems to predict a better response to ursodeoxycholic acid (3) . however, the specificity and sensitivity of this imaging modality for cfld has never been studied. given the technical difficulties with the study, it has been superceded by mri, ct and us for routine hepatic imaging and is not suitable as a screening test for early cfld. liver disease is increasingly recognized as a major cause of morbidity and mortality in patients with cystic fibrosis (cf). the typical hepatic manifestation of cf is focal biliary cirrhosis and results from biliary obstruction and progressive periportal fibrosis; it is the most clinically relevant cf-associated hepatic problem, since extension of the initially focal lesions may lead to multilobular biliary cirrhosis, portal hypertension and related complications (1, 2) . surveys on cf patients with liver disease have reported rapid progression of cirrhosis to portal hypertension and high mortality rates due to complications of cirrhosis and lung disease (3, 4) . in a retrospective study spanning a 26-year period, of 44 children with cf investigated for cirrhosis, esophageal varices developed in 86% and in half of these patients esophageal bleeding occurred early during the second decade of life; liver failure occurred in 36 % of them at a mean age of 15 years (3). in another retrospective study, over a period of 28-year, 16/31 cf patients with multilobular biliary cirrhosis died as a result of respiratory (10 cases) and hepatic (6 cases) complications (4). more recently information regarding natural history of liver disease in cf patients have been obtained through prospective studies in carefully monitored cf patients (5, 6) . in a cohort of 177 consecutive cf patients identified over a ten-year period and followed for a median time of 14 years at the cf center of the university of milan, there were 48 incident cases with no incidence peak in any age group (5). we estimated that crude incidence of liver disease in our whole cf population was 27%, corresponding to 2.4 cases per 100 patient-years. median age at time of diagnosis of liver disease was 7 years (range: 2 months to 18 years) and in 14 of the 48 patients with liver disease (29%) this complication was diagnosed during the first five years of life. in our cohort, after the age of 10 years, incidence declined and, by the age of 12, liver disease had developed in 45 out of 48 patient. liver disease should be therefore considered a relatively early complication of cf, which may be susceptible to prophylactic strategies. the 48 patients with liver disease (treated with ursodeoxycholic acid since 1988) were followed for a median time of 8.5 years (range: 1 month to 20 years), corresponding to 407 patient-years. cirrhosis was already present at time of diagnosis of liver disease in 5 of these patients (10%) and developed in 12 additional patients during follow-up after a median period of 5.1 years from time of diagnosis of liver disease (range: 6 months to 15 years). incidence rate of cirrhosis was 4.5 cases per 100 liver disease patient-years (95% ci: 2.3 to 7.8). among the 17 patients with liver cirrhosis who were altogether detected, 13 (76%) developed portal hypertension (incidence: 28.8 cases per 100 cirrhotic patient-years, 95% ci: 15.4 to 49.3), 4 (24%) esophageal varices (incidence: 3.2 cases per 100 cirrhotic patient-years, 95% ci: 0.9 to 8.3), 1 (6%) developed liver decompensation (incidence: 0.4 cases per 100 cirrhotic patient-years, 95% ci: 0 to 2.0) and underwent liver transplantation at the age of 10 yrs. at the end of follow-up, of the 45 patients with liver disease who were still alive 31 had not developed liver cirrhosis after a median time of 7.9 years (range: 2 to 15 years) from time of diagnosis of liver disease. present evidence concerning efficacy of ursodeoxycholic acid on patient survival in cf is still inconclusive (7), but we cannot exclude that treatment with this bile acid also contributed to the favourable clinical course we have observed. in order to establish the impact of liver disease on the outcome of cf, we also compared the main clinical outcomes of patients who developed liver disease with those observed in the group of patients who did not (5) . no significant differences between patients with or without liver disease were observed in the occurrence rates of clinically relevant end-points, like respiratory failure, need of oxygen therapy or number of hospitalisations during the last two years of follow-up. these data indicate that liver disease does not expose cf patients to higher risk of developing respiratory failure or other major outcome events. mortality from any cause also did not differ significantly among patients with different liver status (death rate ratio 0.44), indicating lack of association between liver disease and mortality in cf. other studies addressing cf-related liver disease prospectively (6) have also provided evidence that this is a relatively early complication of cf, and that its clinical course may be milder than previously reported by retrospective studies (3, 4) . in a cohort of carefully managed swedish cf patients who had been followed-up for more than a decade, liver disease developed before adolescence in 35 % of cases and in none of the patients this occurred in adulthood. rate of progression was slow, as also indicated by histologic changes at repeat liver biopsy during the follow-up (6) . in summary, liver disease associated with cf develops in a significant percentage of cf patients, usually before or at puberty, displays a slowly progressive course and until the most advanced stages are attained, it does not seem to influence nutritional status and severity of pulmonary involvement. only in a minority of patients, often in the pediatric age, liver disease may represent the main clinical problem and its progression may be unusually rapid. the role of genetic modifiers in determining this variability in terms of severity is presently under study (8) . the most prevalent cf-causing mutation is a three base pair deletion that results in the loss of phenylalanine 508 (f508) in the first nucleotide binding domain (nbd1) of cftr. over ninety percent of all cf patients carry at least one delta f508 allele (1) . not surprisingly, the molecular and cellular phenotypes of this mutation have been the focus of intensive study over the past fifteen years. this work has established that the mutant cftr does not efficiently traffic to, nor accumulate at, the plasma membrane (2) due to a disruption of nbd1 folding (3; 4) and destabilization and rapid turnover of the minor fraction that does reach the membrane (5). the inefficiently folded delta f508 cftr is recognized by cellular quality control systems and marked for degradation by the proteasome (6; 7) . the lack of properly folded, native cftr at the apical membrane underlies the aberrant fluid and electrolyte homeostasis that define the cf pathology. delta f508 cftr that does fold and reach the plasma membrane retains some function as a chloride channel (8; 9). thus, promoting the folding of delta f508 cftr would be predicted to at least partially ameliorate the pathology (3; 10). in this regard, conditions that promote folding and stabilize native structures, such as reduced temperature (11) and addition of osmolytes (12) (13) (14) have been shown to improve mutant cftr folding and partially restore function. however, treatments such as these, as well as alterations of cellular chaperones that assist in folding, lack specificity and the consequences of chronic alterations of these parameters on cellular function remain unknown. more specificity could theoretically be achieved if ligands that bind tightly to the native, but not unfolded cftr could be identified (3; 10; 15). the favourable binding energy for these ligands would thereby be coupled to the otherwise unfavourable folding of mutant cftr (15) . ideal compounds would promote folding and potentiate cftr function although weak anatagonists could also have utility. detailed structural knowledge of cftr and its domains provides a context for understanding not only the functional mechanics of cftr, but also the role of f508 in folding and further suggest sites which could be targeted for ligand binding. the recent solution of the murine nbd1 at high resolution using x-ray diffraction methods provides the first such structural information (16) . the structure reveals that f508 is exposed on the surface of the nbd in an alpha-helical sub-domain. replacement of the phenylalanine with any other amino acid has no measurable effect on the folding or final structure of the isolated nbd, indicating that it is the loss of the backbone that leads to inefficient nbd folding (17) . in contrast, charged or bulky side chains at the 508 position interfere with the the later steps in the folding of the full-length cftr. together with the surface exposure of the 508 position, these results indicates that f508 may be at an interface between the nbd and other domains of cftr, most likely the tmds. these results have implications for understanding the function of cftr and the development of ligands to promote mutant cftr folding for therapeutic benefit. structural predictions suggest that cftr, a member of the abc superfamily of transport atpases, consists of two homologous halves, each comprised of six transmembrane (tm) helices and a nucleotide binding domain (nbd1 and nbd2) that are connected by the regulatory (r) domain (1). this complex, multidomain structure, conceivably, renders the post-translational folding of cftr sensitive to point mutations, while ensures its regulation by phosphorylation and atp-binding/hydrolysis. depending on the expression system examined, 20-80% of the newly synthesized wt cftr degrades at the endoplasmic reticulum (er), as coreglycosylated folding intermediate (2) (3) (4) . the remaining 20-80% of the cftr undergoes an atp-dependent posttranslational conformational maturation (3) (4) (5) and attains an export competent, folded conformation that is incorporated into transport vesicles at the endoplasmic reticulum (er). during traversing the cis/medial golgi, the high mannose-type n-linked oligosaccharides of cftr (core-glycosylated form) are converted to complex-type oligosaccharides (2) . it was proposed that the atp-dependent conformational maturation of the nascent, core-glycosylated cftr is a prerequisite for entering the distal stages of the secretory pathway from the er (3). this conclusion was verified by examining the in vivo and in vitro protease susceptibility of the early folding intermediates and the fully mature cftr either in its core-or complex-glycosylated form (3, 6) . the conformational maturation of cftr is facilitated by an array of chaperone interactions. accordingly, mutations perturbing the folding process of cftr alter the association/dissociation dynamics of these chaperones and vice versa; modulation of chaperone activity has an effect on cftr folding (7) . in the cf gene more than 1000 mutations have been identified, leading to impaired biosynthesis, processing, activation and stability of cftr or a combination of these (8) . missense mutations, including the most frequent one, deletion of phenylalanine at position 508 (∆f508) in the nbd1, are believed to interrupt the post-translational folding of cftr (2) (3) (4) 6) and target the core-glycosylated intermediate for degradation via the ubiquitin-proteasome pathway at the er (9,10). while exposure of er-retention signals, may also contribute to the inability of folding intermediate(s) to exit the er (11), the consequence of ∆f508 at the molecular/structural level remains elusive. numerous model systems, represented by synthetic peptides and isolated nucleotide binding domains, harboring the wild-type sequence or the ∆f508, have been applied to unravel the structural impact of the mutation. in our approach we used systematic amino acid substitution of the f508 residue in the context of the full-length cftr to investigate the role of the side-chain and back-bone structure of f508 on the biogenesis, stability and interdomain interaction of the channel. hydrophobic amino acids with side chain volumes comparable to, but not larger than phenylalanine supported the post-translational folding and stability of cftr. on the other hand, charged amino acids, as well as proline and glycine substitutions were non-permissive for both folding and stabilization of the channel. while deletion of f508 caused limited conformational defect in the nbd1, similar to non-permissive amino acid substitutions, it substantially compromised the conformation of nbd2 and its interaction with nbd1 (unpublished data). these and other results, discussed in the symposium, strongly suggest that hydrophobic side chain interactions of f508 are indispensable to ensure normal folding nbd2 and thus to achieve global stabilization of the native conformation of cftr. reduced temperature or chemical chaperones can partially rescue the folding defect of ∆f508 cftr (12) the majority of patients with cystic fibrosis (cf) express a mutant cystic fibrosis transmembrane conductance regulator (cftr) protein that is defective in folding (1). the misfolded protein is retained in the endoplasmic reticulum (er) and is not trafficked to the cell surface (misprocessed mutants) (2). these patients would benefit from therapy that can cause the mutant cftr protein to fold properly and be delivered to the cell surface in a functional form. recently, we discovered a strategy for correcting misfolded defects in p-glycoprotein (p-gp) that has direct application to cf. p-gp and cftr are both members of the atp-binding cassette (abc) family of proteins. the abc proteins generally have two transmembrane domains (tmds) and two nucleotide-binding domains (nbds). mutations in p-gp at equivalent positions to those in cftr that cause it to be misprocessed also caused the mutant p-gp to misfold and be retained in the er. the misprocessed p-gp mutants, however, could be induced to fold properly when they were expressed in the presence of a specific drug substrate (3). the protein was then trafficked to the cell surface where it was fully active. therefore, p-gp is a useful model system for studying how processing mutations interfere with folding of abc proteins. the next objective was to understand how the misprocessed cftr and p-gp mutants were different from the mature wild-type proteins and how drug substrates the mutant channel to the cell surface. biochemical assays revealed that the ∆f508 mutation not only impairs the gating kinetics, but also decreases the channel stability by tenfold at the cell surface (13) . to unravel the cellular machinery responsible for the recognition and elimination of the mutant from the plasma membrane, the internalization and recycling rates of cftr were determined. whereas native cftr recycled from sorting endosomes back to the cell surface, misfolding by ∆f508 and ∆70 mutations prevented recycling and facilitated lysosomal targeting of cftr by promoting its ubiquitination. rescuing the peripheral folding defect or down-regulating the e1 ubiquitin-activating enzyme stabilized the mutant cftr without interfering with its internalization. these and other observations, in concert with the preferential association of the mutant cftr with ub-binding proteins (e.g. hrs and stam-2) and other components of the ub-dependent endosomal sorting machinery (e.g. tsg101), establish a functional link between ub-modification and lysosomal degradation of misfolded cftr from the cell surface (13) . our data provide evidence for a novel cellular mechanism of cf pathogenesis in case of ∆70 cftr. the results also imply that efficient relocation of the ∆f508 cftr from the er to the cell surface will require the conformational stabilization of the mutant. finally, these observations suggest a paradigm for the quality control of membrane proteins in general, involving the coordinated function of the ubiquitination and ub-dependent endosomal sorting machinery in post-golgi compartments. act as specific chemical chaperones to rescue the misprocessed mutants. initial studies suggested that processing mutations affect the structure of p-gp (4, 5) or cftr (6) by trapping the mutant proteins in the er in "loosely-folded" conformations that resemble the immature core-glycosylated form of the wild-type protein. these trapped proteins are inactive. one folding step that may be sensitive to the presence of processing mutations is the establishment of correct domain-domain interactions between the two tmds and the two nbds. to test whether processing mutations affect domain-domain interactions, we constructed a pgp/cftr(⌬f508) chimera in which the predicted third ␣-helix in nbd1 of p-gp was replaced with the equivalent region from cftr containing the ⌬f508 mutation. it was found that the ⌬f508 mutation caused misprocessing of the p-gp/cftr chimera and loss of activity by disrupting packing of the tm segments in the tmds (7) . it also appeared that the ⌬f508 mutation disrupted interactions between nbd1 and the first cytoplasmic loop connecting tm segments 2 and 3. more recently, we showed that processing mutations could also disrupt interactions between nbd1 and nbd2 (loo et al., 2004, submitted) . these results indicate that processing mutations may interfere with folding of abc proteins by hindering the proper interactions between the domains. the drug substrates (specific chemical chaperones) may act by stabi-lizing proper domain-domain interactions during the folding process. mutations in cftr profoundly influence the function of respiratory epithelial cells, with resultant mucous accumulation, inflammation and infection. cftr plays numerous roles in cellular function, interacting directly and indirectly with cellular proteins and processes that regulate many aspects of epithelial cell biology. in order to identify pathways by which cftr influences cell function, and to explore the effects of cftr on cellular processes per se, we have generated a series of transgenic mice (1) lacking cftr (fabp-cftr, cftr-/-), (2) overexpressing human cftr in lung epithelial cells (sp-c-hcftr), and (3) expressing the human ∆508 cftr (sp-c-∆508 cftr, cftr-/-, fabp-hcftr). all of the mice are viable and without overt pulmonary pathology. rna microarray analysis of the lung tissue was compared in lungs from adult mice of each genotype using the jeffrey a. whitsett, m.d. and yan xu, ph.d. affymetrix system. rnas differentially expressed were cross-compared. comparison of sp-c-∆508 cftr, cftr-/-vs. cftr-/-demonstrated a strong, positive correlation (r = 0.938), and a weak, negative correlation (r = -0.372) with the sp-c hcftr overexpression, indicating human cftr ∆508 did not correct the abnormalities in gene expression characteristic of cftr deficiency. genes regulating inflammation, neutrophil chemotaxis, as well as fluid and electrolyte transport were induced in cftr-/-and sp-c-∆508 cftr, cftr-/-mice, while those regulating cell growth, cell matrix and adhesion, and angiogenesis were significantly decreased. increased expression of human cftr (wild type) in the ∆508 cftr induced genes in the heat shock protein family, indicating that this subset of genes was induced in response to cftr or misfolded ∆508 cftr. dnajb1 and hsp105 rnas were increased by human cftr and human ∆508 cftr, perhaps indicating a response to excess or misfolded cftr protein. genes involved in the endoplasmic reticulum response pathway (erad) were not induced by overexpression of cftr or ∆508 cftr in vivo. while rnas in the ∆508 and cftr deficient mice were quite similar, several genes involved in host defense including s100a8, s100a9, h2-eb1, h2-dmb1, h2-t10, and c-fos were induced in cftr-/-, but decreased in the ∆508 mutant mice. several genes regulating lipid homeostasis were uniquely influenced by the ∆508 cftr (scd-1, lipocalin, and carboxyesterase-1). in summary, increased expression of cftr and ∆508 cftr similarly induced genes in the heat shock protein (hsp) pathway, but did not induce erad transcriptional responses. genomic responses to cftr deficiency were, in general, not corrected by the ∆508 cftr. cftr influences genes regulating host defense, inflammation, and fluid and electrolyte transport by processes likely mediated by its direct interaction with other cellular proteins and by influencing adaptive responses to the lack of cftr. the absence of cftr activity from the cf airway epithelium results in abnormal regulation of ion transport mechanisms leading to a reduced volume of airway surface liquid (asl) producing mucus dehydration and decreased mucociliary clearance, both hallmarks of cf lung disease. the consequences of these physiological alterations in the lung eventually result in chronic bacterial infection, mostly notably pseudomonas aeruginosa, and exaggerated neutrophil-mediated inflammation, eventually leading to airway obstruction and death. a logical therapeutic strategy for cf lung disease would restore cftr function to the airway epithelium and gene transfer strategies remain one approach towards this goal. at present, the regions of the cf lung that would require cftr expression for restoration of normal physiological function and reduction of disease symptoms are not well established. in normal human lung, the ciliated epithelial cells of the surface epithelium are considered to express cftr. however, restoration of normal ion transport functions to cf airway epithelium after delivery of cftr exclusively to ciliated cells has not yet been achieved to test this hypothesis. the possible involvement of the submucosal glands in the pathogenesis of cf lung disease suggests that cell-types other than ciliated cells may also require cftr expression for normal function (1). however, the predominance of ciliated cells in the upper and lower airways especially in cf infants who have not developed chronic airway inflammation, and the accessibility of the ciliated surface epithelium to intralumenal-delivered therapeutics identifies ciliated cell-types as attractive targets for cf lung therapeutic strategies. a critical factor for cf gene transfer strategies has been overcoming the low transduction efficiency for intralumenal delivered vectors. there are several extracellular barriers to gene transfer vectors in human airway that may result in inefficiency of gene transfer, including: the mucociliary clearance system; the glycocalyceal barrier; the absence of most viral receptors from the airway lumen; and, the slow rate of lumenal endocytosis in airway epithelial cells. although strategies to circumvent these barriers with commonly available vectors are under investigation, i.e., retargeting vectors to new apical surface receptors, transiently opening tight junctions or using new serotypes of the common vectors, the results so far published have not shown significant improvement. novel vector-types are also being assessed for airway gene transfer that may be more efficient at breaching epithelial cell apical barriers. for example, sendai virus, human coronavirus 229e, and lentiviral vectors pseudotyped with ebola virus or sendai virus envelope proteins have shown improved gene transfer efficiency to airway epithelial cells in vitro and/or in vivo (2) (3) (4) (5) . however, the usefulness of these vectors remains to be determined. we have chosen to investigate the utility of the human parainfluenza viruses (piv) as gene transfer vectors since these viruses exclusively infect ciliated cells of the human airway epithelium after intralumenal delivery. to test these vectors, we used an in vitro model of human pseudostratified, mucociliary airway epithelium (hae) that recapitulates the morphologic and phenotypic characteristics of the in vivo human airway epithelium. recent studies have revealed that this model system displays the phenotypic differences that occur between cf and non-cf airway epithelium, i.e., reduced chloride ion transport, hyperabsorption of sodium ions, the failure to regulate the depth of the asl, and, the dehydration of secreted mucus that results in cilial dysfunction and mucostasis. we have previously shown that human recombinant parainfluenza virus (hpiv) efficiently infects human ciliated airway epithelial cells from the lumenal surface. to test our hypothesis that cftr expression in ciliated cells may restore normal physiological function to cf epithelia, we generated a hpiv that expressed cftr (hpiv-cftr) and systematically tested the effects of cftr expression in cf hae. western analyses performed 48 hrs after apical surface inoculation of cf hae with hpiv-cftr, hpiv expressing gfp (hpiv3-gfp) or vehicle control alone showed that only hpiv-cftr produced expression of fully glycosylated "band c" cftr. immunodetection of cftr protein in histological sections of cf hae inoculated with hpiv-cftr localized cftr to the apical surface of ciliated cells in regions corresponding to the microvilli of the ciliated cells. to determine whether cftr expression in cf ciliated cells resulted in chloride ion channel activity, cf hae were assayed in ussing chambers after viral inoculation. compared to control cf hae (vehicle or hpiv-gfp treated), inoculation with hpiv-cftr resulted in a significantly increased forskolin-mediated activation of cftr chloride channel activity [1.5 ± 0.1 _a/cm 2 for control (n = 18) and 23 ± 2.5 _a/cm 2 for hpiv-cftr (n = 9)]. to determine whether this level of cftr expression in ciliated cells was sufficient to restore the altered fluid transport of cf hae, we assessed the regulation of asl depth by measuring the rate of absorption of liquid across the apical surfaces of cf hae 48 hrs after inoculation with vehicle control alone or hpiv-gfp or hpiv-cftr. in control cf hae, the asl stabilized at 3.1 ± 0.3 _m (n = 12), a depth associated with decreased mucus transport. however, in cf hae inoculated with hpiv-cftr, liquid absorption ceased at a depth of 7.3 ± 0.3 _m (n = 12), a depth similar to that maintained by normal hae (7.4 ± 0.6 _m, n = 7). a hallmark of cf lung disease is mucostasis, a parameter that is recapitulated in cf hae. therefore, to determine if the restoration of fluid transport to cf hae by cftr expression in ciliated cells was sufficient to allow the transport of mucus secretions, we measured the capacity of cf hae to transport mucus after inoculation with hpiv-cftr. for control cf hae, rotational velocity of mucus transport was low with little or no mucus transport (1 ± 1 _m/sec, n = 5) compared to significant mucus transport in cf hae inoculated with hpiv-cftr (21 ± 2 _m/sec, n = 5). therefore, hpiv as a gene delivery vehicle has circumvented a major barrier to the effective use of gene transfer vectors, i.e., the restricted uptake of vectors from the apical surface. we have demonstrated that expression of cftr in ciliated cells of cf hae resulted in restoration of the ability of the epithelial cells to regulate the depth of the asl to levels that were sufficient for effective mucociliary transport. whether, expression of cftr in ciliated cells in the airway epithelium in vivo will be sufficient to restore these physiological indicators of cf lung disease remains to be determined. since hpiv-cftr is the most efficient and efficacious vector that we have so far tested for cftr gene transfer and results in correction of pathophysiological consequences of the cftr defect, efforts will be directed at generating derivatives of hpiv vectors that can be safely delivered to the human airway in vivo. the genetic etiology of human respiratory diseases is being increasingly emphasized as a means of better understanding disease pathogenesis, with the ultimate goal of improving preventive strategies, diagnostic tools and therapies. considerable effort and expense is currently being expended in attempts to detect genetic loci contributing to both 'simple' (single-gene) and 'complex' human diseases. association and linkage studies comprise the two dominant strategies, the former aiming to find disease predisposing alleles at the population level and the latter focusing on familial segregation. although both strategies have compelling strengths, association analyses are currently more widely conducted and likely to spread even further in the future, particularly in the pharmacogenetics domain. facilitating these studies are technical developments in molecular genetics and in the use of gene-specific variants derived from the human genome. in addition, extensive catalogues of anonymous dna sequence variants across the human genome have begun to be compiled. some large-scale, population-based human samples have been or are expected to be collected (e.g., epic, isis, million women study, mrc/wellcome trust biobank uk), and the use of dna variants in drug development is expanding. the coupling of high-throughput molecular technology, large numbers of genetic variants, and large samples offers some new opportunities for understanding the etiological basis of many diseases, including cystic fibrosis (cf). modifier genes are classically defined as genes that have small quantitative effects on the level of expression of another gene. studies of modifier genes therefore attempt to detect the sometimes subtle modulation of the main effects of a major locus regulating a disease (the cf transmembrane conductance regulator [cftr] gene in the case of cf). such effects may modify disease severity and progression. a broad spectrum of disease severity exists in cf, and it has become increasingly clear that cftr genotype does not account for the wide variance in the clinical spectrum of cf phenotype. many family-based and population-based study designs are possible to attempt detection of modifier genes in cf. this symposium will review the statistical concerns related to genetic studies of modifier genes and will discuss study design issues related to the investigation of modifier effects in gene discovery projects. university of western australia. phenotypic heterogeneity of cf lung disease is not explained by intragenic mutations in cftr, and likely reflects genetic heterogeneity at other loci, plus environmental effects. a multi-center study of gene modifiers for cf lung disease is underway to identify non-cftr genetic contributions to the variability of cf lung disease. the study is designed as a case-control association study in unrelated patients with the same cftr genetic background (homozygous ∆f508). the analysis focuses on comparing the prevalence of alleles and genotypes in patients who have "severe" (lowest 25 th percentile for age) versus "mild" (highest 25 th percentile) lung disease. we have enrolled 864 subjects, who are segregated into three groups: 1) 320 young subjects with "mild" lung disease (age 15-28 yrs), 2) 255 older subjects with "mild" disease (>age 29 yrs), and 3) 289 subjects with "severe" disease (age 8-25 yrs). longitudinal analysis of spirometric measures covering five years up to enrollment (average (20 measures per patient) has been performed by a mixed model analysis with empirical bayes estimates of individual slopes and intercepts. the intercepts (at birth) for the fev 1 (% pred.) for these three groups are 116, 112, and 108 (% pred.), respectively. the rates of decline (%/year) for the two groups of mild subjects were similar (-1.2 and -1.0), whereas the severe cohort had a more rapid rate of decline (-3.7). the mild versus severe groups also differed in body mass index (bmi). the bmi z-score was nearnormal (-0.11 and -0.4) for the younger and older mild lung disease cohorts, respectively, but much worse (-1.28) for severe patients. the prevalence of pseudomonas aeruginosa during the 3 years up to enrollment was similar for all three groups (-86-88%). thus, cf patients in this protocol are characterized as "mild" and "severe", and other key clinical variables are defined. genotypes in 815 subjects have been completed for selected snps in genes previously studied as modifiers of cf lung disease, including alpha-antiprotease, ace, β-adrenergic receptor 2, gstm1, gstm3, gstp1, il-10, mbl2, nos3, tnfβ and tgfβ1. results show a striking association of snps in tgfβ1 with severe lung disease (p = 0.0004; odds ratio >2). snps flanking tgfβ1 show no association, which implies that tgfβ1 is the causative modifier gene, as has been reported for asthma and copd. ongoing studies are testing other genetic variants in several pathogenic cascades in cf related to infection and inflammation. *reporting for the gene modifier study group. supported by cff knowle00a0, nih r01 hl68890, and nih r00046. garry r. cutting, md institute of genetic medicine, johns hopkins university, baltimore, md usa genetic, environmental, and stochastic factors are the three major factors contributing to phenotype variation. for single gene disorders the nature of the defect in the disease-causing gene is a major contributor to variation. however, in most single gene disorders, including cf, the disease-causing gene is not the sole determinant of variation indicating an important role for the other major factors. this point is highlighted by the observation that cystic fibrosis patients with identical cftr genotypes have considerable variability in the severity of lung disease (1). study of related affected individuals provides a method to dissect the contributions of genetic background, environmental and stochastic factors to phenotype variability (2). this is achieved by comparing the degree to which monozygous (mz) twins, dizygous (dz) twins and siblings are similar to each other for selected traits. if pairs of mz twins are found to be highly correlated (e.g. if one member of the twin pair has a trait; the other twin also has the trait) then one could attribute this similarity to genes (since they share 100% of their genes) or the environment that they share (since they are together in utero and grow up together in the same household). if dz twins are examined for the same trait and found to have lower rates of correlation than mz twins, then genetic factors can be implicated since dz twins share only 50% of genes but have the same degree (or nearly so) of shared environment as mz twins. on the other hand, similar high levels of correlation among mz and dz twins implicates the environment that they share. finally, siblings share 50% of genes like dz twins but have a lower degree of shared environment since they are born at different times (although still raised in the same household). thus, siblings provide an opportunity to test the role of environment on genetically similar individuals. a trait that shows similarly high levels of correlation among twins but a lower level of correlation among siblings would implicate environmental rather than genetic factors for the trait under question. the european twin and sibling study has demonstrated the utility of this approach. this group reported that genetic factors independent of cftr can influence phenotype severity by altering chloride secretion (3;4). to increase our understanding of the role of these major factors, a cf twin and sibling study is being conducted in the united states. over 100 participating centers located throughout the united states have contributed patient information to the study. at the current time, 50 pairs of monozygous twins, 23 pairs of dizygous twins, and 239 sibships have been enrolled into the study. several themes are emerging from the u.s. twin and sibling study. first, variation in pulmonary disease, as measured by fev 1 % predicted is minimal among twins. in fact, mz and dz twins show similarly high levels of correlation for this parameter suggesting that genes other than cftr have a limited role in differences in lung disease severity. furthermore, lower levels of correlation for fev 1 % predicted among siblings both by cross-sectional and longitudinal analysis supports the concept that genes are not a major factor, but that sharing of environment plays an important role. this conclusion is consistent with observations from cftr genotype-cf phenotype studies over the past decade. however, the small number of enrolled dz twins in both studies reveals a limitation in this approach. analysis of a larger collection of siblings will allow rigorous evaluation of the role of environment, and the specific factors therein, that influence pulmonary outcome. second, evaluation of gi manifestations of cf in twins and siblings has revealed an important role for genetic factors. previous studies have documented familial clustering of meconium ileus (mi) and a locus for this trait has been mapped on chromosome 19 (5) . mz twins display a high rate of concordance for mi (92%) while dz twins have a lower rate (22%) strongly implicating genetic factors, as has already been noted. liver disease and cf-related diabetes mellitus follow the same pattern suggesting that genetic factors also underlie these traits. third, some twins and siblings exhibit considerable difference in the age that they acquire bacterial infections. most impressive is the observation that mz twins can differ by several years in the initial age of acquisition of pseudomonas aeruginosa or burkholderia cepacia. since mz twins share 100% of genes and environment, the difference in acquisition of these bacteria suggest a substantial role for stochastic factors. in summary, study of affected twins and siblings permits an assessment of the contribution of genetic factors to trait variation. this is an important step prior to the investment of considerable effort to identify modifier genes for particular traits. genomic analysis is now widely used to probe the basis for disease and how disease may be expressed in individual patients. the attempt to identify specific disease-associated mutations, polymorphisms, or transcript patterns currently underpins much of our thinking about disease mechanisms, but the overall complexity of genebased data and the limited amount of functional annotation continue to present challenges in the interpretation of these experimental results. going forward, it will become increasingly important to look beyond single genes to genetic networks and ultimately to the integration of these data with quantitative measures of biological function at the system level to discover key molecules that can explain and classify clinically-relevant phenotypes. we have taken a systems biology approach to discovery that is based on the concurrent analysis of gene expression in combination with two other large-scale data streams: metabolomics and quantitative histomorphometry. metabolomics uses mass spectrometry to measure changes in the relative abundance of endogenous small molecular weight biochemicals (e.g. amino acids, sugars, lipids) present in cells, tissue and biofluids. quantitative histomorphometry uses computationally intensive image analysis of normal and pathological histology slides to quantify tissue structures and identify phenotypic changes in response to toxicity or progress of disease. the assessment of the integrated cellular state using biochemical profiles and quantitative tissue analysis is an important complement to the large-scale molecular state of a biological system. we are currently applying this approach to the study of drug-induced liver injury and to the discovery of novel drug targets and biomarkers for type ii diabetes. hepatotoxicity: acetaminophen (apap) overdose remains one of the most common causes of hospital admissions for acute liver toxicity. the initiating mechanism is believed to involve hepatic metabolism of apap to a reactive oxidative species, napqi, which subjects the liver to oxidative stress, resulting in depletion of glutathione. oxidative stress is also believed to play a pivotal role in the hepatotoxicities of a wide range of chemical and pharmaceutical agents. therefore, apap may serve as a useful model toxicant to elucidate the mechanism of liver injury. single oral doses of apap producing gross liver pathologies ranging from no histopathological change to frank necrosis were administered to rats. tissue and biofluid samples were analyzed by lc/ms (tof) with an esi source in either positive or negative mode. mass spectra at each retention time were matched to a library of known standards using proprietary software and linked to metabolic pathways. analysis of individual metabolites at the tissue level revealed major perturbations in pathways associated with known injury and repair mechanisms -e.g. depletion of glutathione and cystathionine (oxidative stress), decreases in nad and various nucleotides (nucleic acid repair), and a decrease in cdpcholine (phospholipid turnover). several of these decreases in liver metabolites were also reflected in urine, suggesting that they might serve as useful biomarkers for early detection of liver disease. time and dose-related changes were also observed in various other biochemicals that were not predicted by current knowledge. type ii diabetes: as a disease model, obesity, insulin resistance and hyperglycemia were induced by feeding c57bl/6 mice high-fat diets. responses were monitored over 16 weeks. high-fat fed obese mice were chosen that demonstrate either hyperinsulinemia or symptoms of type ii diabetes and were then compared to control mice fed a normal diet and demonstrating normal weight gain, fasting plasma insulin levels and fasting blood glucose levels. the mice selected at each time point were sacrificed and 14 tissues (liver, skeletal muscle, pancreas, white adipose tissue, heart, kidney, brain, testes, skin, tongue, stomach, intestine, spleen, and tongue) were harvested and frozen in liquid nitrogen until processed for rna isolation and metabolic profiling with adjacent sections fixed in formalin for histological processing. by combining data sets, we are able to identify novel and important drug targets and biomarkers that would be overlooked in a conventional target discovery processes. this unique approach to the discovery of novel drug targets and biomarkers is based upon the hypothesis that tissue structure, which is at the core of most diagnosis, holds the key to unlocking the link between genes, biochemicals, function, and disease. through correlations, differential analysis, and knowledge of gene function and pathways, we are able to identify biologically relevant drug targets and biomarkers that support this hypothesis. co-variant analysis of gene expression and tissue feature data identified different type of genes than were found using gene expression analysis alone. comparing the ontological classifications of the 303 genes that were up or down regulated greater than 2-fold, and 306 genes were closely correlated to liver features, fasting glucose, or insulin at sacrifice revealed, but there were only 56 genes in common. comparing the molecular function and biological processes ontological classifications associated with these two sets also shows distinct differences in the classes of gene families represented in the individual groups. many of the genes identified through co-variant analysis were altered <2-fold and therefore would have been lost in conventional analyses. in conclusion, these results indicate that a systems biology approach combining gene expression analysis with biochemical profiling and quantitative tissue analysis can help identify disease mechanisms, drug targets and biomarkers that other gene-only approaches often overlook. we are indebted to diathegen (athens, oh) who is a strategic research partner in the diabetes target discovery program. this research was funded in part by the national institute of standards and technology -advanced technology program (nist atp), award #70nanb2h3009. pseudomonas aeruginosa is an interesting organism since it provides a prototype for a nutritionally diverse, free living organism that is found in many microenvironments such as soil, water, plants, and animals, and because it is a common cause of hospital-acquired and cystic fibrosis (cf) patient infections, in part because of its high intrinsic resistance to many antibiotics. the most important recent development in p. aeruginosa research was the publication of the genome sequence in august 2000 (stover, kc, et al. nature 406:959-964). however a genome sequence is only as good as the usage we put it to. to permit us to capitalize on and reach beyond the genome sequence with maximum efficiency, we and other laboratories have been developing genomic tools. these include bioinformatics, whole genome knockout and gene fusion libraries, and microarrays. a major bioinformatics resource has been provided by the pseudomonas genome web site (www.pseudomonas. com), which is an attractive, information rich resource, maintained with funding from the cystic fibrosis foundation. this site includes all known information about each of the genes in the prototype strain pao1, permitting investigators to find genes of interest through key word searching or browsing through the genome. other resources are being continually developed, e.g. the recent development of psortb has permitted a prediction of the location of every gene in the genome and especially key secreted or surface genes that may be involved in pathogenesis. using the tactic of fusing fragments of the p. aeruginosa genome to an n-terminal signal sequence lacking alkaline phosphatase gene, we have confirmed several hundred of these predictions. whole genome knockout libraries are of tremendous value in pathogenesis. for example one can use such libraries to determine if a particular mutation attenuates virulence. in addition to the established library, we have made a knockout library that consists of random gene fusions to the promoter-less luxabcde cassette. this library is not as comprehensive as the library published by jacobs et al (pnas usa 100:14339-44; , but creates in addition to knockouts, a reporter system for expression of the particular gene into which the lux cassette, encoding light production, has been inserted. thus one can also study regulatory events related to pathogenesis. by far the most utilized genomic tool has been microarrays, which permit the analysis of the expression of every gene in the genome simultaneously. many of the known regulatory events in pseudomonas that are connected to pathogenesis are being probed through the use of microarrays. as the tool-kit for post-genomic studies grows, we are making rapid in-roads into understanding how this complex organism responds to life outside and in the host. funding through the functional pathogenomics of mucosal immunity program grant, provided through genome prairie and inimex pharmaceuticals is gratefully acknowledged. in the chronic airway infections that afflict cystic fibrosis patients p. aeruginosa live in matrix-encased groups known as biofilms (2, 6, 8) . biofilms are often thought of as communities of bacteria because the cells occupy a defined territory, they can exhibit coordinated behavior facilitated by communication, and because key biofilm characteristics (like their marked resistance to killing) cannot be generated by the bacteria living as individuals (9) . the functioning of many types of biological communities is enhanced by diversity. diversity is beneficial because the presence of diverse subpopulations increases the range of environmental conditions in which some community members will survive, or even thrive (1, 7, 10) . we have observed that growing wild-type p. aeruginosa in biofilms for 2-7 days produced considerable colony morphology variation. planktonic (free-swimming) batch cultures grown to log, stationary, or late stationary phase produced no variants. the variant phenotypes we studied were heritable, suggesting genetic changes produced them. a prime candidate for mediating such variation is reca. this protein is involved in phase variation and adaptive mutation, both which can be triggered by specific environmental cues (3, 5) . inactivation of reca dramatically reduced biofilm-induced colony variation and this defect was complemented with chromosomally inserted reca. the involvement of reca, which could mediate genetic change anywhere in the chromosome, led us to hypothesize that biofilm-induced diversity could extend to other functions. to test this, we examined the effect of biofilm growth on additional phenotypes, and found that biofilm growth produced heritable variation in several other traits. furthermore, some variants exhibit specialized functions in biofilms. isolates from cf airway infections have also been observed to undergo phenotypic diversification (4) . our data suggest that shortterm biofilm growth can rapidly produce genetic diversification of p. aeruginosa. this diversity may increase the stability of biofilm communities and speed the evolution of the resident bacteria. despite advances in the assessment and management of pain in children, there are still subpopulations of pediatric patients for which pain and its management has not been well studied. children with cystic fibrosis (cf) are such a population. pain and discomfort associated with cf may be secondary to direct involvement of the organs (e.g., abdominal cramping and pleuritic chest pain), physiologic changes that may occur related to organ involvement (e.g., headaches from hypercarbia and sinusitis, musculoskeletal pain), or due to treatment such as procedural pain. historically, pain management has not been included in an overall treatment plan, and commonly used analgesics such as opioids have long been avoided in the cf population because of the fear they may compound respiratory difficulties. only a few studies have focused on assessment or management of acute or chronic pain symptoms in children and adolescents with cf. the first published report of chronic pain in a relatively older and sicker population with cf was conducted via a retrospective chart review of patients who died and patients who were referred to a pain treatment service. this study demonstrated that patients with cf primarily complained of chest pain and headaches but back pain, abdominal pain, and limb pain also occurred regularly. 1 in the patients referred for treatment of chronic pain, safe and effective management with opioid analgesics was achieved. another study, focused on musculoskeletal complications experienced by pediatric patients with cf, found through clinical assessment, serology, and bone scan that 21 of 125 (17%) patients had joint pain. 2 finally, pain and dyspnea have been shown to be a common symptom at the end of life in children with cf. 3 opioids were effective in managing these symptoms in terminal patients. although these studies provide evidence of the type and nature of pain associated with cf, to our knowledge, there has not yet been any published report of the multidimensional experience of acute and chronic pain in a broad, un-referred population of children and adolescents with cf. unrecognized and untreated pain in children with cf may be associated with increased morbidity, poorer prognosis, and decrements in their overall quality of life. therefore, research to characterize the full spectrum of the pain experience from the patient's point of view may help to identify areas where pain management intervention is needed. our research group initiated a study, the dch pain in cf study, to characterize the experience of acute and chronic pain and its impact on the quality of life in children with cf. forty-six children ages 8-17 (m = 13.0 years; 52% male) with cf were recruited at one university-based and two community-based pediatric cf clinics. a previously validated survey instrument collected standardized data regarding the location, frequency, intensity, duration, associated emotional distress, and perceived functional limitations of chronic pain, and the intensity of procedural pain. findings demonstrated that chronic pain was a regular experience for children and adolescents with cf, occurring at least weekly in 46% of children. commonly reported locations of pain were: abdominal/pelvic region pain (50%); chest pain (37%); head pain (33%); leg pain (15%); and arm/hand pain (13%). most children (71%) reported the intensity of their pain as being mild and lasting for "less than 1 hour" (65%). however, a small subgroup of patients (15%) reported pain lasting onehalf day or longer and 11% reported experiencing moderately intense pain. there were no significant differences in pain reports by age or gender. pain location and frequency were related to pulmonary function and perceived limitations. patients with chest pain had lower fev 1 % (p<.05) than those patients with no chest pain. these patients also had more perceived functional limitations due to pain (p<.001) than patients without chest pain. patients with weekly pain had more perceived functional limitations (p<.0001) than patients with less frequent pain. the majority of patients reported experiencing mild procedural pain. a small number of patients reported experiencing severe pain from blood draws (7%), picc line placement (9%), gastrostomy tube placement (8%), pfts (2%), throat cultures (8%), and chest physiotherapy (3%). patients who experienced higher intensity of pain from picc line placement were more likely to also experience higher intensity of chronic pain (p<.05). patients who experienced higher intensity of pain from patrick hubbard, r.n., b.s.n the purpose of this study was to develop a web-based education program tailored to patients with cf who may be experiencing pain, and to investigate, via the website, the pain experiences of those patients by studying their pain reports, disability, and coping strategies. this study was a cross-sectional survey of cf patients. recruitment efforts focused on informing patients of the website and research study. a sample of 18 participants was recruited. three separate questionnaires were employed: a demographic questionnaire, the pain disability index, and the pain response inventory. approximately half of the sample reported experiencing pain episodes on a daily basis lasting two hours or less. the average intensity of a pain episode was reported to be in the moderate range. participants reported pain disability highest in areas of recreation, occupation, and social activities. the most commonly used coping strategies included active and accommodative coping techniques such as problem solving, acceptance, and self-encouragement. pain management is clearly a problem for some young adults with cf. this study provided information about the areas of disability caused by pain, as well as common coping strategies utilized by patients. the findings of this study provide direction for the future of pain management in cf patients. pfts and chest physiotherapy were more likely to experience chronic chest pain (p<.05). the majority of children reported that medication (41%), rest (63%), relaxation (41%), heat or cold (39%), distracting activities (36%), and family/friends (39%) provided some pain relief. twenty-four percent of patients reported taking no medication for pain. of those taking medication to treat pain, acetaminophen, nsaids, or a combination of the two was used. none of the patients reported taking opioids for pain management. it was interesting to note that all the patients who reported head pain were taking some medication, mostly nsaids. children with cf appear to experience pain, especially abdominal, chest, and head pain, on a regular basis. the finding of frequent chest and head pain is consistent with the work of ravilly et al. 1 the majority of patients in our study described their pain as mild and of relatively short duration, however a significant number of patients seem to have pain of long enough duration to potentially have a negative impact on daily functioning. a good example of this is that children with chest pain have reduced fev 1 % and more perceived functional limitations as a result of the pain. our study provides further insight into the experience of pain for patients with cf with mild to moderate disease who were seen as outpatients. it is important to note that even in a less sick population, problems with chronic pain were very common. evidence suggests that pain assessment should be a routine part of the clinical care of children and adolescents with cf. further research is clearly needed to better understand the sources of pain and how best to provide relief. pharmacological interventions were minimal in this population and possibly contribute to the under treatment of pain. effective pain management may serve an integral role in reducing morbidity and improving quality of life in children with cf. figure 1 . relationships between pain, coping, and disability. assessment of health related quality of life (hrqol) in children and adolescents with cystic fibrosis (cf) is important to better understand disease and treatment-related factors that impact function and well-being, and to evaluate the effectiveness of therapies and methods of drug delivery. limited data exist concerning the hrqol of youth with cf, although the recent development and validation of cf specific measures of hrqol for children and adolescents 1 will likely increase the use of qol measurement in clinical trials. measurement of how symptoms, such as pain, impact on hrqol is essential, but these data have not yet been reported in children and adolescents with cf. there are preliminary data that patients with cf do in fact experience and/or suffer from pain related to their disease or complications of their disease. in the only published study of chronic pain in cf, headaches, chest pain, back pain, abdominal pain, limb pain, arthritis, and neuropathic pain were experienced by the majority (84%) of an older sicker pediatric population with cf. 2 it is well documented that chronic pain can have a significant influence on hrqol of youth by impacting a variety of normal daily activities, such as school attendance and social interactions as well as psychological functioning . 3 in addition, pain may limit a child's ability to take deep breaths, cough, and tolerate pulmonary toilet exercises that are so impor-tant for patients with cf. therefore, our research group initiated a study, the dch pain in cf study, to examine the impact of pain on the hrqol of children and adolescents with cf. a convenience sample was recruited from the cf center at doernbecher children's hospital (dch). the study sample consisted of 46 children and adolescents with cf (24 males, 22 females) and their parents; n = 30 child subjects (ages 8-13 years) and n = 16 teen subjects (ages 14-17 years). participants completed two survey instruments to measure hrqol and pain characteristics. the cystic fibrosis questionnaire (cfq) 1,4 is a disease-specific quality of life measure, with different versions for children, adolescents, and parents. the cfq teen version includes 12 subscales (the child version includes 8 subscales) to assess cf specific symptoms and perceptions of hrqol. higher scores indicate better quality of life. pain characteristics were measured using a self-report questionnaire assessing frequency, location, duration, intensity, and emotional upset due to pain. adequate reliability and validity have been reported in the investigators' previous research with this pain questionnaire. 5 in addition, to assess disease severity, forced expiratory volume in 1 second (fev 1 percent) was obtained from the most recent clinic visit. as expected, findings demonstrated that having frequent pain (at least once/week) produced decrements in the hrqol of children and adolescents with cf. specifically, children with frequent pain reported increased treatment constraints compared to children with less frequent pain. adolescents with frequent pain, on the other hand, reported negative impact on their hrqol in many areas including physical and role functioning, vitality, eating disturbances, respiratory and digestive symptoms, as well as overall perception of their health, compared to adolescents with less frequent pain. cf specific symptoms, in particular, respiratory and digestive symptoms, were associated with more frequent, longer lasting, and more intense pain that is often in more than one location for both children and adolescents. several specific pain locations were related to decreased hrqol. in particular, having chest pain was related to decrements in physical, emotional, and role functioning, increased treatment constraints, respiratory symptoms, and poorer overall health perception. the presence of abdominal/pelvic pain was associated with more digestive symptoms. disease severity was also associated with hrqol; a significant positive association was found between fev 1 percent and respiratory symptoms. children and adolescents with cf, who are seen in an outpatient setting, experience frequent pain that is associated with decrements in their hrqol. in the dch pain in cf study, adolescents experienced the greatest negative impact on their hrqol from frequent pain. moreover, patients experiencing chest or abdominal pain were particularly at risk for having more cf specific symptoms and reduced hrqol. our study highlights the need for future research to identify methods to reduce pain and improve quality of life, particularly in adolescent patients with cf. effective pain management is likely to include both pharmacological and psychological interventions. the role of biobehavioral or psychological interventions for cf has received limited research attention. a recent systematic review 6 identified only eight studies of psychological interventions for cf, and none were focused on pain relief. however, the three studies that used biofeedback, music, and massage therapy to assist with physiotherapy found some effectiveness. 6 such interventions may also play a potential role in providing pain relief to these children and adolescents. pain relief should constitute an important treatment goal for youth with cf, as adequate pain treatment is expected to lead to broad improvements in health-related quality of life. pain in patients with cystic fibrosis (cf) results from discomfort arising from the disease (e.g., chest pain, gastrointestinal complaints, headaches, renal stones, arthritis, and end-stage disease), and caused by therapies (e.g., chest physiotherapy, intravascular access, and surgery.) characterization of an individual's pain is necessary in order to treat it effectively. types of pain include acute, recurrent, and chronic. patients' experience of pain is dependent not only on its physical nature, but also on their emotional, psychological, and spiritual states. social factors usually contribute to the impact of pain in patients' lives. evaluation of pain involves review of physical and psychological factors. patients should be asked regarding the onset, duration, and frequency of the pain. the nature of the pain can be described by its location, distribution, quality, and intensity. psychological factors that impact the experience of pain include anxiety, depression, substance abuse, and certain behaviors, e.g., reinforcement by caregivers who attend to the patient when pain is reported. in order to gain insight into how patients perceive their pain, they should be queried about their beliefs regarding the reason for the pain, its meaning, when it is most bothersome, what would change when the pain improves, and their expectations of the therapy for pain. hypnosis is useful in the treatment of pain because it can affect perception of pain, and psychological interactions with the experience of pain. hypnosis can be defined as an altered state of consciousness, characterized by a heightened state of suggestibility, which allows instruction and alteration of perception based on patients' specific needs. hypnosis may block the pain signal by allowing the triggering of a substitute signal through focus on an alternative thought, which usually is pleasant. hypnosis can reduce acute pain, chronic pain, depression, and anxiety, and can decrease dependence on pain medications. it is more effective in controlling pain than placebo, relaxation, distraction, or alpha-feedback. self-hypnosis for the control of pain can be taught by a qualified hypnotherapist or members of the cf care team who have been trained in hypnotherapy, e.g. physicians, nurses, social workers, or psychologists. many patients can be instructed in 1 or 2 sessions, which require 5-50 minutes. some patients need several additional sessions, especially if there is a large psychological component to their presentation. steps for instruction in hypnosis can include a pre-hypnotic interview, induction, deepening, hypnotic suggestions, and trance termination. techniques that can be used in hypnosis to help improve patients' mastery of their pain experience include egostrengthening, dissociation, time distortion, amnesia, positive visualization, and validation. self-hypnosis can be used to reduce the intensity or develop complete anesthesia at the site of the pain. the pain sensation can be altered, e.g., changing a sharp pain to a dull, warm, or tingling sensation. patients also can learn to relocate pain to a part of the body where it is not as bothersome. the patient was an 11-year-old with cf who became agitated whenever he required phlebotomy, which typically required 30 minutes, as he became very upset, cried, and struggled. the patient explained that he was frightened by the thought of a needle in his arm. use of a topical anesthetic was not helpful, probably because it did not alleviate his anxiety about the procedure. the patient was interested in learning how to use selfhypnosis to help himself. a single 10-minute instruction session was offered by his cystic fibrosis physician. hypnosis induction was achieved by rolling his eyes back as far as possible, inhaling deeply, and closing his eyes as he exhaled. deepening was achieved with the suggestion that the patient imagine being in a favorite place. the patient was coached to imagine what he might see, hear, smell, feel, and taste there. in order to promote dissociation, it was explained that the more he paid attention to his different senses the more relaxed he could become. to help with his comfort control during phlebotomy, the patient was given the hypnotic suggestion that he imagine his antecubital fossa was covered by a magic sleeve that reduced his perception of pain at that site. following hypnosis, the experience was validated when the patient was asked to pinch his antecubital fossa and reported no discomfort. he was congratulated for his success, thus providing ego-strengthening, and encouraged to use self-hypnosis as he saw fit, which reinforced his sense of self-mastery. subsequently, the patient was proud and almost eager to undergo phlebotomy with use of hypnoanalgesia alone. he reported feeling more confident. in later years the patient used self-hypnosis to reduce or eliminate headaches and abdominal pain, as well as to gain insight into how to cope better with stressful situations. hypnosis is best taught by those who have had specific training in hypnosis and hypnotherapy. instruction to uninterested patients is likely to be of little benefit because the efficacy of hypnosis is affected by the patients' desires. hypnosis sometimes can be used instead of medications for the treatment of pain, but oftentimes its optimal role is to augment the effectiveness of medical therapy. in conclusion, self-hypnosis for patients with cystic fibrosis can improve mastery of their response to the disease, cooperation with therapy, and comfort for patients, family members and the health care team. the primary defect in cfrd is insulin deficiency. abnormal glucose tolerance is present in the majority of adult cf patients because of fibrosis-related partial islet destruction. progression to overt diabetes, however, appears to require diminished function of the remaining beta cells, and this may be related to the same genetic defect that causes diminished beta cell function in type 2 diabetes. insulin resistance must also play a role. in several different studies, insulin resistance has been reported to be decreased, normal, or increased. it is likely that these contrary observations have been related to different degrees of illness severity in the patient populations that were studied. why do we care if cf patients have diabetes? cfrd contributes to morbidity and mortality in cf. a prospective study followed pulmonary function for four years in 152 patients separated at baseline by ogtt testing into three groups: normal glucose tolerance, impaired glucose tolerance and diabetes without fasting hyperglycemia. at baseline, the groups did not differ in age, weight, pulmonary function or bacterial colonization. the rate of decline in pulmonary function over the fouryear observation period was directly related to the severity of glucose tolerance abnormalities at baseline. pulmonary function deteriorated more rapidly in patients with igt than those with ngt, and patients with diabetes without fh had the greatest decline. when patients were grouped at baseline based on the amount of insulin secreted during the ogtt, the degree of pulmonary decline over the subsequent four years was directly related to the degree of insulin deficiency at baseline, suggesting a cause and effect relationship between insulin deficiency and cf clinical deterioration. insulin is a potent anabolic hormone that plays a pivotal role in carbohydrate, protein and lipid metabolism. insulin deficiency may contribute to morbidity and mortality in cf by promoting an overall catabolic state. mounting evidence supports the use of insulin to improve nutritional status and thus pulmonary function in cf. a retrospective survival analysis at the university of minnesota in 1988 found that while nearly 60% of cf patients without diabetes were still alive at 30 years of age, less than 25% of those with diabetes reached this age. since 1988, we have been aggressively treating all patients with fasting hyperglycemia with insulin. a new survival analysis was performed on 1081 patients followed at the university of minnesota over the last 25 years. median survival time after a diagnosis of cfrd was 5.4 years, but after the first 5 years the survival curve was less precipitous, suggesting that the patients dying in the first few years after diagnosis were probably sicker than the long-term survivors. median survival was 49.5 years for non-diabetic males, 47.4 years for diabetic males, 47.0 years for non-diabetic females, and 30.7 years for diabetic females. thus, the diagnosis of diabetes had a dramatic negative impact on survival in women but not men with diabetes. this did not appear to be related to bmi, genotype, pulmonary pathogens, steroid use, or pregnancy. women had worse pfts at the time of diagnosis. we hypothesize that gender-related hormonal factors may modulate the impact of diabetes on cf prognosis, with either a deleterious effect of estrogen or a beneficial anabolic effect of testosterone. holley f. allen, md department of pediatrics, baystate medical center children's hospital, springfield, ma cystic fibrosis related diabetes mellitus (cfrd) is different in etiology, course, and treatment than type 1 or type 2 diabetes. as life expectancy cf patients increases, so does the prevalence of cfrd. evidence to support standardized recommendations for screening and diagnosis of cfrd is just emerging; such evidence to support specific treatment modalities is nearly non-existent. cfrd occurs in cf subjects with pancreatic insufficiency, and increases with age, reaching a prevalence rate of over 30% in those over age 30 (1) . estimates of the prevalence of cf vary from center to center, likely related to the strategies used for screening and diagnosing cfrd. cfrd is caused by insulinopenia and is exacerbated by insulin resistance. initially it has an indolent course with absence or intermittent occurrence of the classic symptoms of hyperglycemia (polyuria and polydipsia) mostly during episodes of acute illness. ketoacidosis is extremely rare. clinical status has been shown to decline prior to the development of overt symptoms of diabetes (2;3). to prevent the catabolic state which is present during insulinopenia, screening for abnormalities of glucose metabolism is advocated by most experts in the field. ogtt is the gold standard for detecting abnormal glucose metabolism. the issues around recommendation of universal ogtt screening for cf patients arise primarily because: 1)ogtt is felt to be a cumbersome test to perform, 2)intermediate results such as impaired glucose tolerance (igt) fasting<110, 2-hr impaired (140-200 mg/dl) may normalize in subsequent tests, and 3) no solid evidence shows that the asymptomatic cf patients who have diabetic glucose tolerance without fasting hyperglycemia (dgt), (fasting<110, 2-hr>200mg/dl) benefit from treatment. although other tests including hba1c, fbg and actual bg are frequently used by physicians caring for cf patients (4), none have consistently demonstrated the sensitivity required of a screening test. hba1c has been in the normal range in 16%(5) to 70%(6;7) of subjects at the time of cfrd diagnosis. numerous groups have evaluated use of a combination of fbg and hba1c and have reported that they are not reliable in identifying cfrd(8) (2) (9). annual ogtt screening for cf subjects over the age of 10 has been advocated by several experts in the field (8) . increasing numbers of u.s. centers are screening with ogtt. a cfrd consensus conference in 1998(10), however, did not recommend universal ogtt screening, but rather an algorithm based on random plasma glucose in asymptomatic subjects ongoing research showing benefit from early detection of cfrd is needed to justify universal ogtt screening. the goal of treatment of cfrd is to achieve optimal nutritional and clinical status by maintaining near normoglycemia, to avoid both short and long term complications. because cfrd is a disease characterized by insulin insufficiency, insulin is generally the treatment of choice. early in the development of cfrd, significant insulin secretory capacity remains. most problems (hyperglycemia, decreased protein synthesis) occur in the post-prandial state, suggesting a possible role for short acting insulin secretagogues. sulfonylureas have been used by some with success comparable to insulin early in the disease process (11) . sulfonylureas bind with the sulfonylurea receptor (sur), which like cftr is a member of the atp-binding cassette superfamily of proteins. theoretical concern exists because of evidence that sulfonylurea like drugs bind to and inhibit the cftr. because of these concerns, sulfonlyureas should only be used by practitioners very aware of the risk benefit ratios. many insulin treatment regimes are available. the approach to insulin therapy needs to be tailored to the subject's preference, stage of disease, and ability to adhere to a particular regime. 1. maximum flexibility with best post-prandial glycemic control is achieved using rapid acting insulin administered at the time of food consump dana s. hardin, m.d. cf related diabetes (cfrd) is a frequent clinical problem. the north american cf foundation has greatly improved the knowledge and management of this disease by providing research funding, hosting the cfrd consensus conference and providing educational opportunities and written materials for cf caregivers. although the current clinical initiatives are appropriate for many cf patients, particular patient subsets may have unique needs. our group has had experience with both research and clinical management of two such groups, hispanics with cf and pregnant women with cf, and will review what we have learned. a relatively unexplored method of treating patients with cfrd is the insulin pump. we have just completed a study of its use in cfrd and will share our findings as part of this seminar. we studied 9 pregnant women and found that 88% developed gestational diabetes (gdm) by 12 weeks gestation, 99% by 24 weeks gestation and 100% by week 32 gestation. our studies of the metabolic changes caused by pregnancy revealed that pregnant cf women have lower insulin levels, higher hepatic glucose production and more peripheral and hepatic insulin resistance than normal pregnant women. insulin secretion did not increase during pregnancy, hepatic glucose production increased and insulin resistance worsened. we believe that these defects contribute to earlier presentation and greater incidence of gdm in cf. we also measured protein turnover and learned that pregnant women with cf are more catabolic than normal pregnant women and that catabolism worsened during pregnancy. catabolism correlated with less gain in lean mass in the pregnant cf women. furthermore, despite markedly greater caloric intake, pregnant women with cf had significantly less weight gain than normal pregnant women. based on our findings, we recommend that women who are actively seeking pregnancy be screened for cfrd. during pregnancy, we recommend an oral glucose tolerance test (glucose load 100 grams) be administered each trimester to any woman not yet diabetic. women who are diabetic should be treated with insulin to improve glycemic con-trol, improve weight gain and prevent muscle loss from on-going catabolism. although cystic fibrosis (cf) occurs less often in hispanics than in caucasians, clinicians have long recognized that the disease progresses more quickly, and the morbidity and mortality are higher, in this patient group. these observations have been supported by several research studies. we hypothesize that one major reason for increased morbidity and mortality in hispanic cf patients is increased glucose intolerance occurring at a younger age. we believe the earlier development of cfrd and glucose intolerance is secondary to underlying insulin resistance, a hallmark of type 2 diabetes, which is worse in hispanics. our review of medical records supports our hypothesis and describes an incidence of normal random glucose levels in less than 5% of hispanic children and 0% of hispanic teens. data from moran et al collected at university of minnesota indicate normal glucose tolerance occurs in 57% of children and 36% of teens. to date we have performed measures of insulin secretion and insulin resistance using a frequently sampled ivgtt in 12 hispanics with cf. we have found that insulin resistance is greater in these patients than in agematched caucasians. another possible factor affecting increased risk of cfrd in hispanics may be advanced clinical disease. these patients tend to be sicker. previous studies have identified a mechanistic role of cytokines, specifically tnf-_, in the development of insulin resistance, and increased hepatic glucose production. elevated levels of il-6 and tnf-_ have been associated with protein catabolism. we measured tnf-α in 19 cf children and found higher levels in the hispanics (tnf-( = 98 ± 10 pg/ml) than in the caucasian children (tnf-_ = 76 pg/ml). tnf-_ levels correlated with protein catabolism. furthermore we have found poorer longitudinal growth and weight gain in hispanic children, another indicator of worsened clinical status. the general recommendation for screening for cfrd is to wait until age 13. we recommend that all hispanic carol brunzell r.d., l.d., c.d.e. maintenance of a healthy body weight and optimal nutritional status are critical to survival for the patient with cf. with the additional diagnosis of cfrd, nearnormal blood sugar control is essential to normalize metabolism of macronutrients and to facilitate weight gain and weight maintenance (1). these patients are at risk for diabetic microvascular disease, so optimal control of blood glucose is imperative for prevention (2, 3, 4) . the risk of macrovascular disease necessitating the typical lowfat, low-sodium diet restrictions for type 1 and type 2 diabetes do not apply to the patient with cfrd as macrovascular complications appears to be non-existent at the present time (5, 6) . however, one study showed that patients with cfrd had a 4% prevalence of elevated cholesterol and 16% prevalence of elevated triglycerides, suggesting that this recommendation may change as these patients live longer (7) . the typical diet for cf, high-calorie, high-fat, and high-sodium remains essential to optimize weight and nutritional status. to meet the increased energy demands of cf many patients require some form of nutrition support in the form of oral or gastrostomy-delivered supplements. fluctuations in appetite make a rigid meal plan unrealistic. prescribing a complicated meal plan in addition to an already complicated medical regimen is not necessary today due to the dramatic improvements in insulin types and delivery systems. the need for simplicity is imperative. the cfrd consensus committee recommended matching insulin to carbohydrates for maximum flexibility as one approach to management (1) the use of carbohydrate counting is a simplified way to superimpose some diet organization onto the traditional cf diet and works well with the use of rapid-acting insulin to optimize blood sugar control. the rationale for carbohydrate counting is based on the relative effects of carbohydrate, protein, and fat on blood glucose, with dietary carbohydrate having the most significant effect compared to protein and fat. (8, 9) the advent of insulin pump therapy and the insulin lantus tm has made carbohydrate counting user-friendlier as many patients now administer rapid-acting insulin according to the amount of carbohydrates they plan on eating. one study showed excellent glycemic control in patients with type 1 diabetes using 1 unit of regular insulin for each 10 grams of carbohydrate consumed. glycemic control was maintained over a wide range of carbohydrate ingested and was not affected by the glycemic index, fiber, caloric or lipid content of the meals (10) . to confirm the approximate carbohydrate-to-insulin ratio, self-monitoring of blood glucose (smbg) data and food records for at least 3 days are recommended, with pre-meal and frequent 2-hour post-prandial glucose values to verify the ratio. most patients require approximately 0.5-2.0 units rapid-acting insulin per 15 grams carbohydrate (11) . while using fixed doses of insulin may not be the best approach, patients receiving fixed doses of insulin need to be consistent with their carbohydrate in conjunction with the time action of the insulin injected (1). currently, most patients who have cfrd without fasting hyperglycemia are not treated with insulin unless they are unable to maintain an appropriate weight or if pulmonary function is declining more rapidly than expected. at the university of minnesota, the strategy in diabetes not children ages 5 and over receive an ogtt at least yearly, and random blood glucose levels should be checked during every acute illness. further study may indicate a need for pre-emptive treatment for these children who are at high-risk for cfrd. the insulin pump has been utilized for treatment of type1 diabetes, including pediatrics. the pump's benefits include adjustable basal coverage and the ability to give bolus insulin for all meals and snacks without giving an injection. cf patients require high caloric intake. constant carbohydrate intake (a method for regulating blood sugar when traditional insulin is given) is not recommended for these patients. for this reason, the insulin pump may provide ideal therapy for cfrd. we have just completed a study on the use of the insulin pump in 9 cfrd patients. we found that as compared to multiple injections per day of subcutaneous insulin, the pump improved weight gain and lowered hemoglobin a1c. reduced protein catabolism was also noted. all patients but one requested insulin pump therapy at the conclusion of the study. based on our findings we believe the pump is a beneficial treatment for cfrd. as part of this seminar, we will share our experience regarding basal and bolus settings for the pump. treated with insulin is to spread carbohydrates throughout the day to minimize large carbohydrate loads without reductions in total calories. patients obtain a blood glucose profile periodically at home to monitor status. patients with impaired glucose tolerance are at high risk of progressing to cfrd. the risks of igt in the general population with regards to cardiovascular disease do not appear to be of concern for the patient with cfrd (5,6). the risk of microvascular disease with igt is not known at present. unlike diet strategies used in the general population, which recommended weight loss and a low fat diet for people with igt, it is never appropriate to recommend weight loss or a restriction of fat and calories in cf patients. the only potential restriction may be to minimize the excessive consumption of regular soda or other sweetened beverages, and to try to maximize intake of more nutrient-dense foods to prevent weight loss. spreading carbohydrates throughout the day may also be beneficial. it is internationally recognised that urinary incontinence (ui) is a common problem for women with cf. there is a reported prevalence of between 35-68% (1,2,3) . this compares to an approximate 8-13% prevalence of ui in healthy young women (4, 5) . ui is also reported as a problem for male cf patients (6) . there is an increased prevalence of ui described in children with cf (3). a wide-ranging age at onset is reported, from 5 to 45 years of age (1, 3) . whilst acknowledging the symptoms of urinary leakage in children and adults with cf, this report will focus on the possible mechanisms of ui in women with cf. the mechanism for preservation of continence is known to be a complex interaction of the muscles, fascia and ligaments of the abdomino-pelvic capsule and a competent urinary sphincter. in the general population risk factors for urinary leakage include female gender, obesity, genetic causes such as collagen weakness and conditions which increase intra-abdominal pressure (iap) such as constipation, vaginal delivery and persistent cough (7) . leakage may be related to features of urge and/ or stress incontinence. urge incontinence occurs when leakage is accompanied or preceded by urgency, whilst stress incontinence occurs when the pressure within the bladder exceeds that of the maximum urethral closing pressure. it is necessary to perform urodynamic assessment in order to provide a diagnosis of genuine stress incontinence (8) . the aetiology of ui in cf has not been fully evaluated. cough is recognised as the major cause of leakage in cf, where long spells of intense coughing are seen (1, 3, 9) . normally in healthy subjects the pelvic floor muscle (pfm) contracts prior to voluntary abdominal muscle activity suggesting a pre-programmed response to iap (10) . however in the presence of increased stress to the pelvic floor (pf), for example with chronic cough, the continence threshold may be exceeded. indeed chronic cough is recognised as a poor prognostic indicator for success of treatment of ui (11) . coughing often occurs in a position of spinal flexion in which the urethral closing pressure is diminished. abnormal posture, which is increasingly reported in cf, may be implicated in the mechanism of urinary leakage (12, 13) . increased kyphosis, over strengthened upper abdominals and a weaker transversus and multifidus are reported. it is recognised that the muscles of the abdomino-pelvic capsule play a role both as postural and respiratory muscles and therefore it is possible that dysfunction of any of these synergistic actions may contribute to the symptoms of urinary leakage (14) . all of these factors may reduce the ability of the pf musculature to provide timely, co-ordinated and sufficiently strong contraction to maintain adequate urethral closing pressure in response to increased iap. invasive urodynamic studies are not routinely performed on cf women complaining of leakage as patients are surprisingly symptom tolerant and reluctant to be assessed (1, 3) . in the absence of a definitive diagnosis it is therefore important to recognise other potential causes of ui in relation to both the muscular and neural aspects of continence. altered muscle metabolism in female athletes with cf is described and this may suggest differences in the quantity and quality of muscle in cf patients (15) . autonomic neuropathy in cf is also reported and may disturb the autonomic control of bladder filling and voiding (16) . the elevated inflammatory cytokines evident during pulmonary exacerbation may also have an effect on the muscle strength, as shown in-patients with copd (17) . in summary, ui in cf is a widely reported problem and it may be considered over simplistic to suggest that chronic cough is solely responsible for the mechanism of leakage. multifactorial causes should be considered when posing the question of why ui occurs in cf. physiotherapist, spec. resp diseases, bsc, lund cf team, lund university hospital, lund, sweden many children, adolescents and young adults with cystic fibrosis (cf) are reported as having a hyperinflated chest, stiff intervertebral and costovertebral joints, bad posture, thoracic kyphosis and back pain [1] [2] [3] [4] [5] [6] . the reported wedging or compression of vertebrae [3, 5] in adult cf populations may be caused by the ergonomic burden of the thoracic kyphosis in combination with osteoporosis [7] . pulmonary hyperinflation is a response to the clogging and collapse of airways in an attempt to keep airways open and ventilated, i.e. to keep the functional residual capacity (frc) at the level of the closing vol-ume. most often the degree of hyperinflation follows the degree of obstruction. with hyperinflation, expansion of the chest is accomplished by contracted inspiratory muscles. expiratory flow and expired volume is controlled by eccentric contractions of inspiratory muscles and inspiration is accomplished by concentric contractions, but from an unfavourable point on the length/tension curve. the more hyperinflated the chest, the closer the inspiratory muscles get to active insufficiency. atelectasis occurs if airways are not kept open, resulting in decreased ventilation distribution, and further increased breathing frequency, minute ventilation and work of breathing. accessory muscles become involved in the process of hyperinflation. as hyperinflation increases and as ventilation becomes more insufficient these muscles are progressively more involved in the mechanics of breathing. accessory muscles originate from the chest and insert on the upper limbs, cervical region and skull. concentric contraction assists in the inspiratory movements required of the severely hyperinflated chest when upper limbs and head are fixed while sitting upright, leaning forward with elbows on a table and the head resting in the hands [8] . all soft tissues shorten when not stretched to full range regularly. shortening of the musculoskeletal tissues in the hyperinflated chest results in the elevated and protracted shoulders, the thoracic kyphosis, cervical lordosis and the decreased mobility of the chest [1, 9] that have been reported. a stiff, hyperinflated chest makes effective airway clearance therapy impossible. it has been shown that bad posture, thoracic kyphosis and back pain are partly reversible if properly treated [1, 6, 9, 10] . but trying to regain what has been lost can be difficult. it is time-consuming, uncomfortable for the patient and an added burden to the cf care package. thoracic kyphosis due to wedged vertebrae cannot be rehabilitated, but rather increases the load on the adjacent vertebrae. therefore preventing a stiff chest and bad posture is of great importance. some simple physical exercises which maintains muscle length and strength and joint mobility as an incorporated part of the daily physiotherapy programme from the very beginning can make a big difference in the long run. physical loading in upright positions stimulates bone accretion, which may reduce the risk of osteopenia/osteoporosis and the risk of spontaneous fractures including wedging and compression of vertebrae. if physical exercise aiming to preserve physical function is included in the treatment, patients can maintain good posture and chest mobility, even if lung disease should progress. nowadays most cf centres recommend physical activity to their patients. however, children and adolescents with cf, despite having good lung function, have been shown to be engaged in less vigorous spontaneous physical activity than their non-cf peers [11] . whether this is due to less spare time caused by time consuming therapy, to protective parents or health care system, or to the disease itself can be discussed. simply recommending patients to be physically active is obviously not enough. they probably need more active guidance and continuing encouragement to become and remain physically active. all children need physical activity to develop motor maturity and body awareness. if children with cf are given the opportunity to experience pleasure and satisfaction during physical activity, much may be gained for future outcomes. the different types of exercises that should be included in a physical exercise pro-gramme from the very beginning or as soon as possible are [12, 13] : • chest mobility activities/exercises using movements around a vertical, sagittal and horizontal axis • shoulder mobility exercises, especially elevation and external rotation • muscle-strengthening activities/exercises, especially for postural muscles • working capacity training/activities/exercises exercises must never be uncomfortable. for infants, toddlers, children and adolescents, the activities/ exercises must be stimulating, enjoyable and age-appropriate. activities/exercises should always be individualized and provided at appropriate times in different settings. team sports/activities offer parts of what is to be included in the cf care and provide the added benefits of normal social interaction. good chest mobility allows effective airway clearance therapy. good posture probably reduces the risk of back pain and spinal complications. good posture contributes to positive body image and self-esteem. there is definitive evidence that nutritional status is the single most important factor that determines aerobic and anaerobic exercise performance in subjects with cystic fibrosis (cf) 1,2,3 . however, diminished exercise capacity has been reported in subjects with normal nutritional status 4 , 5 . there are several factors that can limit skeletal muscle function in cf. this can be considered in the following categories: intrinsic abnormalities in the skeletal muscle cells of patients with cf have been demonstrated in the mitochondria of fibroblasts and leucocytes and include increased calcium concentration, lower nicotinamide adenosine dehydrogenase activity (respiratory chain enzyme complex) 6 and a higher ph optimum of nicotinamide adenosine dehydrogenase 7 . moser and colleagues have suggested that there is a muscle related abnormality in oxygen metabolism in patients with cf 8 . studies utilizing 31-p magnetic resonance spectroscopy 5, 9 during exercise have demonstrated inefficient and sub-optimal aerobic and anaerobic muscle metabolism in subjects with cf and good nutritional status. the mechanism of this remains unclear. the class of the cftr mutation may be a factor in determining muscle function 10 . subjects with a cftr mutation belonging to either class i or ii have a lower peak aerobic capacity and anaerobic power compared to those with class iii, iv or v cftr mutations. in addition, compared to the dd angiotensin converting enzyme (ace) gene polymorphism, the ii polymorphism has been shown to be associated with less end organ damage in subjects with cf 11 and better anabolic response to exercise training programs in healthy subjects 12 . girls with cf have a lower peak aerobic capacity 13 and anaerobic power 10 . this gender difference is not unique to cf and have been attributed to differences in androgen levels, efficiency of fat metabolism, ratio of type i to type ii muscle fibres and total muscle mass. a significantly reduced breathing reserve is associated with reduced exercise capacity 14 and habitual activity 15 . while nutritional status is a major determinant of habitual activity 15, 16 , increasing habitual activity as part of a training program can improve muscle 17 . arthropathy occurs in up to 8% of patients with cf and limit activity. this can lead to deconditioning 18 . although nutritional status is a primary determinant of muscle function, there are several other factors that can limit optimal performance. larry c. lands, m.d., ph.d. with increased life expectancy for cf patients, we are required to pay attention to organ systems beyond the respiratory and gastrointestinal systems, whose dysfunction can cause significant long-term morbidity. many authors have documented significant decreases in bone mineralization in cf patients 1-3 , with increased risk for fracture and pain. there are two distinct phases with respect to the skeletal system. in the growing child, especially in the critical peripubertal and pubertal periods, the goal is to achieve maximal bone accretion 4 . maximal bone mass is achieved by the early 20's and so a great emphasis must be made during the early growth phases. once adulthood is achieved, the goal is to maintain this bone bank. there are many influences on bone, including genetic, nutrition, body habitus, hormonal status, and exercise patterns 5 . one of the most significant factors affecting ultimate bone mass is genetics. this is also true for patients with cf, where there is a significant relationship between a mother and child's bone mineral density 6 . the other major way in which parents can influence bone mineralization is through the encouragement of a healthy diet. a variety of population-based studies have demonstrated the positive influence of vegetable and fruit intake and the supply of vitamins k and c, and minerals, such as magnesium 5 . certainly, calcium and vitamin d intake are very important. sources of calcium include milk, tofu, and bok choy. these essential elements are particularly important during the phase of bone accretion in the first two decades of life 7 . other factors that play important roles in bone accretion and maintenance include sex hormones and inflammatory factors. cf patients are at risk for delayed puberty and thus delay in bone maturation. at the other end of the spectrum, premature menopause and andropause can lead to loss of bone mineral 8 . production and circulation of pro-inflammatory cytokines, such as il-1 and 6, and tnf-_, can affect the balance between osteoblastic bone formation and osteoclastic bone resorption in favor of bone resorption 9 . activity, both the amount and type, has a major impact on both bone accretion and retention 10 . a recent study from australia highlighted these factors 11 . the australian population is particular in that there is lots of sun exposure, promoting vitamin d formation, and the population is oriented towards being physically active. in this population, adolescent patients maintained good calcium intakes and vitamin d levels and were as physically active as their contemporaries. yet their femoral neck bone densities were reduced, possibly due to the higher inflammatory level. in the adults, reductions in physical activity also played a significant role in reducing bone densities. in a healthy pediatric population, differences in the amount of time spent being physically active affects bone mineralization 12 . in cf patients, both lung function and maximal exercise ability, which partially reflects the degree of physical activity, influence bone mineralization, such that higher lung function and exercise capacity are associated with greater bone density 13 . since both lung function and peripheral skeletal muscle function influence exercise ability in both healthy individuals and cf patients 14 , greater muscular ability will thus positively influence bone mineralization. of the exercises which most promote bone growth and maintenance, weight bearing exercises are primordial 10;15-17 . this is applicable to both children and adults. in summary, there are multiple influences on bone accretion and maintenance, with cf patients facing the additional challenges of chronic inflammation. it is important that a major effort be made to replete the bone reserves by early adulthood. this makes the peripubertal, pubertal, and post-pubertal periods the most critical. physical activity, especially impact activities and weight-bearing, plays an important role in achieving optimal bone reserves and maintaining these throughout life. phosphorylation of the cystic fibrosis transmembrane conductance regulator (cftr) by protein kinases is thought to be an absolute prerequisite for opening of cftr channels. in addition, nucleoside triphosphates were shown to regulate opening of phosphorylated cftr. cftr contains multiple sites of phosphorylation by camp-dependent protein kinase (pka) and protein kinase c (pkc) [1] . it was also shown that the membrane-associated cgmp-dependent protein kinase isoform ii is able to phosphorylate cftr [2, 3] . recently it was reported that in human sweat gland ducts endogenous cftr is activated by heterotrimeric g proteins via a camp-independent pathway [4] . the question arose if alternative pathways exist to activate cftr, apart from protein phosphorylation. therefore we tested the effect of phosphatidylinositol 4,5-bisphosphate (pip 2 ), a known regulator of ion channels and transporters [5] , on plasma membrane patches of oocytes, heterologously expressing human cftr. our study demonstrates that phospholipids, like pip 2 , enable kinase-independent activation of cftr, resulting in atp-responsiveness of pip 2 -treated cftr [6] . pip 2 alone is not sufficient to open cftr but atp opens nonphosphorylated cftr after application of pip 2 . the effect of pip 2 is independent of protein kinases, as pip 2 activates cftr in the complete absence of mg. phosphatidylinositol (pi) and phosphatidylinositol monophosphate (pip) activate cftr less efficiently than pip 2 . pip 2 application to phosphorylated cftr may inhibit the cftr chloride current. we suggest that regulation of cftr by pip 2 is a previously not recog-nized, alternative mechanism to control cftr-mediated chloride conductance. supported by the max-planck-society and the german research foundation(dfg). cftr channels help determine the rates of chloride and fluid transport across the epithelia affected in cystic fibrosis. early studies revealed two general modes of regulation, one mediated by secretagogues that elevate camp and activate cyclic-amp dependent protein kinase (pka), and another mediated by agonists that activate protein kinase c (pkc) and/or mobilize cell calcium. maneuvers that elevate camp induce sustained secretion across dog trachea and other preparations whereas global stimulation of pkc by phorbol esters leads to robust secretion lasting 20-30 min followed by profound inhibition 1 . the stimulatory effect of pkc is due, in part, to activation of apical cftr channels 2 whereas its antisecretory action probably reflects inhibition of basolateral potassium conductance, which is needed to maintain the driving force for apical chloride exit. a direct role of cftr in phorbol ester-stimulated secretion is implied by the presence of many consensus sequences for protein kinase c (pkc) phosphorylation on the r domain which, like the pka sites, are highly conserved across species. the physiological significance of the pkc sites is less obvious than for pka sites, however, since exposing inside-out membrane patches to pkc in combination with the diacylglycerol analog 1,2dioctanoyl sn-glycerol (dic8) causes only a slight increase in channel activity (a few % compared to pka). this weak response can not be attributed to the use of dic8 as lipid activator because similar results are obtained when pkc is added in a cocktail containing phosphatidylserine and diacylglycerol under conditions that increase pkc activity by 100-fold. rather than acting alone, pkc phosphorylation seems to regulate cftr gating primarily by enhancing the rate and magnitude of its response to pka, effects which are most noticeable in patches after channel activity has been allowed to run down. pkc phosphorylation of cftr channels in vivo is undoubtedly regulated by hormones and/or transmitters but appears partially "constitutive" in cultured cells, perhaps a reflection of high pkc activity in cells soon after they are removed from medium containing serum and its growth factors. cftr regulation is often studied using potent activators (eg forskolin, cpt-camp, phorbol esters, etc), but to assess the physiological role of a pathway it is preferable to expose cells to a "first messenger" (eg vip, acetylcholine, neurokinin) and block the pathway of interest. pharmacologic inhibitors, antisense oligonucleotides, dominant-negative mutants and small interfering rnas have all been used in various preparations, although specificity is always a concern. chelerythrine and gö6976 were both used to establish the role of pkc in stimulation of cftr by pka 3 , but it is now clear that chelerythrine is less potent and less specific than gö6976 or the other bisindoylmaleimides (eg bis1 or bis10 4;5 ) and should be avoided. interactions between signals could arise if an agonist activates more than one pathway or if multiple secretagogues stimulate the cell simultaneously. the role of such interactions in regulating cftr has not been studied but may be important in vivo since several transmitters are often co-released and cells also receive inputs from circulating hormones and locally-generated agonists such as atp, adenosine and no. sorting out the relative contributions and crosstalk between these signals remains a major challenge, however a key factor will probably lie in the subcellular localization of receptors and signaling molecules. the fact that phorbol esters have opposing actions at the apical and basolateral membranes itself implies that transport is controlled by multiple pkc isozymes having restricted distributions. pkc signaling involves activation by phospholipid and/or calcium, translocation to the site of action (eg plasma membrane), and binding to a receptor for activated c-kinase (rack) 6 . racks are ideally suited to orchestrate localized signaling responses because they are located near pkc sub-strates and are specific for particular pkc isozymes. during secretion, basolateral uptake of chloride into human tracheal cells by sodium-potassium-chloride cotransporters is regulated by the pkc_ and pkc_ whereas apical chloride channels in the calu3 cell line are regulated by pkc_ 7;8 . pkc_ binds to rack1, which in turn binds the scaffolding protein ebp-50 (ezrin-radixin-moesin binding phosphoprotein of 50 kd); 9 ). regulation of these associations appears complex since phosphorylation of a pkc site on the pdz1 domain of ebp-50 can disrupt the pdz1-cftr interaction 10 . together these findings raise the possibility that when activated, pkc could potentially regulate its own targeting to cftr. finally, choosing the right preparation will be important for physiological studies of pkc signaling as cell lines may differ in their expression of receptors, anchoring proteins, pkc isozymes, and apical-basolateral polarization. using first messengers rather than artificial activators such as phorbol esters should minimize artifacts caused by hyperstimulation, and isozyme-specific translocation inhibitor and activator peptides are available which act specifically on pkc_ or other pkc isozymes. these tools in combination with mutant channels that lack pkc sites but are fully competent for gating (eg the "6ca revertent" mutant 11 ) should provide new insights into the regulation of cftr by pkc. cftr is a chloride channel in the atp-binding cassette (abc) transporter protein family and contains its defining features, two membrane-spanning domains and two nucleotide-binding domains (nbds) (1, 2) . like other abc transporters cftr can bind and hydrolyze atp. earlier work has shown that enzymatic activity is required for normal channel gating (3, 4) . but this has long seemed puzzling because ion flow is passive without a fixed stoichiometric relationship to atp hydrolysis. furthermore, no other ion channel is known to require the energy of atp hydrolysis for gating. therefore we asked whether cftr had another atpdependent enzymatic activity that released less energy. in previous work we found that a recombinant nbd2 polypeptide had atpase activity (atp ( adp + p i ) in the presence of atp alone. but when amp was also present, the nbd2 polypeptide functioned as an adenylate kinase (atp + amp ( adp + adp) and its atpase activity was suppressed (5) . under physiologic conditions an adenylate kinase reaction is reversible and releases very little energy (6) . we therefore tested the hypothesis that cftr has adenylate kinase activity that gates the channel. we did five sets of experiments. we first asked whether ap 5 a (p 1 ,p 5 -di(adenosine-5') pentaphosphate), a well characterized inhibitor of adenylate kinases that does not inhibit atpases, would inhibit cftr. we found that ap 5 a inhibited cftr currents by reducing the channel opening rate. our data with different di(nucleoside) polyphosphates indicate that the inhibition involved simultaneous binding to one of cftr's two atp binding-sites and to an amp binding-site. second we asked whether amp would influence cftr gating. we found that amp did not evoke currents in the absence of atp but could increase currents at non-saturating atp concentrations by increasing the channel opening rate. amp altered the relationship between atp concentration and current from one with no apparent cooperativity between the two atp-sites to one with positive two-site cooperativity for atp, indicating a different gating mechanism. third we asked whether the effect of amp involved atp:amp phosphotransfer. to address this question we tested the effect of amp-nh 2 (adenosine 5'monophosphoramidate), an amp analog that cannot act as a phosphate acceptor. we found that amp-nh 2 did not mimic the effect of amp but inhibited gating produced by atp alone (i.e., atpase-dependent gating). amp, but not atp, reduced amp-nh 2 inhibition, indicating that the inhibition occurred because amp-nh 2 bound to the amp-site, which is distinct from the atp binding-site. fourth we asked whether adp would alter cftr gating through the readily reversible adenylate kinase reaction. we found that adp inhibited atp generated cftr currents. in addition, like amp, it induced positive twosite cooperativity for atp. adp-nh 2 (adenylyl 5´-phosphoramidate), an adp analog that does not allow phosphotransfer, and gdp, which does not bind to an adenylate kinase amp-site, failed to induce positive cooperativity. these results support the conclusion that phosphotransfer between two bound nucleotide diphosphates induced positive cooperativity. fifth we tested the nbd contribution to cftr adenylate kinase activity. we found that the mutations k1250a and d1370n in nbd2 and the homologous nbd1 mutations k464a and d572n reduced the potency of atp to stimulate cftr currents, indicating a reduction in atp binding and hydrolysis. the mutations k1250a and d1370n also abolished current stim-ulation by amp and current inhibition by ap 5 a while k464a and d572n did not. these differential effects suggest that nbd2, and not nbd1, contains the adenylate kinase activity that gates the channel. in addition, we studied the effects of the cf-associated mutation n1303k in nbd2. this mutation did not reduce the potency of atp to stimulate currents, but abolished amp-induced current stimulation and ap 5 a inhibition, indicating that this mutation may be related to the amp-site. in summary, our data indicate that cftr has adenylate kinase activity that regulates channel gating. when functioning as an adenylate kinase, cftr showed positive cooperativity for atp suggesting its two nucleotide-binding domains may dimerize. thus, channel activity could be regulated by two different enzymatic reactions, atpase and adenylate kinase. both activities share a common atp binding-site in the second nucleotide-binding domain. our data indicate that it is the interaction of amp with the amp-site that induced adenylate kinase activity and suppressed atpase activity. the amp k m of 73±20 µm for cftr gating suggests that cftr will function as an adenylate kinase in vivo. thus, at physiologic nucleotide concentrations adenylate kinase activity, rather than atpase activity may control gating, and therefore involve little energy consumption. this work was supported by the nhlbi (hl29851-21 and hl1234-05), the deutsche forschungsgemeinschaft (ra682/3-2 and ra682/5-1) and the hhmi. phosphorylation of cftr in the r domain by protein kinase a (pka) is essential for the channel to be active. however, after phosphorylation, atp is required to open phosphorylated cftr channels (gadsby and nairn, 1999; zou and hwang 2001) . atp-dependent gating of cftr is studied in inside-out membrane patches excised from nih3t3 or cho cells heterologously expressing wild-type or mutant cftr. the opening rate of the channel follows a simple michaelis-menten function and the closed time histograms show a negative exponential component (zeltwanger et al., 1999) . both of these observations are consistent with the idea that cftr gating is not in microscopic equilibrium (i.e., injection of free energy from atp hydrolysis drives the gating transition). since the open time is drastically prolonged when lysine 1250 (k1250) or glutamate 1371 (e1371), both critical for atp hydrolysis, is mutated, it is reasoned that atp hydrolysis at nbd2 closes the channel (zou and hwang 2001) . several biochemical studies indicate that atp binding at nbd1 is extremely tight (szabo et al., 1999; alexandrov et al., 2002; basso et al., 2003) presumably because nbd1 does not hydrolyze atp (lewis et al., 2004) . if atp is occluded in nbd1 (i.e., the offrate is extremely slow), association and dissociation of atp at nbd1 cannot account for the electrophysiologically observed, millisecond-to-second gating transitions (vegani et al., 2003) . by default, atp opens the channel by binding to nbd2. thus, nbd2 controls both opening and closing of cftr. although ample experimental results have supported the role of nbd2 in channel closing, kinetic evidence for its role in channel opening is lacking. since adp competitively inhibits atp-dependent opening of the channel, we reason that determining the site where adp binds to affect the channel opening rate will identify the nbd that controls channel opening. homology model of cftr's nbd2 was built by using the framework of the crystal structure of nbd1 from mouse cftr (lewis et al., 2004) . according to our homology model, tyrosine 1219 (y1219) was identified as the aromatic amino acid that interacts with the adenine ring of atp at nbd2. mutating y1219 to glycine (y1219g) shifts the atp dose-response to the right with an apparent affinity >50 fold of that for wild-type channel. adp inhibition was also dramatically reduced with this mutant. these results are consistent with the idea that atp as well as adp binds at nbd2 to modulate channel opening. it thus appears that nbd2 can open and close the channel. does nbd1 play any role in channel gating? biochemical and crystallographic studies of nbds in bacterial abc transporters suggest that binding of two atp molecules at the interface of two nbds drives nbd dimerization smith et al., 2002; chen et al., 2003) . if an equivalent molecular motion happens in cftr during opening-closing transition, the free energy of atp binding at nbd1 should contribute to the overall energetics of association-dissociation of nbd dimer. we found that adp can increase the closing rate of the channel (also see weineich et al., 1999) , suggesting that adp may act at another binding site other than nbd2. mutation of lysine 464 at nbd1 also shortens the open time and the amp-pnp-locked open time (powe et al., 2002) , indicating that atp binding at nbd1 affects the energetic stability of the open (or the locked-open) state. based on these results, we propose a model that entails energetic coupling of atp binding (nbd1) and hydrolysis (nbd2) in controlling cftr gating. using automobile as an analogy, we picture that phosphorylation of the r domain serves as the ignition mechanism. atp binding at nbd1 acts as the clutch that controls the speed of the energy-consumption engine at nbd2. 4 . cftr null mice have more inflammation and higher mortality than normal mice after airway infection with pseudomonas aeruginosa 5 . because airway epithelial cells (aec) express cftr and orchestrate inflammation, they may mediate dysregulated inflammation in cf. hyper-inflammation in cf could result from pro-inflammatory gene expression in aecs that is excessive, prolonged, and/or without stimulation. many in vitro models of aecs expressing a cf or non-cf phenotype demonstrate increased il-8 release by cf cells either spontaneously or after exposure to inflammatory stimuli. the models include aec lines from cf patients complemented with wildtype cftr 6 and non-cf aec lines with decreased cftr function due to expression of the cftr regulatory domain or antisense oligonucleotides 7, 8 . in one cf cell line, il-8 release was prolonged after removal of the inflammatory stimulus 8 . increased constitutive or induced il-8 release has also been reported from primary cf aecs, including both surface cells cultured under submerged or airliquid interface conditions and gland cells [9] [10] [11] . the observation that multiple cf model systems from different laboratories display greater pro-inflammatory gene expression provides strong support for the intrinsic hyper-inflammatory hypothesis. altered expression of inflammatory mediators other than il-8 has been demonstrated in cf aecs, including gm-csf, il-6, il-10, rantes, and nitric oxide synthase-2 8, [12] [13] [14] . altered regulation of multiple mediators suggests modulation of common signaling pathways by mutant cftr. the nf-b pathway appears to be an important target, likely via effects on upstream regulatory proteins [15] [16] [17] . other signaling pathways may also function improperly in cf cells, including the tgf-␤1/smad and jak-stat cascades 14, 18 . thus, altered regulation of diverse signaling pathways have been found in cf epithelial cell models by several groups, and these may provide unique therapeutic targets for controlling airway inflammation in cf. multiple studies do not clearly support the hypothesis that inflammation in intrinsically dysregulated in cf. for example, greater il-8 and neutrophils found in cf bal samples could easily be explained by defective mucus clearance rather than a hyper-inflammatory response. furthermore, many cftr-expressing tissues in patients with cf are not inflamed, suggesting that any hyper-inflammatory phenotype must be airway-specific. although multiple in vitro studies suggest hyper-inflammation in cf aecs, others show no change or even lower secretion of pro-inflammatory mediators 13, 19 . most studies were done with immortalized cell lines, which are inherently unstable and acquire differences other than cftr status over time, making them difficult to control for experimentally. complementation with wildtype cftr inconsistently affects inflammatory responses 10, 20 . primary aecs freshly removed from inflamed cf airways release more il-8, but this phenotype is lost in culture suggesting an acquired, rather than intrinsic, response 10, 12 . recent studies of well-differentiated primary aecs show significant person-to-person variation, but few differences in cf versus non-cf cells 10, 11, 20 . taken together, it is clear that hyper-inflammation has stimulus-and condition-specificity that is highly variable between cell models 10, 11 . another concern is that potential bias in the literature against negative studies may over-represent positive outcomes. finally, despite numerous investigations, one can argue that a solid mechanistic link between mutant cftr expression and altered regulation of inflammation has not yet been established. while cftr mutation links to disease pathogenesis and effects on the host response are hotly debated, there is essentially universal agreement that severe and sustained inflammation impairs pulmonary function and ultimately destroys the cf lung. anti-inflammatory therapy slows the progression of cf lung disease, confirming the importance of airway inflammation, but current therapeutic approaches are nonspecific and entail risks 21, 22 . research directed towards understanding the regulation of ongoing airway inflammation in cf will suggest more selective treatments to mitigate lung damage without adversely affecting host defense and pathogen clearance. the authors acknowledge colleagues whose work could not be cited due to page limitations. cf airway inflammation is typified by the presence of neutrophils and their protein products, including elastase, myeloperoxidase and matrix metalloproteinases, in the airways. neutrophils are attracted to the airway by interleukin (il)-8, a chemokine which is released from epithelial cells and macrophages as part of the innate immune response to infection. besides il-8, epithelial cells and macrophages are capable of producing other pro-inflammatory cytokines such as tumor necrosis factor (tnf)-␣, il-1␤, and il-6, each of which are increased in the airways of patients with cf. the gene expression of il-8 and other pro-inflammatory proteins is regulated largely by transcription factors of the nuclear factor (nf)-b and activator protein (ap-1) families. in the context of the il-8 promoter, the nf-b site serves as a cis-acting response element to diverse stimuli including tnf␣, il-1␤, pkc␦ and rhinovirus (unpublished data), whereas the ap-1 site serves as a basal level enhancer (1). the basic nf-b complex is a dimer of two rel family members, p50 (nf-b1) and p65 (rel a). the ap-1 complex is a dimer of fos and jun family transcription factors. in unstimulated cells, nf-b is sequestered in the cytoplasm by ib proteins. phosphorylation and degradation of ib allows nf-b translocation to the nucleus, where it regulates gene transcription by binding to specific sequences of dna. the "classical" signaling pathway to nf-b activation includes successive phosphorylation and activation of nf-b activating kinase (nik) and ib kinase (ikk). ikk consists of two catalytic subunits (ikk-␣ and -␤) and a regulatory subunit (ikk␥). while ikk␣ and ikk␤ contain similar kinase domains with essentially identical activation loops, they are functionally distinct. recent studies suggest that ikk␤ serves as the target for pro-inflammatory signals, whereas ikk␣ plays a critical role in development (2, 3) . the most potent ikk activator is the serine-threonine kinase nik. nik is required for activation of nf-b by non-typeable h. influenzae (4) and micrococci (5) . we have shown that successive activation of nik and ikk␤ is required for il-8 transcription induced by ligation of the asialogm1 (6), the putative glycolipid receptor for p. aeruginosa. ikk␤ and the mitogen-activated protein (map) kinase kinases share structural elements, including the position of two activation loop serine phosphoaccepting sites. thus, it was found that map kinase/extracellular signal regulated kinase (erk) kinase kinase (mekk) phosphorylates and activates ikk (7) . mekk1 is activated by tnf␣ and il-1, and dominant negative mekk1 inhibits ikk␤ and nf-b activation (7, 8) . the nf-b signaling pathway in cf could be activated either by cytokines, infection, or endoplasmic reticulum (er) stress. tnf␣ and il-1␤ are potent activators of nf-b-dependent gene expression. nf-b activation is required for maximal il-8 expression in response to p. aeruginosa (9) (10) (11) . these studies also noted a requirement for the map kinases, which in turn function as upstream activators of ap-1 (1, 6) . recent attention has focused on the mechanisms by which p. aeruginosa engagement of asialogm1, a glycolipid which lacks transmembrane and intracellular domains, elicits host cell responses. asialogm1 ligation promotes atp release from the host cell, which is followed by successive activation of a nucleotide receptor, phospholipase c, ca 2+ flux and erk, a map kinase family member (12) . also, it has recently been shown that asialogm1 is associated with toll-like receptor (tlr)-2, and that stimulation with p. aeruginosa flagella mobilizes tlr2 into a lipid raft receptor complex containing the tlr downstream effectors myeloid differentiation (myd)-88, il-1 receptor-associated protein kinase (irak)-1 and tnf receptor associated factor (traf)-6 (13). the pathway from traf-6 to nf-b activation has not been identified, but may involve transforming growth factor-␤-activated kinase (tak)-1 and nik (5, 14) . infants with cf show airway inflammation without apparent infection, suggesting that inflammatory signaling pathways may be primarily upregulated in cf. consistent with this, tracheas from cf fetuses grafted into severe combined immunodeficient mice show increased intraluminal il-8 and leukocytic infiltration compared to those from normal fetuses (15) . on the cellular level, airway epithelial cell lines derived from cf patients show increased basal and stimulated nf-b activation and il-8 expression vs. corrected cells (7, 16) . similar observations have been made in cf airway gland cells (17) . expression of ⌬f508 cftr, an incorrectly folded protein which partially accumulates in the er, but not g551d cftr, which is trafficked normally to the epithelial cell surface, increases nf-b transcriptional activity in chinese hamster ovary cells (16) , suggesting that cftr misfolding, with subsequent er stress, is responsible for dysregulated inflammatory signaling in cf. overload of the er with mis-or unfolded proteins leads to the unfolded protein response (upr), an integrated pathway leading to the expression of molecular marc b. hershenson, m.d. chaperones, attenuation of protein synthesis, and erassociated degradation (18) . if these adaptive responses do not correct the protein folding defect, cells undergo apoptosis. in addition, a distinct but poorly-defined pathway leads to nf-b activation, perhaps via calcium mobilization or the generation of reactive oxygen species. the upr and er stress in cf cells have not been carefully studied. acute inflammation is a normal, protective response to tissue injury that, in the ideal circumstance, leads to removal of injurious stimuli and damaged cells or matrix and results in restoration of normal structure and function. in most forms of acute inflammation, neutrophil influx is an early and essential component of the process. after reaching a peak the influx ceases and the emigrated inflammatory cells are removed as a key component of the resolution process. emigration of monocytes into the inflamed lung is slower and appears critical to the neutrophil removal and resolution process. ultimately the excess macrophages are also removed. this simple scheme raises a number of important questions, including: 1) what initiates the decline in neutrophil accumulation?. 2) what causes the later influx of monocytes (1)? 3) by what mechanisms are the neutrophils recognized and designated for removal? 4) how are they removed? 5) how are the excess mononuclear phagocytes cleared from the lung? in chronic, persistent inflammatory responses this normal, self-limited sequence is disrupted, perhaps because of persistent stimuli, initiation of new forms of stimulation, or abnormalities in the resolution. persistent and recurrent neutrophil influx in cf may encompass all three of these abnormal effects. neutrophils are short-lived cells and usually undergo spontaneous apoptosis within a few hours of release from the bone marrow. emigration into the lung can alter this apoptosis in complicated ways that can both delay or accelerate the process, but only by a few hours (2) . the apoptotic neutrophils are recognized and cleared locally due to their ingestion by macrophages and tissue cells, including epithelial cells. this is normally a very efficient process; so efficient that detection of significant numbers of apoptotic cells within a tissue may be considered to suggest some defect in the clearance process. for example, only a few percent of the neutrophils being cleared during resolution of bacterial pneumonia or ards are seen to be apoptotic. by contrast, examination of cf patients revealed up to 40% apoptotic neutrophils (3). apoptotic cell recognition and removal in vivo occurs quietly and unlike uptake by other phagocytic processes, is both non-inflammatory and actively anti-inflammatory (4) . instillation of such cells into an ongoing inflammatory reaction in the lung enhances resolution (5) . while a large number of molecules have been implicated in this recognition, a specific receptor for phosphatidylserine (the phosphatidylserine receptor, psr) appears to be critical for these anti-inflammatory and anti-immunogenic effects. in its absence, alternative receptors may in fact induce pro-inflammatory mediators and/or the lack of apoptotic cell clearance results in necrotic death with pro-inflammatory consequences. these observations led to the possibility that in cf, apoptotic cell clearance was ineffective, leading to persistence of the apoptotic neutrophils, increased necrosis and liberation of pro-inflammatory cell contents, as well as abrogation of the normal anti-inflammatory consequences of apoptotic cell recognition. all of these would lead to persistence of the neutrophilic phase of inflammation. in seeking the mechanism for defective apoptotic neutrophil clearance, the known presence of high levels of active elastase in cf airways suggested a likely possibility. apoptotic cell recognition receptors, particularly the psr, are highly susceptible to proteolysis. elastolytic cleavage of these in the airways would result in blockade of both apoptotic cell clearance as well as shutdown of the pro-inflammatory mediator generation (3) . extending this concept more generally, the possibility arises that during the induction of acute inflammation, active neutrophil elastase liberated during the early phase of neutrophil influx, can cleave and inactivate the psr, thereby providing a window of time during which the acute inflammatory reaction can proceed. as vascular permeability increases, influx of protease inhibitors would inactive the elastase, now allowing incoming monocytes to mature into macrophages that are free to express their psr and initiate the anti-inflammatory resolution phase. in cf this puts a great deal of weight on the persistent presence of active elastase. to some extent, a positive feed-back loop from non-clearance of the apoptotic neutrophils, cytolytic release of their elastase content, with more suppression of apoptotic cell clearance, all in the presence of a high bacterial load, could drive a self-sustaining neutrophilic inflammation. this does not, how-ever, explain how it all got going in the first place, leading to questions about genetically driven alterations in apoptotic cell recognition and response as potential explanations for the very early post-natal infiltration of cf airways with neutrophils. although there may still be uncertainty about whether misfolded protein or other intracellular consequences of cftr defects initiate some inflammation in the lungs of cf patients, it seems clear that infection plays a major role in stimulating inflammation in the airways. there also seems to be a consensus that the response to infection in the cf airway is dysregulated and excessive (1) . several studies in mice with cftr mutations have shown excessive responses to the prototypic inducer of inflammation, lipopolysaccharide (lps). most investigators have reported that huge quantities of pro-inflammatory cytokines, chemokines, and low molecular chemoattractants are found in lung secretions of cf patients, mice with mutations in cftr that have been challenged with lps or bacteria, and the supernatants of cultured cells with defective or blocked cftr. decreased production of anti-inflammatory mediators such as lipoxins (2) and il-10 (3) is also likely to be important in the dysregulation of the inflammatory response in the cf lung. there is considerable evidence that the nf-b dependent proteins, tnf, il-1, il-6 and il-8 are over-produced in cf and contribute to both intrapulmonary and systemic morbidity (reviewed in 3). the major inflammatory effector cell in the cf airway is the neutrophil (pmn). besides a role for il-1 and tnf in priming pmn, they induce expression of adherence molecules which facilitate migration of pmn into the airway; and il-8 is itself an important chemoattractant. additional chemoattractants important in cf are the lipoxygenase product, ltb 4 , and the complement fragments c5a and c5a desarg (3, 4) . the pmn themselves contribute additional ltb 4 . more importantly, pmn products including active proteases, long stranded dna, and oxidants all have deleterious effects on airway function and structure which are out of proportion to their necessary role in controlling infection. these pmn products contribute to airway obstruction, induce nf-b activation and additional pro-inflammatory cytokine production, and impair phagocytic defense mechanisms. thus, the pmns initiate and perpetuate a vicious cycle of infection, inflammation and airway destruction which eventually claims the life of the patient (3,4). the paradigm above illustrates an array of targets for anti-inflammatory therapy. the role of infection as the major stimulus for the inflammatory process emphasizes the importance of controlling infection and reducing the burden of bacteria with antibiotics and airway clearance techniques/therapies. numerous studies have documented decreases in inflammatory mediators after "cleanouts", confirming the importance of limiting this stimulus. while we all hope that correction of the basic defect will terminate the cycle of infection and inflammation, if chronic infection and airway damage are already established, this may not be possible. inhibition of intracellular signaling pathways such as the i-b/nf-b pathway or correction of putative cf-related dysregulation of this pathway would reduce many of the proinflammatory cytokines and chemokines, and would thus hit many lung and systemic targets by reducing tnf and il-8 (3). aspirin, nsaids, and corticosteroids all inhibit nf-b activation (5, 6) , and ibuprofen and prednisone have shown beneficial effects in cf (7, 8) . the pleotropic effects of cytokines might suggest that inhibiting any individual pro-inflammatory cytokine may not be very effective. however, inhibition of tnf alone has been very effective in rheumatoid arthritis (ra), which has several features in common with cf, especially the role of pmn in destruction of the end organ (9) . interestingly, methotrexate, the mainstay of ra treatment, has shown promising effects in small studies in cf (10) . inhibiting production of individual pathways like 5-lipoxygenase is also possible, and inhibitors of individual chemoattractant receptors are being studied. although preventing the pmn influx might seem to be a better strategy than attempting to neutralize their products one-by-one, the success of dnaase illustrates the efficacy of the latter approach. similar efforts could be directed at elastase and pmnderived oxidants. in evaluating anti-inflammatory therapy in cf, it is important to differentiate short-term improvement in symptoms from long-term preservation of lung function (11) . a two-tiered approach is envisioned by the cff tdn aim program, in which preliminary 30 day studies looking for efficacy in reducing inflammatory mediators in induced sputum will be used as a screening tool to select the best agents for larger, long-term studies of efficacy in preventing loss of lung function. this strategy offers our best chance of fairly evaluating new agents. any time one contemplates interfering with inflammatory mechanisms that also play important roles in host defenses, there is fear of allowing infection to get out of control. this has not occurred in trials of ibuprofen or corticosteroids in cf, but problems with infection, particularly activation of old tb in patients over 65, have been seen with the use of tnf inhibitors in ra (9) . some of the concerns with these agents and other anti-inflammatories may be relevant to cf, but some may not. adverse effects frequently occur in organs other than the lung and may or may not be related to that mechanism of action of the drug which is believed to be important in cf. observation of the latter should lead to development of more specific agents in which the therapeutic effect is maintained while the action(s) causing the adverse effects is eliminated. specifically modifying the drug and/or route of delivery to decrease access to sites of toxicity while preserving effects on the therapeutic targets is the principle behind the use of inhaled corticosteroids for asthma, but these drugs seem to have only limited efficacy in cf. just as the long-term therapeutic effects must be considered, long-term adverse effects must also be weighed. it is particularly worrisome that "catch-up" growth has not occurred in cf patients who were given prednisone in earlier trials (12) . although there are many reasons to believe that anti-inflammatory therapy should a part of every cf patient's regimen, recent cff surveys suggest that use of these agents, particularly ibuprofen, is actually decreasing. this seems ironic in the face of the confirmation of the efficacy of ibuprofen presented by schlucter et al at this meeting. development of newer strategies, realistic assessment of the risk of adverse effects, development of better targeted agents, and better understanding of the role of inflammation in lung damage are all necessary for determining the appropriate place for anti-inflammatory therapy in cf. a small-molecule discovery program was established to identify inhibitors of wildtype cftr and activators of ∆f508-cftr (1). primary high-throughput screening is done using automated instrumentation, a collection of >200,000 diverse drug-like small molecules, and a cellbased assay utilizing a halide-sensitive green fluorescent protein (2) . two interesting classes of small-molecule cftr inhibitors were identified. the compound cftr inh -172 contains a 2-thioxo-4-thiazolidinone core (3) . cftr inh -172 reversibly inhibits cftr clchannel function with k i in the range 0.3-5 µm, depending on cell type/membrane potential. the mechanism of cftr inhibition by cftr inh -172 involves stabilization of the closed channel state with prolonged mean channel closed times by patch-clamp analysis (4), probably as a consequence of cftr inh -172 binding to the first nucleotide binding domain of cftr. cftr inh -172 did not inhibit calciumor volume-activated chloride channels or the atp-binding cassette protein mdr-1, and produced little toxicity in cell culture and mouse models. a single intraperitoneal dose of 0.25-1 mg/kg cftr inh -172 inhibited cholera toxin-induced intestinal fluid secretion in closed-ileal loop models in mice and rats (3, 5) . cftr inh -172 was also found in mice to reproduce the nasal epithelial ion transport defect in cystic fibrosis (6) , and in pig and human airways to reproduce cystic fibrosis defects in submucosal gland fluid secretion (7) . analysis of rodent pharmacology indicated slow renal elimination without metabolism, and efficient liver uptake with enterohepatic recirculation (8) . the in vitro and in vivo results provide the rationale for further evaluation of thiazolidinone-type cftr inhibitors as antidiarrheals and agents for pharmacological creation of cystic fibrosis animal models. a second class of small-molecule cftr inhibitors, glycine hydrazides, was identified using a screen designed to identify compound that might act at the external cftr surface (9) . the compound n-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (glyh-101) reversibly inhibited cftr clconductance in <1 min. whole-cell current measurements revealed voltage-dependent cftr block by glyh-101 with strong inward rectification, producing an increase in apparent inhibitory constant k i from 1.4 µm at +60 mv to 5.6 µm at -60 mv. apparent potency was reduced by lowering extracellular clconcentration. patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms. glyh-101 inhibitory potency was independent of ph from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility ~1 mm in water. topical glyh-101 in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. in a closed-loop model of cholera, intralu-minal 2.5 µg glyh-101 reduced by ~80% cholera toxin-induced intestinal fluid secretion. compared to cftr inh -172, glyh-101 has much greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. glycine hydrazides may be useful as nonabsorbable antidiarrheals in enterotoxic-mediated secretory diarrheas. ∆f508-cftr 'potentiators' (correctors of defective gating) and 'correctors' (correctors of cellular misprocessing) were identified using frt epithelial cells expressing human ∆f508-cftr and an ultra-high halide sensitive green fluorescent protein. initial screening revealed a tetrahydrobenzothiophene class of ∆f508-cftr potentiators that corrected defective ∆f508-cftr chloride channel gating at concentrations down to 100 nm (10) . recent additional screening hasidentified 2 novel classes of potentiators with good efficacy and medicinal profiles. after structure-activity analysis and optimization, one class of compounds corrected defective ∆f508-cftr gating at concentrations down to 10 nm. in cell attached patch-clamp experiments, the potentiators evoked increased channel open probability >5-fold by reduction of interburst closed time. stimulation of chloride secretion was confirmed in low temperature-rescued human bronchial epithelial cells from ∆f508 cf subjects. two novel features of the new class of ∆f508-cftr potentiators include amplifed response (synergy) with camp agonists, and correction of other cftr gating mutants including g551d-cftr. screening of ∆f508-cftr expressing cells cultured at 37 o c for correctors of defective cellular processing produced 3 classes of small-molecules with favorable medicinal properties that conferred greater chloride conductance to ∆f508-cftr expressing cells than low temperature rescue. complex glycosylation was confirmed as measured by immunoblot analysis, as was plasma membrane expression as measured using an external epitope-tagged cftr. correction was seen within 3-6 hours after compound addition and persisted for up to 18 hours after washout. a unique feature of some correctors was their ability to confer partial correction of defective ∆f508-cftr gating, possibly by improved ∆f508-cftr folding. the small-molecule potentiators and correctors maybe useful in therapy of cf caused by ∆f508 and possibly other mutations. cystic fibrosis (cf) is a fatal genetic disease caused by mutations in the cf transmembrane conductance regulator (cftr) protein, a protein kinase a-regulated anion channel in the apical membrane of many epithelial tissues, including the airway. approximately 70% of cf patients are homozygous for a single mutation that causes a deletion of phenylalanine at position 508 (∆f508-cftr). this mutation causes improper folding of the protein, deceasing both channel cell surface density and gating. either directly or indirectly, ∆f508-cftr impairs ion transport, fluid secretion and mucociliary clearance, resulting in chronic infection, inflammation and eventual loss of lung function. our strategy for clinical treatment of cf is to discover drugs to increase cell surface expression and/or increase anion transport for mutant cftr, including ∆f508-cftr. the hypothesis is that such pharmacological "rescue" of cftr would restore airway ion transport and ameliorate or slow the deterioration of lung function. agents such as 4-phenylbutyrate and genistein have been demonstrated to increase the density and gating of ∆f508-cftr in recombinant cells, respectively. however, these agents have other biological effects and have not been chemically optimized for activity on cftr or for human pharmaco-kinetics. furthermore, these agents show little effect on chloride transport in primary airway cultures from cf lungs, and therefore likely lack either the potency or the efficacy to be effective drugs in humans. we have pursued a targeted approach to discover drugs that improve ∆f508-cftr function. to identify starting points for chemistry optimization, we employed fluorescence-based assays of membrane potential in 3t3 cells expressing recombinant ∆f508-cftr. approximately 200,000 compounds were screened to identify small molecules that either increased ∆f508-cftr gating in the presence of pka stimulation (so-called "potentiators") or increased ∆f508-cftr trafficking to the apical membrane (so-called "correctors"), or both. in addition to activity in the fluorescence assays, both potentiators and correctors were assayed for the ability to increase chloride transport in ussing chamber measurements using monolayers of primary human bronchiolar epithelial cell from the airway of ∆f508-cftr patients (∆f508-hbe). potentiators were prioritized if they exhibited drug-like chemical features and efficacy and potency greater than or equal to genistein. correctors were prioritized based on drug-like features and efficacy greater than or equal to temperature correction, which has been previously shown to partially reverse the defect in vitro. based on these criteria, distinct chemical series have been identified for correctors and potentiators. chemical optimization is aimed at improving both in potency and efficacy measured in both the fluorescence assay and (f508-hbe chloride transport with the goal of achieving greater than 10% of ion transport observed in normal airway. several thousand molecules have been synthesized in the program to date. the presentation will provide an update on recent progress in lead optimization and describe some of the key challenges moving toward compounds suitable for clinical testing. the most common mutation associated with cf is ⌬f508, which results in the in-frame deletion of the single codon encoding a phenylalanine residue at position 508. this mutation accounts for ~69% of all cf alleles, and ~90% of cf patients carry at least one copy of ∆f508 cftr. the protein encoded by the (f508 cftr allele has been classified as a processing mutant and as such, it is retained in the endoplasmic reticulum through interactions with elements of the er's quality control chaperone machinery and tar-geted for subsequent degradation in the proteasome. however, investigators have identified a number of in vitro manipulations that allow cohorts of newly synthesized ∆f508 cftr protein to depart the er, undergo post-synthetic processing in the golgi complex and be delivered to the cell surface. the resultant "rescued" surface population of ∆f508 cftr proteins are able to function as chloride channels with properties that are similar, but not identical, to those of the wild type polypeptides. in light of this behavior, numerous efforts aimed at developing small molecule pharmacotherapies for cf are focused on identifying compounds that can either stabilize the tertiary structure of the ∆f508 protein, or that can interfere with the interactions between the mutant protein and er chaperones. by preventing these interactions, the newly synthesized mis-folded but functional ∆f508 cftr protein might be allowed to escape recognition by the mechanisms that are responsible for its retention and ultimate degradation. we have previously shown that compounds that inhibit the endoplasmic reticulum ca-atpase (serca) can induce the release of er-retained _f508 cftr, which is delivered to the cell surface and able to function. many compounds that inhibit the ca-atpase are potentially toxic, and are thus not suitable as therapeutic candidates. curcumin is a non-toxic ca-atpase inhibitor that can be administered to humans safely in very large quantities. in preliminary studies the oral administration of curcumin to homozygous ∆f508 cftr mice in mg/kg doses com-parable to those well tolerated by humans resulted in alterations of these animals' characteristic nasal potential difference (npd) defect, such that there is a decrease in the baseline npd and the appearance of an isoproterenol response. furthermore, this treatment altered the electrical properties of gastrointestinal epithelia in these animals as demonstrated by the rectal potential difference assay. these effects are not observed in mice homozygous for a complete knockout of the cftr gene. curcumin also induces the functional appearance of _f508 cftr protein in the plasma membranes of transfected bhk cells. taken together, our initial studies suggest that curcumin is a nontoxic compound that can partially correct defects associated with the homozygous expression of ∆f508 cftr. however, in a number of follow up studies using different in vitro cell models and in vivo mouse models have not resulted in similar changes in ion transport properties. the difference in these in vivo and in vitro studies will be discussed. lastly, the findings from the first clinical study will be presented. an estimated one-third of mutations underlying human disorders result in premature termination of translation (nonsense or stop mutations) 1 . nonsense mutations account for approximately 10% of the total mutant alleles in cystic fibrosis (cf) patients (cystic fibrosis mutation database), however, in certain populations the incidence is relatively high. among ashkenazi jews, nonsense mutations account for 64% of all cftr alleles 2,3 . these mutations are associated with a severe form of the disease due to no cftr chloride channel function. aminoglycosides, in addition to their antimicrobial activity, can promote read-through of nonsense mutations in eukaryotic cells by allowing synthesis of full-length proteins 4, 5 . aminoglycosides were shown to restore the cftr function in cell lines and patients carrying the nonsense mutation w1282x 6-8 . in a recent randomized, double blind, placebo-controlled, crossover clinical trial 9 we showed that the aminoglycoside gentamicin can increase the expression of fulllength cftr and correct the electrophysiological abnormalities, in cf patients carrying the w1282x nonsense mutation. however, in these studies several patients did not respond. here we studied the molecular basis for the variable response in cf patients carrying the w1282x mutation. a wide variability in the level of cftr nonsense transcripts was found among these patients (n = 10). this variability correlated with the response of the patients to gentamicin treatment as demonstrated by nasal potential difference (npd) measurements. all the patients with relatively high transcript levels showed a significant reduction in basal potential difference and/or a significant response to chloride-free isoproterenol solution, indicating functional restoration of cftr following gentamicin treatment. in contrast, the patients with markedly reduced levels (n = 2, <26%) of cftr transcripts did not correct the abnormal basal potential difference nor the chloride transport, indicating no response to the gentamicin treatment. we then analyzed the effect of the cftr transcript levels on the response to gentamicin treatment in two cell lines (cfp15a and cfp15b) from unrelated cf patients carrying the w1282x mutation. analysis of the cftr transcript levels revealed a significant difference between the cells, such that in cfp15a the level was 4.5 fold higher than in cfp15b. chloride efflux measurements in cfp15a and cfp15b cells revealed no forskolinstimulated chloride efflux, indicating that the cftr channels in both cell lines are inactive. following treatment with 50 _g/ml gentamicin, restoration of the cftr function was demonstrated only in cfp15a cells. following treatment with 200 _g/ml gentamicin, chloride efflux was detected in both cell lines. these results indicate that the level of cftr nonsense transcripts correlates with the response to gentamicin, similarly to the findings in the patients. the level of nonsense transcripts is regulated by several factors, among which is the nonsense mediated decay (nmd) pathway. we hypothesized that inhibition of nmd would increase the level of nonsense transcripts available for read-through, and thus would improve the response to gentamicin. hence we treated the cfp15a and cfp15b cells with cycloheximide (chx), a potent inhibitor of nmd. this treatment significantly increased the level of nonsense transcripts. a direct inhibition of nmd by sirna directed against the nmd factors hupf1 and hupf2, resulted in a similar increase in the level of cftr transcripts. we further analyzed the effect of nmd inhibition on the cftr chloride efflux. fro this we studied the effect of sirna against hupf1 and hupf2 on the cftr function. the results showed a significant higher chloride efflux in response to gentamicin, indicating that inhibition of nmd can improve the response to gentamicin. our results suggest that nmd plays a role as a genetic modifier of the response to aminoglycoside treatment in cf and many other inherited diseases. lung transplant needs to be considered for all cf patients once they develop end-stage lung disease. our goal should be that no cf patients should die without having had the option of considering lung transplantation. with still limited availability of lung grafts, up to 15 -40% of cf patients will die while awaiting transplant. thus timing of referral for transplant is the issue -bal-ancing risks of death of cf lung disease with risks of transplantation; identifying those ill enough to benefit from transplant but well enough to survive the procedure. selection of candidates at the appropriate level of illness is therefore the challenge for the transplant team. for the cf team, it is obviously better to err on the side of a premature referral. as it may take time for the patient to prepare for the idea of transplant, this time has to be factored into the equation as well. early referral to provide information for patient and family and to assess for transplant (but not necessarily listing) is likely the best option for patients. early referral should not mean premature transplantation and the system should have the flexibility to move patients on and off an active waiting list to "fine tune" or optimize the timing of transplant. the major difficulty is that cf is an unpredictable disease; the patients are young and can withstand severe dysfunction and treatment has a huge effect on disease stabilization. as patients become more ill, adherence to therapy can improve and intensification of treatment often occur. prediction of patients at high risk of dying has proven to be elusive -several models have shown we are better at predicting who will survive over who will die. mayer-hamblett et al used the cf foundation national patient registry to find predictors of mortality (fev 1 , burkholderia cepacia, pseudomonas aeruginosa, hospitalizations, use of iv antibiotics) which were incorporated into a multivariate logistic regression model. 2 this model did not significantly improve on the previous model developed in the toronto cf population where fev 1 < 30 % predicted alone was the most significant predictor of mortality (kerem et al 3 ). liou et al. also used data from the cf foundation national patient registry and from unos (united network for organ sharing) to develop a multivariate logistic regression model to predict 5-year survival and they showed that patients with a 5-year survival of < 30 % showed maximal survival benefit from transplantation. 4 however, this still does not help predict the timing of referral to transplant. in addition to our inability to accurately predict at what stage a person should be referred and listed for transplant, we have to contend with other variables. predicted long term survival depends on survival post transplantation plus the waiting time to transplant. survival post transplant depends mainly on b. cepacia status (the species cenocepacia or genomar iii appears to result in higher immediate post operative mortality)positive or negative). the waiting time to transplant depends on country and centre. in the usa, the previous organ allocation policy depended on body size and blood type and accrued active waiting time on the national waiting list. in canada, waiting list time is affected by medical necessity giving much more flexibility for patients who suddenly deteriorate. a similar style of practice is present in many european countries. waiting list of less than 1 year in toronto allows for later referral when patients are more ill (which can be easier to predict) and patients feel more "ready" at time of referral as they have a lower functional status. from a practical point of view, we must use the information and models that we have available, recognizing differences depending on our patient population, using predictors (fev 1 , rate of decline in fev 1 , bacteriology, co 2 retention, hypoxia, clinical stability (hospitalizations, use of iv antibiotics), and functional status (6minute walk test)). all cf physicians will recognize older, clinically stable patients with slow decline in fev 1 who may remain with fev 1 below 30 % predicted for decades. thus, it is imperiative for the cf clinician to communicate this information to the transplant center, because they have their finger on the pulse of each individual patient. the reality is that afterall, the transplant pulmonologists focus more on post transplant after care than on pre transplant-care. thenus, the goal of the cf centre is to maximize medical therapy and follow patient frequently to fine tune. rehabilitation programs improve functional condition and provide an early warning system for clinical deterioration. ongoing screening for cf complications which may impact the post transplant course (eg. cf related diabetes, osteoporosis) should not be overlooked. the concept of transplant is frightening and at the time of referral, patient may not feel that this is necessary for them. emotional and psychological preparation of the patient and family is required. linking the patient and family with another cf patient of similar age and life experience who has had a transplant is a helpful strategy. close links between cf centre and transplant centre lead to optimal care for patients with hopefully an enhanced ability to minimize deaths on the waiting list. we performed 21 transplants in ventilator dependent patients from july 1988 to january 2000. selected pretransplant characteristics of these patients are described in table 1 . the pretransplant characteristics of the nonventilated patients and the stable ventilated patients were compared and no significant differences were found. the 5 unstable ventilated patients were all acute retransplantations for early graft failure and represented such unique and unpredictable situations that statistical comparisons with stable patients did not make sense. the single re-transplant in the stable group was a woman who developed bronchiolitis obliterans syndrome after an en-bloc double lung transplant for copd and was relisted 17 months after her first transplant. living lobar lung transplantation was developed as a procedure for patients considered too ill to await cadaveric transplantation. the procedure involves using a lower lobe from each of two donors for each recipient. 138 living lobar lung transplants have been performed in 133 patients between 1993 through june 2004. 90 patients were adults (mean age 27) and 43 were pediatric (mean age 13.9). the primary indication for transplantation was cystic fibrosis (85%). at the time of transplantation, 72.4% of patients were hospitalized and 21.1% were intubated. 1, 3, and 5 year actuarial survival among living lobar recipients was 70, 54, and 45% respectively. there was no difference in actuarial survival between adult and pediatric living lobar recipients (p = 0.65). there were 66 deaths among living lobar recipients, with infection being the predominant cause (53.4%), followed by obliterative bronchiolitis (12.7%), and primary graft dysfunction (7.9%). the overall incidence of acute rejection was 0.8 episodes per patient. 78% of rejection episodes were unilateral. age, gender, indication, donor relationship, preoperative hospitalization status, use of preoperative steroids, and hla a, b, and dr typing did not influence survival or rejection. however, patients on ventilators preoperatively had significantly worse outcomes (odds ratio 3.06, p = 0.03; kaplan-meier, p = 0.002), while those undergoing retransplants had an elevated risk of death (odds ratio 2.50). we examined the perioperative outcomes associated with the first 253 donor lobectomies performed. there have been no perioperative or long-term deaths. 80.2% of donors (n = 203) had no perioperative complications, while fifty (19.8%) had one or more complication. the incidence of intraoperative complications was 3.6%. complications requiring reoperation occurred in 3.2% of donors. 15.0% of donors had other perioperative complications; the most serious were two donors who developed pulmonary artery thrombosis, while the most common was the need for an additional thoracostomy tube or a thoracostomy tube for ≥ 14 days for persistent airleaks and/or drainage. right sided donors were more likely to have a perioperative complication than left sided donors (odd ratio 2.02, p = 0.04), likely secondary to right lower and middle lobe anatomy. these results support the continued use of living lobar lung transplantation in patients deemed unable to await a cadaveric transplant. we consider retransplants and intubated patients to be at significantly high risk due to the poor outcomes in these populations. our experience has shown donor lobectomy to be associated with a relatively low morbidity and no mortality. this is important if this procedure is to be considered an option at more pulmonary transplant centers, given continued organ shortages and differences in philosophical and ethical acceptance of live organ donors. the postoperative length of intubation, intensive care unit stay, and overall hospital stay were significantly longer for the stable ventilated patients as compared to non-ventilated patients. when the 16 stable ventilated transplant recipients were compared with the 479 non-ventilated recipients, the difference between the survival curves failed to reach statistical significance with p = 0.15. finally, the survival difference between stable and unstable ventilator dependent recipients was significant with p = .013. acute lung retransplantation for severe graft dysfunction is a high risk strategy that has led to no long term survivors in our limited experience in five patients. in each case the recipient fulfilled two criteria: acute lung dysfunction of such severity that the surgeon and pulmonologist agreed that survival was unlikely, and generally intact physiologic systems without systemic infection or multisystems organ dysfunction. other strategies considered in this dire situation have been ecmo and profound hypothermia but the experience in these modalities is equally anecdotal and the results equally poor. lung transplantation for patients in stable respiratory failure is more promising. in our sixteen stable ventilated patients there were no postoperative hospital deaths and a long-term survival that is not statistically different from the general population of our program's transplant recipients. these patients do seem to be more prone to prolonged intubation and hospitalization after the transplant, but the results indicate that these problems are surmountable and that long term results are acceptable. our practice continues to be the same as described in this paper: we will consider transplantation in stable patients who have declined on the waitlist but we are skeptical of and generally discourage transplantation for acute lung dysfuction, regardless of the etiology. the medical consequences of delayed diagnosis in cf can be thought of as short term or long term. in the short term, infants with cystic fibrosis (cf) are subject to a wide range of complications if diagnosis is delayed. the most striking complications are related to severe nutritional deficiencies and to hypoelectolytemia. infants with cf can present with severe protein calorie malnutrition closely resembling kwashiorkor. these infants have low albumin levels, edema, anemia and hepatomegaly from steatosis. they are also developmentally delayed at diagnosis. trace metal deficiencies have also been described including severe zinc and selenium deficiencies. zinc deficiency is associated with severe, weeping rash and immune dysfunction. selenium deficiency can lead to congestive heart failure. deficiency of alpha tocopherol, fat soluble vitamin e, is associated with severe anemia. vitamin k deficiency can lead to intracerebral hemorrhage. (1) linoleic acid deficiency likely coexists with other severe nutritional abnormalities and can cause rash. hypoelectrolytemia can lead to lethargy, poor intake and seizures. a death related to hypoelectolytemia and seizures in an infant with unsuspected cystic fibrosis has been reported. (2) the causes of nutritional deficiency and hypoelectolytemia in infants with cf are well understood and help explain the short term consequences of delayed diagnosis. because of exocrine pancreatic insufficiency, fat and protein malabsorption occur in most infants by two months of age. (3, 4) trace element metabolism is also abnormal by two months of age. albumin and fat soluble vitamin levels in young infants inversely correlate with the degree of nutrient malabphilip m. farrell, m.d., ph.d. during the past 25 years, numerous investigations have identified medical and psychosocial benefits associated with early diagnosis of cf through neonatal screening. research has included both observational studies and a randomized controlled trial. the following list of potential benefits provides a compelling rationale for universal newborn screening using the trypsinogen/dna(cftr) method. 1. prevent deaths of undiagnosed patients-save lives! 2. improve access-avoid geographic and fiscal barriers 3. avoid disparities related to gender, race and ethnicity 4. prevent protein-energy malnutrition and stunted growth 5. prevent prolonged micronutrient deficiencies such as e and k 6. reduce risk for cognitive dysfunction due to malnutrition 7. preempt bronchopulmonary disease and pa infection* 8. provide genetic risk information ("counseling") to parents 9. reduce costs for diagnosis and possibly treatment 10. enhance clinical research opportunities while improving quality * create the opportunity to initiate respiratory therapy before irreversibility develops i.e., the "point of no return," which will eventually facilitate prevention of lung disease sorption. we also know that impaired weight gain after birth is correlated with malabsorption. hypoelectolytemia arises from sweat electrolyte loss. sweat electrolytes are abnormally high in cf within a week after birth. early diagnosis rather than delayed diagnosis along with treatment of pancreatic insufficiency and supplementation of salt intake might therefore avoid short term complications of cf in infancy. two lines of evidence support this notion. the most important evidence that early diagnosis and treatment can avoid complications in infants with cf comes from the wisconsin trial of newborn screening. this trial has clearly demonstrated that growth is improved in the short term (as well as the long term) in infants identified through newborn screening compared to those identified after symptoms appear. (5) the second line of evidence stems from observational studies of infants identified through newborn screening showing that nutrient levels and growth improve with treatment. impaired growth is a long term consequence of that cf that is clearly related to delayed diagnosis. (5) since improved pulmonary outcome in cf has been related to improved growth, (6, 7) it might be expected that delayed diagnosis could lead to worse pulmonary outcome. the one study that has looked at this carefully however showed worse pulmonary outcome in patients identified through newborn screening compared to those with delayed diagnosis. (8) there were special circumstances dealing with environmental pseudomonas exposure that made this study difficult to interpret, however. thus further studies of short term and long term medical consequences of delayed diagnosis are needed. to further explore the short and long term medical consequences of delayed diagnosis of cf we examined data from the cf foundation registry from 2000, 2001, and 2002. short term consequences examined included presentation with hypoproteinemia or hypoelectrolytemia, as well as growth status pseudomonas culture positivity, and hospitalization in the year of diagnosis. long term medical consequences included growth status, pseudomonas culture positivity, pulmonary function and hospitalization. we specifically examined four modes of diagnosis (delayed diagnosis, newborn screening, meconium ileus and prenatal diagnosis). more than 2500 new diagnoses were studied. we found that stunting, wasting and hospitalizations in the year of diagnosis were three times as common in infants with delayed diagnosis compared to infants identified through newborn screening. occurrence of hypoproteinemia was significantly more common in infants with delayed diagnosis as was pseudomonas culture positivity. we examined long term consequences by age group (2-5 years, 6-10 years, and 11-20 years). delayed diagnosis was associated with stunting and wasting at essentially each age, higher percentage of mucoid pseduomonas at each age, and decreased fev1 (absolute and percent predicted) at ten years of age. hospitalization rate was higher in the delayed diagnosis group than the newborn screened group at two age ranges. examination of the cf registry therefore supports the notion that delayed diagnosis of cf is associated with significant short term and long term medical consequences. these data strongly support the need for early diagnosis and treatment to improve outcome in cf. cystic fibrosis (cf) is not readily diagnosed without newborn screening. it has few unique features, is very rare, and varies in its presentation.(1) in the united states, half of all persons with cf were diagnosed after six months of age. (2) the median delay in diagnosis is well over one year in parts of the united states where newborn screening is not universal. the impact of delayed diagnosis of cf on families is poorly described. in the united states, most studies have been concerned with the clinical features of those with delayed diagnoses and have rarely explored the impact on the mother, father and siblings.(e.g., 3-6) a few bibliographic accounts of families with a child with cf have been written.(e.g., 7, 8) outside the u.s., however, studies have found that misdiagnosis leads to increased anxiety, guilt and anger, and mistrust of the medical profession, and late diagnosis leads to more negative feelings about the pre-diagnostic period and less confidence in the medical profession.(e.g., 9, 10) we aimed to describe the impact of delayed diagnosis on u.s. families with cf at different life stages. we requested diagnostic stories from individuals on the cf research, inc. e-mail list in september 2003. we obtained 20 diagnostic stories and used an additional 20 stories found on the cystic-l listserv dating back to 1997. we condensed the stories into that of a single family and qualitatively described the affected child's symptoms at different life stages and the medical and family responses to these symptoms. we provide likely impacts on health and family resulting from a diagnosis had it occurred at different ages. we found that the age at which a person with cf was diagnosed can have large, irreversible negative impacts on that person's health status, quality of life, longevity, compliance with medical regimens, self-image, family structure, and major life decisions. familial relationships were seriously impacted. the stress around not knowing what is wrong was damaging to the family. economic losses can be expected. strong views about incompetence in the medical care profession abound. these negative effects on families were likely to become compounded the later the diagnosis of cf was made. we found that long-term parental guilt around not finding out sooner was difficult to avert altogether or remove later. delayed diagnoses means that opportunities were lost to make informed decisions about health care, employment, housing, insurance, reproduction, and other matters. even if untrue, it was common for parents to feel like appropriate steps could have been taken to prevent their child's lung disease, for example, if only they had known sooner about the diagnosis. a few parents who had a first child diagnosed on account of a second child being diagnosed early in life via newborn screening indicated that it was far better knowing the diagnosis than being tormented by not knowing it. it was extremely hard to plan for the future when it was unclear what was wrong with the child. there was parental regret, anger and pity for their older child who was not able to benefit in the way the younger child can through prevention and focused cf medical care, which works best for persons who present early without damaged lungs or nutritional deprivation. in nearly all of the delayed diagnosis stories we reviewed, parents and persons with cf would end with the hope that in the future others could avoid the pain they suffered of not having known sooner. 1. how should we understand our collective use of "early" and "late" diagnosis of cf as we consider the ethics of newborn screening? in most medical communities, often repeated case reports become part of our explanatory narrative, such that they express the shared understanding of our work. these explanatory narratives form a common language by which we express normative assumptions about the appropriate roles of patients and doctors, and the proper approach to the treatment and diagnosis of cf. case reports about "late" diagnosis of cf are one such common explanatory narrative. among ourselves, we often tell the story of a child or young adult diagnosed with cf only after being seen in a specialist clinic. we all recognize this story immediately, and it has a kind of defining quality for those of us in the cf community: i doubt anyone who has come to a nacf conference could make it through the conference without either hearing or telling one version of this story. we tell it to each other even if we have just met, when the talk turns to our patients, and we commonly use this story, in one form or another, as a cautionary tale to our trainees. why do we tell each other this story so often? from a simple point of view, it's not so surprising, because the story makes us look smart-in particular, it confirms us as better clinicians than whoever saw the patient before us, and lets us comfortably and collectively shake our heads in acknowledging that another doctor could have missed the obvious diagnosis. perhaps the frequent telling of the late diagnosis story is a just a harmless form of self-congratulation among colleagues, much like a group of carpenters telling stories about a non-carpenter neighbor's inept attempt at building a tree house for his child. but perhaps there is more to it. in thinking about this story, we should first admit that the frequency of the story does not reflect the frequency of the event, that is, we are not simply telling the late diagnosis story so often because it is such a common one. the sort of late diagnosis reflected in the story happens far less often than, say, the first acquisition of pseudomonas, or the onset of cf related diabetes, or for that matter, the death of a patient from cf. like many narratives, the moral of the story of "late" diagnosis is more complicated than it first appears. in this case, the "late" diagnosis story stands in for a complex set of assumptions about life with cf, cf clinical care, and the role of the cf physician. our head-shaking reaction to the narrative of late diagnosis reflects our assumption that the patient's life would walter m. robinson, md, mph children's hospital, boston and harvard medical school analysis. this improves the detection sensitivity from 94% to approximately 99%. additionally, the direct confirmation of cf from the newborn screen by identification of two alleles increased from 41% with ∆f508 analysis only to 64% with multiple mutation analysis. 7. since the introduction of newborn screening for cf in wisconsin, the total number of annual sweat tests has decreased from 1670 in the year 1991 to 804 in the year 2000. 4 concomitant with this decrease in the number of sweat tests has been a decrease in the number of institutions performing sweat tests from 44 in the year 1991 to 15 institutions in the year 2000. the cost savings of a fewer number of sweat tests accounts for 77% of the cost to operate wisconsin's cf neonatal screening program. 8. in order to achieve excellent nutritional outcomes, we exceed the cff recommendation of quarterly clinic visits by utilizing every 6-8 week clinic visits in infants <1 year of age. newborn screening provides the opportunity for better outcomes, and excellent follow-up care is essential to achieve outstanding results. always be better if we had made the diagnosis sooner. that the story is much more complicated is evident in the outpatient clinic of every cf physician; the contemporary heterogeneity of the clinical course of cf, and the presence of a wide variety of clinical conditions at every age, simply belies the assumption that in any individual case a "late" diagnosis always means more suffering than an "early" diagnosis. and if the history of cf care has a cautionary tale for us, it is that our current therapies and models of care may well have unintended consequences, and that we cannot see ourselves as always wiser in our use of various therapies than our forebears. in short, it may be that more therapy at the youngest possible age is in every case a good idea, but then again it may not. the late diagnosis narrative also supports an historically inaccurate view of the process of diagnosis; it is the state of diagnostic technology, rather the presence or absence of symptoms, which is the strongest determinant of the timing of diagnosis. before the reliability of the sweat test was established, no child could be diagnosed with cf in the absence of characteristic symptoms; in this situation, there are no pre-clinical diagnoses, that is, there are no "early" diagnoses, and all "late" diagnoses are likely to be a reflection of unfamiliarity with the disease. as the diagnostic accuracy of the sweat test advances, we begin to see its use first to confirm a clinical suspicion based on signs and symptoms, then to establish the diagnosis in the face of a confusing set of signs and symptoms, and finally to screen for a diagnosis to detect the pre-symptomatic disease. in a very real sense, the screening aspect of the sweat test transforms the healthy into the diseased before the appearance of any symptoms, and in so doing redefines the boundary of "early" diagnosis. if neonatal screening is adopted, we can only expect further reconfiguring of the boundaries of early and late diagnosis, so that a child diagnosed at six months of age would then be seen as a "late" diagnosis and the asymptomatic only child with a cf genotype will be unable to live the quiet life of the undiagnosed. considerations of newborn screening often center on parental anxiety and its function as either a benefit or burden of screening programs. a variety of types and causes of anxiety are put forth as amenable by (or caused by) newborn screening: • anxiety about the presence of disease in an apparently healthy infant • anxiety about the lack of diagnosis for the symptoms seen in a sick infant • anxiety about the detection of life threatening illness in the apparently healthy, asymptomatic infant • anxiety about the detection of life threatening, as opposed to self-limited, illness in the sick infant • anxiety about future reproduction • anxiety about the blame for passing on a genetic illness • anxiety about the implications of the carrier state of the newborn it is perhaps peculiarly modern that we are debating the role of a public health program in promoting or alleviating anxiety. yet anxiety is a slippery diagnosis, and as in the late diagnosis story, our attribution of anxiety to others may reflect our own concerns rather than the concerns of others. how ought we as clinicians assess these claims and counterclaims about anxiety, and how ought we to weigh the relief or creation of anxiety when assessing the ethics of newborn screening? parts although cfrd is different than type 1 or type 2 diabetes, the development of diabetes-induced complications is similar. these complications include eye, kidney, circulation and nerve problems, and are caused by high blood glucose levels. thus the goals of cfrd management are similar to those of people with all forms of diabetes and help prevent development of complications from diabetes. insulin is an important anabolic hormone which prevents protein catabolism and studies have demonstrated catabolism in both adults and children with cf, and in both the fed and fasted state. protein catabolism is assoappropriate nutrition is essential to management of patients with cystic fibrosis. many cannot maintain appropriate weight or growth without supplemental nutrition support. initiation of such support is facilitated by lifelong education of patients and their parents as to the importance of good nutrition in the management of cf. dieticians play a role in every cf visit and families are exposed to information about gastrostomy-tubes long before they become necessary. nutrition failure is identified using the guidelines established by the consensus report on nutrition for pediatric patients with cystic fibrosis and the cf adult care consensus conference. patients are initially evaluated for manageable causes of nutrition failure, including poor oral intake, uncontrolled malabsorption, and increased nutritional needs. poor oral intake can result from many causes, including chronic sinusitis, chronic pulmonary infections, onset of cf-related diabetes, and gastrointestinal causes (e.g., gastroesophageal reflux or distal intestinal obstructive syndrome). depression can also contribute to poor intake. malabsorption may result from inadequate or inappropriate enzyme use, cholestasis, short bowel syndrome, or small bowel overgrowth. the presence of pulmonary or sinus infections may lead to increased nutritional needs which the patient may not be able to meet orally. if oral supplementation and medical management fail, gastrostomy tube placement must be considered to restore normal nutritional status and normal growth. percutaneous endoscopic gastrostomy placement is generally successful. patients may then receive nocturnal drip feedings over 8-10 hours as supplement. aggressive control of reflux and monitoring for abnormal glucose tolerance is important. preoperatively, we counsel patients and families that gastrostomy tube placement is usually permanent in cf patients and will allow normal activity. we review their pulmonary status and may request an ng tube feeding trial to demonstrate efficacy. patients are admitted after placement to allow pain management, provide classes for parents and caregivers, and maintain good pulmonary function after the procedure. g-tube feedings are usually an elemental or semielemental formula, with enzymes used at bedtime and available if the patient arouses at night. am glucoses are monitored for a time after feeding introduction. good follow-up is essential to monitor progress and reduce complications. patients may experience pain or nausea during feedings, diminished daytime appetite, no weight gain despite gastrostomy tube feedings, onset of insulin requirement, or complications of the gastrostomy itself. malnutrition and nutritional growth failure in cf is a common and important issue in cf management because of its adverse effects on long term outcome. optimal standard of care in most cases requires meeting the excess energy, protein and vitamin needs of cf patients via overcoming malabsorption with appropriate pancreatic enzyme replacement therapy (pert), the use of oral supplements, sometimes helped by behavioral therapy, and /or, in some cases, enteral nutrition supplements (en). there are, however, some cf -related complications , where conventional nutritional support is not enough, may fail, or is impossible to maintain. these include neonatal meconium ileus with consequent short bowel syndrome, cf related liver disease with liver synthetic dysfunction, severe lung disease with frequent exacerbations, chronic lung disease or pulmonary failure, cfrelated diabetes, and some cases of poor intake and/or compliance, particularly if these are manifest during critical phases of growth such as infancy or adolescence. various adjuncts to nutritional therapy have been studied and may be considered in these circumstances (1) . firstly, maximising pert with acid suppression or increasing duodenal bicarbonate may enhance absorption of nutrients (2, 3) .however, there is insufficient evidence to indicate whether there is an improvement in nutritional status, lung function, quality of life, or survival (4). secondly, enhancing en with specific supplements, eg trophic factors such as glutamine for short bowel syndrome (5), and branched chain amino acids for liver disease may give added benefit (6) . thirdly, improved protein turnover, and efficiency of energy/fuel use and consequent wight gain and growth may be achieved by adjunctive growth hormone therapy (7) . other adjuncts, such as igf-1, and megestrol acetate have been evaluated with more variable outcomes. .finally, where en is inadequate, or impossible, the use of parenteral nutrition may be necessary, and has documented benefits (10) . the latter is particularly useful in pre-lung transplant patients, where improving/ maintaining nutrition may have an important bearing on outcomes. questions as to when and how these various adjuncts are best utilised are at present incompletely answered, but it does appear that any or all approaches are preferable to continuing to starve the patient in the face of progressive undernutrition. etiology and pathogenesis some organic digestive disturbances in early life the diagnosis of cystic fibrosis: a consensus statement cluster analysis classification and regression trees chest physiotherapy -the mechanical approach to anti-effective therapy in cystic fibrosis effectiveness of home vs hospital care in the routine treatment of cf schwachman-kulczcki score and resting energy expenditure in cf effect of exercise and physiotherapy in aiding sputum expectoration in adults with cystic fibrosis chest physiotherapy, gastro-oesophageal reflux, and arousal in infants with cystic fibrosis european respiratory society task force on the use of nebulizers use of nebulised saline and nebulised terbutaline as an adjunct to chest physiotherapy inhaled hypertonic saline increases sputum expectoration in cystic fibrosis liver involvement in cystic fibrosis hepatobiliary complications of cystic fibrosis outcome of liver cirrhosis in cystic fibrosis. management of portal hypertension liver cirrhosis in cystic fibrosis. therapeutic implications and long-term followup giunta a and the italian group for the study of ursodeoxycholic acid in cystic fibrosis. ursodeoxycholic acid for liver disease associated with cystic fibrosis: a double-blind multicenter trial natural history of liver disease in cystic fibrosis ursodeoxycholic acid for cystic fibrosis-related liver disease (cochrane review) genotype-phenotype correlation in cystic fibrosis: the role of modifier genes cystic fibrosis genotype/phenotype consortium. correlation between genotype and phenotype in cystic fibrosis novel uses to study complex traits and genetic diseases residual chloride secretion in intestinal tissue of deltaf508 homozygous twins and siblings with cystic fibrosis. the european cf twin and sibling study consortium chloride conductance and genetic background modulate the cystic fibrosis phenotype of delta f508 homozygous twins and siblings detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13 chronic pain in cystic fibrosis the musculoskeletal complications of cystic fibrosis end-of-life care in cystic fibrosis cystic fibrosis foundation. (2003) about cf. retrieved methodological issues in webbased research ethical and legal aspects of human subjects to research on the internet graphics visualization usability center tenth www user survey research and the web: eyeballs or smiles? designing and implementing web-based surveys chronic pain in cystic fibrosis pain in infants, children, and adolescents self-hypnosis for patients with cystic fibrosis hypnosis, theodore roosevelt, and the patient with cystic fibrosis hypnosis in treatment of clinical pain: understanding why hypnosis is useful adjunctive non-pharmacological analgesia for invasive medical procedures: a randomized trial hypnosis and hypnotherapy with children handbook of hypnosis for professionals abnormal glucose metabolism in cystic fibrosis diabetes mellitus associated with cystic fibrosis influence of the development of diabetes mellitus on clinical status in patients with cystic fibrosis identification and treatment of cystic fibrosis-related diabetes: a survey of current medical practice in the diagnosis of cystic fibrosis related diabetes: a selective approach in performing the oral glucose tolerance test based on combination of clinical and biochemical criteria glucose tolerance in patients with cystic fibrosis: five year prospective study diagnosis of diabetes in cystic fibrosis and thalassemia major diagnosis, screening and management of cystic fibrosis related diabetes mellitus diabetes mellitus in danish cf patients: prevalence and late diabetic complications diabetic microangiopathy in patients with cystic fibrosis the interaction of two diseases: diabetes mellitus and cystic fibrosis generalised atherosclerosis in an adult with cf and diabetes mellitus (abstract) lipid metabolism in adults with cystic fibrosis abnormal lipid levels in cystic fibrosis effect of protein ingestion on the glucose and insulin response to a standardized oral glucose load ingested protein has little effect on glucose concentration or rate of glucose appearance in people with type 2 diabetes (abstract #97) effects of meal carbohydrate content on insulin requirements in type 1 diabetic patients treated intensively with the basal-bolus (ultralenteregular) insulin regimen cystic fibrosis related diabetes: an approach to diagnosis and management clinical guidelines for physio. management of sui back pain and spinal deformity in cystic fibrosis kyphosis and fractures in children and young adults with cystic fibrosis increased rate of fractures and severe kyphosis: sequelae of living into adulthood with cystic fibrosis musculoskeletal involvement in cystic fibrosis vertebral deformities and low bone mineral density in adults with cystic fibrosis: a cross-sectional study posture and cystic fibrosis impact of lung inflammation on bone metabolism in adolescents with cystic fibrosis postural relief of dyspnoe in severe chronic airflow limitation: relationship to respiratory muscle strength physiotherapy for respiratory and cardiac problems, 3 rd edn chest mobilization and respiratory function habitual physical activity in children and adolescents with cystic fibrosis physiotherapy in infants and young children with cystic fibrosis: current practice and future developments international physiotherapy group for cystic fibrosis (ipg/cf): physiotherapy in the treatment of cf effects of nutritional status on exercise performance in advanced cystic fibrosis respiratory and peripheral muscle function in cystic fibrosis determinants of aerobic and anaerobic performance in cystic fibrosis exercise performance and breathing patterns in cystic fibrosis: male-female differences and influence of resting pulmonary function muscle function and resting energy expenditure in female athletes with cystic fibrosis the km of nadh dehyrogenase is decreased in the mitochondria of cystic fibrosis cells mitochondrial nadh dehyrogenase in cystic fibrosis muscle size and cardiorespiratory response to exercise in cystic fibrosis peripheral muscle weakness and exercise capacity in children with cystic fibrosis the relationship between genotype and exercise tolerance in children with cystic fibrosis end organ dysfunction in cystic fibrosis angiotensin converting enzyme gene insertion/ deletion polymorphism and response to physical training exercise performance and breathing patterns in cystic fibrosis: male-female differences and influence of resting pulmonary function multiple factors that limit exercise capacity in cystic fibrosis activity levels and relationship to lung function and nutritional status in children with cystic fibrosis habitual activity in children and adolescents with cystic fibrosis a randomized controlled trial of a 3 year home exercise program in cystic fibrosis musculoskeletal manifestatons in cystic fibrosis low bone mineral density in adults with cystic fibrosis osteoporosis and osteopenia in adults and adolescents with cystic fibrosis: prevalence and associated factors histomorphometric analysis of bone biopsies from the iliac crest of adults with cystic fibrosis the role of physiotherapy in the prevention and treatment of osteoporosis exercise, bone and nutrition bone mineral density and bone acquisition in children and young adults with cystic fibrosis: a follow-up study milk intake and bone mineral acquisition in adolescent girls: randomised, controlled intervention trial sex steroids and body composition in men with cystic fibrosis impact of lung inflammation on bone metabolism in adolescents with cystic fibrosis acquisition of optimal bone mass in childhood and adolescence bone mineral density in australian children, adolescents and adults with cystic fibrosis: a controlled cross sectional study a six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: the university of saskatchewan bone mineral accrual study decreased bone mineral density in normal-growing patients with cystic fibrosis analysis of factors limiting maximal exercise performance in cystic fibrosis prospective ten-month exercise intervention in premenarcheal girls: positive effects on bone and lean mass highimpact exercise and bones of growing girls: a 9-month controlled trial former exercisers of an 18-month intervention display residual bmd benefits compared with control women 3.5 years post-intervention: a follow-up of a randomized controlled high-impact trial identification of the cystic fibrosis gene: cloning and characterization of complementary dna isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cgmp-dependent protein kinase ii apparent affinity of cftr for atp is increased by continuous kinase activity camp-independent phosphorylation activation of cftr by g proteins in native human sweat duct the complex and intriguing lives of pip2 with ion channels and transporters protein kinase-independent activation of cftr by phosphatidylinositol phosphates phosphorylation-regulated clchannel in cho cells stably expressing the cystic fibrosis gene phosphorylation by protein kinase c is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase a specificity and mechanism of action of some commonly used protein kinase inhibitors the bisindolylmaleimide gf 109203x is a potent and selective inhibitor of protein kinase c intracellular transport mechanisms of signal transducers the first nucleotide binding domain of cystic fibrosis transmembrane conductance regulator is a site of stable nucleotide interaction, whereas the second is a site of rapid turnover prolonged nonhydrolytic interaction of nucleotide with cftr's nh2-terminal nucleotide binding domain and its role in channel gating a tweezers-like motion of the atp-binding cassette dimer in an abc transports cycle control of cftr channel gating by phosphorylation and nucleotide hydrolysis structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator cooperative, atp-dependent association of the nucleotide binding cassettes during the catalytic cycle of atp-binding cassette transporters mutation of walker-a lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide binding domains atp binding to the motor domain from an abc transporter drives formation of a nucleotide sandwich dimer nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. different patterns in the two nucleotide binding domains on the mechanism of mgatp-dependent gating of cftr cl -channels dual effects of adp and amp-pnp on cftr channel kinetics show binding to two different nucleotide binding sites gating of cftr by nucleoside triphosphates: quantitative analysis of a cyclic gating scheme gating of cftr by atp hydrolysis: structure and function early pulmonary inflammation in infants with cystic fibrosis the relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients inflammation and infection in naive human cystic fibrosis airway grafts excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with pseudomonas aeruginosa diverse pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8 overproduction of the cftr r domain leads to increased levels of asialogm1 and increased pseudomonas aeruginosa binding by epithelial cells proinflammatory cytokine responses to p. aeruginosa infection in human airway epithelial cell lines altered cytokine production by cystic fibrosis tracheal gland serous cells inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis cytokine secretion by cystic fibrosis airway epithelial cells altered respiratory epithelial cell cytokine production in cystic fibrosis chemokine expression in cf epithelia: implications for the role of cftr in rantes expression in vivo alterations of ifn regulatory factor-1 and pias1 protein levels in cystic fibrosis epithelium activation of nf-b by adherent pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells exaggerated activation of nf-b and altered ib-␤ processing in cystic fibrosis bronchial epithelial cells high susceptibility for cystic fibrosis human airway gland cells to produce il-8 through the ib kinase ␣ pathway in response to extracellular nacl content reduced smad3 protein expression and altered transforming growth factor-␤1-mediated signaling in cystic fibrosis epithelial cells reduced interleukin-8 production by cystic fibrosis airway epithelial cells interleukin-8 production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-␣ and respiratory syncycial virus stimulation alternate-day prednisone reduces morbidity and improves pulmonary function in cystic fibrosis effect of high-dose ibuprofen in patients with cystic fibrosis regulation of human airway epithelial cell il-8 expression by map kinases abnormal morphogenesis but intact ikk activation in mice lacking the ikk␣ subunit of the ib kinase ikk␣ provides an essential link between rank signaling and cyclin d1 expression during mammary gland development activation of nf-b by nontypeable hemophilus influenzae is mediated by tlr2-tak1-dependent nik-ikk␣/␤-ib␣ and mkk3/6-p38 map kinase signaling pathways in epithelial cells micrococci and peptidoglycan activate tlr2-myd88-irak-traf-nik-ikk-nf-b signaling pathway that induces il-8 transcription hershenson mb. signaling intermediates required for nf-b activation and il-8 expression in cf bronchial epithelial cells coordinate regulation of ib kinases by map kinase kinase kinase 1 and nf-b inducing kinase ras and mekk-1 coregulate ap-1 and nf-bmediated gene expression in airway epithelial cells activation of nf-b via a src-dependent ras-mapk-pp90 rsk pathway is required for pseudomonas aeruginosa-induced mucin overproduction in epithelial cells pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells cf pathogens activate ca 2+ -dependent map kinase signaling pathways in airway epithelial cells atp transduces signals from asgm1, a glycolipid that functions as a bacterial receptor tlr2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells the kinase tak1 can activate the nik-ib as well as the map kinase cascade in the il-1 signalling pathway inflammation and infection in naive human cystic fibrosis airway grafts activation of nf-b in airway epithelial cells is dependent on cftr trafficking and cl (-) channel function selective upregulation of il-8 expression in cf bronchial gland cells in vivo and in vitro signal transduction from the endoplasmic reticulum to the cell nucleus monocyte retention and migration in pulmonary inflammation. requirement for neutrophils differential roles for alpha(m)beta(2) integrin clustering or activation in the control of apoptosis via regulation of akt and erk survival mechanisms elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/ paracrine mechanisms involving tgf-beta, pge2, and paf phosphatidylserine-dependent ingestion of apoptotic cells promotes tgf-beta1 secretion and the resolution of inflammation cystic fibrosis in adults drug discovery in academia high-affinity activators of cftr chloride conductance identified by high-throughput screening thiazolidinone cftr inhibitor identified by highthroughput screening blocks cholera-toxin induced intestinal fluid secretion altered channel gating mechanism for cftr inhibition by a high-affinity thiazolidinone blocker prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule cftr inhibitor cftr involvement in nasal potential differences in mice and pigs studied using a thiazolidinone cftr inhibitor a small molecule cftr inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways pharmacology and antidiarrheal efficacy of a thiazolidinone cftr inhibitor in rodents discovery of glycine hydrazide pore-occluding cftr inhibitors: mechanism, structure-activity analysis and in vivo efficacy nanomolar affinity small-molecule correctors of defective ∆f508-cftr chloride channel gating nonsense-mediated mrna decay in health and disease highly variable incidence of cystic fibrosis and different mutation distribution among different jewish ethnic groups in israel cystic fibrosis in jews: frequency and mutation distribution suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics g-418 and paromomycin aminoglycoside suppression at uag, uaa and uga codons in escherichia coli and human tissue culture cells suppression of a cftr premature stop mutation in a bronchial epithelial cell line a pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis gentamicin-induced correction of cftr function in patients with cystic fibrosis and cftr stop mutation developing cystic fibrosis lung transplant referral criteria using predictors of 2-year mortality prediction of mortality in patients with cystic fibrosis survival effect of lung transplant among patients with cystic fibrosis living lobar transplantation lung transplantation of ventilator dependent patients recipient selection international guidelines for the selection of lung transplant candidates lung transplantation is warranted for stable, ventilator dependent recipients early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth longitudinal evaluation of bronchopulmonary disease in children with cystic fibrosis bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis cognitive function of children with cystic fibrosis: deleterious effect of malnutrition references 1. merelle me, griffioen rw, dankert-roelse je. cystic fibrosis presenting with intracerebral haemorrhage dehydration deaths in infants and young children pancreatic function in infants identified as having cystic fibrosis in a neonatal screening program pancreatic insufficiency, growth, and nutrition in infants identified by newborn screening as having cystic fibrosis early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth. wisconsin cystic fibrosis neonatal screening study group investigators and coordinators of the epidemiologic study of cystic fibrosis. growth and nutritional indexes in early life predict pulmonary function in cystic fibrosis stature as a prognostic factor in cystic fibrosis survival bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis the diagnosis of cystic fibrosis cystic fibrosis foundation cystic fibrosis diagnosed after age 13. twenty-five teenage and adult patients including three asymptomatic men diagnosis of cystic fibrosis during adolescence clinical features associated with a delayed diagnosis of cystic fibrosis delayed diagnosis of us females with cystic fibrosis the spirit of lo. an ordinary family's extraordinary journey dueling with cystic fibrosis: finding the chloride defect early versus late diagnosis: psychological impact on parents of children with cystic fibrosis effects of newborn screening of cystic fibrosis on reported maternal behaviour newborn screening for cystic fibrosis in wisconsin: nine years experience with routine trypsinogen/dna testing genetic counseling and neonatal screening for cystic fibrosis: an assessment of the risk communication process sweat chloride concentrations in infants homozygous or heterozygous for f508 cystic fibrosis analysis of the costs of diagnosing cystic fibrosis with a newborn screening program comprehensive analysis of risk factors for acquistion of pseudomonas aeruginosa in young children with cystic fibrosis pediatric clinics of north america managing cystic fibrois related diabetes (cfrd). a teaching manual for cystic fibrosis related diabetes. the cf foundation enteral tube feeding for cystic fibrosis consensus report on nutrition for pediatric patients with cystic fibrosis cystic fibrosis adult care: consensus conference report optimising nutrition in cystic fibrosis gastric acid suppression and treatment of severe exocrine pancreatic insufficiency omeprazole, a proton pump inhibitor, improves residual steatorrhoea in cystic fibrosis patients treated with high dose pancreatic enzymes drug therapies for reducing gastric acidity in people with cystic fibrosis.cochrane database syst rev trophic and cytoprotective nutrition for intestinal adaptation, mucosal repair, and barrier function nutritional support in children with end-stage liver disease: a randomized crossover trial of a branched-chain amino acid supplement growth problems and growth hormone treatment in children with cystic fibrosis effects of glutamine and recombinant human growth hormone on protein metabolism in prepubertal children with cystic fibrosis improved growth and clinical, nutritional, and respiratory changes in response to nutritional therapy in cystic fibrosis s20.4 when enteral nutrition is not enough -adjuncts to nutrition support in cf newborn screening for cystic fibrosis (cf) has been performed as part of a randomized, controlled trial for patients born in wisconsin between april 15, 1985 and june 30, 1994 . in the randomized, controlled trial, the study investigators were informed of a positive neonatal screen (immunoreactive trypsinogen [irt] in the first six years of the study and irt/dna in the last three years of the study) for patients in the early diagnosis group. the study investigators informed the primary care physician of the positive newborn screen and the infant was brought to madison or milwaukee (the two study centers) for sweat testing at six weeks of age. in july 1994, cf newborn screening was added to the panel of routine neonatal screening tests. over the past decade, we have learned the following lessons from the routine cf newborn screening program. 1 1. initially in the routine neonatal screening program, the newborn screen collection card had a check-off box for meconium ileus. if this box was checked, then dna analysis for the ∆f508 mutation was performed regardless of the trypsinogen level. the rationale for this was to detect cf patients who theoretically could have a low trypsinogen level after surgery for meconium ileus. however, in the first 3 1/2 years of the routine cf newborn screening program, there were a total of 1266 infants in which meconium ileus was checked on the newborn screening specimen card. the staff at newborn nurseries were possibly checking this box erroneously for meconium stained amniotic fluid. in confirmed cases of cf with meconium ileus, there were no infants in which the trypsinogen level was less than the cut-off value. therefore, the meconium ileus box on the newborn screening form was removed. 2. initially in the routine newborn screening program, the written recommendation for an infant with a positive cf newborn screen was to have a sweat test performed at four weeks of age. although the two cf centers in the state of wisconsin were listed on the report, it was not explicitly stated that sweat testing should occur at a cf center. this led to two difficulties: a. families were required to wait until the baby was four weeks of age for a sweat test. during this time, they encountered anxiety about the possibility that their child could have cf. b. a number of sweat tests were performed at hospitals that were not cf centers. this led to questions about the accuracy of the sweat testing procedure. additionally, families were not receiving genetic counseling in these non-cf center settings.these issues were addressed by revising the positive cf neonatal screening report. the report no longer states that the infant should be four weeks of age for a sweat test and, therefore, there is now less of a delay from the time that the primary care physician notifies the family of the positive newborn screening test until the time that a sweat test is performed. additionally, the newborn screening report now states that a quantitative pilocarpine iontophoresis sweat test should be performed at cf centers or affiliate centers listed on the report. as a result of these changes, in the calendar year 2003, there was only one infant who had a sweat test outside of a cf core center or affiliate center. 3. we are aware that families are anxious when they learn that their infant has a positive neonatal screen (specifically, an elevated trypsinogen and one cf mutation).to help allay the parents' anxiety 2 , a letter has been created that accompanies the positive cf newborn screening report to the primary care physician. this letter is intended to be given to the parents and emphasizes that there is only a possibility of cystic fibrosis. the letter briefly describes the sweat test and that no special preparation is needed. contact information for the cf core and affiliate centers and the state newborn screening coordinator is provided. additionally, the letter directs the parents to a website that we have created that provides useful information about cf newborn screening <http://www.pediatrics.wisc.edu/childrenshosp/cf/nbs.html>. within this website is a video entitled "understanding newborn screening for cystic fibrosis". all of these educational efforts have significantly decreased the number of phone calls that we receive from panicked parents. 4. from our experience in the cf newborn screening program, it is clear that the traditional decision levels for sweat chloride (<40 meq/l is normal, 40-60 meq/l is borderline, and >60 meq/l is diagnostic of cystic fibrosis) is not applicable to infants. we have demonstrated that sweat chloride levels of >30 meq/l in an infant is suspicious for cf. extended mutation analysis in infants with a sweat chloride of >30 meq/l almost invariably reveals two cf mutations. this agrees with previous data. 3 5 . infection control measures are of utmost importance, as clinic exposures in a small waiting room (110 sq. ft.) was a risk factor for acquisition of pseudomonas aeruginosa. 5 6. cf newborn screening has evolved from an algorithm of irt/∆f508 analysis to irt/multiple (25) mutation ciated with insulin deficiency and resistance to insulin's anti-catabolic effects on whole body protein turnover. protein catabolism is an especially important consideration in the malnourished cf child and adult. malnourishment, principally loss of muscle mass, has been associated with worsened morbidity and mortality. insulin lowers blood sugar levels and functions as an anabolic agent by increasing cellular uptake of ingested nutrients and conversion of the nutrients to energy. insulin is the principal hormone preventing protein catabolism. the 1998 cf consensus conference recommended that insulin be the only medical therapy prescribed for cfrd with fasting hyperglycemia. the recommendations were based on the review of existing medical literature. the use of insulin may be especially important in a malnourished patient with cf, and several studies have documented weight gain and improvement of catabolism with insulin. insulin use can be made more convenient by using insulin injection devices and insulin pumps. there are many types of insulin and potential insulin regimens available for treating cfrd, and therapy should be individualized. however, there are several principles to guide the clinician in choice of therapy. 1. the regimen should as closely as possible fit the patient's life-style. 2. meal coverage should be provided to prevent postprandial hyperglycemia. 3. the regimen should confer as low a risk as possible for the development of hypoglycemia. 4. in most cases, good glycemic control can be obtained using of multiple injections. the broad classes of insulin are "rapid-acting," "short-acting", "longer-acting" and basal insulin. insulin action (when it peaks in activity, and how long it lasts) may vary from person to person. there is also some variability from one day to the next in the same person. when deciding on an insulin regimen, three types of coverage should be considered.• meal coverage (bolus insulin): a normal pancreas secretes insulin as a "bolus" to cover the meal (specifically the carbohydrates) ingested. rapid-acting or short-acting insulin is given before meals to mimic this extra insulin "bolus." generally, the best way to dose pre-meal insulin dose is to account for the carbohydrate content of the meal. the normal pancreas makes a small amount on insulin at all times. these low levels of insulin are called "background" or "basal" insulin. generally long-acting insulin such as ultralente (lasts approximately 12-24 hours and has a small peak) or lantus (lasts 24 hours and has no peak) are used. most people with cf make some insulin, thus they often do not require as much basal insulin as people who have diabetes but do not have cf. however, protein catabolism is higher in malnourished patients; therefore basal insulin is an important part of the insulin regimen in these patients. in general a starting dose which minimizes the risk of hypoglycemia is 0.1 units per kilogram of weight per day. this dose should be increased as needed, but hypoglycemia should be avoided.• correction: when the blood glucose level is too high, rapid-acting insulin is given in addition to the usual insulin dose to "correct" the blood glucose level. correction is especially important during illness and to prevent hyperglycemia-related diabetes complications. in general, when a patient is normally insulin sensitive, one unit of rapid-acting insulin will lower the glucose level approximately 50 mg/dl. night-time enteral feeding are particularly useful in the malnourished cf patient, and some may only have high blood sugar levels during these feedings. hyperglycemia at this time is best managed by a combination of longeracting (nph or lente) plus short-acting insulin (regular), at the start of the feeding. a starting dose which minimizes the risk of hypoglycemia is 0.1 units per kilogram per dose of regular and 0.1 units/kg/dose of nph. the cf consensus conference on diabetes did not recommend that oral agent be used to treat cfrd with fasting hyperglycemia (the only glucose tolerance category for they recommended mandatory treatment). there are multiple types of oral agents, and a few small studies have documented efficacy in cfrd. research is needed to test potential oral therapies. key: cord-023311-7wqdlha4 authors: nan title: oral session date: 2010-11-24 journal: respirology doi: 10.1111/j.1440-1843.2010.01864.x sha: doc_id: 23311 cord_uid: 7wqdlha4 nan introduction rheumatic heart disease, predominantly mitral stenosis is a chronic disease that produces an increase in the left atrial pressure and consequently venous pulmonary hypertension. preoperative lung function which could be obtained from spirometry can evaluate respiratory reserve in cardiopulmonary patients who will undergo surgery. however, data on the use of spirometry in predicting the rate and extent of regression of preoperative pulmonary artery hypertension is limited. methods we determined the usefulness of preoperative lung function by spirometry in predicting regression of pulmonary hypertension after surgical correction of mitral stenosis among 20 patients who underwent mitral valve surgery at philippine heart center from july to december 2009. results among the twenty patients included in the study, one had normal spirometry and another one had mild obstructive abnormality. majority of the patients (18/20) had restrictive abnormality. nineteen patients had regression of pap. among them, 18 patients were noted to have restrictive abnormality and one with normal spirometry. there was only one patient who did not have regression of pap and found to have a mild obstructive abnormality. correlation of the severity of restrictive lung defect with the change in pap classifi cation among nineteen patients showed lack of correlation with a spearman coeffi cient of 0.041 and p-value of 0.863. (figure 1 ) conclusion this study showed that majority of rhd patients particularly mitral stenosis will have a preoperative spirometric abnormality of restrictive pattern. among the study group, almost all patients (19/20) will have regression of pulmonary hypertension after surgery except for one patient with obstructive lung abnormality. though results were not signifi cant, we cannot conclude that preoperative lung function is not predictive of regression of pulmonary hypertension after surgical correction of mitral stenosis due to inadequacy of sample size. thus, further investigation is warranted. introduction drug-induced interstitial lung disease (ild), particularly pulmonary fi brosis, is a serious clinical concern and myofi broblasts have been suggested to play a major role, with it recently being revealed that some of these myofi broblasts are derived from lung epithelial cells through epithelial-mesenchymal transition (emt). in this study, we used cultured epithelial cells to examine the emt-inducing abilities of drugs known to induce ild clinically. methods induction of emt in cultured lung epithelial cells were monitored by up-regulation of the expression of myofi broblast marker proteins and downregulation of the expression of epithelial cell marker proteins. the severity of lung injury and fi brosis in mice was assessed by various methods, such as histopathologic evaluation, histochemical analysis of collagen and determination of hydroxyproline. results emt-like phenotypes were induced by a771726, an active metabolite of lefl unomide having an inhibitory effect on dihydroorotate dehydrogenase (dhodh). smad interacting protein 1 (a transcription factor regulating emt) and the notch-signaling pathway were shown to be involved. when the cultures were supplemented with exogenous uridine, the a771726-induced emtlike phenotypes disappeared. likewise, an a771726 analog without inhibitory activity on dhodh produced no induction, suggesting that this process is mediated through the inhibition of dhodh. in vivo, administration of lefl unomide stimulated bleomycin-induced emt-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fi brosis, both of which were suppressed by co-administration of uridine. conclusion these fi ndings suggest that lefl unomide-dependent exacerbation of bleomycin-induced pulmonary fi brosis is mediated by stimulation of emt of lung epithelial cells, providing the fi rst evidence that drug-induced pulmonary fi brosis involves emt of these cells. we consider that this lefl unomide-dependent exacerbation of bleomycin-induced pulmonary fi brosis provides a suitable animal model of drug-induced ild, which is important to establish not only a clinical protocol for its treatment but also an assay system that will facilitate screening in order to eliminate candidate drugs with the potential to produce this type of side effect. introduction to observe the infl uence of arsenic trioxide on the bleomycininduced pulmonary fi brosis in rats and its mechanisms. methods pulmonary fi brosis was induced in sprague-dawley (sd) rats by intratracheal instillation of bleomycin(blm). the rats of the treatment group, the steroid group and model group were intraperitoneally injected with arsenic trioxide(ato), dexamethasone or normal saline(ns)respectively, while the control rats received ns both intratracheally and intraperitoneally. the effects of interference were evaluated by median survival time, hydroxyproline level in lung, semi-quantitative grading of alveolitis and pulmonary fi brosis and quantititative analysis of collagen in lung (masson's trichrome stain). apoptosis index (ai) of lung was detected by using the terminal transferase dutpdigoxygenin nick end-labeling (tunel) method and the results of the immunohistochemical staining of some cytokines were quantitatively analyzed. results ato might (1) prolong the median survival time of blm-induced pulmonary fi brosis rats at some extent; (2) attenuate the alveolitis and pulmonary fi brosis, reduce hydroxyproline level and collagen deposition in lung tissue; (3) increase the ai of lung tissue at a certain phase; and decrease the levels of transforming growth factor-β1 (tgf-β1) and tissue inhibitor of metalloproteinase-1 (timp-1), increase the content of interferon-γ(ifn-γ) signifi cantly. conclusion ato might attenuate blm-induced pulmonary fi brosis in rats via increasing the ai of lung tissue. introduction combined pulmonary fi brosis and emphysema (cpfe) is a syndrome involving both emphysema and diffuse parenchymal lung disease with fi brosis on chest computed tomography (ct). the clinical characteristics of cpfe have been described; however, the differences between the syndrome and interstitial pneumonia (ip) or chronic obstructive pulmonary disease (copd) are not fully understood. the purpose of this study was to clarify the differences in respiratory resistance and reactance using a forced oscillation technique. methods the subjects included 26 patients with cpfe, 27 with ip, and 21 with copd. respiratory resistance and reactance were measured using most-graph-01 (chest mi co., ltd., tokyo, japan), and pulmonary function tests were also performed on the same examination day. results the fev1 and fev1/fvc values were signifi cantly lower in copd patients compared to those with cpfe and ip. there was no signifi cant difference in vc between cpfe, ip, and copd patients. the carbon monoxide transfer coeffi cient values were signifi cantly lower in cpfe and copd patients compared to those with ip. resistance at 5 hz (r5: cmh20/l/s) was significantly elevated in patients with copd (cpfe, 3.48; ip, 4.20; and copd, 4.56, respectively, p < 0.05 for cpfe vs. copd), while r20 was elevated in patients with ip and copd compared to those with cpfe (cpfe, 2.53; ip, 3.47; and copd, 3.24, respectively, p < 0.05 for cpfe vs. ip and for cpfe vs. copd). the resonant frequency (hz), a parameter of reactance, was signifi cantly higher in copd patients compared to cpfe and ip patients (cpfe, 8.76; ip, 9.09; and copd, 15.50, respectively, p < 0.05 for cpfe vs. copd and for ip vs. copd). conclusion cpfe patients exhibited no airfl ow limitation or restrictive impairment, but showed severe gas exchange abnormality similar to that in copd patients. the absence of an elevation of respiratory resistance or reactance refl ects homogenous ventilatory mechanics in cpfe, thereby differentiating it from ip and copd. these results suggest that cpfe is a distinct syndrome differing from ip or copd. pneumonectomy is a surgical removal of a lung. it poses several adverse consequences as it substantially diminishes diffusion capacity by reducing total number of alveoli and vasculature available for gas exchange. the challenge is to maintain adequate gas exchange following resection of the lung tissue. literature revealed a good prognosis for pneumonectomized infant. there is enhancement of diffusion capacity in remnant lung through generation of new pulmonary gas exchange units. this was evidenced by normal lung volumes of the pneumonectomized infants after 20 years. in this article, we present a 22 day old female who was noted to be tachypneic, with chest indrawing and subcostal retractions. chest roentgenogram done revealed collapsed right lung, dextrocardia with hyperinfl ated and hyperluscent left lung. 2d echo showed no anatomic anomaly, except for dextroposed heart probably secondary to the collapsed right lung. impression then was congenital cystic adenomatoid malformation versus congenital lobar emphysema, left lung. extensive work-up was done. chest ct scan showed overly infl ated and hyperluscent lung segment arising from left lower lobe which is characteristic of a congenital lobar adenomatoid malformation. lung perfusion scan demonstrated diminished perfusion of the left lung with differential contribution to the total perfusion of the 38% left lung and 62% right lung. patient then underwent open thoracotomy with pneumonectomy of the left lung. biopsy revealed congenital cystic adenomatoid malformation type ii. three months after the surgery, she has gained weight, not receiving any medications and is symptom free. introduction despite the importance of infection and infl ammation in the pathogenesis and management of bronchiectasis, there are few published data on lower airway microbiology and cellularity in these children. methods children attending a single centre (1992 to 2009) with non-cystic fi brosis bronchiectasis who underwent bronchoalveolar lavage (bal) within 4 weeks of diagnosis were identifi ed. the point prevalence of infection (>105 colony-forming units (cfu)/ml of respiratory bacterial pathogens), its effects upon airway cellularity and the impact of clinical and demographic variables on infection risk were evaluated. results of 113 children with bronchiectasis, 44 (39%) had bal evidence (>105 cfu/ml) of infection, which was frequently polymicrobial and caused mostly by haemophilus infl uenzae, streptococcus pneumoniae and moraxella catarrhalis. in contrast, pseudomonas aeruginosa was uncommon and mycobacterial and fungal species were undetected. upper airway commensal organisms were also isolated in large numbers (>105 cfu/ml) from 24 (21%) bal cultures. the median (interquartile range; iqr) bal fl uid total cell counts (tcc × 106/l) and neutrophil percentages were signifi cantly higher in those with, than without, infection [tcc 730 (320-4140) vs 280 (143-1131); p = 0.001 and neutrophil percentage 76% (22-94) vs 38% (12-80); p = 0.01 respectively]. only age at diagnosis was associated with infection. conclusion bal microbiology of children with newly diagnosed bronchiectasis substantially differs from adults. children have marked airway neutrophilia, particularly when bacterial loads were high. younger children were more likely to have a lower airway infection at diagnosis. the role and interactions of respiratory bacterial fl ora in initiating and progressing airway damage in bronchiectasis requires further study. the radiological defi nition of airway dilatation and bronchiectasis in children has substantial limitations. bronchoarterial ratio is a commonly used criterion to defi ne airway dilatation despite the lack of normative pediatric data. the objective of our study was to determine the range of normal bronchial to accompanying arterial diameter ratio on high resolution ct scan of the chest in children and compare it with the available adult data. methods children undergoing mdct chest for non-pulmonary conditions at a single centre were prospectively identifi ed. high resolution reconstruction was performed on those included and both airway and vessel diameters were measured in the upper and lower lobes of both lungs. mean bronchoarterial (ba) ratio was calculated for each included child and its correlation with age assessed. results forty one children were included, the mean (sd) ba ratio was 0.626 (0.068) (range 0.437 to 0.739). this ratio was clinically similar though statistically lower than comparable adult data [combined mean (sd) 0.676 (0.12); p = 0.01]. no correlation was found with age in our cohort (r = −0.21, p = 0.19). there was no difference in the ratio based on laterality or lobe. conclusion in pediatric age-group, the airway is signifi cantly smaller than the adjoining vessel. using the radiological criteria of ba ratio greater than one to defi ne bronchial dilatation would under estimate the presence and extent of bronchiectasis leading to delayed and missed diagnosis. this highlights the need to redefi ne the criteria for bronchial dilatation in children. introduction sleep disordered breathing, especially obstructive sleep apnea syndrome, has been found to be associated with endothelial dysfunction in both adult and paediatric populations. however, the role of non-apnoeic snoring on endothelial function has not been investigated. methods children aged 6-18 years with habitual snoring were recruited from our sleep disorder clinic. non-snoring controls were recruited from participants of a community growth survey. all participants underwent nocturnal polysomnography (psg) and ultrasonographic fl ow mediated dilation (fmd) evaluation on the same day. fasting blood was taken for glucose level and lipid profi le determination. subjects with an obstructive apnoea hypopnoea index (oahi) < 1 but reported by parents to have habitual snoring (at least 3 nights per week) in the past 12 months were defi ned as primary snorers (ps). those having an oahi < 1 without habitual snoring in the past 12 months were grouped as non-snorers. children with body mass index of greater than the 85th percentile of the local reference were defi ned as overweight. subjects were divided into groups of normal weight, overweight, non-snorers and ps for comparisons. results in total, 161 children, of whom 78 were boys, with a mean (sd) age of 11.8 (2.8) years were recruited. sixty-six of them were ps. subjects with ps had signifi cantly reduced fmd than non-snoring controls for both the normal weight group (p = 0.040) and the overweight group (p = 0.039) ( table 1) . multivariate linear regression model showed that primary snoring (p < 0.001) were independently associated with fmd after controlling for possible confounders including overweight, gender, baseline vessel diameter and log-transformed oahi. conclusion primary snoring in children is independently associated with impaired endothelial function. introduction children with cyanotic congenital heart disease live with baseline oxygen saturations in the mid 70s, so hence they exist on the steep part of the oxyhemoglobin dissociation curve. these patients are at increased risk for the hemodynamic variations occurring during apneas/hypopneas. longterm outcomes for children with congenital heart disease could be adversely affected since the etiology of pulmonary hypertension is believed to be secondary to the hypoxia and hypercarbia seen in chronic airway obstruction paired with the sympathetic overstimulation caused by frequent sleep arousals. methods a prospective two part questionnaire for the screening of sdb for pediatric patients was performed. part one consists validated pediatric sleep questionnaire (psq). part two consisted of subjective assessment of the subject's cardiovascular and respiratory symptom. all odd ratios of greater than 1 with p-values less than 0.05 were considered signifi cant covariates. results a total of 206 children met the inclusion criteria and were included in the fi nal analysis. the prevalence of sleep disordered breathing (sdb) was high at 63.1%. among the factors analyzed, an increased frequency of pulmonary diseases (greater than 7 times/year) was statistically correlated with increased psq scores (p = 0.002). likewise, early palliative repair (p = 0.001) was statistically associated. a high total cardiac score is almost four times associated with increased psq ratings (p = 0.018). conclusion increased frequency of pulmonary diseases and early palliative repair was statistically correlated with increased pediatric sleep questioner scores. a high total cardiac score is almost four times associated with increased psq ratings. hence patients with congenital heart disease and sleep disordered breathing are more likely to have worse cardiac symptoms. patient with congenital heart disease shoukl be routinely examined for the presence of sleep disordered breathing because these sub group of pediatric patients are 7x have more high risk for developing sleep disordered breathing. introduction atelectasis, is a common pulmonary complication of patient who underwent open heart surgery. deep breathing exercise is one of the interventions implemented to prevent the occurrence of this complication postoperatively. among preschoolers however, making them perform this breathing exercise and maintaining compliance is a challenge since children in this age group have a short attention span and may get bored easily. with this problem at hand, the investigators conceptualized an innovative technique, blowing bubbles as a breathing exercise, in order to prevent atelectasis among post open heart surgery preschoolers. methods this study is an open-label randomized control trial that compared blowing bubbles with the traditional deep breathing exercise among preschoolers who underwent open heart surgery. it took 4 months to complete the study and there were 64 patients screened but only 60 qualifi ed based on the inclusion/exclusion criteria. thirty were assigned randomly to the blowing bubbles group and 30 to the deep breathing exercise. atelectasis as documented on chest x-rays was the outcome measured. results out of the 30 participants in the deep breathing group, 8 developed atelectasis while in the blowing bubbles group, 1 out 30 had atelectasis this generated a p-value of 0.026 which is statistically signifi cant, favoring the blowing bubbles group. furthermore, risk analysis showed an absolute risk reduction of 23.34% and a relative risk of less than 1 which means that atelectasis is less likely to occur in the blowing bubbles group in comparison to the traditional breathing exercise group. conclusion blowing bubbles signifi cantly reduces the occurrence of atelectasis among post-open heart surgery preschoolers as opposed to deep breathing exercise. the use of blowing bubbles as a deep breathing modality incorporated through play activity is recommended among preschoolers. introduction severe acute respiratory syndrome (sars) is a novel contagious respiratory infection caused by the sars coronavirus (sars cov). in adults, a mortality rate of 10% has been reported, and respiratory complications can occur in up to 20% of survivors. the disease pattern is different in children [1, 2] but prevalence of longer term respiratory complications in children is unknown. the aim of this study was to investigate the aerobic capacity of children at 3 years after the diagnosis of sars. methods twenty-seven patients (mean age of 17.7 years) who completed both pulmonary function and maximal aerobic capacity (peak vo 2 ) tests at 6 and 15 months after the acute illness were invited for re-assessment. they underwent anthropometric assessment, full pulmonary function and exercise treadmill test. subjects with abnormal hrct at 15 months underwent repeat scanning. results at this 36-month assessment, 6 subjects refused to take part, and the main reasons of refusal were work commitments or time clashes with school activities. the remaining 21 subjects (43% female) provided complete pulmonary function and exercise data. pulmonary function test was normal in all patients. peak vo 2 , peak oxygen pulse, and ventilatory anaerobic threshold (vat) at this assessment were signifi cantly higher than that recorded at 6 and 15 months. ventilatory effi ciency (ventilatory equivalents for oxygen, ve/vo 2 ) and perfusion to the lungs (end-tidal partial carbon dioxide pressure, petco 2 ) signifi cantly improved since 15 months and maintained at 36 months. though peak vo 2 further improved at 36 months in patients with persistent or without radiological abnormalities, their values were 65% and 81% respectively of normal controls. conclusion this study is the most comprehensive report of post-sars exercise responses in children and adolescents. improvements in aerobic capacity over a period of 36 months after the initial illness were demonstrated, but the values remained suboptimal when compared to normal reference. introduction domestic mites are an important source of indoor allergens responsible for the development of allergic diseases worldwide. to date, there is no local epidemiology data on the allergic sensitization to domestic mites among adult patients with asthma and allergic rhinitis. methods from november 2009 to june 2010, we prospectively recruited adult patients with asthma and/or allergic rhinitis from an urban-based specialist medical centre in penang, malaysia, carefully profi ling their allergic sensitization to domestic mites by means of skin prick tests and clinico-demographic details. of the 60 patients [mean age (ci) 29 years (26-32); 35% male] recruited, skin allergic rates to dermatophagoides pteronyssinus, d. farinae, blomia tropicalis and tyrophagus putrescentiae were 71%, 75%, 70% and 56% respectively. there was no signifi cant difference in these rates among patients with asthma, allergic rhinitis or both. there were signifi cant associations between the number of people living in the same house with rates of d. pteronyssinus (p = 0.05) and d. farinae (p = 0.02), and the frequency of bed sheet changing with the rate of tyrophagus putrescentiae (p = 0.02). with younger age, there were also signifi cant higher allergic rates with d. pteronyssinus (p < 0.006), d. farinae (p < 0.001), b. tropicalis (p < 0.002) and tyrophagus putrescentiae (p = 0.04). conclusion our preliminary data shows a high prevalence of allergic sensitization to domestic mites in our local adult patients with asthma and/or allergic rhinitis. the fi ndings have implication on allergen control with the view of disease mitigation. introduction a study was carried out to compare the effi cacy and sensory perception of mometasone furoate and ciclesonide aqueous nasal spray in moderate-severe allergic rhinitis. methods a single blind study of 3 months on patient of both sexes, >18 years, diagnosed as moderate-severe allergic rhinitis as per aria workshop(2008)1, with skin test positivity to at least two aeroallergens was carried out on 52 patients. patients were divided into two, i.e. mometasone (group a) and ciclesonide groups (group b). group a received 200 microgram/day of mometasone furoate and group b received 200 microgram/day of ciclesonide, nasal spray once daily in the morning. the evaluation was made at 0, 2, 4, 8, 12 weeks by total nasal symptoms score (tnss), visual analogue scale (vas) and sinonasal outcome test-22 (snot-22). sensory perception of both nasal steroids was carried out on initial visit before allocating groups, employing a sensory perception questionnaire with a 14 sensory items (100 points scale). patients were given one nasal spray, and immediate and after two minutes response was noted in questionnaire. after 30 minutes of washout period, second drug was given and response was noted in similar way. results after 3 months, both mometasone and ciclesonide signifi cantly decreased nasal symptoms as assessed by tnss (p-value = 0.003) and vas (p-value = 0.004) and improved quality of life signifi cantly as assessed by snot-22 (p-value = 0.003). however there was no statistically signifi cant difference among two drugs. the sensory perception, in favour of mometasone was observed immediately after drug administration, than ciclesonide by providing more comfort during administration, less irritation, odor prference (all p-value 0.002 to 0.004). however, after 2 minutes of drug application, there was no signifi cant difference among both drugs in strength of taste, amount of medication rundown and irritation. the overall acceptance by patients was for mometasone over ciclesonide (p-value = 0.003). conclusion both mometasone and ciclesonide adequately controlled symptoms of allergic rhinitis in 3 months. the sensory perception preference for many of the sensory attributes in mometasone group were in favour of mometasone. thus, mometasone has equal effi cacy but slightly better acceptability over ciclesonide in the treatment of allergic rhinitis. results of 1460 patients, 62.3% were male and 37.7% females. 79% patients had used alternative asthma therapies: homeopathy, ayrurveda and yoga with poor results and 23.8% had used multiple therapies prior to visit our centre. patients reported being afraid of acute attack (42.8%) and hospitalization (28.8%). although inhalers were used by indian patients in 54.7% but still oral drugs were used regularly by 41.6% patients. compared to 27.1% in 2002 only 6.1% were inhaler naïve (t-value 0.00018). only 540/850 (63.5%) patients were using spacer with mdi's and 48% (260/540) being able to demonstrate correct use. common errors seen in mdi's use were: a) slow and steady inhalation (48.1%) and b) breath holding after deep inhalation (42.5%). formoterol and budesonide dpi was considered most effective by indian patients in controlling disease when asked to rate their devices and drugs. when counseled by experts 86% were sure to be regular on treatment but 6 month latter 61% remained regular (t-value 0.00007). fear of addiction (32.7%) and cost of therapy (25.4%) were cited causes for noncompliance. conclusion indian patients use alternative therapy for asthma treatment before coming to tertiary centre and still prefer oral therapy. despite extensive education being afraid of attacks become noncompliant due to fear of addiction and cost of therapy. the oesophagus and airways have a common origin. refl uxrelated respiratory symptoms may be triggered by aspiration of gastric refl uxate into airways or a vagally mediated oesophago-tracheo-bronchial. this association has not been reported previously in sri lanka. the aim of this study was to describe the association between gastro-oesophageal refl ux (gor) events and respiratory symptoms in a cohort of adult asthmatics in sri lanka. methods thirty stable, mild asthmatics (american thoracic society criteria) underwent dual-sensor ambulatory oesophageal ph monitoring. respiratory symptoms (cough, wheeze, diffi cult breathing, chest tightness) during monitoring were recorded and correlated with refl ux events. results both proximal and distal gor parameters were signifi cantly higher in asthmatics than controls (p < 0.050; mann-whitney u-test). however, there was no difference in any parameter between asthmatics with and without respiratory symptoms. abnormal proximal acid refl ux was documented in 66.7% and distal refl ux in 73.3% of 30 asthmatics. of 102 respiratory symptoms in all asthmatics, majority (72%) were cough episodes. in total, 93% of coughs, 81% of wheeze and all of chest tightness was refl ux-associated, where in most, refl ux events preceded respiratory symptoms. of 15 asthmatics with respiratory symptoms, acid exposure was normal in 4 (26%), abnormally high in proximal oesophagus in 9 (60%) and abnormally high in the distal oesophagus in 11 (73%) and abnormal at both levels in 8 (53%). most refl ux events in asthmatics occurred in the upright position. conclusion asthmatics have more gor and associated respiratory symptoms than non-asthmatic volunteers, with refl ux episodes preceding respiratory symptoms in most cases. distal gor and upright acid exposure was more prominent than proximal gor. in 1999, fi rst population-based studies to determine the magnitude of the asthma problem have been carried out in bangladesh, to defi ne the prevalence of asthma and to identify the risk factors of asthma in bangladesh. after 10 years, same study carried out to fi nd out trends of asthma in bangladesh. methods a cross-sectional study was conducted from january 1999 to august 1999 on 5642 people and same study carried out from november 2009 to april 2010 on 5256 subjects. data collected from stratifi ed randomly selected primary sampling units of all 64 districts. face-to-face interviews were performed with housewives or other guardians at the household level using a structured questionnaire. results in 1999, the prevalence of asthma (wheeze in the last 12 months) was 6.9% (95% ci: 6.2-7.6) whereas in 2010 it is 7.67% (95% ci: 7.59-7.75). in 1999, prevalence of other asthma defi nitions were: ever wheeze (lifetime wheeze) 8.0% (95% ci: 7.3-8.7); perceived asthma (perception of having asthma) 7.6% (95% ci: 6.9-8.3); doctor diagnosed asthma (diagnosis of asthma by any category of doctor either qualifi ed or unqualifi ed) 4.4% (95% ci: 3.9-4.9). in 2010, ever wheeze (lifetime wheeze) 8.6% (95% ci: 8.52-8.69); perceived asthma (perception of having asthma) 8.31% (95% ci: 8.23-8.40); doctor diagnosed asthma (diagnosis of asthma by any category of doctor either qualifi ed or unqualifi ed) 6.26% (95% ci : 6.20-6.33).the prevalence of asthma in children (5-14 years) was higher than in adults (15-44 years) (7.3% versus 5.3%; odds ratio [or] = 1.41, 95% ci: 1.09-1.82). trends of asthma in bangladesh remains, almost static over last 10 years although at present prevalence is more in adults than children. in adults (15-44 years) all categories were slightly higher than in children (5-14 years) (7.04% versus 6.25%; odds ratio [or] = 1.17, 95% ci: 1.037-1.215). it is found to be significantly higher in house-holds with one to fi ve members than in larger households (or = 1.172, 95% ci: 0.91393-1.50432). the poor two quintiles (or = 1.4598, 95% ci: 1.1386-1.8707) and illiterate group (or = 1.2107, 95% ci: 0.87418-1.65278) and primary level of education (or = 1.4336, 95% ci: 1.10874-1.84769) were more vulnerable to asthma attacks than the highincome group and more educated people, respectively. conclusion asthma has increased from 7 million people to 11 million over last 10 years although prevalence is almost similar. introduction secretory gvpla2 is an inducible protein and an essential signaling molecule for airway infl ammation and airway hyperresponsiveness in immunosensitized and lps-induced ali in mice. however, identifi cation of secretory gvpla2 in human airway diseases has not been identifi ed previously. methods donors were classifi ed as non-asthmatic, asthmatic, copd or ipf from prior medical records. identifi cation of gvpla2 in airway microsections was quantifi ed by immunofl uorescence analysis. expression of gvpla2 in was analyzed using criteria for intensity scoring in a single-blinded method. in separate studies, airway smooth muscle cells (asmc) obtained from asthmatic and non-asthmatic subjects (regional organ bank of illinois) were cultured within 24 h from death. adhesion was assessed by measuring the eosinophil peroxidase activity of adherent eosinophils to asmc. inhibition of adhesion was assessed using neutralizing mabs against surface adhesion molecules and mab against gvpla2. results gvpla2 was abundantly expressed in airway smooth muscle, epithelium and endothelium of patients with a history of asthma compared to non-asthmatic. low expression of gvpla2 was observed in tissues from copd and ipf subjects. in cultured asthmatic asmc, surface icam-1 and vcam-1 also were upregulated. activation of asthmatic asmc with methacholine caused release of gvpla2, which corresponded to augmented eosinophil adhesion; mcl-3g1, a mab against gvpla2, prevented these responses. blockade of surface β1-/β2-integrin on eosinophils or its counter-ligands on asmc blocked also the adhesion. conclusion our data demonstrated that gvpla2 is highly expressed in asthmatic asm but not in patients having, no history of asthma, copd or ipf. gvpla2 secreted from activated asmc augments eosinophil adhesion; mcl-3g1 specifi cally blocked the cell-cell ligation. these data are the fi rst demonstration that the upregulated eosinophil adhesion to the surface of asthmatic asmc is linked directly to the secretory gvpla2. based on our fi ndings, it is likely that the asmc, which is the natural source of gvpla2, regulates airway infl ammation and airway hyperreactivity, which are hallmarks of asthma. supported by nih grant hl-85779 and uk gsk center of excellence in asthma. introduction currently, there is still a lack of operational research analyzing the infl uence of an adequate tb curriculum in medical school. this study, aims to determine the effi ciency of integration of tb program into the medical school curriculum, measured through the knowledge, attitudes, and practices of medical clerks on tb. methods a questionnaire-based survey on the knowledge, attitudes and practices on tuberculosis was conducted among medical clerks (fourth year medical students) in the ust hospital. in total, 240 questionnaires were randomly distributed, of which 199 (83% response rate) of the questionnaires were returned fully accomplished. this was done over a period of one week. the questionnaire used was developed by hong, et al 29; huebner, et al 30; and yu, et al 31. and modifi ed by the present authors. results a total of 94.5% (n-188/199) of the clerks believed that sputum exam and culture are still the standard diagnostic modalities; quadruple therapy for 6-8 months as the standard treatment regimen. in total, 98.5% (n = 196/199) realized the magnitude of the problem of tb in the philippines; half of them rated the directly-observed therapy, short course (dots) program as good. in order to avoid infection, 94% wear masks, 47.7% keep their distance, and only 28.6% open windows. only 12.6% would add two drugs to the current regimen of a patient with suspected drug resistance; while 37.2% responded with adding just one drug. conclusion the integration of tb program in the curriculum of ust medical students is effective in enhancing knowledge and improving attitudes of the medical clerks. however, there is still a need to stress the importance of other practices aside from wearing masks in order to avoid infection, and to clarify issues on drug resistance. the study was undertaken to assess the feasibility of diagnosing pulmonary tuberculosis (ptb) by collecting two sputum samples on a single day (1-day protocol) and to compare the same with the national policy of collecting two samples on consecutive days (2-day protocol). methods five hundred and thirteen individuals with cough exceeding 2 weeks were screened for pulmonary tuberculosis (ptb) by collecting three sputum samples, viz. day-1 spot sample, sample collected 1 hour after the fi rst sample and next day morning sample. for the 2-day protocol, performance of the fi rst and third samples were considered, while the 1-day protocol was evaluated using the two day-1 samples. staining and microscopy were undertaken by two different technicians in a blinded manner. results out of 513 patients, 40 patients defaulted on second day. of the total number of patients recruited, 124 (24.2%) were smear-positive. the 2-day protocol was capable of detecting 121 patients (97.6%), whereas in the 1-day protocol 118 patients (95.2%) were smear-positive (p = 0.3). of the drop-out patients, 7 (17.5%) were smear-positive. comparing the variation in results between spot and morning samples, collection of morning sample exhibited no signifi cant benefi t over the collection of a second spot sample. conclusion because the 2-day protocol does not lead to a statistically signifi cant diagnostic difference compared to the 1-day protocol, the latter can be adopted as an alternative protocol, particularly in subjects who are more likely to default. introduction tuberculosis, an important preventable and treatable cause of death, is a major health problem worldwide. detecting patients with active pulmonary tuberculosis is an important component of tuberculosis control as early appropriate treatment renders these patients noninfectious and interrupts the chain of disease transmission. sputum microscopy remains the test of choice as initial work-up for symptomatic patients with tuberculosis. however, in patients with a compatible clinical picture, sputum smears do not always reveal acid-fast bacilli. patients symptomatic for tuberculosis but are found to be smear-negative are recommended to undergo further tests including fi beroptic bronchoscopy and sputum induction. the latter, however, is invasive and more costly. this study aims to compare the sensitivity and specifi city of sputum induction and bronchscopy in the diagnosis of sputum smear-negative tuberculosis by means of meta-analysis. methods computer search was done to obtain studies meeting inclusion criteria. the sensitivity, specifi city and other measures of accuracy were pooled using forest plots. diagnostic odds ratios were obtained. summary receiver operating characteristic curves were used to summarize overall test performance. funnel plots and egger regression analysis were used to examine for publication bias. results five prospective studies comparing diagnostic accuracy of fi beroptic bronchsocopy and sputum induction to diagnose sputum smear negative tb were obtained. the pooled summary indeces showed that for bronchial lavage, the sensitivity is 0.49 (95% ci, 0.42 to 0.59) while specifi city is 0.79 (95% ci, 0.75 to 0.84). whereas for sputum induction, the sensitivity is 0.47 (95% ci, 0.40 to 0.54) and specifi city is 0.83 (95% ci, 0.79 to 0.87). the summary dor for bronchial lavage was 3.27 (95% ci, 2.17 to 3.92) while the summary dor for sputum induction was 429 meaning sputum induction test had a higher level of overall accuracy (95% ci, 291 to 634). conclusion sputum induction has comparable sensitivity and specifi city and higher level of overall accuracy compared to bronchial lavage in diagnosing for sputum smear negative tuberculosis. introduction much of tuberculosis control is based on the current understanding of factors that infl uence transmission of mycobacterium tuberculosis and that lead to active tuberculosis among persons who acquire the infection. one of these activities, contact investigation, is intended to identify persons who have acquired tuberculosis infection from a newly discovered active case, thereby enabling targeting of preventive treatment to a group at high risk of developing active tuberculosis, this being the main goal of activity. methods the charts of close contacts of mdrtb patients enrolled in the programmatic mdrtb management of lcp phdu from january 2008 to june 2009 was reviewed. results there were 920 contacts of 244 culture and dst-confi rmed mdrtb patients identifi ed from january 2008 to june 2009. among these contacts, 733 (78.8%) were traced and underwent evaluation and screening tests. among the contacts >5 years old, 193 (23.83%) had a positive chest x-ray, 25 (3.95%) were afb +, 18 (2.80%) were positive for mtb culture and sensitivity. in contacts <5 years old, 42 (38.2%) had positive chest x-ray results, but none had positive results on afb smear, and mtb culture and sensitivity. tuberculosis was identifi ed in 42 of contacts <5 years old and 75 of contacts >5 years old. there were 10 mdrtb cases identifi ed (8 confi rmed by culture and dst, and 2 treated empirically), all from contacts >5 years old. all identifi ed mdrtb cases were treated with category iv regimens under pmdt, while other tb (non-mdrtb) cases were managed under dots. conclusion contact tracing remains a helpful tool in public health programs at the lung center of the philippines. although the average contact per index is 3.8, 78.8% were successfully traced, which is comparable to other studies abroad. among the screening tools, the chest x-ray was the most commonly utilized and also the most productive; afb smear, tuberculin test, and mtb culture were performed in less than 15%. among identifi ed contacts, mdrtb was noted in 1.57%. introduction active pulmonary tuberculosis (tb) requiring intensive care is rare but known to be of poor outcome. the present study aimed to describe the characteristics of patients with this condition and to identify the mortality rate and risk factors that predicts in-hospital mortality. methods from january 2008 to december 2009, 55 patients were admitted to tuberculosis intensive care unit (tbicu) of mackay memorial hospital, taipei, taiwan. among these, 38 patients were enrolled and were followed up for 180 days. incidence of in-hospital deaths was documented in the medical records and all possible parameters contributing to mortality were collected for analysis. results the patients' median age was 73 years (range 28-90 years). the median length of intensive care unit stay was 15 days (range 2-172 days) and the median duration of mechanical ventilation was 13 days (range 0-172 days). overall in-hospital mortality was 63% (24/38). sepsis and shock were independently associated with in-hospital mortality. conclusion these data indicated a high mortality of patients with active tuberculosis requiring intensive care, especially in those with sepsis and shock. introduction we have shown that two commonly used prediction model s (va and mayo) estimate poorly the probability of malignancy of solitary pulmonary nodules (spn) in the philippines. this is due to a large proportion of spn arising from tuberculosis (tb). in the philippines, and possibly for other countries with a high tb-burden, our clinical prediction model has a better estimate to the probability of spn than both the va and mayo. methods we developed a prediction model to identify malignant lung nodules based on clinical data and radiographic characteristics among patients with spn identifi ed retrospectively (october 2006 to march 2008) in our institution. univariate and multiple logistic regression analysis were used to identify independent clinical variables. we applied the model to a new set of spn patients (april 2008 and august 2008) and described its accuracy by comparing the predicted probability of malignancy with the fi nal diagnosis. we constructed receiver operating characteristic (roc) curves and reported 95% confi dence interval. calibration was done by dividing the sample into fi ve equal groups based on predicted probability and plotting the median probability of each quintile against the observed frequency of malignancy for that group. results seventy-six spn patients were included in the development of the prediction model, where size, margin and smoking history were found significant in the multivariate analysis. prevalence of malignancy was 33%. the area under the receiver operating characteristic (roc) curve was 0.92; 95% confidence interval (ci) of 0.77-0.95. the equation was obtained based on the identifi ed predictors. fifty-eight patients with spn were included in the validation sample. prevalence of malignancy was 36%. the roc curve was 0.91; 95% c.i. of 0.85-0.97. median predicted probabilities in all quintiles were lower than the observed frequency of malignant nodules, probably refl ective of the validation sample's higher prevalence of malignancy. conclusion the local clinical model appeared to be suffi ciently accurate to inform clinical decisions about the choice and interpretation of subsequent diagnostic tests. the accuracy of the local clinical prediction model was similar to that reported in its development. introduction in a high-burden country for pulmonary tuberculosis like philippines, it's not uncommon for intracthoracic masses be treated empirically with anti-tuberculosis regimen. we aim to describe patients' profi les, determine outcomes of empiric anti-tuberculosis treatment for such lesions. methods we monitored patients with intrathoracic mass given empiric antituberculosis regimen until "end-of-treatment," decision to pursue diagnosis, or mortality. a 1-year prospective, observational, open-label, descriptive, cohort study, in a tertiary government hospital. percentage association analysis was done at end of the study. results in total, 20 patients presenting with intrathoracic mass lesions on chest x-ray/chest ct scan were treated empirically with anti-tuberculosis medicines without histopathologic evidence suggestive of pulmonary tuberculosis for the mass. patients' choices, clinical and fi nancial status were factors considered by physicians in the decision for empiric treatment. there were 15 males, 5 females, with average age 47 years. most common chief complaints were cough (50%), pain (20%), hemoptysis (15%), shortness of breath (15%). 11 patients had pulmonary consult within 3 months of initial radiography. histology of mass was confi rmed within 3 months of pulmonary consult in 12 patients. 4 patients had the histopathology prior to starting empiric anti-tb treatment, which revealed non-specifi c fi ndings. a total of 16 patients were treated empirically prior to attempts for histologic diagnosis. two of these patients never had diagnostics due to fi nancial constraints. while 14 patients went on to pursue histopathology, which revealed underlying malignancy in eight patients. malignancy was seen more on males, older age (≥40 years), signifi cant smoking history, larger mass size (∼7-10 cm). seven patients had clinical/radiographic improvement, two patients died, three were lost to follow-up. conclusion our study suggests no role for empiric anti-tb treatment for intrathoracic masses, even in a high-burden country like philippines. we should vigorously pursue and search for defi nite histological diagnosis, as it will translate to cost-effectiveness, avoidance in delayed diagnosis, early institution of appropriate therapeutic management. we recommend further studies with larger sample size, to characterize patients' profi les, do subset analysis, identify who may need anti-tuberculosis treatment. introduction the use of viruses as targeted cancer therapy has shown signifi cant promise for novel anticancer therapy. actually, a small number of enteroviruses, such as coxsackievirus a (cva) and echovirus, have been reported to possess oncolytic activities against various human malignancies. however, a single intratumoral administration of cva in vivo induces severe progressive muscle paralysis necessitating euthanasia of mice. in this context, we discovered that coxsackievirus b (cvb) displayed a high level of tropism and lytic activities for human lung cancer cell lines as a result of screening of representative 39 enteroviruses. cvb specifi cally destroyed both human non-small and small cell lung carcinoma via surface virus receptors of coxsackievirus and adenovirus receptor (car) at a multiplicity of infection (moi) of 0.001, whereas it did not destroy normal lung cells at even a higher moi of 10. the mts cell proliferation assay also supported those results. furthermore, our in vivo study showed that consecutive intratumor injections of cvb remarkably inhibited the growth of subcutaneously pre-established lung tumors, with signifi cantly more increased survival than untreated mice (p < 0.001). surprisingly, in metastatic tumor mice model, intratumoral cvb injection into primary tumors in the right fl ank also signifi cantly retarded the growth of pre-established contra-lateral tumors compared with untreated mice. according to the results of fragmented parp detection assay, the oncolytic effects of cvb against tumors could be partially attributed to their apoptosis as well as cellular degenerative destruction. furthermore, fl ow cytometric analyses showed that cvb could possess an immuno-stimulatory ability through robust infi ltrated dendritic cells maturation in treated tumors. moreover, none of mice died of cvb administration, suggesting the feasibility of clinical trials in the future, although analyses of serum biochemistry revealed moderate hepatic dysfunction due to cvb administration. conclusion our fi ndings suggest that intratumor cvb administration could be a novel therapeutic modality against not only primary human lung cancer but also metastasized lesions. introduction radio frequency ablation (rfa) is a technique that employs high-energy radio frequency waves to destroy non-small cell lung cancer. the radio frequency ablation probe, le-veen multiple array needle electrodes, is placed inside a tumor and opened like a tiny umbrella with 10 curved prongs that spring into the surrounding tumor tissue. with this tool tumor cells are somewhat heated until they boil and become inert. methods patients with medically inoperable or unresectable single nodule nsclc underwent treatments, in 3 different centers of bangladesh. multimodality treatment was mode of management. on the basis of intention to treat, patients were divided into fi ve groups for fi ve mode of treatment. group 1: percutaneous rfa (n = 120); group 2: rfa followed by radiotherapy (n = 80); group 3: chemotherapy with rfa (n = 86); group 4: radiotherapy alone (n = 65); and group5: chemo radiation (n = 35) during 9-year period (2000-2009). patients' characteristics, local recurrences and overall and disease-free survivals were compared. results in total, 386 patients were selected for study since december 2000. mean size of tumors were 5 ± 3.1 (range 2.3-8.2 cm). follow-up range was from 2 to 52 months, median 23.5 months. survival rate of group 1: only percutaneous rfa was 83% at 1 year, 63% at 2 years and 42% at 3 years; for group 2: rfa and ebrt 96% at 6 months, 80.8% at 1 year, 62.4% at 2 years, and 51.1% at 3 years; group3: patients treated rfa with chemo therapy 80% at 1 year, 63% at 2 years and 52% at 3 years; group 4: with radiotherapy alone 55% at 1 year, 33% at 2 years, and 20% at 3 years; for group 5: similar patients treated with chemoradiation 61% at 1 year, 45% at 2 years and 22% at 3 years. irrespective of stages, patients with tumor size 5 cm (n = 25) had an average survival 17 ± 5 months. local recurrence occurred in 2.8% having tumors size 5 cm. 19 developed pneumothorax and 35 had lung infections, 7 of them had fetal. a total of 24 patients died of co morbid diseases while 77 died of disease progression within 3 years following rfa and ebrt or chemotherapy or rfa alone. conclusion the rfa followed by ebrt or rfa along with adjuvant or neo adjuvant chemotherapy for inoperable nsclc has a relatively low rate of complications that are easily managed and above all survival has improved compared with other combination therapy, i.e. chemoradiation. nb: rfa plus chemotherapy was only applied in stage ii and iii. introduction patients with chronic or debilitating illness such as lung cancer usually accompanied by some form of emotional responses such as denial, anxiety, and depression. clinician should be aware of these condition for better lung cancer management. methods hamilton rating scale for anxiety and depression questionnaire were administered to all patients diagnosed with lung cancer in dept pulmonology-respiratory medicine university of indonesia/persahabatan hospital consecutively. follow-up evaluation will be done to evaluate anxiety/depression after a 3-month therapy. results among 30 lung cancer patients , 14 (46%) has anxiety, 12 patients (40%) has depression and all patients with depression also has anxiety. these conditions commonly found in male than female (10 out of 24 male patients (42%) vs. 2 out of 6 female patients (33%)). further evaluation are underway to evaluate the degree of these disorders and other factors correlate with these disorder. conclusion depression and anxiety were also found in lung cancer patients and need further evaluation and attention from clinician. to compare the preoperative classifi cation of lung carcinoma made on histological specimens by fiberoptic bronchoscopy(fob)with the postoperative classifi cation made on resected specimen and how often was used term of nsclc. methods we reviewed the records 116 patients who had a diagnosis ofthe lung cancer made by fi beroptic bronchoscopic biopsy (at yedikule chest hospital, istanbul in 2009) and who underwent a lung resection.postoperative histological classifi cation were made according to the who classifi cation of the lung tumours. results fifty one of 84 squamous carcinoma, 1 of 13 adenocarsinoma and 8 of 9 carcinoid tumours were correctly typed with the small biopsy obtained by fob. forty eight patients who had a diagnosis of lung cancer established by fi breoptic bronchoscopy were labelled as nsclc, 69%, 25% and 6% of them were classifi ed squamous carcinoma , adenocarcinoma and other tumour type respectively with examination of tissue obtained by surgical resection. conclusion accurate cell typing by specimens obtained at fi breoptic bronchoscopy may be extremely diffi cult.if clearcut morphological criteria can not be satisfi ed , the diagnosis of "lung cancer ,non-small cell" type should be made. introduction the nsclc patients who experienced good clinical responses even sometimes dramatic responses to egfr-tkis gefi tinib or erlotinib will inevitably develop acquired resistance. however, the clinical defi nition of acquired resistance is not clear. we investigated the clinical characteristics of acquired resistance to gefi tinib in nsclc retrospectively. methods we analyzed four hundred and forty nsclc patients who had taken gefi tinib more than 6 months duration. all clinical data were obtained from 11 centers of korean molecular lung cancer group (kmlcg). the timing, clinical manifestations, and the association of egfr genotype were analyzed in the aspect of acquired resistance development. the mean duration of gefi tinib prescription was 12.3 + 7.1 months. signifi cant predominance in female (63.2%) and non-smoker (68.9%) was noted. among the patients who examined egfr genotype, the mutation rate was 54.0 % (68/126), relatively lower than expectation. the relative ratio of local vs. systemic progression is 62.8%:38.2% and symptomatic progression rate is 63.8%. the survival time after the development of acquired resistance is 9.3 + 9.5 months. conclusion these retrospectively analyzed clinical data for the development of acquired resistance to gefi tinib will help set up the clinical defi nition of acquired resistance to egfr-tki. introduction numerous studies have documented overall effectiveness and safety of chemical pleurodesis using variety of agents. although reports regarding complications post-talc pleurodesis were minimal, concerns on the adverse effect profi les remains, especially on occurrences of serious and life threatening respiratory insuffi ciency and ards following talc pleurodesis. methods records of patients admitted at lcp who underwent talc pleurodesis from january 2008 to december 2009 were reviewed and all complications post pleurodesis were noted. results a total of 96 charts of patients admitted at lcp who underwent pleurodesis from january 2008 to december 2009 were reviewed. the mean age was 57 ± 14 y/o with male predominance compared to female at 65 (67.7%) and 31 (32.3%) respectively. of the total procedures evaluated, 43 (44.7%) involved all post procedure complications, 30 (31.25%) patients developed minor complications while 13 (13.54%) had major complications. there was no statistically signifi cant association noted with age, sex, smoking history, co-morbid illness, underlying disease, and method of pleurodesis, while chest tube drainage time more than 7 days was noted to be associated with greater incidence of major complications which was statistically signifi cant. most common minor complications were fever (21.9%), followed by tachycardia (20.8%), chest pain (13.5%) and dyspnea (9.4%). the top 3 major complications were hypoxemia, hypotension and pneumonia. there were 2 (2.1%) patients who died post pleurodesis that is believed to be related to ards following talc slurry. conclusion talc pleurodesis is an effective agent for chemical pleurodesis but not without adverse effects. cardiovascular complications are potentially avoidable by proper patient selection and preparation prior to talc pleurodesis. respiratory failure and ards are rare but serious complications that should be promptly recognized and addressed rapidly and effectively. introduction nebulized antibiotic is an established safe and effective therapy for bronchiectasis. gentamicin are considered among the most useful classes of antibiotics for treating pseudomonas aeruginosa infections.the major drawback of aminoglycosides is the need for their relatively high dose intravenous administrations which carries the potential systemic toxicity.when gentamicin is given intravenously in maximum safe doses, only relatively low sputum concentration are achievable. these limitations can be circumvented by direct delivery of aerosolized antibiotic to the airways. methods this study was carried out in nidch dhaka. in total, 65 patients were taken initially for the study. introduction immune-modulator nutrition may decrease mortality among patients who are mechanically ventilated due to severe community acquired pneumonia (cap). methods we compared an immuno-modulator nutrition and standard feeding formula to determine the effect on in-hospital mortality as well as 30 days mortality among mechanically ventilated patients due to severe cap. the mean number of ventilator days, icu stay, as well as clinical parameter (clinical pneumonia infection score (cpis) and pao 2 /fio 2 ratio from arterial blood gas) was also compared. in total, 38 eligible patients were randomized, double blind, to receive either immuno-modulator nutrition (supportan sp) or standard feeding formula (sf). follow-up was done on day 1, 4 and 7 on cpis and pao 2 /fio 2 ratio. results primary outcome was mortality. no signifi cant difference noted between the two groups (p = 0.64; 95% ci: −0.18 to 0.28). the 30 day mortality on 32 patients revealed 2 patients (12.5%) died on sf group and 1 patient (6.25%) on sp group (p = 0.52; 95% ci: −0.14 to 0.28). the mean ventilator days on the sf group and sp group was 7.44 days and 5.47 days respectively (p = 0.15; 95% ci: −0.75 to 4.69), although not signifi cant, it suggests a trend favoring sp group. the mean icu stay in the sp group was noted to be signifi cantly shorter (7.05 days) than in the sf group (10.84 days) (p = 0.04; 95% ci: 0.19 to 7.48). the cpis and pao 2 /fio 2 ratio were done on day 1, 4 and 7. on day 1, the mean pao 2 /fio 2 ratio was still signifi cantly higher on the sp group (p = 0.0001; 95% ci: −93.54 to −36.79); while the mean cpis was still the same with baseline (p = 0.43; 95% ci: −0.58 to 1.32). on day 4, no significant difference was noted (p = 0.63; 95% ci: −109.85 to 66.87 and p = 0.46; 95% ci: −1.26 to 2.18, respectively), as well as those on day 7 (p = 0.47; 95% ci: −81.71 to 168.05 and p = 0.47; 95% ci: −2.07 to 4.67, respectively). conclusion we found no difference on mortality between sf and sp group. however, it suggests trend of earlier extubation and signifi cant shorter in icu stay, in patients who received immuno-modulator nutrition. introduction several equations to predict lung function of individuals of different population are available. however it is desirable that lung function laboratories use reference equations that most closely describe the population they test. the objective of the study was to develop a prediction equation for the malaysian population. methods spirometry was performed on a total of 1483 "healthy", lifetime non smoker volunteers (386 males and 1097 females) all measurements met the ats acceptability and reproducibility criteria. prediction equations were derived for both men and women for fvc and fev1. the equations were validated on a new group of subjects (n = 532, 222 males and 310 females) who met the same inclusion and exclusion criteria as the main cohort. introduction patients with severe asthma (experiencing previous hospital admissions and/or daily symptoms) have occasionally been seen with poor or weak complaints. several studies have analyzed the psychiatric status of the patients with severe asthma, but few studies have the japan respiratory society (jrs) has proposed new predicting scores, called the i-road system for hospital acquired pneumonia (hap) in 2008. depending on the presence of the parameters listed below, patients with hap were stratifi ed into those with high, moderate or low-risk. the high-risk group was defi ned as patients with three or more of the following risk factors: 'malignant tumor or immunocompromised status', 'impaired consciousness', 'requiring fraction of inspired oxygen (fio 2 ) > 35% to maintain spo 2 > 90%', 'male aged 70 years or older, or female aged 75 years or older' and 'oliguria or dehydration'. the moderaterisk group was defi ned as patients with any of the secondary risk factors as follows: 'crp ≥ 20 mg/dl' and 'extent of infi ltration on cxr covers at least 2/3 of one lung'. the low-risk group was defi ned as all other patients. the aim of this study was to confi rm whether i-road is useful in predicting severity of cap and hcap. methods all patients with an admission diagnosis of cap and hcap from january 2009-july 2009 were reviewed. clinical and laboratory features at presentation in electrical medical records were used to calculate severity scores using the curb-65 (2004), a-drop (2005) and i-road (2008). results consecutive 314 patients (53% cap) of mean age 77.5 years were included in the analysis. nineteen (11.7%) patients with cap and seventeen (11.7%) patients with hcap died. the roc analysis for predicting mortality at 30 days showed that i-road score has similar predictive accuracy for short-term mortality to curb-65 and a-drop in patients with cap, but shortterm mortality of the patients with hcap are not similar to them. conclusion the jrs i-road could be used to assess severity of cap, and gives similar results to curb-65 and a-drop. os 08-05 introduction numerous asthma and copd patients repeatedly return to hospital ed for urgent therapeutic care despite referral to their primary care provider. this study was aimed to identify these respective populations with frequent ed visits and assess the current therapeutic management of acute exacerbation of asthma and copd at hospital kuala lumpur, malaysia. methods the demographic and medical data was prospectively collected and recorded in march 2010 using convenient sampling, and then descriptively analyzed using spss version 16. appropriate statistical analysis were applied with p < 0.05 was considered as signifi cant. the study recruited 126 patients (male 51.6%) with 87.3% asthmatics and 12.7% copd. malays signifi cantly presented to ed the most (65.1%) followed by indians (25.4%) and chinese (7.1%). most patients were between 17-64 years old (72.2%) with mean age of 41.5. about 17.5% were smokers (16.7% ex-smokers) with an average duration of smoking 26.73 years and 21.95 pack-years. for occupational data, 52.5% were belonging to non-professional group. about 85.5% asthma and 87.5% copd patients had visited ed last year with average visit of 3.44 and 7.3 times and mean number of hospitalization was 1.68 and 1.33, per year, respectively. about 51.6% patients without scheduled appointments and 15.1% were not on any prescribed medications for asthma or copd. among patients with prescribed medications, 99.1% were on saba and 66.4% on inhaled corticosteroids. respiratory infections remained as main triggering factors of admission (43.7%), followed by weather (30.2%) and air pollution (5.6%). average duration of treatment was about 50 minutes with mean direct therapy cost of rm15 per patient excluding the standard admission fee. whil hr monitoring, the level of oxygen saturation and pefr were signifi cantly improved post treatment. oxygen-driven nebulised saba and iv hydrocortisone were the mainstay of treatment. however, the use of an anticholinergic as a step up approach in nebulised treatment was underused. most discharged patients were given oral saba (87.3%) and prednisolone (88.1%). pefr measurement was not practiced post treatment regularly. conclusion limited number of staff contributed towards omitted monitoring steps. involvement of ed pharmacists in respective therapeutic management is highly suggested. introduction fluid volume is an important factor when considering pleural drainage. however, there is limited literature regarding accurate quantifi cation of pleural effusion by ultrasonography. in a study by visperas et al, they quantifi ed the pleural effusion volume by measuring the length, width, and depth of the fl uid, while the patient was in a sitting upright position, and the actual volume drained. they postulated a linear regression equation to quantify estimate the pleural fl uid volume that will be drained. introduction insomnia disorder is the most common sleep disorder which affected more than 18 300 000 people in bangladesh. 7 600 000 people develop chronic insomnia disorder with symptoms of diffi culty falling asleep for more than 30 minutes and last for more than 1 month. patients with insomnia disorder in bangladesh took 2 900 000 sleeping pills each year for insomnia disorder. however, there were still some side effects about sleeping pills such as allergy, amnesia, hallucination etc. because of the side-effects of modern medicine and because of the inability of modern medicine to cure insomnia disorder, international scientifi c interest has re-focused on the traditional uses of medicinal plants to fi nd effective cure for insomnia disorder as well as hundred of other disorders. a study of the traditional health practitioners in the habiganj district of bangladesh suggested that some of the herbal medicines prepared from medicinal plants might be quite effective for insomnia disorder. methods information was collected through a series of interviews with the traditional health practitioners, rural and urban people. field notes were recorded on the medicinal plants and their uses; following the methodology of bhat et al. (1990) and martin (1995) . the identifi ed medicinal plant specimens were stored at the bangladesh national herbarium; under the author's collector series. results the following medicinal plants or plant parts were found to be used as remedy for insomnia disorder: cyrtandra cupulata ridl., bacopa monnieri (l.) pennell, ocimum gratissimum l., lawsonia inermis l., cinnamomum camphora (l.) sieb., aconitum napellus l., datura metel l., mimosa pudica l., achyranthes aspera l., piper betle l., randia dumetorum (retz.) poir., ficus glomerata roxb., nigella sativa l., agaricus albolutescens zeller, ipomoea aquatica forssk., stephania japonica (thunb.) miers, withania somnifera (l.) duna, cannabis sativa l., calamus rotang l., uraria picta (jacq.) dc., sesamum indicum l., asparagus racemosus willd., abrus precatorius l., and brassica napus l. conclusion information on indigenous use of medicinal plants has led to discovery of many medicines in use today. it is important that modern scientifi c studies be conducted on these medicinal plants towards isolation and identifi cation of compounds through which insomnia disorder can be effectively treated. introduction existing data on the association between sleep duration and blood pressure in adolescents are inconsistent and confl icting. this study aims to determine the relationship between sleep duration and 24-hour ambulatory blood pressure in adolescents. methods subjects aged 10-17.9 years were recruited from the community. they underwent nocturnal polysomnography (psg) and 24-hour ambulatory blood pressure monitoring (abpm) on the same day. daytime, nocturnal and 24-hour average systolic and diastolic blood pressure (sbp and dbp) were converted to z score with reference to height and gender according to normal reference. a 7-day sleep diary was completed prior to psg. daily sleep duration was defi ned as the average of nocturnal sleep duration plus the average of daytime nap duration over 1 week. subjects with body mass index (bmi) greater than the 85th percentile of the local normal reference were classifi ed as overweight. those who were overweight and had an obstructive apnoea hypopnoea index (oahi) greater than or equal to 5 were excluded from the analysis. results one hundred forty one subjects (86 boys) with a mean (sd) age of 14.3 (1.8) years were recruited. they were divided into 4 groups according to their daily sleep duration (<7.5 vs. 7.5-8.5 vs. 8.5-9.5 vs. > 9.5). subjects with shorter sleep duration tended to have higher daytime sbp (p < 0.001), nocturnal sbp (p = 0.002) and 24-hour sbp (p < 0.001). similar results were found after converting the bp data into z score (p = 0.005, 0. introduction some studies have shown decreased plasma and hair zn in human asthmatics which may indicate a state of zinc defi ciency. we have shown, in a well characterized murine model of allergic airway infl ammation, that there were marked losses of zinquin-stainable (labile) zn in the infl amed airway epithelium (ae); when these mice were placed on low zn diets, there was excessive cell death in the ae, and increased airway infl ammation. we have proposed that labile zn protects the ae from premature cell death and loss of this zn contributes to the ae fragility and infl ammation in asthma. a screen of whole lung gene expression of the two major families of zip infl ux transporters and znt effl ux transporters, indicated a marked loss of znt4 in the infl amed lungs. the hypotheses being tested here are 1) ae zn is normally stored in vesicles within the apical cytoplasm, 2) znt4 is responsible for transporting zn into these bodies and 3) loss of znt4 expression in asthma may result in a failure of zn to be sequestered. methods human nasal epithelial brushings were obtained from healthy, consenting donors. lung tissue was obtained from balb/c mice sensitized and aerosol-challenged with ovalbumin or saline (controls). distributions of zinc were determined by zinquin fl uorescence or in vivo selenite autometallography (se-amg). distributions of znt4 were determined by immunofl uorescence. results zinquin fl uorescence of human and murine ae indicated abundant labile zn with a vesicle-like pattern of staining in the apical cytoplasm. se-amg confi rmed the presence of apical zn vesicles at an ultrastructural level. there was strong immunolabelling for znt4 in the same region. furthermore, there was an almost complete loss of znt4 protein in the bronchial epithelium of mice with allergic airway infl ammation. conclusion colocalization of znt4 with labile zn in ae may indicate a role for this zinc transporter in replenishing zn storage pools in this tissue. loss of znt4 protein during airway infl ammation would then result in failure to sequestrate zn, depleting critical storage pools of zn in the lung and airways, leading to increased ae damage and cell death. this work was supported by nhmrc project grant 519206. introduction community-acquired pneumonia remains a major cause of mortality in developed countries. there is much discrepancy in the literature regarding factors infl uencing the outcome in the elderly population. methods data were derived from a multicentre prospective study initiated by the german competence network for community-acquired pneumonia. patients with community-acquired pneumonia (n = 4,647; 2,298 aged < 65 years and 2,349 aged ≥65 years) were evaluated, of whom 71.4% were hospitalised and 28.6% treated in the community. clinical history, residence status, course of disease and antimicrobial treatment were prospectively documented. microbiological investigations included cultures and pcr of respiratory samples and blood cultures. factors related to mortality were included in multivariate analyses. results the overall 30-day mortality was 6.6%. elderly patients exhibited a signifi cantly higher mortality rate that was independently associated with the following: age; residence status; confusion, urea, respiratory frequency and blood pressure (curb) score; comorbid conditions; and failure of initial therapy. increasing age remained predictive of death in the elderly. nursing home residents showed a four-fold increased mortality rate and an increased rate of gram-negative bacillary infections compared with patients dwelling in the community. conclusion the curb score and cerebrovascular disease were confi rmed as independent predictors of death in this subgroup. age and residence status are independent risk factors for mortality after controlling for comorbid conditions and disease severity. failure of initial therapy was the only modifi able prognostic factor. introduction hospital-acquired pneumonia (hap) attributes to 15% of all nosocomial infections. mortality rate is as high as 30-70%. guidelines for the management of adults with hap were recently updated. despite the emergence of evidence-based medicine, the use of these guidelines in daily clinical practice is still limited. currently, there is no literature published regarding the impact of adherence to the guidelines and clinical outcomes of hap. methods a total of 40 patients (male: 50%; female: 50%) admitted and diagnosed with hap at our center were followed up to investigate the rate of adherence of physicians on the diagnosis and treatment of hap based on level i and ii ats/idsa 2008 recommendations and to determine its association with outcome (mortality, mechanical ventilation, icu stay, hospital stay). adherence to diagnostics and therapeutic management were computed per patient. management of patients was classifi ed as adherent if it meets more than 70% of the guidelines that should be enforced. results in this cohort, 55% of the physicians adhered to the currently recommended guidelines. age, gender, and co-morbid conditions such as hypertension, diabetes mellitus, copd, ckd and cerebrovascular disease were not statistically associated with the outcome of the study. a total of 30% of the subjects were eventually mechanically ventilated (p = 0.525). a total of 5% of patients who adhered to the recommendations consequently died during hospitalization (p = 0.073). similarly, univariate analysis of variance revealed that there is no signifi cant association of adherence to length of icu stay and hospital stay (p = 0.807, 0.802 respectively). of the level i and ii current recommendations, request of blood culture showed signifi cant association with adherence (p = 0.045). however, logistic regression analysis showed that there is no association of adherence in doing blood culture to mortality. conclusion this analysis showed that compliance with the currently recommended ats/idsa recommendations is 55%. blood culture is the most signifi cantly associated recommendation. rate of endotracheal intubation, length of icu and hospital stay and mortality however was not signifi cantly associated with adherence. introduction reportedly high arsenic level in drinking water causes increased mortality and morbidity in adult copd patients. arsenic related health hazards include respiratory symptoms with decreased lung function added to skin lesion. currently 70 million people of bangladesh are at potential risk of consuming arsenic contaminated drinking water and a major section of them showed many symptoms including alteration of lung function. methods the present study was conducted on chronic arsenicosis patients in selected areas of bangladesh to assess lung function status by measuring fvc, fev1, fev1/ fvc% & pefr. in total, 120 subjects of 20-50 years of age of both sexes were selected. 40 apparently healthy subjects were selected from non arsenic residency as well as not exposed to arsenic in their tube-well water and were grouped as healthy control. of 80 subjects from area exposed to arsenic contaminated tube-well water, 40 were patients of chronic arsenicosis with skin lesions were considered as experimental group, whereas 40 subjects without skin lesions were regarded as exposed control. results the mean measured values of the lung function parameters of nonarsenic exposed healthy control and exposed control were within normal ranges. but these values were signifi cantly lower in chronic arsenicosis patients with skin lesions. the parameters showed negative correlation with age, arsenic concentration in tube-well water but positive correlation with duration of the consumption. but these relationships were not statistically significant. all the patients of arsenocosis complained about respiratory symptoms in the morning. conclusion the present study reveals that arsenicosis patients are suffering from respiratory insuffi ciency and symptomatic respiratory illnesses. in addition, populations consuming higher arsenic concentration in drinking water are at the risk of lung function impairment and ultimately may lead to respiratory disorders, though it would be better to draw a defi nite conclusion from a further study involving large sample size. introduction despite of the detailed study of community-acquired pneumonia, the role of atypical microorganisms such as m. pneumoniae, c. pneumoniae and l. pneumophilla is not still defi ned. also there are some discussions about role of the associations of these bacteria with the other so-called typical microorganisms as s. pneumoniae and h. infl uenzae as well as the place of the viral pathogens in community-acquired pneumonia ethiology structure. the aim of our research was to defi ne the etiology of the community-acquried pneumoniae in young adults (17-34 years, fi rst group) and to compare the results with the data gained in aged patients (<60 years, second group). methods the 300 young and 300 aged patients with community-acquired pneumonia were screened with bacteriological, disk-diffusion with mic, pcr and other methods. antimicrobial agents resistance was checked to nccls standards and the clonality of the isolates was checked by pfge and mlst for the most frequent clones. results bacterial associations were defi ned in 55% versus 72% in the second group. m. pneumoniae was identifi ed in 39% vs 19%, c. pneumoniae 33.4% vs 24%. the bacterial pathogens were represented with the species s. pneumoniae (58.44%/42%), h. infl uenzae (15.06%/21%), m. catarrhalis (26.23%/7%). among the viral pathogens the most often was metapneumovirus in young adults (23%), and infl uenzae virus in aged patients (18%). the most prevalence bacteria were genotyped and there were revealed the relations between several isolates of m. pneumoniae and s. pneumoniae existing as association in several cases of different age groups what proved the epidemiological character of the spread of this association. so, the most frequent clone of s. pneumoniae was recognized as st156, as well as st214. pfge typing of atypical microorganisms also revealed the spread of the several clones. conclusion some changes in etiology structure of community-acquired pneumonia seems to be connected with the changes in immunology peculiarities of different age groups, as well as with the other epidemiology reasons. introduction despite of the detailed study, the role of acinetobacter baumanii pathogen as the a ubiquitous opportunistic nosocomial pathogen is still appreciated. the most of epidemiological aspects of this infection are still discussed though the problem of the microbiology charecteristics of this pathogen are of keen microbiology interest. it is often isolated in immunocompromised hosts in different forms of hospital-acquired infections, but more often it was recognized as the main pathogen agent of hospital-acquired pneumonia. the aim of our research was to establish clinical signifi cance of a. baumanii in development of hospital-acquired pneumonia, to defi ne its epidemiology and to characterize antimicrobial agents resistance pattern. methods we made 1-year surveillance of all hospital-acquired pneumonias (hap) in adult patients in the main clinics of vladivostok (hospital ð1, ð2), defi ned etiology with standard microbiology methods. all isolates of a. baumanii were tested for antimicrobial agents resistabce according to nccls. the strains with the same antimicrobial agents resistance pattern were checked to clonality by pulsed-fi eld gel electrophoresis (pfge). results during 2008-2009, we studied all cases of hap in 450 adult patients (<60 years) admitted to icu and revealed that a. baumanii has taken the second place in etiology structure (19.1%, 94 cultures from 86 patients). the fi rst place was in pseudomonas aeruginosa (29.3%, 132 strains) and the third one was in stenotrophomonas maplthophila (11.7%, 53 strains). mostly (58 strains, 61.7%), a. baumanii was isolated as monoinfection, but in other cases it was isolated in association with another strains of a. baumanii, or p. aeruginosae, s. palthophila, s. aureus, e. feacalis, e. cloaceae. there were defi ned lower resistance to ciprofl oxacin. the clonality research revealed about 8 genotype clusters what could allow to suggest the genetic relatedness of the isolates. conclusion acinetobacter baumanii should be studied to defi ne the role in hospital-acquired infections, as well as it confi rms the fact that the importance of local surveillance programs in correctly guiding empiric therapy and local intervention programs in attempt to reduce antimicrobial resistance. introduction community acquired pneumonia (cap) is the most common cause of death associated with infectious disease. locally, it is the leading cause of morbidity and the fi fth cause of mortality according to the department of health. the initial management decision after diagnosis is to determine the site of care: outpatient, hospitalization in a medical ward, or admission to an icu. the decision to admit the patient is the most costly issue in the management of cap, because the cost of inpatient care for pneumonia is up to 25 times greater than that of outpatient care. it is a well documented fact that signifi cant variation in admission rates among hospitals and among individual physicians occurs. physicians often overestimate severity and hospitalize a signifi cant number of patients at low risk for death. because of these issues, interest in objective site-of-care criteria has led to attempts by a number of groups to develop such criteria. the two foremost criteria are the british thoracic society criteria (curb-65) and the pneumonia severity index (psi). the idsa/ats committee preferred the curb-65 criteria because of ease of use and because they were designed to measure illness severity more than the likelihood of mortality. patients with a curb-65 score > 2 are not only at increased risk of death but also are likely to have clinically important physiologic derangements requiring active intervention. these patients should usually be considered for hospitalization. therefore, the study was done to determine and compare mortality rates of the admitted cases of community acquired pneumonia assessed by either curb-65 criteria or the philippine clinical practice guidelines on cap, and to determine the applicability of curb-65 as a site-of-care tool in the admission of patients with community acquired pneumonia either at the wards or the intensive care unit. methods all patients seen at the emergency room and out-patient department with the diagnosis of community acquired pneumonia were included in the study. thorough history-taking and physical examination was taken by the er/opd fellow whom would determine if the patient has pneumonia. subsequently, laboratories (cxr, cbc, bun, abg) was requested. randomization was done for severity assessment: one group was assessed via the curb-65 criteria, while the other group was assessed using the philippine clinical practice guidelines for cap. severity assessment was done by the er fellow together with the investigator not more than one hour of the patient's arrival at the er/opd. patient was followed-up by the investigator within 24 hours of admission (ward or icu) and until discharge or death. results a total of 206 patients diagnosed with community acquired pneumonia (cap) were included in the study. the age range for the curb group is from 18 to 100 years of age with a mean age of 62.33 ± 18.25 years, while in the cpg group, 16 to 97 years of age with a mean age of 57.20 ± 20.85 years. no significant difference were noted (p = 0.564). no signifi cant difference were also noted in the gender of both groups (p = 0.889). there was a signifi cant difference noted in the presence of comorbidities (p = 0.001) between the two groups, 70.9% and 47.6%, curb group and cpg group, respectively. the presence of previous ptb treatment, cardiovascular disease and copd, ranks as the three most common comorbidity. dyspnea (94.7%), cough (85%) and fever (72.3%) were the three most common symptoms noted. there were no signifi cant difference noted in these symptoms (p = 1.00, 0.697, 1.00, respectively). with regards to the physical fi ndings: crackles (89.3%), tachypnea (44.7%), wheezes (22.3%) were the three most common signs noted. no signifi cant difference were noted in most of the signs, except for "tachypnea" and "hypotension" (p = 0.000, 0.0007, respectively). there were no signifi cant difference in the radiographic fi ndings between both groups. no signifi cant difference were also noted in the complete blood count results be it leukocytosis (p = 0.314), anemia (p = 0.477) and leukopenia (p = 1.00). there is a signifi cant difference in the blood urea nitrogen (p = 0.00002). no signifi cant difference was also noted in the arterial blood gas result: hypoxemia (p = 0.109) and hypercapnia (p = 0.499). for the curb-65 group, more than half of the population was assessed to have a score of 2 (in-patient), 54 (52.4%). for the cpg group, more than half was assessed to be under the moderate risk, 58 (56.3%). all of the patients assessed in the lower severity class, either thru the curb-65 or the cpg, had been discharged improved. the overall mortality rate per group was: 5.8% for the curb-65 group, 6 out of the 103 patients, and 1.9% for the cpg group, 2 out of the 103 patients. mortalities were noted only on those with higher severity ratings. on further determination of mortality rate per level of severity, it revealed that those with a curb-65 score of ≥3 has a mortality rate of 28.6% (6 out of the 21 patients), while those on the cpg, 22.2% (2 out of the 9 patients). conclusion in this study, we determined that all of the mortality came from the higher severity levels: curb-65 score ≥ 3 (28.6%), cpg-high risk (22.2%), none from the lower severity ratings the curb-65 criteria is a useful site-of-care tool, though, the usage of curb-65 criteria does not offer additional benefi ts compared to the use of the cpg, in fact because of familiarity of physicians with the latter, they are more adept in using it. introduction acute exacerbation has been a major complication of interstitial lung disease (ild). the rapid recognition of a bacterial pneumonia and an acute exacerbation of underlying ild appears to be clinically important for proper treatment. procalcitonin (pct), a precursor peptide of the hormone calcitonin is commonly detected at elevated levels under bacterial infection conditions. this study was to assess whether or not serum procalcitonin levels were useful as a biomarker for the differential diagnosis of ild exacerbation from bacterial pneumonia. methods we had planned a prospective observational study. our study enrolled 21 ild patients who had presented with recently progressive dyspnea, and newly infi ltrates of the chest in underlying ild. results nine of them evidenced bacterial pneumonia with high pct level. serum pct levels in ild exacerbation group were signifi cantly lower than in the pulmonary infection group (the mean value 0.05 ng/ml vs 0.91 ng/ml, 95% [ci]). sensitivity, specifi city, and negative predictive value of serum pct level were 88.9%, 100%, 92.3% respectively. conclusion the fi ndings of this study suggest that serum pct value is useful in the differential diagnosis of bacterial pneumonia from exacerbation in patients with interstitial lung disease. introduction it has been well confi rmed that malnutrition and muscle wasting are important extra-pulmonary manifestations of chronic obstructive pulmonary disease (copd), which are recognized as contributing to an increase in morbidity and decrease in quality of life. myostatin, a transforming growth factorbeta superfamily member, is mainly expressed in skeletal muscle and has been characterized as a negative regulator of skeletal muscle mass. studies have showed that myostatin is implicated in several diseases involved in muscle wasting and cachexia. recently, there is evidence of myostatin secretion and circulation in animals and human blood, and more recently, studies have shown that intramuscular myostatin expression accelerated in copd patients; while levels of circulating myostatin in copd remain unclear. we therefore analyzed serum myostatin levels to investigate the relationship between circulating myostain levels and nutritional depletion and muscle wasting in copd; and the relationship between circulating myostain levels and systemic cytokines such as tumor necrosis factor (tnf)-α and interleukin (il)-6 in copd. methods fifty-four male patients with stable copd and twenty-one agematched, healthy, male control subjects participated in the study. total-body skeletal muscle mass (smm) were calculated according to a validated formula by using body weight, height and age. nutritional status was evaluated by anthropometric measurements and serum protein levels; the former included body mass index (bmi), triceps skin-fold thickness (tsf) and mid-upper arm circumference (mac), and the latter included serum prealbumin, transferrin and albumin. serum levels of myostatin, tnf-α and il-6 were detected by elisa. results out of the patients with copd, 38 ones (59.25%) had malnutrition, with bmi less than 21 kg/m 2 . serum levels of myostatin were signifi cantly elevated in copd patients compared with controls (11.51 ± 4.99 ng/ml vs. 6.97 ± 1.70 ng/ml, p < 0.01), and the levels were even much higher in those with malnutrition (12.31 ± 5.34 ng/ml). however, smm was signifi cantly decreased in copd patients compared with controls (20.66 ± 2.44 kg vs. 24.95 ± 2.35 kg, p < 0.01). all the nutritional parameters except of prealbumin were signifi cantly decreased in copd patients compared with controls, with each p < 0.05. there is an inverse correlation between myostatin levels and smm (r = −0.437, p = 0.001) and a positive correlation between myostatin and tnf-α levels (r = 0.317, p = 0.019) in copd group, but no correlation between myostatin levels and serum proteins concentrations. conclusion this study demonstrates that circulating myostatin levels were elevated in patients with copd and that the elevated myostatin levels are closely related with malnutrition and muscle wasting in patients with copd. introduction chronic obstructive pulmonary disease (copd) is defi ned by airfl ow limitation that is not fully reversible and no medication exists that clearly improves the mortality. oxidative molecules, in particular superoxide anion, are believed to play an important role in copd-associated abnormal infl ammatory response due to increase in the level of pro-infl ammatory cytokines and chemokines and pulmonary emphysema due to proteases/antiproteases imbalance and apoptosis. superoxide dismutase (sod) catalyses the dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxifi ed to oxygen and water. lecithinized sod (pc-sod) has overcome a number of previous clinical limitations of sod, including low tissue affi nity and low stability in plasma. in this study, we examine the effect of pc-sod on elastase-or cigarette smoke-induced pulmonary emphysema, animal models for copd. methods severity of pulmonary emphysema in mice was assessed by various methods, such as the number of leucocytes (neutrophils, lymphocytes and alveolar macrophages) in bronchoalveolar lavage fl uid (balf) and enlargement of airspace (increase in mean linear intercept). lung mechanics were assessed by a computer-controlled ventilator. the pulmonary level of superoxide anion was estimated by electron spin resonance analysis and the level of 8-hydroxy-2′-deoxyguanosine. results not only intravenous administration but also inhalation of pc-sod suppressed elastase-induced pulmonary emphysema and dysfunction. inhalation of pc-sod showed therapeutic effect against elastase-induced pulmonary emphysema and dysfunction even when it administered after the development of emphysema. inhalation of pc-sod suppressed elastase-induced increase in the pulmonary level of superoxide anion and apoptosis. inhalation of pc-sod also suppressed elastase-dependent activation of proteases and induction of expression of pro-infl ammatory cytokines and chemokines. we also found that inhalation of pc-sod suppressed cigarette smoke-induced pulmonary emphysema and dysfunction. conclusion results suggest that pc-sod protects against pulmonary emphysema through decreasing the pulmonary level of superoxide anion and resulting inhibition of infl ammation, apoptosis and activation of proteases. we propose that inhalation of pc-sod is therapeutically benefi cial for copd. introduction tiotropium is a recently developed inhaled anticholinergic in the management of copd, exhibiting a prolonged duration of action and a kinetic selectivity to specifi c muscarinic receptors, leading to a more effective bronchodilator response at once-daily dosing. we determined the effi cacy of long-term tiotropium use on clinical endpoints such as mortality, exacerbations, and hospitalizations compared to inhaled long-acting beta-2 agonists among patients with copd. methods search methods rcts were identifi ed from electronic databases. bibliographies and relevant reviews were also searched. the date of the last search is august 27, 2009. selection criteria rcts among patients with copd comparing tiotropium monotherapy with inhaled labas with at least 6 months follow-up were selected for inclusion. data on all-cause mortality, mortality from pulmonary causes, mortality from cardiovascular causes, rates of hospitalizations and exacerbations were identifi ed. data collection and analysis three investigators evaluated and extracted relevant data. any disagreements were discussed and consensus decisions were made. the data were analyzed using revman 5. studies were pooled to yield odds ratios and were reported using 95% confi dence intervals. results from 27 rcts, 3 clinical trials with a total of 2,552 patients met inclusion criteria. tiotropium did not reduce the odds of all-cause mortality (or 0.48 95% ci 0.06 to 3.58) compared to inhaled labas. subgroup analysis also shows that tiotropium did not reduce the odds of mortality from pulmonary causes (or 0.63 95% ci 0.24 to 1.64) but shows a trend that might increase mortality from cardiovascular causes (or 2.17 95% ci 1 to 4.71). tiotropium reduced the odds of hospitalizations from all causes (or 0.75 95% ci 0.56 to 0.99) and showed a trend towards benefi t in reducing the odds of exacerbations (or 0.88 95% ci 0.75 to 1.04). conclusion tiotropium did not reduce all-cause mortality among patients with copd compared to inhaled labas, although it showed a possible trend towards harm in causing cardiovascular mortality. it also reduced hospitalizations, although it did not decrease the odds of exacerbations among patients with copd. background patients from asia may have different outcomes compared to those from other backgrounds. we therefore examined outcomes in the subgroup of asian patients in the uplift trial. . more patients will be recruited until august 2010 in vietnam and singapore as well as in the above nine countries. this abstract contains an interim analysis. the recruited patients were mostly male (93.6%) and elderly (mean age, 69.3 years). according to gold criteria, severity of airfl ow limitation was mild in 13.2% of patients; moderate in 44.0%; severe in 33.2%; very severe in 9.5%. a total of 92.7% of patients were exposed to cigarette smoking; 56.8% to dusty jobs. a total of 29.3% of patients had symptoms of "chronic bronchitis -phenotype" viz. chronic cough with phlegm; 58.0% had wheezing in the last 12 months. a total of 89.0% of patients were on regular medications, e.g. inhaled steroid combined with long-acting beta-agonist, theophylline, shortacting beta-agonist, tiotropium, combined inhaler of salbutamol and ipratropium, and inhaled steroid alone, in decreasing order. in the past one year, 45.9% patients required/ were prescribed antibiotics for acute exacerbations and 31.1 % required/were prescribed oral steroids. of this cohort, 26% patients had unscheduled/ emergency visits to the er or were hospitalized. conclusion this interim report showed that asian copd patients are heterogeneous. a high proportion was exposed to dusty environments at their job sites and many were cigarette smokers. further studies are ongoing to fi nd out other characteristics of copd phenotypes including the infl uence of dusty job environment in the development and progression of copd in asia. introduction it is widely recognized that copd is not only a lung-based disease, but also a systemic disorder with systemic infl ammation, which further aggravates the disease progression at acute exasperation (aecopd). it is important to fi nd a systemic biomarker which is lung-specifi c and can be used to indicate the severity of the disease in the stable state (scopd) and at exacerbation. c reactive protein (crp) and tumor necrosis factor (tnf)-a have been attracted attention as they can refl ect the systemic burden of infl ammation, while they are not lung-specifi c. surfactant protein d (spd) is produced and secreted by alveolar type ii epithelial cells and clara cells. recently study has reported that spd can be used to track disease progression and predict clinical outcomes in copd. the present study was aimed to determine the value of spd as a biomarker of systemic infl ammation in staging the severity of copd and diagnosis of the exacerbation. methods we recruited three groups of subjects: patients experiencing aecopd (n = 38), patients with scopd (n = 71) and controls with normal lung function (n = 60). elisa was used to analyze serum spd, crp and tnf-a levels. the bode scoring system was employed to evaluate health status of patients with copd, which included the four variables: body mass index, degree of airfl ow obstruction, dyspnea and exercise capacity. conclusion the present study confi rms that circulating spd levels are higher in copd and closely related with health status of the patients and severity of the disease; moreover, circulating spd can be regarded as a valid biomarker of systemic infl ammation and a potential diagnostic biomarker for aecopd. methods we reviewed all the records of bronchoscopies in our hospital from february 1, 2007 to january 31, 2010 and identifi ed 288 patients diagnosed subsequently with primary lung cancer whose sputum cytology was negative or not performed prior to bronchoscopy. a total of 26 patients with pulmonary tuberculosis were also identifi ed whose prebronchoscopic sputum acid-fast stains were negative or not performed. we reviewed the result of pathological examination of bronchoscopic specimens and postbronchoscopic sputum for the lung cancer patients and the result of mycobacterial culture of these specimens for the tuberculosis patients. of the 288 lung cancer patients, postbronchoscopic sputum cytology was performed in 238 patients and gave a positive result in 35 (14.7%) patients. the postbronchoscopic sputum was the only diagnostic specimen in 4 (1.7%) patients. meanwhile, postbronchoscopic sputum culture was performed in 20 of the 26 tuberculosis patients and was positive for m. tuberculosis in 10 (50%) patients. of these 10 patients, the culture of specimens obtained by bronchoscopy was negative in 5 patients, 3 of whom also had a negative result of prebronchoscopic sputum culture. conclusion postbronchoscopic sputum cytology has a limited role for the diagnosis of primary lung cancer and should not be performed in terms of cost-effectiveness. but its culture seems to provide additional information for the diagnosis of pulmonary tuberculosis and further studies are needed to determine when to obtain postbronchoscopic sputum specimens. for other malignant tumours -in 3 (2.1%) patients. in 13 (8.5%) patients with benign pathology 26 procedures were performed for tracheomalacia -in 3 (23.1%), for tracheobronchial fi stula -in 3 (23.1%), for endobronchial lipoma -in 1 (7.7%) and for other pathology -in 6 (46.1%) patients. there were 94 (54.7%) unilateral, 32 (18.6%) -tracheal, 31 (18.0%) -tracheobronchial and 15 (8.7%) -bilateral bronchial procedures. on 71 (41.3%) occasions procedure was elective, on 95 (55.2%) -urgent and 6 (3.5%) times it was performed as an emergency. eight patients required stent replacement. stenting was performed under general anaesthesia using combination of rigid and fi beroptic bronchoscopy and in majority with intraoperative x-ray control. uncovered and covered ultrafl ex stents (boston scientifi c) have been mainly used. results there was no intra-operative mortality. eleven (7.9%) patients died in hospital prior to discharge and 6 of them died within 24 hours after procedure. in all these patients urgent or emergency stenting was performed. tumour debulking and/or cryotherapy were required on 52 occasions after stenting. hospital stay ranged from 1 to 32 (mean -4.47, median -3) days. in benign group there was one hospital death in a patient with tb stricture. in patients with malignant tumours total survival ranged from 1 to 1678 (mean -138.23, median -55) days, in elective subgroup -from 6 to 1678 (mean -180.16, median -56) and in urgent subgroup -from 1 to 1146 (mean -103.98, median -49) days. all patients had signifi cant improvement of distressing symptoms. conclusion tracheobronchial stenting is rapid and effective technique of palliation in patients with malignant or in selected cases of benign tracheobronchial stenosis. it provides better outcome if performed electively. stent occlusion may be controlled by endobronchial cryotherapy. introduction in the clinical evaluation of airway disease, fi beroptic bronchosopy (fob) is a crucial tool in the diagnosis. however it is invasive, time-consuming and requires sedation. endobronchial lesions may be seen in both fi beroptic bronchoscopy and mdct virtual bronchoscopy (vb). the extensive image data acquired with vb permits the simulation of the internal as well as external appearance of the tracheobronchial tree. the objective of the study is to compare mdct virtual bronchoscopy with fiberoptic bronchoscopy in the detection and characterization of tracheobronchial (airway) pathology, particularly to determine the overall agreement in the fi ndings in both modalities. methods patients who underwent ct scan of the chest and then underwent fi beroptic bronchoscopy were included in the study. each patient was assessed into 5 levels: trachea, carina, right mainstem bronchus, left mainstem bronchus, tracheobronchial branches. these levels were examined using virtual bronchoscopy for presence/absence of obstructive, endoluminal and mucosal lesions and compared with the actual fi beroptic fi ndings. the sensitivity and specifi city, ppv and npv of vb were determined with fob as the gold standard. results a total of 13 patients were included in the study. sixty-fi ve (n = 13; 5 levels) levels were observed, of which 48/65 (74%) accounted for the same evaluation using fob and that of the vb. the sensitivity of vb in diagnosing endoluminal lesion was noted to be 40%, with specifi city of 95%. virtual bronchoscopy overestimated obstructive lesions with two false positive results and detected two false negative patients with endoluminal mass. none of the mucosal abnormalities (mucosal erythema, edema, etc.) are detected using vb. conclusion virtual bronchoscopy may be comparable with fi beroptic bronchoscopy in the localization, morphologic description of tracheobronchial lesions. however, it has limited capability to reconstruct subtle mucosal irregularities. consequently, it is prone to artifactual reconstruction for mucosal changes that may render a false positive fi nding. overall, virtual bronchoscopy best complements the fi beroptic bronchoscopy in thorough evaluation of the tracheobronchial pathologies and cannot obviate the need for this invasive approach in the diagnosis of early mucosal changes within the airway. results of 20 patients 16 (80%) were male. all patients had multiple comorbidities which include severe copd = 13(65%), 12 (60%) patients each had heart failure, respiratory failure and anaemia,hypoalbuminemia = 14(70%), active tuberculosis = 8(40%), pah = 4(20%), cad, crf, cld in 3 patients and age > 65 years was in 16 (80%) patients. all patients underwent ct thorax and fob and after localizing bleed underwent bae within 72 h of admission. in 3/20 (15%), bae could not be done on account of technical reasons. immediate control of bleeding was achieved in 17/20 patients (85%). in total, 15/17 patients (88.2%) reported no rebleed till 24 months. none developed early rebleed (<1 month) while 2/17 (11.7%) develop late rebleed (>6 months). one managed conservatively and other underwent lobectomy. only complication of bae was transient dysphagia in 1/17 (5.8%). conclusion bae is safe and effective in immediate and long term control of massive haemoptysis in elderly patients with multiple comorbidities and should be considered even in such high risk group. introduction histologic specimens obtained by endobronchial ultrasoundguided transbronchial needle aspiration (ebus-tbna) often provide valuable information for diagnosis or management decisions. besides the conventional 22-gauge needle, a 21-gauge needle is now available for this procedure. the purpose of the present study was to compare the histologic specimen retrieval yields of ebus-tbna using 21-gauge and 22-gauge needles for sampling hilar/mediastinal lesions. methods sixty patients with hilar/mediastinal lymphadenopathy or a tumor adjacent to the central airway were enrolled in this study and randomized to undergo ebus-tbna using a 21-or 22-gauge needle. each histologic specimen obtained by ebus-tbna on the initial two punctures of each patient (total 120 punctures) was interpreted separately and categorized by an experienced pathologist as follows: i, diagnostic; ii, nondiagnostic but adequate (e.g. lymphoid tissue); iii, nondiagnostic and inadequate (e.g. clot); and iv, no specimens. results median targeted lesion size in shortest diameter on ct in the group using a 21-gauge needle and a 22-gauge needle was 23.5 mm and 21 mm, respectively. prevalence of malignancy on primary disease in each group was 77% and 83%, respectively. the specimens obtained by 21-gauge needle were interpreted as i in 35, ii in 8, iii in 15 and iv in 2. the specimens obtained by 22-gauge needle were judged to be i in 34, ii in 13, iii in 7 and iv in 6. the sampling yield of adequate histologic specimens (i and ii) obtained by the 21-and 22-gauge needle was 72% and 78% (p = 0.4). no complications were associated with the procedures. conclusion histologic specimens can be obtained with a high sampling yield by either of the needles. our study could not show any difference in sampling yield between the two needles. introduction diagnostic tuberculosis using acid fast bacilli (afb) microscopy and conventional lowenstein jensen (lj) culture remain the cornerstone but the sensitivity of these traditional methods is quite low, especially in the samples containing small number of organism. there is a need for rapid, sensitive and accurate detection of these organisms in clinical specimens to hasten the administration of appropriate antimycobacterial therapy and prevent the spread of infection in the community. sputum smear-negative pulmonary tuberculosis (ssn-ptb) is a common problem faced by clinicians. performing fi ber optic bronchoscopy (fob) and subjecting the bronchoalveolar lavage (bal) material to diagnostic methods of smear and mycobacterial culture appears to be helpful in the diagnosis of ssn-ptb. radiological and pulmonary function outcome of children with sars longer term follow up of aerobic capacity in children affected by severe acute respiratory syndrome (sars) the usefulness of virtual bronchoscopic navigation has been established for the diagnosis of small peripheral lesions. exclusive software (bf-navi) from olympus medical systems is commercially available, but because no function to display extra-airway structures has been installed, this could not be indicated for testing where the purpose is mediastinal/hilar lymph node aspiration. using an improved version of the software from olympus medical systems to permit lymph node visualization, this study evaluated the aspiration support function. methods before testing, size and position of lymph nodes for aspiration were established on mpr images of ct data. on the virtual navigation image, these were displayed in a transparent ellipse from the airway. besides rotation and back and forth movements, changes in angle of the bronchoscope tip are simulated, and a function is also available to move the virtual image fi eld up, down, left, and right. using these, the navigation image was compared with the real image, and the lymph nodes were aspirated. in 8 patients with enlarged mediastinal/hilar lymph nodes, transbronchial needle aspiration (tbna) without using an ultrasound probe, or ebus-tbna, was selected for aspiration. results lymph nodes were aspirated under navigation in all patients. the diagnosis was primary lung cancer in fi ve patients, metastatic tumor in one patient, and sarcoidosis in two patients. depth perception was diffi cult for the transparently displayed lymph nodes, particularly #2 and #4, and judging anterior-posterior relationships was diffi cult when nodes were superimposed. conclusion some room for improvement thus remains in the display method. however, this offers future promise as diagnostic support technology. key: cord-017016-twwa9djm authors: tomashefski, joseph f.; dail, david h. title: aspiration, bronchial obstruction, bronchiectasis, and related disorders date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_5 sha: doc_id: 17016 cord_uid: twwa9djm the conducting airways play a pivotal role in the spectrum of pulmonary pathology, not only as conduits for injurious agents to enter the lung, but also as an anatomic compartment that is affected by a diverse array of primary or secondary bronchocentric diseases. this chapter discusses aspiration and bronchial obstruction in detail, with emphasis on the aspiration of toxic, infective, or particulate matter. lung abscess, a frequent complication of obstruction or aspiration, is also reviewed. both aspiration and lung abscess are reconsidered within the context of pulmonary infectious disease mainly in chapter 8 on bacterial infections, and to some extent in the chapters on mycobacterial (chapter 9), fungal (chapter 10), and parasitic diseases (chapter 14). the conducting airways playa pivotal role in the spectrum of pulmonary pathology, not only as conduits for injurious agents to enter the lung, but also as an anatomic compartment that is affected by a diverse array of primary or secondary bronchocentric diseases. this chapter discusses aspiration and bronchial obstruction in detail, with emphasis on the aspiration of toxic, infective, or particulate matter. lung abscess, a frequent complication of obstruction or aspiration, is also reviewed. both aspiration and lung abscess are reconsidered within the context of pulmonary infectious disease mainly in chapter 8 on bacterial infections, and to some extent in the chapters on mycobacterial (chapter 9), fungal (chapter 10), and parasitic diseases (chapter 14) . bronchiectasis, which is frequently grouped with other forms of obstructive lung disease (see chapter 24) , is discussed in this chapter as a major pathway of airway remodeling that may be of inflammatory, postobstructive, or congenital etiology, and is, itself, an important sequela of aspiration. the topic of bronchiectasis cannot be considered without reference to inflammatory lesions of the small airways, which may follow or precede the development of bronchiectasis (see chapter 25) . the present chapter also discusses a variety of pulmonary disorders that may simulate, initiate, or complicate bronchiectasis. involvement of the large airways in systemic diseases such as amyloidosis (chapter 21), sarcoidosis (chapter 18), collagen vascular diseases, including relapsing polychondritis (chapter 20), connective tissue disorders such as marfan's syndrome (chapter 20), or as a complication of environmental dust exposures (chapter 26) is reviewed in each of their respective chapters. an excellent review of bronchiectasis in systemic diseases is that by cohen and sahn.l a discussion of asthma and its related conditions of mucoid impaction, bronchocentric granulomatosis, and allergic bronchopulmonary aspergillosis can be found in chapter 15. unique congenital lesions of the airways in 84 addition to the enigmatic intralobar sequestration are discussed respectively in chapters 6 and 7 on pediatric lung pathology. a variety of degenerative or so-called metabolic disorders that affect the large airways, such as tracheobronchopathia osteoplastica, are reviewed in chapter 21. finally, bronchial tumors are extensively covered throughout volume 2, which is devoted to pulmonary neoplasia. aspiration is the inhalation of liquid or solid materials into the lower respiratory tract, usually from the oral or nasal cavities, oropharynx, esophagus, or stomach. logically, the course of aspiration is determined by such laws of physics as inertia and gravity. larger, more solid materials, and finer more liquid materials, all follow the straightest and most dependent course after they enter the trachea. as explained in chapter 2, the right mainstem bronchus continues on a more direct course than the left (20 to 30 degrees compared to 40 to 60 degrees for the left mainstem bronchus); the wider angle of the left bronchus allows it to extend around the heart. larger, more solid objects that pass the larynx often lodge in the right mainstem bronchus, while smaller solid objects most frequently continue into the right lower lobe bronchus. 2 this has been well demonstrated in the aspiration of foreign objects by children, generally in the age group of 1 to 3 years. 3 ,4 during this age range, children examine almost everything by placing items into their mouths. in both children and adults, larger objects are sometimes stopped at the larynx and may be expelled by strong coughing. in adults, sudden death due to laryngeal obstruction by aspirated food (most frequently meat) has been termed the cafe coronary.s more than 80% of aspirated foreign bodies occur in children, and among all age groups only 5% are spontaneously expectorated. 6 ,7 sharper objects may perforate a bronchus and cause bleeding, or even penetrate the pleural cavity and cause pneumothorax. 8 a foreign body may migrate within the bronchi and cause wandering infiltra tes. 9 young children most frequently aspirate peanuts, beads, and other fragments of wooden or plastic toys. peanuts and sunflower seeds lead the list in western countries, whereas in arabic countries children most often aspirate melon seeds. 8 older children may inhale flowering grass fragments, which wedge themselves into more distal bronchi and resist expectoration. in any age group, teeth, fragments of bone ( fig. 5.1) , food, blood clots, tissue fragments, nasal pack components, lipids from oily nose drops or orally administered cathartics, bacterial fragments, and gastric content most commonly enter the lung. noguchi et a1. 10 reported a subacute reaction to mud aspiration in a victim of near-drowning, while aspiration of sand in children has been reported to cause a radiographic "sand bronchogram.,, 11 drowning, often thought of as occurring in fresh or salt water, has also occurred in large vats of beer, wine, liquid chocolate, and other interesting concoctions. a literary example of this is shakespeare's richard iii, in which the duke of clarence is finally dispatched in a large vat of wine (the "malmsey butt,,). 12 brock 13 .1 4 in 1942, beautifully illustrated the mechanics of aspiration with abscess formation, which most often followed the dependent course described. once within the lung, finer and more fluid ingredients flow into the first dependent orifices that are encountered. in the supine position, these are most often the posterior segment of the upper lobe and superior segment of the lower lobe ( fig. 5.2 ). in the more upright position, material flows preferentially into the basilar segments of the lower lobes. the basilar segments divide rather evenly, and localization within these segments is not as discrete as in other areas of the lung. when a person is in the lateral decubitus position, the axillary branches of the subsegments of apical and posterior upper lobe bronchi are favored. the more anterior portions of the lung are usually spared the effects of aspiration, unless aspiration occurs in the prone position, as in near-drowning. aspiration need not only be from external sources. rupture of large fluid-filled abscess cavities, tuberculous cavities, or other cysts might be followed by intrabronchial aspiration of infective or other types of material into dependent zones (see fig. 9 .7 in chapter 9). the most common conditions predisposing to aspiration include impaired consciousness, most frequently from alcohol, drugs, or anesthesia, followed by central nervous system disorders (e.g., epilepsy, stroke, dementia) or neuromuscular diseases. 4 next in frequency is aspiration secondary to obstructing masses or other functional defects of the esophagus or stomach. episodes of aspiration are eventually confirmed in about 85% of children, but in only about 30% of adults. 3 ,ls the difference in documentation is probably related to the altered level of consciousness in adults causing temporary amnesia. the common clinical manifestations of an aspirated foreign body constitute a triad of cough, wheezing or rhonchi, and decreased air entry. 3 the wheezing that is sometimes associated with aspirated foreign bodies in children may be ameliorated with theophylline, leading to diagnostic confusion with asthma. 16 early experimental animal studies showed that aspiration of material into the lungs regularly occurs when materials are placed in the nares or accessory sinuses during anesthesia. 17 ,18 myerson 19 found blood immediately postoperatively in the tracheobronchial tree in 79% to 100% of humans who underwent tonsillectomy, including those under general or local anesthesia. several experimental studies have also proven that normal adults aspirate with some regularity. quinn and meyer20 in 1929 introduced lipiodal (iodinated poppy seed oil) into the noses of sleeping subjects and found the material often entered the lungs. amberson 21 in 1937 reported placing barium in the mouths of normal subjects during sleep, with similar results. radiologists sometimes observe aspiration while performing upper gastrointestinal tract barium studies (see below). as reviewed by bartlett, 22 various markers placed in the stomach the night prior to surgery have been identified in lungs sampled during surgery the next day in 7% to 16% of patients. 23 • 24 huxley and associates 2s refined these techniques by placing indium-ll1 chloride in the posterior nasopharynx periodically during sleep. on lung scanning, this tracer was found in the lungs of 45% of normal individuals and in 70% of those with some alteration of the central nervous system. those in the normal group who did not aspirate were fitful sleepers who tended not to enter deep sleep. the implication is that most normal people who enter deep sleep, aspirate. nasogastric and oropharyngeal tubes, including endoscopes and tracheostomy tubes, increase the risk of aspiration. 26 normal individuals tend to clear such occult aspirations without difficulty or sequelae. an acute cough reflex is most important, but also valuable are intact mucociliary activity and alveolar macrophage response (see chapter 3) . the pathologic effects of aspiration are dependent on the character, volume, and frequency of the aspirated components. in this chapter, food and medicinals, gastric acid, lighter hydrocarbons, and heavier oils are separately considered. aspirated bacteria are covered in the section on abscess formation. retained squamous if. tomashefski, jr. , and d.h. dail cells from meconium in newborns are a sign of in utero distress. squamous cells in the lungs of adults are an indicator of oral, oropharyngeal, or esophageal aspiration ( fig. 5.3 ). in the absence of other evidence of aspiration, finding mixed bacteria in lung tissue is very suggestive of aspiration of oral content. lung injury following aspiration has been subdivided by marik 27 into aspiration pneumonitis and aspiration pneumonia. aspiration pneumonitis refers to acute chemical lung injury due to inhaled gastric acid with or without injury due to aspirated particulate matter, whereas aspiration pneumonia is an infectious process resulting from the inhalation of oropharyngeal secretions colonized by pathogenic bacteriay aspiration of gastric acid, a major cause of acute respiratory distress syndrome (ards), has been well studied in humans and in experimental animals. 22 in experimental models, it has been suggested that a ph of 2.4 or lower and a significant quantity of acid (estimated to be 20 to 25ml in an adult human or 1 to 4mllkg in an experimental animal) are necessary to induce a chemical pneumonitis.27 when gastric acid with methylene blue was put into anesthetized dog tracheas, dye was visible on the pleural surface 12 to 18 seconds later. 28 atelectasis oflung tissue was noted in 3 minutes. human studies date from the classic study of mendelson 29 in 1946, and acute gastric acid aspiration is sometimes called the mendelson syndrome. mendelson studied 61 cases of massive gastric aspiration in obstetric patients (0.15% incidence) under ether anesthesia. respiratory distress occurred soon after aspiration, with accompanying cyanosis, tachypnea, and tachycardia. bronchospasm occurred in most of his patients. chest radiographs initially showed rather widespread mottled densities, most of which cleared by 7 to 10 days. only eight of 61 patients became infected. this study was conducted before antibiotics were readily available, but other studies in humans with or without antibiotics or steroids have confirmed his findings. in general, later studies have found a lower incidence (about 30%) of bronchospasm, more frequent early temperature elevation, and increased mortality (in the range of 30% to 60%) despite therapy. hypotension and hypoxemia occurred more commonly than in mendelson's series. 22 the combination of acid and particulate aspiration exacerbates alveolar capillary injury. 30 bynum and pierce 3 ! studied 50 patients with welldocumented gastric acid aspiration. in their series, all these events followed altered consciousness, most often by a sedative drug overdose or a general anesthesia. as in mendelson's29 experience, respiratory symptoms developed rapidly and were very similar in all patients despite eventual outcome. three clinical outcomes were described: 12% died shortly after aspiration; 62% had rapid clinical and radiographic clearing on the average of 4.5 days; and 26% had rapid improvement followed by deterioration relating to bacterial infection. of this latter group 60% died, whereas 28% of the whole group died; death occurred between day 1 and 16, averaging 7.2 days. these authors found initial steroid or antibiotic therapy did not affect eventual outcome. in both humans and animals, edema, congestion, hemorrhage, and degeneration of bronchiolar lining cells and alveolar type i and ii cells follows early in the course (fig. 5.4) . after 4 hours alveoli are filled with polymorphonuclear neutrophils (pmns) and fibrin. hyaline membranes are formed by 48 hours, providing a histologic picture of diffuse alveolar damage (dad) (see chapter 4) . resolution begins at about 72 hours and may either lead to figure 5.4 . gastric acid aspiration. acute effects show hemorrhagic necrosis of lung parenchyma. complete restoration of alveoli or leave some residual scarring. of course repeated aspiration may lead to combined acute, subacute, and chronic appearances. in his review, bartlett 22 referred to this rapid and irreversible type of injury as comparable to a "flash burn," and noted that little can be done to prevent injury once acid has made contact with the lung (fig. 5.4 ). the reactions just described plus fluid extravasation help to dilute the acid, as do buffering components from serum and cell breakdown products, but these only occur after injury. more recent studies have indicated that the mechanism of lung injury following acid aspiration extends beyond the direct chemical effects of acid to involve various inflammatory mediators including tumor necrosis factora (tnf-a), interleukin-8 (il-8), adhesion molecules, and cyclooxygenase and lipoxygenase products. 32 -35 the role of reactive oxygen species, and the adverse effect of oxygen administration after an episode of aspiration were demonstrated experimentally by nader-djalal et al. 36 a primary role is currently placed on neutrophils and complement in mediating lung injury in this setting. 27 ,32,37.38 thus, aspiration pneumonitis represents a biphasic response composed of early-onset direct pulmonary injury due to acid, followed by delayed injury due to acute inflammation. 26 ,38 low-grade chronic aspiration of gastric content may escape easy detection. these occult aspirations may lead to interstitial fibrosis, and perhaps account for the 20% to 54 % incidence of associated and unexplained pulmonary fibrosis in patients with esophageal abnormalities, most commonly hiatal hernia or simple reflux,39,40 the role of reflux in asthma, chronic bronchitis, chronic cough, recurrent pneumonia, cystic fibrosis, and sudden infant death syndrome has been reviewed by allen et al. [see also section on cholesterol (endogenous lipid) pneumonia in this chapter].41 children, particularly those aged 1 to 5 years, are likely to ingest various lighter hydrocarbons, mostly lighter volatile petroleum distillates. these products include kerosene, turpentine, and other paint thinners; furniture or shoe polish; lighter fluid; gasoline; dry cleaning fluids; and some insecticides. the toxicity is greater with those products that disperse most easily, specifically those that cause the greatest decrease in surface tension, or have the least viscosity or the highest volatility. 42 although ingestion precedes aspiration, eade and associates 43 nicely reviewed the reasons that aspiration is the most important toxic pathway of injury. symptoms develop rapidly and radiographs often show localized pulmonary infiltrates, often in the aspiration zones mentioned earlier. experimentally, the lethal dose by figure 5.5. lentil bean. a. at top is thick outer coat; below, cotyledon compartments with starch cells. b. cellulose framework of legume cotyledon compartments is birefringent under ingestion alone is much higher than that usually ingested by persons who subsequently aspirate. sizable doses of distillates have been placed in the stomachs of experimental animals whose esophagi were ligated, and these animals did not suffer pulmonary toxicity. the pulmonary changes, almost identical to those of gastric acid aspiration, include diffuse congestion, hemorrhage, edema, hyaline membrane formation, and bronchopneumonia. atelectasis occurs early, apparently by direct toxic effect of these light hydrocarbons on s urfactan t. 44 about 75% of affected children have abnormal chest radiographs, but only 25% to 40% have pulmonary signs or symptoms. 43 ,45 in the past, death has been reported in about 2% to 10% of cases, but in two large series death occurred in 0.3% and 1 %, respectively.43a5 death usually occurs within 24 hours of exposure. most survivors experience few sequelae. various food particles, such as skeletal muscle, fat tissue, or fragments of bone, may be aspirated and identified histologically in lung tissue. cooking or digestion may result in poorly defined particles that appear foreign but defy further definition. as was well demonstrated by knoblich 46 and others,47-49 portions of legume seeds are one of the better markers of food aspiration. the legumes most commonly eaten are various peas, beans, and peanuts; because they are relatively inexpensive and nutritious, they occur in many products. (the word lentil is sometimes used in these references, but it is also j.f. tomashefski, jr., and d.h. dail polarized light. c. degenerated aspirated vegetable material retains compartmental structure and stains strongly with gomori methenamine silver. the name of a specific type of bean). the legume seed ( fig. 5 .5) consists of a thick cellulose outer coat, the cellulose walls of the inner food storage compartments, and the starch cells contained within these food compartments. cooking softens the outer shell and cell walls of the beans or peas and allows easy disruption of the content, with a resultant jelling effect of the starch particles (called "thickening" in cooking). the cellulose walls of the outer coat and starch compartments are more difficult to totally disrupt or digest, and therefore act as both a chronic irritant and a good marker of aspiration. aspiration of these fragments in both experimental animals and humans produces an acute exudative response within 24 hours (fig. 5.6 ), followed by a foreignbody giant cell reaction ( fig. 5.7) . these cell wall fragments are gomori methenamine silver (gms) positive, and usually birefringent under polarized light (fig. 5.5 ). the glycogen compartments, when intact, are vividly periodic acid-schiff (pas) positive ( fig. 5.7 a) . starch cells may be mistaken histologically for parasite larvae (fig. 5.6b ).47 at about 10 days (experimentally) an organized granulomatous reaction occurs around the aspirated particles, and eventually the starch cells disappear, leaving only the cellulose fragments. 48 the walls of carrots, onions, and most nonlegumes digest more readily and do not give rise to as much exuberant chronic reactions as seen with legumes. they do, however, undergo the same type of early changes if aspirated, and initiate acute and subacute pneumonia during digestion of the starch cells. some of the most offensive aspirated food fragments have undergone alterations in preparation, and some of the worst combinations are cooking oils and salts, as, for example, an aspirated potato chip ( fig. 5 .7b). eventually these areas become small fibrotic or fibrocalcific nodules, which may appear almost as degenerated parasites or as sclerosed blood vessels ( fig. 5.8 ). they may appear as small hyalinized granulomas or possibly as entrapped calcospherites, usually within a fibrous stroma. 46 at times the conditions for aspiration are chronic, and recurrent aspiration leads to the acute and chronic changes together or in close proximity in the same specimen. 50 respiratory bronchioles exhibit marked remodeling associated with proliferative bronchiolitis obliterans, foreign-body granulomas, and entrapped food particles. 51 the macroscopic appearance is that of scattered yellow miliary nodules that are reminiscent of miliary figure 5.8. chronic reaction from aspirated lentil beans. two hyalinized starch cells may be mistaken for fibrosed, obliterated blood vessels. 53 matsuse and colleagues 52 have designated this condition as diffuse aspiration bronchiolitis, which they observed in 31 of 4880 consecutive autopsies (0.64%). the mean age of patients with this condition was 81.2 years. affected individuals frequently were bed-ridden or had dysphagia due to underlying neurologic disorders. 52 high-resolution computed tomography (hrct) may show a striking pattern of centrilobular miliary opacities. 54 in chronically ill or hospitalized patients, aspirated medicinal products, such as intact or partially digested pharmaceutical tablets, may be associated with aspiration pneumonitis. inert tablet components such as microcrystalline cellulose, talc, and crospovidone may be identified histologically in conjunction with aspirated food, pneumonia, and foreign-body reaction (see chapter 26) 55 microcrystalline cellulose, like legume components, is brightly birefringent on polarization, and also positive with gms stain. 55 it can be distinguished from vegetable particles, however, by its fiber-like, or "matchstick-like" appearance. 55 aspirated tablet filler components, which reside within alveoli and bronchioles, usually can be discriminated from identical particles introduced by illicit intravenous injection of aqueous tablet suspensions, which localize within small pulmonary arteries or in a perivascular, interstitial distribution (see chapter 26) . aspirated sodium (or calcium) polystyrene sulfonate (kayexalate), a potassium-binding cation exchange resin, has a distinctive histologic appearance characterized by large dark eosinophilic or basophilic, angulated, "glassy" particles, that sometimes appear striated ( fig. 5.11 ). [56] [57] [58] [59] duct. b. angulated, "glassy" kayexalate particles have elicited a foreign-body giant cell reaction. kayexalate particles are weakly birefringent, and positive with pas, acid-fast, and gram stains, but negative with von kossa stain. 60 kayexalate has also been identified in tissue sections by infrared microspectrophotometry.6l in humans and experimental animals, kayexalate has been shown to produce a necrotizing or organizing pneumonia. 6l ,62 more frequently, kayexalate is a cause of mucosal ulcers of the gastrointestinal tract. 60 ,63 kayexalate histologically closely resembles the less frequently encountered material cholestyramine. 60 cholestyramine, however, is more opaque and pink rather than red on acid-fast staining. 60 the aspiration of an intact ferrous sulfate tablet may induce severe, potentially fatal, bronchial mucosal ulceration and hemoptysis. 64 -66 the endoscopic appearance of the ulcerated bronchus typically is of a golden-brown discoloration ( fig. 5.12a ).6465 histologically, ulceration, necrosis, foreign-body reaction, and brown or yellow pigment that stains blue with prussian blue stain may be seen ( fig. 5.12b ). the pathogenesis of this syndrome, colorfully termed "iron lung," is thought to be a chemical burn induced by oxidation of iron from the ferrous to the ferric form. 64 the radiographic contrast material barium sulfate (bas04) is also readily visualized by chest x-ray in patients who aspirate this material. barium is a fairly inert white powder that tends to produce minimal functional lung impairment. grossly, following barium aspiration, lung parenchyma is chalky, tan-gray, and slightly indurated ( fig. 5.13a ). histologically, fine, golden-tan, refractile, weakly birefringent particles of barium sulfate are present within the cytoplasm of alveolar macrophages. with chronicity, or upon repeated aspiration, barium-laden macrophages migrate into the interstitium (fig. 5.13b ). inhalation of barium in the industrial setting (barytosis) is discussed in chapter 26. activated charcoal is sometimes given therapeutically for oral drug overdose. however, if the airway is not protected, charcoal may be aspirated in copious amounts, causing "charcoal lung," in which coarse black carbon particles obstruct small airways. 55 oils that may be aspirated include mineral oils such as used in nose drops and cathartics, vegetable oils used in cooking, and animal oils such as cod liver oil or fat-soluble vitamin preparations. mineral oil, derived from petroleum products, is the most common agent of exogenous lipid pneumonia. 68 oil aspiration was first described by laughlen 69 in 1925 in a child who received oily oral and pharyngeal preparations for diphtheria; it was confirmed by him experimentally. the role of oil-based contrast media used for bronchoscopy was well reviewed by spencer. 70 animal oils cause a more severe inflammatory reaction than mineral or vegetable oils, and this difference appears related to the number of free fatty acids and increased viscosity of animal oi1. 42 ,71.72 mineral oils are fairly inert, as they have no fatty acids, and are rapidly emulsified and consumed by pulmonary macrophages. vegetable oil droplets may remain in alveoli for months without eliciting significant reaction, but eventually, due to low-grade chronic irritation, they cause scarring. animal and mineral oils, but only rarely vegetable oils, may be seen in regional lymph nodes. 71 aspiration of oils commonly occurs in older individuals, who may take oily nose drops or cathartics at bedtime. aspiration most frequently gravitates to the basilar segments of the lower lobes suggesting these patients usually sleep in a more upright position or experience aspiration before reclining.73 because of its weakly irritative nature, mineral oil can enter the tracheobronchial tree without stimulating glottic closure or cough reflex. 74 only two of 14 cases documenting exogenous lipid pneumonia at autopsy had reported significant clinical symptoms during life. 75 because these oils float in the stomach, it is possible they also are aspirated via reflux from the stomach,z2.75 in unselected autopsy series, oil aspiration has been documented in 2.5% to 14.6% of adults. 75 .76 other oily products that have been incriminated as being aspirated in the lung include fragments of lip balm, burning fats (an occupational exposure), a rapid drying agent in spray enamel paint, oils applied to tobacco products (blackfat tobacco), and possibly hair spray.77-81 children may aspirate oily medications if they are force-fed these while resisting and crying violently.82 atypical mycobacteria, particularly rapid growers such as mycobacterium jortuitum or m. chelonae, have been reported associated with oil aspiration pneumonia (see chapter 9) . [83] [84] [85] [86] [87] symptoms of lipid aspiration include fever (39% of patients), weight loss (34%), cough (64%), and dyspnea (50%), although in one large study lipid pneumonia was an incidental finding, without associated symptoms, in 41 % of patients. 68 lung function tests may indicate either obstructive or restrictive changes. computed tomography (ct) scans usually show alveolar consolidation or groundglass opacities in the lower lobes. subfissural clear zones may be interposed between densities, creating a "sandwich effect" on ct scan. 68 grossly, lungs affected by oil aspiration are often gray to yellow and rather solid (fig. 5.14a ). dense localized fibrotic lesions (paraffinomas) may grossly mimic cancer or complicated pneumoconiosis. 88 ,89 occasionally oily droplets exude from the cut surface. microscopically the lipid droplets are often dissolved by tissue processing. 71 one exception is cod liver oil, which remains as salmoncolored droplets on hematoxylin and eosin stain after tissue processing. fats may be seen with rapid watersoluble or oil red 0 stains on frozen section ( fig. 5 .15d); variably sized fat droplets and varying numbers of multinucleate giant cells are present ( fig. 5 .15a-c). when only a small amount of fat has been aspirated, the reaction may be contained in alveolar macrophages. this is most commonly seen in mild degrees of aspiration with a diluted fatty substance, as might be seen with milk aspiration. when larger doses of thicker and more toxic oils are aspirated or when oil aspiration is repeated, the areas become densely fibrotic with reduction of the background lung architecture ( fig. 5.14a ).89 occasionally, cor pulmonale results,90.91 transbronchial biopsies may provide enough tissue to make this diagnosis. precise identification of specific lipids can be determined by infrared spectrophotometry. 88 in the differential diagnosis is artifactual collapse of lung around remnant air bubbles (see fig. 1 .2c in chapter 1). exogenous lipid pneumonia can usually be distinguished histologically from endogenous lipid chronically ingested mineral oil as a laxative. note yellow oil layered on the surface of fluid. (cholesterol) pneumonia by subdivisions within fat droplets, coarse cytoplasmic vacuoles in macrophages, multinucleated foreign-body giant cell response, and, in more chronic cases, a greater degree of chronic inflammation and fibrosis with destruction of background lung parenchyma in exogenous lipid pneumonia ( fig. 5.15 ). at times some lipid is incorporated/entrapped in the interstitium ( fig. 5.15c ), resembling a similar appearance in diffuse pan bronchiolitis and xanthomatous bronchiolitis obliterans (see below and chapter 25). diffuse panbronchiolitis is centered more on terminalrespiratory bronchioles and is composed mostly of finely vacuolated fat. sputum cytology or cytologic aspiration specimens have been used to confirm lipid pneumonia. on bronchoalveolar lavage an oily layer is sometimes present on the surface of the collection tube ( fig. 5.14b ). in 1950, losner et al.,92 using oil stains, found lipid-rich macrophages in 19 of 20 suspected cases in contrast to two of 45 control patients. more recently, corwin and irwin 93 restudied this situation using bronchoalveolar lavage in various lung diseases including aspiration, hemoptysis, cancer in the lung (either primary or secondary), bronchiectasis, interstitial fibrosis, and sarcoidosis. when compared to normal lungs, samples from diseased lungs in general contained increased fat-filled macrophages. these authors warned that the simple presence of fatty macrophages in cytology preparations is not diagnostic of lipid pneumonia; however, the quantity of lipid was more abundant in aspirators than in these other groups. their figure 5.15. oil aspiration. a. subacute effects of oil aspiration show varyingly sized fat droplets, inflammation, lymphoid aggregates, and fibrosis. most of the lung architecture has been obliterated. b. higher power view shows foamy lipid-filled macrophages and multinucleate foreign-body cells. c. in the chronic state, oil droplets are still seen within the interstitium with "aspirator" group consisted mainly of patients with a history of upper gastrointestinal tract disease, including reflux in most. in young children the presence of numerous (>50) oil red o-positive lipid-laden macrophages on tracheal aspirate is highly specific for aspiration. 94 corwin and irwin emphasized that the size of the fat droplets in lavage fluid cannot be used to distinguish endogenous from exogenous lipid pneumonia. wherever it occurs, an abscess is a localized accumulation of inflammatory cells, initially having abundant neutrophils, that is usually accompanied by tissue destruction. in the lungs, "cross-country" necrosis occurs during the formation of an abscess (figs. 5. 16 bronchi, and arteries. in contrast, cavities that are more chronic and more slowly formed, such as tuberculous cavities, often leave remnants of fibrotic bronchopulmonary rays coursing through the cavity itself (see chapter 9) . some more slowly forming nontuberculous abscesses can do this, but most of the abscesses in the lung have an acute initial phase that destroys most of the tissue in the area ( fig. 5.17b ). bronchi frequently connect with abscess cavities, allowing drainage of the necrotic material, leaving an empty or partially empty cavity with or without an air/fluid level on chest radiograph (figs. 5.18 and 5.19). occasionally, inflammation seals off all such bronchial connections, resulting in a solid mass that may be suspected of being a tumor. adjacent organization in acute and subacute abscesses often accounts for an enlarged surrounding radiographic density (figs. 5.17 and 5.18). there are many etiologies for cavity formation in the lung, and abscess formation is but one of them. other pulmonary abscess formation, which were already well known by 1922, were summarized in this review. by 1936, a total of 2114 cases had been published. 97 aspiration is the most common cause of lung abscess. other instigators of this type of damage include b. gross specimen. note cavity is mostly drained, with freshappearing, thin lining without fibrous wall. note also persistence of some trabeculae, presumed bronchopulmonary rays, and variable but narrow surrounding inflammatory reaction. penetrating trauma, postoperative states, obstruction, hemorrhage or infarction, necrotizing pneumonia, infected emboli, infection of a preexistent cyst or bulla, or extension from nearby infected areas in the mediastinum, chest wall, diaphragm, or infradiaphragmatic locations. nonaspiration types of pulmonary abscesses of course do not follow the aspiration patterns of distribution, but occur as their coexistent factors dictate. for example, if there is an infarct or an obstructing tumor, infection would occur in the affected areas. septic emboli are hematogenously spread, and resultant abscesses are often multiple, small, and peripherally distributed (see fig. 28 .29 in chapter 28). as aspiration is the principal cause of pulmonary abscess formation, it is reasonable that conditions that favor abscess formation are identical to those favoring aspiration. 98 the locations are similar; men are more frequently affected than women; and the right lung is involved twice as often as the left. 98 -103 the posterior segments of the right upper lobe and superior segments of the right lower lobe are involved most frequently, followed by the corresponding segments on the left side. 103 the single most commonly associated event is an alteration of consciousness; the second most frequent association is poor dental hygiene; and the third is an immunodeficient status. poor dental hygiene was noted in cases without other apparent causes of pulmonary abscess in the early studies between 1927 and 1936.103-108 the spectrum of bacteria involved in abscess formation is almost identical to that of endogenous oral flora. 109 -114 moreover children and edentulous older people do not often develop lung abscesses.ll5 in children cases caused by aspiration must be separated from other cases of cavitary necrosis, such as pneumatoceles in primary staphylococcal pneumonia. 116 -1l8 anaerobic bacteria are the only organisms cultured in about one half to two thirds of lung abscesses; in the remaining cases either aerobic or facultative aerobic bacteria are isolated, or no bacteria are culturedys the anaerobic bacteria most frequently found are peptostreptococci (i.e., gram-positive anaerobic cocci), pigmented gram-negative bacilli (including bacteroides), and the fusobacteria.98.1l9.120 spirochetes were described morphologically in earlier studies and seemed to be significant, as they were present in the growing rims of necrosis; however, they have not been mentioned much recently, perhaps because they are difficult to culture.los. 121 about half of cultures with anaerobes also contained aerobic bacteria capable of necrosis, specifically staphylococcus, streptococcus, haemophilus, pseudomonas, klebsiella, and escherichia spp.122 patients with predominantly anaerobic pulmonary abscesses often present with indolent symptoms, in contrast to those with necrotizing aerobic abscesses. the latter may be more common in nosocomial-acquired lung abscess. 9s -98 immunocompromised patients often acquire gram-negative necrotizing pneumonias and vasoinvasive necrotizing fungal pneumonias, both of which may lead to cavitation. one clue to aspirated bacteria in lung abscesses is finding mixed-type organisms on smear gram stain, tissue gram stain, or culture. the oral cavity abounds with mixed bacteria and is estimated to contain some 200 different types of organisms. 121 this is one reason sputum cultures are notoriously difficult to interpret, and why even oral contamination of a bronchoscope interferes with most lung cultures. 122 transtracheal and transthoracic needle aspirations, however, correlate well with blood culture results. i 19. 123 the third most frequent factor in abscess formation is host response. factors that compromise normal host defenses include alcohol ingestion, diabetes mellitus, renal failure, malnutrition, malignancy, and other debilitations, along with treatment with immunosuppressive agents for any reason. patients with these factors do more poorly with pulmonary abscesses (and most other insults) than those without. [124] [125] [126] pathologically, acute cavities have only a thin transition zone into the reactive adjacent lung parenchyma (see numbers of neutrophils and macrophages, along with tissue necrosis. the nearby lung parenchyma has variable findings depending on the rapidity of spread. rapidly growing cavities necrose nearby lung parenchyma, destroying any early attempts at organization. there may be adjacent hemorrhage, exudate, and fibrin extravasation. in those that are slightly more stable, beginning organization occurs in the surrounding lung parenchyma, sometimes chronic cavities heal with a thin fibrous border and may retain a coagulum of necrotic debris in their lumen. this apparently indicates that the nearby bronchi have been sealed off. chronic cavities may resolve by collapse and fibrosis, or may remain open. in the open variety they may become reepithelialized, first with a squamous lining and then with ciliated respiratory lining. 127 in the latter case, the distinction from bronchiectasis or bronchocele may be somewhat confusing, but multiple bronchial connections in a chronic abscess cavity distinguish these entities. spontaneous healing may occur, but healing is greatly aided by appropriate antibiotic dosage. weiss l28 noted in appropriately treated and monitored cavities that 13% of cavities disappeared by 2 weeks of therapy, 44% by 4 weeks, 59% by 6 weeks, and 70% by 3 months. surgery is sometimes indicated for nonhealing cavities or when complications develop, such as hemoptysis, persistent sepsis, bronchopleural fistulas, or empyema. the incidence of abscess formation has greatly decreased during the past 50 years, partly because antibiotics are available and frequently used early in pulmonary infections, and partly because factors leading to of fibrin (2) resting on cellular granulation tissue (3). b. wall of chronic abscess cavity composed of dense, fibrotic, mature granulation tissue. abscess formation are better understood; for example, surgery is avoided with the patient in the upright position or on patients with food in the stomach. in the pre antibiotic era approximately 34% of the patients treated either conservatively or by surgery died, and another one third had chronic residual lung disease. 97 more recently the prognosis is much better, but the mortality associated with established abscess formation remains in the range of25% ? although the term gangrene of the lung has been applied to necrotizing, sometimes putrid, pneumonia in any location, it has more recently been used more specifically to indicate massive necrosis and sloughing of lung associated with severe infection. this entity almost always involves the upper lobes, usually on the right side, and radiographically evolves through typical changes of diffuse infiltrate into multiple cystic spaces that become confluent, leaving a crescent of lung density compressed medially, or occasionally laterally, with a final walled-off area of pus and necrotic lung in an otherwise empty structureless space. curry and colleagues l29 and penner et a1. 130 attribute the first description of pulmonary gangrene to laennec in the 1820s. phillips and rao,l3l who reported four cases, refer to sir william osler's132 description of diseased lung "converted into a horribly offensive greenish, black mass, torn and ragged in the centre" (fig. 5.21) . the patients may cough up large pieces of necrotic lung, which in one case was therapeutic. 130 these fragments histologically show ghosts of lung parenchyma and thrombosed vessels. vasculitis has been described in some reports. 129 ,130 assorted microorganisms cultured from these cases include klebsiella pneumoniae, pseudomonas aeruginosa, haemophilus injluenzae, staphylococcus aureus, streptococcus pneumoniae, and mucor species, 131 anaerobic bacteria probably also playa significant role. 130 in their reviews, phillips and rao l3l and penner and colleagues130 note that similar predisposing factors as those with community-acquired pneumonia, such as aspiration and abscess formation, pertain to this entity, but the location helps distinguish it from the other typical sites of aspiration, when in the upper lobes, it appears to progress through necrotizing pneumonia with thrombosis of arteries (pulmonary and bronchial) and veins, [129] [130] [131] although not strictly abiding by the foregoing definition (of localization in upper lobe), in one case total unilateral lung gangrene was attributed to hilar vessel involvement following treatment of a massive hilar recurrence of hodgkin's disease. 133 pulmonary gangrene is life threatening, and surgical removal of necrotic lung tissue if. tomashefski, jr. , and d.h. dail figu re 5.21. pulmonary gangrene, lung parenchyma is greenish-black, necrotic, and cavitated in this specimen from a patient with central bronchogenic carcinoma, bronchial obstruction, pulmonary artery invasion, and thrombotic occlusion. note hyperemic rim between necrotic lung and apical lung parenchyma. proteus, e coli, and enterococcus cultured, is often indicated since only a few patients survive with antibiotic therapy alone,134 an entire lung, or even one lobe, may rotate around the hilar structures and cause congestion, hemorrhage, or infarction,135 cases of torsion involving only single lobes most commonly occur postoperatively when a portion of ipsilateral lung has been removed,135,136 torsion is an infrequent event. a review of the literature in 1994 by schamaun 137 documented 17 cases of postoperative torsion, five posttraumatic occurrences, and four spontaneous events. in a poll of british thoracic surgeons, however, 35 of 117 responders (30 % ) had encountered at least one instance of torsion,137 torsion has also been documented to occur in transplanted lungs or as a result of pneumothorax or mass lesions. 138 ,139 due to the simultaneous compromise of pulmonary and bronchial arteries and the pulmonary vein, fatal gangrene may occur (see above), the entity of obstructive, golden, or endogenous lipid pneumonia is most commonly seen secondary to tumor obstruction of large airways, but any of the causes of obstruction can lead to obstructive pneumonia. 140 the obstructive effect accounts for a much larger infiltrate on the usual chest radiograph than is caused by tumor alone. the involved area is primarily supplied by the affected bronchus, and the larger the obstructed bronchus, the greater the area involved. however, even when obstruction occurs in the smallest bronchioles, there may be secondary effects in the centriacinar regions. cholesterol pneumonia may also spread into the adjacent nonobstructed segment, and occasionally throughout the lobe. 141 the latter pattern of disseminated spread is often associated with more poorly differentiated or cavitated carcinomas. 141 the involved lung is reduced in size, but not to the extent expected in simple atelectasis. the difference is due to the infiltration by abundant inflammatory cells. the microscopic hallmark of obstructive pneumonia is flooding of air spaces initially by edema followed by fat-filled, finely vacuolated, so-called foamy alveolar macrophages (fig. 5.22a golden-yellow color, hence the term golden pneumonia. 142 obstructed secretions, increased cell breakdown products, and possibly leakage from vessels and interstitium, may give rise to the fat seen in this characteristic reaction. as these products are derived from the lung, this is called endogenous lipid pneumonia. early in its course the alveolar outlines are well defined though distended with foamy macrophages. if the pneumonia is rapidly reversed, lung function may return. gradually, permanent damage ensues, including fibrosis and vascular sclerosis, and it is then difficult to restore lung function even though the obstruction may eventually be reversed. some degree of intraalveolar organization may also be present in approximately 57 % of cases. l44 features of superimposed infection, such as acute inflammation, necrosis, or abscesses are seen in a minority of cases. 142 in contrast to exogenous (aspiration) lipid pneumonia, endogenous lipid pneumonia is characterized by finely vacuolated fat, absence of a foreign-body response, and minimal inflammation and fibrosis of the underlying lung architecture (figs. 5.15 and 5.22b,c). sometimes there are changes that suggest pulmonary alveolar lipoproteinosis (see below). 145.146 at times parenchymal changes very similar to those just described may be present, although no obstruction of bronchus can be identified, so-called idiopathic cholesterol pneumonia. 147 ,148 in 1949, robbins and sniffen 148 described 11 cases of chronic nonobstructive cholesterol pneumonia, 10 of which (91 %) occurred in men aged 32 to 67 years, the only female being a 12-year-old girl. in extent, five of their cases involved most of the lobe and six cases included a portion of one or more segments, often with pleural adhesions; some had small abscess cavities. the involved areas were wedge-shaped with their bases on the pleura and were described as bright yellow; they were accounted for histologically by abundant, finely vacuolated foamy macrophages, but otherwise these areas presented as mass effects. mucoid or mucopurulent exudate filled some bronchioles and bronchi, but no other cause of obstruction was present. bronchiectasis was absent, although focal necrotizing bronchitis was noted. the lobar distribution was not documented. these authors argued against aspiration as they noted the usual aspiration to be more diffuse, often multifocal, and more often seen in lower lobes. lawler able foreign material in 11 (37%) and strongly suspected aspiration in another six for a total of 57% in this series. all except two cases (82 %) of confirmed aspiration were solitary lesions. it seems reasonable that at least some of both chronic organizing pneumonia and idiopathic cholesterol pneumonia, even when not so confirmed, may be the result of aspiration, usually involving ingredients other than exogenous lipid. secondary chronic organizing pneumonia, involving large portions of a lobe or presenting as a mass lesion, is not to be confused with the distinctive form of interstitial lung disease termed cryptogenic organizing pneumonia (bronchiolitis obliteransorganizing pneumonia), which is further discussed in chapter 4. fisher et al. 153 have described a series of patients (six children [<13 years of age] and two adults) with progressive diffuse interstitial lung disease having a combination of histologic features including endogenous lipid pneumonia, interstitial cholesterol granulomas, and patchy alveolar proteinosis (fig. 5.23 ). three patients in this study had severe combined immunodeficiency, two had pulmonary hypertension, and one each had cystic fibrosis (cf), trisomy 10q, and ventricular septal defect (vsd), or lysin uric protein intolerance. all patients exhibited delayed growth, five had digital clubbing, six had depressed appetite or anorexia, five were anemic, and three experienced hemoptysis. six of eight patients also had evidence of gastroesophageal reflux, which the authors suggest is important in the pathogenesis of this condition. 153 pulmonary function tests in four patients showed either restrictive or mixed restrictive and obstructive physiology. chest x-rays predominantly showed nodular pulmonary opacities, while bronchiectasis and perihilar or mild hazy parenchymal infiltrates occurred in one patient each. 153 the mechanism whereby gastroesophageal reflux might elicit this interesting triad of histologic findings is through recurrent micro aspiration of gastric content with associated bronchospasm (see discussion of alveolar proteinosis in chapter 21). 153 the differential diagnosis of cholesterol pneumonia also includes drug reactions, notably reactions to amiodarone. the clinical history and documentation of amiodarone therapy should facilitate the correct diagnosis (see chapter 22 and fig. 22.8) . while the foamy macrophages in both of these disorders resemble each other histologically, electron microscopy demonstrates more abundant osmiophilic dense bodies and giant lamellar bodies in many different cell types in amiodarone toxicity compared to similar but smaller inclusions within macrophages in endogenous lipid pneumonia.146.153~156 compared to endogenous lipid pneumonia, amiodarone toxicity also encompasses a prominent inflammatory response that may include fibrosis, organizing pneumonia and diffuse alveolar damage (see chapter 22) . 156 other drugs that have been implicated as a cause of endogenous lipid cholesterol granulomas not only are associated with endogenous lipid pneumonia and pulmonary alveolar proteinosis, but also have been attributed to pulmonary hypertension, organizing hemorrhage, and a unique case of excessive consumption of apples.160-162 kay and colleagues 163 suggest that cholesterol granulomas in patients with pulmonary hypertension are more likely due to other concomitant processes characterized by type ii pneumocyte hyperplasia and degeneration. atelectasis is the collapse of aerated lung. most often it is caused by internal bronchial obstruction of the air flow (absorption atelectasis) (see fig. 15 .2 in chapter 15), but it may result from external compression of the lung, such as by empyema, mesothelioma, constrictive pleural fibrosis, or tumors, or by internal compression, secondary to a bulla, tumor, or other space-occupying lesions (compressive atelectasis). pneumothorax is an important cause of atelectasis in the ipsilateral lung. atelectasis may also be caused by a change in metabolism or surface-wetting balance such as with hyaline membrane disease, ards, infection, or gastric acid or other aspiration with loss of pulmonary surfactant. it may also have vascular causes as with embolism, postoperative splinting, obesity (e.g., pickwickian syndrome), or secondary to nerve or muscle dysfunction of diaphragm or chest wall. following complete airway obstruction, atelectasis occurs when alveolar oxygen and nitrogen are absorbed. atelectasis is also commonly seen around chronic inflammatory reactions such as bronchiectasis, and may be part of the sequence of events leading to bronchiectasis (see below). pathologists must be cautious, however, because most often, when observed histologically, atelectasis is artifactual. upon the release of negative pressure, air escapes from the lung when the thorax is opened or when lung tissue is excised, and this collapse may cause confusion with preexisting atelectasis (see fig. 1 when bronchial obstruction is partial, it may easily lead to air trapping, as discussed in chapter 15 (see asthma). obstruction of segmental bronchi usually does not cause atelectasis because of preserved collateral ventilation. a collapsed lung may be an isolated cause of fever, but is also a frequent site for superinfections, such as in postoperative patients. 164 chronic atelectasis may lead to irreversible scarring. the special situation of rounded atelectasis is discussed in chapter 27 on asbestos-related pathology. bronchiectasis simply defined refers to dilatation of bronchi. included in this broad definition are conditions such as traction bronchiectasis secondary to parenchymal scarring; airway dilatation accompanying parenchymal loss as in emphysema; or reversible dilatation, which may be seen radiographically in atelectasis or pneumonia. 165 -172 a more selective definition of bronchiectasis, and the type usually understood by pathologists, is irreversible fixed airway dilatation associated with inflammation and destruction of bronchial matrix components. 173 • 174 bronchiectasis can be further categorized as localized or diffuse/multifocal. causes of localized bronchiectasis, the most important of which is airway obstruction, are listed in table 5 .1. localized bronchiectasis may also have an infectious etiology, most notably pulmonary tuberculosis (see fig. 9 .13 in chapter 9). obstructive bronchiectasis is most commonly seen beyond endobronchial tumors (fig. 5.24 ), but foreign bodies, concretions such as broncholiths, secretions such as inspissated mucus in mucoid impaction and allergic bronchopulmonary aspergillosis (see chapter 15), strictures, or compression as by tumor or enlarged nodes may play a role. rarely, lack of cartilaginous support with airway collapse, bronchial atresia, or mucosal webs may be associated with bronchiectasis. obstructive bronchiectasis occurs anywhere obstruction occurs, but there are some localizing factors in a few of these conditions: the upper lobe in allergic aspergillosis or with primary epithelial tumors, which are more common in this site; the middle lobe with its tendency toward airway compression (middle lobe syndrome); and localized bronchiectasis governed by the usual routes of aspiration (covered earlier in this chapter). there are many exceptions, and bronchoscopy is usually indicated in both children and adults to diagnose the type of obstruction. localized bronchiectasis is often successfully treated by surgical resection or elimination of the cause of bronchial obstruction. diffuse or multi focal bronchiectasis is usually of the nonobstructive type, the major causes of which are listed in table 5 .2. it is this type that is more frequently a cause of significant chronic obstructive pulmonary disease (copd) and respiratory failure. nonobstructive bronchiectasis occurs most frequently in the basal segments of the lower lobes, often sparing the superior segment and the anterior basal segment. it is found more than twice as frequently in the left lower lobe as in the right. 17s -j77 next in frequency are the right middle lobe and its counterpart, the lingula. these areas of the lung may have the poorest drainage. the upper lobes may be involved but are usually not solely involved by nonobstructive bronchiectasis. tuberculosis more selectively causes bronchiectasis in the upper lobes, and cystic fibrosis should be considered in cases of upper lobe involvement without a definitive etiology. about one third of the cases of nonobstructive bronchiectasis have bilateral involvement. 176.178,179 macroscopically bronchiectasis typically involves the second to the eighth order of segmental bronchi, sparing the larger, more proximal airways, which are protected by a firmly supporting cartilaginous network. 179 -181 more distal airways are often obliterated or effaced as the number of bronchial divisions is reduced (fig. 5.25 ).173,182 within a bronchopulmonary segment there may be patchy involvement (see fig. 1.9 in chapter 1). there is also apparent loss of more distal lung parenchyma as the dilated airways approach the visceral pleura ( fig. 5.25) . 173, 182 bronchiectasis has been divided into many different patterns grossly and radiographically. the most widely applied classification is that suggested by reid l82 : (1) saccular (cystic), (2) cylindrical (fusiform or tubular), and (3) varicose. in the saccular form, the distal extensions of the bronchi are more dilated than proximal portions, described by reid as "globular ballooning." usually, the second-to fourth-order bronchi are involved. cylindrical bronchiectasis consists of evenly enlarged tubular dilatation of bronchi, usually involving the sixth-to eighthorder bronchi. is i on bronchograms dilated bronchi come to an abrupt, square-ended termination thought to be caused by impacted endobronchial secretion.173 the varicose type describes focal dilatation separated by more narrow areas (see fig. 1.9 in chapter 1). these various gross patterns were previously best visualized by bronchography, and are not specific for any given etiology, although certain clinical-pathologic correlations have been made (see below). whitwell 175 integrated histologic features into his classification of follicular, saccular, and atelectatic bronchiectasis. follicular bronchiectasis most frequently begins in childhood as the sequela of viral infections, most notably adenovirus. 183 saccular bronchiectasis, in whitwell's series, was often found to be postinfective or idiopathic. the pathogenesis of atelectatic bronchiectasis was linked to lobar bronchial obstruction, often by enlarged lymph nodes. congenital bronchiectasis, purportedly due to cartilage deficiency in the bronchial walls, is a controversial entity discussed elsewhere in this chapter under the williams-campbell syndrome. 180 ,1s1,184 not to be confused with congenital bronchiectasis are those hereditary conditions, such as cystic fibrosis or primary ciliary dyskinesia, that predispose to the subsequent development of progressi ve bronchiectasis (see below). 174, 180 patients with bronchiectasis typically present clinically with forceful cough, purulent sputum production, wheezing, and recurrent pneumonia in the bronchiectatic zones. wet bronchiectasis refers to abundant inflammation and mucus hypersecretion, whereas dry bronchiectasis refers to minimal sputum production. dry bronchiectasis is most common in the upper lobes, is often of the cylindrical type, and probably relates to better drainage in this zone. hemoptysis commonly presents as blood-streaked sputum, but may be massive and life-threatening. frequently purulent sinusitis accompanies bronchiectasis and may contribute to its development. the chest radiograph is usually abnormal in bronchiectasis, including specific features of ring-like shadows due to dilated airways seen on end, or of tram lines when the airways are visualized longitudinally. iss high-resolution ct scan is the best current modality for diagnosing bronchiectasis, revealing airways that are dilated relative to the adjacent blood vessels, lack of airway tapering, constrictions along the path of the airway, and terminal balloon-like cysts. is5 ,is6 pulmonary function tests show obstructive changes with reduced forced expiratory volume in 1 second (fev1)/ forced vital capacity (fvc) and frequently airway hyperresponsiveness. 185 various theories have been proposed to explain nonobstructive bronchiectasis. inflammation seems to best account for the changes that are observed, including the fact that the involved zones of lung are those most difficult to drain. it was known even in the 1930s and confirmed in subsequent decades that respiratory infection often preceded bronchiectasis. 175 ,176,187-192 usually older children and young adults have the well-developed disease pattern, but also have a history of infections before the age of 2 or 3 years, with recurrent respiratory problems dating from this time. 187 many patients may appear stable and do well for some time, and then develop a progressive course of recurrent infections and systemic toxicity. 174, [177] [178] [179] [180] [181] [182] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] in approximately 50% of cases of bronchiectasis, however, a specific inciting factor is not identified-so-called idiopathic bronchiectasis . 195 in these patients, childhood respiratory infections especially those likely to have produced bronchiolitis obliterans, are presumed to have initiated the process of bronchiectasis. 173 ,195,196 viral infections may be important in many cases. 167 ,194,196-203 glauser and associates 204 noted in an extensive review that measles and pertussis immunizations probably have played a significant role in decreasing the incidence of bronchiectasis. bacteria also playa significant role, both in primary infections (see following) and in superinfections or reinfections in areas of previous injury. aggressive treatment of pediatric pulmonary infections with antibiotics has helped to make bronchiectasis a disappearing disease. 204 historically, the impact of immunizations and antibiotics on the declining incidence of bronchiectasis occurred at about the same time, and it is difficult to differentiate their effects; nonetheless, this association supports the role of early infection in initiating bronchiectasis. excluding cases of kartagener's syndrome, the concurrence of sinusitis and bronchiectasis is greater than expected. as early as 1929, quinn and meyer20 noted a 58% incidence of chronic sinusitis in cases of bronchiectasis. aspiration of infective material from the sinuses may playa role. however, another study noted a 15% incidence of sinusitis in cases with less than 5 years of symptoms of bronchiectasis compared to the 44% incidence in all cases of bronchiectasis. 179 h. infiuenzae, a common pathogen of the upper respiratory tract, is also found with some regularity in lung cultures from patients with bronchiectasis. anaerobic bacteria, reflecting endogenous oral flora , may also be cultured from bronchial secretions. long-term antimicrobial treatment may be required for complete eradication of these organisms. the role of recurrent infection in perpetuating and aggravating bronchiectasis cannot be overemphasized. this has been documented in children and adults. the dilatations of the bronchial contours, their irregularities, their relative stenosis at the proximal end, altered secretions and exudate, surface mucosal ulcerations, and metaplasia all playa role in hampering adequate drainage. a vicious cycle ensues as the injured area perpetuates further injury, leading to increased damage and progressive bronchiectasis. necrotizing inflammation involves bronchial walls and adjacent parenchyma (fig. 5.26 ) . some scarring probably takes place in healing, with retraction of surrounding tissue. retraction occurs circumferentially, and bronchial dilatation results. as noted, the more distal bronchi and bronchioles are often destroyed. there is also general lung contracture due to atelectasis of involved zones, while nonaffected lobes may undergo compensatory hyperinflation (fig. 5.26) . the basic principles of fibrosis and contraction also apply to traction bronchiectasis seen in interstitial fibrosis and honeycombing.172 traction bronchiectasis is usually not as marked as primary bronchiectasis, and is localized and most severe in the peripheral subpleural zones where fibrosis is often most prominent (see chapter 19) . 172 grossly, the involved lung tissue is usually atelectatic, gray-blue, shrunken, and rubbery. there may be zones of golden or obstructive pneumonia, and sometimes these zones form layers around the dilated bronchial tubes. it may be difficult or impossible to adequately inflate such a chronically contracted specimen. the involved bronchi are dilated instead of following their smoothly contoured courses as they extend peripherally. these dilated bronchi almost reach the pleural surface and run in a somewhat parallel or radial fashion without interbronchial connections (figs. 5.25 and 5.26). partially or totally circumferential thin folds in the mucosa extend internally from the wall and are seen as transverse infolded pleats on the bronchial cast (see fig. 1.9 in chapter 1). these give the appearance of webs or bands of mucosa. there are variably sized outpouchings, larger ones between the remnant bronchial cartilages, and dilated smaller pits that appear to be dilated submucosal glands. grossly, elastic fibers can be seen still running through the wall, but these are more widely separated than is normal because of the stretched diameter of the bronchus. in wet bronchiectasis there is thickening of the wall, and mucinous, granular, semisolid material accumulates within the lumen (fig. 5.27 ). occasionally this material hardens and even calcifies (see broncholithiasis, below). in dry bronchiectasis the wall is thin, almost translucent, and gray-pink without mural thickening. microscopically the respiratory mucosa may be intact, show squamous metaplasia, or be ulcerated or inflamed (fig. 5.28 ). the bronchial walls are usually chronically inflamed. submucosal glands and surface goblet cells are not prominent and may decrease, although they may occasionally increase. elastic tissue is preserved except in areas of necrosis (fig. 5.28 ). smooth muscle is usually present and often shows some degree of hypertrophy; occasionally this is atrophic. cartilage seems less obvious and occasionally is eroded, but most often appears normal histologically. in advanced saccular bronchiectasis cartilage is markedly reduced or absent. 173 ,205,206 neutrophils, macrophages, and desquamated and mucinous debris are present in the bronchial lumen in wet bronchiectasis. acute inflammatory cells may infiltrate the bronchial wall or the adjacent lung parenchyma depending on the status of inflammation and active infection at the time of lung removal. as these patients are subject to recurrent infections, acute pneumonia may also be present. lymphocytes and plasma cells usually predominate in bronchial wall and surrounding lung tissue. in follicular bronchiectasis, hyperplastic lymphoid follicles may appear to constrict the bronchial lumens (fig. 5.29 ).175 there may be a degree of obstructive pneumonia correlating with the gross yellow color. small granulomas are present in a few cases, apparently as a reaction to inspissated material within the bronchi. if granulomas are extensive or present in the adjacent lung parenchyma, in more normally contoured segmental and subsegmental bronchi, or in lymph nodes, one must consider fungal or mycobacterial infections. if granulomas are confined to the injured areas, one must also consider aspiration. bronchioles are often constricted or obliterated beyond the dilated bronchi (fig. 5.30 ). other small airways may be dilated and sometimes mucus-filled probably because of their obstruction at the junction with the larger bronchi. foci of carcinoid atypical proliferation (tumorlets) occur with some frequency in bronchiectasis (see chapter 36) . bronchial arteries respond to sustained inflammation, and may exceed 1 mm in diameter. 207 ulceration of these systemic arteries accounts for the bright-red appearance of hemoptysis. the right middle lobe and occasionally its left-sided counterpart the lingula, have lobar bronchi that branch from their parent supply at a more acute angle than most other dividing bronchi (see chapter 2) . the middle lobe bronchus is relatively narrow, and there are frequently moderately prominent nodes in the angle of bifurcation that may compress and further constrict the bronchus. the subcarinal node may even approach this angle. several authors have also suggested there is less effective collateral ventilation in the middle, compared to the adjacent upper lobe. 208 .209 because of these anatomic characteristics there is a greater tendency toward middle lobe and lingular atelectasis, inflammation, nonspecific scarring, broncholith formation, and bronchiectasis-collectively termed middle lobe syndrome (mls) .2io in addition to peribronchial lymphadenopathy, mls can result from numerous disorders including asthma, tuberculosis, foreign bodies, cf, broncholiths, endobronchial silicosis, cardiovascular and of bronchiolar wall. white arrow indicates cross section of separate, obliterated airway (patient with cf) (elastic van gieson stain). bronchopulmonary malformations, and allergic bronchopulmonary aspergillosis. 2 11-213 the pathologic findings in resected lung specimens of 21 patients with middle lobe syndrome have been comprehensively described most recently by kwon and colleagues,212 and are delineated in table 5 .3. although the histologic findings are nonspecific, a combination of bronchiectasis, bronchiolitis, and atelectasis is typical. 212 in this series, a mechanical obstruction (broncholith) was identified in only one patient. 212 as early as 1966, culiner 208 also recognized bronchial patency in most cases of middle lobe syndrome. the current understanding suggests that mls is due to recurrent infection related to poor lung drainage, possibly associated with intermittent obstruction of the precariously situated bronchi in the setting of reduced collateral ventilation of the middle lobe.208.212 broncholiths represent calcified material in the airways.214-220 they most commonly are calcified lymph nodes that compress bronchi and either partially or completely erode through the bronchial walls (fig. 5.31 ).217.221 they then may be expectorated (lithopytsis) (fig. 5.32) calculus (scale equals 1 cm). c. rare yeast-like organisms, consistent with histoplasma, were identified in the necrotic center of the broncholith (gomori methenamine silver stain). or aspirated and cause hemorrhage or obstructive changes, including cough, atelectasis, pneumonia, abscess formation, bronchiectasis, or air trapping. broncholiths form less often from chronic reaction to retained aspirated material or eroded fragments of calcified or ossified bronchial cartilage. 18o they may also occur with retained mucus as in bronchiectasis. 180 historically, "spitting stones" dates back to descriptions by aretaeus, galen, and aristotle. 218 although usually less than 1 cm in diameter, a record-sized calculus of 139g c/3ib) occurred in a patient who also had produced multiple sand-like or melon-seed-sized calcified particles. 214 the pathognomonic finding of lithoptysis is fairly rare and was seen in only two of 43 (5%) cases by faber et al. 218 and six of 41 (15%) cases by schmidt et au 16 the regional nodes usually calcify from old granulomatous disease, and tuberculosis is the most common etiology worldwide while histoplasmosis is the most common etiology in the united states. 219 other infectious agents include coccidioides, cryptococcus, actinomyces, or no cardia. 219 ,22o,222,223 the latter two organisms probably represent superinfections of necrotic debris.224 silicotic lymph nodes may also cause a similar reaction. 225 ,226 men and women are about equally affected, and although calcified nodes may occur at any junction of the bronchial tree, they are 2 to 6.5 times as common on the right side, and favor the anterior superior segment of the upper lobe and the bronchus intermedius, along with the right middle lobe bronchus, where they may produce the middle lobe syndrome. 217 ,227,228 the superior segment of the lower lobe is also a site of occurrence. occasionally, erosive calcified nodes may cause bronchopleural fistulas and are the most common cause of bronchoesophageal fistulas, [228] [229] [230] retraction diverticula of the esophagus may also occur secondary to peribronchial fibrosis and calcified mediastinallymph nodes associated with broncholiths. 218 ,231 calcified nodes have also been studied with ct scans.232 in the retrospective series by conces et al.,227 of 15 patients with ct-proven broncholiths, 11 (73 % ) had juxtabronchial calcified nodes identified on chest radiographs. calcified intraparenchymal nodules were seen radiographically in only four (27%) patients. bronchoscopy is less accurate in detecting calcifications, ranging from 28% to 56% of cases. rarely, calcifying tumors such as an ossifying bronchial carcinoid or endobronchial hamartoma can cause confusion (see chapters 36 and 40) . 232, 233 histologically broncholiths appear similar to calcified fibrocaseous lymph node lesions. the outer surface of the often sharp-edged calculus may be coated with inflammatory exudate or, in cases of actinomyces superinfection, eosinophilic rays (splendore-hoeppli phenomenon). 221, 223 the gms stain may disclose histoplasma yeast forms in the centrally necrotic area of the calculus (fig. 5.32b) . 224, 234, 235 the airway in which the calculus is lodged is typically stenotic, with mural fibrosis and chronic inflammation. within the chest, fistulas may be bronchopleural, bronchocutaneous, bronchomediastinal, or bronchoesophageal in their connections. an aortobronchial fistula is a rare (and often fatal) complication of previous aortic or cardiac surgery,236 bronchopleural fistulas are the most common form and often are secondary to surgery, such as from a leaking postoperative bronchial stump. other causes include necrotizing pneumonia or abscess, penetrating wounds, eroding granulomatous disease, penetrating broncholiths, or malignancies. see chapter 6 for congenital causes. extrathoracic bronchial fistulas include connections with bile ducts, pancreas, and other assorted sites. bronchocele means one or more dilated bronchi filled with fluid, which may be mucinous (bronchomucele) or purulent (bronchopyocele ).237 this condition is caused by stenosis or occlusion of the proximal end of dilated sac(s), and therefore differs from bronchiectasis and mucoid impaction, in which proximal ends are generally still patent. it may be either congenital, or early or late acquired, usually of postinflammatory nature but sometimes of malignant nature.238 localized emphysema, which occurs around the bronchocele, may be caused either by inflammation early in lung growth with continued traction-type effects on nearby lung, or by sustained air-trapping due to airway obstruction. 237 ,239.24o many cases are reported as bronchial atresia.241-245 bronchocele/ atresia may present in adults or children and typically affects the left upper lobe. a characteristic ct appearance is that of a branching mass surrounded by hyperlucency. 246 an irregularly cylindrical (sometimes branched) thin-walled cyst (fig. 5,33a) grossly and histologically resembles a bronchocele lined by respiratory or squamous epithelium. 238 ,24o,242 occasionally a scar or intrabronchial web proximal to the lesion represents the remnant atretic or occluded bronchus. the adjacent bronchial arteries may appear hypertrophic, especially if there have been recurrent infections (fig. 5.33b) , bronchocele may be a relative of saccular bronchiectasis and may be the etiology for some so-called intraparenchymal bronchogenic cysts (see below and chapter 6).247 mucoid impaction may also be related to an allergic effect, often to noninvasive aspergillus (see chapter 15), usually does not have proximal bronchial stenosisocclusion, and has more eosinophils and cellular debris in the mucus, in addition to intraluminal hyphae. bronchocele/atresia is distinguished from intralobar seques bronchogenic cysts are closed sacs lined by respiratory mucosa, usually with bronchial glands, smooth muscle, and cartilage in their walls. they often represent congenital fragments that drop off or are remnants of the original budding of the lungs from the primitive endodermal canal. they are most common in the middle mediastinum where they account for 10% to 15% of all primary mediastinal masses but can be seen as isolated masse es) in the lung. within the lung, some may form as bronchoceles as discussed previously. a series of 86 cases of bronchogenic cyst, with 66 (77%) in the mediastinum and 20 (23%) in the lung, was presented by st. georges et al. 248 from montreal. a similar distribution was recorded in adult patients by patel and colleagues. 249 of interest, 75% to 90% of those in the lung were symptomatic at the time of operation, most often because of infection or bronchial obstruction,z48,249 although suspected, a preoperative diagnosis was not correctly made in any case in the large montreal series. 248 the presence of bronchial epithelial cells on trans bronchial fine-needle aspiration (fna) was found not to be specific for the diagnosis of bronchogenic cyst. 249 most occur in the lower lobes, but all lobes may be affected.248-z50the ct appearance is that of a well-defined hypertrophic bronchial arteries (ba). lumen of cyst is at top (movat stain). ovoid lesion, with surrounding mosaic and band-like linear attenuation consistent with emphysema and bronchiolar metaplasia/fibrosis. 250 a bronchioloalveolar cell carcinoma arising in a bronchogenic cyst in a 37-year-old woman has been reported as a rare association.251 bronchogenic cysts are uncommon in adults and are further discussed in children in chapter 6. bronchorrhea is arbitrarily defined as production of more than 100ml of sputum per day.252 although it is a clinical symptom, pathologists may ponder the differential diagnosis if faced with this history on a specimen request card. bronchorrhea may be idiopathic, or secondary to chronic bronchitis, bronchiectasis, scleroderma, asthma, mucinous bronchioloalveolar carcinoma, metastatic mucinous adenocarcinoma, tuberculosis, or relapsing polychondritis.252-258 cytology exams, cultures, or trans bronchial biopsies may help evaluate at least some of these possibilities. cystic fibrosis is a prototypic example of bronchocentric inflammation and bronchiectasis and the most common lethal genetic disease among caucasians, having a frequency of approximately 1 in 2500 live births.259-261 the molecular defect of this autosomal recessive disorder was discovered in 1989 to involve mutations in a 1480 amino acid polypeptide, the cystic fibrosis transmembrane conductance regulator (cftr), encoded by a gene on the long arm of chromosome 7. 262 ,263 over 1000 different mutations of the cftr gene have so far been identified, but the most frequent mutation worldwide and the most severe genetic lesion, is the deletion of phenylalanine at position 508 of cftr (af508), accounting for over 70% of affected patients. 26o , 264 cystic fibrosis transmembrane conductance regulator functions as a cell membrane-associated, cyclic adenosine monophosphate (camp)-regulated chloride channel, which also has regulatory activity on the absorption of sodium through a separate epithelial channel (enac).264-266 the structure of cftr is schematically depicted in figure 5 .34. mutations in cftr have been grouped into six major types, each of which may present phenotypically as cf: (1) lack of synthesis of cftr; (2) defective processing of cftr such that it does not reach the cell membrane; (3) aberrant regulation of ion transport due to dysfunctional cftr; (4) abnormal conductance of chloride ions; (5) partly defective production and processing; or (6) accelerated turnover at the cell surface ( fig. 5.35) . 259, 261, 267, 268 the af508 mutation is a type 2 defect in which abnormal cftr is sequestered within cellular organelles leading to reduced insertion into the cell membrane, markedly limiting the ability of chloride to cross the membrane. 269 in epithelial cells of bronchi, biliary tract, and intestine, impaired transport of intracellular chloride and its accompanying water molecules leads to dehydration of ductal and lumen secretions. 26o in bronchial epithelium there is also enhanced intracellular absorption of sodium ions, which further dehydrates secretions within the airway lumen. 26o ,264,266 in contrast, the uptake of extracellular chloride is inhibited in sweat ducts, causing an elevation of sweat chloride concentration, a key diagnostic indicator of cf. 260 ,270 the manifestations of cf are protean, involving nearly every organ system either directly or secondarily. the correlation between genotype and phenotypic expression is best exhibited for pancreatic function and is relatively poor for pulmonary manifestations. 267 ,271,272 however, certain mutations such as a455e or the ivs8 5t allele are associated with relatively mild lung disease that may initially present in adults.m-276 a unifying feature of the pathophysiology of cf is impaction of viscid secretions in exocrine gland ducts leading to cardinal manifestations such as intestinal obstruction (e.g., meconium ileus); pancreatic acinar atrophy and fibrosis with consequent metabolic insufficiency (due to intestinal malabsorption); organ maldevelopment (e.g., congenital bilateral absence of the vas deferens); hepatic fibrosis (focal biliary cirrhosis); and infection associated with mucus stasis (e.g., infective bronchitis).26o,277 pulmonary involvement is usually the lungs in cf are structurally normal at birth. dilatation of mucous gland ducts followed by intrabronchial mucus stasis are the earliest pulmonary lesions seen in infants. 261 ,279,28o it has long been recognized that patients with cf are predisposed to lung infection. 28 1.282 current hypotheses suggest that susceptibility to infection may be related not only to entrapment of bacteria in thick bronchial secretions, but also possibly to abnormal binding and reduced uptake of bacteria by epithelial cells, or impaired epithelial antimicrobial protection provided by defensins (natural antibiotics of the innate immunity system).259.283-289 even in infants without apparent infection, however, bronchoalveolar lavage studies document ongoing bronchial inflammation associated with increased levels of endobronchial il-8, a potent cytokine that recruits neutrophils into the inflammatory response, and relatively decreased levels of il-lo, an inhibitor of proinflammatory cytokines. 29 g-294 it is as yet undetermined whether or not intrinsically exaggerated inflammatory responses are the direct result of mutations in cftr. 295 infection and inflammation stimulate bronchial mucus secretion leading to a vicious cycle of worsening airway infection and obstruction, progressing to chronic bronchitis, bronchiolitis obliterans, and bronchiectasis. 286 the chronic pulmonary complications of cf evolve from the airway disease.277 hyperinflation or collapse is the direct result of bronchial obstruction. air trapping and postinflammatory cystic lesions underlie an increased susceptibility to recurrent pneumothorax. pulmonary hypertension and cor pulmonale derive from sustained hypoxia, while hemoptysis is a direct effect of bronchiectasis and bronchial artery hypertrophy. 277 endobronchial infection tends to occur in sequential fashion, initiated by s. aureus, followed by h. injluenzae, and finally by p. aeruginosa (mucoid strains).260.2 8 submucosal glands are enlarged and chronically inflamed behind ducts that are obstructed by dense, inspissated, eosinophilic secretion (a characteristic, but not pathognomonic feature of cf) (fig. 5.36 ).300-302 although bronchial smooth muscle in individual patients may appear hypertrophic, its mean volume density is within the normal range. 300 in patients with cf-associated lung disease, saccular bronchiectasis is usually present beyond 4 months of age.27r although all bronchopulmonary segments may be affected, bronchiectasis tends to be more severe in the upper lobes (fig. 5.37 ).301.303.304 blind-ended ectatic airways, devoid of cartilage, are surrounded by atelectatic, chronically inflamed, and fibrotic parenchyma (see fig. 5 .27).206 bronchial mucosa is frequently denuded or ulcerated leaving the bronchial surface lined by highly vascular granulation tissue that is rich in histiocytes (see fig. 5 .28). in severe disease, bronchi terminate in large, juxtapleural, thin-walled cavities that present radiographically as contiguous, bubble-like cysts. intrapleural blebs or emphysematous bullae are less common forms of cystic lesions, which contribute to an increased incidence of pneumothorax.305.306 extensive acute and chronic bronchiolitis and bronchiolar mucoid impaction impart a finely nodular texture to the parenchymal surface and account for a micronodular radiographic appearance ( fig. 5.38 ).307 bronchiolitis obliterans, predominantly of the constrictive type, contributes importantly to airway obstruction, and likely precedes the development of bronchiectasis (see fig. 5 .30).301.308 occasionally, occlusion of respiratory bronchioles by polypoidal protrusions of fibroblastic tissue accompanies interstitial and organizing pneumonia?09 small airway density decreases with age and is most significantly reduced in patients with hypercapnia. 310.311 the lung parenchyma is grossly indurated by multifocal, bronchocentric chronic pneumonia and fibrosis, with features of both organizing pneumonia and endogenous lipid (cholesterol) pneumonia. a variable degree of acute bronchopneumonia may also be seen at autopsy. some patients who are colonized by burkholderia cepacia undergo an accelerated decline due to acute necrotizing pneumonia (see figs. 8.61 and 8.62 in chapter 8). other patients colonized by burkholderia follow a more protracted course, similar to those colonized by p aeruginosa. [312] [313] [314] fungi and nontuberculous mycobacteria may also colonize cf airways and contribute to lung destruction. 288 ,315,316 bhargava and colleagues 317 identified fungal organisms histologically in 21 % of cf patients retrospectively studied at autopsy. the dilated, obstructed airways of cf patients are predisposed to fungal colonization, accounting for an increased prevalence of allergic bronchopulmonary aspergillosis (abpa) of approximately 2.0%.318 infrequently the pathologic features of abpa, including bronchocentric granulomatosis, are superimposed on chronic cf-associated airways disease (see fig. 15 .19 in chapter 15).277 in cf patients with non tuberculous mycobacterial infections (often due to mycobacterium avium or rapidly growing strains like m. chelonae or m. abscessus), necrotizing fibrocaseous granulomas may be present. 315 ,316,319,320 pulmonary lesions are most likely to be found in patients with repeatedly positive sputum cultures for mycobacteria (see chapter 9) . 320 the pulmonary vascular changes of cf-associated lung disease are usually pronounced, chronic hypoxia and inflammatory changes contribute to pulmonary artery medial hypertrophy and intimal fibrosis of muscular pulmonary arteries and medial myxoid degeneration of elastic arteries.321-326 postmortem arteriograms often show abnormally tapered arteries with a reduced background haze (see fig. 1 .8 in chapter 1).277 morphometric studies provide evidence of a decreased density of arteries, which correlates inversely with the degree of right ventricular cardiac hypertrophy,321 the dropout of arteries may be related to impaired postnatal growth or to vascular destruction secondary to chronic hypoxia or sustained inflammation. 321 right ventricular cardiac hypertrophy, seen at autopsy in approximately 84% of cf patients older than 3 years of age, is a direct consequence of pulmonary artery remodeling and associated pulmonary hypertension. 321 bronchial arteries also undergo significant hypertrophy as a response to sustained bronchial inflammation, bronchiectasis, and bronchocentric abscesses (fig. 5.39 ).327-329 the source of hemoptysis in cf patients is most frequently the delicate capillaries within airway granulation tissue (see fig. 5 .28),330,331 occasionally, mucosal ulcers erode into hypertrophied bronchial vessels leading to life-threatening massive hemoptysis (fig. 5.39 ). interventional bronchial artery embolization of metal coils, polyvinyl alcohol (ivalon), or gelfoam particles is undertaken to induce thrombosis and control bronchial artery bleeding. 331 ,332 degenerated remnants of embolized polyvinyl alcohol may surround stenotic or occluded bronchial arteries in patients who have undergone this procedure. 333 bronchopulmonary arterial anastomoses may further allow the paradoxical entry of small embolized particles into the pulmonary arterial circuit (see fig. 28 .42 in chapter 28). 334 other less frequently reported complications of cystic fibrosis include systemic amyloidosis, intralobar sequestration, and anaerobic lung abscess. 335 -338 emphysema is usually a minor feature, localized to bronchiolocentric scars or as paraseptal emphysema in the upper lung zonesys,30s,329 cystic fibrosis patients with indwelling venous access devices may surreptitiously inject aqueous suspensions of psychoactive pharmaceutical tablets leading to pulmonary artery obstruction due to embolized tablet filler materials (see chapter 26).339 primary ciliary dyskinesia (pcd) is an autosomal recessive disorder, occurring in approximately 1 of 15,000 to 30,000 persons, characterized by the absence or dysregulation of ciliary movement mainly due to ultrastructural defects in the ciliary axoneme. (see chapter 2)?40-344 cilia on the respiratory epithelial surface play an important role in propelling mucus, bacteria, and inhaled particulate debris out of the lung (see chapter 3). as a result of impaired clearance due to ciliary malfunction, patients with pcd are predisposed to chronic sinusitis, serous otitis, and recurrent bronchopulmonary infections beginning in early childhood. 345 ,346 primary ciliary dyskinesia has also been implicated as a cause of neonatal respiratory distress syndrome. 347 ,348 male patients are usually infertile due to poor flagellar motility of sperm. 345 approximately 50% of patients with pcd also have situs inversus secondary to abnormal rotation of embryonic epithelia consequent to the lack of ciliary movement. 343 the syndromic triad of situs inversus, sinusitis and bronchiectasis was first proposed by kartagener in 1933, and is now designated kartagener's syndrome (fig. 5.40 ).349.350 while pcd is an important cause of bronchiectasis, the prognosis is generally more favorable than that of cf. 340 in 1976 afzelius 340 and pedersen and mygind 341 were among the first to recognize that ultrastructural abnormalities of ciliary dyne in arms were associated with kartagener's syndrome. originally termed immotile cilia syndrome by afzelius, it is now recognized that there are numerous structural variations that may contribute to if. tomashefski, jr., and d.h. dail pcd, and that cilia are not always immotile?51 when compared to normal (fig. 5.40a) , the most commonly observed ultrastructural defects are the complete absence of dyne in arms or the selective absence of either inner or outer arms (fig. 5.40b ). other derangements of the axoneme contributing to pcd include defective or absent radial spokes (fig. 5.41d ), transposition of microtubules (fig. 5.41c ) (well seen in longitudinal sections of cilia), central microtubular agenesis, absence of nexin links, agenesis of cilia, or rarely, bizarre cystic dilatation of ciliary shafts. 352 -356 nonspecific findings such as ciliary blebs, megacilia, compound cilia, and displaced microtubules may accompany the more specific defects, but are also frequently present in inflammatory airway disease of diverse causes including infectious bronchitis, cf, or air pollution. 35 6-361 some patients with structurally normal cilia and a normal ciliary beat frequency may develop the clinical manifestations of pcd due to ciliary disorientation, resulting in uncoordinated ciliary motion (fig. 5.40e ).362,363 ciliary disorientation has also been described in individuals with infectious bronchitis (including cf), but the degree of disorientation is usually not as great as in those in whom the defect is primary, and the disorientation secondary to infection typically resolves after effective antibiotic treatment. 299 the diagnosis of pcd is established by ultrastructural analysis of respiratory epithelium in conjunction with typical clinical manifestations, exclusion of other causes of chronic airway inflammation, and documentation of abnormal ciliary motion by phase contrast microscopy.344.364,365 mucosal samples obtained by endoscopic biopsy or brushing are examined by transmission electron microscopy. in patients with pcd, nasal mucosal samples are reflective of bronchial changes when most cilia are abnormal. when only few cilia are structurally abnormal in a patient in whom the diagnosis of pcd is highly suspected, a bronchial sample is required. 366 abnormalities in sperm flagella may differ in type and quantity from those of respiratory cilia within the same patient, suggesting separate genetic control of axone mal structures at differing loci? the lung pathology in pcd is postinfective in appearance. both saccular and cylindrical bronchiectasis may be present with the predominant histologic pattern of follicular bronchiectasis. 175 ,350,370 neither bronchial mucus stasis nor squamous metaplasia is prominent chronic interstitial pneumonitis, peribronchial fibrosis, and atelectasis accompany the bronchiectatic changes. 349 ,370,371 studies to date suggest that pcd is a genetically heterogeneous disorder. the molecular basis of pcd has been localized in a few instances to mutations in the human dyne in axonemal heavy chain (dnah5) located on chromosome 5, or in the intermediate dynein chain gene 1 (dnail) on chromosome 9,372-374 a mutation in the dyne in axonemal heavy chain type 11 (dnahll) has been associated with pcd and situs inversus, without evident ultrastructural ciliary changes, 372 ongoing studies on genetically engineered knockout mice may uncover other genetic defects associated with pcn,372,375 in 1970 donald young, a urologist, reported a series of patients with obstructive azoospermia, 54 % of whom had associated respiratory conditions including bronchitis and bronchiectasis,376 this condition was initially designated as berry-perkins-young syndrome and later shortened to young's syndrome. 377 patients, nearly all had chronic cough, sputum production, and recurrent pulmonary infections. bronchiectasis and chronic sinusitis were each present in about two thirds of patients. in one study it was estimated that young's syndrome accounted for approximately 3% of all patients who presented with bronchiectasis of unknown etiology (equivalent to the prevalence of cf and slightly greater than that of pcd). 195 azoospermia in young's syndrome is the result of retention of semen in an enlarged epididymal head. motility studies have demonstrated impaired upper airway mucociliary transport; however, ciliary beat frequency and ultrastructure are normal. 377 ,378,382,383 in patients with young's syndrome, sweat chloride concentration and the electrical potential difference across the nasal epithelium are normal. 384 the respiratory symptoms in young's syndrome have been suggested to be the result of altered viscoelastic properties of airway secretions, but the basic molecular defect is unknown. 385 an association with mercury toxicity has been hypothesized. 386 friedman and colleagues 380 evaluated mutations of cftr in a cohort of patients with young's syndrome and found that the prevalence of mutations did not differ significantly from the expected carrier frequency in the general population. pulmonary involvement is generally less severe than in cf. 379 bronchiectasis tends to occur at an early age and predominantly involves the lower lobes. pulmonary function tests indicate mild obstruction with decreased fev1 and increased residual volume, although a number of patients have undergone lung resections for bronchiectasis, the pathologic features of bronchiectasis in young's syndrome have not been well described, and it is uncertain if there are any distinctive histopathologic changes. from a diagnostic standpoint it is important to exclude cf and pcd, each of which may be clinically misclassified as young's syndrome?87,388 the distinguishing characteristics among these three conditions are presented in table sa . williams-campbell syndrome is a rare disorder in which extensive loss of bronchial cartilage is associated with diffuse cystic bronchiectasis without other recognized predisposing factors. 184 ,402 the clinical presentation that commences in infancy may include cough, dyspnea on exertion, cyanosis, and clubbing.403 on chest radiograph large thin-walled cysts reside in hyperinflated lungs. highresolution ct scan characteristically shows central, cystic, thin-walled airways that collapse upon expiration. 404.405 the clinical course is one of recurrent pulmonary infections leading to respiratory failure. patients may survive into adulthood and require lung transplantation. 406 as described in the original report by williams and campbell and substantiated in subsequent morphologic studies, the lungs grossly exhibit extreme saccular and cystic bronchiectasis (fig. 5.43a ).406-409 microscopically, dilated airways have very thin walls with minimal inflammation (fig. 5.43b ). cartilage is absent or markedly deficient from the fourth to the eighth divisions of subsegmental bronchi. first-and second-order bronchi usually have a normal cartilage investment. pan acinar emphysema or emphysema localized to the peribronchial zone is usually also present.406.407 bronchiolitis obliterans has also been reported.403.407 the williams-campbell syndrome has been considered to be the result of a congenital absence of cartilage in the subsegmental airways. morphologic studies documenting absent cartilage and insignificant inflammation, and rare reports of familial occurrence have been used to support this view.402.409.410 however, given the propensity for cartilage loss in acquired saccular bronchiectasis of diverse etiologies, the williams-campbell syndrome remains a controversial entity, and its congenital origins have yet to be proven beyond question. 175.205.206.407 the williams-campbell syndrome has not been associated, nor is it to be confused, with congenital lobar emphysema, in which cartilage is focally deficient, usually in upper lobe bronchi, leading to bronchial collapse and air trapping (see also chapter 6).411 tracheobronchomegaly (tbm) is a condition of marked dilatation of the trachea and major bronchi, often associated with recurrent respiratory infections. tbm can be congenital, or at least evident in early life, in which it is termed mounier-kuhn syndrome (see chapter 6) . it has occurred in several cases of ehlers-danlos syndrome, suggesting it may be related to poor elastic support, or perhaps to loss of other matrix components as in chondromalacia. 412 tracheobronchomegaly occurs in adults, mostly in men in their fourth and fifth decades, and can be an acquired condition secondary to sustained inflammation affecting the trachea, such as in chronic tracheobronchitis secondary to tobacco abuse, cystic fibrosis, trauma, emphysema, or pulmonary fibrosis. 412, 413 a comprehensive review and an intriguing study of various pulmonary fibrotic reactions associated with this entity was reported by woodring et al. 412 these investigators evaluated the tracheal diameter on plain chest radiography in a series of 34 cases of fibrotic lung reactions, and found enlargement of the trachea in 10 (29%). the associated lung diseases were idiopathic pulmonary fibrosis and sarcoidosis in four patients each, and progressive histoplasmosis in two patients. in seven of these patients as well as in nine of 24 patients (38%) who did not meet initial radiographic criteria for tracheal dilatation, tracheomegaly developed or progressed over time, tracheobronchomegaly was usually associated with moderate-to-severe restrictive pulmonary defects, and it was proposed that shrinkage of the lung tissue retracts all adjacent spaces including the trachea in a manner similar to traction bronchiectasis. 412 regardless of the cause oftbm, airway dilatation may extend distally, bronchomegaly simulates bronchiectasis, probably impairs lung clearance, and promotes recurrent bronchopulmonary infection, which paradoxically may induce secondary bronchiectasis. 412 roditi and weir 414 identified tracheobronchomegaly in 17% of patients with evidence of bronchiectasis on ct scan, thereby emphasizing the frequent association and possible causal connections between these two conditions. 414 tracheobronchomegaly is predominantly a radiologic diagnosis, and its pathologic features have not been well characterized. associated radiographic features include marked tracheal wall thinning, scalloping due to mural infolding, bronchial diverticula, and collapse on expiration. 415 . 416 the diagnostic criteria of tbm by ct scan are a tracheal diameter of greater than 3cm (measured 2cm above the aortic arch) and diameters of 2.4 and 2.3 cm for the right and left main bronchi, respectively.415al? tracheobronchomegaly must be distinguished from saber-sheath trachea, seen in some patients with emphysema, in which there is a decrease in tracheal coronal diameter and increased sagittal diameter. 415 .4 1 8 patients with immune deficiency, especially hypogammaglobulinemia due to x-linked agammaglobulinemia or common variable immunodeficiency (cvid), are predisposed to develop bronchiectasis secondary to recurrent pulmonary infections. 419 -421 chronic pulmonary disease is the most common long-term complication in patients with hypogammaglobulinemia. 422 common variable immunodeficiency, a heterogeneous immunodeficiency syndrome characterized by depressed levels of serum immunoglobulin g (igg) and defective antibody response to antigen challenge, is associated with sinusitis, recurrent pneumonia, and chronic sputum production in up to 90% of patients.423 patients with cvid also have an increased incidence of autoimmune diseases, and as with other primary immunodeficiency syndromes, a tendency toward lymphoproliferative disorders (see chapter 32) . 422, 424 in this population there is a spectrum of lung abnormalities including interstitial fibrosis (>80% of patients), pneumonia, lymphoid interstitial pneumonia (lip), sarcoidosis-like granulomatous disease (10%), lung abscess, and bronchiectasis. 422 ,425 bronchiectasis is the most common radiologic finding and may be identified in over 30% of patients by chest x-ray, and in up to 80% of patients by hrct. 424 ,426 by hrct bronchiectasis may be either focal or multilobar, and is of the cylindrical or rarely cystic type. 420 ,426 the lower and middle lobes tend to be predominantly involved.420.424 although mucociliary clearance is impaired in these patients, ciliary ultrastructure is normal. 427 there is little information on the histopathology of bronchiectasis in cvid. 42 8. 429 hill and colleagues 429 noted severe bronchiectasis, emphysema, fibrosis, and granulomas in the lung explant of a 37-year-old man with cvid. no unique features of bronchiectasis were described. patients with cvid are treated with immunoglobulin replacement therapy, which reduces the severity and frequency of respiratory infections. symptomatic bronchiectasis, identified by hrct scan, has also been reported in patients with hiv disease in whom it is associated with rapidly progressive airways obstruction. 43o , 431 king and colleagues 432 correlated airway dilatation on ct scan with increased neutrophils on bronchoalveolar lavage. a single report of a transbronchial biopsy showed only nonspecific lymphocytic peribronchiolitis. 431 the pathogenesis of bronchiectasis in hiv patients is likely consequent to bronchial damage from recurrent pneumonia and bacterial bronchitis in this immunosuppressed population. 430 .433 frequently cultured microorganisms include h. injluenzae, p. aeruginosa, and s. pneumoniae. 430 ,433 bronchiectasis also occurs as a complication of lung transplant-associated immunosuppression and bronchiolitis obliterans and is further discussed in chapter 23. 434.435 rheumatoid arthritis symptomatic bronchiectasis is estimated to occur in 1 % to 3% of patients with rheumatoid arthritis (ra), although with hrct scan, up to 30% of patients with ra can be shown to have cylindrical bronchiectasis. 185 .4 3 6,437 early autopsy studies of patients with ra provided a prevalence of bronchiectasis of 0% to 12%. [438] [439] [440] [441] in some studies bronchiectasis typically preceded the development of arthritis, leading to the interesting hypothesis that chronic suppurative airway disease is involved in the pathogenesis of ra.442-444 shadick and colleagues,444 however, reported 23 patients with bronchiectasis and ra, of whom 18 developed bronchiectasis as a late complication of severe ra. bronchiectasis may also be more frequent in patients with ra-associated sjogren's syndrome.442a45 bronchiectasis associated with ra cannot be adequately ascribed to either traction bronchiectasis or therapeutic immunosuppression. 436 ,444 the morphologic features of ra-associated bronchiectasis are not well documented. by ct scan, cylindrical bronchiectasis primarily involves the middle and lower lung zones. 437a44 , 445 bronchiectasis is part of the spectrum of lung involvement in patients with inflammatory bowel disease (ibd), ulcerative colitis more so than crohn's disease (see also chapter 20) .446,447 suppurative bronchiectasis may also develop after proctocolectomy for either of these conditions. 447 ,448 in the 1993 literature review of camus et al.,446 bronchiectasis was identified in six patients with ulcerative colitis out of 33 patients (18%) with ibd-associated lung disease 446 histologically a dense cuff of lymphocytes typically occupies the submucosa, and squamous metaplasia replaces the overlying epithelium (fig. 5.44 ). the chronic inflammatory infiltrate involves bronchial glands and ducts; however, lymphoid germinal centers are usually absent, distinguishing ulcerative colitis-associated bronchiectasis from the usual pattern of follicular bronchiectasis (fig. 5 .44b).175,446 neutrophils infiltrating the mucosa and spilling into the dilated bronchial lumen impart a suppurative appearance in some cases. 44 6--449 direct immunofluorescence staining of bronchial biopsies in three patients with ulcerative colitis showed deposits of immunoglobulin and complement in bronchial structures. 448 lung biopsy in patients with crohn's disease and bronchiectasis may show features of either granulomatous bronchiolitis or suppurative-appearing acute bronchiolitis (see fig. 20 .27 in chapter 20).450 inhaled steroids were of durable benefit in patients with ibd-associated chronic bronchitis, but less so in patients with bronchiectasis. 446 speculations on the pathogenesis of bronchiectasis in ibd are presented in a provocative editorial by stockley.451 bronchiectasis is reported as a late sequela of heroinassociated pulmonary edema. 452 the bronchographic features include diffuse or localized cylindrical and varicose bronchiectasis. 453 itis are etiologic factors in some patients, and bronchial ulceration and foreign-body giant cells have been observed at autopsy.454 other cases of diffuse bronchiectasis in heroin users appear to be unrelated to aspiration. 453 see chapter 31 for other pathologic features of heroin toxicity. in adults, severe direct chemical injury such as ammonia gas inhalation or aspiration can cause bronchiectasis. 455 -457 delayed-onset bronchiectasis has also been described starting 12 years after smoke inhalation. 458 bronchiectasis in systemic diseases foreign body inhalation foreign body in the airway: a review of 202 cases quinn ceo aspiration emergencies the cafe coronary: sudden deaths in restaurants thoracic and cardiovascular surgery with related pathology inhaled foreign bodies migratory pulmonary infiltrates secondary to aspirated foreign body muddy lung idowu 0. sand aspiration: a case report shakespeare, the complete works. fort worth: harcourt brace observations on the anatomy of the bronchial tree, with special reference to the surgery of lung abscess. guy's bronchial embolism and posture in relation to lung abscess. guy's aspirated foreign bodies in the tracheobronchial tree: report of 250 cases bronchial foreign body vs. asthma studies on the lymph drainage of the accessory nasal sinuses the pulmonary aspiration of particulate matter pulmonary aspects of tonsillectomy under general anesthesia the relationship of sinusitis and bronchiectasis aspiration pneumonia silent" regurgitation and aspiration during anesthesia aspiration of food and vomit pharyngeal aspiration in normal adults and patients with depressed consciousness aspiration pneumonia. recognizing and managing a potentially growing disorder aspiration pneumonitis and aspiration pneumonia bosomworth pp aspiration pneumonitis the aspiration of stomach contents into the lungs during obstetrical anesthesia pulmonary aspiration of gastric contents in anaesthesia pulmonary aspiration of gastric contents acid aspiration-induced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms synergism between leukotriene b4 and thromboxane a2 in mediating acid-aspiration injury tumor necrosis factor-alpha mediates acid aspiration-induced systemic organ injury intercellular adhesion molecule-1 mediates acid aspiration-induced lung injury reactive oxygen species contribute to oxygen-related lung injury after acid aspiration acid aspiration induced lung injury. new insights and therapeutic options experimental murine acid aspiration injury is mediated by neutrophils and the alternative complement pathway incompetency of the gastric cardia without radiologic evidence of hiatus hernia: the diagnosis and management of 71 cases pulmonary fibrosis associated with tracheobronchial aspiration: a study of the frequency of hiatal hernia and gastroesophageal reflux in interstitial pulmonary fibrosis of obscure etiology gastroesophageal reflux and chronic respiratory disease hydrocarbon pneumonitis effects of furniture polish on pulmonary surfactant roentgenographic changes in the lungs of children with kerosene poisoning pulmonary granulomatosis caused by vegetable particles: so-called lentil pulse pneumonia foreign body reaction to inhalation of lentil soup: giant cell pneumonia pulmonary nodular granulomatosis caused by inhaled vegetable particles diffuse miliary granulomatosis of the lungs due to aspirated vegetable cells case records of the massachusetts general hospital. case 9-1987 zuzarte 1, tomashefski if lr. the histologic spectrum of aspiration bronchiolitis obliterans (abo). an autopsy study importance of diffuse aspiration bronchiolitis caused by chronic occult aspiration in the elderly miliary pulmonary nodules due to aspirated vegetable particles. 1 pediatr lentil aspiration pneumonia: radiographic and ct findings the pulmonary pathology of illicit drug and substance abuse the microscopic appearance of a sodiumpotassium exchange resin in histologic sections calcium polystyrene sulfonate: an unusual cause of inhalation pneumonia chaplin al histologic occurrence of polystyrene sulfonates kayexalate (sodium polystyrene sulfonate) aspiration necrosis of the gastrointestinal tract in uremic patients as a result of sodium polystyrene sulfonate (kayexalate) in sorbitol sodium polystyrene sulfonate (kayexalate) aspiration. histologic appearance and infrared micro spectrophotometry analysis of two cases sodium polystyrene sulfonate pneumonitis upper gastrointestinal tract injury in patients receiving kayexalate (sodium polystyrene sulfonate) in sorbitol. clinical, endoscopic, and histopathologic findings iron lung": distinctive bronchoscopic features of acute iron tablet aspiration bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration charcoal lung. bronchiolitis obliterans after aspiration of activated charcoal exogenous lipid pneumonia: a retrospective multicentre study of 44 cases in france pneumonia following nasopharyngeal injections of oil pathology of the lung the reaction to oils and fats in the lungs toxicological studies on hydrocarbon: ix. the aspiration hazard and toxicity of hydrocarbons and hydrocarbon mixtures lipoid pneumonia exogenous lipoid pneumonia lipoid pneumonia in a veterans' hospital incidence of lipoid pneumonia in a survey of 389 chronically ill patients lipid pneumonia: a new etiology inhalation lipoid pneumonia from burning fats. a newly recognized industrial hazard acute lipoid pneumonitis due to aspiration of pressurized paint droplets the lipoid pneumonia of blackfat tobacco smokers in guyana lung disease due to abuse of hair spray lipoid pneumonia: review of the literature with a case report infection of lungs by "saprophytic" mycobacteria in achalasia of cardia, with report of fatal cases showing lipoid pneumonia due to milk stergus 1. death due to mycobacterium fortuitum group iv atypical mycobacterium infection occurring in association with mineral oil granuloma of lung atypical mycobacterial infection complicating mineral oil pneumonia aspiration and occult esophageal disorders paraffinoma confirmed by infrared spectrophotometry case records of the massachusetts general hospital. case 33-1999 lipoid (mineral oil) pneumonia and cor pulmonale due to cardiospasm chronic cor pulmonale associated with lipoid pneumonia diagnosis of lipoid pneumonia by examination of the sputum the lipid-laden alveolar macrophage as a marker of aspiration in parenchymal lung disease the cytologic evaluation of lipid-laden alveolar macrophages as an indicator of aspiration pneumonia in young children thoracic and cardiovascular surgery with related pathology abscess of the lung treatment of lung abscess with report of 100 consecutive cases lung abscess: pathogenesis, diagnosis and treatment lung abscess revisited: a survey of 184 cases primary lung abscess lung abscess: a study of the results of treatment based on 90 consecutive cases lung abscess: an analysis of the massachusetts general hospital cases from 1943 through 1956 lung abscess experimental aspiration abscess fuso-spirochaetal diseases of the lungs putrid abscess of the lung following dental operations etiologic factors in the pathogenesis of putrid abscess of the lung the symptomatology of putrid abscess of the lung the microbiota of the gingival crevice area of man-ii: the predominant cultivable organisms the micro biota of the gingival crevice area in man-i: total microscopic and viable counts and counts of specific organisms anaerobic bacteria: role in disease. springfield: charles c thomas microorganisms indigenous to man bacteriology and treatment of primary lung abscess bacteriologic flora of aspiration-induced pulmonary infections a clinical consideration of abscesses and cavities of the lung lung abscess in childhood primary lung abscess in childhood anaerobic (putrid) lung abscess in adolescence anaerobic bacterial infections of the lung anaerobic infections lung abscess should fiberoptic bronchoscopy aspirates be cultured? diagnostic accuracy of transtracheal aspiration bacteriologic studies aspiration pneumonia, necrotizing pneumonia, and lung abscess clinical classification and analysis of 97 cases of lung abscess lung abscess: a changing pattern of disease the lining of healed but persistent abscess cavities in the lung with epithelium of the ciliated columnar type cavity behavior in acute, primary nonspecific lung abscess pulmonary gangrene: radiologic and pathologic correlation pulmonary gangrene. a complication of bacterial pneumonia gangrene of the lung gangrene of the lung total unilateral lung gangrene in hodgkin's disease: treatment by thoracostomy gangrene of the lung: successful medical management of three patients lung lobe torsion following lobectomy lung torsion: survival of a patient whose hemorrhagic infarcted lung remained in situ after detorsion postoperative pulmonary torsion: report of a case and survey of the literature including spontaneous and posttraumatic torsion lung torsion after lung transplantation: evaluation with helical ct an unusual case of lung torsion obstructive pneumonitis of neoplastic origin. an interpretation of one form of so-called atelectasis and its correlation according to presence or absence of sputum lipoid pneumonia in lung cancer: radiographic and pathological features obstructive pneumonitis: a pathologic and pathogenetic reappraisal xanthomatous bronchiolitis obliterans with cholesterol pneumonia organizing pneumonia adjacent to lung cancer. frequency and clinico-pathologic features pulmonary phospholipid accumulation distal to an obstructed bronchus endogenous lipid pneumonia and alveolar proteinosis-type changes in the vicinity of non-small cell lung cancer: histopathologic, immunohistochemical, and ultrastructural evaluation cholesterol pneumonitis correlation between the roentgenologic and pathologic findings in chronic pneumonitis of the cholesterol type idiopathic cholesterol pneumonitis localized organizing pneumonia: its resemblance to carcinoma. a review of its clinical, roentgenographic and pathologic features organization of pneumonic exudates aetiology of unresolved pneumonia coexisting endogenous lipoid pneumonia, cholesterol granulomas, and pulmonary alveolar proteinosis in a pediatric population: a clinical, radiographic, and pathologic correlation in vitro studies of the foamy macrophage of postobstructive endogenous lipoid pneumonia in man electron microscopic demonstration of lysosomal inclusion bodies in lung, liver, lymph nodes, and blood leukocytes of patients amiodarone lung toxicity: a human and experimental study pulmonary histiocytosis simulating desquamative interstitial pneumonia in rats receiving oral iprindole effects of chlorphentermine on the rat lung electron microscopy of chlorphentermine lung pulmonary parenchymal cholesterol-ester granulomas in patients with pulmonary hypertension pulmonary lipogranulomatosis due to excessive consumption of apples diffuse storage of vegetal wax hydrocarbons of dietary origin aetiology of pulmonary cholesterol-ester granulomas bacteriological studies in pulmonary atelectasis reexpansion of atelectatic lower lobe and disappearance of bronchiectasis pseudo bronchiectasis the pathologic anatomy of influenza. based chiefly on american and british sources bronchiectasis: a bronchographic study of 60 cases of pneumonia the reversal of advanced bronchiectasis reversible bronchiectasis reversible bronchial dilatation: report of a case traction bronchiectasis in end-stage pulmonary fibrosis pathology of pulmonary disease. philadelphia: lippincott textbook of pulmonary diseases whitwell e study of pathology and pathogenesis of bronchiectasis the natural history of bronchiectasis. a clinical, roentgenologic and pathologic study bronchiectasis: a study of prognosis based on a follow-up of 400 cases surgical diseases of the chest. philadelphia: lea and febiger bronchial obstruction, bronchiectasis, and related disorders 179 pathology of the lung chronic airflow obstruction reduction in bronchial subdivision in bronchiectasis latent adenoviral infection in follicular bronchiectasis generalized bronchiectasis associated with deficiency of cartilage in the bronchial tree bronchiectasis in children pathology of bronchiectasis bronchiectasis: study of pathology of 16 surgical lobectomies for bronchiectasis factors causing bronchiectasis: their clinical application to diagnosis and treatment bronchiectasis: a neglected disease long-term follow-up of bronchiectasis if. bronchiectasis: update of an orphan disease an investigation into causative factors in patients with bronchiectasis bronchiolitis obliterans, bronchiectasis and other sequelae of adenovirus type 21 infection in young children pneumonia following influenza (at camp pike, ark) crofton 1. respiratory tract disease. diagnosis and treatment of bronchiectasis. i. diagnosis crofton 1. respiratory tract disease. bronchiectasis. ii. treatment and prevention non-tuberculous juvenile bronchiectasis: a viral disease? bronchiectasies secondaires a une adenovirose: etude radiologique, virologique et anatomique d'une observation chronic lung damage caused by adenovirus type 7: a ten-year follow-up study bronchiectasis: a review of 187 cases in children with follow-up pulmonary function studies in 58 the cartilage of the intrapulmonary bronchi in normal lungs, in bronchiectasis, and in massive collapse destruction and loss of bronchial cartilage in cystic fibrosis enlargement of the bronchial arteries and their anastomoses with the pulmonary arteries in bronchiectasis the right middle lobe syndrome, a nonobstructive complex collateral ventilation and the middle lobe syndrome middle lobe syndrome the middle lobe syndrome and its quasi variants middle lobe syndrome. a clinicopathological study of 21 patients right middle lobe atelectasis associated with endobronchial silicotic lesions lloyd 11. broncholiths. with report of four cases the surgical implication of broncholithiases current concepts of an ancient disease bronchiolithasis: bronchoscopic vs. surgical management a review of twentyseven cases etiology of bronchiolithiasis case records of the massachusetts general hospital. case 14-2002 bronchiolithiasis due to histoplasma capsulatum subsequently infected by actinomyces letter: broncholithiasis in silicosis tracheobronchial obstruction due to silicosis bronchiolithiasis: ct features in 15 patients egg-shell silicotic calcification causing bronchoesophageal fistula broncholithiasis: a neglected cause of bronchoesophageal fistula acquired bronchoesophageal fistula of benign origin traction diverticula of the esophagus in the middle lobe syndrome broncholithiasis: review of the causes with radiologic-pathologic correlation ct demonstration of an ossifying bronchial carcinoid simulating broncholithiasis schwarz 1. broncholithiasis. produced by histoplasmosis complications of the arrested primary histoplasmic complex postoperative aortic fistulas into the airways: etiology, pathogenesis, presentation, diagnosis, and management bronchocele and blocked bronchiectasis the syndrome of bronchial mucocele and regional hyperinflation of the lung developmental origin of cystic, bronchiectatic and emphysematous changes in lungs. a new concept the syndrome of bronchial mucocele and regional hyperinflation of lung: report of four cases atresia of the bronchus to the apical posterior segment of the left upper lobe congenital bronchial atresia. a report of 4 cases and a review of the literature bronchial atresia of the left upper lobe atresia of an apical bronchus of the left upper lobe-report of three cases bronchial atresia computer tomography in bronchial atresia clinical spectrum of bronchogenic cysts of the mediastinum and lung in the adult presentation and management of bronchogenic cysts in the adult intrapulmonary bronchogenic cyst: ct and pathologic findings in five adult patients bronchioloalveolar carcinoma arising in a bronchogenic cyst biochemistry and rheology of sputum in asthma a case of pituitous catarrh crofton 1, douglas a. respiratory diseases bronchorrhea in a case of alveolar cell carcinoma bronchorrhea-a presenting feature of active endobronchial tuberculosis pang 1. relapsing polychondritis presenting with bronchorrhea cystic fibrosis cystic fibrosis identification of the cystic fibrosis gene: cloning and characterization of complimentary dna identification of the cystic fibrosis gene: chromosome walking and jumping ion channels-basic science and clinical disease macromolecular interactions and ion transport in cystic fibrosis cystic fibrosis transmembrane conductance regulator. structure and function of an epithelial chloride channel a clinical perspective of cystic fibrosis and new genetic findings: relationship of cftr mutations to genotype-phenotype manifestations new approaches to cystic fibrosis cystic fibrosis f508del patients have apically localized cftr in a reduced number of airway cells laboratory tests for the diagnosis of cystic fibrosis fibrosis genotype-phenotype consortium. correlation between genotype and phenotype in patients with cystic fibrosis cftr expression and organ damage in cystic fibrosis a cystic fibrosis mutation associated with mild lung disease a mild variant of cystic fibrosis mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens lung disease associated with the ivs8 5t allele of the cftr gene the pathology of cystic fibrosis the lung in cystic fibrosis. a quantitative study including prevalence of pathologic findings among different age groups quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants the pathogenesis of fibrocystic disease of the pancreas. a study of 36 cases with special reference to the pulmonary lesions the bronchial mucous glands-their hypertrophy and change in intracellular mucus similarity of the tracheobronchial mucous glands and epithelium in infants with and without cystic fibrosis the innate immune system in cystic fibrosis lung disease the genesis of cystic fibrosis lung disease pathogenesis of bacterial bronchitis in cystic fibrosis current understanding of the inflammatory process in cystic fibrosis: onset and etiology how mutant cftr may contribute to pseudomonas aeruginosa infection in cystic fibrosis lung infections associated with cystic fibrosis bacterial infections and inflammation in the lungs of cystic fibrosis patients bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation cystic fibrosis lung inflammation: early, sustained and severe inflammatory cytokines in cystic fibrosis lungs normal bronchial epithelial cells constitutively produce the antiinflammatory cytokine interleukin-lo, which is downregulated in cystic fibrosis early pulmonary inflammation in infants with cystic fibrosis cytokine secretion by cystic fibrosis airway epithelial cells immunohistopathologic localization of pseudomonas aeruginosa in lungs from patients with cystic fibrosis. implications for the pathogenesis of progressive lung deterioration basal-like cells constitute the proliferating cell population in cystic fibrosis airways ultrastructural features of respiratory cilia in cystic fibrosis ciliary disorientation in patients with chronic upper respiratory tract inflammation a morphometric study of bronchial and bronchiolar walls in children quantitative aspects of lung pathology in cystic fibrosis the central bronchial glands in cystic fibrosis, a morphometric, clinicopathologic study regional distribution of macroscopic lung disease in cystic fibrosis pulmonary cystic fibrosis in the adult: early and late radiologic findings with pathologic correlation pulmonary air cysts in cystic fibrosis: relation of pathologic features to radiologic findings and history of pneumothorax pneumothorax in cystic fibrosis cystic fibrosis: state of the art cystic fibrosis of the pancreas: structural changes in peripheral airways the pathologic characteristics of interstitial pneumonia in cystic fibrosis. a retrospective autopsy study clinical findings and lung pathology in children with cystic fibrosis age as a factor in the distribution of lower-airway conductance and in the pathologic anatomy of obstructive lung disease pseudomonas cepacia colonization among patients with cystic fibrosis. a new opportunist pseudomonas cepacia associated pneumonia in cystic fibrosis. relation of clinical features to histopathologic patterns of pneumonia burkholderia cepacia complex infection in patients with cystic fibrosis nontuberculous mycobacteria in cystic fibrosis isolation of rapidly growing mycobacteria in patients with cystic fibrosis the pathology of fungal infection and colonization in patients with cystic fibrosis allergic bronchopulmonary aspergillosis in cystic fibrosis-state of the art: cystic fibrosis foundation consensus conference mycobacterium abscessus infection in cystic fibrosis. colonization or infection? nontuberculous mycobacteria in cystic fibrosis. an autopsy study the pulmonary circulation in cystic fibrosis pulmonary hypertension in cystic fibrosis. a description and morphometric analysis of the pulmonary vasculature cor pulmonale in cystic fibrosis. a morphometric analysis new findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension pulmonary hypertension and cor pulmonale in cystic fibrosis of the pancreas medial mucoid lesions of the pulmonary artery in cystic fibrosis, pulmonary hypertension, and other disorders the bronchial arteries in cystic fibrosis selective bronchial arteriography in patients with cystic fibrosis and massive hemoptysis cystic fibrosis in adults. an autopsy study pulmonary changes and cor pulmonale in mucoviscidosis bronchial artery embolization in cystic fibrosis: technique and long-term results a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis longterm histopathologic follow-up of bronchial arteries after therapeutic embolization with polyvinyl alcohol (ivalon) in patients with cystic fibrosis intrapulmonary shunts in cystic fibrosis secondary (aa) amyloidosis in cystic fibrosis. a report of three cases systemic amyloidosis complicating cystic fibrosis pulmonary intralobar sequestration in a patient with cystic fibrosis lung abscess in cystic fibrosis recreational use of psychoactive drugs by patients with cystic fibrosis a human syndrome caused by immotile cilia absence of axone mal arms in nasal mucosa cilia in kartagener's syndrome the immotile-cilia syndrome. a congenital ciliary abnormality as an etiologic factor in chronic airway infections and male sterility immotile cilia primary ciliary dyskinesia-diagnostic and phenotypic features evidence for congenitally nonfunctioning cilia in the tracheobronchial tract in two subjects immotile cilia syndrome in persons with and without kartagener's syndrome kartagener syndrome: an uncommon cause of neonatal respiratory distress? immotile cilia syndrome: a new cause of neonatal respiratory distress zur pathogenese der bronchiektasien: bronchiektasien bei situs viscerum inversus bronchiectasis with situs inversus primary ciliary dyskinesia cilia with defective radial spokes. a cause of human respiratory disease transposition of ciliary microtubules. another cause of impaired ciliary motility absence of nexin links as a possible cause of primary ciliary dyskinesia severe bronchiectasis in patients with "cystlike" structures within the ciliary shafts central microtubular agenesis causing primary ciliary dyskinesia nasal ciliary ultrastructure and function in patients with primary ciliary dyskinesia compared with that in normal subjects and in subjects with various respiratory diseases ultrastructural nasal pathology in children chronically and sequentially exposed to air pollutants ciliary defects in healthy subjects, bronchiectasis, and primary ciliary dyskinesia ultrastructural ciliary defects in children with recurrent infections of the lower respiratory tract atypical bronchial cilia in children with recurrent respiratory tract infections. a comparative ultrastructural study random ciliary orientation. a cause of respiratory tract disease ciliary disorientation alone as a cause of primary ciliary dyskinesia syndrome clinico-pathological evaluation of ciliary dyskinesia: diagnostic role of electron microscopy computer-assisted analysis of radial symmetry in human airway epithelial cilia: assessment of congenital ciliary defects in primary ciliary dyskinesia do nasal ciliary changes reflect bronchial changes? an ultrastructural study kartagener's syndrome with motile cilia and immotile spermatozoa: axone mal ultrastructure and function dissimilar expression of axone mal anomalies in respiratory cilia and sperm flagella in infertile men fertility in men with primary ciliary dyskinesia presenting with respiratory infection a reappraisal of kartagener's syndrome report of a family with two cases of kartagener's triad and two additional cases of bronchiectasis among six siblings genetic regulation of cilia assembly and the relationship to human disease homozygosity mapping of a gene locus for primary ciliary dyskinesia on chromosome 5p and identification of the heavy dynein chain dnah5 as a candidate gene germline mutations in an intermediate chain dynein cause primary ciliary dyskinesia investigation of the possible role of a novel gene, dpcd, in primary ciliary dyskinesia surgical treatment of male infertility lung mucociliary clearance in patients with young's syndrome obstructive azoospermia: respiratory function tests, electron microscopy and the results of surgery young's syndrome. obstructive azoospermia and chronic sinopulmonary infections screening young syndrome patients for cftr mutations respiratory tract disease and obstructive azoospermia ciliary function in young's syndrome rutland i mucociliary function, ciliary ultrastructure, and ciliary orientation in young's syndrome measurement of nasal potential difference in adult cystic fibrosis, young's syndrome and bronchiectasis viscoelastic properties of sputum from patients with young's syndrome was young's syndrome caused by exposure to mercury in childhood? cystic fibrosis, young's syndrome, and normal sweat chloride young's syndrome (a case report) alpha-i-antitrypsin deficiency presenting as bronchiectasis alpha-i-antitrypsin deficiency presenting as bronchiectasis lieberman i bronchiectasis and homozygous alpha-1 antitrypsin deficiency distribution of alpha-l antitrypsin alleles in patients with bronchiectasis bronchiectasis in patients with alpha-l antitrypsin deficiency. a rare occurrence? pathological findings in alpha-l antitrypsin deficiency alpha-1 antitrypsin deficiency: evaluation of bronchiectasis with ct for the alpha i-antitrypsin deficiency registry study group. the bronchopulmonary pathology of alpha-1 antitrypsin (aat) deficiency: findings of the death review committee of the national registry for individuals with severe deficiency of alpha-1 antitrypsin selective iga deficiency and pi zz-antitrypsin deficiency: association with recurrent sinopulmonary infections, emphysema and bronchiectasis chronic airflow obstruction chronic obstructive lung disease. a comparison between clinical, roentgenologic, functional and morphologic criteria in chronic bronchitis, emphysema, asthma and bronchiectasis an assessment of the anatomical factor in cor pulmonale in emphysema the non-respiratory bronchioles in pulmonary emphysema familial bronchiectasis generalized bronchiectasis due to extensive deficiency of bronchial cartilage erasmus i case 4: williams-campbell syndrome congenital bronchiectasis in an adult lung transplantation for williams-campbell syndrome congenital bronchiectasis due to deficiency of bronchial cartilage (williams-campbell syndrome) mortality following adenotonsillectomy in a patient with williams-campbell syndrome familial congenital bronchiectasis: williams-campbell syndrome probable familial congenital bronchiectasis due to cartilage deficiency (williams-campbell syndrome) congenital lobar emphysema acquired tracheomegaly in adults as a complication of diffuse pulmonary fibrosis radiologic and pathologic abnormalities of the trachea in older patients with cystic fibrosis weir 1. the association of tracheomegaly and bronchiectasis using ct to diagnose nonneoplastic tracheal abnormalities: appearance of the tracheal wall a 38-year-old man with tracheomegaly, tracheal diverticulosis, and bronchiectasis mounier-kuhn syndrome): ct diagnosis sabre-sheath" trachea: relation to chronic obstructive pulmonary disease recurrent respiratory infections in a family with immunoglobulin a deficiency bronchiectasis in hypogammaglobulinaemia-a computed tomography assessment igg subclass deficiencies associated with bronchiectasis common variable immune deficiency: respiratory manifestations, pulmonary function and high-resolution ct scan findings pulmonary manifestations of hypogammaglobulinaemia respiratory disorders in common variable immunodeficiency pulmonary abnormalities in patients with primary hypogammaglobulinemia radiologic findings of adult primary immunodeficiency disorders. contribution of ct mucociliary clearance in patients with immunoglobulin deficiency respiratory dysfunction in patients with common variable hypogammaglobulinemia heart lung transplantation in a patient with end stage lung disease due to common variable immunodeficiency bronchiectasis in hiv disease accelerated obstructive pulmonary disease in hiv infected patients with bronchiectasis bronchial dilatation in patients with hiv infection: ct assessment and correlation with pulmonary function tests and findings at bronchoalveolar lavage bacterial bronchitis and bronchiectasis in human immunodeficiency virus infection pathologic pulmonary alterations in long-term human heart-lung transplantation postmortem findings in lung transplant recipients use of high resolution computed tomography of the lungs in patients with rheumatoid arthritis airways involvement in rheumatoid arthritis. clinical, functional, and hrct findings visceral lesions associated with chronic infectious (rheumatoid) arthritis lung lesions in rheumatoid arthritis pulmonary disease associated with rheumatoid arthritis convalescent care in chronic arthritis bronchiectasis and rheumatoid arthritis: a clinical study pulmonary lesions and rheumatoid arthritis bronchiectasis. a late feature of severe rheumatoid arthritis high resolution computer tomography of the lung in lifelong nonsmoking patients with rheumatoid arthritis the lung in inflammatory bowel disease bronchiectasis following colectomy for crohn's disease chronic bronchial suppuration and inflammatory bowel disease unexplained bronchopulmonary disease with inflammatory bowel disease noninfectious lung pathology in patients with crohn's disease commentary: bronchiectasis and inflammatory bowel disease the impact of substance abuse on the respiratory system bronchiectasis: a cause of pulmonary symptoms in heroin addicts pulmonary complication of heroin intoxication. aspiration pneumonia and diffuse bronchiectasis bronchiectasis following ammonia burns of the respiratory tract: a review of two cases fatal anhydrous ammonia inhalation bronchiectasis following pulmonary ammonia burn said sl bronchiectasis and progressive respiratory failure following smoke inhalation acknowledgments. the authors are deeply appreciative to diane gillihan for expert secretarial assistance, vince messina for photography, and the staff of the brittingham memorial library for bibliographic support. key: cord-017248-a37t31u1 authors: nan title: alphabetic listing of diseases and conditions date: 2010-05-17 journal: handbook of autopsy practice doi: 10.1007/978-1-59745-127-7_17 sha: doc_id: 17248 cord_uid: a37t31u1 part ii begins with a list of special histologic stains, their for use and their corresponding references. at the end of this list is a procedure for removal of formalin precipitate from tissue sections. diseases. there may also be a list of possible associated conditions. these entities are generally linked pathogenetically to the main disease entry. any asterisk after a related disease indicates that that disorder is also listed as a disease entry. many disease entries will be followed by a three-column table that provides the reader with a listing of the pathologic findings to be expected with the disease as well as the prosection and dissection procedures necessary to demonstrate those findings. it is expected that routine hematoxylin-eosin stains will be done on all sections submitted for histologic examination. special stains will be recommended in the procedures column of the tables, when indicated. any table immediately following the two columns of disease entries always refers to the disease in the right column. prepare smears of undiluted blood. obtain blood for molecular studies for preservation of small intestinal mucosa and for preparation for study under dissecting microscope, see part i, chapter 2. submit sample for histologic study. submit stool for chemical analysis. record weight and submit sample for histologic study. freeze liver for molecular studies record appearance of spine (see also chest roentgenogram). for removal and specimen preparation, see chapter 4. request luxol fast blue stain. for removal and specimen preparation, see chapter 5. below-normal weight in infants. kyphoscoliosis. very low concentrations of cholesterol and decreased triglycerides; serum~-lipoprotein or absent; a.-lipoproteins present. acanthocytosis (spiny red cells). gene mutations (4) . abnormal shape of villi; vacuolation of epithelial cells. fatty stools fatty changes. gene mutations (4) systemic manifestations of malabsorption syndrome* and of vitamin a deficiency. * kyphoscoliosis. axonal degeneration of the spinocerebellar tracts; demyelination of the fasciculus cuneatus and gracilis (2) . possible involvement of posterior columns, pyramidal tracts, and peripheral nerves. atypical retinitis pigmentosa (2) with involvement of macula. angioid streaks (3) . synonym: cerebral abscess. note: for microbiologic study of tissues and abscesses, see part i, chapter 7. include samples for anaerobic culture. it is best to study the brain after fixation but if specimen is examined fresh, aspirate and prepare smears of abscess content. photograph surface and coronal slices of brain. request giemsa stain, gram stain, pas stain, and grocott's methenamine silver stain for fungi. external examination if there is evidence of trauma, see also under "injury, head." prepare roentgenograms of chest and skull. submit for microbiologic study. for removal and specimen preparation, see chapter 4. for microbiologic study, photography, and special stains, see under "note." for exposure of venous sinuses, see chapter 4. sample walls of sinuses for histologic study. for exposure of paranasal sinuses, mastoid cells, and middle ears, see chapter 4. for removal and specimen preparation, see chapter 5. procedures depend on suspected lesions as listed in right-hand column. skin infections in upper half of face. edema of forehead, eyelids, and base of nose, proptosis, and chemosis indicate cerebral venous sinus thrombosis. * trauma; craniotomy wounds. skull fracture and other traumatic lesions. for possible intrathoracic lesions, see below under "other organs." the national transportation safety board (ntsb)* has authority over aircraft wreckage and the legal authority to investigate and to determine the cause of air crashes. (1) the dead are the responsibility of the medical examiner or coroner. local police will seal off the area of the crash. other than for the purpose of determining that death has occurred, no one should be allowed to approach the bodies or any objects until the identification teams and the medical examiner or coroner have taken charge. the sudden influx of bodies after a commercial air carrier accident and the request for speedy identification of the victims would overburden almost any institution. managing such a disaster is eased by writing a contingency plan beforehand. temporary morgue facilities may have to be established near the scene of the crash. refrigerated trucks may serve as storage space. a practical approach is to deal first with those bodies that seem to be the easiest to identify, in order to narrow the field for the more difficult cases. if bodies are scattered, their locations can be referenced to stakes in the ground or spray paint on pavement; only then should these bodies (or parts) and personal effects be collected. for large-scale crashes a locations can be referenced to a string-line grid benchmarked to gps coordinates. records and diagrams of the relative positions of victims are prepared during this phase. if bodies are still within the airplane, their positions are recorded, and photographed. the personnel of the medical examiner or coroner can augmented by d-mort team staffed by forensic pathologists, anthropologists, dentists, morgue technicians, and investigators supplied by the national disaster medical system. ** the airline will provide a list of the passengers and the federal bureau of investigation (fbi) disaster team will make itself available to take and identify fingerprints and aid in the acquisition of other identifying data such as age, race, weight, height, and hair color and style. if dental records can be obtained, this provides one of the most certain methods of identification. a medical history indicating amputations, internal prostheses, or other characteristic surgical interventions or the presence of nephrolithiasis, gallstones, and the like will be helpful. fingerprints (and footprints of babies) should be taken in all instances. wallets with identification cards,jewelry, name tags in clothing, or other personal belongings may provide the fastest tentative identification. the medical examiner may elect to autopsy only the flight crew but not the passengers of an aircraft crash. however, the grossly identifiable fatal injuries should be described, photographed, and x-rayed. this may reveal identifying body changes. if comparison of somatic radiographs, dental records, fingerprints, or photographs do not identify the victim, dna comparison must be considered. burned or fragmented bodies of passengers and the bodies ofcrew members, and particularly the pilots, must have a complete autopsy, including roentgenographic and toxicologic examinations, which must always include alcohol and carbon monoxide determinations. internal examination might reveal a coronary occlusion, or roentgenograms may disclose a bullet as evidence that violence preceded the crash. in some airplane crashes, particularly in light airplane accidents, suicide must be considered. in such cases police investigation is required to determine if the pilot exhibited suicidal ideation in the recent past.. when resources permit, autopsies should be performed on all deceased occupants of aircraft crashes, including passengers, in order to distinguish among blunt impact trauma, smoke inhalation, and flash fires as causes ofdeath, and to answer future questions concerning pain and suffering, intoxication, and sequence of survivorship. after a crash victim has been identified, the coroner or medical examiner will issue a death certificate. if remains of a decedent cannot be found, a judge can, upon petition, declare a passenger dead and sign a death certificate prepared by a medical examiner. *phone # ofntsb command center: 202-314-6000 **phone # of dmort: 800-872-6367. entry should be followed. usually, the circumstances that led to drowning are not apparent from the autopsy findings but can be reconstructed from reports of witnesses and the police. because the reflex drive to seek air is triggered by hypercarbia, not hypoxia, loss of consciousness and drowning can ensue after hyperventilation and breath-holding by experienced swimmers who then drown without a struggle. there are no specific autopsy findings. a search for trauma, including a posterior neck dissection, should be made in all instances. head and cervical injuries may be responsible for loss of consciousness and drowning, usually in individuals diving into shallow water. toxicologic examination as described below for scuba diving accidents is always indicated. with scuba diving fatalities, investigation of the equipment and circumstances is usually more important than the autopsy. scuba fatalities should be studied by or with the aid of diving experts-for instance, members of a diving club or shop (not the one providing the gear used by the decedent) or the u.s. navy. (1) careful investigation of the scene and study of reports of witnesses and the police are essential. the investigation should ascertain the site of diving (currents and other underwater hazards), the estimated depth, the water temperature (exposure to cold), and a description of water clarity. electrocution should be considered if the site has electric underwater cables (see "injury, electric"). cerebral concussion should be considered if explosives were used in the vicinity. knowledge of the method of recovery of the body and the type of resuscitation efforts can aid in the interpretation of apparent wounds. the medical history of the diving victim should be sought, as it may lead to a diagnosis for which the autopsy is typically silent, such as seizure disorder, or may reveal asthma, emphysema, or chronic bronchitis, all of which increase the risk of air trapping and arterial air embolism. although drowning may be the terminal event in some scuba deaths, the investigation should be focused on the adverse environmental and equipment factors that place a capable swimmer at risk of drowning (see "embolism, air" and "sickness, decompression"). because scuba divers risk arterial air embolism if they ascend with a closed glottis, on can attempt to document gas bubbles at autopsy, but their interpretation is problematic: bodies recovered immediately are subjected to resuscitation efforts, which can by themselves produce extra-alveolar air artifacts, and bodies not recovered immediately tend to be found in a putrefied condition, full of postmortem gas. in the remaining cases, the pathologist must consider the potential of introducing artifactual gas bubbles by the forcible retraction of the chest plate and by sawing the calvarium. the following procedures apply primarily to scuba diving accidents. interrogation of witnesses is important; the behavior and complaints of the decedent, if any, might help distinguish between a natural death by heart disease and an unnatural death by air embolism. external examination eyes and ears head (skull and brain) chest blood (from heart and peripheral vessels) heart tracheobronchial tree and lungs a procedures photograph victim as recovered and after removal of wet suit and other diving gear. record condition of clothing and gear. impound all diving equipment for study by experts, particularly scuba tank, breathing hoses, and regulators. residual air in tank should be analyzed. record color of skin (including face, back, soles, palms, and scalp). palpate skin and record presence or absence of crepitation. record extent and character of wounds. prepare histologic specimens. record appearance of face (including oral and nasal cavities) and of ears. prepare roentgenograms. if air embolism must be expected, as in the presence of pneumomediastinum, follow procedures described under "embolism, air." for evaluation of findings, see also above under "note." if decompression sickness (caisson disease) is suspected, also prepare roentgenograms of the elbows, hips, and knees. otoscopic examination. funduscopic examination. save vitreous for possible toxicologic and other studies. for removal of brain, see chapter 4. record contents of arteries of the circle of willis and its major branches and basilar artery. strip dura from base of skull and from calvarium. for removal and specimen preparation, see chapter 4. for demonstration of pneumothorax, see under "pneumothorax". if gas is visible in coronary arteries, photograph. photograph and aspirate gas in heart chambers. submit samples of heart blood and peripheral blood for toxicologic study and drug screen. examine lungs in situ. save bronchial washings for analysis of debris. fresh dissection is recommended. if decompression sickness is suspected, prepare sudan stains from fresh-frozen lung sections. complete toxicologic sampling should be carried out (see chapter 13). record nature of gastric contents. remove neck organs toward end of autopsy. for posterior neck dissection, see chapter 4. incise tongue. for removal, see chapter 4. for removal, see chapter 4. for removal, prosthetic repair, and specimen preparation, see chapter 2. consult roentgenograms. in decompression sickness, fatty change of liver, and ischemic infarctions of many organs. interstitial emphysema. aspiration (see above). trauma to cervical spine. mottled pallor of tongue after air embolism. contusion of tongue after convulsive chewing. nitrogen bubbles in spinal cord arteries may occur after rapid ascent. air embolism;' cerebral edema in decompression sickness. aseptic necroses (infarcts, "dysbaric osteonecrosis"), most often in head of femur, distal femur, and proximal tibia. infarcts indicate repeated hyperbaric exposures. nitrogen bubbles in and about joints and in periosteal vessels ("bends") occur during rapid ascent. related terms: automobile accident; motorcycle accident. note: a visit to the scene can make the interpretation of the autopsy findings easier. the vehicle can also be inspected in a more leisurely fashion at the impound lot. this is particularly useful for correlating patterned injuries with objects in the vehicle. most vehicular crashes occur as intersection crashes or because a vehicle with excessive speed left a curved road. the medical examiner or coroner should gain a basic understanding of the crash mechanism so that informed descriptions can be rendered, e.g., "impact to the b pillar of the decedent's automobile by the front of a pickup truck which failed to stop for a stop sign at an intersection, resulting in a 2-feet intrusion into the cabin; restraint belts not employed; air bag deployed; extrication required which took 15 minutes." police are responsible for determining mechanical and environmental risk factors for the crash and for determining some human risk factors such as suicidal or homicidal intent. the pathologist determines other risk factors for crashes such as heart disease, a history of epilepsy, and intoxication by carbon monoxide, drugs, and alcohol. suicide as a manner of death should be considered when a single-occupant vehicle strikes a bridge abutment or a large tree head-on, with no evidence of evasive action or braking. in such a situation, the standard police traffic investigation should be supplemented of interviews of the victim's family and friends. the ambulance run sheet is an invaluable source of observations that often are not available from the police. this document should be acquired in all instances, even if the paramedics determined that death occurred and did not transport. the basic autopsy procedures are listed below. most traffic victims who die at the scene or who are dead on arrival at the hospital died from neurogenic shock caused by wounds of the head or vertebral column, or from exsanguination from a tom vessel or heart. as such, they have little lividity, and little blood is found in the vehicles. presence ofintense lividity may indicate suffocation or heart disease as a cause of death. if postural asphyxia is suspected, the first responders to the scene should be interviewed to determine the position of the decedent in the vehicle, and the vital signs, ifany, ofthe decedent from the time of the crash to the time of extrication. posterior neck dissection is indicated in these instances. if manifestations of heart disease, intense lividity, and absence oflethal wounds suggest that a crash occurred because the driver was dead, other drivers on the road may have observed that the victim was slumped at the wheel before the crash. the determination of heart attack at the wheel is usually simple, because most such victims realize that something is wrong, and bring the vehicle to a stop at the side of the road, or coast gently into a fixed object. in such instances, damage to the vehicle is minor, and wounds to the decedent are usually trivial. while pattemed wounds can often be matched to objects (see below), patternless wounds usually cannot be visually matched to specific objects, although an opinion can sometimes be given as to what object was struck, based on the direction of motion and position ofthe body with respect to the vehicle. impacts with the a-pillar produce narrow vertical zones of facial laceration and fractures extending from forehead to jaw. tempered glass shatters into small cubes on impact, and leaves so-called "dicing" wounds, which are abraded cuts arranged in a somewhat rectilinear pattern. windshield glass leaves shallow, abraded, vertically oriented cuts on the face or scalp. with pedestrians, the lower extremities are of particular forensic interest, to determine the height and direction of impact from vehicles that left the scene. scalp hair and blood should be collected from such "hit and run" victims and from occupants of a suspect car if police have a question as to which occupant was the driver; these exemplars can be compared to fibers and tissue recovered from the vehicle in question. likewise, foreign material in wounds can sometimes be matched to suspect vehicles, and should be sought and retained as evidence. for pedestrians, the distance between the impact point on the lower extremities and the soles of the feet should be recorded. the legs should be opened to inspect tibial fractures; cortical fractures initiate propagation opposite to the side of impact, where they usually have a pulled-apart appearance, and then splinter the cortex at the side of impact. abrasions are better impact markers than contusions, because subcutaneous blood extravasation can be caused not only by impact to the skin, but also from blood extravasating from underlying fractures. if no cutaneous abrasions or fractures of the leg bones are found, the skin of the legs should be incised to expose contusions. fracture descriptions should include location in the bone (e.g., proximal metaphysis or shaft), whether the fracture is complete or incomplete, and whether the fracture is displaced or distracted. lacerations of intervertebral disks, facet joint capsules, and ligamenta flava should not be loosely termed "fractures." the presence or absence of blood extravasation in soft tissue adjacent to the fractures should be recorded, and its volume estimated if it appears severe enough. venous air embolism from tom dural sinuses cannot be diagnosed without a pre-autopsy chest radiograph or an in situ bubble test. if an x-ray machine is readily available, an anterior-posterior chest radiograph should be obtained in every traffic victim who dies at the scene or after a failed resuscitation attempt. if a hemothorax is suspected, the rib cuts should be placed further lateral and the chest plate reflected so that the internal mammary vessels can be inspected before the chest plate is removed. after measuring and removing the bloody effusion, the underlying serosal surfaces should be inspected for defects. lacerations of the heart and aorta will be obvious. tamponaded lacerations of the aorta, around which the adventitia still holds, must be noted as such. if no lacerations are found at the usual sites, lacerations of the azygous veins must be considered, especially in association with fracture dislocations of the thoracic vertebral column; other sites are the internal mammary arteries, especially with fractures of ribs i and 2 or of the sternum, and intercostal arteries with displaced rib fractures. only after the serosal defect is identified should the organs be removed, because that procedure creates many more holes in the serosa. for that reason, as much information as possible should be gained by in situ observation. the only evidence of concussion of the heart may be a cardiac contusion or a sternal fracture. the usual clinical history suggests cardiovascular instability that is not associated with craniocerebral trauma and which does not respond to the infusion of intravenous volume agents. the autopsy assistant may saw but should not retract the skull cap and remove the brain. the pathologist should observe in situ whether shallow lacerations of the pontomedullary junction with stretching of the midbrain are present. these lesions cannot be distinguished from artifact by examining the brain later. thus, only after appropriate in situ inspection should the pathologist remove the brain. a posterior neck dissection is required if no lethal craniocerebral or cardiovascular trauma is found, or if suffocation is suspected; neck trauma must be ruled out to diagnose suffocation in a traffic fatality. sudden death in a patient with seemingly trivial wounds may be caused by undiagnosed trauma of the craniocervical articulation. a posterior neck dissection is required in these instances. the diagnosis of diffuse axonal injury of the brain in victims with no appreciable survival interval requires that suffocation be ruled out and that no resuscitation from a cardiac arrest has been attempted. clinicians are quick to apply the label "closed head injury" when a victim of a traffic crash has cerebral edema on a computerized axial tomogram of the head, even if no cerebral contusions, scalp contusions, or skull fractures are evident. this may be a misinterpretation, because cerebral edema can be caused by hypoxic encephalopathy made evident after resuscitation from a cardiac arrest, or from hypoxia caused by suffocation. procedures possible or expected findings record presence of lividity. photograph all external wounds; measure all lacerations and any abrasions or contusions with a pattern. collect scalp hair and blood (see below) from victims of hit and run accidents. collect foreign material in wounds. intense lividity and absence of lethal wounds may indicate that the crash occurred because the driver was dead from heart disease or suffocation. wound documentation. patterned injuries often sometimes be matched to objects in or about the vehicle (the most common patterned wound is that from tempered glass; see above under "note"). impact patterns in pedestrians may help to reconstruct the accident. hair and blood of the victim may be matched to transfer evidence on a vehicle suspected of having left the scene. part ii / diseases and conditions internal examination of body cavities heart and great vessels abdomen skull and brain; neck soft tissue compartments at any location prepare roentgenograms of chest is cases with head impact and skull fractures. collect samples for toxicologic study from all victims, including passengers. create pleural window to detect pneumothorax. if blood is seen, examine internal mammary vessels (see under "note"). measure volume of blood in cavity bleeds, and note whether chambers of heart and great vessels are collapsed or filled. record evidence of cardiac contusion, sprain of intracardiac inferior vena cava, laceration of pericardial sac, and fracture of sternum. laceration of heart or great vessels (measure volume of blood). follow routine procedures for dissection of heart and great vessels (see chapter 3) . in situ bubble test to confirm venous air embolism. record evidence of trauma and volume of blood in peritoneal cavity; estimated volume of blood in retroperitoneal soft tissues. autopsy assistant may saw the skull but pathologist should inspect brain in situ and remove it personally. for removal and specimen preparation of brain, see chapter 4. record brain weight. posterior neck dissection is indicated if there is no craniocerebral or cardio-vascular trauma, or if suffocation is suspected. record evidence of trauma and estimate volume of blood. venous air embolism.' evidence of alcohol or drug intoxication. pneumothorax, hemothorax, e.g., after laceration of internal mammary vessels. evidence of significant hemorrhage. indirect evidence of cardiac concussion. evidence of exsanguinating wounds. evidence of cardiovascular disease that may have felled the driver before the crash. in european countries, the concentration is expressed in promille (grams per liter). in the united states, it has become customary to refer to concentration by percentage (grams per deciliter), and values in these units have been written into legislation and included in the uniform vehicle codes. unless qualified, the use of promille or percentage does not indicate whether the result of the analysis is weight/weight, weight/ volume, orvolume/volume. another common way ofexpressing concentration, milligrams per deciliter, has also been used to indicate alcohol concentrations. the method ofexpressing concentration must be clearly specified whenever the alcohol level is mentioned. the desired expression canbe derived from the toxicologic report by using the following equation: i,000~g/ml =100mg/dl =0.10g/dl =21.74 mmolll =1.0 promille =0.10% what is the legal interpretation of alcohol (ethanol) intoxication? objective impairment of driving ability is observed at threshold blood alcohol concentrations of .035-.040 g/dl. as of august 2005 all states and the district of columbia have adopted laws that make it criminal offense for a driver to operate a motor vehicle with a blood alcohol concentration of 0.08 g/ dl or greater. many states have an enhanced penalty for high concentrations such as 0.15 g/dl or above. several states have zero tolerance laws, under which drivers who are minors are legally operating only if their blood alcohol concentration is 0.02 g/dl or less, and in some states, not detectable at all. blood alcohol concentrations obtained at autopsy are valid until putrefaction begins. specimen tubes with sodium fluoride should be used, and the specimen should be stored in the refrigerator. if the air space above the blood samples in the container is large, alcohol can evaporate and a falsely low blood alcohol level can result. putrefactive changes before autopsy or during storage may cause a falsely high blood alcohol concentration. ethanol can be produced in the specimen container; this is more likely in the absence of a preservative. because fluoride inhibits bacteria far more than fungi, higher fluoride concentrations are required for the inhibition of fungal growth (4) . although there is no major difference in the alcohol concentrations ofblood samples from the intact heart chambers and the femoral vessels (5), autopsy samples from pooled blood in the pericardial sac or pleural cavity are unsatisfactory. we therefore recommend that blood be withdrawn from peripheral vessels. is there normal "endogenous" blood alcohol (ethanol) in a living person? blood alcohol concentrations are generally believed to be negligible in the absence of ingested alcohol. "endogenous" ethanol in human blood exists at a concentration of about 0.0002 g/dl, which is below the limit of detection for most methods (6) . first in such a list would be postural asphyxia, for example, in drunks who fall asleep face down. also, depressant drugs in the tricyclic, analgesic, barbiturate, and benzodiazepine classes all potentiate the effect of alcohol (7) . also included in such a list would be infancy and childhood; ischemic heart disease;' chronic bronchitis and emphysema;' other chronic debilitating diseases; poisoning with carbon tetrachloride' or carbon monoxide;' and other causes of hypoxia.' how can one estimate blood alcohol (ethanol) concentrations from vitreous, urine, or tissue alcohol levels and from alcohol in stomach contents? the ratio of serum, plasma, urine, vitreous, and various tissues has been compiled by garriot (8) . the values may vary considerably. for vitreous, the ratios varied from 0.46-1.40. these variations may depend on whether blood alcohol concentrations were increasing or decreasing at the time of death. most other body fluids and tissues showed ranges closer to 1. most urine values were above the blood alcohol concentrations. in another study (9) , the blood/vitreous (bn) ratio in the early absorption phase was 1.29 (range, 0.71-3.71; sd 0.57) and in the late absorption and elimination phase, the bn ratio was 0.89 (range, 0.32-1.28; sd 0.19). blood ethanol concentrations probably can be estimated using b =1.29v for early absorption and b = 0.89v for later phases. a urinelblood ethanol ratio of 1.20 or less indicates that the deceased was in the early absorption phase. how can one use alcohol (ethanol) concentrations in postmortem specimens to estimate the blood alcohol concentration at various times before death? with certain limitations, one can base calculations of this kind on the assumption that the blood alcohol level decreases from its peak at a fairly constant rate of 0.015-q.018g/dl/h until death (10) . if blood is not available, conversion factors (see above) must be used. alcoholics have been reported to metabolize at a rate of up to 0.043 g/dl/h (6) . example: the driver of an automobile drinks at a party until midnight. he leaves his host at about 1:30 a.m. and is involved in a head-on collision at 2:15 a.m. he dies in the emergency room at 6:35 a.m. there are multiple injuries and the patient exsanguinates. the autopsy is done at 1:30 p.m. although this appears quite unlikely, let us assume that no satisfactory blood sample was obtained before death and that no blood or plasma expanders were given. if under such circumstances the alcohol concentration in the vitreous was found to be 0.157 g/dl, what was the alcohol concentration in the blood at the time of the accident? vitreous and blood alcohol concentrations may be assumed to have remained unchanged after death. therefore, the blood alcohol level at the time of death must have been approx 0.157 (vitreous humor alcohol) x 0.89 (conversion factor, see above) = 0.14g/dl. the time interval between the accident (2:15 a.m.) and death (6:35 a.m.) is 4 hand 20 min or 4 1/3 h. if we assume that the decedent was not an alcoholic and that the blood alcohol concentration was decreasing from its peak at a constant rate of 0.015 g/dl/h, then the concentration at the time ofthe accident is estimated to have been 0.14 (concentration at time of death) + (4 1/3 x 0.015) = 0.140 + 0.065 = 0.205 g/dl or 0.2%. the blood alcohol concentration at the time of the accident could have been lower if the victim stopped drinking later than 1h or 1 1/2 h before the accident. in the latter case, the peak alcohol level would have occurred after the accident, reflecting the time to absorb the latest drink. the blood alcohol concentration at the time of the accident could have been lower or higher if the time when the patient stopped drinking, the time of the accident, or the time of the death is uncertain. the blood alcohol concentration at the time of the accident could have been higher if the victim was a chronic alcoholic. the elimination rate in such persons may be as high as 0.040 mg/dl, which would change the figures in our example above to 0.140 + (4 1/3 x .040) =0.140 + 0.173 = 0.313 g/d1 or 0.3%. only rough estimates are possible. first, the peak blood alcohol level must be determined or calculated, as described in the previous paragraphs. tables (see below) are available that relate blood alcohol level to the minimal amounts of whiskey, wine, or beer that must have been consumed (10) . however, tables of this type are often based on the minimum amount of alcohol circulating in the body after specific numbers of drinks; such tables do not yield reliable results if used conversely. furthermore, inasmuch as drinking and elimination of alcohol may take place concomitantly, over a longer period the total amount of alcohol consumed may have been much greater than the tables would indicate. it cannot be lower. according to these tables, 6 pints of ordinary beer or 8 fl oz of whiskey would be the minimal amounts needed to produce a blood alcohol level of about 200 mg/dl in a person weighing 140-180 pounds. the total body alcohol can be calculated from the blood alcohol level by using widmark's formula: average concentration of alcohol in entire body = .68 concentration of alcohol in the blood in a person weighing 70 kg, the blood alcohol concentration would be increased 50 mg/dl (0.05%) by the absorption of 1oz of ethanol (20z of 100-proof whiskey). strength of alcohol is measured in "proof'; absolute alcohol is 200 proof. therefore, in the united states, alcohol content as volume percent is half the proof (for example, 100-proof whiskey contains 50% alcohol by volume). the alcohol content of various beverages is shown in the following table. approximate alcohol content in various beverages t toata from glaister, rentoul e. medical jurisprudence and toxicology, 12th ed. e & s livingstone, edinburgh, 1966 with permission. twithin 1 h after consumption of diluted alcohol (approx 15%) on an empty stomach, assuming body weight of 140-180 pounds (63.6-81.7 kg) reproduced from (11) with permission. *one ounce (about 30ml) of whiskey or 120z (about 355ml) of beer. what is the toxicity of alcohol other than ethanol? in general, the toxicity increases as the number of carbon atoms in the alcohol increases. thus, butyl alcohol is two times as toxic as ethyl alcohol: but isopropyl alcohol is only twothirds as toxic as isobutyl alcohol and one-half as toxic as amyl alcohol. primary alcohols are more toxic than the corresponding secondary isomers (10) . anemia, hemolytic synonyms and related terms: acquired hemolytic anemia; extracorpuscular hemolytic anemia; hereditary hemolytic anemia (hereditary elliptocytosis, pyropoikilocytosis, stomatocytosis. spherocytosis); immunohemolytic anemia; intracor-puscular hemolytic anemia; microangiopathic hemolytic anemia; spur cell anemia. possible associated conditions: disseminated intravascular coagulation;* eclampsia;* glucose-6-phosphatase deficiency (g6pd); hemolytic uremic syndrome;* malignant hypertension; lymphoma* and other malignancies; paroxysmal nocturnal hemo-globinuria; sickle cell disease;*thalassemia;* thrombotic thrombocytopenic purpura.* (see also below under "note.") note: hemolysis also may be caused by conditions such as poisoning with chemicals or drugs, heat injury, snake bite,* or infections or may develop as a transfusion reaction* or be secondary to adenocarcinoma, heart valve prostheses (see below), liver disease (see below), renal disease, or congenital erythropoietic porphyria. * procedures prepare skeletal roentgenograms. jaundice; skin ulcers over malleoli. in young patients: thickening of frontal and parietal bones with loss of outer table ("hairon-end" appearance); paravertebral masses caused by extramedullary hematopoiesis; deformities of metacarpals, metatarsals, and phalanges. osteonecrosis* of femoral heads. remove and place in fixative as early as possible in order to minimize autolysis (alternatively, formalin can be injected in situ; see below). samples should include oxyntic corpus and fundus mucosa. record weights. submit tissue samples for histologic study. record weight of thyroid gland. for removal and specimen preparation, see chapter 4. request luxol fast blue stain. for removal and specimen preparation, see chapter 5. if there is a clinical diagnosis of anemia-related amblyopia, follow procedures described under "amblyopia, nutritional." jaundice. manifestations of malnutrition. * stomatitis with cheilosis and perianal ulcerations due to folic acid deficiency. chronic exfoliative skin disorders. vitiligo. macrocytosis; poikilocytosis; macroovalocytes; hypersegmentation of leukocytes; abnormal platelets. atrophic glossitis with ulcers. pharyngoesophagitis (folic acid deficiency). previous total or subtotal gastrectomy. carcinoma of stomach. autoimmune gastritis (diffuse corporal atrophic gastritis) with intestinal metaplasia. crohn's disease;* sprue;* other chronic inflammatory disorders; jejunal diverticula; intestinal malignancies; fish tapeworm infestation; previous intestinal resection or blind intestinal loop; enteric fistulas. hepatosplenomegaly. alcoholic liver disease. * giant epithelial cells. hyperthyroid goiter; thyroiditis. demyelination of cerebral white matter (in advanced cases). demyelination in posterior and lateral columns of spinal cord, most frequently in thoracic and cervical segments. demyelination of peripheral nerves. retinal hemorrhages; demyelination of optic nerves. hypercellular; megaloblastic. myeloproliferative disorder. brain other organs if mycotic aneurysms are expected and microbiologic studies are intended, follow procedures described below under "aneurysm, mycotic aortic." request verhoeff-van gieson, gram, and grocott's methenamine silver stains. for cerebral arteriography, see chapter 4. if arteriography cannot be carried out, rinse fresh blood gently from base of brain until aneurysm can be identified. record site of rupture and estimated amount of extravascular blood. for paraffin embedding of aneurysms, careful positioning is required. expected findings depend on type of aneurysm. mycotic aneurysms are often multiple and deep in brain substance. berry aneurysms are the most frequent types and often are multiple. most frequent sites are the bifurcations and trifurcations of the circle of willis. saccular atherosclerotic aneurysms are more common than dissecting aneurysms, which are very rare. with congenital cerebral artery aneurysm: coarctation of aorta;* manifestations of hypertension;* and polycystic renal disease. with mycotic aneurysm: infective endocarditis;* pulmonary suppurative processes; and pyemia. aneurysm, dissecting aortic (see "dissection, aortic.") aneurysm, membranous septum of heart note: for general dissection techniques, see chapter 3. most aneurysms ofthe membranous septum probably repre-sent spontaneous closure of a membranous ventricular septal defect by the septalleafiet of the tricuspid valve. aneurysm, mycotic aortic note: (i) collect all tissues that appear to be infected. (2) request aerobic, anaerobic, and fungal cultures. (3) request gram and grocott methenamine silver stains. (4) no special precautions are indicated. (5) no serologic studies are available. (6) this is not a reportable disease. chest and abdominal organs aorta other organs submit blood samples for bacterial culture. en masse removal of adjacent organs is recommended. photograph all grossly identifiable lesions. aspirate material from aneurysm or para-aortic abscess and submit for culture. prepare sections and smears of wall of aneurysm and of aorta distant from aneurysm. request verhoeffvan gieson and gram stains. septicemia and infective endocarditis. * streptococcus, staphylococcus, spirochetes, and salmonella can be found in mycotic aneurysm. para-aortic abscess. septic emboli with infarction or abscess formation. aneurysm, syphilitic aortic part ii / diseases and conditions heart and aorta other organs en masse removal of organs is recommended. for coronary arteriography, see chapter 10. request verhoeff-van gieson stain from sections at different levels of aorta, adjacent great vessels, and coronary arteries. see also under "syphilis." aneurysm usually in ascending aorta. may erode adjacent bone (sternum). syphilitic aortitis may cause intimal wrinkling, narrowing of coronary ostia, and shortening of aortic cusps. disruption of medial elastic fibrils. aortic valvulitis and insufficiency;* syphilitic coronary arteritis; syphilitic myocarditis. external examination aorta prepare chest and abdominal roentgenograms. open aorta along line of blood flow, or bisect into anterior and posterior halves. photograph tear(s). measure bloody effusions in body cavities. measure or estimate amount of blood in mediastinum. request verhoeff-van gieson stain. cutaneous impact trauma. mediastinum widened by hemorrhage in case of tarnponaded dissection. a bleed into a body cavity of less-thanexsanguinating volume should point to an alternate mechanism of death such as neurogenic shock or lethal concussion; a posterior neck dissection may be required in such instances. microscopy may show transmural rupture, false aneurysm, or localized dissection. angiitis (see "arteritis, all types or type unspecified.") angina pectoris note: see under "disease, ischemic heart" and chapter 3. angiokeratoma corporis dittusum (see "disease, fabry's.") angiomatosis, encephalotrigeminal (see "disease, sturge-weber-dimitri.") angiopathy, congophilic cerebral synonyms and related terms: beta amyloid angiopathy due to~-amyloid peptide deposition (~a4) (associated with alzheimer's disease; hereditary cerebral hemorrhage with amyloid angiopathy of dutch type; or sporadic beta amyloid angiopathy); hereditary cerebral amyloid angiopathy, due to deposition of other amyloidogenic proteins such as cystatin c (icelandic type) and others (e.g., transthyretin, gelsolin) (1). procedures possible or expected findings request stains for amyloid, particularly congo red, and thioflavine s (examine with polarized and ultraviolet light, respectively). request immunostain for~a4. some tissue should be kept frozen for biochemical studies. multiple recent cerebral cortical infarctions or small cortical hemorrhages, or both, or massive hemispheric hemorrhages, both recent and old. amyloid deposition in leptomeninges and cortical blood vessels. senile plaques are usually present. in some cases, angiopathy is part of alzheimer's disease. * other organs a prepare material for electron microscopy. electron microscopic study permits definite confirmation of diagnosis. organs and tissues may be minimally affected by amyloidosis. anomaly, coronary artery possible associated conditions: with double outlet right ventricle; persistent truncal artery; tetralogy of fallot;* and transposition of the great arteries.* note: coronary artery between aorta and pulmonary artery, often with flap-valve angulated coronary ostium. coronary artery may communicate with cardiac chamber, coronary sinus, or other cardiac veins, or with mediastinal vessel through pericardial vessel. saccular aneurysm of coronary artery with abnor-mal flow, infective endarteritis of arteriovenous fistula, and myocardial infarction may be present. ifone or both coronary arteries originate from pulmonary trunk, myocardial infarction may be present. heart perform coronary angiography. if infective endarteritis is suspected, submit blood sample for microbiologic study. ectopic origin of coronary arteries or single coronary artery. sudden death. for a detailed description of possible additional findings, see above under "note." anomaly, ebstein's (see "malformation, ebstein's") anorexia nervosa note: sudden death from tachyarrhythmias may occur in advanced cases and thus, autopsy findings may not reveal the immediate cause of death. external examination all organs record height and weight, and prepare photographs to show cachectic features. record abnormalities as listed in righthand column. follow procedures described under "starvation." record weight of endocrine organs and submit samples for histologic study. cachexia, often with preserved breast tissue; hirsutism; dry, scaly, and yellow skin (carotenemia). mild edema may be present. parotid glands may be enlarged. manifestations of starvation.* ovaries tend to be atrophic; other endocrine organs should not show abnormalities. synonyms: cutaneous anthrax; gastrointestinal anthrax; pulmonary (inhalational) anthrax. note: (1) collect all tissues that appear to be infected. this is a reportable disease. bioterrorism must be considered in current cases. external examination and skin blood photograph cutaneous papules, vesicles, and pustules. prepare smears and histologic sections. submit samples for bacteriologic study. submit sample for serologic study. disseminated anthrax infection may occur without skin lesions. edema of neck and anterior chest in nasopharyngeal anthrax. anthrax septicemia. see above under "note." part ii i diseases and conditions lungs gastrointestinal tracts and mesentery neck organs record character and volume of effusions. after sampling for bacteriologic study (see above under "note") perfuse one or both lungs with formalin. extensive sampling for histologic study is indicated. extensive sampling for histologic study is indicated. photograph meningeal hemorrhage in situ. pleural effusions;* hemorrhagic mediastinitis; anthrax pneumonia (inhalational anthrax; woolsorter's disease). histologic sections reveal hemorrhagic necrosis, often with minimal inflammation and gram-positive, spore-forming, encapsulated bacilli. gastrointestinal anthrax with mucosal edema and ulcerations. hemorrhagic mesenteric lymphadenitis. tongue, nasopharynx, and tonsils may be involved. hemorrhagic meningitis (hemorrhage tends to predominate). external examination distal colon and rectum photograph perineum. measure depth of anal pit, if any. dissect distal colon, rectum, and perirectal pelvic organs in situ (as much as possible). search for opening of fistulous tracts from lumen. use roentgenologic study or dissection, or both, to determine course of tract. absence of normally located anus; anal dimple. abnormal termination of the bowel into the trigone of the urinary bladder, the urethra distal to the verumontanum, the posterior wall of the vagina, the vulva, or the perineum. aortitis note: see also under "arteritis" and "aneurysm, ascending aortic." heart and aorta other organs and tissues remove heart with whole length of aorta and adjacent major arteries. record width and circumference of aorta at different levels. describe and photograph appearance of intima and of orifices of coronary arteries and other aortic branches. submit multiple samples for histologic study and request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. secondary aortic atherosclerosis or intimal fibroplasia. widening of aorta; syphilitic aneurysm. * giant cell aortitis; rheumatoid aortitis; syphilitic aortitis; takayasu's arteritis.* manifestations of rheumatoid arthritis, * syphilis,* systemic sclerosis,* hodgkin's lymphoma, and many other diseases associated with vasculitis. external examination brain spine and spinal cord other organs prepare roentgenogram of spine. for removal and specimen preparation, see chapter 4. for removal of spinal cord and specimen preparation, see chapter 4. expose nerve roots. record appearance and photograph spinal cord in situ. submit samples of spinal cord and inflamed tissue for histologic study. request gram, gomori's iron, and grocott's methenamine silver stains. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. signs of previous spinal surgery or lumbar puncture (myelography). evidence of previous trauma or previous myelography. cerebral arachnoiditis. fibrous arachnoidal adhesions and loculated cysts. tuberculosis;* syphilis;* fungal or parasitic infection. systemic infection (see above). ascending urinary infection or other manifestations of paraplegia. arch, aortic, interrupted synonym: severe coarctation. note: the basic anomaly is a discrete imperforate region in the aortic arch, with a patent ductal artery joining the descending thoracic aorta. type a interruption is between the left subclavian and ductal arteries; type b between the left subclavian and left common carotid arteries; and type c (rare) between the left common carotid and brachiocephalic (innominate) arteries. for general dissection techniques, see part i, chapter 3. possible associated conditions: bicuspid aortic valve (with type a); di george syndrome* with thymic and parathyroid aplasia (with type b); hypoplasia of ascending aorta (with all types); persistent truncal artery (truncus arteriosus); ventricular septal defect. arrhythmia, cardiac note: see also under "death, sudden cardiac." toxicologic studies may be indicated, for instance, if digitalis toxicity (see "poisoning, digitalis") is suspected. if a cardiac pacemaker had been implanted, the instrument should be tested for malfunction. arteriosclerosis (see "atherosclerosis.") arteritis, all types or type unspecified synonyms and related terms: allergic angiitis and granulomatosis (churg-strauss);* allergic vasculitis; anaphylactoid purpura* and its synonyms; angiitis; buerger's disease;* cranial arteritis; giant cell arteritis;* granulomatous arteritis (angiitis); hypersensitivity angiitis; infectious angiitis; necrotizing arteritis; polyarteritis nodosa;* rheumatic arteritis; rheumatoid arteritis, syphilitic arteritis; takayasu's arteritis;* temporal arteritis; thromboangiitis obliterans; and others (see also below under "note"). note: autopsy procedures depend on (1) the expected type of arteritis, such as giant cell arteritis,* polyarteritis nodosa,* or thromboangiitis obliterans (buerger's disease*); and (2) the nature of suspected associated or underlying disease, such as aortic arch syndrome,* beh~et's syndrome,* cogan's syndrome, degos' disease,* dermatomyositis,* erythema nodosum and multiforme,* goodpasture's syndrome,* polymyositis, rheumatic fever, * rheumatoid arthritis,* syphilis,* and other nonspecific infectious diseases, systemic lupus erythematosus,* systemic sclerosis (scleroderma),* or takayasu's disease. for histologic study of blood vessels, verhoeff-van gieson stain or a similar stain is recommended. temporal and ophthalmic arteritis. arteritis of ciliary and retinal vessels. clinically, polymyalgia. anemia. arteritis, takayasu's synonyms: aortic arch syndrome; pulseless disease. external examination heart, aorta, and adjacent great vessels kidney eyes and optic nerve brain for in situ aortography, clamp distal descending thoracic aorta and neck vessels as distal as possible from takeoff at aortic arch. remove heart together with aorta and long sleeves of neck vessels. for coronary arteriography, see chapter 10 (method designed to show coronary ostia). test competence of aortic valve. open aortic arch anteriorly and measure (with calipers) lumen at origin of great neck vessels. photograph aorta and neck vessels and submit samples for histologic study. request verhoeffvan gieson stain. submit tissue for histologic examination. for removal and specimen preparation, see chapter 5. for removal and specimen preparation, see chapter 4. facial muscular atrophy and pigmentation. narrowing at origin of brachiocephalic arteries. dilated ascending aorta. narrowing of coronary arteries at origins. myocardial infarction. aortic insufficiency. * aortic atherosclerosis. thromboses of brachiocephalic arteries. giant cell arteritis. * diffuse mesangial proliferative glomeulonephritis (1) . atrophy of optic nerve, retina, and iris; cataracts; retinal pigmentation. ischemic lesions. artery, patent ductal synonym: patent ductus arteriosus. note: the basic anomaly is persistent postnatal patency of the ductal artery, usually as an isolated finding (in 75% of cases in infants, and in 95% in adults). it is more common in premature than full-term infants and at high altitudes than at sea level. possible complications in unoperated cases include congestive heart failure, * plexogenic pulmonary hypertension,* ductal artery aneurysm or rupture, fatal pulmonary embolism,* or sudden death. in some conditions, such as aortic atresia* or transposition with an intact ventricular septum,* ductal patency may be necessary for survival. possible associated conditions: atrial or ventricular septal defect;* coarctation ofthe aorta;* conotruncal anomalies; necrotizing enterocolitis in premature infants; postrubella syndrome; and valvular or vascular obstructions. artery, persistent truncal synonym and related terms: type i, pulmonary arteries arise from single pulmonary trunk (in 55%); type 2, pulmonary arteries arise separately but close-by (in 35%); type 3, pulmonary arteries arise separately but distal from one another (in 10%). note: the basic anomaly is a common truncal artery, with truncal valve, giving rise to aorta, pulmonary arteries, and coronary arteries, usually with a ventricular septal defect. interventions include complete rastelli-type repair, with closure of ventricular septal defect, and insertion of valved extracardiac conduit between right ventricle and detached pulmonary arteries. possible associated conditions: absent pulmonary artery (in 15%); atrial septal defect (in 15%); absent ductal artery (in 50%); coronary ostial anomalies (in 40%); di george syndrome;* double aortic arch; extracardiac anomalies (in 25%); interrupted aortic arch* (in 15%); right aortic arch (in 30%); truncal valve insufficiency (uncommon) or stenosis (rare); trun-cal valve with three (in 70%), four (in 20%), or two (in 10%) cusps. heart and great vessels if infective endocarditis is suspected, follow culture procedures for endocardial vegetation described in chapter 10. request verhoeff-van gieson stain. infective endocarditis,* usually of truncal valve. late postoperative conduit obstruction. postoperative late progressive truncal artery dilation with truncal valve insufficiency. hypertensive pulmonary vascular disease. cerebral abscess,* if right-to-ieft-shunt was present. arthritis, all types or type unspecified note: for extra-articular changes, see under the name of the suspected underlying conditions. infectious diseases that may be associated with arthritis include bacillary dysentery, * brucellosis, * gonorrhea, rubella,* syphilis, * tuberculosis, * typhoid fever, * and varicella. * noninfectious diseases in this category include acromegaly,* beh<;et's syndrome,* felty's syndrome,* gout,* rheumatoid arthritis,* and many others, too numerous to mention. remove synovial fluid and prepare smears. submit synovial fluid for microbiologic and chemical study. for removal of joints, prosthetic repair, and specimen preparation, see chapter 2. for removal and specimen preparation, see chapter 5. in the polyarticular variant, facial asymmetry may be noted. rheumatoid factor positive in some cases. pericarditis.* interstitial pneumonitis; pleuritis. (see also under "arthritis, rheumatoid.") lymphadenopathy. splenomegaly. monarthritis or severe, erosive polyarthritis; see also under "arthritis, rheumatoid" and above under "externalexamination and skin." ankylosing spondylitis* may be present. chronic iridocyclitis. see "arthritis, rheumatoid." arthritis, rheumatoid synonyms and related terms: ankylosing spondylitis;* felty's syndrome;* juvenile rheumatoid arthritis* (still's disease); rheumatoid disease; and others. possible associated conditions: amyloidosis;* polymyositis (dermatomyositis*); psoriasis;* sjogren's syndrome;* systemic lupus erythematosus;* systemic vasculitis, and others. subcutaneous rheumatoid nodules on elbows, back, areas overlying ischial and femoral tuberosities, heads of phalangeal and metacarpal bones, and occiput. deformities and subluxation of peripheral joints (see also below under "joints"). subaxial dislocation of cervical spine may be cause of sudden death. pneumothorax;* pleural empyema.* t-cell abnormalities (1) . bacteremia. positive rheumatoid factor. rheumatoid granulomas in myocardium (septum), pericardium, and at base of aortic and mitral valves; constrictive pericarditis;* aortic stenosis;* coronary arteritis. systemic vasculitis (arteritis*). rheumatoid granulomas in pleura and lung (with pneumoconiosis*); bronchopleural fistula; rheumatoid pneumonia with interstitial pulmonary fibrosis and honeycombing; bronchiectasis;* bronchiolitis with cystic changes; pulmonary arteritis. pneumoconiosis* in caplan arthrogryposis (2) may be a primary muscle disease, or it may involve abnormalities of the brain, spinal cord, and/or peripheral nerves. etiologies are numerous, as are the modes of inheritance. critical to making the appropriate diagnosis is the collection of muscles from various sites for routine histology, muscle histochemistry, and electron microscopy. portions of peripheral motor nerves must also be prepared for histology and electron microscopy. abdominal cavity intra-abdominal lymphatic system puncture abdominal cavity and submit fluid for microbiologic study. record volume of exudate or transudate and submit sample for determination of fat and cholesterol content. prior to routine dissection, lymphangiography (see below) may be indicated. possible associated conditions: with pulmonary aspergillosis-bronchiectasis; * bronchocentric granulomatosis;* sarcoidosis;* tuberculosis. * with systemic aspergillosisleukemia;* lymphoma;* and other conditions complicated by immunosuppression (l, 2) . other organs a carefully make multiple parasagittal sections through the unperfused lungs. culture areas of consolidation. if diagnosis was confirmed, perfuse lungs with formalin. prepare histologic sections from walls of cavities, cavity contents, and pneumonic infiltrates. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. assault note: all procedures described under "homicide" must be followed. asthma note: spray death* may occur in asthma sufferers from pressurized aerosol bronchodilators. record thickness and position. perfuse one lung with formalin. because mucous plugs may block bronchial tree, attach perfusion apparatus to pulmonary artery or to bronchus and pulmonary artery. monitor perfusion to ensure proper inflation. prepare photograph of fixed cut section. submit samples of pulmonary parenchyma and bronchi for histologic study. request azure-eosin and verhoeff-van gieson stains. record weight and thickness of walls. leave attached to stomach. photograph and submit samples for histologic study. eczema. conjunctival hemorrhages and subcutaneous emphysema may be present after fatal attack. pneumothorax;* mediastinal emphysema. low diaphragm (see below). increased igeconcentrations in fatal asthma; postmortem tryptase determination is of doubtful value in this regard (1) . hypertrophy. low position of diaphragm. hyperinflated lungs. thick-walled bronchi with prominent viscid mucous plugs. typical microscopic inflammatory changes (2) . asthmatic bronchitis with eosinophilic infiltrates. bronchocentric granulomatosis.* pulmonary atherosclerosis with breakup of elastic fibers. paucity of ecosinophils in mucous (6) . cor pulmonale. refl ux esophagitis (3) . peptic ulcer. * pneumatosis of small intestine; emphysema of colon. centrilobular congestion and necrosis. petechial hemorrhages in hypothalamus; necrosis of cerebellar folia; anoxic changes in cortex, globus pallidus, thalamus, sommer's sector of hippocampus, and purkinje cells of cerebellum. suspected changes in anterior hom cells of spinal cord in patients with asthma-associated poliomyelitis-like illness (hopkins syndrome) (4). allergic polyps and other allergic inflammatory changes (5) . increased erythropoiesis. atresia, aortic valvular synonym: aortic atresia; aortic atresia with intact ventricular septum; hypoplastic left heart syndrome. note: the basic anomaly is an imperforate aortic valve, with secondary hypoplasia ofleft-sided chambers and ascending aorta. for possible surgical interventions, see two-stage norwood and modified fontan procedures in chapter 3. possible associated conditions: atrial septal defect* (or patent foramen ovale, usually restrictive); dilatation of myocardial sinusoids thatcommunicate with coronary vessels; dilatation of right atrium, right ventricle, and pulmonary trunk; fibroelastosis ofleft atrial and left ventricular endocardium; hypertrophy of ventricular and atrial walls; hypoplastic left atrium, mitral valve, left ventricle, and ascending aorta; mitral atresia* with minute left ventricle; patent ductal artery (ductus arteriosus); small left ventricle with hypertrophic wall; tubular hypoplasia of aortic arch, with or without discrete coarctation. synonyms and related terms: congenital biliary atresia; extrahepatic biliary atresia; infantile obstructive cholangio-pathy; syndromic (alagille's syndrome) or nonsyndromic paucity of intrahepatic bile ducts ("intrahepatic" biliary atresia). possible associated conditions: alpha]-antitrypsin deficiency;* choledochal cyst;* congenital rubella syndrome;* polysplenia syndrome* (1); small bowel atresia; trisomy 17-18; trisomy 21; turner's syndrome;* viral infections (cytomegalovirus infection;* rubella*). dissect extrahepatic bile ducts in situ or leave hepatoduodenalligament intact for later fixation and sectioning (see below). record appearance and contents of gallbladder and course of cystic duct. in postoperative cases, submit sample of anastomosed hepatic hilar tissue for demonstration of microscopic bile ducts. remove liver with hepatoduodenalligament. prepare horizontal sections through ligament and submit for histologic identification of ducts or duct remnants. prepare frontal slices of liver and sample for histologic study. request pas stain with diastase digestion. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. jaundice. congenital rubella and other viral infections. alpha]-antitrypsin deficiency;* defects in bile acid synthesis. chromosomal abnormalities. in atresia of the hepatic duct, the gallbladder will be empty. in isolated atresia of the common bile duct, the gallbladder contains bile but it cannot be squeezed into the duodenum. atresia or hypoplasia of bile duct(s); choledochal cyst(s). biliary drainage created by kasai operation. obliterative cholangiopathy (2) . intrahepatic cholelithiasis; postoperative ascending cholangitis; secondary biliary cirrhosis; giant cell transformation; paucity of intrahepatic bile ducts. pas-positive inclusions in alphal-antitrypsin deficiency.* polysplenia syndrome* (1) with malrotation, situs inversus, preduodenal portal vein, absent inferior vena cava, anomalous hepatic artery supply, and cardiac defects. for other abnormalities outside the biliary tree, see under "possible associated conditions"). nephromegaly (3) . atresia, cardiac valves (see "atresia, aortic valvular," "atresia, mitral valvular," "atresia pulmonary valvular, with intact ventricular septum," "atresia, pulmonary valvular, with ventricular septal defect," and "atresia, tricuspid valvular.") atresia, duodenal possible associated conditions: with membranous obstruction of the duodenum-annular pancreas; atresia of esophagus* with tracheoesophageal fistula; congenital heart disease; cystic fibrosis;* down's syndrome;* hirschsprung's disease; imperforate anus* or other congenital obstructions of the intestinal tract (1); intestinal malrotation; lumbosacral, rib-, and digitllimb anomalies; single umbilical artery; spinal defects; undescended testis (1). see also under "atresia, small intestinal." the basic anomaly is an imperforate pulmonary valve, with a hypoplastic right ventricle. in unoperated cases, ductal closure is the most common cause of death. for possible surgical interventions, see modified blalock-taussig shunt, mod-ified fontan procedure, and pulmonary valvulotomy in chapter 3. for general dissection techniques, see chapter 3. possible associated conditions: dilated myocardial sinusoids that may communicate with epicardial coronary arteries or veins; patent ductal artery (ductus arteriosus); patent oval foramen (foramen orale); tricuspid atresia with minute right ven-tricle; tricuspid stenosis with hypoplastic right ventricle (in 95%); tricuspid insufficiency with dilated right ventricle (in 5%). synonym: tetralogy of fallot with pulmonary atresia. note: the basic anomaly is atresia of the pulmonary valve and ofvariable length ofpulmonary artery, and ventricular septal defect (membranous or outlet type), with overriding aorta, and with pulmonary blood supply from ductal or systemic collateral arteries. for possible surgical interventions, see rastelli-type repair and unifocalization of multiple collateral arteries in chapter 3. possible associated conditions: right ventricular outflow tract a short blind-ended pouch (70%) or absent (30%); atresia of pulmonary artery bifurcation, with nonconfluent pulmonary arteries; right aortic arch (40%); atrial septal defect (50%); persistent left superior vena cava; anomalous pulmonary venous connection; tricuspid stenosis or atresia; complete atrioventricular septal defect; transposed great arteries; double inlet left ventricle; asplenia, polysplenia, or velocardiofacial syndromes; dilated ascending aorta, with aortic insufficiency. related term: jejuno-ileal atresia. possible associated findings: esophageal atresia* with tracheoesophageal fistula; lumbosacral, rib-, or digit/limb anom -alies; undescended testes (l) . note: see also under "atresia, duodena1." fascia lata, blood, or liver these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter 9) . intestinal tract for mesenteric angiography, see chapter 2. leave mesentery attached to small bowel, particularly to the atretic portion. trisomy 21. multiple atresias; proximal dilatation; volvulus; malrotation; meconium impaction; other evidence of cystic fibrosis. anorectal malformation (l) . annular pancreas (1). atresia, tricuspid valvular note: the basic anomaly is an absent right atrioventricular connection (85%) or imperforate tricuspid valve (15%), with a hypoplastic right ventricle (100%), muscular ventricular septal defect (90%) that is restrictive (85%), and a patent oval atresia, urethral foramen (80%) or secundum atrial septal defect (20%). for possible surgical interventions, see modified fontan or glenn procedures in chapter 3. for general dissection techniques, see chapter 3. possible associated conditions: juxtaposed atrial appendages; large left ventricular valvular orifice; large left ventricular chamber; persistent left superior vena cava; pulmonary atresia; transposition of the great arteries (25%), with aortic co-arctation (35% of those); anomalies of musculoskeletal or digestive systems (20%); down's,* asplenia, or other syndromes. heart aorta and cervical arteries brain if infective endocarditis* is suspected, culture using the method described in chapter 7. for dissection of carotid and vertebral arteries, see chapter 4. for removal and specimen preparation, and cerebral anteriography, see chapter 4. if a foreign body is discovered during a medicolegal autopsy or if the discovery of a foreign body may have medicolegal impli-cations (e.g., presence of a surgical instrument in the abdominal cavity), the rules of the chain of custody apply. for the handling of bullets or bullet fragments, see "injury, firearm." if analysis offoreign material is required, commercial laboratories may be helpful. bolus (see "obstruction, acute airway!') burns note: fatal bums should be reported to the medical examiner's or coroner's office. the questions to be answered by the pathologist depend on whether the incident was accidental, sui-cidal, or homicidal, and whether the victim survivied to be treated in the hospital. a pending death certificate should be issued if the fire and police investigators are not sure of the circumstances at the time of the autopsy. for electrical bums, see under "injury, electrical." for victims who were treated at the hospital, autopsy procedures should be directed toward the discovery or confirmation of the mechanism of death, such as sepsis or pulmonary embolism.* death can be caused primarily by heart disease, with other-wise minor bums and smoke inhalation serving as the trigger that leads to lethal ventricular arrhythmia. because carbon monoxide concentrations are halved approx every 30 min with 100% oxygen therapy, the pathologist must obtain the first clinical laboratory test results for co-hemoglobin. soot can be detected with the naked eye 2 or 3 d after inhalation of smoke. ambulance records should be examined to determine whether a persistent coma might have been caused by hypoxic encephalopathy following resuscitation from cardiac arrest at the scene. admission blood samples should be acquired to test for cohemoglobin and alcohol. this may not have been done in the emergency room. persons suffering from chronic alcoholism succumb to fire deaths more often than persons who do not drink. a very high initial serum alcohol concentration suggests a risk factor for the fire and presence of chronic alcoholism. patients with chronic alcoholism typically are deprived of alcohol when they are in the bum unit and this can cause sudden, presumably cardiac, death,just as it occurs under similarcircum-stances, not complicated by bums. under these circumstances, the heart fails to show major abnormalities. this mode of dying seems to have no relationship to the presence or absence of liver disease. if the body is found dead and charred at the scene, prepare whole body roentgenograms, before and after removal of remanants of clothing. see also under "identification of the body" and "external examination" in chapter 13). one or two fingerpads may yield sufficient ridge detail for identification. if this is not possible, ante-and postmortem somatic and dental radiographs must be compared for identification, or dna comparison must be used. external examination, heart and lungs abdominal cavity and liver see below under "cardiomyopathy, dilated." record volume of ascites. record actual and expected weight of liver. request iron stain. see below under "cardiomyopathy, dilated." alcoholic cirrhosis and alcoholic cardiomyopathy rarely coexist. however, in genetic hemochromatosis,* cirrhosis and heart failure are common findings. cardiomyopathy, dilated (idiopathic, familial, and secondary types) note: for general dissection techniques, see chapter 3. external examination heart other organs and tissues record actual and expected weights. record ventricular thicknesses and valvular circumferences. evaluate relative atrial and ventricular chamber sizes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. note: huntington's disease maps to the short arm of chromosome 4. the gene is widely expressed but of unknown function; it contains a cag repeat sequence, which is expanded (range, 37 to 86) in patients with huntington's disease. a sensitive diag-nostic test is based on the determination of this cag sequence, which can be done on fresh-frozen tissue or blood (1) . in the absence of genetic confirmation, sampling of organs and tissues cannot be excessive because a complex differential diagnosis must be resolved. note: disseminated intravascular coagulation (dic) often is a complication of obstetrical mishaps such as abruptio placentae or amniotic fluid embolism,* or it complicates malignancies (such as adenocarcinomas or leukemia*) or bacterial, viral, and other infections. other conditions such as aortic aneurysm* or hemolytic uremic syndrome* are known causes also. ifthe nature of the underlying disease is known, follow the procedures under the appropriate heading also. note: this is a cause of diarrhea. microscopic colitis is associated with older age; collagenous colitis is associated with female sex (1). the colon is grossly normal but microscopically, increased lymphocytes in the lamina propria and a subepithelial band of collagen is found. if only the lymphocytic infiltrate is found, the term "lymphocytic colitis" or "microscopic colitis" should be applied. a trichrome stain should be ordered in all instances, because the collagen band may be difficult to see without the special stain. i death, anaphylactic synonym: generalized anaphylaxis. note: autopsy should be done as soon as possible after death. neck organs should be removed before embalming. if death is believed to be caused by drug anaphylaxis, inquire about type of drug(s), drug dose, and route of administration (intravenous, intramuscular, and oral or other). this will determine proper sampling procedures-for instance, after penicillin anaphylaxis. allergy to bee stings, wasp stings, fire ants, and certain plants may also be responsible for anaphylaxis. however, envenomation also can be fatal in the absence of anaphylaxis. external examination search for injection sites or sting marks. if such lesions are present, photograph and excise with 5-cm margin. freeze excised tissue at -70â°c for possible analysis. prepare chest roentgenogram. foam in front of mouth and nostrils. swelling of involved tissue. antigen-antibody reaction in involved tissues. antibodies against suspected antigen. laryngeal edema may recede soon after death. foamy edema in trachea and bronchi; diffuse or focal pulmonary distention ("acute emphysema") alternating with collapse; pulmonary edema and congestion; accumulation of eosinophilic leukocytes. eosinophilic leukocytes in red pulp. death, anesthesia-associated. note: there are many possible causes of anesthesiaassociated death that are not drug-related, such as acute airway obstruction* by external compression, aspiration, arrhythmia of a heart not previously known to be diseased, tumor, or an inflammatory process. some ofthe complications are characteristically linked to a specific phase of the anesthesia, and many are not revealed by customary morphologic techniques. the task for the pathologist charged with investigating an anesthesia-associated death is to reconstruct the chain of physiologic events culminating in cessation of vital signs. autopsy morphology plays a supporting role; the main investigations center around the record left by the anesthesiologist, testing of anesthesia equipment, and toxicological testing. a consulting anesthesiologist can divine much more information from the anesthesia and recovery room records than can the pathologist, and can suggest avenues of further investigation. therefore, the most important step in these autopsies is to obtain the anesthesiaassociated records and to secure the consulting services of an independent anesthesiologist. the changes in the vital signs during and after anesthesia will help to focus the investigation toward a cardiac mechanism ofdeath or depression ofbrainstem function as a terminal mechanism. when information is gathered about drugs and chemical agents that have been administered or to which the victim may have had access, the pathologist must keep in mind that some non-medical chemicals and many drugs are known to affect anesthesia. drugs and their metabolic products, additives, stabilizers, impurities, and deterioration products (one of which can be carbon monoxide) may be present and can be identified in postmortem tissues. therefore, all appropriate body fluids and solid tissue should be submitted for toxicological examination. if the anesthetic agent was injected into or near the spinal canal, spinal fluid should be withdrawn from above the injected site into a standard toxicologist's collection tube with fluoride preservative. if the anesthetic agent was injected locally, tissue should be excised around the needle puncture marks at a radius of 2-4 em. serial postmortem analysis of specimens may permit extrapolation to tissue concentration at the time of death. the time interval between drug administration and death sometimes can be calculated from the distribution and ratio ofadministered drugs and their metabolic products. for a review of anesthetic death investigation, see ref. (1) . halothane anesthesia and some other anesthetic agents may cause fulminant hepatitis and hepatic failure. the autopsy procedures suggested under "hepatitis, viral" should be followed. note: for special autopsy procedures in postoperative deaths, see chapter 1. in some instances, procedures described under "death, anesthesia-associated" may be indicated. for a review of investigational procedures and autopsy techniques in operating-room-associated deaths, see ref. (1) . if the autopsy will involve anatomy or dissection techniques that are unfamiliar, the pathologist should not hesitate to invite the surgeon to the autopsy. in patients who develop a cerebral infarction after open heart surgery, arterial air embolism should be considered as a possible cause. the diagnosis often must be based on excluding other causes because the air has been absorbed prior to death. if a patient dies rapidly, the hospital records may be incomplete or scanty. for example, if a patient bleeds to death despite attempted repair of hepatic lacerations, hospital records may not suffice to reach the correct cause-of-death opinion; personal accounts from the surgeon and anesthesiologist may be needed. autopsy data on patients dying following thoracic surgery may be found in ref (2) . d death, restaurant (see "obstruction, acute airway.") death, sniffing and spray related terms: glue sniffing; sudden sniffing death syndrome. note: no anatomic abnormalities will be noted at autopsy. sudden death may occur after cardiac dysrhythmia or respiratory arrest. procedures possible or expected findings lungs brain if poison had been inhaled at the time when death occurred, tie main bronchi. submit lungs in glass container for gas analysis. submit samples of small bronchi for histologic study. for removal and specimen preparation, see chapter 4. submit samples of fresh or frozen brain for toxicologic study. submit samples in glass containers (not plastic) for toxicologic study. trichloroethane, fluorinated refrigerants, and other volatile hydrocarbons are most often involved in the "sudden sniffing death syndrome." spray death may occur in asthma sufferers using pressurized aerosol bronchodilators. freons and related propellants may also be responsible for sudden death. toxic components of glue-such as toluene-accumulate in the brain of glue sniffers. also present in various glues are acetone, aliphatic acetates, cyclohexane, hexane, isopropanol, methylethyl ketone, and methylisobutyl ketone. aerosols may occlude the airway by freezing the larynx. carbon tetrachloride sniffing may cause hepatorenal syndrome (see also under "poisoning, carbon tetrachloride"). death, sudden unexpected, of adult note: medicolegal autopsies are usually indicated, and appropriate procedures should be followed. ifanaphylactic death is suspected, see also under that heading. for all unexpected deaths, the pathologist should learn the circumstances of the death, in order to determine whether the mechanism of death was rapid or slow, and to guide the selection of ancillary tests. whenever paramedics attended a person, the run sheet should be obtained to look for a history of recent drinking or ofchronic alcoholism may be an important clue. the combination of a history ofalcoholism, a negative test for ethanol, and absence ofcardiovascular disease, should suggest alcohol withdrawal as the cause ofa sudden death. the list of"possible or expected findings" below is not complete. for general toxicologic sampling, see chapter 13. possible associated conditions: atrial septal defect;*bicuspid aortic valve;* coarctation,* hypoplasia, or interruption (type a) of aortic arch; coronary artery from main pulmonary artery; right atrial arch; patent ductal artery;* right pulmonary artery from ascending aorta; subaortic stenosis;* tetralogy of fallot;* ventricular septal defect. * (in approx 50% of the cases, one or more of these associated conditions are found.) defect, atrial septal note: the basic anomaly is a defect of the atrial septum, usually at the oval fossa (in 85%). possible complications in unoperated cases include atrial arrhythmias, congestive heart failure; paradoxic embolism; plexogenic pulmonary hypertension â«10%), and pulmonary artery aneurysm. possible surgical interventions include surgical and transcatheter closure of defect. for deficiency, vitamin c synonyms: hypovitaminosis c; scurvy. external examination and skin other organs bones, joints, and soft tissues record extent and character of skin lesions; prepare sections of skin. describe appearance of gums, and prepare sections. record evidence of bleeding. for removal, prosthetic repair, and specimen preparation of bones and joints, see chapter 2. hyperkeratotic hair follicles with perifollicular hemorrhages (posterior thighs, anterior forearms, abdomen); petechiae and ecchymoses (inner and posterior thighs); subcutaneous hemorrhages. gingivitis. in rare instances, gastrointestinal or genitourinary hemorrhages. hemorrhages into muscles and joints. subperiosteal hemorrhages occur primarily in distal femora, proximal humeri, tibiae, and costochondral junctions (scorbutic rosary). deficiency, vitamin d synonyms: hypovitaminosis d; rickets. note: features or rickets may be found in familial hypophosphatemia (vitamin d-resistent rickets; fanconi syndrome). vitreous or blood (serum) other organs prepare skeletal roentgenograms. in infants with suspected rickets, record size of anterior fontanelle and shape of head; state of dentition; and shape of costochondral junctions, wrists, long bones, and spine. submit samples for calcium, magnesium, and phosphate determination. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. weigh parathyroid glands and submit samples for histologic study. submit samples of intestine for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. in infantile rickets, diagnostic sites for histologic sampling are costochondral junctions, distal ends of radius and ulna, and proximal ends of tibia and humerus. for adults, see under "osteomalacia." in infants, rachitic changes at costochondral junctions; in adults, osteoporosis* and osteomalacia*-with or without pseudofractures (milkman's syndrome (1) . note: the term spinocerebellar degeneration encompasses a variety of lesions whose classification is controversial. a new approach has come from linkage analysis and molecular biology. for instance, friedreich's ataxia, the classic form of hereditary ataxia, is due to an intronic expansion of a gaa tri-nucleotide repeat. other forms are also identified by their specific gene loci. neuropathologic examination still is important and ample sampling is suggested, which should include cerebral cortex, basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, subthalamic nucleus, midbrain (red nucleus and substantia nigra), pons (pontine nuclei), spinal cord (at cer-vical, thoracic, and lumbar levels), optic tract, optic nerves with lateral geniculate nucleus, and sensory and motor peripheral nerves. for removal and specimen preparation, see chapter 5. enlargement of head. poor demarcation between cortex and gelatinous white matter. extensive demyelination and vacuolation of white matter, particularly subcortically. optic atrophy. degeneration, striatonigral (see "atrophy, multiple system.") related term: thirst. note: possible underlying conditions not related to inaccessibility of water include bums, exposure to heat, gastrointestinal diseases, recent paracentesis, renal diseases, and use of diuretic drugs. see also under "disorder, electrolyte(s)." external examination vitreous urine prepare histologic sections of blisters, ulcers, or skin abrasions. submit sample for sodium, chloride, and urea nitrogen determination. skin turgor may be decreased and eyes may be sunken. microscopic changes help to decide whether skin lesions are antemortem or postmortem. sodium concentrations more than 155 meqll, chloride concentrations more than 130 meq/ and urea nitrogen concentrations between 40 and 100 meq/dl indicate dehydration. absence or minimal amount of urine. dementia (see "disease, alzheimer's.") drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." drug abuse, cocaine note: cocaine is spontaneously hydrolyzed by blood esterases, even after death. however, one of its major metabolite, benzoylecgonine, is routinely identifiable by immunoassay screening tests. when cocaine is abused concurrently with heroin or other depressant drugs, it may be difficult to ascribe deth to a single agent, unless circumstances clearly point to a rapid cardiac mechanism or a slow brainstem depression mechanism. note: if narcotic paraphernalia and samples of the drug itself are found at the scene of the death, they should be submitted for analysis. helpful information about the nature of a drug may be obtained from witnesses. state crime laboratories may provide much assistance. if name of drug is known, see also under "poisoning,..." the slang name of a drug may be insufficient for identification because these names often are used for different compounds at different times of places. opoid narcotics can be injected intravenously, or subcutaneously, or snorted. death may occur with such speed that the bodies may be found with needles and syringes in the veins or clenched in the hands. drug abuse may be associated with a multitude of local (see below) or systemic complications, including malaria* and tetanus. * as stated in chapter 13, for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subclavian vein. the authors prefer the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. it may be pru-dent to add naf to some of the samples. related term: childhood dermatomyositis (or polymyositis) associated with vasculitis; dermatomyositis (or polymyositis) associated with neoplasia or collagen vascular disease; primary idiopathic dermatomyositis; primary idiopathic polymyositis. possible associated conditions: carcinoma (lung, stomach, intestine, and prostate in males; breast, ovary, and uterus in females; miscellaneous sites in both sexes); lymphoma* (rare) and other malignancies (1); lupus erythematosus;* mixed connective tissue disease; progressive systemic sclerosis;* rheumatoid arthritis;* sjogren's syndrome;* and others. vasculitis of childhood polymyositis (dermatomyositis). external examination and skin heart lungs esophagus and gastrointestinal tract photograph grossly involved skin. prepare sections of involved (anterior chest, knuckles, knees) and grossly uninvolved skin and subcutaneous tissue. prepare roentgenograms. submit samples from myocardium for histologic study. perfuse one lung with formalin. submit samples from all segments for histologic study. arteritis* and phlebitis* with thrombosis, fibrosis, and infarctions. steatohepatitis and manifestations of diabetes mellitus* may be found (2) . myositis with muscular atrophy and fibrosis; vasculitis in childhood cases. polyneuropathy (rare) (5). arthritis. diabetes mellitus synonyms: type i (insulin-dependent or juvenile-onset) diabetes mellitus; type ii (insulin-independent or adult onset) diabetes mellitus; secondary diabetes mellitus (e.g., due to drugs or pancreatic disease). note: in infants of diabetic mothers, macrosomia and congenital malformations must be expected. record size and weight of placenta and total weight and length, crown to rump length, and crown to heel length of infant. compare with expected measurements (see part iii). expected histologic finding in-clude hyperpla-sia with relative increase ofb cells of the islands of langerhans with interstitial and peri-insular eosinophilic infiltrates, decid-ual changes of the endometrium, enhanced follicle growth in the ovaries, and leydig cell hyperplasia. possible associated conditions: acanthosis nigricans; acro-megaly;* amyotrophic lateral sclerosis; * ataxia telangiectasia;* fanconi's anemia;* friedreich's ataxia;* gout;* hemochro-matosis; *hyperlipoproteinemia; * hyperthroidism;* obesity;* turner's syndrome;* and many others, too numerous to mention. note: the term "caroli's syndrome" often is used for cases that also show histologic features of congenital he-patic fibrosis or other manifestations of fibropolycystic liver disease,* whereas the name "caroli's disease" refers to idiopathic dilatation of intrahepatic bile ducts, without associated abnormalities. possible associated conditions: choledochal cyst* and related extrahepatic biliary abnormalities (1); congenital hepatic fibrosis; * cysts of kidneys (renal tubular ectasia or medullary sponge kidney; autosomal-recessive polycystic kidney disease, and rarely, autosomal-dominant polycystic kidney disease [2] )* and of pancreas. record volume of effusions. prepare smears of fresh blood or of buffy coat, or make thick-drop preparation. submit sample for xenodiagnosis or animal inoculation and for serologic study. record weight. in chronic chagas' disease, perfuse intact heart with formalin (chapter 3) and slice fixed heart in a frontal plane so as to create anterior and posterior halves. prepare photographs. histologic samples should include conduction system. include several sections of atrial (auricular) walls for histologic study of autonomous ganglia. perfuse at least one lung with formalin. leave affected hollow viscera intact and fill with formalin. cut fixed organs in half, photograph, and cut histologic sections on edge. record liver weight and submit samples for histologic study. record weight. prepare photographs of abnormalities. weigh and examine. prepare histologic sections. for removal and specimen preparation, see chapter 4. autopsy is desirable in suspected cases because the diagnosis can only be firmly established after neuropathologic examination. serologic studies are not available. unfortunately, all tissues (not just the brain and spinal cord) may remain infectious even after prolonged fixation and histologic processing. thus, the autopsy recommendations for most other infectious diseases do not apply here. this is a reportable disease in some states. special precautions are indicated and therefore, the procedures described here should be followed strictly (1) (2) (3) (4) : all persons in the autopsy room must wear disposable long-sleeved gowns, gloves, and masks. contamination of the autopsy table should be prevented by covering it with a disposable, non-permeable plastic sheet. autopsy generally should be restricted to the brain. if organs in the chest or abdomen need to be examined, this is best done in situ. to prevent aerosolization of potentially infectious bone dust, a hood or other protective device should be used while opening the skull with a stryker saw. after completing the autopsy, instruments and other potentially contaminated objects should be autoclaved in a steam autoclave (1 h at 134â°c). porous load is considered more effective than gravity displacement autoclaves. immerse autopsy instruments in distilled water before and during autoclaving, in order to protect them from corrosion. ifno autoclave is available, chemical disinfection (see below) is a satisfactory alternative. disposable items should be put in a container for infectious hospital waste and ultimately incinerated. contaminated objects not suitable for autoclaving (such as the stryker saw) should be soaked with a2nnaoh solution for 1 h (alternatively, 1 nnaoh may be used for 2 h). contaminated surfaces should be thoroughly washed with the same solution. aluminum should be treated for 2 h with a fresh 5% naoci (sodium hypochlorite) solution with at least 20,000 ppm free chloride. wash waters should be collected; if no autoclave is available, 2 n naoh or >4 volumes of 5% sodium hypochlorite bleach should be added to the water and left for a minimum of 2 h before being discarded. before removing the body from the autopsy room, it should be sponged with 5% sodium hypochlorite. to deactivate cjd infectivity, tissue blocks, 5 mm or less in thickness, should be fixed in formalin in a formalin-totissue ratio of at least 20: 1 for at least 48 h and then soaked in concentrated formic acid (95-100%) for i h, followed by another 48 h of formalin fixation. the fixation fluid should be collected and decontaminated, as described earlier for wash water. glassware and tissue carriers should also be decontaminated as previously described. after this deactivation, the tissue blocks can be processed in a routine fashion. at any stage of these procedures, special care must be taken to avoid cuts with potentially contaminated glassware, blades, or other objects. parenteral exposure to potentially contaminated material also should be avoided. remains of patients who have died of the disease should not be accepted for anatomy teaching for students. if specimens are prepared for pathology collections, they should be handled with great caution. morticians and mortuary workers should be warned of possible hazards posed by tissues of patients with transmissible spongiforme encephalopathies; they should be advised about proper use of disinfectants. clinical laboratories that receive autopsy tissues or fluids must be warned about the infectious nature of the material. if possible, decontamination should be done at the site where the autopsy was done. for the shipping of potentially infected material, see chapter 15. increased concentrations of nse (5). spongiforme changes, astrocytosis, neuronal loss, amyloid plaque formation, prp deposition, and proliferation of activated microglia (6). cerebrospinal fluid brain submit sample for neuron-specific enolase (nse). for removal and specimen preparation, see chapter 4 and above under "note." submit fresh-frozen material for confirmation of diagnosis by histoblot technique on protease k-digested frozen tissue or western blot preparations on brain homogenates. immunohistochemical localization ofprp and hla-dr protein on paraffin-embedded tissue is possible. disease, demyelinating (see "degeneration, spongy, of white matter," "encephalomyelitis, all types or type unspecified," "leukodystrophy, globoid cell," "leukodystrophy, sudanophilic," "sclerosis, multiple;' and "sclerosis, schilder's cerebral.") disease, diffuse alveolar synonym: diffuse pulmonary disease. note: autopsy procedures are listed under the more specific diagnoses, such as "hemosiderosis, idiopathic pulmonary," "lipoproteinosis, pulmonary alveolar," "microlithiasis, pulmonary alveolar," "pneumonia, lipoid," and "syndrome, goodpasture's." glycosphingolipid storage in cornea; lens opacities; dilated vessels in conjunctiva and lens; thrombi in blood vessels (5). disease, fibropolycystic, of the liver and biliary tract note: "fibropolycystic disease of the liver and biliary tract" comprises a group of well defined conditions, which may occur together and hence need a collective designation. the conditions include autosomal-recessive (infantile) and auto-somal dominant (adult) polycystic disease of the liver; caroli's disease or syndrome;* choledochal cyst,* congenital hepatic fibro-sis,* multiple biliary microhamartomas, and related disorders. for autopsy procedures, see also under more specific designations. disease, glycogen storage synonyms: andersen's disease or brancher deficiency (glycogenosis, type iv); cori's or forbes' disease (glycogenosis, type ill); cyclic amp dependent kinase (type x); glycogen synthetase deficiency (type 0); hers' disease (glycogenosis, type vi); mcardle's disease (glycogenosis type v); phosphorylase b kinase deficiency (types ixa, b, and c); pompe's disease (glycogenosis, type it); tarui disease (glycogenosis type vii); von gierke's disease (glycogenosis, type ia); x-linked glycogenosis (type vill). note: if the diagnosis had not been confirmed prior to death, samples of liver, skeletal muscle, blood, and fascia (for fibroblast culture, see below) should be snap-frozen for enzyme assay, which will determine the specific deficiency. types ia and b, iii, vi, and hepatic phosphorylase b kinase deficiency (types ixa, b and c) are hepatic-hypoglycemic disorders, whereas types v and vii affect muscle energy processes. type ii also affects the musculature, whereas type iv may cause cirrhosis and death in infancy from extreme hypotonia. determination of type of glycogenosis usually can be based on (i) pattern of glycogen storage in liver, (2) presence or absence of nuclear hyperglycogenation in liver, (3) cytoplasmic lipid in liver, (4) presence or absence of liver cirrhosis, and (5) presence or absence of glycogen and basophilic deposits in skeletal muscles. possible associated conditions: fanconi syndrome* or gout* with type ia glycogenosis; neutropenia, recurrent infections, and crohn's disease with types ib or ie. glycogen primarily in retinal ganglion cells and ciliary muscle. glycogen in sympathetic nerve ganglia and neurons of cranial nerves in type vii. gouty arthritis. disease, graft-versus-host note: this disease occurs most commonly after bone marrow transplantation. the disease has also occurred after transfusion of viable lymphocytes, for example, to patients with cancer or leukemia. * in patients with graft-versus-host disease (gvhd), autopsy also may reveal recurrence of the underlying disease such as leukemia. possible associated conditions: alphal-antitrypsin deficiency;* amyloidosis;* ankylosing spondylitis;* primary sclerosing cholangitis;* sjogren's syndrome. * see also below under "possible or expected findings." note: in many instances, either chronic ulcerative colitis or crohn's disease* had been diagnosed clinically, but sometimes, the distinction is difficult to make, even at autopsy. many features described below occur in chronic ulcerative colitis but some manifestations of crohn's disease or conditions that may occur in all types of inflammatory bowel disease also are listed so that both positive and negative findings can be recorded properly. osteoporosis;* ankylosing spondylitis;* arthritis of peripheral joints; periarthritis; hypertrophic osteoarthropathy;* tendinitis (particularly of ankle and achilles tendons). disease, iron storage (see "hemochromatosis.") related terms: atherosclerotic heart disease. note: the most common anatomic finding at autopsy in subjects older than 30 yr is coronary atherosclerosis. unusual under-lying or associated conditions include chronic aortic stenosis or regurgitation; coronary artery anomalies; coronary artery dissection; coronary embolism; coronary ostial stenosis (due to calcification of aortic sinotubular junction or, rarely, to syphilitic aortitis); coronary vasculitis (for instance, in polyarteritis nodosa* or acute hypersensitivity arteritis); hyperthyroidism,* gastrointestinal hemorrhage; * hypothyroidism, * idiopathic arterial calcification of infancy; intramural coronary amyloidosis; pheochromocytoma, polycythemia vera; * pseudoxanthoma elasticum,* radiationinduced coronary stenosis; severe pulmonary hypertension (with right ventricular ischemia); sickle cell disease;* and others. if bypass surgery had been performed, see "surgery, coronary bypass." macular rash (4). multifocal fibrinopurulent pneumonia with sparing of the bronchi and bronchioles. exudate is rich in phagocytes, fibrin, and karyorrhectic debris. synonym: lyme arthritis note: this infection is caused by the spirochete, borrelia burgdoiferi, which is transmitted from rodents to human by the hard deer ticks, ixodes dammini, 1. ricinus, and others. brain and spinal cord for removal and specimen preparation, see chapter 4. request luxol fast blue stain for myelin. symmetric and zonal demyelination in corpus callosum, anterior commissure, optic chiasm, optic tracts, and white matter of frontal lobes. external examination and skin; oral cavity lungs aorta record distribution of skin lesions and submit tissue samples for histologic study. for preparation of angiograms of the pulmonary arterial and venous vasculature, see chapter 2. if aneurysm or dissection is present, follow procedures described under those headings. telangiectatic (often papular) lesions most commonly found in cheeks, scalp, nasal orifices, oral cavity, ears, neck, shoulders, fingers, toes, and nail beds. cyanosis and clubbing may be prominent. arteriovenous malformations/fistulas. aneurysm; * aortic dissection. * if cirrhosis is present, prepare angiograms of hepatic arteries and veins (chapter 2). photograph and prepare sections of angiomatous lesions. note: parkinson's syndrome is caused by conditions that may simulate parkinson's disease; these include carbon monoxide* and manganese poisoning, corticobasal degeneration, druginduced parkinsonism, huntington's disease, multiple system atrophy,* progressive supranuclear palsy* (steele-richardson-olszewski syndrome), space-occupying lesions (rare), trauma (dementia pugilistica), and causes related to tumors and vascular diseases. brain for removal and specimen preparation, see chapter 4. histologic sections should include midbrain (substantia nigra), upper pons (locus ceruleus), medulla, nucleus basalis (substantia innominata), and basal ganglia. if parkinsonian syndrome was diagnosed, follow procedures described under the name of the suspected underlying condition (see above under "note"). depigmentation of substantia nigra and locus coeruleus; neuronal loss and reactive gliosis; eosinophilic intracytoplasmic inclusion bodies (lewy bodies) in some of the surviving neurons; no significant changes in basal ganglia. disease, pelizaeus-merzbacher synonyms: sudanophilic (orthochromatic) leukodystrophy. brain and spinal cord for removal and specimen preparation, see chapter 4. request luxol fast blueipas stain for myelin and bielschowsky's stain for axons. prepare frozen sections for sudan stain. brain generally atrophic. myelin loss in centrum ovale, cerebellum, and part of brain stem, with a tigroid pattern of residual myelin near vessels. axons are preserved. diffuse gliosis with relatively few lipoid-containing macrophages, compared to the myelin loss. lipoid material stains with sudan. brain and spinal cord for removal and specimen preparation, see chapter 4. request silver stains (bielchowsky or bodian stain). histochemical stains in pick's cells and bodies reveal phosphorylated neurofilaments, ubiquitin, and tubulin. some tissue should be kept frozen for biochemical studies. severe cerebral atrophy, involving primarily frontal and anterior temporal lobes (knifeblade atrophy; walnut brain). microscopically, severe neuronal loss accompanied by astrocytosis. characteristic argyrophilic, intracytoplasmic inclusions (pick's bodies), particularly in hippocampus and swollen, distended "ballooned" neurons (pick's cells). these changes are not always present. external examination, skin, and adipose tissue blood cerebrospinal fluid heart liver and kidneys brain, spinal cord, and peripheral nerves eyes submit sample for determinaion of phytanic acid concentration and for molecular studies. for obtaining a sample, see chapter 7. sample for histologic study. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 5. ichthyosis. phytanic acid accumulation in adipose tissues. phytanic acidemia, mutation of phyh or pex 7 (2). increased protein concentrations. cardiomyopathy.* phytanic acid accumulation. axonal neuropathy. retinitis pigmentosa. hypoalphalipoproteinemia. lymphadenopathy with diffuse deposition of cholesterol esters. premature atherosclerotic cardiovascular disease (1). hepatosplenomegaly with foam cells. enlarged tonsils with characteristic orange discoloration. polyneuropathy (2) . in adults, corneal infiltrates. foam cells. request pas stain. in granulomas, bacilli are not always pas positive (2) . section all grossly involved tissues for histologic examination. submit section for electron microscopy. emaciation. hyperpigmentation, particularly of exposed skin and in scars. hyperkeratosis. arthritis involving ankles, knees, shoulders, and wrists. ascites; fibrinous peritonitis. * nodules in peritoneum containing sickle-form particlecontaining cells (spc cells submit sample for determination of sodium, potassium, chloride, glucose, urea nitrogen, and creatinine concentrations. calcium and phosphate concentrations can also be tested. if sample is small, indicate priority for testing. if indicated, submit sample for chemical study. submit tissue samples for histologic study. considerably increased or decreased values for sodium (more than 155 meqll or less than 130 meqll) and chloride (more than 135 meqll or less than 105 meqll) indicate that changes were present before death. for further interpretation, see chapter 8. postmortem electrolyte concentrations are quite unreliable. may be useful for calcium determination. vacuolar nephropathy (vacuolar changes in proximal convoluted tubules) in potassium deficiency (may also occur after infusion of hypertonic solutions). disorder, hemorrhagic (see "coagulation, disseminated intravascular," ''disease, christmas:' ''disease, von willebrand's," "hemophilia," and "purpura,.â�¢â�¢") disorder, inherited, of phagocyte function note: several conditions represent phagocyte function disorders. autopsy procedures for one of these disorders can be found under "disease, chronic granulomatous." consult this entry for other phagocyte function disorders. synonyms and related terms: fabry's disease* (angiokeratoma corporis diffusum); gangliosidosis;* gaucher's disease;* glycogenosis,* type ii; leukodystrophies (krabbe's or globoidcell,* metachromatic leukoencephalopathy*); mucopolysaccharidoses* (hunter, hurler, morquio, and sanfilippo disease); mucolipidosis; niemann pick disease* (type a, b, c, or sphingomyelinase deficiency); neuraminidase deficiency; neuronal ceroid lipofuscinosis (batten's disease or kufs' disease). hypopharyngeal pulsion diverticulum (zenker's diverticulum) at lower margin of inferior constrictor muscle of pharynx. traction diverticulum at midesophagus after an inflammatory process-for instance, tuberculous lymphadenitis. epiphrenic diverticulum may also occur. luxtacardiac or juxtapyloric diverticulum. heterotopic tissue in meckel's diverticulum, with or without peptic ulceration. colonic muscular hypertrophy and stenosis, usually in sigmoid colon. diverticulitis with perforation, fistulas, or peritonitis. * diving (see "accident, diving (skin or scuba).") related terms: dry drowning; fresh-water drowning; near-drowning; salt (sea)-water drowning (see the following table). primary drowning ("immediate drowning") deaths occurring within minutes after immersion, before or without resuscitative measures deaths from hypoxia and acidosis caused by glottal spasm on breath holding. there may be no evidence of water entering stomach or lungs and no appreciable morphologic changes at autopsy. note: the diagnosis is one of exclusion. the pathologist should help the police to determine: i) how did the person (or dead body) get in the water, and 2) why could that person not get out of the water? it is not enough to ask if a person could swim but investigators should find out how well (what strokes did the victim know?) and how far he or she could swim. the inquiry must include the depth of the water and must address hazards such as undertow or underwater debris, and the behavior deaths occurring from within 30 min to several weeks after resuscitation, because of metabolic acidosis, pulmonary edema, or infective or chemical pneumonitis deaths from hypoxia and acidosis caused by obstruction of airway by water related to: hypervolemia hemolysis hyponatremia hypochloremia hyperkalemia of the victim immediately before submerging. deaths of adults in bathtubs and swimming pools are usually from natural, cardiac causes, or they are suicides, unless the victim was drunk. diatom tests (1) have not proven useful in the united states but there is enthusiasm for such tests among european pathologists. the distinction between hyponatremic deaths in fresh water and hypernatremic deaths in salt water derives from experimental studies; in practice, one cannot reliably predict the salinity of the immersion medium from autopsy studies. because many bodies of drowning victims are recovered only after the body floats to the surface, decomposition will often obscure even the nondiagnostic findings such as pleural effusions, which are often associated with drowning. external examination and skin (wounds) organ samples for diatom search serosal surfaces and cavities if identity of drowning victim is not known, record identifying features as described in chapter 13. prepare dental and whole-body roentgenograms. submit tissue samples for histologic study of wounds. inspect inside of hands. collect fingernail scrapings. record appearance and contents of body orifices. record features indicative of drowning. photograph face from front and in profile. take pictures of all injuries, with and without scale and autopsy number. remove vitreous for analysis. if diatom search is intended, clean body thoroughly before dissection to avoid contamination of organs and body fluids with algae and diatoms (see below). submit sample for toxicologic study. sample early during autopsy, before carrying out other dissections. use fresh instruments for removal of specimens to avoid contamination. submit subpleural portion of lung: subcapsular portions of liver, spleen, and kidneys; bone marrow; and brain. store samples in clean glass jars. for technique of diatom detection, see below. record volume of fluid in pleural spaces. photograph petechial hemorrhages. photograph layerwise neck dissection if strangulation* is suspected. open airways posteriorly, and photograph, remove and save mud, algae, and any other material in tracheobronchial tree. record size and weight of lungs. there may be wounds that were inflicted before drowning occurred-for instance, in shipwrecks or vehicular and diving accidents. other wounds may be inflicted after deathfor instance, from ship propellers or marine animals. sometimes, premortem and postmortem wounds can be distinguished histologically. object (hair?) held by hands in cadaveric spasm. cutis anserina and "washerwoman" changes of hands and feet are of no diagnostic help. foreign bodies; semen (see also under "rape"). foam cap over mouth and nose. in the autopsy room, water running from nose and mouth is usually pulmonary edema or water from the stomach. high concentrations of alcohol indicate intoxication (see under "alcoholism and alcohol intoxication"). evidence of alcohol intoxication may be found. diatoms may occur in the liver and in other organs of persons who have died from causes other than drowning. comparison with diatoms in water sample from area of drowning may be helpful. penny-sized or smaller hemorrhages may indicate violent respiratory efforts or merely intense lividity. presence of pleural fluid suggests drowning. for diatom detection (l) , boil 2-5 g oftissue for 10--15 min in 10 rnl of concentrated nitric acid and 0.5 rnl of concentrated sulfuric acid. then, add sodium nitrate in small quantities until the black color of the charred organic matter has been dispelled. it may be necessary to warm the acid-digested material with weak sodium hydroxide, but the material must soon be washed free from alkali to avoid dissolving the diatoms. the diatoms should be washed, concentrated, and stored in distilled water. for examination, allow a drop of the concentrate to evaporate on a slide, and then mount it in a resin of high refractive index. all equipment must be well-cleaned, and distilled water must be used for all solutions. there are several variations and adaptations of this method. drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." ductus arteriosus, patent (see "artery, patent ductal.") synonyms and related terms. achondroplastic dwarf; asexual dwarf; ateliotic dwarf; micromelic dwarf; normal dwarf; pituitary dwarf; true dwarf; and many other terms, too numerous to mention. external examination bones and joints record height and weight. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter 2. growth retardation. abnormal growth of epiphyseal cartilage with enlargement of metaphysis. long bones and pelvis most commonly affected. cavernous hemangiomas (maffucci's syndrome). see above under "external examination." chondrosarcoma. dyscrasia, plasma cell note: these conditions are characterized by abnormally proliferated b-immunocytes that produce a monoclonal immunoglobulin. multiple myeloma, * plasma cell leukemia, plasma-cytoma, and waldenstrom's macroglobulinemia* as well as heavy-chain diseases and monoclonal gammopathies of unknown type belong to this disease family. amyloidosis* is closely related to these conditions. for autopsy procedures, see under "amyloidosis," "macroglobulinemia," or "multiple myeloma" and under name of condition that may have caused the plasma cell dyscrasia. such conditions include carcinoma (colon, breast, or biliary tract), gaucher's disease,* hyperlipoproteinemia, * infectious or noninfectious chronic inflammatory diseases, and previous cardiac surgery. synonym: shigella dysentery. note: (i) collect all tissues that appear to be infected. blood bowel eyes joints submit sample for culture and for serologic study. submit sample of feces or preferably bloodtinged mucus for culture. if bacteriologic diagnosis has already been confirmed, pin colon on corkboard, photograph, and fix in formalin for histologic study. submit sample of vitreous for study of sodium, potassium, chloride, and urea nitrogen concentrations. for removal and specimen preparation of eyes, see chapter 5. for removal, prosthetic repair, and specimen preparation, see chapter 2. escherichia coli septicemia. colitis with microabscesses; transverse shallow ulcers and hemorrhages, most often in terminal ileum and colon. dehydration* pattern of electrolytes and urea nitrogen. serous arthritis* of knee joints is a late complication. external examination record extent of pigmentation, facial features, and primary and secondary sex characteristics. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter 2. record size of apertures of cranial nerves in base of skull. unilateral skin pigmentation and precocious puberty in females (albright's syndrome), less commonly in males. synonyms and related terms: becker's muscular dystrophy; congenital muscular dystrophy; duchenne's progressive muscular dystrophy; dystrophinopathy; em-ery-dreifuss mucular dystrophy; facioscapulohumeral dystrophy; limb girdle dystrophy; myotonic muscular dystrophy. external examination record pattern of scalp hair. record status of skeletal musculature. obtain sections for histologic examination. dystrophin staining of the sarcolemma is absent in duchenne's muscular dystrophy and patchy in becker's dystrophy. frontal baldness (in myotonic muscular dystrophy). atrophy and wasting of muscles (generalized or local: predominantly distal in myotonic muscular dystrophy). pseudohypertrophy of calf muscles in duchenne's muscular dystrophy. dystrophic changes include variations in fiber size, fiber degeneration and regeneration, peri-and endomysial fibrosis, and fatty replacement of muscle. the liver, especially the right lobe, is the most common site of involvement. secondary infection or calcification may be present. the lung is the second most common site of involvement. fluid and air may be visible on the roentgenogram. cysts may be present in the abdominal cavity, muscles, kidneys, spleen, bones, heart, and brain. eosinophilia. edema, angioneurotic synonym: angioedema. note: possible causes and suggested autopsy procedures are described under "death, anaphylactic." related term: silo-filler's disease. n<yfe: this condition is causedby inhalationoftoxic gases, such as oxides of nitrogen (silo-filler's disease) and phosgene (coci 2 ). see also "bronchitis, acute chemical" and "poisoning, gas." (1) gunshot and shotgun wounds of the head involving dural sinuses; (2) injury to large veins, particularly cranial sinuses (during neurosurgical procedures) and veins ofthe neck (knife wounds, surgery) or uterus (criminal abortion); (3) insufflation offallopian tubes (particularly in pregnancy or during menstrual period); (4) infusion of blood components or crystalloid; (5) malfunctioning of dialysis machines; (6) subclavian vein catheterization in the semi-fowler position; and (7) fracture in the hub of a central venous catheter used for parenteral nutrition. possible causes of arterial air embolism include: (1) open heart surgery involving the aorta, left atrium, or left ventricle; (2) positive-pressure ventilation in newborn infants; and (3) underwater ascent with closed glottis (see accident, diving) ifair embolism is suspected, the autopsy should be performed as soon after death as possible. decomposition gases may be produced within a few hours. roentgenography of the whole body may detect large quantities of air, and the roentgenograms may serve as a guide to the most advantageous way of dissection. the postmortem diagnosis of arterial air embolism is made largely on the basis of medical history and circumstances. the autopsy serves to rule out competing conditions. the diagnosis of venous air embolism is made by chest roentgenography before the autopsy (1). the air in the right chambers of the heart can be confirmed at autopsy by aspiration into a syringe. the formerly recommended procedure of clamping the internal mammary vessels below the sternoclavicular joints, and then cutting across the sternum distal to these clamped vessels so that the sternoclavicular joint area remains intact, is designed to prevent the production of a vacuum that pulls air into the veins. the procedure is not necessary if the air is welldocumented by roentgenography before autopsy. at autopsy, a large fatal pulmonary air embolism is readily apparent. the right atrium and ventricle are distended with fine, frothy, brightred blood, which also may distend the pulmonary arteries and superior vena cava. microbiologic examination ofblood and pericardial sac contents may help to rule out the presence of gas-forming bacteria that may simulate air embolism (fig. ii-i). however, the presence of putrefactive emphysema in any part of the body points toward a bacterial origin of the gas. differentiation of air and decomposition gases at the autopsy table with the pyrogallol test is described below. a 2% pyrogallol solution is prepared (it should be waterclear). two lo-ml syringes (syringe a and syringe b) are loaded with 4 ml of the pyrogallol solution in each, without permitting any air to enter the system. immediately before the solution is used, 4 drops of 0.5 n naoh is aspirated through the needle of syringe a to adjust the ph to about 8 (1 drop per 1ml of solution); the mixture will turn faint yellow. six ml of gas is then aspirated from the heart or blood vessels. the needle is immediately sealed with a cork or replaced by a cap, and the syringe is vigorously shaken for about 1 min. in the presence of air, the pyrogallol solution will turn brown. if the solution remains clear, decomposition gases were present. in the latter instance, 4 drops of 0.5 n naoh and 6 ml of room air should be aspirated into syringe b, which is then also sealed and shaken for 1 min. the mixture should turn brown, thus serving as a fig. 11â·1 . gas-forming bacteria simulating air embolism. the pericardial sac is opened and filled with water. the heart is kept submerged with a pair of scissors. the coronary arteries have been incised with a scalpel. note gas bubbles and foam on the water surface. no discoloration of 2% pyrogallol was noted. blood cultures were positive for enterococcus organisms. microscopically, gram-negative rods were found in most tissues. control that the pyrogallol solution had been properly prepared. syringe b may also serve as a reserve. if only one syringe is used, the decomposition gas can be expel1ed and room air can be aspirated for the control test. if only small amounts of gas can be aspirated, the volume of the pyrogallol solution should be decreased so that the gas-fluid volume ratio is at least 3:2. if no bacterial gas formation is present, the edges of the pericardial incision are elevated and the pericardial sac is fil1ed with water. clamping of the ascending aorta and venae cavae prevents the escape of gas into these vessels. the heart is held under water while the coronary arteries are incised, and the escape of bubbles is recorded. when the right coronary artery is opened, care must be taken that the right atrium is not incised. air in the coronary arteries indicates systemic embolism. the heart chambers are then incised. when there is gas in any of the arteries or heart chambers, gas bubbles rise to the surface of the water in the pericardial sac ("bubble test"). sometimes the vessels have to be somewhat compressed in order to cause the gas to escape. because large amounts of air or other gases cause the heart to float, it must be kept submerged before the vessels and chambers are incised. basically the same procedure is used for demonstrating the presence of gas in the superior or inferior vena cava and the pelvic veins (for example, in cases of criminal abortion). in this situation, the abdominal cavity is filled with water and the inferior vena cava and its tributaries are incised. in support of the diagnosis of systemic arterial air embolism, the skull vault may be removed without puncturing the meninges, so that the cerebral arteries can be inspected for gas bubbles. the demonstration of gas bubbles in the meningeal vessels and in the circle of willis is meaningful only when the neck vessels are still intact and the internal carotid artery and basilar artery have been clamped before the brain is removed. in acute cases, gas bubbles will be visible within the cerebral vessels. they are released under water when the clamps are removed and the vessels are slightly compressed. for the collection of gas from blood vessels or cavities, a system oflittle quantitative reliability is an air-tight, water-filled glass syringe with a needle. the needle is inserted into the vessel or cavity in question and gas is carefully aspirated. a combined qualitative and quantitative method has been described by kulka (2,3) (see fig. 11 -2). he devised an apparatus for gas collection and described it as shown in fig. 11 -2 and caption. the entire system is filled with mineral oil so that, when the funnel is level with the upright bottle, the oil fills only about half of the funnel. in operation, the funnel is first raised to a position 30-40cm above the level of the upright bottle. all the cocks are opened and the position is held until every trace of gas has been driven from the system through the needle, which is thereby coated on the inside by a film of oil. after all air has been expelled, the cocks are closed and the funnel is lowered to its original position. as a precautionary measure and control, the air-tightness of the whole system should be tested before operation. this is done by inserting the needle into musculature or skin and attempting aspiration in the manner described in the next paragraph. to make the test, the bottle is inverted and the needle is inserted into the cavity in question. when the needle is in position, all cocks are opened. the funnel is lowered about 70-90 cm, or until adequate suction is created. this aspirates the contents of the cavity, which may consist of air or other gases, either pure or mixed with blood or other liquid. any gas or liquid entering this system may be observed through the wall of the short bent glass tubing. in a positive test, gas bubbles will collect in the bottle above the level of the oil. if desired, this gas can be saved for further examination by closing all the cocks and returning the bottle to its upright position (4). the volume ofintravenous air needed to cause death in adults depends of the cross sectional area of the cardiac cavity or great vessel that is the site of the gas lock, which in tum depends on the position of the decedent at the time of the embolism (5). 100 ml of gas can pass through an intravenous hub in just a few seconds. small amounts of air entering the systemic circulation may cause death within minutes. delayed air embolism with fatal outcome may also occur. other organs if purpura is present, prepare photographs and record extent. submit sample (from right atrium) for microbiologic study. collect blood from right atrium and right ventricle. after the heart has been removed, allow blood in pulmonary vessels to pool in the pericardial sac. centrifuge this blood and submit sample of flocculent layer above buffy coat for microscopic study (1) . submit a section of lung for bacteriologic study. dissect pulmonary arteries; prepare histologic sections of all lobes; request mucicarmine stain and the aldan blue and phloxinetartrazine stain of lendrum. also request sudan stain on frozen sections. skin purpura. vernix, lanugo hairs, and meconium can be found in pericardial blood pool. meconium-type material in blood vessels. in histologic sections, squamous epithelium, meconium, and fat from vernix caseosa. complete or incomplete lower uterine tear; chorioamnionitis. manifestations ofdisseminated intravascular coagulation* and fibrinolysis. intrauterine pneumonia. large amounts of debris in the blood vessels of all sections of the lungs may be considered to be lethal if there is no other cause of death. small amounts in one or more blocks of pulmonary tissue are more likely incidental (1). a small uterine tear is more likely followed by a fatal amniotic fluid embolization than is a large tear, which may result in fatal hemorrhage or fibrinogen depletion. chorioamnionitis, intrauterine pneumonia, and positive lung cultures of the mother indicate infection of the amniotic fluid. record weight and sample for histologic study. for removal and specimen preparation, see chapter 4. photograph horizontal sections through brain, brain stem, and spinal cord. prepare frozen sections and request sudan stain. prepare frozen sections of one-half of gland and paraffin sections of the other half. petechial hemorrhages of skin (chest, neck, and face). wounds; other traumatic lesions. bone fractures. petechiae of conjunctivas and retinas. fat may accumulate on surface ofblood pool. no useful technique is available for estimating the amount of fat globules in the blood. fat emboli in lumen of small vessels and in pulmonary air spaces. severe fatty changes may be the cause offat embolism. fractures are the most common cause of fat embolism. petechial hemorrhages, fat emboli (2). abdominal cavity submit sample of subphrenic exudate for gram stain and cultures. record location and volume of subphrenic exudate. possible causes of subphrenic empyema include appendicitis, cholecystitis, * diverticulitis, intrahepatic abscess, pancreatitis,* ruptured viscus; penetrating abdominal wound(s), perforated ulcer of stomach or duodenum,* and other conditions. pleural cavities and lungs e procedures record volume of effusion or exudate in pleural space. basal pleuritis and pneumonia, adjacent to empyema. encephalitis, au types or type unspecified synonyms and related terms: acute disseminated encephalomyelitis;* acute hemorrhagic encephalitis; acute infective encephalitis or encephalomyelitis; acute poliovirus encephalitis or encephalomyelitis; amoebic encephalitis; arbovirus encephalitis (japanese encephalitis; eastern encephalitis, western encephalitis, venezuelan equine encephalitis, st. louis encep-halitis); bulbar encephalitis;* brain stem encephalitis;* herpes encephalitis (cytomegalovirus encephalits, epstein-barr virus encephalitis, varicella-zoster encephalitis); herpes simplex ence-phalitis; hiv encephalitis; measles encephalitis; measles inclusionbody encephalitis; postinfectiousencephalitis; postvac-cinal encephalitis; progressive multifocal leukoencephalitis or leukoencephalopathy; rabies* encephalitis; subacute encephalitis; subacute sclerosing panencephalitis; viral encephalitis, west nile encephalitis and other terms (1), too numerous to mention. see also under "note" and under "possible or expected findings." note: if the condition that caused the encephalitis is known, see also under that heading. if the cause of the encephalitis is unknown, submit samples of tissue for microbiologic and toxicologic study, particularly if there is a suspicion of lead poisoning.* see also under "encephalitis, brain stem," "encephalomyelitis,...," "encephalopathy" and "myelopathy, myelitis." encephalitis, brain stem synonyms and related terms: brain stem abscess; infectious brain stem encephalitis; listeria monocytogenes brain stem encephalitis; viral brain stem encephalitis. note: see also under "encephalitis, limbic." brain for removal and specimen preparation, see chapter 4. necrotizing encephalitis, with or without abscess formation (1 enterocolitis, other types or type undetermined note: a multitude of infectious and noninfectious agents may cause inflammation of the small bowel, large bowel, or both. if the condition is not listed under "colitis," "enteritis," or "enterocolitis" or under another specific heading such as "dysentery, bacillary," obtain sufficient material for microbio-logic and histologic study to identify organisms such as clos-tridium, chlamydia, shigella, salmonella, yersinia, helicobacter, verotoxic e. coli, and others. if lymphogranuloma venereum,* or tuberculosis* are suspected, see also under these headings. see also under "disease, inflammatory bowel" and "disease, crohn's." synonyms and related terms: c. difficile colitis; darmbrand; hemorrhagic necrosis (gangrene; infarction) of intestine; ischemic enteritis or enterocolitis;* neutropenic enterocolitis;* pseudomembranous colitis. note: the name "pseudomembranous enterocolitis" is descriptive; the condition may be infectious, ischemic, or both. if the intestinal changes are clearly ischemic, see above under "enterocolitis, ischemic." if the cause is in doubt and if pseudomembranes can be identified, follow the procedures described here. (l) collect all tissues that appear to be infected. for other infectious intestinal diseases, see under specific names, such as "enterocolitis, neutropenic" or "enterocolitis, staphylococcal." intestinal tract and mesentery other organs collect material from pseudomembranes for culture and for c. difficile toxin assay. sample intestinal wall with pseudomembranes for histologic study. if the condition is suspected to be caused by ischemia, follow procedures described above under "enterocolitis, ischemic." note: myoclonic seizures also have been described in a number of progressive encephalopathies with complex neurological symptoms, such as gmi and gm2 gangliosidosis,* and niemann-pick* and krabbe's disease but also acquired disorders, including alzheimer's disease,* creutzfeldt-jakob epilepsy, myoclonus (continued) disease,* posthypoxic encephalopathy, and subacute sclerosing panencephalitis. mitochondrial encephalomyopathy also can present with myoclonus epilepsy and a mitochondrial myopathy with ragged red fibers (merrf syndrome) in skeletal muscles. lafora-body-type material in the heart, liver, retinas, peripheral nerves, skeletal muscles, and sweat gland ducts (especially axillary). ragged red fibers in mitochondrial myopathies. epilepsy, symptomatic note: possible causes or underlying conditions include cerebrovascular diseases, congenital malformations ofthe brain, degenerative and demyelinating diseases of the brain, head injury,* intracranial and cerebral infections, toxic or metabolic disorders (alcoholism,* barbiturate,* carbon monoxide,* and lead poisoning,* hemodilution, hypocalcemia, or hypoglycemia),* and tumors of the brain. * f failure, congestive heart note: coronary atherosclerosis and manifestations of ischemic heart disease, * valvular heart disease, congenital cardio-vascular diseases, and manifestations of systemic or pulmonary hypertension are the most frequent findings in patients dying of or with congestive cardiac failure. other causes include cardiomyopathies* and secondary myocardial disease (such as amyloid or pericardial constriction). if the cause of the congestive cardiac failure is unknown or not immediately evident after dissection of the heart and of the great vessels, myocardium and other appropriate tissues may be submitted for microbiologic study-including viral cultures-and for electron microscopy. specimens can also be snap-frozen for possible immunofluorescent, biochemical, or histochemical studies, particularly of the myocardium. possible causes of congestive cardiac failure are too numerous to mention. dilatation of heart, with or without mural thrombosis. myocardium may be soft, normal, or firm. pulmonary congestion, with or without hemosiderosis; congestion of viscera with organomegaly. other organ manifestations include bowel edema or hemorrhagic enteropathy (without mechanical vascular occlusion) and zonal hepatic steatosis, fibrosis, or necrosis, with or withoutevidence of liver failure. acute renal tubular necrosis may be present also. synonyms and related terms: acute kidney failure; chronic kidney failure; renal failure; uremia. note: if acute kidney failure had been diagnosed, the autopsy procedures will depend on the expected causes, such as poisoning with ethylene glycol,* lead,* mercury,* or methyl alcohol;* disseminated intravascular coagulation* and its various underlying conditions; glomerulonephritis* and its various underlying conditions; diabetes mellitus;* or multiple myeloma. * the procedures described below deal primarily with chronic renal failure. if the patient had had dialysis, see also under "dialysis (for chronic renal failure )." if transplantation had been carried out, see also under "transplantation, kidney." lassa fever is a highly communicable disease and autopsy studies are not recommended in the usually available surround-ings. if lassa fever is suspected, contact the state health department and the centers for disease control and prevention, atlanta, ga, for disposition and further studies (1).ifan autopsy is done, disinfection can be accomplished by washing instruments with 0.5% phenol in detergent (i.e., lysol), 0.5% hypochlorite solution, formalin, or paracetic acid. for shipping procedures, see chapter 15. this is not a reportable disease. synonyms: borreliosis; louseborne (epidemic) relapsing fever; tickborne (endemic) relapsing fever. note: (i) collect all tissues that appear to be infected. (2) rat/mouse inoculation with infected blood is the most sensitive method for the detection of the organism. consult the state health department. (3) before the autopsy, consultation with the microbiology laboratory is advised. (4) request direct dark-field examination and giemsa or wright stain. (4) no special precautions are indicated. (5) serologic studies are of questionable value due to the antigenic variability of the organism. (6) fever, scarlet note: usually, this disease is a nonfatal group a streptococcal tonsillitis and pharyngitis with skin rash. potentially fatal complications include suppurative otitis media,* mastoiditis, and pharyngeal abscess (1), with or without septicemia. (1) collect all tissues that appear to be infected. (2) atresia, but may also be seen with viral myocarditis, type ii glycogen storage disease (pompe disease) and carnitine deficiency. thus, a metabolic disorder must be considered. it is ideal to perform the autopsy as soon after death as possible (1) . urine plasma for removal, prosthetic repair, and specimen preparation, see chapter 2. malnutrition (1) muscle necrosis (clostridial myonecrosis) and accumulation of gas; little leukocytic infiltration. other organs g procedures depend on expected findings or grossly identified abnonnalities as listed in right-hand column. see also above under "note" and under "skeletal muscles." synonyms and related terms: acute postinfectious glomerulonephritis (nonstreptococcal postinfectious glomerulonephritis;*minimalchangedisease;mesangialproliferativeglomerulonephritis;* focal and segmental glomerulosclerosis with hyalinosis (focal sclerosis); poststreptococcal glomerulonephritis; idiopathic nephrotic syndrome; iga nephropathy (berger's disease); membranous glomerulonephritis; membranoproliferative glomerulonephritis; mesangial proliferative glomerulonephritis; rapidly progressive glomerulonephritis (associated with systemic infectious or immunologic multisystem diseases; drug idiosyncrasy; or as primary crescentic glomerulonephritis or superimposed on another primary glomerular disease). possible associated conditions: acquired immunodeficiency syndrome (aids);* alport's syndrome;* amyloidosis; anaphylactoid purpura; bee stings; chronic allograft rejection; dennatomyositis;* dennatitis herpetiformis; diabetes mellitus;* drug dependence;*fabry's disease;* goodpasture's syndrome;* guillain-barre syndrome;* henoch-schonlein purpura;* hemolytic uremic syndrome;* infective endocarditis;* leprosy;* malig-nancies;mixedconnectivetissuedisease;myxedema;polyarteritis nodosa;* rheumatoid arthritis;* preeclamptic toxemia; renovascular hypertension; sarcoidosis;* serum sickness;* sjogren's syndrome;*syphilis;*systemiclupuserythematosus;*systemic sclerosis;* thrombotic thrombocytopenic purpura;* thyroiditis;* vasculitis; viral hepatitis;* wegener's granulomatosis;* and many other conditions. urate deposits in scleras and corneas. granulocytopenia note: midline granulomas may belong to the angiocentric immunoproliferative lesions, which are related to lymphomatoid granulomatosis* and malignant lymphoma. the name "idiopathic midline granuloma" should be reserved for the few cases without evidence of malignant lymphoma or wegener's granulomatosis* (1). the diagnosis of idiopathic midline granuloma also can be ruled out if studies reveal fungal organisms or features of leishmaniasis;* leprosy,* rhinoscleroma, pseudotumor of the orbit or tuberculosis. * complications of nasal cocaine abuse also may mimic midline granuloma (2). eosinophilic pneumonitis (degenerating eosinophils with charcot-leyden crystals) and granulomas. angiitis (mostly arteritis), typically with giant cells in tunica media (1) . findings resemble those in polyarteritis nodosa. * heart (2), grastrointestinal tract (3), skin, muscles, and joints are commonly involved. however, renal disease is often (but not always) mild or absent. necrotizing vasculitis of small arteries and veins is present, with extravascular granulomas and eosinophilic infiltration of vessels and perivascular tissues. optic neuritis may be found (4). may be affected by the vasculitis (4). pcr studies on paraffin sections via rna in situ hybridization may confirm presence of epstein-barr virus-positive b-cell proliferations combined with dense t-cell accumulations (3) (4) (5) . the condition closely resembles angiocentric t-/nk cell lymphoma (3). infiltration of lymphocytoid cells, plasma cells, and macrophages with necroses and granulomatous features, which are found primarily in the vicinity of blood vessels. special stains may reveal evidence of infection. lymphoreticular and granulomatous infiltrates (see "lung"). lymphoreticular and granulomatous infiltrates (see "lung"). characteristic infiltrates may be present in all organs and tissues. involvement of spleen, lymph nodes, and bone marrow is uncommon. in rare instances, the disease is confined to the abdomen. in most instances, characteristic infiltrates are present (6). 4 septicemia; circulating immunoglobulin complexes, and antiendothelial antibodies (7). angiocentric granulomatosis (4) hemorrhage, intracranial (see under "hematoma, subdural" and under name of suspected underlying condition, such as "aneurysm, cerebral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemorrhage, subarachnoid (see under name of suspected underlying condition, such as "aneurysm, cere-bral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemosiderosis, idiopathic pulmonary note: this diagnosis is made by exclusion; involvement of organs other than the lungs (see below under "kidneys") suggests another disease. hepatitis, chronic synonyms and related terms: autoimmune hepatitis; chronic viral hepatitis b (with or without d) and c. note: the term "chronic hepatitis" is not a complete etiologic diagnosis and related names such as chronic active (aggressive) hepatitis, chronic active liver disease, and chronic per-sistent hepatitis are obsolete. most cases in these categories represent autoimmune hepatitis or chronic viral hepatitis b or c. many other liver diseases, including drug-induced hepatitis, inborn errors ofmetabolism (e.g., alphal-antitrypsin deficiency* or wilson's disease*), developmental disorders, and chronic biliary diseases such as primary biliary cirrhosis or primary sclerosing cholangitis also may present as chronic hepatitis. ifchronic hepatitis was the cause ofdeath, submassive hepatic necrosis or cirrhosis* is usually present, often with manifestations ofportal hypertension,*hepatic encephalopathy, hepatorenal syndrome,* and with ascites or spontaneous bacterial peritonitis. possible associated conditions: see also below under "possible or expected findings." conditions that may be associated with hepatitis c include (1): autoimmune hepatitis; behcet's disease; diabetes mellitus (type 2);* glomerulonephritis;* guillain-barre syndrome;* idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; iga deficiency; lichen planus; mixed essential cryoglobulinemia; mooren's corneal ulcers; polyarthritis; porphyria cutanea tarda;* thyroiditis. * note: coinfection with other hepatitis viruses (e.g., c and g) and/or systemic viruses such as the immunodeficiency virus are common, particularly in drug addicts (1, 2) . in many cases of fulminant hepatitis, tests for known hepatitis viruses are negative (3, 4) . homicide note: not all of the following procedures can be carried out or will be required in all cases, nor will this checklist be sufficient in all instances. consult also the entries indicating the cause of death, such as "injury, firearm" or "injury, stabbing." if the victim is an infant, see all under "infanticide." before the body arrives or before autopsy is begun: 1. investigate the scene where the body was found and where the crime may have been committed (these may be two separate locations). if this is not possible, study the report and photographs submitted by the investigator or the police. study all available information. request previous medical records and roentgenograms of the victim. 2. emphasize to first responders and autopsy personnel that the body of the victim should not be undressed, washed, or otherwise disturbed until it has been inspected by the pathologist. embalming is not permitted before completion of the autopsy. advise first responders or transporters to put paper bags over the hands of the victim. this will protect possible evidence, such as hair from the assailant. 3. prepare record sheets to document the time of arrival and release of the body; chain of custody for the body and specimens; the name, age, sex, and other information about the victim; the names ofthe technician, pathologist and guests; and the specimens taken. 4. prepare roentgenographic and photographic equipment. after body arrives but before autopsy is begun: 1. if the body is left unguarded-for instance, overnight-a lock should be placed on the refrigerator or cool room where the deceased is kept, and the key should be retained by the technician. 2. if the pathologist is not accustomed to a busy autopsy room, he or she should feel free to ask all persons other than the pathologist(s), technicians, and law enforcement personnel who are not immediately involved in the actual performance of the autopsy to leave the morgue. 3. the body temperature of the victim can be recorded at the autopsy facility, but this is rarely useful, particularly if the victim seems to have been deadfor longer than a day ortwo or ifthe body had previously been stored in arefrigeratoror cool room. insert thermometer deep into the anus (about 7-8cm). record temperature and manner and time of procedure. 4. aspirate vitreous from one or both eyes and store the specimen in a refrigerator. 5. prepare roentgenograms. in each case, a decision must be made whether roentgenograms should be made of the entire body or only of parts of it-or not at all-and whether they should be made before the victim is undressed, after the victim is undressed, or at both times. 1. take overall survey photographs that depict the anterior surfaces of the body as it is on arrival to the facility, before any undressing, manipulation for roentgenographs, or cleaning. 2. take close-up photographs of any trace evidence such as fibers or flakes of gunpowder before undressing or cleaning. 3. photograph the face of the victim for identification purposes after it has been cleaned of any blood and foreign matter. to avoid perspective distortion from wide angle lenses, the lens should be about 4 ft from the face. 4. photograph all injuries and other forensically significant findings after blood and foreign matter have been cleaned off the body. a moist towel is useful; brushes are too abrasive. two photographs should be taken of each finding. one should include the autopsy number and a scale. a second photograph should be taken without any extraneous objects. collection and documentation of evidence: 1. fingerprinting can be done before or after the autopsy but should be done in all homicide cases. in cases involving close contact between assailant and victim, fingernail scrapings or clippings (use a new clipper) and hair with roots should be collected, and its source should be identified (hair pulled from scalp, axillae, and pubis is identified as that of the victim; hair in hands or under fingernails or on clothing of victim may be from the assailant). hair exemplars can be collected in cases of homicide by firearm but are rarely of use. if the body is decomposed or mutilated or for any other reason has not been identified, prepare roentgenograms ofthe head, neck, and torso before the autopsy (the radiographic appearance is altered by the autopsy). these can be used for comparison purposes if antemortem films are located. if films of the extremities are needed they can be prepared after the autopsy. dental roentgeno-.grams are taken, usually after the autopsy, when investigators have located antemortem dental records. detailed descriptions and sizes of clothing, with photographs of clothing, can be useful then there is no putative identity. 3. in cases in which sexual assault may have been involved, collect pieces of clothing that may contain seminal fluid stains. follow procedures described under "rape." 4. preserve clothing or part of clothing as evidence. wet clothing should be dried before it is stored in labeled and sealed bags. the autopsy: 1. the measured length and weight, extent of rigor, and color and distribution of livor should be recorded, along with the state of preservation, nutrition, and hydration. do not omit examination of the hands, particularly of the volar surfaces. 2. if air embolism is suspected, see procedure described under part ii "embolism, air". if pneumothorax is suspected, see procedure described under part ii "pneumothorax". if there is evidence of strangulation or other neck injury, perform a layerwise dissection of the neck that includes the hyoid bone and tongue. 3. prepare diagrams of wounds and identify their location by anatomic region. for wounds of the torso, state the distance from the soles of a foot and the distance laterally from to the right or left of the midline. 4. submit samples of wounds for histologic study when there is any question of the age of the wounds. 5. the records should show when body fluids and tissues were collected for laboratory analysis, the volume and appearance of such specimens, and what was done with them. if most of the toxicology specimens are collected immediately after the internal examination has commenced, the time of the internal examination serves as the collection time. in all instances, the following items should be collected: blood ofvictim for dna comparison and toxicologic study (determination of alcohol, carbon monoxide, and drug concentrations will be requested most frequently); vitreous; urine; gastric contents; at least one solid organ specimen for toxicologic study (liver and brain have the most comparison data if an extracerebral congenital malformation is suspected, follow procedures described under "malformation, arnold-chiari." if cerebrospinal fluid is aspirated with a syringe, record volume. record weight of brain; record size of brain in relation to inner dimensions of skull. describe size of ventricles. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. if a surgical shunt is present, its location should be recorded and both ends of the implanted specimen that was used for shunting may be submitted for microbiologic study. if the shunt is not patent, record site and nature of obstruction. related terms: antibody-mediated hydrops fetalis; erythroblastosis fetalis;* nonimmune hydrops fetalis. note: the classic example of antibody mediated (rh incompatibility) hydrops fetalis is hemolytic disease of the newborn (erythroblastosis fetalis*). however, nonimmune hydrops fetalis also may be caused by hematologic disorders, e.g., alpha-thalassemia* (1) or it may have no known cause. infec-tions, e.g., with human parvovirus bl9 (2), cytomegalovirus, or syphilis;* heart and vascular diseases (3) (cardiac tumors, cardi-omyopathy,* myocarditis,* arterial calcification, and others); storage disease (4); tumors (including neonatal leukemia*); and many other fetal (e.g., congenital chylothorax* or lymphatic dysplasia; pulmonary sequestration and cystic adenomatoid malformation) or maternal conditions, e.g., maternal thyrotoxicosis, also may cause nonimmune hydrops fetalis. if coronary insufficiency or myocardial infarction is suspected, follow procedures described under "disease, ischemic heart." for coronary arteriography, see chapter to. record distribution of atherosclerotic lesions in aorta. samples for histologic study should include aorta, coronary arteries, and peripheral arteries. see also above under "heart." submit samples of head, corpus, and tail for histologic study. if applicable, see also under "diabetes mellitus." for special procedures and stains, see above under "heart." other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 4. obesity* is a common finding. eruptive xanthomas (palms, elbows, knees) in some but not all types of hyperlipoproteinemia. most prominent in apoprotein cii deficiency. xanthomas of tendons (knees, elbows, dorsum of hands), xanthelasmas, and arcus comeae in familial hypercholesterolemia. gangrene of lower extremities (see below under "arteries"). severe coronary atherosclerosis and myocardial infarcts in type 3 hyperlipoproteinemia and multiple lipoprotein-type hyperlipidemia. atherosclerosis of abdominal aorta and its branches and of carotid arteries. coronary atherosclerosis (see above). pancreatitis* in familial apoprotein cii deficiency. foam cells with triglycerides in liver, spleen, and bone marrow in familial lipoprotein lipase deficiency. manifestations of diabetes mellitus* and hypothyroidism* in some cases of type 3 hyperlipoproteinemia. cerebral infarct (stroke*) in type 3 hyperlipoproteinemia. hypernatremia (see "disorder, electrolyte(s).") hyperoxaluria synonyms and related terms: oxalosis; primary hyperoxaluria type i (a1anineglyoxylate aminotransferase deficiency); primary hyperoxaluria type ii (d-glyceric acid dehydrogenase deficiency); secondary hyperoxaluria (see under "note"). note: in ethylene glycol poisoning,*oxalatecrystals in media ofsmall arteries, with associated ischemic lesions. similardeposits may occur after long-term hemodialysis (1). these conditions must be distinguished from the genetic disease. also, oxa-iate nephropathy may be a complication of short bowel syndrome. if patient with congenital hyperoxaluria underwent liver or combined liverlkidney transplantation (2) , see also under these headings. remove vitreous for biochemical evalvation. submit tissue (i.e., fascia lata) for karyotype analysis. polycystic ovaries, testicular adrenal rests (4 note: there are no diagnostic autopsy findings for hypoxia. possible causes of acute hypoxia include anesthesia-associated death,' compression of the torso by a vehicle, diving accident,' exposure to toxic gases-forinstance, carbon monoxide poisoning;' mechanical failure ofan oxygen supply system, as in an airplane; and sudden mechanical airway obstruction,' as in aspiration ofa foreign body orstrangulation. causes ofchronic asphyxia include prolonged exposure to high altitude and chronic pulmonary disease. profound medial hypertrophy of small pulmonary arteries and of pulmonary veins from chronic exposure to hypoxia. acute pulmonary edema may also occur in chronic hypoxia. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. malnutrition. empty and collapsed colon; small amounts of gray and dry meconium in terminal ileum; meconium masses in dilated and hypertrophied mid-ileum; liquid contents in proximal intestine (1); appendicitis; bowel perforation. glandular inspissation and atrophy of intestinal mucosa. volvulus, infarction, necrosis, perforation, peritonitis, and acquired intestinal atresia may be present. absence of neural plexuses in congential megacolon* (hirschsprung's disease). manifestations of cystic fibrosis, with or without cirrhosis;* biliary atresia* (3). immunodeficiency (see "syndrome, acquired immunodeficiency" and "syndrome, primary immunodeficiency.") impression, basilar note: basilar impression constitutes an upward bulging of the margins of the foramen magnum. when occipital condyles are displaced above the plane of the foramen magnum, basilar invagination is present. possible associated conditions: klippel-feil syndrome;* osteogenesis imperfecta;* osteomalacia;* paget's disease of bone;* rheumatoid arthritis; rickets; syringobulbia; syringomylia. * compression and secondary ischemic injury to lower brain stem and spinal cord. see also above under "skull and spine" and under "possible associated conditions." incompetence,â�¢â�¢â�¢ (see "insufficiency,..â�¢") related terms: battered-child syndrome; child-abuse or child-neglect death. note: vitreous should not be aspirated until the skull has been opened and trauma ruled out, because there is a risk of artifactual damage to the retina. in the presence of craniocerebral trauma the pathologist can opt to examine the eyes by removal, fixation, and histologic study; or retinal photography. follow general procedures described under "homicide" and, if necessary,. the procedures described under "rape." external examination see above under "note." prepare roentgenograms of entire body. see above under "note." in other situations, or after study of the fundus, submit sample of vitreous for chemical and possible toxicologic study. umbilical cord attachment prepare histologic sections of umbilicus. and end of umbilical cord blood submit sample for toxicologic study. consider retaining dried blood on filter paper for possible dna testing for paternity investigation. if there is a possibility that the cadaver represents a stillbirth, see part ii, "stillbirth". if infant is thought to have died shortly after birth, determine the location of air in the intestinal tract; roentgenograms may be helpful. save stomach contents and record amount. other organs see also under "homicide." in the abused child, bruises, hematomas, burn marks, or other patterned injuries. in any child, diaper rash, mongolian spots, self-inflicted fingernail scratches, skin infections, and emaciation. in the previously battered child, fractures in various states of healing. in hypertonic dehydration,' sodium concentrations more than 150meq/l. this may be caused by organic disease, improper medical treatment, or physical neglect. if infant was born alive, inflammatory changes may be found, depending on survival interval. for the hydrostatic lung test, see "stillbirth." the test is unreliable. extraneous material in air passages indicates that child was alive. air reaches the stomach after 15 min, the small intestine after 1-2 h, the colon after 5-6h, and the rectum after l2h. there is no difference in the speed of gas propulsion between full-term and premature infants. bacterial gas production and previous resuscitation attempts are potential sources of errors. in questionable stillbirth, presence of milk proves that infant was alive and had been nursed. traumatic lesions and signs of neglect may be present. unilateral ulcers in visceral zoster. in rare instances, diffuse meningoencephalitis may occur. limited necrotic and inflammatory lesions in the cord or brain stem at the level of the affected ganglion are common. posterior hom myelitis (3). ganglion cell necrosis; lymphocytic infiltration, hemorrhage, and, later, fibrosis. as a rule, only one ganglion is severely involved, but less severe lesions may occur in the ganglia that are immediately adjacent. diffuse lymphocytic infiltration; demyelination; axonal destruction; fibrosis. conjunctivitis; keratitis; iridocyclitis; retrobulbar neuritis; neuroretinitis; occlusion of retinal vessels. evidence of hematologic malignancies (2) or other conditions, as listed above under "note." infection, middle ear (see "otitis media.") infection, pneumocystis carinii synonym: pneumocystosis. note: (1) collect all tissues that appear to be infected. (2) this organism cannot be cultured at present but is frequently associated with viral, bacterial, or fungal infections that can be diagnosed by culture. (3) for rapid staining of aspirates, use gram-weigert stain, which will stain cysts of pneumocystis carinii and also fungi and bacteria. pneumocystis carinii is best demonstrated with grocott's methenamine silver stain. (4) no special precautions are indicated. (5) usually, no serologic studies are available. (6) this is not a reportable disease. injury, fire (see "burns.") injury, firearm note: protect all drains of autopsy table, which will prevent accidental loss of bullets or bullet fragments. recovery of bullet fragments and pellets can also be improved by passing tissue fragments, blood, and other appropriate materials through a fine nonmetallic sieve. caution must be exercised because sharp edges andjagged projections ofbullets and bullet fragments may cause injury (1). this applies particularly to the black talon bullet. bullets and bullet fragments should not be touched with forceps or other metal instruments that may produce artifactual markings; they must be placed in properly labeled evidence containers, and the chain of custody must be preserved. from a birdshot wound, at least 10 pellets should be recovered. the firearms examiner will divide the total pellet weight by 10 and derive median pellet weight. from a buckshot wound, all pellets should be recovered. in many of these cases, procedures described under "homicide" must also be followed. do not excise wounds before completion of autopsy (see below). as an academic exercise, excised firearm wounds may be used later for analysis of metal traces by neutron activation or for tests for carboxyhemoglobin. in practice, however, a high quality photograph is adequate to establish the presence or absence of gunpowder stippling or soot deposition toward the end of establishing the range of fire. the dimensions of the stippled areas and soot deposits should be recorded. after completion of external examination, dry the wet clothing and keep as evidence. clothing may also be used for possible test firing or for stain, powder, or particle analysis. external examination if identity of victim is unknown, follow procedures described in chapter 13. prepare initial roentgenograms of the body regions that have been shot, before disrobing of body, and then lateral films of body regions with bullets. follow up with films of other body regions as needed in the course of autopsy. if the body is decomposed to the extent that the external examination cannot be relied upon to determine cutaneous gunshot perforations, prepare roentgenograms of the entire body before autopsy. record location and number of bullet holes in all layers of garments, indicating whether the involved area is bloodstained or shows gunpowder or soot. if there are several cutaneous perforations, number or letter each consecutively and refer to these numbers in all records. location of bullet holes should be described by recording distance from soles of feet or top of head, and from midline of body. prepare diagrams and photograph holes, with and without labels. photographs should show surrounding garments and extent of blood stains. in hairy areas, shave around bullet wounds for better photographic documentation. for evaluation of distance from where a shot had been fired, see fig. 11 additional roentgenograms during the autopsy may be needed to find bullets embolized to the femoral veins or down the spinal canal. systemic roentgenograms may reveal bullets from obscure entrance wounds, particularly in decomposed bodies, old bullets, bullets in the skin flaps reflected at autopsy, or bullets external to the body in clothing. bullet(s) may have been arrested in hollow viscus or blood vessel and may have been transported to distant sites by peristalsis or bloodstream ("bullet embolus"). bullets may be deflected in unexpected directions from bony surfaces. soot ("smudging") and gunpowder stippling may occur around entrance wound, depending on the muzzle-to-skin distance. there may also be an impression from a recoiling handgun or from a power piston. wounds may be stellate, round, jagged, or slit-like, with or without wide margins of abrasion. primer residues on hand of suicide victim after use of a handgun. fatal secondary injuries, particularly hemorrhages. alcohol intoxication is a frequent finding. fig. 11â·3 . bullet wounds. differences in the appearance of cutaneous wounds of entrance and of exit and differences in wounds of entrance according to the distance between muzzle and skin at the moment of fire. entrance wounds often differ from exit wounds in that the former are usually surrounded by a narrow zone of abrasion. if the muzzle of a gun is in close contact with the skin at the moment of fire, the combustion products will be blown into the wound and will not be visible on the surface. for close-range wounds, the stippling of the skin around the wound, produced by particles of burned and unburned powder, becomes progressively more dispersed as the range of fire increases and is ordinarily not perceptible if the range has been greater than 24 inches ( synonyms and related terms: acute radiation syndrome; chronic (delayed) radiation injury; radiation enterocolitis; radiation nephritis; radiation pneumonitis. note: procedures and expected findings depend on type of radiation damage, whether acute or chronic, localized or whole-body irradiation. if suspected radiation injury was associated with the administration of radionuclides such as 32p, 131 1, or 198au, follow procedures suggested in chapter 11. in fatal 355 whole-body irradiation, findings are most likely related to bone marrow injury, and the suggested procedures and expected findings are those described under "pancytopenia." record extent of oropharyngeal and intestinal ulcerations and hemorrhages. late complications include malignant tumors (carcinoma, leukemia,* lymphoma*), manifestations of hypothyroidism,* and cataracts. organ changes in localized radiation injury depend on site of irradiation (lung, brain, kidney, intestine). the skin is involved in both acute (erythema) and chronic (atrophy, epilation) radiation injury. related terms: cutting injury; knife injury. note: in many of these cases, procedures described under "homicide" must also be followed. insuffiency, adrenal synonyms and related terms: adrenocortical insufficiency; polyglandular autoimmune syndrome (type i, usually in childhood, with parathyroid insufficiency and chronic mucocutaneous moniliasis; type ii, usually in adults, with two or more autoimmune endocrine disorders such as thyroiditis and diabetes mellitus*); primary adrenal insufficiency (addison's disease); secondary adrenal insufficiency (in hypothalamic-pituitary disease or steroid induced); x-linked adrenal hypoplasia. is caused by anatomic changes (see below under "adrenal glands"), drugs (enzyme inhibitors or cytotoxic drugs), or acth-blocking antibodies. possible associated conditions: aids with systemic infections (1); antiphospholipidantibody syndrome (2); autoimmune hepatitis; chronic lymphocytic thyroiditis; type i diabetes mellitus;* hypoparathyroidism;* hypothyroidism;* megaloblastic anemia;* surgical procedures, such as heart surgery or orthopedic procedures. large cell lymphoma (5). should be removed as soon as possible (before embalming), either from cervical midline incision or from chest (neck of cadaver must be well-extended). photograph obstruction before larynx is opened, and inside of larynx and epiglottis after larynx is is opened in posterior midline. make gram-stained touch preparations. make histologic sections of larynx and epiglottis. hemophilus injluenzae (cannot always be isolated from larynx synonyms and related terms: acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, and hairy cell leukemia. multiple subtypes have been identified; they are characterized by cell surface markers, chromosomal abnormalities, staining reactions, and morphologic features. note: at the time of autopsy, most leukemias have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent of the disease and the presence of complications. if the leukemia had not been classified or if the features might have changed from the time of the last work-up (e.g., in suspected cases of superimposed diffuse large cell lymphoma [richter's syndrome]), material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation,* see also under that heading. possible associated conditions: agnogenic myeloid metaplasia with myelofibrosis; bloom's syndrome;* cutaneous mastocytosis; down's syndrome;* immunodeficiency syndromes* (bruton's disease; louis-bar syndrome; wiskott-aldrich syndrome); infantile genetic agranulocytosis; klinefelter's syndrome;* lymphoma;* multiple myeloma;* polycythemia;* and many others. for sampling and specimen preparation, see chapter 4. increased protein concentration. material in sediment stains red with toluidine blue. metachromatic material. arylsulfatase-a deficiency. excessive loss of myelin with large amounts of metachromatic material (see above under "urine") in white matter and also in some neurons. demyelination with metachromatic material (see above under "urine"). leukoencephalopathy, progressive multifocal possible associated conditions: aids* and other immunosuppressed states; carcinoma; malignant myeloproliferative or lymphoproliferative disorder; sarcoidosis;* tuberculosis. * procedures submit portion of fresh brain for viral culture. fix remainder of brain and spinal cord in formalin and submit samples for histologic study. in situ hybridization for ic virus is available on paraffin-embedded tissue. small patches of demyelination with tendency to form confluent areas in the cerebral white matter. white matter necrosis may be present. eosinophilic intranuclear inclusions occur in affected oligodendroglia cells. subsequently, large, bizarre astrocytes develop. no inflammatory (lymphocyte) cell reaction is present. lightning (see "injury, lightning.") lipoproteinemia (see "hyperlipoproteinemia.") lipoproteinosis, pulmonary alveolar synonym: idiopathic alveolar lipoproteinosis. note: the condition has been described in allografts (1); in such cases, see also under "transplantation, lung." elephantiasis of penis, scrotum, or vulva (in chronic cases); skin rash (l) and conjunctivitis (in acute cases); genital ulcers. suppurative or fibrosing inguinal, iliac, or pelvic lymphadenitis, with or without sinus tracts. rectal strictures and fistulas in chronic cases (2) . systemic involvement in the acute stage with pericarditis,* and arthritis,* and meningitis,* proctitis (3) lymphoma synonyms and related terms: aids-related lymphoma; adult t-cellieukemiallymphoma; angioimmunoblastic lymphadenopathy; b-celllymphoma; burkitt's lymphoma; cutaneous t-cell lymphoma (includes mycosis fungoides and sezary syndrome); hodgkin's disease; non-hodgkin's lymphoma (low grade, intermediate grade, high grade, all with multiple subtypes too numerous to list, which vary in natural history); natural killer cell neoplasms; post-transplant lymphoproliferative disorders (ptld); t-cell lymphoma. note: at the time of autopsy, most lymphomas have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent ofthe disease and the presence of complications. if the lymphoma had not been classified or if the features might have changed from the time of the last work-up, material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation, * see also under that heading. for current terminologies of hodgkin's disease and non-hodgkin lymphomas as well as for staging criteria, appropriate hematologic textbooks should be consulted. possible associated conditions: acquired immunodeficiency diseases (e.g., after organ transplantation; human immunodeficiency virus-l infection); autoimmune disease (e.g., celiac sprue,*rheumatoid arthritis,* systemic lupus erythematosus,* or sjogren's syndrome*); chemotherapy with or without radiation treatment; epstein-barr virus infection; human t-cell leukemia virus infection; inherited diseases with immunodeficiency (e.g., klinefelter syndrome; * common variable immunodeficiency disease, wiskott-aldrich syndrome); radiation. the lesions are found most commonly in the urinary bladder and other parts of the urinary tract, but may also occur at many other sites, including skin (1) and upper respiratory tract (2). note: (i) collect all tissues that appear to be infected. (2) usually, cultures are not indicated. (3) request giernsa or wright stain. tissue blocks should be rinsed of blood and be as thin as possible before fixation in refrigerated buffered and neutral 10% formalin solution. this is to avoid precipitationofformalin pigment that may be confused with malaria pigment. the formalin solution should be used in a ratio of 100 parts formalin to one part tissue and should be agitated every few hours. if one is dealing with tissues that contain formalin pigment, this pigment may be removed with a bleaching solution. (4) usually, no special precautions are indicated. (5) possible associated conditions: histoplasmosis* and other chronic fungal infections; immune connective tissue diseases such as rheumatoid arthritis* (l) ; malignancies (l); sarcoidosis;* silicosis;* tuberculosis* (l) . note record location and extent of skin changes or skin defects on back. prepare skeletal roentgenograms. if meningitis is suspected, submit sample of cerebrospinal fluid for culture. dse the posterior approach to remove the spinal cord. atrophic skin over meningocele, lacking rete pegs and skin appendages; skin defect in complete rachischisis. bony defects of spine. in meningocele and spina bifida occulta, arachnoid and dura herniate through the vertebral defect. spinal cord and roots are generally not involved. lumbosacral mass in meningomyelocele, with a highly vascular mass (area medullovasculosa) in spina bifida aperta. neural defects in complete rachischisis. diastematomyelia, hydrocephalus (1) . pyelonephritis;* enlarged urinary bladder ("neurogenic bladder"). external examination heart lungs procedures prepare chest roentgenogram. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. record weights. prepare photographs and roentgenograms of fresh and fixed lung specimens. perfuse one lung with formalin. for preparation of paper-mounted sections, see chapter 2. submit one lobe for microbiologic and chemical study. decalcify tissue for histologic study. request van gieson's, hale's colloidal iron, pas, and sudan stains. miliary mottling in lower lung fields. mitral stenosis* may be a cause of microlithiasis (mostly intra-alveolar ossification). * cor pulmonale and heart failure (1) (l) , see also under that heading. (1) collect all tissues that appear to be infected. (2) viral isolation, especially with ebv, is not diagnostically useful, due to long incubation periods. (3) note: these diseases are characterized by a deficiency of a variety of hydrolases, resulting in accumulation of gly-cosaminoglycans (mucopolysaccharides) and glycolipids within lysosomes of fibroblasts, macrophages, white cells, and parenchymal cells of many organs (1, 2) . mucopolysaccharides are also excreted in the urine. the general approach is similar in all types. formalin may dissolve all of the stored material and leave empty vacuoles in the involved cells. therefore, frozen sections should be utilized or tissues should be fixed in absolute alcohol. the accumulated material will show intense metachromasia if stained with toluidine blue. it will also stain with pas, alcian blue, and colloidal iron. oil red a will also stain the material from frozen sections. for specimen preparation for elec-tron microscopy, see chapter is. characteristic external features include dwarfism; thickened long bones; coarse facial features; coarse hair; macrocephaly; prognathism; hypertelorism; malformed teeth, and scaphocephalic skull with hyperostosis of sagittal suture; short neck; chest deformity; umbilical and inguinal hernias; lower thoracic and lumbar gibbus; genu valgum and coxa valga; pes planus and other joint deformities; wide hands (clawhands) and feet. coarse thickened skin, covered with lanugo-like hair. hyperlordosis; ovoid deformities of vertebrae; odontoid hypoplasia; large, shoe-shaped sella turcica; kyphosis; hypoplasia of femoral heads; osteoporosis.* if patient underwent bone marrow transplantation (3), see also under that heading. chronic upper respiratory infections. large cytoplasmic granules in neutrophils. storage of mucopolysaccharides in osteocytes, chondrocytes, and fibroblasts of periosteum, tendons, fasciae, and other connective tissues; dysostosis multiplex. other tissues histologic sampling cannot be excessive. (1) collect all tissues that appear to be infected. (1), see also under that heading. blood; other organs and tissues record extent of skin lesions and prepare photographs; prepare sections of affected and unaffected skin. request gram stain of sections and smears. submit samples of blood and grossly affected organs or tissues for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. extensive epidermal necrosis. shedding of granular and horny layers of epidermis. septicemia; staphylococcal infection of nose, throat, ears, eyes, heart valves, urogenital tract, and other sites. bronchial epithelial detachment and bacterial pneumonia (2). synonyms and related terms: acute kidney failure; *acute tubular necrosis; lower nephron nephrosis. note: the morphologic diagnosis of this condition may be difficult to discern from autolysis. the features that suggest tubular necrosis are: tubular dilation with epithelial flattening, intertubular edema and necrotic epithelial cells in the collecting ducts. the autopsy should be performed as soon as possible. needle specimens of the kidneys obtained in the immediate postmortem period may yield acceptable material. ifnephrotoxic drugs or chemicals are thought to be responsible for tubular necrosis, submit samples for toxicologic study (see also under synonyms and related terms: multiple endocrine neoplasia (men), type 1 (parathyroid hyperplasia or adenoma; pancreatic islet cell hyperplasia, adenoma, or carcinoma; pituitary hyperplasia or adenoma); or type 2a (medullary thyroid carcinoma, parathyroid hyperplasia or adenoma; and pheochromocytoma); or type 2b (medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, and marfanoid features). note: in men, type 1, foregut carcinoids and subcutaneous and visceral lipomas also may be found. in type 2a, cutaneous lichen amyloidosis may be observed. mixed syndromes include (i) familial pheochromocytoma and islet cell tumor, (2) von hippel-lindau syndrome, pheochromocytoma and islet cell tumor, (3) neurofibromatosis* with features of men1 or 2, and myxomas, spotty skin pigmentation, and generalized endocrine overactivity (carney complex). in all instances, the autopsy should be done as soon as possible so that tissues for biochemical study can be frozen without delay. nephritis note: see under specific designation, such as "glomerulonephritis" and "pyelonephritis," or under name of suspected underlying condition, such as "gout" or "lupus erythematosus, systemic." nephroblastoma (see "tumor of the kidney(s).") synonyms and related terms: renal stones; urolithiasis. possible associated conditions: carcinomatosis; cushing's syndrome;* cystinuria;* fanconi syndrome;* hyperoxaluria;* hypervitaminosis d;* gout;* multiple myeloma;* osteoporosis;* polycystic renal disease;* primary hyperparathyroidism;* rheumatoid arthritis* (1); sarcoidosis* (2). kidneys ureters, urinary bladder, and urethra other organs remove kidneys, ureters, and urinary bladder en bloc. excise and bivalve kidneys to reveal renal pelves. open ureters. record size, number, and appearance of stones, and save for chemical analysis (4). record heart weight. dissect and record weights of all parathyroid glands. other procedures depend on suspected underlying conditions, as listed above under "possible associated conditions." nephropathy note: see under name of suspected underlying condition, such as "diabetes mellitus," "disorder, electrolyte(s)" (hypercalcemia, potassium depletion), "gout," "hypertension (arterial), all types or type unspecified," or "poisoning,.. ." (heavy metal). if kidney failure was present, procedures under that heading should also be followed. renal tissue may need decalcification or fixation in water-free solution. (2) note: in this condition, lesions in the brain and cranial nerves, spinal cord, and spinal roots may be schwannomas (including bilateral vestibular schwannomas); multiple meningiomas; gliomas (generally of spinal cord), mostly ependymomas (75%) and pilocytic astrocytomas; or schwannosis of spinal dorsal root entry zones. intracortical meningioangiomatosis; glial hamartia (intracortical, basal ganglia, thalamus, cerebellum, and dorsal horns of spinal cord) and cerebral calcifications also may be found. cutaneous abscess (j). acute necrotizing nocardial pneumonia with abscesses or sinus formation into surrounding tissues; empyema. pulmonary and mediastinallymph nodes other organs submit samples for culture and histologic study. submit samples from all organs and tissues with suspicious gross lesions for culture and histologic study. poorly encapsulated abscesses of any organ; endocarditis (2) . related term: total parenteral nutrition. note: air embolism* or blood loss may have occurred if line became detached from the catheter hub. metabolic complications such as fluid overload or disturbances of acid-base and electrolyte balance often cannot be diagnosed reliably at autopsy. the disease(s) that may have necessitated parenteral nutrition therapy are not considered here; they include the acquired immunodeficiency syndrome (aids);* cancer cachexia; inflammatory bowel disease; liver or kidney failure;* severe pancreatitis;* short bowel syndrome, and others. obstruction, acute airway synonyms and related terms: aspiration; bolus death; "cafe coronary"; croup; restaurant death. note: ifpermission has been obtained, remove neck organs through straight incision from chest to chin to avoid dislodging a foreign body. ifdissection has to be accomplished from chest, neck of unembalmed cadaver should remain well extended during procedure. inspect larynx and trachea from above and below, respectively, before opening them carefully along the posterior midline. for removal, prosthetic repair, and specimen preparation, see chapter 2. submit samples of osteocartilaginous and synovial tissues for histologic study; sagittal or frontal saw section through spine provides for best routine evaluation. heberden's nodes at interphalangeal joints of fingers. degenerative changes, primarily of spine, hip joints, and knee joints. histologic and macroscopic degeneration of cartilage; exposure of subchrondral bone; formation of marginal osteophytes; bone cysts; synovial fibrosis; hypertrophic synovitis. o procedures if artificial joints (e.g., hip or knee) had been implanted, record state of implant site. loose joint prostheses. synonyms: hypertrophic pulmonary osteoarthropathy; pac-hydermoperiostosis (idiopathic hypertrophic osteoarthropathy [1j). note: in all instances, an underlying disease must be identified; they include cardiac disease (cyanotic congenital heart dis-ease with right-to-left shunt; infective endocarditis*); gastroeso-phageal reflux (3); neoplasms of lungs, esophagus, intestine, and liver; chronic liver disease; inflammatory bowel disease;* pulmonary disease (e.g., abscess;* bronchiectasis;* cystic fibrosis;* emp-yema;* emphysema;* lipoid pneumonia* (4); pneumocystis pneumonia; sarcoidosis;* tuberculosis*); hyperthyroidism. * external examination elastic arteries other organs prepare roentgenograms of extremities. for removal, prosthetic repair, and specimen preparation, see chapter 2. record presence of aneurysms (thoracic aorta, subclavian) or arteriovenous fistula of brachial vessels. if abdominal aortic aneurysm had been surgically repaired, search for evidence of graft infection (2) . procedures depend on expected findings or grossly identified abnormalities as listed above under "note." clubbing of fingers or toes (see below under "elastic arteries"); swelling of extremities. periosteal new bone formation in distal shafts of bones of forearms and legs. all bones of extremities may be involved. see above under "external examination." unilateral clubbing. clubbing of toes but not of fingers. swelling; fistulas. bone defect(s) and focal osteosclerosis. bacterial or fungal infections, most common in metaphyseal region of bones; paravertebral or psoas abscess in osteomyelitis of spine. bacterial or fungal osteomyelitis, with or without cavitation and fistulas. trauma, surgery, insertion of prosthesis, generalized (1) or contiguous infection, inflammatory bowel disease (2), diabetes mellitus,* and peripheral vascular diseases, deep vein thrombosis (3). synonyms and related terms: aseptic necrosis of bone; avascular necrosis of bone; idiopathic osteonecrosis; perthes' dis-ease; postfracture osteonecrosis; renal transplant associated osteonecrosis. note: possible underlying conditions include chronic alcoholism,* decompression sickness,* diseases that have been treated with high doses of corticosteroids (1), gaucher's disease,* human immunodeficiency virus infection* (2), tuberculosis* (1), sickle cell disease, * systemic lupus erythematosus,* tuberculosis* (2), and bisphosphonate therapy (3). other organs submit sample for biochemical study. for removal, prosthetic repair, and specimen preparation, see chapter 2. if osteonecrosis is suspected because one of the possible underlying conditions (see above) is present, prepare saw sections through femoral heads, medial femoral condyles, and heads of humeri. procedures depend on suspected underlying conditions, as listed above under "note." osteopetrosis synonymsand related terms: albers-schonberg disease; autosomal-dominantosteopetrosis; carbonic anhydrase-it deficiency; malignant infantile osteopetrosis (1); marble bone disease. note: manifestations of renal tubular acidosis may have been a clinical complication. possible associated conditions: bone marrow transplantation* (for infantile osteopetrosis). anemia, recurrent infections, bleeding, and bruises may have resulted from myelophthisis associated with osteopetrosis. upper airway obstruction in malignant infantile osteopetrosis may have necessitated tracheostomy (1). in heritable osteoporotic disorders of connective tissue (osteogenesis imperfecta,* marfan' s syndrome,* and others) are pre-sented separately. for"osteomalacia," see above. for "osteodystrophy," see under "failure, kidney." possible associated conditions: acromegaly;* chronic alco-holism;* chronic obstructive pulmonary disease; chronic kidney failure* (1); cushing's syndrome;* debilitating disease (various kinds, often with immobilization); diabetes mellitus* (2); epilepsy;* hyperthyroidism (2);* hypogonadism; malabsorption syndrome;* malnutrition;* primary biliary cirrhosis;*rheu-matoid arthritis;* scurvy;* steroid therapy or anticonvulsant medication. normal parathyroid glands in uncomplicated cases. hyperparathyroidism* (1) (without osteitis fibrosa) may be present, however. features of osteitis fibrosa (osteoclastic osteoporosis; see "hyperparathyroidism") or osteomalacia* exclude the diagnosis of uncomplicated osteoporosis. osteoporosis, which may be localized, occurs in various neoplastic (e.g., mastocytosis) and inflammatory diseases. external examination brain and base of skull with middle ears examine external ear canal. for removal and specimen preparation, see chapter 4. culture any lesion appearing to be infectious. draining, foul-smelling greasy material associated with acquired cholesteatoma. bacterial or viral infection, with or without mastoid osteitis. neck abscess, sinus thrombosis (1) and brain abscess* and meningitis* may complicate otitis media. otosclerosis (1,3) note: otosclerosis may be a measles-virus-associated disease (1) and therefore, studies to rule out a past infection may be indicated. for removal and specimen preparation, see chapter 4. for current methods of temporal bone studies, see ref. (2) . spongy bone in the capsule of the labyrinth. trabeculae of woven bone show pagetoid changes. if stapedectomy had been done, a prosthesis may be in place. brain is externally normal or mildly atrophic. characteristic is midbrain atrophy with aqueduct dilatation and depigmentation of the substantia nigra. neuronal loss with reactive gliosis, associated with neurofibrillary tangles (globose tangles). primarily affected are globus pallidus, subthalamic nucleus, red nucleus, substantia nigra, tectum, periaqueductal gray matter, and dentate nucleus (grumose degeneration). palsy, pseudobulbar (see "disease, motor neuron.") related terms: acute pancreatitis; alcoholic pancreatitis; chronic (or chronic fibrosing) pancreatitis; interstitial pancreatitis. note: some causes of pancreatitis such as adverse drug reactions (e.g., due to azathioprine, furosemide, or estrogens) cannot be diagnosed at autopsy. possible associated conditions: acute fatty liver of pregnancy; apolipoprotein cli deficiency; cystic fibrosis; *hyperparathyroidism;* kidney transplantation. * status post endoscopic retrograde cholangiopancreatography. synonyms and related terms: calcifying panniculitis; histiocytic cytophagic panniculitis; mesenteric panniculitis (mes-enteric lipodystrophy); "sclerema neonatorum." note: the term weber-christian disease described systemic panniculitis with fever, bleeding, pulmonary and pancreatic lesions, and other abnormalities also involving lungs and pancreas, among others. however, this condition is not a specific entity and thus, the name has become obsolete. the term histiocytic cytophagic panniculitis is more descriptive and preferred. leukemia* and subcutaneous t-celllymphoma* may closely simulate panniculitis. possible associated conditions: alphaj-antitrypsin deficiency;* dermatomyositis;* rheumatoid arthritis;* scleroderma and morphea; sjogren's syndrome;* systemic lupus erythematosus.* external examination, skin, subcutaneous tissue, and breasts chest heart lungs liver and spleen record extent and character of skin lesions. record size, location, and gross appearance of subcutaneous nodules. submit tissue samples for histologic study of grossly unaffected skin, skin lesions, and subcutaneous nodules. request verhoeff-van gieson, gram, and grocott's methenamine silver stains. request s-l 00 protein stain. ascertain that cell infiltrates are not leukemic or lymphomatous (1) . dimpling of skin; necroses; fistulas. panniculitis with subcutaneous nodules in trunk, breasts, and thighs (5) . calcification may occur in breast tissue but also at other sites (e.g., in kidney failure*). pancreatic enzymeinduced fat necroses associated with severe pancreatitis. widespread hardening of fat tissue with rupture of fat cells and crystal formation in "sclerema neonatorum." focal traumatic panniculitis also may occur in newborns. s-ioo stain negative in panniculitis but positive in rosai-dorfman disease (sinus histiocytosis with massive lymphadenopathy). mediastinal panniculitis. pericarditis;* interstitial myocarditis. * pneumonia;* pleuritis. fat embolism. * features of alphal-antitrypsin deficiency* (2) . fatty changes of liver; splenitis. intestinal mucosal erosions and ulcers, with or without perforation. massive gastrointestinal hemorrhage in histiocytic cytophagic panniculitis. blind intestinal loop (3). for sampling and specimen preparation, see chapter 4. prepare samples for electron microscopy. vacuolar myopathy. (1) . external examination, skin, and mouth record extent of skin lesions; photograph; submit multiple samples for histologic study, preferably of areas that are free of secondary infection. prepare sections for immunofluorescent staining. bullous and other lesions of scalp, eyelids, nose, axillae, umbilicus, inframammary areas, back, hands, groins, genitalia, anus, knees, and feet. similar lesions may occur in the mouth. acantholysis is suprabasal or near granular layer. igg deposits on the surfaces of keratinocytes. note: (i) collect all tissues that appear to be infected. collect inguinal, popliteal, axillary, and supraclavicular lymph nodes for aerobic culture. photograph enlarged lymph nodes, and prepare smears of fresh cut surfaces. submit consolidated areas for microbiologic study (see above under "note"). perfuse both lungs with formalin. submit samples of pulmonary tissue and bronchial lymph nodes for histologic study. submit samples of grossly abnormal organs, exudates, or drainage fluids for culture and gram stain. hemorrhagic necrosis; variable suppuration. severe hemorrhagic edema; pneumonia with lobular to lobar features. large areas of necrosis teeming with organisms and minimal to suppurative inflammation. plasmacytoma (see "myeloma, multiple.") platybasia note: possible causes or associated conditions include arnold-chiari malformation;* basilar impression* orinvagination; fusion ofatlas to the foramen magnum; klippel-feil syndrome;* malpositioning of odontoid process; osteitis deformans (paget's disease of bone*); osteogenesis imperfecta;* osteomalacia;* rickets; syringobulbia, syringomyelia. * skull and spine prepare roentgenograms of skull and cervical spine. flattening of the base of the skull (angle formed bythe planeoftheclivus andtheplane of the anterior fossa exceeds 135â°). pleuritis (see "effusion(s) and exudate(s), pleural.") pleurodynia, epidemic synonyms: bornholm disease; devil's grip; epidemic myalgia; epidemic myositis. blood heart and pericardium submit samples for viral culture. sample for histologic study and for viral cultures (coxsackievirus a and b; echoviruses). if pericardia! fluid can be obtained, submit for viral culture also. cultures may be negative and search for viral rna by in situ hybridization may be indicated. chest organs other organs dissect thoracic duct and its tributaries (see chapter 3). perfuse one or both lungs with formalin. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit samples of cystic lesions for histologic study. submit samples of cystic lesions for histologic study. gas in thoracic duct. emphysema* of lungs. necrotizing enterocolitis in premature infants. pyloric stenosis, colitis (5), redundant sigmoid colon, ischemic bowel disease. mucosal pseudolipomatosis (6) . usually, cysts are subserosal in adults and located between muscularis mucosae and muscularis propria in infants and children. mucosa may be inflamed. extraintestinal cysts, as listed above under "abdomen." cysts may also occur in vaginal mucosa. include bright-field and polarized light microscopy, transmission electron microscopy, scanning or transmission electron microscopy with energy dispersive x-ray analysis (2), ion beam instrumentation, and secondary ion mass spectrometry. the last three methods are time-consuming, require much skill and expensive equipment, and do not lend themselves well to quantitation. ideally, bulk analysis and in situ microanalysis should be used in combination. analyses can be conducted in specialized laboratories at the following centers (for charges and other information, consult the appropriate laboratories): prepare chest roentgenogram. submit sample for microbiologic study. refrigerate sample for possible immunologic study. submit one lobe or samples of all lobes for bulk analysis and for in situ microanalysis (see above under "note"). microincineration may permit preliminary dust analysis. for demonstration of asbestos fibers, see chapter 2 and ref. (4) . submit one lobe or segment for microbiologic study. for pulmonary arteriography and bronchography, see chapter 2. prepare roentgenograms of entire lungs and slices. perfuse one lung (or both, if only routine studies are intended) with formalin. submit samples for histologic study of nodular dust lesions, diffuse fibrotic lesions, grossly uninvolved areas, bronchi, and bronchopulmonary lymph nodes. for preparation of paper-mounted sections, see chapter 2. prepare samples for transmission and scanning electron microscopic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. alveolar rupture with dissection of air into the mediastinum in neonates with respiratory distress (see "syndrome, respiratory distress, of infant") and in adult patients ventilated on volume respirators. external examination prepare chest roentgenogram. photograph mediastinum. explore major veins and record compression in cases of tension pneumomediatinum. roentgenograms provide the best permanent record of a pneumomediastinum. air bubbles in mediastinal soft tissues. part ii / diseases and conditions abdomen and neck organs perfuse one lung with formalin. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. search for evidence of trauma of other lesions that may have allowed air to enter soft tissues. intubation injury (j); perforation or rupture of major bronchus, trachea, or esophagus. alveolar rupture, e.g., in asthma* (2), may not be discernible. bronchiolitis obliterans (3), interstitial pneumonia* (4) and other lung conditions also may lead to pneumomediastinum. dissection of air from lesions in abdomen (e.g., retroperitoneal colonic perforation [5] ) or in neck area. pneumonia, all types or type unspecified note: if the type of underlying infection is known, follow procedures suggested under the name of the infectious disease. if the etiologic agent of the pneumonia is unknown, proceed as follows: (l) collect all tissues that appear to be infected. and grocott's methenamine silver stains. (4) special precautions may be required (see chapter 6). (5) serologic studies may be helpful once a specific etiologic agent is suspected. thus, collect serum at the time of autopsy or procure serum that was collected prior to death. (6) this may be a reportable disease. related terms: acute interstitial pneumonia (hamman-rich syndrome); desquamative interstitial pneumonia (dip); idiopathic organizing pneumonia (or "bronchiolitis obliterans with patchy organizing pneumonia [boop]" or "cryptogenic organizing pneumonitis"); idiopathic pulmonary fibrosis; lymphoid interstitial pneumonitis (lip); nonspecific interstitial pneumonia (nsip) (or "cellular interstitial pneumonia" [eip]); usual interstitial pneumonia (uip); fibrosing alveolitis; pulmonary alveolitis; and many others. note: the conditions listed under "related terms" are histologic variants of idiopathic interstitial pneumonia. vip is synonymous with idiopathic pulmonary fibrosis (ipf) (1). diffuse alveolar damage is the histologic finding in patients with the adult respiratory distress syndrome* (ards) causing interstitial and intra-alveolar fibrosis. this is an acute condition, referred to as acute interstitial pneumonia when it occurs as an idiopathic form of rapidly progressive interstitial pneumonia. possible associated conditions: acquired immunodeficiency syndrome* (aids) in patients with with lip or nonspecific interstitial pneumonia (2,3); trauma and shock in ards. secondary pulmonary fibrosis from inhalants (extrinsic allergic alveolitis or pneumonia; pneumoconiosis*), in drug-induced pneumonia, hypersensitivity pneumonitis (microgranulomatous hypersensitivity reaction of lung), rheumatoid arthritis,* sjogren's syndrome,* and other collagen-vascular diseases; primary biliary cirrhosis in patients with nonspecific interstitial pneumonia (2). external examination postmortem chest roentgenograms provide the only reliable and permanent record of a pneumothorax and its main complication, mediastinal shift due to a tension pneumothorax (fig. 11-4) . if roentgenograms cannot be prepared, a reasonably reliable diagnosis still can be made if the prosector inserts a needle through the lateral chest wall. the needle should be connected to a water-filled flask. ifa pneumothorax is present, gas bubbles appear in the flask as shown in fig. 11 -5. one can also expose the intact parietal pleura and observe if the lung tissue is separated from the parietal pleura by gas. incising the thorax at the base of a water-filled skin pocket is the least reliable method. infants can be totally submerged under water before the chest cavity is incised. however, care must be taken not to make an accidental incision into the underlying lung tissue. poisoning, all types or type unspecified note: if a specific substance is suspected-for instance, arsenic or ethylene glycol-follow procedures described under the appropriate entry. similar entries can be found for poisons whose general character or source is known-for instance, gas or mushroom poisoning. for some substances, the appropriate entry can be found under "abuse,...," "death,...," or "dependence, ..." in all instances, routine sampling of toxicologic material should be done as described in chapter 13. if no specific substances can be incriminated, the toxicologist must be provided with all available clinical information, as shown in chapter 13. in most cases of fatal poisoning, the coroner or medical examiner must be notified. poisoning, alkaloid note: see under specific name of alkaloid-for instance, "dependence, cocaine," "poisoning, atropine," "poisoning, digitalis," or "poisoning, strychnine." only a fraction of this large group of plant poisons has been listed. whether the specific name of the alkaloid is known or unknown, complete toxicologic sampling is recommended. poisoning, antimony ote: toxicologic material should be submitted for anal ysis, as suggested under "poisoning, arsenic." iatrogenic antimony toxicity may occur after treatment with antimony compounds for conditions such as filariasis, fungal infections, and schistosomiasis.* external examination and eyes pharynx and gastrointestinal tract heart, liver, and kidneys record skin changes and eye abnormalities. for toxicologic sampling of contents, see chapter 13. submit tissue samples for histologic study. request frozen sections for sudan stain. if exposure was from du t in smelting work, dermatitis and conjunctivitis may be present. severe gastroenteritis in acute poisoning. if victim drank antimony trichloride, ulcerative pharyngitis and gastritis may be present. fatty changes of myocardium and hepatic and renal parenchyma. poisoning, arsenic note: toxicologic material will be contaminated by bringing it in contact with fluids. keratinized tissues take up arsenic from solutions. put plastic bags over hands of victim. if exhumed bodies are investigated for arsenic poisoning, include material from surrounding soil and coffin along with tissues submitted for chemical analysis. interpretation of toxicologic findings: after fatal poisoning, arsenic concentrations in the liver tend to exceed 0.5-1.0 mg/ioo g wet tissue. in acute poisoning, arsenic concentrations in hair may reach 3 !1g/g (1) and in nails 8 !1g/g. organs collect all contents, particularly in accidental poisoning in children. body dry and warm after death. mydriasis. iatrogenic atrioventricular block (1) . fruits of atropa belladonna or seeds of datura stramonium may be found. no characteristic findings. poisoning, barbiturate(s) note: this type of poisoning has become uncommon. barbiturates may cause sudden death. in all instances, concomitant alcohol intoxication* must be ruled out. standard toxicologic sampling is sufficient. blood bile urine esophagus and stomach liver and brain procedures submit samples of blood from portal vein and peripheral veins or heart for toxicologic study. refrigerate for possible toxicologic study. record total volume and ph value. request tests for protein, glucose, and ketones; request drug screen. submit all contents and record their character. analyze for barbiturates and alcohol. submit samples for toxicologic study. evidence of alcohol intoxication.* poisoning by other addictive drugs. gritty residues of unabsorbed tablets, powder, or capsules. mucosal corrosion, ulceration, and discoloration from capsules may occur. concentration of barbiturate in parenchyma important for interpretation. poisoning, bismuth note: accidental poisoning is common (industrial exposure or drugs with soluble bismuth compounds). search also for other heavy metals. acute kidney failure* may be the cause of death. external examination and oral cavity stomatitis with bluish black discoloration of gums; loose teeth; sticky white membranous patches in mouth and throat. jaundice. protein casts and tubular epithelial cells in sediment. gray or black mucosal membranes; swelling of mucosa; intestinal ulcers that may be perforated. hemosiderosis. fatty changes; hemosiderosis. fatty changes; renal tubular degeneration with amorphic basophilic deposits in epithelium of convoluted tubules. hemosiderosis. see above under "external examination." for removal and specimen preparation, see chapter 4. peripheral neuritis. synonyms: bromine poisoning; bromism. note: the lethal dose is about 0.2 g in children and i g in adults (ingested). after fatal methyl bromide poisoning, headspace gas chromatography revealed a subclavian blood concentration of 3.0 microgram/ml whereas inorganic bromide concentrations were 530 micrograms/ml in the blood (1). tissue concentrations were lower than those in the blood. toxicologic samples should include liver and kidneys. chemical bums on face and conjunctivitis indicate direct exposure; skin pustules over body and nodose bromoderma of the legs indicate bromism. blood is best suited for bromide determination. after ingestion of bromide, necrosis with brown discoloration of mucosa of upper gastrointestinal tract may be present. after inhalation of bromide, swelling and inflammation ofmucous membranes in upper and lower respiratory tracts may be present. there may be pulmonary edema. pneumonia occurs in bromism. organs to be analyzed for cd should have no contact with water or be contaminated with blood; they should be sealed in polyethylene bags. cd leaks into fixation fluid. postmortem blood concentrations are very high and no indicator of the antemortem values (1). external examination and oral cavity blood urine gastrointestinal tract other organs procedures submit sample for toxicologic study. submit sample for determination of cadmium concentration. submit sample for toxicologic study and a portion of one lobe for microbiologic study. perfuse one lung with formalin. fix bowel as soon as possible. collect renal tissue for light microscopic and electron microscopic study. sample for toxicologic study. submit samples for histologic study also. poisoning, carbon monoxide note: if the victim had been in a fire, see also under "bums." carbon monoxide poisoning may rarely be responsible for automobile accidents. relatively low carboxyhemoglobin concentrations may contribute to death if there is concomitant poisoning-forinstance, with alcohol or drugs, particularly sedatives. anemia, atherosclerotic heart disease, and chronic pulmonary disease also increase sensitivity to carbon monoxide. ifblood had been withdrawn at time of hospital admissionfor instance, for crossmatching-submit this for carboxyhemoglobin determination. if no blood can be obtained, see under "heart, kidneys, and other organs." for quick-orienting qualitative tests, for quantitative methods of carbon monoxide determi-nation, and for interpretation of toxicologic findings, see below. request also determination of hemoglobin concentrations and of blood alcohol. request drug screen. for shipping of blood and tissues for carbon monoxide determination, see chapter 15. it should be noted that losses of up to 60% of the original saturation occurred when blood was kept in uncapped container at room temperature for 2 yz wk or at 4â°c for 3 wk. external examination blood heart, kidneys, and other organs brain record color of fingernails, particularly in heavily pigmented persons in whom lividity is difficult to discern. record appearance of blood and submit sample of postmortem blood for toxicologic study. see also above under "note." if no blood can be obtained, prepare water extract of spleen, kidneys, or other organs. request determination of carbon monoxide content and of carbon monoxide-binding capacity of this mixture. submit tissue samples for histologic study. for removal and specimen preparation, see chapter 4. pink skin and fingernails; bullous edema of skin; decubital ulcers. blood tends to be cherry red. for interpretation of toxicologic findings, see below. necrosis of papillary muscles in the heart or myocardial infarction may occur. renal tubular degeneration may also be found. acute kidney failure* has been observed after rhabdomyolysis complicated by compartment syndrome (2) . hemorrhagic necrosis of basal ganglia (lenticular nucleus in globus pallidus); diffuse petechial hemorrhages in white matter; cerebral edema. acute hydrocephalus in infants (3) . many methods of carbon monoxide determination have been described. currently, carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spectrophotometry or gas chromatography. in hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. pink discoloration of skin and organs usually indicates the presence of more than 30% carboxyhemoglobin (but rule out cyanide poisoning* and exposure to cold*). in a healthy, middle-aged person, a carboxyhemoglobin concentration greater than 50-60% is usually fatal. if the victim was anemic or suffered from chronic lung disease, particularly emphysema* or atherosclerotic heart disease, the concentration may be lower. in association with alcohol, sedatives, and other drugs, carboxyhemoglobin levels may also be much lower and yet fatal. a heavy cigarette smoker may have a carboxyhemoglobin concentration of 8-10%, and higher levels may occur in police officers and other persons exposed to automobile exhaust in dense traffic. if the victim survived the carbon monoxide poisoning for several hours, postmortem blood samples usually will fail to show the presence of carboxyhemoglobin. in these instances, blood taken at the time of admission to the hospital may still be available and of particular value. if the victim had spent 1 h in fresh air before death, 40-50% of the carbon monoxide will have been removed, and 8-10% will have been removed during each subsequent hour. even though clearance may be complete, death may still occur-primarily from brain damage and infectious complications in prolonged coma. or delayed by only a few hours, particularly after inhalation of carbon tetrachloride. sudden death probably is caused by cardiac dysrrhythmia. alcohol concentrations should be determined in all cases or, if death was delayed, evidence of drinking at the time of exposure should be sought. alcohol considerably increases the hazards of carbon tetrachloride. note: see also under "bronchitis, acute chemical" and under "poisoning, gas." hydrochloric acid is sold by plumbing supply houses and pool supply companies as muriatic acid. it is a liquid. chlorine is a water-soluble gas. as supplied for pool sanitation, liquid chlorine is usually acidic. for convenience, chlorine and hydrochloric acid are discussed here together. prepare photographs of face. conjunctivitis and cyanosis in chlorine gas poisoning; burns of lips from hydrochloric acid. see also above under "larynx and trachea." photograph opened esophagus and stomach. sample for histologic study, particularly if there is doubt whether a perforation was antemortem or postmortem. for removal and specimen preparation, see chapter 4. severe pulmonary edema, bronchopneumonia, and swelling of mucous membranes in chlorine poisoning. arterial thrombosis may occur. pulmonary fibrosis may develop after prolonged survival. swelling and ulceration of mucous membranes in chlorine poisoning; acute laryngotracheitis. tracheoesophageal perforation. corrosion of mucosa with thickening, hemorrhage, and blackish discoloration after ingestion of hydrochloric acid. antemortem and postmortem perforation may occur. glomerular capillary thromboses. hemorrhages in white matter (1). poisoning, cyanide synonym: hydrocyanic acid (hydrogen cyanide) poisoning. note: hydrocyanic acid (hydrogen cyanide, hcn) is a water-soluble gas. its salts, sodium cyanide and potassium cyanide are sold as "eggs" to the jewelry industry. hydrocyanic acid is formed when cyanide salts are dissolved in acidic solutions. containers from which the poison might have been ingested or inhaled should also be submitted for toxicologic examination. for cyanide screening tests in the autopsy room and for interpretation offindings, see below. caution: stomach may still contain cyanide gas, formed by acidic reaction of cyanide salt. it may be best to open the stomach under a hood (1). the odor is quite characteristic for cyanide poisoning but most persons are unable to smell this odor. it is helpful to know in advance if any person in an office or laboratory can smell cyanide. forensic pathologists who can smell the compound state that it has its own specific odor, which differs from the often quoted smell of bitter almonds. autopsies also can be done in a negatively pressured isolation room (1). for odor, see above under "note." bright red skin color is not always present. corrosion around mouth and in oral cavity may be found after ingestion of potassium or sodium cyanide. blood is fluid and sometimes bright red. if potassium or sodium cyanide was ingested, brown-red mucosal corrosion may be present in stomach or in upper digestive tract. pulmonary edema. for odor, see above under "note." if death was not instantaneous, there may be hyaline thrombi in small blood vessels, minute hemorrhages, and necroses of lenticular nuclei. this test can be used for blood and gastric contents (2) . dip squares of filter paper in a small amount ofsaturated picric acid. let these squares dry until barely moist. place a drop ofthe material to be tested-e.g., blood or gastric contents--on a piece of paper. let material dry for a moment, and then place one drop of 10% sodium carbonate in the center of the material to be tested. ifcyanide is present, a reddish purple color will chromatograph out from the material. the higher the concentration of cyanide the more blue the color will be. it is possible to recognize whole blood because the blood turns a rather dark brown and the reddish to purple color is clearly visible. high concentrations of sulfide interfere by giving a false-positive test. another screening test is done as follows (3) . dip filter paper into normal blood. then treat the paper with potassium chlorate, whereupon brown methemoglobin forms. place this preparation into the fluid suspected of containing cyanide (e.g., blood, gastric contents, pulmonary edema fluid). if bright red cyanmethemoglobin forms, the reaction is positive. if the concentration of cyanide in the stomach is high and the concentration in the lungs is low, cyanide was ingested. alternatively, if the pulmonary cyanide concentration is high and the concentrtaion in the gastric contents is low, hydrogen cyanide most likely was inhaled. occasionally, minimal cyanide levels will be present in decomposed bodies. related term: digoxin toxicity. note: certain drugs may interfere with correct digitalis determination. blood heart vitreous procedures submit sample of peripheral blood for digoxin radioimmunoassay. freeze fresh myocardium for digitalis extraction. submit sample for digoxin radioimmunoassay. poisoning, drug(s) (see "dependence, drug(s), all types or type unspecified" or under "poisoning,â�¢â�¢â�¢" followed by specific name of drug.) poisoning, ethanol (ethyl alcohol) (see "alcoholism and alcohol intoxication," "cardiomyopathy, alcoholic," "disease, alcoholic liver," "syndrome, fetal alcoholic," and syndrome, wernicke-korsakorr.") poisoning, ethylene glycol related term: antifreeze poisoning. note: pulmonary and cerebral manifestations are the main findings in acute poisoning, and renal tubular necrosis is the primary finding in chronic poisoning. for general toxicologic sampling, see chapter 13. calcium oxalate crystals can be demonstrated in routine histologic sections but also in scanning electron micrographs of thick deparaffinized sections. eyes blood urine for removal and specimen preparation, see chapter 5. in acute cases, submit sample for ethylene glycol determination; in chronic cases, request determination of calcium concentrations. prepare sediment. papilledema; optic nerve atrophy. ethylene glycol in serum (i) . protein casts; calcium oxalate crystals (2) . crystals are light yellow and birefringent, arranged as sheaves, rhomboids, or prisms. poisoning, food related terms: bacillary dysentery* (shigella food poisoning); botulism;* clostridium perfringens food poisoning; favism; mushroom poisoning;* salmonella food poisoning; staphylococcal food poisoning. note: if cause of food poisoning is unknown, submit suspected food for aerobic and anaerobic cultures, gram stain of smears, and routine toxicologic study. this should include tests for heavy metals (antimony, cadmium, and lead) that may have leaked from old cooking utensils. test for the presence of staphylococcal enterotoxin are done only in specialized laboratories. if botulism is suspected, follow procedures described under that heading. mushroom poisoning also is listed as a separate entity. if salmonella food poisoning is suspected, see under "fever, typhoid." see also under "enteritis" or "enterocolitis" or under another specific heading such as "dysentery, bacillary." obtain sufficient material for microbiologic and histologic study to identify organisms such as chlamydia, clostridium (type f strains), salmonella, shigella, verotoxic e. coli, yersinia, and others. external examination gastrointestinal tract submit contents for aerobic and anaerobic cultures (see above under "note"); prepare smears of contents for gram stain. submit samples for histologic study. debilitated states; patients in extremes of life. enteritis or enterocolitis. poisoning, gas note: anesthesia-associated death, * carbon monoxide poisoning,* and sniffing and spray death* are presented under the appropriate headings. procedures discussed here deal with other volatile substances, including chemical irritants such as ammonia (nh 3 ), chlorine or hydrochloric acid poisoning (see also under that heading); methylene chloride, phosgene (coci 2 ), sulfurous acid (h 2 s0 3 ), or sulfur dioxide (s02) ' gases from body cavities, heart chambers, or blood vessels can be removed as described under "embolism, air." gases can also be trapped with a rubber dam after cutting organs under water. samples from various organs should be shipped in hermetically sealed nonplastic containers or in analyzing solutions. external examination, and oral cavity blood larynx and trachea record extent of chemical bums. submit sample for gas analysis. in many instances, inhaled gases can be demonstrated chromatographically in gas from head space above sealed blood specimen. chemical bums in and around mouth or conjunctivas. chemical bums. other organs if gas was inhaled and is to be analyzed, submit intact lungs with bronchi ligated in airtight, nonplastic container to laboratory that can conduct gas analysis. if survival was short, formalin perfusion of lungs is not recommended; it may cause artifactual ballooning and internal ruptures of organ. submit samples of tissue for routine histologic study. see above under "note." chemical pneumonia; pulmonary edema. after longer survival, obliterating fibrous bronchiolitis, chronic bronchitis, and saccular bronchiectasis* may occur. poisoning, glycol (see "poisoning, ethylene glycol.") poisoning, halogen (see "fluorosis," "poisoning, bromide," "poisoning, chlorine or hydrochloric acid," "poisoning, gas?, and "poisoning, iodine!') poisoning, heavy metal (see "poisoning, antimony," "poisoning, arsenic," "poisoning, cadmium," "poisoning, lead," poisoning, mercury," "poisoning, thallium!') poisoning, insecticide (see "poisoning, organophosphate(s)*) poisoning, iodine related terms: lugol's solution; tincture of iodine. for toxicologic sampling, see chapter 13. external examination and eyes urine, blood, and parenchymal organs lungs and upper respiratory tract stomach record color of skin and extent of corrosive lesions. submit samples for toxicologic study. submit samples for histologic study. submit contents for toxicologic study; photograph mucosa; prepare histologic sections. see above under "stomach." perioral corrosive lesions; yellow discoloration of skin; conjunctivitis after exposure to vapors. acute inflammation of respiratory tract after inhalation of vapors. corrosive gastritis; if starch was used as antidote, gastric lining will be bluish. histologically, well-preserved mucosa is present because of in vivo fixation. mucosa may show same changes as stomach. swelling of tubular epithelium. synonyms: propanol; rubbing alcohol. procedures see under "alcoholism and alcohol intoxication." nonspecific autopsy findings: visceral congestion; pulmonary and cerebral edema. poisoning, lead note: lead-free syringes and lead-free polyethylene containers should be used. blood lead concentrations can be determined by inductively coupled plasma mass spectrometry (1). for screening methods, see ref. (2) . this is a reportable disease in some states. external examination, oral cavity, and hair bluish lead line at gingival margin in victims with poor oral hygiene. external examination, oral cavity, and hair for diagnosis of chronic plumbism, analysis of scalp hair may be useful. analysis is done by neutron activation (see also under "poisoning, arsenic"). remove samples with lead-free syringe (see above under "note") or 20-ml vacutainer tubes (becton, dickinson and company). do not add anti-coagulant or preservative. for coliection procedures, see above under "blood" and under "note." request also determination of coproporphyrin concentrations. submit sample for histologic study; submit remaining tissue for toxicologic analysis. submit with contents for toxicologic study, particularly in acute poisoning. submit sample from each kidney for histologic study; submit remaining tissue of both kidneys separately for toxicologic study. submit at least 10 g of fresh bone for toxicologic study. for removal and specimen preparation, see chapter 4. poisoning, lsd (d-lysergic acid diethylamide) (see "abuse, hallucinogen(s).") poisoning, lye related terms: ammonium hydroxide poisoning; calcium oxide or quicklime poisoning; poisoning by alkaline corrosives; potassium hydroxide poisoning; sodium hydroxide poisoning. external examination and oral cavity blood neck organs, esophagus, trachea, and lungs record extent of oral, perioral, and other facial corrosive injuries. photograph lesions. prepare histologic sections of tissue from inside of lips or mouth. submit sample for toxicologic study. after removal of heart, remove neck organs with hypopharynx, esophagus, larynx, and trachea. leave stomach attached to esophagus. open pharynx and esophagus along posterior midline. in acute cases, formalin perfusion of lungs is not recommended; it may cause artifactual baliooning and internal ruptures of organ. lye bums on face and chest in acute cases; scars and manifestations of malnutrition* in chronic cases. sweliing, edema, and necrosis of mucous membranes in acute poisoning. fibrosis and strictures in chronic cases. bronchitis* and bronchopneumonia. submit specimen from pharynx for histologic study. for removal and specimen preparation, see chapter 4. submit sample of brain for toxicologic study. exfoliative dermatitis. blue line at gingival margin; hypertrophy of gum; acute and chronic gingivitis; exfoliation and loss of teeth (2) . degeneration of myocardium. increased concentrations of mercury (1) . congestion. erosive gastritis and colitis. increased concentrations of mercury (1) . degeneration of proximal tubules; calcifications. chronic kidney failure* may be the cause of death. induration of mucosa. increased concentrations of mercury (1) . cortical hemorrhages. cholinesterase activity will be low. pulmonary edema if poison was inhaled. airways may contain aspirated material. cholinesterase activity can be determined reliably even after decomposition and embalming. clinically, guillain-barre syndrome has been observed after poisoning with organophosphate. in acute poisoning, the cholinesterase levels may be 25% of the normal values (see below). the cholinesterase levels in the blood are not affected by the duration of the postmortem interval; measurement may be attempted even on decomposed or exhumed bodies. severe fatty changes; periportal necroses. fatty changes in myocardium, skeletal muscles, and other organs. for toxicologic sampling (gastric contents, urine, blood, brain, and other organs), see chapter 13. rigor mortis after fatal strychnine poisoning may occur very soon after death and may be very severe (opisthotonos); it may persist until decomposition sets in. congestion of viscera; no characteristic morphologic autopsy findings. acute pancreatitis has been observed (1). high strychnine concentrations (also demonstrable in exhumed bodies [2] ). poisoning, thallium note: for toxicologic sampling, see below and chapter 13. thallium is used as a rodenticide and pesticide, and has some industrial applications. retrobulbar neuritis. chapter 4 and 5. submit brain for toxicologic study. submit sections of brain and optic nerves for histologic study. submit specimens for toxicologic study (take from lower extremity). for removal, prosthetic repair, and specimen osteomalacia;* osteomyelofibrosis. preparation of bones, see chapter 2. for preparation of sections and smears of bone marrow, see chapter 2. submit samples for toxicologic study. synonym: acute anterior poliomyelitis. note: the disease has been nearly eliminated in the usa but not in many other countries. (1) collect all tissues that appear to be infected. ( for removal and specimen preparation, see chapter 4. in acute cases, submit portions of brain and spinal cord for viral culture. neurogenic atrophy of skeletal muscles in areas of paralysis. electrolyte disorder. * hypertensive heart disease; myocarditis.* aspiration or bronchopneumonia (or both); edema; atelectasis; embolism;* alveolar wall necrosis (acute or organizing diffuse alveolar damage) after oxygen toxicity. acute ulcers. acute gastric dilatation; acute gastroduodenal ulcers; gastrointestinal erosions and hemorrhages. dilatation of colon; perforation of cecum. urolithiasis and nephrolithiasis,* pyonephrosis and pyelonephritis. * phlebothrombosis of legs, most commonly on left side. necrosis of anterior hom cells of spinal cord, with neuronophagia and perivascular inflammatory reaction. old lesions show neuronal loss and gliosis. medulla ("bulbar polio") and other areas of brain stem, cerebellum, and cerebrum, particularly the motor cortex, may be affected in various degrees. part ii / diseases and conditions pregnancy note: in some instances, procedures described under "death, abortion-associated," "embolism, amniotic fluid," or "toxemia of pregnancy" may be indicated. synonym: werner syndrome. external examination and skin abdomen procedures record volume of intraperitoneal fluid; prepare smears; submit samples of peritoneum for histologic study. colloid carcinoma of stomach or colon. well-differentated adenocarcinoma of ovary or, rarely, of appendix; ruptured appendiceal mucinous adenoma is the most common cause of peritoneal adenomucinosis. pseudotumor cerebri synonym: benign intracranial hypertension; meningeal hydrops. note: this condition, which generally affects young, obese females, is characterized by symptoms and signs of increased intracranial pressure without a demonstrable cause. hence, intra-cranial mass lesions, infections, and related conditions should be excluded. such conditions include adrenal insufficiency;* guillain-barre syndrome* (increased colloid-osmotic pressure); hyperadrenalism; hypervitaminosis a* (e.g., after treatment of acne); hypoparathyroidism;* hypothroidism;* infectious mono-nucleosis;* lyme disease; pregnancy; sydenham's chorea; throm-bus ofthe lateral or superior sagittal sinus (otitic hydrocephalus); and wiskott-aldrich syndrome. submit samples of all accessible organs and tissues, with or without gross lesions. submit material for electron microscopy. for removal and specimen preparation, see chapter 5. for removal, prosthetic repair, and specimen preparation, see chapter 2. heart small bowel liver procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. fix the bowel as soon as possible. record weight. submit samples for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. aortitis involving ascending aorta and aortic valve with regurgitation; mitral valve and adjacent myocardium and conduction system also may be involved. sprue-like changes with loss of villi. fibrosis or cirrhosis and other abnormalities, particularly in patients who had been taking methotrexate. psoriatic arthritis and spondylitis. synonyms and related terms: allergic purpura; anaphylactoid purpura; hypersensitivity vasculitis. external examination and skin rabies note: (i) in most instances, an autopsy limited to the brain is sufficient to confirm the diagnosis. (2) rabies virus is especially infectious and thus, universal precautions should be strictly followed (see chapter 6) . avoid the use of scalpels whenever possible. the generation ofaerosols should be assidusee above under "note." if a complete autopsy is done, sample heart, lungs, kidneys, pancreas, submaxillary salivary glands, and adrenal glands; freeze or refrigerate sampled tissues and submit for viral study (see below under "brain and spinal cord"). obtain serum for serologic studies. freeze or refrigerate cerebral tissue immediately after removal and submit frozen to special laboratory for fluorescent antibody staining. take these sections from hippocampus or brain stem. place the refrigerated tissues in 50% neutral glycerol saline solution for preservation. fix remaining brain and spinal cord tissue in 15% formalin and submit for histologic study. as an alternative to freezing of tissue, immunoperoxidase methods for detection of rabies viral antigens can now be applied to formalin-fixed tissue (1 in almost all instances, the procedures described under "assault" and under "homicide" must also be followed. see also refs. (1) and (2) . for the evaluation of bite marks, photographs with and without scales and black and white film should be used. a forensic dentist is required to compare the victim's bite marks to the teeth of suspects. swabs of possible sperm or other fluids must be sent to the crime lab for proper evaluation. external examination other organs and tissues genital organs comb pubic hair over a towel and then pull sample; also, pull samples of hair from head. collect fingernail clippings and place in containers marked "right" and "left." collect blood, hair, fibers, and residues of urine or saliva and/or semen that may have stained the victim's clothing or that may be found on the skin of the victim. for study of stains and scrapings, see below. introduce sterile dry cotton swab into the posterior vault of the vagina or-preferablyinto the cervical canal. prepare smear on glass slide and send to crime laboratory for identification of sperm (see also above under "note" using a sterile cotton swab moistened with sterile saline, lift suspected dried spermatic fluid from hair or skin of external genitalia, perineum, buttocks, or other sites. stains on clothing will be extracted by forensic laboratory personnel. let specimens air dry in absence of sunlight and then place in paper bags or envelopes and seal. smears are made for the demonstration of spermatozoa, cytologic changes, bacteria, and other possible findings (some crime labs prefer to make their own smears from the swabs). other samples are used for the determination of acid phosphatase, as described below. usually, the survival time of morphologically recognizable spermatozoa in the vagina is less than 24 h, but on occasion this may be much longer. thus, within the first 24 h after coitus, 64% of cervical smears have been found to be positive for spermatozoa. at day 10, spermatozoa have been found in 13% of the smears. obviously, if the assailant had had a vasectomy, spermatozoa will not be found at any time. spermatozoa have been found in the vagina of some women several days or weeks after death. dried stains (see above) may give positive results after longer intervals. acid phosphatase activities greater than 5 bodansky units (for method, see above) indicate that probably less than 12 h have elapsed since the time of intercourse (i) . in another study, vaginal swab specimens showed acid phosphatase activities of more than 2,000 king if there is evidence of parotid involvement, other salivary glands should also be studied. parotid gland can be biopsied from scalp incision. submaxillary gland can be removed with floor of mouth. for sampling and specimen preparation, see chapter 4. for removal, prosthetic repair, and specimen preparation, see chapter 2. chronic meningitis; space-occupying lesions (submeningeal nodular granulomas). granulomatosis. iridocyclitis; chorioretinitis; papilledema; posterior uveitis; keratoconjunctivitis sicca; conjunctival follicles; cataracts. involvement of lacrimal glands and other orbital tissues (2) . sarcoidosis of parotid gland is common. sarcoid neuropathy and sarcoid myopathy. cystic changes, primarily of small bones of hands and feet. sarcoidosis of joints (3). synonyms and related terms: bilharziasis; schistosoma haematobium infection; schistosoma japonicum infection; schis-tosoma mansoni infection. note: unless specifically stated, the changes listed below refer to chronic schistosomiasis mansoni and japonica. (1) collect all tissues that appear to be infected. (2) request direct examination for schistosoma. the following procedures have been described and recommended (1). for demonstration of schistosoma eggs, compress 4-mm tissue fragments-for instance, mucosa of the urinary bladder-between glass slides. if this gives negative results, digest a 5-g portion of tissue in potassium hydroxide. for the recovery of adult worms of schistosoma mansoni and schistosoma haematobium, remove the viscera en bloc and rinse with water. separate the intestines from the mesentery. subsequently, perfuse the portal vein system, the liver, and one lung with saline (2) . then pass the perfusion fluid through a monofilament nylon cloth with an aperture size of 180 11m. submerge the cloth in water and examine with a dissecting microscope. fix worms in formalin solution. examine the intestinal mucosa directly. from the urinary bladder, ureters, and surrounding connective tissue, worms can be recovered as follows. inject water into the tissue until the tissue increases 2 or 3 times in thickness. then compress the tissue gently with a glass plate. cut slices 0.1-0.2 cm in thickness. compress these slices between the glass plate and the stage of a dissecting microscope and examine for the presence ofadult worms. many worms also will be present in the fluid expressed from the tissue as it is cut. for the counting of eggs in tissues, urine, and feces, see ref. (1) . immunodiagnostic methods (elisa and imrnunoblot) also have been developed prepare skeletal roentgenograms. there are no diagnostic laboratory tests but it still is advisable to save a blood sample. record heart weight. test competence of valves (chapter 3). open heart in line of blood flow. photograph and measure valvular lesions. prepare sections of valves, myocardium, conduction system (if there was a history of heart block), and ascending aorta. perfuse at least one lung with formalin. submit any consolidated area for microbiologic study. fix intestines as soon as possible. abnormalities as listed in right-hand column. sample for histologic study and request amyloid stains or renal parenchyma. prepare roentgenograms of spine, sacroiliac joints, symphysis ossium pubis, and manubriosternal, sternoclavicular, and humeroscapular joints. if spine cannot be removed in its entirety, it can be split in midline and one half can be removed, with costovertebral and costotransversal joints. hip joints should be exposed. histologic sections should include synovia and periarticular tissue. secondary and peripheral osteoarthritis* may be present. leukemia* developed in some patients who had had radiation treatment (1950 or earlier). acute anterior uveitis. (2) request fungal culture. (3) request grocott's methenamine silver stain. (4) record weight of all organs (for expected weights, see part iii). submit samples of all major organs, including endocrine glands, lymphatic and fat tissue, bone, and bone marrow for histologic study. hunger edema; skin changes secondary to vitamin deficiencies. hypoproteinemia. atrophy; hemosiderosis of spleen. degree of atrophy varies from organ to organ; atrophy of fat tissue, lymphoid tissue, and gonads usually is most pronounced. severe infections, such as tuberculosis,* may be present without having been apparent clinically. steatohepatitis, nonalcoholic (nash) note: the morphologic findings in the liver are indistinguishable from those in alcoholic liver disease* (1). follow autopsy procedures described under "disease, alcoholic liver." if the patient had received a liver transplant, procedures described under "transplantation, liver" should be followed also. possible associated conditions: celiac sprue (rare); diabetes mellitus* (type 2); hyperlipidemia; malnutrition* from bulemia (rare); morbid obesity with liver failure particularly after episodes of rapid weight loss (e.g., after recent gastroplasty); lipodystrophy; mild obesity;* short bowel syndrome (rare); total parenteral nutrition. * stenosis, congenital valvular pulmonary related terms: isolated (pure, simple, or dome-shaped) pulmonary stenosis. note: the pulmonary valve is usually acommissural in isolated congenital pulmonary stenosis. with other coexistant congenital heart disease (such as tetralogy), the pulmonary valve is usually bicuspid and hypoplastic, but may be unicommissural or dys-plastic and tricuspid. heart and great vessels; peripheral pulmonary arteries brain procedures culture any grossly infected valve. for general dissection techniques, see chapter 3. record weight of heart, thickness of ventricles, and annular circumferences. for removal and specimen preparation, see chapter 4. infective endocarditis* of pulmonary valve and tricuspid valve; infective endarteritis at bifurcation of pulmonary trunk. it is preferable to have autopsies of fetuses and stillborns performed by pathologists experienced in perinatal pathology. if such personnel are not immediately available, the attending pathologist may nevertheless collect important information. if attempts to induce abortion appear to have caused the death of the mother, see "death, abortion-associated." the placenta should be immediately procured from the delivery room should it not arrive in the laboratory with the fetus. this will avoid the possibility of the placenta being discarded by the delivery room staff. no fetal autopsy is complete without a careful examination of the placenta (see part i, chapter 2). pathologic changes of placenta that are causative of stillbirth are summarized in ref. 1 . fascia lata or other aseptically obtained tissue should be collected for tissue culture for karyotype analysis. if the fetus is at all autolyzed, then the fascia lata cells may not grow in tissue culture. therefore, if the fetus shows any evidence of autolysis, tissue should be taken aseptically, from placenta immediately beneath the chorionic plate. a portion ofplacenta and liver should also be snap-frozen for possible molecular analysis. the initial stage of the autopsy should include photography and radiography, taking anterior-posterior and lateral views. the photographs will record the degree of maceration, which can be roughly correlated with the duration of fetal demise before delivery [1] external measurements should include body weight, circumference ofhead, chest and abdomen, crown-rump length, crown-heel length, and foot length. these measurements are compared with tables of standards for normal fetuses (see part iii of this book). assessment of growth retardation may be based on these data. a careful external examination should search for abnormalities such as jaundice, bulging fontanel, cranial bone softening, hyper-or hypotelorism, choanal atresia, external ear anomalies, cleft lip, cleft palate, macroglossia, micrognathia, colobomata, cystic hygroma, shortened neck, contractures, omphalocele, gastroschisis, abnormal external genitalia, anal atresia, absent vagina, sacral pits, open neural tube defects, hemihypertrophy, syndactyly, clinodactyly, transverse palmar creases, or incomplete descent of testes. the organ bloc may be removed in the manner similar to the adult, using the technique of letulle (see chapter 2) . if the thyroid gland is noted to be in its usual location and if it appears normal, then the tongue may be left in the body. in macerated fetuses, it is suggested that the organ bloc be fixed overnight in formalin solution prior to dissection. to aid adequate fixation, the following simple steps may be done: i) wash the organ bloc thoroughly prior to fixation; 2) place multiple transverse cuts through the liver and lungs; 3) dissect the posterior leaves of the diaphragm away from the adrenal glands and kidneys; 4) bivalve the adrenal glands and kidneys in the coronal plane; 5) instill formalin in the lumen of the intestine, using a syringe; and 6) gently instill formalin into both ventricles of the heart, being careful to avoid the ventricular septum. the procedure for dissection of the organs is similar to that of the adult except: 1) the venous and arterial connections of the heart, including the patency of the ductus arteriosus must be determined before the heart is removed; 2) the esophagus must be opened posteriorly prior to its complete removal so that esophageal atresia or tracheo-esophageal fistula may be recognized and photographed prior to further dissection; 3) the location of the appendix and of the testes should be recorded. until the intestine has been examined for stenosis or atresia, the mesentery should be left attached. the degree of autolysis as seen grossly and with histologic examination of both the fetus and the placenta can be used to estimate the duration of time between fetal death and delivery (2, 3, 4) . trichrome stain is useful for better visualizing histologic features in severely autolyzed tissue. to remove the brain, benecke's technique of one of its modifications may be used. stillbirth vs livebirth. a decision has to be made whether the infant was born alive or was stillborn. the hydrostatic lung test, described in the previous edition, appears unreliable. the presence of gas in the lungs does not rule out stillbirth. after death, air can be introduced into the lungs, or putrefaction gases might be present. however, air artificially introduced after death will not distend the alveoli and can be squeezed out, whereas this does not seem to be the case after active ventilation. the distribution of fat in the fetal zone of the adrenal cortex may indicate whether intrauterine death was acute, more prolonged, or chronic (3). this is particularly helpful if the stillborn baby is macerated. if the mother died also, see under "death, abortion-associated" strangulation note: in many instances, procedures described under "hom-icide" must also be followed. if a rope or some other material had been used (see also under "hanging"), leave ligature in place until autopsy can be done. see also under "hypoxia." toxicologic sampling, particularly for alcohol, should be done in all instances (chapter 13). results than body fluids. (2) universal precautions should be strictly followed. the generation of aerosols should be minimized. to sterilize tissue surfaces in preparation for culture, swab the surface with povidone iodine. avoid searing the tissue surface since this will produce an aerosol. keep no more than one scalpel in the dissecting area at anyone time. have an assistant available with clean, gloved hands to receive specimens in containers, so as to minimize the degree of contamination on the outside of the containers. for cleaning procedures and related information, see chapter 6. (3) hiv-l in tissue may be demonstrated using polymerase chain reaction (pcr), immunohistochemistry, in situ hybridization, or immunofluorescence. (4) for immunocytochemical and molecular studies, fix tissue in ethanol or carnoy's solution. the virus can be identified using pcr in most tissues, whether fresh, frozen, or fixed in ethanol. (5) serologic tests as well as direct fluorescent antibody tests are avail-able for many of the expected infections. (6) this is not a reportable disease. external examination and skin record body weight and evidence of lipodystrophy following aids medications. record and photograph any skin lesions. examine oral cavity. if the diagnosis is in doubt, samples can be submitted for enzyme immunoassay. cachexia. severe loss of subcutaneous fat in face and extremities, associated with large fat deposits on upper back and upper abdomen ("protease paunch"). cutaneous kaposi's sarcoma (l) . test may be positive for many weeks after death (2) . syndrome, myelodysplastic synonyms and related terms: chronic myelomonocytic leukemia;* refractory anemia; refractory anemia with excess of blasts; refractory anemia with excess of blasts in transformation; refractory anemia with ringed sideroblasts; refractory dysmyelopoietic anemias. note: the myelodysplastic syndromes are represented by a heterogeneous group of normocytic anemias, often with neutropenia, thrombocytopenia, and monocytosis. for expected bone marrow changes, see above under "synonyms and related terms." autopsy procedures are similar to those recommended for most cases of leukemia, with particular attention paid to intercurrent infections and thrombocytopenic hemorrhages. in all instances, material should be collected using aseptic technique for tissue culture for chromosome analysis. common findings in these conditions are deletion of the long arm ofchromosome 5, deletion of chromosome 5 or 7, or trisomy 8. syndrome, nephrotic note: see under name of suspected underlying condition, such as amyloidosis,* anaphylactoid purpura,*diabetes mellitus,* glomerulonephritis,* goodpasture's syndrome,* heavy metal poisoning, hemolytic uremic syndrome,* infective endo-carditis,* polyarteritis nodosa,* syphilis, * or systemic lupus erythematosus. * if accelerated hypertension or constrictive pericarditis is the suspected underlying condition, see under "hypertension (arterial), all types or type unspecified" or "pericarditis," respectively. in all instances, the renal veins and the inferior vena cava should be opened in situ. "en masse" removal of organs is recommended for this purpose. if thrombosis is found, record exact location and size of clot and submit sample of clot with wall of veins for histologic study. see also under "thrombosis, venous." coronary atherosclerosis and its complications seem to be increased in patients with the nephrotic syndrome. submit sample for bacteriologic, viral, and serologic study. record weight and submit samples for histologic study. if heart block was present, submit samples of conduction system for histologic study (chapter 4). submit consolidated areas for microbiologic study. perfuse at least one lung with formalin. for removal of urethra, see chapter 2. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 5. consult roentgenograms (see above under "external examination, skin, and oral cavity"). keratoderma (keratosis blennorrhagica) with vasculitis (2) of sole of feet, of palms, and of circumcised glans penis. arthritis* of knees, ankles, and metatarsal and midtarsal joints, with or without ankylosis. osteoporosis.* usually, sterile culture. pericarditis; * myocarditis.* aortic valve lesions. pleuritis and pneumonia.* pulmonary fibrosis involving upper lobes. erosions, papules, and plaques in urethral or bladder mucosa. enteritis. thrombophlebitis. conjunctivitis; multifocal choroiditis; acute anterior uveitis; iritis; keratitis. arthritis* (see above under "external examination, skin, and oral cavity") resembling rheumatoid arthritis. * nonspecific synovitis. for removal and specimen preparation, see chapter 5. syndrome, reye's note: hypoglycemia* may have been present, but that condition is difficult or impossible to confirm after death. an immediate autopsy is indicated (see below under "liver" and "pancreas"). vitreous blood urine heart lungs submit sample for sodium, chloride, and urea nitrogen determination. submit sample for microbiologic (viral) and serologic study, for ammonia and bilirubin determination, and for determination of salicylate levels if there is a history of treatment with this drug. obtain sample for biochemical and toxicologic analysis. record weight. request frozen sections for sudan stain. submit fresh tissue for bacterial and viral culture. request paraffin sections and frozen sections for fat stain (see above under "heart"). influenza;* varicella.* manifestations of liver failure. evidence of salicylate administration. assay for organic acids should rule out acylcoenzyme a dehydrogenase deficiency, and toxicity, e.g., of acetaminophen and valproic acid (l) . cardiomegaly; fatty changes of myocardium and patchy myocytolysis. viral pneumonia rarely present. acute interstitial pneumonia;* bronchitis;* hemorrhages. lipid-laden histiocytes in alveoli. record body weight and length, stature, and distribution of head, axillary, and pubic hair. record and prepare photographs of features of face and neck. prepare skeletal roentgenograms. submit samples of breast tissues for histologic study. these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter 9) . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit for histologic study. submit samples of all portions for histologic study. if biliary atresia is suspected, follow procedures described under that heading. dissect kidneys and ureters in situ and record findings. submit samples of gonads or-if gonads cannot be identified-equivalent ridges on mesosalpinx for histologic study. also submit samples of endometrium, cervix, and vagina. record weight and submit sample for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 5. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for sampling and specimen preparation, see chapter 4. peroxidase-labeled gastrin antibodies seem to react with cells in all gastrinomas. aberrant gastrinoma may occur at hilus of spleen. metastases are found in regional lymph nodes and liver. there may be manifestations of multiple endocrine neoplasia. * secondary syphilis. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody techniques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. (4) in adult autopsies, no special precautions are indicated (see below under "liver"). (5) serologic studies are available from local and state health department laboratories. (6) this is a reportable disease. external examination record and prepare photographs of all abnormalities listed in right-hand column. prepare smears and sections of acute lesions; prepare sections of older skin lesions or anogenital mucosal lesions. hunterian chancre in primary syphilis; condylomata lata. noduloulcerative gummas and scarring in later stages. cerebrospinal fluid lymph nodes heart and aorta other organs and tissues prior to 20 wk gestation, the destructive effects of syphilis may not be seen. gummata are rare in neonates. tabes dorsalis is also uncommon. serologic diagnosis is difficult in the neonate because of transplacental transfer of maternal igg antibodies. acquired syphilis (syphilis in adulthood) is presented above under a separate heading. (1) collect all tissues that appear to be infected. (2) culture methods are not available, but animal inoculation can be performed. consultation with a microbiology laboratory is recommended. (3) special stains for treponema pallidum rarely are positive except with material from fresh lesions of primary or secondary syphilis and of syphilitic hepatitis of the newborn. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody tech-niques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. (4) in neonates, special precautions are indicated (see below under "liver") (5) serologic studies are available from local and state health department laboratories. (6) this is a reportâ· able disease. (i) collect all tissues that appear infected. (2) request aerobic and anaerobic cultures. however, the presence of tetanus bacilli established in culture is not diagnostic, since spores of c. tetani frequently contaminate wounds. record body weight and length and appearance of wound(s); photograph and excise wound(s) for histologic study. record evidence of parenteral drug abuse, especially subcutaneous injection (i.e., "skin popping"). prepare chest roentgenogram. evidence of weight loss; subcutaneous abscesses. tetanus may occur in drug addicts. tension pneumothorax* after mechanical ventilation. submit sample for microbiologic study. submit any consolidated area for microbiologic study. bacterial meningitis must be ruled out. aspiration and bronchopneumonia; embolism;* atelectasis. tetany note: see under name ofsuspected underlying conditions, such as hyperparathyroidism,* malabsorption syndrome,* or vitamin 0 deficiency. * respiratory or metabolic alkalosis* cannot be confirmed after death; determination of blood ph is not helpful because acidity increases rapidly after death. the concentration of serum phosphates also increases after death. synonym: large ventricular septal defect with pulmonary stenosis* or atresia.* note: the basic anomaly consists of subpulmonary stenosis, ventricular septal defect, overriding aorta, and secondary right ventricular hypertrophy. for general dissection techniques, see chapter 3. surgical interventions include modified blalock-taus-sig subclavian-to-pulmonary arterial shunt; complete repair with patch closure of ventricular septal defect, and reconstruction of right ventricular outflow tract (with a patch or with an extracardial conduit). possible associated conditions: origin of left pulmonary artery from aorta; minor abnormalities of the tricuspid valve; absent ductal artery (25%); atrial septal defect* (in 20%; pentalogy of fallot); bicuspid pulmonary valve;* dextroposition of aorta; double aortic arch; hypoplastic pulmonary arteries; patent ductal artery;* patent oval foramen; complete atrioven-tricular septal defect* (usually with down's syndrome*); persis-tent left superior vena cava; pulmonary valve atresia (see "atresia, pulmonary valve, with ventricular septal defect"); right aortic arch (25%); second ventricular septal defect; origin ofleft anterior descending (lad) or right coronary artery (rca) from contralateral aortic sinus or coronary artery (5%); syndrome with absent pulmonary valve and massively dilated pulmonary arteries (rare). chest cavity heart lungs other organs record course of superior vena cava and of its tributaries. record course of thoracic aorta and of its main branches. record origin of pulmonary arteries. if infective endocarditis is suspected, follow procedures described under that heading (chapter 7). for coronary arteriography, see chapter 10. perfuse both lungs with formalin. request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. persistent left superior vena cava. right aortic arch with or without right-sided ductal artery; double aortic arch; absent ductal artery. origin of the left pulmonary artery from descending aorta. for possible additional findings, see above under "note" and under "possible associated conditions." infective endocarditis* (usually of pulmonary or aortic valve). see also above under "possible associated conditions." marked right ventricular hypertrophy. right ventricular dilatation and fibrosis with late postoperative right-sided heart failure or sudden death due to arrhythmia. old in situ thrombosis of small pulmonary artery branches. paradoxic embolism; cerebral abscess. * thalassemia synonyms and related terms: congenital hemolytic anemia; alpha-thalassemia; beta-thalassemia major (cooley's anemia); beta thalassemia minor (beta-thalassemia trait). note: the changes described below are observed primarily in beta-thalassemia major. unless the cause of the renal vein thrombosis and the nature of the complicating or underlying renal disease are known, follow procedures described under "glomerulonephritis." procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. in infants, manifestations of dehydration. * pulmonary embolism. * tumor of the kidney* (renal cell carcinoma); aortic aneurysm; lymphadenopathy; other mass lesions. inferior vena cava thrombosis involving orifice or whole length of renal veins. tumor thrombus (e.g., from renal cell carcinoma). femoral vein thrombosis. rare venous malformations also may cause renal vein thrombosis (l) . hemorrhagic infartion. parenchymal renal disease with nephrotic syndrome,* including amyloidosis* and vasculitis* (these last conditions may be associated with renal vein thrombosis). acute gastroenteritis in infancy. hyperparathyroidism,* pregnancy,* trauma, and other conditions. thrombosis, venous note: for peripheral venous thrombosis, the autopsy procedures are essentially similar to those described under "phlebitis." see also under "hypertension, portal," "syndrome, budd-chiari," "thrombosis, cerebral venous sinus," "thrombosis, lateral sinus," and "thrombosis, renal vein." thymoma (see "'fumor of the thymus.") thyroiditis synonymsand related terms: chronic fibrosing thyroiditis (riedel's struma); chronic thyroiditis with transient thyrotoxicosis; hashimoto's thyroiditis; pyogenic thyroiditis; subacute (granulomatous, giant cell, de quervain's) thyroiditis. possibleassociatedconditions: withhashimoto's thyroiditisautoimmune (chronic) hepatitis,*megaloblastic anemia,*rheumatoid arthritis,*sjogren's syndrome,*and systemic lupus erythematosus.* with riedel's struma-retroperitoneal fibrosis,* sclerosing cholangitis,* sclerosing mediastinitis,*and other conditions with idiopathic fibrosis. with subacute thyroiditis-viral infection. submit sample for determination of protein concentration; record appearance of sediment. for removal and specimen preparation, see chapter 4. thromboses of small vessels. glomerulonephritis.* hemorrhagic necroses. proteinuria; abnormal sediment. abruptio placentae, small placenta with accelerated maturation of villi; infarcts; fibrinoid necrosis of decidual arterioles. thrombotic occlusion of small vessels with hemorrhagic necroses; anterior lobe of pituitary gland may contain necroses (see also "syndrome, sheehan's"). submit samples for histologic study. if infection is expected, submit material for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. transposition, complete, of the great arteries note: the basic anomaly is the origin of the aorta from the right ventricle, and of the pulmonary artery from the left ventricle, with a shunt, and usually with a right anterior aorta. there are four major types: (1) with an intact ventricular septum (65%), (2) with a ventricular septal defect* (20%), (3) with a ventricular septal defect and subvalvular pulmonary stenosis* (10%), and (4) with an intact ventricular septum and subvalvular pulmonary stenosis* (5%). for general dissection techniques, see chapter 3. interventions include atrial septostomy or septectomy; mustard or senning atrial switch procedure; rastelli-type repair with a valved extracardiac conduit; and jatene arterial switch procedure with lecompte maneuver. possible associated conditions: abnormal origin ofcoronary arteries (10%); atrial septal defect* (5%); coarctation of the aorta;* interrution of the aortic arch;* juxtaposition of atrial appendages (4%); overriding aorta (5%); overriding pulmonary artery (10%); patent ductal artery;* patent oval foramen; subvalvular pulmonary stenosis* (15%); tubular hypoplasia ofthe aortic arch;* ventricular septal defect* (30%), often mal-alignment type (50%). synonyms: atrioventricular and ventriculoarterial discordance; l-transposition. note: the basic anomaly is a mirror-image ventricular inversion, with a left anterior aorta, and with blood flow from right atrium to left ventricle to pulmonary artery, and from left atrium to right ventricle to aorta. for general dissection techniques, see chapter 3. interventions include patch closure of the ventricular septal defect; relief of pulmonary stenosis; relief of tricuspid insufficiency; and insertion of pacemakers. possible associated conditions: anterior and posterior av nodes (100%), prone to develop complete heart block; dysplasia or epstein's malformation* ofleft-sided tricuspid valve (40%); mirror-image epicardial coronary artery distribution (100%); right-sided mitral valve anomalies; subvalvular pulmonary stenosis* (40%); ventricular septal defect* (65%). kinyoun's, or other acid-fast stains. polymerase chain reaction for mycobacterial dna may be helpful in the differential diagnosis ofgranulomas (1). (4) universal precautions should be strictly followed and aerolization should be avoided. (5) reliable serologic studies are not available. a definite diagnosis requires isolation of the organism. (6) this is a reportable disease. cavitary, fibrocalcific, miliary, bronchial, and other types of pulmonary tuberculosis; granulomas in hilar lymph nodes. most organs and tissues may be involved, including liver, pancreas, spleen, kidneys, adrenal glands and other endocrine glands, gonads, and lymph nodes. tuberculous meningitis; tuberculoma. iridocyclitis or panophthalmitis. tuberculous arthritis and synovitis (hips, spine, knee); tuberculous osteomyelitis (anterior aspect of vertebrae; metaphysis of long bones). part ii / diseases and conditions procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. chronic ulcerative colitis may be associated with carcinoma of bile ducts, typically arising in psc.* metastases common in regional lymph nodes and lungs. i for removal, prosthetic repair, and specimen preparation and decalcification procedures, see chapter 2. if osteoblastic metastases appear to be present, search for primary tumor in prostate and breast, carcinoid tumors and hodgkin's lymphoma. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. cachexia. evidence of hypercalcemia (particularly in presence of osteolytic metastases). metastases to bone (e.g., from carcinoma of prostate, breast, bronchi, thyroid gland, kidney, or urinary bladder) usually involve red bone marrow. therefore, distal extremities are rarely involved by metastatic tumors in adults. metastases, commonly in lungs. metastatic calcification in patients with hypercalcemia. eosinophilia. myopathy.* metastases in liver and regional lymph nodes. see also above under "possible associated conditions." malakoplakia (rare) (2) . part ii / diseases and conditions other organs and peripheral arteries tumor(s). if tumor is within a heart chamber (usually a myxoma), record extent of mobility and capability of tumor to obstruct a valvular orifice. submit samples of tumor(s) for histologic study and, particularly if the tumor is of unknown type, for immunohistochemical study and electron microscopy (chapter 15). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. metastatic carcinoma or melanoma. secondary cardiac effects by tumors include carcinoid heart disease, amyloid heart disease in multiple myeloma, and lymphocytic myocarditis in pheochromocytoma. treatment effects such as adriamycin cardiotoxicity may be noted also. tumor (myxoma) emboli in pulmonary or peripheral vessels (1) . multiple aneurysms* of cerebral and other arteries may rarely be associated with atrial myxomas. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. part ii / diseases and conditions if there was evidence of endocrine activity (see above), snap-freeze portion of fresh tumor for hormone assay. perfuse lungs with formalin. sample neoplastic and non-neoplastic tissue for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 2. see above under "possible associated conditions." note that hypoglycemia* generally cannot be confirmed at autopsy. nonbacterial thrombotic endocarditis.* carcinoma may be associated with asbestosis or other types of pneumoconiosis,* chronic bronchitis,* emphysema,* interstitial pneumonia,* and many other bronchopulmonary diseases. see above under "possible associated conditions." metastases (regional lymph nodes, liver, bones, brain, and many other sites) and metastatic calcification. migratory thrombophlebitis.* encephalomyelitis;* cerebellar corticoid degeneration;* subacute spinocerebellar degeneration. * crooke cell hyperplasia. myasthenic syndrome (eaton-lambert syndrome); myopathy;* dermatomyositis.* peripheral neuropathy. see above under "external examination and skin." bones (with red marrow) are common sites of metastases. record presence or absence of testes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. thmor of the soft tissues the possible sites and characteristics of soft tissue tumors vary so much that no universally applicable autopsy techniques can be presented. in all instances, the size, weight, and location of the tumor(s) must be recorded and tissue must be sampled for histologic study. if the tumor had not been classified prior to death, samples should be snap-frozen for immunohistochemical study. other samples should be prepared for electron microscopic study. evidence of paraneoplastic syndromes (see below) may require additional procedures. possible associated conditions: only a few paraneoplastic syndromes or systemic complications can be presented here. for the type of soft tissue tumor that was associated with each condition, see title of reference. kasabach-merritt syndrome (1) (thrombocytopenia, microangiopathic hemolytic anemia, and acute or chronic coagulopathy associated with a rapidly enlarging hemangioma); liver function abnormalities (2); neurofibromatosis (3); osteomalacia (4). heart esophagus, stomach, and duodenum other organs and tissues t in most instances, detennination of vitreous sugar concentration and of blood group is not indicated. inspect valves and prepare photographs and sections of vegetations. leave esophagus and part of duodenum attached to stomach. submit samples of tumor and of grossly uninvolved stomach for histologic study. request pas stain and warthin-starry stain for identification of h. pylori. portions of endocrine gastric tumor should be snap-frozen for immunohistochemical study and possible hormone assay. record location of tumor metastases. other organs procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for dissection of the thoracic duct (for search of tumor cell clusters), see chapter 3. tumor of the uterus (with cervix) possible associated conditions: acquired immunodeficiency syndrome* (aids) (1) if peritonitis is present, submit exudate for bacteriologic study. record volume of exudate and location of perforation. see "disease, ischemic heart." other procedures depend on grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. perfuse at least one lung with formalin. for celiac arteriography, see chapter 2. open stomach and duodenum in situ and record site of perforation or penetration. record measured or estimated volume of blood in gastrointestinal tract. rinse ulcer with saline to locate eroded vessel(s). pin stomach and duodenum on corckboard (serosa toward board) and fix specimen in formalin before sectioning. prepare histologic sections of ulcer(s) and of remainder of stomach. request warthin-starry stain for h. pylori. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. free air in abdomen suggests perforation of ulcer. peritonitis. * perforation of ulcer. coronary atherosclerosis and manifestations of coronary insufficiency are commonly associated with peptic ulcer disease. in rare instances, pericardial fistula may be present from ulcer in hiatus hernia. peptic ulcers may be associated with emphysema,* tuberculosis,* and other chronic pulmonary diseases. vasculitis (see "aortitis," "arteritis, .â�¢.," "phlebitis," and "purpura,â�¢â�¢â�¢") ventricle, double inlet left (1) synonyms: single functional ventricle; univentricular heart; univentricular atrioventricular connection; holmes heart. note: the basic anomaly is the connection of both atrioventricular valves to the left ventricle, often with transposed great arteries and a restrictive ventricular septal defect. there are four major types, based on the ventriculoarterial connection: (1) with congenitally corrected transposition (60%); (2) with complete transposition (30%); (3) with normally related great arteries (holmes heart; 5%); and (4) double outlet, persistent truncal artery, or pulmonary atresia (5%). for general dissection techniques, see chapter 3. possible associated conditions: bicuspid pulmonary valve; bilateral mirror-image mitral valves (without tricuspid morphology); subvalvular aortic stenosis,* often with hypoplasia, coarctation, or interruption of the aortic arch; subvalvular pulmonary stenosis;* dual av nodes and progressive heart block in patients with congenitally corrected transposition. ventricle, double outlet right synonym: origin of both great arteries from right ventricle; taussig-bing anomaly. note: the basic anomaly is the origin of the aorta and pulmonary artery primarily from the right ventricle, usually with a ventricular septal defect, and often with subpulmonary stenosis. for general dissection techniques, see chapter 3. possible associated conditions: complete atrioventricular septal defect (often with asplenia syndrome); muscular discontinuity between aortic and mitral valves; right ventricular infundibular stenosis; ventricular septal defect* that may be subaortic, subpulmonary, doubly committed, or remote. examine histologically with h&e and oil red a stains. hepatomegaly and splenomegaly, jaundice. deficiency of acid lipase (1). foam cells in the lamina propria. hepatomegaly, severe steatosis with cholesterol clefts in hepatocytes and kupffer cells. fibrosis. splenomegaly with lipid-laden foam cells. symmetric enlargement with dystrophic calcifications, giving the adrenals a gritty texture. vacuolated cells of the inner zona fasciculata and entire zona reticularis with cholesterol clefts. foam cells may be found in the lungs, bone marrow, lymph nodes, vessel walls, as well as schwann and ganglion cells. new insights into lipoprotein assembly and vitamin e metabolism from a rare genetic disease angioid streaks associated with abetalipoproteinemia familial hypobetalipoproteinemia: genetics and metabolism aviation pathology scuba diving accidents: decompression sickness, air embolism aetiology and occurrence of diving injuries. a review of diving safety scuba decompression illness and diving fatalities in an overseas military community diving-related emergencies bodies found in water achalasia and squamous cell carcinoma of the esophagus: analysis of 241 patients esophageal achalasia and adenocarcinoma in barrett's esophagus: a report of two cases and a review of the literature a technique for necropsy evaluation of stenosis of the foramen magnum and rostral spinal canal in osteochondrodysplasia mutation analysis of the men i gene in multiple endocrine neoplasia type i, familial acromegaly and familial isolated hyperparathyroidism. i increased incidence of neoplasia in females with acromegaly benign and malignant tumors in patients with acromegaly pathology of acromegaly ethanol in sequestered hematomas stages of acute alcoholic influence/intoxication electrolyte imbalance in alcoholic liver disease collection and storage of specimens for alcohol analysis analysis for alcohol in postmortem specimens disposition of alcohol in man medicolegal aspects of alcohol. garriott jc blood, urine and other tissue specimens for alcohol analysis relationship between postmortem blood and vitreous humor ethanol levels larson cpo alcohol: fact and fallacy gradwohl's legal medicine left ventricular hypertrophy is more prominent in patients with primary aldosteronism than in patients with other types of secondary hypertension intracranial aneurysm and hemorrhagic stroke in glucocorticoid-remediable aldosteronism association of adrenal medullae and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional consideration amyloidosis: recognition, confirmation, prognosis, and therapy electron microscopy of primary and secondary cutaneous amyloidosis and systemic amyloidosis ocular amyloidosis current status review: cerebral amyloid spinal cord compression by amyloid deposits the burden of "sticky" amyloid: typing challenges quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain kidney involvement in takayasu arteritis pathogenesis of rheumatoid arthritis splenic involvement in rheumatic diseases aspergillus sinusitis in patients with aids: report of three cases and review invasive aspergillosis in systemic lupus erythematosus aspiration (see "obstruction, acute airway aspergillus endocarditis, myocarditis and pericarditis complicating necrotizing fasciitis. case report and subject review aspergillus-related lung disease biliary atresia and the polysplenia syndrome: its impact on final outcome biliary atresia nephromegaly and elevated hepatocyte growth factor in children with biliary atresia coccidiomycosis pneumonia in a nonendemic area associated with inftiximab codeine (see "dependence, drug[s], all types or type unspecified all types or type unspecified (see "enterocolitis, other types or type undetermined chronic ulcerative (see "disease, inflammatory bowei cytologic diagnosis of cryp -tococcus neofonnans in hiv-positive patients cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis hypereosinophilia in disseminated cryptococcal disease cryptococcosis-a review of 13 autopsy cases from a 54-year period in a large hospital cryptosporidiosis in persons with hiv infection broncho-pulmonary cryptosporidiosis in four hiv-infected patients cryptosporidiosis and inflammatory bowel disease sclerosing cholangitis associated with chronic cryptosporidiosis in a child with a congenital immunodeficiency disorder cyanide (see "poisoning, cyanide cyst(s), choledochal choledochal cyst-dinical features and classification cyst(s), liver (see "disease, fibropolycystic, of the liver and biliary tract pulmonary related terms: congenital cystic adenomatoid malformation; congenital pulmonary lymphangiectasis; intralobular bronchopulmonary sequestration. references infection or calcification of cysts; pyelonephritis;* perinephric abscess. obstructive uropathy in recessive polycycstic renal disease, congenital hepatic fibrosis is present and cystic bile ducts may be present in dominant cases (5). see above under "possible associated conditions the pathology ofhuman renal cystic disease cystic kidney: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis hepatic fibrosis associated with hereditary cystinosis: a novel form of noncirrhotic portal hypertension ophthalmic manifestations and histopathology of infantile nephropathic cyctinosis: report of a case and review of the literature pathological investigations of deaths following surgery, anaesthesia and medical procedures clinical outcome versus post-mortem finding in thoracic surgery: a lo-year experience order from american registry of pathology sales office intra-alveolar pulmonary siderophages in sudden infant death: a marker for previous imposed suffocation a contribution to a possible differentiation between sids and asphyxiation sudden infantdeath syndrome (sids)-standardized investigations and classification: recommendations investigation of the sudden deth of infants: a multicenter analysis alpha l-antitrypsin deficiency-associated panniculitis: resolution with intravenous alpha i-antitrypsin administration and liver transplantation clinical manifestations of alpha l-antitrypsin deficiency risk of hepatobiliary disease in adults with severe alpha i-antitrypsin deficiency (pizz): is chronic viral hepatitis b or c an additional risk factor for cirrhosis and hepatocellular carcinoma? alpha i-antitrypsin deficiency and inflammatory bowel disease severe alphal-antitrypsin deficiency (piz homozygosity) with membranoproliferative glomerulonephritis and nephrotic syndrome, reversi-bleafterorthotopic livertransplantation klippel-feil syndrome associated with malfonned larynx. case report klippel-feil syndrome in association with a craniocervical dennoid cyst presenting as aseptic meningitis in an adult: case report klippel-feil syndrome accompanied by an aneurysm of the non-coronary sinus of valsalva the hereditary ataxias clinical and genetic characterizations of 16q-linked autosomal dominant spinocerebellar ataxia (ad-sca) and frequency analysis of ad-sca in the japanese population cocain-related medical problems. consecutive series of 233 cases direct cocaine cardiotoxicity demonstrated by endomyocardial biopsy the pathology and etiology of cocaineinduced heart disease ischemic colitis related to cocaine abuse hepatic dysfunction accompanying acute cocaine intoxication high incidence of malignancies in patients with dennatomyositis and polymyositis: an li-year analysis partial lipodystrophy associated with juvenile dennatomyositis: report of two cases chronic pneumomediastinum and subcutaneous emphysema: association with dennatomyositis spontaneous esophageal rupture in adult dennatomyositis polyneuropathy in juvenile dermatomyositis combined glucose and lactate values in vitreous humor for postmortem diagnosis of diabetes mellitus diabetes is related to cerebral infarction but not to ad pathology in older persons hepatic lesions resembling alcoholic liver disease relationship between cardiomyopathy and liver disease in chronic alcoholism cyanamide-associated alcoholic liver disease: a sequential histologic evaluation caroli's disease: 1977-1995 experiences caroli 's disease and autosomal dominant polycystic kidney disease: a rare asso-ciation? spontaneous rupture of a bile duct and its endoscopic management in a patient with caroli's syndrome bartonella henselae endocarditis in an immunocompetent adult cat-scratch disease and bacillary angiomatosis attack, transient cerebral ischemic" and "infarction, cerebral!') cholesteryl ester storage disease: hepatopathology and effects of therapy with lovestatin clinical, biochemical and histological analysis of seven patients with cholesterol ester storage disease cutaneous manifestations of chronic granulomatous disease. a report of four cases and review of the literature aspergillus endocarditis in chronic granulomatous disease colitis complicating chronic granulomatous disease. a clinicopathological case report hypertrophic cranial pachymeningitis with spinal epidural granulomatous lesion ocular pathologic findings of chronic granulomatous disease of childhood review: creutzfeldt-jakob disease survival of creutzfeldt-jakob disease virus in formol-fixed brain tissue guidelines for high risk autopsy cases: special precautions for creutzfeldt-jakob disease tissue handling in suspected creutzfeldt-jakob disease and other human spongiforme encephalopathies (prion diseases) cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of creutzfeldt-jakob disease autopsy case of sporadic creutzfeldt-jakob disease presenting with signs suggestive of brainstem and spinal cord involvement metastatic crohn's disease: a rare cutaneous manifestation crohn's disease with pulmonary involvement in a 3 year old boy mesenteric fibromatosis asso-ciated with crohn's disease cholangiocarcinoma and crohn's disease the pancreas as a site of granulomatous inflammation in crohn's disease dermatomyositis associated with crohn's disease 1994 cardiocyte storage and hypertrophy as a sole manifestation of fabry's disease. report on a case simulating hypertrophic non-obstructive cardiomyopathy an atypical variant of fabry's disease with manifestations confined to the myocardium pulmonary involvement in fabry disease cerebrovascular complications offabry's disease high incidence of thrombosis in fabry's disease oral involvement in chronic graft versus host disease after allogenic bone marrow transplantation massive pericardial effusion complicating the course of chronic graft-versus-host disease (cgvhd) in a child with acute lymphoblastic leukemia following allogeneic bone marrow transplantation the histological spectrum of pulmonary graft-versushost disease in bone marrow transplant recipients bronchiectasis in bone marow transplantation oncocytic metaplasia of bile duct epithelium in hepatic gvhd immune-mediated myelopathy after allogeneic marrow transplantation gunther's (see ''porphyria, congenital erythropoietic heavy-chain synonyms and related terms: gamma heavy-chain inflammatory bowel disease (first of two parts) takayasu's arteritis associated with idiopathic ulcerative colitis cerebrovascular complications ofinflammatory bowel disease pathology of inflammatory bowel disease legionnaires' disease source of infection for sporadic legionnaires' disease: a review legionnaire's disease associated with macular rash; two cases severe skin loss after meningococcal septicemia: complications in treatment osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review rhabdomyolysis during the subacute stage of meningococcal sepsis primary meningococcal arthritis in a child: case report and literature review chondrosarcoma as a complicating factor in paget's disease of bone lymphoma arising in paget's disease osteoarthritis and paget's disease sickle cell disease sickle cell disease and sudden death: a retrospective/prospective study of 21 autopsy cases and literature review vascular lesions of the liver in sickle cell disease. a clinicopathologic study in 26 living patients whipple's disease and "tropheryma whippelii periodic acid-schiff-negative granulomatous lymphadenopathy in patient with whipple's disease. localization of the whipple bacillus to noncaseating granulomas by electron microscopy serial imaging studies of cerebral whipple's disease: from onset to end the wilson's disease gene is a putative copper transporting p-type atpase similar to menke's gene the liver biopsy diagnosis of wilson's disease. methods in pathology hepatolenticular degeneration (wilson's disease) venous air embolism in homicidal blunt impact head trauma: case reports a practical device for demonstrating air embolism amniotic fluid 3. kulka w. laboratory methods and technical notes: a practical device for demonstrating air embolism proof of fatal air embolism venous air embolism from head and neck wounds hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease liver involvement in hemorrhagic fever with renal syndrome hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hanta virus pulmonary syndrome lassa fever in the united states. investigation ofa case and new guidelines for management lassa fever: review of virology, immunopathogenesis, and algorithms for control and therapy early diagnosis of lassa fever by reverse transcription-pcr rheumatic pneumonia: reappearance of a previously recognized complication of rheumatic fever acute rheumatic fever and poststreptococcal acute glomerulonephritis caused by t serotype 12 streptococcus scleritis, uveitis, and glaucoma in a patient with rheumatic fever comparison of clinical features and pathologic findings in fatal cases of typhoid fever during the initial and later stages of the disease typhoid fever complicated by acute renal failure and hepatitis: case reports and review hemophagocytosis and granulomas in the bone marrow of a child with down syndrome diseases involving intrahepatic bile ducts idiopathic eosinophilic esophagitis with stricture formation in a patient with longstanding eosinophilic gastroenteritis eosinophilic gastroenteritis presenting with colitis and cholangitis eosinophilic gastroenteritis presenting with acute pancreatitis idiopathic midline destructive disease: does it 3. mendenhallwmetai.lethalmidlinegranoloma-nasalnaturalkillerff-celi exist? nasal cocain abuse mimicking midline granuloma pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with epstein-barr virus infection recurrent bilateral spontaneous pneumothoraces associated with pulmonary angiocentric immunoproliferative lesion lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications wegener's granulomatosis: histological documentation of common and uncommon manifestations in 216 patients necrotizing granulomatosis of the breast wegener's granulomatosis with gangrene of toes pathology of pulmonary granulomatous vasculitis. sarcoidosis gunshot (see "injury, firearm splenic involvement in rheumatic diseases wegener's granulomatosis, acute laryngotracheal airway obstruction and death in a 17-year-old female: case report and review of the literature antiendothelial antibodies in patients with wegener's granulomatosis: prevalence and correlation with disease activity and manifestations. i rheumatoi2007 transfusion-transmitted disease references flattening, broadening, and possible fusion of villi. phlegmonous inflammation and edema, mainly in ileocecal region encephalitis.* leukopenia; thrombocytopenia; aplastic anemia (4) (pancytopenia*) synovitis (arthritis*) hepatitis g virus infection in hepatitis c virus-positive patients co-infected or not with hepatitis b virus and/or human immunodeficiency virus hepatitis g and c coinfection in children tumor of the liver diaphragmatic related terms: congenital diaphragmatic hernia fulminant hepatitis in patients undergoing liver transplantation: evidence for anon-a, non-b, non-c marrow transplantation for hepatitis-associated aplastic anemia: a follow up oflong-term survivors langerhans cell histiocytosis research. past, present, and future an update on c1onality, cytokines, and viral etiology in langerhans cell histiocytosis langerhans cell histiocytosis in adults pulmonary langerhans cell granulomatosis central nervous system disease in langerhans cell histiocytosis immunohistochemical analysis oflangerin in langerhans cell histocytosis and pulmonary inflammatory and infectious diseases pathological approach to the diagnosis of hydrocephalus thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report infectious causes ofhydrops fetalis human parvovirus b19 infection associated with hydrops fetalis cardiac abnormalities associated with hydrops fetal is gmi-gangliosidosis presenting as nonimmune hydrops fetalis: a case report a case of recurrent infantile polycystic kidney associated with hydrops fetalis the pathologist and the hydropic placenta, fetus, or infant chronic diarrhea associated with thymoma and hypogammaglobulinemia (good's syndrome) systemic amyloidosis in a patient with hypogammaglobulinemia. i hcv infection in patients with primary defects of immunoglobulin production encepahomyelitis in primary hypogammaglobulinemia retinitis pigmentosaand hypogammaglobulinemia intestinal obstruction in the newborn with congenital syphilis meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis biliary atresia and meconium ileus associated with nieman-pick disease postmortem cranial mri and autopsy correlation in suspected child abuse cytomegalovirus-associated pseudotumor simulating gastric malignancy in acquired immuno-deficiency syndrome: a case report with review of literature bone marrow fibrin ring granulomas and cytomegalovirus infection cutaneous reactions following herpes zoster infections: report of three cases and a review of the literature herpes zoster and internal malignancy posteriorhorn varicella-zoster virus myelitis death or injury caused by electrocution myocardial hemorrhagic necrosis in delayed death from electrocution safety in bullet recovery procedures: a study of the black talon bullet lightning injuries: prognostic signs of death lightning injuries adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by nocardia asteroides in a patient with acquired immunodficiency syndrome ards and adrenal insufficiencyassociated with the antiphospholipid antibody syndrome adrenal insufficiency from bilateral adrenal hemorrhage after total knee replacement surgery non-hodgkin's lymphoma presenting with adrenal insufficiency and hypothyroidism: an autopsy case report a case ofprimary unilateral adrenal burkitt-like large cell lymphoma presenting as adrenal insufficiency effect of growth hormone on fatty liver in panhypopituitarism increased fracture frequency in adult patients with hypopituitarism and gh deficiency pituitary abscess the liver: an atlas and text of ultrastructural pathology intubation (see "injury, intubation iodine (see "poisoning, iodine attack, transient cerebral ischemic" and "infarction, cerebral heart (see "disease, ischemic heart isomorism (see "syndrome, polysplenia and asplenia a study on performance of two serological assays for diagnosis of leprosy detection of mycobacterium leprae infection by pcr pathology of eye in leprosy references intervillous abscesses containing grampositive, non-acid-fast bacilli bacteria are predominantly intracellular. enterocolitis. listeria monocytogenes can be cultured from meconium. generalized lymphadenitis. disseminated abscesses and/or granulomas, particularly after transplacental infection listeria monocytogenes empyema in an hiv infected patient fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis liver abscesses due to listeria monocytogenes yust 1. brainstem abscess and meningitis due to listeria monocytogenes in an adult with juvenile chronic arthritis pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions the liver in systemic lupus erythematosus: pathologic analysis of 52 cases and review of japanese autopsy registry data chronic pancreatitis: a complication of systemic lupus erythematosus disseminated perivenous necrotizing encephalomyelitis in systemic lupus erythematosus: report of an autopsy case skeletal muscle lymphocytic vasculitis in systemic lupus erythematosus: relation to disease activity clinically occult avascular necrosis of the hip in systemic lupus erythematosus storage histiocytes and hemophagocytosis: a common finding in the bone marrow of patients with active systemic lupus erythematosus extensive medium vessel vasculitis with sle: an unsual association cutaneous manifestations of sexually transmitted ing cause of proctitis in men who have sex with men lymphogranuloma venereum as a cause cutaneous malakoplakia in a patient with acquired immunodeficiency syndrome (aids) extensive malakoplakia ofthe nasopharynx: management of a rare disease malakoplakia and colorectal adenocarcinoma coinfection is common in measles associated pneumonia giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings intestinal neuronal dysplasia thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report disease, meningococcal encephalitis, all types or type unspecified" and "meningitis mercury (see "poisoning, mercury with myelofibrosis synonym: idiopathic myelofibrosis acute cardiac tamponade associated with pericardial extramedullary hematopoiesis in agnogenic myeloid metaplasia methyl alcohol) (see "poisoning, methanol (methyl alcohol) thrombotic thrombocytopenic (see "purpura, thrombotic thrombocytopenic epstein-barr virus in inflammatory diseases of the liver and liver allografts: an in situ hybridization study fulminant hepatitis due to epstein-barr virus infection the mucopolysaccharidoses: a clinical review and guide to management biochemical diagnosis of mucopolysaccharidoses: experience of 297 diagnoses in a 15-year period (1997-1991) mucormycosis synonym: phycomycosis; zygomycosis. note: diseases that may be complicated by mucormycosis include bums,* diabetes mellitus,* leukemia,* lymphoma,* and tuberculosis cardiac involvement in mucopolysaccharidosis: effects of allogeneic bone marrow transplantation mitral and aortic regurgitation in 84 patients with mucopolysaccharidosis toxic epidermal necrolysis possibly linked to hyperacute graft-versus-host disease after allogeneic bone marrow transplantation pulmonary complications in toxic epidermal necro-iysis: a prospective clinical study references possible or expected findings pyelonephritis;* nephrocalcinosis; granulomas; tumor infiltrates; manifestations of the conditions listed below under "other organs renal stones in patients with rheumatoid arthritis nephrolithiasis as a presenting feature ofchronic sarcoidosis: a prospective study nephrolithiasis and risk of hypertension posterior lens opacities; retinal hamartomas. peripheral neuropathy with focal schwannomatous changes or onion-bulb-like schwann cell or perineural cell proliferation neurofibromatosis type i. in: pathology and genetics of tumours of the nervous system neurofibromatosis type 2. in: pathology and genetics oftumours ofthe nervous system neurological complications involving the central nervous system in neurofibromatosis type i primary cutaneous nocardia asteroides infection after heart transplantation native valve endocarditis due to nocardia-like organisms hepatobiliary complications of total parenteral nutrition total parenteral nutrition: a histopathologic analysis of the liver changes in 20 children obesity and breast cancer risk nonalcoholic steatohepatitis obesity cardiomyopathy: pathogenesis and pathophysiology bennett cpo pachydennoperiostitis in childhood unilateral hypertrophic osteoarthropathy associated with aortofemoral graft infection gastroesophageal reflux and esophagitis-associated hypertrophic osteoarthropathy hypertrophicosteoarthropathy caused by lipoid pneumonia achondroplasia" and dyschondroplasia, oilier's osteogenesis imperfecta synonyms and related terms: lobstein's syndrome osteogenesis imperfecta congenita; 01 type i, ii, and iii; osteogenesis imperfecta cystica hypercalcemia in osteogenesis imperfecta treated with pamidronate gastrointestinal problems in patients who have type-iii osteogenesis imperfecta osteomalacia related terms: osteoporosis;* renal osteodystrophy; rickets. note: many possible causes of osteomalacia may not be apparent at autopsy, e.g., sodium fluoride or diphosphonate toxicity or use of anticonvulsant drugs osteomalacia associated with adult fanconi syndrome: clinical and diagnostic features epstein-barr virus in b-celllymphomas associated with chronic suppurative inflammation osteomyelitis and osteonecrosis in inflammatory bowel disease references hyperlipidemia; hyperuricemia. fracture or traumatic dislocation of hip see above under "note osteomyelitis and osteonecrosis in inflammatory bowel disease osteonecrosis and human immunodeficiency virus infection report of fifteen cases. therapeutic recommendations malignant infantile osteopetrosis: otolaryngological complications and management renal tubular acidosis and osteopetrosis with carbonic anhydrase ii deficiency: pathogenesis of impaired acidification spondylolysis in children who have osteopetrosis osteopetrosis with arnold chiari malformation type 1 and brain stem compression osteoporosis and atherosclerosis in chronic renal failure endocrine disorders and osteoporosis otosclerosis: a measles virus associated inflammatory disease correlations between pathologic changes in the stapes and conductive hearing loss in otosclerosis the aetiology of otosclerosis: a review of the literature etiologic links between chronic pancreatitis and pancreatic cancer hyperparathyroidism and pancreatitis during pregnancy pancytopenia related terms: agranulocytosis;* aplastic anemia; fanconi's anemia.* note: some causes of pancytopenia such as adverse drug reactions (e.g., due to antimetabolites, sulfa drugs thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis retinopathy as asys-temic complication of acute pancreatitis references mesenteric panniculitis necrotizing pancreatitis. * vasculitis with thrombi; adrenocortical infarctions. vasculitis with thrombi and infarctions. relapsing polychondritis (4). see also above under "possible associated conditions subcutaneous panniculitic t-cell lymphoma is a tumor of cytotoxic t lymphocytes abecassism. alpha i-antitrypsindefi-ciencyassociated panniculitis: resolution with intravenous alpha l-anti-trypsin administration and liver transplantation blind loop syndrome: an unusual cause of panniculitis. j am acad paracoccidioidomycosis synonyms: paracoccidioides brasiliensis infection cutaneous panniculitis and relapsing polychondritis: two cases normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides paraneoplastic pemphigus a case of pemphigus vulgaris with esophageal involvement rheumatoid constrictive pericarditis (clinical conference) benign paroxysmal (see "fever, familial mediterranean pneumatosis intestinalis in pediatric acquired immunodeficiency syndrome pneumatosis cystoides intestinalis: an unusual complication of systemic amyloidosis pneumatosis intestinalis in children with primary combined immunodeficiency pneumatosis cystoides intestinalis with abdominal free air in a 2-year-old girl after allogeneic bone marrow transplantation pneumatosis intestinalis: a review pneumatosis coli: a proposed pathogenesis based on study of 25 cases and review of the literature collagenous inorganic dust pneumoconiosis, diffuse type: aluminosis; asbestosis; chronic pulmonary berylliosis; talcosis; collagenous inorganic dust pneumoconiosis, nodular type: silicosis; noncollagenous inorganic dust pneumoconiosis (includes mixed-dust fibrosis): baritosis; china clay pneumoconiosis; chromite pneumoconiosis; coal worker's pneumoconiosis; fuller's earth pneumoconiosis; hematite or magnetite miner's lung; siderosis or welder's lung; stannosis; organic dust pneumoconiosis: byssinosis. note: in addition to the aforementioned classic forms of pneumoconiosis, many other airborne substances have been impli-cated in recent years, for example, cerium uncommon causes ofoccupational interstitial lung diseases rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review diagnostic criteria for chronic beryl1ium disease (cbd) based on the uk registry 1945-1991 pneumocystis carinii (see "infection, pneumocystis carinii this condition is diagnosed by inspection, palpation, and roentgenography. tension pneumomediastinum may autopsy evaluation of asbestos exposure: retrospective study of 135 cases with quantitation of ferruginous bodies in digested lung tissue atypical mycobacteriosis as a complication of talc pneumoconiosis silicosis, mixed dust pneumoconiosis, and lung cancer complications associated with the use ofthe esophageal-tracheal combitube pneumomediastinum: an unusual complication of asthma in a young man spontaneous pneumomediastinum in a patient with bronchiolitis obliterans after bone marrow references i. katzenstein a-l, myers j. idiopathic pulmonary fibrosis: clinical relevance of pathologic classification update on lymphoid interstitial pneumonitis lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis histologic diagnosis of extrinsic allergic alveolitis role of electron microscopy in interstitial lung disease detennination of arsenic in hair using neutron activation arsenic intoxication associated with tubulointerstitial nephritis atrioventricular block after administration of atropine in patients follow-ing cardiac transplantation death following intentional methyl bromide poisoning: toxicological data and literature review pulmonary edema, alveolar wall damage, and interstitial pneumonia after acute inhalation. severe pulmonary fibrosis may develop in chronic cases. gastroenteritis after nonlethal food poisoning. degeneration of proximal tubules and proteinuria in acute poisoning degenerative changes of liver and myocardium problems in the analysis of cadmium in autopsied tissues elevated urinary cadmium concentrations in a patient with acute cadmium pneumonitis cadmium-associated renal disease the effects of storage conditions on the stability of carbon monoxide in postmortem blood acute renal failure, poisoning, carbon tetrachloride synonym: tetrachloromethane poisoning. note: toxicologic sampling of body fluids and organs should be done routinely in all cases. in many instances, however, death occurs i wk to 10 d after exposure, and by this time no carbon tetrachloride is demonstrable. death may be sudden compartment syndrome, and systemic capillary leak syndrome complicating carbon monoxide poisoning southern je acute hydrocephalus following carbon monoxide poisoning prevention of occupational cyanide exposure in autopsy prosectors gradwohl's legal medicine references possible or expected findings digoxin values in digitalis toxicity are greater than 2 ng/ml (1) digoxin concentration may be higher or lower than concentration in serum, depending on how long before death drug was taken (i) digoxin concentrations in postmortem specimens after overdose and therapeutic use a fluorimetric determination ofmyocardial digoxin at autopsy, with identification of digitalis leaf, digitoxin and gitonin ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis ethylene glycol poisoning: case report of a record-high level and a review lead concentrations in human plasma, urine and whole blood trace metals (lead and cadmium screening) an emg case report oflead neuropathy 19 years after a gunshot injury aminoaciduria and glycosuria following severe childhood lead poisoning risk of nervous system cancer among workers exposed to lead demonstration of mercury in the human brain and other organs 17 years after metallic mercury exposure spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism. oral surg oral med oral pathoi1997 acute chemical pancreatitis associated with nonfatal strychnine poisoning homicide by strychnine poisoning coronary arteritis, with or without aneurysms and infarctions, primarily in childhood. myocardial hypertrophy secondary to hypertension. * minimal or no involvement by polyarteritis nodosa; considerable involvement in other types ofnecrotizing vasculitis (see "arteritis, all types or type unspecified") with or without formation of aneurysms and infarctions, may occur in all organs. the liver may show bile duct injury and rarely, nodular regenerative hyperplasia (8) more frequently involved in giant cell elastic arteritis.* polyarteritis of small muscular arteries, including vasa nervorum. arthritis* may rarely be present with swollen joints. infarctions;* subarachnoid hemorrhage; arteritis of cerebral arteries. papillitis; retinal hemorrhages polyarteritis nodosa-like vasculitis in human immunodeficiency virus infection the coexistence of familial mediterranian fever and polyarteritis nodosa: report of a case microscopic polyarteritis during polymyalgia rheumatica remission development of polyarteritis nodosa in the course of inactive systemic lupus erythematosus bone marrow pathology in relapsing polychonditis: high frequency of myelodysplastic syndrome relapsing polychondritis associated with subclinical sjo-gren's syndrome and phlegmon of the neck giant cell arteritis and polymyalgia rheumatica posterior scleritis and polymyalgia rheumatica polymyalgia rheumatica in patients with ankylosing spondylitis: a report of 5 cases inflammatory changes of hip synovial structures in polymyalgia rheumatica polymyositis (see "dermatomyositis polyneuritis (see "syndrome, guillain-barre familial, and related syndromes related terms: cowden's disease (multiple hamartoma syndrome); familial colonic polyposis gardner's syndrome; non-polyposis syndrome (hereditary nonpolyposis colorectal cancer syndrome) sampling for histologic study depends on expected findings or grossly identified abnormalities as listed in right-hand column. (see also below under "soft tissues syndrome; mucocutaneous pigmentations (buccal, perioral, priorbital, distal extremities) in peutz-jeghers syndrome;* papules in face and oral mucosa in cowden's disease; tumors of skin and subcutis (see below under "soft tissues gardner's syndrome. osteomas or exostoses (mandible, calvara desmoid in familial adenomatous polyposis malignant potential in intestinal juvenile polyposis syndromes ampullary carcinoma in familial adenomatous polyposis adenoma of the common bile duct in gardner's syndrome may cause relapsing acute pancreatitis orbital osteoma in gardner's syndrome myelopathylmyelitis," and "syndrome, guillain-barre.") references possible or expected findings hypertrichosis; erythrodontia; scarring and mutilation of hands and face. hemolytic anemia. erythrocytes contain large amounts of uroporphyrin i; nonnoblasts and reticulocytes exhibit intense red fluorescence uroporphyrin i and coproporphyrin i in high concentrations. splenomegaly (may have been treated by splenectomy) correction of congenital erythropoietic porphyria by bone marrow transplantation successful cord blood stem cell transplantation for congenital erythropoietic porphyria (gunther's disease) congenital erythropoietic porphyria associated with nephrotic syndrome atypical red cell inclusions in congenital erythropoietic porphyria possible associated conditions: adverse drug reaction; chronic alcoholism;* chronic hepatitis c (1); human immunodeficiency virus infection porphyria cutanea tarda and human immunodeficiency virus: two cases associated with hepatitis c porphyria cutanea tarda and antibodies to hepatitis c virus an unhappy triad: hemochromatosis, porphyria cutanea tarda and hepatocel1ularcarcinoma-a case report variegate porphyria possible associated conditions: ebstein's malformation of tricuspid valve. references possible or expected findings chronic eczematous skin lesions or superficial scarring; nail changes. perivascular pas-positive hyaline (2) brown protoporphyrin deposits with red to yellow birefringence with a maltese cross configuration in hepatocytes, kupffer cells, and bile canaliculi erythropoietic protoporphyria cytofluorometry as a diagnosis of protoporphyria recessive inheritance of erythropoietic protoporphyria with liver failure pseudogout synonym: calcium pyrophosphate dihydrate (cppd) deposition disease puncture grossly affected joints and submit sample of synovial fluid for crystal analysis under compensated polarized light (2) references possible or expected findings punctate calcium deposits in kneejoints and, less commonly, in joints of hips, ankles, shoulders, or wrists or in symphysis ossium pubis and intervertebral disks. arthritis* with synovitis. crystals of calcium pyrophosphate dihydrate in periarticular tissue (1) and synovial fluid. cartilage contains calcium salts of pyrophosphate, hydroxyapatite, and orthophosphate. alkaptonuria;* gout calcium pyrophosphatedihydratedeposition disease presenting as tumoral calcinosis (periarticularpseudogout) calcifications in dermis; calcifications of blood vessels. diaphragmatic hernia.* hypertensive cardiomegaly. characteristic plaques in pericardium and endocardium, with or without mitral valve involvement. coronary atherosclerosis may have been a causeofangina(2).coronarythrombosisand myocardial infarction may be present also. accelerated atherosclerosis; calcification of peripheral vessels. gastrointestinal hemorrhage;* peptic ulcer degeneration of bruch's membrane with retinal hemorrhages; sclerosis of choroid vessels calcification of elastic fibers in pseudoxanthoma elasticum new report of severe coronary artery disease in an eighteen-year-old girl with pseudoxanthoma e1asticum. case report and review of the literature hivrelated thrombotic thrombocytopenic purpura: report of 2 cases and a review of the literature beham-pyelonephritis synonyms and related terms: acute ascending pyelone angiotropic large cell lymphoma presenting as thrombotic micro-angiopathy (thrombotic thrombocytopenic purpura) splenic pathology in thrombotic thrombocytopenic purpura emphysematous pyelonephritis in diabetic patients nephrobronchialfistulaandlungabscessresulting from nephrolithiasis and pyelonephritis a case ofan enterorenal pyothorax (see "empyema, pleurai.") fistula and pyelonephritis with airin renal pelvis systemic amyloidosis secondary to pyonephrosis. resolution after nephrectomy medicolegal investigation of death the laboratory's role in detecting sexual assault toluidine blue in the detection at autopsy of perineal and anal lacerations in victimes of sexual abuse sarcoidosis presenting as heart disease sarcoidosis and its ocular manifestations rheumatic features of sarcoidosis cutaneous sarcoidosis: differential diagnosis schistosoma mansoni and s. haematobium infections in egypt. i. evaluation of techniques for recovery of worms and eggs at necropsy. am i a quantitative post-mortem study of schistosomiasis mansoni in man immunodiagnosis of schistosomiasis. screen with fast-elisa and confinn with imrnunoblot hepatic connective tissue changes in hepatosplenic schistosomiasis schistosomiasis in women: manifestations in the upper reproductive tract sclerosis, systemic amyotrophic lateral (see "disease, motor neuron diffuse (see "sclerosis, schilder's cerebrai multiple synonyms and related terms: acute and subacute variants: acute multiple sclerosis (marburg type), acute necrotizing myelopathy; concentric sclerosis (ba16 type); concentric lacunar leukoencephalopathy; encephalitis periaxialis diffusa (schilder's type; see also next entry silicone ganuloma in acral skin in a patient with silicone-gel implants and systemic sclerosis organizing diffuse alveolar damage associated with progressive systemic sclerosis bayle 0, fiessinger in. brain involvement in scleroderma: two autopsy cases skeletal muscle pathology in systemic sclerosis mechanisms of scleroderma-induced lung disease thberous sclerosis complex and subependymal giant cell astrocytoma bilateral temporal arachnoid cysts associated with tuberous sclerosis poisoning, alkaloid accident, diving [skin or scuba scurvy (see "deficiency, vitamin c shigellosis (see "dysentery, bacillary shock related terms: anaphylactic shock; bacteremic shock; many others. note: see also under name of suspected underlying condition, such as "bums intestinal histological and ultrastructura1 inflammatorychanges in spondyloarthropathy and rheumatoid arthritis the sacroiliac joint in the spondyloarthropathies granulomatous ileitis in a patient with ankylosing spondylitis sporotrichosis synonym: sporothrix (sporotrichum) schenckii infection. note: (1) mckiernan 1. partial lipodystrophy in coeliac disease coeliac disease and lymphoma: current status review: nonalcoholic steatohepatitis alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease non-alcoholic fatty liver disease in the metabolic syndrome the placenta in stillbirth estimating the time of death in stillborn fetuses: i. histologic examination of fetal organs: an autopsy study of 150 stillborns estimating the time of death in stillborn fetuses: ii. histologic evaluation ofthe placenta; a study of71 stillborns estimating the time ofdeath in stillborn fetuses: iii. external fetal examination; a study of86 stillborns fat distribution in the adrenal cortex as an indication of the mode of intrauterine death stimulant(s) (see "dependence, drug(s), all types or type undetermined cutaneous manifestations of the acquired immunodeficiency syndrome aids: cardiac findings from 115 autopsies the liver in hiv infection neuropathology of the spinal cord in the acquired immunodeficiency syndrome postmortem enzyme immunoassay for human immunodeficiency virus bk virus-associated renal failure among hiv patients -2100 or write to american registry ofpathology sales office pathology of acquired immunodeficiency syndrome (aids) in children thymus involution in the acquired immunodeficiency syndrome micrencephaly in children congenitally infected with human immunodeficiency virus-a gross-anatomical morphometric study a rapid method for detecting the predominant ashkenazi jewish mutation in the bloom's syndrome gene bloom syndrome: multiple retinopathies in a chromosome breakage disorder severe eczema in a patient with di-george's syndrome digeorge's syndrome orthe iii -iv pharyngeal pouch syndrome: pathology and a theory of pathogenesis basal ganglia calcification and psychosis in 22q 11.2 deletion syndrome down's synonyms and related terms: mongolism possible associated conditions: acute lymphocytic, my references epicanthal folds; cleft lip/palate; high arched palate; furrowed tongue; rhagades. depressed nasal bridge small, broad or fiat nose; slanted palpebral fissures; fiat occiput; brachycephaly; palmar "simian crease"; short fifth middle finger ventricular septal defect;* complete atrioventricular septal defect* (1). duodenal stenosis and atresia; imperforate anus. microcephaly; poorly developed secondary gyri hypoplastic brain stem, medulla and cerebellum; polymicrogyria; neurofibrillary tangles; meningomyelocoele. acute lymphocytic leukemia; acute myelocytic leukemia; acute megakaryocytic leukemia cardiovascular disease in down syndrome possible or expected findings hyperelasticity of skin, bruises or scars, hemorrhages, and hyperpigmentation. lipomatous pseudotumors that may be calcified or ossified. normal or abnormal amounts and fragmentation of elastic tissue. abnormalities of collagen. dislocation of hip, shoulder, patella, radius, or clavicle. loose-end clavicles; spondylolisthesis mitral valve prolapse; aortic insufficiency.* aortic dissection, aneurysm, ectasia, occlusion (1). various congenital anomalies. congenital anomalies of gastrointestinal and genitourinary tracts. bleeding from various organ sites vascular ehlers-danlos syndrome: imaging findings note: this syndrome belongs to a large family (with more photograph abnormal features as listed in right-hand column. record appearance of external genitalia. prepare skeletal roentgenograms. procure long bones and vertebral bodies short-limb dwarfism* with narrowing of the rib cage; polydactyly; dysplasia of fingernails; thin and sparse hair; premature eruption of teeth; defective dentition; eye abnormalities; upper lip bound down by multiple frenula. cryptorchidism; epispadias; hypospadias. chondrodysplasia with acromelic micromelia (shortening of the distal segment of the limb); fusion of capitate and hamate bones of wrist; defects of lateral aspect of proximal tibia see lesions listed above under "external examination empty sella synonyms and related terms: primary empty sella syndrome; secondary empty sella syndrome (e.g., after surgical removal or spontaneous infarction of pituitary adenoma) chorea, philic pneumonia: histopathologic features in nine cases. am rev resp hereditary ascariasis and hookworm chronic eosinophilic pneupophosphatemic vitamin d-resistant rickets; galactosemia;transport defect; tyrosinemia. * excludes fan-1988 weight and external measurements; record and photograph abnormalities as listed in right-hand column. prepare skeletal roentgenograms. radiographs of long bones. submit for tissue culture for possible enzyme analysis. possible or expected findings redlblond hair, fair skin (diminished pigmentation); dehydration;* stigmata of hypothyroidism;* delay of sexual maturation. rickets; osteomalacia.* positive rheumatoid factor. various types of pneumonia after splenectomy, presence of accessory spleen may account for treatment failure. manifestations of portal hypertension.* lymphadenopathy of mediastinal and paraaortic lymph nodes. anemia;* neutropenia sinusoidal lymphocytosis of the liver in felty's syndrome with a review of the liver involvement in felty's syndrome splenic involvement in rheumatic diseases felty's and pseudo-felty's syndrome nodular regenerative hyperplasia of the liver in rheu-matic diseases: report of seven cases and review of the literature fetal alcohol syndrome: craniofacial and central nervous system manifestations goodpasture's syndrome anti-glomerular basement membrane glomerulonephritis: a morphologic study of 80 cases gronblad-strandberg (see "pseudoxanthoma elasticum acute inflammatory polyradiculoneuropathy; guillain-barre-strohl syndrome hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: pathophysiology and treatment cyclosporin-induced hemolytic uremic syndrome: factors that obscure its diagnosis enterohemorrhagic escherichia coli 0157:h7: an emerging pathogen hemolytic uremic syndrome as a presenting form of hiv infection hemolytic uremic syndrome in prostatic carcinoma diabetes secondary to genetic disorders. baillieres suprasellar tumors of maldevelopmental origin in klinefelter's syndrome. a report of two cases klippel-feu synonym: congenital fusion of cervical vertebrae primary yolk sac tumor of the prostate in a patient with klinefelter's syndrome extragonodal germ cell tumors are often associated with klinefelter syndrome organs and tissues procedures possible or expected findings external examination prepare roentgenograms of chest, neck, and head skull, spine, brain, and spinal cord myasthenia gravis disorders with dysraphia (see below), fusion of cervical vertebrae. congenital elevation of the scapula (sprengel's deformity) chiari malformation;* basilar impression;* meningomyelocele; platybasia;* spinal cord compression; weight and length; record and photograph abnormalities as listed in right-hand column. record weight and sample for histologic study. if renal transplantation (3) had been carried out, see also under that heading. follow procedures described under "glomerulonephritis retinal dystrophy (1) and other retinal changes the cardinal manifestations of bardet-biedl syndrome, a form of laurence-moon-biedl syndrome laurence-moon-biedl syndrome accompanied by congenital hepatic fibrosis pediatric renal transplantation in laurence-moon-biedl syn-drome possible or expected findings typical skeletal proportions. arachnodactyly; pectus excavatum genu recurvatum; dislocation of joints diaphragmatic hernia. * infective endocarditis. * atrial septal defect. * myxomatous transformation of mitral ring. mitral valve prolapse. aortic and pulmonary dilatation and valvular insufficiency.* aortic dissection* with dissection of adjacent vessels. ascending aortic aneurysm multiple cysts (see "cyst(s), pulmonary") aortopulmonary fistula: an uncommon complication in dystrophic aortic aneurysm peripartum acute myocardial infarction in marfan's syndrome aneurysm of the cervical internal carotid artery associated with marfan's syndrome--ease report noonan's syndrome in association with acute leukemia lymphatic dysplasia in a neonate with noonan's syndrome noonan syndrome: a clinical description emphasizing the cardiac findings cerebral arteriovenous malformation in noonan's syndrome clinical variability in a noonan syndrome family with a new ptpnii gene mutation peutz-jeghers syndrome bilateral large-cell calcifying sertoli cell tumor of the testes in peutz-jeghers syndrome: a case report ovarian tumors associated with the peutz-jeghers syndrome robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome 4g33-gter congenital heart disease in the pierre robin syndrome glossoptosis-apnea syndrome chiari i malformation, caudal regression syndrome, and pierre robin syndrome: previously unreported combination risk factors for squamous cell carcinoma of the esophagus heterotaxy syndrome; ivemark's syndrome possible associated conditions: with asplenia: right isomerism (bilateral mirror-image right-sided symmetry) of heart, lungs, and abdominal viscera; common atrium; total anomalous pulmonary venous connection; absent coronary sinus; complete atrioventricular septal defect;* subpulmonary stenosis;* pulmonary valve atresia;* midline symmetric liver; malrotation of bowel; absent spleen. with polysplenia: left isomerism (bilateral mirror-image left-sided symmetry) ofheart and lungs, with variable sidedness ofabdominal viscera primary immunodeficiencies reiter's syndrome-like patternin aids-associated psoriasiformdermatitis reference pneumothorax;* pneumomediastinum;* pneumoperitoneum. septicemia. right ventricular hypertrophy and/or dilatation. intubation trauma and mural edema and hemorrhage in trachea; hyaline membrane disease intubation trauma. hemorrhages or other evidence of birth trauma; germinal matrix hemorrhages in premature infants; infarctions of subcortical white matter in term infants coalson 11. pathology of bronchopulmonary dysplasia pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia inborn errors of metabolism and reye's syndrome: differential diagnosis prevalence and non-specificity of microvesicular fatty changes liver histopathology in clinical reye syndrome ultrastructural study of intranuclear inclusions in the exo-crine pancreas in reye's syndrome neuropathologic findings in reye syndrome sanfilippo's (see "mucopolysaccharidosis scheie's (see "mucopolysaccharidosis segawa's (see "syndrome, dystonia severe acute respiratory (sars) note: this highly contagious illness, caused by a coronavirus (sars-cov)details, see biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects pathology and pathogenesis of severe acute respiratory syndrome pulmonary involvement in primary sjogren's syndrome polymyositis and sjogren's syndrome associated with bronchiolitis obliterans organizing pneumonia the gastrointestinal manifestations of sjogren's syndrome clinicopathological factors relating malignant lymphoma with sjogren's syndrome acute transverse myelopathy and cutaneous vasculopathy in primary sjogren's syndrome primary localized cutaneous amyloidosis with unusual clinical features in a patient with sjogren's syndrome disease, parkinson's erythema multiforme man related terms: armadillo disease; continuous muscle fiber activity; neuromyotonia autoimmune central nervous system paraneoplastic disorders: mechanisms, diagnosis, and therapeutic options sudden infant death (sids) (see "death, sudden unexpected, of infant.") syndrome, superior vena cava related term: superior vena cava obstruction continue dissection of veins into neck and axillas. record and photograph site of thrombosis or of compression by surrounding pathologic conditions. submit samples for histologic study. benign or malignant tumors; fibrosing mediastinitis;* postradiation fibrosis; infectious disease (tuberculosis,* histoplasmosis*); thoracic aortic aneurysm;* chronic constrictive pericarditis;* chest trauma; arteriovenous fistula between ascending aorta and superior vena cava toxic shock synonyms and related terms: staphylococcal scarlet fever. note: (1) collect all tissues this is a reportable disease. premature aging; increased number of pigmented nevi; infantile sex organs; webbed neck; broad chest with wide spacing of nipples. short fourth metacarpal; abnormal epiphyseal fusions; osteochondrosis-like changes of spine bicuspid aortic valve;* coarctation of aorta. * rarely other anomalies (1) decreased/absent follicles. intestinal telangiectases. biliary atresia. * horseshoe kidney; double ureters. streak gonads without germ cells or follicles hashimoto's thyroiditis. * manifestations of diabetes mellitus,* hypertension,* or thyrotoxicosis. neuroblastoma and related tumors (2). keratoconus acute aortic dissection, aortic insufficiency, and a single coronary artery in a patient with turner's syndrome neuroblastoma and related tumors in turner's syndrome turner's syndrome associated with bilateral retinal detachments weil's (see "leptospirosis related terms: alcoholic wernicke's encephalopathy; korsakoff's psychosis; nonalcoholic wernicke's encephalopathy (1,2); and specimen preparation, see chapter 4. for selection of histologic samples, see right-hand column melissas 1. wernicke's encephalopathy after vertical banded gastroplasty for morbid obesity syndrome, respiratory distress, of infant.") syndrome, wiskott-aldrich (see "syndrome, primary immunodeficiency malformation, ebstein's" and "preexcitation, ventricular alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease cerebro-hepato-renal syndrome; infantile refsum's disease. * note: this congenital familial cholestatic syndrome results from impaired assembly of peroxisomes and has its main manifestations in the brain, liver, and kidneys postmortem findings and prenatal diagnosis ofzellwegersyndrome. case report alcoholism and alcohol intoxication" and "disease, alcoholic liver possible associated condition: multiple endocrine neoplasia primary cardiac gastrinoma causing zollinger-ellison syndrome localization, syphilis, acquired synonym: treponema pallidum infection. note: congenital syphilis is presented below under a separate heading and surgical management of gastrinoma fundic argyrophil carcinoid tumor in a patient with sporadic-type zollinger-ellison syndrome nonimmune hydrops fetalis due to congenital syphilis associated with negative intrapartum mater-nal serology screening syringomyelia synonyms and related term: hydromyelia; idiopathic syringomyelia; secondary syringomyelia; syringobulbia. possible associated conditions: with idiopathic syringomyelia-arnold chiari malformation, type i;* basilar impression;* 2. oppenheimer eh, dahms bb. congenital syphilis in the fetus and neonate congenital syphilis: detection of treponema pallidum in stillborns with secondary syringomyelia-intramedullary gliomas (ependymoma, pilocytic astrocytoma) and vascular tumors; spinal arachnoiditis and pachymeningitislisted in right-hand column. prepare roentgenograms of spine and joints. for removal and specimen preparation, see chapter 4. for histologic sampling, see right-hand column. hypertrophy of body parts. muscle atrophy of upper extremities and hands cervical spinal cord is swollen and tense, with cavitation (syrinx) containing clear fluid. wall of cavity consists of degenerated glial and neural elements, with marked gliosis. spinal cord parenchyma is markedly compressed. see also above under hydrops fetalis caused by alpha-thalassemia: an emerging health care problem limb defects in homozygous alpha-thalassemia: report of three cases poisoning, thallium thirst (see "dehydration thromboangiitis obliterans (see "disease, buerger's purpura, thrombotic thrombocytopenic" and "syndrome, hemolytic uremic the pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome a spectrum in the pathology of toxoplasmosis in patients with acquired immu transfusion (see "reaction to transfusion bone marrow note: if the patient had symptoms of acute or chronic graft-versus-host disease (gvhd), see "disease, graftversus-host morphology and diagnostics of human toxoplasmosis fatal myocardial coinfection by toxoplasma gondii and parvovirus b 19 in an hiv patient if there is evidence of infection, submit material for microbiologic studies. if there is evidence of recurrent leukemia or lymphoma, sample as described under those headings. if the patient had symptoms of thrombotic thrombocytopenic purpura (tip) or hemolytic uremic syndrome (hus), see these headings. record average size. fix specimens in b-plusâ®. make touch preparations. request giemsa or wright stain. for preparation of sections and smears (imprints), see chapter 2. manifestations of graft-versus-host disease (e.g., scleroderma-like changes).* septicemia. interstitial pneumonia* and cytomegalovirus infection* or human herpesvirus 6 infection (1). bacterial, fungal bacterial or fungal infections in gvhd.* other manifestations of gvhd. * recurrent leukemia,* lymphoma* (including posttransplant, epstein-barr virus associated lymphoproliferative disorder). recurrent solid tumor, e.g., carcinoma of breast, lung (small cell carcinoma), ovary lymphomatous or leukemic infiltrates myeloid cells may be absent in marrow graft rejection or nonimmunologic marrow graft failure. references hematomas; hemorrhagic necroses; infarcts; bacterial or fungal infections leukoencephal-opathy; vascular siderocalcinosis; neuro-axonal spheroids (6) human herpesvirus 6 infection and associated pathogenesis following bone marrow transplantation pulmonary veno-occlusive disease in an adult following bone marrow transplantation. case report and review of the literature acute renal failure following bone mar-row transplantation transplantation-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome thrombotic microangiopathies associated with drugs and bone marrow transplantation record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record weight, ventricular thickness, and valve circumferences. take multiple sections from all areas of myocardium. cut coronary arteries in cross sections; request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. left ventricular hypertrophy, from cyclosporine-related hypertension. acute transplant rejection (for grading, see ref. 1). chronic transplant vasculopathy with coronary artery stenosis (2) opportunistic infection (1). pneumonia a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group autopsy findings in cardiac transplant patients: a lo-year experience fulminant toxoplasmosis following heart transplantation banff schema for grading liver allograft rejection: an international consensus document record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record lung weights separately. if lung infection is suspected, submit material for microbiologic possible or expected findings cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. opportunistic infection (in patients with a single lung transplant, infections may involve references the nontransplant lung, as well). chronic bronchitis* and bronchiectasis.* acute rejection (rare); chronic airway rejection (obliterative bronchiolitis); chronic vascular rejection. (for grading of rejection, see ref 1.) post-transplant lymphoprolijerative disorder a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group recurrent salmonella enteritidis and hepatic tuberculosis tularemia synonyms and related terms: francisella tularensis infection; pasteurella tularensis infection cerebral abscesses complicating tularemia meningitis endocrine (see "neoplasia, multiple endocrine any type note: ifthe tumor had been treated by surgery, irradiation, chemotherapy, or other means (the most common situation possible associated conditions: with adrenocortical adenoma-conn's syndrome with adrenocortical carci-noma-cushing's syndrome;* hypoglycemia;* virilization. with pheochromocytoma-cerebellarhemangioblastoma ery-throcytosis; hypercalcemia; hypertension,* multiple enarteries record body weight and abnormal features. photograph skin changes. record heart weight and thickness of ventricles. procure multiple sections of myocardium possible associated conditions.") focal myocarditis. * hypertensive cardiovascular disease (see "hypertension myocardial infarction without severe coronary artery disease (with pheochromocytoma) asystematicreviewoftheassociationbetween barrett's esophagus and colon neoplasm malakoplakia and colorectal adenocarcinoma an unusual case of colonic mixed adenoendocrine carcinoma: collision vs composite tumor. a case report and review of the literature submit lymph nodes for histologic study. remove neck organs with tongue together with esophagus. open pharynx and esophagus in posterior midline. if fistulas and abscesses are suspected, dissect esophagus but leave attached to mediastinum, stomach and diaphragm. record width of lumen of esophagus at various levels. photograph and record size and location of tumor; sample tumor and uninvolved esophagus for histologic study. request pas stain of uninvolved esophagus (sample from all levels). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. eczematoid dermatitis with adult renal cell carcinoma. cushing's syndrome,* galactorrhea or feminization or masculinization in some cases of renal cell carcinoma. evidence of hyperalcemia. pulmonary tumor embolism after invasion of renal vein and inferior vena cava. acquired renal cystic disease prolactin, renin, and prostaglandins in some renal cell carcinomas. see above under "possible associated conditions wilms tumor associated with polycythemia: case report and review of the literature renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents acquired cystic kidney disease byler's disease, carcinoid syndrome,*cirrhosis* of any type (mostly nonbiliary), congenital hepatic fibrosis, * cushing's syndrome,* erythrocytosis, genetic hemochromatosis, * glycogen storage disease (type 1),* hepatitis b or c virus infection, hereditary tyrosinemia (type i),* hypercalcemia, hypercholesterolemia, hypoglycemia,* neurofibromatosis,* osler-rendu-weber disease* (hereditary hemorrhagic telangiectasia), polycythemia,* porphyria (acute intermittent and porphyria cutanea tarda), * pseudohyperparathyroidism,* and thorium (thorotrast) deposition. with bile duct carcinoma (cholangiocarcinoma}-clonorchis sinensis or opisthorchis viverrini infection, fibropolycystic liver and biliary tract disease,* hepatolithiasis, hypercalcemia, pri-mary sclerosing cholangitis,* and thorium (thorotrast) deposition. with hepatoblastoma-alpha-fetoproteinemia, cardiac and renal malformations, cleft palate, diaphragmatic hernia,* down's syndrome,* familial colonic polyposis,* hemihypertrophy, nephroblastoma. with angiosarcoma-thorium (thorotrast) deposition. see also below under "possible or expected findings describe and photograph cut surfaces. sample tumor and nonneoplastic liver for histologic study. if thorium deposition is suspected, prepare roentgenograms of liver slices and submit samples for energy-dispersive x-ray microanalysis of paraffin sections. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. cachexia. precocious puberty. feminization and gynecomastia in rare cases of hcc. spider angiomas; clubbing of fingers. see above under "possible associated conditions thorotrast storage may cause cirrhosis, hcc, bile duct carcinoma, or angiosarcoma. see above under "possible associated conditions tumor of the lung or bronchus possible associated conditions: abnormal concentrations of hormons or other metabolites in blood and tumor tissue (adrenocorticotropic, antidiuretic, growth hormone, parathyroid-like substances, or 5-hydroxyindolacetic acid); acanthosis nigricans s syndrome;*dermal hyperpigmentation; feminization; hyperglycemia; hypercalcemia; hypoglycemia;* hypokalemia; hyponatremia; precocious puberty. for syndromes affecting the brain, peripheral nerves or muscles, see below under "possible or expected findings organs and tissues procedures possible or expected findings external examination and skin record body weight. record abnormal features as listed in right-hand column; prepare photographs. prepare histologic sections of normal and grossly abnormal skin clubbing of fingers; spider angiomas. for other rare tumor-related changes, see above under "possible associated conditions thmor of the pancreas possible associated conditions: with carcinoma of the exocrine pancreas--cushing's syndrome;* diabetes mellitus* (rare); hypercalcemia; hyperglycemia with islet cell tumor-abnormal concentrations of hormons in blood and tumor tissue (see below under "pancreas"); features. dermal hyperpigmentation. for other rare tumor-related changes, see above under "possible associated conditions biliary obstruction caused by tumor in head of pancreas. islet cell tumor with adrenocorticotropic hormone, gastrin, glucagon, insulin, or other peptide hormones. see above under "possible associated conditions paraneoplastic pemphigus associated with pancreatic carcinoma pancreatic cystadenocarcinoma in peutz-jeghers syndrome testes may have been surgically removed to achieve androgen deprivation. urinary obstruction and hydronephrosis. * osteoblastic metastases hemolytic uremic syndrome in prostatic carcinoma thmor of the small intestine possible associated conditions: acquired immunode oncogenic osteomalacia associated with prostatic cancer gardner's syndrome) submit sample of non-neoplastic small bowel for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above. cachexia. mucocutaneous pigmentations associated with peutz-jeghers syndrome. * carcinoid tumor. lymphoma (see also above under "possible associated conditions"). familial polyposis or related syndrome. * celiac sprue. * crohn's disease. * see above under "possible associated conditions kasabach merritt syndrome in infants paraneoplastic hepatopathy associated with soft tissue sarcoma neurofibro-thmor of the spinal cord possible associated conditions: with angioma-cerebellar hemangioblastoma; segmental cutaneous vascular nevi; with matosis in children with soft tissue sarcoma tissues record abnormal features; photograph and submit nevi for histologic study. for removal and specimen preparation, see chapter 4. see also below under "vertebral column bone erosion and calcification of spinal canal. vertebral hemangiomas may be associated with arteriovenous malformation of spinal cord. manifestations of von hippel-lindau disease. * thmor of the stomach possible associated conditions: hypoglycemia;* megaloblastic anemia;* skin changes (as listed under "possible or expected findings)weight. record abnormal features; photograph and submit samples of normal and abnormal skin for histologic study. possible or expected findings cachexia; lower leg lymphoedema (3) metastases common in liver, retroperitoneal and supraclavicular lymph nodes (virchow's node), ovaries (krukenberg tumor), and peritoneal cui de sac (blumer's shelf') carcinoma of the stomach with hyperkeratosis palmaris and plantaris and acanthosis of the esophagus gastric lymphoma ofmucosa-associated lymphoid tissue and helicobacter pylori gastric signet-ring cell carcinoma: unilateral lower extremity lymphoedema as the presenting feature thmor of the testis possible associated conditions: demyelinating neuropathy record location, size, and weight of both testes and of testicular tumor. submit samples of tumor and of ininvolved testis and epididymis for histologic study. snap-freeze tumor tissue for hormone assay. possible or expected findings cachexia. cryptorchism; hypospadia. gynecomastia. increased concentrations of alphafetoprotein and human chorionic gonadotropin testicular microlithiasis. secondary testicular tumors (metastases to the testes) are rare, except in association with leukemia* in children. references pulmonari embolism.* paraneoplastic diseases as listed above under "possible associated conditions chronic inflammatory demyelinating polyneuropathy (clop) associated with seminoma dermatomyositis associated with intratubular germ cell tumor and metastatic germ cell cancer testicular cancer in association with developmental renal anomalies and hypospadias submit samples of lymph nodes, spleen, peyer's plaques, and bone marrow for histologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. dermal hyperpigmentation. pemphigus foliaceus (rare). hypogammaglobulinemia and other immunoglobulin abnormalities. usually, thymoma presents as an infiltrating, anterior mediastinal mass that rarely metastasizes. carcinoid tumor, malignant lymphoma* (hodgkin's disease), and metastases from carcinomaofthe breast* and other tumors also may occur in this location herpes simplex*) and fungal infections (e.g., candidiasis*) due to thymoma-related immunodeficiency (3). manifestations of paraneoplastic diseases and conditions as listed above under "possible associated conditions thrombotic thrombocytopenic purpura accompanied by transient pure red cell aplasia and thymoma glomerulonephritis associated with myasthenia gravis thymoma and cellular immune deficiency in an adolescent possible associated conditions: with papillary carcinoma-familial adenomatous polyposis thyroid carcinoma associated with familial adenomatous polyposis an association between acromegaly and thyroid carcinoma thyroid carcinoma causing fatal laryngeal obstruction prostate cancer metastasis to thyroid gland schistosomiasis and the risk of bladder cancer in human papilloma virus infection (2) or herpesvirus infection (3) with invasive cervical carcinoma. erythropoietin may be found in some tumors. thrombosis. * myopathy* may be associated with carcinoma of the cervix. cerebellar cortical degeneration* may be associated with carcinoma of the uterus. manifestations of uremia (see "failure, kidney") invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients association of risk factors for cervical cancer and human papilloma viruses in invasive cervical cancer association of herpesvirus infection with the development of genital cancer tyrosinemia type ii: a challenge for ophthalmologists and dermatologists hereditary tyrosinemia type i (chronic form): pathologic findings in the liver richner-hanhart syndrome (tyrosinemia ii): early diagnosis of an incomplete presentation with unusual findings acute pancreatitis-associated acute gastrointestinal mucosal lesions: incidence, characteristics, and clinical significance uncinariasis (see "ancylostomiasis uremia (see "failure, kidney from: handbook of autopsy practice urticaria pigmentosa of childhood (see ''mastocytosis, systemic acute aortic dissection;* aneurysma(s) of cerebral arteries; aortic insufficiency;* calcific aortic stenosis,* coarctation of the aorta;* infective endocarditis;* shone's syndrome; turner's syndrome. * organs and tissues procedures possible or expected findings heart if infective endocarditis is suspected, see chapter 7. open heart in cross-sections (see chapter 3). photograph aortic valve and test valvular competence note: reye's syndrome* is a possible postviral complication of varicella that must be distinguished from varicella encephalitis. varicella may also cause exacerbation of tuberculosis.* (i) collect all tissues that appear infected bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions varicella-zoster virus infection in children with underlying immunodeficiency virus infection group a beta-hemolytic streptococcal epiglottitis as a complication of varicella infection optic neuritis: a rare complication of primary varicella infection hsv-l-induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement varicella with acute motor axonal neuropathy descending necrotizing mediastinitis in a child with chicken pox double outlet right ventricle with intact ventricular septum respiratory syncytial (see "pneumonia, all types or type unspecified vitamin a (see ''deficiency, vitamin a" and "hypervitaminosis a vitamin bt (thiamine) (see "syndrome, wernicke-korsakott vitamin bt2 (see "anemia, megaloblastic deficiency deficiency, vitamin d" and "hypervitaminosis d waldenstrom's macroglobulinemia (see ''macroglobulinemia, waldenstrom's.) waterhouse-friderichsen syndrome (see "disease, meningococcal wegener's granulomatosis (see "granulomatosis, wegener's werdnig-hottman disease (see "disease, motor neuron acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate which may be secondarily infected by bacteria. dark field microscopy will identify the organism expected findings mucosal ulcers, resembling those of typhoid fever, primarily of distal ileum and colon. villous shortening, crypt hyperplasia with mucosal microabscesses. microabscesses in regional mesenteric lymph nodes zellweger's syndrome (see "syndrome mucormycosis" and procedures under "diabetes mellitus abscesses and granulomas may occur in all organs and tissues. granulomatous lesions in central nervous system (4) and eyes (5). fungal osteomyelitis* that may be multifocal, including sites such as metacarpals and metatarsals. external examination other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. osteosclerosis of vertebrae, ribs, clavicles, pelvic bones, scapulae, skull, and metaphyseal ends of femur and humerus. osteomyelofibrosis or osteoreticulosis; rarely, panhyperplasia of bone marrow; increase of megakaryocytes.changes associated with chronic granulocytic leukemia* or with polycythemia vera* may imitate idiopathic myelofibrosis. widespread extramedullary hematopoiesis (1) with splenomegaly. infectious diseases, including tuberculosis;* gouty arthritis; ascites and other manifestations of portal hypertension;* or hepatic vein thrombosis (budd-chiari syndrome*). cardiac tamponade (1) . synonyms and related terms: hepatic porphyria; protoporphyrinogen oxidase deficiency (1) .note: the manifestations of the disease closely resemble those in porphyria cutanea tarda and hereditary coproporphyria. measurements of porphyrins and porphyrin precursors are the only clearly distinguishing features. for autopsy procedures, see under "porphyria cutanea tarda." fibrin and platelet thrombi, with or without microaneurysms and purpura in kidneys, adrenal glands, pancreas, heart, and brain; lesions may also be present in liver; spleen (3), lymph nodes, muscle, bone marrow, and synovium. fibrin thrombi can be demonstrated with labeled antihuman fibrin antibodies. lesions in precapillary arterioles. hypertrophic osteoarthropathy* (with pleural mesothelioma).pleural effusions. * asbestosis may be complicated by pleural mesothelioma. other organs and tissue sample bladder tumor and uninvolved urinary bladder for histologic study.cachexia. hydronephrosis* (obstructive uropathy) and hydroureter, usually caused by distal ureteral obstruction. recurrent nephrolithiasis* or pyelitis may have been present and is a risk factor for urinary bladder carcinoma.chronic urocystitis; infestation with schistosoma haematobium (1) (uncommon in north america). manifestations of uremia (see "failure, kidney"). key: cord-019063-mcxbl8mv authors: vijayan, vannan k. title: diagnosis of pulmonary parasitic diseases date: 2013-06-05 journal: parasitic diseases of the lungs doi: 10.1007/978-3-642-37609-2_1 sha: doc_id: 19063 cord_uid: mcxbl8mv the protozoal and helminthic parasites that traverse the respiratory tract during their life cycles can cause lung diseases, though the most common habitats of these parasites are the gastrointestinal tract and the blood or lymphatic circulations. these diseases are commonly encountered in the tropical regions of the world. however, parasitic lung diseases are increasingly being reported from other parts of the world due to an increase in the occurrence of immunosuppression (acquired immunodeficiency syndrome, organ transplantations, the use of immunosuppressive drugs) and transcontinental travel. the lung diseases that may result from these infections range from asymptomatic phase to life-threatening acute respiratory distress syndrome. these diseases can also mimic common respiratory diseases such as bacterial pneumonias, pulmonary tuberculosis, lung cancer, bronchial asthma, interstitial lung disease, and pulmonary hypertension. the diagnosis of parasitic lung diseases is a challenge to physicians, if they are not aware of the entity or these diseases are not investigated properly. the diagnosis of these diseases is based on the identification of the causative organism in the stool, sputum, other body fluids, or tissue specimens. radiological imaging studies of the thorax including chest radiographs, high-resolution computerized tomograms, and ultrasonograms may aid in the diagnosis. in certain situations, invasive investigations such as fiberoptic bronchoscopic evaluation (transbronchial lung biopsies and bronchoalveolar lavage studies) and thoracic surgical procedures (thoracoscopy and open lung biopsy) may be required for a diagnosis and also to exclude other lung diseases. serologic and molecular diagnostic methods are being developed for accurate diagnosis of the parasitic diseases. pulmonary parasitic lung diseases are commonly diagnosed in countries where the prevalence of parasitic infection is high. however, there is an increase in the number of patients diagnosed as parasitic lung diseases recently, even in countries of low prevalence of parasitic infection which demands an awareness of such diseases in these countries. this increase in diagnosis in countries with low prevalence with parasitic infections has been attributed to an increase in the numbers of immunosuppressed individuals due to various reasons, organ transplantations and global travel [ 1 ] . the parasites can cause a wide spectrum of lung diseases varying from mild self-limiting bronchitis to life-threatening acute respiratory distress syndrome [ 2 ] . in addition, parasitic lung diseases may mimic diseases such as bacterial pneumonias, pulmonary tuberculosis, bronchial asthma, lung cancer, interstitial lung disease, and pulmonary hypertension. both protozoal and helminthic parasites can cause lung diseases and helminthic lung infections are important causes of eosinophilic lung diseases [ 3 ] . though the treatment of parasitic lung diseases is with specifi c antiparasitic drugs, the physicians treating such diseases should be competent in tackling the specifi c issues related to lung injury and sequel that may follow such infections. the lung diseases that may result from infections with parasites are listed in table 1 .1 [ 4 ] . the important protozoal parasites that cause pulmonary diseases are entamoeba histolytica , leishmania donovani , malarial parasites ( plasmodium vivax , p . falciparum , p . malaria , p . ovale , and p . knowlesi ), babesia spp. ( babesia microti and babesia divergens ), and toxoplasma gondii . amebiasis results from ingestion of mature entamoeba histolytica cysts in fecally contaminated food, water or from hands. infection with entamoeba histolytica can lead to intestinal colonization, colitis or extraintestinal manifestations resulting from the hematogenous spread of infection from the intestine. about 90 % of intestinal colonization is with nonpathogenic species, entamoeba dispar and entamoeba moshkovskii . invasive amebiasis occurs in 10 % of persons colonized with e . histolytica . patients with amebic colitis present with several-week history of cramping abdominal pain, weight loss, and watery or bloody diarrhea. extraintestinal amebic infection can manifest as amebic liver abscess, splenic abscess, brain abscess, empyema, and pericarditis. nearly 80 % of cases of amebic liver abscesses occur in the right lobe. the most common complication of amebic liver abscess is rupture into the pleural space resulting in pleuropulmonary amebiasis [ 5 ] . the main symptoms in pleuropulmonary amebiasis are fever, cough, hemoptysis, right upper quadrant abdominal pain, and chest pain. some patients may present with respiratory distress and shock. lung abscess, hepatobronchial fi stula, and bronchopleural fi stula with pyopneumothorax have also been reported. expectoration of anchovy saucelike pus indicates amebiasis [ 6 ] . the fi ndings of elevated hemidiaphragm, tender hepatomegaly, pleural effusion, and basal pulmonary involvement are suggestive of pleuropulmonary amebiasis. amebiasis can be suspected in patients with a history of immigration from or travel to developing countries and many patients give history of dysentery and alcoholism. amebiasis is commonly diagnosed by microscopy and cysts or motile trophozoites can be identifi ed on a saline wet mount of a stool specimen [ 7 ] . microscopic examination of fresh stools, sputum or pleural pus, rectal smears or rectosigmoidoscopy materials, pus from liver abscesses, and colonic biopsy samples may reveal motile trophozoites, even though it is a relatively specifi c test but is not sensitive for the identifi cation of e . histolytica . the presence of ameba in the stool does not indicate that the disease is due to pathogenic e . histolytica as two other nonpathogenic species found in humans ( e . dispar and e . moshkovskii ) are indistinguishable morphologically [ 8 , 9 ] . this method can, therefore, give false-positive results if entamoeba dispar or entamoeba moshkovskii infection is present. however, it has been reported recently that several different genotypes of e . dispar can be potentially responsible for tissue damage similar to that observed with e . histolytica [ 10 ] . a nonpathogenic entamoeba gingivalis which is present in the oral cavity has to be differentiated from entamoeba histolytica in sputum samples. a combination of serological tests with identification of the parasite by antigen detection by pcr is the best approach to diagnosis. in vitro culture by inoculation of portions of stool, liver abscess, or empyema fl uid into sterile culture media and incubating it at 37 °c is also useful in the diagnosis of amebiasis. the culture media is examined for the growth of amebic trophozoites, which, if present, can be seen on the walls of test tubes or in debris [ 11 ] . antibody detection and antigen detection are other important immunodiagnostic methods. indirect hemagglutination (iha) test is used for routine serodiagnosis of amebiasis. antigen consists of a crude soluble extract of axenically cultured organisms. the enzyme immunoassay (eia) test detects antibody specifi c for e . histolytica in approximately 95 % of patients with extraintestinal amebiasis, 70 % of patients with active intestinal infection, and 10 % of asymptomatic persons who are passing cysts of e . histolytica . detectable e . histolytica -specifi c antibodies may persist for years after successful treatment, so the presence of antibodies does not necessarily indicate acute or current infection. specifi city is 95 % or higher and false-positive reactions rarely occur. e . histolytica -specifi c antigen detection may be useful as an adjunct to microscopic diagnosis in detecting parasites and to distinguish between pathogenic and nonpathogenic infections. detection of circulating antigens in the serum has been found to be an important advancement in the diagnosis [ 12 ] . polymerase chain reaction (pcr) assays are useful for the differentiation of e . histolytica , e . dispar , and e . moshkovskii and for genetic typing of isolates [ 13 , 14 ] . however, these tests are time-consuming and expensive and, hence, are not practical in areas endemic for amebiasis. infection with leishmania donovani causes visceral leishmaniasis and is transmitted by various species of phlebotomus , the sand fl y [ 15 ] . there are no clinical symptoms and signs that are pathognomonic of visceral leishmaniasis (vl) or kala-azar. the symptoms and signs suggestive of visceral leishmaniasis are irregular fever, weight loss, enlargement of liver and spleen, and anemia. pneumonitis, septal fi brosis, pleural effusion, and mediastinal adenopathy are reported in patients coinfected with human immunodeficiency virus (hiv) [ 16 ] . leishmaniasis has also been reported in lung transplant patients [ 17 ] . leishmania amastigotes can be found in the alveoli, pulmonary septa, and bronchoalveolar lavage (bal) fl uid. diagnosis of leishmaniasis is by the demonstration of the parasites in bone marrow aspirates and by the identifi cation of specifi c dna sequences in tissues by molecular biology techniques [ 18 ] . the differential diagnosis of leishmaniasis includes malaria, cirrhosis with portal hypertension, miliary tuberculosis, brucellosis, histoplasmosis, lymphoma, and leukemia [ 19 ] . the diagnosis of leishmaniasis is based on the microscopical demonstration of leishmania amastigotes in the relevant tissue aspirates or biopsies such as bone marrow, spleen, lymph nodes, or liver, skin slit smears, or in the peripheral blood buffy coat [ 19 ] . the smears can be stained with romnowsky's, hematoxylin-eosin, or immunoperoxidase stains. the amastigote stage seen in clinical samples is known as leishman-donovan (ld) bodies. the amastigotes observed in the smears have to be differentiated from "dot"-like structures (e.g., histoplasma spp., platelets) by looking for the characteristic size (2-4 mm in diameter), shape (round to oval), and the internal organelles (the nucleus and kinetoplast). culture of these specimens on solid nnn medium will demonstrate promastigotes. immunological methods of diagnosis include indirect fl uorescent test (ifa), direct agglutination test (dat), and enzymelinked immunosorbent assay (elisa) to detect antibodies against leishmania [ 20 ] . antigen detection tests are better means of diagnosis of active leishmaniasis [ 21 ] . the antigen detection is the ideal test in immunocompromised patients, where antibody response is very poor. pcr is found to be the most sensitive and specifi c molecular test and is useful in molecular epidemiological studies besides diagnosis [ 22 ] . malaria is caused by the obligate intraerythrocytic protozoa of the genus plasmodium and is primarily transmitted by the bite of an infected female anopheles mosquito. five species of malarial parasites ( plasmodium vivax , p . falciparum , p . malariae , p . ovale , and p . knowlesi ) infect man [ 23 ] . the main symptoms of malaria are periodic fever, chills, malaise, headache, abdominal pain, and vomiting, usually manifesting 10-15 days after mosquito bite. anemia and splenomegaly are other important fi ndings in malaria. falciparum malaria is the most deadly type. the pulmonary manifestations range from cough to severe and rapidly fatal non-cardiogenic pulmonary edema and acute respiratory distress syndrome (ards) ( fig. 1.1 ) [ 24 ] . acute lung injury and ards have also been reported to occur in infection with p . vivax and p . ovale [ 25 , 26 ] . there has been no convincing evidence for the existence of true malarial pneumonitis, and if it occurs, it may be due to viral and secondary bacterial infections. diffusing capacity was signifi cantly impaired in patients with severe malaria. in addition to ards, falciparum malaria can cause many other severe complications such as cerebral malaria, acute renal failure, severe anemia, thrombocytopenia, bleeding, and gastrointestinal, hepatic, and metabolic complications [ 27 ] . microscopic examination of the giemsa-stained blood smears is the gold standard for the diagnosis of malaria. microscopic diagnosis is based on staining thick and thin blood fi lms on a glass slide to visualize the malaria parasite [ 28 ] . it is inexpensive, able to differentiate malaria species and quantify parasites. the detection threshold in giemsa-stained thick blood fi lm has been estimated to be 4-20 parasites/μl. plasmodium species can be correctly recognized in thin blood fi lm. sometimes malarial parasites cannot be detected in peripheral blood smear, but malaria pigments may be seen in circulating phagocytic leukocytes. this is a pathognomonic sign of recent infection. the parasite count, number of circulating pigment-containing phagocytes, and the presence of late asexual stages of the parasite observed in the blood smear are all positively correlated with a fatal outcome. bone marrow aspirate can also demonstrate malarial parasites, if thin smears of the peripheral blood do not show the parasites. quantitative buffy coat (qbc) method involves staining parasite deoxyribonucleic acid (dna) in micro-hematocrit tubes with fl uorescent dyes (e.g., acridine orange) and its subsequent detection by epifl uorescent microscopy. the parasite nuclei fl uoresces bright green and the cytoplasm appears yellow-orange [ 29 ] . rapid diagnostic test (rdt) is a device that detects malaria antigen in a small amount of blood, usually 5-15 μl, by immunochromatographic assay with monoclonal antibodies directed against the target parasite antigen and impregnated on a test strip [ 30 , 31 ] . the result, usually a colored test line, is obtained in 5-20 min. histidine-rich protein 2 (hrp-2) which is specifi c for p . falciparum is the most common malaria antigen targeted [ 32 ] . plasmodium lactate dehydrogenase (pldh) enzyme is the other group of targeted antigens. monoclonal antibodies against pldh and aldolase enzymes are available for the detection of plasmodium spp. (pan malaria). hrp-2 often persists in the patient's blood for weeks after successful treatment. molecular methods such as pcr allow the specifi c amplifi cation of a selected region of the malarial genome. this is a specifi c and sensitive method and permits genotyping. drug-resistant parasites and mixed infections can be detected by pcr using single nucleotide polymorphism. a pcr-based detection of plasmodium falciparum in human urine and saliva samples has been described. the antibodies against asexual blood stages of malaria parasite can be detected by the immunofl uorescence assay (ifa). serological tests are useful in epidemiology surveys and are not suitable for the acute diagnosis of malaria [ 32 ] . babesiosis is caused by hemoprotozoan parasites, babesia microti and babesia divergens [ 33 ] . man gets the infection by the bite of an infected tick, ixodes scapularis , and can also be infected from a contaminated blood transfusion [ 34 ] . the parasites attack the red blood cells and can be misdiagnosed as plasmodium . the symptoms are fever, drenching sweats, tiredness, loss of appetite, myalgia, and headache. acute respiratory distress syndrome occurring a few days after initiation of medical therapy is the important pulmonary manifestation [ 35 ] . chest radiological features include bilateral infi ltrates with an alveolar pattern and thickening of the septa. the peripheral blood smears may show, in addition to ring forms, tetrads inside the red blood cells. these tetrads, known as maltese cross formations, are pathognomonic of babesiosis because they are not seen in malaria [ 36 ] . specifi c diagnosis is made by examination of a giemsastained thin blood smear, dna amplifi cation using pcr, or detection of specifi c antibody [ 33 ] . toxoplasmosis is caused by one-celled protozoan parasite, toxoplasma gondii . cats are the primary carriers of the organism [ 37 ] . man gets the infection by eating parasitic cyst-contaminated raw or undercooked meat, vegetables, or milk products. the symptoms of toxoplasmosis are fl u-like syndrome, enlarged lymph nodes, or myalgia. chronic toxoplasmosis can cause chorioretinitis, jaundice, encephalitis, and convulsions. pulmonary toxoplasmosis has been reported with increasing frequency in patients with hiv infection. toxoplasma pneumonia can manifest as interstitial pneumonia/ diffuse alveolar damage or necrotizing pneumonia [ 38 ] . diagnosis of toxoplasmosis is based on the detection of the protozoa in body tissues. antibody levels can be increased without active disease. a real-time pcr-based assay in bal fl uid has been reported in immunocompromised hiv-positive patients [ 39 ] . the important helminthic parasites that cause lung diseases include cestodes ( echinococcus granulosus and echinococcus multilocularis ), trematodes ( schistosoma haematobium , schistosoma mansoni , schistosoma japonicum , and paragonimus westermani ), and nematodes ( ascaris lumbricoides , ancylostoma duodenale , necator americanus , strongyloides stercoralis , wuchereria bancrofti , brugia malayi , brugia timori , dirofi laria immitis , dirofi laria repens , toxocara canis or cati , and trichinella spiralis ). the parasite species that cause hydatid disease in man are echinococcus granulosus and echinococcus multilocularis . the adult e . granulosus resides in the small intestine of the defi nitive hosts, mainly dogs. the intermediate hosts including man are infected by ingestion of eggs excreted in the feces of the dogs. primary pulmonary cystic hydatid disease is usually single. multiple cysts may be seen in 20 % of cases and may be unilateral or bilateral [ 2 ] . secondary metastatic pulmonary cystic hydatid disease may occur by the rupture of a liver cyst in vena caval circulation or a heart cyst in the right ventricular cavity. patients are asymptomatic in the initial stages of infection. pulmonary symptoms include cough, fever, dyspnea, and chest pain [ 40 ] . signs and symptoms can occur due to compression of adjacent tissue by the cysts. rupture of the cysts into a bronchus may result in hemoptysis and expectoration of cystic fl uid containing parasite membrane and can cause anaphylactic shock, respiratory distress, asthma-like symptoms, persistent pneumonia, and sepsis [ 41 , 42 ] . rupture of the cyst into the pleural space can result in hydropneumothorax, pleural effusion, and empyema. diagnosis of pulmonary hydatid cyst is based on thoracic imaging studies (chest radiography, thoracic computerized tomography (ct), and thoracic ultrasonography) [ 43 ] and an uncomplicated cyst presents as a well-defi ned homogenous round (cannonball) opacity that may be lobulated by contiguous bronchovascular axes ( fig. 1.2 ) [ 2 ] . chest radiographs show solitary or multiple round opacities that may mimic lung tumors. ct is helpful in doubtful cases, because the internal structure of the cyst can be analyzed and its density measured. ct is also useful to assess the state of the neighboring parenchyma and to evaluate the whole thorax and abdomen for associated cystic lesions or anomalies. ultrasonography using a portable ultrasound scanner has been found as reliable, inexpensive, and rapid technique in community-based screening surveys for cystic hydatid disease. the crescent sign, cumbo's sign (onion peel sign), water lily sign, and air-fl uid level are seen on chest radiography and computed tomography (ct) [ 41 ] . inverse crescent sign, signet ring sign, and serpent sign are recognized as features of pulmonary hydatid cysts in computerized tomogram. laboratory tests are complementary to clinical and imaging investigations. eosinophilia and elevated immunoglobulin e (ige) levels are seen when the hydatid cyst ruptures [ 40 ] . serologic tests are less sensitive in patients with lung hydatid disease than in those localized in liver. falsepositive tests may be observed in patients suffering from other helminthic infections. immunologic tests may be helpful to confi rm the hydatid origin of a cystic lesion and permit the serologic monitoring of medically or surgically treated patients [ 44 , 45 ] . pulmonary alveolar echinococcosis (ae) is due to hematogenous dissemination from hepatic lesions. the liver is the fi rst target of the parasite, with a silent and long incubation period (5-15 years). exogenous proliferation causes infi ltration of adjacent tissues and pressure necrosis. it can metastasize to distant organs mainly to lungs, brain, and bones [ 46 ] . lung involvement results from metastatic dissemination or direct extension through the diaphragm of hepatic echinococcosis with intrathoracic rupture into the bronchial tree, pleural cavity, or mediastinum. direct extension to the right atrium through the inferior vena cava with recurrent episodes of pulmonary embolism has also been reported. imaging studies with radiography, ultrasonography, ct, and magnetic resonance imaging (mri) may help in the diagnosis of metastatic lung disease [ 2 ] . biopsy may be needed to confi rm the diagnosis [ 47 ] . serologic the schistosomes that cause human disease are schistosoma haematobium , schistosoma mansoni , and schistosoma japonicum . the fi nal habitat of s . haematobium is urinary bladder vesicle beds and of s . mansoni and s . japonicum is the mesenteric beds. the schistosome eggs are passed in urine ( s . haematobium ) or in feces ( s . mansoni and s . japonicum ) by the infected humans. the parasites can cause schistosoma dermatitis at the site of skin penetration. pulmonary schistosomiasis can manifest clinically as an acute form and a chronic form [ 49 ] . acute symptoms can develop 3-8 weeks after skin penetration [ 50 ] . the acute form, also known as katayama syndrome, presents with fever, chills, weight loss, diarrhea, abdominal pain, myalgia, and urticaria and is seen in nonimmune patients [ 51 ] . pulmonary manifestations include shortness of breath, wheezing and dry cough. patients with chronic schistosomiasis present with features of pulmonary hypertension and cor pulmonale [ 52 ] . massive hemoptysis and lobar consolidation and collapse have been reported in schistosomiasis [ 53 ] . hepatosplenomegaly due to portal hypertension has been reported in patients infected with s . mansoni or s . japonicum [ 49 ] . chest radiographic abnormalities range from multiple nodules to diffuse interstitial infi ltrates. small pulmonary nodules in ct have been described in acute schistosomiasis [ 54 ] . diagnosis of chronic schistosomiasis is based on the demonstration of eggs in stool or urine by direct microscopy or rectal/bladder biopsy [ 55 ] . multiple examinations of specimens are required in mild and chronic infections. in active infections, eggs contain live and mature miracidia. the incubation period of the infection is usually 3 months and hence eggs can be detected after 3 months of last known contact with fresh water. peripheral blood eosinophilia with mild leukocytosis, abnormal liver function test results and elevated ige levels are reported in acute schistosomiasis. hyperglobulinemia is observed in chronic schistosomiasis. serological tests with elisa are available, but cannot differentiate active from past infections [ 56 ] . bronchoscopy and transbronchial biopsy may reveal eosinophilic pneumonitis. paragonimiasis is a food-borne zoonoses and is caused by infection with paragonimus species and manifests as subacute or chronic infl ammation of the lung. though more than 50 species are known to cause paragonimiasis in man, the main species that cause paragonimiasis is paragonimus westermani . adult worms live in the lungs and the eggs are voided in sputum or feces. the man gets infection, when raw or undercooked crabs or crayfi shes infected with infective metacercariae are ingested. the parasite from the human gut passes through several organs and tissues to reach the lung. pulmonary paragonimiasis manifests as fever, chest pain, chronic cough, and bloodtinged sputum [ 57 ] . the cough is dry at fi rst and later productive with blood-stained, rusty-brown tenacious sputum. occasionally, there is profuse hemoptysis. pulmonary paragonimiasis is confused with tuberculosis as the symptoms in both diseases are similar. chest radiographs may show infi ltrative, nodular, and cavitating shadows. pleural effusion or pneumothorax is an important fi nding in paragonimiasis [ 58 , 59 ] . ct scan may show single or multiple nodules in the lung parenchyma, calcifi ed spots and pleural thickening with interlobar pleuritis, and pleural effusion. mri may show conglomerated lesions with hemorrhage or tunnel signs. defi nitive diagnosis is based on the demonstration of eggs in sputum samples, bal fl uid, or lung biopsy specimens. ascaris lumbricoides is the most common intestinal helminthic infection. respiratory symptoms in ascariasis are due to larval pulmonary migration, airway hyper-reactivity, and bronchospasm. symptomatic pulmonary disease may range from mild cough to loffl er's syndrome [ 61 ] . loffl er's syndrome is a self-limiting infl ammation of the lungs and is associated with blood and lung eosinophilia. this syndrome can occur as a result of parasitic infestations (especially ascariasis in children) and exposure to various drugs. patients may present with general symptoms of malaise, loss of appetite, fever lasting 2-3 days, headache, and myalgia. the respiratory symptoms include chest pain, cough with mucoid sputum, hemoptysis, shortness of breath, and wheezing [ 62 ] . there may be rapid respiratory rate and rales can be heard on auscultation. leukocytosis particularly eosinophilia is an important laboratory fi nding. chest radiographs demonstrate unilateral or bilateral, transient, migratory, and non-segmental opacities of various sizes. these opacities are often peripherally situated and appear to be pleural based [ 63 ] . the severity of symptoms will depend upon the larval burden. rarely chronic eosinophilic pneumonia or symptoms of upper airway obstruction can occur. a diagnosis of pulmonary disease due to ascariasis can be made in an endemic region in a patient who presents with dyspnea, dry cough, fever, and eosinophilia. sputum may show charcot-leyden crystals and the chest radiograph may reveal fl eeting pulmonary infi ltrates. because of the occurrence of respiratory symptoms during larval pulmonary migration, stool examination usually does not show ascaris eggs and stool samples may be negative until 2-3 months after respiratory symptoms occur, unless the patient was previously infected. however, larvae can sometimes be demonstrated in respiratory or gastric secretions [ 64 ] . it has been suggested that measurement of ascaris -specifi c igg4 by elisa may be useful in the serodiagnosis of ascariasis [ 65 ] . hookworm disease in humans results from infections with two species, ancylostoma duodenale and necator americanus . during pulmonary larval migration, patients may present with fever, cough, wheezing, and transient pulmonary infi ltrates in chest radiographs. this is associated with blood and pulmonary eosinophilia [ 3 ] . the other characteristic feature is iron defi ciency anemia due to chronic blood loss [ 66 ] . in severe hookworm anemia, patients may present with fatigue, exertional dyspnea, poor concentration, and cardiac murmurs. during massive infection from oral ingestion of hookworm larvae, patients can present with nausea, vomiting, cough, dyspnea, and eosinophilia, and this condition is termed as wakana disease. prominent gastrointestinal symptoms in hookworm disease are abdominal pain, nausea, anorexia, and diarrhea. a direct microscopical examination of stool demonstrates the presence of characteristic hookworm eggs. concentration method may be used when the infection is light. eosinophilia in the peripheral blood is a prominent fi nding. a peripheral blood smear examination will reveal microcytic hypochromic anemia. a polymerase chain reaction (pcr) to differentiate between a . duodenale and n . americanus has been developed [ 67 ]. strongyloides stercoralis is seen worldwide and the unique feature of the life cycle of s . stercoralis is that it can complete its life cycle either in the human host or in the soil. it has been observed that 15-30 % of chronically infected people may be asymptomatic. although symptoms in individuals with chronic strongyloides stercoralis infection are usually mild, it can persist for many years due to autoinfection. this may occasionally progress to the hyperinfection syndrome with high mortality especially in immunosuppressed individuals [ 68 , 69 ] . the relative risk of s . stercoralis infection is increased in elderly men and patients who had recently used corticosteroids, had a hematologic malignancy, and had prior gastric surgery. other risk factors include chronic lung disease, use of histamine blockers, or chronic debilitating illness. strongyloidiasis is a chronic relapsing illness of mild to moderate severity characterized by gastrointestinal complaints (diarrhea, pain, tenderness, nausea, vomiting), peripheral blood eosinophilia, and hypoalbuminemia. pulmonary signs and symptoms include cough, shortness of breath, wheezing, and hemoptysis. in patients at high risk for strongyloidiasis, adult respiratory distress syndrome and septicemia due to intestinal transmural migration of bacteria can occur as a result of hyperinfection or disseminated strongyloidiasis [ 70 , 71 ] . in addition, acute anemia, acute renal failure, and systemic infl ammatory response syndrome are also reported in hyperinfection. strongyloidiasis can manifest as eosinophilic pleural effusion in both immunocompetent and immunocompromised individuals. rare pulmonary manifestations include acute respiratory failure due to respiratory muscle paralysis, granulomatous reaction in the lung with interlobular septal fi brosis, and pulmonary microcalcifi cations. a paradoxic therapeutic response of asthma to glucocorticosteroids, in which bronchial asthma symptoms worsened after treatment with parenteral corticosteroids, has been described in patients with strongyloides superinfections [ 72 ] . exacerbations of chronic obstructive pulmonary disease and worsening of symptoms in idiopathic pulmonary fi brosis have also been reported in strongyloides stercoralis infection. in immunocompetent patients with strongyloidiasis, the parasite load is usually low and the larval output is irregular. as a result, the diagnosis of strongyloidiasis by examination of a single stool specimen using conventional techniques usually fails to detect larvae in up to 70 % of cases [ 73 ] . the diagnostic yield can be increased by examination of several stool specimens on consecutive days. examination of stool by agar plate culture method was found to be superior to direct smear and modifi ed baermann technique [ 74 , 75 ] . strongyloides stercoralis larvae can be demonstrated in duodenal aspirate. in disseminated disease, larvae and adult parasites can be seen in sputum, urine, bronchoalveolar lavage fl uid, and other body fl uids [ 76 ] . a serological test using centers for disease control (cdc) enzyme immunoassay (eia) for detection of antibodies to strongyloidiasis was found to have a sensitivity of 94.6 % in patients with proven infection [ 77 ] . tropical pulmonary eosinophilia (tpe) results from immunologic hyper-responsiveness to human fi larial parasites, wuchereria bancrofti and brugia malayi [ 78 -80 ] . tpe is a systemic disease involving mainly the lungs, but other organs such as liver, spleen, lymph nodes, brain, and gastrointestinal tract may also be involved. the disease occurs predominantly in males, with a male to female ratio of 4:1, and is mainly seen in older children and young adults between the ages 15 and 40 years. the systemic symptoms include fever, weight loss, and fatigue. patients with tpe usually present with respiratory symptoms that include paroxysmal cough, breathlessness, and wheeze and chest pain [ 81 , 82 ] . the symptoms occur predominantly at night, but can persist during the day. severe cough can lead to fractured ribs. sputum is usually scanty, viscous, and mucoid. the sputum often shows clumps of eosinophils, and rarely charcot-leyden crystals are observed. on examination, patients are often breathless. bilateral scattered rhonchi and rales may be heard on auscultation [ 61 , 78 ] . leukocytosis with an absolute increase in eosinophils in the peripheral blood is the hallmark of tpe. spontaneous fl uctuations in the eosinophil count can occur. absolute eosinophil counts are usually more than 3,000 cells/mm 3 and may range from 5,000 to 80,000 [ 83 ] . microfi lariae are rarely seen in the peripheral blood. as patients with tpe especially from endemic areas can be simultaneously infected with other helminthic parasites, stool examination may reveal ova or larvae of other helminthes ( ascaris , ancylostoma , whipworm, and strongyloides ) in 20 % of patients with tpe. this observation does not deter the physician from making a diagnosis of tpe, if other conditions for diagnosis are fulfi lled. the chest radiological features of tpe include reticulonodular shadows predominantly seen in mid and lower zones and miliary mottling of 1-3 mm in diameter often indistinguishable from miliary tuberculosis (fig. 1.3 ) . twenty percent of patients have a normal chest radiograph. in patients with a long-standing history, a few patients have honeycomb lungs. radiological improvement occurs on specifi c therapy with dec, but some degree of radiological abnormality persists in some patients. lung function tests reveal mainly a restrictive ventilation defect with superimposed airway obstruction [ 84 , 85 ] . single breath carbon monoxide transfer factor (tlco) is reduced in 88 % of untreated patients with tpe. the reduction in tlco is due to reduced pulmonary membrane diffusing capacity (dm) [ 86 ] . the criteria suggested for the diagnosis of tpe are (a) appropriate exposure history (mosquito bite) in an endemic area of fi lariasis, (b) a history of paroxysmal nocturnal cough and breathlessness, (c) chest radiographic evidence of pulmonary infi ltrations, (d) leukocytosis in blood, (e) peripheral blood eosinophils more than 3,000 cells per cu mm, (f) elevated serum ige levels, (g) elevated serum antifi larial antibodies (igg and/or ige), and (h) a clinical response to diethylcarbamazine citrate [ 87 , 88 ] . pulmonary dirofi lariasis is a zoonotic infection caused by fi larial nematodes, dirofi laria immitis and dirofi laria repens . humans are accidental hosts of this parasite which is transmitted to man by the mosquito. the parasites are usually seen in the pulmonary artery where they produce an embolism ultimately leading to the formation of a pulmonary nodule or "coin lesion" [ 89 ] . nearly 50 % of subjects infected with dirofi lariasis are asymptomatic. clinical symptoms are chest pain, cough, fever, hemoptysis, and dyspnea. ct scan may show a well-defi ned nodule with smooth margin connected to an arterial branch [ 90 ] . positron emission tomography scan can demonstrate hypermetabolic activity in a pulmonary infarct secondary to dirofi lariasis [ 91 ] . a pcr-based diagnosis of d . repens in human pulmonary dirofi lariasis has been reported [ 92 ] . a defi nitive histopathological diagnosis of pulmonary dirofi lariasis can be made tissue specimens obtained by wedge biopsy, by video-assisted thoracoscopy, or rarely by fi ne needle biopsy. toxocara larva migrans syndromes are important zoonotic infections. certain nematode parasites entering into an unnatural host (e.g., man) may not be able to complete their life cycle and their progress is arrested in the "unnatural host." the common parasites that cause visceral larva migrans (vlm) and eosinophilic lung disease in man are a dog ascarid ( toxocara canis ) and less commonly a cat ascarid ( toxocara cati ) [ 93 ] . human toxocariasis occurs in all parts of the world wherever there is a large pool of infected dogs. visceral larva migrans (vlm) is characterized by leukocytosis and eosinophilia. the larva induces a granulomatous reaction in the tissues containing eosinophils and multinucleated giant cells. larvae can get encapsulated within the granuloma where they are either destroyed or persist for many years in a viable state. granulomata are found in the lungs, liver, central nervous system, and eyes. later fi brosis and calcifi cation occur. larval antigens can cross-react with human a and b blood group antigens. visceral larva migrans is usually reported in young children with a history of pica. a history of exposure to puppies or dogs supports the diagnosis of vlm. these children usually present with fever, cough, wheezing, eosinophilia, and hepatomegaly. however, most of the children infected with toxocara spp. are asymptomatic. the main symptoms in patients with visceral larva migrans are fever, cough, wheezing, seizures, anemia, and fatigue. pulmonary manifestations are reported in 80 % of cases and patients may present with severe asthma [ 94 ] . scattered rales and rhonchi are heard on auscultation. there will be intense blood eosinophilia. skiagram chest may reveal focal patchy infi ltrates. in some cases, severe eosinophilic pneumonia may lead to respiratory distress [ 95 ] . other clinical features include generalized lymph node enlargement, hepatomegaly, and splenomegaly. skiagram chest may show patchy infi ltrates. nonspecifi c changes include hypergammaglobulinemia and elevated isohemagglutinin titers to a and b blood group antigens. serological tests by elisa method using excretory-secretary proteins obtained from cultured t . canis may be useful in the diagnosis. cross reactivity with other helminths limits the usefulness of this test in endemic areas. detection of ige antibodies by elisa and toxocara excretory-secretary antigens by western blotting procedure have also been reported for diagnosis [ 96 , 97 ] . however, serodiagnostic procedures cannot distinguish between past and present infections. histopathological examination of lung or liver biopsy specimens may demonstrate granulomas with eosinophils, multinucleated giant cells, and fi brosis. since man is not the defi nitive host of toxocara sp., eggs or larvae cannot be demonstrated in the feces. human trichinellosis is an important food-borne zoonosis. the most important species that infect man is trichinella spiralis . the parasite has a direct life cycle with complete development in one host (pig, rat, or man). man gets infection from raw and partially cooked pork, when infected pig's muscle containing larval trichinellae is eaten by man. the common symptoms of trichinellosis are muscle pain, periorbital edema, fever, and diarrhea [ 98 ] . pulmonary symptoms include dyspnea, cough, and pulmonary infi ltrates. dyspnea may be due to the involvement of diaphragm [ 99 ] . leukocytosis, eosinophilia, and elevated levels of serum muscle enzymes (creatine phosphokinase, lactate dehydrogenase, aldolase, and amino transferase) are important laboratory fi ndings. an enzyme-linked immunosorbent assay (elisa) for detection of anti-trichinella antibodies using excretory-secretary antigens may be useful in the diagnosis. a defi nitive diagnosis can be made by muscle biopsy (usually deltoid muscle) that may demonstrate larvae of t . spiralis [ 99 ] . is the incidence of parasitic lung diseases increasing, and how may this effect modern respiratory medicine? emerging and established parasitic lung infestations immunopathogenesis and treatment of eosinophilic lung diseases in the tropics parasitic lung infections progress in research on entamoeba histolytica pathogenesis thoracic amoebiasis value of microscopy in the diagnosis of dysentery associated with invasive entamoeba histolytica diagnosis of amebiasis in bangladesh comparison of pcr, isoenzyme analysis and antigen detection for diagnosis of entamoeba histolytica infection human amebiasis: breaking the paradigm? a new liquid medium for xenic cultivation of entamoeba histolytica and other lumendwelling protozoa laboratory diagnosis of amebiasis genomic dna differences between pathogenic and non pathogenic entamoeba histolytica development of multiplex real-time polymerase chain reaction for detection of entamoeba histolytica, entamoeba dispar, and entamoeba moshkovskii in clinical specimens bronchopulmonary and mediastinal leishmaniasis: an unusual clinical presentation of leishmania donovani infection leishmaniasis among organ transplant recipients detection of leishmania infantum kinetoplast dna in laryngeal tissue from an immunocompetent patient new developments in diagnosis of leishmaniasis comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis latex agglutination test for the detection of urinary antigens in visceral leishmaniasis molecular diagnosis of leishmaniasis malaria prevention in short-term travelers lung biology in health and disease: tropical lung diseases acute lung injury and other serious complications of plasmodium vivax malaria acute respiratory distress syndrome in a case of plasmodium ovale world health organization. guidelines for the treatment of malaria. geneva: world health organization laboratory diagnosis of malaria infection-a short review of methods malaria diagnosis: a brief review rapid diagnostic tests for malaria parasites rapid diagnostic testing for malaria a review of malaria diagnostic tools: microscopy and rapid diagnostic test (rdt) co-infections acquired from ixode ticks acute respiratory failure in patients treated for babesiosis diagnosis of pulmonary infection with toxoplasma gondii in immunocompromised hiv-positive patients by real time pcr clinical and diagnostic management of toxoplasmosis in the immunocompromised patient eosinophilic lung disease in the tropics parasitic diseases of the respiratory tract spontaneous rupture of lung echinococcal cyst causing anaphylactic shock and respiratory distress syndrome expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans usefulness of four different echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (uhd) and postsurgical follow-up of patients treated for uhd pulmonary cystic echinococcosis echinococcus multilocularis disseminated alveolar echinococcosis mimicking a metastatic malignancy the immunodiagnosis of echinococcus multilocularis infection pulmonary schistosomiasis pulmonary manifestations of early schistosoma infection in nonimmune travelers katayama fever; an acute manifestation of schistosomiasis schistosomiasis associated pulmonary hypertension a diagnostic dilemma of right lower lobe collapse caused by pulmonary bilharsiasis acute schistosomiasis in nonimmune travelers: chest ct fi ndings in 10 patients pathophysiology, diagnosis, and treatment detection of circulating antigens in patients with active schistosoma haematobium infection studies on clinical manifestations, diagnosis and control of paragonimiasis in china pulmonary paragonimiasis: an evaluation of roentgen fi ndings in 38 positive sputum patients in an endemic area in thailand clinicoradiologic features of pleuropulmonary paragonimus westermani on kyushu island evaluation of human igg subclass antibodies in the serodiagnosis of paragonimus heterotremus tropical parasitic lung diseases strongyloidiasis and other intestinal nematode infections eosinophilic lung disease (or how to slice pie) ascariasis and hookworm possible approach for serodiagnosis of ascariasis by evaluation of immunoglobulin g4 response using ascaris lumbricoides somatic antigen nature and causes of hookworm anemia differentiation between the human hookworm ancylostoma duodenale and necator americanus using pcr-rflp biology and immunology of human strongyloidiasis disseminated strongyloides stercoralis in human immunodefi ciency virus-infected patients: treatment failure and review of literature pulmonary manifestations of strongyloidiasis strongyloides stercoralis infestation associated with septicemia due to intestinal transmural migration of bacteria corticosteroidinduced asthma: a manifestation of limited hyperinfection syndrome due to strongyloides stercoralis diagnosis of strongyloides stercoralis infection a simple modification of the baermann method for diagnosis of strongyloidiasis evaluation of three methods for laboratory diagnosis of strongyloides stercoralis infection diagnosis of pulmonary strongyloidiasis by bronchoalveolar lavage serology and eosinophil count in the diagnosis and management of strongyloidiasis in a non-endemic area tropical pulmonary eosinophilia acute tropical pulmonary eosinophilia: characterization of the lower respiratory tract infl ammation and its response to therapy tropical pulmonary eosinophilia: analysis of antifi larial antibody localized to the lung pulmonary eosinophilia: progress in respiration research tropical pulmonary eosinophilia re-examination of the diagnostic criteria of tropical pulmonary eosinophilia the course of lung function in treated tropical eosinophilia diffusing capacity in acute untreated tropical eosinophilia pulmonary membrane diffusing capacity and capillary blood volume in tropical eosinophilia persistent lower respiratory tract infl ammation associated with interstitial lung disease in patients with tropical pulmonary eosinophilia following treatment with diethylcarbamazine how to diagnose and manage common parasitic pneumonias? public health aspects of dirofi lariasis in the united states pulmonary dirofi lariasis: computed tomographic fi ndings and correlation with pathologic features pet fi ndings in pulmonary dirofi lariasis detection of dirofi laria (nochtiella) repens dna by polymerase chain reaction in embedded paraffi n tissues from two human pulmonary locations highlights of human toxocariasis visceral larva migrans associated with the hypereosinophilic syndrome and the onset of severe asthma acute respiratory failure due to toxocara infections evaluation of an immunenzymatic assay detecting specifi c anti-toxocara immunoglobulin e for diagnosis and post treatment follow up of human toxocariasis application of the western-blotting procedure for the immunodiagnosis of human toxocariasis clinical aspects of infections with trichinella spp tropical infectious diseases: principles, pathogens and practice key: cord-006888-qfnukav4 authors: nan title: irish thoracic society annual scientific meeting, ramada hotel, belfast: 7th–8th november 2008 date: 2008-10-21 journal: ir j med sci doi: 10.1007/s11845-008-0235-y sha: doc_id: 6888 cord_uid: qfnukav4 nan welcome to the irish thoracic society annual scientific meeting 2008. we are delighted that the meeting has made a return to belfast this year and in honour of this we've put together a programme that we feel sure will make for a highly interesting and worthwhile experience. a central feature will be the presentation of original research in both oral and poster form, showcasing the wide range of important and innovative work being carried out throughout the island. the focus of this year's symposium is lung cancer and we are delighted to welcome a panel of leading international speakers who will share their knowledge and insights on this important topic. additional guest lectures on ciliary dyskinesia and asthma by distinguished specialists in both fields complete a varied and, we hope, highly stimulating programme. it is my pleasure to welcome you to the irish thoracic society annual scientific meeting 2008. on behalf of the irish thoracic society i wish to thank dr. jackie rendall for her outstanding work in conjunction with the its office in organising this year's programme. thanks to her sterling efforts we look forward to a rewarding and stimulating meeting. 2008 has been a busy year for the irish thoracic society and i would like to take this opportunity to reflect on some of the highlights. february saw the publication of the inhale report, 2 nd edition (ireland needs healthier airways and lungs -the evidence). compiled by the its in conjunction with dr neil brennan and dr terry o'connor, the report underlines the serious resource deficits that still exist in respiratory health-care. clearly a lot more work is needed in this area and the society will continue advocating for a respiratory strategy to tackle the imbalance. throughout the year the society has made representations on a broad range of issues including copd, tuberculosis, critical care services and lung cancer. with respect to the latter, the irish thoracic society lung cancer sub-committee has been working with the national cancer control programme towards the development of improved services for lung cancer care. the society has also been represented on the national copd strategy group and the national tb advisory committee and we look forward to the respective reports on this work. significant headway has been made in the area of education and research. the irish thoracic society boehringer ingelheim research fellowship, launched last year, has recently been awarded for a second time, promising valuable contributions to respiratory research from two very worthy projects in the coming years. our ability to communicate with members has improved dramatically thanks to a radical upgrade of the irish thoracic society website -www.irishthoracicsociety.com. this provides information on the society's activities and other relevant issues. many delegates will have become familiar with its facilities for on-line registration and submission of abstracts in the lead-up to the meeting and we trust they have proven convenient and user-friendly. password protected members areas are designed for more specialist interest content and we encourage members to help us develop these areas further as a resource for sharing information and discussion of issues. we recognise the important role our members continue to play in all these activities. in order to sustain our efforts, the continued support of members and the expansion of our membership base is vital. i would also like to take this opportunity to thank our partners in the pharmaceutical and medical equipment sectors. their support over the years has been central to the society's development and is now more important than ever -we look forward to continued collaboration in 2009 and beyond. dr. jj gilmartin, president, the irish thoracic society patient's with copd are known to have decreased levels of activity. this study looks at free-living activities as measured by the sense-ware armband to determine if there was a relationship with standard exercise field tests for this patient population. thirty one patients with copd were recruited: men (n = 14), female (n = 17). ethical approval and written consent was obtained. the senseware ò armband was worn for seven consecutive days and distance on the shuttle walk test was measured. pearson's correlations were undertaken using spss version 12. the mean age of the study population was 67.5 yrs (±7.4) with a mean fev 1 45% (±41). the shuttle walk test (swt) was 212 m (±121). free-living activities as measured by; physical activity level (pal) of 1. patients low levels of activities in daily life as measured by the senseware ò armband parameters correlates with the shuttle walk test making it a reliable outcome measure. the shuttle walk test can provide us with valuable insight into the patient's free-living activities and sleep pattern. international literature points to inequity in the provision of palliation in favour of patients with cancer despite the high mortality associated with copd. chronicity is associated with biographical disruption, loss and shifting relationships with healthcare professionals all of which may influence the nature of palliative care in advanced copd. a three phased project is underway aimed at developing palliative care for patients with copd. the purpose of phase one is to identify palliative care needs of these patients. a mixed method research design was employed for phase one involving health status measurement and qualitative interviews. inclusion criteria were those patients who were hospitalised for exacerbation of copd. the patient sample was mainly derived from one hospital over a one year period with a primary diagnosis of copd. gatekeepers were in place to protect confidentiality. data was collected using the st george's respiratory questionnaire(sgrq), hospital anxiety and depression scale (hads), and the medical research council (mrc) dyspnoea scale. in a 2nd round of interviews, the questions are open and semi-structured. interviews were undertaken in patients' homes. an integrated analysis is underway of data represented in different forms using spss and nvivo software. twenty six patients were interviewed. ages ranged from 52-85 yrs (mean = 69). anxiety and depression scores averaged 8.4 and 6.7 respectively. scores of [11 indicating moderate and severe levels were found in 10 cases for anxiety and 4 for depression. average sgrq scores = 62.4 indicating significant impact on quality of life. themes from qualitative data include a catastrophic diagnostic event along the illness trajectory, ambivalence towards opd visits and rigid daily routine to control breathlessness. issues emerged regarding recruitment, the construing of palliative care in copd and articulation of experiences of quality of care. conclusion: preliminary findings suggest significant disability and lay expertise; isolation; anxiety; impact on relationships and poorly articulated fears of the future. unmet palliative care needs are evident and challenge nursing to find appropriate ways of construing palliative care in copd. patients with severe copd are likely to have repeated exacerbations and early mortality. in ventilated patients herpes simplex virus-1 (hsv-1) is frequently identified and is associated with an increased mortality. we determined the frequency of hsv-1 in copd patients (stable and exacerbated) and if it was associated with disease severity and mortality. methods: stable and exacerbated copd patients were recruited. spirometry was performed. sputum was obtained and lysed by ddt. nucleic acids were extracted and specimens were tested for hsv-1 and gapdh using real-time pcr. results: one hundred and thirty six patients with exacerbations of copd and 68 stable patients were recruited. hsv-1 was detected in 19% of copd patients during an exacerbation and 13% of stable copd patients. no significant differences in hsv-1 copy numbers were seen on comparison of these groups. detection of hsv-1 was associated with increasing copd disease severity, p \ 0.005. the presence of hsv-1 during exacerbations was associated with increased mortality, p \ 0.05, predominantly from respiratory causes, p = 0.05. conclusion: hsv-1 is frequently detected in the sputum of copd patients. it is more commonly found in patients with severe airways disease and its presence during exacerbations is associated with increased mortality. pulmonary rehabilitation (pr) is associated with symptomatic and physiologic improvements in patients with copd. however, biologic effects on systemic inflammatory and profibrotic cytokines are unproven. thirty two patients with moderate or severe copd (age 66.1 ± 9.63 y, fev 1 42 ± 17.9% predicted) were recruited to a pr programme. cardiopulmonary exercise testing was performed before and after the programme. serum c-reactive protein (crp), tumour necrosis factor-alpha (tnf-a), interleukin-6 (il-6), transforming growth factor-beta (tgf-b) and oxidative burst were measured before exercise, at peak exercise and at recovery. there were statistically significant improvements in all domains of the st georges respiratory questionnaire, chronic respiratory disease questionnaire and hospital anxiety and depression questionnaire. there were no significant changes in crp or tnf-a associated with exercise or pulmonary rehabilitation. exercise was associated with a surge in oxidative burst. endurance exercise was associated with an increase in il-6 (p = 0.0227) that was attenuated by pulmonary rehabilitation (p = 0.6271). incremental exercise was associated with an increase in tgf-b (p = 0.0388) that was attenuated by pulmonary rehabilitation (p = 0.1441). demonstrating biological effects of pr has proved elusive to date. this is the first study to demonstrate modulation of both circulating inflammatory and profibrotic cytokines by pr in patients with copd. queen's university, 2 royal victoria hospital, 3 belfast city hospital, n. ireland beta cryptoxanthin is a pro-vitamin a carotenoid which is reported to be a good biomarker of fruit and vegetable intake. we hypothesised that levels of serum beta cryptoxanthin would be related to fev 1. in 1991, 2745 men aged 50 to 59 years were recruited into the belfast arm of the prospective epidemiological study of myocardial infarction (prime). we describe the cross-sectional analysis of the 1208 men who had a valid spirometry trace and plasma sample at 10 year follow-up. beta cryptoxanthin levels were measured using hplc analysis. fev 1 values at 10 years were modelled using simple linear regression, and adjusted for covariates. serum beta cryptoxanthin levels were positively correlated with fev 1 (r = 0.23, p \ 0.0001). for each nanomole per litre increment in serum beta cryptoxanthin levels, fev 1 was 2.22 mls greater. following adjustment for the covariates, for each nanomole per litre increment in serum beta cryptoxanthin levels, fev 1 was 1.26 mls greater (95%ci 0.78 to 1.75, p \ 0.0001). serum beta cryptoxanthin levels are positively correlated with fev 1 . this suggests that in this population a moderate increase in serum beta cryptoxanthin levels (achievable by a modest increase in dietary intake of fruit and vegetables) may have a protective effect on lung function. the study was undertaken to evaluate organ utilization in the republic of ireland. a retrospective review of potential donors from january 2006 to august 2008 was performed. donors organ were selected according to criteria set by the international society of heart and lung transplantation (ishlt), abo group compatibility and predicted tlc. this included a donor age \ 45 years old, a satisfactory history, a normal chest radiogram, arterial blood gases (abg) of [40 kpa (300 mmhg) (fio2 of 100% and a peep of 5). 227 potential offers for organ donation occurred. the median donor age was 43 years (range 1-72), the mean period of mechanical ventilation was 3 days (range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . sixty percent (137 offers) were declined on the basis of ishlt criteria, 20% were declined because of age, 35% due to poor blood gases, 35% due to abnormal chest x-ray and 10% because of chest trauma. sixty-three offers were evaluated at the donor site. five percent (11 offers) were excluded because of size and hla crossmatch constraints, these were offered to uk transplant. 17 lungs (7.5%) were successfully transplanted. organ donation in republic of ireland is high (22 per million population), however lung utilisation is low. the pathway to increase number of lung transplantation may include a framework for optimising donor physiology. the use of endoscopic ultrasound with fine-needle aspiration (eus-fna) is well established in diagnosing and staging non-small cell lung cancer with positron emission tomography (pet) positive posterior mediastinal lymph nodes. the sensitivity of eus-fna ranges between 83 and 96%. it is less invasive and has lower complication rates when compared to surgical staging of mediastinal nodes. this study aims to describe our initial experience of eus-fna in lung cancer. eus-fna was used prospectively for the assessment of pet positive mediastinal lymph nodes between january 2007 and july 2008. when eus-fna did not show malignant invasion, a confirmatory mediastinoscopy was done. endpoints were performance of eus-fna, morbidity and length of hospital stay. 23 patients underwent eus-fna during the study period for both diagnosis and staging. 20 patients had positive lymph node invasion and 3 had no evidence of malignant invasion on eus-fna. negative cytology on the latter 3 was confirmed on mediastinoscopy giving eus-fna a sensitivity of 100% for the study period. it upstaged the disease in 15 patients. eus-fna is reliable, non-surgical tool for mediastinal staging. it reduces the need for surgical staging procedures in lung cancer patients with suspected mediastinal involvement. the limitation of this study is the poor documentation of the lymph node stations that were sampled. the role played by the innate immune system in determining survival in non-small-cell lung cancer (nsclc) is unclear. the aim of this study was to investigate the prognostic significance of cytotoxic t-lymphoctye infiltration in nsclc. immunohistochemistry was used to detect cd8 + t-lymphocytes in the tumor islets and tumor stroma in 179 patients with surgically resected nsclc. quantification of immune infiltration was performed using a novel automated image analysis algorithm. univariate cox regression analysis, kaplan-meier analysis and the log-rank test were used to illustrate differences in overall survival according to the expression of tumour to stroma lymphocyte infiltration ratio. lymphocytes were detected in both the tumour islets and stroma in all patients. we used the median of tumor:stroma cd8 + infiltration ratio as a threshold to dichotomise patients to either high or low infiltration rate. results showed that those with a higher intratumoral lymphocyte infiltration had a significantly better survival compared to those with a low tumour/stroma infiltration ratio (p \ 0.001). microlocalization of infiltrating cytotoxic t-lymphocytes is a powerful predictor of outcome from surgically resected nsclc. the biologic explanation for this and its implications for the use of adjunctive treatment require further evaluation. 1.9 ebus-tbna for lung cancer -initial irish experience accurate mediastinal staging is essential in lung cancer patients under consideration for surgical resection. compared to mediastinoscopy or l anterior mediastinotomy (the gold standard), ebus-tbna is less invasive and may be carried out during diagnostic bronchoscopy, offering the potential for one-stop diagnosis and staging. ebus commenced in sjh in mid 2007. we retrospectively analysed the first 74 cases, 66 (89%) of whom had tbna. 44 patients with known or suspected lung cancer had ebus-tbna of n2 glands. cytology was positive for malignancy in 30/44 (68%). in the 14 patients where cytology was negative, 2 had no further follow up. of the other 12, only 3 were proven node positive, but 7 were confirmed node negative at mediastinoscopy/surgery and 2 had resolution of nodes at repeat ct. therefore, in these patients ebus-tbna had a negative predictive value of 75%, sensitivity 91% and overall accuracy 93%. in 3 patients with primary paratracheal mass, ebus-tbna was positive in all 3. of a total 33 lung cancer cases where ebus-tbna was positive, this was the only positive sample in 15 (45%). in 68% of patients ebus-tbna obviated the need for more invasive sampling of the mediastinum-this is one-stop diagnosis and staging. the national patient safety agency (npsa) (may 2008) highlights the role of ultrasound guidance for pleural drain procedures. we review our practice from a district general hospital. pleural studies were identified from the hospital pas and the radiology database. case notes of those who had their effusions drained by catheter were studied. 37 patients (m:f 25:12; age 24-87 [mean 64.9]) had 50 drains placed in the period august 2007 through july 2008. 33 were in patients; 3 electively admitted for the procedure and 1 remained an outpatient. 38 (76%) drains were sited under ultrasound guidance, 8 (16%) placed on ward (3 of these marked in ultrasound) and 4 (8%) sited in ct. 28 patients had single drain placement, 6 had 2 drains; 2 had 3 drains and 1 patient had 4 drains. 42 (84%) drains were placed by career radiology staff, 8 (16%) by ward based staff. consent was documented on 12 (24%) occasions. 28 (56%) drains were flushed regularly as instructed by radiologist. average time in situ was 11 days (range 2-81 days). 7 (19%) patients were discharged home with drains in situ. 3 patients died with drains in place. drain removal was performed by hospital ward staff. we conclude that most drain placements conform to npsa recommendations. consent is poorly documented. this will be addressed by implementation of a chest drain management chart. protocol for the care of pleural-sited catheters has been written. ultrasound is more sensitive than clinical examination or chest x-ray for determining the presence of pleural fluid and helps guide thoracentesis. we report our initial experience with 50 chest ultrasound examinations performed at the bedside by the respiratory consult service for assessment of pleural effusion. 24 (48%) of referrals were from the oncology/haematology service, 24 (48%) from other medical teams and 2 patients from general surgery. on 44 (88%) of ultrasound examinations pleural fluid was detected and drainage was performed. in 20/44 (45%) fluid was drained by aspiration alone and in the remaining 24 (55%) a seldinger chest drain was placed. 30 patients (68%) had malignant or paramalignant exudative effusions (13 with lung primary; 13 with metastatic cancer from other sites and 4 with lymphoma), 6 (13%) had parapneumonic effusions and there were 4 transudates, 1 hemothorax and 3 unexplained exudates. the procedure was well tolerated by all patients. one patient had a small post-aspiration pneumothorax on chest x-ray that required no further intervention and there was one drain misplacement. ultrasound is safe, portable and sensitive for detection of pleural fluid. it can be used at the bedside by respiratory physicians to guide management of pleural effusion. in an effort to standardise treatment of primary spontaneous pneumothorax (psp) and secondary spontaneous pneumothorax (ssp), the british thoracic society (bts), in 2003, published the first evidence based guidelines for management of this condition. the objective of this audit was to assess compliance with the bts guidelines in amnch. retrospective hipe data, chart and radiology review of all spontaneous pneumothoraces admitted to amnch during 2007. there were 29 spontaneous pneumothoraces admitted to hospital in the year studied (17 psp's and 12 ssp's). of the psp's: mean age was 26.2 years; male:female ratio was 15:2; 14 were classified as large and 3 as small; 1 attempted aspiration; 15 intercostal drains were inserted with an average drain time in situ of 2 days; mean drain calibre was 20 fr. of the ssp's: mean age was 48.3 years; male:female ratio was 10:2; underlying pulmonary disease was copd in 8 and cystic fibrosis in 4 (2 patients); 7 were classified as large and 5 as small; none were aspirated; 6 intercostal drains were inserted with an average drain time in situ of 3 days; mean drain calibre was 22 fr. conclusion: bts guidelines are not being adhered to in amnch. in particular, aspiration, which is the recommended first line treatment in psp, is an underutilised therapeutic procedure. on average, the calibre of intercostals drain used was too large and not in keeping with the guidelines which recommend initial placement of small calibre (10-14 fr) drains. it is important to note that our data reflects patients admitted to hospital, and does not include patients managed and discharged from the emergency department. penetrated chest trauma is potentially fatal if not promptly addressed. we aim to review the incidence, demographics and the outcome of patients with napct who attended a single level-iii trauma centre. we conducted a retrospective study of all napct's presented to cork university hospital between 1997-2007.our definition of napct is non accidental injury of chest involving sharp objects penetrating skin and muscular layers with or without injury to deep structure above the level of the diaphragm requiring hospital admission. 149 patients with napct wounds were admitted to c.u.h from january 1997 to december 2007. there were 24 females (17.8%) and 125 males (83.2%) with a median age of 30 years (range 14-93). the average length of stay in hospital was 7 days.the highest incidence was 9.5/100000 population (n = 19), which was in 1997. there was a decreasing trend in the following nine years. the incidence in 2007 was 3.8 injuries /100000 population (n = 14 patients). the incidence of napct's was low and the annual incidence is decreasing over the last 10 years. young men were the commonest victims. pleural infection is a difficult management issue with approximately 40% patients requiring surgical intervention and a 20% mortality rate. early diagnosis and appropriate therapy is vital to reducing morbidity, mortality and health care costs. this is an audit of the management of pleural infection in the ulster hospital from february to april 2008 as per bts guidelines. pleural infection was diagnosed as pleural fluid with ph \ 7.2, ldh [ 1000 iu/l, glucose \ 2.2 mmol/l. eight patients were identified, 5 (62.5%) male with a mean (sd) age 51 (±12) years. all patients had pleural fluid sampling within 24 hours of suspected infection. all patients had chest drains inserted with median (iq range) duration of drainage 7 (6-22) days. positive bacterial culture from pleural fluid was obtained in 2 (25%) patients (haemophilus influenzae, enterococcus) and from sputum in 3 (37.5%) patients (h.influenzae, coliforms, klebsiella). antibiotic therapy was as per bts guidelines. median (iq range) hospital stay was 28 (15-41) days while surgical intervention was required in 3 (37.5%) patients. one patient died at day 13 from a non-respiratory complication. this audit demonstrates appropriate management of pleural infection but shows that pleural infection remains a difficult management problem with significant morbidity and mortality. twenty-two patients died awaiting lung transplantation. the highest mortality rate was among those patients with idiopathic pulmonary fibrosis n = 10. the mode of death was acute exacerbations of ipf. 8 cf patients, 2 emphysema patients and 1 sarcoid patient, 1 patient with bronchiectasis died awaiting lung transplantation. these data indicate that the mortality rate of patients awaiting lung transplantation is highest amongst patients with idiopathic pulmonary fibrosis suggesting that early referral on the basis of a gas transfer less that 40% predicted as per international guidelines should be considered. approximately 900 lung cancers are diagnosed annually in northern ireland, however despite recent advances in the management of this disease little impact has been made on survival rates 1 . the short interval from diagnosis to death and the complex problems that are a reality for many of these patients make it imperative that health care professionals gain a greater understanding of the experiences of those living with this disease. this qualitative study aimed to explore the experiences of patients and carers living with a lung cancer diagnosis within a northern ireland context. a secondary aim was to describe participants' experiences of service delivery in order to identify any areas for improvement. a semi-structured in depth interview process was used and a purposive sample of 23 participants was identified, comprising 12 patients with advanced lung cancer and 11 carers. the central emerging theme of ''living with dying'' highlighted the struggle of dealing with a poor prognosis and the pervasive uncertainty associated with an unpredictable disease trajectory, whilst trying to maintain some quality of life and positivity. the findings confirm the need for effective support and information pathways that provide timely and responsive services which embrace the holistic needs of patients and carers. 1 the total number of patients was 196 and complete data was available on 141 (72%) patients. the mean age was 65 (18-92). there were 124 (63%) male and 72 (37%) female patients. the mean duration from first radiological investigation suspicious for lung cancer to first procedure, first diagnostic procedure and first treatment regardless of modality were 19, 29 and 54 days respectively. the mean interval from first procedure to first diagnostic procedure, and mean interval from first diagnostic procedure to first treatment were 10 and 25 days respectively. there were 183 (93%) patients with confirmed cancer diagnosis and 38 (21%) of them went for curative surgery. in conclusion, our practice in management of lung cancer is consistent with the bts guidelines. transbronchial needle aspiration (tbna) is a bronchoscopic technique that enhances the diagnosis and staging of patients with thoracic cancers and other diseases. while endobronchial ultrasound guided-tbna (eus-tbna) is the gold standard, many units do not have access to the technology and expertise required. this study explored the clinical utility of tbna in the diagnosis and staging of patients with thoracic cancer in a university hospital. we studied the yield of tbna in patients diagnosed with thoracic cancers in our institution from september 1st 2006 to august 31st 2007. from 114 patients with thoracic cancer, 18 patients had tbna performed. eleven specimens (61%) were diagnostic (4 from hilar nodes and 11 from subcarinal nodes) and 6 of the 11 (55%) were the only positive diagnostic specimen, avoiding the need for further diagnostic procedures such as transthoracic needle biopsy and mediastinoscopy in these patients. three of the eleven cases were small cell lung carcinoma, 5 were squamous cell carcinoma and 3 were adenocarcinoma. while eus-tbna is the gold standard for the diagnosis and staging of patients with thoracic cancer, tbna provides a clinically useful alternative in units that are not equipped to provide this service. however, dedicated training in the use of tbna should preclude the routine use of this procedure in bronchoscopy units. types of cancers in our study are as follow: poorly differentiated small cell cancer 14 (all smokers), squamouss cell cancer 13 (all smokers), nonsmall cell ca 11 (3 nonsmokers), adenocarcinoma 10 (all were smokers), nonclassified 8, wide spread metastasis 3, carcinoid tumour 2.and one patient presented with mesothelioma transformed to sarcomatous changes. among 6 nonsmokers 3 had nonsmall cell cancer, 1 mesthelioma, 1 metastatic lung cancer, 1 was unclassified. 37 patients underwent bronchoscopy, bronchoscopic biopsies, 34 bronchial washings sent,14 patientst under went ct guided biopsies, 2 had mediastinoscopy and lymph node biopsy to confirm diagnosis. 26 patients were given chemotherapy and 4 got radiotherapy. at the time of diagnosis 12 patient were referred for surgery, among them 7 had lobectomies, 3 pneumonectomies, 1 wedge resection, 1 deemed unresectable perioperatively. 26 patients were given chemotherapy and 4 got radiotherapy. 18 patients deemed unsuitable for treatment. majority of these patients referred by general practitioners and the early diagnosis determined the final out come of the patients. there needs to be high index suspicion for early patients refrall in the presence of risk factors for lung cancer. 2.11 audit of lung mass/nodule service regional hospital department of respiratory medicine, midland regional hospital mullingar a lung mass/nodule service was established in 2004. service was provided by consultant respiratory physician and respiratory nurse specialist. we performed a retrospective audit on this service and held regular weekly mdt meetings involved oncology service in tullamore and st james's lung cancer service to monitor the progress of all possible lung cancers. aims of study: aim of the service is to provide prompt access to the patients, their investigations, management and proper follow up. in last five years 395 patients were reviewed. 226 (57%) were male. average age was (range 21-95). number of patients evaluated each year was 46 in 2004, 80 in 2005, 85 in 2006, 80 in 2007, and 103 in 2008 (jan-aug). more detailed analysis was performed of a subgroup this population; all patients who presented from jan-june 2006. total number of patients was 36. 30(83%) presented with lung mass, 6 (17%) with pulmonary nodule, 6 (17%) had associated pulmonary infiltrates, 4 (11%) haemoptysis, 1 (3%) enlarged mediastinal lymph nodes. 28 (78%) had bronchoscopy, 5 (14%) ct/us guided biopsy, 1 (3%) thoracentesis, 1 deemed unsuitable for further evaluation. 27 (75%) were diagnosed with bronchogenic carcinoma (22 non small cell cancer 61%, (4 squamous cell cancer 11.11%, 5 adenocarcinoma 13.88%, 13 undifferentiated 36.11%), 5 small cell cancer 13.88%, 1 sarcoidosis (2.77%), and 8 had either an initial nondiagnostic evaluation or assigned to follow-up ct scan protocol (as per fleischner society guidelines 2005). of the lung cancer patients, 15 (42%) were referred for chemotherapy/radiotherapy, 4 (11%) for surgery, 4 (11%) for palliative management. of the 8 (22%) assigned to the follow-up protocol, 1 was subsequently diagnosed with cancer. lung mass/nodule service facilitates early diagnosis of lung cancer and subsequent assignment to appropriate therapy; it also provides a coordinated careful follow-up of lung nodules. many patients with lung cancer are symptomatic from diagnosis, 1 and quality of life (qol) may be maximised through use of specialist palliative care in parallel with other treatments. patients are at increased risk of psychological disorders such as depression and anxiety. 2 this study explored anxiety, depression and qol of a small group of patients (n = 5), predominantly male (66.7%), mean age 74 years, using the marie curie ''breathing space'' outpatient clinic over a four week period. ''breathing space'' is a nurse led multidisciplinary clinic using integrative, person-centred care to maximise qol of patients with lung cancer through weekly assessments and interventions to enhance breathing, stamina, relaxation, mood, independence and well-being. a prospective survey design incorporated qualitative and quantitative approaches using semi structured interviews at baseline and after four weeks. qualitative data explored patient expectations and experiences of clinic attendance. most reported preconceived fears about the clinic due to poor information which were later dispelled. anxiety, depression and quality of life scores improved for this small sample. the core elements of ''breathing space'' may contribute to improve qol for patients with lung cancer. further work is needed on a larger sample to confirm the effect on anxiety and depression. we performed a retrospective analysis of procedures done in the endoscopy/bronchoscopy unit in a dublin teaching hospital from 2004 to 2008. since 2004, all data in the unit has been entered live to an electronic patient record, replacing hand written reports. this excludes procedures carried out in areas other than the endoscopy unit, such as icu, hdu, bone marrow transplant unit etc. a total of 50144 procedures were carried out, which consisted of upper gi endoscopy 22,984 (46%), colonoscopy 16,095 (32%), bronchoscopy 3,681 (7.5%), cystoscopy 5%, ercp 5%, sigmoidoscopy 2.5%, trus biopsy 1.5%, ileoscopy 0.5%. during bronchoscopy, the following procedures were performed; tbbx 735 (20%), endobronchial bx 565 (15.5%), tbna of mediastinal glands or peripheral lesions 715 (20%). indications for bronchoscopy were; mass on the chest radiograph 20%, haemoptysis 6%, consolidation 5%, persistent symptoms with normal chest radiograph 2.5%, pleural effusion 2%, recurrent infections 1%, hoarseness 0.5%, stridor 0.2%, others 6%. focal endobronchial tumours were present in 529 (15%) cases, the vast majority of which were cancer. excessive haemorrhage occurred in 86 (2.5%), which was controlled in all cases with standard measures. the incidence of pneumothorax post transbronchial biopsy was \1%. mortality was zero. an electronic database provides a useful tool for audit of clinical practice. bronchoscopy is the third most common procedure performed in an endoscopy unit of a major teaching hospital in dublin. the most common pathological diagnosis in this unit is lung cancer. the workplace smoking ban protects adults from secondhand smoke (shs/ets) but there is now concern about the protection of children from (shs) in the home or in cars. we evaluated the levels of shs/ ets that might be experienced by a child in the back of a car, where the driver is smoking. a particle was located in the back of a car at the height of a child's car seat. measurements were recorded prior, during, and 5 minutes after smoking had stopped. this was repeated with the drivers window open, and with it closed. these results show that particulate levels rise significantly with active smoking. the levels are significantly higher when the driver's window is closed during smoking, however even with the window open the levels are significantly elevated. post smoking levels are higher than the levels during smoking with the window open. these results are comparable with those reported from the us. children in the back seat of cars with the driver smoking are subject to very high levels of ets, which persist even after the smoking has stopped. having the driver's window open reduces the exposure, but it is still significantly higher than background levels. cells (gift from dieter c. gruenert, usf) were pretreated ±5% cse and then stimulated with lps; cytomix 1 (tnf-a, il-1b, lps) or cytomix 2 (tnf-a, il-1b, ifn-c) and il-8 release measured. exposure to cse significantly reduced the stimulated il-8 release in all cases in the cfbe41o-cells (cytomix 1: 883.2 ± 143.2 pg/ml, +cse 575.2 ± 185.4 pg/ml; cytomix 2: 3646 ± 520.1 pg/ml, +cse 1780 ± 304.9 pg/ml; lps-pa 100 lg/ml 470.5 ± 80.9 pg/ml, +cse 281.7 ± 64.9 pg/ml; all p \ 0.05). however, il-8 release from 16hbe14o-cells was only significantly inhibited basally and after stimulation with cytomix 2 (cytomix 2; 1573 ± 215.7 pg/ml, +cse 1159 ± 343.5 pg/ml; p \ 0.05). these data indicate that the response of the cf cell line to cse differs from that of the normal cell line. cse inhibits via tlr4 in a549 cells causing a reduction in both basal and lps stimulated il-8 release. this would provide a rationale for reduced responses to cytomix 1 or lps. further studies will examine the effect of cse on tlr4 in our cell lines. 1 previous studies have demonstrated improved lung health in bar workers, and reduction in cardiac morbidity following such bans. we sought to evaluate medical admissions before & following implementation of the ban, to assess for any impact on respiratory and cardiovascular admissions. data were obtained for all medical emergency admissions to our institution in the 24-month periods from january 2002-december 2003, and january 2005-december 2006 using the hospital in-patient enquiry system. data were examined for trends in respiratory and cardiovascular disease between the study periods. medical admissions increased over the study period (02/03 n = 5386; 05/06 n = 6352). however, there was a decrease in the proportion of admissions due to pneumonia (rr 0.74), asthma (rr 0.86), spontaneous pneumothorax (rr 0.78), stroke (rr 0.79), and unstable angina (rr 0.70). admissions with copd increased (rr 1.4). these changes were seen in smokers & non-smokers. no significant mortality impact was observed. the proportion of medical admissions for respiratory and cardiovascular disease decreased in the years following the implementation of the smoking ban. any impact on chronic diseases may take many more years to become apparent. the ban on smoking in public places came into force in northern ireland on the 30th april 2007. the objective was to look at the impact of the smoking ban on smoking prevalence in diabetic patients a year before and a year after the smoking ban. a retrospective analysis of smoking habit data held on a computerised data base (the diamond system) a year before and a year after the introduction of the smoking ban. the data of 1807 diabetic patients was analysed. there were 59% male and 41% female. type 1 diabetics were 19% and 81% of the patients had type 2 diabetes mellitus. a year before the smoking ban, 250 (14%) were smokers-60% male, 40% female. the prevalence of smoking in type 1 male diabetic was 19% and in type 1 female diabetic 18%. the prevalence of smoking in type 2 male diabetics was 13% and in females 12%. one year after the smoking ban 249 (14%) were smoking-61% male and 39% female (p [ 0.5). to date, there has been no statistical difference in the number of patients with diabetes mellitus who smoke since the introduction of the smoking ban. the ban on smoking in public places came into force in northern ireland on the 30th april 2007. our objective was to look at the impact of the smoking ban on blood pressure control in a group of patients with diabetes mellitus. a retrospective analysis of smoking habit data held on a computerised data base (the diamond system) was performed, one year before and one year after the introduction of smoking ban. the data of 1807 diabetic patients was analysed, 59% male and 41% female. type 1 diabetics were 19% and 81% of the patients had type 2 diabetes mellitus. mean blood pressure of the non-smokers was 135/ 73 mmhg before the smoking ban and 131/71 mmhg one year after the introduction of smoking ban. the mean blood pressure of smokers was 133/72 mmhg before and 133/72 mmhg a year after the ban (p [ 0.5). there was a small improvement in the blood pressure control of nonsmoking diabetic patients a year after the ban, however it was not statistically significant. overall, blood pressure control was at target. the law prohibiting workplace smoking improved the respiratory health of dublin bar workers. however, non-smoking bar workers living with a smoker are exposed to cigarette smoke at home. (2007) . for this study 39 barworkers were studied. current smokers (n = 5), and asthmatics (n = 4) were excluded from analysis. 9 (30%) lived with a smoker, and 21 (70%) did not. all barworkers had similar baseline co levels, but those without exposure at home had a more significant reduction 1 yr post ban. breathing symptoms in those without home exposure were lower at baseline, while ent symptoms were similar in both groups. fev1 remained constant in those not exposed to cigarette smoke at home, while it declined (not significantly) in those with home exposure. exposure to cigarette smoke in the home continues to put nonsmokers at increased risk of significant respiratory symptoms. smokefree homes would bring improved health. smoking adversely affects the health of patients with cf. study aims: to determine active and passive smoking exposure among adult irish cf patients. methods: cf patients attending cuh completed a questionnaire relating to personal smoking and second-hand smoke (shs) exposure, correlated with pulmonary function and exacerbation-rate data. results: 91 patients (51 male) completed the questionnaire (table 1) . 7.6% were currently smokers. 5.5% admitted to having tried smoking at some time. in the never-smoked group (n = 79), 38% were currently exposed to shs; mean duration of exposure was 19.1 pack-years (95%ci: 6.9-31.3), while 27% had previous shs exposure; mean duration of exposure was 13.9 pack-years. 35% were never exposed to shs. in those currently exposed, the source was from a parent in 52.6% and a sibling in 18.4%. anthropometric data and exacerbation rates were similar between groups, but smokers showed a trend towards better lung function. a large level of exposure to shs exists among irish cf patients, with a clinically relevant proportion actively smoking. this study identifies a need for more aggressive smoking cessation strategies for both patients and caregivers. supported by cfai. a non-probability sampling of self-identified glc was recruited using electronic and print media advertisements between december 2006 and march 2007. 1,648 respondents completed the questionnaires. otc data for the same period was analysed (n = 4,000 respondents). appropriate statistical analyses were performed to compare the mean differences in smoking rates between these two surveyed populations across age, gender and socio-economic groups (ses). adjusted current rates in glc were 26% and 24.6% in general population (p = 0.99) and ''heavy'' smoking prevalence was 44.1% in glc and 36.6% in general population (p = 0.02). upper ses glcs are ''heavy'' smokers compared to general population of similar ses group (p = 0.01). glcs (\25 years) were ''heavy'' smokers compared to general population of same age-groups (p = 0.01). no significant gender differences were observed. more glc were ''heavy'' smokers than the general irish population, but current smoking rates among the glc in ireland were not significantly different from the general population. a sampling frame of 731 post-primary schools was used to randomly select schools for the 2007 issac study. 2,809 children (13-14 years) completed the isaac questionnaire. smoking prevalence was based on children's self-reported answer to the question ''if you travel by car does anyone smoke cigarettes in the car [yes/no]? for part 2, we used ''upgreen counters'' for 3 vantage points: a shopping car park (saturday 5-6 pm); near a school (monday 1-2 pm); a busy sub-traffic junction (monday 5-6 pm), and ''moving cameras'' for the 4th vantage point, the civic offices (monday 7-10 am). smoking prevalence in cars was 14.8% in ireland. for the four vantage locations, the prevalence rates were: 6.5% (n = 34/489), 2.2% (n = 11/503), 3.1% (n = 37/1184), and 4.3% (n = 13/300), respectively. smoking in cars is a public-health policy issue. our findings show that children are regularly exposed to second-hand-smoke in cars in ireland and this demands legislation. in march 2006 we performed a pilot study to determine the feasibility of a pulmonary outreach programme in the midlands area. there are currently two similar programmes in ireland, but this was the first in a rural setting. our programme has two pathways of care: patients discharged \96 h received home visits on 3 consecutive days and were followed up for 14 days (early discharge programme, edp); if discharged [96 h due to unsuitability for the edp, they were reviewed for the first two weeks by specialist respiratory support within the community (outreach programme op). all patients were subsequently enrolled into pulmonary rehab. to date, 242 patients have been enrolled to pop. 59% were male; mean age 72 years; majority had severe disease (30.7% stage ii, 30.7 iii, 22.8% iv). 29.9% required ltot, 6% home-nppv. 24.4% were active smokers. 12.8% were readmitted within the first 2 weeks. the mean length of inpatient stay was 2.5d for the edp, and 4.5d for op; the average los nationally is 8.6 days. overall there were substantial financial savings. thus, rural pulmonary outreach programmes are feasible as they lead to a reduction in hospital los, improved patient knowledge of their disease, and are cost effective. the physical and psychological symptom burden associated with patients with advanced copd has been compared to that of patients dying with cancer. traditionally palliative care services have focussed on people with cancer, however recently the american thoracic society have endorsed the concept that palliative care should be available to patients at all stages of their illness [1] endorsing the who palliative care definition [2] . the irish hospice foundation and hse undertook a study in 2008 examining how all levels of palliative care can be extended to people with copd. challenges identified for introducing palliative care for people with copd include the uncertainty of the copd disease trajectory, the tension between delivering hope and planning for the inevitable, the lack of comprehensive respiratory services and the need for further education and research. the development of a model of care for patients with stage iii / iv copd providing a clear pathway of access to all levels of palliative care, the production of information and educational material, the requirement that all palliative care services are accessible to copd patients as required and the need for further collaboration between respiratory and palliative care services are key recommendations in the report of the study. school of pharmacy, queen's university 1 , and mater hospital 2 , belfast introduction: self-management plans for copd is derived from success in asthma. patients may benefit from the early intervention following selfmanagement plan 1 . methods: 173 patients (67 y; 54% females) with mod-severe copd, were randomly assigned to an intervention group (86) and usual care (87). a pharmacist delivered an education program on disease state, medications, home exercise and breathing techniques. a booklet and a customised action plan for acute exacerbations (antibiotics and steroids) were given. follow up was at three months by telephone and a six months scheduled visit. the eq-5d health status and sgrq were administered to all patients. outcomes included admissions, a/e visits and quality of life. results: at 6 months the intervention group had reduction in both admissions [34 (43%) vs 15 (19%); p = 0.01], and a/e visits [43 (53%) vs 21 s447 (25%); p \ 0.010]. on the sgrq there was improvement in the symptom (-7.9; p = 0.01), impact (-7.6; p = 02). and total score (-5.6; p = 0.05). physical activity scores did not improve. the difference in the eq-5d scores improved both vas scale [54.6 vs 47.3; p = 0.004], and utility scale [0.51 vs 0.43; p = 0.062]. this ongoing study indicates that a clinical pharmacy led management programme can reduce the need for hospital care in patients with moderate-to-severe copd and improve aspects of their health related quality of life. copd is a leading cause of morbidity and mortality worldwide. exacerbations of copd result in frequent hospitalisation and account for 70% of the costs associated with the disease. our objective was to identify risk factors which predict relapse requiring readmission following an exacerbation of copd. from 2001 to 2007, 348 consecutive exacerbations of copd admitted to hospital were prospectively studied. baseline demographics, number of hospitalisations in the previous year, oxygen use and smoking history were assessed. breathlessness and quality of life scores were recorded and oxygen saturations and spirometry measured. rehospitalisation data was collected at day 14, 6 weeks and 3 months. during the follow up period, 46 patients (13%) were readmitted by day 14, 81 (23%) were admitted by six weeks and 106 (37%) were admitted by three months. logistic regression analysis identified hospitalisation in previous 12 months (p = 0.03, or 2.25, ci 1.1-4.8) and borg score 3 or higher (p = 0.04, or 2.15, ci 1.0-4.7) predicted readmission in 75% of patients at day 14. home oxygen use (p = 0.001, or 3.28, ci 1.6-6.5), pack year [ 50 (p = 0.008, or 3.13, ci 1.4-7.3) and borg score [ 3 (p = 0.001, or 3.31, ci 1.6-6.8) predicted 6 week admission in 68.9%. admission in the previous year and borg score of c 3 predict early relapse, while home oxygen use, pack-year history c50 and borg score of c3 predict later relapse following an acute exacerbation of moderate copd. aecopd is an inflammatory lung disease associated with systemic consequences. a systematic analysis was undertaken of alpha-1 antitrypsin (a1at), c-reactive protein (crp) and procalcitonin (pct) in the serum of patients with aecopd and matched inflammatory controls (cellulitis), pre-and post-antibiotic therapy. a1at and crp are acute phase proteins. pct, a serum calcitonin precursor, is also raised in bacterial infections. venous samples from 12 patients were analysed in this prospective study. amongst the 6 aecopd and 6 controls (cellulitis), were 7 males and 5 females (aged 37 to 80). crp(mg/l) levels were elevated in cellulitis (mean ± std error, 49.1 ± 10.4) and aecopd (30.3 ± 14.0) patients prior to treatment. a1at(lmol/l) levels were also significantly elevated in cellulitis (34.2 ± 4.6) and aecopd (29.8 ± 3.7) patients. following intravenous antibiotic therapy, crp levels fell in cellulitis (11.4 ± 4.6) and copd (8.0 ± 5.9) patients. similarly, a1at values fell in cellulitis (29.9 ± 4.1) and in aecopd (23.8 ± 3.2) patients. pct (ng/ml) levels were not elevated in all individuals with either cellulitis (4/6) or aecopd (2/6), but did decrease significantly in those that were elevated following antibiotic therapy. crp and a1at levels are significantly elevated during aecopd and cellulitis. both levels fell significantly post antibiotic treatment (p = 0.0015 for crp and p = 0.0015 for a1at). pct levels, when elevated, were reduced post antibiotic therapy. these data suggest that aecopd elicits a systemic response similar to a non-respiratory infection and this response to treatment can be monitored using biomarkers. in copd there is a cytotoxic t-cell infiltrate in the airway mucosa. it has been suggested that a virus may be a co-factor. we have recently shown high levels of ebv in severe disease 1 . we wanted to establish if it was present in early disease. we recruited 44 smoking (53 pack y) subjects (58y) with early copd with mean fev 1 1.84(66%) and 45 smoking (41 pack y) unobstructed smokers (49y) with mean fev 1 2.58(92%). none of the subjects had used inhaled or oral steroids. nose and throat swabs were taken. induced sputum was obtained using hypertonic saline. total nucleic acids were extracted, and ebv dna was detected using taqman quantitative pcr. results: ebv was detected more often in the copd (23/45 swabs and 33/43 sputum) than the control (13/45 swabs and 20/42 sputum). p = 0.05 and 0.007 for swabs and sputum respectively (fisher's exact test). there was a wide range of copy numbers which were not different among those who were positive. conclusion: ebv is present more frequently in early copd than in unobstructed smoking controls. ebv is known to be a cyclical herpes virus which comes and goes. it may have a role in the pathogenesis of copd. background and method: niv is a valuable treatment for hypercapnic respiratory failure. [1] transcutaneous co2 monitors(tosca) has provided novel approach to monitor these patients.. we assessed niv service and the use of transcutaneous co2 monitors in our tertiary care center. charts of patients attended from july 2006 to dec 2007 were retrospectively evaluated. data was retrievable on 32 from total of 34 patients. there mean age was 64 ± 14yrs. 19 were female. copd was in 75%, decompensated obesity/hypoventilation was 18% and neuromuscular/chest wall deformity was 7%. fev1 (mean) was 47.57% -16.26%. 97% had type 2 and 1 (3%) had type 1 respiratory failure. their average-ph was (7.37 -0.05). mean pco2 was (8.16 -1.69). only 47% of patients had repeat blood gas analysis at 1-2 hour, tosca was used to monitor non-invasive ventilation in 26 (82%) of patients. the average number of use per patient was 4.46 -2.88. there length of stay(avg) was 15.61 days. their mean ipap and epap were 14.24 and 5 respectively. 3(8%) patients were commenced niv in hdu. 3(8%) died due to respiratory failure. conclusion: 82% of patients were successfully monitored with tosca. we conclude that tosca is a valuable tool for monitoring patients at ward level. respiratory assessment unit, crest directorate, st. james's hospital, dublin 8 as part of its transformation programme, the hse established a group in september 2007 to develop a national strategy for the management of copd. the aim of this survey was to capture information regarding the availability and range of physiotherapy services for persons with copd in ireland and thus inform the report of the national copd strategy group. the survey was emailed to 120 physiotherapy managers across care settings in november 2007. the survey sought information relating to the range of physiotherapy services for persons with copd provided at each site, as well as perceived service deficits and existing/potential innovations in practice. data were analysed using descriptive statistics. fifty-seven sites responded to the survey. no formal joint services between acute hospitals and pccc were reported. pulmonary rehabilitation programmes (prps) were available in 12 sites only. service deficits reported related to lack of appropriate treatment space, lack of specialised respiratory staff, the absence of prps, and lack of interaction between acute hospitals and community services. physiotherapy services for persons with copd vary greatly across sites and settings. prps are not widely available and are primarily hospital based. there is a need for wider availability of joint hospital/ community based initiatives such as copd outreach and prps. pulmonary rehabilitation has established efficacy, but patients often require follow-up care or maintenance. there are few studies that explore the patients' experience of pulmonary rehabilitation and maintenance. also, there are no guidelines for health professionals as to what constitutes effective maintenance for clients who complete pulmonary rehabilitation. the study aim was to explore patients' perceptions of pulmonary rehabilitation and the maintenance options provided to them. a qualitative, exploratory descriptive design used focus groups to collect data. the purposive sample (n = 25), had a diagnosis of either copd or bronchiectasis, and had attended a pulmonary rehabilitation programme within the last year. a focus group schedule using open ended questions and prompts was designed. discussions were transcribed verbatim and burnard's (1991) thematic content analysis was used to guide data analysis. the dynamics of group participation and peer support were identified as important incentives for patients. increased confidence and personal achievement were described as outcomes. the reasons for non-participation in maintenance were also elucidated by patients. this study provides an important contribution in relation to the experience of patients and the findings enhance current quantitative studies. patients' experience of outcomes and expectations has the potential to influence future services. following recommendations from the hse transformation programme 2006, a multidisciplinary committee was established to develop a national strategy for the management of copd. as a member of the committee representing anail, the author conducted a survey to establish the range of inpatient and outpatient services provided by respiratory nurses for persons with copd. a questionnaire was devised to gather information regarding the provision of services such as inhaler technique, oxygen assessments, copd outreach programmes, pulmonary rehabilitation programmes (prp's) and palliative care services for persons with end stage copd. thirty-five members of anail were surveyed in october 2007. data were analysed using descriptive statistics. a response rate of 57% was achieved. inpatient and outpatient services such as respiratory nurse reviews, oxygen assessments, self management plans were provided by more than 70% of respondents. of those who replied 100% provided inhaler technique education, 63% can refer persons with copd for prp, 25% run an outreach programme and 15% providing limited palliative care services. a number of current innovations and deficits within the services provided by respiratory nurses were highlighted. the contribution made by specialist nurses to the acute and chronic respiratory service is reflected by this survey. the inspiratory fraction-inspiratory-to-total-lung capacity (ic/ tlc) is an independent risk factor for mortality in chronic obstructive pulmonary disease (copd) 1 . ic/tlc b 25%predicted is associated with significantly shorter survival. little data exists about the effect of pulmonary rehabilitation (pr) on survival in copd 2 . the purpose of this study is to examine the effect of pr on ic/tlc. 64 patients (mean age 65.3 ± 9.5), with clinical evidence of copd (mean fev 1 45.7 ± 17%predicted, mean ic/tlc 0.29 ± 0.11%predicted) were enrolled in an 8 week pr programme, consisting of twice-weekly sessions of exercise and education. assessments/re-assessments consisted of lung function (spirometry, diffusion, sniff nasal inspiratory pressure, capacity), exercise tests (shuttle, treadmill) and quality-of life-questionnaires (qol). 10 patients, re-assessed at 6 months, demonstrated improvements in exercise and qol compared to baseline (p \ 0.001). these patients were divided into groups-group 1 ic/tlc b 25% predicted (n = 4), group 2 ic/tlc [ 25% predicted (n = 6) at baseline. there were no between-group differences in improvements in exercise or qol at 6 months. group 1 ic/tlc improved at 6/12 from baseline 0.17% predicted to 0.22% predicted, and group 2 from 0.33 to 0.36% predicted (not significant). although the results are not significant there appears to be a trend in improved ic/tlc following pr. this study should be repeated with a larger sample size. department of medicine, midland regional hospital, mullingar, ireland primary objective was to assess the appropriateness of our hospital admissions for copd exacerbations as per nice guidelines. we also assessed the quality of their outpatient copd medical care. all copd related admissions mar-may 2007 were prospectively reviewed. 16 variables as per nice guidelines were considered with one point for each variable. a score of zero was considered an inappropriate admission while c1 was appropriate. 50 patients were included. mean age was 74 (54-91), 26 (52%) were male. 49 (98%) patients were admitted as per guidelines, 1 (2%) patient met no criteria. commonest variables present were: poor level of activity 34 (68%), significant co morbidities 28 (56%), inability to cope at home 24 (48%). least common variables were: impaired level of consciousness 8 (16%), cyanosis 6 (12%), acute confusion 4 (8%). 48 (98%) received antibiotics. 48 (96%) had spirometry performed for diagnosis. 19 out of 20 smokers (98%) were offered cessation advice. 45 (90%) were appropriately on inhaled steroids. 12 out of an eligible 24 (50%) were enrolled in pulmonary rehab. mean los was 8.6 days, and there was linear relationship between length of stay and guideline score. there was excellent compliance with nice guidelines for copd admissions. quality of outpatient care was good in the domains evaluated. up to 30% of copd hospital inpatients will be readmitted within 4 weeks of discharged. specific predictive markers of readmission are not currently in routine clinical practice. in this study, we assessed a remote monitoring system for continuous readout of patients' pulse rate and o 2 saturation (biancamed, ireland). a cohort of normal volunteers (n = 5) and copd patients (n = 10) were enrolled and full remote monitoring and psychological profiling (via a modified hospital anxiety and depression score (hads)) of patients' well being was performed. in controls and patients, mean percentage of recording time was 78% (range: 23%-100%) and 59% (range: 10%-99%) respectively. principal reasons for loss of recording were a) patients moving out of range of monitor, b) non-compliance due to impracticality and/or discomfort while wearing the device, and c) accidental slippage of the oximeter probe. analysis of time of o 2 saturation below 90%, 85%, 80% and 75% per hour revealed significant improvement over time in 90% of patients. one patient subsequently readmitted showed a significant deterioration prior to admission. in conclusion, this system shows potential in the early identification of copd patients who clinically deteriorate at home. in 2006, 29% of respiratory inpatient discharges related to copd. information on hospital services for copd patients was required for the development of a national strategy. a survey on relevant staffing, wards and diagnostic units, policies and practice and access to specialist services was distributed to acute hse hospitals via hospital networks. 26 hospitals responded (72%). written policies are in place for management of copd (46%), non invasive ventilation (niv) (73%) and long term oxygen therapy (58%). niv is provided in the emergency department (ed) (38.5%), medical assessment unit (mau) (15.4%), icu (82.6%), hdu (19.2%), respiratory ward (19.2%), all medical wards (26.9%). in almost two thirds of hospitals, all inpatients with copd can access respiratory nurse specialists, smoking cessation officers and palliative care services. access by ed/mau patients is possible in 54%, 27% and 12% of hospitals respectively and by gp referral in 27%, 15% and 4%. ten hospitals have pulmonary rehabilitation programmes (38%), five have onward referral mechanisms and four were planning a programme. the waiting time for programmes is up to one year. three hospitals have outreach programmes in place. this survey highlights the variation in hospital based services for copd patients and opportunities for service development. in 2007 the health services executive established a steering group to develop a national strategy for the management of copd. this study aims to describe the range of services available to copd patients and ease of access from the primary care perspective. a postal survey was distributed to a random sample of 500 gps by the icgp. data was analysed using excel. 121 valid questionnaires were returned (response rate 24.2%) from practices in 23 counties. 53.7% have access to spirometry within their own practice. a practice nurse usually conducts the test (64%) and a gp interprets the results (83%). patients are unable to access patient support groups (21.1%), pulmonary rehabilitation (23.9%), rapid access respiratory clinics (37.6%) or community options for management of an exacerbation-home based (49.1%) or local community unit/district hospital (30.9%). waiting times are up to six months for physiotherapy and pulmonary function testing and up to one year for respiratory consultant review, long term oxygen therapy assessment and pulmonary rehabilitation. this survey highlights geographical variation and gaps to be addressed for a shift to occur towards a community-based, responsive, flexible service for copd patients. oxygen therapy is an important treatment option for patients with severe copd, as long term continuous therapy (ltot). information on relevant community resources for ltot was required for the development of a national copd strategy. a survey was distributed by e-mail via each local health office (lho) manager (32), covering activity and costs of aids and appliances, policy and procedure. data was analysed using excel. twenty two responses were received from 14 local health areas (44%). there were wide population differences in the rate of ltot between areas, from 50-289/100,000. home oxygen can be prescribed by hospital consultant, gp, respiratory nurse specialist or physiotherapist. arrangements for follow-up of patients on long term home oxygen vary considerably. half of respondents have difficulty with the level of detail provided on home oxygen prescriptions. 64% have a policy on provision of portable oxygen cylinders. 86% are aware of arrangements for ongoing maintenance of oxygen appliances. cost of ltot in 2006 was estimated to be in excess of €4million. long term oxygen is an important copd therapy but is costly and has potential for harm. standardised practices are required for its use in the community. spirometry is the gold standard for diagnosis of copd. additional pulmonary function tests (pfts) can also assist in management. details of respiratory diagnostic resources in ireland were required to inform the national copd strategy. a questionnaire was developed in conjunction with the irish association of respiratory scientists and circulated to members via email. questions focused on staffing, workload, waiting times, tests available, referral sources and educational activities. ten laboratories responded (27%). pft activity ranged from 1,419 to 12,837. minimum waiting times ranged from 0 days to 3 weeks and maximum from four days to eight weeks. laboratories accepted referrals for basic pfts from respiratory consultants (all), other hospital consultants (all), respiratory nurse specialists (50%), emergency departments (90%), medical assessments units (60%) and gps (60%). tests confined to respiratory team/other consultant referrals included bronchial provocation, 6 minute walk, long term oxygen therapy and fitness to fly assessments. five hospitals participated in training relevant to copd in the hospital and two in the community. additional copd services included participation in pulmonary rehabilitation and outreach programmes. respiratory diagnostic laboratories are predominantly resourced for hospital referrals. examples are provided where scientists also provide a service to the community, for diagnostic tests and education. patients with copd have higher blood levels of markers of inflammation such as tumour necorsis factor (tnf-a), interleukin 6 (il-6) interleukin 8 (il-8) and c-reactive protein (crp). these are independent risk factors for decreased lung function and are associated with increased symptoms such as shortness of breath and respiratory rate. recently, tools to measure activity have been developed which continuously record patient free living activity and sleep. we hypothesized that there may be a relationship between levels of systemic inflammation and measures of free-living activities. thirty one patients were recruited: men (n = 14), female (n = 17). ethical approval and written consent was obtained. venous blood samples were taken (il-6, il-8, tnf-a and crp which were logged for normal distribution). a senseware ò activity monitor was worn for 7 consecutive days and the st.george's respiratory questionnaire were measured. pearson's correlations were undertaken using spss version 12 s451 mean age of 67.5yrs (+/-7.4) with an fev1 or 45(+/-41) and a mean smoke pack history of 46 (+/-32). a medium negative correlation was found between lgcrp and physical activity duration [r = -0.44, n = 24, p = 0.03]. a large correlation was found between the lgcrp and the st. georges respiratory questionnaire [r = .5, n 23, p = 0.015]. this was also reflected in the impact section of the questionnaire [r = 0.49, n = 23, p = 0.018]. c-reactive protein blood levels appear to be inversely correlated to free-living activities and quality of life. these data suggest that the measure of crp may be an important factor to include in the assessment of the severity of copd. during the months of july and august 2008, in a prospective study, patients were transferred from the adelaide and meath hospital within 4 days of their acute admission with copd, to peamount hospital for airc. 19 patients were enrolled: 8 males with a mean age of 66.6 yrs and a mean fev1 of 1.23 l (63%). the mean length of stay (los) in the acute hospital was 4.93 days and the mean los in peamount hospital was 13.7 days, with a total mean hospital stay of 18 days. we hypothesise that this extended hospital stay and targeted respiratory care will improve patients overall quality of life, breathlessness, and exercise capacity, and reduce their dependency on the acute hospital service and re-admission rates. these patients will be followed up over the next year as a continuation of this study. the miners' disability score (mds), developed during the compensation process for uk miners, utilises a ten-point scale. the medical research council(mrc) dyspnoea scale, previously validated using the incremental shuttle walking test(iswt), utilises a five-point scale which may be less discriminating. we aimed to validate the mds as a score of respiratory disability in copd patients. patient data (mds/iswt/endurance shuttle walking test(eswt)) from our pulmonary rehabilitation programme were initially analysed (n = 214; median fev 1 = 1.04 l; mean age = 69 yrs). subsequently, 40 inpatients (median fev 1 = 0.70l; mean age = 72.7 yrs) had baseline mrc dyspnoea grade, mds, and manchester respiratory activities of daily living score (mradl) determined. degree of association between variables was assessed using the spearman rank correlation. mds correlated well with iswt (rs = -0.69, 95%ci -0.75 to -0.61), but not with eswt. fev 1 was not associated with mds grade. mds correlated well with mrc dyspnoea grade (rs = 0.79, 95%ci 0.63 to 0.88). mrc grade and mds correlated well with mradl (mrc rs = 0.-0.72, 95%ci -0.85 to -0.53; mds rs = -0.85, 95%ci -0.92 to -0.73) score. the mds showed a more favourable association. the mds is a valid measure of respiratory disability that could be used to complement fev 1 and may provide an accurate reflection of performance status and disability in patients with copd. copd is an unremitting disease that impacts negatively on quality of life. the aim of this study was to compare functional capacity (fc); a measure of weight 9 distance over six minutes, with standard tools used in the assessment of patients with stable copd. forty one patients with severe copd: fev1 50% ± 20% predicted were recruited: men (n = 19), women (n = 23). senseware ò armbands were worn for seven days to quantify their average daily steps. ethical approval and written consent were obtained. pearson's and spearman's correlations were performed using spss version 12. functional capacity was significantly associated with mean daily steps: men (r = 0.79, p = 0.0001) women (r = 0.54, p = 0.008), shuttle walk test: men (r = 0.73, p = 0.0001) women (r = 0.54, p = 0.007) and fev 1 in men only (r = 0.61, p = 0.013). there was no relationship between fc and borg: men (r = 0.12, p = 0.268) women (r = 0.15, p = 0.491) or the saint-george respiratory questionnaire: men (r = -0.16, p = 0.528) women (r = 0.01, p = 0.973). we found that quantifying ''free-living'' measures is an important dimension of functional status not ordinarily captured and that functional capacity is a reliable outcome measure for assessing stable copd. the only gender difference identified was in male fev 1. patients (n = 22, mean age 66) admitted to castle hill hospital with acute exacerbation of copd were studied. patients were either treated with standard therapy plus 300 mg erdosteine bd (n = 13) or standard therapy alone. (n = 9) and followed up at day five and day ten. there was no significant improvement in subjective measures of breathlessness. at day 10 subjective cough frequency was reduced by 50% in the +erd group as compared with deterioration in the -erd group. fev1 increased by 70 ml -erd group and 600 ml in the +erd group. hacc 24 hour recordings on nine patients revealed 344 coughs on day one falling to 114 coughs by day five. there was a 76% reduction in cough frequency on the +erd group and 67% reduction in the -erd group. cough counting may be a useful objective marker to judge the success or failure of treatment strategies in acute exacerbation. chronic obstructive pulmonary disease is a lung disease characterized by chronic airflow obstruction that is not fully reversible measured using spirometry. the aim of this audit was to assess use of spirometry in diagnosis of copd in primary care. two hundred questionnaires were sent to primary care practices, seventy nine were completed. questionnaires identified which practices used spirometry. information was obtained on who performed spirometry within the practice, what training had been received, what criteria for screening for copd was utilised and general information on management of copd. we found 46% of practices had a spirometer. the most common reasons for not were cost involved (29%) and lack of confidence in interpreting results (31%). spirometry was performed most commonly by practice nurses (51%), interpretation of results was largely done by general practitioners (73%). only 5% had received recognised training in spirometry. the largest group of patients screened were symptomatic smokers over 45 years old, however only 40% of patients screened had spirometry performed. these data indicate that we need to promote training in the use of spirometry for the diagnosis and management of copd in primary care in ireland. physiological responses to pulmonary rehabilitation (pr) are measured using a variety of clinical exercise tests. we compared incremental with endurance cardiopulmonary exercise testing (cpet) in a series of patients attending a pr programme. thirty two patients with moderate or severe copd (age 66.1 ± 9.63 y, fev 1 42 ± 17.9% predicted) were recruited to an 8-week pr programme. exercise capacity was assessed using incremental cpet before and after pr in 14 patients and endurance cpet (at 75% of the peak incremental cpet workload) before and after pr in 16 patients. among the incremental exercise group, there were no significant differences in vo 2max (mls/min) (p = 0.2734), vo 2max (mls/kg/min) (p = 0.4434), vco 2max (mls/min) (p = 0.9999) or maximum workload achieved (watts) (p = 0.3835) before and after pr. among the endurance exercise group, there was a significant difference (p = 0.0003) in exercise duration (468 vs 815 seconds), but no differences in vo 2max (mls/min) (p = 0.5236), vo 2max (mls/kg/min) (p = 0.8250) or vco 2max (p = 0.3013) (mls/min) before and after pr. incremental cpet is a poor tool to measure physiological changes in exercise capacity associated with pr. endurance cpet is the more ideal test, demonstrating significant increases in endurance time associated with pr despite unchanged peak oxygen consumption and carbon dioxide production. cardiopulmonary exercise testing (cpet) provides a global assessment of the integrative exercise responses involving the pulmonary, cardiovascular, haematopoietic, neuropsychological, and skeletal muscle systems, which are not adequately reflected through the measurement of individual organ system function. this case report looks at how cpet makes the initial diagnosis of mcardle's syndrome. a 20 year old man initially presented to the cardiologists complaining of muscle fatigue after a short period of sustained exertion. all his cardiac investigations were normal. deconditioning would have explained the young mans symptoms adequately. as such, he was sent for cardiopulmonary exercise testing (cpet) to differentiate between poor aerobic conditioning and a possible pathological aetiology. the patient managed to exercise for six minutes and the test was limited by muscle fatigue. there was early failure in the aerobic metabolic pathway with a significantly reduced vo 2 max (oxygen uptake-aerobic metabolism) and the absence of a corresponding rise in the vco 2 signalling a concurrent failure of the anaerobic pathway. these results pointed towards a rare muscle enzyme deficiency. diagnosis was confirmed in the conventional way using a muscle biopsy. this case represents a unique and non invasive way of diagnosing a rare and often under diagnosed enzyme deficiency and underlines the versatility and diagnostic value of cpet. non-invasive ventilation (niv) is increasingly provided at ward level with implications for skills and practice development, support and inter-professional decision-making. despite recommendations by the british thoracic society (1) that niv can be provided outside of the intensive care unit, use of niv at ward level remains problematic, presenting particular contextual challenges to care. a qualitative research study was undertaken, involving focus group interviews with nursing staff (n = 10) and individual semistructured interviews with doctors (n = 4) from specialised (respiratory) and non-specialised units in a regional teaching hospital. a number of support issues were identified. niv was considered a time-consuming procedure, with a perception of inadequate staffing levels at ward level. access to experienced medical and nursing support was viewed as an integral part of niv service provision. knowledge gaps exist at local level specifically in relation to inadequate education and training. clinical practice guidelines for niv were recommended to guide practice. this research study sought to inform practice development, specifically the greater acceptance and use of niv at ward level, through examining care issues. the themes expressed in the findings point s453 towards the need for review of present service provision, particularly in the areas of education, training and guideline development. pulmonary function laboratories interface with all medical disciplines. there is anecdotal evidence that pulmonary function tests (pfts) are often requested inappropriately. an audit was undertaken in the pulmonary function laboratory, belfast city hospital to determine how many referrals were appropriate, the origin of each referral and the designation of the referrer. the audit randomly considered 965 requests over a six-month period. requests were reviewed by a clinical scientist and a consultant chest physician. a request was deemed inappropriate if tests unlikely to contribute to the patient's management were sought, or if tests were omitted that should have been requested. the requests originated from the following main specialities: respiratory medicine (35%), general surgery (22%), general medicine (14%) and haematology (9%). sixty-seven percent of referrals were made by junior doctors (junior or senior house officers) and 76% of these were appropriate. thirteen percent of requests were made by consultants of which 83% were appropriate. only 76% of respiratory referrals were appropriate. overall 77% of requests were considered appropriate, however there was significant variability among disciplines (65%-85%). the results indicate that many pft requests are inappropriate. additionally, the quality of respiratory referrals is not better than nonrespiratory referrals. consultant requesting does not guarantee correct referral. the findings have both resource and educational implications. a phenomenological approach enabled the researcher to gain an insight into the participants lived experiences and uncover their stories. the researcher is a respiratory nurse specialist and therefore has a particular interest in this area. a husserlian phenomenological approach with bracketing of preconceived ideas underpinned the chosen methodology. a total of seven interviews were transcribed by the researcher in this study. the participants were patients on long term non invasive ventilation. data was generated using unstructured interviews, which were tape-recorded. data was analysed using colaizzi's framework. beginning the therapy, process of adjustment to the therapy and gaining a new independence were the major themes identified within the study. this study is small however; the findings have implications for nursing practice, education and management locally and highlighted areas that require further research. dysregulation of pulmonary inflammation has been proposed as contributing to airways disease in cystic fibrosis (cf). the aim of this project was to compare two t helper-1 cytokines (interleukin (il)-8 and il-18) for their relative stability, activity and interaction with glycosaminoglycans (gags) which are highly abundant in the cf lung. bronchoalveolar lavage fluid (balf), serum and sputum pre-and post-nebulised hypertonic saline (hts) were collected from cf patients and compared to balf and serum from non-cf controls. western blots and elisas were used to visualize and quantify cytokine levels respectively. il-18 was undetectable within cf balf and was shown to be degraded by neutrophil elastase. as a biological consequence significantly reduced levels of il-2 were secreted by jurkat t lymphocytes (p = 0.0328). il-18 was competitively displaced from gags by il-8, which binds gags via electrostatic interactions. exposure of cf balf to hts or treatment of cf patients with hts displaced il-8 from gag matrices rendering the chemokine susceptible to proteolytic cleavage and reducing the chemoattractant capacity of cf sputum. in conclusion, gags possess the ability to influence the cytokine profile of the cf lung promoting a neutrophil dominated immune response and hts treatment may improve resolution of this inflammation. human cathelicidin, ll-37, a 37 amino acid antimicrobial peptide produced by neutrophils and respiratory epithelium has been shown to have antimicrobial activity as well as possess immunomodulatory properties. we have investigated this potential immunomodulatory effect of ll-37 using lps stimulated thp-1 monocytes. effects of ll-37 on the lps signalling pathway were investigated using western blot and elisa. ll-37 was shown to inhibit the degradation of ijba and ijbb during lps stimulation, whilst preventing the phosphorylation of ijba, ikk, stat-1, akt, c-jun and atf-2. cytokine data showed a partial reduction in lps induced il-8 and tnf-a with 10 lg/ml ll-37. further investigation revealed that lps induced cytokine production could be reduced to control levels when 10 ng and 100 ng of lps was used to challenge cells in the presence of 10 lg/ml of ll-37. washing of cells following pretreatment with ll-37 abolished ll-37's inhibitory effects on lps-induced il-8 production when compared to unwashed samples. results suggest that ll-37 is exerting its anti-inflammatory effect primarily by neutralising lps activity as nearly all these effects can be inhibited by higher ll-37:lps ratios. exposure of bacteria such as p. aeruginosa, growing within a biofilm in the lungs of cf patients, to antibiotics during treatment of recurring pulmonary exacerbations, may result in the development of antibiotic resistance. the aim of this study was to compare biofilm formation and antibiotic susceptibility of matched p. aeruginosa isolates cultured from cf sputum before and after antibiotic treatment of an acute exacerbation of pulmonary infection. biofilm formation (24 hours) by 10 matched pairs of p. aeruginosa isolates, cultured from sputum samples prior to commencing and at the end of antibiotic treatment, was assessed by total viable count using the calgary biofilm device. all isolates formed biofilms with no differences in biofilm formation apparent between any of the matched pairs of isolates. prior to commencing antibiotic treatment, p. aeruginosa isolates from 8 (caz), 10 (tob), 8 (pip/taz) and 6 (mer) patients were susceptible. following antibiotic treatment, the susceptibility status of isolates changed from sensitive to resistant for 3 (caz), 2 (tob), 1 (pip/taz) and 3 (mer) patients. these results indicate that antibiotic treatment had no effect on the ability of p. aeruginosa isolates to form bacterial biofilms but in some patients resulted in the development of antibiotic resistance. cause of death. paradoxically, neutrophils are recruited into the lungs but fail to clear infections. the question that this project will address is; are cf neutrophils intrinsically abnormal? within this study we shall focus on neutrophil membrane proteins and present the first proteome study on normal and cf membranes. a pure neutrophil membrane fraction was prepared by sucrosedensity ultracentrifugation. the solubilizing power of nonionic and zwitterionic detergents as membrane protein solubilizers for twodimensional electrophoresis was investigated. ief was performed with immobilized ph gradients. optimized solubilization of membrane proteins was achieved by combining the zwitterionic detergent chaps (4%) or sb3-12 (2%) with the nonionic detergent triton x-100 (2%). excellent reproducibility of protein-spots was observed on ph linear gradient strips (4-7 and 6-11), allowing for comparative studies. with our now optimized protocol we propose to screen circulating neutrophils from cf patients during periods of exacerbation, and to look for quantitative changes in membrane protein expression (up-regulation, down-regulation or post-translational changes) using a stable-isotope labeling approach. data arising from this project will identify candidate proteins that could be used as biomarkers and/or contribute to a better understanding of disease progression in cf. neutrophil dominated inflammation characterises acute lung injury, pneumonia, copd, cystic fibrosis and bronchiectasis. factors modulating neutrophil mediated inflammation may have important therapeutic potential in these conditions. the anti-inflammatory protein, secretory leukoprotease inhibitor (slpi), is a non-glycosylated molecule produced by epithelial cells, macrophages and neutrophils. this study aims to enhance our knowledge of the anti-inflammatory effects of slpi and to investigate the relationship between slpi and the human neutrophil. neutrophils were purified from whole blood and subcellular fractionation performed employing sucrose gradients and ultracentrifugation techniques. translocation of slpi to the outside of the cell post pma(10 ng/ml) or fmlp(10 -6 m) activation was assessed by western blot analysis. our experimental results confirm the findings of sallenave et al [1] and demonstrate that slpi resides within the neutrophil cytosol. however, contrary to previously published data we have found that slpi does not co-localise with lactoferrin in the secondary granules [2] (figure 1 ). cytosolic spli migrated as a dimer on sds-page and upon cell activation translocated to the outside of the cell in predominantly monomeric form. our results may support the concept that slpi orchestrates diverse effects within the neutrophil, with monomer and dimer forms of the molecule possessing distinct anti-inflammatory modes of action. the primary cause of morbidity and mortality is infection by gramnegative bacteria such as pseudomonas aeruginosa, resulting in chronic airway inflammation characterized by release of interleukin (il)-8. to avoid the innate immune system, p. aeruginosa can undergo genetic changes [1] , such as modification of the lipid a component of the lipopolysaccharide (lps) structure [1] . the aim of this study was to compare the pro-inflammatory response of various types of purified lps isolated from cf patients with that of commercially available lps. human (hte) and cf (cfte) tracheal epithelial cells at *80% confluency were serum starved (24 h), then stimulated with lps from sigma (laboratory stain) or isolates that differed in their lipid a structure: pak8 (mild cf), se22 (severe cf), se4 (infant cf), bronc5 (bronchiectasis) and il-8 release measured. in order to achieve similar il-8 release, sigma lps was required at 1000-fold higher concentrations than cf lps isolates (ug/ml vs. ng/ml). there was a differential response to lps between hte and cfte cells: se22 and pak8 strains induce a higher response in cfte cells when compared to hte. in conclusion, the inflammatory response to p. aeruginosa is dependent upon strain and environment, which may be due to changing lipid a structures. we investigated the ability of secreted bacterial proteinases from three pathogens (burkholderia multivorans, burkholderia cenocepacia, and pseudomonas aeruginosa) involved in chronic bacterial infections in cystic fibrosis to degrade various host defence-related molecules. these included secretory leukocyte proteinase inhibitor (rhslpi), alpha-1 antitrypsin (aat), secretory iga (siga), igg, lactoferrin and lysozyme. host defence-related molecules were co-incubated with cell-free bacterial supernatants from 48 hour biofilm cultures from all three pathogens under investigation. no degradation of aat, siga, igg, and lactoferrin was observed for any of the organisms. only one out of 18 isolates tested demonstrated the ability to degrade lysozyme. all isolates of b. multivorans (n = 4) and p. aeruginosa (n = 4) were able to degrade rhslpi however, out of five bacterial isolates tested for b. cenocepacia only two demonstrated a limited ability to degrade the molecule with [95% of the protein band still remaining intact at the end of the experiment. this study demonstrates that the majority of the host defence molecules investigated are resistant to degradation by bacterial proteinases from b. multivorans, b.cenocepacia and p. aeruginosa when grown as a biofilm. however, rhslpi was vulnerable to significant degradation which could result in aberrant serine proteolysis in regions of the lungs containing biofilm growth. children are ten times more sensitive to radiation-induced cancer than adults. we aimed to determine the cumulative radiation exposure associated with imaging in a paediatric population with cf, to identify contributing factors and to suggest ways of reducing their lifetime radiation exposure. medical and radiology records were reviewed. effective radiation dose (msv) and cumulative lifetime radiation doses were calculated for each patient using national radiological protection board (uk)data files. 77 patients, mean age 9.5 (1-19.25) years with a total follow up time of 658 person years, had 1485 chest radiographs, 215 abdominal radiographs and 57 computerized tomography (ct) scans, including 51 thoracic ct scans. average cumulative radiation exposure per patient was 6.2 (0.04-25) msv. radiation exposure increased with age (p = 0.0014) and with increasing numbers of cts (p = 0.0004). radiation dose was significantly increased in the subgroup who presented with meconium ileus (p = 0.004, independent of age).radiation dose was not significantly related to lung disease severity (measured as forced expiratory volume in 1 second (fev 1 ). radiation exposure in our cf population compares favourably with other tertiary centres worldwide. radiation dose can be minimised by reducing frequency of scans and altering scanning technique. a number of mirna expression profiling studies have shown mir-126 to be highly expressed in rat and human lung. tom1 a predicted target of mir-126 has been shown to interact with tollip and proposed as a negative regulator of il-1b and tnf-a signalling pathways. the aim of this study was to validate tom1 as a target of mir-126 and elucidate its role in tlr4 and il-1 signalling pathways in cystic fibrosis (cf) versus non-cf airway epithelial cells. expression of mir-126 and tom1 were evaluated by qpcr. overexpression of premir-126 was performed by reverse transfection and tom1 was subsequently detected by western blot. mir-126 was found to be down-regulated (p = 0.034) and tom1 mrna significantly up-regulated (p = 0.0021) in cf bronchial cells when compared to their non-cf counterparts. overexpression of mir126 in cf cells led to a decrease in tom1 protein production. this data shows that mirna is differentially regulated in cf airway epithelial cells and that tom1 is a target of mir-126 and may have an important role in regulating innate immune responses in the cf lung. case 2: 20 y.o. male with recurrent infective exacerbations was noted to experience more severe and longer exacerbations compared to other similar patients. common variable immunodeficiency (cvid) was diagnosed based on low iga, igm, igg1, igg3 and lack of antibody response to pneumovax. treatment with ivig has commenced. case 3: 17 y.o. male who experienced severe anxiety during transition to adult cf care. obsessive compulsive disorder was recognized as exemplified by patient using 500 alcohol wipes weekly to clean himself. he has responded well to cognitive behavioural psychotherapy. we conclude that physicians should appreciate the spectrum of coexisting conditions that are separate to a diagnosis of cf and can contribute to morbidity and mortality. cfrd adversely affects pulmonary function however diabetic control did not significantly impact function any further. this finding warrants larger prospective studies to confirm that a diagnosis of cfrd impacts pulmonary function but that diabetic control may not. with improving cf survival, fertility issues emerge. this descriptive study assesses knowledge & approaches to fertility information provision in cf care. prospective anonymous questionnaires were mailed to a male cf cohort (n = 50). sections included demographics, fertility knowledge, investigation & personal relationships. response rate was 32% (n = 16). mean age 24 years (range 19-35, sd3.98). all knew that cf affected fertility but only 68.8% (n = 11) were able to provide explanations. of this group, 45.5% (n = 5) provided the correct explanation. 50% (n = 8) have discussed fertility with a healthcare professional and half (n = 4) selfinitiated this. mean discussion age was 21.9 years (range 18-26, sd2.6). one third stated preference for earlier discussion. 87.5% (n = 7) who had discussions were satisfied with information provided. commonest first source where patients heard of infertility was written material (37.5%, n = 6). three-quarters of respondents (n = 12) requested further fertility information. the preferred source was written material (43.8%, n = 7). 12.5% (n = 2) have had semen analysis & all remaining (n = 14) would accept an opportunity for this if offered. all respondents were aware of infertility however most unaware of explanation. few have formally discussed fertility. the majority want further information (preferred method written material) & an opportunity for semen analysis. this study identifies significant gaps existing in sex education during provision of cf care. a. sahadevan, s.h. chotirmall, a.k. mann, p. branagan, c. gunaratnam, n.g. mcelvaney discovering predictors of mortality in cf within ireland has therapeutic implications. we aim to determine factors predicting mortality in an irish cf cohort. a retrospective analysis of clinical, microbiological and radiological parameters in deceased cf patients over an 8-year period (2001-2008) was conducted (n = 15). this was age matched to a living cf cohort. spss version 16.0 was used-chi-squared and independent student t-testing applied. mean age 22.4 years (sd +/-4.4, range 16-31) [deceased group] and 22.1 years (sd +/-4.2, range 16-31) [living group]. 40% (n = 6) and 33.3% (n = 5) were female in the deceased and living groups respectively. within the deceased cohort, 73.3% (n = 11) had abnormal liver function (p = 0.011), 93.3% (n = 14) grew pseudomonas (p = 0.002) and 86.7% (n = 13) had candida in sputum (p = 0.046). correspondingly, in the living cohort 26.7% (n = 4) had abnormal liver tests, 40.0% (n = 6) and 53.3% (n = 8) respectively grew sputum pseudomonas and candida species. the deceased had poorer lung function (p \ 0.001), weight (p \ 0.01) and bmi (p \ 0.001). mean fev1 was 1.43 litres and mean weight 11.3 kilograms less than that of the living cohort. poor pulmonary function (fev1, fvc), abnormal liver function, sputum culture of pseudomonas and candida spp and suboptimal nutrition (weight, bmi) were all predictors of mortality in our cohort. abnormal lfts are common in cf. we aim to determine any relationship between abnormal lfts & pulmonary function in a cf cohort. 52 cf patients were included during the 12-month study (2006-07). serum bilirubin, alanine aminotransferase (alt), alkaline phosphatase (alkp) & international normalised ratio (inr) were obtained in the outpatient clinic when exacerbation free. lung function (fev1) was concurrently determined. spearman (nonparametric) correlation was applied where appropriate. mean bilirubin was 8.5umol/l (range 2.5-24 umol/l) and mean alt 32.7 iu/l (range 11-256 iu/l). 53.8% (n = 28) had both above average bilirubin and alt of which 15 (53.6%) and 20 (71.4%) respectively in the bilirubin and alt groups had fev1 abnormal liver function did not impact fev1 however inr showed negative correlation. this may relate to malabsorption of fat soluble vitamins or be explained by a residual coagulopathic state following recurrent pulmonary exacerbations. osteoporosis & vitamin malabsorption contribute to poor nutritional status in cf. we aim to determine the effect of vitamin d deficiency and bone fragility on pulmonary function. systematic random sampling of an outpatient cf cohort was studied. pulmonary function (fev1), vitamin d status (serum) and bone fragility (z-score on dexa) was determined. chi squared analysis was applied to results (spss version 16.0). 38 patients were included in the study (age 19-32). 47.4% (n = 18) exhibited vitamin d deficiency. of these, a single patient had normal lung function (fev1 [ 80% predicted), 5 reduced function (fev1 50-80% predicted), 11 markedly reduced function (fev1 30-49% predicted) and 1 patient fev1 \ 30% predicted (p = 0.05). vitamin d deficiency was commoner in males (n = 10). within the cohort, 8 patients had normal bmd (z-score [ -1), 25 had osteopenia (z-score -1-2.5) & 5 had osteoporosis (all male) (z-score [ -2.5) (n = 38). in those with vitamin d deficiency (n = 18), 1 patient had osteoporosis & a further 12 osteopenia (66.7%) (p = 0.046). vitamin d deficiency is characterized by lower fev1 & bmd (osteopenia) however osteoporosis was present in cases of normal vitamin d levels. our study suggests the role bone health plays in the cf ''gender gap'' may be overestimated. biopsy showed nodular glomerulosclerosis (ngs) occurring in the absence of diabetes mellitus, amyloidosis and any other known cause of ngs. a recent paper has suggested that the pathogenesis of ngs in normoglycaemic, non-diabetic cf patients is similar to that of classic diabetes induced ngs and may be mediated by the development of advanced glycosylation end products (age). in cf, chronic pulmonary infection/inflammation, in combination with reduced glutathione levels contribute to an oxidative state and increased levels of age and to s100/calgranulin. it is postulated these ligands interacting with rage resulting in the formation of nodular glomerulosclerosis in patients with cystic fibrosis. we conclude that increasing life spans of cf patients may lead to an increased identification of proteinuric renal disease, the aetiology of which may include this newly described pathological process. this is the first case described in a european cystic fibrosis population and the fourth case worldwide. nebulised hypertonic saline is an effective and safe therapy for cf lung disease. however reports show over 10% of patients cannot tolerate this treatment, and up to 36% of patients are totally noncompliant when using standard nebuliser units. positive expiratory pressure nebulizer devices splint open the airways and have a more controlled rate of nebulisation. we tested if patients who had failed hypertonic saline via standard nebuliser units could tolerate this therapy via a pep nebulizer. we prospectively recruited 4 adult cf patients over a 4 month period, who had previously failed hypertonic saline trials and commenced them on hypertonic saline via a pep nebulizer. patients completed a questionnaire on tolerability of the new device. notes were examined retrospectively on mean time to intravenous antibiotic usage pre and post therapy and mean time to next exacerbations. there was a subjective reduction of over [50% noted in coughing, chest tightness and bad taste using the pep nebulizer, with all 4 patients tolerating this form of treatment. in this small study we found an absolute reduction in antibiotic usage of 70% post hypertonic saline usage and a 3 fold increase in time to next exacerbation post nebulized pep treatment. hypertonic saline administered via a pep nebulizer may be a novel therapeutic strategy for patients who cannot tolerate hypertonic saline through a standard nebulizer. chronic lung infection with p. aeruginosa is responsible for most of the morbidity and mortality in patients with cf. cross infection involving the epidemic strains liverpool (les), manchester (mes), midlands 1 (mid1) and clone c has been documented. regular genotyping is recommended to assess distribution of genotypes. genotyping is not currently performed in the belfast trust. the aim of this study is to perform molecular typing of isolates. the results will inform future strategies for laboratory screening, and infection control. 92 p.aeruginosa isolates collected during routine clinics were typed using pulsed field gel electrophoresis (pfge), restriction patterns were analysed using bionumerics. a multiplex pcr (1) was used to type 110 isolates. 92 samples were typed by both methods and results compared. 42% of isolates were defined by pfge as the les genotype. 4.3% of adult isolates were defined as clone c. no mes or mid1 isolates were reported. there was 96.7% correlation between pfge and pcr results. this study reports prevalence of the les strain in the ni cf population. it is recommended that patients with cf infected by p.aeruginosa have all isolates of varying phenotypes genotyped. pcr detection of les isolates will be a useful tool for screening. having successfully derived a method to culture nasal epithelial cells (necs) from cystic fibrosis (cf) and non-cf subjects, the aim of this study was to characterise the electrophysiological responses of these cells. cells from f508del/f508del patients and non-cf controls were used for patch-clamp investigation. cultured cells were separated and plated on glass coverslips chambers. culture medium was replaced with standard external solution (ses) before establishing whole-cell configuration. membrane ion currents were recorded using patchclamp. once a stable current was achieved, amiloride and forskolin were applied to the cells to elicit their responses. the f508del/ f508del cells responded to amiloride by rapid reduction in wholecell current, when forskolin was added to the bath it had little or no effect on the cell current amplitude in the cf cells (n = 6). in the non-cf cells however, there was a response to the addition of forskolin. these responses to both amiloride and forskolin were as expected and demonstrate that this cell model of nasal epithelial cells obtained from nasal brushings proves to be a very feasible model for future studies of cf and can be used as an ideal model for research into the nature of action of numerous cf drugs. it has been shown that females with cystic fibrosis (cf) have worse lung function compared with cf males. gender and bmi have been correlated with lung function in adults. we aimed to show a similar trend in a paediatric population. we studied children aged 10 years and older attending our cf unit. fev1, height and weight were measured at the clinic when patients were at their baseline. of 30 patients, 21 were males (70%) and 9 were females (30%).14 of the males had fev1 [ 80% (67%), 4 had fev1 50-80% (19%) and 3 with fev1 \ 50% (14%). within the female subgroup, 3 had fev1 [ 80% (33.3%), 3 had fev1 50-80% (33.3%) and 3 had fev1 \ 50% (33.3%) (p = 0.26). bmi was divided into 2 groups; \ 10th percentile indicating poor nutrition that may affect lung function and [ 10th percentile (table 1) . no statistical difference was found (p = 0.34). there was no statistical difference between genders with regards to lung function although there was a trend in favour of males. in our group of well nourished patients, there was no correlation between bmi and fev1. this is in contrast to adult data in a similar study. piperacillin-tazobactam induced fever is well documented in patients with cystic fibrosis. here, we report adverse reactions which occurred in three patients treated with piperacillin-tazobactam over a three month period. setting: tertiary care, academic medical centre (beaumont hospital, dublin, ireland). patients and methods: three patients were evaluated retrospectively for piperacillin-tazobactam induced fever and evidence of bone marrow suppression. results: two of our series had evidence of drug induced fever (using criteria by young et al.). both patients had a mean duration of piperacillin-tazobactam exposure of 9.5 days with an average temperature of 38.3 c. fever resolved in both patients within 72 hours of discontinuation of the antibiotic. neither had evidence of a septic focus (determined by cxr, blood cultures, ivc tip analysis or msu). two of our series developed bone marrow suppression, one becoming pancytopenic requiring bone marrow biopsy, the other becoming transiently neutropenic. both recovered within 72 hours of cessation of offending agent. components of the new iv piperacillin-tazobactam preparation (ph buffers or stabilising agents) may be involved in the development of these late reactions. objectives: cystic fibrosis patients suffer from chronic bacterial colonisation and repeated exacerbations. one of the most challenging elements of treating these patients is that they develop antibiotic resistance. the aim of this study was to assess if changes in antibiotic sensitivity were related to the number of exacerbations (noe). we compared the noe requiring intravenous antibiotics with respiratory cultures at the time. the sensitivities of pseudomonas to 5 antibiotics were analysed from 2004 to 2007. we correlated the changes in sensitivities with the noe they had in this period. a univarious analysis was performed using a non-parametric test. noe was used as a dependent variable. thirty-two patients were included. in the piperacillin/tazobactam group, 38% became resistant with a mean noe of 6.25. (p = 0.016) no resistance developed with colomycin. 84% of patients developed resistance to gentamicin. 50% of patients developed resistance to ceftazidime with average noe of 7.2. (p = 0.005) only 9% of patients developed resistance to ciprofloxacin. the findings of this study showed considerable variations with antibiotic sensitivities. twenty-one patients demonstrated some change in sensitivities, and eleven with none. those with antibiotics resistance had a higher noe compared to those with constant sensitivities. slpi is an anti-inflammatory antiprotease that negatively regulates tlr2, 4, and 9. we examined the effect of the viral rna mimic polyic on cytokine production by evaluating responses it induced in airway epithelial cells; we then evaluated slpi's effect on these responses. we performed selective inhibition studies to identify the receptor by which polyic induces its effects. rna was isolated from cystic fibrosis (cf) and non-cf bronchial epithelial cells and used in quantitative rtpcr reactions with genefev1 \ 50% 0 (0%) 6 (24%) s461 specific primers to each receptor. cells were stimulated with polyic in time course and dose response experiments and il-8 and interferon (ifn)-beta production quantified by elisa. the effect of pre-treatment with slpi or receptor inhibitors was evaluated. polyic induced il-8 but not ifn beta production. il-8 production was inhibited by pretreatment with slpi but not by inhibitors to pkr, rig1, or mda5. further rtpcr experiments demonstrated deficiency of phosphatase shp-1, important for interferon production. polyic induces expression of the proinflammatory cytokine il-8 via a mechanism involving tlr3. slpi inhibits this effect. polyic does not induce ifn beta production in airway cells. shp-1 deficiency demonstrated is a proposed mechanism for this effect. objective: in cystic fibrosis the mechanisms that lead to initial bacterial colonization, the development of a sustained and predominantly neutrophilic inflammatory response, and the ultimate destruction of the lung over decades remains unclear. here we investigate whether humoral autoimmunity could play a role in pathogenesis of cystic fibrosis. circulating autoantibodies in plasma of cystic fibrosis patients (n = 20) and of healthy controls (n = 10) were studied using immunofluorescense using hep2 cells as a substrate. selected samples were studied using immunofluorescense on primary bronchial epithelial cells. eight out of 20 cf patients presented igg autoantibodies against hep2 epithelial cell line and against primary bronchial epithelial cells. there was no apparent correlation between fluorescence intensity and autoantibody titers and the disease intensity. the fluorescence pattern was in all cases mixed (speckled and nucleolar). igg autoantibodies with avidity for bronchial epithelium are present in some patients with cystic fibrosis. this suggest that autoreactive adaptive responses directed against bronchial epithelium may be important in aetiology of the disease and warrant further investigations. the role of pulmonary rehabilitation (pr) has not been widely investigated in patients with diagnoses other than copd. the aim of this study was to investigate the effects of an 8 week pr programme in patients with bronchiectasis. seventeen patients with a diagnosis of bronchiectasis were recruited from respiratory consultant clinics. all patients underwent an 8-week programme (16 supervised sessions) of exercise training and education. subjects were assessed at baseline and on programme completion on measures of exercise capacity and quality of life. data were analysed using minitab version 14. thirteen patients (10 female, 3 male) completed the programmemean age 64.94 (sd = 10.14) years, mean %predicted fev1 75.56 % (sd = 21.66). after eight weeks, there was a significant increase (p = 0.045) in the incremental shuttle walk test distance of 54.6 metres (95% ci: 1.5 to 107.7 m). there was a trend toward a statistically significant improvement (p = 0.05) in the st george's respiratory questionnaire impacts subscale although there was no statistically significant improvement in the overall score (p = 0.38). results of this observational study support the potential role of pr in patients with bronchiectasis. robust trials are necessary to assess the effect of such programmes on a range of outcomes, as well as the efficacy of individual programme components. pkcd genetically depleted mice had baseline capillary filtration coefficient (kfc) compared to their wild type counterparts. there was no difference between these groups.similarly, there was no difference between wet-to-dry ratio's at baseline or in response to hydrostatic challenge.however, pkcd knockout mice experienced a significant protective effect when challenged with lps for 24 hours injected intraperitoneally compared to their wild type. this correlated with reduction in neutrophil recruitment to the lung as well as significant attenuation of histological evidence of lung injury. however, there was no significant difference in the respective cytokine profiles. pkcd plays a central role the development of acute lung injury following exposure to lps and may represent a potential therapeutic target in attenuating acute lung injury. the precise mechanisms remains to be elucidated, however it is likely mediated through impaired neutrophil response. mycobacterium tuberculosis (mtb) is responsible for almost 2 million deaths annually (who). the success of the bacillus is largely due to its ability to evade the host immune response. mycobacteria survive s462 within macrophages by blocking fusion of phagosomes and lysosomes, thus avoiding exposure to antimicrobial peptides and enzymes present in lysosomes. il-10 inhibits the progression of phagosome maturation in murine macrophages, however little is known about the role of il-10 on phagosome maturation in human macrophages. pma-differentiated thp-1 cells were seeded at 1.0 x 10 5 cells/ml on glass coverslips. monocyte derived macrophages (mdms) were isolated from buffy coats obtained from the irish blood transfusion board. cells were treated with anti-il-10 monoclonal or isotype control antibody, infected with live or killed gfp-bcg and pkh67green-labeled m. tuberculosis h37ra, and incubated with lamp-1 antibody and alexa 594 fluorescent stain. the colocalisation of mycobacteria-containing phagosomes with lysosomes, as identified by lamp-1, was determined by confocal microscopy. colocalisation of mycobacteria-containing phagosomes with acidified lysosomes was infrequent in untreated thp-1 cells, 21% ± 6.95. however colocalisation increased when killed mycobacteria were internalised (51.35% ± 10.26). similarly in cells treated with anti-il-10 colocalisation increased to 52% ± 8.98. mdms treated with anti-il-10 and infected with gfp-bcg showed a significant increase in phagosome maturation compared to untreated mdms. this data suggests il-10 suppresses the ability of macrophages to proceed with phagosome maturation, favouring survival of mycobacteria within the host. the prevalence of tuberculosis (tb) in ireland is not decreasing and management is becoming more complex with a multi-cultural population and increased drug resistance. there are new presentations (e.g. tb associated with biological agents). shorter rotations for junior doctors may be associated with less familiarity with tb management. we reviewed the appropriateness of our practice by examining the management of 24 randomly selected patients attending our clinic. there was an equal gender balance and 54% were non-nationals. forty-two percent had pulmonary tb. significant deficiencies in management and documentation were identified. bcg status was not recorded in 18 cases. incomplete data was available on the patients' hiv status. while all patients received pyridoxine prophylaxis, 19% of patients receiving ethambutol did not have an ophthalmology review. only 67% of patients received ethambutol. one patient (with multi-drug resistant tb) was transferred to another centre for negative pressure isolation. of the 23 patients who were treated, one did not complete their treatment. this review indicated a need for improved documentation of bcg, hiv status and attention to issues such as ophthalmology review. to achieve this we have developed a clinical care pathway for management of tb in our clinic. background: northern ireland has consistently had a low tb prevalence (3.6/ 100,000). 1 however, there has been an increase in cases of mdr-tb admitted to the rvh since 2006. retrospective chart review of mdrtb cases. six patients were admitted between 2006-2008. five were suspected to have mdr-tb on admission based on epidemiological risks; two had prior tb treatment; one was a contact of mdrtb; two were from countries of high mdr prevalence. four patients had primary mdrtb. the rifampicin resistance probe was positive in all cases. susceptibility testing showed isolates to be resistant to a median of 4 drugs. all were susceptible to second line injectable agents, and 5/6 were susceptible to quinolones. one patient was hiv co-infected. patients converted their sputum to culture negative in a median of 7 weeks (range 4-18) and were considered for discharge when they had 2 negative cultures 1 month apart. hospital admissions were prolonged due to drug toxicities and/or social issues (median hospital stay 6 months, range 1-11). discussion: due to globalisation, even countries with low tb prevalence need to manage mdrtb. management of these patients is complex, and significant toxicities were seen. prolonged isolation also has significant resource implications. a 33 year old male was admitted from the local psychiatric unit with apparent pneumonia. a chronic schizophrenic and heavy smoker, he had spent some 15 years in institutional care in various facilities. he was treated with high flow humidified oxygen and nebulised bronchodilators. 48 hours later he was found to be sputum smear positive for aafb. he was isolated and the infection control team mobilised. he turned out to have a fully sensitive m.tb organism and responded well to treatment. bts tb guidelines were followed and all staff and patients in the same ward bay as the patient were informed and letters sent to all gps. one patient contact had oesophageal carcinoma and was a chronic alcoholic. he developed post operative pleural effusion (tb culture positive) some 6 months post exposure. a second contact (also alcoholic) was investigated as possible lung cancer some 30 months after exposure and was smear positive for aafb. both patients died on treatment for their tb. all 3 tb isolates were identical. the index case is alive and well. this highlights the danger of even short delays in diagnosis and appropriate isolation of tb patients. streptococcus pneumonia is a leading cause of invasive diseases which will pose an important health problem in ireland. efficacy of pneumovax is between 50 and 70% (ref 1). the vaccination would prevent severe pneumococcal infections (ref 2). the purpose of the audit was to determine the rate of update of pneumovax among the chronic respiratory group and to identify reasons why the vaccination may not have been administered. subsequently, address ways of improving current policy. patients who attended the respiratory outpatient clinic in st. james's hospital from july 2007 till jan 2008 were questioned whether they have the pneumovax given at any stage of the life and ever receive booster dose; and reasons why if they had not been vaccinated. a total of 147 patients were interviewed. 49.7% were male and 50.3% were female. mean age of male was 61.2 and mean age of female was 59.8. approx 70% of patient has obstructive airflow diseases. 37% has received pneumovax at some stage while 63% never received pneumovax. the main reason (93.6%) is poor awareness of the importance of the vaccine. the main implication from the audit is to raise awareness of pneumovax via campaign, reminding gp; and the funded pneumovax clinic. nursing home acquired pneumonia (nhap) is an important subset of healthcare associated pneumonia. we have prospectively compared three different pneumonia severity scores: pneumonia severity index (psi), modified bts score and naughton score and several inflammatory markers including procalcitonin levels to determine their ability to predict fatality in nhap. this study was carried out on fifty patients presenting to our hospital over a two year period, july 2005 to june 2007 inclusive. the case fatality rate was 20%. the modified bts score most accurately identified fatal outcome (3 n = 5; 4 n = 3; 5 n = 2). psi and naughton scores were poor predictors with 40% of deaths characterized as psi category 2 or 3. procalcitonin levels co-related well with disease severity as measured by the psi and modified bts scores. none of the inflammatory markers accurately predicted patients at low risk of death from nhap. we conclude that the different pneumonia severity scores and several commonly used inflammatory markers fail to accurately predict patients at low risk of death from nhap. the adelaide and meath hospital, tallaght opened in 1998 and celebrates its 10th anniversary this year. we sought to assess changes in the incidence, demographic characteristics, and microbiological profile of tb infection in our institution over this period. patients diagnosed with active tb in the years 1999 & 2007 were included. patients were identified using laboratory, public health, and hipe records. demographic, clinical, and microbiological data were obtained by retrospective chart review. there was marked growth in the incidence of tb over this period our data emphasizes the re-emergence of tb as a major public health issue in ireland, and highlights immigration as a major contributing factor. the emergence of resistant infection has not to date been seen in our institution, but can be anticipated in the near future. we report a case of a 68-year-old white male who presented with one month history of pleuritic chest pain. chest radiograph demonstrated left upper lobe cavitation. bronchoalveolar lavage was smear positive for acid-fast bacilli and culture grew pan-sensitive mycobacterium tuberculosis complex. standard anti-tuberculous treatment with rifater and ethambutol was instituted; however the patient developed a severe cutaneous reaction after 4 weeks. skin biopsy demonstrated findings consistent with allergic dermatitis. the rash resolved after discontinuation of therapy; however it recurred after sequential re-exposure to rifampicin, ethambutol, isoniazid and moxifloxacin. the patient underwent desensitization to rifampicin; ethambutol and isoniazid using modified penicillin protocols. titration to target dosing with each individual drug was achieved allowing reinstitution of standard therapy with rifater and ethambutol. the sequential desensitisation processes were each complicated by the reoccurrence of a mild rash, which was controlled by oral prednisolone. to our knowledge this is the first reported case of hypersensitivity to four anti-tuberculosis agents with a favourable response to a strategy of sequential rapid oral desensitisation. introduction: tb is a major infectious disease. in n.ireland the incidence remains low but is increasing. in 2007 66 cases were notified, 17 from the southern trust. a tb nurse was appointed in september 2006. this audit was undertaken to review the tb clinic and optimise the service provided. a retrospective 6 month case note audit. all aspects of the service were reviewed; nationality of those attending, use of interpreter services, the problem of missed appointments and the reason for failure to attend. contact tracing and mantoux testing were assessed as this is a new role for the tb nurse. compliance with treatment in the light of mdrtb cases. results: 36 patients attended the tb clinic in the 6 month period. 21(58.3%) were not native of n.ireland. there have been 3 cases of mdrtb in the trust in the last year which has posed particular operational problems. tb remains an active problem in the southern trust. patient attendance at the clinic may be optimized by having appointment letters in the patients own language and introducing services at peripheral sites. lack of negative pressure ventilation facilities is an ongoing problem. pharmacy dispensing of full treatment course has improved compliance. we sought to analyze differences between patients with latent, pleural and pulmonary tuberculosis (tb) attending a dedicated tb clinic in the mercy university hospital, cork from july 05 to june 07. two hundred and sixty nine patients were referred to the clinic. one hundred twenty eight (48%) had active tuberculosis, 79 (29%) had latent tuberculosis infection (ltbi), 57 (21%) had an alternative diagnosis and 5 (2%) had atypical mycobacterial infection. among those with active tuberculosis, 97 (76%) had pulmonary tb and 31 had extra pulmonary tb, of whom 19 had pleural tb (61%). in this group of patients with tb infection, female sex and foreignborn status were more frequent in those with ltbi compared with those with pulmonary tb. in contrast, smoking was more prevalent in patients with pulmonary tb. 16% of patients with pulmonary tb were asymptomatic. mantoux induration was less in those with pleural tb compared with those with pulmonary tb and ltbi. directly observed therapy was implemented predominantly in patients with pulmonary tb and at a rate considerably short of world health organisation recommendations across all groups. the purpose of this study was to determine if the lysophospholipids sphingosine 1-phosphate (s1p) and lysophosphatidic acid (lpa), which have been implicated in allergy, induce up-regulation of adhesion molecules and eosinophil chemoattractants in an in vitro cholinergic nerve cell model, imr-32 cells. s1p and lpa act mainly via g-protein coupled receptors s1p 1-5 and lpa 1-3 respectively. eosinophils accumulate at innervating cholinergic nerves in fatal asthma and in animal models of asthma and adhere to nerve cells in culture via intercellular adhesion molecule-1 (icam-1). the methods used were real-time pcr and western blotting. s1p 1 , s1p 3 , lpa 1 , lpa 2 and lpa 3 were expressed on imr-32 cells. both s1p and lpa induced erk phosphorylation and erkand g i -dependent up-regulation of icam-1 expression in imr-32 cells, with differing time courses. lpa also induced erk-and g i -dependent up-regulation of the eosinophil chemoattractant, ccl-26. the eosinophil granule protein eosinophil peroxidase (epo) induced erk-dependent up-regulation of transcription of s1p 1 , lpa 1 , lpa 2 and lpa 3 . thus s1p and lpa, acting via g i -coupled nerve cell receptors, induce up-regulation of adhesion molecules and chemoattractants which stimulate eosinophil accumulation and adhesion to cholinergic nerve cells. in turn, epo induces up-regulation of s1p and lpa receptors, potentially perpetuating s1p-and lpa-induced effects. asthma and rhinitis are characterised by eosinophilic inflammation. however, recent studies indicate that eosinophils are not essential for clinical symptoms, but instead exert a remodelling effect on the local tissues. we propose that neural remodelling involving the bmp pathway, enhancing a cholinergic phenotype, is a potential mechanism of airway remodelling in asthma. imr32 cells behave like cholinergic neurons when cultured with sodium butyrate. we exposed imr32 cells to eosinophil granule proteins and to bone morphogenetic proteins 6 & 7 and harvested the cells. proteins were separated into fractions and western blot analysis was performed. rna was isolated, converted to copy dna, and analysed using quantitative pcr. we found that major basic protein, but not eosinophil peroxidase, produced a down-regulation of bmpreceptor1a (bmpr1a) gene expression (41% reduction at 4hrs, p = 0.005). mbp decreased bmpr1a in membrane protein and increased bmpr1a within the nuclear protein. co-incubation of bmp7 with mbp attenuated expression of the bmp pathway transcription target id1 and of choline acetyltransferase. co-incubation with bmp6 & mbp produced increased expression of id1 and choline acetyltransferase. these results indicate that eosinophil granule proteins change bmp receptor balance, producing a downstream effect on cholinergic gene expression and therefore on the cholinergic phenotype of cells. exercise-induced bronchoconstriction (eib) has a reported prevalence of 11-50%. this study aimed to measure for the first time, the prevalence of eib and asthma in professional rugby players and to demonstrate the utility of a sport specific field-test as a screening tool in field-sport. prospectively a cohort of senior international rugby players underwent spirometry before and after a rugby-specific exercise challenge. exercise intensity levels were also assessed. a fall in forced expiratory volume in one second (fev 1 ) c 10% from baseline after exercise challenge was considered diagnostic of eib. during analysis, players were divided into two groups: those with airflow obstruction (ao) and those without (nao). ao airflow obstruction, nao no airflow obstruction forty-two players were tested. table 1 summarises the spirometric results. twelve players (29%) had a history of, or were diagnosed with, airflow obstruction (ao group). seven players had a previous diagnosis of asthma and were on inhaled treatment, of these; 57% (n = 4) had eib after exercise despite regular inhaled therapy. five players (12%) were newly diagnosed with eib. eib is more prevalent in professional rugby players than in the general population. a pre-existing diagnosis of asthma with regular inhaled therapy does not preclude eib. sports-specific field-testing is a useful method of screening in players. ulster hospital, dundonald, 3 regional immunology service, royal hospitals, belfast. asthma is a major risk factor of anaphylactic deaths in children with peanut allergy. peanut allergy is a lifelong condition but some children outgrow their coexistent asthma. it is currently not known whether children who have outgrown their asthma symptoms have ongoing eosinophilic airways inflammation. exhaled nitric oxide is recognised as a non-invasive marker of eosinophillic airways inflammation. the aim of our project was to examine the levels of exhaled nitric oxide in peanut allergic children. children with peanut allergy were recruited at the ulster hospital and royal belfast hospital for sick children, northern ireland. exhaled nitric oxide levels (eno) were measured using the niox mino in all children. results: 94 children were enrolled over a 9 month period, age range 4 to 15 years (median 10 years). 30 (32%) had no history of wheeze, 8 (8%) had outgrown asthma, 37 (39%) had current active asthma and 20 (21%) had occasional wheeze within the last year but were not taking any regular asthma medication. levels of eno were significantly elevated in those with outgrown asthma and those with occasional wheeze but no regular asthma medication (p \ 0.05). exhaled nitric oxide levels were elevated in children with a history of asthma outgrown and those with current 'untreated' asthma. this would suggest ongoing allergic airways inflammation. our study gives a rationale for checking eno in children with peanut allergy. consideration should then be given to starting inhaled corticosteroid therapy in peanut allergic children with elevated exhaled nitric oxide levels. guideline-defined asthma control, physical activity and bmi omalizumab has been shown to be effective in severe persistent allergic. this study describes our local experience in a group of carefully selected asthmatic sufferers. a retrospective audit was performed on 21 patients with severe persistent allergic asthma who fulfilled the criteria for omalizumab therapy. only those who were regarded as omalizumab-responders were included in this analysis (n = 15). the primary outcome measures for the study were acute hospital admissions, exacerbation rates, reliever usage and change in fev 1. these data were analysed for the six month period prior to commencement of omalizumab and for the six months following. the results showed that as a group acute hospital admissions reduced by 85%, with a corresponding reduction in exacerbation rates of 60%. mean fev 1 increased by 261 ml and reliever usage was reduced by 76%.this is consistent with that reported in the literature. omalizumab has proven effective in our local population of carefully selected severe asthmatics. introduction: difficult to treat asthma (dta) is associated with frequent symptoms despite therapy with high dose inhaled corticosteroids (ics). adolescent asthma presents special difficulties given the associated development issues. we sought to determine objective features of dta in this group. methods: all patients (11-18 yrs) attending the adolescent asthma clinic were reviewed. clinical data was collected at referral. dta was defined as requiring high dose ics (bdp) of [800 mcg/day (\12 yr) or [1600 mcg/day (12-17 yrs). the dta group was compared with remainder on low dose ics. of a total of 70 (25 f:45 m) patients, 8 (11%) had dta with a mean ics of 1425 mcg/day. there were no significant differences in steroid rescue, bmi, fev1/fvc, serum ig or eosinophil level, dust mite responsiveness, eczema, rhinitis, or smoking. dta patients were more likely to have elevated eno (50 vs. 38; p = 0.05) and lower ige level (223 vs. 901; p = 0.025). co-existing conditions more likely in dta included grass allergy (p = 0.12), any food allergy (p = 0.009), gord (p = 0.026) and vcd (p = 0.023). conclusions: dta in adolescents was associated with higher eno and lower ige levels, grass and food allergy, gord and vcd. identification of these features early can potentially facilitate more comprehensive and effective management. omalizumab is a monoclonal ige antibody which reduces asthma exacerbations. it is only cost effective in severe asthmatics with recurrent exacerbations. to assess the outcome of omalizumab treatment in severe asthmatic patients in the respiratory department of connolly hospital. a retrospective chart review of 9 asthmatic patients treated with omalizumab. baseline demographics, omalizumab dose and frequency, other asthma medications, fev1, exacerbation rates, and side effects were reviewed. male to female ratio was 2:8. mean age was 49.9 years. mean ige level prior to omalizumab was 345.6u/ml. mean fev1 prior to treatment was 69.6%. mean fev1 post treatment was 83.2%. prior to omalizumab five patients were on step 5 of gina treatment guidelines, 4 patients were on step 4. four patients reduced their shortacting beta agonist requirements, one patient was weaned to a lower dose of steroid and 1 patient increased their inhaled steroid dose during the treatment period. patients had recurrent exacerbations prior to treatment. mean number of exacerbations during year of treatment was 1.5. the most common side effect experienced was joint pains. treatment with omalizumab reduced exacerbation rates in these severe poorly controlled asthmatics. airway sensory hyperreactivity (shr) is an important clinical feature in chronic cough and is characterised by bouts of coughing triggered by relatively innocuous stimuli including exposure to aerosols, scents and changes in air temperature. these abnormal sensory responses are often what distress a patient most about their condition 1 . the aim of this study was to determine the prevalence and clinical features of shr in patients with chronic cough. we undertook a retrospective case review of 200 sequential referrals to the belfast city hospital cough clinic. we defined shr + as those individuals reporting cough provoked by one or more of the following; 1) change in air temperature (thermoactivation), 2) exposure to aerosols, scents, odours (chemoactivation), 3) talking, laughing or singing (mechanoactivation). we compared shr + with shr-patients across a range of variables using chi-square and analysis of variance as appropriate. 135 charts were available for review. we identified shr + in 85 (63%) with significantly more females in the shr + (82% versus 54%, p = 0.001). no other features including age, cough duration, cough aetiology, atopic status, preceding urti or pc 20 reliably distinguished shr + from shr-patients. these preliminary results suggest shr is a common problem especially among females with chronic cough. omalizumab is a humanized monoclonal antibody to ige which prevents binding to fceri receptor. it is known to increase eosinophilic apoptosis and inhibit the th2 immune response culminating in a reduction in eosinophil recruitment, activation and tissue migration. omalizumab use in the management of css however has been reported in one case to ameliorate the asthmatic component of the disease and to significantly lower plasma eosinophil counts at three months. our patient, mb, a 45 year old man diagnosed with css 7 years ago manifesting with uncontrolled asthma, severe pan-sinusitis with recurrent nasal polyposis, eosinophilic gastoenteritis histologically confirmed with duodenal biopsy and subacute bowel obstruction. initial eosinophil count was 0.92 9 10 9 /l., with an elevated ige level of 559 u/ml. autoantibodies were negative. mb's extrapulmonary symptoms were well contolled with oral steroids and azathioprine. efforts to minimize oral steroid doses were hampered by persistent asthma and rhinosinusitis as well as a relapse of eosinophilic gastroenteritis in 2003. omalizumab was commenced in june 2008. after 16 weeks, apreciable improvements in asthmatic and sinusitis symptomes were noted. average peak flow improved from 520 to 600 l/m and a reduction in peripheral eosinophil count from 0.9 9 10 9 to 0.41 9 10 9 /l. a paediatric asthma telephone clinic (patc) was set up in our hospital to provide follow up for children with mild asthma. structured telephone interviews with children aged between 1 and 16 and their carers who received detailed asthma education, was conducted and subsequent appropriate clinical action initiated. the aim of this study is to assess the effectiveness of the patc for medical surveillance by the asthma nurse. a retrospective review of case notes of the children referred to the patc was conducted. unscheduled use of health care & use of antibiotics or steroids between time of the patc and the next out patient clinic were recorded. pulmonary function was compared pre and post the patc. descriptive statistics were carried out and a paired t test was used to detect any significant change in lung function. forty one patients were referred to the patc with asthma. only 9 (22%) had an unscheduled visit to the gp and no patient presented to s468 the emergency department. there was no significant change in pulmonary function. routine follow up of children with mild asthma and their carers by an asthma nurse via a structured telephone consultation can be considered an alternative to face to face follow up. further evaluation comparing the patc to an outpatient clinic and assessment of child/ carer satisfaction could confirm this. approximately 26,548 (17.9%) patients received inhaled shortacting beta2 agonists in combination with a regular standard-dose inhaled corticosteroid.. a further 5,044 (3.4%) patients were also prescribed a regular inhaled long-acting beta2 agonist (salmeterol or formoterol). 2506 patients (6.2%) on combination therapy were coprescribed four different asthmatic treatments inclusive of oral prednisolone. approximately 5177 (3.5%) of the patients prescribed a respiratory drug were co-prescribed nicotine replacement therapy. in total there were 9,728 patients prescribed a mucolytic drug in combination with a respiratory drug b) there were significant levels of coprescribing of salbutamol with beta blocking agents at 15.2% (95%ci: 15, 15.4 ). in addition 9.5% (95%ci: 9.0, 10.0) of the patients prescribed theophylline in 2006 were also prescribed ciprofloxacin. c) levels of co-prescribing with antibiotics was 22%. the antibiotics coprescribed were augmentin, clarithromycin, cephalosporins and ciprofloxacin. assessing changes in asthma control is difficult. peak flow diaries are not completed and history is subject to recall bias. with a view to developing an electronic asthma management system, we attempted to use breath acoustics to assess changes in respiratory status. spirometry and breath sounds were simultaneously recorded in asthmatic subjects during histamine challenge. breath sounds were recorded by a microphone over the trachea. data was collected from seven male and four female subjects with a mean age 29.9yrs. acoustic features were extracted from the breath sounds and evaluated for a correlation with the percentage change in fev1. the highest correlation occurred between the duration of exhalation and percentage change in fev1(r = -0.505). fev1 showed a correlation with number of wheezing episodes (r = -0.382), median wheeze frequency (r = -0.364), maximum wheeze duration (r = 0.278), frequency of maximum duration wheezing group (r = -0.096) and mean frequency of the wheezes(r = -0.292). using these features together (combined in a linear discriminant classifier) a 5% drop in fev1 was detected with a sensitivity of 80% and specificity of 84%. the results of this study suggest a strong relationship between duration of exhalation and fev1.the study also showed that combining acoustic features can be beneficial in detecting a decrease in fev1. the liver disease of alpha-1 antitrypsin deficiency (aatd) is associated with endoplasmic reticulum(er) stress. seps1 is a selenoprotein that through a chaperone activity decreases er stress. we aimed to determine the effect of seps1 on er stress in this condition by measuring activity of the grp78 promoter and levels of active atf6 as markers of the unfolded protein response in hepg2 cells transfected with zaat transgene. we investigated levels of nfjb activity, a marker of the er overload response. to determine the effect of selenium supplementation on the function of seps1 we investigated glutathione peroxidase activity, grp78 promoter and nfjb activity. we also investigated the anti-inflammatory effect of selenium through the 15-deoxy-d 12,14 -prostaglandin j 2 pathway(15d-pgj2) and checked selenium levels in a population of zz and mm phenotypes for aatd. seps1 reduced levels of active atf6. overexpression of seps1 also inhibited grp78 promoter and nfjb activity and this effect was enhanced in the presence of selenium supplementation. increased 15d-pgj2 concentrations were found in selenium supplemented cells. we demonstrated serum selenium levels to be in the low normal range in the patients tested. this data demonstrates a role for seps1 in this conformational disease and suggests a possible therapeutic potential for selenium supplementation. alpha-1 antitrypsin (a1at) is a glycoprotein synthesised chiefly in the liver and functions as the most important antiprotease in the lung and also demonstrates anti inflammatory properties. it has previously been demonstrated that a1at is packaged along with neutrophil elastase within the primary granules of these cells [1] . thus there remains a paradox as to why an enzyme and cognate inhibitor would simultaneously compartmentalize, potentially impeding protease antimicrobial activity. this aim of this study was to reevaluate the localisation of a1at within the neutrophil. compartmentalisation of a1at within the neutrophil was established by sub-cellular fractionation, western blot analysis and confocal immunofluorescence. our data clearly show that a1at is a genuine outer membrane protein of neutrophils associated with cholesterol-and sphingolipidenriched membrane domains called lipid rafts. we have observed that treatment of neutrophil membranes with phosphatidylinositol-specific phospholipase c (piplc) or high nacl concentrations removed a1at from the neutrophil membrane indicating that localization of a1at in lipid rafts is mediated by electrostatic interactions to a glycosylphosphatidyl-inositol (gpi) linked membrane protein. further studies will address the relevance of neutrophil associated a1at and may support the theory that the anti inflammatory effects of a1at are not simply related to modulation of serine proteases activity. aat deficiency (aatd) is a hereditary disorder, resulting from mutations in the serpina1 gene, classically presenting with earlyonset emphysema and liver disease. the most common mutation associated with aat deficiency is the z mutation, with the s mutation also associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. world health organisation guidelines advocate screening patients with copd, asthma, cryptogenic liver disease and first degree relatives of known aatd patients. zz aatd patients on the national alpha-1 registry (n = 61, 49.3 +/-1.3 years, 39 male, 22 female) were compared to a cohort of mm copd patients (n = 100, 60.4 +/-1.3 years, 40 male, 60 female). mean aat levels in the zz group were 0.127 +/-0.013 g/l compared to 1.393 +/-0.03 g/l in the mm copd cohort. the mean fev1 for all zz patients was 63.0 +/-4.2% compared to 62.8 +/-2.6% for mm copd patients. however, when zz cases identified by family screening were removed, the mean fev1 of the zz cohort was lower than the mm group (55 +/-4.8%, p = 0.005, compared to mm group). when mm and zz groups were stratified by smoking status, zz smokers had mean fev1 of 51.0 +/-4.4% compared to 82.6 +/-6.7% for never smokers, while mm smokers had mean fev1 of 60.9 +/-3.7% compared to 65.6 +/-3.5% for never smokers. these findings underline the clinical significance of the zz phenotype and smoking in the development of copd. ). in the present study we examined the immunomodulatory activity of aat and investigated whether nadph-oxidase activation via the g-protein coupled n-formyl-methionyl-leucyl-phenylalanine (fmlp) receptor was inhibited by aat. oxygen (o 2 ) consumption was quantified using a clark-type oxygen electrode and o 2 production was determined by superoxide dismutase (sod)-inhibitable reduction of cytochrome c. both the rate of o 2 consumption and o 2 production elicited by fmlp (10 -6 m) was significantly inhibited in the presence of aat (1 lm). in addition, inhibition of o 2 production was dose dependent and almost completely inhibited by 7.7 lm aat. mechanisms of inhibition were investigated and found to be mediated through a decrease in intracellular camp. levels of camp at 15 seconds post fmlp stimulation were elevated to 1.3 ± 0.2 pmol/10 7 neutrophils, whilst co-treatment with aat (7.6 lm) reduced camp levels to 0.06 ± 0.1 pmol/10 7 cells. in conclusion, the observed inhibition of neutrophil nadphoxidase activity by aat, is further evidence supporting a role for this molecule as an anti-inflammatory mediator. alpha-1 antitrypsin (aat) is a serum glycoprotein that inhibits proteases, and is produced mainly by hepatocytes. it is particularly important in dampening the action of neutrophil elastase, which can damage the lungs. aat deficiency results from both a qualitative and a quantitative deficiency of the protein which predisposes to the development of emphysema, chronic bronchitis, bronchiectasis, and liver disease. we investigated 95 patients registered on the irish alpha-1 database as aat deficiency mz phenotype. the information gathered from the database was supplemented with chart reviews for clinical information and pulmonary function tests. mz patients had a mean fev1% predicted of 81.9 +/-3.3%. we note that there is a negative correlation between cigarette pack years and fev1% predicted (r 2 = 0.18). the serum level of aat does not necessarily correlate negatively with fev1 (r 2 = 0.02). nearly 60% of mz patients were detected by family screening of known aat deficient patients. it remains uncertain whether mz patients are predisposed to aat deficiency sequelae when compared to the general population. we aim to settle this uncertainty by comprehensively describing the characteristics of this cohort of patients. this may represent a change in the way mz patients are managed and could implicate earlier preventative measures to decrease the likelihood of developing emphysema. alpha-1 antitrypsin (aat) is produced by hepatocytes, and is the most important antiprotease in the lung. aat deficiency (aatd) is a hereditary disorder resulting from mutations in the aat gene, presenting with emphysema in adults and liver disease in childhood. who guidelines advocate a targeted strategy in screening copd, non-responsive asthma, and cryptogenic liver disease patients and also relatives of known aatd patients. the most common phenotype associated with disease is zz followed by sz. a chart review of aatd patients on the national alpha-1 registry was performed on zz (n = 61) and sz (n = 12) patients. the mean age at diagnosis for zz patients was 43.6 +/-2.0 years for males and 42.2 +/-2.6 years for females. we demonstrate that zz individuals identified as a result of family screening have significantly increased fev1 (78.5 +/-6.9%, 47.3 +/-2.4 years) when compared to zz patients identified by targeted symptomatic screening (55.0 +/-4.8%, 52.0 +/-1.3, p = 0.0062). zz and sz patients who smoked had significantly decreased lung function compared to nonsmoking zz and sz and that a positive correlation between pack years and fev1 exists. our results emphasize the need for increased awareness and early detection of asymptomatic aatd. identification of patients from a targeted detection programme should include aggressive family screening and allow the initiation of preventative measures before significant lung disease has occurred. thirty-three females age 19-75 mean 52 and 57 males, age 31-82, mean 56 underwent cpx. mean bmi in females was 27.8 (5.7) and 28.1 (4.9) in males. most tests were done because of unexplained dyspnoea (51). other reasons for requesting cpx were: assessment for fitness for lung cancer surgery (13); assessment of fitness in patients with sarcoid (11); cardiac transplant assessment (3); pre surgical assessment for other major surgical procedures (6). the data on the patients who underwent cpx for unexplained dyspnoea was analysed. mean bmi was 29.3. thirty-two of these patients had normal lung function. as a group these patients were extensively investigated before coming for cpx, 22 had ct scans performed, 7 had lung perfusion scanning, 18 had echocardiography. forty-one of the 51 patients had sub maximal tests with no evidence of cardiac or respiratory disease, the test being limited by reconditioning. in conclusion, cpx is a valuable tool in this district general hospital with various reasons for requesting the test. in patients with unexplained dyspnoea, it may be prudent to request cpx at an earlier stage in the investigative journey. sarcoidosis is a multisystemic disease of unknown aetiology. prognostic biomarkers are not part of routine clinical practice. our hypothesis is that enhanced activity for myofibroblast differentiation in sarcoidosis at the initial diagnosis and is associated with an adverse prognosis and the development of pulmonary fibrosis. in addition we investigate the role of tgf-b in this process. fifty patients with biopsy proven sarcoidosis (stage i n = 28, stageii n = 15, stage iii n = 7) were enrolled. bronchoalveolar lavage (bal) samples were obtained at initial evaluation. primary lung fibroblasts (ccd-19lu) were incubated with bal samples and a-smooth muscle actin (asma) mrna expression as a marker of myofibroblast differentiation was assessed via rt-pcr. asma mrna was significantly elevated up to 350% (above control) in patient's samples. we also found a significant correlation between asma mrna expression and progression of pulmonary disease in sarcoidosis, (p \ 0.005). to investigate the role of tgf-b contributing to this enhanced myofibroblast differentiation, we coincubated bal samples with saturating concentrations of anti-tgf-b antibody and found a 38% reduction in this biological activity, (p \ 0.005). in conclusion we demonstrate firstly enhanced capacity of bal samples to induce myofibroblast differentiation, secondly it is of prognostic significance and finally tgf-b is a significant contributor to this biological activity (fig. 1 ). we present a case of cs seen recently at our hospital and the management undertaken for this 54 yr old, the son of a tuberculosis specialist in south africa. his main symptom was exertional syncope with evidence of conduction abnormality on ecg, poor cardiac function on echocardiogram with normal coronary angiogram. an exercise stress test induced ventricular tachycardia. a dual chamber pacemaker and a defibrillator were placed. he had several hospital admissions over a year with ventricular tachycardia/ ventricular fibrillation. cardiac ablation followed electrophysiological studies. cardiac biopsies confirmed cs. there was no evidence of pulmonary or eye involvement. we discuss diagnostic tests and criteria and also treatment options. corticosteroids are believed to control the inflammation and fibrosis, preventing cardiac dysfunction. we closely follow his progress to assess long term effect of steroid treatment especially on his arrhythmia. idiopathic pulmonary fibrosis (ipf) is a progressive disease frequently associated with terminal respiratory failure. the insight patients with ipf have into their disease process and prognosis is unknown. this may lead to delayed communication regarding end-of-life issues. we studied insight into disease and attitudes towards invasive ventilation (iv) amongst a group of ipf patients and compared these outcomes with copd patients with a comparable severity of disease. ten patients with ipf and eight patients with copd were studied. spirometry, the 6 minute walk test and arterial blood gas were recorded as markers of severity. patient insight into disease and attitudes towards iv were surveyed using a newly developed questionnaire. 50% of patients with ipf felt they had sufficient information about their illness compared to 87% of copd patients. 10% of ipf patients had discussed prognosis with their doctor, versus 50% with copd. if the need arose, 20% of ipf patients wanted iv compared with 62% of copd patients. only one patient felt that the issue of iv should not be discussed. there is a deficiency in knowledge regarding disease process and prognosis among ipf patients. end of life issues and iv should be carefully addressed with these patients. coeliac disease is a gluten sensitive enteropathy that results in a chronic malabsorptive disorder. the disorder is rarely associated with pulmonary conditions though the link between the gut and lung remains obscure. conditions include the rare association with pulmonary haemosiderosis (hamilton lane syndrome) and for the first time to the best of our knowledge, pulmonary alveolar microlithiasis(pam). we also describe a literature review of the pulmonary associations of coeliac disease. pulmonary haemosiderosis is a form of pulmonary haemorrhage syndrome progressing to pulmonary fibrosis. we describe pulmonary haemosiderosis and capillaritis in a 23 year old female coeliac, an association known as hamilton lane syndrome. treatment involved corticosteroids and a gluten free diet with resolution of her symptoms. pam is a rare disease where minute calculi are found in alveoli resulting in progressive pulmonary fibrosis. no association with coeliac disease has previously been published. we describe a case of a 62 year old female coeliac who over 20 years developed pulmonary fibrosis secondary to pam. there is no effective treatment and individuals may progress to transplantation. coeliac disease should be considered in patients presenting with pulmonary haemorrhage/ haemoptysis and in patients with interstitial lung disease where histological findings are consistent with pam. the vdi at 0 and 12 months with pulmonary involvement were significantly higher (p = 0.02, p \ 0.001 respectively), but not at 6 month. a vdi [ 5 has been associated with a 6 fold increase in mortality. 27 (69%) with pulmonary involvement had a vdi [ 5 at initial presentation compared to 8 (26%) (p \ 0.001) without pulmonary involvement. the percentage with vdi [ 5 in the pulmonary patients reduced at 6 and 12 months (58% and 43% respectively, p = 0.05). 37 (94%) of patients with pulmonary involvement at presentation had eu-vas criteria for generalised and severe subgroups compared to 14 (36%) without pulmonary involvement (p \ 0.001). 3 of 4 patients died had pulmonary involvement; one was attributed to vasculitis. we conclude that pulmonary involvement at presentation is highly predictive of severe organ damage (vdi [ 5) at initial presentation and 1 year and more extensive disease activity (bvas (all stages) and euvas) in anca-associated vasculitis. background: levels of adenosine can be rapidly increased during settings of inflammation and acute lung injury. these increases in adenosine have been implicated in pathological progression of lung diseases, as well as, protection against acute lung injury. we tested the effects of elevated adenosine levels on endothelial barrier function in vivo and in vitro. methods: intracellular levels of adenosine were elevated in rodents through inhibition of adenosine deaminase with pentostatin, and lung edema was measured in models of acute lung injury caused by a-naphthylthiourea (antu). the degree of antu-induced lung edema, as measured by lung wet to dry weight ratios and filtration coefficients (k f ), was significantly diminished in the rodents given pentostatin either before or after the antu injury. in vitro analyses using pulmonary artery endothelial cells plated on a monolayer demonstrated that adenosine receptor a 2a and a 2b agonist, n-ethylcarboxamidoadenosine (neca), improved permeability. while no significant effects were noted with adenosine receptor a 1 , a 2a , a 2b , or a 3 inhibitors or adenosine transporter inhibitors alone, we noted a significant attenuation of the adenosineinduced barrier function enhancement in the presence of adenosine receptor a 2a and a 2b antagonists, and adenosine transporter inhibitor, nitrobenzylthioinosine (nbti). adenosine enhances the pulmonary endothelial barrier function in acute lung injury through interaction with a 2a and a 2b receptors. childhood interstitial lung disease (child) comprises a spectrum of heterogenous disorders characterised by tachypnoea, radiological diffuse pulmonary infiltrates and abnormal histology. pathologies largely unique to children presenting in the first two years of life are now appreciated and up to 10% of cases may be inborn errors of surfactant metabolism leading to surfactant dysfunction 1 . we report our experience of child in 12 infants presenting to the royal belfast hospital for sick children over 15 years and describe the presentation, investigations and clinical outcome in this group. all children presented with chronic tachypnoea, hypoxaemia, crackles, indrawing and failure to thrive. differential diagnoses of cystic fibrosis, aspiration, immunodeficiency and cardiac disease were considered and excluded in all cases. nine children had a hrct scan, the commonest finding being a 'mosaic' or diffuse interstitial pattern and four underwent lung biopsy, none of which resulted in a definitive diagnosis. eleven children were treated with inhaled corticosteroids and three with additional systemic steroids and all have experienced clinical resolution over time. the recent identification of the genes responsible for surfactant dysfunction disorders may, in future, obviate the need for a lung biopsy and be diagnostic in approximately 10% of children. screening of 164 patients with contrast echocardiography, thoracic computerised tomography (ct) and cerebral magnetic resonance imaging (mri) has identified 88 patients with definite hht, 72 (82%) of whom had epistaxis, 70 (80%) had telangiectasia and 72 (82%) had a first-degree relative with hht. contrast echocardiography and/or ct were performed in 86 patients, identifying 27 patients (31%) with pulmonary arteriovenous malformations (pavms). nineteen patients with single or multiple pavms had 28 embolization procedures performed, with 1-6 pavms embolized per procedure. mri was performed in 78 (89%) patients but no cerebral arteriovenous malformations (cavms) were diagnosed. hht incidence in ireland is thought to be 1 in 5-10,000, suggesting that there are many more undiagnosed cases nationally. internationally published data suggest a prevalence of 15-35% for pavms and 10-15% for cavms in patients with hht. while the prevalence of pavms in our group is consistent with these data, the prevalence of cavms is not, suggesting that irish patients with hht may differ genotypically and phenotypically from those in other countries. atrial and c-type natriuretic peptides (anp, cnp) are known vasodilators in many vascular beds. the role of npr-c in natriuretic peptide (np) mediated pulmonary vasodilation remains unknown. furthermore the role of endothelium in mediating vasoactive effects of np is controversial. using isolated ventilated-perfused lungs to monitor pulmonary artery (pa) pressures upon exposure to increasing doses of angiotensin ii in the presence of vehicle or anp, cnp, or the selective npr-c ligand, canf. additionally, using isolated pa rings constricted with phenylephrine, concentration dependent relaxations were measured in response to nps in endothelium intact/denuded vessels. results: anp and cnp but not canf significantly attenuated the vasoconstrictive properties of angiotensin ii in isolated perfused lung. similarly, canf had no vasodilatory effect in constricted pa rings. anp and cnp both vasodilated the pa rings. however, only cnp had endothelium dependent vasodilation at doses higher than 10-8 m. the endothelium dependent vasodilation was completely abolished by pretreatment with l-name, no synthase inhibitor, and iberiotoxin, a k + channel blocker. pretreatment with 18 a-glycyrrhetinic acid (18a-ga), a myoendothelial gap junction inhibitor, and indomethacin, a cyclo-oxygenase inhibitor, had no effect on endothelium dependent vasodilation. conclusion: npr-c plays limited role in np mediated pulmonary vasodilation. the endothelium dependent effect of cnp is mediated by no and bkca channels. pulmonary embolism with potential fatal consequences is common amongst acutely ill medical patients. it is recognised that venous thromboembolism (vte) prophylaxis is underutilised in this population. we postulated that an education campaign could increase its usage. we prospectively studied consecutive medical admissions for one month before and after an educational intervention to see if the rate of thromboprophylaxis prescribed increased in patients who warranted such prophylaxis as recommended by the american college of chest physicians (accp) guidelines. 1 prior to an educational intervention, we studied ninety-nine consecutive medical admissions. thirty-six patients in this group met criteria for prophylaxis and had no contraindication to prophylaxis. nineteen (52.8%) of these patients received prophylaxis and seventeen (47.2%) did not receive prophylaxis. the data were presented to the medical staff and the guidelines were discussed (educational intervention). subsequently, of 115 consecutive medical admissions, 49 met the criteria for prophylaxis. thirty-six (73.5%) of these patients received thromboprophylaxis. compliance with the accp guidelines therefore rose from 52.8% to 73.5% (p = 0.041; fisher's exact test) of those eligible following the educational intervention. our data supports the use of education to increase adherence to guidelines which may improve outcome in acutely ill medical patients. venous thromboembolic disease is a major cause of morbidity and mortality amongst medical inpatients. prophylactic therapy with low molecular weight heparin, unfractionated heparin, and graded compression stockings has been shown to significantly reduce risk of pe & dvt. we sought to assess compliance with current international guidelines in a medical inpatient population, as well as the impact of a simple, prominent educational poster at ward and emergency department level. data was collected using a proforma derived from current accp guidelines. collection was performed on 2 dates, before and one month after intervention. 150 patients were assessed on each date. at baseline, prophylaxis was indicated in 64% (n = 96) of patients. of these 48% (n = 47) received appropriate therapy. compliance was best among specialist disciplines such as stroke medicine. acute s474 medical teams fared less well. among the post-survey cohort, prophylaxis was indicated in 61% (n = 92), and prescribed in 58 (n = 63%) (p = 0.52 for comparison) of these. vted prophylaxis is under prescribed. our results suggest that simple educational measures can improve compliance with established international guidelines. however, more interactive methods may yield greater benefits. pulmonary arteriorovenous malformation (pavm), a rare cause of hypoxia, are familial in 70% manifesting as osler weber rendu (owr) syndrome and are associated with embolic stokes in 18% (1, 2) . this report highlights a case of hypoxia secondary to pavm with a family history of embolic strokes not associated with owr. a 45 year old man was admitted with dyspepsia. admission chest x-ray identified a left mid zone mass. he had no medical history. his mother died from a stroke aged 51 and had an ''abnormality in her lung''. his brother died at age 47 from an embolic stroke. shortly after admission, he complained of dyspnoea. physical exam revealed oxygen saturations of 88%, tachypnea and tachycardia. oxygen saturations improved to 90% on 100% oxygen. he had no telangectasia or other signs of owr syndrome. ct pulmonary angiogram displayed a large pavm in left lower lobe. a contrast echo identified a large extracardiac right to left shunt. endoscopy confirmed oesophagitis secondary to excessive alcohol intake. he was discharged on aspirin pending review by cardiothoracic surgery for definitive treatment of his pavm to prevent worsening of his symptomatic hypoxia and reduce his risk of an embolic stroke. it has been hypothesised that fatigue of the genioglossus muscle contributes to collapse of the upper airway in osas. in this study, fatigability of the genioglossus was compared in 3 healthy control subjects (aged 27-42) and 3 osas patients (aged 41-64; apnoea/ hypopnoea frequency 24-87/hr) using surface electromyographic (emg) signals recorded with a novel intra-oral electrode. emg signals were recorded during sustained isometric tongue protrusion at 30% of maximum voluntary contraction. endurance time was the point at which force fell 10% below the target level. muscle fibre conduction velocity (cv), an index of fatigue, was estimated from two adjacent emg signals and the rate of muscle fibre cv decrease during each contraction was calculated. the mean endurance time was lower in patients (90.7 ± 59.3 secs) than controls (261.7 ± 74.7 secs). in addition, the mean rate of decrease of muscle fibre cv, when normalised to an initial value of one, was greater in patients (0.13 ± 0.09 percent per second) than in controls (0.06 ± 0.03 percent per second). together, these results suggest increased susceptibility to genioglossus fatigue in untreated osas patients. furthermore, this approach provides a means of examining the role of fatigue in the pathophysiology of osas. obstructive sleep aponea (osa) is characterised by recurrent collapse of the airway during sleep leading to reduced or complete cessation of airflow despite respiratory effort, causing fragmented sleep and excessive daytime sleepiness. sleep deprivation is thought to be responsible for up to 20% of road traffic accidents. we examined data on the occupation of 99 consecutive patients treated for osa with continous positive airway pressure (cpap) in our institution. specifically the numbers driving vehicles as the primary part of their occupation. drivers made up 21% (21) of the total. this included taxi drivers (8), bus drivers (6) and train drivers, delivery persons (3) and drivers of other vehicles (2) . this data is of major importance given that the rate of osa in the populaiton is estimated at 2-4%, whilst drivers making up 21% of our diagnosed osa polulation. it has major implications for service provision, the safety of all road users and those legislating in this regard. to avoid expensive sleep laboratory diagnosis of osa, we provide a home-based monitoring service with overnight oximetry and/or five channel limited sleep study. we wished to review these studies to assess their usefulness in reaching a diagnosis and to look at practical difficulties that may arise. we reviewed all oximetries and sleep studies, in patients with suspected osa, requested by one consultant in 12 months period in st. john's hospital limerick and the mid-west regional hospital. of the 155 oximetries, 10.9% were suggestive of mild, 18.7% moderate and 12.2% severe osa. 29% were normal, 10.3% borderline and 18.7% malfunctioned. 24 patients did not attend their first appointment. 90% of patients were waiting less than 4 weeks. of the 60 limited sleep studies, 36.6% were suggestive of mild, 6.6% moderate and 16.6% severe osa. 39.9% were normal. 12 patients (19.9%) had prior non-diagnostic oximetries. there were a large number (30) of non-attendants for sleep study; we attribute this to longer waiting time and lack of secretarial back-up. there was minimal damage to the equipment. 56 patients were started on long term cpap, to date nearly 80% are persisting with this. this confirms that home diagnosis of osa is both feasible and practical. both conditions represent a significant burden to the health service in terms of diagnosis, treatment and management. volunteers agreed to undergo a home limited cardiopulmonary sleep study and to interview with questionnaires including the epworth score. studies were manually scored to determine the apnoea hypopnoea index. results: 25 volunteers were recruited, 2 were excluded due to incomplete studies and 1 withdrew consent. 22 subjects (5 female) were analysed, mean age: 50yrs (range 26-73), mean bmi: 37 (±5.8). significant osahs (ahi [ 5) was found in 4 females (80%) and 15 male (88%) subjects, 86% overall. conclusion: osahs is very common in this group of patients without being the primary reason for attendance. this would suggest that osahs remains under-diagnosed particularly in the context of cardiovascular disease. we suggest that there should be routine screening for osahs in this patient group. to assess newly diagnosed patients with osahs for cardiac dysfunction by echocardiography. background: osahs is a common condition occurring in approximately 4% of men and 2% of women. it is associated with an increased morbidity and mortality from cardiovascular disease. newly diagnosed patients (ahi [ 5) attending the sleep clinic in st. james's hospital were sent for echo to determine evidence of early heart changes in this group. results: 20 patients were scanned (4 female). average age: 52 years, (range 32-75 yrs) with average weight of 108 kg (+/-25) evidence of diastolic dysfunction was found in 1 female (25%) and 12 of the male subjects (75% the nf-jb inflammatory pathway is selectively activated by intermittent hypoxia in vitro and in patients with obstructive sleep apnoea syndrome (osas). 1 nitric oxide (no) acts as an important signalling molecule in several biological processes including inflammation. we hypothesise that no plays a critical role in regulating the microenvironment of intermittent hypoxia and in modulation of the associated nf-jb response. serum nitrite and nitrate levels were measured in 55 osas patients with no other medical disorder and 24 healthy controls (body mass index-and age-matched). levels in osas patients were remeasured following continuous positive airway pressure (cpap) therapy. we also investigated the effect of no on transcriptional events initiated by intermittent hypoxia in an in vitro model. serum nitrite and nitrate levels did not differ between osas patients and controls (p = 0.232 and p = 0.376 respectively). nitrite levels in osas patients increased significantly following cpap therapy (5.34 lm compared to 2.92 lm (p = 0.012)), indicating increased endothelial nitric oxide synthase (nos) activity. in our translational in vitro model no increases oxygen bioavailability in hypoxia through mitochondrial inhibition and decreases intermittent hypoxia-induced nf-jb activity. conversely, nos inhibition increases nf-jb activity. the findings support a role for no in regulating the inflammatory response to intermittent hypoxia. continous positive pressure (cpap) is an effective therapy for obstructive sleep aponea (osa). it's efficacy is limited by several factors including variable compliance with therapy. compliance is defined as cpap usage of greater than 4 hours more than 70% of nights. studies of predictors of compliance have shown conflicting results. data on 99 patients with osa treated with cpap in our institution was examined including aponea-hyponea index (ahi), oxygen desaturation index (odi), minimum oxygen saturation, epworth sleepiness score (ess) and compliance rates. the overall compliance rate was found to be 62%, with overall compliance at 2 weeks following initiation of therapy 61.8%. this rate compared with compliance of 57.2% at 3 months. this suggests that 2 week compliance rates are a good marker of longer-term compliance. obstructive sleep aponea (osa) is characterised by recurrent collapse of the airway during sleep leading to reduced or complete cessation of airflow despite respiratory effort. this leads to sleep fragmentation through multiple arousals with poor sleep and has been associated with major co-morbidities including impaired cognition, poor quality of life, and increased risk of accidents. evidence is emerging that osa is an independent risk factor for adverse cardiovascular outcomes. we treat over one hunderd patients for osa with continous positive airway pressure (cpap) in our institution. patient data including body mass index, neck circumference, aponea-hyponea index (ahi), oxygen desaturation index (odi), lowest oxygen (o2) saturation, epworth sleepiness score (ess) was collected. neck circumference was correlated with markers of disease severity and found to be a predictor of more severe disease as defined by higher ahi and lowest o2 saturation. no correlation was found with odi or ess. we propose that this is a useful and easily obtained marker of severity of osa. prompt management of exacerbations is a cornerstone of effective treatment of cystic fibrosis. it is therefore imperative that patients promptly report changes in symptoms to their physician/cf team. we sought to clarify which symptoms patients would report to the clinical team and when they would report them. an anonymous questionnaire was sent to a random sample of 40 stable adult patients with cystic fibrosis (20 male and 20 female). predictors of early response were evaluated using logistic regression. 68% returned questionnaires that were suitable for analysis. for all symptoms a significant number of patients would not contact the medical team before their next routine out-patient appointment or even report it at all (table 1 ). there was a wide variation in the time to alerting the clinical team between and within symptoms. females were significantly more likely to report a change in small volume haemoptysis or cough before their next out-patient appointment. younger patients were more likely to report small and large volume haemoptysis. delayed patient responsiveness to changes in respiratory symptoms is a barrier to prompt management of exacerbations in cf. supported by: health research board, cf association of ireland. bronchoalveolar lavage can be used to investigate pulmonary aspiration in children by measuring pepsin concentration, however, it is invasive. sputum induction is a potential non-invasive way of obtaining samples. we aimed to: (1) assess safety and feasible of measuring pepsin in inducted sputum in children and (2) determine whether the sputum induction procedure caused gastro-oesophageal reflux with refluxed pepsin contaminating samples. children with no respiratory or gastroesophageal symptoms were recruited (n = 21, range 4-16 years). following spirometry, sputum induction was carried out and pepsin concentration measured by an 'in house' elisa. spirometry was repeated and any complications noted. only one child (aged 4) produced no sample, however two other 4 year olds did complete sputum induction. no adverse effects were reported. one child required a sabutamol nebuliser following a 10% decrease in fev 1. 17 of the 20 sputum samples (85%) were positive for pepsin. sputum induction appeared a safe procedure in children. it is well tolerated by children and can be successfully carried out in children as young 4 years of age. however, the analysis of pepsin in sputum obtained by induction is not useful in the investigation of respiratory associated gastroesophageal reflux disease as 85% asymptomatic children have positive samples. immunodeficiency may result in recurrent pulmonary infection leading to chronic lung damage and bronchiectasis. the aim of our study was to identify immunodeficiency in idiopathic bronchiectasis and the parameters that correlate with disease severity. 150 patients with idiopathic bronchiectasis were assessed. patients were recruited over a one year period from the respiratory out-patient and in-patient service. serum immunoglobulins, igg subclasses and pneumococcal antibody levels were measured. clinical, physiological, microbiologic and radiologic data was obtained. patients with igg subclass deficiency (iggsd) were significantly more likely to be hospitalised with a respiratory exacerbation as compared with the immunocompetent group (p \ 0.05). iggsd was associated with lower fev1 (p \ 0.05), more severe obstructive airways disease (p \ 0.05) and persistent sputum purulence (p \ 0.05). iggsd correlated with low pneumococcal antibody levels. there was no significant difference in bronchiectasis severity or lobar involvement on high resolution ct. our findings support the hypothesis that patients with iggsd and documented bronchiectasis suffer more severe exacerbations even in the contaxt of radiologically mild disease. patients with evidence of bronchiectasis radiologically should have serum immunoglobulins and igg subclasses performed. this cohort is high risk for progressive lung damage in the setting of recurrent severe exacerbations and should be identified early to ensure optimal management. macrophages undergo apoptosis after infection with m. tuberculosis (mtb). this macrophage response deprives the bacillus of its niche cell, and supports the host response through better antigen presentation.virulent strains of mtb do not cause apoptosis at low multiplicities of infection (moi) which may contribute to this pathogen's ability to survive long-term in macrophages. we investigated the ability of mtb to cause macrophage cell death at a high moi (10-20) which is likely to represent the high bacillary burden seen in the later stages of infection. the mechanism of cell death was analysed by fluorescent microscopy and nucleosome elisa. macrophages infected with virulent (h37rv) mtb displayed several features typical of apoptosis including dna fragmentation and exposure of phosphatidylserine. however, mitochondrial cytochrome c release and nuclear fragmentation were not observed, suggesting that mtb-induced cell death differs from classical apoptosis. cell death was significantly reduced (p \ 0.001) following treatment with serine protease inhibitors (aebsf and tpck) but was not effected by the caspase inhibitor zvad-fmk. in summary, mtb triggers a novel serine protease-dependent macrophage cell death pathway which may facilitate dissemination in the later stages of infection. a better understanding of this macrophage response may direct new vaccine and treatment options. 266 patients had microbiological confirmation of tb, 259 were culture positive and 7 patients were pcr positive. the mean (sd) age was 37(15). male:female ratio 4:5. of the189 pulmonary cases, bronchoscopy (bronchial washings/ biopsy) was required to make a microbiological diagnosis in 58 cases (31%). of the 259 cases where sensitivity data was available, there were 27 cases (10.4%) of drug resistant tb. of these, 12 (44%) were mono-resistant, 6 (22%) were poly-resistant and 9 (33%) were multidrug-resistant tb. various factors including the presence of a resistant organism determined duration of treatment and site of care. six patients had disease in multiple sites the high rate of drug resistant tb in this population has implications not only for the management of index cases but also for the management of contacts and the management of latent tb in the population as a whole. resource limitations have raised interest in portable monitoring systems that can be used to improve access to the diagnosis of obstructive sleep apnoea syndrome (osas). this prospective study compares a combined electrocardiogram and oximetry recorder (holter-oximeter) in an unattended home setting against attended polysomnography for detection of osas. 31 subjects (28 male:3 female) with suspected osas underwent attended polysomnography (psg) in hospital and subsequent unattended evaluation at home with a holter-oximeter (nemon dr180 +). an algorithm for estimation of the (apnoea-hypopnoea index) ahi using only a single lead of electrocardiograph (ecg) and the oximetry trace had previously been developed using a database of psg recordings. osas was defined as ahi c 15, non-osas as ahi \ 5, and 5 b ahi \ 15 as indeterminate. evaluation methodology was in accordance with the recommended american academy of sleep medicine guidelines (flemons et al. 2003) . sensitivity and specificity were 100%. positive and negative likelihood ratios were [20 and 0 respectively. estimated ahi agreed closely with psg ahi (r = 0.83; p \ 0.001). combined holter-oximeter monitoring compares well against polysomnography for identifying osas and is potentially a suitable device for home screening of sleep apnea in a population suspected of having osas. current international guidelines suggest the combined use of pre test probability scores, d dimers and ct pulmonary angiography (ctpa) for the management of suspected pulmonary embolism (pe). our aim was to audit adherance to international guidelines for the management of suspected pe in a teaching hospital. a retrospective audit was performed on all ctpa's carried out in merlin park hospital from the 1/1/07 to the 30/4/08.104 patients had clinical notes available for analysis. only 7% of patients had a documented well's or geneva score. ctpa confirmed pe in 17% of cases overall. when a well's score was calculated in these patients, 33% were high risk, 33% were intermediate risk and 28% were low risk. a third of patients in the low and intermediate risk groups had no d dimers measured. half of patients in the high risk group had an inappropiate d dimers. no patient with a confirmed pulmonary embolism had a normal d-dimers. 64% of patients had d dimers measured and 12.5% of these of were normal. all ctpa's in this subgroup were negative for pulmonary embolism. when a well's score was calculated, retrospectively, from clinical notes 9 patients were in an low or intermediate risk group where ctpa was not indicated. we conclude that pre test probability scores are underutilised. it confirms that patients with low or intermediate risk of pe and negative ddimers should not require ctpa. it also highllights that d-dimers should be used appropriately in low or intermediate risk groups. common variable immunodeficiency (cvid) is a primary immunodeficiency syndrome characterised by diminished serum immunoglobulin levels and impaired antibody responses. cvid may present as granulomatous disease and masquerade as ''sarcoidosis''. we have previously described four patients referred to us with a diagnosis of sarcoidosis with granulomatous disease on biopsy who in fact had cvid. sarcoidosis is more typically associated with hypergammaglobulinaemia. we have evaluated serum immunoglobulin levels in 57 patients with granulomatous disease on biopsy with clinical and radiological pattern compatible with sarcoidosis. thirty seven (65%) were male. mean age of presentation was 43.3 (sd +/-13, range 26-72). 25% of patients demonstrated hypergammaglobulinaemia (n = 14). 23% (n = 13) demonstrated low igm levels. 4% (n = 2) demonstrated an isolated low igg level. a single patient had low igg and igm levels and has been further evaluated for cvid. 37% of patients had low/ normal iga levels (n = 21). in conclusion, hypergammaglobulinaemia was less common than anticipated in this cohort of patients with granulomatous disease believed to be secondary to sarcoidosis. patients with a putative diagnosis of sarcoidosis should have immunoglobulin levels determined to exclude cvid. in chronic asthma, goblet cell hyperplasia and decreased ciliogenesis are characteristic features which may be influenced by th2 cytokines (eg il-9 and il-13). in vitro basal mucociliary differentiation and differences in paediatric epithelial cells (normal & asthmatics) exposed to il-13 were studied. blind non-bronchoscopic bronchial brushings obtained from children were differentiated at air liquid interface for 28 days. cells s480 were treated with 20 ng/ml il-13 and 2 ng/ml il-13. transepithelial resistance (ter), number of ciliated (anti a 1 -acetylated tubulin antibody) and goblet cells (muc5ac + ) were assessed as a measure of tissue differentiation. both asthmatics and normal cell cultures formed well differentiated pseudostratified epithelium (ter [ 500 x/cm 2 ). asthmatic cultures expressed significantly more goblet cells (50.6%, sd = 15.9) when compared with non-asthmatic cultures (23.6%, sd = 6.3) under basal culture conditions (p = 0.036). significant more goblet cells are seen in asthmatic cultures when chronically exposed to il 13 (20 ng/ml and 2 ng/ml) when compared with identically treated nonasthmatic cultures (p \ 0.05). asthmatic cultures expressed significantly less ciliated cells (15.1%, sd = 2.4) when compared with nonasthmatic cultures (24.7%, sd = 5.8) under basal culture conditions (p = 0.0381). asthmatic cells differentiate at basal conditions with higher proportion of goblet cells and decreased number of ciliated cells when chronically exposed to il-13. combining inhaled corticosteroids with long-acting b 2 -agonists results in improved asthma symptom control and fewer asthma exacerbations compared to inhaled corticosteroids alone. however, there are limited data as to whether these beneficial effects are due to enhanced antiinflammatory actions, or whether such combination therapies impact on airway remodeling in asthma. we sought to determine the effects of inhaled budesonide/formoterol combination therapy, versus inhaled budesonide alone or inhaled placebo, on allergen-induced airway responses, airway inflammation and airway remodeling. fourteen asthmatic subjects with dual responses after allergen inhalation were included in this prospective randomized, double-blind, 3-period cross-over study. outcomes included asthmatic responses, changes in airway responsiveness and sputum eosinophilia, measured before and after allergen challenge, and numbers of airway submucosal myofibroblasts measured before and after study treatment. combination treatment attenuated the maximal early asthmatic responses and also resulted in a 2-doubling dose attenuation of allergen-induced airway hyperresponsiveness compared to budesonide or placebo treatment (p \ 0.01). allergen-induced increases in sputum eosinophils and in submucosal tissue myofibroblasts were significantly reduced by combination treatment (p \ 0.05), but not by budesonide alone when compared to placebo. inhaled budesonide/formoterol combination therapy attenuated allergen-induced airway responses and provided greater anti-inflammatory effects than either budesonide alone or placebo. combination therapy also attenuated the allergen-induced increases in submucosal myofibroblast numbers, suggesting that clinical benefits associated with such combination treatment may relate to effects on airway remodeling. the purpose of this study was to determine the effects of sub-cytotoxic concentrations of eosinophil peroxidase (epo) on expression and sub-cellular localisation of the linked cell growth and cell cycle mediators, focal adhesion kinase (fak), cyclin-dependent kinase inhibitor p27 kip and the epidermal growth factor receptors egfr and erbb2. eosinophils exert many of their inflammatory effects in allergic disorders by degranulation and release of cationic granule proteins including epo. in sub-cytotoxic concentrations, eosinophil granule proteins increase transcriptional expression of various growth factors in airway cells. the methods used were real-time pcr, western blotting of membrane, nuclear and cytoplasmic cell fractions and immunoprecipitation. epo induced a concomitant time-dependent egress of fak and p27 kip from the cell nucleus to the cytoplasm. immunoprecipitation indicated a physical association between fak and p27 kip , implying that fak acts as a nuclear-cytoplasmic shuttle for p27 kip . epo also induced up-regulation of expression of egfr and erbb2. our results imply that epo potentially induces cell proliferation, by up-regulating egfr and erbb2 and cell cycle, by driving p27kip from the nucleus. this has implication for the role of eosinophils in tissue remodelling and turnover in conditions from asthma to cancer. alpha-1 antitrypsin (a1at) deficiency predisposes individuals to early onset emphysema and is a debilitating disease in which neutrophils play a central role. it is becoming more evident that a1at possess key anti inflammatory properties and the aim of this project was to examine the possible role of a1at in modulating neutrophil chemotaxis. western blot and facs analysis was utilised to examine the effect of a1at on release of cd16b, a key molecule in chemotaxis and adhesion, from the neutrophil membrane. the effect of a1at on neutrophil migration using il-8 (1-40 ng/2.5 9 10 5 cells) was quantified employing a multiwall chemotaxis chamber. our experimental results revealed that a1at (27.5 lm) prevented the release of cd16b from the neutrophil membrane. inhibition of il-8 chemotaxis was dose dependent and almost completely inhibited by 3.4 lm aat. in addition, neutrophils of a1at deficient (zz) individuals displayed decreased levels of cd16b. this study highlights the importance of serum levels of a1at for modulating neutrophil activity and aims to evaluate whether infused a1at possess the ability to bind and govern the activity of circulating neutrophils in vivo. aat deficiency (aatd) is a hereditary disorder, resulting from mutations in the serpina1 gene, and classically presents with earlyonset emphysema and liver disease. the most common mutation causing aatd is the z mutation, with the s mutation also associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. the world health organisation advocates screening copd, poorly-controlled asthma, cryptogenic liver disease patients and first degree relatives of known aatd patients. 2,600 individuals with copd, asthma, or cryptogenic liver disease were screened in the national targeted detection programme. 1,000 healthy individuals from the tcd biobank were genotyped for s and z alleles. targeted screening identified 33 zz, 37 sz, 12 ss, 358 mz, 228 ms, and 12 mi individuals, yielding gene frequencies of 0.055 and 0.09 for s and z respectively. biobank screening of 1,000 healthy individuals identified 98 ms, 46 mz, 2 sz and a single ss case, yielding gene frequencies of 0.053 and 0.022 for s and z. the allele frequencies for s and z in ireland were previously estimated at between 0.02-0.04 and 0.005-0.015 (1). our pilot study shows s and z alleles occur at higher frequencies, suggesting 2,900 zz individuals and over 700,000 carriers on the island of ireland. the z mutation is more clinically significant with a higher penetrance than s in the groups we have evaluated. inspiratory-to-total lung capacity ratio predicts mortality in patients with chronic obstructive pulmonary disease joint accp/aacvpr evidence-based clinical practice guidelines sarcoidosis: clinical update hereditary hemorrhagic telangiectasia pulmonary arteriovenous malformations: techniques and long-term outcome of embolotherapy obstructive sleep apnoea in a patient with bilateral carotid body tumours, a unique case references obstructive sleep apnea caused by carotid body tumor: case report obstructive sleep apnea due to a carotid body paraganglioma continuous positive airway pressure therapy compliance rates across three sleep centres we present a unique case of a patient who presented with obstructed sleep apnoea (osa) caused by bilateral carotid body tumours.the patient was treated during a 4 year period, during which time he required non-invasive ventilation at night via continuous positive airways pressure (cpap). he also underwent surgical resection of his right and left carotid body tumours sequentially while on cpap. during family screening his daughter was discovered to have a carotid body tumour but without osa.at presentation, his apnoea-hypopnoea index (ahi) was 19.6, and his epworth score was 19 out of possible 24. after resection of both carotid body tumours, his ahi score fell to 4.3, and he was symptomatically improved and he was able to return to work.this unusual case highlights the need to investigate patients with osa for an underlying treatable cause. to our knowledge, this is the first report of bilateral familial carotid body tumours causing osa; to date osa has only been reported with unilateral carotid body tumours. 1,2 to determine long-term compliance rates for patients prescribed continuous positive airway pressure (cpap) therapy from three sleep centres. having 'limited study' devices now in use for 2-3 years now, we have the opportunity to compare cpap compliance rates between sleep centres using these, and established centres using full polysomnography (psg).a population-based analysis of 371 patients prescribed cpap therapy over a 3-year period 2005-7, with compliant vs. non-compliant status determined in june 2008.it is possible to achieve long-term compliance rates of [ 75% with limited study systems and correct follow-up care. however, factors such as patient education and follow up, along with a high level of home service, are vital.2.138 is brain natriuretic peptide a good marker for obstructive sleep apnea and the efficacy of continuous positive airway pressure therapy in obstructive sleep apnea patients? the aim to assess natriuretic peptide levels n-terminal pro b-type and b natriuretic peptide in congestive heart failure (chf) patients with/without obstructive sleep apnea (sa). the effect of cpap on natriuretic peptides in a sleep apnea population was assessed.a blind study in a known population, sleep apnea status was unknown. biosyn triage measured bnp, roche 2010 measured nt bnp. both were measured in 34 congestive heart failure patients (7 female, 20 male) 17 with obstructive sleep apnea (2 female, 15 male) and 22 normal patients (10 female, 12 male).bnp is a good marker for chf. nt probnp is a good marker for chf, it could distinguish chf patients from chf osa patients. cpap didn't reduce natriuretic peptide concentrations. aim: to show that osahs is a common feature in patients with metabolic syndrome. background: metabolic syndrome has been described as a constellation of risk factors for cardiovascular disease. the who places the incidence at 21% of the population. osahs occurs in 2-4% of males and females. key: cord-023211-kt5gt26t authors: nan title: poster session abstracts date: 2007-08-29 journal: pediatr pulmonol doi: 10.1002/ppul.20700 sha: doc_id: 23211 cord_uid: kt5gt26t nan causing mutation is ∆f508, making studies of nbd1 essential in understanding cftr function. crystal structures have been solved of wild-type (wt) and mutant cftr nbd1, including variants containing ∆f508 (lewis et al. embo j. 2004; lewis et al. j. biol. chem. 2005; thibodeau et al. nat. struct. mol. biol. 2005) . cftr nbd1 contains a regulatory insertion (ri) and a regulatory extension (re) that contain pka phosphorylation sites. although the nbd core is very similar in the various structures, the ri and re in some structures of human wt nbd1 differ by 180°rotations, indicating that they are mobile. we have used nmr spectroscopy to study wt and ∆f508 nbd1 in the phosphorylated (phospho) and non-phosphorylated (non-phospho) states, and bound to different regions of cftr. despite their similar structures, differences are observed in the nmr spectra of wt and ∆f508 nbd1. these changes, possibly due to altered dynamics or interactions of the ri and re with the nbd core, will be discussed. the inherent stability and function of nbd1 could be affected by these changes, accounting for part of the ∆f508 defect. different interactions between wt and ∆f508 nbd1 with the icls could also affect channel function. to identify residues comprising the icls, we generated a homology model of cftr based on the crystal structure of sav1866 in which nbd1 and nbd2 form a productive dimer (dawson and locher. nature. 2006) . our data on a peptide corresponding to the first intracellular loop (icl1) indicate that binding to nbd1 requires phosphorylation of the ri. our homology model shows that the icl1 binding site is buried when the ri is bound to the nbd core. nmr data comparing phospho and non-phospho nbd1 indicates that phosphorylation of the ri and re disrupts their interactions with the nbd core, likely exposing the site for icl1 binding. the structure of the molybdate transporter modb2c2, with the nbds in an open conformation, shows icl1 binding is cognate nbd at a different interface (hollenstein et al. nature. 2007) . icl1 in modb2c2 interacts with the nbd near y93, the analogous residue to f508 in cftr. we will show binding data of icl1 with wt and ∆f508 nbd1 in different states and also present resonance assignments of nbd1 to map the icl1 interaction sites. since the sav1866 and modb2c2 structures were solved in different states, the different icl interactions may represent structural changes during the reaction cycle. the ∆f508 mutation may thus affect intramolecular associations in certain conformations, which would account for another part of the ∆f508 defect. probing differences in interactions of wt and ∆f508 nbd1 is critical for understanding the molecular basis of normal cftr function and of the altered cftr function in cystic fibrosis. shsps target ∆f508 cftr for erad via the sumo pathway ahner, a. 1 ; brodsky, j.l. 2 ; frizzell, r.a. 1 1. cell biology and physiology, university of pittsburgh, pittsburgh, pa, usa; 2. biological sciences, university of pittsburgh, pittsburgh, pa, usa the most frequent disease-causing mutation in cystic fibrosis (cf) is a deletion of phenylalanine at position 508 (df508) in the first nucleotide binding domain of cftr. essentially all of the mutant protein, as well as 70% of wild type (wt) cftr, is retained in the er and targeted for erassociated degradation (erad) by the 26s proteasome. key mediators of cftr folding and degradation are molecular chaperones, which help protein substrates fold, but can target them for degradation if folding efficiency is compromised. to identify factors modulating erad of cftr we performed a microarray analysis, screening for transcription profile differences between yeast expressing cftr and control strains, and identified enhanced transcript levels for the small heat shock protein hsp26 in yeast expressing cftr. we then demonstrated that cftr degradation was blocked in strains lacking the genes encoding the two yeast shsps, hsp26 and hsp42 (1) . to examine whether shsps regulate cftr biogenesis in mammalian cells, we asked which of the 10 human shsp homologues are endogenously co-expressed with cftr using rt-pcr and western blot analysis. we detected 5 shsps, including alphaa-crystallin and hsp27, in calu-3 and primary hbe cells. co-expression of alphaacrystallin or hsp27 together with wt or df508-cftr in hek293 cells selectively decreased the steady state levels of df508-cftr. pulse-chase experiments showed that the rate of df508-cftr degradation was enhanced when either shsp was over-expressed, but wt cftr biogenesis was unchanged. co-immuno-precipitation experiments in hek 293 cells revealed that alphaa-crystallin and hsp27 interacted preferentially with df508-cftr. thus far, our results suggest that shsps selectively increase df508-cftr's accessibility to proteasome-mediated degradation pathways. previously, hsp27 was reported to interact with ubc9, the sumo (small ubiquitin-like modifier) conjugating enzyme (2) . sumo is covalently linked to its substrates, often at sumoylation consensus sites; similarly to ubiquitin, this occurs through a series of thiol transfer reactions. we confirmed this interaction by co-immuno-precipitation and found that ubc9 as well as the sumo specific protease, senp1, are expressed in airway epithelial cells. over-expression of ubc9 decreased, while over-expression of senp1 increased df508-cftr protein levels. pulse-chase experiments indicated that these enzymes regulate selectively the degradation of df508-cftr, similarly to the shsps. preliminary studies in vitro and in vivo indicate that cftr, and particularly purified nbd1, is sumoylated and that hsp27 facilitates this process. mutational analysis of the 3 sumoylation consensus sites within cftr suggests a cycle of sumo modification that facilitates wt cftr biosynthesis but targets df508-cftr for degradation, probably because the balance between sumo addition and removal is impaired by an excessive interaction of shsps with the mutant protein. [supported by grants from the nih and the cystic young, a. physiology and biophysics, chicago medical school, north chicago, il, usa efficient retrieval of wtcftr from the cell surface is mediated by components of the clathrin mediated endocytic pathway; however many aspects of cftr endocytosis remain to be elucidated. we have previously shown that endocytosis of wtcftr is dependent upon recognition of a tyrosinebased motif in the carboxyl tail of cftr by the ap2 endocytic adaptor complex. in contrast to wtcftr, less is known about the mechanisms whereby mutant cftr undergoes internalization. according to one model, df508 cftr undergoes accelerated endocytosis compared to wtcftr, whereas others have argued that endocytic rates of df508 cftr are comparable to those of wild-type cftr. to further define the function of proteins involved in the initial steps of wt and mutant cftr endocytosis, we investigated the role of the gtpase dynamin. dynamin has been proposed to play a key role in endocytosis by facilitating the scission of nascent clathrin coated vesicles from the cell surface. until recently, very few tools were available to modulate clathrin mediated events. we took advantage of dynasore, a newly discovered small molecule, non-competitive inhibitor of the gtpase dynamin. dynasore acts as a potent, rapid and reversible inhibitor of endocytic pathways known to depend upon dynamin, by blocking coated vesicle formation within seconds of dynasore addition. endocytosis of wtcftr was inhibited rapidly and efficiently upon addition of dynasore. furthermore, within minutes of dynasore washout, endocytosis of wtcftr resumed completely. also upon dynamin washout, normal trafficking and recycling of wtcftr was also observed. temperature correction of df508 causes expression of cftr at the cell surface, however such 'rescued' df508 cftr is not stabilized upon returning cells to 37c. application of dynasore prevented such rapid loss of mutant cftr from the cell surface. since maintenance of mutant cftr at the cell surface is critical for effective therapeutic intervention in patients with cf, our results provide insight into additional potential targets for pharmacological manipulation for the treatment of cf. richardson, j.m.; thibodeau, p.h.; watson, j.; thomas, p.j. physiology, ut southwestern medical center, dallas, tx, usa protein misfolding is the basis for a multitude of human diseases; however, the mechanisms underlying misfolding are not well understood. most cases of cystic fibrosis are associated with mutations-including the most common, deletion of phenylalanine 508 (∆f508) in nbd1-that interfere with the folding of the cystic fibrosis transmembrane conductance regulator (cftr). the resulting loss of functional cftr causes the disease. thus, elucidating how ∆f508, affects the folding of cftr is critical to understanding the pathology of the disease. cftr is composed of five domains: two integral membrane transmembrane domains (tmds), a regulatory domain (r), and two nucleotide binding domains (nbds). ∆f508 occurs in the n-terminal nbd1. both the murine wildtype and ∆f508 nbd1 can be expressed in bacteria and purified to near homogeneity. while the soluble expression yield of ∆f508 is lower than the wild type under identical conditions, ∆f508 achieves a native conformation similar to wild type as monitored by a variety of hydrodynamic and spectroscopic characteristics such as analytical size exclusion chromatography, circular dichroism, and fluorescence. recently, a non-native, but folded, species has been detected under mildly denaturing conditions. far-uv cd reveals a time and temperature dependent conversion from a mixed alpha/beta native conformation to a less helical non-native conformation, while fluorescence measurements reveal a parallel blue shift in the peak emission intensity from 343 to 330 nm. this non-native species is in a more open conformation as determined by both limited proteolysis and the change in retention time on analytical size exclusion chromatography. the conversion to this state is inhibited by the native state ligand (atp) and by the presence of the second site suppressors (g550e, r553m, and r555k). notably, the ∆f508 nbd1 protein populates the non-native state under milder conditions than the wild type nbd1. these studies reveal the properties of the native state and its conversion to a partially folded state that is affected by the ∆f508 mutation. this data highlight the proximal affect of the disease causing mutation and provide a means of assessing strategies designed to correct the defect. this work is supported by the cystic fibrosis foundation through a postdoctoral training grant and by the nih we have previously demonstrated that the stability of cftr is negatively modulated by the cftr interacting protein, cal. cal competes with nherf in binding to the pdz motif at the c-terminus of cftr and directs cftr trafficking to the lysosome. recently, silencing of cal or overexpression of nherf has been shown to restore chloride transport activity in ∆f508-cftr cell lines. to identify the molecular machineries involved in cal dependent degradation of cftr, we investigated the role of the cal interacting snare protein syntaxin 6 (stx6). stx6 protein expression and function were manipulated by sirna silencing, over-expression, and a dominant-negative mutant. we also performed a functional study of chloride transport activity in cystic fibrosis bronchial epithelial cell line cfbe41o-stably expressing either wt-cftr or ∆f508-cftr. the endogenous stx6 and cal protein expressions were knocked down by sirna techniques in multiple human cell lines including hek293, hela cell stably expressing ∆f508-cftr and cfbe41o-stably expressing either wt-cftr or ∆f508-cftr. in hek293 cells, knockdown of either stx6 or cal augments the expression of transfected gfp-tagged cftr (gfp-cftr), which is consistent with the observation that overexpression of either stx6 or cal enhances the degradation of cftr. stx6 interaction with cal was confirmed by a co-immunoprecipitation assay. because of this interaction, we hypothesize that stx6 negatively regulates cftr protein stability by interacting with cal which in turn binds to the c-ter-minal pdz motif of cftr. consistent with his hypothesis, stx6 knockdown has no effect on protein levels of a cftr mutant lacking the pdz motif (gfp-cftr∆trl). more importantly, cal knockdown eliminates the inhibitory effect of stx6 overexpression on gfp-cftr. a cell surface biotinylation assay and confocal microscopy showed significant increases in plasma membrane expression of cftr in stx6 knockdown cells without gross changes in the expression pattern. furthermore, consistent with its trans-golgi localization, stx6 knockdown has no effect on gfp-∆f508-cftr expression. however, ∆f508-cftr increases significantly in stx6 knockdown, temperature-rescued cells. to assess the effect of stx6 in bronchial epithelial cells, stx6 was knocked down in cfbe41o-stably expressing wt-cftr. consistent with the observations in hek293 and hela cells, the expression of untagged wt-cftr is also augmented. knockdown of stx6 increases cftr expression in a does-dependent manner, reciprocal to the expression level of stx6, without changing the level of expression of cal or gapdh. furthermore, stx6 knockdown lead to a does-dependent increase in cftr mediated short-circuit current that is stimulated by forskolin and inhibited by cftr chloride channel inhibitor cftrinh-172. more importantly, in temperaturerescued cfbe41o-stably expressing ∆f508-cftr, stx6 knockdown tripled the cftr mediated short-circuit chloride current. these results not only delineate the effect of stx6 on post-golgi trafficking of cftr and its dependence on cal-mediated pdz-based interaction, but also points to it as a potential therapeutic target in conjunction with the er-quality control based rescue of ∆f508-cftr. supported by the cystic fibrosis foundation and the national institutes of health. seavilleklein, g. 1 ; evagelidis, a. 2 ; amer, n. 1 ; chappe, f. 1 ; hanrahan, j. 2 ; chappe, v. 1 1. physiology & biophysics, dalhousie university, halifax, ns, canada; 2. physiology, mcgill university, montreal, qc, canada cftr channel activity is regulated by phosphorylation and de-phosphorylation of the r-domain. direct pkc phosphorylation at specific sites in the r domain and nbd1 is essential and modulates cftr activation by pka. however, the molecular mechanism by which phosphorylation of the r domain induces channel activity remains unknown. we have reported biochemical evidence for the role of phosphorylation in domain-domain association using a deltar-split construct encoding the front (tmd1-nbd1) and back (nbd2-tmd2) halves of cftr as separate polypeptides (1) . in agreement with the inhibitory role of the r domain, iodide effluxes and patch-clamp experiments confirmed that deltar-split channels were functional and constitutively active without camp stimulation. co-transfecting a plasmid that directs expression of the r domain restored the pka-dependence of re-assembled channels. co-immunoprecipitations of the 2 halves of the deltar-split with a gst-r domain fusion protein in vitro or with the cotransfected r domain in bhk cells revealed that pka phosphorylation enhances the r domain binding to the rest of the channel. we have now investigated the effect of pkc phosphorylation on the r domain binding. our data demonstrate that pkc phosphorylation also induces a strong binding of gst-r domain in vitro or co-transfected r domain in vivo when bhk cells are stimulated with pma. interestingly, the combination of pkc+pka phosphorylation induced a stronger binding than pka alone. mutation of all consensus pkc sites on the r domain to alanine (7ca) eliminated specific pkc phosphorylation but did not alter pka phosphorylation in gst-rd. western blotting and confocal microscopy experiments confirmed that expression levels of all 3 domains of the split-7ca/rd are unchanged compared to split-rd and co-localize at the plasma membrane of bhk cells. basal activity of the split-7ca/rd was similar to that of the split-rd in iodide efflux experiments. however, activation by camp+ibmx was delayed and significantly reduced (36.92 % of split-rd and 13.85% of wt-cftr activation). these results confirm the functional re-assembly of the 7ca-rd with the front and back halves of the channel and the inhibitory role of the r domain. in co-immunoprecipitation experiments, the same level of interaction was observed between the 7ca/rd and rd with the split halves suggesting that basal, inhibitory interaction of the r domain is independent of pkc phosphorylation. however, 7ca/rd binding with the split channel was not increased by pkc or pka stimulation. we conclude that pkc phosphorylation is essential for pka-dependent binding of the r domain to the rest of the channel, and that the combined effect of these kinases further strengthens the r domain interaction. these structural changes are consistent with the essential role of pkc sites for cftr activation by pka as previously reported (2) . supported by cihr, nshrf, dmrf & ccff. g.seavilleklein was supported by nserc. (1) the in vivo folding mechanism of multidomain membrane proteins, including the cystic fibrosis transmembrane conductance regulator (cftr), is poorly understood. cftr, a member of the abc transporter superfamily, contains two symmetrical halves, each consisting of a membrane spanning domain (msd1 and msd2) and a cytosolic nucleotide binding domains (nbd1 and nbd2), connected by the regulatory (r) domain. to address whether cftr follows the archetypical domain-wise folding of multidomain soluble polypeptides and/or the cooperative domain folding mechanism, first we determined domain combinations that are necessary and sufficient to escape the endoplasmic reticulum (er) quality control. one-, twoor three-domain assemblies failed to be processed efficiently in cells, measured by biochemical and morphological assays. the smallest folding unit that escaped the er quality control was composed of a four-domain assembly, containing msd1, nbd1, r or msd2 as linear or split polypeptide. as a second approach we showed that cf-causing point mutations in the msd1, nbd1 or msd2 provoked the er retention of the full-length cftr with severe conformational defect in the nbd2, measured by limited proteolysis and immunoblotting, using domain spacific antibodies. the posttranslational folding kinetics of the four-domain folding unit was comparable to that of cftr, suggesting that cooperative domain assembly is essential for the channel biogenesis. this mechanism provides an explanation for the processing defect caused by numerous cf mutations and outlines a possible paradigm for cftr folding in living cells. cftr in post-golgi compartments. our results, jointly, uncover the role of n-glycans as a critical structural determinant of cftr conformational maturation/stability in a chaperone-independent manner. karamyshev, a.l. 1 ; patrick, a.e. 1 ; johnson, a.e. 2 ; thomas, p.j. 1 1. ut southwestern medical center at dallas, dallas, tx, usa; 2. texas a&m university, college station, tx, usa cystic fibrosis (cf) is caused by mutations in cftr (cystic fibrosis transmembrane conductance regulator). cftr, a member of the abc transporter family, is a multispanning membrane protein critical to chloride and water movement in secretory epithelia. over one thousand mutations, in all parts of the cftr sequence, have been identified, many of which disrupt the function of the protein to cause the disease. some of the disease-associated mutations in the n-terminal portion of cftr were predicted to alter cotranslational interactions with factors involved in the membrane integration of cftr required for its proper function. to test this hypothesis, a photoreactive probe was incorporated into different positions of the n-terminal portion of cftr by introducing amber stop codons and translating these messages in vitro in the presence of the amber suppressor trna n ε -(5-azido-2 nitrobenzoyl)-lys-trna amb . ribosome-associated nascent chains of a specific length containing the modified amino acid at a defined position were produced using truncated mrnas. some of these cftr nascent chains photocrosslinked with proteins of approximately 50 and 100 kda molecular mass. crosslinking to the ~50 kda protein was observed only after the tm1 span had emerged from the ribosomal tunnel. moreover, truncation analysis demonstrated that tm1 is necessary for this interaction. in evaluating a range of nascent chains, the extents of photocrosslinking to the ~50 kda and 100 kda proteins often varied inversely. immunochemical and knockdown/depletion studies indicate the ~50 kda protein is srp54, a subunit of the signal recognition particle involved in targeting ribosome nascent chain complexes to the translocon in the endoplasmic reticulum membrane. highlighting the functional relevance of this interaction, in cells with reduced srp levels, nonglycosylated forms of cftr accumulate and total cftr levels decrease. notably, some cf-causing mutations in tm1 also reduce crosslinking to srp. these data reveal that mutational inhibition of nascent cftr association with srp likely contributes to or mediates some types of cf. aleksandrov, l.; aleksandrov, a.a.; riordan, j.r. the initial reversible steps of atp binding to the first (nbd1) and second (nbd2) nucleotide binding sites of cftr are divalent cation independent and dependent, respectively (aleksandrov et al, j biol chem 277, 15419-25, 2002) . subsequent to the initial binding, either mg or mn drive rapid hydrolysis at the second site while promoting trapping of the nucleotide at the first site. this occlusion at the first site of functional wildtype cftr is somewhat similar to that which occurs when the catalytic glutamates in both of the hydrolytic sites of p-glycoprotein are mutated. this occluded state has been proposed to be the result of dimerization of the two nbds and represent a transient intermediate formed during atp hydrolysis (tombline and senior, j bioenerg biomembr 37, 497-500, 2005) . to test the possible relevance of this interpretation to cftr, we have now characterized the process by which nbd1 occludes [ 32 p]-8n 3 atp and [ 32 p]-8n 3 adp. we have now found that only n 3 atp and not n 3 adp can be bound initially at nbd1 in the absence of mg. consistent with this mg requirement for the ndp binding, n 3 adp or adp is able to compete for the n 3 atp binding site only when the divalent ion is present. despite the lack of a requirement for mg for atp binding, retention of the nucleotide triphosphate at the binding site at 37°c was dependent on the cation. however, at reduced temperature (4°c), n 3 atp remains locked in the binding pocket with virtually no reduction over a 1 hour period even in the absence of mg. these apparently paradoxical temperature effects hint that different molecular events can contribute to the occlusion of nucleotide at nbd1. that the occlusion process occurs exclusively at nbd1 and does not depend on its interaction with nbd2, was shown by experiments in which identical behavior was exhibited by ∆nbd2 constructs of cftr. thus, nucleotide trapping by wild-type cftr, unlike that by mutant p-glycoprotein, is accomplished by events at a single domain (nbd1), rather than by dimerization of the two nbds. (supported by the nih and cff). cftr consists of five domains or regions, two regions that span the cell membrane (the tmds), two cytoplasmic nucleotide-binding domains (the nbds), and a regulatory (r) domain. the r domain is unique to cftr: without phosphorylation of this domain, the cftr chloride channel does not open. the tmds and the nbds of cftr are similar to those found in many atp-binding cassette proteins, the majority of which are transporters. the domains are fused in a single polypeptide chain, linked in the sequence: tmd1, nbd1, r domain, tmd2, nbd2. the first nbd is the site of the location of the mutation that is responsible for the development of cystic fibrosis in the majority of human patients (loss of phenylalanine 508). although there is a structure for nbd1, we are still lacking a structure for the entire cftr protein. we have employed electron microscopy to provide low-to medium-resolution structures for the complete cftr protein which was expressed in bhk cells and purified by affinity chromatography. using single particle analysis combined with site-specific labelling using 1.8nm diameter ni-nta nanogold, we have identified the locations of nbd2 and the region around residue 696 of the r-domain within the low resolution 3d structure. by a process of elimination, this has also allowed the identification of nbd1 and the region occupied by the two tmds. these low resolution studies have now enabled us to interpret medium resolution 3d structural data obtained for cftr. progress in obtaining two-dimensional arrays (crystals a single molecule thick) should allow higher resolution structural data to be obtained for the entire cftr protein -sufficient for the identification of the transmembrane alpha helices. these data should allow a much clearer picture of the transmembrane channel formed by cftr and will inform molecular modelling approaches aimed at the development of novel drugs for the treatment of cystic fibrosis. we explored the gating process by introducing cysteines along the entire lengths of various transmembrane domains and extra-cellular loops, and studying their ability to chemically modified. we study the changes in their reactivity in closed and open channel states to characterize the changes in transmembrane domain conformations during gating. using membrane impermeant mts-reagents, we have characterized the conformational changes in tm1-ecl1, ecl3-tm6, ecl4-tm8, and ecl6-tm12 of cftr. our results suggest that atp binding induces modest changes in the transmembrane domains and extracellular loops. channel opening involves a rearrangement of the tm helices leading to decreased hydration of residues near the extracellular end. this conformational change is either a part of, or allosterically coupled to the gating mechanism. 14 physiological role of nherf1 in the regulation of cftr raghuram, v.; yang, y.; yaemsiri, s. laboratory of kidney and electrolyte metabolism, nhlbi/nih, bethesda, md, usa in the apical membrane of epithelial cells, cftr seems to exist within a multiprotein complex, in which its activity is regulated by interactions with other proteins. the c-terminus of cftr contains a pdz-binding motif, which binds to several pdz domain-containing proteins, including members of na+/h+ exchanger 3 regulatory factor (nherf) family, nherf1/ebp50, nherf2/e3karp, cap70/nherf3, nherf4, and cal/pist/gopc. pdz domain proteins act as scaffolds to assemble signaling proteins to form signaling complexes. these signaling complexes are central to achieve specificity and efficiency of signal transduction. nherf1 and nherf2 have a c-terminal erm domain, which interacts with cytoskeleton-associated erm proteins. this interaction may anchor cftr to the actin-cytoskeleton and restrict its movement within the membrane. in addition, several functional roles have been proposed for cftr-pdz domain interactions, including as an adapter to promote efficient pka phosphorylation of cftr, apical targeting and recycling of cftr, regulation of single-channel activity, organization of β-adrenergic, and lysophosphatidic acid-2 receptor signaling complex, and trafficking of romk channels. our current understandings of the role of cftr-pdz interactions are mostly obtained from heterologous expression studies. very few studies have addressed the role of pdz domain proteins in native epithelial cells or in organ physiology. we have used rna interference techniques to produce a knockout or knockdown (kd) calu-3 cell lines using a lenti-viral vector for stable suppression. using four different shrnas, we have generated four stable nherf1-kd cell lines with varying levels of nherf1 suppression. in these cells, nherf1 expression ranged from 10-40% of calu-3nt cells (control cells). in nherf1-kd cells, steady-state cftr protein level was greatly reduced, whereas its apical localization was unaffected. we also found structural defects in the apical microvilli. in polarized nherf1-kd epithelia, membrane-permeable camp analogs were unable to stimulate cftr mediated short-circuit currents. however, forskolin induced response was nearly normal despite reduced cftr expression. examinations of βadrenergic-, and adenosine-receptor signaling pathways suggest that nherf1 plays a unique role in organizing the adenosine receptor signaling pathway. abnormal retention of the df508 cftr mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis. a drug discovery alliance between cfft and biofocus dpi was initiated with the aim to identify novel genes which upon shrna-mediated knockdown were able to efficiently restore df508 cftr activity. a high-throughput assay based on an yfp halide reporter was developed in a human cystic fibrosis bronchial epithelial cell line (cfbe41o-), whereby the rate of halide influx directly correlated to df508 cftr activity. biofocus dpi's proprietary adenoviral shrna libraries totalling 11,334 viruses were screened in this functional assay and yielded 753 hits. these 753 hits were repropogated to generate new adenoviral stocks and rescreened at three concentrations on the yfp halide reporter assay. 39 genes were eliminated due to potential cytotoxic effects, and hit calling was performed on the remaining 714 hits. a total of 315 duplicate hits were confirmed (44%). 7 of the confirmed hits had more than one shrnas which targeted the respective gene. 36 confirmed hits were stronger in restoring df508 cftr function than the most potent positive control: syntaxin-8. in order to elucidate which of these 315 hits induce halide influx via direct rescue and activation of df508 cftr, the yfp halide reporter assay was performed in the presence or absence of the cftrinh172. these cftr dependent hits will move forward into subsequent validation experiments whereby hits will be categorised into potentiators and correctors. the final goal of this program is to prioritize targets for entry into drug discovery. restoring expression and/or function of mutated cftr provides a means to treat cystic fibrosis. biofocus dpi -a galapagos company -in collaboration with the cystic fibrosis foundation therapeutics (cfft) has established a drug target discovery project that will enable the selection of proteins that rescue mutated cftr. by screening biofocus dpi's proprietary adenoviral shrna libraries in a high-throughput primary functional assay for cftr activity in human cells, we have identified and confirmed 315 hits out of 11,334 that entered the primary screen. to further validate these proteins as potential drug targets, we have set up a series of experiments and assays focused on the rescue of cftr expression and its maturation by knock-down of specific targets. 1) in order to establish whether the knock-down of a specific target was able to promote df508-cftr plasma membrane localization, we performed immunofluorescence stainings of df508-cftr in a patient-derived human cell line carrying the df508-cftr mutation (cfbe41o-) grown in air-liquid interface culture. a transduction protocol to deliver shrna adenovirus to this polarized airway cell model system has been optimized and preliminary results showed increased levels of expression of df508-cftr at the plasma membrane level upon knock-down of syntaxin-8, our positive control. experiments performed at 27 οc also demonstrated redistribution of df508-cftr to the apical plasma membrane at low temperature. we will show the results of all the shrna hits in this assay. 2) changes in the post-translational maturation levels of df508-cftr upon knock-down of specific proteins were evaluated by determining the accumulation of band b and c by using a western blot approach. in this way, we can select targets able to promote post-translational maturation of df508-cftr and/or its escape from er in the cfbe41o-cell line. accumulation of band b and c indicates maturation of cftr and will be considered as a beneficial effect. preliminary results show increased accumulation of band b upon knock-down of specific targets and overexpression of wild-type cftr induces band c accumulation. experiments performed at 27 οc also demonstrated increased band b expression. we will show the effect of all the shrna hits in this assay on band b and c expression. we believe that the results obtained by the combination of those two approaches will guide us in obtaining very strong drug targets for entry into a drug discovery phase. because gene therapy as a corrective measure for cf is possible in the foreseeable future, it is important to understand whether the global effects of gene introduction will have significant detrimental impact on cell function. to address this question, global gene mrna expression profiles were created with the affymetrix u133a & b chip sets. wild type cftr (n=1), ∆f508 (n=1) and g551d (n=1) were transfected in separate ib3 cell cultures using lipofectamine 2000™. control expression profiles were generated from untreated ib3 cells; one from this experimental set and five from previous control experiments. differential gene expression was determined by anova using a 10% false discovery rate threshold. genespring was used for data analysis and visualization. genego was used to determine which functional categories were over represented in the list and further assisted in interpretation of the results. rt-pcr was used to confirm differential expression of selected genes and examine the response in more detail. all transfected constructs produced very similar gene expression profiles quite distinct from the untransfected cells. 256 probe sets representing 228 uniquely named genes were significantly different between transfected and untransfected cells. cftr mrna expression in all forms was increased an average of 525-fold. transfection of ib3-1 cells resulted in upregulation of a variety of genes involved in several basic processes: inflammation, protein folding and cytoskeleton framework regulation. genego canonical pathways with a significantly enriched number of genes from the data set involved glucocorticoid signaling and regulation, cytoskeleton remodeling and adhesion plus a variety of other signaling pathways including il8, chemokines, g-protein and mapk cascades. cell processes overrepresented in the list were predominately associated with protein folding, inflammatory responses and cytoskeleton regulation. geneontology categories over represented in the list were regulation of signal transduction, receptor mediated endocytosis and protein folding. the proinflammatory interleukins il6 and il 8 were upregulated 2.5 fold and 4 fold respectively. the genes most upregulated, samd9 (15-fold) and oasl (12 fold), are not well annotated. as controls, rt-pcr analysis of cells treated with either lipofectamine 2000™ alone or any form of cftr dna alone did not show upregulation of il6, il8, samd9 or oasl. in cells transfected with lipofectamine containing dna, upregulation of il6 (n=3), oasl (n=3) and samd9 (n=3) was confirmed by rt-pcr. oasl is known to interact with methyl cpg-binding protein 1, a dna transcription repressor acting at methylated promoter regions. thus it is possible that the global effects noted here leading to dramatic upregulation of two novel genes oasl and smad9 could be previously unrecognized consequences of gene therapies. cystic fibrosis (cf) is a disease which is caused by mutations of the cf gene, which codes for the cftr chloride channel. the ∆f508-cftr mutation results in a mutant protein that undergoes transcription but fails to localize to the apical membrane, where it normally functions as a camp-regulated clchannel. however, clchannel activity can be detected at the plasma membrane when ∆f508-cftr is overexpressed, synthesized at a reduced temperature, or in the presence of chemical chaperones. thus, strategies that facilitate the translocation of ∆f508-cftr to the plasma membrane by increasing cftr transcription may be beneficial for the treat-ment of cf. previous studies performed using fluorescence halide efflux measurements and short-circuit current voltage clamp have shown that treatment with pparγ (peroxisome proliferator activated receptor gamma) agonists, such as pioglitazone and fll (fmoc-l-leucine), resulted in an increased biosynthesis and trafficking of ∆f508-cftr to the cell surface. this effect was at least partially due to increased ∆f508-cftr expression. further studies were designed to investigate the effect of pparγ agonists on cftr promoter activity. two firefly luciferase-conjugated cftr promoter plasmids, containing either 0.79 kb or 3.9 kb fragments, were transfected together with a control plasmid containing a renilla luciferase in human alveolar basal epithelial (a549) cells. the ratio of the firefly to renilla luminescence was used to measure cftr promoter activity. pparγ agonists increased cftr expression acting both on 0.7 kb and 3.9 kb promoter fragments. these results were confirmed in quantitative rt-pcr studies. transcription factor binding site analysis using current online programs and matrices showed three putative ppre (peroxisome proliferator response element) consensus sequences in the cftr promoter. the interaction of these sites with pparγ was shown using emsa (electrophoretic mobility shift assay). interestingly, none of these consensus sequences appear in the 0.7 kb construct, which still maintained a significant pparγ activation response. therefore, we hypothesized that a novel pparγ agonist effector site(s) is involved in transcription activation of cftr. to characterize this novel site(s), a series of deletion constructs to the promoter region were created, using mutation-introduced restriction sites in the original promoter construct. the new constructs ranged from 0.61 kb to 24 base pairs upstream of the transcription start site. in preliminary studies we found that at least one previously uncharacterized pparγ effector site was present in the 0.61 kb promoter fragment that regulated cftr expression. additional experiments were performed using emsa to determine the proteins and cofactors which controlled the optimal conditions for cftr expression. the results of this study show that pparγ agonists, currently prescribed to treat hyperglycemia and improve peripheral insulin resistance, may also have additional benefits for the treatment of cf. our studies also show that pioglitazone and other pparγ agonists, by increasing cftr expression, are potent activators of clflux in epithelial cells expressing ∆f508-cftr. this work was supported by grant from the canadian cystic fibrosis foundation decreased by 10 µm gsno. the effect of gsno on hop expression was post-transcriptional. in cfbe41o-cells transfected with sirna duplex specific for hop (48 hr), hop expression was modestly inhibited; however, gsno dramatically augmented the inhibition. we next showed that knockdown of endogenous hop by sirna increased the steady-state levels of immature and mature forms of cftr. though overexpression of hop did not significantly affect cftr expression (immature and mature forms increased 1.3 ± 0.1 and 1.2 ± 0.3 fold respectively), in the presence of 10 µm gsno, expression of both immature and mature cftr forms significantly increased in the hop overexpressing cells (3.6 ± 0.4 and 2.7 ± 0.2 fold respectively). next, we treated cfbe41o-cells with a pulse of gsno (10 µm; 4 hr); isolated cytosol, er and golgi fractions; immunoprecipitated cftr from these fractions; and studied whether hop, co-immunoprecipitated with cftr, was s-nitrosylated (using a biotin substitution technique followed by streptavadin isolation). we found that sno-modified hop coimmunoprecipitated with ∆f508 cftr in the cytosol and er at baseline; and that gsno decreased the association between sno-modified hop and ∆f508 cftr. we speculate that the effect of gsno to decrease hop-cftr association may 1) result from increased hop s-nitrosylation; and 2) be permissive for ∆f508 cftr maturation. these data may be of relevance to the development of no donor-based therapies for cf. supported by cf foundation and the nih. mouse models for cystic fibrosis (cf) provide new possibilities to study disease pathogenesis, to correlate genotype and phenotype, and to test novel cf therapies. we use the cftr tm1eu mice homozygous for the f508del mutation in a congenic fvb background as a tool in pre-clinical testing of therapeutic strategies aimed at the correction of the f508del folding and trafficking defect. these mice show characteristic cf ion transport abnormalities such as reduced camp-and cgmp activated intestinal chloride secretion and an increase of the amiloride-sensitive sodium absorption in nasal epithelium. we developed an ex vivo assay for the rescue studies of the f508del trafficking defect in murine intestine by proteasome inhibitors based on periodical measurements of camp-activated transepithlial chloride currents in the ussing chamber. muscle-stripped ileum was incubated at 37 0 c in william's e glutamax medium supplemented with insulin (10µg/ml) and dexamethasone (20µg/ml), in the presence of vehicle or proteasome inhibitor. a partial gain of chloride secretory function was achieved by incubating the tissue for 2-4h at 37 0 c in the presence of the proteasome inhibitors (pi) epoxomicin (1µm), mg132 (10µm), nip-(leu)3-vinyl and adalys(bio)ahx3l3vs (both 2µm) and bortezomib (3nm). exposure of the tissue for 6h at 37 0 c to alln (20µm) revealed a functional recovery up to wt levels. immunohistochemical and western blot analysis of all pitreated tissues showed the appearance of cftr positive crypt cells and mature band c cftr protein. rescue by alln remained detectable following inhibition of the er to golgi transport by brefeldin a. our ex vivo experiments demonstrate that proteasome inhibitors can enhance the amount of mature, functionally active murine f508 cftr mutant protein in intact tissue by preventing proteolytic degradation and increasing its expression at the cell surface. in vivo treatment of homozygous f508del mice with bortezomib at a dose of 0.1mg/kg/day for 3 days resulted in a high morbidity. administration of a lower dose (0.04mg/kg/day ) was well tolerated and no mortality or significant weight loss could be observed. as outcome parameters for the in vivo treatment with pis we used a salivary secretion assay and bioelectric measurements of intestinal and nasal epithelium in the ussing chamber. there was no difference in isoproterenol-induced salivary gland secretion or and in cftr-mediated intestinal chloride currents between control -and bortezomib-treated animals. however we found a reduction (1.7 fold) of the amiloride-sensitive short circuit current (isc) in the nasal epithelium of bortezomib-treated mice, indicating a moderate correction of the enacmediated sodium hyper absorption, that was not paralleled by a gain of the forskolin-mediated transepithelial chloride secretion. current in vivo experiments focus on the effects of pis upon short term (2h-6h) rather than chronic treatment, to allow a direct comparison with the ex vivo treatment data. supported by grants from the cf foundation, bethesda, the sophia foundation and the dutch stomach-liver-intestine foundation. rotterdam, netherlands; 2. physiology and biophysics, university of alabama birmingham, birmingham, al, usa; 3. physiology, school of medical sciences, university of bristol, bristol, united kingdom recent progress in the development of small molecule correctors and potentiators capable of restoring cftr function have increased the need for pre-clinical test models including cultured airway epithelial cells from human cf patients as well as cf mouse models. the validity of cf mice as a surrogate for human cf patients depends on the assumption that human and mouse cftr, despite their limited sequence homology (78%) and differences in open probability (p o ) and channel subconductance state, behave similarly in their response to pharmacological correctors and potentiators. to verify this assumption, we used the 125 iodide efflux technique to compare the cftr chloride channel activity in (i) cho cells stably transfected with mouse f508del-cftr, (ii) nih-3t3 cells stably expressing human f508del-cftr, and (iii) their wt-counterparts. forskolin (10µm)-stimulated iodide efflux was very low in both mouse and human f508del cftr cells (0.5 respectively 2 % of wt values) and was stimulated to a similar extent (3.5 and 3.8fold) by the cftr potentiator genistein (100 µm). preincubation for 14 h at 26 0 c, or at 37 0 c in the presence of the vertex corrector cfcor-325 (3 µm) caused a 3-4 fold increase in forskolin-activated 125 i-efflux in both cell types that was further enhanced by 2.6-3 fold upon addition of genistein. functional correction was paralleled by the appearance of mature, complex-glycosylated mouse or human cftr on western blots. surprisingly, two other, structurally unrelated compounds known to correct the gating defect in hcftr, i.e. the vertex potentiator cfpot-532 (10 µm) and the uncharged nppb analogue nppb-am (20 µm; cf. wang w et al. 2005 ; j biol chem 280: 23622) mimicked the effect of genistein on hf508del-cftr but failed to potentiate mf508del-cftr in the 3t3 cells. both cfpot-532 and nppb-am likewise failed to potentiate forskolin-stimulated, cftr-mediated transepithelial chloride currents ex vivo in muscle-stripped ileal mucosa from homozygous f508del-cftr tm1eu mice (congenic fvb) mounted in ussing chambers under conditions in which genistein (100µm) enhanced the current response by 2-3 fold. in contrast, preincubation of the tissue in william's e glutamax medium for 14h at 26 0 c, or for 5 h at 37 0 c in the presence of cfcor-325 (10µm) enhanced the forskolin+genisteininduced transepithelial clsecretion up to 70-100% of the response in cftr+/+ littermates, and was associated with the appearance of band c on western blots. these results indicate that mouse and human f508del-cftr (1) respond to low temperature incubation or to a small molecule corrector with a similar gain in surface expression and function, supporting the use of cf mouse models for in vivo tests of cftr correctors; (2) respond differently to various classes of cftr potentiators, in line with their pronounced differences in gating behaviour, and emphasizing the specificity of cftr-potentiator interaction. further studies focussing on human/mouse cftr chimera and aimed to identify the domain(s) accounting for the differential response to the cftr potentiators are in progress. cell apoptosis is a highly regulated process which is central to the maintenance of pulmonary vascular homeostasis. recent investigations implicated cftr in the regulation of intracellular sphingolipid levels. our laboratory demonstrated that an imbalance in endothelial sphingolipid levels triggers apoptotic cell death. we therefore hypothesized that a dysregulated cftr function in human lung endothelial cells inhibits programmed cell death in response to stress. methods: patch clamp analysis was used to verify the expression of cftr on human lung endothelial cells and the effect of specific inhibitors. we inhibited cftr with dpc, cftr inh -172, and nppb or specific sirna followed by treatment with prototypical stressors that induce endothelial cell apoptosis, staurosporine, h 2 o 2 , or tnf-α. apoptosis was quantified by caspase-3 activity or annexin/ propidium iodide staining. lipid extraction was performed by a modified bligh and dyer method and intracellular sphingolipids were measured by tandem mass spectrometry. results: patch clamp analysis confirmed functional cftr expression on endothelial cells and a complete inhibitory effect of dpc and nppb but no effect of dids. as expected, treatment with staurosporine, a general protein kinase inhibitor and h 2 o 2 , as an oxidative stress stimulus resulted in significant endothelial cell apoptosis. similarly, tnf-α treatment (20 ng/ml) in combination with cycloheximide caused activation of caspase-3 associated with a significant upregulation of the pro-apoptotic sphingolipid ceramide. knock-down with specific cftr sirna was validated with western blot and whole cell patch clamp analysis. dpc, nppb, and cftr inh -172 consistently and significantly inhibited apoptosis in human lung and primary mouse lung endothelial cells. caspase-3 activity was decreased by 38-95% (% decrease in caspase units/µg/min) in hlmvec treated with h 2 o 2 after pretreatment with dpc (90%), cftr inh -172 (95%), and nppb (38%). however, we observed that dids, a non-specific chloride channel inhibitor also inhibited staurosporine induced caspase-3 activation (93%), suggesting the possible involvement of other chloride channels. in conclusion, our data suggest a role of cftr in modulating endothelial cell apoptosis in response to various stresses. the absence of functional cftr impaired stress-induced apoptosis, a process which may be mediated via alterations in sphingolipid trafficking. this abnormal cftr function in cf leading to aberrant cellular responses to stress may perpetuate an activated, pro-inflammatory phenotype of the lung vasculature. in turn, aberrant vascular activation may have a significant impact to the abnormal airway remodeling and persistence of a chronic inflammatory state characteristic of cf. supported by: cystic fibrosis foundation clinical fellowship training grant hegedus, t. 1, 2 ; serohijos, a.w. 1, 3 ; dokholyan, n.v. 1 ; he, l. 1,2 ; riordan, j.r. 1, 2 1. department of biochemistry and biophysics, unc at chapel hill, chapel hill, nc, usa; 2. cystic fibrosis treatment and research center, unc at chapel hill, chapel hill, nc, usa; 3. department of physics and astronomy, unc at chapel hill, chapel hill, nc, usa cftr (cystic fibrosis transmembrane conductance regulator) is a camp dependent chloride channel. the phosphorylation of its 200 amino acid r domain by protein kinase a (pka) is obligatory for channel gating under normal conditions. r domain contains multiple pka phosphorylation sites, which participate in activation, but no specific site is essential for channel function. in spite of numerous studies of the role of r domain in cftr regulation, the mechanism is largely unknown. one reason for this is the lack of information on r domain structure and its interactions with other parts of cftr. it has been shown that r domain lacks well-defined secondary structural elements and is intrinsically disordered. in this study, we used computational methods to explore the structure and function of the disordered r domain. our results show a specific disorder-order pattern in r domain, which is conserved among species even though the primary structure is not conserved. this result implies a role of the disorder-order pattern in r domain function and provides a basis of a novel working model. to gain some insight into the three dimensional structure of r domain, we per-formed ab initio folding of r-domain using discrete molecular dynamics with an all-atom force field. as disordered domains can not be characterized with a single structure, an ensemble was generated by selection of structures with the lowest energy from a large number of folding simulations. these structures were clustered to determine the dominant r domain conformations. they reveal features of secondary and tertiary structure, the positions of phosphorylation sites in 3d, and residue contacts within the r region that might be important for structure and function. to computationally confirm the mechanism proposed in our working model, an ensemble of phosphorylated r domain structures was also produced and characterized. supported by nih (r01dk051619), cff (dokhol07i0), and novartis (06123815). physiology system that utilises the planar patch clamp technique (finkel et al 2006) . this format utilises perforated patch clamp recording of 384 wells in parallel, permitting the generation of up to 4000 data points per day. we have employed this system to develop assays to detect modulators of mutant cftr. human cftr-g551d and df508, over expressed in bhk and cho cells respectively, were cultured to approximately 80% confluence before harvesting. df508 cells were temperature corrected at 26°c for 72 hours prior to experimentation to facilitate channel trafficking to the plasma membrane. cells were placed on the quattro system and whole cell chloride currents measured using asymmetric chloride selective solutions such that the chloride reversal potential was significantly different from that of leak currents (0mv). currents were assessed using a voltage ramp from -100 to +100mv applied prior and 10 min subsequent to compound addition. to validate the assays we used the prototypical cftr potentiator, genistein, which was added simultaneously to cells under study with 10 µm forskolin. after assessing the effects of genistein, the specific cftr inhibitor glyh101, was added at 30 µm to confirm the currents were mediated via activation of cftr. the ec 50 values determined for genistein were 3.8 ± 0.7 µm (mean ± s.e.m, n=7) for df508 and 16.3 ± 2.6 µm (mean ± s.e.m, n=4) for g551d. these values agree with literature values (bulteau-pignoux et al 2002 ) . two alternative potentiators, phenylglycine-01 (pg-01) and vrt-532 were also tested in the presence of 10 µm forskolin and were active against both df508 and g551d. the ec 50 values for pg-01 were 1.1 ± 0.1µm (mean ± s.e.m, n=10) for df508 and 3.2 ± 0.5µm (mean ± s.e.m, n=8) for g551d. the ec 50 values for vrt-532 were 2.8 ± 1.2µm (mean ± s.d, n=3) for df508 and 20.8 µm (mean, n=2) for g551d. these values agree with published data from ion transport studies (pedemonte et al 2005; van goor et al 2006) . in summary the ionworks quattro can be used to identify compounds which modulate the activity of cftr. this assay has a number of advantages over other formats in that it is direct, sensitive and employs voltage control of the cells under study. these assays can be used for primary screening of small to medium compound sets or as a secondary screen to triage the output from higher throughput indirect formats such as membrane potential. references it is very well agreed that ∆f508 cftr is recognized as a misfolded protein by the er quality control and targeted for degradation by the proteasome. during the course of experiments for aav gene therapy for cf we created an aav2/5 vector expressing a truncated cftr insert (∆264) driven by a chicken beta actin promoter (raav-cb-∆264-cftr). ∆264-cftr contains the normal coding sequence of wt cftr except that it is missing the first 264 amino acids. we observed that cos7 cells transiently transfected with raav-cb-∆264 cftr express cftr at significantly lower levels as compared to cells similarly transfected with a wt-cftr construct. to examine why, cells were transfected with raav-cb-∆264-cftr and treated for 16 hours with 5 µm of proteasome inhibitor, mg132. in the cells treated with mg132, ∆264-cftr protein expression was significantly increased. because lysosomal inhibitors did not have a significant effect, the data suggest that a large frac-tion of ∆264-cftr protein is degraded in the proteasome. to evaluate the rate of degradation, cells were treated with cycloheximide. time course experiments showed that ∆264 is more rapidly degraded than ∆f508-cftr. importantly, in cos7 cells cotransfected with ∆f508 and ∆264-cftr, there was a significant increase in the mature c-band of ∆f508 cftr compared to cells transfected with ∆f508 alone. this suggests that ∆264-cftr enhanced the maturation of ∆f508 from b to c bands. enhanced maturation of cftr from b band to c band also occurred when ∆264-cftr was transfected into hela cells stably expressing ∆508 cftr (hela ∆508) and cfbe 41o-cells stably expressing ∆508 (cfbe 41o-∆508). a number of proteins participate in the er to determine the fate of cftr. ∆f508-cftr is known to associate with retrograde translocation protein complex on the er membrane containing vcp (valosin-containing protein) and derlin 1. vcp is also found in aggresomes containing ∆f508-cftr in association with cytosolic histone deacetylase, hdac6. we hypothesized that since ∆264 is more efficiently degraded as compared to ∆f508-cftr it may have higher affinity with vcp protein complex. we verified that ∆264-cftr interacts more readily with vcp and also has a higher degree of association with hdac6 as compared to ∆f508-cftr. our data suggest that ∆264 is more readily degraded by the proteasome as compared to ∆f508-cftr and lacks biosynthetic arrest in the er. ∆264-cftr binds more readily to vcp and associates avidly with hdac6, two outcomes that are expected to enhance proteasomal degradation. by engaging key quality control proteins, ∆264-cftr allows more ∆f508-cftr to mature. our data suggest that truncated forms of cftr (∆264) may be useful for cf gene therapy by affecting the maturation of endogenous cftr. ∆f508, the most common disease causing cftr mutation, has impaired conformational maturation during its biosynthesis, and thus is unable to exit the endoplasmic reticulum (er). a well-defined di-acidic er exit code has been identified within the first nucleotide binding domain (nbd1) of wildtype cftr (jcb 167: 65-74, 2004) , the disruption of which prevents cftr from efficiently exiting the er. this is consistent with the role of the diacidic code in the copii-mediated cargo concentration process. however, it is not known whether other types of er-to-golgi trafficking signals play a role in cftr trafficking in the early secretory pathway. even less is known about the trafficking signal(s) that are responsible for the rescue of ∆f508 cftr by a number of means. to achieve a better understanding of the trafficking signals dictating the export of cftr from the er, we constructed a series of cftr mutants to probe the potential functional targeting signals within both wild-type and ∆f508 cftr. it has been shown that nbd2 is not required for the export of wild-type cftr from the er. we further found that an internal deletion mutant lacking membrane spanning domain 1 (msd1) and nbd1-r but retaining the n-terminal cytoplasmic region, msd2 and nbd2 does not have sufficient signal to allow er export, suggesting that the signal(s) that are responsible for cftr export largely reside within the nbd1-r. unlike ∆f508 cftr, the di-acidic code mutants are not temperature-sensitive. dissecting the di-acidic code revealed that both acidic residues contribute to er export, and that simultaneous substitution of both leads to a much decreased export of cftr. we found that the temperature-dependent export of ∆f508 relies on the presence of the di-acidic code within the nbd1, suggesting that this code has a functional role in the temperature-dependent rescue of ∆f508 cftr. in our effort to further examine the role of the di-acidic code in the rescue of ∆f508 cftr by second site mutations, we found that ∆f508 cftr rescue by the simultaneous disruption of two of the arginine-framed tripeptides (afts) is also dependent upon the di-acidic code. surprisingly, we found that while nbd2 is not necessary for the export of wild-type cftr, it is required for the aft-mediated rescue of ∆f508 cftr, suggesting a role for cytoplasmic inter-domain interactions in the aft-mediated rescue of ∆f508 cftr. a detailed biochemical analysis of these mutants provides valuable insights into the roles of different cftr "trafficking" motifs in the er-to-golgi transport of cftr, the specific conformational defects of ∆f508 cftr and the potential molecular mechanism underlying the rescue of ∆f508 cftr. supported by cff. sonawane, n.d. 1 ; zhao, d. 1 ; galietta, l. 2 ; zegarra-moran, o. 2 ; verkman, a. 1 1. medicine, university of california san francisco, san francisco, ca, usa; 2. genetica molecolare, istituto gianina gaslini, genova, italy cftr inhibitors are predicted to prevent intestinal fluid secretion in enterotoxin-mediated secretory diarrheas such as cholera. cftr inhibitors that are not absorbed across the intestinal wall are attractive for diarrhea therapy because they may provide safe oral therapy. we previously discovered low affinity glycine hydrazide (glyh, ic50 ~5 µm) cftr inhibitors that block cftr at its external pore (muanprasat et al., j. gen. physiol. 2004; 124:125-137) . in order to increase inhibitor potency and prevent washout during severe secretory diarrhea, we synthesized a series of glycocalyx interactive cftr inhibitors containing a malonic acid hydrazide (malh) cftr pore blocking moiety linked to a lectin (sonawane et al., gastroenterology 2007; 132:1234 -1244 . lectin conjugation improved cftr inhibitory potency by ~100-fold (ic50 to 50 nm). high-affinity cftr inhibition was abolished by malh-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. fluorescently labeled malh-lectin remained membrane-bound for >6 hours after washout, whereas washout occurred in a few minutes without the lectin. malh-lectins blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice with ec50 50-100 pmol, and greatly reduced mortality in a suckling mouse model of cholera. we recently synthesized mono-and divalent cftr inhibitors consisting of malh coupled via a disulfonic stilbene linker to flexible mono and bifunctional polyethyleneglycols (pegs) of molecular size 0. 2, 0.75, 2, 3, 5, 10, 20, 40 and 100 kda, with calculated solution lengths of 1-23 nm, with the larger size pegs potentially spanning cftr dimers or inducing their formation. ic50 for inhibition of cftr chloride current was 10-15 µm for monovalent malh-pegs, but substantially lower and size-dependent for divalent malh-pegs, decreasing from 1.5 µm to 300 nm with increasing peg size. the mechanisms responsible for the improved and size-dependent potency of divalent malh-pegs were studied by whole cell, single-channel patch-clamp and by functional analysis of multivalent malh-conjugated dextrans and asymmetric divalent pegs. whole-cell experiments revealed reversible voltage-dependent block of cftr currents, with outward (positive) currents being more strongly blocked. in outside-out patch-clamp experiments, inhibitors caused a reduction of the mean open time. for malh-peg20kda-malh (2 µm) , the mean open time decreased from 189 ± 44 to 97 ± 29 ms. the effect on currentvoltage relationship and channel kinetics are consistent with a mechanism involving occlusion of the cftr pore from the extracellular side. luminally added divalent malh-pegs blocked by > 90% cholera toxin-induced fluid secretion in mouse intestinal loops with ic50s of <10 pmol/loop, and greatly reduced mortality in a suckling mouse model of cholera. nonabsorbable, multivalent cftr inhibitor-macromolecule conjugates may be useful as anti-secretory agents in the treatment of enterotoxin-mediated diarrheas. supported by cff and nih. activation of the cystic fibrosis transmembrane conductance regulator (cftr) clchannel is primarily controlled by pka-dependent phosphory-lation of the r domain. once the r domain is phosphorylated, atp binding and enzymatic activity at two nucleotide-binding domains (nbds) open and close the cftr clchannel. we hypothesized that r domain phosphorylation regulates cftr activity by modulating atp interactions within two nbds. to test this hypothesis, we studied the activity of wild-type cftr and five variants with deletions of portions of the r domain between residues 708 and 835. to alter atp-dependent channel gating, we tested three cftr stimulators, pyrophosphate (pp i , 2 mm), 2'deoxy-atp (2'datp, 1mm) and caatp (1mm) . consistent with previous studies, pp i , 2'datp and caatp all increased current of wild-type cftr about two-fold. each stimulator produced a similar increase in the r domain variants. the similar effects of stimulators on the channel activities of wild-type cftr and r domain variants suggest that the r domain does not have a major role in regulating atp-dependent channel gating. the r domain deletions did not alter the single-channel current amplitude of the r domain variants. however, r domain variants missing the sequence between residues 784 and 835 markedly reduced the open state probability, suggesting that this region is required for normal gating. these data suggest that the r domain does not control cftr activity by modulating atp interactions with nbd binding sites. instead, we speculate that the c-terminal part of r domain might participate in the channel-gating machinery downstream of atp regulation. supported by the cystic fibrosis foundation and howard hughes medical institute. haggie, p.m.; verkman, a. medicine and physiology, cvri, u.c.s.f., san francisco, ca, usa it was recently reported that phagolysosomes of alveolar macrophages from cf mice acidify in a cftr-dependent manner and that defective phagolysosomal acidification impairs bactericidal activity (di et al., nat. cell biol. 2006, 8:933-944) . these findings suggested a unifying hypothesis for cf disease progression: defective phagolysosomal acidification in cf macrophages permits the initiation and promotes progression of bacterial infection in the lungs. to reassess the central finding of that study we measured phagolysosomal ph using a fluorescent ph indicator containing oregon green® 488 (pka ~4.7) and tetramethylrhodamine covalently bound to zymosan. phagolysosomal ph was insensitive to cftr inhibition (10 µm cftrinh-172) in j774 macrophages (ph 5.08 ± 0.08 vs. 5.10 ± 0.03), alveolar macrophages from mouse lung (ph 5.34 ± 0.05 vs. 5.37 ± 0.07), and alveolar macrophages from human lung (ph 5.30 ± 0.08 vs. 5.35 ± 0.07). phagolysosomal ph in alveolar macrophages from ∆f508 cf mice was not significantly different from that in alveolar macrophages from wild-type mice (ph 5.34 ± 0.05 vs. 5.40 ± 0.06). to account for their finding of defective phagolysosomal acidification in alveolar macrophages, di et al. reported that lysosomal acidification was cftr-dependent and that fusion of lysosomes to phagosomes (at ~20 min after phagocytic uptake) was responsible for phagosomal acidification. we measured lysosomal acidification in j774 macrophages using a dextran-conjugate containing oregon green® 488 and tetramethylrhodamine and found that acidification was not impaired by cftr inhibition (ph 4.48 ± 0.03 vs. 4.42 ± 0.03) . we also measured the kinetics of phagosomal acidification using a zymosan-conjugate containing 5-(and-6)-carboxyfluorescein (pka ~ 6.4) and tetramethylrhodamine. phagosomal acidification in j774 macrophages and murine alveolar macrophages began within 3 min of phagocytosis and reached steady-state by 10-15 min, in agreement with prior data in murine peritoneal and bone marrow-derived macrophages. acidification kinetics in j774 macrophages was not altered by cftr inhibition, nor was acidification kinetics different in wild-type vs. cf alveolar macrophages. our findings do not support the conclusion that phagolysosomal acidification in alveolar macrophages is cftr-dependent, nor that it is impaired in cf. the mechanism of phagosomal acidification proposed by di et al. is not in accord with our data or precedents in the literature. because phagolysosomal acidification is central to the proposed mechanism linking defective cftr chloride channel function with cf lung disease, our results do not support the involvement of cftr in defective macrophage function in the pathogenesis of cf lung disease. supported by cff and nih. pared to that of wild-type cftr. the hsp70 family of molecular chaperones play important roles in the protein quality control process within the er. hsp70 atpase activity is regulated by multiple co-chaperones such as hsp40, bag-1 and hspbp1. hsp105, a high-molecular-weight member of the hsp70 family was recently shown to display nucleotide exchange activity for hsp70s in vitro. furthermore, hsp105 was identified as a component of a cftr-associated multiple protein complex using a global proteomic approach (cell 127: 803-815, 2006) . in an attempt to explore the role of hsp105 in cftr conformational maturation in the er, we over-expressed the co-chaperone in hek293 cells and quantitatively analyzed its effect on the maturation and degradation of cftr. consistent with its role as a nucleotide exchange factor for hsp70s, over-expression of hsp105 inhibits the er export of wild-type cftr and promotes its degradation. however, in striking contrast, over-expressing hsp105 stabilizes ∆f508 cftr and promotes its er export at reduced temperature. this effect is less pronounced at physiological temperature. the apparently opposite effects of hsp105 on wild-type and ∆f508 cftr maturation and quality control, suggests distinct conformational maturation pathways for the two cftr molecules, and reveals a specific role for hsp105 in regulating ∆f508 cftr refolding. such conclusion is reinforced by rnai experiments and further supported by quantitative co-immunoprecipitation. rnai-mediated down-regulation of hsp105 expression destabilizes ∆f508 cftr and reduces its export at reduced temperature, and hsp105 shows more extensive association with the er form of ∆f508 cftr than that of wild-type cftr. further analysis revealed that hsp105 not only modulates the chaperone activities of hsp70s but also alters their steady state levels within the cell, creating secondary effects on cftr maturation and quality control. further studies are necessary to achieve a better understanding of the machinery, pathway and mechanism of ∆f508 cftr conformational maturation at reduced as well as physiological temperatures, and this in turn will provide critical insights and key factors that are of potential value to the rescue of the trafficking defect of ∆f508 cftr. farinha, c.m. 1, 2 ; pissarra, l.s. 1 ; amaral, m.d. 1, 2 1. department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; 2. ctr hum genet, nat inst health, lisboa, portugal the most frequent mutation in the cystic fibrosis (cf) gene, f508del, causes retention of its protein product, f508del-cf transmembrane conductance regulator (cftr) in the endoplasmic reticulum (er) as a core-glycosylated intermediate that is rapidly degraded. therefore, most f508del-cftr fails to traffic to the plasma membrane, where wild-type (wt) cftr normally functions as a chloride (cl-) channel. retention, however, is not due to lack of function, since the mutant still retains some function if it reaches the membrane. instead, it results from misfolding which is recognized by the er quality control (erqc) in which many intervenients, including molecular chaperones, participate. identification of molecular partners involved in the disposal of f508del-cftr to the proteasome is therefore crutial. it was recently shown that casein kinase ii (ck2), a pleiotropic constitutively active protein kinase involved in several processes, such as neoplasia, cell survival and viral infections, binds wt-cftr near the f508 residue, phosphorylating its first nucleotide binding domain (nbd1) at s511 [1] . interestingly, deletion of f508 abrogates this interaction, which is the first described f508del-dependent protein-protein interaction. our aim here was to identify whether ck2 interaction affects the early steps of cftr biogenesis, turnover and processing. to this end, novel bhk cells were produced which stably express wt-or f508del-cftr in which the consensus residue s511 was substituted by either a neutral (alanine -s511a) or an acidic residue (aspartate -s511d). initial biochemical analyses of these cell lines revealed that: 1) cftr proteins bearing d or a at position 511 are processed; 2) despite producing equivalent levels of cftr transcripts, s511a expressing lines consistently show lower levels of cftr protein. metabolic labelling and pulse-chase experiments followed by cftr immunoprecipitation were also performed in these lines. after quantification of bands b (immature form) and c (mature form) of cftr, these preliminary results show that substitution of s511 (by a or d) does not affect the turnover or processing of either wt-or f508del-cftr. the effect of ck2 inhibition on the turnover and processing of cftr was also studied, by incubating cells with 20 µm of the ck2 inhibitor tetrabromobenzotriazole (tbb) for 90 min. pulse-chase experiments under these conditions show that: 1) the steady-state levels of both wt-and f508del-cftr are reduced; 2) the turnover of wt-cftr (but not of f508del-cftr) is increased; and 3) processing of wt-cftr is decreased. such data are consistent with a putative stabilizing role for ck2 upon wt-cftr [1] . however, in our preliminary results this effect does not appear to be dependent on residue 511 (nor on the putative charge added by the kinase on this residue), thus suggesting that this effect may be indirect. further studies are underway to identify the mechanism by which ck2 affects the turnover and processing of cftr. 1. treharne et al (2007) pissarra, l.s. 1 ; xu, z. 2 ; farinha, c.m. 1, 3 ; sheppard, d.n. 2 ; amaral, m.d. 1, 3 1. department of chemistry and biochemistry, faculty of sciences, university of lisboa, lisboa, portugal; 2. dep physiology, univ bristol, school med sciences, bristol, united kingdom; 3. ctr hum genet, nat inst health, lisboa, portugal g550e and 4rk (the simultaneous mutation of four arginine-framed tripeptides (afts): r29k, r516k, r555k and r766k) are second site mutations that rescue the processing and function of f508del-cftr [1, 2] . these revertant mutations rescue f508del-cftr from retention within the endoplasmic reticulum by distinct mechanisms: g550e likely alters the conformation of the first nucleotide-binding domain (nbd1), whereas 4rk plausibly allows f508del-cftr to escape er retention/retrieval mediated by afts [3] . both g550e and one of the afts (r555k) lie close to the residue g551, where a common cf-causing mutation g551d occurs, generating a correctly localized clchannel with a severe gating defect. our aim here was to assess whether the revertants g550e and 4rk influence the folding, processing and gating behaviour of the g551d mutation, by employing biochemical and functional approaches. to test this idea, we introduced the g551d mutation into cftr cdnas containing either g550e or 4rk in the pnut vector and stably expressed these constructs in bhk cells. preliminary results from biochemical studies indicate that the mature fully-glycosylated form of cftr protein (band c) of both g550e-g551d-cftr and 4rk-g551d-cftr were present at similar levels to those of g551d-cftr. analysis of cftr-mediated iodide efflux from these cells revealed that g550e is unable to rescue the functional defect of g551d. however, 4rk generated an efflux of iodide larger than that elicited by cells expressing g551d, albeit smaller than that of cells expressing wild-type (wt) cftr. consistent with these data neither 4rk nor g550e rescued the defective channel gating of g551d-cftr. however, both revertants caused a small increase in g551d-cftr activity by attenuating the prolonged interburst interval of g551d. we conclude that f508del and g551d disrupt cftr channel gating by distinct mechanisms. altogether, our data suggest that at least when in cis with the g551d mutation, the afts (together or individually) might have a direct effect on cftr channel gating. this raises the possibility that 4rk, in addition to its well-described effect on trafficking, may act on cftr structure and/or folding, as previously suggested for r555k [4] . work supported by grant pocti/sau/mmo/58425/2004 and pluriannual funding of cigmh (fct, portugal) and the uk cf trust. l. pissarra was a recipient of bd/9095/2002 doctoral fellowship (fct, portugal) . [1] carvalho ac, gansheroff lj, teem jl (2002) j biol chem 277, 35896-35905. [2] chang xb, cui l, hou yx, jensen tj, aleksandrov aa, mengos a, riordan jr (1999) mol cell 4, [137] [138] [139] [140] [141] [142] [3] roxo-rosa m, xu z, schmidt a, neto m, cai z, soares cm, sheppard dn, amaral md (2006) proc natl acad sci usa 103, 17891-17896. [4] teem jl, carson mr, welsh mj (1996) improves ∆f508-cftr intracellular trafficking in cf epithelial cells, although the mechanism by which this occurs is not clear. to identify gene products with atlered abundance in response to 4pba, we performed a differential display rt-pcr screen on rna isolated from ib3-1 cf epithelial cells treated with 4pba for 0-24 hours. we isolated a cdna encoding stip-1, a putative human stat-3 (signal transducer and activator of transcription-3) interacting protein and confirmed that 4pba causes transiently increased stip-1 mrna and protein abundance after 8 hours of exposure. stip-1 was originally described as a scaffold protein that is required for ligand-dependent activation of stat-3. stip-1 is also identical to elongator protein 2 (elp-2), a subunit of the multicomponent elongator complex that stimulates rna polymerase ii activity. interestingly, recent data suggests that elongator may also regulate polarized secretion (rahl, et al. (2005) , mol. cell, 17:841-853). we therefore hypothesized that stip-1/elp-2 would regulate ∆f508-cftr intracellular trafficking. in ib3-1 cells, stip-1/elp-2 associates with ∆f508-cftr in coimmunoprecipitation experiments after 4pba treatment. in ib3-1 cells overexpressing stip-1/elp-2, immunofluorescence experiments suggested that ∆f508-cftr trafficked to the plasma membrane even in the absence of 4pba treatment. stip-1/elp2 co-localized with markers of the golgi (58 kda golgi protein) and exocytic vesicles (snap-25) when overexpressed in these cells. in contrast, overexpression of a deletion mutant of stip-1/elp-2 lacking the wd domain, or stat-3 binding region, blocks the improvement of ∆f508-cftr trafficking in response to 4pba in immunofluorescence and surface biotinylation experiments. this stip-1/elp-2 deletion mutant did not co-localize with golgi or exocytic vesicle markers. furthermore, immunofluorescence experiments also suggested that 4pba causes colocalization of stip-1/elp-2 and elp-1, another member of the elongator complex that is often defective in familial dysautonomia. these data are consistent with stip-1/elp-2 regulating ∆f508-cftr intracellular trafficking in response to 4pba. this regulation may occur in the context of 4pba stimulating assembly of elongator, which in turn may promote trafficking of exocytic vesicles carrying ∆f508-cftr to the plasma membrane. supported by grants from niddk. fold [1, 2] . however, functional studies demonstrate significant differences in the gating behaviour of hcftr and mcftr [3] . a powerful approach to investigate cftr structure and function is to examine interspecies differences and identify regions of conservation and divergence. to understand the structural basis for the functional differences between hcftr and mcftr, we generated hmcftr chimeras containing mcftr domains on an hcftr backbone. for this purpose, we replaced all or part of nbd1, nbd2 or the rdomain of hcftr with the equivalent regions of mcftr and investigated their biochemical and functional properties. the in vivo folding of these hmcftr chimeric proteins was indirectly evaluated from their maturation status, after their stable expression in novel bhk cell lines. like wt-hcftr, most chimeric proteins were processed within the cell. however, two chimeras failed to mature: clone 12b (mnbd1, amino acid (aa) residues 518-585) and clone 114c (mnbd2, aa 1260-1412). we compared the murine sequence of these two chimeras with that of hcftr to determine the respective physico-chemical distances (pcds) of their aa changes. changes with higher pcd values were selected and in vitro mutagenesis performed to introduce these aa alterations into hcftr cdna. for clone 12b the selected changes were: e527q, e528q (pcd = 29); s531t (pcd = 58); k536q (pcd = 53), i539t (pcd = 89) and k584e (pcd = 56). for clone 114c the changes were: p1290t (pcd = 38), k1302q (pcd = 53), y1307n (pcd = 143), s1311k (pcd = 121), c1344y (pcd = 194), d1394g (pcd = 94) and e1409d (pcd = 45). biochemical analyses of these mutants stably expressed in bhk cells revealed that for clone 12b, e527q, s531t, k536q and i539t were processed, whereas k584e was not. for clone 114c, p1290t, k1302q, y1307n, d1394g and e1409d were processed. iodide efflux showed that s531t, k536q, i539t, e1409d and d1394g were functional, but not k584e. we consider that with additional functional analyses, our approach will identify critical residues responsible for conformational changes and hence, functional differences between hcftr and mcftr. work supported by the bbsrc and grant pocti/mgi/47382/2002 (fct, portugal) and pluriannual funding of cigmh (fct, portugal) . ac dapaula is a recipient of phd fellowship sfrh/ bd/17475/2004 (fct, portugal we recently reported that the upr decreases endogenous wild-type (wt) cftr expression. as a follow up of these studies, we investigated the role of the folding deficient, ∆f508 cftr on upr induction and identified a mechanism by which the upr decreases cftr expression. for these studies, we developed a cell line expressing recombinant ∆f508 cftr on the endogenous wt background (calu-3 ∆f) and established individual clones with different ratios of endogenous (wt) to recombinant (∆f508 cftr) expression. two clones which express 1:1 (calu-3 ∆fc3) and 1:8 (calu-3 ∆fc5) ratios of wt to ∆f508 cftr and cfpac-1 cells expressing endogenous ∆f508 cftr were tested as models. the upr was constitutively active in calu-3 ∆fc5 cells only. in calu-3 ∆fc3 induction of ∆f508 cftr expression resulted in upr activation, indicating that high expression of ∆f508 cftr is required for upr induction. furthermore, following pharmacological induction of the upr, endogenous cftr mrna decreased to undetectable amounts both in calu-3 ∆f and cfpac-1 cells. the decrease in cftr mrna levels was not the result of shortened mrna half-life. in contrast, using a human cftr promoter driven reporter (pcftr-pluc), we demonstrate suppression of the cftr promoter when the upr is activated. considering that correction of ∆f508 cftr is the main therapeutic approach for cf, it is important that ∆f508 cftr expressed at low levels, such as in vivo, does not activate the upr. however, our results also reveal that ∆f508 cftr correctors have to be tested for upr activation, since transcriptional inhibition in this setting may contribute to inefficient rescue in native cells or in vivo. genistein, a naturally occurring tryrosine kinase inhibitor, at low concentrations (50µm) stimulates wt -cftr-mediated chloride secretion in a variety of cell and tissue types by an adenosine 3',5'-cyclic monophosphate (camp)-independent pathway. genistein has been shown to potentiate the channel function of ∆f508-cftr and g551d-cftr channels by prolonging channel open time (hwanget al., am j physiol cell physiol 273: c988-c998, 1997; illeck et al. am j physiol cell physiol 277: c833-c839, 1999) patch clamp studies have provided indirect evidence that genistein may bind directly to cftr and potentiate channel open probability by modifying the function of the nucleotide binding domains (weinreich et al., pflügers arch 434: 484-491, 1997) . the goal of the present study was to determine whether genistein activated purified and reconstituted cftr protein as an initial step toward defining its molecular mechanism of action. the effect of genistein on purified and reconstituted wt-cftr was first studied following its reconstitution into planar lipid bilayers. in the presence of 100µm atp, phosphorylated cftr exhibited a low open probability (po= 0.12). the addition of 50µm genistein, caused a significant increase to the open probability of cftr (po=0.76). using an electrogenic flux assay on a population of purified and phosphorylated cftr molecules, we determined that 50 µm genistein caused a potentiation (70% increase) of chloride channel activity in the presence of 100 µm atp. these results suggest that this macroscopic assay of purified cftr function is sufficiently sensitive to detect the effect of this potentiator. currently, the consequences of genistein on the channel activity and atpase activity of purified reconstituted ∆f508-cftr and g551d-cftr are being assessed. preliminary results suggest that in the presence of low atp concentrations (10-100 µm),50µm genistein inhibits the atpase activity of ∆f508-cftr, suggesting that it interacts directly with the nucleotide binding domains to alter their function. previous studies indicated an acute coordination between the activities of cystic fibrosis transmembrane conductance regulator (cftr) chloride channel and the amiloride sensitive epithelial sodium channel (enac) so that both these channels are either activated or deactivated in a synchronous fashion in the human sweat duct (reddy et.al, 1999) . however, the mechanisms responsible for such cooperativity between these ion channels are unknown. previous studies indicated that: cytoplasmic ph controls cftr activity through effects on phosphorylation (reddy et.al. 1998) . at ph 6.0, cftr chloride conductance was reduced to 10±5 ms/cm 2 , but incresed to 42±7 ms/cm 2 at ph 8.0 (n=number of ducts=5). enac channel activity is also a function of cytosolic ph in heterologous expression systems (chalfant et.al, 1999) . the objective of this study was to test the hypothesis that the cytosolic ph may mediate the cooperative effects that occur between cftr and enac. we used basolaterally a-toxin permeabilized apical membrane preparations of native human sweat duct which expresses enac and cftr robustly as an experimental system. we showed that while luminal ph had no effect, changes in cytosolic ph acutely affected enac activity. that is, acidic ph inhibited, while basic ph activated enac activity. alkalinizing cytosolic ph from 6.8 to 8.5 increased enac conductance (genac) by 35±1.8 ms/cm 2 (n=6). ph regulation of enac activity appears to be independent of cftr and endogenous kinase activities because basic ph stimulated enac: a.) after deactivating cftr by removing camp and atp in normal ducts, b.) in the absence of cftr in cf ducts, and c.) after blocking endogenous kinase activity with the non-specific kinase inhibitor, staurosporine. na + /h + exchanger (nhe) may mediate changes in cytosolic ph as a function of intracellular na + activity. nhe mrna is expressed in the sweat duct and cytosolic ph responds to changes in na + gradients across the basolateral membrane. conceivably, when transport conditions are favorable and intracellular na + is low, alkaline ph would allow enac and cftr to cooperatively admit na + and clthrough the apical membrane. when conditions are unfavorable and intracellular na + is excessive, acid ph would limit na + and clentry to protect the cell from disruptive changes in cell volume. thus, changes in cytosolic ph may play a crucial role in coordinating the activities of enac and cftr during transepithelial salt transport. acknowledgements: kirk taylor and sucharitha madireddi for expert technical assistance. funded by nih-ro1 de14352, nih-ro1hl08042 and the nancy olmsted trust. the cystic fibrosis transmembrane conductance regulator (cftr) is an anion channel that is normally expressed in the plasma membrane but is mislocalized in cf. to monitor trafficking we inserted super ecliptic phluorin, a ph-sensitive variant of green fluorescent protein (gfp) (miesenbock g, de angelis da, rothman je (1998) nature 394, 192-5) into the fourth extracellular loop of cftr. this construct, which we call cftr-phluor, has weak fluorescence intracellularly that increases ~10-fold when it is inserted into the plasma membrane and the phlour becomes exposed to more alkaline extracellular ph . thus cftr-phluor should be less fluorescent than cftr with the normal gfp fused to the n or c termini when situated in intracellular vesicles. despite the insertion of an entire gfp in the middle of the channel protein, cftr-phluor trafficked to the membrane and patch clamp studies revealed little change in unitary conductance or open probability compared to wild-type cftr. cftr-phluor enabled visualization of vesicle insertion events at the plasma membrane by total internal reflection fluorescence (tirf) microscopy. faintly fluorescent vesicles containing cftr were observed under the plasma membrane and eventually fusing with it to generate a burst of fluorescence. phluorin inserted at the same position in ∆f508-cftr, the predominant mutant in cf, was retained in the er where the phluor remained partially quenched. when the folding defect was corrected by culturing cells at 29°c or treating them with known correctors, ∆f508-phluor trafficking was partially restored and total cell fluorescence increased. this enabled a quantitative analysis of ∆f508-cftr correction based on the increase in fluorescence in individual cells by flow cytometry. cftr-phluor may be most useful for microscope-based high-content screening studies of cftr and ∆f508-cftr trafficking. support: the breathe program, canadian cf foundation, canadian institutes of health research and cf foundation therapeutics (usa). tukaye, d.n.; guggino, w.b. physiology, johns hopkins university, baltimore, md, usa type iii secretion system (t3ss) toxins of p. aeruginosa are important virulence factors in p. aeruginosa infections. one of the t3ss toxins, exos, has been shown to facilitate uptake and invasion of p. aeruginosa at airway surface epithelium. in mouse models of pseudomonas pneumonia, infection with p. aeruginosa exos+ strains caused increased levels of fluid in lungs as determined at autopsy in contrast to infection with p. aeruginosa exosstrains. the exact molecular mechanism underlying these observations is not known. exos is a bifunctional protein with gtpase activating protein (gap) activity at the n terminal domain and adp ribosyl transferase (adprt) activity at the c terminal domain. we found that exos-gap activity increases total cellular levels of mature (c band) wild type (wt) cftr in cfbe41o-cells as measured by western blot analysis. this increase was mediated by decreased delivery of wt cftr for lysosomal degradation. in contrast, exos-gap failed to increase total levels of ∆508 cftr (b band). interestingly, exos-gap increased total levels of ∆508 cftr, bands c and b, following rescue with 4-phenylbutyrate. this indicates that targets of exos-gap exist in cftr trafficking beyond the er degradation pathway. exos-gap also brought about a corresponding increase in surface levels of mature wt cftr as measured by surface biotinylation. in conclusion, we have shown that p. aeruginosa t3ss exos-gap, upregulates total and surface levels of mature wt cftr by modulating cftr trafficking beyond er, most likely by decreasing lysosomal degradation. the decrease in lysosomal degradation could be occurring in part by inactivation of small molecular weight g-proteins involved in delivery of wt cftr to lysosomes. increases in total and hence surface levels of cftr could in part explain increased amounts of fluids seen in p. aeruginosa pneumonia mouse model treated with exos+ strains. the post-maturational trafficking and localization of cftr is regulated by a wide variety of proteins. among the most prominent are several pdz domain proteins that bind to the c-terminal residues of cftr. in particular, two of these proteins, nherf1 and cal, have been shown to mediate opposing effects on the apical membrane levels of the disease-associated ∆f508-cftr mutant, by controlling the balance between endocytic recycling and lysosomal degradation. in particular, whereas overexpression of nherf1 rescues ∆f508-cftr at the cell surface, overexpression of cal has the opposite effect on cftr. cal-mediated downregulation of cftr requires a functional pdz binding site. we have now shown that suppression of endogenous cal cooperates with temperature rescue to stabilize functional ∆f508-cftr channels at the apical membrane in polarized bronchial epithelial cells. this work supports the hypothesis that the cal pdz binding site is a therapeutic target for treatment of cystic fibrosis. a more direct test of this hypothesis will require competitive inhibitors that can efficiently block the cal:cftr interaction. in addition, given the large-number of protein-protein interactions formed by both the cal pdz domain and the cftr c-terminus, these inhibitors should ideally exhibit bidirectional selectivity, neither disrupting the favorable nherf1:cftr interaction, nor strongly displacing other membrane proteins that interact with cal. using a fluorescence polarization assay and peptide-array technology, we have now identified a high-affinity, cal-selective peptide inhibitor. compared to cftr, this sequence binds cal more tightly, but nherf1 more weakly. furthermore, we have shown that the cal:cftr interaction is the weakest among the known cal-binding proteins, and that cftr should thus be susceptible to selective displacement from the cal binding site. finally, we have designed cell-permeable peptides that allow us to test the hypotheses (a) that such compounds will specifically disrupt the cal:cftr interaction and (b) that such targeted stereochemical inhibition will stabilize the functional cell-surface expression of ∆f508-cftr in airway epithelial cells. the pathways for the endoplasmic reticulum associated protein degradation (erad) of misfolded proteins in the mammalian cells are incompletely understood. we investigated the role of molecular chaperones in erad for the cystic fibrosis transmembrane conductance regulator (cftr) and its common folding mutant, ∆f508cftr. we found that hsp70 and its cochaperone hdj-2 interacted significantly with ∆f508cftr and wt-cftr in airway epithelial cells. hsp70 interacted with the immature form of cftr while hdj-2 associated with both immature form and ubiquitylated cftr. structure-function studies showed that hdj-2 recognized ubiquitylated cftr via its zn-binding domain. immunoprecipitation and gst-fusion protein pulldown experiments revealed that hdj-2 interacted with ubiquitin. in steady-state, over-expression of hdj-2 elicited increasing both immature and mature forms of wt-cftr, but it resulted in decreasing immature form of ∆f508cftr. pulse-chase studies showed that co-expression of hdj-2 promoted ∆f508cftr degradation, reducing its half-life from 85 to 50 min. in contrast, hdj-2 expression did not significantly affect wt-cftr biogenesis. these data are consistent with our finding that hdj-2 shows a selective physical interaction with ∆f508cftr, which translates into a functional discrimination between the mutant and its wt counterpart. the hpd motif within the j-domain of hdj-2, which is necessary for the co-chaperone to bind hsp70 and stimulate its atpase activity, was required for hdj-2 mediated ∆f508cftr degradation. mutation of the hpd motif retarded the degradation of ∆f508cftr and increased the steady-state levels of ∆f508cftr 3-fold. a role for hsp70 in hdj-2 mediated ∆f508cftr degradation was tested in which hsp70 knockdown increased ∆f508cftr expression 3-4 fold. hjd-2 regulates ∆f508cftr maturation and knockdown of endogenous hdj-2 promoted ∆f508cftr and its camp-dependent anion conductance in airway epithelial cells. in contrast, we were unable to detect maturation of ∆f508cftr in hsp70 knockdown experiments, indicating that the maturation of ∆f508cftr mediated by reduced hdj-2 expression may be independent of hsp70 function. taken together, our data demonstrate that hdj-2 is a molecular sensor that can detect differences in the folding related to the ∆f508cftr maturation. these data also highlight a novel pathway for hdj-2 linked, ubiquitin-dependent degradation of ∆f508cftr. reduction of hdj-2 expression or its association with ∆f508cftr promotes maturation of this mutant and therefore represents a potential therapeutic approach for cystic fibrosis. [supported by nih and cf foundation] how the loss of cftr function results in cholesterol accumulation within the cell is currently unknown. cftr activation is driven by the camp and we propose that cf cells respond to the loss of cftr activity by increasing the camp pathway in order to increase cftr expression. to test this hypothesis, epithelial cells were treated with the phosphodiesterase-3 (pde3) and pde4 inhibitors milrinone and rolipram, respectively. inhibition of pde4 function with rolipram in wildtype cells leads to peri-nuclear free cholesterol accumulation identical to what is observed in cf epithelial cells as viewed by filipin staining, strongly implicating a camp-dependent pathway in the regulation of cholesterol trafficking. the pde3-selective inhibitor milrinone had no effect on cholesterol trafficking suggesting specificity for the pde4 pathway. our preliminary data support this hypothesis in that both cf-model 9hteo-pcepr cells and mouse nasal epithelium (mne) from cftr -/-mice exhibit reduced protein expression of the campspecific phosphodiesterases pde4 compared to wt controls. the proposed consequence of a chronic amplification in camp signaling is altered cholesterol transport regulation. to address this potential role of pde4 in cholesterol trafficking, free cholesterol was visualized using filipin staining in wild type cells that were treated with rolipram, a pde4-specific inhibitor. conversely, treatment of cf-model pcepr cells with the pka inhibitor rp-camps significantly improves cholesterol processing, further pointing to the involvement of the camp pathway. we propose that the camp pathway influences cholesterol processing through the regulation of β-arrestin-2 (βarr2) according to the premise that chronic increase in the camp pathway would initiate an elevation in βarr2 expression. βarr2 is an important regulator of adrenergic receptor recycling and organelle trafficking. because βarr2 is pivotal in regulating endocytotic recycling pathways, it could also impact cholesterol processing. this predicted increase in βarr2 protein expression is observed in both cf-model pcepr cells and cftr -/-mne compared to respective controls. over expression of βarr2 in wt epithelial cells leads to cf-like peri-nuclear cholesterol accumulation further implicating a role for βarr2 in the development of this phenotype. altered cholesterol trafficking in cftr would lead one to expect different βarr2 localization throughout the cell. further understanding of the implications of altered camp signaling and its relationship to aberrant cholesterol accumu-rationale: we recently reported that vcp (valosin containing protein) physically interacts with gp78/amfr (autocrine motility factor receptor) to couple retrograde translocation of ∆f508-cftr to proteasomal degradation (vij et al. jbc 2006) . recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded proteins termed the aggresome. histone deacetylase-6 (hdac6) is a unique cytoplasmic deacetylase capable of interacting with ubiquitin and mediating the accumulation of ∆f508-cftr in aggresome bodies. hypothesis: vcp competes with hdac6 to dissociate ∆f508-cftr perinuclear aggregates. methods: ib3-1 (∆f508/w1282x) cf bronchial epithelial cells were transiently transfected with ∆f508-cftr and vcp-gfp constructs and treated with a hdac6 inhibitor (tubacin), proteasome inhibitor (ps-341 or mg-132), lysosome inhibitor (baflomycin a1) or nil-tubacin (control) for 4, 8 or 12 hrs. the effect of these inhibitors on ∆f508-cftr and vcp:∆f508-cftr interactions were quantified by immunoprecipitating these protein complexes followed by immunoblotting with vcp, hdac6 or clathrin antibody. ∆f508-cftr levels were measured by metabolic labeling using tran-35s-label (250 µci/ml) for a 30 min pulse and 2 hrs chase. the subsequent effects of these inhibitors on vcp localization was detected by fluorescence microscopy of gfp moiety. results: the co-immunoprecipitation experiments showed that hdac6inhibition by 10µm tubacin (12 hrs) promotes vcp:∆f508-cftr and prevents hdac6:∆f508-cftr interaction. the proteasome inhibition (mg132 10µm) resulted in maximal hdac6:∆f508-cftr at 4hrs with minimal at 12 hrs while vcp:∆f508-cftr increased overtime (4 to 12hrs). adding tubacin (10µm; 4 hrs) reversed the proteasomal inhibitor effects. in pulse-chase experiments, mg-132 increased the accumulation of cftr bform and poly-ubiquitinated-∆f508-cftr as compared to the untreated control. tubacin suppressed the levels of mg-132 induced poly-ubiquinated-∆f508-cftr as well as ∆f508-cftr b-form. the vcp localization by microscopy showed the accumulation of vcp-gfp in perinuclear aggregates in the presence of proteasome inhibitor (ps-341 10µm). the 10µm tubacin blocked these ps-341 induced perinuclear aggregates. we observed that vcp is associated with endocytic protein-complexes containing clathrin in the presence of ps-341 indicating cytosolic re-localization of vcp. we further confirmed the cytosolic re-localization of vcp-gfp in the presence of lysosome inhibitor (bafilomycin a1 10µm) by fluorescent microscopy. conclusion: we found that a small molecule inhibitor of aggresome function prevents hdac6:∆f508-cftr interaction, promotes vcp:∆f508-cftr interaction, and suppresses the accumulation of poly-ubiquinated-∆f508-cftr. wang, y. 1 ; jiang, y. 1 ; zhu, n. 1 ; feng, x. 1 ; yang, h. 1, 2 ; ma, t. 1, 2 1. membrane channel research laboratory, northeast normal university, changchun, china; 2. biopharmaceutical center, liaoning normal university, dalian, china deletion of the codon encoding the phenylalanine residue at position 508 (∆f508) in the cystic fibrosis transmembrane conductance regulator (cftr) is the most common mutation causing cystic fibrosis (cf). the ∆f508 mutation results in a cftr protein with impaired folding, trafficking and gating in human and rodents. the consequences of ∆f508-cftr mutation in other species have not been studied. the purpose of the present study was to characterize the ∆f508 mutant of porcine cftr in cell culture model. cdna sequence encoding full-length porcine cftr (pcftr) was cloned from the lung by rt-pcr using primers designed according to porcine genomic sequences in two bac clones (genbank accession no. ac092497 and ac092478) containing all exons of the porcine cftr. pcftr encodes a 1482-amino-acid protein that shows 92.3% identity to human cftr. the phenylalanine residue at position 508 is conserved in pcftr. functional analysis of pcftr stably expressed in fisher rat thyroid (frt) epithelial cells by short-circuit current assays indicated a campactivated cl-channel similar to human cftr except that the sensitivity of pcftr to the specific cftr inhibitor cftrinh-172 is 25-fold lower than that of human cftr. a ∆f508 mutation of porcine cftr was generated by site-directed mutagenesis. surprisingly, ∆f508-pcftr expressed as a func-tional chloride channel activated by forskolin in stably transfected frt cells. western blot analysis of cos7 and bhk cells transiently transfected with cmyc-tagged ∆f508-pcftr indicated a protein pattern identical to that of wildtype pcftr. immunofluorescence analysis of the transiently transfected cells demonstrated similar plasma membrane expression pattern of ∆f508-pcftr and wildtype pcftr. these results clearly demonstrated unimpaired cellular processing of ∆f508-pcftr. however, the sensitivity of ∆f508-pcftr to forskolin activation (ic50~2.5 µm) was dramatically lower than that of wildtype pcftr (ic50~0.5 µm), indicating a defect of channel gating by pka phosphorylation. the present study provided the first evidence that the ∆f508 mutation in mammalian cftr does not result in impaired cellular processing that is closely associated with cf phenotype. we have optimized airway epithelial cell spheroid cultures for functional measurements of cftr chloride conductance in order to screen smallmolecule activators and inhibitors. spheroids, which are sealed spheroidal monolayers of airway surface epithelial cells with diameter 75-250 micron, were generated within 2 to 4 days after nasal brushing and suspension culture. yields of up to 2000 spheroids were obtained from nasal brushing of a non-cf or cf subject. approximately 3,000 spheroids could be prepared from one million freshly isolated human bronchial epithelial cells. we compared several electrophysiological and fluorescence methods to assay cftr function in spheroids. whole-cell current was measured in single spheroids following pipette immobilization and micropipette puncture of the solutionexposed apical cell membrane. large, forskolin-stimulated whole-cell chloride currents were measured in non-cf spheroids. as an independent approach, transepithelial potential difference was measured by micropipette insertion into the spheroid lumen. for fluorescence detection, cell cytoplasm was stained with the halide indicators mqae or lzq. fluorescently stained spheroids were immobilized on a coated coverglass or in a custom microfluidic chamber. halide flux across the apical surface was measured in response to chloride-nitrate or iodide-chloride solution exchange, in the absence and presence of camp activators. the fluorescence assay is suitable for medium-throughput screening of ∆f508 correctors and potentiators, with definitive verification of cftr chloride channel function by whole-cell current analysis. an airway spheroid cultures provide a powerful approach to prioritize the efficacy of ∆f508 correctors because of their rapid generation without prolonged culture, and their suitability for quantitative analysis of cftr function. supported by cff and nih. background and aims: cftr has been shown to be expressed and functional in ventricular cardiomyocytes. however, to date, no physiological role for cftr in the heart has been established. the aim of this study was to determine if cftr plays a role in the regulation of cardiomyocyte contraction rate. methods: cardiomyocyte contraction rate was assessed using the neonatal mouse beating assay. ventricular myocytes were isolated from neonatal mice and cultured for 2-3 days until they formed a functional in sitium. contraction rates were captured by video imaging and analyzed by metamorph in the presence and absence of beta-adrenergic receptor stimulation by isoproteronol (10 µm). assessment of cftr activity was determined by pharmacological inhibition by glibenclamide (100 µm), dpc (500 µm), and cftr inh-172 (10 and 20 µm). the role of k atp channels was assessed by 5-hd (100 µm). results: baseline contraction rate of vehicletreated (dmso or h 2 o) cardiomyocytes was unchanged. addition of isoproteronol caused a significant increase in contraction rate, which was sustained for at least 30 minutes. in contrast, application of glibenclamide to beating ventricular myocytes significantly, but transiently, inhibited cardiomyocyte contraction rate, with recovery occurring within 20 minutes. glibenclamide did not affect the initial increase in contraction rate caused by isoproteronol stimulation, but inhibited sustained increases in isoproteronol-stimulated contraction rate. to determine if this was an effect of k atp channel inhibition we examined the effect of 5-hd on contraction rate. in contrast to glibenclamide, 5-hd had no inhibitory affect on basal or isoproteronol-stimulated contraction rate, indicating glibenclamide's inhibitory effect was due to its inhibition of cftr. to further verify this, we examined the effect of two other cftr inhibitors, dpc and cftr inh-172 on ventricular cardiomyocyte contraction rate. both dpc and cftr inh-172 elicited similar responses as glibenclamide, with a quick, but transient inhibition of baseline contraction rate and inhibition of sustained isoproteronol-stimulated increases in cardiomyocyte beating. summary and conclusions: using neonatal murine ventricular myocyte cultures inhibition of cftr leads to a significant, but transient inhibition of baseline ventricular cardiomyocyte contraction rate. additionally, while inhibition of cftr did not affect initial increases in beta-adrenergic receptor-stimulated contraction rate, cftr inhibition did prevent sustained increases in contraction rate. the data suggest that cftr plays a role in modulating ventricular cardiomyocyte contraction rate during resting and adrenergic-stimulated conditions. drevillon, l. 1,2 ; tanguy, g. 1, 2 ; hinzpeter, a. 1,2 ; arous, n. 1,2 ; goossens, m. 1,2 ; fanen, p. 1, 2 1. génétique, inserm u.841, créteil, france; 2. faculté de médecine, université paris 12, créteil, france cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cftr protein. the aim of this study was to find cellular proteins capable of interacting with cftr and modifying its processing or its trafficking. we have used a genetic screen in yeast to identify such proteins and identified commd1 that preferentially interacted with the third cytoplasmic loop of cftr. commd1 is a regulator of copper transporter atp7b in hepatocyte and sodium epithelial channel enac, but its exact biochemical function and physiological relevance remain elusive. here, we report that first, commd1 interacted with wild-type and f508del-cftr in epithelial cells and second, commd1 subcellular distribution was not only both nuclear and cytoplasmic, but also in cytoplasmic vesicular compartments. we wondered if commd1 was involved in the processing and/or the trafficking of cftr protein in epithelial cells. we demonstrated that commd1 was not involved in cftr processing but that cell surface expression of wild-type cftr at the plasma membrane was cut by half when commd1 expression was 90% reduced by rna interference. we drew the conclusion that commd1 is a major component of cftr trafficking and/or recycling at the plasma membrane, precise characterization of commd1 in these vesicular compartments is under process to decipher its function in cftr trafficking. commd1 has been labelled as the prototype of a newly described protein family that plays a role in inhibiting nf-kappab signalling. moreover, subcellular distribution of commd1 was different in cf and non cf cells, nuclear form of commd1 being increased in f508del cells compared to wild-type cells. thus, this opens up a new area of investigation about commd1 nuclear function. finally, these data indicate that commd1 is involved in multiple cellular processes of outstanding interest in cf pathophysiology. understanding how its modulation modifies transepithelial transport and inflammation in cf versus non cf cells should give new therapeutic clues to reduce exaggerated inflammation and improve fluid secretion in cf patients. supported 1 ; fanen, p. 2 ; galietta, l.j. 1 1. lab. of molecular genetics, istituto giannina gaslini, genova, italy; 2. inserm u.841, creteil, france class iii cystic fibrosis mutations cause reduced cltransport by impairing the process of cftr channel opening. typical class iii mutations include g551d, g1349d, and f508del, all of them affecting nucleotide binding domains. it has been reported that mutations residing in other regions of the cftr protein may also reduce channel activity. we investigated the extent of loss of function caused by such mutations, in particular by missense mutations localized in cftr intracellular loops, and the sensitivity to pharmacological stimulation with cftr potentiators. to determine cftr activity, plasmids carrying the coding sequence for wild type or mutant cftr were co-transfected in cos-7 cells together with the halide-sensitive yellow fluorescent protein yfp-h148q/i152l. the rate of cftr-dependent anion transport was measured after short-term stimulation with forskolin alone or in combination with potentiators (felodipine, phenylglycine pg-01, or sulfonamide sf-01). processing of cftr protein was assessed by examining glycosylation status of cftr by immunoprecipitation experiments. when stimulated with forskolin alone, cftr mutants showed the following anion transport compared to the wild type protein: i148t (45 %), i175v (153 %), q179k (54 %), e193k (9 %), g970r (6 %), d1152h (29 %). interestingly, all mutants having a reduced anion transport responded to stimulation with potentiators pg-01, sf-01, and felodipine (5 µm) with an increase in anion transport that approached the level of normal cftr protein. the correction of defective cftr activity by potentiators was confirmed in frt cells expressing e193k and d1152h mutants using short-circuit current measurements. our results indicate that some missense cf mutations, like e193k and g970r, really cause a severe reduction in cftr activity. others, like d1152h, cause only a partial loss of function which may explain their association with a milder form of the disease. conversely, some other amino acid changes (with the extreme example of i175v that seems to cause a gain of function) do not impair cftr activity and therefore may represent polymorphisms and not cf-causing mutations. interestingly, all mutants having a mild or more severe anion transport defect were sensitive to potentiators. this finding indicates that cftr potentiators have a wide efficacy on many class iii mutants and therefore may represent a promising therapeutic strategy for a significant number of cf patients. supported by cfft and telethon-italy. cftr, a cl-channel membrane protein, is a member of the atp-binding cassette (abc) super-family. mutations in cftr result in a misfolded protein which fails to mature, leading to impaired flow of cl-ions across the membrane of the epithelial cells lining the airway, and to cf. the most common mutation leading to cf is df508. currently available structural information relevant to cftr modeling includes low resolution structures of cftr (20å) and p-gp (8å) and high resolution structures of other abc transporters as well as nucleotide binding domains (nbd) . of particular relevance is the newly published structure of the bacterial multidrug abc transporter, sav1866, whose topology can serve as a starting point for cftr modeling. as a first step towards the development of cf therapeutics, epix has undertaken the modeling of the 3d structure of cftr (excluding the r domain) in its conducting (i.e., open-channel, nbd-dimer) state, using our proprietary membrane-protein modeling technology (predicttm) in combination with other modeling tools. to date, we have modeled the cytosolic part of the receptor, namely the nbds:icls construct and work is ongoing on modeling the transmembrane (msd1:msd2) region. the now available partial model has been scanned for putative binding sites for df508-cftr correctors/potentiators. several potential sites have been located, and epix has initiated its in silico screening technology in order to identify potential drug candidates. this work is supported by the cfft. the deletion of a phenylalanine residue at position 508 (f508del) in the first nucleotide-binding domain (nbd1) of cftr is the principal cause of cystic fibrosis (cf). the altered interaction of f508del cftr with endoplasmic reticulum (er) quality control proteins, primarily chaperones, promotes its proteasomal degradation. however, it is believed that crucial cftr-interacting proteins (cips) remain unknown [1] . moreover, there is little information currently available on the strength of cip-cftr interactions. our goals here are two-fold: 1) to quantify cftr-cip interactions; and 2) to isolate and characterise unidentified cips. to address our first goal, we used surface plasmon resonance (spr; biacore tm ) to quantify real-time binding of hsc70 (a well-known cip) to bacterially expressed wt-and f508del-nbd1. hsc70/hsp70 was covalently immobilised onto the surface of carboxymethyl dextran (cm5) sensor chips (500 µm) and the real-time binding of purified nbd1 and control proteins quantified. in control experiments, anti-hsc70 antibody 1b5 (20 nm; against residues 373-650) bound specifically to immobilised hsc70 with high affinity (k d , 0.46 ± 0.07 nm; n = 3) whereas bovine serum albumin (15 µm) did not interact (n = 10). given the difficulties associated with expression of hnbd1 carrying the f508del mutation [2] it was not used in our spr analyses. instead, we used purified murine (m) nbd1 to quantify the impact of f508del on the strength of the hsc70-nbd1 binding. in the presence of low concentrations of atp (≤5 um), the affinity of nbd1 binding to immobilised hsc70 was strengthened when f508 was deleted (wt, k d app , 1.20 ± 0.16 µm; f508del, k d app , 0.43 ± 0.11 µm; n = 3; p < 0.05). interestingly, raising the concentration of mgatp weakened the binding of nbd1 (0.5 µm) to hsc70 (wt, k i , 29 µm atp; n = 2). furthermore, deletion of f508 dramatically increased the concentration of mgatp required to destabilise the nbd1-hsc70 interaction (f508del, k i , 221 µm atp; n = 2). in summary, we used spr to demonstrate that: (i) nbd1 of cftr binds specifically to hsc70, and (ii) the f508del mutation enhances the association of nbd1 with hsc70. we are presently investigating the effect of co-chaperones and small molecules on the interaction of hsc/hsp70 with wt-and f508del-nbd1. to identify novel cips, purified nbd1 was immobilised onto metalaffinity resin and used to capture cips from epithelial cell lysates. by this approach, cips were captured from human respiratory cell (calu-3) lysates and analysed by 2d-electrophoresis. relevant protein spots so far identified by mass spectrometry include: 1) raichu404x (thr/ser kinase); 2) profilin 2 isoform b; 3) annexin a5; 4) ifapsoriasin (intermediate filament-associated ca 2+ regulatory protein); 5) mgc35308 (member of the er reticulon family). current analyses are underway to determine their functional roles in cftr trafficking and function. [1] amaral ( billet, a. 1 ; melin, p. 1 ; norez, c. 1 ; bilan, f. 2 ; vandebrouck, c. 1 ; mettey, y. 3 ; becq, f. 1 1. physiologie, cnrs umr 6187, poitiers, france; 2. gmas umr 6187, poitiers, france; 3. faculté medecine et pharmacie, poitiers, france in order to gain a better insight into the structure-activity relationship (sar) of cftr protein, we have functionally characterized a cystic fibrosis mutation: the cftr-g622d, identified in patients with oligospermia (vankeerberghen et al., 1998) . we examined cftr chloride channel activity by patch-clamp analysis, in whole cell configuration, using cos-7 cells transiently transfected with gfp-cftr-wt or with the mutant gfp-cftr-g622d. cftr channels were activated by 10 µm forskolin (fsk), and, after stable activation 10 µm cftrinh-172 was added. as gfp-cftr-wt, g622d chloride channels elicit a time and voltage independent current in presence of 10 µm fsk. gfp-cftr-g622d current density at +40mv (52.08 ± 5.8 pa/pf) is 1.5 fold less than gfp-cftr-wt current density at +40mv (76.67 ± 9.3 pa/pf). g622d mediated currents are blocked by 10 µm of the specific cftr-inhibitor, cftrinh-172 (ma et al., 2002) . as expected, gfp-cftr-wt channels are stimulated by 100 µm of the potent activator 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (mpb-91, derand et al., 2001 ) with a current density at +40 mv of 127.44 ± 25.49 pa/pf. interestingly, no activation of g622d channels is recorded in presence of mpb-91: difference between currents recorded in basal condition or in presence of mpb-91 is not significant. using iodide efflux experiments, we showed that cftr-g622d mutant does not respond to several benzo[c]quinolizinium derivatives. however, the mutant channel can be activated by forskolin, ibmx and genistein. thus, the mechanisms of activation by xanthine and isoflavone are not affected by the mutation. these results show that: (1) cftr-g622d channels are functional: activation by camp pathway, inhibition by cftrinh-172. (2) camp chloride currents elicited by cftr-g622d protein are weaker than cftr-wt. (3) cftr-g622d channels are refractory to benzo[c]quinolizinium activation. the replacement of the glycine by a negative charged amino acid in position 622 affects cftr chloride channel activity and prevents activation by benzo [c] quinolizinium. this amino acid g622, localized in the interface of nbd1 and r domain between two β sheets (callebaut et al., 2004) seems to have a crucial role in pharmacological activation of cftr by mpbs. further experiments will be performed to evaluate the role of the charge and of the nature of the amino acid at position 622 in cftr protein. supported by vaincre la mucoviscidose and cnrs. cysteine string protein (csp) is a j-domain containing protein that regulates the extent of wild type cftr maturation. over-expression of csp blocks the formation of mature, glycosylated (band c) cftr and increases the level of immature (band b) cftr, whereas knockdown of csp elicits a marked increase in cftr maturation (zhang et al., j. biol. chem. 281: 11312, 2006) . thus, csp appears to block the exit of cftr from the er. when the er exit of cftr is blocked by a different mechanism, i.e. the expression of mutant sar1-gtp (sar1 h79g interferes with the formation of copii vesicles), the steady-state level of immature cftr is two-fold greater than that resulting from csp over-expression. this finding suggests that csp, in addition to blocking er exit of cftr, may facilitate the degradation of the immature protein. in agreement with this concept, csp expression increased cftr ubiquitylation. a mutation within the conserved hpd motif of the j-domain (h43q) disrupts the ability of csp to interact with and stimulate the atpase activity of hsp70. csp h43q does not block cftr maturation (above ref); in addition, the level of immature cftr was ~60% higher when h43q csp was over-expressed than the level observed with mutant sar1. this finding suggests that the pro-degradative effect of csp on immature cftr requires hsp70 binding/activation. chip (carboxy terminus of hsp70-interacting protein) is an e3 ubiquitin protein ligase that can target wt and ∆f508 cftr for proteasome-mediated degradation (meacham et al., nature cell biol. 3: 100, 2001) . we sought to determine weather chip was involved in the csp-mediated degradation of cftr. indeed, co-immunoprecipitation (co-ip) experiments showed that chip associated with csp. also, csp h43q, which has reduced interaction with hsp70, showed a similar level of chip interaction by co-ip, suggesting that their association did not depend on hsp70 as a linker between the two proteins. a csp mutant that is truncated after the cysteine string domain, which is its site of membrane attachment, blocked cftr maturation as did wt csp; however, this mutant did not decrease cftr band b levels like wt csp did. in addition, the csp mutant missing its cterminus did not associate with chip. these findings further implicate chip in the csp-mediated degradation of immature cftr, and they suggest that the c-terminus of csp is a chip binding domain. when a dominant-negative mutant of chip, which lacks the u-box needed for chip-mediated ubiquitylation (chip∆ub), was co-expressed with cftr, csp was no longer able to facilitate the degradation of cftr in the er. these data show that it is possible to separate the effect of csp on cftr maturation from its ability to mediate the degradation of immature cftr. thus, csp blocks the exit of cftr from the er, and this requires a j-domain mediated activation of hsp70 but not the csp c-terminus. the action of csp on cftr degradation requires both a functional j-domain and the csp c-terminus, and this may require a trimeric complex involving csp, hsp70 and chip. [ the cystic fibrosis transmembrane conductance regulator (cftr) represents the main clchannel in the apical membrane of epithelial cells for camp-dependent clsecretion. mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of cftr is crucial to design future medicament for protein therapy. recently, we reported on the synthesis and screening of a small library of 6-phenylpyrrolo [2,3b] pyrazines (named rp derivatives) evaluated as activators of wild-type cftr, g551d-cftr and f508del-cftr clchannels (noël et al, 2006) . this preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of cftr (rp-107 and rp-108). here we studied structure-function relationship of more than 190 compounds prepared by chemical synthesis, and the subsequent activation of cftr channels. within the 6-phenylpyrrolo [2,3-b] pyrazines family, we observed by iodide efflux technique that rp-173 bearing a 2hydroxyphenyl substituant is more potent (ec 50 = 16 nm) than rp-107 having 4-hydroxyphenyl substituant (ec 50 = 150 nm). by whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of rp-173 activate linear chloride current in cho cells stably transfected with human wild-type cftr. this current was inhibited by 10 µm of cftr inh -172. we also found significant stimulation of short circuit current (i sc ) by rp-173 (ec 50 = 9 nm) on colon of cftr +/+ but not of cftr -/mice mounted in ussing chamber. stimulation of i sc by rp derivatives was inhibited by glibenclamide. the structural analogue , was less potent (ec 50 = 347 nm). as for rp-107 compound, we found that the 7-nbutyl chain is crucial for rp-146 and rp-173 activity, and that the 2-hydroxyphenyl compounds without 7-n-butyl chain are unactive on cftr. in this study, we showed that the presence of an hydroxyl group at position 2, 3 or 4 of the pyrrolopyrazine cycle determined the highest activity on cftr. the most potent compound is the 7-n-butyl-6-(2-hydroxyphenyl)5hpyrrolo [2,3b] pyrazine . the potency of these agents indicates that compounds in this class may be of therapeutic benefit in cftr-related diseases, including cystic fibrosis. the most common mutation causing cystic fibrosis (cf) is the deletion of f508 in the cftr gene, which results in inefficient trafficking of the cftr protein from the endoplasmic reticulum to the plasma membrane. the surface expression of the cftr is usually quantified biochemically with western blot or functionally with electrophysiological assays. however, these methods are labor intensive. we explore using fluorescence-based techniques for monitoring cftr surface expression. wild-type cftr was tagged at the amino terminus with green fluorescent polypeptides (gfp-cftr). the gfp-cftr was either transiently or stably expressed in chinese hamster ovary (cho) cells. the cells were then treated with a red fluorophore that specifically labels plasma membranes. the fluorescent images were acquired with an olympus laser scanning confocal microscope. two image channels were taken simultaneously. the green channel was excited with a 488nm laser, and signals within 500-545nm were collected. the red channel was excited with a 559nm laser, and signals within 570-670nm were collected. for a single focal plane, each image was composed of 1024*1024 pixels. threshold values were given to both green and red channels. pixels with an intensity value above the threshold were marked as green/red pixels. the green pixels in the first channel represent the signal of gfp-cftr, and the red pixels in the second channel represent the location of the plasma membrane. thus, pixels that are both green in the first channel and red in the second channel are the cftr surface expression, and denoted as yellow pixels. the percentage of successfully trafficked cftr is thus quantitatively estimated by the ratio of yellow to green pixels in the single plane. alternatively, a stack of images from the same cell at different depth was collected and analyzed. the total amount and surface portion of cftr expression are then determined by summing the correspondent pixels. the percentage of cftr surface expression was then calculated and will be verified both biochemically and functionally. the approach is modified to study deltaf508 cftr trafficking. we anticipate that the method can be used to screen cftr correctors from a small or intermediate sized chemical database. supported by cfft and nih. the most common mutation of the cystic fibrosis transmembrane conductance regulator (cftr) gene (deletion of phe-508 (∆f508) in the first nucleotide binding domain (nbd1)) causes retention of the ∆f508 protein in the endoplasmic reticulum (er). ∆f508 mutation prevents conformational maturation of cftr protein without altering profoundly the local structure of nbd1, but possibly by disrupting the interaction between nbd1 and nbd2. however, the individual role of nbd1 and nbd2 in biogenesis, folding, maturation and membrane stability of a full length cftr is still under debate. using splicing by overlap extension method (soe by pcr), we generated six recombinant cftr proteins with inverted or suppressed nbds. the native boundary domains of nbd1 and nbd2 were respected for all mutants (riordan j.r. et al. 1989 ) and their c-terminal tails preserved. the constructs were transiently or stably expressed in cos-7 and bhk cells respectively. immmunodetection and immunolocalization assays confirmed that deletion of nbd2 domain (n1-cftr) did not alter the trafficking or the plasma membrane expression of cftr. both processes were abolished in the presence of ∆f508 mutation. however, using whole cell patch-clamp configuration, we did not detect any camp-stimulated clcurrent. pulse-chase experiments established that the turnover of n1-cftr is 3-4 fold faster (t1/2~ 5h) than its wild type counterpart (t1/2~ 18h). when we replaced nbd2 with nbd1 (2n1-cftr), we could detect a faint plasma membrane expression associated with a weak cl-channel activation. more surprisingly, the t1/2 of 2n1-cftr coreglycosylated form was around 12h. when nbd1 was deleted (n2-cftr) or substituted with nbd2 domain (2n2-cftr), the expression and functional pattern were similar to cftr ∆f508. furthermore, we showed that the n1-cftr folding was not attenuated as demonstrated by protease susceptibility and sdsresistant thermoaggregation tendency, supporting the notion that n1-cftr membrane stability requires nbd1-nbd2 interaction. altogether, our results suggest that nbd1 domain is essential to form a foldable cftr that satisfies er quality control (erqc) but correct inter-domain assembly is mandatory for its stability at plasma membrane. the results are also in agreement with previous suggestion that ∆f508 mutation alters the interactions between nbds and transmembrane domains of cftr. supported 1 1. inserm u845, inserm, paris, france; 2. inserm u845, inserm, paris, france; 3. inserm u845, inserm, paris, france; 4. inserm u845, inserm, paris, france; 5. inserm u845, inserm, paris, france; 6. inserm u845, inserm, paris, france; 7. inserm 845, inserm, paris, france; 8. inserm u845, inserm, paris, france the physiological role of cftr in renal epithelium is not known. in the proximal tubule, cftr co-localizes with the sodium-phosphate cotransporter npt2a, a protein involved in phosphate (pi) reabsorption. the aim of our work is to determine if there is a functional interaction between cftr and npt2a. to this end, the arnm coding for cftr and the arn coding for npt2a (bearing a myc tag in c-ter) were injected in xenopus laevis oocytes. electrophysiological or biochemical experiments were performed 3 days after arns injection. currents induced by 1mm pi (ipi) were measured in voltage-clamped (-50 mv) oocytes that expressed npt2a alone, or in oocytes co-expressing cftr + npt2a. for immunoprecipitation experiments, 24-1 cftrab and myc ab were used. results ipi was significantly reduced in oocytes expressing cftr + npt2a compared to that measured in oocytes expressing npt2a alone. using a two-bath electrodes configuration during voltage-clamping did not change this observation. this result suggests that when expressed in oocytes, cftr is in functional interaction with npt2a. kinetic analysis (ipi as a function of pi concentration) of npt2a showed a reduction in vmax but not in phosphate km. this suggests that npt2a membrane expression and/or activity is altered when cftr is co-expressed. co-immunoprecipitation experiments performed on protein lysate derived from oocytes co-expressing cftr and npt2a suggested an interaction between the 2 transporters. using a pka activating cocktail as a pkc activator induced cftr-mediated currents in cftr+npt2a expressing oocytes. in these oocytes, ipi is decreased by pkc activation (as expected from npt2a regulation) but is increased shortly after exposure to the pka activating cocktail. the increase of ipi observed under this condition is at variance to that expected from classical npt2a regulation. in oocytes expressing npt2a alone, both pka activation and pkc activation reduced ipi. these observations suggest that in oocytes, cftr expression modifies npt2a regulation. our study suggests that, after expression in xenopus laevis oocytes, cftr interacts functionally with npt2a, and modifies its regulation. an interaction is also suggested by the co-immunoprecipitation of cftr and npt2a. these results cannot discriminate between a direct or an indirect (via a third protein) interaction. to determine if an intracellular chloride concentration change may participate to the functional interaction, we are now measuring in cftr+npt2a expressing oocytes intracellular chloride activity (using chloride-sensitive microelectrodes) during the use of the pka activating cocktail, and measuring ipi when pka is stimulated in the presence of a specific cftr inhibitor. cystic fibrosis (cf), the most commonly inherited lethal pulmonary disorder in caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (cftr). to date, more than 1000 mutations were identified in cftr and were associated with clinical disease. a phenylalanine deletion at position 508 accounts for 70% of cf genotypes in caucasian populations, and determines cftr misfolding and degradation by proteasome. consequently, limited cftr abundance leads to multiorgan disease, affecting the lung, pancreas, gut, liver, sweat glands and the reproductive organs. the past several years have witnessed an explosion of information regarding the identification and elucidation of molecules and pathways that are regulated by cftr or that regulate cftr activity. genomics and proteomics technologies now offer the opportunity to examine global alterations in the mrna and protein expression patterns of cf cells and tissues to elucidate the pathways linking defective cftr to clinical disease. here we describe systems biology methods that integrate heterogeneous datasets, including protein-protein interaction networks, gene expression and mass-spectrometry profiles, and mutation and genetic variation information, in order to identify the regulatory circuits and active subnetworks that are responsible for the progression and the severity of cf disease. the study will focus on correlations that define the key events in the cftr folding and trafficking pathways that lead to pathogenesis and dysfunctions, and could provide insights and targets for intervention and therapy. the number of cftr cl-channels in the plasma membrane, and thus the transepithelial cl-secretion is controlled, in part, by the endocytic trafficking of cftr. rab gtpases regulate the endocytic trafficking by acting as molecular switches that cycle between the gdp-bound (i.e. inactive) and the gtp-bound (i.e. active) state, associate with target membranes, and recruit downstream effectors. rab5 facilitates cftr endocytosis and rab11a facilitates the recycling of internalized cftr to the plasma membrane. furthermore, rab7 and rab9 facilitate cftr trafficking to the lysosome. the mechanism of sorting internalized cftr for either recycling to the plasma membrane or degradation in the lysosome is not well understood. recent evidence demonstrates that protein sorting occurs in rab4 specific sorting endosomes. presence of cftr in rab4 compartment has been previously demonstrated in human airway epithelial cells by electron microscopy. yet, the role of rab4 in cftr trafficking has not been determined in these cells. rab4 did not control cftr trafficking in fibroblasts (bhk cells). however, it negatively regulated the plasma membrane expression of cftr in the colorectal cells . the goal of this study was to elucidate the role of rab4 in the endocytic trafficking of cftr in polarized human airway epithelial cells (calu-3 cells and cfbe41o-cells stably expressing wt-cftr or deltaf508-cftr). endogenous rab4 coimmunoprecipitated with cftr in calu-3 cell and cfbe41o-cells. to determine if rab4 plays a role in cftr trafficking, cfbe41o-cells were transfected with either the flag-tagged, dominant negative (dn) rab4 mutant deficient in gtp binding (gdp-locked; flag-rab4-n121i) or with a non-specific cdna control (gfp). if rab4 sorts internalized cftr to the recycling pathway, the dn rab4 should inhibit recycling, and, thus, should decrease the plasma membrane expression of cftr. if, on the other hand, rab4 sorts internalized cftr for degradation the dn rab4 should inhibit cftr degradation, increase cftr recycling and thus, should increase the expression of cftr in the plasma membrane. the dn rab4 increased cftr expression in the plasma membrane as determined by cell surface biotinylation. these data are consistent with the role of rab4 in sorting internalized cftr for degradation. similar to overexpression of the dn rab4, silencing endogenous rab4 with double stranded small interfering rna specific for the human rab4 sequence increased the plasma membrane expression of cftr and increased the cftr mediated cl-secretion across polarized cfbe41o-cells in ussing chamber experiments. the plasma membrane half-life of deltaf508-cftr is decreased compared to wt-cftr. however, after silencing rab4, the plasma membrane half-life of deltaf508-cftr was similar to that of wt-cftr, as determined by cftr disappearance from the plasma membrane following incubation with cycloheximide. our data are consistent with the role of rab4 in sorting the internalized cftr for degradation and are consistent with the changing paradigm for the role of rab4 in protein trafficking (supported by the nih grant 1 p20 rr018787 and the cff swiate03qo). the conformational changes underlying activation of phosphorylated cftr by atp remain unclear. in the present study we assessed the utility of labeling endogenous cysteine(s) in cftr using an environmentally-sensitive fluorescent probe alexa-488 in monitoring such changes in structure. these studies were performed using purified and functionally reconstituted wild-type cftr. we labeled pka-phosphorylated cftr with 138 µm alexa-488 maleimide prior to reconstitution in phospholipids liposomes. changes in fluorescence intensity were monitored in a suspension of proteoliposomes following addition of 1 and 5 mm mg-atp or 5 mm mg-amp-pnp in order to evaluate nucleotide dependent changes in conformation. in order to identify the labeled cysteines, the protein was subjected trypsin-mediated proteolysis and analysis by mass spectrometry. we have shown that cftr is labeled using alexa-488 maleimide. a significant increase in fluorescence emission occurred in alexa-488 labeled cftr of 17 ± 5 (au) relative to empty liposomes one minute after the addition of activating nucleotide, 1 mm mg-atp (n = 3). the non-hydrolyzable analogue, 1mm mg-amp-pnp, failed to cause an increase in fluorescence of alexa-488-labeled cftr. however, the higher concentration of 5 mm mg-amp-pnp evoked an increase in fluorescence similar in magnitude to that of 5 mm mg-atp suggesting that the fluorescence response reflects structural changes relating to atp binding to cftr. analysis by mass spectrometry identified the labeled residue as c647. this cysteine resides at the junction between nbd1 and the "r" domain in a region which has been suggested to be highly dynamic on the basis of multiple crystal structures. our studies suggest that this flexible region around c647 of cftr undergoes conformational change upon mg-atp binding such that it moves the alexa 488 fluorophore into a relatively more polar environment. this finding presents a first case where conformational changes in the cftr nbds induced by atp binding could lead to changes in the conformation of the r domain. further, these studies suggest that fluorescence methods can be used to probe dynamic conformational changes in purified reconstituted cftr. acknowledgements cystic fibrosis is caused by mutations in the apical chloride channel cftr with 90% of patients carrying at least one allele of the ∆f508 mutation. this mutant form of cftr is characterized by a trafficking defect in which it fails to exit the endoplasmic reticulum (er). we have previously reported that ∆f508 cftr is found in complex with hsp90 and its co-chaperones in cell extracts suggesting that either this mutant form of the protein accumulates in on-pathway folding intermediates which result in its accumulation in the er or that these intermediates include inhibitory factors leading to retention of ∆f508 cftr in this compartment. we have examined the role of one of these hsp90 co-chaperones, fk506 binding protein 8 (fkbp8), in the life cycle of cftr. fkbp8 is a unique member of the immunophilin family of peptidyl-prolyl isomerases (ppiases) which mediate the cis/trans interconversion of peptidyl-prolyl bonds, which is thought to represent a rate limiting step in protein folding. in order to establish a functional role for fkbp8 in cftr stability and trafficking, we modulated the expression level of this protein in human bronchial epithelial cells endogenously expressing either wild type or ∆f508 cftr. a knockdown of fkbp8 by sirna resulted in a destabilization of both wild type and ∆f508 cftr as well as a concomitant loss in channel activity of wild type and temperature corrected ∆f508 cftr. conversely, over expression of fkbp8 resulted in a stabilization of the er glycoform of ∆f508 cftr in both mammalian and yeast systems. these data suggest that fkbp8 is an essential component of the hsp90-mediated folding machinery for both wild type and ∆f508 cftr, which supports the hypothesis that ∆f508 cftr accumulates in the er in an on-pathway folding intermediate. dmh cftr folding involves a series of sequential, although potentially overlapping steps that involve i) formation, orientation and integration of (tms), ii) helical packing, iii) folding of cytosolic and extracytosolic domains, and iv) establishing proper domain-domain contacts. this process begins cotranslationally and is facilitated by the sec61 er translocation machinery and a diverse group of lumenal and cytosolic chaperones. the most common inherited cftr mutation, deltaf508, disrupts one or more early steps along this folding pathway. a major obstacle in understanding how deltaf508 causes cf is that the native folding environment, comprised of lipids, cytosolic and er proteins, is not amenable to traditional biochemical and biophysical approaches. to overcome this problem we are developing a flu-orescence based analytical approach that enables one to directly access nascent ribosome-attached folding intermediates at virtually any stage of synthesis. the strategy requires incorporation of fluorescent probes into the substrate protein, which provide a highly selective means to monitor structural features at sub-nanomolar concentrations in highly complex biological mixtures. polypeptides are synthesized in vitro from truncated mrna templates to generate uniform cohorts of ribosome-bound nascent chains. progressive stages of folding can then be evaluated using mrna templates of different length. as proof-of-principle we have shown that fully synthesized gfp variants can be readily trapped in an unfolded state and complete their folding only after the entire nascent peptide has exited the large ribosomal subunit. this approach enabled us to measure real-time folding kinetics of extended peptide domains following synchronous ribosome release and compare fluorophore maturation of four fluorescent protein variants with different spectral properties cfp, gfp, venus and mcherry. to extend this approach to cftr and other general protein substrates, we are employing fluorescence resonance energy transfer (fret) using "donor" and "acceptor" probes incorporated simultaneously into the substrate protein. an in frame n-terminal fusion to cfp provides the "donor" fluorophore, while the "acceptor" fluorophore is incorporated at an engineered stop codon using a synthetic modified aminoacyl-suppressor trna. we are currently developing a 'molecular ruler' to correlate the efficiency of energy transfer with changes in the relative distance between probes in ribosome-bound and ribosome-released cftr folding intermediates. fret-based experiments are underway to evaluate folding of both n-terminal (1-80aa) and nbd1 (387-674aa) domains. these studies will provide new insight into the mechanism of cotranslational cftr domain folding and provide a potential means to identify agents that correct the deltaf508 folding defect. relationships between hypoxia, ado release, and prostenoid production in ariway cells are not defined. in the present studies, we examined mechanisms governing hypoxia-induced production of prostenoids in cfbe41o-and calu-3 monolayers. using elisa-based detection of prostenoid and leukotriene production, both calu-3 and cfbe41omonolayers treated with ado (10 µm) or arachidonic acid (aa, 10 µm, the parent molecule of prostenoids and leukotrienes) for 16 hrs led to increases in pge 2 levels (1198-1283 ± 64-145 pg/mg of total proteins vs controls 732 ± 89 pg/mg in calu-3 cells, 756-839 ± 8-12 pg/mg vs controls 408 ± 42 pg/mg in cfbe41o-cells, p<0.05 in both airway cell types compared with control). in contrast, ltb 4 and ltc 4 release were not stimulated by both maneuvers. we next used sirna techniques to knockdown a2b adenosine receptor expression, and demonstrated durable knockdown of a2b ar expression to~25% of scrambled sirna-treated controls following lipid-based transfection with sirna directed against the a2b ar in cfbe41o-cells. following sirna knockdown of a2b ar expression, cells were exposed to hypoxic conditions (mucosal volume = 2 ml in 12-well plate), normoxic conditions (mucosal volume = 0.2 ml, fio 2 = 0.21), and ado (10 µm). hypoxic stress led to high levels of mucosal pge 2 production (1613 ± 102.8 pg/mg vs controls 495 ± 19.6 pg/mg, p<0.01) that was sensitive to a2b ar knockdown (743 ± 70.6 pg/mg, p<0.01). exogenous ado also stimulated pge 2 production, but this effect was small relative to hypoxia. we next examined basolateral effects of ado and aa on transmucosal clsecretion in cfbe41o-and calu-3 cells grown as monolayers on permeable supports and conducted ussing chamber analysis under voltage clamp conditions. addition of ado and aa to the basolateral membrane (10 µm) activated transmucosal clsecretion [18.94 ± 4.07 µa/cm 2 (ado) and 14.30 ± 2.6 µa/cm 2 (aa) for calu-3 cells; and 12.65 ± 2.31 µa/cm 2 (ado) and 18.96 ± 1.24 µa/cm 2 (aa) for cfbe41o-cells expressing wtcftr] that was sensitive to basolateral blockade with barium cl -(10 mm). isc by either agonist was stimulat-ed in the presence and absence of apical glybenclamide (200 µm), adenosine deaminase (ada -2u/ml) and hexokinase (hexo -2u/ml), confirming that the effects were specific for the basolateral membrane and could be accomplished with or without cftr activity. these results demonstrate that hypoxia is sufficient to stimulate pge 2 production through a2b ars, and that both ado and aa activate basolateral k + channels to promote transepithelial clsecretion. the findings further support a model in which ado (and prostenoids) regulate cltransport through effects on both the apical and the basolateral membrane, and elucidate molecules that couple the two membranes as part of the normal clsecretory response. hypoxia would be predicted to increase local prostenoid production through ado release and stimulation of a2b ars, and further highlight a unifying model by which local hypoxia promotes inflammation in airway epithelia. supported by the nih and cff. low levels of tissue oxygen -observed in lung diseases such as cystic fibrosis -initiate a signaling cascade resulting in altered transcription of genes possessing a hypoxia response element (hre). we recently used gene chip mrna array, quantitative rt-pcr, protein analysis, and functional (short circuit current) assays to show that cftr expression is strongly repressed by hypoxia in vitro and in vivo. among several thousand human mrnas suppressed by low ambient oxygen, cftr was among the most strongly inhibited (by 10-20 fold) in a cell model system. however, cftr and the vast majority of other repressed genes lack a traditional hre. because hypoxia inducible factor (hif) acts primarily as a transcriptional activator, the mechanisms underlying hypoxia mediated suppression of mammalian genes are not well understood. we therefore tested the hypothesis that micrornas play a central role during transcriptional regulation of genes such as cftr. micrornas are evolutionarily conserved, short noncoding rna sequences believed important for repression of gene expression. some reports have estimated that at least 30% of protein-coding genes are regulated by micrornas. thousands of micrornas have been predicted in the human genome by available algorithms (e.g. palgrade, mirscan, and promir) and several hundred experimentally validated. the cftr gene contains 16 predicted target sites for 13 micrornas in the 3'utr, including hsa-mir-383 (nucleotides 1309-1331 of the cftr 3'utr), hsa-mir-449 (nt 1443-1463) and hsa-mir-509 (nt 1041-1063). however, no study to date has examined the effect of micrornas on cftr message levels, or the influence of micrornas during the cftr response to environmental perturbations such as hypoxia. we therefore performed a global, genome-wide analyses of both mrnas and micrornas expressed during hypoxia (using air/liquid or liquid/liquid interface culture systems) in ht29 (colonic epithelial) cells that robustly express cftr. we found that approximately 9% of micrornas were significantly altered by hypoxia, as judged by mirna profiling (ambion mirvana™ bioarray 1566v2, asuragen microrna expression profiling service). comparison of these hypoxia-related micrornas with expression profiles of their predicted targets indicated a much lower level of correlation than has been previously hypothesized by others. we show that none of the micrornas predicted to regulate cftr are altered in ht29 (in response to hypoxia). these findings are therefore consistent with the notion that highly expressed genes in a particular tissue type are not co-expressed with their predicted regulatory micrornas. our data also indicate that micrornas do not mediate the hypoxic repression of cftr via the 3' utr. supported by nih and cff. 1 1. physiology, mcgill university, montreal, qc, canada; 2. biochemistry, mcgill university, montreal, qc, canada the ∆f508 mutation impairs cftr maturation and trafficking to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. using our highthroughput trafficking assay, we previously showed that the quinoline km60, a structural analogue of sildenafil, corrects the ∆f508-cftr processing defect and leads to a significant increase in cftr surface expression after 2 h treatment with 10 µm and after 24 h treatment with 10 nm according to detection of mature cftr in western-blots. additional studies have now been carried out with other preparations to assess the functionality of ∆f508-cftr following rescue by km60 treatment. different time and concentration exposures were tested on the cftr function using iodide efflux assays with both bhk and human bronchial epithelial cells over-expressing ∆f508-cftr. exposure to 10 µm km60 for 2 h partially restored iodide efflux responses to 10 µm forskolin + 50 µm genistein in both cell line. after 24 h incubation, rescue of the mutant protein in bhk cells still required 10 µm, whereas 100 nm was sufficient in the human cell line cfbe. iodide efflux assays were performed at different times following km60 wash out to functionally assess the stability of the rescued mutant protein. camp-stimulated iodide efflux was still detectable 6 h after removing km60, but responsiveness was lost after 24 h in both cell types. electrophysiological studies were performed to examine the channel activity of ∆f508-cftr rescued by km60 treatment. incubation with 10 µm km60 for 48 h increased camp-responsive ∆f508-cftr current density in hek293 whole-cell patches ~6-fold. the conductance was time-and voltage-independent, anion selective, and strongly inhibited by glibenclamide. incubating polarized cfbe monolayers with 20 µm km60 increased the forskolin + genistein-stimulated short-circuit current after 48 h, and this response was inhibited by cftrinh172 (10 µm). permeabilization of the basolateral membrane with nystatin confirmed the apical location of the forskolin + genistein-stimulation. ussing chamber experiments also revealed trafficking correction in ileum that had been excised from cftrtm1eu mice homozygous for ∆f508. when the ileum was pre-incubated with 20 µm km60 for 4-6 h, the forskolin + genistein-stimulation of short-circuit current was significantly increased when compared to untreated intestine. these results confirm partial correction of ∆f508-cftr by km60 in unpolarized cells and in human polarized epithelial cell monolayers and ex-vivo intestine isolated from ∆f508-cftr mice. this work was supported by the breathe program funded by the ccff and cihr, cihr, cfft, and génome québec. primary sequence and overall structure of human cftr overlaps significantly with murine, rabbit and other homologues, a feature that has limited generation of conformation specific reagents to address cftr folding. because regions of ∆f508 cftr exposed extracellularly may exhibit aberrant topology (jbc 279:39620-7), probes that identify cftr from the extracellular surface could be useful to 1) verify plasma membrane localization of ∆f508 cftr, 2) discriminate wild-type from mutant protein, and 3) provide a means of determining whether small molecule "correctors" of the ∆f508 processing lead to a native (wildtype) cftr configuration. our laboratories are investigating phage-display techniques suitable for detecting properly folded extracellular domains of cftr. two large phage libraries encoding 7 or 12 amino acid sequences (approximately 10 9 phage per library) were panned against extracellular regions of cftr in hela cells. following incubation in blocking solution containing pbs and 1% bsa at 4°c, unbound fraction was incubated with recombinant helas expressing wild-type or temperature corrected ∆f508 cftr (protein expression verified biochemically and functionally). bound bacteriophage was dislodged after multiple washings by exposure to an acidic elution buffer and amplified to increase copy number (five rounds of antigen selection for each library, eur j biochem 268:2004-12). supernatants from microtiter wells expressing phage were analyzed for binding to parental (no cftr), wt or ∆f508 cftr expressing hela cells. using this method we obtained enrichment of phage at the surface of cells expressing wt or ∆f508 cftr. an example using a 7-amino acid sequence (10 10 phage) with specificity for ∆f508 cftr (representative of four repeat experiments) is shown in a binding assay with 10 6 hela cells/condition. reagents such as these can be further optimized by constructing second generation libraries using core recognition sequences (derived from the initial screen) and surrounding the core epitopes with random amino acid diversity. these experiments are intended to identify peptides with high binding affinity (nm binding constants) that recognize native cftr epitopes exposed extracellularly. because there is evidence that low temperature corrected ∆f508 cftr in the plasma membrane is misfolded, these probes may ultimately distinguish wild-type cftr from the surface targeted ∆f508 mutant and be useful as drug discovery reagents. supported by cff and nih. bridges, r.; nagubadi, s.; thakerar, a.; jia, y.; bradbury, n.a. physiology and biophysics, rosalind franklin university of medicine and science, north chicago, il, usa the goal of this project was to compare several reported correctors of ∆f508-cftr biosynthesis for their efficacy on chloride channel activity under a standard set of experimental conditions. fisher rat thyroid (frt) cells were transfected with ∆f508-cftr and a stable cell line selected with g418. cells were grown on transwell filters and studied under short circuit current (i sc ) conditions in ussing chambers. i sc measurements were performed at 27°c. the basolateral membrane was permeabilized with α toxin and chloride currents were measured with a serosal to mucosal chloride gradient. atp (1 mm) and gtp (100 µm) were added to the serosal bath. cftr channel activity was stimulated with camp (100 µm), ibmx (100 µm) and genistein (30 µm). cells were treated with correctors or vehicle (dmso) at 24 and 48 hours before the i sc measurements. a subgroup of cells were temperature corrected at 27°c for 48 hours for comparison. because only modest currents were observed even in the temperature corrected cells (3 -6 µa/cm 2 ) cells were treated for 48 hours with 5 mm sodium butyrate and 30 nm dexamethasone to enhance the observed currents. under these conditions temperature corrected cells displayed stimulated chloride currents of ~100 µa/cm 2 that were 6-8-fold greater than cells maintained at 37°c. correctors c4 (n5' ]bithiazolyl-2'-yl]-benzamide) and c1 (6-(1h-benzomidazol-2-ylsulfanylmethyl)-2-(6-methoxy-4-methyl-quinazolin-2-ylamino)pyrimidin-4-ol) caused a modest rescue of ∆f508-cftr chloride currents compared to cells maintained at 37°c (c4, 1.7-fold; c1, 1.3-fold) but far below the effect of temperature correction. rescue by c4 was concentration dependent with a significant observable effect at 300 nm and a maximum effect at 1 µm. rescue by c1 was observed at only 3 µm, lower concentrations had no effect and higher concentrations caused an inhibition of the stimulated chloride currents. compounds c2 -piperazin-1-yl]-ethyl}-4-piperidin-1-yl-quinazoline) (1 to 10 µm), c3 (4-cyclohexyloxy-2-{1[4-(4methoxy-benzensulfonyl)-piperazin-1yl]-ethyl}-quinazoline) (1 to 10 µm) and 4 phenylbutyrate (3 mm) did not significantly increase the stimulated chloride currents. all four compounds c1 -c4 caused significant decreases in the transepithelial resistances at the higher concentrations (3 -10 µm) suggesting some degree of cytotoxicity. our results indicate only c1 and c4 demonstrate efficacy in frt cells and that these effects are modest compared to temperature correction. supported by the cystic fibrosis foundation. cftr contains two membrane-spanning domains (msd), two nucleotide-binding domains (nbd), and a regulatory domain that require proper assembly for chloride channel activity. the most prevalent disease causing mutation, cftr∆f508, arises from deletion of phenylalanine 508 in the nbd1 domain. the cftr∆f508 mutant is translated and inserted into the endoplasmic reticulum (er) membrane, but is unable to attain its native state and accumulates in a kinetically trapped conformation that is degraded by the ubiquitin proteasome system (1) . the nature of the misfolded cftr∆f508 intermediate that is selected for premature degradation is not clear and is a topic of great interest (2) . recent work in the cyr lab indicate that defects in cftr and cftr∆f508 folding are sensed by two different e3 ubiquitin ligase complexes that are located in the er membrane and cytosol (3) . the membrane-associated derlin-1/rma1 e3 complex appears to recognize early folding defects of cftr that may involve assembly of msd2 into a complex with the amino-terminal regions of cftr. in contrast, the cytosolic hsc70/chip e3 complex appears to sense folding defects that occur after synthesis of the nbd2 domain. misfolded cftrs recognized at either checkpoint are retained and degraded via the proteasomal pathway. the presence of dual protein quality control (qc) checkpoints suggests a mechanism by which the folding status of cftrs membrane and cytosolic domains are sequentially monitored prior to its escape from the er. currently little is known about how these erqc complexes sense misfolded substrates. small-molecule chemical correctors have recently been identified which rescue the folding defect of cftr∆f508 in model cell culture systems. in the presence of these chemical correctors a subset of cftr∆f508 is able to localize to the cell surface and retain channel function. however, how the chemical correctors rescue cftr∆f508 folding is not clear. we have examined the extent to which the molecules correct folding defects that are recognized by the derlin-1/rma1 or hsc70/chip qc checkpoints. in addition, because it is unknown at what point of cftr biogenesis that the chemical correctors act to facilitate folding, we have determined how the chemical correctors affect the folding of different biogenic intermediates. our results indicate that the correctors act in a post-translational manner to influence assembly of cftr sub-domains. interactions with members of the qc pathway have also been used to monitor the folding and assembly process of cftr in the presence of chemical correctors. ultimately the results from these studies will enhance our knowledge of how chemical correctors permit passage of the cftr mutants past different erqc checkpoints. supported by -cystic fibrosis foundation combination therapy has proven successful in treating a wide variety of human diseases, including cancer, infectious disease, and diabetes. historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effects. we have developed a combination high-throughput screening (chts™) technology to systematically and efficiently find synergistic combinations that target multiple pathways underlying disease biology, and that may be developed into new therapeutics. we are applying chts technology to discover and develop combination therapeutics for the treatment of cystic fibrosis. we have optimized two high throughput screening assays for identifying correctors and potentiators of the cftr (cystic fibrosis transmembrane protein regulator) ∆f508 mutation. one assay uses the flipr membrane potential dye (fmp) and the other uses genetically modified yellow fluorescent protein yfp h148q/i152l to measure changes in halide flux through ∆f508 cftr. both assays were optimized and validated on the flipr tetra using the panel of cf correctors, potentiators and inhibitors provided by the cf foundation. using our automated chts, we systematically evaluate pairwise combinations of a library of ~2,000 approved drugs and other bioactive molecules to identify novel disease-relevant biological interactions. candidate single agents and combinations are tested in secondary assays and prioritized for testing in animal efficacy models. combination therapies can act on multiple pathways in a coordinated way. chts may prove to be a useful tool for identifying combination therapeutic candidates with novel multi-target mechanisms and a way to enhance co-therapy regimens for the treatment of cystic fibrosis. supported the highly variable clinical phenotypes of cf airway disease suggest that a number of genetic factors, other than cftr, play a role in its pathophysiology. some of these modifier genes are expected to play a role in the endoplasmic reticulum quality control (erqc), since the major defect caused by f508del is misfolding, retention and degradation of the mutant protein by this cellular surveillance system. the nematode caenorhabditis elegans is an excellent multicellular genetic model, which has been successfully used in studies of human diseases. its ~20,000-genes genome has been fully sequenced, it has short life-span (2-3 weeks) and life cycle (~ 3.5 days) and it is easily cultured and amenable for gene disruption, both by knockout or rnai. our goal is to generate a c. elegans model for the cftr folding defect, so as to take advantage of this model for the identification of genes involved in cftr folding and/or degradation. to this end, and because no cftr orthologue has been described in c. elegans, we constructed two previously described human p-gp-/cftr chimeras (p-gp/wt-cftr and p-gp/f508del-cftr) [1, 2] to be used as an in vivo folding substrate in this organism. the two chimeric cdnas and intact human p-gp cdna were cloned into the c. elegans ubiquitous expression vector ps235 and injected into mutant nematode strains for multidrug resistance genes [3] . the effect on the nematodes phenotype was evaluated by an assay of heavy metal sensitivity (2.0 mm arsenite), as described [3] . our quantitative results show that the p-gp/wt-cftr chimera increases the resistance to arsenite when injected into the pgp-1/pgp-3 c. elegans double mutant, whereas p-gp/f508del-cftr causes no effect. these preliminary results indicate it is possible to generate two distinct nematode phenotypes caused by each of the transgenic chimeras (p-gp/wt-cftr and pgp/f508del-cftr). they also suggest that the same folding defect impairing f508del-cftr function in cf may be responsible for the loss of heavy metal resistance function by the p-gp/f508del-cftr chimera. these chimeras are currently under test in pgp-1/pgp-3 double and in pgp-1/pgp-3/mrp-1 triple c. elegans mutants. analysis of these strains by rt-pcr showed that the mrnas from the two p-gp/cftr chimeras have low expression levels. this led us to produce more robust constructs, including: 1) to test chimera expression under the c. elegans endogenous pgp-1 or intestinal-specific (potent) promoters; and 2) to develop p-gp chimeras with a full cftr-nbd1. these c. elegans models will be used in genome-wide rnai screens to identify genes involved in the erqc of the p-gp/cftr chimeras. work channel gating of cftr is regulated by atp binding and hydrolysis at the nbds and by pka phosphorylation at multiple sites, primarily within the intrinsically disordered regulatory (r) region. phosphorylation at multiple sites is required for full activation of channel function, but no one specific phosphorylation site is required. the phospho-regulation of r region interactions with nbd1 likely contributes to the regulation of cftr channel function by modulating r region interactions with nbd1 at the nbd1/nbd2 dimerization interface, consistent with nbd1 crystal structures that include the regulatory extension (re) comprising the first 20 residues of the r region (1). this regulation likely occurs via a dynamic complex where multiple segments of nonphosphorylated r region with αhelical propensity transiently engage nbd1 and are released. pka phosphorylation disrupts r region α-helical propensity and decreases binding of each interacting segment (2) . regulation of the ∆f508 cftr by pka is altered, with the rate of channel activation by pka 7-fold slower than for wild-type (3). we hypothesize that the dynamic interactions between the r region and nbd1 are affected by the ∆f508 mutation. to characterize the differences in r region binding between wild-type and ∆f508 nbd1 at residue-level resolution, we use nuclear magnetic resonance (nmr) techniques. experiments are performed using either r region or nbd1 samples labeled with nmr-active nuclei and monitoring their structural changes upon the addition of unlabeled binding partner, allowing us to confirm r region binding to nbd1 at the nbd1/nbd2 dimerization interface and to identify other potential interaction surfaces on both the r region and nbd1. together, these experiments will allow us to characterize the dynamic associations between the r region and nbd1 in order to better understand the molecular basis for the pathogenesis associated with ∆f508 cftr. funded by the canadian institutes for health research, the canadian cystic fibrosis foundation, and the us cystic fibrosis foundation 1. lewis, h.a., et al, embo j. 23, 1-12 (2004) . 2. baker, j.m.r., hudson, r.p., kanelis, v., choy, w-y, thibodeau, p.h., thomas, p.j., and forman-kay, j.d. submitted. the ∆f508-cftr mutation, the most common gene mutation in cystic fibrosis (cf), results in diminished plasma membrane expression of cftr, leading to loss of functional cftr and altered mucociliary clearance. this impairment promotes chronic infection of cf patients by the opportunistic pathogen, pseudomonas aeruginosa. we previously reported that a secreted protein from p. aeruginosa (cftr inhibitory factor (cif)) reduces the wt-cftr and ∆f508-cftr-mediated chloride secretion and the plasma membrane expression of cftr by decreasing endocytic recycling of cftr. the aim of the current study was to investigate the mechanism by which cif is secreted by p. aeruginosa and delivered to human airway epithelial cells (cfbe41o-). in this study, we show that cif is packaged in outer membrane vesicles (omv) for secretion from p. aeruginosa. interestingly, cif secretion via omv was increased when the bacteria were exposed to airway epithelial cells. changes in cif protein level or bacterial growth did not account for the increase in cif secretion. purification and application of p. aeruginosa omv to airway epithelial cells caused a decrease in plasma membrane expression of cftr. the decreased plasma membrane cftr expression was followed by increased ubiquitination and lysosomal degradation of cftr. cif delivery to the airway epithelial cells via omv showed a more robust decrease in plasma membrane cftr expression, in comparison with cif delivery as purified protein. to investigate the mechanisms whereby cif enters epithelial cells, optiprep gradient fractionation experiments were conducted in polarized airway epithelial cells. in airway epithelial cells treated with p. aeruginosa omv, cif was associated with airway cell lipid rafts. pharmacological inhibition of lipid raft formation with filipin iii blocked the decrease in cftr plasma membrane expression upon treatment with omv. these studies demonstrate that cif is released by p. aeruginosa via omv and enters the airway epithelial cells through lipid rafts. upon entry into the epithelial cell, cif increases the ubiquitination and degradation of cftr and redirects cftr trafficking from the recycling pathway to the lysosomal-degradative pathway. these data suggest that chronic infection of p. aeruginosa in the cf lung may reduce the efficacy of therapeutics developed to increase plasma membrane expression of the mutant ∆f508-cftr (supported by the nih 5 ro1hl074175-06 and 5 t32-dk007301-29). 1 1. physiology, dartmouth medical school, hanover, nh, usa; 2. microbiology and immunology, dartmouth medical school, hanover, nh, usa we have identified and cloned a secreted protein from pseudomonas aeruginosa (p. aeruginosa pa14 and clinical isolates), designated cftr inhibitory factor (cif). cif reduces the apical membrane expression of cftr and inhibits the cftr-mediated chloride ion secretion by human airway epithelial cells expressing wt-cftr and df508-cftr. cif does not have general effects on protein trafficking, as the localization and expression of gp114, na + -k + -atpase, and the transferrin receptor were not affected. cftr is a member of the atp-binding cassette (abc) transporter superfamily. abc proteins are atp-dependent transporters involved in exporting a wide variety of cytotoxic agents across the plasma membrane. p-glycoprotein (pgp; abcb1, mdr1 gene product), also an abc transporter, is one of the major drug-efflux pumps expressed in normal tissues, as well as in many human cancers. over-expression of pgp results in reduced intracellular drug concentration and reduced cytotoxicity, conferring multi-drug resistance (mdr) to cancer cells. the aim of the current study was to examine whether cif also affects pgp expression in the plasma membrane, which could be exploited as a potential therapeutic strategy for restoring the sensitivity to pgp-transported drugs in cancer chemotherapy. cif significantly reduced the apical expression of pgp in mdck cells stably transfected with gfp-tagged pgp (mdck-gfp-mdr1 cells) and in caco-2 and calu-3 cells, which express endogenous pgp in the apical plasma membrane. the drug sensitivity of mdck-gfp-mdr1 cells to doxorubicin, a pgp substrate, was evaluated in the absence and presence of cif by measuring the ec 50 . cif reduced the cytotoxicity of doxorubicin by 100-fold. neither cif nor vehicle alone had a cytotoxic effect. the drug sensitivity of the parental cell line, mdck-c7, that expresses 4-fold less pgp than the transfected mdck cells, to doxorubicin was also increased by 20-fold. by contrast, cif had no effect on the ec 50 values of cisplatin (a substrate of mrp2) and etoposide (a substrate of mrp1), suggesting that the alteration of the sensitivity to doxorubicin was due to decreased apical expression of pgp, but not other abc transporters. these results suggest that although cif may be an obstacle to therapeutic attempts to restore the apical expression of cftr in the cf lung, it could be useful for the development of a novel class of inhibitors of pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs (supported by the nih (5 ro1 hl074175)). hamai, h. 1 ; keyserman, f. 1 ; worgall, t.s. 1, 2 1. pathology, columbia university, new york, ny, usa; 2. pediatrics, columbia university, new york, ny, usa cystic fibrosis (cf) is characterized by an inflammatory state and susceptibility to chronic lung infections. a common clinical finding is dyslipidemia characterized by altered plasma free fatty acid patterns, low plasma hdl levels and increased phospholipase a2 (pla2) activity. it is not clear how cftr mutations relate to lipid abnormalities. it was shown that defective cftr is associated with decreased uptake of sphingolipids (sl). sl homeostasis is tightly regulated and metabolites are relevant to lipid metabolism and cf. sl synthesis (sls) correlates with activity of srebp, a key transcription factor of lipid metabolism; ceramide-1-phosphate, a sl metabolite, is a regulator of pla2 that is highly activated in cf. we investigated the hypothesis that sls is increased in cf. experiments were carried out in airway epithelial cell lines that express defective cftr (c38 / ib3), no cftr (16hbe sense / antisense) and overexpress cftr (a549 cells infected with adcftr or adcontrol). sls was assessed using radioactive tracers 3h-serine (de-novo sls) and 3h-sphinganine (recycling pathways). ceramide and sphingomyelin mass were determined enzymatically. neutral and alkaline sphingomyelinase activity was assessed using 3h-sphingomyelin as substrate. srebp mediated gene transcription was assessed using sre-promoter assays. cholesterol synthesis was evaluated by incorporation of 3h-acetic and 3h-mevalonic acid. cholesterol mass was determined by gas-chromatography. abca-1 expression was determined by western blot analysis. pla2 activity was measured using fluorescent phospholipid substrates. sls was post-transcriptionally increased in cells expressing defective or no cftr through the de-novo pathway (133 % ± 15% for ib3; 78%± 12% for 16hbe-antisense; p<0.05 ) and the recycling pathways (38% ± 11% or ib3; 27% ± 12 % for 16hbe-antisense; p<0.05). ceramide mass was increased by 50 % (± 18 %) in 16hbe antisense, 36 % ± 9% in ib3 cells. sphingomyelin mass was increased 2-fold in ib3, up to 3-fold in 16hbe antisense cells. neutral and alkaline sphingomyelinase activity was increased up to 3-fold in both cell lines. sre-mediated gene expression was increased up to 2-fold in ib3 and by 50 % in 16hbe-antisense cells. overexpression of cftr in a549 decreased sls and srebp activity up to 40%. free cholesterol synthesis was increased by 60 % in ib3 and by 30 % in 16hbe antisense. expression of the cholesterol efflux receptor abca-1 was decreased in ib3 and 16hbe antisense compared to controls. sl mass, sre-mediated gene transcription and pla2 activity were decreased following incubation with inhibitors of sls in ib3 and 16hbe antisense cells. conclusion: defective cftr or lack of cftr expression correlates with 1) increased sls synthesis and sl mass; 2) increased sremediated gene transcription and free cholesterol synthesis; 3) decreased expression of the abca-1 cholesterol efflux receptor. inhibition of sls decreases sre-mediated gene transcription and pla2 activity. dysfunctional sls is a newly recognized pathway associated with defective cftr and a possible therapeutic target in cf. the ability to make accurate, reproducible measures of mucociliary and cough clearance (mcc/cc) in cf patients is critical to assessment of new therapies designed to improve mcc/cc function, and thus decrease pulmonary infections and decline in lung function. for example, using mcc/cc methods developed in our laboratory, we showed that 2-week treatment with aerosolized hypertonic (7%) saline (hs) led to a sustained increase in mcc and improved lung function in cf patients (donaldson et al, n eng j med 2006; 354:241) . the mcc/cc technique involves patient inhalation of radiolabeled particles followed by gamma camera scanning to determine the rate of particle movement from the lungs. while a few cf centers have shown the capability to measure mcc/cc, the techniques across these centers are not standardized, making comparison of results difficult. it is vital to develop standard, simplified techniques for these measures to make larger cf patient populations available for such studies. to standardize a radiolabeled particle inhalation technique that can be easily used by other cf centers, we have compared three protocols in healthy and cf subjects. protocol #1 was identical to that used in recent studies, and incorporates a devilbiss 646 jet nebulizer to generate 5 µm mmad (gsd = 2.0), aqueous droplets containing suspended tc99m-sulfur colloid particles. tidal volume and inhaled/exhaled flow rates were guided by a volume signal displayed on an oscilloscope. protocol #2 was further standardized and simplified to allow use at other centers. this protocol utilized the same nebulizer, but triggered by a commercial dosimeter (spira electro 2, finland) during inhalation as the subject breathed in time to a metronome (30 breaths/min) at flow rate of 0.5l/sec. subjects controlled inspiratory flow rate by feedback from a digital flow readout on the dosimeter. repeat measurements of mcc/cc were made in 8 healthy subjects and 5 mild cf patients to compare mcc/cc variables following radioaerosol inhalation using protocols #1 and #2. there were no differences in particle deposition (i.e. central to peripheral ratio, or "c/p"), or clearance of particles through 90 minutes and 24 hours in either subject group. these data suggest that the new methodology (protocol #2) will be useful and easily employed by other cf centers to test effectiveness of new therapies for cf. the third protocol (#3) was then tested in five of the healthy subjects. this protocol used a nebulizer that generated very large droplets (9.5 um mmad, gsd =2.0) and subjects inhaled at very slow flow rates (~80ml/sec). it was designed to provide greater bronchial airway deposition and thus an "improved signal" for mcc/cc evaluation. indeed, this methodology produced a larger average c/p and significantly greater particle clearance from the lung compared to the other standardized methodology (protocol #2) (c/p = 1.77 vs. 1.60; 90min clearance = 25% vs. 18%; 24hr clearance = 54% vs. 35%, respectively). interestingly, the greater fraction of clearance between 90min and 24hr may also reflect heightened ability to assess clearance from smaller airways, which could be particularly important in cf. protocol #3 will be tested further in cf patients. supported by cf foundation. previous clinical trials in cystic fibrosis (cf) indicate that anti-inflammatory therapy probably will not result in improvement in lung function, but will slow the rate of decline. this imposes constraints on study design for new anti-inflammatory agents, requiring that they use many patients over long periods. it is highly desirable to design a strategy for evaluation of antiinflammatory agents that will allow for the selection of only the most promising agents for phase iii trials. sputum induction (si) samples lower respiratory tract secretions and permits measurement of inflammatory markers. the purpose of this study was to assess the measurement of inflammatory markers in induced sputum as one such strategy by using ibuprofen as the test agent because it has demonstrated clinical benefit in cf. if changes in inflammatory markers are detectable in ibuprofen treated patients, then si might serve as a quick, noninvasive method by which to select the most promising anti-inflammatory agents for further study. methods: in this twoarm (ibuprofen and no treatment), open-label, parallel group study, 114 cf patients >/= 10 years of age with mild to moderate lung disease were screened at 16 sites. si was performed on days 0, 14, 42 and 56. patients in the ibuprofen group received drug on days 14-42. 82 subjects met eligibility criteria. 40 were randomized to ibuprofen and 40 to no treatment. 63 subjects (32 ibuprofen and 31 no treatment) completed the study through day 42 and comprise the intent-to-treat efficacy population. a maximum of 60 subjects (32 ibuprofen and 28 no treatment) and a minimum of 47 subjects (23 ibuprofen and 24 no treatment) across all markers had acceptable inflammatory marker values at both days 14 and 42. within group and between group comparisons were made. results: with respect to the within group comparisons (before and after ibuprofen), most inflammatory markers slightly decreased in the ibuprofen group and increased in the no treatment group. for ibuprofen patients, mean changes were most apparent for il-6 (-0.13 log pg/ml, p=0.04) and % neutrophils (pmns) (-4%, p=0.073). with respect to the between group comparisons (ibuprofen vs. no treatment), differences were strongest for il-6 (0.03 log pg/ml, p=0.0237), percent pmns (7%, p=0.085), and absolute pmn count (0.14 cells/ml, p=0.166). fourteen days after discontinuing ibuprofen, inflammatory mediators tended to increase. there was no difference in aes between the two groups, and si was generally safe. conclusions: 1. overall, si is well-tolerated in patients >/= 10 years, 2. a 4-week treatment period may not be long enough to study anti-inflammatory drugs, 3. with respect to changes in inflammatory markers associated with anti-inflammatory therapy, the effect size is small and the variance of the effect is small in clinically stable patients. 4. measuring inflammatory markers in induced sputum has potential as a screening tool for studying anti-inflammatory drugs in cf, 5. further studies of si in cf are warranted. supported by cystic fibrosis foundation therapeutics, inc. in preparation for our gene therapy clinical trial programme we are currently assessing a number of sputum biomarkers including viscosity and elasticity, total solids and dna content and 24 hr sputum weight. we tracked and correlated these biomarkers in cf patients (12 years and over) during a course of iv antibiotics (ab) by collecting samples on several occasions [visit (v)1: at the start of ab treatment, v2: at the end of ab treatment (generally after 2 weeks) and interim periods]. to ensure adequate reproducibility of the results viscosity/elasticity measurements were carried out in triplicate using a csl 100 rheometer which required a comparatively large volume (5 mls) of spontaneously expectorated sputum. because of this requirement paired samples could only be obtained from approximately 50% of the patients. there was no change in viscosity/elasticity (n=13), solid or dna (n=15) content when comparing samples at the beginning and end of iv ab. in contrast, 24 hr sputum weight was significantly (p<0.05) lower at the end of the ab treatment (visit 1: 57.74±14.58 g, visit 2: 49.41±24.01 g, n=16). there was a strong and significant correlation between solid content and viscosity/elasticity (r=0.8, p<0.0001) and a modest correlation between 24 hr sputum weight and % predicted fev1 (r=-0.42, p<0.05) and 24 hr sputum weight and patient scored symptom severity (r=0.48, p<0.001). surprisingly, sputum dna content did not correlate with viscosity/elasticity, despite being generally thought of as a contributor to viscosity, which may in part be related to the assay not being able to discriminate between free and cell-enclosed genomic dna.in summary, after a course of iv antibiotics which lead to significant subjective and objective (fev1) improvement the overall quantity of expectorated sputum was significantly reduced but, based on analysis available to date, none of the other parameters (viscosity, elasticity, solid and dna content) changed significantly. considering the difficulties we encountered in collecting sufficient sputum during this period of an exacerbation, sputum viscosity/elasticity measurements may not be feasible parameters to measure in a gene therapy trial to which stable patients re likely to be recruited. this study was funded by the cf trust. chronic neutrophilic inflammation is a feature of cystic fibrotic (cf)related inflammation and serves as a significant contributor to morbidity and mortality associated with the condition. recently, our group has described a novel collagen-derived neutrophil chemoattractant, proline-glycine-proline (pgp), in a murine model of lipopolysaccharide-induced inflammation. the purpose of this study was to explore both the presence of this peptide in sputum from cf patients and the determination of a proteolytic system involved in pgp generation. we show that lower airway secretions from cf subjects have 30-fold increase in pgp levels compared to normal controls and that cf sputum (p<0.05) is able to generate pgp from intact type i and ii collagen 100-fold above that seen from normal control sputum ex vivo (p<0.01). we further demonstrate that production of pgp is dependent on two specific matrix metalloproteases (mmps), mmp-8 and mmp-9. finally, we show that the generation of pgp can be significantly inhibited (80-90% inhibition) by the use of either mmp-8 or mmp-9 specific inhibitors (p<0.01 versus no inhibitor); a nonspecific mmp inhibitor (doxycycline) also demonstrates significant attenuation of pgp production. the determination of the requirements of these proteases in pgp generation allows for the identification of logical targets for disease-modifying therapeutics in cf. 1 1. cfrc, uab, birmingham, al, usa; 2. cfrc, university of north carolina, chapel hill, nc, usa cystic fibrosis (cf) lung disease is characterized by chronic neutrophilic inflammation. the discrete collagen breakdown product prolineglycine-proline (pgp) is a potent neutrophil chemoattractant thought to be generated by a specific proteolytic cascade present during cf pulmonary exacerbation (nat med 12(3):317, 2006) . high-mobility group box 1 (hmgb1) is a potent inflammatory mediator found in sepsis, rheumatoid arthritis, and other inflammatory diseases characterized by neutrophilic inflammation. the role of pgp and hmgb1 as pmn attractants in cf is unknown. in this study we utilized specimens derived both from humans and scnn1b-transgenic mice (βenac mice) in which overexpression of the βenac subunit in the airways leads to sodium hyperabsorption and airway surface liquid depletion, mimicking the cf muco-obstructive phenotype. pgp was quantified using tandem ms. neutrophil chemotaxis was measured in vitro following preincuabtion of sputum or balf with anti-hmgb1 neutralizing antibody. murine balf was assayed for hmgb1 by western blot and elisa. human sputum was similarly evaluated after normalizing for total protein concentration. in vivo hmgb1 activity was measured after intratracheal instillation in wt mice. βenac mice had 60% higher cell counts and a greater percentage of pmns (37.3% vs. 0.6%) in bal samples than wild-type (wt) littermates (p<0.005, n=15/genotype). pgp was elevated in balf of scnn1b animals (48.0 vs. below limits of detection in wt controls, p<0.001, n=11), increased in serum from cf subjects (834 vs. 340 pg/ml normal controls, p<0.005, n=5), and detected at high levels in cf sputum (54,700 pg/ml). balf screened for hmgb1 by western blot revealed elevated levels in βenac mice (p<0.05 by densitometry, n=10/genotype); elisa confirmed 52% greater hmgb1 concentration (61.8 vs. 40.6 ng/ml, p<0.02). hmgb1 was also detected by western blot in human cf sputum (19 of 23) and at higher levels than secretions from normal subjects (p<0.05 by densitometry; n=23 cf, 4 control). purified hmgb1 induced dose-dependent pmn chemotaxis in vitro (peak 50-250 ng/ml, p<0.05, n=5) and cf sputum caused potent chemotaxis (7-fold over control, p<0.005, n=12) that was inhibited by preincubation with hmgb1 antibody (p<0.005). balf from βenac mice (but not wt controls) was also chemotactic (2-fold greater than media control, p<0.01, n=5) and inhibited by anti-hmgb1 antibody (p<0.05). exogenous hmgb1 (25 ng) administered intratracheally to lps resistant mice (c3hej, toll-4 receptor mutant) and balb/c mice caused pmn influx in balf at 24 hours (c3hej: 9.0 x 10 5 pmns after hmgb1 vs. 2.0 x 10 5 after vehicle, p<0.02; balb/c: 3.5 x 10 5 vs. 0.8 x 10 5 , p<0.01), and generation of pgp (189.7 vs. 80.7 pg/ml in c3hej, p<0.05, n=3) . in summary, pgp and hmgb1 are elevated in balf of βenac mice that present with cf-like lung pathology; in cf airway secretions, hmgb1 is present in vivo at concentrations that induce neutrophil chemotaxis in vitro and pgp production in vivo, and is inhibited by blocking antibody. the role of pgp and hmgb1 in cf deserve further evaluation as they may be potential therapeutic targets of dysregulated inflammation. esther, c.r. 1 ; jasin, h.m. 2 ; collins, l.p. 3 ; boysen, g. 3 ; boucher, r.c. 2 1. pediatric pulmonology, university of north carolina at chapel hill, chapel hill, nc, usa; 2. cf research center, univeristy of north carolina at chapel hill, chapel hill, nc, usa; 3. center for environmental health and susecptibility, univeristy of north carolina at chapel hill, chapel hill, nc, usa biomarkers of airway inflammation are needed in cystic fibrosis (cf) to aid clinical management and assess the efficacy of novel therapies. we describe a simple and non-invasive method to measure airway purines, signaling molecules previously shown to be biomarkers of neutrophilic airway inflammation. airway secretions were obtained using exhaled breath condensate (ebc) collection, a technique that was easily performed in the outpatient setting on children as young as three years. the purines adenosine and amp in ebc were measured using ultra-sensitive liquid chromatography/tandem mass spectrometry (lc/ms/ms). in addition, we used lc/ms/ms to simultaneously measure urea as a dilution marker to control for the known dilutional variability of airway secretions within ebc. detection of adenonosine, amp, and urea was optimized using positive mode ms with selected reaction monitoring, and stable isotope dilution was utilized to improve quantification. detection was linear with concentration over four orders of magnitude, with coefficients of variation <15%. the method was sensitive, with limits of detection for adenosine and amp (~0.1 nm) and urea (~0.5 µm), below expected ebc concentrations. applying the methods to ebc demonstrated that detection of purines and urea was reliable, with intraclass correlation coefficients greater than 0.9 between duplicate measures (n=7). we also measured purines and urea in ebc samples collected prospectively from children with cf (n=17) and healthy controls (n=6) during regular clinic visits. all samples were lyophilized and reconstituted to increase concentration 10-fold. adenosine, amp, and urea were detected in all samples. neither adenosine nor amp levels differed between groups, but urea was significantly lower in cf (6.6 ± 2.3 µm) versus healthy control (15.1 ± 4.9 µm, p=0.02), suggesting that airway secretions were more dilute in cf samples. when dilutional variability was controlled using purine to urea ratios, the amp to urea ratio was found to be elevated in subjects with cf (0.50 ± 0.14) compared to healthy controls (0.09 ± 0.03, p=0.015), consistent with previous findings. the amp to adenosine ratio was also elevated in cf (4.3 ± 1.9) compared to control (0.87 ± 0.30), although the difference did not reach statistical significance in this small sample set (p=0.19). experiments are ongoing to increase sample size and assess ebc purine levels before and after treatment of a cf exacerbation. these results demonstrate that lc/ms/ms analysis of ebc provides a non-invasive method to measure purine biomarkers of cf airway inflammation. given the flexibility of ms, this methodology could also prove applicable for study of other small molecule biomarkers. importantly, we also show that dilution of airway secretions in ebc may be altered in cf, and lc/ms/ms can be used to control for this variability through measurement of urea as a dilution marker. introduction: mucociliary clearance and antimicrobial peptide activity may be affected by airway surface liquid (asl) ph, which is regulated by epithelial ion transport. asl ph is abnormal in disease states in which il-17a is elevated, including cystic fibrosis (cf), and the il-17a receptor is expressed at the basolateral surface of bronchial epithelium. therefore, we investigated the effects of il-17a on vectorial ion transport in well-differentiated hbe cells. methods: hbe cells were grown with an apical air interface and incubated with il-17a at 0 or 50 ng/ml in the basolateral medium for 48 hours prior to being studied with standard short-circuit current (isc) techniques. results: il-17a treated cells had a minimal increase in resting and amiloride-sensitive isc compared to control cells, and had a dosedependent, statistically significant increase in forskolin-stimulated isc. at a dose of 50 ng/ml, il-17a doubled forskolin-stimulated isc (10.5 ± 1.72 µa/cm2 for control vs. 20.3 µa/cm2 for il-17a). the increased isc was not bumetanide-sensitive, but was sensitive to acetazolamide and dnds, and was not present in hco 3 --free solutions, suggesting it was due to hco 3 secretion. to investigate whether this hco 3 secretion was cl --dependent, we studied hbe cells in cl --free solutions, and there was no difference between untreated and il-17a treated hbe cells. to test the hypothesis that il-17a promoted cl -/hco 3 exchange, we performed experiments in hbe cells mounted in cl --free solutions, and after addition of amiloride and forskolin, 30 mm nacl was added to the mucosal bath and 30 mm nagluconate was added to the serosal bath. under these conditions, one would predict a decrease in isc due to mucosal to serosal clmovement down its electrochemical gradient. in untreated hbe cells, we saw a decrease in isc followed by a recovery. in il-17a treated cells there was a sustained increase in isc, suggesting that movement of cldown its electrochemical gradient resulted in exchange of clfor an anion at the apical membrane. in the absence of hco 3 -, both untreated and il-17a treated cells responded to claddition to the mucosal bath with decreases in isc. to assess the cftr dependence of the il-17a-induced isc, we stimulated primary cf hbe cells treated with il-17a. in cf cells, there was a minimal increase in amiloride-sensitive isc (30.6 ± 3.35 µa/cm2 for control vs. 37 ± 8.24 µa/cm2 for il-17a ) and in forskolin-stimulated isc (1.2 ± 0.56 µa/cm2 for control vs. 3.4 ± 1.16 µa/cm2 for il-17a ). conclusion: the proinflammatory cytokine il-17a induces cl -/hco 3 exchange in hbe cells. notably, the observed cl -/hco 3 exchange appears to be cftr-independent. however, cftr activation is required for maximum cl -/hco 3 exchange as demonstrated by the smaller isc generated in cf cells. these data suggest that the cytokine milieu of the airway epithelium can alter ion transport and potentially asl physiology. furthermore, they suggest that cftr may interact with or regulate novel proteins in the presence of inflammation. supported by cff and nih periciliary fluid balance is maintained by the coordination of sodium and chloride (cl-) channels in the apical membranes of the respiratory epithelia. in the absence of the cystic fibrosis transmembrane conductance regulator (cftr), cl-secretion is diminished and sodium reabsorption becomes exaggerated. activation of non-cftr-dependent cl-channels can provide an alternate pathway for cl-secretion in the airways and gastrointestinal tract. the ph and voltage-dependent type-2 cl-channel (clc-2) is expressed in airway epithelial cell luminal membranes. we hypothesize that topically applied clc-2 agonists may restore cl-secretion in cystic fibrosis (cf) murine airways. using in vivo nasal potential difference measurements, we quantified clc-2-mediated cl-transport in both cf and wild-type mice during nasal perfusion with lubiprostone (a prostone compound and specific clc-2 agonist; sucampo, pharmaceuticals, inc., bethesda md) or vehicle control. wild-type (c57bl6, n = 9) and cf knock-out (cfko; n=10(jax stock# 002364)(jackson laboratories, bar harbor, me) mice were sedated and intubated to protect the lower airways from aspiration of perfusate. nasal and subcutaneous bridges were connected to fluid-filled silver chloride electrodes. baseline (in ringer's), amiloride-inhibited, low cl-(cl-free, gluconate-substituted ringer's with amiloride), and low cl-plus lubiprostone (with increasing concentrations of lubiprostone or vehicle) mice were perfused and potential differences were measured with a high impedance voltmeter (world precision instruments, sarasota fl) and continuously recorded. a clear dose-response relationship was detected in both wild-type and cf mice. at 20 µm lubiprostone, wild-type mice showed hyperpolarization of -7.8 ± 3.4 mv and cf mice responded with -4.7 ± 1.5 mv hyperpolarization. a paired t-test of low cl-perfusion and lubiprostone perfusions revealed significant (p<0.001) differences in both genotypes. five clc-2 cfko mice were similarly tested and showed no response to lubiprostone (+1.1 ± 1/5 mv). cftr inhibitor-172(50 µmol) (calbiochem, san diego ca) added to the low cl-perfusion in 6 additional wild-type mice eliminated the low cl-response but did not abolish the lubiprostone response, confirming that clc-2 is present and independent of cftr regulation. we conclude that direct application of a clc-2 agonist in the cf murine upper airways restores near normal levels of cl-secretion. cftr is an apical membrane chloride channel whose activity is required to maintain airway surface liquid (asl) volume and efficient mucociliary clearance. cftr is activated by camp dependent stimulation of protein kinase a. however, studies have suggested that the actin cytoskeleton may also be required for regulation of cftr, by acting as a structural element in second messenger compartmentalization (1) . to determine whether an intact cytoskeleton is required for asl volume homeostasis, adenosine (ado), atp, or isoproterenol (iso) (all at 200 µm) were added apically to human bronchial epithelial cultures (hbec's) before or after the cytoskeleton had been disrupted by cytochalasin d exposure (5µm for 1h). in control cultures ado, atp and iso all resulted in a significant increase in asl height (∆asl) within 10 min (∆asl; ado, 6.2±1.2µm; atp, 7.2±2.3µm; iso, 8.3±1.3µm). however, in cytochalasin d exposed cultures ado was without effect while atp and iso were still able to evoke an increase in height (∆asl; ado, -0.7±1.2µm; atp, 7.4±3.1µm; iso, 10.2±3.1µm). however, we showed that a2b receptor function was not compromised by cytoskeleton disruption as camp levels were seen to increase both after addition of adenosine and adenosine in the presence of cytochalasin d. thus, the effect of cytochalasin d on asl height appeared specific for adenosine-mediated cl-secretion. to further test the effect of cytochalasin d on the relationship between adenosine and cftr, we designed a fluorescent resonance energy transfer (fret) pair of cfp-cftr (labeled at the n-terminus) and yfp-a2b receptor (labeled at the c-terminus). addition of adenosine (100µm) increased fret by 2.3 times (% efficiency; control, 15.2±2.3%, ado, 35.3±3.5%) compared to the vehicle control (n=13). cytochalasin d exposure (5 µm for 1 h) completely inhibited the increase in fret that was observed after adenosine addition (p<0.01). in contrast, cytochalasin d had no effect on fret between cftr and the β2adrenoceptor. to determine whether cytochalasin d treatment (5 µm for 1 h) had any direct effect on cftr protein levels and localization we used a cell line stably expressed with an exotope-tagged cftr (hb8 cells) (2) . cftr protein levels were found to be significantly reduced as compared to nontreated controls by both western blot analysis and chemiluminescence detection (n=3). cells were also stained with an antibody against the external cftr exotope and imaged by confocal microscopy and a significant decrease in staining at the plasma membrane was observed after cytochalasin d treatment (n=3). we conclude that cytochalasin d exposure decreases cftr protein levels and specifically inhibits adenosine-mediated regulation of cftr activity in the airways, leading to disruption of asl homeostasis and inhibition of mucociliary clearance. numerous studies have reported evidence that cftr is expressed in the apical membrane of serous cells in the airway submucosal glands. thus, it has been reasoned that (1) cftr plays some role in normal anion and fluid secretion by airway glands and (2) loss of this channel's function in cystic fibrosis (cf) airways plays some role in the etiology of cf lung disease. a recent study by joo et al. (jbc 277:50710-50715, 2002) indicates that cf airway glands lose the ability to secrete fluid when treated with forskolin or vip but maintain the ability to secrete when treated with muscarinic agonists. this result suggests that vip and muscarinic agonists mediate secretion by cftr-dependent and cftr-independent pathways, respectively. the present study was undertaken to explore the dynamics of fluid secretion by individual submucosal glands to determine if further differences in their responses to these two agonists could be distinguished. pig tracheas were obtained from a local slaughterhouse. after removal of the cartilage, tissues were mounted in a warm (37°c) observation chamber that permitted exposure of the submucosa to krebs solution. the mucosal surfaces of the airways were dried with a stream of warm dry air and then layered with mineral oil. liquid secreted from the gland duct openings formed spherical aqueous droplets within the oil layer, and their volumes were estimated from spatial dimensions taken from sequential digital images. liquid secretion rates were estimated for individual glands present within a 28.9 mm 2 region of interest. rates were determined for two experimental protocols. for one group of tissues, secretion rates were determined for a control period, followed by a period of exposure to 1 µm vip, and then a period of exposure to both vip and 10 µm acetylcholine (ach). in the second group, the order of vip and ach treatment was reversed. these agonist concentrations were expected to produce near maximum rates of secretion. when the agonists were applied first, secretion rates were significantly (p<0.05) increased from the control period by both ach (2.01±0.44 nl/min to 4.52±0.38 nl/min) and vip (2.26±0.63 nl/min to 4.64±0.78 nl/min). the secretion rates for individual glands were highly variable with both agonists (ach: 0.11-19.20 nl/min; vip: 0.03-32.21 nl/min). when ach application followed vip treatment, the mean secretion rate was significantly increased further to 6.13±0.87 nl/min; however, when vip application followed ach treatment, the secretion rate was only marginally and insignificantly increased to 4.97±0.48 nl/min. we conclude that both ach and vip are efficacious secretagogues for porcine airway glands and that the rate of secretion from individual glands to either agonist is very heterogenous. because vip has no effect following stimulation by ach, we speculate that ach induces secretion by activating all transporters relevant to vip-induced secretion including cftr. however, ach must also activate channels and/or transporters not induced by vip since its effects are additive to vip. this most likely includes activation of a non-cftr anion channel. ( serous cells are thought to serve as the principal anion and fluidsecreting cells of airway submucosal glands. mucous cells primarily secrete gel-forming mucins. these respective physiological roles for these two cell types imply that the mucous cells, which secrete the thick mucus gel, should lie downstream of the serous cells so that their watery secretions can flush the mucins out of the ducts and onto the airway surface. early studies indicated that such arrangements existed within glands but reports of cell distributions have been descriptive rather than quantitative. because of resurgent interest in serous cell function, we revisited this issue to formally document the distribution of these cell types within the secretory tubules of the submucosal glands as well as at the surfaces of the glands, where serous cells are most accessible for study. a piece of porcine lung, containing a 5mm diameter bronchus, was treated with formalin fixative, and 250 consecutive 5µm sections were taken. the apices of virtually all gland epithelial cells, except the cells lining the collecting and ciliated ducts, stain positive for pas indicating that these cells are either serous or mucous cells. however, pas staining does not allow distinctions to be made between these two cell types. consequently, all slide sections were stained with hematoxylin and eosin. with these stains, mucous cells were identified as cells containing lucent granules in their apices whereas serous cells were considered to be cells where the apical cytoplasm stained a uniform dark reddish color. first, the distribution of serous and mucous cells in the secretory tubules were determined. the acini of individual tubules were located in the slide sections, and cells were identified and counted in consecutive sections that approached the collecting ducts until the tubules were no longer distinguishable as discrete tubes. secretory tubules from 6 individual glands were studied. in the acini, serous cells accounted for 95.0±3.2% of total cells whereas mucous cells accounted for 3.8±2.2% of total cells (remaining cells were not distinguishable as either cell type). there was a significant (p<.05) negative correlation of serous cell numbers with distance from the acini so that at 35µm from the acinus serous cells accounted for only 73.4±13.5% of total cells. there was a coincident significant positive correlation with mucous cell numbers so that at 35µm from the acini mucous cells accounted for 20.1±10.5% of total cells. next, we examined the cell distribution at the adventitial and mucosal surfaces and at the lateral margins of the glands. serous cells accounted for 82.7%, 85.4%, and 96.9% of the total cells at these respective locales. mucous cells represented 15.7%, 10.1%, and 2.4% of total cells in these same respective regions. we conclude that serous cells are by far the dominant cell type in secretory tubules of porcine submucosal glands. mucous cells are rare in acini but increase in frequency with distance from the acini. the outer surfaces of the glands are dominated by serous cells as well. at the lateral margin of the glands, serous cells outnumber mucous cells by 20-to-1. (supported by nih hl63302) . many of the membrane transporters that participate in this process have been identified; however, the identity of the class of kchannels that maintains resting membrane potential and/or cell membrane polarization during the secretion process remains poorly characterized. liquid secretion by porcine submucosal glands is insensitive to numerous k + channel blockers including ba 2+ , tetraethylammonium, apamin, charybdotoxin, iberiotoxin, clotrimazole, penitrem a, 4-aminopyridine, and quinidine. recently, a new class of k + channels, the "tandem-pore" or k 2p channels, have been described. potassium channels of this class are insensitive to many traditional k + channel inhibitors but are blocked by local anesthetics, such as lidocaine and bupivacaine. additionally, many k 2p channels are sensitive to changes in extracellular ph. in the present study, we considered whether k 2p channels might participate in the liquid secretion response to acetylcholine (ach). intrapulmonary bronchi, excised intact from the lungs of pigs, were cleared of accessible luminal liquid, cannulated, and treated with 10 µm ach to induce secretion. paired airways were pretreated for 1 h with 3 mm bupivacaine. bupivacaine blocked 90.4±1.8% of the liquid secretion response to ach. the ic 50 for the bupivacaine inhibition was approximately 220 µm. the inhibitory effect of bupivacaine was not due to nonspecific toxicity since tissues exposed to bupivacaine for 1 h and then washed with fresh inhibitor-free buffer recovered to 80% the control achinduced secretory rate. the bupivacaine effect was not due to inhibition of voltage-gated na + channels in intrinsic neurons since 1 µm tetrodotoxin did not inhibit ach-induced secretion. no significant effect was seen in the secretion rate when the extracellular solution ph was lowered from 7.43±0.01 (normal hco 3 --buffered krebs gassed with 5% co 2 ) to 6.60±0.01 by gassing with 40% co 2 gas (balance o 2 ). while we recognize the relative nonspecificity of this agent, the inhibitory effects of bupivacaine on liquid secretion by porcine bronchi are consistent with k 2p channel participation in this secretory response. the failure to demonstrate inhibition with acidification of extracellular solution could signify a k 2p channel subtype that is insensitive to low extracellular ph. ( 4 1. novartis, horsham, united kingdom; 2. rosalind franklin university, chicago, il, usa; 3. mount sinai medical center, miami, fl, usa; 4 . genomics institute of the novartis foundation, la jolla, ca, usa enac activity in the human airway epithelium has been reported to be partially-sensitive to the broad spectrum trypsin-like protease inhibitors aprotinin and placental bikunin (bridges et al., 2001 am j physiol 281:l16-23; donaldson et al., 2002 jbc 277:8338-45) . a low molecular weight inhibitor of the airway channel activating protease (cap) would represent a potential therapeutic approach to attenuating enac function in cystic fibrosis (cf) lung disease. the aims of the current study were to: (1) further characterise the in vitro efficacy of cap inhibitors in human bronchial epithelial cells (hbes), (2) to evaluate the relevance of a cap mechanism to the regulation of enac function in vivo, and (3) to establish whether the inhibition of the airway cap can modulate mucociliary clearance in vivo. primary cultures of hbes (normal and cf), cultured under air-liquid interface conditions, demonstrated an amiloride-sensitive short circuit current (isc), that was sensitive to aprotinin but that was insensitive to inhibition by sbti, α1-anti-trypsin and α1-pdx. the amiloride sensitive isc was also attenuated by the low molecular weight cap inhibitor nvp-qau145 (hbc276) with an ic50 value of approximately 30nm. nvp-qau145 caused a time dependent inhibition of the isc with a t 1 ⁄ 2 of approximately 20 min and this effect could be reversed by the addition of excess trypsin. in vivo, aprotinin attenuated the guinea pig tracheal potential difference (tpd) from -10.0±0.8mv to -4.7±0.6mv (n=4-11) with an ed50 value of 40pmoles/kg, measured at 2 hours following intra-tracheal instillation. the tpd was unaffected by either sbti or α1-anti-trypsin at doses up to 1500pmoles/kg. the combination dosing of an enac blocker with aprotinin did not attenuate the tpd beyond the effect observed with either agent alone, consistent with aprotinin attenuating enac function in this model system. intra-tracheal instillation of nvp-qau145 attenuated the tpd from -10.3±1.0mv to -4.7mv with an ed50 value of 3µg/kg (n=6-8). in the sheep, the administration of nvp-qau145 into the airways by aerosolisation of a 1mg/ml solution resulted in a 3-4 fold enhancement of the rate of clearance of 99mtc sulfur colloid from the lungs compared with the vehicle control. these studies indicate that the cap mechanism of enac regulation can translate through in vitro human assays, into in vivo models. cap inhibitors can attenuate tpd in the guinea pig in vivo and enhance the rate of mucociliary clearance in the sheep, thereby representing an approach to the therapeutic regulation of enac function in cf lung disease. cotton, c. pediatrics, physiology and biophysics, case western reserve university, cleveland, oh, usa cystic fibrosis is caused by mutations in the gene that encodes cftr, a camp-activated apical membrane anion channel. loss of cftr-mediated anion conductance is a primary defect in cf but secondary defects such a sodium hyperabsorption are also implicated in cf pathophysiology. it is generally accepted that mucociliary clearance is compromised in cf airways due to reduced fluid secretion and/or increased fluid absorption. the recent generation of a transgenic mouse model with enac overexpression that exhibits cf-like lung disease highlights the importance of sodium hyperabsorption. therapies designed to reduce mucus production and viscosity, increase lumenal water content, stimulate fluid secretion, and inhibit fluid absorption are under development for treatment of cf airways. although controversial, several recent studies have demonstrated that serca pump inhibitors partially correct the trafficking defect associated with delta f508 mutant cftr. the goal of this work is to determine if serca pump inhibitors affect enac-mediated sodium absorption. a renal collecting duct epithelial cell line and primary cultures of well-differentiated human tracheal epithelial cells (air/liquid interface culture) were used for these studies. amiloride-sensitive short circuit current was determined as a measure of enac-mediated sodium absorption. quantitative rt-pcr was used to evaluate expression levels for each of the 3 subunits of enac (alpha, beta, and gamma). epithelial monolayers were treated for 18-24hours with serca pump inhibitors (thapsigargin, 100-500 nm; dbhq,10-50 um; and curcumin, 10-50 um). treatment with serca pump inhibitors decreased amiloride-sensitive short circuit current by 50-80%. in contrast, campstimulated short circuit current was not reduced by treatment with serca pump inhibitors. the steady-state levels of alpha, beta, and gamma enac were reduced by 60-80% in renal epithelial monolayers treated with thapsigargin whereas beta and gamma enac were reduced by 80-90% with no significant change in alpha enac in airway epithelial cells. the results of these studies demonstrate that sustained depletion of endoplasmic reticulum calcium stores and/or elevation of intracellular calcium by inhibition of serca pump activity reduces enac mrna expression and enac-mediated sodium absorption. thus, inhibition of serca pump activity in airway epithelial cells of cf patients that carry the delta f508 mutation may provide dual benefit by promoting delivery of mutant cftr to the membrane 1 1. pediatrics, national jewish medical and research center, denver, co, usa; 2. integrated department of immunology, national jewish medical and research center, denver, co, usa; 3. department of medicine, cystic fibrosis/pulmonary research and treatment center, the university of north carolina at chapel hill, chapel hill, nc, usa increased cytosolic calcium ([ca 2+ ] i ) initiated by the release of stored ca 2+ from the endoplasmic reticulum (er) is a key signal to elicit a wide range of essential cellular responses, including secretory functions of the respiratory epithelium that are modified in cf. serca pumps are responsible for (re)filling the er ca 2+ stores, and serca blockers such as thapsigargin are very potent and commonly used pharmacological triggers of ca 2+ -signals. modulation of the activity of sercas can profoundly affect ca 2+ homeostasis. although defective calcium homeostasis is a characteristic of several pulmonary diseases including cf, the role of serca is unknown. lung tissue samples (bronchus and distal lungs) from normal (n=2) and cf subjects (n=2) were evaluated by immunohistochemistry. serca2 expression was decreased in the bronchial and bronchiolar epithelia of cf. non-cf and cf bronchial epithelial cell line pairs including calu-3 and jme/cf15, c-38 and ib3-1, 16hbe14o-(16hbe), cfbe41o-(cf41o-) and cfbe45o-(cf45o-) were also probed. given certain limitations of such cells, several consistent findings still emerged. a 65% and 75% decrease in serca2 expression was observed in cf41o-and cf45ocells as compared to 16hbe cells. immunocytochemical studies in these cells confirmed that the serca2 was localized in the er and that the decreased serca expression was not associated with decreased er content. reduced serca2 expression and activity (3.51±0.27 vs 1.76±0.23 and 2.59±0.29 pmol/min/mg protein in 16 hbe vs. cf41o-and cf45ocells respectively) was observed in the purified er membranes from cf cell lines. northern blot analysis revealed a parallel reduced mrna expression as well. decreased serca2 was accompanied by increase in the low affinity isoform serca3 (0.95±0.20 vs. 1.39±0.10 and 1.68±0.32 arbitrary serca2 intensity/b-actin in respectively) . we have also evaluated a limited number of primary airway epithelial cells isolated from lung samples of normal and cf subjects for serca2 expression. serca2 expression in polarized tracheobronchial epithelial cell lysates from cf subjects was decreased by about 60% as compared to those from normal subjects (1.86±0.56 vs. 5.68±1.24 arbitrary serca2 intensity/b-actin units in cf and normal respectively, n=3). expression of serca2 could also be suppressed by inhibiting cftr with cftr inh 172 in normal human bronchial epithelial cells ( various studies indicate that the airway surface fluid possess an antibacterial system based on the combined action of lactoperoxidase, h 2 o 2 , and scn -(thiocyanate). the enzyme lactoperoxidase (secreted by submucosal glands) utilizes h 2 o 2 and scnto generate oscn -(hypothiocyanite) a molecule with antimicrobial activity. h 2 o 2 is produced by dual oxidases expressed on the apical membrane of airway epithelial cells whereas scnis transported across the epithelium through anion transporters and channels. in particular, scntransport seems to occur through cftr and other ca 2+dependent clchannels. therefore, reduced scntransport in the airways of cystic fibrosis patients may contribute to impaired antimicrobial activity. in a recent study, we have found that cytokines, in particular il-4, cause a strong increase in the ability of cultured bronchial epithelia to transport scnfrom the basolateral to the apical membrane. this effect is mediated by upregulation of ca 2+ -dependent clchannels and of the slc26a4 (pendrin) anion transporter. these findings suggest that under proinflammatory conditions the activity of the scn -/h 2 o 2 /lactoperoxidase system is potentiated. we evaluated the antimicrobial activity of airway surface by seeding bacteria on the apical membrane of human bronchial epithelia grown with an air liquid interface on a porous membrane. an inoculum corresponding to 1,000 cfu of s. aureus was added to the cells in 25 µl of saline solution with and without lactoperoxidase (6.5 µg/ml). experiments were performed in the presence or absence of scn -(100 µm) in the basolateral solution. bacteria were recovered after four hours and plated on agar plates for colony counting. our preliminary results show that simple addition of lactoperoxidase to the apical surface decreases bacterial survival. in addition, bacterial killing is strongly enhanced by prestimulation of cells for 24 hours with il-4 (10 ng/ml). the effect of lactoperoxidase is dependent on h 2 o 2 as it is prevented by addition of catalase and is absent in cell-free experiments when h 2 o 2 is omitted from the reaction mixture. surprisingly, the presence of scnin the basolateral compartment did not increase bacterial killing and, in some cases, appeared to generate a protective effect. our results suggest that lactoperoxidase, in the presence of h 2 o 2 , is an effective antimicrobial molecule. the contribution of scnand the mechanism of the potentiation caused by cytokines is less clear and requires further investigation. a possibility is that, in the absence of scn -, lactoperoxidase generates an oxidant molecule that is more toxic to bacteria than oscn -. elucidation of this mechanism and comparison between cf and non-cf epithelia is in progress to assess the role of cftr and of other anion channels. supported by cfft and telethon-italy. cf patients become infected with pseudomonas aeruginosa, which release flagellin into the airway surface liquid to activate toll-like receptor 5 and proinflammatory signaling. flagellin has been shown to inhibit na absorption by airway epithelia. we tested flagellin on cl secretion and proinflammatory signaling (nf-κb activation) by calu3 cells, a cftrexpressing, serous-like airway gland cell line. calu3 cells were grown on filters and either mounted in ussing chambers (clamped to zero mv) in the presence of a serosa-to-mucosa gradient of [cl] for measurements of transepithelial cl secretion (i cl ) or treated with an adenovirus expressing nf-κb-controlled luciferase to assay nf-κb activation. flagellin (10 -7 g/ml) on either the apical or basolateral surface of cells increased apparent anion secretion that began in 3-10 mins and increased over 20-30 mins by 20-50 µa/cm 2 . this i cl was blocked by glibenclamide and glyh101, indicating that it resulted at least in part from activation of cftr. flagellin also stimulated fluid secretion by intact human tracheal glands. flagellin activated nf-κb (luciferase assays) in both calu-3 cells and in the cf cell line cf15, while i cl was stimulated in calu3 but not in cf15 cells. flagellin-stimulated i cl in calu-3 cells was blocked 50% by sb202190 (a p38 mapk blocker) and by a similar amount by wortmannin (pi3 kinase blocker). interleukin 1β and the tlr2-agonist pam3cys also activated i cl . the effect of flagellin was not due to increases in cytosolic [ca 2+ ] (ca i ) because flagellin did not alter ca i . in contrast to the slow effects of flagellin, pam3cys and il1β, atp rapidly increased i cl (within secs, up to 80 µa/cm 2 ) followed by slower decrease to steady i cl = 20-40 µa/cm 2 that had a similar time signature as the increases in ca i . forskolin (to increase cytosolic [cyclic amp]) increased i cl within 2 mins to a steady value = 50-80 µa/cm 2 . flagellin had small or no effects on i cl following maximal stimulation with either atp or forskolin. atp and forskolin on their own had no effect on nf-κb. flagellin increased phosphorylation of p38 mapk and of akt (down-stream kinase phosphorylated by pi3k) with a time course similar to the increase in i cl . flagellin-activated nf-κb was reduced by roughly 50% by either wortmannin or sb202190. these results indicated that tlr agonists and inflammatory cytokines stimulate both nf-κb and proinflammatory processes and also cftr-dependent cl and fluid secretion by airway gland cells. these responses are mediated in part by activation of both p38 mapk and pi3 kinase. flagellin-tlr5-activated increases in cftr-mediated cl secretion and reduction in enac-mediated na absorption will increase fluid secretion into the airways, which may facilitate bacterial removal by the mucociliary escalator and thereby reduce the proinflammatory stimulus. in cf it is expected that bacteria will activate tlr signaling to trigger innate immune responses, but cl and water secretion and the resulting "bacterial flush," will be missing, leading to sustained inflammation. (1, 2) . treatment with hs improved several measures of lung function including mucociliary clearance. in one study amiloride was seen to have a negative impact on the benefit of hs (2) . the long duration of action of hs and the paradoxical effect of amiloride are surprising and require further investigation. aims: our goal was to study the effect of exposure to a hypertonic challenge (hc) on amiloride sensitive na+ transport (ina) in primary cultures of human bronchial epithelial (hbe) cells from non-cf and cf patients. methods: hbe cells were grown at an air liquid interface and studied under short circuit current conditions. cells were challenged with hs by exchange of the apical or basolateral baths. in some experiments the timedependent effect of a small (30 µl) isosmotic or hs volume at the airway surface was tested on ina and apical osmolality. results: application of hypertonic nacl and mannitol solutions from either the mucosal or serosal sides of the epithelium inhibited ina. the degree of inhibition was a saturable function of the imposed hypertonicity with an ic50 of 379 (mannitol) and 398 (nacl) mosmol/kg h2o and a maximal inhibition of 97%. the inclusion of amiloride (10-100 µm) did not affect the hc-induced reduction in ina. the inhibition in ina by hc was rapid in onset and accompanied by a fall in the transepithelial resistance and the total transepithelial capacitance as expected for cell shrinkage. the inhibition in ina was only partially reversible (~20%) after returning to isotonic solutions for 45 min. exposure of the airway surface to hypertonicity (680 mosmol/kg h2o) inhibited ina by 72-79% at 30 min of exposure. as osmotically-driven water moved from the serosal to the mucosal side, the apical volume increased concomitant with a decrease in its osmolality and a recovery of ina. the osmolality declined exponentially and reached the isosmotic value after ~4 hrs. the recovery of ina lagged behind the recovery of apical osmolality by 80, 20 and 10% at 0.5, 2 and 3 hrs of incubation, respectively. after 4 hrs of airway hc exposure, the osmolality and ina recovered completely. our results demonstrate that hc causes a rapid, dramatic and prolonged decrease in na+ absorption and that continuous presence of amiloride had no effect on the inhibition of ina or the recovery of apical osmolality. hc also causes the epithelium to shrink. no appreciable differences were observed between cf and non-cf hbe cells. conclusions: we propose cell shrinkage together with the continued influx of na+ increases intracellular na+ ([na]i) and lead to an inhibition of na+ transport. elevated [na] i is a known inhibitor of epithelial na+ channels (3). the sustained nature of the inhibition in na+ transport to a hc may help explain the longer than expected duration of action of hs in the clinical trials (1, 2) . explanation of the paradoxical effects of amiloride observed in a clinical trial (2) requires further investigation. references: 1. n engl j med 354:229-240,2006; 2. n engl j med 354:241-250, 2006; 3. physiol rev 77:359-396, 1997 reduced airway surface liquid (asl) volume resulting from enac mediated na+ hyperabsorption and cftr-mediated hyposecretion plays a critical role in cf lung disease pathogenesis. however, the mechanisms involved in enac/cftr regulation are poorly understood. using human bronchial epithelial cultures (hbecs) under thick film conditions, i.e. in the ussing chamber with the asl washed away, adenosine (ado) directly stimulates cl-secretion. however, in a cl-free environment, ado has no effect on the amiloride-sensitive current, which is mediated by enac. mean change in i sc following 100µm ado to the apical chamber was 0.05 µa/cm 2 from baseline compared to vehicle alone (-0.17 µa/cm 2 ;ns;n=3) in hbecs. mean delta amiloride (100µm) was -5.32 and -5.38 µa/cm 2 respectively (ns; n=3 each). in contrast, under thin film conditions we have previously shown that ado stimulates sustained asl secretion via activation of cftr in the presence of the protease-inhibitor aprotinin, whereas trypsin pre-treatment abolished this secretion, likely by activating enac and abolishing the electrical driving force for cl-secretion 1 . further, ado-mediated asl secretion is significantly increased if hbecs (thin film conditions) are left with intact asl for 24 h, an action that is abolished by acutely washing the apical surface 1 . thus, we hypothesised that a soluble enac inhibitor was secreted into the asl under thin film conditions, which could accumulate sufficiently to inhibit enac and provide the necessary driving force for cftr-mediated cl-secretion after ado-addition. to search for such an inhibitor, we incubated asl with trypsin-coated beads and identified bound proteins by mass spectrometry (albumin-coated beads were used as a control to exclude non-specific binding). the major identified protein was plunc, a protein that is secreted into the asl both in vivo and in vitro which has no current known function. secretion of plunc into hbec asl was confirmed by western blot. to test whether plunc could alter asl volume homeostasis, we made an anti-plunc shrna retroviral construct which we used to infect hbecs. unlike the control anti-luciferase shrna infected hbecs, anti-plunc shrna-infected hbecs exhibited >90% knockdown of plunc and a failure to regulate asl volume (ctrl, 9.0±1.8µm; 5±0.9µm; n=5) . when co-expressed with enac in oocytes, plunc inhibited enac currents by 78% (n=12). cftr function in contrast was unaffected by plunc expression (n=10). thus,it is likely that ado stimulates cftr mediated cl-secretion but is not involved in enac regulation. however, experiments were performed in a very simplified environment i.e. static cultures with little mucosal atp or mucus and these findings will need to be expanded and further evaluated in native tissues. we conclude that plunc, rather than ado may be the soluble mediator in the asl which regulates enac function by reducing its activity. 1. tarran, r. et al., j. gen. physiol., 2006. 127 (5) airway surface liquid (asl) absorption is mediated by epithelial na + channels (enac), which establish an osmotic gradient that drives fluid absorption. we and others have recently reported that a protease / anti-protease balance regulates enac in normal human bronchial epithelial cells (hbe) and provides a mechanism for auto-regulation of asl volume. in cf, this balance is disturbed, leading to constitutive proteolytic activation of enac and the pathological na + hyper-absorption characteristic of the disease. to determine if channel activating protease expression is regulated by changes in asl volume and is altered in cf, we examined prostasin expression in control and cf hbe under basal and asl volume expansion conditions using western blotting. prostasin migrates as 37 kda and 40 kda bands in apically biotinylated proteins, apical secretions, and whole cell lysates of primary hbe. following apical aprotinin exposure, only the 40 kda prostasin molecular species was present in the biotinylated proteins, suggesting that the proteolytic conversion of prostasin zymogen to active enzyme occurs on the cell surface. following asl volume expansion, cell surface prostasin expression increased by > 55% (p=0.007, n=12 tissue donors, >2 cultures from each). as our recent studies indicated that increased proteolytic activation of enac occurs in cf airway epithelia, we next compared prostasin expression in cf and control hbe cells. prostasin expression in the apical biotinylate of cf hbe was 1.5 fold greater than in control hbe (p=0.025, n=9 tissue donors). furthermore, the ratio of the 37 kda (active enzyme) to 40 kda (zymogen) prostasin molecular species was 2.5 fold greater in cf (p=0.009), indicating that increased activation of prostasin occurs in cf airway epithelium. we next determined whether increased prostasin activation in cf may reflect the deficiency of a protease inhibitor. serpin e2, also known as protease nexin-1 (pn-1), forms an ~82 kda complex with prostasin and permanently inactivates its protease activity. while no significant difference in pn-1 expression was observed between control and cf hbe, pn-1 was found to contribute to prostasin regulation by (i) forming an inactive prostasin complex, (ii) inhibiting the amiloride-sensitive short-circuit current across hbe, and (iii) preventing conversion of prostasin zymogen to active enzyme. these findings demonstrate that cellular mechanisms coordinate prostasin expression and activity with asl volume. accordingly, at times when the asl volume is high, prostasin expression increases, and this presumably augments na + and asl absorption to absorb the excess luminal fluid. these regulatory mechanisms govern the apical membrane expression and processing of zymogen to active enzyme. because prostasin is incapable of autocatalysis, these findings support the existence of a proteolytic cascade that controls enac activity during asl volume homeostasis, perhaps reflecting the recently reported matriptase-prostasin cascade. furthermore, the increased expression of prostasin in cf suggests that abnormal regulation of prostasin contributes to na + hyper-absorption in cf airways. supported by the nih, cff, and ala. we report that annexin 2 (anx 2)-s100a10 forms a functional camp/pka/calcineurin (can)-dependent complex with cftr. cell stimulation with forskolin/ibmx significantly increases the amount of anx 2-s100a10 that reciprocally co-immunoprecipitates with cell surface cftr and can a. pre-inhibition of the cells with pka or can inhibitors attenuates the interaction. furthermore, we find that the acetylated peptide (stvheilcklsleg, ac1-14), but not the non-acetylated equivalent n1-14, corresponding to the s100a10 binding site on anx 2, disrupts the anx 2-s100a10/cftr complex. analysis of dids and cftr inh172 -sensitive currents, taken as indication of the outwardly rectifying cl-channels (orcc) and cftr-mediated currents, respectively, showed that ac1-14, but not n1-14, inhibits both the camp/pka-dependent orcc and cftr activities. can inhibitors (cypermethrin, cyclosporin a) discriminated between orcc/cftr by inhibiting the cftr inh172 , but not the dids, sensitive currents, by more than 70%. furthermore, peptide ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. our data suggests that the anx 2-s100a10/cftr complex is important for cftr function across epithelia. the content and water (h2o) mobility in airway epithelium is tighly coupled to airway function. in cystic fibrosis (cf), h2o epithelial permeability is reduced in airways. the demand of noninvasively imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical and functional phenotyping in the genetically altered mice. magnetic resonance microscopy (mrm) is a noninvasive, inherently three-dimensional (3d) imaging technique capable of visualizing anatomical structures in the mouse and allows for interpretation of complex spatial relationships between substructures and h2o. in this study, we explore different mr contrast parameters and signal-to-noise ratios at a 30 µm pixel size to characterize microstructure and h2o mobility in ex vivo trachea of cf transmembrane conductance regulator (cftr)-deficient (cftr knockout, cftr tm1unc ) mice and their aged-matched wt littermates. this study is performed using a bruker mrm system at 11.7 tesla. we demonstrate for the first time the ability of 3d-mrm to map the h2o content and mobility in trachea epithelium. from the 3d-mrm videoimages, differential h2o content was visualized in different levels of trachea in wt and cf mice. t2 mrm images depicting the h2o rotational mobility which is related to environmental viscosity of trachea epithelium will be also shown. finally, this 3d-mrm imaging method is a valuable method for measuring h2o content and permeability in airways and can serve for assessing the effects of drugs on h2o mobility in cf airways. supported by grants from inserm, upmc-paris6, cnrs, and the french cystic fibrosis association (vlm). cystic fibrosis (cf) is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein resulting in progressive lung oxidative damage. in this study, we evaluated the role of cftr in the control of ubiquitin-proteasome activity (ups system) and the nf-κb / iκb-αsignaling after lung oxidative stress. we exposed cftr deficient (cftr-/-) and wild type mice for 64 h to hyperoxia-mediated oxidative stress. cftr deficient mice exhibited significantly higher lung proteasomal activity than cftr+/+ animals after oxidative stress. this was accompagnied by a strong reduction of lung caspase-3 activity and an absence of degradation of nf-κb inhibitor iκb-α. in vitro, human cftr-deficient lung cells also exhibited higher proteasomal activity and a lack of increased nf-κbdependent transcriptional activity compared to cftr-sufficient lung cells after oxidative stress. furthermore, inhibition of the proteasomal activity by mg132 in cftr-deficient lung cells restored the nf-κb/iκb-α signaling to that of cftr-sufficient lung cells. inhibition of caspase-3 by z-dqmd in cftr-sufficient lung cells mimicked the response profile of increased proteasomal degradation and lowered nf-κb transcriptional activity of cftr-deficient lung cells when exposed to oxidative stress. all together, these results suggest that cftr is a crucial molecule in regulating proteasomal degradation and nf-κb activity in lung epithelium under oxidative stress. staphylococcus aureus, one of the major pathogen involved in airway infections, releases in the airway lumen virulence factors that may impair the airway epithelial functionality. to date, the effect of s. aureus virulence factors on the loss of electrolyte homeostasis of the airway epithelium has not been investigated. we have previously shown that the combination of a corticosteroid and a long-acting beta 2 agonist attenuated the airway epithelial cell inflammatory response induced by s. aureus virulence factors. the aim of the present work was to investigate the effect of s. aureus virulence factors on the airway epithelial tightness and on the chloride efflux of airway epithelial cells incubated or not with salmeterol hydroxynaphthoate 2×10 -7 m and fluticasone propionate 1×10 -8 m (sm/fp). the airway epithelial tightness was assessed by immunocytochemistry, western blotting and transepithelial resistance measurement. the chloride efflux was evaluated by dynamic imaging using the 6-methoxy-n-(3-sulfopropyl) quinolinium (spq) fluorescent probe. s. aureus (strain 8325-4) virulence factors were obtained by growing bacteria at 5×10 8 cfu/ml for 18h at 37°c. the bacteria supernatant containing s. aureus soluble virulence factors was then centrifuged, filtered and diluted at 2% in cell culture medium (a concentration that did not alter cell viability). human airway epithelial cells (mm39) were incubated with 2% s. aureus virulence factors for 1h and then co-incubated or not with sm/fp for 4h. we observed that the incubation with 2% s. aureus virulence factors alone did not significantly alter the epithelial integrity assessed by the expression of tight junction proteins and transepithelial resistance measurement. however, when the cells were incubated with sm/fp alone or with 2% s. aureus virulence factors plus sm/fp, the expression of tight junction proteins was significantly increased (p<0.05) as compared to cells incubated with s. aureus virulence factors. interestingly, the chloride efflux in airway epithelial cells was significantly decreased in a time-dependent way by s. aureus virulence factors (3 fold decrease after a 4h incubation). the co-incubation of airway epithelial cells with s. aureus virulence factors and sm/fp prevented the s. aureus virulence factorsdependent chloride efflux decrease. our results demonstrate that bacterial virulence factors induce the loss of the electrolyte homeostasis and suggest that the treatment by the combination of a corticosteroid and a long-acting beta 2 agonist may preserve the airway epithelial functionality. supported by association vaincre la mucoviscidose and glaxo-smithkline nilsson, h.e. 1 ; dragomir, a. 1 ; ahlander, a. 1 ; johannesson, m. 2 ; roomans, g.m. 1 1. medical cell biology, uppsala university, uppsala, sweden; 2. women's and children's health, uppsala university, uppsala, sweden inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions on mucociliary clearance (mcc) is unclear. one explanation has been suggested, namely that hypertonic solutions open tight junctions, which may lead to increased water transport followed by an increased airway surface liquid (asl) volume. furthermore, the role in cftr-mediated hco3-conductance in regulating asl ph has led us to investigate if ph changes may have an effect on the tightness of tight junctions. the effect of osmolarity was investigated on the 16hbe14o-cell line by the addition of nacl, nabr, licl, mannitol or xylitol (295-700 mosm). the effect of ph was investigated on the 16hbe14o-, calu-3 and t84 cell lines as well as the cystic fibrosis cell line cfbe41o-. transepithelial resistance was measured as indicator of the tightness of the cultures. cell-cell contacts and morphology were investigated by immuno-fluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. the electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mosm on, when the hyperosmolar exposure was gradually increased from 295 to 700 mosm, whereas the non-electrolyte solutions caused a decrease in transepithelial resistance from 700 mosm on. immunofluorescence micrographs showed weaker staining for the proteins zo-1, claudin-4 in treated samples compared to the control. the ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and with electrolyte solutions opening a larger proportion of tight junctions than non-electrolyte solutions. it was noted that during the exposure of the cultured cells to the hyperosmolar solutions, the ph of the medium increased from 7.4 to 8.0. 16hbe14o-cells exposed to both a rise in ph and hyperosmotic stress showed an overall lower teer and a significantly reduced ability to recover from stress compared to cultures at a ph hold constant at 7.4. without exposure to hyperosmolar solutions, a rise in ph caused a significant decrease in transepithelial resistance in 16hbe14o-cells, calu-3 and t84 cell lines but not in the cfbe14o-cell lines, where the reaction was significantly less and delayed. in conclusion, hyperosmolar solutions caused a reversible opening of the tj in 16hbe14o-cell cultures with electrolytes having stronger effects than non-electrolytes, where one of the effects on mcc may be due to increased water transport across the leaky paracellular space. an increase in ph caused a significant decrease in teer in the healthy cell lines compared to the cfbe41o-cell line. we speculated that an impaired alkalinisation of the apical fluid due to a defective cftr also will cause the tight junctions to react different to other external stimuli, such as osmolarity, compared to healthy cells, which is also indicated by preliminary experiments on the effect of nacl on teer in cfbe41o-cultures. extracellular nucleotides regulate surfactant secretion in alveoli and mucociliary clearance in airway epithelia, but the mechanism(s) of their release and their regulatory pathways remain incompletely understood. previously, we showed that hypotonic swelling of a549 epithelial cells induces ca 2+ -dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. in this study, we examined sources of intracellular ca 2+ ([ca 2+ ] i ) elevation evoked by acute 50% hypotonic stress and the role of autocrine purinergic signaling in ca 2+ -dependent atp release. we found that atp release does not directly involve ca 2+ influx from extracellular spaces, but depends entirely on ca 2+ mobilization from intracellular stores. the [ca 2+ ] i response consisted of slowly-rising elevation representing mobilization from thapsigargin (tg)-insensitive stores and a superimposed rapid spike due to ca 2+ release from tg-sensitive endoplasmic reticulum (er) stores. the latter could be abolished by hydrolysis of extracellular tri-and di-nucleotides with apyrase; blocking p2y 2 /p2y 6 receptors of a549 cells with suramin; blocking udp receptors (p2y 6 ) by ppads; emptying tg-sensitive stores downstream with 1 µm tg or 10 mm caffeine in ca 2+ -free extracellular solution; or blocking the ca 2+ -release inositol 1,4,5-triphosphate (ip 3 ) receptor channel of the er with 75 µm 2aminoethyl diphenylborinate. these results demonstrate that the rapid [ca 2+ ] i spike results from the autocrine stimulation of ip 3 )/ca 2+ -coupled p2y 2 /p2y 6 receptors, which accounts for ~70% of total ca 2+ -dependent atp release evoked by hypotonic shock. our study reveals a novel paradigm in which atp release is amplified by the synergistic autocrine/paracrine action of co-released uridine and adenosine nucleotides. we suggest that a similar mechanism of purinergic signal propagation operates in other cell types. (this study was supported in part by the canadian institutes of health research and the canadian cystic fibrosis foundation (ccff). s.t. was the recipient of a ccff studentship). the maintenance of a thin liquid layer on the airways and alveoli surfaces is essential for normal lung physiology.yet, the mechanism sensing the height of the layer remains obscure. in this study, we examined atp secretion from human lung a549 epithelial cell monolayers mounted in a closed, flowthrough chamber (0.5 mm height) and found that passage of an air bubble over the monolayer caused transient (<1.5 min) but significant atp release (600 to 2,000 pmoles/10 6 cells). air bubble-induced atp release was reduced by~70% from cells loaded with the intracellular ca 2+ chelator bapta-am, and was completely abolished in n-methylmalemide-treated (1 mm) cells, suggesting the involvement of ca 2+ -dependent exocytosis. fura-2 intracellular ca 2+ ([ca 2+ ] i ) imaging experiments revealed transient [ca 2+ ] i elevation during the passage of an air bubble over the cell monolayer, but the [ca 2+ ] i response did not involve non-specific ca 2+ influx from the extracellular space, e.g. due to cell damage, since similar responses were observed in ca 2+ -free extracellular solution. ethidium bromide staining did not disclose any cell damage in these experiments. confocal fluorescence microscopy study showed reversible cell deformation (flattening) of 10% to 40% in height in the monolayer part in contact with the air bubble and confirmed that cell integrity was entirely preserved. these experiments demonstrate that in close proximity to the air-liquid interface (i.e. between the air bubble and the wet cell surface), surface tension forces are transmitted directly on cells, causing their mechanical deformation, elevation of [ca 2+ ] i and subsequent atp release. we propose that a similar mechanism may operate in vivo in the airways, where surface tension forces acting directly on exposed epithelial cell surfaces may provide a fail-proof mechanism to protect proper airway surface liquid volume via mechanosensitive, ca 2+ -dependent atp release and purinergic modulation of fluid secretion. this mechanism may be defective in cystic fibrosis due to excessive mucus layer covering the airways. introduction : cystic fibrosis is a genetic disease that reflects the consequences of mutations in the cystic fibrosis transmembrane conductance reg-ulator (cftr) gene, affecting anionic transport in epithelia. slc26 family members are anionic transporters involved in cl-and hco3-absorption or secretion in epithelia. in addition, the activation of some slc26 family members by cftr has been demonstrated (nature cell biology, 2004, 6: 343-350) . slc26a9 is a poorly characterized member of this family, being the solely expressed in lung. putative interaction domains with cftr are also present in the slc26a9 protein. in this study, we have investigated the transport properties of slc26a9 (human) to determine the functional and pharmacological characteristics of this transporter. methods : to this aim, electrophysiological studies (two-electrode voltage clamp or current clamp methods and intracellular ph measurements using ph sensitive microelectrode) were performed in xenopus laevis oocytes expressing slc26a9 proteins. complementary (36cl) transport studies were also undertaken. results : the protein expression results in the appearance of an anionic current showing a linear current/voltage relationship. 36cl influxes experiments confirmed the induced cl-permeability following slc26a9 protein expression. the sequences of conductivity, cl->i-> no3-≥ gluconate > so4 2-and selectivity (px/pcl), i-> cl-= no3-> gluconate > so42-are found. the hco3-conductance mediated by the slc26a9 protein expression is low. using co2/hco3-containing ringer solutions, no intracellular phi changes were detectable in conditions (low chloride in external medium) favoring the cl-/hco3-exchange whereas phi changes (alkalinization) were observed with the expression of the ae1 exchanger. however, phi changes could be detected in conditions largely favoring the driving force for hco3-entry. dids and ns 3623 inhibited slc26a9 associated currents. the specific cftr inhibitor (cftrinh172) or glybenclamide had little effect. elevation of intracellular camp (a cftr activator) was also ineffective while maneuvers increasing intracellular calcium blocked the slc26a9 associated currents. conclusions : our study demonstrates that slc26a9 presents an electrogenic anion conductance (characteristics of an anionic channel) when expressed in xenopus oocytes (however a transporter functioning as a ncl-/hco3-exchanger can not be excluded). this function (channel) has also been described for another member of this family, slc26a7 (j biol. chem, 2005, 280: 6463-6470) . the physiological role of slc26a9, present in the bronchial cells of airway epithelia and its potential interaction with cftr has to be precised in situ or in oocytes and in mammal expression systems. medicine, women's & children's hospital, nth adelaide, sa, australia; 2. cf research & treatment center, university of nth carolina at chapel hill, chapel hill, nc, usa; [3] [4] [5] [6] [7] [8] jasri, hyogo, japan; 4. physics and synchrotron science, monash university, clayton, vic, australia; 5. paediatrics, university of adelaide, adelaide, sa, australia non-invasive imaging of lung (e.g. hrct, mri, pet) is a valuable modality for detection and monitoring of the effects of cf in human airways, but resolution is limited; e.g. airway hrct detects airways no smaller than ~ 1.5mm dia. detection of wall or lumen changes in smaller airways, or the detection and monitoring of induced airway disease in live rodent models, demands significantly greater resolution coupled with rapid image capture. we report substantially increased airway resolution is achievable using synchrotron phase-contrast x-ray and can produce three-dimensional reconstructions encompassing the smallest mouse lung airways. nembutal-anaesthetised c57bl/6 mice (~ 20 gm) were imaged at the spring-8 synchrotron in hyogo, japan. 2-d phase-contrast airway images were obtained on ccd detectors (1.1 µm pixels) with a 90 cm phase-contrast propagation distance at 25 kev; nose and trachea were imaged at 15 or 30 sec intervals (100-300 ms exposures) over 45 mins. mice killed with nembutal overdose were imaged in axially aligned segments to obtain 3dimensional ct data voume of approximately 20x20x15 mm3 with 12x12x12 um3 voxels of nose, trachea, and lung (hammamatsu ccd detector). volume renderings were produced using volview or osirix software. live, 2-d nasal or tracheal imaging revealed airway-surface activity in some live mice consistent with dynamic mucociliary clearance activity. individual ct slices revealed the fine detail in the mouse lung, with dynamic ("fly through") sequences of lung ct slices permitting visual identification and tracking of lung tree branching from trachea (~ 1mm dia) into small airways (approx 100 um dia). adjustment of x-ray contrast, opacity, and range in these volume reconstructions permitted selective display of the mouse lung conducting-airway tree and airway surfaces. selected regions of the lung could be examined statically, or in rotation through different orientations in high-resolution 3-d. synchrotron phase contrast x-ray provides a new option for non-invasive imaging of intact mouse lung, at a resolution that permits examination of small conducting airways in mice. combined with volume-reconstruction software these ultract images can provide a powerful option for understanding the structure-function relationships produced by airway disease. ultra-ct 3-d studies are underway to compare lung airways at high resoltuion in normal and transgenic mice having altered airway pathophysiology. the epithelial sodium channel, enac, has a vital role in the function of the pulmonary epithelia and significantly contributes to the pathophysiology of the cf airway. thus, strategies to repair mutant cftr dysfunction must also consider the influence of such repair on enac functional expression. the ∆f508 trafficking repair agent 4-phenylbutyrate modulates the expression of the 70 kda molecular chaperones hsc70 and hsp70 in cf epithelial cells. we therefore assessed the role of these chaperones in the regulation of enac trafficking. in xenopus oocytes, we previously observed that overexpression of hsc70 inhibits murine enac (menac) functional and surface expression. in contrast, hsp70 can either enhance or inhibit menac functional and surface expression depending on the extent of overexpression [goldfarb et al. (2006), proc. natl. acad. sci. 103:5817-22 ]. here, we tested the hypothesis that these differential effects of hsc70 and hsp70 on menac expression also occur in epithelial cells. mdck cell lines with stable expression of α-ha, β-v5, γ-myc-menac and tetracyclineinducible expression of either hsc70, atpase-deficient hsc70, hsp70 or atpase-deficient hsp70 containing myc/his epitope-tags were selected. all epitopes were fused to the respective c-termini. these cells were grown as high-resistance (>500 ω/cm 2 ) monolayers on permeable supports. doxycycline-induced overexpression of hsc70 decreased amiloride-sensitive i sc and the whole cell content of α-haand β-v5-menac. in contrast, lower amounts of tetracycline-induced hsp70 overexpression increased amiloridesensitive i sc and whole cell α-haand β-v5-menac expression; these effects were absent at higher levels of hsp70 overexpression. these data are consistent with our previous data in xenopus oocytes. interestingly, the atpase-deficient chaperones had the opposite effect on menac functional expression. modest doxycycline-induced expression of atpase-deficient hsc70 increased amiloride-sensitive i sc in these cells, while modest overexpression of atpase-deficient hsp70 decreased amiloride-sensitive i sc . these effects may result from "dominant negative" interference with hsc70 and hsp70 function, respectively. these data are consistent with hsc70 and hsp70 having different effects on menac functional expression in epithelial cells, and that these effects are dependent upon the atpase activity of the respective chaperones. supported by grants from niddk. we have studied survival, airway epithelia bioelectrics and lung pathology of mice over-expressing various combinations of the 3 epithelial na + channel (enac) subunits (α, β and γ, genes scnn1a, scnn1b and scnn1g). we generated double-transgenic mice and crossed them with single-transgenic mice to obtain litters comprising all 8 possible genotypes. survival analysis revealed that overexpression of βenac in combination with either αenac or γenac significantly reduced survival in comparison to wild-type (wt) littermates. strikingly, at 3 days of age, all genotypes were represented according to the expected mendelian proportion, except for the triple transgenic αβγenac, which was significantly under represented. in utero studies are ongoing to understand if over-expression of αβγ affects the fetuses or the newborn pups during the first hours post-partum. due to the high mortality of mice over-expressing βenac subunit combinations, we studied tracheas from pups at 3 days of age. as previously reported, mice overexpressing β, but not the α or γ enac subunits, exhibit airways hyperabsorption of na + and lung pathology (mall et all 2004) . the βenac tracheas exhibited na + absorption [as measured by the change in short circuit current (isc) in response to amiloride in the ussing chamber] that was ~5.4 fold greater (∆isc 24.6±3.9 µa×cm -2 , n=9, means±sem) than wt (4.6±0.8 µa×cm -2 , n=7). over-expressing αβ or αγ enac subunits resulted in rates of na + absorption that did not differ significantly from those of βenac or γenac subunit alone, respectively. however over-expressing the β and γ enac subunits together resulted in an amiloride sensitive isc that was ~13.7 fold greater (∆isc 63.1±8.2 µa×cm -2 , n=9) than wt. we also studied the tracheal bioelectrics of the few 3 day-old αβγenac pups available. tracheas from the αbγ pups exhibited na + absorption that was ~16.7 fold greater (∆isc 77±8.2 µa×cm -2 , n=3) than wt. due to the small sample size, we cannot determine if this response is significantly different from the response of the βγenac pups. analysis of lung pathology in 3 day-old pups revealed that all the combinations that exhibited increased airway na + absorption and decreased survival in comparison to wt littermates, e.g. αβ, βγ and αβγ, also presented with alveolar space enlargement, maybe due to postobstructive air trapping, and airway epithelia necrotic degeneration associated with ab-pas negative bronchial obstruction. from these data, it appears that the rate of airway na + absorption negatively correlates with the survival of the pups. thus, it is likely that the mucus and airway surface liquid is more dehydrated as a function of the rates of na + absorption (aβγ > βγ > αβ, β > wt) which produces gradual failure to survive due to asphyxia secondary to airway obstruction. supported by nih scor p50 hl060280 choi, j. 1,2 ; joo, n. 1 ; wu, j. 1 ; krouse, m. 1 ; wine, j.j. 1 1. cystic fibrosis research laboratory, , stanford, ca, usa; 2. otorhinolaryngology, yonsei university, seoul, south korea submucosal glands, which produce most airway mucus, are mainly controlled by parasympathetic pathways that stimulate glands via airway intrinsic neurons distributed along the airway walls. the intrinsic neurons can be stimulated via axon reflexes from receptors in the mucosa. in previous experiments, it was shown that capsaicin applied to the mucosa stimulated gland secretion in the upper tracheas of wt but not cftr -/-mice (ianowski et al., j physiol, 2007, 580, 301) . it seemed important to assess a possible role of capsaicin-sensitive pathways in pigs and humans. we obtained pig tracheas following acute experiments carried out for other reasons, and human airways following lung transplants from donor tracheas and from the excised lungs of the transplant recipients. secretion from individual submucosal glands was quantified optically by time-lapse digital imaging of the growth of spherical bubbles of mucus in an oil layer in humans, commercial chili oil (5 µm dispersed in 20 µm mineral oil) stimulated mucus secretion from submucosal glands from both donors (n = 46 glands from 9 subjects, 0.30 ± 0.24 nl/min/gland) and disease control subjects (n = 45 glands from 7 subjects, 0.25 ± 0.16 nl/min/gland). however, there were no responses to chili oil in submucosal glands from cf subjects (n = 36 glands from 5 subjects, 0.01 ± 0.01 nl/min/gland). in pigs, we determined concentrationresponse relations for gland secretion in response to purified capsaicin (sigma). the threshold was ~1 µm and the ec50 was 19.01 ± 4.9 µm. the response to 100 µm capsaicin was partially blocked by high dose ttx (>1 µm). as in humans, the capsaicin response required cftr, because it was blocked ~70% by cftrinh-172, (n = 29 glands, from 4 pigs, p < 0.05). cftrinh-172 is presently the most specific inhibitor of cftr that can be used with glands, where we do not have access to the apical membrane. in ferrets, intrinsic airway neurons express acetylcholine, vip and sp, often colocalized within the same neurons (dey et al., am j respir cell mol biol, 1996, 14, 207) . in pigs, all three of these transmitter systems appear to be involved in gland secretion to capsaicin, because the response was partially blocked by the nk-1 receptor blocker l703606 (oxalate salt, 1µm), the vip receptor inhibitor l8k (10µm), and the muscarinic receptor blocker atropine (1µm); each of these blocked 36 to 59 % of the response (n = 21 to 39 glands from 3 to 5 pigs). indeed, most of the local pathways are probably damaged, which may account for the relatively small responses to strong stimulation. relatively few experiments have been carried out in intact airways with uninterrupted innervation and circulation, but in those experiments the glands were highly responsive to modest mucosal stimuli (reviewed in wine, auto neurosci, 2007, 133, 35) . hence, we hypothesize that (1) reflex stimulation of glands plays an important role in lung innate defenses; (2) defects in these responses is an important reason that cf airways are prone to infection, and (3) local reflexes may assume greater importance for maintaining the mucosal defenses of transplanted lungs. supported by niddk ro1-51817 (jjw), cff and cfri. most airway mucus arises from submucosal glands, which express cftr in their ciliated ducts and in serous cells. the airway glands secrete to agonists that either increase intracellular ca2+ (e.g. acetylcholine) or camp (e.g. vip).substance p(sp) also stimulates airway gland liquid secretion in pig (trout l et al., am j physiol lung cell mol physiol. 2001, 281,:l639) , suggesting that sensory afferents or local neurons that express sp play a role in stimulating submucosal glands. we used pharmacological methods to dissect the secretory mechanism of sp-induced gland secretion in comparison with responses to carbachol. tracheas were obtained from pigs after acute surgeries carried out for other reasons. the ventral mucosa with underlying glands was dissected from the cartilage, pinned mucosal side up at the gas/bath interface of a physiological chamber, and covered with oil. secretions from individual glands could be visualized as spherical bubbles in the oil, and secretion rates determined by optical monitoring of bubble diameters. concentration-response relations for gland secretion were determined for sp (19-42 bubbles at each concentration from 9 pigs). the threshold was ~100 nm for sp and the ec50 was 1.76 ± 0.57 µm. the maximum secretion rate to sp was 1.17 ± 0.21 nl/min.gland, which is only ~ 1/3 of the maximal secretion rate to carbachol (3.43± 0.29 nl/min.gland). the inhibition profile for responses to sp (10 µm) was quite different from those to carbachol (200 nm, a value chose to mimic the secretion rate to 10 µm sp). gland secretory responses to 10 µm sp (0.75 ± 0.17 nl/min.gland, 41 glands from 4 pigs) were strongly inhibited by 20 µm cftrinh-172 (0.34 ± 0.10 nl/min.gland, 36 glands from 4 pigs, p<0.05), and by 25 µm clotrimazole (0.28 ± 0.06 nl/min.gland, 31 glands from 4 pigs), whereas 100 µm niflumic acid did not inhibit (0.81 ± 0.25 nl/min.gland, 32 glands from 3 pigs). in contrast, secretory response to 200 nm carbachol (0.61 ± 0.22 nl/min.gland, 36 glands from 3 pigs) were only weakly inhibited by cftrinh-172 (0.49 ± 0.13 nl/min.gland, 29 glands from 3 pigs, p<0.05), and by clotrimazole (0.53 ± 0.16 nl/min.gland, 33 glands from 3 pigs, p>0.05), but niflumic acid, which was ineffective with sp, inhibited the carbachol response (0.44 ± 0.13 nl/min.gland, 41 glands from 3 pigs, p<0.05). gland secretion to sp depends at least partially on intracellular ca2+ release, because the response was partially inhibited by 50 µm bapta-am (0.33 ± 0.09 nl/min.gland, 21 glands from 2 pigs, p<0.05). there was no additional effect of sp on top of 1 µm carbachol, but a subthreshold level of sp (50 nm) showed synergic response (0.44 ± 0.22 nl/min.gland, 21 glands from 3 pigs) with subthreshold level of vip (10 nm). we are presently testing how sp activates cftr-dependent mucus secretion. in initial experiments to determine if pkc was being recruited, however the pkc inhibitor (gf 109203, sigma) did not suppress sp-induced mucus secretion. in contrast, the nkcc inhibitor bumetanide (100 µm) markedly suppressed secretion to sp (0.22 ± 0.06 nl/min.gland, 23 glands from 2 pigs, p<0.05). supported by niddk ro1-51817 (jjw), cff and cfri. mucus obstruction of airways is considered the most vicious agent of morbidity and mortality in cystic fibrosis (cf). to provide optimal defense of the small airways the volume/thickness of airway surface fluid must be controlled according to physiological demand. to date we have no clear understandings of how these fluids are maintained in a steady state between two pathological extremes (too little or too much) in native small airways. earlier studies have attempted to measure the ion transport properties of small airways of sheep (al-bazzaz et. al. 2001 ) and pigs (ballard et. al. 1992 ), but the complicated branching structure of small airways may have compromised electrical signals. to avoid dissection trauma wang et. al. (2005) examined the electrophysiological properties of undissected intact small airways from pigs in vitro by microperfusing bronchioles (diam.1-2 mm) embedded in the lung parenchyma; however, these studies were limited to luminal manipulations and transbronchiolar electrical potentials only. to more rigorously define small airway properties, we designed a special micro ussing chamber ( area ≈1 mm 2 ). we excised and opened small airways (diam.1-2mm) of pig lung that we mounted as a flat sheet over a pvc supporting filter (10-20 µm holes). both sides of the tissue could then be bathed with different solutions at 37°c. the luminal side was isolated by pressing a pipette (≈1 mm diam., tip fire polished) on to the apical surface of the tissue until it sealed electrically. constant current pulses (0.5-3.0 µa) were passed across the tissue. transepithelial potential (tep) and resistance in bilateral 150 mm nacl ringers were, -1.3 ± 0.1 mv and 83.1 ± 8.1 ω.cm 2 (mean ± se) respectively. when 150 mm naglu ringers replaced the luminal solution, tep increased significantly to -36.7 ± 1.5 mv (n=30, p< 0.0005) and resistance increased to 173.8 ± 21.0 ω.cm 2 (n=30, p< 0.0005. adding forkskolin (fsk, 10 µm) plus ibmx (0.1 mm) hyperpolarized the tep to -40.22 ± 1.99 mv (n=16, p<0.002), but decreased the resistance to 142.9 ± 28.2 ω.cm 2 (n=16, p< 0.0001). luminal amiloride (10 µm) depolarized the tep to -32.6 ± 1.6 mv (n=25, p<0.0001) but significantly increased resistance to 215.7 ± 26.6 ω.cm 2 (n=25, p<0.0005). these results show that this system supports measurements of ion transport properties of airways smaller than 1 mm diameter where cf lung pathology is thought to originate. these airway epithelia express a very high constitutively active clconductance and are apparently capable of secretory as well as absorptive functions. amiloride sensitive na + conductance (≈ 43 ms/cm 2 ) was not significantly affected by fsk added to activate secretion. the observation that stimulation with fsk did not significantly suppress na + conductance (≈ 41 ms/cm 2 ) (p= 0.4) may suggest that separate groups of reabsorptive and secretory cells comprise small airway epithelia. mucins are class of proteins uniquely characterized by large glycosylated domains. these proteins likely play a key role in cystic fibrosis (cf) lung disease as major constituents of mucus. in airways of both humans and mice, the gel-forming secreted mucins are represented by muc5ac and muc5b, and the transmembrane mucins are represented primarily by muc1, muc4, and muc16. we have been interested in the role of the transmembrane mucins in airways and have previously demonstrated that lack of muc1 in a knock-out mouse model (provided by s. gendler) shows increased susceptibility to adenoviral-mediated gene transfer (stonebraker, j. virology, 2004) . to expand upon these findings, mice deficient for muc4 were developed using embryonic stem cell technology by deleting exon 1 of the mouse muc4 gene, which contains the atg start codon and signal peptide. rna analysis reveals loss of exon 1 in homozygous mutant mice and immunohistochemistry using a vntr antibody indicated a loss of full length muc4 protein. muc4 homozygous mice are viable and do not develop any spontaneous, readily detectable disease phenotype. although the muc4 deficient mice do have reduced fertility consisting of both reduced litter size and number, all major tissue systems where the muc4 protein is easily detected, including the eye, respiratory tract, intestine, and reproductive tracts, appear normal in year old mice after histological analyses. in contrast to the results we reported in the muc1 mice, the muc4 deficient mice do not show an increase in adenoviral mediated gene transfer, suggesting that muc4 is not involved in the barrier aspect of the airway glycocalxy, at least for adenoviral infection. interestingly, quantitative real-time pcr analysis revealed up-regulation of muc16 protein in the trachea of the muc4 deficient mice, suggesting that muc16 may be able to compensate for loss of muc4. the expression of muc4 in human ciliated cells as revealed by recent antibody studies prompted us to evaluate the potential role of muc4 in ciliary function. ciliary beat frequency (cbf) was monitored in a shear chamber using microscopy image analysis. cbf under baseline conditions for this study (low shear stress) from muc4 deficient trachea (c57bl/6j congenic n5) was significantly lower than that for littermate controls (8.86 vs. 11.53, sem ±1.31, p = 0.046). application of shear resulted in increased cbf in both groups, although the wt response to shear was smaller in magnitude. thus, muc4 may have a role in supporting basal cbf and may make cbf less vulnerable to stimulation by shear, perhaps by reducing the inherent friction of ciliary motion. this finding underscores the usefulness of this model for dissecting out the normal roles that the transmembrane mucins play in airway biology. supported by nih (hl066973) and cff (r026-cr02). enac (joo et al. jbc 279, 2004 and jbc 281, 2006) . in a search for a model to test the hypothesis we found that ussing chamber short-circuit current(i sc ) measurements with freshly obtained airway mucosa provided useful information about enac activity in response to various drug treatments. in these studies we observed an as yet unidentified pathway that regulates surface enac activity. airway mucosa from sheep tracheas displays a large, amiloride/benzamil-sensitive i sc prior to stimulation. when stimulated with basolateral carbachol, the enac i sc was almost abolished, as indicated by a sustained decrease (77.1 ± 3.4%, n=14) in i sc after an initial increase and left a small effect to subsequent amiloride or benzamil. in contrast, when carbachol was given after benzamil, it again produced a peak response followed by a sustained increase (~30%, n=4) in the i sc . enac inhibition by carbachol was dose dependent with a minimal sensitivity between 0.1~1 µm and was slowly reversible. apical 100 µm atp also inhibited enac i sc by 48.4 ± 8.1% (n=3) and carbachol on top of atp further reduced the enac i sc by 93.6 ± 3.9% (n=3). however, 10 u/ml of apyrase pretreatment, in attempt to breakdown atp, failed to inhibit carbachol-induced enac inhibition, indicating that atp release is not on obligatory component of carbachol inhibition of enac. carbachol inhibition of enac could result from a direct action of carbachol on the surface epithelia, by activation of airway intrinsic neurons, or by activation of the glands, which were intact in these preparations and are strongly stimulated by carbachol. rabbit tracheas, which lack airway submucosal glands, displayed large enac currents and these were not inhibited by cholinergic stimulation (n=3). carbachol-induced enac inhibition, like gland stimulation but unlike stimulation of neurons, was mediated by muscarinic receptors since it was eliminated by atropine but not by the nicotinic receptor antagonist, hexomethonium bromide (n=3). however, inhibition of gland secretion with hepes/bumetanide did not eliminate the ability of carbachol to inhibit enac. the effects of carbachol on surface na + transport in our study are consistent with previous observations in microperfused sheep bronchiole preparations (al-bazzaz, ajp-lung 11, 1994). although we do not yet know the mechanism, it would be physiologically efficient if parasympathetic, cholinergic activation of airway gland secretion was accompanied by enac inhibition, which would minimize the fluid absorption by surface epithelia and increase clearance. it will be important to determine if cholinergic inhibition of enac is present in human tissues, and if so, if it is defective in cf. supported by cff and nih. introduction: lung disease accounts for more than 95% of the current morbidity and mortality associated with cf. the majority of research aiming to elucidate the reason(s) of severe lung disease has focused on airway surface liquid (asl) layer homeostasis or composition. the cftr gene encodes a camp-regulated clchannel located on the apical side of the epithelium and is instrumental in maintaining a hydrated asl layer and promoting effective mucociliary clearance (mcc). in patients with class i or class ii cftr genetic mutations, normal cftr clchannel activity in the epithelium is impaired resulting in na + hyperabsorption and consequently in the generation of a thick and static overlaying asl layer creating an environment that favors progressively worsening bacterial infections. in this study we investigated the potential contributory role of altered respiratory cilia function to inefficient mcc in cf airways allowing for bacterial infestations. our ex vivo model system was the nasal airways of the cftr gene knockout (cftr tm1unc ) mouse in which key aspects of the phenotype of human cf lung disease, such as dehydrated asl layer and goblet hyperplasia, are observed. methods and results: ciliary beat frequency (cbf) was measured using a dual temperature controlled perfusion chamber, differential interference contrast microscopy and high speed digital video analysis system. we evaluated the cbf of nasal septa explants from homozygote cf (affected) and non-affected littermate wild type (wt) and heterozygote mice congenic on the c57bl/6 background. the basal cbf of homozygote cf mice (12±3 hz, n=7) was significantly lower than that of wt mice (28±5 hz, n=7) and also heterozygote mice (19±3 hz, n=17) (p<0.05, anova, snk test). to determine whether the dehydrated asl layer was still present on the surface of the cf mouse nasal airway epithelium and as such impeding normal cilial beating, we fixed nasal septa (n=3) of cf and wt mice immediately following isolation using the pfc/oso 4 fixation method to preserve the fragile asl layer and compared the height of the asl layer to nasal septa (n=3) from cf and wt mice fixed with pfc/oso 4 immediately following cbf analysis. electron microscopy analysis revealed that although the asl layer was present at the time of isolation, there was no asl layer following the perfusion of solutions necessary for the cbf measurements. this finding demonstrated that the activity of cilia was not impaired in the cf nasal airway epithelium due to the presence of a dehydrated asl layer. cbf frequency was also significantly reduced in three different codes of mouse ciliated nasal airway epithelial cultures derived from affected cf mice compared to those derived from non-affected wt mice. conclusion: we show that the absence of the cftr gene product in the cf knockout mouse nasal airway epithelium is associated with significantly decreased basal cbf compared to the non-affected wt and heterozygote mice. this observation potentially provides an additional mechanism as to why lung disease persists in human cf lungs. ml and mba contributed equally. clinical studies have linked increased sputum and peripheral blood neutrophil mpo activity with increased airflow obstruction in cystic fibrosis (cf) patients of the same age, gender, airway bacterial flora, and cftr genotype. variations in the tgf-β1 gene associated with increased tgf-β1 production have been linked to worse airflow obstruction in cf patients of similar age, gender and cftr genotype. we hypothesized that in the presence of mpo, tgf-β1 production would increase in airway epithelial cells. we obtained normal human airway epithelial cells (hae) and cultured them in trans-wells. tgf-β1 mrna was measured by pcr. tgf-β1 protein was detected by immunofluorescence staining and immunoprecipitation. we found increased tgf-β1 mrna after hae were exposed to mpo (at activities found in cf sputum) normalized to mrna of constitutive beta actin. under identical conditions, tgf-β1 protein expression was increased in hae. we conclude that neutrophil mpo present on the apical surface of cultured hae induces transcription and protein synthesis of tgf-β1. these results suggest that mpo induction of airway epithelial tgf-β1 is one molecular system linking the chronic neutrophilic endobronchitis seen in cf and subsequent airway wall fibrosis. are more oxidatively stressed than normal. as oxidative stress (os) is a potent activator of nf-κb, these data supported our hypothesis that cftr dysfunction causes alterations in the expression and/or modification of redox related proteins thereby enhancing activation of nf-κb. delineation of this interaction will produce good therapeutic targets. in this report we examine the role of os in cf inflammation. methods: to address our hypothesis, we used both in vitro and in vivo models of cf. our airway epithelial cell models are the 9hteo-pcep and pcep-r cell pair; and the 16hbeo-sense (s) and antisense (as) cell pair. additionally, we examined whole lungs from 8-12 week old b6.129s6-cftr tm2mrc mice (r117h mutants back-crossed into the c57bl6j background). proteins were prepared from homogenates of whole samples, run on 2-d gels, and their densities compared. the gel spots of differentially expressed proteins were excised, subjected to in-gel trypsin digestion, and identified by lc ms/ms. for biochemical analysis, we tested for the levels of h 2 o 2 , glutathione, and lipid peroxidation. we correlated these data with measurements of nf-κb activity (elisa and cytokine production), and the activity of nrf-2, an antioxidant (ao) response pathway transcription factor. we studied cultured cells in the presence and absence of inflammatory stimulation (tnf-α/il-1β), and compared non-stimulated cf mouse lungs to lungs from normal litter mates. results: our proteomic analysis revealed that in the absence of inflammatory stimulation, a paradoxical decrease in ao proteins exists in cf cells by 2 or more fold compared to matched pairs. this is despite a significant increase in intracellular os, which we confirmed by 3 different biochemical measures. activity of nrf-2 was decreased by ~30% in cf cells vs. normal. the data predict an increase in h 2 o 2 , which is a potent activator of nf-κb, and we confirmed both a significant elevation in h 2 o 2 (up 3-5 fold in cf) and a related significant increase in nf-κb activity, as assessed by elisa for nuclear p50, and il-6 and il-8 production. when we analyzed whole lungs from cf mice for antioxidant protein levels, we similarly observed decreases vs. normal littermates. when cells were stimulated, differences in protein expression and oxidative stress between cf and normal were further enhanced, corresponding to an excessive activation of nf-κb and increased production of il-6 and il-8. treatment with the antioxidants nac or selenium decreased the activation of nf-κb to normal levels. members of the clc family of proteins form either voltage-gated chloride channels or cl -/h + exchangers; nine subtypes are found in mammals. among these, clc-2 is one of the most ubiquitous variants, which is expressed in several of the types of epithelial cells that also express the cystic fibrosis transmembrane conductance regulator (cftr). it has been postulated that clc-2 may be a suitable alternative chloride pathway in these cells; however the contribution of clc-2 to chloride transport in these cells is poorly understood. this is partly due to the lack of appropriate pharmacological tools that are capable of specifically inhibiting clc-2. we describe here the isolation, from venom of the scorpion leiurus quinquestriatus hebraeus, of the first peptide inhibitor of any clc channel. this toxin, which we have named gatx2, specifically inhibits the clc-2 channel. gatx2 bears primary sequence identical to that of a toxin thought to serve as a k + channel inhibitor, although no molecular target for this toxin was previously known. a homology model of gatx2 reveals that it adopts a similar fold to other scorpion toxins. gatx2 was prepared via solid-phase chemical synthesis, and the synthetic toxin inhibits clc-2 with higher affinity than any other inhibitor of clc-2 or any other chloride channel. the k d for gatx2mediated inhibition of clc-2 in multichannel patches is only 11 pm at v m = -100 mv, and 80 pm at -160 mv. single channel experiments show that gatx2 does not alter single channel amplitude, but may alter channel gating. these experiments provide the basis for developing gatx2 into a useful tool that can be used to define the role of clc-2 in airway and intestinal epithelial cells. furthermore, the high affinity and specificity of inhibition suggest that gatx2 interacts intimately with the channel, and thus may the exploitation of alternative chloride channels to bypass the defect in cftr-mediated chloride secretion is an established therapeutic strategy in cf. in search of such potential therapeutic targets, we assessed camp/pkadependent chloride secretion across excised nasal epithelium of cftr null mice and wildtype controls, by measuring short-circuit currents (isc) in an ussing chamber set-up. all experiments were performed in the presence of amiloride to block the contribution of epithelial sodium channels to the isc. in cftr null mice of mixed background (c57bl/6;129), the camp agonist forskolin activated a bumetanide-sensitive rise in isc that was comparable in magnitude to the cftr-mediated isc response of littermate controls (118±30 vs. 90±21 µa/cm 2 , respectively). the forskolin-induced isc in these null mice was inhibited by the general chloride channel inhibitor dpc, but appeared insensitive to the cftr inhibitors cftrinh-172 and glibenclamide, the orcc and calcium-activated chloride channel inhibitor dids, and the clc-2 inhibitors cadmium and pre-activated omeprazol. moreover, the isc response to forskolin greatly exceeded the response to the calciumlinked agonists ionomycin and carbachol. intriguingly, in nasal epithelium of cftr null mice with a different genetic background (fvb), no such forskolin-inducible secretory pathway was evident (cftr-/-: 7±4 vs. cftr+/+: 98±23 µa/cm 2 ). when, in nasal epithelium of cftr null mice (c57bl/6;129), the basolateral membrane electrical resistance was negated by nystatin treatment, and in the presence of a transepithelial chloride gradient, forskolin failed to elicit a rise in isc, suggesting that camp/pka signaling targets a basolaterally located ion transport system. indeed, in intact epithelium, addition of chromanol 293b, a selective inhibitor of camp/pka-activated potassium channels, to the serosal bath, blocked the forskolin-induced rise in isc. we tentatively conclude that the hyperpolarization of nasal epithelial cells, which ensues from camp/pka-induced potassium efflux across the basolateral membrane, drives chloride exit across the apical membrane via a constitutively active anion conductive pathway that is absent from fvb cftr null mice. rt-pcr and immunostaining demonstrated that, among the candidate anion channels that fit the above pharmacological profile, clc-ka, but not clc-kb, was expressed in nasal epithelium of both cftr null mice and controls (c57bl/6;129), together with the accessory protein barttin. importantly, immunostaining suggested a strong increase in clc-k protein abundance in the cftr null mice, as compared to wildtype animals. on the basis of these findings, we postulate that clc-ka channels may serve as cftr bypass channels in nasal epithelium of cftr null mice that, albeit indirectly, are responsive to camp/pka signaling. future studies aim to characterize clc-ka and barttin in the airways from cf patients and controls, and to identify the post-transcriptional mechanism resulting in the up-regulation of clc-ka protein in cftr null mice. in the disease cystic fibrosis (cf), the most common mutation f508del results in endoplasmic reticulum retention of misfolded cf gene proteins (cftr). the endoplasmic reticulum (er) f508del-cftr protein retention is dependent upon chaperone proteins, many of which require ca 2+ for optimal activity. an increase in cytosolic free ca 2+ concentration is dependent on the activity of ion channels in the membrane of er and on extracellular ca 2+ influx. this influx, named capacitative ca2+ entry (cce), induced by calcium store depletion, represents the major ca 2+ influx mechanism in cells (putney et al., 1986) . during the cce, ca 2+ influx can be mediated by one or several of the 7 isoforms of transient receptor potential canonical channels (trpc) (putney et al., 1986) . aims & methods: in the present work, we studied the cce in human cf tracheal gland serous cfkm4 cells compared to non cf human tracheal mm39 cells. for this, we characterized the molecular identity of trpc channels responsible of cce by rt pcr technique and we compared global ca 2+ influx in cf and non cf cells by using fluo4-am probes. moreover, we measured the single channel activity of plasma membrane trpc channels by cellattached patch-clamp configuration in cf and non cf cells. we also studied the cce in cf cells after correction of the abnormal f508del-cftr trafficking by miglustat (2h at 100 µm of n-butyldeoxynojirimycin, nb-dnj) (norez et al., 2006) and low temperature (24h at 27°c) (denning et al., 1992) . results: we detected by rt-pcr technique the presence of trpc1 and trpc6 in both cell lines and we observed that cce was increased 2-fold in cf-km4 cells compare to non-cf mm39 cells. following stimulation by histamine, we recorded elementary ca 2+ currents with similar amplitude (0.5pa at -60mv) and conductance (8ps) whatever the cells tested (cf-km4 or mm39 cells). however, we observed a 2-fold increase of the npo (number of channels in each patch × open probability of single channel) in cf cells compare to non-cf cells. in f508del-cftr corrected-cf cells, the ca 2+ influx stimulated by the ca 2+ stores depletion was reduced to a similar level to non cf mm39 cells. the permanent contact of the airway epithelium with the external milieu induces frequent injuries caused by inhaled pathogens and particles. regeneration of the wounded airway epithelium needs to respect its prior structure in order to restore its defence functions. the underlying molecular mechanisms associated with the regeneration of the human airway epithelium are poorly described. the aim of our study was to determine, using a global expression approach, the transcriptional profile of human airway cells during the epithelial regeneration process. human airway epithelial cells collected from nasal polyps were seeded on type iv collagen-coated porous membranes, cultured up to confluence in liquid-liquid(ll) conditions, then put at the air-liquid interface (ali) allowing epithelial mucociliary differentiation. rna were extracted from epithelial cells at 70% confluence in ll conditions (epithelial cell proliferation and migration stage, step i), after 5 days in ali conditions (beginning of epithelial cell differentiation, step ii), when first ciliated cells appeared (step iii) and when cultures were completely differentiated (step iv). our results demonstrated that between step i and step ii, 127 genes were significantly modulated (ratio r<-3 or r>3, p<0.001), among them 74 were activated and 53 were repressed. between step ii and step iii, 127 transcripts were up-regulated (tubulin, dynein, stath, microtubule associated proteins…) and only one gene (fatty acid desaturase-1) was repressed. finally, between step iii and step iv, except stath which was~12-fold down-regulated, we observed only few genes differentially and slightly modulated. all along the regeneration process, between step i and step iv, 305 genes were significantly modulated with 84% of activated and 16% of repressed genes, among them a family of genes encoding proteins involved in the ciliary differentiation (foxj1, tektin, dynein, tubulin, …), extracellular matrix proteins (collagen, laminin, …) and inflammatory-related genes like cytokines, growth factors and their receptors (ccl20, vegfc, csf3, il32, il13ra2…). by elucidating the specific transcriptome involved in each step of the human airway epithelium regeneration, our study could allow to better understand the different cellular and molecular mechanisms, as well as the chronological events involved in these processes, and will further be applied to the study of the regeneration process in cystic fibrosis airway epithelium. supported by french association vaincre la mucoviscidose. electrolytes may be transported either through cells, the transcellular pathway, or between cells, the paracellular pathway. most research has focused on the channels and transporters of the transcellular pathway, while much less is known about paracellular transport. paracellular transport is regulated by tight junctions. claudins, the main component of tight junctions, are necessary and sufficient for tight junction formation and are important in establishing ion selectivity. the goal of this study was to further investigate the role of tight junctions in human airway epithelia. real-time pcr identified claudins 1, 3, 4, 7, 10, and 16 in primary human airway epithelia. immunocytochemistry localized claudins 1, 3, 4, and 7 to tight junctions. to measure the relative ion selectivity of tight junctions, we studied well-differentiated human airway epithelia in ussing chambers. to minimize transcellular transport, we used cf epithelia, which lack cftr and blocked enac with amiloride. we found the pna/pcl is 1.55 ± .34, suggesting tight juctions are more permeable to cations than anions. the relative cation selectivity was na+ > k+ > cs+ > tma+ > mg2+ and the relative anion selectivity was cl-> br-≥ i-> no3-> gluconate-. we compared these in vitro results to in vivo studies in people with cf and measured nasal voltage in the presence of amiloride. we found the pna/pcl is 1.2 ± 0.1. tight junctions were slightly more selective for cations than anions in vitro and in vivo. to determine the effect of specific claudins on the selectivity of the paracellular pathway, primary human airway epithelia were infected with adenovirus expressing claudin 4 or gfp. epithelia overexpressing claudin 4 had a higher transepithelial resistance (ter) and the pna/pcl was 1.11. however, infection efficiency was only 22%. this lead us to investigate if transformed human airway epithelial cells lines may be a better model to study permeability. cufi cells also express claudins 1, 3, 4, 7, 10, and 16. furthermore, like the primary human airway, cufi cells are more selective for cations; pna/pcl = 1.38 ± .14. future studies will investigate the effect of claudin expression on the paracellular permeability of these cells. the results will help identify the properties of a pathway critical to epithelial transport and barrier functions. altered adenosine (ado) signaling is associated with chronic lung disease and increased asl ado levels are thought to drive inflammation by stimulation of a2b receptors (a2b-r) in airway epithelia, which can activate nfκb. when measured by lavage, asl ado is ≤200 nm. due to its relatively low affinity to ado, a2b-r are thought to be inactive under normal conditions (ec50 is ~1.5 µm), and only stimulated during chronic lung disease. however, we have previously demonstrated that the ado-receptor (ado-r) antagonist 8-spt inhibits asl volume regulation, and that a2b-r is in close proximity to cftr. thus, we hypothesized that the asl ado concentration close to the apical membrane is higher than measured by asl lavage in bulk solution. we tested this hypothesis by looking for ado-r expression and function in well-differentiated human bronchial epithelia cultures (hbecs) and in vivo. by pcr analysis, we found that a2b-r was the only ado-r expressed in hbecs. we then measured asl height over 8 h ± ado-r antagonists by xz confocal microcopy when asl height was preset to ~5 µm at t=0. at 30 µm, caffeine is a non-specific ado-r antagonist, which inhibited asl volume regulation (8 h asl height was 4.1 µm vs. 8.2 µm for controls, n=6). similar inhibition was observed with alloxazine, which is relatively specific for a2b-r (n=6). in contrast, dpcpx, zm241385 & mrs241385, which inhibit a1, a2a & a3-r respectively had no effect on asl volume regulation. asl height regulation was also ablated by the addition of adenosine deaminase (ada) to the asl (n = 6). we next attempted to recapitulate the changes in asl height seen under control conditions, i.e. increase from 5 to 8 µm over the initial 2 h followed by a plateau (the remainder of the 8 h) by adding the non-hydrolyzable ado analogue neca to the asl in the presence of ada. 100 nm neca was insufficient to regulate asl volume and height remained at 5 µm over 8 h. in contrast, addition of 1 µm neca to the asl resulted asl volume regulation that was indistinguishable from control conditions (n=6), suggesting that near-membrane asl is much higher than has been measured by lavage. to confirm that a2b-r stimulates cftr chloride secretion, we measured transepithelial potential differences of hbecs mounted in ussing chambers. a dose-dependent increase in cl-secretion was observed by ado addition with an ec50 of 1.6 +/-0.09 µm, similar to the concentration of neca required to regulate asl height. to demonstrate which ado-r are expressed in human airways in vivo, we used laser capture to obtain ciliated cells from frozen tissue sections of excised donor bronchi. relative expression of a2b-r by qpcr was found to be 4.42 +/-0.30 fold higher than the a1, a2a-r, or a3 receptors (n=3). to confirm that ado-r activate cftr in vivo, we measured nasal potential differences. we found that ado activated chloride secretion at similar levels to isoproterenol. the effect of ado was inhibited by pre-exposure to 30 µm caffeine (ado, 20.2 +/-2.5 mv; ado + caffeine, 5.6 +/-4.9mv). we conclude that a2b-r has an important role in asl homeostasis through regulation of cftr and it is active in human airway epithelia under normal physiological conditions. through osmotic forces, hypertonic saline (hs) may increase the volume of cf airway surface liquid, restore mucus clearance, and improve lung function. the na + channel antagonist amiloride is predicted to increase the duration of action of hs. however, patients taking amiloride with hs showed less clinical benefits than patients taking placebo with hs. in vitro data suggested that amiloride inhibits transepithelial water flux (pf) in cystic fibrosis airway epithelia. we have extended this study by looking at the effects of amiloride on aquaporins in a heterologous expression system (mdck cells) and have used immunofluorescence to determine which aquaporins are expressed in human airways in vivo and in vitro. inhibition of enac by aprotinin pretreatment had no effect on hs-induced pf (n=6), suggesting that amiloride did not exert its actions on pf by inhibiting enac. however, since hs-induced pf was also inhibited with hgcl 2 , we speculated that amiloride directly blocked an aquaporin. to test this hypothesis, we measured the rate of cell shrinkage of wild-type mdck cells, and those stably-expressing aqp1 or 5, or expressing aqp3 (after 24 h exposure to a mildly hypertonic media; 400 mosm). following mucosal exposure to a 450 mosm hypertonic ringer solution, mdck cultures grown to confluency on permeable filters shrunk by 8% after 30 s (n = 6), as measured by xz confocal microscopy following staining with calcein-am. in contrast mdck/aqp1 or mdck/aqp3 cultures shrunk by 20% over the same period and mdck/aqp5 cultures shrunk by 35% (all n=6). a 10 min pretreatment with 200 µm amiloride abolished hypertonic ringer-induced cell shrinkage in mdck/aqp5 cultures (93% at 30 s; n=6) but had no affect on cell shrinkage in aqp1 or aqp3 expressing cultures. amiloride is fluorescent and can be imaged in epithelia by xz confocal scanning under uv excitation. amiloride was not internalized immediately (<10 s) before hypertonic ringer addition and did not block aqp5-mediated cell shrinkage (69% at 30 s; n = 6). however, a 10 min pretreatment with amiloride caused it be internalized and at this time aqp5-induced cell shrinkage was blocked, suggesting that amiloride blocks aqp5 intracellularly. to confirm that aqp5 is present in airway epithelia, we performed immunostaining with an aqp5 antibody and found that aqp5 is highly expressed at the apical membrane of ciliated cells in human bronchial tissue and well-differentiated bronchial cultures. we conclude that amiloride blocks aqp5, which is abundantly expressed in the superficial airway epithelia. supported we have previously observed that cftr, annexin 1 (a1) and cytosolic phospholipase a2 (cpla2) are partially recruited in detergent insoluble microdomains (dim) upon proinflammatory stimulation. this is prevented by specific inhibition of cftr. a1 participates in the regulation of inflammation by inhibiting cpla2 activity. we have also shown that a1 expression is decreased in cftr-/-mouse tissues. we postulate that cftr, a1 and cpla2 participate in the regulation of inflammation by their dynamic interaction within dim. in this study we aimed to (i) demonstrate and characterize this interaction, and (ii) identify other potential partners of this complex in dim. by coimmunoprecipitation, we examined the interaction between cftr, a1 and cpla2 in human pulmonary epithelial cells calu3. to search for additional partners we developed a new proteomic approach based on double sds-page (dsds-page), which is compatible with membrane protein analysis. calu3 cells and human epithelial bronchial cfbe cells (expressing either wt or f508delcftr) were subjected or not to proinflammatory conditions (tnfα). dim were isolated by density gradient and analyzed by dsds-page. cftr immunoprecipitated with a1 and cpla2 in calu3 cells. dsds-page analyses showed significant differential expression of cytokeratin 18, actin and protein disulfide isomerase (pdi, involved in protein folding) in dim between control and proinflammatory conditions in calu3 cells, and between wt and f508del cfbe cells, as determined by mass spectrometry. in conclusion, cftr, a1 and cpla2 interact in basal conditions. proinflammatory treatment causes differential expression of cytoskeletal proteins and pdi depending on the expression of normal and mutated cftr. this work suggests that cftr could participate in the regulation of inflammation within a complex with a1 and cpla2. cytoskeletal proteins and pdi could be involved in the dynamics of this complex. this could be related to the abnormal inflammatory response characteristic of cf. we acute and chronic inflammation, common features of cf airways disease, have been linked to higher atp release and changes in atp metabolic patterns in vivo, e.g., in induced sputum and exhaled breath condensate from cf patients and bronchoalveolar lavage fluid from inflamed lobes (esther, ped pulm sppl 29, 2006) . we, therefore, investigated the contribution of airway epithelia per se to the raised atp concentrations on cf airway surface during inflammation. when exposed to supernatant of mucopurulent material (smm) from cf airways for 48 h, or infected with respiratory syncytial virus (rsv) 72 h prior to the study, basal atp release from well-differentiated primary human bronchial epithelial (hbe) cultures did not differ from that of control cultures. however, basal atp release was increased 6 weeks after rsv infection when inflammation had waned (as indexed by il-8 secretion). the increase in basal atp release correlated with increased basal udp-glucose and mucin release, and goblet cell metaplasia. these data indicate that the increased basal atp release following rsv infection reflected increased goblet cell secretion. a different pattern was observed for stimulated atp release. hbes exposed to smm for 48 h, or infected with rsv 72 h prior, released 2-3 times more atp following hypotonic challenge than control cultures (okada, ped pulm sppl, 2006) . the increase in peak atp concentrations correlated with the degree of inflammation as indexed by il-8 secretion and the increase in intracellular calcium (ca 2+ i ) mobilization in response to hypotonic challenge, but not with the goblet cell number. ca 2+ i mobilization in response to hypotonic challenge in control hbes was almost completely due to extracellular ca 2+ i influx which was inhibited by antagonists of a transient receptor potential v4 (trpv4) ca 2+ channel. in contrast, an additional component of atp-induced ca 2+ i release from endoplasmic reticulum (er) was observed in smm-treated hbes. the increase in hypotonicity-induced atp release in smm-treated hbes compared to control hbes was almost completely sensitive to chelation of ca 2+ i by bapta (10 µm; 1 h), or inhibition of exocytosis by brefeldin a (5 µm; 2.5 h), monensin (10 µm; 18 h) or n-ethylmaleimide (1 mm; 15 min), whereas these reagents were ineffective against hypotonicity-induced atp release from control hbes. these data suggest a positive feedback loop between the increased atp release linked to ca 2+ i -dependent exocytosis and the increase in atp-induced ca 2+ i mobolization linked to increased er ca 2+ i stores (ribeiro, jbc 280, 2005) in inflamed hbes. in conclusion, we propose that basal and hypotonicity-induced atp release from hbes are regulated in distinct mechanisms during inflammation. basal atp release reflects continuous exocytosis processes independent of ca 2+ i mobilization and differs as a function of cell type (ciliated vs goblet cell), whereas acute inflammation upregulates hypotonicity-induced atp release via increased ca 2+ i mobilization and ca 2+ i -dependent exocytosis. we conclude that airway epithelia exhibit multiple mechanisms of increasing atp release into the lumen in response to inflammation. supported by cff grant okada06i0 to sfo and grants from nih. the main biological role of vitamin d is in control of bone and mineral metabolism, but it has also been discovered to have many immune system functions. vitamin d, a fat soluble vitamin, is poorly absorbed in cf leading to low serum levels. we sought to investigate the involvement of vitamin d in the control of inflammation in a cf model. methods cf tracheal epithelial cells (cfte) were pretreated with vitamin d (1, 25 [oh]2d3) or control and exposed to p aeruginosa lipopolysaccharide (lps) for 24 hrs before collecting the cell supernatants. cytokine levels in the supernatants were assessed by cytokine array and subsequent elisas specific for il-6, il-8 and mcp-1. vitamin d was measured in the plasma of 37 children with cystic fibrosis aged 3 months to 18 years. neutrophil numbers, neutrophil elastase (ne) activity and il-8 concentrations were measured in the bronchoalveolar lavage (bal) of these children. children with cystic fibrosis were deemed to be non-colonised, intermittently colonised or chronically colonised with p aeruginosa on the basis of the leeds criteria. pretreatment with vitamin d significantly reduced secretion of il-6, il-8 and mcp-1 from cftes after challenge with p aeruginosa lps in a dose dependant manner. plasma vitamin d levels were highest in the noncolonised group and lowest in the chronically colonised group with intermediate levels in the intermittently colonised group. bal neutrophil counts, ne activity and il-8 concentration showed a reciprocal pattern. conclusions vitamin d acts on cf lung epithelial cells to protect against excessive cytokine production in response to lps. levels of vitamin d are inversely related to both p aeruginosa colonisation status and bal markers of inflammation. low levels of vitamin d may contribute to advancing lung disease in children with cystic fibrosis. more aggressive correction of vitamin d deficiency may have a role in protecting against excessive inflammatory response to infection. sleeping in a mist tent has been used as a treatment for cystic fibrosis (cf) patients, in order to hydrate the viscous mucus and make it easier to be removed by coughing (matthews et al., 1968) . however, the efficiency of the method has been questioned, and its use was largely discontinued. with a new method to measure the ion content of human nasal fluid the effect of sleeping in a moisture tent on the ion content of nasal airway surface liquid (asl) in cf-patients and healthy controls was determined. the cf-patients and healthy controls spent a night (8h) in a mist tent, and samples of the nasal asl were taken before the experiment, after the period in the tent, and then at each hour during 4h after the persons had left the tent. samples of nasal fluid were collected on sephadex g-25 beads that had been mounted on strips of filter paper, which were then put into the nasal cavity of the cf patients or controls for 10-15 min. the strips were removed, and the beads were isolated, dried, and analyzed by x-ray microanalysis (vanthanouvong et al., 2006) . the concentration of na, cl, and k in the nasal fluid of cf patients was 132, 147, and 50 mm, respectively, before the patient entered the tent, significantly higher than the levels in the nasal fluid of the controls (na 118 mm, cl 107 mm, and k 19 mm). during the period in the tent, the ion content decreased, to levels of na 44 mm, cl 88 mm, and k 19 mm (cfpatients) and na 32 mm, cl 45 mm, and k 9 mm (controls) immediately after leaving the tent. after leaving the mist tent the ion levels in the nasal fluid increased, reaching after 4 h values of na 131 mm, cl 176 mm, and k 59 mm (cf-patients) and na 133 mm, cl 157 mm and k 37 mm (controls), which was for both groups higher than before entering the tent. no major changes in the ion content of the asl occurred after 4h. the salt content of the asl may be relevant in cf, since the asl is known to contain anti-bacterial proteins, defensins, which are sensitive to the salt concentration (bals et al., 1998) . hence, the higher salt concentra-tions in the asl of cf-patients may have negative consequences for the anti-bacterial defense system in the lungs, and conversely, the decrease in ion concentrations, caused by spending time in a mist tent, may have positive effects. (we have previously shown that none of our patients was chronically colonized with pseudomonas aeruginosa while regularly sleeping in mist tents.) however, with currently used procedures, the effect of sleeping in a mist tent on the ion content of the nasal asl is short-lived. bals background: antibody microarrays for clinical applications have been anticipated for more than a decade but the technology has only recently become sufficiently mature for reproducible, robust detection of low abundance proteins. the advantage of this new technology is the high throughput, parallel detection of identified, known, low abundance proteins. antibody microarray results are usually given in terms of ratios between two samples (e.g., "experiment" and "control"). however, given the printing and calibration issues inherent with antibody microarrays, this ratio'ing approach precludes statistically valid inter-array comparisons of the individual protein concentrations. we have therefore developed and implemented a different calibration strategy using a benchmark mixture that is applied to every array as an internal standard, thus controlling for any differences in antibody activities as well as printing imperfections. the amount of protein bound to each spot is determined relative to the respective protein in the internal standard. since all samples in the study are compared to the same internal standard, this semi-quantitative approach permits the comparison of multiple samples. all data for the same antibody on multiple arrays can then be used to calculate the average and standard deviation for a given population (such as patients or control groups) in a parallel fashion. methods: cf lung epithelial cultured cells ib3-1 and the cftrrepaired ib3-1/s9 were biosynthetically labeled with a 1 hour pulse of 35 [s]methionine, and then chased with cold methionine for 2, 4 or 6 hours. we then isolated total protein from each sample, and labeled the proteins with cy3. labeled proteins were applied to clontech® antibody arrays (507 antibodies printed in duplicates) according to the manufacturer's protocols. the internal standard, consisting of a mixture of cell cultures and tissue extracts known to contain the cognate protein for each antibody on the array, was labeled with cy5. the data were then used to compare the cf and the corrected cells in terms of amount, biosynthesis rate and degradation half-life (t 1/2 ) for each one of the 500 features on the array. data analysis: for each spot on the array the ratio of cy3/cy5 (after background subtraction) was calculated, normalized and stored for further analysis. for "pulse-chase" experiments the ratio of 35 [s] counts to cy3 at each time point gives a measure of the fraction of newly synthesized (radiolabeled) to total protein (dye labeled) bound to each given antibody on the array. the addition of the cy5 labeled internal standard enables the calibration of these totals, allowing the comparison all the spots for any given antibody on multiple arrays. furthermore, the internal standard calibration makes it possible to compare not only t 1/2 values but also to calculate the relative degradation rates in different cell lines even when the amounts of that protein are different in the "control" and "experiment" cells. the new approach quantitatively identifies and subjects to analysis the effect of the cftr mutation on protein biosynthesis and degradation of the cellular signaling proteome. supported by nih no1-hv-28287 (hbp) and ro1-53051 (hbp) in cf patients, defective apical chloride secretion, due to lack of functional cftr, leads to dehydration of airway surface liquid (asl) and mucus plugging. this defect is likely ameliorated, in part, by an alternative route of chloride conductance, the calcium activated chloride channel (cacc) . female gender appears to be associated with more rapid decline of lung function and earlier death in cf. this suggests that gender differences in hormonal environments might influence asl height regulation, thus modifying the pulmonary phenotype. we hypothesized that changes in estrogen concentrations might alter asl hydration by affecting cacc. consistent with this, we have shown that estrogen attenuates increases in intracellular calcium and asl height that follow purinergic receptor activation in primary cultures of human bronchial epithelial cells (hbecs, see accompanying abstract by hengrui sun et al). here, we report correlations between estrogen levels and nasal ion transport in vivo and further probe hormonal effects on ion transport and asl height regulation in vitro. in vivo studies: in 10 healthy, spontaneously menstruating females, nasal potential difference (npd) was measured (both nostrils) on two occasions during a single monthly cycle. based on a history of the 3 most recent menstrual cycles, we measured npd on a day when estrogen levels were predicted to be low (generally 8-9 days following onset of menses) and subsequently on the day when the pre-ovulatory estrogen surge was predicted. the recording electrode was positioned at the location where maximal stable basal pd was detected and changes in pd recorded continuously during sequential perfusion with 1] ringer's solution, 2] ringer's solution containing amiloride (to inhibit sodium absorption via enac), 3] modified (low chloride) ringer's containing amiloride and finally 4] a similar solution also containing utp (to activate cacc). as predicted, blood estrogen levels were higher on the "pre-ovulatory" day (0.44±0.01 vs 0.10±0.09 nm, p<0.001). utp-stimulated change in pd was significantly lower on the study day when estrogen levels were elevated (13.5±1.77 vs 18.9±0.7 mvs, p<0.05). further, the amiloride-sensitive component of basal pd was higher on this day (14.1±1.7 vs 9.4±2.8 mvs, p<0.05). the change in pd in response to perfusion with a low chloride solution was not different. in vitro studies: expression of estrogen receptor (er) α (but not β) was confirmed in non-cf and cf hbecs by real time pcr. erα was detected in hbecs from males and female donors. in cf and non-cf hbecs mounted in modified ussing chambers, exposure to 10 nm e 2 was associated with a significant (~25%) reduction in utp-stimulated short circuit current. e 2 inhibited the atp-stimulated increase in asl height in male and female hbecs and this asl regulatory phenotype was not reproduced by testosterone or progesterone. in conclusion, estrogen inhibits utp-activated chloride secretion in vivo and in vitro suggesting that elevated pre-ovulatory estrogen levels may further impair mucociliary clearance in females with cf. all data mean±sem. supported by cff leroy matthews award [1] . data demonstrating an upregulation of caccs when cftr is absent or defective [2, 3] have triggered further studies of these channels and prompted further elucidation of the functional (and possibly also physical) interactions between these two channels. although searched for long, the molecular identity of caccs is still under debate. the role of bestrophin family proteins as putative candidates for caccs is discussed controversially. bestrophin 1 (best-1) has been proposed to form ca 2+ activated clchannels in epithelial cells. moreover, caccs have been shown to support cell proliferation, namely in the development of cancer [4] . here, our goal is two-fold: 1) to investigate the correlation between expression levels of best-1 and epithelial cell proliferation; and 2) to pursue a search for interacting protein partners of best-1 to better understand the role and function of bestrophin(s) in epithelial cells. to study the correlation between best-1 expression and cell proliferation, we analyzed two populations of the t84 colonic carcinoma cell line with very different proliferation rates: the original (t84-slow) and the spontaneously transformed t84 cells (t84-fast). we observed that t84-slow cells have a small proliferation rate and express low amounts of best-1, while t84-fast cells express high levels of this protein. best-1 is spontaneously active in t84-fast cells. best-1-rnai inhibited ca 2+ activated clconductance and cell proliferation, therefore establishing a novel role of bestrophins in cell proliferation, which may be of relevance for the regeneration of the epithelia in cf and also for alternative therapies for cf. for the second objective, two putative cytoplasmic domains of best1, polyhistidine -tagged (phis) n-term (aa 1-30, best1-n) and c-term (aa 291-584, best1-c), were cloned into the pet-sumo bacterial expression vector. these domains were then immobilized onto metal-affinity resin to capture interacting proteins from human t84 whole cell and sub-cellular lysates. protein-containing fractions recovered from best1-n/c-coated and blank resins were subjected to 2d-gel and protein identification. it is expected that functional characterization of the interaction between the captured proteins and best-1 will give new insights into the biological role, plausibly of relevance to cf. work supported by dfg-sfb699-a7 grant (germany) and pluriannual funding of cigmh (feder-eu and fct, portugal extracellular nucleotides acting on epithelial cell surface purinergic receptors regulate the mucociliary clearance process that removes noxious materials from the lung. however, how epithelial cells release nucleotides into the airways remains largely unknown. cftr has been suggested to mediate atp release in airway epithelia, but conclusive demonstration remains elusive. calu-3 is a human airway derived cell line often described as a model of airway gland serous cells. at least 70% of cells in a monolayer expressed high levels of cftr protein and activity at the apical membrane. atp, adenosine, and udp-glucose, the naturally occurring agonists for the p2y2, a2b, and p2y14 receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity using radiolabeling and fluorescence hplc-based techniques, and real-time atp measurements. atp (and udp-glucose) was found to be released constitutively and selectively onto the mucosal surface of calu-3 cells. however, the specific cftr inhibitor 172 (10 µm, 10-30 min) failed to decrease atp concentration in the mucosal bath of resting calu-3 cells. forskolin (10 µm, 10 min) stimulated cftr channel activity, but did not affect basal levels of atp (or udpglucose) release. in contrast, elevation of intracellular calcium using ionomycin (1-10 µm, 1-10 min) significantly increased atp (and udp-glucose) release into the mucosal (but not serosal) bath of calu-3 cells. calciummediated nucleotide release enhancement was not abolished by cftr inhibitor 172. maneuvers like pre-incubation in nominally free-calcium solution, bapta (10 µm, 30 min), or cytochalasin d (5µm, 30 min) to disrupt actin cytoskeleton, which inhibited regulated exocytosis in calu-3 cells, also reduced calcium-mediated increase in nucleotide release. calu-3 monolayers presented a fraction of cells (<30%) exhibiting negligible cftr immunostaining signal, but displaying airway goblet cell mucin granules. remarkably, stimulation of atp and udp-sugar release with ionomycin resulted in concomitant enhanced exocytotic secretion of muc5ac. these results suggest that cftr is not involved in regulation of atp release in airway epithelial cells. more likely, nucleotides and nucleotide-sugars residing in the biosynthetic pathway involved in modification of apically targeted glycoconjugates (e.g. mucins) are released to the extracellular environment via calcium-triggered exocytosis. supported although the regulation of na+ transport is relatively well described in normal and cf airway epithelial cells, the impact of inflammation and infection, two major components of cf lung disease, on na+ transport has not been studied extensively. significant amounts of evidence suggest that bacterial by-products may influence na+ transport in airway epithelium. furthermore, the secretion of exoenzyme s by pseudomonas aeruginosa is enhanced during an exacerbation of lung disease in cystic fibrosis. in the past few years, our laboratory has shown that exposition of lung epithelial cells to p. aeruginosa lps leads to a rapid decrease in activity and expression of enac channel. the aim of our study was to evaluate the mechanism involved in the modulation of enac expression and activity by lps. primary culture of lung epithelial cells grown on filters were treated with lps (15µg/ml) for different time (30min, 1, 2, 4h). at each time point, the total and amiloride-sensitive short circuit current (isc) was determined in ussing chamber. in parallel, the cell proteins were collected to investigate by western blot the signalling pathways involved in this response to lps. a 4 hours lps treatment leads to a significant decrease in alpha and gamma enac mrna to 44% and 53% of control respectively. similarly, the amiloridesensitive current was decreased by 53% of the current in untreated cells after a 30 min exposure to lps. by western blot, we demonstrated the strong implication of the pi3k/akt pathway in response to lps in the regulation enac mrna and the potential implication of tyrosine/kinase receptors signalling pathway through mtor in the regulation of enac channel activity. in conclusion, we demonstrate that inflammatory molecules can modulate the expression and function of the na+ transport mechanism in lung epithelial cells and could contribute to the modulation of airway surface fluid in the chronic infectious process associated with cf. serous acinar cells are thought to be crucial for secretion of airway surface liquid by submucosal glands in large airways, but the molecular mechanisms of serous cell ion and fluid transport are not well understood. to address this, we isolated murine nasal serous acinar cells and studied them using simultaneous dic and quantitative fluorescence microscopy to track changes in cell volume and intracellular concentrations of ions involved in fluid secretion ([cl -] i ; spq) and its regulation ([ca 2+ ] i ; fura-2). previous results indicated that serous acinar cell volume changes were indicative of solute efflux (shrinking) and solute influx (swelling), reflecting the secretory state of the cell. acinar cells stimulated with 100 µm carbachol (cch) exhibited a rapid increase in [ca 2+ ] i and subsequent ~ 20 % cell shrinkage due to efflux of ~ 67 % of cell clcontent and parallel loss of k + and osmotically obliged h 2 o. because murine serous cells are sites of apical cftr expression in the airway, we tested the requirement for cftr in cch/ca 2+activated shrinkage/clefflux. acinar cells isolated from either cftr tm1unc-/mice or wt cells pre-treated with cftr inh 172 exhibited identical rates and magnitudes of ca 2+ -induced cell shrinkage and clloss compared to wt control cells. resting [cl -] i was also identical in wt and cftr tm1unc-/serous acinar cells (65 ± 4 and 66 ± 2 mm, respectively), suggesting similar levels of resting clconductance. thus, murine serous acinar cells can secrete kcl/h 2 o by ca 2+ -dependent mechanisms in the absence of functional cftr, in agreement with studies of intact glands suggesting cch-induced glandular fluid secretion is cftr-independent. wt serous acinar cells were stimulated with camp-agonists, including 15 µm forskolin, 100 µm isoproterenol, 200 µm adenosine, and 3 µm vip. however, no camp-induced shrinkage was observed, even in the presence of a cocktail of inhibitors to block solute uptake. to determine if cftr activation could enhance cch/ca 2+ -induced cell shrinkage at a sub-optimal [cch], serous acinar cells were pre-treated with forskolin followed by stimulation with 1 µm cch in the continued presence of forskolin. however, similar rates and magnitudes of cell shrinkage were observed in forskolin-treated and control cells. these results suggest that the magnitude of the cftr clconductance in murine serous acinar cells is much smaller than the ca 2+ -activated clconductance, below the resolution of these optical assays. more sensitive electrophysiological approaches are being used to determine the molecular identity of the non-cftr clconductance(s) and the role of cftr in murine serous acinar cell ion transport. females with cf exhibit more rapid decline in fev1 than males, which reduces their life span. normal airway epithelia regulate airway surface liquid (asl) volume and mucus transport by secretion through both cftr and the ca2+-activated cl-channel (cacc), and inhibition of either of these pathways in isolation does not abolish mucus transport. however, a loss of both pathways is predicted to result in mucus plugging which contributes to lung disease. despite a loss of cftr, cacc is functional in cf airways due to mechanical stimulation of atp release and activation of the p2y2 pathway which serves to raise intracellular ca2+. we hypothesized that e2 may inhibit cacc, leaving females with cf more prone to lung disease than males due to a greater depletion of asl volume. using confocal microscopy, we found that acute pre-exposure with e2 inhibited atp induced asl secretion in normal and cf human bronchial epithelial cultures (hbecs) from both male and female donors. the ic50 for e2-inhibition of atp-induced asl secretion was 0.75 nm. to better understand how estrogen receptors (ers) interact with the p2y2 pathway, we transiently transfected er-α or er-β linked to an orange fluorescent protein in an er null bhk cell line. with the co-transfection of ha tagged p2y2-r, we observed that e2 addition did not induce internalization of p2y2-r, neither did it alter the internalization induced by atp. we then measured total cell inositol phosphates (ip) in cf airway cells +/-e2 pretreatment. cell ip levels were not different between groups (n=9), suggesting that the effect of e2 is downstream of ip production. the investigation of the changes of the intracellular ca2+ (ca2+i) by recording fura-2 emission ratio over time in er-α or er-β expressed cells provided clues. after 10 nm e2 exposure, the fura-2 emission ratio in untransfected cells (n=7) or er-β transfected cells (n=7) increased by 0.6 following 10 um atp addition. this increase was significantly inhibited by~40% in er-α transfected cells (n=7), providing good evidence that er-α is respon-sible for inhibiting the p2y2 pathway. we next measured ca2+i in normal hbecs. while acute 10 nm e2 addition had no affect on basal ca2+i levels, which were around 370 nm, this maneuver reduced the ca2+i response to 70 nm following atp addition (both n=9), suggesting that e2 addition altered ca2+ signaling in response to atp. thapsigargin induces ca2+i release from the endoplasmic reticulum, but the magnitude of ca2+i release after thapsigargin addition was not altered by e2 pretreatment, indicating that e2 works further up stream to affect the p2y2 signaling pathway. together, these results suggest that e2 directly interacts with the p2y2 signaling pathway through er-α by altering ca2+i. we propose that this interaction leaves females more vulnerable to infections due to a reduced asl volume during periods of high e2, such as occur prior to ovulation. the nasal potential difference (or npd) is a sensitive, real time bioelectric assay of cftr-dependent and independent ion transport that is a commonly used study endpoint in early clinical stages of drug development. technical limitations of the assay must be controlled to facilitate its use in multicenter trials and limited intra-site variability. two forms of equipment are commonly employed to evaluate nasal potential difference in humans: silver-silver chloride electrodes (agcl) with saline bridges or calomel electrodes with agar bridges; however, these techniques have never been directly compared. we repeatedly evaluated (within 7 days) the basal potential difference of five normal subjects using the two available techniques, then compared variance and repeatability of the measures. basal pd was measured during perfusion of ringer's solution, and measured at 0.5, 1.0, 1.5, 2.0, and 3.0 cm within the inferior meatus, as indicated in the cf-tdn standard operating procedures. mean basal pd were within 0.8 mv using the two techniques, with very similar coefficients of variation (cvar; 0.36 vs. 0.32 in agcl vs. calomel, respectively). maximum basal pds were also similar between the two methodologies (mean difference 1.5 mv; cvar 0.32 and 0.28, respectively). the correlation between first and second measures of mean pd was highly reproducible, and similar between the two techniques (r=0.94 vs. 0.91 in agcl vs. calomel, respectively); however, when each individual basal pd measure was compared, correlation between first and second pd using the agcl device appeared superior (r=0.90 vs. 0.60 respectively; n=25 measures per device). comparison of values obtained in cf subjects are currently in progress and will also be reported. next, we examined diagnostic npd tracings in 12 cf subjects for whether the effect of amiloride might influence values obtained in the contralateral nare. an effect of cross-contamination by amiloride or other agent (gene therapy vectors or other nasal administered compounds, for example) could influence accurate measure of npd values. when basal pds were performed in both nostrils prior to amiloride exposure in either nostril, mean basal pd between nares remained stable (<1 mv, p=ns for change). however, if the basal pd in one nostril was measured after amiloride exposure in the contralateral side, mean pd depolarized by 12 mv (from -50 to -38 mv; p<0.01), indicating the presence of a 'cross-nostril' amiloride effect that might potentially affect interpretation of this endpoint if not performed in a sequence designed to avoid amiloride exposure. in summary, we show that calomel and agcl electrodes perform similarly in repeated measures from the same normal subjects, and that improper npd sequence can adversely affect basal pd measures. other potential impediments to the pd measurement (e.g. solution temperature, tonicity, ionic content, and sources of electrical offset) will also be discussed. these findings are relevant to the optimizing the use of the npd assay in clinical trials. supported by nih, cff, and cfft. the gene defective in patients with cystic fibrosis (cf) is the cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel in the apical membrane of epithelia, but alteration of expression and function of many genes undoubtedly contributes to the pleiotropic manifestations of cf. a hallmark of cf in airway epithelia is the hyperabsorption of sodium through the epithelial sodium channel (enac) in the apical membrane followed by extrusion through the na,k-atpase in the basolateral membrane. this results in airway surface liquid (asl) dehydration, mucus buildup and bacterial infection characteristically observed in the lungs of cf patients. previous studies have shown that the na,k-atpase is increased in cf airway epithelia, consistent with increased sodium transport along the enac/na,k-atpase axis. we have previously demonstrated that fxyd5, a member of a small family of proteins known to regulate the na,k-atpase, is increased in s489x cf mouse lung and nasal epithelia and in primary human tracheal epithelial cells (hte) after treatment with an inhibitor of cftr (172). recent evidence has indicated that enac activation occurs through modification of the serine protease-protease inhibitor balance in asl. thus, we hypothesized that enac-mediated increases in sodium absorption may be partially responsible for the increase in fxyd5 expression observed in cf airway epithelia. we now report that increasing asl volume on hte cells upregulated fxyd5 expression (p<0.01) after 24 hours, an effect that was further increased after trypsin addition and abrogated by the serine-protease inhibitor aprotinin. treatment of hte cells with nystatin also increased fxyd5 expression measured by quantitative rt-pcr (p<0.01). based on these results and the observation that mice overexpressing enac beta-subunit exhibit cystic fibrosis-like lung pathophysiology, we examined fxyd5 expression in the lungs of mice overexpressing the scnn1b-transgene. contrary to our expectations, fxyd5 lung expression was decreased as assessed by quantitative rt-pcr and immunoblot analysis (p<0.005), indicating fxyd5 may be alternatively regulated in epithelia under conditions of acute versus chronic sodium hyperabsorption. therefore, we measured fxyd5 expression in an epithelial cell culture model using an inducible, tri-cistronic vector capable of expressing enac alpha, beta and gamma subunits. we determined that fxyd5 expression is decreased after chronic enac induction. we conclude that fxyd5 expression is coordinately regulated based on acute versus chronic enac-mediated sodium absorption and suggest that fxyd5 is increased in cystic fibrosis airway epithelia as a result of an imbalance between increased inflammatory signaling and altered ion transport. picher, m.; van heusden, c. cystic fibrosis center, university of north carolina, chapel hill, nc, usa airway infection by the respiratory syncytial virus (rsv) is considered an important cause of pulmonary exacerbation and hospitalization in cystic fibrosis (cf) children. this disease is characterized by functional mutations in the cf transmembrane regulator (cftr). the inability of cftr -/-mice to clear rsv is associated with exaggerated inflammatory responses. a relationship was recently established between airway inflammation and chronically-elevated airway adenosine. this signaling molecule regulates key aspects of lung defenses, including bacterial clearance and inflammatory cells. high adenosine levels measured in bronchoalveolar lavage fluid of asthmatic or cf patients cause airway inflammation, remodeling and fibrosis in animal models. the objective of this study was to investigate the longterm effects of rsv infection on adenosine regulation and inflammatory responses in human airway epithelia. methods. polarized primary cultures of bronchial epithelial cells from healthy subjects and cf patients were exposed to vehicle, uv-inactivated or active green fluorescent protein (gfp)-rsv. the cultures were monitored 21 days for infection (fluorescence microscopy), adenosine levels (fluorescence chromatography), the release of cytokines regulating adenosine tnfα) and enzyme activities (ecto 5'-nucleotidase [ecto 5'-nt: amp ç adenosine] and adenosine deaminase 1 [ada1: adenosine ç inosine]). results. (a) both normal and cf cultures exhibited detectable rsv infection over 21 days, with peak density on day 3-4. (b) we demonstrate that rsv enhances the release of all five cytokines from normal cultures over 21 days. in contrast, cf cultures responded to rsv by a transient decrease in il-1β, il-4 and tnfα (peak on day 3), but a sustained decrease in il-12, and a sustained increase in il-13, secretion. (c) on normal epithelia, rsv transiently stimulated ecto 5'-nt (peak on day 3) but caused a sustained increase in ada1 activity, resulting in chronically-low adenosine levels. in cf, rsv raised adenosine levels by the combined effects of a sustained increase in ecto 5'-nt activity and the lack ada1 response. (d) chronically-elevated adenosine generated by ada1 inhibition [erythro9-(2-hydroxy-3-nonyl)adenine] stimulated the secretion of all cytokines, except il-12. conclusion. these data support a complex relationship between adenosine and cytokine regulation in human airway epithelia. the il-13-adenosine amplification pathways identified in rsv-infected cf epithelia are in agreement with animal models and their key role in chronic airway inflammation/remodeling. on the other hand, the data also suggest that ada1 activity is influenced by il-12, known to stimulate the expression of a major protein anchoring soluble ada1 to epithelial surfaces: cd26. altogether, this study suggests that an rsv infection in cf children may accelerate the progression of the disease by setting the stage for airway adenosine-cytokine amplification pathways. supported by the cystic fibrosis foundation (picher 05g0). cystic fibrosis (cf) airway submucosal glands are defective in mucus secretion rate (jayaraman, pnas, 2001, 98, 8119; joo, j biol chem, 2002, 277, 50710; joo, pediatr. pulmonol., 2003, suppl. 70, 281; wine, proc ats, 2004, 1, 47; thiagarajah, faseb j, 2004, 18, 875; song, ajp cell, 2005; salinas, faseb j, 2005, 19, 431) . one hypothesis is that loss of cftr results in reduced secretion of electrolytes leading to underhydration of mucins, thereby altering the mucus properties. it has been generally believed that the cf mucus in response to cholinergic agonists is thicker and more viscous. however, comparisons of pure gland mucus are difficult because of variability in gland secretion rates and a decline in mucus solids with repeated stimulations (wu, pediatr. pulmonol. 2006, suppl. 29, 252) . in the present study, we directly compared the solid content of airway submucosal gland mucus between control and cf individuals. human donor tracheas (hn) or disease control bronchi (dc) were dissected and mounted as previously described but without mineral oil. tissue was kept warmed and humidified in a chamber with two layers of saturated 37 degree c 95/5% co 2 /o 2 water vapor. the mucosal surface was blotted dry and coated with a thin layer of dimethylpolysiloxane to prevent re-absorption by the surface epithelium. the tissue surface was covered with a cover slip, leaving minimal air space between it and the surface to further reduce evaporation. a 12 µl droplet of 2% nacl was placed at the side of every tissue as a standard to assess evaporation and measurement error. any basal secretions were removed prior to stimulation with 10 µm carbachol. mucus secretion from individual glands was pooled using forceps and then drawn into a constant bore microcapillary (drummond) that had been alcohol-cleaned, dried, and weighed. stimulation time was allowed to vary so that at least 2 µl of mucus was collected. after mucus collection, both the standard and the bath solution were refreshed. mucus filled capillaries were weighed with a mettler microbalance before and after overnight 80 degree c oven drying to obtain nonvolatile solid content. the average solids measured in six repeated measures of 2% salt solutions was 2 ± 0.2 % with no trend over time. results for hn (n=5) and dc (n=3) did not differ and were pooled as 'control'. the initial control secretion yielded 6.5 ± 0.8 % solids (mean ± sem, n=8 subjects) and required 24 ± 3 min to produce adequate mucus. the initial cf secretion gave 9.1 ± 0.5 % solids (n=4) in 31 ± 2 min. the second control secretion yielded 5.6 ± 0.5 % solids (n=8) and required 40 ± 5 min, whereas the second cf secretion gave 7.4 ± 0.9 % solids (n=4) and required 56 ± 7 min. in summary, cf gland mucus solids content was 40% higher than control following the first stimulation (p = 0.05). after the second stimulation, cf solids content was 32% higher than the control (p = 0.08). solids contents of controls observed in our experiment are much higher than was found for mucus collected from airway lumens after 2 hr stimulation (c. 2%, trout, am j physiol, 1998, 274, l258) . supported by niddk ro1-51817 (jjw) and cff. in patients with cystic fibrosis (cf) as well as in some animal models of cf lung disease, higher levels of prostaglandin e2 (pge2) have been detected compared to control subjects. it is not clear whether this excess of pge2 contributes to the airway pathology of cf, or whether it may serve a protective function against airway constriction. inhaled pge2 has been tried in asthma and may be of benefit as a bronchodilator. interestingly, pge2 has shown benefit in aspirin-sensitive asthmatics, a population that shares some features with the cf airway phenotype including nasal polyposis. the mechanism of airway relaxation to pge2 is mediated primarily by ep2 receptors signaling through camp. in cf, beta-adrenergic bronchodilators are commonly used, but are often only partially effective at reversing bronchospasm. beta-agonists and pge2 both signal through g-proteins coupled to camp and pka signaling, thus pge2 may offer another treatment when beta-agonists are ineffective at reversing bronchospasm in cf. in mouse tracheas, the beta-agonist isoproterenol (iso) will partially relax a pre-contracted airway, but 47 ± 2% of contractile force remains (n=13). in contrast to the limited relaxation with iso, constricted mouse airways demonstrate significantly greater relaxation to pge2, with only 18 ± 3% of force remaining (n=9, p<0.001 compared to iso). this difference in relaxation responses suggests different intracellular signals are activated by each agonist despite both signaling through pka. to investigate phosphorylation events in response to iso or pge2, we developed a new method for perfusing isolated mouse lungs. using this technique, tissues were perfused with buffered solution containing 32p for radiolabeling. lungs were then stimulated with either iso or pge2, and phosphoproteins were compared from each condition. radiolabeling was effective under all three conditions. targets were chosen from each group (unstimulated, iso, or pge2 treated) that showed phosphorylation or dephosphorylation, and proteins were identified by mass spectrometry. over 20 differentially phosphorylated proteins were identified, and two of these (rho gdi 1 and inositol-requiring protein 2) were selected as candidates to be tested in the context of relaxation to pge2 compared to iso. by identifying signaling targets involved in airway relaxation to pge2, it may be possible to activate beneficial airway relaxation responses without the limitations seen in iso relaxation and without activating pge2 responses other than relaxation that may link pge2 to the airway pathology seen in cf. the parental ∆f508/∆f508 cf bronchial epithelial cell line (cfbe41o-) was compared to clones of cfbe41o-complemented with 6.2kb wild type (wt) or 4.7kb ∆f508 cftr cdna in terms of their ion transport characteristics. the transepithelial resistance (rt) (~310 (cm2) observed in the parental cfbe41o-was maintained in the complemented cells. two clones complemented with wt cftr cdna (c7-6.2wt and c10-6.2wt) demonstrated rt ~315 ωcm2, while another clone (c4-4.7∆f) complemented with the ∆f508 cftr cdna showed an rt of ~340 ωcm2. two stable clones expressing wt cftr were selected based on electrophysiological characteristics and stability of cftr expression. forskolin-stimulated cl secretion in clone c7-6.2wt was on average 13.4±2.5 µa/cm2 and 41.3±25.3 µa/cm2 in clone c10-6.2wt. furthermore small camp-stimulated transepithelial current of 4.7±0.7 µa/cm2 was observed in clone c4-4.7∆f indicating that ∆f508 cftr can traffic to the plasma membrane if present at high enough levels at physiological temperatures. the role that cftr plays in ca-activated cl conductance was investigated following treatment of the cells with atp in parental cfbe41o-and wtcftr6.2-cfbe41o-monolayers. in symmetrical cl-containing solutions, mucosal application of atp (100 µm) elicited a small, transient cl secretory response in the parental cfbe41ocells (8.5±2.1 µa/cm2), while the magnitude of the atp-stimulated cl current was markedly enhanced in cftr-corrected cfbe41o-cells with peak currents at 29.8±6.4 µa/cm2. recordings done in presence of a serosal-tomucosal cl gradient to increase the driving force for cl secretion showed a transient atp-dependent activation of cl currents in parental cfbe41ocells with peak cl currents of 33.3±3.8 µa/cm2 (n=30). cftr corrected cfbe41o-cells showed a sustained activation of cl currents of similar magnitude (42.7±10.5 µa/cm2; n=16). atp-stimulated cl currents were effectively blocked by the cftr inhibitor glyh101 (20µm). histochemical analysis showed prominent epithelial characteristics in all clones. these observations indicate that i) the apical driving force is limiting for atp-stimulated cl secretion and that ii) cftr participates in the cl secretory response to atp. furthermore, the findings suggest that these novel clonal isolates of the cfbe41o-bronchial epithelial cell line can be useful for the investigation of cf therapies. this work was supported by grants from the cff, cfri, nih, elizabeth nash foundation, and pacfi. the lactoperoxidase (lpo) system functions in normal airways to prevent infection and may be compromised in cystic fibrosis (cf) due to defective transport of its substrate thiocyanate. a computational model of normal airway surface liquid suggests that hydrogen peroxide levels regulate lpo system activity and that the lower thiocyanate in cf airways may result in elevated levels of hydrogen peroxide that is likely produced by duox the major nadph oxidase in airway epithelial cells. to investigate the regulation of duox by bacterial products, passage 1 normal human airway epithelial cells were grown and redifferentiated at the air liquid interface and treated for 12, 24 or 48h with either pseudomonas aeruginosa flagellin or lps, applied apically. treated and mock treated cultures were analyzed for levels of duox and lpo mrna and protein, and for hydrogen peroxide production. quantitative pcr was performed using taqman kits, protein was measured by western blots, and hydrogen peroxide was quantified using amplex red-hrp coupled fluorescence. the data showed the duox 2 and lpo mrna but not duox 1 mrna, were significantly upregulated at all measured time points following apical treatment of the cultures with purified flagellin compared to mock treated. at 24 h duox 2 was increased 7.7 +/-3.8 fold and lpo was increased 2.3 +/-0.7 fold (means +/-s.e.m., n = 5 lungs, 1-3 cultures of each lung). muc5ac mrna was also upregulated by flagellin treatment (4.9 +/-1.6 fold, n = 5 lungs, 1-3 cultures of each lung). apical treatment with lps had no effect on any tested mrna and induced minimal il-8 secretion in these cultures. western blots using anti-duox antibodies (courtesy of f. miot, brussels, belgium) showed corresponding increases in duox protein following flagellin treatments. hydrogen peroxide production by flagellin treated cultures was increased in proportion to increases in duox 2 mrna. application of flagellin alone to the apical surface of previously untreated cultures did not increase hydrogen peroxide production. thus, pseudomonas flagellin up-regulates duox 2 and results in higher activity in normal human airway epithelial cells perhaps serving to increase innate host defense activity of the lpo system. chloride ion in phagolysosomes is an essential substrate for neutrophils to produce hypochlorous acid (hocl), an important bactericidal agent. we have previously shown that cftr was present in the phagolysosomal membrane of neutrophils and the cftr defect led to deficient chlorination of ingested pseudomonas aeruginosa (pa) (painter et al, biochemistry, 45:10260-10269, 2006) . these results suggested that chloride accessibility to the phagolysosomal lumen might be impaired in cf neutrophils thereby affecting neutrophil-mediated bacterial killing. to understand the role of chloride ion in the process, we first assessed the effect of extracellular chloride concentration on pa killing by normal neutrophils under iso-osmotic conditions. surprisingly, bacterial killing was strongly dependent on extracellular chloride concentration. the initial rate of killing of pa was~2 fold less in a chloride-free medium than a physiological chloride medium. killing efficiency increased as the chloride concentration was raised in a dosedependent fashion and plateaued at~60 mm chloride concentration. to determine what percent of the chloride-dependent killing was oxidantdependent, we applied the cftr inhibitor glyh-101, the myeloperoxidase (mpo) inhibitor aminobenzoic acid hydrazide (abah) and the nadph-oxidase inhibitor diphenylene iodium chloride (dpi), respectively to bacterial killing assays. the results showed that the killing of pa could be divided into three discernible components: (1) a component accounting for about 20-25 % of the total killing rate that was inhibited by glyh-101, abah or dpi; (2) a second component amounting to~25-30% that was chloride-dependent and partially sensitive to glyh-101 and; (3) a third accounting for~50% of the total killing activity that was not sensitive to oxidant inhibitors, or extracellular chloride concentration. dpi and abah had no effect on killing of pa in the absence of extracellular chloride suggesting that most of the oxidant-mediated killing activity toward pa was due to hocl. none of the inhibitors had any significant effect on phagocytosis. finally, the rate of pa killing by cf neutrophils was found to be significantly lower (30-40 %) than that seen for normal neutrophils and was comparable to the effect of glyh-101 on killing of pa by normal neutrophils. the data suggest that cftr plays a significant role in killing of pa by normal neutrophils and, when defective as in cf, may compromise the ability of neutrophils to efficiently kill pa. this research was supported by a cff grant to gw. 1%. the cf group demonstrated a significantly higher frequency of pseudomonas respiratory infections than the non-cf group. interestingly, no significant differences were detected in any infections from other systems including blood, sinuses, skin, wounds, oral cavity, bowel, eyes, peritoneal cavity, and urinary tract. moreover, the cf lung-transplant patients had significantly less time free from pseudomonas infections in the transplanted lungs. conclusion normal lungs transplanted into cf patients had significantly higher susceptibility to pseudomonas infections than those transplanted into non-cf patients, which strongly suggests that defective host defense mechanism(s) beyond the respiratory system contribute to cf lung pathogenesis. pyocyanin (n-methyl-1-hydroxyphenazine, pcn) is produced by pseudomonas aeruginosa and is found in pulmonary secretions of infected cf patients at concentrations up to 100 µm. pcn is a redox-active compound that generates superoxide and hydrogen peroxide in the presence of cellular reductants. since cftr was reported to be modulated by h 2 o 2 , we hypothesized that pcn, through its ability to redox cycle and to produce h 2 o 2 , would affect cftr-mediated cl transport. we therefore tested pcn and h 2 o 2 for effects on cltransport (transepithelial short-circuit currents in ussing chambers measured with serosal-to-mucosal clgradient) and cytosolic redox potential (imaging microscopy on cells transfected with a redox sensitive gfp, rogfp1) using cftr-corrected cf bronchial epithelial cells (wtcftr6. . under resting conditions, acute exposure of the apical epithelial surface to 100 µm pcn elicited a small clsecretory response (7.3±0.9 µa/cm 2 , n=10) that was completely blocked by the cftr inhibitor glyh101 (20 µm). in contrast, in presence of the camp-elevating agonist forskolin, pcn caused a time-dependent decline of forskolin-stimulated cftr clcurrents by 86%. exposure to h 2 o 2 (100 µm) elicited a more pronounced clsecretory response than pcn in resting cells and h 2 o 2 -stimulated clcurrents peaked at 47.4±5.8 µa/cm 2 (n=10). h 2 o 2 was less effective than pcn at inhibiting forskolin-stimulated cftr clcurrents and currents declined by 33%. both pcn and h 2 o 2 oxidized the cytosolic redox potential from a resting value of -270 mv by similar amounts (48±8 mv for pcn vs. 44±10 mv for h 2 o 2 ), but rates of oxidation were faster for h 2 o 2 (2.5±0.1 mv/min) than for pcn (1.1±0.2 mv/min). inhibition of camp-stimulated cftr clcurrents by oxidation was a linear function of the cytosolic redox potential, but pcn (2 µa×cm -2 ×mv -1 ) was more potent than h 2 o 2 (0.69 µa×cm -2 ×mv -1 ). cf bronchial epithelial cells homozygous for ∆f508-cftr did not show a clsecretory response to pcn or h 2 o 2 . these data suggest that cftr-corrected cf bronchial epithelial cells respond acutely to oxidative stress by turning on cftr activity probably as part of the innate host defense response and this mechanism is absent in cf airways. furthermore, prolonged exposure to pcn and h 2 o 2 is detrimental for the proper function of the camp-regulated cftr clconductance. we conclude that oxidative stress in p. aeruginosa-infected airways is a key factor that needs to be considered for successfully correcting cftr function in cf airways. supported by nih (nccam p01 at002620), cff (fischer07g0) the lung is continually exposed to environmental agents and pathogens. the lung epithelial lining fluid (elf) is first to encounter these agents and gsh is a major antioxidant found in this apical fluid. the cystic fibrosis transmembrane conductance regulator protein (cftr) is the only known gsh transporter that maintains lung elf gsh levels. cystic fibrosis (cf) patient have low levels of gsh in their elf and have copious and viscous mucus. hypertonic saline is used in cf patient to help clear mucous and improve lung function however the mechanism(s) by which it does so are poorly understood. the purpose of these studies was to examine whether hypertonic (3.0%) saline can modulate apical gsh levels and protect lung epithelial cells against the oxidant, t-butyl hydroperoxide (t-booh). we used human lung epithelial cell lines sufficient (c38) and deficient (ib3) in cftr. the cftr deficient ib3 cells have 40% lower basal levels of apical gsh as compare to cftr sufficient c38 cells. cells were exposed separately and in combination to 3% saline, and t-booh (100 um) for 48 hours. the cftr deficient ib3 cells were much more sensitive to t-booh-mediated oxidative injury as measured by lactate dehydrogenase (ldh) release. hypertonic saline exposure was associated with an increase in apical gsh levels in both cftr sufficient and deficient cells and decreased t-boohmediated oxidative injury in both cells lines. hypertonic saline increased gsh in the apical compartment, which appeared to be largely cftr mediated. we examined this issue in mice given a clinically used dosing regimen of 7% hypertonic saline nebulized twice daily and examined changes in elf gsh levels 12 hours after last treatment. mice receiving the hypertonic saline treatment had significantly higher gsh levels in their elf than untreated controls. this data suggests that cftr and gsh adaptive responses play an important role in lung's reaction to oxidants. we propose that factors, which interfere with the lung's capability to mount and maintain an adequate adaptive apical gsh response, may compromise and sensitize the lung to oxidative injury. (supported in part from nih grant hl075523). the absence of functional cftr in airway epithelia of cf patients leads to abnormal airway surface liquid, which favors chronic bacterial infection. this chronic infection in cf lungs is associated with an exaggerated inflammatory response characterized by abnormal cytokines secretion and massive lung neutrophils infiltration. we have previous recapitulated the abnormal inflammatory response in cftr-/-(cf) mice by repeated administration of pseudomonas aeruginosa lipopolysaccharide (lps) (ped. pulmonology, supp. 29, 2006) . we demonstrated that the bronchoalveolar lavage (bal) fluid of cf and heterozygous (het, cftr+/-) littermate control mice when compared to the bal from wt mice shows 1) higher numbers of neutrophils and 2) higher concentrations of 6 cytokines (il-1alpha, il-6, kc, mcp-1, gm-csf, m-csf) as assessed using multiplex analysis of 22 selected cytokines. of note, airway epithelial cells from cf patients are known to have elevated secretion of some of these cytokines. however to date, the contribution of immune cells to these abnormal cytokine levels has not been well characterized. this study aims to investigate the contribution of cf macrophages in this altered cytokine profile/response. we examined cytokine secretion from two different macrophage populations: alveolar macrophages (am) obtained from the bal fluid and bone marrow (bm)-derived macrophages differentiated in presence of m-csf. both macrophage populations were cultured in the presence or absence of lps, and the supernatants were tested for the concentration of the 6 cytokines that we had shown to be abnormal in vivo. these macrophage populations were examined from cf, het and wt mice. in absence of lps exposure, bm-derived macrophages from cf mice have lower levels of il-6 and kc compared to wt and het mice. in contrast, am of cf mice have higher levels of il-6 and kc than am of wt mice. this difference may be due to am's prior exposure to antigens in vivo whereas the bm-derived macrophages are a truly naïve population. interestingly, after lps stimulation, il-1a, il-6, mcp-1, kc, g-csf and gm-csf are higher in cf macrophages compare to wt and het mice suggesting that cf affected macrophages display an altered inflammatory response when compared to wt and het. the abnormal production of these cytokines was also detected at the transcriptional level using quantitative rt-pcr analysis. in conclusion, our data support the hypothesis that cftr-null macrophages may have an impaired cytokine profile at baseline as well as after stimulation. furthermore, cftr deficient macrophages may contribute directly to the abnormal inflammatory response in cf. (supported by cff, nih, niddk) cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx-890 is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx-890 effectively inhibits ne activity in cf sputum sol (ic50 = 766 pm). however the physical barriers posed by cf whole sputum may prevent dx-890 accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. the lipid portion of pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles) increases the fluidity of whole sputum. ( aim we aimed to show that, by reducing the surface tension of sputum, bles would enhance the anti-elastase effect of dx-890 in whole sputum. methods whole sputum (0.2g per well in a 12-well plate) was pre-incubated with 20 µl saline as control, 20 µl dnase (100µg/ml) or 20 µl bovine lung extract surfactant (bles, 100 µg/ml) for 30 minutes at 37 °c followed by addition of 20 µl dx-890 (50 µm or 100 µm) for 1 h at 37 °c. the contents of each well were aspirated, added to 1 ml nacl (0.9%) and centrifuged at 10,000 g for 30 min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by 50 µm dx-890 (p=0.0071) and 100 µm dx-890 (p=0.0009). 50 µm dx-80 inhibited significantly more ne when sputum was pre-treated with bles (p=0.0173) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p=0.0398) whereas treatment of sputum with bles did not have this effect. conclusion dx-890 effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles enhanced the anti-elastase effect of dx-890 and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx-890 inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. bovine lung extract surfactant, bles™ was a kind gift from bles biochemicals inc., on, canada. dx-890 was a kind gift from dyax corp, ma who also funded part of this research. table 1 : results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n=27 experiments. cystic fibrosis lung disease is associated with an excess of free neutrophil elastase (ne) in the airway arising from persistent neutrophil inflammation. dx-890 is a potent small protein inhibitor of neutrophil elastase that could be a useful therapy for diseases involving neutrophilic inflammation such as cystic fibrosis and copd. dx-890 effectively inhibits ne activity in cf sputum sol (ic50 = 766 pm). however the physical barriers posed by cf whole sputum may prevent dx-890 accessing and inhibiting ne ensconced within the mucus. the mucolytic dnase acts by cleaving extracellular dna in mucus thereby reducing viscosity. however dna binds and inactivates ne and therefore dnase treatment of mucus results in an acute increase in active ne. pulmonary surfactant acts as a lubricant and our group has previously shown that bovine lung extract surfactant (bles™) increases the fluidity of whole sputum. ( we aimed to show that, by reducing the surface tension of sputum, bles™ would enhance the anti-elastase effect of dx-890 in whole sputum. methods whole sputum (0.2 g per well in a 12-well plate) was pre-incubated with 20 µl saline as control, 20 µl dnase (100 µg/ml) or 20 µl bovine lung extract surfactant (bles™, 100 µg/ml) for 30 minutes at 37 °c followed by addition of 20 µl dx-890 (50 µm or 100 µm) for 1 h at 37 °c. the contents of each well were aspirated, added to 1 ml nacl (0.9%) and centrifuged at 10,000 g for 30 min. the resulting sputum sol was assayed for ne activity using the colorimetric substrate n-meosuc-aapv-pna. results ne activity in whole sputum was significantly inhibited by 50 µm dx-890 (p=0.0071) and 100 µm dx-890 (p=0.0009). 50 µm dx-80 inhibited significantly more ne when sputum was pre-treated with bles™ (p=0.0173) treatment of sputum with dnase caused a significant increase in ne activity compared to untreated sputum (p=0.0398) whereas treatment of sputum with bles did not have this effect. conclusion dx-890 effectively inhibits elastase in cf sol. pre-treatment of cf whole sputum with bles™ enhanced the anti-elastase effect of dx-890 and treatment of sputum with bles alone did not cause an increase in ne activity in contrast to treatment with dnase. dx-890 inhibitory capacity in whole sputum could be improved by co-treatment with surfactant which increases fluidity of sputum and could allow greater access of drugs to sputum components. we would like to thank prof. f. possmayer, on, canada and bles biochemicals inc., on, canada for the bovine lung extract surfactant, bles™. dx-890 was a kind gift from dyax corp, ma who also funded part of this research. table 1 results are summarised as neutrophil elastase activity µg/ml and show the mean and sem of n=27 experiments. cf patients infected with p. aeruginosa (pa) have increased levels of proinflammatory cytokines, such as tnf-α, il-1β, il-6, and il-8; but have low levels of the anti-inflammatory cytokine, il-10 in their airways. the proinflammatory mediators in the cf airway recruit a large number of polymorphonuclear neukocytes (pmns), which release high levels of neutrophil elastase and human neutrophil peptides and damage the lungs. the intense neutrophil-dominated inflammatory response associated with chronic pa infection results in progressive airway obstruction, the cause of death of over three-quarters of cf patients. recent studies highlight the significance of sphingolipid metabolism in cell growth, differentiation, membrane traffic, apoptosis, senescence, and immune regulation. acid sphingomyelinase (asm) plays a critical role in sphingolipid metabolism by hydrolyzing sphingomyelin to ceramide. ceramide self-associates to form ceramide-enriched membrane platforms(also called lipid rafts). these platforms have been shown to be involved in the internalization of pa; induction of apoptosis and immune regulation. our in vitro and in vivo studies of asm confirm that there is a loss of induction of asm after pa infection in both cf bronchial epithelial cells and cftr knock out mice as compared with controls. in vitro, we examined the asm activity in human bronchial epithelial cells with normal cftr expression (s9 cells) and with mutant cftr expression (ib3 cells ) after pa infection at 0, 10, 30, 60, 180, and 300 min. asm activity was up-regulated in s9 cells following pa infection after 60 min and kept increasing until 300 min, while asm induction was lost in ib3 cells. asm protein and ceramide levels also increased in s9 cells following pa infection compared with ib3 cells. in our in vivo studies, cftr knock out mice and control c57bl/6 mice were both administered various doses of pa orally (2x106 cfu to 5x107 cfu) . six hours after pa infection, lung homogenates from the c57bl/6 mice indicated increasing asm protein and ceramide production, which corresponded to the dose of pa given. in contrast, asm protein and total ceramide production remained at baseline levels in the lung homogenates of cf mice regardless of the dose of pa given. rna interference (sirna) assays were performed by transfecting a 21 bp pre-designed sirna specific for human asm in s9 cells. these cells were then infected with pa for 5 hrs to see if they had similar il-8 level compared with infected ib3 cells. we demonstrated a significantly elevated il-8 expression in s9 cells tranfected with asm sirna compare with s9 cells transfected with a scramble sirna (p<0.005). our results suggest that the loss of asm induction, caused by mutant cftr, is involved in pa clearance and contributes to the exaggerated proinflammatory cytokine response in cf airway. we are conducting research on defining the roles of asm and the sphingolipids in pa infection; cell signaling; as well as cytokine induction, we are also looking at the therapeutic potential of aav.asm vectors in pa-infected cf bronchial epithelial cells and cftr knock out mice. work supported by nhlbi introduction: new therapies are critically needed to treat cystic fibrosis (cf) lung disease, which is characterized by chronic infection and inflammation. novel therapies to target airway inflammation are likely to improve the clinical outcome in cf. as yet there is no consensus regarding the molecular pathway(s) mediating inflammation in the cf lung. objective: to establish whether toll-like receptor (tlr) signalling is the key molecular pathway responsible for the inflammation seen in cf lung disease and to determine whether inhibitors of tlr signalling can reduce this damaging inflammatory response. methods and results: to quantify the airway inflammatory response, we used established cf and cf-corrected lung epithelial cells (ib3-1 and c38). since artefacts may alter the inflammatory phenotype of cell lines, we validated all findings with fresh peripheral blood mononuclear cells (pbmcs) from cf patients (n=23) and healthy controls. we measured the inflammatory response (il6, il8, tnfα) of these cells following stimulation with: (i) cf pathogens (p. aeurginosa, b. multivorans, b. cenocepacia); (ii) purified tlr ligands. both cf respiratory epithelial cells and cf pbmcs had a greatly increased inflammatory response compared to matched controls when stimulated with whole bacteria and purified tlr ligands (p<0.05). interestingly, the cf airway cells responded most vigorously to the tlr5 ligand, flagellin (p<0.001). to investigate tlr5 as a potential anti-inflammatory target, we found that tlr5 mrna (p<0.001) and protein (p<0.05) were expressed at a higher level in cf compared to cf-corrected lung epithelial cells. finally, to determine the contribution of tlr5 activation to cf lung inflammation we exposed the airway cells to wild-type (pak & pao1) and isogenic flagellin-deficient strains of p. aeruginosa (pak∆flic, pao1∆flic, pao1∆flge & pao1∆flim) . strikingly, the absence of the tlr5-flagellin interaction significantly reduced the proinflammatory response of cf respiratory epithelial cells to p. aeruginosa (p<0.001). we show that tlr-mediated innate immune responsiveness is increased in both cf respiratory epithelial cells and fresh blood cells from cf patients. moreover, inhibition of the tlr5-flagellin interaction markedly reduced proinflammatory response of cf respiratory epithelial cells following exposure to predominant cf pathogen p. aeruginosa. these data suggest that tlr5 activation may represent a novel anti-inflammatory target for improving cf lung disease. supported by the canadian cf foundation. cf airway cells (ib3-1) were stimulated with wild-type (pak) and flagellin-deficient p. aeruginosa (pak∆flic). introduction: genetic polymorphisms for tgf-β 1 have recently been identified as a significant modifier of cf lung disease severity. however, at present little is known about protein measurements of tgf-β 1 in bronchoalveolar lavage fluid (balf) or serum obtained from pediatric cf patients during an acute respiratory exacerbation. hypothesis: tgf-β 1 in cf balf and serum is increased in association with markers of cf lung disease severity, and is decreased after completion of iv antibiotic (iv abx) therapy. study design and methods: in a descriptive, prospective study of children with cf presenting for hospital admission to treat a pulmonary exacerbation, balf specimens were obtained from patients undergoing clinically-indicated flexible bronchoscopy on admission. balf was immediately analyzed for total and differential cell counts with the cell-free balf supernatant stored at -80°c. serum specimens were obtained at the initiation and conclusion of iv abx and similarly stored at -80°c. measurement of total tgf-β 1 utilized a commercial elisa kit. t-test statistical analysis assumed significance at p<.05. results: balf was obtained from 8 cf patients with a mean age of 8.5±1.9 years (range 1-16 years). three balf cultures were pseudomonas aeruginosa positive (psa+). balf tgf-β 1 levels ranged from 88-309 pg/ml (mean 168±26.4 pg/ml) with the highest tgf-β 1 measured in a patient with abpa and culture positive for m. abscessus. in balf, higher neutrophil counts (greater than the median, 2.58x10 6 pmn/ml) were associated with increased balf tgf-β 1 (214±31.9 pg/ml vs. 121±28.8 pg/ml, p<.05). serum tgf-β 1 was collected in 8 cf patients with a mean age of 7.5±2.3 years (range 1-18 years), four of whom were psa+. serum tgf-β 1 ranged from 21.4-99.7 ng/ml (mean 56.6±9.23 ng/ml). in both balf and serum, neither patient age nor psa culture status was associated with increased tgf-β 1 at admission. however, previous hospitalization in the last 12 months was significantly associated with an elevated admission tgf-β 1 in both balf (233±38.2 pg/ml vs. 122±23.8 pg/ml, p<.05) and serum (75.8±8.19 ng/ml vs. 37.3±9.09 ng/ml, p<.02). after completing the clinically-indicated course of iv abx, 3/4 psa-cf patients had a reduction in serum tgf-β 1 (mean 69.5±11.4 ng/ml to 48.1±4.52 ng/ml, p=ns). in contrast, 3/4 psa+ cf patients had an increase in serum tgf-β 1 (mean 43.7±12.5 ng/ml to 62.3±17.1 ng/ml, p=ns) after completing iv abx therapy. conclusions: tgf-β 1 is measurable in balf and serum from pediatric cf patients and varies in association with clinical parameters. these preliminary results support our hypothesis that increased tgf-β 1 may be associated with more severe lung disease as evidenced by 1) association of increased tgf-β 1 in balf and serum obtained from cf patients with previous hospitalization in the last 12 months, and 2) association of increased tgf-β 1 in balf with increased cellular inflammation. the direction of change in serum tgf-β 1 after iv abx therapy may be influenced by the presence or absence of pseudomonas infection. acknowledgments: supported by cff (harris07ao). paula murphy and cassidy henegar provided technical support. background: cystic fibrosis is one of the leading genetic causes of end stage lung disease for which the treatment is lung transplantation. however, mortality due to chronic rejection known as obliterative bronchiolitis (ob) remains high due to small airway obliteration. ob is associated with increased levels of matrix metalloproteinase-8 (mmp-8), an interstitial collagenase, expressed by polymorphonuclear cells (pmns). pmns are among the first inflammatory cells to be detected in ob but very little is known about the role of pmn mmp-8 or the mechanisms used by pmns to migrate through extracellular collagen matrices. objective: to identify the molecular mechanism for neutrophil recruitment into the airway. we hypothesize that mmp-8 promotes accumulation of pmns in ob lesions by promoting their migration through the extracellular matrix protein barriers by its pericellular collagenase activity. method: the heterotopic airway transplant model was used to study the role of neutrophil mmp-8 in promoting pmn migration through extracellular matrices. mhc-disparate balb/c tracheas were subcutaneously transplanted into c57bl/6 wild-type (wt) or mmp-8 deficient (mmp-8-/-) mice. to further dissect the mechanism of pmn migration in vitro their migration through collagen gels was studied. collagen gels were made in transwells using rat-tail collagen providing a highly cross-linked thick collagen barrier. bone marrow derived pmns from wt or mmp8 -/-mice were fluorescently labeled and stimulated to induce the surface expression of mmp-8. pmns were placed on thick collagen gels in traswells. the transwells had polycarbonate membranes at the bottom of the gels. the pore size of the membranes did not allow pmns to pass through into the lower chambers that had flmp a chemo attractant. at 4 hours and 24 hours the gels were fixed and the polycarbonate membrane at the bottom of the collagen gel was gently peeled off. pmns on the membrane were counted under a fluorescent microscope. results: significantly decreased migration of pmns into the airway lumen was noted in the mmp-8 deficient mice (p<0.05) 2 weeks post-transplantation compared to wt mice in the heterotopic airway transplant model. in addition there was more than a three-fold increase in the pmns outside the lumen in the extracellular collagen matix (p<0.02). wt-pmns were able to penetrate collagen barriers in greater numbers both at 4 and 24 hours compared to mmp8 -/-pmns in vitro (p < 0.002 for 4 hours and 0.001 for 24 hours). in addition, mmp inhibitor gm 6001-treated wt cells demonstrated decreased migration through collagen gels (4 hours p<0.008 and 24 hours p<0.008). conclusions: mmp-8 -/-mice has significantly less pmns in the airway lumen due to decreased migration through collagen matrices providing protection against ob. this has great significance for conditions such as cystic fibrosis, lung transplant rejection (obliterative bronchiolitis) and other inflammatory diseases mediated by pmns. mmp-8 blockade can potentially be a novel therapeutic target to decrease neutrophil efflux into the airways and thus reduce airway inflammation. background: our laboratory has demonstrated alterations in cf-related regulation of cholesterol processing. hydroxycholesterols (oxysterols) are oxygenated derivatives of cholesterol formed from autoxidation of cholesterol and from de novo synthesis. oxysterols are known to be key regulators of cholesterol metabolism and cellular signaling. the role of oxysterols in the cellular pathways involved in cholesterol processing in cystic fibrosis has not been elucidated. based on our previous findings of increased de novo cholesterol synthesis in cf tissues, we hypothesize that oxysterol production in cf would be positively impacted. increased oxysterol production may lead to abnormalities in cellular signaling associated with oxidative stress and may influence the inflammatory response in cf. the goals of this research are to investigate the effect of oxysterols on inflammation in cf and to determine if there is a difference in oxysterol production in cf compared to wild type controls. results: the goal of these studies is to determine if cf cells respond uniquely to oxysterols compared to control cells. initial studies demonstrate that cf-model 9/hteo-pcepr cells failed to respond to 25-oh cholesterol (25-oh) with regards to nfκb activation. control 9/hteo-pcep cells, however, exhibited a significant 2-fold increase in nfκb activity upon 25-oh stimulation (2.1-fold +/-0.1 increase from il-1, tnf alpha alone). however, il-8 promoter activation in response to 25-oh was not significantly altered in either cf or wt cells (2.5-fold +/-2.3 (cf) increase from il-1, tnf alpha alone, 1.8-fold +/-2.3 (wt) increase from il-1, tnf alpha alone). in order to verify il-8 promoter results, il-8 protein production was measured directly in response to pseudomonas aeruginosa. il-8 protein level in response to 25-oh was increased in cf cells (1.4-fold) and decreased in wt cells (1.2-fold), although not significantly in either group. oxysterol content and de novo synthesis are currently being examined in vivo in cf and wild type mouse models. mice lacking cftr on a c57bl/6 background and wild type controls were obtained from our animal core and the amount of oxysterol present in these mouse models is currently being studied. conclusion: these findings suggest that endogenous oxysterol production or other components of oxidative stress are pre-stimulating nfκb activation via the oxysterol-sensitive pathway. interestingly, although oxysterols robustly activate nfκb in wt cells, oxysterol-dependent pathways are not involved in il-8 production. these data suggest that il-8 is not a primary cause for increased oxysterol mediated inflammation. elucidating the role of oxysterols in cf will be an important contribution to our understanding of the inflammatory pathways in cf and will contribute to the growing body of evidence for aberrant cholesterol regulation in cf. this work is supported by grants from the cff and the nhlbi. cystic fibrosis (cf) is a common genetic disorder caused by a mutation in the cftr, a chloride transporter of the abc transporter family. innate immunity, the first line in host defense against infectious agents, is abnormal in cf patients. cf patients are commonly colonized with bacterial pathogens, which is the major clinical problem of this disease. despite a marked difference in the clinical outcome amongst cf patients infected with pseudomonas aeruginosa and burkholderia cepacia, the mechanisms under-lying these differences remain unclear. understanding the innate immune response, specifically the role of cftr, in p. aeruginosa and b. cepacia infections will lead to new insights into mechanisms that limit these infections. our objective is to develop a cf model in the simple organism, the worm, c.elegans, and to test the role of the cftr orthologue, mrp-4, in host defense. using this model, we have compared the survival of c. elegans between virulent p.aeruginosa (pa14) and b.cepacia (bc). our experiments have demonstrated that, when compared to growth on their natural food source, e.coli op50, worms fed on pa14 and bc die prematurely. specifically, worms survive up to 140 hours on op50, whereas pa14 fed worms die with in 40 hours. death on pa14 also requires active infection, as heat inactivation of these bacteria destroys their killing capacity. two bc strains have been tested, a virulent strain (25416) and an avirulent strain (17616). worms infected with bc show an intermediate phenotype, surviving approximately 100 hours. our future work will define whether synergy occurs during coinfection with p.aeruginosa and b.cepacia, as would be suggested by clinical data. in addition, we will define the transcriptional changes induced in c.elegans fed on pa14 and bc, and use these data to identify common and pathogen specific responses to these infections. finally, mrp-4 knock-down worms will be generated using rna interference, and the role of the cftr orthologue in infection will be tested. in conclusion, c.elegans offers a genetically tractable model organism in which the innate immune response to pseudomonas aeruginosa and burkholderia cepacia can be studied. in addition, this simple model system will allow us to define the role of the cftr orthologue in host defense. supported serine proteases released from neutrophils are considered central to the pathogenesis of cf lung disease, and have been obvious therapeutic targets. although intracellular serine proteases are important in host defense and bacterial killing, extracellular enzymes may contribute to bacterial persistence and promote airway inflammation. neutrophil elastase (ne) digests key opsonins present in the lung and has been shown to disrupt phagocytosis, allowing the bacteria to persist in the face of established pulmonary inflammation. in addition we have found that other proteases, like cathepsin g (cg), an abundant serine protease found in human and murine neutrophils, may also have other, critical roles in development of cf lung disease. indeed, using murine models of endobronchial inflammation, cg inhibits airway defenses and interferes with the host's ability to clear pseudomonas aeruginosa from the lung with effects quite distinct from ne. to test the hypothesis that differences in bacterial killing are due to defects in innate defenses created by excess, unopposed cg at the apical surface of the airway epithelium, we have examined profiles of proteins secreted into the airway lumen by epithelial cells, which are necessary for p. aeruginosa clearance and susceptible to cg proteolysis. analysis of lavage fluid utilizing 2-d page indicated a total of 88 spots which were increased in pooled lavage fluid from 6 infected cg-deficient mice as compared to 6 wild-type littermates. tandem mass spectrometry analysis identified several novel proteins that were cleaved by cg, including inter-alpha inhibitor protein, selenium-binding protein, sec14-like protein, beta-2-glycoprotein, and annexin a5. another protein candidate, serum amyloid p component (sap), is a novel opsonin and may be involved in airway defenses. immunoblot analy-sis demonstrates that cg cleaves sap. these results suggest that cg, like ne, plays an important role in the pathogenesis of lung diseases. we have previously demonstrated in ∆f508-cf mice that in vivo treatment with azithromycin (azm) attenuates cellular infiltration in baseline and lipopolysaccharide (lps)-induced inflammation, and inhibits proinflammatory cytokine release in induced inflammatory condition (legssyer et al, 2006) . this study aimed at investigating macrophages isolated from wild type (wt) or ∆f508-cf mice (van doorninck et al, 1995) and the effect of azm on these cells. purified peritoneal and alveolar macrophages were stimulated with lps (p. aeruginosa, 0.1µg/ml) plus ifnγ (0.1µg/ml), with or without azm (1µg/ml). macrophage inflammatory status was investigated by assessing different pro-and anti-inflammatory mediators at mrna level 6h after stimulation. pro and anti-inflammatory markers seemed to be reduced in naive alveolar cf macrophages. by contrast, a trend to overexpression of these markers was seen in naive peritoneal cf cells. pro-inflammatory status, as assessed by il-1β, was induced in stimulated alveolar and peritoneal cf macrophages and in naive peritoneal cf cells. moreover, nos-2 was found to be overexpressed in stimulated alveolar cf macrophages while il-10 was downregulated. azm significantly reduced il-1β expression in stimulated alveolar macrophages. in conclusion, peritoneal and alveolar macrophages exhibit distinct phenotype. a pro-inflammatory status was more prominent in stimulated cf alveolar and peritoneal cells. interestingly, azm modulates il-1β overexpression only in cf stimulated alveolar macrophages, supporting the antiinflammatory activity of this macrolide and identifying, at least partly, alveolar macrophages as possible target cells for its effects. supported it has been recently reported by perez and coll. that the inhibition of function of w/t cftr produces an inflammatory profile that resembles that observed in cf patients [am j physiol lung cell mol physiol, 2007] , whereas we demonstrated that correction of f508del cftr function with mpb-07 down modulates the pseudomonas aeruginosa (p.aeruginosa) dependent expression of the pro-inflammatory mediators il-8 and icam-1 in cf bronchial cells [am. j. resp. cell. mol biology, 2007] . since both evidence support a direct link between cftr function and inflammatory response in respiratory epithelial cells, we extended our investigation to other f508del cftr correctors, such as miglustat [norez et al, febs letters, 2006] , which is an approved drug for gaucher disease, in comparison with an isomer without any correcting effect, namely nb-dgj. human bronchial ib3-1 (cftr f508del/w1282x), cufi-1 (cftr f508del/f508del) and nuli-1 (cftr w/t) cells were exposed to the laboratory strain of p.aeruginosa (pao1) or tnf-alpha or il-1beta and the transcription of icam-1 and il-8 were quantitated by real time rt pcr. cftr function was assayed by single-cell fluorescence imaging, using the potential-sensitive probe disbac2 [renier et al, hum. gene ther. 1994 ]. analysis of binding of nf-kb and ap-1 transcription factors to labelled dna target was performed with electrophoretic mobility shift assay (emsa) [borgatti et al, j. biol. chem. 2003 ]. miglustat significantly reduced the expression of il-8 by 90% in ib3-1 and by 70% in cufi-1 cells and of icam-1 by 80% in ib3-1 and by 50% in cufi-1 cells, upon infection by pao1. in parallel, correction of f508del cftr function was observed after miglustat treatment in both ib3-1 and cufi-1 cells. miglustat had no major effects on overall binding activity of transcription factors nf-kb and ap-1, activated by pao1 in ib3-1cells. the inflammatory response induced by tnf-alpha or il-1beta was also significantly reduced in ib3-1 and cufi-1 cells treated with miglustat. nb-dgj, which is not a corrector of function of f508del cftr, down modulated the induction of il-8 and icam-1 in ib3-1 and cufi-1 cells. in addition, both miglustat and nb-dgj reduced the inflammatory response to pao1 in non cf nuli-1 cells. in conclusion, miglustat and nb-dgj have an anti-inflammatory effect in bronchial cells independently of the correction of f508del cftr, by interfering with the pro-inflammatory signaling downstream the receptors for pathogens and pro-inflammatory cytokines. since miglustat is already approved for the treatment of gaucher disease and other glycosphingolipidoses, it may be an interesting candidate to ameliorate lung inflammation in cf patients. background: the primary cause of mortality and morbidity in cystic fibrosis (cf) is lung disease, which is characterized as a chronic cycle of infection and inflammation dominated by a neutrophilic infiltrate and increased levels of circulating mediators such as il-8. a wide variation in the rate of progression of lung disease is observed in cf. hypothesis: we postulate the presence of a brisk systemic inflammatory response in cf patients and that this systemic response reflects the magnitude and progression of lung disease. methods: lung function and clinical data were obtained from 164 cf patients treated and followed in the adult cf clinic, st paul's hospital. forced expiratory volume at 1s (fev1) and forced vital capacity (fvc) values dating back to 2000 were used in regression equations to group patients into quartiles based on their decline in predicted fev1%. rate of decline in lung function was compared with clinical characteristics and various systemic inflammatory biomarkers, including wbc, band cell count, crp, il-1β, il-6, il-8, mcp-1, and gm-csf results: the quartiles exemplified significantly different rates of decline in fev1/fvc% values. subjects with a rapid decline in lung function were younger and were colonized/infected more commonly with methicillinresistant staphylococcus aureus (mrsa) (p<0.05). in addition, altered calcium, magnesium, and albumin levels were found in subjects with increased rates of decline in lung function (p<0.05). preliminary results show that patients with cf have significantly elevated levels of wbc (10.94e9/l), crp (8.708e3ng/ml), and il-8 (48.65 pg/ml) in serum when compared to control subjects (p<0.05). there were trends in relationships between levels of il-6, il-8 and mcp-1 and rates of decline in lung function, however none reached statistical significance. conclusion: there is a variable rate in the decline in lung function among cf patients and one factor may be a heterogeneous systemic inflammatory response. circulating pro-inflammatory mediators may not only impact progression of lung disease, but could also be novel biomarkers to monitor and assess disease severity in cystic fibrosis. nutritional status in addition effects the rate of decline in lung function perhaps by modifying the inflammatory response. this research is supported by the bc lung association. overexpression of the epithelial na + channel β subunit (protein = βenac, gene = scnn1b) in transgenic mice results in cf-like lung pathology, characterized by neonatal mortality, mucus obstruction and airway inflammation (mall et al., nat. med. 2004) . breeding βenac mice into gene-deleted mice enables quick and efficient determination of the specific pathways relevant to the development of lung pathology. tnfα is a pleiotropic pro-inflammatory cytokine released by many different cell types, including t cells, macrophages, granulocytes, and epithelial cells. tnfα levels are significantly elevated in bronchoalveolar lavage (bal) from βenac mice in comparison to wild-type (wt) littermates. we crossbred βenac mice (heterozygous, inbred line c57-6608) with tnfα knockout mice, generating four types of mice: wt, tnfα +/-; wt, tnfα -/-; βenac, tnfα +/-; and βenac, tnfα -/-. all mice had comparable, high survival, ranging between 80-90%. the lack of tnfα did not prevent neutrophil and eosinophil infiltration and did not significantly modify the lung pathology characteristic of βenac mice, namely mucus plugs, mucous secretory cell hyperplasia, airway inflammation and emphysema. the absence of tnfα in bal from βenac, tnfα -/-mice, in comparison to βenac, tnfα +/mice, was confirmed by luminex cytokine assay. the levels of kc, a potent neutrophil chemoattractant, were significantly elevated in bal fluid from βenac mice, regardless of the presence or absence of tnfα. in wt mice, no granulocytic infiltrate or morphologic changes were observed and tnfα and kc were undetectable. similarly, the pathologic changes in βenac mice were not mitigated by crossbreeding to knockout mice for tnfα receptor 1 (tnfαr1), the major mediator of biologic responses classically attributed to tnfα. these results suggest that tnfα per se does not have a critical pro-inflammatory role in the development of inflammation in the βenac mouse model. the production of alternative cytokines may compensate for the loss of tnfα bioactivity in the tnfα and tnfαr1 -/-mice. since tnfα has been suggested to play a role in the regulation of enac, ussing chamber studies are underway to test whether the bioelectric features of tracheobronchial epithelia from βenac mice are altered by the absence of tnfα in vivo. these data suggest that a tnfα-independent signaling cascade links airway surface dehydration to airway inflammation in the βenac mouse model. innovative pharmacological approaches to control the excessive neutrophil infiltrates into the bronchial lumen of cf patients are thought to be beneficial to reduce the extensive airway tissue damage. the activation of expression of proinflammatory genes by p.aeruginosa with bronchial epithelial cells is a central mechanism to be targeted with novel therapies. medicinal plants from the socalled traditional asian medicine are attracting a growing interest because of their potential safety, already tested in large scale applications in human diseases. however, due to the presence of different active principles in each plant extract, whose multifunctional effects may even result contradictory, understanding the effect of each component is mandatory to pursue selective and reproducible applications. a panel of medicinal plant extracts have been firstly screened for their capacity to interfere in the binding of nuclear transcription factor proteins (tf) with dna consensus sequences identified in the promoters of the pro-inflammatory genes, thus for their potential inhibitory action on gene expression. extracts from several medicinal plants have been screened for their ability to interfere with the tf nf-kb, ap-1 and creb, which are induced by p.aeruginosa, and some of them, such as emblica officinalis (eo), aegle marmelos (am), polyalthia longifolia (pl), have been shown to inhibit tf/dna interactions, opening the possibility of potential applications to downregulate expression of pro-inflammatory genes. extracts from eo, am, pl were tested in ib3-1 cf bronchial cells exposed to the p.aeruginosa laboratory strain pao1. eo, am and pl strongly inhibited the pao1-dependent transcription of il-8 in ib3-1 cells. pyrogallol, one active principle of eo, was tested in ib3-1 cells, where it inhibited the transcription of the neutrophil chemokines il-8, gro-alpha and gamma, of the intercellular adhesion molecule icam-1 and the pro-inflammatory cytokine interleukin 6, similarly to the whole eo extract, whereas a second active principle from eo, namely 5hydroxy-isoquinoline, had no effect. similar results were obtained with eo and pyrogallol in the monocyte-derived macrophage cell line thp-1 exposed to pao1. in conclusion, extracts from plants of the traditional medicine can inhibit expression of pro-inflammatory genes and screening active principles purified from medicinal plants could result useful to identify safe and innovative pharmaceutical molecules to control lung inflammation in the lung of cf patients. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando 2005 -malattie rare e della povertà. massive infiltrates of neutrophils in the mucosal wall and lumen of the conductive airways of cf patients contribute to the progressive lung func-tion decline by releasing different proteases responsible for the progressive airway tissue damage. bacterial products and pathogens themselves within the mucopurulent material of the airway surface fluid induce the activation of transcription factors such as nf-kappab, ap-1, sp1, nf-il6, nf-at, elk-1, creb resulting in expression of chemo/cytokine genes driving the recruitment of leukocytes inside the bronchial lumen. to find new treatment options focused on the reduction of neutrophil chemotaxis, without abolishing the immune response against pathogens, we are exploring the transcription factor (tf) "decoy" strategy, in which oligodeoxynucleotides (odn) mimicking the consensus sequences for the tfs proteins identified in the promoters of different chemo/cytokines are delivered inside the cell in order to interfere with gene transcription. cf bronchial epithelial cells ib3-1 have been exposed to the (i)p.aeruginosa(/i) strain pao1. a clear pao1-dependent activation of tfs such as nf-κb, sp1, ap-1, nf-at, nf-il6 was confirmed by electrophoretic mobility shift assay (emsa). in parallel, transcription of genes involved in innate immune response has been quantified by real-time rt pcr. transcription factor "decoy" odns directed against the consensus sequences identified in the promoters of different genes have been designed and validated by testing their interference in tf protein/dna binding assays. transfection of ib3-1 cells with hiv-1 ltr and igk chain nf-κb odn "decoys" complexed with lipofectamine, performed 30 hrs before challenge with pao1, was shown previously to inhibit strongly pao1-dependent transcription of il-8 but not gro, il-1β, il-6 and icam-1. therefore other tf "decoy" odns have been also tested: a) odn for nf-κb from il-8 promoter inhibited il-8, gro-gamma and il-6 by 50%, b) odn for sp1 from hiv-1 genome inhibited il-6 by 50%, c) odn for ap-1 from il-8 promoter inhibited both il-8 and gro-gamma by 50%. a tf "decoy" molecule designed as peptide-dna chimera mimicking the consensus sequence of hiv ltr nf-κb strongly and selectively inhibited il-8 transcription. in conclusion, transcription of chemo/cytokines induced by (i)p.aeruginosa(/i) in cf bronchial epithelial cells (i)in vitro(/i) can be inhibited with different efficiency and selectivity by tf "decoy" molecules. these results provide useful hints for a gene-targeted anti-inflammatory approach and add further information on the regulation of expression of pro-inflammatory genes induced by (i)p.aeruginosa(/i) in bronchial epithelial cells. supported by italian cystic fibrosis research foundation and by fondazione cariverona -bando 2005 -malattie rare e della povertà. background: cf is characterized by hypersecretion of the pro-inflammatory cytokine il-8 from airway epithelial cells. however the mechanism by which il-8 gene expression is dysregulated in cf is not known. the expression of cytokine and chemokine genes is known to be regulated at multiple mechanistic steps including transcription, mrna decay, translation, and various post-translational steps. sequence-specific mrna degradation is now recognized to be an important site controlling the expression of several chemokine mrnas. this selective behavior is conferred by cisacting elements in the mrna composed of au-rich sequences (ares). the regulatory function of ares is thought to be mediated via rna-binding proteins that specifically recognize the are motifs. the steps required for mrna decay is comprised of deadenylation, decapping and body decay. deadenylation or removal of the poly (a) tail by poly(a)-specific ribonuclease (parn) appears to be the first and perhaps the rate-limiting step. this is accompanied by decapping or enzymatic removal of the 5' methylate guanosine cap. subsequently, exonuclease activities in the 5' to 3' or 3' to 5' direction predominate to degrade the remaining mrna. hypothesis: we have hypothesized that defects in transcription as well as modulation of the post-transcriptional stability of il-8 mrna might contribute to hyper-production of il-8 protein in the cystic fibrosis. methods: il-8 mrna stability was assessed in cystic fibrosis ib3-1 lung epithelial cells and in aav-cftr-repaired ib3-1/s9 cells, by measuring residual il-8 mrna levels at various intervals of time after addition of actinomycin-d to the culture. protein s100 extracts, prepared from the cf cell line as well as the cftr-corrected cell line, were analyzed by western blot. the expression levels of the various factors known to participate in are-mediated mrna decay, including ttp (an are-binding protein), parn and the exosome were compared in the two sets of s100 extracts. results: we find that the levels of il-8 mrna in the cf cells are greater than the levels found in the cftr-repaired cells. in addition, we found that the rate of decay of il-8 mrna in cf cells was significantly less than that in the repaired cell line. the levels of ttp, parn and exosome proteins were significantly reduced in the cf cells compared to the cftrrepaired controls. however, expression of ttp in ib3-1 cells causes significant destabilization of il-8 mrna. conclusion: we conclude that the high levels of il-8 protein expression in cf lung epithelial cells can be partly due to enhanced stability of the il-8 mrna. consistently, the actual levels of il-8 mrna are higher in the cf cells compared to controls. understanding the mechanism by which ttp promotes enhanced destabilization of il-8 mrna in cf cells may be important for developing novel therapeutic targets to alleviate the pulmonary pathophysiology of this disease manifested by hyper-secretion of il-8 protein. understanding the mechanism of airway remodeling could lead to the identification of novel therapeutic targets for the prevention of irreversible lung damage in cf. we have investigated transcriptional responses to epithelial injury in a mouse model of cf that was developed in our laboratory (f508del, cftr tm1eur fvb backcross f12). to induce transient epithelial lung injury, homozygous normal and mutant age matched littermates were treated in parallel with naphthalene (200 mg/kg ip) or carrier (corn oil) as control (n= 4x12). naphthalene causes an almost complete selective ablation of clara cells overnight, followed by migration of resistant ciliated cells and rapid proliferation of undifferentiated progenitor cells. two and seven days after treatment, lungs were collected for histological and transcriptome analysis. based on a previous affymetrix microarray analysis (n=6), a quantitative pcr array was designed containing 56 genes differentially expressed after naphthalene injury. as expected, naphthalene injury results in a transient increase of cell proliferation markers (mki67, cdc20, cdc2a etc), and a strong (90%) reduction of clara cell markers (cc10/scgb1a1; claudin10/cldn10; cyp2f2). in addition, we have now identified twenty-five genes that are increased (p< 0.001) two-to fifty-fold after naphthalene injury in both normal and mutant mice. this includes known and novel markers of epithelial tissue injury such as timp1, retlna, mmp8, serpin3n, and lipocalin. in particular, a group of egf receptor agonists is strongly induced: amphiregulin (areg, 13 fold), epiregulin (ereg 50 fold) and heparin binding egf (3 fold). amphiregulin mrna can be detected by in situ hybridization only in airway epithelial cells after injury. these egfr agonists are involved in stimulating epithelial repair, but can also activate cells in the underlying mesenchyma. indeed, we observe a substantial increase of major extracellular matrix mrna's two days after injury (co1a1: 5-fold, col3a1: 10-fold, elastin: 3-fold). whereas expression levels were substantially reduced in normal animals seven days after injury, in cf mutant animals two to three fold higher levels were observed for these three genes at day seven (p<0.04). conclusions. we have designed and validated a gene array that can be used to study distal lung injury and repair. egfr agonists produced by epithelial cells are the most prominent growth factors after injury, involved in both epithelial repair and extracellular matrix production by mesenchymal cells. ecm production in cf mouse lung is sustained compared to normal mice, suggesting an inherent tendency towards fibrosis in cf lungs. our data suggest that the mesenchymal egf receptor and regulation of its agonists are important future targets of novel therapeutic strategies. supported by eec 6th fw projects eurocarecf, improved precision the mucosa of the proximal airways defends itself and the lower airways from inhaled irritants, allergens, and microbial and viral infections by several mechanisms. sensory nerves monitor the luminal microenvironment and trigger reflexes in the central nervous system (cns) that alter breathing, induce cough and stimulate mucus secretion when challenged with noxious stimuli. sensory nerves also release the tachykinins substance p (sp), neurokinin a and calcitonin gene-related peptide through axon reflexes in neighboring tissues, and these locally released tachykinins stimulate mucus secretion by binding to neurokinin 1 receptors on submucosal glands. recently we reported that local fluid secretory responses to noxious stimuli are dependent on the clchannel cftr in mouse airway submucosal glands and are defective in glands from cftr knockout mice (ianowski et al., j physiol, 2007, 580, 301) . we have now tested the effects of sp directly and examined the possible role of cftr in mediating these responses using tracheas from congenic wild-type and cftr knockout mice (cftr m1unc /cftr m1unc ) that had been bred onto c57bl/6 j or balb/c genetic backgrounds. we compared single gland secretion rates using optical methods as described previously. the cftr genotype of each mouse was assessed by using pcr to amplify genomic dna from tail clippings obtained at age 18 days. after the cftr knockout mice were weaned, intestinal obstruction was minimized by supplementing their water with peglite. capsaicinoids (chili pepper oil) increased fluid secretion from glands of wild-type mice from 10 ± 2 pl/min (n=9 tracheas, 17 glands) to 50 ± 10 pl/min (n=4 tracheas, 9 glands, anova p<0.05, tukey-kramer multiple comparison test p<0.05). this response was abolished by exposing the basolateral surface of the tracheas to l-732,138 (10 µmol/l), a known sp (nk-1) receptor antagonist (15 ± 5 pl/min, not different from control, tukey-kramer multiple comparison test p>0.05). secretion was stimulated from 4 ± 1 pl/min to 100 ± 30 pl/min (n=12 tracheas, 55 glands, student's t-test p<0.05) by the direct application of sp, and this response was strongly inhibited by pre-incubation with the cftr inhibitor cftrinh172 (saturating concentration, nominal 100 µmol/l; n=6 tracheas, 21 glands). finally, submucosal glands from cftr knockout mice failed to secrete when exposed to sp (1 µmol/l) whereas wild-type littermates were responsive. these results indicate that sp mediates local responses to capsaicinoids through a cftr-dependent mechanism. loss of this local regulation in cf may contribute to the susceptibility of cf airways. support: canadian cf foundation, canadian institutes of health research, nih (dk51817), and the cff (usa). chronically infected/inflamed cf human bronchial epithelia (hbe), or normal hbe exposed to supernatant from mucopurulent material (smm) from human cf airways, exhibit expansion of the endoplasmic reticulum (er) ca 2+ stores and amplification of ca 2+ -mediated inflammatory responses. we have shown that infection/inflammation of hbe triggers an unfolded protein response (upr) coupled to mrna splicing of x-box binding protein-1 (xbp-1). spliced xbp-1 (xbp-1s) is a transcription factor that promotes er expansion to augment the protein folding capacity during increased protein synthesis. because we have shown that hbe inflammation couples to increases in protein synthesis, we hypothesized that hbe infection/inflammation-induced er ca 2+ store expansion is mediated by xbp-1s. to test this hypothesis, we constructed four retrovirally-tranduced stable 16hbe14o-cell lines containing empty vector (ev), xbp-1 unspliced (xbp-1u), xbp-1s, or dominant negative xbp-1 (dn-xbp-1). cells were grown to confluence under an air-liquid interface and er ca 2+ store expansion and il-8 secretion studied in the absence or following 24 hr mucosal pbs or smm exposure. consistent with our hypothesis, expression of xbp-1s in the absence of stimulation induced er expansion and increased mucosal utp-sensitive er ca 2+ stores ( to confirm that these effects were mediated by upr activation, cells were transfected with a upr response element luciferase reporter plasmid, and luciferase activity was measured in the absence or presence of the upr inducer tunicamycin (tm). cells expressing xbp-1s exhibited higher baseline luciferase activity than cells expressing ev or xbp-1u, whereas luciferase activity was decreased in the dn-xbp-1 expressing cells. furthermore, while tm increased luciferase activity in cultures expressing ev or xbp-1u, its effect was blocked in dn-xbp-1 expressing cells. these findings suggest that xbp-1s is the major trigger of er ca 2+ store expansion, which mediates the amplified ca 2+ -dependent inflammatory response in infected/inflamed airway epithelia. although it remains to be established whether upr-dependent xbp-1s is a beneficial or a maladaptive response in infected/inflamed airways, therapies aimed at manipulating this upr pathway may be beneficial for patients with chronic inflammatory lung diseases. luminal exposure of well-differentiated normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases the secretion of inflammatory mediators and triggers an unfolded protein response (upr) mediated by the mrna splicing of the x-box binding protein-1 (xbp-1). spliced xbp-1 (xbp-1s) is a transcription factor that expands the endoplasmic reticulum (er) and protein secretory pathway during er stress induced by accumulation of unfolded proteins due to increases in protein synthesis. our previous studies in primary cultures of hbe and 16hbe14o-cells stably expressing empty vector, inactive unspliced xbp-1 (xbp-1u), xbp-1s or a dominant negative xbp-1 (dn-xbp-1) suggest that induction of xbp-1s by smm exposure promotes er ca 2+ store expansion, which mediates a ca 2+ -dependent hyperinflammatory response. the present studies were designed to determine if pseudomonas aeruginosa pak strain (p.a.) would reproduce the effects of smm in both in vitro and, importantly, in vivo models. for in vitro studies, hbe were exposed to broth (tsb) or to a 20% p.a. extract for 24 hr, and inflammation, xbp-1s, and er ca 2+ store expansion investigated by il-8 secretion, southern blots and calreticulin expression, respectively. similar to smm, p.a. induced a 4.5 fold increase in il-8 secretion coupled to a 2 fold increase in xbp-1s and larger er ca 2+ stores as compared with tsbexposed hbe. these data demonstrate the link between p.a. infection, upr activation and xbp-1s in vitro. we next tested the relevance of these findings in vivo. wild-type mice airways were challenged with pbs or 10 6 cfu of p.a., and airway epithelial er density was assessed by calreticulin staining 24 hr later. in comparison with pbs challenges, p.a.-challenged mice exhibited airway epithelial er expansion, which correlated with the degree of airway inflammation, based on the presence of inflammatory cells. to test whether this p.a.-induced er expansion was linked to xbp-1s, we utilized "er stress activated indicator" (erai) mice expressing a fusion protein consisting of xbp-1u and the fluorescent protein venus. in erai mice, upr activation-dependent xbp-1 splicing leads to venus expression; hence, venus fluorescence is an index of xbp-1s. consistent with our hypothesis that p.a. infection-triggered inflammation would induce upr-dependent xbp-1s, venus fluorescence, after 24 hr challenge with 10 6 cfu of p.a., was increased in inflamed as compared with non-inflamed airways. these findings suggest that 1) airway epithelia respond to bacterial infection-induced inflammation by up-regulating the er ca 2+ stores and 2) activation of the xbp-1s pathway by bacterial infection may be relevant to airway inflammatory responses in vivo. funded by the cff. we have shown that luminal exposure of well-differentiated primary cultures of normal human bronchial epithelia (hbe) to supernatant from mucopurulent material (smm) from human cf airways increases total cellular protein synthesis, which reflects the increased secretion of inflammatory factors induced by the infectious and inflammatory process. in the present studies, we first investigated whether these hbe responses to smm were linked with an increased metabolic rate by measuring lactate accumulation into the serosal media. smm increased lactate production, and this effect was maximal within 24 hrs (9.0+1.0 vs. 13.2+0.8, and 7.1+0.9 vs. 13.1+0. 4 mmol/l in 24 and 48 hr pbs vs. smm, respectively; n=5-10), suggesting that the increase in protein synthesis couples to a hyper-metabolic state in infected/inflamed hbe. in agreement with this notion, 6 or 24 hr smm, as compared with pbs exposure, induced the expression of genes associated with amino acid transport and metabolism (n=4). in addition, 6 or 24 hr smm exposure up-regulated genes involved in oxidative stress (n=4). we hypothesized that these hbe responses were linked to an unfolded protein response (upr) mediated by activation of the pkr-like er kinase/pancreatic eif2α kinase (perk)-induced activating transcription factor 4 (atf4), since this pathway has been shown to confer protection against amino acid loss and oxidative stress in other cells. we first tested whether smm induced activation of perk/atf4 in hbe by performing western blot analyzes of the components of this pathway. twenty-four hr smm exposure induced perk activation, as indexed by phosphorylation of perk, in comparison with pbs-exposed hbe. on the other hand, total perk protein levels were unchanged in smm-treated hbe. phosphorylation of eif2α, the downstream effector of perk, and increased atf4 protein levels, which depend on the phosphorylated status of eif2α, provided additional evidence that the perk/atf4 pathway was activated by smm. these data are consistent with the hypothesis that induction of atf4 is triggered by upr activation resulting from increased synthesis of inflammatory factors. we next utilized rna microarrays to test whether atf4 target genes were induced by smm. six or 24 hr smm exposure induced atf4 target genes (e.g., ero1, an oxido-reductase that provides protection against the accumulation of endogenous peroxides during er stress; stanniocalcin 2, whose expression is associated with anti-apoptotic functions; and heme oxygenase 1). these findings suggest that 1) activation of the upr-dependent atf4 pathway is a compensatory component of the airway epithelial adaptive response to luminal infection/inflammation, and 2) activation of the atf4 pathway protects against inflammation-induced amino acid loss and oxidative stress by up-regulating genes involved in amino acid transport/metabolism and oxidative stress responses. unraveling the functions of atf4 should help determine if therapies targeted to manipulate pathway activity would be likely to improve lung function in patients with cf or other chronic inflammatory airway diseases. funded by the cff. miller, t.j.; perez, a.; qian, y.; davis, p. pediatrics, case western reserve university, cleveland, oh, usa fxyd5 is a cell surface protein originally identified in a screen for molecular markers of tumorigenesis. increased fxyd5 expression was found in tumors from stomach, thyroid, colon, pancreatic, breast and lung cancers and correlated with down-regulation of e-cadherin and poor patient prognosis. recent studies have shown that overexpression of fxyd5 promotes cell motility, decreases cell-cell attachment and increases tumor metastasis. fxyd5, also known as dysadherin, is a member of small family of proteins known to regulate the na,k-atpase. we now report that fxyd5 is upregulated in cystic fibrosis (cf) airway epithelia and modulates wound healing. we show by immunohistochemistry and immunoblot analyses that fxyd5 is increased in the lungs of s489x cf mice, and demonstrate an almost 3-fold increase in fxyd5 expression in the nasal epithelia of cf mice compared to wild-type littermates (p<0.001). furthermore, we show that fxyd5 is upregulated in nasal scrapings from human cf patients compared to controls (p<0.02). immunofluorescence data show that flag-tagged fxyd5 co-localizes with the na,k-atpase in epithelial cells, suggesting that fxyd5, similar to other members of the fxyd family, may regulate na,k-atpase function. it has previously been shown that expression and localization of the na,k-atpase is required for efficient polarization and suppression of cell motility in epithelial cells. the recurrent remodeling of pulmonary epithelium as a result of bacterial infection in cf requires that airway epithelial cells polarize and migrate to wound sites in order to maintain lung integrity. thus we hypothesized that fxyd5 may be involved in wound healing after infection. laser-capture microdissection and microarray analysis of murine lung epithelia after 3 hours treatment with p. aeruginosa indicated a significant, 5-fold increase in expression of fxyd5 that was confirmed by immunoblot analysis. others have shown that fxyd5 may mediate expression of mcp-1, a critical determinant in monocyte recruitment, through activation of the nf-kb pathway. treatment of human tracheal epithelial (hte) cells with a cftr inhibitor (172) confirmed that loss of cftr function correlated with increased fxyd5 expression by quantitative rt-pcr (p<0.001), an effect that was abrogated with treatment of pdtc, an inhibitor of nf-kb (p<0.01). we speculated that fxyd5-induced increases in cell motility may be due in part to phosphorylation at serine 163. in a murine airway epithelial cell wound healing model, serine to alanine (s163a) mutations at serine 163 inhibited wound healing compared to wild type fxyd5 overexpression, whereas aspartic acid (s163d) mutations increased wound healing (p<0.005). immunoblot and immunofluorescence analyses of these mutants suggest phosphorylation at ser163 regulates membrane localization. we conclude that fxyd5 is increased in cystic fibrosis epithelia due to increased inflammatory mediators and suggest that fxyd5 may modulate airway epithelia wound healing after infection with p. aeruginosa through phosphorylation at ser163. the inflammatory response to bacterial infection in the cf airway is exaggerated compared to normal, leading to the accumulation of millions of necrotic neutrophils. extracellular neutrophil elastase (ne) activity in the cf airway not only compromises innate defences by cleaving opsonins and reducing ciliary activity, but amplifies the inflammatory response by stimulating expression of the neutrophil chemoattractant il-8 and increases mucus production, in addition to degrading the tissue matrix leading to fatal bronchiectasis. ne therefore represents an important target for the development of new therapies. however, this strategy requires consideration of the normal physiological function of this enzyme, since previous studies in knock-out mice indicated an essential role for ne in cell migration, bacterial phagocytosis and killing. our approach was to use the intracellular neutrophil elastase inhibitor gw311616a to 'knock-out' ne activity in neutrophils in normal human blood and test the function of isolated cells in chemotaxis, bacterial phagocytosis and killing assays. whole normal human blood was incubated with gw311616a, or pbs control, for 1h at 37°c. neutrophils were isolated by sedimentation of red blood cells (rbc) on dextran 70, purification on lymphoprep and hypotonic lysis to remove contaminating rbc. chemotaxis towards il-8 was measured using a modified micro-boyden chamber. phagocytosis was assayed by the depletion of staphylococcus aureus (sa), pseudomonas aeruginosa (pa) and e. coli (ec) in supernatants following culture in a 10:1 ratio with pmn for 2h at 37°c. supernatants were diluted and remaining organisms were plated on agar and grown overnight to count viable colonies. bacterial killing was assayed by incubating bacteria and cells in a 1:1 ratio for 15 minutes to allow phagocytosis, washing off remaining organisms, and incubating for 4h at 37°c. neutrophils were lysed with water and lysates plated on agar to test for bacterial growth overnight. results; gw311616a inhibited intracellular ne dose-dependently and at 10 µm 0.168±0.062 % ne activity remained (n=5). there was no significant effect of 10 µm gw311616a on neutrophil chemotaxis, bacterial phagocytosis or killing of any organism compared to pbs-treated controls. phagocytosis data is shown in the table. thus, in the absence of ne activity, human neutrophils remain wellequipped with other defence molecules including myeloperoxidase and defensins to successfully maintain the role of the neutrophil in innate immunity. however, mouse neutrophils which lack defensins require ne activity for optimal intracellular bacterial killing, and mice are not a perfect model for studies of human infection. the development of novel inhibitors of ne to treat lung disease in cf therefore remains an important goal. supported by the cf trust of great britain. background: sphingolipid signalling may differ between individuals with cf and healthy controls. the response to bacterial inflammation is different, and uptake and inactivation of sphingosine-1-phosphate, an intracel-lular pro-inflammatory mediator, is reduced in cf cells. it may therefore continue to act on g-protein coupled receptors in the plasma membrane. (boujaoude et al, j biol chem 2001) . furthermore, ceramide originating from basolateral sphingomyelin hinders augmentation of cftr-mediated anion conductance across the apical membrane, resulting in reduction of transepithelial airway anion secretion (ito et al, bbrc 2004) . aim of study: to determine if there is a difference in the levels of alkaline, neutral or acid sphingomyelinase (smase), or in the levels of neutral or acid ceramidase, in the intestinal or bronchial mucosa and some other tissues, between wildtype, homozygous (+/+) and heterozygous (+/-) delta-f508 cftr mice. methods: enzyme activities (duan and nilsson meth enzymol 2000) were determined in intestine (and content) divided into four regions, liver, lungs, kidney and spleen from deltaf508-cftr mice (+/+) and controls (wildtype, +/-). results: there was an increased amount of neutral ceramidase in spleens from deltaf508-cftr mice (+/+) in comparison to control mice (p=0.0278). no other significant differences were seen. conclusion: delta-f508 mutation did not influence the levels of alkaline smase and neutral ceramidase acting as ectoenzymes, or the levels of intracellular smases and ceramidases, which may all generate bioactive sphingolipid metabolites in intestine and lungs. the implications of the increased level of neutral ceramidase in spleen are not known. in children with cystic fibrosis (cf) there is a clear correlation between the development of chronic p aeruginosa infection and acceleration in the decline of lung function. when chronically present, p aeruginosa takes on a mucoid phenotype and is impossible to eradicate. prior to this, when colonisation is intermittent, it is possible to eradicate it with aggressive antibiotic regimes. we sought to examine the degree of inflammation and innate defence status in the lungs of children with cystic fibrosis in various stages of colonisation by looking at a range of proteases, innate defence proteins and markers of inflammation in broncho alveolar lavage (bal). children with cf were allocated to one of three groups in relation to p aeruginosa infection; chronically colonised, intermittently colonised and non-colonised, on the basis of the leeds criteria. bal was collected as per ers guidelines as part of each patient's routine clinical care. bal was collected from control patients undergoing elective non-pulmonary surgical procedures. differential cell counts in bal were performed manually. secretory leukocyte protease inhibitor (slpi), elafin, alpha-1 antitrypsin (a1at) and lactoferrin concentrations were measured by elisa. neutrophil elastase activity and cathepsin activity were assayed by colorometric activity assays. fifty two patients were included in the study ranging in age from 3 months to 18 years (11 chronic, 10 intermittent, 17 non colonised and 14 controls). neutrophil counts, neutrophil elastase activity and cathepsin activity were markedly increased in children chronically colonised with p aeruginosa compared to those in the intermittent and non colonised groups. in contrast, levels of the antiproteases slpi and a1at and the antimicrobial peptides elafin and lactoferrin were highest in the control group and decreased as colonisation progressed, with levels in the chronically colonised group markedly lower than those with intermittent colonisation. this study demonstrates that in children with chronic p aeruginosa colonisation, there is a marked decrease in antiproteases and antimicrobial factors and a marked increase in protease activity and neutrophil influx in comparison with those who are non-colonised or intermittently colonised. these findings underline the importance of careful microbiological surveillance and early aggressive treatment of p aeruginosa infection in children with cf in order to avoid chronic colonisation. background : abnormal bronchial angiogenesis is responsible for hemoptysis in cystic fibrosis (cf). expression of vegf-a in airway epithelium induces bronchial angiogenesis in animal models. we have recently found that vegf-a and egf receptors (egfr) are increased in the airway epithelium of subjects with advanced cf lung disease. aims: to examine the effects of pa bacterial products and egfr inhibition on vegf synthesis in airway epithelium. methods: culture of non cf (nci-h292) and cf (cfte29o-) human airway epithelial cell lines. stimulation with pa lipopolysaccharide (lps). assessment of vegf mrna and protein by rt-pcr and elisa. use of chemical inhibitors, blocking antibodies and sirna. results: pa lps increased vegf gene expression and protein production time-and dose-dependently in both cells lines. using chemical inhibitors, we show that egfr and erk1/2 activation are required for lpsinduced vegf production. using blocking antibodies to egfr and its ligands, we show that tgf-alpha-dependent egfr activation mediates pa lps-induced vegf gene and protein synthesis. using pharmacological inhibitors (an ros scavenger and an nadph oxidase inhibitor) and using small interfering rna of dual oxidase (duox) 1 and tnf-alpha converting enzyme (tace),we show that lps-induced vegf upregulation is dependent on duox1-mediated ros release and tace activation. thus, pa products induce vegf synthesis in airway epithelium via a duox1-ros-tace-tgf-alpha-egfr-erk1/2 cascade. conclusions: these results describe a novel pathway by which bacterial products induce angiogenic signaling in cf and non cf airway epithelium. background: unlike bronchoalveolar lavage (bal), the airway mucosa has been under-investigated in cystic fibrosis (cf), despite the fact that irreversible airway wall changes (bronchiectasis) are a feature of endstage disease. cf is characterized by a neutrophil-dominated inflammation in bal, but little is known about the pattern of inflammation in the airway mucosa, especially in children with relatively early stage disease. we aimed to assess whether the pattern of inflammation seen in cf bal was also found in the airway mucosa in cf children. methods: to date, endobronchial biopsies and bal from 46 children (0-16 years) with cf and 8 control children (0-16 years) without lower respiratory disease have been assessed. bal cell differential was assessed on may-grünwald-stained cytospins. endobronchial biopsies were stained for neutrophils (neutrophil elastase, ne), t-(cd3) and b-(cd20) lymphocytes, eosinophils (eg2), and macrophages (cd68). area profile counts of immunopositive cells in subepithelial tissue were performed by investigators blinded to disease group. results: all cell types were increased in cf bal compared to controls. cf bal was characterized by an abundance of neutrophils (837 x 10 3 /ml vs. 3 x 10 3 /ml in controls, p<0.0001) with moderate numbers of lymphocytes (48 x 10 3 /ml vs. 3 x 10 3 /ml in controls, p<0.001). in contrast, cf subepithelial tissue was characterized by a lymphocytic infiltrate (961 cells/mm 2 vs. 575 cells/mm 2 , p<0.01) with only very few neutrophils (12 cells/mm 2 vs. 0 cell/mm 2 , p<0.05). the lymphocytic infiltrate in cf consisted mainly of t lymphocytes (91%). eosinophil counts in subepithelial tissue did not differ between cf and controls. for all cell types, there was no correlation between counts in bal and counts in subepithelial tissue. conclusions: in contrast to the neutrophil-dominated inflammation in the airway lumen, cf is characterized by a lymphocytic inflammation in the airway mucosa. the lymphocytic infiltrate consists mainly of t lymphocytes, the pathophysiological function of which may be important and is being investigated in future work. support: ers long-term fellowship and swiss national foundation grant to nr the epithelium serves as a barrier to the penetration of foreign antigens, particles, and infectious agents across the airway. the integrity of this barrier is dependent, in part, upon the apical junctional complex consisting of the tight junction (tj) and the adherens junction. alterations in tj permeability have been linked to the pathogenesis of inflammatory bowel disease and this increased intestinal permeability may actually precede the onset of chronic inflammation. in cystic fibrosis (cf), the airway lumen is filled with high concentrations of inflammatory cells, bacteria, and inflammatory mediators. since tj barrier function can be significantly reduced by inflammatory mediators, we hypothesized that measures that enhance airway tj barrier function will decrease airway responses to the continuous presence of inflammatory mediators in the lumen. to test this hypothesis, we examined the relationship between lung inflammation and epithelial permeability in vivo using a lipopolysaccharide (lps) model of lung inflammation. pseudomonas aeruginosa lps was instilled intratracheally into the lungs of c57bl/6 mice which were then euthanized at 24, 48 and 72 hrs. lung inflammation was assessed by total cell counts using a hemacytometer and differential counts by wrights staining of cytospin preparations of the bronchoalveolar lavage fluid (balf). lps increased total cell counts and neutrophil concentrations that peaked at 48 hours after lps administration, compared to saline controls. measurements of the proinflammatory murine cytokine kc in balf, by a cytokine antibody bead technique, showed an increase in murine kc at 48 hr following lps administration, which correlated with the substantial increase in neutrophil concentration. changes in lung permeability with inflammation were assessed by elisa measurements of the levels of serum protein murine albumin in balf. correlating with changes in cellular inflammation and murine kc levels, albumin concentration peaked at 48 hr after lps administration. this increase was subsequently resolved, consistent with the restoration of barrier function. an examination of frozen sections of lung from lps-treated animals showed a redistribution of the tight junction protein zo-1 consistent with the disruption of barrier function. since the p38 map kinase signaling pathway has been implicated in lps-induced airway inflammation, and an inhibitor of this kinase, sb203580 has been shown to reduce this inflammation, the effect of this inhibitor on barrier function is being investigated. in initial studies, sb203580 appears to reduce total and neutrophil cell counts by 50% in vivo. the effect of sb203580 on murine albumin concentrations in balf and on tj protein localization is currently being evaluated. a reduction in these parameters will be used as indices of improved barrier function with sb 203580. these studies will determine whether a reduction in lung inflammation correlates with a restoration of barrier function. the degree of protection provided by the p38 map kinase inhibitor sb 203580 could have important implications for inflammatory lung diseases such as cf. background: chronic pulmonary inflammation in cf is characterized by a robust neutrophil response associated with airway damage and failure to eliminate the pathogen, p. aeruginosa (pa). pa is a highly adaptable opportunist which quickly develops resistance to antimicrobials. thus, the development of specific immunotherapy targeting the neutrophil recruitment without ablating the host's immune response to infection or promoting pathogen resistance would be ideal. our group has identified il-17 as a prime target for the development of immunotherapy due to the central role that the il-23/il-17 proinflammatory axis plays in neutrophil recruitment. however il-17 does not mediate the early neutrophil recruitment seen in response to infection. hypothesis: il-23, acting synergistically with il-1, is critical to early neutrophil recruitment during pulmonary pa infection. the primary effector cells are the il-23-producing antigen presenting cells: alveolar macrophages (ams) and myeloid dendritic cells (dcs). methods: wt and il-23-deficient mice were infected with pa at 1x10 6 cfu/50ul by intratracheal (it) inoculation for 3 hours. bal inflammatory cell counts, and cytokines and chemokines were measured. am and dc cultures were infected for 3 hours in vitro and supernatant cytokines/chemokines were measured by luminex and elisa; il-23 levels were measured by taqman. these studies were designed to elucidate the role of il-23 in the early neutrophil peak and define am-and dc-mediated cytokine/chemokine production. recombinant murine il-23, il-1, and il-23 + il-1 were instilled via it into wt and il-23-deficient mice. bal inflammatory cell counts and cytokines/chemokines were measured at 3 hours. these studies were designed to elucidate the role of il-23 and il-1 in the early neutrophil peak and define am-and dc-mediated cytokine and chemokine production. results: at 3 hours post-infection, il-23 deficient mice had significantly lower percent neutrophils (p<0.02) and lower mip1α, kc, and il-6 (p<0.001) in the bal. il-17 was undetectable. there was no significant difference in bacterial load that could account for these cytokine/chemokine differences. infected wt ams elaborated significantly more mip1a, gm-csf, mcp-1, il-1, g-csf, ip-10, kc and il-6 than the il-23-deficient ams (p<0.001) and the response was inoculum-dependent (p<0.01). dcs elaborated no il-1 and exhibited il-23-dependent differences in mip-1α and mcp-1 production (p<0.05). in vivo studies of il-23 and il-1 effect demonstrated a synergistic increase in bal neutrophil recruitment (p<0.01) and cytokine and chemokine induction (p<0.01). conclusion: the first wave of neutrophil recruitment seen during pa infection is il-23-dependent and il-17-independent. ams and dcs are critical to il-23 and il-1 indcution of this neutrophil recruitment. these studies identify il-23 as a key mediator of neutrophil recruitment in the early stages of infection as well as the proximate mediator in the il-23/il-17 pro-inflammatory axis and suggest il-23 as a potential target for anti-inflammatory therapy in the treatment of pa pulmonary infection. supported by the cystic fibrosis foundation, american lung association and the nih the airways are under constant assault from air-borne pathogenic material. despite the intake of up to 100,000 bacteria per hour, the airways are sterile below the larynx in healthy individuals. the task of maintaining this sterility falls to the airway surface liquid layer (asl), the protective twophased system consisting of the viscoelastic mucus layer and the periciliary layer (pcl) through which cilia beat, sweeping the mucus layer away from the lungs. the mucus layer, which is responsible for trapping pathogenic material, is comprised of mucins (high molecular weight glycoproteins), cellular debris, dna, neurtifils, and more than 100 other proteins. this chemically heterogeneous mixture forms a viscoelastic gel that is thick enough to trap pathogens of various sizes and surface chemistries, while not sticking to the underling cellular / cilia layer, allowing the transport of trapped pathogenic materials away from the lungs. the performance of this trapping / transportation system is defined by the rheological properties of the mucus layer and the force imparted on the mucus. therefore, understanding how mucins and other chemical components of the mucus layer interact with each other to form a successful mucus gel (i.e. one that is cleared from the airways) is crucial to understanding airway defense. here we present the results of physical and chemical composition studies of sputum samples collected from patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf). the rheological properties of each sample was assayed using parallel plate rheolometry, probing the materials non-linear viscoelastic properties such as viscosity, elasticity, and yield stress. the physical properties of the sample are then correlated to the sample's chemical properties such as percents solids (divided between salts, proteins, and mucins), as well as the relative concentrations of the key airway mucins muc 5b and muc 5ac. our results indicate that the physical properties of sputum are not well predicted from the total amount of biosolids in a given sputum sample, but by the relative concentrations of muc 5b and muc 5ac and the interactions of these molecules with themselves and the other proteins present. further, we establish that the heterogeneous physical properties within a given sputum sample correlate to differences in the muc5b and muc 5ac concentrations. the gene modifier study (gms) was established as an effort to identify potential genetic modifiers of cystic fibrosis pulmonary disease and survival. during the course of this study over 1000 delta f508 homozygous cf patients classified as having mild lung disease, severe lung disease, or increased survival, have submitted both clinical data and blood samples for single nucleotide polymorphism (snp) analysis. the original snp analyses have shown a significant association between variants in the endothelin receptor a (ednra) gene, and cf survival, most markedly in female cf patients. sixteen additional snps within and around the ednra gene have now been genotyped, and have implicated the 5' and 3' untranslated regions of the gene as having the most significant association with pulmonary disease in females (p<0.000001), suggesting quantitative differences as a possible mechanism for the association with pulmonary phenotype. we are in the process of saturating the 5' and 3' regions of ednra with an additional 20 snps to further delineate the genetic association. ednra binds endothelin-1 (et-1) in airway smooth muscle cells, causing increased cell proliferation, smooth muscle contraction, and stimulation of inflammatory molecules. because each of these effects is known to be deleterious to the cf lung, we hypothesize that the ednra variants found more commonly in "severe" cf females ("severe" alleles) are marking increased ednra expression compared to alleles found more commonly in "mild" cf females ("mild" allelels). because the genetic association was strongest in cf females, we used the matinspector software to analyze 2kb of ednra promoter sequence and found several putative binding sites for both estrogen and progesterone. we then used a brdu assay to measure cell proliferation after stimulation with both estrogen and progesterone. these experiments showed that the asm cells with the "severe" 5' ednra genotype proliferated at levels approximately twice that of the asm cells with the "mild" 5' ednra genotype following stimulation with either estrogen or progesterone. using a single base extension protocol and quantitative pcr on human airway smooth muscle cells, we were also able to compare ednra expression from the "severe" allele, and the "mild" allele. these comparisons of ednra expression demonstrate that expression levels appear to be approximately 20% higher from alleles found more frequently in the "severe" cf females. in addition, preliminary data suggest that stimulating the asm cells with estrogen increases ednra expression by approximately 10 fold, and like the cell proliferation experiments, these increases are most pronounced in cell lines with the "severe" genotypes. these data suggest that the "severe" genotypes are marking alleles with increased expression, perhaps due to estrogen binding, that leads to increased et-1 functional effects that over time are deleterious to the cf lung. cf patients do present with variable spectra of lung disease, of which infections are most life-threatening. β-defensins have an antimicrobial activity against a broad spectrum of microorganisms and are chemotactic agents for cells of the adaptive immune system, and therefore assist in combating these infections. β-defensins 2-6 are part of a repeat region. this repeat region is polymorphic between individuals and therefore the dosage of these defensin genes/proteins varies. we developed a real time pcr assay to quantify the number of βdefensin repeats in this region. appropriate controls are needed for an accurate quantitative assay. therefore we made 6 concatemeric constructs with 1 copy of defb1 and a particular number of defb4 copies, which ranged from 1 to 6 copies. using these controls as standards, the number of defensin repeats could be accurately determined in dna samples. we then tested 146 f508del homozygous cf patients from belgian (57 patients), czech (52 patients) and south-italian (37 patients) origin. the diploid number of repeats varied between 3 and 10. for each patient group, a higher number of repeats was found in the group of patients with milder disease (fev1 >70%) compared to the group of patients with more severe disease (fev1 < 70%) (student t test, p-values of 0.0006, 0.03 and 0.019 respectively). moreover, in our cohort of 112 belgian cf patients, cf patients of 25 years or older have a significant higher number of repeats than the cf patient group below 25 (p-value 0.05). to evaluate this at the functional level, we cultured nasal epithelial cells from 3 individuals with a low number of repeats (i.e. 3 or 4 repeats) and 4 individuals with a high number of repeats (i.e. 8 repeats). the cells where grown in air liquid interface cultures. after differentiation, the cells were stimulated with 10ng tnfα. in cells with a high number of repeats, defb4 expression, as measured by the extent of transcription, was strongly upregulated by tnfα. in cells with a low number of repeats, defb4 was not upregulated (p-value = 0.015). we also tested the antimicrobial activity of epithelial cells. we challenged epithelial cells from 3 individuals (8 repeats) with a laboratory strain (pa01) of pseudomonas aeruginosa and a clinical isolate (30 -300 cfu), either in combination with tnfα or without tnfα. after 3h, surviving bacteria were counted by a plating out method. cells that were stimulated with tnfα 12h prior to the bacterial challenges were more bactericidal. the clinical strain was more vulnerable to the surface liquid than the laboratory strain. in epithelial cells from individuals having a low number of repeats, these effects were very variable from individual to individual. in summary, the β-defensin region is a modulator of cystic fibrosis lung disease. the pro-inflammatory response in cultured epithelium cells strongly correlates with the number of β-defensin repeats. cells with a higher number of repeats respond to tnfα treatment, which in turn results in a better antimicrobial activity of the surface liquid. rationale: studies of affected twins and siblings demonstrate that modifier genes are major contributors to variation in cystic fibrosis (cf) lung disease severity. we performed genome wide linkage analysis to identify regions likely to contain modifier genes affecting severity of cf lung disease. methods: 683 individuals with cf from 360 families were analyzed. pulmonary function data were collected from patient chart review and were supplemented with data from the us cystic fibrosis foundation patient registry. to minimize environmental variation, only data obtained while subjects were living with an affected twin or sibling were analyzed. the pulmonary phenotypes were defined using the best cf-specific percentile for fev1 (kulich, et al) within the last year of available pft data as a crosssectional measure (maxfev1cf%) and using two longitudinal measures: the lifetime average cf-specific % for fev1 (avgfev1cf%) and the estimated percent-predicted fev1 at age 20 (estfev1%pred@20yrs, schluchter, et al). longitudinal measures were derived from a minimum of 4 years of pft data. short tandem repeat markers (strs) were typed in all affected individuals and their parents (marshfield genotyping center: 402 markers or decode genotyping center: 1030 markers). two-point and multipoint linkage analyses were performed using sequential oligogenic linkage analysis routines (solar). results: patients represented the spectrum of lung disease severity, with maxfev1cf%'s ranging from 0 to 1, mean 0.70 ± 0.26 and avgfev1cf% ranging from 0.01 to 0.99, mean 0.59 ± 0.24. the maxfev1cf% was predictive of avgfev1cf% (r=0.89, p<0.0001) for the 486 individuals for whom both measures were available. the two longitudinal measures were also highly correlated (r=0.80, p<0.0001). linkage was found at chromosome 5 for all three phenotype definitions. peak multipoint lod scores on chromosome 5 occurred at 196 cm for maxfev1cf% and avgfev1cf% (lod 3.0 and lod 3.4, respectively) and at 191 cm for estfev1%pred@20yrs (lod 2.8). single point lod scores on chromosome 5 peaked at marker aaat072 (3.3 for maxfev1cf%, 3.4 for avgfev1cf%, and 1.88 for est-fev1%pred@20yrs). the region of linkage encompasses approximately 6 megabases near the telomere of chromosome 5q. conclusions: chromosome 5 appears to contain one or more genetic modifiers of cf lung disease severity. supported by the nhlbi, cff and genome canada through the ogi. cystic fibrosis-related diabetes (cfrd) is the most common extrapulmonary complication of cf and is an increasingly important contributor to morbidity and mortality as cf patients live longer. while pancreatic fibrosis and loss of exocrine and endocrine tissue are common in cf, 20-30% of cf adults develop defects in insulin secretion and accumulation of islet amyloid polypeptide, features typical of type 2 diabetes (t2dm) in the general population. to test whether modifier genes play a role in cfrd, we compared concordance rates for cfrd in 68 pairs of monozygous (mz) twins, 22 sets of dizygous (dz) twins, and 470 sets of 2 or more siblings (1176 individuals with cf). criteria for defining cfrd included physician diagnosis, treatment with insulin/oral agent, and 2 episodes of glucose ≥200 mg/dl. mz twins were highly concordant for cfrd (9 of 12 pairs, 75%). the young age of dz twin recruits precluded analysis of this group in isolation (0 of 4 pairs were concordant). twelve of 71 (14%) sibling pairs were concordant for cfrd. with heritability defined as: h 2 =2*(mz concordance -dz concordance), and including siblings as a proxy for dz twins, heritability is estimated as ~1.0. the same results were obtained considering only same-sex dz twins and siblings, correcting for differences in age and duration of clinical follow-up, or restricting analysis to ∆f508 homozygotes. these data support a significant role for one or more modifier genes in development of cfrd. we then tested whether cfrd correlated with a strong family history of adult-onset diabetes (at least 1 first-degree or 2 second-degree relatives on the same side of the family). of those reporting family history of diabetes, 20 of 69 had cfrd, compared to 34 of 271 with no family history (or=2.84 [1.5-5.4 ]; p=0.001). this correlation persisted after adjusting for age, sex, and pancreatic insufficiency (or=2.6; p=0.028). thus, family history of diabetes correlated with increased risk of cfrd. we then tested whether variants in tcf7l2, a transcription factor in the wnt signaling pathway, that have been reproducibly associated with t2dm in the general population were associated with cfrd in our study subjects. genotyping of four single nucleotide polymorphisms associated with t2dm (rs4506565, rs7903146, rs12243326, rs12255372, here termed snpa-d) and transmission disequilibrium testing (tdt) of 53 parent-parent-child trios revealed significant overtransmission for snpb (31:16, p=0.03) and snpc (28:15, p=0.047), and possible overtransmission for snpa (33:22, p=0.1) and snpd (31:18, p=0.06). in every case, the tcf7l2 allele overtransmitted to patients with cfrd is the same allele that confers increased risk for t2dm. furthermore, individuals with cfrd who were homozygous for risk alleles were diagnosed at a significantly earlier age (average 14.6 vs. 20.1; p=0.03). these data support a key role for modifier genes in development of cf-related diabetes, and demonstrate that cfrd and type 2 diabetes may share disease mechanisms such as alterations in wnt signaling. supported by nih dk076446, hd27799, dk44003 and hl68927, and cf foundation grant cuttin06p0. cystic fibrosis (cf) phenotypes and survival are highly variable among df508 homozygous patients, pointing to the existence of modifier genes and/or environmental factors that contribute to this disease. studies to identify genetic modifiers of cf are being carried out using dna from homozygous df508 cf patients. important clinical features, such as severity of lung disease, liver disease and meconium ileus (mi) status, are well defined. tgfβ1 has been previously identified as a modifier of cf lung disease (drumm et al., nejm, 353(14) : [1443] [1444] [1445] [1446] [1447] [1448] [1449] [1450] [1451] [1452] [1453] 2005) , but it does not explain all of the genetic heterogeneity in this population. current evidence suggests that mucus is involved in the progression of cf, making the muc genes prime candidates as modifiers of lung disease and/or other phenotypes. methods: our approach to evaluate muc genes utilizes both variable number tandem repeat (vntr) polymorphisms and single nucleotide polymorphisms (snps). vntr polymorphisms in the muc2 and muc5ac genes (n=405 and n=366 patients, respectively) were detected by southern blotting under conditions fully optimized to maximize the allele size resolution. to minimize gel to gel variation, a genomic dna mixture with the most common muc2 and muc5ac alleles was used as internal markers. accuracy and reproducibility were evaluated by duplicating the southern on the critical dna samples for muc2 and muc5ac genes (n=85 and n=63 patients, respectively). fifty snps in muc5ac, muc5b, muc2, muc1 and muc4 genes are being tested using illumina technology in 808 cf patients. results: preliminary analysis suggests there are significant differences between cf patients with "severe" and "mild" lung disease for both muc2 and muc5ac allele distribution, which is mainly driven by the male population; exhaustive statistical data analysis still is underway. the vntr data also suggest significant association between the larger muc2 allele size and cf patients with mi. the ongoing vntr analysis will be complemented by muc gene snps being genotyped. conclusions: initial results indicate that we can reproducibly characterize muc2 and muc5ac vntr alleles. additional characterization of muc2 and muc5ac vntr alleles, coupled to snp data, will allow us to better define the significance of muc gene variations as modifiers of different cf phenotypes. supported by cff perezv06g0 (jpv), cff knowle00a0 (mk), cff r026-cr02 (wko), nih rr00046, r01 hl68890, and cff drumm00a0. reporting for the gene modifier study group (mrk). we have previously reported that βenac transgenic mice, which overexpress the beta subunit of the amiloride sensitive sodium channel (scnn1b) specifically in the airways, share common features with cf, including increased enac activity, reduced airway surface liquid, mucus accumulation and obstruction, inflammation, and death. interestingly, analysis of this model also suggested the existence of potential genetic modifiers of phenotype severity, and we speculated that identification of these modifiers would provide novel insights into disease phenotype. to establish a set of reagents that could be used to uncover genetic modifiers, we have bred βenac transgenic mice from two independent founder lines (6608 and 6047, b6:c3 background) onto several strains of inbred mice, including c57bl/6n, c3h/hen, balb/cj, fvb/j, and 129/svj. these studies revealed dramatic phenotypic differences as measured by survival (0 to 80%) among strains and between lines. all lines thus far tested show ~2-3 fold increases in amiloride sensitive short-circuit current as measured by ussing chambers in the trachea. complete phenotypic characterization of c57bl/6n line 6608 at backcross generation 12 reveals high survival (66±5% in comparison to 54±4% of the mixed b6:c3 background), yet the mice maintain the pulmonary features of the originally reported mice, including increased mucus plugging, mucous cell hyperplasia, neutrophilic and eosinophilic inflammation peaking at early timepoints (5 days -2 weeks). lymphocytic nodules, which are not commonly seen in 4-6 weeks-old animals with mixed strain background, are a common feature in the c57bl/6n congenic line. emphysema and early airway epithelial cell necrosis, two phenotypes initially not strongly associated with transgene expression, are also observed. line 6608 on balb/cj and c3h/hen backgrounds has reduced survival compared to the c57bl/6n background, and generation 8 backcross lines are now being evaluated in these two strains for other phenotypic characteristics. analysis of backcross data from line 6047, which has low survival on all genetic backgrounds tested to date, including c57bl/6n, suggested that the transgene may have integrated onto a c3h locus with a dominant negative effect on survival. genetic analysis using genome-wide snp genotyping revealed a region of chromosome 4 linked to the transgene in line 6047 (lod score > 3.0). further analysis of this region is underway. in summary, backcrossing onto different genetic backgrounds is revealing genetic modifiers for phenotypes in the βenac overexpressing model. characterization of these phenotypic and genetic differences should provide clues about the mechanisms relevant to disease development. furthermore, inbred lines with variable phenotype will likely be an important reagent for the cf community as the utility of this model is evaluated in future studies. supported by nih (scor p50 hl60280) and cff (mall04go, oneal07go). introduction: new york state screens newborns with immunoreactive trypsinogen levels within the top 5% of all infants, for 32 common cf gene mutations. infants found to be heterozygote carriers are referred to a cf center to determine sweat chloride concentration. the proposed abnormal sweat chloride value for this group of newborns is ≥ 40 mmol/l, ≥ 3 standard deviation (sd) above the mean (farrell, 1996) . this retrospective study reports on the mean sweat chloride value + 3 sd in cf heterozygote newborns who have been referred for evaluation to suny upstate medical university cystic fibrosis center, and regarding the genotype of these infants with abnormal sweat chloride levels. method: from october, 2002 to december, 2006, 404 infants were referred for positive cf screening, and 300 of these (75%) were identified as heterozygote carriers by the screening program. at our center, these patients underwent pilocarpine iontophoresis, followed by collection of sweat (≥ 20 µl) in macroduct ® coils. sweat testing was performed successfully in 279 of the 300 (93%) patients. the patients' age (mean ± sd) at the time of sweat testing was 45.8 ± 19.0 days. for infants with an initial sweat chloride level ≥ 24 mmol/l, the sweat test was repeated within a week and a complete gene sequencing was requested (quest laboratory, ca). results: the sweat chloride level (mean ± sd) in newborns who were heterozygous for a cf mutation (excluding those who were found to have an additional mutation or deletion) was 10.4 ± 4.5 mmol/l (n = 273). the mean + 3 sd was 24 mmol/l, which defined our minimal value for an abnormal test. eleven infants had sweat chloride values of 27-91 mmol/l (table) ; 6 of them (55%) were subsequently diagnosed with cf by complete gene sequencing. four of the 5 remaining patients (80%) had ∆f508 mutation coupled to the 5t variant on the opposite chromosome 7 (sweat chloride levels, 24-41 mmol/l). conclusions: the reference range for sweat chloride in cf heterozygote infants appears to be significantly lower for some centers than previously reported. thus, each cf center should consider evaluating the cutoff values for the test at their site. moreover, the 5t polymorphism may account for sweat chloride elevations in heterozygote infants. method: we reviewed the charts of all 28 patients with cf who were referred as a result of the new york state newborn screening program to the suny upstate medical university cf center from october, 2002 through april, 2007. we included the patients who were identified by the state as heterozygote carriers of one of 32 common cf gene mutations, and whose second mutation was identified only after complete cf gene sequencing. results: seven of the 28 patients (25%) met inclusion criteria. six of the 7 patients (86%) were compound heterozygous for a novel or a rare cf gene mutation. one patient was compound heterozygous for a large deletion in the cf gene. the genotype and clinical status of the 7 patients are shown in table. all patients have been pancreatic sufficient to date. conclusions:the clinical effect of compound heterozygosity as a result of novel or rare cf gene mutations appears to be mild in early childhood. prior to newborn screening (with the exception of the patient with cftr deletion), these patients may not have been diagnosed with cf in the first few years of life. these patients may have subclinical airway inflammation and thus benefit from early treatment. patients with cf manifest symptoms in the pancreas, respiratory tract, male reproductive tract and sweat gland due to mutations in cftr. patients with non-classic cf have disease in a subset of these organ systems. most non-classic cf patients have two disease-causing mutations in cftr and at least one mutation permits residual cftr function. a subset of non-classic cf patients have only one cf-causing mutation after screening for a panel of common cf-causing mutations or following mutation scanning of the coding region of cftr. these patients present a diagnostic dilemma and a challenge for genetic counseling. we evaluated 10 cf patients with only one cf-causing mutation identified after a screen of 97 cftr mutations (3 patients) or scanning of the coding region of cftr (7 patients). nine of these patients, including one set of siblings, have non-classic cf with borderline or elevated sweat [cl -] plus lung disease (table) . one patient is pancreatic insufficient and has classic cf. many of these patients have features which are consistent with cftr dysfunction including a cf-like nasal potential difference (npd),p. aeruginosa infection, or congenital bilateral absence of the vas deferens (cbavd), suggesting that they have a second cftr mutation. mutations that are not detected by screening methods include insertions or deletions, mutations outside of the cftr coding region that affect rna splicing or expression, or mutations in the coding region of cftr that were missed by screening methods. to exclude the third possiblity, dna sequencing of the 27 exons and flanking introns of cftr was performed. a second mutation was identified in the coding region of cftr in 6 of the 10 patients; 3 had screening for 97 known cftr mutations (genzyme), while the remaining 3 had comprehensive scanning of the coding region of cftr by modified tgge (ambry). each of the mutations identified by sequencing has been previously described in patients with cf and is predicted to cause cftr dysfunction. these results reaffirm that patients with one cftr mutation who have biochemical and clinical features of cf are likely to have a second mutation in the coding region of cftr. thus, we suggest sequencing cftr in patients with only one mutation after mutation screening before employing other more complex genotyping methods (insertion/deletion or rna analysis). diagnostic criteria for confirming cf in symptomatic individuals includes two positive sweat tests or two known disease-causing cftr mutations. accurate sweat testing is performed at accredited cf centers, while cftr testing is available through national and specialty genetic labs. most labs offer analysis of a basic panel of cftr mutations as recommended by the american college of medical genetics (acmg), while specialty laboratories may offer an expanded panel or full sequencing/scanning. we report on one center's use of these methodologies to confirm the diagnosis of cf presenting in adulthood. the charts of 20 patients diagnosed with cf at 〉18 years of age were reviewed. 19 were sweat tested. all were genotyped. results: twelve (63%) pts had two positive sweat tests (>60 mmol/l). an additional five (25%) had at least one borderline result (40-59 mmol/l). two pts had negative results (35-39 mmol/l). one refused sweat testing because dna analysis through the acmg panel had confirmed the diagnosis prior to initial consultation. genotyping results for the 20 pts are summarized in the table below. rare mutations identifiable only through gene sequencing accounted for 18/40 alleles (45%) in our population. importantly, among this group, only one pt had a negative sweat test, a suggestive 39 mmol/l. two pts with positive sweat tests and clinical symptoms failed to reveal any cf mutations after sequencing. we continue to follow pts without genotypic confirmation, based on their clinical presentation and sweat chloride levels, and have recommended additional evaluation, including nasal potential difference studies in our series of adult-diagnosed patients, sweat test results were positive, borderline, or suggestive in all cases tested. sweat testing costs $100-$250 and results are ready in a day. genotyping costs $400-$2500 and takes several weeks. we acknowledge that circumstances may arise where reliable sweat testing is not conveniently available; but in our series, genotyping with the acmg panel would have diagnosed 10% of pts; using an expanded panel would have diagnosed 35%. genotyping is an important tool for genetic counseling, determination of eligibility for research studies, furthering knowledge of cftr dysfunction and cf pathophysiology, and for confirming a cf diagnosis after borderline or suggestive sweat test results. based on our findings and the dramatic difference in cost, we conclude that sweat testing should remain the first approach in the diagnostic workup of adult patients with a clinical presentation suggestive of cf. in colorado, 318 infants with cf (non-meconium ileus) have been diagnosed with cf by a two tiered immunoreactive trypsinogen (irt/irt) based newborn screening approach. the irt/irt algorithm has been recently adopted by other screening programs with two mandatory screening tests. while most infants in colorado have been successfully identified, the program has had a missed case rate of approximately 5%. the more common approach to cf newborn screening is the irt/dna method in which the blood spot of infants with an initial elevated irt is tested for the most common cf mutations. the initial irt cutoff is lower in the irt/dna programs than in the irt/irt programs, resulting in a lower missed case rate. the considerable number of carriers identified through the irt/dna approach puts a significant burden on the genetic counseling community, as carriers are identified at a rate of 1/20-1/25 of positive irts we propose an irt/irt/dna newborn screening algorithm that will maximize sensitivity and specificity while minimizing the number of identified carriers. using new database technologies in the newborn screening lab we will be able to identify those infants with an elevated first irt (>60ng/ml, approximately 97th percentile). all infants with an irt >60ng/ml will have a repeat irt on their second state mandated blood-spot. if the second screen is also elevated (>60ng/ml), the blood spot will be tested using a panel of 43 mutations, including mutations specific to the hispanic community. infants with one or two cftr mutations will have a sweat test to confirm the diagnosis, or rule out cf. we compared the projected statistics of our current method irt/irt to the new irt/irt/dna method, and to irt/dna is presented in the table, based on 70,000 births per year in colorado. four infants (1.5%) identified under the current irt/irt protocol would not have been identified by the mutation panel proposed in the new algorithm, out of 288 genotyped, non-meconium ileus infants. three of these missed cases are hispanic. two would be identified using an extreme irt cutoff of the 99.9th percentile (150ng/ml). the projected missed case rate would be <0.7% (0.2 -2.5%, 95% ci), using the irt/irt/dna algorithm, with 23 carriers identified, maximizing both sensitivity and specificity. this algorithm may provide a better alternative to the irt/irt screening methods in states with two mandatory screening tests, and has advantages over both the irt/irt and irt/dna methods. newborn screening for cystic fibrosis (cf) is rapidly expanding and has been implemented in at least 30 states. although most newborn screening assays are done using biochemical testing, many laboratories screening for cf include both biochemical and molecular testing of multiple alleles in the cystic fibrosis transmembrane conductance regulator gene. in response to the growing need for proficiency testing (pt) materials for molecular testing, the centers for disease control's newborn screening quality assurance program (nsqap) in collaboration with the university of wisconsin school of medicine and public health, the johns hopkins hospital, and case western reserve university, created a repository of dried-blood spot specimens with known mutations in the cftr gene to be used in a pt program. twenty milliliters of blood was collected voluntarily from adult donors with cf and sent to the nsqap laboratory. each specimen was adjusted to a hematocrit of 55% before being spotted onto whatman 903 paper (75 µl per spot), dried, and stored at -20°c with desiccant. proficiency testing (pt) panels consisted of 5 to 7 blind-coded specimens from adult donors. the panels were sent quarterly to laboratories worldwide that test specimens for cf using molecular methods. laboratories were asked to report the genotype, method used, and the presumptive clinical assessment of each specimen. twenty-two laboratories participated during both quarters 1 and 2, 2007. the laboratories used 16 different methods ranging from in-house assays to commercially available kits. most reporting laboratories tested the following 3 alleles -∆f508, g542x, and g551d. another twelve alleles were detected by most participants. nine more alleles were common among commercially available kits. laboratories were evaluated based on the clinical assessments. mutations that were not detected by a particular method were not evaluated. overall, the laboratories performed well. data compiled from both quarters demonstrated that there was 1 incorrect clinical assessment and 2 amplification failures. developing a pt program for dna-based testing is complicated by the number of methods and different alleles each laboratory chooses to test. though molecular testing for cf may be complex, pt monitors the laboratory's ability to test multiple alleles, including uncommon alleles, the limitations of various assays, and the different algorithms used for screening. the repository will also allow storage and access to rare specimens that may be useful for future research but are not readily available. who did could not reproduce. genetic counseling focused on a patient's parents, who were counseled about their recurrence risk at the time of the child's diagnosis. today, cf is a disease of adulthood. in 2002, >40% of cf patients in the us were >18; by 2010 it will be >50%. together with advances in assisted reproductive technology (art), reproduction and recurrence risk are now important issues for adolescent and young adult cf patients. methods: a 19 item questionnaire was developed from the results of prior semi-structured interviews with 18 cf patients age 16-25 years. knowledge based questions (medical issues, inheritance, and reproductive options/risks) as well as communication patterns (preferred resources for learning about cf and preferred people with whom to talk about reproductive issues) were addressed. recruited from the uab cf clinic population, 51 patients age 15-29 (mean 21), 24 male (47%), 27 female (53%), completed the questionnaire. results: regarding autosomal recessive inheritance of cf, only 33% knew that two carriers have a 25% chance of having a child with cf, and 25% knew that two carriers have a 50% chance of having a child who is a carrier. however, 82% knew that two carriers could have a child who did not have cf, and 52% knew that two carriers could have a child who did not carry cf. on their own reproductive risks, 59% knew that a cf patient had a 0% chance of having a child with cf if their partner was not a carrier, but only 26% knew that all their children would be carriers even if their partner was not a carrier. in the scenario of a cf patient with a cf carrier partner, 44% knew that a child had a 50% chance of having cf, and 24% knew that a child had a 50% chance of being a cf carrier. most patients knew about their reproductive potential, as 96% responded that cf patients are able to have children. however, when asked about whether the chance for having children was different for males and females with cf, 65% answered that it was more difficult for males, 8% that it was more difficult for females, and 27% answered "not sure." while 62% reported that they knew that there were options for male cf patients who wanted to have children, only 26% knew of art. conclusions: despite widespread availability, the lack of knowledge of adolescents and young adults with cf about the genetics of their disease continues. furthermore, these patients are unaware of both modern technologies that could enable them to have biological children and the risk of those children having cf. this study illustrates the changing needs of patient education as medical knowledge progresses. cf patients would benefit from further genetic knowledge and counseling to enable them to make informed decisions about reproduction as they mature into adulthood. center at the university of minnesota is one of three sites in the state providing confirmatory testing and follow up services for newborns identified by screening. while cf nbs identifies children with cf, most of the infants with positive screening results are carriers. our goal is to provide genetic counseling to every cf nbs patient seen at our center, and we believe that a protocol incorporating genetic counseling in the initial care plan for infants both with and without cf is imperative. the literature has shown that families who obtain genetic counseling through the cf nbs process recall genetic information more easily and accurately and are more likely to have testing to determine parental carrier status. for parents of a child that is determined to be a cf carrier, it is especially important to find the optimal method and timing of genetic counseling as many of these families are from several hours away and therefore less likely to return to clinic. to provide genetic information and emotional support for the families of infants screening positive on cf nbs, our center has a genetic counselor who serves as the cf nbs coordinator and clinical contact for the family. this allows many opportunities to speak with the genetic counselor and ask questions, as well as learn about their child's diagnosis or carrier status and the subsequent carrier testing recommendations for the infant's parents and families. to assess the success and impact of the minnesota cystic fibrosis center's nbs follow-up program and the incorporation of genetic counseling, an anonymous questionnaire was developed for parents of infants who were seen at our center due to a positive cf nbs result. questionnaires were mailed to parents of all infants seen at our center for a sweat test and genetic counseling due to a positive cystic fibrosis newborn screening result. as of the last mailing, this totals 54 families. two questionnaires were returned due to incorrect address and 28 questionnaires were returned answered, indicating a response rate of 54% (28/52). responses overwhelmingly indicated that parents were satisfied with our center's algorithm for the cf nbs follow-up program and found the information and support provided through genetic counseling to be a useful and recommended portion of the program. as cf nbs continues, it is critical that we learn about the patient's experience with genetic counseling and the nbs program, as well as identify areas needing improvement. genetic counseling is vital to the comprehensive success of our center's program, and we will report on the responses gathered from the families identified through cf nbs this first year, as well as discuss the lessons learned from setting up such projects on a state-wide basis. background & aims: aberrant splicing and nonsense mediated decay (nmd) lead to dysfunctional mrnas by skipping exons and to a reduced number of functional mrna respectively. both mechanisms have a strong quantitative aspect and may determine whether a cf patient develops a classic or atypical disease phenotype. in order to approach these highly important questions we wanted to establish a new quantitative real-time pcr based assay which allows allele specific quantification on cdna level. using this assay we like to determine the exact proportions of the f508del and non-f508del cftr mrna in cf patients compound heterozygous for the f508del mutation (for example in cf patients carrying the f508del and a nonsense mutation such as the r553x). material & methods: materials: we used genomic dna (gdna) and total rna (extracted from white blood cells and nasal epithelial cells respectively) from cf patients with compound heterozygosity for the f508del mutation, homozygosity for f508del mutation and from healthy individuals (controls). methods: the lasq (ligation dependent allele specific quantification) assay comprises 4 reactions: 1. reverse transcription of cftr mrna into cdna using gene specific primers (all rna specific). 2. overnight hybridization (12-16h) of the cftr cdna with either the f508del specific or the wt specific oligo probe pair provided by jan schouten (mrc holland). 3. ligation of the hybridized oligo pairs using the ligase 65 enzyme from mrc holland. 4. quantitative real-time pcr of the allele specific ligation products on the lightcycler (roche). results: in order to establish the lasq assay we first validated it using gdna instead of cdna as template. mixing experiments were performed to verify the accuracy of the assay. in brief, gdna (c=20mg/l) of a f508del homozygous and a f508del compound heterozygous cf patient were mixed in such a manner that 0.1, 0. amplification products of the f508del and the wt allele (both 328bp long) were analyzed by gel electrophoresis (page) and direct sequencing (abi 3100) to control specificity. our results using gdna and cdna showed that there occurs unspecific hybridization/ ligation for both probe pairs. the proportion of unspecific amplification products varies between 0.01 and 0.005 and increases the lower the initial number of templates is. however, the specificity of this assay can be significantly improved by increasing the hybridization temperature and/or decreasing the ligation time. conclusion: although some minor limitations concerning allele specificity the lasq assay has been proven to be an accurate, reliable and reproducible method for allele specific quantification and may be applied for several important questions in cystic fibrosis such as the exact determination of the amount of nmd of cftr mrna containing a premature termination codon (ptc) or the allele specific determination of aberrant splicing of cftr mrna . background & aims: as clinical presentation varies significantly among cf patients with the same genotype, e.g. in the f508del homozygous, it is evident that factors in addition to the cftr genotype such as modifier genes, are involved in determining disease severity. however, only one of several previously postulated modifier genes, the tgfβ1 gene, could recently be confirmed in a large association study. hence the identification of new modifier genes is a very important task in order to find new explanations for the heterogeneity of pulmonary disease in cf patients. we decided to search for new potential modifier genes applying a quantitative proteomic approach comparing the proteomes of a wild type (16hbe14o-) and a f508del homozygous bronchial epithelial cell line (cfbe41o-). the main goal of this study is the identification of up or down regulated proteins in the cfbe cell line which may act as modifiers of cf disease. material & methods: materials: we used two bronchial epithelial cell lines, e.g. a wild type (16hbe 41o-) and a f508del homozygous (cfbe 14o-) cell line which we obtained from dr. gruenert (california, usa). additionally, we also used nasal cells from f508del homozygous cf patients obtained either from nasal brushings or nasal polyps. methods: proteome analysis was performed by making 2d-gels using high sensitive staining protocols (ruthenium and deep purple). quantitative analysis was accomplished applying the powerful dige (difference in gel electrophoresis) method. for each dige experiment we made 4 gels whereby each cell line was twice labelled with cy3 and cy5 (=4 technical replicates). finally, identification of the protein spots was done by the use of a maldi-tof mass spectrometer. results: in a first step we established the proteomes of the two bronchial epithelial cell lines. we were able to optimize protein extraction and 2d gel electrophoresis in such a manner that the proteomes of the two cell-lines looked very similarly and the assignment of spots could easily be done. protein spots from both cell lines were analyzed using our mass spectrometry (maldi-tof ms) and allowed the identification of more than 60 different proteins so far. in a next step we quantitatively compared the proteome of the two cell lines using the 2d-dige method leading to the identification of 8 proteins which are down regulated and 5 proteins which are up regulated at least twofold in the cfbe cell line. out of the aforementioned 13 differently expressed proteins 3 could already be identified because they were among the 60 previously determined proteins. while glutathione s-transferase p and protein s100-a11 (s100 calcium binding protein) were down regulated (3.13 and 2.08-fold respectively), superoxide dismutase was up regulated (2.62-fold) in the cfbe cell line. conclusion: comparative quantitative proteomics using the dige method is a promising tool in search of potential new modifier genes which may unravel one of the key problems in cf: the large heterogeneity of pulmonary disease in f508del homozygous patients. background: cystic fibrosis is one of the most common autosomal recessive disorders among caucasians, and manifests a wide range of disease severity. although this range of disease expression can be attributed, in part, to specific mutations within the cftr gene, much of this variability has not been adequately explained. from the gene modifier study (gms-a multicenter study of 1,306 cf patients), ten genes were tested as potential modifiers of cf, and the tgfβ1 codon 10 cc genotype was associated with lung disease severity. objective: to test whether the adverse codon 10 cc genotype is associated with higher circulating levels of tgfβ1, compared to the tt genotype in cf patients and healthy controls. if true, then a link will be established between genotype, disease severity, and circulating levels of tgfβ1, and have implications for novel treatment of cf patients. methods: the study includes 60 clinically stable cf patients and 60 healthy control subjects equally distributed between the cc and tt genotypes. the cf patients enrolled are age 8 and older, of both genders, and all ethnicities that fulfilled the standard diagnostic criteria for cf, using the genetic information from the gms. healthy controls are age 18 and older, caucasian males and females, obtained from the environmental polymorphism registry (epr), a dna registry of 20,000 self-reported normal volunteers. we genotyped 485 blood samples from the epr to define 30 healthy control subjects for each of the cc and tt cohorts. subjects have a blood draw of 35 ml. blood is divided into 4 tubes and each tube is used to measure a different parameter: cbc with differential; tgfβ1 levels in platelet poor plasma by quantikine human tgfβ1 elisa kit; tgfβ1 levels in the buffy coatwhich includes platelets; and tgfβ1 mrna levels in lymphocytes from the buffy coat, using real-time pcr roche light cycler. cbc with differential is performed to quantitate lymphocytes and platelets in order to reference tgfβ1 protein and rna levels to the number of circulating blood cells. analyses will include graphical comparisons between the two groups, chi square analysis, student's two-sample t test, and one way analysis of variance (anova). results: pilot studies show that elisa tgfβ1 measurements are reproducible and mrna levels can be quantified. we have currently enrolled 14 cc genotype and 26 tt genotype of the 60 cf subjects and 11 cc genotype and 19 tt genotype of the 60 healthy controls. blood samples have been collected and processed. conclusion: genetic variants that predispose to more severe cf disease are potential targets for new therapies. we are testing the hypothesis that the adverse (codon 10) cc genotype is likely to reflect increased transcription and/ or tgfβ1 protein synthesis/ secretion. if true, "anti-tgfβ1" therapies could provide a novel therapy in cf. *reporting for the gene modifier study group. supported by cff knowle00a0, cff drumm00a0, gcrc rr00046, nih 5r01 hl68890. aim: centralized periodic evaluations of data from the screening laboratories and cf centers by afdphe (french association for screening and prevention of infant handicaps), were analysed to optimize the efficiency of the program. methods: the strategy combined d3 irt assay/dna analysis (kit elucigen cf-30 arms) /d21-fail-safe irt. revised irt-cut off levels were decided in order to maintain the percentage of positive screens around a 0.5% target. a questionnaire yearly sent to the cf centers collected the cf false negative cases. results: from 2002 to december, 31 2005, 625 cf cases were detected through nbs (2 717 905 screened infants). the i period (p) (n=553 248); d3-irt:60 µg/l and d21-irt: 30 µg/l showed a) 0.82% infant above the d3 cut-off, generating an excess of costly dna tests b) 20% had a d21-irt above the cut-off leading to a very high number of st (n=808) with an extremely low rate of cf (n=1). by increasing slightly both d3/d21 irt cutoff levels (65 µg/l, 40 µg/l) during the ii p (n=1 172 868) a) the number of positive screens decreased to 0.64% b) 11.5% of infants with elevated d21-irt had to be referred for st (n=768) with 9 cf diagnosis. since the risk to had true cf remained very low among infants with no detected mutations, during the iii p (n= 991 789), d21-irt concerned only the ones with d3-irt>100 µg/l and the percentage of infants requiring a st was reduced to 2.5% (n=146, 5 cf). the incidence of cf detected during these 3 p did not vary significantly (1/4289-1/4635-1/4081). another point of concern was the false negative cohort; with a follow-up period over 18 months, 23 cf were detected on clinical symptoms (3.4%) at a mean age of 10 months. only 3 were directly related to the modifications of the strategy. conclusion: centralisation of data made possible changes in the flow charts of the screening strategy to limit the number of false positive cases without significant alteration of the global performance of the program. there were three infants of mixed aa/caucasian origin diagnosed with cf, all had ∆508/genotype on screening. mutation data on the 53 additional aa cf patients followed at the 11 cf centers in new york was collected. six patients have not been genotyped. there were 6 patients who were homozygous for ∆508, 14 patients had only one mutation identified and 5 were not found to have any cf mutations. ∆508(27%) and 3120+1g>a(11%) were the most common mutations. there are five cf patients (including 3 infants diagnosed by nbs) with mixed racial origin who are not included in this analysis. mutations results: the median (range) mbl plasma level was 2.46 (0.04-11.14) µg/ml in cf patients, compared to 1.43 (0.02-11.30) µg/ml in controls. mbl2 genotype frequencies were similar in patients and controls. lung function level was not correlated with mbl genotype or plasma level. the frequency of colonization with pseudomonas aeruginosa was 41% in mbldeficient (xa/o and o/o genotype) children with cf and 15% in mbl-sufficient (a/a and ya/o genotypes) children (p=0.04). we found a trend of a decreased age of first onset of colonization with staphylococcus aureus, haemophilus influenza and pseudomonas aeruginosa in mbl-deficient cf patients (p=0.19, p=0.10, and p=0.14, respectively). conclusions: mbl-deficiency was associated with an increased frequency of pseudomonas aeruginosa colonization in children. mbl deficiency was not associated with lung function deterioration. in a larger cohort we hope to confirm that mbl deficiency influences age at onset of bacterial colonization in cf patients. acknowledgments although there is some evidence that cftr gene mutations may be associated with respiratory diseases, little is known about the relationship between cftr gene mutations and idiopathic bronchiectasis (ngiam et al. 2006) . we have recently showed that a rare allele (-33g>a) in the minimal cftr promoter, previously reported in patients with idiopathic bronchiectasis ( using supershift assays, we demonstrated that these transcription factors bind cftr promoter in vitro. a functional analysis, by using co-transfection assays with expression vectors of each transcription factor in pulmonary epithelial cells, showed that nrf2, irf1 significantly decrease cftr expression, whereas irf2 and sp1 increase it. in an attempt to further elucidate the mechanisms involving these factors in the cftr transcriptional regulation, for instance, to determine whether these factors could interact together in order to regulate cftr transcription, we started several experiments such as co-immnoprecipitation, multiple co-transfection and rnai. taken together, these data evidence that the variant -33a in cftr promoter should be considered as an important risk factor in bronchiectasis pathogenesis. furthermore, we have identified a novel regulatory complex on the minimal cftr promoter, which will enlighten the understanding of the transcriptional regulation of the cftr gene. a better knowledge of cftr cis-and trans-acting elements will allow to consider new approaches to modulate and/or control more specifically cftr expression. this work is supported in part by the association vaincre la mucoviscidose. (nat genet, 1999) . a recent study reports strong genetic influences for mi (blackman et al., gastroenterology, 2006) , but it did not replicate the modifier locus on chr. 19q13. inconsistency of results may reflect the variability of data reporting and different classifications of mi (i.e. surgically or medically treated). we tested the accuracy of reporting of mi on case report forms (crfs), as compared to primary source documents. methods: the crf for the gms has a checklist for past medical history. for example, we requested "yes" or "no" for mi (an obstruction of the terminal ileum at birth), but did not require source documents or identification of the type of treatment. to evaluate crf reporting of mi, we requested source documents for patients with reported mi, including a surgical or medical treatment report. if a written report from the time of birth was unavailable, a clinic note, detailing mi at birth, treatment, and evidence of a surgical, abdominal scar (if applicable), was required. verbal confirmation by the patient and evidence of a surgical scar was also accepted, if no written documentation was available. results: on crfs, 233 of 1371 patients (17%) were initially reported to have mi. to date, source documentation has been obtained for 140 patients, and 112 of those (80%) have been confirmed to have mi (92% by written report, 8% by verbal report). of the 112 with confirmed mi, 92 (82%) had surgery, and 20 (18%) had medical treatment. there were 10 false reports of mi (7.1%). mi could not be confirmed or refuted for 18 patients (12.9%) because of insufficient information (n=16) or confounding circumstances (n=2). to date, documentation for 243 patients who were reported to have no mi has been obtained, and no false negatives have been found. additional documentation for mi from other sites is expected. conclusion: at least 7%, and perhaps as many as 18%, of the reports of mi on crfs were inaccurate. gene modifier studies must include rigorous documentation of mi to ensure an accurate correlation between phenotype and genotype. studies should also characterize different classifications (i.e. surgical vs. medical treatment) to assist the detection of gene modifiers of mi. reporting for the gene modifier study group (mrk); supported by cff knowle00a0, cff drumm00a0, nih 5r01 dk66368, nih 5r01 hl68890, and gcrc rr00046. any mutation that disrupts or diminishes the efficiency of the splicing process will have an impact on disease manifestation. in the cystic fibrosis (cf) transmembrane conductance regulator (cftr) gene more than 1,500 mutations were identified, most of them being disease-causing [1] . among these,~42% are classified as missense and about 13% are classified as splicing mutations, given that they disrupt the consensus splice sites [1] . however, the splicing mutation concept is evolving, as it nowadays also includes mutations other than just those within or close to the consensus splice sites, rendering prediction of mutation consequences a hard task. nevertheless, it is still very important for the clinical settings to determine the functional effect of gene mutations. our aim here was to study the effect of i1234v, a rare cftr missense mutation in nbd2 (exon 19), directly in native tissues of a cf patient bearing f508del in the other allele, so as to gain insight on how it influence the disease outcome. to look for a possible effect at the rna level, total rna was extracted from native nasal cells and colonic tissue, and cdna producd using random primers. rt-pcr amplification was performed in the region spanning exons 18-20, being one of the primers fluorescently labelled. the products were analysed as described before [2] . the i1234v(3832a>g) mutation which creates both a novel acceptor and a novel donor, was found here to cause alternative spliced cftr transcripts lacking the last 18 nucleotides of exon 19, thus showing that only the novel donor is used in vivo. moreover, our data show that no normal (only alternatively spliced) transcripts result from this allele. we have also analysed the i1234v mutation at the protein level by producing a stable bhk cell line expressing the i1234v-cftr, after generating the respective mutant cdna construct by site-direct mutagenesis. protein expression and function was determined by immunoblot and iodide efflux assay, respectively. results show that i1234v-cftr protein is processed and functional. however, given the above-described absence of normally spliced mrna coding for this cftr variant, we have to conclude that this protein is not produced in vivo. indeed, since only the mrna coding for cftr lacking the last six aminoacids of exon 19 was detected, we are currently characterizing in vitro the proprerties of this truncated protein. altogether, our data clearly demonstrate that the functional effect of this mutation is not due to the amino acid change but to abnormal splicing. in conclusion, characterization of the consequences of mutations in native affected tissues is important, not just because this provides unexpected information about the mechanisms underlying the basic defect but also for disease diagnosis and prognosis. (darrah et al, nacfc 2006) . the purpose of this study was to examine the influence of common genetic variation in the endothelin pathway on the cf phenotype. methods: patients were recruited from the cf clinics in dublin, belfast and seattle. serial clinical data were abstracted from medical charts and local clinical databases. studentized residuals of maximum fev1, after adjusting for age, gender and height, was the phenotype of interest. twentyone maximally informative tagsnps were identified in the edn1, edn3, ednra and ednrb genes using the niehs environmental genome project and were genotyped using the illumina beadarray system. genotype and haplotype analysis were carried out using helixtree genetic analysis software. results: clinical and genetic data were available on 544 cf patients (239 from seattle and 305 from dublin/belfast). in the combined cohorts, tagsnps in the ednra gene were significantly associated with differences in cf lung disease severity (p=0.000026). this was observed in both the irish cohort (p=0.0049) and the seattle cohort (p=0.00507). the effects were independent of gender and cftr genotype. four common haplotypes (haplotype frequency>5%) were identified in the ednra gene. there was significant association between ednra haplotypes and cf lung disease severity (table 1 ). there was no association between genetic variants in ednrb, edn1 and edn3 and cf lung disease severity. conclusions: the ednra gene is a genetic modifier of the cf phenotype. our findings were seen in two independent cohorts and verify existing associations found in other populations. the endothelin pathway may be a novel therapeutic target for the treatment of cf lung disease. background: cystic fibrosis (cf) is a recessive "monogenetic" disorder, but there is heterogeneity of lung disease severity and survival reflecting environment and non-cftr genetic modifiers. the unc/cwru multisite gene modifier study (gms) identified patients who were "severe" or "mild" as teenagers or young adults and pertinent cross-sectional data was collected; however, we did not collect all pertinent information about early (age <8 years) clinical features. objective: we sought to retrospectively evaluate the early clinical features of "severe" and "young mild" patients enrolled in the gms. methods: we obtained all cff registry data available on 596 patients. there were 303 "severe" (worst 25 th percentile of birth cohort, age range 8-25) and 293 "mild" (best 25 th percentile, age range 15-28). initial analyses focused on cross-sectional plots of patient age versus multiple clinical features, including age at diagnosis, hospitalizations, cdc height and weight percentiles, presence/absence of ps. aeruginosa from respiratory cultures, and fev 1 (% pred). results: there were 15.5 and 17.6 years of cff registry data per patient for "severes" and "milds," respectively. preliminary results indicate "severe" patients were diagnosed earlier in life than "mild" patients (mean: 1.2 vs. 1.9 years, p=0.004). this difference is greater after omitting patients who had meconium ileus (diagnosis: 1.4 vs 2.3 years, "severes" and "milds" respectively, p=0.003). between 1 and 7 years of age, "severe" patients were hospitalized more frequently than "mild" patients and, from age 8 onwards, the disparity in frequency of hospitalization increased. "severes" and "milds" had similar cdc height percentiles (~35 th percentile) until age 8, at which point percentiles increased more for "mild" versus "severe" patients. by age 3, "severe" patients already had lower cdc weight percentiles than "milds" (32 nd versus 41 st percentile) and this disparity increased throughout adolescence. from age 1 to 8, "severe" patients had a 2-3 fold higher prevalence of ps. aeruginosa than "mild" patients. as early as 6 years of age, fev 1 (% pred) was ~30 points lower in "severes" as compared to "milds." however, some "severe" patients had normal lung function at ages 6-8 and overlapped with mild patients; thus phenotyping of lung severity for young patients is not optimal. all of these results were similar for males and females. summary: retrospective analysis of the cff registry data indicates that patients classified later in life as being "severe" experience a worse course of disease from early in life. understanding the early clinical course may prove helpful in defining surrogate phenotypes for modifier studies, and help define appropriate therapeutic targets. cystic fibrosis is mainly caused by small molecular defects of the cftr gene; despite the genotype is defined in the majority of patients, a number of cf cases still remain uncharacterised. the cf mutation database lists more than 35 large rearrangements that may escape detection using pcrbase techniques. the innogenetics assay, the dhplc and sequencing screening showed a mutation detection rate of 92.6% in our population. we report here the results of mlpa screening for ctfr gene rearrangements, performed on the unidentified alleles of our cf patients. our sample population consists of 691 unrelated italian cf patients (for a total of 1378 alleles), followed at cf centres of lombardia region. mlpa analysis was performed in 40 patients who still had one or two unidentified alleles after extensive analysis of cftr gene. all patients studied had classical clinical cf symptoms. subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, salt loss syndrome and gastro-intestinal findings. we characterized 10 different deletions and a new duplication (dup promoter-ex.3). thus, 26.2% (21/80) tested alleles had a large gene rearrangement. the deletion of exons 22-23 (6/80) was the most frequent in our cohort. all patients had positive sweat chloride values (above 60meq/l), except the patient carrying duplication who has borderline sweat chloride value. out of 24 patients, 6 (25%) had fecal elastase levels consistent with a preserved pancreatic function: of these patients, 3 had mild mutation, 1 had severe, and 2 had unknown mutation. six patients present liver involvement. the results of the present study could indicate that compound heterozygosity for large rearrangements in cftr gene, is strongly associated with severe pancreatic disease as mutations in classes i, ii, and/or iii. l997f is a missense substitution which changes from leucine to phenylalanine at position 997, resulting from a g/c transition at position 3123 in exon 17a of the cftr gene. it has been described in patients with disseminated bronchiectasis, recurrent idiopathic pancreatitis, sarcoidosis, newborns with hypertripsinemia,. recently derichs et al. reported one healthy 3-year-old girl homozygous for l997f; therefore the pathogenic role of the variant is still unclear. in this study we present 6 subjects compound heterozygotes with l997f. no other mutations have been identified after molecular analysis performed using sequencing analysis of the whole coding region of cftr gene and mlpa technique in order to search for gene rearrangements.none of them presented ivs8 5t allele. three of them had a severe mutation in trans (r553x, 2183aa>g, and n1303k), while the other three had a mild mutation on the complementary allele (d1152h, r334q, and r334w). individuals with a severe mutation in trans presented a remarkably different clinical picture compared to those with a mild mutation in trans. the formers were diagnosed at a mean age of 2.8 years, had an average sweat chloride of 55.9 meq/l; besides, all of them have respiratory symptoms, staphilococcus aureus in sputum cultures (one had pseudomonas aeruginosa as well), and in two the chest x-rays was abnormal. the three l997f/ps mutation individuals were diagnosed at a mean age of 20.1 years, had an average sweat chloride of 39.1 meq/l; besides none of them had respiratory symptoms, abnormal chest-xrays, or positive sputum cultures, but two had a history of pancreatitis. these data seem to suggest that l997f cannot in any case be considered a neutral polymorphism. the variability of its clinical expression seems to be influenced by the mutation in trans. further studies are needed in order to support our results. expression of the cystic fibrosis transmembrane conductance regulator (cftr) is tightly regulated both spatially and temporally, yet the molecular mechanism of this regulation is not well understood. because no tissue-specific regulatory elements were recognized in the basal cftr promoter, the crucial cis-regulatory elements are likely to be located elsewhere within the cftr locus. a number of the non-coding regions of the cftr gene were found by our laboratory to contain dnase i hypersensitive sites (dhs), suggesting the presence of regulatory elements at these sites. studies presented here investigate the role of an intron 1 dhs (dhs1) in the tissue-specific regulation of cftr gene transcription. the elucidation of molecular mechanisms underlying the temporal and spatial expression of cftr may aid in developing more specific targeted therapies for cystic fibrosis (cf). footprinting analysis of cftr intron 1 revealed a protected region within the core of dhs1 at 185 + 10 kb. in silico analysis of this sequence uncovered a number of transcription factors binding motifs, including a consensus binding sequence for hepatocyte nuclear factor 1 (hnf1). we have previously identified a role for this factor in the regulation of cftr expression. results of electrophoretic mobility shift assays (emsa) demonstrate that hnf1α specifically binds to the motif in cftr intron 1 in vitro. in addition, chromatin immunoprecipitation (chip) analysis of cells expressing both cftr and hnf1α factor shows that this factor binds to the intron 1 site in vivo. when cloned as an enhancer, the dhs1 element was found to augment minimal cftr promoter activity in a luciferase reporter based assay. this increase in luciferase activity was abolished when two nucleotides within the core hnf1 binding site were mutated, suggesting a functional role for hnf1α in cftr gene transcription. further experiments are underway to determine whether additional transcription factors can be recruited to the core of the intron 1 dhs regulatory element and can interact with hnf1 and the cftr basal promoter to modulate tissue-specific cftr gene expression. the promoter of the cftr (cystic fibrosis transmembrane conductance regulator) gene is not solely responsible for its complex pattern of expression. to identify potential regulatory elements for cftr we previously mapped dnase i hypersensitive sites (dhs) across 400 kb spanning the locus. in addition to intronic dhs, a number of sites were observed that flank the cftr gene. we hypothesized that these may include insulator elements that establish the chromatin expression domain, within which the cftr gene is regulated. insulators are elements that shield against the effects of regulatory elements from adjacent genes, and they may also block silencing of integrated transgenes. using a well-established insulator assay, in which dna regions of interest are assayed for their ability to interfere with communication between a chicken β-globin enhancer and a neomycin resistance gene, we demonstrated that two dhs, at -20.9 kb from the cftr translational start site, and at +15.6 kb from the 3' end of the gene, exhibit enhancer blocking activity comparable to known insulator elements. electrophoretic mobility shift assays (emsa), demonstrated in vitro binding of the well-characterized insulator protein ctcf (ccctc-binding factor) at the -20.9 kb dhs. this was confirmed in vivo in both cftr-expressing and non-expressing cell types using chromatin immunoprecipitation (chip). in contrast, although the +15.6 kb dhs did not bind ctcf, we obtained in vitro evidence for the interaction of other factors that may be involved in insulator activity. furthermore, chip analysis of histone modifications across the cftr locus revealed striking differences between the -20.9 kb and +15.6 kb dhs, again suggesting mechanistic differences between these elements. the characterization of insulator elements flanking the cftr locus may be of direct practical relevance in the design of vectors for effective cf gene therapy. one of the problems encountered in gene therapy protocols is the relatively rapid loss of expression from the cftr cdna once it is introduced into mammalian cells. incorporation of the -20.9 kb and +15. aminoglycosides, particularly tobramycin, are primary antibiotics used to treat the airway infections in cystic fibrosis patients. lifetime, systemic exposure to these antibiotics is significant and can be associated with significant nephro-and ototoxicity. these toxicities have the potential to significantly reduce the quality of life in the aging adult cf population. we previously reported an incidence of aminoglycoside ototoxicity of greater than 50% with sensitive audiometric studies (i.e. pure tone audiometry and distortion product otoacoustic emissions). these studies indicated considerable variability in ototoxicity in patients with similar systemic aminoglycoside exposure. the literature suggests that genetic variability in mitochondrial dna can partially explain these differences. single nucleotide polymorphisms (snps) in the mitochondrial 12srrna gene are associated with aminoglycoside ototoxicity. we genotyped 90 unselected adult cf patients for four of the mitochondrial 12s-rrna polymorphisms associated with aminoglycoside ototoxicity by direct sequencing of dna harvested from peripheral blood. four patients exhibited polymorphisms in this gene. two patients possessed the a1555g transition, while another patient each revealed a polymorphism at a827g and c1494t transitions. the patient with the a827g polymorphism died prior to audiometric testing. both patients with the a1555g had audiometric studies consistent with moderate to severe ototoxicity. while the patient with the c1494t had mild aminoglycoside ototoxicity. all four patients had severe airway obstruction and at least one full course of parenteral tobramycin at 8mg/kg/d. there was no history of toxic serum levels aminoglycosides during this therapy. both patients with the a1555g polymorphism had a family history consistent with the expected maternal inheritance pattern associated with the mitochondrial 12s-rrna gene. we found no evidence of the delt961 polymorphism in this population. these studies indicate a higher than expected frequency (4%) of mitochondrial 12s rrna polymorphisms associated with aminoglycoside ototoxicity. these studies demonstrate that genetic screen-ing provides valuable susceptibility information and may change clinical decision-making. support: rising hope foundation/national cf foundation. cystic fibrosis is a genetic autosomal recessive disease that is caused by deleterious mutations in the cftr gene. in its most severe form, cf results in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency. in fact, cf is the most frequent cause of pancreatic insufficiency in humans. carriers of cf (i.e., heterozygotes) do not express any of these classic symptoms. recent studies indicate that significant numbers of non-cf patients diagnosed with congenital bilateral absence of the vas deferens (cbavd) or idiopathic chronic pancreatitis (icp) are compound heterozygotes and carry two cftr mutations of varying severity. in dogs, especially german shepherds and rough-coated collies, exocrine pancreatic insufficiency (epi) has been observed with symptoms similar to those seen in human patients. epi in dogs is most often caused by pancreatic acinar atrophy (paa), a degenerative disease of the exocrine pancreas that has been shown to be inherited as an autosomal recessive trait. the locus responsible for canine paa has not been identified to date. we considered the possibility that cftr mutations might be responsible for this disease. a dog that had been diagnosed with paa and one that was a known carrier of paa were screened for cftr mutations. our samples also included dogs diagnosed with idiopathic pancreatitis as well as healthy dogs. we have established protocols for detecting putative mutations in the canine cftr gene using temporal temperature gradient gel electrophoresis (ttge). in the dog with paa and in the carrier of paa, our screening methodology identified mutations in 8 of the 27 amplicons that span the 27 exons of the cftr gene. dna sequencing revealed silent mutations in exons 17, 26 and 27. in exons 17 (l888) and 26 (l1402), single nucleotide polymorphisms converted ctt codons into ctc codons, both leucine codons. in exon 27 (p1447) another single nucleotide polymorphism converted a ccc proline codon into a cct proline codon. the other identified mutations (amplicons 15, 16 and 17, 20, 22, 24, 25, 26 and 27) were found in intronic sequences and have no predicted effect on cftr expression or function. based on these findings, we conclude that cftr mutations are not responsible for paa and the epi that it causes in dogs. hypothesis: specific polymorphisms in genes of the innate immunity may modify the severity and progression of cf pulmonary disease and influence pseudomonas aeruginosa colonisation. methods: single nucleotide polymorphisms (snps) analysis of multiple genes contributing to the innate immunity ( (mbl2), masp (mbl associated serine protease) 1ç3, fcn (ficolin) 1ç2, lbp (lipopolysaccharide -binding protein), cd14, tlr (toll-like receptor 1ç10) ) was performed in 116 cf patients (age 6-44 years). spirometric data and bacterial colonisation status with pseudomonas aeruginosa were collected retrospectively. a decline in fev1 of 14 % over a 6 years period and a percent predicted fev1 value of 70 % were used to discriminate mild from more severe affected adult cf patients (≥ 16 years). the frequency of single and combined snps in well-defined subgroups of the cf population was compared. results: in adult cf patients with a mean fev1 lower than 70%, the combinations of snps of cd14 (promoter) with tlr 10 (promoter), fcn 1 with tlr 5 (exon 6), and masp 3 with tlr 9 (promoter) were more common than in adult cf patients with mean fev1 > 70 % (odds ratio (or) 6,2 (95% confidential interval (ci) : 1,7-22,2 ) , or 6, 2 (95 % ci: 1, [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] 2) and or 5,6 (95% ci: 1,7-18,4) respectively). the frequency of combination snps of masp2 with tlr2 and masp2 with tlr4 was significantly higher in cf adults with more than 14 % decline in fev1 compared to cf adults with less than 14 % decline in fev1 (or 6,5 (95% ci: 1,6-25,8) and or 5,3 (95% ci: 1,5-18,6) respectively). however, the single snps of masp2, tlr2, tlr4 were not more frequently found. in pseudomonas aeruginosa colonised cf patients, an increased frequency of combined snps of tlr1 (exon 4) with tlr6 (exon 1), lbp (exon 10) with tlr6 (exon 1) and mbl2 with tlr 6 (exon 1) (or 3,6 (95% ci: 1,3-10,1), or 3,6 (95% ci: 1,3-10,1), or 3,4 (95% ci: 1,1-10,2) respectively) was found. in contrast, for single snp tlr6 (exon 1) no statistically significant differences were found between the groups (or : 2,0 ( 95% ci: 0,9-4,6)). in general, the odds ratios for the combined snps were higher than for the single snps and remained significant when the different groups were subdivided according to cftr genotype. conclusion: certain combinations of snps of genes of the innate immunity are more frequently found in cf patients with a lower fev1, a stronger decline in pulmonary function and pseudomonas aeruginosa colonisation. however, for the single snps this trend was less pronounced or absent. congenital bilateral absence of the vas deferens (cbavd) is a rare condition associated with mutations in cftr. cbavd lacks extra-genital organ system abnormalities; it has been viewed as a mild (variant) form of cf. the disease serves as an excellent model for studying the clinical and experimental ramifications of marginal cftr activity, and the functional thresholds necessary to confer a cf-related phenotype. recently, drumm et al. (nejm, 2005) found that the codon 10 cc genotype of tgfβ-1 is associated with more severe pulmonary disease, particularly in individuals homozygous for ∆f508 cftr. tgfβ is also known to play a critical role during normal development and differentiation of the human vas deferens (han-nema et al., horm res, 2007) . because 70% of cbavd subjects carry at least one defective cftr allele (but millions of males worldwide are heterozygous for cftr mutations without developing cbavd), the present study was designed to test whether tgfβ polymorphism might serve as a genetic modifier underlying isolated vas deferens loss in the setting of cbavd. we sequenced the 5' end of the tgfβ gene in eighty cbavd individuals possessing at least one cftr mutation, and compared genotype frequency with results previously published from demographically similar controls. when cc frequency in cbavd subjects was compared to pooled estimates from earlier trials, no significant differences were seen using a non-dominant model. compared to frequencies reported by arkwright et al. and garrote et al., a greater number of homozygous cc subjects (23.8% vs. 11.4%, p<0.005) was observed. our preliminary results therefore indicate that if tgfβ-1 codon 10 polymorphism is associated with cbavd penetrance, it does not represent the major modifier of disease phenotype. in order to evaluate this possibility further, we plan analysis of a larger (matched) control set of cbavd and non-cbavd individuals, studies of tgfβ-1 activity in vas deferens epithelial cells with and without cftr, and experiments to investigate the effects of tgfβ on cftr biogenesis and function. supported by nih and cff. newborn screening (nbs) for cf has been successful in identifying cf infants, particularly those who have positive sweat tests immediately following the positive screening result. tracking by personnel at the central nbs program facilitates resolution of diagnostic status because cf nbs presents a challenge to cf center (cfc) systems and primary care providers when apparently asymptomatic infants have inadequate (quantity not sufficient (qns)), borderline or indeterminate sweat tests. short-term follow up is more problematic when the screening algorithm requests earlier sweat tests and defines a lower cutoff for indeterminate sweat test results than is used for older infants, children and adults. the massachusetts (ma) cf nbs workgroup chose a low cutoff for indeterminate results of 30 meq/l up to 6 months of age, reverting to the accepted 40 meq/l cutoff thereafter. follow up by the central nbs program identified infants whose diagnostic status remained unresolved more than 6 weeks after a qns or borderline sweat test and eliminated the need for individual cfcs to follow infants tested at more than one cfc. the program sent informational letters and a reminder recommendation to finalize the diagnosis to primary care providers of infants with unresolved diagnoses. of the 189 cf infants identified by ma cf nbs, 25 had an initial sweat result that was indeterminate and another 20 had an initial result that was qns. the central screening program has the capacity to track infant and child outcomes across cfcs; for example, we have been tracking growth outcomes of all cf infants identified by ma cf nbs, despite 31 of the 189 cf infants having moved from one cfc to another during their first 5 years of life. tracking and outcomes data will be presented. when more than one cfc operates within a screening program, centralized tracking ensures capture of quality data. despite several educational campaigns cf is often not diagnosed in early infancy in poland (the mean age of diagnosis 42 months, median is 12 months). after several years of discussions,in september 2006 a neonatal screening for cf supported by the ministery of health was started as the third obligatory neonatal screening in poland. more than 124,000 neonates were screened in the first seven months using the existing screening infrastructure and spare blood spots after pku and congenital hypothyreosis screening. the protocol was two stage irt/dna (covering the 47 most common mutations in the polish population). the final diagnosis was based on sweat tests (conventional pilocarpine-iontophoresis and conductometric nanoduct) as well as clinical examination. values of chloride in sweat >40 mmol/l and nacl >80 mmol/l were considered diagnostic for cf. 12 children were confirmed as cf. although delta f508 is the most common mutation in the polish population, only two infants were delta f508 homozygotes, six were delta f508 heterozygotes and the remaining 8 had different mutations (3 children had 3849+10kbc>). half of the diagnosed group was still pancreatic sufficient. the children had confirmed cf diagnosis at the mean age of 6 weeks. only 3 children were free from any radiological changes in lungs. the most common pathogen in this group was staphylococcus aureus. at the first visit information for parents about cf was given along with physiotherapy and dietary education. anthropometric measurements, airways bacteriology and elastase -1 in stool were performed. psychologist consultations were available. neonatal screening for cf enabled earlier diagnosis and the introduction of complex therapy in comparision with symptomatic screening. objectives: the clinical course of cystic fibrosis (cf) varies widely among patients carrying the same cftr gene mutations supporting that additional genetic modifiers could affect the cf phenotype. as inflammation is a central contributor to the pathogenesis of cf lung disease, genes involved in the inflammatory response are potential modifier candidate genes. methods: we examined the influence of 13 polymorphisms in 7 genes involved in the inflammatory response (tnf, il-1β, il-1 receptor antagonist (rn), il-6, il-8, il-10 and tgf-β1), on disease progression in a group of 329 caucasian children with cf. the genotypes were tested for an association with changes in lung function tests, pseudomonas aeruginosa colonization and nutritional status by multivariable analysis. results: we found a significant association between tgf-β1 +869t/c variants and decline in lung function measured as the forced expiratory volume in one second (fev1) and the forced vital capacity (fvc) (p=0.004 and 0.02 respectively). il-8 gene polymorphisms (-251a/t, +396g/t and +781t/c) were associated with the occurrence of pseudomonas aeruginosa colonization (p<0.02). conclusions: this study suggest that il-8 variants may influence pseudomonas aeruginosa colonization in cf, however this need further confirmation. moreover, we provide additional support to the results of other trials and strongly suggest an association between tgf-β1 variants and lung disease phenotype in cf. if such a role of tgf-β1 is confirmed by functional studies, it may have important clinical impact for the identification of patients at risk to develop more severe respiratory manifestations and for the development of new therapeutic strategies aimed at adequately balancing tgf-β1 production and action introduction: cystic fibrosis (cf) is the most common monogenic disease in caucasian population, its frequency is about one in 2500 live born. the estimated frequency in hispanic population is about one in 8500, with differences in mutation frequency especially for low frequent alleles. heterogeneity in pulmonary manifestations cannot be explained by the cftr mutation genotype. recent studies are focused in the study of modifier genes. these are genes different from the primary cftr gene that could influence the fq phenotype, acting trough mechanisms like inflammation, repair and remodeling, protease-anti protease balance, innate immune response, etc. this work shows some advances in cf diagnosis and modifier genes analysis in mexican population. methods: we tested the effectiveness of innolipa cftr36 kit (innogenetics) in 23 clinically diagnosed cf patients. thirty six frequent mutations in cftr gene were analyzed by pcr and reverse hybridization with allele specific oligonucleotides probes in dna samples extracted from blood or oral swabs. for frequency analysis of modifier genes we tested about seventy dna samples from a dna bank of patients previously tested positive for cftr mutation. the polymorphism analysis of modifier genes was made by pcr-rflp. for alpha 1 antitrypsin (aat), z and s alleles were analyzed according stanford and cols. (1999) . for tumor necrosis factor alpha (tnfα) -308 polymorphism in the promoter region was analyzed according chen and cols. (2006) . results: mutation was detected in in 20 of 23 patients (87% of effectiveness). df508 allele frequency was 52% (caucasian 60-70%) followed by 2789 +5g-a, s549n, g542x, g85e and 1078delt (20%). 28% of the mutant alleles remain undetected. preliminary results in modifier genes show low frequency for aat mutant alleles (1.4% for aat s and z alelle respectively) and 8.7% for tnf-308. conclusions: df508 alelle frequency was 52%, and we found the low fequency alleles s549n and 2789+5g-a. frequency of modifier genes mutant alleles in cf patients are low. this work is in progress of selection and recruitment of patients and controls, as well as standardization of the other genes modifiers, along with some other biomarkers that will allow to understand the physiopathology of cf and to define a possible genetic risk profile of severe pulmonary disease. over 1300 putative cystic fibrosis (cf)-causing mutations have been reported to the cf mutation database and almost half (~630) are rare missense mutations that are predicted to substitute a single amino acid. to determine if any of these rare missense mutations cause cf by altering localiza-tion of cftr, we examined mutations in the cytosolic loops of cftr since localization signals have been identified in cytosolic loops of other ion channels. amino acids alignments comparing human cftr sequence to that of 36 species revealed that cytosolic loop 4 (cl4) was the most conserved loop by identity. furthermore, cl4 had the most naturally observed mutations: 33 of the 68 amino acids were found to be mutated in cf patients and 10 sites had multiple missense mutations at the same amino acid. in addition, different substitutions of the same cl4 amino acid have been associated with different disease consequences, such as the missense mutations in the arginine at codon 1070. patients with the r1070p mutation have pancreatic insufficient (pi)-cf, those with r1070w mutation have pancreatic sufficient (ps)-cf or cbavd, while patients with r1070q primarily had pi-cf. to determine whether the r1070 mutations cause disease by affecting cftr localization, we used the flp-in system to create stable polarized mdck-gfp-cftr cells to produce isogenic clones with cftr expressed from the same integration site. confocal microscopy studies of stable mdck-frt-gfp-cftr-r1070 mutant cell lines revealed that cftr-r1070p was cytoplasmic, cftr-r1070w was apical and cytoplasmic, and cftr-r1070q was apical. to confirm these observations, quantitative biotinylation studies were performed. biotinylation assays of mdck cells stably expressing cftr-r1070p confirmed that this mutant was absent from the apical surface, cftr-r1070w had a low level of fully glycosylated protein at the apical membrane, while cftr-r1070q had fully glycosylated protein at the apical membrane comparable to wild-type cftr. thus, cftr-r0170p and cftr-r1070w displayed properties in polarized cells that were distinctly different from wild-type cftr consistent with their associated cf phenotypes. however, the profile of cftr-r1070q (apical localization and cl-channel function (seibert et al. 1996 , mickle et al. 2000 ) appears inconsistent with a pi-cf phenotype. indeed, re-analysis of cftr genes bearing r1070q revealed that 11 of 12 carried a second mutation in cis (s466x). in nine of these patients in whom cftr genotype and pancreatic status is known, presence of the nonsense mutation s466x is associated with the pi-cf phenotype. since nonsense mutations are known to cause severe gene dysfunction by promoting decay of their rna transcript, we concluded that the s466x mutation rather than r1070q is responsible for the pi-cf phenotype. the finding of s466x explained the otherwise enigmatic functional studies of r1070q and clinical observations in patients bearing this mutation. this study also demonstrates that stable expression of cftr mutants in polarized mdck cells using the flp-in system provides a useful screen for evaluating the disease potential of rare missense mutations. the clinical course of cf is characterized by recurrent pulmonary infections and chronic inflammation. in cf patients, up-regulation of toll-like receptor-2 (tlr2) gene in airway epithelial cells is believed to enhance proinflammatory responses towards bacterial tlr2 ligands. we have recently shown that decreased methylation (demethylation) of the tlr2 promoter is responsible for cf-related up-regulation of tlr2 in bronchial epithelial cells (shuto. t. et al., faseb j, 2006) . however, the molecular mechanisms responsible for dna demethylation-dependent tlr2 gene upregulation in cf cells remain unknown. here, we identified minimum region of the tlr2 promoter critical for its expression at -120/+110, which contains one putative binding site for sp1. sp1 inhibitor mithramycin a treatment reduced tlr2 promoter activity and its expression in cf bronchial epithelial cells (cfbe41o-), suggesting the importance of this sp1 site for the tlr2 gene regulation. moreover, bisulfite sequence analysis revealed the hypomethylation of adjacent this sp1 binding motif within tlr2 promoter in cfbe41o-cells, implying the cf-specific demethylation of adjacent sp1 binding motif. although mithramycin a rarely affected basal expression of tlr2 gene in 16hbe14o-cells (non-cf bronchial epithelial cells), mithramycin a inhibited tlr2 expression in 5-azacytidine (dna-demethylating agent)-treated 16hbe14o-cells. taken together, our results suggest that sp1 is crucial for dna demethylation-induced gene upregulation of tlr2 in cf bronchial epithelial cells. mucus hypersecretion due to goblet cell metaplasia is a critical feature of cf lung disease, affecting both mucociliary clearance and drug delivery. various studies in cf lungs and cf primary cultures have shown increased expression of muc5ac, the main gel-forming mucin produced by goblet cells; however, little is known about the role of muc5ac in the progression of lung disease. to determine whether overexpression of muc5ac alone is sufficient to induce lung pathology, we generated a mouse model overexpressing muc5ac. the entire muc5ac cdna (minus 26% of the predicted vntr sequence, final size ~9kb) was cloned and tagged with an internal gfp epitope at a site close to the vntr to preserve the integrity of mucin domains. gfp-muc5ac was linked to the rat ccsp promoter to drive specific airway expression, primarily in clara cells since these cells are capable of secreting mucins. blastocyst injections were used to generate transgenic founder lines in c57bl/6 background. although muc5ac-gfp mrnas were expressed in transgenic mice, lung histology did not show significant increases in inflammation, ab/pas-positive cells, or luminal secretion compared to wild-type littermates. gfp fluorescence from freshly excised lung was weak but immunohistochemistry showed that the distribution of gfp correlated with the expected expression pattern, with the majority of positive cells localized to the airways and few positive cells in the alveolar space. at higher magnification, gfp-positive granules were observed in dome-shaped cells that were also positive with cc10 antibody, suggesting proper muc5ac-gfp packaging in clara cells. bronchoalveolar lavage samples separated by agarose gel western blots and probed with gfp antibody confirmed that each line secreted muc5ac-gfp, with a migration pattern comparable to a mucin. to further characterize the secreted transgenic mucin, a pool of lavage samples was separated into supernatant and pellet. the pellet was resolubilized in 4m guanidine. both elements were passed through a s1000 size-exclusion column. analysis by light scattering and refractometry to determine molecular weight and concentration revealed that the solubilized pellet contained an enormous but compact complex (920 x 106 g/mol and 320nm radius of gyration). gfp-rich fractions were pas-positive, indicating proper glycosylation of the transgenic muc5ac-gfp. cscl density gradient studies revealed that the gfp-rich fractions had a high molecular weight but did not appear to be a prominent part of the megacomplex retrieved in lavages, unlike other tested mucins. in conclusion, we showed that the overexpressed muc5ac-gfp was a high-molecular-weight protein that was glycosylated, packaged in clara cells and formed multimers. lack of discernable phenotype in the transgenic lungs suggests that mucin overproduction alone is not sufficient to trigger goblet cell metaplasia and mucus accumulation, supporting the protective role of mucins. currently we are crossing the muc5ac-gfp with the scnn1b mouse model to study muc5ac-gfp under dehydrated conditions and we are conducting allergen-challenges in the transgenic mice. results of such studies will provide novel insights into the role of secreted muc5ac in development of lung disease in these models. supported by grants from the cf foundation. the major characteristic in cystic fibrosis (cf) is acquirement of chronic lung infections with p. aeruginosa. once established the chronic infection can only be suppressed, not eliminated. the cf patients harbour one single strain of p. aeruginosa which only rarely is replaced by other strains. during the chronic lung infection there is a year long mutual impact of the bactaria on the host, and of the host response and antibiotics on the bacteria, resulting in a continous adaptation of the bactaria in the lungs of the cf patients with selection for different clones. in contrast, mouse models of the lung infection in cf is limited to two weeks! therefore, we introduced a new strategy infecting different groups of balb/c mice with pulsed field gel electrophoresis-(pfge) identical clones isolated from one cf patient during 23-years of p. aeruginosa lung infection. 1) infecting the mice with non-mucoid isolate showed that the early isolates were significantly better in establishing an infection with reduced clearance of the bacteria from the lungs (p<0.04), increased dissimination of the macroscopic pathology (p<0.002) and a more acute type and a higher degree of inflammation (p<0.05) as compared to mice infected with a late isolate. in addition, the pulmonary cytokine response was comparable with observations in cf where gm-csf and il-10 correlated to a milder disease, whereas g-csf, mip-2 (il-8 analog), and il-1b correlated to severe disease. 2) infecting the mice with the mucoid pfge-identical isolates from the same cf patient revealed a signicantly higher mortality in mice infected with the late isolates (p<0.005). in addition, the pulmonary g-csf and mip-2 response increased in the groups of mice that were infected with the late isolates as compared to the early isolate (p<0.05). moreover, the g-csf and mip-2 increased during the first three days of infection with the late mucoid isolates (p<0.05), indicating an impairment of the host response in controlling the lung infection. 3) when tobramycin treatment was initiated 1h after infection of mice with a mucoid isolate, the number of bacteria were cleared or reduced to a significantly lower level (p<0.05), and a significant decrease of both g-csf and mip-2 at day one, two and three was observed (p<0.03). in contrast, when treatment was initiated after 24h the number of bacteria did not change significantly, and the reduction in inflammatory cytokine response became significant for both late isolates only at day three of treatment (p<0.03). moreover, cytokine levels were significantly lower in the 1h as compared to the 24h group (p<0.04) confirming the importance of early and aggressive antibiotic treatment in cf. non-mucoid isolates were reduced during the first three days of infection independent of treatment. in conclusion, we have implemented a clinical important cf-time perspective (years) in experimental p. aeruginosa lung infection, useful for patho-physiological and treatment studies. cystic fibrosis (cf) is a complex disease that affects multiple organs and results in a wide range of phenotypes in humans. mouse models of cf display many of the phenotypes observed in human patients and provide necessary tools to dissect this multifaceted disease. however, due to the involvement of cftr in various organ systems, the ability to discern the contribution of each organ system to each phenotype of cf is unknown. for example, we have found that mouse growth is dramatically reduced compared to wildtype, and mechanisms ranging from neuroendocrine disruption, intestinal malabsorption and cacchexia have been proposed. we have found that cf mice consume more calories per gram body weight than non-cf mice and intestinal absorption of dietary lipids is similar between the two. cf animals absorb 94% and non-cf 97% (p > 0.5). to identify the source of the growth deficit, it would be advantageous to restrict expression of cftr in specific tissues to determine each tissue's contribution to the phenotype. to accomplish this, we have created a conditional, null murine cftr allele. a conditional null allele is one in which the gene of interest is induced to be non-functional in either a specific tissue or cell type, or at a specific developmental time. examination of cftr absence on a tissue-by-tissue basis will greatly facilitate our understanding of the disease. the conditional cftr allele was created by "floxing" exon 10 of cftr, known to be critical for function, with flanking loxp sites that allow for the deletion of exon 10 when bacterial recombinase cre is present. to date, mice stably transmitting the floxed allele have been created. these founder mice have been crossed with mice expressing cre from the promoter of the villin gene, which restricts transcription of cre to intestinal epithelium. first generation animals show appropriate recombination and deletion of exon 10 in the gut. however, these first generation animals are heterozygous for the floxed allele. we are currently backcrossing these animals with the founders to generate animals with homozygous deletion of cftr in the intestinal epithelium. similar crosses are planned to target other tissues. this new animal model of cf will be available to the cf research community to further our understanding of cf pathophysiology. this work was supported by a grant from the cf foundation increased enac-mediated na+ absorption is a hallmark of cystic fibrosis (cf) airways. we demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) depletion and reduced mucus clearance causing a spontaneous cflike lung disease with high pulmonary mortality, and airway mucus obstruction, mucous cell metaplasia and chronic airway inflammation in adult survivors (mall et al., nature med.10:487, 2004) . the aim of this study was to identify the initiating lesions and investigate the natural history of lung disease caused by asl depletion in βenac overexpressing (βenac-tg) mice. to achieve this goal, we performed longitudinal studies on lung morphology, airway mucus obstruction, bronchoalveolar lavage inflammatory cell counts, expression levels of mucins (muc5ac, muc5b, muc4) and proinflammatory cytokines (tnfα, kc, ifnγ, il-13, eotaxin-1), lung volume, lung mechanics, and airway na+ transport in neonatal to adult βenac-tg mice and wild-type littermate controls. we show that airway mucus obstruction in βenac-tg mice originated in the trachea in the absence of mucous cell metaplasia in the first days of life, and was associated with hypoxic degeneration and necrosis of airway epithelium, and death. in surviving βenac-tg mice, mucus obstruction extended into the lungs and was accompanied by secondary mucous cell metaplasia, increased expression of muc5ac, muc5b and muc4, and airway inflammation with transient increases in macrophages and tnfα, in eosinophils and il-13, and persistent increases in neutrophils and kc in the lung. βenac-tg mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. several disease surrogate markers waned in parallel with βenac transcript levels and airway na+ absorption in adult βenac-tg mice indicating that the level of enac overexpression was critical in determining disease severity. in summary, our results provide several novel insights into the in vivo pathogenesis of lung disease caused by asl depletion. we show that (i) asl depletion is sufficient to initiate severe airway mucus obstruction in the absence of mucous cell metaplasia and/or mucus hypersecretion; and (ii) airway na+ hyperabsorption and mucus plugging-induced hypoxia is associated with airway epithelial necrosis constituting a mechanism that initiates airway inflammation in the absence of infection. further, our results point to a novel link between airway na+ hyperabsorption, eosinophilic inflammation, and emphysema. the high frequency of allergic airway inflammation and emphysema in cf patients warrants further studies of the mechanisms that link asl depletion with these associated pathologies. the balance of airway surface liquid (asl) at a height compatible with efficient clearance of mucus is a process regulated by two main pathways: the secretion of clvia cftr, and absorption of na + via the epithelial na + channel (enac). to study this interplay, an immortal cell line capable of growing at an air liquid interface and having properties of human bronchiolar epithelium would be desirable for its simplicity in preparation and ease of molecular manipulation. an immortal human bronchial adenocarcinoma cell line (hbac) was infected with a lcfsn retrovirus carrying either cftr cdna (hbac cftr ) or the blank vector (hbac mock ). 8-10 day monolayers, grown on transwell clear membranes, were mounted in ussing chambers and studied under open-circuit conditions. to study regulation of clsecretion, the equivalent short circuit current was calculated in the presence of amiloride (apical, 100 µm). in hbac mock cells, forskolin (10 µm, basolateral) had no effect upon i sc . in contrast, hbac cftr cells demonstrated a robust increase in i sc ; a rapid increase to a peak (∆~20 µa cm 2 ), followed by relaxation to a stable and sustained plateau. genistein (50 µm) also stimulated i sc in hbac cftr (∆i sc 13 µa cm 2 ) but not hbac mock cells. amiloride resistant basal current was consistently higher, by 2-3 µa cm 2 , in hbac cftr versus hbac mock cells, presumably reflecting low basal cftr activity. we next examined the ability of endogenous receptors present in hbacs to regulate transfected cftr. stimulation of i sc in response to apical adenosine (100 µm) in hbac cftr was qualitatively and quantatively similar to the forskolin response. while the above experiments provided evidence to suggest that hbac cftr cells possess the bioelectric capability of sustaining anion transport via nucleoside-stimulated cftr activity, it does not guarantee a resultant vectorial flow of fluid. in the final series of experiments, we examined whether adenosine was indeed able to stimulate secretion across the hbac-cftr monolayer by assaying asl height. monolayers were loaded apically with fitc-labeled dextran (20 µl), and the majority of dye was then aspirated. monolayers were then allowed to equilibrate over a 2 hour period, and asl measured by xz scanning on a leica confocal microscope. ~100 µm adenosine and ~10 µm benzamil was then added apically in pfc and asl height measured after 10 and 30 minutes. in both hbac mock and hbac-cftr cells, the basal asl height was ~4 µm. over 30 minutes asl height of hbac mock cells did not change in response to adenosine. in contrast, adenosine stimulated hbac cftr cells demonstrated a doubling of asl height (to 8 µm) over the same period. this response is consistent with the prolonged increase in i sc observed upon stimulation with adenosine. in summary, the hbac model which displays only absorptive currents endogenously, can be transformed into a cell line in which cftr-mediated secretion can be stimulated and sustained by endogenous receptor systems present in the apical membrane. this new model will allow us to study cftr-mediated fluid transport in a defined manipulatable system. despite the identification of the cftr channel more than 15 years ago, many questions remain about its regulation and in vivo function. in order to better understand how cftr functions in vivo, numerous studies have searched for and identified potential interacting partners but to date, there is little evidence linking any of the known cftr interactors to its activity. during a forward genetic screen in zebrafish, aiming to identify genes regulating endodermal organ development, we identified a mutant, which we named baobab (bao), after the african tree that accumulates water during the wet season, which exhibits a dramatically enlarged fluid-filled gut tube. most interestingly, we have shown that various cftr inhibitors reduced the appearance of enlarged guts in bao mutant embryos, suggesting that the fluid accumulation phenotype resulted from an increase in cftr activity. to test whether increased cftr activity resulted from changes in cftr protein levels or localization we raised antibodies against zebrafish cftr. western-blot and immunolocalization studies revealed that the channel was expressed and localized as in wild type in bao mutant embryos indicating that the increased cftr function most likely resulted from the activation of the channel. these data, together with the recessive character of the mutation, suggest that the bao protein is a negative regulator of cftr activity. we have positionally cloned the bao locus and isolated the affected gene. bao encodes a cytosolic protein that has not been previously shown to regulate cftr. in agreement with the pharmacological data pointing to a gut-specific function of bao, in situ hybridization studies showed that bao is higly expressed in gut. we are currently investigating how the bao protein regulates cftr function. in addition, we are also investigating the function of cftr during zebrafish development. these studies lay the groundwork to take advantage of the zebrafish system to further investigate cftr biology. we establish a genetic model system for studying the regulation of the cftr channel that will contribute to the understanding of the pathophysiology of cystic fibrosis and several intestinal secretory conditions. acknowledegements: this work was supported by an embo long-term postdoctoral fellowship to mb and by nih grants to d.y.r.s.. we thank a. verkman for providing cftr inhibitors and k. brand for technical assistance. the trachea of the adult cf mouse (cftr tm1unc or cftr tm1kth ) expresses little cftr and exhibits neither a defect in camp-mediated clsecretion nor hyperabsorption of na + , both signature abnormalities in human cf airways. we generated a mouse exhibiting hyperabsorption of na + in the airways by over-expressing the β subunit of the epithelial na + channel (βenac, gene scnn1b). βenac mice exhibit airway pathology (decreased mucociliary transport, mucus plugging, and airway inflammation) similar to human cf airways. to determine whether inactivation of cftr in βenac mice results in a more severe phenotype, we generated a cf/βenac mouse by crossing the ∆f508 (c57bl/6n) mouse (cf mouse) with the βenac mouse (c57bl/6n). survival analysis showed a drastic reduction in survival for cf/βenac mice in comparison to cf mice or βenac mice. we studied the airway bioelectrics of 3 day-old pups of each genotype. the basal short circuit current (i sc ) was ~5 fold greater in wild-type (wt) (50 ± 13 µa . cm -2 ; mean ± sem, n=4) vs. cf (12.2 µa . cm -2 , n=2) neonatal tracheas. as previously reported, the basal i sc in βenac preps was significantly elevated (99.2 ± 14 µa . cm -2 , n=7) vs. wt tracheas. however, the basal i sc in the cf/βenac tracheas (26.6 ± 19 µa . cm -2 , n=4) was intermediate between the cf and the βenac tracheas. the amiloride-sensitive i sc was elevated similarly in the tracheas of the βenac and cf/βenac pups and was ~ 3x greater than that exhibited by the wt or cf tracheas. the post-amiloride i sc was strikingly reduced in both cf and cf/βenac tracheas compared to the wt and βenac tracheas. we suggest that the post-amiloride i sc reflects constitutive cftr-mediated clsecretion, which may help hydrate airways in the neonate. unlike the tracheas of adult cf mice, neonatal cf tracheas exhibited the classic defect in camp-mediated clsecretion, with the forskolin response in the cf and cf/βenac tracheas being reduced ~10fold compared to the response in the wt or βenac preps. the response to utp (ca ++ -mediated clsecretion) was similar in the four genotypes. absence of cftr-mediated clsecretion does not appear to cause airway pathology in neonatal cf mice, but when this defect is coupled with na + hyperabsorption, it results in a significant increase in neonatal mortality. due to lack of cftr-mediated clsecretion, cf/βenac mice appear to experience greater mucus adhesion, producing death due to asphyxiation. in addition, the tracheas of cf mice (especially congenic c57bl/6n) often appear constricted or collapsed due to a cartilaginous defect resulting in increased compliance. this anatomical defect does not appear to be detrimental to the cf mouse. however, in cf/βenac pups, that accumulate thick, sticky mucus in their tracheas, this tracheal constriction may contribute to their asphyxiation. these results demonstrate the importance of both abnormal na + absorption and decreased clsecretion in the pathogenesis of cf lung disease. supported by nih scor p50 hl060280. recently, we reported that species-specific differences in the airway biology of raav transduction exist between mice and human (am j respir cell mol biol, 2006, 34:56-64) . these differences significantly interfere with the use of mice as surrogate models for human lung gene therapy with certain raav serotypes. to survey which species might be an optimal animal model for raav preclinical testing, we utilized differentiated airway epithelia grown at an air-liquid interface (ali) from five species (human, non-human primate [nhp] , pig, ferret, and mouse) to test the efficiency of transduction with three serotypes of raav (type 1, 2, and 5). our results demonstrated significant species-specific differences in raav transduction biology in airway epithelia. the serotype preferences of raav transduction from the apical membrane in human, ferret, and pig epithelia was raav1>raav2≈raav5, different from that seen in mouse epithelia (raav1>>raav5>>raav2). surprisingly, the profile of raav transduction with these three serotypes in nhp rhesus monkey airway epithelia (raav2≈raav5>>raav1) was less similar to human than pig and ferret. additionally, raav1 lacked a bias in polarity of transduction in human airway epithelia, however, in nhp epithelia, raav1 transduced the basolateral membrane 10-fold more effectively than the apical membrane. furthermore, differences in the properties of raav1 receptors between these species was also seen. using neuraminidase treatment of the apical surface of ali cultures, we tested whether n-linked sialic acids were potential receptors for raav1 binding and/or transduction. these studies demonstrated removal of n-linked sialic acids inhibited raav1 transduction of human, nhp, and mouse airway epithelia, but not ferret and pig. however, raav1 binding in this setting did not correlate with changes in transduction; neuraminidase treatment dramatically reduced raav1 binding to nhp epithelia, but enhanced binding to human epithelia (~100 fold). these findings suggest viral binding to airway epithelial cells may not be a major barrier for raav1 transduction. evidence that proteasome-modulating agents enhancing raav transduction 2-3 logs for all serotypes and species tested, suggest that the ubiquitin/proteasome pathway represents a common intracellular block in raav transduction of airway epithelia. these findings support the notion that viral interactions at the surface of airway epithelial cells (with co-receptors and receptors) influence intracellular processing aav virions that can alter the efficiency of productive transduction. enac surface density in cortical collecting duct (ccd) epithelial cells is established by camp/pka regulated insertion and retrieval of the channel at the apical membrane, together with the recycling of internalized enac back to the apical surface (butterworth et al. (2005) journal of general physiology 125: 81-101). ubiquitination by the nedd4-2 ligase elicits endocytosis of surface enac, which may lead to its degradation. however, enac recycling to the apical surface requires ubiquitin removal by a deubiquitinating enzyme (dub). using a chemical probe approach, we identified ubiquitin carboxy-terminal hydrolase (uch-l3) as the predominant dub in vesicular endocytic and recycling compartments in mouse ccd epithelia. specific inhibition of uch-l3 (10µm of 4,5,6,7-tetrachloroindan-1,3-dione) in filter-cultured ccd cells resulted in a rapid decline in enac currents, which was accelerated by forskolin stimulation, suggesting that uch is responsible for recycling enac to the apical surface. knockdown of uch-l3 significantly reduced both basal and camp-stimulated enac currents (from 7.7±1.2µa/cm2 in control sirna treated to 1.5±0.7µa/cm2 in uch-l3 knockdown cells). to determine whether the same dub was responsible for the regulation of enac in human bronchial epithelial (hbe) airway cells, differentiated cultures derived from cf and non-cf lung tissue were treated with the uch inhibitor. to address the possibility that protease cleaved enac may be regulated differently from unprocessed channels, differentiated hbe cultures were either maintained at air/liquid interface or were submerged under liquid/liquid conditions to alter the cleavage state of enac, as we have previously described (myerburg et al. (2006) journal of biological chemistry 281: 27942-49). in all cases, no significant difference in enac-mediated na+ transport was observed between control and uch-l3 inhibited conditions. these data are in sharp contrast with the mechanisms of enac regulation observed in the ccd model system, and they support our previous findings that enac turnover and mode of regulation in hbe are distinct from the recycling mechanisms defined in the ccd epithelia (butterworth et al. primary human tracheobronchial epithelial (htbe) cells cultured at an air-liquid interface (ali) regulate ion transport, mucin secretion, and cilia beating to recapitulate directional mucus clearance, a key innate defense mechanism that fails in cystic fibrosis (cf). thus, ali htbe cell cultures are crucial for studying cf pathogenesis and for developing/validating novel therapies. different research centers use alternative protocols that enable reproducible creation of ali htbe cell cultures. however, limited primary htbe cell availability has driven the creation of cognate cell lines, accomplished mainly by the introduction of viral oncogenes. viral oncogenes disrupt normal cell physiology causing cellular hyperplasia, abundant apoptosis, genomic instability and, sometimes, poor polarization/differentiation, rendering the cells less useful than primary cells for assessing cftr function. furthermore, genetically unstable cell lines will continuously acquire changes independent of cftr genotype. bmi-1 is a polycomb group protein that controls cell cycle and cell identity via epigenetic regulation of chromatin, maintaining stem cells by suppressing expression of p16, an inhibitor of cyclin dependent kinases. bmi-1 expression has been used to create cell lines that recapitulate normal cell structure and function better than viral oncogene-immortalized cell lines. using hiv lentiviral vectors, we introduced bmi-1 and the catalytic subunit of telomerase to enhance the growth of 3 different ∆f508 homozygous cf and 3 non-cf primary human airway epithelial cell preparations. all 6 new cell lines grew for at least 40 population doublings, while their normal counterparts senesced prior to a maximum of 20 doublings. at passage 14-15, the new cell lines had a diploid karyotype compared to grossly abnormal chromosomes in cells immortalized by viral oncogenes. ussing chamber analysis of ali cultures at passage (p) 14-15 revealed variable transepithelial resistances among the cell lines ranging from 125 ->1000 ω*cm2, but short circuit current (isc) responses stimulated by forskolin and inhibited by cftr172 were true to the cell's genotype and comparable to early passage primary cells, except for one non-cf cell line with relatively low cftr isc's at p15. amiloride sensitive and utp-stimulated isc's were present but were more variable in magnitude in comparison to the currents observed in low passage primary cells. ali cultures exhibited a pseudostratified morphology with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells and occasional ciliated cells. all cf and non-cf cell lines in culture produced similar levels of il-8 at baseline and cf and non-cf cells equally increased il-8 secretion in response to il-1β, tnfα and the tolllike 2 receptor agonist, pam3cys. these novel cell lines will help fill gaps currently hindering cf research and therapeutic development. supported by cystic fibrosis foundation grant randel04g0. we recently considered that domestic dogs could represent a large natural reservoir to search for cftr mutations and, ultimately, to identify and breed animals with cf traits. the canine genome has been completely sequenced, and the canine cftr gene, found on chromosome 14, consists of 27 exons spread over 163 kb. an animal model that faithfully expresses human cf lung pathology is critical for the development of effective treatments for this most lethal aspect of cf disease. while transgenic mouse models of cf replicate numerous aspects of cf disease, they unfortunately do not develop the characteristic lung pathology including mucus plugging of airways, chronic bacterial infections, and bronchiectasis. the reason for this is not completely understood but it is likely related to significant differences in lung morphology between these species. to date, no non-murine genetic model of cf has been developed, and naturally-occurring, cf-causing mutations have not been identified in any non-human animal species. temporal temperature gradient gel electrophoresis (ttge), developed as a screening tool to identify sequence variations and mutations in the human cftr gene, was used to screen dogs for cftr mutations. dna was purified from whole blood received from veterinary clinics treating dogs for a variety of ailments. we have performed ttge analysis on dna from 179 dogs, which includes 24 dogs with pancreatitis and 4 dogs with bronchiec-tasis, and have found hundreds of potential mutations amongst the thousands of amplicons tested. ttge can detect single base pair changes within a fragment of dna. as the temperature increases, the migrating dna becomes partially melted, creating single stranded regions (internal bubbles and branched ends) that decrease its mobility through the gel. the dna fragments have distinct melting profiles that are sequence dependent; therefore, changes in sequence of even a point mutation can alter the profile and result in a change in the rate of migration through the gel that can distinguish wild type from even a homozygous mutant. thus far 65 exonic mutations have been sequenced but none have caused changes to cftr at the amino acid level (i.e., the mutations have all been silent). we conclude that ttge is a sensitive and effective method for screening large canine populations for cftr mutations. ( introduction: the search for "correctors" of defective protein processing of ∆f508 cftr has led to the development of high throughput screening assays to find compounds that promote the surface activity of the mutant protein. by necessity, primary screening assays typically use cell lines expressing recombinant ∆f508 cftr. however, "hits" identified in primary screening require scrutiny for false positives whose activity is either dependent on the ∆f508 cftr expression system or insufficiently robust to correct processing in the complex physiological setting of a native epithelium. to this end, we have developed a secondary functional assay that measures the shortcircuit current (isc) responses in organ cultures of gallbladder mucosa from ∆f508 cftr mice (cftr tm1kth ). previous studies have shown that murine ∆f508 cftr recapitulates the protein processing defect including the capability to increase processing when mucosa is incubated at reduced temperature (27°c) (j. clin. invest. 98:1304 invest. 98: , 1996 . gallbladder mucosa was chosen for this assay because the murine gallbladder can be removed aseptically for organ culture, the epithelium is essentially a monolayer, and the mucosal wall is transparent which facilitates morphological assessment. methods: using either wild-type (wt), ∆f508 cftr (df) mutant or cftr knockout (cftr ko) mice, excised gallbladders were opened longitudinally, rinsed and positioned on loose nylon mesh. the preparation was mounted between two halves of a polycarbonate culture cup with 4.5 mm aperture and placed in submersion culture for 0 -2 days at either 37°c or 27°c using supplemented ham's f12 medium mixed 50:50 with nih 3t3 fibroblast-conditioned dmem containing 2% calf serum. after incubation, the culture cups were placed in custom designed ussing chambers for measurement of isc responses to a cocktail of 10 µm forskolin and 100 µm ibmx (camp isc) at 37°c. contribution of calcium-activated clchannels to the camp isc was eliminated by addition of the distilbene dids (100 µm) to the luminal bath. results: the mean ∆camp isc responses (in µa/cm 2 ) after two days incubation were as follows: wt at 37°c = 21.1 ± 4.1 (n = 4); df at 37°c =3.9 ± 0.3 (n = 5); df at 27°c = 13.6 ± 2.7 (n = 6); and cftr ko at 27°c = 2.8 ± 0.1 (n = 3). in a pairwise comparison, the wt (37°c) was significantly greater than the df (37°c) and cftr ko (27°c) but not significantly different from df (27°c). conclusions: incubation of ∆f508 cftr mutant murine gallbladder mucosa at 27°c for two days in organ culture restored approximately 65% of the cftr-dependent isc responses to camp stimulation. the temperature-dependent effect is consistent with correction of murine ∆f508 cftr processing, indicating a physiological screening assay that is useful for preclinical testing of candidate drugs targeted at correcting the protein processing defect of ∆f508 cftr. supported by cfft and nih. the lack of a large animal model that replicates the lung disease found in humans with cystic fibrosis is a major impediment to understanding disease pathogenesis and the development of effective therapies. although cystic fibrosis is a multi-system disease, airway infection and inflammation currently cause most of the morbidity and mortality in patients. several homozygote null and missense cftr mutations have been developed in mice, but they do not exhibit the lung phenotype observed in humans. in contrast to mice, the structure and physiology of porcine lung and airways closely resemble those of humans. therefore, the goal of this project is to develop a swine model for cystic fibrosis by combining gene targeting and nuclear transfer to produce a pig with a cftr null genotype. we employed homologous recombination to disrupt exon 10 of pig cftr with a neomycin resistance cassette. we generated primary male fetal fibroblasts that were positive for cftr-targeting at one allele as detected by multiple pcr screens. genomic southern blot analysis confirmed proper gene targeting and revealed most clones were free of random integration events. these cells were used for somatic cell nuclear transfer that resulted in the birth of ten cftr null heterozygote piglets. subsequent breeding of these male pigs to wild-type females has yielded at least 9 female cftr null heterozygotes. breeding male and female heterozygotes will provide cftrnull homozygotes. our recent published and preliminary studies demonstrate that mouse slc26a9, a newly discovered member of slc26 family of anion exchangers, is expressed on the apical membrane of tracheal epithelial cells and gastric surface epithelial cells and in the tubulovesicle membranes of gastric parietal cells. the functional properties of mouse slc26a9 were examined in xenopus oocytes and in stably transfected cultured cells. in xenopus oocytes injected with the slc26a9 crna and studied with the voltage and/or ph sensitive microelectrodes, the rank order for anion selectivity at +80mv was cl->i-> no3->gluconate > sulfate, and that of the selectivity (px/pcl) was i-> cl-> no3-= gluconate > sulfate. inhibitor profile studies demonstrated that nppb at 100 µm inhibited the current at 5 min by 40% (p<0.001 compared to control period, n=6). the effect of nppb was partly reversible. dids at 500 µm had no significant effect on the slc26a9-mediated current (p>0.05 compared to control period, n=6). niflumic acid at 100 µm inhibited the current at 5 min by ~38% (p<0.001 compared to control period, n=5) which was partly reversible. cftr inh 172 at 5µm inhibited the current at 5 min by ~23% (p<0.005 compared to control period, n=5) which was reversible. clamping currents in a 85 mm k-gluconate medium and using 10 mm k-cl or k-bicarbonate revealed very low bicarbonate conductance for slc26a9 compared to that of cl-. no significant phi changes were observed in oocytes when favoring cl-exit by substituting perfusate cl-with gluconate in the presence of co2/hco3-. conversely we observed significant alkalinization in ae1-expressing oocytes in the same experimental solutions. interestingly, in hek 293 cells stably transfected with the mouse slc26a9, a significant intracellular alkalinization was observed upon switching to a chloride-free perfusate. the alkalization in slc26a9-expressing cells was inhibited by ~55% in the presence of 500 µm dids. baseline intracellular ph was significantly increased in slc26a9expressing hek293 cells vs. non transfected cells. however, baseline phi was comparable in control and slc26a9-expressing cells in the presence of 100 µm eipa, indicating the activation of endogenous nhe by slc26a9. in conclusion, slc26a9 displays characteristics of an anionic channel in xenopus oocytes, with low bicarbonate permeability. in mammalian expression systems, slc26a9 can also function in cl-/hco3-exchange mode with moderate sensitivity to inhibition by dids. we propose that slc26a9 plays an important role in anion (chloride) secretion and/or ph regulation in tracheal and gastric epithelial cells. no cell lines exist at present that robustly express endogenous wt and ∆f508 cftr on the same genetic background. zinc finger nucleases (zfns) allow the introduction of specific genetic changes at loci within the human genome (nature 435: [646] [647] [648] [649] [650] [651] 2005) . we have been using this technology in an attempt to generate human cells carrying the major cf-causing genotype -∆f508 -at the endogenous cftr locus. such lines could be expected to provide better understanding of the ∆f508 maturational processing defect, as well as new model systems for cell based assays of ∆f508 repair. we performed a bioinformatic analysis of cftr exon 10, and designed multiple distinct zfns against the ∆f508 dna sequence. all of these nucleases were assembled, cloned into mammalian expression vectors, and assayed for dna binding affinity and specificity. the zfns were then assayed for endogenous cftr locus editing in calu-3 and ht-29 cells using a novel, massively parallel methodology applied for the detection of genome editing events. the effort demonstrated that specific zfn pairs successfully bind to, and cleave, within exon 10 of the endogenous cftr locus in both cell types. we will describe experiments that yielded optimized zfns for the cftr locus, and a robust viral platform to transiently deliver genome editing reagents to the human pulmonary cell line, calu-3. supported by cff. the localization of slc26 anion transporters, as well as their function vary among members. some (slc26a2, slc26a3, slc26a6, slc26a11) are widespread, implying specific physiological function in diverse tissues. others are expressed only in a single epithelia, e.g., slc26a5 (prestin) in the inner ear. we have previously found that slc26a9 is a highly electrogenic cl --hco 3 exchanger with a large anion conductance. to determine the possible physiologic roles of slc26a9, we developed a chicken antibody (designed against sulfate transporter and anti-sigma domain, stas, of slc26a9) to localize the protein. to test the antibody specificity for functional protein, we inject xenopus oocytes with crna for slc26a6, slc26a7 or slc26a9 and documented appropriate ph and membrane potential changes. only oocytes functionally expressing slc26a9 were stained using our primary slc26a9 antibody. further, specific staining was eliminated when either pre-immune serum or blocking peptide was used. slc26a9 was only found in epithelia. in the respiratory system, slc26a9 was in the apical membrane of nasal epithelial cells and tracheal submucosal glands. membranes of calu-3 cells, derived from human tracheal submucosal glands, stained indicating that our antibody also recognizes the human slc26a9 protein. slc26a9 is found in the proximal parts of the gastrointestinal tract (esophagus, stomach), with lesser amounts in the small intestine and colon. however, slc26a9 is not present in liver. slc26a9 is present in ducts of secretory epithelia such as salivary glands and pancreas. in the urinary tract, slc26a9 is localized to the renal proximal tubule apical membrane but not in urinary bladder. in the reproductive system, we found slc26a9 in epithelia of the uterus and seminal vesicles but not the vas deference or the prostate. finally, slc26a9 is found on the eye surface, i.e., corneal epithelial cells. together, these results and the varied slc26a9 physiology suggest that several epithelial function models will need to be re-evaluated to incorporate an electrogenic cl --hco 3 exchanger and/or another apical anion channel. (support: romero-06g0, sindic-06f0). a tgfβ-1 polymorphism modifies severity of cystic fibrosis lung disease in the setting of ∆f508 cftr homozygosity (drumm et al., nejm, 2005) . the mechanism(s) underlying this observation, and whether elevated or repressed tgfβ are responsible, are not known. in addition, the pathogenic role of tgfβ in other cf organs and in murine models of the disease has not been well studied. because the tgfβ codon 10 cc genotype is associated with worsened cf pulmonary prognosis, and since high levels of tgfβ are also known to suppress cftr mrna expression and apical cftr protein expression in colonic epithelia (howe et al., exp cell res, 2004) , we developed a mouse model to test tgfβ/cftr interactions that may directly influence cf phenotype. we bred ∆f508 heterozygous mice (cftr tm1kth , ∆f508 cftr) against the tgfβ receptor-deficient murine strain (mtrii28). mtrii28 mice express a dominant-negative mutant form of the tgfβ type ii receptor which lacks the intracellular (kinase) receptor domain. the receptor binds tgfβ, but fails to transmit a tgfβ dependent signal. the tgfβ receptor transgene was inserted downstream of a metallothionein-derived promoter and expressed in numerous murine tissues following induction with 25 mm znso 4 in drinking water. an antibody raised against active tgfβ (lc-1-30-1, gift of dr. kathy flanders, national cancer institute) was used to establish tgfβ in murine tissues including lung and intestinal tract of the mtrii28 strain. rt-pcr confirmed high level transgene expression similar to gapdh internal control. nasal potential difference measurements in cftr +/+ mice expressing the tgfβ transgene indicated intact camp dependent cltransport following induction of the dominant-negative receptor. homozygous cf animals deficient for tgfβ signaling have recently been obtained and exhibit the expected litter sizes and distribution of the ∆f508 allele. studies of cf homozygous mice following induction with znso 4 are in progress, and will be used to determine whether a failure to signal through tgfβ in vivo 1) influences the severity of cf intestinal disease, 2) alters airway or intestinal bioelectric findings characteristic of cf, or 3) changes survival, histopathology or other features of the disease. these experiments are intended to provide an in vivo means of investigating tgfβ signaling and resultant cf modifier effects in vivo. supported by nih and cff. objective: to determine if the abpa phenotype observed in cftr mice was preventable by corrective gene replacement, we delivered aav5 cftr to the lung. in addition to further assess whether cftr-/-lymphocytes alone where responsible for the phenotype, we performed adoptive transfers of cftr-/-lymphocytes into cftr+/+ mice and proceeded to expose the mice to the abpa model. design/methods: cftr-/-mice underwent it administration of raav5 cftr or raav5gfp. all mice were then sensitized with 20ug of crude af antigen via intraperitoneal route. non-injected mice were sensitized without prior virus exposure. challenges were performed on all mice, using an af solution delivered by aerosol inhalation in a closed chamber on 3 consecutive days. serum ige levels were then obtained, as well as bronchoalveolar lavage fluid for differential cell counts, histologic and cytokine analysis. adoptive transfer experiments were done using spleens cells isolated from af sensitized delaf508-/-and f508+/+ mice and transfer into c57/bl6_rag-/-mice. after 7 weeks post transfer c57/bl6_rag-/-mice with f508-/-and f508+/+ lymphocytes were then challenged with af. results: following challenge with af, raav5-cftr mice had significantly lower total serum ige levels (17,251) as compared to raav5-gfp controls (26,892) (p=0.037). analysis of af-specific ige also revealed a two-fold reduction in the mice receiving the corrective gene therapy. curiously, gene transfer to the lung lowered the secretion of inflammatory cytokines from activated splenocytes of raav5-cftr when compared to raav5-gfp controls. these results suggest that correction of immune cells in the lung that traffic to the spleen may be partially responsible for the attenuation of the phenotype. in addition the transfer experiments showed that the hyper-ige syndrome can be partially transferred into b6rag-/-by f508-/-splenocytes. conclusions: correction of cftr deficiency by aav lung transfer attenuates the inflammatory phenotype associated with abpa. epithelial cells lining the vas deferens express cyclooxygenase (cox) 2 when exposed to testosterone in vivo, although no functional link to ion transport has been established. we hypothesized that testosterone modulates cftr-mediated anion secretion across vas deferens epithelia that is stimulated via cox-dependent pathways. vas deferens epithelial cells were isolated from human and porcine tissues and cultured as monolayers on permeable supports until assayed in modified ussing chambers. rna was isolated concurrently for semi-quantitative gene expression analysis. bradykinin (bk) caused an increase in anion secretion (measured as short circuit current) and this effect was inhibited by indomethacin, which indicates cox dependency. testosterone treated monolayers exhibited a bkinduced response that is 58% greater than paired vehicle treated monolayers. qrt-pcr revealed that cox-2 mrna is 19 times more abundant than cox-1 mrna in cultured vas deferens epithelial cells. the effects of bk on ion transport are inhibited by the b2 receptor antagonist hoe140 while the b1 receptor agonist des-arg9-bk produced no effect. prostaglandin e2 and selective agonists of the ep4 (1-oh-pge1) and ep2 (butaprost) receptors produced concentration dependent increases in short circuit current whereas sulprostone, an agonist of ep1 and ep3 receptors, had no effect. taken together, these results suggest that testosterone plays a key permissive role for bk-stimulated anion secretion by inducing cox-2 expression. bradykinin acts at the b2 receptor to increase cox-2 activity and the synthesis of prostaglandins that ultimately bind to ep receptors to promote cftr-dependent hco3-and cl-secretion that would be expected to modify the environment for sperm. the results further suggest that developmentally associated increases in testosterone levels during gestation and puberty will produce physiological changes in vas deferens luminal environment that are likely compromised in cystic fibrosis patients. supported by cystic fibrosis foundation schult06po and ksu cobre nih rr-17686. background: recent studies in cystic fibrosis (cf) suggest a prominent t h 2 profile; a facet that may interfere with normal bacterial clearance. this phenotype appears to increase secondary to infection with aspergillus fumigatus or pseudomonas aeruginosa and also, may interfere with macrophage activation. these same characteristics are present in cftr-deficient mice (cftr s489x -/-fabp-hcftr (+/+), hereafter designated cf mice). as a study of pathogenesis of cf related diabetes (cfrd), we previously observed increased il-10 and splenocyte proliferation in cf mice subject to streptozotocin-induced hyperglycemia. herein, we hypothesized that differences in glucose concentration would increase t h 2 profiles of splenocytes in vitro. methods: cf mice and c57bl/6j mice (b6 mice; n=3/group) were sacrificed at 14 wk of age. splenocytes were collected and cultured at the following glucose concentrations: 10, 15, 20 and 25 mm. each mouse and glucose concentration was stimulated with cona, lps and cd3/cd28. supernatants were collected at 48 hr and measured by luminex analysis. statistical analysis was performed by a one-way anova. results: stimulation by cona produced increased levels of il-10, il-4, tnf-a, gm-csf and il-2 in splenocytes of cf mice (p<0.01 for all). stimulation by lps produced decreased concentrations of il-10 in cf mice(p<0.05). stimulation of cf mice by cd3/cd28 produced decreased concentrations of il-2 (p<0.001). interestingly, no difference was observed in cytokine elaboration as a function of glucose concentration. data are presented in table as fold increase of cytokine concentration for cf mice compared to b6 mice. discussion: profound differences in cytokine production were measured in stimulated splenocyte responses of cf in comparison to b6 mice. the profiles did support the notion for a prominent t h 2 response, with the potential to interference of normal macrophage activation. in addition, differences in cytokine production profiles appear related to the nature of adaptive and innate stimuli provided (cona vs. lps). the absence of influence by glucose concentration in vitro suggests that local/immediate concentration of glucose is not relevant to prolonged expression of cytokine responses to stimuli. further studies on the impact of chronic hyperglycemia and its relation to immune response are necessary. an improved understanding of these mechanisms holds hope for a better overall outcome in patients with cf and cfrd. studies from different laboratories to characterize ∆f508 cftr clchannel activity have yielded disparate results, likely due to variations in technique, cell type, or method of analysis. in the present study, we compared ∆f508 cftr activation in two well characterized polarizing cell models. fisher rat thyroid (frt) cells have been used in high-throughput drug screening to identify ∆f508 cftr active agents, whereas cfbe41ocells have been transduced to provide robust ∆f508 cftr expression in an airway epithelial background. ∆f508 cftr biogenesis following low temperature correction was compared, indicating qualitatively similar levels of cftr transcripts and band b and c cftr. in paired studies in ussing chambers, distinct differences in isc were seen following exposure to cftr activating stimuli. in frt cell monolayers grown at 37°c, forskolin (20 µm) or genistein (50 µm) were strong stimuli of isc, producing mean isc of 13.0 +/-6.6 (forskolin) and 18.6 +/-7.2 (genistein) µa/cm 2 . regardless of exposure sequence, both agents induced equivalent changes in maximal current (59% of 42.2 µa/cm 2 by forskolin when administered prior to genistein; 54% of 44.7 µa/cm 2 by forskolin in converse order). growth at 27°c for 24 hours increased currents approximately 3.7 fold, but relative contributions of the two stimuli were similar to cells grown at 37°c (42% of total current stimulated by forskolin). in cfbe41o -∆f508 cells grown at 37°c, forskolin-dependant currents were minimal and genistein-stimulated currents small (4.2 +/-1.4 µa/cm 2 , compared to zero current in parental, nontransduced cfbe41ocells, p<0.05). in temperature-corrected ∆f508 cfbe41ocells, forskolin was a poor stimulus of cl-transport relative to genistein, contributing 15% to the total current produced by both agents (p<0.001). genistein-stimulated currents did not require pre-treatment with forskolin to produce maximal effects (28.8 µa/cm 2 with genistein alone vs. 31.1 µa/cm 2 after forskolin and genistein), and forskolin did not cause further isc when added after genistein (0 µa/cm 2 ). levels of camp did not account for these differences, since forskolin markedly increased camp in both models. to determine whether excessive phosphodiesterase (pde) or phosphatase (pp) activity limited ∆f508 cftr activation by forskolin in the cfbe41o -∆f508 cells, monolayers were pretreated with the nonspecific pde inhibitor papaverine (100 µm) or the pp2 inhibitor endothall (400 µm), each sufficient to activate isc in cfbe41oexpressing wtcftr (papverine: 59.8 vs. 1.3 µa/cm 2 with vehicle, p<0.005; endothal: 40.1 vs. 8.8 ua/cm 2 , p=0.05). neither agent restored isc in ∆f508 cfbe41oby forskolin (0.8 mv difference, p=ns). these results indicate that activity of ∆f508 cftr in polarizing monolayers is exquisitely dependant on the model chosen for study. substantial differences in ∆f508 cftr behavior in two commonly used cell models, and suggest that the failure of camp to activate ∆f508 cftr in cfbe41ocells is due to cell-specific factors that may 1) influence ∆f508 cftr folding and tertiary structure, or 2) favor interactions of ∆f508 cftr with inhibitory binding partners that limit activation by camp. adverse immune responses to viral vectors or vector-encoded proteins may hinder the practical application of gene therapy. to date, little effort has been devoted to determining the immunological consequences of topically delivered lentiviral vectors to respiratory epithelia. previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (gp64-fiv) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to murine nasal epithelia following a single application of gp64-fiv. longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than 11 months without significant decline. by histological analysis, surface epithelial cells were transduced following a single nasal application of gp64-fiv expressing beta-galactosidase. importantly, we observed that 7 consecutive doses of gp64-fiv delivered over 7 consecutive days greatly increased the number of beta-galactosidase positive epithelial cells. using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days resulted in a linear additive increase in gene expression. in addition, we performed studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. in control mice, we observed that giving 4 nasally instilled doses (2 weeks apart) of an e1/e3 deleted ad5 vector resulted in significantly attenuated expression after the second dose. the attenuated expression correlated to the production of neutralizing antibodies. in contrast, using the same delivery protocol, 4 doses of gp64-fiv resulted in gene transfer without loss of expression of the forth dose. no significant production of neutralizing antibodies was observed following the lentiviral delivery protocol. we further report the additive increase in reporter gene expression following 7 doses of lentiviral vector delivered over 7 consecutive weeks (1 dose/week), as well as the lack of production of systemic and local neutralizing antibodies. gp64-fiv efficiently transduces and persistently expresses a transgene in respiratory epithelia and has the potential for repeat administration without eliciting adverse immune responses. these data have important implications for the application of lentiviral gene therapy to pulmonary diseases. uniform delivery of gene therapy vectors to all lung lobes is important for cf gene therapy. however, this important objective has not been achieved in large animals. to address this obstacle, we have developed an approach to specifically deliver vector into all lung lobes. in this strategy, an intracorporeal nebulizing catheter (aero-probe) is inserted into a bronchoscope to permit visual targeted aerosolization of vector specifically into each lung lobe. using this approach, 2 ml of 0.1% lpc (to transiently open tight junctions) containing 1x1012 vp of hdad-k18lacz was sequentially aerosolized into each of the six major lung lobes of a baboon. aerosolization was triggered by the ventilator upon inspiration at 0.1 ml/pulse at a rate of 8 to 10 pulses/min. halfway through the procedure a very slight, transient and fluctuating decrease in oxygen saturation of no more than 2% was noted that did not warrant supplemental oxygen. the entire procedure was otherwise uneventful and well tolerated with no clinical manifestations of toxicity. no changes in chest x-rays taken at 6 h, 24 h and at necropsy 72 h post-vector were observed compared to baseline. x-gal staining of the lungs at 72 h revealed extensive transduction of the epithelium in the large and small airways in all six major targeted lung lobes. x-gal histochemistry revealed substantial transduction that was exclusively restricted to the airway epithelial cells and submucosal glands, the target cells for cf gene therapy. assessment of the proximal major bronchi from each lung lobe revealed that~20% of the airway epithelial cells were transduced in the left upper lobe (lul), ≥ 90% were transduced in the lml, lll, rul, rml and rll, and~80% were transduced in the accessory lobe. approximately 70% of the airway epithelial cells in the tracheal sections examined were transduced. we also investigated the duration of pulmonary transgene expression. in these studies, we aerosolized a hdad expressing the baboon α-fetoprotein from the k18 promoter into the lungs of baboons. by measuring serum bafp levels, we found that pulmonary transgene expression from transduced airway epithelial cells can be detected for at least 177 days post-vector. these results demonstrate for the first time that exceedingly high levels of transduction of the airway epithelial cells and submucosal glands throughout all lung lobes in a large animal can be achieved with negligible toxicity resulting in long term trangene expression and should pave the way towards successful clinical cf gene therapy. inefficient gene transfer after repeat administration of most viral vectors has limited their suitability for cf gene therapy. however, lentiviral vectors are reported to be less immunogenic. this property combined with their capacity to integrate into the genome of transduced cells and, therefore, allowing gene expression for the life-time of the cell (approximately 3 months for airway epithelial cells and theoretically unlimited for stem cells) justify further investigation of these vectors for cf gene therapy. to ensure high transduction efficiency in the lung without the need for any pre-conditioning strategies we pseudotyped a simian immunodeficiency virus (sivagm)-based vector with the sendai virus f and hn proteins (f/hn-siv-gfp) and transduced mouse nasal epithelium (4x10e8 tu/mouse). at this titre transduction efficiency along the nasal septum was up to 5% when measured 10 and 30 days after transduction (n=4/time-point). the vast majority (approx. 80%) of gfp positive cells across all time-points were respiratory epithelial cells followed by neuronal and sustenticular cells (approx. 20%) in the olfactory epithelium. interestingly, we observed two types of duration patterns of gfp expression in mouse nose. in some mice the number of gfp positive cells decreased over time (day 30: 218±17 cells/section, n=4, day 360: 19±17 cells/section, n=6). however, in other mice strong gfp expression persisted for up to 360 days (day 360: 159±81 cells/section, n=4), significantly longer than the proposed life-span of airway epithelial cells. in order to support the idea of f/hn-siv integration into nasal respiratory stem/progenitor cells we artificially induced cell division after siv vector transduction by damaging the nasal tissue with the detergent (polidocanol), which strips of the epithelium within a few hours, while retaining the basal cell layer. the epithelium completely regenerates over the course of a few days. seven and 28 days after the vector transduction (4x10e8 tu/mouse, n=3) the nasal tissue was perfused with 2% polidocanol (10 µl/mouse) and gene expression was analyzed 4 weeks after the last detergent treatment. gfp expression was detectable in various cell types of the regenerating epithelium including ciliated and basal cells. importantly, gfp-expressing cells were more clustered after polidocanol treatment, possibly indicating origination from a common progenitor. these data suggest that, f/hn-siv integration may have occurred into differentiated airway epithelial cells as well as into stem/progenitor cells involved in the regeneration of the airway epithelium. introduction: hypersecretion of mucus is a major cause of airway obstruction in cystic fibrosis, asthma and chronic bronchitis. though mucus is a complex, non-homogeneous mixture of secretions, one group of its constituents, the mucous glycoproteins or mucins, contributes greatly to the obstruction associated with airway diseases. currently, there are a limited number of therapeutic agents available for controlling mucin overproduction and hypersecretion. the objective of this study is to develop a sensitive, cost-effective technique for reliably quantifying total mucin concentration in a robust mucin-secreting cell line for use in high throughput screening of small molecule compounds with potential to inhibit mucin secretion. methods: an enzyme-linked lectin assay (ella) was adapted using a lectin from helix pomatia (hpa), which binds specifically to n-acetylgalactosamine residues. bovine sub-maxillary mucin was used for test development and later, as a mucin standard. the test sample was "sandwiched" between hpa pre-coated onto 96-well microtiter plates and biotinylated hpa lectin. the plate was developed with a fluorescent hrp substrate, and measured at 590 nm using a standard fluorescence plate reader. using the ella, a number of carcinoma (hm3, a431, ls174, nci-h292) and immortalized lung epithelial (16hbe, cfsme) cell lines were tested to determine their responsiveness to various secretagogues. preliminary experiments indicated that the nci-h292 cells, derived from a human mucoepidermoid lung carcinoma, were most suitable for adaptation to high throughput studies. nci-h292 cells were seeded into 96-plates at a density sufficient to produce confluence in approximately 48 hours. then, individual wells were incubated for 1 hour in either control or secretagogue-containing media. supernatants were collected and mucin concentration of individual wells was determined using the ella. results: optimization of the ella provided a sensitive calibration curve from 150 ng/ml to 2.5 mg/ml. mucin secretory ratios (sr) were calculated by comparing mucin release from test and control wells. significant (p ≤ 0.05) secretory ratios were obtained following incubation with utp at 400 µm (3.17 ± 0.64), vip at 300nm (3.60± 1.41), carbachol at 2.4 mm (2.17± 1.2), interleukin-1β at 5 ng/ml (2.74± 0.47) and heparin-binding egf-like growth factor at 90 ng/ml (2.49± 0.53); all data expressed as sr ± sem, n = 6. histamine at 54 µm did not result in significant mucin secretion. conclusion: a relatively simple and economical assay has been developed for high throughput screening of small molecules for detecting their effects on mucin secretion. cystic fibrosis (cf) is the most common lethal genetic disease in north america, where it occurs with a frequency of one in ~2200 live births. it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene, the most common being the deletion of phenylalanine 508 (delf508). the delf508 mutation causes a folding defect that inhibits maturation and trafficking to the plasma membrane, however the mutant still functions as a chloride ion channel when delivered to the plasma membrane, and partial rescue (15-20%) may be sufficient to alleviate disease symptoms. thus cftr is an attractive target for the development of pharmaceutical therapies. we recently described a novel, cell-based assay for protein trafficking which directly monitors expression of delf508 cftr on the surface of baby hamster kidney (bhk) cells and is suitable for high throughput screening of small molecule libraries. in this study we have extended the approach to natural products by studying 720 extracts derived from marine sponges that were collected from the coasts of indonesia and papua new guinea. sponges are a particularly rich source of novel bioactives and secondary metabolites. iterative use of our high throughput assay and chemical deconvolution of active fractions yielded four alkaloids jmcg1102, jmcg1103, jmcg1104 and jmcg1105. these compounds caused a detectable increase in delf508 cftr trafficking to the plasma membrane within 6 h, and after exposure to 10 pm for 24 h all four increased surface delf508 cftr expression 10-30% and enhanced camp-stimulated halide conductance. we tested a series of analogues of the alkaloids and found three others jmcg1106, jmcg1107 and jmcg1108 that also caused trafficking correction and functional ion channels in the plasma membrane. these results were confirmed in functional studies of the human bronchial epithelial cell-line cfbe stably overexpressing delf508 cftr. these compounds increased surface delf508 cftr by 15-80% within 6 h when treated with 1nm. these results identify a promising group of delf508 cftr trafficking correctors that are now being actively pursued as potential pharmacotherapeutics for cystic fibrosis along with other hits from compound library screening campaigns. work supported as part of the breathe program jointly funded by the ccff and cihr, and grants from cystic fibrosis foundation therapeutics, (cfft). kim chiaw, p. 1,2 ; bear, c.e. 2 background/rationale: biosynthetic misprocessing of the major cf mutant ∆f508-cftr has been proposed to result from the disruption in domain-domain interactions (du et al. nature struct and mol biol, 2005) that may lead to the exposure of di-arginine (rxr) retention/retrieval motifs. simultaneous site directed mutagenesis of all four rxr motifs to rxk or kxr motifs overcame retention and promoted cell surface expression of ∆f508-cftr (chang et al. mol cell, 1999) . we hypothesize that it is possible to compete inhibitory interactions with rxr motifs exposed in ∆f508-cftr by delivering synthetically derived cftr peptides bearing one or a combination of rxr motifs (cf-rxr), each conjugated to cell penetrating peptides (cpp) to permit intracellular delivery. results: confocal immunofluorescence microscopy confirms that alexa-488-tagged cpp-cf-rxr peptides can be taken up rapidly (within 10 min) by bhk cells stably expressing recombinant human ∆f508-cftr. fluorophore-conjugated peptides appear to be concentrated in vesicular compartments, consistent with the current hypothesis that cpp-peptides enter cells into endosomal vesicles via macropinocytosis from which efflux into the cytosol occurs (wadia et al. nature med, 2004) . we determined that treatment of cell cultures with cpp-cf-rxr peptide at a final concentration [1 µm] for 90 minutes is effective in inducing cell surface functional expression of human ∆f508-cftr in bhk cells as observed by a 2.3-fold increase in cyclic amp-mediated iodide efflux over control ∆f508-cftr (n=6; p < 0.01). this rescue is specific to the cf-rxr peptide as controls (cpp alone or peptides bearing a kxk motif in place of rxr) show no functional rescue of ∆f508-cftr (n=6; p > 0.05). preliminary cyclic ampmediated iodide efflux results also indicate that ∆f508-cftr functional rescue by cpp-cf-rxr peptide is as effective as temperature (27 o c) rescue. although we hypothesize that the peptide acts to facilitate trafficking of the major mutant to the cell surface, immunoblotting studies fail to show a clear conversion of the er-form (band b) to the fully processed, band c form of the protein. currently, we are developing more sensitive assays of cell surface expression as well as evaluating the possible efficacy of this peptide in potentiating function of ∆f508-cftr. discussion: future efforts include investigating the relative efficacy of peptides corresponding to the four different rxr motifs in rescuing ∆f508-cftr trafficking. the promising candidate described above is currently being evaluated with respect to functional rescue of the major mutant in primary human bronchial epithelial cells obtained from patients homozygous for ∆f508-cftr (joseph zabner and phil karp, university of iowa) and in cftr-∆f508 mice. these studies may provide the basis for future pre-clinical studies of peptide based therapies of cystic fibrosis (supported by ccff (breathe program grant) and cihr -(strategic training program in the structural biology of membrane proteins linked to disease)). several promising methods of promoting ∆f508 cftr escape from er degradation increase cftr chloride channel activity at the cell surface. however, increasing data from a number of laboratories indicate that in both epithelial and non-epithelial cell lines expressing ∆f508 cftr, the rescued chloride channel is unstable at the cell surface compared to wild-type cftr. small molecule correctors have therefore become a widely used approach to enhance ∆f508 cftr function at the cell surface. in most cases, the exact mechanism of these promising compounds has not been established. in the present study, we use stably transfected, polarized cfbe41o-cells to elucidate the effect of two of these compounds, cfcor-325 and corr-4a, on the surface stability of wild type and low temperature rescued ∆f508 cftr. specifically, we cultured cells expressing ∆f508 cftr at 27c for 48 hours to establish a large pool of surface-expressed ∆f508 cftr; we then monitored cftr internalization rates and half-life with and without small molecule treatment using surface biotinylation-based assays, performed as described previously (jurkuvenaite et al. j. biol. chem. 281:3329, 2006) . as a control, internalization and half-life of transferrin receptor was also followed. our results indicate that both cfcor-325 and corr-4a slow ∆f508 cftr endocytosis from ~30%/2.5 min to ~5%/2.5 min, and this is similar to wild type cftr internalization rates. neither wild type cftr nor transferrin receptor internalization rates were affected by corrector treatment, suggesting that the effects of the correctors were specific for ∆f508 cftr. in the surface half-life studies, both correctors doubled ∆f508 cftr halflife from 2 to 4 hours, but they did not correct the half-life to wild type levels (~8 h). again, the correctors had no effect on either wild type cftr or transferrin receptor surface half-lives. our results suggest that small molecule correctors may not only rescue cftr from erad during biogenesis, but can contribute to the stabilization of ∆f508 cftr at the cell surface, and this effect seems to be ∆f508 cftr-specific. the ∆f508 mutation in cftr causes defects in chloride channel gating and cellular processing. cell cultures models suggest the need for both a potentiator and corrector to restore ∆f508-cftr channel function and processing, respectively. previously, we identified several classes of potentiators, including 'phenylglycines', which probably bind to cftr at the cell surface and restore normal channel gating (pedemonte et al., mol. pharm. 2005; 67:1797 -1807 . we also identified several classes of correctors, including 'bisaminomethylbithiazoles', which may bind to ∆f508-cftr in the er and partially restore its folding and plasma membrane targeting (pedemonte, et al., j. clin. invest. 2005; 115:2564 -2571 . these compounds were identified from screening a diverse collection of 150,000 small molecules; the most active potentiator was phenylglycine pg01 {2-[(2-1h-indol-3-ylacetyl)-methyl-amino]-n-(4-isopropylphenyl)-2-phenyl-acetamide} and the most potent corrector was the bisaminomethylbithiazole corr-4a {n(2-[5-chloro-2-methoxyphenylamino]-4'-methyl-4,5'-bithiazol-2'yl) benzamide}. here we synthesized and characterized a hybrid molecule (corr-4a-linker-pg01) containing both corrector and potentiator fragments. based on sar data of potentiators and correctors, a hybrid molecule was designed that incorporated an enzymatically hydrolysable linker to generate and deliver the potentiator and corrector-linker fragments. using synthetic organic chemistry we synthesized a hybrid molecule containing a pg01-oh moiety and a corr-4a-linker-cooh moiety, linked with an ethylene glycol spacer through an ester bond. the potentiator and corrector fragments (after cleavage) had low micromolar potency or better. however, the intact hybrid molecule was inactive, likely because of its large size (990 daltons) and hence poor cell penetration. as proof-of-principle evidence that the hybrid molecule can be hydrolyzed to the active fragments, treatment with carbonic anhydrase or rodent stool specimens gave the appropriate potentiator and corrector-linker fragments identified by lcms. smallmolecule corrector-potentiator hybrids have potential utility as single drug therapy for cf caused by the ∆f508 mutation. supported by cff and nih. cftr is a chloride channel gated by atp binding and hydrolysis at its two nucleotide binding domains (nbd1 and nbd2). the two nbds may dimerize in a head-to-tail configuration, as observed in other abc transporters, forming two atp binding pockets (abp1 and abp2), with the atp molecules buried at the dimer interface. abp2, formed by the walker a and b motifs of nbd2 and the signature sequence of nbd1, was proposed as the site critical for the atp-dependent opening of the cftr channel, while abp1 (consisting of the walker a and b motifs of nbd1 and the signature sequence of nbd2) may contribute to the stabilization of the open channel conformation. g551d, the third most common cf-associated mutation, is characterized by a very low open probability, and patients carrying this mutation present a severe phenotype. this mutation is located in the signature sequence of nbd1 (and thus in abp2). we have recently characterized this mutant channel and found that its low activity is atp-independent. this behavior corroborates with the idea that the occupancy of abp2 by atp is crucial for catalyzing the opening of the channel. interestingly, we now found that a high affinity atp analog, n 6 -(2-phenylethyl)-atp (p-atp), increases g551d currents primarily by increasing the open time of the channel. this effect of p-atp is reduced when p-atp was applied together with atp, suggesting a competition between atp and p-atp for a common binding site. introducing the mutation y1219g (located at abp2), which reduces by more than 50-fold the atp-binding affinity in wild-type channels, does not alter the effect of p-atp in the g551d/y1219g mutant. in contrast, when we introduce the mutation w401g (located at abp1) in the g551d background, the effect of p-atp is reduced remarkably, suggesting that abp1 is the binding site where p-atp binds to increase the activity of g551d channels. these new results further confirm the idea that nucleotide binding at abp1 could stabilize the open channel conformation. since the competition experiments show that atp and p-atp are readily exchanged in the binding site, we conclude that the atp molecule is not occluded in abp1, at least under the g551d background. our observation that p-atp enhances the g551d activity by binding at abp1 implicates that abp1 can potentially be a target for drugs to bind and increase the channel activity. supported by cff grant to s. bompadre and nih grants to t.-c. hwang (hl53445r01, dk55835r01) and s. bompadre (dk75408k01). van goor, f.; hadida, s.; grootenhuis, p. vertex pharmaceuticals, san diego, ca, usa several cf-causing mutations, including ∆f508, g551d, and r117h, cause gating defects in cftr that decrease the open probability (po) of the channel, resulting in decreased cl-secretion across epithelia of multiple organs, including the lung. a therapeutic strategy for the treatment of cf is to develop pharmacological potentiators that increase po leading to increased fluid transport in affected tissues. vx-770 was identified through cell-based screening and chemistry optimization as a potent potentiator of cftr, including ∆f508-, g551d-, and r117h-cftr. in single-channel recordings of cftr expressed in fisher rat thyroid cells, vx-770 increased the po of ∆f508-cftr from 0.11 ± 0.05 to 0.45 ± 0.06. the po of g551d increased from 0.08 ± 0.03 -0.52 ± 0.19. these levels are similar to the po of wild-type cftr, indicating that vx-770 restores normal gating at the single channel level. to assess the potency and efficacy of vx-770 in native airway cells, cl-secretion was monitored in ussing chamber studies using human bronchial epithelia (hbe) cultured from the bronchi of cf and non-cf donors. in the absence of the potentiator vx-770, cftr-mediated clsecretion in ∆f508/∆f508or g551d/∆f508-hbe reached a maximum amplitude of 2.6 ± 0.5 and 2.7 ± 0.2 ua/cm2, respectively. this is ~5% of that observed in wild-type-hbe (56 ± 6 ua/cm2) and consistent with the low level of residual cftr activity reported in some individuals with severe cf disease. in ∆f508/∆f508-hbe, vx-770 caused a 2-fold increase (peak response; 5.5 ± 0.5 ua/cm2) in cftr mediated cl-secretion with an ec50 of 33 ± 8 nm, whereas in g551d/∆f508-hbe, cl-secretion increased 14fold (peak response; 36 ± 3 ua/cm2) with an ec50 of 600 ± 200 nm. the improved efficacy observed in g551d/∆f508-hbe is consistent with a higher cell surface density of g551d-cftr compared to ∆f508-cftr, which has defective trafficking to the cell membrane. vx-770 was found to be highly selective for cftr and to have good oral bioavailability in multiple species with a half-life of 6 -14 hours. the biological and pharmacokinetic data support the clinical development of vx-770 for the treatment of cystic fibrosis. we would like to acknowledge the cfft for their support and dr. joseph pilewski for providing cf hbe. the pari eflow ® rapid nebulizer is a member of the "soft mist" inhaler class for the administration of aerosolized antibiotics that eliminates the need for a compressor. objectives: to compare the pharmacokinetics and safety of tobramycin inhalation solution (tobi ® ) delivered via the pari eflow ® rapid electronic nebulizer vs the pari lc ® plus jet nebulizer with compressor. methods: we compared tobramycin levels in sputum and serum, incidence of bronchospasm and frequency of adverse events (ae) of 300 mg tobi, administered twice-daily for 2 weeks from these two delivery systems in a randomized, crossover study in 25 cystic fibrosis patients. blood and sputum samples were collected over a dosing interval after the first and last dose on each device (days 1 and 15). tobramycin serum and sputum area under the concentration-time curves (auc) were derived and the therapeutic ratio (mean sputum auc / mean serum auc) calculated. pulmonary function tests were performed before and 30 min after nebulization on days 1 and 15, and ae were recorded. results: the total nebulization time was reduced by half for the pari eflow rapid vs pari lc plus (7.3 ± 1.7 min vs 17.7 ± 3.8 min [p<0.0001] on day 1, and 8.0 ± 1.8 vs 16.3 ± 2.7 [p<0.0001] on day 15, respectively). tobramycin predose serum levels >2 µg/ml or cmax >12 µg/ml, predefined as a potential for increased risk of systemic toxicity, were not exceeded in any patient during use of either nebulizer. as tabulated below, mean systemic exposure to tobramycin from the pari eflow rapid on day 15 was slightly lower, by 14%, whereas mean sputum exposure was higher, by 80%, compared with the pari lc plus. consequently, the therapeutic ratio was nearly 2-fold higher for the pari eflow rapid due mainly to higher sputum concentrations. adverse events occurred in 16 patients using the pari lc plus nebulizer (primarily headache and abdominal pain) and in 19 patients using the pari eflow rapid nebulizer (primarily headache, cough, dyspnea). on day 15, no clinically relevant bronchospasm (defined as ≥20% decrease in fev 1 ) was observed for either treatment. the mean percentage change in fev 1 at 30 mins from predose on day 15 was -2.76% for the lc plus and -1.27% for the eflow rapid. conclusions: tobi delivered via the pari eflow rapid electronic nebulizer required shorter delivery times with similar safety and a higher therapeutic ratio (sputum/serum exposure) compared with the pari lc plus jet nebulizer. sponsored by novartis pharma ag which acknowledges support from pari gmbh introduction alpha-1 antitrypsin (aat) is a protein protecting lung tissues by inhibition of neutrophil elastase. the function of neutrophil elastase is to digest bacteria and other foreign objects in the lungs. in a person deficient of aat, the neutrophil elastase acts uncontrolled, destroying healthy tissue. the result of the damage is emphysema, which progresses if not treated. patients show a specific impaired breathing pattern of short inhalation followed by prolonged exhalation. current therapy consists of weekly i.v. infusion of aat of about 60-90 mg/kg body weight. by this method only about 2% of the administered dose is estimated to reach the lungs. since aat can currently be derived only from human blood serum by an expensive purification process, the worldwide supplies are limited and do not allow to treat all patients. an improvement of the delivery efficiency is highly desirable. inhaled treatment would offer a targeted therapy by delivering aat directly to the lungs and allow for a better use of the limited drug. the eflow ® , a novel electronic nebulizer, is more efficient than conventional nebulizers. it operates by means of a perforated vibrating membrane, applying no or only little stress to the nebulized substances. hence it is well suited for the pulmonary delivery of proteins. it has been shown previously, that a 2% aat solution can be nebulized by eflow ® without a loss of biological activity. we investigated two eflow ® configurations comparing the in-vitro delivered dose for different breathing patterns. method a highly purified 2% aat preparation (kamada ltd, rehovot, israel) was aerosolized with the eflow ® electronic nebulizer. initial studies were conducted using the 30l configuration of eflow ® . delivery performance is compared to a specially designed 30xl configuration, with increased aerosol chamber volume. aerosol delivery efficiency was determined by breath simulation using an emphysema breathing pattern (tidal volume 450 ml, 17 breaths/min, inh:exh ratio = 1:2.5) generated by a pari compas™ breath simulator. as reference, a standardized regular breathing pattern (tidal volume 500 ml, 15 breaths/min, inh:exh = 1:1) was also investigated. the respirable fraction (drug in droplets < 5 and 3.3 µm) was determined using the andersen cascade impactor at 28.3 l/min. samples were analysed for aat activity by an elastase inhibition assay. when the eflow ® 30l configuration was investigated for the standardized breathing pattern, a delivered dose (dd) of 70 ±3% of the loaded dose (92mg) was found. this value was reduced to 50 ±1% when the emphysema pattern was applied. with the new eflow ® 30xl configuration an improved dd of 81 ±3% was obtained. furthermore, when the emphysema pattern was applied, the dd did not drop strongly, but remained at values above 68%. with 83% of aerosol droplets in the respirable size range (<5µm) and 42% <3.3 µm, a high % of aat can be expected to reach both, the central and peripheral lungs of patients. conclusion inhaled aat has the potential to significantly improve the efficiency of aat replacement therapy. a customized eflow ® electronic nebulizer produced delivered doses > 60% even when breathing patterns of patients with impaired breathing capabilities were mimicked. the formulation is optimized for rapid aerosol administration through use of higher drug concentrations and is taste-masked. this study was conducted to evaluate the aerosol performance of novel higher dosed levofloxacin inhalation solutions in a customized eflow ® electronic nebulizer. methods two concentrations of mp-376 (50 mg/ml and 30 mg/ml of levofloxacin) were nebulized at fill volumes of 2 and 4 ml using the eflow ® 35l nebulizer configuration. the in-vitro delivered dose (dd) was determined by breath simulation using a standardized breathing pattern (tidal volume 500 ml, 15 breaths/min, inh:exh ratio = 1:1) generated by a pari compas™ breath simulator. the geometric droplet size distribution was determined by laser diffraction (ld). the aerodynamic droplet size distribution was determined using the andersen cascade impactor (aci). the invitro respirable dose (rd) was calculated by multiplying the respirable fraction (rf, %droplets < 5µm) derived from the cascade impaction experiment and the dd derived from breath simulation. nebulization time was determined by the electronic shut-off of the eflow ® control unit. all tests were conducted for three devices in duplicate, each (n=6). the eflow ® electronic nebulizer delivered mp-376 at both concentrations and fill volumes with equal efficiency, obtaining in-vitro dds of around 60±5 %. the average nebulization times were between 3 and 4 minutes for the 2 ml fill volume and between 7 and 8 minutes for the 4 ml fill volume and were also independent of the concentration in the investigated range. determination of the mass median diameter (mmd) by ld resulted in values between 3.5 and 4.1 µm. values of the mass median aerodynamic diameter (mmad) obtained by cascade impaction were similar (between 3.7 and 4.3 µm). the average respirable fraction was in the range between 70% and 75% for both experimental methods. conclusion the eflow ® electronic nebulizer can be customized to deliver high doses of mp-376. the short nebulization times achieved are patient friendly and should support high patient compliance. mp-376, with the potential for once daily administration, provides significant advantages over currently avail-able aerosol antibiotics. clinical evaluation of mp-376 delivered by the eflow ® nebulizer in cf patients is in progress. lavange, l. 1 ; engels, j. 2 ; accurso, f.j. 3 1. university of north carolina, chapel hill, nc, usa; 2. inspire pharmaceuticals, inc., durham, nc, usa; 3 . university of colorado, denver, co, usa introduction: percent change from baseline in a continuous outcome variable, such as lung function, is a useful descriptive measure in therapeutic clinical trials. while often favored by clinicians as the primary efficacy measure, the use of percent change as the basis for statistical comparisons raises a number of issues. defined as 100*(post-pre)/pre, where pre and post represent baseline and follow-up values, respectively, percent change is a ratio of random variables and as such, does not follow a normal distribution. analyzing percent change with standard methods can result in inefficiences and may be inappropriate without a suitable transformation (e.g., logarithmic). furthermore, the treatment effect estimated on the transformed scale is difficult to interpret. the purpose of this abstract is to describe an alternative method for analyzing percent change and illustrate its utility in cf clinical trials. methods: probability plots are generated to compare the distributions of percent change in fev 1 from baseline to study endpoint among treatment groups. the plots are similar to kaplan-meier curves, with percent change on the horizontal axis in lieu of survival time. the percentage of patients on the vertical axis depicts the cumulative percentage of patients who had a percent change at least as great as the corresponding value on the horizontal axis. a log-rank test statistic provides an overall test for any difference in distributions among treatment groups. the methods are illustrated using data from a 28-day, phase 2, multicenter, randomized, double-blind, placebo-controlled clinical trial of denufosol in cf patients with a screening fev 1 ≥75% predicted. results: a probability plot of denufosol (active doses combined, n=68) vs. placebo (n=21) is shown below. more denufosol patients (43%) experienced improvement compared to placebo (33%). the log rank test, adjusted for study site, showed that the overall distributions of percent change in fev 1 between denufosol and placebo were significantly different (p-value=0.029). conclusions: the proposed methods provide a convenient means for assessing differences in percent change and may be useful in cf clinical trials. comparisons of both a descriptive and inferential nature can be made with minimal assumptions, thereby avoiding the pitfalls in analyzing percent change with standard techniques. the methods are easy to implement with existing software packages (e.g., sas). trial supported by inspire pharmaceuticals, inc. and cf foundation therapeutics, inc. chen, x. 1,2 ; davis, p. 1 compacted dna nanoparticles formulated with pegylated polylysine and plasmid dna transfect airway cells in vivo at high efficiency, so they have great potential for gene therapy. the mechanism by which dna nanoparticles enter the cell and get expressed is not well understood. we previously showed that rhodamine labeled dna nanoparticles enter primary human tracheal epithelial (hte) cells within 15 min, and accumulate in the nucleus in 1 hr, where the transgene can be expressed. dna nanoparticles do not enter via clathrin-mediated endocytosis (cme), which leads to the degradation of internalized material in lysosomes. further characterization of the kinetics and trafficking pathway may allow us to improve the formulation of the nanoparticles and facilitate its gene delivery. as they do in hte cells, rhodamine labeled dna nanoparticles enter hela and 16hbeo-, an immortalized human bronchial epithelial cell line, within 15 min, and by 1-4 hr accumulate in the nucleus, especially the nucleolus, where they colocalize with nucleolin. expression of the gfp reporter gene in the nanoparticles was observed at 18 hr, which confirms the functionality of the labeled dna nanoparticles. cellular entry is energy dependent, as little or no intracellular rhodamine was observed at 4°c by 4 hr. we also applied rhodamine labeled nanoparticles and biotin-conjugated transferrin, a marker for cme, simultaneously. little colocalization was observed during a 15 min to 4 hr time course, so the nanoparticles do not follow this pathway. we then found a direct and strong relationship of cell surface nucleolin and the ability of the cells to take up and express dna nanoparticles. nucleolin directly binds to dna nanoparticles with kd=25.9 nm. manipulations of cell surface nucleolin in hela cells affect transfection of dna nanoparticles with a positive correlation. we therefore costained nucleolin in both hela and 16hbeo-cells treated with the rhodamine labeled dna nanoparticle. the rhodamine fluorescence colocalizes with nucleolin both in the cytoplasm at early time points and in the nucleus/nucleolus at 1 hr and 4 hr, which further supports that nucleolin might be associated with the nanoparticles during their trafficking. to confirm this association, we performed uptake experiments with ms-3, a mouse monoclonal antibody against nucleolin, alone or with rhodamine labeled nanoparticles over a 15 min to 4 hr time course. the ms-3 antibody enters hela cells at similar kinetics to the nanoparticles. at 30 min, we observed substantial amount of cytoplasmic staining, while nuclear staining tends to increase by time until 4 hr. it has little or no colocalization with transferrin in the cytoplasm, which suggests that this antibody also enters the cell via a pathway other than cme. in contrast, it showed extensive colocalization with dna nanoparticles in both cytoplasm and nucleus at all time points, which suggests that cell surface nucleolin and dna nanoparticles are associated or at least in close vicinity during their trafficking inside the cell. therefore, we suggest that dna nanoparticles enter the cell by binding to cell surface nucleolin and enter the cell via the same pathway as nucleolin antibody. chen, x. 1,2 ; davis, p. 1 dna nanoparticles are non-viral gene delivery vectors in clinical trial for treating genetic disorders including cystic fibrosis. we previously discovered that cell surface nucleolin serves as a receptor for the dna nanoparticles, and is important for their gene delivery efficiency. as nucleolin has no signal sequence or membrane-spanning domain, it is not clear how nucleolin are expressed on the outer surface of plasma membrane or what signals increase its surface delivery. we initially observed that the transfection efficiency of dna nanoparticles in hela cell decreases by 72.2% following 24hr serum-free medium treatment, which reduces cell surface nucleolin by 35.7% as determined by cell surface biotinylation and streptavidin bead pulldown. since serum depletion inhibits cell proliferation and affects cell cycle progression, we examined the level of cell surface nucleolin at different stages of cell cycle. hela cells were arrested at s phase by high concentration of thymidine, and allowed to progress synchronously through cell cycle after removing this block. surprisingly, we observed substantial increase of cell surface nucleolin at the onset of m phase, about 8 hours after release of thymidine block. it has been reported that nucleolin is phosphorylated by a cell cycle dependent kinase (cdk) cdc2, and has eight consecutive cdk phosphorylation sites. therefore it is appealing to speculate that phosphorylation by cdc2 might increase the targeting of nucleolin to the cell surface. we then determined the potential export signal in nucleolin by serial deletions. deletion of c-terminal 584 aa of nucleolin, including the c-terminal glycine/arginine rich (gar) domain and the four rna recognition motifs (rrm) does not affect its arrival at the cell surface. the n-terminal 123 aa of nucleolin is sufficient to target a gfp protein to the cell surface. in contrast, when we further delete the 8 cdk phosphorylation sites from aa 69 to 123, cell surface expression of nucleolin is significantly diminished. furthermore, nucleolin lacking the n-terminal 68 aa is not present on the cell surface. therefore, phosphorylation on the cdk sites may serve as a signal to enhance the cell surface expression of nucleolin. there has been recent interest in dry powder inhaled mannitol as a therapeutic agent in patients with cystic fibrosis (cf). it is has been shown to increase mucociliary clearance (mcc) by airway rehydration. whilst there has been one short term clinical trial of mannitol in adult subjects [1] , to date no studies have been conducted looking at its potential as a therapy in children with cf. it could be argued that children may have the potential to benefit most from a therapeutic agent that acts relatively proximally in the cf pathogenic pathway. the aim of this study was to determine acute tolerability of inhaled mannitol in children with cf. we recruited 39 children (aged 8 to 18 years) with cf. inclusion criteria were either rhdnase treatment or an fev 1 > 40% and < 70% of mean predicted normal value. 39 bronchial provocation challenges with incrementally increasing doses (5, 10, 20, 40, 80, 160, 160mg) of dry powder mannitol were carried out. subjects were pre-treated with bronchodilator 15 minutes prior to the challenge (400 mcg of salbutamol or 1 mg of terbutaline). fev 1 was measured following each dose increase up to a maximum cumulative dose of 475mg. oxygen saturation monitoring was carried out throughout. a positive challenge was defined by a drop in fev 1 of >15% from baseline. these subjects received bronchodilator treatment and had spirometry repeated at 15 minute intervals until fev 1 returned to within 5% of baseline. those children with a negative challenge had spirometry repeated 15 minutes post-completion of the challenge results mean baseline fev 1 was 68% predicted (45-94, sd 11.7). 9/39 subjects (23%) had a positive challenge. mean pd 15 (dose of mannitol required to cause a 15% reduction in fev 1 ) was 262mg (411-107, sd 104). the mean time to complete the challenge was 26 minutes (17-37, sd 5.0) for negative challenges and 27 minutes (15-40, sd 8.0) to pd 15 for those subjects with a positive challenge. we found no association between a positive challenge and age, sex, weight, height, baseline fev 1 , pseudomonas aeruginosa colonization, bronchodilator reversibility, previous aspergillus fumigatus sen-sitization, atopy or corticosteroid use. there was a non-significant trend for lower fef 25-75 (means 0.99 versus 1.40; p=0.099) and higher prevalence of aspergillus fumigatus cultured in sputum at baseline (5/9 versus 7/30 children; p=0.066) amongst those children who went on to have a positive challenge. there was no significant drop in oxygen saturation in either group. although cough was common during the challenge, other adverse events were infrequent. we find that 23% of children with cf could not tolerate inhaled mannitol as compared with 12% of adult subjects as reported in the previous study [1] . we could not identify factors predictive of a positive mannitol challenge in these patients. the most common cause of cystic fibrosis (cf) is the deletion of phenylalanine 508 (∆f508) in the cf transmembrane conductance regulator (cftr) chloride channel [1] . a major problem with ∆f508 cftr is that the protein is defective in folding so that little mature protein is delivered to the cell surface [2] . expression of ∆f508 cftr in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein [3] [4] [5] [6] . unfortunately, the efficiency of correctorinduced maturation of ∆f508 cftr is low and other approaches are needed to increase the therapeutic value of correctors. we postulated that expression of ∆f508 cftr in the presence of multiple correctors that bound to different sites in the protein may have an additive effect on maturation. in support of this mechanism, we found that expression of p-glycoprotein processing mutants (cftr's sister protein) in the presence of two compounds that bind to different sites (rhodamine b and hoechst 33342) had an additive effect on maturation. therefore we tested whether expression of ∆f508 cftr in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. it was found that the combination of the quinazoline vrt-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ∆f508 cftr (about 40% of total cftr was mature protein) compared to expression in the presence of a single compound. the additive effect of the correctors on ∆f508 cftr maturation suggests that they may directly interact at different sites of the protein. the use of multiple correctors has the potential to increase the therapeutic value of pharmacological chaperones. pharmacol. 70, 297-302. the cftr protein is expressed at the surface of the airway epithelium, where it plays a critical role in maintaining airway hydration by secretion of chloride. for gene therapy of cf lung disease to be successful, it is critical that the endogenous transgene be expressed in the correct cell type and at levels sufficient to restore normal function. the expression of high levels of cftr has resulted in cftr mediated chloride secretion in a wide range of experimental systems. however, the cell type or types that need to be corrected and the level of cftr expression required to ameliorate the pulmonary manifestations of cf are not yet clear. because cftr has been localized to the ciliated cells of human airway epithelial cells, we have been investigating the use of ciliated cell-specific promoters to improve the efficiency and safety of gene therapy for cf and other airway diseases. in previous studies a fragment of the human foxj1 promoter was shown to target transgene expression specifically to ciliated cells. however, expression of cftr from this promoter in transgenic cf mice did not significantly improve the cf phenotype, as measured by the nasal potential difference (pd) technique (ostrowski et al, 2007) . this may be due to the inability of this promoter to correct the olfactory epithelium, which comprises approximately 50% of the mouse nasal cavity. in cf mice, the olfactory epithelium exhibits sodium hyperabsorption and an absence of cftr mediated chloride secretion, similar to the respiratory epithelium (grubb et al, 2007) . in this work, we used a fragment of the ciliated cell-specific promoter foxj1 to drive cftr expression specifically in human ciliated cells. replication deficient adenovirus expressing either egfp or cftr from the foxj1 promoter were used to transduce well-differentiated cultures of human cf cells following treatment with c10 to disrupt tight-junctions. cultures were studied 48 hours after treatment, and those treated with ad-foxj1/egfp showed strong expression of egfp that was dependant on ciliated cell differentiation. rna analysis demonstrated strong expression of cftr from the foxj1 promoter, and western blotting demonstrated levels of protein that were much greater than the level in normal airway cells (>100-fold). immuno-localization of cftr with specific antibodies resulted in a strong signal at the apical membrane of ciliated cells. cultures treated with ad-foxj1/cftr demonstrated a significant increase in forskolin-stimulated short circuit current (isc; 5.0 +/-0.8 µa/cm2, mean +/-sem, n=9), that was approximately 8-fold greater than the response in ad-foxj1/egfp treated cultures (0.6 +/-0.2 µa/cm2, n=9). the increase in isc was blocked by inh172, an inhibitor of cftr mediated secretion. under these conditions, ad-foxj1/cftr restored approximately 30% of the forskolin response of normal human airway cells (15.1 +/-0.3 µa/cm2, n=3). because patients expressing low levels of normal cftr mrna (5-20%) have mild disease symptoms, these studies demonstrate that the incorporation of the ciliated cell-specific foxj1 promoter into gene therapy vectors may be useful for treatment of cf. supported by nhlbi ro1 hl70199 and the cff. there is a compelling need for safe and effective ai therapy for cf lung disease. low-dose methotrexate (mtx) has been used to treat inflammatory diseases and its use has been reported in cf. encouraging results previously described regarding mtx in cf prompted this study. the study objective was to determine if mtx safely reduces inflammation in the airways of cf patients. methods: this was a single-center, open label study of mtx in stable cf patients with mild to moderate lung disease. baseline levels of neutrophils, free elastase, pro-inflammatory cytokines, and bacteria were determined from an induced sputum specimen. subjects received 15 mg/m2/week of oral mtx (single dose). after 12 weeks of treatment, a second induced sputum specimen was obtained for the same inflammatory indices. within subject comparisons (end of treatment vs baseline) were performed for the following primary endpoints: total white cell and neutrophil counts, percent neutrophils, and concentrations of free elastase, il-8, il-6, tnf-α, and il-1β. sputum quantitative microbiology was obtained at baseline and end of therapy. routine laboratories and spirometry were performed monthly. pharmacokinetic testing was performed on the final day of treatment. results: thirteen subjects were screened with 8 started on mtx. six completed the protocol and 2 withdrew early secondary to adverse events (gastrointestinal [gi] complaints and pulmonary exacerbation). five of 6 subjects completing the protocol had declines in pulmonary function: mean change in fvc was -0.257 l (range -0.47 to -0.09); mean change in fev1 was -0.15 l (range -0.29 to -0.06); change in fef25-75 was 0.01 l (range -0.25 to 0.22). mean change in weight was -2.7 pounds (range -12 to 7.5). mean change in free elastase was 62.4 µg/ml (range -68.8 to 406.4; sd 177.0). mean change in il-8 was 121511 pg/ml (range -122248 to 238400; sd 121511). similar results were obtained for the other cytokines. analysis of cytology specimens is pending. esr and crp did not show significant changes. two subjects completing the protocol had significant gi side effects, including 1 requiring admission for severe abdominal discomfort. quantitative microbiology specimens revealed an increase in colony counts in 2 subjects, decreases in 3, and mixed results in the 6th. other safety laboratories were not remarkable. pharmacokinetic studies are pending. complete statistical analysis and safety assessment are also underway. conclusions: the small sample size of this study precludes definitive conclusions regarding the safety or efficacy of mtx. however, the data suggest that 1) induced sputum inflammatory indices can be used to assess an ai in clinically stable patients, and 2) mtx therapy may not be beneficial, and may be difficult to tolerate as along-term ai therapy for cf lung disease. additional analyses of cell counts and microbiology from this study are ongoing, and may provide additional insight into the effect of mtx in the cf lung. sponsored by the cf foundation , a cellular component of many gram negative bacteria (e.g. pseudomonas aeruginosa), is a common airway stimulus. dampening the inflammatory response in cf reduces the progressive decline in lung function, so anti-inflammatory agents have become both a cornerstone of cf clinical care and a focus of therapeutics research development. reactive oxygen species [ros] can play a role in proinflammatory signaling, including the activation of nuclear factor κb [nfκb] . research has demonstrated increased oxidative stress in cf epithelial cells, potentially highlighting one of the mechanisms responsible for the exuberant inflammation observed. we have previously shown that cddo, a novel anti-inflammatory agent, significantly limits nfκb activation in cftr deficient cell culture models at nanomolar concentrations. using cf mice, we have also shown that intra-tracheal cddo limits neutrophil accumulation and the concentrations of proinflammatory cytokines and chemokines in bronchoalveolar lavage [bal] fluid in response to lps. the synthetic triterpenoids have been shown to activate the nrf2 transcription factor, thereby inducing several genes involved in redox balance. we now present data demonstrating that cddo may inhibit inflammation in models of cf pulmonary disease by reducing the oxidative stress within cells. methods: b6.129s6-cftr tm2mrc cf mice received 25µl of 10µm cddo in pbs with 3%dmso or vehicle (control) daily. drug was administered intratracheally with an atomizer (penncentury) for three days before stimulation. in separate experiments, all mice received 1µg lps or free pseudomonas aeruginosa intratracheally. mice were sacrificed by co2 asphyxiation and cardiac puncture 20 hours after stimulation and bal, serum and lung tissue were obtained. the lungs were incubated in lysis buffer containing protease inhibitors and immediately frozen. upon thawing, the tissue was homogenized and whole lung protein extracted. one mg of lung protein from each experimental animal was separated by two dimensional gel electrophoresis. gels were then imaged and analyzed with pdquest to identify protein spots that were differentially expressed in drugtreated mice compared with controls. spots of interest were excised from the gels, trypsin digested and analyzed by liquid chromatography/tandem mass spectroscopy. results: over 120 proteins with greater than 2-fold differences in expression were identified. we categorized proteins appearing in repeated analyses and identified several affecting intracellular redox regulation; including glutathione s-transferase mu1 and 2, peroxiredoxin 5 and 6, enolase 1 (alpha non-neuron), contrapsin, alpha-1 antiproteinase and cu/zn superoxide dismutase. these proteins were expressed at significantly greater concentration in the lungs of mice treated with cddo rather than vehicle. conclusion: cddo exhibits anti-inflammatory effects in mouse models of cf pulmonary disease and one potential mechanism for this effect may be the upregulation of reducing proteins to combat oxidative stress. methods: lpc (15 ul of 0.1%, 0.3% or 1.0% (w/v) in pbs) was administered (trans-orally via a cannula) to trachea of c57l/b6 mice. in sheep (1 of 5 completed) we targeted delivery of 4.5 ml of 0.3% lpc dissolved in pbs to the main bronchus of the right lung at branch 9 in a 3 month old sheep (bronchoscope, via tracheostomy). one hour later 30 ul (mice) or 0.45 ml (sheep) of a lv-lacz vector (3.12x109 tu/ml) was administered in the same manner. one week post-exposure lungs were inflation-fixed in situ, removed, stained using x-gal, sectioned, and counterstained with saf-o or h/e. results: no lacz gene expression (blue cells) was found in lungs of control (pbs-treated) mice or in the left (untreated side) of sheep lung. blue cells were found in scattered punctuate groups, or in lines of stained cells in mice and sheep. mice given the highest lpc dose (1.0%) had extensive gene transfer in larynx, trachea, carina, and in large, middle and small airways of most lobes of the lung, reaching 79% airway perimeter cell transduction in one animal. with decreasing lpc dose the distribution and number of blues cells was reduced, but transduced epithelial cells including nonciliated columnar cells, ciliated cells, and basal cells were seen in all cases. in sheep, blue cells appeared in the right main bronchus between branch 6 and branch 15, and in the branch 9 airway, and with highest expression found near branch 8 and 9. cross-sections revealed that primarily ciliated columnar cells and basal cells (and no goblet cells or macrophages) were transduced. conclusions: lv gene transfer into mouse or sheep lung can be enhanced by pretreatment with lpc. in mice this early data suggests an lpc dose-dependency. for sheep, we await further results to confirm the encouraging finding in this first study; however, it does confirm that gene transfer into the airways of large animals with a lung size similar to humans can also be achieved using lpc pre-treatment and a vsv-g pseudotyped lentivirus. the transduction of both ciliated and basal lung epithelial cells in-vivo in both models is an encouraging funding for the development and understanding of our continuing efforts to produce life-long airway gene transfer suitable for a cf airway gene therapy. supported by: nh&mrc, usa cff, sa channel 7 crf, philanthropic donations. increased airway na+ absorption is a characteristic abnormality in cystic fibrosis (cf), and is thought to play a key role in the pathogenesis of cf lung disease. we have previously demonstrated that mimicking na+ hyperabsorption by overexpression of βenac in mouse airways results in airway surface liquid (asl) volume depletion and reduced mucus clearance causing a spontaneous cf-like lung disease with high pulmonary mortality, and airway mucus plugging, mucous cell metaplasia and chronic airway inflammation in surviving βenac-transgenic (βenac-tg) mice (mall et al., nature med.10:487, 2004 ). in the present study, we used βenac-tg mice to test if inhibition of increased airway na+ absorption by the classic enac blocker amiloride has therapeutic effects on cf-like lung disease in vivo. specifically, we determined the effects of 'early' and 'late' enac blocker intervention on mortality, airway mucus obstruction and inflammation by starting intrapulmonary amiloride therapy in βenac-tg mice either at birth, i.e. prior to the onset of lung disease, or after mucus obstruction and inflammation had established. to achieve this goal, newborn, 5 day, or 4 week old βenac-tg mice and wild-type littermate controls were treated by intranasal instillation of amiloride (10 mmol/l; 1µl/g body weight, tid) or vehicle (h2o) alone for a period of 14 days. initial deposition studies showed that this treatment protocol resulted in pulmonary amiloride concentrations sufficient for inhibition of enac. during amiloride therapy, growth and survival were monitored, and any loss in body volume due to diuretic side effects was substituted by subcutaneous injection of isotonic saline (nacl 0.9%). after the 14 day treatment period, mice were euthanized, bronchoalveolar lavage (bal) was performed to determine inflammatory cell counts, and lungs were processed for histology and morphometry to quantitate airway mucus obstruction and mucous cell metaplasia. we show that amiloride significantly reduced pulmonary mortality by~70% (p < 0.001), when therapy was started in newborn βenac-tg mice. further, early amiloride treatment significantly reduced airway mucus content (p < 0.05), mucous cell numbers (p < 0.05), and bal eosinophils (p < 0.001) compared to vehicle treated βenac-tg mice. in contrast, amiloride therapy had no benefits on airway mucus obstruction, mucous cell metaplasia or inflammation, if treatment was started in 5 day, or 4 week old βenac-tg mice with established lung disease. taken together, our results are consistent with previous human studies where amiloride lacked therapeutic benefits in cf patients with established lung disease, and demonstrate for the first time that early inhibition of na+ hyperabsorption is an effective therapy for cf-like lung disease in vivo. these results warrant an evaluation of more potent and longer acting amiloride derivatives in chronic lung disease in mice, and a clinical evaluation of preventive enac blocker therapy in human trials. supported by cff (mall04g0) and ec (mext-2004-013666 objective: hypertonic saline aerosol delivered intranasally is currently being studied to enhance mucociliary clearance. there is evidence of patients' reluctance to use concentrations above 3% due to potential discomfort. our study was performed to determine the short term tolerance of 3.5% and 7% hypertonic saline versus normal saline delivered intranasally via a nebulizer/compressor system (pari sinustartm, pari respiratory equipment, midlothian, va). methods: using the sinustartm nasal aerosol delivery system, we administered 3 concentrations of saline solution, (0.9%, 3.5%, 7%) to 18 healthy, adult volunteers for 5 minutes each. a washout period of 5 minutes between treatments allowed volunteers to wipe their nose and cleanse their mouth with water. a 6 question self-administered questionnaire was completed following each treatment using a 9 point scale (1=high tolerability; 9=low tolerability). burning sensation, cold, cough, throat irritation, runny nose, and overall comfort were measured. the order of treatments was randomized and volunteers were blinded to the concentration. data: for all measurements 3.5% and 7% were equally well tolerated compared to normal saline with burning, cough, and throat irritation measuring 2.2 or below. no variable measured more than 3.8. 17 out of 18 volunteers stated they would continue this treatment on a regular basis. conclusions: our study indicates that during the time of treatment nasal aerosol delivery of hypertonic saline up to 7% is well tolerated in healthy adults. there appears to be no issue of discomfort associated with hypertonic saline that would prevent nasal aerosol treatment compliance. our overall research goal is to discover new drugs for clinical treatment of cystic fibrosis (cf) that will correct the biochemical defect in the predominant cf mutation, the ∆f508 form of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which accounts for over 67% of all cf cases. this mutation leads to a misfolded protein which is rapidly degraded as well as changes in its function and half-life at the cell surface. however, it has been proposed that only a fraction of the normal surface expression level is needed to provide a clinically significant impact on the disease. thus, from a pharmacological standpoint, strategies that can partially but effectively correct these defects would be expected to have a clear clinical benefit to these cf patients, and these strategies may also be applicable to other cftr mutations. we previously demonstrated that several members of a particular class of related drugs, the anthracyclines, anthraquinones, and anthracenediones, significantly increased cftr cell surface expression and function in cell culture. in particular, we demonstrated that a non-cytotoxic concentration of doxorubicin (dox), a model anthracycline drug, significantly increased total cellular and membrane-associated cftr protein levels and cftr-associated chloride currents in human colon cancer t84 cells, and also caused a two-fold increase in ∆f508 cftr-associated chloride current in a canine mdck-c7-derived cell line that expresses a stably transfected copy of human ∆f508 cftr. our previous studies also demonstrated that dox is able to impart structural integrity to ∆f508 cftr, increasing its half-life and decreasing its proteolytic sensitivity, which is indicative of efficient folding. additionally, two other structurally related drugs, i.e., the anthracycline, daunorubicin, and the anthraquinone, mitoxantrone, were shown to have similar effects on ∆f508 cftr expression. in the current work, we have extended these effects to two other related aza-anthracenedione molecules, bbr2778 (pixantrone) which is a cancer chemotherapy drug with lower non-target toxicity than dox and which is in phase iii clinical trials, and a structurally related analog, bbr2828, which is essentially non-cytotoxic with a 100-fold lower cytotoxic potency than bbr2778. both compounds increased cftr-associated chloride currents to a similar extent as dox in cfbe human airway epithelial cells expressing ∆f508 cftr, as measured by ussing chamber experiments. the ability of the non-cytotoxic analog bbr2828 to do so is particularly important, since it indicates that correction of the ∆f508 cftr defect is not directly tied to the toxicity of this class of compounds per se but rather is a result of other structural features. thus, it is likely that this and/or other non-toxic analogs in this chemical class can be developed that have potential as clinically useful agents for treatment of cf in patients. supported by a cystic fibrosis foundation (cff) grant to jwh and rm, and by the cff-supported dartmouth cf program. cystic fibrosis (cf) is caused by mutations in cystic fibrosis transmembrane conductance regulator (cftr). cftr is not only an epithelial chloride channel, but it also regulates other transporter and ion channels, including epithelial sodium channels (enac) and aquaporin water channels. in patients with cf, absent or dysfunctional cftr results in abnormal electrolyte and fluid content on the epithelial surface. treatments intended to normalize ion transport in cf airways through non-cftr dependent pathways represent attractive approaches to alleviate the underlying pathologic defect. calcium (ca2+)-activated cl-channels have been proposed as rescue channel for the cyclic amp-dependent cftr cl-channels, offering a target for cf pharmacotherapy. increases in cytosolic ca2+ concentration activate epithelial cl-channels, but inhibit epithelial na+ channels, which is also beneficial in correcting the hyperabsorption of na+ in cf. herein we developed a high throughput screening (hts) assay for screening compound libraries with the intention of finding compounds which can increase intracellular ca2+. in the pilot primary screening, we screened a mssp library which contains 2000 known bioactive compounds and natural products. 7 compounds were classified as the real hits after the secondary screening validation with the hit percentage 0.35%. we have found that the ec50 of these 7 compounds are less than 100um in dose-response studies and none of those compounds were toxic to the cells. we have further discovered that those compounds stimulate cl-secretion through activating ca2+ dependent cl-channels in cf and non-cf human airway epithelial cells in short-circuit current experiments. in summary, our data suggest that modulation of intracellular ca2+ is a target for cf therapy. the compounds identified by our study will provide possible therapeutic leads in the treatment of cf. non-viral gene delivery particles with various synthetic polymer coatings have been developed for cystic fibrosis (cf). intracellular trafficking and in vivo tissue distribution of these particles must be carefully monitored to guide rational design of efficient delivery system. fluorescent semiconductor quantum dots (qds) allow sensitive, long-term and multi-target imaging in cellular environment, and so are a promising tool in tracing gene delivery materials in vitro and in vivo. however, application has been limited by the lack of efficient and reproducible techniques of qd bioconjugation. here, we describe a method of labeling dna nanoparticles with tunable qds that may enable us to study gene delivery in vitro and in vivo. highly fluorescent zns-coated cdse qds were first synthesized and encapsulated in amine-containing phospholipid micelles. the non-viral vector system we used is based on a polymer backbone consisting of 30 lysines with a cysteine on the n-terminus (ck 30 ) which is conjugated to polyethylene glycol (peg). this polymer vector condenses dna plasmids into stable nanoparticles that efficiently transfect airway epithelial cells in vivo. to introduce the aminecontaining qds, we utilized a hetero-bifunctional peg with an nhs ester at one end, which was first reacted with the amine groups of the qds and a maleimide group at the other end which was subsequently reacted with the thiol group in ck 30 . excess reagents were removed from the final conjugates qd-peg-ck 30 by filtration. conjugation was first confirmed by 0.5% agarose gel electrophoresis. free qds were positively charged and migrated toward the cathode. pegylated qds migrated significantly slower than free qds, and qd-peg-ck 30 conjugates migrated at still a different rate. to further demonstrate conjugation, we biotin-labeled the ck 30 and assessed the binding of qd-peg-ck 30 -biotin to avidin coated agarose beads by monitoring the yield of fluorescent beads. qds, pegylated qds, and physical mixture of qds and ck 30 -biotin were also incubated with the agarose beads as controls. fluorescent beads were detected only when qd-peg-ck 30 -biotin was present. to directly evaluate the ability of qd-peg-ck 30 to bind double stranded dna, we also immobilized double stranded dna fragments to agarose beads. qd-peg-ck 30 , but not unconjugated qd bound to the immobilized dna fragments as determined by the yield of fluorescent beads. qd-peg-ck 30 was then used to compact luciferase reporter plasmids. electron microscopy images of the compacted dna showed that qds were integrated into some dna nanoparticles. we transfected hela cells with qd-labeled and unlabeled dna nanoparticles. luciferase assay and toxicity assay indicated that qd-labeled dna nanoparticles transfected hela cells, though with less reporter gene expression than unlabeled nanoparticles and more toxicity than uncompacted qd. in conclusion, we have developed a method to conjugate qd to dna nanoparticles. improvement of the quality and biocompatibility of the qd may be required for further in vitro and in vivo studies. this system is also versatile. other less toxic optical makers can be readily tested. this study was supported by dk58318 and the cff. in cystic fibrosis (cf) the epithelial sodium channel (enac) hyperactivity plays a role in the pathogenesis of chronic lung disease. the missing enac regulation by the cf transmembrane conductance regulator (cftr) causes increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the perciliary liquid layer and to the consequent inhibition of mucus clearance. we developed a hiv-based lentiviral (lv) vector containing a sirna cassette to efficiently knockdown the expression and activity of enac in human respiratory cells. background: sildenafil has been implicated in the relocation of cystic fibrosis transmembrane conductance regulator (cftr) protein. the effect was observed in vitro and in the presence of doses roughly 300 times larger than those commonly used for treating erectile dysfunction. aim: to evaluate in vivo therapeutic efficiency of clinical doses of sildenafil and vardenafil, two approved type v phosphodiesterase inhibitors, for correcting chloride transport defect in ∆f508 mice. methods: we measured transepithelial potential difference in vivo across the nasal mucosa as a readout for sodium and chloride conductance. the effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in df508/df508 and normal homozygous mice. results: in df508/df508 mice, chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected 1 h after sildenafil administration. a more prolonged effect, persisting at least for 24 h, was observed with vardenafil. the forskolin response was increased after sildenafil and vardenafil in both normal and df508 mutant animals. no effect on sodium conductance was detected in any group of animals. conclusion: our results provide preclinical evidence of effectiveness of both drugs for correcting chloride transport defects in the cf. acknowledgments: this work was supported by grants from the french cf association, vaincre la mucoviscidose and by an educational grant from pfizer belgium. there is a strong interest in developing small molecules able to correct the phenotypic effects of cystic fibrosis (cf) mutations. many mutations (e.g. ∆f508) impair the function of cftr protein, by altering the protein targeting to the plasma membrane and/or by causing an abnormally low open channel probability. drug-like organic compounds may restore the correct membrane localization ("correctors") or increase channel activity ("potentiators") of mutant cftr. in the last years, various research teams have identified molecules with activity as correctors (corr-4a, vrt-325, miglustat, curcumin) or as potentiators (tetrahydrobenzothiophenes, phenylglycines, sulfonamides, vrt-532, 1,4-dihydropyridines). however, these compounds have been tested using different assays and the results are sometimes controversial, with marked differences in declared efficacy and potency of compounds. we have tested a panel of correctors and potentiators to directly compare their effects under the same experimental conditions. for this purpose, we have used the functional assay based on the halide-sensitive eyfp-h148q/i152l to measure ∆f508-cftr activity in frt and a549 cells. the assay for potentiators consisted in stimulation with the test compound (0.02 -60 µm) plus forskolin (on cells previously incubated at 27°c for 24 hours). the assay for correctors consisted in 24 hours incubation of ∆f508 cells with test compounds and then determination of cftr activity in the presence of forskolin plus genistein (50 µm). our results indicate that all potentiators are active in our assay with a comparable maximal effect but with values of potency that vary significantly among compounds. the potency order measured in frt cells was: dhp-194 = pg-01 (ka~50 nm) > sf-01 (ka = 140 nm) > dhp-229 = dhp-226 (ka~180 nm) > act-2b (ka = 250 nm) > felodipine (ka = 900 nm) > vrt-532 (ka = 2.6 µm) > genistein (ka = 18.5 µm). a similar order of potency was found also in a549 cells expressing ∆f508. the activity of correctors showed a more marked dependence on cell line. while the potency was comparable between frt and a549 cells, the maximal effects showed clear differences. in frt cells, corr-4a, corr-4b and corr-3c generated a maximal effect that was 1.5 -2-fold higher than that obtained by incubating the cells at low temperature. conversely, vrt-325 and vrt-640 were less effective (maximal correction equivalent to 50-75 % of low temperature rescue). in a549 cells, all compounds were instead less effective than low temperature, with vrt-325 being the molecule eliciting the highest activity (50-60 % of low temperature). our results indicate the 1,4-dihydropyridine dhp-194 and the phenylglycine pg-01 among the most potent activators of the mutant cftr channel. the similar behavior of potentiators in two different cell lines is consistent with the assumption that all potentiators act with a similar mechanism, by interacting with the cftr protein itself. in contrast, the cell line dependence of correctors suggests that they act with indirect mechanisms, possibly by interacting with proteins involved in cftr biosynthesis and trafficking. supported by cfft and telethon-italy. we also thank cfft and rfums for providing chemical compounds. lung damage in cystic fibrosis (cf) patients is determined by mucus accumulation, pseudomonas aeruginosa infection and chronic inflammation. extracellular gsh is a scavenger of free radicals produced by neutrophils in inflamed tissues. glutathione transferases (gst) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. clinical beneficial effects have been reported in cf patients following treatment with the macrolide azythromicin (azm); anti-inflammatory properties have been proposed as possible mechanism. the aim of this study is to investigate the regulation of the gstt1 and gstm1 activity and expression by azm. reductions of about 25% and 40% on gst enzymatic activity were detected in ib3-1 and 2cfsmeo-cells respectively. gsts mrna expression in cf airway epithelial cell lines was analysed by quantitative pcr (qpcr). the level of gstt1 and gstm1 basal expression in cf cells ib3-1 was significantly higher than in isogenic non-cf cells c38. we found statistically significant decreases of gstt1 and gstm1 mrna of about 30% and 25% respectively in ib3-1 cells after treatment with azm for 24 hours, restoring the levels observed in c38 cells. in 2cfsmeo-cells after exposure to azm we observed 50% and 45% reductions in gstt1 and gstm1 mrna respectively. the macrolide jm, known to lack clinical anti-inflammatory properties, had no significant effects on gstt1 and gstm1 mrna expression in all cell lines. furthermore, azm did not alter the mrna expression levels of gstp1, a glutathione-s-transferase not differentially expressed in cf and isogenic non-cf cells. decreased expression of 50% and 85% of gstt1 protein has been detected by immunoblotting in ib3-1 and 2cfsmeo-cells, respectively, following treatment with azm. in the same conditions we found a drastic reduction of protein level of gstm1 in both cf cell lines. finally, gsts activity and the expression of gstt1 and gstm1 proteins in cf cells, were reduced approximately to the same level detected after treatment with interleukin 10 (il-10), an anti-inflammatory cytokine, shedding light on a possible correlation between gsts inhibition and antiinflammatory properties of azm. the effects of azm described in this study suggest that downregulation of gstt1 and gstm1 expression may result in increased availability of intracellular gsh making cf cells less susceptible to oxidative stress induced by chronic inflammation. inhibition of gstt1 and gstm1 might provide a therapeutic approach for limiting the effects of inflammation critical for lung damage in cf patients. this study is supported by italian cf research foundation; comitato di vicenza-associazione veneta per la lotta contro la fibrosi cistica; azienda ospedaliera verona, italy. tradtrantip, l.; padmawar, p.; yangthara, b.; verkman, a. activation of cftr chloride conductance by gpcrs involves camp elevation and pka-mediated cftr phosphorylation. we developed a 'pathway screen' in which cftr-mediated iodide influx is used as a read-out of gpcr activation. the cell-based fluorescence assay utilizes multiply transfected epithelial cells expressing wildtype cftr, yfp-h148q/i152l and a specified gpcr. we recently used this assay to identify a new class of nanomolar-affinity, 5-aryl-4-benzoyl-3hydroxy-1-(2-arylethyl)-2h-pyrrol-2-one vasopressin-2 receptor antagonists (yangthara et al., mol. pharm. 2007, in press ). additional screening of 100,000 diverse small molecules yielded 4 novel chemical classes of inhibitors of cftr activation. the potential molecular targets of pathway inhibitors include the gpcr, gs or gi proteins, adenylyl cyclase, phosphodiesterase, pka and cftr. a series of target identification studies was done to classify the new pathway inhibitors, which involved the use of agonists acting at different sites in the activation pathway, and specific site-of-action assays. the pathway screen yielded 3 new small-molecule cftr inhibitors that are unrelated to thiazolidinone and glycine hydrazide inhibitors. one interesting class of pathway inhibitors, thiophenecarboxylates, represent the first small-molecule phosphodiesterase activators, which strongly reduce camp and cgmp concentration in many cell types. the best thiophenecarboxylate greatly reduced intestinal fluid secretion in closed loop mouse models of cholera and travelers' diarrhea, and slowed cyst growth in a model of polycystic kidney disease. other pathway inhibitors, which are potential effectors of g-proteins and pka, are under evaluation. the gpcr-linked cftr pathway screen developed here is useful for high-throughput parallel identification of small-molecule inhibitors of multiple targets in the cftr activation pathway. potential uses of the inhibitors identified here include therapy of secretory diarrheas, polycystic kidney disease, and cyclic nucleotide-dependent tumor growth, as well as pharmacological creation of cf-phenotype in ex vivo human tissues and animal models. supported by cff and nih. inhibitors of intestinal caccs are predicted to have anti-secretory effects in certain diarrheas, and activators of airway and intestinal caccs are of potential use in cystic fibrosis therapy (activation of 'alternative' chloride channels). the purpose of this study was to identify small-molecule cacc inhibitors and activators that target caccs directly, rather than ubiquitous upstream processes such as calcium signaling. we screened a collection of 100,000 chemically diverse small molecules using a novel high-throughput screening assay involving lentiviral introduction of a yfp-based halide sensor in cacc-natively expressing human epithelial cells. cacc inhibitors were identified from iodide influx following cacc simulation by carbachol/atp. we identified five classes of cacc inhibitors with micromolar potency, including tetrahydro-cyclopentaquinolines, and 3-aryl-5-(trifluoromethyl)-pyrazoles each of which was unrelated to known transport modulators. two classes of compounds inhibited calcium-activated halide flux following stimulation by multiple types of agonists, including thapsigargin and calcium ionophores, and by patch-clamp analysis appear to target cacc directly. structure-activity analysis of 137 analogs of 'hits' yielded compounds with improved potency, which have been resynthesized and characterized for use in assays of antidiarrheal effiacy in rodent models of viral and drug-induced secretory diarrheas. screening for caccselective activators that act in a sustained manner (non-transiently) was accomplished using a similar cell-based fluorescence assay, but instead testing for increased halide influx. several classes of putative cacc activators with micromolar-potency were identified in screening of 100,000 small molecules, whose mechanism-of-action and specificity are under investigation. small-molecule modulators of cacc function that target caccs directly have potential clinical applications, and may be useful in defining the physiological roles and molecular identity of caccs. supported by cff and nih. introduction: inhaled hypertonic saline 〈hs〉improves lung function and decreases pulmonary exacerbations in older children and adults with cf. initiating therapeutic interventions in the youngest patients, particularly those that target the underlying cf defect of airway surface liquid volume depletion, has potential to preserve lung function and improve prognosis. subbarao et al performed baseline, post-albuterol and post-hs lung function testing in infants using the raised volume rapid thoracoabdominal compression technique 〈rvrtc〉and demonstrated no significant drop in lung function 〈 pediatric pulmonology, 2007〉. however, performing three sets of rvrtc maneuvers under the same sedation could prove difficult. before conducting a therapeutic trial of hs in this population, a simplified protocol must be possible at multiple centers. we sought to evaluate a simplified approach as well as to analyze changes in lung function and clinical findings after acute administration of hs. methods: in this ongoing study, clinically stable children with cf between the ages of 4 months and 4 years inhale 2.5 mg of albuterol prior to sedation with chloral hydrate. rvrtc and plethysmography are then performed before and after inhalation of 5 ml of 3‰ hs. fvc, forced expiratory volume in 0.5 seconds 〈fev 0.5 〉, fef 25-75 , frc, rv/tlc ratio, respiratory rate, oxygen saturation, and chest exam findings are recorded. predefined stopping criteria include a 20% drop in fev 0.5 or in oxygen saturation to below 90‰. results: six subjects 〈mean age 1.6 ± 0.9 years〉 have completed the protocol with 3‰ hs. comparison of post-albuterol lung function to that obtained 15 minutes after 3‰ hs revealed no changes in mean fvc 〈561 vs 562 ml; p=0.93〉, mean fev 0.5 〈426 vs 427 ml; p=0.89〉, mean fef 25-75 〈801 vs 817 ml/sec; p=0.59〉, frc 〈measured after each inhaled therapy in 3 of 6 subjects; 260 vs 260 ml; p=1.0〉, or rv/tlc 〈measured after each inhaled therapy in 3 of 6 subjects; 0.35 vs 0.34; p=0.77〉. respiratory rate, oxygen saturation and chest exam were unchanged. conclusions: results from this study demonstrate that a two-step protocol may used to evaluate the safety of hs. based on these findings, acute administration of 3‰ hs is safe in children ages 4 months to 4 years with cf. despite the known improvement in mucociliary clearance, preliminary findings demonstrate a lack of an immediate response in lung volume measures. given the demonstrated benefits in older children and adults, a multicenter therapeutic trial of hs is warranted. supported by the cystic fibrosis foundation. methods and results: we have characterized aav serotypes 1-9 in addition to twenty novel vectors isolated from human or macaque tissues to transduce the murine airway epithelium in vivo. vectors [1e+11 genome copies (gc)/mouse] expressing α-1-antitrypsin (aat) and βgalactosidase (β-gal) were co-instilled into the mouse lung or nose. transgene expression levels were monitored by assaying aat concentration in serum as well as the number and cell-types positive for (β-gal) expression in lung and nasal airways. of all vectors tested aav5 and aav6 were the two most efficient vectors in conducting airways. when these aav vectors (2e+11 gc) were subsequently evaluated on human ciliated airway epithelial cultures (haec), in contrast to our findings in mouse airways, aav5 failed to transduce haec, whereas aav6 resulted in~10% of the haec expressing transgene. since aav6 was the most efficient vector in mouse and human airway epithelium we performed structure-function analyses of the aav6 vector capsid and found two atypical capsid residues that were unique in otherwise conserved positions (f129, k531). to generate a potentially fitter vector, residue f129 was mutated to its conserved state. residue k531 was found to confer lung tropism and was left unchanged. the resulting vector, aav6.2, transduced mouse lung and nasal airway with greater efficiency than all aav vectors tested. the increased transduction efficiency of this vector was also observed (~20%) in haec derived from six different human subjects. to continue our preclinical studies in a more relevant model, aav2/6.2 expressing egfp was tested in ciliated cultures derived from macaque airways and showed 40-50% of cells expressing egfp one week after inoculation with 2e+11 gc. confocal microscopy revealed that aav6.2 targeted a significant number of ciliated cells: the airway cell-types that likely require cftr expression in cf patients. aav6.2 expressing rhesus α-fetoprotein (rhafp) was then inoculated in the nasal airways of a rhesus macaque and transduction evaluated by monitoring concentration of rhafp in the nasal lavage fluid. we have found that rhafp expression remained high (~300 ng/mg) and stable for at least 72 days. conclusion: the enhanced transduction efficiency of aav2/6.2 vector in human and macaque airway cultures and its ability to stably transduce the nasal airway of a rhesus macaque in vivo demonstrates that aav2/6.2 is a good candidate gene transfer agent for the efficient expression of cftr in human cf airway epithelium. submitted for presentation at the american society of gene therapy. supported by gsk, cff, cfpo1, p30, mtcc. pharmacological correction of ∆f508 cftr cellular processing is a potential therapeutic strategy for cystic fibrosis. recent high-throughput screening has identified synthetic small molecules, such as bisaminomethylbithiazoles (corr-4), which partially restore chloride permeability in ∆f508 mutant cells. the purpose of this study was to examine the utility of natural compounds (chinese medicinal herbs) as ∆f508 correctors. a herbal compound fraction library was constructed from 500 herbs most frequently used in traditional chinese medicine (tcm) that are believed to contain therapeutic compounds for a broad spectrum of human diseases including lung disease. for construction of the tcm fraction library, crude herbal extracts were first prepared by 95% ethanol extraction on soxhlet reflux apparatus followed by automated fractionation by preparative hplc. eighty fractions were collected from each of the 500 herbs. each fraction contained 1 to 17 (average 10.4) individual compounds as determined by analytical hplc. collected fractions were dried and 1 milligram of the material was dissolved in 200 µl dmso to generate 5 mg/ml solutions in 96-well plates. each 96-well plate contained 80 fractions from one herb. high-throughout screening was done using the frt cell-based fluorescence assay developed previously (j. clin. invest. 115:2564 -71, 2005 . of 16,000 fractions screened, 87 active fractions from 12 herbs were identified, with 26 positive fractions verified in secondary screening. the positive fraction did not increase halide transport in control non-transfected cells, and halide transport in ∆f508-corrected cells was fully abolished by cftrinh-172. we have fractionated some of the most active fractions by preparative hplc to identify which compound(s) conferred activity. for example, in one fraction there were 14 single compounds, 2 of which conferred corrector activity with ic50s < 5 µm and efficacy comparable to that of low temperature rescue. these results demonstrate the feasibility of ∆f508-cftr corrector discovery from natural compounds. further fractionation, characterization and structure determination are in progress. the unexpectedly high 'hit'rate for the natural compounds suggests their further exploration in cf therapy. cystic fibrosis (cf) is the most common genetic disease affecting the caucasian population, with an incidence of approximately one in three thousand births. cf transpires as a result from a mutation in the cystic fibrosis transmembrane conductance regulator protein (cftr), which regulates ion transport across epithelial membranes. subsequently, patients afflicted with cf have an abnormally excessive incidence of chronic lung infection, with organisms such as pseudomonas aeruginosa. because cystic fibrosis is characterized by chronic bacterial infections, excessive neutrophil recruitment to the lungs, and a coinciding increase in pro-inflammatory cytokine production and nuclear factor-kappa b (nf-κb) activation, we hypothesized that exogenous addition of the nf-κb inhibitor iκbα might ameliorate this phenotype. we cloned the human iκbα gene as well as a mutated iκbα gene into plasmids with chicken-beta actin hybrid promoters. we then tested the new plasmids, paav2.cb-hiκbα and paav2.cb-hiκbαm, in vitro in the presence and absence of pseudomonas aeruginosa infection in the ib3-1 and s9 cell lines. both plasmids produce iκbα at high levels as shown by enzyme linked immunosorbant assays (elisas). we also show that paav2.cb-hiκbα transfected ib3-1 cells, after infection with pseudomonas aeruginosa, express significantly reduced levels of interleukin (il)-1β (2 fold, p<0.0001), il-8 (13 fold, p<0.0001), and tnf (2 fold, p=0.0007) as well as nf-κb activation (2 fold, p<0.0001) compared to p. aeruginosa-infected ib3-1 controls as determined by human cytokine and nf-κb phosphoprotein bio-plex assays; cytokine expression and nf-κb activation levels in infected paav2.cb-hiκbα transfected ib3-1 cells were between levels found in infected ib3-1 and s-9 cells, excluding il-8 levels which were below s-9 levels of expression. flavonoids are among the most potent cftr modulators known. equol [7hydroxy-3-(4'-hydroxyphenyl)-chroman] is a product of intestinal metabolism of dietary isoflavones such as daidzein, and has been of recent interest in studies of cancer, cardiovascular risk, and neurologic disease. equol is metabolically stable, and 49% circulates in the free (non-protein bound) form, which is considerably greater than the proportion of free daidzein (19%). structural differences such as modification of ring c (e.g. saturation at c-2/3, and absence of carbonyl group at c-4) and an absent hydroxyl at position c-5 distinguish equol from compounds previously reported to modulate cftr activity. in prior work by our center, we showed that equol activates wt and ∆f508 cftr in membrane patches excised from bhk cells and in cfbe41o-monolayers studied in ussing chambers. activation by equol occurred only after r-domain phosphorylation in wt and ∆f508 cftr constructs, but was independent of rdomain phosphorylation in ∆r cftr, indicating activity may be related to dimerization of the nbds or other domain-domain interaction. because a molecule that alters nbd interactions might have effects on the aberrant processing of ∆f508 cftr, we screened equol and other flavonoids by preincubating compounds with cfbe41o-cells expressing ∆f508 cftr for 18 hours, and then tested for rescue of cftr dependent cl-channel activity after exchange of media solution. we found that preincubation with equol (50-100 µm) induced rescue of short-circuit current compared to vehicle treated cells (12 vs. 4 µa/cm2, p<0.001, n=8). we then evaluated 24 hour preincubation with equol (50um) for biochemical evidence of cftr processing correction in ∆f508 cfbe41o-cells grown in polarizing conditions. immunoprecipitation and in vitro phosphorylation demonstrated minimal formation of band c compared to vehicle treated cells, but adaptation of a more sensitive avidin label/biotinylation assay specific for surface localized cftr revealed clear evidence that equol preincubation led to cftr at the plasma membrane. next, we examined equol in hela cells stably transduced with ∆f508 cftr. preincubation of equol led to dose-dependent increases in halide transport measured by fluorescence-based halide efflux (spq) after stimulation by genistein (0.50 and 0.95 fluorescence slope (∆%/sec) with equol 5 and 50 µm respectively, compared to 0.2 ∆%/sec in cells pretreated with vehicle alone; p<0.005, n=120-180 cells/condition). immunohistochemical staining of ∆f508 hela cells for cftr with 24-1 c-terminus antibody showed rescue of surface localized protein with equol (50 µm) preincubation for 16 hours, while vehicle treated cells showed only perinuclear staining. in summary, we show functional, biochemical, and immunohistochemical evidence that the naturally occurring flavonoid equol corrects the ∆f508 processing defect in two model systems. a naturally occurring agent that both activates and corrects ∆f508 cftr deserves further exploration as a potential cf therapeutic, and may lead to new insights regarding domain-domain interactions that influence the activation and biogenesis of the mutant channel. supported by nih and cff. mote ∆f508 cftr maturation has been identified. although several small molecule agents have been described that overcome ∆f508 cftr processing defects in specific cellular models, few studies have directly compared the activity of temperature corrected and chemically corrected ∆f508 cftr in polarized cell systems. in the present study, we examined chemical and temperature corrected activity of ∆f508 cftr. correctors included all members of the cfft modulator library (c1, c2, c3, and c4; rosalind franklin university, chicago, il). in a screen using ∆f508 cfbe41omonolayers, maximal corrector activity across the two model systems exhibited a rank order of c4 >> c3 > c2 = c1, using forskolin (20 µm) and genistein (50 µm) stimulated isc as a sensitive test for ∆f508 cftr activity at the plasma membrane. no change in isc was observed in matched control (parental) cells lacking ∆f508 cftr expression. based on these results, we further defined the activity of c4 in ∆f508 cfbe41oand frt model systems, including dose/response and time dependence for peak isc rescue. in cfbe41o-cells, exposure to 2 µm c4 for 8 hrs produced maximal reproducible correction of ∆f508 cftr processing, with loss of activity following prolonged or high concentration exposure; in frt cells, peak effects were seen at 24 hours. ∆f508 cftr activity following small molecule treatment qualitatively mirrored temperature correction (27°c growth for 24-48 hrs) in both cell types. maximal currents produced by stimulation with forskolin (20 um) and genistein (50 µm) in ∆f508 frt monolayers following c4 pretreatment were 83% of that produced by temperature correction (p<0.001). forskolin was responsible for 57% of maximal current in frt ∆f508 cells following chemical correction and 43% of maximal currents following temperature correction. in ∆f508 cfbe41o-cells, maximal currents following chemical correction were 22% of that produced by temperature correction (p<0.001). forskolin was responsible for 10.8% of maximal currents in cfbe41o -∆f508 cells following chemical correction and 15.6% of maximal currents following temperature correction. these results illustrate dose and time response with a small molecule corrector in two polarizing epithelial model systems, and provide reassurance that observations based upon ∆f508 cftr following low temperature incubation are relevant to functional analysis after chemical correction. similarity in activation properties between chemical and low temperature correction suggest it is unlikely that the two maneuvers result in ∆f508 cftr with significantly different structural properties. the studies also indicate fundamental differences in ∆f508 cftr behavior in frt compared to cfbe41ocells, and emphasize the importance of identifying an agent that can restore camp dependent regulation to bronchial epithelial cell types. supported by the nih, cff and cfft. participants: n=38 patients (21 randomised to azm and 17 to placebo) who had successfully completed a course of intravenous antipseudomonal antibiotics immediately before the trial (mean age: 23.7 years; mean fev1: 62% of predicted). measurements and results: after treatment (mean dose of 21.2 mg/kg body weight once a week) pulmonary function declined in both groups compared to baseline (i.e. after cessation of iv antibiotics). the azithromycin group had signifcantly better results regarding the mean changes in serum crp (azm: +0.9 mg/l, placebo: +21.6 mg/l, p=0.019), lipopolysaccharide binding protein in serum, lbp (azm: +0.9 µg/ml, placebo: +7.0 µg/ml, p=0.015), serum interleukin-8 (azm: -3.1 pg/ml, placebo: +2.9 pg/ml, p=0.001) and alginate in sputum (azm: +85 µg/ml, placebo: +353 µg/ml, p=0.048). quality of life (german version of the cfq) showed significantly better results after azm in adolescents and adults. azithromycin was well tolerated with no increase in treatment-related adverse events. conclusion: once-weekly azithromycin ameliorated inflammatory reactions and improved quality of life. a decline of pulmonary function after cessation of intravenous antibiotics could not be prevented, however. this study has been sponsored pfizer gmbh, germany this open-label, multicenter study was conducted in the usa and australia to evaluate the clinical responsiveness of a patient-reported outcome measure, the cfq-r respiratory scale, by determining the minimal clinically important difference (mcid) following a 28-day course of tobramycin inhalation solution (tis). cf patients (n=84 [≥6 to <18 yrs, n=56; ≥18 yrs, n=28]) with pseudomonas aeruginosa and clinical symptoms predictive of a pulmonary exacerbation (increased cough, increased sputum /chest congestion, decreased exercise tolerance or decreased appetite) were enrolled. three efficacy measures were included: 1)change in forced expiratory volume in 1 second (fev1) from baseline (day 0) to end of treatment (day 28) or end of study (day 42); 2)one question about change in respiratory function (days 28 and 42; global rating of change questionnaire, respiratory domain, grcq rd;0 =no change;7 =maximal improvement or worsening);and 3)change in cfq r-respiratory scale (day 28, 42). average change from baseline fev1 (mean [standard deviation, sd]) was 5.1% (16.5) at day 28 and 4.9% (16.0) at day 42. based on grcq-rd at day 28, 22 patients (28%) reported no change in respiratory symptoms (score <1.1), 32 (41%) a minimal change (≥1.1 to <3.1), 16 (20%) a moderate change (≥3.1 to <5.1), and 9 (11%) a large change (≥5.1). mean (sd) change from baseline cfq-r respiratory was 5.7 (19.2) at day 28 and 5.9 (20.7) at day 42. at day 28, change in cfq-r was moderately correlated with change in fev1 and with grcq-rd; the correlation was stronger for patients with baseline fev1 <75% of predicted fev1 values (see table) . mean change from baseline on the cfq-r respiratory scale was 10.0 at day 28 for patients with grcq-rd scores indicating minimal change in symptoms (≥1.1 to <3.1; n=30); this provided an estimate of the mcid for the cfq-r respiratory scale for those in exacerbation. this mcid value was consistent with estimates from distribution-based methods( 1 ⁄2 sd and standard error of measurement). the cfq-r was responsive to changes in pulmonary symptoms in patients in mild exacerbation following tis treatment; the mcid in this population was 10.0 points. responses on the cfq-r-respiratory scale were moderately correlated with changes in fev1. funded by gilead sciences, inc. this was an open-label, multicenter study conducted in the usa. we determined the minimal clinically important difference (mcid) for the cfq-r, respiratory scale following a 28-day course of tobramycin inhalation solution (tis) in patients with cf and chronic pa infection (n=140, 14 children [<13 yrs]). patients had received ≥3 courses of tis (mean = 5.4) within the previous year, however their respiratory symptoms were stable at study entry, with forced expiratory volume in 1 second (fev1) between 25% to 75% of predicted values. efficacy measures included: 1) percent change in fev1 (l) from baseline (day 0) to treatment end (day 28); 2) a single question about change in respiratory function (day 28; global rating of change questionnaire; respiratory domain, grcq-rd; 0 =no change, 7 =maximal improvement or worsening); 3) change in cfq-r-respiratory scale (day 0 to 28); and 4) change in log10 pa colony-forming unit (cfu) density in sputum (day 0 to 28). the mcid for cfq-r was estimated using three methods: 1) change in cfq-r (day 0 to 28) for the patient subset with minimal change in respiratory function, as determined by the grcq-rd at day 28; 2) cfq-r standard error of measurement (sem) from a validation sample), and 3) 1 ⁄2 standard deviation (sd) of the cfq-r respiratory scale scores. at day 28, change from baseline fev1 (mean [sd]) was 1.0% (11.3), change from baseline cfq-r was -0.6 (12.6), and change in log10 pa cfus was -0.4 (1.5) . based on the grcq-rd at day 28, 37 patients (41%) reported no change in respiratory symptoms (score <1.1), 40 (44%) a minimal change (≥1.1 to <3.1), 10 (11%) a moderate change (≥3.1 to <5.1), and 4 (4%) a large change (≥5.1). pearson r-values for the correlation of change in the cfq-r-respiratory scale and change in fev1, log10 cfus and grcq-rd were 0.1, 0.08, and 0.46, respectively. estimates of the mcid for cfq-r-rd ranged from 4.6 to 5.9 for adults/adolescent patients (table) . in patients with cf who had no immediate need for antipseudomonal therapy at study entry, the cfq-r-respiratory scale appeared responsive to changes in patient disease perception following 28 days of tis treatment; the mcid for cfq-r was approximately 5 points for the adolescent/adult patient population. funded by gilead sciences, inc. ex vivo chloride secretion measurements (intestinal current measurement, icm) in cf patients have been established over the past 15 years to study the cftr-basic defect in more functional detail. modified micro-ussingchambers are used to registrate the transepithelial short-circuit current (isc) in freshly obtained human rectal suction biopsies as a measure of ion transport after stimulation with secretagogues. hereby, the cftr clchannel, its amount of residual function in cf and alternative cl-channels can be investigated by a standardised protocol.in the course of the development of cftr pharmacotherapeutics as well as agents activating alternative cl-channels, icm may function as an useful outcome parameter in preclinical and clinical trials. it is easy to perform repeatedly at all patients ages and comprises the safety advantages of an ex vivo method which is relevant especially for early study phases. aim of this study was to describe reference values and quantify the intraindividual variability of different icm parameters. methods: a total of n=574 rectal biopsies from n=212 individuals; with pancreatic insufficient (pi)-cf (n=22; mean age 14.5 years), pancreatic sufficient (ps)-cf (n=9; 10.8 years), excluded cf by icm diagnostics (n=169; 12.8 years) and healthy control (n=12; 26.3 years) were included into analysis. for calculation of intraindividual variability, 2-4 biopsies/patient were compared with respect to basal tissue resistance (rt basal), basal open circuit potential difference (pd basal), basal short circuit current (isc basal) and the isc responses to stimulation with carbachol (10-4 mol/l, serosal), 8-bromocyclic monophosphate (camp) (10-3 mol/l, mucosal+serosal) + forskoline (10-5 mol/l, serosal) and histamine (5x10-4 mol/l, serosal).results:we determined icm reference values for the groups of pi-cf (isc basal 23.4 ± 18.1 µa/cm 2 , ∆isc carbachol 0.4 ± 3.6 µa/cm 2 ,∆isc camp/forskoline 2.0 ± 2.9 µa/cm 2 ,∆isc histamine -3.1 ± 4.6 µa/cm 2 );ps-cf (isc basal 45.6 ± 31.5 µa/cm 2 ,∆isc carbachol 3.4 ± 6.8 µa/cm 2 ,∆isc camp/forskoline 8.0 ± 10.4 µa/cm 2 ,∆isc histamine 6.3 ± 10.8 µa/cm 2 ),and healthy control (isc basal 39.0 ± 26.1 µa/cm 2 , ∆isc carbachol 27.3 ± 7.1 µa/cm 2 , ∆isc camp/forskoline 15.2 ± 10.6 µa/cm 2 , ∆isc histamine 27.4 ± 15.2 µa/cm 2 ). for the total cohort, mean coefficients of variation were: rt basal 29%, pd basal 48%, isc basal 49%, ∆isc carbachol 59%, ∆isc camp/forskoline 64%, ∆isc histamine 80%. conclusion:this first comprehensive analysis of the intraindividual variability of icm basal tissue and cl-secretion parameters provides the basis for the method as an useful outcome measure for future clinical trials aiming to rescue the cftr basic defect. possible effects of pharmacological therapeutics in cf relevant human epithelia have to be adequately interpreted with respect to subject variability and laboratories reference data. ex vivo cl-secretion measurements have the potential of being an essential step in the evaluation process of cftr-correcting/potentiating agents on their way from laboratory screening to the application in human cf tissue without any risk of toxicity. center, placebo-controlled, double-blinded pilot study we assessed safety and tolerability of 2.5 mg/d moli1901 versus placebo (normal saline) administered by inhalation (pari lc plus) once daily for 28 days. patients included were ≥16 years of age in phase i and ≥12 years of age in phase ii, with a fev 1 >60% predicted and stable lung disease. overall, 12 subjects received moli1901 and 6 placebo. exclusion criteria included abpa, b. cepacia infection and severe liver disease. the study involved 7 clinic visits over a period of 8 weeks to assess adverse events, spirometry, pulse oximetry and quality of life. a total of 111 adverse events (aes) were observed in 16 subjects (101 ae in 12 subjects receiving moli1901), with only 1 (productive cough) in the moli1901 group being of severe intensity. the most frequent aes related to the study medication were (productive) cough (43x) and dry throat/throat irritation (14x), and most of these resolved within 1 hour after inhalation. in the moli1901 group no significant ae, defined as a decline of fev 1 ≥20% from baseline accompanied by symptoms, a decrease in oxygen saturation to <90% or a fall of 8% from baseline requiring therapeutic intervention, or a change in safety parameters judged to be clinically significant was observed. this trial was not primarily designed to show efficacy; however, the median change in fev 1 from day 1 to day 56 was -3% in the placebo group, and +2% in the moli1901 group, and this difference was significant (wilcoxon test, p=0.0217). similarly, there was a significant difference between the median change in fef 25-75% from day 1 to day 56 in the placebo group as compared to the moli1901 group (-10% vs. +1%, p=0.029). no significant changes were observed for the other study days or for fvc and pulse oximetry. moli was well tolerated in this trial, with the observed aes generally being mild and of short duration. these encouraging explorative results are currently being further evaluated in explorative and confirmatory trials. introduction: results from published data elucidate that microbes found in the upper respiratory tract are similar or the same as those found in the lower airways of cf patients. inhaled, aerosolized drug delivery to the lower respiratory tract is an established treatment route. however, drug delivery systems capable of depositing drug to the paranasal cavities are not yet established and require evidence of deposition and efficacy. pari developed the vibrent™ paranasal drug delivery system to enable the aerosol and drug to penetrate into the nose and sinuses. objectives: this study was conducted to demonstrate that the pari vibrent™ pulsating drug delivery system is capable of ventilating the human paranasal sinuses of 3 healthy volunteers. methods: 81mkr-gas was continuously ventilated through the nasal tract of three healthy non-smokers in front of a single-head gamma camera (diacam, siemens, germany), using the pari vibent™ pulsating drug delivery system. the nebulizer was coupled to the right nostril and a flow resistor to an output tube was inserted into the left nostril. during ventilation with the krgas (about 10 sec) the subject closed their soft palate to transmit the pulsation and to prevent penetration into the lower respiratory tract. the gas supply of the vibrent™ was directly taken from the 81mkr-gas generator output channel. kr-gas ventilation imaging was performed with and without pulsation. serial images were recorded with anterior and lateral views. additionally, mri (magnetic resonance imaging) lateral slices of the subjects' head were recorded. the gamma camera images were superimposed to the mri images by adjusting the spatial resolution. with no pulsation from the vibrent™ no ventilation of sinus cavity was visualized by gamma camera images and radioactivity was detected in the nose only. when pulsation was added the maxillary sinuses can be visualized in the gamma camera images of all volunteers. conclusions: without pulsation no ventilation was observed. gas penetration to the paranasal sinuses can be demonstrated using the pulsating action of the pari vibrent™, potentially enabling drug delivery via aerosols. this confirms results of in vitro studies using a cast model. 81mkr-gas ventilation of the nasal cavities during 10 sec breath holding in front of a planar gamma camera head (anterior) using the pari vibrent without (w/o, left image) and with (w, right image) the pulsation system. the delivery and the exhaust tubing of the kr-gas are shown together with the outline of the head, obtained from mri pictures. introduction: pediatric patients with cf were previously studied in clinical trial studies of denufosol, a novel selective p2y 2 agonist that enhances ciliary beat frequency and activates chloride secretion to hydrate the airways in the lung. pediatric patients are often discouraged from participation in clinical trials until later stages of drug development. aims: in order to evaluate the safety experience with denufosol in this population, we have retrospectively examined integrated data for pediatric cf patients with mild to moderate pulmonary disease that participated in three phase 2 studies. demographic and baseline characteristics in addition to safety and tolerability results for 115 cf patients aged 5-18 years old are reported. methods: three phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel group studies were conducted. patients were randomized to receive either denufosol (20, 40 or 60 mg) or placebo (normal saline) tid for 28 days by inhalation. only study 1 included denufosol 40mg, while all 3 studies included denufosol 20mg and 60mg. the fev 1 predicted normal required to be eligible was >75% (study 1); 60%-90%, inclusive (study 2); >60% (study 3). all three studies included a one-week pre-randomization period during which reproducibility of fev 1 (l) ±12% was required in order to be randomized to double-blind study medication. patients were allowed to use bronchodilators, dornase alfa and corticosteroids in studies 1, 2 and 3. patients were allowed to use oral antibiotics including macrolides and inhaled tobramycin solution in studies 2 and 3. results: a total of 115 cf patients 5-18 years old were randomized and dosed in three 28-day studies. eighty-one received denufosol (active doses combined) and 34 received placebo. demographics were similar for all treatment groups -denufosol pediatric patients had a mean (sd) age of 11.8 (±3.70) years old compared to placebo pediatric patients who had a mean (sd) age of 11.9 (±4.05) years. denufosoltreated patients were 59% male and placebo-treated patients were 53% male. the mean (sd) percent predicted fev 1 at baseline was similar between treatment groups [89.9% (±15.13) and 92.0% (±14.28) for denufosol and placebo, respectively]. the overall incidence of treatment emergent adverse events (ae) was similar between treatment groups (84% denufosol, 82% placebo). the most common ae was cough, reported by 48% and 47% of patients that received denufosol and placebo, respectively. seven percent of denufosol and 6% of placebo patients prematurely discontinued from the study due to aes. there were no differences in compliance with administration of study drug (96% in patients given denufosol and 100% in patients given placebo). conclusion: doses of denufosol up to 60mg given tid for 28 days were well tolerated in pediatric cf patients 5-18 years old. these data demonstrate that inclusion of patients 5-18 years old is feasible regardless of administration of denufosol or placebo. a longer term phase 3 study of denufosol in cf patients >5 years old in north america is currently ongoing. acknowledgements: this research was funded by inspire and the cystic fibrosis foundation. objective: pulmonary delivery of anti-infectives provides the potential to attain pk-pd indices exceeding those which can be achieved with systemic dosing. mp-376 is a novel formulation of levofloxacin that enables delivery of high concentrations over a short period, and provides taste masking. the objective of this study was to: i) determine the safety of aerosol doses of mp-376 and, ii) determine pharmacokinetics of levofloxacin in serum, urine, and sputum following aerosol doses of mp-376 using the pari eflow nebulizer in normal healthy volunteers (nhv) and patients with cystic fibrosis (cf). methods: nhv and patients with stable cf were enrolled in a single within-subject ascending dose study of 3 dose levels (loaded doses of 78, 175, and 260 mg) of inhalational mp-376 (levofloxacin solution for inhalation) or placebo. study participants were monitored for safety and changes in pulmonary function. serum, urine, and sputum (cf patients only) samples were collected at various times following the dose and assayed for levofloxacin concentration using hplc. in addition, each participant received a single iv dose of levofloxacin to determine the systemic bioavailability following aerosol mp-376. noncompartmental and compartmental methods were used to determine serum, sputum and urinary pharmacokinetic parameters. pharmacokinetic deconvolution methods were used to determine the amount of the dose remaining in the lung over time. results: this study is ongoing. dosing in 8 nhvs (6 active mp-376, 2 placebo) has been completed. there were no serious adverse effects, and no significant changes in pulmonary function were noted. preliminary pharmacokinetic data from 4 nhvs show a proportional increase in serum levofloxacin concentrations with increasing aerosol mp-376 dose; serum aucs were 3.1, 1.9, 5.1, and 8.9 mg-h/l for the iv dose, low, mid, and high aerosol doses, respectively. deconvolution of the serum levofloxacin concentrations from aerosol mp-376 and iv dosing shows absorption of drug from lung over time. conclusion: preliminary results show that mp-376 is well tolerated following aerosol administration in normal volunteers. serum concentration data following aerosol dosing suggests that absorption of levofloxacin into the systemic circulation is a major route of elimination from the lung. studies in cf patients are in progress and will be presented. methods: a 10 mg/kg aerosol dose of mp-376 was given using a microspray aerosol generation device. when placed just above the tracheal bifurcation, and activated, it delivers a bolus aerosol dose to the lung. a 10 mg/kg intravenous (iv) dose of lvx was given as a slow bolus into the lateral tail vein. plasma lvx was determined in all rats up to sacrifice at 1, 2, 3, or 6 hours, at which time rats were humanely euthanized and bronchialalveolar lavage (bal) performed. fluids were analyzed for lvx concentration using an hplc/ms method. results: the plasma lvx versus time profiles of both iv and aerosol doses were best described by a two compartment model. the plasma auc after an iv dose of levofloxacin and an aerosol dose mp-376 were similar (3.79 mg᭹hr/l vs. 3.72 mg᭹hr/l, respectively) suggesting near 100% bioavailability from the lung. after an aerosol dose, the mean residence time (mrt) was prolonged when compared to the intravenous dose (0.88 vs. 0.79 hours). this delay in absorption was associated with an increase in bal lvx auc0-6h in bal (1.6 mg᭹hr/l vs. 8.3 mg᭹hr/l for iv vs. aerosol dosing, respectively). conclusion: these data show that levofloxacin is highly available to plasma following a single microspray aerosol dose of mp-376. the aerosol dose does produce a slightly longer mean residence time in plasma, suggesting delayed, but complete absorption from the lung; this delay in absorption was associated with increase in bal aucs. these data suggest that high concentrations of lvx can be attained in lung fluids following an aerosol dose of mp-376. auc: area under the time-concentration curve; cl: clearance; cmax: maximum concentration; f: bioavailability; mrt: mean residence time. methods: animals were assigned to one of five exposure groups for all studies. for the 28-day toxicology studies, animals were exposed for up to four hours daily for twenty-eight consecutive days, followed by a 28-day recovery period. respiratory function and cardiovascular safety were conducted on the first and last day of the 28-day study in dogs. rats were exposed to 5, 10, or 20 mg/kg/day, and dogs were exposed to 5, 10, or 15 mg/kg/day. at the conclusion of the study, necropsy was performed and all respiratory tissues were harvested, weighed, and underwent gross and microscopic examination. a separate dog respiratory safety study was conducted in which dogs were exposed to 5, 10, or 20 mg/kg. results: four week repeat dose inhalational exposure of mp-376 at target doses up to 15 mg/kg/day in dogs and 20 mg/kg/day in rats was not associated with any test article-related changes in any respiratory tissues. aerosol administration of mp-376 at doses of up to 20 mg/kg in dogs was found to have no acute effects on minute volume, tidal volume, respiratory rate, or ecg. conclusion: local effects due to the inhalational administration of levofloxacin formulated as mp-376 were not observed in either rats or dogs. these data suggest that the risk of respiratory toxicity from nebulized doses of mp-376 is low. methods: bacteria were grown overnight in mueller-hinton broth (mhb) at 37°c and then sub-cultured into fresh mhb and allowed to reach log phase (4 hours). female swiss mice were infected under anesthesia by intratracheal instillation of 0.05 ml of a 2 x 106 cfu/ml bacterial suspension. ip doses were selected to provide lvx exposures (as auc) comparable to that obtained with systemic dosing regimens. for the p. aeruginosa infection, treatment was initiated by either the ip or aerosol route 2 hours post-infection. mice were euthanized 1 and 4 hours after the start of treatment, lungs harvested, and bacterial counts in lung determined. results: lvx plasma pharmacokinetic profiles were nearly identical following intraperitoneal or aerosol dosing. the geometric mean log cfu/lung pair (sd) for the p. aeruginosa are shown in the table below: in both the k. pneumoniae and p. aeruginosa lung infection studies, aerosol administration was more effective than systemic administration; for k. pneumoniae, the extent of bacterial killing at 24 hrs was ca. 1.4 log cfu greater with aerosol mp-376 than with systemic lvx. conclusion: aerosol administration of mp-376 produces a greater extent of bacterial killing than systemic dosing of lvx in mouse models of pneumonia due to k. pneumoniae and p. aeruginosa. the majority of morbidity and mortality in cystic fibrosis patients is caused by chronic and persistent lung infections especially with pseudomonas aeruginosa. since galactosyl ceramide had been previously shown to be involved in pseudomonas internalization, ceramide levels in the plasma of cf patients were assessed and compared to healthy volunteers using hplc followed by mass spectrometry. the results demonstrated that cf patients displayed significantly lower levels of several ceramide species. also, cftr-knockout mice displayed diminished ceramide levels in cf related organs (lung, pancreas, and ileum) and plasma compared to wildtype controls. treatment with a semi-synthetic retinoid (fenretinide), which was previously reported to induce ceramide in neuroblastoma cell lines, was able to increase ceramide concentrations in cf related organs in cftr-knockout mice to the levels of wildtype mice. treatment also dramatically improved the ability of cftr-knockout mice to control pseudomonas infection. following infection with pseudomonas-impregnated agar beads, fenretinide treated cftr-knockout mice were able to clear bacterial infection as efficiently as wildtype mice. overall, these findings not only documented a novel deficiency of ceramide in cf patients but also demonstrated a pharmacological means to correct this defect in cftr-knockout mice. our data provides a strong rationale for clinical intervention that may benefit cystic fibrosis patients suffering from cf lung disease. recent reports show that adult bone marrow-derived stem cells can localize to and acquire phenotypic and functional markers of lung epithelium. these findings raise the novel possibility of stem cell therapy for multitude of lung diseases. however, only small numbers of adult marrowderived stem cells localize to lung and it is not clear whether these cells will be clinically useful. we investigated whether mesenchymal stem cells (mscs) obtained from human cord blood might have increased potential to participate in structural lung remodeling. cord blood was obtained from normal deliveries at the university of vermont. mononuclear cells were isolated and plastic adherent cells were expanded and characterized as mscs according to international society for stem cells research (isscr) criteria. following systemic (2x106 cells/mouse by tail vein) administration to sublethally irradiated (1.4 gy) immunodeficient (nod/scid) mice, lungs harvested 1 day, 2 weeks, 1 or 3 months later demonstrated small numbers of human b2-microglobulin positive cells in the airway epithelium at all time points. small number of cells was found also stain positive for pancytokeratin (pan-ck) and rarely, we identified cells of b2-microglobulin+/pan-ck+ in the airway epithelium of these mice after 2 weeks. we are currently characterizing the phenotype of these cells with ccsp and cftr but these data suggest that cb-mscs may be a potential alternative source of stem cells for use in lung remodeling. high-throughput screening (hts) and other drug development programs have identified cftr activators and potentiators that require secondary evaluation and mechanistic confirmation. we recently evaluated the cfft modulator library (rosalind franklin university, chicago, il) and found some, but not all, cftr potentiators induced potent phosphorylation of the regulatory-domain (r-d), conventionally viewed as the first step in cftr activation. to assess whether this observation has functional significance regarding ion channel activation by these agents, we evaluated cftr potentiators in cfbe41o-and fisher rat thyroid (frt) cells stabily transduced with ∆f508 cftr. cells were studied in modified ussing chambers under control conditions and after correction of ∆f508 cftr misprocessing using either low temperature (27οc x 48 hrs) or pre-incubation with the chemical corrector c4. total currents were determined following potentiator (at reported ec 50 ), forskolin (2 µm), and genistein (50 µm) stimulation. in temperature corrected ∆f508 cfbe41ocells, potentiator 1 (p1, cfpot-532), an agent that does not induce r-d phosphyorylation, caused modest activation when acutely administered (5 µm) (15.5 vs 1.1 µa/cm 2 in vehicle treated cells, p < 0.005). importantly, this agent potentiated forskolin mediated short-circuit current (isc: 9.8 vs 1.5 µa/cm 2 , p < .05). forskolin accounted for 43% (vs. 4% with vehicle) of total current, demonstrating potent rescue of camp dependent cftr activity, an effect not previously reported in this cell type (p<0.005). in contrast, two cftr potentiators that induce r-d phosphorylation, p8 (uccf-029, a benzoflavone intermediate, 2 µm) and p10 (uccf-152, an isoxazole, 50 µm) induced modest direct activation of cftr (5.3 and 6.1 µa/cm 2 respectively, p=0.10 and p<0.01), but no evidence of forskolin potentiation (p8: 2.3 µa/cm 2 , 10% of total stimulated current p=ns; p10: 0.4 µa/cm 2 , 1%, p=ns). in ∆f508 frt cells grown at low temperate, p1 elicited strong potentiation of forskolin mediated currents (121.7 vs. 46.7 µa/cm 2 with vehicle, p <0.01), and modest direct cftr activation (8.7 vs. 0.51 µa/cm 2 , p < 0.05). p8 and p10 were limited to minimal (not significant) activation and no evidence of forskolin potentiation. in cfbe41oand frt cells pretreated with c4 (to chemically correct ∆f508 cftr processing), p1 again potentiated forskolin mediated current (101.3 vs. 43.2 µa/cm 2 with vehicle, p<0.001 in the frt model) but did not directly activate i sc . findings with p8 and p10 were otherwise as seen with low temperature corrected cells. in summary, in ∆f508 cftr polarized epithelia, p1 both activates and potentiates cftr activity (with potentiation being the predominant effect, a unique observation in cfbe41ocells), while p8 and p10 confer activation of cftr without forskolin potentiation. given that p8 and p10 induce r-d phosphorylation while p1 does not, our findings suggest that agents that do not confer phosphorylation of the r-d may be better suited to rescue the endogenous camp mediated component of i sc . screening of potential therapeutic agents for effects on r-d phosphorylation may help predict utility at restoring ∆f508 chloride channel activity. supported by nih and cff. activation of cftr is conventionally viewed as a two step process: pka-regulated phosphorylation of multiple sites within the regulatory domain (r-domain), followed by atp dependent gating mediated by binding sites at the interface of the two nucleotide binding domains (nbds). cftr 'potentiators', small organic molecules that overcome mutant cftr gating defects at the cell surface, have been proposed as therapies for cf. although a number of these agents are advancing to the clinical testing phase, their mechanism(s) of action are not well understood. as a step towards better characterizing cftr potentiators available through the cfft modulator library or other resources, we are developing standardized biochemical and functional assays to evaluate the r-domain during cftr activation in living cells. we have previously described a gel-shift method by which phosphorylation of isolated r-domain (residues 635-836) can be monitored. using this method, we have confirmed that one potentiator, p1 (cfpot-532), does not induce phosphorylation of the r-domain (4% of forskolin response, n=7, p = ns). unexpectedly, two potentiators, p8 (uccf-029, a benzoflavone intermediate) and p10 (uccf-152, an isoxazole), robustly confer phosphorylation of the r-domain (p8: 32% of forskolin response, n=8, p = 0.002, p10: 37% of forskolin response, n=8, p = 0.004). we found the phosphorylation conferred by either p8 or p10 could be inhibited with the pka inhibitor h89 (10 µm). maximal stimulation of phosphorylation occurs within 2 minutes, indicating time dependence similar to forskolin. importantly, neither p8 nor p10 increased total cellular camp, a finding confirmed by a number of other laboratories. the results may therefore implicate compartmental inhibition of cftr-associated phosphotases (eg. pp2a) or phosphodiestereases (eg. pde4) as an underlying mechanism by which isoxazole or certain flavone-derivative compounds stimulate cftr. to further test this hypothesis, p1, p8 and p10 are being examined for effects on ∆r-cftr chloride channel activity. these studies provide a means by which novel cftr potentiators can be biochemically categorized based on r-domain phosphorylation, a measure of the first step of cftr activation. compounds working through distinct mechanisms may have particular relevance to certain cftr mutations, and could provide synergy in the clinical setting. supported by nih and cff. mucociliary clearance (mcc) is an innate defense mechanism that protects the lungs from bacteria and viruses. mcc requires maintenance of a thin layer of airway surface liquid (asl) to eliminate inhaled particles. the asl volume is tightly regulated by a balance of ion and water transport across the airway epithelia. in cf, the loss of cftr clsecretion coupled with unregulated na + absorption via enac results in asl volume depletion and impaired mcc. although significant progress has been made in the identification of the basic disease mechanism in cf, therapeutic approaches that address abnormal cftr biogenesis are not currently available. the extracellular nucleotides atp and utp are important mediators of asl volume and mcc. in the airways, secreted atp acts on the g-protein coupled p2y 2 receptors to fine-tune mcc via the regulation of apical ion transport, ciliary beating, and mucin secretion. we have previously demonstrated that atp controls asl volume by inhibiting absorption through enac and increasing secretion through apical membrane chloride channels. p2y 2 receptor agonists are good candidates to treat cf. however, the rapid hydrolysis of atp and utp on the airway surface of cf patients limits the effectiveness of this approach. consequently, inspire pharmaceuticals has developed di-nucleotide molecules which retain the ability to activate p2y 2 receptors, but are more resistant to hydrolysis by ectonucleotidases. denufosol, inspire's lead compound for cf treatment, potently activates p2y 2 receptors to stimulate cl -/water secretion, ciliary beating, and mucin release in epithelial tissues. however, the stability and potency of denufosol has not been determined using human bronchial epithelial (hbe) cells. in primary cultures of hbes, denufosol was significantly more stable than utp. under thin-film conditions, the initial hydrolysis rate of a therapeutic concentration of denufosol (1 mm) was 0.08 nmol × min -1 × cm -2 with a half-life of approximately 1 hour. by comparison, denufosol is more than 10 times more stable than utp on the mucosal surface of hbes. importantly, the increased stability of denufosol translated into an increase in efficacy for this compound in vitro. equipotent concentrations of denufosol and utp significantly increased asl height. however, denufosol produced maximum asl height increases that were both greater (77.6% versus 33.2% maximum increase over basal asl for denufosol and utp, respectively) and longer-lasting (72.2% versus 16.6% increase over basal asl following a 60 minute application of denufosol and utp, respectively) compared to utp. furthermore, the asl height increases are specific for denufosol as the addition of apyrase blunts the response to utp, but not denufosol. our data demonstrate that denufosol is more stable than utp on the mucosal surface of human airway epithelia, which results in larger and more sustained increases in asl volume. recent phase ii clinical trials show that administration of denufosol over 28 days was well tolerated and associated with improved lung function in mild cf patients. taken together, these data suggest that denufosol is a promising candidate molecule for cf therapeutics. mucociliary clearance (mcc) is the primary airway host defense against chronic exposure to infectious and noxious agents. mcc is dependent upon ciliary beating and the volume and composition of the airway surface liquid (asl). asl volume is regulated via isotonic fluid transport which is dominated by na + absorption in the superficial epithelium. in cf, unregulated na + absorption through enac drives asl volume depletion and a subsequent decline in mcc. the accumulation of mucus in the airways of cf patients supports persistent and life-threatening bacterial infection. increasing airway surface hydration represents a promising therapeutic approach for treating cf. in vivo, aerosolized osmotic agents such as hypertonic saline (hs) improve mcc and lung function in cf patients. however, the benefits derived from hs treatments are transient, as nacl is relatively rapidly absorbed by airway epithelia. previously, donaldson et al. (nejm 2006) tested the hypothesis that blocking na + absorption with the enac inhibitor amiloride would increase the efficacy and extend the benefit of hs. surprisingly, amiloride blunted the response to hs, which resulted from a previously unrecognized property of amiloride to inhibit aquaporin-mediated water transport. while potent inhibitors of enac would likely be of great benefit to the cf treatment milieu, the compounds then available were not adequate for this purpose. parion sciences has developed novel 2-substituted pyrazinoylguanidine compounds that selectively inhibit enac and are >100-fold more potent than amiloride. in the present study, we evaluated the ability of two compounds, 552-02 and 680, to increase asl volume under thin film conditions. in primary cultures of human bronchial epithelial cells (hbes), 552-02 alone produced a small, but significant increase in asl height. in cf cultures exposed to phasic motion that simulates the shear stress generated by normal tidal breathing, 552-02 alone increased asl heights by 46.7%. additionally, we tested the effects of 552-02 pre-addition in combination with 4% hs. as expected, hs alone produced a rapid and substantial increase in asl height (671.7% by 10 minutes) which declined to 50% bỹ 1 hour post-treatment. pre-treatment of cultures with 552-02 prior to hs was both more potent (867.8% by 10 minutes) and longer-lasting (50% initial response at ~4 hours) than hs alone. on normal hbes cultures, the 680 compound alone increased the basal asl height by 107.7% following a 2 hour exposure. similar to 552-02, 680 in combination with hs likewise extended the longevity of the hs effect (t1/2 ~4 h versus ~1 h for 680 + hs and hs, respectively). by blocking na + absorption, the parion compounds alone increased asl volume in both normal and cf hbes. strikingly, when used in combination with hs, the parion compounds enhance and sustain the increase in asl volume associated with hs alone. our data demonstrate that the parion compounds alone are sufficient to increase basal asl volume. furthermore, these data provide a proof-of-concept that combinational therapies utilizing osmotic agents and compounds that regulate ion transport will provide therapeutic benefits to cf patients. methods: a total of 24 patients (with fev 1 ≥40% of predicted) received 500 mg of arikace by inhalation for 14 days. drug was administered using the pari lc star nebulizer. laboratory parameters, adverse events and pulmonary function tests were collected for all study subjects in order to determine safety and tolerability of arikace™. sputum samples were collected to determine changes in bacterial density, and amikacin pharmacokinetic parameters were assessed at selected time points in urine, serum and sputum specimens. change in fev 1 , fev 1 % predicted, fef 25-75 % and fvc relative to baseline and change in log 10 cfu on days 7 and 14 were assessed. results: on day 7, 14 and 21, the observed change for fef 25-75 % was 0.49 (p < 0.001), 0.42 (p = 0.02) and 0.34 l/sec (p = 0.04), respectively. on day 7 and 14, the observed change for fev 1 was 0.24 (p = 0.002) and 0.13 l (p = 0.10), respectively, and was 7.49 (p <0.001) and 4.38 l/sec (p = 0.03) for fev 1 % predicted. significant relationships (p ≤ 0.05) between log 10 cfu and serum auc:mic ratio, and between changes in log 10 cfu and fev 1 , fev 1 % predicted and fvc were identified. treatment was safe and well tolerated with the most frequent adverse events being dyspnea and headache of mild to moderate severity. conclusion: inhaled arikace™ 50 mg/ml was well tolerated and in select patients improved pulmonary function. together these clinically relevant changes from baseline likely represent drug effect and warrant further development of liposomal amikacin for inhalation in patients with cf. allergic bronchopulmonary aspergillosis (abpa) is a disease caused by hypersensitivity to aspergillus fumigatus (af). the prevalence of abpa is approximately 1%-15% of patients with cf and can contribute to worsening of their pulmonary disease. the treatment of abpa consists of high-dose oral corticosteroids for many months and may include also antifungal antibiotic such as itraconazole. the clinical effectiveness of corticosteroids is usually shown by an improvement in clinical symptoms and radiological parameters, as well as a reduction in total ige. the prolonged period of systemic steroids may cause significant side effects such as cushingoid appearance, hypertension, glucose intolerance and more. over the past 5 years, we have used pulse high-dose methylprednisolone in 5 patients with cf. method:all 5 patients were diagnosed as suffering from abpa by the standard criteria as the may have mucoid impaction or central bronchiectasis on chest radiography, an elevated ige (>417 iu/ml), the presence of specific ige anti af and an elevated eosinophil count. the patient may have a positive skin prick test to af allergen. they were treated once a month with iv pulse high-dose methylprednisolone (10mg/kg/a day) for 3 days once a month for several months until the total ige decreased to normal values. three patients were treated also with itraconazole. in 2 patients itraconazole was discontinues due to side effects. results: we treated 5 patients age 10-35 years old (3m/2f). four patient are pancreatic insufficient and 1 pancreatic sufficient. ige was 622+/-371 (119-1213) at the time of diagnosis and decrease to 55+/-64 (57-89). fev1 increased from 65+/-16 to 78+/-15. the patients also gained weight and improved their chest x-ray finding. side effect were minor and mainly during the treatment days and resolved 1-2 days after each treatment and included malaise during the infusions, glucose intolerance on infusion days in one case, and hypertension in another case. conclusion: iv pulse steroid treatment should be considered in abpa as it was found effective with fewer side effects that should be expected from prolonged oral corticosteroid treatment. lung pathology in individuals with cystic fibrosis (cf) is linked to sodium hyper-absorption. the defective regulation of the epithelial sodium channel enac is thought to be a major contributor to reduced airway surface liquid (asl) volume and impaired mucociliary clearance of the airways. thus, strategies designed to inhibit enac function may result in clinical benefit. rna interference (rnai), mediated by short, double-stranded rna molecules, can be used to target complementary rna sequences for degradation via the rna induced silencing complex (risc). we investigated the possibility of using rnai to reduce expression of enac in the mouse lung, as proof of principle for a strategy to reduce sodium hyperabsorption in the cf lung. potent sirna molecules capable of efficient knockdown of enac alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney m1 cells (50,000 cells per 24-well; 20pmol sirna complexed with lipofectamine 2000; 48 hour harvest) in which enac expression was quantified using real-time rt-pcr. approximately 80% knockdown of each enac subunit was observed with the most potent sirna molecules in this system. subsequently, sirna molecules were delivered to the lungs of female balb/c mice via hydrodynamic tail vein injection (40 µg naked sirna, n=5-6). after 24 hours, lungs were harvested, rna extracted and enac mrna measured using real-time taqman pcr. whereas, enac subunit mrna levels in mice treated with a negative control sirna were not different from untreated mice (p < 0.05), delivery of enac alpha and beta-specific sirna molecules resulted in a reduction to 56.5 ± 12.3 % or 33.3 ± 4.7 % of the expression levels observed in untreated mice, respectively (p < 0.05, mann-whitney u). interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent enac gamma-specific sirna molecule led to no reduction in enac gamma expression compared with untreated controls (p > 0.05). these data show proof of principle that enac expression can be reduced in the lung using sirna. further work is needed to assess the functional consequences of inhibiting enac in the lung. gene therapy for cystic fibrosis lung disease will likely require longterm transgene expression; however, in the lung, many gene transfer agents have resulted in only transient gene expression. previously we have shown that the choice of enhancer/promoter elements has a strong influence on the duration of reporter gene expression following delivery of non-viral vectors to the mouse lung (gill et al., 2001 , gene ther, 8, 1539 . however, it is also possible that other elements of plasmid vector design play an important role. using a clinically relevant mouse lung aerosol model, we have studied the effect of varying the plasmid cpg-dinucleotide content on the duration of expression from plasmids that had an identical enhancer/promoter sequence. firstly we constructed a cpg-free plasmid vector containing a synthetic cpg-free luciferase gene under the transcriptional control of the human cmv enhancer/ef1α promoter. secondly, we constructed a similar vector containing an identical enhancer and promoter but with a small number of cpg motifs, in the luciferase gene (97 cpgs) and in the backbone sequence (52 cpgs). we then investigated the level and duration of transgene expression following aerosol delivery of these plasmids complexed with the genzyme lipid gl67 to the lungs of balb/c mice (2.5 mg/ml pdna, 6mm gl67, 10ml, pari lc+ nebuliser). total lung extracts were assayed for luciferase activity at days 1, 2, 7, 14 & 28 (n=6 per time-point). the cpgcontaining plasmid initially directed approximately 2-fold higher levels of reporter gene expression than the cpg-free plasmid (p<0.05 both days 1 and 2 post-delivery). however, while expression from the cpg-containing plasmid subsequently fell slowly to background levels, expression from the cpg-free plasmid increased 2-fold to day 7, and remained at the peak levels observed with the cpg-containing plasmid until the end of the experiment at day 28 (cpg-free plasmid 4-fold, 16-fold and 50-fold higher expression at days 7, 14 and 28 post-delivery respectively; p<0.05 for each). these data suggest that the ability of a promoter/enhancer sequence to direct long-term expression in the mouse lung is dependent by the sequence of the plasmid backbone. one explanation for this observation is that a specific, but as yet unidentified, sequence exists in the cpg containing backbone that triggers transcriptional silencing. alternatively, the well-described host inflammatory response to cpg-containing dna, may be involved and the cpg content of the vector could be responsible for these differences. we are currently evaluating the potency and duration of effect of cftr expressing, cpgfree, plasmids in late-stage pre-clinical studies prior to the initiation of a further round of clinical studies of non-viral gene transfer in cf patients. background: novel approaches to cf therapy by improving/restoring cftr chloride channel function using gene therapeutic tools or small molecules are currently undergoing phase i and phase ii clinical trials. there remains a great need for accurate and practical methods of measuring cftr function in vivo to act as primary outcome measures of efficacy. we propose that an in vivo assay measuring cftr function in sweat glands offers several potential advantages over the nasal potential difference test. objective: to develop a reliable test of sweat gland function that is capable of measuring the range of cftr function in vivo. methods: we are performing repeated measures of sweat gland function in healthy controls (n=10), obligate heterozygotes (n=10), pancreatic sufficient (cfps, n=10) and insufficient cf patients (cfpi, n=10). sweat secretion is stimulated by iontophoresis (30µa/cm2) with 1% pilocarpine. to elucidate the most sensitive and discriminatory parameter of sweat gland function within this study group we are performing simultaneous measurements of: a) the transglandular potential difference of a stimulated skin area using an ecg electrode (espd) and b) single sweat gland potential differences in single sweat glands (spd). using a wescor® collector cup we also measure: c) sweat secretion rate and (d) sweat chloride concentration (sweat cl-). results: we report interim results of our ongoing study (table) . sweat rate was not different between the groups, but a gender difference was observed (mean±sd; 12 male: 1.6 ± 0.7µl/min/cm2; 15 female: 0.9 ± 0.5µl/min/cm2, p<0.001) as previously described. our preliminary data show that espd as well as sweat cl-allow good discrimination among healthy controls, cfps and cfpi (p<0.01 and p<0.05 respectively). spd show similar trends to espd but with greater overlap (differences not significant). conclusion: these encouraging preliminary results, particularly the espd method and sweat cl-following stimulation with a lower iontophoretic current, justify further efforts to complete enrolment of subjects and to further refine technical challenges such as minimizing the effect liquid junction potentials across the ecg electrode. results will also be compared to the classical sweat test using higher iontophoresis current and npd measurements in these patients. this study is supported by the ccff and genome canada. ino-4995, a prodrug, has been demonstrated to inhibit nasal potential difference (pd)in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. as such it is being developed as potential therapeutic for cystic fibrosis. ino-4995 cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino-4913) with the ether-linked octyl group is expected to be more slowly metabolized. however, the precise kinetics of the uptake of ino-4995, its conversion to its active metabolite, and methods. confluent cultures of hela or t84 cells were incubated for varying periods of time ranging from 10 min to 3 hrs with medium containing [ 3 h] ino-4995 (2 x 10 6 cpm/ml) and 5µm ino-4995 cold carrier to ascertain the rate of uptake. in pulse chase experiments after cultures were incubated for 2 hours, they were washed and incubated with media without radiolabel for varying periods of time. after the indicated times, the media was removed and the cells were washed, harvested, extracted and the aqueous and organic extracts subjected to sax and reversed phase hplc respectively. results ino-4995, a prodrug, has been demonstrated to inhibit nasal pd in human cf nasal airway epithelia and cf mice and this effect is more potent with repeated dosing. ino-4995 cell entry is facilitated by its hydrophobic propionoxy(methyl)ester protecting groups which can be hydrolyzed by intracellular carboxyesterases after the prodrug enters the cell. once the protecting groups are removed, the drug (ino-4913) with the ether-linked octyl group is expected to be more slowly metabolized. however, it is not known whether the mucus present in cf airways could impede its access to airway epithelia or whether ino-4995 would cross the epithelial layer to enter the bloodstream. here, in order to address these issues we measured its uptake into human tracheal epithelial sheets in the presence and absence of mucus and compared its transepithelial permeation to mannitol. we studied the uptake and transepithelial fluxes of [ 3 h]ino-4995 in primary cultures of human tracheal epithelium with transepithelial resistance of from 500 to 1000 ohms.cm 2 . the mucosal surface of some tissues was rinsed three times with pbs to remove mucus for comparison with control cells which were not rinsed. elisa for human airway mucins performed on the rinsings confirmed that three rinses sufficed to remove the mucosal mucous blanket. the development of long-term, safe and efficacious gene therapies for lung disease will require efficient gene delivery to stem cells which produce the differentiated progeny of affected epithelial tissues. recombinant lentiviral vectors are being considered for gene therapy applications for the lung because they can efficiently transduce a wide variety of stem and progenitor cell types. however, it is of concern that strong constitutive viral promoters have produced oncogenesis in x-scid patient recipients of retroviral vectors in part due to the disregulation of proto-oncogenes by the strong viral promoter/enhancer located near the integration site. we propose that regulation of transgene expression to the tissue or cell type of interest will enhance the safety of gene therapy for respiratory disease. in this study we have investigated the potential for respiratory specific transgene expression from integrated lentiproviruses in cell lines in vitro and in mice in vivo. the lentiviral vectors constructed for this study contained regulatory elements predicted to produce lung specific transgene expression: the surfactant protein c promoter (spc) for alveolar epithelial type ii cell (aecii) expression, the clara cell 10kd protein (cc10) for clara cell expression in the airway, and the jaagskiete sheep retrovirus (jsrv) promoter for expression in both cell types. transgene expression from the spc and cc10 vectors was restricted to aecii and clara cell lines respectively, while expression from the jsrv vector was observed in multiple respiratory and non-respiratory cell types. following intra-tracheal delivery of lentivector supernatant to mice, transgene expression was observed in aecii from the spc lentivector (n=8 mice), and in clara cells from the cc10 promoted lentivector (n=6 mice). transgene expression was not detected in non-respiratory tissues following intravenous delivery of cc10 and spc lentiviral vectors to neonatal murine recipients (n=4 and n=7 respectively). in summary, incorporation of genomic regulatory elements from the spc and cc10 genes resulted in respiratory specific transgene expression in vitro and in vivo. these vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders such as cf. cystic fibrosis (cf) is associated with chronic pulmonary inflammation and progressive lung dysfunction, associated with the formation of oxidants derived from neutrophil myeloperoxidase (mpo). indeed, respiratory tract levels of mpo are extensive, and have been found to correlate with decreases in respiratory parameters or disease severity in cf. mpo-derived oxidants thus may play a role in perpetuating the progressive lung dysfunction associated with cf. based upon these premises, we reasoned that mpo could represent a novel therapeutic target for the treatment of inflammatory diseases such as cf. herein we report that substituted urea and thiourea compounds are potent inhibitors of mpo. utilizing tetramethylbenzidine (tmb) as a classical heme peroxidase substrate, we screened a large number of structurally varied urea and thiourea derivatives. alkyl, cycloalkyl, and aromatic ureas and thioureas all proved to be effective mpo inhibitors, however, with a high degree of variability in potency. of those compounds examined, substituted phenylthioureas proved to be the most potent mpo inhibitors. various ortho-, meta-, and para-substituted phenylthioureas (ptus) were then assessed for inhibitor potency. the most potent inhibitor of mpo was 2-chlorophenylthiourea (ic50 = 700 nm, ki = 320 nm). the potency of ortho-ptus as mpo inhibitors showed a linear relationship with ortho sigma substituent constants (reflecting resonance, steric and inductive effects). moreover, cyclic voltametry experiments with ortho-substituted ptu compounds revealed a linear relationship between inhibitor potency and thiourea oxidation potential. this correlation is indicative of a substituent effect on the relative ease of sulfur oxidation within the thiourea moiety. this finding suggests that the redox-active sulfur plays a crucial role in mpo inhibition, perhaps via a direct interaction with the heme of mpo. thioureas proved to also inhibit the chlorinating activity of mpo, whereas sustituted ureas were not effective in this regard. both ureas and thioureas were also effective inhibitors of dityrosine formation by mpo. these findings will help facilitate the rational design and optimization of mpo inhibitors with even greater potency, which could serve as novel therapeutic agents for the treatment of cf. gene therapy is the transfer of a normal copy of the cftr gene and should, in theory, provide a long-term cure for cf. the development of prenatal screening programs for cystic fibrosis has provided an opportunity to identify cf patients in utero, and may provide an opportunity to treat the disease before birth, prior to the onset of disease symptoms. the long-term goal of our study is to investigate the therapeutic potential of delivering the normal cftr gene to epithelial stem cells in fetal cf mice by in utero administration of recombinant lentiviral vectors carrying the normal cftr gene. this would provide us with the unique opportunity to "correct" the chloride channel defect in the epithelial stem cells, which give rise to the epithelial cells in the tissues affected by cf at a very early stage. the aim of the current study is to optimize the in utero gene delivery of recombinant lentiviral vectors, carrying an egfp reporter gene to epithelial stem cells in fetal mice. mice have been used for these studies, as mouse strains with a variety of cf mutations are available. in all studies, the uterus was exteriorized following a mid-line laparotomy, and the injections were made directly into the amniotic fluid of individual fetuses. in the first studies, the survival of pregnant females and pups following saline injection at e16.5 (n=2) and e17.5 (n=3) was evaluated. all five females survived the procedure and an average of 54% (26/48) of the pups present at the time of injection were born alive. this experiment demonstrated that the surgical procedures were reasonably safe. in the next experiments, we injected 5.0ul lentiviral supernatant (~4.5x107iu/pup) into the amniotic fluid of each pup and evaluated the effect of the gestational age pups on transduction efficiency. the pregnant mice were allowed to carry the fetuses to term. at p0, p7 and p28, 2-3 pups of each litter were sacrificed and skin, trachea, lung, stomach and intestines were harvested for analysis of gene transfer and egfp transgene expression. all but one pup (11/12) from the e12.5 time point aborted prior to parturition. from the e14.4 and e16.5 injections, 6/12 and 7/7 pups were born naturally, respectively. at e14.5 and e16.5, proviral sequences were observed in the skin, trachea and stomach, although the level of transduction varied among individual fetuses and tissues. immunofluorescence analysis on the skin of one mouse with a high proviral copy number demonstrated that some of the egfp positive cells were also positive for vimentin or f4/80, markers of mesenchymal cells and macrophages, respectively but negative for pancytokeratin. our preliminary studies suggest that in utero gene therapy with lentiviral gene delivery may be safe, however ongoing studies will provide more in depth analysis on safety and feasibility of delivering the normal cftr cdna to epithelial stem cells in the tissues affected by cf in utero. future studies will investigate the therapeutic potential of delivering a lentiviral vector containing the cftr gene to fetal pups in a murine model of cf. supported background & objectives: cystic fibrosis is characterized by progressive loss of lung function resulting from chronic bacterial infection and concomitant airway inflammation. while a number of bacteria are known pathogens in cf, mounting evidence suggests that the lungs of cf patients harbor numerous bacterial species, and that interactions within this ecosystem may affect outcomes. however, the composition of this community is not well characterized. we therefore initiated work to profile the bacterial diversity of the lungs of cf patients with end-stage disease by 16s rrna gene sequencing. methods: genomic dna was purified from snap-frozen explanted lung tissue of 21 randomly selected cf patients undergoing transplantation in toronto between 1998 and 2006. bacterial 16s rrna genes were amplified from each sample using broad-spectrum pcr primers, and pcr products used to generate 16s rdna libraries for each patient. 16s rdna profiles for each patient were generated by automated dna sequencing of up to 96 randomly selected clones per library, and tentative species assignments made by comparison to known 16s rrna gene sequences using phylogenetically-based analyses. results: sequence analysis of 1514 16s rdna sequences from this preliminary set of 21 patients (avg. = 72 sequences/patient; range = 36-96) demonstrates a range of complexity of the microflora in cf lungs, with some patients showing a single dominant organism and others showing multiple co-existing species. in total, over 60 species have been identified to date in this set of patients. these include species identified previously by clinical microbiology, including pseudomonas aeruginosa (detected in 20/21 patients), and burkholderia species (detected in 4/5 patients known clinically to be infected). numerous organisms not previously reported in the lung were also identified, including several from the taxonomic order burkholderiales. interestingly, while organisms commonly associated with cf were detected in patients transplanted throughout the year, a cluster of four plant-associated species demonstrated striking seasonal variation in rates of detection. conclusions: the lungs of cf lung transplant patients harbor a diverse bacterial community. sequencing of 16s rrna genes is a powerful method to profile this diversity. correlation of these profiles with clinical information may help identify new patterns of infection and important interactions between species in this community. introduction and aims: pulmonary infection, primarily with pseudomonas aeruginosa, is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. we have demonstrated that anaerobic bacteria, belonging primarily to the genera prevotella, veillonella, propionibacterium and actinomyces, can be cultured from the sputum of cf patients. the aim of this study was to determine the antibiotic susceptibility of these anaerobic isolates grown planktonically and as biofilms. methods: the planktonic susceptibility of anaerobic isolates to antibiotics used in the treatment of cf (meropenem, piperacillin/tazobactam) as well as to antibiotics traditionally used to treat anaerobic infection (clindamycin, metronidazole and ampicillin) was examined using e-test® strips. selected anaerobic isolates were grown as biofilms for 4 or 24 hours in 96 well trays prior to challenge with antibiotic. bacterial biofilm formation was determined using crystal violet. results: the planktonic susceptibility of 39 anaerobic isolates (14 prevotella, 5 veillonella, 5 propionibacterium, 8 actinomyces and 7 additional isolates) comprising 9 different genera was determined. although all of the anaerobic isolates examined were sensitive to meropenem when grown planktonically, high levels of resistance to the anti-anaerobic drugs, metronidazole and clindamycin were observed. examining susceptibility by genera, the veillonella were the most resistant to piperacillin/tazobactam and the propionibacterium the most resistant to metronidazole. furthermore, several isolates, including 4 of the 14 prevotella isolates examined, were found to be resistant to multiple antibiotics. the susceptibility of anaerobic biofilms to antibiotics was found to be isolate and antibiotic dependent. generally antibiotic treatment of 4 hour old biofilms was often able to eradicate biomass and prevent biofilm formation; however, antibiotic treatment after 24 hours of biofilm formation with up to 100x the mic of an antibiotic often failed to eradicate the biofilm. conclusion: these results suggest that alternative antibiotic treatment regimes may be necessary to treat cf pulmonary infection if anaerobes are present. these results also suggest, based on in vitro susceptibility, that meropenem and not metronidazole or clindamycin would be the most effective antibiotic in treating any anaerobes present in cf pulmonary infection. early eradication protocols for the first appearance of pseudomonas aeruginosa (psa) have become standard practice in many cystic fibrosis (cf) clinics. the details of such protocols often vary significantly between clinics, making it important to compare efficacy from one approach to another. however, comparison of reported results is currently very difficult. this is not only because of variations in completeness of reporting, but especially because calculation of average time to regrowth cannot by definition include patients who remain free of psa regrowth at the time the "average" is calculated (nor those in whom a re-appearance of psa is found to be due to a different strain). such results would be better expressed using "time to event" (tte) statistics (analogous to "life tables") and different protocols can be better compared using this methodology. we report for the first time, results from our psa early aggressive eradication protocol for the period 1995-2007, expressed using tte statistical analysis. children are seen in clinic on average 4 times yearly for full assessment, and at each clinic visit sputum or pharyngeal cultures obtained by the physiotherapist. standard treatment for "first growth" pseudomonas aeruginosa consists of 2 weeks of intravenous antibiotics (piperacillin 600 mg/kg/day plus tobramycin 12 mg/kg/day) followed by 3 weeks of oral ciprofloxacin (20-30 mg/kg/day) and inhaled colymycin (100 mg bid) for 6 months. a total of 145 treatment courses have been completed for the period 1995-2007 with a 90% clearance rate, defined as 3 or more consecutive negative cultures for pseudomonas aeruginosa over 6 months. since the initiation of this protocol, the percentage of patients colonized with psa in our cf pediatric clinic has declined from 44% in 1995, down to 15% in 2007. subsequent regrowth of psa occurred in 1/3 of patients. treatment courses may then be repeated a second, third, or fourth time. to determine whether these repeat psa isolates are newly acquired or identical to the previous isolate, rapd typing of these isolates is routinely assessed. a total of 44 isolates have been rapd tested and have identified the same strain as the previous isolate in 16 (36%) and a different strain in 28 (64%)treatment courses. the average time to regrowth for those who had a recurrence of psa following this treatment protocol was 24 months, but this does not include those patients who have had no further growth of psa for periods up to 10 years. to reflect the results for all patients, including those who have not as yet had further regrowth of psa, we have constructed tte curves. these measures can be used to compare results using our protocol with results from other approaches to clearing pseudomonas aeruginosa in cf. retsch-bogart, g.z. 1 inhaled antibiotics have been part of the therapeutic armamentarium for patients with cf for decades. we studied the effect of inhaled aztreonam on respiratory symptoms in cf in a phase 3, double blind, placebo (pl) controlled trial of azli, a novel formulation of aztreonam, which enrolled 164 patients with cf from 67 centers in the us, canada, australia, and new zealand. all patients have completed study participation. inclusion criteria included age ≥ 6 years, pa in sputum or throat swab, fev1 ≥ 25% to ≤ 75% predicted, and no use of anti-pseudomonal antibiotics in the previous 14 days. following a 14 day screening period, patients were treated for 28 days with either azli 75 mg or pl. study drug was administered tid using the pari eflow® electronic nebulizer after pre-treatment with bronchodilator. concomitant standard cf therapies were allowed, with the exception of anti-pseudomonal antibiotics, azithromycin and hypertonic saline. the primary endpoint was change from baseline to day 28 in scores from the cystic fibrosis questionnaire-revised (cfq-r) respiratory domain, a validated patient-reported measure of respiratory symptoms. other efficacy measures included change in pulmonary function (fev1, fvc, and fef25-75), number of hospital days and number of courses of iv, oral or inhaled anti-pseudomonal antibiotics. microbiological endpoints included change in pa bacterial density in sputum, change in susceptibility of pa to aztreonam, and emergence or disappearance of other pathogens. safety evaluations included adverse events, airway reactivity (defined as acute decrease in fev1 ≥ 15% at 30 minutes after dosing) and clinical chemistry and hematology. complete efficacy and safety results from this trial will be presented. salam, a.p.; orchard, c.; wee, a.; hodson, m. introduction: oral ciprofloxacin is often used for pseudomonas aeruginosa (psa) infections in patients with cystic fibrosis (cf) not requiring intravenous antibiotics. oral chloramphenicol is commonly used at the royal brompton hospital (rbh) as an alternative to ciprofloxacin, but less so in other cf centres due to concerns about aplastic anaemia and uncertainty regarding effectiveness. this study aimed to address three questions: 1) what proportion of psa is sensitive to ciprofloxacin and/or chloramphenicol? 2) were there any adverse haematological effects with the use of oral chloramphenicol at rbh? 3) how effective is chloramphenicol in comparison to ciprofloxacin? study design: we carried out a retrospective review from the rbh cf database (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) together with analysis of patients' notes. methods: 1) psa sensitivities to ciprofloxacin and chloramphenicol were recorded from sputum samples taken in late 2004. 2) all blood dyscrasias in patients who had received chloramphenicol from 1985 to 2004 were reviewed. 3) the database was searched for patients who had received a course of either oral chloramphenicol or ciprofloxacin from 2004 to 2005. 25 patients from each group were randomly selected. fev 1 , fvc and oxygen saturations pre and post treatment completion were analysed, as were markers of symptom improvement. results: 1) 216 sputum samples from 216 patients grew psa. some were sensitive to only one antibiotic, and there were some cross-sensitivities, but in total 35.46% were sensitive to chloramphenicol and 54.25% to ciprofloxacin. 2) 648 patients received oral chloramphenicol and 755 oral ciprofloxacin from 1985-2004. multiple courses were often given, but usually no more frequent than 3 monthly with chloramphenicol. over 20 years, 4 cases of blood dyscrasia were recorded, none due to chloramphenicol, and all recovered. 3) in the ciprofloxacin group: the mean oxygen saturations improved by 0.25 % (p 0.672), the mean fev 1 improved by 5.3% (p 0.126), and the mean fvc by 3.35% (p 0.202). in the chloramphenicol group: the mean oxygen saturations improved by 0.48% (p 0.118). ), the mean fev 1 improved by 4% (p 0.438) and the mean fvc improved by 5.9% (p 0.047). 56% of patients in the chloramphenicol group and 56% of patients in the ciprofloxacin group reported symptom improvement. a significant proportion of psa is sensitive to chloramphenicol and in over 20 years there have been no documented cases of related aplastic anaemia. clinical improvements with choramphenicol comparable to ciprofloxacin were also recorded. our data shows chloramphenicol is safe and may be the oral antibiotic of choice in ciprofloxacin allergy or psa resistance to ciprofloxacin. a randomised prospective study is needed to further evaluate the efficacy of chloramphenicol in comparison to ciprofloxacin. pseudomonas aeruginosa lung infections are one of the main factors causing disease in cf patients. at present the generally recommended treatment is repeated courses of antibiotics. this has resulted in fewer infections and a markedly improved prognosis for the patients. but it has also severe drawbacks such as antibiotic resistance, disturbed normal flora and allergenicity. antibiotic resistance is an emerging problem world wide and alternatives are urgently needed. specific chicken antibodies, anti-pseudomonas igy, have potential both as an alternative and a complement to antibiotics (pediatr pulmonol 2003; 35:433) . anti-pseudomonas igy prevents p. aeruginosa from adhesion to epithelial cells and has affinity for flagellin in vitro. humans do not produce anti-igy antibodies and oral administration of igy is generally regarded as safe. the risk that bacteria should develop resistance to igy is extremely low. in total, 21 cf patients have received antipseudomonas igy for up to 12 years. since november, 2003 the drug is given to a group of cf patients (at present 14 patients) on individual permissions granted by the swedish mpa and reimbursed by the swedish government. in our first study about the effect of igy there were 2.5 positive cultures/100 months, compared to 13.7/100 months in a control group. up to december 31, 2006, nearly five years later, the effect is maintained with 2.4/100 months for the whole study period. we are at present compiling the data from the control group during the same period. only two siblings in the igy-treated group have been chronically colonized (with an identical p. aeruginosa strain), which still is non-mucoid after 4.5 and 1.5 years. during the whole treatment period there have been no cultures positive for mucoid p. aeruginosa or b. cepacia and other pathogens (s. maltophilia, a. xyloxidans, atypical mycobacteria and a. fumigatus) have only appeared sporadically -possibly due to the relatively low use of antibiotics. all patients have preserved pulmonary functions and nutritional status. there have been no adverse events. thus treatment with anti-pseudomonas igy has diminished the number of positive cultures and delayed the onset of chronic infection. the need for antibiotics is reduced as well as their adverse side effects. in conclusion, igy is an important complement to antibiotics for prevention of p. aeruginosa infections in cf. introduction and aims: we have previously shown by culture that the lungs of clinically stable cystic fibrosis (cf) patients are not only colonised by commonly recognised aerobic bacteria, such as pseudomonas aeruginosa, but also by a range of potentially pathogenic anaerobic species. the aim of this study was to determine whether anaerobes are also present in the sputum of cf patients with an acute exacerbation of pulmonary infection. methods: sputum samples were collected and processed using strict anaerobic bacteriological techniques, prior to commencing and at the end of antibiotic therapy, from 32 adult cf patients admitted for treatment of an acute exacerbation of pulmonary infection. bacteria within the samples were detected by plating on selective agars, quantified by total viable count and identified by pcr and sequencing of 16s ribosomal rna genes. as a control induced sputum samples were collected, using hypertonic saline, from 20 healthy volunteers who did not have cf and processed using similar methods. results: anaerobes from a range of species including prevotella, veillonella, propionibacterium, actinomyces and gemella were detected in high numbers (up to 6 x 10 7 cfu/g of sputum) from all patients prior to commencing antibiotic therapy with the predominant primary pathogens (p. aeruginosa or b. cepacia complex) detected in similar numbers. anaerobic bacteria were also detected in sputum samples at the end of antibiotic treatment and for 22/32 (69%) patients they were present in lower numbers than detected before antibiotic treatment. moreover, in 11/32 (34%) patients there was greater than a one log reduction in the total viable count of anaerobes. similar data for detection of the predominant aerobic pathogens was also collected for 27 of these patients. p. aeruginosa or b. cepacia complex was present in lower numbers following antibiotic treatment in 22/27 (81%) patients and in 13/27 (48%) patients there was more than a one log reduction in the total viable count. anaerobes were not detected in the induced sputum samples from 4 volunteers and in the remaining samples they were present in much smaller numbers, ranging from 10 2 to 10 5 cfu/g of induced sputum, than detected in cf sputum. the most commonly isolated bacteria from the induced sputum samples were the actinomyces and streptococcus, with prevotella cultured from only 2 samples. no veillonella and propionibacterium species were isolated from the induced sputum samples and combined with the low frequency of isolation of prevotella species, these results strongly suggests that the prevotella, veillonella and propionibacterium species recovered from cf sputum samples are not oral contaminants. conclusion: these results indicate that anaerobes are present in large numbers within the cf lung during an acute exacerbation of pulmonary infection. their presence could be of important clinical relevance to cf patients as they may contribute significantly to the inflammatory process. airway infection and inflammation cause the majority of morbidity and mortality in cystic fibrosis (cf). the microbiology of cf is complex. not only are the routinely identified pathogenic bacteria present, increasingly a number of other bacteria are associated with pulmonary disease. in addition, the normal microbiota likely contribute to airway disease in cf via intracellular signaling, but remains largely unstudied. the application of culture independent methods for bacterial identification in cf airway secretions provides for the first time the ability to characterize the bacterial communities in the cf airway comprehensively and efficiently. here we present the results of a two-year study of cf airway samples to describe the bacterial communities present by ribosomal rna sequences. we have examined 231 airway specimens from 117 subjects (82 cf, 35 controls). these results provide a unique perspective on cf airway microbiology. bacterial communities identified in sputum were more complex than communities identified in bronchoalveolar lavage fluid (balf). however, pulmonary status determined the level of complexity, with higher complexity observed during stable pulmonary function. we identified a collection of anaerobic bacteria that were present at high levels during pulmonary exacerbation in a subset of cf subjects. detection rates ranged from 8-50% of cf subjects examined depending on the specific organism (table 1 ). these organisms of interest are suspected to be involved in pulmonary exacerbation especially when cultures for standard cf pathogens are negative (~20% in our center). preliminary data will be presented from quantitative real-time pcr experiments to track these organisms. we also will present the application of new meth-ods for community comparisons. this analysis provides the ability to look at how clinical information from individuals with cf corresponds to the microbial communities that occur in the airway. overall, the results point to a much richer bacterial community associated with the cf airway than previously thought. the results also point to new opportunities to understand the differences within the cf population, and thus potentially improve individual patient outcomes. cystic fibrosis (cf) patients are characterized by persistent microbial colonization of the airways and recurrent pulmonary infection. however, due to the domination of bacterial communities present in cf sputum by a small number of species (typically pseudomonas aeruginosa), culture-independent approaches to characterize the depth of bacterial diversity in cf sputum have yielded limited information. here we describe application of a novel microarray, the 16s rrna phylochip to comprehensively describe the bacterial diversity present in temporal sputum samples of 3 cf patients who experienced periods of exacerbation (defined as hospitalization) and remission. in addition, these samples permitted assessment of bacterial community dynamism during periods of antimicrobial administration in these patients. phylochip analysis identified 980 bacterial taxa present across these patient samples, including a large number of known human pathogens. antimicrobial administration caused a profound decrease in bacterial diversity in all patients examined. despite this, a sizeable fraction of each community remained stable or proliferated during the period of antimicrobial administration, suggesting many of the bacterial taxa present were resistant to the antimicrobials administered. taxa exhibiting this behavior included p. aeruginosa, arcobacter cryaerophilus, streptococcus constellatus and burkholderia mallei amongst other pathogens. these results suggest that complex polymicrobial communities exist in cf sputum that exhibit significant antimicrobial driven dynamism and consist of multiple antimicrobial resistant pathogens. future studies will focus on whole community gene expression to determine which virulence systems are activated in response to bacterial community perturbations and contribute to pathogenic processes in cf airways. the lungs of chronically infected cystic fibrosis (cf) patients have been intensively studied. it is well documented that pseudomonas aeruginosa is the predominant pathogen in cf (høiby 1974 , baurnfeind et al.1987 , koch 2002 , and that p. aeruginosa accumulates in heaps (aggregates), detected in cf sputum (høiby 1977) and intraluminal in the lung (baltimore et al. 1989) . the bacterial density is highest in the bronchi (potts et al 1995) and these p. aeruginosa microcolonies are embedded in a matrix (lam et al. 1980) . the morbidity of the infection is due to an extensive and ongoing pmn response which apparently does not eradicate the bacteria, instead leading to slow degradation of the lung. in addition, even highest deliverable doses of antibiotics fail to clear the bacteria completely, though survival has significantly increased the last 30 years due to aggressive anti p. aeruginosa therapy (koch and høiby 2000) the present study was performed to investigate the significance of the aggressive therapy for the distribution of p. aeruginosa and how p. aeruginosa is organized and persists in the cf lung. the material used was: autopsies from dead short term colonized cf patients (n=12) obtained before today's aggressive antibiotic treatment , and explanted lungs from long term p. aeruginosa infected cf patients (n=3) (i.v. antibiotics every 3rd month since 1976 + inhalation of colistin daily since 1987. histological sections of the lungs were investigated using he stain, gram stain, alcian blue, antibodies against alginate and p. aeruginosa specific pna-fish combined with bacuni pna-fish and with dapi as counter stains. due to the high specificity of the pna-fish probes employed in this study, our results provide strong evidence for that before the aggressive antibiotic therapy, p. aeruginosa infected and destroyed the cf lung due to fast spreading into the respiratory zone. today antibiotics suppress but can not eradicate the bacteria from the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time. the conductive zone serves as a reservoir; here the bacteria are organized in microcolonies embedded in puss, a trait which is independent on the time course of the infection and amounts of antibiotics. these microcolonies consisted solely of p. aeruginosa. in addition, we found no bacteria adhering to the epithelial tissue. the pus consisted mainly of leukocytes, surrounding the microcolonies. a smear of dna and dead leukocytes were detected just around the microcolonies, possible due to the quorum sensing dependent rhamnolipid killing recently described by us (jensen et al. 2007) . we conclude that p. aeruginosa persists in the cf lung due to its ability to create microcolonies i.e. biofilms. within these biofilms the bacteria are protected against antibiotics and the host defence. respiratory viral infections, including influenza (flu) and respiratory syncytial virus (rsv) contribute to the morbidity of children with cf. additional human respiratory viruses have been identified in children such as human metapneumovirus (hmpv) and coronavirus (cov), but these have not been examined in cf patients. in our laboratory, pcr assays have been developed to identify both traditional and newer respiratory viruses in healthy and immunocompromised children. these assays are usually performed on nasal swabs or nasal wash samples. however, oropharyngeal (op) and sputum samples are routinely obtained in cf for bacterial culture. the goals of this trial are to determine whether these available samples can be used for viral pcr studies and to examine the epidemiology of respiratory viruses in cf. for this single center observational trial, 44 cf patients, ages 6.1 to 17.7 yrs (mean 12.8 yrs), have been recruited. subjects provide op and nasopharyngeal (np) samples at quarterly clinic visits and with acute pulmonary exacerbations requiring hospitalization; sputum is also collected if patients can expectorate. np and op samples are pooled when both are available. viruses assayed are flu a and b, rsv, parainfluenza (piv) 1, 2, 3 and 4, hmpv, cov, adenovirus (adv), and rhinovirus (rhv). subjects are being followed for two respiratory viral seasons. we report here the methodology of viral pcr detection in cf sputum, as well as the results of viral detection during the first respiratory viral season in the seattle community. the mean length of time subjects were followed was 0.84 yrs (median: 0.92 yrs, range: 0.18-1.18 yrs). overall, 47 samples (20% of the 235 samples analyzed) were positive. twenty-four subjects (69.1%) had at least one viral infection detected and 8 (18.2%) had two viruses at different visits. both sputum and pooled op/np samples were available at 55 visits and were concordant in 80% of cases. among the discordant cases, it was more common for sputum to be positive and op/np to be negative (9.1%) than the converse (3.6%). the most frequently identified viruses were rhv (32 isolations); piv3 and cov were the next most prevalent with 4 and 3 isolations, respectively. in conclusion, sputum viral pcr shows promise for the diagnosis of both traditional and novel viral infections in cf. as part of ongoing clinical trials in cf, non-fermenting gram negative bacilli (nfgnr) identified at site laboratories are often sent to the tdn core microbiology laboratory for confirmation of identification. among nfgnr from cf patients, p. aeruginosa are the most frequently isolated and also are acknowledged to be the easiest to identify. however, in a survey of 428 "p. aeruginosa" isolates sent to the laboratory in the past year, pcr identified 30 (7%) as alternate species. these included: non-aeruginosa pseudomonads, stenotrophomonas maltophilia, achromobacter xylosoxidans, ralstonia spp., alcaligenes spp., brevidimonas spp., and bordetella bronchiseptica. identification of these nfgnr can be difficult; however, p. aeruginosa is an important isolate for the clinical microbiology laboratories in all cf centers to be able to identify. many methods are available including conventional biochemical testing and rapid identification kits. in the tdn core laboratory we previously demonstrated the importance of combining phenotypic identification (morphology plus a short set of biochemical tests) and genotypic identification (16s rdna sequencing of v3 and/or pcr using three primer sets) and developed an algorithm for testing. in this study, we examined all of the cf cultures performed in the clinical microbiology laboratory at chrmc over the past two years to determine the number of isolates for which it was necessary to perform genotypic identification in nfgnr. among this collection of 182 nfgnr from clinical cultures sent to the microbiology laboratory from the seattle children's hospital cf center, pcr identified 43 isolates as burkholderia cepacia complex. next most commonly identified were a. xylosoxidans (28), p. aeruginosa (23) and non-aeruginosa pseudomonas spp. (23). three isolates remained unidentified, even with pcr sequencing of the v3 determinant. the clinical microbiology laboratory at chrmc has served as the core microbiology laboratory for tdn clinical trials for 8 years and thousands of cf samples are processed annually. the result is that there is increased experience with cf isolates among the laboratory personnel. however, even with experienced technologists, a significant number of nfgnr required genotypic identification in the past two years. thus, it is important for clinical microbiology laboratories with less experience in the identification of cf nfgnr to utilize additional molecular techniques or send problematic isolates to a reference laboratory. based on these results we propose an algorithm for the most efficient means of identifying nfgnr from cf samples using a combination of tests including phenotypic (colony morphology, biochemical testing) and genotypic identification (pcr, sequencing). background: the epidemiology of s. aureus has been changing in both cf and non-cf patients. in non-cf patients, methicillin-resistant s. aureus (mrsa) has traditionally caused infections in patients with risk factors such as hospitalization, surgery, and anterior nares colonization. recently, community-acquired (ca-) mrsa infections have increased among patients without known risks. in cf, colonization of the anterior nares with methicillin-susceptible s. aureus (mssa) is associated with transmission between patients and household members. the prevalence of respiratory tract colonization/ infection with mrsa has increased in cf, but little is known about risk factors for mrsa, including possible transmission among families, and if strains are ca-or healthcare-acquired. methods: this is a case-control, multicenter study to investigate potential risk factors for mrsa among children with cf 1-17 years of age. subjects are enrolled from 3 cf centers in the new york metropolitan area that have a mrsa prevalence ranging from 11% to 21%. cases are children with a positive respiratory tract culture for mrsa within the past 5 years. controls (2 age and center matched per case) are negative for mrsa. the aims of the study are: [1] to determine the point prevalence of anterior nares colonization with s. aureus in children with cf and their household members; [2] to assess potential risk factors for mrsa by administering a survey to the parents of subjects inquiring about factors such as crowding, pets, and participation in contact sports and by reviewing the medical record for antibiotic use, hospitalization, and surgery; [3] to determine potential transmission of s. aureus within families and centers by assessing the molecular epidemiology of strains using pulsed field gel electrophoresis and meca type of mrsa isolates. results: to date, 42 cf subjects (12 cases, 30 controls), mean age 8.9 years, and 100 household members (1 to 5 per subject) have been enrolled. preliminary data demonstrate positive anterior nares cultures among cases, controls, and household members to be 7/12 (58%), 7/30 (23%), and 24/100 (24%), respectively. of the 38 s. aureus isolates, 27 were mssa and 11 were mrsa (5 cases, 3 household members of cases, and 3 household members of controls). during the past 6 months, 4 household members have been hospitalized and one has stayed overnight in a nursing home; 4 had staphylococcal infections and 2 had skin infections. data collection and molecular analysis are ongoing. conclusions: we expect this study to provide insight into risk factors for respiratory tract colonization/ infection with mrsa, the role of ca-mrsa in cf, and potential strategies to prevent mrsa. aims: 1. revise infection control guidelines for the institution 2. establish outpatient infection control policies consistent with those used for hospital inpatients 3. cohort young and recently diagnosed cf patients without pseudomonas or mrsa to clinic days separate from colonized patients 4. establish a protocol for eradication of mrsa in newly colonized patients. methods: working with representatives from hospital epidemiology, the cf center, inpatient units, outpatient clinics, respiratory therapy, and cf families, a uniform infection control policy for cf patients in all settings (inpatient, outpatient, home care) was drafted and revised. arrangements were made to cohort newly diagnosed patients and children <5 years free of pseudomonas and mrsa to separate clinic days from colonized patients. review of the 41 mrsa positive patients in our pediatric population, for whom data was available, showed median age of acquisition of 8 years (range 1 mo to 18 yrs). for 3 patients the first culture obtained at diagnosis or transfer from another center was mrsa positive (1 mo, 1 yr, 4 yr). an eradication protocol for newly acquired mrsa has been developed involving treatment with oral and topical antibiotics and phisohex or clorox washes for 5 days, plus changing disposable respiratory care equipment at the beginning and end of the treatment period. surveillance cultures of nose, groin, and airway or sputum are done after completion of the treatment protocol and re-treatment initiated if still positive for mrsa. conclusion: using a three-tiered approach of infection control guidelines, cohorting, and mrsa eradication protocol we expect to decrease our overall rate of mrsa and protect our newly diagnosed and young cf patients from acquisition. objective: studies have shown that respiratory pathogens can be transmitted within cf centers. in a recent cross-sectional study at 7 cf centers, we assessed the rate of bacterial shedding by cf patients during office visits and noted that 6% of patients carried respiratory flora on their hands. the current study was performed to assess the effectiveness of alcohol-based rubs on hand carriage of respiratory pathogens by patients during the course of office visits. methods: four bacterial organisms were chosen for study: pseudomonas aeruginosa (pa), staphylococcus aureus (sa) [including methicillin-resistant (mrsa) strains], stenotrophomonas maltophilia (sm) and burkholderia cepacia complex (bcc). at the beginning of clinic visits, hands of study patients were cultured using the "glove-juice" technique (gjt) [1] . hand hygiene was then performed using an alcohol-based rub. at the end of the visit, hands were again sampled using the gjt. samples were sent to the microbiology laboratory at dartmouth-hitchcock medical center for culture and identification of study organisms. hand and respiratory tract isolates were compared using pulsed field gel electrophoresis (pfge). recovery experiments were also performed to assess the sensitivity of the gjt. results: samples were collected from 100 encounters (50 adult; 50 pediatric). median encounter time was 72 min (range 60.5-170.0 min). recovery experiments determined that the level of detection for each study organism using the gjt was ~10 2 colony forming units. confirmation of matches between hand and respiratory isolates was determined by pfge. the number of patients with study organisms recovered from the respiratory tract was: 38 pa, 31 sa, 21 mrsa, 8 sm, and 2 bcc. the overall hand contamination rate with a matched respiratory tract isolate at either culturing time was 14% (95% ci, 8.6-22.2%). before use of the alcohol rub, hand contamination was 9.0% (95% ci, 4.9-16.2%). in those patients with hand contamination by respiratory pathogens prior to alcohol rub, 50.0% (95% ci, 23-77%) had negative cultures at the end of the clinic visit. however, there was strong evidence of hand contamination during the encounter, despite hand hygiene, as the overall rate at the end of visits was 8.0% (95% ci, 4.2-15.0%). there was a trend toward increased hand contamination in those suffering from pulmonary exacerbations versus clinically stable patients (28.6% [95% ci, 11.8-55%] vs. 11.6% [95% ci, 6.5-20%], p=0.10). conclusions: contamination of patient hands by respiratory tract pathogens is observed in the outpatient setting in approximately 14% of patients. a single application of an alcohol-based hand rub, while safe and inexpensive, has limited antiseptic effectiveness during the full course of a typical cf clinic visit. repeated use of alcohol-based hand rubs during outpatient encounters would likely be more effective. these data support recent recommendations in the cf foundation consensus guidelines for infection control [2] (nthi) is the most common initial bacterium infecting the airways in cystic fibrosis (cf). nthi infection usually precedes pseudomonas aeruginosa infection in many chronic lung diseases, including cf, emphysema, diffuse panbronchiolitis, and immotile cilia syndrome. it is hypothesized that nthi acts as a gateway organism paving the way for subsequent infection with p. aeruginosa, however, the mechanism by which this occurs remains poorly understood. we used a novel co-culture model of persistent nthi biofilm infection on airway epithelial cultures to study epithelial immune responses following nthi biofilm infections. objective: we hypothesized that prolonged exposure to nthi biofilms causes airway epithelia to become tolerant to inflammatory stimuli and show subsequent decreased innate immune responses. methods: nthi were inoculated onto the apical surface of calu3 air-liquid interface epithelia and grown in co-culture. rna from 3 sets of paired airway epithelial cultures at 4, 24, 48 and 96 hours both with and without nthi inoculation was isolated and hybridized on custom affymetrix chips. data were normalized with rma and data biases were removed using the mixed model anova method. differentially expressed gene analyses were performed using anova, and pathways analyses were performed using gene set enrichment analysis (gsea). to test the hypothesis that prolonged nthi infection induces tolerance, epithelial cultures were pre-treated with either 4 days of nthi or 4 days of pbs before stimulation with pbs, il1β (100 ng/ml) or 10 6th cfus of live p. aeruginosa before subsequent measurement of epithelial responses. results: both rma and gsea analyses of microarray data showed many innate immune and pro-inflammatory responses at early timepoints that largely returned to baseline after 4 days of co-culture, despite an increasing apical nthi biofilm infection. stimulation with il1β or p. aeruginosa after 4 days of nthi infection resulted in significantly decreased il8 production compared to uninfected controls. nthi treated cultures subsequently infected with p. aeruginosa also displayed increased tight junction integrity and resistance to cell culture death. conclusions: persistent nthi infections on airway epithelia resulted in increased tolerance to bacteria and inflammatory stimuli and increased resistance to bacterial toxicity. airway epithelial tolerance may contribute to a failure to clear subsequent bacterial infections. these phenomena would help to explain how early infections with nthi may promote chronic bacterial infections and p. aeruginosa acquisition in cf and other nthi related airway diseases. the colonization of cf airways by p. aeruginosa leads to intractable and persistent lung infections that resist permanent eradication by antibiotics. the lack of efficacy of current therapies is believed to be due to the formation of p. aeruginosa antibiotic resistant biofilms in the cf airways. however, little is known about biofilm formation on human airway epithelial cells. thus, we designed a continuous flow, live cell imaging system to grow p. aeruginosa biofilms on glass or on the apical surface of polarized human cells. cfbe41ohuman airway epithelial cells homozygous for the ∆f508 mutation (cfbe), and cfbe41o-cells complemented with wt-cftr (cfbe+wt-cftr) were grown as polarized monolayers at an air-liquid interface and gfp-labeled p. aeruginosa were applied to the apical surface of the cells. after 6 hours biofilms did not form on glass but large bacterial macrocolonies developed on cfbe cells. surprisingly, biofilms also developed on cfbe+wt-cftr cells: however, p. aeruginosa biomass on cfbe+wt-cftr cells was significantly reduced compared to cfbe cells (0.60±0.12 µm 3 /µm 2 vs 1.58±0.20 µm 3 /µm 2 ). the minimal bactericidal concentration for tobramycin was >10,000 µg/ml for p. aeruginosa grown on cfbe and cfbe+wt-cftr cells. these results explain why tobramycin given clinically, which is~1,000 µg/ml in bronchoalveolar fluid collected from cf patients, fails to eradicate the persistent infection by p. aeruginosa. p. aeruginosa also formed biofilms on human bronchial epithelial cells, which produce mucus. indeed, biomass was similar on cfbe+wt-cftr cells, which do not produce mucus, and human bronchial epithelial cells. to determine if human airway epithelial cells secrete factors that facilitate biofilm formation, conditioned medium was collected from cfbe and cfbe+wt-cftr cells and applied to p. aeruginosa grown on glass. under these conditions, biofilm formation was dramatically facilitated, with a biomass of 0.02 ± 0.01 µm 3 /µm 2 when p. aeruginosa was grown in conditioned medium collected from cfbe+wt-cftr cells and 0.11 ± 0.02 µm 3 /µm 2 when grown in conditioned medium collected from cfbe cells. the secreted factor(s) were greater than 5 kda, as assessed by size exclusion. our results suggest that human airway epithelial cells secrete factor(s) that facilitate biofilm formation by p. aeruginosa, in the presence and absence of mucus, and that secretions from cf cells are more effective in promoting biofilms than secretions from non-cf cells. the ability of non-cf cells to facilitate biofilm formation appears to contradict the observation that non-cf lungs do not harbor p. aeruginosa. we propose that the innate immune response in the non-cf lung effectively eliminates p. aeruginosa from the lung, thus, biofilms do not form even thought the airway epithelial cells secrete factors that have the potential to facilitate biofilm formation. however, in cf when mucociliary clearance is compromised, p. aeruginosa accumulates in the lung and secretions by cf airway epithelial cells greatly enhances the formation of drug resistant biofilms (supported by the nih (ro1 ai51360, ro1-hl-074175, p20-rr018787, t32-dk-07301), and the cff (stanto97ro, anders06fo). ma, l. 1 ; parsek, m.r. 2 ; wozniak, d.j. 1 1. microbiology and immunology, wake forest university health science, winstonsalem, nc, usa; 2. university of washington, seattle, nc, usa bacteria in natural, industrial and clinical settings live in surface-associated communities termed biofilms, which are the source of persistent infection and resistance to antimicrobial treatment. biofilm development is initiated by the attachment of planktonic cells to a surface, followed by formation of microcolnies, and finally disperses swimming cells from microcolnies to occupy a new surface. to maintain the community structure, bacteria in a biofilm are usually enmeshed in an extracellular polymeric matrix, which consists of nucleic acids, proteins, and polysaccharides. exopolysaccharides (eps) have been known as a component of the biofilm matrix for years. however, little is know about how the eps matrix forms and develops and whether an eps can form a matrix independently. in the present report, we use lectins that specifically detect the mannose or galactose structure in pseudomonas aeruginosa psl eps. this technique allows us to visualize psl eps on the bacteria cell surface and in the biofilm matrix. our results indicate that psl eps is likely anchored on the cell surface in a helical pattern and clearly forms a matrix, which holds bacteria in the biofilm and on the surface. in a flat multiple-layer biofilm, psl eps is equally distributed in the entire biofilm. however, microcolonies reveal peripheral staining of psl eps with minimal staining of matrix in the center of the microcolonies. instead, this region has swimming cells indicating a biofilm development stage prior to dispersion. more strikingly, this area also has concentrated dead cells and/or extracellular dna, which fills up the viod spaces in the lower center of microcolonies at the stage prior to dispersion. these data provides a plausible mechanism for how p. aeruginosa sacrifices a portion of cells to make void spaces and free another portion of cells for future dispersion. in addition, our data also show that psl eps matrix and dna matrix are not overlapping, which suggests that these two components of the matrix work coordinately to encase bacteria in the biofilm. finally we show that the psl eps matrix is present in biofilms formed by mucoid p. aeruginosa, and formation of the psl eps matrix is independent of the production of alginate and pel, two eps that also contribute to p. aeruginosa biofilm formation. overall, our data indicates that psl eps functions as a primary scaffold, holding biofilm cells together in the matrix. moreover, our data along with published literature suggest a possible model for how p. aeruginosa biofilms persist in cystic fibrosis patients. 1 1. clinical microbiology, rigshospitalet, copenhagen, denmark; 2. biocentrum, technical university of denmark, kgs. lyngby, denmark; 3. department of paediatrics, copenhagen cystic fibrosis centre, rigshospitalet, copenhagen, denmark pseudomonas aeruginosa predominates chronic lung infections in patients with cystic fibrosis (cf). a hallmark of chronic p. aeruginosa lung infections in cf patients is the presence of mucoid p. aeruginosa biofilms surrounded by numerous polymorphonuclear leukocytes (pmns) in the lower airways. how p. aeruginosa escapes the bactericidal pmns is not fully understood. several virulence factors of p. aeruginosa is controlled by quorum sensing (qs); a density dependent mode of inter-bacterial communication based on signal transmitter molecules. active qs is present during chronic p. aeruginosa lung infections in cf patients and we have previously demonstrated a qs-regulated tolerance of biofilm bacteria to the antimicrobial properties of the pmns. the precise qs-regulated effect on the pmns is, however, unknown. in the present study, further elaboration using flow cytometry and microscopy revealed that qs-competent p. aeruginosa (pao1 and ∆rhli lasi complemented with c4-hsl and 3-oxo-c12-hsl) induces rapid necrosis of the pmns. this mechanism was also observed in in vitro biofilms and in mouse lungs infected with p. aeruginosa embedded in alginate. by using hplc fractionation the toxicity could be ascribed to a single substance. using lc-ms and 2d nmr this substance was identified as rhamnolipid b. in accordance, concentrations of rhamnolipid exceeding 200 µg/ml were found in the toxic supernatants from wild-type p. aeruginosa (pao1) whereas non-toxic supernatants from qs-deficient knock-out mutants (∆rhlr lasr and ∆rhli lasi) and from ∆rhla mutants contained low amounts of rhamnolipids approaching 0 µg/ml. our results demonstrate the potential of the qs system to facilitate infections with p. aeruginosa by utilizing rhamnolipid to disable a major first line of defense of the host -the pmns. furthermore, our study emphasizes the inhibition of qs as a target for the treatment of infections with p. aeruginosa. carlsson, m. 1,2 ; pettersson, a. 1 ; andersson, c. 1 ; wieslander, j. 4 ; eriksson, l. 3 ; segelmark, m. 2 ; hellmark, t. 2 1. dept of microbiology, immunology, and glycobiology, university of lund, lund, sweden; 2. department of nephrology, lund university, lund, sweden; 3. heart lung division, the cystic fibrosis center, lund university hospital, lund, sweden; 4. wieslab analys ab, lund, sweden the clinical consequence of chronic pseudomonas (p) aeruginosa colonization in cystic fibrosis (cf) varies between individuals for unknown reasons. auto-antibodies against bactericidal/ permeability increasing protein (bpi-anca) are associated with poor prognosis in cf. we hypothesize that there is a correlation between the presence of bpi-anca, the biological properties of the colonizing bacteria and the clinical conditions of the hosts. we have compared isolates of p aeruginosa from two groups of cf patients: one with positive serum levels of bpi-anca and deteriorating lung disease, and one with negative bpi-anca levels and stable clinical conditions. epithelial cells (a549) and isolated polymorphonuclear granulocytes (pmns) were stimulated with the clinical isolates and cell death was analyzed with flow cytometry. interleukin-8 (il-8) released into the supernatant was measured by elisa. we found that the anca associated strains in most cases showed a pyocyanin negative phenotype. these strains also induced less inflammatory response than the non-anca associated strains as shown by the number of necrotic cells and il-8 release, yet elevated compared to control. we conclude that colonization with strains of p aeruginosa that induce a weak inflammatory response is associated with unfavourable outcome in cf. we speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying pmns in the airways, contributing to the progressive lung disease seen in many cf patients. 2 1. pulmonary critical care, northwestern university, chicago, il, usa; 2. microbiology/immunology, northwestern university, chicago, il, usa; 3. pulmonary medicine, children's memorial hospital, chicago, il, usa; 4. pediatrics, children's memorial hospital, chicago, il, usa purpose: most cystic fibrosis (cf) patients are infected with pseudomonas aeruginosa (pa) and have progressive loss of lung function. among individual patients, however, there are marked differences in rates of lung function deterioration. although the reasons for this variability are not completely understood, it is likely that microbiological factors play a role. type iii secretion in pseudomonas aeruginosa (pa) isolates from non-cf patients have been associated with poorer outcome. the aim of this study was to determine whether there is an association between type iii secretion properties and deterioration in lung function in cf. methods: we prospectively enrolled cf children and adults over 3 years. demographics, clinical characteristics, spirometry and a respiratory culture were obtained at the first visit. the age at time of first postive pa culture was also determined. subsequently, spirometry and respiratory cultures were obtained and clinical characteristics recorded every 6 months until december 2006. from each sputum culture 5 individual isolates were selected for evaluation and type iii secretion was evaluated for each isolate by western blot analysis. the children were subcohorted into those newly infected (1st positive pa culture) or those chronically infected (minimum 2 sputum pa (+) >1 year duration). results: there were 100 patients evaluated, of which 31 were adults and 69 were children. ninety percent of our population cohort was caucasian and 53% were female. overall 63% of the patients were on tobi and 72% on macrolides chronically. the mean age at time of a first pa positive culture was 11 years for the chronically infected subjects(adults and children) and 6 years for the 1st time infected subcohort. a total of 2409 sputum samples were collected for the current study. the prevalence of type iii secretion properties for the overall population was 21.11%. foe adults, 73 of 807 (9.7%) pa isolates were type iii secretion positive compared to 346 of 1602 isolates (26.8%) in chronically infected children and 48 of 90 isolates(53.3%) from newly infected children. at study entry the mean fev1 % predicted was 68 +/-24 for the whole cohort. for the adult subcohort the fev1% predicted was 60% +/-26%, for chronically infected children it was 72% +/-21% and newly infected children is was 82%+/-22%. the average annual rate of fev1 % change was -0.67% for adults, -2.42% for chronically infected children and +4.62 for newly infected children. analysis on the relationship between type iii secretion pa isolates and fev1% decline is ongoing and will be presented at nacf. conclusion: prevalence of type iii secretion in pa isolates from cf patients decreases with age. there is a slower decline in fev1% in chronically pa infected adults compared to chronically pa infected children. the relationship of type iii secretion to fev1 % change and pulmonary exacerbations will be presented at nacf. a limited number of bacterial species -in example pseudomonas aeruginosa, staphylococcus aureus, burkholderia cepacia complex, stenotrophomonas maltophilia -is typically found in the cf lung. these facultative anaerobic bacteria exhibit the capability to switch from aerobic to anaerobic metabolism, which enables them to survive in the hypoxic environment in the cf lung [1] . absence of oxygen would also favour the growth of fastidious anaerobes and, indeed, sequencing of 16s rrna resulted in the detection of strict anaerobic species in cf sputum [2] . thus, we identified and quantified strict anaerobes in cf sputum using conventional anaerobic microbiological methods, examined the recovery rate of identical species in sputum samples from the same patients, and determined the antibiotic susceptibilities. 92 sputum samples were collected from 8 cf children and 31 cf adults (mean age 25.6 ± 8.5 yrs). samples were incubated aerobically on columbia agar, supplemented with 10% sheep blood, and anaerobically on brain heart infusion agar and schaedler agar supplemented with 5% mutton blood, for up to 7 days. bacteria were identified (rapid ana ii identification system, remel, lenexa, ks) and cfu's were determined by dilution plate counting. fastidious anaerobes (141 strains) were submitted to e-test ® susceptibility testing (ab biodisk, solna, sweden) using the anaerobically active antibiotics ceftazidime, clindamycin, meropenem, metronidazole, and piperacillin/tazobactam. in 75.0% of the patients, the following strict anaerobes were detected with a mean of 6.3x10 6 cfu/ml (range 2.5x10 4 to 2.5x10 7 cfu/ml): peptostreptococcus spp., clostridium spp., actinomyces spp., prevotella spp., wolinella spp., propionibacterium spp., streptococcus spp., lactobacillus spp., gemella spp., bacteroides spp. and eubacterium spp., whereas p. aeruginosa, s. aureus and b. cepacia revealed 7.1x10 7 cfu/ml (range 5.0x10 5 to 3.5x10 8 cfu/ml). in 30 sputum samples contaminated with p. aeruginosa together with strict anaerobes 5.2x10 7 ± 7.8x10 7 cfu/ml p. aeruginosa were counted, whereas in 9 samples with p. aeruginosa without strict anaerobes only 1.7x10 7 ± 1.8x10 7 cfu/ml were found (p=0.03). for s. aureus, no significant difference was observed. identical strict anaerobic species were detected in 12 out of 19 patients with two or more repeated sputum samples (63%). e-test® sensitivity testing for strict anaerobes yielded high sensitivity for meropenem (only 5.7% resistant strains), piperacillin/tazobactam (20.6%), and clindamycin (24.8%), but not ceftazidime (49.0%) or metronidazole (50.4%). high numbers of strict anaerobes are present in the majority of cf patients. possibly, strict anaerobes promote growth of p. aeruginosa but not s. aureus. the high persistence of identical anaerobic strains reflects chronic lung infection and may be caused by their increased resistence against standard antibiotics such as ceftazidime. [ in sputum in the cf lungs bacteria such as pseudomonas aeruginosa have to metabolize anaerobically [1] . for anaerobic energy generation, p. aeruginosa can use nitrate and arginine, but also pyruvate which is produced from glucose via anaerobic glycolysis [2] and can be metabolized to lactate and vice versa. in order to investigate if p. aeruginosa may benefit from externally produced lactate, we measured the concentration of lactate in p. aeruginosa, staphylococcus aureus, burkholderia cenocepacia and polymorphonuclear neutrophils in vitro and in cf sputum. in sputum samples of 25 cf patients and in neutrophils (3 x 10 7 /ml) from healthy donors lactate concentrations were determined. in addition, p. aeruginosa (starting with 1 x 10 7 cfu/ml in tryptone soy broth), s. aureus (3 x 10 6 cfu/ml), and b. cenocepacia (8 x 10 6 cfu/ml)were grown aerobically (0 through 24 hrs) and anaerobically (1 through 4 days). l-lactate was measured spectrophotometrically (detection limit: 0.1 mmol/l), and total lactate gaschromatographically. aerobic and anaerobic gene expression of p. aeruginosa strain pao1 was determined using affymetrix® microarrays. lactate concentrations in cf sputum amounted to 3.0 ± 3.1 mmol/l (range 0.2 to 14.1 mmol/l). concentrations were similar in sputum samples colonized with p. aeruginosa, s. aureus (3.3 ± 3.7 vs. 2.8 ± 2.3 mmol/l, p=0.67) and b. cenocepacia (1.9 mmol/l). neutrophils produced 3.2 mmol/l. in all samples exclusively l-lactate was found. during in vitro experiments, p. aeruginosa did not generate any lactate at all, neither aerobically nor anaerobically. in contrast, anaerobically grown s. aureus produced up to 2.8 mmol/l lactate, and b. cenocepacia up to 10.0 mmol/l. a p. aeruginosa suspension [1x10 7 cfu/ml] spiked with 10 mmol/l l-lactate did not change its concentration, indicating that p. aeruginosa does not metabolize lactate. similar results were obtained in our gene chip experiments: after three days of anaerobic growth, the genes encoding for the lactate dehydrogenases were downregulated (pa0927 ldha -10.78 fold, pa2382 llda -2.81 fold) or unchanged (pa4771 lldd 1.2 fold). in contrast, the genes encoding for pyruvate decomposition to acetyl coa (pa3416 and pa3417, both encoding for pyruvate dehygrogenase e1 components) were upregulated by 208 and 47 fold, respectively. we could demonstrate that p. aeruginosa does not benefit from externally produced lactate. we confirmed the important role of pyruvate metabolism for anaerobic p. aeruginosa energy generation. whether lactate production of neutrophils, s. aureus or b. cenocepacia contributes to cf lung pathophysiology still remains to be investigated. references: [1] macleod, d. 1 ; barker, l. 1 ; gurgel, j. 1 ; kenney, t. 1 ; burns, j. 2 ; baker, w. 1 1. gilead sciences, inc., seattle, wa, usa; 2. university of washington, seattle, wa, usa antibiotic resistance may severely limit therapeutic options in individuals with cystic fibrosis (cf) or bronchiectasis. because of frequent antibiotic treatment courses, resistance continues to emerge, even to newer agents. treatment with multiple antibiotics in a single aerosol formulation may be a promising approach to slow development of resistance. fosfomycin is a phosphonic acid antibiotic that is bactericidal against both gram positive and gram negative organisms. fosfomycin inhibits the first committed step in the synthesis of peptidoglycan, suggesting cross resistance to other cell wall acting antibiotics will not occur. the aminoglycoside tobramycin is one of the most commonly used antimicrobials in cf, with potent activity against gram negative bacteria and the majority of staphylococcus aureus isolates. a 4:1 (wt/wt) fixed combination of fosfomycin:tobramycin (gs-9310/11) was used to determine the in vitro susceptibilities of a panel of respiratory pathogens: cf pseudomonas aeruginosa (100), s. aureus (16), haemophilus influenzae (16), stenotrophomonas maltophilia (17) and burkholderia cepacia complex (20), including minimal inhibitory concentration (mic) and time-kill experiments in the absence and presence of 2% porcine mucin. synergy was evaluated using the checkerboard method, and spontaneous resistance mutation frequencies were determined in antibiotic-containing agar (4x, 8x and 16x mic). in vivo drug efficacy was examined using a rat agar bead pneumonia model of either p. aeruginosa or s. aureus. all experiments compared gs-9310/11 to fosfomycin and tobramycin as single agents. gs-9310/11 had a lower mic 90 than tobramycin for the s. aureus strains, 75% of which were methicillin resistant (mrsa). for p. aeruginosa, gs-9310/11 had a lower mic 50 and mic 90 than fosfomycin alone, but tobramycin was more active than either. for h. influenzae and s. maltophilia, gs-9310/11, fosfomycin and tobramycin had similar mic 50 and mic 90 . b. cepacia complex were resistant to all three drugs. results in the presence of mucin were similar. time-kill studies showed a more rapid and prolonged killing of s. aureus and p. aeruginosa by gs-9310/11 compared with either agent alone at the same drug concentrations. gs-9310/11 was bactericidal and exhibited concentration-dependent killing. synergy studies showed no antagonism between fosfomycin and tobramycin, and the majority of p. aeruginosa and all of the s. aureus tested demonstrated indifference for the combination. at 4x mic concentrations the mutation frequency of gs-9310/11 was at least 2-3 logs lower than tobramycin and 2-6 logs lower than fosfomycin alone for s. aureus. for p. aeruginosa the mutation frequency of gs-9310/11 was 2-3 logs lower than fosfomycin and 1-2 logs lower than tobramycin. in the rat pneumonia model, gs-9310/11 and tobramycin alone demonstrated bactericidal killing of p. aeruginosa; both were more active than fosfomycin alone. in vivo killing of s. aureus by gs-9310/11 was also demonstrated. gs-9310/11 appears to have advantages over single agents for the treatment of both gram positive and gram negative bacterial lung infections in cf and bronchiectasis. britton, l.j. 1 antibiotic resistance is becoming a major problem in the treatment of pulmonary exacerbations in cystic fibrosis (cf). organ-isms that are resistant to multiple antibiotics infect the airways of an estimated 25-45% of adults with cf.1 as a result of the growing resistance in cf patients, many centers have been performing synergy testing of sputum cultures in addition to the conventional culture and sensitivity testing. the purpose of our investigation was to determine if antibiotic synergy studies and the use of synergistic combinations of antibiotics improve therapeutic outcomes in cystic fibrosis patients with acute pulmonary exacerbations. methods: this study is a retrospective chart review of cf patients who had antibiotic synergy testing performed while hospitalized for respiratory tract infections. eligibility criteria included cf patients hospitalized with respiratory tract infections from 1998 to 2005. laboratory data from each hospital admission was reviewed for synergy among those antibiotics commonly used against pseudomonas aeruginosa. a review of medical charts ascertained each patient's pulmonary function (determined by fev1 before and after antibiotic therapy), weight z-score, organism cultured from sputum sample, time to next hospital admission, and the antibiotic(s) actually prescribed. primary endpoints were determined to be the change in fev1 and the time to next admission. results: four hundred seventy-five hospital admissions were analyzed. a total of 48 cystic fibrosis patients, age birth to 36 years, were included in the study. patients receiving antibiotic synergy experienced a significant decrease in mean time to next admission (50 days with synergy vs. 68 days without synergy, p=0.004). no significant difference was found in the change in fev1 before or after antibiotic therapy, with an increase of 8.72% with synergy vs. 9.28% without synergy (p=0.585). patients infected with non-mucoid p. aeruginosa experienced 60 days to the next hospital admission, while patients infected with mucoid p. aeruginosa experienced 56 days to the next hospital admission (p = 0.45). no statistically significant difference was observed between the synergy and non-synergy groups in regards to nutritional status and lung function prior to antibiotic therapy. speculations: antibiotic resistance in cystic fibrosis patients increases the morbidity and mortality caused by this disorder with each exacerbation the patient experiences. synergistic antibiotic therapies should improve patient outcomes through more efficient bacterial eradication and increased time to next hospital admission; however, we were unable to substantiate this assumption based on the results from this study. synergistic antibiotics did not show an improvement in the therapeutic outcomes of days to next admission or change in fev1. background: expectorated sputum (es) technique is currently the most frequently used method for routine assessment of lower airway infection in patients with cystic fibrosis (cf). induced sputum (is) using hypertonic saline (hs) has been successfully used in cf patients unable to produce sputum spontaneously, but only limited data are available comparing the diagnostic yield of expectorated versus induced sputum in cf patients. while ultrasonic nebuliser have been used in the majority of studies, new high output jet nebulisers may offer a suitable alternative technique to induce sputum expectoration. aim: to assess the feasibility of sputum induction using a pari e-flow nebulizer and to compare the diagnostic yield of is and es in children with cf . methods: this is a preliminary report of an ongoing study in routine clinical care in sputum producing children with cf. es is being obtained before sputum induction. subsequently, sputum induction is performed using stepwise inhalation of nebulized 5 ml of 3%, 4% and 5% hypertonic saline with an e-flow nebulizer (pari, starnberg, germany). lung function is assessed by portable spirometry before the procedure and after inhalation of each saline concentration. results: so far 40 cf patients (18 females) with a mean age of 13.6 years (range 8 to 18 years) and fev1 between 38-121% predicted (mean 82%) have been included in the study. all subjects provided es samples and all produced sputum after induction. discrepancies in cultures between es and is samples were seen in 9 cases (22.5%). in 6 cases additional cf pathogens were found in is samples, whereas in 2 cases es yielded additional organisms compared to is. the number of distinct pathogens was similar in the remaining patient, but different bacteria were found with the 2 techniques. in 4 cases p. aeruginosa was detected only with one of the 2 techniques (2 es versus 2 is). the spectrum of side effects was similar to previous reports using other nebulizer systems, with throat irritation being the most common adverse event of the is technique. all patients who had symptoms during the procedure became asymptomatic at the time of discharge from the clinic 20-30 minutes after the procedure. 4 patients did not finish all 3 cycles of sputum induction procedure due to symptoms of shortness of breath and/or drop in fev1>20% from baseline (in 3 patients); all reversed after salbutamol inhalation. vomiting occurred in one patient. 3 patients refused to complete the procedure due to unpleasant taste and/or throat irritation. conclusion: these preliminary results show discrepancies between expectorated sputum and induced sputum cultures in a significant proportion of cf patients. this may be explained by the previously described regional differences in lower airway infection in cf airways rather than by a higher diagnostic yield of one of the techniques. the results also demonstrate that the e-flow system is an efficient and safe device for sputum induction in sputum producing children with cf. seidler, m.j.; salvenmoser, s.; müller, f.c. dept. pediatrics iii, university heidelberg, pediatric pulmonology, cystic fibrosis centre & infectious diseases, heidelberg, germany background: the preferred growth form of bacteria is a biofilm. s. aureus, h. influenzae, and p. aeruginosa can produce an extracellular matrix (ecm) with implications in cystic fibrosis (cf) lung disease. the biofilm can protect against host defenses and antimicrobials. a. fumigatus is a frequent colonizer of the cf respiratory tract and can cause allergic bronchopulmonary aspergillosis (abpa). while antifungals in vitro are active against a. fumigatus, in vivo antifungal therapy is often complicated or resistance is observable. the aim of this study was to investigate the ability of a. fumigatus to form a biofilm-like matrix in vitro on polystyrene (ps), human bronchial epithelia cells (16hbe) and human bronchial epithelia cells with f508del/f508del (cfbe41o-). methods: a. fumigatus atcc #9197 was incubated in rpmi at different ph and concentrations of fbs. temperature, production time and different flow conditions were varied on ps, 16hbe and cfbe41o-. dry weight measurement and antifungal drug susceptibility testing was performed. scanning electron microscopy (sem) and confocal scanning laser microscopy (cslm) images were analyzed. results: the thickest biofilm was produced on ps with rpmi (+10% fbs, ph=6.5) at 35°c for 72h slightly rocking. biofilm dry weight on ps was 2.2 mg after 4 h and 8.3 mg after 72 h. the dry weight of produced biofilm exceeded 7.4 mg on 16hbe and 7.7 mg on cfbe41o-cells after 72h of biofilm production. there was no significant difference in dry weight increase between the cell lines and ps. planktonic a. fumigatus was susceptible to itraconazole (0.125µg/ml), voriconazole (0.063µg/ml) and amphotericin b (1µg/ml). aspergillus in biofilm was resistant against all drugs (>32µg/ml). the sem pictures displayed a network of hyphal structures and matrix at 12h. characteristic flow channels were observed at 72h. cslm images displayed conidia and hyphal structures embedded in matrix formations. a-alexafluor dyed polysaccharides of the cell wall and of the ecm in the biofilm. three dimensional constructs of the cslm pictures displayed biofilm on 16hbe and proofed viability of the cells after 48h co-incubation. differences in biofilm production between 16hbe and cfbe41o-were not significant. conclusions: a. fumigatus is able to form a biofilm structure in vitro on ps, 16hbe and cfbe41o-. a biofilm-like matrix produced by a. fumigatus was evidenced by dry weight measurement, sem, cslm and antifungal drug resist-ance in comparison to planktonic cells. potential clinical implications of a. fumigatus biofilm formation in vivo require further attention and investigations. etherington, c. 1 ; peckham, d. 1 ; conway, s. 1 ; hall, m. 2 ; denton, m. 2 1. seacroft hospital, regional adult cf unit, leeds, united kingdom; 2. microbiology department, leeds teaching hospitals, leeds, united kingdom susceptibility testing results are not predictive of clinical response to antibiotic therapy in chronic pseudomonas aeruginosa infections in cystic fibrosis. we assessed the impact of reducing the number of routine susceptibility tests performed on clinical outcome in these cases. in june 2006 we introduced a protocol of limiting susceptibility tests to p. aeruginosa isolates obtained from respiratory samples taken at the commencement of antibiotic therapy, when there was evidence of clinical failure, or routinely if not tested in the previous three months. at all other times, isolates were identified and reported as normal but p. aeruginosa isolates were not subjected to susceptibility tests. between 1st june and 30th november 2006 p. aeruginosa, was isolated on at least one occasion from 193 patients attending our adult cf unit. in this six month period we reduced the number of susceptibility tests by 56% (from a projected 2,231 tests on 872 samples to an actual 972 tests on 427 samples). this resulted in projected savings of $4,777 in consumables and 170 hours (costed at $8,871) of laboratory staff time per annum, a total saving of $13,649 (£6,500) per annum. we assessed the response to intravenous antibiotic treatment between the study period in 2006 and the same period in 2005. no significant differences in median change of fev1, fvc, crp, white cell count, weight, or duration of intravenous antibiotics were observed. for cf units sending regular, routine sputum samples, a reduction in the number of susceptibility tests performed in cases of chronic p. aeruginosa can be carried out without impacting on clinical outcomes. we report our preliminary results for a total of 233 strains obtained from 112 patients (85 patients harbored more than 2 strains). these patients were classified in 3 groups of respiratory insufficiency according to their fev1: severe in 31 patients (fev1<40%), moderate in 36 patients (fev1: 40-60%) and mild in 45 patients (fev1>60%). the clonal distribution was analyzed for the different strains of sa (1 to 6) isolated from patients sputum. these strains were analyzed for their antibiotic susceptibility and typed by pulsed-field electrophoresis gel (pfge) after smai digestion of chromosomal dna. thirty seven patients (33%) were colonized with mrsa. nine patients were both colonized with mrsa and mssa. among mrsa strains, 43/61 (70%) were also resistant to more than three other antibiotic family. strains harboring minor differences in the banding pattern (>80% similarity as assessed by the dice coefficient) were considered clonal. our results show that 83% of the patients were colonized with a single persistent strain during the year of follow-up. consecutive isolates with different pfge profiles were obtained from only 14/80 patients (17%). pfge analysis revealed that mrsa isolated from 27 patients were grouped in 8 clusters. these results revealed a possible clonal relationship between mrsa isolated from different patients with cf. we did not find any difference in the distribution of sa strains in our cf patients among the 3 groups of respiratory insufficiency. the study is ongoing in our adult cf population and in necker pediatric cf centre in paris. giusti, r. 1 ; furfaro, s. 2 1. pediatrics, long island college hospital, brooklyn, ny, usa; 2. research, lumina fund, new york, ny, usa palivizumab(synagis) is a humanized monoclonal antibody to rsv. the 2006 redbook acknowledges that some patients with cystic fibrosis may be at increased risk of rsv infection but that there is insufficient data to determine the effectiveness of this therapy. the objective of this study was to assess: 1)practice patterns of cf physicians in the us and canada 2)the severity of rsv disease in cf infants during the past rsv season. 3)if there is a standard of care concerning the use of synagis for cf infants. methods a questionnaire was developed using a web-based commercial vendor. an embedded web link was distributed via an e-mail sent to all us and canadian pediatric cf center directors. respondents clicked on a link to respond to the survey and results were automatically tabulated in real-time. completed responses were received from 84 center directors (73 us and 11 canadian) for a response rate of 63% in us and 44% in canada. most responders (70%) have prescribed synagis for infants with cf in the first rsv season, however only 38% routinely prescribed synagis for all infants with cf. only 35% expressed having had difficulty in obtaining insurance approval for this medication. many physicians indicated that synagis was frequently prescribed by the general pediatrician and that infants living at long distances from a cf center may be hospitalized at local hospitals. these issues may affect the accuracy of the data and result in an underestimate of actual synagis prescription and hospitalization rates. there were 456 infants diagnosed with cf in the past year and 172 of these infants were reported as symptomatic. there were 74 cf infants (16%) reported as having a documented rsv infection and 13 (18%) of these infants had received synagis. there were 31 infants (42%) with documented rsv infection who responded to outpatient management.there were no deaths but 40 patients were hospitalized and 8 of these had received synagis. of the 3 infants that required admission to an icu 2 had received synagis. of the 12 patients who were noted as having persistent chest x-ray changes 5 had received treatment with synagis. there were 36 infants with persistent wheezing and 13 of these infants had been treated with synagis. conclusions despite the limitations of a retrospective survey, this data demonstrates that rsv can cause significant and prolonged pulmonary disease and is a significant precipitating factor resulting in hospitalization of cf infants. the data also notes that many cf infants infected during the past rsv season have a mild illness and respond to outpatient management. a surprising finding of this survey is that infants at cf care centers where synagis was prescribed for all cf infants continued to have significant rsv related hospitalizations, persistent wheezing or prolonged chest x-ray abnormalities. there are different opinions among cf physicians concerning the routine use of synagis and currently the data suggests that there is not a standard of care concerning the prophylaxis of cf infants with synagis. this results of this survey should encourage physicians to prospectively study the efficacy of synagis prophylaxis in preventing hospitalization, persistent wheezing and chest x-ray abnormalities in cf infants. milani, a. 1 ; cisbani, g. 1 ; macchi, r. 1 ; vidal-aroca, f. 2 ; bertoni, g. 1 1. biomolecular sciences and biotechnology, university of milan, milano, italy; 2. basilea pharmaceutica ltd., basel, switzerland with ever increasing frequency, we now observe several examples of bacteria being resistant to every clinically available drug. therefore this urgently calls for the development of novel and improved antibiotics that may escape the extant mechanisms of bacterial resistance. one recent and promising development of the genome-wide search for target functions for antibiotics led to the identification of essential genes of pathogens by interfering antisense rnas. the first step is the construction of shotgun antisense libraries (sals) as follows. genomic dna is extracted from the bacterial strain of interest, fragmented by shearing into short pieces of dna, blunt-ended and cloned in an expression vector under the control of a regulatable promoter. the library is then reintroduced into the cognate bacterial strain and screened by replica plating colonies both in the presence and the absence of an inducer of the vector promoter. by this method, insert sequencing of clones showing conditional growth phenotypes is expected to lead to the identification of essential genes that can be silenced via antisense rna activity. we adopted this technology in order to generate a panel of essential functions of the cystic fibrosis-related opportunistic pathogen pseudomonas aeruginosa. so far, we tuned the protocol for sal generation in p. aeruginosa and identified a number of sequences conferring different levels of growth inhibition we are now characterizing these putative antisense rnas in order to define the minimal sequence able to cause the toxic effect and, on the other hand, to understand the cellular role of their targets. pseudomonas aeruginosa (pa) is an extremely versatile microbe with a vast array of pathogenic and metabolic mechanisms that allow it to form persistent infections in select patient populations, especially patients with cystic fibrosis. an ineffective immune response is considered partially to blame for failure of pa eradication. we have discovered that some strains of pa express peptidylarginine deiminase (padi) activity. padi is an enzyme that post-translationally modifies peptidylarginine to peptidylcitrulline with ammonia as a byproduct. our lab has shown that human padi can modulate the immune system through downregulation of tlr4 and ikk-gamma signaling. characterization of the pa padi will offer insights into a completely novel method of immune modulation by pseudomonas aeruginosa. using a widely published colorimetric assay for padi activity we have found the specific activity of crude pa cell lysates is very low. however this is similar to the only other known prokaryotic padi described in porphyromonas gingivalis. a protein homology search with the porphyromonas padi has revealed the likely genetic locus of pseudomonas padi in a 4.5 kb operon that appears in the genome of the pathogenic pseudomonas isolate pa14. this operon appears to contain two candidate genes for padi activity based on conserved motif searches (padi 1 and padi 2). both have been cloned into expression vectors, partially purified by affinity tag technology and tested in the colorimetric padi assay. curiously padi 1 autocitrullinates itself while padi 2 has not shown activity. reaction conditions and substrate specificities for pa-padi are not like either the human or porphyromonas padi. furthermore analysis of up to 64 clinically diverse strains has demonstrated 15% carry the gene for padi 1. this work is only the second description of padi activity in any prokaryote. pa padi could clearly have a dramatic impact on the local inflammatory milieu if it can access the same targets as human padi. the description of this activity in pseudomonas will advance our knowledge of the human-pathogen relationship and give insight into new therapies. which functions by translocating toxins into the cytoplasm of host cells. these toxins cause disease by damaging the surrounding host tissue, promoting dissemination of the organism and paralyzing the phagocytic mechanism of macrophages. pcrv is a factor required for the translocation of the toxins. the bases of these studies were to evaluate pcrv as a protective antigen in "p. aeruginosa" pneumonia and cldc as a vaccine adjuvant. methods: mice were vaccinated 1, 2, or 3 times with the pcrv antigen combined with either aluminum hydroxide (alum) or cldc by various routes of administration. efficacy of vaccination was evaluated by challenge with "p. aeruginosa" and evaluation of survival and/or measurement of various parameters associated with lung injury. results: increase in median survival time was highly significant when cldc/pcrv was compared with cldc or pcrv alone. following 2 subcutaneous administrations cldc/pcrv showed an increase in median survival time (58 hours versus 43 hours) and overall survival benefit following 3 intraperitoneal administrations (100% versus 80%). mice with anti-pcrv antibody levels above 10µg/ml were significantly protected. conclusion: the investigators establish the efficacy of cldc/pcrv vaccines via several parenteral routes of administration compared to no treatment as well as cldc and pcrv-only controls. differences were demonstrated between performance of cldc/pcrv and alum/pcrv in measures of lung injury, median survival, and overall survival. the results correlated with antibody levels and histological examination of the lung tissues. importantly, these studies indicate that protection can be achieved against "p. aeruginosa" infection by targeting an antigen associated with the type iii secretion system. background: there has been a recent increase in the number of reported cases of acute renal failure (arf) in cystic fibrosis (cf). our group have undertaken a national survey, which measured the incidence risk of arf in cf patients at between 4.6 and 10.5 cases / 10,000 cf patients / year. we have now conducted a case control study to determine which factors which are associated with an increased risk of arf. methods: in our initial survey we confirmed 24 cases of arf, in cf patients from 20 uk cf centres, presenting between 1997 & 2004. using the uk cf database, we identified sex and age (within 6 months) matched controls. informed consent was sought from the control patients, or their parents, for access to the case notes and clinical data were extracted. analysis of risk factors was by conditional logistic regression, using stata (version 9) and by fisher's exact test. results: there were 24 cases of arf (12 male, median age 10y, range 4m-32y) and 28 controls (17 male, 9y, 10m-32y). in the group of patients with arf, 21/24 had received an aminoglycoside at the time of their episode of arf or in the preceding week, compared with only 3 of the controls for the same time period (p<0.001). the median number of days of aminoglycoside in the year prior to the index case developing acute renal failure was 24 (7-99) for cases and 0 (0-47) for controls. conditional logistic regression showed that the odds ratio for arf per each day of aminoglycoside was 1.06 (95% ci 1.01 to 1.11, p=0.02 it is well-known that pa senses the environment and changes its phenotype. for instance, it produces greater amounts of the extracellular polysaccharide alginate in the cf lung, characterized by a microaerobic environment. little is known about the changes in protein secretion induced by oxygen limitation in pao1, the proto-typical pa laboratory strain. no data are available on this regard about pa clinical strains. our work was aimed to study the differential regulation of proteins secreted by pa strains grown in microaerobic or aerobic conditions. a pa clinical isolate and pao1 were grown overnight in aerobiosis and in microaerophilic conditions. the supernatants were collected and proteomic analysis was carried over by two-dimensional capillary chromatography -tandem mass spectrometry (mauri et al., faseb j 2005) to evaluate the differential protein expression. in the pa clinical isolate, we identified 66 proteins down-regulated and 42 proteins upregulated in aerobic conditions in comparison with microaerophilic culture while in pao1 16 proteins were down-regulated and 30 were up-regulated. 6 proteins were down-regulated and 12 up-regulated both in the clinical and in the laboratory strain. these proteins can mediate different biological functions since they are enzymes, heat shock proteins, chaperones, proteins involved in adaptation, motility and in the transport of small molecules. among all these proteins, as the alkaline metalloproteinase is associated with tissue invasion not only by causing rupture of epithelial tight-junctions but also by degrading several chemokines, we decided to validate its up-regulation observed in aerobic conditions by zymography. we found that the proteolytic activity of the supernatants of pa grown in aerobic conditions was higher than in microaerophilic culture both for the laboratory and clinical strain, indicating the functional relevance of data obtained by proteomic analysis. the identification of proteins differentially regulated in aerobiosis and oxygen limitated conditions in pa laboratory and clinical strains might be helpful for the knowledge of the mechanisms of colonization and lung damage due to pa in cf patients. for instance, the validation of the upregulation of the alkaline metalloproteinase in aerobic condition may shed light on these mechanisms of cf lung disease. further studies are in progress to evaluate the function of other bacterial exoproducts regulated in this model and to extend the analysis to other clinical strains. supported by the italian cystic fibrosis research foundation (ffc-grant#17/2006), comitato di vicenza dell'associazione veneta per la lotta contro la fibrosi cistica and azienda ospedaliera di verona, italy. cystic fibrosis (cf) sufferers are subject to repeated lung infections most commonly with the bacterium pseudomonas aeruginosa. in spite of antibiotic treatment p. aeruginosa tends to become established giving rise to persistent chronic infection. in the lung environment, the bacterium grows as a highly structured biofilm consisting of a complex community of cells embedded within a self-secreted polysaccharide matrix. investigations of p. aeruginosa biofilm growth using model strains have elucidated mechanisms which appear to govern the biofilm life-cycle. our research aims to test whether observation of these mechanisms in planktonic culture can be related to the efficiency of biofilm formation. biofilm initiation has been linked to cell motility and in particular to the presence of flagella and pili, which are thought to be important for cell attachment and the formation of microcolonies. on testing a large, genetically diverse group of clinical p. aeruginosa isolates retrieved from the lungs of cf patients we found no definitive correlation between the degree of motility of an isolate in planktonic culture and its ability to form a biofilm in vitro. the development of biofilm architecture has been demonstrated in model systems to be coordinated by the production and secretion of n-acylhomoserine lactone (ahl) quorum sensing molecules. however among the clinical isolates tested we observed no obvious correlation between the amount of ahls produced in planktonic culture and the extent of biofilm formation. overall we have found observation of phenotypic characteristics in planktonic culture to be poor predictors of efficient biofilm formation. a proteomics approach was adopted to provide further insight into the physiology of biofilm growth of p. aeruginosa isolates by comparison with planktonic growth of the same isolates. two genetically unrelated clinical isolates, demonstrated as being capable of efficient in vitro biofilm formation, were selected from our culture collection on the basis of their diverse phenotypic characteristics when cultured planktonically. one displays both twitching and swimming motilities, is mucoid and expresses ahls, while the other is non-motile, non-mucoid and no ahls have been detected in planktonic culture. we have developed a simple flow-through bioreactor to provide sufficient quantities of biofilm for proteomic analysis. gel-based and gel-free techniques were employed to study protein expression patterns for both biofilm and planktonic cultures of each isolate by mass spectrometry. proteins specific for each growth phase could be detected and may prove suitable biomarkers for monitoring the physiological status of biofilm forming strains when a larger bank of clinical isolates are examined. liposomal amikacin (arikace tm ) is a liposome-encapsulated form of amikacin that is formulated to treat chronic p. aeruginosa infections in cystic fibrosis patients. these liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. a key aspect of the activity of the formulation is the ability to penetrate to the sites of pseudomonas biofilm-like growth in the lung. experiments were designed to investigate the penetration of liposomes into p. aeruginosa biofilms and in vitro activity. methods and results: model liposomes of the same size and lipid composition as liposomal amikacin (arikace tm ) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. a mucoid strain of pseudomonas aeruginosa (pa3064) was used to establish biofilms in rectangular optical grade glass flow cells. biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. images indicated significant penetration of liposomes into the interior of biofilms under these conditions. the rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. liposome concentrations were higher near the periphery than the interior. however, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. penetration of liposomes was observed under flow or static conditions. in a "washout" experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. the penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent dna content assay. similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. liposomes similar to liposomal amikacin (arikace tm ) readily penetrate into biofilms of pseudomonas aeruginosa and may even have enhanced binding to biofilms. this binding along with localized release can explain the substantial efficacy observed in animal models. coates, a.l. 1 adherence to recommended therapy in cf has always been a challenge, in part, due to the time demands of the daily therapy. while twice daily inhaled tobramycin for those infected with pseudomonas aeruginosa (pa) has become an accepted standard of care, as much as 40 minutes a day may be consumed inhaling 300 mg in 5 ml of tobramycin (tobi ® ) from the pari lc plus ® breath enhanced jet nebulizer. the purpose of this study was to determine if equivalent levels of pulmonary deposition could be achieved in a much shorter time period using 1.5 ml of a more concentrated (100 mg/ml) tobramycin solution delivered by a perforated vibrating membrane nebulizer (eflow ® membrane configuration 35l) both, developed by pari pharma; germany. methods with a goal to study 7 children and 8 adults, to date, the subjects are 4 children 10 years and older and 2 adult males, all with an fev1 > 50% predicted, with stable cf. all were receiving inhaled tobramycin for positive sputum cultures of pa. following pretreatment with albuterol, they inhaled both preparations on two occasions with 99m tc-dtpa added to the tobramycin in the nuclear medicine facility. in vitro preliminary work demonstrated that the radiolabel tracked with both formulations of tobramycin. deposition was measured by a gamma camera taking both tissue attenuation and mucociliary clearance during nebulization into account (pediatric pulmonol suppl 29: a343; 2006) . in order to have a continuous rate of deposition, the pari lc plus ® was run for a timed 10 minutes and then both the total deposition and time of nebulization "scaled up" from in vitro testing when the nebulizer was run to dryness. this was done by multiplying the deposition by the total output when run to dryness divided by the total output in 10 minutes. (blood samples were taken for quantification of tobramycin in the serum but not yet analysed). the rate of output per minute was calculated from the 10 minute run and the total time was total output from in vitro testing divided by the rate of output. the eflow ® pro-vides a continuous output and stops automatically at dryness. quality assurance was the agreement between total radioactivity pre nebulization (in the nebulizer) and post which included the subject, the nebulizer, the connectors and the expiratory filter. the pari lc plus ® delivered 38.1±6.1 mg in 15.8±1.6 minutes compared to 42.7±10.2 mg in 4.2±1.2 minutes for the eflow ® . only the time of delivery was significantly different with p<0.0001(paired t-test). tolerability of the treatment was comparable for both inhalation regimes, but the shorter treatment was preferred by all patients. these results demonstrate the possibility of delivering equivalent levels of tobramycin in much shorter periods of time into the lungs of cf patients when using eflow ® , a very efficient electronic nebulizer. this time saving may improve adherence to recommended therapy. (pediatrics 2006; 118:1260) . in order to properly interpret op cultures from nbs infants, especially those with non-classic cf mutations, we need to know the op flora of non-cf infants. we obtained op cultures from 100 healthy infants under 1 yr of age. op specimens were plated on standard cf culture media. exclusion criteria included a first degree relative with cf, respiratory illness at the time of culture, or positive newborn screen for cf. data on cigarette smoke exposure, animal exposure, and exposure to hot tubs/swimming pools was collected. 57 samples have been collected to date. in healthy, non-cf infants, the most common finding is non-specific mixed gram negative and positive growth. however, 3 infants have grown pa (ages 3 months, 10 months, and 10 months). many infants have grown multiple organisms. the following bacteria have been found in (n) number of infants: s. aureus (10), e. coli (8), e. cloacae (6) , h. flu (4), klebsiella (4), pseudomonas aeruginosa (3), h. parainflu (3), unidentified non-lactose fermenting (4), other (9). data on 100 infants including correlation to environmental factors will be presented. conclusion: non-cf infants commonly have s. aureus and many gram negative organisms including pa in their oropharynx. these results may have some bearing on interpetting colonization and clearance of pa in infants identified through nbs and in epic study participants. #300) ). identification of smg by sputum cultivation represents a significant challenge because the organisms are phenotypically diverse, grow poorly on routine culture media, and are very difficult to discriminate from other members of the oropharyngeal flora. we have developed a solid media for the selective isolation of smg from sputum. the value of the media is highlighted by the identification of smg as the quantitatively dominant organism in sputum samples of three cf patients admitted to hospital for an acute pulmonary exacerbation. in all three, the smg species failed to be identified on routine or selective media currently described for the culture of cf-specific sputum pathogens. antibiotic treatment directed against the smg correlated with clinical resolution of acute symptoms as well the reduction of smg on daily serial sputum cultures during hospitalization. this novel selective media makes use of antibiotics (colistin, sulfadiazine and oxolinic acid) inhibitory to the growth of principal cf pathogens and much of the usual oropharyngeal flora. smg agar utilizes a colorimetric indicator to uniquely identify smg colonies. the sensitivity and specificity of the selective media has been evaluated by molecular methods using terminal restriction fragment length polymorphism analysis. smg organisms do not respond well to anti-pseudomonal therapy, therefore proper detection and culture-directed antibiotic therapy is paramount. we believe that smg represent significant respiratory pathogens in cf, and because of the inability to effectively culture and identify smg they have largely gone unrecognized. pseudomonas aeruginosa releases substantial amounts of the blue antibiotic pigment pyocyanin. in presence of a reductant (such as nadph), pyocyanin redox-cycles and generates superoxide and h 2 o 2 . in infected cf airways, pyocyanin concentrations can reach high micromolar levels and contribute to oxidative stress of the airways. the structural basis of the pyocyanin molecule that underlies the redox-cycling with reductants of the airways is not clear. we therefore investigated i) the ability of physiologically or pharmacologically relevant reductants of the airways to support the redox-cycle activity of pyocyanin, and ii) the molecular features of pyocyanin that support redox cycling. dose-and time-dependent h 2 o 2 production by pyocyanin was measured by amplex red oxidation in presence of horseradish peroxidase. rates of h 2 o 2 production by 100 µm pyocyanin in presence of 150 µm reductant were: nadph (392 pmole/min) > l-ascorbate (108 pmole/min) > reduced glutathione (41 pmole/min) > α-tocopherol (12 pmole/min). in contrast, lipoic acid, genistein, or resveratrol did not significantly support pyocyanin-mediated h 2 o 2 production. in absence of a reductant, pyocyanin showed no measurable formation of h 2 o 2 . to identify the structural characteristics of the pyocyanin molecule that allow for its redox-cycling activity we synthesized a number of new pyocyanins containing electron-donating or electron-withdrawing substituents. functional assays were performed in presence of l-ascorbate as reductant. molecular substituents that donated electrons to the positively charged core of pyocyanin, either by hyperconjugative or resonance effects, reduced the h 2 o 2 output of the corresponding pyocyanin. in contrast, a closely related analog (2-hydroxyphenazine-5,10dioxide) showed significantly increased activity (2.2x compared to pyocyanin) suggesting that the electron-withdrawing effect of the n-oxide functionality led to an increase in the redox-cycle activity. these data indicate that the functional characteristics of pyocyanin as a redox-cycling compound are governed by its positively charged core. in the airways, pyocyanin is predicted to utilize several reductants that are present in the airway surface liquid or intracellularly, thus contributing to cf airway disease. because pyocyanin utilizes a variety of reductants, it appears prudent to test whether inhaled small-molecular cf therapeutics support pyocyanin function. supported by nih (hl-071829, p01 at002620), cfri, philip morris usa inc and philip morris international, and cff (fischer07g0). taccetti, g.; braggion, c.; ravenni, n.; zavataro, l.; neri, a.; festini, f.; campana, s. meyer hospital, university of florence, cf center of tuscany, florence, italy for practical purposes, after early eradication treatment, at least three consecutive negative respiratory cultures over a 6-month period would indicate that the organism has been eradicated (cf trust guidelines). this recommendation is based on opinion/clinical experience of respected authorities in the absence of directly applicable studies of good quality. aims: using molecular biological techniques, we evaluated whether this 6-month interval is really trustworthy for distinguishing between regrowth of the same strain, suppressed but not eradicated by treatment, and new pseudomonas aeruginosa (pa) colonization. patients and methods: cystic fibrosis (cf) patients were treated with oral ciprofloxacin and nebulized colistin at detection of pa. all pa colonization episodes were recorded in an appropriate database. molecular study of each bacterial isolate from each colonization episode was performed with the rapd-pcr. results: between 1998 between -2006 of patients in follow-up in our center had repeated pa colonization. the patients' mean age at first pa colonization was 78 ± 4.9 months. a total of 78 episodes was observed (mean of 2.75 episodes per patient, median of 2, range 2-7). molecular typing on 78 strains indicated that 66 (84.6%) were a different genotype from later colonization episodes while 12 (15.4%) isolates had the same genotype as those of the preceding episode. the same genotype as preceding colonization was observed in 7 (46.6%) of 15 isolates from patients in which a successive colonization was verified in less than 6 months, and was verified in 8 (12.6%) of 63 strains in patients having a successive colonization in over 6 months (or=6.01). colonization was due to a genotypically diverse strain in 54% of cases where colonization occurred within 6 months of eradication. during the observation period 7 (25%) of 28 patients acquired chronic pa infection. conclusion: re-colonization by pa following eradication therapy is mostly (84%) caused by strains with a different genotype, suggesting acquisition from an external source. a short pa-free period is mainly due to transient suppression of pa growth, and true eradication followed by acquisition of a new pa genotype occurred in most cases only after a pa-free period longer than 6 months. those patients with a pa-free interval of less than 6 months had a six-times higher chance that the pa was not eradicated compared to those with a germ-free interval of over 6 months. this evidence demonstrates that the definition of successful eradication should be reconsidered, taking into account additional parameters such as molecular analysis. cystic fibrosis (cf) patients appear to have an increased risk of urolithiasis. while a number of possible explanations for this have been proposed and investigated, no definitive mechanism has yet been demonstrated. as cf patients frequently get respiratory tract infections they regularly receive ciprofloxacin treatment, often given in doses well in excess of conventional prescription regimes. there are occasional reports of ciprofloxacin crystals urine and stones in the urinary tract. here we investigate the hypothesis that ciprofloxacin excreted in urine might act as a promoter of crystallisation of calcium or magnesium salts and thereby increase the risk of kidney stone disease. the effect of ciprofloxacin was tested in artificial urine (au). in vitro crystallisation was tested using a 96 well plate turbidity method, to identify a metastable limit of oxalate concentration (ml) and a growth and nucleation parameter, the turbidity rate index (tri). the nucleation ph of urine was examined by tritrating oxalate free au through a ph range of 5.3 to 9.0 and monitoring the solution/suspension turbidity. in au at ph 6.0, ciprofloxacin, at 300, 600, 1000 or 1800mg/l, had no detectable effect on initiation of calcium oxalate crystallisation (ml) or its progress (tri) (n=6 for each concentration). when au with 6mm ca, 3mm mg and 25mm po4 was titrated there were two distinct nucleation points; a slow event starting at about ph6.7 and a much faster event at about ph 7.4. omitting ca or mg confirmed that the first event was due to calcium phosphate precipitation and the second to struvite. including ciprofloxacin at 400, 500 or 600mg/l did not alter these nucleation ph values, but the magnitude of the turbidity rise showed that the ciprofloxacin co-precipitated with the struvite. ciprofloxacin at 1000mg/l and without ca or mg began to precipitate at ph 6.2 and could be held in solution until ph 7.2 when ca and mg were included. even at high concentrations, ciprofloxacin does not influence calcium oxalate crystallisation. nor does it promote calcium phosphate or struvite precipitation; on the contrary, while the calcium and magnesium remain in solution, they help to prevent precipitation of the ciprofloxacin itself. urinary ciprofloxacin does not appear to act as a stone or crystal promoter. pseudomonas aeruginosa is a significant cause of mortality in cystic fibrosis (cf) sufferers. cf patients were thought to acquire p. aeruginosa from the environment; however genotyping over recent years has revealed clonal strains in sputa from cf patients in the uk, australia, and canada that are transmitted person to person or from a common source. one clonal strain, australian epidemic strain-1 (aes-1), (formerly melbourne epidemic strain, m16 or pi) currently infects up to 40% of patients in five cf clinics on the eastern seaboard of australia. most cf clonal strains have been associated with increased virulence not fully explained by greater antibiotic resistance. both genotypic and phenotypic differences have been postulated as important in enabling transmission of clonal strains. in order to compare the expression profile of aes-1 to the type strain p. aeruginosa pao1, the cf research group at the university of sydney compared the genome expression data of four clonal aes-1 isolates and pao1, when grown as planktonic and as 72-hr biofilm cultures. in aes-1, a set of 22 significantly differentially expressed genes (all downregulated) were identified, including the quorum sensing genes lasa, lasb and rhll. in contrast, both upregulated and downregulated genes were differentially expressed in pao1 biofilm compared to pao1 planktonic culture. expression data was validated using quantitative real-time pcr. to compare biofilm growth at the phenotypic level, the four clonal strains and pao1 were grown as 72-hr biofilms in a double-blind study, and the size of ten randomly selected biofilms per isolate, stained with syto® 9 green fluorescent stain was measured. at 72 hr, the biofilms formed by aes-1 isolates were significantly larger (ca. 18-fold) than pao1 (average size: 33389±14211µm 2 vs 1791±498µm 2 )(p<0.01). the average thickness of three biofilms per isolate, measured by confocal microscopy, showed aes-1 biofilms to be approximately 1.5-fold thicker than those formed by pao1 (12.6±0.2µm vs 8.5±1.4µm). the general gene downregulation observed in aes-1 biofilms suggests an adaptation to the cf host, while a larger biofilm would provide for more effective bacterial dispersal. thus the transmissibility of aes-1 may be linked to enhanced biofilm formation upon colonisation. background -objective: whilst influence induced by bacterial colonization in cystic fibrosis (cf) is established, risk induced by fungal colonization is less defined. prevalence of other species than aspergillus sp. or candida sp., and factors associated with fungal presence are also poorly documented. our preliminary study aimed to determine which fungal species were present in sputum collected from adult cf patients, and which factors were associated with fungal presence. methods: in a monocenter, transversal prospective study, 21 cf adult patients were included to determine fungal presence in sputum using semi selective growing media. clinical parameters (shwachman score, respiratory function, nutritional status, gastro-oesophageal reflux, pancreatic insufficiency and diabetes); therapeutics used (including oral or intravenous antibiotics, systemic or inhaled corticosteroids or bronchodilatators, antifungal treatments); microbiological data of bacterial colonization and environmental parameters (potted plants or domestic animals presence) were determined for each patient. correlation between fungal, clinical, environmental, therapeutic or microbiological data was evaluated by mann-whitney non parametric u test. results: 16 patients (76%) presented with fungal presence in sputum. 61% presented with yeasts species, 52% with moulds. aspergillus fumigatus and candida albicans were the predominant species in moulds and yeasts respectively, but less common mould species such as exophiala dermatidis or paecilomyces variotii were also recovered. factors associated with fungal presence were pancreatic insufficiency (p=0,04); malnutrition (p=0,039), bacterial colonization and inhaled corticosteroids. candida albicans was correlated with more severe shwachman score (p=0,019), bacterial colonization (p=0,017), notably with pseudomonas aeruginosa (p=0, 03) , and intravenous antibiotics use (p=0,015). moulds species were significantly associated with inhaled corticosteroids (p=0,049). antifungal use was associated with frequent resistance to azoles treatments (4 resistant isolates out of 5 patients treated). conclusion: fungal presence in cf appears frequent. some species could have been previously overlooked due to diagnosis difficulties. the effect of corticosteroids on moulds species, already found in other pathologies, appears important in cf. influence of fungal presence on cf course needs prospective studies, in order to establish if patients could benefit from antifungal treatments or preventive measures. dren on a ventilator without a previous diagnosis of cystic fibrosis is unknown. the aim of our study was to investigate the prevalence of these microorganisms in routine sputum cultures in young children on a ventilator in a pediatric intensive care unit (picu). methods: from all ventilated children aged 0-3 years admitted from 1998-2004, sputum culture results obtained from tracheal aspirates within the first week of admission were retrospectively analysed. three patient subgroups were identified: respiratory failure due to pulmonary disease (group 1), ventilation after elective surgical procedures (group 2) and other ventilated children (group 3). children with a previous intensive care admission or cf were excluded. the cf database was checked (2007) to identify any children with a new diagnosis of cf that were included in the study. results:12.1 % of all ventilated children had a positive sputum culture with one of the "cf-bacteria" s. aureus, h. influenzae and p. aeruginosa these were found mainly in group 1 (see table) . 27/54 of these children were admitted for treatment of respiratory syncytial virus (rsv) bronchiolitis. a sweat test was performed in 7 children, all admitted with pulmonary disease and because of co-existing clinical signs or symptoms. one sweat test was positive and subsequently cf was diagnosed in this child. no other children out of the study group have since then been diagnosed with cf. as the population in the northern part of the netherlands is very stable, it is unlikely a diagnosis of cf has been missed. conclusion: s. aureus and h. influenzae are cultured frequently in ventilated children without cf, especially when ventilated because of pulmonary disease. the opportunistic pathogen p. aeruginosa causes both acute and chronic human infections. the balance between systemic infection and mortality or chronic persistence and morbidity depends on complex relationships in which the immunological status and genetic potential of the host but also the bacterial biodiversity are determinant factors. p. aeruginosa extensive genetic adaptation and microevolution have been repeatedly observed in chronic infections of cf patients in contrast to what it is documented in acute infections. whether these p. aeruginosa clonal variants differ in their pathogenic potential is not yet known. a total of 19 clinical p. aeruginosa isolates from six cf patients which carried unique clonal lineage from the onset of colonization over 4-18 years were selected (bragonzi et al, 2006; montanari et al, 2007) . five p. aeruginosa environmental strains, which represent the source of acquisition for cf patients, and two laboratory strains pao1 and pa14 were also used as references. multiple genotypic analysis of sequential p. aeruginosa isolates which included pfge, atchip and multilocus snps showed intraclonal diversity with genome rearrangements, variations in pathogenic islands and acquisition of prothoadaptive mutations in the muc genes. the largest divergence was observed between the completely sequenced reference strains pao1 and pa14, the latter represented in our panel by three cf isolates. p. aeruginosa virulence has been assessed by monitoring its capacity to induce bacteremia and to establish chronic infection in a murine model (bragonzi et al, 2005) . overall, p. aeruginosa environmental strains increased five times the risk of bacteremia when compared to clinical strains (test of proportion: p<0.001). the high risk of mortality was also evidenced for p. aeruginosa cf strains isolated at the onset of the infection when compared with those isolated after years of colonization (p=0.002) indicating that environmental strains or newly acquired strains were similar in their virulence. p. aeruginosa clinical strains isolated after years of colonization increased chronic persistence and reduced the risk of bacteremia (p=0.002) when compared to strains isolated at the onset of the infection. the strain pa14 was found to be lethal in contrast to p. aeruginosa clonal strains of clinical origin that established chronic persistent infection in the murine lung. furthermore, our data showed that adaptive traits commonly associated with the chronic p. aeruginosa infections in cf patients, such as transition to the mucoid phenotype, did not confer a selective advantage to bacterial cells in colonizing the murine lung (p=0.294). these results suggest that p. aeruginosa pathogenicity is independent of the strain's genotype but rather genetic adaptation to the cf airways plays an important role in the development of persistence and in the resistance to host defences. supported by telethon and italian cf research foundation. beringer, p. the combination of antipseudomonal beta-lactam and aminoglycoside antibiotics is frequently prescribed during acute pulmonary exacerbations. since the introduction of the beta-lactam compounds there has been numerous reports citing altered pharmacokinetics of several compounds (but not all) within the beta-lactam class. recently it has been suggested that substrate specificity for the renal transporter pgp may explain the variability in renal drug clearance observed in patients with cf. pgp is structurally related to cftr and evidence suggests it serves a complementary role in modulating alternative chloride channel function. in a cftr knockout model mdr1 expression was reported to be increased nearly four-fold. in an effort to elucidate the potential contribution of pgp to the renal clearance of drugs in patients with cf, we conducted a controlled clinical trial comparing the pharmacokinetics of fexofenadine (fx) in patients with cf and age matched healthy volunteers (hv). fexofenadine is not significantly metabolized and is a relatively specific substrate for the membrane transporters pgp and oatp. probenecid (pb) is a selective inhibitor of oatp and was used pharmacologically to block the activity of this transporter in-vivo. our hypothesis is that if pgp is upregulated within the renal tubules of patients with cf we would expect to see an enhanced renal clearance of fexofenadine in these patients when compared with control subjects. methods: 16 (n=8 cf, 8 hv) subjects underwent this prospective controlled study which fx was received alone or in combination with probenecid (pb). iothalamate was given each day to measure glomerular filtration rate (gfr). blood and urine samples were obtained at specified times over 12 hours each study day for determination of fexofenadine and iothalamate concentrations. fexofenadine and iothalamate concentrations were assayed using liquid chromatography-tandem mass spectrometry and hplc-uv respectively. pharmacokinetic analysis was performed using a 1-compartment cumulative urinary excretion model with the adapt ii software. differences between groups were determined using a paired t-test or mann whitney-u. results: 16 patients were included, cf patients were slightly older and had lower body mass index when compared to hv, (mean±sd, 27±5.6 vs. 33.4±7 years p=0.0499, 20.03±1.6 vs. 25.2±2.2 p=0.0019 respectively) but did not differ in gfr (120±21.7 vs. 108±25 ml/min-1.73m 2 p=0.38 respectively). no significant differences were found between cf and hv in clr of fx alone and fx in combination with pb clr (93.3±20 vs. 83.3±23.3 ml/min-1.73m 2 p=0.31 and 38.3±10 vs 36.7±23.3 ml/min-1.73m 2 p=0.79 respectively). conclusion: the results of this study indicate the enhanced clearance of certain antibiotics previously reported in patients with cf do not appear to be due to upregulation of renal tubular pgp or increased gfr. maceachran, d.p. department of microbiology and immunology, dartmouth medical school, hanover, nh, usa p. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (cf) patients. shortly after birth cf patients are colonized by p. aeruginosa, which leads to a lifelong chronic infection. the mechanisms behind p. aeruginosa colonization of the cf lung are still poorly understood, however several secreted toxins have been associated with this phenomenon. cf is characterized by a loss of mucocilliary clearance, a key component of innate immunity in the lung, and an increase in sputum viscosity as a result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). we have previously shown that the novel p. aeruginosa secreted protein cif is capable of reducing apical membrane expression of cftr. we have begun to characterize cif expression and have identified a key regulator of cif transcription. mutations affecting the divergently transcribed putative tetr family repressor encoded by the pa2931 gene result in a significant increase in cif expression. furthermore, we have demonstrated that the pa2931 gene product is capable of binding the promoter region located immediately upstream of the cif-containing operon. this work has been supported by the nih t32-dk007301-29. ryall, b. 1 ; davies, j.c. 2, 4 ; wilson, r. 3 ; shoemark, a. 3 ; williams, h.d. 1 1. division of biology, imperial college london, london, united kingdom; 2. department of gene therapy, imperial college london, london, united kingdom; 3. host defence unit, royal brompton hospital, london, united kingdom; 4. paediatric respiratory medicine, royal brompton hospital, london, united kingdom pseudomonas aeruginosa is the most important respiratory pathogen in patients with cystic fibrosis (cf) and non-cf bronchiectasis. the factors which enable p. aeruginosa to establish chronic, debilitating respiratory infections are unclear. however p. aeruginosa is one of a limited number of bacteria that is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. this study examined whether hydrogen cyanide is produced by p. aeruginosa in cf and non-cf bronchiectasis airway infection. cyanide concentration was measured in fresh sputum from cf and non-cf bronchiectasis patients with and without p. aeruginosa lung infection using a cyanide ion sensing electrode. cyanide was detected in sputum from 15/25 cf and non-cf bronchiectasis patients with current p. aeruginosa infection, whereas it was not detected in any of the 10 patients without this organism (p<0.01). maximum lev-els were 130µm (mean ±se: 72 ± 6.6 µm), which compares with levels of above 40µm which would be considered toxic in blood. concurrent lung function data were available on all 21 p. aeruginosainfected cf patients; the group with measurable sputum cyanide (n=11) was not different from those without (n=10) on the basis of age, gender or p. aeruginosa phenotype (mucoid/ non-mucoid). however, those with detectable cyanide had significantly poorer lung function than those without (fev1% predicted: 26.8±3.8% versus 46.0±6.7%, p<0.01; fvc% predicted: 44.4±4.9% versus 60.1±7.7, p<0.05). we have shown that cyanide is detectable at clinically significant levels in sputum from cf and non-cf bronchiectasis patients infected with p. aeruginosa and is associated with significantly impaired lung function. we have recently reported a dose regimen for once-daily tobramycin dosing in patients with cystic fibrosis which was based on a retrospective study (lam w et al., j antimicrob chemother 2007) . we present here an interim analysis of an ongoing prospective study to evaluate the proposed dosing in paediatric patients with cystic fibrosis. as part of this protocol pharmacokinetic parameters were determined in cystic fibrosis (cf) patients receiving intravenous antibiotic therapy for a pulmonary exacerbation. serum creatinine, audiometric testing, respiratory cultures and fev 1 were assessed at baseline and after a 2 week treatment period. serum tobramycin concentrations were interpreted by clinical pharmacists performing therapeutic drug monitoring (tdm) to optimize dosing. serum tobramycin levels were drawn after the first dose, repeated with each dose adjustment and weekly thereafter. the proposed sample size of this study is 60 patients; an interim analysis was performed to identify trends for pharmacokinetic and safety outcomes from 18 patients admitted from november 2006 through april 2007. seven (38.9%) patients achieved the target c max of 25-35mg/l, 10 (55.6%) patients remained under the target range and 1 patient was above the target range with the 1st dose. with 2nd, 3rd, and 4th tdm performed for either routine weekly levels or dose adjustment levels, 6, 16, and 17 patients met the target c max respectively. the percentage of patients who achieved target c max increased to 88.9 % with the 3rd set of tdm levels and 94.44 % with the 4th set of tdm levels (p-value<0.0001). there were no significant changes in k e , t 1/2 and v d /kg for each patient from 1st to 2nd set of tdm levels over time. compared to the previous retrospective study the mean v d /kg was significantly higher (0.327l/kg versus 0.267l/kg, p=0.0021) and the mean t 1/2 was significantly shorter ( 1.73 hrs versus 2.14 hrs; p<0.0015). none of the patients' serum creatinine increased ≥ 50 % from baseline. baseline audiometric tests were within normal limits in 12 (70.6 %) patients. p. aeruginosa was isolated from respiratory specimens of 6 patients on initial culture. of the 5 patients with follow-up cultures, 3 patients grew p. aeruginosa sensitive to tobramycin, demonstrating no decrease in sensitivity. our preliminary results suggest the pharmacokinetic data differ from the expected profile suggested by the retrospective study. all study patients treated according to the guidelines were clinically well after their course of therapy and did not experience clinically significant toxicity from once-daily tobramycin dosing. the once-daily tobramycin dosing will continue to be evaluated until the proposed sample size is achieved for final analysis. anderson, g.g.; o'toole, g.a. dartmouth medical school, hanover, nh, usa pseudomonas aeruginosa chronically colonizes the lungs of individuals with cystic fibrosis (cf). evidence suggests that this infection progresses through distinct phases, wherein initial colonizing strains undergo a switch to mucoidy that corresponds to formation of bacterial microcolonies in the airways and life-long persistence. currently, the environmental signals and mechanisms regulating conversion from the acute, highly virulent form to a less virulent biofilm state remain obscure. to investigate these issues, we developed a tissue culture model system for growth of p. aeruginosa biofilms on cf-derived human airway epithelial cells in vitro. biofilms grown on these cells appear similar to those found in cf airways as well as to abiotic biofilms. eventually, epithelial cells were killed by the p. aeruginosa biofilms. however, we discovered that addition of the antibiotic tobramycin preserved the monolayer integrity for at least 24 hours. bacteria were not completely eliminated by this treatment, suggesting that tobramycin influenced the virulence of the biofilm bacteria. microarray analysis of cf cell grown biofilms treated with tobramycin revealed marked alterations in transcriptional profiles compared to untreated controls. two tobramycin-induced genes were identified from this screen which, when deleted, exerted altered virulence phenotypes on airway epithelial cells in culture. quantification of cytotoxicity of these mutants revealed either increased or decreased ability to kill the epithelial cells. complementation with full-length copies of these genes restored wild-type function. these studies suggest a complex relationship between acute and chronic forms of p. aeruginosa, and that antibiotic treatment may influence transitions between these modes. it is this interplay that may determine the virulence status of the microorganism upon initial infection as well as during cf exacerbations. neville, m.; sardaryan, g.; ejaz, s.; scotto, a.w.; gupta, r. transave, inc., monmouth junction, nj, usa purpose: during the course of treatment, patients with cystic fibrosis and chronic pseudomonas aeruginosa infections in their lungs may be treated with nebulized tobramycin (tobi®). reduction in treatment times or dosing frequency may improve compliance and outcome. nebulized arikace™ may offer advantages to tobramycin by producing sustained lung levels of drug, reducing dosing frequency and increasing antimicrobial efficacy. herein are presented biodistribution data and efficacy data in a chronic rat model of p aeruginosa comparing arikace™, soluble amikacin and tobramycin. method: female rats (charles river) received (~6 mg / kg) of arikace™, soluble amikacin and tobramycin by inhalation. test articles were aerosolized in a pari lc star nebulizer at 30 psi using devilbiss compressors (sunrise medical) for 80 min. 3-4 rats were euthanized at time 0 and 6 hr post inhalation by co2 asphyxiation. blood was collected by a cardiac puncture. serum was collected from coagulated blood by centrifugation and stored at 4oc until analysis for drug content. in addition lungs, kidneys, intestinal contents, urine and brains were harvested. tissues and biological samples were homogenized in saline and antibiotic concentrations determined by tdx analyzer (abbott). chronic lung infections were produced in rats by intratracheal instillation of agar beads containing p. aeruginosa. three days post instillation, rats (n=12/group) were treated with aerosolized saline or once daily with arikace™, or twice daily with tobramycin (total daily dose = 6 mg /kg) for 14 days. rats were euthanized 3 days post final dose and the pseudomonas cfu counts per lung were determined. results: the biodistribution and pharmacokinetics of aerosolized arikace™ was significantly different from that of amikacin and tobramycin. the primary difference was the amount of antibiotic retained in the lungs. rats that inhaled soluble amikacin cleared approximately 84% of the amikacin from the lungs after 6 hr with concomitant 86% increase in drug levels in the urine. rats that inhaled arikace™ cleared only 41% of the deposited amikacin from the lungs after 6hrs. this initial clearance of drug most likely reflected the amikacin released during nebulization. extended clearance of arikace™ from the lungs was measured by recovery of amikacin in the urine at later time points. in a separate longer-term study comparing arikace™ and tobramycin, auc (0-168 hr)for arikace™ in the lung was 56233 ug*hr/g while only 1150 ug*hr/ g for tobramycin which demonstrates a 4-fold increase in retention of antibiotic in the lung with arikace™. eradication of p. aeruginosa from the lungs of rats with arikace™ was equal to tobramycin (given equivalent daily doses). however, arikace™ reduced the number of cfu in the lungs with a single daily exposure to the same degree as inhalation of tobramycin twice a day. conclusions: inhalation of arikace™ achieved higher concentrations and increased retention time of the antibiotic in the lungs of rats when compared to inhaled amikacin or tobramycin. it is expected that these properties of arikace™ will reduce dosing frequency and improve antimicrobial efficacy in patients. nielsen, x.c. 1 ; johansen, u.r. 1 ; nørregaard, l. 1 ; vandamme, p. 2 ; høiby, n. 1 1. clinical microbiology, rigshospitalet, copenhagen, denmark; 2. laboratory for microbiology, university of ghent, ghent, belgium background: the burhkolderia cepacia complex (bcc) is a diverse group of bacteria with at least 9 genomovars (gv) or species. accurate species identification is necessary for better understanding of the epidemiology and pathogenesis of bcc. species identification based on phenotypic characters is difficult when it is based on few if any differences. crossed immunoelectrophoresis (cie) has been performed routinely to identify all burkholderia and other difficult isolates in our department (høiby et al.: "taxonomic application of crossed immunoelectrophoresis." int. j. syst. bact. 37 (1987): 229-240.) . in this study, we compared the results obtained by cie with those obtained by the standard molecular biological method using reca gene pcr and rflp to identify bcc at the species level (golden standard). methods: between 1996 and 2005, burkholdreia isolates were recovered from 34 patients in the two danish cystic fibrosis (cf) centres. 1. cie method: immunological cross-reactivity between antigens from the reference strains and the strains isolated from our cf patients were compared by employing cie and rabbit standard-antibodies (purified igg) raised against water-soluble antigens from reference strains of the bcc. species identification was made based on matching coefficient (mc). 2. reca gene-based identification: bcc specific primers were used to amplify reca gene. species-specific pcr is then performed using b. multivorans specific primers to identify the b. multivorans gv ii. rflp patterns were generated for those non-multivorans bcc by digesting the bcc-specific pcr products with haeiii. the patterns were then compared with the patterns generated from reference strains in the database. pulsed field gel electrophoresis (pfge) genotyping was performed for all 34 strains to detect patient-topatient transmission. results: 32/34 isolates were bcc-specific pcr positive. species-specific pcr identified 29 isolates as b. multivorans (gv ii). the other three isolates were identified as b. cenocepacia (gv iii) based on rflp patterns. two isolates with negative bcc-specific pcr reactions were identified as b. gladioli by cie (confirmed by reference laboratory in belgium). results from cie showed that strains from bcc were immunologically closely related with only a few non-cross-reactive antigens. compared with the standard reca gene-based method, cie method resulted in correct identifications in 27 isolates (79.4 %), misidentifications in 4 isolates (11.8 %) and uncertain identifications in 3 isolates (8.8%). genotyping by pfge showed unique patterns for all isolates except for those from two sisters. this suggested that patient-to-patient transmission of bcc among the danish cf patients has remained very rare. conclusions: reca gene-based pcr and rflp is a quick and reliable method for identification of bcc at the species level. cie proved useful in initial classification of bcc strains, but additional information is obtained by the reca gene-based species identification and pfge method. background: in childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. in this setting, recurrent exposure to low numbers of pseudomonas aeruginosa occurs routinely. chronic infection by p. aeruginosa is associated with a significant increase in morbidity and mortality in cf, and is believed to occur by formation of a biofilm in the cf airway. previously, we have shown a significant neutrophil dependent enhancement of p. aeruginosa biofilm density that is greatest for low initial concentrations of p. aeruginosa. biofilm enhancement is facilitated via neutrophil-derived dna and f-actin, which form a framework that p. aeruginosa can exploit for growth. dna and factin polymerize via positively charged molecules such as histones. p. aeruginosa then associates with these polymers, which results in increased early biofilm density, displaying an increase of over 400 fold at 24 hours compared to p. aeruginosa in the absence of neutrophils. hypothesis: compounds that disperse dna and/or f-actin can disrupt neutrophil-enhanced biofilms. methods: poly(aspartic acid), a negatively charged amino acid chain with the capacity to disrupt f-actin, was examined singly and in combination with dnase and/or antibiotics (ciprofloxacin and tobramycin). the nunc-tsp system was used to allow for high throughput assessment of the density of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. the biofilm assay is independent of cellular settling, as the reactor is vertically suspended in the culture. p. aeruginosa strains tested were pao1, and two isogenic cf isolates recovered from an initial infection (early), and following established infection ~4 years later (late). biofilms were formed by incubation of p. aeruginosa with human neutrophils for 24 to 48 hours in rpmi with 2% heat inactivated platelet poor plasma. results: antibiotics were incapable of disrupting biofilms. poly(asp) significantly disrupted neutrophil-induced biofilms. in the presence of neutrophil products this action is sensitive to proteolytic degradation, but can be protected by the presence of protease inhibitors. dnase also disrupts a biofilm formed in the presence of human neutrophils. the combination of poly(asp) + dnase resulted in an increase in biofilm disruption for a 24 hour biofilm. biofilms allowed to form for 48 hours were more resistant to disruption by dnase and poly(asp) as single agents, but the combination of both demonstrated a synergistic effect. similar disruption of biofilms were observed when pao1 was compared to early and late cf strains. conclusions: pseudomonas aeruginosa biofilms formed in the presence of neutrophils in vitro can be disrupted by agents that disperse the f-actin and dna framework. support: cff, max and yetta karasik foundation cystic fibrosis (cf) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. the black yeast, exophiala dermatitidis (synonym wangiella dermatitidis) has been found with increasing frequency in sputum specimens of cf patients, with reported isolation rates ranging from between 1.1 and 15.7%. at present, no species-specific diagnostic pcr exists to aid with the clinical laboratory detection and identification of this organism. a novel species-specific pcr-based assay was developed for the detection of e. dermatitidis, based on employment of rdna operons and interspacer (its) regions between these rdna operons. two novel primers, (designated exdf & exdr) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of exophiala, as well as with other commonly described yeasts and filamentous fungi within cf sputum, including candida, aspergillus and scedosporium. pcr employing exdf (forward primer [16-,mer], 5'-ccg cct att cag gtc c-3') and exdr (reverse primer [16-mer], 5'-tct ctc cca ctc ccg c-3'), was employed and optimized on extracted genomic dna from a well characterized culture of e. dermatitidis, as well as with high quality genomic dna template from a further 16 unrelated fungi, including candida albicans, c. dubliniensis, c. parapsilosis, c. glabrata, scedosporium apiospermum, penicillium sp., aspergillus fumigatus, aspergillus versicolor, pichia guilliermondii, rhodotorula sp., trichosporon sp., aureobasidium pullulans, fusarium sp., mucor hiemalis, bionectria ochroleuca, gibberella pulicaris. results demonstrated that only dna from e. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. subsequent direct employment of this primer pair detected this yeast in the sputum of 2/50 (4%) adult cf patients, employing a nested pcr approach with panfungal outer flanking primers of the its1-its2 region. these two patients were the only patients who were previously shown to have a cultural history of e. dermititidis from their sputum. e. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan-and multiresistant bacterial pathogens from sputum, namely pseudomonas aeruginosa and burkholderia cepacia complex organisms, hence this species-specific pcr assay may help detect this organism from cf sputum more specifically and rapidly. overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of exophiala dermatitidis, as an emerging fungal agent in patients with cf. cystic fibrosis patients are particularly susceptible to infection by strains of pseudomonas aeruginosa and burkholderia cepacia complex. since 2001, the pseudomonas genome database has been a resource for peer-reviewed, continually-updated annotations for the pseudomonas aeruginosa pao1 reference strain's genome and, more recently, comparative analyses to several closely-related pseudomonas species. in order to facilitate better cross-strain and cross-species genome comparisons, we have developed or are incorporating methods to improve the identification of orthologs (genes diverged due to speciation) and identify genes undergoing unusual selection. our analysis of several completely sequenced pseudomonas genomes has revealed that approximately 5% of ortholog predictions by the classic "reciprocal best blast hit method" are likely incorrect. we have also performed an analysis of unusually large intergenic regions in p. aeruginosa pao1 that appear to be essential (according to saturation transposon mutagenesis) or involved in virulence (according to signature tagged mutagenesis). we identified at least 20 new putative protein-coding regions and non-coding rnas that were not previously annotated and may play critical roles in microbial pathogenesis or viability. we are also focusing our attention on facilitating better cross-strain comparisons between recently sequenced burkholderia cepacia complex genomes through development of a new online burkholderia genome database. in addition to improved ortholog prediction, this new database will provide access to a very flexible boolean search feature that allows researchers to search and compare annotations within or between the genomes of burkholderia strains, returning annotations from multiple genomes suitable for simultaneous viewing and downstream analyses. we have also included new, very accurate protein subcellular localization predictions for the deduced proteome from each of these genomes -predictions that can aid the identification of new cell surface or secreted therapeutic targets or vaccine candidates. further comparative analyses with other newly-sequenced, related strains should provide insight into strain-specific features that may be exploited to better understand virulence and antimicrobial resistance exhibited by cf-relevant pathogens. funding provided by cystic fibrosis foundation, therapeutics (usa). popova, a.p.; verma, r.; zanni, r.l.; sembrano, e. pediatrics, monmouth medical center, long brnach, nj, usa background: cystic fibrosis (cf) is a chronic, progressive, genetic disease leading to poor lung function, chronic bronchial infection with certain bacteria and recurrent pulmonary exacerbations. despite evidence of inflammation within the airway, it is unclear which circulating inflammatory biomarker best reflects the airway inflammation and lung function in cf. objective: to determine the utility of c-reactive protein (crp) value in evaluating patients with cystic fibrosis pulmonary exacerbation. design: a retrospective chart review was carried out on 87 patients followed at the cystic fibrosis center at the children's hospital. information about serum crp values, pulmonary function test results(pft), presence of pulmonary exacerbation as documented in the chart, sputum culture results, current medications, as well as demographic data, genetic compositions and weight percentiles between january 2005 and march 2007 was collected from the charts and was analyzed. results: of 87 patients whose charts were reviewed, 5 were excluded because they underwent lung transplant, 3 were excluded because they had other mild infection, not diagnosed as pulmonary exacerbation. for 31 patients crp was not measured and they were also excluded from the analysis. total of 48 patients were included in the study. for 9 patients information about crp was obtained on more than one occasion. the patients were divided into groups based on the presence of pulmonary exacerbation and elevation of crp values -in the group with pulmonary exacerbation, 10 patients had elevated crp and 23 had normal crp and in the nonexacerbation group 1 had elevated crp and 26 had normal. fisher's exact test (two -tail) was applied (p=0.043) and confirmed statistically significant elevation of crp in patients with pulmonary exacerbation of cystic fibrosis. the means and standard deviations of the crp values for the patients without pulmonary exacerbation and the patients with pulmonary exacerbation were calculated and analyzed using independent group t-test (two tail). mean crp value for the exacerbation group was 16.34 with standard deviation of 33.72 and mean crp value for the non-exacerbation group of 1.83 with a standard deviation of 2.43. statistically significant difference between the two means was noted (p=0.019). conclusion: serum crp values in patients with cf pulmonary exacerbation were significantly higher than in patients without pulmonary exacerbation. serum crp may be a useful marker of pulmonary exacerbation in children. in the absence of pulmonary exacerbation, cf is not associated with elevated crp values in children. the findings of this study could aid in identifying patients with pulmonary exacerbation earlier and initiate appropriate therapy sooner. further studies are needed to determine whether crp values are associated with the severity of the underlying disease, to what extent the pft are affected, the age of the patient, the type of bacterial pathogen, and genetic factors. background pulmonary infections with nontuberculous mycobacteria (ntm) are commonly associated with structural lung diseases or airways clearance disorders such as cystic fibrosis and primary ciliary dyskinesia. we follow approximately 100 individuals with ntm pulmonary disease who share common clinical features but have no identifiable systemic immune defect or clearly defined predisposing conditions. the pulmonary disease and clinical presentation suggest these individuals may have disorders of ion transport or ciliary function. while some cftr mutations and ciliary abnormalities are difficult to detect through standard genetic and ultrastructural analyses, in vivo physiologic tests of ion transport and measures of ciliary function may allow us to identify ion transport abnormalities in subjects with no identified cftr abnormalities, determine the functional significance of novel cftr mutations, and distinguish disorders of ciliary function from other airways clearance disorders. methods we evaluated the medical and family histories, chest and sinus ct's, sputum cultures, cftr genotypes, and sweat chloride levels of 15 individuals with nontuberculous mycobacterial pulmonary disease and 3 individuals with chronic airways disease and bronchiectasis. we systematically tested the pulmonary function (pft), nasal nitric oxide production (nno), and nasal potential difference (npd) of each subject. nasal mucosal scrape biopsies were performed and sent to unc-chapel hill for assessment of ciliary ultrastructure as part of the genetic diseases of mucociliary clearance consortium. pulmonary function, nno, and npd were measured on 8 healthy controls. results all subjects had sino-pulmonary symptoms consistent with those associated with cf and pcd. 5 subjects had low nno (27.2 ± 20.7 nl/min) consistent with levels reported in subjects with pcd (<100 nl/min, noone 2004). one subject with variant cf (∆f508/r347h) displayed low basal pd (-46.1 mv), elevated na+ absorption (34.2 mv), and no cftrmediated cl-conductance (-0.6 mv). 5 subjects with cftr mutations, 1 subject with isolated elevated sweat chloride (72 mmol/l), and 1 subject with isolated borderline sweat chloride (44 mmol/l) displayed normal basal potential difference (-12.4 ± 5.1 mv) and na+ absorption (8.0 ± 4.4 mv) but decreased cftr-mediated cl-conductance (-9.3 ± 5.5 mv) relative to controls (-15.0 ± 9.1 mv). conclusions ntm patients heterozygous for cftr-mutations or with elevated sweat chloride (and no identified mutations with coding region sequencing of cftr) may have detectable subtle differences in cftr clconductance, while measurement of nno may identify disease associated with ciliary abnormalities. these findings suggest subtle abnormalities in airways clearance may predispose to airway infection with environmental organisms such as ntm. support: intramural funds, national institute of allergy and infectious diseases muhlebach, m. 1 ; goodrich, j. 2 ; sutton-shields, t.n. 2 ; wedd, j.p. 3 ; henegar, c. 1 ; miller, m.b. 4 ; gilligan, p.h. 4 1. dept. of pediatrics, unc chapel hill, chapel hill, nc, usa; 2. university of north carolina hospitals, chapel hill, nc, usa; 3. school of public health, university of north carolina hospitals, chapel hill, nc, usa; 4. dept. pathology and laboratory medicine, university of north carolina hospitals, chapel hill, nc, usa introduction: proportion of methicillin resistant (mrsa) vs. susceptible sa infections increased from 36% in 1992 to 64% in 2003 in us hospitals. similarly, the prevalence of mrsa in the community has risen in recent years. these community acquired (ca) mrsa isolates are genotypically different, have different antibiotic susceptibility, and often carry the pvl virulence gene. this virulence gene is associated with more invasive, mostly skin infections in healthy individuals. reports on impact of mrsa in cf show conflicting data. 1 , 2 . the aim of this study was to determine the prevalence of ca-mrsa strains at our pediatric and adult cf center and to assess the impact of these infections on clinical outcome. methods: all mrsa isolates recovered from routine clinical cultures, were prospectively collected and analyzed for 18 months (10/05 -4/07). using molecular assays to determine the pvl status and to identify the sccmec (staphylococcal cassette chromosome) type, the organisms were classified as ca or ha. classification as ca mrsa was based on presence of pvl and sccmec iv type. clinical outcomes included lung function measurements in patients reliably performing spirometry and nutritional parameters (bmi%). patients who had undergone lung transplant were excluded as their lung functions vary depending on transplant outcome. results: of the 104 patients identified in this 19 month period, 83% had ha and 17% ca mrsa. neither current age (mean 18.1 yrs. in ha vs.14.6 years in ca) nor age at acquisition of mrsa (14.6 vs. 13 years) was significantly different. sixty-eight % of all mrsa infected patients had chronic p. aeruginosa infection, (70% in the ha and 51% in ca, relative risk 2.3 of having ha in presence of chronic p. aeruginosa infection). crosssectional comparison of patients infected with ca vs. ha acquired strains showed no difference in nutritional status (bmi 37 vs. 38%). lung function (fev1 and fef25-75) was not different in ca vs. ha patients, neither when including all mrsa infected patients or those who had no concomitant infection with other organisms (n=22). we will present further comparisons to age and gender matched children with ossa infection in the final report. conclusion: comparison to ossa in matched patients will assess clinical outcomes of infections with mrsa. initial data collected at our center did not show a significant difference in outcomes between patients infected with ca vs. ha mrsa. a multicenter study would most efficiently assess the impact on clinical outcomes and determine regional differences of mrsa patterns (ca vs. ha) in different cf centers. ref : the stringent response is a global regulatory mechanism used by bacteria to adapt to nutrient limitation and other environmental stresses, and is mediated by the signaling molecules (p)ppgpp (phosphorylated guanosine nucleotides). increases in (p)ppgpp repress metabolic processes and growth, and regulate genes involved in long-term stress and starvation survival in several species ((i)e. coli, m. tuberculosis(i)), bacterial multicellular behavior ((i)myxococcus xanthus(i)), and pathogenesis ((i)p. aeruginosa(i)). while many studies have investigated bacterial functions (such as attachment and motility) involved in biofilm development, less is known about the physiological adaptation occurring in biofilm growth. when cells grow to high densities entrapped in a polymer matrix, they must adapt to heterogeneous micro-environments where gradients in nutrients, oxygen and metabolic waste exist. nutrient may thus become depleted as they are consumed by overlying cells, and some biofilm subpopulations are likely starved. we hypothesized that the stringent response mediates adaptation to nutrient starvation in (i)p. aeruginosa(i) and is important for biofilm formation. we tested a (i)rela(i) deletion mutant unable to synthesize (p)ppgpp in response to amino acid starvation, and its isogenic wildtype parent pao1. we measured the starvation survival of planktonic bacteria in phosphate buffered saline and observed that the (i)rela(i) has a 10 to 100fold greater loss in viability compared to wild type after 48h. we also tested biofilm formation in a static biofilm assay using polystyrene peg lids and estimated biomass accumulation by crystal violet staining. in this system, the (i)rela(i) accumulates 5 times less biomass than wild type. in a flow-cell reactor system, the wildtype strain forms complex mushroom-like biofilm structures, but the (i)rela(i) mutant forms flat, thin biofilms. we determined cell attachment to polystyrene by crystal violet staining, and to glass surfaces by direct visualization under microscopy. the attachment to both surfaces is similar in the (i)rela(i) and wildtype strains, and the strains have identical planktonic growth rates. our findings suggest that the stringent response is involved in starvation survival, a physiological condition relevant to growth in biofilm. furthermore, (i)rela(i) inactivation impairs biofilm formation, suggesting that the stringent response may be an important stress adaptation in biofilm. belfast, united kingdom; 2. northern ireland public health laboratory, belfast city hospital, belfast, united kingdom; 3. northern ireland regional adult cf unit, belfast city hospital, belfast, united kingdom; 4. department of respiratory medicine, queen's university, belfast, united kingdom pseudomonas aeruginosa (pa) is a clinically significant bacterial pathogen responsible for increased morbidity and mortality in patients with cystic fibrosis. small non-coding micro(mi)rna species (generally 80~100nts or less) in prokaryotes are involved in numerous cellular processes as is the case with eukaryotes. as in eukaryotes, these mirnas act by base pairing with target mrnas imposing translational and stability changes of mrnas and thus culminating in the control and regulation of target mrnas, crucial for bacterial stress responses and virulence to changing environments surrounding the infected zone or host cell (e.g. human airway epithelial cells). we recruited a commercial kit (biodynamics dynaexpress mirna cloning kit av, tokyo, japan) and isolated mirnas from a variety of pseudomonas pathogens from plant, soil / agricultural environment (pseudomonas syringae -ps), type strain (pseudomonas aeruginosa -pa) and a collection of clinical isolates of pseudomonas aeruginosa (cfpa) from adult cf patients, who were chronically infected with p. aeruginosa. the results on the mirna clones obtained exhibited a wide variation in the occurrence of mirnas in this bacterium. the putative mirnas obtained, cfpa in particular, yielded small non-coding rnas of sizes from as little as 16nts to well over 45nts. an initial search using the bioinformatic tool srnapredict (www.waldorlab/tufts.edu) has not as yet, fully revealed the genomic annotations for these novel srna sequences in the inter-generic regions of the pathogenic pseudomonad group of bacteria. we present our data on these unknown cellular mirnas cloned from cfpa isolates from cf patients and discuss their significance as novel regulators to bacterial stress responses and in the context of the bacterium's involvement as the predominant pulmonary pathogen, associated with cystic fibrosis. airway function is diminished in infants with cf diagnosed clinically but whether this is true for those identified by newborn screening remains unclear. we investigated whether lung function is diminished in infants with cf diagnosed by newborn screening and if this occurs in association with pulmonary infection. methods:lung function was measured in asymptomatic infants with cf following sedation with chloral hydrate (60-100mg/kg) using the raised volume rapid thoracic compression technique at an inflation pressure of 30cmh 2 0. we measured forced vital capacity (fvc), forced expired volume after 0.5 seconds (fev 0.5 ) and forced expired flow at 75% expired fvc (fef 75 ) which were then expressed as z scores. 1 broncho-alveolar lavage (bal) was performed under general anesthesia within forty-eight hours following infant lung function testing. three separate aliquots of 1ml/kg sterile saline were instilled into the right middle lobe and a further aliquot in the left lingula and the bal fluid analyzed for bacterial, fungal and viral pathogens using routine culture techniques. results: bal was successful in all 24 infants studied aged 0.2 to 2.1 yrs (median 1.0 yrs) and lung function in 21 infants. median (range) z scores for fvc, fev 0.5 and fef 75 were -1.3 (-2.7 to 1.6), -2.0 (-5.0 to 0.6) and -1.7 (-3.5 to 0.47) respectively. these decrements in lung function were all significant (p<0.01). bal cultures were positive in 12 (50%) infants of whom 4 (17%) had evidence of infection with s. aureus and 1 (4%) with p. aeruginosa. in 12 infants there was no growth in bal or growth only of mixed oral flora. viruses were not identified in any infants. there was no association between any of the lung function parameters and pulmonary infection detected by bal. lung function was successful in a subgroup of 7 of 9 infants who were less than 0.5 yrs (median age 0.3 yrs) when tested. diminished airway function was identified in this younger subgroup even though only one infant had evidence of pulmonary infection with respiratory organisms in bal. in this younger subgroup median fev 0.5 z score was -1.6 which was significantly below that predicted (95% ci: -2.3,-0.2 and p=0.03). conclusions: airway function is diminished in infants with cf diagnosed by newborn screening and occurs irrespective of infection identified by bal. lung function is abnormal in very young infants before pulmonary symptoms develop and when no evidence of pulmonary infection can be detected in bal. 1 inhaled high-dose tobramycin appears to transiently clear p. aeruginosa (pa) from airway secretions in young children with cf, though inflammation may not be markedly reduced (am j resp crit care med 2003; 167:841) . because inhaled antibiotics may not reach some areas of infection, we hypothesized that intravenous (i.v.) antibiotics may be more effective than inhaled high-dose tobramycin for reducing lower airways inflammation in cf children with early pa infection. study design: we initiated a single-center, randomized, prospective study comparing the effects of two antibiotic treatment regimens on bronchoalveolar lavage fluid (balf) inflammatory markers. clinically stable cf children with a recent isolate of pa from surveillance cultures are randomized to receive 4 weeks of tobi® (group 1) or 2 weeks of i.v. ceftazidime and tobramycin (group 2). balf is obtained just before treatment, and repeated 4-6 weeks after completion of treatment. the primary study outcome is change in balf % neutrophils (pmn). multiple secondary outcomes are assessed including quantitative bacterial cultures and cytokines in balf. this report summarizes interim analysis at the halfway point of the study. results: to date, 18 subjects have enrolled. five subjects (3 in group 1, 2 in group 2) dropped out after the initial bronchoscopy due to a decision by the primary cf physician to use other treatments based on balf culture results (2) , or because of the development of a new respiratory illness before the second bronchoscopy (3). a total of 5 adverse events (1 significant, unrelated to protocol) were reported in 3 subjects, none resulting in a change of protocol request by the irb or data safety monitor. of the 13 subjects completing the protocol to date, 5 were in group 1, and 8 were in group 2. the groups were well matched in terms of age, pa in initial balf, and initial balf %pmn. a majority of subjects in each group were children with their first isolation of pa on surveillance cultures. there was a tendency for group 2, but not group 1, to have mild reductions in balf %pmn (-9 ± 6%) after treatment. interim efficacy analysis did not suggest a significant difference between the treatment groups at this stage. similar changes were seen in group 2 for pmn/ml (-589 ± 351 x 10 3 /ml) and several cytokines including gm-csf (-54 ± 27 pg/ml) and mcp-1 (-368 ± 231 pg/ml). two group 2 subjects were unable to have stable i.v. access established and were treated with oral ciprofloxacin and inhaled tobi® for 2 weeks as alternative systemic antibiotics, per protocol. dropping these subjects from group 2 data did not change data trends. conclusions: the protocol appears to have adequate safety. parents of children with first-time pa infection may be more likely to consent to treatment randomization, than those with repeated pa infection. interim data analysis suggests that continuation of the study toward the original sample estimate is appropriate. acknowledgements: supported by cff(noah04a0) and nih (gcrc r00046). we thank tracy callahan rn, lupe haynes rrt, justin hubbard, paula murphy, cassidy henegar, nancy lee rn, benjamin butler rn, and sheree berckmans rn for logistical and technical support. methods: our local cf database was searched for patients born 1985-2000. patients were included in the analysis if they were followed in our cf center before the age of 5. patients were separated into two birth cohorts, born 1985-1992 (cohort 1) and 1993-2000 (cohort 2). yearly peak fev1% predicted from ages 6-12 were obtained using knudson reference equations. yearly peak bmi% and weight-for-age% at each age were also obtained. type 3 tests of fixed effects and repeated measures analyses were used to assess mean peak bmi%, mean peak weight-for-age%, mean peak fev1% predicted at first pft, mean peak fev1% predicted from ages 6-12, and mean rate of change (slope analysis) in peak fev1% predicted from ages 6-12. chi-square test for association was used to analyze differences in demographic parameters. wilcoxon rank-sum test was used to assess differences in age at diagnosis. wilcoxon non-parametric test was used to analyze differences in mean clinic visits per year. chi-square testing with a two sided alpha of p <0.05 was used to determine significance. results: 144 cf patients were included in the analysis. there were no significant differences between birth cohorts 1 and 2 for age of diagnosis (p=0.94), sex (p=0.3792), race (p=0.35), genotype (p=0.16), pancreatic insufficiency (p=0.61), or the mean number of clinic visits per year from ages of 0-5 (p=0.33). there was a significant difference in the mean number of clinic visits per year from ages of 6-12 (p=.0009; see table 1) . birth cohort had a significant effect on the rate of decline in lung function from ages 6-12. birth cohort and age had significant effects on the mean weight-for-age%, mean fev1% predicted at first pft, and mean fev1% predicted from ages 6-12. sex had a trend for significant effects on lung function parameters. adjusted for age and sex, there were significant differences between birth cohorts 1 and 2 in mean bmi% and mean weight-for-age% at ages 6 and 12, mean fev1% predicted at first pft, mean fev1% predicted from ages 6-12, and in the mean rate of decline in fev1% predicted (see table 1 ). conclusions: improvements in lung function and nutritional outcomes in our cf center are associated with an increase in the mean number of clinical visits per year for cf patients ages 6-12. the frequency of clinical follow-up is a marker of cf care that may have positive impacts on cf outcomes. background: current management of cf airway disease includes the use of key pharmacological therapies designed to combat the chronic cycle of obstruction, infection, and inflammation. randomized controlled trials have demonstrated that maintenance therapy with azithromycin (azm), dornase alfa (dnase), and tobramycin for inhalation (tob) each improves pulmonary function and reduces the frequency of exacerbations in patients with p. aeruginosa. the purpose of this study was to determine if cf patients in an uncontrolled environment (clinical setting), experience a reduced rate of pulmonary function decline when receiving azm + dnase + tob compared to those not on all three therapies. methods: adult cf patients were selected based on positive sputum cultures for p. aeruginosa, pulmonary function test results (fev 1 % predicted) for the past year, and receiving one of the key pharmacological therapies. in addition data was collected on cfrd, depression, medication adherence, airway clearance, and nutritional status. patients were stratified according to baseline pulmonary function: mild (fev 1 ), moderate (fev 1 of 40-70%), and severe (fev 1 <40%). patients were then sub-grouped as to whether they were receiving all 3 or ≤ 2 of the key therapies. the annual rate of decline in % predicted fev 1 was determined using linear regression on non-exacerbation data and was categorized as stable (<2.3% decline), intermediate (2.3-4.1%) , or rapidly declining (> 4.1%). time to intermediate or rapid decline in pulmonary function was determined using cox proportional hazard modeling. results: 107 patients (54 males/53 females, median age 28 years) were included in this study; 28 with mild, 42 with moderate, and 35 with severe pulmonary disease. overall, the mild and moderate groups experienced median declines of 2.12% and 1.50%, while the severe group experienced a 2.41% improvement in fev 1 % predicted respectively. the use of regimens containing azm therapy significantly delayed time to intermediate or rapid decline in lung function in the mild group (median 210 days vs. 49.5 days, hr=0.19, p=0.013). similarly, regimens containing dnase + azm + tob significantly delayed time to intermediate or rapid decline in lung function in the mild group (median 231 days vs. 105 days, hr=0.27, p=0.048). no significant differences were seen in the moderate or severe group for patients receiving all 3 or individual therapies. con-clusion: administration of pharmacological regimens containing dnase + azm + tob, or azm + tob or azm + dnase significantly delay progression of pulmonary disease in cf patients with mild lung disease. sawhney, v. 1 ; arthur, b. 2 ; seltzer, r. 3 ; kraynack, n.c. 4 1. internal medicine, akron general medical center, akron, oh, usa; 2. pediatrics, akron children's hospital, akron, oh, usa; 3. biostatistics, neoucom, rootstown, oh, usa; 4. pulmonology, akron children's hospital, akron, oh, usa early and efficient diagnosis of pulmonary exacerbations (pex) in patients with cystic fibrosis (cf) is crucial in the management of cf. in oct 2004, we implemented a pulmonary exacerbation scoring (pes) system to uniformly and consistently identify pex in cf patients as part of a quality improvement project at the cf center at akron children's hospital. we have previously reported improved pulmonary function outcomes in pediatric patients at our center after implementation of the pes. we now describe the impact of the pes on clinical practice in the management of pex and pulmonary function in our adult cf patients. the pes was developed by our multidisciplinary cf team after an extensive literature review and was implemented in oct 2004. the pes has been previously described and consists of 13 clinical questions divided into three domains: systemic and pulmonary symptoms and signs, and objective measurements. these elements are scored individually and a cumulative pes of ≥ 5 (range 0-17) is considered a pex and treatment with antibiotics is recommended. the course and choice of antibiotic regimen is left to the physician's discretion. we examined median percent predicted fev 1 decline from quarterly percent predicted fev 1 measurements obtained from port cf. we calculated this for all the patients seen in our adult cf clinic (age>19) from oct 2004 till sep 2006 during which the pes was in use. we compared this rate of decline to median percent predicted fev 1 decline for the same cohort of patients over the preceding two years (oct 2002 till sep 2004 , during which the pes was not in use. the percent utilization of the pes was calculated individually for all adult cf care providers. the influence of individual components of pes in decision to treat was also studied. we found no significant difference between the rates of decline in percent predicted fev 1 during the pre-pes and post-pes periods (mean = -.25, sd = 1.58 vs mean = -.59, sd = 1.66, p=0.29) as measured by paired samples t-test. logistic regression analysis was done to evaluate the relative importance of each pes component in predicting decision to treat a pex. we found that changes in fev 1 (or=1.87), cough (or=2.64), sputum (or=3.28), and chest examination (or=1.51) are most likely to affect the decision to treat where as dyspnea (or=0.62) has the little effect on decision to treat. we found a high degree of correlation between adult provider use of the pes and an independent chart reviewer (correlation coefficient 0.66-0.87; p<0.001). in contrast to cf patients ages 6-18 years, we found no significant effect of use of the pes on pulmonary function in our adult population, despite the high frequency of use by our adult providers. it is unclear why this is the case given the robust effect of the pes on our pediatric population. this study suggests that a simplified score that includes fev 1 , cough, sputum, and chest exam may be useful in identifying a pex in adult cf patients. special thanks to tasha capozzi for her help with chart review. this project was supported by the akron children's hospital foundation. objective chronic pulmonary infection with pseudomonas aeruginosa is the main cause for morbidity and mortality in cystic fibrosis (cf) patients. tobramycin and colistin, widely used inhaled antibiotics in cf patients, were found to affect elastase activity in vitro. we showed recently that cxcr1 on neutrophils mediates bacterial killing, but is cleaved in cf airways by elastase. therefore, we examined the effects of inhalation with tobramycin and colistin on cxcr1 expression and antibacterial capacities by airway neutrophils in cf patients in vivo. methods cxcr1 expression was quantified by flow cytometry on neutrophils in peripheral blood and induced sputum of 25 cf patients without inhalation therapy, 25 cf patients with tobramycin and 25 cf patients with colistin inhalation therapy. the longitudinal effect of a 2 weeks tobramycin or colistin inhalation period on cxcr1 expression, free elastase levels and bacterial killing by airway neutrophils was assessed. results inhalation with tobramycin increased, while inhalation with colistin decreased cxcr1 expression on neutrophils in induced sputum of cf patients. neutrophils isolated from sputum of cf patients after tobramycin therapy had higher, whereas patients after colistin therapy had lower bacterial killing capacity and α-defensin release as compared to neutrophils before antibiotic inhalation therapy. conclusions tobramycin and colistin differentially modulate the pulmonary host defense in cf patients in vivo via cxcr1 on neutrophils. these findings may have clinical implications when considering antibiotic treatment in cf patients. sharp, j. 1 ; sheehan, d. 1 ; ren, c.l. 2 1. pediatrics, women & children's hospital, buffalo, ny, usa; 2. pediatrics, university of rochester, rochester, ny, usa background: airway infection and inflammation occur early in infants with cf. the raised volume/rapid thoracoabdominal (rv/rtc) technique is more sensitive than older infant pulmonary function test (ipft) methods in detecting airflow obstruction in these patients. however, there are limited data regarding lung function measured by rv/rtc in infants with cf nbs. we hypothesized that abnormalities of lung function measured by rv/rtc are present in cf nbs infants. to test this hypothesis, we reviewed the ipft data from cf nbs infants followed at our cf centers. methods: this study was a retrospective chart review. we identified infants with cf nbs followed at our cf centers who had had at least 1 ipft performed in the first year of life. ipfts were performed using the collins ipl. forced expiratory flows were measured using rv/rtc as described by feher et al (j appl physiol 80:2019 , 1996 and fractional lung volumes were measured by whole body plethysmography (bp) as described by castile, et al (ped pulm 30:215, 2000) . data from the cf infants were compared to normal data reported by jones, et al (ajrccm 161:353, 2000) for rv/rtc and castile, et al for bp. results: we reviewed data from 22 infants. their mean age was 9.7 m (range=4-24). pancreatic insufficiency was present in 20 (91%) infants, and 3 (12.5%) had been treated for pseudomonas infection. their pft findings are summarized in the table. fvc and fev0.5 were not significantly different from the normal predicted values. however, the mean fef75 and fef25-75 z scores were significantly below the mean of normal infants (p=0.02 and 0.03 respectively). gas trapping was present, as evidenced by significantly increased frc, rv, and rv/tlc (p<0.001 for all 3 measures). six infants (27%) had an fef75 z score of < -2.0, and 20 (91%) had an frc > 100% predicted. conclusions: infants with cf nbs have abnormal lung function early in life. our results suggest that pfts in cf nbs infants can be useful in detecting early changes in lung function. we speculate that early therapeutic interventions may improve lung function in this group of patients. table. pft findings in infants with cf nbs. forced expiratory flows are reported as z scores, while fractional lung volumes are reported as % predicted. objective: clinicians use pfts to assess effectiveness of therapy in hospitalized cf patients. however, the variability of multiple pft efforts has not been described in this group of patients. previous study of within day variation of pfts has been reported in healthy adults and children (enright, 2004, and nickerson, 1980) and clinically stable outpatients with cf (nickerson, 1980, and cooper, 1990) . nickerson and cooper found coefficient of variations (cv) for fvc of 1.9-6%, fev 1 of 2.4-5.3%, and fef 25-75 of 7.4-9.3% for adults and children with cf. no one has studied the within day variation of pfts in cf patients during pulmonary exacerbations. our objective was to determine the within day variation of pfts during hospitalizations for cf pulmonary exacerbations. we hypothesized that within day variation would improve during treatment. methods: we retrospectively reviewed pft data for all patients admitted to childrens' hospital and regional medical center for a cf pulmonary exacerbation in 2005. patients performed pfts under the direction of experienced pulmonary function technicians. the test was complete when the patient performed 3 efforts that met ats/ers criteria for acceptability. if the patient was unable to produce 3 efforts because of coughing, 2 efforts were accepted if the fvc and fev 1 were within 5% or 150 ml of each other. for analysis, we included only those pfts: (1) with at least 2-3 flow-volume curves that fulfilled ats/ers criteria, and (2) were performed within 3 days of hospital admission or discharge. for patients with multiple admissions, we used pft data for their first admission only. for patients with admission and discharge pfts available, we compared the coefficient of variation (cv) for pfts (fvc, fev 1 , ). there were at least 8 days between admission and discharge pfts. the wilcoxon rank-order test was used to compare the cv for admission and discharge pfts. data: sixty-one patients were admitted 114 times in 2005. excluding repeat hospital admissions, 17 patients had individual pft trials available at the time of admission and 13 patients at the time of discharge. seven admission and 2 discharge pfts included only 2 trials. three patients at admission, and 2 patients at discharge, were unable to produce pft curves that met ats/ers criteria, despite previously having done so when well. nine patients had paired admission and discharge pfts from the same hospitalization. the cv for fvc at discharge was significantly better than at admission, p = 0.02 (see table) . conclusions: within day variation of pfts during hospitalizations for cf pulmonary exacerbations is comparable to published outpatient data. cv for fvc improves significantly during exacerbation. changes in fvc > 4%, fev 1 > 3%, and fef 25-75 >10% can be considered a significant spirometric response to therapy in hospitalized cf patients. background: lung clearance index (lci), a measure of ventilation heterogeneity, can be calculated from multiple breath washouts of inert gas. in cross sectional studies it is a more sensitive measure of early airway dysfunction in cf than spirometry alone. in normal subjects, the technique is reproducible, with a narrow normal range of 5.9-7.5. we hypothesized that it may be helpful in detecting change longitudinally, and may have value in assessing novel therapeutic interventions such as the upcoming gene therapy by the uk cfgt consortium. methods: patients aged 10 years and over, presenting with an exacerbation requiring intravenous antibiotics, were recruited. lci was assessed in triplicate by multiple breath washout of 0.2% sulphur hexafluoride (sf 6 ), using a novel gas analyser (innocor, denmark) and custom-built software within the first 24 hours of starting treatment. the test was repeated at the end of parenteral antibiotic therapy and again 4-10 weeks later when clinically stable. patients were recruited at three sites and assessed using standardised equipment and a data analysis protocol. results: paired lci measurements were available in 33 patients, mean age 24 (range 11-44) years, 18 male. in a further 4 patients, technically acceptable washouts could not be obtained at one or other time point. mean (sd) lci improved from 14.7 (2.9) to 13.9 (2.4), p<0.005. in 11/33 patients there was an improvement in lci of >10% after antibiotics. 2 patients showed a >10% deterioration in lci. mean fev 1 was significantly greater after treatment (2.34 v 2.11 l/s, p<0.05, n=18). there was a weak correlation between percent change in fev 1 and lci (r 2 =0.33, p<0.05). 26 patients also had lci measured at the follow up. mean (sd) lci at this visit was 13.9 (2.5), p<0.05 vs visit 1, no significant difference vs visit 2. conclusions: lci can be used to demonstrate changes in the lung after antibiotic therapy. this is the first study in cf to demonstrate a lon-gitudinal improvement after intervention in a marker of ventilation heterogeneity, and offers different but complimentary information to spirometry. the higher sensitivity of lci may make possible measurement of improvement in mild patients after novel therapies such as gene therapy. this work was funded by the cystic fibrosis trust. aim: inhaled hypertonic saline positively affects sputum production in patients with cystic fibrosis (cf) by improving mucociliary clearance. this study was conducted to compare the aerosol delivery performance of various nebulizers upon aerosolization of hypertonic saline (hyper-sal™) 3.5% and 7%, available as preservative-free, sterile 4 ml solution in single dose, patient friendly blow-fill seal vials. method: 4 ml hypertonic saline (hyper-sal™) 7% was nebulized by three jet nebulizers the pari lc plus®, lc star® and hudson rci micro mist as well as the eflow® for hypertonic saline (hs) generating aerosols via a perforated vibrating membrane. hs 3.5% was characterized in the eflow®hs. droplet size distribution patterns were assessed by laser diffraction at a flow rate of 15 l/min and aerosol delivery performance by breath simulation tests mimicking a sinusoidal breathing pattern. the delivered dose (dd) was determined by gravimetric analysis of the inhalation filters. inhalation and exhalation filters were changed after 2 min and the drug delivery rate (ddr) was calculated as nacl found on inhalation filters per min. respirable drug delivery rate (rddr; ddr x respirable fraction <5 µm) indicates how much nacl can reach the lungs per minute. results: aerosol characteristics of the different nebulizers after aerosolization of 4 ml hs 7% are summarized in the table. data for hs 3.5% nebulized by the eflow®hs were as follows: dd= 61.1%, ddr= 9.3 %, nebulization time = 6.6 min and comparable to data for hs 7%. the lower recoveries for the jet nebulizers are probably associated with increased evaporation and longer nebulization times. conclusion: about 18 -59% of the charged sodium chloride from 4 ml hs was delivered and 57% -76% of these droplets were in a respirable size range <5 µm resulting in vitro respirable doses of about 10% -40% of the label claim. ddr and rddr are about 3-to 10-fold higher for eflow®hs indicating a much higher delivery efficiency compared to conventional jet nebulizers. further studies will be needed to demonstrate that about 2 ml hs may be sufficient to deliver a comparable dose to the lungs using an eflow®hs compared with jet nebulizers. this bears the potential to further reduce nebulization time and increase acceptance by patients. objectives: individuals with cf frequently have complicated and time consuming treatment regimens consisting of inhaled antibiotics, mucolytics, and airway clearance. patients and clinicians may have difficulty determining which specific therapies are effective for any individual patient or whether a particular treatment is causing adverse effects. this study assessed the utility of home spirometry for providing an objective measure of pulmonary status in between scheduled clinic visits. methods: consecutive adults with cf were recruited at a single academic medical center and were asked to measure fev1 twice daily for six months using the piko-1 meter (nspire health™, llc). 35 subjects agreed to participate and 24 completed the study. subjects were given diaries to record changes in respiratory symptoms and changes in medications during the study period. subjects were evaluated in clinic at least every three months and fev 1 values obtained in the pulmonary function lab were compared to home fev 1 from the same day using linear regression and bland-altman analyses. graphical displays of home fev 1 measures were visually inspected for changing trends and these were compared with medication changes. results: the mean fev 1 measured at home was 2.14±0.2l. the mean fev 1 in the pulmonary lab was 2.35±0.22l. the home fev 1 was significantly correlated with the clinic value from the same day, r 2 =0.94, p<0.001. fev 1 measures fell within the limits of agreement (±2 sd) by bland-altman plot. in 7 of 24 subjects (29.2%) there was at least one sustained change in fev 1 that correlated with either stopping or starting a particular treatment. the figure shows two significant changes in fev 1 in one patient related to medication changes. there is an increase in fev 1 at the initiation of aerosolized 7% saline and a fall in fev 1 that corresponded to inhaled tobramycin use. the improvement on hypertonic saline increased this patient's enthusiasm for this treatment. conclusions: home fev 1 monitoring is feasible and appears to provide accurate measurement of pulmonary function. it can provide clinicians with objective data to tailor treatments without necessitating a clinic visit. feedback from home monitoring may improve medication adherence and may allow treatments to be individually tailored based on patient responses to the medications. 1 1. respiratory unit, royal hospital for sick children, glasgow, united kingdom; 2. diagnostic imaging, nhs greater glasgow & clyde, glasgow, united kingdom; 3. medical statistics, royal hospital for sick children, glasgow, united kingdom; 4. medical physics department, gartnavel hospital, glasgow, united kingdom background: there have been calls for caution regarding the suggested use of regular chest computed tomography (ct) scans for monitoring disease progression in cf 1 . consideration of the associations of higher ionizing radiation exposure must be made before regular chest cts are performed on the wider cf population. aim: we tested the hypothesis that higher ionizing radiation exposure from chest radiographs (cxr) and chest cts would be associated with markers of severity including gender, genotype (class i/ii or iii-v), height sds, weight sds, bmi, fev 1 sds, length of hospital admission (days), scottish index of multiple deprivation (simd) 2 scores and pa/bc microbiological infection (chronic or intermittent colonisation with pseudomonas aeruginosa and/or burkholderia complex). methods: effective doses of ionizing radiation (msv) from cxrs and chest cts (including hr scans) were determined for all cf children at our centre between 1st january 2003 and 11th july 2006 (184 weeks). the most recent anthropometric and lung function data to july 2006 were used. simd scores report a data zone that encompasses ~750 individuals and comprises 31 indicators in six domains (current income, employment, health, education, housing and access). results: 134 cf patients (67 male:67 female) underwent 1853 radiation exposure procedures (mean of 3.9 procedures per patient per year). the average effective doses were calculated for 1806 of the 1853 procedures (97.5%) including 1591 cxrs and 35 chest cts. lung function data were available on 89 patients (66%). univariate analysis revealed that higher ionizing radiation exposure was significantly associated with fev 1 sds (p<0.001), length of stay (p<0.001) and pa/bc infection (p<0.001). the regression line indicates that for each drop of two fev 1 sds, each child receives an extra 3msv in radiation exposure from cxrs and chest cts alone. similarly, each child receives an extra 2msv in radiation exposure from cxrs and chest cts for every 100 days of hospital admission. multivariate analysis with backward selection demonstrated that length of hospital admission (p<0.001) and pa/bc infection (p=0.05) were associated with higher ionizing radiation exposure. discussion: in cf, higher ionizing radiation exposure from cxrs and chest cts in childhood are associated with poor lung function, pa/bc infection and length of hospital admission but not other indicators of severity such as gender, severe genotype, anthropometric measures or socio-economic status. using effective doses of ionizing radiation as an outcome marker in cf is an alternative means of assessing the impact of the disease and its management on the child. background: exhaled volatile compounds have attracted attention as potential non-invasive biomarkers of chronic lung disease. early sustained airway inflammation and bacterial infection are characteristic features of pulmonary involvement in cystic fibrosis (cf). the cellular response is dominated by neutrophils, which have been shown to generate oxygen-and halogen-derived radicals as part of their host defense armamentarium. aims: to gather pilot data on the pulmonary output or uptake of selected organic trace gases in cf patients and healthy controls; and to explore the impact of pulmonary exacerbations and antibiotic treatment. methods: samples of ambient air and exhaled breath were collected from 30 children, adolescents and young adults with cf and from 39 healthy controls. the patients' age ranged from 9-29 years and their fev 1 between 21-114 % predicted, 27 were pseudomonas positive. twelve patients were studied at the start of i.v. antibiotic treatment for a pulmonary exacerbation; repeated sampling after the course was performed in 7 subjects. all samples were analyzed on a customized gas chromatography system with flame ionization and electron capture detectors. after determining the mixing ratios of a variety of volatile organic compounds, alveolar gradients were calculated from the corresponding exhaled and ambient values. results: compared to healthy control subjects, cf patients showed a significantly lower respiratory output of methanol, ethanol, acetaldehyde and dimethylsulfide but a higher net release of benzene, methylchloride, trichloromethane and trichloroethene. these differences were most pronounced in patients with acute exacerbations. a relevant scatter and overlap between groups was noted for ethane, pentane, acetone, isoprene, toluene, and tetrachloromethane. after antibiotic treatment, the output of benzene, methylchloride and trichloromethane decreased by 40-250%. conclusion: although important pathways remain incompletely understood, the local biochemical milieu in cf lungs may be informative and accessible through non-invasive breath testing. in this context, the diagnostic potential of exhaled organic traces gases (especially oxygenated, halogenated and aromatic compounds) should be further explored. supported by: german research foundation (dfg ba 1895/3-2). toy, e. 2 ; weiner, j. 2 ; sacco, p. 1 ; duh, m.s. 2 1. health economics, novartis pharmaceuticals, east hanover, nj, usa; 2. analysis group, inc., boston, ma, usa objective: to review up-to-date economic outcomes data in patients with cystic fibrosis (cf), and in particular, costs related to respiratory infection by pseudomonas aeruginosa (pa), which is the leading cause of morbidity and mortality in cf patients. methods: a systematic search of the medline database from 1990-2007 was conducted to identify major and review articles that contained the terms "cystic fibrosis" and "cost" and which were published in english language journals. selected conference abstracts were searched, and additional articles were identified through bibliographies of retrieved articles. recent articles that contained economic data on antibiotic and mucolytic therapies were selected for in-depth review. cost estimates were converted to 2006 us dollars for comparability. results: approximately 300 articles fulfilled the initial search criteria, and 27 were selected for in-depth review. articles were divided into four categories: economic impact of inhaled tobramycin (2 articles), the effect of home-vs. hos-pital-based antibiotic therapies for pulmonary exacerbations (4 articles), economic impact of recombinant human deoxyribonuclease (rhdnase) (10 articles), and cost-of-illness for cf (11 articles). inhaled tobramycin (actual or recommended dose of 300 mg/5ml) has been associated with reductions in health care costs; these cost savings offset between 37% and 57% of the cost of this therapy. antibiotic therapy for pulmonary exacerbations generally resulted in lower health care costs when administered in a home setting rather than a hospital setting. use of rhdnase led to reduction in health care costs, with higher reductions observed in patients with higher levels of use; the cost savings offset between 17% and 38% of the drug cost. cost-of-illness studies have been conducted in 7 different countries; the economic estimates varied widely across studies due to differences in treatment patterns, health systems, methodologies, and patient populations. most cost-of-illness studies were retrospective observational studies from the perspective of a hospital or third-party payer, and the cost of cf ranged from $9,000 to $64,000 per patient per year. the largest cost categories included hospitalizations, out-patient visits, rhdnase and antibiotics, while disease severity and presence of pa infection were common determinants of cost. cost-of-illness studies have underestimated societal costs because they have rarely considered patient time costs and none have considered indirect costs (e.g., burden on lost productivity or informal caregivers). conclusions: studies show that inhaled tobramycin and rhdnase can partially offset medical costs; home-based iv antibiotic therapy is likely to reduce costs; and direct costs associated with cf can be high but vary widely across countries and analytical methodologies. areas for future research include direct comparisons of inhaled antibiotic therapies, examination of the relationship between treatment adherence and economic outcomes, and estimation of societal cost-of illness. this study was sponsored by novartis pharmaceuticals corporation, east hanover, nj. background: sputum interleukin 8 (il8) and myeloperoxidase (mpo) are advocated as markers for infective exacerbations in cf. we have previously shown that the neutrophil protein, calprotectin, is a sputum biomarker of disease activity in cross sectional and longitudinal studies. in the present study we evaluate longitudinal measurement of sputum and serum calprotectin during infective exacerbation. methods: sputum and venous blood samples were taken at the beginning (visit 1) and end of a course of iv antibiotics (visit2), and at time of clinical stability following the infective episode (visit 3). processed sputum supernatant was analysed by elisa for calprotectin, il8 and mpo. serum calprotectin was assayed by elisa. results: 36 paired sputum samples samples were available for visits 1 and 2 and 25 paired samples for visits 2 and 3. all data are presented as mean(sem). sputum calprotectin decreased from 1123(134) to 852(136) µg/ml, p<0.05, from visit 1 to 2. there was a non-significant increase to 1052(159) µg/ml at visit 3. sputum il8 did not change from visit 1 to 2 [13.3(1.5) 6(4.2) ]. serum calprotectin decreased from 51(9.5) to 16.5 (2.5) ng/ml, p<0.0001 (n=35 paired samples) from visit 1 to 2. at visit 3 there was an increase to 18(2) ng/ml, p<0.05 (n=28). discussion: calprotectin appears a valuable marker both in sputum and serum for tracking changes in lung inflammation during an exacerbation of cf and may be more sensitive to change than il8 or mpo. on stopping antibiotic therapy sputum and serum calprotectin increase implying that this marker may be sensitive to small sub-clinical changes in lung inflammation. these data suggest that sputum and serum calprotectin might be employed to assess responses to drug therapies in cf such as gene therapy. this research was funded by the cf trust uk. chronic lung disorders are usually associated with a hypoxia driven increase in red cell mass. however, patients with cystic fibrosis often have normal or decreased haemoglobin levels. the present prospective observational study in cystic fibrosis (cf) patients was performed to determine which factors were involved in alterations in the hematopoetic response to corresponding arterial oxygen pressure. sixty adult patients (age 21-51) with stable cf were included. they all had vitamin a, d, e and k but no vitamin b12 supplementation. 25 patients were on oral fe2+ (100mg/day). resting arterial blood gases, lung function, complete blood counts, parameters of iron status, crp, sputum microbiology and serum erythropoietin were measured at recruitment and after 3 and 6 months. patients had varying degrees of pulmonary functional impairment and 9% were hypoxemic (arterial oxygen pressure < 60 mm hg). low-grade systemic inflammation (crp > 0.5 mg/dl) was present in 40% of the patients, who all had bacterial colonization. none of the patient had erythrocytosis and 12 patients had anemia . there was no significant difference in iron status between patients with or without chronic iron supplementation and erythropoietin levels were normal. during the 6 months observation period no significant changes occurred. the patients exhibited an impaired erythropoietic response to hypoxemia with normal or low hematocrit in spite of chronic lung disease. linear multivariate regression analysis (table) revealed crp and iron levels but neither iron substitution, nor erythropoietin levels nor lung function parameters as independent determinant of haemoglobin levels. cf may be associated with anemia of variable severity as expression of the chronic inflammation present in these patients. the therapeutic consequences are to treat the underlying inflammation rather than to supplement iron. variables included in initial model: background improvement in the treatment and survival of females with cf has led to many leading a normal adult lifestyle, and some can now expect to raise a family. however, pregnancy can have a profound impact on wellbeing, and caring for a young child can affect compliance with cf treatment. to look at this further, we surveyed the experience of pregnancy and motherhood in cf women attending our large adult cf centre (230 patients). methods we reviewed the records of 10 cf women who chose to remain pregnant (12 babies [7 male] from 11 pregnancies), looking at changes in lung function, bmi, respiratory exacerbations during pregnancy, clinic attendance, and oral and iv antibiotic therapy (home and inpatient care) during pregnancy and also in the first year post delivery. we devised a questionnaire to examine how patients coped with pregnancy and the demands of motherhood, focussing on changes in compliance with therapy, worries regarding the effect of cf treatments on the baby, and support given by the cf team. results at conception mean fev1 was 82% predicted (range 61 to 107) and mean bmi 21 (range 18 to 26).there were no miscarriages or neonatal mortalities and all children remain well, but the sickest mother died at 4 years. mean fev1 following pregnancy was 73% [range 51 to 113] (p=0.01), with 1 mother losing 21% by one year post delivery. at one year, bmi was unchanged (mean 21, range 16 to 27). rates of respiratory exacerbations and iv antibiotics were similar pre and during pregnancy, although during the first year post delivery 2 mothers avoided hospital based treatment preferring home care. six mothers' clinic attendances increased during and post pregnancy (1 attended outpatients 17 times in the first year post delivery). nine mothers worried about the effects of cf medication on their unborn child: the remaining mother was in her 2nd pregnancy. all managed their health differently during pregnancy: whilst 6 increased compliance, worked harder to stay healthy, and increased clinic attendances; 4 avoided clinic, postponed treatment, avoided any medication in the first trimester, and reduced oral antibiotic use to prevent harm to their unborn child. following delivery, 7 mothers spent less time on their own health due to motherhood demands, and 8 missed treatments during the first year: 4 missed all except iv antibiotics. seven mothers ceased chest clearance and nebuliser therapy, and whilst 4 missed oral therapies only 1 avoided iv therapy. one mother who returned to work early felt this had contributed to her post natal depression. nine mothers indicated that cf team support was adequate, but the remaining mother felt that she should have been offered more frequent home visits post delivery due to lack of family support. conclusions the experience of pregnancy in our centre has positive outcomes for most mothers. however, compliance with treatment was affected and a number of mothers lost significant pulmonary function due to pregnancy. our study has shown that the demands of motherhood especially in the first year challenge the management of their own health. we offer a proactive service to women considering pregnancy, with early participation of the multidisciplinary team, and have increased home visit frequency to during the first year post-delivery. geller, d.e.; kesser, k.c. research, nemours children's clinic, orlando, fl, usa background: alpha-1 antiprotease (α-1ap) is being developed as an inhaled drug in cf to inactivate excess elastase in the lungs. it is estimated that tens of milligrams of α-1ap need to be deposited in the lungs to be effective. delivering a high dose with conventional jet nebulizers may be too time-consuming. we speculated that slowing and prolonging inspiration could optimize α-1ap delivery. the i-neb® adaptive aerosol delivery (aad® system, respironics) is a portable, electronic, battery-powered, mesh nebulizer that uses the aad algorithms to deliver drug in the first portion of inspiration. interchangeable mouthpieces allow the i-neb to be used in a conventional tidal breathing mode (tbm) or in the target inhalation mode (tim) that guides the patient to inhale very slowly. the low residual volume after nebulization reduces drug waste and optimizes efficiency. we compared delivery efficiency of α-1ap in vitro with the i-neb in the tbm vs. 2 tim modes. methods: we studied 3 new i-neb devices using tbm (tidal volume [vt] 0.5l, respiratory rate [rr]=15, i-time=2 seconds), tim-6 (vt 1.6l, rr=6, i-time=6 sec), and tim-9 (vt 1.6l, rr=4, i-time=9 sec) using a breath simulator. nebulizers were filled with 0.5 ml α-1ap (50 mg/ml). nebulized drug was captured on a filter and measured via spectrophotometer (inhaled dose), and particle size was measured with an insitec laser system. results: median particle sizes for the 3 modes were similar (4.4-4.8 µm) . the dose emitted from the i-neb was very high, and similar between modes (82-90% of nominal dose). predicted lung doses for these experiments were calculated from a previous scintigraphy study (nikander 2006 ) that showed 63% of the emitted dose deposited with tbm and 73% with tim. predicted lung dose of α-1ap (as % of nominal) and delivery times were tbm: 56.6% in 7.5 min, tim-6: 59.9% in 4.4 min, and tim-9: 64.5% in 2.5 min. discussion: the i-neb has a very high predicted lung dose with both tbm and tim. however, tim reduces the time of administration to as little as 1/3 that of tidal breathing. if larger predicted lung dose is necessary to see a clinical effect, either the initial drug volume or drug concentration may be increased. we conclude that slow, deep, controlled inspirations using i-neb is a very efficient method to deliver α-1ap, and is faster than tidal breathing. we also speculate that tim has the additional potential advantage of better distribution of drug in the lungs because of slower flow rate and particle velocity. objective: early inflammation was observed in infants with cf regardless the occurrence of bacterial infection. lung function (lf) is a tool to assess bronchial inflammation consequences. few data are available on the pronostic value of impaired lf in infancy. methods: in infants, airflow obstruction (t ptef/te , curve shapes, tidal volume, functional residual capacity (frc, nitrogen washout technique) were assessed with the sensor medics 2600 and interpreted with stocks reference values. at 6y, fev 1 and pulmonary flows (spiro 2000, nova medical sc, zapletal reference values) and frc he , were measured. symptomatic children were defined when cough, wheeze or rattle were recurrent. all symptomatic infants have been treated with inhaled steroids as any infantile wheezer. results: 30 screened infants (11 ∆f508/∆f508, 14∆f508/other, 5 other/other) were investigated at mean age 16mo and 6 years. 16 (54%) had respiratory symptoms (rs); the mean tidal volume was normal (9.5ml/kg), 19 (64%) had a marked obstructive curve shape, 9 (30%) a decreased t ptef/te , and 7 (23%) hyperinflation. then, 7 (23%) had normal lf, 16 (53%) ao, 3 (10%) hyperinflation and 4 (13%) both. at age 6, 50% had rs, and lf with mild alteration as mean flows ≥ 95%, and frc he = 97.7%. accurate analyses showed that 10 (33%) had normal lf, 9 (30%) ao and 11 (47%) small airway diseases (only distal ao and/or hyperinflation). frc in infancy and at age 6 were correlated (r=0.579; p=0.03). conversely, no correlation between ao parameters in infancy and at age 6 was found. the lf alteration was independent of the genotype, bacterial colonization and recurrent symptoms at both ages. conclusion: ao in infancy is frequent as found in half of the screened cf infants. the lack of correlation with the ao observed at age 6 suggested that ao in infants might be due to a non specific inflammation (i.e viral), transient and then relevant from a treatment. hyperinflation, although mild, is conversely an early marker of the cf disease as persistent between infancy and age 6. we describe the efficacy of a novel system using an electronic real-time distant home monitoring of symptoms and spirometry in cf patients with the aim of early detection of p exs patients and methods: this is a 6 month prospective observational study. all patients were provided with a set consisted of a mobile phone-palm pc (the x daii, o2-uk) and with a spirometer (vitalograph) with an attachment to the pc. the pc contained a purpose-built software on which cf symptoms and spirometric values were recorded once daily. these were then sent on a real time to a computer server where the data were plotted on a time-magnitude graph. the site was accessed by a password-permitted cf physicians. patients were asked to score four symptoms (cough, sputum, breathlessness and fatigue) once a day and to record at least three attempts of spirometry. these were accepted by the software only if they met the ats criteria. preset criteria for p exs were stipulated. when the recordings met the criteria for p exs patients were invited to come to the cf clinic for review and were managed by either watchful waiting, oral or iv antibiotics. a prodrome phase is deemed to be present when a patient had an increase in one or more symptom for one week prior to the p ex. twenty one evaluable patients were included in the study. in 19 (90%) there were an increase in one or more than one recorded symptoms throughout the study. a total of 50 p exs median 1.2 per patient were detected within the 6 months study period. in 21(42%) p exs, symptom increase and/or decline in fev1 met the preset criteria for p ex and in 29 cases patients presented despite that symptom changes were not sufficient to meet the pre-set definition of p ex. prodromal phase prior to p exs were seen in 36 (72%) patients. adherence and technical issues were the main two problems throughout the study. distant daily monitoring of patients symptom is easy and sensitive. it can be used to monitor symptoms and early detect p exs. the vast majority of our patients have an increase in their symptoms even when they perceive their disease to be stable. a prodormal phase preceded p ex is seen in the majority of patients. the pre-set criteria for defining p ex may have been too stringent. the research was funded by o2 telecommunication, uk the monitoring system used in the study. a combination of a mobile phone-palm computer (x daii, o2 uk) and a spirometer (vitalograph uk) .. the aim of this research was to examine whether systemic inflammation is present in adult patients with cf during their stable status. patients and methods: blood samples were obtained from a total of 17 cf patients 6 female, mean age 23, range(18-26)11 males mean 23.5 range15-36 and from 32 control subjects, 20 female, mean age 21.3, range(21-40)12males,mean age27.1yrs, range(21-33) all cf patients were included when they were at least 2 weeks away from any pulmonary exacerbations. all control subjects were not known to have any pulmonary or systemic disease. venous blood samples were taken and spun at3000 rpm.separated sera was aliquted and frozen at -70 degrees c. they were analysed in batches for 6 cytokines il-1b,il-6,il-8, il-10, ilp70,and tnf-alpa using a cba human inflammatory kit(bd bioscience). whitney-u test was used to compare values of serum cytokines between stable cf patients and control subjects.. serum il-6 and il-8 were raised in cf patients compared to control subjects. the mean value for il-6 was 8.2pg/ml (sd 2.0) for patients and 2.3pg/ml (sd 0.3) for controls, p<0.0001. similarly, the mean value of serum il8 was 29.6pg/ml (sd 6.7) in cf patients compared to 8.6pg/ml (sd 0.64) in the control group, p<0.0001 (figure below). there was no difference between cf patients and control with regards to serum il-1b, il-10 and il-12p70. conclusion: serum il-6 and il-8 were raised in cf patients compared to an agematched control subjects. inflammatory markers were raised in the sera of cf patients even during disease stability. even during disease stability, the lungs remain an inflamed organ which could explain the decline in lung function tests and the countinued daily systemic symptoms experienced by patients with cf. serum il6 and il8 in cf patients with stable disease compared to the control group (please see abstract body) we have shown that even during disease stability, an increase in inflammatory markers are present in the sera of cf patients compared to an agematched control group. this longitudinal study examines changes in serum inflammatory markers during p exs. we investigated 22 cf patients 6 female, mean age 23, range15-36 years. their mean baseline fev1 was 62.8% of predicted (range 27.7-99 %). a baseline venous blood sample was withdrawn and sera were stored at -70 degree c from 22 patients during disease stability. out of those there 17 experienced p exs. blood samples were obtained at the beginning and at the end of the p exs.the following inflammatory markers were analysed: white blood counts, c -reactive protein (crp), il-6, il-8, il-10, il-1b, il-12p70 and tnf-α. patients recorded their symptoms and their fev1 on a daily basis on an electronic diary and the data were beamed daily to a research centre where data were analysed. p ex was defined as 1). an increase in two symptoms for 3 successive days, or 2). an increase in 2 symptoms and 10 % reduced fev1 from baseline over 3 successive days or 3). 10% decline in fev1 over 3 successive days or 4).when a patient felt that he/she was going through a p ex. paired t tests were used to analyse changes in inflammatory markers. there was no change in the number of circulating neutrophils, lymphocytes or eosinophiles at the start compared with the end of p ex. there was an increase in mean crp values at the start of p ex and reduction at the end of p ex, but this did not attain statistical significance. mean (sd) of serum il6 increased from 8.8pg/ml (2.8) at baseline to 16.18pg/ml (4.0) at the start of p ex (p=0.007) and returned to base line 8.88pg/ml (2.5) at the end of p exs (p=0.012 compared to values at the start of p ex). (figure 1) there were no changes in the values of serum il-8, il-10, il-1b, il12p70 or tnf-α at the start and at the end of p exs. serum il6 increased at the start of p ex and returned at the baseline at the end of p ex. crp was the closest inflammatory marker to follow that pattern. none of the other inflammatory markers changed with the diagnosis of p ex's. this and other studies call for the price of analysis of il-6 to be reduced to become available in routine clinical practice. background. abpa can be a therapeutic challenge in children with cf. aim was to describe the tolerability of voriconazole and nebulised liposomal amphotericin in the treatment of abpa in children with cf. methods. we performed a retrospective case note review of voriconazole and nebulised liposomal amphotericin use, in the treatment of abpa in children with cf, at our centre. results. 7 children diagnosed with abpa according to the current criteria, failed to improve despite previous treatment with itraconazole and steroids, and received voriconazole as a second line agent. the dose of voriconazole was as recommended by the manufacturer's summary of product characteristics and the duration of treatment was two weeks. significant side effects noted in 4 children. observed side effects included visual hallucinations (n=1), photopsia (n=2), sleep disturbances (n=2), severe headache (n=2), influenza like symptoms (n=1), gastrointestinal side effects (n=1) and elevation of alkaline phosphatase (n=1). of these 4 children, one child tolerated voriconazole when concomitant treatment with omeprazole was withdrawn. a second course of voriconazole, given at a lower dose and without the loading dose was tolerated by two children. voriconazole blood levels were not measured in any patient. we used nebulised liposomal amphotericin in 4 of these 7 children. one child received amphotericin as a second line agent when voriconazole had to be stopped because of significant side effects. 3 children had active abpa despite treatment with first line (itraconazole and steroids) and second line (voriconazole) agents and received amphotericin as a third line agent. amphotericin was used at a starting dose of 2mg nebulised twice daily, with weekly increments as tolerated, up to a maximum dose of 15mg twice daily for four weeks. the starting dose and all subsequent increments in dose were supervised. all children received salbutamol immediately before nebulised amphotericin. the standard formulation available for intravenous infusion was made up into single doses for nebulisation by the pharmacy. this was stored at 4 0 c and diluted with water for injection for use with the nebuliser. the only side effect, experienced by all 4 children was a dry irritant cough at the start of nebulisation that settled soon without need for discontinuation of the medication. improvement in clinical symptoms, lung function and total serum ige was noted in all 7 children. the improvement in fev1 and fvc, expressed as mean (range) of percentage predicted were 18 and 20 respectively. the mean (range) fall in serum total ige and aspergillus specific ige were 1395ku/l (240-3299) and 11 mg/l (4-18) respectively. conclusion. voriconazole use was associated with significant side effects. avoidance of drugs that may potentially result in interaction is important. omitting a loading dose and using a lower maintenance dose may help selected patients tolerate voriconazole. nebulised liposomal amphotericin was tolerated by our children with cf and abpa who did not tolerate or responded poorly to voriconazole. recent studies have shown that high resolution computed tomography (hrct) is a more accurate method of evaluating early lung disease in cystic fibrosis (cf) than pulmonary function tests (pfts) or chest x-rays. since young children can not perform the breathing maneuvers required for high quality hrct, lung volume control is necessary. positive pressure facemask ventilation using chloral hydrate allows full inspiration and end exhalation images, but requires special expertise. controlled ventilation can also be produced with general anesthesia (ga). ga has not been reported in children undergoing hrct for cf research. the advantages of ga over sedation include rapid onset, faster recovery and reliability of scan completion. the undesirable effects of sedation are unpredictability and the higher failure rates. additionally, failed sedation contributed significantly to parental dissatisfaction. (malviya, et al., 2000) . the primary outcome of our study was to examine the safety of using ga for hrct scans and the quality of images obtained. a secondary outcome was parental satisfaction. methods: participants were recruited from a larger nih/cffti funded multi-site randomized clinical trial focusing on behavior and nutrition treatment to improve growth in preschoolers with cf. hrct in subjects 2 to 5 years old (mean age 38 ±10 months) was performed with ga. parents accompanied the child during induction and left after the child was asleep prior to the intravenous (iv) being placed. after induction of anesthesia, a proseal®, specialized laryngeal mask airway (lma), was placed which allowed ventilation with higher pressures and the ability to suction the stomach. volumetric thin section ct scans were obtained. ga using inhalation (sevoflurane) and/or iv (propofol) agents was used and 6 out of 7 families who were eligible (85% recruitment rate) participated in the sub-study. results: the quality of the hrct scans in all 6 subjects were optimal. there were no anesthetic complications upon emergence and none of the patients experienced any side effects after follow-up phone calls at 24 hours. parents also expressed that the clinical interpretation of the hrct scan provided beneficial information about their child's current lung disease status. the mean time from completion of scan to discharge was 38.8±15 minutes. conclusions: the use of ga in radiology departments is becoming more common in pediatric centers. our initial findings of this study demonstrate that ga can be safely administered for children with cf. in addition to very good quality scans being obtained, parental satisfaction was also noted. the use of the proseal® lma for hrct may improve the image quality and aid in the diagnosis of early lung disease in preschoolers with cf. supported by nih grant r01 dk054915 and a cf foundation clinical research grant. whey protein is rich in sulfhydryl (sh) groups and is recognized for its ability to increase glutathione and reduce oxidative stress. hyperbaric pressure treatment of whey protein increases its digestibility, promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects (zavorsky et al, int j vit nutr res, 2007) , and reduces in vitro production of il-8 (vilela et al, inter immnuopharmacol, 2006) . we present here the initial results of a pilot study of supplementation with pressurized whey in cf patients. children and adults with cf were supplemented with hyperbaric whey protein for 1 month (20 gm/day in patients less than 18 years of age, 40 gm/day in older patients). anthropometric measures, pulmonary function, serum c-reactive protein (crp) and plasma lipid peroxides were measured before and after supplementation. the results of the first 11 patients to have completed the study are reported, with further data to be presented. in 7/11 subjects weight increased and in 7/11 subjects lean body mass increased. there were no significant changes in fev1 or fev/fvc ratio. in 7/11 patients the crp fell (4/7 by more than 50%) and in 2/11 the crp increased. in the remaining two patients, the crp was undetectable at baseline and did not change at follow-up. plasma lipid peroxides decreased in the three patients in whom it could be measured (2/3 to undetectable levels). these preliminary results suggest that supplementation with pressurized whey protein can reduce systemic inflammation in cf patients and has the potential to beneficially modulate the inflammatory response in cf patients. supported by the breathe initiative of the canadian cf foundation with pseudomonas aeruginosa. soon after its introduction, the mesh technology based pari eflow® nebulizer gained preference over jet nebulization in the cf community because of its short nebulization time, portability and noiseless operation. concerns were raised about the durability of the device due to expected vulnerability of the mesh. the aim of this study was to compare the particle size distributions in the aerosol, delivered doses and output rates from the eflow and the pari lc plus®(lc+)with paritur-boboy compressor before and after use in daily practice. lc+ nebulizers in use by the adult patients taking part in the study were taken in for testing and new eflows, cleaning instructions and a disinfection device were provided. after 6 months use (3 cycles of 2x28 doses tsi: 300 mg/5 ml), the eflows were collected by the local cf center. particle size distributions were measured at a constant flow rate of 30 l/min with a laser diffraction apparatus. because of uncertainty regarding the cleaning procedure after last use, 8 additional e-flows were collected from the same patients after another period of 6 months use. conclusions: new eflows produce slightly larger droplets in a narrower size range than new lc+ nebulizers. after use in daily practice, droplet size distribution changes for both devices. output rate (delivered dose per minute) decreases for both devices. for used eflows delivered dose is lower than for used lc+ nebulizers, because the eflow is programmed to switch off after 10 min, even when nebulization is still incomplete (9 out of 21 tested eflows). this may result in a considerable reduction of delivered dose. however, when a mesh is replaced, performance is the same as that of a new device, suggesting that the changes in performance originate in the vibrating mesh. for daily practice these results indicate: (1) the performance of both nebulizers changes during use but the changes are smaller for eflow than for lc+ (2) changes in droplet size distribution are relatively small for both devices;in contrast with output rate and delivered dose (3) proper functioning of eflow can be improved by timely replacement of the mesh, ultimately when eflow switches off after 10 minutes. without replacement, the delivered dose is unpredictable. mean values for size distribution, delivered dose, nebulization time and output rate (x 50 is volume median diameter) backgrounds: lung surfactant protein a (sp-a) belongs to the family of collagen containing, c-type lectins, called collectins. sp-a plays a critical role in the innate immune defense of the lung. sp-a binds calcium dependent to carbohydrate structures on pathogens via its carbohydrate-binding domain (crd). cystic fibrosis (cf) lung disease is characterized by chronic inflammatory and proteolytic processes in the airways, leading to destruction of the lungs and early death. aims: we hypothesized that sp-a might have a reduced function in cf. this might result from sp-a degradation by neutrophil proteases, an altered macromolecular organization of sp-a, or inhibition of crd-binding by airway components. methods: we studied bronchoalveolar lavage fluid (balf) from 31 clinically stable patients with cf and from 13 children suffering from bronchitis. balf samples were purified by carbohydrate-binding assay on fucose attached to sepharose resin with and without calcium. in the absence of calcium sp-a cannot bind via its crd. each fraction was analysed for sp-a and igg by immunoblotting. macromolecular organization of sp-a was determined by gel chromatography. results: only 49 % of total sp-a of cf balf bound to the fucose column, whereas 92 % bound from the bronchitis balf. the structural organization of the sp-a oligomers did not differ between the non-binding and the binding fractions of both groups. 4 of the cf balf had sp-a at 27 kd that did not bind to fucose. these balfs had a high percentage of neutrophils (> 80%). in vitro human leucocyte elastase decreased sp-a to less than 25% of its initial levels and sp-a fragments at 27 kd were detected. conclusions: neutrophil proteases degrade sp-a in cf lungs. the carbohydrate dependent binding of sp-a is diminished due to a reduced function of the crd. these alterations contribute to the reduced ability in cf to remove specific pathogens from the lungs. chiron, r. 1,6 ; murris-espin, m. 2, 6 ; crampette, l. 3,6 ; varrin, m. 4 ; didier, a. 2, 6 ; wallaert, b. 5, 6 ; chanez, p. 1, 6 ; leroy, s. 5, 6 1. crcm, chu, montpellier, france; 2. crcm, chu, toulouse, france; 3. orl, chu, montpellier, france; 4. iurc, chu, montpellier, france; 5. crcm, chu, lille, france; montpellier, france in cystic fibrosis (cf) patients, upper airways involvement is frequent but its impact and relationships on the course and severity of the disease has been rarely investigated. objective: to relate upper airways involvement with lower airways parameters and quality of life in 91 cf patients. methods: clinical and radiological prospective study of subjects with cf at 3 french cf centres. clinical rhino-sinusitis (rs) was defined by the presence of more than 3 episodes of nasal symptoms during the last year, current nasal obstruction or rhinorrhea. nasal investigations included: anterior rhinoscopy, sinus ct scan analysis and rs quality of life questionnaire. lower airways were assessed using a thoracic ct scan (bhalla score), spirometry and sputum culture and a cf quality of life questionnaire (cfq14+). results: 91 (47f/44m) patients aged 25.4 (14-71) years old were enrolled. endoscopy demonstrated polyposis in 12% and nasal hyper secretions in 67.5% of the patients. spirometry, bhalla score, sputum colonization did not discriminate patients with (n=61) or without rs (n=30), and regardless the presence of polyps. cfq14+ was significantly lower in patients with of rs (p=0.04). conclusions: rs involvement altered quality of life in cf patients, although, they do not have a more severe pulmonary disease. a specific attention and treatment to rs should be paid in cf patients. the impact on different outcomes deserves to be examined in longitudinal studies. as part of the uk cystic fibrosis gene therapy consortium "tracking study", ebc was measured (ecoscreen, jaeger) in adult and paediatric patients presenting with an infective exacerbation requiring iv antibiotics. exacerbation was defined by pre-determined criteria. ebc was repeated at the end of parenteral antibiotic therapy and again 2-4 weeks later. patients were recruited at three sites and assessed using standardised equipment. samples were obtained according to ers guidelines. exhaled breath ph was assayed (shindengen ph meter, camlab, uk) immediately after collection without a de-aeration step. results: paired ebc measurements at the start and end of antibiotic therapy were available in 40 patients. mean age was 24yrs (range 11-44). data from 6 patients were excluded because at least one ph measurement was >7, suggesting salivary contamination. for the remaining 34 patients mean ebc (sd) ph increased from 5.9 (0.5) to 6.1 (0.3), p<0.01. of these patients, 28 also had measurements available at a third visit, 2-4 weeks after completion of antibiotics. 2 were excluded because of salivary contamination. no difference in mean(sd) ebc ph was found (6.1 (0.4) vs. 6.1 (0.4)) conclusions: ebc ph increases after treatment with antibiotics, and may offer a non-invasive means of assessing airways inflammation in chest exacerbations. further work is required to follow the longitudinal change in ebc ph and other inflammatory markers in clinically stable patients, as ebc may be a useful tool in assessing response to novel treatments, such as gene therapy. this work was funded by the cystic fibrosis trust. background: children with cystic fibrosis (cf) undergo multiple procedures exposing them to ionizing radiation in hospital. some specialist centres have advocated regular chest computed tomography (ct) as a means of better quantifying lung pathology. the clinical benefit of this approach is unproven and previous calculations of risk have failed to account for other ionizing radiation exposure procedures 1 . aim: to quantify the exposure of ionizing radiation in the pediatric cf population of a tertiary level children's hospital. to determine the potential difference in exposure of introducing biannual chest ct scans. methods: effective doses of ionizing radiation from chest (cxr) and abdominal (axr) radiographs, fluoroscopy and cts (sinuses and chest including hr scans) were determined for cf children at our centre between 1st january 2003 and 11th july 2006 (184 weeks). the maximal effect was tested i.e. introduction of biannual chest ct scans from age 2 years abolishing the requirement for cxrs and the assumption was made that axrs, sinus cts and fluoroscopy rates were unchanged (median exposure values calculated for age groups 0-<1year, 1-<2year, 2-<4year, 4-<8year, 8-<12year, >12year) . results: cf patients (n=134, gender ratio 1:1) underwent a mean of 13.8 ionizing radiation exposure procedures per patient over the study period (total of 1853 procedures). the average effective ionizing radiation doses (msv) were calculated for 1806 (97.5%) procedures. each cf child received a mean (median) effective radiation dose of 1.47 (0.04) msv/year with a range of 0.004 -25.76msv/year. the reason for the skewed distribution was identified by analysing the proportions of the procedures to the total effective dose in this population: fluoroscopy 83.2%, chest ct 7.1%, sinus ct 6.3%, cxrs 2.5%, axrs 0.7% and hrct 0.2%. it was calculated that these patients undergo 3.4 cxrs per patient per year. we calculated that an average patient diagnosed by newborn screening could expect to receive 1.24msv from ionizing radiation procedures between birth and age 16. biannual ct chest scans in addition to median exposures for each age group from axrs, sinus cts and fluoroscopy would potentially result in 14.34 msv by age 16, an 11 fold increase. discussion: there is a 1 in 100 lifetime risk of developing a solid or haematological cancer with each exposure to 100 msv of ionizing radiation 2 . although fluoroscopy forms the most significant contribution to a pediatric cf patient's ionizing radiation exposure in our centre, introducing biannual chest cts would markedly increase exposure. as cf survival improves, cf physicians must be cognisant of the potential health cost of survival. references : previous studies have used generic measures of hrqol, which are not sensitive to important changes that may occur as a result of treatment. this study assesses the impact of iv antibiotic treatment for a pulmonary exacerbation on both generic and disease-specific hrqol of children and adolescents with cf. method: participants included 45 patients (m age = 13.4 years) from the cincinnati children's hospital and university of florida cf centers. fiftyfour percent of the sample were male. patients completed two hrqol measures, the pedsql™ (generic) and cfq-r child or teen/adult version (cf-specific), which have excellent reliability and validity. these measures were completed within 48 hours of hospital admission (pre) and 48 hours of discontinuation of iv antibiotics (post). note that approximately 32% of patients went home on iv antibiotics and 68% remained in the hospital for approximately 2 weeks. results: significant improvements were found in pulmonary functioning (fev1 % predicted) pre to post iv antibiotic treatment (paired t (44) = -6.9, p < .0001), with an average of 14% increase in fev1 % predicted. paired t-tests were conducted to examine changes in hrqol scores, using holm's procedure to correct for multiple comparisons. significant improvements were found on the cfq-r respiratory (paired t (45) = -5.3, p < .0001) and weight scales (paired t (23) = -4.1, p < .0001). trends were also noted for improved emotional functioning (paired t (45) = -1.8, p = .08) and vitality (paired t (23) = -1.9, p = .07), and worse treatment burden (paired t (45) = 1.7, p = .09). on the pedsql, only emotional functioning improved significantly (paired t (44) = -2.6, p < .05). conclusions: results of this study confirm the effectiveness of iv antibiotics for the treatment of pulmonary exacerbations, with significant improvements found for both pulmonary functioning and hrqol. these results also highlighted the importance of using a disease-specific hrqol instrument, which proved to be more sensitive than the generic measure. on the cfq-r, significant improvements were observed in respiratory symptoms and weight, aspects of functioning that are not assessed on generic measures. fukushima, l.k.; hsu, e.; woo, m.s. division of pediatric pulmonology, childrens hospital los angeles, los angeles, ca, usa do hispanic and caucasian cf patients share the same frequency of pulmonary exacerbations? increased frequency of pulmonary exacerbations is linked to increased risk for cf morbidity and mortality. we have previously reported that pediatric hispanic cf patients have greater mortality than our caucasian cf patients who attend the same accredited cf center. we performed a retrospective review of cf pulmonary exacerbations (defined as physician prescription of intravenous or oral antibiotic therapy) in our cf patients who routinely attended the cf clinic during the period of january 1, 2005 through december 31, 2006. data collected included demographics, number of intravenous antibiotic courses and number of oral antibiotic courses. unpaired student's t-test was used to compare age of the groups and chi-square was used to compare use of intravenous and oral antibiotics between hispanic and caucasian cf patients as well as between males and females. during the study period, there was a total of 197 cf patients (106 males:91 females; mean age 10.8 ± 5.7 years). of the 197 patients, 82 (42%) were classified as hispanic (46 males:36 females; mean age 11.0 ± 5.3 years) and 99 (50%)were classified as caucasian (49 males:50 females; mean age 10.8 ± 6.2 years; p=0.42, ns). hispanic cf patients (51 patients had 299 episodes; 26 males:25 females) had significantly greater number of pulmonary exacerbations (received treatment with intravenous and/or oral antibiotics) than caucasian cf patients (35 patients had 182 episodes; p = 0.0006). hispanic cf patients who required intravenous antibiotic therapy were significantly younger (12.3 ± 5.1 years vs 15.0 ± 5.9 years; p<0.00001) than the caucasian cf patients who were treated with intravenous antibiotics. there was no significant difference in age between hispanic and caucasian cf patients who received oral antibiotic courses (11.2 ± 5.3 years vs 13.1 ± 6.4 years; p=0.08, ns). gender did not have a significant impact on pulmonary exacerbation risk in our population (hispanic iv use by gender p=0.82; hispanic oral use by gender p=0.12; caucasian iv use by gender p=0.38; caucasian oral use by gender p=0.37). we conclude that hispanic cf patients had increased incidence of pulmonary exacerbations than caucasian cf patients who attend the same cf clinic. hispanic patients who were treated with intravenous antibiotics were younger than the caucasian cf patients. we speculate that hispanic ethnicity has a major impact on the incidence of pulmonary exacerbations. other factors, such as modifier genes, environmental exposures, or inflammatory responses, may play a role in the increased risk of pulmonary exacerbations in the hispanic cf population. introduction: one of the main goals of the multidisciplinary team of the cf association of minas gerais, brazil is to find strategies to preserve lung tissue from the bacterial exacerbation in cystic fibrosis patients. objective: to investigate clues on onset of bacterial exacerbation. to our knowledge, no studies were found regarding the identification of the early warning signs of bacterial exacerbation with cf patients. we wanted to know whether cf patients present early warning signs before onset of fever and/or breathing difficulties. methods: telephone interviews were carried out with the 330 cf patients registered in the association, by a physiotherapist academic who did not know the patients and just ask two questions. the first question was about the use of antibiotics during the year of 2006 and the second was related to early signs before the onset of fever. results: from the 330 patients, only 207 patients replied. patients who were using antibiotics therapy during 2006 and had fever: 117/207 patients who were using antibiotics and did not have fever: 37/207 patients who had fever and did not use antibiotics: 9/207 patients who did not have fever and did not use antibiotics: 44/207 the results revealed the following warning signs and also that some patients had more than one sign. tiredness, quietness, slowness (51/117); increased cough (27/117); lack of appetite (19/117); sticky sputum (13/117); sleepiness (12/117); headache (14/117); increased excitement, nervousness (3/117); crybaby (11/117); dry mouth (1/117); clammy (1/117); difficult to walk (1/117); gastric upset (5/117); itchy throat (6/117); abdominal pain (7/117) ; itchy eyes (10/117); breathing difficulties (7/117); tremulous (3/117); paleness (3/117); burning ear (1/117); chest pain (1/117); hissing (1/117) ; and syncope(1/117). conclusions: it is helpful to know these early warning sings. we assume that the earlier the diagnosis of the exacerbation is made, the easier it is to resolve the problem and the less destruction of the lung tissues. reference: lung line, national jewish center for immunology and respiratory medicine 1988. background: cystic fibrosis patients are experiencing increasing survival and are being treated with more chronic therapies. how these changes are reflected in day to day clinical symptoms has not been evaluated. objective: we examined data from the epidemiologic study of cystic fibrosis, a large longitudinal observational study, to characterize the change in respiratory symptoms over time. for each year between 1995 and 2005 data from patients with at least one visit recorded as occurring while clinically stable were included. data from all visits, including sick as well as stable, were used for each year in which at least one clinically stable visit was recorded. patients were separated into age groups less than 6, 6 to 12, 13 to 17, and 18 or older. note that in 2003 data collection changed, potentially confounding the results. results: the average number of patients per year was 16393. the percent of patients with no reported cough progressively increased over time (table) . results were similar for pateints with no reported sputum production at clinically stable visits. when sick visits were added there were similar results. the frequency of cough reported at greater than 90 percent of visits progressively decreased over time (table) . again the results for sputum production were similar. these findings were seen in all age groups although older patients are more likely to experience these symptoms. conclusions: these data suggest that cf patients are experiencing fewer respiratory symptoms of cough and sputum production over the last decade. this improvement has occurred in patients with either intermittent or chronic symptoms. how clinical care has impacted on these symptoms has yet to be determined. also, more people are being diagnosed with cf at an earlier age or with milder disease, potentially resulting in the entire cf population having fewer symptoms. lung disease in children with cf begins in early childhood, with intermittent and then chronic infection, associated with an exaggerated inflammatory response. the extent to which inflammation contributes to bronchiectasis has not yet been identified. the aim of this study was examine relationships between inflammation and early lung damage methods: twenty eight children with cf were assessed at diagnosis and annually thereafter with a bronchoalveolar lavage (bal). bal fluid was used to assess micro-organisms and inflammation, including total and differential cell counts. a three slice hrct technique (inspiration and expiration) was performed under general anesthesia at age 3-6y, immediately preceding the annual bal. hrct scans were scored by an independent and experienced radiologist using a modification of the brody score. results. twelve children (43%) had evidence of bronchiectasis on hrct. inflammation present in bal at 12 months of age predicted bronchiectasis at 3-6 y. total cell count at 12 mo, predominantly neutrophils, was higher in those who developed bronchiectasis compared to those who did not (0.81 vs 0.43 x10 6 cells/ml bal; p=0.047). the total number of infections detected in bal was not related to bronchectasis. however, children with bronchiectasis had a higher incidence of pseudomonas aeruginosa within the first 3 years of life (50% vs. 25%). conclusions: bronchiectasis is evident within the first five years of life and appears to be associated with an exaggerated inflammatory response and the early acquisition of pseudomonas aeruginosa. supported by: cystic fibrosis foundation (usa), national health and medical research council (australia). fortunately the survival of patients with cystic fibrosis has improved remarkably over the last few decades. many patients can life quite "normal". therefore family planning becomes more important. several reports docu-mented the good maternal and fetal outcome in pregnant women with cystic fibrosis. besides the data from the french cf registry no longterm data about the impact of paternity of male patients with cystic fibrosis are available until now. in a retrospective analysis we gathered data about all our male patients who became father while being followed-up in our cystic fibrosis center for adults. from 43 male patients, 6 patients became father between 1999 and 2006. patient no. 1 became father twice (second time father of twins); patients no. 6 became also father of twins .we looked at fev1 % 1 year prior to childbirth, perinatal and 1 and 5 years after the childbirth. for the fev1% one year prior to birth as well as for fev1% 1 and 5 years after birth, we calculated the difference to the fev1% at time of birth. between 1999 and 2006, 7 male adult patients with cystic fibrosis became fathers of a total of 9 children, all by the assistance of reproductive techniques. their fev1% are shown in table 1. although the male patients are not directly influenced by a pregenancy (as the pregnant women are by growth of the uterus), there seems to be more than ususal differences in this lung function parameter. there were also more fluctuations in fev1 per year. the biggest differences in fev1% were seen 1 year after birth of their children. further investigations in a larger group (in cooperation with other centers) are planned; especially with a focus on the frequency of infections and quality of life compared to male patients without children. intravenous (i.v) aminoglycosides are widely used in cystic fibrosis (cf) patients as treatment for pulmonary exacerbations. in an effort to reduce undesiderable effects of these antibiotics it is recommended to measure drug serum levels. recent experience suggests that tobramycin (to) may be administered as a single daily dose with equal efficacy as multiple doses but less risk of nephrotoxicity. aim: we evaluated the efficacy of once-daily i.v to treatment to improve pulmonary function expressed as forced expiratory volume in one second (fev 1 ) in 15 cf patients with pulmonary exacerbations compared to the same group of patients who were previously treated with to in multiple daily i.v doses, as historic control. we evaluated baseline and peak drug serum levels of to administered once daily compared to multiple dose levels. methods: all patients were treated with i.v to in combination with a beta-lactam antibiotic for two weeks. respiratory exacerbation was defined according to cff criteria. all cf patients were colonized by non-fermentative gram-negative bacteria. serum concentrations of to were measured (fluorescent polarisation immunoassays) before the fifth infusion (basal) and 30 minutes after the fifth infusion (peak). reference to blood levels were considered to be ≤ 2 mg/l (basal) and <12 mg/l (peak) for multiple daily doses, and ≤ 1 mg/l (basal) and 20-30 mg/l (peak) for single daily dose. treatment efficacy was evaluated on the basis of improved clinical condition and increased fev 1 values compared to the beginning of therapy. results: 15 patients with respiratory exacerbation were evaluated, with a mean (± ds) age of 27.05 years (± 6.09; median 25.83, range 16.8-39.4) . 14 out of 15 patients were chronically colonized by pseudomonas aeruginosa. the total mean (± sd) daily dose of to administered in multiple doses was 410 (± 68.66) mg whereas the mean single dose was 406.6 (± 37.1) mg. of those patients given multiple doses of to, only one patient (6.6%) had basal to values outside the range and 4 patients (26.6%) had peak values outside the range. of those patients given a single daily dose, no patients had basal to values outside the range whereas 12 out of 15 (80%) had peak values of less than 20 mg/l and 3 patients out of 15 (20%) had peaks between 20 and 30 mg/l. the mean (± sd) increase in fev 1 was 5.6% (± 9.09) in patients treated with a single dose and 7% (± 9.41) in those treated with multiple doses. the mean (± sd) period between fev 1 measurements before and after exacerbation was 59.53 days (± 53.4, median 37, range 6-201) in case of monodose therapy, and 36.93 (± 24.79, median 29, range 17-105) in case of therapy with multiple doses. conclusions: both single and multiple-dose i.v to therapy caused a comparable fev 1 increase and were effective in resolving the respiratory exacerbation. although clinical improvement was observed, the mean i.v to dose of 400 mg/day in patients treated with once-daily i.v to determined peak values between 20 and 30 mg/l in 20% of subjects. intravenously administered aminoglycosides are efficacious for the treatment of pulmonary infections of cystic fibrosis (cf) patients, with the principal adverse effects of these molecules being acoustic nerve damage and nephropathy. damage to the eighth cranial nerve varies from 1.8 to 32% in cf patients treated with multiple daily doses of aminoglycosides. recent experience suggests that tobramycin can be administered as a single daily dose, resulting in less nephrotoxicity but equal efficacy. patients and methods: we evaluated the prevalence of oto-and nephrotoxicity due to aminoglycosides administered to cf patients followed at the tuscan regional center where we are currently following 192 patients. cochlear damage was evaluated using tonal audiometry (amplifon amplaid 171 s). renal damage was evaluated by measuring patients' creatinine clearance. results: 96 (50%) patients (52 males and 44 females) between the ages of 9 and 43 (mean age 22.5 years, median 21, sd 9) were tested with audiometry. all these patients had been repeatedly treated with multiple daily doses of intravenous aminoglycosides. 15 (7.8%) (5 males and 10 females) out of 192 patients between 3 and 45 years (mean age 24.8, median 24.2, sd 13.1) had their creatinine clearance measured. we found that 7 patients (7.2%) had an abnormal audiometry, typically attributable to aminoglycoside ototoxicity (high frequency deficit). two patients required a hearing aid. one patient with normal cochlear function had labyrinth deficit. one out of 15 patients tested had pathological creatinine clearance values. conclusions: our data indicate that 8 (8.3%) of our patients had damage to the eighth cranial nerve due to repeated multiple daily doses of aminoglycosides. we recommend that cf patients under aminoglycoside therapy be routinely given audiometric and creatinine clearance exams in an attempt to reduce the incidence of undesirable side effects of these drugs. davies, j. 1, 6 ; voase, n. 1, 6 ; dewar, m. 2,6 ; mullard, k. 1,6 ; gammie, f. 1, 6 ; saunders, c. 1, 6 ; horsley, a. 2, 6 ; gray, r. 2, 6 ; macleod, k. 3, 6 ; somerton, l. 1,6 ; higgins, t. 1,6 ; donovan, j. 1, 6 ; cornish, n. 1, 6 ; hansell, d. 4, 6 ; aziz, z. 4, 6 ; ashby, d. 5 ; geddes, d. 1, 6 ; greening, a. 2, 6 ; cunningham, s. 3,6 ; innes, a. 2,6 ; alton, e. 1, 6 1. gene therapy, imperial college, london, united kingdom; 2. western general hospital, edinburgh, united kingdom; 3. royal hospital for sick children, edinburgh, united kingdom; 4. royal brompton hospital, london, united kingdom; 5. queen mary, university of london, london, united kingdom; 6. uk cf gene therapy consortium, edinburgh, london, oxford, united kingdom the improving clinical status of patients with cf and the slow rate of decline of conventional outcome measures such as fev 1 necessitate the development of clinically-relevant surrogate outcome assays for use in clinical trials. in our forthcoming clinical trial of cftr gene therapy, the uk cf gene therapy consortium will use both established, clinically-available assays and more novel measures designed specifically for this purpose. both groups of assays are being subjected to rigorous testing prior to use to establish their utility as disease biomarkers. in this study, we examined their performance in the context of an infective exacerbation treated with iv antibiotics. this abstract will report the response of established, clinically-available assays; available data from novel assays will be reported separately. children (12 years and above) and adults with cf were recruited from three centres at the time of a clinician-defined infective exacerbation requiring iv antibiotic treatment. exclusion criteria included fev 1 <30% predicted and requirement for supplemental oxygen. a panel of assays was performed at the start and end of treatment, which was most commonly 14 days. data are presented as mean (sd) or median (range) and differences compared with either parametric or non-parametric paired analysis as appropriate. clinical assays included spirometry, sputum microbiology, sputum cell count and differential, serum inflammatory markers (crp, white blood cell (wbc) count,) and chest ct. patients also completed a symptom score sheet. to date, 40 patients (mean age 24.2 [7.5] years, 21 male) have paired data available from the start and end of the course of ivs. at baseline, 24 were infected with p. aeruginosa, 8 with b. cepacia complex organisms and 14 with s. aureus (2 mrsa). significant changes from baseline were observed in fev 1 (53.3[15.1] the clinical response to any novel intervention, for example, cftr gene therapy is difficult to predict. prior testing of experimental assays in a study such as this provides data on the variability of the measurements within the disease population and the degree of change observed with an intervention known to lead to clinical benefit. this should aid the design of rational, powered clinical trials. funded by the cf trust influenza can worsen cf and lead to serious complications and mortality. influenza vaccine is safe and effective in cf patients. in france, annual influenza vaccination is recommended for all cf patients aged over 6 months and for healthcare workers in regular and prolonged contact with high-risk patients. objective: to estimate the influenza vaccination coverage rate for 2005-2006 season in cf patients and healthcare workers in cf care centres of the great south region of france. methods: a multicentric observational study between february and september 2006 was conducted. healthcare workers were contacted by telephone and were questioned about their 2005-2006 influenza vaccination status, and the reason for not getting vaccinated. for patients aged over 6 months whose vaccination booklet was available, the physician filled in a written questionnaire. results: a total of 128 professionals were interviewed. the survey included 33.6% of doctors, 27.3% of nurses, 9.4% of dieticians, 9.4% of physiotherapists, 9.4% of social workers and 4.7% of psychologists. the overall influenza vc rate was 59.4% and varied considerably according to profession: 81.4% of doctors, 66.7% of physiotherapists, 58.3% of dieticians, 50% of psychologists, 48.6% of nurses and 16.7% of social workers. the overall influenza vc rate in cf patients was 80.6% (86.5% in children (age < 18 years old) versus 70.2% in adults; p<0.05). receiving a voucher for free vaccination from the national insurance increased the influenza vc rate. for healthcare workers, the main reason for non vaccination was that flu was considered as a benign disease (useless vaccine); for patients, it was a lack of time. conclusion methods: adult patients with cf were studied. patients completed a standardised questionnaire. the questionnaire identified which changes and duration of symptoms would cause them to contact the cf team and/or change treatment. results: all patients would contact the cf team for haemoptysis but 40% would wait for more than 24 hours and 20% would wait for more than 48 hours before doing so. up to 60% of patients would wait a week or more before contacting the cf team because of sputum purulence. 20% of patients would not contact the cf team if they developed new chest pain. conclusion: there appears to be a disconnect between recognizing a change in symptoms and the length of time before acting on the change. this has major implications for the delivery of cf care. supported by cystic fibrosis association of ireland. aim: to determine the efficacy of standardized desensitization protocols in treating antibiotic allergies in adults with cystic fibrosis (cf). background: dependence on high dose and multiple combination intravenous (iv) antibiotics to treat pulmonary exacerbations in adult cf patients has resulted in an increased frequency of allergic reactions when compared with the general population. (1, 2) strategies to address these allergies are essential to maintain effective antibiotic treatment in this population. antibiotic desensitization is the process by which one induces immune tolerance by incremental exposure to the antibiotic, which may induce stabilization of mast cells. the mechanism remains undetermined but may be mediated via ige or memory t-cells. prior to the study period, 9 standardized antibiotic desensitization protocols were developed by an allergist/immunologist based on published reports and were approved through the hospital's pharmacy and therapeutics committee. methods: the toronto cf database was accessed to generate a list of hospital admissions for iv antibiotics during the 5-year study period (1999) (2000) (2001) (2002) (2003) (2004) . patients who underwent desensitization were identified and each of their case notes underwent retrospective analysis. data were collected with respect to age, organism requiring antibiotic therapy, antibiotic allergy requiring desensitization, reactions during desensitization, reactions post desensitization, treatments required to treat these reactions, and the completeness of antibiotic therapy post desensitization. statistical analyses were performed using graph pad prism®. results: during the study period the cf unit dispensed and initiated approximately 600 courses of iv antibiotics. a total of 63 desensitizations were performed in 22 patients (17 female). although some patients were desensitized to only one drug or to the same drug more than once, there were 43 patient-drug combinations with a median of 2 (+/= 1.2) unique combinations per patient. of the 63 desensitizations protocols initiated, 7 patients developed documented reaction to the antibiotic during desensitization however only 1 desensitization protocol was not completed due to an adverse event. there were no episodes of anaphylaxis either during or after desensitization. a total of 9 patients developed documented reactions during the subsequent antibiotic course -5 required termination of the antibiotic and 4 completed the course of iv antibiotics with management of allergic type reactions with antihistamines. conclusions: standardized desensitization protocols were initiated in 22 patients during the study period. the success rate for desensitizations in these patients was 79% however when the data is analyzed for true allergic phenomenon, the success rate rises to 90%. this demonstrates the efficacy of these standardized protocols in the treatment of ige-mediated allergic reactions to antibiotics in the adult cf patient. references: 1. burrows, j., toon, m., bell, s., (2003) . antibiotic desensitization in adults with cystic fibrosis. respirology, 8: 359-364. 2. ramesh, s. (2002) . antibiotic hypersensitivity in patients with cf. clin rev allerg immun, 23: 123-139. surgery for symptomatic disease. there is a long-held suspicion that poorly-controlled chronic sinusitis negatively impacts the respiratory status of cf patients, but whether sinus surgery improves the clinical course of cf lung disease is unclear. aim: assess the impact of functional endoscopic sinus surgery (fess) on respiratory exacerbation rate and lung function in adult cf patients methods: this is a single-center, retrospective study based on chart review. during the study period, 1999-2006, 40 adult patients underwent 55 separate fess procedures. this abstract contains results so far from the analysis of 26 patients that underwent a total of 37 surgeries. primary outcomes were 1-year change pre to post fess in pulmonary function parameters and pulmonary exacerbation rate. results: at the time of surgery, the mean fev1 was 62% of predicted (ppd), the mean fvc 79.6 ppd, and the median age was 24.0 years (range 18.0 -46.0). 56.1% of patients were female. the median number of iv antibiotic courses for respiratory exacerbations in the 12 months prior to fess was 1 (range 0-5) compared to 2 (range 0-5) in the year after surgery (p = .04). further,there were fewer days on intravenous (iv) antibiotics for respiratory exacerbations in the 12 months prior to fess than in the 12month post-operative period (median 14 vs. 23 days), but this difference was not statistically significant (p = .09). the use of oral antibiotics for respiratory flares was comparable in the 12 months before and after fess (median 122 vs. 104 days, respectively, p = 0.53). the median number of hospital admissions and median number of days hospitalized for respiratory exacerbations did not differ in the 12 months pre vs. post-fess (p = 0.70 and p = 0.77, respectively). the mean fev1 and fvc in the 12 months pre and post surgery did not differ significantly (61.9 vs. 57.7 ppd, p = 0.13, and 77.4 vs. 76.0 ppd, p = 0.11). the linear rate of change in fev1 and fvc did not differ significantly in the 12 months prior to and following fess (-.01 l/month vs. +.01 l/month, p = .16 and -.01 l/month vs. +.02 l/month, p = 0.11, respectively). there were no significant differences (p > 0.10) for any of the above comparisons when the analysis was limited to 6 months before and after fess. there were no significant differences (p > 0.10) in antibiotic use and hospitalizations when the analysis was limited to the 29 surgeries in which patients were experiencing both respiratory and sinus symptoms. this latter group had lower fev1 and fvc ppd in the 12 month post-fess period (59.9 vs. 56.7 ppd, p = .001, and 75.4 vs. 72.9 ppd, p = .03, respectively). however, the rate of decline in fev1 and fvc in the 12 months before and after surgery did not differ significantly ( p = 0.17 and p = 0.18, respectively). conclusion: fess did not significantly affect the rate of respiratory exacerbations or rate of decline in pulmonary function in adults with cystic fibrosis. cumulative decline in lung function as measured by fev 1 in cystic fibrosis (cf) pulmonary exacerbation is a major cause of cf-related morbidity and mortality. high-dose extended interval aminoglycoside (hdei ag) use may be more effective in improving lung function than traditional multiple daily dosing in cf-related pulmonary exacerbation. intermittent dosing of beta-lactam antibiotics are often used with hdei ag in patients with cf pulmonary exacerbation, which does not optimize betalactam pharmacodynamics. the primary purpose of this study is to determine the effect of hdei ag plus continuous infusion beta-lactam (cibl) on patients' return to best baseline fev 1 from the previous 12 months. this study was conducted at university of kentucky healthcare. this was a concurrent observational trial with patients serving as their own historic controls via review of the medical record. all pediatric and adult patients with cf respiratory disease hospitalized with an acute pulmonary exacerbation between november 1, 2005 and may 1, 2007, with pseudomonas aeruginosa in their respiratory culture and were followed for at least one year were included. excluded were patients with known hearing disability or renal insufficiency or an inability to reliably perform spirometry (ie.less than six years old). patients served as their own historic controls via conventional treatment (control) versus hdei ag plus cibl (treatment). the primary endpoint was to determine the percent of patient courses that return to best baseline fev 1 in the last twelve months after treatment with hdei ag plus cibl. secondary endpoints included determination of patient courses that returned to mean baseline fev 1 in the last twelve months after study treatments, and description of beta-lactam dosing characteristics. thirteen patients were included in the pilot analysis which totaled 21 patient courses (mean 1.6 courses per patient). average patient age was 24 ± 11.1 years with a mean fev 1 of 42%; 61.5 % were female. after hdei ag plus cibl no patients showed a return to their best baseline fev 1 in the previous 12 months with the exception of one patient whose value was unchanged. average overall percent change in best baseline fev 1 group was -11.0 ± 7.8 at follow up. 62% of patient courses exhibited a return to their mean baseline fev 1 . average overall percent change in mean fev 1 baseline was 1.8 ± 6.8 at follow up. five of the 8 (62.5%) study patients who had serum beta-lactam concentrations obtained were able to achieve at least four times the mic of the target organism. more data will be collected on subsequent courses and follow up lung function. our preliminary data suggest that our patients lost 11% of their best fev 1 with an acute exacerbation and returned to 1% above mean baseline after treatment with hdei ag and cibl therapy. this evidence suggests that this might be an alternative to intermittent beta-lactam therapy. follow up data on remaining patients may provide sufficient evidence to validate this hypothesis. respiratory exacerbation in cystic fibrosis patients is characterized by increased sputum that may become more purulent. the detection of the exacerbation is based mainly on clinical subjective parameters. fev1 measurements are accepted as gold standard to be used for the life-time of the patient but do not adapt fast enough after resolution of an exacerbation. we compared parameters that are affected by the uneven distribution of ventilation in the lung due to increase sputum. methods: a zen body plethysmograph was used to measure tlc, fev1 and dlco (co/ch3 mixture). tlc was also calculated by the volume of ch3, the angle of the slope of phase iii was calculated in 20 cf patients at the beginning of the respiratory exacerbation and after 2 weeks of antibiotic treatment. patients were 17 -30 years old (12m/8f). results: fev1 changed by 9.8+7.41% at the end of the antibiotic treatment, the tlc(pleth) however was stable and changed by only 0.9+8.7%. the rv/tlc decreased by 23.8+ 23.4% and the tlc(gas) was increased by 5+36.6%. the difference between tlc (pleth) and tlc (gas) before and after the antibiotic treatment decreased from 24.7+13.6% to 10.2+7.1 %. conclusion: the changes in the parameters affected by the unevenness of distribution of ventilation (m/p reflecting the increased sputum in the airway) are more pronounced than fev1 and may be used as a more sensitive parameter for assessing the course of respiratory exacerbation. fibreoptic bronchoscopy was performed in 78 children aged 6-16 years: 27 with cf, 16 with non-cf bronchiectasis (bx), 24 with asthma, and 11 control children without lower respiratory disease. endobronchial biopsies were taken and stained with haematoxylin and eosin. asm content, myocyte number and size were quantified using stereology. results: the volume fraction of asm in subepithelial tissue (median; iqr) was 0.04 (0.02-0.05) in controls. by comparison, it was 0.12 (0.6-0.21, p<0.01) in cf, 0.16 (0.04-0.21, p<0.01) in bx, and 0.27 (0.12-0.49, p<0.0001) in asthma. myocyte number (cells per mm 2 of reticular basement membrane) was 1944 (1596-6318) in controls, 4504 (2838-8962, ns) in cf, 4971 (3476-10057, ns) in bx, and 8204 (5270-11749, p<0 .05) in asthma. myocyte size (um 3 , mean; sd) was 1948 (386) in controls, 3364 (890, p<0.01) in cf, 2857 (873, p<0.05) in bx, and 3249 (801, p<0.01) in asthma. in cf, the volume fraction of asm in subepithelial tissue was related to myocyte number (r=0.88, p=0.001), but not to myocyte size. conclusions: asm remodeling occurs in cf children. this, however, is not disease-specific and is also found in non-cf bronchiectasis and in asthma. both myocyte hypertrophy and hyperplasia contribute to increase in asm in cf. support: ers long-term fellowship and swiss national foundation grant to nr purpose: ct scans are increasingly being ordered on a routine basis in patients with cystic fibrosis. while prior studies have investigated their clinical utility in children, there is limited and conflicting information on their utility in adults. the purpose of this study was to determine the relationship between clinical disease severity as assessed by spirometry and ct findings as well as initial ct score ability to predict change in lung function in a nonselected group of adult cf patients. methods: a retrospective review of 45 cf patients (mean age 30.2 +/-8.5, male 62%) who had undergone a routine chest ct was performed. the ct scans were scored using the brody method by a blinded reader with the degree of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal disease, and air trapping assessed individually and a composite score generated. the ct metrics were first compared with spirometric test that were temporally related to the date of the ct scan, and then with future spirometric tests. results: for the cohort the patients had a wide range of severity of disease with the average fev1% 42.9 +/-21.9, and the average fvc% was 63.5 +/-20.7. no significant correlation was found between initial fev1% and total ct score (0.051 p value 0.739), or initial fvc% and total ct score (-0.082 p value 0.594). for ct metrics and future spirometry, the average time from ct to pft's was 671 days +/-528 with median of 484 days. predictors for a significant decline in pft's were a decreasing bmi and male sex (p-values 0.015 and 0.035 respectively). further, for patients with a low fev1% (<40% at baseline), the fvc% change over time was associated with total ct score, male gender, and bmi (p-values 0.006, 0.007, and 0.027 respectively) conclusions: in adult cf patients, ct scan findings did not correlate with lung function. however in longitudinal analysis, bmi and gender had an effect on fvc change. for patients with low lung function, total ct score did have an association with change in fvc along with bmi and gender. pulmonary exacerbations (pes) are episodes of worsening of respiratory symptoms that commonly occur in patients with cystic fibrosis (cf). in clinical trials of new therapies in cf, pulmonary exacerbation is widely used as a primary or secondary endpoint. despite its importance, no single clearly agreed upon definition exists. some are based on patient reported symptoms while others require hospitalization or antibiotic prescription. use of different definitions makes it difficult to compare the results across studies and also to plan future studies. moreover, the rate of pes (proportion of patients with at least one exacerbation over a given time-period) is affected by baseline patient characteristics such as age, fev 1 and medication use at the start of the clinical trial; factors which also contribute to the difficulty in comparing trials. using data from the cf therapeutics development network (tdn) data bank, we examined several pe definitions and baseline characteristics for their effect on pe rates. we find that pulmonary exacerbation rates vary substantially depending on both the pe definition and the baseline characteristics. for example, in one study, the proportion of subjects hospitalized and/or prescribed antibiotics during the 6 month treatment period was 29%, while the proportion who met a symptoms based criteria for pe during the same period was 65%. we also examined the implications of varying control group exacerbation rates on sample size requirements for clinical trials and find that the control group pe rate has a large impact. for illustration, we assume a two group study with two-sided significance level 0.05 and 80% power. if the control group rate is 30%, then approximately 200 subjects per group are required to detect a relative rate reduction of 40% (corresponding to a treatment group rate of 18%). however, if the control group rate is 65%, then only approximately 70 subjects per group are required to detect the same relative rate reduction of 40% (corresponding to a treatment group rate of 39%). thus, a pe definition that selects for a greater pe rate in the control group will require fewer study subjects to detect the same relative rate reduction given that all other variables remain equal. sample size requirements can be further reduced by analyzing the number of events or the time to first event instead of the proportion of subjects who have at least one pe during a given time period. supported by cystic fibrosis foundation therapeutics, inc. (ram-sey03y0), the national center for research resources (ncrr mo1-rr00037) and csl behring. briglia, h.; hilliard, j.; chmiel, j.f.; krenicky, j.; konstan, m.w. pediatrics, case western reserve university, cleveland, oh, usa cystic fibrosis (cf) is characterized by a vicious cycle of obstruction, infection and inflammation. the inflammatory response, which is profound and excessive relative to the burden of bacteria, is characterized by a massive neutrophil (pmn) influx. cf patients often experience intermittent declines in lung function associated with increases in both bacterial burden and pmn influx. these pulmonary exacerbations require treatment with antibiotics. there are substantive issues in the identification of pulmonary exacerbations and the assessment of therapies. a marker which indicates the inflammatory state of the lung would be useful to identify infective/inflammatory exacerbations, as opposed to worsening due to pulmonary vascular disease, or simply upper airway infection (cold), and might guide therapy for intensity and duration. g-csf and gro-α are cytokines produced in the lung by a variety of cells, including macrophages and epithelial cells. g-csf is involved in the proliferation, differentiation and activation of pmn precursors, while gro-α has known chemotactic and activating effects on pmns. transfer of these cytokines through the basolateral membrane into the bloodstream results in increased pmn production and activation in the bone marrow. the plasma concentrations of g-csf and gro-α might serve as potential biomarkers for a pulmonary exacerbation. in this pilot study we measured g-csf and gro-α in the plasma of healthy volunteers (n=3), clinically stable cf patients (n=23) and cf patients experiencing a pulmonary exacerbation, both before (n=14) and following (n=10) 10-14 days of antibiotic (abx) therapy. cytokines were measured using commercially available elisa kits (r&d systems, minneapolis, mn) . t-tests were performed on natural log transformed cytokine data (sigmastat v3.5, systat software inc., san jose, ca). ᭹ the lack of a significant difference between g-csf and gro-α plasma levels in healthy and clinically stable cf patients suggests that these cytokines are not indicators of chronic inflammation. ᭹ both cytokines are increased during pulmonary exacerbations in patients with cf and decrease to baseline values following antibiotic therapy, suggesting that these cytokines might serve as potential biomarkers for cf pulmonary exacerbations. ᭹ although not statistically significant, there was a trend towards an inverse correlation between both cytokines and fev 1 during pulmonary exacerbations in this small study (data not shown). ᭹ further studies are warranted to increase our understanding of g-csf and gro-α as potential biomarkers of pulmonary exacerbation in cystic fibrosis. this work was supported in part by the cystic fibrosis foundation through an institutional research development grant. pletcher, s.d. 1 ; koff, j.l. 2 ; kleinhenz, m. 2 1. otolaryngology, university of california, san francisco, san francisco, ca, usa; 2. medicine, university of california, san francisco, san francisco, ca, usa introduction: while sinus disease is common in adult patients with cystic fibrosis (cf), the severity of sinus symptoms and relationship of sinus disease to other manifestations of cf are relatively unknown. objectives: to evaluate the severity of sinus symptoms in adult patients with cf and correlate these findings with other cf manifestations. methods: twenty-six consecutive adult patients with cystic fibrosis were surveyed during routine clinic visits for sinus specific and overall quality of life outcomes using both the sino-nasal outcome test (snot-20) and the cystic fibrosis questionaire (cfqr). analysis of snot-20 and cfqr scores were compared to %fev1 and sputum culture growth of pseudomonas for all patients. results: snot-20 scores ranged from 0.1 to 3.1 with a mean of 1.17 on a 0-5 point scale. for the purpose of data analysis, the patients were divided into two 13 patient cohorts: group 1 with minimal sinus symptoms (snot-20 < 1.0) and group 2 with mild to moderate sinus symptoms (snot-20 > 1.0). the mean %fev1 for group 1 was significantly higher than the mean %fev1 for group 2 (71.5% vs. 49.9% respectively, p<0.007). patients in group 2 were more likely to have sputum culture growth of pseudomonas although this trend did not reach statistical significance (92% vs 62%, p=0.16). cfqr scores differed significantly among the two groups with group 2 patients reporting more disability in the physical, role, vitality, body, eating, health, respiratory, weight, and digestion domains but not in the emotion, social, and treatment domains. conclusions: this cohort of adult cf patients show minimal to moderate symptoms of sinonasal disability. those patients with mild to moderate sinonasal symptoms have decreased pulmonary function and decreased overall quality of life compared to cf patients with minimal sinus symptoms. 1, 2 1. internal medicine, university of iowa hospital and clinics, iowa city, ia, usa; 2. translational lung imaging program, university of iowa hospitals and clinics, iowa city, ia, usa; 3. pulmonary and critical care medicine, mount sinai medical center, miami beach, fl, usa mucociliary transport is an important component of airway host defense against inhaled particles and microbial pathogens. mucociliary clearance is hypothesized to be defective in cystic fibrosis allowing for airway colonization and infection. current methods for measuring mucociliary transport and clearance lack the ability to measure mucociliary transport in specific proximal and distal airway segments. using a 64-slice high resolution ct scanner (somatom 64, siemens), we are developing a method to track radiopaque particle movement in the swine airway. swine were induced with ketamine/acepromazine, intubated, and then anesthesia maintained with propofol infusions. animals spontaneously breathed humidified air with the endotracheal tube cuff deflated. radiopaque teflon/bismuth trioxide disks (1 mm diameter, 0.8 mm thick) were instilled via a catheter into the airways. serial ct images (0.6 mm slice thickness and slice increment of 0.5 mm) were obtained every 2 minutes for a total of 20 minutes. airway tree segmentation was performed using pulmonary workstation 1.1 (vida diagnostics, iowa city, ia). this program allows for fully automated airway tree segmentation. baseline mucociliary transport rates (2-dimensional) were 15.6 ± 2.6 mm/min. following aerosol delivery of human sputum leukocyte elastase (150 mu), mucociliary transport was markedly diminished (5.0 ± 1.1 mm/min) at 45 minutes. thus far, we are able to measure mucociliary transport down to the 3rd generation airways. future studies include more distal deposition and measurement of particle movement. in summary, this novel method measures mucociliary transport in defined airways and will allow us to examine the effect of altered airway epithelial function on mucociliary transport. lung transplant. purpose: to design a ct scoring system to quantify structural abnormalities on ct scans from cf patients with severe advanced lung disease (sald) and to investigate the correlation between ct scores and survival. materials and methods: ct scans of 57 cf patients screened for lung transplant between 1990 and 2005 were collected from 3 transplant centres. all scans were scored with the brody ii scoring system. to design a new scoring system sensitive for the specific changes in sald, a panel of 3 experts reviewed a random set of 10 ct scans on eligible items to be used in a pilot analysis. the resulting sald scoring system consisted of four items: bulla/cysts; areas with consolidation/mucous; areas with hypo perfusion/air trapping and hyper/normal perfusion. in each ct slice (10 mm spacing) and for each of the items the surface area of corresponding lung tissue involved was estimated on a 0-100% scale. total surface area for the 5 items and each slice added up to 100%. the final sald score was a mean volume estimate of abnormal and normal lung tissue. results: pilot analysis of a set of 25 ct scans showed a a spectrum of abnormalities, ranging from predominantly bronchiectasis to predominantly mosaic perfusion. a moderate correlation was found between the sald components inflammation and hypo perfusion/ air trapping and brody score (p=0.004, r=0.55 and p=0.006, r=0.536). the inter-observer variability of both scoring systems was comparable. (ri ranging from 0.70 to 0.83) analysis of the complete cohort with the brody ii score showed a significant correlation with survival with a hazard ratio of 2.4 (95% ci of 1.07 to 5.33) for dying on the waiting list for every ten point increase in brody ii score. (p=0.02). the brody component scores for bronchiectasis, airway wall thickening and parenchyma showed a similar correlation with hazard ratios (95% ci's) of respectively 1.9 (0.98-3.73), 1.9 (1.03-3.35) and 5.3 (1.44-19.2) for every ten point increase in score. (p=0.044, 0.028 and 0.01). the preliminary results with the brody score suggest that it is useful to include ct information in prediction models. it is likely that the predictive value of the ct can for survival can be further improved using the dedicated sald-ct score. scoring of the remaining scans according to the sald system is ongoing and will be completed shortly. snell, g. 2 1. physiotherapy, the alfred, melbourne, vic, australia; 2. allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; 3. epidemiology and preventative medicine, monash university, melbourne, vic, australia; 4. radiology, the alfred, melbourne, vic, australia background despite the widespread use of airway clearance (ac) techniques to clear excessive secretions and improve lung function, little is known about their efficacy following lung transplant (lt). this study aimed to compare the effects of two ac strategies (proactive vs reactive) on a range of clinical outcomes following lt. methods a prospective randomized trial design with stratification for suppurative pre-lt disease was used. consecutive patients at 1 month post lt were eligible for inclusion. subjects were excluded if medically unstable, ventilator dependent or had a contraindication to performing positive expiratory pressure (pep) therapy. patients performed ac using pep either twice daily (proactive strategy) or only in the presence chest infection (reactive strategy). lung function (fev 1 and fvc), chest radiography (brasfield score), exercise capacity (6 minute walk) and bronchoscopic airway characteristics (anastomotic healing, patency and secretions) were assessed at 1, 2 and 3 months post lt. patient adherence and satisfaction were measured. results of 60 consecutive patients, 36 (18 in each group) were recruited and completed the study. both groups improved lung function (fev 1 72 ± 4% to 81 ± 4 % p<0.0001; fvc 69 ± 3% to 81 ± 3% p<0.0001), brasfield scores (17.8 ± 0.5 to 19.8 ± 0.5 p<0.002) and 6 minute walk (451± 16m to 545 ± 16m p<0.0001) over the study period. no significant differences between groups for any outcome were found. the vast majority had fully healed, 100% patent anastomoses without secretions at 3 months. there were no significant differences between airway characteristics and incidence of chest infection. adherence to both strategies was high (84% proactive, 100% reactive). conclusion in the absence of significant differences in outcomes, it is recommended that ac only be performed in the presence of chest infection based on greater treatment cost and treatment time required by a proactive strategy. further studies in those with reduced anastomotic patency and in those with recurrent chest infections later post-transplant are warranted. supported by an alfred trusts grant. munro, p.e. 1 ; button, b. 1, 2 ; bailey, m. 3 ; holland, a. 1, 4 ; snell, g. 2 1. physiotherapy, the alfred, prahran, vic, australia; 2. allergy, immunology and respiratory medicine, the alfred, melbourne, vic, australia; 3. epidemiology and preventative medicine, monash university, melbourne, vic, australia; 4. school of physiotherapy, la trobe university, melbourne, vic, australia background the optimal duration and structure of pulmonary rehabilitation (pr) for lung transplant (lt) recipients is not known. this study aimed to describe changes in functional outcomes in lt recipients who participated in a post lt pr program at the alfred, melbourne, australia. methods prospective, repeated measures design. functional exercise capacity (six minute walk test-6mwt), lung function (fev 1 , fvc) and quality of life (sf 36) were assessed at 1, 2 and 3 months following lt. following discharge, all subjects attended a 1 hour supervised outpatient exercise training class 3 days per week until 12 weeks post lt and 6 education sessions. patients with post-operative complications were excluded. data were analysed using descriptive statistics and anova with repeated measures. results 60 consecutive lt recipients from sept 2003 to mar 2005 were assessed for inclusion. 36 (50% male) subjects, mean age 46 ± 14 yrs were recruited and completed the study. 81% had undergone bilateral lt. 33% had cystic fibrosis, 31% chronic obstructive pulmonary disease. significant improvements were demonstrated in 6mwt (451 ± 16m to 545 ± 16m, p<0.001), fev 1 (72 ± 4 % to 81 ± 4%, p<0.0001), fvc (74 ± 4% to 78 ± 4%, p<0.0001) and all quality of life domains, p<0.05. the greatest changes in 6mwt and lung function were seen between 1 and 2 months. conclusion functional exercise capacity, lung function and quality of life improved significantly over the first 3 months in lt recipients who participated in pr at our institution. these data will allow benchmarking with other centres and program structures. supported by an alfred trusts grant. does gender play a role in perception of cf quality of life domains before and after lung transplantation? the cf quality of life (cfq-r) for patients м14 years of age is a disease-specific instrument. previous studies using this instrument have reported that cf females have higher scores in weight and body perception than cf males. to determine the impact of lung transplantation on gender-based perception of quality of life, we reviewed the cfq-r on all cf patients м14 years of age and who underwent successful lung transplantation at our center. cfq-r was administered 1-3 months prior to transplant surgery and then 3 months after lung transplantation while the patients were in the outpatient clinic setting. domains measured: physical, role, vitality, emotion, social, body image, eating, treatment burden, health status, weight, respiratory symptoms, and digestion. the cfq-r responses were computer-scored and domain scores were then generated (score 0-100, 100=better). during the 2 1 ⁄2 year study period, 14 cf patients underwent successful lung transplantation (5 males:9 females; mean age 16.1 ± 2.5 years). prior to lung transplantation, female cf patients had lower physical domain scores but higher scores in health status perceptions than the male cf lung transplant candidates (see graph). all patients had marked improvement in all domains 3 months after lung transplantation (see graph). after transplant surgery, cf males had higher scores than cf females in emotion and in eating domains. scores for body image domain were similar for both genders before and 3-months after lung transplantation. we conclude that although there were differences in physical and health domain perceptions prior to lung transplantation, both male and female pediatric cf patients had marked improvement in all domains after lung transplantation. however, female cf lung transplant recipients had lower emotion and eating domain scores after transplantation. we speculate that lower scores in these domains may reflect increased post-traumatic stress and depression in female lung transplant recipients. further study on cf quality of life in pediatric lung transplant recipients is on-going. background cf liver disease (cfld) with severe cirrhosis develops in 5-10% of patients, usually within the first 2 decades of life. most patients with cfld suffer from complications of portal hypertension but hepatocellular function usually remains intact for many years, even decades. patients with cfld referred for lung transplantation (tx) may be offered lung tx, lung-liver tx or be excluded from lung tx. however, variceal hemorrhage due to portal hypertension can be managed by banding, sclerotherapy or shunt procedures without liver tx. the aim of this study was to examine outcome of cfld patients who underwent lung tx and the subsequent progression of cfld, particularly with the use of potentially hepatotoxic immunosuppressive drugs. we conducted a retrospective cohort study to compare cf patients undergoing lung tx with and without cfld. b. cepacia-negative cf patients undergoing lung tx at toronto general hospital from 1987-2007 were eligible for inclusion. liver cirrhosis was defined by histology, esophageal varices on endoscopy or imaging evidence of splenomegaly. each patient with cfld was matched for age and year of tx with 3 cf patients without cfld. demographic data, survival and liver function tests (lfts)(ast, alt, alp, inr, albumin, protein at week 1, month 1, 2, 3, 6, 9, 12, 24, 36, 48 and 60 post tx) were obtained by chart review. results of the 2 groups were compared using unpaired t-test (parametric data) or mann-whitney u test (non-parametric data). results 110 b. cepacia-negative cf patients underwent lung tx over this period, 6 (5.5%) having cfld with cirrhosis. data from the 6 cfld patients vs. 18 matched cf patients without cfld were analyzed. no significant difference was found between the groups in pre-tx age, gender, bmi, pi status, cfrd status, fev1% predicted, 6-minute walk distance or lfts. in the cfld group albumin was lower 1 week post-tx (18.2+1.7 g/l vs. 22.2+3.9 g/l, p<0.026), alp was higher 1 month post lung tx (389.5 vs. 137.9, p<0 .033) and alt was higher 60 months post-tx (26.7+8.3 u/l vs. 16.0+4.5 u/l, p<0.026). there was no significant difference in the other blood tests. azathioprine was changed to mycophenolate mofetil in 2 cfld patients due to liver test abnormalities. 1 cfld patient developed hepatic encephalopathy and ascites 4 years post lung tx and is being assessed for liver tx. no difference was seen in the number of episodes of acute rejection or post-tx survival (66.7% vs. 61.1% alive at 5 years). discussion cfld patients undergoing lung tx did not have a worse prognosis than patients without cfld. transient elevation of lfts were seen in the immediate post-tx period but settled either spontaneously or after discontinuation of azathioprine. we conclude that selected patients with cirrhosis due to cfld may be safely offered lung tx without concomitant liver tx. infectious mononucleosis, commonly called mononucleosis, or "mono," is an illness caused by the epstein-barr virus. mononucleosis has been nicknamed the "kissing disease" because the epstein-barr virus commonly is transmitted in saliva during kissing. however, sneezes and coughs also can transmit the virus occasionally. the epstein-barr virus (ebv) permanently infects more than 90% of the people on earth, but it causes symptomatic mononucleosis only in a small number. in developed nations, such as the united states, mononucleosis most often occurs between the ages of 15 and 25. unfortunately ebv can not only infect, but can also transform, b cells after transplant and may lead to lymphoma with a resulting ~50% mortality rate. the source of ebv is presumed to be passenger lymphocytes in the donor tissue and those at greatest risk are patients who are ebv naive before transplant. since most cf patients with endstage lung disease in the developed world are now being referred for transplant given the success of this intervention, we reviewed our entire lung transplant database to determine the nature of ebv infection and the likelihood of developing lymphoma following lung transplantation. of 272 first-time lung transplants, 178 (65%) had cf. ebv serology was not available in 27 (15 cf, 12 others) before transplant. 34 of 163 (21%) cf patients were ebv seronegative before transplant as compared to 4 of 82 (5%) patients with other end stage lung diseases (composed mostly of copd and ipf) in the control population (chi square p < 0.001). the cf ebv seronegative cohort was 24 (sd 9) years old on average, an age when most seroconversion has already occurred in our society. of the cf ebv seronegatives, 27 survived beyond 3 months and were thus at risk for post transplant lymphoma. of these 27, 8 developed lymphoma (incidence = 30%) in comparison to 1 of the 4 ebv seronegative controls (incidence = 25%, p = ns). all lymphomas were stage iv at presentation and the majority (9 of 11) arose in the first post transplant year. the cf ebv seronegatives who developed lymphoma did not differ from those who did not with regard to levels of immunosuppression (cyclosporine, methylprednisolone or prednisone) or doses of antiviral therapy (i.e., ganciclovir which is active against both cmv and ebv). in addition, the cessation of anti-lymphocyte antibody induction therapy in the summer of 1995, somewhat surprisingly, had no impact on the development of lymphoma. two cases of lymphoma developed in cf ebv seropositives (incidence = 1.2%) and no cases of lymphoma developed in the control ebv seropositives (p = ns). thus ebv seronegativity raises the lymphoma risk 25 fold in cf patients (p < 0.001). two of the 11 (18%) cases of lymphoma resulted in death (one cf and one other) and one cf patient probably died of complications related to chemotherapy even though he was tumor free at that time. lymphoma outcome was not stage or clonality dependent. in conclusion, cf lung transplant recipients are at an increased risk for lymphoma largely due to their lack of exposure to ebv before transplant. kissing more before transplant may lower the risk of lymphoma afterward. university hospital uzb, brussels, belgium; 2. chest medicine, erasme university hospital, brussels, belgium; 3. dermatology, erasme university hospital, brussels, belgium; 4. gynaecology, erasme university hospital, brussels, belgium; 5. gastroenterology, erasme university hospital, brussels, belgium; 6. pathology, erasme university hospital, brussels, belgium hpv infection is an underestimated phenomenon in organ transplantation (tx) recipients, being unable during immunosuppression (is) to mount an adequate anti-viral immune response and risking genital/anal warts (condylomata acuminata) as well as pre-cancerous/cancerous lesions. we retrospectively assessed the incidence/treatment of genital/anal hpv-associated lesions in cystic fibrosis (cf) lung tx recipients. the files of all 74 tx patients transplanted in the ulb center between 1988 and 01/2007 (38 men/36 women, median age 24±7.5, range 8-45 yrs) were reviewed. maintenance is consisted in chronic triple therapy combining a calcineurin inhibitor, a cell cycle inhibitor and corticosteroids. median survival was 46.5 ± 44.4 months (range 0-186). ten of 60 sexually active patients (17%) who survived ≥12 months post-tx developed hpv-associated genital/anal lesions: 3 men (35-41 yrs) and 7 women (25-41 yrs). genital/anal hpv-pcr proven condylomata were diagnosed between 62-97 months in the men and between 14-90 months post-tx in the women. all 10 underwent local treatment (cryotherapy ± laser ± topical imiquimod), 3/10 patients underwent multiple treatments under general anesthesia. recurrence of the condylomata was high. one male patient presented high-grade anal dysplasia and 3 women moderate to high-grade cervical dysplasia; 2 underwent conization and one complete hysterectomy after 2 conizations. one of the females presented both condylomata and a high grade cervical dysplasia. these retrospective data in a cf population indicate that 1) hpv infection may cause significant morbidity in young subjects with chronic disease needing organ tx. it may even compromise life expectancy after tx. 2) data on the effectiveness of treatment strategies -including topical immunomodulators -have to be collected 3) the potentially protective effects of the now available hpv vaccination in females and males? with chronic disease at risk for future organ tx, when administrated before sexual activity and before tx, should be rapidly evaluated in a multi-center effort. ballmann, m. 1 ; pfister, e. 2 ; schlueter, k. 1 ; becker, t. 3 ; melter, m. 2 ; junge, s. 1 1. pediatric pulmonology and neonatology, medical school, hannover, germany; 2. pediatric nephrology and hepatology, medical school, hannover, germany; 3. visceral and transplant surgury, medical school, hannover, germany liver disease is an important comortality and comorbidity in cf patients even in the pediatric age group. liver transplantation (ltx) is an accepted option in end stage liver disease. nevertheless the clinical outcome in cf patients is still under discussion. here we reported the clinical outcome 12 months after isolated liver tx in pediatric cf-patients.since 2000 n=9 ltx were performed in children (male n=7, female n=2; age (mean±sd) 14,1±3 years) . immunosuppressive therapy was done with ciclosporin ± basilixumab and steroid. we followed pulmonary function, cn-score, nutritional status (bmi z-score) and body com-position, airway cultures and igg.results:all were still alive 12 months after ltx. before ltx all patients had a mild to moderate pulmonary disease: fvc(%pred) 92,2±16 (71,4%-119), fev1(%pred) 88,7±16,1 (68-103), mef25(%pred) 61, 7± 28,5 (35-114) , cn-score 8±4,3 points. nutritional status: bmi(mean) 17,2±1,8% (range: 15,3%-20,3%, z-score: 0,88±0,86), upper arm circumference(cm)(mean±sd) 17,8±0,9, triceps skin fat fold(cm) 5,2±1,2. body composition: 13,5±7,2% of bodyweight were fat. one year after ltx all patients were still in a stable pulmonary situation: fvc(%pred) 93,9±14 (range:75,5%-114%), fev1(%pred) 88,6±12,9 (range:68%-108%), mef25(%pred) 61,6±30,4 (range:24,7%-106%), cn-score 7,5±4,8 points. there weren't any significant changes in the airway microbiology under immunosuppressive therapy (steroid, ciclosporin±basiliximab), the serum igg levels declined significant from 17,4±3,9g/l to 9,8±2,3/l(p<0.05). the growth over the year was 6,9±5 cm and the increase of weight come to 5,1±6,1kg, while the bmi didn't increase in this first year after transplantation. certainly we found an increase of the body fat mass (to 18,2±3,8% of body weight), of the upper arm circumference (to 20±4,2cm) und of the triceps skin fat fold (to 11,4± 8,1cm) .conclusion:liver transplantation is an effective therapy even in children with cf related liver disease and stabilise pulmonary function and improves nutritional status in patients with cf and mild or moderate pulmonary involvement before ltx. (neglia jp et al., n engl j med 1995; 332:494-499) , and colon cancer has been described in young adults with cf (chaun h et al., can j gastroenterol 1996; 10:440-442) . three of 62 lung transplant (ltx) recipients from our center developed colon cancer after successful bilateral lung transplant for end-stage lung disease. data from these 3 individuals are given in table below . an additional 8 recipients with cystic fibrosis were screened via colonoscopy. colonic polyps were detected in 2 and included lesions up to 3 cm in diameter. five years after a colonoscopy that had shown a 4 mm polyp, one patient underwent a 2nd colonoscopy that revealed multiple polyps, including a 3 cm diameter sessile polyp in the sigmoid colon. colonic malignancies appear to arise from mucosal polyps, and screening via colonoscopy can detect and remove premalignant lesions. lung transplant recipients have an increased risk of gi malignancies and tumor surveillance is impaired by post-transplant immunosuppression, colonoscopic screening should be considered for transplant recipients with cystic fibrosis. additionally, colonic malignancy should be considered as a potential cause of unexplained gastrointestinal symptoms such as constipation. nossent, g. 1 ; kastelijn, e. 1 ; teding van berkhout, f. 1 ; zanen, p. 1 ; van den bosch, j. 2 ; van de graaf, e. 1 1. respiratory dis, university medical centre, utrecht, netherlands; 2. respiratory diseases, st. antonius hospital, nieuwegein, netherlands objective: in the netherlands lung allocation is based on waiting time. to prioritize patients with a poor prognosis and to reduce waiting list mortality the high urgency (hu) status was introduced in 2001. to be considered for hu status there has to be a limited life expectancy on clinical judgement and all three transplantation centers have to audit this decision. the aim of this study is to analyze the potential effect of las on patients with cystic fibrosis (cf) who became hu or died on the waiting list. it would be expected that patients who became hu had a higher las. methods: from 2001 till 2007 in all cf patients placed on the hu waiting list, the median las (25th, 75th) was calculated at the moment of listing and at the moment the patients became hu. also for the cf patients who died on the waiting list the mean las (sd) was calculated at the moment of listing. results: from the 187 patients on the waiting list 52 patients had cf. of these patient 19 were placed on hu list and 4 died on the waiting list. in the cf patients that became hu the median las on the moment of listing was 35. 1 (33.9;35.9) . at the moment they were placed on the hu waiting list the median las was 35. 5 (34.8;37.4)(p= 0.2) . the median las of the 4 patients who died on the waitinglist was 35.3 (2.77) . this las did not differ significantly from the las of listing of the patients that became hu (p= 0.2). conclusion: the las does not detect the deterioration in patients with cf as diagnosed by clinical judgement. this may be due to the exclusion of specific prognostic factors of cf in the calculation of the las. besides that, the las does not differentiate cf patients on the waiting list with a high chance of mortality. introduction: lutx recipients have one of the highest rates of ia in solid organ transplantation causing high morbidity and mortality rates. nowadays antifungal prophylaxis is extensively used but clinical trials are rare. methods: all patients who received lutx in our hospital were included in this retrospective study. aspergillus airway colonization was defined as aspergillus cultured twice from airway specimens in the absence of ia or tracheobronchitis (tb). definition of ia and tb was according to literature. after the death of 2 cf-patients due to ia, a prophylaxis protocol was introduced containing avoidance of use of the cell saver during the operation in pretx colonized patients to avoid hematological fungal spreading and targeted prophylaxis with itraconazole or voriconazole in all pre-and posttx colonized patients during 3 months and inhaled amphotericin during hospital admission. results: a total of 93 lutx were performed (in 92 patients) (35% cf patients) from 15-7-2001 until 31-12-2006. 23 patients (24.5%) were colonized with aspergillus pretx and 16 patients posttx. only 4 patients were colonized before and after tx. before introduction of the protocol 4 patients (4/69=5.7%) (50% cf patients) developed an aspergillus infection: 3 (4.3%) patients developed a. fumigatus tb and 1 (1.4%) patient suffered from ia: cerebral a. fumigatus infection (proven ia). two patients, both cf patients, (2/4=50%) died due toaspergillus infection (cerebral and tb).after introduction of the protocol only one a. fumigatus tb occurred (1/24= 4.1%) but no invasive fungal infection. conclusions: the rate of fungal infections in our lutx patients was comparable with data from literature. since introduction of azole prophylaxis protocol and avoidance of use of cellsaver no ia occurred. fischer, a. 1 ; müllinger, b. 2 ; arendt, t. 1 ; bernhardt, t. 3 ; hannah, k. 4 ; scheuch, g. 1 1. activaero gmbh, gemuenden, germany; 2. biological products, bayer healthcare, leverkusen, germany; 3. activaero gmbh, gauting, germany; 4. talecris biotherapeutics, research triangle park, nc, usa background patient compliance during a study is an important factor in view of assessing the clinical effect of a treatment. this is especially true when patients administer the drug at home. usually, patient records, counting of returned doses or mechanical counters are used to track compliance, which may be biased by the study subjects. this report is about an aerosol study in cf which used a device for controlled breathing (akita® inhalation system, activaero, germany). the device works with a patient-individual smart card that records every single breath during a treatment including a date/time stamp in an encrypted manner. each patient's inhalation protocol can be displayed by loading the smart card into the proprietary "compliance manager" software. methods for this report, the data set of a recent controlled study (griese et al., 2007) was analyzed. 72 cf-patients were instructed to inhale with the akita inhalation system at home for 42 days. after the study, the smart cards were returned to activaero, and the inhalation records on the chip were analysed. we analysed the compliance of patients who participated at least 21 days (59 out of 72 patients, others deemed to be drop-outs . most of the patients showed a dsc which is lower than the tdc, indicating that they had missing treatment days, which were compensated by additional inhalations on other days. we found 9 patients with a dsc more than 10% lower than their tdc (max difference: 23.81%). conclusion this aerosol study with home treatment demonstrated a high compliance rate for tdc and dsc. this information is more reliable for compliance than study participation in days alone. for future studies it is recommended to define in the protocol not only a minimum of study participation in days but also a minimum in tdc and dsc. in addition the compliance thresholds may be defined with regard to the drug's pharmacokinetic profile. in general, the inhalation record of the akita smart card provides an unbiased view of the inhalations treatments during a study especially when the subjects perform the inhalations at home. compliance calculations as shown above may be performed and linked to other outcomes of a study for validation. compliance data like these may also be used routinely by the treating physician in order to guide and supervise his patients. introduction: adaptive aerosol delivery (the i-neb ® aad ® system) has been developed to deliver precise, reproducible doses of aerosol into inhalation. i-neb utilizes a medication chamber, which incorporates a metering chamber to deliver a preset (metered) dose of aerosol. inhalation of hypertonic (7%) saline has been shown to aid mucociliary clearance in patients with cystic fibrosis. [1] we performed an in vitro test to determine the emitted, delivered and residual doses of hypertonic saline from the i-neb aad system with 0.5 ml metering chamber, and a conventional jet nebulizer (lc plus ® ) with a 4 ml fill. the results were then entered into a model to predict the likely lung dose from the in vitro tests, in order to determine the equivalent i-neb dose to the 4 ml conventional jet nebulizer fill. methods:an i-neb device configured to operate at power level 10 (of 15) was fitted with a metering chamber designed to deliver a preset dose of 0.5 ml. the i-neb was weighed, loaded with 1 ml of 7% saline and run on the cen simulated breathing pattern. this process was repeated using an lc plus nebulizer loaded with 4 ml of 7% saline. aerosol was collected on a filter placed between a harvard pump and the device. emitted dose and residual dose were determined by gravimetric output, and delivered dose was determined by ion analysis. all tests were conducted in triplicate. results: as shown in table 1, the emitted dose approximated the delivered dose for i-neb, whereas for lc plus the emitted dose was approximately twice the delivered dose, due to the wastage of aerosol upon simulated exhalation. as i-neb has a delivered dose of 513 mg, and i-neb has been shown to deposit 62.8% of the dose in the lung, [2] this would result in a lung dose of 322 mg. the pari lc plus has a higher delivered dose, but the overall lung deposition is only 47% of the fill volume, i.e. 613 mg. [3] conclusion: the results of this test suggest that two treatments would be required in order to deliver an equivalent lung dose of hypertonic saline from an i-neb device fitted with a 0.5 ml metering chamber, compared with a 4 ml fill in an lc plus jet nebulizer. references introduction: inhaled colistimethate sodium can be used for the eradication of pseudomonas aeruginosa in patients with cystic fibrosis. the tonicity of colistimethate sodium inhalation solution has been linked to the occurrence of bronchospasm in some patients. it has been suggested that this bronchospasm can be avoided by using an isotonic solution of colistimethate sodium. [1] this can be complicated when using conventional jet nebulizers, since evaporation during nebulization tends to change the tonicity of the solution remaining in the nebulizer. however, because very little evaporation occurs within the i-neb medication chamber during nebulization, the tonicity of the loaded solution at the beginning and end of nebulization remains almost constant. aim: we investigated the effects of various saline concentrations upon the tonicity of two concentrations of colistimethate sodium solution (0.5 miu/ml and 1 miu/ml). methods: diluent containing sodium chloride concentrations of: 0% (water for injection), 0.225%, 0.45%, 0.675%, and 0.9% (normal saline) were used to reconstitute vials of colistimethate sodium (promixin ® , profile pharma, uk). each vial was reconstituted with either 1 or 2 ml of diluent to make up 1miu/ml or 0.5miu/ml colistimethate sodium solution, respectively. the tonicity of each diluent concentration/colistimethate sodium concentration combination was made up and tested in triplicate using an advanced micro osmometer (advanced instruments, norwood, usa). the results were plotted on a graph along with a line of best fit, to determine the diluent concentration that gave a mean tonicity closest to that of an isotonic solution. results: tonicity increased linearly with increasing concentrations of saline solution for both 1miu/ml and 0.5miu/ml concentrations. the tonicity of the 1miu/ml solution was approximately 116 mosm higher than the 0.5miu/ml solution across the range of saline concentrations tested. the best fit line passed through the isotonic solution point (290 mosm) closest to the 0.45% saline concentration point. conclusions: the diluent that produces the closest approximation of isotonicity for the mean of 0.5miu/ml and 1miu/ml colistimethate sodium concentrations was 0.45% saline. references 1. transave, inc., monmouth junction, nj, usa; 2. activaero, gmbh, gauting, germany arikace™ is an inhalation formulation of a liposomal amikacin suspension that is designed to treat chronic pseudomonas aeruginosa infections in cystic fibrosis patients. liposome encapsulation of amikacin reduces nonspecific binding of this cationic aminoglycoside drug to the negatively charged mucus and biofilm surfaces and allows penetration and delivery of packets of highly concentrated drug to the otherwise protected bacteria. as nebulized and delivered to the lungs, arikace™ comprises 65% liposomal amikacin and 35% 'free' amikacin that is not entrapped by liposomes; free drug is produced by liposome leakage during nebulization. this profile provides an initial high peak concentration of amikacin followed by a sustained level as drug leaks from the liposomes. nebulized arikace™ with this profile was evaluated previously in human clinical studies using the lc star® nebulizer. although the 0.3 µm liposomes of arikace™ are efficiently loaded with drug, the expected effective dose in humans will require relatively large volumes of drug solution to be administered. to minimize treatment time and improve patient convenience we sought to find a nebulizer that would efficiently deposit drug and produce aerosol at a high output rate, while still producing the same level of free drug during nebulization. methods: nebulization of liposomal amikacin was compared using several nebulizers, including 2 jet nebulizers: lc star® and lc sprint® (both from pari), and 3 electronic mesh nebulizers: microair® ne u22v (omron), aeronebgo® (nektar), and eflow® (pari). additionally, meshes of different pore sizes were examined for the microair® and eflow® devices. output rates were measured by gravimetric differences. droplet size distribution was assessed by a laser light scattering method and by cascade impaction; mass median aerodynamic diameters (mmad), geometric standard deviation (gsd), and fine particle fraction (fpf) (i.e., % droplet mass <5 µm). amikacin association with the liposomes was measured using a centrifugation-filtration assay. results: in terms of output rate, the order of performance was eflow® (~0.5 g/min) > lc sprint® > lc star® > aeronebgo® > microair®. while the aeronebgo® and microair® mesh nebulizers performed well with saline, their performance declined dramatically when nebulizing the liposomal amikacin solution, and, in fact, the microair device clogged. in additional studies with the eflow® where different meshes were compared, the 40l mesh was selected as it provided ideal aerosol properties (mmad = 3.7 µm, gsd = 1.7, fpf >70%) with a ratio of entrapped to free amikacin of approximately 65%/35%. importantly, from in vitro breath simulation studies it is estimated that about twice of the nominal drug dose is expected to deposit in the lungs compared to the lc star®. conclusions: for nebulization of liposomal amikacin, the eflow® configured with a 40l mesh provided optimum aerosol characteristics with a high output rate and ideal size for distribution throughout the lung. the eflow® reduces dose administration time not only because of its faster output rate but also because its higher deposition factor greatly reduces the amount of drug needed. zeman, k.l.; bennett, w.d. cemalb, university of north carolina, chapel hill, nc, usa many inhaled, therapeutic drugs have their site of action in the conducting airways, but may deposit elsewhere in the respiratory tract, resulting in unwanted side effects and waste. the quantity and location of particle deposition in the respiratory tract depends on both the particles' aerodynamic size and breathing pattern. in 8 subjects with normal lung function, we evaluated three methodologies for their ability to deliver radiolabeled particles (99mtc-labeled sulfur colloid) to the conducting airways: 1. inhalation of 5 µm mmad particles (devilbiss 646 jet nebulizer) using a resting tidal volume and flow (mean 0.43 l and 0.39 lps); 2. inhalation from the same nebulizer using a very large inspiratory capacity volume and higher flow rate (mean 2.6 l and 0.52 lps); and 3. inhalation of larger 9.5 µm mmad (heart jet nebulizer) particles with a large tidal volume and very low flow rate (mean 0.89 l and 0.08 lps). gamma scintigraphy gave an estimate of % conducting airway deposition (%cad) by multiplying the percent of activity in the lungs immediately post-deposition relative to the total deposition (i.e. lung+mouth +esophagus+stomach) times the percent of activity cleared from the lungs over 24 hours. %cad was 17%, 14%, and 31% for the three methods, respectively, significantly greater for the large particle, very slow inhalation technique when compared to the other two methods. the amount deposited in the mouth and larynx was 52%, 34 and 33%. in addition, we evaluated protocols 1 and 3 above in a preliminary group of 3 cf patients. for protocol 1, mean inhaled volume and rate was 0.48 l and 0.44 lps. mean inhaled volume and flow were 0.73 l and 0.078 lps for protocol 3. the %cad for the two protocols 1 and 3 were 16% and 25%, respectively. the amount deposited in the mouth and larynx was 41% and 33%. higher therapeutic value of a medication delivered to the airways where the primary defect is associated with cf disease, and with fewer losses to the extrathoracic airways, can be obtained by using a "very large particle/slow inhaled flow" routine when compared to normal or higher flow breathing associated with typical nebulizers. supported by cf foundation. positive effects of physical conditioning in cystic fibrosis (cf) have been reported for quality of life (qol) in addition to effectis on fitness and lung functions. the objective of this study was to identify the effects of different modes of training on qol in cf. 58 cf-patients (age 12-40 yr., fev1 >35%pred.) were randomized into 5 groups: germany: unsupervised homebased training, 3 hours per week, free choice of training mode and means (ut, n=18) and controls (con1, n=10); switzerland: aerobic training (at, n=11) or weight training (wt, n=11) in a fitness center, 3 x 30 min per week, or controls (con2, n=8). subjects in the training groups were asked to train for 6 months. at study entry and after 3 and 6 months, qol was assessed by questionnaire and maximal physical working capacity (pwc, %pred) was determined during cycle ergometry (godfrey protocol). changes from baseline values were calculated and anova for repeated measures was used to test for differences among groups. weight training resulted in a significant decrease in qol (generic dimension) at 3 and 6 months compared with all other training modes including controls. there was no significant difference between at or ut and controls in qol during the program. however, there was a positive association between the change in pwc and the change in qol (r=0.32, p<0.05) in the entire sample. in conclusion, physical training may not always result in an increase in ql in cf. possibly, the decrease in qol in the wt group may have resulted from tiredness induced by the training. thus, a different outcome might have been found with less intense training. positive effects may be achieved by improving aerobic fitness. supported introduction: exercise and exercise training programs are felt to be important to improve aerobic exercise capacity, muscle strength, lung function, and quality of life in cf-patients. recent studies in children and adults with cf have demonstrated an increase of motor performance after exercise training. less is known about trainability of motor performance in younger children with cf. the aim of this study was to assess the effects of a training program on motor performance in preschool children with cf. methods: 19 preschool children with cf (10 female and 9 male patients, mean age 5.2 yrs; range 4-6 yrs, fev1%pred. 100,1±15.6 rage from 65.8-123.1 ) participated. at baseline (t1) and at the end of the training program (t2) the "motorik test (mot)" was used to assess motor performance and to evaluate training effects. components of motor performance tested with the mot are balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques. the duration of training was 4-6 weeks (3 times/week, 45 minutes per session) with different kinds of sport activities. results: at t2, strength and balance had increased significantly (p<0.05). no other components of the mot increased, but the increases in strength and balance were enough to cause a significant (p<0.05) increase overall motor performance, as indicated by an increase of 10.7% in mq. the mq is the sum of all 18 test-items of the mot. no correlation was found between lung function and motor performance. discussion: to our knowledge this was the first study that examined the effects of an exercise training program on motor performance in preschool children with cf. main findings of this study were that a training program of 4-6 week of duration improved some aspects of motor performance. the improvements may be explained by different kinds of sports activities during training when compared with activities before training. classification of motor performance of the tested children was normal (mq =3.1±0.8). this could be the reason that only some aspects of motor performance increased. parents of the children were asked about habitual physical activity at home. all children were physically active and some of the children participated in organized sports. a recent study showed that with an increase in age lower motor performance in children and adolescent with cf was seen compared to healthy children. we speculate that time spent in physical activity in preschool children with cf is comparable to healthy children. conclusion: an exercise program offered to preschool-children with cf may lead to an improvement in motor performance. pre-school children with cf have normal motor performance. to prevent a decline of motor performance, exercise programs should be available for preschool children. gruber, w. 1 ; braumann, k.m. 2 ; orenstein, d.m. 3 ; huels, g. 1 1. dept. for sports and exercise, clinic sattelduene for children and adolescent, nebel/amrum, germany; 2. institute for sport and exercise medicine, university of hamburg, hamburg, germany; 3. school of medicine, university of pittsburgh, pittsburgh, pa, usa; 4. dept. of medicine, clinic sattelduene for children and adolescent, nebel/amrum, germany introduction: a decline in physical activity with age is one of the main problems in patients with cf. as a consequence, lower exercise capacity and a more rapid decline in lung function may be seen in those patients who are physical inactive. furthermore, motor performance is lower in children and adolescents with cf compared to healthy children of the same age. little attention has been paid to younger children with cf and motor performance. the aim of the study was to compare motor performance in preschool-children with cf with healthy children of the same age. methods: 19 children with cf (mean age 5.2 yrs; range 4-6 yrs) and 21 healthy children (mean age 5.5 yrs; range 4-6 yrs) participated. the "motorik test" (mot) was used to assess motor performance. the mot consisted of 18 test-items which evaluated seven different components of motor performance (balance, agility and flexibility, strength, coordination, fine motor skills, reactivity, accuracy of movement). lung function in patients with cf was measured by spirometric techniques results: results of the mot showed no difference either for single testitems or for parameter "motorische quotient (mq)" (p>0.05). this parameter classified motor performance of all aspects tested with the mot. classification of children with cf and of healthy children was normal. mean value for parameter mq (cf: 3.1±0.8 vs. healthy 3.29±0.7) was slight higher in patients with cf than in healthy children (p>0.05). differences between healthy and cf for different components of motor performance were not significant (p<0.05) discussion: the mot is a reliable and valid test method in germany to determine motor performance in younger children. to our knowledge this was the first study to examine motor performance in preschool-children with cf. furthermore, we compared patients with cf and healthy children. we observed no differences between groups for motor performance. in older children with cf, lower motor performance has previously been found compared to healthy children of the same age. we do not have any information about the level of habitual activity in tested healthy children. we therefore speculate that time spent in physical activity in cf is similar to that of healthy children at this age. a recent study showed a decline in physical activity in cf when age and problems during physical activity increased. we think that motor performance is normal in children with cf up to an age of 6 yrs. when age increased, time spent in physical activity decreases (treatment, school) and therefore motor performance decreases. conclusion: motor performance is normal in preschool-children with cf compared to healthy children. to prevent a decline or to maintain motor performance it is recommended to motivate cf-children from early infancy to participate in sports activities or other physical activities to maintain or to increase their physical fitness. 1 1. respiratory medicine, the hospital for sick children, toronto, on, canada; 2. respirology, st. michael's hospital, toronto, on, canada; 3. respiratory medicine, montreal children's hospital, montreal, qc, canada purpose: the habitual activity estimation scale (haes) questionnaire has been shown to be a feasible tool to measure physical activity however the reliability has yet to be determined in cf populations. we therefore assessed the reliability and validity of the haes questionnaire in paediatric and adult cystic fibrosis (cf) populations. methods: fourteen (7 male, 7 female) patients aged 16.2 + 4.2 yrs with cystic fibrosis from the cf clinics at the hospital for sick children and st. michael's hospital participated in this study. participants were clinically stable at the time of the study (fev1 > 70% predicted) and participating in their 'habitual' physical activity. to assess test-retest reliability, patients completed each of the haes (schneiderman-walker et al., j pediatr. 2005; 147 (3):321-6.), and a validated 3-day activity diary (bratteby et al., eur j clin nutr. 1997; 51(9) :585-91), and wore an actigraphtm tri-axial accelerometer for two consecutive weeks. validity was assessed by comparing the activity results of each of the three instruments over a single week time period. results: intra-class correlation coefficient estimates of reliability for the haes, diary and accelerometer were 0.72 (p<0.0001), 0.76 (p<0.0001), 0.63 (p<0.0001), respectively. validity was estimated by comparing the results from each instrument using a one-way anova with block design. there were no overall differences among the participants' activity results as estimated by the haes, diary and accelerometer. furthermore, there were no significant differences between activity measures among the 3 instruments when broken into morning, afternoon, or evening periods, or between measures from weekday or weekend days. while there were no significant differences among the 3 instruments when recording 'very active' intensity levels, they did detect differences during the inactive / somewhat inactive (p = 0.02) and somewhat active (p = 0.001) intensity levels. conclusion: the findings of this study suggest that the haes questionnaire is a reliable and valid instrument that can be used to assess activity levels in patients with cystic fibrosis. in agreement with others, our patients found it easiest to recall and record accurately those periods in which they are participating in vigorous activity. acknowledgement: this research was supported by the canadian cystic fibrosis foundation. 1 1. respiratory medicine, hospital for sick children, toronto, on, canada; 2. respiratory medicine, montreal children's hospital, montreal, qc, canada; 3. respirology, st. michael's hospital, toronto, on, canada objective: previous work showed a relationship between habitual physical activity (hpa) and decline of lung function (fev1) in cf females (sneiderman-walker, j peds 147:321,2005) . this study only included a small number of patients and the follow-up period was limited to 18 months. the purpose of this study was to evaluate daily activity levels and decline in fev1 in a larger cohort over a longer period of time. methods: a total of 187 (n= 99 female) patients with cf (age 7-25 yrs) were studied over a six year period at the hospital for sick children, st. michael's hospital and montreal children's hospital. at each quarterly cf clinic visit, patients performed pulmonary function tests to determine fev1 and completed the habitual activity estimation scale (haes). weekday total activity (wdta), expressed as hours/day (h/day), was calculated from the sum of 'very active' (wdva) and 'somewhat active' (wdsa) categories as previously described. a repeated measures linear regression was performed to evaluate the relationship of fev1 and hpa over time. results: at baseline, mean fev1 was 85% predicted (range 29-119% pred). mean baseline wdta was 5.3 h/d (wdva = 2.3 h/d and wdsa = 3.0 h/d) similar to that previously reported. subjects were divided into high and low activity groups based on the group median wdta. those in the low activity group had a significantly (p=0.004) steeper rate of decline (-1.47 % predicted per year) compared to those in the high activity group (-1.06 % predicted per year) . unlike in the in the previous study this difference was observed in males as well as females. conclusions: six year follow-up of this patient cohort has shown that higher activity levels are clearly associated with a slower rate of decline of fev1. we are currently evaluating whether strategies aimed at increasing physical activity will have an impact on lung function decline in cf patients. background: knowledge of exercise principles, benefits, and techniques has not been assessed in children with cystic fibrosis (cf) or their parents. regular exercise is a recommended cf treatment regimen that is often not followed, but one which might improve cardiovascular fitness, lung function, sputum clearance, and health-related quality of life. further, aerobic fitness, measured as peak oxygen uptake (vo 2 ), is positively related to survival in cf. construction of a reliable and valid measure of exercise knowledge is necessary to assess level of knowledge and study its relationship to exercise behavior. the aim of this study was to develop and test a measure of exercise knowledge for pediatric patients with cf and their parents. methods: we constructed a 22-item exercise knowledge test (ekt) and then examined its psychometric properties. a comprehensive review of the exercise literature guided the formulation of objectives and development of items. to establish content validity, a panel of experts (cf physician, two exercise physiologists, and a clinical cf nurse) evaluated the objectives and test items for relevance, clarity, and accuracy. after feedback from the panel, we revised the ekt through an iterative process. in consultation with a reading level expert, we wrote the ekt items so that the test could be administered to children and adults. a measurement and evaluation expert also reviewed the test, and additional revisions were made. during routine clinic visits, the ekt was administered to 50 subjects with cf and 50 parents. the cf subjects included 31 boys and 19 girls, age 8 to 18 years, fev 1 range: 26-123 % predicted. the parents had an average age of 41 years and most (n=44) were female. results: the ekt includes 22 items, 12 true-false and 10 multiple choice. it is self-administered, yields a single summed score, and has a flesch-kincaid 3rd grade reading level. the means were 17.96 + 2.7 (82% correct) and 20.24 + 1.4 (92% correct) for the cf subjects and parents, respectively; 74% of the parents scored between 20 and 22. internal consistency reliability estimates were .64 for the children and .26 for the parents, reflecting both the multidimensionality of the material and the lack of variability in the parent scores. item difficulty ranged from 0.50 to 0.98 for the cf subjects and from 0.64 to 1.00 for the parents. in both groups, the discrimination values were positive for all items with difficulty values less than 1.00. conclusions: we were able to develop a measure of exercise knowledge with an acceptable reading level for children with cf and their parents. this sample of cf subjects and parents showed substantial exercise knowledge. the ekt can be a useful tool to identify gaps in exercise knowledge in cf patients and their parents, develop educational programs to increase knowledge, and examine how knowledge relates to exercise behavior in children with cf. supported by cystic fibrosis foundation therapeutics, inc. purpose: although physical activity is routinely recommended for adolescents with cystic fibrosis, past research suggests that many patients do not regularly participate in physical activity. there may be a variety of reasons, both related to disease and otherwise, for this inconsistent participation. in this study, qualitative methods were used to describe factors that facilitated or served as barriers to physical activity in adolescents with cf and compared these factors to those in a healthy peer group. methods: ten subjects with cf and ten subjects without cf aged 13-17 volunteered for this study. each subject participated in two, open-ended semi-structured interviews with 3-4 weeks between each interview. subjects were questioned about their current and past physical activity as well as the benefits and barriers to participation. the adolescents with cf were also asked about their perceptions of physical activity on their own health and other adolescents with cf. interviews were transcribed and coded by three investigator groups. analysis was conducted using the grounded theory approach at two points in the study, following each interview and again at the completion of all interviews. at the first point, individual interviews were coded using line-by-line coding, and similar information was grouped into categories. in the second interview, categories were confirmed and subjects were also asked to elaborate or clarify points from the first interview. interviews were continued until themes reached a saturation point. upon completion of all interviews, data for each group were organized by question. data categories appearing throughout the interviews became major themes describing facilitators and barriers to exercise. the major themes were identified by the three researcher groups and then through collaborative group analysis. results: the central theme for both groups was perceived importance of physical activity for health benefits. for the cf group, facilitators and barriers were both psychological and physical. physical benefits were categorized as either relating to their general health (such as feeling energized) or as benefiting their disease (such as improving breathing). in the non-cf group, facilitators included social factors in addition to general health-related and psychological benefits. in the non-cf group, barriers were categorized as either internal or external, with the internal barriers being similar to the cf group (physical and psychological), however, the non-cf participants also indicated external barriers (such as time) not articulated by the cf group. conclusions: in general, we found very similar facilitators and barriers to physical activity in adolescents with cf and those without. however, the adolescents with cf strongly indicated that physical activity would have positive effects on their physical health. further work is needed to determine ways to accentuate facilitators and remove barriers to improve physical activity levels in adolescents with cf. this project was supported by a research grant from the cardiovascular & pulmonary section, apta. lloyd, e. 1 ; dodd, m. 2 1. paediatric cystic fibrosis service, central manchester and manchester children's university hospital, manchester, united kingdom; 2. adult cystic fibrosis centre, wythenshawe hospital, university hospital of south manchester nhs foundation trust, manchester, united kingdom background: training in the skills of communication and self care are considered important components of transitional care1. physiotherapists work to involve families and young people to encourage this independence. the cf manager training programme2 (cfmtp) is a recognised tool for measuring age appropriate tasks in cf care. aim: the aim of the study was to survey the current levels of autonomy in all patients with cystic fibrosis in the north west of england from birth to 18 years to provide a baseline measure for encouraging autonomy in preparation for transition. method: three areas of the cfmtp were selected for the survey: physiotherapy (p), inhalation therapy ( it) and communication (c) for the 10 age groups: 0-1. 5, 1.5-3, 3-5, 6-7, 7-8, 8-9, 9-10, 11-12, 13-15,and 16+ years. physiotherapists from all the north west cf clinics were invited to assess all the patients under their care. data collected were age, gender and whether the patients skills fell "below", "same" or "above" the age appropriate self management tasks as described in the cfmtp. 'not applicable' was indicated where the skills did not apply. results: 14/16 clinics responded. 401out of a total of 474 patients in the nw were assessed, three patients were excluded. population by clinic ranged from 6-133. population by age category: 15 (0-1.5 years) 24 (1.5-3years) 45 (3-5 years) 25 (6-7 years) 36 (7-8 years) 16 (8-9 years) 45 (9-10 years) 50 (11-12 years) 78 (13-15 years and 64 (16+ years). 213/398 (54%) were male. for p (n=390), it (n=261), c (n=367), 32%, 40%, 22% = below, 59%, 51%, 65% = same, 9%, 9%, 13% = above respectively. there were no gender differences. conclusions: overall scores revealed that 2/3rd of patients were gaining age appropriate independence in all areas. there was the least autonomy with it. it was rare for a child to be setting up and cleaning equipment regularly at 7-8years, but this continued throughout the adolescent years. overall 35% were 'below' for p and it; this was highest after the age of 11years. autonomy with physiotherapy appeared slow to develop. some categories in cfmtp were different from our practice for e.g. patients are not invited to be seen alone in our clinics until 13 years v 9-10 years and this may account for the lower scores in communication below this age. this study has highlighted that some of our patients are not fully independent in the time up to transfer. we need to encourage independence from an early age with all aspects of care and integrate the process into our routine clinical practice. refs 1 mcdonagh and viner bmj 2006; 332:435-6, 2. parents' guide to the cf manager training programme. children's hospital of eastern ontario planner, s. 1 ; morrison, l. 2 1. physiotherapy, gartnavel general hospital, glasgow, united kingdom; 2 cystic fibrosis (cf) causes a deterioration in lung function and exercise tolerance. assessing exercise capacity is therefore essential in order to monitor changes in disease severity. the 6-minute walk test (6mwt) and 3 minute step test (3mst) are validated and widely used outcome measures for identifying exercise capacity in patients with cf. exercise testing is part of annual review of all patients in the west of scotland. previous studies identified that the 3mst was a sensitive measure of exercise tolerance, but did not successfully challenge patients with mild or moderate lung disease as defined by fev(sub)1(/sub)>40% (morrison 2005) . further analysis of the 3mst, where step height was increased to 8 inches did not show any difference in sao(sub)2(/sub). decline or rate of perceived exertion (rpe) in patients with mild disease. the chester step test (cst) was developed to assess aerobic fitness which is used in the uk cardiac population for exercise prescription (sykes 1999) . it is a 10 minute progressive, sub-maximal test with a variable step height (6-12inches) , making it suitable for those with a wide range of exercise capacities. in addition, cst results extrapolate to a mets value (oxygen utilisation: 1met = rate of oxygen utilisation at rest) as an outcome measure which can facilitate exercise prescription based on established values for physical activities. we aim to determine whether the cst is a more sensitive measure of exercise capacity than the 6mwt or 3mst when considering decrease in sao(sub)2(/sub) and rpe. 13 patients ( aged 16-49 mean fev(sub)1(/sub)2.39l) with mild-moderate cf performed the cst and 6mwt and results were correlated with fev(sub)1(/sub). decrease in sao(sub)2(/sub), increase in hr and rpe scores were also compared between the cst, 6mwt and recent 3mst data. patients were exercised to a symptom limited maximum which differed from the cardiac cst where patients are exercised to 80% of maximal hr. the cst appears to be more challenging as only 4 patients completed it, all with an fev(sub)1(/sub)of >50% predicted, whereas in the 3mst and the 6mwt all patients completed the test. mets levels for the cst were weakly correlated with fev(sub)1(/sub)(r=0.456) and the 6mwt (r=0.29) . this could be explained by the narrow range of mets data available or the size of the sample population. there were significantly greater rises in hr (p= 0.01, p= 0.01), decrease in sao(sub)2(/sub) (p=0. 03, p=0.0001) and rpe (p=0.04, p=0.013) , between the cst, 3mst and 6mwt respectively, suggesting that the cst is a more sensitive measure of cardiorespiratory effort. conclusions the cst appears to be a more challenging exercise test for those with mild to moderate disease and therefore may be a better reflection of exercise capabilities. cst results are useful for prescribing exercise and monitoring programmes appropriately for this patient group. further study could determine whether the cst is also suitable for those with severe disease. lee, a.l. 1 ; button, b. 1 ; holdsworth, m. 1 ; holland, a. 2 1. physiotherapy, the alfred, melbourne, vic, australia; 2. latrobe university, melbourne, vic, australia musculoskeletal pain is prevalent in cystic fibrosis (cf). while the chest and back are frequently reported regions of acute or chronic pain, it is unclear if pain in these areas is related to disease severity or respiratory symptoms. manual therapy and postural advice has been shown to alleviate pain, but the effects of a combined approach of musculoskeletal physiotherapy and massage has not been studied. the aim of this study was to identify the primary regions of musculoskeletal pain, the relationship between pain severity and respiratory symptoms and to examine the effect of musculoskeletal physiotherapy techniques and soft tissue therapy, including remedial massage on pain and ease of breathing (eob) in adults with cf. one hundred and twenty-nine adults with cf (70 with acute exacerbation, 24 post lung transplant) aged 31 ± 9 years (mean ±sd) with fev(sub)1(/sub) of 51± 21% participated in this study. following assessment of primary pain regions, each subject underwent a single treatment session including spinal joint / intercostal mobilisation and soft tissue therapy. pain and eob on a visual analogue scale were measured before and after treatment. changes were compared using paired-samples t-tests. pain was frequently reported in the thoracic spine region (38% of subjects), followed by the shoulder region (31%), cervical spine region (16%) and chest wall region (9%). equivalent ratings of pain and eob prior to treatment were reported for all regions of pain. eob rating prior to treatment were worse in those with low bmi (r=-0.21, p=0.02) and low fev(sub)1(/sub) (r=-0.24, p=0.01). a single treatment session was associated with reduction in pain (95%ci 1.6 to 2.1, p<0.001) and improvement in eob (95%ci 0.4 to 0.7, p<0.001), irrespective of clinical status. improvement in pain was equivalent for all primary pain regions. however, greater improvement in eob was evident in subjects with shoulder pain compared to other regions (95%ci -1.1 to -0.02, p=0.04). a combination of musculoskeletal physiotherapy techniques and soft tissue therapy reduces musculoskeletal pain and improves eob in adults with stable cf, during an acute exacerbation and post lung transplantation. service cystic fibrosis is characterized by an excessive production of airway secretions responsible for bronchial obstruction and recurrent infections of the respiratory tract. the transport by ciliary activity and cough may be dependent on the unsteady rheological properties of the respiratory mucus such as thixotropy and shear-thinning properties which correspond to a decrease of viscosity with time or flow rate, respectively. we have previously shown that respiratory mucus with high shear-thinning and thixotropic properties is better transported by the cough mechanism (zahm et al, eur. respir. j., 1991) . to promote mucus clearance and to finally expectorate the mucus by a cough manoeuvre, an innovative device, the airhelp, has been developed and aimed to improve chest physiotherapy effectiveness. during a prolonged expiratory manoeuvre, the airhelp delivers negative pressure variations (maximal amplitude: -4 kpa) at an oscillatory frequency of 13 hz. the aim of the present work was to test the effect of the airhelp device on the in vitro clearance of mucus by airflow. a plexiglass tube of 9 mm in diameter and 210 mm in length has been connected by one part on the airhelp and by the other part on a 30 l thank mimicking the lung compliance. a 100 µl drop of mucus simulant at different concentrations (viscogum at 0.25% or 0.5% with actigum at 0.5% to 1.75%) or of respiratory mucus, was deposited within the plastic tube and the distance travelled by the sample under a 5sec-airflow was measured. twelve respiratory mucus samples collected from cf patients were analyzed and the viscosity of the samples was measured before and after the airhelp experiment. we observed a significant (p < 0.03) and negative relationship (r = -0.77) between the viscosity of mucus simulants and oscillatory airflow transport: the lower the viscosity, the higher the mucus transport by oscillatory airflow. using cf respiratory mucus samples, we observed that without the oscillatory airflow, no transport occurred, whereas when the samples were submitted to the oscillatory airflow, a significant increase in mucus transportability was measured. in addition, the viscosity of the cf respiratory mucus samples was significantly decreased after oscillatory airflow treatment (485 ± 148 pa.s to 85 ± 55 pa.s, p <0.02). these results demonstrate that the mucus transport efficiency by an oscillatory airflow is dependent on mucus viscosity and that the improvement in mucus transport is related to the thixotropic and shear thinning properties of the airway mucus. we conclude that the airhelp device may improve chest physiotherapy effectiveness. potter, e. 2 ; nufer, j. 2 ; cullina, j. 2, 4 ; quinton, h. 3 ; jain, m. 1, 4 ; mccolley, s.a. 1, 2 1. northwestern university feinberg school of medicine, chicago, il, usa; 2. children's memorial hospital, chicago, il, usa; lebanon, nh, usa; 4. northwestern memorial hospital, chicago, il, usa socioeconomic status (ses) significantly impacts health outcomes in cystic fibrosis (cf). the cystic fibrosis foundation (cff) is currently using median income estimated by zip code from the 2000 us census of the population for ses risk adjustment of data reported publicly from the cff patient registry. median income estimated by zip code is an "ecologic" variable that may misclassify patient and family income. in order to more fully assess the impact of ses on cf health outcomes, the cff began to ask centers to collect data on household income directly from patients and families. these data were captured for 6.5% of patients and families in 2005 and 16.1% in 2006 in the national registry. we report a method of collecting ses data from children and adults implemented at the children's memorial hospital and northwestern university cystic fibrosis center, and compare directly collected income to income based on zip code. methods: a short survey with the questions on household composition, income, and education level from the cff patient registry was developed. a letter from the center director and adult program director was mailed to patients and families informing them of the survey's purpose and confidentiality measures, i.e. responses would be seen only by the individual entering the data and identified by cff id number. the survey was distributed to cf patients and families in clinic by clerical staff. completed surveys were placed in an envelope and sealed by the patient or family. both directly collected income and income from zip code was classified into 9 groups (<$10k, 10-20k, 20-30k, 40-50k, 50-60k, 60-70k, 70-80k, 80-90k, and >90k) . percent concordance and spearman's rank correlation were calculated. 2005 data were used for patients who provided data in 2005; otherwise, 2006 data were used. each patient's data was used only once in the analysis. results: 202 unique patients were identified, 127 pediatric and 75 adult patients. eighteen percent of pediatric and 39% of adult patients preferred not to answer income questions or did not complete the survey. the mean income by zip code was $60 k for patients who did not report income data and $61k for the entire group. for the 150 responses, reported income data was moderately correlated with income by zip code (rho=0.48, p<0.001). concordance between the 9 categories was 15%. the correlation was stronger for pediatric patients (rho=0.53, p<0.001) than for adults (rho=0.27, p=0.07). re-classifying income as "low", "medium" and "high" (<$40k, $40-70k, >$70) led to a concordance of 40%. conclusion: survey methods that protect patient and family confidentiality result in good return rates of full ses data from patients and families. zip code derived median income shows moderate concordance with reported income. comparing the associations of the 2 forms of income data with important cf outcomes needs to be done in a larger data set. we acknowledge the cystic fibrosis foundation, gerald o'connor, kathryn sabadosa, and participating patients and families. the patient registry provided a unique opportunity to assess changes in the epidemiology of respiratory pathogens in cf. while the incidence of p. aeruginosa remained stable, the prevalence decreased, suggestive of the impact of antibiotic eradication strategies. in contrast, the increasing incidence and prevalence of s. aureus and mrsa may reflect improved microbiological surveillance and laboratory techniques. the trends noted for b. cepacia complex may reflect successful implementation of infection control strategies. future studies are needed to better define the association between these observed trends and improving care for cf patients. vanderwyden, a. 1, 2 ; drumm, m.l. 1, 3 ; schluchter, m. 2 1. pediatrics, case western reserve university, cleveland, oh, usa; 2. epidemiology and biostatistics, case western reserve university, cleveland, oh, usa; 3. genetics, case western reserve university, cleveland, oh, usa in the genetic modifier study (gms) of lung disease of cystic fibrosis (cf), patients, homozygous for the ∆f508 mutation, were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (fev1) and a linear model was fit from these patients' longitudinal data (drumm 2005 , schluchter 2006 . while these models successfully dichotomized patients for the association study, we assessed whether a single slope model could be improved. here we have begun to compare models with one, two and three slopes, each representing different age ranges, for their use in this type of association study. as an example, interleukin-8 (il-8), a neutrophil chemoattractant, associates with the severe lung inflammation seen in cf patients. we have observed the tt genotype of the il-8 snp rs4073, correlated with severe pulmonary function in the gms sample of 808 patients, relative to the aa and at genotypes of this snp. in a replicate study of 268 patients, with cftr genotypes associated with exocrine insuffiency, longitudinal linear mixed models were used to further characterize the decline in lung function over time as associated with the il-8 snp, rs4073. three linear regression models of fev1% predicted of patients were fit over age and compared with likelihood ratio tests. the three models differed with respect to the number of nodes. the first model had a single slope while the second and the third were piecewise linear models, with nodes at age 15, and ages 15 and 25, respectively. age 15 was chosen as the first node, as by this age most cf patients have reached puberty, an event believed to coincide with changes in pulmonary function. the second node, age 25, was arbitrarily chosen as a point in the third decade, as survivor effect becomes a significant issue during this period. when compared to the single slope and two slope models, the three slope model with nodes at age 15 and age 25 better explained the pulmonary function of this study (p<0.001, -2loglikelihood chi-squared tests). when the explanatory variable, il-8 rs4073 genotype, was added, the single slope model and the three slope model were again compared. the three slope with nodes at age 15 and age 25 again resulted in a better fit of fev1% predicted over age as correlated with rs4073 genotype (p<0.001, -2loglikelihood chi-squared test). estimates of fev1% predicted at ages 6, 15, 25, and 30 for each rs4073 genotype (aa, at/ta, or tt) were determined using the three slope model (p<0.001 for all estimates). other explanatory variables, like gender and survival (p<0.001), were added to further elucidate the relation between il-8 genotype and lung disease phenotype. we propose to use this piecewise three slope model with nodes at age 15 and age 25 to look at the pulmonary function of patients in the cf foundation registry data to better explain the decline in fev1% predicted over time seen in cf patients. (supported by hl-68890 and grants from the cf foundation) stephenson, a.l. 1 purpose: the purpose of this study is to determine the annual incidence of hospitalizations for individuals with cf living in ontario and to examine predictors of hospitalization, specifically gender. methods: this is a retrospective cohort study from 1993 to 2002, using newly linked clinical and administrative databases. the canadian cf foundation patient data registry (cpdr), contains detailed clinical information on all cf patients receiving care at one of 38 accredited cf centres across canada. the institute for clinical evaluative sciences (ices), holds linked administrative databases containing information on all publicly reimbursed health care services delivered in ontario. canadian institute for health information (cihi) discharge abstracts database (dad), ontario health insurance plan (ohip), and homecare databases were used for this study. the cpdr was probabilistically linked (automatch software) to the ices administrative database using name, gender and date of birth. all individuals were then linked to the cihi dad, ohip, and homecare databases using unique numeric identifiers. with these databases, longitudinal records for each individual were created. cihi-dad, ohip, and homecare claims data were used to calculate the number of hospitalizations per year for pulmonary infections. males and females were analyzed separately to elucidate any gender disparities. other predictor variables include age, geographical location, number of clinic visits, socioeconomic status (income quintile) as well as clinical variables such as pulmonary function tests, nutritional status, diabetes mellitus, sputum bacteriology and pancreatic status. results: the cpdr contained data on 4244 individuals with cf followed within canada between 1993-2002. of those individuals, 1452 could be found within the ices administrative databases using probabilistic linkage. of those 1452 individuals matched to the administrative databases, 1170 (45.6% female) had 7244 hospitalizations. a total of 312,015 ohip claims were made during this 10-year period which represents any encounter between an individual with cf and a physician in ontario. multivariable analysis of hospitalization predictors is in progress. zhang, z.; lai, h. uw-madison, madison, wi, usa the 2002 cff practice guidelines recommend adjusting for genetic potential when evaluating height status in children with cf. however, calculation of adjusted height percentile is not recommended due to the complexity of method involved in such calculation. instead, a simple method to estimate a target height based on mid parental height was recommended. however, this target height includes a 10-cm confidence interval, which spans most of the channels on the growth chart. therefore, a child's unadjusted height may be considerably below the target height, yet remains above the lower bound of the target height range. in addition, there is a paucity of data on the associations between adjusted height to lung disease outcomes to justify whether adjustment for genetic potential is necessary. the objective of this study is to compare two methods of incorporating genetic potential in identifying short stature, namely, the cff's target method and the method developed by himes et al (pediatr 1985; 75:304-313) , which applies positive adjustments to children with short parents and negative adjustments to those with tall parents. data from 1986-2005 cff registry were utilized. a total of 558 children born between 1984-1988 who had complete height data from age 2-18 years and their parental height data were analyzed. short stature was defined by unadjusted height < 10th percentile, unadjusted height below the lower bound of cff's target height, or himes' adjusted height <10th percentile. our results showed that adjusted height percentiles are consistently lower than unadjusted height percentiles, with an average difference of 0.2 z-score unit between ages 2-14 years and 0.3 z-score unit between ages 15-18. proportionately more children were classified as short stature based on himes method compared to unadjusted height (31% vs. 26%, p = 0.0003). in contrast, proportionately fewer children were below the lower bound of cff's target height (23%) compared to unadjusted height < 10th percentile (26%), p < 0.001. among children with average parents, unadjusted and adjusted height percentiles were similar. however, among children with short parents (< 25th percentile), the percentage children with of short stature decreased from 68% with unadjusted height to 35% with himes' adjusted height and 14% with cff's target height method. among children with tall parents (> 75th percentile), the percentage of short stature increased from 7% with unadjusted height to 32% with himes method and 35% with cff's target height method. taken together, these results demonstrate that, without incorporating genetic height potential, 2-5 times more cf children who have short parents would be classified as short stature, while 5 times fewer cf children who have tall parents would be classified as short stature. further analyses show that, compared to unadjusted height percentile, himes adjusted height percentile is more sensitive to, and has stronger association with, percent predicted fev-1. in conclusion, our findings provide evidence that genetic potential should be incorporated in evaluating short statue in cf children, particularly for children with short or tall parents. the cff's target height method and himes method differ significantly in identifying short stature in cf children with short parents. cohort study of individuals in the cff patient registry from 1995-2005. all individuals 6 years of age or older and diagnosed before age 45 were included. the new, persistent mrsa infection cohort was defined by having at least two cultures negative for mrsa over a two-year lead-in period followed by at least two mrsa positive cultures. individuals with only one mrsa culture (transient infection) were excluded. we developed multiple linear regression models using generalized estimating equations to assess the effect of mrsa on fev 1 %predicted (%fev 1 ) and rate of change of %fev 1 . results: during the study period 5,955 individuals cultured mrsa. of these, 2,025(34%) cultured mrsa only once (transient infection) and were excluded from subsequent analysis. 3,116 individuals met the criteria for new and persistent mrsa infection. 16,088 met the criteria for never having mrsa resulting in a total study cohort of 19,204. participants were followed for a median of 10 years. the median time to first isolation of mrsa in the persistent mrsa cohort was 5.5 years and the mean number of positive mrsa cultures per patient was 7.5. a comparison of baseline characteristics between the persistent mrsa and never mrsa cohorts revealed the mrsa cohort to be younger (13.1 vs 15.0 years p<0.001), equivalent in lung function (%fev 1 79.1% vs 78.3% p=ns), and with higher likelihood of methicillin sensitive staph aureus colonization (45% vs. 39.3% p<0.001). 5.5 years into the study period (the median time of acquisition for new mrsa), the mrsa cohort had a lower %fev 1 (68.1% vs 71.1% p<0.001), and averaged twice as many hospital admissions per year (0.88 vs 0.44 p<0.001) and home iv courses per year (0.40 vs 0.21 p<0.001). in an unadjusted analysis, the presence of mrsa was associated with a mean %fev 1 that was 9.8% lower than the mrsa cohort (95%ci 9.6%-10.0%). however, after adjusting for sex, age, pancreatic status, p. aeruginosa, and b. cenocepacia, the presence of mrsa was associated with a mean %fev 1 3.3% lower (95%ci 3.1%-3.5%). most importantly, an analysis investigating the interaction between mrsa and time, after adjusting for confounders, indicated no statistically nor clinically meaningful difference in the mean rate of lung function decline between the two groups. conclusions: approximately one-third of patients who culture mrsa do so only once and do not subsequently develop persistent mrsa infection. mrsa infection occurs in a group with more severe structural lung disease as measured by fev 1 , and frequent hospitalizations and iv antibiotics are strongly associated with mrsa acquisition. after adjusting for confounders, mrsa infection is not statistically significantly associated with a more rapid decline in %fev 1 over time. simmonds, n.j. 1, 2 ; macneill, s. 2 ; cullinan, p. 2, 1 ; hodson, m.e. 1, 2 1. department of cystic fibrosis, royal brompton hospital, london, united kingdom; 2. national heart and lung institute, imperial college, london, united kingdom introduction: disentangling the influence different factors have on long-term survival in cf is complex. there are a myriad of influences (including environmental, healthcare-related, psychological and socioeconomic) which may be important, as there is generally a poor genotype-phenotype correlation. the aim of this case-control study was to identify these factors. the cf database at the royal brompton hospital was used to identify long-term survivors. they were classified as cases and were patients who had reached 40 years of age (without transplantation). each case was agematched with at least one control who died of cf (or a cf-related condition) or was transplanted before reaching 30 years of age. late diagnosis patients were excluded. conditional logistic regression models were used to identify potential influences on survival. results: 78 cases and 152 controls were analysed, producing 1811 matched pairs. 97% of the 230 patients were pancreatic insufficient. of the many factors investigated, those resulting in increased probabilities of survival included: increased body mass index (or=1.52, 95% ci 1.35-1.72), fev 1 (or per 5% increase=1.62, ) at transfer to the adult clinic (after adjusting for age and sex) and the use of oral antibiotics before attending the adult clinic (or=3.01, 1.38-6.56) after adjusting for age at first attendance. factors associated with a reduced probability of survival included: cf diagnosis <5 years old (or=0.46, 0.28-0.76); initial presentation of chest symptoms or malabsorption (or=0.44, 0.27-0.71 and or=0.38, 0.24-0.61, respectively); referral from a paediatric clinic in a deprived area (or=0.12, 0.04-0.34); pneumothorax before transfer to the adult clinic (or=0.03, 0.005-0.24) after adjusting for age at first attendance; and s. aureus or p. aeruginosa colonisation before 16 years of age (or=0.36, 0.14-0.89 and 0.18, 0.06-0.58, respectively). factors not influencing survival included: sex, h. influenzae colonisation before 16 years of age, development of diabetes before 16 years of age, major haemoptysis before transfer to the adult clinic, parents' occupation, number of siblings (cf and non-cf) and school achievements. in a very carefully matched study we failed to identify major non-clinical determinants of long-term survival in early diagnosis cf. findings suggest that the majority of significant factors were directly related to optimal physical parameters, the use of oral antibiotics, avoiding bacterial colonisation and a low pneumothorax rate. adler, a. 1, 2 ; bilton, d. 2 ; haworth, c. 2 ; gunn, e. 2 ; shine, b. 3 1. addenbrooke's cambridge university hospitals, cambridge, united kingdom; 2. papworth hospital, papworth everard, united kingdom; 3. oxford centre for diabetes and endocrinology, oxford, united kingdom background and aims: although the prevalence of diabetes in patients with cf (cfrd) is high, few prospective studies exist, and even for genet-ic factors, cross-sectional studies may bias the magnitude of associations. this study sought to identify risk factors for cfrd from a predominantly white cohort of individuals with cf in the united kingdom. methods: 8,029 individuals aged 0 -64 were identified from the uk cf database, a registry of patients coordinated by the uk cystic fibrosis trust with data from over 44 specialists centres from england, scotland and wales, countries which provide medical care free of charge. of the patients, 5,196 had no diabetes at baseline (1996-2004) , and also had at least one follow-up annual visit. diabetes was defined as either a physician diagnosis of diabetes, a blood glucose of >11.1 mmol/l two hours following an oral glucose tolerance test, or treatment with insulin or oral hypoglycemic drugs. follow-up was calculated as time from baseline to the first detection of new diabetes or censoring. risk factors included clinical, genetic (functional classes i,ii,iii vs iv,v), and anthropometric characteristics measured at baseline. survival analyses were limited to patients with complete data. cox proportional hazards modeling was used to estimate the magnitude of the association between potential risk factors and incident diabetes. one-way interactions with sex were tested. results: the median age of entry into the cohort was 12 years and bmi was 17.9 kg/m2. 54% were male, and 96% were white. 526 patients developed diabetes during 15,718 person-years of follow-up (mean 3.0 years); the incidence was 3.4% per year. patients who developed diabetes were more likely in univariate analyses to be older, have a high body mass index (bmi), bacterial pulmonary infections, liver and pulmonary function abnormalities, poor oxygen saturation, a history of organ transplantation, supplemental feeds, and take pancreatic enzymes or bile acids. in addition, patients with class iv,v genotypes were less likely to develop diabetes relative to patients with other genotypes, as were patients screened for cf at birth. not associated with incident diabetes was age at diagnosis of cf and ethnicity. in multivariate analyses (n= 4,004) age (hazard ratio 1.02 per year, 95% ci 1.01 -1.03), female sex (1.67 (95% ci 1.39-2.00), % predicted forced expiratory volume in 1 second (1.019, 1.014 -1.024 per 1% decrement), dose of pancreatic enzyme replacement (1.33, 1.10 -1.62), above vs below median dosage, use of bile acid supplements (1.51, 1.20 -1.91) , and genotype class i,ii, iii (5.60, 2.08 -15.1) relative to class iv/v. these associations were not confounded by bmi or history of transplantation. there were no significant interactions between sex and other risk factors. conclusions: the study confirms the high incidence of cfrd, and shows that patients with class iv/v genotypes are less likely to develop diabetes independent of other cf-related complications. females are disproportionately at risk for diabetes which is not explained by a higher prevalence of known risk factors nor by effect modification. cystic fibrosis children are at increased risk for hearing disorders due aminoglycoside exposure, mucopurulent upper respiratory tract infections, and increased inflammatory responses. pure tone audiometry is the standard instrument used to determine hearing loss. however, it can be expensive and time-consuming to use audiometry for routine hearing screening. hearing questionnaires have been used as a screening tool to assess possible hearing loss in otherwise healthy populations. what are the results of hearing questionnaires administered to pediatric cf patients? does cf patient age or ethnicity increase the likelihood of hearing loss as determined by a hearing questionnaire? a hearing questionnaire (english and spanish versions) was administered to pediatric cf patients over a 2 week period at our accredited cf center. data collected included demographic information. inclusion criteria: age м10 years and in baseline health status. possible hearing loss was defined as at least 3 "yes" responses (>3) to the 14 quesions administered to the patients. fisher's exact test was used to perform the analyses. a total of 20 cf patients were seen in the cf clinic during the study period. 14 cf patients completed the hearing questionnaire. mean age was 14.3 ± 3.6 years (median 13.6 years). there were 9 males and 5 females; 10 were classified as hispanic and 4 were classified as caucasian. 7 patients were older than 13.6 years (the median age) and 7 patients were less than 13.6 years. 3 patients who were older than age 13.6 years had a score of >3 and 4 patients < age 13.6 years had scores >3 (p=1.0, ns). thus, 7 of 14 cf patients (50%) were classified as having possible hearing loss as per their questionnaire scores. 6 of 10 hispanic cf patients had hearing scores >3 and 1 of 4 caucasian patients had hearing score >3 (p=0.56, ns) . we found that a significant proportion (50%) of our cf children may require formal hearing evaluation. we speculate that hearing questionnaires may be useful to screen for hearing loss in the cf patient population. hispanic cf patients have increased morbidity and mortality risks compared to caucasian cf patients who attend our accredited cystic fibrosis center in southern california. is the increased morbidity/mortality risk due to decreased access to outpatient cf care? we performed a retrospective review of all patient visits to our cf clinic between january 1, 2005 through december 31, 2006 . data collected included demographic information (age, ethnicity, gender) , and the number of clinic visits during the study period. data was analyzed by unpaired student's t-test as well as by chi-square. during the study period, a total of 197 cf patients (106 males:91 females; mean age 10.8 ± 5.7 years; range 3 months to 21 years) were routinely followed in our cf clinic. these patients had a total of 1806 outpatient visits to the cf clinic (9.2 clinic visits/patient over the 2 year study period). there were 82 hispanic cf patients (46 males:36 females; mean age 11.0 ± 5.3 years) and 99 caucasian cf patients (49 males:50 females; mean age 10.8 ± 6.2 years; p=0.42, ns). there was no difference in gender distribution between the 2 group (p=0.52, ns). hispanic patients had a greater number of cf clinic visits (11.2 visits/patient) as compared to caucasian patients (7.9 vists/patient; p=0.03) during the same period. we conclude that the increased morbidity and mortality of hispanic cf patients in california cannot be attributed to barriers to outpatient care. in fact, hispanic cf patients were seen more frequently in cf clinic compared to caucasian cf patients who attend the same clinic. we speculate that hispanic cf patients are seen more frequently in cf clinic due to increased severity of cf disease manifestations. duguépéroux, i. 1 ; scotet, v. 1 ; audrézet, m. 1, 2 ; blayau, m. 3 ; parent, p. 4 ; journel, h. 5 ; boisseau, p. 6 ; férec, c. 1, 2 1. inserm u 613, brest, france; 2. dep. of genetics, brest, france; 3. dep. of genetics, rennes, france; 4. unit of medical genetics, brest, france; 5. unit of medical genetics, vannes, france; 6. dep. of genetics, nantes, france aim: the aim of this study was to describe 15-year experience of prenatal diagnosis (pd) for cf in brittany (western france) and to assess its impact on incidence. method: we registered, by the genetic laboratories of our region, all the pds performed in women living in brittany over the period 1991-2005. we described the number of pds made for each reason (way by which the one-in-four risk was identified: previous affected child, family testing, echogenic bowel, etc). we reported the proportion of cf-affected fetuses and of consecutive terminations, and assessed the incidence modification due to pd. results: over the 15-year period, a total of 253 pds were performed in couples living in brittany. most of them were done in couples already having cf child(ren) (n=167, 66.0%). extended testing in families -a practice largely proposed in brittany -led to the identification of 18 new onein-four risk couples among the relatives of cf patients who opted for pd 38 times over the study period (15.0%). the 48 other pds were made in couples without previous history of cf. the one-in-four risk was mainly identified following the detection of an echogenic bowel during pregnancy ultrasound examination (21 pds were done directly following the positive ultrasound (8.3%), whereas 19 others were done for subsequent pregnancies (7.5%)). the other pds were consecutive to the detection of an heterozygote through newborn screening (n=6, 2.4%) or for an other reasons (n=2, 0.8%). over the study period, the number of pds per couple varied between one and five, the mean being 1.6. overall, a total of 88 cf fetuses were identified, among whom 77 were terminated (87.5%). the inclusion in the incidence calculation of these 77 pregnancy terminations led that to an incidence modification of 28.8% over the study period. conclusion: this study shows that pd for cf is commonly used in brittany and highlights the impact of family testing and of routine ultrasound examination of pregnancies in that region. supported context: hispanics with cystic fibrosis (cf) in the united states experience an 85% increased annual risk of death from cf compared to non-hispanic patients. when adjusted for socioeconomic status this disparity persists. studies characterizing this at-risk population do not exist. objective: to characterize the center-reported us hispanic population with cystic fibrosis through a cystic fibrosis foundation (cff) patient registry analysis and to elucidate factors present within this hispanic population that may affect outcomes. design: retrospective cross sectional study of the 2004 cff patient registry. patients: all 22,714 patients in the 2004 cff patient registry were included in the analysis. there were no exclusion criteria. the hispanic population is defined as those who were entered by their respective care center as hispanic ethnicity regardless of race. main study measures: prior to analysis, study measures were selected to demographically and genetically characterize the hispanic cf population. genotype, state of residence, mean age and mean age of diagnosis were obtained in the entire population. maternal education (for patients <18 years of age) and insurance status data were captured as indicators of socioeconomic status. complication rates, fev1 percent predicted (under 18 years of age) and absolute fev1 (over 18 years old of age) were obtained as measures of clinical status. t-tests and logistic regression analyses were used to compare measures between hispanic and non-hispanic groups. odds ratios (or) and 95% confidence intervals are reported. results: 1511 center-reported hispanic patients with cf were identified. over 50% of hispanic patients resided in california, florida, texas or new york. the most common genotype was delta f508 homozygous, accounting for 32% of the population. mean age was 12.3 years +/-9.2 years for hispanic patients and 17.3 years +/-12.1 years for non-hispanic patients. hispanic patients were diagnosed at an earlier age (2.8 years vs. 3.3 years, p=0.005). they were more likely to have mothers with education at less than a high school level (or 5.25 (4.12, 6.71) ) and have government insurance (or 2.73 (2.45, 3.04) ). complication patterns differed between the two groups; non-hispanics were more likely to have complications reported that were related to depression (or 1.94 (1.48, 2.54) ), bone health (or 1.82 (1.41, 2.41) ) and cf related diabetes (or 1.9 (1.6, 2. 3)) compared to hispanics. hispanics however, were more likely to have complications reported from cf liver disease (or 1.31 (1.1, 1.56) ). pulmonary function, as measured by fev1 percent predicted in children (p<0.001) and absolute fev1 for adults (p=0.01) were lower for hispanic patients. conclusions: differences exist between hispanics and non-hispanics with cf with respect to maternal education, insurance status, complications rates and pulmonary function. the lower reported rates of depression, bone complications and cf related diabetes may represent ascertainment bias. further study is needed into the etiology of health outcome disparities in this population, and to design interventions for this high-risk population. mastella, g.; baldo, e.; forneris, m.; furnari, m.; lucidi, v.; manunza, d.; marinelli, i.; messore, b.; neri, a.; raia, v.; salvatore, d.; buzzetti, r.; cairo group, italian cf research foundation, verona, italy with our work we reviewed the international scientific literature coming from cf registries worldwide. our aims were: to verify what has been produced in scientific literature thanks to the material derived from fc registries and to see which clinical problems have been tackled and which clinical questions were answered correctly and exhaustively. a search in medline and embase produced 160 articles (our strategy was "cystic fibrosis"[mesh] and ("registry"[mesh]) or registr$) updated to the 04/30/07). out of these articles 88 have been selected by two independent assessors on the basis of their pertinence (primary studies that used data taken from a formally established registry, at least national, to test some hypothesis of research). each article has been examined with the help of a pre-defined form that in particular evaluated skills of different registry data, selected clinical characteristics of the study populations and statistical methods. more than one half of cases data came from usa-cff foundation patient registry, the remaining coming from canadian, uk, french, italian, german, and other european registries. the main focuses of the articles were included in the following topics: 1) incidence/prevalence of the disease and survival (30 studies); 2) neonatal screening, growth and nutrition (18); 3) genotype/phenotype correlation/ different ethnical groups and twin/brothers (12); 4) complications and outcomes (abpa, diabetes, nasal polyposis, pregnancy and paternity, etc) (19); 5) microbiology (9). our assessment scores were good/excellent for 62/88 studies for the relevance of the problem addressed; 54/88 for the usefulness of the outcomes and 46/88 for the usefulness of the implications for research from the italian registry. thirty-two articles gave a significant contribution to the analysis of the studied problem, but 47 of them left a partial uncertainty, while 9 left complete uncertainty. cf registries are a very important source of data to provide a powerful tool for clinical and research questions. however, further comparable studies have to be planned to assess registry data as a platform for improvement in clinical practice. vandenbranden, s.l. 1 ; mcmullen, a. 7 ; konstan, m. 4 ; morgan, w. 3 ; wagener, j. 6 ; schechter, m. 2 ; watts, k. 1, 5 ; mccolley, s. 1, 5 1. children's memorial medical center, chicago, il, usa; 2. emory university, atlanta, ga, usa; 3. university of arizona, tuscon, az, usa; 4. case western reserve university, cleveland, oh, usa; 5. northwestern university feinberg school of medicine, chicago, il, usa; 6. university of colorado, denver, co, usa; 7. university of rochester, rochester, ny, usa background: although life expectancy in cf has dramatically improved over the last 20 years, most of the improvement is due to improved survival during early childhood. adolescence and young adulthood continue to be a time of worsening pulmonary disease and mortality. study objective: data from a large longitudinal observational study, the epidemiologic study of cystic fibrosis (escf), was examined to characterize lung function decline in subjects with cf during the periods preceding and after the age of 18. design: escf data from 2136 individuals with cf collected during the period of 1994-2005 were analyzed. inclusion criteria required individuals to have a minimum of 5 encounters in each of the 3.5 year time periods before and after the age of 18. the cohort was strati-fied by disease severity based on the best fev 1 at the 18th year (+/-6 months). one hundred thirty seven subjects had severe lung disease (fev 1 <40% predicted), 668 had moderate-severe lung disease (fev 1 40-<70 % predicted), 1049 had mild lung disease (fev 1 70-<100% predicted), and 282 had very mild disease (fev 1 >100% predicted) the study compared the annualized rate of fev 1 decline during the adolescent period, defined as ages 14-17.5 years, and in the young adult period defined as 18.5-22 years. results: in the entire cohort, the annualized rate of decline was less in the young adult period than in the adolescent period (p<0.001). the most dramatic improvement in slope of decline was seen in the severe group, and significant improvements were also noted in the moderate group (p<0.001). the mild group demonstrated insignificant change; the very mild group was the only group that demonstrated a more negative annualized rate of the decline in young adulthood than in adolescence (p=0.027). conclusions: these data suggest that, overall, young adults have a slower rate of decline in fev 1 in the period after age 18. this "stabilizing" of lung function is most prominent in those individuals with moderate-to-severe lung disease. only those with very mild lung disease have a slightly more rapid rate of decline between the ages of 18 and 22. overall there is less variation in the rate of fev 1 decline across disease strata after the 18th birthday. further analysis is underway to better understand the factors leading to these findings. 1 1. department of paediatrics, university of florence, cystic fibrosis centre, florence, italy; 2. italian cystic fibrosis microbiology group, florence, italy; 3. institute of medical microbiology and hygiene, tübingen, germany mrsa is now a worldwide public health problem, due to the increasing rate of infection in several settings as well as in cf patients. mrsa was first recognized as being acquired from hospitalized patients (ha-mrsa), but the onset of mrsa infection outside the hospital setting, in communityassociated strains (ca-mrsa), has recently been described with increasing frequency. ha-mrsa are known to be responsible for infections in hospitalized patients, although highly virulent ca-mrsa are increasingly reported worldwide as the cause of severe outbreaks, replacing ha-mrsa strains in nosocomial infections. little evidence is available about the characterization (sccmec type) and epidemiology of community-acquired mrsa (ca-mrsa) or hospital-acquired mrsa (ha-mrsa), transmissibility, antibiotic susceptibility and virulence in the cf population. the present multicenter study investigated the susceptibility pattern, epidemiology, and sccmec type of mrsa strains isolated from nine italian cf centers. the susceptibility pattern was determined by the disk diffusion method and pulsed field gel electrophoresis (pfge) was performed for epidemiological purposes. the sccmec type in order to identify hospital-and community-acquired mrsa strains (ha-mrsa and ca-mrsa respectively) was determined with molecular methods, and the presence of gene encoding panton-valentine leukocidin (pvl) was tested. during the study period, 181 mrsa strains were isolated from 178 out of 2362 (7.5%) patients attending these cf centers. of the sccmec type representing ha-mrsa, our data showed an high prevalence of sccmec i (49.4%) while the prevalence of sccmec ii, (reported as the most represented among ha-mrsa) was only 1.1%. furthermore a high prevalence (31.4%) was found of sccmec iv (suggestive of ca-mrsa strains). epidemiological analysis showed that 31 (17.1%) out of 178 analyzed mrsa strains belong to the same pfge clone shared among six centers, belonging to the sccmec type iv, suggestive of ca-mrsa isolates. twenty-four (77.4 %) out of these 31 strains were sccmec type iv, indicative of ca-mrsa, as suggested by the good susceptibility rate to trimethoprim-sulfamethoxazole and rifampin. surprisingly all the isolates belonging to this epidemic clone were negative for the pvl genes. these results show that ca-mrsa is now spreading in the cf population, documenting the first epidemic of ca-mrsa in cf patients who are considered at risk for ha-mrsa acquisition due to frequent hospitalizations. these data show a peculiar picture of mrsa epidemiology indicating that further studies are required to clarify the role of ca-mrsa in global epidemiology, their pathogenicity and clinical impact on cf patients. we thank the fondazione per la ricerca sulla fibrosi cistica-onlus for its grant (ffc#12 2006) . there are concerns regarding an increased risk of cancer in patients with cystic fibrosis (cf). this study aimed to review the occurrence of malignant disease in a large population of adult patients with cf. the case notes of 372 patients attending the leeds adult cf unit were reviewed. demographics and data regarding a diagnosis of malignant disease were recorded. a total of eight patients (3 male, 5 female) were diagnosed with a malignant condition over the 10 year period. the median (range) age of diagnosis of a malignant condition was 30.5 years (23-55 years). five patients were diagnosed with malignancy post-transplantation. within this group of patients there were two cases of lymphoproliferative disease, one case of basal cell carcinoma, one case of liver cancer and one case of small cell lung cancer related to the donor lung. the median (range) time to diagnosis post-transplantation was two years (1-6 years) with a median (range) age of 26 years (23-55 years). three patients were diagnosed with a malignant condition without solid organ transplantation. this group consisted of one case of pancreatic carcinoma, one case of oesophageal carcinoma and one case of bowel carcinoma. the median age of diagnosis in this group of patients was 44 years (27-55 years) . prior to transplantation we found all malignant diagnoses were related to the gastrointestinal tract. post-transplantation we found a wider variety of malignant diagnoses, with lymphoproliferative disease being most common. awareness of the increased risk of malignant disease is important and appropriate investigations should be undertaken particularly in patients with atypical symptoms. background: patient outcomes can be improved by implementing evidence-based guidelines for initiating therapies. in our cf center, practitioners have traditionally prescribed medications without a centralized database to screen patients for evidence-based criteria. goals: the primary goal of this project was to increase cf practitioner, family and patient awareness of evidence-based medication guidelines to facilitate discussions regarding therapies. the secondary goal of this quality improvement (qi) project was to determine if cf practitioner prescribing patterns would be altered by increasing awareness. methods: the cf steering committee at cincinnati children's hospital medical center (cchmc) identified oral azithromycin, inhaled tobramycin, and dornase alpha as target medications for this qi project. an evidence-based medicine committee was formed, including a parent advocate, to direct the project. a consensus was reached among cf practitioners for when to initiate therapy discussions. dornase alpha was recommended for cf patients ages six and older. inhaled tobramycin was recommended for cf patients ages six and older with chronic pseudomonas (defined as 2 positive cultures in past 12 months). oral azithromycin was recommended for cf patients ages six and older, with chronic pseudomonas, and a weight of 25 kilograms or more. the cf database was screened to determine each patient's medication eligibilities. eligibilities were reviewed weekly at our cf chart conference. medication specific family and patient handouts were developed, reviewed by the parent advocate, and given to eligible families and patients on arrival to clinic. data was collected to document the number of discussions facilitated at clinic visits, the decisions regarding medications, and the number of new treatment prescriptions that were facilitated. after seven weeks of the initiative, statistical analysis was conducted using mcnemar's test for differences. a chi-square test with a two sided alpha of p <0.05 was used to determine significance. results: 148 of the 203 cf patients in our center were eligible for pulmonary medications using our evidence-based criteria. there were 253 individual prescribing opportunities in these 148 patients (taking each medication for each patient as a unique prescribing opportunity). at the start of the project, 64.3% (163/253) of the prescribing opportunities were fulfilled. 50% (74/148) of patients were prescribed all of their recommended medications. during the first seven weeks of the project, cf practitioners discussed initiating new pulmonary medications with 40 cf families and patients. the proportion of prescribing opportunities fulfilled increased from 64.3% (163/253) to 75.5% (191/253) (p value < .0001; 95% ci 65.3%-82.8%). the proportion of patients prescribed all of their recommended medications increased from 50% (74/148) to 65.5% (97/148) (p value < .0001; 95% ci 57.8%-80.0%). conclusions: increasing awareness of evidence-based pulmonary medication recommendations with cf practitioners, families and patients facilitates therapy discussions, increases the proportion of therapies prescribed and increases the proportion of patients on all recommended therapies. purpose: our purpose was to optimize lung function (fev1) by consistently and aggressively diagnosing and treating pulmonary exacerbations (pex) in all cf patients at our center. it was important to work on this because our families wanted standardized care and we felt our patients' fev1's were suboptimal. methods: our core llc team included two parents of cf patients and one adult patient. we met weekly, received coaching every other week, and learned and applied quality improvement techniques. physicians began monthly meetings, and monthly meetings of the entire cf team continued. the fab met every other month. we identified our specific aims, and approached them systematically. the providers, starting with criteria used by several authors, agreed upon the definition of pex and created a pulmonary exacerbation score sheet (pexss). we selected a score of 4 as the threshold for defining a pex. after using the pexss for three months, all providers reviewed the individual pexsss and validated our original choice of 4. run charts were produced and reviewed periodically to display each provider's percent of pexsss used. when the use of the pexss was not standardized after a year, it was reprinted on bright yellow paper to make it stand out. to educate the families and patients about the early symptoms of pex, a refrigerator magnet was designed and distributed that included all pex criteria. results: we standardized the use of our pexss by all providers on all patients seen in cf clinic with ongoing measurement of its use and regular feedback. many patients and families gained greater knowledge about what a pex was, and the importance of early recognition and treatment. the cf nurses note that 1) many patients call with milder pex symptoms than they did before, 2) providers treat pex earlier, 3) many patients call with their pex score as well as their symptoms. we hospitalized more patients for pex after this project began, with an average of 62 patients/year in the 3 years prior, and 84 in 2006. the median fev1 % predicted of our 6conclusions: participating in the llc changed the culture and functioning of our cf team. limitations to our study include: 1) fev1 change may not have been due solely to this project, 2) the groups who's fev1's were averaged over the various years are different. we will continue to optimize and standardize 1) prevention of pex, 2) inpatient care, 3) care of pex in cf providers' private offices, and 4) follow up of patients with pex. patient adherence to airway clearance techniques (act) in the treatment of cystic fibrosis is known to be sub-optimal resulting in deterioration of lung function. the global aim of our project was to improve median fev1. the specific aim was to improve adherence to act. we believed that these goals could be acheived by implementing a program which included re-education of airway clearance techniques (react) for our cystic fibrosis patients. methods: the initial phase of this project began with the administration of an anonymous survey to patients/families. this survey was designed to assess act practices, knowledge of the rationale for performing act and barriers that prevent patients/families from adhereing to act. the next step included an in-clinic questionnaire and patient/family demonstration of current aerosol therapy and act. based on individual results patients were identified as: adherent with correct technique, adherent with incorrect technique or non-adherent. we defined adherent as correct performance of act one to two times daily as prescribed. then all patients entered the react program which included a standardized tutorial on proper act and correction of techniques if necessary. in the non-adherent group barriers to performing act were discussed and problem solving techniques were used to overcome barriers. the patients in the non-adherent group were re-evaluated in clinic monthly while the adherent patients returned on an every two month schedule. results: analysis of our initial survey revealed that while the majority of patients/families reported performing act daily, the duration of treatments was less then medically prescribed. many patients/families reported barriers which decreased adherence to therapy. to date 85% (74/87) of our patients have completed the in-clinic questionnaire and entered the react program. the results of these evaluations and patient demonstrations revealed: 37% (27/74) were adherent with correct technique, 3% (2/74) were adherent with incorrect technique and 60% (44/74) were non-adherent. in the six months since the implementation of the react program, patients/families are self-reporting an increased adherence to act and the median fev1 for our center has increased form 84.7% (2005 cff registry data, ages 6-18 years) to 87.4% (cff registry, first quarter, 2007, ages 6-17 years). conclusion: while adherence to act in our center was poor, we were able to improve adherence and ultimately improve the median fev1 by implementing a program of re-education for act (react). we believe re-assessment of adherence to therapy and reviewing correct technique is necessary at every clinic visit. continuous quality improvement (cqi) techniques have recently been employed in medicine to improve quality of care. although there is evidence that cqi improves efficiency in hospitals, little is known about effects of cqi on improving patient outcomes. we have previously reported creation of a unique nutritional risk pathway (nrp) to care for cystic fibrosis (cf) patients who are at nutritional risk. the goal of this study is to assess perceived impact and objective clinical efficiency of our nrp. patient data was recorded from all children attending the cf clinic over an 18-month observation period (between sep 05 and mar 07). bmi percentiles were assessed and nutritional risk zones -green: acceptable (bmi > 25%ile), yellow: at risk (bmi 10-25%ile) or red: at urgent risk (bmi < 10%ile) -assigned at each visit. the visit intervals were every 4 to 12 weeks based on severity of nutritional risk. patients that remained in the red zone for an average of 3 to 6 months with no improvement were considered for gastrostomy tube (gt) placement. the cf dietitian updated the data weekly and e-mailed quarterly to cf physicians for review of their patients' status. patient/family perceptions and understanding of the nrp was assessed by questionnaire. of the 164 children in the study, 29 (18%) were identified to be at nutritional risk and were enrolled into the nrp: 19 in red and 10 in yellow zones; 13 (45 %) male, 16 (55%) female; 18 (62%) age < 12 yrs, 11 (38%) age ≥ 12 yrs. over the 18-month observation period, 21 (72%) showed improvement (7 moved from red to yellow, 7 from red to green and 7 from yellow to green zones). thirteen (81%) females improved, whereas 8 (62%) males improved. in the under 12 yrs age group, 17 (94%) improved compared to 4 (36%) in the ≥ 12 yrs age group. as a result of participation in the nrp, 9 (31%) children had gt placed: six of the 9 showed improvement with four moving from the red to green zone. from the survey responders, 80% stated they understood the meaning of the cf nrp. all responders were extremely to somewhat motivated to focus on their child's nutrition when asked to return for growth monitoring or follow-up visits. having specific goals for weight gain, calorie requirements and bmi were considered extremely helpful by 100% of families. when gt placement for their child was recommended, 40% felt they lacked all the necessary information to make a decision. our new cf nutrition education booklet was not received by 40% of families. some of the physicians who received the quarterly nutritional patient data provided positive feedback and found this information beneficial for patient care. nutritional cqi in our cf center utilizing a specific nrp and monitoring of nutritional risk status resulted in improved patient outcomes. overall perceptions of our nrp were positive from both patients and cf physicians. these results suggest that this cqi strategy improved clinical outcomes in our patient population. patients with cf require frequent healthcare visits to optimize growth, initiate early interventions and delay progression of lung disease. the cff recommends evaluation at a cf center at least quarterly. as part of a llciv quality improvement (qi) initiative, our cf team set a goal to increase the percent of cf patients attending clinic 4 or more times a year to 90% or greater by developing strategies to monitor and improve adherence rates. methods: we reviewed cf patients who attended clinic 3 times or less in 2005 to determine patterns of non-adherence in regards to age, gender, distance from center, season, physician or insurer. a patient survey was used to assess clinic attendance barriers from a patient/family perspective. a brainstorming session identified other barriers. a fishbone diagram was drafted into 4 barrier categories: external, internal, communication, and patient/family perceptions. after reviewing all barriers, we concluded the most effective improvements could be made to internal clinic barriers. clinic processes were then assessed including making of appointments, reminder letters/calls, a time cycle analysis, and number of cf appointments available per patient per physician. our family advisory board participated by focusing on the importance of 4 or more clinic visits in their newsletter and by assignment of a member to the llciv team. we discovered a lack of a standardized process to follow-up (fu) patients who "did not keep appointments" (dnka). a spreadsheet was created to monitor physician dnka rates. a primary fu strategy was devised to identify patients who dnka through daily auto-emails to the cf office assistant and utilization of a rescheduling decision tree for reappointment of these patients by our appointment center. two secondary fu strategies were developed. the first involves the clinical coordinator printing a monthly report off port cf entitled "patients due a visit" then emailing this list to the cf team to contact and reappoint. the second involves the cf social worker tracking patients seen per quarter on a spreadsheet and emailing the spreadsheet quarterly to the cf team as well as posting on our datawall. results: in 2004 , and 2006 , 35%, 56%, and 89% of our patients attended clinic 4 or more times, respectively. in 2004 in , and 2006 , 33%, 25%, and 23% of our patients were at nutritional risk, respectively. in 2004 and 2005, mean fev1% predicted was 88.3% and 90.4%, respectively. conclusion: the number of cf patients attending clinic quarterly at our center increased by implementing processes to track dnka patients and reappoint them to clinic promptly. however, some of the early improvement in clinic attendance was partially due to an increased awareness of our qi efforts by our clinic staff, cf team and patients/families. we speculate that improvements in nutritional status and lung function are at least partially related to more frequent clinic attendance by our patients. continued monitoring of the number of dnka patients and patients who attend clinic quarterly is required to sustain these gains in improved patient outcomes. data when we first measured patient/family satisfaction in 2003, 31 parents and 14 children were given questionnaires to measure their satisfaction with services provided at the clinic. at that time, responses in our survey were very positive. with regards to our new model we received very positive feedback, encouraging us to continue with the model. parents and children liked the elimination of duplication in questions and they also like having the whole team involved in the meeting and problem-solving. final results for our second satisfaction questionnaire will not be available until june of this year as the questionnaires are being given at clinics over a 3 month period this spring. the children complete a questionnaire with the help of their parents if needed and the parents also complete a survey. results to date are very positive for both the parents and the children. of the 14 children who have completed the survey to date, all stated they would like to continue to meet with the group as a whole as opposed to meeting individually with team members. of the 14 parents surveyed to date, all are either very satisfied or satisfied with the group format. conclusion: we have not yet completed the survey but results to date clearly show that both the patients and their families are happy with the round table model. results will be compiled early this summer and would be ready for the poster and/or presentation in the fall. the cff clinical care guidelines recommend routine clinic visits for assessments, interventions, monitoring, education and counseling every 3 months or more often as indicated. adherence to these guidelines is important in ensuring better clinical outcomes. in reviewing our cf center data from the 2003 cff registry report, 46.2% of pediatric patients (6-18 yrs.) and 47.8% of adults (18 yrs. and older) adhered to the guidelines. national goals were not provided then; instead data was given for the top ten cf centers (80.9% and 78.5% for pediatric and adult patients respectively) as well as the national rates (55.5% and 44.8% for pediatric and adult patients respectively). our cf care center acknowledged the need to improve adherence to these guidelines and at the beginning of the year 2005, embarked on a quality improvement project to address this issue. following the pdsa (plan-do-study-act) steps, we agreed on the objective of increasing the percentage of pediatric and adult patients adhering to the guidelines by at least 50% and at the same time, to exceed the national average in 1 year or by the end of the year 2005. the physicians and social worker obtained feedback from patients and/or families during a clinic visit regarding barriers in complying with scheduled clinic visits. suggestions were elicited to overcome these barriers. the team members then identified barriers in meeting the guidelines and implemented measures to improve compliance with clinical care guidelines. among these measures were sharing the information about clinical care guidelines and importance of adherence through our cf newsletter and at every clinic visit, reminder calls to patients or caregivers at least 2 days prior to the visit, appointments for the next visit given prior to discharge from the clinic, and same day calls to patients who failed to keep their appointment for rescheduling. our social worker identified patients with difficulties or problems in consistently keeping appointments and helped address these problems. progress made in this endeavor was shared with parents and caregivers during the "parents' night" held in the fall of 2006. indeed, with consistent implementation of these measures, we have seen improvement in the percentage of patients adhering to guidelines. in our 2005 cf care center data, 68.2% of pediatric patients and 89.5% of adult patients adhered to recommended clinical care guidelines, approximating and in fact for the adults, exceeding, our target of 50% increase in the percentage (48% and 87% increase respectively for pediatric and adult patients). these data were better than the national average for 2005 (63.9% and 48.3% for pediatric and adult patients respectively) but for pediatric patients, still 22% below the national goal of 90%. adults approximated the national goal of 90%. we are committed to continually engage in the process of quality improvement in further improving adherence to clinical care guidelines and to hold on to the gains. weight z-scores and height z-scores were monitored prospectively for all infants, children, and adolescents at a pediatric outpatient cf clinic during an ongoing, multidisciplinary quality improvement (qi) initiative. all cf team members participated in this qi project which emphasized the importance of good nutrition and adequate growth for optimal lung health. specific strategies for improvement included education to families at each visit with a written nutrition action plan, increased emphasis on liquid supplements and enteral feeds as needed, and a consist message from all cf clinic staff in encouraging attention to nutrition. the study objective was to determine if multidisciplinary qi interventions to improve nutritional status had any effect on weight z-scores and/or height z-scores over a 3-year period. the null hypothesis was that z-scores for weight and height would either not improve or worsen. inclusion criterion for this study was attendance at cf oupatient clinic during the first 5 months of the qi initiative (time 1) with a subsequent clinic visit 3 years later (time 2). patients not attending clinic at both time 1 and time 2 were excluded. a cohort of 119 patients (53 male, 66 female) met the inclusion criterion. the mean time 1 to time 2 interval was 3.05 ± .11 years (range 2.70 to 3.40 years) with mean patient age at time 1 of 6.75 ± 4.55 years (range .10 to 15.80 years). pancreatic enzyme replacement therapy was used for 112 (94.1%) of the cohort. paired sample t-tests indicated significant improvements for weight z-score (p <.000, t (118) = 6.16) and height z-score (p <.000, t (118) = 4.49) after the 3-year intervention. at time 1, 89 children (74.9%) were underweight or less than 50th percentile while 30 children (25.2%) were normal-weight, at or more than 50th percentile for age. mean differences in both weight and height z-scores in the initially underweight patients were greater than those for the normal-weight patients over the 3 year interval. no significant differences were found for the initially normal weight-for-age children between time 1 and time 2 for weight z-score ( p = .83) or height z-score (p =.37). children who were not underweight at time 1 grew normally, without acceleration over the 3 yr intervention. therefore, we conclude that a longterm multidisciplinary qi project to enhance nutritional status resulted in significantly improved weight z-scores and height z-scores for those individuals who were initially underweight for age. the most common cause of morbidity and mortality for patients with cystic fibrosis (cf) is pulmonary exacerbations. these exacerbations lead to hospital admissions and treatment with intravenous (iv) antibiotics. all cf patients at our facility are admitted to a teaching service which includes house officers and students. standardized order forms are used to improve accuracy and timeliness of medications and therapies. the efficiency of this process was brought into question in 08/2006, when several parents complained about a delay in the receipt of first dose iv antibiotics after hospital admission. the objectives of this study were to determine what were the actual delivery times of the iv antibiotics to the patients (both retrospectively and prospectively), and areas where improvements could be made. our predetermined target time was < 3 hours. two analyses were performed in order to answer the objectives. the first analysis was done in a retrospective fashion. this analysis included cf patients admitted from 5/1/2006-9/30/2006. a total of 44 patients were included. total median time from admission to receipt of first dose of iv antibiotics was analyzed. a second collection of blinded prospective data was performed between 10/1/2006-12/31/06. only the pulmonologist, pharmacist collecting data, and pharmacy clinical manager were aware of the data collection. a total of 15 patients were included in the prospective project. each part of the process model were recorded and analyzed so that areas of improvement could be realized. the median time from hospital admission to first dose of antibiotics was 7 hours (range 0-31 hours) (retrospective cohort). the prospective cohort median time from hospital admission to first dose of antibiotics was 6 hours (range 67 min-11 hrs). the median time from admission to order entry by pharmacy: 2 hrs, 24 min (range 35 min-4 hrs, 11 min). the median time from the medication order being written by medical practitioner to order entry by pharmacy: 55 minutes (range 3 min-3 hrs, 20 min). the median turn-around time by the hospital pharmacy of order entry until reaching the nursing unit: 19 min (range 3 min-50 min). the median time required by the nursing staff to administer the antibiotic: 2 hrs, 23 minutes (range 29 min-6 hrs). this project was initiated in response to parental concerns. from the results of the retrospective and prospective analyses, it was determined that a delay is occurring in the receipt of iv antibiotics, which translates to hours in the hospital without therapy. this is both inconvenient and uncomfortable to the family, and wasteful of medical resources. the findings of this project were presented to the pharmacy and therapeutic committee of the hospital, to the nursing directors, and to the parents of cystic fibrosis patients. the delay is greater than expected before this project was initiated. as a result of these findings a "fast track" medication order set was developed to expedite initiation of inpatient therapy. four key areas included in this order set are sputum, iv access, diet, and iv antibiotics. we speculate that time to first drug delivery will decrease and patient satisfaction with improve. the pediatric intermountain cystic fibrosis center (imcfc) provides multidisciplinary cystic fibrosis (cf) care to 220 pediatric patients from utah and parts of idaho, wyoming, and nevada. historically the imcfc has embraced early intervention. as new therapies have been added to the cf armamentarium it has become necessary to have a method of keeping track of which patients are candidates for various therapies and also which medications have been prescribed, utilized or even should be discontinued. in order to address this quality issue, the imcfc decided to develop and implement the use of a medication tracker (mt). the imcfc decided to focus on key therapies which were felt to be at risk for variability in prescriptive practice. the five therapies included were: pulmozyme<®>, tobi<™>, hypertonic saline(hs), azithromycin and asthma medications (inhaled corticosteroids and/or leukotriene receptor antagonists). ibuprofen was intially included and later removed from the mt. prior to implementing the mt, the imcfc met to review literature, medication trackers from other centers, and develop consensus among team members. six versions of the mt have been developed between 1/1/2007-4/30/2007 . each mt has been tested in a pdsa cycle and revised. since initating the mt, 188 patients have been seen at least once. there have been 264 mt encounters. the mt is used only after the first visit to the imcfc and reflects changes to established cf care. there have been a total of 76 changes in prescribed therapies in 66 different patients(35% of patients seen). a minority of patients were receiving therapies that did not meet our pre-determined consensus for prescription. the most common therapies in this category were tobi™ and azithromycin. primary reasons cited for prescribing were: chronic suppressive antibiotic use, pseudomonas positive and <6 years old, and significant changes on chest imaging. reasons patients were not prescribed clinically indicated therapies were: patient refusal, participation in clinical trials, cost, and lack of compliance with other therapies. changes in asthma therapy included the discontinuation of medication in 17 encounters and the initiation of therapy in 8. the use of a specific cf patient mt allows consistent practice in selection of therapies. it can be used by all team members and helps to identify discrepancies in therapy. assuring all patients receive information and are considered for new therapies is increasingly important as our treatment options expand. the mt also highlights the need to consider discontinuing therapies. we speculate the mt will become an increasingly valuable tool over time and improve patient care. rationale: recent reports document increasing prevalence of antimicrobial resistance across a range of pathogens in patients with cf. the increase in multidrug resistance of these organisms complicates the therapeutic management of these patients. objective: to describe patterns of antibiotic use among physicians treating cf patients. methods: for the original purpose of evaluating site characteristics that act as effect modifiers on the relative efficacy or cost of novel therapies, we administered a survey to acquire information on practice patterns and characteristics of physicians participating in a clinical study of an investigational drug for patients with cystic fibrosis. chi-square and spearman rank test were used to evaluate associations between physician characteristics and patterns of reported antibiotic use. results: fifty-nine physicians, representing 59 study sites, completed the survey. ninety percent reported a medical specialty of pulmonology and 98% reported practicing at a teaching hospital. on average, respondents reported that about half of their practice consisted of patients with mild cf (defined by fev1 ≥ 75% of predicted normal.). forty-two percent of respondents reported prescribing oral antibiotics and 61% of respondents reported prescribing inhaled antibiotics for maintenance therapy for at least 50% of their mild cf patients. eight-five percent and 53% of respondents reported prescribing iv antibiotics for pulmonary exacerbation and pulmonary 'clean out', respectively, with about a third of these cases being prescribed for outpatient iv use. physicians that prescribed iv antibiotics for pulmonary clean out were more likely to pre-scribe oral (p= .04) and inhaled antibiotics (p=.02) for maintenance therapy than physicians that did not prescribe iv antibiotics for pulmonary clean out. conclusions: antibiotic use for maintenance therapy and iv antibiotic use for pulmonary exacerbation and pulmonary clean out acted as 'complements' rather than 'substitutes' as there was a group of physicians who reported less use of antibiotics. our findings warrant further research as well as additional exploratory analyses with a larger and broader sample to examine associations between physician characteristics and patterns of antibiotic use. understanding the relationship between physician characteristics and antibiotic use has the potential to influence the approach to management of cf patients in an environment of increasing antimicrobial resistance. introduction: hypertonic saline (hs) has been shown to increase mucus clearance and improve lung function in cf, and is now increasingly being used as part of routine therapy. one of the goals of our cf center quality improvement (qi) initiative is to increase use of hs among all eligible patients. objectives: to assess adherence to treatment with hs, parental opinions on treatment, perception of side effects, and the effect of initiating hs on the use of other nebulized medications in pediatric cf patients started on hs therapy during 2006. methods: 86 patients under 18 years of age were identified as initiating hs therapy during 2006. a telephone survey of their parents using a 10-item multiple-option questionnaire was administered in april 2007 as part of our cf center qi initiative. results: 56 parents (65%) completed the survey: 39 (70%) patients were still using hs. of those still reporting use of hs, 16 (41%) were using it twice a day; 20 (51%) once a day; and 6 less often than daily. among the 17 patients who discontinued hs, 8 (47%) stopped following recommendations from their pulmonologist; 5 (29%) stopped due to side effects; 3 (18%) felt hs took too long to administer; and 3 (18%) felt hs solution was too difficult to mix. the most common side effect reported was cough in 33 patients (59%); 19 (34%) reported no side effects. the majority of patients (64%) spent 10-30 minutes per day on hs therapy. thirteen (23%) patients discontinued another nebulized medication when they initiated hs; 11 (85%) of those stopped pulmozyme. reasons for stopping pulmozyme included: time to nebulize both medications was too long (54%), perception that hs was working better for their symptoms (23%); and doctor's instructions (23%). overall, 33 (59%) parents felt hs improved their child's symptoms; 16 (29%) saw no difference; and 2 (4%) believed hs made symptoms worse. there was a significant difference in the proportion of patients that reported improvement of their symptoms in the group using hs twice a day (15/16, 93%) when compared to those using it once a day (11/20, 55%) (p=0.022). discussion: the majority of patients continued to report the use of hs therapy several months after our initial intervention, with the most parents reporting improvement in their child's symptoms. physician recommendation was the most common reason cited for discontinuation of hs therapy. further study of physician opinions on hs therapy, particularly the timing of initiation and discontinuation, is warranted. one-half of patients reported using hs only once a day; however those reporting twice daily use were more likely to report subjective improvement of symptoms. since treatment complexity in cf increasing, determining the effectiveness of once daily hs therapy, either alone, in place of, or in combination with other therapies such as pulmozyme is essential. long-term follow up of these hs-initiated patients will focus on effects on exacerbations, pulmonary function, and hospitalizations. the uab/children's hospital (chs) cf center participated in learning and leadership collaborative ii. as part of the qi journey, the chs cf qi team thoroughly evaluated the current system of care in an effort to identify areas of needed improvement. one area assessed was clinic attendance. it was discovered that 21% of patients failed to attend clinic appointments every week. according to cff care guidelines, patients with cf should be seen a minimum of 4 times each year at the cf center. the cff has also found that those centers that see their patients more frequently have improved outcomes for fev1 and nutritional status. aim to decrease the number of missed appointments at the chs cf clinic. the chs cf center administered a qi survey to identify the reasons patients and families were missing appointments. this survey was a 7 item questionnaire that evaluated: reasons for missed appointments, if the families rescheduled missed appointments, difficulties with rescheduling, the need for reminder calls, distance traveled to the cf center, and insurance status. results 59 completed surveys were collected. 41% of the families reported medicaid as their insurance provider. 74% of families live more than 50 miles away from the cf center. 27% of the families confirmed that they had missed an appointment in cf clinic over the last 12 months. the reasons for the missed appointments were widely variable; however, 15.8% of those who missed appointments reported that they did not have access to transportation to get to the appointment. overall, families did not report difficulties in rescheduling appointments. 68% of those that missed appointments identified medicaid as their insurance which can be correlated with lower income families and 87.5% of those that missed lived greater than 50 miles away from the cf center. 83% of families reported that a reminder call would be helpful for them. interventions after evaluation of the survey data, the center quickly initiated reminder calls for cf clinics. a student employed by the cf center made these telephone calls to the patients scheduled for clinic every week. the majority of the time the student caller left a message for the family. with the implementation of this simple intervention, missed appointments have decreased from 21% to 14% which is a 30% improvement rate. reminder calls are now a routine part of the cf center's system of care. other ongoing interventions include increased involvement of primary care doctors for patients lost to follow up for more than 6 months and a system for quickly rescheduling patients. next steps in addition to reminder calls, the cf team will now determine where patients who routinely miss appointments live. if there are pockets of patients in common areas of the state, the team will attempt to maximize services for those patients such as linking them with community transportation resources, community health advocates, or providing satellite clinics. this will be accomplished through geomapping of the alabama cf population based on zip code. we designed a scoring system to uniformly identify patients experiencing a pulmonary exacerbation. the elements of this pul-monary exacerbation score (pes) have been previously described. at the time this project began the median fev 1 % predicted of the cf patients at our center 6-18 years old was below the national average (2004 cf registry data). our hypothesis was that greater uniformity of identification of pulmonary exacerbations, would improve pulmonary function in the pediatric age group at our center. this abstract describes continued improvement in the pulmonary function of this age group at our center now that the pes has become standardized in our clinical practice. methods: beginning in october 2004, a pes was calculated for all patients presenting to our center age 6-18 years of age. any patient with a pes of > 5 was defined as having a pulmonary exacerbation, and treatment with antibiotics was recommended. the patient's cf physician dictated the ultimate decision for treatment and specific course of treatment. median fev 1 of all patients age 6-18 years was calculated quarterly to determine the effect of the pes use on pulmonary function. in october 2006 the pes was incorporated into our cf clinic medical record and its use was standardized. the use of the pes, the adherence to the recommendation for treatment, and quarterly fev 1 of the population has continued to be monitored. results: from 10/2004 to 9/2006 (the duration of the original qi project), 1067 patient visits occurred, with a pes calculated on 968 (utilization of 86.7%). 327 patients were treated for a pulmonary exacerbation of the 357 patients with a pes of > 5 (adherence of 91.6 %). 64.9% of exacerbations were treated with oral and/or inhaled antibiotics, and 35.1% were treated with intravenous antibiotics. since standardization of the pes in 10/2006, 348 visits have occured. use of the score has decreased slightly (83.4% utilization), however adherence to the score has increased (92.1 % of pes > 5 have been treated). use of oral vs. iv antibiotics has been similar (69.5% oral vs. 30.5% iv). the median fev 1 of our patient population has improved from an average of 83.7 % predicted during the 24 months before implementing the pes to an average of 88.5% predicted during the ensuing 24 months of the original qi project (a 5.7 % improvement). since standardization of the pes in 10/2006, the average median fev 1 of the population has continued to increase to 90.7% predicted (an 8.4% increase from the baseline period). this improvement in median fev 1 is reflected in our cf registry data. summary: implementation of a pulmonary exacerbation score, designed to encourage uniform identification of pulmonary exacerbations in patients with cf, is associated with an improvement in median fev1 in children 6-18 years of age. standardization of the pes has resulted in continued improvement in pulmonary function of this population at our cf center. this work has been supported in part by the cff and akron children's hospital. our cf family council parent and patient advisory group has been involved in the development and implementation of this project. utilizing data from the 2005 cff patient registry, we determined that our center has had nutritional outcomes consistently below the national average for pediatric and near the average for our adult patients. as a result, we developed the global aim to improve and maintain optimal growth and nutrition in all patients at our cf center. our specific aims included: 1)to record growth parameters at all outpatient visits; 2)to identify patients at nutrition risk, categorizing them based on anthropometric data; 3) to develop and implement a specific nutrition plan based on nutritional category; and 4) to educate staff, patients,and families on the benefits of normal growth and nutrition. we surveyed our patients to determine their overall attitudes toward nutrition and nutritional care received at our center. we discovered that many patients and families did not feel that nutrition was addressed at every cf visit nor that they had a nutritional plan at each encounter. this led to the development of an intake form/home-going sheet for our patients to complete that included data elements necessary for assessment of nutrition and that clearly outlined a nutrition action plan. a standardized nutritional assessment score (nas) was devised that incorporated weight, height/length, bmi and weight for length percentiles as well as weight loss, failure to gain or maintain, and failure to remain in the expected growth channel. the nas was developed after examining the medical literature, reviewing work done by other qi teams, and acquiring input from our pediatric gastroenterologist and endocrinologist. we then developed nutritional algorithms to address nutritional intake, malabsorption, evaluation for cfrd, and short stature. these four algorithms were used to develop a nutrition action plan for each individual. beginning in march 2007, a nutritional category (optimal/acceptable, concerning, at risk or failure) was assigned to each patient at each visit based on their nas. an individualized nutrition action plan was developed with each patient. at the time of project implementation, patients age 2-20 at our center had a median bmi %ile of 52.9% with 54.5% of our patients with a bmi <50th%ile. our patients > 20 years had a median bmi of 22.1 with 52.5% having a bmi<50th%ile. after one month of implementation of the nas, 60% of our patients had been categorized with 13% of our patients having individual nutrition action plan developed. to track our progress, we will monitor median bmi, % of patients with bmi <50%ile, and % of our patients in each nutritional category quarterly using portcf. our goal is to have all our patients assessed and a nutrition action plan implemented by the end of 2007. we have developed a nutritional assessment score and nutrition treatment algorithms for pediatric and adult patients with cf as part of a qi initiative to improve nutritional status of patients at our center. the project has been supported, in part, by akron children's hospital. we are indebted to lori lundquist, a parent of two of our cf patients, who has been an integral part of our nutriton qi team. in germany there are about 8,000 cf patients who are usually treated in special centres by a team of trained and experienced health care professionals. the result of this structured approach to patient care, including frequent clinical assessments, monitoring, and aggressive interventions is an improved mean survival and a higher quality of life of pediatric and adult cf patients. however, a recent survey showed that german reimbursement schemes cover only about 50 % of resources used within these centres. objective to assess and evaluate direct costs of outpatient treatment of cf patients in germany. a micro-costing approach was used to record resource use data directly within representative cf centres. results of the evaluation may be used to initiate and support discussions between health care providers and insurances about adequate reimbursement schemes in germany. outpatient care was evaluated in seven different centres for pediatric and adult cf patients. patient reported outcomes, clinical patient data, resource utilisation, and physician related time consumption for treatment was recorded for every patient during one representative month in 2006. cost data for staff, materials, medical equipment, rooms, etc. were assessed within the respective centres. data for 326 cf patients were collected. about half of them were children and adolescents, and half of them were adults. a comparison of the resource uses to the actual remuneration of these services indicates that only about 50 % of the costs are covered by the reimbursement system. correlation analysis identified significant cost drivers like the age of the patient, comorbidities like pancreatic insufficiency and hepatobiliary complications, lung function (% fev1), and the presence of certain pathogens in the lungs. as the actual reimbursement covers only about 50 % of the resource usage costs in germany, the existence of the cf centres and sufficient treatment for cf patients is uncertain in the future. in order to ensure the existence of the centres it would be necessary to agree to a cost-covering reimbursement at minimum. this may be based on a lump-sum payment that could be differentiated for pediatric and adult centres or according to comorbidities or clinical parameters. the data calculated in this study could be used to trigger and support discussions between health care providers and insurances about a new cost-covering reimbursement system for the out-patient treatment of cf patients in germany. the impact of the new us cystic fibrosis foundation (cff) nutrition practice guidelines, i.e., discontinuing the use of percentage of ideal body weight (%ibw) to define "nutritional failure" and setting body mass index (bmi) below the 50th percentile to define "nutritional risk", on evaluating nutritional care practices is unclear. methods: data from 14702 children reported to the 2002 cff patient registry were analyzed to compare the rates of malnutrition among 113 cf centers. results: nationally, eliminating %ibw < 90% as a criterion for underweight resulted in a 6.2% reduction (from 33.1% to 26.9%) in "nutritional failure" rate. misclassification of "nutritional failure" by %ibw < 90% ranged 1.2-15.6% among 113 centers and was greater for centers having a larger proportion of tall patients. one-third of centers switched to a different tertile ranking, after correcting for misclassification by %ibw. use of bmi < 50th percentile led to the classification of 56.8% of patients as "nutritional risk". more than half of the centers had different tertile rankings on "nutritional risk" compared to "nutritional failure". a total of 5.1% (0-12.5% among 113 centers) of patients who had height < 5th percentile but bmi ≥ 50 percentile were not considered at nutritional risk. the cystic fibrosis questionnaire-revised (cfqr) is a disease specific quality of life measure that is currently undergoing clinical validation and may prove to be a useful adjunct to provider assessments. we proposed the routine use of the cfqr in a busy adult cystic fibrosis (cf) clinic would assist in identifying patients in need of limited clinic resources. this performance improvement project focused on new and infrequently-seen patients (i.e. fewer than three outpatient clinic visits/year) to determine the frequency of patients with domain scores that fall more than one standard deviation below our clinic mean. methods: the cfqr is routinely self-administered to all adult cf patients in clinic at least once every year. these assessments occur after vital signs are obtained and prior to any other clinical assessments. patients were deemed to be at baseline if there were no changes made to their pharmacologic or chest physiotherapy regimens. the mean and standard deviation of each domain of baseline cfqr assessments were determined. the frequency of rarely seen and new patients with domain scores less than one standard deviation below the mean was determined. results (202). of these 84 baseline assessments, forty five were obtained from patients who were rarely seen or new to the clinic. 48.9% (n=22) of these patients had one or more domain scores falling more than one standard deviation below the mean. 35.5% (n=16) of these patients had two or more domain scores below these cutoffs. 20% (n=9) of the rarely evaluated patients had domain composite scores (i.e. the sum of the individual domains) that fell below the standard deviation cutoff. the most common outlying domains in these infrequently seen or new patients were body (10), weight (10), physical (7), health perceptions (7) and the composite domain (9). conclusions: the cfqr is easy to administer and score during routine adult cf clinical visits. as many as 35% of our infrequently evaluated patients had multiple scores that were more than one standard deviation below our clinic mean. we conclude that the cfqr is a useful adjunctive measure to provider assessments and can help focus limited personnel resources. aminoglycoside antibiotics are known to be toxic to the inner ear. however, they continue to have a leading role in the treatment of certain infections and in the treatment of pulmonary exacerbations in patients having cystic fibrosis (cf). while there is ample evidence in the literature of ototoxicity, and there are established protocols for monitoring the effects of toxic agents on hearing, less is known about the prevalence of vestibular toxicity among patients who are exposed, and there are no commonly accepted protocols for monitoring vestibular system function. oscillopsia and unsteadiness when standing and walking are the two most commonly reported and disturbing symptoms associated with bilateral loss of vestibular system function. oscillopsia is the perception that viewed stationary objects or surroundings move coincident with head movement. when it is severe, oscillopsia can prevent an individual from having clear vision with even the slightest head movement. the unsteadiness that accompanies vestibular loss can range from mild to disabling. because of the referral of a number of patients to the vestibular testing center (vtc) at the university of michigan in whom severe bilateral vestibular loss was evident secondary to apparent aminoglycoside toxicity and our concern about quality of life issues, we initiated a quality improvement clinical protocol with our cf patients. vestibular system function and hearing are evaluated at the time of admission for pulmonary exacerbations and coincident with the three month follow-up visit with the pulmonologist. our goal is to understand the incidence of ototoxicity in this patient population, and to determine how best to measure incremental changes over time. the ultimate desired outcome is to investigate the efficacy of protective agents for limiting the toxic impact of these drugs on hearing and vestibular function. to date, we have completed vestibular testing on 30 patients with cf ranging in age from 10-56 years, some of whom were referred prior to the initiation of this monitoring program. of the sample, 4 individuals have completely normal vestibular test results, and 2 of the 4 had no prior aminoglycoside exposure. of the remaining patients, 10 have bilateral vestibular loss ranging from mild to severe, two have evidence of a unilateral vestibular loss, and each of the others has non-lateralizing evidence of vestibular involvement. we have also documented change in the three individuals we have seen for repeat testing. specifically, repeat testing has shown incremental decline in vestibular function with repeated exposure to aminoglycosides. interestingly, only 6 individuals in the group have documented hearing loss, and although most report that they do not and have never had any problems with dizziness or balance, many patients have evidence of oscillopsia or abnormal postural control test results. it is clear from our limited data that it is important to monitor vestibular system function whenever potentially toxic agents are used. while monitoring hearing is also warranted, our data suggest that a monitoring program that includes only hearing is insufficient. background: the correlation between bmi, lung function, and longterm outcomes in cystic fibrosis patients has been well documented. based on this data the cf foundation (cff) has established adult patient care guidelines for bmi of 22 or greater for women and 23 or greater for men. in our cf center implementation of nutrition therapies in cf adult patients has been difficult due to lack of acceptance of proposed interventions. due to the importance of optimal nutrition to attain the cff recommendation for bmi, university of cincinnati adult cf center implemented a weight management quality improvement protocol to improve the nutritional status of patients below the cff recommended bmi. our aim is to improve to the nutritional outcomes in our center through patients' acceptance of our nutrition interventions. strategies: as part of the weight management protocol we developed a nutrition action plan, nutrition algorithm, and weight management education record and learning log. the action plan took a systematic approach to weight management, classifying patients into different risk categories based on bmi. patients were categorized as mild nutritional insufficiency (bmi 20-21), moderate nutritional insufficiency , and severe nutritional insufficiency . each category required a specific action to address weight loss or inability to gain weight. areas of focus for the action plan include; intake assessment through computerized dietary analysis, assessment of pancreatic function, pancreatic enzyme replacement therapy (pert) regimen assessment, 72 hour stool collection for malabsorption, assessment of glycemic control, assessment for gerd, and enteral/parental feeding. the dietitian completed a chart review and nutrition assessment with the patients and classified them into one of the three nutritional risk categories. eleven patients with a bmi below 19 were initially identified. nine (82%) of the eleven patients with a bmi of 19 or less have been approached with the protocol and algorithm. patients completed a three day food journal and oral intake was assessed via computerized dietary analysis. oral enteral supplements were recommended 2-3 times per day. g-tube placement was discussed and an educational material on tube feeding was distributed. the protocol and algorithm guided identification and assessment of contributing factors to weight loss. results: of eighty patients seen in the adult center since the beginning of october, thirty-seven of the patients (46%) have a bmi below 22 and eleven patients (14%) have a bmi below 19. out of the nine patients approached with the protocol 55% (n= 5) completed and returned the three day food journal. of the patients that completed their food journal 60% (n= 3) agreed to a peg placement, one of the patients is still contemplating peg placement while sorting through some psychosocial issues, and the other patient has had a differential diagnosis that might negate the necessity for a peg placement. conclusion: we developed a systematic approach to weight management within our adult cf program. this systematic approach to weight management, which includes patient involvement and specific education, has improved the overall acceptance of nutrition interventions in our adult population. the inpatient care of adults with cystic fibrosis can be a challenging given complicated medical regimens, specialized respiratory care orders, and unique dietary requirements. a large proportion of these patients are admitted to academic centers with rotating house staff that may have never treated an adult with cystic fibrosis. to improve the quality of care of our adult patients we have implemented a standardized electronic order set, a recurring educational program, and a "pocket card" to help the house staff. the adult cystic fibrosis program at vanderbilt cares for over 160 patients, with 7-12 inpatient admissions per month. vanderbilt's inpatient order system is completely electronic. we have devised an order set based on our agreed standard of care for inpatients with pneumonia. the order set guides the house staff through most aspects of inpatient care including culturing, antibiotic choice and dosing, dietary consultation, nutritional recommendations, and respiratory care orders. this order set not only educates the house staff on our standard of care but also provides direct access to each order they may wish to enter. we have reiterated this educational process with a pocket sized handout that guides them through the typical issues in adults with cf as well as provide phone numbers to members of the cf team. these resources are presented in short talk given at the beginning of each month to incoming house staff. a preliminary data analysis suggests that over 90% of all adults with cf are admitted using some component of this order set. an informal survey of the house staff also concludes that the pocket card and lectures have been helpful. all three aspects of this quality improvement have been implemented since december of 2006. we plan to formally test the impact of this intervention when a meaningful number of house staff have been through the service. * background: malnutrition is a common complication of cystic fibrosis (cf). the median bmi percentile in cf patients between the ages of 2 and 20 years is 44.8% but ranges between various centers from 23.6 to 65.9 percent.1 previous research has shown a trend toward improvement of pulmonary disease and a significant improvement in weight, height, and bmi z-scores after nutritional supplementation via a gastrostomy tube2; furthermore, fev1 is positively correlated with bmi percentile.1 higher weight at three years of age predicts better pulmonary function at six years of age.3 the effect of gastrostomy tube feedings on pulmonary exacerbation frequency has not been well defined. hypothesis: the incidence of pulmonary exacerbations in children will decrease after the initiation of supplemental gastrostomy feedings. methods: a retrospective chart review of all pediatric patients seen at the cmh cf center, and who received gastrostomy tubes between 1997 and 2007 was performed. data was evaluated in six month time intervals for two years prior and two years after gastrostomy tube placement. we assessed weight, height and bmi percentiles, fev1 percent predicted, number of courses of exacerbation therapy (intravenous and oral) and microbiologic data. results: we identified 20 patients, 14 females and 6 males, with ages ranging from 11 months to 14 years of age (6.82 years mean) at time of gastrostomy tube insertion. all patients had increases in their weight-forage percentile (mean of all patients of 13.83 percentile pre to 31.23 percentile post, p<0.05), height-for-age percentile (16.78 percentile pre to 27.03 percentile post, p<0.05), and bmi percentiles (24.23 percentile pre to 45.98 percentile post, p<0.05). the mean fev1 percent predicted declined overall from 85.5% to 78.1% (2.2 percent predicted per year) over four years in 10 patients. there was a trend of exacerbation reduction after gastrostomy tube placement from 153 to 121 total courses (p=0.06). this decline in exacerbations after gastrostomy tube placement was most dramatic in those patients growing pseudomonas aeruginosa without other organisms, with a decline from 90 total courses to 60 total courses in 10 patients (p=0.05). discussion: weight, height and bmi percentile improved after initiation of gastrostomy tube feedings. lung function (fev1 % predicted) decreased at the rate predicted in spite of gastrostomy feedings. there is a trend toward decrease in exacerbation frequency after improved nutrition with gastrostomy tube feedings, especially in patients infected with pseudomonas aeruginosa. limitations of this study include the small sample size, relatively short follow-up time, and the retrospective nature of the study which did not allow assessment of adherence. conclusion: improved nutrition through gastrostomy tube placement may decrease the frequency of pulmonary exacerbations. evidence of an association of improved pulmonary function with improved nutritional status has motivated aggressive nutritional intervention. percutaneous endoscopic gastrostomies (pegs) are employed increasingly in cystic fibrosis to optimize nutritional status. we reviewed our center data over the past 15 years to assess changes in our peg practice and effects on nutritional and pulmonary outcomes. we hypothesized that our change in practice to earlier peg intervention improved patient's nutritional and pulmonary outcomes. we conducted a case controlled retrospective study of pediatric cf patients spanning the past 15 years divided into two periods: p1 (peg placed before 1996) and p2 (peg placed 1996 or later) compared with their respective age/sex matched controls. we compared peg vs. control cf patients with respect to age, nutritional and pulmonary status. we compared weight, height and bmi directly preceding peg placement and at approximately one-year post peg. we compared available data for best yearly fev1 and fvc before peg and at 1, 5 and 10-years post-peg. we then compared data between the two time periods. results: peg patients in p2 (n=38; mean age 6.6 + 5.1 yrs.) had lower bmi ( z = -1.2 v. -0.2, p<0.001) pre-peg placement compared to controls. by 1 year post peg, the bmi normalized and equaled controls (z=-0.4 v. -0.1, p=0.1). peg patients' fev1 %ile prior to placement was lower than controls (88 v. 94, p=0.2) as was their fvc %ile (97 v. 103, p=0.2) and remained lower at 1 year post-peg. following peg placement, absolute bmi increased (0.9kg/m2, p=0.009), as did bmi z-score (0.6, p=0.01). we did not see significant changes in pulmonary function tests (pft). peg patients in p1, (n= 12; mean age 10.7 + 2.3 yrs.) also had lower bmi (z= -1.6 v. -0.3, p= 0.0001) pre-peg than controls. at 1 year post-peg, the peg patients still had a significantly lower bmi (z = -0.7 v. -0.2, p=0.08). fev1 was lower in peg patients compared to controls in pre-, 1year, and 5-year post-peg data (p<0.05). variability in pfts and small sample size limited our analysis. pre-peg weight, height, bmi, fev1 and fvc were lower in p1 patients compared to the younger p2 patients. at 1-year post-peg, similar bmi zscores (-0.7, p=0.99) was obtained between the two periods. variability in pfts among the patients within each period prevented valid comparisons, although p2 tended to have better pft values. conclusions: peg placement was effective at improving nutritional status in patients with cystic fibrosis. placement in younger patients with better pre-peg nutritional and pulmonary status allowed more complete nutritional recovery. we speculate that improved height z-scores with apparently stable fev1 is consistent with increased pulmonary growth. demonstration of an attenuated rate of pft decline to correlate with this earlier intervention will require a larger population and longer observation period. a multi-center longitudinal outcome study would help determine optimal timing and criteria for peg intervention. 1 1. pediatric gastroenterology and hepatology, university medical center groningen, groningen, netherlands; 2. biochemistery, erasmus medical center, rotterdam, netherlands background: ursodeoxycholic acid (udca) treatment is frequently applied for cystic fibrosis-related liver disease (cfld). it has been hypothesized that udca is beneficial through its choleretic activity, i.e. its capacity to induce bile flow. the hepatic expression of the cftr protein is restricted to the cholangiocytes lining the bile ducts. cholangiocytic cftr is involved in the generation of ductular bile flow. it is not known to what extent the choleretic activity of bile salt treatment depends on expression of competent cftr protein, and whether or not udca differs in this respect from other bile salts. we evaluated the role of cftr in the acute and chronic choleretic effect of bile salt treatment, through comparative studies in cftr-null, homozygous f508del, and corresponding wild type control (wt) mice. methods: bile flow was determined after gallbladder cannulation. bile flow during the first 30 minutes after acute interruption of the enterohepatic circulation was regarded as basal bile flow. after 30 min, taurocholic acid (tca) or tauroursodeoxycholic acid (tudca) were iv administered in stepwise increasing dosages, up to 1200 nmol/min/100g bw, to cftr-null mice and wt littermates fed a standard chow diet. other cftr-null, homozygous f508del mice and their respective wt littermates were fed either the standard chow, or the same diet supplemented with cholic acid (ca, 0.5wt%) for 3 weeks. finally, cftr-null mice and wt littermates were fed the standard chow or the same diet supplemented with ursodeoxycholic acid (udca, 0.5 wt%) for 3 weeks. : upon a regular chow diet, the basal bile flow was similar in cftr-null and homozygous f508del mice, compared to their respective wt littermates (cftr-null, 6.3±1.8 vs. 6.2±0.1; and, f508del(∆/∆), 5.4±2.8 vs. 8.5±10.8 µl/min.100 g bw, resp.; ns). iv administration of tca or tudca to cftr-null mice and wt littermates increased bile flow to similar extents. dietary ca treatment increased basal bile flow significantly less in cftr-null and homozygous f508del mice than in their respective wt littermates (cftr-null, 13.7±3.0 vs. 17.9±1.7, p<0.05; and, f508del(∆/∆), 20.9±5.5 vs. 26.2±6.2 µl/min.100 g bw, p=0.06; resp.). interestingly, dietary udca treatment increased basal bile flow more profoundly than ca treatment, and to similar levels in cftr-null mice and wt littermates (+~500%, 29.0±5.9 vs. 31.0±4.4 µl/min/100 g bw, resp.; ns). conclusion: upon chronic treatment, udca is highly choleretic in mice, independently of the presence of functional cftr. the independence of functional cftr is udca specific, since the choleretic activity of chronic ca treatment is diminished in cftr-null and homozygous f508del mice. we speculate that this specific, cftr-independent choleretic effect of chronic udca treatment could be therapeutically important for cystic fibrosis. most cystic fibrosis (cf) patients have mild to moderate focal portal tract changes (ductal obstruction and cholangitis) and hepatosteatosis. etiology of severe liver disease (ld)with cirrhosis and portal hypertension which occurs in 5-7% of cf patients is unknown. the c57bl/6j congenic cf mice develop progressive cf-like ld. as a useful therapeutic model, we have shown that dietary addition of docosahexaenoic acid (dha) significantly ameliorates inflammation but has little effect on ductal obstruction (am j physiol gastrointest liver physiol 292: g839-g848, 2007) . we hypothesized that treatment with udca,a hydrophilic bile acid will ameliorate portal tract ductal obstruction and have an additive effect with dha in preventing ld. methods: thirty-day old wild-type and cf littermates are fed peptamen and either olive oil or dha, with and without 0.4% udca (8 groups,10 mice/group). mice are killed following 90 days of treatment and bile salts, serum liver enzymes and serum and tissue lipids analyzed. h&e stained liver tissues are coded and assessed blindly by an expert hepatopathologist (jp) for evidence of hepatosteatosis, inflammation, bile duct obstruction, fibrosis and other signs of liver pathology. an arbitrary scale of 0 to 4 is used, with 0 representing no pathology and 4 being the most severe. we present interim statistical analysis performed for pathology results only for untreated mice (wt, n=7; cf, n=6); treated mice with dha, (wt, n=7; cf, n=6), treated mice with udca (wt, n=7; cf, n=7) and treated mice with udca and dha (wt, n=6; cf, n=6). results: mean pathology scores from groups of mice are pooled to examine the effects of genotype, treatment and diet (table) . at present,the bile,lipid and liver biochemical test data are insufficient to perform statistical analysis. udca-treated mice have significantly increased bile duct obstructed scores compared to the untreated mice which subanalyzed (not shown) reveals increased bile duct obstruction in the wild-type littermates and no effect in cf mice. hepatosteatosis and inflammatory scores in the untreated mice (fed olive oil) are increased compared to the mice treated with dha. summary: we anticipate completing and performing final analysis within six months. however, these preliminary data confirm that dha reduces inflammation in c57bl/6j mice livers and suggest that udca therapy has an adverse effect in wt mice and is not effective in ameliorating liver disease in cf mice. udca with dha therapy does not appear to have an additive effect in ameliorating cfld in this mouse model. supported by axcan pharma inc. background the cf mouse intestine has an innate response associated with small intestinal bacterial overgrowth (sibo). a previous affymetrix genechip analysis of the cf mouse intestine showed altered expression of several eicosanoid metabolic genes. eicosanoids are biologically active arachidonic acid metabolites with a variety of pro-and anti-inflammatory actions as well as effects on mucus production, electrolyte transport, and gastrointestinal motility, all of which are of potential importance to the cf phenotype of the intestine. methods wild type (wt) and cf mouse (cftr tm1unc ) littermates congenic on the c57bl/6j background were fed an elemental liquid diet (peptamen). intestinal rna was isolated for quantitative rt-pcr of expression levels of genes involved in eicosanoid metabolism to confirm and extend the genechip analysis. various eicosanoids were measured in small intestinal lavage fluid using specific enzyme immunoassays. results we confirmed microarray results by qrt-pcr that the following genes were differentially expressed in the cf small intestine relative to wt: cyp4a10 (cytochrome p450 4a10; 20-hete synthesis; 5% of wt); ltb4dh (leukotriene b 4 dehydrogenase; metabolizes prostaglandins and leukotrienes; 22% of wt); cyp2c40 (cytochrome p450 2c40; 16-hete synthesis; 25% of wt); ltc4s (leukotriene c 4 synthase; ltc 4 synthesis; 25% of wt); hpgd (hydroxyprostaglandin dehydrogenase 15; metabolizes prostaglandins; 30% of wt); and pla2g5 (group 5 phospholipase a 2 ; potentiates arachidonic acid generation; 500% of wt). non-significant or less than 2-fold changes in gene expression were measured for: cyclooxygenase (cox) genes, pge synthase, pgi synthase, and 5-and 15-lipoxygenases. as measured by their stable metabolic products, pge 2 and pgf 2α were significantly increased in the cf mouse intestine (600% and 1190% of wt, respectively). the following eicosanoids were significantly decreased in the cf mice: 12-hete (20% of wt), 15-hete (19% of wt) and 20-hete (4% of wt). there were not significant changes in the levels of pgi 2 , pgd 2 , ltb 4 , cys-lts, or lxa 4 . the changed levels of eicosanoids are generally consistent with the differential gene expression. conclusions there are significant changes in expression of eicosanoid metabolic genes and certain eicosanoids in the cf mouse small intestine. the increase in pla2g5 and decrease in cyp expression levels are expected to make more arachidonic acid available for eicosanoids except hetes. decreases in degradative genes (hpgd, ltb4dh) will prolong availability of prostaglandins. altered levels of eicosanoids are expected to play important roles in the pathophysiology of the cf intestine. elevated pge 2 may contribute to increased mucus secretion which is characteristic of cf. pgf 2α and pge 2 regulate intestinal motility and small intestinal transit is slower in cf patients and in cf mice. the hetes are mostly known for their effects on vascular tone and electrolyte transport in the kidney but are less-well characterized in the intestine. their dramatic decrease in the cf intestine may affect blood flow, electrolyte transport, intestinal motility, or other gi functions. the specific effects of the altered eicosanoid metabolism in cf remain to be explored. supported by cff grant delisl05g0. for the scandinavian study consortium. background: progressive pulmonary disease, correlating with igg levels, is a major cause of morbidity and mortality in cf patients. recently, immunosuppressive effects of vitamin d and a potential role of vitamin d deficiency in the pathophysiology of autoimmune diseases have been described. we investigated whether vitamin d deficiency could contribute to the chronic proinflammatory state characterizing cf and the higher occurrence of immune-hyperreactivity-related conditions in cf patients. methods: multiple linear regression analysis, taking the confounding factor of age into account, was used to evaluate the relationship between cross-sectionally determined vitamin d variables and: lung function parameters, inflammatory parameters and immune hyperreactivity markers in 898 patients followed at the cf centers in sweden, norway and denmark. vitamin d intake was based on a 7-day precoded dietary food record, calculated in the national food databases. findings: in the population of all patients included in the study (n=898), blood vitamin d positively correlated with fev1 (p<0,01) as well as with fvc (p<0,01). negative correlation was found between blood vitamin d and igg (p<0,001), supplemented vitamin d per kg bw and igg (p<0,001), total vitamin d intake per kg bw and igg (p<0,05). additionally, lower blood vitamin d levels were related to both presence of pathological ogtt (p<0,05) and occurrence of cfrdm (p<0,05). correspondingly, food vitamin d (p<0,05), food vitamin d per kg bw (p<0,05), supplemented vitamin d (p<0,05), supplemented vitamin d per kg bw (p<0,001), total vitamin d intake (p<0,01) and total vitamin d intake per kg bw (p<0,01) all correlated negatively with hba1c values. most of the studied vitamin d variables were lower in patients with endomysial antibodies positivity (n=14) and higher in patients with abpa (n=4) than in the rest of cf population studied (ns). interestingly, in children (n=442) blood vitamin d correlated negatively with transglutaminase levels (p<0,05) and there was also a significant negative correlation between hba1c and vitamin d intake. surprisingly, in adult cf patients (n=456) iga positively correlated with total vitamin d intake (p<0.05) and total vitamin d intake per kg bw (p<0.05). in the population of stockholm cf-center patients (n=165), where it was possible to allow for the influence of both age and compliance, negative correlation between blood vitamin d and igg (p<0.01) was found. after allowing for age, compliance showed significant correlation only with igg (p<0.001) and esr (p<0.05), and with none of the rest of the studied variables. conclusions: vitamin d deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of dm and celiac disease in cf patients. new recommendations for vitamin d supplementation and monitoring of blood vitamin d levels are needed, in order to improve immunological balance and decrease the need for anti-inflammatory therapy in cf patients. supported by swedish heart lung foundation, stiftelsen frimurare-barnhuset i stockholm, norwegian and swedish cystic fibrosis associations, and by an unrestricted grant from solvay pharma. von drygalski, a.; biller, j.a. medical college of wisconsin, milwaukee, wi, usa anemia is associated with increased morbidity and mortality in other chronic diseases, but little is known about anemia in cf. recognition and correction of anemia in cf might lead to better outcomes. chronic inflammation, iron deficiency and malabsorption may be important mechanisms contributing to the anemia in cf patients. we hypothesized that anemia might be correlated with poor lung function. methods: clinical charts and portcf.org visit logs of 218 patients of all ages (1 m to 61 yrs) with cf were reviewed for as many years as charts permitted (range 1-7 yrs). the following data were extracted: cbc, iron studies, pulmonary function tests (pfts), vitamins a, d, and e levels, creatinine, pertinent medical history, and current medications. anemia was defined by age-and gender-specific world health organization (who) criteria. patients were considered anemic if low hemoglobin(hb) was present on two separate occasions at least two months apart, or average annual hb met who criteria. pfts in 176 patients (all >6 yrs) included forced expiratory volume (fev1) and forced vital capacity (fvc) as percent predicted of normal and were considered a reflection of patient performance. the most representative annual pft from at least 4 annual tests was chosen for analysis since acute infection adversely influences daily performance. results: 61 of 218 patients were anemic (prevalence 28%). prevalence of anemia increased with age from 11.5% in patients < age 18 to 58% in patients > age 40 and was significantly higher in patients with vitamin deficiencies. 90% vitamin-deficient patients vs. 59.5% non-vitamin deficient patients were anemic (p=0.02). mean hb was 10.1 mg/dl (range 6.6-12.9). roughly two thirds of patients had moderate and severe anemia (hb <11 mg/dl) with no gender difference in regard to prevalence or severity identified. complete iron studies were available in 16/48 patients. 7 were identified as iron deficiency anemia (ida), 4 as anemia of chronic illness(aci), and 3 as combined; 2 others had renal failure. in 32 patients, iron studies were incomplete, but renal failure, hemoptysis, hematochezia and solid organ transplants were contributing factors in half. pfts obtained in 176 patients ≥ 6 yrs were compared in anemic and non-anemic patients. mean fev1 and fvc at all ages were statistically significantly poorer in the anemic patients (p <0.005). complete iron studies were also available in 9 non-anemic patients with impaired lung function. 8/9 patients had ferritins <35 ng/ml suggesting relative iron deficiency. conclusions: the prevalence of anemia in cf patients is high and increases with age. in patients for whom iron studies were available, ida was the prominent underlying mechanism, followed by aci or a combination of both. in addition, a group of non-anemic patients with poor lung function had relative iron deficiency. the strikingly significant association between anemia and poor pulmonary function as well as the higher prevalence of anemia in vitamin deficient patients uncover anemia as significant co-morbidity in cf. identification of iron deficiency in anemic as well as in non-anemic patients with poor lung function could be important since iron supplementation might allow hb levels to increase as an appropriate response to hypoxia. oral administration of dha to a cf mouse model corrected the lipid imbalance and reversed certain pathological manifestations in tissues affected by cf. the aim of this study was to investigate the metabolism of la through the n-6 pathway, and to determine the effect of dha supplementation on la metabolism in cultured cells with a cf phenotype. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. cells were supplemented with various concentrations of la (ranging from 25 to 200 µm) and dha (10, 20, and 40 µm) for one week. cellular lipids were extracted from confluent monolayers, and fatty acids were methylated and analyzed by gc-ms. metabolism through the n-6 pathway was studied by incubating the cells with 4.6 µm [ 14 c] la for 4 hours, and quantitating its downstream metabolites by hplc. results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: 10.8±0.2, cf: 8.2±0.1%, p<0.01; dha in wt: 2.0±0.2, cf: 0.6±0.1%, p<0.01, mean ± sem for each). la supplementation resulted in an increase in la and aa levels in both wt and cf cells. at most la concentrations tested, la levels were significantly lower and aa levels were significantly higher in cf cells, indicating an increased production of aa from la in cf cells. the study of [ 14 c] la metabolites showed an increased production of multiple downstream fatty acids including 18:3n-6 (3.1 fold), aa (4.3 fold), and 22:5n-6 (2.7 fold) in cf cells compared to wt cells, further substantiating an increased metabolism through the n-6 pathway. supplementing 16 hbe cells with dha for 1 week resulted in a significant increase in la levels and a corresponding decrease in aa levels in wt and cf cells. when supplemental dha was added in combination with la, dha inhibited the la-derived increase in aa levels in cf cells and normalized them to the wt values. the formation of [ 14 c] la metabolites was inhibited after 24-hour supplementation with dha. this inhibitory effect of dha was greatest on aa production, and it was more prominent in cf than in wt cells (wt: 3.7 fold, cf: 12.4 fold decrease in aa production). conclusions: decreased la levels in cf are at least in part related to increased la metabolism through the n-6 pathway. the fact that dha supplementation decreased the flux from la to fatty acid metabolites in the n-6 pathway, especially to aa, may partially explain dha's mechanism of therapeutic benefit. these data would also suggest that dha combined with la in the diet may normalize fatty acid metabolism in cf patients. background: decreased levels of linoleic acid (la) and docosahexaenoic acid (dha) have been found in the blood and tissues of cf patients. studies on cultured cf cells and cftr-/-mice have indicated that decreased la levels in cf might be related to its increased metabolism through the n-6 pathway. other potential mechanisms of the observed decreased la and dha levels in cf could be related to altered cellular uptake of these two essential fatty acids. the goal of this study was to investigate the uptake of la and dha into cultured cf cells, and their distribution among the major lipid classes. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. the uptake of la and dha into the cells was studied by incubating the cells with 0.45 µm [ 14 c] la or [ 14 14c] dha for 1 hour and 4 hours, followed by measurement of radioactivity in the supernatant and cell lysate. lipid fractions from the cells (total phospholipids, triglycerides, cholesteryl esters, and free fatty acids) were separated by thin layer chromatography (tlc), and the fatty acid composition was determined by gc-ms after methylation. incorporation of la and dha into cellular lipid fractions was determined by incubating the cells with 4.5 µm [ 14 c] la or 9.1 µm [ 14 c] dha for 1 hour and 4 hours, followed by tlc separation of lipids and measurement of radioactivity associated with the various fractions. (all data are mean±sem). results: the levels of la and dha were significantly decreased in cf cells compared to wt cells (la in wt: 10.8±0.2, cf: 8.2±0.1%, p<0.01; dha in wt: 2.0±0.2, cf: 0.6±0.1%, p<0.01). fatty acid composition analysis of lipid fractions indicated that la levels were significantly decreased in the phospholipid and triglyceride fractions of cf cells (80.9% and 42.3% of the wt values, respectively), and dha levels were significantly decreased in the phospholipid fraction of cf cells (21.2% of the wt values). the uptake of la was significantly higher into cf cells compared to wt cells at 1 hour (wt: 14.5±0.1, cf: 35.5±0.9 dpm/µg protein, p<0.01) and 4 hours (wt: 34.7±2.9, cf: 100.8±5.0 dpm/µg protein, p<0.01). the uptake of dha was also significantly higher in cf cells compared to wt cells at 1 hour (wt: 46.3±8.4, cf: 131.2±8.7 dpm/µg protein, p<0.01) and 4 hours (wt: 140.7±12.9, cf: 276.8±12.0 dpm/µg protein, p<0.01). incorporation of la and dha was increased in the phospholipid, cholesteryl ester, and free fatty acid fractions of cf cells at 4 hours. dha, but not la, incorporation was significantly increased in the triglyceride fraction of cf cells. conclusions: low la and dha levels in cf cells are associated with a decrease in cellular lipid fractions, the largest of which is in total phospholipids. there is an increased uptake of la and dha into cf cells, and into most lipid fractions of these cells. the decreased levels of la and dha in cf cells, despite their increased cellular uptake and esterification in lipid classes, indicate that there is increased mobilization of these fatty acids in cultured cf cells. perez, a.; issler, a.; davis, p.b. pediatrics, cwru, cleveland, oh, usa even though lung disease continues to be the primary cause of death in cystic fibrosis (cf), the considerable increase in life expectancy of the cf patient over the last decades, has made management of the diverse gastrointestinal and nutritional complications in the adult cf increasingly important. malnutrition and fatty acid deficiency has been a hallmark of cf and its impact in long-term lung health has been established. data obtained by us using affymetrix mg-u74av2 arrays from ileum and liver of pristine 8-week-old male mice bearing the y122x cf mutation and their normal littermates, indicates that there is a profound disturbance in genes associated with lipid metabolism, many of which are regulated by the peroxisome proliferator-activated receptor-gamma (pparγ), a ligand-regulated transcription factor that regulates lipid metabolism and possess anti-inflammatory properties. we hypothesize that lack of cftr function results in altered expression of pparγ regulated genes in the gut and liver, leading to the fatty acid metabolism abnormalities observed in cf. pparγ mrna and protein levels were measured in ileum, colon, liver, and fat of 8 week old female mice bearing the cf mutation s489x and its wt littermates (c57bl/6j background), and fabpcftr mice (gut corrected, mixed background) after the administration of ethanol or pioglitazone (a pparγ agonist, 30 mg/kg) by gavage every 24h for 2 days, sacrificed 24h after last dose. highest pparγ mrna levels were found in fat, followed by colon, liver, and lowest in ileum. at basal state, mrna levels were higher only in fabp ileum vs. wt and fabp colon vs. cf. after treatment, higher expression was seen in cf and fabp ileums and cf liver vs. controls and wt treated mice. however, there were striking differences at the protein level, especially in the colon. at basal state, pparγ was localized, mainly at the surface of the epithelia in wt, in crypts in cf, and mainly in crypts but extending towards the surface in fabp mice. upon treatment, pparγ location in wt colon moved almost exclusively to the crypts, and in the cf and fabp mice, although expression remained mainly in crypts, pparγ could now be seen at the surface. changes in pparγ protein were also seen in ileum and especially liver, between wt and cf, at basal and upon treatment, but changes were subtle and will require more sensitive methods to further examine them. we also examined mrna and protein levels for several genes involved in fat processing that could be related directly or indirectly to pparγ. mrna and protein levels for aqp1 and cubilin, and protein levels for megalin, apoa-1, and apob-48 were reduced in the ileum of cf mice compared to wt. our data indicates a pparγ dysfunction in the gastrointestinal system of cf mice, which could be responsible for the gastrointestinal manifestations observed in cf. understanding the association between pparγ, cftr, and fat processing may help us to develop new therapies directed towards improving the nutritional status of cf patients that, in the long run, will influence their lung health. cf is characterized by low linoleic acid (la) and docosahexaenoic acid (dha) levels. although this could be due to a primary abnormality in fatty acid biosynthesis, this could also be explained by alterations in phosphatidylcholine (pc) formation. pc are formed by two pathways. the most utilized is the de novo conversion of choline to pc via the cdp-choline pathway, which preferentially forms pc containing saturated and monounsaturated fatty acids. in the other pathway phosphatidylethanolamine (pe) is converted to pc through three methylation steps utilizing the enzyme phosphatidylethanolamine n-methyltransferase (pemt). this pathway preferentially forms pc containing polyunsaturated fatty acids. thus decreased pemt activity would be predicted to result in decreased dha and la levels. this is supported by results seen in pemt knockout mice (watkins et al, j nutrition, 2003) showing decreased dha and la levels, and variable to increased levels of arachidonic acid (aa). furthermore, innis et al have shown that cf patients have decreased choline levels making them more dependent on the pemt pathway. we hypothesized that alterations in fatty acid levels in cf may be due at least in part to decreased pemt activity. methods: cftr -/mice (cf) and wt littermates were fed with either a diet containing choline (control) or, in order to mimic choline status in humans, an otherwise matched choline deficient (cd) liquid diet for 7 days. choline levels were measured using a kit. livers were perfused and microsomes prepared. to measure pemt activity, microsomes were incubated with radioactive s-adenosylmethionine in the presence of dimethyl pe. the pc product was extracted and radioactivity measured. fatty acids from liver and pancreas were analyzed by gc/ms. results: there was no difference in choline levels in serum or liver comparing wt and cf mice. there was no difference in pemt activity in livers from cf mice compared to wt on control diet. cf mice on cd diet had decreased pemt activity in the liver compared to wt mice on cd diet (wt:26145±1893; cf:16515±1240 cpm/mg protein/min; p<0.001 conclusion: induction of choline deficiency in cf mice to mimic the situation in cf patients, leads to decreased pemt activity. this resulted in low la levels compared to wt mice in both the liver and pancreas and indicates that the fatty acid abnormality in cf is at least in part due to defective pc metabolism through the pemt pathway. (4995). urolithiasis appears to be more common in adult cystic fibrosis (cf) patients than in the general population. a variety of urinary findings have been observed which could account for this, including low volumes, raised oxalate and low citrate, but not all the reports are consistent. most cf patients have pancreatic insufficiency and a plausible explanation for an elevated oxalate excretion in these patients is malabsorbed fat in the intestinal tract sequestering calcium and permitting more oxalate to be absorbed from their food. if this were the mechanism for the elevated stone risk in cf patients then we would expect to observe differences in stone incidence and oxalate excretion between cf patients with exocrine pancreatic sufficiency (ps) and pancreatic insufficiency (pi). methods diagnosis of cf is by clinical and genetic criteria and ps or pi status is based on faecal elastase measurement. 39 patients and 14 healthy volunteers (hv) provided 24h urines for analysis of volume, ph, ca, mg, na, k, po 4 , oxalate, citrate, creatinine, urea. supersaturations were calculated using equil2. there is a history of renal stone disease in 20 out of 307 cf patients attending our unit. 286 patients have pi and include 19 of the stone formers. the remaining stone former is amongst the 21 ps patients. no significant differences in urine chemistry or derived supersaturations were found between the cf patients without a history of stones that were ps (n=10) or pi (n=11). nor were any such differences found when stone forming cf patients were included (ps, n=11; pi, n=18) or when the healthy volunteers were included in the ps group (ps, n=25; pi, n=18). amongst the cf patients, only citrate and creatinine excretions were significantly different between stone formers (n=8) and non stone formers (n=21) (medians (mmol/24h), citrate, 1.4 vs. 2.3, p=0.019; creatinine, 9.7 vs. 12.4, p=0.032) . the difference in creatinine excretion can be accounted for by the difference in body mass of these two groups (median (kg) 54.8 vs. 62.8, p=0.045) compared to the hv, the cf patients with a history of stones had significantly lower citrate excretion and higher calcium oxalate supersaturation (medians, citrate (mmol/24h), 2.9 vs. 1.4, p=0.002; 4.4 vs. 8.1, p=0.037) conclusions there is no evidence to suggest that the prevalence of stones is different in the ps and pi groups nor could we detect any difference in excretion of oxalate or any other urine variable related to pancreatic sufficiency. comparing those with and without stones, the only consistent finding was a lower citrate excretion by stone formers. between the hv and stone formers this was also associated with a significantly reduced calcium oxalate supersaturation. our evidence does not support the hypothesis that fat malabsorption by the majority of cf patients contributes to an elevated risk of renal stone disease. we undertook assessment of the use of a hypopharyngeal sensor for detection of laryngopharyngeal reflux in infants and children. gastric reflux in the airway, or supraesophageal reflux, commonly takes a gaseous form that cannot easily be measured using conventional technology. the miniaturized ph sensor at the tip of the dx-ph probe is the only sensor able to measure ph in the airway. fifteen infants and children referred to the pediatric pulmonary center for assessment of chronic cough, hoarse voice, or uncontrolled asthma unresponsive to usual medical intervention underwent 6-48 hour to placement of the dx-ph probe in supplementation of radiologic assessment of gastresophageal reflux. the probe was placed transnasally and visualzed in the hypopharynx with the red light at the tip. drops in ph <5 were correlated with food and symptom diaries. positive results were found in 10/15 patients, leading to surgical intervention in three patients. one patient with cystic fibrosis age 3 months with failure to thrive underwent study for symptoms of cough. upper gi demonstrated no gross gastroesophageal reflux or anatomic obstruction.gastric emptying scan was mildly prolonged. outpatient supraesophageal ph probe demonstrated drops to ph<4 associated with respiratory symptoms. patient underwent nissen and g tube placement with resolution at six week follow-up.the patient returned at age six months with onset cough and underwent repeat study. there were no drops in ph and the cough responded to oral steroid burst. the device was tolerated in 13/15 patients with early removal in one young adult with endstage neuromuscular disease and in an infant with "colic" due to a hysterical parental reaction.as a result of our experience we have incorporated the use of supraesophageal ph monitoring into our practice, with particular attention to infants with persistent asthma and children with histories suggestive of gastric asthma. we have now incorporated it into our vivometrics lifeshirt, an ambulatory physiologic monitoring device which will allow us to examine supraesophageal ph and its relationship to sleep apnea, cough, tachycardia, tachypnea, and hypoxemia. aim: monitoring and adjusting dose requirements of pancreatic enzyme replacement therapy (pert) are an integral part of the dietetic assessment of patients with cf. we investigated characteristics of enzyme use in our adult clinic population and determine the extent to which inappropriate enzyme use contributed to poor nutritional and clinical state. method: information was collected using an annonymous self-administered questionnaire developed to measure patient practice, knowledge and beliefs on pert. exclusion criteria included pancreatic sufficiency, <1500 units lipase/kg/d, and fev1 <30%. results: 49 out of potential clinic population of 72 patients completed the questionnaire (16-54y, 55% male, fev1 31-125%). 67% of participants reported to never miss enzymes with meals; this was considerably lower for snacks (35%). those patients who omit enzymes with meals also missed enzymes with snacks (r2=30%, p<0.001). a more appropriate use of pert was observed in patients with lower as opposed to higher bmi. despite intensive dietetic input 29% of patients missed pert with foods containing fat and 20% took pert inappropriately with food and drink that did not contain fat. the results identified 5 potentially better practices for measuring pert behaviour and knowledge: 1) taking pert with all meals, 2) taking pert with majority of snacks 3) swallowing capsules intact rather than splitting them open, 4) carrying enzymes around with them and 5) the ability to titrate pert in accordance to the fat content of food. patients were scored on the basis of the above criteria to differentiate between prudent enzyme users and those who are compromising therapy. in conjunction with their nutritional status score (bmi and gastrointestinal symptoms) risk for intervention can be assessed. discussion:underweight patients have more optimal enzyme use, suggesting greater dietetic involvement in these patients. schall et al 2006 also found this to be the case in children. the findings emphasised the need for targeted and effective input in patients where problems are less obvious. the questionnaire has been a useful research tool, and has been adapted as a screening tool for dietitians to gain a subjective perspective of patient's enzyme management and identify patients who need support. the combination of patient's pert usage and their nutritional status could help capture and identify risk objectively and quickly and allows resources to be allocated most effectively. schall .2), nutritional failure, measured as bmi<20, (p>0.99), or lung transplant status (p>0.3) between patients with and without colonic wall redundancy. there was no significant difference in cftr function, per sweat chloride values, between groups (p>0.9). conclusion: we report a previously undescribed colonic wall abnormality seen on ct in patients with cystic fibrosis: proximal colonic wall redundancy was found in 39% of adults with cystic fibrosis, but not in children, appeared to be chronic, and was associated with non-∆f508 cftr gene mutations, particularly the g542x mutation. recognition of the ct appearances will prevent erroneous diagnosis of acute colonic disease in this patient population, and stimulate investigation of a biologic basis for this finding as a recognizable feature of adult cystic fibrosis. intro: infants and children with cystic fibrosis, both pancreatic sufficient and insufficient, are at risk for developing hyponatremia due to an excess loss of salt though skin during sweating. these children should receive daily supplemental sodium. children without cf require 2-4 meq/kg of sodium daily; and one could argue children with cf need more. a historical recommendation is 1/8 tsp table salt daily for infants; this contains 11 to 13meq of sodium based on the density of the table salt. parents may measure the salt inaccurately, and it may be difficult logistically to distribute the salt throughout the day. if supplementation is given as sodium chloride solutions distributed through pharmacies, as suggested in a recent consensus conference, co-pays or full payments may be required. our aim is to provide a simple, inexpensive, and more accessible method for accurately giving sodium supplementation. do pre-packaged salt packets contain a precise amount of sodium and thus could be recommended to fulfill the daily requirement of sodium in patients with cf? method: pre-packaged table salt packets were collected with permission from four national fast food restaurants and one commercial salt producer. contents (sodium chloride) of ten packets from each source were weighed individually. measurements were made using a scientific scale with the ability to measure up to 1/10,000 of a gram. weights of sodium chloride from individual packets were converted to meq sodium. means, ranges and standard deviations were calculated for the data using microsoft office excel 2003™ program. see table 1 conclusions: there is some variability in the content of pre-packaged salt packets, both between and within brands, though some brands have less variance than others. measurements showed that packages contained mean values of approximately 11 to 13 meq of sodium. there is a range in the sodium dose recommendations, and spot urine sodium can be used to determine if patients are sodium depleted. pre-packaged salt packets lack the precision of pharmacological dosing but are inexpensive, practical, and supply a reasonably predictable dose of sodium. background: ten to forty percent of people with cf have vitamin d deficiency secondary to pancreatic insufficiency. obtaining optimal vitamin d levels (25-ohd) has been a challenge. for vitamin d deficient individuals, current supplementation strategies (50,000 iu weekly for 2 months) have been mostly unsuccessful. objectives: to identify individuals at risk for vitamin d deficiency and to evaluate the efficacy of a 2 week repletion course of high dose ergocalciferol in children and young adults with cf. methods: as part of a quality improvement initiative, a prospective cohort study was performed from january to april 2007. phase i included querying our cf practice database to identify patients who were due for annual routine blood tests or who had recently documented levels of 25-ohd less than 30 ng/ml. in phase ii, 50,000 iu of daily ergocalciferol was prescribed for a 14 day period as part of routine inpatient or outpatient care. during phase iii, a post treatment 25-ohd level was obtained. baseline subject characteristics were obtained at entry and included age, gender, pubertal status, pancreatic status, and fev 1 %. post 25-ohd levels were classified as sub therapeutic (<30 ng/ml), therapeutic (30-50), high therapeutic (50-100), or potentially toxic (>100). a paired t test was performed to evaluate pre and post intervention differences. the impact of age, gender, and lung function on the response to vitamin d supplementation were also analyzed. all values are expressed as mean ± standard deviation. results: eighteen individuals with cf participated in the study. the mean age was 17 years with a range of 6 to 25 years. 66.7% were male, and 100% were pancreatic insufficient requiring pancreatic enzyme replacement. fev 1 % was 64.9 ± 26.4%. all 18 participants had 25-ohd levels less than 30 ng/dl pre-treatment. 25-ohd levels increased from 20.5 ± 6.2 to 57.8 ± 21.1 (p<0.001). 17 of the 18 participants (94.4%) became therapeutic over the two week interval. an increase of 37.3 ± 22.0 (ng/ml) or 214% was seen in the two week period. no correlation was seen on the extent of increase in 25-ohd and baseline lung function. pre and peri pubertal individuals had a greater increase in 25-ohd levels than post pubertal individuals (55.5 ± 21.7 vs 28.3 ± 16.3, p<0.05) although the magnitude of change did not reach significance. the degree of response also appeared related to age (r=-0.47). no impact of gender was seen. seven individuals achieved normal therapeutic values while 10 were in the high therapeutic range. no participants had toxic levels. conclusions: the results of this study demonstrate that high dosing of ergocalciferol over a 14 day period is an effective strategy in achieving therapeutic levels of vitamin d. it is unclear whether a pubertal effect on the degree of response exists or if this response is merely age related. further research is needed to evaluate whether this strategy is able to maintain therapeutic levels after completion of the intervention. additional studies that monitor compliance and evaluate responders and non-responders are needed. foundation consensus committee established guidelines for optimizing bone health in individuals with cf, including screening for vitamin d insufficiency and a treatment protocol for achieving and maintaining normal serum vitamin d concentrations. however, since the guidelines were published, there has been additional research assessing various vitamin d treatment regimens and their effect on serum vitamin d concentrations. in addition, vitamin d assay variability has recently been well documented. the objective of this study was to evaluate variability of vitamin d surveillance methods as well as treatment protocols used to treat low vitamin d concentrations at cf centers. methods: a survey was created via survey monkey, and was sent to the cf nutrition listserv, which includes over 200 cf healthcare providers. spss version 13.0 was used for statistical analysis. results: fifty-nine listserv members responded to the survey. the majority (72%) of respondents measured 25 oh vitamin d concentrations, and 75% considered patients to be vitamin d deficient when serum concentrations were below 30 ng/ml. however, 27% were monitoring only 1, 25 oh vitamin d or a combination of both concentrations. there was considerable variability in vitamin d supplementation protocols for treating low 25 oh vitamin d concentrations. only 39% were following the consensus guidelines of 50,000 units of ergocalciferol once/week for ages 5 through adults. one third indicated that they followed consensus guidelines for patients less than age 5; however nearly half of the remaining two thirds were not sure what is prescribed or have not observed low vitamin d concentrations in this age group. fifty-two percent of respondents were aware of vitamin d assay variability. despite this knowledge, the majority were unaware of the type of vitamin d assay used to measure serum vitamin d concentrations at their institution. conclusions: there is considerable variability in the measurement and treatment of vitamin d concentrations across cf centers despite guidelines provided via a cff consensus report. the consensus guidelines for measuring vitamin d concentrations and treatment of vitamin d insufficiency should be revisited based on recent studies and expert opinion. children with cystic fibrosis (cf) who suffer from sub-optimal growth are offered overnight enteral feeding via gastrostomy tube. this study aimed to assess the nutritional impact of overnight feeding over a 2 year period using a retrospective study design. data, including height, weight, fev1, pancreatic function were collated for all patients with cf who had a gastrostomy tube placed in the past 4 years. data were collected at baseline and then every 6 months for 2 years post-gastrostomy insertion. data for height, weight and body mass index (bmi) were converted into z scores using centre for disease control 2000 reference ranges and the lms method (1, 2) . a total of 21 patients (7.7% of the clinic population) were identified as having a gastrostomy tube. two were no longer receiving feeds and there were incomplete data for 6 subjects. on placement of the tubes, there were substantial deficits in nutritional status with mean z scores for height -0.9 (sd 1.02), weight -1.43 (sd 0.71) and bmi -1.1 (sd 0.58), with compromised lung function (mean fev1 69%, range 44-86). using a repeated measures anova with fev1 as a co-variate, there were no significant differences in weight, height or bmi from baseline to 2 years (at 2 years post placement: mean z scores for height -1.1 (sd 0.59), weight -1.36 (sd 0.56) and bmi -0.86 (sd 0.75). fev1 remained stable over time. these results indicate that gastrostomy feeding can potentially halt the decline in nutritional status that is a feature of cf. however, patient expectations that over night enteral feeding will lead to an increase in nutritional status need to be sensitively managed. background : cystic fibrosis (cf) is the most common life threatening autosomal recessive disorder in caucasians.following a landmark paper by corrie et al a high fat,high calorie diet has promoted a normal growth pattern.improved nutritional status together with prevention and early treatment of respiratory infections has contributed to improved survival. objective: the aim of this study was to assess the relationship between lean body mass and disease severity in children with cf. methods: the nutritional status and body composition of 108 children with cf was measured using a harpenden stadiometer, calibrated electronic scales and a ge lunar prodigy densitometer. the following indices were calculated; body mass index (bmi), fat mass (expressed as logarithm % total body fat (ln%tbf) and fat free mass (ffm). body composition data were expressed as z-scores using dutch reference values. correlations were performed between indices of nutrition (bmi, height, ffm, tbf) z scores and markers of disease severity (percent predicted fev 1 and shwachman kulczycki (sk) scores).the nutritional status of these children was also compared with 154 healthy controls. statistical analyses were performed using microsoft excel 2000. results: there was a weak but significant correlation between sk scores and height z score and ffm (r =0.41, p<0.0001 & r =0.54, p= <0.0001 respectively). there was also a weak but significant correlation between % predicted fev 1 and bmi and ffm (r=0.39, p <0.0001, r= 0.49, p<0.0001). height and weight z scores were significantly lower in children with cf (-0.42 and -0.59 respectively) than in control subjects (0.25 and 0.41 respectively) with p <0.0001 in both groups. conclusion: this study demonstrates that there is an association between indices of nutrition and disease severity in children with cystic fibrosis. muscle mass (ffm), assessed by dual energy x-ray absorptiometry (dxa) correlates with lung function and with sk scores. the strongest correlation between markers of disease severity (sk scores and % predicted fev 1 ) was with ffm. ffm may be expected to be associated with respiratory function tests.however,sk scores are a more general assessment of well being and less obviously directly related to muscle mass. ffm and growth may reflect long term nutrition and health in contrast to bmi and ln%tbf which may reflect acute deterioration or short term nutritional intervention. therefore ffm may prove to be a useful tool to assess quality of nutrition and may be predictive of respiratory and general decline. body composition assessment has an important role in chronic conditions such as cystic fibrosis, in order to identify nutritional depletion and evaluate nutritional interventions. it is important that reliable and accurate methods are used. the usefulness of non-invasive methods, such as skinfold thickness measurements (sft) to measure change in body composition has not been evaluated in cf. this study aimed to compare changes in fat-free mass (ffm) and fat mass measured using sft and dual-energy x-ray absorptiometry (dxa) in adults with cf. methods: 57 adults with cf (60% male, 88% pancreatic insufficient, mean age at baseline 30.3 (sd 7.9) years, mean fev 1 at baseline 63.2 (sd 21.3) % predicted) were studied. they underwent body composition assessment using dxa and sft, at baseline and a mean of 3.6 (sd 0.4) years later. durnin and womersley equations were used to estimate ffm and fat mass from sft. estimates of change in ffm and fat mass obtained using sft were compared with dxa using paired t-tests, univariate analysis and bland and altman analysis. results: at baseline, mean ffm was 49.5 (sd 9.0) kg; mean fat mass was 10.8 (sd 5.8) kg and mean bmi was 21.2 (sd 2.4) kg/m 2 . mean ffm, fat mass and bmi were not significantly different at follow-up, indicating no change in nutritional status for the population overall. individual change in ffm ranged from -4.3 to +5.9 kg, while change in fat mass ranged from -4.2 to +5.8kg. the table shows mean change (∆) in ffm and fat mass by each method, correlations (r 2 ) between sft and dxa, and the 95% limits of agreements (loa) between sft and dxa for change in ffm and fat mass. mean change in ffm and fat mass estimated using sft did not differ significantly from dxa. mean bias between the methods was small, and overall correlations between sft and dxa were strong for changes in both ffm, suggesting good agreement for the overall population. however the 95% loa between the two methods were wide for change in both ffm and fat mass. this suggests that sft will not accurately predict changes in body composition in all cf patients. conclusion: sft measurements do not accurately estimate change in ffm or fat mass over time in individual adult cf patients compared with dxa. body composition changes measured using sft tended to more closely reflect changes detected by dxa in male patients compared with females. caution should be exercised when interpreting the results of serial measurements of body composition using sft in individuals with cf. analysed were significantly correlated with age-cml or srage levels (age, gender, bmi, presence of cf-related diabetes mellitus (cfrd) or hba1c level). conclusions: serum levels of ages are elevated in adults with cf. elevation is not restricted to those with cfrd. the levels of srage reflect ability to respond to this pathway and are associated with poorer lung function. modification of the diet in cf may reduce this mediator and may have the potential to modify lung and renal injury. mouse models of cf generated by expression of improperly processed cftr (∆f508) or lacking cftr expression (ko) demonstrate selective induction of sult1e1 in the liver (falany et al., biochem. j. 364:115, 2002) . sult1e1 is responsible for the sulfation and inactivation of β-estradiol (e2) at physiological concentrations. the increase in sult1e1 expression is specific to the hepatocyte, whereas cftr is selectively expressed in cholangiocytes. the induction of sult1e1 activity is associated with changes in levels of e2regulated proteins in cftr (-/-) liver (li and falany, j cystic fibrosis 6:23, 2006) . increased sult1e1 activity is correlated with low body weight and decreased igf-1 message levels in livers of cftr(-/-). the mechanism for the induction of sult1e1 in hepatocytes by cholanigiocytes lacking cftr expression, and the mechanism for the inhibition of igf-1 expression by increasing sult1e1 expression was investigated in human cells. human mmnk-1 cholangiocytes in which cftr expression was inhibited by short interfering rna (sirna) induced sult1e1 expression in human hepg2 hepatocytes when cocultured in a permeable membrane separated system. sult1a1 expression was not altered in this model. the data suggests that loss of cftr function in cholangiocytes is capable of selectively modulating sult1e1 expression in hepatocytes. to investigate the ability of increased sult1e1 activity to modulate expression of igf-1, hepg2 cells were stably transformed with sult1e1/pcdna3 to activity levels intermediate to the levels observed in cftr(-/-) mice. the increased sult1e1 activity was associated with decreased igf-1 message expression in the hepg2 cells. since a major pathway for igf-1 regulation involves growth hormone (gh) stimulation of stat5b phosphorylation, the effects of e2 and increased sult1e1 activity on gh stimulated stat5b phosphorylation were examined. stat5b activation was identified using immunoblot analysis with a rabbit anti-tyrosine phosphorylated-stat5b antibody. treatment of control hepg2 with 10 nm e2 prior to the addition of gh increases stat5b phosphorylation. in hepg2 cells expressing increased sult1e1 activity, the stimulation of stat5b phosphorylation by 10 nm e2 was significantly decreased. e2 had an apparent rapid action on stat5b phosphorylation that is not attenuated by the estrogen receptor antagonist, ici 182,780. e2 was effective at increasing stat5b phosphorylation when applied to hepg2 cells 15-30 min before gh. increased stat5b phosphorylation was observed in control hepg2 cells at 1 nm e2 although 10-fold higher e2 concentrations were required in the sult1e1-hepg2 cells consistent with the increased e2 sulfation. no differences were observed in total stat5b in any of the studies. this study demonstrates that human cholangiocytes with low levels of functional cftr are capable of inducing sult1e1 expression in human hepatocytes and the increase in e2 sulfation inhibits the gh stimulation of igf-1 expression via a decrease in stat5b phosphorylation. supported by grants from the cystic fibrosis research, inc. and nih (gm38953). williams, j.e. 1 ; benden, c. 2 ; jaffe, a. 2 ; suri, r. 2 ; wells, j.c. 1 ; fewtrell, m.s. 2 1. mrc childhood nutrition unit, institute of child health, london, united kingdom; 2. portex respiratory medicine unit, great ormond street hospital, london, united kingdom background: patients with cystic fibrosis (cf) are at high risk of malnutrition. to our knowledge, no study so far has employed a reference fourcomponent model(sup)1(/sup)(4cm) to assess body composition (bc) in cf children, which allows accurate evaluation of both fat mass (fm) and the components of fat-free mass (ffm; mineral, protein, water) and most studies have been cross sectional. methods: 53 cf subjects, aged 8-12 yrs, were compared with age-and sex-matched healthy controls for assessment of bc using a reference 4cm. comparison between groups was performed using paired t-tests and general linear models to adjust for age, height and pubertal status. in addition fm index (fmi; fm/height2) and ffmi (ffm/height2) standard deviation scores (sds) were calculated using measurements performed in a reference population of 415 healthy children aged 4 to 19 yrs. repeat measurements were made in 31 children with cf (15 boys) after 2 yrs and the change in fmi sds and ffmi sds investigated. results: for the initial measurement at baseline, boys with cf (n=26) were significantly shorter (mean (se) height sds: -0.7 (0.20, p<0.01) compared to uk 1990 reference data; girls with cf were lighter, bmi sds (-0.6 (0.2), p<0.05). at baseline, comparison of cf children with pair-matched controls indicated that there was no difference in the boys but cf girls had less fm (cf minus control) (-2.6 (0.8)kg, p<0.01) and mineral mass (-0.1 (0.0)kg, p<0.05) after adjustment for age, height and puberty. fmi sds adjusted for age and puberty was also significantly lower in girls with cf (-0.7 (0.3), p<0.01) whereas ffmi sds was not. for comparison with the large reference population there was no difference in the boys at either time point for fmi sds and ffmi sds (paired t-test). for girls however, there was a significant difference at both baseline fmi sds (-0.8 (1.0), p<0.01), ffmi sds (-0.8 (1.1), p<0.05) and 2yrs fmi sds (-0.9 (1.3), p<0.05) and ffmi sds (-0.7 (1.0), p<0.05). paired t-test analysis between baseline and 2 yr followup measurements indicated that fmi sds and ffmi sds were not significantly different at each point in time in either boys and girls. conclusion: at baseline, the bc of cf boys appears to be within normal range, but the cf girls already have lower fm and mineral than their healthy pair matched controls, even when adjustment for size and puberty is made. follow-up measures in 31 cf children indicated that these descriptions of nutritional status of cf boys and girls remained consistent over the following 2 years, with no evidence of any further deterioration in girls. however, nutritional surveillance is important in both sexes in cf at around puberty to address potential deterioration and this study suggests that the 4cm is a useful tool for this purpose. 1fuller background: although lung function (lf) in children with cystic fibrosis (cf) has been reported to be related to their nutritional status, particularly body fat, the latter has usually been assumed from anthropometric measurements and not measured using an appropriate reference method. habal, h. 1 ; al-turkmani, m. 1 ; freedman, s. 2 ; laposata, m. 1 1. pathology, massachusetts general hospital and harvard medical school, boston, ma, usa; 2. medicine, bidmc and harvard medical school, boston, ma, usa background: increased dietary intake of fatty acids is recommended for cf patients. it is unknown if this results in increased incorporation of all fatty acids-saturated, monounsaturated, and polyunsaturated-into cells with a cf phenotype to a greater extent than into non-cf cells. docosahexaenoic acid (dha) supplementation of cftr-/-mice has been shown to reverse the pathologic cf phenotype. aim: to investigate whether the uptake of multiple predominant fatty acids and the release of membrane-bound arachidonic acid (aa) are altered in cultured epithelial cells with a cf phenotype; in addition, to determine whether fatty acid uptake is selectively inhibited by dha supplementation. methods: sense (wt) and antisense (cf) cftr 16hbe cells were cultured in mem containing 10% horse serum. aa release from membrane phospholipids (pls) was studied by incubating the cells with 2 µm [ 14 c] aa for 15 min, and then with serum free medium containing 0.2% fatty acid free human serum albumin in the presence or absence of 100 µm adenosine for 15 min, followed by measurement of radioactivity in the supernatant. the uptake of fatty acids into the cells was studied by incubating the cells for different time points with 0.04 µm [ 14 c] linoleic acid (la), [ 14 c] aa, [ 14 c] dha, [ 14 c] palmitic acid or [ 14 c] oleic acid, in the presence and absence of 10 µm or 20 µm dha, followed by measurement of radioactivity in the cell lysate. results: basal and adenosine-stimulated aa release from membrane pls was significantly higher in cf cells compared to wt cells (basal wt:113.7±1.1, cf:202.4±12.8 dpm/10 6 cells, p<0.01; stimulated wt:207.8±11.4, cf:468.7±49.1 dpm/10 6 cells, p<0.01; mean±sem for each). the uptake of la, aa, dha, palmitic acid and oleic acid were significantly higher into cf cells compared to wt cells. dha significantly decreased the elevated uptake of these fatty acids into cf cells, but not into wt cells. data are shown in the table below. conclusion: in this cell culture model, these data demonstrate that aa uptake and release are increased in cf cells, which may suggest elevated mobilization of this fatty acid by cf cells. our data also show that cftr dysfunction leads to increased uptake of all tested fatty acids into the cells. downregulation of this increased uptake may be one mechanism by which dha exerts its therapeutic benefits. low bmi in cf correlates directly with worsening lung function and is an independent risk factor for mortality. the causes for this decreased bmi are incompletely understood but could be related to levels of the orexigenic peptide ghrelin, and to levels of leptin, an anorexigen. studies assessing plasma levels of leptin in cf have shown contradictory results, while the data on ghrelin in cf are scarce. we assessed both leptin and ghrelin plasma levels in mild (fev1 > 75% predicted), moderate (fev1 45% to 74% predicted), and severe (fev1 < 45 % predicted) adult cf patients. we compared these levels in cf subjects to 20 age matched controls with normal bmi (range 19.5 to 24.5 kg/m2). we conclude that leptin levels are decreased, while ghrelin levels are increased only in cf patients with severe disease and low bmi. plasma levels of ghrelin and leptin did not differ from normals in those with mild and moderate disease. gastroesophageal reflux (ger) is common in cf before and after lung transplant (ltx). laparascopic fundoplication (lapf) is used when patients fail on conservative medical therapy. aims: to review longer term clinical outcomes of lapf in patients with cf in terms of lung function, body mass index (bmi), reflux symptoms, patient satisfaction with surgery and complications. method: retrospective review of patient records and patient questionnaire to gather relevant information. results:thirty-two patients with cf underwent lap.f recently; 15 transplanted (ltxgroup), 8 female (mean age 40 [21-67]) years, and 17 not transplanted (cf group), 14 female (35 ). the median time between ltx & lapf was 654 (414; 1133) days, range 145-1838. endoscopy was undertaken in 27 patients prior to surgery and all underwent 24hr ph monitoring. complete (nissen) lapf was undertaken in 50% patients and a partial (toupet) in the other 50%. total median time in the operating theatre was 143(81-232) minutes. the average length of stay in hospital (in days) in the ltxgroup: pre-op. was 7.81; post-op. was 7.82 days; and in the cf group was: 5.2 and 12.5 respectively. lung function (fev1 and fvc in percent predicted) and bmi were measured at minus(-)100, plus (+)100, +200 and +300 days from lapf. patient satisfaction with lapf was a mean of 95% in the ltx group and 85% in the cf group and improvement in reflux symptoms of 90% in both groups. nocturnal cough reduced markedly in the cf group and there was an overall improvement in quality of life in both groups. complications consisted of 2 port site hernias, 1 failed lapf with re-operation 20 months later; 2 esophageal dilatations and 1 patient with lower esophageal sphincter hypotonia. there were 3 deaths on day 19, 253 and 382 post fundoplication. one was in a non-transplanted patient with recurrent episodes of hemoptysis. she succumbed to a catastrophic hemoptysis episode not related to lapf. the other 2 were in transplanted patients. conclusions: in this relatively small series of patients there was a small but significant drop in fev1 in the ltx group 300 days after lapf, but not in the cf group who remained stable throughout the review period. there was a significant drop in bmi in both groups after lapf, but the cf group returned to pre lapf values at +300 days. patient satisfaction with the procedure was high. fundoplication is an expensibe complicated procedure not without risk. a long term prospective study is warranted. historically this phenomenon has been attributed to intestinal malabsorption and cachexia as a consequence of chronic lung infection. however, cf mice exhibit similar growth and weight deficits but are not afflicted with malabsorption or lung infection, suggesting other mechanisms are participating as well. we hypothesize that small size is in part a result of increased β-oxidation in adipose stores. to test this we utilized a mouse model to study lipid metabolism. our study includes female cf mice with a null mutation in the cftr gene. these mice are congenic on the c57bl/6j background as well as age matched with their controls (wt) to 50-70 days. liver, adipose and skeletal muscle (calf) were dissected from wt and cf mice in a fed state. tissues were immediately flash frozen in liquid nitrogen and used for rna and protein lysate harvest. quantitative pcr (qpcr) was performed using primers for the lipogenic isoform of acetyl-coa carboxylase 1α (acc1α) in the liver and adipocytes, whereas the non-lipogenic isoform acc2β was assessed in the skeletal muscle. primers for acyl coa oxidase (aox), carnitine palmitoyltransferase 1 (cpt1) and long acyl-coa dehydrogenase (lcad) were used as gene markers for β-oxidation steps in the peroxisome, carnitine shuttle and mitochondrion, respectively. western blots were performed to detect acc (active) and phospho-acc (inactive) forms. the results show that there is a decrease in the total amount of acc protein in the fat as well as the skeletal muscle of the cf mice and the ratio of acc:phospho-acc is substantially lower in the cf tissues. phosphorylation and/or decreased expression of acc results in decreased expression of malonyl coa, leading to a cascade of events that increase β-oxidation. in addition, increased expression of aox and lcad, two downstream β-oxidation genes, is also observed. hyperactivity of the β-oxidation pathway could account for the decreased adipose stores seen in cf mice compared to the wt. such patterns of expression are reminiscent of starvation. caloric intake and intestinal absorption of dietary lipid were measured and cf animals consumed approximately 110% the calories of wt animals (per gram body weight) and lipid absorption was not significantly different (94% for cf, 97% for wt), indicating the cf animals have sufficient access to nutrition. we will continue to investigate fatty acid synthesis and degradation pathways during both the fed and fasted states to delineate the mechanism. this research was supported by grant hl 68883. effect of gender on dha levels in p and rbc with lower values for male cf patients. acknowledgments: this work was supported by the belgian cf association (ablm), the french cf association (vlm) and by a grant from frs (ucl). data are expressed as means ± sd. * and indicate a significant difference with controls and cf ps patients respectively, p<0.022. background: deficit of antioxidant systems and increased oxidative stress have been demonstrated in cf patients with respect to healthy controls. the origin of this imbalance is known to be multifactorial. objective: the aim of our study was to assess changes in antioxidant systems and oxidative stress parameters in cf pediatric patients in presence or absence of pancreatic insufficiency material and methods: we recruited 70 patients (37 with pancreatic insufficiency pi and 33 with pancreatic sufficiency ps) attending the cystic fibrosis centre of turin (2005) (2006) . during the annual routine control visit were performed antropometric measurements, pulmonary function tests (fev1), and were collected fasting blood samples and last 3 days food record. vitamin e, a, c, selenium, glutathione (gsh, gshpx, gssg), dehydroascorbic acid, pcr were assessed. data were analysed using the two sample t-test with equal variances. results: there are not statistically significant differences between two groups for sex, age and fev1. 33/37 of pi patients and 8/33 of ps patients took oral vitamin supplementation. serum levels of alpha-tocopherol and beta-carotene resulted within the local reference range either in pi or ps patients showing no significative differences. the mean serum levels were respectively suboptimal and optimal for vitamin e and a (biesalsky '97). pi and ps patients did not show significant intergroup differences of water soluble and enzymatic antioxidants levels as well. serum levels of vitamin c, selenium, erythrocyte reduced glutathione (gsh) and glutathione peroxidase activity (gshpx) resulted within the local reference range. plasma vitamin c concentrations resulted slightly inferior to the optimal values (biesalsky '97) in both groups. oxidative stress parameters (erythrocyte glutathione disulfide-gssg, dehydroascorbic acid) and inflammatory parameters (pcr) did not differ significantly, presenting values within the local reference range, in both groups. nutritional data revealed significantly reduced zscore for weight (p<0.05) and height (p<0.01) in pi patients vs ps patients. our data did not evidence significant differences in total energy (kcal/kg) and macronutrients (% of total kcal) intake in both pi and ps patients. dietary intake of antioxidant vitamins resulted slightly superior the rda (larn-1996) presenting no significant intergroup differences. conclusions: this study shows that pancreatic insufficiency do not influence oxidant/antioxidant balance in cf patients at least in paediatric age. until innovative supplementation guidelines will be proposed for application, nutritional education and monitoring and correct dietary habits are useful to optimize antioxidant status in cf patients. there are more than 1400 cf mutations that are responsible for the spectrum of disease severity in cystic fibrosis (cf). the most common, f508del mutation, is associated with pancreatic insufficiency (pi), and a classic phenotype. . yet even within a group of f508del homozygotes, there is a small minority of patients (2.3%) who are not taking pancreatic supplements, and are therefore presumably pancreatic sufficient (ps) (data from 2002 * cff registry). this observation prompted us to look more closely at enzyme use in cf patients with non-f508del homozygous genotype. we reviewed enzyme use in cf patients who were fully genotyped and whose enzyme use was documented in the 2002 * cf registry. heterozygotes with one or two non-f508del mutations were included in this analysis. using the assumption that the milder mutation is dominant, if the patient was ps and heterozygous with one f508del mutation, the second mutation was classified as ps; if the person was pi, the second mutation was classified as pi. we also reviewed the pancreatic status and genotype of patients participating in the wisconsin newborn screening study. in this randomized control trial (rct), pancreatic status was determined by 72hour fecal fat analysis. enzyme use was extracted from the database for the year 2002. there were 14494 patients in the 2002 cff registry with a total of 27 different mutations. twenty-one of these mutations were associated with pi. this finding correlated well with the per cent of patients who were on enzyme supplements (range of per cent patients on enzymes by mutation 100-88%). however when we looked at the six mutations associated with ps, the distribution of patients on enzymes vs. not, was much closer. see table below. possible reasons for enzyme supplement use by patients with ps mutations include a) treatment for recurrent pancreatitis, b)the fact that this is a cross-sectional analysis and patient may be on enzymes temporarily, c) patient may truly be pi, d) overuse of enzymes due to unreliable test to measure pancreatic status in patient, e) patients who are on enzymes are older, and have changed from ps to pi phenotype. this analysis suggests a need for better assessment of pancreatic status in patients with cf. * this analysis was originally done by request for the ecfs conference at garda on cftr genetics, and focused on the suggested "33 main mutations", and will be updated with the 2005 cff registry data. (supported by nih grants dk072126, dk34108, mo1 rr03186, mo1 rr00058) number of patients in rct with each mutation: 7,3,1,1,3,0 respectively. dutta, a. 1 ; woo, k. 1 ; fitz, j.g. 2 ; feranchak, a.p. 1 1. department of pediatrics, ut southwestern medical center/children's medical center, dallas, tx, usa; 2. medicine, ut southwestern, dallas, tx, usa extracellular atp is an important signaling molecule contributing to bile formation by the liver through binding biliary epithelial cell (cholangiocyte) membrane p2 receptors and stimulating cl-efflux. importantly, atp appears to work through cl-channels unrelated to cftr, suggesting it may be a potential way to modulate bile flow in cf liver disease. however, the signaling pathways linking receptor binding to channel activation in cholangiocytes are unknown. consequently, the aim of the present study was to identify the pathways responsible for atp-stimulated increases in [ca2+]i and membrane clpermeability in a human biliary epithelial cell model. studies were performed in mz-cha-1 human biliary cells; [ca2+]i was measured by fura-2 and membrane cl-currents by patch-clamp techniques. results: exposure of cells to atp (100 µm) resulted in a rapid increase in [ca2+]i to 1054.23 ± 244.90 nm (n=6) which was abolished by prior depletion of intracellular ca2+ by thapsigargin (37.94 ± 14.50 nm; n= 7), but unaffected by removal of extracellular ca2+ (egta, 819.00 ± 211.94 nm; n= 7). in parallel studies, atp (50 µm) increased current density from -1.99 ± 05 pa/pf to -13.20 ± 2.00 pa/pf (n= 19) . currents reversed at 0 mv (ecl-), were outwardly rectifying, and were inhibited by the cl-channel inhibitor nppb (-1.81 ± 1.71 pa/pf, n= 6), but not the cftr inhibitor cftrinh172. atp failed to activate currents after depletion of intracellular ca2+ stores by bapta-am (-0.70 ± 0.72 pa/pf, n=3). the p2y receptor antagonist suramin inhibited atp-stimulated increases in [ca2+]i (50.04 ± 8.17 nm; n= 7) and cl-channel activity (-2.25 ± 0.80 pa/pf; n= 6). however, the p2x receptor antagonist brilliant blue g did not affect the magnitude of atp-stimulated cl-currents (-14.63 ± 1.81 pa/pf, n=4). together these findings suggest that atp activates cl-currents primarily through p2y, but not p2x, receptors in human biliary cells. since p2y are g-proteincoupled receptors and modulate [ca2+]i by phospholipase c (plc) generation of inositol 1,4,5-trisphosphate (ip3), a series of experiments were designed to directly assess the effects of ip3 receptor inhibition or stimulation on atpstimulated cl-currents. first, the plc inhibitor, u73122 blocked atp-stimulated cl-currents (0.86 ± 0.60 pa/pf; n= 3). second, the specific ip3 receptor blocker 2-apb, inhibited both the atp-stimulated increase in [ca2+]i (34.36 ± 7.33 nm, n= 4) as well as membrane cl-currents (-0.13 ± 0.55 pa/pf, n= 6). lastly, intracellular dialysis with purified ip3 during whole-cell patch clamp activated cl-currents with identical properties to those activated by atp (-9.40 ± 1.23 pa/pf, n=4). together these studies demonstrate that extracellular atp stimulates ca2+-activated cl-channels in cholangiocytes primarily through p2y receptor binding and plc/ip3-dependent release of intracellular ca2+ stores. thus, the p2y-ip3 receptor signaling complex represents a potential target to increase cholangiocyte secretion, and hence augment bile flow, in the treatment of the cholestatic liver disease associated with cf. supported by grants from the nih niddk and the cff to a. feranchak. background: supplemental pep therapy is used to treat cf patients with epi to assist with digestion, food absorption. this open-label, multipledose, single-treatment, multicenter study evaluated efficacy and safety of eur-1008 for malabsorption treatment in young patients with cf and epi. eur-1008 is a novel, zero-overfill, highly-stable formulation of porcinederived pancreatic enzymes, and was specifically developed for use in very young children allowing the product to be sprinkled on food. methods: eligible patients were < 7 years with cf and epi, in acceptable nutritional status, needing peps and clinically stable. patients consumed a standard cf recommended diet, could not take drugs affecting gastric ph or motility. the trial involved a 7-day dose stabilization period and a 7-day treatment period. patients switched from their baseline enzyme treatment without washout. the optimal dose of eur-1008, determined during dose stabilization, was used during treatment. primary endpoint compared malabsorption of fat after eur-1008 vs. previous treatment. clinical symptoms were also evaluated, as was physicians' and parents' judgment on the control of malabsorption signs and symptoms. safety measures included aes, physical exams, vital signs and changes in clinical laboratory findings, including cholesterol levels and fat-soluble vitamins. results: nineteen patients (12 male) enrolled and completed all study phases. mean age was 3.9 years (range 1-6). percentage of responders (patients without steatorrhea (<30% fecal fat content) and without signs and symptoms of malabsorption after 1 and 2 weeks of treatment) was 52.6 at baseline, 68.4 (p=0.375 vs. base) after stabilization and 57.9 (p =0.999 vs. base) at study end. frequency and oily stools showed a significant decrease vs. baseline at study end. the mean number of stools per day was 1.82 stools at screening and 1.45 stools during treatment (p<0.001). the mean proportion of stool samples with visible oil or grease at screening was 11.10% and 4.73% during treatment (p=0.001). a statistically significant improvement was seen in the incidence of moderate bloating. physicians characterized 37% of patients as "improved" in the control of epi signs and symptoms vs. previous therapies and 63% as "unchanged" at the end of the treatment phase, while parent assessments were 47% "improved" and 53% "unchanged." vitamin k absorption (measured through pivka) improved with eur-1008 treatment over prior pep treatment measured at baseline. eur-1008 was safe and well tolerated, with minimal aes. no aes led to discontinuation of study drug interruption. no incidences of uric acid toxicity or fibrosing colonopathy following eur-1008 treatment. no trends were seen in lab parameters, physical examination, or vital signs. conclusion: eur-1008 is effective, safe, well-tolerated in the treatment of epi in young cf patients. treatment controlled malabsorption and clinical symptoms of epi in young patients with cf. safety and efficacy of eur-1008 in this trial is consistent with the pivotal phase iii trial. there is an association between the presence of cf and alterations in fatty acid metabolism, consisting of a decrease in linoleate (18:2n-6). more recently, a deficiency of docosahexaenoate (22:6n-3, dha) has arisen as a prominent fatty acid abnormality in cf. an established breeding colony of exon 10 cftr knockout (cftr-/-, cf, n=14) and wild type (wt, n=7) mice was used for this study. all mice were weaned at 23 days of age and then raised on peptamen and water until 30 days of age, and then continued for 10 days with 15 ml/day of peptamen to homogenize the nutritional status. after this period, some mice (n=3 per group) were subjected to dha treatment (40 mg/day) for one week. after sacrifice, pancreatic tissue was collected, homogenized and extracted. lipid classes were separated by sequential elution in aminopropyl columns. the eluted fractions were transmethylated and injected into a hewlett-packard 5890a gas chromatograph with a flame ionization detector for quantitative analysis. the vast majority of fatty acids were mainly distributed in phospholipids, except those with 18-carbons. wt and cf mice were studied a gender gap in the survival of cystic fibrosis (cf) is well-documented. however, little is known whether there is a gender différence in pubertal growth pattern in cf children. delayed and attenuated pubertal growth are commonly observed in adolescents with cf. however, characterizing height velocity (hv) pattern is very challenging because of errors associated with calculating hv, e.g. interpolation and extrapolation between 2 heights measured in irregular intervals, as well as difficulties in determining the location and the magnitude of peak height velocity (phv). in this study, we applied a new statistical method, namely, the semi-parametric shape-invariant model, to characterize the hv pattern of cf children. this method is based on the assumption that all individuals' growth curves have a common shape. therefore, this method attempts to shift and stretch (squeeze) individual's growth data until they can be modeled by the common shape curve. the semi-parametric shape-invariant model has the advantage of fitting measured heights directly, thereby eliminating the errors associated with calculating hv. the study population included 996 cf children who had height measurements between ages 2 to 18 years documented in the 1986-2002 cff registry. because fitting each individual height curve is computing-intensive, 100 boys and 100 girls were randomly chosen for this preliminary analysis. our results showed that girls exhibited a typical growth deceleration during late preadolescence, followed by accelerated hv and a single phv during adolescence (figure) . unexpectedly, cf boys exhibited a notable hv peak during late preadolescence in addition to the typical adolescent phv (figure) . overall, the mean magnitude and age of phv was 7.4 cm at 14.6 years for boys and 6.6 cm at 11.9 years for girls. when compared to phv of non-cf children based on tanner's reference (pediatr. 1985, 107:317) , cf boys showed a greater delay in the age of phv while cf girls showed a greater attenuation in the magnitude of phv. further analyses are being performed to characterize the hv patterns of all 996 children to confirm these results. (supported by nih-dk02891). anelli, m.; foresti, r.; peloso, l.; ortenzi, g. medical affairs, eurand, pessano con bo, italy background: given their inherent instability, the dose units of all pancreatic enzyme preparations (peps) have always been "overfilled" to compensate for enzyme degradation over time and assure at least 90% of the label lipase content at the end of their shelf life. this overfill issue was first examined by whitehead who reported overfills ranging from 14 to 39% after analyzing several commercially available peps (whitehead am. pharm weekbl sci. 1988) . the usp presently allows for pep overfills up to 65%. thus, the actual active lipase content of a pep capsule labelled at 10,000 iu might vary from 9,000 to 16,500 units according to its "freshness." as a result, patients taking peps receive a product with variable potency, which can lead to efficacy issues, increased pill burden, unnecessary drug interactions, and product switching. the fda has also noted the potential safety risk posed by overfilling and, in a recently published guidance, it imposed a zero overfill requirement for all peps to be marketed in the usa after april 2008. methods: over a period of two years, we ran a series of tests similar to those conducted by whitehead to evaluate overfill on 16 commercial peps currently available in the u.s. and europe. results: no finished product was formulated to 100% of the labelclaimed lipase enzyme activity. overfill on commercially available peps ranged from 7% to 47%, with a median value of 32%. the peps analyzed were 10 to 12 months old on average; therefore, the actual amount of overfill is likely underestimated. conclusion: our findings confirm those reported earlier by whitehead and demonstrate that none of the currently available peps complies with the zero overfill requirement of the fda guidance. these findings highlight a potential cause of suboptimal therapy with currently available peps. references: -whitehead am. study to compare the enzyme activity, acid resistance and dissolution characteristics of currently available pancreatic enzyme preparations. pharm weekbl sci. 1988 feb 19; 10(1): 197-201. -food and drug administration. guidance for industry: exocrine pancreatic insufficiency drug products -submitting ndas. april 2006. heubi, j. 1 ; boas, s.r. 2 ; blake, k. 3 ; nasr, s.z. 4 ; woo, m.s. 5 ; graff, g.r. 6 ; hardy, k.a. 7 ; amaro-galvez, r. 8 ; latino, m. 9 ; lee, c. 10 1. children 's hospital medical center, cincinnati, oh, usa; 2. chicago cf care, glenview, il, usa; 3. nemours, jacksonville, fl, usa; 4. michigan u, ann arbor, mi, usa; 5. children's h, los angeles, ca, usa; 6. penn state h, hershey, pa, usa; 7. children's h, oakland, ca, usa; 8. texas u, tyler, tx, usa; 9. eurand, milan, italy; 10. eurand, vandalia, oh, usa background: supplemental pep therapy helps prevent maldigestion and malabsorption in cf patients. eur-1008 is a novel, zero-overfill, highly-stable formulation of porcine-derived pancreatic enzymes developed to treat epi in cf patients. this phase iii, randomized, doubleblind, placebo-controlled, two-treatment, crossover, multicenter trial evaluated the efficacy and safety of eur-1008 to treat epi in cf patients. methods: patients with confirmed cf and epi, age ≥7 years, good nutritional status and weight ≤ 70 kg were enrolled. no drugs affecting gastric ph or motility were allowed. after open-label dose titration, patients were randomized to receive eur-1008 or placebo over a week-long period. following another open-label normalization, all patients were crossed over to the alternative treatment arm. primary endpoint was change in coefficient of fat absorption (cfa) following oral administration of eur-1008 vs. placebo. secondary endpoints were change in coefficient of nitrogen absorption (cna), cholesterol, fat soluble vitamins, body weight, bmi and epi symptoms following oral administration of eur-1008 vs. placebo. safety endpoints included aes, clinical laboratory parameters, physical exams and vital signs. results: thirty-four patients were enrolled (17 female); 32 were evaluated. mean age was 15.4 years (range 8-23). eur-1008 treatment was associated with statistically significant (p<0.001) increases in both cfa and cna vs. placebo. eur-1008 treatment was associated with decreases in pivka ii (an indicator of vitamin k status). epi symptoms consistently improved during eur-1008 treatment, including a statistically significant reduction in stool frequency and fewer soft and watery stools. treatment with eur-1008 improved the signs and symptoms of malabsorption even in the small subset of patients with cfa values greater than 80% under placebo treatment. there were no notable changes in serum lipids, vitamin a and e values, mean weight or bmi in patients who received eur-1008. eur-1008 was safe and well-tolerated. there were no unexpected or significant differences in the frequency or type of aes between eur-1008 and placebo, and no trends were seen in lab parameters or vital signs. no patient discontinued from the study due to an ae. two serious aes were considered unrelated to the study drug and both resolved. conclusion: in this randomized, double blind, placebo-controlled study, eur-1008 was safe, well tolerated and effective in the treatment of cf patients with epi, with clinically and statistically significant improvements in cfa, cna, and epi signs and symptoms in the absence of any concomitant treatment affecting gastrointestinal motility and ph. the therapeutic benefit of eur-1008 was seen even in patients with very high cfa values (>80%). the relative infrequency of cystic fibrosis related diabetes (cfrd), until recently, means little is known about hypoglycaemia ('hypos') associated with its treatment. insulin is frequently taken by those who have cfrd, for whom the risk of hypoglycaemia may be high due to dramatic changes in insulin sensitivity that can occur with pulmonary exacerbations and a decreased ability to secrete glucagon. a number of studies involving people with type 1 diabetes mellitus (t1dm) have reported on their experiences of hypoglycaemia, but whether results from such investigations apply to cfrd is unclear. an exploratory investigation was conducted to compare the type, frequency and severity of hypoglycaemic symptoms experienced by patients who had cfrd with patients who had t1dm. method a cross sectional study was conducted, involving adults (18-60 years) with t1dm or cfrd, recruited from two hospitals in england. a questionnaire, sent to 265 patients with t1dm and 145 with cfrd, included investigator-developed items about knowledge and frequency of hypoglycaemia and the edinburgh hypoglycaemia scale (ehs), a standardised measure that assesses autonomic (e.g. sweating, hunger) and neuroglycopenic (e.g. confusion, poor co-ordination) symptoms associated with hypoglycaemia. questionnaire data were entered into spss for analysis. comparisons were conducted using t-tests, mann whitney tests or chi square tests as appropriate. results questionnaires were returned by 60 patients with t1dm and 52 with cfrd. five of those with t1dm were over 60 years of age and, therefore, excluded from analysis. almost all participants had experienced a 'hypo' (98%). comparisons between the two groups are reported in table 1. conclusion patients with cfrd reported fewer and tended to have less severe episodes of hypoglycaemia compared to those with t1dm. fewer neuroglycopenic symptoms in the former could be related to their shorter duration of diabetes or to how cfrd was managed; patients with cfrd were given shorter acting insulin, making them less prone to 'hypos' during the night, and had a high sugar diet to ensure weight maintenance. differences between the groups could also be related to specific characteristics of these two forms of diabetes. table 1 : data from questionnaire insulin resistance. the purpose of this study was to investigate the relationship between pem and the risk of developing glucose intolerance and/or cfrd and the effect on lung function in children ≤21 years of age whose history suggests pem. patients whose diagnosis of cf was suggested by failure to thrive(ftt) and whose growth parameters remain <10% for weight and height for age were identified as having incurred pem. a retrospective analysis of our cf registry database was performed. 173 cf patients ≤21 years old were identified. 32 of 173 patients(18.5%) had cfrd. 103(59.5%) of these plotted <10% for weight and height (pem)and 23(22.3%) were diagnosed with cfrd. further review of the 103 patients showed 35 whose diagnosis of cf was suggested by ftt and 10 (28.6%) of those 35 had cfrd. statistical analysis did not show a clinical significance, but a strong relationship is implied. pulmonary function, fev1%, was significantly lower in the pem/cfrd patients (mean fev1 63.5% ± 24.36%) than the pem only group, (mean fev1 88.17% ± 22.14%)(p=0.0001). this review provides further support that patients who have pem are at increased risk of developing cfrd. a significant risk was not established; however a strong association is suggested. assessment of a larger cohort of patients may show a stronger relationship. findings from this study would suggest that further research into the physiological relationship between pem and cfrd should be initiated. cfrd screening should be considered at a younger age than the current cff recommended age of >14 years. diagnosing cfrd/glucose intolerance early may contribute to improve growth parameters, preserve lung function, and improve survival. based on this review, our center is initiating cfrd screening for all patients ≥ 10 years, and considers screening for all pem patients ≥6 years. background: patients with cf are thought to have a rapid postprandial rise in plasma glucose which may be followed by a delayed and prolonged insulin response and hypoglycemia. we sought to estimate the prevalance, cumulative one year incidence, and factors associated with hypoglycemia during oral glucose tolerance testing in non-diabetic adults with cf. we also compared the prevalence of hypoglycemia to a geographically matched cohort of young adults without cf. methods: we performed a prospective study of 161 individuals aged over 16 years with cf followed in clinic providing population-based, specialized care in cambridgeshire, england. we excluded patients with previously-diagnosed diabetes or taking anti-diabetic medications. we evaluated 108 individuals who underwent 75 g 2 hour oral glucose tolerance testing (ogtt) in 2004. of these, 65 individuals had neither new diabetes nor hypoglycemia in 2004 and were retested using ogtt in 2005. we testing the association between biochemical and anthropometric factors measured in 2004 with the prevalence of hypoglycemia in 2004, and incident hypoglycemia in 2005. biochemical hypoglycemia was defined as a blood glucose between <4.0 mmol/l (72 mg/dl), but greater or equal to 2.5 mmol/l (45 mg/dl) and severe hypoglycemia as a value lower than 2.5 mmol/l on either the fasting or post glucose challenge value. for comparison, we used data from the cambridgeshire-based young ely study of 183 individuals age 30 -48 years who underwent 75 g ogtt testing in 1994 -1995 . results: in 2004 , in patients with cf, the prevalence of fasting hypoglycemia was 5.5%. no patients had fasting severe hypoglycemia. following glucose challenge, 20.2% and 2.8% of patients had glucose values consistent with hypoglycemia and severe hypoglycemia respectively. there were no diferrences in age, hepatic, pulmonary or renal function, but patients with hypoglycemia at two hours were five times more like to be male (odds ratio 5.1, 95% ci 1.7 -15.6) and more likely to have a higher bmi. the prevalence of a 2 hours value of < 4.0 mmol/l in patients with cf at 22.9% (25/109) was not different than in controls 20.7% (38/183). of patients who did not have hypoglycemic readings in 2004, at one year, 1 patient developed fasting hypoglycemia and 14 individuals developed post-challenge hypoglycemic for a cumulative incidence 21.5%. of these 6.2 % (4) had values in the range of severe hypoglycemia. there were no differences between age, sex, bmi, liver, renal, or pulmonary function in those who did and did not develop hypoglycemia. conclusions: hypoglycemia appears common in patients with cf but is also common in a healthy population. male sex and bmi were associated with post load hypoglycemia in patients with cf in cross-sectional analyses only. further research needs to assess whether these low blood glucose values are associated with symptoms. it is well recognised that cystic fibrosis related diabetes (cfrd) is a poor prognostic indicator in cf, and therefore early recognition is imperative to allow effective treatment in an attempt to prevent the associated decline in pulmonary function and nutritional status. although the oral glucose tolerance test (ogtt) is the accepted method of detecting diabetes mellitus in non cf individuals, glucose intolerance and lack of insulin can be variable in cf patients such that the reliability of ogtt in the diagnosis of cfrd has been questioned. under these circumstances, other methods of monitoring glucose intolerance, such as serial post prandial glucose monitoring (sgm), may be more appropriate. to look at this further, we prospectively compared ogtt with sgm in a group of adult cf patients. fourteen patients with no previous history of cfrd ( 127], 9 df508 homozygous, 9 male) admitted for acute pulmonary exacerbations were evaluated. all were exocrine pancreatic insufficient, and 1 was on long term oral steroids. eleven patients received 30mg/day oral prednisolone in addition to iv antibiotic therapy for the duration of their admission. ogtt was performed in the standard fashion (ingestion of 1.75g glucose/kg body weight [maximum 75 g] within 5 minutes, after an overnight fast). sgm was performed 2 hours post prandially and before bedtime over the period of the admission. the local ethics committee approved the study and informed consent was obtained from all patients. results sgm revealed elevated postprandial blood glucose values in all patients. there were 83 episodes (mean 6 per patient [0 to 18]) with glucose >11.1 mmol/l (frank diabetes, who criteria) and 86 episodes (mean 6 per patient [1 to 13] ) with glucose 7.8 to 11.1 mmol/l (impaired glucose tolerance, who criteria). however, ogtt revealed impaired glucose tolerance in only one patient (2 hour glucose value 9.8 mmol/l). in the remaining patients, ogtt revealed normal 2 hour glucose values (mean 5.0 mmol/l [range 3.8 to 6.9]). fasting plasma glucose values were also normal (mean 4.5 mmol/l [3.5 to 5.9]) in all patients. we have shown a high prevalence of hyperglycaemia in our adult patients admitted for pulmonary exacerbations that was only detected using serial glucose monitoring. this study suggests that sgm may be more sensitive at detecting early cfrd than the ogtt. further work needs to be carried out to look at the best methods of diagnosing cfrd, to facilitate early treatment and thereby improve the prognosis. the diagnosis of cystic fibrosis related diabetes (cfrd) is associated with a decline in pulmonary function and nutritional state and is a poor prognostic indicator: the risk of its development increases with age. furthermore, the development of cfrd is due to a gradual loss of insulin production, and the disease may therefore be preceded by a period of glucose intolerance. despite this, the incidence of hyperglycaemia in cf patients is unknown. to study this further, we monitored the blood glucose profiles of adult cf patients admitted for the treatment of a pulmonary exacerbation. we looked at 60 consecutive cf patient admissions for iv antibiotic therapy to our large adult unit. of these, 28 had established cfrd and were excluded. the remaining 32 patients (mean age 23 years [range 17 to 54], 22 male) formed the study population. none were on long term oral steroids, but 28 received 30mg/day prednisolone as part of their routine exacerbation treatment. all patients underwent blood glucose measurement 2 hours after each meal and before bedtime as part of a standard assessment protocol in our clinic. thirty patients (94%, 26/28 on steroids) demonstrated post prandial hyperglycaemia: 14 exhibited values between 7.8 and 11.1 mmol/l (impaired glucose tolerance, who criteria) and 16 >11.1 mmol/l (frank diabetes, who criteria). there were no significant differences in age, genotype, fev1, fvc, bmi, or duration of hospital stay between the two groups. of the hyperglycaemic patients, 28 (93%) had abnormal glucose levels detected before bed time, significantly more than 2 hours post breakfast (23%, p<0.001), post lunch (60%, p<0.002), but not post dinner (80%, p=0.16). this study has shown a high prevalence of hyperglycaemia in adult cf patients treated for a pulmonary exacerbation who were not otherwise known to suffer from cfrd. nearly all our patients demonstrated glucose intolerance later in the day, when the recommended screening test for cfrd (the fasting oral glucose tolerance challenge) cannot be used. post prandial glucose monitoring of such patients during times of stress may therefore be advisable, allowing the introduction of insulin treatment at an earlier stage in the disease process. whittaker, l.a.; tilluckdharry, l.; christian, r. medicine, university of vermont, burlington, vt, usa objective: bone disease is a recognized complication of cystic fibrosis (cf). there are currently no guidelines for bone mineral density (bmd) testing or tools for fracture risk assessment in cf adults. we piloted a survey designed to identify cf-specific risk factors for low bone density and fracture. methods: a 45 question survey was completed by patients prior to measurement of bmd at the hip, spine and radius by densitometry (dxa). the survey was comprised of questions on calcium and vitamin d intake, exercise, family history, medications, reproductive health, ultraviolet exposure, cf related diabetes (cfrd), weight loss, severity of cf lung disease and quality of life. z scores (bmd adjusted for age and gender) were used so that male and female data could be analyzed collectively. patient perceived health (pph) was rated by four questions and totaled for the pph score, which ranged from 4-12 with the highest score indicating the best health. functional status (fs) was rated by three questions that reflect the degree to which cf interferes with school, work or play. the total fs score ranged from 3-9, with the highest score representing the least interference with life. questions on pph and fs were derived from a validated clinical assessment tool, the cf questionnaire (cfq) (1) . results: all cf adult patients (n=46, age 18 and older) were offered participation in the study. dxa of total hip and lumbar spine was performed on 39 patients (85%); 30 (65%) of these patients also completed the survey. 51% of patients had low bone density on dxa (z score <-1). 62 % of male cf adults had low bone density vs 39 % of females. fracture was reported by 57% of patients who completed the survey (64% female and 50% male). lower pph score was associated with lower z score at the hip and spine. lower fs score was associated with lower z score at the hip and spine. low body mass index was associated with low z scores at both hip and spine. patients with bmi below 22 kg/m2 were most likely to have osteopenia. conclusions: osteopenia and history of fracture were common in our adult cf population regardless of gender. bmi, pph and fs predicted bone density. risk stratification by a cf-specific bone health survey may guide bone density screening strategies for cf adults. prospective studies in cf adults are needed to determine whether a cf-specific survey can predict fracture risk as well as bone density. references : as life expectancy in cystic fibrosis (cf) increase, osteoporosis has become more prevalent. vitamin d status is one of many factors that contribute to optimal bone health. previous data has shown, the majority of cf patients are deficient in vitamin d despite being prescribed daily cf specific vitamin therapy. previous research has also shown that the current recommendations for vitamin d supplementation and repletion provided by the cystic fibrosis foundation bone health consensus (2002) are not sufficient to achieve optimal vitamin d levels in the majority of patients. moreover, the recommendations fail to sustain levels in the optimal range for bone health. purpose: we demonstrate a successful supplementation algorithm for individualized high dose vitamin d with continued high dose maintenance therapy which achieves and sustains appropriate levels. methods: all patients followed at the pediatric center are prescribed routine cf vitamin supplementation. vitamin d levels are obtained as part of routine clinical care via mass spectroscopy. if serum vitamin d level is <30 ng/dl, vitamin d therapy with ergocalciferol is initiated one time per week. patients < 5 years of age receive 12000 iu/ dose and > 5 years of age receive 50000 iu/dose. at the next routine quarterly visit, vitamin d levels are re-assessed via mass spectroscopy to determine efficacy of once weekly supplementation. if levels are > 30ng/dl, patients continue high dose supplementation 1 time a week as maintenance therapy. however, if levels remain <30ng/dl, the ergocalciferol is increased by 1 time a week in 3 month increments until adequate levels are obtained. once adequate serum vitamin d levels are obtained, patients remain on the level of supplementation needed to achieve levels >30ng/dl as maintenance therapy. all patients are instructed to take vitamin d and their cf vitamins with enzymes and food to optimize absorption. results: after implementation of the algorithm, 70.5% of patients, with varying levels of supplementation, had vitamin d levels > 30ng/dl (mean=38.4 ng/dl). of the patients that achieved goal vitamin d levels, 58% were able to achieve and sustain adequate vitamin d levels without additional vitamin d supplementation while on standard cf vitamins (mean vitamin d level = 35.9 ng/dl). the remaining patients required chronic maintenance vitamin d: 17% required vitamin d supplementation once per week with a resulting mean vitamin d level of 45.2ng/dl, 15.9% required maintenance vitamin d supplementation twice per week with a resulting mean vitamin d level of 39.9 ng/dl, and, 3.6% required vitamin d supplementation 3 or more times per week with a resulting mean vitamin d level of 42.3 ng/dl. another 4% of patients take vitamin d supplements other than ergocalciferol and were not included in this study. conclusions: individualized vitamin d repletion and maintenance therapy as outlined in our algorithm is a successful mechanism to obtain optimal vitamin d levels in patients with cf. inhaled tobramycin (29%). sixty-two percent reported using at least 5 different types of oral medications on that day including pancreatic enzymes (85%), oral antibiotics (33%), anti-reflux medication (49%), azithromycin (47%), anti-histamine (29%), and a decongestant (10%). twenty percent reported taking at least one medication for pain. forty-nine percent reported performing airway clearance at least twice during the day with 35% using a vest, 18% performing standard chest physiotherapy, and 15% using a flutter or acapella device. there was no difference in the reported number of medications or the reported time needed to complete therapies based on respondent age. although respondents with severe lung disease (fev1 <40% predicted) reported a higher median number of therapies (8 vs. 7, p=0.05), the reported time for completing therapies was not associated with fev1 (p=0.4). conclusion: daily treatment burden and complexity in all adults with cf is high, and is only marginally increased in those with worse pulmonary function. given this already high load of daily therapies, efforts to assess the treatment burden of new cf therapies are warranted. the impact of this high treatment burden on overall health related quality of life for adults with cf needs to be assessed. objective: the cystic fibrosis foundation (cff) has a network of over 115 accredited care centers throughout the united states. data is collected from these care centers to help better understand the disease and describe changes in survival, standards of care, and health outcomes. when the cff was founded in 1955 few children survived to school age. now the predicted survival extends beyond the mid 30's (cff, 2005) . with this change has come the need for age appropriate care in many areas including reproduction. the adult cystic fibrosis center at morristown memorial hospital presently follows 68 adults. of those, 3 women(4%) with cf have had children affected with cystic fibrosis. each woman was diagnosed after her child. the cf data registry does not have any means of identifying if a patient has an affected child. as the average age of survival of cf patients has increased, so has the likelihood of cf patients having cf offspring. what is the estimated incidence of cf patients having cf affected children attending accredited cf centers in the united states? method: questionnaires were sent to care centers and satellite centers via standard mail or facsimile based on information from the cff directory. one month after the last questionnaire was mailed, the cf foundation sent out a reminder email to the nurse coordinators. questions asked were as follows: 1) name of center 2) do you presently care for any patients with cf who have given birth to, or fathered a cf affected child? 3) how many patients at your center have had cf affected children? 4) was the parent diagnosed before or after pregnancy was confirmed? 5) if parent's diagnosis came after conception, was this after child was diagnosed? 6) was the parent's diagnosis a result of prenatal testing? results: a total of 144 questionnaires were mailed or faxed. this netted a result of 50 responses. after email reminder, an additional 68 questionnaires were returned for a total of 118 responses, an 82% response rate. name of center was checked to remove duplicate information. the number of parents with cystic fibrosis who have had children with cf numbered 62. of those, 27 (44%) were diagnosed before pregnancy; 35 (56%) were diagnosed after pregnancy. of those diagnosed after pregnancy, the majority (86%) were diagnosed as a result of their child's diagnosis. only 3 (8%) were diagnosed as a result of prenatal testing. conclusion: based on the responses from accredited cf care centers, 62 patients with cf have children also affected with this disorder. prior to conceiving their child, 44% knew that they had cf. having cf did not appear to be a deterrent to delivering cf offspring. this study did not look at the age of the parent or child; they may have conceived their child before genetic screening was common for spouses. as the median survival of this population increases, the incidence of cf adults with cf children may also increase. the cff may choose to add a question to the cf data registry, identifying cf patients with cf offspring. the potential physical and psychosocial impact can then be tracked. anderson, a.; popli, k.; stewart, j.; heslop, k.; gascoigne, a.; bourke, s. adult cystic fibrosis centre and fertility centre, royal victoria infirmary, newcastle upon tyne, united kingdom as the outcome of patients with cf improves fertility issues become increasingly important. almost all men with cf are infertile because of azoospermia due to congenital absence of the vas deferens. these men can attain genetic paternity by sperm aspiration from the epididymis or testis with intracytoplasmic sperm injection (icsi), but few men with cf seek fertility treatment to assess their knowledge and attitude towards fertility issues a questionnaire was sent to 71 men with cf: 50(70%) responded; 13(19%) declined to give information and 37(53%) replied in full. overall 75% thought that nearly all men with cf were infertile, 25% did not know how many were affected, and 66% could describe the exact nature of infertility. only 50% knew that treatment for infertility was available: 33% of these thought it was rarely successful and 33% were unsure of success rates. although 75% had thought of having children at some stage, only 15% had sought investigation of infertility. two men had children, one by icsi and one by donor insemination. overall 67% feared that the child could have cf and 53% worried that their health would affect their ability to function as a parent. in terms of receiving advice 57% thought that this should be given by a fertility specialist, 27% by a cf physician and 16% by a primary care physician. with regard to fertility treatment 44% thought that decisions should be made by the patients and their partners, 26% by the patient alone, and 30% by a doctor. the reluctance of men with cf to seek fertility treatment appears to be due to many factors including lack of knowledge of available treatments, fears about the child inheriting cf, and concerns that their health would adversely affect their role as a parent. in addiition to information provided by the cf team advice from a specialist in fertility treatment may help to improve the provision of reproductive counselling to these patients. nash, e.f. 1,2 ; coonar, a.s. 3 ; stephenson, a.l. 1, 4 ; delgado, d.h. 5 ; singer, l.g. 2, 4 ; tullis, e. 1, 4 ; chaparro-mutis, c. studies have shown varying degrees of right ventricular (rv) dysfunction in adult cf patients with severe lung disease. left ventricular (lv) dysfunction is much less common with conflicting reports as to its prevalence. pulmonary hypertension (ph) is commonly seen, the severity correlating with fev 1 . one group reported that ph was more prevalent in b. cepaciacolonized patients, albeit in a small sample. the aim of this study was to determine the true prevalence of ph and cardiac dysfunction in cf patients with severe lung disease. we carried out a retrospective cohort study of all adult cf patients referred to the lung transplant program at toronto general hospital from 1987-2007. adult patients referred with non-cf bronchiectasis were included for comparison. demographic data, lung function, 6-minute walk, micro-biology, doppler echocardiography and resting and stress radionuclide ventriculography (muga) results were analyzed by students t-test (parametric) or mann whitney u test (non-parametric). results 244 adult cf patients (61% non-b. cepacia, 39% b. cepacia) and 25 non-cf bronchiectasis patients were included. data from these 3 groups are summarized in the table. we found higher 6 minute walk and bmi values and a higher percentage of males in the b. cepacia compared to non-b. cepacia group. ph was present in 53% of the cf patients in whom pasp could be obtained. b. cepacia patients had lower first pass rvef compared to non-b. cepacia patients. 51% of the b. cepacia patients had an abnormal lvef response to exercise (defined as <5% increase on exercise) compared to 42% of the non-b. cepacia patients. we found a high prevalence of ph as well as rv and lv dysfunction in cf patients with severe lung disease. b. cepacia-colonized patients had worse rv function compared to non-b. cepacia-colonized patients on first pass radionuclide ventriculography. *non-cf bronchiectasis group older than cf groups (p<0.001) b. cepacia cf group higher than non-b. cepacia cf group (p<0.01) b. cepacia cf group lower than non-b. cepacia cf group (p<.001) perobelli, s. 1 ; dorazio, c. 1 ; assael, b. 1 ; tamanini, a. 2 ; castellani, c. 1 1. cystic fibrosis center, verona, italy; 2. molecular biology laboratory, verona, italy cf neonatal screeening (nbs) can identify neonates with elevated irt, normal or borderline sweat chloride concentrations, and one cf mutation plus an additional cftr sequence variant, or, alternatively, two cftr sequence variants. the combination of hypertrypsinogenemia and two cftr gene defects is consistent with the presence of a cftr-related disorder (cftr-rd). although these neonates are usually healthy at diagnosis, the long-term phenotypical consequences may be highly variable. such variability makes impossible to predict the clinical outcome, and provide satisfactory genetic counselling for the family. the diagnosis of such a vague condition could potentially cause considerable family stress. this study aimed at assessing how parents of infants diagnosed with a cftr-rd through nbs perceive their children's health. a questionnaire for parents was designed, consisting of three sections. the first section asked about information given to the family in the neonatal period, and about emotional reactions to the cftr-rd communication of diagnosis. the second section investigated the present perception of the child's health, and the parents level of anxiety/concern. the third section focused on the impact of the diagnosis on family planning, on relationships inside the couple and with the child, and on emotional distress; this section was concerned also with the use of carrier testing in relatives. parents were also asked to fill in a standardized form on child behaviour (achenbach child behaviour check-list). the questionnaire was distributed to two populations, the parents of 18 children diagnosed with cftr-rd through nbs (group a), and the parents of 18 children diagnosed with cf through nbs as the life expectancy of children with cf increases new issues arise that have not been dealt with in the past. in the past,issues related to the support and care of a young adult with cf were rarely raised because only small numbers of children with cf lived into adulthood. however, with an increasing young adult population, young adults without family support have surfaced in significant numbers and their needs should be identified and understood by the cf center care team. these young adults face the same range of issues others with cf face related to health insurance, government benefits, housing, education and employment. however, they do not have a family to help them wiht support, health insurance benefits and care needs. method in october 1998, the cf legal information hotline ("hotline") began to provide free and confidential information to people with cf, family members and healthcare providers in the u.s. the hotline is staffed by attorneys, beth sufian and james passamano(bs and jp) and is accessed by dialing a toll free number or by e-mail. information about the hotline is available at cf care centers, in cf publications and on the web. information regarding the content of each call is recorded on an intake form and includes caller name, address, phone number, age of person with cf, cf center attended, and information related to the caller's question, attempts made to access legal information prior to calling the hotline, relation of access to information to access to care. calls and e-mails are tracked by seven major categories which are divided into subcategories. if a caller has more than one question, the call is tracked using the caller's primary issue. results from november 2006 to april 2007 the cf legal information hotline has received 42 calls from young adults with cf who do not have family support and have issues related to obtaining health insur-ance, government benefits, educational oppornties or employment opportunities. as well as issues related to lack of support for care. the hotline has also received 33 calls from cf care centers team members with questions related to the needs of young adults without family support. over an eight year period the cf hotline received _______calls. the categories for the calls were: social security benefits (1, 324) , health insurance (1, 163) , educational (434), employment (422), long term disability benefits (190), family law (59) and miscellaneous legal issues (95). discussion people with cf face obstacles to care if they are unaware of their legal rights especially in the areas of health insurance and government benefits. healthcare providers are busy addressing the medical needs of patients and are often not well equipped to provide detailed information about the legal rights of their patients. the hotline provides important information to both patients and healthcare providers that can optimize both patient care and outcomes. access to legal information can result in individuals obtaining health insurance, medicaid, medicare or other state coverage that in turn results in better access to treatment. legal information can also lead to important modifications in the educational or employment environment for the individual with cf. these modifications can be critical in allowing those individuals access to important care and treatments. providing information that can help individuals obtain social security or long term disability benefits can result in a monthly benefit and health insurance that then allows the individual to devote more time to their healthcare. many people with cf and healthcare providers lack information about the legal rights of individuals with cf. understanding the legal rights of people with cf can reduce obstacles to obtaining care, optimize education and employment, and increase access to retirement benefits. further work needs to be done to identify ways to make individuals with cf and healthcare providers aware of the hotline. the cf hotline provides a unique service to cf patients and healthcare providers by providing information regarding the legal rights of people with cf. identification of ways to assist young adults without family support should be identified and cf center xcare teams shoudl be given the tools to allow them to help their patients access government assistance and other programs that can help make sure that the young adults have acces to care and support they need to live with cf. the dvd gives healthcare staff, patients and families a rare glimpse of ten couples with cystic fibrosis (cf) interacting at a cf care center-sponsored 'couples support group'. they discuss the impact this forum has in their daily lives for gainingo information about how cf affects their lives as individuals, and as couples, and how the 'non-cf partner' learns from others. o skills at networking and personal coping strategies. o reflections on 'family life and cf', and o observations on ways cf care centers can join them in greater health partnership. lastly, this dvd provides a 'starter kit' for ways to establish such a group. one of the realities of a life-threatening illness can be that of social isolation, and of separation from others who share the same disease. having the opportunity of participate in a support group can significantly reduce that sense of isolation and give the participants an ability to share experiences and give support to others with this disease. cystic fibrosis as an illness has growing ranks of 'survivors' who live longer, fuller lives, thanks to the advances made on multiple-research fronts. historically, adults with cystic fibrosis have not had much occasion to share person-to-person experiences within the structure of their cf care centers. justifiably, with the potential for cross-infection, the cystic fibrosis foundation has urged caution for the cf population in having face-to-face interactions. in 2004, our cf care center recognized this need and sponsoredwith guidance from staff facilitators-an on-going adult support group. prior to the formation of the group pre-and on-site group ic protocols were developed. at the pre-group stage, the physician initially certifies that the patient is free from aggressive pathogens. each time the patient rsvp's permission to attend a group session, the physician must 'recertify' this status. on-site protocols emphasize a number of ic precautions to minimize crossinfection. the center recognized that by developing such a support group, 'patient and family centered care' could be enhanced. a goal in the group's formation was to create 'mutually beneficial partnerships among patients, families, and healthcare providers'. group structure/successes to date: the group is limited to 20 participants. two cf care center staff act as facilitators. this is a support, not a therapy group. compliance with therapies is reported to have increased as one outcome of this forum. formal topics are purposefully not predetermined; the group's success is predicated on the personal responsibility of each participant to bring topics/issues from their daily life experiences. the group has gained identity and trust and has evolved over three years into two separate groups: an individual cf group and a couples cf group. ownership of these groups by those who attend is apparent. both the care center and the couples support group are forums for learning 'how to live with cf'. future goals will call for using identified quality of life tools to measure psychosocial and emotional growth in individuals through their participation over time in the group. sawicki, g. 1 ; asher, d. 1 ; dill, e. 2 ; sellers, d. 2 ; robinson, w. 2 1. children's hospital, boston, ma, usa; 2. education development center, newton, ma, usa background: advance care planning (acp) is an important tool to promote alignment of the patient's care with his or her needs, goals and values, and is particularly useful if the patient's ability to make decision becomes compromised. the purpose of acp is to develop a treatment plan promoting a high quality of life for those near the end of life. such planning is especially appropriate in persons with life limiting disease. minimal research exists on the advance care planning process of adults with cf.<p>methods: in the fifth survey wave of the project on adult care in cystic fibrosis (pac-cf), an ongoing longitudinal panel study of cf adults from 10 cf centers in the us, participants were asked to report on their opinions and experiences with advance care planning. the survey asked about opinions on advance care planning documents, communication about advance care planning with family and clinicians and whether a directive had been completed. clinical data were obtained from the participating centers. preliminary bivariate analyses were conducted to examine factors associated with completing an advance care directive.<p>results: 233/303 (77%) surveys were completed. the mean age was 34 ± 13 years, 63% were female, and the mean fev1 (% predicted) was 69 ± 28. 77% reported that they had spoken to someone about the care they would want if they became too ill to make their own decisions in the future, 65% had thought about whom they would want as their healthcare proxy, and 60% reported having specific wishes about the types of medical treatment they want or would not want if they became too ill to make decisions for themselves in the future. however, only 33% reported completing a healthcare proxy form, living will or any other type of written instructions concerning advance care directives. 80% of respondents reported feeling comfortable talking to their clinician about acp. 28% of subjects said that their cf clinicians have asked them about acp, and only 11% reported that they have discussed acp with their cf clinician. the following characteristics were significantly related (p<.05) to completion of an advance care directive: female gender (38% vs. 25%), age (mean 37 years vs. 33 years), having a college degree (41% vs. 23%), being married currently or in the past (38% vs. 25%), having children (41% vs. 28%), having diabetes (48% vs. 31%), having specific wishes about treatment decisions (46% vs. 15%), and reporting that a clinician had discussed acp with them (59% vs. 24%).<p>discussion: though the majority of cf adults report thinking about, communicating with family and deciding on their advance care wishes, only a minority report completing any legal documentation supporting their decision. additionally, very few cf adults report being asked about acp by their clinicians and even fewer report discussing acp with them. formulating specific wishes and discussing acp with a clinician are strongly associated with completing an advance care directive, suggesting that if clinicians were more active in talking to their patients about acp, patients are much more likely to complete advance care directives. efforts to improve clinician communication with cf adults around the issues of advance care planning are needed. objectives: caregiver involvement in children's cf treatments fosters better adherence and improved health outcomes. several studies have suggested that parents of children with cf report elevated symptoms of depression (quittner et al., 1998) , however, there is no data on the effects of caregiver depression on adherence. adherence to treatment is difficult to measure in patients with cf, and prior research has relied primarily on self-report, which is likely to be inflated. the purpose of this study was to examine the effects of caregiver depression on adherence to enzymes using both parent-reported and electronically monitored enzyme adherence over a three-month period. in addition, we examined the relationship between depression, rates of enzyme adherence and short-term changes in weight. methods: as part of a larger intervention study at 3 cf centers, 89 children with cf ages 1 to 11 and their parents were recruited. parents completed a standardized measure of depression (cesd) at enrollment and were provided with mems caps that recorded the date and time of each bottle opening. three months later, the electronic data from the pill caps was downloaded and parents completed a structured interview reporting their child's adherence to all components of the cf treatment regimen. only the section addressing enzyme adherence was included in this analysis. standard health outcomes, such as weight and height, were also assessed at enrollment and 3 months later. results: preliminary analyses indicated that caregivers reported elevated levels of depression, with 30% scoring in the clinical range. rates of adherence to enzymes were poor (43% at home and 48% at school). caregiver depression was negatively associated with adherence, with depressed caregivers demonstrating lower rates of adherence (11 percentage points). adherence to enzymes was associated with changes in weight, with a 100% adherence translating into 5 percentile points of weight gain. final analyses will also include parent-reported adherence to enzymes. conclusions: a substantial number of parents scored in the clinical range on a depression screening measure. in addition, depression was associated with poorer adherence. rates of adherence to enzymes were surprisingly low, both at home and at school. furthermore, poor adherence was associated with a decrease in weight three months later. caregiver depression appears to be under-diagnosed and these results suggest that screening and intervention may be warranted. adherence to enzymes should also be targeted in clinical interventions. funding was provided by nih grant #hl69736 of non-compliance with medical treatment and greater behavioral and emotional distress. adherence is particularly problematic during adolescence, however, few family-based interventions have been developed to target adherence behaviors in this population. the goal of this study was to compare the effects of behavioral-family systems therapy (bfst), an empirically-supported treatment, to both family education (fe) and standard care (sc). methods: as part of a larger intervention study, 117 adolescents with cf ages 10 to 17 and their parents completed the conflict behavior questionnaire (cbq) and the parent-adolescent relationship questionnaire (parq) developed by robin and foster (1989) . adolescents completed these measures for each parent and both parents, if available, completed them with respect to their teen. the problem-solving, communication, and beliefs subscales from the parq were administered. participants were then randomly assigned to one of three treatment arms: bfst, fe or sc. families in the bfst group received ten 90-minute sessions over a 6 month period, including family problem-solving, communication skills training, and cognitive restructuring. families assigned to the fe group received ten 90minute psychoeducational sessions over 6 months aimed at increasing knowledge about cf and its management. those in the sc group received their usual care at the cf center. the cbq and parq were completed pre, post and 6, 12 and 18 months following the intervention. results: adolescents reported that the bfst intervention, relative to the other two, improved their communication with their primary caregivers (p < .03) and caregivers improved their communication with their adolescents (p < .02). in addition, caregivers reported improved problem-solving with their adolescents (p < .04). on the cbq, bfst reduced both adolescent and primary caregiver conflict and fe reduced caregiver conflict. no significant improvements were found for those in sc. conclusions: preliminary findings suggest that bfst is effective in improving communication skills and reducing conflict in adolescents with cf and their caregivers. future analyses will evaluate the effectiveness of bfst and fe on adherence and other family functioning measures over the course of the study. funding was provided by nih (hl #47064) background: cases of diabetes among people with cystic fibrosis (cf) have increased as life expectancy for these patients improves, yet the impact of this additional illness on daily functioning is under-researched. to explore this issue, a study was conducted comparing the views of patients with either cystic fibrosis related diabetes (cfrd) or type 1 diabetes mellitus (t1dm) about being diagnosed and living with diabetes. methods: qualitative research was used because the study was concerned with understanding diabetes from patients' perspectives. purposive sampling was employed to achieve maximum variation in terms of duration and type of diabetes experienced by participants. data were collected via semi-structured interviews, all of which were taped and transcribed verbatim. recruitment continued until no new ideas or insights emerged from additional participants. a framework approach to analysis was adopted. this involved coding and summarising interview data into charts to explore and develop main themes. initial analysis was undertaken by two researchers (st and cd) and amended after comments from the remaining authors. results: interviews, carried out with 11 cfrd and 12 similarly aged t1dm patients, lasted for an average of 40 minutes (range 20-60 minutes). the following themes were derived from the data collected: evolving vs fracturing identity; diabetes in context; self-management motivators. for patients with cfrd, diagnosis represented a progression in their health status, which called on them to adapt existing treatment regimens to accommodate this additional condition. in contrast, interviewees with t1dm had to re-evaluate their previous sense of self as 'healthy' and adjust to manag-ing a long-term complaint. these individuals were more likely to talk about diabetes in relation to a range of competing commitments (e.g. work or family related) and to describe feeling psychologically low due to diabetes compared to patients with cfrd, the latter depicting demands from their primary illness (cf) as a major obstacle to caring for diabetes. participants with cfrd recalled feeling lucky when told they would not face strict dietary restraints, which they associated with other forms of diabetes, and seemed less concerned about diabetic complications than those with t1dm. for interviewees with t1dm, a desire to reduce future health risks motivated their self-management efforts, whilst those with cfrd were driven by the negative effect poor control of diabetes had on their chest and weight. conclusions: both sets of interviewees found diabetes time consuming and, on occasions, frustrating to accommodate into daily life. findings from the study act as a reminder that patients manage their condition in the real world, against a plethora of other demands on their time and energy. in the narratives of participants with cfrd, these demands were often related to the existence of their primary illness. ing the course of development. the purpose of this study is to examine rates of medication adherence across a wide age span using an objective measure of adherence and to compare adherence to health outcomes. methods: patients with cf age 6 years or older who are prescribed azithromycin, colistin, hypertonic saline, pulmozyme, and/or tobi for at least one year are eligible. recruitment is ongoing. the previous year's medication refill records were requested from participant-identified pharmacies. a medication possession ratio (mpr) was calculated for each drug and was defined as the sum of all days of medication supply received during the 12 months divided by the number of days the medication was prescribed. values were truncated to 100% and averaged across all medications to obtain a composite score. medical records were abstracted to identify the prescribed drug regimen and health outcomes (e.g., lung function, exacerbations, bmi) over the same time period. results: thus far, 100 patients have joined the study (80% participation rate), resulting in 213 pharmacy records requested (86 unique pharmacies). of the first 48 participants with complete pharmacy data, the mean age was 21.5 years (sd=11.9; range= 7-69) and 56% were female. the table presents mpr data for each drug and composite score overall and stratified by child, adolescent, and adult participants. the complexity of the drug regimen and age were significantly correlated with the composite score (rho= -.39 and rho= -.29 respectively; p<.05). future analyses will compare the composite score and each drug's mpr with health outcomes. con-clusions: participants had suboptimal medication adherence similar to that reported in the literature. as mpr provides the maximal possible level of adherence, actual adherence may be even lower. poor adherence spanned the age groups and was associated with regimen complexity. children had the highest adherence, but also the least complicated drug regimen. these results suggest that obtaining pharmacy records is a viable means to objectively assessing medication adherence. moreover, results suggest that interventions targeting medication adherence may be a strategy for improving health outcomes, particularly for adolescents and adults. * fewer than 5 participants within this cell 564 (cf) whose exchange capacities for oxygen and carbon dioxide are already diminished. lack of rem and delta sleep have significant implications for the daytime functioning of pediatric patients with cf, potentially leading to impaired memory and attention which would reduce their ability to complete typical activities, such as school work and disease-specific tasks related to self care and adherence. based on these clinic findings, the authors are beginning a prospective study on the effects of sleep on neuropsychological functioning, well-being and compliance in pediatric patients with cf. future research should examine the relationship between decreased slow wave, rem sleep and psychosocial outcomes, including attention, memory, and health-related quality of life. there are diagnostic challenges related to non-classic cf (knowles and durie, 2002) particularly patients presenting in adolescence or adulthood with obstructive azoospermia, chronic sinopulmonary disease and chronic or acute, recurrent pancreatitis. practitioners are often unable to provide clarity to patients about diagnosis, disease course, and prognosis. potential harms and benefits of relaying a clear or unclear diagnosis may be psychological rather than physical and have long-term implications for mental and social well-being. the aim of the current study is to assess the psychological impact of diagnostic information for adults presenting with a cf phenotype, be it confirmatory (cf or most likely not cf) or unclear. method: we administered self report measures pertaining to psychological state, cognitive appraisal and uncertainty in a welldefined cohort of adult patients. presenting symptoms of patients include chronic sinopulmonary disease, obstructive azoospermia, pancreatitis, and those presenting with more than more cf phenotype. patients completed the self-report measures on two occasions: at the time of diagnostic testing and 6 months after being counseled and notified of the diagnostic results. at the time of the initial assessment, the subjects were unaware of the test results and had not been seen by a cf physician. results: we provide interim observations on 70 patients (sinopulmonary (n=34), obstructive azoospermia (n=20), pancreatitis (n=4), and multiple phenotypes (n=6) who completed the initial assessment. at the time of diagnostic testing, the level of depression, hostility, anxiety and interpersonal sensitivity were elevated in each group (t score > 65); male patients reported significantly greater depression, anxiety and interpersonal sensitivity than female patients. compared to published normative data of patients with mixed chronic illnesses, patients presenting with sinopulmonary or pancreatic disorders reported significantly more uncertainty(p < .05). to date, 26 of these patients have completed the measures 6 months after receiving the diagnostic information. preliminary review of results shows that the level of depression, hostility, anxiety and interpersonal sensitivity remained elevated while the degree of uncertainty decreased. among the 26 patients who completed assessment at 6 months, 17 patients were told that it was unlikely that they had cf, 7 were told that they had mild cf, while 2 were given an unclear diagnosis. those who were told that they did not have cf reported more uncertainty than those who were told they had cf (p < .05). detailed analyses related to outcome diagnosis will be conducted at the time of study completion (october 2007) . when this study is complete, the information will assist in our understanding of the psychological impact of a genetic diagnosis at an older age, identify issues confronting those individuals and help to establish appropriate paradigms for delivering complex information about genetic diseases. supported by grants from niddk [scor] and the canadian cf foundation. (1). in accordance with these guidelines, we report our experience in using our nutrition action plan (nap) in patients with suboptimal nutrition. objective: to determine if implementing our nap would improve the bmi of these patients. methods: our nap is a written contract between the patient, caregiver and cf team. for clarity of nutritional goals, the nap is based on the colors of a traffic light. the red zone indicates a bmi less than the 25th percentile and poor nutritional status. the yellow zone indicates a bmi between the 25th and 49th percentile and fair nutritional status. the green zone indicates a bmi greater or equal to the 50th percentile and desired nutritional status. within each color zone, specific recommendations to improve nutritional status are listed. the ultimate goal for each patient is reaching the green zone. the caregiver, patient and nutritionist review the current nutritional status and a mutually accepted weight gain goal is established. the goal is recorded on the nap which is then signed by the nutritionist and patient. this contract establishes a commitment to achieve the goal by the next clinic visit. a reward system is implemented in conjunction with the nap. for patients reaching their goal, a previously agreed upon prize is given. patients achieving some weight gain but falling short of their goal receive a gift such as candy or small toys that serve as an incentive for continued weight gain. patients chosen for inclusion into this study demonstrated a bmi less than the 50th percentile; those on growth hormone, appetite stimulants or chronic systemic steroids were excluded. results: twenty-one patients, 12 male and 9 female, with a mean age of 11.6 years (range 3 to 18 years) were included in this study. fifteen patients demonstrated an improvement in their bmi with use of the nap: 7 improved by 1-10%; 2 improved by 11-20%; 3 improved by 21-30%; 1 improved by 31-40%; 2 improved by 41-50%. six participants had a negative change in bmi which we attribute to family upheaval, missed appointments with the cf team, exacerbations of disease and non-adherence. conclusion: we intervened in 21 patients with a bmi less than the 50th percentile using a nap and a reward system. the results of this pilot study suggest that our nap and reward system may be of benefit in achieving positive gains in bmi. reference: (1) previously it was rare for women with severe cf to undertake pregnancy and doctors tended to advise against pregnancy if the forced expiratory volume in one second (fev1) was less than 60%. the attitudes of doctors and patients are changing and many women living their lives with cf nowadays chose to undertake pregnancy despite the severity of their health problems. over the last 15 years 102 females (age range 16-56 years) have attended the newcastle adult cf centre. there have been a total of 25 pregnancies involving 20 women with 24 live births (1 ectopic). a further 3 women had termination of unwanted pregnancy. mean age at the time of pregnancy was 21 years. we compared an age-matched cohort of never pregnant women to see if there were differences in key variables making successful pregnancy more likely. the mean fev1 was 61% (range 30-112%) and 58% (range 10-91%) of predicted in the pregnant and non-pregnant group respectively (p=0.36); rates of chronic pseudomonas aeuriginosa infection were 14 (70%) and 12 (57%) respectively (p=0.52). 2 cases of burkholderia cenocepacia were identified in the non pregnant group. there were no cases in the pregnant group (p=0.21); 7 (35%) had diabetes with 5 patients having cf related diabetes prior to pregnancy and 2 patients developing gestational diabetes; this compared with 9 (21%) of non pregnant women (p=0.75); there was impaired nutritional status within both groups with a mean bmi of 18.9 pre pregnancy (range 14-27) and 20.1 in the non pregnant group (range 14-31) (p=0.28); 5 (25%) and 3 (14%) patients had had previous gastrostomy feeding (p=0.45). pancreatic insufficiency was present in 17 (85%) and 19 (90%) patients in respective groups (p=0.66). no child had cf but one child had complex congenital heart disease. there were no maternal deaths but 2 patients subsequently underwent lung transplantation and 2 patients died leaving young children (ages 5 and 7 years). 2 patients in the non-pregnant group have also died. conclusion the profile of women with cf undertaking pregnancy is now similar to the overall cf population. the woman's decision to undertake pregnancy is often not directly related to the severity of her disease. amongst those who successfully completed pregnancy maternal and fetal outcomes are generally favorable. objectives: health-related quality of life (hrqol) instruments provide important information on disease progression and are increasingly used as patient-reported outcomes (pros) for behavioral and pharmacological trials (goss & quittner, in press) . recent advances in medical care have improved the long-term stability of pulmonary functioning, which has made it more difficult to detect small improvements in fev1% predicted. new outcome measures are needed; the cystic fibrosis questionnaire-revised (cfq-r; quittner et al., 2004) , a disease-specific hrqol measure, has shown promise in this regard. the purpose of this study was to examine the longitudinal relationships between fev1% predicted, weight percentiles, and cfq-r scores for adolescents with cf. methods: as part of a larger study evaluating adherence interventions, 117 adolescents with cf, ages 10 to 17, and their parents were enrolled at six centers. parents reported on their adolescents using the cfq-parent version, and adolescents completed the cfq-teen/adult version as a selfreport. measures were completed pre and post treatment and at 6, 12, and 24 month follow-up visits. spirometry and weight data were collected at these same time points. results: empirical bayes estimates were used to examine change over time. preliminary results indicated that adolescents' scores on the cfq-r respiratory scale were significantly and positively correlated with changes in pulmonary functioning. parents' scores on the cfq-r vitality scale were also correlated with changes in pulmonary functioning, with higher cfq-r vitality scores associated with better lung function. in addition, parents' scores on the cfq-r weight scale were significantly and positively correlated with changes in adolescents' weight. conclusions: these results support the validity and sensitivity of the cfq-r to changes in both pulmonary functioning and weight for adolescents with cf. additional analyses will be conducted to determine the relative stability of hrqol scores over time and to examine other cfq-r scales that may reflect positive changes in adolescent functioning (e.g., reduced treatment burden background: development of cf specific patient symptom tools as outcome measures is a critical step toward the development of potential new therapies for cf. we have developed a novel instrument to assess respiratory symptoms in cf patients. methods: we conducted 25 in-depth qualitative interviews using the day reconstruction method and 9 cognitive debriefing interviews (3 adults, 4 youth, and 2 parents) at two cf programs, the university of washington and children's hospital and regional medical center. interviews were conducted until no new symptoms were mentioned by interviewees (i.e., data saturation). the interviews were audio-recorded, transcribed, coded and analyzed for themes. results: six pulmonary symptoms were identified in the interviews: cough, sputum production, wheeze, chest tightness, difficulty breathing/shortness of breath and fever. in addition, the most commonly cited activity and emotional impacts identified in the interviews were also included on the questionnaire. these emotions included: frustration, sadness/depression, irritability, worry, difficulty sleeping, and activities included time spent sitting or lying down, reduction of usual activities, and missing school or work. in all, eight symptom items were selected for inclusion on the self-administered questionnaire (see table) . as a result of the cognitive debriefing interviews, we changed initial language and adjusted the response options on some of the items in order to create greater distinctions between them. no important issues were felt to be omitted by patients who were interviewed. conclusions: using qualitative inductive methodology, we have created a novel patient reported outcome measure for cf. further study will be required to assess validation of the instrument. or more signs and symptoms occurred for more than two days, antibiotics were ordered with physician input and phone follow-up in 3 days. if symptoms occurred less than two days, chest clearance was increased with phone follow-up in 24 hours. the initial algorithm (ia) was piloted by 2 nurses on 25 patient calls on the emr and reviewed for adherence. subsequently, the cf phone note was revised, expanding the symptoms to include gi issues, last visit date and last hospitalization. the revised algorithm (ra) was used for a one month trial period with the entire nursing staff. a total of 22 patient calls relating to pulmonary exacerbation were again reviewed for adherence to the algorithm. results: the review of 25 phone notes following the ia found 14 (56%) of the phone notes to be adherent to the assessment and treatment plan on the algorithm. several exceptions included calls received for gi issues, medications, complaints of general pediatric symptoms, and lab results. the phone follow-up within 24-72 hrs was also difficult to accomplish secondary to nursing responsibilities and difficulty locating the patient. the review of calls following the ra found 19 (86%) notes were adherent to the algorithm. discussion: the revised algorithm and cf phone note for pulmonary exacerbation improved consistency in nursing assessment of signs and symptoms, evaluation of the current plan of care, and triggered appropriate interventions based on symptoms and length of illness. this improvement was particularly pronounced among the part-time rns who found it easier to obtain a more accurate history of patient's illnesses without asking for assistance from other cf team members. since the algorithm and cf phone note are newly implemented, tracking for consistency with the cf phone note will continue, revisions will be made as needed, and phone notes for conditions other than pulmonary exacerbation may be developed. methods: in 2000, helen devos children's hospital integrated research coordinators (rc) from the spectrum health research department into the cf care team. this model incorporates two key aspects: 1. a shared resource of experienced, centrally educated and trained research coordinators (rc) with three rc's having a primary cf focus 2. incorporation of rc's into helen devos cf care centers' multidisciplinary team for weekly contact with patients. using the cf registry consent as a starting point of research discussion with the patients and families, the rc's present new and upcoming opportunities and answer questions about research. with attendance at local and national cf meetings, rc's have expanded their cf knowledge, networked with other rc's and explored new research trials. our site also integrated education about various aspects of research into both the clinical staff and cf family education meetings. a review of the research database was completed to assess our cf center's research program. results: over the 7 years reviewed, participation in clinical research trials has seen significant growth and patient participation has exceeded 250 patients enrolled into clinical studies. conclusion: utilizing a team of dedicated rc's from the research department has proven to be effective in advancing our cf center's clinical research program. discussion: to meet the growing demand of patients needed to participate in clinical trials, cf centers will need to provide research education to both the patients and families and the clinical team, improve on skills needed to identify and enroll our patients into clinical trials and include the rc's into the multi-disciplinary cf patient care team. centralized research represents an excellent model for cf centers that want to excel in clinical research. integration of rc's into the cf care team, along with education initiatives for both clinical staff and patients and families has led to successful participation and enrollment in clinical trials. further studies need to be done to better identify barriers that patients and families have with participation in research. the goal of "cystic fibrosis transition care" is to ensure that youth with cystic fibrosis (cf) are adequately prepared to participate in the management of their health condition into adulthood and as they graduate to the adult health care system. in canada, it is unknown if, or to what extent, cf transition care is practiced in individual clinics. we therefore surveyed all canadian pediatric cf clinic coordinators (n=27)using a brief standardized questionnaire; 23 surveys (85%) were completed and returned for analysis. findings: all 23 responding clinics transfer their pediatric patients to a distinct adult cf clinic, however almost half of these share one or more team members between the two clinics. transition care is recognized and practiced by 74% of responding clinics: 22% follow a formal transition program, and 52% follow an "informal program". "informal" practices vary widely from one clinic to another. formal programs were created by 5 individual clinics, and share the common properties of being based on current research, having set goals, and utilizing a tool to document the process. the average age of transfer to the adult clinic is 18 years. most clinics have "rare exceptions only" to delaying the age of transfer, the most commonly cited reason being "very ill/palliative patient". a small number of clinics also cited "intellectually challenged patient" and "reluctant patient" as reasons to delay transfer. only 30% of pediatric clinics hold a formal "transition" or "graduation" clinic, which allows adult team members to be introduced to youth before the actual transfer of care takes place. conclusions: although sharing team members between pediatric and adult clinics may ease the change from pediatric to adult care, it can also create barriers to the transition process. therefore, it is encouraging to learn that the majority of canadian pediatric cf clinics view adult care as distinct from pediatric care, and that there is a consistent age of transfer across the country. transition preparedness is also recognized as an important component of care, as evidenced by the number of clinics following a formal or informal transition process. it remains undetermined, however, whether in general there is adequate preparation for adult care. with only 22% of clinics following a formal transition process (and 26% not following any type of transition program), further assessment seems warranted and may reveal inadequacies requiring remediation. future direction: to strengthen cf transition care in canada, a "patient readiness to transition" questionnaire has been developed and will be administered to canadian cf patients transferring to adult cf clinics in 2007. analysis of this questionnaire will provide insight into how well canadian cf clinics are preparing their youth for transfer to adult life, and may provide directions for further improvement. nearly all females with cf now survive into adulthood, and advice regarding pregnancy and contraception is becoming increasingly important as part of their sexual health education. we set out to identify the level of knowledge of these issues in patients attending our large adult cf unit. using a structured questionnaire, we surveyed 49 consecutive cf females (age range 17 to 42 years) attending routine cf clinics. we asked about knowledge and usage of contraception, and issues relating to pregnancy and its possible effect in cf, ensuring the opportunity for a one to one discussion with the individual on completion of the questionnaire. only 29 (60%) claimed to have had previous advice relating to contraception and 23 (47%) pregnancy education. of these, 23 (81%) stated that this was from adult cf nurse specialists, 17 (57%) from adult cf physicians and the remainder from other sources (general medical practitioners, written literature, and the internet). only 7 patients (16%) recalled that they had been given information in the paediatric sector before transition. the majority of all advice was given verbally. of the 29 (60%) who were using some form of contraception (10 [33%] condoms, 9 [30%] combined pill, 3 [11%] depot injection, 2 [7%] a coil, and 6 [19%] the mini pill); 10 (33%) had experienced problems, and in some cases patients had been misinformed about the reliability of their contraceptive choice. as regards pregnancy, 2 (4%) had undergone previous termination and 8 (16%) already had children. thirty six (73%) had considered becoming pregnant but 46 (93%) said they would discuss this with a member of the cf team beforehand: 35 (71%) were aware this might impact on their health. of these, 32 (91%) believed a reduction in lung capacity to be the biggest possible problem. other problems identified were alteration of medication, tiredness, and weight loss or gain. overall, 29 (60%) felt they did not have enough information regarding pregnancy in cf and 25 (51%) that insufficient contraceptive advice was given, expressing a wish for further knowledge. conclusion this survey has shown that a significant number of adult females with cf require further education about contraception and pregnancy. in some cases the advice they had already been given may not have been appropriate for patients with cf. furthermore, few patients appeared to have had effective counselling in the paediatric sector, despite the risk of pregnancy. we are working with patients and paediatric colleagues to improve the education in this important area for our women with cf. the state of minnesota added cystic fibrosis to the newborn screening panel on march 1, 2006. one-year follow-up to date reveals 286 infants have screened positive, of the screen positive, 23 have been diagnosed with cystic fibrosis, 30 infants are pending sweat chloride tests and evaluation. of this population 8 infants are being followed by the minnesota cf center. the university has a comprehensive, interdisciplinary team available to provide care to infants, children and adults with cystic fibrosis. in response to newborn screening for cystic fibrosis, the center has implemented a prophylactic care program to include clinical care through early intervention and education within the first two weeks of life. benefits of collaborative care have been previously identified in the literature, showing improved quality of care with increased patient satisfaction, lower mortality, and improved outcomes. infants who are identified by newborn screening and receive earlier treatment have the potential for improved physical health and development. the inclusion of comprehensive genetic consultation as an integral component of cf education, allows for informed reproductive decisions. the newborn screening intervention plan is supervised by a nurse practitioner and consists of initial evaluation, initial therapy implementation, and a teaching/consultation schedule, which covers a minimum of four appointments scheduled over 2 weeks. follow-up visits are scheduled within 24 hours of referral and then at one week, 10 days and 2 weeks post referral. the schedule is adjusted based on infant acuity and family needs. initial evaluation consists of the confirmatory sweat chloride testing and/or genetic mutation analysis, serum laboratories, chest x-ray, stool for fecal elastase, and nasopharyngeal culture. general, supportive teaching with evidence from the literature is provided. preliminary results to date demonstrate that our infants have had no pulmonary exacerbations, no pulmonary hospitalizations with pulmonary functions obtained in over half of the infants. nasopharyngeal cultures obtained at the time of ascertainment demonstrate newborn screening patients have positive cultures for staphylococcus aureus, haemophilus influenzae, escherichia coli, streptococcus pneumoniae, klebsiella pneumoniae, acinetobacter junii and alcaligenes faecalis. one infant was culture positive for pseudomonas aeruginosa at the first out-patient clinic visit. this demonstrates colonization of organisms prior to clinic exposure. through minnesota's early intervention and education plan, the families will be more adherent to prescribed care, which will result in improved cf infant outcomes as monitored by number of exacerbations, pulmonary functions and potential complications. a detailed outline of medical intervention, education, and outcomes will be presented. cf-pediatric centre we developed a malabsorption blood test (mbt) using 2 odd-chain length fatty acids, pentadecanoic acid (pa) and heptadecanoic acid (ha), to assess fat absorption in subjects with cf. pa is a free fatty acid, while ha requires hydrolysis of triheptadecanoic acid (tha) by pancreatic enzymes for absorption. objective: to determine the mbt reproducibility in healthy subjects and subjects with cf. methods: subjects with cf ingested a liquid test meal including 2 fats (pa and tha) on 3 occasions at least 5 days apart. for each subject, a standard dose of pert (80,000 lipase units) or the subject's usual dose (if higher) was given with each test meal. serum was analyzed for pa and ha levels at baseline and then hourly for 8 hours. a non-compartmental pharmacokinetic analysis was performed using pa and ha concentration-time data from subjects with cf. c max was directly observed from the individual subject profiles, and auc was calculated using the linear trapezoid rule. summary statistics (mean±s.d.) were calculated for all parameters. reproducibility of the mbt was also assessed in healthy adult controls tested on 3 occasions. between subject (bsv, %) and within subject variability (wsv, %) was calculated for pa, ha and the ha/pa ratio. results: bsv (%) and wsv (%) of pa and ha absorption are presented in the table for 9 subjects with cf (age 17.8±3.9 y, 4 females) and 6 healthy adult subjects (age 25.8±3.8 y, 4 females). as expected, the mbt had greater reproducibility in healthy controls than in subjects with cf. in healthy controls, the reproducibility for pa and ha absorption was comparable; there is less wsv than bsv, and the ha/pa ratio has the best reproducibility. in subjects with cf, pa and the ha/pa ratio show less wsv than ha. conclusion: wider variability in fat absorption in subjects with cf reflects the complex interactions of the biliary, pancreatic and intestinal factors background: the international, multi-center gene modifier study of cystic fibrosis liver disease (cfld) initially enrolled 133 patients with severe liver disease (i.e. cirrhosis and portal hypertension). for the initial population despite severe liver disease with portal hypertension, many of these patients had normal liver biochemical function tests and/or inr (24-68%), depending on the test. the mean age of enrollment into the study was 20 years, with the mean age of cfld diagnosis of 11 years. candidate gene testing revealed an increased prevalence of the alpha-1 antitrypsin z allele in cfld patients, particularly in females, as compared to cf patients (>15 yrs.) without cfld, and an association with tgfβ1 variants (-509 and codon 10) in males smoothing reference centile curves: the lms method and penalized likelihood hepb) and inactivated hepatitis a (hepa) vaccines in cystic fibrosis (cf) patients chronic liver disease (cld) remains the second mortality cause in cf. we evaluated the immunogenicity of hepa-and hepb-vaccines in cf-patients because it is described lower in patients with cld. patients and methods: blood samples of 153 cf-patients aged 0.2-57.8y (mean 13.5y) were tested twice (time interval 1y) by chemiluminescent microparticle immunoassay to asses the presence of hbs-ab and ha-igg, and their vaccination status was recorded. seronegative patients for hbs-ab and/or ha-igg were vaccinated in between tests with engerix®, havrix® or twinrix®. results: 115/120 (95.8%) patients tested positive for hbs-ab. 59/115 cld (9 steatosis and 10 cirrhosis). 3/115 tested positive at first but negative at present data are incomplete for hbs-ab and ha-igg for 33 and 70 patients. conclusions: 1. immunogenicity of hepa-and hepb-vaccines is comparable in cf-patients and healthy subjects. 2. cf-patients are at risk for cld and seroconversion must be checked after vaccination. 3. vaccination records are important as numbers of antibodies may decline beneath the detection limit in time methods: lf was assessed in 53 children with cf, aged 8-12 yrs, using forced expiratory volume (fev(sub)1(/sub)% predicted). body composition was measured using a reference four-component model(sup)1(/sup) (4cm) allowing accurate evaluation of both fat mass (fm) and the components of fat-free mass dual-energy xray absorptiometry (dxa; lunar prodigy, g.e. medical systems) was used to measure fat, lean (non mineral) and bone mass and skin-fold thicknesses (bicep, tricep, sub-scapular) were measured. strength of relationship was assessed using pearson's correlation coefficient (r) and significant body composition components were fitted into a regression model. difference in fev(sub)1(/sub) between the sexes was assessed with an independent t test. results: boys with cf did not differ from the reference population conclusion: our results confirm that body fat, but not ffm or bone mass, are related to the severity of impaired lf in children with cf. it is likely that the significant association between fev(sub)1(/sub)and fat in girls but not boys reflects the poor body composition of the girls with cf. longitudinal follow-up of these children should indicate whether this sex difference persists after puberty. given the fact that prognosis is worse in girls the sex difference we've identified merits more attention. 1fuller nj et al. four-component model for the assessment of body composition in humans: comparison with alternative methods and evaluation of the density and hydration of fat-free mass conclusions: these results show that pancreatic status plays an important role in relation with pufa status in cf patients and particularly in (the initial fatty acids in the n-6 and n-3 pathways) in neutral lipids, nonesterified fatty acids, and phospholipids this may, in some way, account for the fact that 18:2n-6 is more readily metabolized, and therefore depleted, in cf animals than in wt mice. conclusion: dha supplementation increased the localization 539 does an integrated clinical and nutritional approach prevent this case control study examines the impact of clinical approach on pre and post-cfrd clinical course. 48 patients with cfrd (mean age 25.9+5.9yrs) matched to 48 cf controls (25.9+6.3yrs) for age, sex and pseudomonal status had parameters of clinical status and nutritional intervention recorded annually from six years pre diagnosis of cfrd to two years post. weight and body mass index (bmi) were lower at all time points to diagnosis of cfrd (ns) but were stable as a % of control values intravenous antibiotic treatment intensified, peaking at one year post diagnosis 47.7 days/yr (cfrd) v 34.7 (cf) ns but there was no difference in nebulised antibiotic use. an aggressive clinical approach prevents nutritional decline and delays respiratory decline until the year preceding diagnosis of cfrd data show mean (sd) or % 1,2 ; battezzati with advancing age insulin secretory defects and insulin resistance cause glucose intolerance and diabetes in an increasing proportion of cystic fibrosis (cf) patients. prediction of diabetes development in cf is made difficult by unique features: many patients are normoglycemic or even hypoglycemic after overnight fast, and there are repeated changes of glucose tolerance status from normal to diabetes and vice-versa, for many years and for unclear reasons. aim of the study was to detect predictive factors of definitive cf related diabetes (cfrd) development in patients undergoing oral glucose tolerance test (ogtt) evaluations routinarily. methods. starting from 2002, all patients followed at the cf center in milan aged >10 years and without established cfrd undergo ogtt yearly. among those who received their first ogtt between 2002 and 2004, 14 had developed definitive diabetes by 4 µu/ml, and 1.4±0.1 vs 1.4±0.2 ng/ml). glucose (p=0.010) and insulin aucs (p=0.030) were the most important predictive variables, respectively directly and inversely related to cfrd development (pseudo r 2 for the model: 0.581, p<0.001). glycated hemoglobin and baseline glucose concentrations were directly related with outcome at univariate analysis whereas c-peptide concentrations were inversely related. in contrast, no relationship emerged between insulin-sensitivity indexes and outcome. anthropometric (weight, height z scores, bmi) and pulmonary function indexes were also unrelated. conclusions. insulin secretory defects are an important determinant of subsequent cfrd developement response rate was 18/18 in group a and 17/18 in group b. group a included 5 males and 13 females, mean age was 52 months, sd 22.34; group b included 4 males and 13 females, mean age 52 months, sd 20.91. followgroup b parents do ("poor-very poor" health 6% vs 47%, p<0.002), but over time health perception in parents of children with cf improves, and the gap vanishes ("poor-very poor" health in last year 6% in group b). post-diagnosis anxiety, depressive symptoms, sleep disturbances, mood changes, and nervousness are less frequently reported in group a (62% vs 94% p<0.002), and the diagnosis was considered to affect the parent-child relationship more in group b than in a (p<0.01). the two groups did not differ in the assessment scores of internalizing, externalizing and total behavioural/emotional problems. however, group b performed worse than the general control population average (mean 12.65 sd 6.72 vs mean 9 sd 6.6, p<0.04). thirty-five% of parents in group a and 53% in group b changed their family planning projects following the diagnosis physical health and anxiety symptoms: does monitoring mediate the relation? results: physical health, monitoring, and anxiety symptoms were related. children who perceived themselves to be relatively healthy were less likely to have an information processing style characterized by high levels of monitoring (r = -.39, p < .05), and they reported fewer trait anxiety symptoms (r = -.50, p < .01). monitoring, as hypothesized, was associated with increased trait anxiety (r = .52, p = .001). low bmi, a potential indicator of poor nutritional status and physical health among youth with cf, also was related to higher monitoring (r = -.34, p = .05) and trait anxiety (r = -.29, p = .10). tests of mediation indicate that monitoring partially mediated the relation between poor health and trait anxiety for both child report of physical health (goodman test: z = 1.86, p = .06) and for bmi (goodman test: z = 1.67, p = .09), but not when parental report or fev1 were used to assess youth's physical health, or when parental report of youth's internalizing symptoms was used as the outcome. conclusion: youth who tend to generally scan for and are attentive to changes in their environment, which may include their internal physical environment, were found to report more anxiety. moreover, this monitoring style appears to partially mediate the relation between physical health and anxiety among youth with cf. if monitoring is found to be a risk factor leading to an increase in future anxiety symptoms among youth with cf data was analysed using a thematic analysis: "framework" 3 results: young people say that befriending is fun, offers opportunities for new experiences, a confidant outside the family, and gives them a boost psychologically. carers see befriending as confidence building for their children, providing time out for themselves, and helping with the big questions. befrienders see their role as mentoring, broadening young people's horizons and providing a safe place physically and emotionally. challenges include: forming and ending relationships, having multiple befrienders, ongoing support and training for befrienders, maintaining boundaries, sibling rivalry, and cost. conclusions: befriending is a new innovation in cf, and has the potential to make a difference to young people's lives. careful planning at the outset, ongoing support for befrienders and regular evaluation are essential factors in ensuring its success good practice in befriending services for people with learning disabilities qualitative research practice sage london 552ଙ in addition, there was high use of non-cf vitamins (n=102) and protein shakes (n=88) which were not included as a cam therapies. when prayer was excluded (which was used by 63% of respondents), 53.1% of the patients are still using some form of cam. the most commonly used cam therapies in cf were relaxation therapies (n=83), massage therapy (n=61), chiropractor care (n=59), herb/plant product therapies (n=53), homeopathy (n=19), and yoga (n=19). an analysis of the relaxation therapies revealed that deep breathing exercises (n=80) were the most common and frequently combined with other relaxation techniques: meditation (n=43), progressive relaxation (n=31) and guided imagery (n=24). the most common herb/plant products used were: echinacea (n=33), garlic (n=21), selenium (n=20), ragweed or chamomile (n=17) and ginseng (n=14). the use of possible cf specific cam therapies were fish oils/omega fatty acids (n=37), glutathione (n=13), docosahexaenoic acid (n=11) and curcumin (n=9). in conclusion, cam is widely used by the cf population with "prayer for health" being the most common modality, however, many patients are utilizing multiple cam therapies (data not presented) within the framework of a weighted satisfaction model of quality of life, we investigated the importance ratings of adolescent and adult patients regarding disease-specific aspects of living with cf. method: 108 outpatients (aged 15-47 years, m=27.0, sd=7.9; fev1 20-125%, m=62.6, sd=26.2) repeatedly filled in the cf-specific module of the questions on life satisfaction (flz-cf) to measure satisfaction with nine cf-specific aspects of life in relation to the subjective importance of each life domain. a ranking list of the most important life domains across the study group was determined, and intra-individual changes of the importance scores were analysed. associations of importance ratings with changes of pulmonary functioning (fev1%) were examined. results: the most important aspects (% of "very important" or "extremely important" answers) are sleep (96.2%), integration of therapy into daily routine (94.4%), breathing (89.8%), gastrointestinal functioning (86.9%), and eating (86.9%), less important is understanding by others (44%) 1,5 ; durieu, i. 4,5 1. département d'information médicale, hospices civils de lyon dr. von hauner children`s hospital helios hospital, e.v. behring, berlin, germany supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute cystic fibrosis foundation therapeutics inc. symptoms 571 patient-reported respiratory symptoms in cystic fibrosis: initial validation children's hospital and regional medical center the first author is supported by a second year clinical fellowship from razvi, s. 1 ; quittell, l.m. 1 ; sewall, a. 2 ; marshall, b.c. 2 ; saiman, l. 1 1. pediatric pulmonary medicine, columbia university, new york, ny, usa; 2. cystic fibrosis foundation, bethesda, md, usa significance: significant improvements have been made in diagnostic and therapeutic strategies for cf patients in recent decades. we hypothesized that these changes could potentially impact cf respiratory microbiology. thus, we examined longitudinal trends in the annual incidence and prevalence of cf respiratory pathogens from 1995 to 2005.methods: cf foundation patient registry data, estimated to include 90% of the cf population in the u.s., was utilized in this analysis. patients were included if results from at least one respiratory culture were in the registry from january 1995 to december 2005. patients were excluded after organ transplantation. to avoid misclassification of incident cases, a retrospective review of registry data from 1985 to 1994 was performed to establish the culture status of included patients prior to 1995. thus, incident cases were subjects with first detection of a given pathogen in a given year. prevalent cases were defined as subjects with at least one positive respiratory culture for a given pathogen in a given year. all submitted culture results were included.results: the number of cf patients submitting registry data increased from 19,735 in 1995 to 23,347 in 2005 . the proportion of subjects meeting inclusion criteria remained relatively constant (85% to 91% per year). the median age of the study cohort increased from 13.1 years in 1995 to 15.1 years in 2005. during the study period, the incidence of hemophilus influenzae remained stable (10.3% to 10.6%) as did the prevalence (15.3% to 17.3%). the incidence of pseudomonas aeruginosa ranged from 20.8% in 1996 to a peak of 24.2% in 2004. the prevalence of p. aeruginosa declined from 60.4% in 1995 to 56.1% in 2005. there is a trend for both an increasing incidence and prevalence of staphylococcus aureus; the incidence increased from 21.7% in 1995 to 33.2% in 2005 and the prevalence increased from 37.0% in 1995 to 52.4% in 2005. the age specific prevalence of s. aureus remained highest in children aged 6-17 years. the incidence of methicillin-resistant s. aureus (mrsa) increased from 0.1% in 1995 to 7.0% in 2005, with a parallel increase in prevalence from 0.1% to 17.2%. the highest prevalence of mrsa was noted in subjects 18 years of age and older. the incidence of burkholderia cepacia complex decreased from 1.3% in 1995 to 0.9% in 2005, while the prevalence remained relatively stable (range 2.9% to 3.6%). both the incidence and prevalence of stenotrophomonas maltophilia increased (incidence: 2.6% to 6.4%, prevalence: 3.5% to 12.4%).this retrospective study aims at assessing the respiratory function and the cystic fibrosis (cf) co-morbid conditions according to the patients' nutritional status.method: data were collected from the french observatory (onm) 2004 (n=4533) (vlm, ined). the last year data for weight (w), height (h), fev1, fvc were extracted from the database (exclusion criteria: transplan-tation); were also analysed the genotype, pancreatic enzymes consumption, pseudomonas aeruginosa (pa) colonization, cirrhosis, diabetes, pulmonary transplantation and mortality (sas 9.1). we used international age-adjusted standards for bmi z-score for children (c) and bmi for adults (over 18 years) (a) (cole tj bmj 2000) to define underw (uw), normal w (nw), overw (ow) and obesity (ob) .results: data are available for 2647 patients in the c cohort and for 1498 patients in the a cohort (36 %). nutritional status subgroups prevalence (%) is: 6.1, 87.1, 5.6 and 1.2 in c and 37.3, 57.7, 4. 3 and 0.7 in a for respectively uw, nw, ow and ob. mean age in a is significantly increasing with bmi (p<.0001). frequency of 508 del/508 del in c and a is lower in ow/ob (p<.02) as well as the use of pancreatic enzymes (p<.001). ow/ob patients have the best fev1 and fvc values whatever the gender and the age, with significantly less pa colonization in a (p<.01). we could identify a positive correlation between the pulmonary function and bmi. the cf co-morbid conditions demonstrated a lower prevalence of diabetes (12/257) and cirrhosis (5/257) in ow/ob. none of the ow/ob c or a are on a transplant waiting list versus 1.7 % in uw/nw (n=69/4042) and none died versus 1 % in uw/nw (n=42/4042).conclusion: the observed prevalence of ow and ob in cf is respectively 5.2 and 1 % whereas the french obepi 2006 study collected 29 and 12 %. our results suggest that increased bmi is associated with better fev1, fvc, lower prevalence of pa, cirrhosis and diabetes. the potential risks of chronically high bmi have not been studied in this population yet, but justify further investigations in this longer life expectancy cohort. materials and methods: we retrospectively identified abdominal ct scans of 38 consecutive patients (19 females, 19 males, mean age of 28 years) with cystic fibrosis and a control group of 38 consecutive patients (21 females, 17 males, mean age of 40 years) scanned as potential renal donors. three readers reviewed all scans and recorded the presence and location of colonic wall redundancy, and the wall thickness of the ascending, transverse, and descending colon. clinical information on cystic fibrosis patients, including cftr gene mutations, was queried from our cystic fibrosis patient registry database. additionally, medical records of all cystic fibrosis patients were reviewed to determine the indication for abdominal ct. results: colonic wall redundancy was seen exclusively in patients with cystic fibrosis, and was noted in 11 of 38 patients each for reviewers 1, 2, and 3 (p<0.05). colonic wall redundancy was seen in 11 of 28 adult patients with cystic fibrosis (39%), but was not seen in any children age 17 or younger (p<0.05). excellent agreement was found for the ct identification of colonic wall redundancy among readers (kappa=0.91, p<0.001). cystic fibrosis patients with colonic wall redundancy had significantly thicker ascending colonic walls (mean 4.0 mm) vs. those without wall redundancy (mean 1.8 mm) or controls (mean 1.2 mm), (p=0.03). three patients with colonic wall redundancy had follow-up ct, and all showed temporal stability (mean of 50 months). among adult cystic fibrosis patients, cftr gene mutations were available for 10 of 11 patients with and 13 of 17 without colonic wall redundancy. while the common ∆f508 mutation was the predominant mutant allele among patients with normal colons at ct (only 3 of 17 patients, or 23%, had an identified mutation other than ∆f508 on either allele), a higher prevalence of less common non-∆f508 mutations was seen patients with colonic wall redundancy (7 of 10 patients, or 70%, p<0.05). the g542x mutation was seen exclusively in patients with colonic wall redundancy (3 of 10 patients, or 30%, p=0.06). there was no significant difference in the proportion of patients with abdominal pain (>0.7), pancreatic insufficiency (p>0.99), diabetes mellitus (p>0.1), history of meconium ileus background: airway inflammation in cf is associated with marked remodelling and bronchiectasis. pro-inflammatory mediators such as advanced glycation end-products (ages) and the soluble receptor for age (srage) may perpetuate this response in the lung and in other organs such as the kidney 1 . the total body burden of ages reflects exogenous sources from the diet, endogenous production by the body, tissue degradation and renal clearance, which may be reduced in renal impairment. the accumulation of endogenous age is accelerated in conditions of high oxidative stress and inflammation, and ages have been implicated in the pathogenesis of diabetic nephropathy. the burden and significance of ages in cf has not been determined, but if elevated, dietary modification of ages may represent a novel anti-inflammatory approach for cf. the aim of this study was to determine serum levels of age and srage, and identify clinical correlates of age and srage levels.methods: adults with cf (n=58, 35 males, 86% pancreatic insufficient, mean age 33.9±7.7 years (range 23 to 62 years), mean fev 1 %predicted 58.7±22.6%predicted (range 20-110)) and healthy adults (n=24, 13 male, mean age 32.7±9.2 years (range 21-55 years)) were studied. cf participants provided 1-3 serum samples each over a six month period, while healthy controls provided a single sample. serum was analysed for levels of advanced glycation end-product (age-cml) and soluble receptor for age (srage) (elisa). for each cf participant, levels in the multiple samples were averaged, and the median for the study sample reported. clinical data, including bmi, presence of cf-related diabetes mellitus (cfrd) and serum hba1c level were collected from the medical record. mann-whitney tests were used to compare cf and control levels, while spearman rank correlations were used to identify clinical correlates of age and srage.results: prevalence of cfrd was 27.6%. mean bmi was 21.8±2.7kg/m 2 and mean hba1c level was 5.9±1.1%. median (iqr) levels for age-cml were 1650 (1158, 1986) background: osteopenia is diagnosed in cystic fibrosis (cf) using dual-energy x-ray absorptiometry (dxa). areal bmd from dxa is subject to error when bones are smaller in volume than reference standards (t-scores). normalization of bone size by use of zscores in cf is controversial and not widely utilized, thus comparing to larger bone areas. reports in cftr-deficient mice (cf mice) reveal osteopenia when measured by dxa. we hypothesized that use of pqct, which eliminates bias of size, would more accurately analyze bmd.methods: femurs were collected at necropsy from 4 cf mice and 4 c57bl/6j (b6) mice at 6 wks, and 7 cf mice and 7 b6 mice at 14 wks (all female) for pqct. time points were chosen to coincide with pre-pubertal and adult ages for comparison to human disease. total mineral, trabecular and cortical densities were measured. student's t-test was used to detect significant differences (p<0.05).results: femur measurements from both cf mice and b6 mice are listed as mean ± sd in the table (below). length was greater in b6 mice compared to cf mice at 6 and 14 wks. total area at the metaphysis and diaphysis were greater in b6 mice at 6 and 14 wks. data are consistent with larger bones in b6 mice. total mineral density 1 , trabecular density 2 and cortical density 3 , however, were greater in cf mice compared to b6 ( 1 6 & 14 wks, 2 14 wks, 3 6 wks).discussion: our study demonstrates greater bmd in cf mice when volumetric data are analyzed and size differences are accounted for by use of pqct. these findings persisted at adult age, suggesting a normal deposition of bone. in clinical management, dxa imaging is readily available compared to pqct, and can be used as a predictor of bmd and fracture risk. correct reference ranges must be utilized to minimize erroneous values secondary to size. z-scores allow correction for differences in bone area and size, even in adults. more studies into understanding bone mineral deficit and tools of measurement are needed in cf. meanwhile, bone measurements by radiographic imaging must be taken in context of overall health, pubertal progression, and size of individuals. gainesville, fl, usa; 2. nemours children's clinic, orlando, fl, usa; 3. suny upstate medical university, syracuse, ny, usa; 4. case western reserve university, cleveland, oh, usa; 5. genentech, san fancisco, ca, usa background: greater growth rates, bmi and fat-free mass are associated with improved lung function in individuals with cf (pedreira, et al. pediatr pulmonol. 2005) . current treatments for weight gain have focused on nutritional supplements and appetite stimulation, and not underlying issues of catabolism or chronic disease. we hypothesized that anabolic effects of gh would not only improve weight and height, but lbm as well.methods: sixty-seven prepubertal children with cf and height less than or equal to the 10th percentile were randomized to daily gh (n=35) or observation (n=32) for a period of 1 year, followed by an off-treatment observation period of 6 months. children randomized to gh received somatropin injections daily at 0.3 mg/kg/wk. height and weight were measured every three months. height was evaluated as height standard deviation score (sds) to control for differences in age and sex. lbm was measured by dexa scan at time zero, at 6 and 12 months, and at the end of the 6-month observation period. in addition, for lbm, change from baseline was calculated for subjects for whom the same dexa equipment was used at both time points. the preliminary results of the first 12 months of the study are presented here.results: data for 27 subjects in the observation arm and 29 subjects in the gh arm, who completed 12 months, are available. as shown in the table below (listed as mean ± sd), gain in height, weight and lbm were significantly greater in the gh treated group than in the observation group over the 12 month period.discussion: gh improves growth in prepubertal children with cf as measured by height sds. in addition, gh significantly improves weight gain and this gain is, in part a result of the significantly greater increase in lbm in the gh-treated group than in the observation group. the relationship between the improvement in lbm and other outcomes in cf deserves further exploration. the national cystic fibrosis(cf) registry database notes cystic fibrosis related diabetes mellitus(cfrd) as a complication in 15.6% of cf patients at all ages. cfrd is associated with poorer nutrition and increased pulmonary morbidity. the cause of cfrd is not completely known but insulin resistance may be associated with fibrotic damage to pancreatic islet cells due to chronic inflammation. other studies suggest protein energy malnutrition(pem) in early life leads to impairment of insulin secretion and cystic fibrosis-related diabetes (cfrd) accounts for increased morbidity and mortality in patients with cf and occurs in approximately 30% of patients by the age of 30 years. cfrd net (network for epidemiology and trials) is a consortium of four large uk cf centres caring for 1052 adult patients with cf. the aim of this group is to undertake research into the diagnosis, investigation and management of cfrd. in this abstract, we estimate the prevalence of diabetes in a screened population of adults with cf. methods annual review data were collected on attending patients with cf aged 16 and above during 2006. 75g ogtts were performed after fasting for at least 8 hours on patients without known diabetes. each ogtt was categorised as either normal (2 hour glucose 3.5 -7.8mmol/l), impaired (2 hour glucose ≥ 7.8mmol/l and < 11.1mmol/l) or diabetic (2 hour glucose ≥ 11.1 mmol/l). all patients with a diabetic ogtt were followed up with serial bm monitoring to determine whether or not they had cfrd. fasting plasma glucose (fpg) was considered to be elevated ≥ 7.0 mmol/l, isolated impaired fasting glucose (igf) was defined as a value between 6.1 mmol/l and 7.0 mmol/l and hypoglycaemia was defined as a blood glucose <3.5mmol/l. in addition, we obtained information on age, sex and prescription of anti-diabetic medications subsequent to ogtt. in three of four centres, hba1c was routinely performed on all patients. of 1052 patients (median age 26 years, 57% male), 344 (33%) had established diabetes and were therefore excluded from further screening. of the remaining 708 patients, 392 (55%) underwent formal ogtt testing. in this latter group 34 (9%) had a diabetic ogtt, 58 (15%) an impaired ogtt and 49 (13%) had evidence of reactive hypoglycaemia at 2 hours. of the 34 patients with a diabetic ogtt, 28 (82%) only had an abnormal 2 hour value, 4 (12%) only had an elevated fpg and 1 (<1%) had both. all patients with diabetic ogtts underwent blood glucose monitoring and 20 (59%) went on to treatment with hypoglycaemic agents in the calendar year. none of the 4 patients with an isolated elevated fpg had diabetes.hba1cs were available in 23 of 34 newly diagnosed patients with diabetes (in the centres which performed hba1c). in this group median hba1c was 6.1%. this study confirms the high prevalence of diabetes among screened patients and the growing burden of diabetes management in adult cystic fibrosis clinics. it further highlights the importance of performing screening for diabetes in this population; the majority of patients were identified on the basis of abnormal 2 hour values. despite this only half of all suitable patients underwent ogtts in the four centres committed to screening. these patients merit further study, as previous work suggests that the decline in clinical status occurs several years before diabetes becomes apparent. cystic fibrosis related diabetes (cfrd) occurs in up to 40 % of patients with cystic fibrosis (cf), the incidence rising with increasing age. the likelihood of developing long-term complications secondary to diabetes increases with poor glycaemic control and duration of diabetes. as survival has improved for people with cf those who develop cfrd may live with diabetes for several years. aim: to establish the frequency of diabetic complications in patients with cystic fibrosis related diabetes.method : patients with cfrd attending the adult cf service at the royal brompton hospital between april 2006-march 2007 were screened for diabetic complications and cardiovascular risk factors. a total of 78 patients (male/female: 44/34) including 10 post transplant patients were screened. mean age was 33.1 years (17-52), mean hba1c 8.0% (5.6-16.2% ) and average duration of diabetes was 7.3 years (<1-38 years).as immunosuppressive therapy can also cause many of the complications associated with diabetes the results are presented separately for nontransplant and post transplant patients (table 1) . 9 patients in total had retinopathy: 7 background retinopathy,1 proliferative retinopathy undergoing laser therapy and 1 maculopathy. the average duration of diabetes in those with background retinopathy was 5.8 years with only 1 patient having diabetes for >10 years. the patient with proliferative retinopathy (non-transplant) had been diabetic for 15 years. a raised creatinine level was identified in all transplanted patients with microalbuminuria but none of the non-transplanted patients. 1 patient (nontransplant) had macroalbuminuria. 2 post transplant patients were on treatment for hypertension and a further 6 patients (2 post transplant and 4 nontransplant) had elevated blood pressure (>140/90) at screening requiring follow-up. none of the patients had evidence of cardiovascular disease or stroke.conclusion: macrovascular complications were not seen. microvascular complications occurred but were less common than the reported incidence in type 1 and type 2 diabetes. this may reflect the relatively short duration of diabetes (mean 7.8 years) of patients in this study. a further study comparing cfrd patients with a non-cf diabetic control group of similar duration of diabetes is warranted. cystic fibrosis (cf) is a disease that leads to serious disturbances in nutritional status and bone calcification. comparison of two methods for assessment of bone mineralization: dexa and hand radiograms in diagnosing osteopenia or osteoporosis. study was performed in a group of 26 cf patients (10f, 16m), aged 7-30 yrs. nutritional status was assessed using bmi, cole's index and bmc. radiograms of non-dominant hand were assessed according to normalized optical density comparing to aluminum standard. bone density was also assessed using dexa. for statistical analysis, backward stepwise binary logistic regression (wald's test) was used. analysis of data revealed that using bmc, cole's index and hand radiograms markers we can diagnose bone mass disturbances (z score <1sd) with precision up to 84.62% comparing to dexa. sensitivity and specificity of this method was respectively 86.67% and 81.82%. false negative results were obtained in 2 patients and false positive were also in 2 patients. hand radiograms method could be an alternative for dexa in screening of bone density disturbances in cf patients. the study was partly supported by grant of ministry of science and higher education no 2 p05e 041 28.in a very recent publication(1) a high rate of fasting (13%) and reactive hypoglycaemia (15%) was described in a group of n=129 cf patients older than 8 years who received an oral glucose tolerance test. reactive hypoglycaemia was related to the 120minute glucose concentration in the ogt test. we were surprised by the high percentage of asymptomatic hypoglycaemic situations in cf patients. as a part of a prospective intervention study in cf patients with cfrd, we screened more than 1400 patients 10 years or older with cf for cfrd. in this multi-centres population ogt was performed in the morning after overnight fasting according to who standards. using the same definition for fasting hypoglycaemia (glucose < 60mg/dl; 3.3mmol/l) and reactive hypoglycaemia (glucose < 50mg/dl; 2.8mmol/l at 120minute during ogt test)as in the other study we calculated the percentage of cf patients with fasting or reactive hypoglycaemia in our cohort. results: ogt were done in 1495 patients with cf. age(mean±sd)19,6±8,6 years; bmi z-score -0.73±1,1;height z-score -0,8±1,1 and weight z-score -1,0±1,3. ogt was categorised according to ada criteria. normal ogt (n=782 all ; fasting hypoglycaemia (fh) n=21(2,7%), reactive hypoglycaemia (rh)n=18 (2,3%),ifg (n=329 all, fh n=0, rh n=5 (1,5%), igt(n=131,fh n=2(1,5%), rh n=0),fgt (n=75 all, fh n=0,rh n=0) and diabetes mellitus (n=187 all, fh n=3(1,7%)). fh was observed in 26 (1,7%) and rh in 23(1,5%) out of 1495 ogt tests. there were no difference related to age, bmi z-score, height z-score and weight z-score comparing those patients with fh or rh to those without fh or rh in all categories of ogt tests.in this large multi-centres cohort of cf patients we were neither able to confirm the high percentage of fasting nor of reactive asymptomatic hypoglycaemia which was reported recently on a small group of cf patients. nutritional status measured by bmi z-scores, height and weight z-scores were unaffected by hypoglycaemia in our cohort as noticed also in the small group. as the ogt tests in the other study were done in a single centre it might by that a centre specific situation influenced the frequency of hypoglycaemia in that small group. we conclude that large numbers of investigations might be needed to come up with firm conclusions related to frequency of specific aspects of glucose disturbance in cf patients.fh does not exclude diabetes mellitus or igt. as survival from cf improves, surveillance to identify and treat complications associated with longevity is an important component of management. renal disease has been reported in adults with cf. risk factors may include cf-related diabetes mellitus (cfrd) and use of nephrotoxic medications. diabetic nephropathy has been observed in the absence of cfrd 1 , highlighting the need for screening for renal impairment. this study aimed to measure renal function in a sample of adults with cf, and to compare estimated glomerular filtration rate (egfr) and urinary creatinine clearance (urcrcl) as markers of renal function.methods: adults with cf aged 25 years or over (n=29, 66% male, 83% pancreatic insufficient, mean age 36.7±9.1 years) underwent screening for renal impairment. of these patients, 15 (9 males) had cfrd, and 14 (10 males) had no history or clinical features of cfrd. 24 hour urine collections were analysed for urcrcl, with renal impairment being defined as <90ml/min. serum creatinine level was used to calculate egfr (mdrd formula), which was classified renal function as normal or mildly impaired (>60ml/min); moderately impaired (30-59 ml/min) or severely impaired (<30ml/min). sensitivity of egfr for identification of renal function was calculated using urinary creatinine clearance as the reference method. unpaired t-tests were used to compared cfrd with non-cfrd patients.results: mean urcrcl was 105±35ml/min, and 8 eight patients (27.6%) had renal impairment (<90ml/min). of these, only 3 had egfr suggestive of renal impairment. the sensitivity of egfr for identifying renal impairment in this sample was 38%. a further 2 patients had moderately impaired egfr, but normal urcrcl. five of the 8 patients with impaired urcrcl had cfrd. age, gender, cfrd, fev 1 %predicted and hba1c level did not correlate with urcrcl. the table compares cfrd patients with non-cfrd patients. hba1c level was higher in cfrd patients. there were no other significant differences in clinical or renal function parameters.conclusion: renal impairment, is common in the adult cf population and is not confined to patients with cfrd. screening using egfr is poorly predictive of impaired urcrcl in this population. these results suggest that surveillance to monitor renal function is indicated in the adult cf population, including in patients without cfrd, and that determination of urcrcl should be included in the screening process. cystic fibrosis (cf) patients suffer from pancreatic insufficiency resulting in malabsorption of fat soluble vitamins including vitamin d. many fac-tors contribute to low bone mass and fractures in patients with cf; however, chronic vitamin d deficiency plays a major role. the prevalence of vitamin d deficiency has been reported as high as 81% in some specialized care centers. in addition, reports of occult vertebral fractures have been reported as high as 25%. we sought to determine the prevalence of vitamin d deficiency (defined as 25-hydroxyvitamin d (25(oh)d) < 30 ng/ml) and of vertebral fractures at our cf center. we obtained irb approval to review the records of all patients seen at our cf center during [2004] [2005] . we collected information related to bone health including 25(oh)d, bone mineral density and lateral spine or chest x-rays to examine for the presence of a vertebral fracture. we reviewed the records of 185 subjects who were seen at our center during the study period. the mean age of the subjects was 29 ± 9 years. subjects had a mean bmi of 21.2 ± 3 and an fev1% predicted of 64.4 ± 25%. the percentage of subjects having an annual 25(oh)d level checked was only 57% and 62% in 2004 and 2005, respectively. the mean 25(oh)d was 22.7 ± 10 and 24.9 ± 10 ng/ml in 2004 and 2005. the prevalence of vitamin deficiency was 78% and 73% in 2004 and 2005. about one quarter of the subjects had bone mineral density testing with half of the tests showing osteopenia or osteoporosis. twenty-seven percent of subjects had vertebral abnormalites detected on lateral chest x-ray. we sought to determine whether any factors were associated with vitamin d deficiency. we found that taking a multivitamin did not significantly protect against vitamin d deficiency. however, not taking a supplement containing vitamin d other than a multivitamin was associated with a 54% risk of vitamin d deficiency (p=0.04). subjects having 25(oh)d levels determined in the winter or spring was associated with a 30% risk of vitamin d deficiency (p=0.05). in summary, we found annual testing for vitamin d status was inadequate in our cf center and that when 25(oh)d level was determined, over 70% of subjects were vitamin d deficient. nearly one quarter of our adult patients already had evidence of vertebral compression fracture seen in lateral chest x-ray. we urge greater screening for vitamin d deficiency in the cf population. effective protocols to prevent and/or treat vitamin d deficiency are urgently needed in the cf population. improved vitamin d status in cf patients is one factor that may reduce the high prevalence of vetebral fractures. support for this study was provided by proctor and gamble pharmaceuticals background: more cf patients are surviving into adulthood, in part due to the increasing use of new therapies and more aggressive management of chronic respiratory and gi disease. as a result, the recommended daily treatment regimens for most cf adults are both complex and time consuming. objective: to assess the self-reported daily treatment burden of cf therapies in a cohort of adults with cf. methods: in the sixth survey wave of the project on adult care in cf (pac-cf), an ongoing longitudinal panel study of adults with cf from 10 us cf centers, respondents were asked to report the type of medications, inhaled therapies, and airway clearance therapies they used during the day prior to completing the survey, as well as estimate the time generally required to complete each type of therapy.results: of the 204 respondents (response rate 69%), the median number of therapies reported was 7 (iqr 5-9) and the median reported amount of time usually spent on treatments was 90 minutes per day (iqr 50-150 minutes). forty-eight percent reported using 5 or more different inhaled therapies or using inhaled therapies 5 or more times during that one day. the most commonly reported inhaled therapies were a bronchodilator (61%), pulmozyme (49%), an inhaled steroid (32%), hypertonic saline (30%), and objective: caring for a child with cf is stressful and often leads to adverse effects for both children and caregivers. support from family and friends can help reduce the adverse effects of chronic stress, however, there is little research documenting types of support provision as well as sources of support. moreover, social support may be related to important health outcomes. this study described the types of support provided to caregivers of children with cf and examined relationships between support and health outcomes.method: as part of a larger intervention study to improve adherence to medical regimens, 89 children with cf ages 1 to 11 and their caregivers were enrolled across 3 cf centers in florida: university of florida, nemours children's clinic, and all children's hospital. caregivers completed the norbeck social support questionnaire (nssq; norbeck, 1980) in addition to other measures at baseline and follow up. this questionnaire provides information about the number and type of people providing support, as well as its type and quality.results: caregivers received the majority of support from their families, spouses and friends and characterized these relationships as longer in duration, with more frequent provision of support. other individuals who provided support included the cf team, religious leaders, counselors or psychologists, and work associates. overall, caregivers rated family members and spouses as providing the greatest amount (i.e., "quite a bit") of emotional support. spouses received the highest ratings for tangible support; they were perceived as providing "quite a bit" of support in comparison to families (i.e., moderate) and friends (i.e., a little). in terms of size of the support network, caregivers in this study had similar networks to those of a normative sample of healthy adults. in addition, there was no difference in amount of emotional or tangible support received by these caregivers. preliminary baseline results indicated that social support was positively related to both adherence, children's growth (i.e., height and weight percentile), and family income.discussion: caregivers of young children with cf reported similar support networks as other healthy adults. however, there was variability in the amount of support provided by source, with spouses providing the greatest amount. social support appeared to be a protective factor, and lack of support was related to negative health consequences for their children. more attention should be focused on the potentially beneficial effects of social support.funding was provided by nih grant #69736 befriending is reported to be valued by those who have been befriended, offering opportunities for social activities and new experiences and can impact positively on self confidence and self esteem 1. whilst government policy supports befriending, few schemes collect evidence to demonstrate the effectiveness of these services 2. young people with cf may suffer social isolation due to chronic illness and treatment demands and a befriend-ing service was offered to those considered socially vulnerable in the edinburgh area.this project aims to evaluate the effectiveness of this befriending service on young people with cf and their carers. methods liverpool, united kingdom; 2. child mental health, university of liverpool, liverpool, united kingdom; 3. child health, university of liverpool, liverpool, united kingdom; 4. psychology, university of miami, miami, fl, usa; 5. psychology, birkbeck college, university of london, london, united kingdom; 6. mathematics and statistics, university of lancaster, lancaster, united kingdom; 7. psychiatry, university of manchester, manchester, united kingdom introduction: existing treatments for cystic fibrosis (cf) are time-consuming and labour intensive and biomedical advances are likely to lead to further novel interventions. there is concern amongst clinicians that a high care burden associated with these interventions may compromise the wellbeing of caregivers, reduce adherence to the treatment protocol and increase the likelihood of inadvertent errors in the delivery of interventions. at present, there is no measure of 'treatment burden' and therefore no way to assess the impact of new and increasingly more complex interventions conducted by lay caregivers at home. furthermore, trials are hampered by the lack of measurable patient-reported outcomes beyond a broad quality of life assessment. to address this gap we have developed a measure of treatment burden for cf using qualitative methods (focus groups, action research, in-depth interviews) with participating parents and a working group of cf team professional staff. this measure addresses the time, effort, meaning and ease of management for caregivers of children with cf up to 13 years and post the first year following a diagnosis. methods: we report here the pilot phase of the validation of this instrument, which involved (i) cognitive interviewing with n=16 caregivers; and, (ii) n=30 caregivers completing the clcf instrument together with a quality of life measure at 2 time points. this yielded data for stability, reliability and coherence of the hypothesized constructs. results: the clcf takes 15 minutes to complete. face validity and acceptability are established. for the first pilot sample the age of the child ranged from 1.5-11.5 years. treatments took, on average 72 minutes per day to complete and an average of 16 minutes of this was devoted to administering enzymes particularly for carers of infants. a prominent concern amongst these parents was their child's height and weight and their efforts were directed at optimising growth. for the child however, management of nebulised medications and physiotherapy were more frequently flagged as a concern. these data suggest this is a reliable, coherent and useful, although complex tool. conclusion: the caregiver challenge for cf cannot be understood simply in terms of time and effort involved in delivery of treatments. it also involves contextualising interventions for their meaning at multiple levels of explanation for the individual concerned. structural equation modelling would be an appropriate way to proceed with the main validation when such complex relationships occur between latent variables and would serve as a confirmatory factor analysis for the hypothesised relationships. sponsored by: royal liverpool children's nhs trust and university of liverpool. funded by: national institute for health research (nihr) research for patient benefit programme.introduction: the french cf practice recommendations, published at the end of 2002 in parallel with the creation of cf reference centres, advise that each patient should be seen at least every 3 months at a cystic fibrosis reference centre.objective: to investigate the impact of these recommendations on the effectiveness of the follow-up of patients at the four reference centres in the rhône-alpes region.methods: all patients with cystic fibrosis attending one of the four cf centres between 1996 and 2005 were retrospectively included. the total number of visits was recorded for each patient and each year of the study period. to determine the evolution slope for each patient followed for at least 3 years, a negative binomial regression of number of visits versus time was carried out (confirmed with a repeated model). to estimate the impact of the recommendations, the analysis was restricted to patients with at least two visits before and after 2003 and a second model was adjusted with a new intercept in 2003 to estimate the change in slope as of this point.results: a total of 650 patients were included in the cohort. the average number of visits per patient rose from 3.68 in 1996 to 4.96 in 2005 (p<0.0001). the proportion of patients with at least 4 visits per year increased from 38% to 70%. the negative binomial regression for the 537 patients having had at least 3 years follow-up confirmed this trend with an average slope of +0.07 (sd=0.18). a total of 352 patients were evaluable for the change in slope. no significant change in trend was observed in 2003: only 34% of patients had a higher rate of growth (this change was significant for only 7 subjects). at the last follow-up visit, patients with increasing rates of number of visits were older (21yrs vs. 17yrs, p<0.0001), had lower %fev1 (68 vs. 75, p=0.02), had a similar average number of visits before 2003 (3.7 vs. 3.9, p=0.33), and a similar weight-for-age z-score (-0.58 vs. -0.46, p=0.50) .conclusion: the number of visits per patient is regularly increasing. since the publication of the practice recommendations in 2003, the growth has tended to slow down: clinicians were already convinced by the need for closer follow-up and had begun to increase the rate of visits. methods: patients > 13 years who presented for routine healthcare voluntarily completed four surveys: 1) the revised eating attitudes test (eat), validated for cf patients by abbott and colleagues, where higher scores reflect worse attitudes towards eating; 2) the rosenberg self-esteem scale (rse), where higher scores reflect better self-esteem; 3) the body image scale (bis), developed for cf patients by wenninger and colleagues, where higher scores reflect better body image; 4) the cystic fibrosis questionnaire (cfq), developed by quittner and colleagues, where higher scores reflect better hrqol. also, fev1%, body mass index (bmi), and pancreatic sufficiency or insufficiency (based on the need for pancreatic enzymes) was recorded. regression analyses controlling for age, gender and bmi were used to examine the associations between the surveys. results: this study included 37 patients with 41 % males and a mean age of 26 years. the eat was negatively associated with the rse (p=0.015, adjusted r 2 =0.127), bis (p=0.001, adjusted r 2 =0.221) and cfq (p=0.015, adjusted r 2 =0.122). the rse was positively associated with the cfq (p=0.001, adjusted r 2 =0.346). the bis was positively associated with the rse (p=0.001, adjusted r 2 =0.529) and cfq (p=0.001, adjusted r 2 = 0.540). neither bmi nor pancreatic function was associated with the surveys (p=ns). fev1% was positively associated with the cfq (p=0.028, adjusted r 2 =0.106) but was not associated with the other surveys.conclusions: more negative attitudes towards eating predict worse selfesteem and body image, while more positive body image predicts better self-esteem. also, attitudes towards eating, self-esteem and body image are significant predictors of hrqol with body image being the most important predictor.clinical importance: hrqol is an important clinical outcome measure in cf. clinicians need to be sensitive to attitudes towards eating, self-esteem and body image in cf patients, because they are important predictors of hrqol. riekert, k.a. 1 ; mogayzel, p.j. 2 ; bilderback, a. 1 ; hale, w. 1 ; boyle, m.p. 1 1. medicine, johns hopkins university, baltimore, md, usa; 2. pediatrics, johns hopkins university, baltimore, md, usa background: existing research suggests that self-reported adherence to all aspects of the regimen is likely suboptimal and objective measures suggest even poorer adherence. there is little empirical data on how much adherence is necessary to achieve desired health outcomes. moreover, there is limited data on how adherence changes dur-objective: cf may be associated with pain attributed to several etiologies. this study evaluates the prevalence of pain symptoms in adult cf patients and the influence on patients` lives.methods: 243 patients of 6 adult cf centers in germany completed a validated, self report questionnaire during a routine clinic visit assessing characteristics of chronic pain (prevalence, duration, location, quality and intensity of pain symptoms). furthermore, the impact of pain on different aspects of life was explored using a numeric scale from 1 to 10 with 10 being the worst. every-day-life was divided into 8 categories: duties at home, recreation, social activities, occupation, sexual life, autonomy, vital activities and cf-therapy. the average intensity of pain within the last 4 weeks was correlated to fev1, bmi and age.results: 243 patients (119 male) completed the questionnaire. the age was 18-65 years (mean 28.13±8.13). 182 patients (75%) aged 18-65 years (mean 28.51±8.23)reported pain within the last 3 months. bmi was 14.7-38.3 kg/m2 (mean 20.4±3.1), fev1 12-142% (mean 54.7±24.5). if asked for pain within the last 4 weeks 174 (72%) reported painful episodes lasting from 1-28 days with a mean of 9.8 days (±7.8). most patients described pain occurring at more than one site with the head being the most localized site, followed by chest and abdomen. concerning the quality of pain 85.4% characterized their pain episodes as attacks whereas 8.8% reported them as continious, 5.8% had continious pain combined with pain attacks. 179 patients desribed the intensity of pain as 5.77 (±2.35) on a scale from 0 to 10 with 10 being the most severe pain. the category of life negatively influenced most of all by pain episodes was recreation, followed by occupation and duties at home. female patients were more limited in their acitivites by pain symptoms than male patients with the highest difference being reported in sexual life. there was no correlation of the average intensity of pain within the last 4 weeks to fev1, bmi or age.conclusion: the prelevance of pain in cf is often underestimated. painful episodes can be the cause of worsening the quality of life for adult cf patients. assessment of pain should be routinely performed as part of care in cf centers. objective. sleep has been examined in a number of pediatric conditions, with impaired sleep resulting in worse neurobehavioral functioning (beebe, 2005) . recent research has shown that slow wave sleep is critical for memory (marshall, helgadottir, molle, & born, 2006) . to the authors' knowledge, no studies have examined slow wave and rapid-eye-movement (rem) sleep in pediatric patients with cystic fibrosis. methods. retrospective medical record review revealed two patients who underwent clinically indicated polysomnography (psg) who reported feeling tired and lacking energy. patients were females with cystic fibrosis, ages 12 and 14 years. results. psg revealed an increase in stage 2 sleep for both patients, resulting in increased risk and observed hypopneic episodes. patient -a experienced 16.3% in stage 1, 65.6% in stage 2, and 0% in stages 3 and 4 combined. this patient also had no rem sleep. patient -b experienced 12% in stage 1, 67.8% in stage 2, and 10% in delta wave sleep, with no rem sleep. respiratory disturbance index (rdi) for patients a and b were calculated at 3.6 and 3.5, respectively. respiratory effort related arousal (rera) contributed to rdi by 1.9 and 1.1 points in patients a and b, respectively. in addition, endtidal co2 for patient a and b were maxed at 50 mm hg and 48 mmhg, respectively. their epworth sleepiness scales were scored at 8 and 10 for patients a and b, respectively. conclusions. structure of sleep was abnormal in both patients with decreased slow wave (delta) sleep in patient b and lack of delta sleep in patient a. neither patient experienced rem sleep. compensatory increased stage 2 (nrem) sleep could cause respiratory related abnormalities, such as hypopnea and apnea in patients with cystic fibrosis background: understanding and advancing the application of tools to measure patients' symptoms is critical to advancing our evaluation of potential new therapies used to treat cf. we performed initial evaluation of the measurement properties of a cf-specific respiratory symptom daily questionnaire. methods: we planned to enroll 60 cf subjects, stratified by age, at three cf programs, the university of washington medical center and children's hospital and regional medical center in seattle and mary bridge children's hospital in tacoma in a prospective assessment of a novel cf specific respiratory questionnaire. cf subjects 2 years and older were eligible for enrollment. patients (parents for children under the age of 12) completed a daily symptom questionnaire during two 7-day periods of clinical stability and one 7-day period when patients were ill for a total of 21 days. the ill state began when patients/parents sought medical attention for respiratory symptoms. the questionnaires were completed using a secure web-based application or via paper for those patients without access to the internet. two health related quality of life measures (generic and cf specific) were completed by the subjects at the end of each 7-day period to assess the relationship between the novel questionnaire and health related quality of life. patients also used pedometers during the well and ill states to assess relationship between symptoms and activity level.results: 52 cf subjects have been enrolled to date. at the time of this interim analysis, 34 patients had completed 384 questionnaires. of this total, only 2 individual questionnaire entries have been missed in 2 patients (<1% of possible questionnaires). interim review of cross-sectional data suggests clear differences in symptom reporting between the ill and well periods. examples include 46% (13/28) noted difficulty breathing and 32% (9/28) noted tightness in the chest in the preceding 24 hours during the initial well period compared to 70% (14/20) noting difficulty breathing and 70% (14/20) noting tightness in the chest during the ill period.conclusions: interim evaluation of this novel instrument demonstrates feasibility of deploying this instrument via the internet with an extremely high completion rate. using a cross-sectional analysis, the instrument can discriminate between the ill and well state. further data will be presented regarding within patient variability and discrimination of the instrument.supported by the cystic fibrosis foundation leroy matthews physician scientist award, the national heart, lung, and blood institute (hl72017-03), national institute of health (rr-00037-39), cystic fibrosis foundation therapeutics inc. background: improved communication in today's health care delivery system is a critically important aspect of patient care. nurses are in an effective position to improve communication due to their close interaction with patients. as a result, they are able to identify potential problems, communicate them to the healthcare team and improve patient care and satisfaction.hypothesis: improving communication between the patient/family and the health care team will improve patient/family satisfaction.method: as a member hospital partnering with the institute for healthcare improvement's (ihi) initiative, transforming care at the bedside (tcab), the children's hospital of philadelphia (chop) introduced the use of daily patient goal sheets (dpgs) as a vehicle to improve communication between members of the healthcare team, patients and families and the dpgs was refined to meet the needs of an inpatient medical unit to which cf patients are admitted. using rapid plan-do-study-act (pdsa) cycles, a multidisciplinary team engaged in small tests of change in different stages. initially, patient goals and discharge criteria generated in daily rounds were posted in each room on easel paper. then large "whiteboards" were placed in each patient room for documenting patient goals, discharge criteria, names of the care team members and questions from patients and families. as a final step in the process, we shifted the health care team rounds to the patient's bedside and implemented bedside shift to shift nurse report and safety checks. patient satisfaction was continuously monitored over this process using the press ganey survey.results: over an eight month period, the press ganey survey results for the unit showed an average increase of 10%. we attribute this increase to better information sharing among patient, family and hospital staff regarding the plan of care for the patient's hospitalization. the health care team also had an improved dialog with the patients/families. conclusion: patient/family and healthcare team communication and satisfaction were improved using an organized and stepwise pdsa process to develop daily patient goal sheets on a medical unit. background: the uab/chs pediatric cf center provides care for approximately 300 patients. the pulmonary division has 5 full-time rns and 3 part-time rns who rotate phone triage for all calls (including cf) of patients seen by the 9 faculty physicians. historically, patient calls received during office hours are taken by the receptionist and put into the emr, then returned by the rn on call. the rn contacts one of the staff md's, gives report of the patient's condition,treatment orders are received, and the patient notified. there are several issues with this phone triage system, which include inconsistencies in rn and md response as well as differences in documentation. global aim: to improve the consistency among rns in phone triage of sick cf patients.method: an algorithm for pulmonary exacerbation phone triage was developed by the cf center director and lead rns to improve consistency in symptom assessment and treatment plan. a specific list of systemic and pulmonary signs and symptoms was formed including length of illness. if three objective: patients with cf often require intravenous antibiotics for treatment of pulmonary exacerbations. patients often receive peripherally inserted central catheter (picc) lines or totally implantable venous access devices (tivads) for venous access. few studies have examined complications of tivad implantation and little published data exists concerning picc line complications in cf patients. this study sought to assess the complication rates of both tivad and picc lines as well as to identify possible risk factors for developing complications.methods: this retrospective study included patients from 3 cf centers in northern new england. data was obtained from each patient's local medical record, port cf, and patient interviews. demographic data was recorded for all cf patients between 1/1/03-6/1/06. for each tivad or picc line, the following information was recorded: type of line placed, history of prior line placement during the study period, patient age, history of use for blood draws, method of line flushing, and status of the line at the end of the study (if still in use). complications were defined as catheter occlusion, vascular thrombosis/stenosis, infection, or other local inflammatory reactions.results: data was collected for 237 pediatric and 155 adult cf patients during the defined study period. seventy-three tivads and 356 picc lines were placed during the study period in 205 patients. the tivad and picc line complication rates were 33% (24/73) and 6.5% (23/356), respectively. in pediatric patients, 27% (6 of 22) tivads and 6.3% (13/205) picc lines had a complication. of the 6 tivad complications, three were systemic infections and three were catheter occlusions. of the 13 picc line complications, there were two venous thromboses, one line occlusion, two systemic infections, and nine minor incidents of localized phlebitis. in adults, complications were recorded in 35% (18/51) of tivads and 6.6% (10/151) of picc lines. of the 18 tivad complications, there were five systemic infections, seven catheter occlusions, and four venous thromboses. of the 10 picc line complications, there were three venous thromboses, three line occlusions, and four minor incidents of localized phlebitis. all adult patients who developed a deep venous thrombosis(dvt) associated with tivad implantation were homozygous for the deltaf508 mutation. the presence of diabetes or burkholderia cepacia complex(bcc) lung infection was associated with dvt with odds ratios of 5.91 (95% ci 1.16-30.1) and 5.11 (95%ci 0.93-28.1), respectively. conclusions: complications of picc lines were uncommon and usually minor. the rate of tivad complication observed was more common over the lifetime of the catheter and was similar to previously published reports. we identified potential risk factors for the development of dvt associated with tivads and picc lines, specifically cf related diabetes, bcc infection, and homozygous deltaf508 genotype. the mechanism by which these factors are associated with catheter complications is unclear and warrants further investigation.